Synthetic Peptides

SYNTHETIC PEPTIDES
Advances in Molecular Biology Series Editor Allan Jacobson, University of Massachusetts Medical Center Editorial Advisory Board Joan Brugge, ARIAD Pharmaceuticals, Inc. Henry Erlich, Roche Molecular Systems Stanley Fields, University of Washington Larry Gold, NeXstar Pharmaceuticals, Inc.
The Yeast Two-Hybrid System, edited by Paul L. Bartel and Stanley Fields A Comparative Methods Approach to the Study of Oocytes and Embryos, edited by Joel D. Richter Synthetic Peptides: A User's Guide, Second Edition, edited by Gregory A. Grant
SYNTHETIC PEPTIDES A User's Guide

Second Edition
Edited by
Gregory A. Grant
OXPORD
UNIVERSITY PRESS 2002
OXTORD
UNIVERSITY PRESS
Oxford New York Auckland Bangkok Buenos Aires Cape Town Chennai Dar es Salaam Delhi Hong Kong Istanbul Karachi Kolkata Kuala Lumpur Madrid Melbourne Mexico City Mumbai Nairobi Sao Paulo Shanghai Singapore Taipei Tokyo Toronto and an associated company in Berlin
Copyright 2002 by Oxford University Press, Inc.

Published by Oxford University Press, Inc. 198 Madison Avenue, New York, New York 10016 www.oup.com Oxford is a registered trademark of Oxford University Press All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior permission of Oxford University Press. Library of Congress Cataloging-in-Publication Data Synthetic peptides: a user's guide / edited by Gregory A. Grant.2nd ed. p. cm. (Advances in molecular biology) Includes bibliographical references and index. ISBN 0-19-513261-0 1. Peptides Synthesis. 2. Protein engineering. I. Grant, Gregory A., 1949- II. Series. QP552.P4 S963 2002 572'.6545- dc21 2001036643
987654321 Printed in the United States of America on acid free paper
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Contents
Contributors \
ix
Synthetic Peptides: Beginning the Twenty-first Century 3 Gregory A. Grant Peptide Design Considerations 10 Michael L. Moore & Gregory A. Grant Principles and Practice of Solid-Phase Peptide Synthesis Gregg B. Fields, Janelle L. Lauer-Fields, Rong-qiang Liu, & George Barony Evaluation of the Synthetic Product Gregory A. Grant 220 93
Applications of Synthetic Peptides 292 Victor J. Hruby & Terry O. Matsunaga Index 377
Contributors
eorge Barany epartments of Chemistry and Laboratory Medicine and Pathology niversity of Minnesota Minneapolis, Minnesota 55455
Victor J. Hruby Department of Chemistry University of Arizona Tucson, Arizona 85721
regg B. Fields epartment of Chemistry and Biochemistry and the Center for Molecular Biology and Biotechnology lorida Atlantic University oca Raton, Florida 33431
Janelle L. Lauer-Fields Department of Chemistry and Biochemistry and the Center for Molecular Biology and Biotechnology Florida Atlantic University Boca Raton, Florida 33431
regory A. Grant epartment of Molecular Biology and Pharmacology Washington University School of Medicine t. Louis, Missouri 63110
Rong-qiang Liu Departments of Chemistry and Laboratory Medicine and Pathology University of Minnesota Minneapolis, Minnesota 55455
x Contributors Michael L. Moore Smith-Kline Beecham Pharmaceuticals King of Prussia, Pennsylvania 19406
Terry O. Matsunaga Department of Pharmacology and Toxicology School of Pharmacy University of Arizona Tucson, Arizona 85721
SYNTHETIC PEPTIDES
1
Synthetic Peptides
Beginning the Twenty-first Century
Gregory A. Grant
This second edition of Synthetic Peptides is being published at the beginning of the twenty-first century and marks nearly 100 years since the beginnings of the chemical synthesis of peptides. From the first decade of the twentieth century up to the present time, the evolution of the development, analysis, and use of synthetic peptides has been steady and remarkable. It has been about 10 years since the first edition of this book was published. Much remains unchanged, such as the basic principles of peptide structure, the basic chemistry for assembling a peptide chain, and many of the techniques used to evaluate synthetic peptides. However, during that time we have seen a switch from primarily the use of Boc chemistry for routine synthesis to that of Fmoc chemistry. Mass spectrometry has also matured with the development of more user-friendly and affordable instrumentation to the point that it is now the premier analytic method for synthetic peptides. Methods for the production of very long peptides, such as chemoselective ligation, are maturing, although they are still not an everyday thing for most peptide chemists, and better chemistries for producing peptides with "posttranslational modifications," such as phosphates, sugars, and specific disulfide bonds, are now well within reach. As a result, you will find many sections of this book largely unchanged, but you will also find
3
Synthetic Peptides
many new sections that document the developments of the last ten ears with the inclusion of new information and methodologies. However, a good feeling for what the beginning of the twenty-first entury offers can best be appreciated by considering the developments that have led to this point.
Emil Fischer introduced the concept of peptides and polypeptides and resented protocols for their synthesis in the early 1900s (Fischer, 902, 1903, 1906). Although others also made contributions in those ays, most notably Theodor Curtius, the work of Fischer and his olleagues stands out, and he is generally regarded as the father of eptide chemistry. An excellent account of the history of peptide ynthesis which treats the subject much more comprehensively than an be attempted here can be found in a book by Wieland and Bodanszky (1991). Fischer's place in the history of synthetic peptides s eloquently summed up in one sentence from that book which simply tates, "To Emil Fischer we owe the systematic attack on a field of atural substances that had previously been avoided by chemists." In hose early days, the chemistries developed by Fischer and others led o the production of molecules containing as many as 18 amino acids, uch as Leucyl (triglycyl) leucyl (triglycyl) leucyl (octaglycyl) glycine Fischer, 1907). Nonetheless, the syntheses performed were difficult nd limited to simple amino acids, and progress was slow for many ears. Then, the discovery of an easily removable protecting group, the arbobenzoxy group, by Bergmann and Zervas in 1932 (Bergmann nd Zervas, 1932) provided new impetus by opening the way to the se of polyfunctional amino acids. As a result, the synthesis of naturally ccurring small peptides such as carnosine (Sifferd and du Vigneaud, 1935) and glutathione (Harington and Mead, 1935) were soon chieved. Almost 20 years later, the synthesis of an active peptide ormone, the octapeptide oxytocin, by du Vigneaud (du Vigneaud et l., 1953) was acclaimed as a major accomplishment and spurred the dvancement of peptide synthesis once again. The synthesis of the 39 esidue porcine adrenocorticotropic hormone in 1963 (Schwyzer and Sieber, 1963) by solution-phase segment condensation methods was iewed as no less than sensational at the time; and in 1967, Bodanszky nd colleagues succeeded in synthesizing the 27 residue secretin pepide by solution phase stepwise addition methods (Bodanszky and Williams, 1967; Bodanszky et al., 1967). These accomplishments in eptide synthesis were considered to be monumental at the time in hat they were exceptionally difficult undertakings that pushed the revailing technology to its limits. In 1963, Bruce Merrifield published a landmark paper (Merrifield, 1963) describing the development of solid-phase peptide synthesis. This technique, for which he was awarded the Nobel Prize in
Synthetic Peptides
Chemistry in 1984, was responsible, more than anything else, for opening the way to the widespread use of synthetic peptides as reagents in chemical and biomedical investigations. Theodor Wieland (Wieland, 1981) once described the advances in peptide chemistry in the following way: "[T]he synthesis of glutathione opened the door to peptide synthesis a crack, and the synthesis of oxytocin pushed the door wide open." To extend that analogy, the introduction of the solid-phase method blew the door off its hinges. Since that time, many other developments, such as improved synthesis chemistry, automated instrumentation for the unattended production of peptides, and improved purification and analytical methods have contributed to our ability to exploit the potential of synthetic peptides. Not only has there been an explosion in the number of synthetic peptides being produced, but reports of the synthesis of larger and larger peptides (Gutte and Merrifield, 1969, 1971; Clark-Lewis et al., 1986; Nutt et al., 1988; Schneider and Kent, 1988, Muir et al., 1997), some exceeding 100 residues in length, are becoming more commonplace. Now, and for the last 10 years or so, synthetic peptides are available not only to those actively involved in developing the field, but to virtually every investigator in any field who perceives a need for them and wishes to pursue their use. This book is intended primarily for those investigators who wish to utilize synthetic peptides in their research but who themselves are not already intimately involved in the field. This group would encompass researchers who either simply want access to synthetic peptides as tools, or who wish to become actively involved in the production of the peptides, themselves. As such, it strives to provide practical background information, answer basic questions, and address common problems relating to all aspects of synthetic peptides. At the same time, however, it is intended to be current, comprehensive, and sophisticated in its treatment of the subject and should be of general interest to researchers and educators at all levels. The process of obtaining a synthetic peptide for use in biochemical or biomedical research involves several discrete steps which can be represented diagrammatically as shown in figure 1-1. The process starts with the design of the peptide and follows through with the chemical synthesis, evaluation, and purification of the product, and finishes with application or the actual use of the peptide in an experimental situation. As indicated in the diagram, the process can be viewed as pivoting on the evaluation step which actually is part of a more comprehensive evaluation-purification cycle. It is at this point that the product is characterized and its disposition determined. That disposition can be either (1) use of the peptide as is, (2) additional purification and subsequent evaluation, eventually leading to experimental usage, or (3) changes in either the design or synthesis
Synthetic Peptides
Figure 1 -1. Diagram of the steps involved in obtaining a synthetic peptide.
protocols, or both, followed by reiteration of the latter steps of the process. Each subsequent chapter of this book deals with one major aspect of this process, that is, design, synthesis, evaluation/purification, and application. The book begins in chapter 2 with a discussion of the fundamental aspects of peptide structure and uses that as a foundation to build into both basic and more advanced considerations in the design of synthetic pep tides. The design process is a constantly evolving one which is something of an art in itself. In many cases, there are no hard-andfast rules and function usually dictates design. Perhaps the most commonly asked question from general users of synthetic peptides is how to choose a sequence for the production of antibodies. This chapter contains an expanded section on this aspect that draws from what has been learned from the last 10 years. In addition, a section on combinatorial peptide libraries has been added. This is an area that presents great promise for screening very large numbers of peptides for de novo discovery as a complement to rational design and for optimizing structure-activity relationships. Chapter 3 deals with the synthesis chemistry itself. It presents a logical and straightforward explanation of the solid-phase method of peptide synthesis and discusses the overall state of the chemistry today. This chapter places particular emphasis on the Boc and Fmoc protection strategies as the two most useful approaches available and provides specific recommendations for undertaking routine synthesis. It is immediately evident that the chemistry is at the same time very sophisticated and complicated, yet relatively straightforward in the overall approach and amenable to routine procedures. Yet, one must always be mindful that, although routine synthesis using a uniform set of reagents is commonly performed successfully, a vast array of problems can occur. At the same time, there are a large number of alternatives that can be utilized to prevent or circumvent these problems. This chapter therefore presents both recommended "recipes" for routine
Synthetic Peptides
synthesis and more advanced chemical strategies and considerations for the maturing practitioner. This chapter also contains a useful section on the automation of solid-phase synthesis. It gives an informative overview of the range of instruments available today and illustrates the choice of capabilities, from single peptide synthesis to as many as 96 at one time and from microgram to gram quantities. Chapter 4 deals with the evaluation of the finished peptide. It presents a discussion of the latest methods available for the routine characterization of the synthetic product and illustrates their utilization through examples of the evaluation of actual peptides. The message that should be very clear from this chapter is that although the chemistry can in many cases be preprogrammed on modern peptide synthesizers, and these machines do most of the work that used to be done laboriously on the benchtop, the process is by no means foolproof or trivial. Many problems from a large variety of sources can be manifest even in a "routine" synthesis and the final product can never be taken for granted. Every peptide produced must be rigorously evaluated with the ultimate goal being the proof that the peptide obtained is the one intended. In addition, problematic residues and peptide solubility are very important considerations in the evaluation and use of the peptides, which are very often ignored or overlooked. This chapter also contains a more in-depth description of mass spectrometry as an evaluation tool and highlights its use in determining the sequence of a peptide. Chapter 5 presents an excellent survey of the use and application of synthetic peptides. It not only illustrates the diversity in the use and applications of synthetic peptides in modern research but, perhaps, serves as a basis from which new applications and ideas may develop. These include the antigenic and immunogenic use of synthetic peptides, the use of peptides as enzyme inhibitors, structure/function studies involving synthetic peptides, peptide-based vaccines, antisense peptides, and the use of peptides for affinity labeling of receptors or "acceptors" and their use in structure/function studies of receptors. Undoubtedly, some uses for synthetic peptides have not been included, but the areas that are discussed are among the more exciting and successful applications being investigated today. Furthermore, many of the concepts and approaches discussed can be easily adapted to other systems and areas of exploration. Also, since the use very often dictates the design, many aspects of peptide design are discussed in this chapter and, as such, it complements chapter 2 very nicely. Peptide synthesis has come a long way from the beginning of the twentieth century to the present day. It has spanned the twentieth century and has enhanced the passing of that period. As we start the new millennium, it is tempting to speculate on what may lie ahead. However, history has told us that such attempts invariably fall far
Synthetic Peptides
short of the eventual reality. After all, even Merrifield admitted in a 1986 article (Merrifield, 1986) that he did not foresee the impact of his technique: "From the accumulated data presented, I conclude that the solid phase synthesis of peptides up to 50 or somewhat more residues can be readily achieved in good yield and purity; this is a far better situation than I could have expected when this technique was first proposed" (p. 345). Undoubtedly, in the future great things will be accomplished in peptide synthesis and in the use of the peptides themselves. It is, perhaps, sufficient to have had the privilege of taking part in a small bit of that process.
References
Bergman, M., and Zervas, L. (1932). Uber ein allgemeines Verfahren der Peptid-Synthese. Ber. Dtsch. Chem. Ges. 65:1192-1201. Bodanszky, M., and Ondetti, M. A. (1966). Peptide Synthesis, New York, John Wiley and Sons. Bodanszky, M., and Williams, N. J. (1967). Synthesis of secretin I. The protected tetradecapeptide corresponding to sequence 14-27. J. Am. Chem. Soc. 89:685-689. Bodanszky, M., Ondetti, M. A., Levine, S. D., and Williams, N. J. (1967). Synthesis of secretin II. The stepwise approach. J. Am. Chem. Soc. 89:6753-6757. Clark-Lewis, I., Aebersold, R., Ziltener, H., Schrader, J. W., Hood, L. E., and Kent, S. B. H. (1986). Automated chemical synthesis of a protein growth factor for hemopoietic cells, Interleukin-3. Science 231:134-139. Fischer, E. (1902). Ueber einige Derivate des Glykocolls Alanins und Leucins. Ber. Dtsch. Chem. Ges. 35:1095-1106. Fischer, E. (1903). Synthese von Derivaten der Polypeptide. Ber. Dtsch. Chem. Ges. 36:2094-2106. Fischer, E. (1906). Untersuchungen iiber Aminosauren, Polypeptide, und Proteme. Ber. Dtsch. Chem. Ges. 39:530-610. Fischer, E. (1907). Synthese von Polypeptiden XVII. Ber. Dtsch. Chem. Ges. 40:1754-1767. Gutte, B., and Merrifield, R. B. (1969). The total synthesis of an enzyme with ribonuclease A activity. J. Am. Chem. Soc. 91:501. Gutte, B., and Merrifield, R. B. (1971). The synthesis of ribonuclease A. J. Biol. Chem. 246:1922. Harington, C. R., and Mead, T. H. (1935). Synthesis of glutathione. Biochem. J. 29:1602-1611. Merrifield, R. B. (1963). Solid phase peptide synthesis I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149-2154. Merrifield, R. B. (1986). Solid phase synthesis. Science 232:341-347. Muir, T. W., Dawson, P. E., and Kent, S. B. H. (1997). Protein synthesis by chemical ligation of unprotected peptides in aqueous solution. Meth. Enzymol. 289:266-298.
Synthetic Peptides
Nutt, R. F., Brady, S. F., Darke, P. L., Ciccarone, T. M., Colton, C. D., Nutt, E. M., Rodkey, J. A., Bennett, C. D., Waxman, L. H., Sigal, I. S., Anderson, P. S., and Veber, D. F. (1988). Chemical synthesis and enzymatic activity of a 99 residue peptide with a sequence proposed for the human immunodeficiency virus protease. Proc. Natl. Acad. Sci. U.S.A. 85:7129-7133. Schneider, J., and Kent, S. B. H. (1988). Enzymatic activity of a synthetic 99 residue protein corresponding to the putative HIV-1 protease. Cell 54:363-368. Schwyzer, R., and Sieber, P. (1963). Total synthesis of adrenocorticotrophic hormone. Nature 199:172-174. Sifferd, R. H., and du Vigneaud, V. (1935). A new synthesis of carnosine, with some observations on the splitting of the benzyl group from carbobenzoxy derivatives and from benzylthio ethers. J. Biol. Chem. 108:753-761. Vigneaud, V. du, Ressler, C., Swan, J. M., Roberts, C. W., Katsoyannis, P. G., and Gordon, S. (1953). The synthesis of an octapeptide amide with the hormonal activity of oxytocin. J. Am. Chem. Soc. 75:4879^880. Wieland, T. (1981). From glycylglycine to ribonuclease: 100 years of peptide chemistry. In Perspectives in Peptide Chemistry, A. Eberle, R. Geiger, and T. Wieland, eds., Basel, Karger, pp. 1-13. Wieland, T., and Bodanszky, M. (1991). The World of Peptides: A Brief History of Peptide Chemistry, Berlin, Springer-Verlag.
Peptide Design Considerations

Michael L Moore Gregory A. Grant
Peptides have become an increasingly important class of molecules in biochemistry, medicinal chemistry, and physiology. Many naturally occurring, physiologically relevant peptides function as hormones, neurotransmitters, cytokines, and growth factors. Peptide analogs that possess agonist or antagonist activity are useful as tools to study the biochemistry, physiology, and pharmacology of these peptides, to characterize their receptor(s), and to study their biosynthesis, metabolism, and degradation. Radiolabeled analogs and analogs bearing affinity labels have been used for receptor characterization and isolation. Peptide substrates of proteases, kinases, phosphatases, and aminoacyl or glycosyl transferases are used to study enzyme kinetics, mechanism of action, and biochemical and physiological roles and to aid in the isolation of enzymes and in the design of inhibitors. Peptides are also used as synthetic antigens for the preparation of polyclonal or monoclonal antibodies targeted to specific sequences. Epitope mapping with synthetic peptides can be used to identify specific antigenic peptides for the preparation of synthetic vaccines, to determine protein sequence regions that are important for biological action, and to design small peptide mimetics of protein structure or function. A number of peptide hormones or analogs thereof, including arginine vasopressin, oxytocin, luteinizing hormone releasing hormone
10
11
LHRH), adrenocorticotropic hormone (ACTH), and calcitonin, have lready found use as therapeutic agents, and many more are being nvestigated actively. Peptide-based inhibitors of proteolytic enzymes, uch as angiotensin converting enzyme (ACE) and human immunoeficiency virus (HIV) protease, have widespread clinical use, and nhibitors of renin and elastase are also being investigated for herapeutic use. Finally, peptides designed to block the interaction f protein molecules by mimicking the combining site of one of the roteins, such as the fibrinogen receptor antagonists, show great herapeutic potential as well. With the development of solid-phase peptide synthesis by Bruce Merrifield (1963) and the optimization of supports, protecting groups, nd coupling and deprotection chemistries by a large number of esearchers, it has become possible to obtain useful amounts of peptides n a more or less routine basis. With the increasing ease of synthesizing eptides, it has become all the more important to understand the nderlying principles of peptide structure and physical chemistry that overn solubility, aggregation, proteolytic resistance or susceptibility, econdary structure stabilization or mimicry, and interaction with onpolar environments like lipid membranes. The design of any specific peptide depends primarily on the use for which it is intended as well as on synthetic considerations. A number f aspects of the peptide sequence and structure can be manipulated to ffect solubility, proteolytic resistance, or stability, and the preilection to adopt specific secondary structures to produce peptides with specific properties. It is this topic that will be addressed in this hapter. The distinction between what constitutes a peptide and what contitutes a protein becomes increasingly fuzzy as peptides increase in ength. An operational definition in the context of this chapter might e that a peptide is any sequence that the researcher can conveniently ynthesize chemically. Although such proteins as ribonuclease A (124 mino acids) (Hirschmann et al., 1969; Yajima and Fujii, 1981), acyl arrier protein (74 amino acids) (Hancock et al., 1972), and the HIV rotease (99 amino acids) (Nutt et al., 1988; Wlodawer et al., 1989) ave been chemically synthesized, most peptide synthesis generally nvolves peptides of 30 amino acids or less. The underlying principles pplied to peptide design will be dependent in some degree on peptide ength, especially those relating to peptide secondary structure. For xample, shorter peptides do not tend to exhibit preferred solution onformations and possess a large amount of segmental flexibility. If econdary structure is important in a small peptide's design, it must be pproached by chemical modifications designed to decrease conformaional flexibility. As peptides increase in length, they have a greater endency to exhibit elements of secondary structure, with a consequent
Synthetic Peptides
ecrease in segmental flexibility. In such cases, secondary structure can ten be induced by optimizing the peptide sequence.
rotein and Amino Acid Chemistry
he physical and chemical properties of proteins and peptides are etermined by the nature of the constituent amino acid side chains nd by the polyamide peptide backbone itself. Twenty protein amino cids are coded for by DNA, which are translationally incorporated to proteins. Amino acids can be modified in the protein postanslationally to yield new amino acids. Also, many peptides are ynthesized enzymatically rather than ribosomally, especially in lower ukaryotes, and those peptides often contain highly unusual amino cids.
rotein Amino Acids
he structures of the 20 primary protein amino acids (those coded for y DNA) are given in table 2-1 along with their three-letter abbreviaons, one-letter codes, and a general grouping by physical properties. mino acids generally can be divided into hydrophobic and hydrohilic residues. The hydrophobic residues include those with aliphatic de chains, such as alanine, valine, isoleucine, leucine, and methionine, nd those with aromatic side chains, such as phenylalanine, tyrosine, nd tryptophan. The hydrophilic residues include amino acids with (1) eutral, polar side chains, such as serine, threonine, asparagine, and utamine; (2) those with acidic side chains, such as aspartic acid and utamic acid; and (3) those with basic side chains such as histidine, sine, and arginine. It can be appreciated that these categories are not ntirely exclusive. Alanine, with its small aliphatic side chain, and ycine can be found in hydrophilic regions of peptides and proteins. onversely, the long alkyl chains of lysine and arginine can give those sidues an overall hydrophobic character with just the terminal harged group being hydrophilic. Two amino acids, cysteine and proline, have special properties that et them apart. Cysteine contains a thiol moiety that can be oxidatively oupled to another cysteine thiol to form a disulfide linkage. Bisulfides e the principal entities by which peptide chains are covalently linked gether to stabilize secondary or tertiary structure or to hold two fferent peptide chains together. Although the disulfide form is the ost stable form under normal aerobic conditions, free thiols are also resent in some proteins, where they often serve as ligands for metal helation, as nucleophiles in proteolytic enzymes, such as papain, or as arboxyl activators in acyl transferases. The secondary amino acid roline has specific conformational effects on the peptide or protein
Peptide Design Considerations 13 Table 2-1 Amino acid structures and properties
Glycine Gly G
Alanine Ala A Neutral, hydrophobic, aliphatic
Valine Val V
Isoleucine He I
Leucine Leu L Neutral, hydrophobic, aliphatic
Methionine Met M
Phenylalanine Phe F
Tyrosine Tyr Y Neutral, hydrophobic, aromatic
Tryptophan Tip W
(continued)
14
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Table 2-1 (Continued)
Serine Ser S
Threonine Thr T Neutral, hydrophilic
Asparagine Asn
Glutamine Gin Q Neutral, hydrophilic
Aspartic Acid Asp D
Glutamic Acid Glu E
Acidic, hydrophilic
Histidine
His H
Lysine Lys K Basic, hydrophilic
Arginine Arg R
Cystetne Cys C Thiol-containing
Proline Pro P Imino acid
15
Figure 2-1. Some amino acids formed by post-translational modification.
backbone both because of its cyclic structure and because of the alkylation of the amino group. It often plays an important role in stabilizing or influencing the secondary structure of proteins, as will be discussed later. Some amino acids can be modified enzymatically after incorporation into certain proteins to give rise to new amino acids, some of which are shown in figure 2-1. In collagen, for example, proline can be hydroxylated to yield fra/w-4-hydroxyproline (Hyp). Glutamic acid is carboxylated by a vitamin K-dependent carboxylase to yield y-carboxyglutamic acid (Gla) in a number of proteins involved in blood coagulation, where the malonyl moiety of the Gla residue is thought to be important in providing a bidentate ligand for calcium ions. The hydroxyl functions of tyrosine, serine, and threonine can be reversibly phosphorylated by kinases and phosphatases, a process thought to be an important regulator of biological activity in the target proteins. The hydroxyl of tyrosine may also be sulfated in peptide hormones such as gastrin and cholecystokinin. One important nonenzymatic transformation of a protein amino acid occurs with glutamine, which is chemically unstable at the amino terminus of a peptide or protein in aqueous solution. Glutamine will spontaneously cyclize to form pyroglutamic acid (pyrrolidone carboxylic acid, also shown in figure 2-1). This transformation typically occurs when an internal glutamine residue s exposed by proteolysis of an X-Gln bond (where X represents any amino acid) or during protein sequencing. All amino acids (except for glycine) are chiral molecules with an asymmetric center at the a-carbon. The protein amino acids all have
16
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the L absolute configuration at this center, as shown in the structure at the top of table 2-1. The L does not refer to the direction of optical rotation but, rather, to having the same stereochemical arrangement as L(-)-glyceraldehyde. In the more unambiguous Cahn-Ingold-Prelog convention, L-amino acids have the S absolute configuration (except cysteine, for which this configuration is defined as R). Two amino acids, threonine and isoleucine, have a second asymmetric center at the /J-carbon, as shown in table 2-1. Threonine has the R absolute configuration at the ^-carbon; that is, it is 2-(5)-amino-3(7?)hydroxybutanoic acid. Isoleucine has the S absolute configuration at the /J-carbon; that is, it is 2-(5)-amino-3-(5)-methylpentanoic acid. Because there are two asymmetric centers in the molecule, four stereoisomers exist for each of these amino acids. The o-amino acid has the opposite configuration at both asymmetric centers; D-isoleucine is the 2-(R)-3-(R) isomer, for example. The trivial prefix allo is used to denote inversion at only one asymmetric center. Thus D-allo-isoleucine is the 2-(R)-3-(S) isomer. o-allo-Isoleucine occasionally forms as an artifact during hydrolysis of isoleucine-containing peptides or proteins. Because it is a diastereomer of isoleucine rather than an enantiomer, it will appear as a separate peak in amino acid analyses. Nonprotein Amino Acids Literally hundreds of naturally occurring amino acids exist that are not found in proteins. Some of them, like y-aminobutyric acid, have important functions as neurotransmitters. Others, like ornithine, appear as intermediates in metabolic pathways or, like dihydroxyphenylalanine, are precursors to amino acid-derived products, catecholamines in this case. By far the largest number of nonprotein amino acids are found in prokaryotes and lower eukaryotes, especially in algae, sponges, yeasts, and fungi, although peptides with unusual amino acids have been isolated from chordates, such as tunicates, as well. These amino acids are incorporated into peptides by enzymatic synthesis rather than ribosomally. More than 700 of these nonprotein amino acids are known, and the structural variations are immense (Hunt, 1985). Those amino acids and the peptides containing them are usually the products of secondary metabolism, and their function in the producing organism is often obscure. Those unusual amino acids can confer unusual biological activities on peptides that contain them. Such peptides have been the basis either of biochemical tool molecules or of antibiotics, immunomodulators or antineoplastic agents that have therapeutic utility. Nonprotein amino acids can be roughly categorized into a few basic structural types, representative members of which are depicted in
17
Figure 2-2. Some nonprotein amino acids.
figure 2-2. Some nonprotein amino acids are simply the enantiomeric D-amino acid analog of a protein L-amino acid, for example, o-alanine and o-glutamic acid, which are important constituents of the proteoglycan bacterial cell wall. Others have a normal a-amino acid structure but with a novel side chain. The side chain can be a simple alkyl group, such as in norvaline, or it can be quite unusual, such as in 4-()butenyl-4(/?)-methyl-Ar-methyl-L-threonine (MeBmt), a critical constituent of the immunosuppressive peptide cyclosporin A (Riiegger et al., 1976). Some nonprotein amino acids deviate from the normal a-amino acid structure and are methylated on their amine function, such as the jV-methylleucine (MeLeu) of the tunicate-derivated antineoplastic peptide didemnin B (Rinehart et al., 1981), or on their acarbon, such as the aminoisobutyric acid (a-methylalanine, Aib) of the ionophoretic antibiotic peptide alamethicin (Payne et al., 1970; Pandey et al., 1977). In addition, there are large numbers of amino acids in which the amino group is not on the a-carbon but at some other position in the molecule. Sometimes several of these features are combined in one amino acid, such as statine [3-(5')hydroxy-4-(5)-amino-6methylheptanoic acid, Sta], the critical residue in the fungal protease inhibitor pepstatin (Morishima et al., 1970). Side-Chain Interactions Side chains interact with each other, with the amide peptide backbone, with bulk solvent, and through noncovalent interactions, such as
Synthetic Peptides
drogen bonds, salt bridges, and hydrophobia interactions. Cysteine o participates in a covalent interaction, disulfide bond formation.
drogen Bonds and Salt Bridges proteins, polar side chains tend to be extensively solvated. Acidic sp and Glu) and basic (Lys, Arg, and His) residues generally are und on the protein surface with the charged ends of the side chains ojecting into the bulk solvent, although the alkyl portion of the Lys d Arg side chains is usually buried. Internal charged residues are most invariably involved in salt bridges, where acidic and basic side ains are either directly bonded ionically to each other or connected a single intermediary water molecule (Baker and Hubbard, 1984). onionic polar residues (Ser, Thr, Asn, Gin, and Tyr at the phenolic droxyl) are also extensively hydrogen-bonded, either to bulk solvent to backbone, other side-chain groups, or to specifically bound water olecules (Thanki et al., 1990). In helices, the side chains of Ser, Thr, d Asn often make specific hydrogen bonds to the carbonyl oxygen the third or fourth residue earlier in the sequence, which may help stabilize helical segments (Gray and Matthews, 1984). In shorter ptides, side-chain hydration occurs mostly through the bulk solvent, hough the ability to form low-energy intramolecular hydrogennded structures or salt bridges may be an important factor in the sociation of peptides with macromolecular targets or receptors. Consideration of solvation and desolvation effects is a potentially tical, but often overlooked, aspect of peptide design and structuretivity relationships. One case will serve to illustrate the importance solvation in interpreting structure-activity studies. Bartlett and workers prepared a series of thermolysin inhibitors that were sed on the peptide substrate sequence Cbz-Gly-Leu-Leu-OH but ntained a phosphoryl moiety in place of the Gly carbonyl function Morgan et al., 1991). The phosphoryl moiety was designed to mimic e tetrahedral transition state for amide bond hydrolysis and was ggested by a naturally occurring glycopeptide inhibitor of thermosin, phosphoramidon, which contains a similar phosphoryl group. he inhibitors that were prepared are shown in table 2-2. Compound which contains a phosphoramidate linkage (PO2NH), was found to a potent inhibitor. X-ray crystal structure analysis of the enzymehibitor complex showed a hydrogen bond between the phosphormidate NH and a backbone carbonyl in the enzyme. Compound 2 ntained a phosphonate linkage (PO2O). It was unable to form the me hydrogen bond as compound 1 because it lacks the correspondg hydrogen. It was found to be three orders of magnitude less potent an compound 1, but X-ray crystal analysis showed that it bound to e enzyme in an identical fashion (except for the absence of the
19
Table 2-2 Phosphorus-containing thermolysin inhibitors
Compound
X
NH O
CH2
Ki(nM)
9.1
1 2 3
9000
10.6
hydrogen bond). Compound 3 contained a phosphinate linkage (PO2CH2). Like compound 2, it could not form that same hydrogen bond. Unlike compound 2, however, it was essentially equipotent with the phosphoramidate 1. The explanation comes from consideration of solvation and desolvation effects. Both the phosphoramidate (1) and the phosphonate (2) will be solvated in aqueous solution and require desolvation to bind to the enzyme active site. The phosphoramidate (1) can recover some of the energy required for desolvation by forming a hydrogen bond with the enzyme, but the phosphonate (2) cannot. The phosphinate (3) is somewhat less polar due to the presence of a methylene group instead of an amine or oxygen moiety. It is correspondingly less solvated in aqueous solution, and, therefore, it requires less desolvation when it binds to the enzyme. The net change in free energy in going from solution to enzyme-bound states is roughly comparable for compound 1 (with the PO2X group going from solvated to solvated states) and compound 3 (going from unsolvated to unsolvated states) and is more favorable than for compound 2 (going from solvated to unsolvated states).
Hydrophobic Interactions
Just as hydrophilic residues tend to be solvated, hydrophobic side chains have an equally strong tendency to avoid exposure to the aqueous environment. This effect is largely entropic, reflecting the unfavorable free energy of forming a water-hydrocarbon interface, where the side chain would penetrate the aqueous solvent (Tanford, 1973; Burley and Petsko, 1988). It has long been recognized that the interiors of soluble proteins are highly hydrophobic (Kauzmann, 1959)
20
Synthetic Peptides
and that proteins fold in such a way as to minimize the exposure of hydrophobic side chains on the protein surface. It has been proposed that protein folding proceeds first through the formation of hydrophobic clusters which direct further folding of the peptide chain into the various low-energy secondary structures, such as helices and ^-structure (Rose and Roy, 1980). The importance of hydrophobic interactions to protein stability has been studied by comparing the susceptibility to denaturation of a series of proteins in which site-specific mutagenesis was used to modify single residues in the protein sequence (Kellis et al., 1988). It was found that the absence of even a single methyl group (He to Val substitution) destabilized the protein by 1.1 kcal/mol, underscoring the important cumulative effect of hydrophobic interactions on overall protein structure and stability. Although the sequestering of nonpolar residues away from the aqueous environment is largely an entropy-driven process, specific interactions of hydrophobic side chains occur as well. These are typically induced dipole-induced dipole interactions, known as van der Waals or London interactions (Burley and Petsko, 1988). Although they are much weaker than the salt bridges and hydrogen bonds involving polar residues, they can be important in local secondary structure and protein interactions. For example, a number of DNA-binding proteins that function biologically as dimers share an unusual sequence, featuring a leucine every seventh residue in a 30-residue segment and having a relatively high probability of helical structure. This sequence would create a helix in which the leucine residues occupied every other turn of the same side of the helix. The structure was termed a "leucine zipper" based on the hypothesis that the leucines from such a helix in two monomers could interdigitate like a zipper, holding the monomers together (Landschutz et al., 1988). Although it was subsequently found that the Leu residues do not interdigitate, but rather align themselves parallel to each other in pairs along the helical interface (O'Shea et al., 1989), the term leucine zipper has remained, and it is an important structural motif in hydrophobic protein-protein interaction. Aromatic residues have an inherent dipole with the electron-rich 7r-cloud lying parallel to and above and below the plane of the ring and with positively polarized hydrogen atoms in the plane of the ring (Burley and Petsko, 1988). Although aromatic rings in proteins do interact with each other, the arrangement is not typified by the parallel stacking of base pairs in DNA helices. Rather, the edge of one ring interacts with the face of the other in a roughly perpendicular arrangement (Burley and Petsko, 1985), allowing a favorable interaction between the positively polarized hydrogens and the negatively polarized 7r-cloud. Peptides, in general, are not long enough to allow the hydrophobic residues to arrange themselves in such a way that they are totally
Peptide Design Considerations 21
shielded from solvent. This occurrence undoubtedly contributes to the poor aqueous solubility of many peptides compared to proteins. Many peptides require the presence of strong organic co-solvents like dimethylsulfoxide (DMSO), dimethylformamide (DMF), or ethanol to achieve sufficient solubility for biological testing, which may be a limiting factor in some biological test systems. Peptides that have substantial hydrophobic character also tend to aggregate with increasing concentration. Solubility generally increases with peptide length because of the peptide's consequent ability to adopt stable secondary structures and to segregate nonpolar residues.
Disulfide Bonds, Thioesters, and Thioethers Unlike side-chain interactions of other residues that are noncovalent in nature, cysteine residues form a number of covalcnt linkages with other amino acid side chains. The most common of these is the disulfide, which involves oxidative coupling of two cysteine thiol groups to form cystine (figure 2-3). Like the amide bond, the sulfur-sulfur bond in a disulfide is not freely rotatable. Rather, it exists in one of two rotamers, with torsional angles in the vicinity of either +90 or -90. The entire disulfide moiety, CH-S-S-CH2, can rotate as a unit by simultaneous rotation about the side-chain angles xl (NH-CaH-CH2-S) and x2 (CaH-CH2-S-S). In proteins, disulfide bonds are important in the stabilization of tertiary structure. In disulfide-containing peptides like oxytocin, vasopressin, and somatostatin (see figure 2-4), the disulfide has proportionally greater importance in maintaining a biologically active conformation because the peptides are too small to maintain a stable conformation otherwise. Replacement of the cysteine residues with isosteric alanine residues results in a dramatic loss in biological activity (Walter ct al., 1967; Polacek et al., 1970; Sarantakis et al., 1973). A great deal of conformational flexibility still remains in cyclic disulfide-containing peptides like vasopressin, and it does not exhibit a single preferred conformation (Hagler et al., 1985), but the disulfide does constrain the peptide to folded conformations that otherwise would have a low probability for linear peptides. Cysteine residues can also form covalent thioestcr and thioether linkages. The thioester is found as the activated form of acyl groups in acyl transferases and as an acyl enzyme intermediate in thiol proteases such as papain. Thioesters also are found in the reactive binding sites of complement C3b and a2-macroglobulin, where the tetrapeptide sequence Cys-Gly-Glu-Glu contains a thioester involving the Cys-1 thiol and the Glu-4 side-chain carboxylate (figure 2-3) (Sothrup-Jensen et al., 1980; Tack et al., 1980; Howard, 1981). Cysteine forms aliphatic thioether linkages, such as in lanthionine, a
Synthetic Peptides
Figure 2-3. Disulfide, thioester, and thioether linkages.
Figure 2-4. Disulfide-containing peptides.
constituent of the peptide antibiotics subtilin (Alderton and Fevold, 1951) and nisin (Berridge et al., 1952). Aromatic thioether linkages also occur, such as in the Cys-Trp conjugate found in mushroom toxins such as amanitin and phalloidin (Wieland, 1968). A novel thioether Cys-Tyr conjugate recently has been identified in the enzyme galactose oxidase (Ito et al., 1991).
The Amide Bond
The polyamide peptide backbone is also an important contributor to overall protein and peptide structure. A substantial double-bond character is found in the carbon-nitrogen peptide bond due to the resonance structure shown in figure 2-5(a). This structure gives the amide bond several characteristics that are important in peptide and protein structure. The amide bond is flat, with the carbonyl carbon, oxygen, nitrogen, and amide hydrogen all lying in the same plane. No free rotation occurs about the carbon-nitrogen bond because of its partial double-bond character (the barrier to rotation is about 25 kcal/mol). The torsional angle of that bond, CD, is defined by the peptide backbone atoms Ca-C(O)-N-Ca. Because of the partial double-bond character, there are two rotational isomers for the peptide bond: trans (&>=180) and cis (a> = 0), as shown in figure 2-5(b). The lower-energy isomer is the trans peptide bond, which is the isomer generally found for all peptide bonds not involving proline. In the case of amide bonds involving proline, the energy of the trans X-Pro bond is somewhat elevated, and both the difference in energy between cis and trans isomers and the barrier to rotation is lowered. Proline-containing peptides thus will often exhibit cis-trans isomerism about the X-Pro bond. This can be detected by nuclear magnetic resonance (NMR) studies because the chemical shifts of some hydrogens, especially those on the proline 5-carbon, are often different. The equilibrium ratio of cis to tans isomers and the rate of isomerization are highly dependent on the exact peptide sequence. Empirically, it has been found that the cis content of X-Pro peptides generally increases when X is a bulky, hydrophobic amino acid (Harrison and Stein, 1990). It has been appreciated only recently that cis X-Pro peptide bonds may be important in proteins as well. The X-ray crystal structure of several proteins has revealed specific cis X-Pro peptide bonds (Frommel and Preissner, 1990). Proline isomerization has also been implicated as a slow step in protein refolding (Kim and Baldwin, 1982). The resonance forms of the amide bond give it one other characteristic that is extremely important in peptide and protein structure. The
24
Synthetic Peptides
Figure 2-5. Characteristics of amide bonds.
amide bond is quite polar and has a significant dipole moment, which makes the amide carbonyl oxygen a particularly good hydrogen-bond acceptor and the amide NH a particularly good hydrogen-bond donor. Hydrogen bonds involving the peptide backbone are an important stabilizing factor in protein secondary structures. Peptide bonds have a strong tendency to be solvated, by either bulk solvent or specifically bound water molecules, or by internal hydrogen bonds. This is especially true in the hydrophobic interior of soluble protein molecules. Peptide bonds of regular secondary structures, such as helices and ^-sheets, are internally solvated by the hydrogen bonds that stabilize those structures. The peptide bonds of the so-called random coil, or irregular structures, also participate in an extensive network of hydrogen bonds involving internal polar side chains, bound water molecules, and backbone interactions. The polarity of the amide bond also can impart a net dipole to regular structures containing peptide bonds, such as helices. The overall dipole points from the amino terminus (positive partial charge) to the carboxyl terminus (negative partial charge) in a helix, which can be an important factor in protein tertiary structure as well as a contributor to catalytic activity in enzymes. The dipole can be used to stabilize interactions with substrates or to modify the pKa, or nucleophilicity, of catalytically active residues (Knowles, 1991).
Protein Structure
Protein structure is organized in several hierarchical levels of increasing structural complexity. The most basic level is the primary structure, or amino acid sequence, of the protein. Secondary structure deals with the folding up of short segments of the peptide, or protein chain, into regular structures such as a-helices, /3-sheets, and turns. Tertiary structure describes how the secondary structural elements of a single protein chain interact with each other to fold into the native protein structure. Quaternary structure involves the interaction of individual protein subunits to form a multimeric complex. In the design of peptides, we are concerned mainly with the primary and secondary structures. The primary structure, or sequence, of a protein, is readily obtainable by chemical sequence analysis of the isolated, purified protein or by translation of the corresponding DNA coding sequence. Since it is easier to sequence DNA than to sequence protein, most new protein sequences are now obtained in this way. Computer programs are available, which will be discussed later (see below, Prediction of Protein/Peptide Structure), to help analyze and compare protein primary structures. Protein secondary structure has also become increasingly well understood, to the point that it can be predicted or optimized on the basis of the primary sequence reasonably well by using computer algorithms. In addition, several techniques have been applied to either induce or stabilize secondary structure in peptides, which will be discussed in more detail later (see below, Conformational Design and Constraint).
Protein Secondary Structure
Chains of amino acids can fold into several types of regular structures that are stabilized by intrachain or interchain hydrogen bonds in the amide backbone. Helices and turns are formed from continuous regions of protein sequence and are stabilized by intrachain hydrogen bonds. Sheets are formed from two or more chains, which are separated by intervening sequences (regions of secondary structure as well), and are stabilized by interchain hydrogen bonds. The parts of a protein sequence that do not appear in helices, sheets, or turns are said to be in random coil, which is meant to imply only that no regular, repeating structure exists. It should not be taken to mean that the conformation of the protein is random in those regions, because in any individual protein, the random coil sequences are just as highly ordered and reproducibly formed as other regions of secondary structure. The conformation of the peptide backbone can be described by three torsional angles: 0, which is the angle defined by C(O)-N-Ca-C(O); V> which is defined by N-Ca-C(O)-N; and the amide torsional angle a>,
26
Synthetic Peptides
Figure 2-6. Torsional angles of the peptide backbone, (a) Backbone torsional angles 0, ijs, and a>. (b) Newman projections of torsional angles 0 and ^r.
which was discussed previously (see above, The Amide Bond). These angles are shown graphically in figure 2-6(a). The convention in protein chemistry is that these angles are defined with respect to the peptide backbone. The angle 0 is 180 when the two carbonyl carbons are trans to each other, and the angle iff is 180 when the two amide nitrogens are trans to each other as in figure 2-6(b). The angle i/r, therefore, is + 180 compared to the usual chemical definition. A fully extended peptide chain, however, would have backbone torsional angles all of 180, just like a fully extended carbon chain.
Helices One of the most common types of secondary structure in proteins is the helix in which amino acid residues are wrapped around a central
Peptide Design Considerations 27 Table 2-3 Torsional angles for helices

<
a-Helix (right-handed) a-Helix (left-handed) 310 Helix Collagen helix
*
-47
-57
57
-60 -51 -76 -45 -78
47
-30 153 127 148 149
Polyproline
Angles taken from IUPAC-IUB Commission on Biochemical Nomenclature (1970).
axis in a regular pattern. Given planar, trans peptide bonds, helices will be uniquely defined by their 0 and V angles. Several types of helices are found in protein structures, the a-helix being the most common. These helices and their characteristic torsional angles are listed in table 2-3. Helices are also characterized by the number of residues per turn and the hydrogen bonding pattern, specifically the number of atoms in the cyclic structure formed by the hydrogen bond. The or-helix is a 3.613 helix, meaning there are 3.6 residues per turn of the helix, and 13 atoms are included in each repetitive hydrogen-bonding structure. Likewise, a 3j 0 helix has three residues per turn and 10 atoms in the cyclic hydrogen-bonded structure. These are the two most relevant helices for protein and peptide structure. There is also a handedness to helical structure, which is defined by the screw sense of the helix. Looking down the helical axis from the amino terminal end, the peptide backbone can be traced from amino terminus to carboxyl terminus in a clockwise sense in a right-handed helix, which is the form that generally occurs. Helical structures are stabilized by intrachain hydrogen bonds. In a helix, the carbonyl bonds and amide NH bonds lie parallel to the helix's axis with the carbonyls pointing downward, in the direction of the carboxyl terminus of the helix, and the NHs pointing upward, in the direction of the amino terminus. In an a-helix, the carbonyl of any residue i is hydrogen-bonded to the amide NH of the / + 4 residue; that is, the residue four amino acids farther down along the peptide chain. In a 310 helix, the rth residue carbonyl is hydrogen-bonded to the NH of the i + 3 residue. These relationships are shown graphically in figures 2-7 and 2-8 for six-residue segments of a and 3i 0 helices as viewed from the side, perpendicular to the helical axis. For clarity, only the peptide backbone atoms are shown. The helix forms a poly amide cylinder. The uniform arrangement of carbonyl and NH groups imparts a strong dipole moment to the helix,
28
Synthetic Peptides
Figure 2-7. Backbone structure of an a-helix. Note that the NH and carbonyl groups are aligned parallel to the axis of the helix. Each hydrogen-bonded segment contains 13 atoms.
with the positive end at the amino terminus and the negative end at the carboxyl terminus. The side chains of the residues are arranged radially outward from the helix, looking down the helix axis (figure 2-9). Because all the backbone amide groups are involved in intrachain hydrogen bonds, the interactions of helices with other peptide chains or small molecules occurs predominantly through side-chain interactions (hydrophobic interactions, salt bridges, and hydrogen-bonding interactions; see above, Side-Chain Interactions) and interactions with the helix dipole itself. Because of this arrangement of intrachain hydrogen bonds, the amide bonds of the helix can be thought of as being internally solvated, resulting in the helix being more readily accommodated in a nonpolar environment, such as a lipid bilayer. The membrane-spanning regions of transmembrane proteins are generally thought to be helical, with the axis of the helix perpendicular to the plane of the membrane (Eisenberg, 1984; Wickner and Lodish, 1985). The membrane poreforming peptide antibiotics, such as alamethicin, also have a highly helical structure, and a high degree of helix potential exists in the
29
opposite directions. Both can be found in protein structures. These two arrangements are shown diagrammatically in figure 2-10. The ^-structure can be thought of as forming a surface or sheet, although there is a slight twist to it because the peptide backbone is
30
Synthetic Peptides
Figure 2-9. View of an a-helix looking down the helical axis. The residue side chains, shown in black, all project radially outward from the helix.
not fully extended (typical angles are given in table 2-4). The side chains extend above and below the rough plane of the sheet, with every other side chain on one surface. Because the interchain hydrogen bonds between two adjacent chains involve only every other residue, there is the possibility of larger structures forming that involve many strands. Such extended /J-structures are common features in proteins. The twist of the ^-structure also allows sheets to fold into cylindrical structures, called ^-barrels, which is another common structural motif.
Turns Strands of a-helix or ^-structure do not extend indefinitely in proteins but rather fold back on themselves. Several regular structures, called turns, are involved in changing the direction of the peptide chain. Turns are classified by the number of residues that are involved in the regular structure; /6-turns contain four amino acid residues, while y-turns contain three residues. Each structure is stabilized by a hydrogen bond extending across the turn, in effect holding the two ends together. The first residue of a turn is usually designated as /. In a /J-turn, the hydrogen bond is between the carbonyl of the / residue and the NH of the / + 3 residue, giving the equivalent of a 10-membered ring. There
Figure 2-10. Structure of /J-sheet. Amide bonds lie in the plane of the sheet, with side chains alternately projecting above and below the plane of the sheet, (a) Parallel sheet, with both strands running in the same direction, (b) Antiparallel sheet, with strands running in opposite directions. may be an additional hydrogen bond between the NH of the i residue and the carbonyl of the / + 3 residue, as if they were an incipient antiparallel ^-structure. In a y-turn, the hydrogen bond is between the carbonyl of the i residue and the NH of the i + 2 residue, giving the equivalent of a seven-membered ring. (Turns are sometimes named for the number of atoms in the ring structure formed by the hydrogen bond. Thus a /J-turn would be a C10 turn and a y-turn a C7 turn.) Table 2-4 Torsional angles for ,0- structure
4>
Parallel -sheet Antiparallel /6-sheet
y,
113 135
on
-119 -139
Angles taken from IUPAC-IUB Commission Biochemical Nomenclature (1970)
32
Synthetic Peptides
Table 2-5 Torsional angles for turns

i+ l 4>
Type I Type I' Type II Type II' Type III Type III' y-Turn Inverse y-turn
-60
tfr
i+2
<t>
-90 90 80 -80 -60 60
iff
0 0 0 0 -30 30
-30
60
-60
30
120 -120 -30
60
-60
60
70 to 85 -70 to -85
30
-60 to -70 60 to 70
Angles taken from Smith and Pease (1980).
Several types of /Murns are possible. Given planar amide bonds, ft and y turns are uniquely defined by the torsional angles of the internal residues / + 1 and, in the case of ft-turns, i + 2. Table 2-5 lists the torsional angles associated with the various types of ft- and y-turns. Note that there is an inverse form for each turn in which the sign of the internal torsional angles is reversed. Note also that the type III /J-turn is equivalent to a 310 helix structure. Figure 2-11 depicts the backbone of types I, I', II, and II" ft-turns and figure 2-12 depicts that of y- and inverse y-turns. Turns have several important structural features. The peptide backbone in a turn forms a rough plane that contains the intramolecular hydrogen bond. In a /3-turn, the amide bond between the i + 1 and i + 2 residues lies perpendicular to this plane. Since it is not part of the hydrogen-bonding structure of the turn, its hydrogen bonding needs must be satisfied elsewhere. In proteins, /3-turns are most often found on the protein surface, where the / + 1 / + 2 peptide bond can be solvated by bulk solvent. When such a turn is located in the (generally hydrophobic) interior of a protein, it is often solvated by a bound water molecule (Smith and Pease, 1980). Besides the amide bond, the side chains of the / + 1 and i + 2 residues, which form the "corners" of the turn, are also particularly exposed. In small peptides, such as vasopressin or somatostatin, where a turn has been postulated based on the cyclic nature of the peptide chain, the corner residues are implicated as important elements in the expression of the peptide's biological activity (Rivier et al., 1976; Walter et al., 1977). Side chains will adopt either a pseudo-axial or pseudo-equatorial arrangement around the cyclic hydrogen-bonded turn structure. One diagnostic criterion for the importance of a turn involves changing the chirality of a corner residue to allow the side chain to be in the more
33
Figure 2-11. (a) Type I jS-turn. (b) Type I' /J-turn. (c) Type II /Mum. (d) Type II' /3-turn. The turn is stabilized by a hydrogen bond from the carbonyl of the /th residue to the NH of the / + 3 residue. The amide bond between the i + 1 and i + 2 residues is perpendicular to the plane of the turn. The side chain of the i + 1 residue in a type I' and II' turn is in a pseudo-axial orientation. Substitution of a o-amino acid would give the more stable pseudo-equatorial orientation. The NH and side chain of the / + 2 residue of the type II' turn are both on the same side of the ring, an arrangement in which a proline residue could be accommodated as well. The NH and side chain of the i + 2 residue of a type I' turn are on opposite sides of the ring, which would exclude the possibility of a proline residue at that position.
stable equatorial configuration without otherwise altering the peptide backbone structure. In a type II' /J-turn, for example, the / + 1 side chain is in a pseudo-axial configuration (see figure 2-11). Replacement of this residue with a o-amino acid should allow for equatorial placement of the side chain while the peptide backbone remains unchanged, lowering the overall free energy of that structure and making it more stable. Replacing the i + 1 residue with a glycine, with no side chain, should have a similar effect.
34
Synthetic Peptides
Figure 2-12. Structure of a j/-turn (a) and inverse y-turn (b). The turns are stabilized by a hydrogen bond from the carbonyl of the /th residue to the NH of the / + 2 residue. The side chain of the / + 1 residue adopts a pseudo-axial orientation in the j/-turn and a pseudo-equatorial orientation in the inverse y-turn.
Amphipathic Structures Given the regular placement of side chains in helical or ^-structures, it is possible to obtain forms in which the polar side chains are segregated on one face of the structure and hydrophobic side chains are on the other. This type of arrangement is termed an amphiphilic, or amphipathic, structure. In an ideal amphipathic helix, the hydrophobic residues are all found on one side of the helix cylinder, and the hydrophilic residues are found on the other side of the cylinder. In the linear sequence,
35
these hydrophilic and hydrophobie residues will be interdispersed, so it can achieve the desired arrangement when folded into a helix. One idealized amphipathic helix, designed by Kaiser and coworkers as a model for apolipoprotein A, has the helical sequence Lys-Leu-GluGlu-Leu-Lys-Glu-Lys-Leu-Lys-Glu-Leu-Leu-Glu-Lys-Leu-LysGlu-Lys-Leu (Fukushima et al., 1980). The arrangement of side chains can best be shown schematically in a helix wheel projection, which is depicted looking down the helix axis with the side chains arranged radially outward, as shown in figure 2-13(a). In /J-structure, the side chains alternate above and below the plane of the sheet. In an amphipathic strand of /3-structure, every other residue in the linear sequence would be hydrophobie and the remaining residues would be hydrophilic. This arrangement would give a structure in which one face of the sheet would be hydrophobie and the other face would be hydrophilic, as in figure 2-13(b). In proteins, amphipathic structures can be important structural elements, allowing the hydrophobie residues to be sequestered in the interior of the protein while a hydrophilic face is presented to the aqueous environment at the protein surface. This arrangement can be reversed in transmembrane proteins, with amphipathic helices clustered so that the hydrophobie face is exposed to the lipid environment and the hydrophilic faces are sequestered in the interior of the protein, forming a hydrophilic pore, or channel, in the membrane. The recognition that amphipathic structures are important to the biological activity of smaller peptides is due in large part to the pioneering work of E. T. Kaiser. He recognized potential amphipathic helices in such membrane-active peptides as apolipoprotein A and mellitin, as well as peptide hormones such as calcitonin, corticotropin releasing factor, and /S-endorphin, which interact with membranebound receptors. Structure-activity studies of synthetic analogs of these peptides have demonstrated the importance of their amphipathic structures by designing sequences optimizing amphipathicity or replacing it with nonhomologous sequences adopting the same amphipathic structure (Kaiser and Kezdy, 1984). Examples of the design of amphipathic peptides will be discussed later (see below, Hydrophobicity Measurements).
Prediction of Protein/Pcptide Structure Protein Sequence Homology The primary sequence of a peptide, or protein, is its most fundamental structural element. It has long been presumed that all the information content of a proteinfolding, translocation, and functionare encoded within the amino acid sequence of the primary translation
Figure 2-13. (a) Helical wheel projection of a designed amphipathic helix. The residues with hydrophilic side chains are segregated on one face of the helix, while the hydrophobic residues are segregated on the other, (b) Side view of one strand of an amphipathic /J-sheet. The hydrophilic side chains project on one side of the sheet, while the hydrophobic side chains project on the other side. The puckering of the backbone, which gives rise to the often-used name ^-pleated sheet, is clearly evident in this side view.
37
product. Many proteins can be denatured reversibly and renatured with complete recovery of biological activity, lending credence to this concept. The situation in protein biosynthesis is more complex, because nascent peptides can begin to interact with cellular proteins, such as signal recognition particles (which mediate protein translocation across biological membranes), chaperonins, and heat shock proteins (which are involved in protein folding and assembly) even before the full-length translation product has come off the ribosome (Landry and Gierasch, 1991). The elements involved in the recognition of the nascent polypeptide chain by these signal recognition particles and chaperonins still must derive from the primary sequence of the peptide chain. Although the rules relating protein sequence to folding, translocation, and function are not well understood, the ability to compare protein sequences and determine homology is quite useful. Homology can help to suggest protein class or function in newly identified protein sequences. Identification of a homologous protein for which structural information is known, particularly X-ray crystal structures, can aid in the construction of three-dimensional models for the protein structure. A case in point was the early identification of the HIV-1 protease as an aspartic protease, based on sequence homology to other aspartic proteases, and the construction of a model structure, based on the known X-ray crystal structures of fungal aspartic proteases, such as penicillopepsin and rhizopus pepsin (Pearl and Taylor, 1987). Our ability to search protein databases for protein similarities is based on the pioneering effort of Margaret Dayhoff at the National Biomedical Research Foundation (NBRF), who began publishing compilations of protein sequences during the 1960s (Dayhoff et al., 1965). Today, several electronic databases are available that are updated on a regular (daily) basis such as the NBRF protein database, the Swiss-Prot database and the Gen-Bank database. These databases, along with software to search for sequence homology, such as BLAST, are available over the internet on an interactive basis. Some particularly useful sites for this purpose are the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/), the ExPASy Molecular Biolgy server of the Swiss Institute of Bioinformatics (http://www.expasy.ch/), and the European Molecular Biology Laboratory Protein and Peptide Group (http://www.mann.embl-heidelberg.de/Default.html). In addition, a comprehensive discussion of computational methods for protein sequence analysis with specific instructions can be found in Michaels and Garian, 1995. With the tremendous advances in nucleotide sequencing, many new protein sequences are available from a translation of the nucleotide sequence rather than from conventional protein sequencing techniques.
38
Synthetic Peptides
Two basic operations exist for sequence homology searching. One is a matching function that allows searching for occurrences of a specific sequence. For example, one could search the protein database for all occurrences of the sequence Arg-Gly-Asp-Ser. The number of mismatches allowed in the sequence can be specified, allowing retrieval of such sequences as Arg-Ala-Asp-Ser. It is also usually possible to specify a list of amino acids that would be considered a match at a given position in the sequence, for example, Arg-Gly-(Asp or Glu)Ser. Finally, one can also search for patterns like Ser-X-X-Tyr-Pro when one is interested in sequences in which a Ser is found three residues upstream from a Tyr-Pro sequence and where the nature of the intervening amino acids (X) is not important. BLAST is a modern version of a sequence searching algorithm available through the National Center for Biotechnology Information that is widely used today. A detailed description and instructions for its use can be found in Wolfsberg and Madden, 1995. Although matching is useful for identifying regions of contiguous sequence, many proteins may have homologous regions separated by sequences of varying lengths or may have small gaps or insertions within a region of homology. For this reason, an alignment algorithm is used to compare two entire protein sequences and to determine the best fit of the two sequences. Several variations occur on sequence alignment algorithms: one is based on methodology originally derived by Needleman and Wunsch (1970), and another (FASTA), developed by Pearson and Lipman (Lipman and Pearson, 1985; Pearson and Lipman, 1988), is optimized for speed, something of concern when one wants to check a new sequence against the entire protein database. Modern algorithms have become more and more complex and their inner workings are beyond the scope of this chapter. However, a basic illustration of how they were originally designed can be considered. Most methods relied on the use of a scoring table or matrix that assigns a numerical value to each pair of amino acids. The simplest scoring table would have 1 for identical amino acids and 0 for any other combination. If you were to create a matrix with the sequence of one protein running along the columns and that of the other protein running along the rows, each cell (ij) would be either 0 or 1 depending on whether residue / from the first protein matched residue j from the second protein. If the two proteins were identical, there would be 1's all along the diagonal of the matrix. If there were more limited regions of homology, they would appear as stretches consisting mainly of 1's along or parallel to the diagonal. Any offset from the diagonal reflects gaps or insertions of amino acids into one of the protein sequences. This often indicates the presence of variable length loops of sequence that connect conserved regions of secondary structure in homologous proteins.
39
It is readily apparent that the scoring table does not have to be limited to values of 0 and 1. The comparisons can be graded so that a conservative substitution (e.g., valine for isoleucine) would have a high score, and substitution of a very dissimilar amino acid (such as aspartic acid for isoleucine) would have a very low score. A commonly used scoring table (Dayhoff et al., 1978) takes into account conservative substitutions, the frequency of appearance of amino acids, the frequency of single base mutations to change the genetic three-base code from one specific amino acid to another, and the conservation of rare amino acids (like tryptophan) or structurally significant amino acids (like cysteine). Scoring values range from a high of 17 (conservation of the relatively uncommon tryptophan in a sequence) to 2 (conservation of the commonly occurring and not generally structurally significant alanine) to lows of 6 to 8 (substitution of the structurally significant cysteine). This sort of table is ideal for studying the evolutionary relatedness of various proteins. If one were interested in convergent evolution, where a peptide or protein might have independently evolved to a similar structure and function, one could construct a scoring matrix which emphasized chemically conservative substitutions. The regions of sequence homology are not the only regions of interest or importance in protein function. For example, if there are species differences in the activity of homologous proteins, the structural basis for these differences may lie in the regions of nonhomologous amino acid sequence. In addition, loops, such as those connecting conserved regions of secondary structure, have a high probability of lying on the protein surface and may serve as important sites for post-translational modification or as antigenic epitopes (see below, Epitope Mapping and Production of Protein-Specific Antibodies from Peptides).
Secondary Structure Prediction
Protein sequence alignment alone cannot suggest any of the structure of a protein unless a homologous protein has been studied by X-ray crystallography, nuclear magnetic resonance, circular dichroism or any of the other physical methods of protein structure determination. Structural information of this type is not available for most protein sequences. However, algorithms have been developed that allow prediction of secondary structures based on the primary sequence of the protein. Secondary structure prediction algorithms are generally available as part of the protein sequence analysis software packages such as that found at the ExPASy site mentioned above. They are based on statistical analyses of the frequency of residues or sequences being found in particular secondary structures in a dataset of known protein structures. Prediction of overall secondary structure is
Synthetic Peptides
enerally good, but specific details, such as exact helix boundaries, may be less reliable. A comprehensive discussion of secondary strucure prediction methods with specific instructions can be found in Krystek et al., 1995b. Perhaps the most widely used secondary structure prediction cheme is that of Chou and Fasman (1978). It is based on a statistical nalysis of the frequencies of individual amino acid residues being ound in specific secondary structures (a-helix, /2-structure or ^-turn) ompared to the overall frequency in which they appear in protein equences. This allows each amino acid to be assigned a numerical robability of being found in a given secondary structure and to e classified as a strong helix-former, helix-breaker, weak ^-former, -breaker, and so on. The Chou-Fasman probabilites for /3-turns are sted in table 2-6. Since an a-helix or ^-structure is a regular repeating ne, each position in it is essentially equivalent. To a first approximaion, then, the probability of a given amino acid appearing in a helix or ^-structure is not dependent on the position it would occupy in that tructure. Each amino acid, therefore, has a single probability value or a-helix or ^-structure. Since the conformational requirements for ach position in a /?-turn are different, however, each amino acid will ave a different probability of appearing in each of the four positions f a /?-turn. Secondary structure is predicted by applying a series of empirically etermined rules to the primary sequence. Helices are predicted by earching for nucleation sitesclusters of residues with a high helix orming potential. The helix propagates out in both directions until a luster of helix-breaking residues is encountered. There are other rules or locating helix boundaries, such as the general tendency for egatively charged amino acids (Asp and Glu) at the amino terminal nd (where they help stabilize the helix dipole) and positively charged mino acids (His, Lys and Arg) at the carboxyl terminal end. /3-sheet tructure is predicted in a similar fashion. Turns are predicted for a equence of amino acids i to / + 3 by considering the sum of their osition-dependent probability values. In practice, algorithms upplied with protein database software generally evaluate helix otential, /6-sheet potential, and /3-turn potential (also, sometimes elix and sheet boundary conditions) separately and plot each against esidue number. Helix and sheet potential are averaged over a userefined window of residues (typically four) and normalized. Average robabilities greater than one indicate a propensity for that sequence o adopt that type of secondary structure. The graphical output llows regions with a high probability of adopting helix, ^-structure, r /J-turns to be readily identified, and plotting of residues often found t helix or sheet boundaries helps to define the extent of secondary tructures.
Peptide Design Considerations Table 2-6 Hydrophobia and /?-turn indices of amino acids Amino acid Arginine Lysine Aspartic acid Glutamic acid Asparagine Glutamine Histidine Proline Tyrosine Tryptophan Serine Threonine Glycine Alanine Methionine Cysteine Phenylalanine Leucine Valine Isoleucine Symbol (Arg, R) (Lys, K) (Asp, D) (Glu, E) (Asn, N) (Gin, Q) (His, H) (Pro, P) (Tyr, Y) (Trp, W) (Ser, S) (Thr, T) (Gly, G) (Ala, A) (Met, M) (Cys, C) (Phe, F) (Leu, L) (Val, V) (He, I) Hydrophobicity value3
41
/J-Turn propensity13
-4.5 -3.9 -3.5 -3.5 -3.5 -3.5 -3.2 -1.6 -1.3 -0.9 -0.8 -0.7 -0.4 1.8 1.9 2.5 2.8 3.8 4.2 4.5
0.95 1.01 1.46 0.74 1.56 0.98 0.95 1.52 1.14 0.96 1.43 0.96 1.56 0.66 0.60 1.19 0.60 0.59 0.50 0.47
'Kyte and Doolittle (1982). 'Chou and Fasman (1978).
It often happens that there are ambiguities in the assignment of secondary structure to regions that may have high probabilities of adopting either helix or ^-structure. There are additional rules by which these ambiguities can be resolved, including considering which type of secondary structure has the highest numerical probability for that sequence, the length of the sequence (or-helices, being more compact structures, typically are longer than regions of ^-structure), and so forth. Gamier et al. (1978) developed a method, again based on a statistical analysis using a known set of protein structures, in which position-dependent probability values are used to generate a numerical value which specifies helix, ^-structure, j0-turn, or random coil for each residue of the protein. An algorithm based on this method is also often included in protein sequence analysis software packages. It is not currently possible to predict the secondary structure of a protein from the sequence alone with absolute certainty. These predictive methods generally have an overall accuracy of about 70% when applied to proteins of known structure that are not in the "knowledge set" used to generate the predictive rules, that is, about 70% of the secondary structure present is predicted in an overall sense. Helical and turn structures are reasonably well predicted, probably in
42
Synthetic Peptides
part because they require only one strand of peptide, while /?structures, which require the presence of at least one complementary strand, are not as well predicted. It must be remembered that these methods rely on statistical analyses and empirically determined rules. Predictions of overall helical content or /3-sheet content may be reasonably accurate, but details such as boundaries are generally much less precise. There are many sequences that are ambiguous as to secondary structure preferences and, indeed, many peptide sequences are able to adopt different conformations or structures depending on their environment, solvent, and so on. Also, while random coil or irregular structure can be inferred from the absence of any propensity to form a regular secondary structure, there is no conformational or structural information available for such sequences to aid in model building. Finally, prediction of secondary structure in and of itself does not necessarily yield much information on tertiary structure or protein topology. Secondary structure prediction is best utilized as a guide in model building or in selection of peptide targets in conjunction with other techniques, such as sequence homology, chemical modification, proteolytic digestion, site-specific mutagenesis, and so on.
Hydrophobicity Measurements Given that soluble proteins fold in such a way as to minimize the exposure of hydrophobic side chains to the aqueous medium, it is possible to infer portions of the protein sequence that are likely to be buried or to be exposed on the protein surface by examining the relative hydrophilicity or hydrophobicity of the protein sequence. A comprehensive discussion of hydrophobicity methods with specific instructions can be found in Krystek et al., 1995a. The method most widely available in protein sequence analysis software is that of Kyte and Doolittle (1982). They devised a set of values representative of the free energy change of each amino acid side chain going from a polar to a nonpolar environment. These values are averaged over a window (generally six to nine residues), and these average hydropathy values are plotted against the residue number of the first residue in the window. Positive values indicate hydrophobic segments of the protein, and negative values indicate hydrophilic regions. Segments of the protein chain with a strongly hydrophobic character correlate well with the interior regions of the protein, and those with a strongly hydrophilic character correlate well with regions on the surface. The Kyte-Doolittle hydrophobic propensity values are listed in table 2-6. An example of the graphical output from a KyteDoolittle analysis is shown in figure 2-14 (pages 44-45). Hydrophilicity analysis of protein sequences has been used to predict antigenic determinants or epitopes in proteins. As one might
43
intuitively expect, most antigenic determinants in proteins (though not all) are found in regions exposed on the surface of the protein molecule. Hopp and Woods (1981) modified a set of hydrophilicity values for amino acids ultimately derived from octanol-water partition coefficients, used them to calculate the hydrophilicity of protein sequences over a six-residue window, and plotted the results against the residue number. They found a good correlation between peaks of hydrophilicity and the known antigenic sequences of several proteins, although not all highly hydrophilic segments corresponded to known antigenic regions, and not all antigenic determinants corresponded to the highest hydrophilic peaks. The region of highest hydrophilicity did always correspond to an antigenic determinant in the set of 12 proteins examined. This method is useful for examining proteins of unknown structure, both to suggest peptides for epitope mapping studies or antibody production and also to look for other probable surface features, such as receptor binding sites, enzyme cleavage sites, or sites for post-translational modification. From the earlier discussion on amphipathic secondary structures (see above, Amphipathic Structures), it is clear that sequences can exhibit an overall hydrophobic or hydrophilic profile that is rather flat and yet fold into structures that present very hydrophobic or hydrophilic faces. In order to evaluate this type of behavior, Eisenberg et al. (1982) developed the concept of a hydrophobic moment, which can be calculated for a protein sequence having a given secondary structure. For a helix, the hydrophobicity of a side chain is represented by a vector projecting outward from the helix, and its magnitude reflects the magnitude of the hydrophobicity. Its direction is determined by its position in the helix; successive side chains will be offset from each other by 100 in an a-helix. Hydrophilic side chains give rise to a hydrophobic vector of the same magnitude but pointing 180 back from the side chain projection. A hydrophobic moment can be calculated by averaging the magnitude and direction of these vectors over a window of residues. A large moment reflects the segregation of hydrophobic residues along one face of the helix and hydrophilic on the other. A similar calculation can be applied to /3-sheet structure. In this case, the angular offset for succeeding residues is 160. Eisenberg et al. (1982) calculated hydrophobic moments for a series of helices in 26 different proteins of known structure and compared those values to the average hydrophobicity. The moment and average hydrophobicity were calculated for the entire helix sequence, so each different helix sequence gave a single value for moment and hydrophobicity. Buried or membrane-spanning helices tended to have large hydrophobicity and low hydrophobic moment, as one might intuitively expect, since they exist in a uniformly hydrophobic
Figure 2-14. Graphical representation of the results of analysis of a protein sequence for /J-turn propensity (a) using the method of Chou and Fasman (1978), and hydrophobicity (b) using the method of Kyte and Doolittle (1982).
46
Synthetic Peptides
environment. Amphiphilic helices have low overall average hydrophobicity but possess a large hydrophobia moment. They are found at interfaces between hydrophobic and hydrophilic environments, on the surface of proteins, or in peptides such as mellitin, which associate with plasma membranes. The asymmetric distribution of polar and nonpolar side chains allows these structures to interact favorably with both polar and nonpolar environments when they are situated at the interface. In practice, with proteins of unknown structure, a hydrophobic moment is calculated over a small window, typically six residues, first assuming an a-helical structure and then assuming a /3-sheet structure (Eisenberg et al., 1987). These are both plotted against residue number and compared with hydrophobicity and Chou-Fasman secondary structure calculations. In that way, one can identify probable amphipathic helices and ^-structures and probable membranespanning helices.
Application of Sequence Analysis to Modeling and Design
Construction of detailed molecular models of peptide or protein structure based on sequence data is most fruitful when there is an X-ray crystal structure of a related protein available to use as a template. When the relatedness of the template protein to the protein of unknown structure is relatively distant, the use of secondary structure prediction, hydropathy measurements, and a certain amount of intuition also come into play. This convergent approach is illustrated in the following examples. The complement protein C5a is a 74-residue protein that is released by proteolysis of a larger precursor during the complement activation cascade. It is thought to be involved in inflammatory responses by recruiting monocytes and polymorphonuclear leukocytes by chemotaxis and activating them to release free radicals and degradative enzymes. Inhibitors of C5a activity can have therapeutic application in the treatment of inflammatory diseases such as rheumatoid arthritis. In an attempt to develop C5a antagonists, it would be very useful to have a model of the C5a structure. At the time, there was no X-ray crystal structure of C5a available, but there was a crystal structure of the related complement protein, C3a. Greer (1985) used this structure as a template on which to construct a model of C5a. The initial difficulty was that the sequences of C5a and C3a were only about 36% identical. Greer chose to begin the alignment with the central region of C3a, which was well defined in the crystal structure, and chose an alignment with C5a that would not cause any deletions or insertions in the helical regions. This alignment would not otherwise
47
be considered the optimal one strictly on the basis of sequence. The model built using this alignment showed that the internal residues, which form the core of the structure, are, in fact, highly conserved, and that the residues on the surface are not conserved. Thus, while the overall similarity between the two proteins is only modest, the residues important for defining the structure are highly conserved. However, they do not appear adjacent to each other in the linear sequence, so sequence homology does not reveal this close relationship. The second difficulty encountered was that the C3a amino terminus did not appear in the structure, possibly because it was too flexible in the crystal to give rise to substantial electron density. Thus, there was no template for the amino terminus of C5a. Secondary structural analysis suggested that the amino terminus of C5a had a strong potential for helical structure. When that region was folded into a helix, it was found to be strongly amphipathic, so it was reasonable that the hydrophobic face of this helix interacted with an accessible hydrophobic region elsewhere on the C5a molecule. One such hydrophobic patch was located on the surface in a region where the residues were highly conserved in human and porcine C5a and where the amino terminal helix could be docked. This yielded a working model for the C5a structure where the core structure was identical to that of C3a, but the surface residues had considerable difference, which is consistent with the two proteins' distinct biological activities. The structure was also consistent with known pieces of structure-activity data. Porcine and human C5a will both bind to human C5a receptor, and there is a region on the surface of both molecules that is relatively conserved. This region is spatially near the C-terminal Arg residue, which is important for biological activity but not for binding to the receptor. When the threedimensional solution structure of C5a was determined using 2-D NMR techniques and distance geometry methods (Zuiderweg et al., 1988), the proposed model was found to be in very good agreement with the deduced solution structure with respect to both the core structure, the helical conformation, and placement of the amino terminal sequence. The carboxyl terminus of the protein, however, seems to be highly mobile in solution and does not adopt a fixed conformation. A similar approach was used to create initial structural models for the human immunodeficiency virus (HIV-1) protease. HIV-1 protease is responsible for the proteolytic maturation of the HIV structural proteins, reverse transcriptase, integrase, and RNase H, which are encoded by the gag and pol genes. It was initially known that many related retroviruses encode such a protease that processes the gag and pol translation products and that the HIV gag and pol proteins
48
Synthetic Peptides
were indeed produced as polyprotein precursors. An approximate location in the genome for the protease was determined by protein sequence homology comparisons of the gag and pol regions of a number of retroviruses, considering especially two highly conserved tripeptide sequences (Asp-Thr-Gly and Ile-Ile-Gly) (Yasunaga et al., 1986). This was confirmed, and the exact location of the protease and its primary sequence were unambiguously determined when the gene was subsequently cloned and expressed (Debouck et al., 1987). The tripeptide sequence, Asp-Thr-Gly, which is conserved in retroviral proteases, is also a conserved sequence in aspartyl proteases, and, in fact, represents the catalytically active aspartic acid residues of the active site. Aspartyl proteases are relatively well-studied enzymes, and a number of crystal structures are available. They consist of two domains, each of which contributes one Asp to the active site. The two domains have a fair degree of sequence homology, and both domains have a similar three-dimensional structure, suggesting that aspartyl proteases arose by gene duplication and fusion (Tang et al., 1978). The retroviral proteases are only about half the size of eukaryotic aspartyl proteases and contain only one Asp-Thr-Gly sequence. Pearl and Taylor (1987) hypothesized that the retroviral protease represents one lobe of an aspartyl protease, and that the protein forms a symmetrical dimer as the active protease. Using a combination of protein sequence comparisons among a large number of aspartyl proteases and secondary structure prediction, they were able to construct a model for the HIV-1 protease as a symmetrical dimer based on the crystal structure of the fungal aspartyl protease endothiapepsin. The dimeric nature of the HIV-1 protease and its identification as an aspartyl protease was later confirmed experimentally (Meek et al., 1989). The model predicted the general structure of the protease as four strands of /3-sheet and a central hydrophobic cleft formed at the dimer interface that contains the substrate binding site and the active site aspartyl residues. This overall structure was confirmed when X-ray crystal structures of the HIV-1 protease were obtained (Lapatto et al., 1989; Wlodawer et al., 1989), although some important structural details varied. For example, the model was based on a sequence that incorrectly located the mature amino terminus of the protease and, therefore, failed to predict the intermolecular ^-structure between the amino terminus of one monomer and the carboxyl terminus of the other monomer, which forms an important part of the dimer interface. Nevertheless, early recognition that the HIV-1 protease was likely to be a symmetrical dimer with a symmetrical active site led to the design of novel, symmetrical inhibitors of high potency (Erickson et al., 1990).
49
Peptide Design Based on Sequence It has been a long-standing goal in peptide chemistry to be able to design short peptides that mimic some important aspect of protein structure or function. The peptides can be designed to be agonists of a biologically active protein by encompassing those residues necessary for activity. Antagonists can be designed using peptides containing only those residues required for binding to a receptor, a substrate, or a required protein subunit. (Fibrinogen receptor antagonists, which fall into this category, will be discussed in more detail later; see below, Cyclization.) When one has antibodies against the protein that neutralize the protein's activity, epitope mapping (or locating the antibody combining sites) can help identify protein sequence portions involved in the biological activity. Synthetic peptides based on the sequences of neutralizing antigens can be used as synthetic antigens in the preparation of antisera and vaccines. Conversely, antibodies can be produced with synthetic peptides that recognize specific proteins simply from a knowledge of the gene sequence without ever having isolated the protein. Designing peptide agonists based on protein sequences is a formidable task. With increasing size of the polypeptide chain, it becomes increasingly likely that residues important for biological activity will be in close proximity in the three-dimensional structure but widely separated in the primary sequence. This makes design of a small peptide that can mimic that arrangement problematic, especially when there may not be any three-dimensional structural information available about the target protein. The case is often easier with peptides of moderate length, since there is often a minimum active fragment that retains a substantial portion of the biological activity. This can be illustrated with the case of gastrin-releasing peptide and bombesin. Gastrin-releasing peptide (GRP) is a 27-amino-acid peptide originally isolated from porcine gut on the basis of its ability to cause release of gastrin (McDonald et al., 1979). Its C-terminus has a striking homology with bombesin, a 14-residue peptide isolated from frog skin (Anastasi et al., 1971), as well as a number of related peptides of amphibian origin. The sequences of GRP and bombesin are shown in table 2-7. Both GRP and bombesin produce a number of biological responses, including hypothermia or lowering of the core body temperature in rats exposed to cold. A series of truncated analogs was prepared and assayed for hypothermia activity in order to determine the minimum sequence necessary for agonist activity (table 2-7; Rivier and Brown, 1978; Marki et al., 1981). Deletions from the conserved Cterminus impaired activity. In contrast, the peptides could be shortened considerably from the amino terminus without loss of
50
Synthetic Peptides
Table 2-7 Gastrin-releasing peptide and bombesin analogs

Peptide
GRP Bombesin
Sequence A-P-V-S-V-G-G-T-V-L-A-K-M-Y-P-R-G-N-H-W-A-V-G-H-L-M-NH2 <E-Q-R-L-G-N-Q-W-A-V-G-H-L-M-NH2 <E-Q-R-L-G-N-Q-W-A-V-G-H-L-M-OH <E-Q-R-L-G-N-Q-W-A-V-G-H-NH2 Ac-G-N-Q-W-A-V-G-H-L-M-NH2 Ac-N-H-W-A-V-G-H-L-M-NH2 Ac-H-W-A-V-G-H-L-M-NH2 Ac-W-A-V-G-H-L-M-NH2
Relative activity
0.28 1.00 <0.01 <0.01 1.00 0.50 1.00 0.04
<E = pyroglutamic acid.
activity, and full activity was obtained with just the C-terminal octapeptide sequence. Peptides derived from an internal protein sequence may require blocking groups at the amino and/or carboxyl terminus to mask the presence of charges that are not present in the native protein. For example, the HIV-1 protease mentioned earlier (see above, Application of Sequence Analysis to Modeling and Design) is an endopeptidase whose substrate is a viral polyprotein. One of the cleavage sites is encompassed by the heptapeptide Ser-Gln-Asn-Tyr-Pro-ValVal, with cleavage occurring between the Tyr and Pro residues (Moore et al., 1989). A series of peptides of varying sizes were prepared either with 7V-acetyl and carboxamide blocking groups or free amino or carboxyl termini and their substrate activity compared. The results are shown in table 2-8, where Km is the Michaelis binding constant, kcat is the catalytic rate constant, and kcat/Km is a measure of catalytic efficiency. The fully blocked heptapeptide (4), the free amino peptide amide (5), and the acetyl peptide free acid (6) all have fairly similar activities. Removal of the C-terminal Val residue (peptide 7) causes a substantial loss in kcat and catalytic efficiency, and removal of both Val residues (peptide 8) results in an inactive peptide. Truncation at the amino terminus causes a slight loss in activity in hexapeptide 9 and pentapeptide 11, with a complete loss of activity when tetrapeptide 13 is reached. The free amine hexapeptide 10 is as active as its acetylated parent, but the free amine pentapeptide 12 again shows a substantial loss in kcat and catalytic efficiency. These data collectively suggest that the active site of the HIV-1 protease will accommodate a hexapeptide, stretching three residues on either side of the cleavage site. Charged groups and the presence of additional residues have little effect on substate activity since they extend out of the active site. The loss of activity in uncovering the free amino group in the pentapeptide 12 is probably due to the presence of a
Peptide Design Considerations 51 Table 2-8 HIV-1 protease substrates

^m
K-cat
Compound
Sequence Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 H-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-OH Ac-Ser-Gln-Asn-Tyr-Pro-Val-NH2 Ac-Ser-Gln-Asn-Tyr-Pro-NH2 Ac-Ala-Asn-Tyr-Pro-Val-Val-NH2 H-Ala-Asn-Tyr-Pro-Val-Val-NH2 Ac-Asn-Tyr-Pro-Val-Val-NH2 H-Asn-Tyr-Pro-Val-Val-NH2 Ac-Tyr-Pro-Val-Val-NH2 H-Tyr-Pro-Val-Val-NH2
(mM)
(sec"1)
kcal/Km (HIM"' -s" 1 )
4 5 6 7 8 9 10 11 12 13 14
5.5 1.2 5.0 4.9

neg. 13.5
54
14.0 25.7 0.49 neg.
9.8
11.7
5.1
0.090 neg. 0.69
9.3
29.7
18
17.1 12.8 neg. neg.
37
0.88 neg. neg.
1.6 2.2
0.068 neg. neg.
charged group in the generally hydrophobic active-site cleft. This interpretation of the extent of the enzyme active site is confirmed by X-ray crystal structures of co-crystals of the protease containing substrate-based inhibitors bound in the active site (Miller et al., 1989; Wlodawer et al., 1991). Peptide based protease substrates can be made more sensitive and easier to measure by the incorporation of fluorescent groups. Matayoshi et.al. (1990) made an HIV protease substrate that incorporated two fluorescent groups that internally quenched each other by resonance energy transfer. This substrate had the structure, DABCYL-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-EDANS. In this case, the fluorescence donor is EDANS (5-(2-aminoethylamino)-naphthalene sulfonic acid) and the quenching acceptor is DABCYL (4dimethylamino-azobenzene 4'-carboxylic acid). When the substrate is cleaved at the Tyr-Pro bond by the protease, the two fluorescent groups are no longer held in close proximity so the quenching of the EDANS signal by DABCYL is significantly decreased. The resulting increase in fluorescence emission is used to monitor the course of the reaction. In this case, the fluorescence increased by 40-fold upon cleavage of the peptide and a Km of 0.1 mM and a kcat of 4.9 s^1 was determined for HIV protease. It thus appeared that the presence of the EDANS and DABCYL groups confered improved substrate binding properties over those found for the peptides in table 2-8. This is not always the case, however. Incorporation of fluorescent groups into a peptide substrate for poliovirus 3C protease (Weidner and Dunn, 1991) reduced the rate of cleavage by approximately 50-fold. The addition of a charged moiety, remote enough from the active site that it does not interfere with catalytic activity, can be advantageous
52
Synthetic Peptides
in terms of solubility. Succinyl groups are often added to the amino terminus of enzymes' substrates, for example, the chymotrypsin substrate succinyl-Ala-Ala-Pro-Phe-/?-nitroanilide (DelMar et al., 1979). This modification not only improves solubility of hydrophobic peptides but also blocks potential aminopeptidase degradation at the amino terminus.
Epitope Mapping and Production of Protein-Specific Antibodies from Peptides Epitope Mapping One of the most common applications of peptides based on protein sequences is epitope mapping. Determination of antibody combining sites on proteins is used to study the mechanism of immunogenicity of proteins as well as to determine protein topology (on the assumption that most epitopes will be located at the protein surface). If there are monoclonal antibodies that block the protein's activity, epitope mapping can help determine the parts of the protein sequence involved in the expression of biological activity. If the protein is derived from a pathogenic organism, peptides based on neutralizing epitopes can be the basis of synthetic vaccines. Determination of an antigenic epitope relies on preparation of a peptide representing a portion of the protein sequence and demonstrating competition with the protein in antibody binding. There are several approaches to determining which sequences to examine as potential epitopes. The complete approach would require synthesizing overlapping short peptides, generally hexapeptides, that cover the entire protein sequence. Several techniques for multiple solid-phase peptide synthesis make this approach possible (see chapter 3). Houghten (1985) devised a method in which small amounts of aminoacyl resin are sealed in small polystyrene mesh bags called "T-bags." The bags can be labeled with an indelible marker for identification. The common steps of washing, deprotection, and neutralization can be performed together for a number of these T-bags, which are then separated only for the coupling of the individual amino acids and at the end of the synthesis for HF (hydrogen fluoride) cleavage. While there is still a fair amount of labor involved, it is possible to turn out 50-mg quantities of a large number of peptides in a relatively short time. Houghten's method is advantageous because the peptides can be isolated, purified, and characterized, but characterization clearly becomes rate-limiting if a sufficiently large number of peptides is being prepared. An alternative approach has been developed by Geysen et al. (1988). The peptides are synthesized on the tips of polystyrene pins
53
arranged on a support such that the tips can be immersed in the wells of a standard 96-well microtiter plate. As with Houghten's approach, steps such as washing and deprotection are carried out in common while the couplings take place in the microliter wells. In the original conception, an ELISA (enzyme-linked immunosorbent assay) type assay was used so that the results could be read colorimetrically in microliter plates. The pins were incubated with the probe antibody, washed, incubated with the conjugate antibody (horseradish peroxidase coupled to anti-IgG for the appropriate species in which the probe antibody was raised), washed again, and then immersed in wells containing the enzyme substrate. Color developed only in those wells where the pins bound the original probe antibody. A large amount of labor is involved in this synthesis approach, also, but an extremely large number of peptides can be prepared in a short amount of time. The advantage to this approach is that, in theory, the pins can be reused with another probe antibody if the original peptide-antibody complex can be disrupted. The disadvantage is that the peptides remain attached to the pins and cannot be characterized; there is the possibility that the derivatized polystyrene matrix of the pin could affect the interaction of peptide and antibody. A recent modification to this method involves the attachment of a protected Lys-Pro to the pins and peptide assembly on the e-amino group of the lysine. The peptide can be released from the pins by mild base treatment, giving the free peptide with a pendant diketopiperazine moiety (Bray et al., 1990). One approach to limiting the number of peptides that must be prepared for epitope mapping has already been mentioned, namely, the use of a hydropathy profile of the protein sequence (see above, Hydrophobicity Measurements). This approach is based on the supposition that most epitopes will be found on the protein surface and that relatively hydrophilic stretches of the protein sequence are more likely to be on the surface. One disadvantage of this method is that the hydropathy profile does not give an unambiguous or very detailed indication of protein topology. It is also true that all hydrophilic regions of the protein do not necessarily correspond to epitopes, that all epitopes are not hydrophilic or even on the protein surface, and that the major antigenic regions are not necessarily the most hydrophilic. The more typical approach to epitope mapping involves using protein biochemistry and molecular genetics techniques to create fragments of the protein, narrowing down the location of the epitope by determining successively smaller fragments that cross-react with the target antibody. With modem techniques and the judicious use of restriction endonucleases, it is often possible to clone and express the protein of interest along with a library of partial sequences.
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Using sensitive methods of detection such as Western blotting, it is possible to rapidly identify fairly small regions of protein sequence as containing a particular epitope, which can then be refined further through the use of synthetic peptides. A typical case is the identification of the principal neutralizing epitope of the HIV envelope protein. The HIV envelope protein, gp!20, is the viral ligand for the CD4 receptor protein on T4 lymphocytes and is involved both in binding of the virus to target cells and in fusion of the cellular and viral membranes, allowing viral genetic material to enter the cell. Antisera to gp!20 have been shown to block both infectivity and fusion (Allen et al., 1985; Veronese et al., 1985). Using protein and peptide fragments produced by recombinant techniques, limited proteolysis and peptide synthesis, the major neutralizing epitope was localized with both polyclonal antisera (Rusche et al., 1988) and a monoclonal antibody (Matsushita et al., 1988) to a 24-residue peptide which occurs in a 36-residue disulfide loop on the gp!20 molecule. Synthetic peptides corresponding to portions of this loop were prepared, guided partially by sequence homology comparisons using the sequences of gp 120 from different isolates of HIV-1. An octapeptide sequence (IleGln-Arg-Gly-Pro-Gly-Arg-Ala) located at the middle of the loop was shown to be the minimum sequence that could block the effects of neutralizing antibodies and that, when coupled to a carrier protein, could itself generate neutralizing antisera (Javaherian et al., 1989).
Producing Protein-Specific Antibodies
Antibodies that recognize intact proteins can be produced with synthetic peptides whose sequences are based on short stretches of the protein sequence. There are certain considerations, based on general properties of protein structure, that go into the design of appropriate synthetic peptides for this purpose. This section will concern itself mainly with these design considerations while more detailed treatment on the their production can be found in a number of good references (Grant, 1999; Tarn and Spetzler, 1997). A synthetic peptide that will be successful for the production of antiserum that will recognize an intact protein should be based on a stretch of sequence that is exposed to solvent. Moreover, that sequence should be a continuous stretch of amino acids. Although discontinuous epitopes certainly exist, designing a synthetic peptide to recognize them is much more complex. An inspection of known protein structures indicates that their surfaces are populated with a predominance of charged and polar residues (Creighton, 1993). Therefore, it is reasonable to assume that stretches of protein sequences that contain proportionately higher levels of these amino acids will be found on the surface of proteins.
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Turns or loop structures tend to be more useful for antibody production because they are generally found on the surface of proteins connecting larger arrays of helices and sheets and they consist of continuous stretches of amino acids. Many amino acid residues in helices and sheets are also found exposed to solvent, but since these structures are also usually found in larger arrays such as entended sheets and helical bundles, they are less appealing for this purpose. These simple considerations form the basis for predicting and designing synthetic peptides for this purpose. The main problem is determining which part of the protein satisfies these criteria. Many different indices have been devised which predict hydrophilicity, hydrophobicity, secondary structure, segmental mobility, side-chain accessibility, and sequence variability (see Van Regenmortel et al., 1988). All of these methods generally tend to yield similar results. A good general approach is to use an index of hydrophilicity (which is usually just an inversion of an index of hydrophobicity) along with a prediction of secondary structure. The results are then compared and areas of high turn propensity and hydrophilicity are identified. The ExPASy web site of the University of Geneva offers free access to a variety of different programs over the internet at http://expasy.hcuge.ch/tools/. Note that these procedures were developed for, and work best with, proteins that are water soluble and composed of a single globular structure. They may not be as successful with multi-subunit proteins or membrane proteins which may have large sections shielded from the solvent. An effective immunogen can usually be produced by coupling the peptide to a carrier protein or by synthesizing a "multiple antigenic peptide" or MAP (see below). MAPs are assembled on a branched lysine core producing either four or eight identical peptides branching from the a. and s amines of the terminal lysines of the core.
General Procedure
A general procedure for selecting potential sequences is outlined here.

1. Using the selected algorithms, compute the hydropathy index and the tendency for /3-turns of your protein sequence. Use a "window" of 7 or 9 and give equal weight to each amino acid. A window size of 9 includes four amino acids on each side of the central amino acid for which the value is computed and which is the simple average of the values for each amino acid in the window. 2. Compare the results of the two analyses and look for areas of sequence that are high in turn tendency and high in hydrophilicity (or low in hydrophobicity). These areas will generally be sequences high in Lys, Arg, His, Asp, Glu, Ser, Thr, Asn, Gin, Pro, and Tyr content.
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3. Examine the sequences for glycosylation site motifs and discard any sequences which contain them unless you know that the protein is not glycosylated. Amino-linked carbohydrate chains can occur at Asn-X-Ser or Asn-X-Thr sequences. Hydroxyl-linked carbohydrate chains do not appear to have a set motif but there are now some programs available that aid in their prediction. 4. The immediate amino-terminal and carboxyl-terminal regions of proteins are often exposed to solvent. If these areas appear to be hydrophilic in nature, they should be considered as acceptable candidates. 5. Mimicking the charge state of the ends of the pep tide may be helpful in certain instances. For instance, unless the peptide is at the immediate amino or carboxyl end of the protein, its ends will be in the peptide linkage to the rest of the protein and thus will not be protonatable. Synthesis of the peptide with blocked termini (acetyl-amino or carboxylamide) may be helpful in mimicking this situation by reducing the presence of potentially undesirable charge effects at the peptide termini. However, note that it is important to make sure the peptide has sufficient additional charge for solubility. 6. Peptides of 10 to 15 residues are usually adequate for the production of antibodies. Longer peptides are more difficult and expensive to make and usually unnecessary. Peptides do not necessarily need to be rigorously purified before conjugation, especially for polyclonal antibody production, since the antisera will be selected on the basis of specificity as well as titer. 7. Including a cysteine residue at either the amino or the carboxyl end of the peptide can be very helpful for cross-linking to carrier protein with heterobifunctional reagents. An example of an analysis using the ExPASy web site is shown in figure 2-14 for the 99-residue HIV-1 protease. The figure shows an analysis using the Chou and Fasman /3-turn propensity index (a) and the Kyte and Doolittle hydropobicity index (b). One area in particular, that around residue 41, shows both a high level of hydrophilicity (negative hydrophobicity) and high propensity for a j8-turn. This sequence, which is NLPGKWKPK, contains a high level of the amino acids expected on surface turns (see step 2 above) and would be an excellent candidate for producing an antibody to this protein.
Designing the Immunogen A synthetic peptide is seldom able to produce a sufficient immunogenic response on its own. A common solution to this is to chemically couple the synthetic peptide to a carrier protein which will boost the immune response. A less used but equally effective approach is the direct synthesis of a covalent multimer of the simple peptide sequence. This is called a "multiple antigenic peptide" or MAP (Tarn and Spetzler, 1997). Unlike simple peptides, where additional chemical manipulations after synthesis are required for coupling to a carrier
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protein, a MAP is complete and ready for immunization at the conclusion of the synthetic protocol. However, MAPs are more difficult to produce as completely homogeneous synthetic products, more difficult to analyze post-synthetically, and they may also be more prone to insolubility problems. Both four- and eight-branched MAPS peptides have been found to be effective, but four-branched MAPS are less prone to synthesis problems and are easier to characterize. Synthetic peptides produced as immunogens should be checked for homogeneity by analytical HPLC (high-performance liquid chromatography) and correct mass by mass spectrometry. The characterization of MAPs can be more problematic due to their multi-branched nature (Mints et al., 1997). If each of the four to eight peptide chains of a MAPS molecule have only a small percentage of modification at any particular residue, the aggregate will produce a relatively broad spectra or distribution. This can compromise HPLC and mass spectrometric analysis by producing broadly eluting peaks and heterogeneous spectra. However, this feature of MAPs usually does not tend to compromise their ability to form antigens to the proper peptide since the correct sequence is usually present in high enough concentration that a significant amount of the specific antibody is produced among the polyclonal population. Amino acid analysis, which is less sensitive to multiple small differences, may give a more reasonable assessment of the composition of the MAP. Design and Use of Combinatorial Peptide Libraries With the use of combinatorial techniques, it is now possible to screen peptide libraries potentially representing millions of peptide sequences to optimize peptide structure-activity relationships (SAR) for receptor or enzyme targets or even to discover ligands for targets whose natural ligands are unknown. Peptides are ideal molecules for the application of combinatorial technologies. Synthetic protocols are highly optimized and largely independent of peptide sequence. There are extremely sensitive and powerful analytical methods available for determining peptide composition and sequence. In fact, combinatorial chemistry was first conceived with peptides (Geysen et al., 1986, 1987) and most of the methodologies in use today were adapted from peptide technology. With the ability to screen large libraries of diverse peptide sequences, design based on specific amino acid properties becomes much less important. The major factors influencing peptide library design derive from the specific techniques employed. While a comprehensive review of combinatorial chemistry is beyond the scope of this chapter, a brief discussion of some of the most widely used peptide combinatorial techniques and their underlying assumptions, limitations and utility will be useful.
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Methods of Synthesis and Elucidation of Sequence Because of the ease of handling, peptide combinatorial libraries are most often prepared using solid phase peptide synthesis, either on traditional polystyrene supports (beads, pins or crowns) or on hydrophilic supports such as polyethylene glycol (PEG)-polystyrene (TentaGel or ArgoGel) or special-purpose supports like PEG-polyacrylamide (PEGA). An extensive listing of supports is found in Hudson (1999a). While a number of highly ingenious approaches have been developed for combinatorial chemistry, the utility of combinatorial chemistry is largely in screening, and the most straightforward methods have generally proven adequate. The simplest way to introduce a diverse set of amino acids at a given coupling step is to utilize an equimolar mixture of protected amino acids during the coupling step, and this is in fact how the first peptide combinatorial libraries were prepared. One can verify by sequencing or by amino acid composition that essentially equimolar amounts of amino acids can be incorporated at any given position in a peptide sequence (Ostresh et al., 1994; Rutter and Santi, 1989). It is important to note that an equimolar representation of amino acids at each position in a combinatorial mixture does not necessarily mean that each possible peptide sequence is also present in the mixture at equimolar amounts. Results from deconvolution of these mixtures really represent concensus sequences, and there is a basic implicit assumption that each position in a peptide sequence can be optimized independently of the other positions in the sequence. To the first approximation, this is not an unreasonable assumption for peptides, especially at the level of screening where combinatorial chemisty is most useful. A more rigorous way to prepare mixtures in which every possible sequence is equally represented is the split-and-mix technique described independently by Furka (1991) and Lam et al (1991). The general scheme for split-and-mix synthesis is shown in figure 2-15. The process is conceptually straightforward and uses traditional solidphase peptide synthesis. To create a library with 10 different amino acids, for example, the resin is split into 10 equal aliquots and each aliquot is coupled separately with one of the 10 different amino acids. The couplings can be monitored for completeness and recoupled as necessary. The 10 aliquots are then recombined, thoroughly mixed and then split again into 10 aliquots for the second round of coupling. Each aliquot now contains an equimolar mixture of the 10 different aminoacyl resins. The process can be repeated until the library is of the desired size. There are two important things to bear in mind about split-and-mix libraries. The first is that the splitting or portioning of resin introduces
Figure 2-15. Split-and-mix approach to combinatorial library synthesis, illustrated for a 5 x 5 library. If the number of resin beads is large compared to the number of aliquots the resin is split into, each aliquot will contain an equimolar distribution of amino acids at each step. Note that all possible sequences are represented. At the end of the process, each resin aliquot contains a mixture of peptides with a common TV-terminal residue. If the aliquot is cleaved, a mixture of peptides will be produced. Note also that each resin bead contains a unique peptide sequence. If the beads can be segregated and assayed individually, information can be obtained for individual sequences rather than for mixtures.
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an element of statistics into the synthesis. An equimolar distribution of the various peptidyl resins will be obtained only if the number of beads is appreciably larger than the number of theoretical sequences present. Because the number of theoretical sequences increases exponentially with the length of the combinatorial peptide, this can become an issue quite quickly. Using all 20 naturally occurring amino acids, a peptide with four variable positions generates 160,000 theoretical sequences; for five variable positions, the number of theoretical sequences is 3,200,000. There have been a number of calculations as to what fold excess of beads over theoretical sequences is necessary; for a probability that 95% of theoretical sequences are actually contained in the library, one needs at least a five-fold excess of beads over theoretical sequences. Standard 100-200 mesh peptide synthesis resin contains about 106 beads per gram of resin (Burgess et al., 1994). For the example of a peptide library with five variable positions and using all 20 naturally occurring amino acids, it would require starting with 16 grams of resin to ensure that 95% of those 3.2 million theoretical sequences were actually represented! For this reason large peptide libraries are usually prepared as consensus libraries, which will be discussed below. The second property of split-and-mix libraries is that each individual bead contains just a single peptide sequence. If the resin from a split-and-mix synthesis is cleaved in bulk, it will yield a mixture of peptides, just as libraries prepared using mixtures of amino acids. If the beads can be assayed on an individual basis, however, individual peptide sequences can be obtained directly. Single-bead screening will be discussed in more detail below.
Iterative Deconvolution
The most general method for determining active sequences from screening mixture libraries is iterative deconvolution, which is illustrated in figure 2-16. A set of mixtures in which one of the variable positions is defined in each mixture is assayed and the optimal residue(s) at that position are determined. Each of the variable positions is probed in turn by the synthesis of ever smaller mixtures in which each of the optimal residues is held fixed. At the end of the process, individual peptide sequences are obtained. This methodology was the initial deconvolution technique introduced in the first combinatorial libraries and has been used extensively and successfully (Berk et al., 1999). There are a few points to consider about iterative deconvolution. The first is that, in the first round of screening, very large mixtures are being evaluated. If the activity has very stringent sequence requirements, a very small portion of the library components will be active and the assay will need to be sufficiently sensitive to detect what may
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Figure 2-16. Iterative deconvolution for a hexapeptide library containing 20 different amino acids. At the first round each mixture is quite large, so the assay must be sensitive enough to detect what may be a weak activity in the mixture. Activity should increase at each successive round as optimized residues are held fixed and the number of peptides in each mixture decreases. The deconvolution process can be speeded up by holding two residues fixed at each round rather than one. This requires more robust parallel synthesis technology since in this example each library will now consist of 400 mixtures rather than 20, meaning that 400 individual syntheses rather than 20 are needed at each step. However the size of the mixtures also decreases by a factor of 20 at each round, so the assay sensitivity requirements are also reduced.
be very weak activity in the mixture. More seriously, deconvolution may also be confounded if the opposite is true and there are many weakly active peptides in the mixture, in which case the activity of the mixture is the sum of those weak activities (Terrett et al., 1995a, b). Specific activity does not increase with successive rounds of iterative deconvolution and optimized individual sequences cannot be identified. Finally, since resynthesis is required for iterative
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deconvolution, it does require additional effort. And, since the specific sublibraries which will be resynthesized in the second round of deconvolution will be selected based on activity against a specific target and are not likely to be of use if the original library is screened against a second target, the overhead of resynthesis makes iterative deconvolution less attractive in a screening paradigm in which the same libraries are screened against multiple targets. Consensus Libraries Since large peptide libraries tend in fact to be consensus libraries, that is, libraries in which only a fraction of the possible sequences are present but in which there is an equal distribution of amino acids at each variable position, it can be more efficient to determine consensus sequences directly. There are two ways in which this is possible. The first involves assaying the entire library at once and requires some method to separate the active peptides from the inactive. The active peptides can then be sequenced as a mixture, providing a consensus sequence. This has been successfully applied to the determination of kinase substrates by Cantley and coworkers (Songyang et al., 1994; Nair et al. 1995). A peptide library in which the phosphate acceptor residue is fixed at a specific position within the sequence is incubated with kinase and ATP. Phosphorylated peptides are then separated from the nonphosphorylated peptides by careful column chromatography on an iron (Songyang et al., 1994) or molybdate (Nair et al. 1995) column, which selectively retains the phosphorylated peptides. Careful elution of the retained peptides followed by sequencing of the mixture provides consensus sequences which, when resynthesized individually, have provided highly active substrates. A similar result is obtained with affinity selection, in which a library is passed through a target which is immobilized in some fashion (Chu et al., 1995) (covalently linked to a column or focused by isoelectric focusing, for example). Peptides which interact with the target elute later and can be collected and sequenced. The advantage of this method is that extremely large libraries can be probed in a single experiment, minimizing both effort and biological reagent. The disadvantages are that not every system is amenable to the separation of the active peptides, and the target must be sufficiently promiscuous to recognize a consensus sequence. The concentration of any one peptide in such a mixture is exceedingly small, so it is relatively unlikely to detect a peptide if the sequence requirements for activity are very stringent.
Scanning Libraries
A specific type of consensus library is the scanning library (Pinilla et al., 1992; 1994). In this configuration there is one sublibrary for each
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Figure 2-1 7. Scanning library for a pentapeptide sequence using the set of 20 naturally occurring amino acids. The library consists of 5 x 20 = 100 mixtures. Each mixture consists of 20 x 20 x 20 x 20 = 160, 000 peptides. Screening the 100 mixtures identifies a consensus sequence. The complete library represents 3.2 million sequences and can be deconvoluted without resynthesis by assaying only 100 mixtures. The assay must be sufficiently sensitive to detect activity in a mixture of 160,000 peptides. Targets with extremely stringent sequence requirements may be less amenable to this approach.
amino acid at each variable position in the peptide, as shown in figure 2-17. Thus for a library using all 20 amino acids at each of five variable positions, there would be 20 x 5 = 100 sublibraries, each with a different amino acid fixed at one of the five variable positions. The efficiency of this arrangement is again readily apparent. A 20 x 5 library encodes 3.2 x 106 sequences, but they can be evaluated in 100 assays. The results of the assay would identify the best amino acid(s) at each of the various postions, allowing construction of a consensus sequence(s). The same caveats apply here as with other large mixtures (in the example each sublibrary would consists of 160,000 theoretical components) in that the target must be promiscuous enough to recognize a consensus sequence.
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One-Bead, One-Compound Screening The product of split-and-mix bead libraries is a mixture of beads each one of which bears a single peptide sequence. If the beads can be assayed individually, one can get results for single peptide sequences rather than results extrapolated to single peptides from assays performed on mixtures of sequences. This is an important point if the particular peptide interaction has a high degree of sequence stringency where identification from mixture screens is less likely. Beadassociated assays do however impose a relatively high technological barrier. The simplest application of one-bead, one-compound screening is an on-bead assay in which the peptides remain attached to the bead, a technique first applied in probing antibody specificity (Lam et al., 1991). This requires the use of a hydrophilic support like TentaGel. In this case the beads were treated in bulk with a fluorescently labeled antibody, but any double-labeling technique would be appropriate. Beads bearing an active epitope will light up and can be physically picked with tweezers or a capillary using a dissecting microscope. The peptide sequence is determined by removing the antibody with a guanidine wash followed by microsequencing. Although PEG-PS resins are typically lower loading than polystyrene resins, each bead still typically contains 100-300 pmol of peptide, which is more than sufficient for solid-phase microsequencing, which generally requires less than 10 pmol of material. On-bead analysis has also been used to probe enzyme specificity. In the case of kinases (Wu et al., 1994), beads are incubated in bulk with the kinase and 32P-ATP to specifically label substrate-bearing beads. The beads are then washed and immobilized in a dilute gelatin matrix onto autoradiographic film or a phosphorimager to detect and pick the labeled beads. Typically this process is applied several times until the labeled beads are sufficiently dispersed to allow individual beads to be picked. In the case of proteases, fluorescencequenched substrates have been prepared on resin (Singh et al., 1995). These substrates contain a fluor at the C-terminal end and a quencher at the TV-terminal end, so that the intact peptides on the resin are not fluorescent. Endo proteolysis releases the quencher into solution and the C-terminal fragment which remains attached to the resin bead then fluoresces, allowing substrate containing beads to be picked. If proteolysis is not allowed to proceed to completion, microsequencing reveals both the complete substrate sequence as well as the cleavage site. Application of this method requires that there be no residual fluorescence on the resin support, which unfortunately is not the case with the PEG-PS resins, prompting Meldal (1994) to design a PEG-polyacrylamide (PEGA) resin specifically for this purpose.
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The advantage of these on-bead assays is that they can be performed in a single experiment with bulk resin, since the labeled beads can be selected out of the mixture. One caveat with on-bead assays is that the support may influence the interaction with the target in unpredicatable ways. Another caveat is that while the peptide loading on the bead may be only 100-300 pmol, this can still translate into a concentration approaching millimolar levels, so it is quite possible to select low-affinity interactions which may not be reproduced when soluble versions of the same peptides are prepared. Not every biological target is suitable for on-bead screening, so various techniques have been developed to remove the peptide from the bead before assaying. Ideally, this is accomplished in such a way that some residual peptide remains on the bead for identification by solid-phase sequencing. The trick is being able to perform the cleavage in such a way that the peptide still remains associated with the resin until it is released at the time of the assay. Cleavage of the resin in situ in the assay is usually not possible because of the harsh nature of most linkage cleavage conditions. Lerner (Quillan et al., 1995) has developed an interesting system in which peptides prepared on typical polystyrene resins are treated with neat gaseous trifluoroacetic acid (TFA), which effects a small amount of cleavage. Vacuum removal of the TFA leaves the cleaved peptide adsorbed to the resin beads. This technique has been used in melanophore-based assays for G-protein coupled receptor (GPCR) ligands. Pigmentation of frog skin melanophores is under the control of a GPCR system and transient expression of an exogenous GPCR into melanophores allows a simple visual read-out of ligand activity. Resin is adhered to plastic wrap, cleaved with TFA vapor and then layered over melanophores cultured in soft agar. Sufficient peptide diffuses out from the bead to cause a local response in melanophores immediately adjacent to beads containing an active ligand, allowing those beads to be picked and sequenced. A more general approach to partial release of peptides from resin involves use of multiple selectively-cleavable linkers on the resin such that under some conditions a controlled amount of peptide can be released from the resin beads while retaining sufficient peptide covalently attached to allow for subsequent micro sequencing (Patek and Lebl, 1999). With judicious choices of linkers, it is possible to design a resin from which peptides can be released under several different orthogonal conditions (Lebl et al., 1993). This allows small aliquots of beads to be assayed in one well. Beads from active wells can then be rearrayed as singles for a second round of cleavage to determine the individual active beads, which can then be selected for microsequencing. It is also possible to effect total cleavage of the peptide from the resin if the resin beads are tagged in some way to encode the peptide
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sequence. The tags are some other type of molecule which is synthesized onto the resin and which can be detected in some orthogonal release and/or analysis. Chemical tags which are selectively cleaved and identified by electron capture gas chromatography (Ohlmeyer et al, 1993) or HPLC (Ni et al., 1996) have been described, as have polynucleotide tags, which are decoded by PCR (polymerase chain reaction) (Brenner and Lerner, 1992; Nielsen et al., 1993). Tagging adds an additional synthetic step for each peptide coupling in order for the tag to be introduced at each step and so increases the synthetic effort required. However tagging can allow identification of peptides which are otherwise not amenable to identification by sequencing (e.g., those containing blocked amino termini, unusual amino acids or D-amino acids, etc.) The main disadvantage with bead libraries in general is that due to the statistical nature of such libraries it is necessary to sample many more beads than there are theoretical components in the library in order to be confident that the majority of theoretical sequences have in fact been assayed. This is not as big a problem with on-bead assays, which can be performed on bulk bead samples, but becomes a major logistical problem when beads must be arrayed individually so that released peptides may be assayed in solution but still associated with the bead of their origin. Arraying is very time-consuming and can be technically challenging depending on the exact format of the assay. There can also be an increased requirement for biological reagent, since the number of assays required is minimally three times the number of theoretical compounds in order to have a 95% chance that each peptide sequence has been sampled at least once, if individual beads are being arrayed and assayed. This requirement is often reduced by arraying multiple beads per well for the first round of assay and then rearraying individual beads only from the active wells. This can substantially reduce the number of assays required, but does require additional bead handling since active mixtures need to be rearrayed, and requires a mechanism for controlled partial release of peptides from the resin, adding to the synthetic overhead.
Parallel Synthesis An alternative to mixture library synthesis is parallel synthesis of peptides. Geysen introduced synthesis on polystyrene pins which were arranged to match the spacing of standard 96-well microtiter plates (Geysen et al., 1984). Each individual well could contain a different amino acid during coupling and the pins could be washed and deprotected in parallel. This method has been used extensively to prepare overlapping peptide sequences for epitope mapping of proteins, for example. An alternative method for parallel synthesis
which took advantage of the efficiency of split-and-mix synthesis was introduced by Houghten and involved the use of polypropylene mesh bags (T-bags) to hold the synthesis resin (Houghten, 1985). T-bags were labeled with india ink (the only marking system which could withstand repeated exposure to organic solvents used in peptide synthesis). T-bags could be combined in a single flask for common operations like deprotection and washing and could be segregated for coupling reactions. A variation on this which is more amenable to automation was introduced by Nicolaou et al. (1995) which relied on glass-encapsulated radiofrequency tags which were used to identify polypropylene mesh cannisters which contain the synthesis resin. An automated system for tracking and sorting these rf-tagged cannisters has been commercialized by Irori Quantum Microchemistry.! The use of libraries of individually synthesized peptides offers advantages over mixture libraries in that the identity of peptides is already known from the synthesis and there is no need for deconvolution or identification by sequencing or reading chemical tags. In addition, there is the opportunity to characterize the peptides, verify sequence or structure, purify if necessary, and quantitate the biological response since exact weight or concentrations of the peptides can be determined. The disadvantage is that the number of samples to be processed multiplies exponentially as sites of diversity are added and can require considerable resources to accommodate large numbers of peptides. Parallel synthesis is probably best applied to situations where a relatively small number of positions are being varied.
Other Aspects of Peptide Design
Enzymatic Stability
For simple peptides, the serum half-life and biological activity is often limited by proteolytic hydrolysis of susceptible peptide bonds. To make these peptides pharmacologically or physiologically useful, it is necessary to increase their serum half-life by increasing their resistance to proteolysis. The exact pathway for proteolysis depends on the peptide in question, and determining the metabolic fate of a given peptide can be a difficult and tedious process. However, inspection of the peptide sequence can usually provide insight into the most likely sites for proteolytic degradation. There are two types of proteolytic enzymes: exopeptidases, which cleave amino acids from either end of a peptide chain, and endopeptidases, which cleave peptide bonds in the middle of a peptide chain. Exopeptidases include aminopeptidases, which cleave the TVterminal amino acid from a peptide chain; carboxypeptidases, which cleave the C-terminal amino acid from a peptide chain; and dipeptidyl
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carboxypeptidases like angiotensin converting enzyme (ACE), which cleave the C-terminal dipeptide from a peptide chain. Their recognition sites include the charged amino or carboxyl termini of the peptide, and they may show side-chain specificities as well. Endopeptidases generally have specificities for a particular amino acid side chain or a discrete sequence. The most relevant endopeptidase activities to consider are trypsin-like, in which Lys-X or Arg-X bonds are cleaved; chymotrypsin-like, in which Phe-X, Trp-X or bulky aliphatic-X bonds are cleaved; and post-proline cleaving enzyme, in which Pro-X bonds are cleaved. There are many ways to stabilize a peptide against specific proteolysis. One approach is as simple as acetylating the amino terminus to block aminopeptidase action because aminopeptidases require the presence of a free a-amino group. Another approach involves modifying or deleting an amino acid side chain or inverting its chirality (most proteolytic enzymes have greatly reduced activity against D-amino acid residues). The susceptible peptide bond itself may be altered as well. Introduction of steric bulk along the peptide backbone by substitution of methyl groups for a-hydrogens or amide hydrogens reduces the rate of enzymatic hydrolysis adjacent to those substitutions. Peptide bonds can also be substituted for by a variety of hydrolytically inert isosteric structures such as trans olefins. All of these approaches involve some modification to the peptide structure that can have effects on biological activity apart from any effects on proteolytic stabilization. Modifications that reduce susceptibility to proteolytic cleavage can, however, have a profound effect on the in vivo potency of peptides. Arginine vasopressin (AVP) is a naturally occurring peptide hormone produced in the pituitary and exhibits antidiuretic activity in vivo. The antidiuretic activity from a bolus injection of AVP has a very short duration of action, suggestive of metabolic instability. Indeed, immunoreactive AVP disappears rapidly from the circulation after a bolus injection and is degraded rapidly upon exposure to serum in vitro (Edwards et al., 1973). The structure of arginine vasopressin is given in figure 2-18 (compound 15). From the structure, several potential sites for enzymatic degradation can be identified. The peptide has a free amino group and might be susceptible to aminopeptidase action. There is an arginine residue at position 8, which could be a site for tryptic-like cleavage. The ammo-terminal hexapeptide is cyclized by means of a disulfide bond. Cyclic peptides are often resistant to proteolytic degradation due to the conformational constraints imposed by cyclization. Bisulfides, however, can be opened by reduction or by interchange reactions with sulfhydryl containing molecules such as glutathione (y-glutamyl-cysteinyl-glycine). In addition, most small cyclic peptides require the cyclic structure for biological activity since it folds them into conformations that are otherwise relatively unlikely for
69
Number
Compound
15 16 17 18 19
H2N H HzN H H
S S S S CH2
Arg Arg
D-Arg D-Arg
AVP
dAVP DAVP dDAVP [Asu^-AVP
Arg
Figure 2-18. Structure of arginine vasopressin and analogs.
linear peptides. AVP is no exception, and the linear Ala-1,6 analog is devoid of antidiuretic activity (Walter et al., 1967). Figure 2-18 shows some AVP analogs that were modified in ways that would expectedly increase the stability of the peptide. Analog 16 (dAVP) is the desamino version, which would be expected to be resistant to any aminopeptidase activity. Analog 17 (DAVP) is the D-Arg-8 peptide, which should be resistant to tryptic-like cleavages. Analog 18 (dDAVP) contains both modifications together. Analog 19 is a dicarba analog, one in which the disulfide is replaced with a nonreducible ethylene bridge; it is a desamino analog as well. The biological activities of these analogs are given in table 2-9. The ADH (antidiuretic hormone) activity is a measure of antidiuretic potency in vivo in the rat. AVP has 332 International Units of antidiuretic activity per milligram of peptide (Sawyer et al., 1974). The desamino peptide 16 exhibits a five-fold enhanced in vivo potency compared to AVP (Bankowski et al., 1978), suggesting increased metabolic stability. The D-Arg-8 analog 17 also retains good in vivo antidiuretic
Table 2-9 Biological activity of modified arginine vasopressin analogs Number

15
Compound
ADH (IU mg~')
A"bind (HIM)
K^ (nM)
AVP
dAVP DAVP dDAVP [Asu''6]-AVP
332
1745
0.44
16
17
1400 3000
0.1 2800 3450
18 19
114 955
1274
1.4
0.4
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Synthetic Peptides
potency (Zaoral et al., 1967a). The doubly substituted analog 18 shows roughly additive effects for the individual substitutions (Zaoral et al., 1967b). The desamino-dicarba analog 19 also shows enhanced potency in vivo, roughly equivalent to the disulfide analog 16 (Hase et al., 1972). The peptide dDAVP (compound 18) not only exhibits enhanced in vivo antidiuretic activity but has a five-fold-longer serum half-life after bolus injection and is completely stable to incubation with plasma in vitro (Edwards et al., 1973), establishing the utility of enhancing metabolic stability. What is even more remarkable can be found by comparing the in vitro activity of these analogs. Vasopressin acts in the kidney by binding to specific receptors activating adenylate cyclase. Both the binding to rat kidney membrane preparations and the hormone-dependent activation of adenylate cyclase are listed in table 2-9. AVP has a subnanomolar binding affinity and activation constant (Hechter et al., 1978). In contrast, both DAVP and dDAVP show more than three orders of magnitude decrease in receptor affinity and activation constant (Roy et al., 1975; Butlen et al., 1978). The increased metabolic stability of dDAVP more than makes up for a tremendous loss in intrinsic activity at the receptor, allowing this analog to exhibit enhanced antidiuretic potency in vivo to the extent that it is clinically useful for the treatment of diabetes insipidis. This is a remarkable result and underscores the importance of metabolic stability in the expression of the in vivo biological activity of pep tides. There are more exotic ways to stabilize individual peptide bonds against proteolysis. These approaches involve replacing the amide linkage with a proteolytically resistant linkage. Some peptide bonds, like the trans double bond or the thioamide (C=SNH), are isosteric with the amide bond and preserve their geometry while others, like the ethylene (CH2CH2) or thiomethyl ether linkages (CH2S), preserve the connectivity and chain length only. All of them alter the polarity and hydrogen bonding potential of the amide bond as well as imparting resistance to proteolysis. A number of these substitutions were examined in the endogenous opiate peptide leucine enkephalin, which has the sequence H-Tyr-GlyGly-Phe-Leu-OH. Leu-enkephalin is susceptible to a number of enzymatic cleavages including aminopeptidase cleavage of the TyrGly bond (Hambrook et al., 1976), carboxypeptidase cleavage of the Phe-Leu bond (Marks et al., 1977), and dipeptidyl carboxypeptidase cleavage of the Gly-Phe bond (Malfroy et al., 1978). Biological activity requires the free a-amino group and the L-tyrosine side chain at position 1 (Frederickson, 1977), so desamino or o-Tyr substitutions are not useful to decrease aminopeptidase degradation. Substitution of o-Ala for Gly-2 gives an analog with somewhat higher biological activity than the parent compound (Kosterlitz et al., 1980) and
71
Figure 2-19. Enkephalin analogs with modified peptide bonds.
improved resistance to proteolysis (Miller et al., 1977). However, in the quest to further improve metabolic stability, a number of peptide bond replacements were also examined, some of which are summarized in figure 2-19. The results from amide-bond replacement differ with the particular amide bond being replaced. Replacement of the Tyr-Gly bond with a trans olefin isostere, which retains the
72
Synthetic Peptides
geometry of the amide bond, provides analog 20, which retains full brain-receptor-binding affinity (Hann et al., 1982). The case here is not simple because the thioamide-containing analog 21 does not retain binding affinity despite the thioamide preserving the geometry of the amide bond (Clausen et al., 1984). Further, the thiomethyl ether analog 22, which does not preserve the amide geometry, does retain some binding affinity, but it behaves more like an antagonist than an agonist at low doses in a functional assay (inhibition of electrically induced contraction of guinea pig ileum; Spatola et al., 1986). The Gly-Gly bond in Leu-enkephalin cannot be replaced by a trans olefin (analog 23; Cox et al., 1980), ethylene (analog 24, which is an analog of the other naturally occurring enkephalin sequence, methionine enkephalin; Kawasaki and Maeda, 1982), or thiomethyl ether moiety (analog 25; Spatola et al., 1986), but substitution of a thioamide (analog 26) gives a peptide with enhanced binding and biological activity (Clausen et al., 1984). The most forgiving bond in terms of replacement is the Phe-Leu bond, which can be replaced with either a thioamide (analog 27; Clausen et al., 1984) or a thiomethyl ether (analog 28; Spatola et al., 1986) with retention of binding affinity. The lack of a requirement of a Phe-Leu amide bond for biological activity led to some more innovative peptide bond replacements. Analog 29 contains an a-aza-amino acid (aza-leucine), which gives rise to an acyl hydrazide linkage rather than an amide bond. The aaza-amino acid has no chiral center. This analog again retains full biological activity (Dutta et al., 1977). In the o-Ala-2 cnkephalinamide analog 30. amide bonds are retained, but the sense of the Phe-Leu and Leu-NH2 amide bonds is reversed, introducing what are called retroamide bonds. The phenylalanine carboxyl group has been replaced by an amino group and the leucine amino group by a carboxyl group to form a NHCO linkage rather than a CONH peptide bond. The same reversal of functional groups is made at the Leu-NH2 peptide bond. (Since both amino and carboxyl groups of the Leu-5 residue have been "exchanged," it is now in reality o-leucine, but the relative projection of the leucine side chain from the peptide backbone has been preserved.) The o-Ala substitution should block aminopeptidase action, and the retro-peptide bonds should block chymotryptic-like cleavages. The modified peptide exhibits both enhanced potency in vitro and prolonged activity in vivo compared to D-Ala-2 enkephalinamide (Chorev et al., 1979). Conformational Design and Constraint Conformational considerations in peptide synthesis remain one of the greatest challenges in the design of biologically active peptides. In
73
addition to the nature of the side chains, the three-dimensional shape assumed by a peptide when interacting with its target, be it receptor, antibody, or enzyme, determines its biological activity. Unlike proteins, peptides of under 15 or so residues in length tend not to exhibit a stable or even a preferred solution conformation. There is generally too little hydrophobic character in a short peptide that can be sequestered from the polar environment by folding. The peptide backbone and polar side chains will have a driving force to be solvated, and entropic considerations will generally favor solvation by the bulk solvent. Thus, there will usually be an ensemble of conformational states in solution. If biological activity involves only one discrete conformer, this conformational ensemble essentially represents a dilution of the biologically active species. The problem is most acute for peptides designed to mimic a portion of a protein structure. In their native environment, these peptide sequences can rely on the protein's structural rigidity to hold them in a particular conformation, while as free peptides they have no such constraining influence. Even for analogs of linear peptide hormones, which have evolved to express high biological activity despite conformational mobility, favorable conformational constraints can impart an appreciable increase in biological activity. Conformational constraints can be divided conceptually into the three following categories: Local constraints involve restricting the conformational mobility of a single residue in a peptide; regional constraints are those which affect a group of residues that form some secondary structural unit, such as a /3-turn; global constraints involve the entire peptide structure. Peptides that are long enough to adopt stable solution conformations can have conformational preferences optimized globally by modifying the peptide sequence in accordance with the empirical rules of secondary structure prediction such as those devised by Chou and Fasman (1978; see above, Secondary Structure Prediction). This approach does not "freeze" a peptide into a particular secondary structure, but it stabilizes that structure relative to random coil or other structures, increasing the number of peptide molecules that would be found in that conformation at any given time. Peptides that are too small to adopt stable conformations on their own require covalent modifications to introduce local or regional conformational constraints. These constraints typically involve addition of sterically bulky substituents adjacent to a rotatable bond to restrict its mobility or the incorporation of cyclic structures. It is impossible to cover all the approaches that have been taken to introduce conformational constraints into peptides, but some of the more successful approaches will be discussed below.
74
Synthetic Peptides
Figure 2-20. (a) Helix wheel projection of a mellitin 1-20 helix. There is some segregation of hydrophilic and hydrophobic residues, but the arrangement is interrupted by Gly and Ala residues and by proline-14, which destabilizes the helix. The basic hexapeptide tail hangs off the helix.
Globally Optimized Secondary Structure It is possible to design peptides of 15 to 20 residues in length that will exhibit a preference for adopting helical structures in solution. The most successful applications involve the design of peptides containing amphiphilic helices, such as the bee venom peptide mellitin. Mellitin is a 26-residue peptide with the sequence Gly-Ile-Gly-Ala-Val-Leu-LysVal-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-LysArg-Gln-Gln-NH2 with the biological activity of binding to and lysing erythrocytes (Habermann, 1972). The lytic activity requires the basic C-terminal hexapeptide, but the amino-terminal 20-residue peptide can still interact with phospholipid bilayers and membranes (Schroder et al, 1971). This suggested to Kaiser and coworkers that
75
Figure 2-20. (b) Helix wheel projection of the idealized amphiphilic lytic pep tide based on the mellitin structure. The hydrophobic face is composed solely of leucine residues while the hydrophilic face is composed of serine and glutamine residues. There is little sequence homology in the two helical sequences 1-20, but the length of the amphipathic helix has been preserved while the amphiphilicity has been optimized. Tryptophan-19 and the basic hexapeptide tail have been retained.
the amino-terminal 20 residues might have formed an amphiphilic helix that bound at the membrane interface and held the basic hexapeptide tail responsible for the lytic activity close to the membrane surface where it could interact (DeGrado et al., 1981). A helical wheel projection of mellitin 1-20 is shown in figure 2-20(a). There are several problems with this as an ideal amphiphilic helix. The Gly residues and especially the Pro-14 residue tend to reduce the helical propensity of the pep tide. Also, several of the Gly residues are found on the hydrophobic face. DeGrado et al. (1981) designed an idealized sequence for the 1-20 region in which the hydrophobic residues were replaced
76
Synthetic Peptides
with Leu, and the hydrophilic residues were either Ser or Gin; the Cterminal basic hexapeptide was not modified. The resulting idealized helix is shown in figure 2-20(b). The idealized peptide had an identical profile of activity compared to mellitin, but it interacted with lipid monolayers more strongly, formed tetramers at a lower concentation, and lysed erythrocytes at a lower concentration than mellitin, which agrees with the formation of a better amphiphilic helix than mellitin.
Local Conformational Constraints The simplest local constraints that can be placed on a given residue involve the substitution of a methyl group for a hydrogen adjacent to a rotatable bond. For example, replacing the a-hydrogen on alanine with a methyl group gives aminoisobutyric acid (Aib; figure 2-21). The greater steric bulk of the methyl group reduced the rotational freedom of the two adjacent peptide backbone angles 0 and T/T (see figure 2-6; see above, Protein Secondary Structure). In the case of Aib, the low energy backbone torsional angles are 0 = ^ ~ 60, which are the backbone angles associated with helical structure (Marshall and Bosshard, 1972). a, a-Diethylglycine, with its bulkier substitution at the a-carbon, tends to constrain the backbone angles 0 and ^ to approximately 180, angles typical of a fully extended chain (Benedetti et al., 1988). Some other local constraint-producing amino acids are shown in figure 2.21. Substitution of a methyl group for the hydrogen on the amino group, to give A^-methy 1-alanine for example, has an effect on the cis-trans ratio of the amide bond, lowering the relative energy of the cis isomer. It has only a modest constraining effect on its own torsional angle 0, but it affects the angle V of the preceding residue, constraining it to large positive values typical of those found in extended or ^-structure (Marshall and Bosshard, 1972). Proline is a special case of TV-methyl amino acids because its own angle 0 is constrained to about 80 by being contained in the five-membered pyrrolidine ring. Proline is often found in reverse turn structures, occupying the i + 1 position in Type II and III' /3-turns, the / + 2 position in Type II' and III /J-turns, and in inverse y-turn conformations (Smith and Pease, 1980). An interesting proline congener is 5,5-dimethylthiazolidine-4-carboxylic acid (Dtc), a /?,/3-disubstituted thioproline analog. Dtc is conformationally similar to proline, except that the y-turn is disfavored in Dtc because of steric interaction between the /J-methyl groups and the carbonyl carbon. Also, it was suggested as a probe for proline-containing inverse y-turns (Samanen et al., 1990). Constraint-producing amino acid substitutions are typically used as probes for the biologically active conformation. If the constrained peptide retains good biological activity, then it can be
Figure 2-21. Some constraint-producing amino acids.
inferred that the particular backbone constraint is compatible with the biologically active conformation. Negative results are more difficult to interpret because the additional steric bulk producing the constraint may also interfere with the peptide binding to its target molecule regardless of conformational effects. This approach has been applied to a number of peptide hormones including arginine vasopressin (Moore et al., 1985), bradykinin (Turk et al., 1975), and angiotensin (Samanen et al., 1989). In most cases, the constrained analogs do not exhibit any better potency than their unconstrained analogs, in spite of the reduction in conformational mobility. This may be due in part to the fact that these modifications do not eliminate conformational mobility but only reduce it to some extent. The results of incorporation of constraint-producing amino acids can also be somewhat capricious. TV-methyl or a-methyl substitutions, for example, have converted angiotensin agonists into antagonist analogs (Pena et al., 1974; Turk et al., 1976). In addition to providing conformational constraints, TV-methyl and a-methyl amino acids can impart other properties to peptides, such as increased resistance to proteolysis, and have been successfully used in the design of metabolically stable and orally active renin inhibitors (Pals et al., 1986; Thaisrivongs et al., 1987).
Regional Constraints Turns are a prominent feature in peptide secondary structure that have often been implicated as important elements for biological activity, as discussed previously (see above, Turns). Turns or chain reversals must
78
Synthetic Peptides
Figure 2-22. y-Lactam turn mimetic. Ball-and-stick of y-lactam mimetic showing /S-turn conformation; compare with figure 2-ll(d).
occur in cyclic peptides or in peptides that occur as short loops in proteins, but linear peptides can fold into conformations containing turns as well. There are steric constraints placed on the side chains and backbone torsions of the corner residues in a turn. Gly and Pro residues most readily accommodate these constraints, and their appearance in a linear sequence is suggestive of a potential turn structure. A number of investigators have designed turn mimetics, cyclic moieties designed to replace the internal residue(s) of a turn while maintaining the overall geometry associated with the turn (Nagai and Sato, 1985; Huffman et al., 1988; Kahn et al., 1988). The mimetic most frequently applied to peptide design is the y-lactam of Freidinger (1981; figure 2-22), which induces a Type II' /Rurn conformation. The y-lactam has successfully been applied to the peptide hormone, luteinizing-hormone-releasing hormone (LHRH), whose sequence is shown in figure 2-23. LHRH contains a Gly residue at position 6. When substituted by a o-Ala, the biological activity increased, while the L-Ala analog exhibited low activity (Monahan et al., 1973). This outcome suggests that the Gly residue might be occupying the i -f 1 position of a Type II' /Rurn consisting of the sequence Tyr-Gly-LeuArg. As discussed previously (see above, Protein Amino Acids), a
79
Figure 2-23. LHRH and y-lactam-containing analog.
o-amino acid at that position would have an equatorial placement of the side chain, while an L-amino acid would have the less favorable axial placement of the side chain. Freidinger et al. (1980) prepared the y-lactam-containing peptide shown in figure 2-23, in which the corner Gly-Leu residues were replaced by the lactam structure. The constrained peptide proved to be more potent than the parent LHRH, both in vivo and in an in vitro assay, suggesting that conformational stabilization is indeed responsible for the improved activity.
Cyclization The simplest way to introduce a conformational constraint into a linear peptide is by cyclization, either through disulfide bond formation or by coupling the a- or Lys e-amino groups with the carboxyl terminus of an Asp or Glu side chain. Cyclization usually requires peptides of moderate length to adopt some sort of folded conformation consistent with bringing the two ends together, but the exact nature of the conformation(s) induced may not be predictable. As mentioned previously, even a cyclic disulfide hexapeptide still has a great deal of conformational mobility (Hagler et al., 1985). Nevertheless, cyclization greatly reduces the number of accessible conformations compared to the linear peptide and, in favorable cases, can substantially improve or alter peptide biological activity. One case in point involves the preparation of small peptide fibrinogen receptor antagonists. Platelets express a cell surface glycoprotein, gpIIb/IIIa, which binds to the extracellular matrix protein fibronectin and to the plasma protein fibrinogen. Fibrinogen expresses multiple platelet-binding domains and causes aggregation of activated platelets. This adhesive process is important in the formation of blood clots, and inhibitors of this process are potentially useful as
80
Synthetic Peptides
Table 2-10 Fibrinogen receptor binding peptides

Antiaggregator IC50 (JIM)
91 33 16 4.1
Compound Sequence Fibrinogen 31 32 33 34 35 Ac-Arg-Gly-Asp-Ser-NH2 Ac-Cys-Arg-Gly-Asp-Ser-Cys-NHa Ac-Cys-Arg-Gly-Asp-Cys-NH2 Ac-Cys-Arg-Gly-Asp-Pen-NH2 Ac-Cys-MeArg-Gly-Asp-Pen-NH2
gpIIb/IIIa binding (UM) 0.043

4.2 5.3
0.78 N.D.
0.36
0.027
antithrombotic agents. Both fibrinogen and fibronectin are extremely large proteins, and their three-dimensional structures are not known. The binding domain on fibronectin was localized to a tetrapeptide sequence Arg-Gly-Asp-Ser by the use of a proteolytic fragment of fibronectin and a series of synthetic peptides based on secondary structure predictions for the sequence of the fragment (Pierschbacher and Ruoslahti, 1984). Small peptides containing this Arg-Gly-Asp-Ser sequence were shown to block binding of platelets both to fibronectin and to fibrinogen, but at concentrations in the 100 mM range (Plow et al., 1985). In order to try to improve binding affinity, Samanen and coworkers (1991) prepared the series of cyclic analogs shown in table 2-10. Both inhibition of aggregation of activated platelets and binding affinity to the gpIIb/IIIa receptor reconstituted in liposomes were measured. The smallest peptide containing the binding sequence is Ac-Arg-Gly-AspSer-NH2 (31), which has about 100 JIM antiaggregatory activity and a binding affinity for the reconstituted receptor of 4 JIM, 100-fold less potent than fibrinogen itself. Enclosing this sequence in a disulfide loop (32) has only a modest effect on activity, and removing the Ser from the sequence (33) enhances activity and binding affinity further. Replacing the C-terminal cysteine with penicillamine (Pen, ft, /J-dimethyl-cysteine; compound 34), which introduces an additional conformational constraint to the side chain and disulfide, increases the activity still further. The addition of an TV-methyl group to the Arg residue (35), which adds yet another conformational constraint, now
gives a compound with submicromolar antiaggregatory activity and binding affinity, which is now comparable to that of fibrinogen itself. The end result is a suitably constrained pentapeptide with the binding properties and affinity of a very large protein.
Summary We have seen examples of peptides designed to mimic protein epitopes or binding sites, and we have seen examples of small peptides that have been modified for enhanced proteolytic stability or for conformational constraints. The exact nature of the design of biologically active peptides depends to a large extent on their intended use. However, several general principles should be kept in mind. Peptides are generally much more flexible and much less soluble than proteins. Smaller peptides do not tend to adopt any preferred solution structure, and longer peptides, which can exhibit strong tendencies to fold into a particular secondary structure, are still in equilibrium with partially folded structures. Peptides are less likely to fold in such a way as to shield hydrophobic regions from solvent exposure, contributing to both the lack of stable solution structures and to insolubility. Conformation, solubility, proteolytic resistance, and other properties are modified by altering the nature of the peptide backbone, the side chains, or both. Even small modification of one residue in a peptide may have a large effect on biological activity, conformation, solubility, or proteolytic susceptibility. Finally, because peptides are synthesized chemically rather than ribosomally, one is not necessarily limited to the use of the 20 naturally occurring amino acids in designing analogs. There are hundreds of synthetically available amino acids with a wide variety of structure, functional groups, charge, polarity, hydrophobicity, or chemical reactivity that can be incorporated to tailor a given peptide to a specific property or use.
Note 1. Irori Quantum Microchemistry, 9640 Towne Centre Drive, San Diego, CA 92121. (619) 546-1300. References Alderton, G., and Fevold, H. L. (1951). Lanthionine in subtilin. J. Am. Chem. Soc. 73:463-464. Allen, J. S., Coligan, J. E., Barin, F., McLane, M. F., Sodroski, J. G., Rosen, C. A., Haseltine, W. A., Lee, T. H., and Essex, M. (1985). Major glycoprotein antigens that induce antibodies in AIDS patients are encoded by HTLV-III. Science 228:1091-1093.
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Anastasi, A., Erspamer, V., and Bucci, M. (1971). Isolation and structure of bombesin and alytensin, two analoguous active peptides from the skin of the European amphibians Bombina and Alytes. Experentia 27:166-167. Baker, E. N., and Hubbard, R. E. (1984). Hydrogen bonding in globular proteins. Prog. Biophys. Mol. Biol. 44:97-179. Bankowski, K., Manning, M., Haldar, J., and Sawyer, W. H. (1978). Design of potent antagonists of the vasopressor response to arginine-vasopressin. J. Med. Chem. 21:850-853. Benedetti, E., Barone, V., Bavoso, A., Blasio, B. D., Lelj, F., Pavone, V., Pedone, C., Toniolo, C., Leplawy, M. T., Kaczmarek, K., and Redlinski, A. (1988). Structural versatility of peptides from Ca'a-dialkylated glycines. I. A conformational energy computation and X-ray diffraction study of homo-peptides from C^-diethylglycine. Biopolymers 27:357-371. Berk, S. C., Rohrer, S. P., Degrade, S. J., Birzin, E. T., Mosley, R. T., Hutchins, S. M., Pasternak, A., Schaeffer, J. M., Underwood, D. J., and Chapman, K. T. (1999). A combinatorial approach toward the discovery of non-peptide, subtype-selective somatostatin receptor ligands. J Combinatorial Chem. 1:388-391. Berridge, N. J., Newton, G. G. F., and Abraham, E. P. (1952). Purification and nature of the antibiotic nisin. Biochem. J. 52:529-535. Bray, A. M., Maeji, N. J., and Geysen, H. M. (1990). The simultaneous multiple production of solution phase peptides; assessment of the Geysen method of simultaneous peptide synthesis. Tet. Lett. 31:5811-5814. Brenner, S., and Lerner, R. A. (1992). Encoded combinatorial chemistry. Proc. Natl. Acad. Sci. USA 89:5381-5383. Burley, S. K., and Petsko, G. A. (1985). Aromatic-aromatic interaction: a mechanism of protein structure stabilization. Science 229:23-28. Burley, S. K., and Petsko, G. A. (1988). Weakly polar interactions in proteins. Adv. Protein Chem. 39:125-189. Burgess, K., Liaw, A. I., and Wang, N. (1994). Combinatorial technologies involving reiterative division/coupling/recombination: statistical considerations. J. Med. Chem. 37:2985-2987. Butlen, D., Guillon, G., Rajerison, R. M., Jard, S., Sawyer, W. H., and Manning, M. (1978). Structural requirements for activation of vasopressin-sensitive adenylate cyclase, hormone binding, and antidiuretic actions: effects of highly potent analogues and competitive inhibitors. Mol. Pharmacol. 14:1006-1017. Chorev, M., Shavitz, R., Goodman, M., Minick, S., and Guillemin, R. (1979). Partially-modified retro-inverso enkephalinamides. Science 204:12101212. Chou, P. Y., and Fasman, G. D. (1978). Prediction of the secondary structure of proteins from their amino acid sequence. Advances Enzymol. 47:45148. Chu, Y.-H., Kirby, D. P., and Karger, B. L. (1995). Free solution identification of candidate peptides from combinatorial libraries by affinity capillary electrophoresis mass-spectrometry. J. Am. Chem. Soc. 117:5419-5420. Clausen, K., Spatola, A. F., Lemieux, C., Schiller, P. W., and Lawesson, S.-O. (1984). Evidence of a peptide backbone contribution toward selective
83
receptor recognition for leucine enkephalin thioamide analogs. Biochem. Biophys. Res. Comm. 120:305-310. Cox, M. T., Gormley, J. J., Hayward, C. F., and Peter, N. F. (1980). Incorporation of trans-olzfimnc dipeptide isosteres into enkephalin and substance P analogs. J. Chem. Soc. Chem. Comm. 800-802. Creighton, T. E. (1993). Proteins: Structure and Molecular Properties, 2nd ed., New York, W. H. Freeman. Dayhoff, M. O., Eck, R. V., Chang, M. A., and Sochard, M. R. (1965). Atlas of Protein Sequence and Structure, Silver Spring, Md., National Biomedical Research Foundation. Dayhoff, M., Schwartz, R. M., and Orcutt, B. C. (1978). Atlas of Protein Sequence and Structure, Silver Spring, Md., National Biomedical Research Foundation. Debouck, C., Gorniak, J. G., Strickler, J. E., Meek, T. D., Metcalf, B. W., and Rosenberg, M. (1987). Human immunodeficiency virus protease expressed in Escherichia coli exhibits autoprocessing and specific maturation of the gag precursor. Proc. Natl. Acad. Sci. USA 84:8903-8906. DeGrado, W. F., Kezdy, F. J., and Kaiser, E. T. (1981). Design, synthesis and characterization of a cytotoxic peptide with mellitin-like activity. J. Am. Chem. Soc. 103:679-681. DelMar, E. G., Largman, C., Brodrick, J. W., and Goekas, M. C. (1979). A sensitive new substrate for chymotrypsin. Anal. Biochem. 99:316-320. Dutta, A. S., Gormley, J. J., Hayward, C. F., Morley, J. S., Shaw, J. S., Stacey, G. J., and Turnbull, M. T. (1977). Enkephalin analogues eliciting analgesia after intravenous injection. Life Sci. 21:549-562. Edwards, C. R. W., Kitau, M. J., Chard, T., and Besser, G. M. (1973). Vasopressin analogue DDAVP in diabetes insipidis: clinical and laboratory studies. Br. Med. J. 3:375-378. Eisenberg, D. (1984). Three-dimensional structure of membrane and surface proteins. Ann. Rev. Biochem. 53:595-623. Eisenberg, D., Weiss, R. M., and Terwilliger, T. C. (1982). The helical hydrophobic moment: a measure of the amphilhilicity of a helix. Nature 299:371-374. Eisenberg, D., Wilcox, W., and Eshita, S. (1987). Hydrophobic moments as tools for analysis of protein sequences and structures. In Proteins, Structure and Function, J. J. LTtalien, ed., New York, Plenum Press, pp. 425-436. Erickson, J., Neidhart, D. J., VanDrie, J., Kempf, D. J., Wang, X. C., Norbeck, D. W., Plattner, J. J., Rittenhouse, J. W., Turon, M., Wideburg, N., et al. (1990). Design, activity, and 2.8 A crystal structure of a C2 symmetric inhibitor complexed to HIV-1 protease. Science 249:527-533. Frederickson, R. C. A. (1977). Enkephalin pentapeptides: a review of current evidence for a physiological role in vertebrate neurotransmission. Life Sci. 21:23^2. Freidinger, R. M. (1981). Computer graphics and chemical synthesis in the study of conformation of biologically active peptides. In Peptides, Synthesis, Structure, Function, Proceedings of the Seventh American Peptide Symposium, D. H. Rich and E. Gross, eds., Rockford, 111., Pierce Chemical Co.
84
Synthetic Peptides
Freidinger, R. M., Veber, D. F., Perlow, D. S., Brooks, J. R., and Saperstein, R. (1980). Bioactive conformation of luteinizing hormone-releasing hormone: evidence from a conformationally constrained analog. Science 210:656-658. Frommel, C., and Preissner, R. (1990). Prediction of prolyl residues in cisconformation in protein structures on the basis of amino acid sequence. FEES Lett. 277:159-163. Fukushima, D., Kaiser, E. T., Kezdy, F. J., Kroon, D. J., Kupferberg, J. P., and Yokoyama, S. (1980). Rational design of synthetic models for lipoproteins. Ann. N.Y. Acad. Sci. 348:365-373. Furka, A., Sebestyen, F., Asgedom, M., and Dibo, G. (1991). General method for rapid synthesis of multicomponent peptide mixtures. Int. J. Peptide Protein Res. 37:487^93. Gamier, J., Osguthorpe, D. J., and Robson, B. 1978. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J. Biol. Chem. 120:97-120. Geysen, H. M., Meloen, R. H., and Barteling, S. J. (1984). Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci. USA. 81:3998-4002. Geysen, H. M., Rodda, S. J., and Mason, T. J. (1986). A priori delineation of a peptide which mimics a discontinuous antigenic determinant. Mol. Immunol. 23:709-715. Geysen, H. M., Rodda, S. J., Mason, T. J., Tribbick, G., and Schoofs, P. G. (1987). Strategies for epitope analysis using peptide synthesis. J. Immunol. Methods 102:259-274. Geysen, H. M., Rodda, S. J., and Mason, T. J. (1988). A synthetic strategy for epitope mapping. In Peptides, Chemistry and Biology, Proceedings of the Tenth American Peptide Symposium, G. R. Marshall, ed., Leiden, Escom. Grant, G. A. (1999). Synthetic peptides for the production of antibodies that recognize intact proteins. Current Protocols in Protein Science, 18.3.118.3.14, New York, John Wiley and Sons. Gray, T. M., and Matthews, B. W. (1984). Intrahelical hydrogen bonding of serine, threonine and cysteine residues within a-helices and its relevance to membrane-bound proteins. J.. Mol. Biol. 175:75-81. Greer, J. (1985). Model structure for the inflammatory protein C5a. Science 228:1055-1060. Habermann, E. (1972). Bee and wasp venoms. Science 177:314-322. Hagler, A. T., Osguthorpe, D. J., Dauber-Osguthorpe, P., and Hempel, J. C. (1985). Dynamics and conformational energetics of a peptide hormone: vasopressin. Science 227:1309-1315. Hambrook, J. M., Morgan, B. A., Ranee, M. J., and Smith, C. F. C. (1976). Mode of deactivation of the enkephalins by rat and human plasma and rat brain homogenates. Nature 262:782-783. Hancock, W. S., Prescott, D. T., Marshall, G. R., and Vagelos, P. R. 1972. Acyl carrier protein. XVIII. Chemical synthesis and characterization of a protein with acyl carrier protein activity. J. Biol. Chem. 247:6224-6233. Hann, M. M., Sammes, P. G., Kennewell, P. D., and Taylor, J. B. (1982). On the double bond isostere of the peptide bond: preparation of an enkephalin analogue. J. Chem. Soc. Perkin 1:307-314.
85
Harrison, R. K., and Stein, R. L. (1990). Substrate specificities of the peptidyl prolyl cw-trans isomerase activities of cyclophilin and FK-506 binding protein: evidence for the existence of a family of distinct enzymes. Biochemistry 29:3813-3816. Hase, S., Sakakibara, S., Wahrenburg, M., Kirchberger, M., Schwartz, I. L., and Walter, R. (1972). 1,6-Aminosuberic acid analogs of lysine- and arginine- vasopressin and -vasotocin. Synthesis and biological properties. J. Am. Chem. Soc. 94:3590-3600. Hechter, O., Terada, S., Spitsberg, V., Nakahara, T., Nakagawa, S., and Flouret, G. (1978). Neurohypophyseal hormone-responsive renal adenylate cyclase. III. Relationship between affinity and intrinsic activity in neurohypophyseal hormones and structural analogs. J. Biol. Chem. 253:3230-3237. Hirschmann, R., Nutt, R. F., Veber, D. F., Vitali, R. A., Varga, S. L., Jacob, T. A., Holly, F. W., and Denkewalter, R. G. (1969). Studies on the total synthesis of an enzyme. V. The preparation of enzymatically active material. J. Am. Chem. Soc. 91:507-508. Hopp, T. P., and Woods, K. R. (1981). Prediction of protein antigenic determinants from amino acid sequences. Proc. Natl. Acad. Sci. USA 78:38243828. Houghten, R. A. (1985). General method for the rapid solid phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA 82:51315135. Howard, J. B. (1981). Reactive site in human o^-macroglobulin: circumstantial evidence for a thiolester. Proc. Natl. Acad. Sci. USA 78:2235-2239. Hudson, D. (1999a). Matrix assisted synthetic transformations: a mosaic of diverse contributions. I. The pattern emerges. J. Comb. Chem. 1:333-360. Hudson, D. (1999b). Matrix assisted synthetic transformations: A mosaic of diverse contributions. II. The pattern is completed. J. Comb. Chem. 1:403^157. Huffman, W. F., Callahan, J. F., Eggleston, D. S., Newlander, K. A., Takata, D. T., Codd, E. E., Walker, R. F., Schiller, P. W., Lemieux, C., Wire, W. S., and Burks, T. F. (1988). Reverse turn mimics. In Peptides, Chemistry and Biology, Proceedings of the Tenth American Peptide Symposium, G. R. Marshall, ed., Leiden, Escom. Hunt, S. (1985). The non-protein amino acids. In Chemistry and Biochemistry of the Amino Acids, G. C. Barrett, ed., London, Chapman and Hall. Ito, N., Phillips, S. E. V., Stevens, C., Ogel, Z. B., McPherson, M. J., Keen, J. N., Yadav, K. D. S., and Knowles, P. F. (1991). Novel thioether bond revealed by a 1.7 A crystal structure of galactose oxidase. Nature 350:87-90. Javaherian, K., Langlois, A. J., McDanal, C., Ross, K. L., Eckler, L. I., Jellis, C. L., Profy, A. T., Rusche, J. R., Bolognesi, D. P., Putney, S. D., and Matthews, T. J. (1989). Principal neutralizing domain of the human immunodeficiency virus type 1 envelope protein. Proc. Natl. Acad. Sci. USA 86:6768-6772. Kahn, M., Lee, Y.-H., Wilke, S., Chem, B., Fujita, K., and Johnson, M. E. (1988). The design and synthesis of mimetics of peptide /3-turns. J. Mol. Recognition 1:75-79.
Synthetic Peptides
Kaiser, E. T., and Kezdy, F. J. (1984). Amphiphilic secondary structure: design of peptide hormones. Science 223:249-255. Kauzmann, W. (1959). Some factors in the interpretation of protein denaturation. In Advances in Protein Chemistry, C. B. Anfinsen, Jr., M. L. Anson, J. T. Edsall and F. M. Richards, eds., New York, Academic Press. Kawasaki, K., and Maeda, M. (1982). Amino acids and peptides II. Modification of glycylglycine bond in methionine enkephalin. Biochem. Biophys. Res. Comm. 106:113-116. Kellis, J. T., Jr., Nyberg, K., Sali, D., and Fersht, A. R. (1988). Contribution of hydrophobic interactions to protein stability. Nature 333:784-786. Kim, P. S., and Baldwin, R. L. (1982). Specific intermediates in the folding reactions of small proteins and the mechanism of protein folding. Ann. Rev. Biochem. 51:459^89. Knowles, J. R. 1991. Enzyme catalysis: not different, just better. Nature 350:121-124. Kosterlitz, H. W., Lord, J. A. H., Paterson, S. J., and Waterfield, A. A. (1980). Effects of changes in the structure of enkephalins and narcotic analgesic drugs on their interactions with ^- and 5-receptors. Br. J. Pharmacol. 68:333-342. Krystek, S. R. Jr., Metzler, W. J., and Novotny, J. (1995a). Hydrophobicity profiles for protein sequence analysis. In Current Protocols in Protein Science, 2.2.1-2.2.13, New York, John Wiley and Sons. Krystek, S. R. Jr., Metzler, W. J., and Novotny, J. (1995b). Protein secondary structure prediction. In Current Protocols in Protein Science, 2.3.1-2.3.20, New York, John Wiley and Sons. Kyte, J., and Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105-132. Lam, K. S., Salmon, S. E., Hershg, E. M., Hruby, V. J., Kazmierski, W. M., and Knapp, R. J. (1991). A new type of synthetic peptide library for identifying ligand-binding activity. Nature 354:82-84. Landry, S. J., and Gierasch, L. M. (1991). Recognition of nascent polypeptide for targetin and folding. TIBS 159-163. Landschutz, W. H., Johnson, P. F., and McKnight, S. L. (1988). The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 240:1759-1764. Lapatto, P., Blundell, T., Hemmings, A., Overington, J., Wilderspin, A., Wood, S., Merson, J. R., Whittle, P. J., Danley, D. E., Geoghegan, K. F., Hawrylik, S. J., Lee, S. E., Scheld, K. G., and Hobart, P. M. (1989). X-ray analysis of HIV-1 proteinase at 2.7 A resolution confirms structural homology among retroviral enzymes. Nature 342:299-302. Lebl, M., Patek, M., Kocis, P., Krchnak, V., Hruby, V. J., Salmon, S. E., and Lam, K. S. 1993. Multiple release of equimolar amounts of peptides from a polymeric carrier using orthogonal linkage-cleavage chemistry. Int. J. Peptide Protein Res. 41:201-203. Lipman, D. J., and Pearson, W. R. (1985). Rapid and sensitive protein similarity searches. Science 227:1435-1441. Malfroy, B., Swerts, J. P., Guyon, A., Roques, B. P., and Schwartz, J. C. (1978). High affinity enkephalin-degrading peptidase in brain is increased after morphine. Nature 276:523-526.
87
Marki, W., Brown, M., and Rivier, J. E. (1981). Bombesin analogs: effects on thermoregulation and glucose metabolism. Peptides 2, Suppl. 2:169-177. Marks, N., Grynbaum, A., and Neidle, A. (1977). On the degradation of enkephalins and endorphines by rat and mouse brain extracts. Biochem. Biophys. Res. Comm. 74:1552-1559. Marshall, G. R., and Bosshard, H. E. (1972). Angiotensin II. Biologically active conformation. Circ. Res., Suppl. II 30-31:11-143-150. Matayoshi, E. D., Wang, G. T., Krafft, G. A., and Erickson, J. (1990). Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer. Science 247:954-958. Matsushita, S., Robert-Guroff, M., Rusche, J., Koito, A., Hattori, T., Hoshino, H., Javaherian, K., Takatsuki, K., and Putney, S. (1988). Characterization of a human immunodeficiency virus neutralizing monoclonal antibody and mapping of the neutralizing epitope. J. Virol. 62:2107-2114. McDonald, T. J., Jornvall, H., Nilsson, G., Vagne, M., Ghatei, M., Bloom, S. R., and Mutt, V. (1979). Characterization of a gastrin releasing peptide from porcine non-antral gastric tissue. Biochem. Biophys. Res. Comm. 90:227. Meek, T. D., Dayton, B. D., Metcalf, B. W., Dreyer, G. B., Strickler, J. E., Gorniak, J. G., Rosenberg, M., Moore, M. L., Magaard, V. W., and Debouck, C. (1989). Human immunodeficiency virus 1 protease expressed in Escherichia coli behaves as a dimeric aspartic protease. Proc. Natl. Acad. Sci. USA 86:1841-1845. Meldal, M., Svendsen, I., Breddam, K., and Auzanneau, F.-I. (1994). Portionmixing peptide libraries of quenched fluorogenic substrates for complete subsite mapping of endoprotease specificity. Proc. Natl. Acad. Sci. USA. 91:3314-3318. Merrifield, B. (1963). Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149-2154. Michaels, G., and Garian, R. (1995). Computational methods for protein sequence analysis, in Current Protocols in Protein Science, 2.1.1-2.1.18, New York, John Wiley and Sons. Miller, M., Schneider, J., Sathyanarayana, B. K., Toth, M. V., Marshall, G. R., Clawson, L., Selk, L., Kent, S. B. H., and Wlodawer, A. (1989). Structure of complex of synthetic HIV-1 protease with a substratebased inhibitor at 2.3 A resolution. Science 246:1149-1152. Miller, R. J., Chang, K.-J., Cuatrecasas, P., and Wilkinson, S. (1977). The metabolic stability of enkephalins. Biochem. Biophys. Res. Comm. 74:1311-1317. Mints, L., Hogue Angeletti, R., and Nieves, E. (1997). Analysis of MAPS Peptides. In ABRF News, Vol. 8, J. Rush, ed., Bethesda, Md., Association of Biomolecular Resource Facilities, pp. 22-26. Monahan, M. W., Amoss, M. S., Anderson, H. A., and Vale, W. (1973). Synthetic analogs of the hypothalamic luteinizing hormone releasing factor with increased agonist or antagonist properties. Biochemistry 12:4616-4620. Moore, M. L., Huffman, W. F., Bryan, W. M., Silvestri, J., Chang, H.-L., Marshall, G. R., Stassen, F., Stefankiewicz, J., Sulat, L., Schmidt, D.,
88
Synthetic Peptides
Kinter, L., McDonald, J., and Ashton-Shue, D. (1985). Vasopressin antagonist analogs containing or-methyl amino acids at position 4. In Peptides: Structure and Function, Proceedings of the Ninth American Peptide Symposium, C. M. Deber, V. J. Hruby and K. D. Kopple, eds., Rockford, 111., Pierce Chemical Co. Moore, M. L., Bryan, W. M., Fakhoury, S. A., Magaard, V. W., Huffman, W. F., Dayton, B. D., Meek, T. D., Hyland, L., Dreyer, G. B., Metcalf, B. W., Strickler, J. E., Gorniak, J. G., and Debouck, C. (1989). Peptide substrates and inhibitors of the HIV-1 protease. Biochem. Biophys. Res. Comm. 159:420-425. Morgan, B. P., Scholtz, J. M., Ballinger, M. D., Sipkin, I. D., and Bartlett, P. A. (1991). Differential binding energy: a detailed evaluation of the influence of hydrogen bonding and hydrophobic groups on the inhibition of thermolysin by phosphorus-containing inhibitors. J. Am. Chem. Soc. 113:297-307. Morishima, H., Takita, T., Aoyagi, T., Takeuchi, T., and Umezawa, H. (1970). The structure of pepstatin. J. Antibiot. 23:263-265. Nagai, U., and Sato, K. (1985). Synthesis of a bicyclic dipeptide with the shape of a jS-turn central part. Tet. Lett. 26:647-650. Nair, S. A., Kim, M. H., Warren, S. D., Choi, S., Songyang, Z., Cantley, L. C., and Hangauer, D. G. (1995). Identification of efficient pentapeptide substrates for the tyrosine kinase pp60c-src. J. Med. Chem. 38:4276^4283. Needleman, S., and Wunsch, C. (1970). A general method applicable to the search for similarities in the amino acid sequence of two proteins. J. Mol. Biol. 48:443^153. Ni, Z.-J., Maclean, D., Holmes, C. P., Gallop, M. A. (1996). Encoded combinatorial chemistry: binary coding using chemically robust secondary amine tags. Methods Enzymol. 267:261-272. Nicolaou, K. C., Xiao, X.-Y., Parandoosh, Z., Senyei, A., and Nova, M. P. (1995). Radiofrequency encoded combinatorial chemistry. Angew. Chem. Int. Ed. Eng. 34:2289-2291. Nielsen, J., Brenner, S., and Janda, K. J. (1993). Synthetic methods for the implementation of encoded combinatorial chemistry J. Am. Chem. Soc. 115:9812-9813. Nutt, R. F., Brady, S. F., Darke, P. L., Ciccarone, T. M., Colton, C. D., Nutt, E. M., Rodkey, J. A., Bennett, C. D., Waxman, L. H., Sigal, I. S., Anderson, P. S., and Veber, D. F. (1988). Chemical synthesis and enzymatic activity of a 99-residue peptide with a sequence proposed for the human immunodeficiency virus protease. Proc. Natl. Acad. Sci. USA 85:7129-7133. Ohlmeyer, M. H. J., Swanson, R. N., Dillard, L. W., Reader, J. C., Asouline, G., Kobayashi, R., Wigler, M., and Still, W. C. (1993). Complex synthetic chemical libraries indexed with molecular tags. Proc. Natl. Acad. Sci. USA 90:10922-10926. O'Shea, E. K., Rutkowski, R., and Kim, P. S. (1989). Evidence that the leucine zipper is a coiled coil. Science 243:538-542. Ostresh, J. M., Winkle, J. H., Hamashin, V. T., and Houghten, R. A. (1994). Peptide libraries: determination of relative reaction rates of protected amino acids in competitive couplings. Biopolymers 34:1681-1689.
89
Pals, D. T., Thaisrivongs, S., Lawson, J. A., Kati, W. M., Turner, S. R., DeGraaf, G. L., Harris, D. W., and Johnson, G. A. (1986). An orally active inhibitor of renin. Hypertension 8:1105-1112. Pandey, R. C., Cook, Jr., J. C, and Rinehart, Jr., K. L. (1977). High resolution and field desorption mass spectrometry studies and revised structures of alamethicins I and II. J. Am. Chem. Soc. 99:8469-8483. Patek, M., and Lebl, M. (1999). Safety-catch and multiply cleavable linkers in solid-phase synthesis Biopolymers 47:353-363. Payne, J. W., Jakes, R., and Hartley, B. S. (1970). The primary structure of alamethicin. Biochem. J. 117:757-766. Pearl, L. H., and Taylor, W. R. (1987). A structural model for the retroviral proteases. Nature 329:351-354. Pearson, W. R., and Lipman, D. J. (1988). Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85:2444-2448. Pena, C., Stewart, J. M., and Goodfriend, T. C. (1974). A new class of angiotensin inhibitors: N-methylphenylalanine analogs. Life Sci. 14:1331. Pierschbacher, M. D., and Ruoslahti, E. (1984). Cell attachment of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 309:30-33. Pinilla, C., Appel, J. R., Blanc, P., and Houghten, R. A. (1992). Rapid identification of high affinity peptide ligands using positional scanning synthetic peptide combinatorial libraries. BioTechniques 13:901-905. Pinilla, C., Appel, J. R., Blondelle, S. E., Dooley, C. T., Eichler, J., Ostresh, J. M., and Houghten, R. A. (1994). Versatility of positional scanning synthetic combinatorial libraries for the identification of individual compounds. Drug Dev. Res. 33:133-145. Plow, E. F., Pierschbacher, M. D., Ruoslahti, E., Marguerie, G. A., and Ginsberg, M. H. (1985). The effect of Arg-Gly-Asp-containing peptides on fibrinogen and von Willebrand factor binding to platelets. Proc. Natl. Acad. Sci. USA 82:8057-8061. Polacek, I., Krejci, I., Nesvada, H., and Rudinger, J. (1970). Action of [1,6-dialaninej-oxytocin and [l,6-di-serine]-oxytocin on the rat uterus and mammary gland in vitro. Eur. J. Pharmacol. 9:239-248. Quillan, J. M., Jayawickreme, C. K., and Lerner, M. R. (1995). Combinatorial diffusion assay used to identify topically active melanocyte-stimulating hormone receptor antagonists. Proc. Natl. Acad. Sci. USA 9:2894-2898. Rinehart, Jr., K. L., Gloer, J. B., Cook, Jr., J. C., Miszak, S. A., and Scahill, T. A. (1981). Structures of the didemnins, antiviral and cytotoxic depsipeptides from a Caribbean tunicate. J. Am. Chem. Soc. 103:1857-1859. Rivier, J. E., and Brown, M. R. (1978). Bombesin, bombesin analogues, and related peptides: effects on thermoregulation. Biochemistry 17: 1766-1771. Rivier, J., Brown, M., and Vale, W. (1976). D-Trp8 somatostatin: an analog of somatostatin more potent than the native molecule. Biochem. Biophys. Res. Comm. 65:746-751. Rose, G. D., and Roy, S. (1980). Hydrophobic basis of packing in globular proteins. Proc. Natl. Acad. Sci. USA 77:4643^647. Roy, C., Earth, T., and Jard, S. (1975). Vasopressin-sensitive kidney adenylate cyclase. J. Biol. Chem. 250:3149-3156.
90
Synthetic Peptides
Ruegger, A., Kuhn, M., Lichti, H., Loosli, H.-R., Huguenin, R., Quiquerez, C., and Wartburg, A. V. (1976). Cyclosporin A, ein immunosuppressiv wirksamer Peptidmetabolit aus Trichoderma polysporem Rifai. Helv. Chim. Acta 59:1075-1092. Rusche, J. R., Javaherian, K., McDanal, C., Petro, J., Lynn, D. L., Grimaila, R., Langlois, A., Gallo, R. C., Arthur, L. O., Fischinger, P. J., Bolognesi, D. P., Putney, S. D., and Matthews, T. J. (1988). Antibodies that inhibit fusion of human immunodeficiency virus-infected cells bind a 24-amino acid sequence of the viral envelope, gp!20. Proc. Natl. Acad. Sci. USA 85:3198-3202. Rutter, W. J., and Santi, D. V. (1989). Preparation of peptide mixtures and selecting peptides with specific properties. PCT Int. Appl., 57 pp. Samanen, J., Narindray, D., Cash, T., Brandeis, E., Adams, J., W., Yellin, T., Eggleston, D., Debrosse, C., and Regoli, D. (1989). Potent angiotensin II antagonists with non-/5-branched amino acids in position 5. J. Med. Chem. 32:466-472. Samanen, J., Zuber, G., Bean, J., Eggleston, D., Romoff, T., Kopple, K., Saunders, M., and Regoli, D. (1990). 5,5-Dimethylthiazolidine-4carboxylic acid (Dtc) as a proline analog with restricted conformation. Int. J. Peptide Protein Res. 35:501-509. Samanen, J., Ali, F. E., Romoff, T., Calvo, R., Sorenson, E., Bennett, D., Berry, D., Kostler, P., Vasko, J., Powers, D., Stadel, J., and Nichols, A. (1991). SK&F 106760: Reinstatement of high receptor affinity an a peptide fragment (RGDS) of a large glycoprotein (fibrinogen) through conformational constraints. In Peptides 1990: Proceedings of the 21st European Peptide Symposium, E. Giralt and D. Andreu, eds., Leiden, Escom. Sarantakis, D., McKinley, W. A., and Grant, N. H. (1973). The synthesis and biological activity of Ala3'14-somatostatin. Biochem. Biophys. Res. Comm. 55:538-542. Sawyer, W. H., Acosta, M., Balaspiri, L., Judd, J., and Manning, M. (1974). Structural changes in the arginine vasopressin molecule that enhance antidiuretic activity and specificity. Endocrinology 94:1106-1115. Schroder, E., Liibke, K., Lehmann, M., and Beetz, I. (1971). Peptide syntheses. XLVII. Mellitin. 3. Hemolytic acitivty and action on surface tension of aqueous solutions on synthetic mellitins and their derivatives. Experientia 27:764-765. Singh, J., Allen, M. P., Ator, M. A., Gainor, J. A., Whipple, D. A., Solowiej, J. E., Treasurywala, A. M., Morgan, B. A., Gordon, T. D., and Upson, D. A. (1995). Validation of screening immobilized peptide libraries for discovery of protease substrates. J. Med. Chem. 38:217-219. Smith, J. A., and Pease, L. G. (1980). Reverse turns in peptides and proteins. In CRC Critical Reviews in Biochemistry, G. D. Fasman, ed., Boca Raton, Fla., CRC Press. Songyang, Z., Blechner, S., Hoagland, N., Hoekstra, M. F., Piwnica-Worms, H., and Cantley, L. H. (1994). Use of an oriented peptide library to determine the optimal substrates of protein kinases. Curr. Biol. 4:973-982. Sothrup-Jensen, L., Petersen, T. E., and Magnuson, S. (1980). A thiol-ester in a2-macroglobulin is cleaved during proteinase complex formation. FEBS Lett. 121:275-279.
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Spatola, A. F., Saneii, H., Edwards, J. V., Bettag, A. L., Anwer, M. K., Rowell, P., Browne, B., Lahti, R., and von Voightlander, P. (1986). Structure-activity relationships of enkephalins containing serially replaced thiomethylene amide bond surrogates. Life. Sci. 38:1243-1249. Tack, B. F., Harrison, R. A., Janatova, J., Thomas, M. L., and Prahl, J. W. (1980). Evidence for the presence of an internal thiolester bond in third component of human complement. Proc. Natl. Acad. Sci. USA 77:57645768. Tarn, J. P., and Spetzler, J. C. (1997). Multiple antigen peptide system. Methods Enzymol. 289:612-637. Tanford, C. (1973). The Hydrophobic Effect, New York, John Wiley & Sons. Tang, J., James, M. N. G., Hsu, I. N., Jenkins, J. A., and Blundell, T. L. (1978). Structural evidence for gene duplication in the evolution of the acid proteases. Nature 271:618-621. Terrett, N. K., Gardner, M., Gordon, D. W., Kobylecki, R. J., and Steele, J. (1995a). Combinatorial synthesisthe design of compound libraries and their application to drug discovery. Tetrahedron 51:8135-73. Terrett, N. K., Bojanic, D., Brown, D., Bungay, P. J., Gardner, M., Gordon, D. W., Mayers, C., and Steele, J. (1995b). The combinatorial synthesis of a 30,752-compound librarydiscovery of sar around the endothelin antagonist, FR-139,317. Bioorg. Med. Chem. Lett. 5:917-22. Thaisrivongs, S., Pals, D. T., Lawson, J. A., Turner, S. R., and Harris, D. W. (1987). a-Methylproline-containing renin inhibitory peptides: In vivo evaluation in an anesthetized, ganglion-blocked, hog renin infused rat model. J. Med. Chem. 30:536-541. Thanki, N., Thornton, J. M., and Goodfellow, J. M. (1990). Influence of secondary structure on the hydration of serine, threonine and tyrosine residues in proteins. Prot. Eng. 3:495-508. Turk, J., Needleman, P., and Marshall, G. R. (1975). Analogs of bradykinin with restricted conformational freedom. J. Med. Chem. 18:1139-1142. Turk, J., Needleman, P., and Marshall, G. R. (1976). Analogs of angiotensin II with restricted conformational freedom including a new antagonist. Mol. Pharmacol. 12:217-224. Van Regenmortel, M. H. V., Briand, J. P., Muller, S., and Plaue, S. (1988). Synthetic polypeptides as antigens. In Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 19, R. H. Burdon and P. H. van Knippenberg, eds., Amsterdam, Elsevier. Veronese, F. D., DeVico, A. L., Copeland, T. D., Oroszlan, S., Gallo, R. C., and Sarngadharan, M. G. (1985). Characterization of gp41 as the transmembrane protein coded by the HTLV-III/LAV envelope gene. Science 229:1402-1405. Walter, R., Rudinger, J., and Schwartz, I. L. (1967). Chemistry and structureactivity relations of the antidiuretic hormones. Am. J. Med. 42:653-677. Walter, R., Smith, C. W., Mehta, P. K., Boonjarern, S., Arruda, J. A. L., and Kurtzman, N. A. (1977). Conformational considerations of vasopressin as a guide to development of biological probes and therapeutic agents. In Disturbances in Body Fluid Osmolality, T. E. Andreoli, J. J. Grantham and F. C. Rector Jr., eds., Bethesda, Md., American Physiological Society.
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Weidner, J. R., and Dunn, B. M. (1991). Development of synthetic peptide substrates for the poliovirus 3C proteinase. Arch. Biochem. Biophys. 286:402-408. Wickner, W. T., and Lodish, H. F. (1985). Multiple mechanisms of protein insertion into and across membranes. Science 230:400-407. Wieland, T. (1968). Poisonous principles of mushrooms of the genus Amanita. Science 159:946-952. Wlodawer, A., Miller, M., Jaskolski, M., Sathyanarayana, B. K., Baldwin, E., Weber, I. T., Selk, L. M., Clawson, L., Schneider, J., and Kent, S. B. H. (1989). Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease. Science 245:616-621. Wlodawer, A., Miller, M., Swain, A. L., and Jaskolski, M. (1991). Structure of three inhibitor complexes of HIV-1 protease. In Methods in Protein Sequence Analysis, Jornvall, Hoog and Gustavsson, eds., Basel, Birkhauser Verlag. Wolfsberg, T. G., and Madden, T. L. (1995). Sequence similarity searching using BLAST. In Current Protocols in Protein Science, 2.5.1-2.5.29, New York, John Wiley and Sons. Wu, J., Ma, Q. N., and Lam, K. S. (1994). Identifying substrate motifs of protein kinases by a random library approach. Biochemistry 33:1482514833. Yajima, H., and Fujii, N. (1981). Studies on peptides. 103. Chemical synthesis of a crystalline protein with the full enzymic activity of ribonuclease A. J. Am. Chem. Soc. 103:5867-5871. Yasunaga, T., Sagata, N., and Ikawa, Y. (1986). Protease gene structure and env gene variability of the AIDS virus. FEBS Lett. 199:145-150. Zaoral, M., Kolc, J., and Sorm, F. (1967a). Amino acids and peptides. LXX. Synthesis of D-Arg8- and D-Lys8 vasopressin. Coll. Czech. Chem. Comm. 32:1242-1248. Zaoral, M., Kolc, J., and Sorm, F. (1967b). Amino acids and peptides. LXXI. Synthesis of l-Deamino-8-D-y-aminobutyrine-vasopressin, l-Deamino-8D-lysine-vasopressin and l-Deamino-8-D-arginine-vasopressin. Coll. Czech. Chem. Comm. 32:1250-1257. Zuiderweg, E. R. P., Henkin, J., Mollison, K. W., Carter, G. W., and Greer, J. (1988). Comparison of model and nuclear magnetic resonance structures for the human inflammatory protein C5a. Proteins: Struct. Funct. Genet. 3:139-145.
Principles and Practice of Solid-Phase Peptide Synthesis

Gregg B. Fields Janelle L Lauer-Fields Rong-qiang Liu George Barany
ptides play key structural and functional roles in biochemistry, armacology, and neurobiology, and are important probes for search in enzymology, immunology, and molecular biology. The mino acid building blocks can be among the 20 genetically encoded residues, or else unusual ones, and the sequences can be linear, clic, or branched. It follows that rapid, efficient, and reliable ethodology for the chemical synthesis of these molecules is of utmost terest. A number of synthetic peptides are significant commercial or armaceutical products, ranging from the sweet dipeptide L-Asp-Lhe-OMe (aspartame) to clinically used hormones such as oxytocin, renocorticotropic hormone, calcitonin, and gonadotropin releasing rmone (GnRH) super-agonists. Synthesis can lead to potent and lective new drugs by judicious substitutions that change functional oups and/or conformations of the parent peptide. These include troduction of TV- or C-alkyl substituents, unnatural or D-amino ids, side-chain modifications including sulfate or phosphate groups carbohydrate moieties, and constraints such as disulfide bridges tween half-cystines or side-chain lactams between Lys and Asp or lu. Commercially important products that evolved from such studies clude protease inhibitors, such as captopril and other angiotensin nverting enzyme (ACE) inhibitors, peptidomimetic HIV protease
93
94
Synthetic Peptides
inhibitors, and the somatostatin analog lanreotide. Most of the biologically or medicinally important peptides which are the targets for useful structure-function studies by chemical synthesis comprise under 50 amino acid residues, but occasionally a synthetic approach can lead to important conclusions about small proteins (full or domains) in the 100-200 residue size range. Methods for synthesizing peptides are divided conveniently into two categories: solution (classical) and solid-phase pep tide synthesis (SPPS). The classical methods have evolved since the beginning of the twentieth century, and they are described amply in several reviews and books (Wimsch, 1974; Finn and Hofmann, 1976; Bodanszky and Bodanszky, 1984; Goodman et al, 2001). The solid-phase alternative was conceived and elaborated by R. B. Merrifield beginning in 1959, and has also been covered comprehensively (Erickson and Merrifield, 1976; Birr, 1978; Barany and Merrifield, 1979; Stewart and Young, 1984; Merrifield, 1986; Barany et al., 1987, 1988; Kent, 1988; Atherton and Sheppard, 1989; Fields and Noble, 1990; Barany and Albericio, 1991; Fields et al., 1992; Gutte, 1995; Fields, 1997; Lloyd-Williams et al., 1997; Chan and White, 2000; Kates and Albericio, 2000). Solution synthesis retains value in large-scale manufacturing, and for specialized laboratory applications. However, the need to optimize reaction conditions, yields, and purification procedures for essentially every intermediate (each of which has unpredictable solubility and crystallization characteristics) renders classical methods time consuming and labor intensive. Consequently, most workers now requiring peptides for their research opt for the more accessible solid-phase approach. In this chapter, revised with respect to an edition published a decade ago, we discuss critically the scope and limitations of the best available procedures for solid-phase synthesis of peptides. Developments and trends in the field considered new and mentioned briefly in the previous edition have now faced the test of time, and text has been adjusted accordingly. Literature citations are weighted toward detailed reports with full experimental descriptions, with some bias toward those describing procedures with which we and our collaborators have laboratory experience. Overview of Solid-Phase Strategy The concept of SPPS (figure 3-1) is to retain chemistry proven in solution (protection scheme, reagents), but to add a covalent attachment step (anchoring) that links the nascent peptide chain to an insoluble polymeric support. Subsequently, the anchored peptide is extended by a series of addition (deprotectionj coupling) cycles, which are required to proceed with exquisitely high yields and fidelities. It is the essence of the solid-phase approach that reactions are driven to
Principles and Practice of Solid-Phase Peptide Synthesis 95
Figure 3-1. Stepwise solid-phase synthesis of linear peptides. An 7Va-derivatized amino acid is attached to an insoluble support (resin) via a linker moiety. The A^-protecting group (A) is then removed selectively (deprotection). The next amino acid, which is again ^"-protected, is coupled to the amino acid-linker support. These deprotection/coupling cycles are repeated until the desired sequence of amino acids is generated. Finally, the peptide-linker support is cleaved to obtain the peptide as a free acid or an amide, depending on the chemical nature of the linker. Ideally, the cleavage reagent also removes the amino acid side-chain-protecting groups (B), which were chosen to be stable to the repetitive 7Va-deprotection conditions. See text for further details, discussion, and variations.
completion by the use of excess soluble reagents, which can be removed by simple filtration and washing without manipulative losses. Because of the speed and simplicity of the repetitive steps, which are carried out in a single reaction vessel at ambient temperature, the major portion of the solid-phase procedure is readily amenable to
Synthetic Peptides
tomation. Once chain elaboration has been accomplished, it is cessary to release (cleave) the crude peptide from the support der conditions that are minimally destructive towards sensitive resies in the sequence. Finally, there must follow prudent purification d scrupulous characterization of the synthetic peptide product, in der to verify that the desired structure is indeed the one obtained. An appropriate polymeric support (resin) must be chosen that has equate mechanical stability, as well as desirable physicochemical operties that facilitate solid-phase synthesis (see below, Polymeric pport). In practice, such supports include those that exhibit signifint levels of swelling in useful reaction/wash solvents. Swollen resin ads are reacted and washed batchwise with agitation, and filtered her with suction or under positive nitrogen pressure. Alternatively, lid-phase synthesis may be carried out in a continuous-flow mode, pumping reagents and solvents through resins that are packed into lumns. The usual batchwise resins often lack the rigidity and ength necessary for column procedures. More appropriate supports, hich are usually, but not always, lower in terms of functional capay, are obtained when mobile polymer chains are chemically grafted to, or physically embedded within, an inert matrix. Regardless of the structure and nature of the polymeric support osen, it must contain appropriate functional groups onto which e first amino acid can be anchored. In early schemes which still ve considerable popularity, chloromethyl groups are introduced to a polystyrene resin by a direct Friedel-Crafts reaction, following hich an 7Va-pro tec ted amino acid as its triethylammonium or cesium lt is added to provide a polymer-bound benzyl ester. Subsequently, it as recognized that greater control and generality is possible by use of andles," which are defined as bifunctional spacers that on one end corporate features of a smoothly cleavable protecting group. The her end of the handle contains a functional group, often a carboxyl, at can be activated to allow coupling to functionalized supports, for ample ones containing aminomethyl groups. Particularly advantaous, though more involved to prepare, are "preformed" handles, hich serve to link the first amino acid to the resin in two discrete eps, and thereby provide maximal control over this essential step of e synthesis (see below, Attachment to Support). The next stage of solid-phase synthesis is the systematic elaboration the growing peptide chain. In the vast majority of solid-phase ntheses, suitably Na- and side-chain-protected amino acids are ded stepwise in the C * N direction. A particular merit of this ategy is that the best practical realizations have been shown experientally to proceed with only negligible levels of racemization. A emporary" protecting group is removed quantitatively at each step liberate the 7Va-amine of the peptide-resin, following which the next
97
incoming protected amino acid is introduced with its carboxyl group suitably activated (see below, Formation of Peptide Bond). It is frequently worthwhile to verify that the coupling has gone to completion by some monitoring technique (see below, Monitoring). Once the desired linear sequence has been assembled satisfactorily on the polymeric support, the anchoring linkage must be cleaved. Depending on the chemistry of the original handle and on the cleavage reagent selected, the product from this step can be a C-terminal peptide acid, amide, or other functionality. The cleavage can be conducted so as to retain "permanent" side-chain-protecting groups, and thus yield protected segments which, once purified, are suitable for further condensation. Alternatively, selected "permanent" groups can be retained on sensitive residues for later deblocking in solution. However, the most widely used approach involves final deprotection carried out essentially concurrent to cleavage; in this way, the released product is directly the free peptide. Finally, in what is formally a hybrid approach that has come to the fore quite recently, free peptide segmentsthemselves prepared by solid-phase synthesiscan be modified with suitable activating groups and then ligated selectively, in solution or even on the solid phase, to create larger structures. Protection Schemes The preceding section outlined the key steps of the solid-phase procedure, but dealt only tangentially with combinations of "temporary" and "permanent" protecting groups, and the corresponding methods for their removal. The choice and optimization of protection chemistry is perhaps the key factor in the success of any synthetic endeavor. Even when a residue has been incorporated safely into the growing resinbound polypeptide chain, it may still undergo irreversible structural modification or rearrangement during subsequent synthetic steps. The vulnerability to damage is particularly pronounced at the final deprotection/cleavage step, since these are usually the harshest conditions. At least two levels of protecting group stability are required, in so far as the "permanent" groups used to prevent branching or other problems on the side chains must withstand repeated applications of the conditions for quantitative removal of the "temporary" TV'-amino protecting group. However, structures of "permanent" groups must be such that conditions can be found to remove them with minimal levels of side reactions that might affect the integrity of the desired product. The necessary stability is often approached by kinetic "finetuning," which is a reliance on quantitative rate differences whenever the same chemical mechanism (usually acidolysis) serves to remove both classes of protecting groups. An often-limiting consequence of such schemes based on graduated lability is that they force adoption of
98
Synthetic Peptides
relatively severe final deprotection conditions. Alternatively, orthogonal protection schemes can be used. These involve two or more classes of groups that are removed by differing chemical mechanisms, and therefore can be removed in any order and in the presence of the other classes. Orthogonal schemes offer the possibility of substantially milder overall conditions, because selectivity can be attained on the basis of differences in chemistry rather than in reaction rates.
"Temporary" Protection of JV-Amino Groups

Boc Chemistry
The so-called standard Merrifield system is based on graduated acid lability (figure 3-2, in a modern improved version). The acidolyzable "temporary" TV-terf-butyloxycarbonyl (Boc) group is introduced onto amino acids with either di-terJ-butyl dicarbonate (Boc2O) or 2(?er?-butoxycarbonyloxyimino)-2-phenylacetonitrile (Boc-ON), in aqueous 1,4-dioxane containing NaOH or trie thy lamine (Et3N) (Bodanszky and Bodanszky, 1984). The Boc group is stable to alkali and nucleophiles, and removed rapidly by inorganic and organic acids (Barany and Merrifield, 1979). Boc removal is usually carried out with trifluoroacetic acid (TFA) (20-50%) in dichloromethane (DCM) for 20-30 min, and, for special situations, HC1 (4N) in 1,4-dioxane for 35min. Deprotection with neat (100%) TFA, which offers enhanced peptide-resin solvation compared to TFA-DCM mixtures, proceeds in as little as 4min (Kent and Parker, 1988; Wallace et al., 1989). Following acidolysis, a rapid diffusion-controlled neutralization step with a tertiary amine, usually 5-10% Et 3 N or ,/V,.Af-diisopropylethylamine (DIEA) in DCM for 3-5 min, is interpolated to release the free A^-amine. Alternatively, Boc-amino acids may be coupled without prior neutralization by using "in situ" neutralization, that is, coupling in the presence of DIEA or NMM (Suzuki et al., 1975; Schnolzer et al., 1992; Sueiras-Diaz and Horton, 1992; Alewood et al., 1997; Miranda and Alewood, 1999). "Permanent" side-chain-protecting groups are ether, ester, and urethane derivatives based on benzyl alcohol, suitably "fine-tuned" with electron-donating methoxy or methyl groups or electron-withdrawing halogens for the proper level of acid stability/ lability. Alternatively, ether and ester derivatives based on cyclopentyl or cyclohexyl alcohol are sometimes applied, as their use mitigates the levels of certain side reactions. These "permanent" groups are sufficiently stable to repeated cycles of Boc removal, yet cleaved cleanly in the presence of appropriate scavengers by use of liquid anhydrous hydrogen fluoride (HF) at 0C or trifluoromethanesulfonic acid (TFMSA) at 25 C (see below, Cleavage). The 4-methylbenzhydrylamine (PAM) or 4-(hydroxymethyi)phenylacetic acid (MBHA)
Figure 3-2. Merrifield protection scheme for solid-phase synthesis, based on graduated acidolysis. Temporary A^-amino protection is provided by the Boc group, removed at each step by the moderately strong acid TFA. Permanent Bzl and cHex type side-chain-protecting groups, and the PAM linkage, are then cleaved simultaneously by HF or other strong acids, with a free peptide acid being formed in high yield.
anchoring linkages are similarly "fine-tuned" to be cleaved at the same time (see below, Attachment to Support).
Fmoc Chemistry A mild orthogonal alternative is constructed using Carpino's baselabile "temporary" A^-9-fluorenylmethyloxycarbonyl (Fmoc) group (figure 3-3). The optimal reagent for preparation of Fmoc-amino acids is fluorenylmethyl succinimidyl carbonate (Fmoc-OSu), applied in a partially aqueous/organic mixture in the presence of base; the alternative procedure involving derivatization by Fmoc-chloride is
Figure 3-3. A mild two-dimensional orthogonal protection scheme for solid-phase synthesis. Temporary A^-amino protection is provided by the Fmoc group, removed by the indicated base-catalyzed /3-elimination mechanism. Permanent /Bu-based side-chain-protecting groups and the HMP/PAB ester linkage are both cleaved by treatment with TFA to yield the free peptide acid. A third dimension of orthogonality may be added with an acid-stable, photolabile anchoring linkage (details in text).
100
Synthetic Peptides
accompanied by unacceptable levels (2-20%) of Fmoc-dipeptide formation (Pacquet, 1982; Sigler et al., 1983; Lapatsanis et al., 1983; Tesser et al., 1983; Ten Kortenaar et al., 1986; de Lisle Milton et al., 1987; Fields et al., 1989). Removal of the Fmoc group is achieved usually with 20-55% piperidine in jV./V-dimethylformamide (DMF) or N-methylpyrrolidone (NMP) for 10-18 min (Atherton et al., 1978a,b; Chang et al., 1980a; Albericio et al., 1990a; Fields and Fields, 1991); piperidine in DCM is not recommended, as an amine salt precipitates after relatively brief standing. The base abstracts the acidic proton at the 9-position of the fluorene ring system; /6-elimination follows to give a highly reactive dibenzofulvene intermediate which is trapped by excess secondary amine to form a stable, harmless adduct (Carpino and Han, 1972). The Fmoc group may also be removed by 2% l,8-diazabicyclo[5.4.0]undec-7-cnc (DBU) in DMF; however, this reagent is recommended for continuous-flow syntheses only since the dibenzofulvene intermediate does not form an adduct with DBU and thus must be washed rapidly from the peptide-resin (Wade et al., 1991). For batch syntheses, a solution of DBUpiperidine-DMF (1:1:48) is effective, as the piperidine component scavenges the dibenzofulvene (C. G. Fields et al., 1993b; Kates et al., 1996). After Fmoc removal mediated by base, the liberated 7V"-amine of the peptide-resin is free and ready for immediate acylation without an intervening neutralization step (compare to previous paragraph on Boc chemistry). "Permanent" protection compatible with jV-Fmoc protection is provided primarily by ether, ester, and urethane derivatives based on /ert-butanol. These derivatives are cleaved at the same time as appropriate anchoring linkages, by use of TFA at 25 C. Scavengers must be added to the TFA to trap the reactive carbocations which form under the acidolytic cleavage conditions.
A problem that occurs during preparation of Fmoc-amino acids is the precipitation of either the Fmoc-OSu reagent or the base (NaHCO3 or Na2CO3) upon mixing of the organic (1,4-dioxane, aceLone, DMF, and acetonitrile have been proposed) and aqueous co-solvents. This problem is best overcome by use of aqueous dimethoxyethane with Na2CO3 (Fields et al., 1989; Nagase et al., 1994). A solution of Fmoc-OSu (3.0 mmol) in dimethoxyethane (lOmL) is added slowly to the amino acid (2.0mmol) dissolved in 10% aqueous Na2CO3 (lOmL); final yields of Fmoc-amino acids after workup are in the 75-95% range. Another successful procedure (Han et al., 1996b) involves addition of a solution of Fmoc-OSu (1.1 mmol) in acetonitrile (2.0mL) to a solution of amino acid (1.0 mmol) plus triethylaminc (2.5 mmol) in water (2.0 mL); yields are in the
8 0 & ra n ge.
101
The Fmoc group has been shown to be completely stable to treatment with TFA, HBr in acetic acid (HOAc), or HBr in nitromethane for 1-2 days (Carpino and Han, 1972). Somewhat less stability was found in dipolar aprotic solvents (Atherton et al., 1979). Fmoc-Gly was deprotected after 7 days in dimethylacetamide (DMA), DMF, and NMP to the extent of 1%, 5%, and 14% respectively. Although these low levels of decomposition are considered relatively insignificant, it is nevertheless prudent to purify (by distillation at reduced pressure) the aforementioned solvents just before use. With NMP, Fmoc group removal is attributed directly to the presence of methylamine as an impurity (Otteson et al., 1989). The addition of HOBt (0.01-0.1 M) greatly reduces the detrimental effect of methylamine (Albericio and Barany, 1987); Fmoc-Gly-HMP-rcsin was <0.05% deprotected after 12h in NMP containing 0.01 M HOBt (Fields et al., 1990). Fmoc-amino acids can be stored in purified or "synthesis-quality" NMP with little decomposition for 6-8 weeks, in the dark at 25 C (J. Kent et al., 1991).
"Permanent" Protecting Groups for Reactive Amino Acid Side Chains Once the means for yV a -amino protection has been selected, compatible protection for the side chains of trifunctional amino acids must be specified. These choices are made in the context of potential side reactions, which should be minimized. Problems may be anticipated either during the coupling steps or at the final deprotection/ cleavage. For certain residues (e.g., Cys, Asp, Glu, Lys), side-chain protection is absolutely essential, whereas for others, an informed decision should be made depending upon the length of the synthetic target and other considerations. Most solid-phase syntheses follow maximal rather than minimal protection strategies. Almost all of the useful A^-Boc and Na-Fmoc protected derivatives can be manufactured in bulk, and are found in the catalogues of the major suppliers of peptide synthesis chemicals. The most widely used "permanent" protecting groups for the trifunctional amino acids have been listed (table 3-1), together with information on how derivatives are prepared, conditions for their intentional deblocking, and conditions under which the indicated side-chain protection is either entirely stable, or prematurely cleaved by reagents used for peptide synthesis.
A useful resource to find amino acid derivatives is the Peptide Synthesis Database located on the World Wide Web at http://ChcmLibrary. BRI.NRC.CA/home.html; this site is searchable and contains links to commercial suppliers.
Table 3-1 Amino acid side-chain protection for solid-phase peptide synthesis3
Side-chain Protected amino protecting group acid derivatives Compatible with Boc chemistry Benzyl Asp/Glu(OBzl) Stability Removal3 Reagent(s) for introduction Reference(s)3
TFA
strong acid
Bzl-OH + H+
Ser/Thr/Tyr(Bzl) Hyp(Bzl) 2-Adamantyl
TTA base TFA
strong acid strong acid
Bzl-Br + base Bzl-Br
Barany & Merrifield, 1979* Bodanszky & Bodanszky, 1984V Tarn & Merrifield, 1987* Yajima et al., 1988* Kiso etal., 1989* Weber & Nitschmann, 1978 Okada & Iguchi, 1988*1
Asp(O-2-Ada)
TFA piperidine 1MHC1
strong acid
Ada-2-OH + DCC
Cyclohexyl
Asp(OcHex)
TFA
strong acid
cHex-OH + carbodiimide + DMAP or cHex-OH + H+ Fm-OTos + base Fm-OH + DCC/DMAP
Tarn & Merrifield, 1987* Tarn et al., 1988*1* Yajima etal., 1988* Penke & Toth, 1989* Kiso et al, 1989* Albericioefa/., 1990c**e Albericioera/., 1990c*'*'e
9-Fluorenylmethyl Cys(Fm) Asp/Glu(OFm) TFA HF TFA HF piperidined piperidine TBAF
2,6-Dichlorobenzyl
Tyr(2,6-Cl2Bzl)
TFA
strong acid
2,6-Cl2Bzl-Br + base
Erickson & Merrifield, 1973av Tarn & Merrifield, 1987* Yajima etal., 1988* Kiso etal, 1989*
2-Bromobenzyloxycarbonyl
Tyr(2-BrZ)
TFA
strong acid
2-BrZ-ONp + base
Yamashiro & Li, 1973*-* Tarn & Merrifield, 1987*
2-Chlorobenzyloxycarbonyl
Lys(2-ClZ)
TFA
strong acid
2-ClZ-OSu + base
Erickson & Merrifield, 1973b*!* Bodanszky & Bodanszky, 1984* Tarn & Merrifield, 1987* Kiso etal, 1989* Albericioefa/., 1990c*'*'e
9-Fluorenylmethyloxycarbonyl
Lys(Fmoc)
TFA Pd(0)
piperidine TBAF
Fmoc-N3 + MgO
(continued)

Side-chain protecting group 4-Toluenesulfonyl Protected amino acid derivatives His(Tos) Stability TFA Removal3 Reagent(s) for introduction Reference(s)3 Stewarte? a/., 1972* Barany & Merrifield, 1979* van der Eijk et al, 1980* Bodanszky & Bodanszky, 1984* Tarn & Merrifield, 1987*
strong acid Tos-Cl + base Ac2O/pyridine HOBt Tos-Cl + base
Arg(Tos) Mesitylene-2-sulfonyl Arg(Mts)
HBr/TFA TFA TFA strong acid
Mts-Cl + base
Yajima etal, 1978*'* Tarn & Merrifield, 1987* Yajima etal, 1988* Chillemi & Merrifield, 1969*'* Stewart et al, 1972* Tarn & Merrifield, 1987* Applied Biosystems, Inc., 1989a* Brown etal, 1982*'* Tarn & Merrifield, 1987* KisoetaL, 1989*
2,4-Dinitrophenyl
His(Dnp)
acids
thiophenol
l-fluoro-2,4dinitrobenzene + base
Benzyloxymethyl
His(Bom)
TFA
strong acid
ClCH2OBzl
Formyl (on AT)
Trp(CHO)
TFA HFC
TFMSA piperidine HP
HCO2H
Bodanszky & Bodanszky, 1984* Tarn & Merrifield, 1987* Yajima et al., 1988*
Cyclohexyloxycarbonyl
Trp(Hoc)
TFA DffiA piperidine
HF+pcresol HF+ 1,4 butanedithiol + p-cresol

MMA DMF-SO3 HP NHJ TMSBr + thioanisole
Hoc-Cl + tetran-butyl ammonium hydrogensulfate + NaOH H202
Nishiuchietfa/., 1996*'*
Sulfoxide (on thioether)
Met(0)
TFA base HF
Houghton & Li, 1979* Bodanszky & Bodanszky, 1984* Tarn & Merrifield, 1987* Fujiietal., 1987* Beck & Jung, 1994* Ferrer et al, 1999* Erickson & Merrifield, 1973a*'* Barany & Merrifield, 1979* Tarn & Memfield, 1987* Fujii etal., 1987* Matsueda & Walter, 1980* Albericioera/., 1989b* Rosen etal., 1990*
4-Methylbenzyl
Cys(Meb)
TFA
strong acid Tl(Tfa)3
Meb-Br + base
3-Nitro-2-pyridinesulfenyl
Cys(Npys)
TFA HFC
thiols HF HOBt@ piperidine
Npys-Cl
(continued)

Side-chain protecting group Compatible with Fmoc chemistry tert-Butyl Protected amino acid derivatives Asp/Glu(O?Bu) Stability base Pd(0) base Pd(0) base Pd(0) piperidine Removala TFA Reagent(s) for introduction C4H8/H+ Reference(s)a Change al, 1980b* Bodanszky & Bodanszky, 1984* Meienhofer, 1985* Loffet etal., 1989* Lajoie et al., 1990*'e Greene, 1991*
CH3
CH3-C-
CH3
Ser/Thr/Tyr(fBu)
TFA
C4H8/H+
Hyp(fBu) 4,4-Dimethyl-2,6dioxocyclohexylidene-3-methylbutylaminobenzyl ester
TFA
C4H8m+
Asp/Glu(Dmab)
hydrazine
DBU
Pd(0)
Dmab-OH + DCC
Chan etal, 1995*-*
TFA
terf-Butyloxycarbonyl
Lys(Boc)
base
TFA
Boc2O or Boc-ON
Bodanszky & Bodanszky, 1984* Meienhofer, 1985* Atherton & Sheppard, 1989*-* White etal., 1992* Rinikerâ/., 1993* Fields & Fields, 1993*
His(Boc) Trp(Boc)
piperidine TFA base TFA
Boc2Of Boc2O
4,4-Dimethyl-2,6-dioxocyclohex- 1 ylidene-ethyl
Ly s(Dde)
piperidine TFA
hydrazine
2-acetyldimedone +Fmoc-lys
Bycroft et a/., 1993*' * Bloomberg et al., 1993* C.G. Fields et a/., 1993a* Rohwedderef al., 1998* Chhabra etal., 1998*'*
4,4-Dimethyl-2,6-dioxocyclohex- 1 ylidene-3-methylbutyl
Lys(i vDde)
piperidine hydrazine DBU Pd(0) TFA
dimedone + isopentanoic acid + DCC/DMAP +Fmoc-Lys Mtt-Cl + Et3N +(CH3)2SiCl2
4-Methyltrityl
His(Mtt)
piperidine dilute TFA hydrazine Pd(0) piperidine hydrazine Pd(0) dilute TFA
Earlosetai, 1991d*'*
Mtt-Cl + Et3N + (CH3)3SiCl
Aletras etal., 1995*'*
4-Methoxy-2,3,6-trimethylbenzenesulfonyl
Arg(Mtr)
piperidine TFA8
2,3,5-trimethyl- Fupnoetal., 1981*'* anisolee Atherton & Sheppard, 1989* + C1SO3H
(continued)

Side-chain protecting group 2,2,5,7,8-Pentamethyl-chroman-6sulfonyl Protected amino acid derivatives Arg(Pmc) Stability Removala Reagent(s) for introduction 2,2,5,7,8pentamethylchroman + C1SO3H Reference(s)3 Ramage & Green, 1987*'*
piperidine TFA
2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl
Arg(Pbf)
piperidine
TFA
2,2,4,6,7pentamethyldihydrobenzofuran + C1SO3H
Carpino etal., 1993*'* Fields etal, 1993*
Triphenylmethyl
His(Trt)
piperidine 1MHC1 piperidine
TFA
Trt-Cl + (CH3)2SiCl2 Trt-OH + BF3-Et2O
Sieber & Riniker, 1987* Barlos et al, 1982* Photaki et al., 1970* Bodanszky & Bodanszky, 1984* Meienhofer, 1985* Greene, 1991* Sieber & Riniker, 1991*'*
Cys(Trt)
HOAc
TFA
Hg(II)
I2
Asn/Gln(Trt)
piperidine 1MHC1 piperidine
TFA
Trt-OH + Ac,O/H+ Trt-Cl+ Et3N
Ser/Thr
TFA + TIS
Barlos et al, 1987*'* Barlos et al., 1991b* Barlos etal., 1998*
terr-Butoxymethyl
His(Bum)
piperidine TFA
ClCH2O?Bu
Colombo el al., 1984*'
2,4,6-Trimethoxybenzyl
Asn/Gln(Tmob) Cys(Tmob)
piperidine
TFA
piperidine TFA
Tmob-NH2 + DCC/HOSu Tmob-OH + TFA
Weygand et al., 1968a* Hudson, 1988b* Munson et al., 1992"' *
Methoxytrityl
Cys(Mmt)
piperidine hydrazine Pd(0)
dilute TFA/TES
Mmt-Cl
Badosetal., 1996'*
(continued)

Side-chain protecting group Protected amino acid derivatives Stability Removal3 Pd(0) Reagent(s) for introduction Al-OH +(CH3)3SiCl or H2S04 Aloc-Cl Reference(s)3 Belshaw et al., 1990* Greene, 1991*'* Lyttle & Hudson, 1992*'* Lyttle & Hudson, 1992*'*
Compatible with both Boc and Fmoc chemistries Allyl Asp/Glu(OAl) acids base
Allyloxycarbonyl
Lys(Aloc)
acids base
Pd(0)
9-Xanthenyl
Asn/Gln(Xan)
base
strong acid TFA
Xan-OH + HOAc
Dormant al., 1972*'* Stewart & Young, 1984* Tarn & Merrifield, 1987* H&netal., 1997b*-*
Acetamidomethyl
Cys(Acm)
piperidine acids
Hg(II) I2 Tl(Tfa)3
Acm-OH/TFA
Trimethylacetamidomethyl
Cys(Tacm)
piperidine acids piperidine acids
Hg(II)
Tacm-OHATFA
I2
AgBF4 thiols phosphines fBu-SH rBu-SCN
Veber et al., 1972*'* Bodanszky & Bodanszky, 1984* Atherton et al., 1985a* Albericio et al, 1987a*|C Tarn & Merrifield, 1987* Albericio et al., 1987*'g Fujii etal, 1987* Brady etal., 1988* McCurdy, 1989* Andreu et al, 1994* Sakakibara, 1999* Yoshida era/., 1990* Kiso etal ,1990*'* Wiinsch & Spangenberg, 1971* Atherton et al, 1985a* Romani et al, 1987*
terf-Butylsulfenyl
Cys(SriBu)
The protecting group structure drawn in the left column does not include the functional group that is being protected through a point of attachment at the far right of the structure. The order corresponds, by category, to first mention in the text. Abbreviations are provided in the second column. Conditions or reagents listed under "Removal" are intended for quantitative deprotection (see "Cleavage"), unless marked by "@", in which case the removal is an unacceptable side reaction that indicates an incompatibility with the protecting group. "Strong acid" means HF, TFMSA, or equivalent reagents (see "Cleavage"). TFA cleavages are best carried out in the presence of appropriate scavengers (see "Cleavage"). References marked by " * " refer to preparation of the side-chain derivative; references marked by "#" refer to stability and lability of each derivative. b Boc-Thr(Bzl) is best prepared by the procedure of Chen et al., 1989. c Stable to "high" (90%) HF, but removed by "low" HF-scavengers; see "Cleavage". d Complete deprotection requires 4h treatment by 50% piperidine in DMF. e One-pot synthesis of the Fmoc-amino acid side-chain protected derivative. f Fmoc-His(Boc) is prepared by reacting (BocÔ with commercially available Fmoc-His(Fmoc). g Arg(Mtr) deprotection by TFA can be slow, especially in multiple Arg(Mtr)-containing peptides. See "Cleavage". h Tmob-amine is reacted with a carboxyl protected Asp or Glu to produce Asn(Tmob) or Gln(Tmob), respectively.
12
Synthetic Peptides
The side-chain carboxyls of Asp and Glu are protected as benzyl OBzl) esters for Boc chemistry and as tert-butyl (OtEu) esters for moc chemistry. A sometimes serious side reaction with protected sp residues involves an intramolecular elimination to form an asparmide, which can then partition in water to the desired a-peptide and e undesired by-product with the chain growing from the /3-carboxyl Bodanszky and Kwei, 1978; Barany and Merrifield, 1979; Tarn et al., 988). Aspartimide formation is sequence dependent, with Asp(OBzl)ly, -Ser, -Thr, -Asn, and -Gin sequences showing the greatest tenency to cyclize under basic conditions (Bodanszky et al., 1978; odanszky and Kwei, 1978; Nicolas et al., 1989); the same sequences e also quite susceptible in strong acid (Barany and Merrifield, 1979; ujino et al., 1981; Tarn et al., 1988). For models containing sp(OBzl)-Gly, the rate and extent of aspartimide formation was subantial both in base (100% after lOmin treatment with piperidineMF [1:4], 50% after 1-3 h treatment with Et 3 N or DIEA) and in rong acid (a typical value is 36% after 1 h treatment with HF at 5C). By comparison, sequences containing Asp(OrBu)-Gly are omewhat susceptible to base-catalyzed aspartimide formation (11% ter 4h treatment with piperidine-DMF [1:4]) (Nicolas et al., 1989), ut do not rearrange at all in acid (Kenner and Seely, 1972). Sequence ependence studies of Asp(O?Bu)-X pep tides revealed that piperidine ould induce aspartimide formation when X = Arg(Pmc), Asn(Trt), sp(OfBu), Cys(Acm), Gly, Ser, Thr, and Thr(/Bu) (Yang et al., 994; Lauer et al., 1995), and also showed that aspartimide formation an be conformation-dependent (Dolling et al., 1994). This side rection can be minimized by including O.!M HOBt in the piperidine olution (Lauer et al., 1995), or eliminated by using an amide backone protecting group (e.g., 2-hydroxy-4-methoxybenzyl) for the resiue in the X position of an Asp-X sequence (Quibell et al., 1994). To minimize the acid-catalyzed imide/or ft rearrangement side action, Boc-Asp may be protected with either the 2-adamantyl O-2-Ada) (Okada and Iguchi, 1988) or cyclohexyl (OcHex) (Tarn et ., 1988) groups. The base-labile 9-fruorenylmethyl (OFm) group fers orthogonal side-chain protection for Boc-Asp/Glu (Bolin et ., 1989; Albericio et al., 1990c; Al-Obeidi et al., 1990), while the alladium-sensitive allyl (OA1) group (Belshaw et al., 1990; Lyttle nd Hudson, 1992) offers orthogonal side-chain protection for both oc- and Fmoc-Asp/Glu. The ./V-[l-(4,4,-dimethyl-2,6-dioxocycloexylidene)-3-methylbutyl]aminobenzyl (Dmab) group can be used r quasi-orthogonal protection of Asp during Fmoc chemistry, as mab is stable to piperidine-DMF (1:4) and can be removed selecvely with 2% hydrazine in DMF (Chan et al., 1995). The side-chain hydroxyls of Ser, Thr, and Tyr are protected as Bzl hers for Boc and either tEu or Trt ethers for Fmoc SPPS. In strong
113
cid, the Bzl protecting group blocking the Tyr phenol can migrate o the 3-position of the ring (Erickson and Merrifield, 1973a). This ide reaction is decreased greatly when Tyr is protected by the 2,6ichlorobenzyl (2,6-Cl2Bzl) (Erickson and Merrifield, 1973a) or 2romobenzyloxycarbonyl (2-BrZ) (Yamashiro and Li, 1973) group; onsequently, the latter two derivatives are much preferred for Boc SPPS. No corresponding C-alkylation occurs in Fmoc chemistry. The e-amino group of Lys is best protected by the 2-chlorobenzylxycarbonyl (2-C1Z) or Fmoc group for Boc chemistry, and reciproally by the Boc group for Fmoc chemistry. The 2-C1Z group offers the esired acid stability for Boc chemistry, by comparison to the benzylxycarbonyl (Z) and other ring-chlorinated Z groups. Branching due o premature side-chain deprotection by TFA is avoided, but the 2C1Z group is still readily removable by strong acids (Erickson and Merrifield, 1973b). Orthogonal side-chain protection for both Bocnd Fmoc-Lys is provided by the palladium-sensitive allyloxycarbonyl Aloe) group (Lyttle and Hudson, 1992; Kates et al., 1993b). In addiion, l-(4,4-dimethyl-2,6-dioxocyclohex-l-ylidene)ethyl (Dde) sidehain protection of Lys during Fmoc chemistry allows for selective eprotection with 2% hydrazine in DMF (Bycroft et al., 1993). Lys(Dde) has been used successfully for the synthesis of branched ep tides and pep tide templates (Bycroft et al., 1993; C. G. Fields et l., 1993a; Grab et al., 1996; Xu et al., 1996). The isovaleryl derivative f Dde, that is, the ivDde group, may prove advantageous because of s enhanced stability to piperidine (Chhabra et al., 1998). A further electively removable side-chain protecting group for Lys is 4-methylrityl (Mtt), which is labile to 1% TFA-triisopropylsilane in DCM Aletras et al., 1995). The highly basic trifunctional guanidino side-chain group of Arg may be protected or unprotected (i.e., protonated). Appropriate enzenesulfonyl derivatives are the 4-toluenesulfonyl (Tos) or mesitylne-2-sulfonyl (Mts) groups in conjunction with Boc chemistry, and ither 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), 2,2,5,7,8-penamethylchroman-6-sulfonyl (Pmc), or 2,2,4,6,7-pentamethyldihydroenzofuran-5-sulfonyl (Pbf) with Fmoc chemistry. These groups most kely block the w-nitrogen of Arg, and their relative acid lability is Pbf > Pmc > Mtr Mts > Tos (Fujino et al., 1981; Green et al., 1988; Carpino et al., 1993). The Tos and Mts groups are removed y the same strong acids that cleave Bzl-type groups. The Mtr roup may require extended TFA-thioanisole treatment (2-8 h) for emoval, while Arg(Pmc) and Arg(Pbf) are deprotected readily by 0% TFA (<2h). A number of other Arg protecting groups have een proposed, particularly A^-mono or A/^-bis-urethane derivaves. However, based on current information, the aforementioned enzenesulfonyl derivatives seem to offer the best prospects of clean
Synthetic Peptides
orporation of Arg without the production of contaminating nithine (Orn) at a later stage (Rink et al., 1984). Due to the high pKa of the guanidino group (~12.5), it can be proted selectively by protonation with HC1 or HBr. Successful SPPS ng protonated Arg requires a proton source (i.e., HOBt) for all bsequent coupling steps. This protocol is recommended to suppress ermolecular acylation of the guanidino group, which would lead to n formation (Atherton et al., 1984; Atherton and Sheppard, 1989). A common side reaction of most Boc/Fmoc-Arg derivatives is lactam formation (Barany and Merrifield, 1979; Rzeszotarska and asiukiewicz, 1988). During carbodiimide activation, 5-lactam forman (intramolecular aminolysis) competes with peptide bond forman (intermolecular aminolysis). Acylations in the presence of HOBt e commonly used to inhibit S-lactam formation, ^"-protected Arg rivatives may also be coupled as preformed esters, from which actam side products have been separated from the desired ester or to use (Atherton et al., 1988b). However, preformed esters will dergo conversion to the <5-lactam relatively shortly after dissolving DMF (D. Hudson, unpublished results). Activated His derivatives are uniquely prone to racemization during pwise SPPS, due to an intramolecular abstraction of the proton on e optically active a-carbon by the imidazole 7r-nitrogen (Jones et al., 80). Racemization could be suppressed by either reducing the basiy of the imidazole ring, or by blocking the base directly (Riniker and eber, 1988). Consequently, His side-chain-protecting groups can be egorized depending on whether the r- or jr-imidazole nitrogen is ocked. The Tos group blocks the NT of Boc-His, and is removed by ong acids. However, the Tos group is also lost prematurely during PS steps involving HOBt; this allows acylation or acetylation (durg capping) of the imidazole group, followed by chain termination e to N'm -> 7Va-amino transfer of the acyl or acetyl group (Ishiguro d Eguchi, 1989; Kusunoki et al., 1990). Therefore, HOBt should ver be used during couplings of amino acids once a His(Tos) idue has been incorporated into the peptide-resin. An HF-stable, hogonally removable, ^-protecting group for Boc strategies is the 4-dinitrophenyl (Dnp) function. Final Dnp deblocking is best carried t at the peptide-resin level prior to the HF cleavage step, by use of ophenol in DMF (see box below). The r-nitrogen of Fmoc-His can protected by the Boc and triphenylmethyl (Trt) groups. When His is T -protected by the Boc group, the basicity of the imidazole ring is duced sufficiently so that acylation by the preformed symmetrical hydride (PSA) method proceeds with little racemization (Atherton d Sheppard, 1989). His(Boc) is reasonably stable to repetitive base atment (Atherton and Sheppard, 1989). His(Trt) is completely ble to piperidine, but removed with TFA (Sieber and Riniker,
115
Dnp removal from His(Dnp)-containing peptide-resins is achieved by use of 20 mmol thiophenol per mmol His(Dnp) residue. The peptide-resin is suspended in DMF (5 mL gram" of resin), thiophenol is added, and the reaction proceeds for 1 h at 25 C. After thorough washing of the Boc-peptide-resin with DMF, H2O, ethanol, and DCM, the 7Va-Boc group is removed and the peptide is cleaved with HF or TFMSA. If Dnp groups still remain, the peptide is dissolved in 6M guanidine -HC1, 50 mmol Tris acetate, pH 8.5 (10-20mg peptide per mL solution), then deprotected by adding 2-mercaptoethanol to 20% (v/v) and treating for 2h at 37 C. The peptide should then be purified immediately by gel filtration or HPLC (Applied Biosystems, Inc., 1989a).
1987). The Trt group reduces the basicity of the imidazole ring (the pKa decreases from 6.2 to 4.7), although racemization by the PSA method is not eliminated completely (Sieber and Riniker, 1987). Since Dnp and Trt 7VT-protection do not allow PSA coupling with ow racemization, it is recommended that the appropriate derivatives be coupled as preformed esters or in situ with carbodiimide in the presence of HOBt (Sieber and Riniker, 1987; Riniker and Sieber, 1988). Boc-His(Tos) is coupled efficiently using benzotriazolyl N-oxyrisdimethylaminophosphonium hexafluorophosphate (BOP, 3 equiv.) n the presence of DIEA (3 equiv.); these conditions minimize racemization and avoid premature side-chain deprotection by HOBt Forest and Fournier, 1990). Blocking of the 7r-nitrogen of the imidazole ring has been shown to be effective in reducing His racemization (Fletcher et al., 1979). The Nn of His is protected by the berizyloxymethyl (Bom) and tert-butoxymethyl (Bum) groups for Boc and Fmoc chemistry, respectively. Couplings using A^-protected Boc-His(Bom) or Fmoc-His(Bum) PSAs result in racemization-free incorporation of His (Brown et al., 1982; Colombo et al., 1984). HF deprotection of His(Bom) and TFA deprotection of His(Bum) liberates formaldehyde, which can modify usceptible side-chains (see below, Cleavage). The carboxamide side chains of Asn and Gin are often left unproected in SPPS, but this approach leaves open the danger of dehydraion to form nitriles upon activation with in situ reagents. On the other hand, acylations by activated esters result in minimal side-chain dehydration (Barany and Merrifield, 1979; Mojsov et al., 1980; Gausepohl et al., 1989b) (see below, Formation of Peptide Bond). Nitrile formaion is also inhibited during in situ carbodiimide acylations when HOBt is added (Mojsov et al., 1980; Gausepohl et al., 1989b) (see below, Formation of Peptide Bond). However, the presence of HOBt does not effectively inhibit N"-protected Asn dehydration during BOP n situ acylations (Gausepohl et al., 1989b).
16
Synthetic Peptides
Boc- and Fmoc-His are prepared by the general procedures described earlier for Boc- and Fmoc-amino acids. Crude Boc-His is dissolved in methanol (MeOH)-O.l M pyridinium acetate buffer (pH 3.8) (1:1) and purified by ion-exchange chromatography over Dowex 50 (H+ form) using a pH gradient from 3.8 to 5.8 (Kawasaki et al., 1989). Crude Fmoc-His s purified by washing with H2O and hot MeOH (Kawasaki et al., 1989). A^-protected Boc- and Fmoc-His derivatives are prepared by straightforward reactions of appropriate side-chain derivatizing reagents with either TV^-protected or free His (see references in table 3-1). TV^-protected derivatives are prepared by protecting His-OMe at the A^-amino position (by the Z or Boc group) and TV1 (by the Boc group), derivatizing N* with the appropriate chloromethyl ether (simultaneously removing the 7VT-Boc group), and saponifying the methyl ester with NaOH (Brown et al., 1982; Colombo et al., 1984). The Na-Z group is removed by hydrogenolysis and replaced by the Fmoc group (Colombo et al., 1984).
At the point where an A^-amino-protecting group is removed om Gin, the possibility exists for an acid-catalyzed intramolecular minolysis which displaces ammonia and leads to pyroglutamate foration (Barany and Merrifield, 1973, 1979; DiMarchi et al., 1982; rlowska et al., 1987). Cyclization occurs primarily during couplings, s ^"-protected amino acids and HOBt promote this side reaction DiMarchi et al., 1982). Consequently, it is recommended that the coming residue that is to be incorporated onto Gin be activated as nonacidic species, for example, PSA or a preformed ester (see below, ormation of Peptide Bond). Although conditions are available for the safe incorporation of sn and Gin with free side chains during SPPS, there are compelling asons for their protection. Side-chain-protecting groups such as #-xanthen-9-yl (Xan), 2,4,6-trimethoxybenzyl (Tmob), and Trt miniize the occurrence of dehydration (Mojsov et al., 1980; Hudson, 990c; Gausepohl et al., 1989b; Sieber and Riniker, 1990; Han et al., 996a) and pyroglutamate formation (Barany and Merrifield, 1979), nd may also inhibit hydrogen bonding that otherwise leads to secndary structures which substantially reduce coupling rates. nprotected Fmoc-Asn and -Gin have poor solubility in DCM and MF; solubility is improved considerably by Tmob, Trt, or Xan sidehain protection. The Xan group does not entirely survive the TFA eprotection conditions in Boc chemistry (Dorman et al., 1972; tewart and Young, 1984). The highly sensitive side-chain of Trp is best protected by the Vw-formyl (CHO) or cyclohexyloxycarbonyl (Hoc) group for Boc hemistry and the Nm-Eoc group for Fmoc chemistry. Trp(CHO) is eprotected at the peptide-resin level by treatment with piperidineMF (9:91), 0C, 2 h, prior to HF cleavage (Fields et al., 1993);
11 7
In peptides where several Asn, Gin, His, and Cys residues are close in sequence, it may be worthwhile to limit the global use of Trt side-chain protection. Interspersing side-chain unprotected Asn and/or Gin residues in such congested sequences should limit difficult couplings due to steric hindrance. In addition, Asn or Gin adjacent to Trp should be left unprotected, since the Tmob, Trt, and Xan side-chain-protecting groups can modify Trp during TFA deprotection/cleavage (Southard, 1971; see also Cleavage, below, for additional references).
he formyl group is also removed by 20-25% HF in the presence of imethyl sulfide and 4-thiocresol (see below, Cleavage). The Hoc roup is removed by HF and does not require the presence of thiols Nishiuchi et al., 1996). Unprotected Trp may be incorporated by Boc hemistry when 2.5% anisole plus either 2% dimethyl phosphite or ndole are added to the TFA deprotection solution (Stewart and Young, 1984; Hudson et al., 1986). During the TFA cleavage proceure in Fmoc chemistry, the Boc group protecting the side chain of rp is removed partially, with the indole tied up as an vV-carbamate nvolving a molecule of carbon dioxide. Complete deprotection occurs n aqueous solution (Franzen et al., 1984; White, 1992). The intermedite carboxy-indole is relatively resistant to alkylation (White, 1992; Riniker et al., 1993; Johnson et al., 1993) and sulfonation (White, 992; Choi and Aldrich, 1993; Fields and Fields, 1993). The side chain of Met generally survives cycles of Fmoc chemistry, ut protection during Boc chemistry by the reducible sulfoxide funcon is often advisable. Smooth deblocking of Met(O) occurs in 205% HF in the presence of dimethyl sulfide (see below, Cleavage), or y TV-methylmercaptoacetamide (MMA) (10 equiv.) in 10% aqueous HO Ac at 37 C for 12-36h (Houghten and Li, 1979), NH4I-dimethyl ulfide (30 equiv. each) in TFA at 0 C for 3 h (Fujii et al., 1987, Ferrer t al., 1999), trimethylsilyl bromide (TMSBr) and 1,2-ethanedithiol EDT) under anhydrous conditions (Beck et al., 1994), or DMF SO3-EDT (5 equiv. each) in pyridine-DMF (1:4) at 20C or 1 h (Futaki et al., 1990b). DMF SO3-EDT treatment of Met(O) an be carried out only while hydroxyl residues are side-chain proected, as free hydroxyls will be sulfated (Futaki et al., 1990a). The most challenging residue to manage in peptide synthesis is Cys, which for some applications is required in the free sulfhydryl form and or others as a contributor to a disulfide linkage (Barany and Merrifield, 1979; Andreu et al., 1994; Annis et al., 1997). Another ssue is the selective formation of multiple disulfides by the concurrent se of two or more classes of Cys protecting groups (see below, PostTranslational Modifications and Unnatural Structures). Compatible with Boc chemistry are the 4-methylbenzyl (Meb), 4-methoxybenzyl
18
Synthetic Peptides
Mob), acetamidomethyl (Acm), trimethylacetamidomethyl (Tacm), erf-butylsulfenyl (S?Bu), 3-nitro-2-pyridinesulfenyl (Npys), and 9luorenylmethyl (Fm) /3-thiol protecting groups; compatible with Fmoc chemistry are the Acm, 4-methoxytrityl (Mmt), Tacm, S/Bu, Trt, Tmob, and Xan groups. The Mmt, Trt, Tmob, and Xan groups re labile in TFA; due to the tendency of the resultant stable carboium ions to realkylate Cys (Photaki et al., 1970; Munson et al., 1992; Han et al., 1996b), effective scavengers are needed (see below, Cleavage). The Meb group is optimized for removal by strong acid Erickson and Merrifield, 1973a); Cys(Meb) residues may also be conerted directly to the oxidized (cystine) form by thallium (III) rifluoroacetate [Tl(Tfa)3], although some cysteic acid forms at the ame time. Cys(Npys) and Cys(Fm) are stable to acid, and cleaved espectively by thiols and base. The Acm and Tacm groups are acidnd base-stable, and removed by mercuric (II) acetate [Hg(OAc)2] in queous acidic media (Veber et al., 1972; Sakakibara, 1999), or TFA olutions of either silver tetrafluoroborate (Fujii et al., 1989) or silver rifluoromethanesulfonate (Tamamura et al., 1998) in the presence of nisole. Metal-mediated deprotections are followed by treatment with H2S or excess mercaptans to free the /J-thiol. In multiple Cys(Acm)ontaining peptides, mercuric (II) acetate may not be a completely ffective removal reagent (Kenner et al., 1979). Mercuric (II) acetate an modify Trp, but can be used safely with Trp-containing peptides when an acidic solvent, for example, 50% aqueous HOAc, is used Nishio et al., 1994). Alternatively, Cys(Acm) residues are converted irectly to disulfides by treatment with I2 or Tl(Tfa)3 in solution Kamber 1971; Fujii et al., 1987; Andreu et al., 1994) or on-resin Albericio et al., 199la; Munson and Barany, 1993; Andreu et al., 1994; Edwards et al., 1994; Annis et al., 1997). Finally, the acid-stable tEu group is removed by reduction with thiols or phosphines Romani et al., 1987). C-Terminal esterified (but not amidated) Cys residues are racemized y repeated piperidine deprotection treatments during Fmoc SPPS. ollowing 4 h exposure to piperidine-DMF (1:4), the extent of racemization found was 36% D-Cys from Cys(S/Bu), 12% D-Cys from Cys(Trt), and 9% D-Cys from Cys(Acm) (Atherton et al., 1991). Racemization of esterified Cys(Trt) was reduced from 12% with piperdine-DMF (1:4) to only 2.6% with 1% DBU-DMF after 4h treatment (Atherton et al., 1991; Wade et al., 1991). Additionally, the steric indrance of the 2-chlorotrityl linker has been shown to minimize acemization of C-terminal Cys residues (Fujiwara et al., 1994). More recently, a novel side-chain-anchoring strategy that combines S-Xan protection and xanthenyl (XAL) handle for anchoring amides as been applied to prevent C-terminal Cys racemization. Detachment f completed peptides anchored through the Cys side chain can be
rried out under mild conditions, either by acidolysis or by applicaon of oxidative reagents (Barany, 1999). The above-mentioned proaches also circumvent formation of 3-(l-piperidinyl)alanine byoducts, a sometimes serious side reaction with C-terminal cysteine ukszo et al., 1996) depending on the Cys protecting group (SfBu is rticularly bad; this explains in retrospect a previously mysterious nding by Eritja et al. [1987]) and anchoring linkage (HMP/PABpe is problematic; 2-ClTrt and XAL are relatively safe). Cys is also susceptible to racemization during Fmoc SPPS. When plying protocols for Cys incorporation which include phosphonium d aminium salts as coupling agents (BOP, HBTU, HATU, and yAOP), as well as preactivation in the presence of suitable additives HOBt and HOAt) and tertiary amine bases (DIEA and NMM), 5% racemization is observed (Han et al., 1997a). These high levels of cemization are generally reduced by avoiding preactivation, using a eaker base (such as collidine), and switching to the solvent mixture MF-DCM (1:1). Couplings with less than 1% racemization were rformed successfully using either BOP plus HOBt, HBTU plus OBt, or HATU plus HOAt, in all cases in the presence of collidine, thout preactivation, and in DMF-DCM. Alternatively, the Pfp ester a suitable Fmoc-Cys derivative can be used. Under the aforemenoned optimized conditions, Cys protecting groups Tmob, Trt, Xan, d Acm do not enhance the risk of racemization (Han et al., 1997a). TV-Terminal Cys residues are modified covalently by formaldehyde, berated during HF deprotection of His(Bom) residues (see below, eavage). Additional difficulties, often poorly understood, have isen with a range of protected Cys derivatives in a variety of applitions (Barany and Merrifield, 1979; Atherton et al., 1985b; Andreu al., 1994). As is clear from the preceding discussion, the Boc and Fmoc groups ve risen to the fore as the most widely used and commercially viable V^-amino-protecting groups for SPPS. A plethora of other 7Va-aminootecting groups, some illustrating remarkably creative organic emistry, have been proposed over the years (figure 3-4). Among ese, the 2-(4-biphenyl)propyl[2]oxycarbonyl (Bpoc) (Wang and errifield 1969; Kemp et al., 1988), 2-(3,5-dimethoxyphenyl)propyl[2]ycarbonyl (Ddz) (Birr et al., 1972; Voss and Birr, 1981), and 4ethoxybenzyloxycarbonyl (Moz) (Wang et al., 1987; Chen et al., 87) groups are removed in dilute TFA, the dithiasuccinoyl (Dts) arany and Merrifield, 1977; Barany and Albericio, 1985; Albericio d Barany, 1987; Zalipsky et al., 1987; Planas et al., 1999) and 3tro-2-pyridinesulfenyl (Npys) (Matsueda and Walter, 1980; Wang al., 1982; Ikeda et al., 1986; Hahn et al., 1990) groups are removed thiolysis, the 6-nitroveratryloxycarbonyl (Nvoc) group is removed photolysis (Patchornik et al., 1970; Fodor et al., 1991), and
3-4. Alternative JV"-amino protecting groups for SPPS. otected nitrogen which is part of the amino acid is shown dface.
121
methylsulfonyl)ethoxycarbonyl (Msc) (Tesser and Balvert-Geers, 5; Camarero et al., 1998; Canne et al., 1999) and the 2-[4-(methylonyl)phenylsulfonyl]ethoxycarbonyl (Mpc) (Schielen et al., 1991) ups are base-labile. Chemistries relying on these protecting groups implicit in the cited references, but otherwise are beyond the scope he present article.
ymeric Support
term "solid phase" often conjures misleading images among the nitiated. Supports that lead to successful results for macromolecule thesis are far from static, and due to the need for reasonable capaes, it is rare for solid-phase chemistry to take place exclusively on aces. The resin support is quite often a polystyrene suspension ymer cross-linked with 1% of 1,3-divinylbenzene; the level of funcnalization is typically 0.2-l.Ommol/g. Dry polystyrene beads have average diameter of about 50 um, but with the commonly used vents for peptide synthesis, namely DCM and DMF, they swell to 6.2-fold in volume (Sarin et al., 1980). Thus, the chemistry of d-phase synthesis takes place within a well-solvated gel containing bile and reagent-accessible chains (Sarin et al., 1980; Live and nt, 1982). Polymer supports have also been developed based on concept that the insoluble support and peptide backbone should of comparable polarities (Atherton and Sheppard, 1989). A resin of olymerized dimethylacrylamide, A",N'-bisacryloylethylenediamine, acryloylsarcosine methyl ester (typical loading 0.3mmol/g), comcially known as polyamide or Pepsyn, was synthesized to satisfy criteria (Arshady et al., 1981). Under the best solvation conditions both polystyrene and polyamide supports, reaction rates approach, generally do not reach, those attainable in solution. It has been wn for a polystyrene carrier that macroscopic dimensions of both dry and solvated beads change dramatically once an appreciable el of peptide has been built up (Sarin et al., 1980). Thus, for this cific case, reactions continued to occur efficiently throughout the rior of a peptide-resin that was four-fold the weight of the starting port. A fertile area of inquiry has been the testing of supports with croscopic physical properties and possibly other characteristics difng from 1% cross-linked polystyrene and polyamide gel beads. se include membranes (Bernatowicz et al., 1990), cotton and er appropriate carbohydrates (Frank and Doring, 1988; Lebl and hler, 1989; Eichler et al., 1989), controlled-pore silica glass (Buttner al., 1988), linear polystyrene chains covalently grafted onto dense -F particles (Tregear, 1972; Kent and Merrifield, 1978; Albericio et 1989a), polyethylene sheets (Berg et al., 1989), or "augmented
Synthetic Peptides
face polyethylene prepared by chemical transformation" SPECT) (Cook and Hudson, 1996, 1997; Adams et al., 1998a; dson, 1999). Supports developed specifically to withstand the k-pressures that arise during continuous-flow procedures have n low-density, highly permeable inorganic matrices with polyamide bedded within. These embedded matrices include polyamide-kieselhr (known commercially as Pepsyn K) (Atherton et al., 1981b) and yamide-Polyhipe (Small and Sherrington, 1989). Pepsyn K has a ical loading of O.lmmolg" 1 , while Polyhipe loadings range from to l.Smmolg" 1 . The aforementioned materials have been largely pplanted by polyethylene glycol-polystyrene graft supports which ell in a range of solvents and have excellent physical and mechanical perties for both batchwise and continuous-flow SPPS (Hellermann al., 1983; Zalipsky et al., 1985, 1994; Bayer and Rapp, 1986; Bayer al., 1990; Barany and Albericio 1991; Barany et al., 1997; Kates al., 1998; Gooding et al., 1999). These latter materials are marketed PEG-PS, TentaGel, or ArgoGel, and the loadings are usually -0.4 mmolg"1, but higher loadings can be achieved by branching ategies. Also suitable for both batchwise and continuous-flow PS are poly(Ar-[2-(4-hydroxyphenyl)ethyl]acrylamide) (core Q), yethylene glycol dimethylacrylamide (PEGA) resins, and crossked ethoxylate acrylate resin (CLEAR). Core Q is available at h loading capacities (Smmolg" 1 ) (Epton et al., 1987; Baker et al., 90). PEGA resins, which consist of poly(N,Af-dimethylacrylamidebisacrylamido polyethylene glycol-co-monoacrylamido polyethye glycol) (Meldal, 1992; Renil et al. 1998), are freely permeable to cromolecules. CLEAR is suitable for both peptide and organic id-phase synthesis, due to its excellent swelling behavior in polar d nonpolar solvents ranging from water to DCM (Kempe and rany, 1996).
achment to Support
most all syntheses by the solid-phase method are carried out in the -> N direction, and therefore generally start with the intended Cminal residue of the desired peptide being linked to the support her directly or via a suitable handle. Anchoring linkages have n designed so that eventual cleavage provides either a free acid amide at the C-terminus, although, in specialized cases, other useful groups can be obtained. The discussion that follows focuses on kers that are either commercially available, readily prepared, and/or special interest (table 3-2); more complete listings are available arany et al., 1987; Fields and Noble, 1990; Barany and Kempe, 97; Songster and Barany, 1997; Blackburn, 2000).
Table 3-2 Resin linkers and handles3

Linker/Handle/Resin
4-Chloromethyl resin
Cleavage conditions
strong acid
Resulting C-terminus
acid
Reference(s)
Gutte & Merrifield, 1971 Stewart & Young, 1984
4-Hydroxymethylphenylacetic acid (PAM)
strong acid
acid
Mitchell etal, 1978 Tarn et al, 1979
3-Nitro-4-(2-hydroxyethyl)benzoic acid (NPE)
piperidine DBU
acid
Eritja etal, 1991 AlbericioefaZ., 1991b
9-(Hydroxymethyl)-2-fluoreneacetic acid (HMFA)
piperidine
acid
Mutter & Bellof, 1984 Liu et al., 1990 Sakakibara, 1999
2-Hydroxypropyl-dithio-2 -isobutync acid (HPEI)
CM' or P(CH2CH2C02H)3
acld
Brugidou & Mery, 1994
4-Nitrobenzophenone oxime resin
HOPip AA" +N(nBu)4 AA-imj
acidb
DeGrado & Kaiser, 1982C Findeis & Kaiser, 1989 Scarr & Findeis, 1990
a'16
(continued)

Linker/Handle/Resin Cleavage conditions
hv (350 nm)
Resulting C-terminus Reference(s)

acid Wang, 1976
4-Alkoxybenzyl alcohol resin
TFA
acid
Wang, 1973C Luefa/., 1981
4-Hydroxymethylphenoxyacetic acid (HMPA/PAC)
TFA
acid
Sheppard & Williams, 1982
3-(4-Hydroxytnethylphenoxy)propionic acid (PAB)
TFA
acid
Albericio & Barany, 1985
3-Methoxy-4-hydroxymethylphenoxyacetic acid
dilute TFA
acid
Sheppard & Williams, 1982
4-(2',4'-Dimethoxyphenylhydroxymethyl) phenoxymethyl resin
dilute TFA
acid
Rink, 1987
2-Methoxy-4-alkoxybenzyl alcohol resin (SASRIN)
dilute TFA
acid
Merglerera/., 1988a
2-Chlorotrityl chloride resin
dilute TFA or HO Ac
acid
Bailo&etal., 1989 Barlosefa/., 1991a
4-(4-Hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB)
dilute TFA
acid
Florsheimer & Riniker, 1991
5-(4-Hydroxymethyl-3,5-dimethoxyphenoxy)valeric acid (HAL)
dilute TFA
acid
Albericio & Barany, 1991
Hydroxy-crotonyl-aminomethyl resin (HYCRAM)
Pd(0) NMMor dimedone
acid
Kunz & Dombo, 1988 GuibeetaL, 1989 Lloyd-Williams etal, 199 Ib
3-Nitro-4-hydroxymethylbenzoic acid (ONb)
hv (350 nm)
acid
Rich & Gurwara, 1975C Giralt etal., 1982 Barany & Albericio, 1985 Kneib-Cordonierefa/., 1990 Chao el al., 1993
4-[l-Amino-2-(trimethylsilyl)ethyl]-phenoxyacetic acid (SAL)
moderate acid
amide
(continued)
Linker/Handle/Resin
4-Methylbenzhydrylamine resin (MBHA)
Cleavage conditions
strong acid
Resulting C-terminus
amide
Reference(s)
Matsueda & Stewart, 1981 Gaehde & Matsueda, 1981
4-(2',4'-Dimethoxyphenylaminomethyl)phenoxymethyl resin
dilute TFA
amide
Rink, 1987
4-(4'-Methoxybenzhydryl)phenoxyacetic acid (Dod)
TFA
amide
Stiiber et al, 1989
3-(Amino-4-methoxybenzyl)-4,6-dimethoxyphenylpropionic acid
dilute TFA
amide
Breipohl et al, 1989
5-(4-Aminomethyl-3,5-dimethoxyphenoxy)valeric acid (PAL)
TFA
amide
Albericio & Barany, 1987b Albericioefaf., 1990a
5-(9-Aminoxanthen-3-oxy)valeric acid (XAL)
dilute TFA
amide
Sieber, 1987c,c Barany & Albericio,1991 Han era/., 1996b
3-Nitro-4-aminomethylbenzoic acid (Nonb)
hv (350 nm)
amide
Hammer et al., 1990
4-(2'-Aminoethyl)-2-methoxy-5-nitro-phenoxypropionic acid
hv (356 nm)
amide
Holmes & Jones, 1995
Structural diagrams are oriented so that the resin or point of attachment to support is on the far right, and the site for anchoring the C-terminal amino acid residue is on the far left. Benzyl ester linkages may also be cleaved by a range of nucleophiles to create acids, esters, or other derivatives. See text discussion under "Cleavage," and consult Barany and Merrifield (1979) and Barany et al. (1987) for further examples. b Cleavage by aminolysis results in 0.6-2% racemization (DeGrado and Kaiser, 1980). c Reference for historical reasons; preparation of linker/resin has been improved in later references.
128
Synthetic Peptides
Note that several handles (table 3-2) have a free or activated carboxyl group that is intended for attachment to the polymeric support. Such handles are most frequently coupled onto supports that have been functionalized with amino groups. Aminomethyl-polystyrene resin is optimally prepared essentially as described by Mitchell et al., (1978), except that methanesulfonic acid (0.75g per 1 g polystyrene) is preferred as the catalyst instead of the originally described TFMSA (S. B. H. Kent and K. M. Otteson, unpublished results). Amino groups are introduced onto a variety of polyamide supports by treatment with ethylenediamine to displace carboxylate derivatives (Atherton and Sheppard, 1989). All else being equal, there are significant advantages to those anchoring methods in which the key step is amide bond formation by reaction of an activated handle carboxyl with an amino support, since such reactions can be readily made to go to completion. This approach allows control of loading levels, and obviates difficulties that may arise due to extraneous or unreacted functionalized groups. As indicated earlier, the best control is achieved by coupling "preformed handles," which are protected amino acid derivatives that have been synthesized and purified in solution prior to the solid-phase anchoring step. Peptide Acids For Boc chemistry, the most common approach to peptide acids uses substituted benzyl esters which are cleaved in strong acid at the same time as other benzyl-type protecting groups are removed (figure 3-2). The classical procedures starting with chloromethyl-rcsin are still favored by many (Gutte and Merrifield, 1971; Gisin, 1973; Stewart and Young, 1984), although preformed handle approaches with 4(hydroxymethyl)phenylacetic acid (PAM) (Mitchell et al., 1978; Tarn et al., 1979; Clark-Lewis and Kent, 1989) are preferable for a number of applications. Other resins and linkers proposed for Boc chemistry are cleaved by orthogonal modes, allowing their use for the preparation of partially protected peptide segments (see below, Auxiliary Issues). The 4-(2-hydroxyethyl)-3-nitrobenzoic acid (NPE) (Eritja et al., 1991; Albericio et al., 1991b) and 9-(hydroxymethyl)-2-fluoreneacetic acid (HMFA) (Liu et al., 1990; Sakakibara, 1999) linkers are cleaved by bases (see below, Cleavage), The HMFA linker is also cleaved by free A^"-amino groups from the peptide-resin; the addition of HOBt during SPPS inhibits premature cleavage from this source (Liu et al., 1990). A 4-nitrobenzophenone oxime resin (DeGrado and Kaiser, 1980, 1982; Findeis and Kaiser, 1989; Scarr and Findeis, 1990) yields a peptide acid upon cleavage by either yV-hydroxypiperidine (HOPip) or amino acid tetra--butylammonium salts [AA~ + N(nBu) 4 ] (Findeis and Kaiser, 1989; Lansbury et al., 1989;
129
Sasaki and Kaiser, 1990). Note that in the latter mode of oxime resin cleavage, the penultimate residue of the desired peptide is the one initially attached to the support. Finally, the acid-stable 2-bromopropionyl (a-methylphenacyl ester) linker (Wang, 1976) is of interest because it can be cleaved by photolysis (A. = 350nm). For Fmoc chemistry, peptide acids have been generated traditionally using the 4-alkoxybenzyl alcohol resin/4-hydroxymethylphenoxy (HMP/PAB) linker (Wang, 1973; Lu et al., 1981; Sheppard and Williams, 1982; Colombo et al., 1983; Albericio and Barany, 1985; Bernatowicz et al., 1990), which is cleaved in 1-2 h at 25 C with 50100% TFA. The precise lability of the resultant 4-alkoxybenzyl esters depends on the spacer between the phenoxy group and the support. The HMP/PAB moiety can be established directly on the resin, or can be introduced as a handle; preformed handles are best coupled to aminofunctionalized resins as their 2,4,5-trichlorophenyl or 2,4-dichlorophenyl activated esters, sometimes in the presence of HOBt (Albericio and Barany, 1985;Bernatowiczetal., 1990; Albericio and Barany, 1991). A number of supports and linkers are available that can be cleaved in dilute acid; under optimal circumstances these can be used to prepare protected peptide segments retaining side-chain tert-buty\ protection. These include 3-methoxy-4-hydroxymethylphenoxyacetic acid (Sheppard and Williams, 1982), 4-(2',4'-dimethoxyphenyl-hydroxymethyl)phenoxy resin (Rink acid) (Rink, 1987; Rink and Ernst, 1991), 2-methoxy-4-alkoxybenzyl alcohol (SASRIN) (Mergler et al., 1988a), 2-chlorotrityl-chloride resin (Barlos et al., 1989b; Barlos et al., 199la), 4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB) (Florsheimer and Riniker, 1991), and 5-(4-hydroxymethyl-3,5dimethoxyphenoxy)valeric acid (HAL) (Albericio and Barany, 1991). Because of its acute acid lability, and in order to prevent premature loss of peptide chains, the Rink acid linker is used in conjunction with 7Va-protected amino acid preformed symmetrical anhydrides or esters in the presence of excess DIEA (3 equiv.) (Rink and Ernst, 1991). The HMP/PAB and SASRIN linkers are available as the corresponding chlorides or bromides (Colombo et al., 1983; Mergler et al., 1989a; Bernatowicz et al., 1990). Linkers for preparing peptide acids that are compatible with both Boc and Fmoc chemistries include hydroxy-crotonyl (HYCRAM) (Kunz and Dombo, 1988; Kunz, 1990; Lloyd-Williams et al., 1991), which is cleaved by Pd(0) catalyzed transfer of the allyl linker to a weak nucleophile, and 3-nitro-4-hydroxymethylbenzoic acid (ONb) (Rich and Gurwara, 1975; Giralt et al., 1982; Barany and Albericio, 1985; Kneib-Cordonier et al., 1990), which is cleaved photolytically at l = 350nm. All of the anchoring linkages that ultimately provide peptide acids are esters; rates and yields of reactions for ester bond formation
130
Synthetic Peptides
(table 3-3) are less than those for corresponding methods for amide bond formation (see below, Formation of Peptide Bond). Consequently, compromises are needed in order to achieve reasonable loading reaction times and substitution levels, while ensuring that the extent of racemization remains acceptably low. As a point of departure, esterification of TV-protected amino acid PSA catalyzed by 1 equiv. of 4-dimethylaminopyridine (DMAP) in DMA results in significant (1.5-20%) racemization (Atherton et al., 1981a). In general, racemization levels can be reduced to acceptable levels (0.21.2%) when catalytic (0.06 equiv.) amounts of DMAP are used and loadings are performed with carbodiimides in situ (Mergler et al., 1988a), sometimes in the presence of TV-methylmorpholine (NMM) (0.9 equiv.) (D. Hudson, personal communication). Alternatively, in situ carbodiimide loading with HOBt (2 equiv.) and DMAP (1 equiv.) at low temperature (0-3 C) provides a good compromise of minimized racemization (0.1-0.3%) and reasonable loading times (16h) (van Nispen et al., 1985). No racemization was detected (< 0.05%) when in situ loadings were carried out at 25 C with only 7V,A^'-dicyclohexylcarbodiimide (DCC) (4 equiv.) and HOBt (3 equiv.), without DMAP (Grandas et al., 1989). Esterifications of Fmoc-amino acids mediated by Ar,./v"-dimethylformamide dineopentyl acetal (Albericio and Barany, 1984, 1985), 2,6-dichlorobenzoyl chloride (DCBC) (Sieber, 1987a), diethyl azodicarboxylate (DEAD) (Sieber, 1987a), or 2,4,6-mesitylene-sulfonyl-3-nitro-l,2,4-triazolide (MSNT) (Blankemeyer-Menge et al., 1990) have all been reported to suppress racemization, as is the case with preformed Fmoc-amino acid 2,5-diphenyl-2,3-dihydro3-oxo-4-hydroxythiophene dioxide (OTDO) esters (Kirstgen et al., 1987, 1988), Fmoc-amino acid chlorides (Akaji et al., 1990a), or Fmoc-amino acid jV-carboxyanhydrides (NCAs) (Fuller et al., 1990). The well-established cesium salt method (Gisin, 1973) also allows loading of JV"-protected amino acids to chloromethyl linkers and resins with low levels of racemization (Colombo et al., 1983; Mergler et al., 1989b) while effectively preventing alkylation of susceptible residues (Cys, His, Met) (Gisin, 1973). Bromomethylated-linkers may be loaded directly by Boc-amino acids in the presence of KF (Tarn et al., 1979) or by Fmoc-amino acids in the presence of DIEA (Bernatowicz et al., 1990), in each case with little racemization. While premature removal of the Fmoc group during loading can result in dipeptide formation, the efficient ester bond formation methods described in this paragraph minimize this side reaction. However, care should be taken when preparing cesium salts of Fmoc-amino acids, as Cs2CO3 may promote partial removal of the Fmoc group. Of the ester bond formation methods discussed here, the most generally applicable are esterification of A^-protected amino acids in situ by carbodiimide (DCC or DIPCD1) in the presence of catalytic amounts of DMAP (0.06-0.1 equiv.), or by
Table 3-3 Formation of ester bonds to attach TV*-protected amino acids to linkersa
Reagents(s) Boc-AA-OH : DCHA Boc-AA-OH : KF DMF-dineopentyl acetal Fmoc-AA-OH : DMAP : HOBt : DCC Fmoc-AA-OH : DCBC : pyridine Linker8 Br-PAM* Br-PAM* HO-HMP'" HO-HMP" HO-HMP" Stoichiometriesb Conditions ' 2:2:1 DMF (4 h, SO C +14 h, 25 C) 2.2: 1.1 : 1 CH3CN (18-48 h) 1.7 : 1.7 : 1 DMF (72 h) 2:2:4:2:1 2: 2 : 3.3 : 1
DMF (18 h, 0C) DMF (1 5-20 h)
cd e
Racemization (%)e
<0.1 N.R. <0.05
0. 1 - O.3 0.1 -0.7 Arg(Mtr) < l.O His(Trt) 27.0 His(Bum) 2.2 Cys(Acm) 1.2 Cys(Trt) 4.O 0.3 Cys(Acm) 1.6 0.2- 1.0 lie 1.2 Cys(Acm) 4.O Cys(Trt) 18.3 His(Trt) 26.O <0.05
Loading (%)e
86 -100 67 - 75 47 55-66 His(Bum) 16
Reference Mitchell etal., 1978 Tame/ at., 1979 Albericio & Barany, 1984 Albericio & Barany, 1985 van Nispen et al., 1985 Sieber, 1987a
Fmoc-AA-OH : DEAD : Ph3P Fmoc-AA-OH : DCC : DMAP
HO-HMP" HO-SASRIN"
3:3:3:1
1.5 : 1.2 : O.O10.1
THF (16 h, OC) DMF-DCM (1:3) (2O h, O C)
53 -61 -8O
Sieber, 1987a Mergler e/a/., 1988a Merglere/o/., 1989b
Fmoc-AA-OH : DCC : HOBt
HO-HMP"
4:4:3:1
DMA (17 h)
Fmoc-AA-OH : MSNT : Melm Fmoc-AA-OH : DIEA Fmoc-AA-OH : DIEA Fmoc-AA-O' CV" Fmoc-AA-O' Cs* Fmoc-AA-O' Cs* : Nal Fmoc-AA-OTDO : DIEA Fmoc-AA-Cl Fmoc-NCA : NMM
a
HO-HMP" Br-HMP' Cl-Trt" C1-CH2-" Cl-HMP" Cl-SASRIN" HO-HMP" HO-HMP" Rink acid"
2 : 2 : 1.5 : 1 1.1 : 1 : 1 0.3-1.0: 2.5 : 1.6 1 :1 2 :1 1.5-3.O : 1 : 1 4O mM : 1 : 1 5 :1 3 : 0.02 : 1
DCM1 (O.5 h, twice) His(Bum) CHC13

DMF (2-3 h) DCE (O.5 h)
0-0.6 Cys(StBu) 2.1 Asp(OtBu) 1.4 <O.l <O.O5
6O- 94 Arg6 Asn31 Gin 29 Pro 54 72 - 1OO 61 - 99 61 -99 TrpSl 72-95 89-98 N.R. 81 - 97
Grandas et al., 1989a
Blankemeyer-Menge et al., 199O Bematowiczero/., 199O Barlosetal., 1991a Gisin, 1973 Colombo et al., 1983 Merglercra/., 1989b Kirstgen etal., 1987 Kirstgen & Steglich, 1989 Akajietal., 199Oa Fuller etal., 199O
DMF(16h, 50 C) DMA (15-24 h, 50 C) DMA (24 h) DCMf (1-2 h) He 10 h pyridine-DCM (2:3) (1 h) toluene' (0.5-1 h)
N.R. O.O1 - O.O7 O.I -0.7 Cys(Trt) 2.5 <0.2 Cys(Acm) 1.0 <O.5 Met 1.7 Ala 0.7 <0.1
-100 N.R.
The column entitled "Linker" distinguishes between ester bond formation first to provide a preformed handle (*) (often the preferred route, as discussed further in the text) and ester bond formation directly to the linker-resin (#). b Stoichiometries (equivalents) are stated in the same order as the reagents are listed, followed last by the linker or resin. c When publications state overnight reactions, this table indicates 18h. d Reactions are at room temperature (25 C) unless stated otherwise. e Values are representative for incorporation of most amino acids. Individual amino acids falling outside of the general range are also listed. f The solvent must be dry, or yields decrease dramatically.
132
Synthetic Peptides
A^jV-dimethylformamide dineopentyl acetal. For esterifications in the presence of DMAP, time and temperature should be carefully mediated, and HOBt (1-2 equiv.) may be included. Fmoc-His and Fmoc-Cys derivatives are particularly difficult to load efficiently while suppressing racemization. Low-racemization loadings have been documented using the cesium salts of FmocHis(Trt) (0.4% D-His) and Fmoc-Cys(Acm) (0.5% D-Cys) (Mergler et al., 1989b), and for Fmoc-His(Bum) (0.3% o-His) esterification by MSNT (Blankemeyer-Menge, 1990). Since the last-mentioned result is undoubtedly due to the fact that the Bum group blocks N* of His, it may be noted that a more efficient loading procedure involves Fmoc-His(Bum) (2 equiv.) esterified in situ by N,N'-diisopropylcarbodiimide (DIPCDI) (2 equiv.) and DMAP (0.16 equiv.) in DCM-DMF (1:3) for 1 h (Fields and Fields, 1990). The best reported results for loading Fmoc-Cys(Trt) and -Cys(S/Bu) are 2.1% D-Cys during Fmoc-Cys(S?Bu) esterification by MSNT (BlankemeyerMenge, 1990) and 2.0-2.5% D-Cys during Fmoc-Cys(Trt) cesium salt loading (Mergler et al., 1989b) or in situ Fmoc-Cys(Trt) loading with DCC and HOBt (Atherton et al., 1991). Loading of FmocCys(Acm), Fmoc-Cys(Trt), and Fmoc-His(Boc) to bromomethylated-linkers in the presence of DIEA has been reported to result in <0.1% D-isomers (Bernatowicz et al., 1990). Racemization may be avoided by anchoring Cys to the resin via its side chain. For this purpose, Fmoc-Cys-OrBu is condensed with 5-(9-hydroxyxanthen-2oxy)valeric acid (XAL) to provide the Fmoc-Cys(2-XAL4)-O?Bu preformed handle, which is subsequently coupled to an amino functionalized PEG-PS resin (Han et al., 1997b; Barany, 1999). Preformed handle coupling was performed using DIPCDI/HOBt (4 equiv. each) or BOP/HOBt/NMM (4, 4, and 8 equiv., respectively) for 2h. Peptide Amides Most anchoring linkages that ultimately provide C-terminal peptide amides in a useful and general manner are benzhydrylamide derivatives. The attachment step is a direct coupling of an AT"-protected amino acid via its carboxyl to an appropriate benzhydrylamineresin, with eventual cleavage at a different locus providing the desired carboxamide. The 4-methylbenzhydrylamine (MBHA) linkage has been "fine-tuned" with an electron-donating 4-methyl group (Matsueda and Stewart, 1981; Gaehde and Matsueda, 1981; Adams et al., 1998b) to cleave in strong acid with good yields, yet is completely stable to the conditions of Boc chemistry. The benzhydrylamide system has also been "fine-tuned" with electron-donating methoxy groups to create the TFA-sensitive 4-(2',4'-dimethoxyphenylaminomethyl)phenoxymethyl (Rink amide) (Rink, 1987),
1 33
4-(4'-methoxybenzhydryl)phenoxyacetic acid (Dod) (Stiiber et al., 1989), and 3-(amino-4-methoxybenzyl)-4,6-dimethoxyphenylpropionic acid (Breipohl amide) (Breipohl et al., 1989) linkers for use in Fmoc chemistry. Other structural themes are compatible with Fmoc chemistry and provide anchoring linkages which cleave in TFA to give peptides amides. These include the 5-(4-aminomethyl-3,5-dimethoxyphenoxy)valeric acid (PAL) (Albericio et al., 1990a) and 5-(9-aminoxanthen-3-oxy)valeric acid (XAL) (Han et al., 1996b) handles, both of which have highly desirable features by direct comparison to alternative structures. Also, the photolabile 3-nitro-4-aminomethylbenzoic acid (Nonb) handle is an option with both Boc and Fmoc chemistries (Hammer et al., 1990), as is the 4-(2'-aminoethyl)-2-methoxy-5-nitrophenoxypropionic acid variant (Holmes and Jones, 1995). Dod, Breipohl amide, XAL, PAL, and Nonb handles in their N-protected (usually Fmoc) forms are attached to the appropriate aminofunctionalized supports by in situ activation with DIPCDI or BOP/ DIEA in the presence of Dhbt-OH or HOBt (Stiiber et al., 1989; Breipohl et al., 1989; Albericio et al., 1990a; Hammer et al., 1990; Han et al., 1996b). Esterification of ^"-protected Asn and Gin can be sluggish (Barany and Merrifield, 1979; Wu et al., 1988; Fields and Fields, 1990). As an alternative, Boe-Glu(OH)-OBzl has been coupled (via an unprotected y-carboxyl side-chain) to benzhydrylamine resin, with HF cleavage yielding a peptide containing C-terminal Gin (Li et al., 1976). In parallel fashion, Fmoc-Asp(OH)-O;Bu or Fmoc-Glu(OH)-O?Bu have been coupled (via an unprotected ft- or y-carboxyl side chain) to PAL, Rink amide, or Breipohl amide, with TFA cleavage yielding peptides containing C-terminal Asn or Gin (Albericio et al., 1990b; Breipohl et al., 1990; Fields and Fields, 1990).
The substitution level of Fmoc-amino acid-resins is determined by quantitative spcctrophotometric monitoring following piperidine deblocking. Fmoc-amino acid-resin (4-8 mg) is shaken or stirred in piperidine-DMF (3 :7) (0.5 mL) for 30min, following which MeOH (6.5 mL) is added, and the resin is allowed to settle. The resultant fulvene-piperidine adduct has UV absorption maxima at A. = 267 nm (e = 17,500 M"1 cm'1), 290 nm (e = 5800 M'1 cnT1), and 301 nm (R 7800 M"1 cm"1). For reference, a piperidine-DMF-MeOH solution (0.3 : 0.7: 39) is prepared. Spectrophotometric analysis is typically carried out at 301 nm, with comparison to a free Fmoc-amino acid (e.g., Fmoc-Ala) of known concentration treated under identical conditions. The substitution level (mmol/g) = (A301 x 10ft umol/ mol x 0.007 L)/(7800 M"1 cm~' x 1 cm x mg of resin) (Meienhofer et al., 1979; D. Hudson, unpublished results).
1 34
Synthetic Peptides
A logical extension/generalization of side-chain anchoring strategies is the concept of backbone anchoring (backbone amide linker: BAL) (Jensen et al., 1998). As implemented in concert with Fmoc chemistry, the ultimate C-terminal residue (with its a-carboxyl either suitably protected or modified to a different end group, but its a-amino function free) is added to an aldehyde precursor of PAL via a reductive amination reaction (Jensen et al., 1998; Alsina et al., 1999b). A BAL variation compatible with Boc chemistry has also been described (Bourne et al., 1999a, b). The cited papers should be consulted to learn special considerations and the range of applications associated with BAL. Formation of Peptide Bond There are currently four major kinds of coupling techniques that serve well for the step wise introduction of N"-protected amino acids for solid-phase synthesis. In the solid-phase mode, coupling reagents are used in excess to ensure that reactions reach completion. The ensuing discussion will skirt some of the rather complicated mechanistic issues and focus on practical details. Recommendations for coupling methods are included in tables 3-4 and 3-5. In situ Reagents See figure 3-5. The classical example of an in situ coupling reagent is /V,Ar'-dicyclohexylcarbodiiinide (DCC) (Rich and Singh, 1979; Merrifield et al., 1988). The related AT,AT'-diisopropylcarbodiimide (DIPCDI) is more convenient to use under some circumstances, as the resultant urea co-product is more soluble in DCM. The generality
Table 3-4 General Boc chemistry SPPS

Description Deprotection Wash3 Activation and coupling Solvents/Chemicals Time
TFA DMF
Boc-amino acidb (5 equiv.) HBTU (4.75 equiv.) :DIEA (7.5 equiv.) in DMF or Boc-amino acid (5 equiv.) HATU (4.75 equiv.) :DIEA (7.5 equiv.) in DMF
2 x 1 min 1 min flow 2 min 10 min 2 min 5-10 min 1 min flow
Wash3
a
DMF
DCM washes may be included to prevent acid catalyzed side reactions of Gin; however coupling efficiency for certain amino acids may suffer (Schnolzer et al., 1992, Alewood et al., 1997). b Boc-Asn and -Gin are side-chain protected.
135
Table 3-5 General Fmoc chemistry SPPS

Description Wash Deprotection Wash Activation and coupling Solvents/Chemicals Time
3 x 1 min 20 min 3 x 1 min 5 min 45 min 5 min 45 min
NMP piperidine: NMP (l:4) a NMP Fmoc-amino acid (4 equiv.) in NMP DIPCDI (4 equiv.) : HOBt (4 equiv.) in DMF
or
Fmoc-amino acidb (4 equiv.) in NMP BOP (3 equiv.) : NMM (4.5 equiv.) : HOBt (3 equiv.) in DMF Fmoc-amino acidb (4 equiv.) in NMP HBTU (3.6 equiv.): DIEA (7.2 equiv.): HOBt (4 equiv.) in DMF or Fmoc-amino acidb (4 equiv.) in NMP HATU (3.6 equiv.): DIEA (7.2 equiv.): HOAt (4 equiv.) in DMF or Fmoc-amino acid preformed ester (4 equiv.) in NMP NMP
5 min 45 min 5 min 45 min 60 min 3 x 1 min
Wash
a b
0.1 M HOBt may be added to reduce aspartimide formation and resulting side reactions. Fmoc-Asn and -Gin are side-chain protected.
of carbodiimide-mediated couplings is extended significantly by the use of either 1-hydroxybenzotriazole (HOBt) or l-hydroxy-7-azabenzotriazole (HOAt) as an additive, either of which accelerates carbodiimide-mediated couplings, suppresses racemization, and inhibits dehydration of the carboxamide side chains of Asn and Gin to the corresponding nitriles (Konig and Geiger, 1970a, 1973; Mojsov et al., 1980; Carpino et al., 1994). Within the past decade, protocols involving benzotriazol-1 -yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), 2-(lH-benzotriazol-l-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazoll-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HATU), 2(1 H-benzotriazol-1 -yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), and 2-(2-oxo-1 (2H)-pyridyl)-1,1,3,3-bispentamethyleneuronium tetrafluoroborate (TOPPipU) have achieved popularity. (Interestingly, X-ray crystallographic analysis has shown that the solid-state structures of HBTU and HATU are not tetramethyluronium salts, but guanidinium TV-oxide isomers [Abdelmoty et al., 1994].) BOP, HBTU, HATU, TBTU, and TOPPipU require a tertiary amine such as NMM or DIEA for optimal efficiency (Dourtoglou et al., 1984; Fournier et al., 1988; Ambrosius et al., 1989; Gausepohl et al., 1989a; Seyer et al., 1990; Fields et al., 1991; Knorr et al., 1991; Reid and Simpson, 1992; Carpino et al., 1994). HOBt has been reported to accelerate further the rates of BOP- and HBTU-mediated couplings (Hudson, 1988; Fields et al., 1991). In situ activation by
Figure 3-5. In situ coupling reagents and additives for SPPS. Note that HBTU and HATU are shown in both their tetramethyluronium and guanidinium jV-oxide salt forms.
cess HBTU or TBTU can cap free amino groups (Gausepohl et al., 992; Story and Aldrich, 1994); it is not known whether HOBt can ppress this side reaction. Acylations using BOP result in the liberaon of the carcinogen hexamethylphosphoramide, which might limit use in large scale work. The modified BOP reagent benzotriazole-1-oxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyBOP) berates potentially less toxic by-products (Coste et al., 1990). otocols have been reported for the use of BOP to incorporate de-chain unprotected Thr and Tyr (Fournier et al., 1988, 1989). A veat to using PyAOP, PyBOP, and PyBroP is that some commercial urces are contaminated with small amounts of pyrrolidine, and this ntaminant can be incorporated to form pyrrolide by-products in ose cases when reactions of the activated carboxylates are particurly slow, for example, some cyclizations or couplings to relatively eak nucleophilic amines (Alsina et al., 1999a). The side reaction does ot occur when the reagents are recrystallized before their use in couing reactions.
ctive Esters
ee figure 3-6. A long-known but steadfast coupling method involves e use of active esters. The classical 2- and 4-nitrophenyl esters (ONo d ONp, respectively), used in DMF, allow relatively slow but dehyation-free introduction of Asn and Gin (Mojsov et al., 1980). ONo nd ONp esters of Boc- and Fmoc-amino acids are prepared from CC and either 2- or 4-nitrophenol, and the undesired nitrile conminant is easily separated (Bodanszky et al., 1973, 1980). 7V-hydroxyccinimide (OSu) esters of Fmoc-amino acids have been used ccessfully in SPPS (Fields et al., 1988), but are not recommended r general use due to formation of succinimidoxycarbonyl-yS-alanineV-hydroxysuccinimide ester (Gross and Bilk, 1968; Weygand et al., 968b). Workers later concentrated on pentafluorophenyl (OPfp), benzoazyl (OBt), 3-hydroxy-2,3-dihydro-4-oxo-benzotriazine (ODhbt), nd substituted 1-phenylpyrazolinone enol esters. Boc- and Fmocmino acid OPfp esters are prepared from DCC and pentafluorohenol (Kisfaludy et al., 1973; Penke et al., 1974; Kisfaludy and chon, 1983) or pentafluorophenyl trifluoroacetate (Green and erman, 1990). Although OPfp esters alone couple slowly, the addion of HOBt (1-2 equiv.) increases the reaction rate (Atherton et al., 988a; Hudson, 1990b). Fmoc-Asn-OPfp allows for efficient incororation of Asn with little side-chain dehydration (Gausepohl et al., 989b). OBt esters of Fmoc-amino acids are formed rapidly, with OBt plus DIPCDI, and are highly reactive (Harrison et al., 1989; elds et al., 1989), as are Boc-amino acid OBt esters (Geiser et al.,
138
Synthetic Peptides
Figure 3-6. Activated TV"-protected amino acids. W is either Boc or Fmoc, Y is the structure specified next to the abbreviation of the active ester derivative.
1988). A^-protected amino acid ODhbt esters suppress racemization and are highly reactive, in similar fashion to OBt esters (Konig and Geiger, 1970b). Preparation of ODhbt esters, with Dhbt-OH and DCC, is accompanied by the formation of the by-product 3-(2-azidobenzoyloxy)-4-oxo-3,4-dihydro-l,2,3-benzotriazine (Konig and Geiger, 1970c). Fmoc-amino acid ODhbt esters are far more stable than HOBt esters, and can therefore be isolated from the side product
before use (Atherton et al., 1988b). Fmoc-amino acid l-(4-nitrophenyl)-2-pyrazolin-5-one (Hpp), 3-phenyl-1 -(4-nitrophenyl)-2-pyrazolin5-one (Pnp), and 3-methyl-l-(4-nitrophenyl)-2-pyrazolin-5-one (Npp) esters also allow for rapid couplings (Hudson, 1990a; Johnson et al., 1992). Competition experiments have shown ester reactivity to be usually Pnp > Hpp ~ Npp > ODhbt > OPfp > OSu > ONp > ONo (Hudson, 1990a, b; Johnson et al., 1992), although Hpp esters were found to be superior to Pnp and Npp esters for "difficult" couplings (Johnson et al., 1992). Both Fmoc-Tyr and Fmoc-Ser have been incorporated successfully as preformed active esters without side-chain protection (Fields et al., 1989; Otvos et al., 1989a). Preformed Symmetrical Anhydrides See figure 3-6. Preformed symmetrical anhydrides (PSAs) are favored by some workers because of their high reactivity. They are generated in situ from the corresponding ^-protected amino acid (2 or 4 equiv.) plus DCC (1 or 2 equiv.) in DCM; following removal of the urea by filtration, the solvent is exchanged to DMF for optimal couplings. Detailed synthetic protocols based on PSAs have been described for Boc (Merrifield et al., 1982; Yamashiro, 1987; Geiser et al., 1988; Kent and Parker, 1988; Wallace et al., 1989) and Fmoc (Chang et al., 1980a; Heimer et al., 1981; Atherton and Sheppard, 1989) chemistries. The use of the PSA procedure to introduce Boc/Fmoc-Gly or -Ala occasionally results in inadvertent coupling of a diglycyl or dialanyl unit (Merrifield et al., 1974, 1985; Benoiton and Chen, 1987). Also, side-chain unprotected Asn and Gin, all Arg derivatives, and 7VT-protected His should not be used as PSAs due to potential side reactions discussed previously (see above, Protection Schemes). The solubilities of some Fmoc-amino acids make PSAs a less-than-optimum activated species. Not all Fmoc-amino acids are readily soluble in DCM, thus requiring significant DMF for solubilization. Optimum activation conditions, which require neat DCM (Rich and Singh, 1979), cannot be achieved. In addition, the resulting Fmoc-amino acid PSAs are even less soluble than the parent Fmoc-amino acid (Harrison et al., 1989). Preformed Fmoc-vV-carboxyanhydrides (NCAs) are stable derivatives which do not have to be generated in situ. Their reactivity is comparable to PSAs. In addition, NCAs are thermally stable,
Efficient couplings using Boc-amino acid PSAs are critically dependent on the concentration of the activated species in solution. It has been recommended that Boc-amino acid PSA couplings proceed at a concentration of 0.15 M, and that double couplings be standard practice for syntheses of >50 residues (Kent and Parker, 1988).
140
Synthetic Peptides
Fmoc-amino acids have variable solubility properties in relatively nonpolar solvents, such as DCM. Fmoc-Asp(OfBu), -Glu(OfBu), -Ile,-Leu, -Lys(Boc), -Ser(fBu), -Thr(?Bu), and -Val are soluble in DCM, while Fmoc-Ala, -Gly, -Met, -Trp, and -Tyr(^Bu) require the presence of a more polar co-solvent (e.g., DMF) for solubilization. Fmoc-Asn, -Gin, -His(Bum), and -Phe require at least 60% DMF to remain in solution. Conversion of Fmoc-amino acids to the corresponding PSAs results in poorer solubilities in nonpolar solvents. Thus, whether Fmoc-amino acids are coupled in situ or as preactivated species, relatively polar solvents (DMF or NMP) should be used to ensure that all reactants are in solution.
allowing for couplings at elevated temperatures. Such protocols can be advantageous for coupling sterically hindered amino acids. NCAs are sensitive to moisture and should therefore be used in dry solvents (Fuller et al., 1992). Acid Halides A^*-protected amino acid chlorides have a long history of use in solution synthesis. Their use in solid-phase synthesis has been limited, as the Boc group is not completely stable to reagents used in the preparation of acid chlorides. The Fmoc group, however, survives acid chloride preparation with thionyl chloride (Carpino et al., 1986), while both the Fmoc and Boc groups are stable to acid fluoride preparation with cyanuric fluoride (Carpino et al., 1990, 1991a; Bertho et al., 1991). For derivatives with fBu side-chain protection, only the acid fluoride procedure can be used (Carpino et al., 1990). Fmoc-amino acid chlorides and fluorides react rapidly under SPPS conditions in the presence of HOBt/DIEA and DIEA, respectively, with very low levels of racemization (Carpino et al., 1990, 1991b, 1992). Fmoc-amino acid fluorides are especially effective for coupling to N-alkyl amino acids (Wenschuh et al., 1994, 1995). For convenience, tetramethylfluoroformamidinium hexafluorophosphate (TFFH) can be used for automated preparation of Fmoc-amino acid fluorides (Carpino and El-Faham, 1995). Monitoring A crucial issue for stepwise solid-phase peptide synthesis is the repetitive yield per deprotection/coupling cycle. There are a number of ways of monitoring these steps, including some with a possibility for "real-time" feedback based on the kinetics of appearance or disappearance of appropriate soluble chromophores measured in a flow-through system. Most accurate and meaningful are qualitative and quantitative tests for the presence of unreacted amines after an
acylation step. Such tests should ideally be negative before proceeding further in the chain assembly. For certain active ester methods, the leaving group has "self-indicating" properties in so far as a colored complex is noted for as long as unreacted amino groups remain on the support. These various techniques reveal that high efficiencies can indeed be achieved in stepwise synthesis. The best known qualitative monitoring methods are the ninhydrin (Kaiser et al., 1970) and isatin (Kaiser et al, 1980) tests for free A^-amino and -imino groups, respectively, where a positive colorimetric response to an aliquot of peptide-resin indicates the presence of unreacted 7V"-amino/iniino groups. These tests are easy, reliable, and require only a few minutes to perform, allowing the chemist to make a quick decision on how to proceed. A highly accurate quantitative modification of the ninhydrin procedure has been developed (see Box). Other monitoring techniques exist that are generally nondestructive (noninvasive) and can therefore be carried out on the total batch. Resin-bound W-amino groups can be titrated with picric acid, 4,4'dimethoxytrityl chloride, bromophenol blue dye, and quinoline yellow dye. Picric acid is removed from resin-bound amines with 5% DIEA in DCM, and the resulting chromophore quantitated at X = 362 nm (Hodges and Merrifield, 1975; Arad and Houghten, 1990). For trityl monitoring, 4,4'-dimethoxytritylchloride and tetra-H-butylammonium perchlorate are reacted with the resin, released with 2% dichloroacetic acid in DCM, and quantitated at 498 nm (Horn and Novak, 1987; Reddy and Voelker, 1988). The effect of the dilute acid on Fmocamino acid side-chain protecting groups and linkers has not been
Free amino groups are quantitated based on their reaction with ninhydrin to produce Ruhemann's purple. Three reagent solutions are required: solution 1 is phenol ethanol (7: 3), solution 2 is 0.2 HIM KCN in pyridine, and solution 3 is 0.28 M ninhydrin in ethanol. A sample of Boc-peptide-resin (2 5mg) is incubated with 75 ^L of solution 1, lOO^L of solution 2, and 75(iL of solution 3 for 7min at 100C (Sarin et al., 1981; Applied Biosystems, Inc., 1992). For Fmoc-peptide-resins, premature removal of the Fmoc group (by pyridine) is minimized by adding 2-3 drops (20-40 uL) of glacial HO Ac to each resin sample and heating the reaction mixture for 5min instead of 7min (Applied Biosystems, Inc., 1989b). Immediately following the designated incubation time, 60% aqueous ethanol (4.8mL) is added to each sample with vigorous mixing. Once the peptide-resin has settled, the absorbance of each sample solution is read at X = 570 nm; 60% ethanol is used as a reference. The concentration of free amino groups (mmolg^ 1 ) (A570 x 106 umol/mol x 0.005 L)/ (15,000 M"1 cirT1 x 1 cm x mg of resin).
142
Synthetic Peptides
reported. For bromophenol blue and quinoline yellow monitoring, the dye is bound to free amino groups following deprotection, then displaced as acylation proceeds. Quantitative monitoring can be carried out at A = 600 and 495 nm for bromophenol blue and quinoline yellow, respectively (Krchnak et al., 1988; Flegel and Sheppard, 1990; S. Young et al., 1990). Gel-phase nuclear magnetic resonance (NMR) spectroscopy has been proposed for direct examination of resin-bound reactants (Epton et al., 1980; Giralt et al., 1984; Butwell et al., 1988), but would appear to suffer from problems of sensitivity, expense, and time needed to accumulate data. On-resin monitoring may also be achieved by NMR using the magic angle spinning method with a nanoprobe (Fitch et al., 1994). Magic angle spinning allows for the possible recording of high resolution NMR spectra of the resin-bound growing peptide chain and analysis of the synthesis. As an alternative to quantify resin-bound species, soluble reactants or co-products can be analyzed. Continuous measurement of electrical conductivity can be used to evaluate coupling and Fmoc deprotection efficiencies (Nielson et al., 1989; Fox et al., 1990; McFerran et al., 1991). The progress of Fmoc chemistry can be evaluated by observing at A = 300-312 nm the decrease of absorbance when Fmoc-amino acids are taken up during coupling, and by the increase in absorbance when the Fmoc group is released with piperidine (Chang et al., 1980a; Atherton and Sheppard, 1985, 1989; Frank and Gausepohl, 1988). Monitoring a decrease in Fmoc-amino acid concentration at A = 300 nm can be complicated when OPfp esters are utilized (Atherton et al., 1988b). More straightforward acylation monitoring is possible when Fmoc-amino acid ODhbt, Hpp, Pnp, and Npp esters are used. During the coupling of Fmoc-amino acid ODhbt esters, the liberated Dhbt-OH component binds to free 7V"-amino groups, producing a bright yellow color, which diminishes as acylation proceeds (Cameron et al., 1988). Real-time spectrophotometric monitoring proceeds at A 440 nm (Cameron et al., 1988). In a similar way, ionization of the leaving group from Fmoc-amino acid Npp esters by free A^-amino groups results in a blood red color (Hudson, 1990a). As coupling proceeds the color change (to gold) can be monitored at A = 488 nm. Unfortunately, continuous-flow monitoring is inherently insensitive for direct judgment of reaction endpoints. Assuming an initial twofold excess of activated incoming amino acid, absorbance will drop from 2.00 units to 1.05 units or 1.01 units respectively with 95% or 99% coupling. It is difficult to distinguish accurately between 1.05 and 1.01. In contrast, if unreacted resin-bound components are titrated for the same two cases, the five-fold difference between 0.05 and 0.01 is easily noted. The sensitivities of techniques monitoring resin-bound components is limited by nonspecific binding or irreversible reactions
143
of the titrant with the protected peptide or resin, which contribute to background readings despite complete reactions. Invasive monitoring of both synthetic efficiency and amino acid composition of peptide-resins can be achieved by a powerful quantitative variation of the Edman sequential degradation, called "preview analysis" (Tregear et al., 1977; Matsueda et al., 1981; Kent et al., 1982). Crude peptide-resins are sequenced directly; each Edman degradation cycle serves to identify a primary amino acid residue and "preview" the next amino acid in the sequence. Because preview is cumulative, quantitation of peaks after a number of cycles indicates the average level of deletion peptides and thus the overall synthetic efficiency. Since the linkers as well as most side-chain protecting groups used in Boc chemistry are stable to the conditions of Edman degradation, sequencing is a true solid-phase process; moreover, identification of amino acid phenylthiohydantoins requires a set of protected standards (Simmons and Schlesinger, 1980; Steiman et al., 1985). Preview sequence analysis of peptide-resins made by Fmoc chemistry requires initial TFA cleavage, followed by immobilization (covalent or noncovalent) of the crude peptide on a suitable support (Kochersperger et al., 1989; C. G. Fields et al., 1992, 1993c). Most side-chain-protecting groups used in conjunction with Fmoc chemistry are not stable to the conditions of Edman degradation; hence, the usual free side-chain phenylthiohydantoin standards can be used. Recently, on-resin mass spectrometry (MS) via the MALDI (matrix-assisted laser desorption/ionization) technique has been developed (Egner et al., 1995). This method can be used as a semi-on-line monitoring technique during SPPS (Talbo et al., 1997). An especially elegant approach for on-resin MS is to use the photolabile a-methylphenacyl ester linker, which is cleaved directly upon laser photolysis and MALDI ionization (Egner et al., 1995; Fitzgerald et al., 1996). The technique of "internal reference amino acids" (IRAA) is often very useful to measure accurately yields of chain assembly and retention of chains on the support during synthesis and after cleavage (Matsueda and Haber, 1980; Atherton and Sheppard, 1989; Albericio et al., 1990a). In addition, amino acid analysis of peptide-resins may be used to monitor synthetic efficiency; the advent of microwave hydrolysis technology may permit rapid analysis (Yu et al., 1988).
Automation of Solid-Phase Synthesis
A significant advantage of solid-phase methods lies in the ready automation of the repetitive steps (see tables 3-4 and 3-5). The first instrument for synthesis of peptides was built by Merrifield, Stewart, and Jernberg (1966). Numerous models for both batchwise and continuous-flow operation at various scales of operation are now commercially available.
144
Synthetic Peptides
Some of these instruments include features to facilitate monitoring (compare to previous section). Supported procedures have also been introduced for generation of large numbers of (usually related) peptide sequences in a reasonably short time, although with some sacrifices in the usual standards for purity and characterization. Automated Synthesizers, 1-3 Simultaneous Syntheses The PE Biosystems Model 431A/433A can utilize either Boc or Fmoc chemistries, with reaction mixing by vigorous vortex or gas bubbling. The 431A/433A uses fully automated HBTU + HOBt in situ cycles (Fmoc chemistry in NMP), called FastMoc (Fields et al., 1991). The synthesis scale is from 0.1 to 0.25mmol, with microprocessor-control via an internal computer. Advanced ChemTech Models 90 and 400 use either Boc or Fmoc chemistries, with reaction mixing by nitrogen bubbling or oscillation. Couplings for the Models 90 and 400 can be in situ, preactivation to form symmetrical anhydrides or HOBt esters, or by preformed Pfp esters. Pharmaceutical considerations are fulfilled by the Model 400 (Birr, 1990a, b), as it can utilize lOOg or more of resin. The Rainin/Protein Technologies PS3 features coupling by in situ BOP with Fmoc-amino acids only (all prepackaged), at scales from 0.1 to 0.5 mmol with nitrogen bubbling for reaction mixing. CS Bio offers several synthesizers that accommodate both Boc and Fmoc chemistries. Models 036, 100, 136, and 536 differ primarily in their flexibility (i.e., the 036 adds up to four amino acids in a single run while the 136 adds up to nine) and synthesis scales (0.12.5mmol, 0.05-lmmol, 0.1-2.5mmol, and 0.2-25mmol, respectively). Models 936S and 936 are pilot plant instruments, with scales of up to 600mmol and 12.5 mol, respectively. The just-mentioned instruments are designed for batchwise syntheses. Continuous-flow instruments (Fmoc chemistry only) include the PerSeptive Biosystems 9050 Plus and Pioneer and the PE Biosystems 432A (Synergy). The PerSeptive Biosystems 9050 Plus (Kearney and Giles, 1989) is an automated instrument with a synthesis scale of 0.05-5.0 mmol and flow rates typically from 5-15mL/min. The automated Pioneer Peptide Synthesis System has two columns for synthesis and can operate on a scale of 0.02-2.0 mmol. The PE Biosystems 432A is a single column automated instrument with a synthesis scale of 25 umol. Automated and Semi-automated Multiple Peptide Synthesizers The Advanced ChemTech 348 Q synthesizes up to 48 pep tides simultaneously. Either Boc or Fmoc chemistry is used, and the synthesis
145
scale is 5-150 umol. The CS Bio System 10 synthesizes 10 peptides simultaneously on a 0.1-0.5 mmol scale using either Boc or Fmoc chemistry. The Zinsser Analytic PepSy utilizes four independent pipetting probes to synthesize up to 96 peptides simultaneously via Fmoc chemistry. The ABIMED/Gilson Model AMS 422 (Gausepohl et al., 1990, 1991) uses Fmoc chemistry to synthesize up to 48 peptides on a scale from 5-50 umol, with activation by in situ PyBOP. A single robotic arm dispenses reagents, while resins are contained in fritted polypropylene tubes. In similar fashion, the Shimadzu PSSM-8 uses a single robotic arm to synthesize up to eight peptides simultaneously. The Rainin/Protein Technologies Symphony/Multiplex uses Fmoc chemistry to synthesize up to 12 peptides simultaneously. Synthesis scale is 5-100 umol, and peptide-resin cleavage is performed on the instrument. The Spyder Instruments Compas 768 uses tilted plate centrifugation to prepare up to 768 peptides per run by Fmoc chemistry. The PerSeptive Biosystems Pioneer (described above) has a multiple peptide synthesis accessory that allows for the synthesis of 16 or 32 peptides on a 0.005-1.0 mmol scale. Tyler Research Corporation offers the semi-automated SAMPS (Semi-Automatic Multiple Peptide Synthesizer), which synthesizes up to 100 peptides using Fmoc chemistry on a 10-200 umol scale. The manual Multiple Peptide Synthesis TeaBag method can synthesize up to 120 peptides simultaneously, using either Boc or Fmoc chemistry, and coupling by PSAs or in situ DCC/DIPCDI (Houghten et al., 1986; Beck-Sickinger et al., 1991). The "TeaBag" method has been semi-automated (BeckSickinger et al., 1991). Cambridge Research Biochemicals markets the Pepscan/PIN method (Geysen et al., 1984; Hoeprich, et al., 1989; Arendt et al., 1993), which uses either Boc or Fmoc chemistry, and coupling in situ with DCC or BOP/HOBt, to synthesi/e up to 96 peptides (10-100 nmol) simultaneously. Finally, the Affymax Parallel Chemical Synthesis system (VLSIPS, for "very large-scale immobilized polymer synthesis"), using the photolabile Nvoc ^"-protecting group and preformed HOBt esters, can be used for simultaneous synthesis of an extraordinarily high number of related peptides (e.g., 1024 = 210 by 10 stages requiring 2.5h each) (Fodor et al., 1991). Automated Solid-Phase Organic Synthesizers Recent years have seen a dramatic increase in the variety of organic reactions, beyond peptide synthesis, that have utilized solid-phase methodologies. Concurrently, instrumentation to automate solidphase organic chemistry has been developed. These instruments differ from conventional peptide synthesizers primarily in their durability to a range of caustic reagents. The Zinsser Analytic Sophas M can accommodate a variety of reactors, from 96-well plates to 25 mL reaction vessels.
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Synthetic Peptides
It can perform up to 864 syntheses in a single run, and allows for heated reactions up to 150C. The Sonata/Pilot from Rainin/Protein Technologies performs large scale (0.1-50mmol) peptide and organic synthesis with heating and cooling capabilities. Advanced ChemTech has several "biomolecular" synthesizers, including the 357FBS, 384HTS, 396MBS, 440MOS, 496MOS, and Labtech Synthesis System. The 357FBS synthesizes up to 36 compounds on a scale of 5-250 umol. The 384HTS, 440MOS, 496MOS, and LabTech contain four 96-well reactor blocks, one 40-well (8 mL per well) reactor block, one 96-well reactor block, and one 96-well reactor or solution phase block, respectively, and each has a controlled temperature range of -70 to 150C. The 396MBS accommodates a 96-well reactor block with a synthesis scale of 5-1000 umol. The PE Biosystems Solaris 530 Organic Synthesis System has 48-position reagent racks, synthesis modules, and product racks, and uses eight liquid-handling tips.
Cleavage
Boc SPPS is designed primarily for simultaneous cleavage of the peptide anchoring linkage and side-chain-protecting groups with strong acid (HF or equivalent), while Fmoc SPPS is designed primarily to accomplish the same cleavages with moderate strength acid (TFA or equivalent). In each case, careful attention to cleavage conditions (reagents, scavengers, temperature, and times) is necessary in order to minimize a variety of side reactions. Considerations for separate removal of acid-stable side-chain-protecting groups has been covered earlier (see above, Protection Schemes). Nonacidolytic methods for cleavage of the anchoring linkage, each with certain advantages as well as limitations, may also be used in conjunction with either Boc or Fmoc chemistries. Hydrogen Fluoride (HF) Treatment with HF cleaves PAM or MBHA linkages and simultaneously removes the side-chain protecting groups commonly applied in Boc chemistry (figure 3-2), that is, Bzl (for Asp, Glu, Ser, Thr, and Tyr), 2-BrZ or 2,6-Cl2Bzl (for Tyr), cHex (for Asp), 2-C1Z (for Lys), Bom or Tos (for His), Tos or Mts (for Arg), Xan (for Asn and Gin), and Meb (for Cys) (Tarn and Merrifield, 1987; Applied Biosystems, Inc., 1992). HF cleavages are always carried out in the presence of a carbonium ion scavenger, usually anisole (10%, v/v). For cleavages of Cys-containing peptides, further addition of 4-thiocresol (1.8%) is recommended. Additional scavengers, such as dimethyl sulfide and 4-cresol, are used in conjunction with a two-stage "low-high HF" cleavage method that provides extra control and thereby better product
147
HF cleavage procedures require a special all-fluorocarbon apparatus. The standard method uses HF-anisole ( 9 : 1 ) (1 mL per 20 umol peptide) at 0"C for 1 h, with the addition of 2-mercaptopyridine (10 equiv.) for Met-containing peptides. For Cys- and Trp-containing peptides, HF-anisole-dimethyl sulfidc^t-thiocresol ( 1 0 : 1 : 1 : 0 . 2 ) is recommended. Following cleavage, HF is evaporated carefully under aspirator suction with ice bath cooling, and most of the anisole is removed by vacuum from an oil pump. The vessel is triturated with ether to remove benzylated scavenger adducts; at the same time, the ether facilitates transfer of the cleaved resin (with trapped peptide) to a fritted glass funnel. Next, 30% aqueous HOAc (twice, 1.5mL per 20 umol peptide) is used to rinse the cleavage vessel and extract the resin on the fritted glass filter. The combined filtrates are diluted with H7O to bring the HOAc concentration to < 10%, and the peptide is usually recovered by lyophilization (Stewart and Young, 1984; Tarn and Merrifield, 1987; Stewart, 1997). "Low-high" HF cleavages are carried out following the detailed description of the original and later publications (Tarn et al, 1983; Tarn and Merrifield, 1985, 1987).
purities (Tarn and Merrifield, 1987). Both Trp(CHO) and Met(O) can be dcprotected under "low HF" conditions [20-25% HF-0 to 5% 4thiocresol-70 to 80% dimethyl sulfide, 0 C for 1 h; 4-thiocresol is necessary only for Trp(CHO)] (Tarn and Merrifield, 1987). In the presence of the large levels of dimethyl sulfide used in "low HF" conditions, Tyr(Bzl) undergoes little C-alkylation (Tarn and Merrifield, 1987). In strong acid, the y-carboxyl group of Glu can become protonated and lose water. The resulting acylium ion is then trapped either intramolecularly by the TV-amide nitrogen to give a pyrrolidone or (more likely) intermolecularly with the commonly used scavenger anisole (Feinbcrg and Merrifield, 1975). This serious problem can be controlled by attenuation of the acid strength (i.e., "low HF" conditions) and caution with regard to cleavage temperature (Tarn and Merrifield, 1987). Strong acid can also cause an TV O acyl shift in Ser- and Thrcontaining peptides resulting in the thermodynamically less stable O-acyl species (Fujino et al., 1978). This process can be reversed for simple cases by treating the cleaved, deprotected peptide with 5% aqueous NH4HCO3, pH 7.5, 25 C for several hours, or in 2% aqueous NH 4 OH, 0 DC, 0.5 h (Barany and Merrifield, 1979); reversal of the TV - O acyl shift in multiple Ser- and Thr-containing peptides may be more problematic. HF-liberated Boc groups can modify Met residues (Noble et al., 1976); therefore, the A^-Boc group should be removed prior to HF cleavage. HF deprotection of His(Bom) liberates formaldehyde, resulting in methylation of susceptible side chains and cyclization of TV-terminal Cys residues to a thiazolidine (Mitchell et al., 1990; Gesquiere et al., 1990; Kumagaye et al., 1991). These side reactions
148 Synthetic Peptides
may be inhibited by including in the HF cleavage mixture a formaldehyde scavenger, for example, resorcinol (0.27 M), or Cys or CysNH2 (30-90 equiv.), and purifying the peptide immediately after cleavage (Mitchell et al., 1990; Kumagaye et al., 1991). Serious Trp alkylation side reactions has been observed during workup after HF cleavage of peptides containing Cys or Met adjacent to Trp; the problem may be mitigated by adding free Trp (10 equiv.) as a scavenger during cleavage (D. Hudson, unpublished results) or during the initial lyophilization (Ponsati et al., 1990a).
Other Strong Acids The alternative strong acids listed here, and further examples elsewhere (Barany and Merrifield. 1979; Stewart, 1997), are very likely to promote the same side reactions just described for HF. TFMSA (1 M)-thioanisole (1 M) in TFA cleaves PAM or MBHA linkers (Tarn and Merrifield, 1987; Bergot et al., 1987). and simultaneously removes many side-chain-protecting groups used in Boc chemistry, for example, Mts (for Arg), Bzl (for Asp, Glu, Ser, Thr), cHex (for Asp), Meb (for Cys), 2-C1Z (for Lys), 2-BrZ or 2,6-Cl2Bzl (for Tyr), and Bom or Tos (for His) (Tarn and Merrifield, 1987), without requiring a special apparatus. A "low-high" method can be used with TFMSA, in similar fashion to HF (Tarn and Merrifield, 1987). Tetrafluoroboric acid (HBF4) (1 M)-thioanisole (1 M) in TFA offers a similar range of sidechain deprotcction as TFMSA (Kiso et al., 1989; Akaji et al., 1990b). Trimethylsilyl bromide (TMSBr) and trimethylsilyl trifluoromethanesulfonate (TMSOTf) have also been used for strong acid cleavage and deprotection reactions, which arc accelerated by the presence of thioanisole as a "soft" nucleophile (Yajima et al., 1988; Nomizu et al., 1991). TMSBr (1 M)-thioanisole (1 M) removes Mts (for Arg), Bzl (for
For TFMSA cleavages, peptide-resin (100 mg) is stirred with thioanisole (187 uL) and 1,2-ethanedithiol (EOT) (64p.L) in an ice bath for lOmin. TFA (1.21 mL) is added, and after equilibration for lOmin, TFMSA (142 uL) is added slowly. Cleavage and deprotection proceeds for 1 h (unless the MBHA linker is being cleaved, in which case cleavage proceeds for 1.5-2.5h). During cleavage and deprotection, the ice bath is removed, but precautions are taken to ensure that the temperature of the reaction does not increase rapidly. Ambient temperature (25 C) is reached in about 15min. Subsequently, the cleavage mixture is filtered through a fritted glass funnel directly into methyl ;Bu ether, to rapidly precipitate the peptide and remove the acid and scavengers. The precipitated peptide should be washed with methyl ?Bu ether and dried under vacuum overnight (Bergot et al., 1987; Fields and Fields, 1991; C. G. Fields et al., 1993a).
Asp, Glu, Ser, Thr, Tyr), 2,6-Cl2Bzl (for Tyr), and 2-C1Z (for Lys), as well as reducing Met(O) to Met (Yajima et al., 1988). Although not specifically stated, TMSBr-thioanisole probably deprotects His(Tos), Cys(Meb), and Tyr(2-BrZ). TMSOTf (1 M)-thioanisole (1 M) (a.k.a. DEPRO) additionally removes Bom from His (Yajima et al., 1988) and efficiently cleaves PAM or MBHA linkages (Akaji et al., 1989; Nomizu et al., 1991). Stable Cys side-chain protection should be used during TMSBr-thioanisole cleavages. Trifluoroacetic Acid (TFA) The combination of side-chain-protecting groups, for example, rBu (for Asp, Glu, Ser, Thr, and Tyr), Boc (for His and Lys), Bum (for His), Tmob (for Asn, Cys, and Gin), Trt (for Asn, Cys, Gin, and His), and/or Xan (for Asn, Cys, and Gin), and anchoring linkages, for example, HMP/PAB or PAL, commonly used in Fmoc chemistry (see figure 3-3) are simultaneously deprotected and cleaved by TFA. Such cleavage of ?Bu and Boc groups results in tert-butyl cations and fer^-butyl trifluoroacetate formation (Jaeger et al., 1978a, b; Low et al., 1978a, b; Lundt et al., 1978; Masui et al., 1980). These species are responsible for /<?r/-butylation of the indole ring of Trp, the thioether group of Met, and, to a very low degree (0.5-1.0%), the 3'-position of Tyr. Modifications can be minimized during TFA cleavage by utilizing effective ferr-butyl scavengers. An early comprehensive study showed the advantages of 1,2-ethanedithiol (EOT) (Lundt et al., 1978); this dithiol has the additional virtue of protecting Trp from oxidation that occurs due to acid-catalyzed ozonolysis (Scoffone et al., 1966). To avoid acid-catalyzed oxidation of Met to its sulfoxide, a thioether scavenger such as dimethyl sulfide, ethyl methyl sulfide, or thioanisole should be added (Guttmann and Boissonnas, 1959; King et al., 1990; Guy and Fields, 1997). TFA deprotection of Cys(Trt) is reversible in the absence of a scavenger (Photaki et al., 1970). Realkylation by the Trt carbocation can occur readily following TFA cleavage if solutions of crude peptide in TFA are concentrated by rotary evaporation or lyophilization (D. Hudson, unpublished results). EOT, triethylsilane, or triisopropylsilane are recommended as efficient scavengers to prevent Trt reattachment to Cys (Pearson et al., 1989); this recommendation extends to prevent reattachment of Tmob as well (Munson et al., 1992). TFA deprotection of His(Bum) liberates formaldehyde, in similar fashion to HF deprotection of His(Bom) (Gesquiere et al., 1992). Cyclization of JV-terminal Cys residues to a thiazolidine is only partially (60%) inhibited by even complex TFA/scavenger mixtures, such as reagent K (see discussion below). The indole ring of Trp can be alkylated irreversibly by Mtr, Pmc, and Pbf groups from Arg (Sieber, 1987b; Harrison et al., 1989; Riniker
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Synthetic Peptides
and Hartmann, 1990; King et al, 1990; Fields and Fields, 1993), Tmob groups from Asn, Gin, or Cys (Gausepohl et al., 1989b; Sieber and Riniker, 1990), and even by some TFA-labile ester and amide linkers (Atherton et al., 1988a; Riniker and Kamber, 1989; Albericio et al., 1990a; Gesellchen et al., 1990; C. G. Fields et al., 1993c; G. B. Fields et al., 1993). The extent of Pmc modification of Trp is dependent upon the distance between the Arg and Trp residues (Stierandova et al., 1994). Cleavage of the Pmc group may also result in 0-sulfation of Ser, Thr, and Tyr (Riniker and Hartmann, 1990; Jaeger et al., 1993). Three efficient cleavage "cocktails" for Mtr/Pmc/Pbf/Tmob quenching, and preservation of Trp, Tyr, Ser, Thr, and Met integrity, are TFA-phenol-thioanisole-EDT-H2O (82.5:5:5:2.5:5) (reagent K) (King et al., 1990; Van Abel et al., 1995), TFA-thioanisole-EDTanisole (90:5:3:2) (reagent R) (Albericio et al., 1990a), and TFAphenol-H2O-triisopropylsilane (88 : 5:5:2) (reagent B) (Sole and Barany, 1992; Van Abel et al., 1995). Studies on Trp preservation during amino acid analysis (Bozzini et al., 1991) has led to the development of reagent K', where EDT is replaced by 1 -dodecanethiol (Guy and Fields, unpublished results). Water is an essential component of reagents K and K', but phenol is necessary only with multiple Trp-containing peptides (King et al., 1990). Thioanisole, a soft nucleophile, accelerates TFA deprotection of both Arg(Mtr) and Arg(Pmc). Triethylsilane (4 equiv.) in MeOH-TFA (1:9) has been reported to efficiently cleave and scavenge Pmc groups (Chan and Bycroft, 1992). Given a choice for Arg protection, Pmc/Pbf is preferred because it is more labile, and gives less Trp alkylation during unscavenged TFA cleavage; the
Cleavage and side-chain deprotection of peptide-rcsins assembled by Fmoc chemistry is carried out in TFA, in the presence of carefully chosen scavengers. The text discussion should be consulted with respect to peptide sequence and potential side reactions. It is recommended that small scale cleavages (< lOmg peptide-resin) be performed and analyzed before proceeding to large scale cleavages. Standard cleavages of HMP/PAB, Dod, and PAL linkers, and simultaneous side-chain deprotection, proceed by stirring peptide-resin (50-200 mg) in 2mL of the appropriate, freshly prepared cleavage cocktail for 1.5-2.5h at 25 C. The resin is filtered over a medium fritted glass filter and rinsed with 1 mL of TFA. The combined filtrate and TFA rinse are either (a) precipitated with methyl ;Bu ether (~-50mL) or (b) dissolved in ~40mL H2O and extracted six times with ~40mL methyl rBu ether. Following (a), the mixture is centrifuged at 3000 rpm for 2min and decanted. The peptide pellet is dispersed with a rubber policeman, washed thoroughly with methyl ;Bu ether, and dried overnight in a lyophilizer (King et al., 1990; Albericio et al., 1990a). Following (b), the H2O layer is concentrated in vacuo and loaded directly to a semipreparative HPLC column and the peptide is purified.
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recommendation for Pmc/Pbf is particularly appropriate for sequences containing multiple Arg residues (Green et al, 1988; Harrison et al., 1989; King et al., 1990). The use of Boc side-chain protection of Trp also significantly reduces alkylation by Pmc/Pbf groups (Choi and Aldrich, 1993; Fields and Fields, 1993). The Trt group, instead of Tmob, is suggested for Asn/Gln side-chain protection in Trp-containing peptides, as Trt cations are easier to scavenge (Sieber and Riniker, 1990). Nonacidolytic Cleavage Methods Benzyl ester anchoring linkages can be cleaved usefully under nonacidic conditions. An interesting alternative to standard HF cleavages for Boc chemistry is catalytic transfer hydrogenolysis (CTH), which removes Bzl side-chain protecting groups (from Asp, Glu, Ser, Thr, and Tyr) and cleaves benzyl ester anchors to provide a C-terminal carboxyl group (Anwer and Spatola, 1980, 1983). Peptide-resin (Ig) is treated initially with palladium (II) acetate (1 g) in DMF (13 mL) for 2h; ammonium formate (1.3g) is then added, and the reaction proceeds for an additional 2h (Anwer and Spatola, 1983). CTH can reduce Trp to octahydrotryptophan (Mery and Calas, 1988). Benzyl ester linkages may also be cleaved by 2-dimethylaminoethanol (DMAE)-DMF (1:1) for 70 h, with subsequent treatment of the peptide-DMAE ester by DMF-H2O (1:5) for 2 h yielding the peptide acid (Barton et al., 1973). For some applications when peptide amides are required, benzyl ester-type linkages are treated with NH3 in anhydrous MeOH, 2-propanol, 2,2,2-trifluoroethanol (TFE), or MeOH-DMF for 2-4 days at 25 C (Atherton et al., 198 Ic; Stewart and Young, 1984; Story and Aldrich, 1991), although these conditions will also convert Asp(OBzl) and Glu(OBzl) residues to Asn and Gin, respectively. Additionally, ethanolamine in DMF or MeOH cleaves benzyl ester linkages (8^10 h, 45-60 C) to provide an ethanolamidated peptide C-terminus (Prasad et al., 1982; Fields et al., 1988, 1989; Prosser et al., 1991). Nucleophilic cleavages of benzyl esters can be accompanied by side reactions, including racemization of the C-terminal residue (Barany and Merrifield, 1979). On the other hand, base cleavages of the NPE and HMFA linkers appear to be quite safe and general. Peptide acids are obtained upon treatment with either piperidineDMF (1:4) or DBU (0.1 M)-l,4-dioxane, after 5min (for HMFA) to 2h (for NPE) at 25C (Liu et al., 1990; Albericio et al., 1991b). Palladium-catalyzed peptide-resin cleavage is used for the HYCRAM linker (Kunz and Dombo, 1988; Guibe et al., 1989). The fully protected peptide-resin (0.1 mmol of peptide) is shaken for 6-18 h under N2 or Ar atmosphere in 8mL of DMSO-THF-0.5M aqueous HC1 (2:2:1), in the presence of 50-190 equiv. of either NMM (for
152
Synthetic Peptides
Following synthesis, peptide-resins should be well dried, and then stored in a desiccator at 4 C with the jV-terminus protected. As a general practice, peptides should never be stored in the solid state after being lyophilized from moderate or strong acid; deamidation, among other side reactions, may proceed rapidly (see below, Auxiliary Issues). If stored in solution following purification, peptides should be used for biological or chemical studies as soon as possible. Met-containing peptides oxidize rapidly upon storage in solution, especially when repeated freeze-thawing occurs (Stewart and Young, 1984), while Asn-containing peptides can hydrolyze spontaneously in solution (see below, Auxiliary Issues).
Boc-peptides) or dimedone (for Fmoc-peptides) as well as 0.015 equiv. of tetrakis(triphenylphosphino)palladium(0) catalyst. The reaction mixture is filtered and washed with DMF, DMF-0.5M aqueous HC1 (1:1), and DMF. The nitrate and washings are combined and evaporated to minimal volume; the peptide is then precipitated with methyl ?Bu ether (Orpegen, 1990; Lloyd-Williams et al., 1991). The crude peptide acid (0.7mmol scale) can be converted to an amide by dissolving in dry DMF (50 mL) at 25 C, adding NMM (80 uL), cooling to -20 C, adding isobutylchloroformate (90 uL) and, after 8min, 25% aqueous NH 4 OH (0.3 mL), and stirring for 2-12 h. The solution is evaporated and the peptide amide dried over P2O5 in vacuo (Orpegen, 1990). Photolytic cleavage at A. = 350 nm under inert (N 2 , or Ar) atmosphere is used for the ONb, 2-bromopropionyl, and Nonb linkers. The most efficient photolysis of the ONb and Nonb linkers is achieved when peptide-resins are swollen with 20-25% 2,2,2-trifluoroethanol in either DCM or toluene (Giralt et al., 1986; Kneib-Cordonier et al., 1990; Hammer et al., 1990). Photolytic cleavage yields after 9-16h range from 45-70% for relatively small peptides (five to nine residues). The 2-bromopropionyl linker is cleaved in DMF, with a yield of 70% after 72 h for a tetrapeptide (Wang, 1976). Photolabile linkers combining the o-nitro and a-methyl themes may cleave more rapidly and with higher yields (Holmes and Jones, 1995). Post-Translational Modifications and Unnatural Structures Peptides of biological interest often include structural elements beyond the 20 genetically encoded amino acids. This section summarizes the best current methods to duplicate by chemical synthesis the post-translational modifications achieved in nature, including the alignment of half-cysteine residues in disulfide bonds. This section also covers a set of unnatural structures which are of considerable interest for peptide drug design, namely lactams (head-to-tail, side-chain-to-side-chain,
etc.), and lastly describes the steps needed to elicit good antibody production from synthetic peptides. Hydroxylated Residues Hydroxyproline (Hyp) has been incorporated successfully without side-chain protection in both Boc (Felix et al., 1973; Stewart et al., 1974) and Fmoc (Fields et al., 1987; Fields and Fields, 1992) SPPS. Alternatively, the usual hydroxyl protecting groups Bzl (Cruz et al., 1989) for Boc and fBu (Becker et al., 1989; Grab et al., 1996) for Fmoc can be used. Smaller peptides can be synthesized efficiently without side-chain protection on Hyp (Fields and Noble, 1990), but larger peptides containing multiple Hyp residues should use the appropriate (Bzl or ?Bu) side-chain protected Hyp derivative (Grab et al., 1996; Henkel et al., 1999). Hydroxylysine (Hyl) has been incorporated in SPPS as FmocHyl(Boc, 0-te^-butyldimethylsilyl [TBDMS]). This derivative was prepared by (a) protecting the A^-amino group of copper-complexed Hyl by treatment with Boc2O, (b) disrupting the copper complex, (c) protecting the A^-amino group by treatment with 9-fluorenylmethyl chloroformate, (d) protecting the carboxyl group by treatment with benzyl bromide, (e) blocking the side-chain hydroxyl group with tertbutyldimethylsilyl trifluoromethanesulfonate, and (f) hydrogenation to remove the benzyl group (Broddefalk et al., 1998). Hyl cannot be incorporated without side-chain protection of the hydroxyl group, as activation of an unprotected Hyl derivative leads to intramolecular lactone formation (Koeners et al., 1981; Remmer and Fields, 1999). y-Carboxyglutamate Acid-sensitive x-carboxyglutamate (Gla) residues have been identified in a number of diverse biomolecules, such as prothrombin and the "sleeper" peptide from the venomous fish-hunting cone snail (Conus geographus). Fmoc chemistry has been utilized for the efficient SPPS of the 17-residue "sleeper" peptide, with the five Gla residues incorporated as Fmoc-Gla(O/Bu)2 (Rivier et al., 1987). Cleavage and sidechain deprotection of the peptide-resin by TFA-DCM (2:3) for 6 h resulted in no apparent conversion of Gla to Glu. Phosphorylation Incorporation of side-chain-phosphorylated Ser and Thr by SPPS is especially challenging, as the phosphate group is decomposed by strong acid, and lost with base in a /3-elimination process (Perich, 1990). Boc-Ser(PO3Ph2) and Boc-Thr(PO3Ph2) have been used,
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Synthetic Peptides
where HF or hydrogenolysis cleaves the peptide-resin, and hydrogenolysis removes the phosphate phenyl groups (Perich et al., 1986; Arendt et al., 1989). Fmoc-Ser(PO3Bzl,H) and Fmoc-Thr(PO3Bzl,H) can be used for Fmoc syntheses provided appropriate precautions are taken (Wakamiya et al., 1994; White and Beythien, 1996; Nagata et al., 1997; Perich, 1997; Perich et al., 1999). Alternatively, peptideresins that were built up by Fmoc chemistry to include unprotected Ser or Thr side chains may be treated with a suitable phosphorylating reagent, for example, Ar,A'-diisopropyl-bis(4-chlorobenzyl)phosphoramidite or dibenzylphosphochloridate, to produce a phosphite triester intermediate. This phosphitylation step is then followed by oxidation with MCPBA, ?BuOOH, or aqueous iodine/THF to produce the desired phosphorylated residue(s) (Perich, 1997). The phosphorylated peptide is then obtained in solution, following simultaneous deprotection and cleavage with TFA in the presence of scavengers (Otvos et al., 1989a; de Bont et al., 1990). Side-chain-phosphorylated Tyr is less susceptible to strong acid decomposition, and is not at all base-labile. Thus, SPPS has been used to incorporate directly Fmoc-Tyr(PO3Me2) (Kitas et al., 1989), Fmoc-Tyr(PO3Bzl2) (Kitas et al., 1991), Fmoc-Tyr(PO3fBu2) (Perich and Reynolds, 1991), Fmoc-Tyr(PO3H2) (Ottinger ct al., 1993; Xu et al., 1996; Chan et al., 1997; Lauer et al., 1997), and Boc-Tyr(PO3H2) (Zardeneta et al., 1990). Syntheses incorporating Fmoc-Tyr(PO3Bzl2) use 2% DBU in DMF for TV-ammo deprotection, as piperidine was found to remove the benzyl protecting groups from phosphate (Kitas et al., 1991), whereas the /Bu phosphate group is inert to piperidine-mediated dealkylation during Fmoc removal steps (Perich, 1997). BOP/PyBOP and HBTU have been successfully applied for coupling of Fmoc-Tyr(PO3Me2), Fmoc-Tyr(PO3?Bu2), and FmocTyr(PO3Bzl2). DCC and DIPCDI are not recommended, since interaction with the phosphodiester causes undesired activation (Perich, 1997). TFMSA or TMSBr can be used for peptide-resin cleavage and removal of the methyl phosphate groups without 0-dephosphorylation (Kitas et al., 1989; Zardeneta et al., 1990), while TFA is used for removal of the benzyl and /Bu phosphate groups (Kitas et al., 1991). For most applications, however, the direct use of side-chainunprotected Fmoc-Tyr(PO3H2) (Ottinger et al., 1993; Xu et al., 1996; Perich et al., 1999) is particularly convenient and reliable. Coupling is achieved by a BOP/HOBt protocol, and the base used for all subsequent coupling steps needs to be DIEA. An intramolecular side reaction may occur during coupling, leading to pyrophosphate linked byproducts (Ottinger et al., 1996; Garcia-Echeverria, 1995). Net Tyr phosphorylation can be achieved after chain assembly (Tyr residues incorporated with free phenolic side-chain) by phosphite triester treatment using the dialkyl jV^V-diethylphosphoramidates
155
(?BuO)2PNR2, (BzlO)2PNR2, or (TmseO)2PNR2 (R' = ethyl or isopropyl) followed by MCPBA, /BuOOH, or aqueous iodine/THF oxidation. If Cys or Met residues are present in the peptide sequence, the use of /BuOOH is preferred to minimize oxidation of these residues (Perich and Johns, 1988). Formation of H-phosphonates during the course of phosphitylation/oxidation routes, together with protocols to minimize occurrence of this side reaction, have been documented (Xu et al., 1997; Perich, 1998). Sulfation Gastrin, cholecystokinin, and related hormones contain sulfated Tyr; thus, incorporation of this residue into synthetic peptides is of great interest. Synthesis of Tyr sulfate-containing peptides is difficult, due to the substantial acid lability of the sulfate ester; also, most sulfating agents are more reactive towards the hydroxyls of Ser or Thr with respect to the phenol of Tyr. While there is an elegant history of success in solution chemistry (Beacham et al., 1967; Ondetti et al., 1970; Pluscec et al., 1970; Wunsch et al., 1981), this brief discussion focuses on the best SPPS approaches. Side-chain-unprotected Tyr can be incorporated by Boc or Fmoc chemistry and sulfation while the otherwise fully protected peptide remains anchored to the support is achieved by use of pyridinium acetyl sulfate (Fournier et al., 1989). Base or acid-promoted deprotection/cleavages follows, under conditions carefully optimized to avoid or minimize desulfation. Alternatively, sulfated Tyr can be incorporated directly by use of Fmoc-Tyr(SOJ-Na+)-OPfp, Fmoc-Tyr(SO^ Na+), or FmocTyr(SOj Ba^~2) in situ with BOP/HOBt (Penke and Rivier, 1987; Penke and Nyerges, 1989, 1990; Han et al., 1996b). A brief and carefully optimized acidolytic deprotection/cleavage is then used to minimize desulfation. PEG-PS resins functionalized with xanthenylamide (XAL) handles are the solid supports of choice for this application, as cleavage requires only 1-5% TFA. Cholecystokinin containing a sulfonated Tyr (CCK-8) was synthesized successfully using a 15min treatment with 5% TFA, providing the desired deprotected and cleaved peptide 95% pure (Han et al., 1996b). O-Sulfated Ser and Thr residues can be obtained by simply incorporating Fmoc-Ser/Thr(SO3H) into the peptide chain. If an Mtr or Pmc-protected Arg is present in the peptide, the sulfated Ser and/or Thr can alternatively be obtained by cleaving the peptide with 50% TFA in DCM without any scavengers. Under these conditions the sulfate moiety of the Arg-protecting group will be transferred to the hydroxyl groups in a yield of up to 60% (Jaeger et al., 1993). The sulfated and unsulfated peptides can then be separated via HPLC or ion-exchange chromatography (Jaeger et al., 1993).
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Synthetic Peptides
Glycosylation Methodology for site-specific incorporation of carbohydrates during chemical synthesis of peptides has developed rapidly. The mild conditions of Fmoc chemistry are more suited for glycopeptide syntheses than Boc chemistry, as repetitive acid treatments as well as final cleavage with a strong acid such as HF can be detrimental to sugar linkages (Kunz, 1987, Meldal, 1994). Fmoc-Ser, -Thr, -Hyl, -Hyp, and -Asn have all been incorporated successfully with glycosylated side chains. Glycosylation is typically performed in the presence of Lewis acids with glycosyl bromides or trichloroacetimidates for Ser, Thr, and Hyp, or by coupling glycosylamines onto Asp (to produce a glycosylated Asn). The side-chain glycosyl is usually hydroxyl-protected by either acetyl (Ac) or benzoyl (Bz) groups (Paulsen et al., 1988, 1990; Jansson et al., 1990; de la Torre et al., 1990; Bardaji et al., 1991; Kihlberg et al., 1997; Malkar et al., 2000), although some SPPS have been successful with no protection of glycosyl hydroxyl groups (Otvos et al., 1989b, 1990; Filira et al., 1990). Glycosylated residues are incorporated as preformed Pfp esters or in situ with DCC/HOBt or HATU/HOAt (Paulsen et al., 1988; Filira et al., 1990; Jansson et al., 1990; Meldal and Jensen, 1990; Otvos et al., 1990; Paulsen et al., 1990; Bardaji et al., 1991; Jensen et al., 1996; Malkar et al., 2000). These sugars are relatively stable to Fmoc deprotection by piperidine, morpholine, or DBU (Paulsen et al., 1988, 1990; Jansson et al., 1990; Meldal and Jensen, 1990; Otvos et al., 1990; Bardaji et al., 1991; Meldal et al., 1994; Jensen et al., 1996; Kihlberg et al., 1997; Malkar et al., 2000), brief treatments (2h) with TFA for side-chain-deprotection and peptide-resin cleavage (Paulsen et al., 1988, 1990; Filira et al., 1990; Meldal and Jensen, 1990; Jansson et al., 1990; Otvos et al., 1990; Bardaji et al., 1991), and palladium treatment for peptide-resin cleavage from HYCRAM (Kunz and Dombo, 1988). Deacetylation and debenzoylation may be performed with hydrazine-MeOH (4:1) prior to glycopeptide-resin cleavage (Kunz, 1987; Bardaji et al., 1991). Removal of acetyl and benzoyl groups is usually carried out in solution using catalytic methoxide in methanol. Some side reactions, including ^-elimination and Cys-induced degradation of the peptide backbone (Paulsen et al., 1988; Peters et al., 1995; Vuljanic et al., 1996; Elofsson et al., 1997), may occur, but if the apparent pH is controlled carefully to be about 10, these deprotections are generally "safe" (Meldal et al. 1994; Jensen et al., 1996). Acid-labile protecting groups have been developed for the carbohydrate moiety, such as TBDMS, ter;-butyldiphenylsilyl (TBDPS), and isopropylidene (Elofsson et al., 1997). The use of DBU in combination with silyl protection should be avoided, due to DBU-induced decomposition of the glycopeptide (Christiansen-Brams et al., 1993).
Disulfide Bond Formation In the majority of cases, intramolecular disulfides or simple intermolecular homodimers have been formed from purified linear precursors by nonspecific oxidations in dilute solutions. An even number of Cys residues are brought to the free thiol form by removal of the same 5-protecting group, following which disulfide formation is mediated by molecular O2 (from air), potassium ferricyanide [K3Fe(CN)6], DMSO, or others from a lengthy catalogue of reagents (Hiskey, 1981; Stewart and Young, 1984; Tarn et al., 1991b; Andreu et al., 1994; Annis et al., 1997; Albericio et al., 2000). Accomplishing the same end, but proceeding by a different mechanism, the polythiol can be treated with a mixture of reduced and oxidized glutathione, which catalyze the net oxidation by thiol-disulfide exchange reactions (Ahmed et al., 1975; Lin et al., 1988; Pennington et al., 1991; Andreu et al., 1994; Annis et al., 1997; Albericio et al., 2000). These procedures, which require scrupulous attention to experimental details, have often given the desired disulfide-containing peptide products in acceptable yields. However, even under the best conditions, significant levels of dimeric, oligomeric, or polymeric material are observed. The nonmonomeric material has usually proven to be difficult to "recycle" by alternating reduction and reoxidation steps. A more sophisticated approach, which also requires dilute solutions, involves selective pairwise co-oxidations of two designated free or protected sulfhydryl groups. Such reactions are best carried out in intramolecular fashion because, if the paired groups are on separate chains, there is the problem that homodimers form along with the desired heterodimer. If the thiols have already been deblocked, oxidation follows using procedures mentioned earlier. The prototype oxidative deprotections involve I2 treatments on Cys(Trt) or Cys(Acm) residues (Kamber et al., 1980). These reactions are carried out in neat TFE, MeOH, 1,1,1,3,3,3-hexafluoroisopropanol, HOAc, DCM, or chloroform, or mixtures of the these solvents, and often proceed in modest to high yield; however, side reactions have been observed at Trp residues resulting in Trp-2'-thioethers (Sieber et al., 1980) and /?-3-oxindolylalanine (Casaretto and Nyfeler, 1991). Pairwise oxidative removal of appropriate Acm or Tacm Cys protecting groups with Tl(Tfa)3 or methyltrichlorosilane (in the presence of diphenylsulphoxide) also furnishes the disulfide directly (Fujii et al., 1987; Kiso et al., 1990; Akaji et al., 1991; Albericio et al., 2000). However, Trp and Met must be side-chainprotected during such treatments (see, for example, Edwards et al., 1994). Liberation of Acm by Tl(Tfa)3 can also result in a S -> O Acm shift, a side reaction that can be suppressed by glycerol (Lamthanh et al., 1993). As a final example in this category, Cys(Fm) residues form
158
Synthetic Peptides
disulfides directly upon treatment with piperidine (Ponsati et al., 1990b). Most general, but also most demanding in terms of the range of selectively removable sulfhydryl protecting groups required, are unsymmetrical directed methods of disulfide bridge formation (Barany and Merrifield, 1979; Hiskey, 1981). For example, Cys(Acm) or Cys(Trt) residues in peptides can be reacted with methoxy- or ethoxycarbonylsulfenyl chloride to form Cys(Scm) or Cys(Sce) residues, respectively, which are attacked by the free thiol of a deprotected Cys residue to form a disulfide bond (Kullmann and Gutte, 1978; Mott et al., 1986; Ten Kortenaar and van Nispen, 1988). The 5'-(A'-methyl-Af-phenylcarbamoyl)sulfenyl (S-Snm) group is generated and used in the same manner, but is preferred due to its enhanced stability and the specificity of its reactions (Schroll and Barany, 1989; Chen et al., 1997). Disulfide bonds may also be formed by a free thiol attack of Cys(NBoc-NHBoc) residues, which are prepared by treatment of Cys with azodicarboxylic acid di-fBu ester (Romani et al., 1987). Cys(Npys) residues form disulfides upon reaction with deprotected Cys residues (Bernatowicz et al., 1986). Directed methods are particularly suited for linking two separate chains. As already alluded, directed methods require at least two classes of selectively removable Cys protecting groups; the same holds true for experiments aimed at controlled formation of multiple disulfide bridges by sequential pairwise deprotection/co-oxidations. An overriding concern with all such chemical approaches is to develop conditions that avoid scrambling (disulfide exchange). Oxidation by 12 in TFE allows for selective disulfide bond formation between Cys(Trt) residues in the presence of Cys(Acm) residues; in DMF, I2 oxidation is preferred between Cys(Acm) residues in the presence of Cys(Trt) residues (Kamber et al., 1980). Direct I2 oxidation of Cys(Acm) or Cys(Trt) residues is particularly advantageous in that existing disulfides are not exchanged (Barany and Merrifield, 1979; Kamber et al., 1980; Hiskey, 1981; Gray et al., 1984; Atherton et al., 1985b; Ponsati et al., 1990b). Since the Acm group is essentially stable to HF, a Meb/ Acm combination of protecting groups facilitates selective disulfide formation in Boc chemistry (Gray et al., 1984; Tam et al., 1991a). The Tmob/Acm or Xan/Acm combinations work well for the same purpose in Fmoc chemistry (Munson and Barany, 1993; Hargittai and Barany, 1999). The alternative of carrying out deprotection/oxidation of the Cys residues while the peptide chain remains anchored to a polymeric support is of obvious interest, and has received attention. Such an approach takes advantage of pseudo-dilution, which is a kinetic phenomenon expected to favor facile intramolecular processes and thereby minimize dimeric and oligomeric by-products (Barany and
159
Merrifield, 1979; Andreu et al., 1994). Disulfide bond formation on peptide-resins has been demonstrated by air, K3Fe(CN)6, dithiobis(2nitrobenzoic acid), or diiodoethane oxidation of free sulfhydryls, direct deprotection/oxidation of Cys(Acm) residues by Tl(Tfa)3 or I 2 , direct conversion of Cys(Fm) residues by piperidine, and nucleophilic attack by a free sulfhydryl on either Cys(Npys) or Cys(Scm) (Gray et al., 1984; Mott et al., 1986; Buchta et al., 1986; Eritja et al., 1987; Ploux et al., 1987; Ten Kortenaar and van Nispen, 1988; GarciaEchcverria et al., 1989; Albericio et al., 1991a, 2000; Edwards et al., 1994). The most generally applicable and efficient of these methods is direct conversion of Cys(Acm) or Cys(Trt) residues by I2 (10 equiv. in DMF), Cys(Acm) residues by Tl(Tfa)3 (1.5 equiv. in DMF) (Albericio et al., 1991a; Edwards et al., 1994), and Cys(Fm) residues by (a) piperidine-DMF (1:1) for 3h at 25 C (Ponsati et al., 1990b; Albericio et al., 1991a) or (b) piperidine-DMF-2-mercaptoethanol (10:10:0.7) treatment for 1 h at 25 C, followed by air oxidation in pH 8.0 DMF for 1 h at 25 C (Albericio et al., 1991a). The best solid-phase yields were at least as good, and in some cases better than, the results from corresponding solution oxidations. An ingenious new application of the pseudo-dilution concept to the solid-phase-mediated preparation of intramolecular disulfides was reported recently (Annis et al., 1998). Ellman's reagent, 5,5'-dithiobis(2-m'trobenzoic acid), was bound through two sites to a suitable solid support (PEG-PS, modified Sephadex, or controlled-pore glass). The linear oligo(thiol) precursors are dissolved in acidic aqueous media, and treated with these solid-phase Ellman's reagents. An initial "capture" step ensures that the critical intramolecular displacement that leads to disulfide formation occurs while the intermediate is polymer supported, and thus sequestered from other species in solution that could lead to by-products. Upon completion of the oxidation, the product is readily obtained by a simple filtration, while the solid-phase oxidation reagent is readily recovered and can be recycled. Lactams Intrachain lactams are formed between the side chains of Lys or Orn and Asp or Glu to conformationally restrain synthetic peptides, with the goal of increasing biological potency and/or specificity (reviewed by Kates et al., 1994; see Seyfarth et al., 1995; Limal et al., 1998; and Hargittai et al., 2000 for recent examples). Lactams can also be formed via side-chain-to-head, sidc-chain-to-tail, or head-to-tail cyclization (Kates et al., 1994). The residues used to form intrachain lactams must be selectively side-chain-deprotected, while all side-chain protecting groups of other residues remain intact. Selective deprotection is best achieved by using orthogonal side-chain protection, such as Fmoc
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Synthetic Peptides
and Fm for Lys/Orn and Asp/Glu, respectively, in combination with a Boc/Bzl strategy (Felix et al., 1988a, b; Hruby et al., 1990). A more complicated, but equally efficient approach, is to use side-chain protection based on graduated acid lability (Schiller et al., 1985; Sugg et al., 1988; Hruby et al., 1990). In the Fmoc strategy, orthogonality is achieved with Aloe or Dde protection for Lys and Al or Dmab protection for Asp/Glu (Lyttle and Hudson, 1992; Kates et al., 1993b). Optimized automated conditions for removal of Aloc/Al groups are Pd(Ph3)4 (1 equiv.) in chloroform-acetic acid-NMM (37:2:1) for 2h at 25 C, followed by washing steps with a solution of 0.5% DIEA and 0.5% sodium diethyldithiocarbamate in DMF (Kates et al., 1993a). Cyclization is carried out most efficiently with BOP (3-6 equiv., 2h, 20 C) in the presence of DIEA (6-7.5 equiv.) while the peptide is still attached to the resin (Felix et al., 1988a; Plaue, 1990), taking advantage of the pseudo-dilution phenomenon discussed in the previous section. The three-dimensional orthogonal protection scheme of Fmoc/;Bu/ Al protecting groups is the strategy of choice for head-to-tail cyclizations (Trzeciak and Bannwarth, 1992; Kates et al., 1993b, 1994). In the cited examples, an amide linker is used for side-chain attachment of a C-lerminal Asp/Glu (which are converted to Asn/Gln) and the a-carboxyl group is protected as an allyl ester (Trzeciak and Bannwarth, 1992; Kates et al., 1993b). A more general way to achieve such on-resin head-to-tail cyclizations involves backbone amide linkage (BAL) (Jensen et al., 1998). For side-chain-to-head cyclizations, the Lys/Orn and Asp/Glu protecting groups should be either very acid sensitive or base labile. The A^-terminal residue (head) can simply be introduced as an A^-Boc or W-Fmoc derivative while the peptide-resin linkage and the other sidechain protecting groups are stable to dilute acid or carry a third dimension of orthogonality. Peptide Antigens For the production of antipeptide antibodies, the peptide must be attached to a carrier (van Regenmortel and Muller, 1999). The simplest, although not necessarily most effective, way to accomplish this is to make direct use of peptide-resins in which the side chains have been freed but where the anchoring linkage is stable to the appropriate deprotection conditions. Polyamide-type and polyethylene glycol-polystyrene resins have been applied according to this approach (Chanh et al., 1986; Kennedy et al., 1987; Goddard et al., 1988; Fischer et al., 1989; Bayer, 1991; Kanda et al., 1991), as have synthesis of the peptide directly on beaded cellulose (Englebretsen and Harding, 1994) or protein carriers (Hansen et al., 1993). Peptides may also be synthesized containing topographic immunogenic determinants
161
(Kaumaya et al., 1992) or on a scaffold that, following cleavage and deprotection, is used directly for immunization. The scaffold, consisting of branched Lys residues, is referred to as a multiple antigen peptide system (MAP) (Tarn, 1988; Tarn and Lu, 1990). MAPs may be prepared by either Boc (Tarn, 1988) or Fmoc (Pessi et al., 1990; Drijfhout and Bloemhoff, 1991; Biancalana et al., 1991) chemistries. Yet another approach, which is possible when the three-dimensional structure of a discontinuous epitope is known, involves linking the key resides with spacers designed to confer geometric constraints that will favor native-like conformation (Borras et al., 1999). The more traditional (and still most common) methods for preparing peptide antigens start with free synthetic peptides that have been cleaved from the support and dcprotected. In one variation, the peptide is conjugated to a protein carrier, for example, bovine serum albumin, by coupling mediated by water-soluble carbodiimide (Deen et al., 1990). Alternatively, the peptide can be extended at the C- or Nterminus with a Cys residue, which is subsequently used to form a disulfide bridge with a free sulfhydryl on the carrier. Cystine formation is best achieved by the direct attack of a carrier protein thiol onto a peptide Cys(Npys) residue. Thiol groups are introduced on the carrier protein either by reduction to form free Cys (Albericio et al., 1989b; Ponsati et al., 1989) or by functionalization of Lys using S-acetylmercaptosuccinic anhydride (Drijfhout et al., 1988). Peptide-carrier conjugation is quantitated by monitoring the liberation of the Npys group at A. = 329 nm (Drijfhout et al., 1988). For peptides synthesized by Fmoc chemistry, Boc-Cys(Npys) is incorporated as the TV-terminal residue, thus avoiding additional piperidine treatments which would remove the Npys group (Albericio et al., 1989b).
Auxiliary Issues
This final section of the chapter covers a variety of practical considerations that researchers in SPPS should be aware of. Included are some potential side reactions that do not fit neatly with categories covered earlier, and ways to mitigate the extent of their occurrence. A logical culmination of expertise in SPPS is the successful preparation of long sequences, and we outline current achievements and possible ways to improve on them in the future. Diketopiperazine Formation The free Na-amlno group of an anchored dipeptide is poised for an acidor base-catalyzed intramolecular attack onto the C-terminal carbonyl (Gisin and Merrifield, 1972; Barany and Merrifield, 1979; Pedroso et al., 1986). Base deprotection (Fmoc chemistry) or neutralization
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Synthetic Peptides
(Boc chemistry; at the stage when the third residue is being coupled) can thus release a cyclic diketopiperazine while a hydroxymethylhandle leaving group remains on the resin. With residues that can form cis peptide bonds, for example, Gly, Pro, TV-methylamino acids, or D-amino acids, in either the first or second position of the (C > N) synthesis, diketopiperazine formation can be substantial (Barany and Merrifield, 1979; Pedroso et al., 1986; Gairi et al., 1990). It can also be a problem when the third amino acid residue is added to a BALanchored sequence (Jensen et al., 1998), or when an activated handle leaving group such as o-nitrobenzyloxy is used (Barany and Albericio, 1985). For most other sequences and/or handles, the problem can be adequately controlled. In Boc SPPS, the level of diketopiperazine formation can be suppressed either by removing the Boc group with HC1 and coupling the NMM salt of the third Boc-amino acid without neutralization (Suzuki et al., 1975), or else by deprotecting the Boc group with TFA and coupling the third Boc-amino acid in situ using BOP, DIE A, and HOBt without neutralization (Gairi et al., 1990). For susceptible sequences being addressed by Fmoc chemistry, the use of piperidine-DMF (1:1) deprotection for 5min (Pedroso et al., 1986), or deprotection for 2min with a 0.1 M solution in DMF of tetrabutylammonium fluoride ("quenched" by MeOH) (Ueki and Amemiya, 1987) has been recommended to minimize cyclization. Alternatively, the second and third amino acids may be coupled as a preformed A7-protected dipeptide, hence circumventing the diketopiperazineinducing deprotection/neutralization at the second amino acid. The steric hindrance of the 2-chlorotrityl linker may minimize diketopiperazine formation of susceptible sequences during Fmoc chemistry (Barlos et al., 1989a, b). In the case of BAL strategies, diketopiperazine formation is suppressed by incorporation of the penultimate residue as its jV-trityl (Trt) or A""-Ddz derivative, followed by selective deprotection with very dilute TFA (negligible premature loss of chains at this stage), and finally incorporation of the third residue as its A^-Fmoc derivative under in situ neutralization/ coupling conditions mediated by PyAOP/DIEA in DMF (Jensen et al., 1998).
For continuous-flow Fmoc SPPS, diketopiperazine formation is suppressed by deprotecting for l.Smin with piperidine-DMF (1:4) at an increased flow rate (15mL/min), washing for 3min with DMF at the same flow rate, and coupling the third Fmoc-amino acid in situ with BOP, NMM, and HOBt in DMF (MilliGen/Biosearch, 1990). For batchwise SPPS, rapid (a maximum of 5 rain) treatments by piperidine-DMF (1:1) should be used, followed by DMF washes and then in situ acylations mediated by BOP or HBTU (Pedroso et al., 1986).
Capping Some workers choose to "cap" unreacted chains, thereby substituting a family of terminated peptides for a family of deletion pep tides. In either case, these by-products must ultimately be separated from the desired product. Intentional termination of chains may be carried out when there is an indication of unreacted sites. In the simplest case, capping is carried out with reactive acetylating agents, such as acetic anhydride (Ac2O) or jV-acetylimidazole in the presence of tertiary base (Bayer et al., 1970; Stewart and Young, 1984). An alternative capping reagent is 2-sulfobenzoic acid cyclic anhydride (OSBA). Application of OSBA/tribenzylamine results in a negatively charged amino terminus; the desired product with the positively charged amino terminus may then be isolated after purification by ion-exchange chromatography (Drijfhout and Bloemhoff, 1988). In a reciprocal strategy, chains capped by acetylation are separated by acylating the jV-terminus of the desired peptide with suitable Fmoc derivatives. These derivatives include the 9-(2-sulfo)fluorenylmethyloxycarbonyl (Sulfmoc) group (Merrifield and Bach, 1978), tetrabenzo[a,c,g,i]fluorenyl-17-methyloxycarbonyl (Tbfmoc) group (Ramage and Raphy, 1992; Brown et al., 1993; Ramage et al., 1998), or modified 9-(hydroxymethyl)fluorene-4carboxylates, where the carboxylate at the 4-position is in turn derivatized with Lys, Glu, or 2-aminodecanoic acid (Ball et al., 1990, 1991). After an ion-exchange or reversed-phase purification which separates the completed peptide from all truncated ones, the modified Fmoc group is removed by base. A further option for the purification of Tbfmoc-modified peptide is to take advantage of their extremely high affinity for porous graphitized carbon (PGC). Another variation of the principle uses either -decylsulfonylethyloxycarbonyl or -hexadecylsulfonylethloxycarbonyl groups, homologues of the base-labile Msc group that are retained longer on reversed-phase chromatography by virtue of their hydrophobic arms (Garcia-Echeverria, 1995). Incidental capping has been found to occur during SPPS. Capping via acetylation has been attributed to ethyl acetate breakdown products in Boc-amino acids (Schnolzer et al., 1992). This side reaction may be avoided by lyophilizing Boc-amino acids just prior to use. Deletions The standard explanation for deletion peptides relates to incomplete couplings, which can usually be diagnosed by qualitative or quantitative monitoring tests (see above, Monitoring). In contrast, a chemically plausible side reaction that suggests a different reason for deletion peptides has been elucidated (Kent, 1983). Resin-bound aldehyde groups can form a Schiff s base with deprotected amino groups
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Synthetic Peptides
of the peptide chain. Those amino groups that are involved in a Schiff s base are prevented from acylation by the next incoming protected amino acid. The blockage is not permanent, that is, terminated peptides are not formed by this mechanism. Rather, ready amine exchange of the Schiff s base renders a different set of amino groups temporarily inaccessible at a subsequent coupling, and thus deletion peptides result. The side reaction is not minimized by capping steps (see above); in fact, amines blocked as Schiffs bases contribute to negative ninhydrin tests. The formation of aldehyde sites on polystyrene-resins can be minimized by using a strong acid Friedel-Crafts catalyst during the original functionalization step. In any event, it is crucial to quantify aldehyde concentrations of prepared or purchased resins (Kent, 1983). Acid-Sen si live Side Chains and Bonds Trp is quite sensitive to acid conditions, undergoing reactions with carbonium ions and molecular oxygen (see above, Cleavage). Synthesis of Trp-containing peptides is thus best approached via Fmoc chemistry, where acidolysis is kept at a minimum. Gramicidins A, B, and C (where either three or four of the 15 residues are Trp) have been synthesized efficiently by Fmoc chemistry; acid was avoided entirely throughout the synthesis and final nucleophilic cleavage was achieved with ethanolamine (Fields et al., 1988, 1989), while efficient Fmoc SPPS of indolicidin (which contains five Trp out of 13 residues) used an optimized TFA/scavenger mixture (reagent K) to prevent modification of Trp during acidolytic cleavage and side-chain deprotection (King et al., 1990; van Abel et al., 1995). Fmoc chemistry has also been suggested for incorporation of 2H-labeled amino acids, as the repetitive acidolyses of Boc chemistry can exchange out the 2 H label (Fields and Noble, 1990; Prosser et al., 1991). Finally, Fmoc chemistry may be the better choice for the synthesis of peptides containing the acid-labile Asp-Pro bond. SPPS of baboon /J-chorionic gonadotropin 109145, which contains two Asp-Pro bonds, was reported to be successful by Fmoc chemistry only, as Boc chemistry resulted in acid-promoted destruction of the Asp-Pro bonds (Wu et al., 1988). Imide Formation Asn residues can cyclize to form a succinimide, which can yield both a- and /J-Asp peptides. Imide formation is largely sequence dependent, with Asn-Gly showing the greatest tendency to rearrange (Stephenson and Clarke, 1989). Succinimide formation can be significant for peptides stored in solution (Stephenson and Clarke, 1989;
Patel and Borchardt, 1990a, b). In addition, peptides containing Asn (and even Gin) stored in the solid state with residual acid can undergo deamidation (Ten Kortenaar et al., 1990). Therefore, Asnand Gin-containing peptides should never be stored in a solid form with residual acid present, and samples stored in solution should be monitored carefully for deamidation and decomposition. Solvation and Aggregation Effective solvation of the peptide-resin is perhaps the most crucial condition for efficient chain assembly. Under proper solvent conditions, there is no decrease in synthetic efficiency up to 60 amino acid residues in Boc SPPS (Sarin et al., 1984). The ability of the peptideresin to swell increases with increasing peptide length due to a net decrease in free energy from solvation of the linear peptide chains (Sarin et al., 1980). Therefore, there is no theoretical upper limit to efficient amino acid couplings, provided that proper solvation conditions exist (Pickup et al., 1990). In practice, obtaining these conditions is not always straightforward. "Difficult couplings" during SPPS have been attributed to poor solvation of the growing chain by DCM. Infrared and NMR spectroscopies have shown that intermolecular /J-sheet aggregates are responsible for lowering coupling efficiencies (Live and Kent, 1983; Mutter et al., 1985; Ludwick et al., 1986). A scale of /J-sheet structure-stabilizing potential has been developed for Boc-amino acid derivatives (Narita and Kojima, 1989). Enhanced coupling efficiencies are seen upon the addition of polar solvents, such as DMF, TFE, NMP, and DMSO (Yamashiro et al., 1976; Live and Kent, 1983; Geiser et a]., 1988; Narita et al., 1989; Fields et al., 1990; Fields and Fields, 1991; Hyde et al., 1992; Miranda and Alewood, 1999). It has been suggested that chaotropic salts may be added to organic solvents in order to disrupt ,6-sheet aggregates (Stewart and Klis, 1990; Thaler et al., 1991). Also, using a lower substitution level of resin to avoid interchain crowding can improve the synthesis (Tarn and Lu, 1995). Aggregation also occurs in regions of apolar side-chain-protecting groups, sometimes resulting in a collapsed gel structure (Atherton et al., 1980; Atherton and Sheppard, 1985). In cases where aggregation occurs due to apolar side-chain-protecting groups, increased solvent polarity may not be sufficient to disrupt the aggregate. A relatively unstudied problem of Fmoc chemistry is that the lack of polar sidechain-protecting groups could, during the course of an extended peptide synthesis, inhibit proper solvation of the peptide-resin (Atherton et al., 1980; Fields and Fields, 1991; Bedford et al., 1992). To alleviate this problem, the use of solvent mixtures containing both a polar and nonpolar component, such as THF-NMP (7:13) or TFE-DCM
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(1:4), is recommended (Fields and Fields, 1991). The addition of DMSO (Fields and Fields, 1991; Hyde et al, 1992) or a solvent mixture containing detergents (Zhang et al., 1994) can be effective for disrupting such aggregates. The partial substitution or complete replacement of ?Bubased side-chain-protecting groups for carboxyl, hydroxyl, and amino side chains by more polar groups would also aid peptide-resin solvation (Atherton et al., 1980; Fields and Fields, 1991; Bedford et al., 1992). A different approach to circumvent potential aggregation is the use of reversible protection of the amino acid amide nitrogen. For this purpose, A^-Fmoc, jVa(2-Fmoc-hydroxy-4-methoxybenzyl) [Fmoc (Fmoc-Hmb)] derivatives have been prepared (Johnson et al., 1993). Formation of /?-sheet structure can be minimized by using amide nitrogen protection every sixth to eighth amino acid (Johnson et al., 1993; Hyde et al., 1994). Long Syntheses (>50 Residues) Many impressive long chain syntheses (>50 residues), including ribonuclease A (124 residues) (Gutte and Merrifield, 1971), human parathyroid hormone (84 residues) (Fairwell et al., 1983), interleukin-3 (140 residues) (Clark-Lewis et al., 1986), HIV-1 aspartyl protease (99 residues) (Schneider and Kent, 1988; Nutt et al., 1988; Wlodawer et al., 1989; de Lisle Milton et al., 1992), HIV-1 vpr protein (95 residues) (Gras-Masse et al., 1990), gonadotropin-releasing hormone precursor protein (92 residues) (Milton et al., 1992), Ala-interleukin-8 (77 residues) (Sueiras-Diaz and Horton, 1992), and insulin-like growth factor (70 residues) (Bagley et al., 1990), have been carried out using Boc methodology. There have also been successful long chain syntheses by Fmoc chemistry, including HIV-1 Tat protein (86 residues) (Frankel et al., 1989; Chun et al., 1990; Calnan et al., 1991), preprocecropin A (64 residues) (Pipkorn and Bernath, 1990), ubiquitin (76 residues) (Ramage et al., 1989; Ogunjobi and Ramage, 1990), yeast actin-binding protein 539-588 (50 residues) (King et al., 1990), pancreastatin (52 residues) (Funakoshi et al., 1988), human /6-chorionic gonadotropin 174 (Wu et al., 1989), HIV-1 nucleocapsid protein Ncp7 (72 residues) (de Rocquigny et al., 1991), the "minibody" (61 residues) (Bianchi et al., 1993), bovine pancreatic trypsin inhibitor and analogs (58 residues) (Ferrer et al., 1992; Ferrer et al., 1995; Pan et al., 1995), and "minicollagens" (76-121 residues) (C. G. Fields et al., 1993a, b; Grab et al., 1996). Both chemistries appear susceptible to the same difficult couplings (Meister and Kent, 1983; van Woerkom and van Nispen, 1991; J. Young et al., 1990), and side-by-side syntheses for moderate length chains (~30 residues) are comparable (Atherton et al., 1983; Wade et al., 1986). However, there are two additional considerations when using Fmoc, rather than Boc, chemistry for long chain syntheses. First, the
efficient solvation of hydrophobic side-chain-protecting groups used in conjunction with Fmoc chemistry, which was discussed previously, can become more critical for extended syntheses (Fields and Fields, 1991). Second, deprotection of the Fmoc group can proceed slowly in certain sequences (Atherton and Sheppard, 1985; Larsen et al., 1991). By monitoring deprotection as the synthesis proceeds, one can extend base deprotection times and/or alter solvation conditions as necessary (Ogunjobi and Ramage, 1990). The use of the DBU-piperidine mixture described earlier can also alleviate slow deprotection problems. Segment Condensation The advantages of segment condensation procedures for the synthesis of large peptides have been well described (Barany and Merrifield, 1979; Kaiser et al., 1989; Kneib-Cordonier et al., 1990), but to date there are relatively few examples for polymer-supported procedures. A significant aspect of the problem involves ready access to pure partially protected peptide segments which are needed as building blocks. The application of solid-phase synthesis to prepare the requisite intermediates depends on several levels of selectively cleavable protecting groups and anchoring linkages. Combination of the Boc/Bzl strategy with the 4-nitrobenzophenone oxime resin (DeGrado and Kaiser, 1982; Kaiser et ai., 1989; Lansbury et al., 1989; Sasaki and Kaiser, 1990), base-labile linkers (Liu et al., 1990; Albericio et al., 1991b), palladium-labile linkers (Kunz and Dombo, 1988; Guibe et al., 1989; Lloyd-Williams et al., 1991), or photolabile linkers (Rich and Gurwara, 1975; Albericio et al., 1987b), and of the Fmoc/fBu strategy with dilute acidlabile linkers (Mergler et al., 1988b; Barlos et al., 1989b; Atherton et al., 1990; Albericio and Barany, 1991; Barlos et al., 1991c) or photolabile linkers (Kneib-Cordonier et al., 1990) has proven successful for the generation of 7V"-amino and side-chain-protected segments with free C"*-carboxyl groups. Methods for subsequent solubilization and purification of the protected segments are nontrivial (Atherton et al., 1990; Lloyd-Williams et al., 1991; Albericio et al., 1997; Sakakibara, 1999), and beyond the scope of this review. Solid-phase assembly of protected segments has proven successful for a 44-residue model of apolipoprotein A-l (Nakagawa et al., 1985), human cardiodilatin 99-126 (Nokihara et al., 1989), human gastrin-1 (17 residues) (Kneib-Cordonier et al., 1990), Androctonus australis Hector toxin II (64 residues), A.-Cro DNA binding protein (66 residues) (Atherton et al., 1990), prothymosin a (109 residues) (Barlos et al., 1991c), ^-amyloid protein (42 residues) (Hendrix and Lansbury, 1992; Hendrix et al., 1992), the N-terminal domain of y-zein protein (48 residues) (Dalcol et al., 1995), a variety of triple-helical "minicollagens" (C. G. Fields et al., 1993a, b; Feng et al., 1996a, b, 1997),
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and a template assembled synthetic protein containing three peptide loops (Peluso et al., 1997). Polystyrene crosslinked with 1% 1,3divinylbenzene and polyamide resins have been shown to be suitable supports for solid-phase segment condensations (Albericio et al., 1989c). Individual rates for coupling segments are substantially lower than for activated amino acid species by stepwise synthesis, and there is always a risk of racemization at the C-terminus of each segment. Careful attention to synthetic design and execution may minimize these problems. Chemoselective Ligation As an alternative to the segment condensation approach, methods have been developed by which unprotected peptide fragments may be linked. Known as "chemoselective ligation," the initial strategies resulted in the formation of thioester or oxime bonds between peptide fragments. For example, reaction of a peptide bearing a C-terminal thioacid with a peptide containing an JV-terminal bromoacetyl group results in a synthetic protein product containing a thioester bond (Schnolzer and Kent, 1992; Muir et al., 1997). This approach has been used successfully to construct HTV-1 protease (Schnolzer and Kent, 1992), a four a-helix TASP molecule (Dawson and Kent, 1993), a folded /9-sandwich fibronectin domain model (Williams et al., 1994), and a tethered dimer of HIV-1 protease (Baca et al., 1995). A convenient linker that produces C-terminal thioacids in conjunction with Boc chemistry has been described (Canne et al., 1995). A variation of the thioester approach, in which a peptide C-terminal alkyl thiol is reacted with an W-terminal bromoacetyl or maleimido peptide to form a thioether bond, has been used to construct linked cytoplasmic domains from the aubp3 integrin (Muir et al., 1994), a ^-meander TASP molecule (Nefzi et al., 1995), and a 129-residue tripod protein (McCafferty et al., 1995). Linkers that produce the required modified C-terminal thiols have been described for both Boc (Englebretsen et al., 1995) and Fmoc (Ramage et al., 1996) chemistries. The other approach, in which peptides can be ligated to form oxime bonds (Tuchscherer, 1993; Rose, 1994), require either an aldehyde or N-terminal aminooxyacetyl groups. A peptide aldehyde can additionally be ligated to another peptide containing a weak nucleophilic base, such as hydrazide, TV-terminal Cys, or ^-terminal Thr, to form hydrazone, thiazolidine, and oxazolidine linkages, respectively (Rao and Tam, 1994; Spetzler and Tarn, 1995; Shao and Tam, 1995; Tam and Spetzler, 1997). Chemoselective ligation has been made further attractive by "native chemical ligation," in which an amide bond, rather than a thioester, thioether, or oxime bond, is generated between fragments (Muir et al., 1997); see figure 3-7. The original work employed a peptide bearing a
Figure 3-7. Native chemoselective ligation. Two fragments are assembled initially in the solid phase; in this specific example, one has a C-terminal thioacid and the other has an .V-terminal Cys residue. Both fragments are cleaved, side-chain deprotected, and purified. The thioacid is converted to a thioester, allowing for reaction of the two fragments in aqueous solution to form a thioester bond between them. Spontaneous intramolecular rearrangement of the thioester bond results in a native amide bond between the fragments (suggested mechanism in box). Modifications to the process making it possible to link additional peptide fragments have been described.
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C-terminal thioacid, which was converted to a 5-thio-2-nitrobenzoic acid ester and subsequently reacted with a peptide bearing an ^-terminal Cys residue (Dawson et al., 1994). An intermolecular thioester ensues which undergoes a spontaneous S > N acyl shift, leading to the naturally occurring amide bond and regeneration of the free sulfhydryl on Cys. The method has been further refined so that a relatively unreactive thioester can be used in the ligation reaction, which is then carried out in the presence of thiophenol and benzylmercaptan as thiol exchange reagents (Dawson et al., 1997), or in the presence of 2-mercaptoethanesulfonic acid, which has a less noxious odor (Ayers, et al., 1999). Thioesters are readily prepared by Boc chemistry using a thiopropionic acid spacer attached to BHA-resin (Vlattas et al., 1997) or the water-compatible support PEGA (Camarero et al., 1998). Since thioester linkages decompose in the presence of piperidine used in Fmoc chemistry, alternative deprotection conditions are needed. A solution containing 1-methylpyrrolidine, hexamethyleneimine, and HOBt in NMP-DMSO (1:1) has been suggested (Li et al., 1998; Aimoto, 1999). The problem can also be circumvented completely by introducing the thioester functionality after chain elongation. Very recent developments in this rapidly evolving field include a Fmoc/ BAL strategy to prepare peptide S-benzyl, 5-phenyl, and S-alkyl thioesters (Alsina et al., 1999b), and a novel safety-catch method (Ingenito et al., 1999). Analogues of the glycoprotein diptericin have been assembled (Shin et al., 1999), as well as solid-phase ligation demonstrated (Canne et al., 1999) using the safety-catch approach. It should be noted that amide bonds may also be generated by chemoselective ligation methods which result in thiazolidine linkages via an O -> TV acyl shift to form a hydroxymethyl-thiazolidine (Liu and Tarn, 1994, 1996). Summary The solid-phase method has made the synthesis of peptides widely accessible. The promise of sophisticated commercial automated instrumentation has been fulfilled. The vast majority of solid-phase peptide syntheses of targets containing up to 50 or so residues can be performed with high efficiencies by either Boc or Fmoc stepwise chemistry. For Boc chemistry, the protection strategy applies Bzl-type and compatible derivatives on the side chains, and for Fmoc chemistry, the corresponding is true with rBu and related derivatives. It is important, however, not to lose sight of the fact that each synthetic procedure has limitations, and that even in the hands of highly experienced workers, certain sequences defy facile preparation. Residue-specific side reactions that may lead to failed syntheses include (a) dehydration of Asn and Gin to the respective nitriles (see Protection Schemes and
1 71
Formation of Peptide Bond), (b) racemization of His (see Protection Schemes), (c) aspartimide formation from Asp (see Protection Schemes), (d) alkylation of Trp and Glu (see Cleavage), and (e) acidolysis of Asp-Pro bonds (see Auxiliary Issues). Diketopiperazine-forming sequences (see Auxiliary Issues) and difficult couplings between amino acids with branched side chains (see Auxiliary Issues) are additional, common sources of synthetic problems. In all of the aforementioned examples, deleterious side reactions or other difficulties can be somewhat minimized by careful examination of a peptide sequence prior to synthesis. Appropriate precautions as outlined in this chapter (alternative side-chain-protecting groups, use of additional reagents during coupling or cleavage, etc.) can then be taken. The maturation of high performance liquid chromatography (HPLC) during the 1980s was a major boon to modern peptide synthesis, because the resolving power of this technique facilitates removal of many of the systematic low-level by-products that accrue during chain assembly and upon cleavage. Nowadays, the homogeneity of synthetic materials should be checked by at least two chromatographic or electrophoretic techniques, such as, reversed-phase and ionexchange HPLC, and capillary zone electrophoresis. Also, determination of a molecular ion by mass spectrometry coupled with a mild ionization method is almost de rigeur for proof of structure. Synthetic peptides must be checked routinely for the proper amino acid composition, and in some cases sequencing data are helpful. Spectroscopic measurements, particularly through use of one and two-dimensional nuclear magnetic resonance (NMR), at the least provide insights on structure and purity, and can often give conformational information as well. Improvements in the chemistry of SPPS and allied methods have continued since the first edition of this chapter. The current updated version has touched on most of the key issues and discussed the recent status for each of them. Our enthusiasm and optimism for the importance of peptide synthesis as a tool to elucidate an ever-broadening array of biological processes remains undiminished. Abbreviations Abbreviations used for amino acids and the designations of peptides follow the rules of the IUPAC-IUB Commission of Biochemical Nomenclature in J. Biol. Chem. 247:977-983 (1972). The following additional abbreviations are used: AA, amino acid; Ac4/JGal, 2,3,4,6-tetra-O-acetyl-^-D-galactopyranosyl; Acm, acetamidomethyl; Ada, adamantyl; Al, allyl; Alloc, allyloxycarbonyl; BAL, backbone amide linker; Boc, ferf-butyloxycarbonyl; Boc-ON, 2-?err-butyloxycarbonyloximino-2-phenylacetonitrile; Bom, benzyloxymethyl; BOP,
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benzotriazolyl 7V-oxytrisdimethylaminophosphonium hexafluorophosphate; 2-BrZ, 2-bromobenzyloxycarbonyl; Bum, tert-butoxymethyl; Bzl. benzyl; cHex, cyclohexyl; CLEAR, cross-linked ethoxylate aerylate resin; Cs, cesium salt; 2,6-Cl2Bzl, 2,6-dichlorobenzyl; DBU, 1,8diazabicyclo[5.4.0]undec-7-ene; DCBC, 2,6-dichlorobenzoyl chloride; DCC, ./V^TV'-dicyclohexylcarbodiimide; DCE, 1,2-dichloroethane; DCM, dichloromethane (methylene chloride); Dde, l-(4,4-dimethyl2,6-dioxocyclohex-1 -ylidene)ethyl; DIEA, TV ,,/V-diisopropylethylamine; D1PCDI, A^JV'-diisopropylcarbodiimide; DMA, A^TV-dimethylacetamide; Dmab, N-[\-(4,4,-dimethyl-2,6-dioxocyclohexylidene)-3methylbutyl]aminobenzyl; DMAP, 4-dimethylaminopyridine; DMF, AÂf-dimethylformamide; DMSO, dimethyl sulfoxide; Dnp, 2,4-dinitrophenyl; Dod, 4-(4'-methoxybenzhydryl)phenoxyacetic acid; EDT, 1,2-ethanedithiol; Et 3 N, triethylamine; Fm, 9-fluorenylmethyl; Fmoc, 9-fluorenylmethyloxycarbonyl; Fmoc-OSu, fluorenylmethyl succinimidyl carbonate; HAL, 5-(4-hydroxymethyl-3,5-dimethoxyphenoxy)valeric acid; HATU, C>-(7-azabenzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate or A^-azabenzotriazol-l-yl) (dimethylaminoJmethylenej-A^-methylmethanaminium hexafluorophosphate JV-oxide; HBTU, 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate or /V-[(lH-benzotriazol-l-yl) (dimethylamino)methylene]-Ar-methylmethanaminium hexafluorophosphate A'-oxide; HF, hydrogen fluoride; Hmb, 2-hydroxy-4-methoxybenzyl; HMFA, 9-(hydroxymethyl)-2-fluoreneacetic acid; HMP, 4-hydroxymethylphenoxy; HOAc, acetic acid; HOAt, l-hydroxy-7azabenzotriazole; HOBt, 1-hydroxybenzotriazole; Hoc, cyclohexyloxycarbonyl; HOPip, -/V-hydroxypiperidine; HPLC, high performance liquid chromatography; Hpp, l-(4-nitrophenyl)-2-pyrazolin-5-one; HYCRAM, hydroxycrotonylaminomethyl; Hyp, 4-hydroxy-L-proline; MALDI, matrix-assisted laser desorption/ionization; MBHA, 4-methylbenzhydrylamine (resin); MCPBA, m-chloroperbenzoic acid; Meb, 4-methylbenzyl; Melm, 1-methylimidazole; MeOH, methanol; MMA, ^-methylmercaptoacetamide; Mmt, 4-methoxytrityl; Mob, 4-methoxybenzyl; MS, mass spectrometry; MSNT, 2,4,6-mesitylenesulfonyl-3-nitro-1,2,4-triazolide; Mtr, 4-methoxy-2,3,6-trimethylbenzenesulfonyl; Mts, mesitylene-2-sulfonyl; Mtt; 4-methyltrityl; NCA, 7V-carboxyanhydrides; NMM, A^-methylmorpholine; NMP, jV-methylpyrrolidone; Nonb, 3-nitro-4-aminomethylbenzoic acid; NPE, 4-(2-hydroxyethyl)-3-nitrobenzoic acid; Npp, 3-methyl-l(4-nitrophenyl)-2-pyrazolin-5-one; N.R., not reported; Nvoc, 6-nitroveratryloxycarbonyl (4,5-dimethoxy-2-nitrobenzyloxycarbonyl): ODhbt, l-oxo-2-hydroxydihydrobenzotriazine; ONb, 2-nitrobenzyl ester; ONo, 2-nitrophenyl; ONp, 4-nitrophenyl; OPfp, pentafluorophenyl; Orn, ornithine; OSu, ./V-hydroxysuccinimidyl; OTDO, 2,5-diphenyl-2,3-dihydro-3-oxo-4-hydroxythiophene dioxide; PAB,
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4-alkoxybenzyl; PAL, 5-4-aminomethyl-3, 5-dimethoxyphenoxy)valeric acid; PAM, phenylacetamidomethyl; Pbf, 2,2,4,6,7-pentamethyldihydro-benzofuran-5-sulfonyl; PEG, polyethylene glycol; PEGA, polyethylene glycol dimethyl acrylamide; PGC, porous graphitized carbon; Pmc, 2,2,5,7,8-pentamethylchroman-6-sulfonyl; Pnp, 3-phenyl-l-(4-nitrophenyl)-2-pyrazolin-5-one; PSA, preformed symmetrical anhydride; PyBOP, benzotriazole- 1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate; SASRIN, 2-methoxy-4-alkoxybenzyl alcohol; Scm, S-carboxymethylsulfcnyl; Snm, S'-CJV-methyl-jV-phenylcarbamoyl)sulfenyl; SPPS, solid-phase peptide synthesis; SfBu, tertbutylsulfenyl; Tacm, trimethylacetamidomethyl; TASP, template assembled synthetic protein; TBDMS, /err-butyldimethylsilyl; /Bu, tert-buly\; TES, triethylsilane; TFA, trifluoroacetic acid; TFE, 2,2,2trifluoroethanol; TFMSA, trifluoromethanesulfonic acid; TIS, triisopropylsilane; Tl(Tfa)3, thallium (III) trifluoroacetate; Tmob, 2,4,6-trimethoxybenzyl; TMSBr, trimethylsilyl bromide; TMSOTf, trimethylsilyl trifluoromethanesulfonate; Tos, 4-toluenesulfonyl; Trt, triphenylmethyl; UNCA, urethane-protected ,/V-carboxyanhydride; XAL, 5-(9-aminoxanthen-2-oxy)valeric acid; Xan, 9-xanthenyl; Z, benzyloxycarbonyl. Amino acid symbols denote the L-configuration where applicable, unless indicated otherwise.
ACKNOWLEDGMENTS This revised chapter is re-dedicated to Professors Miklos Bodanzsky and Bruce Merrifield in recognition of the high standards they have set in the development of peptide chemistry. We wish to thank Drs. Fernando Albericio, Robert Hammer, Derek Hudson, Steven Kates, Stephen Kent, and Ken Otteson for helpful discussions and unpublished results, and Dr. Zhenping Tian for his contributions to the earlier edition. Work in the authors' laboratories has been supported over the years by NIH (CA 77402, HL 62427, and AR 01929 to G.B.F. and OM 28934, 42722, 43522, and 51628 to G.B.). References Abdelmoty, I., Albericio, F., Carpino, L. A., Foxman, B. M., and Kates, S. A. (1994). Structural studies of reagents for peptide bond formation:Crystal and molecular structures of HBTU and HATU. Lett. Peptide Sci. 1:5767. Adams, 3. H., Cook, R. M., Hudson, D., Jammalamadaka, V., Lyttle, M. H., and Songster, M. F. (1998a). Improved supports and methods for solid phase mediated transformation. In Innovations and Perspectives, R. Epton, ed., Birmingham, UK, Mayflower Scientific Ltd., pp. 23-30. Adams, J. H., Cook, R. M., Hudson, D. Jammalamadaka, V., Lyttle, M. H., and Songster. M. F. (1998b). A reinvestigation of the preparation, properties and applications of aminomethyl and 4-methylbenzhydrylamine polystyrene resins. J. Org. Chem. 63:3706-3716.
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Adamson, J. G., Blaskovich, M. A., Groenevelt, H., and Lajoie, G. A. (1991). Simple and convenient synthesis of /erf-butyl ethers of Fmoc-serine, Fmoc-threonine, and Fmoc-tyrosine. J. Org. Chem. 56:3447-3449. Ahmed, A. K., Schaffer, S. W., and Wetlaufer, D. B. (1975). Nonenzymic reactivation of reduced bovine pancreatic ribonuclease by air oxidation and by glutathione oxidoreduction buffers. J. Biol. Chem. 250:8477-8482. Aimoto, S. (1999). Polypeptide synthesis by the thioester method. Biopolymers (Pept. Sci.) 51:247-265. Akaji, K., Fujii, N., Tokunaga, F., Miyata, T., Iwanaga, S., and Yajima, H. (1989). Studies on peptides CLXVIII: Syntheses of three peptides isolated from horseshoe crab hemocytes, tachyplesin I, tachyplesin II, and polyphemusin I. Chem. Pharm. Bull. 37:2661-2664. Akaji, K., Tanaka, H., Itoh, H., Imai, J., Fujiwara, Y., Kimura, T., and Kiso, Y. (1990a). Fluoren-9-ylmethyloxycarbonyl (Fmoc) amino acid chloride as an efficient reagent for anchoring Fmoc amino acid to 4-alkoxybenzyl alcohol resin. Chem. Pharm. Bull. 38:3471-3472. Akaji, K., Yoshida, M., Tatsumi, T., Kimura, T., Fujiwara, Y., and Kiso, Y. (1990b). Tetrafluoroboric acid as a useful deprotecting reagent in Fmocbased solid-phase peptide synthesis. J. Chem. Soc., Chem. Commun. 288290. Akaji, K., Tatsumi, T., Yoshida, M., Kimura, T., Fujiwara, Y., and Kiso, Y. (1991). Synthesis of cystine-peptide by a new disulphide bond-forming reaction using the silyl chloride-sulphoxide system. J. Chem. Soc., Chem. Commun. 167-168. Albericio, F., and Barany, G. (1984). Application of A^jV-dimethylformamide dineopentyl acetal for efficient anchoring Na-9-fluorenylmethyloxycarbonylamino acids as p-alkoxybenzyl esters in solid-phase peptide synthesis. Int. J. Peptide Protein Res. 23:342-349. Albericio. F., and Barany. G. (1985). Improved approach for anchoring N"-9fluorenylmethyloxycarbonylamino acids as p-alkoxybenzyl esters in solidphase peptide synthesis. Int. J. Peptide Protein Res. 26:92-97. Albericio, F., and Barany, G. (1987). Mild, orthogonal solid-phase peptide synthesis: Use of ^"-dithiasuccinoyl (Dts) amino acids and N-(iso-propyldithio)carbonylproline, together with p-alkoxybenzyl ester anchoring linkages. Int. J. Peptide Protein Res. 30:177-205. Albericio, F., and Barany, G. (1991). Hypersensitive acid-labile (HAL) tris(alkoxy)benzyl ester anchoring for solid-phase synthesis of protected peptide segments. Tetrahedron Lett. 32:1015-1018. Albericio, F., Grandas, A., Porta, A., Pedroso, E., and Giralt, E. (1987a). One-pot synthesis of S-acetamidomethyl-TV-fluorenylmethoxycarbonylL-cysteine (Fmoc-Cys(Acm)-OH). Synthesis 271-272. Albericio, F., Nicolas, E., Josa, J., Grandas, A., Pedroso, E., Giralt, E., Granier, C., and van Rietschoten, J. (1987b). Convergent solid phase peptide synthesis V: Synthesis of the 14, 3234, and 53-59 protected segments of the toxin II of Androctonus australis Hector. Tetrahedron 43:5961-5971. Albericio, F., Ruiz-Gayo, M., Pedroso, E., and Giralt, E. (1989a). Use of polystyrene-1 % divinylbenzene and Kel-F-g-styrene for the simultaneous synthesis of peptides. Reactive Polymers 10:259-268.
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Albericio, F., Andreu, D., Giralt, E., Navalpotro, C., Pedroso, E., Ponsati, B., and Ruiz-Gayo, M. (1989b). Use of the Npys thiol protection in solid phase peplide synthesis. Int. J. Peptide Protein Res. 34:124-128. Albericio, F., Pons, M., Pedroso, E., and Giralt, E. (1989c). Comparative study of supports for solid-phase coupling of protected-peptide segments. J. Org. Chem. 54:360 366. Albericio, F., Kneib-Cordonier, N., Biancalana, S., Gera, L., Masada, R. I., Hudson, D., and Barany, G. (1990a). Preparation and application of the 5-(4-9-fluorenylmethyloxycarbonyl)aminomethyl-3,5-dimethoxyphenoxy) valeric acid (PAL) handle for the solid-phase synthesis of C-terminal peptide amides under mild conditions. J. Org. Chem. 55:3730-3743. Albericio, F., Van Abel, R., and Barany, G. (1990b). Solid-phase synthesis of peptides with C-terminal asparagine or glutamine. Int. J. Peptide Protein Res. 35:284-286. Albericio, F., Nicolas, E., Rizo, J., Ruiz-Gayo, M., Pedroso, E., and Giralt, E. (1990c). Convenient syntheses of fluorenylmethyl-based side chain derivatives of glutamic and aspartic acids, lysine, and cysteine. Synthesis 119122. Albericio, F., Hammer, R. P., Garcia-Echeverria, C., Molins, M. A., Chang, J. L., Munson, M. C., Pons, M., Giralt, E., and Barany, G. (1991a). Cyclization of disulfide-containing peptides in solid-phase synthesis. Int. J. Peptide Protein Res. 37:402^113. Albericio, F., Giralt, E., and Eritja, R. (1991b). NPE-resin, a new approach to the solid-phase synthesis of protected peptides and oligonucleotides II: Synthesis of protected peptides. Tetrahedron Lett. 32:1515-1518. Albericio, F., Barany, G., Fields, G. B., Hudson, D., Kates, S. A., Lyttle, M. H., and Sole, N. A. (1993). Allyl-based orthogonal solid-phase peptide synthesis. In Peptides 1992, C. H. Schneider and A. N. Eberle, eds., Leiden, Escom, pp. 191-193. Albericio, F., Lloyd-Williams, P., and Giralt, E. (1997). Convergent solidphase peptide synthesis. Methods Enzymol. 289:313-336. Albericio, F., Annis, I., Royo, M., and Barany, G. (2000). Preparation and handling of peptides containing methionine and cysteine. In Fmoc Solid Phase Peptide Synthesis: A Practical Approach, W. C. Chan and P. D. White, eds., Oxford. Oxford University Press, pp. 77-114. Aletras, A., Barlos, K., Gatos, D., Koutsogianni, S., and Mamos, P. (1995). Preparation of the very acid-sensitive Fmoc-Lys(Mtt)-OH. Int. J. Peptide Protein Res. 45:488-500. Alewood, P., Alewood, D., Miranda, L., Love, S., Meutermans, W., and Wilson, D. (1997). Rapid in situ neutralization protocols for Boc and Fmoc solid-phase chemistries. Methods Enzymol. 289:14-29. Al-Obeidi, F., Sanderson, D. G., and Hruby, V. J. (1990). Synthesis of ft- and y-fluorenylmethyl esters of respectively JV-Boc-L-aspartic acid and N"Boc-L-glutamic acid. Int. J. Peptide Protein Res. 35:215-218. Alsina, J., Barany, G., Albericio, F., and Kates, S. (1999a). Pyrrolidide formation as a side reaction during activation of carboxylic acids by phosphonium salt coupling reagents. Lett. Pept. Sci. 6:243-245. Alsina, J., Yokum, T. Y., Albericio, F., and Barany, G. (1999b). Backbone amide linker (BAL) strategy for ^V"-9-fluorenylmethoxycarbonyl (Fmoc)
176
Synthetic Peptides
solid-phase synthesis of unprotected peptide /j-nitroanilides and thioesters. J. Org. Chem. 64:8761-8769. Ambrosius, D.. Casaretto, M., Gerardy-Schahn, R., Saunders, D., Brandenburg, D., and Zahn, H. (1989). Peptide analogues of the anaphylatoxin C3a; synthesis and properties. Biol. Chem. Hoppe-Seyler 370:217227. Andreu, D., Albericio, F., Sole, N. A., Munson, M. C., Ferrer, M., and Barany, G. (1994). Formation of disulfide bonds in synthetic peptides and proteins. In Methods in Molecular Biology, Vol. 35, M. W. Pennington and B. M. Dunn, eds., Totowa, N.J., Humana Press, pp. 91-169. Annis, I., Hargittai, B., and Barany, G. (1997). Disulfide bond formation in peptides. Methods Enzymol. 289:198-221. Annis, I., Chen, L., and Barany, G. (1998). Novel solid-phase reagents for facile formation of intramolecular disulfide bridges in peptides under mild conditions. J. Am. Chem. Soc. 120:7226-7238. Anwer, M. K., and Spatola, A. F. (1980). An advantageous method for the rapid removal of hydrogenolysable protecting groups under ambient conditions; synthesis of leucine-enkephalin. Synthesis 929 932. Anwer, M. K., and Spatola, A. F. (1983). Quantitative removal of a pentadecapeptide ACTH fragment analogue from a Merrifield resin using ammonium formate catalytic transfer hydrogenation: Synthesis of [Asp25,Ala26,Gly27,Gln30]-ACTH-(25-39)-OH. J. Am. Chem. Soc. 48:3503-3507. Applied Biosystems, Inc. (1989a). Removal of 2,4-dinitrophenyl (Dnp) protection from peptides synthesized with Boc-His(Dnp). Peptide Synthesizer User Bulletin 28, Foster City, Calif., Applied Biosystems, Inc. Applied Biosystems, Inc. (1989b). Use of the ninhydrin reaction to monitor Fmoc solid-phase peptide synthesis. Peptide Synthesizer User Bulletin 29, Foster City, Calif., Applied Biosystems, Inc. Applied Biosystems, Inc. (1992). Model 431A Peptide Synthesizer User's Manual, Version 2.0, Foster City, Calif, Applied Biosystems, Inc. Arad, O., and Houghten, R. A. (1990). An evaluation of the advantages and effectiveness of picric acid monitoring during solid phase peptide synthesis. Peptide Res. 3:42-50. Arendt, A., Palczewski, K., Moore, W. T., Caprioli, R. M., McDowell, J. H., and Hargrave, P. A. (1989). Synthesis of phosphopeptides containing Ophosphoserine or 0-phosphothreonine. Int. J. Peptide Protein Res. 33:468^176. Arendt, A., McDowell, J., Hugh, J., and Hargrave, P. A. (1993). Optimization of peptide synthesis on polyethylene rods. Peptide Res. 6:346-352. Arshady, R., Atherton, E., Clive, D. L. J., and Sheppard, R. C. (1981). Peptide synthesis, part 1: Preparation and use of polar supports based on poly(dimethylacrylamide). J. Chem. Soc. Perkin Trans. 1:529-537. Atherton, E., and Sheppard, R. C. (1985). Detection of problem sequences in solid phase synthesis. In Peptides: Structure and Function, C. M. Deber, V. J. Hruby, and K. D. Kopple, eds., Rockford, 111., Pierce Chemical Co., pp. 415^118. Atherton, E., and Sheppard, R. C. (1989). Solid Phase Peptide Synthesis: A Practical Approach, Oxford, IRL Press.
Principles and Practice of Solid-Phase Peptide Synthesis 1 77
Atherton, E., Fox, H., Harkiss, D., Logan. C. J., Sheppard, R. C., and Williams, B. J. (1978a). A mild procedure for solid phase peptide synthesisiUse of fluorenylmethoxycarbonylamino-acids. J. Chem. Soc., Chem. Commun. 537-539. Atherton, E., Fox, H., Harkiss, D., and Sheppard, R. C. (1978b). Application of polyamide resins to polypeptide synthesis: An improved synthesis of /Jendorphin using fluorenylmethoxycarbonylamino-acids. J. Chem. Soc., Chem. Commun. 539-540. Atherton, E., Bury, C., Sheppard, R. C., and Williams, B. J. (1979). Stability of fluorenylmethoxycarbonylamino groups in peptide synthesis: Cleavage by hydrogenolysis and by dipolar aprotic solvents. Tetrahedron Lett. 3041 -3042. Atherton E., Woolley, V., and Sheppard, R. C. (1980). Internal association in solid phase peptide synthesis: Synthesis of cytochrome C residues 66-104 on polyamide supports. J. Chem. Soc., Chem. Commun. 970-971. Atherton, E., Benoiton, N. L., Brown, E., Sheppard, R. C., and Williams, B. J. (1981a). Racemisation of activated, urethane-protected amino-acids by pdimethylaminopyridine: Significance in solid-phase peptide synthesis. J. Chem. Soc., Chem. Commun. 336-337. Atherton, E., Brown, E., Sheppard, R. C., and Rosevear, A. (1981b). A physically supported gel polymer for low pressure, continuous flow solid phase reactions: Application to solid phase peptide synthesis. J. Chem. Soc., Chem. Commun. 1151-1152. Atherton. E., Logan, C. J., and Sheppard, R. C. (1981c). Peptide synthesis, part 2: Procedures for solid-phase synthesis using 7V"-fluorenylmethoxycarbonylamino-acids on polyamide supports:Synthesis of substance P and of acyl carrier protein 65-74 decapeptide. J. Chem. Soc. Perkin Trans. 1:538-546. Atherton, E., Caviezel, M., Fox, H., Harkiss, D., Over, H.. and Sheppard, R. C. (1983). Peptide synthesis, part 3: Comparative solid-phase syntheses of human ^-endorphin on polyamide supports using r-butoxycarbonyl and fluorenylmethoxycarbonyl protecting groups. J. Chem. Soc. Perkin 1'rans. 1:65-73. Atherton, E., Cammish, L. E., Goddard, P., Richards, J. D., and Sheppard, R. C. (1984). The Fmoc-polyamide solid phase method: New procedures for histidine and arginine. In Peptides 1984, U. Ragnarsson, ed., Stockholm, Almqvist & Wikscll, pp. 153-156. Atherton, E., Pinori, M., and Sheppard, R. C. (1985a). Peptide synthesis, part 6: Protection of the sulphydryl group of cysteine in solid-phase synthesis using Na-fluorenylmethoxycarbonylamino acids: Linear oxytocin derivatives. J. Chem. Soc. Perkin Trans. 1:2057-2064. Atherton, E., Sheppard, R. C., and Ward, P. (1985b). Peptide synthesis, part 7: Solid-phase synthesis of conotoxin Gl. J. Chem. Soc. Perkin Trans. 1:2065-2073. Atherton, E., Cameron, L. R., and Sheppard, R. C. (1988a). Peptide synthesis, part 10: Use of pentafluorophenyl esters of fluorenylmethoxycarbonylamino acids in solid phase peptide synthesis. Tetrahedron 44:843-857. Atherton, E., Holder, J. L., Meldal, M., Sheppard, R. C., and Valerio, R. M. (1988b). Peptide synthesis, part 12:3,4-Dihydro-4-oxo-l,2,3-benzotriazin-
178
Synthetic Peptides
3-yl esters of fluorenylmethoxycarbonyl amino acids as self-indicating reagents for solid phase synthesis. J. Chcm. Soc. Perkin Trans. 1:28872894. Atherton, E., Cameron, L. R., Cammish, L. E., Dryland, A., Goddard, P., Priestley, G. P., Richards, J. D., Sheppard, R. C., Wade, J. D., and Williams, B. J. (1990). Solid phase fragment condensationthe problems. In Innovation and Perspectives in Solid Phase Synthesis, R. Epton, ed., Birmingham, UK, Solid Phase Conference Coordination, Ltd., pp. 11 25. Atherton, E., Hardy, P. M., Harris, D. E., and Matthews, B. H. (1991). Racemization of C-terminal cysteine during peptide assembly. In Peptides 1990, E. Giralt and D. Andreu, eds., Leiden, Escom, pp. 243-244. Ayers, B., Blaschke, U. K., Camarero, J. A., Cotton, G. J., Holford, M., and Muir, T. W. (1999). Introduction of unnatural amino acids into proteins using expressed protein ligation. Biopolymers (Pept. Sci.) 51:343-354. Baca, M., Muir, T. W., Schnol/er, M., and Kent, S. B. H. (1995). Chemical ligation of cysteine-containing peptides: Synthesis of a 22 kDa tethered dimer of HIV-1 protease. J. Am. Chem. Soc, 117:1881-1887. Bagley, C. J., Otteson, K. M., May, B. L., McCurdy, S. N., Pierce, L., Ballard, F. J., and Wallace, J. C. (1990). Synthesis of insulin-like growth factor J using A'-methyl pyrrolidinone as the coupling solvent and trifluromethane sulphonic acid cleavage from the resin. Int. J. Peptide Protein Res. 36:356-361. Baker, P. A., Coffey, A. F., and Epton, R. (1990). Accelerated continuous flow ultra-high load solid (gel) phase oligopeptide assembly. In Innovation and Perspectives in Solid Phase Synthesis, R. Epton, ed., Birmingham, UK, Solid Phase Conference Coordination, Ltd., pp. 435-440. Ball, H. L., Grecian, C., Kent, S. B. H., and Mascagni, P. (1990). Affinity methods for purifying large synthetic peptides. In Peptides: Chemistry, Structure and Biology, J. E. Rivier and G. R. Marshall, eds., Leiden, Escom, pp. 435^436. Ball, H. L., Kent, S. B. H., and Mascagni, P. (1991). Selective purification of large synthetic peptides using removable chromatographic probes. In Peptides 1990, E. Giralt and D. Andreu, eds., Leiden, Escom, pp. 323325. Barany, G. (1999). Synthetic approaches to elucidate roles of disulfide bridges in peptides and proteins. In Peptides: Frontiers of Peptide Science, J. P. Tarn and P. T. P. Kaumaya, eds., Dordrecht, Kluwer Academic Publishers, pp. 226-228. Barany, G., and Albericio, F. (1985). A three-dimensional orthogonal protection scheme for solid-phase peptide synthesis under mild conditions. J. Am. Chem. Soc. 107:4936-4942. Barany, G., and Albericio, F. (1991). Peptide synthesis for biotechnology in the 1990's. In Biotechnology International 1990/1991, K. North, ed., London, Century Press Ltd., pp. 155-163. Barany, G., and Kempe, M. (1997). The context of solid-phase synthesis. In A Practical Guide to Combinatorial Chemistry, A. W. Czarnik and S. H. DeWitt, eds., Washington, D.C., American Chemical Society, pp. 51-97. Barany, G., and Merrifield, R. B. (1973). An ATP-binding peptide. Cold Spring Harbor Symp. Quant. Biol. 37:121-125.
179
Barany, G., and Merrifield, R. B. (1977). A new amino protecting group removable by reductioniChemistry of the dithiasuccinoyl (Dts) function. J. Am. Chem. Soc. 99:7363-7365. Barany, G., and Merrifield, R. B. (1979). Solid-phase peptide synthesis. In The Peptides, Vol. 2, E. Gross and J. Meienhofer, eds., New York, Academic Press, pp. 1-284. Barany, G., Kneib-Cordonier, N., and Mullen, D. G. (1987). Solid-phase peptide synthesis: A silver anniversary report. Int. J. Peptide Protein Res. 30:705-739. Barany, G., Kneib-Cordonier, N., and Mullen, D. G. (1988). Polypeptide synthesis, solid-phase method. In Encyclopedia of Polymer Science and Engineering, Vol. 12, 2nd edn., J. I. Kroschwitz, ed., New York, John Wiley and Sons, pp. 811-858. Barany, G., Albericio, F., Biancalana, S., Bontems, S. L., Chang, J. L., Eritja, R., Ferrer, M., Fields, C. G., Fields, G. B., Lyltle, M. H., Sole, N. A., Tian, Z., Van Abel, R. J., Wright, P. B., Zalipsky, S., and Hudson, D. (1992). Biopolymer syntheses on novel polyethylene glycol-polystyrene (PEG-PS) graft supports. In Peptides: Chemistry and Biology, J. A. Smith and J. E. Rivier, eds., Leiden, Escom, pp. 603-604. Barany, G., Albericio, F., Kates, S. A., and Kempe, M. (1997). Polyethylene glycol) containing supports for solid phase synthesis of peptides and combinatorial organic libraries. In Poly(ethylene glycol): Chemistry and Biological Applications, J. M. Harris and S. Zalipsky, eds., ACS Symposium Series 680, Washington, D.C., American Chemical Society, pp. 239-264. Bardaji, E., Torres. J. L., Clapes, P., Albericio, F., Barany, G., Rodriguez, R. E., Sacristan, M. P., and Valencia, G. (1991). Synthesis and biological activity of O-glycosylated morphiceptin analogues. J. Chem. Soc. Perkin Trans. 1:1755-1759. Barlos, K., Papaioannou, D., and Theodoropoulos, D. (1982). Efficient "onepot" synthesis of N-trityl amino acids. J. Org. Chem. 47:1324-1326. Barlos, K., Mamos, P., Papaioannou, D., Patrianakou, S., Sanida, C., and Schafer, W. (1987). Einsatz von Trt- und Fmoc-gruppen zum schutz polyfunktioneller a-aminosauren. Liebigs Ann. Chem. 1025-1030. Barlos, K., Gatos, D., Hondrelis, J., Matsoukas, J., Moore, G. J., Schafer, W., and Sotiriou, P. (1989a). Darstellung neuer saureempfmdlicker harze vom sek.-alkohol-typ und ihre anwendung zur synthcsc von peptiden. Liebigs Ann. Chem. 951-955. Barlos, K., Gatos, D., Kallitsis, J., Papaphotiu, G., Sotiriu, P., Wenqing, Y., and Schafer, W. (1989b). Darstellung geschiitzter peptid-fragmente unter einsatz substituierter triphenylmethyl-harze. Tetrahedron Lett. 30:39433946. Barlos, K., Chatzi, O., Gatos, D., and Stavropoulos, G. (199la). 2Chlorotrityl chloride resin: Studies on anchoring of Fmoc-amino acids and peptide cleavage. Int. J. Peptide Protein Res. 37:513-520. Barlos, K., Gatos, D., Koutsogianni, S., Schafer, W., Stavropoulos, G., and Wenqing, Y. (1991b). Darstellung und einsatz von N-Fmoc-O-Trt-hydroxyaminosauren zur "solid phase" synthese von peptiden. Tetrahedron Lett. 32:471-474.
180
Synthetic Peptides
Barlos, K., Gatos, D., and Schiifer. W. (1991c). Synthesis of prothymosin a (ProTa)a protein consisting of 109 amino acid residues. Angew. Chcm. Int. Ed. Engl. 30:590-593. Barlos, K., Chatzi, O., Gatos, D., Stavropoulos, G., and Tsegenidis, T. (1991d). Fmoc-His(Mmt)-OH und Fmoc-His(Mtt)-OH. Tetrahedron Lett. 32:475-478. Barlos, K., Gatos, D., Hatzi, O., Koch, N., and Koutsogianni, S. (1996). Synthesis of the very acid-sensitive Fmoc-Cys(Mmt)-OH and its application in solid-phase peptide synthesis. Int. J. Peptide Protein Res. 47:148-153. Barlos, K., Gatos, D., and Koutsogianni, S. (1998). Fmoc/Trt-amino acids: Comparison to Fmoc/tBu-amino acids in peptide synthesis. J. Peptide Res. 51:194-200. Barton, M. A., Lemieux. R. U., and Savoie, J. Y. (1973). Solid-phase synthesis of selectively protected peptides for use as building units in the solidphase synthesis of large molecules. J. Am. Chem. Soc. 95:4501-4506. Bayer, E. (1991). Towards the chemical synthesis of proteins. Angew. Chem. Int. Ed. Engl. 30:113-129. Bayer, E., and Rapp, W. (1986). New polymer supports for solid-liquid-phase peptide synthesis. In Chemistry of Peptides and Proteins, Vol. 3, W. Voelter, E. Bayer, Y. A. Ovchinnikov, and V. T. Ivanov, eds., Berlin, Walter de Gruyter & Co., pp. 3-8. Bayer, E., Albert, K., Willisch, H., Rapp, W., and Hemmasi, B. (1990). 13C NMR relaxation times of a tripeptide methyl ester and its polymer-bound analogues. Macromolecules 23:1937-1940. Bayer, E., Eckstein, H., Hagele, K., Konig, W. A., Briining, W., Hagenmaier, H., and Parr, W. (1970). Failure sequences in the solid phase synthesis of polypeptides. J. Am. Chem. Soc. 92:1735-1738. Beacham, J., Bentley, P. H., Kcnner, G. W., MacLeod, J. K., Mendive, J. J., and Sheppard, R. C. (1967). Peptides part XXV: The structure and synthesis of human gastrin. J. Chem. Soc. (C) 2520-2529. Beck, W., and Jung, G. (1994). Convenient reduction of S-oxides in synthetic peptides, lipopeptides and peptide libraries. Lett. Pept. Sci. 1:31-37. Beck-Sickinger, A. G., Diirr, H., and Jung, G. (1991). Semiautomated T-bag peptide synthesis using 9-fluorenylmethoxycarbonyl strategy and benzotriazol-1-yl-tetramethyluronium tetrafluoroborate activation. Peptide Res. 4:88-94. Becker, S., Atherton, E., and Gordon, R. D. (1989). Synthesis and characterization of yU-conotoxin Ilia. Eur. J. Biochem. 185:79-84. Bedford, J., Hyde, C., Johnson, T., Jun, W., Owen, D., Quibell, M., and Sheppard, R. C. (1992). Amino acid structure and "difficult sequences" in solid phase peptide synthesis. Int. J. Peptide Protein Res. 40:300-307. Belshaw, P. J., Mzengeza, S., and Lajoie, G. A. (1990). Chlorotrimethylsilane mediated formation of w-allyl esters of aspartic and glutamic acids. Synth. Commun. 20:3157-3160. Benoiton, N. L., and Chen, F. M. F. (1987). Symmetrical anhydride rearrangement leads to three different dipeptide products. In Peptides 1986, D. Theodoropoulos, ed., Berlin, Walter de Gruyter and Co., pp. 127-130. Berg, R. H., Almdal, K., Pedersen, W. B., Holm, A., Tarn, J. P., and Merrifield, R. B. (1989). Long-chain polystyrene-grafted polyethylene
181
film matrix: A new support for solid-phase peptide synthesis. J. Am. Chem. Soc. 111:8024 8026. Bergot, B. J., Noble, R. L., and Geiser, T. (1987). TFMSA/TFA cleavage and deprotection in SPPS. In Peptides 1986, D. Theodoropoulos, ed., Berlin, Walter de Gruyter and Co., pp. 97-101. Bernatowicz, M. S., Matsueda, R., and Matsueda, G. R. (1986). Preparation of Boc-[S-3-nitro-2-pyridinesulfenyl)]-cysteine and its use for unsymmetrical disulfide bond formation. Int. J. Peptide Protein Res. 28:107-112. Bernatowicz, M. S., Daniels, S. B., Coull, I. M., Kearney, T., Neves, R. S., Coassin, P. J., and Koster, H. (1990). Recent developments in solid phase peptide synthesis using the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group strategy. In Current Research in Protein Chemistry: Techniques, Structure, and Function, J. J. Villafranca, ed., San Diego, Academic Press, pp. 63-77. Bertho, J.-N., Loffet, A., Pinel, C., Reuther, F., and Sennyey, G. (1991). Amino acid fluorides: Their preparation and use in peptide synthesis. Tetrahedron Lett. 32:1303-1306. Biancalana, S., Hayes, T., Toll, L., and Hudson, D. (1991). Immunological and biological activity of octameric peptides prepared by Fmoc methodology. In Twelfth American Peptide Symposium Program and Abstracts, Cambridge, Mass., Massachusetts Institute of Technology, P-234. Bianchi, E., Sollazzo, M., Tramontano, A., and Pessi, A. (1993). Chemical synthesis of a designed /J-protein through the flow-polyamide method. Int. J. Peptide Protein Res. 41:385-393. Birr, C. (1978). Aspects of the Merrifield Peptide Synthesis, Heidelberg, Springer-Verlag. Birr, C. (1990a). The transition to solid-phase production of pharmaceutical peptides. Biochem. Soc. Trans. 18:1313-1316. Birr, C. (1990b). Scale-up and production potential of current SPPS strategies. In Innovation and Perspectives in Solid Phase Synthesis, R. Epton, ed., Birmingham, UK, Solid Phase Conference Coordination, Ltd., pp. 155-181. Birr, C., Lochinger, W., Stahnke, G., and Lang, P. (1972). Der a,a-dimethyl3.5-dimethoxybenzyloxycarbonyl (Ddz)-rest, eine photo- und saurelabile stickstoff-schutzgruppe fur die peptidchemie. Justus Liebigs Ann. Chem. 763:162-172. Blackburn, C. (2000). Solid supports for the synthesis of peptides and small molecules. In Solid-Phase Peptide Synthesis: A Practical Guide, S. A. Kates and F. Albericio, eds., New York, Marcel Dekker, pp. 197-273. Blankemeyer-Menge, B., Nimtz, M., and Frank, R. (1990). An efficient method for anchoring Fmoc-amino acids to hydroxyl-functionalised solid supports. Tetrahedron Lett. 31:1701-1704. Bloomberg, G. B., Askin, D., Gargaro, A. R., and Tanner, M. J. A. (1993). Synthesis of a branched cyclic peptide using a strategy employing Fmoc chemistry and two additional orthogonal protecting groups. Tetrahedron Lett. 34:4709-4712. Bodanszky, A., Bodanszky, M., Chandramouli. N., Kwei, J. Z., Martinez, J., and Tolle, J. C. (1980). Active esters of 9-fluorenylmethyloxycarbonyl amino acids and their application in the stepwise lengthening of a peptide chain. J. Org. Chem. 45:72-76.
182
Synthetic Peptides
Bodanszky, M., and Bodanszky, A. (1984). The Practice of Pep tide Synthesis, Berlin, Springer-Verlag. Bodanszky, M., and Kwei, J. Z. (1978). Side reactions in peptide synthesis VII: Sequence dependence in the formation of aminosuccinyl derivatives from /j-benzyl-aspartyl peptides. Int. J. Peptide Protein Res. 12:69-74. Bodanszky, M., Funk, K. W., and Fink, M. L. (1973). o-Nitrophenyl esters of ?er?-butyloxycarbonylamino acids and their application in the stepwise synthesis of peptide chains by a new technique. J. Org. Chem. 38:3565-3570. Bodanszky, M., Tolle, J. C., Deshmane, S. S., and Bodanszky, A. (1978). Side reactions in peptide synthesis VI: A reexamination of the benzyl group in the protection of the side chains of tyrosine and aspartic acid. Int. J. Peptide Protein Res. 12:57-68. Bolin, D. R., Wang, C.-T., and Felix, A. M. (1989). Preparation of JV-?-butyloxycarbonyl-Oa>-9-fluorenylmethyl esters of asparatic and glutamic acids. Org. Prep. Proc. Int. 21:67-74. Borras, E.; Giralt, E., and Andreu, D. (1999). A rationally designed synthetic peptide mimic of a discontinuous viral antigenic site elicits neutralizing antibodies. J. Am. Chem. Soc. 121:11932-11933. Bourne, G. T., Meutermans, W. D. F., Alewood, P. F., McGeary, R. P., Scanlon, M., Watson, A. A., and Smythe M. L. (1999a). A backbone linker for BOC-based peptide synthesis and on-resin cyclization: Synthesis of stylostatin 1. J. Org. Chem. 64:3095-3101. Bourne, G. T., Meutermans, W. D. F., and Smythe M. L. (1999b). The development of solid phase protocols for a backbone amide linker and its application to the Boc-based assembly of linear peptides. Tetrahedron Lett. 40:7271-7274. Bozzini, M., Bello, R., Cagle, N., Yamane, D., and Dupont, D. (1991). Tryptophan recovery from autohydrolyzed samples using dodecanethiol. Applied Biosy stems Research News, February, Foster City, Calif., Applied Biosystems, Inc. Brady, S. F., Paleveda, W. J., and Nutt, R. F. (1988). Studies of acetamidomethyl as cysteine protection: Application in synthesis of ANF analogs. In Peptides: Chemistry and Biology, G. R. Marshall, ed., Leiden, Escom, pp. 192-194. Breipohl, G., Knolle, J., and Stiiber, W. (1989). Synthesis and application of acid labile anchor groups for the synthesis of peptide amides by Fmocsolid-phase peptide synthesis. Int. J. Peptide Protein Res. 34:262-267. Breipohl, G., Knolle, J., and Stiiber, W. (1990). Facile SPS of peptides having C-terminal Asn and Gin. Int. J. Peptide Protein Res. 35:281-283. Broddefalk, J., Backlund, J., Almqvist, F., Johansson, M., Holmdahl, R., and Kihlberg, J. (1998). T-cells recognize a glycopeptide derived from type II collagen in a model for rheumatoid arthritis. J. Am. Chem. Soc. 120:7676-7683. Brown, A. R., Irving, S. L., and Ramage, R. (1993). Affinity purification of synthetic peptides and proteins on porous graphitised carbon. Tetrahedron Lett. 34:7129-7132. Brown, T., Jones, J. H., and Richards, J. D. (1982). Further studies on the protection of histidine side chains in peptide synthesis: The use of the nbenzyloxymethyl group. J. Chem. Soc. Perkin Trans. 1:1553-1561.
183
Brugidou, J., and Mery, J. (1994). 2-Hydroxypropyl-dithio-2'-isobutyric acid (HPDI) as a multipurpose peptide-resin linker for SPPS. Peptide Res. 7:4047. Buchta, R., Bondi, E., and Fridkin, M. (1986). Peptides related to the calcium binding domains II and III of calmodulin: Synthesis and calmodulin-like features. Int. J. Peptide Protein Res. 28:289-297. Biittner, K., Zahn, H., and Fischer, W. H. (1988). Rapid solid phase peptide synthesis on a controlled pore glass support. In Peptides: Chemistry and Biology, G. R. Marshall, ed., Leiden, Escom, pp. 210-211. Butwell, F. G. W., Haws, E. J., and Epton, R. (1988). Advances in ultra-high load polymer supported peptide synthesis with phenolic supports 1: A selectively-labile C-terminal spacer group for use with a base-mediated ^-terminal deprotection strategy and Fmoc amino acids. Makromol. Chem., Macromol. Symp. 19:69-77. Bycroft, B. W., Chang, W. C., Chhabra, S. R., and Hone, N. D. (1993). A novel lysine protecting procedure for continuous flow solid phase synthesis of branched peptides. J. Chem. Soc., Chem. Commun. 778-779. Calnan, B. J., Biancalana, S., Hudson, D., and Frankel, A. D. (1991). Analysis of arginine-rich peptides from the HIV; Tat protein reveals unusual features of RNA-protein Recognition. Genes and Development 5:201-210. Camarero, J. A., Cotton, G. J., Adeva, A., and Muir, T. W. (1998). Chemical ligation of unprotected peptides directly from a solid support. J. Peptide Res. 51:303-316. Cameron, L. R., Holder, J. L., Meldal, M., and Sheppard, R. C. (1988). Peptide synthesis, part 13: Feedback control in solid phase synthesis: Use of fluorenylmethoxycarbonyl amino acid 3,4-dihydro-4-oxo-1,2,3benzotriazin-3-yl esters in a fully automated system. J. Chem. Soc. Perkin Trans. 1:2895-2901. Canne, L. E., Walker, S. M., and Kent, S. B. H. (1995). A general method for the synthesis of thioester resin linkers for use in the solid phase synthesis of peptide-a-thioacids. Tetrahedron Lett. 36:1217-1220. Canne, L. E., Botti, P., Simon, R. J., Chen, Y., Dennis, E. A., and Kent, S. B. H. (1999). Chemical protein synthesis by solid phase ligation of unprotected peptide segments. J. Am. Chem. Soc. 121:8720-8727. Carpino, L. A., and El-Faham, A. (1995). Tetramethylfluoroformamidinium hexafluorophospate: A rapid-acting peptide coupling reagent for solution and solid phase peptide synthesis. J. Am. Chem. Soc. 117:5401-5402. Carpino, L. A., and Han, G. Y. (1972). The 9-fluorenylmethoxycarbonyl amino-protecting group. J. Org. Chem. 37:3404-3409. Carpino, L. A., and Mansour, E.-S. M.E. (1992). Protected f$- and y-aspartic and -glutamic acid fluorides. J. Org. Chem. 57:6371-6373. Carpino, L. A., Cohen, B. J., Stephens, Jr., K. E., Sadat-Aalaee, S. Y., Tien, J.-H., and Langridge, D. C. (1986). ((9-Fluorenylmethyl)oxy)carbonyl (Fmoc) amino acid chlorides: Synthesis, characterization, and application to the rapid synthesis of short peptide. J. Org. Chem. 51:3732-3734. Carpino, L. A., Sadat-Aalaee, D., Chao, H. G., and DeSelms, R. H. (1990). ((9Fluorenylmethyl)oxy)carbonyl (Fmoc) amino acid fluorides: Convenient new peptide coupling reagents applicable to the Fmoc/tert-butyl strategy for solution and solid-phase syntheses. J. Am. Chem. Soc. 112:9651-9652.
184
Synthetic Peptides
Carpino, L. A., Mansour, E.-S. M. E., and Sadat-Aalaee, D. (1991a). tertButyloxycarbonyl and benzyloxycarbonyl amino acid fluorides: New, stable rapid-acting acylating agents for peptide synthesis. J. Org. Chem. 56:2611-2614. Carpino, L. A., Chao, H. G., Beyermann, M., and Bienert, M. (1991b). ((9-Fluorenylmethyl)oxy)carbonyl amino acid chlorides in solid-phase peptide synthesis. J. Org. Chem. 56:2635-2642. Carpino, L. A., Shroff, H., Triolo, S. A., Mansour, E.-S. M.E., Wenschuh, H., and Albericio, F. (1993). The 2,2,4,6,7-pentamethyldihydrobenzofuran-5sulfonyl group (Pbf) as arginine side chain protectant. Tetrahedron Lett. 34:7829-7832. Carpino, L. A., El-Faham, A., Minor, C. A., and Albericio, F. (1994). Advantageous applications of azabenzotriazole (triazolopyridine)-based coupling reagents to solid-phase peptide synthesis. J. Chem. Soc., Chem. Commun. 201-203. Casaretto, R., and Nyfeler, R. (1991). Isolation, structure and activity of a side product from the synthesis of human cndothelin. In Peptides 1990, E. Giralt and D. Andreu, eds., Leiden, Escom, pp. 181-182. Chan, A. S. H., Mobley, J. L., Fields, G. B., and Shimizu, Y. (1997). CD7mediated regulation of integrin adhesiveness on human T cells involves tyrosine phosphorylation-dependent activation of phosphatidylinositol 3kinase. J. Immunol. 159:934-942. Chan, W. C., and Bycroft, B. W. (1992). Deprotection of Arg(Pmc) containing peptides using TFA-trialkylsilane-methanol-EMS; application to the synthesis of propeptides of nisin. In Peptides: Chemistry and Biology, J. A. Smith and J. E. Rivier, eds., Leiden, Escom, pp. 613-614. Chan, W. C., and White, P. D., eds. (2000). Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford, Oxford University Press. Chan, W. C., Bycroft, B. W., Evans, D. J., and White, P. D. (1995). A novel 4-aminobenzyl ester-based carboxy-protecting group for synthesis of atypical peptides by Fmoc-Bu' solid phase chemistry. J. Chem. Soc., Chem. Commun. 2209-2210. Chang, C. D., Felix, A. M., Jimenez, M. H., and Meienhofer, J. (1980a). Solidphase peptide synthesis of somatostatin using mild base cleavage of Nfluorenylmethyloxycarbonylamino acids. Int. J. Peptide Protein Res. 15:485^194. Chang, C. D., Waki, M., Ahmad, M., Meienhofer, J., Lundell, E. O., and Haug. J. D. (1980b). Preparation and properties of W*-9-fluorenylmethyloxycarbonylamino acids bearing ?er/-butyl side chain protection. Int. J. Peptide Protein Res. 15:59-66. Chanh, T. C., Dreesman, G. R., Kanda, P.. Linette, G. P., Sparrow, J. T., Ho, D. D., and Kennedy, R. C. (1986). Induction of anti-HIV neutralizing antibodies by synthetic peptides. EMBO J. 5:3065-3071. Chao, H. G., Bernatowicz, M. S., and Matsueda, G. R. (1993). Preparation and use of the 4-[l-[Ar-(9-fluorenylmethyloxycarbonyl)-arnino]-2-(trimethylsilyl)ethyl]phenoxyacetic acid linkage agent for solid-phase synthesis of C-terminal peptide amidesdmproved yields of tryptophancontaining peptides. J. Org. Chem. 58:2640-2644.
Chen, S.-T., Weng, C.-S., and Wang, K.-T. (1987). The synthesis of /i-methoxybenzyloxycarbonyl amino acids. J. Chin. Chem. Soc. 34:117123. Chen, S.-T., Wu, S.-H., and Wang, K.-T. (1989). A new synthesis of O-benzylL-threonine. Synth. Commun. 19:3589-3593. Chhabra, S. R., Hothi, B., Evans, D. J., White, P., Bycroft, B., and Chan, W. C. (1998). An appraisal of new variants of Dde amine protecting group for solid phase peptide synthesis. Tetrahedron Lett. 39:1603-1606. Chillemi, F., and Merrifield, R. B. (1969). Use of A^-dinitrophenylhistidine in the solid-phase synthesis of the tricosapeptides 124 146 of human hemoglobin j6 chain. Biochemistry 8:4344-4346. Choi, H., and Aldrich, J. V. (1993). Comparison of methods for the Fmoc solid-phase synthesis and cleavage of a peptide containing both tryptophan and arginine. Int. J. Peptide Protein Res. 42:58-63. Christiansen-Brams, I., Meldal, M., and Bock, K. (1993). Silyl protection in the solid phase synthesis of /V-linked neoglycopeptides: One step deprotection of fully protected neoglycopeptides. Tetrahedron Lett. 34:33153318. Chun, R., Glabe, C. G., and Fan, H. (1990). Chemical synthesis of biologically active tat rram'-activating protein of human immunodeficiency virus type 1. J. Virol. 64:3074-3077. Clark-Lewis, I., and Kent, S. (1989). Chemical synthesis, purification, and characterization of peptides and proteins. In Receptor Biochemistry and Methodology, Vol. 14: The Use of HPLC in Protein Purification and Characterization, A. R. Kerlavage, J. C. Venter and L. C. Harrison, eds., New York, Alan R. Liss, pp. 43-75. Clark-Lewis, L, Aebersold, R., Ziltener, H., Schrader, J. W., Hood, L. E., and Kent, S. B. (1986). Automated chemical synthesis of a protein growth factor for hemopoietic cells, interleukin-3. Science 231:134-139. Colombo, R., Atherton, E., Sheppard, R. C., and Woolley, V. (1983). 4Chloromethylphenoxyacetyl polystyrene and polyamide supports for solid-phase peptide synthesis. Int. J. Peptide Protein Res. 21:118-126. Colombo, R., Colombo, F., and Jones, J. H. (1984). Acid-labile histidine sidechain protection: The ;r-benzyloxymethyl group. J. Chem. Soc., Chem. Commun. 292-293. Cook, R. M., and Hudson, D. (1996). Chemically modified polyolcfin particles for biomolecule synthesis, analysis and display. In Peptides: Chemistry, Structure and Biology, P. T. P. Kaumaya and R. S. Hodges, eds., Kingswinford, Mayflower Scientific Ltd., pp. 39-41. Cook, R. M., and Hudson, D. (1997). Oxidized polyethylene or polypropylene particulate supports. U.S. Patent 5,679,539, 1997. Coste, J., Le-Nguyen, D., and Castro, B. (1990). PyBOP: A new peptide coupling reagent devoid of toxic by-product. Tetrahedron Lett. 31:205208. Cruz, L. J., Kupryszewski, G., LeCheminant, G. W., Gray, W. R., Olivera, B. M., and Rivier, J. (1989). /x-Conotoxin GIIIA, a peptide ligand for muscle sodium channels: Chemical synthesis, radiolabeling, and receptor characterization. Biochemistry 28:3437 3442.
186
Synthetic Peptides
Dalcol, I., Rabanal, F., Ludevid, M.-D., Albericio, F., and Giralt, E. (1995). Convergent solid phase peptide synthesis: An efficient approach to the synthesis of highly repetitive protein domains. J. Org. Chem. 60:75757581. Dawson, P. E., and Kent, S. B. H. (1993). Convenient total synthesis of a 4helix TASP molecule by chemoselective ligation. J. Am. Chem. Soc. 115:7263-7266. Dawson, P. E., Muir, T. W., Clark-Lewis, I., and Kent, S. B. H. (1994). Synthesis of proteins by native chemical ligation. Science 266:776-779. Dawson, P. E., Churchill, M. J., Ghadiri, M, R., and Kent, S. B. H. (1997). Modulation of reactivity in native chemical ligation through the use of thiol additives. J. Am. Chem. Soc. 119:4325-4329. de Bont, H. B.A., van Boom, J. H., and Liskamp, R. M.J. (1990). Automatic synthesis of phophopeptides by phosphorylation on the solid phase. Tetrahedron Lett. 31:2497-2500. de la Torre, B. G., Torres, J. L., Bardaji, E., Clapes, P., Xaus, N., Jorba, X., Calvet, S., Albericio, F., and Valencia, G. (1990). Improved method for the synthesis of O-glycosylated Fmoc amino acids to be used in solidphase glycopeptide synthesis. J. Chem. Soc., Chem. Commun. 965-967. de Lisle Milton, R. C., Becker, E., Milton, S. C. F., Baxter, J. E. J., and Elsworth, J. F. (1987). Improved purities for Fmoc-amino acids from Fmoc-ONSu. Int. J. Peptide Protein Res. 30:431-^32. de Lisle Milton, R. C., Milton, S. C. F., and Kent, S. B. H. (1992). Total chemical synthesis of a D-enzyme: The enantiomers of HIV-1 protease show demonstration of reciprocal chiral substrate specificity. Science 256:1445-1448. de Rocquigny, H., Ficheux, D., Gabus, C., Fournie-Zaluski, M.-C., Darlix, J.L., and Roques, B. P. (1991). First large scale chemical synthesis of the 72 amino acid HIV-1 nucleocapsid protein Ncp7 in an active form. Biochem. Biophys. Res. Commun. 180:1010-1018. Deen, C., Claassen, E., Gerritse, K., Zegers, N. D., and Boersma, W. J. A. (1990). A novel carbodiimide coupling method for synthetic peptides: Enhanced anti-peptide antibody responses. J. Immunol. Methods 129:119-125. DeGrado, W. F., and Kaiser, E. T. (1980). Polymer-bound oxime esters as supports for solid-phase peptide synthesis: Preparation of protected peptide fragments. J. Org. Chem. 45:1295-1300. DeGrado, W. F., and Kaiser, E. T. (1982). Solid-phase synthesis of protected peptides on a polymer-bound oxime:Preparation of segments comprising the sequence of a cytotoxic 26-peptide analogue. J. Org. Chem. 47:32583261. DiMarchi, R. D., Tarn, J. P., Kent, S. B. H., and Merrifield, R. B. (1982). Weak acid-catalyzed pyrrolidone carboxylic acid formation from glutamine during solid phase peptide synthesis. Int. J. Peptide Protein Res. 19:88-93. Dolling, R., Beyermann, M., Haenel, J., Kernchen, F., Krause, E., Franke, P., Brudel, M., and Bienert, M. (1994). Piperidine-mediated side product formation for Asp(OBu')-containing peptides. J. Chem. Soc., Chem. Commun. 853-854.
187
Dorman, L. C., Nelson, D. A., and Chow, R. C. L. (1972). Solid phase synthesis of glutamine-containing peptides. In Progress in Peptide Research, Vol. 2, S. Lande, ed., New York, Gordon and Breach, pp. 65-68. Dourtoglou, V., Gross, B., Lambropoulou, V., and Zioudrou, C. (1984). Obenzotriazolyl-Af,Ar,A'',7\r'-tetramethyluronium hexafluorophosphate as coupling reagent for the synthesis of peptides of biological interest. Synthesis 572-574. Drijfhout, J. W., and Bloemhoff, W. (1988). Capping with O-sulfobenzoic acid cyclic anhydride (OSBA) in solid-phase peptide synthesis enables facile product purification. In Peptide Chemistry 1987, T. Shiba and S. Sakakibara, eds., Protein Research Foundation, Osaka, pp. 191-194. Drijfhout, J. W., and Bloemhoff, W. (1991). A new synthetic functionalized antigen carrier. Int. J. Peptide Protein Res. 37:27-32. Drijfhout, J. W., Perdijk, E. W., Weijcr, W. J., and Bloemhoff, W. (1988). Controlled peptide-protein conjugation by means of 3-nitro-2-pyridinesulfenyl protection-activation. Int. J. Peptide Protein Res. 32:161-166. Edwards, W. B., Fields, C. G., Anderson, C. J, Pajeau, T. S., Welch, M. J., and Fields, G. B. (1994). Generally applicable, convenient solid-phase synthesis and receptor affinities of octreotide analogs. J. Med. Chem. 37:3749-3757. Egner, B. J., Cardno, M., and Bradley, M. (1995). Linkers for combinatorial chemistry and reaction analysis using solid phase in situ mass spectrometry. J. Chem. Soc., Chem. Commun. 2163-2164. Eichler, J., Beyermann, M., and Bienert, M. (1989). Application of cellulose paper as support material in simultaneous solid phase peptide synthesis. Collect. Czech. Chem. Commun. 54:1746-1752. Elofsson, M., Salvador, L. A., and Kihlberg, J. (1997). Preparation of TN and sialyl TN building blocks used in Fmoc-solid phase synthesis of glycopeptide fragments from HIV GP120. Tetrahedron 53:369-390. Englebretsen, D. R., and Harding, D. R. K. (1994). Fmoc SPPS using Perloza beaded cellulose. Int. J. Peptide Protein Res. 43:546-554. Englebretsen, D. R., Garnham, B. G., Bergman, D. A., and Alewood, P. F. (1995). A novel thioethcr linker: Chemical synthesis of a HIV-1 protease analogue by thioether ligation. Tetrahedron Lett. 36:8871-8874. Epton, R., Goddard, P., and Ivin, K. J. (1980). Gel phase I3 C n.m.r. spectroscopy as an analytical method in solid (gel) phase peptide synthesis. Polymer 21:1367-1371. Epton, R., Wellings, D. A., and Williams, A. (1987). Perspectives in ultra-high load solid (gel) phase peptide synthesis. Reactive Polymers 6:143-157. Erickson, B. W., and Merrifield, R. B. (1973a). Acid stability of several benzylic protecting groups used in solid-phase peptide synthesis: Rearrangement of 0-benzyltyrosine to 3-benzyltyrosine. J. Am. Chem. Soc. 95:3750-3756. Erickson, B. W., and Merrifield, R. B. (1973b). Use of chlorinated benzyloxycarbonyl protecting groups to eliminate TV-branching at lysine during solid-phase peptide synthesis. J. Am. Chem. Soc. 95:3757-3763. Erickson, B. W., and Merrifield, R. B. (1976). Solid-phase peptide synthesis. In The Proteins, Vol. II, 3rd edn., H. Neurath and R. L. Hill, eds., New York, Academic Press, pp. 255-527.
188
Synthetic Peptides
Eritja, R., Ziehler-Martin, J. P., Walker, P. A., Lee, T. D., Legesse, K., Albericio, F., and Kaplan, B. E. (1987). On the use of ^-/-butylsulphenyl group for protection of cysteine in solid-phase peptide synthesis using Fmoc-amino acids. Tetrahedron 43:2675-2680. Eritja, R., Robles, J., Fernandez-Forner, D., Albericio, F., Giralt, E., and Pedroso, E. (1991). NPE-resin, a new approach to the solid-phase synthesis of protected peptides and oligonucleotides I: Synthesis of the supports and their application to oligonucleotide synthesis. Tetrahedron Lett. 32:1511-1514. Fairwell, T., Hospattankar, A. V., Ronan, R., Brewer, Jr., H. B., Chang, J. K., Shimizu, M., Zitzner, L., and Arnaud, C. D. (1983). Total solid-phase synthesis, purification, and characterization of human parathyroid hormone-(l-84). Biochemistry 22:2691-2697. Feinberg, R. S., and Merrifield, R. B. (1975). Modification of peptides containing glutamic acid by hydrogen-fluoride-anisole mixtures: yAcylation of anisole or the glutamyl nitrogen. J. Am. Chem. Soc. 97: 3485-3496. Felix, A. M., Jiminez, M. H., Vergona, R., and Cohen, M. R. (1973). Synthesis and biological studies of novel bradykinin analogues. Int. J. Peptide Protein Res. 5:201-206. Felix, A. M., Wang, C.-T., Heimer, E. P., and Fournier, A. (1988a). Applications of BOP reagent in solid phase synthesis II: Solid phase side-chain cyclization using BOP reagent. Int. J. Peptide Protein Res. 31:231-238. Felix, A. M., Heimer, E. P., Wang, C.-T., Lambros, T. J., Fournier, A., Mowles, T. F., Maines, S., Campbell, R. M., Wegrzynski, B. B., Toome, V., Fry, D., and Madison, V. S. (1988b). Synthesis, biological activity and conformational analysis of cyclic GRF analogs. Int. J. Peptide Protein Res. 32:441^154. Feng, Y., Melacini, G., Taulane, J. P., and Goodman, M. (1996a). Acetylterminated and template-assembled collagen-based polypeptides composed of Gly-Pro-Hyp sequences 2: Synthesis and conformational analysis by circular dichroism, ultraviolet absorbance, and optical rotation. J. Am. Chem. Soc. 118:10351-10358. Feng, Y., Melacini, G., Taulane, J. P., and Goodman, M. (1996b). Collagenbased structures containing the peptoid residue Ar-isobutylglycine (Nleu): Synthesis and biophysical studies of Gly-Pro-Nleu sequences by circular dichroism, ultraviolet absorbance, and optical rotation. Biopolymers 39:859-872. Feng, Y., Melacini, G., and Goodman, M. (1997). Collagen-based structures containing the peptoid residue A'-isobutylglycine (Nleu): Synthesis and biophysical studies of Gly-Nleu-Pro sequences by circular dichroism and optical rotation. Biochemistry 36:8716-8724. Ferrer, M., Woodward, C., and Barany, G. (1992). Solid-phase synthesis of bovine pancreatic trypsin inhibitor (BPTI) and two analogues. Int. J. Peptide Protein Res. 40:194-207. Ferrer, M., Barany, G., and Woodward, C. (1995). Partially folded, molten globule and molten coil states of bovine pancreatic trypsin inhibitor. Nature Struct. Biol. 2:211-217.
Ferrer, T., Nicolas, E., and Giralt, E. (1999). Application of the disulfide trapping approach to explain the antiparallel assembly of dimcric rabbit uteroglobin: A preliminary study using short peptide models. Lett. Pept. Sci. 6:165-172. Fields, C. G., and Fields, G. B. (1990). New approaches to prevention of side reactions in Fmoc solid phase peptide synthesis. In Peptides: Chemistry, Structure, and Biology, J. E. Rivier and G. R. Marshall, eds., Leiden, Escom, pp. 928-930. Fields, C. G., and Fields, G. B. (1993). Minimization of tryptophan alkylation following 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis. Tetrahedron Lett. 34:6661-6664. Fields, C. G., Fields, G. B., Noble, R. L., and Cross, T. A. (1989). Solid phase peptide synthesis of [15N]-gramicidins A, B, and C and high performance liquid chromatographic purification. Int. J. Peptide Protein Res. 33:298303. Fields, C. G., Lloyd, D. H., Macdonald, R. L., Otteson, K. M., and Noble, R. L. (1991). HBTU activation for automated Fmoc solid-phase peptide synthesis. Peptide Res. 4:95-101. Fields, C. G., Loffet, A., Kates, S. A., and Fields, G. B. (1992). The development of high-performance liquid chromatographic analysis of allyl and allyloxycarbonyl side-chain-protected phenylthiohydantoin-amino acids. Anal. Biochem. 203:245-251. Fields, C. G., Lovdahl, C. M., Miles, A. J., Matthias Hagen, V. L., and Fields, G. B. (1993a). Solid-phase synthesis and stability of triple-helical peptide incorporating native collagen sequences. Biopolymers 33:16951707. Fields, C. G., Mickelson, D. J., Drake, S. L., McCarthy, J. B., and Fields, G. B. (1993b). Melanoma cell adhesion and spreading activities of a synthetic 124-residue triple-helical "mini-collagen." J. Biol. Chem. 268:1415314160. Fields, C. G., VanDrisse, V. L., and Fields, G. B. (1993c). Edman degradation sequence analysis of resin-bound peptides synthesized by 9-fluorenylmethoxycarbonyl chemistry. Peptide Res. 6:3946. Fields, G. B., ed. (1997). Solid-phase peptide synthesis. Methods Enzymol. 289:1-780. Fields, G. B., and Fields, C. G. (1991). Solvation effects in solid-phase peptide synthesis. J. Am. Chem. Soc. 113:4202-4207. Fields, G. B., and Fields, C. G. (1992). Optimization strategies for Fmoc solid phase peptide synthesis: Synthesis of triple-helical collagen-model peptides. In Innovation and Perspectives in Solid Phase Synthesis--Peptides, Polypeptides and Oligonucleotides7992, R. Epton, ed., Andover, Intercept, pp. 153-162. Fields, G. B., and Noble, R. L. (1990). Solid phase peptide synthesis utilizing 9-fluorenylmcthoxycarbonyl amino acids. Int. J. Peptide Protein Res. 35:161-214. Fields, G. B., Van Wart, H. E., and Birkedal-Hansen, H. (1987). Sequence specificity of human skin fibroblast collagenase: Evidence for the role of collagen structure in determining the collagenase cleavage site. J. Biol. Chem. 262:6221-6226.
190
Synthetic Peptides
Fields, G. B., Fields, C. G., Petefish, J., Van Wart, H. E., and Cross, T. A. (1988). Solid phase synthesis and solid state NMR spectroscopy of [15N-Ala3]-Val-gramicidin A. Proc. Natl. Acad. Sci. USA 85:13841388. Fields, G. B., Otteson, K.M, Fields, C. G., and Noble, R. L. (1990). The versatility of solid phase peptide synthesis. In Innovation and Perspectives in Solid Phase Synthesis, R. Epton, ed., Birmingham, UK, Solid Phase Conference Coordination, Ltd., pp. 241-260. Fields, G. B., Tian, Z., and Barany, G. (1992). Principles and practice of solidphase peptide synthesis. In Synthetic Peptides: A User's Guide, G. A. Grant, ed., New York, W. H. Freeman and Co., pp. 77-183. Fields, G. B., Carr, S. A., Marshak, D. R., Smith, A. J., Stults, J. T., Williams, L. C., Williams, K. R., and Young, J. D. (1993). Evaluation of peptide synthesis as practiced in 53 different laboratories. In Techniques in Protein Chemistry, Vol. IV, R. H. Angeletti, ed., San Diego, Calif, Academic Press, pp. 229-238. Filira, F., Biondi, L., Cavaggion, F., Scolaro, B., and Rocchi, R. (1990). Synthesis of 0-glycosylated tuftsins by utilizing threonine derivatives containing an unprotected monosaccharide moiety. Int. J. Peptide Protein Res. 36:86-96. Findeis, M. A., and Kaiser, E. T. (1989). Nitrobenzophenone oxime based resins for the solid-phase synthesis of protected peptide segments. J. Org. Chem. 54:3478-3482. Finn, F. M., and Hofmann, K. (1976). The synthesis of peptides by solution methods with emphasis on peptide hormones. In The Proteins, Vol. II, 3rd edn., H. Neurath and R. L. Hill, eds., New York, Academic Press, pp. 105-253. Fischer, P. M., Comis, A., and Howden, M. E. H. (1989). Direct immunization with synthetic peptidyl-polyamide resin: Comparison with antibody production from free peptide and conjugates with carrier proteins. J. Immunol. Methods 118:119-123. Fitch, W. L., Detre, G., and Holmes, C. P. (1994). High-resolution 'H-NMR in solid phase organic synthesis. J. Org. Chem. 59:7955-7956. Fitzgerald, M. C., Harris, K., Shevlin, C. G., and Siuzdak, G. (1996). Direct characterization of solid phase resin-bound molecules by mass spectrometry. Bioorg. Med. Chem. Lett. 6:979-982. Flegel, M., and Sheppard, R. C. (1990). A sensitive, general method for quantitative monitoring of continuous flow solid phase peptide synthesis. J. Chem. Soc., Chem. Commun. 536-538. Fletcher, A. R., Jones, J. H., Ramage, W. L, and Stachulski, A. V. (1979). The use of the N(jr)-phenacyl group for the protection of the histidine side chain in peptide synthesis. J. Chem. Soc. Perkin Trans. 1:22612267. Florsheimer, A., and Riniker, B. (1991). Solid-phase synthesis of peptides with the highly acid-sensitive HMPB linker. In Peptides 1990, E. Giralt and D. Andreu, eds., Leiden, Escom, pp. 131-133. Fodor, S. P. A., Read, J. L., Pirrung, M. C., Stryer, L., Lu, A. T., and Solas, D. (1991). Light-directed, spatially addressable parallel chemical synthesis. Science 251:767-773.
191
Forest, M., and Fournier, A. (1990). BOP reagent for the coupling of pGlu and Boc-His(Tos) in solid phase peptide synthesis. Int. J. Peptide Protein Res. 35:89-94. Fournier, A., Wang, C.-T., and Felix, A. M. (1988). Applications of BOP reagent in solid phase peptide synthesis: Advantages of BOP reagent for difficult couplings exemplified by a synthesis of [Ala'5]-GRF(l-29)NH 2 . Int. J. Peptide Protein Res. 31:86-97. Fournier, A., Danho, W., and Felix, A. M. (1989). Applications of BOP reagent in solid phase peptide synthesis III: Solid phase peptide synthesis with unprotected aliphatic and aromatic hydroxyamino acids using BOP reagent. Int. J. Peptide Protein Res. 33:133-139. Fox, J., Newton, R., Heegard, P., and Schafer-Nielsen, C. (1990). A novel method of monitoring the coupling reaction in solid phase synthesis. In Innovation and Perspectives in Solid Phase Synthesis, R. Epton, ed., Birmingham, UK, Solid Phase Conference Coordination, Ltd., pp. 141153. Frank, R., and Doring, R. (1988). Simultaneous multiple peptide synthesis under continuous flow conditions on cellulose paper discs as segmental solid supports. Tetrahedron 44:6031-6040. Frank, R., and Gausepohl, H. (1988). Continuous flow peptide synthesis. In Modern Methods in Protein Chemistry, Vol. 3, H. Tschesche, ed., Berlin, Walter de Gruyter and Co., pp. 41 60. Frankel, A. D., Biancalana, S., and Hudson, D. (1989). Activity of synthetic peptides from the tat protein of human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 86:7397-7401. Franzen, H., Grehn, L., and Ragnarsson, U. (1984). Synthesis, properties, and use of A^-Boc-tryptophan derivatives. J. Chem. Soc., Chem. Commun. 1699-1700. Fujii, N., Otaka, A., Funakoshi, S., Bessho, K., Watanabe, T., Akaji, K., and Yajima, H. (1987). Studies on peptides CLI: Synthesis of cystine-peptides by oxidation of 5-protected cysteine-peptides with thallium (111) trifluoroacetate. Chem. Pharm. Bull. 35:2339-2347. Fujino, M., Wakimasu, M., Shinagawa, S., Kitada, C., and Yajima, H. (1978). Synthesis of the nonacosapeptide corresponding to mammalian glucagon. Chem. Pharm. Bull. 26:539-548. Fujino, M., Wakimasu, M., and Kitada, C. (1981). Further studies on the use of multi-substituted benzenesulfonyl groups for protection of the guanidino function of arginine. Chem. Pharm. Bull. 29:2825-2831. Fujiwara, Y., Akaji, K., and Kiso, Y. (1994). Racemization-free synthesis of C-terminal cysteine-peptide using 2-chlorotrityl resin. Chem. Pharm. Bull. 42:724-726. Fuller. W. D., Cohen, M. P., Shabankareh, M., Blair, R. K., Goodman, M., and Naider, F. R. (1990). Urethane-protected amino acid N-carboxy anhydrides and their use in peptide synthesis. J. Am. Chem. Soc. 112:7414-7416. Fuller, W. D., Krotzer, N. J., Naider, F. R., Xue, C.-B., and Goodman, M. (1993). Urethane-protected amino acid N-carboxyanhydrides^tability, reactivity and solubility. In Peptides 1992, C. H. Schneider and A. N. Eberle, eds., Leiden, Escom, pp. 229-230.
92
Synthetic Peptides
unakoshi, S., Tamamura, H., Fujii, N., Yoshizawa, K., Yajima, H., Miyasaki, K., Funakoshi, A., Ohta, M., Inagaki, Y., and Carpino, L. A. (1988). Combination of a new amide-precursor reagent and trimethylsilyl bromide deprotection for the Fmoc-based solid phase synthesis of human pancreastatin and one of its fragments. J. Chem. Soc., Chem. Commun. 1588-1590. utaki, S., Taike, T., Akita, T., and Kitagawa, K. (1990a). A new approach for the synthesis of tyrosine sulphate containing peptides: Use of the p(methylsulphinyl)benzyl group as a key protecting group of serine. J. Chem. Soc., Chem. Commun. 523-524. utaki, S., Yajami, T., Taike, T., Akita, T., and Kitagawa, K. (1990b). Sulphur trioxide/thiol: A novel system for the reduction of methionine sulphoxide. J. Chem. Soc. Pcrkin Trans. 1:653-658. aehde, S. T., and Matsueda, G. R. (1981). Synthesis of N-fcrt-butoxycarbonyl-(a-phenyl)aminomethylphenoxyacetic acid for use as a handle in solid-phase synthesis of peptide a-carboxamides. Int. J. Peptide Protein Res. 18:451-458. airi, M., Lloyd-Williams, P., Albericio, F., and Giralt, E. (1990). Use of BOP reagent for the suppression of diketopiperazine formation in Boc/Bzl solid-phase peptide synthesis. Tetrahedron Lett. 31:7363-7366. arcia-Echeverria, C. (1995). On the use of hydrophobic probes in the chromatographic purification of solid-phase-synthesized peptides. J. Chem. Soc. Chem. Commun. 779-780. arcia-Echeverria, C. (1995). Potential pyrophosphate formation upon use of jV"-Fmoc-Tyr(PO3H?)-OH in solid-phase peptide synthesis. Lett. Peptide Sci. 2:93-98. arcia-Echeverria, C., Albericio, F., Pons, M., Barany, G., and Giralt, E. (1989). Convenient synthesis of a cyclic peptide disulfide: A type II fiturn structural model. Tetrahedron Lett. 30:2441-2444. ausepohl, H., Kraft, M., and Frank, R. (1989a). In situ activation of Fmocamino acids by BOP in solid phase peptide synthesis. In Peptides 1988, G. Jung and E. Bayer, eds., Berlin, Walter de Gruyter and Co., pp. 241-243. ausepohl, H., Kraft, M., and Frank, R. W. (1989b). Asparaginc coupling in Fmoc solid phase peptide synthesis. Int. J. Peptide Protein Res. 34:287 294. ausepohl, H., Kraft, M., Boulin, Ch., and Frank, R. W. (1990). Automated multiple peptide synthesis with BOP activation. In Peptides: Chemistry, Structure and Biology, J. E. Rivier and G. R. Marshall, eds., Leiden, Escom, pp. 1003 1004. ausepohl, H., Kraft, M., Boulin, C., and Frank, R. W. (1991). A multiple reaction system for automated simultaneous peptide synthesis. In Peptides 1990, E. Giralt and D. Andreu, eds., Leiden, Escom, pp. 206207. ausepohl, H., Pieles, U., and Frank, R. W. (1992). Schiffs base analog formation during in situ activation by HBTU and TBTU. In Peptides: Chemistry and Biology, J. A. Smith and J. E. Rivier, eds., Leiden, Escom, pp. 523-524. eiser, T., Beilan, H., Bergot, B. J., and Otteson, K. M. (1988). Automation of solid-phase peptide synthesis. In Macromolecular Sequencing and
193
Synthesis: Selected Methods and Applications, D. H. Schlesinger, ed., New York, Alan R. Liss, pp. 199-218. Gesellchen, P. D., Rothenberger, R. B., Dorman, D. E., Paschal, J. W., Elzey, T. K., and Campbell, C. S. (1990). A new side reaction in solid-phase peptide synthesis: Solid support-dependent alkylation of tryptophan. In Peptide.t: Chemistry, Structure and Biology, J. E. Rivier and G. R. Marshall, eds., Leiden, Escom, pp. 957-959. Gesquiere, J.-C., Diesis, E., and Tartar, A. (1990). Conversion of TV-terminal cysteine to thiazolidine carboxylic acid during hydrogen fluoride deprotection of peptides containing N-n-Bom protected histidine. J. Chem. Soc., Chem. Commun. 1402-1403. Gesquiere, J.-C., Najib, J., Diesis, E., Barbry, D., and Tartar, A. (1992). Investigations of side reactions associated with the use of Bom and Bum groups for histidine protection. In Peptides: Chemistry and Biology, J. A. Smith and J. E. Rivier, eds., Leiden, Escom, pp. 641642. Geysen, H. M., Meloen, R. H., and Barteling, S. J. (1984). Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci. USA 81:3998-4002. Giralt, E., Rizo, J., and Pedroso, E. (1984). Application of gel-phase 13C-NMR to monitor solid phase peptide synthesis. Tetrahedron 40:4141^1152. Giralt, E., Albericio, F., Pedroso, E., Granier, C., and van Rietschoten, J. (1982). Convergent solid phase peptide synthesis II: Synthesis of the 16 apamin protected segment on a Nbb-resin: synthesis of apamin. Tetrahedron 38:1193 1208. Giralt, E., Eritja, R., Pedroso, E., Granier, C., and van Rietschoten, J. (1986). Convergent solid phase peptide synthesis III: Synthesis of the 44-52 protected segment of the toxin II of Androctonus australis Hector. Tetrahedron 42:691-698. Gisin, B. F. (1973). The preparation of Merrifield-resins through total esterification with cesium salts. Helv. Chim. Acta 56:1476-1482. Gisin, B. F., and Merrifield, R. B. (1972). Carboxyl-catalyzed intramolecular aminolysis: A side reaction in solid-phase peptide synthesis. J. Am. Chem. Soc. 94:3102-3106. Goddard, P., McMurray, J. S., Sheppard, R. C., and Emson, P. (1988). A solubilisable polymer support suitable for solid phase peptide synthesis and for injection into experimental animals. J. Chem. Soc., Chem. Commun. 1025-1027. Gooding, O. W., Baudart, S., Deegan, D. L., Heisler, K., Labadie, J. W., Newcomb, W. S., Porco, J. A., Jr., and van Eikeren, P. (1999). On the development of new poly(styrene-oxyethylene) graft copolymer resin supports for solid-phase organic synthesis. J. Comb. Chem. 1:113122. Goodman, M., Felix, A., Moroder, L., and and Toniolo, C., eds. (2001). Houben-Weyl Volume E22b: Synthesis of Peptidomimetics, Georg Thieme Verlag, Stuttgart, Germany. Grab, B, Miles, A. J., Furcht, L. T., and and Fields, G. B. (1996). Promotion of fibroblast adhesion by triple-helical peptide models of type I collagenderived sequences. J. Biol. Chem. 271:12234-12240.
94
Synthetic Peptides
Grandas, A., Jorba, X., Giralt, E., and Pedroso, E. (1989). Anchoring of Fmoc-amino acids to hydroxymethyl resins. Int. J. Peptide Protein Res. 33:386-390. Gras-Masse, H., Ameisen, J. C., Boutillon, C., Gesquiere, J. C., Vian, S., Neyrinck, J. L., Drobecq, H., Capron, A., and Tartar, A. (1990). A synthetic protein corresponding to the entire vpr gene product from the human immunodeficiency virus HIV-1 is recognized by antibodies from HIV-infected patients. Int. J. Peptide Protein Res. 36:219-226. Gray, W. R., Luque, A., Galyean, R., Atherton, E., Sheppard, R. C., Stone, B. L., Reyes, A., Alford, J., Mclntosh, M., Olivera, B. M., Cruz, L. J., and Rivier, J. (1984). Contoxin GI: Bisulfide bridges, synthesis, and preparation of iodinated derivatives. Biochemistry 23:2796-2802. Green, J., Ogunjobi, O. M., Ramage, R., Stewart, A. S. J., McCurdy, S., and Noble, R. (1988). Application of the ArG-(2,2,5,7,8-pentamethylchroman6-sulphonyl) derivative of Fmoc-arginine to peptide synthesis. Tetrahedron Lett. 29:4341^344. Green, M., and Berman, J. (1990). Preparation of pentafluorophenyl esters of Fmoc protected amino acids with pentafluorophenyl trifluoroacetate. Tetrahedron Lett. 31:5851-5852. Greene, T. W. (1991). Protective Groups in Organic Synthesis, New York, John Wiley and Sons. Gross, H., and Bilk, L. (1968). Zur reaktion von JV-hydroxysuccinimid mil dicyclohexylcarbodiimid. Tetrahedron 24:6935-6939. Guibe, F., Dangles, O., Balavoine, G., and Loffet, A. (1989). Use of an allylic anchor group and of its palladium catalyzed hydrostannolytic cleavage in the solid phase synthesis of protected peptide fragments. Tetrahedron Lett. 30:2641-2644. Gutte, B., ed. (1995). Peptides: Synthesis, Structures, and Applications, Orlando, Fla., Academic Press. Gutte, B., and Merrifield, R. B. (1971). The synthesis of ribonuclease A. J. Biol. Chem. 246:1922-1941. Guttmann, S., and Boissonnas, R. A. (1959). Synthese de 1'a-melanotropine (a-MSH) de pore. Helv. Chim. Acta 42:1257-1264. Guy, C. A., and Fields, G. B. (1997). Trifluoroacetic acid cleavage and deprotection of resin-bound peptides following synthesis by Fmoc chemistry. Methods Enzymol. 289:67-83. Hahn, K. W., Klis, W. A., and Stewart, J. M. (1990). Design and synthesis of a peptide having chymotrypsin-like esterase activity. Science 248:1544-1547. Hammer, R. P., Albericio, F., Gera, L., and Barany, G. (1990). Practical approach to solid-phase synthesis of C-terminal peptide amides under mild conditions based on photolysable anchoring linkage. Int. J. Peptide Protein Res. 36:31-45. Han, Y., Sole, N., Tejbrant, T., and Barany, G. (1996a). Novel JV'-xanthenyl protecting groups for asparagine and glutamine, and applications to ./V"9-Fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis. Peptide Res. 9:166-173. Han, Y., Bontems, S. L., Heygens, P., Munson, M. C., Minor, C. A., Kates, S. A., Albericio, F., and Barany, G. (1996b). Preparation and application of xanthenylamide (XAL) handles for solid phase synthesis of C-terminal
195
peptide amides under particularly mild conditions. J. Org. Chem. 61:6326-6339. an, Y., Albericio, F., and Barany, G. (1997a). Occurrence and minimization of cysteine racemization during stepwise solid-phase peptide synthesis. J. Org. Chem. 62:4307^1312. an, Y., Vagner, J., and Barany, G. (1997b). A new side-chain anchoring strategy for solid-phase synthesis of peptide acids with C-terminal cysteine. In Innovation and Perspectives in Solid Phase Synthesis and Combinatorial Chemical Libraries: Biomedical and Biological Applications, 1996, R. Epton, ed., Kingswinford, Mayflower Scientific Ltd., pp. 385-388. ansen, P. R., Holm, A., and Houen, G. (1993). Solid-phase peptide synthesis on proteins. Int. J. Peptide Protein Res. 41:237-245. argittai, B., and Barany, G. (1999). Controlled syntheses of natural and disulfide-mispaired regioisomers of a-conotoxin SI. J. Peptide Res. 54:468^79. argittai, B., Sole, N. A., Groebe, D. R., Abramson, S. N., and Barany, G. (2000). Chemical syntheses and biological activities of lactam analogues of a-conotoxin SI. J. Med. Chem. 43:4787^1792. arrison, J. L., Petrie, G. M., Noble, R. L., Beilan, H. S., McCurdy, S. N., and Culwell, A. R. (1989). Fmoc chemistry: Synthesis, kinetics, cleavage, and deprotection of arginine-containing peptides. In Techniques in Protein Chemistry, T. E. Hugh, ed., San Diego, Calif., Academic Press, pp. 506516. eimer, E. P., Chang, C.-D., Lambros, T., and Meienhofer, J. (1981). Stable isolated symmetrical anhydrides of TV-fluorenylmethyloxycarbonylamino acids in solid-phase peptide synthesis. Int. J. Peptide Protein Res. 18:237241. ellermann, H., Lucas, H.-W., Maul, J., Pillai, V. N. R., and Mutter, M. (1983). Poly(ethylene glycol)s grafted onto crosslinked polystyrenes, 2: Multidetachably anchored polymer systems for the synthesis of solubilized peptides. Makromol. Chem. 184:2603-2617. endrix, J. C., and Lansbury, P. T. (1992). Synthesis of a protected peptide corresponding to residues 1-25 of the /3-amyloid protein of Alzheimer's disease. J. Org. Chem. 57:3421-3426. endrix, J. C., Jarrett, J. T., Anisfeld, S. T., and Lansbury, P. T. (1992). Studies related to a convergent fragment-coupling approach to peptide synthesis using the Kaiser oxime resin. J. Org. Chem. 57:3414-3420. enkel, W., Vogl, T., Echner, H., Voelter, W., Urbanke, C., Schleuder, D., and Rauterberg, J. (1999). Synthesis and folding of native collagen III model peptides. Biochemistry 38:13610-13622. iskey, R. G. (1981). Sulfhydryl Group Protection in Peptide Synthesis. In The Peptides, Vol. 3, E. Gross and J. Meienhofer, eds., New York, Academic Press, pp. 137-167. odges, R. S., and Merrifield, R. B. (1975). Monitoring of solid phase peptide synthesis by an automated spectrophotometric picrate method. Anal. Biochem. 65:241-272. oeprich, P. D., Jr., Langton, B. C., Zhang, J.-w., and Tarn, J. P. (1989). Identification of immunodominant regions of transforming growth factor a: implications of structure and function. J. Biol. Chem. 264:19086-19091.
Synthetic Peptides
mes, C. P.. and Jones, D. G. (1995). Reagents for combinatorial organic synthesis: Development of a new onitrobenzyl photolabile linker for solid phase synthesis. J. Org. Chem. 60:2318-2319. rn, M., and Novak, C. (1987). A monitoring and control chemistry for solid-phase peptide synthesis. Am. Biotech. Lab. 5 (September/ October): 12-21. ughten, R. A., and Li, C. H. (1979). Reduction of sulfoxides in peptides and proteins. Anal. Biochem. 98:36^6. ughten, R. A., DeGraw, S. T., Bray, M. K., Hoffmann, S. R., and Frizzell, N. D. (1986). Simultaneous multiple peptide synthesis: The rapid preparation of large numbers of discrete peptides for biological, immunological, and methodological studies. BioTechniques 4:522 528. uby, V. J., Al-Obeidi, F., Sanderson, D. G., and Smith, D. D. (1990). Synthesis of cyclic peptides by solid phase methods. In Innovation and Perspectives in Solid Phase Synthesis, R. Epton, ed., Birmingham, UK, Solid Phase Conference Coordination, Ltd., pp. 197-203. dson, D. (1988). Methodological implications of simultaneous solid-phase peptide synthesis 1: Comparison of different coupling procedures. J. Org. Chem. 53:617-624. dson, D. (1990a). New logically developed active esters for solid-phase peptide synthesis. In Peptides: Chemistry, Structure and Biology, J. E. Rivier and G. R. Marshall, eds., Leiden, Escom, pp. 914-915. dson, D. (1990b). Methodological implications of simultaneous solid-phase peptide synthesis: A comparison of active esters. Peptide Res. 3:51-55. dson, D. (1990c). Protecting groups for asparagine and glutaminc in peptide synthesis. US Patent 4,935,536. dson, D. (1999). Matrix supported synthetic transformations. J. Comb. Chem. Part I. 1:333-360; Part II. 1:403-457. dson, D., Kain, D., and Ng, D. (1986). High yielding fully automatic synthesis of cecropin A amide & analogues. In Peptide Chemistry 1985, Y. Kiso, ed., Osaka, Protein Research Foundation, pp. 413-^18. de, C., Johnson, T.. and Sheppard, R. C. (1992). Internal aggregation during solid phase peptide synthesis: Dimethyl sulfoxide as a powerful dissociating solvent. J. Chem. Soc., Chem. Commun. 1573-1575. de, C., Johnson, T.. Owen, D., Quibell, M., and Sheppard, R. C. (1994). Some "difficult sequences" made easy: A study of interchain association in solid-phase peptide synthesis. Int. J. Peptide Protein Res. 43:431^40. da, S., Yokota, T., Watanabe, K., Kan, M., Takahashi, Y., Ichikawa, T., Takahashi, K., and Matsueda, R. (1986). Solid phase peptide synthesis by the use of 3-nitro-2-pyridinesulfenyl (Npys)-amino acids. In Peptide Chemistry 1985, Y. Kiso, ed., Osaka, Protein Research Foundation, pp. 115-120. enito, R., Bianchi, E., Fattori, D., and Pessi, A. (1999). Solid phase synthesis of peptide C-terminal thioesters by Fmoc/f-Bu chemistry. J. Am. Chem. Soc. 121:11369-11374. iguro, T., and Eguchi, C. (1989). Unexpected chain-terminating side reaction caused by histidine and acetic anhydride in solid-phase peptide synthesis. Chem. Pharm. Bull. 37:506-508.
Jaeger, E., Thamm, P., Knof, S., Wtinsch, E., Low, M., and Kisfaludy, L. (1978a). Nebenreaktionen bei peptidsynthesen III: Synthese und charakterisierung von N'"-tert-b\ily\'\erten tryptophan-derivaten. Hoppe-Seyler's Z. Physiol. Chem. 359:1617-1628. Jaeger, E., Thamm, P., Knof, S., and Wiinsch, E. (1978b). Nebenreaktionen bei peptidsynthesen IV: Charakterisierung von C- und C,jV-/erf-butylierten Iryptophan-derivaten. Hoppe-Seyler's Z. Physiol. Chem. 359:1629-1636. Jaeger. E., Remmer, H. A., Jung, G., Metzger, J.. Oberthuer, W., Riicknagel, K. P., Schaefer, W., Sonnenbichler, J., and Zeti, \. (1993). Side reactions in peptide synthesis V: O-sulfonation of serine and threonine during removal of the Pmc and Mtr protecting groups from arginine residues in Fmoc solid phase synthesis. Biol. Chem. Hoppe-Seyler 347:349-362. Jansson, A. M., Meklal, M., and Bock, K. (1990). The active ester N-Fmoc-3O-[Ac4-a-D-Man/)-(l > 2)-Ac 3 -a-D-Man/>-1-]-threonine-O-Pfp as a building block in solid-phase synthesis of an O-linked dimannosyl glycopeptide. Tetrahedron Lett. 31:6991-6994. Jensen, K. J. Hansen, P. R., Venugopal, D.. and Barany, G. (1996). Synthesis of 2-acctamido-2-deoxy-/?-D-glucopyranose O-glycopeptides from Ndithiasuccinoyl-protected derivatives. J. Am. Chem. Soc. 118:3148-3155. Jensen, K. J., Alsina, J., Songster, M. F., Vagner, J., Albericio, F., and Barany. G. (1998). Backbone amide linker (BAL) strategy for solidphase synthesis of C-terminal-modified and cyclic peptides. J. Am. Chem. Soc. 120:5441-5452. Johnson, C. R., Biancalana, S., Hammer, R. P., Wright, P. B., and Hudson. D. (1992). New active esters and coupling reagents based on pyrazolinones. In Peptides: Chemistry and Biology, J. A. Smith and J. E. Rivier, eds., Leiden, Escom, pp. 585-586. Johnson, T., Quibell, M., Owen, D., and Sheppard, R. C. (1993). A reversible protecting group for the amide bond in peptides: Use in the synthesis of "difficult sequences." J. Chem. Soc., Chem. Commun. 369-372. Jones, J. H., Ramage, W. I., and Witty, M. J. (1980). Mechanism of racemization of histidine derivatives in peptide synthesis. Int. J. Peptide Protein Res. 15:301-303. Kaiser, E., Colescott, R. L., Bossinger, C. D., and Cook, P. I. (1970). Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 34:595-598. Kaiser, E., Bossinger, C. D., Colescott, R. L., and Olsen, D. B. (1980). Color test for terminal prolyl residues in the solid-phase synthesis of peptides. Anal. Chim. Acta 118:149-151. Kaiser, E. T., Mihara, H., Laforet, G. A., Kelly, J. W., Walters, L., Findeis, M. A., and Sasaki, T. (1989). Peptide and protein synthesis by segment synthesis-condensation. Science 243:187-192. Kamber, B., Hartmann, A., Eisler, K., Riniker, B., Rink, H., Sieber, P., and Rittel, W. (1980). The synthesis of cystine peptides by iodine oxidation of 5-trityl-cysteine and S-acetamidomethyl-cysteine peptides. Helv. Chim. Acta 63:899-915. Kanda, P., Kennedy, R. C., and Sparrow, J. T. (1991). Synthesis of polyamide supports for use in peptide synthesis and as peptide-resin conjugates for antibody production. Int. J. Peptide Protein Res. 38:385-391.
Synthetic Peptides
tes, S. A., and Albericio, F., eds. (2000). Solid-Phase Peptide Synthesis: A Practical Guide, Marcel Dekker, New York. tes, S. A., Daniels, S. B., and Albericio, F. (1993a). Automated allyl cleavage for continuous flow synthesis of cyclic and branched peptides. Anal. Biochem. 212:303-310. tes, S. A., Sole, N. A., Johnson, C. R., Hudson, D., Barany, G., and Albericio, F. (1993b). A novel, convenient, three-dimensional orthogonal strategy for solid-phase synthesis of cyclic peptides. Tetrahedron Lett. 34:1549-1552. tes, S. A., Sole, N. A., Albericio, F., and Barany, G. (1994). Solid-phase synthesis of cyclic peptides. In Peptides: Design, Synthesis and Biological Activity, C. Basava and G. M. Anantharamaiah, eds., Boston, Birkhaeuser, pp. 39-57. tes, S. A., Sole, N. A., Beyermann, M., Barany, G., and Albericio, F. (1996). Optimized preparation of deca(L-alanyl)-L-valinamide by 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase synthesis on polyethylene glycol-polystyrene (PEG-PS) graft supports, with l,8-diazobicyclo[5. 4.0]undec-7-ene (DBU) deprotection. Peptide Res. 9:106-113. tes, S. A., McGuinness, B. F., Blackburn, C., Griffin, G. W., Sole, N. A., Barany, G., and Albericio. F. (1998). "High-load" polyethylene glycolpolystyrene (PEG-PS) graft supports for solid-phase synthesis. Biopolymers 47:365-380. umaya, P. T. P., Van Buskirk, A. M., Goldberg, E., and Pierce, S. K. (1992). Design and immunological properties of topographic immunogenic determinants of a protein antigen (LDH-C^ as vaccines. J. Biol. Chem. 267:6338-6346. wasaki, K., Miyano, M., Murakami, T., and Kakimi, M. (1989). Amino acids and peptides XI: Simple preparation of A^-protected histidine. Chem. Pharm. Bull. 37:3112 3113. arney, T., and Giles, J. (1989). Fmoc peptide synthesis with a continuous flow synthesizer. Am. Biotech. Lab. 7(9), October:34-44. mp, D. S., Fotouhi, N., Boyd, J. G., Carey, R. I., Ashton, C., and Hoare, J. (1988). Practical preparation and deblocking conditions for N-a-(2-(pbiphenylyl)-2-propyloxycarbonyl)-amino acid (JV-a-Bpoc-XXX-OH) derivatives. Int. J. Peptide Protein Res. 31:359-372. mpe, M., and Barany, G. (1996). CLEAR: A novel family of highly crosslinked polymeric supports for solid-phase peptide synthesis. J. Am. Chem. Soc. 118:7083-7093. nnedy, R. C., Dreesman, G. R., Chanh, T. C., Boswell, R. N., Allan, J. S., Lee, T.-H., Essex, M., Sparrow, J. T., Ho, D. D., and Kanda, P. (1987). Use of a resin-bound synthetic peptide for identifying a neutralizing antigenie determinant associated with the human immunodeficiency virus envelope. J. Biol. Chem. 262:5769-5774. nner, G. W., and Seely, J. H. (1972). Phenyl esters for C-terminal protection in peptide synthesis. J. Am. Chem. Soc. 94:3259-3260. nner, G. W., Galpin, I. J., and Ramage, R. (1979). Synthetic studies directed towards the synthesis of a lysozyme analog. In Peptides: Structure and Biological Function, E. Gross and J. Meienhofer, eds., Rockford, 111., Pierce Chemical Co., pp. 431^38.
199
Kent, J. J., Alewood, P., and Kent, S. B. H. (1991). Peptide synthesis using Fmoc amino acids stored in DMF solution for prolonged periods. In Twelfth American Peptide Symposium Program and Abstracts, Cambridge, Mass., Massachusetts Institute of Technology, P-386. Kent, S. B. H. (1983). Chronic formation of acylation-resistant deletion pep tides in stepwise solid phase pep tide synthesis: Chemical mechanism, occurrence, and prevention. In Peptides: Structure and Function, V. J. Hruby and D. H. Rich, eds., Rockford, 111., Pierce Chemical Co., pp. 99-102. Kent, S. B. H. (1988). Chemical synthesis of peptides and proteins. Ann. Rev. Biochem. 57:957-989. Kent, S. B. H., and Merrifield, R. B. (1978). Preparation and properties of tertbutyloxycarbonylaminoacyl-4-(oxymethyl)phenylacetamidomethyl-(Kel F-g-styrene) resin, an insoluble, noncrosslinked support for solid phase peptide synthesis. Isr. J. Chem. 17:243-247. Kent, S. B. H., and Parker, K. F. (1988). The chemical synthesis of therapeutic peptides and proteins. In Banbury Report 29: Therapeutic Peptides and Proteins: Assessing the New Technologies, D. R. Marshak, and D. T. Liu, eds., New York, Cold Spring Harbor, pp. 3-16. Kent, S. B. H., Riemen, M., LeDoux, M., and Merrifield, R. B. (1982). A study of the Edman degradation in the assessment of the purity of synthetic peptides. In Methods in Protein Sequence Analysis, M. Elzinga, ed., Clifton, N.J., Humana Press, pp. 205-213. Kihlberg, J., Elofsson, M., and Salvador, L. A. (1997). Direct synthesis of glycosylated amino acids from carbohydrate preacetates and Fmocamino acids: Solid-phase synthesis of biomedically interesting glycopeptides. Methods Enzymol. 289:245-266. King, D. S., Fields, C. G., and Fields, G. B. (1990). A cleavage method for minimizing side reactions following Fmoc solid phase peptide synthesis. Int. J. Peptide Protein Res. 36:255-266. Kirstgen, R., and Steglich, W. (1989). Fmoc-amino acid-TDO esters as reagents for peptide coupling and anchoring in solid phase synthesis. In Peptides 1988, G. Jung and E. Bayer, eds., Berlin, Walter de Gruyter & Co., pp. 148-150. Kirstgen, R., Sheppard, R. C., and Steglich, W. (1987). Use of esters of 2,5diphenyl-2,3-dihydro-3-oxo-4-hydroxythiophene dioxide in solid phase peptide synthesis: A new procedure for attachment of the first amino acid. J. Chem. Soc., Chem. Commun. 1870-1871. Kirstgen, R., Olbrich, A., Rehwinkel, H., and Steglich, W. (1988). Ester von 7V-(9-fluorenylmethyloxycarbonyl)aminosauren mit 4-hydroxy-3-oxo-2,5diphenyl-2,3-dihydrothiophen-1,1 -dioxid (Fmoc-aminosaure-TDO-ester) und ihre verwendung zur festphasenpeptidsynthese. Liebigs Ann. Chem. 437^40. Kisfaludy, L., and Schon, I. (1983). Preparation and applications of pentafluorophenyl esters of 9-fluorenylmethyloxycarbonyl amino acids for peptide synthesis. Synthesis 325-327. Kisfaludy, L., Low, M., Nyeki, O., Szirtes, T., and Schon, I. (1973). Die verwendung von pentafluorophenylestern bei peptid-synthesen. Justus Liebigs Ann. Chem. 1421-1429.
Synthetic Peptides
so, Y., Yoshida, M., Tatsumi, T., Kimura, T., Fujiwara, Y., and Akaji, K. (1989). Tetrafluoroboric acid, a useful deprotecting reagent in peptide synthesis. Chem. Pharm. Bull. 37:3432-3434. so, Y., Yoshida, M., Fujiwara, Y., Kimura, T., Shimokura, M., and Akaji, K. (1990). Trimethylacetamidomethyl (Tacm) group, a new protecting group for the thiol function of cysteine. Chem. Pharm. Bull. 38:673-675. as, E. A., Perich, J. W., Wade, J. D., Johns, R. B., and Tregear, G. W. (1989). Fmoc-polyamide solid phase synthesis of an 0-phosphotyrosinecontaining tridecapeptide. Tetrahedron Lett. 30:6229-6232. as, E. A., Wade, J. D., Johns, R. B., Perich, J. W., and Tregear, G. W. (1991). Preparation and use of A^-fluorenylmethoxycarbonyl-O-dibenzylphosphono-L-tyrosine in continuous flow solid phase peptide synthesis. J. Chem. Soc., Chem. Commun. 338-339. eib-Cordonier, N., Albericio, F., and Barany, G. (1990). Orthogonal solidphase synthesis of human gastrin-I under mild conditions. Int. J. Peptide Protein Res. 35:527^538. orr, R., Trzeciak, A., Bannwarth, W., and Gillessen, D. (1991). 1,1,3,3Tetramethyluronium compounds as coupling reagents in peptide and protein chemistry. In Peptides 1990, E. Giralt and D. Andreu, eds., Leiden, Escom, pp. 62-64. chersperger, M. L., Blacher, R., Kelly, P., Pierce, L., and Hawke, D. H. (1989). Sequencing of peptides on solid phase supports. Am. Biotech. Lab. 7(3), March:26-37. eners, H. J., Schattenkerk, C., Verhoeven, J. J., and van Boom, J. H. (1981). Synthesis of 0-(2-0-a-D-glucopyranosyl)-/6-D-galactopyranoside of optically pure <5-hydroxy-L-lysylglycine and 5-hydroxy-L-lysylglycyl-Lglutamyl-L-aspartylglycine. Tetrahedron 37:1763-1771. nig, W., and Geiger, R. (1970a). Eine neue methode zur synthese von peptiden: Aktivierung der carboxylgruppe mit dicyclohexylcarbodiimid unter zusatz von 1-hydroxy-benzotriazolen. Chem. Ber. 103:788-798. nig, W., and Geiger, R. (1970b). Racemisierung bei peptidsynthesen. Chem. Ber. 103:2024-2033. nig, W., and Geiger, R. (1970c). Eine neue methode zur synthese von peptiden: Aktivierung der carboxylgruppe mit dicyclohexylcarbodiimid und 3-hydroxy-4-oxo-3,4-dihydro-1.2.3-benzotriazin. Chem. Ber. 103:2034-2040. nig, W., and Geiger, R. (1973). /V-hydroxyverbindungen als katalysatoren fur die aminolyse aktivierter ester. Chem. Ber. 106:3626-3635. chnak, V., Vagner, J., Safar, P., and Lebl, M. (1988). Noninvasive continuous monitoring of solid-phase peptide synthesis by acid-base indicator. Coll. Czech. Chem. Commun. 53:2542-2548. llmann, W., and Gutte, B. (1978). Synthesis of an open-chain asymmetrical cystine peptide corresponding to the sequence A18~2t-B19'26 of bovine insulin by solid phase fragment condensation. Int. J. Peptide Protein Res. 12:17-26. magaye, K. Y., Inui, T., Nakajima, K., Kimura, T., and Sakakibara, S. (1991). Suppression of a side reaction associated with 7V'ra-benzyloxymethyl group during synthesis of peptides containing cysteinyl residue at the TV-terminus. Peptide Res. 4:84-87.
201
unz, H. (1987). Synthesis of glycopeptides: Partial structures of biological recognition components. Angew. Chem. Int. Ed. Engl. 26:294308. unz, H. (1990). Allylic anchoring groups in the solid phase synthesis of peptides and glycopeptides. In Innovation and Perspectives in Solid Phase Synthesis, R. Epton, ed., Birmingham, UK, Solid Phase Conference Coordination, Ltd., pp. 371-378. unz, H., and Dombo, B. (1988). Solid phase synthesis of peptides and glycopeptides on polymeric supports with allylic anchor groups. Angew. Chem. Int. Ed. Engl. 27:711-713. usunoki, M., Nakagawa, S., Seo, K., Hamana, T., and Fukuda, T. (1990). A side reaction in solid phase synthesis: Insertion of glycine residues into peptide chains via Nim -> N01 transfer. Int. J. Peptide Protein Res. 36:381386. joie, G., Crivici, A., and Adamson, J. G. (1990). A simple and convenient synthesis of w-tert-butyl esters of Fmoc-aspartic and Fmoc-glutamic acids. Synthesis 571-572. amthanh, H., Roumestand, C., Deprun, C., and Menez, A. (1993). Side reaction during the deprotection of (S-acetamidomethyl)cysteine in a peptide with a high serine and threonine content. Int. J. Peptide Protein Res. 41:85-95. ansbury, P. T., Jr., Hendrix, J. C., and Coffman, A. I. (1989). A practical method for the preparation of protected peptide fragments using the Kaiser oxime resin. Tetrahedron Lett. 30:4915-4918. rsen, B. D., Larsen, C., and Holm, A. (1991). Incomplete Fmoc-deprotection in solid phase synthesis. In Peptides 1990, E. Giralt and D. Andreu, eds., Leiden, Escom, pp. 183-185. patsanis, L., Milias, G., Froussios, K., and Kolovos, M. (1983). Synthesis of Ar-2,2,2-(trichloroethoxycarbonyl)-L-amino acids and Ar-(9-fluorenylmethoxycarbonyl)-L-amino acids involving succinimidoxy anion as a leaving group in amino acid protection. Synthesis 671-673. uer, J. L., Fields, C. G., and Fields, G. B. (1995). Sequence dependence of aspartimide formation during 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis. Lett. Peptide Sci. 1:197-205. auer, J. L., Furcht, L. T., and Fields, G. B. (1997). Inhibition of melanoma cell binding to type IV collagen by analogs of cell adhesion regulator. J. Med. Chem. 40:3077-3084. ebl, M., and Eichler, J. (1989). Simulation of continuous solid phase synthesis: Synthesis of methionine enkephalin and its analogs. Peptide Res. 2:297-300. , C. H., Lemaire, S., Yamashiro, D., and Doneen, B. A. (1976). The synthesis and opiate activity of /3-endorphin. Biochem. Biophys. Res. Commun. 71:19-25. , X., Kawakami, T., and Aimoto, S. (1998). Direct preparation of peptide thioesters using an Fmoc solid-phase method. Tetrahedron Lett. 39:86698672. mal, D., Briand, J.-P., Dalbon, P., and Jolivet, M. (1998). Solid-phase synthesis and on-resin cyclization of a disulfide bond peptide and lactam analogues corresponding to the major antigenic site of HIV gp41 protein. J. Peptide Res. 52:121-129.
Synthetic Peptides
n, Y.-Z., Caporaso, G., Chang, P.-Y., Ke, X.-H., and Tarn, J. P. (1988). Synthesis of a biological active tumor growth factor from the predicted DNA sequence of Shope fibroma virus. Biochemistry 27:5640-5645. u, C.-F., and Tarn, J. P. (1994). Peptide segment ligation strategy without use of protecting groups. Proc. Natl. Acad. Sci. USA 91:6584-6588. u, C. F., Rao, C., and Tarn, J. P. (1996). Orthogonal ligation of unprotected peptide segments through pseudoproline formation for the synthesis of HIV-1 protease. J. Am. Chem. Soc. 118:307-312. u, Y.-Z., Ding, S.-H., Chu, J.-Y., and Felix, A. M. (1990). A novel Fmocbased anchorage for the synthesis of protected pep tides on solid phase. Int. J. Peptide Protein Res. 35:95-98. ve, D. H., and Kent, S. B. H. (1982). Fundamental aspects of the chemical applications of cross-linked polymers. In Elastomers and Rubber Elasticity, J. E. Mark and J. Lai, eds., Washington, D.C., American Chemical Society, pp. 501-515. ve, D. H., and Kent, S. B. H. (1983). Correlation of coupling rates with physicochemical properties of resin-bound peptides in solid phase synthesis. In Peptides: Structure and Function, V. J. Hruby and D. H. Rich, eds., Rockford, 111., Pierce Chemical Co., pp. 65-68. oyd-Williams, P., Jou, G., Albericio, F., and Giralt, E. (1991). Solid-phase synthesis of peptides using allylic anchoring groups: An investigation of their palladium-catalysed cleavage. Tetrahedron Lett. 32:4207^4210. oyd-Williams, P., Albericio, F., and Giralt, E., eds. (1997). Chemical Approaches to the Synthesis of Peptides and Proteins, Boca Raton, Fla., CRC Press. ffet, A., Galeotti, N., Jouin, P., and Castro, B. (1989). Tert-butyl esters of TV-protected amino acids with tert-buty\ fluorocarbonate (Boc-F). Tetrahedron Lett. 30:6859-6860. w, M., Kisfaludy, L., Jaeger, E., Thamm, P., Knof, S., and Wiinsch, E. (1978a). Direkte terf-butylierung des tryptophans: Herstellung von 2,5,7tri-ter?-butyltryptophan. Hoppe-Seyler's Z. Physiol. Chem. 359:16371642. w, M., Kisfaludy, L., and Sohar, P. (1978b). terf-Butylierung des tryptophanindolringes wahrend der abspaltung der terf-butyloxycarbonyl-gruppe bei peptidsynthesen. Hoppe-Seyler's Z. Physiol. Chem. 359:1643-1651. , G.-s., Mojsov, S., Tam, J. P., and Merrifield, R. B. (1981). Improved synthesis of 4-alkoxybenzyl alcohol resin. J. Org. Chem. 46:3433-3436. dwick, A. G., Jelinski, L. W., Live, D., Kintanar, A., and Dumais, J. J. (1986). Association of peptide chains during Merrifield solid-phase peptide synthesis: A deuterium NMR study. J. Am. Chem. Soc. 108:6493-6496. kszo, J., Patterson, D., Albericio, F., and Kates, S. A. (1996). 3-(lPiperidinyl)alanine formation during the preparation of C-terminal cycteine peptides with Fmoc/?-Bu strategy. Lett. Peptide Sci. 3:157-166. ndt, B. F., Johansen, N. L., Volund, A., and Markussen, J. (1978). Removal of J-butyl and ?-butoxycarbonyl protecting groups with trifluoroacetic acid. Int. J. Peptide Protein Res. 12:258-268. ttle, M. H., and Hudson, D. (1992). Allyl based side-chain protection for SPPS. In Peptides: Chemistry and Biology, J. A. Smith and J. E. Rivier, eds., Leiden, Escom, pp. 583-584.
203
Malkar, N. B., Lauer-Fields, J. L., and Fields, G. B. (2000). Convenient synthesis of glycosylated hydroxylysine derivatives for use in solidphase synthesis. Tetrahedron Lett. 41:1137-1140. Masui, Y., Chino, N., and Sakakibara, S. (1980). The modification oftryptophyl residues during the acidolytic cleavage of Boc-groups I: Studies with Boc-tryptophan. Bull. Chem. Soc. Jpn. 53:464-468. Matsueda, G. R., and Haber, E. (1980). The use of an internal reference amino acid for the evaluation of reactions in solid-phase peptide synthesis. Anal. Biochem. 104:215-227. Matsueda, G. R., and Stewart, J. M. (1981). A /?-methylbenzhydrylamine resin for improved solid-phase synthesis of peptide amides. Peptides 2:45-50. Matsueda, G. R., Haber, E., and Margolies, M. N. (1981). Quantitative solidphase Edman degradation for evaluation of extended solid-phase peptide synthesis. Biochemistry 20:2571-2580. Matsueda, R., and Walter, R. (1980). 3-nitro-2-pyridinesulfenyl (Npys) group: A novel selective protecting group which can be activated for peptide bond formation. Int. J. Peptide Protein Res. 16:392-401. McCafferty, D. G., Slate, C. A., Nakhle, B. M., Graham, Jr., H. D., Austell, T. L., Vachet, R. W., Mullis, B. H., and Erickson, B. W. (1995). Engineering of a 129-residue tripod protein by chemoselective ligation of proline-II helices. Tetrahedron 51:9859-9872. McCurdy, S. N. (1989). The investigation of Fmoc-cysteine derivatives in solid phase peptide synthesis. Peptide Res. 2:147-151. McFerran, N. V., Walker, B., McGurk, C. D., and Scott, F. C. (1991). Conductance measurements in solid phase peptide synthesis I: Monitoring coupling and deprotection in Fmoc chemistry. Int. J. Peptide Protein Res. 37:382-387. Meienhofer, J. (1985). Protected amino acids in peptide synthesis. In Chemistry and Biochemistry of the Amino Acids, G. C. Barrett, ed., London, Chapman & Hall, pp. 297-337. Meienhofer, J., Waki, M., Heimer, E. P., Lambros, T. J., Makofske, R. C., and Chang, C.-D. (1979). Solid phase synthesis without repetitive acidolysis. Int. J. Peptide Protein Res. 13:3512. Meister, S. M., and Kent, S. B. H. (1983). Sequence-dependent coupling problems in stepwise solid phase peptide synthesis: Occurrence, mechanism, and correction. In Peptides: Structure and Function, V. J. Hruby and D. H. Rich, eds., Rockford, 111., Pierce Chemical Co., pp. 103-106. Meldal, M. (1992). A flow stable polyethylene glycol dimethyl acrylamide copolymer for solid phase synthesis. Tetrahedron Lett. 33:3077-3080. Meldal, M. (1994). Glycopeptide synthesis. In Neoglycoconjugates: Preparation and Applications, Y. C. Lee and R. T. Lee, eds., San Diego, Calif., Academic Press, pp. 145-198. Meldal, M., and Jensen, K. J. (1990). Pentafluorophenyl esters for the temporary protection of the a-carboxy group in solid phase glycopeptide synthesis. J. Chem. Soc., Chem. Commun. 483-485. Meldal, M., Bielfeldt, T., Peters, S., Jensen, K. J., Paulsen, H., and Bock, K. (1994). Susceptibility of glycans to ^-elimination in Fmoc-based Oglycopeptide synthesis. Int. J. Protein Res. 43:529-536.
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Synthetic Peptides
Mergler, M., Tanner, R., Gosteli, J., and Grogg, P. (1988a). Peptide synthesis by a combination of solid-phase and solution methods I: A new very acid-labile anchor group for the solid phase synthesis of fully protected fragments. Tetrahedron Lett. 29:4005^008. Mergler, M., Nyfeler, R., Tanner, R., Gosteli, J., and Grogg, P. (1988b). Peptide synthesis by a combination of solid-phase and solution methods II: Synthesis of fully protected peptide fragments on 2-methoxy-4-alkoxybenzyl alcohol resin. Tetrahedron Lett. 29:4009^012. Mergler, M., Nyfeler, R., and Gosteli, J. (1989a). Peptide synthesis by a combination of solid-phase and solution methods III: Resin derivatives allowing minimum-racemization coupling of ^"-protected amino acids. Tetrahedron Lett. 30:6741-6744. Mergler, M., Nyfeler, R., Gosteli, J., and Tanner, R. (1989b). Peptide synthesis by a combination of solid-phase and solution methods IV: Minimumracemization coupling of 7VQf-9-fluorenylmethyloxycarbonyl amino acids to alkoxy benzyl alcohol type resins. Tetrahedron Lett. 30:6745-6748. Merrifield, B. (1986). Solid phase synthesis. Science 232:341-347. Merrifield, R. B., and Bach, A. E. (1978). 9-(2-Sulfo)fluorenylmethyloxycarbonyl chloride, a new reagent for the purification of synthetic peptides. J. Org. Chem. 43:4808-4816. Merrifield, R. B., Stewart, J. M., and Jernberg, N. (1966). Instrument for automated synthesis of peptides. Anal. Chem. 38:1905-1914. Merrifield, R. B., Mitchell, A. R., and Clarke, J. E. (1974). Detection and prevention of urethane acylation during solid-phase peptide synthesis by anhydride methods. J. Org. Chem. 39:660-668. Merrifield, R. B., Vizioli, L. D., and Boman, H. G. (1982). Synthesis of the antibacterial peptide cecropin A(l-33). Biochemistry 21:5020-5031. Merrifield, R. B., Singer, J., and Chait, B. T. (1988). Mass spectrometric evaluation of synthetic peptides for deletions and insertions. Anal. Biochem. 174:399^14. Mery, J., and Calas, B. (1988). Tryptophan reduction and histidine racemization during deprotection by catalytic transfer hydrogenation of an analog of the luteinizing hormone releasing factor. Int. J. Peptide Protein Res. 31:412-419. MilliGen/Biosearch (1990). Flow rate programming in continuous flow peptide synthesizer solves problematic synthesis. MilliGen/Biosearch Report 7, MilliGen/Biosearch Division of Millipore, Bedford, Mass., pp. 4-5, 11. Milton, S. C. F., Brandt, W. F., Schnolzer, M., and de Lisle Milton, R. C. (1992). Total solid-phase synthesis and prolactin-inhibiting activity of the gonadotropin-releasing hormone precursor protein and the gonadotropin-releasing hormone associated peptide. Biochemistry 31:8799-8809. Miranda, L. P., and Alewood, P. F. (1999). Accelerated chemical synthesis of peptides and small proteins. Proc. Natl. Acad. Sci. USA 96:1181-1186. Mitchell, A. R., Kent, S. B. H., Engelhard, M., and Merrifield, R. B. (1978). A new synthetic route to ter/-butyloxycarbonylaminoacyl-4-(oxymethyl)phenylacetamidomethyl-resin, an improved support for solid-phase peptide synthesis. J. Org. Chem. 43:2845-2852. Mitchell, M. A., Runge, T. A., Mathews, W. R., Ichhpurani, A. K., Harn, N. K., Dobrowolski, P. J., and Eckenrode, F. M. (1990). Problems
associated with use of the benzyloxymethyl protecting group for histidines: Formaldehyde adducts formed during cleavage by hydrogen fluoride. Int. J. Peptide Protein Res. 36:350-355. Mojsov, S., Mitchell, A. R., and Merrifield, R. B. (1980). A quantitative evaluation of methods for coupling asparagine. J. Org. Chem. 45:555560. Mott, A. W., Slomczynska, U., and Barany, G. (1986). Formation of sulfursulfur bonds during solid-phase peptide synthesis: Application to the synthesis of oxytocin. In Forum Peptides Le Cap d'Agde 1984, B. Castro and J. Martinez, eds., Nancy, Les Impressions Dohr, pp. 321-324. Muir, T. W., Williams, M. J., Ginsberg, M. H., and Kent, S. B. H. (1994). Design and chemical synthesis of a neoprotein structural model for the cytoplasmic domain of a multisubunit cell-surface receptor: Integrin arllb ft (platelet GPIIb-IIIa). Biochemistry 33:7701-7708. Muir, T. W., Dawson, P. E., and Kent, S. B. H. (1997). Protein synthesis by chemical ligation of unprotected peptides in aqueous solution. Methods Enzymol. 289:266-298. Munson, M. C., and Barany, G. (1993). Synthesis of a-conotoxin SI, a bicyclic tridecapeptide amide with two disulfide bridges: Illustration of novel protection schemes and oxidation strategies. J. Am. Chem. Soc. 115:1020310210. Munson, M. C., Garcia-Echeverria, C., Albericio, F., and Barany, G. (1992). 5'-2,4,6-trimethoxybenzyl (Tmob): A novel cysteine protecting group for the -/V"-9-fluorenylmethyloxycarbonyl (Fmoc) strategy of peptide synthesis. J. Org. Chem. 57:3013-3018. Mutter, M., and Bellof, D. (1984). A new base-labile anchoring group for polymer-supported peptide synthesis. Helv. Chim. Acta 67:2009-2016. Mutter, M., Altmann, K.-H., Bellof, D., Florsheimer, A., Herbert, J., Huber, M., Klein, B., Strauch, L., and Vorherr, T. (1985). The impact of secondary structure formation in peptide synthesis. In Peptides: Structure and Function, C. M. Deber, V. J. Hruby, and K. D. Kopple, eds., Rockford, 111., Pierce Chemical Co., pp. 397^05. Nagase, H., Fields, C. G., and Fields, G. B. (1994). Design and characterization of a fluorogenic substrate selectively hydrolyzed by stromelysin 1 (matrix metalloproteinase-3). J. Biol. Chem. 269:20952-20957. Nagata, K., Nakajima, K., Wakamiya, T., Togashi, R., Nishida, T., Saruta, K., Yasuoka, J., Kusumoto, S., Aimoto, S., and Kumagaye, K. Y. (1997). Synthetic study of phosphopeptides related to heat shock protein HSP27. Bioorg. Med. Chem. 5:135-145. Nakagawa, S. H., Lau, H. S. H., Kezdy, F. J., and Kaiser, E. T. (1985). The use of polymer-bound oximes for the synthesis of large peptides usable in segment condensation: Synthesis of a 44 amino acid amphiphilic peptide model of apolipoprotein A-l. J. Am. Chem. Soc. 107:7087 7092. Narita, M., and Kojima, Y. (1989). The /3-sheet structure-stabilizing potential of twenty kinds of amino acid residues in protected peptides. Bull. Chem. Soc. Jpn. 62:3572-3576. Narita, M., Umeyama, H., and Yoshida, T. (1989). The easy disruption of the /0-sheet structure of resin-bound human proinsulin C-peptide fragments by strong electron-donor solvents. Bull. Chem. Soc. Jpn. 62:3582-3586.
206
Synthetic Peptides
Nefzi, A., Sun, X., and Mutter, M. (1995). Chemoselective ligation of multifunctional peptides to topological templates via thioether formation for TASP synthesis. Tetrahedron Lett. 36:229-230. Nicolas, E., Pedroso, E., and Giralt, E. (1989). Formation of aspartimide peptides in Asp-Gly sequences. Tetrahedron Lett. 30:497-500. Nielson, C. S., Hansen, P. H., Lihme, A., and Heegaard, P. M. H. (1989). Real time monitoring of acylations during solid phase peptide synthesis:A method based on electrochemical detection. J. Biochem. Biophys. Methods 20:69-75. Nishio, H., Kimura, T., and Sakakibara, S. (1994). Side reaction in peptide synthesis:Modification of tryptophan during treatment with mercury(II) acetate/2-mercaptoethanol in aqueous acetic acid. Tetrahedron Lett. 35:1239-1242. Nishiuchi, Y., Nishio, H., Inui, T., Kimura, T., and Sakakibara, S. (1996). NinCyclohexyloxycarbonyl group as a new protecting group for tryptophan. Tetrahedron Lett. 37:7529-7532. Noble, R. L., Yamashiro, D., and Li, C. H. (1976). Synthesis of a nonadecapeptide corresponding to residues 37-55 of ovine prolactin: Detection and isolation of the sulfonium form of methionine-containing peptides. J. Am. Chem. Soc. 98:2324-2328. Nokihara, K., Hellstern, H., and Hofle, G. (1989). Peptide synthesis by fragment assembly on a polymer support. In Peptides 1988, G. Jung and E. Bayer, eds., Berlin, Walter de Gruyter & Co., pp. 166-168. Nomizu, M., Inagaki, Y., Yamashita, T., Ohkubo, A., Otaka, A., Fujii, N., Roller, P. P., and Yajima, H. (1991). Two-step hard acid deprotection/ cleavage procedure for solid phase peptide synthesis. Int. J. Peptide Protein Res. 37:145-152. Nutt, R. F., Brady, S. F., Darke, P. L., Ciccarone, T. M., Colton, C. D., Nutt, E. M., Rodkey, J. A., Bennett, C. D., Waxman, L. H., Sigal, I. S., Anderson, P. S., and Veber, D. F. (1988). Chemical synthesis and enzymatic activity of a 99-residue peptide with a sequence proposed for the human immunodeficiency virus protease. Proc. Natl. Acad. Sci. USA 85:7129-7133. Ogunjobi, O., and Ramage, R. (1990). Ubiquitin: Preparative chemical synthesis, purification and characterization. Biochem. Soc. Trans. 18:13221323. Okada, Y., and Iguchi, S. (1988). Amino acid and peptides, part 19: Synthesis of jS-1- and /J-2-adamantyl aspartates and their evaluation for peptide synthesis. J. Chem. Soc. Perkin Trans. 1:2129-2136. Ondetti, M. A., Pluscec, J., Sabo, E. F., Sheehan, J. T., and Williams, N. (1970). Synthesis of cholecystokinin-pancreozymin I: The C-terminal dodecapeptide. J. Am. Chem. Soc. 92:195-199. Orlowska, A., Witkowska, E., and Izdebski, J. (1987). Sequence dependence in the formation of pyroglutamyl peptides in solid phase peptide synthesis. Int. J. Peptide Protein Res. 30:141-144. Orpegen (1990). HYCRAM support: Loading and cleavage. Technical Note, Orpegen, Heidelberg, Germany. Otteson, K. M., Harrison, J. L., Ligutom, A., and Ashcroft, P. (1989). Solid phase peptide synthesis with JV-methylpyrrolidone as the solvent for both
Fmoc and Boc synthesis. Poster Presentations at the Eleventh American Peptide Symposium, Foster City, Calif., Applied Biosystems, Inc., pp. 3438. Ottinger, E. A., Shekels, L. L., Bernlohr, D. A., and Barany, G. (1993). Synthesis of phosphotyrosine-containing peptides and their use as substrates for protein tyrosine phosphatases. Biochemistry 32:4354-4361. Ottinger, E. A., Qinghong Xu, Q., and Barany, G. (1996). Intramolecular pyrophosphate formation during Aâ-9-Fluorenylmethyloxycarbonyl (Fmoc) solid-phase synthesis of peptides containing adjacent phosphotyrosine residues. Peptide Res. 9:223-228. Otvos, L., Jr., Elekes, I., and Lee, V. M.-Y. (1989a). Solid-phase synthesis of phosphopeptides. Int. J. Peptide Protein Res. 34:129-133. Otvos, L., Jr., Wroblewski, K., Kollat, E., Perczel, A., Hollosi, M., Fasman, G. D., Ertl, H. C. J., and Thurin, J. (1989b). Coupling strategies in solidphase synthesis of glycopeptides. Peptide Res. 2:362-366. Otvos, L., Jr., Urge, L., Hollosi, M., Wroblewski, K., Graczyk, G., Fasman, G. D., and Thurin, J. (1990). Automated solid-phase synthesis of glycopeptides: Incorporation of unprotected mono- and disaccharide units of N-glycoprotein antennae into T cell epitopic peptides. Tetrahedron Lett. 31:5889-5892. Pacquet, A. (1982). Introduction of 9-fluorenylmethyloxycarbonyl, trichloroethoxycarbonyl, and benzyloxycarbonyl amine protecting groups into O-unprotected hydroxyamino acids using succinimidyl carbonates. Can. J. Chem. 60:976-980. Pan, H., Barbar, E., Barany, G., and Woodward, C. (1995). Extensive nonrandom structure in reduced and unfolded bovine pancreatic trypsin inhibitor. Biochemistry 34:13974-13981. Patchornik, A., Amit, B., and Woodward, R. B. (1970). Photosensitive protecting groups. J. Am. Chem. Soc. 92:6333-6335. Patel, K., and Borchardt, R. T. (1990a). Chemical pathways of peptide degradation II: Kinetics of deamidation of an asparaginyl residue in a model hexapeptide. Pharm. Res. 7:703-711. Patel, K., and Borchardt, R. T. (1990b). Chemical pathways of peptide degradation III: Effect of primary sequence on the pathways of deamidation of asparaginyl residues in hexapeptides. Pharm. Res. 7:787-793. Paulsen, H., Merz, G., and Weichert, U. (1988). Solid-phase synthesis of O-glycopeptide sequences. Angew. Chem. Int. Ed. Engl. 27:1365-1367. Paulsen, H., Merz, G., Peters, S., and Weichert, U. (1990). Festphasensynthese von Oglycopeptiden. Liebigs Ann. Chem. 1165-1173. Pearson, D. A., Blanchette, M., Baker, M. L., and Guindon, C. A. (1989). Trialkylsilanes as scavengers for the trifluoroacetic acid deblocking of protecting groups in peptide synthesis. Tetrahedron Lett. 30:2739-2742. Pedroso, E., Grandas, A., de las Heras, X., Eritja, R., and Giralt, E. (1986). Diketopiperazine formation in solid phase peptide synthesis using palkoxybenzyl ester resins and Fmoc-amino acids. Tetrahedron Lett. 27:743-746. Peluso, S., Dumy, P., Eggleston, I. M., Garrouste, P., and Mutter, M. (1997). Protein mimetics (TASP) by sequential condensation of peptide loops to an immobilised topological template. Tetrahedron 53:7231-7236.
08
Synthetic Peptides
enke, B., and Nyerges, L. (1989). Preparation and application of a new resin for synthesis of peptide amides via Fmoc-strategy. In Peptides 1988, G. Jung and E. Bayer, eds., Berlin, Walter de Gruyter & Co., pp. 142-144. enke, B., and Nyerges, L. (1990). Solid-phase synthesis of porcine cholecystokinin-33 in a new resin via Fmoc strategy. Peptide Res. 4:289-295. enke, B., and Rivier, J. (1987). Solid-phase synthesis of peptide amides on a polystyrene support using fluorenylmethoxycarbonyl protecting groups. J. Org. Chem. 52:1197-1200. enke, B., and Toth, G. K. (1989). An improved method for the preparation of large amounts of w-cyclophexylesters of aspartic and glutamic acid. In Peptides 1988, G. Jung and E. Bayer, eds., Berlin, Walter de Gruyter and Co., pp. 67-69. enke, B., Balaspiri, L., Pallai, P., and Kovacs, K. (1974). Application of pentafluorophenyl esters of Boc-amino acids in solid phase peptide synthesis. Acta Phys. Chem. 20:471^76. enke, B., Zsigo, J., and Spiess, J. (1989). Synthesis of a protected hydroxylysine derivative for application in peptide synthesis. In The Eleventh American Peptide Symposium Abstracts, The Salk Institute and University of California at San Diego, La Jolla, Calif., P-335. ennington, M. W., Festin, S. M., and Maccecchini, M. L. (1991). Comparison of folding procedures on synthetic co-conotoxin. In Peptides 1990, E. Giralt and D. Andreu, eds., Leiden, Escom, pp. 164-166. erich, J. W. (1990). Modern methods of 0-phosphoserine- and 0-phosphotyrosine-containing peptide synthesis. In Peptides and Protein Phosphorylation, B. E. Kemp, ed., Boca Raton, Fla., CRC Press, pp. 289-314. erich, J. W. (1997). Synthesis of phosphopeptides using modern chemical approaches. Methods Enzymol. 289:245-266. erich, J. W. (1998). Synthesis of phosphopeptides via global phosphorylation on the solid phase: Resolution of H-phosphonate formation. Lett. Peptide Sci. 5:49-55. erich, J. W., and Johns, R. B. (1988). Di-terr-butyl-7V,Ar-diethylphosphoamidite and dibenzyl-7V,./V-diethylphosphoamidate: Highly reactive reagents for the "phosphite triester" phosphorylation of serine containing peptides. Tetrahedron Lett. 29:2369-2372. erich, J. W., and Reynolds, E. C. (1991). Fmoc/solid-phase synthesis of Tyr(P)-containing peptides through /-butyl phosphate protection. Int. J. Peptide Protein Res. 37:572-575. erich, J. W., Valerio, R. M., and Johns, R. B. (1986). Solid-phase synthesis of an 0-phosphoseryl-containing peptide using phenyl phosphorotriester protection. Tetrahedron Lett. 27:1377-1380. erich, J. W., Ede, N. J., Eagle, S., and Bray, A. M. (1999). Synthesis of phosphopeptides by the Multipin method: Evaluation of coupling methods for the incorporation of Fmoc-Tyr(PO3Bzl,H)-OH, FmocSer(PO3Bzl,H)-OH, and Fmoc-Thr(PO3Bzl,H)-OH. Lett. Peptide Sci. 6:91-97. essi, A., Bianchi, E., Bonelli, F., and Chiappinelli, L. (1990). Application of the continuous-flow polyamide method to the solid-phase synthesis of a multiple antigen peptide (MAP) based on the sequence of a malaria epitope. J. Chem. Soc., Chem. Commun. 8-9.
eters, S., Lowary, T. L., Hindsgaul, O., Meldal, M., and Bock, K. (1995). Solid phase synthesis of a fucosylated glycopeptide of human factor IX with a fructose-a-(l 0)-serine linkage. J. Chem. Soc. Perkin Trans. 1:3017-3022. hotaki, I., Taylor-Papadimitriou, J., Sakarellos, C., Mazarakis, P., and Zervas, L. (1970). On cysteine and cystine peptides, part V: S-trityland S-diphenylmethyl-cysteine and -cysteine peptides. J. Chem. Soc. (C) 2683-2687. ickup, S., Blum, F. D., and Ford, W. T. (1990). Self-diffusion coefficients of Boc-amino acid anhydrides under conditions of solid phase peptide synthesis. J. Polym. Sci. Polym. Chem. Ed. 28:931-934. Pipkorn, R., and Bernath, E. (1990). Solid phase synthesis of preprocecropin A. In Innovation and Perspectives in Solid Phase Synthesis, R. Epton, ed., Birmingham, UK, Solid Phase Conference Coordination, Ltd., pp. 537541. lanas, M., Bardaji, E., Jensen, K. J., and Barany, G. (1999). Use of the dithiasuccinoyl (Dts) amino protecting group for solid-phase synthesis of protected peptide nucleic acid (PNA) oligomers. J. Org. Chem. 64:7281-7289. Plaue, S. (1990). Synthesis of cyclic peptides on solid support: Application to analogs of hemagglutinin of influenza virus. Int. J. Peptide Protein Res. 35:510-517. Ploux, O., Chassaing, G., and Marquet, A. (1987). Cyclization of peptides on a solid support: Application to cyclic analogs of substance P. Int. J. Peptide Protein Res. 29:162-169. Pluscec, J., Sheehan, J. T., Sabo, E. F., Williams, N., Kocy, O., and Ondetti, M. A. (1970). Synthesis of analogs of the C-terminal octapeptide of cholecystokinin-pancreozymin: Structure-activity relationship. J. Med. Chem. 13:349-352. Ponsati, B., Giralt, E., and Andreu, D. (1989). A synthetic strategy for simultaneous purification-conjugation of antigenic peptides. Anal. Biochem. 181:389-395. Ponsati, B., Giralt, E., and Andreu, D. (1990a). Side reactions in post-HF workup of peptides having the unusual Tyr-Trp-Cys sequence: Lowhigh acidolysis revisited. In Peptides: Chemistry, Structure and Biology, J. E. Rivier and G. R. Marshall, eds., Leiden, Escom, pp. 960-962. Ponsati, B., Giralt, E., and Andreu, D. (1990b). Solid-phase approaches to regiospecific double disulfide formation: Application to a fragment of bovine pituitary peptide. Tetrahedron 46:8255-8266. Prasad, K. U., Trapane, T. L., Busath, D., Szabo, G., and Urry, D. W. (1982). Synthesis and characterization of l-13C-D-Leu12'14 gramicidin A. Int. J. Peptide Protein Res. 19:162-171. Prosser, R. S., Davis, J. H., Dahlquist, F. W., and Lindorfer, M. A. (1991). 2 H nuclear magnetic resonance of the gramicidin A backbone in a phospholipid bilayer. Biochemistry 30:4687^696. Quibell, M., Owen, D., Packman, L. C., and Johnson, T. (1994). Suppression of piperidine-mediated side product formation for Asp(OBut)-containing peptides by the use of Af-(2-hydroxy-4-methoxybenzyl) (Hmb) backbone amide protection. J. Chem. Soc., Chem. Commun. 2343-2344.
10
Synthetic Peptides
amage, R., and Green, J. (1987). NG-2,2,5,7,8-pentamethylchroman-6-sulphonyl-L-arginine: A new acid labile derivative for peptide synthesis. Tetrahedron Lett. 28:2287-2290. amage, R., and Raphy, G. (1992). Design of an affinity-based A^-amino protecting group for peptide synthesis: Tetrabenzo[a,c,g,i]fluorenyl-17methyl urethanes (Tbfmoc). Tetrahedron Lett. 33:385-388. amage, R., Green J., and Ogunjobi, O. M. (1989). Solid phase peptide synthesis of ubiquitin. Tetrahedron Lett. 30:2149-2152. amage, R., Biggin, G. W., Brown, A. R., Comer, A., Davison, A., Draffan, L., Jiang, L., Morton, G., Robertson, N., Shaw, K. T., Tennant, G., Urquhart, K., and Wilken, J. (1996). Methodology for chemical synthesis of proteins. In Innovation and Perspectives in Solid Phase Synthesis and Combinatorial Libraries 1996, R. Epton, ed., Kingswinford, Mayflower Scientific, pp. 1-10. amage, R., Swenson, H. R., and Shaw, K. T. (1998). A novel purification protocol for soluble combinatorial peptide libraries. Tetrahedron Lett. 39:8715-8718. ao, C., and Tarn, J. P. (1994). Synthesis of peptide dendrimer. J. Am. Chem. Soc. 116:6975-6976. eddy, M. P., and Voelker, P. J. (1988). Novel method for monitoring the coupling efficiency in solid phase peptide synthesis. Int. J. Peptide Protein Res. 31:345-348. eid, G. E., and Simpson, R. J. (1992). Automated solid-phase peptide synthesis: Use of 2-(l//-benzotriazol-l-yl)-l,l,3,3,-tetramethyluronium tetrafluoroborate for coupling of ter?-butyloxycarbonyl amino acids. Anal. Biochem. 200:301-309. emmer, H. A., and Fields, G. B. (1999). Problems encountered with the synthesis of a glycosylated hydroxylysine derivative suitable for Fmocsolid phase peptide synthesis. In Peptides: Frontiers of Peptide Science, J. P. Tarn and P. T. P. Kaumaya, eds., Dordrecht, Kluwer Academic Publishers, pp. 287-288. Renil, M., Ferreras, M., Delaisse, J. M., Foged, N. T., and Meldal, M. (1998). PEGA supports for combinatorial peptide synthesis and solid-phase enzymatic library assays. J. Peptide Sci. 4:195-210. ich, D. H., and Gurwara, S. K. (1975). Preparation of a new o-nitrobenzyl resin for solid-phase synthesis of terf-butyloxycarbonyl-protected peptide acids. J. Am. Chem. Soc. 97:1575-1579. Rich, D. H., and Singh, J. (1979). The carbodiimide method. In The Peptides, Vol. 1, E. Gross and J. Meienhofer, eds., New York, Academic Press, pp. 241-314. Riniker, B., and Hartmann, A. (1990). Deprotection of peptides containing Arg(Pmc) and tryptophan or tyrosine: Elucidation of by-products. In Peptides: Chemistry, Structure and Biology, J. E. Rivier and G. R. Marshall, eds., Leiden, Escom, pp. 950-952. Riniker, B., and Kamber, B. (1989). Byproducts of Trp-peptides synthesized on a /7-benzyloxybenzyl alcohol polystyrene resin. In Peptides 1988, G. Jung and E. Bayer, eds., Berlin, Walter de Gruyter & Co., pp. 115-117. Riniker, B., and Sieber, P. (1988). Problems and progress in the synthesis of histidine-containing peptides. In Peptides: Chemistry, Biology,
Interactions with Proteins, B. Penke and A. Torok, eds., Berlin, Walter de Gruyter & Co., pp. 65-74. Riniker, B., Florsheimer, A., Fretz, H., Sieber, P., and Kamber, B. (1993). A general strategy for the synthesis of large peptides: The combined solidphase and solution approach. Tetrahedron 49:9307-9320. Rink, H. (1987). Solid-phase synthesis of protected peptide fragments using a trialkoxy-diphenyl-methylester resin. Tetrahedron Lett. 28:3787 3790. ink, H., and Ernst, B. (1991). Glycopeptide solid-phase synthesis with an acetic acid-labile trialkoxy-benzhydryl linker. In Peptides 1990, E. Giralt and D. Andreu, eds., Leiden, Escom, pp. 418^19. ink, H., Sieber, P., and Raschdorf, F. (1984). Conversion of AfG-urethane protected arginine to ornithine in peptide solid phase synthesis. Tetrahedron Lett. 25:621-624. ivier, J., Galyean, R., Simon, L., Cruz, L. J., Olivera, B. M., and Gray, W. R. (1987). Total synthesis and further characterization of the y-carboxyglutamate-containing "sleeper" peptide from Conus geographus venom. Biochemistry 26:8508-8512. ohwedder, B., Mutti, Y., Dumy, P., and Mutter, M. (1998). Hydrazinolysis of Dde: Complete orthogonality with Aloe protecting groups. Tetrahedron Lett. 39:1175-1178. omani, S., Moroder, L., Gohring, W., Scharf, R., Wiinsch, E., Barde, Y. A., and Thoenen, H. (1987). Synthesis of the trypsin fragment 10-25/75-88 of mouse nerve growth factor II: The unsymmetrical double chain cystine peptide. Int. J. Peptide Protein Res. 29:107-117. omoff, T. T., and Goodman, M. (1997). Urethane-protected N-carboxyanhydrides (UNCAs) as unique reactants for the study of intrinsic racemization tendencies in peptide synthesis. J. Peptide Res. 49:281-292. ose, K. (1994). Facile synthesis of homogeneous artificial proteins. J. Am. Chem. Soc. 116:30-33. osen, O., Rubinraut, S., and Fridkin, M. (1990). Thiolysis of the 3-nitro-2pyridinesulfenyl (Npys) protecting group: An approach towards a general deprotection scheme in peptide synthesis. Int. J. Peptide Protein Res. 35:545-549. zeszotarska, B., and Masiukiewicz, E. (1988). Arginine, histidine and tryptophan in peptide synthesis: The guanidino function of arginine. Org. Prep. Proc. Int. 20:427^64. akakibara, S. (1999). Chemical synthesis of proteins in solution. Biopolymers 51:279-296. arin, V. K., Kent, S. B. H., and Merrifield, R. B. (1980). Properties of swollen polymer networks: Solvation and swelling of peptide-containing resins in solid-phase peptide synthesis. J. Am. Chem. Soc. 102:5463-5470. arin, V. K., Kent, S. B.H., Tarn, J. P., and Merrifield, R. B. (1981). Quantitative monitoring of solid-phase peptide synthesis by the ninhydrin reaction. Anal. Biochem. 117:147-157. arin, V. K., Kent, S. B. H., Mitchell, A. R., and Merrifield, R. B. (1984). A general approach to the quantitation of synthetic efficiency in solid-phase peptide synthesis as a function of chain length. J. Am. Chem. Soc. 106:7845-7850.
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Synthetic Peptides
asaki, T., and Kaiser, E. T. (1990). Synthesis and structural stability of helichrome as an artificial hemeproteins. Biopolymers 29:79-88. carr, R. B., and Findeis, M. A. (1990). Improved synthesis and aminoacylation of /7-nitrobenzophenone oxime polystyrene resin for solid-phase synthesis of protected peptides. Peptide Res. 3:238--241. chielen, W. J. G., Adams, H. P. H. M., Nieuwenhuizen, W., and Tesser, G. I. (1991). Use of Mpc-amino acids in solid phase peptide synthesis leads to improved coupling efficiencies. Int. J. Peptide Protein Res. 37:341-346. chiller, P. W., Nguyen, T. M.-D., and Miller, J. (1985). Synthesis of sidechain cyclized peptide analogs on solid supports. Int. J. Peptide Protein Res. 25:171-177. chneider, J., and Kent, S. B. H. (1988). Enzymatic activity of a synthetic 99 residue protein corresponding to the putative HIV-1 protease. Cell 54:363- 368. chnolzer, M., and Kent, S. B. H. (1992). Constructing proteins by dovetailing unprotected synthetic peptides: Backbone-engineered HIV protease. Science 256:221-225. chnolzer, M., Alewood, P., Jones, A., Alewood, D., and Kent, S. B. H. (1992). In situ neutralization in Boc-chemistry solid phase peptide synthesis: Rapid, high yield assembly of difficult sequences. Int. J. Peptide Protein Res. 40:180-193. chroll, A. L., and Barany, G. (1989). A new protecting group for the sulfhydryl function of cysteine. J. Org. Chem. 54:244-247. coffone, E., Previero, A., Benassi, C. A., and Pajetta, P. (1966). Oxidative modification of tryptophan residues in peptides. In Peptides 1963, L. Zervas, ed., Oxford, Pergamon Press, pp. 183 188. eitz, O., and Kunz, H. (1995). A novel allylic anchor for solid-phase synthesis. Synthesis of protected and unprotected O-glycosylatcd mucin-type glycopeptides. Angew. Chem. Int. Ed. Engl. 34:803-805. eyer, R., Aumelas, A., Caraty, A., Rivaille, P., and Castro, B. (1990). Repetitive BOP coupling (REBOP) in solid phase peptide synthesis: Luliberin synthesis as model. Int. J. Peptide Protein Res. 35:465^72. eyfarth, L., Pineda de Castro, L. F., Liepina, I., Paegelow, I., Liebmann, C., and Reissmann, S. (1995). New cyclic bradykinin antagonists containing disulfide and lactam bridges at the TV-terminal sequence. Int. J. Peptide Protein Res. 46:155 165. hao, J., and Tam, J. P. (1995). Unprotected peptides as building blocks for the synthesis of peptide dendrimers with oxime, hydrazone, and thiazolidine linkages. J. Am. Chem. Soc. 117:3893-3899. heppard, R. C., and Williams, B. J. (1982). Acid-labile resin linkage agents for use in solid phase peptide synthesis. Int. J. Peptide Protein Res. 20:451-454. hin, Y., Winans, K. A., Backes, B. J., Kent, S. B. H., Ellman, J. A., and Bertozzi, C. R. (1999). Fmoc-based synthesis of peptide-" thioesters: application to the total chemical synthesis of a glycoprotein by native chemical ligation. J. Am. Chem. Soc. 121:11684-11689. ieber, P. (1987a). An improved method for anchoring of 9-fluorenylmethoxycarbonyl-amino acids to 4-alkoxybenzyl alcohol resins. Tetrahedron Lett. 28:6147-6150.
Sieber, P. (1987b). Modification of tryptophan residues during acidolysis of 4methoxy-2,3,6-trimethylbenzenesulfonyl groups: Effects of scavengers. Tetrahedron Lett. 28:1637-1640. Sieber, P. (1987c). A new acid-labile anchor group for the solid-phase synthesis of C-terminal peptide amides by the Fmoc method. Tetrahedron Lett. 28:2107-2110. Sieber, P., and Riniker, B. (1987). Protection of histidine in peptide synthesis: A reassessment of the trityl group. Tetrahedron Lett. 28:6031-6034. Sieber, P., and Riniker, B. (1990). Side-chain protection of asparagine and glutamine by trityl: Application to solid-phase peptide synthesis. In innovation and Perspectives in Solid Phase Synthesis, R. Epton, ed., Birmingham, UK, Solid Phase Conference Coordination, Ltd., pp. 577583. Sieber, P., and Riniker, B. (1991). Protection of carboxamide functions by the trityl residue: Application to peptide synthesis. Tetrahedron Lett. 32:739742. Sieber, P., Kamber, B., Riniker, B., and Rittel, W. (1980). Iodine oxidation of S-trityl- and S-acetamidomethyl-cysteine-peptides containing tryptophan: Conditions leading to the formation of tryptophan-2-thioethers. Helv. Chim. Acta 63:2358-2363. Sigler, G. F., Fuller, W. D., Chaturvedi, N. C, Goodman, M., and Verlander, M. S. (1983). Formation of oligopeptides during the synthesis of 9-fluorcnylmethyloxycarbonyl amino acid derivatives. Biopolymers 22:21572162. Simmons, J., and Schlesinger, D. H. (1980). High-performance liquid chromatography of side-chain-protected amino acid phenylthiohydantoins. Anal. Biochem. 104:254-258. Small, P. W., and Sherrington, D. C. (1989). Design and application of a new rigid support for high efficiency continuous-flow peptide synthesis. J. Chem. Soc., Chem. Commun. 1589-1591. Sole, N. A., and Barany, G. (1992). Optimization of solid-phase synthesis of [Ala8]-dvnorphin A. J. Org. Chem. 57:5399-5403. Songster, M. F., and Barany, G. (1997). Handles for solid-phase peptide synthesis. Methods Enzymol. 289:126-174. Southard, G. L. Comments. (1971). In Pe.pt.ides 1969, E. Scoffone, ed., Amsterdam, North-Holland Publishing, pp. 168-170. Spetzler, J. C., and Tarn, J. P. (1995). Unprotected peptides as building blocks for branched peptides and peptide dendrimers. Int. J. Peptide Protein Res. 45:78-85. Steiman, D. M., Ridge, R. J., and Matsueda, G. R. (1985). Synthesis of side chain-protected amino acid phenylthiohydantoins and their use in quantitative solid-phase Edman degradation. Anal. Biochem. 145:91-95. Stephenson, R. C., and Clarke, S. (1989). Succinimide formation from aspartyl and asparaginyl peptides as a model for the spontaneous degradation of proteins. J. Biol. Chem. 264:6164-6170. Stewart. J. M. (1997). Cleavage methods following Boc-based solid-phase peptide synthesis. Methods Enzymol. 289:29^14. Stewart, J. M., and Klis, W. A. (1990). Polystyrene-based solid phase peptide synthesis: The state of the art. In Innovation and Perspectives in Solid
14
Synthetic Peptides
Phase Synthesis, R. Epton, ed., Birmingham, UK, Solid Phase Conference Coordination, Ltd., pp. 1-9. ewart, J. M., and Young, J. D. (1984). Solid Phase Peptide Synthesis, 2nd edn., Rockford, 111., Pierce Chemical Co. ewart, J. M., Knight, M., Paiva, A. C.M., and Paiva, T. (1972). Histidine in solid phase peptide synthesis: Thyrotropin releasing hormone and the angiotensins. In Progress in Peptide Research, Vol. 2, S. Lande, ed., New York, Gordon and Breach, pp. 59-64. ewart, J. M., Ryan, J. W., and Brady, A. H. (1974). Hydroxyproline analogs of bradykinin. J. Med. Chem. 17:537-539. ierandova, A., Sepetov, N. F., Nikiforovich, G. V., and Lebl, M. (1994). Sequence-dependent modification of Trp by the Pmc protecting group of Arg during TFA deprotection. Int. J. Peptide Protein Res. 43:31-38. ory, S. C., and Aldrich, J. V. (1992). A resin for the solid phase synthesis of protected peptide amides using the Fmoc chemical protocol. Int. J. Peptide Protein Res. 39:87-92. iiber, W., Knolle, J., and Breipohl, G. (1989). Synthesis of peptide amides by Fmoc-solid-phase peptide synthesis and acid labile anchor groups. Int. J. Peptide Protein Res. 34:215-221. ueiras-Diaz, J., and Horton, J. (1992). First solid phase synthesis of endothelial interleukin-8 [Ala-IL8]77 using Boc-TBTU chemistry with in situ neutralisation and comparison with synthesis of monocyte interleukin-8 [Ser-IL8]72 using the DCC-HOBt method. Tetrahedron Lett. 33:27212724. ugg, E. E., Castrucci, A. M. de L., Hadley, M. E., van Binst, G., and Hruby, V. J. (1988). Cyclic lactam analogues of Ac-[Nle4]a-MSH4.n-NH2. Biochemistry 27:8181-8188. uzuki, K., Nitta, K., and Endo, N. (1975). Suppression of diketopiperazine formation in solid phase peptide synthesis. Chem. Pharm. Bull. 23:222-224. albo, G., Wade, J. D., Dawson, N., Manoussios, M., and Tregear, G. W. (1997). Rapid semi on-line monitoring of Fmoc solid-phase peptide synthesis by matrix-assisted laser desorption/ionization mass spectrometry. Lett. Peptide Sci. 4:121-127. arn, J. P. (1988). Synthetic peptide vaccine design:Synthesis and properties of a high-density multiple antigenic peptide system. Proc. Natl. Acad. Sci. USA 85:5409-5413. arn, J. P., and Lu, Y.-A. (1990). Synthetic peptide vaccine engineering: Design and synthesis of unambiguous peptide-based vaccines containing multiple peptide antigens for malaria and hepatitis. In Innovation and Perspectives in Solid Phase Synthesis, R. Epton, ed., Birmingham, UK, Solid Phase Conference Coordination, Ltd., pp. 351-370. arn, J. P., and Lu, Y.-A. (1995). Coupling difficulty associated with interchain clustering and phase transition in solid phase peptide synthesis. J. Am. Chem. Soc. 117:12058-12063. arn, J. P., and Merrifield, R. B. (1985). Solid phase synthesis of gastrin I: Comparison of methods utilizing strong acid for deprotection and cleavage. Int. J. Peptide Protein Res. 26:262-273. arn, J. P., and Merrifield, R. B. (1987). Strong acid deprotection of synthetic peptides: Mechanisms and methods. In The Peptides, Vol. 9, S.
215
Udenfriend and J. Meienhofer, eds., New York, Academic Press, pp. 185248. Tarn, J. P., Kent, S. B. H., Wong, T. W., and Merrifield, R. B. (1979). Improved synthesis of 4-(Boc-aminoacyloxymethyl)-phenylacetic acids for use in solid phase peptide synthesis. Synthesis 955-957. Tam, J. P., and Spetzler, J. C. (1997). Multiple antigen peptide system. Methods Enzymol. Tam, J. P., Health, W. F., and Merrifield, R. B. (1983). SN2 deprotection of synthetic peptides with a low concentration of HF in dimethyl sulfide: Evidence and application in peptide synthesis. J. Am. Chem. Soc. 105:6442-6455. Tam, J. P., Riemen, M. W., and Merrifield, R. B. (1988). Mechanisms of aspartimide formation: The effects of protecting groups, acid, base, temperature and time. Peptide Res. 1:6-18. Tam, J. P., Liu, W., Zhang, J.-W., Galantino, M., and de Castiglione, R. (1991a). D-Amino acid and alanine scans of endothelin: An approach to study refolding intermediates. In Peptides 1990, E. Giralt and D. Andreu, eds., Leiden, Escom, pp. 160-163. Tam, J. P., Wu, C.-R., Liu, W., and Zhang, J.-W. (1991b). Disulfide bond formation in peptides by dimethyl sulfoxide:Scope and applications. J. Am. Chem. Soc. 113:6657-6662. Tamamura, H., Matsumoto, F., Sakano., K., Ibuka, T., and Fujii, N. (1998). Unambiguous synthesis of stromal cell-derived factor-1 by regioselective disulfide bond formation using a DMSO-aqueous HC1 system. J. Chem. Soc., Chem. Commun. 151-153. Ten Kortenaar, P. B. W., and van Nispen, J. W. (1988). Formation of openchain asymmetrical cystine peptides on a solid support: Synthesis of pGlu-Asn-Cyt-Pro-Arg-Gly-OH. Coll. Czech. Chem. Commun. 53:2537-2541. Ten Kortenaar, P. B. W., van Dijk, B. G., Peters, J. M., Raaben, B. J., Adams, P. J. H. M., and Tesser, G. I. (1986). Rapid and efficient method for the preparation of Fmoc-amino acids starting from 9-fluorenylmethanol. Int. J. Peptide Protein Res. 27:398-400. Ten Kortenaar, P. B. W., Hendrix, B. M. M., and van Nispen, J. W. (1990). Acid-catalyzed hydrolysis of peptide-amides in the solid state. Int. J. Peptide Protein Res. 36:231-235. Tesser, G. I., and Balvert-Geers, I. C. (1975). The methylsulfonylethyloxycarbonyl group, a new and versatile amino protective function. Int. J. Peptide Protein Res. 7:295-305. Tesser, M., Albericio, F., Pedroso, E., Grandas, A., Eritja, R., Giralt, E., Granier, C., and van Rietschoten, J. (1983). Amino-acids condensations in the preparation of Ara-9-fluorenylmethyloxycarbonylamino-acids with 9-fluorenylmethylchloroformate. Int. J. Peptide Protein Res. 22:125-128. Thaler, A., Seebach, D., and Cardinaux, F. (1991). Lithium-salt effects in peptide synthesis, part II: Improvement of degree of resin swelling and of efficiency of coupling in solid-phase synthesis. Helv. Chim. Acta 74:628-643. Tregear, G. W. (1972). Graft copolymers as insoluble supports in peptide synthesis. In Chemistry and Biology of Peptides, J. Meienhofer, ed., Ann Arbor, Mich., Ann Arbor Sci. Publ., pp. 175-178.
216
Synthetic Peptides
Tregear, G. W., van Rietschoten, J., Sauer, R., Niall, H. D., Keutmann, H. T., and Potts, Jr., J. T. (1977). Synthesis, purification, and chemical characterization of the amino-terminal 1-34 fragment of bovine parathyroid hormone synthesized by the solid-phase procedure. Biochemistry 16: 2817-2823. Trzeciak, A., and Bannwarth, W. (1992). Synthesis of "head to tail" cyclized peptides on solid support by Fmoc chemistry. Tetrahedron Lett. 33:45574560. Tuchscherer, G. (1993). Template assembled synthetic proteins: Condensation of a multifunctional peptide to a topological template via chemoselective ligation. Tetrahedron Lett. 34:8419-8422. Ueki, M., and Amemiya, M. (1987). Removal of 9-fluorenylmethyloxycarbonyl (Fmoc) group with tetrabutylammonium fluoride. Tetrahedron Lett. 28:6617-6620. Van Abel, R. J., Tang, Y.-Q., Rao, V. S. V., Dobbs, C. H., Tran, D., Barany, G., and Selsted, M. E. (1995). Synthesis and characterization of indolicidin, a tryptophan-rich antimicrobial peptide from bovine neutrophils. Int. J. Peptide Protein Res. 45:401-409. van der Eijk, J. M., Nolte, R. J. M., and Zwikker, J. W. (1980). A simple and mild method for the removal of the 7VIm-tosyl protecting group. J. Org. Chem. 45:547-548. van Nispen, J. W., Polderdijk, J. P., and Greven, H. M. (1985). Suppression of side-reactions during the attachment of Fmoc-amino acids to hydroxymethyl polymers. Reel. Trav. Chim. Pays-Bas 104:99-100. van Regenmortel, M. V. H., and Muller, S. (1999). Synthetic Peptides as Antigens: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 28, Amsterdam, Elsevier. van Woerkom, W. J., and van Nispen, J. W. (1991). Difficult couplings in stepwise solid phase peptide synthesis: Predictable or just a guess? Int. J. Peptide Protein Res. 38:103-113. Veber, D. F., Milkowski, J. D., Varga, S. L., Denkewalter, R. G., and Hirschmann, R. (1972). Acetamidomethyl: A novel thiol protecting group for cysteine. J. Am. Chem. Soc. 94:5456-5461. Vlattas, I., Delluureficio, J., Dunn, R., Sytwwu, I. I., and Stanton, J. (1997). The use of thioesters in solid-phase organic synthesis. Tetrahedron Lett. 38:7321-7324. Voss, C., and Birr, C. (1981). Synthetic insulin by selective disulfide bridging II: Polymer phase synthesis of the human B chain fragments. HoppeSeyler's Z. Physiol. Chem. 362:717-725. Vuljanic, T., Bergquist, K. E., Clausen, H., Roy, S., and Kihlberg, J. (1996). Piperidine is preferred to morpholine for Fmoc cleavage in solid phase glycopeptide synthesis as exemplified by preparation of glycopeptides related to HIV GP120 and mucins. Tetrahedron 52:7983-8000. Wade, J. D., Fitzgerald, S. P., McDonald, M. R., McDougall, J. G., and Tregear, G. W. (1986). Solid-phase synthesis of a-human atrial natriuretic factor: Comparison of the Boc-polystyrene and Fmoc-polyamide methods. Biopolymers 25:S21-S37. Wade, J. D., Bedford, J., Sheppard, R. C., and Tregear, G. W. (1991). DBU as an TV"-deprelecting reagent for the fluorenylmethoxycarbonyl group
217
in continuous flow solid-phase peptide synthesis. Peptide Res. 4:194 199. Wakamiya, T., Saruta, K., Yasuoka, J., and Kusumoto, S. (1994). An efficient procedure for solid-phase synthesis of phosphopeptides by the Fmoc strategy. Chem. Lett. 1099-1102. Wallace, C. J. A., Mascagni, P., Chait, B. T., Collawn, J. F., Paterson, Y., Proudfoot, A. E. I., and Kent, S. B. H. (1989). Substitutions engineered by chemical synthesis at three conserved sites in mitochondrial cytochrome c. J. Biol. Chem. 264:15199-15209. Wang, S. S. (1973). /?-Alkoxybenzyl alcohol resin and />-alkoxybenzyloxycarbonylhydrazide resin for solid phase synthesis of protected peptide fragments. J. Am. Chem. Soc. 95:1328-1333. Wang, S. S. (1976). Solid phase synthesis of protected pep tides via photolytic cleavage of the a-methylphenacyl ester anchoring linkage. J. Org. Chem. 41:3258-3261. Wang, S. S., and Merrifield, R. B. (1969). Preparation of some new biphenylisopropyloxycarbonyl amino acids and their application to the solid phase synthesis of a tryptophan-containing heptapeptide of bovine parathyroid hormone. Int. J. Protein Res. 1:235-244. Wang, S. S., Matsueda, R., and Matsueda, G. R. Automated peptide synthesis under mild conditions. In Peptide Chemistry 1981, T. Shioiri, ed., Osaka, Protein Research Foundation, pp. 37^0. Wang, S. S., Chen, S. T., Wang, K. T., and Merrifield, R. B. (1987). 4-methoxybenzyloxycarbonyl amino acids in solid phase peptide synthesis. Int. J. Peptide Protein Res. 30:662-667. Weber, R. W., and Nitschmann, H. (1978). Der einfluss der O-acetylierung auf das konformative verhalten des kollagen-modellpeptides (L-Pro-L-HypGly)10 und von gelatine. Helv. Chim. Acta 61:701-708. Wenschuh, H., Beyermann, M., Krause, E., Brudel, M., Winter, R., Schumann, M., Carpino, L. A., and Bienert, M. (1994). Fmoc amino acid fluorides: Convenient reagents for the solid-phase assembly of peptides incorporating sterically hindered residues. J. Org. Chem. 59:32753280. Wenschuh, H., Beyermann, M., Haber, H., Seydel, J. K., Krause, E., Bienert, M., Carpino, L. A., El-Faham, A., and Albericio, F. (1995). Stepwise automated solid phase synthesis of naturally occurring peptaibols using Fmoc amino acid fluorides. J. Org. Chem. 60:405-410. Weygand, F., Steglich, W., and Bjarnason, J. (1968a). Leicht abspaltbare schutzgruppen fur die saureamidfunktion 3: Derivate des asparagins und glutamins mit 2,4-dimethoxy-benzyl-und 2. 4.6-trimethoxy-benzylgeschiitzten amidgruppen. Chem. Ber. 101:3642-3648. Weygand, F., Steglich, W., and Chytil, N. (1968b). Bildung von ,/V-succinimidoxycarbonyl-j6-alanin-amiden bei amidsynthesen mit dicyclohexylcarbodiimid/AMiydroxysuccinimid. Z. Naturforschg. 23b: 1391-1392. White, P. (1992). Fmoc-Trp(Boc)-OH:A new derivative for the synthesis of peptides containing tryptophan. In Peptides: Chemistry and Biology, J. A. Smith and J. E. Rivier, eds., Leiden, Escom, pp. 537-538. White, P., and Beythien, J. (1996). Preparation of phospho-serine, threonine, and tyrosine containing peptides by the Fmoc method-
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ology using pre-formed phosphoamino acid building blocks. In Innovation and Perspectives in Solid Phase Synthesis and Combinatorial Libraries 1996, R. Epton, ed., Kingswinford, Mayflower Scientific, pp. 557-560. Williams, M. J., Muir, T. W., Ginsberg, M. H., and Kent, S. B. H. (1994). Total chemical synthesis of a folded /3-sandwich protein domain: An analog of the tenth fibronectin type 3 module. J. Am. Chem. Soc. 116:10797-10798. Wlodawer, A., Miller, M., Jaskolski, M., Sathyanarayana, B. K., Baldwin, E., Weber, I. T., Selk, L. M., Clawson, L., Schneider, J., and Kent, S. B. H. (1989). Conserved folding in retroviral proteases: Crystal structure of a synthetic HIV-1 protease. Science 245:616-621. Wolfe, H. R., and Wilk, R. R. (1989). The RaMPS system: Simplified peptide synthesis for life science researchers. Peptide Res. 2:352-356. Wu, C.-R., Wade, J. D., and Tregear, G. W. (1988). ^-Subunit of baboon chorionic gonadotropin: Continuous flow Fmoc-polyamide synthesis of the C-terminal 37-peptide. Int. J. Peptide Protein Res. 31: 47-57. Wu, C.-R., Stevens, V. C., Tregear, G. W., and Wade, J. D. (1989). Continuousflow solid-phase synthesis of a 74-peptide fragment analogue of human /8-chorionic gonadotropin. J. Chem. Soc. Perkin Trans. I: 81-87. Wiinsch, E. (1974). Synthese von peptiden. In Houben-Weyl's Methoden der Organischen Chemie, Vol. 15, parts 1 and 2, E. Miiller, ed., Stuttgart, Thieme. Wiinsch, E., and Spangenberg, R. (1971). Eine neue S-schutzgruppe fur cystein. In Peptides 1969, E. Scoffone, ed., Amsterdam, North-Holland Pub!., pp. 30-34. Wunsch, E., Moroder, L., Wilschowitz, L., Gohring, W., Scharf, R., and Gardner, J. D. (1981). Zur totalsynthese von cholecystokinin-pankreozymin: Darstellung des verknupfungsfahigen "schlusselfragments" der sequenz 24-33. Hoppe-Seyler's Z. Physiol. Chem. 362:143-152. Xu, Q., Zheng, J., Cowburn, D., and Barany, G. (1996). Synthesis and characterization of branched phosphopeptides: Prototype consolidated ligands for SH(32) domains. Lett. Peptide Sci. 3:31 36. Xu, Q., Ottinger, E. A., Sole, N. A., and Barany, G. (1997). Detection and minimization of H-phosphonate side reaction during phosphopeptide synthesis by a post assembly global phosphorylation strategy. Lett. Peptide Sci. 3:333-342. Yajima, H., Takeyama, M., Kanaki, J., and Mitani, K. (1978). The mesitylene2-sulphonyl group, an acidolytically removable AfG-protecting group for arginine. J. Chem. Soc., Chem. Commun. 482-483. Yajima, H., Fujii, N., Funakoshi, S., Watanabe, T., Murayama, E., and Otaka, A. (1988). New strategy for the chemical synthesis of proteins. Tetrahedron 44:805-819. Yamashiro, D. (1987). Preparation and properties of some crystalline symmetrical anhydrides of W-to-f-butyloxycarbonyl-amino acids. Int. J. Peptide Protein Res. 30:9-12. Yamashiro, D., and Li, C. H. (1973). Protection of tyrosine in solid-phase peptide synthesis. J. Org. Chem. 38:591-592.
219
Yamashiro, D., Blake, J., and Li, C. H. (1976). The use of trifluoroethanol for improved coupling in solid-phase peptide synthesis. Tetrahedron Lett.: 1469-1472. Yang, Y., Sweeney, W. V., Scheider, K., Thornqvist, S., Chait, B. T., and Tarn, J. P. (1994). Aspartimide formation in base-driven 9-fluorenylmethoxycarbonyl chemistry. Tetrahedron Lett. 35:9689-9692. Yoshida, M., Tatsumi, T., Fujiwara, Y., linuma, S., Kimura, T., Akaji, K., and Kiso, Y. (1990). Deprotection of the S-trimethylacetamidomethyl (Tacm) group using silver tetrafluoroborate: Application to the synthesis of porcine brain natriuretic peptide-32 (pBNP-32). Chem. Pharm. Bull. 38:1551-1557. Young, J. D., Huang, A. S., Ariel, N., Bruins, J. B., Ng, D., and Stevens, R. L. (1990). Coupling efficiencies of amino acids in solid phase synthesis of peptides. Peptide Res. 3:194-200. Young, S. C., White, P. D., Davies, J. W., Owen, D. E.I. A., Salisbury, S. A., and Tremeer, E. J. (1990). Counterion distribution monitoring: A novel method for acylation monitoring in solid-phase synthesis. Biochem. Soc. Trans. 18:1311-1312. Yu, H.-M., Chen, S.-T., Chiou, S.-H., and Wang, K.-T. (1988). Determination of amino acids on Merrifield resin by microwave hydrolysis. J. Chromatogr. 456:357-362. Zalipsky, S., Albericio, F., and Barany, G. (1985). Preparation and use of an aminoethyl polyethylene glycol-crosslinked polystyrene graft resin support for solid-phase peptide synthesis. In Peptides: Proceedings of the Ninth American Peptide Symposium, C. M. Deber, V. J. Hruby, and K. D. Kopple, eds., Rockford, 111., Pierce Chemical Co., pp. 257-260. Zalipsky, S., Albericio, F., Slomczynska, U., and Barany, G. (1987). A convenient general method for synthesis of N"- or Ar'u-dithiasuccinoyl (Dts) amino acids and dipeptides: Application of polyethylene glycol as a carrier for funtional purification. Int. J. Peptide Protein Res. 30:740-783. Zalipsky, S., Chang, J. L., Albericio, F., and Barany, G. (1994). Preparation and applications of polyethylene glycol-polystyrene graft resin supports for solid-phase peptide synthesis. Reactive Polymers 22:243-258. Zardeneta, G., Chen, D., Wcintraub, S. T., and Klebe, R. J. (1990). Synthesis of phosphotyrosyl-containing phosphopeptides by solid-phase peptide synthesis. Anal. Biochem. 190:340-347. Zhang, L., Goldhammer, C., Henkel, B., Panhaus, G., Zuehl, F., Jung, G., and Bayer, E. (1994). "Magic mixture," a powerful solvent system for solid-phase synthesis of difficult peptides. In Innovation and Perspectives in Solid Phase Synthesis, R. Epton, ed., Birmingham, UK, Mayflower Worldwide, Ltd., pp. 711-716.
Evaluation of the Synthetic Product

Gregory A. Grant
In 1987, an article appeared in the International Journal of Peptide and Protein Research commemorating the 25th anniversary of the development of solid phase peptide synthesis (Barany et al., 1987). While that article dealt with many aspects of peptide synthesis, one statement in particular stands out as exemplifying the rationale for this chapter. It states: "No synthetic endeavor can be considered complete until the product has been adequately purified and subjected to a battery of analytical tests to verify its structure." The characterization or evaluation of a synthetic peptide is the one step in its production and experimental utilization that will validate the experimental data obtained. Unfortunately, it is also the one step that many investigators all too often give too little attention. If the synthetic product, upon which the theory and performance of the experimental investigation is based, is not the intended product, the conclusions will be incorrect. Without proper characterization, the investigator will either have to be lucky, or be wrong. Worse yet, he or she will not know which is the case. Although today the synthesis of a given peptide is often considered routine, the product should never be taken for granted. Peptide synthesis chemistry, although quite sophisticated, is complex and subject to a variety of problems. These problems, which can manifest
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221
emselves as unwanted side reactions and decreased reaction ficiency, are subject to a variety of factors such as reagent quality, compatible chemistries, instrument malfunctions, sequence specific fects, and operator error. Although every effort is made to eliminate eir causes and to plan for potential problems in the design and nthesis steps, it is not always successful and the eventual outcome a synthesis is not always predictable. One must never assume that e final product is the expected one until that has been proven to be e case. To do otherwise may seriously jeopardi/e the outcome of the search. Used and performed properly, the evaluation stage is where the uits of the synthesis are scrutinized and the decision is made to use e peptide as intended, submit it to further purification, or renthesize it and possibly change elements of the design or synthesis otocols. The battery of tests referred to in the above quote are the ols that are used to assure the investigator that he or she has btained the intended peptide and are the main subject of this chapter.
eneral Considerations
efore embarking on a detailed discussion of the techniques and they of peptide evaluation, it is perhaps useful to discuss a few general nsiderations that one should be mindful of within the context of the suing sections. First, homogeneity and correct covalent structure are e basic goals of the synthetic endeavor and are the two properties or ncepts that pervade any and all considerations of the state of the nthetic product. Second, a familiarity with the properties of e amino acid residues may have direct bearing on the design of the ptide and how it is handled. Finally, the decision to use the synthetic oduct as is or to undergo further purification depends on what the ptide will be used for and how much purification and characterizaon is necessary or feasible within the context of that use. That uestion becomes even more relevant from the viewpoint of synthesis cilities which are a major source of synthetic peptides today, and hich produce those peptides for a large group of diverse vestigators. Elements of both time and cost must be considered refully when deciding how much purification and characterization necessary or feasible.
rategy for the Evaluation of Synthetic Peptides
he general strategy for the evaluation of synthetic peptides is esented diagrammatically in figure 4-1. This diagram depicts e general course of the evaluation process as it might be carried ut and indicates the types of analyses that are available and which
222
Synthetic Peptides
Figure 4-1. Stages in the production of a synthetric peptide for experimental use. The common types of analytical techniques that are applicable to each stage are listed on the right.
might be helpful at any particular stage. The individual components of this diagram are discussed in specific sections of this chapter. When approached in a systematic manner, a clear picture of the quality of the synthesis will emerge and will allow an accurate and clear assessment of the peptide product. At each stage, starting with the unpurified material and ending with the homogeneous peptide, the product must be evaluated to ascertain whether the desired purity has been reached and whether the covalent nature of the peptide is correct. The latter stages of the evaluation process comprise a purification-evaluation cycle. This cycle of purification and evaluation may have to be repeated several times before the desired result is obtained and the synthetic chemist feels confident that the product obtained is the one intended. The "battery of analytical tests" generally available for the evaluation of synthetic peptides are the tools that the chemist uses to assess homogeneity and covalent structure. The commonly used techniques are analytical high-performance (or high-pressure) liquid chromatography (HPLC), capillary electrophoresis (CE), amino acid compositional analysis, UV spectroscopy, sequence analysis, and mass spectrometry. Table 4-1 indicates the relative contributions of these tools toward the two goals of assessing homogeneity and covalent
Table 4-1 Advantages and limitations of analytical techniques

Technique Reverse-phase HPLC Capillary electrophoresis Amino acid analysis Advantages Assesses level of heterogeneity Useful for purification Assesses level of heterogeneity Provides assessment based on a second physical principal Accurately quantitates peptide levels Provides mol ratio of amino acids Assists in monitoring tryptophan integrity Useful for monitoring purification Determines linear order of amino acids Very good for detecting deletion and addition sequences Detects presence of some adducts Provides accurate peptide mass Can indicate heterogeneity Can give sequence information Limitations Does not yield structural information Not useful for preparative scale Does not yield structural information Some amino acids not quantitated Accuracy decreases for large peptides Does not distinguish heterogeneous species Does not distinguish heterogeneous species Does not yield structural information Blocked peptides are invisible Cannot sequence long peptides to the end Not reliable for quantitation May destroy or reverse some adducts Not quantitative
Ultraviolet spectroscopy Sequence analysis
Mass spectrometry
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Synthetic Peptides
structure, and summarixes some of their advantages and limitations. Each one makes its own specialized contribution to the evaluation process by providing unique information, and they are generally used in a complementary fashion. Taken as a whole, the information provided by these analytic techniques should be sufficient to allow the investigator to correctly judge the state of the synthetic peptide. Infrared and nuclear magnetic resonance (NMR) spectroscopy can also be useful tools for the evaluation of synthetic peptides, but are usually necessary only in cases where secondary structures or new chemistries are being investigated. In addition, NMR availability is often limited, particularly for core facilities, and is expensive and time consuming. Since these two techniques are generally not needed for routine evaluation, they will not be discussed here. Analytical HPLC or CE is usually the first level of assessment. These two techniques give a relatively rapid overall picture or "feel" for the complexity of the synthetic product in that they reveal the relative number of different species present in the preparation, but they lack direct information about covalent structure. Amino acid analysis quantitates the amount of the peptide and informs whether the requisite amino acids are present at reasonable ratios. However, it is less reliable for certain unstable amino acids such as cysteine, methionine, and tryptophan, and the compositional accuracy decreases as the peptide gets larger. UV spectroscopy monitors the integrity of the aromatic amino acids, particularly tryptophan, and is very useful in monitoring peptide purification, but the amount of information that can be obtained about the covalent state of the peptide in general is very limited. Sequence analysis and mass spectrometry come as close as any to addressing both homogeneity and covalent structure in a single step. However, while these techniques give the best assessment of the covalent nature of the peptide, it must be pointed out that they can lead to incorrect conclusions about the sample if they are relied on exclusively. For example, peptides which are blocked to the Edman chemistry at their amino terminus, either as a consequence of the synthesis chemistry employed or because of an unwanted side reaction, will be completely invisible to sequence analysis. Mass spectrometry is not generally found to be a quantitative technique, so conclusions of relative homogeneity based on mass analysis can be misleading. However, it has generally been found that in a mixture of related species, as would result from a single synthesis run, that the apparent relative level of species is fairly indicative of the yield of those species. Mass spectrometry of peptides has advanced considerably since the first edition of this book was published. Today it is routinely used, sometimes exclusively, as the best and most informative method for analysis of synthetic peptides. Nonetheless, the other analytical techniques listed above can still be
225
useful, often as part of a more in-depth evaluation of a pep tide preparation because certain problems were identified from the mass spectra, or for the more rigorous evaluation of homogeneity and yield. In the examples that follow throughout this chapter, there are instances where incorrect conclusions would have been made if they were based only on the information provided by a single analytical technique. Indeed, in many cases, information from several analytical techniques is necessary in order for the correct conclusion to be made.
Homogeneity and Purification
The assessment of homogeneity of a peptide goes hand in hand with the purification process. First of all, homogeneity is not a characteristic that can be proven directly in that there is no single test that, when met, automatically indicates that a peptide is a clean, single species. Rather, homogeneity is demonstrated by an inability to demonstrate heterogeneity, or the presence of more than one species. This implies, of course, that reasonable attempts have been made to demonstrate heterogeneity under conditions where it should be detected if it is present and that good judgment, based on experience with the techniques used, has been exercised. It often follows that if a particular technique or procedure indicates the existence of multiple species, it also provides either the means to separate and purify the individual species or the evidence upon which a separation protocol can be based. For example, if more than one peptide peak is found using analytical reverse phase HPLC, the individual peptides should be able to be isolated one from the other by collecting fractions of the column effluent and thus physically separating the individual peptides into separate fractions. In contrast, if two sequences show up on automated Edman degradation, the sequencing procedure cannot in itself be used to physically separate the two species, but the sequence information obtained may suggest a separation procedure, say one based on charge differences, that will be productive. Typically synthetic chemists are called on to produce peptides of two general categories, simply referred to as either unpurified or purified. Unpurified peptides consist of either the synthetic product as it comes directly from the synthesis or after only minimal purification is performed, such as desalting (see figure 4-1). Purified peptides, as the name implies, are those which have undergone rigorous purification to homogeneity or near homogeneity. Obviously, the evaluation criteria applied to each of these categories are quite different. The desired state of the peptide and the extent of evaluation needed depend on what the peptide is intended for, or how it will be used. If the peptide is intended to be used as an antigen for producing polyclonal antisera directed against a protein epitope, perhaps a lesser degree of
Synthetic Peptides
rification may be acceptable than for a peptide which will be used detailed conformational studies. Although it may ideally be desirle to purify all peptides to a high degree of homogeneity, there are en practical limitations in the amount of time and effort spent, as ll as fiscal limitations. Moreover, a core laboratory may get requests unpurified peptides from an investigator who intends to do his own rification. It must be emphasized, however, that accepting a lesser gree of purification does not mean sacrificing the integrity of the oduct. The intended covalent structure should surely be present and me minimal criteria, such as mass spectrometry, should be assessed verify the likelihood of that situation.
ovalent Structure
most instances, before the covalent structure of a peptide can be ambiguously determined, the peptide must be relatively homoneous. Although many techniques used for the evaluation of covant structure can in themselves indicate the presence of multiple ecies, those techniques, with the possible exception of mass spectroetry, are not generally able to provide a specific or unambiguous alysis of the covalent nature of each species within a mixture. The two main factors to be evaluated in the assessment of covalent ucture of a peptide are the presence of the correct sequence of amino ids and the presence, or preferably absence, of derivatives or adducts those amino acids. Probably, the most common problem encouned is that of deletion or addition sequences. Deletion sequence pepes are those that contain an internal absence or deletion of an amino d residue brought about by either incomplete coupling to or inmplete deprotection of the a-amino group at a particular cycle regear et al., 1977). Addition sequence peptides are those that ntain two residues of the same amino acid adjacent to each other the sequence when only one was intended at that particular cation. The degree to which deletion and addition sequences occur pends on the conditions of the individual synthesis, but as long as ese do not occur too often during a synthesis and do not represent overwhelming proportion of the total product, the full-length ptide may still be present in appreciable amounts. Deletion sequences due to incomplete coupling, but not incomplete blocking, or other causes (see chapter 3) can be largely eliminated if pping is used. Capping is the chemical derivitization of free a-amino oups, usually by acetylation with acetic anhydride, before deprotecn of the just added residue takes place. This results in an irreversibly ocked peptide that is no longer able to be elongated. Although pped peptides will represent contaminants that will have to be moved from the final product, they are generally easier to separate
227
from the full-length peptide than are deletion sequence peptides which may differ from the full-length peptide by only one or two amino acids. Addition sequences are less likely but are more problematic and cannot be prevented by a routine procedure such as capping. Unwanted modifications usually come in two general forms. They are either side-chain-protecting groups that were not completely removed, or random adducts that were produced as a result of uncontrolled side reactions during the synthesis or cleavage and deprotection steps. Some of the more common side reactions encountered are described in chapter 3 which deals specifically with the synthesis chemistry. Although identification of the exact nature of the adduct may help to prevent it in subsequent syntheses, it is not always possible, or necessary, to do so. If the modified peptides do not represent too high a proportion of the sample, which is often the case, they can often be removed during the purification procedure. A common observation in peptide synthesis is that the crude product will contain a variety of species in addition to the intended product. An acceptable synthesis is one in which there is a predominant component, when analyzed by HPLC or CE, and some number of minor components which can be separated away. The minor components of he synthesis represent side products due to unwanted modifications and inefficiencies in the chemistry. As long as they do not represent an overwhelming proportion of the product, they can be removed without their identity ever needing to be determined. It is only when the unwanted side products become the major species that the chemical protocols or instrument operation needs to be changed.
Residues That Present Potential Problems
There are certain amino acid residues that can be the source of problems in the finished peptide that deserve some consideration. The problems considered here are not meant to include most of those hat result from unwanted reactions during the synthesis, cleavage, and deprotection chemistry as discussed in chapter 3. Rather, they are those that may develop subsequently due to the characteristics of certain amino acid residues in the peptide. They may develop during he work-up of the peptide, or during handling or storage, and present a particular problem in the evaluation steps or the application of the peptide. These amino acid residues are cysteine, methionine, Tryptophan, amino-terminal glutamine, and aspartic acid when it is ollowed by glycine or alanine. The main problem with tryptophan, methionine, and cysteine is hat they are particularly susceptible to oxidation. Of these, methionine and cysteine are the least troublesome because their common oxidation products are usually reversible. When possible, it is perhaps
28
Synthetic Peptides
dvisable to work below neutral pH and with degassed or dexygenated solvents when working up peptides that contain these sidues.
ethionine Methionine is fairly easily oxidized to methioninc sulfoxide. Methionine sulfoxide can be converted back to methionine by eatment with thiol reagents such as dithiothreitol or A'-methylercaptoacetamide (Cullwell, 1987; Houghton and Li, 1979), but -mercaptoethanol is not as effective for this purpose (Houghton nd Li, 1979). Other methods that have been reported to be effective clude NH4-l-dimethylsulnde (Fujii et al., 1987) and DMF-SO3-EDT Futaki et al., 1990). The later should only be performed while hydroyl residues are protected. Peptides that contain methionine sulfoxide nd to elute slightly earlier on reverse-phase HPLC than the corsponding peptide containing methionine due to the more polar ature of the sulfoxide. Thus the possibility of the presence of the ulfoxide derivative can often be spotted in this way. Many laboraries routinely treat methionine-containing peptides with a thiol agent prior to HPLC analysis to check for elution shifts due to the esence of the sulfoxide. This is particularly important if more than ne predominant peak is originally present in the chromatogram. utomated Edman-sequencing instruments that incorporate a ducing agent in their reagents may not detect methionine sulfoxide ecause it will be converted to methionine during the analysis.
ysteine
ysteine is prone to the oxidative formation of disulfide bonds which an result in the presence of multiple species. If a single cysteine sidue is present in a peptide, a dimer can form as a result. owever, if the peptide contains more than one cysteine, disulfide oss-linked oligomeric aggregates can form. Generally speaking, peptides usually emerge from the deprotection ocedure in their fully reduced state. It is only after exposure to air at appreciable amounts of disulfides start to form. The time of air xposure needed can vary from peptide to peptide. If one can avoid the esence of two or more cysteine residues in the same peptide, it is enerally advisable to do so. Also, when using ammonium bicarbonate a solvent, disulfide formation can be diminished initially if it is hilled before adding it to the peptide. Any disulfides that form can be reduced by treatment with a molar xcess (5-100-fold) of dithiothreitol. It should be pointed out, howver, that some complex disulfide cross-linked aggregates can prove to
229
e very resistant to reduction. Peptides linked by intermolecular disfides tend to elute later on reverse-phase HPLC than their monoeric counterparts. The relative elution behavior of peptides with tramolecular cross-links is less predictable.
ryptophan
xidation products of tryptophan are generally not reversible. ommon oxidation products of the indoyl ring of trytophan are xyindoyl, formylkynureninyl, and kynureninyl derivatives. The latter wo result in opening of the five-membered ring of tryptophan and hift the absorbance maximum toward shorter wavelengths. ignificant oxidation of tryptophan residues can also be detected by ass spectrometry. In Edman sequence analysis, a common oxidation roduct of tryptophan elutes just before intact PTH-tryptophan on the PLC. Tryptophan oxidation can result from the conditions under hich the peptide was handled. For instance, the same peptide from a ommon source, can often be observed, by sequence analysis, to be at fferent levels of tryptophan oxidation by investigators at different cations. One possible cause that has been suggested is the exposure f the peptide to small rust particles from equipment used in processg the sample. However, it is also a matter of some conjecture as to hether the oxidation was pre-existing or occurred during the equence analysis itself. Thus, the integrity of the Trp residues can est be monitored by mass spectrometry.
lutamine
When a glutamine residue occurs at the amino-terminus of a peptide, it an cyclize by interaction of the side chain with the a-amino group to orm a lactam structure (figure 4-2). This structure, which is often ferred to as pyroglutamate, renders the peptide inaccessible to dman chemistry so that it cannot be sequenced. This is particularly rone to occur at acid pH but can also occur slowly at neutral or basic H when stored in aqueous solution. This reaction results in a decrease f 17 Da and can easily be seen by mass spectrometry. This same henomenon is sometimes seen with glutamic acid, but is much less ommon.
spartic Acid-X Sequences When using Fmoc chemistry to synthesize peptides, sequences of spartic acid followed by residues with small side chains are articularly prone to cyclic imide formation. Although cyclic imide ormation can also occur at Asp and Asn residues in finished peptides
Figure 4-2. Formation of cyclic imides in peptides. The top two reactions depict potential routes for internal imide formation of Asn and Asp. The bottom reaction depicts the potential cyclization of Gin, and occasionally Glu, when they occur at the amino terminus of a peptide.
231
t\/2 & 53 days for Asp-Gly and ;1/2 % 1.4 days for Asn-Gly), the prolem is particularly evident when Asp residues containing ester based locking groups, such as O-?-butyl, are used in the synthesis ti/i minutes). In practice, this is seen most often for Asp-Gly and Asp-Ala sequences when the Asp side chain is ester protected, but can ccur with other amino acids following Asp as well. Moreover, the egree to which it occurs increases the earlier in the synthesis that it s present. The length of the Asp side chain makes it particularly well suited to nteract with "the peptide backbone by cyclization to a cyclic imide. The yclic imide can subsequently open up to reform the normal Asp conaining peptide with a-peptide linkage or it can also form an iso-Asp eptide with a /3-peptide linkage (figure 4-2). In addition, racemization bout the a-carbon can also occur. In Fmoc synthesis, piperidine an react with the cyclic imide (succinimidyl ring) to form both a- and -piperidides. This results in an adduct that is 67 mass units larger than he expected peptide mass and can be easily seen by mass spectrometry. A second reaction that can occur is chain cleavage due to attack of he main chain carbonyl by the side-chain nitrogen (figure 4-2). This is elatively rare in peptide synthesis because the other reaction is highly avored, however it can become appreciable with bonds such as Aspro where iso-Asp formation is not favored.
eptide Solubility
he solubility properties of synthetic peptides can have profound ffects on their usability and should always be considered before ynthesis. In general, it is advisable to incorporate charged amino cids into a synthetic peptide when possible. Peptides composed comletely of uncharged amino acids tend to be less soluble in aqueous olutions and their solubility will tend to decrease as their size ncreases. The presence of His, Lys, and Arg promote solubility in olutions at a pH where their side chains possess a positive charge. Conversely, the presence of Asp and Glu promote solubility in olutions at a pH where their side chains will be negatively charged. A general rule of thumb for solubility in aqueous solution is that the umber of charges (net) should be at least 20% of the total number of esidues. The contribution of the charge properties of the a-amino and arboxyl groups should also be included. In this regard, it is useful to emember that modification of these end groups, so that they are no onger able to ionize, can significantly effect the solubility properties, specially of small peptides. Long stretches of hydrophobic residues nd the presence of polar uncharged side chains are also factors to be onsidered in assessing solubility. In a "borderline" peptide, their resence can tip the scale one way or the other. However, remember
32
Synthetic Peptides
hat there are always exceptions and that these general rules may not ways be adequate. Some of the common aqueous solutions used for dissolution of eptides are 0.1% trifluoroacetic acid (TFA), 0.1 N acetic acid, and 1 M ammonium bicarbonate. If these are not effective, 25-50% TFA, 0% acetic acid, or the inclusion of acetonitrile or ethanol can help. queous formic acid is another excellent solvent that has been used for eptides, but it has the potential drawback that the peptide may ecome formylated on primary amino groups if formaldehyde, hich is a potential contaminant, is present. In some cases, increasing oncentrations of dimethyl sulfoxide (DMSO) may be helpful. imethylformamide (DMF) and dichloromethane (DCM) are fective nonaqueous organic solubilizcrs of synthetic peptides.
nalysis of the Synthetic Product
When the first edition of this book was published, automated peptide ynthesizers were relatively new and tBoc-based chemistry was used most exclusively. It was not uncommon to experience a wide variety f problems throughout the synthesis procedure, and a number of pproaches for detecting them were used when these problems ccurred. These included resin sampling during the synthesis to etermine the extent of coupling efficiency at each step, on-resin equence analysis, and determination of the peptide-resin mass yield rior to cleavage from the resin. Today, the majority of synthesis is erformed using Fmoc-based chemistry. Because of the higher ficiency and fidelity of this chemistry in most situations, the abovementioned procedures are seldom needed or found useful. However, n-resin sequencing may still be useful on occasion and resin sampling may still be helpful on those occasions when a synthesis proves partiularly problematic. These are discussed briefly later. Today, the two most useful and effective techniques for indicating he initial quality of a synthesis are analytical reverse-phase HPLC and mass spectrometry. These two evaluations are relatively easy to erform and represent the minimum that should always be performed or each synthetic peptide. Analytical HPLC provides a good assessment of the complexity of the unpurified product and mass spectroetry indicates the masses of the species that are present and that can e compared to the expected mass of the desired product. In fact, these same techniques are used throughout the purification f a peptide to assess the homogeneity of the preparation. In addition o these techniques, amino acid analysis is the best method for uantitation of the peptide product and sequencing by either Edman egradation or tandem mass spectrometry are effective in locating the ositions of modifications or deletions. Capillary electrophoresis can
233
be used instead of reverse-phase HPLC for assessing heterogeneity, but there are several disadvantages that lead to its decreased popularity. Although it produces excellent resolution it suffers from a lack ofreproducibility and is difficult to use as a preparative method. A recent overview (Angeletti et al., 1997) of a 6-year study of peptide synthesis by the Association of Biomolecular Resource Facilities (ABRF) discusses the advantages and disadvantages of various analytical techniques in more depth and comes to a similar conclusion. This article is recommended reading for anyone making or using synthetic peptides. Problems can occur either during the synthesis itself or during the procedure used to cleave the peptide from the resin and deprotect the side chains. The ABRF study mentioned above concluded that the majority of problems that they encountered in their studies were a result of the cleavage and deprotection procedures. If a problem does occur, an ability to distinguish at which of these two main steps it manifests itself can be very useful in correcting it. For instance, if a problem occurs during the cleavage or deprotection reaction, it makes no sense to change the synthesis protocol. Resin sampling and on-resin sequencing were traditionally the two main methods used for trying to detect problems during the synthesis itself. However, experience has shown that these techniques can be unreliable and are not generally very revealing. In practice, the general point of origin of most problems are relatively obvious if they are detected during the initial analysis of the cleaved peptide. For instance, deletion or addition products result from problems during synthesis and unremoved or migrated protective groups as well as amino acids modified in other ways generally occur during the cleavage and deprotection process. Initial Criteria for Evaluation of Synthetic Peptides Every peptide, whether it will eventually undergo extensive purification or is intended to be used in a realtively crude form, should be subject to some minimal level of characterization. Generally, the minimal criteria that should be present are (1) a predominate peak with reasonable resolution on analytical HPLC or capillary electrophoresis, and (2) a mass consistent with the presence of the desired peptide. If either of these criteria are not met, there is reason to suspect a problem and the peptide should either be evaluated further or resynthesized. Analytical Electrophoresis and Chromatography Analytical-scale HPLC and capillary electrophoresis (CE) are the two most common and effective techniques used for the assesment of heterogeneity of synthetic peptides. In practice, they complement each other
34
Synthetic Peptides
nce their respective modes of separation are based on different hysical principles. A detailed discussion of the theory and operation these two techniques, which is beyond the scope of this chapter, can e found in several excellent reviews and books (CE: Ewing et al., 989; Sanchez and Smith, 1997. HPLC: Hancock, 1984; Henschen al., 1985; Mant and Hodges, 1990; Chicz and Regnier, 1990; Mant et al., 1997). The most common form of depicting the results of either CE or PLC are measures of absorption of light at a particular wavelength otted versus elution time or fraction number. The most common avelengths used for pep tides are in the range from 210 nm to 30 nm where the polypeptide bond absorbs strongly. If the peptide ontains tryptophan, detection at 280 nm can also be used. However, aution must be excercised when following an elution at 280 nm ecause peptides that do not contain tryptophan, or where the tryptohan has been extensively oxidized, will not be seen. With both CE nd HPLC, a pure peptide is expected to produce a single symmetrical eak. However, it must be kept in mind that that alone is not sufficient iteria for homogeneity. A single symmetrical peak does not uarantee homogeneity (see figure 4-21 below) and a homogeneous ample does not guarantee the intended product (see figure 4-22 elow). Furthermore, reversible oxidation of methionine and cysteine isulfide bonds) can lead to the observation of multiple peaks. These an be misleading but do not, in themselves, necessarily indicate erious chemistry problems. If they are recognized for what they are, ey can usually be dealt with successfully. Finally, as the size of peptides increase, to greater than approxiately 50 residues, the expectation that a homogeneous peptide will rm a single, sharp symmetrical peak tends to decrease. With peptides this size, this has been attributed to the onset of the formation of latively stable, slowly exchanging folded structures which exhibit ightly altered elution characteristics and which will result in a road peak, often with absorption spikes appearing across its profile Kent, 1988; Regnier, 1987). Moreover, in the case of HPLC, the onset f these structures can be enhanced or induced by interaction with the hromatography supports themselves (Regnier, 1987), particularly e hydrophobic reverse-phase supports. This can result in the elution ofile being broad and irregular. Thus, without independent data, correct conclusions regarding heterogeneity can be made.
apillary Electrophoresis apillary electrophoresis, which was first demonstrated in 1981 orgenson and Lukacs, 1981), separates peptides on the basis of
235
heir differential migration in solution in an electric field (Ewing et al., 1989). Like ion exchange HPLC, the separation is a function of the harge properties of the peptide, but the physical basis of separation is ifferent. The separation takes place in a liquid filled capillary tube < 100 urn in diameter) whose ends are placed in buffers exposed to pposite poles of an electric field. Typical running voltages can be rom 5 kV to 30 kV with times from 1 min to greater than 1 hour. Migration takes place due to charge attraction and is recorded when he individual peptide species pass a small window in the capillary which is placed in line with a detector. For peptides, detection is sually by UV absorbance. Actually, migration is due to two comonents. The first is migration of the charged peptide tpward the pole f opposite charge and the second is bulk fluid flow within the apillary that is due to electroendoosmosis. In aqueous solution in used silica capillaries, electroendoosmotic flow is usually toward the athode, so, unless the electrophoretic mobility of an anion in the pposite direction is greater than the osmotic flow, theoretically oth anions and cations can be analyzed in a single run (Young and Merion, 1990). Since the electroendoosmotic flow is the same rate for ll solute molecules (peptides), the separation is a function of the lectrophoretic mobility of the individual species. A common set-up or peptide separation uses a pH 2.0 buffer so that all charged peptides re cationic and thus migrate to the cathode. This also minimizes the ffect of electroendoosmosis. CE is often referred to, somewhat misleadingly, as a higherensitivity technique when compared to analytical HPLC. While it is rue that the actual amount of peptide analyzed is very small when ompared to analytical HPLC (nanograms compared to micrograms), must be remembered that the volume of sample injected into the olumn is also very small (low nanoliters). Thus, the concentration f peptide in the original sample that is needed for analysis is ot much different than that needed for HPLC analysis. Unlike HPLC, which can be scaled up to be used for preparative urposes, CE is mainly useful as an analytical technique. For the nalysis of synthetic peptides, the main advantage of CE is that it rovides a second method, based on a different physical property, or separation of peptide mixtures and thus the assessment of heteroeneity. The resolving power of capillary electrophoresis is demonstrated in igure 4-3 which illustrates the separation of several synthetic peptides hat differ only slightly in their composition. While peptides that differ n the number of charges they carry are resolved by relatively large istances, it can be seen that even peptides with apparent equal charge an be separated.
236
Synthetic Peptides
Figure 4-3. Analysis of synthetic peptides by capillary electrophoresis. The capillary was 50 (im x 60 cm and the sample was run at 30 kV, after a 10s injection, in 100 HIM sodium phosphate buffer, pH 2.0. (Reproduced by permission of Dan Crimmins.)
Analytical High Performance Liquid Chromatography Analytical HPLC separates peptides by their interaction with derivatized insoluble supports, referred to as the stationary phase. In practice, it is completley analogous to conventional "low-pressure" bench-top chromatography. Both conventional chromatography and "high-pressure" or "high-performance" chromatography employ (1) a cylindrical column that contains the derivatized support upon which separation occurs, (2) a means for flowing a modifying solution, referred to as the mobile phase, by which the separation is developed, through the column, and (3) a means to detect the eluting species as they exit from the column. The major difference between high-pressure and low-pressure techniques, and hence the advantage of HPLC, is that HPLC uses columns and pumping systems that can withstand and deliver very high pressures so that very fine particles (3-10 urn mean diameter) can be used as column packing material. The result is superior resolution in relatively short time periods (minutes for HPLC as compared to hours or days for conventional systems). The two general categories of HPLC supports which are used almost exclusively for peptides are ion exchange and reverse phase, with reverse being by far the most widely employed.
237
n-Exchange HPLC paration by ion-exchange HPLC, like its conventional low-pressure unterpart, is based on direct charge interaction between the peptide d the stationary phase. The column support is derivatized with an nic species that maintains a particular charge over a range of pH ere a peptide, or mixture of peptides, can exhibit an opposite arge, depending on their amino acid compositions. When the sample oaded on to the column, the charge interaction binds the peptides to e support. The peptides are eluted from the column by changing her the pH or ionic strength, or both, of the column buffer such at the charge interaction is defeated either through neutralization of e interacting charge groups (pH) or competition with higher conntrations of like charged molecules (ionic strength). Increasing ionic ength with solutions of a salt either in a step-wise fashion or by a adually increasing gradient of concentration is the most common ethod. Thus, ion-exchange HPLC, sometimes referred to as ormal-phase" chromatography, binds the peptide at a relatively w ionic strength and elution is accomplished by progressing to conions of higher ionic strength. Peptides are separated because of their ferential interaction with the ionic support and hence elute at ferent levels of ionic strength. One approach to the ion-exchange paration of synthetic peptides has been the use of strong cationchange columns such as sulfoethyl aspartamide columns (Alpert d Andrews, 1988; Crimmins et al., 1988) which separate peptides the basis of positive charge at acid pH. Sulfoethyl aspartamide CX columns (obtained from the Nest Group) are particularly useful this regard because monomeric peptides elute in a monotonic hion according to their net positive charge at pH 3.0 (Crimmins al., 1988). An example of peptide separation with this column is own in figure 4-4. The peptides used in this figure are the same as ose in figure 4-3 for CE separation. Note that while the patterns are mewhat different and the order of elution is reversed, both give mparable resolution.
verse-Phase HPLC verse phase HPLC is so named because the conditions for elution e essentially the reverse of normal-phase chromatography. Binding through hydrophobic interaction with the column support and ution is accomplished by decreasing the ionic nature, or increasing e hydrophobicity, of the eluanl so that it competes for the hydroobic groups on the column. Column supports are generally hydrorbon alkane chains ranging from four (C4) to 18 (CIS) carbons in ngth, or aromatic hydrocarbons such as phenyl groups. Although any different buffer systems and stationary phases have been
38
Synthetic Peptides
gure 4-4. Strong cation exchange chromatography of synthetic peptides on sulfoethyl aspartimide column. The peptide mixtures are the same as those own in figure 4-3 for CZE separation. The column conditions can be found Crimmins et al., 1988. (Reproduced by permission of Dan Crimmins.)
ported (Snyder and Kirkland, 1979; Hancock, 1984; Henschen et al., 985), the most commonly employed system for peptides is a twouffer system where the column is equilibrated in 0.1 % trifluoroacetic cid for sample loading and elution is produced by an increasing adient of acetonitrile as a water miscible organic modifier. A typical cipe is for reservoir A to contain 0.1% TFA in water and for servoir B to contain 80% acetonitrile in water that has been made 1 % in TFA. The columns are usually eluted with a linear gradient un at between 0.5 and 1% B per minute. Most peptides will elute from verse-phase columns by the time the eluant is 60% acetonitrile. ther buffer recipes and elution protocols can be found in the general ferences in this section. These include the use of 0.1% heptafluoroutyric acid, 0.1% phosphoric acid, dilute HC1, and 5-60% formic cid for low pH (pH 2-4); and 10-100 HIM ammonium bicarbonate, odium or ammonium acetate, trifluoroacetic acid/triethylamine, odium or potassium phosphate, and triethylammonium phosphate r pH 4-8. In addition to acetonitrile, methanol, ethanol, propanol, nd isopropanol have been employed as water miscible eluants Rubinstein, 1979; Mahoney and Hermodson, 1980; Hancock, 1984; eldhoff, 1991).
239
It is generally advisable when using a buffer system for the first time o test the compatability of the components by simple mixing experiments. For example, phosphate is insoluble in high concentrations of cetonitrile and the two are potentially disastrous to use together withut first determining their solubility limits. Such simple experiments ould prevent the loss of an expensive column or a valuable sample. Again, the basis for separation of peptides is that each peptide intercts differently with the column and will thus elute at a different conentration of organic modifier. The resolution of reverse-phase HPLC an be excellent, but one must be constantly mindful that a single peak oes not guarantee homogeneity. This is demonstrated quite conviningly in the chromatograms in figure 4-5 where it can be seen that a ingle symmetrical peak, generated under one set of HPLC conditions, ctually contained two peptides that were separated when the conditions f the chromatography were altered. The two peptides present in this ample differed by only a single glutamic acid residue as a result of a artial deletion during synthesis. In this particular example, the pH of he TFA buffer was raised with triethylamine. Under the basic condiions, the glutamic acid in the peptide becomes negatively charged which in turn alters its interaction with the stationary phase. A word of caution about pH and reverse-phase columns. Many everse-phase columns have a silica-based support to which the onded phase (i.e., the alkane chain) is attached. The bond holding he bonded phase to the silica support is labile to basic pH and these olumns should not be used with basic buffers. However, the columns an usually be used at slightly basic pH (< 8), as shown in the example n figure 4-5, for short periods of time as long as they are returned to cidic conditions at the end of the run. Conditions of high pH or xposure to slightly basic pH for extended periods of time should be voided. Be aware that repeated use at basic pH may gradually destroy he column. Some manufacturers are now offering polymer-based upports that are resistant or unaffected by basic pH. The most common bonded phases used for the reverse-phase eparation of synthetic peptides consist of alkane chains that range n size from 1 to 18 carbon atoms. The most common are C-4, C-8, nd C-18. Because elution from the column is a function of the hydrohobicity of the peptide, and since charge decreases hydrophobicity, ery highly charged small peptides tend to be retained better by longhain bonded supports. Conversely, large hydrophobic peptides tend o exhibit better elution properties from short chain bonded supports. However, in practice, they can often be used interchangeably with little ignificant effect with most peptides. Carbon load, which refers to the amount of bonded phase present er unit of support, also has an effect on the characteristics of a everse-phase column. For instance, two different C-18 columns can
240
Synthetic Peptides
Figure 4-5. Analysis of peptide samples by buffer modulation. Chromatograms are reverse-phase HPLC of the sample depicted in figure 4-21 at two different conditions of pH.
differ significantly in the mass or amount of hydrocarbon chain they contain although they are both C-18 columns. The carbon load not only affects the column's binding capacity, but can also affect retention time. Unfortunately, carbon load is not a parameter often listed by column manufacturers, and usually has to be determined by trial and error or learned by word of mouth. In addition to the type of bonded phase, particle size and pore size are two other parameters which can have significant effect on resolution of peptides. Particle size or mesh size refers to the actual average size of the particles which make up the support. Modern HPLC columns are generally available in particle size ranges from 3-1 Oum. Smaller particle sizes produce better resolution, but they are also more expensive and increase operating pressure, referred to as back-pressure. Commonly, analytical columns (i.e., 4.6 x 250mm) use 3-10nm particles, and preparative columns (i.e., 22 x 250mm utilize 10-um particles. The particles used in reverse-phase chromatography are not solid, but rather contain pores (analagous to a honeycomb) which increases
241
e surface area of the particle and hence the interaction with the ptide. Pore size refers to the average size of the pores within the rticle itself through which solvent and solute pass during interaction th the stationary phase. In order for a molecule to effectively intert with the particle, it must be able to pass through the pores. For ge proteins, pore sizes of 300 A are more effective. This pore size n also be used effectively for most peptides, but for very small ptides (six residues or less) pore sizes of 80-100 A generally produce arper peaks and better resolution. Column age can also have a profound effect on its performance. A lumn that exhibits an extremely high degree of resolution when it is w may lose resolving power as it ages. Thus, shoulders will no longer evident or two similar peptides may co-elute. As a result, heteroneity may go undetected with a column that is operating at less than timal efficiency. In this regard, it is helpful to aquire a peptide ndard that contains closely eluting peaks that can be used to assess lumn performance periodically. When a column starts loosing solving power, it should be replaced.
urification
e crude product obtained from stepwise solid phase peptide nthesis will often contain a variety of by-products in addition to e desired product. These will usually include a family of peptides ntaining deletion or addition sequences generated during the synths itself and a series of peptides containing chemical modifications ually generated during the cleavage and deprotection steps. The two quirements to obtaining the intended product are that it is present in s mixture in sufficient amount and that the other components can successfully removed. In general, using modern synthetic produres, crude peptides of 30-45 residues can be expected to be tween 60-80% target peptide, and those of less than 30 residues ll be between 80% and 95% target peptide. All that is required en, is an effective purification protocol. Most peptides under approximately 50 residues in length can be rified using reverse phase HPLC. In cases where reverse phase is t entirely successful, ion exchange HPLC can be an effective and luable complement to reverse phase techniques. The power of reverse phase HPLC as a purification tool is ustrated in figure 4-6. It shows the reverse-phase HPLC elution ofile of the cleaved product from the synthesis of a 12-residue ptide. It shows the preparative and analytical scale elution profiles the peptide run on a C-18 reverse phase column with a standard etonitrile gradient in 0.1% TFA. The analytical scale run (middle) ows the presence of at least four major species and the preparative
243
ale run (top), which is monitored at both 214 and 254 nm, suggests at the desired peptide is the one which elutes around fraction 34. The gher ratio of A254 signal to A 2[ 4 signal suggests that the later peaks e peptide species that still retain some of the aromatic protecting oups used during synthesis. Aliquots of preparative fractions 32 ough 42 were analyzed for homogeneity with the analytical scale lumn, and fractions 33-37 were pooled to produce the final sample hose analytical profile is shown in the bottom panel. The single mmetrical peak suggests that a homogeneous peptide was obtained m the original sample. This was subsequently verified by mass ectrometry. The process of fraction-by-fraction analysis of complex patterns und in the crude synthetic products by analytical scale HPLC can a very effective approach to purification. This systematic approach en allows the retrieval of the desired product from a synthesis that ould otherwise appear to be too complex.
etailed Characterization of Covalent Structure
e previous sections dealt mainly with the assessment of peptide mogeneity and purification. These methods do not provide any ormation on the covalent nature of the peptide. The determination at the covalent structure of the peptide is correct is no less important an obtaining a peptide free of by-products. After all, some amount thought went into the design of the peptide for a particular periment, and there is much to be lost by performing the experiment th the wrong peptide. Mass spectrometry has become a very valuable tool for the aluation of synthetic peptides and many synthetic chemists now y on it as a routine part of their evaluation process because it ovides the best analysis of the covalent nature of a peptide in a gle technique. Electrospray ionization (ESI) and matrix-assisted er desorption ionization (MALDI) instruments are now readily
gure 4-6. Purification of a synthetic peptide. Top: The elution profile of 0 mg of a cleaved synthetic peptide run on a 21 x 250mm C-18 reverse ase column with a linear gradient from 0-60% acetonitrile in 0.01% luoroacetic acid/water. The column was monitored at 214nm (solid line) d at 254nm (dashed line). Middle: The elution profile of 60|ig of the me peptide preparation run on a 4.6 x 200 mm C-18 reverse column with a ear gradient from 0-60% acetonitrile in 0.01% trilluoroacetic acid/water. ttom: Analytical HPLC of the pool of preparative fractions 33-37. eproduced by permission of John Gorka, Biomolecules Midwest, Inc.)
Synthetic Peptides
ailable and relatively easy to use, and have become the mainstay of e evaluation process. Determination of the overall mass of a synthetic peptide is the most actical and common utilization of mass analysis and is usually suffient to evaluate a synthetic peptide effectively and accurately. owever, the technology now exists to produce useful information on e actual sequence of a peptide (Hunt et al., 1988; Griffin et al., 1990), d thus pinpoint the location of any unintended modification. lthough in practice, evaluation of the mass of the molecular ion is ually sufficient, particularly when used in conjunction with other anatical methods, this section will also discuss sequencing with tandem ass spectrometry.
eptide-Mass Evaluation
he determination that the mass of a synthetically produced peptide is rrect proves in one step that the synthesis was successful and you ve obtained the desired product. It may, in fact, be the only way to etermine the presence of modifications that are unstable or invisible other analytical procedures. A list of mass differences that may e encountered in the analysis of synthetic peptides is provided in ble 4-2. While a detailed discussion of the theory and practice of mass ectrometry in the evaluation of synthetic peptides is beyond the ope of this chapter and is covered in detail elsewhere (McCloskey, 990; Carr and Annan, 1999; Henzel and Stults, 1999), a brief general scussion of the main types of methodologies that have proven to e effective for determination of the mass of synthetic peptides is ppropriate. The development of particle induced desorption techniques opened e way for the determination of the mass of polar, nonvolatile, therally unstable molecules, such as peptides, without prior chemical erivatization. Prior to the development of these procedures, samples ad to be derivatized to affect vaporization without pyrolytic degradaon and analysis was limited to molecular sizes of 1000-1500 Da. The rst particle-induced desorption technique that was effective for other an the smallest of peptides was fast atom bombardment (FAB) mass nalysis (Barber et al., 1981). Although this technique is still available day, it suffers from a severe limitation in its upper mass limit and has een surpassed by more facile techniques. As modern automated ynthesizers and chemistry has improved, it has become possible to utinely synthesize peptides with molecular weights above the range even the best FAB instruments. Since the introduction of FAB, ree other types of instruments have been developed that have the dvantage of being able to analyze large peptides as well as proteins.
245
able 4-2 Mass differences relevant to synthetic peptides Modification 5' Dephospho Dehydration (-H 2 O) Pyroglutamic Acid formed from Glutamic Acid Pyroglutamic Acid formed from Gin Disulphide bond formation (Cystine) Amide formation (C-terminus) Deamidation of Asparagine and Glutamine to Aspartate and Glutamate Methylation (jV-terminus, Nc of Lysine, O of Serine, Threonine of C-tcrminus, N of Asparagine) Hydroxylation (of S C of Lysine, ft C of Tryptophan, C3 or of Proline, ft C of Aspartate) Oxidation of Methionine (to Sulfoxide) Sulfenic Acid (from Cysteine) Sodium Ethyl N, N dimethylation (of Arginine or Lysine) Formylation (CHO) Oxidation of Methionine (to Sulphone) Potassium Acetylation (jV-terminus, N of Lysine, O of Serine) (Ac) Carbamylation A'-Trimethylation (of Lysine) disodium Nitro (NO2) ?-Butyl ester (OfBu) and r-butyl ((Bu) Glycyl (-G-, -Gly-) Sodium + potassium Dehydroalanine (Dha) Alanyl (-A-, -Ala-) Acetamidomethyl (Acm) Glycine (G, Gly) j8-Mercaptoethanol adduct Phenyl ester (Oph) (on acidic) Phosphorylation (O of Serine, Threonine, Tyrosinc and Aspartate, N' of Lysine) Sulfonation (SO3H) Sulfation (of O of Tyrosine) Cyclohexyl ester (OcHex) Homoseryl lactone Dehydroamino butyric acid (Dhb) y-Aminobutyryl 2-Aminobutyric acid (Abu) 2-Aminoisobutyric acid (Aib) /-Butyloxymethyl (Bum) Seryl (-S-, -Ser-) r-Butylsulfenyl (SrBu) Alanine (A, Ala) (continued)
verage mass change
-79 -18 18 17 2 1 1 14 16 16 16 22 28 28 28 32 38 42 43 43 44 45 56 57 60 69 71 71 75 76 76 80 80 80 82 83 83 85 85 85 86 87 88 89
able 4-2 (Continued]

Modification Anisyl Benzyl (Bzl) and benzly ester (OBzl) 1,2-Ethanedithiol (EOT) Dehydroprolyl Trifluoroacetyl (TFA) jV-hydroxysuccinimide (ONSu, OSu) Prolyl (-P-, -Pro-) Valyl (-V-, -Val-) Isovalyl (-I-, -Iva-) t-Butyloxycarbonyl (/Boc) Threoyl (-T-, -Thr-) Homoseryl (-Hse-) Cystyl (-C-, -Cys-) Benzoyl (Bz) 4-Mcthylbenzyl (Meb) Serine (S, Ser) Pyridylethylation of cysteine HMP (hydroxymethylphenyl) linker Thioanisyl Thiocresy 1 Dipthamide (from Histidine) Pyroglutamyl 2-Piperidinecarboxylic acid (Pip) Hydroxyprolyl (-Hyp-) Norleucyl (-Nle-) Isoleucyl (-I-,-He-) Leucyl (-L-, -Leu-) Ornithyl (-Orn-) Asparagyl (-N-, -Asn-) z-Amyloxycarbonyl (Aoc) Prolinc (P, Pro) Aspartyl (-D-,-Asp-) Succinyl Valine(V, Val) Hydroxybenzotriazole ester (HOBt) Dimethylbenzyl (diMeBzl) Threonine (T, Thr) Cystenylation Benzyloxymethyl (Bom) p-Methoxybenzyl (Mob, Mbzl) 4-Nitrophenyl, p-Nitrophenyl (ONp) Cysteine (C, Cys) Chlorobenzyl (ClBzl) lodination (of Histidine[C4] or Tyrosine[C3] Glutamyl (-Q-, -Gin-) Lysyl (-K-, -Lys-) 0-Methyl Aspartamyl Glutamyl (-E-, -Glu-) (continued)
verage mass change 90 90 93 95 96 97 97 99 99 100 101 101 103 104 104 105 105 106 106 106 107 111 111 113 113 113 113 114 114 114 115 115 117 117 117 118 119 119 120 120 121 121 125 126 128 128 129 129
247
ble 4-2 (Continued) Modification A^-(/-Glutamyl)-Glu Norleucine (Nle) Hydroxyproline (Hyp) /3/3-Dimethyl Cystenyl Tsoleucine (I, He) Leucine (L, Leu) Methionyl (-M-,-Met-) Asparagine (N, Asn) Pentoses (Ara, Rib, Xyl) Aspartic Acid (D, Asp) Dmob (Dimethoxybenzyl) Benzyloxycarbonyl (Z) Adamantyl (Ada) p-Nitrobenzyl ester (ONb) Histidyl (-H-, -His-) W-methyl Glutamyl TV-methyl Lysyl O-methyl Glutamyl Hydroxyl Lysyl (-Hyl-) Methyl Methionyl Glutamine (Q, Gin) Aminoethyl Cystenyl Pentosyl Deoxyhexoses (Fuc, Rha) Lysine (K, Lys) Aminoethyl cystenyl (-AECys-) Methionyl Sulfoxide Glutamic Acid (E, Glu) Phenylalanyl- (-F-, -Phe-) 2-Nitrobenzoyl (NBz) Methionine (M, Met) 3-Nitrophenylsulphenyl (Nps) 4-Toluenesulphonyl (Tosyl, Tos) 3-Nitro-2-pyridinesulfenyl (Npys) Histidine (H, His) Arginyl (-R-, -Arg-) 3,5-Dibromo Dichlorobenzyl (Deb) Carboxyamidomethyl Cystenyl Carboxymethyl Cystenyl Methylphenylalanyl Hexosamines (GalN, GlcN) Carboxymethyl cysteine (Cmc) JV-Glucosyl (A'-terminus or Ne of Lysine) (Aminoketose) O-Glycosyl (to Serine or Threonine) Hexoses (Fru, Gal, Glc, Man) Methionyl Sulphone Tyrosinyl (-Y-, -Tyr-) (continued)
erage mass change
130 131 131 131 131 131 131 132 132 133 134 134 134 135 137 142 142 143 144 145 146 146 146 146 146 146 147 147 147 149 149 153 154 154 155 156 158 159 160 161 161 161 161 162 162 162 163 163
ble 4-2 (Continued)

Modification Phenylalanine (F, Phe) 2,4-Dinitrophenyl (DNp) Pentaflourophenyl (Pip) Diphenylmethyl (Dpm) Phospho Seryl 2-Chlorobenzyloxycarbonyl (C1Z) W-Methyl Arginyl Ethaneditohiol/TFA cyclic adduct Carboxy Glutamyl (Gla) Acetamidomethyl Cystenyl Acrylamidyl Cystenyl Arginine (R, Arg) Benzyl Seryl W-Methyl Tyrosinyl p-Nitrobenzyloxycarbonyl (4Nz) 2,4,5-Trichlorophenyl 2,4,6-trimethyloxybenzyl (Tmob) Xanthyl (Xan) Phospho Threonyl Mesitylene-2-sulfonyl (Mts) Tryptophanyl (-W-, -Trp-) jV-Lipoyl- (on Lysine) HMP (hydroxymethylphenyl)/TFA adduct jV-acetylhexosamines (GaWAc, GlcNAc) Tryptophan (W, Trp) Cystine (Cys)2) 4-Methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr) 2-Bromobenzyloxycarbonyl (BrZ) 9-Fluorenylmethyloxycarbonyl (Fmoc) Biotinylation (amide bond to lysine) Dimethoxybenzhydryl (Mbh) Dansyl (Dns) 2-(/)Biphenyl)isopropyl-oxyxarbonyl (Bpoc) "Triphenylmethyl (Trityl, Trt)" Pbf (pentamethyldihydrobenzofuransulfonyl) 3,5-Diiodination (of Tyrosine) O-GlcNAc-1-phosphorylation (of Serine) 2,2,5,7,8-Pentamethylchroraan-6-sulphonyl (Pmc)
erage mass change
165 166 166 166 167 169 170 172 173 174 174 174 177 177 179 179 180 180 181 182 186 188 201 203 204 204 212 213 222 226 226 233 238 242 252 252 266 266
he first of these was plasma-desorption mass spectrometry (PDMS) MacFarlane et al., 1974; Cotter, 1988; Fontenot et al., 1991), which is other particle-induced desorption technique. Although PDMS is ll a viable technique it has been almost completely surpassed by ectrospray-ionization mass spectrometry (ESMS), and matrixsisted laser desorption ionization (MALDI) mass spectrometry.
249
Electrospray ionization introduces a peptide in the liquid state and oduces a scries of multiply charged ions where the charge is carried protonatable groups. At acid pH, the free a-amino group as well as side chains of basic residues such as arginine and lysine carry a sitive charge. This produces a series of ions differing from each her by a single charge which are then analyzed by a computer to culate the molecular weight of the polypeptide. It is this multiple arging phenomenon that has allowed direct molecular weight alysis of large peptides and proteins at high sensitivity (picomol el) and accuracy. An example of a peptide mass spectra produced electrospray ionization is shown in figure 4-7. Note that multiple arging produces multiple signals despite the fact that this is a homoneous peptide. Each signal, or observed mass (m0) in the spectrum is ual to the actual mass of the peptide (m) divided by the number of arges (z) carried by the peptide (m/z). That is, m0 [m + m'(z)]/z, ere m is the mass of the peptide, z is the number of charges, and m' the mass of the charging species. In this case, this is a positive spectra where basic groups on the peptide have been protonated + H+ (PH)+]. Thus, the charging species are protons which have mass of 1. Solving for m produces the relationship, m = [m^z)} - z. ch observed mass peak is fit into this equation and the values for m averaged to produce a final peptide mass. Peptides can also be yzed in negative ion mode where the acidic groups have been protonated (PH -> P" + H+). In this case, m = [m0(z)] + z. In order to perform the calculations described above, referred to as convolution of the spectra, the charge state of each of the signals st first be known. On mass spectrometers where the isotopic peaks sulting from natural abundance of the isotopes) can be resolved, the arge state can be determined from the spacing of the isotopic peaks gure 4-8). For a singly charged peptide, the isotope peaks are 1 m/z t apart. For a doubly charged peptide they are 0.5 m/z units apart d so on. The charge state of each signal can also be determined by relationship of adjacent peaks. If Aw0 is the difference in observed ss between two adjacent peaks and zl and z2 are the charges on the ser and greater charged of the two species, respectively, then z\ (2)/Amo and z2 = m 0(1 )/Amo. In other words, the charged state of an served signal is equal to the mass of its adjacent signal divided by difference in mass between the two signals. The charges calculated this way should be whole numbers. If the charges are not whole mbers, then the two peaks chosen are not truly adjacent peaks for same peptide. Modern mass spectrometers have software that do se calculations automatically but it is a good idea to become miliar with these simple relationships. The deconvolution of the ectrum in figure 4-7 is shown in figure 4-9 and an example of the nual deconvolution is shown in table 4-3. The result of 4005.6 is in
Figure 4-7. An electrospray ionization spectrum of a 33 residue peptide. The peptide was dissolved in 50% acetonitrile/water, 1% acetic acid and injected at lOul/min into a Finnigan LCQ ion-trap mass spectrometer.
Figure 4-8. High resolution spectra (Finnigan LCQ Zoom scan) of peptides with a + 1 (left), +2 (middle), and +3 (right) charge.
Evaluation of the Synthetic Product 253
ble 4-3 Manual deconvolution of the synthetic peptide spectrum shown figure 4-7
z [ffo(z)] z Average mass
6.1 02.3 2.1 8.8 3.5 2.1
3 4 5 6 7 8
4005.3 4005.2 4005.5 4006.8 4007.5 4008.8
4005.3
4006.5
cellent agreement with the calculated mass of 4005.7 for this 33-resie peptide. Note that in the manual deconvolution shown in table 4-3, eater error can be introduced when multiplying the higher charged aks by their charge. Therefore, only the lower-charged peaks (+3, , and +5 in table 4-3) are routinely used for this procedure. Matrix-assisted laser desorption ionization mass spectrometry MALDI-MS) (McCloskey, 1990; Gibson, 1991, and references erein; Henzel and Stults, 1999) employs UV-absorbing matrices ich absorb energy from a laser pulse which in turn ionizes peptides d proteins which have been mixed with the solid matrix. The nsitivity of MALDI is in the femtomol range and has the advantage being less affected by most components commonly found in peptide lutions such as salts. Unlike electrospray, MALDI generally proces a single signal for each peptide that is the singly charged species. owever, on occasion a doubly charges species can also be observed, rticularly for larger molecules. Figure 4-10 shows a MALDI ectrum of the same peptide shown in the electrospray spectra in ures 4-7 and 4-9. The MALDI spectrum shows the (M + H)+ ass of 4006.6, so the measured mass of the peptide is 4005.6 comred to the calculated mass of 4005.7.
quencing Peptides with Mass Spectrometry
th electrospray and MALDI instruments are capable of producing quence information for synthetic peptides which can be very helpful determining the identity or position of an anomoly.
ectrospray Ionization ith electrospray instruments, this is most often accomplished by llision-induced decomposition (CID) of a particular peptide ion.
Synthetic Peptides
gure 4-10. MALDI spectrum of the same peptide in figure 4-7. The ptide was mixed with matrix (a-cyano-4-hydroxy cinnamic acid) and the ectrum run on a Perseptive Biosytems DE-Pro spectrometer.
rticularly useful mass spectrometers for this purpose are instruents that employ an ion-trap. The ion-trap allows a particular ion be easily selected, to the exclusion of all other ions, for collision with inert gas such as helium which causes it to be broken into smaller agments. The instrument then determines the mass of each new fragent and produces a spectrum which can be used to determine the quence of the peptide. Mass spectrometrists have developed a nomenclature to refer to the ptide fragments that are produced which is illustrated in figure 4-11. ith low-energy CID. A peptide can be fragmented in three places ong the polypeptide chain. Fragmentation of side chains is usually
255
Figure 4-11. Nomenclature for ions produced by collision-induced decomposition.
t observed under these conditions. When this occurs at any one ace, two fragments are formed which are named according to the sition of fragmentation and whether the charge is retained on the mino terminal portion or the carboxyl-terminal portion. For ample, in figure 4-11, if fragmentation occurs between the carbonyl rbon and amide nitrogen, the fragments are referred to as b ions and ions. B ions retain charge on the amino-terminal portion of the ptide and y ions on the carboxyl-terminal portion. In ion trap truments, these are the most commonly formed ions. Usually ubly charged ions are selected because they tend to give better gmention and can produce both b and y ions that retain a single arge. An illustration of a single fragmentation for a doubly charged ptide is shown in figure 4-12. For the parent ion, the charges are und on the amino terminus and the side chain of a lysine residue at e carboxy terminus. In the gas phase, the basicity of the amide bond similar to that of amino groups. Thus, the protons can migrate along e peptide backbone with relative ease. Protonation weakens the mide bond and makes it more susceptible to being broken upon lision with helium atoms. When this occurs, two fragments are oduced, a b ion and a y ion, each with a single charge. This occurs oughout the peptide chain so that a series of fragments are oduced that differ by the mass of a particular amino acid residue. is is illustrated in figure 4-13 for a six-residue peptide of sequence l-Thr-Ala-Asp-Phe-Lys, along with an idealized spectra for this gmentation. A residue mass refers to the mass of an amino acid as it occurs in ptide linkage. In other words, it is the free amino acid mass minus ter from the loss of the amino-terminal H and carboxyl-terminal H during formation of the peptide bond. Therefore, each b ion has a ss of the sum of the residue masses plus 1 for the extra proton on e amino terminus. Each y ion has a mass of the sum of the residue sses plus 19 for the extra hydroxyl on the carboxy terminus, the
Synthetic Peptides
Figure 4-12. Mechanism of CID cleavage in a pcptide. The b ion is on the left and the y ion on the right.
tra proton on the amino terminus and an additional proton that mparts a positive charge (see figure 4-14). Note that the additional oton that produces a charge on the y ion can be at the amino rminus or on a side chain. Within a series, each fragment differs ly by the residue mass of the respective amino acids and each set b and y ions are complementary. That is, the sum of their masses dd up to the mass of the parent peptide (i.e., bl +y5, b3 + y3, 5 + yl, etc.). An example of a real spectra generated from a peptide with the quence, EGVNDNEEGFFSAR , is shown in figure 4-15. The soluon is shown below:
2 187 G 1441 13 3 4 286 400 V N 1384 1285 12 11 5 6 7 8 9 10 11 12 13 14 515 629 7588879441091 12381325 1396 1571 D N E E G F F S A R 1171 1056942 813 684 627 480 333 246 175 10 9 8 7 6 5 4 3 2 1 <- y
> 1 130 E 1571 14
The fragmentation is produced from the doubly charged ion m/z = 86. So, the mass of the peptide is 1570 and the mass of the intact ngly charged peptide would be 1571. Note that the y ion series is
Rj
Val 99
R2
Thr 101
R3
Ala
R4
R5
Phe 147
R^
Asp
115
Lys
128
OH 17 = 679 + 2H + = 681
71
1 + R , 100
581 R 2 +R 3 +R 4 + R 5 +R 6 +19 yS 480 R3+R4+RJ+R6+19 y4 409 R4 + RJ+RJ+ 19 y3 294R s +R,i+19 y2 147 R 6 +19 yl
2 l + R j + R 2 201
3 l + R , + R 2 - e R 3 272 1 + R , + R 2 + R3 + R4 387 1 + R, + R., + R3 + R4 + R5 534
gure 4-1 3. Idealized spectra for the C1D of a 6-residue peptide. e composition of the b and y ions found in the spectra are own. R, refers to the residue molecular weight at the /th position.
Figure 4-14. Amino acid residue mass and the masses of the b and y ions. In addition to residue mass, the additional mass at the termini must also be taken into account.
d Decomposition spectra from a Finnigan LCQ ion trap de described in the text. The b and y ions are labeled.
259
early present up to y 11, but that the b ion series is present mainly as hydrated (-18) and deammoniated (-17) species. This is probably e to the large number of E, D, Q, and N residues present in the mino terminal part of the peptide. When this is taken into account, most all of the peaks in the spectra can be accounted for. The peak at 7 is the dehydrated doubly charged parent ion.
ALDI ptide sequencing on MALDI instruments is most often done by ating the peptide with an exopeptidase, such as carboxypeptidase, ior to analysis. The peptidase cleaves the peptide, residue by residue, om one end producing a ladder of masses that differ by one amino id residue. Sequence information can also be obtained from post urce decay data.
ptidase Ladder Sequencing
his approach to peptide sequencing uses an enzyme that will cleave mino acids from the ends of a peptide in a sequential manner atterson et al, 1995; Jimenez et al., 1999). When done properly, e result is a ladder of masses that differ by a single residue mass that the sequence can be read in a straightforward manner. One vantage of this method is that it can be done with a MALDI instruent with only a linear detector. Another advantage is that it is often ore successful in identifying the first one or two terminal amino acids an is collision induced fragmentation on electrospray instruments. A sadvantage is that it is not predictable. Since it employs enzymes that fer in their ability to cleave certain amino acids and in the kinetics amino acid release, a variety of digestion times and enzymes may ve to be used to obtain the best results. However, the experiments e easy to perform and the spectra are easy to read. An example is ven in figure 4-16 and an easy-to-follow protocol is found in Jimenez al., 1999.
st-Source Decay
quencing a peptide by post-source decay can only be done in an strument equipped with a reflector or reflectron which is a device pable of separating fragment ions by flight-time dispersion. A ptide ion that is stable enough to be transported out of the ion urce will often not be stable enough to completely survive the flight the detector and will decompose into fragments in the flight tube. his decomposition process is called post-source decay or metastable cay. When this occurs in the flight tube, all of the fragments will
Figure 4-16. Carboxypeptidase Y ladder sequencing of a peptide. The top spectrum is the undiluted peptide. Dilutions 1 to 5 are increasing dilutions of the enzyme. (Reproduced by permission of Keith Waddell, Applied Biosystems, Inc.)
261
ve the same velocity and will arrive at the detector at the same time. us, they are all detected as having the same apparent mass with a ear detector. A reflector is capable of separating the in-flight decomsition fragments so that their respective masses can be accurately easured. By changing the reflector voltage during an experiment a mplete set of fragments can be seen. Although b and y ions are med during post-source decay, many other ions can also form m fragmentation in other areas of the amino acid, such as the e chains. This can greatly complicate the spectra and make it diffilt, if not impossible, to interpret. Thus, post-source decay analysis is en most productive when it is known what sequence to look for and not usually suited for analysis of unknown sequences.
dditional Analytical Methods for Synthetic Peptides
ass spectrometry is a very powerful tool for the analysis of synthetic ptides and as it has become more affordable and widely available it s tended to overshadow other analytical techniques. Nontheless, ere are several other techniques that are still useful in particular uations or to answer specific questions that should not be ignored. r instance, resin sampling can still be useful to identify particularly oblematic couplings, amino acid analysis is still the best method for ptide quantitation, and Edman sequence analysis can help locate letion sequences and stable adducts if mass spectrometry fails.
sin Sampling
though resin sampling is not commonly used anymore, it can still be eful in determining if particular regions of sequence experience creased coupling efficiencies. Resin sampling is a procedure by hich a small amount of resin is removed from the reaction vessel er the completion of each coupling cycle. Its purpose is to provide measure of the coupling efficiency at each step. In order for a nthesis to proceed smoothly, the coupling efficiency as each new sidue is added must remain high. This is very important because e effect is cumulative. Even if each cycle has a coupling efficiency 95%, a value considered excellent for most organic reactions, the erall efficiency after 10 couplings is only approximately 60%. At % coupling efficiency for each cycle, the overall efficiency after 10 cles is 90%. If one or more cycles has a significantly reduced upling efficiency, the outcome of the overall synthesis can be riously affected, even though all of the other cycles experienced cellent efficiencies. To effectively attempt to correct the problem, it important to know what cycle is causing the problem. Resin mpling can help in providing that answer. Some instruments perform
Synthetic Peptides
sin sampling automatically and deliver the resin to a fraction collecr. The sampling is done at the end of the coupling step but before the blocking step. Once collected, the resin then needs to be analyzed anually for the presence of free a-amino groups. This is most comonly done by the Kaiser test (Kaiser et al., 1970, 1980; Sarin et al., 81) which is a colorimetric ninhydrin analysis specific for primary inhydrin) and secondary (isatin) amino groups. The drawbacks of sin sampling analysis are that it consumes resin and it is time conming to perform on a routine basis. Moreover, it has not been found be consistently reliable. Within certain sequence contexts, some sidues produce a very low color yield with ninhydrin which can ad to incorrect conclusions concerning coupling efficiency. Some boratories still employ the Kaiser test, but many do not. It is now ten reserved for a synthesis where a problem was found during the bsequent evaluation and where one is trying to pinpoint the exact ture of the problem during a resynthesis. In addition to the Kaiser test, other resin sampling chemistries have so been described but are not as widely used today. These include the ilization of picric acid, 2,4,6-trinitroben7enesulfonic acid (TNBS), uorescamine, and chloranil. A description of their use can be found Stewart and Young (1984). The piperidine derivative of Fmoc, hich is formed upon deblocking of the a-amino group of the growing ptide chain, absorbs strongly at 310 nm and can potentially be used monitor the degree of coupling in Fmoc synthesis (Van egenmortel et al., 1988). This method has been incorporated as an tomated protocol in some synthesizers from PE Biosystems.
mino Acid Compositional Analysis
mino acid analysis reveals the content of amino acids in a peptide d quantitates the amount of each. A mole ratio of the amino acids n be calculated from the analysis and compared to the predicted ole ratio based on the intended sequence of the peptide. More mportantly, this is the best method for determining the amount of ptide. With the exception of a few amino acids which do not yield antitatively (see below), the average amount of the amino acids can directly converted into an accurate quantitation of the peptide. eneral references for amino acid analysis with excellent descriptions e available (Ozols, 1990; Dunn, 1995; Crabb et al. 1997). The use of column chromatography for the analysis of amino acids as developed by William Stein and Stanford Moore (Moore and ein, 1948; Moore et al., 1958; Spackman et al., 1958), for which ey won the Nobel Prize in Chemistry in 1972. Their original produre used ion-exchange chromatography on sulfonated polystyrene sins and post-column detection with ninhydrin. This remained the
avaluation of the Synthetic Product 263
ndard procedure for many years, and is still in routine use today. In t, it is still considered by many to be the method of choice when antity is not limiting. A limitation of post-column ninhydrin ection is its limited sensitivity. Even on modern ninhydrin-based truments, levels below approximately 100 pmols do not quantitate curately. In addition, post-column ninhydrin analysis requires detecn at two wavelengths, 570 and 440 nm, for the detection of both ino and imino (proline and hydroxyproline) acids. More recently, post-column o-phthalaldehyde detection has been ed to increase sensitivity to the low picomol range (Roth and mpai, 1973; Benson and Hare, 1979). However, the imino acid, oline, is not detected with o-phthalaldehyde unless it is oxidized th hypochlorite (Bohlen and Mellet, 1979). This procedure also structively reduces the levels of the other amino acids and thus duces sensitivity. With the development of reverse-phase HPLC, veral, more sensitive precolumn derivitization procedures have en developed. These procedures employ chemical derivatization of e amino acids after hydrolysis, but before chromatography, with a agent that reacts quantitatively with the free a-amino group of ino acids and which can be detected by fluorescence, UV, or visible sorbance. These include derivitization with o-phthalaldehyde nes, 1986), dansyl chloride (dimethylaminonapthalene-1-sulfonyl oride) (Tapuhi et al., 1982; Gray et al., 1983; Marquez et al., 86), PITC (phenylisothiocyanate) (Heinrikson and Meridith, 1984; hen and Strydom, 1988), dabsyl chloride (dimethylaminoazonzene-4-sulfonyl chloride) (Chang et al., 1983; Knecht and ang, 1986), DABITC (dimethylaminoazobenzene-4-isothiocyanate) hang, 1983), Fmoc-Cl (9-fluorenylmethyl chloroformate) (Einarsson al., 1983), a combination of o-phthalaldehyde and Fmoc-Cl inarsson, 1985; Blankenship et al., 1989), and e-aminoquinolyl-Ndroxysuccinimidyl carbamate (Cohen and Michaud, 1993).Today veral instrument manufacturers offer one or more chemistries as a ckage. These include Water's Pico-Tag (PITC) and Accu-Tag stem, Varian's Ammo-Tag (Fmoc-Cl) system, and Agilent chnologies amino acid analysis system. An example of an amino d analysis of a 24-residue synthetic peptide using the Accu-Tag tem is shown in figure 4-17 along with a standard chromatogram. e tabulation of the analysis is shown in table 4-4. Since all amino acid analysis techniques analyze the free amino ds, any polypeptide, including synthetic peptides, must first be oken down into their individual amino acids by hydrolysis of the ptide bonds. This is commonly done by subjecting the peptide to N HC1 in either the liquid or gaseous phase. In the original procedure, peptide was dissolved in 6NHC1, the tube was evacuated and led, and hydrolysis was accomplished by incubating the tube at
ure 4-1 7. Amino acid compositional analysis using the Water's cu-Tag sytem. (a) Standard mixture of amino acids (250 pmols each), Analysis of a 24-residue synthetic peptide following hydrolysis in 6N 1.
265
ble 4-4 Ammo acid analysis tabulation of the 24-residue synthetic tide shown in figure 4-17
pmol
147.6 160.5 148.6 56.3 49.5 51.9
idue
Mol ratio
Expected
sp
2.8
3.1 2.9 1.1 0.95
hr er lu ro
ly la ys al Met e eu yr he is ys rg
1.0
3 3 3 1 1 1
155.7 52.3 52.0 212.0

110.9
3.0 1.0 1.0 4.1 2.1
3 1 1 4 2
0C for 24 h (Moore and Stein, 1963). More recently the technique vapor phase hydrolysis has been used (Dupont et al., 1988) where peptide does not come into direct contact with the liquid HC1. This ocedure tends to produce cleaner analyses because nonvolatile conminants in the liquid HC1 no longer contact the peptide. This is more portant for high-sensitivity analyses of very low level samples than s for synthetic peptides where amounts are usually not a problem, it is routinely used for both with excellent results. Vapor-phase drolysis at 150-165 C for I h (Tarr, 1986, Dupont et al., 1988) is o now used routinely with excellent results and with the obvious vantage of saving time. As mentioned earlier, several amino acids are not stable to drolysis in 6NHC1. Serine, threonine, tyrosine, and methionine are tially destroyed and glutamine, asparagine, cysteine, and tryptophan usually completely destroyed. Serine, threonine and tyrosine can be rly accurately determined by carrying out a time course hydrolysis d extrapolating back to zero time. At 110 C, the times are usually 24, and 72 h, and at 150-165 C the times are 1, 2, and 3h. Tyrosine struction can also be decreased by including 0.1% phenol in the drolysis mixture. Phosphoserine, phosphothreonine, and phosotyrosine readily dephosphorylate under normal hydrolysis proceres but can be quantitated with time course analysis using special ocedures (Crabb et al., 1997). Cysteine/cystine and methionine are
Synthetic Peptides
ually determined after performic acid oxidation (Hirs, 1967) as teic acid and methionine sulfone, respectively. Cysteine can o be determined after modification with iodoacetate or 4-vinylidine (Fontana and Gross, 1986; Hawke and Yuan, 1987, Crabb al., 1997) as S-carboxymethyl cysteine or ,S-pyridylethyl cysteine, pectively. Hydrolysis with methanesulfonic acid (Simpson et al., 1976; Crabb al., 1997)), toluenesulfonic acid plus tryptamine (Liu and Chang, 71), mercaptoethanesulfonic acid (Penke et al., 1974), methanefonic acid plus 3-(2-aminoethyl) indole (Simpson et al., 1976), decanethiol (Crabb et al., 1997), or in the presence of thioglycolic d (Matsubara and Sasaki, 1969) have been reported to preserve ptophan, but in practice the results have been found to be variable. kaline hydrolysis (Hugh and Moore, 1972) also preserves tryptoan but destroys other amino acids in the process. It is probably method of choice for accurate tryptophan determination but due its relative difficulty, it is seldom done on a routine basis. The integrity of tryptophan in a polypeptide can be evaluated to me extent by its UV absorbance. Although this does not generally ld accurate quantitation, the absorbance profile of a peptide, nned from 200 to 320 nm, can provide useful information on the te of the tryptophan in a peptide. An example is shown in figure 8 which illustrates a UV scan of a 30-residue peptide containing one ptophan residue and compares it to a scan where the tryptophan is her missing or destroyed. Intact tryptophan absorbs maximally at 0 nm, while its oxidation products absorb at lower wavelengths and h reduced extinction coefficients. 7V-Formyl tryptophan, which is d in t-Eoc synthesis absorbs maximally at 300 nm, so it would be dily apparent if deformylation was not successful. Glutamine and asparagine are completely converted to glutamic d and aspartic acid, respectively, by acid catalyzed deamidation. erefore, the values for Glu and Asp derived from amino acid alysis of an acid hydrolyzed sample represent the Glu plus Gin d Asp plus Asn content, respectively. The rate of peptide bond hydrolysis can also be affected by amino d sequence. This is most apparent when the two aliphatic side chain branched amino acids, Val or He, occur adjacent to one another. In s case, hydrolysis of the peptide bond may not be complete until er 72 h of hydrolysis (3 h for high temperature hydrolysis) so values ermined at earlier time points will be low. Amino acid analysis is particularly well suited for the analysis of nthetic peptides because it is more accurate for smaller molecules h as peptides than it is for larger molecules such as proteins. This due to the inherent error in the procedure. Recent studies (Crabb al., 1990; Tarr et al., 1991) indicate an average error in practice of

5 10 15 20 25 30 TWKPYDAADLDPTENPFDLLDFNQTQPERC
267
Figure 4-18. Amino acid analysis and UV spectroscopy of a tryptophan-containing peptide. The intended sequence is shown along the top. The amino acid analysis data are on the left and the ultraviolet spectra of the original synthesis (top) and the successful resynthesis (bottom) are on the right.
proximately 10% for nmol level analyses and as much as 16-20% pmol level analyses. When analyzing a synthetic peptide at the mol level that contained two aspartic acid residues, the expected or would be approximately 0.2 residues. A calculated value of tween 1.8 and 2.2 residues would quite accurately indicate that ly two residues of aspartic acid were present. If, however, a protein at contained 20 aspartic acid residues was being analysed, the error ould be approximately two residues and give values between 18 and residues per molecule. Synthetic peptides are usually small enough at the inherent error in the analysis is not large enough to cause such ambiguity. Thus, amino acid analysis of synthetic peptides is pected to be quite accurate and any significant deviation from inteal values of residues is usually a sign of heterogeneity in the sample. ere are, however, two general types of exceptions which must be ken into consideration when evaluating a peptide by amino acid alysis. The first comes from the fact that not all amino acids survive
Synthetic Peptides
ble 4-5 Amino acid analysis1 of a 16-residue synthetic peptide

nmol Mol ratio Expected
sidue
Asp
10.9
Thr er Glu ro Gly Ala Cys Val Met He Leu
6.6 4.2
5.7 5.5
2.0 1.2 0.8 1.0 1.0 1.1 0.8 1.1 1.0 1.6
2 1 1 1 1 1 1 1 1 2
6.0 4.2 5.9

5.7 8.7
Tyr he His Lys Arg
10.3
5.1
1.9 0.9
2 1
he peptide was vapor hydrolyzed with 6N HC1 containing 0.1 % phenol for I h at 160C and lyzed on a Beckman 6300 amino acid analyzer.
e hydrolysis procedure quantitatively and the second from the fact at the hydrolysis can remove modifying groups that may be present, ch as side-chain-protecting groups, regenerating the free amino id. Although amino acid analysis provides valuable information in the aluation of a synthetic peptide, it is dangerous to rely on it as the le characterization. The analysis shown in table 4-5 demonstrates s point. The analysis of this 16-residue peptide looks very reasonle when compared to the expected values. The peptide contains one sidue of tryptophan which is not detected, of course, and the two oleucines are adjacent to one another, so the low He value is content with the expected slow hydrolysis of the lie-lie bond. All other lues look good. However, sequence analysis of this peptide revealed at the two aspartic acid residues are not entirely present as free partic acid. Rather, the HPLC chromatograms (figure 4-19) of the TH-amino acids indicate that as much as 60% of the material at the sp positions elute approximately 6 minutes later than aspartic acid, tween the positions for alanine and tyrosine. This example clearly ustrates the presence of unintended modification of the aspartic acid sidues that was not detected by amino acid analysis because the duct was not stable to the hydrolysis procedure and regenerated e free amino acid.
269
gure 4-19. Reverse-phase HPLC analysis of PTH-amino acids from a cle of automated Edman degradation on an Applied Biosystems 477A quencer. Abbreviations: Asp, aspartic acid; DPTU, diphenylthiourea; ?, known amino acid adduct.
dman Sequence Analysis
utomated Edman degradation (Reim and Speicher, 1997; Smith, 97), more commonly referred to as amino acid sequence analysis, obviously well suited for determining the order and identity of mino acids in a synthetic peptide. It can also be very helpful in entifying some, although not all, derivatives of amino acids that ay be present for a variety of reasons (see figure 4-19). The first quence of a polypeptide was determined in 1951 when Sanger termined the sequence of insulin using fluorodinitrobenzene anger and Tuppy, 1951). Repetitive degradation of proteins from e amino-terminal end with phenylisothiocyanate was first reported Edman in 1950 (Edman, 1950) and remains the chemistry of choice sequence analysis today (figure 4-20). The automation of Edman emistry in 1967 (Edman and Begg, 1967), with the introduction of e first "spinning cup" sequencer, marked the beginning of the odem age of sequencing. Later, modifications to the spinning cup quencer greatly improved the sensitivity of sequence analysis unkapiller and Hood, 1978), and the introduction of polybrene unkapiller and Hood, 1978; Tarr et al., 1978), which helped to
Synthetic Peptides
igure 4-20. Edman Chemistry for the sequential degradation of proteins nd peptides. The reaction is carried out in successive cycles with the roduct of each cycle being a PTH-amino acid. The PTH-amino acid dentified at each cycle corresponds to the position from the amino erminus that the amino acid occupied in the peptide.
ysically retain the sample in the cup, extended the length of quence that could be obtained in a single run. Most recently, the velopment of the "gas-phase" sequencer in 1981 (Hewick et al., 81) played a major role in advancing the sequential determination polypeptide structures. The original spinning cup sequencer of Edman and Begg required otein amounts in the 100 nmol range or greater. Later, improveents in the spinning-cup instrumentation allowed sequence analysis
271
as little as l-10nmols of polypeptide, but the introduction of the s-phase sequencer, which could routinely analyze 100 pmol of polyptide, quickly outpaced the utility of the spinning-cup sequencer. day, the spinning-cup sequencer is no longer available and modern quencers are now capable of providing very clear analyses on as little 1 pmol or less of peptide. For a synthetic peptide of 25 residues, this approximately 2.5ng of material. Since sensitivity is not a major ncern for sequence analysis of synthetic peptides because quantity seldom a consideration, synthetic peptides are usually analyzed in 10-100 pmol range. Sequence analysis, as the name implies, reveals the sequence or der of the amino acids in the polypeptide chain. In this regard, e of the technique's main strengths is in revealing the presence of letion or addition sequences which are occasional problems enuntered in synthetic peptides. It can also be used effectively in vealing the presence of side-chain-blocking groups that failed to be moved during the deblocking step. However, it is more effective in s regard for peptides synthesized by t-Boc chemistry than by Fmoc emistry due to the acid lability of the blocking groups used for Fmoc nthesis (see section on on-resin sequencing below). There are also some significant characteristics of sequence analysis ich limit its usefulness. First, unless covalent coupling of the peptide a solid support is employed, it is not always possible to sequence to C-terminal residue of a peptide. This is especially so with longer ptides but can occur with relatively short peptides as well. This is e mainly to a phenomenon known as washout and is brought about the repeated washings of the sample in the reaction chamber with vents in the normal course of the sequencing chemistry. After both e coupling and the cleavage steps of the Edman chemistry, the mple is washed with organic slovents to remove excess reagents, wanted reaction by-products, and the cleaved ATZ-amino acid elf. Although these washings are formulated to remove only the terials of interest and not the peptide itself, some peptide is inriably lost at each cycle. Thus, at some point, the level of peptide maining is below the detection sensitivity of the technique and quence identification stops. Washout is usually more prone to cur with hydrophobic peptides and charged residues near the Cminus tend to reduce washout of the sequentially shortened peptide. Failure to sequence to the C-terminus of a peptide can also be used by rising background due to low-level but repeated internal avage of the polypeptide chain. This occurs because the reaction nnot be kept completely anhydrous. It usually takes many successive cles for the background to build up so that this is usually a problem ly with long peptides. In practice, both washout and rising backound contribute, but for short peptides, the major cause of cessation
Synthetic Peptides
sequencing is washout. For long peptides, sequence identification ops at the point where the effects of the two converge. At a particular int in the sequence analysis, the signal being produced by the quential degradation of the peptide may still be such that it is still thin the detection range of the HPLC. But, if the nonspecific backound has risen so high that it gives comparable signal intensity such at it is impossible to distinguish that due to sequence and that due to mulative background, no additional sequence can be determined. Loss of peptide due to washout can be alleviated to a large extent if e peptide is covalently coupled through its C-terminal residue to an soluble support so that washout does not occur. Automated solidase sequencing was introduced in 1971 (Laursen, 1971) but it has t gained widespread use and there are no automated instruments esently available which use this approach exclusively. However, emically derivatized PVDF (polyvinylidene difluoride) membranes e commercially available for this purpose under the brand name quelon. They come with either isothiocyanate or arylamine groups valently attached to the membranes through which attachment of a ptide can be made either through the amino or carboxyl terminus. tachment through the carboxyl terminus is of course preferable for dman sequence analysis. Any internal carboxyl side chains will also uple to the membrane and result in reduced yields at these positions. owever, the coupling conditions can usually be set up so that some of e carboxyl side chains remain free and a positive identification of the sidue can be made. These membranes are also very useful for quencing radiolabeled phosphorylated peptides (Wang et al., 1988; ebersold et al., 1990; Sullivan and Wong, 1991). Since the osphoryl group of phosphoserine and phosphothreonine is minated from the amino acid during the cleavage step in Edman quencing, especially rigorous conditions are required to extract the osphate ion from the sequencing cartridge and covalent attachment the peptide is critical for success. It is always desirable, and sometimes necessary to verify the quence of a peptide all the way to the C-terminal residue. ccasions where this may be the case is if mass spectrometry is not ailable or if one suspects for some reason that two or more residues ay have been transposed due to an operator error in setting up the nthesizer or a software error in directing the order of addition. If quence analysis of the intact peptide terminates before reaching the -terminal residue, another approach is needed. One approach which s been successful is subdigestion of the synthetic peptide and sequenng the individual peptides after separation on HPLC. The smaller ptides generated by this procedure are usually able to be sequenced their C-terminus, so that all of the sequence of the larger peptide n be verified by analysis of its parts. The major constraint in this
273
proach is the availability of cleavage sites within the peptide such at reasonable sized subpeptides can be generated and recovered. ble 4-6 lists the commonly used cleavage agents and the residues sequences at which they will cleave. General procedures for using ese agents can be found in a number of books and references conned therein (Fontana and Gross, 1986; Wilkinson, 1986; Aitken et , 1989), and in technical bulletins from the suppliers. A second disadvantage of sequence analysis is that it relies on the ailability of standards for identification. That is, identification of the sidue at any particular cycle is dependent on comparison, usually of HPLC peak, to a known compound or standard. This is obviously t a limitation for the normal amino acids or even for the side-chainotected amino acids since standard compounds are readily identified d available. However, it can be severely limiting for unexpected or usual adducts that have formed at some point in the synthesis, such that presented in figure 4-19. While the presence of an unusual peak ay indicate a divergence from normal, it does not provide much eful evidence as to the identity of the adduct. Furthermore, the uation can be further complicated if the unknown adduct co-elutes the position of a standard amino acid. Another major limitation of sequence analysis is that peptides that e chemically modified at their amino terminus will not be detected. is is of particular importance when capping chemistry is employed ring the synthesis. While capping can be very helpful in the purifican process, any capped peptide that is not removed from the sample ll be invisible to sequence analysis. The sequencing chemistry can also reverse some derivatives of mino acids, regenerating the parent amino acid in the process, so at its presence goes undetected. Partial reversal is often seen in onsin sequencing of peptides generated by t-Eoc chemistry. With the otecting groups used for Fmoc synthesis, the reversal can be mplete. Another example of this is methionine sulfoxide. The sulfur om of methionine residues can be oxidzed during synthesis and ndling, usually to the sulfoxide. In fact, methionine sulfoxide is metimes employed directly as a protecting group in the synthesis a peptide. After synthesis, the sulfoxide can be converted back to ethionine by treatment with thiol reagents such as dithiothreitol and -methylmercaptoacetamide (Houghton and Li, 1979; Cullwell, 87). However, if the sulfoxide is present in the peptide, its presence ll go undetected in sequencing because it is converted back to ethionine due to the presence of dithiothreitol in the extraction lvent on many sequencers. Finally, sequence analysis is not very reliable for quantitation, with me residues being detected only at very low levels or not at all. These clude serine, threonine, and cysleine which are degraded to varying
Table 4-6 Common reagents and proteases for cleavage of peptides3

Bond cleaved Arg, Lys-X (X / Pro) Lys-X (X Pro) Arg-X (X / Pro) Glu-X (X ^ Pro), (Asp-X)c Asn-X X-Asp Trp, Phe, Tyr, Leu-X (X Pro) pGlu-Xd Acetyl-Xe Met-X Asn-Gly Asp-Pro Trp-X Agent Trypsin Endoproteinase Lys-C Lysyl Endoproteinase Endoproteinase Arg-C (Submaxillary Protease) Endoproteinase Glu-C S. aureus V8 Protease Asparaginyl Endopeptidase Endoproteinase Asp-N Chymotrypsin Pyroglutamate Aminopeptidase Acylamino Acid Releasing Enzyme Cyanogen Bromide Hydroxylamine Dilute Acid lodosobenzoate BNPS-Skatole ./V-Chlorosuccinimide Reference Wilkinson, 1986 Sourceb Worthington, Pierce Boehringer Mannheim Boehringer mannheim Wako Chemicals Boehringer Mannheim Pierce, Takara Biochemicals Boehringer Mannheim Pierce Takara Biochemicals Boehringer Mannheim Worthington Boehringer Mannheim Boehringer Mannheim Pierce, Takara Biochemicals Pierce, Takara Biochemicals
Masaki et al., 1981a, b Levy et al., 1970 Schenkein et al., 1977 Wilkinson, 1986 Ishii et al., 1990 Maier et al., 1986 Wilkinson, 1986 Shivly, 1986 Tsunasawa and Narita, 1982 Fontana and Gross, 1986 Fontana and Gross, 1986 Fontana and Gross, 1986 Fontana and Gross, 1986
This table is not intended to be exhaustive. Only commonly used agents of high specificity ate listed. For the readers convenience, a limited number of sources are listed. For some, these are the only source; for others, additional sources exist but no attempt has been made to list them all. 0 Occasional cleavage produced at these sites. d pGlu, cyclized Gin at the TV-terminal position. e Removes amino-terminal acetylated residue. Active only on peptides up to 20-30 residues.
275
grees by ^-elimination during sequencing, and tryptophan, which is nsitive to oxidation either during sequencing or during storage and ndling prior to sequence analysis. Although dithiothreitol is often ded to the ATZ-amino acid extraction solvent to trap the reactive mination products of serine, threonine, and cysteine, the yields of ese amino acid adducts are still low and variable. In fact, cysteine en must be derivatized prior to sequencing in order to detect it at . There are many reagents available for this purpose (Gambee et al., 95). Alkylation with 4-vinylpyridine to the S-pyridylethyl derivative n be performed on the sample after it has been applied to the quencer cartridge (Andrews and Dixon, 1987; Hawke and Yuan, 87) and is probably the reagent of choice due to its stability and PLC elution position. Arginine and histidine also tend to produce w yields due to reduced extraction efficiency of the positively arged amino acids and undesirable interactions with incompletely pped HPLC columns. Tn addition to variability in individual PTH-amino acid recovery, e overall initial yield of a sequencing run can also vary. This can be e to several factors, such as the operating efficiency of the sequencer d the chemical properties of the sample itself. Thus, even if only ble PTH-amino acids are considered, the overall yield of the quencing run itself can vary by several fold. In spite of all the limitations listed above, sequence analysis is still a eful technique for the evaluation of synthetic pep tides. What quence analysis does best, and that no other technique can do on routine or available basis, is elucidate the actual linear sequence of mino acids as they occur physically in a peptide. In this regard, quence analysis is outstanding in revealing deletion and addition quences. The role of sequence analysis in finding deletion sequences is ustrated in the example presented in figure 4-21. The product of s synthesis runs as a single major peak on the standard reversease HPLC analysis system, which is 0.1 % TFA with a linear acetitrile gradient, and the amino acid analysis is entirely consistent with e desired product. However, in this case, sequence analysis revealed e presence of two peptides that differed only by a single amino acid. much as 60% of the peptide was present with the deletion of a ngle glutamic acid residue at position number 5. Quite surprisingly, s was not well reflected in the amino acid analysis and again ustrates the danger of relying on a single type of analysis. The utamic acid value may have been more revealing in this case if the ptide contained fewer Glu + Gln residues overall. Subsequent verse-phase chromatography at basic pH, where the glutamate side ain would now be ionized, easily separated the two peptides (figure 5). This example also illustrates how relying on a single set of HPLC
Synthetic Peptides
Figure 4-21. Evaluation of a 20-residue peptide indicating the presence of a deletion-sequence peptide. The expected sequence is shown at the top and the experimentally determined sequence of the sample is shown at the bottom. The amino acid analysis and reverse-phase HPLC analysis of the sample are also shown.
onditions could be misleading unless other analytic procedures are ought to bear, and it also demonstrates how sequence analysis can mplement HPLC as a means of detecting heterogeneity. Mass specometry would also have detected the presence of the two peptides if it d been performed. Figure 4-22 illustrates another example of the ccurrence of a deletion sequence in a synthetic peptide. In this case, e deletion involves a block of residues rather than just a single sidue. This deletion is easily detected by amino acid analysis, but nce it occurs at the C-terminus, Edman sequencing may not be tirely effective. Deletions or additions can occur anywhere in the ptide and can be a much more serious problem with longer peptides here sequencing will not reach to the C-terminus.
n-Resin Sequencing
he growing polypeptide chain can also be analyzed by automated dman degradation while it is still attached to the resin (Kent et al.,
277
Figure 4-22. Evaluation of a 19-residue peptide indicating the presence of a total deletion sequence of more than one residue. The expected sequence is shown at the top and the experimentally determined sequence of the sample is shown at the bottom. The amino acid analysis and reverse-phase HPLC analysis of the sample are also shown. Arrows indicate obvious deviations from that expected and the indicated residues correspond to the missing sequence shown in the box.
82). This technique is most applicable to peptides synthesized th t-Boc chemistry, due to the stability of the protecting groups der acidic conditions. This is often done upon completion of the nthesis, but can be done at any time during the synthesis to assess e quality of the growing peptide. This is often referred to as "preview alysis," since deletion sequences will show up as a preview of residue + 1 in cycle / (see also chapter 3). For example, such an analysis ght be performed several times during the course of synthesis of a ry long peptide. This requires very little resin because of the excellent nsitivity (1 pmol) of modern sequencers. The only requirement is at the resin be removed at a time when the peptide possesses a free amino group, such as after removal of the a-amino protecting group t before the beginning of the next coupling cycle. Because the
Synthetic Peptides
quencing is being performed in the solid phase while the peptide is valently coupled to an insoluble bead, sample washout from the quencer is not a problem. Therefore, the peptide can easily be quenced to its penultimate residue. The C-terminal residue is not ually seen because it is attached to the resin bead by a bond that not cleavable by the Edman chemistry. Since peptides that are sequenced on-resin have not yet been subted to side chain deprotecting procedures, those amino acids whose de chains contain protecting groups will not elute on an HPLC at the me position as the free PTH-amino acids. Generally, the side-chainotected PTH-amino acids elute later than the their unprotected unterparts, and most elute well after the last of the normal PTHmino acids. For this reason, most will not be detected with the andard HPLC program used for routine sequence analysis. For tection of side chain protected PTH-amino acids the gradient and n time needs to be extended (Kent et al., 1982; Kochersperger et al., 89). Note that with some amino acids, particularly Glu, Asp, d Ser, the side-chain-protecting group is unstable to the sequence emistry and some regeneration of unprotected amino acids result. The extended gradients can also be used in routine sequencing of eaved, deprotected peptides since it gives a better picture of other mponents which can be present, particularly side chains that were t successfully deprotected. Some of the more common blocking oups that have been detected still attached to cleaved peptides are syl-arginine and formyl-tryptophan. A list of the relative elution sitions of modified PTH amino acids that may be encountered in e analysis of synthetic peptides is given in table 4-7. A similar approach can also be used for peptides synthesized th Fmoc chemistry (Kochersperger, et al., 1989), but their are veral disadvantages. First, the bond to the resin is not stable der the conditions of the sequencing chemistry, so that the advange of sequencing in the "solid phase" is lost. Secondly, the sideain- protecting groups are less stable to the sequencing chemistry d are removed more readily. This is not necessarily a disadvantage on-resin sequencing but can be a serious drawback in trying to sess the degree of deprotection in cleaved peptides made with Fmoc emistry.
orage and Handling of Synthetic Peptides
he manner in which a peptide is handled and stored can be just as mportant to its integrity and utilization as any of the chemistry used produce it. In theory, most peptides should be stable at neutral pH in ueous solution for extended periods of time. However, contamination th microorganisms or metal ions can cause peptide-bond cleavage.
able 4-7 Relative elution positions of PTH-amino acids on HPLCa

PTH-amino acid Tyr(P) PTC-Gly Asp Asn PTC-Ala Ser Gin Thr Gly Glu Dimethylphenylthiourea (DMPTU) Cys-acetamidomethyl (Acm) Hypro (cis) Ala Hypro (trans) Hisb PTC-Met Ser derivative Cys-3-nitro-2-pyridinesulfenyl (Npys) Asp-0-allyl (OA1) Tyr Ser derivative PTC-Val
etention time min)
Notes stable stable, partial conversion product to ATZ-Gly stable, slight conversion to Asp derivative ~5-10% recovered as PTH-Asp stable, partial conversion product of ATZ-Ala typical ~0-20% recovered, see Ser derivative ~5-20% recovered as PTH-Glu typically 5-25% recovered stable stable, slight conversion to Glu derivative Edman chemistry by-product stable hydroxyl can be cis or trans, Edman chemistry produces both stable hydroxyl can be cis or trans, Edman chemistry produces both stable but may not be completely extracted from sequencer stable, partial conversion product of ATZ-Met breakdown product of serine due to Edman Chemistry Sensitive to DTT. Converts to Cys with successive cycles partially stable, converts to Asp stable breakdown product of serine due to Edman Chemistry stable, partial conversion product of ATZ-Val (continued)
5.8 6.1 6.2 7.1 8.1 8.2 8.8 9.3 9.7 0.5 1.0 2.5 3.0 3.8 4.0 4.6 5.1 5.2 6.8 7.4 7.7 8.8 9.0

Retention time (min) PTH-amino acid Ser-allyloxyxarbonyl (Aloe) Argb PTC-Lys PTC-Leu PTC-Ile Tyr-dimethoxyphosphoryl (OP(OMe)2) Pro Met Val Arg-diallyloxycarbonyl (Aloc)2 derivative Thr-allyloxycarbonyl (Aloe) Asp derivative Diphenylyhiourea (DPTU) Arg-4-toluenesulfonyl (Tos) Glu derivative Lys-allyloxycarbonyl (Aloe) His-2,4-dinitrophenyl (Dnp) Glu-O-allyl (OA1) Trp Asp-O-terf-butyl (O?Bu) Phe Tip-AT-formy! (CHO) Phe-/?Nitrophenyl Cys-allyl (Al) Notes partially stable, converts to Ser derivatives stable but may not be completely extracted from sequencer stable, partial conversion product of ATZ-Lys stable, partial conversion product of ATZ-Ile stable, partial conversion product of ATZ-Ile rapidly converted to Tyr(P) in first few cycles stable usually stable but susceptible to oxidation stable product of conversion of Arg (Aloc)2, may be Arg (Aloe) stable associated with conversion to PTH-Asp Edman chemistry by-product stable associated with conversion to PTH-Glu stable stable stable, slight conversion to Glu derivative typically only partially recovered at inconsistent levels very unstable, converts to Asp stable 20-40% recovered as PTH-Trp stable stable
19.1 19.8 19.9 20.7 20.7 20.8 21.1 21.9 22.3 22.6 23.3 23.7 23.9 23.9 23.3 24.1 24.7 25.1 25.3 25.8 26.1 26.1 26.2 26.2
26.7 27.1 27.4 27.5 27.8 28.2 28.5 28.9 29.2 29.5 30.0 30.1 30.5 30.6 30.6 31.2 31.3 31.7 32.1 32.5 33.4 33.6 33.6 33.8 33.9 34.0 34.3 34.3
He Lys (ptc) Leu Norleucine His-terAutoxymethyl (Bum) Ser-benzyl (Bzl) Cys-allyloxycarbonyl (Aloe) Arg-4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr) Arg-mesitylene-2-sulfonyl (Mts) Cys-ter?-butyl (?Bu) Asp-O-benzyl (OBzl) Arg-diallyloxycarbonyl(Aloc)2 Trp derivative His-3-benzyloxymethyl (3-Bom) Thr-benzyl (Bzl) Tyr-allyl (Al) Glu-O-benzyl (OBzl) Tye-terf-butyl (?Bu) Cys-4-methoxybenzyl (Mob) Thr-ter?-butyl (?Bu) Asp-O-cyclohexyl (OcHex) Lys-chlorobenzyloxycarbonyl (C1Z) Lys-2-chlorobenzyloxycarbonyl (2C1Z) Thr-benzyl (Bzl) derivative Lys-2,4-dinitrophenyl (Dnp) Trp derivative Hydroxyproline-4-benzyl (4-Bzl) Glu-O-cyclohexyl (OcHex)
stable stable, both amino groups are derivatized with PITC stable stable converted to PTH-His with successive cycles partially recovered, remainder converted to Ser derivatives stable partially recovered, converted to PTH-Arg with successive cycles typically 1-5% recovered as PTH-Arg very unstable, converts to Cys -5-20% recovered as PTH-Asp partially stable one of multiple Trp degradation products stable partially stable, products include PTH-Thr and Thr derivatives stable ~ 10-40% recovered as PTH-Glu very stable, converts to Tyr partially stable, products include Ser derivatives very unstable, converts to Thr ~5-20% recovered as PTH-Asp partially stable, some recovered as PTH-Lys (ptc) stable, perhaps 1-2% recovered as PTH-Lys (ptc) produced from Thr-benzyl (Bzl) stable one of multiple Trp degradation products unstable, converts to Hypro ~10^0% recovered as PTH-Glu (continued)
able 4-7 (Continued)

Notes Partially stable, products include Ser derivatives unstable, converts to Hypro converted to PTH-Lys (pts) with successive cycles stable, perhaps 1-5% recovered as PTH-Tyr ~ 10-40% recofered as PTH-Glu one of multiple Trp degradation products
etention time min)
PTH-amino acid Cys-4-methylbenzyl (Meb) Hydroxyproline-4-benzyl (4-Bzl) Lys-9-fluorenylmethyloxycarbonyl (Fmoc) Tyr-2-bromobenzyloxycarbonyl (2-BrZ) Glu-O-9-fluorenylmethyl (OFm) Trp derivative
5.1 5.4 7.8 8.6 8.7 8.9
de-chain-protected amino acids completely or nearly completely unstable to Edman Chemistry converts converts converts converts converts converts converts converts converts converts to to to to to to to to to to His His His Asp/Glu Ser/Tht/Tyr/Hypro Lys/His His/Cys/Asn/Gln Arg Arg Asn/Gln/Cys
is-allyloxycarbonyl (Aloe) is-tosyl (Tos) is-benzyloxycarbonyl (Z) sp/Glu-O-ter/-butyl (O/Bu) er/Thr/Tyr/Hypro-rer/-butyl (?Bu) ys/His-?er?-butyloxycarbonyl (Boc) is/Cys/Asn/Gln-triphenylmethyl (Trt) rg-2,2,5,7,8-pentamethyl-chroman-6-sulfonyl (Pmc) rg-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) sn/Gln/Cys-2,4,6-trimethoxybenzyl (Tmob)
Elution positions are relative and may vary somewhat depending on system and column. Some of these derivatives were originally done with different conditions and are now presented on a typical resin-bound sequencing cycle (ABI "REZ" cycle). Table composed from material taken from G. A. Grant, M. W. Crankshaw, and J. Gorka, Meth. Enzymol. 9, 395 (1997), and C. G. Fields, A. Loffet, S. A. Kates, and G. B. Fields, Anal. Biochem. 203, 245 (1992). Position is variable depending on ionic strength and elution buffer composition.
283
oreover, oxygen can adversely affect tryptophan, cysteine, and ethionine residues, and the presence of metal ions can cause destrucn of tryptophan. Aspartic acid-proline bonds are also extremely nsitive to acid cleavage. Storage of peptides containing this bond extended times in aqueous solutions that are only slightly acidic n lead to significant hydrolysis. Other peptide bonds with aspartic d also tend to be more acid labile than most, but not nearly to degree seen with Asp-Pro. Also in aqueous solution, asparagineycine and aspartic acid-glycine bonds, and in some instances partic acid W-terminal to other short-side-chain amino acid residues er, Thr, Ala, Asn), can cyclize to form an aspartimide intermediate at can undergo spontaneous ring opening to either regenerate e original peptide or produce a /3-aspartyl peptide that possesses o methylene carbons between the backbone amino and carbonyl oup of the residue (see figure 4-2). When this occurs, the structure the peptide is drastically altered. The presence of a /5-aspartyl nd can be indicated by sequence analysis. Because of the additional bon in the backbone, the PTC-peptide is not able to cyclize iciently, and sequencing will stop when such a structure is countered. For the reasons mentioned above, peptides should be stored in ution only for short periods of time. For long-term storage the ptide should be lyophilized from a volatile buffer or solvent and red dessicated at -20 C in either polyethylene, polypropylene or anized glass containers. When containers of dry peptides are moved from the cold, they should be allowed to come to room mperature before they are opened to minimize condensation of ter vapor on the peptide surface. This could contribute to hydrois upon the return of the remainder of the peptide to storage. If a peptide has been stored for an extended period of time, it is not bad idea to re-evaluate it before use. Even "dry" peptides after philization can contain a significant amount of adsorbed solvent ich can contribute to unwanted reactions. An example is the atively fascile deamidation of Asn and Gin when residual acid is esent in the "dry" peptide. Peptides containing amide side chains ould either not be dried down from acidic solution or precaution ould be taken to assure that all acid is removed from the sample ore storage. If peptides are going to be stored for any length of time after nthesis but before they are worked up, they should be desalted ore storage. Many salts and organics can be present as a result of synthesis, cleavage, and deprotection procedures that can have an verse effect upon long term storage. Desalting can be accomplished her by standard low pressure gel filtration chromatography or by eparative reverse-phase HPLC.
Synthetic Peptides
w Much Is Enough?
w much evaluation is necessary? That is a question that is always ed, both by investigators who are having peptides made for them someone else and by the synthetic chemists themselves. In an ideal rld, all synthetic peptides would be subjected to all possible aluation techniques, and there would be no need to ask the question. wever, most investigators are restrained by matters of cost, time, d instrument availability and are therefore often confronted with s question. Practically speaking, most peptides which can be purified to a single mmetrical peak on HPLC or CE and which yield a good mass ctrum will be the intended product. The evaluation should be en at least this far. However, if one procedure indicates a problem ile others do not, it is usually not wise to ignore the one in favor of others. That one procedure is usually trying to tell you something d is sufficient grounds in itself to delve further into the evaluation. It is also not recommended to rely on a single analytical technique your evaluation. We have seen examples where apparent single aks really contained several species and where a reasonable looking ino acid analysis was not truly indicative of the complexity of the mple. Evaluation by sequence analysis alone may reveal only one uence, and while that sequence may be the expected one, other cked species can also be present. There is also a common misnception that mass analysis is all that is necessary if it indicates correct mass ion. It is possible to miss other constituents with s technique for a variety of reasons. The contaminant may produce weaker signal that is not proportional to its actual presence in the mple if it is not desorbed as efficiently or is outside the mass range of instrument. Conversely, the major peptide in regard to total sammay give a very weak signal or none at all. A cumulative 6-year study conducted by the peptide synthesis/mass ctrometry subcommittee of the Association of Biomolecular source Facilities (Angeletti et al., 1997) reinforces many of the nts made above. While its original intention was to assess the pepe synthesis capabilities in core facilities, it offers many enlightening servations pertaining to the strengths and weaknesses of the meths of peptide evaluation. It is recommended reading for anyone gaged in peptide synthesis and evaluation. Unfortunately, no one can say in advance how much evaluation ds to be done. Each peptide is different and each synthesis is ferent. The data need to be evaluated as they are collected in er to make the decision. The answer to the question posed at the ginning of this section can only be "as much as is necessary to nvince a knowledgable person that the product is as it should be."
285
u are the ultimate judge. Hopefully, this chapter, as well as the er chapters in this book, have provided a helpful discussion of means by which to make that decision. How you apply that owledge is up to you.
KNOWLEDGMENTS The author gratefully acknowledges the advice, helpful cussions, and materials provided by Mark Crankshaw and the staff of the shington University Protein Chemistry Laboratory, John Gorka of molecules Midwest, Dan Crimmins of the Department of Pathology at shington University, and the Association of Biomolecular Resource ilities.
References
bersold, R., Pipes, G. D., Wettenhall, R. E., Nika, H., and Hood, L. E. (1990). Covalent attachment of peptides for high sensitivity solid-phase sequence analysis. Anal Biochem. 187:56-65. ert, A. J., and Andrews, P. C. (1988). Cation exchange chromatography of peptides on (2-sulfoethyl aspartamide)-silica. J. Chromatogr. 443:85-96. ken, A., Geisow, M. J., Findlay, J. B. C., Holmes, C., and Yarwood, A. (1989). Peptide preparation and characterization. In Protein Sequencing: A Practical Approach, J. B. C. Findlay and M. J. Geisow, eds., Oxford, IRL Press, pp. 43-68. drews, P. C., and Dixon, J. E. (1987). A procedure for in situ alkylation of cysline residues on glass fiber prior to protein microsequence analysis. Anal. Biochem. 161:524-528. geletti, R. H., Bonewald, L. F., and Fields, G. B. (1997). Six-year study of peptide synthesis. Methods Enzymol. 289:697-717. any, G., Knieb-Cordonier, N., and Mullen, D. G. (1987). Solid phase peptide synthesis: a silver anniversary report. Int. J. Peptide Protein Res. 30:705 739. ber, M., Bordoli, R. S., Sedgwick, R. D., and Tyler, A. N. (1981). Fast atom bombardment of solids (F. A. B.): A new ion source for mass spectrometry. J. Chem. Soc. Chem. Commun. No. 7:325-327. nson, J. R., and Hare, P. E. (1979). o-Phthalaldehyde: fluorogenic detection of primary amines in the picomole range, comparison with fluorescamine and ninhydrin. Proc. Natl. Acad. Sci. USA 72:619-622. nkenship, D. T., Krivanek, M. A., Ackermann, B. L., and Cardin, A. D. (1989). High sensitivity amino acid analysis by derivatization with ophthalaldehyde and 9-fluorenylmelhylchloroformate using fluoresence detection: applications in protein structure determination. Anal. Biochem. 178:227-232. hlen, P., and Mellet, M. (1979). Automated fluorometric amino acid analysis: the determination of proline and hydroxyproline. Anal. Biochem. 94:313-321. r, S. A. and Annan, R. S. (1999). Overview of peptide and protein analysis by mass spectrometry. In Current Protocols in Protein Science, 16.1.116.1.27, New York, John Wiley and Sons.
Synthetic Peptides
ang, J.-Y. (1983). Manual micro-sequence analysis of polypeptides using dimethylaminoazobenzeneisothiocyanate. Methods Enzymol. 91:455-467. ang, J.-Y., Knecht, R., and Braun, D. G. (1983). Amino acid analysis in the picomole range by precolumn derivatization and high-performance liquid chromatography. Methods Enzymol. 91:41^18. cz, R. M., and Regnier, F. E. (1990). High performance liquid chromatography: Effective protein purification by various chromatographic methods. Methods Enzymol. 182:392-421. hen, S. A. and Michaud, D. P. (1993). Synthesis of a fluorescent derivatizing reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Anal. Biochem. 211:279-287. hen, S. A., and Strydom, D. J. (1988). Amino acid analysis utilizing phenylisothiocyanate derivatization. Anal. Biochem. 174:1-16. ter, R. J. (1988). Plasma desorption spectrometry comes of age. Anal. Chem. 60:781A-793A. bb, J. W., Ericsson, L., Atherton, D., Smith, A. J., and Kutny, R. (1990). A collaborative amino acid analysis study from the association of biomolecular resource facilities. In Current Research in Protein Chemistry: Techniques, Structure, and Function, J. J. Villafranca, ed., San Diego, Calif., Academic Press, pp. 49-61. bb, J. W., West, K. A., Dodson, W. Scott, and Hulmes, J. D. (1997). Amino acid analysis. In Current Protocols in Protein Science, 11.9.111.9.42, New York, John Wiley and Sons. mmins, D. L., Gorka, J., Toma, R. S., and Schwartz, B. D. (1988). Peptide characterization with a sulfoethyl aspartimide column. J. Chromatogr. 443:63-71. lwell, A. (1987). Reduction of methionine sulfoxide in peptides using Nmethylmercaptoacetamide. Applied Biosystems User Bulletin, Model 430, No. 17. nn, B. (1995). Quantitative amino acid analysis. In Current Protocols in Protein Science, 3.2.1-3.2.3, New York, John Wiley and Sons. pont, D., Keim, P., Chui, A., Bozzini, M., and Wilson, K. J. (1988). Gasphase hydrolysis for PTC-amino acid analysis. Applied Biosystems User Bulletin, Model 420A, Issue No. 2. man, P. (1950). Method for determination of amino acid sequence in peptides. Acta. Chem. Scand. 4:283-293. man, P., and Begg, G. (1967). A protein sequenator. Eur. J. Biochem. 1:80-91. arsson, S. (1985). Selective determination of secondary amino acids using precolumn derivatization with 9-fluorenylmethylchloroformate and reversed-phase high-performance liquid chromatography. J. Chromatogr. 348:213-220. arsson, S., Josefsson, B., and Lagenkvist, S. (1983). Determination of amino acids with 9-fluorenylmethylchloroformate and revered-phase high-performance liquid chromatography. J. Chromatogr. 282:609-618. ng, A. G., Wallingford, R. A., and Olefirowicz, T. M. (1989). Capillary electrophoresis. Anal. Chem. 61:292A-303A. dhoff, R. (1991). Why not ethanol-based solvents for RP-HPLC of peptides and proteins? In Techniques in Protein Chemistry, Vol. II, J. J. Villafranca, ed., San Diego, Calif., Academic Press, pp. 55-63.
287
ntana, A., and Gross, E. (1986). Fragmentation of polypeptides by chemical methods. In Practical Protein Chemistry: A Handbook, A. Darbre, ed., New York, John Wiley and Sons, pp. 67-120. ntenot, J. D., Ball, J. M., Miller, M. A., David, C. M., and Montelaro, R. C. (1991). A survey of potential problems and quality control in peptide synthesis by the fluorenylmethylcarbonyl procedure. Peptide Res. 4:19-25. jii, N., Otaka, A., Funakoshi, S., Bessho, K., Watanabe, T., Akaji, K., and Yajim, H. (1987). Studies on peptides CLI: Synthesis of cystine-peptides by oxidation of S-protected cysteine peptides with thallium (III) trifluoroacetate. Chem. Pharm. Bull. 26:539-548. taki, S., Yajami, T., Taike, T., Akita, T., and Kitagawa, K. (1990). Sulphur trioxide/thiol: a novel system for the reduction of methionine sulphoxide. J. Chem. Soc. Perkin Trans. 1:653-658. mbee, J., Andrews, P. C., DeJongh, K., Grant, G., Merrill, B., Mische, S., and Rush, J. (1995). Assignment of Cysteine and Tryptophan Residues During Protein Sequencing: Results of ABRF 94 SEQ. In Techniques in Protein Chemistry, Vol. VI, J. Crabb, ed., San Diego, Calif., Academic Press, pp. 209-217. bson, B. W. (1991). A brief overview of mass spectrometric methods for the analysis of peptides and proteins. In Techniques in Protein Chemistry, Vol. II, J. J. Villafranca, ed., Academic Press, San Diego, Calif, pp. 419-425. ffin, P. R., Martino, P. A., McCormack, A. L., Shabanowitz, J., and Hunt, D. F. (1990). Protein and oligopeptide sequence analysis on the TSQ-70 triple quadrupole mass spectrometer. In Current Research in Protein Chemistry: Techniques, Structure, and Function, J. J. Villafranca, ed., San Diego, Calif, Academic Press, pp. 117-126. ncock, W. S., ed. (1984). CRC Handbook of HPLC for the Separation of Amino Acids, Peptides, and Proteins, Vol. I and II, Boca Raton, Fla., CRC Press. wke. D., and Yuan. P. (1987). S-Pyridylethylation of cystine residues. Applied Biosystems User Bulletin for 470A/477A-120A, Number 28. inrikson, R. L., and Meridith, S. C. (1984). Amino acid analysis by reversephase high-performance liquid chromatography: Pre-column derivatization with phenylisothiocyanate. Anal. Biochem. 136:65-74. nschen, A., Hupe, K. P., Lottspeich, F., and Voelter, W., eds. (1985). In High Performance Liquid Chromatography in Biochemistry, Weinheim, VCH. nzel, W. J., and Stults, J. T. (1999). Matrix-Assisted Laser Desorption/ lonization Time-of-Flight Mass Analysis of Peptides. In Current Protocols in Protein Science, 16.2.1- 16.2.11, New York, John Wiley and Sons. wick, R. M., Hunkapiller, M. W., Hood, L. E., and Dreyer, W. J. (1981). A gas-liquid solid phase peptide and protein sequenator. J. Biol. Chem. 256:7990-7997. s, C. H. W. (1967). Determining cysteine as cysteic acid. Methods Enzymol. 11:59-62. ughton, R. A., and Li, C. H. (1979). Reduction of sulfoxides in peptides and proteins. Anal. Biochem. 98:3646. gh, T. E., and Moore, S. (1972). Determination of the trypyophan content of proteins by ion exchange chromatography of alkaline hydrolysates. J. Biol. Chem. 247:2828 2834.
Synthetic Peptides
nkapiller, M. W., and Hood, L. E. (1978). Direct microsequencc analysis of polypeptides using an improved sequenator, a non-protein carrier (polybrene), and high pressure liquid chromatography. Biochemistry 17:2124 2133. nt, D. F., Shabanowitz, J., Yates, J. R., Griffin, P. R., and Zhu, N. Z. (1988). In Analysis of Peptides and Proteins, C. McNeil, ed., New York, John Wiley and Sons, pp. 151-165. ii, S., Abe, Y., Matsushita, H., and Kato, I. (1990). An asparaginyl endopeptidase purified from jack bean seeds. J. Protein Chem. 9:294 295. menez, C. R., Huang, L., Qiu, Y. and Burlingame, A. L. (1999). Enzymatic approaches for obtaining amino acid sequence: On-target ladder sequencing. In Current Protocols in Protein Science, 16.7.1-16.7.3, New York, John Wiley and Sons. nes, B. N. (1986). Amino acid analysis by o-phthaldialdehyde precolumn derivatization and reverse phase HPLC. In Methods of Protein Microcharacterization: A Practical Handbook, J. E. Shively, ed., Clifton, N.J., Humana Press, pp. 121-151. genson, J. W., and Lukacs, K. D. (1981). Zone electrophoresis in opentubular glass capillaries. Anal. Chem. 53:1298-1302. iser, E., Colescott, R. L., Bossinger, C. D., and Cook, P. I. (1970). Color test for the detection of free terminal groups in solid-phase synthesis of peptides. Anal. Biochem. 34:595-598. iser, E., Bossinger, C. D., Colescott, R. L., and Olsen, D. B. (1980). Color test for terminal prolyl residues in the solid phase synthesis of peptides. Anal. Chim. Acta 118:149-151. nt, S. B. H. (1988). Chemical synthesis of peptides and proteins. Ann. Rev. Biochem. 57:957-989. nt, S. B. H., Riemen, M., LeDoux, M., and Merrifield, R. B. (1982). A study of the Edman degradation in the assessment of the purity of synthetic peptides. In Methods in Protein Sequence Analysis, M. Elzinga, ed., Clifton, N.J., Humana Press, pp. 205-213. echt, R., and Chang, J.-Y. (1986). Liquid chromatographic determination of amino acids after gas-phase hydrolysis and derivatization with (dimethylamino) azobenzenesulfonylchloride. Anal. Chem. 58:2375-2379. chersperger, M. L., Blacher, R., Kelly, P., Pierce, L., and Hawke, D. H. (1989). Sequencing of peptides on solid phase supports. Am. Biotech. Lab. 7(3), March: 26-37. ursen, R. A. (1971). Solid phase Edman degradation, an automated peptide sequencer. Eur. J. Biochem. 20:89-102. vy, M., Fishman, L., and Schenkein, I. (1970). Mouse submaxillary gland proteases. Methods Enzymol. 19:672-681. u, T. Y., and Chang, Y. H. (1971). Hydrolysis of proteins with p-tolunesulfonic acid. J. Biol. Chem. 246:2842-2848. acFarlane, R. D., Skowronski, R. P., and Torgenson, D. F. (1974). New approach to the mass spectroscopy of non-valatile compounds. Biochem. Biophys. Res. Comm. 60:616-621. ahoney, W. C., and Hermodson, M. A. (1980). Separation of large denatured peptides by reverse phase high performance liquid chromatography. J. Biol. Chem. 255:11199-11203.
289
ier, G. Drapeau, G. R., Doenges, K. H., and Ponstingl, H. (1986). Generation of starting points for microsequencing with a protease specific for the amino side of aspartyl residues. In Methods of Protein Sequence Analysis, K. A. Walsh, ed., Clifton, N.J., Humana Press. nt, C. T., and Hodges, R. S. (1990). HPLC of peptides. In HPLC of Biological Macromolecules, Chromatographic Science Series, Vol. 51, K. M. Gooding and F. E. Regnier, eds., New York, Marcel Dekker, pp. 301-332. nt, C. T., Kondejewski, L. H., Cachia, P. J., Monera, O. D., and Hodges, R. S. (1997). Analysis of synthetic peptides by high performance liquid chromatography. Methods Enzymol. 289:426^469. rquez, F. G., Quesada, A. R., Sanchez-Jimines, F., and Nunez de Castro, I. (1986). Determination of 27 dansyl amino acid derivatives in biological fluids by reverse-phase high-performance liquid chromatography. J. Chromatogr. 380:275-283. saki, T., Tanabe, M., Nakamura, K., and Soejima, M. (1981a). Studies on a new proteolytic enzyme from Achromobacter lyticus M497-1 I. Purification and some enzymatic properties. Biochem. Biophys. Acta 660:44-50. saki, T., Fujihashi, T., Nakamura, K., and Soejima, M. (1981b). Studies on a new proteolytic enzyme from Achromobacter lyticus M497-1 II. Specificity and inhibition studies of Achromobacter protease I. Biochem. Biophys. Acta 660:51-55. tsubara, H., and Sasaki, R. M. (1969). High recovery of tryptophan from acid hydrolysates of proteins. Biochem. Biophys. Res. Comm. 35:175181. Closkey, J. A., ed. (1990). Mass spectrometry. Methods Enzymol. 193:1960. oore, S., and Stein, W. H. (1948). Photometric ninhydrin method for use in the chromatography of amino acids. J. Biol Chem. 176:367-388. ore, S., and Stein, W. H. (1963). Chromatographic determination of amino acids by the use of automatic recording equipment. Methods Enzymol. 6:819-831. ore, S., Spackman, D. H., and Stein, W. F. (1958). Chromatography of amino acids on sulfonated polystyrene resins. Anal. Chem. 30:1185-1190. ay, B., Lu, H. S., and Gray, R. W. (1983). High-performance liquid Chromatographic separation of Dns-amino acid derivatives and application to protein and peptide structural studies. J. Chromatogr. 270:253266. ols, J. (1990). Amino acid analysis. Methods Enzymol. 182:587-601. terson, D. H., Tarr, G. E., Regnier, F. E., and Martin, S. A. (1995). CTerminal ladder sequencing via matrix-assisted laser desorption ionization mass spectrometry coupled with carboxypeptidase Y time-dependent and concentration-dependent digestions. Anal. Chem. 67:3971-3978. nke, B., Ferenczi, R., and Kovacs, K. (1974). A new acid hydrolysis method for determining tryptophan in peptides and proteins. Anal. Biochem. 60:45-50. gnier, F. E. (1987). The role of protein structure in Chromatographic behavior. Science 238:319-323.
Synthetic Peptides
im, D. F., and Speicher, D. W. (1997). jV-Terminal sequence analysis of proteins and peptides. In Current Protocols in Protein Science, 11.10.111.10.38, New York, John Wiley and Sons. th, M., and Hampai, A. (1973). Column chromatography of amino acids with fluoresence detection. J. Chromatogr. 83:353-356. binstein, M., (1979). Preparative high performance liquid partition chromatography of proteins. Anal. Biochem. 98:1-7. nchez, A., and Smith, A. J. (1997). Capillary electrophoresis. Methods Enzymol. 289:469-478. nger, F., and Tuppy, H. (1951). The amino acid sequence in the phenylalanyl chain of insulin. Biochem. J. 49:463-481. rin, V. K., Kent, S. B. H., Tarn, J. P., and Merrifield, R. B. (1981). Quantitative monitoring of solid phase peptide synthesis by the ninhydrin reaction. Anal. Biochem. 117:147-157. henkein, J., Levy, M., Franklin, E. C., and Frangione, B. (1977). Proteolytic enzymes from the mouse submaxillary gland. Arch. Biochem. Biophys. 182:64-70. ively, J. E. (1986). Reverse Phase HPLC isolation and microsequence analysis. In Methods of Protein Microcharacterization: A Practical Handbook, J. E. Shively, ed., Clifton, N.J., Humana Press, pp. 41-87. mpson, R. J., Neuberger, M. R., and Liu, T.-Y. (1976). Complete amino acid analysis of proteins from a single hydrolysate. J. Biol. Chem. 251:19361940. mith, B. J. (ed.) (1997). Protein Sequencing Protocols, Clifton, N.J., Humana Press. yder, L. R., and Kirkland, J. J. (1979). Introduction to Modern Liquid Chromatography, New York, John Wiley and Sons. ackman, D. H., Stein W. H., and Moore, S. (1958). Automatic recording apparatus for use in chromatography of amino acids. Anal. Chem. 30:1190-1206. ewart, J. M., and Young, J. D. (1984). Solid Phase Peptide Synthesis, Rockford, 111., Pierce Chemical Co. llivan, S., and Wong, T. W. (1991). A manual sequencing method for identification of phosphorylate amino acids in phosphopeptides. Anal. Biochem. 197:65-68. puhi, Y., Schmidt, D. E., Linder, W., and Karger, B. L. (1982). Analysis of dansyl amino acids by reverse-phase high performance liquid chromatography. Anal. Biochem. 127:49-54. rr, G. E. (1986). Manual Edman sequencing system. In Methods of Protein Microcharacterization: A Practical Handbook, J. E. Shively, ed., Clifton, N.J., Humana Press, pp. 155-194. rr, G. E., Beecher, J. F., Bell, M., and McKean, D. J. (1978). Polyquarternary amines prevent peptide loss from sequenators. Anal. Biochem. 84:622-627. rr, G. E., Paxton, R. J., Pan, Y.-C. E., Ericsson, L. H., and Crabb, J. W. (1991). Amino acid analysis 1990: The third collaborative study from the Association of Biomolecular Resource Facilities (ABRF). In Techniques in Protein Chemistry, Vol. II, J. J. Villafranca, ed., San Diego, Calif., Academic Press, pp. 139-150.
291
Tregear, G. W., van Rietschoten, J., Sauer, R., Niall, H. D., Keutmann, H. T., and Potts, J. T., Jr. (1977). Synthesis, purification, and chemical characterization of the amino-terminal 1-34 fragment of bovine parathyroid hormone synthesized with the solid phase procedure. Biochemistry 16:2817-2823. Tsunasawa, S., and Narita, K. (1982). Micro-identification of amino-terminal acetyl amino acids in protein. J. Biochem. 92:607-613. Van Regenmortel, M. H. V., Briand, J. P., Muller, S., and Plaue, S. (1988). In Synthetic Polypeptides as Antigens, Amsterdam, Elsevier, pp. 81-82. Wang, Y. H., Fiol, C. J., DePaoli-Roach, A. A., Bell, A. W., Hermodson, M. A., and Roach, P. J. 1988. Identification of phosphorylation sites in peptides using a gas-phase sequencer. Anal. Biochem. 174:537^17. Wilkinson, J. M. (1986). Fragmentation of polypeptides by enzymic methods. In Practical Protein Chemistry: A Handbook, New York, John Wiley and Sons, pp. 121-148. Young, P. M., and Merion, M. (1990). Capillary electrophoresis analysis of species variations in the tryptic maps of cytochrome C. In Current Research in Protein Chemistry: Techniques, Structure, and Function, J. J. Villafranca, ed., San Diego, Calif., Academic Press, pp. 217-232.
Applications of Synthetic Peptides

Victor J. Hruby Terry O. Matsunaga
e tremendous advances in the development of methods for the sign and synthesis of peptides. pseudo-peptides and related comunds, as well as the corresponding advances in our understanding peptide and protein structure, conformation, topography, and namics provides unique opportunities to apply designed synthetic ptides for an enormous variety of problems in chemistry, biology, and dicine. In addition, if these advances can be coupled to the advances molecular biology and the human genome project, on the one hand, d asymmetric synthesis and catalysis, on the other, it should be posle to provide hitherto unavailable, indeed unthinkable, approaches diverse areas of drug design, behavioral neuroscience, molecular munology, chemotherapy, and a wide variety of other uses. Already it is clear that peptide therapy has enormous potential in ch diverse areas as growth control, blood pressure management, urotransmission, hormone action, satiety, addiction, pain, digestion, production, and so forth. Nature has "discovered" that it can ntrol nearly all biological processes by various kinds of molecular ognition, and that peptides and proteins are uniquely suited for this ntrol because of their enormous potential for diversity and their ique physico-chemical properties. This finding may, perhaps, be st readily understood if one recognizes that, considering only the
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normal eukaryotic amino acids, the number of unique chemical ties for a pentapeptide is 3,200,000 (205), for a hexapeptide it is 00,000 (206), and so on. Considered from this perspective, perhaps not unexpected that Nature has "discovered" that peptides and eins can do it all, from providing structure and motion, to lysis, to information transduction, to growth and maturation, and on. The ability of the immune system in higher animals, including mans, to recognize literally millions of foreign materials made by ure as well as humans, and to get rid of them as part of its survival tegy, is just one example that illustrates the potential of peptideed drugs, therapeutics, and modulators of biological function. Despite the enormous potential of peptides and small proteins for e areas, surprisingly little advantage has been taken of the ential of these molecules as drugs and tools for use in basic and ical research. The reasons given for this are many and include such ors as: (1) the lack of knowledge of most chemists regarding the ctures, conformations, and synthesis of peptides and proteins; (2) putative instability of these compounds in biological systems; (3) putative lack of bioavailability of these compounds (though they found and transported everywhere in the body); (4) their high ency and very small concentrations in most biological systems; and he enormous diversity possible. In fact, many of these problems can overcome or are irrelevant to a particular biological or medical blem. In the case of diversity, this can, in fact, serve as an advan, since it is possible to construct very large libraries of peptides ( -1010 and more) by both biological (e.g., Cwirla et al., 1990; Devlin ., 1990; Scott and Smith, 1990) and chemical (e.g., Furkaetal., 1991; ughten et al., 1991; Lam et al., 1991; Hruby, 1996) methods and to en them for a very wide variety of functions. These new approaches y have been expected to accelerate the use of peptides and peptideved compounds for medical and biological purposes, but full antage of the possibilities is still a long way from realization. n this case, we will provide a brief summary of some of the medical biological applications for synthetic peptides. Much has already n accomplished, but more importantly, current studies are provida framework for much more comprehensive success in the future. eed, it can be anticipated that peptide, protein and peptide-related mpounds will be among the most numerous drugs of the future. If chapter helps you in this process in some small way, we will have omplished our purpose. (For a few recent reviews or overviews of area, see for example: Emmett, 1990; Hruby et al., 1990a,b; Hruby Balse, 2000; Dutta, 1991; Ward, 1991; Rizo and Gierasch, 1992; odman and Ro, 1994; Whittle and Blundell, 1994; Sawyer, 1995; neider and Kelly, 1995; Bonn and Klebe, 1996; Al-Obeidi et al., 8; Strand, 1990.)
Synthetic Peptides
tructure/Function Studies
ne of the principal goals of peptide and protein research is the rational sign of peptide ligands whose chemical, physical, and biological propties can be predicted a priori. It is not possible at this time to propose a neral set of rules that would apply to structure/function studies for all oactive peptides acting at all receptors, enzymes, antibodies, memanes, nucleic acids and other acceptors. However, the goal here is to scuss some general approaches in the design of synthetic peptides that n be used to better understand ligand-receptor/acceptor interactions rough structure/function studies. It now seems clear that there are at least three separate steps tructural states) of the ligand-receptor/acceptor complex which cur during the ligand-receptor/acceptor interaction. First, the gand must be "recognized" by the receptor for binding to take ace. In a second step, a change in conformation of the hormoneceptor/acceptor complex must occur that can result in transduction n the case of an enzyme, a fruitful transition state must be reached) the biological response through a second messenger. Finally, the ormone (products, etc.) must be released from the receptor so that e process can be repeated. The differentiation between binding and ansduction (or inhibitory) states relating to hormone interactions ave been well established as being the result of the hormone exhibitg different conformational and topographical features (Rudinger et ., 1972; Meraldi et al, 1975, 1977; Schwyzer, 1977; Walter, 1977; ruby, 1981a,b). Ideally, one would like to be able to "know" what a receptor/acceptor "looking for" for binding, transduction (transition state), and release urnover) of its complementary binding ligand. In many cases, remarkble progress has been made in these areas. But even with the ability to olate receptors, there is, currently, no method to design ligands with ecific properties a priori. The use of structure/function studies is the assical method for the design of synthetic peptides with specific actives. The ability to perform structure/function studies relies heavily pon the availability of a well-defined ligand-receptor system with senive binding assays and bioassays that can answer questions related to lectivity, binding, and transduction for synthetic peptide analogues.
ystematic Modifications
ructure/function studies usually begin with simple modifications of e native ligand for a receptor or receptor class. The most common of ese are the alanine scan and the glycine scan in which each residue is stematically replaced by an Ala or Gly. The hormone-receptor stem is complex so that, in general, only by individual modifications
295
n one insure that a single substitution is providing the change. This n then be followed by more extensive changes at observed key idues. Thus, a large number of single modifications built upon e another can result in quite sophisticated analogues. In some ses, the modifications in the analogue can be so extensive that it is ficult for an outside observer to see the relation of the analogue ucture to that of the endogenous hormone. In a somewhat related approach, it has been found that many rmones have a minimum sequence that may have full biological tency. Since most peptide analogues are synthesized by either id-phase or solution-phase synthesis, if a minimum active sequence n be found, then it can greatly simplify the synthesis of all subsequent alogues prepared. A great deal of information can often be gleaned m the kinds of studies mentioned, and, in some cases, the separation peptide sequences required for binding and transduction can be deterned. This, in turn, can lead to the design of agonist and antagonist ands. Often, assays that relate modes of action through primary and condary messengers can be examined. In this way, it sometimes is ssible to separate an endogenous ligand's affinity for subtypes of ltiple receptors. A few examples mostly from our laboratory of me of these approaches will now be discussed.
letion Peptides
letion peptides of melanin concentrating hormone (MCH), whose ive structure is Asp-Thr-Met-Arg-c[Cys-Met-Val-Gly-Arg-Valr-Arg-Pro-Cys]-Trp-Glu-Val, showed that only residues 5-15 were cessary for full biological activity (table 5-1) (Matsunaga, et al., 89). Removal of the tryptophan in position 15 lowers the biological tency by more than 10-fold, and this potency continues to decrease th fragments sequentially deleting residues 1-5. These studies sugsted the importance of Trp15 for interaction with the MCH receptor, th the 5-14 fragment having only 1/300 the potency of the native rmone. This is a common observation, namely that for many pepes, neurotransmitters, hormones, cytokines and other bioactive pepes, N- and/or C-terminal truncation leads to shortened analogues, th full bioactivity but often reduced potency. Once the "minimum ive sequence" is found, structural or conformational modifications n restore full potency.
ereochemical Substitutions
ce a minimum bioactive sequence has been determined, it often is nvenient to search for Stereochemical requirements of the receptor/ ceptor with the systematic substitution of D- for L-amino acids. The
Synthetic Peptides
Table 5-1 Relative potencies of MCH fragment analogues as determined by the in vitro fish (Synbranchus marmoratus) skin bioassay
Pep tide
MCH MCH(2-17) MCH(3-17) MCH(4-17) MCH(5-17) MCH(1-16) MCH(2-16) MCH(3-16) MCH(4-16) MCH(5-16) MCH(1-15) MCH(2-15) MCH(3-15) MCH(4-15) MCH(5-15) MCH(1-14) MCH(2-14) MCH(3-14) MCH(4-14) MCH(5-14)
Potency relative to MCH

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.1 0.07 0.05 0.023 0.014
orporation of D-amino acids also can provide clues about the portance of certain secondary structures (e.g., /3-turns, or-helix), also may yield further information about specific residues essary for peptide-receptor interaction as well as contributing bility to analogues against enzymatic breakdown (Hruby, 1982). In one example from our laboratory, the incorporation of D-Phe at ition 7 of [Nle4]a-MSH (melanotropin stimulating hormone) ulted in a melanotropin superagonist (Sawyer et al., 1980). The ive linear peptide has the active sequence Ac-Ser-Tyr-Ser-Metu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2. The substitution was de based on the evidence that heat-alkaline treated a-MSH and e4]a-MSH led to prolonged activity with an unusual amount of emization at the Phe7 position. The [Nle4, D-Phe7]a-MSH analogue s shown to be 60 times more potent than the native hormone with remely prolonged activity and greatly reduced biodegradation. her early examples of the use of D-amino acids include LHRH einizing hormone releasing hormone) agonists and antagonists y et al., 1982; Freidinger et al., 1985), substance P antagonists ercy et al., 1981), oxytocin antagonists (Melin et al., 1983; Lebl et 1985), enkephalin (Morgan et al., 1977) and somatostatin analogues
297
reidinger and Veber, 1984; Torchiana et al., 1978), and many other amples have followed. Indeed a o-amino acid scan is now a standard proach to investigating structural, stereochemical and conformanal requirements for bioactivity of native bioactive peptides (e.g., rieco et al., 2000).
osteric and Other Substitutions
eudo-isosteric replacements are also quite useful for determining ptide-receptor interactions. Amino acids can be replaced with her amino acids of similar size but different electronic or stereoeleconic properties. Such substitutions would include the use of norucine (Nle) for the oxidizable methionine, where a methylene unit used as a replacement for sulfur. This substitution was carried out a-MSH. The resulting [Nle4]a-MSH peptide was shown to be 1.5 2.2 times more potent than the native hormone having methionine the fourth position. This slight increase in potency may, in part, be e result of lack of a methionine oxidation during the bioassays ruby et al., 1980). Other such substitutions include changes in the omatic amino acids tyrosine, tryptophan, phenylalanine, and stidine with various modifications of the aromatic moieties. witching these amino acids might allow study of spatial requirements d/or hydrogen-bonding interactions, nn, or iia interactions and forth. For example, in oxytocin, the importance of the phenolic OH 9 9 the Tyr position was illustrated by the synthesis of [Phe ]oxytocin 2 odanszky and du Vigneaud, 1959) and [Trp ]oxytocin (Kaurov et , 1972), which demonstrate weak agonist activity, while [pePhe2]oxytocin (Rudinger et al., 1972) is an antagonist. Other posbilities include the acidic residues, aspartic acid and glutamic acid, hich might be exchanged for their amide counterparts asparagine d glutamine and vice versa. These types of substitutions might ve a pronounced impact on the analogue's chemical nature because e net charge on the ligand is changed. Similarly, the basic residues sine and arginine also are often interconverted to examine how well e amine functional group fits the receptor relative to the guanidine nctional group. These types of replacements have been very effective the design of analogues of enkephalins (Kruszynski et al., 1980; osberg et al., 1983a), and somatostatins (Freidinger and Veber, 84), /z-opioid selective peptides (Kazmierski and Hruby, 1988), d many others.
onstraint
addition to these more classical approaches, the use of conformanal and topographical constraint has been a very powerful
Synthetic Peptides
roach to peptide ligand design (e.g., for reviews see: Hruby et al., 2; Kessler, 1982; Toniolo, 1990; Hruby, 1990a; Farlie et al., 1995), uding increasing potency, receptor selectivity, providing antagos, and greatly increasing stability against proteolyte biodegradan. We now provide a few examples of these approaches. The incorporation of TV-methyl amino acids has a tendency to conin the x1 angles of amino acids and provide cis-trans isomerism und the amide bond of which they are part. One excellent example n the case of certain [W-MeNle3]CCK-8 (CCK = cholecystokinin) logues, which showed a significant increase in the specificity for the K-B (brain) receptor over the CCK-A (peripheral) receptor. It has n suggested from NMR studies that the enhanced selectivity is the ult of the brain receptors preference for the cis amide bond which is seen in related [Nle3]CCK-8 analogues (Hruby et al., 1990b). other example includes the substitution of A^-methyl-D-phenylnine in the one position of Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-ThrH2 (CTOP). This analogue was shown to be 200 times less potent n [o-Phe^CTOP at the /z-opioid receptor, simply because of the nge in the x1 angle from -60 to 180 (Kazmierski et al., 1988). Nthylalanine was used in place of proline in some bradykinin logues, decreasing their potency (Filatova et al., 1986). A review he literature reveals that many similar examples could be cited. Amino acids that are a-alkylated have also been widely used to mine a receptor's conformational requirements. Besides adding ential additional steric bulk to the peptide, these amino acids, d to induce ft-turns and other secondary structures (De Grado, 88; Toniolo, 1990). One amino acid that has proved to be extremely ful for this purpose is Aib (aminoisobutyric acid) in the place of cine. To study the steric and stereoelectronic nature of a receptor, bulky nproteinogenic amino acids are often incorporated into peptide logues. One of the most widely used replacements is the substitun of penicillamine (Pen, 3-mercapto-o-valine or ft, /3-dimethylteine) for cysteine. This substitution has led to the differentiation ween agonist and antagonist analogues in oxytocin (table 5-2). A es of analogues that have even larger side chains at the j6-carbon o were examined. In these analogues antagonists were obtained only en both hydrogens on the /3-carbon were replaced with alkyl ups. Replacement of only one methyl group was not sufficient antagonism at the uterine receptor. There was little difference in inhibitory properties observed for ft, ^-dimethyl or ethyl analogues d bicyclic [Cppôxytocin. It has been suggested that this antagonis caused, in part, by the restriction of disulfide interchange ween R and S chiralities and to disposition of the x1 and x2 angles the Tyr2 side chain group, and further evidence for this has been
299
le 5-2 Activities of some 1-substituted oxytocin analogues

Activity U/mg
546 803 pA2 = 6.94 91 pA2 = 7.24 pA2 = 7.61 pA2 = 6.86
nalogue3
Reference Chan and Kelly, 1969 Ferrieretal., 1965 Vavreketal., 1972 Sdiulz and du Vigneaud, 1966 Vavreketal., 1972 Lowbridge et al, 1979 Vavreketal., 1972
xytocin Mpa'loxytocin mp]oxytocin D,L-p-MeMpa']oxytocin Dep'joxytocin Cpp'joxytocin Pen'loxytocin
a: p-mercaptopropanoic acid; Dmp: p,p-dimethyl-|J-mercaptopropanoic acid; Dep: P,p-diethylercaptopropanoic acid; Cpp: p-cyclopentarnethyfene-p-mercaptopropanoic acid; Pen: penicillamine. nition: pA2 is defined as the negative logarithm of the molar concentration of a competitive antagonist reduces the response of the agonists by 50 percent of what it would be in the absence of the antagonist
hematically:
E= observed response Em,, = maximum response in absence of antagonist D2 = half maximum concentration i = antagonist concentration a = concentration of agonist
ained using x-constrained tyrosine analogues (Liao et al., 1998). e substitution of cysteine by other larger ft, /?-dialkyl Cys derivatives o has been examined in the development of potent VI and V2 opressin antagonists (Dyckes et al., 1974; Bankowski et al.,1978), selective cyclic enkephalin analogues (Mosberg et al., 1983a,b) and elective somatostatin analogues (Pelton et al., 1985; Kazmierski
Synthetic Peptides
Hruby, 1988). The use of other sterically demanding amino acid chains such as f-butyl, adamantyl, naphthyl, and so on, are almost ess, and many such amino acids have been developed and incorted into peptides.
g Size
cyclic peptides, the optimization of ring size is of great use. The cts of ring size and the necessity of the sulfur atoms in disulfideaining peptides has been the subject of investigation since the est days of bioactive peptide structure-activity studies, and have bably been most completely studied in oxytocin and vasopressin ogues. Replacement of either or both sulfur atoms in oxytocin lted in analogues with agonist activity (for reviews see lost, 7; Hruby and Smith, 1987). Attempts to modify the 20-membered size by addition or deletion of single residues or single side-chain ons or sulfurs led to a 100-fold decrease in potency in all cases. ilar experiments with vasopressin generally resulted in less potent ogues, and the relative effect was largely dependent on the mone receptor examined. One quite successful exploration of size was in the development of somatostatin agonists. Removal ix residues from the ring of somatostatin Ala-Gly-c[Cys-Lys-Asn-Phe-Trp-Lys-Thr-Phe-Thr-Cys] gave rise to the 20-membered ring ogue D-Phe-c[Cys-D-Trp-Lys-Thr-Cys]-Thr-ol, which is superve in inhibiting growth hormone release (Bauer et al., 1983). The was subsequently reduced to the 18-membered cyclic hexapeptide Lpro-Phe-o-Trp-Lys-Thr-Phe I
ch is equipotent to somatostatin in the inhibition of growth mone, insulin, and glucagon release (Veber et al., 1981). In tion, the use of cyclic hexapeptides and heptapeptides with ropriate constraints has been a critical aspect of conformationogical activity studies of many bioactive peptides for many s and is an area that will continue to develop rapidly well into future (for reviews see, for example, Kessler, 1982; Rizo and rasch, 1992).
lic Constraints
formational features in peptides that are important for receptor raction often can be stabilized by introducing cyclic constraints linear peptide ligands (e.g., Hruby, 1982; Kessler, 1982; Hruby ., 1990a; Marshall, 1993). One early example was the development a-MSH superagonists. A proposed /J-turn around Phe7 was
301
bilized by the replacement of Met4 and Gly10 with cysteine residues cyclization to afford the cyclic peptide c[Cys4, Cys10]a-MSH, an logue with much greater potency than the native hormone which o exhibits prolonged activity (Sawyer et al., 1982). In a similar nner, substitution of the Met5 and Gly2 positions of enkephalins h D-Pen, followed by cyclization, led to the highly potent and 8oid receptor selective cyclic peptide, c[o-Pen2, D-Pen5]enkephalin osberg et al., 1983b). Side-chain cyclizations of polypeptides not involving disulfides erally are not found in nature in higher organisms, but they can important tools in structure/function studies with synthetic tides. There are many possibilities for side-chain-to-side-chain lizations, including, but not limited to, amides (lactams), esters tones), ethers, ketones, and so on. Many possibilities have already n examined (Hruby and Boteju, 1997). Perhaps the most common lization, due to its convenience and stability, is the amide bond med between free carboxylate and free amine groups found on no acid residue side chains. This type of cyclization has been d quite successfully in a series of oxytocin analogues. The weak
nocyclic agonist [Mpa1, Glu5, Cys6, Lys8]oxytocin was converted
the highly potent bicyclic antagonist [Mpa1, Glu5, Cys6, Lys8]-
tocin by amide bond formation between Glu5 and Lys8 (Hill, et al., 0). Not only did this cyclization cause antagonist activity, but it o increased the binding to the receptor over 1000-fold. Cyclizations a similar manner were also studied in CCK-8. The monocyclic am analogue Boc-c[Asp-Tyr(SO3H)-Nle-D-Lys]-Trp-Nle-Aspe-NH2 exhibited a significant decrease in potency at the CCK-A ripheral) receptor (Charpentier et al., 1987). A monocyclic bis-lacanalogue of CCK-8 was prepared by replacement of Met28'31 dues with lysine and cyclizing the Ns amino groups with succinic d. This procedure resulted in the highly CCK-B (brain) selective logue (Rodriguez et al., 1990). Based on computer modeling, MSH analogues were prepared incorporating amide cyclizations -Obeidi et al., 1989a,b). This led to the synthesis of the highly ent Ac-c[Nle4, Asp5, D-Phe7, Lys10]a-MSH-(4-10)-NH2 analogue. ide cyclizations have also been studied in the glucagon (Lin et al., 2; Trivedi et al., 2000), enkephalin (Schiller et al., 1985), GnRH tems (Struthers et al., 1984), and many others. Other cyclizations have been reported, including biphenyl-type lizations used in an approach to the synthesis of the antitumor nt bovardin (Bates et al., 1984) and diazobridges in enkephalin logues (Siemion et al., 1980).
Synthetic Peptides
idomimetics
e recently, an increasing emphasis of structure/function studies been directed toward the synthesis and use of ligands containing do-peptides and peptidomimetics (e.g. Hruby et al., 1990a). le it is now possible to stabilize certain conformational features eptides with a high degree of predictive confidence, the use of peptide peptide mimetics as rigid templates for these features of ides and proteins in 3-D space and with appropriate dynamic erties is still problematic. There is also interest in peptides aining novel amino acids (e.g. ^-methyl-2/,6/-dimethylphenylalawhich can allow peptides to maintain their backbone conformabut predictably bias side-chain conformations of the ligand uby et al., 1997). These latter considerations are important because ovo design of nonpeptide peptide mimetics requires careful design iderations, not only of a proper conformational template but also appropriate arrangement of key pharmacophore groups of the ctive peptide on the nonpeptide scaffold. This is particularly ortant in the design of peptide hormones and neurotransmitters, kines, protease inhibitors and many other ligands that interact receptor/acceptor proteins and utilize the side-chain groups as primary sites of molecular recognition or for discrimination ween different possible biological receptor/acceptors (selectivity). le a comprehensive discussion of the design of nonpeptide peptide etics is outside the scope of this chapter, it is critical to know what biologically active conformation is, what the structureactivity ionships are, and what the critical dynamics properties, if any, he peptide are if one is to design de novo a true peptide mimetic. his regard, the role of peptide chemistry, its many manifestations, omes more and more critical to developing a robust approach to peptide peptide mimetic design (see, for example, Hruby et al., 0a; Hirschmann, 1991; Wiley and Rich, 1993; Hruby, 1997; yer, 1997; Hruby and Raise, 2000). Here we will only discuss design since it often involves use of novel amino acids and/or do-peptides. An important aspect of peptide chemistry, particularly with respect harmaceutical development, involves the study of conformational topographical aspects of turns. Reverse-turn regions are ortant aspects of determining whether a molecule will, or will maintain the desired binding potency and transduction or intory potential. Several examples of peptides associated with turn ons are (1) the tripeptide RGD sequence found to bind to various oprotein receptors (Ripka et al., 1993a) inhibiting platelet egation, (2) Gramicidin S, a cyclic, naturally occurring peptide biotic whose biological activity has been shown to be related to
Applications of Synthetic Peptides 303
conformation (Ripka et al, 1993b), a variety of peptides used to hibit HIV proteases (aspartyl proteases) responsible for activity ainst human immunodeficiency virus (Abbenante et al., 1995) and any more (see above). The development of peptide turn-mimetics are multi-fold. Rigid rns designed by alterations in the backbone have been developed aid in the stability of the molecule to enzymatic degradation. ternatively, stabilizations of conformation and/or topography can d in imparting selectivity of the compound with respect to a family of ceptors. For example, the integrin receptors are found on both atelets (GPIIbllla) and on tumor cells (0^3). A knowledge and sign of specific conformational turns may be imperative in selecting r binding to the designated receptor for therapeutic benefit and minating binding to other receptors associated with undesirable de effects. A variety of approaches for synthesizing turn mimetics have been veloped. While some investigators focus upon a specific turn conrmation comprised of specific amino acid sequences, others have voted their attention to more global synthetic procedures to develop verse libraries of turn mimetics. Methodologies directed to the synths of turns is diverse as well. For example, an electrochemical methology has been reported by Moeller, Marshall and coworkers. lomczynska et al., 1996). SÂr chemistry developed in the laborary of Burgess has provided insight into turn conformations as well eng et al., 1998). Alternatively, other investigators have used backne modifications to help stabilize /3-turn conformations. For exame, Kessler and coworkers used this approach to help rigidify the OD conformations (Geyer et al., 1994). This review will elaborate some of these studies. Interestingly, the development of diverse libraries of turn mimetics ing various amino acids in the i, i + 1, / + 2, and i + 3 positions, can viewed as being somewhat complementary to the goals and jectives achieved by using the structure/function approach eluded earlier in the chapter. Although work has progressed to develop verse libraries, indeed, the turn libraries are only diverse in one pect, for the objective of these libraries is, in some instances, to abilize the backbone or conformation of the turn while allowing e topography or side chain to vary. Thus, in essence, the objective to keep phi, psi, and chi angles of the backbone relatively constant d be able to vary only the topography. Hence, one has reduced the ue of turn optimization by a single variable which, in principle, ould aid in understanding the necessary physical properties required r optimal turn mimetic development. Prior to discussing the synthetic aspects of turn conformations, it ould be stated that the evaluation of these mimetics is just as critical
Synthetic Peptides
entifying the correct conformation as the synthesis itself. For this on, strict determination of conformational properties by spectroic and molecular modeling techniques can help to confirm the ormational structure of the analogue. At the cornerstone of e techniques is 2-D NMR spectroscopy. In particular, NOE clear Overhauser Enhancement) spectroscopy in conjunction with elated spectroscopy and heteronuclear spectroscopy are essential in rmining the phi and psi angles of the turn mimetic. In addition, cular mechanics or molecular dynamics routines in conjunction NMR constraints can provide insight into structure. Circular roism and/or crystal structures (if the compound is crystalle) can provide more definitive information of the conformation.
bone Alteration idomimetics can be generated by synthetic backbone alterations. synthesis of pseudo-peptides, whose backbone structure deviates the conventional amide backbone, has been studied in order to mine conformational effects. Work by Geyer et al., performed onformational analysis on a cyclic RGD peptide containing a H2-NH] bond. The pseudopeptide cyclo(-Arg1-Gly2-Asp3-D^[CH2-NH]Val5-) was synthesized on solid phase by first rporating solution-phase reduction of Boc-D-Phe-Val-OH to the esponding Boc-D-Phei/r [CH2-NH]Val-OH with Lawesson's ent. The synthesis appeared particularly advantageous because tion-phase syntheses of the dipeptides are virtually free of racemion, and can be crystallized. The pseudo-dipeptide synthesis was followed by solid-phase coupling to H-Arg(NO2)-GlyObzl)-2-chlorotrityl resin followed by cleavage, cyclization, and otection. The compound was compared to its unmodified parent, o (-Arg1-Gly2-Asp3-o-Phe4Val5-) by solution phase NMR and nsive molecular dynamics simulations. Interestingly, the onated, amine on the reduced peptide bond exhibited strong ogen-bond donor properties, which helped to further rigidify peptide backbone. This yielded a significantly reduced number of ormers relative to its parent counterpart, exhibiting a y-conformawith the o-Phe in the i+ 1 position of the y-turn and a /3H' ormation surrounding the Arg-Gly region. This becomes interestas the pseudo-dipeptide portion of the analogue induces a shift of 3-II turn, compared to the parent peptide, of two positions in a terclockwise direction. This work helped to demonstrate how ction of the backbone can impart both: (1) rigidity to the back, and (2) shift the turn region. This study was consistent with ious work by Ma and Spatola (1993), who noted the ftll' turn on of another pseudopentapeptide.
305
n-Resin Formation of Cyclic fi-Turn Peptidomimetics ith the development of combinatorial and high throughput parallel ethods, the desire to incorporate conformational constraints and, particular, conformational bias, has emerged. Efficient cyclizations n solid-phase supports with predictable conformations, particularly turn conformations, is a priority (Kahn, 1993). Recently, work by the Burgess group (Feng et al., 1998), has cused on ^-turn conformations using SÂr cyclizations on solid pports for the synthesis of /J-turn libraries. Briefly, a Rink amide sin was used as the support followed by synthesis of a prototypical peptide followed by coupling to a fluoro-substituted benzoic acid alogue. Cyclization was initiated by side-chain SN Ar reaction of the st residue displacing the fluoro moiety of the benzoyl compound, elding a macrocyclic analogue. NMR analysis following cleavage as significant for hydrogen bonding of the CO NH being the ring sion point for 10-membered rings formed by the /3-turn analogue. Work in the Ellman laboratory has produced some interesting braries of /3-turn mimetics as well (Virgilio et al., 1994; 1997a,b). sing a Rink amide resin, the / + 1 and / + 2 positions were functionzed with an a-bromo acid and a-Fmoc-protected a-amino acid spectively, with an aminoalkylthiol acting as the constraining backne component. The covalently bound thiol moiety forming the mino alkyl thiol on the analogous / + 3 amine, generated the 9- or 0-membered ring. Thus, different combinations of substitutions at e stereocenters on the / + 1 and / + 2 side chain could be incorpoted while still maintaining the putative /J-turn conformation. A turn nformation was concluded by virtue of NMR coupling constants d searching the cyclic compounds for lowest energy conformers. oupling constants between the central amide NH and the Ca-H of e i + 2 residue in selected compounds was 9.5 Hz. Incorporating this lue to help constrain the backbone </>-dihedral angle, lowest energy nformers were sought using a conformational search analysis. esults indicated that a Type Il'-^-turn best fit the conformer search alysis lending support to the conclusion that ^-turns were indeed ade in these cyclic peptide analogue libraries. A series of 1152 comounds were generated employing the Chiron Mimotope pin apparas for rapid library synthesis. The /Rurns were tested in the fMLF ceptor assay which incorporates the fMLF tripeptide in the receptor ound in the plasma membrane of neutrophils. Results indicated bindg of some of the compound mixtures with two compounds binding ith an IC50 value of 10 um and 13 um respectively. Thus, the libraries peared to generate an ensemble of restricted conformations with ossible lead generation. Shortly thereafter, a similar library based upon the /3-turn was veloped, however, this time incorporating an additional functionality
Synthetic Peptides
he z + 3 position as well (Virgilio et al., 1997a, b). This procedure k advantage of a guanidine based resin (acting both as a support a general base catalyst) which incorporated a disulfide moiety vs. Rink amide resin used previously. Similarly, a library of 1152 pounds were generated and characterized.
trochemical Cyclizations
ther interesting alternative to peptidomimetic turn compounds employed by Slomczynska et al. (1996) who used electrochemical hodology in situ to induce selective anodic amide oxidations to erate an A^-acyliminium cation which is trapped by an intraecular hydroxyl group. Experimenting with a variety of solvents, trodes, and prototype starting materials, Slomczynska and orkers found that optimal conditions for the cyclization of BocPro-OMe to a 6,5-bicyclic turn system required dry acetonitrile, abutyl ammonium tetrafluoroborate as a supporting electrolyte, inum foil electrodes, and a constant current density of mA cm2. Cyclizations were found to be diastereoselective with the onfiguration at the bridgehead being the major isomer. Hydrolysis he methyl ester with 1.1 equivalent of NaOH yielded a mixture of diastereomers due to epimerization of the a-carbon of Ser. wever, separation could still be achieved using flash chromaaphy with silica gel and a chloroform/methanol/acetic acid :5:1, v: v: v) solvent system. Clearly, this is a novel methodology. However, it is not yet clear as ts utility as a methodology for making turn mimetics since side ducts can interfere with product isolation, and the dryness of ent systems are fairly stringent. Nonetheless, the authors provide ery intriguing route to a 6,5-bicyclic turn system.
of Peptides as Enzyme Inhibitors and rapeutic Agents
yme Inhibitors
bition of enzymatic systems remains a prime target for the regulaand control of different disease states. Currently, there are numerPharmaceuticals targeted toward the inhibition of enzymes in (1) cer cells, (2) in bacteria, (3) in the systemic system of humans, and many other systems. For example, 5-fluorouracil and methotrexate commonly used as chemotherapeutic agents by the inhibition of ydrofolate reductase and thymidylate synthetase. Penicillins, native semi-synthetic, which can be viewed as peptidic in nature, are still sidered first-line antibacterials. Similarly, lovastatin, isolated from
Applications of Synthetic Peptides Fibrinogen Thrombin (inhibition by hirudin) Fibrinopeptides and fibrin (monomer)
307
I
Fibrin polymer (soluble)
I
Cross-linked fibrin (insoluble) Figure 5-1 Scheme describing the mechanism of fibrin clot formation and inhibition of thrombin activity by hirudin.
pergillus tereus, is a cholesterol-lowering agent that works by ibiting the conversion of 3-hydroxy-3 methyl-glutaryl Co-A to valonate (Stryer, 1975). More recently, peptides have become attractive as potential therautic agents for targeting specific enzymes or enzyme systems. For ample, hirudin is a 65-amino acid (sulfated on Tyr63) polypeptide ibitor of the serine protease, thrombin, isolated from the salivary nd of the medicinal leech Hirudo medicinalis (Stone and fsteenge, 1986). Interestingly, peptides related to hirudin are eady being tried experimentally in clinical trials for the prevention blood clots in patients with rejoined limbs. Mechanistically, hirudin s as a tight binding inhibitor to thrombin. Thus, once bound, ombin is no longer able to bind to fibrinogen, thus, preventing formation of fibrin monomers, which, in turn, would polymerize form the fibrin (blood) clot (Mann, 1987) (see figure 5-1). netically, it is a slow, tight-binding inhibitor that interacts with ombin in an apparent two-step fashion. The first step is a slow mplexation with thrombin at a remote site while the second step is entially diffusion controlled (4.7 x 108mor' s~'), and involves entially irreversible complexation to the active site (Stone and fsteenge, 1986). Thrombin, being a serine protease enzyme, acts marily by cleaving fibrinogen to fibrin monomers which later casdes into a fibrin clot-forming polymer. For a review of the clotting cade pathway, the reader is referred to a review by Kayser (1983). nce it is a serine protease, it (as do most enzymes of this class) has a ference for cleavage at the carboxy termini of basic residues, partiarly arginine. The synthesis of a peptide affinity label, D-Phe-Prog-CH2-CI acted as an irreversible inhibitor of a-thrombin (Sender
Synthetic Peptides
Fenton, 1984; Walker et al, 1985). This covalent peptidyl-thromcomplex was shown to bind covalently at a histidine moiety and equently blocked the interaction of thrombin with hirudin. Based n the proposal that the C-terminus may be an important binding on of hirudin, Krstenansky and Mao (1987) synthesized by solide methods, the unsulfated C-terminal fragment N-a-acetyl din45_65. They found that this peptide bound to thrombin at a le site with an association rate constant of 10 5 mol~ 1 s~ 1 . In tion, they observed that the synthetic peptide D-Phe-Pip-Arg-/>oanilide was still hydrolyzed by the thrombin-hirudin complex. he same time, the complex was able to inhibit fibrin clot formation. led to the suggestion that the C-terminus portion of hirudin ained the binding domain to complex to the a-thrombin, yet did alter the region about the catalytic active site. This work was owed up later by Mao and coworkers (1988) who synthesized smalC-terminal fragments and determined that the last 10 amino acid dues were sufficient for binding and thrombin mediated clotting. s, once the Phe56 was removed from the C-terminus, no further clotting activity was observed. In addition, replacement of Phe56 either acidic (Glu), hydrophobic (Leu) or enantiomeric (o-Phe) no acids, resulted in complete loss of activity. Therefore, the 10 no acid peptide represented a unique class of noncatalytic site mbin inhibitors. In addition, binding of the C-terminal fragment ced a conformational change in the thrombin when complexed. Work by Dennis and co workers (1990) used site specific muta^9 esis to replace Asn with Met hirudin followed by cyanogen mide cleavage at this single Met site. Their work found, as did o and coworkers, that a conformational change in the complex occur that affects the active site of thrombin. This was shown by ange in the Michaelis constant (Km) for the substrate o-Phe-AlaArg-/?-nitroanilide. hus, the work on the hirundin-thrombin complex via synthesis of ide fragments yielded important information about the mechanof thrombin-induced coagulation pathways. Similar studies in y areas are likely to lead to many clinically valuable peptide rmaceuticals in coming years. In the past decade these studies e led to a literal explosion of peptide and nonpeptide inhibitors hrombin. urning toward the pharmaceutical application based upon vious studies, two approaches have evolved in concert leading to development of an equipotent, stable hirudin analogue for apeutic application. The first incorporated a solid-phase synthetic roach. This resulted in the development of Hirulog analogues. ulogs are a group of synthetic peptides designed to bind bivalently h thrombin at its catalytic site and its anion-binding exosite for
309
rinogen recognition (Maraganore, 1993). They are divided into ee main components: (1) a tetrapeptide, (o-Phe)-Pro-Arg-Pro, hich binds tightly to the active site; (2) a 12-amino acid sequence the C-terminal (residues 53-64), which binds to the anion-binding osite; and (3) a linking sequence comprised of a variable number of ycines. This has led to the synthetic design of Hirulog (Biogen) which s been tested in humans. Parallel work based upon classic structure function studies mbined with research in molecular biology has resulted in the develment of a recombinant analogue of hirudin, [Leu1, Thr3]-63-defohirudin (lepirudin [rDNA], Hoechst). The molecule is identical hirudin with the exception of a recombinant He for Leu in position and the absence of a sulfate group on position 63 of Tyr. Thus, earch in the area of serine protease inhibitors has resulted in the ccessful development of a therapeutic anticoagulant.
ptides as Therapeutic Agents
e development of therapeutic agents based upon peptide design has come an increasingly popular area of research. Previous limitations the development of peptide therapeutics has been based upon the rception that the stability of peptides and peptide-like compounds in e body precluded their utility as effective therapeutics. However, has become increasingly clear that peptides and peptide-like mpounds can and have been developed as effective therapeutics. ost popular of these areas of research revolve around such agents (1) cardiovascular agents where angiotensin converting enzyme hibitors have become a mainstay in managing hypertension; (2) munological agents where drugs such as cyclosporine have literally ade allogeneic heart, kidney, lung, and liver transplants possible to ve lives and prolong life; and (3) vascular agents based upon RGD d RGD-like peptides that are now used clinically as antiplatelet ugs to prevent stroke and myocardial infarcts as well as to detect ots in the vasculature (i.e., Reopro). As the review of these topics ould be too broad for the scope of this chapter, the author would like focus on a single example: diagnostic vascular agents.
agnostic Vascular Agents ver the past two decades, the integrins, a family of cell-surface oteins that mediate cell adhesion, have been a popular target for e development of peptide inhibitors. One particular integrin, the ycoprotein Ilb/IIIa receptor found on platelets, has been the focus extensive research to identify inhibitors of this receptor (Ruoslahti, 91). The a-IIb component of the GP Ilb/IIIa receptors on platelets
Synthetic Peptides
d a peptide sequence HHLGGAKQAGDV found on the terminal oxyl region of the y-chain of fibrinogen (Hawiger, 1995). These nogen/receptor networks are the basis for the formation of platelet ved clots. Platelets are obviously beneficial for the formation of s which are an instrumental aspect of the healing process. wever, platelet clots may also be somewhat detrimental when vasr injury or tears catalyze the clotting process. These can lead to kes and/or myocardial infarcts. In addition, invasive procedures h as angioplasties, rotoablation, or stent placement can also prothe nidus for clot formation leading to restenosis of a vessel lting in an infarct. Consequently, a therapeutic approach to the prevention of clotting hrombosis, would entail the development of an antagonist to fibrien binding to the GPIIbllla receptor. However, in order to do so, molecule had to be designed to be selective for the GPIIbllla ptor versus other integrin receptors found in the body. Work by rborough and coworkers (Scarborough et al., 1993) helped to idenanalogues of RGDW and KGDW that were more selective for the IIbllla receptor. Utilizing a cyclic peptide system similar to that of schbacher and Ruoslahti (1987), Scarborough and coworkers tified acetimidated cyclic compounds of KGDW that were potent highly selective for the GPIIbllla receptor. This resulted in cyclo )-MPR(e-acetimidyl-Lys)GDWPC-NH2 and cyclo (S,S>MPR cetimidyl-Lys)GDWPPen-NH2 which possessed IC50 (inhibitory centrations, 50%) values of 0.55 +0.02m and 0.3 + O.l^m ectively. Eventually, development of even more potent analogues were deped after identifying in the peptidic model that the spatial ximity of the cationic charge on the Arg versus the anionic charge he Asp was vital for activity. This eventually led to a variety of mpounds based upon tyrosine that resulted in low nanomolar and nanomolar IC30s. Since a detailed review of all these compounds is ond the scope of this chapter, the author suggests an excellent ew of this work by Wang et al. (2000). Despite the reduction of potent GPIIbllla analogues down to a le modified tyrosine analog, peptide analogues based upon RGD e found their way as useful drugs in the diagnostic arena. Diatide rmaceuticals based in New Hampshire, has been successful in prong FDA approval for a Tc99m radiolabeled peptide dimer, apcitide (figure 5-2). The agent has been approved as a radiophareutical for deep vein thrombosis imaging. As can be seen in the cture, the compound is comprised of a dimer of a KGD compound uses a homolysine analogue that is substituted with sulfur in the osition. Once again, this homolysine analogue imparts the correct ial proximity between the cationic charge of the homolysine
gure 5-2 Bibapcitide.
alogue and the anionic charge of the aspartic acid to allow the lecule to impart both potency and selectivity for the GPIIb/IIIa eptor.
ceptor Studies: A Brief Overview of the Use of Peptides Studies of Structure-Activity Relationships
haps the most important applications of synthetic peptides are for delineation of receptor types and subtypes and for providing alogues (radiolabeled and otherwise) that can be used for amination of receptor system in vivo. Clearly receptor pharmaogy, physiology, endocrinology, molecular biology and drug velopment are complicated by the fact that there are multiple es and subtypes of different receptors. One may assume that each btype will have a particular functional role in vivo, but the comxities of biological systems, the overlapping redundancies of bioical control, and the plasticity of biological systems in response to anges in environment and in disease state all provided further allenges for peptide science. Thus, the synthesis and design of ptides directed towards receptor type and subtype binding and etics, as well as toward in vitro and in vivo pharmacology are a ical area of current peptide research, and will continue to be so for ny years to come. In this section, we will provide a brief discussion
Synthetic Peptides
the many considerations that go with peptide research in this area oosing an example from our own research interests. One example of a receptor type determination that our group has en involved in is the delta opioid receptor. When the concept of ltiple opioid receptors began to surface in the mid 1970s ughes et al., 1975; Martin et al., 1976; Lord et al., 1977), the quesn arose whether these individual types of opioid receptors might be ponsible for specific pharmacological physiological responses. eculation began to arise as to whether the therapeutic responses the classical alkaloid opiates (e.g., morphine) such as analgesia, cular dilation, euphoria, and so on as well as the adverse reactions, h as respiratory depression, addiction, decreased gut motility leadto constipation, dysphoria, and so on, were elicited by specific eptor types or subtypes. Molecular biology and cloning methods have established the stence for three types of opioid receptors /j,, S, and K. In addition, re now is considerable evidence from extensive pharmacological d physiological studies for "subtype" activities of these receptors, ugh the structural correlates for these subtype activities have still been determined. Furthermore there is now considerable evidence, ecially in the central nervous system (CNS) for neuroplasticity in ponse to various stimulae. In such cases the "bioactivities" of the eptors change in response to their endogenous ligands (neurotranstters, hormones, etc.). Thus ligands that "work" in one circumnce no longer have efficacy in another. Clearly this increases the allenge in designing ligands for their various receptors and receptor ates." One method of peptide design which has been instrumental in definthese receptor types has been the use of conformational constraints luding cyclization (Hruby, 1982; Kessler, 1982; Hruby et al., 1990a, 91a); (for example, see Mosberg et al., 1983a,b for enkephalins). The ionale for the use of conformational and topographical constraints s been to limit the number of conformations the peptide may ume. Decreasing the number of conformations will increase the elihood that a peptide ligand will interact primarily at one receptor ong several. This technique has been shown to be very fruitful in discovery of the ^-selective peptide, DPDPE (Mosberg et al., 83b), the /x-selective peptides, CTOP (Pelton et al., 1985) and CTAP (Kazmierski and Hruby, 1988; Kazmierski et al., 1988), and /^-selective dynorphin analogues (Kawasaki et al., 1990). Prior to describing the design and synthesis of receptor-selective ptides, however, a brief description of the types of bioassays to ferentiate between the different receptor moieties is essential. For example here, we will use studies of the use of opioid receptors d their peptide ligands. As was indicated previously, Lord and
313
workers (Lord et al., 1977) demonstrated a difference in affinities of alkaloid or morphine-like opiates versus the peptidic enkephalins. izing tissues from the guinea pig ileum (GPI) and mouse vas erens (MVD), Lord was able to show a preference of morphineopiates for /x receptors and a preference of enkephalins for 5 eptors. This demonstrated that one could study ligands directed ards specific receptor types. It was later discovered that the D had approximately 80% S receptors, the remainder being /u, K, and the GPI had approximately 30% S (Leslie et al., 1980). n though the receptor populations are not homogeneous for S VD) or S (GPI), one can get a fairly accurate idea of relative ligand ctivity by calculating the GPI to MVD ratio of the corresponding 0 values. Ration < 1 exhibiting // selectivity and > 1 exhibiting /n, ctivity. Of course, these ratios have to be based on absolute ligand ndards which today, we currently use DPDPE (6) and CTOP (/A) as andard" S and // ligands. n addition to these bioassays, binding assays generally have ized membranes from the brain and 3 H- or 125I-labeled highly eptor-selective ligands have been developed.
velopment of <5-Selective Opioid Receptor Peptides
prototypical peptide used as the original starting point for elective peptide design begins with the Leu5-enkephalin and Met"ephalin, since Lord and coworkers (Lord et al., 1977) suggested a ference of both enkephalins for the 5 receptor. Initial design prines which address either shortening or lengthening the enkephalin tapeptides met, in general, with little success with regards to ency (vs. Met3 or Leu5 enkephalin) or selectivity. The second ign principle which was employed, the substitution of the corponding L-amino acid for a D-amino acid, initially met with only or success. Substitution of a o-Ala2 for Gly2 in Leu5 and Met ephalin did not improve the binding character significantly, but did ult in an increase in potency in both the MVD and GPI assay osterlitz et al., 1980). Introduction of a o-Leu in position 5 ADLE) likewise did not cause any great increase in affinity for two binding sites; however, it significantly increased the activity he MVD and reduced it in the GPI such that there was a five-fold rease in preference for the MVD over the GPI. Thus, it became ar that substitution of D-amino acids at positions 2 and 5 were eficial for S receptor selectivity. A major breakthrough in the design of ^-selective peptides evolved m the work of Gacel and coworkers and Fournie-Zaluski et al. cel et al., 1980, Fournie-Zaluski et al., 1981). Starting from struce activity relationships in the enkephalin series, the hexapeptide
Synthetic Peptides
D-Ser-Gly-Phe-Leu-Thr (DSLET) was prepared. Although more potent than DADLE in the MVD (0.58 nM0.20 vs. nM0.09), DSLET was about seven-fold less potent in the GPI us DADLE (DSLET IC50 GPI/MVZ) = 620). Further modificas based upon DSLET as the prototype eventually led to more <5ctive peptides. Replacement of o-Thr2 for o-Ser2 increased the ctivity for the S receptor even more (Gacel et al., 1988a). This followed by the development of the O-tert-butylated analogues Ser2(O-ter/-butyl), Leu5, Thr6]enkephalin (DSTBULET) and [D(0-rer?-butyl), Leu5-Thr6 (0-?erf-butyl)]enkephalin (BUBU) ch further improved upon selectivity (Delay-Goyet et al., 1988; el et al., 1988b). A design theme that proved to be extremely successful in our labora(Hruby, 1982; Hruby et al., 1990a) has been the use of conformaal constraints to study receptor selectivity and, even more ortantly, receptor-site topography. The concept takes advantage he fact that conformational constraints not only can help to generate ptor selectivity but also can aid in determining receptor topography, ause now the number of possible conformations is substantially uced compared to their unconstrained counterparts. Hence, a eased number of degrees of conformational freedom gives one the ed advantage of being able to indirectly study receptor topography xamining the conformational properties of the ligand in solution. Work that led to the development of [o-Pen2, o-Pen5]enkephalin DPE) (Mosberg et al., 1983b) was based upon our knowledge pseudo-isosteric cyclization of side-chain groups in our previous ies with melanotropins. In this case Sawyer et al. (1982), showed substituting the Met4 and Gly' residues by Cys4'10 moieties owed by subsequent oxidation led to a compound that assumes a lar steric space as its linear counterpart, but now becomes conned. This led to a superpotent analogue. Similarly, it was realized the Gly2 and Met5 side chains of enkephalin would be pseudoteric if Cys substitutions were made at these positions. From the nt literature, Sarantakis had found that a [o-Cys2, L-Cys5]ephalin was an effective analgesic in vivo, but no information known about its selectivity until Schiller and coworkers resynthed the compound and found it to be potent but not very selective hiller et al., 1981). The suggestions from other work that o-amino s could be utilized in both the 2 and 5 positions encouraged berg et al. to synthesize both [o-Pen2, o-Cys5]enkephalin DCE) and [o-Pen2, L-Cys5]enkephalin (DPLCE) (Mosberg et al., 3a). These were found to be the most selective S ligands to date. A l permutation led to the synthesis of [o-Pen2, o-Pen5]enkephalin DPE), which has served as the pharmacological standard for ying <-opioid receptor properties for many years.
315
Figure 5-3 Superimposed structures of the two low energy conformations found most consistent with ! H NMR NOESY constraints and vicinal coupling constant data. A series of turn structures (type I, 10, II, lift, III, IIIy3, IV, IV ft, etc.) were used as starting points prior to energy minimization. Note the difference in the helicity of the disulfide bridge.
The development of DPDPE by Mosberg and coworkers also led to ensive biophysical studies to help give us a clue to the topography out the <5-opioid receptor. Hruby and coworkers (Hruby et al., 1988) conducted extensive 2-D MR studies in solution including nuclear Overhauser enhanced MR spectroscopy (NOESY) in 90% HOD and pH 3.1 and 6.0. ese studies revealed some rather interesting dipolar interactions ween the aromatic moieties of Tyr1 and Phe4 and the ft, ^-dimethyl ups of o-Pen2. This direct evidence for transannular interactions Hruby et al. to suggest that an amphiphilic conformation (Hruby al., 1988) may be among the most stable in solution. Starting pririly with coupling constants derived by NMR determinations, comations of all reasonable phi angles consistent with a 14-membered g, were calculated. These starting parameters were in turn energy nimized and finally compared again with the NMR data. Extensive rgy minimization studies led to finding two pairs of low energy nformations which differed primarily by the chirality of the disulfide ure 5-3). Of the two, one of the low energy conformers satisfied all the NMR criteria. The conformation was distinguished by the stence of a type IV /3-turn and the transannular interactions cribed previously. In addition, the molecule contained overall phipathic topographical features. Thus, because of the constrained ure of this peptide ligand, one has a general idea of the 5-opioid eptor topography by virtue of the ligand specific for it. An addinal approach using quenched dynamics was conducted by Pettitt
Synthetic Peptides
Figure 5-4 CPK models of the nine lowest energy conformers derived from quenched dynamics studies on [o-Pen2, D-Pen5]enkephalin (Pettitt et al., 1991). Note that all nine conformers exhibit similar positioning of the aromatic residues above the plane of the backbone ring, while the carbonyl moieties (oxygens are shaded) are pointed toward the opposite side of the backbone ring, giving the molecules their amphiphilic character.
d coworkers (Pettitt, et al., 1991) which yielded a similar set of low ergy conformers that all exhibited the same amphipathic character ich was evident in the earlier work. CPK models of the nine lowest ergy conformers can be seen in figure 5-4. Further important evidence for the low energy conformations cessible to DPDPE were provided when an X-ray crystal structure
317
DPDPE was finally obtained (Flippen-Anderson et al., 1994) lowed shortly thereafter with X-ray crystal structure of analogues DPDPE (Collins et al., 1996). These important studies provided an portunity to compare the conformations obtained by NMR, by olecular dynamics, by computational chemistry, and in the crystale state for both agonist and antagonist biological activity. A major nclusion from these studies is that the 14-membered disulfide ring nformation is the same except the chirality of the disulfide bridge ich could be either right-handed or left-handed, a result consistent th the molecular dynamics studies. Thus it appeared that the conmation of the backbone scaffold of the 14-membered ring for PDPE was the same in both solution and in the crystal and premably also at the 5-opioid receptor. However, to obtain the bioically active conformation and corresponding pharmacophore in D space it also is critical to know the preferred side-chain conforman x1 and x2 angles for the key side-chain groups known to be tical for interaction with the 5-opioid receptor, namely Tyr1 and e4 in DPDPE. Previously, using all four isomers of /?-methylphenylnine (Hruby et al., 1991b) in the 4 position in conjunction with nding and bioassay studies, as well as NMR studies and computanal chemistry (Nikiforovich et al., 1993) we were able to conclude at the gauche (-) x1 conformation was best for high potency and ectivity at the <5-opioid receptor. However, when we utilized the me approach with all four isomers of /3-methyltyrosine in position of DPDPE the biological results were equivocal (Toth et al., 1992). is led us to search for more x1 and x2 constrained amino acids. us we designed and developed an asymmetric synthesis (Qian et al., 95) of all four isomers of ^-methyl-Z'^'-dimethyltyrosine and inrporating them in DPDPE (Qian et al., 1994, 1996). Only one of se isomers [(25I,3/?)/S-methyl-2/,6/-dimethyltyrosine]DPDPE had th high potency and high selectivity for the 5-opioid receptor, and th NMR studies in solution and computational studies showed at the trans x1 conformation of the Tyr was compatible with the oactive conformation at the <5 receptor. Interestingly, this same anague was a weak antagonist at the /u-opioid receptor, while the 5,31S)TMT1]DPDPE analogue was a modestly potent at both the and 8 receptors (Qian et al., 1996) had considerably better analgesic ivity (Bilsky et al., 2000). These results led to the development of a three-dimensional topoaphical model in vector space of the biologically active pharmaphore for the interaction of DPDPE with the 5-opioid receptor henderovich et al., 2000). This then provided a de novo approach the design of a highly potent and receptor selective nonpeptide and for the 5-opioid receptor that had similar structure-activity ationships as DPDPE but different structure-activity relationships
318
Synthetic Peptides
than other putative nonpeptide peptide mimetic analogues with high binding affinity for the 5-opioid receptor (Liao et al., 1998). Clearly appropriate conformational and topographical constraints of bioactive peptides in conjunction with careful biological assay studies, and rigorous conformational studies using spectroscopic and computational studies can provide new insights into structure-activity relationships for ligands that interact with complex biological systems such as the G-protein coupled receptors (GPCRs) of which the 8 receptor is a typical example. In conclusion, using the <5-opioid receptor as an example, one can see how the design of 5-receptor selective analogues has developed from: (1) lengthening and shortening the sequence; (2) systematic substitution of D-amino acids for native L-amino acids; (3) imposing of conformational constraints by pseudo-isosteric replacement; (4) solution phase NMR; (5) X-ray crystallography; (6) molecular mechanics and molecular dynamics calculations; (7) design and asymmetric synthesis of novel topographically constrained amino acids; (8) their incorporation into the cyclic constrained bioactive peptides; and (9) careful binding and bioassay studies can provide detailed insight into the bioactive conformation of peptides which can be used for de novo design of a nonpeptide mimetic. All have helped to develop a "picture" of the 3-D structural requirements of the 6-opioid receptor that can be tested. Clearly, more work needs to be done to refine the picture. Future endeavors incorporating all the above design principles as well as molecular biology and molecular pharmacology techniques may one day soon allow us to isolate and to view the 5-opioid receptor itself as it interacts with its agonist and antagonist ligands. Efforts in that direction have begun to appear (e.g. Salamon et al., 2000).
The Use of Peptides for Affinity Labeling of Receptors
Establishment of some type of irreversible but specific interaction between a ligand and its biological acceptor, termed affinity labeling, is a major approach to identify, characterize, and even isolate receptor molecules for hormones and neurotransmitters. In the case of enzymesubstrate interactions, this approach helps in identifying the chemical constitution (amino acid residues) of the binding and/or catalytic site(s). The most general approach for establishing irreversible binding between a ligand and its acceptor relies on the establishment of a covalent bond between the two entities. Two main methods for achieving this are chemical-affinity labeling and photoaffinity labeling. While both techniques have found their applicability in numerous peptidehormone receptor systems, photoaffinity labeling generally is preferred because of its nondependence on proper juxtaposition as well as the
319
Table 5-3 Reagents for the introduction of maleimido groups in peptides Reagent Reference
presence or absence of certain specific chemical functionalities at the receptor binding site. A common technique for affinity labeling of acceptor molecules involve the use of homo- as well as heterobifunctional cross-linking reagents. These reagents are added to the incubation medium after the binding of a ligand with its acceptor/receptor has been established. This technique leads to a covalent cross-linkage between the ligand and the acceptor. A number of these reagents which utilize a variety of the chemical functionality and photoaffinity approaches, have been developed and are available commercially (see, for example, tables 5-3-5-6). The photoreactive and/or chemically reactive groups used in these bifunctional cross-linking reagents are, in principle, the same that are described in the following discussion on the development and use of chemical-affinity and photoaffinity peptide ligands.
Chemical-Affinity Labels Chemical-affinity labeling involves the establishment of a covalent bond between a ligand and its biological acceptor during their specific binding to each other. Usually, the ligand used in this operation is first modified to bear a chemically reactive group capable of reacting with
Synthetic Peptides
e 5-4 Heterobifunctional reagents for the synthesis of photoaffinity des gent Reference
ve for amino group (may also react with SH groups):
321
Table 5-4 (Continued) Reagent
Reference
Reactive for carboxy group:
Reactive for arginine side-chain functionality:
Reactive for cysteine side-chain functionality:
Table 5-4 (Continued) Reagent

Reactive for tryptophan side-chain functionality:
Reference
Reactive for tyrosine/histidine side-chain functionality:
Carbene generating reagents reactive for amino groups:
any of the chemical functionalities found usually on the acceptor/ eceptor molecule. Most importantly, the nature of this reactive group should be the one that allows this reaction to take place under physiologically compatible conditions, that is aqueous medium, ambient temperature, and pH 6-8 values. These requirements limit the ype of chemical reactions one can efficiently perform under these conditions with functionalities, such as, amino, carboxyl, thiol, midazole, phenol, hydroxyl, and so on, that are usually found on he acceptor protein. Among these, the amino and thiol groups, because of their chemical reactivities under the prescribed conditions, are best suited targets for chemical-affinity labeling. A variety of
323
able 5-5 Radioactive heterobifunctional reagents for specific derivation f amino groups in the peptides for photoaffinity labeling of their espective receptors Reagent Reference
adioactive iodine atoms may easily be incorporated in these molecules.
rivatives capable of reacting with thiol and amino groups are now own and can be efficiently incorporated in the ligand molecule. The lowing section provides a general discussion about some of these emical affinity groups and their incorporation in the peptide-based ands. Perhaps the most germane point while designing these emical-affinity ligands, however, is important consideration in signing the site in the ligand molecule at which one can incorporate e chemically reactive group. Because specific biomolecular recognin and binding is of critical importance for any labeling experiment, e site of derivatization must be one that causes little or no change in e recognition and the binding characteristics of the modified ligand.
iol Reacting Derivatives
e use of affinity labels reactive toward a thiol group generally is mited to acceptor molecules which possess free thiol groups. Most the cysteine residues present in a biomolecule exist in their respece oxidative state, forming a disulfide linkage among a pair of steine residues. In spite of this, the chemistry for thiol sensitive
24
Synthetic Peptides
Table 5-6 Cleavable hetcrobifunctional reagents for derivation of the peptides for reversible labeling of their respective receptors Reagent Reference
erivatives is well developed due to the nucleophilicity of the thiol roup, and can be performed under mild biocompatible conditions. s the result, it is the most favored one for conjugating a biomolecule ither with an insoluble matrix for use in affinity chromatography, or ith another biomolecule for use in diagnostic assays, immunization, ntibody, and vaccine production. Two main strategies followed or reactions with a thiol group are S-alkylation and disulfide bond ormation.
-Alkylating Reactions
Thiols undergo reaction with a-halo ketones and maleimide groups nder physiological conditions. These ketones, particularly the iodond bromo-acetyl groups have been used in many instances for lkylating a SH group in a biomolecule. The order of reactivity mong a-halo ketones is in the order: iodobromo > hloro > fluoro. The reactivity of a-iodo as well as a-bromo haloetones towards -SH functionality is not entirely specific. While a ariety of other functionalities such as water, OH, NH 2 , the imidazole ing of histidine, and so on can react with a-iodo as well as a-bromo etones, the reaction of a-chloroketone with thiols is selective but slow nder physiologically compatible conditions. The a-fluoroketones are ractically inert. Consequently, a-bromo ketones generally are the most favored chemical affinity alkylating reagents. Bromoacetic acid an be coupled using usual methods of peptide synthesis to either Nerminal or side-chain amino function in a peptide ligand to afford this ype of chemical label. For example, such derivatives of a-melanotroin have been prepared and used for conjugation with other large molecular weight protein (Eberle et al., 1977). Unlike a-haloketones the reaction between a thiol and maleimido roup is highly specific. Maleimido groups are fully compatible with he methods of peptide synthesis and purification. A variety of eagents containing maleimido groups are available commercially as ree carboxylic acid or as active esters like ./V-hydroxysuccinimide or pitrophenyl ester (table 5-3), for facile introduction into peptide igands.
Disulfide Bridge Forming Reactions
Commercially available 3-(2-pyridyldithio)propionate (PDF) is a commonly used group that is incorporated into peptide or protein ligands hrough its vV-hydroxysuccinimide ester. The resulting highly stable ffinity ligand undergoes a very specific reaction at pH 6.0-6.5 with hiols to form a disulfide bond with the ligand. This is the method of hoice for conjugating a ligand with another biomolecule or affinity matrix where reversibility is desired. The conjugated ligand can be asily cleaved by reducing the -S-S- bridge with mild reducing agents s dithiothreitol (DTT). Schwyzer and coworkers (Wunderlin et al., 1985a, b) have stablished yet another facile technique for making a disulfide bridge etween a ligand and another biomolecule. In this approach, as emonstrated by conjugating an a-melanotropin analogue with a biomolecule, a bromoalkyl group is first incorporated into the ligand by hemical methods of peptide synthesis. The treatment of the resulting
Synthetic Peptides
able derivative with sodium thiosulfate yields a "Bunte salt" which is eated in situ with a thiol-containing biomolecule resulting in the tablishment of the desired disulfide bridge.
mine-Reacting Derivatives
arbonyl compounds such as aldehydes and ketones react with amino oups of lysine residues to form Schiff s bases that are reversible yet able enough to allow further investigations into affinity labeled proucts. Imidate, isocyanate, or isothiocyanate groups attached to a gand are the other most widely used modalities to target amino nctionalities on the acceptor molecule. The synthesis of peptide gands carrying these groups, however, is not straightforward. hese groups, therefore, have found more use in bifunctional crossnking reagents and are being widely utilized in such studies.
hotoaffinity Labels
hotoaffinity labeling of biological receptors/acceptors for peptide nd protein hormones, neurotransmitters, and so on, is a valuable eans of receptor identification and isolation. In this approach, a emically stable but photolabile group is conjugated to a potent gand. After binding this ligand to its receptor/acceptor site, the comex is subjected to photolysis to generate highly reactive carbenes and trenes which may undergo hydrogen abstraction, insertion, or cloaddition reactions with the adjacent chemical functionalities on e receptor molecule, thereby establishing a covalent bond between the ceptor and the ligand (Bayley, 1983). Because of their high reactivity, e photoaffinity labels have proved to have advantages over the emical affinity labels. Prior to activation, they are stable in aqueous lutions, and most importantly, the reactions exhibited upon phototivation are not dependent on the presence of a nucleophilic center on e receptor for establishing chemical bonds. However, they can offer e disadvantage of nonspecific covalent interactions if they are not ceptor bound at the time of photoactivation. The use of a radiolabeled hotoaffinity ligand in these studies helps to identify by covalent tachment, the site of ligand-receptor/acceptor interaction. The five most common groups of photolabels used are a-diazoetones, diazirines, a, /3-unsaturated ketones, arylazides, and p-nitrohenyl. The singlet or the triplet state of the photogenerated species as own in figure 5-5 tends to determine the type of reaction preferred in gand-receptor cross-linking. Singlet states, in general, exhibit electrohilic reactions such as, insertion, and so on. Triplet states exhibit dical reactions that lead to abstraction of a hydrogen atom Turro, 1980). Nitrenes, due to longer half-lives than carbenes, tend
327
ure 5-5 Alpha-diazocarbonyl, aryl azide, and aryl diazirine groups d their respective photolyzed reactive singlet and triplet states. Adapted m Eberle (1983).
xist in low-energy triplet states which can produce insertion into H bonds (McRobbie et al., 1976a,b). In electrophilic reactions the let nitrenes prefer an O-H or N-H bond over a C-H bond and eby are suggested to be less suitable than carbenes for labeling ptor areas rich in lipids or hydrophobic groups (Bayley and wles, 1978a,b). The a-diazoketones that give rise to a-keto carbenes were the first oaffinity labels used. The active site of chymotrypsin was labeled a derivative of this class of photolabels by Westheimer and coker (Singh, et al., 1962; Chowdhry and Westheimer, 1979). In case of peptides, however, the use of this type of label has been er limited. The main reason has been the inherent chemical revity of a-diazo carbonyl group. The diazo group is a good leaving up in nucleophilic displacement reactions (e.g., reactions with er, -OH, -SH, or -NH2 functionalities). Further, the a-keto carbene erated upon photolysis of a-diazoketone is likely to undergo ff rearrangement reactions thereby lowering the labeling efficy of the photoaffinity labeling of the receptor. Substitution "C-H with "C-COOEt, or "C-CF3, or CC-SO2C6H4CH3 has n reported to increase the stability of the resulting diazocarbonyl pound as well as suppress the Wolff rearrangement (Vaughan and stheimer, 1969; Chowdhry et al., 1976; Chowdhry and Westheimer, 8). This, however, also increases its lability in aqueous medium.
Synthetic Peptides
sorption maxima for a-diazoketones are 250 nm (high a) and 0 nm (low a). Both wavelengths can be used for photolysis. Diazirines, in comparison to a-diazoketones, are chemically inert d generate the same type of reactive carbenes (Bayley and Knowles, 78b). However, on photolysis unsubstituted diazirines may generate ear diazo compounds as intermediates that may also exhibit desired chemical reactivity as described above. Brunner et al. 80), substituted a Ca-H by a Ca-CF3 group to successfully overme this problem. The resulting derivative can be easily photolyzed at 0 nm. The use of such a derivative has been shown by the success in eling membrane components (Brunner and Richards, 1980). The in reason for its limited use, however, stems from the tedious synths of these derivatives. At the same time, it also has been argued that higher reactivity of carbenes than nitrenes capable of facile C-H ertions may be a disadvantage in itself. This high reactivity may se predominant nonspecific insertion reaction into cell membrane ds than with the use of nitrene derivatives. A few examples on the use of a, /3-unsaturated ketones for the otoaffinity labeling of peptides are known. 4-Acetylbenzoyl pentatrin was covalently coupled to albumin by irradiation at 320nm alardy et al., 1974). In this case the photolysis produces a biradical plet state that has selectivity in the abstraction of C-H hydrogens n O-H hydrogens (Turro, 1965). It has been suggested that this otolabel, due to its high lipophilicity, may considerably alter the ding and biological characteristics of short peptides. That alteran is likely to make this type of photolabel unsuitable for many ptide ligands. Arylazides are the best-known labels used in combination with ptides. These are stable to most of the acid and base procedures d in peptide synthesis. That makes them compatible with the nthesis of peptide derivatives. These groups, however, are not stable der reducing conditions such as hydrogenation and exposure to ols. Photolysis of arylazides yield highly reactive nitrenes. The sorption maxima for unsubstituted arylazides is around 260 nm. roduction of an electron withdrawing group, for example, nitro up, in the position meta to the azide group allows one to accomsh photolysis at higher wavelengths. The /j-nitrophenyl group has been used as a photolabel in the form /7-nitrophenylalanine derivatives incorporated in to peptides. This hnique has been successfully used for example, for labeling the ive site of a-chymotrypsin (Escher and Schwyzer, 1974), receptor ding sites for angiotensin II (Escher and Guillemette, 1978), and dykinin (Escher et al., 1981). It, however, failed to label receptors a-MSH which could be labeled by using a /?-azidophenyl group ntaining MSH ligand (Eberle, 1984).
329
orporation of Photolabile Groups in the Peptide rmones and Their Analogues
ch work on structure-activity relationships for peptide hormones ongly demonstrates that introduction of even a minor structural nge for a key residue in the peptide can affect its potency, as well its selectivity towards a particular receptor subtype. However, for ny bioactive peptides, and especially for cyclic conformationally nstrained peptides which have two surfaces, specific sites or surfaces st on these structures where modification of functional groups, even ge structural modifications, does not affect bioactivity or potency g., Sharma et al., 1993). These ancillary sites are particularly useful attaching labeling moieties, with the caveat that their sites of labelmay be somewhat removed from the primary binding site. These tors require careful consideration of the site where a potential up can be incorporated. While not compromising the potency d selectivity towards its biological receptors, the photoaffinity ptide analogues should also exhibit the ability to cross-link with receptors. In general, the methods of incorporating photolabile ups into peptides can be categorized in two main groups. These egories of; (a) de novo synthesis of a particular photoaffinity pepe analogue, and; (b) derivatization of peptides to photoaffinity pepes are discussed below.
Novo Synthesis of Photoaffinity Peptides
s approach generally involves introduction of novel amino acids t also can act as photoaffinity labels, such as the /?-nitrophenylnine (Pnp) residue, in the peptide during its total synthesis. The ished peptide containing a Pnp thus acts as a photoaffinity ligand. ernatively, a Pnp residue, incorporated in a peptide, can be conted to a />-azidophenylalanine (Pap) residue which, as discussed lier, is a more efficient photolabile group. This conversion can be ieved by hydrogenating the Pnp-containing peptide to give the responding /?-aminophenylalanine substituted peptide which is n diazotized and treated with sodium azide to form the Pap bstituted peptide analogue. As shown in figure 5-6, a more direct thod for obtaining a p-amino phenylalanine-substituted peptide ivative is by introducing a benzyloxycarbonyl (Z) protected pH2-phenylalanine residue in the peptide during its synthesis. otoreactive analogues containing Pap have been reported for ny bioactive peptides such as angiotensin II (Escher and illemette, 1978), bradykinin (Escher et al., 1981), a-MSH (Eberle d Schwyzer, 1976).
30
Synthetic Peptides
[Pnp]
[Pap]
Figure 5-6 Alternate synthetic routes for /j-azidophenylalanine [Pap] substituted peptides.
Derivatization of Peptides to Yield Photoaffinity Peptides
A number of heterobifunctional reagents containing the photolabile -azidophenyl group have been developed over the last two decades hat can be incorporated into finished peptides under rather mild onditions (tables 5-4, 5-5, 5-6). The second chemically reactive species n these reagents is designed to react either with an amino, or a arboxyl functionality that commonly occurs as the N- or C-terminal, r on certain side-chain functionalities of some amino acids in the eptide. Some reagents that react with the side chain functionalities f Arg, Cys, Trp, His, and Trp residues also have been developed. Most of these reagents (tables 5-4 and 5-5) have been used for onstructing photoaffinity labels from peptide ligands and are commercially available from various sources. Reagents for derivatizing amino groups such as acid chlorides, acid romides, or/j-nitro phenyl esters of^-azido benzoic acid or/>-azido henylacetic acid can lead to quantitative modifications. So do eagents such as fluoro arylazides, /?-azidophenylisothiocyanates and midates which, in addition, allow the maintenance of the cationic haracteristics of the modified amino group. Some of the reagents, uch as those containing an TV-hydroxysuccinimide ester also provide he choice of varying the length of the spacer between peptide and the hotoreactive azidophenyl moiety. The carboxyl modifying reagents ontain an amino group that could be coupled to the COOH group on
peptide by any of the well-established methods of peptide bond mation. In this case, however, any amino group that may be present the peptide itself should be selectively protected prior to this ction. An arginine specific reagent, />-azidophenyl glyoxal, reacts with the nidino group at pH 7.0-7.5. The product formed, however, is table at alkaline pH regenerating the original guanidino group. steine-specific reagents which contain a-haloketones or maleimide ups, can react quantitatively with the thiol function. However, st cysteine residues are found in peptides in an oxidized disulfide m. The tryptophan-modifying reagent, 2-nitro-4-azido phenylenyl chloride reacts at position 2 of the indole ring in Trp. This gent is also capable of reacting with free Cys (thiol) groups. The group in the azido aryl azo derivative reacts exclusively with the osine or histidine ring. Three carbene generating heterobifunctional reagents that modify amino functionality also are available. These acid chloride or itrophenyl ester reagents are adequately stable for derivatization ctions. A reversible or cleavable class of heterobifunctional reagents also e been developed. The use of these reagents (table 5-6) offers the sibility of cleaving the peptide ligand from its receptor at will under d conditions. The receptor thus separated from the ligand can be d in further studies such as generation of receptor specific antidies, examination of structure, or cell-free reconstitution of the eptor. Normally, the cleavable heterobifunctional reagents tain, in addition to the usual two reactive groups, another group h as a disulfide (-S-S-), glycol (-CHOH-CHOH-), or azo (-N = N-) up. These groups can be cleaved respectively by 10-lOOmM rcaptoethanol at pH 7-9 (mild reductive conditions), 15 mM sodium iodate for 4-10h (oxidative conditions), or sodium dithionite yley 1983). Many other possibilities for affinity labeling a peptide ligand to a tein, nucleic acid, lipid or other receptors/acceptors can be enterned. The development of more robust and sensitive methods to dy these complexes, especially mass spectrometry, Raman spectropy, and other physical methods, suggests that these applications of tide chemistry will continue to be used and developed.
igenic and Immunogenic Uses of Synthetic Peptides
e use of synthetic peptides in the study of the immune response ms from the ability of peptides to mimic similar antigenic sequences proteins. Antibodies raised against an antigen in a native protein bind to the same antigenic sequence in the absence of the rest of
Synthetic Peptides
e protein. With the advent of the Merrifield solid-phase procedure Merrifield, 1963), rapid and selective synthesis of pure peptides has come routine, and is now widely used in the study and prediction of otein antigenicity. Primarily synthetic peptides are used to identify y antigenic epitope regions of proteins. This is achieved by initiating e immune response against the native protein, and then testing the nding of the resulting antisera to synthetic fragments of the protein an appropriate immunoassay, such as the enzyme-linked immunorbent assay (ELISA). Identification of various protein antigens has far-reaching applitions in the fields of biochemistry, immunology, and molecular ology. Short-peptide epitopes can be used in the elucidation of the echanism of the immune response (Smith, 1989), and allows vestigation of the molecular recognition process of the antibody/ ntigen interaction complex (Hinds et al., 1991). The cross-reactivity antibodies raised against synthetic epitopes for the native protein s facilitated the development of new generations of synthetic peptide ccines which are being developed for their enormous potential gainst viral diseases such as foot and mouth disease, hepatitis, and IV. In addition, the cross-selectivity of antipeptide antibodies for the tive protein has been utilized in the isolation and characterization of ne products. Given the ease of synthesis of short peptides, the major fficulty in any study of this nature is deciding which antigenic agments to synthesize. The minimum epitope length of a peptide agment has been indicated to be about five to ten residues, and ptides of those lengths and longer have been used to determine hich regions of a macromolecular structure, such as a protein, will antigenic. Two protocols have been developed to this end. The first these epitope mapping procedures involves the synthesis and testing all possible peptide fragments in the protein of interest. The second otocol is based on prediction of the antigenic regions from the imary sequence of the protein, and hence involves the synthesis of wer peptides. The uses of chemical and biological combinatorial nthesis methods have greatly expanded the number and kind of ptide-related ligands that can be used in this approach. In this chapr we will only outline necessary background material. More recent velopments which are rapidly expanding are outside the scope of is chapter.
ucidation of Antigenic Peptide Sequences in Proteins
ith the introduction of immunological testing of peptides still bound the solid support used in their synthesis (Smith et al., 1977), the oduction of all overlapping peptide fragments in a protein to locate e highly antigenic sequences became attractive (figure 5-7). This
1
(i)
213
H2N-T -T -S -A -G -E -S -A -D -P -(201 amino acids)-T-L-COOH
-T -T -S -A -G -E
(ii) -T -S -A -G -E -S (iii) -S -A -G -E -S -A (iv) -A -G -E -S -A -D
(v) -G -E -S -A -D -P
Figure 5-7 An example of systematic synthesis of all possible overlapping hexapeptides of the Foot and Mouth Disease Virus protein VP1, as utilized by Geyson and coworkers (1986). (Only the first five fragments are indicated.)
tematic protocol was greatly enhanced by the alternative synthetic cedure of Geysen et al. (1984). They prepared peptides on polyylene rods utilizing standard Merrifield solid-phase chemistry. The ker between peptide and support was a polyacrylic acid derivative mpatible with both water and the organic solvent. The polyethylene with attached peptide could then be used as the solid phase in an ISA immunoassay. The feasibility of this procedure was demonated by Rodda and coworkers (1986) who synthesized all possible pta-, octa-, nona-, and decapeptides of the 153-residue protein myobin, and assessed the binding of these to polyclonal antibodies ed against the native protein. In this way, they identified the conuous epitope LKTEAE comprising residues 49-54 of myoglobin as strongest antibody-binding fragment. Although this technique yields a complete systematic epitope mapg it can only be applied to small proteins due to the prohibitive ense and time required for such a procedure. However, an example which a nonselective, systematic, synthetic approach is essential can found in the study of the recognition of T helper cells for processed eign-protein fragments which are located on the surface of macroages (Unanue and Allen, 1987). These fragments are produced by ymatic degradation or denaturation of the native protein, and they not necessarily correspond to the regions of high antigenicity that are ognized by antibodies. Thus, when studying the immune response chanism of T helper cell activation, one cannot easily predict uences that are T-cell epitopes (De Lisi and Berzofsky, 1986; thbard and Taylor, 1988), and the most efficient method of finding antigenic specificity of T-cell recognition is the synthesis of a comte overlapping series of fragments of the protein. T-cells actually bind relatively few antigens due to major histocompatibility complex triction, which is thought to suppress autoimmunity. Finnegan d coworkers (1986) investigated this theory by synthesizing fourteen
Synthetic Peptides
residue overlapping peptides of staphylococcal nuclease, which is a -amino-acid bacterial protein. Binding studies of the overlapping gments to mouse T-cell clones raised against staphylococcal nucleindicated only two strongly antigenic sequences, corresponding to ments 81-100 and 91-110. The observation of so few epitopes for use T cells on a phylogenetically distant bacterial protein suggested t inhibition of autoimmunity is not a major component in the ted repertoire of T-cell antigens.
diction of Protein Antigenic Regions for Antibodies
predicting an epitope region of a protein, the primary consideration hat an antibody will bind only to the surface without disrupting its rall conformation. Hence, a reasonable assumption is that all hly antigenic regions of a protein are on its surface and are well osed, for example, secondary structural elements such as amphihic helices, /S-turns, loops and random-coil regions containing high sities of hydrophilic amino acids. When three-dimensional strucal information is available from X-ray or NMR, the surface acceslity of the protein can be immediately determined (see, for example ning et al. 1986; Thornton et al. 1986). However in the absence of h tertiary structural data, accessible regions can be predicted from wledge of primary structure according to expected surface proper, (see also chapter 2.) of Tertiary Structural Information
e of the first and foremost X-ray studies on antigenic interactions the determination to 2.8 A-resolution of the 3-D structure of an body/antigen complex (Amit et al., 1986). This structure mprised the Fab fragment from a monoclonal antibody bound to antigenic region on lysozyme. One of the key findings of this work that two continuous epitopes of lysozyme were in contact with the body fragment. These were chain residues 18-27 and 116-129 (see re 5-3 of Amit et al., 1986). Although this study graphically monstrated the key epitope-binding sites of an antibody, it also mplified a major drawback in the use of synthetic peptides as antis. It has been calculated that the average contact surface area ween an antigenic globular protein and antibody is 16-20 A in meter (Barlow et al., 1986), and indeed in the aforementioned ay structure study the entire antibody/antigen contact area was wn to be approximately 20 x 30 A. This area constitutes a protein ace that for most cases is too large to contain only one continuous in of residues. This, indicated that protein antigens are primarily de up of a set of "discontinuous" epitopes. Solid-phase peptide
335
nthesis allows the preparation of linear or "continuous" peptide topes only. Thus only partial binding of antibody to linear synthetic ptide epitopes of the native polypeptide can generally be observed ative to the native protein. Despite this drawback, binding between ibodies and continuous synthetic epitopes is substantial enough for ost uses and, at least, for those linear sequences corresponding to the gions of highest antigenicity.
ediction of Antigenicity Based on Primary Structure
y prediction about regions of high antigenicity from primary ucture is concomitant with a prediction about the characteristics surface regions of a protein. Of the surface characteristics of a otein, the most obvious is hydrophilicity. Globular proteins nerally fold, so many of the hydrophobic residues are buried in protein core, while many of the hydrophilic residues are located the protein surface in order to maximize good energetic interactions th the aqueous environment. In order to predict protein tertiary ucture, relative hydrophilicity (or hydrophobicity) scales have en calculated for the 20 naturally occurring amino acids, such that average hydrophilicity over six or seven residues in a chain can be imated. Those segments with the highest average hydrophilicity west hydrophobicity) are then very likely to correspond with posed surface regions and, hence, are potentially highly antigenic. veral relative hydrophilicity scales have been calculated and comred (e.g., Hopp, 1986; van Regenmortel et al., 1988), and generally ve a success rate of 65-75% in locating continuous peptide fragnts of high antigenicity. An example of the Hopp and Woods scale 981) is plotted for myoglobin in figure 5-8a, together with the known topes, and reasonable agreement is observed. A special case of drophilicity in proteins is that of amphipathicity. Secondary uctural regions are said to be amphipathic if one side of the struce is appreciably more polar than the other. Amphipathic or-helices nerally are observed on the surface of proteins, where one half of the ix, as viewed down its axis, contains hydrophilic residues on the otein exterior and the other half comprises hydrophobic residues ich interact with the protein core. (This is particularly well presented using a helical wheel diagramsee figure 5-9.) As such, se regions may be exceptionally antigenic. When the hydrophilic idues are presented to the surface in a secondary structural motif, ch as an amphipathic a-helix, the hydrophilicity scale described ove will only predict a region of intermediate antigenicity, since out half of the residues in the primary sequence are hydrophobic. nce, based on primary sequence alone, these potentially highly antinic sites would be missed. This error has been corrected in some
Hydrophilicity profiles of ulated with the scale of Hopp 81) plotted against residue ack bars above the profile of greatest antigenicity. mobility profile of myoglobin the scale of Karplus and Figures adapted from el et al. (1988).
337
L144RGDLQVLAQKVARTL"9 Figure 5-9 Primary sequence and helical wheel representation of the amphipathic a-helix in VP1 protein of Foot and Mouth Disease Virus. The residues constituting the hydrophobic section of the helix are shown in boldface type.
drophilicity scales, which incorporate prediction of amphipathic ices from the primary sequence (see, for example, De Lisi et al., 86; Sette et al., 1986). The antigenicity of amphipathic a-helices was demonstrated by ff and coworkers (1982). They predicted the presence of seven ices in the foot and mouth disease virus (FMDV) viral protein 1, and assessed the potential antigenicity of these on their amphihic character. The fifth helix in the sequence appeared to have the st amphipathic nature (figure 5-9) and indeed a synthetic hexacapeptide fragment generated from it induced antibodies that utralized the intact virus. Interestingly, a standard Hopp and oods (1981) hydrophilicity plot of this hexadecapeptide predicted o possess limited antigenicity. Another surface characteristic that can be predicted from primary ucture is segmental mobility. The mobility of chains of amino acids n be observed by X-ray or NMR and allows one to calculate an mic temperature factor (B) which is proportional to the mean uare atomic displacement from the equilibrium crystal position. hen calculated for the a-carbons of a protein chain and averaged er segments of six to ten residues, a plot of B versus residue number n be made. Since the segments at the surface will be relatively
Synthetic Peptides
ndered by the rest of the protein, these will generally have a high B or and will correspond to relatively accessible antigenic regions. A ctor scale for the 20 naturally occurring amino acids, parameterfrom 31 protein X-ray structures from the Brookhaven Protein a Bank, has been calculated (Karplus and Schultz, 1985) and an mple profile for myoglobin is shown in figure 5-8b. Notably the ons of high segmental mobility correspond well with those icted to be hydrophilic, and a fair agreement between predicted experimentally observed antigenicity is seen.
logues of Antigenic Peptides
real potential of synthetic peptides is the ability of the researcher esign novel analogues which can mimic or accentuate certain erties of the original biological sequence, based on conformaal information from solution NMR and/or computer aided ecular modeling (Hruby et al., 1990a, 199la). This approach is ly used in the synthesis of potent and selective analogues of ide hormones, neurotransmitters, enzyme inhibitors, cytokins, so on. It has only recently been applied to the conformational ysis of antigenic peptides in studying the molecular recognition esses between antibody and antigen. n an early example in this area, Dyson and coworkers (1988) ied the immunodominant nonapeptide (YPYDVPDYA) from enza virus hemagglutinin, noting that the first four residues of epitope formed a yS-turn in aqueous solution as indicated by ! H R. Assuming that this conformational motif is important for antic recognition, analogues of the pentapeptide YPYDV were ared, in which positions 3 and 4 were substituted for the other natural amino acids. The /J-turn population about Pro2 and tion 3 was assessed by 1- and 2-D NMR in water. In this study nature of the residues in position 3 was found to be most influential abilizing the turn conformation, and in most cases a type II turn favored. Consistent with this finding, YPGDV was observed to uce the highest turn population, as indicated by the low perature dependence of the Asp4 amide proton resonance (table . The Pro-Gly dipeptide is well known to favor a type II reverse (Gierasch et al., 1981). The dependence of antibody binding to nonapeptide on the type of /Rurn conformation about Pro2-Tyr3 further investigated by Hinds et al. (1991). In these studies, a-alkyl vatives of Pro2 were synthesized. In particular the y-spirolactam and a-Me Pro (2) (figure 5-10) analogues were studied for their ty to stabilize turns when compared to their binding affinity for antisera for which YPYDV was proven to be the minimum antic sequence. The cyclic constraint in 1 forces the backbone to adopt
e 5-7 Correlation of the relative turn formation of YPYDV and its ogues with the dissociation constant for equilibrium binding to antibody
'HNMR
M (-p.p.b)
4.6 3.3 1.5 0.0 KA (nM)a
180
ence
DV DV
no binding observed 3.6
s about positions 2 and 3 were determined by observation of the relevant NOEs H NMR. The relative population of the turn is given here by the degree of molecular hydrogen bonding between residues 1 and 4 as indicated by the D4NH ical shift temperature dependence. The lower NH temperature dependencies ate more stable turns. Hence equilibrium binding increases with turn stability, as as the amide NH in position 3 remains intact. asured against antibody DB 19/1 in Hinds et al. (1991).
Figure 5-10 Analogues of YPYDV tested by Hinds et al. (1991). y-Spirolactam (1) shown in the constrained type II y8-turn conformation; a-MePro derivative (2) shown in a type I turn.
Synthetic Peptides
ype II reverse turn as indicated by NOE and the Asp4 NH temperae dependence (table 5-7). The total lack of binding of 1 to either of e antibodies studied appeared to suggest that a type II turn is not vorable for recognition. However, YP[N-Me]YDV also showed no nding affinity which indicates, that the Tyr3 NH is critical for mation of the antibody/antigen complex. a-Methylation of Pro2 2 was shown to favor a type I /8-turn by gas phase molecular odeling, while ] H NMR indicated the presence of a highly stabilized n (table 5-7) which was a mixture of both type I and II turns in ution. The enhanced binding of 2 relative to the native pentapeptide quence was thus attributed to the increased stability of the turn out positions 2 and 3, and this epitope most probably binds in a pe I ^-turn conformation.
ntigenic Peptides in the Isolation and Evaluation of ne Products
pid advances in gene cloning and nucleotide sequencing have made utine the location of genes encoding proteins which have not been lated and for which a function has not been assigned. Since the nonocessed primary sequence of such a protein is determined from the ne, antigenic regions can be predicted as described above. Hence, sing antipeptide antibodies that are selective for the native protein cilitates isolating the putative gene product. The use of this technie has become widespread in the field of protein purification and aracterization and the literature abounds with many variations of application, especially in the detection of viral proteins and protein ermediates. This enormous literature undoubtedly will continue to expand and e use of peptides for this purpose continue to grow because of the any questions that can be addressed, both in terms of isolation and nctional analysis of proteins which have unknown functions and the alysis of protein folding, maturation, post-translational modifican, cellular distribution and other effects which can be examined.
imotopes, Peptide Libraries, and the Future
l of the examples cited so far have been based on protein/antigens of own sequence, and the synthesis of the peptide fragments limited to ntinuous epitopes. An exciting alternative protocol in the search for nthetic-peptide antigens which circumvents these restrictions was oposed by Gey sen and coworkers in 1986. They introduced the ncept of the "mimotope", which is defined as a molecule able to nd to the combining site of an antibody, but which is not necessarily entical to the native epitope that induced that antibody. A mimotope
341
thus mimics the essential binding properties of the native epitope. Moreover, this definition may be extended to the mimicking of discontinuous epitopes, since a linear mimotope may, in theory, contain the major binding residues from more than one continuous epitope, in the correct topographical configuration for binding. Applications of Antigenic Peptides: Development of Peptide Vaccines Over the past two decades, chemists have entertained the use of peptide sequences as potential vaccines for a variety of diseases ranging from viral diseases as hepatitis (Gomara et al., 2000) and AIDS (Cruz et al., 2000; Liao et al., 2000); parasitology (Bueno et al., 2000), and cancer (Appella et al., 1995; Dakappagari et al., 2000; Eisenbach et al., 2000). However, the development of potentially potent and clinically useful vaccines requires further refinement than the methodology described above. Recent developments in the deconvolution of combinatorial libraries to simpler, more-manageable sets, has aided in the identification of peptide-vaccine analogues with both broader recognition and enhanced potency. Strategies employing iterative and positional scanning techniques, have aided in the identification of more potent vaccines against HIV. Work by Boehncke et al. (Boehncke et al., 1993; Berzofsky, 1995) addressed the question of increasing both the interaction of a peptide fragment Tl (KQIINMWQEVGKAMYA, residues 428^43 of HIV-1 1Kb gp!60 envelope protein), a helper T-cell epitope (Cease et al., 1987), with the major histocompatibility complex (MHC) and thereby increasing immunogenicity. For antigens to be effective, they must be complexed to MHCs on the surface of antigen presenting peptides, and only then can T-cell receptors interact with these antigens to elicit the immune response (Ogasawara, 1999). Boehncke et al. hypothesized that MHC complexing may be effected by regions of the sequence that may seem non-essential but actually provide negative or adverse interactions. Hence, there is the possibility of improving binding to MHC and possibly increasing immunogenicity. Working in collaboration with Richard Houghten and employing positional scanning techniques (Dooley and Houghten, 2000), the investigators were able to identify substitutions for Glu in position 436 of the wild-type HIV sequence that resulted in increased binding potency to that of the wild-type sequence. Elimination of the anionic charge at E436 and substitution with an A or Q increased the potency of the immunogen. The increased potency secondary to elimination of the anionic charge was supported by the fact that E436D substitution led to no increase in potency. Immunogenicity studies with the substituted peptides also
342
Synthetic Peptides
confirmed that these agents were more immunogenic at one and two orders of magnitude less peptide versus the wild-type sequence (Berzofsky, 1993). A second question addressed with regards to more effective vaccines is the issue of MHC polymorphism. Because MHC molecules can vary in sequence in the population, response to epitopes or different antigenic determinants can vary. Thus, it was hypothesized that a slightly larger peptide that incorporated sequences for more than one determinant region (or multideterminant regions) could generate a sequence with overlapping antigenic regions that could form a variety of different MHC types. These "cluster peptides" sequences, known to induce helper T-cell activity, were synthesized and linked to Peptide 18 (PI8), a cytotoxic T-cell epitope (Takahashi, et al., 1988) and also part of the V3 loop; a target for neutralizing antibodies (Goudsmit et al., 1988; Palker et al., 1988; Rusche et al., 1988). Results indicated that these multideterminant or cluster peptide constructs were effective at inducing neutralizing antibodies in four different strains of mice, each known to have different MHC types. Control PI8 alone did not induce much of an antibody response and failed to induce neutralizing activity. Thus, these studies demonstrated that the improvement in potency and immunogenicity of peptides could be achieved by new synthetic methodology and cluster design. Other approaches have been attempted to elicit potent and effective vaccine design. For example, retroinverso (all-o or retroenantio) peptides, that is peptides constructed with all o-amino acids and with reverse sequences relative to the wild-type peptide antigenic sequence, have been designed as vaccines for foot and mouth disease (Muller and Briand, 1998) with varying amounts of effectiveness. Clinical trials are now in progress to test the viability of these vaccines. However, the verdict has not yet been finalized as to their clinical and therapeutic applicability. Perhaps by the publishing of the next edition of Synthetic Peptides, the results of these research labors will be known. If one shifts the emphasis from searching for the native epitope of a protein to finding mimotopes of the native epitope, a totally different approach becomes apparent. Searching for a suitable mimotope requires no previous knowledge of the protein sequence or the native epitopes. Instead, having raised antibodies to the native protein, potent mimotopes may be located by binding to a library of randomized peptides incorporating all the 20 natural amino acids in all positions. However, for a library of all possible hexapeptides (the approximate minimum antigenic length), this would require the synthesis and testing of 206 (i.e., 64,000,000) peptides, which until a decade ago was totally impractical. Recently, however, two synthetic procedures for creating
343
such peptide libraries have been presented which offer exciting possibilities for the future. Chronologically the first of these is "light-directed, spatially addressable parallel chemical synthesis" (Fodor et al., 1991). This combines Afa-photolabile protection with photolithography to allow simultaneous multiple-peptide synthesis on a glass microscope slide, using solid phase techniques. This procedure facilitates selective deprotection of the jV-terminus by simply masking one region of the slide during light irradiation. Using a computer-controlled binary masking system, 2" compounds can be synthesized in n steps on the same slide. Since the position of each peptide in the library is known (i.e., the synthesis is spatially addressed), potent mimotopes are easily found in a fluorescence immunoassay simply by the location of the fluorescing regions of the slide. Although this represents, in principle, an efficient procedure of preparing peptide libraries, it relies heavily on an expensive technology that so far not been applied to the preparation of large peptide libraries. The second method for preparing peptide libraries depends entirely on standard solid-phase chemistry. Thus, it is more attractive to peptide chemists. This method relies on "split synthesis" for randomization in the library (Furka et al., 1991; Houghten et al., 1991; Lam et al., 1991, 1992; figure 5-11). A somewhat modified standard beaded solid support is split into an equal number of aliquots, and a single protected amino acid is coupled to each aliquot. After completing the couplings the aliquots are thoroughly mixed together and split again for deprotection and coupling of the next selection of protected amino acids. This process can be repeated as many times as required, and allows the synthesis of larger libraries than the previously described technique. Having completed the library, the mixed peptide resin will contain beads bearing only one peptide sequence each. Location of potent mimotopes is achieved using assay conditions in which the bead bearing the binding peptide will be stained. This bead then can be removed with the aid of a microscope and micromanipulator and the peptide on the isolated bead is then microsequenced. Both of these synthetic technologies provide efficient ways to map antigenic peptides. Use of these approaches have greatly enhanced our understanding of the nature of discontinuous epitopes and the molecular mechanisms of antibody/antigen recognition. Possibly of more importance, peptide libraries have facilitated the discovery of mimotope peptide immunogens which can be used as vaccines. With these methodologies vaccine candidates can be directly mapped from monoclonal antibodies known to neutralize biologically important molecules or pathogens, and numerous other possibilities abound, including applications to oncogenic processes in cancer detection and treatment, autoimmune diseases, viral diseases, and related
344
Synthetic Peptides
Figure 5-11 Scheme for random peptide synthesis using the split synthesis method for a random tripeptide containing S, A or V with a terminal tryptophan added. Diagram from Lam et al. (1991).
areas where the immune response is of critical importance. In all of these cases, peptide libraries have become invaluable tools in the elucidation of receptors of all types and in the general immunochemistry of the future.
Antisense Peptides
In the quest for understanding the laws of Nature, one of the interesting questions that has been asked is whether there may be a structural or functional relationship between the two peptides that could be coded for individually by each of the two opposing strands of a double-stranded DNA stretch. This new area has been referred to as antisense peptides. The term antisense peptide or complementary peptide, therefore, refers to a peptide sequence that could hypothetically be deduced from the nucleotide sequence that is complementary to the nucleotide sequence coding for a naturally occurring peptide, peptide hormone, neurotransmitter, protein, or the sense peptide or protein. Although not
345
studied extensively, some antisense peptides have shown some selectivity in binding with their sense counterparts, and in certain cases have even exhibited surprising biological activity profiles, mediated through the biological receptors for the sense peptide. These initial promising results have not provided any great breakthrough. None the less a brief discussion will be given here. It appears that, originally, this concept was proposed by Mekler (1969). Jones (1972) independently proposed and tested this concept by synthesizing peptides that were antisense to the C-terminal tetrapeptide of gastrins. Because the gene for this tetrapeptide was not known at that time, these authors had to synthesize various permutations and combinations of the possible antisense peptide sequences. It was observed that one of these peptides exhibited inhibitory activity in a gastric-acid-release assay. A theoretical treatment to this concept was later proposed by Biro (1981a,b,c). A thorough investigation into this concept was initiated by Blalock and coworkers (Bost et al., 1985a,b) in a series of studies aimed at investigating the interaction between the antisense and the sense peptide in solid-phase-binding assays. Later on, Chaiken and coworkers (Shai et al., 1987, 1989) developed high-performance affinity chromatography (HPAC) techniques to investigate the binding between antisense and sense peptides. These two techniques remain the primary methods to study the interactions between a pair of sense and antisense peptides. This section, greatly truncated from the original editon, is aimed at providing a brief overview of this concept, highlighting its potential applications in biomedical research as well as various issues concerning the nature of the binding exhibited by a set of antisense-sense peptides.
Antisense Peptides as Internal Images of the Sense Peptides
It was observed by Blalock and coworkers (Blalock and Smith, 1984; Blalock and Bost, 1986) that, in general, the codons for hydrophobic and hydrophilic amino acids on one strand of DNA may be complemented on the complementary strand, respectively, by the codons for hydrophilic and hydrophobic amino acids. Uncharged (slightly hydrophilic) amino acids on the other hand are complemented by uncharged amino acids (table 5-8). Interestingly, either 5' -> 3' or 3' -> 5' reading of the complementary nucleotide sequence displays the same pattern in hydropathicity of the coded amino acids sequence with respect to the same sense peptide sequence. This similarity in the hydropathic profile of the sequences obtained in either way stems from the fact that the middle base of the triplet codon specifies the hydropathic nature of the coded amino acid and this base remain second in both 5' > 3' or 3' > 5' readings. This remarkable pattern in genetic code, termed as
amino acids with their respective hydropathic score, as given by Kyte and Doolittle (1982)
Amino acid encoded by the complementary codon Hydropathic value Codon [5'-3'] AGA AGO CGA CGC CGG CGU AAA AAG GAC GAU GAA GAG AAC AAU CAA CAG CAC CAU CCA CCC CCG Complementary codon [3'-5*] UCU UCC GCU GCG GCC GCA [5'-3'] direction Ser Pro Ser Ala Pro Thr Phe Leu Val lie Phe Leu Val He Leu Leu Val Met Trp Gly Arg Arg Val He Stop Gly Arg Arg Ala Thr Pro Hydropathic score -0.9 -1.6 -0.9 +1.8 -1.6 -0.7 +2.7 +3.7 +4.2 +4.5 +2.7 +3.7 +4.2 +4.5 +3.7 +3.7 +4.2 +1.9 -0.9 -0.4 -4.5 -4.5 +4.2 +4.5 [3'-5'] direction Ser Ser Ala Ala Ala Ala Phe Phe Leu Leu Leu Leu Leu Leu Val Val Val Val Gly Gly Gly Gly Met lie Ser Arg Ser Arg Ser Ser Thr Hydropathic score -0.9 -0.9 +1.8 +1.8 +1.8 +1.8 +2.7 +2.7 +3.7 +3.7 +3.7 +3.7 +3.7 +3.7 +4.2 +4.2 +4.2 +4.2 -0.4 -0.4 -0.4 -0.4 +1.9 +4.5 -0.9 -4.5 -0.9 -4.5 -0.9 -0.9 -0.7
Amino acid
Arg
-4.5
Lvs Asp Glu
-3.9 -3.5 -3.5 -3.5 -3.5 -3.2 -1.6 -1.3 -0.9
uuu uuc
CUG CUA CUU
cue
Asn
Gin His
Pro Tvr Ser
ecu
UAC UAU UCA UCC UCG
ucu
AGC AGU
UUG UUA GUU GUC GUG GUA GGU GGG GGC GGA AUG AUA AGU AGG AGC AGA UCG UCA
Trp
-0.9
UGG
ACC
-0.4 -4.5 -4.5 +1.8 -0.7 -1.6
Thr
-0.7
Gly
-0.4
Ala Met Cys Phe Leu
+1.8
ACA ACC ACG ACU GGA GGC GGG GGU GCA GCC GCG GCU
UGU UGG UGC UGA
ecu
CCG CCC CCA CGU CGG CGC CGA
Cys Gly Arg Ser Ser Ala Pro Thr Cys Gly Arg Ser
+ 1.9
+2.5 +2.7
AUG
UGC UGU UUC
UAC
ACG ACA AAG AAA GAU GAG GAC GAA AAU AAC CAU CAG CAC CAA UAU UAG UAA
His
Ala Thr Glu Lys Stop Glu Gin Lys Stop Gin Tyr Asp His Asn Tyr Asp Asn
uuu
CUA
+2.5 -0.4 -4.5 -0.9 -0.9 +1.8 -1.6 -0.7 +2.5 -0.4 -4.5 -0.9 -3.2 +1.8 -0.7 -3.5 -3.9 -3.5 -3.5 -3.9 -3.5 -1.3 -3.5 -3.2 -3.5 -1.3 -3.5 -3.5
Cys Trp Cys Stop Pro Pro Pro Pro Arg Arg Arg Arg
+2.5 -0.9 +2.5 -1.6 -1.6 -1.6 -1.6 -4.5 -4.5 -4.5 -4.5 -1.3 -0.7 -0.7 -3.9 -3.9 -3.5 -3.5 -3.5 -3.5 -3.5 -3.5 -3.2 -3.5 -3.2 -3.5 -1.3
Tyr
Thr Thr Lys Lys Asp Glu Asp Glu Asn Asn His Gin His Gin Tyr Stop Stop
cue
+3.7
Val
+4.2
He
+4.5
CUG CUU UUA UUG GUA GUC GUG GUU AUA AUC AUU
348
Synthetic Peptides
"anticomplementarity," appears to form the central theme of the concept of antisense peptides. Table 5-9 provides some early examples of peptide systems in which this concept has been applied. As is evident rom these results, binding affinities from 10~3 M to 1CT6 M often exists between a sense peptide and an antisense peptide. The ability of an antisense peptide to recognize its sense counterpart and their anticomplementarity has given rise to the suggestion that an antisense peptide may represent an "internal image" of its sense counterpart. Blalock and coworkers further tested this suggestion by raising antibodies against "HTCA" an antisense peptide for adrenocorticotropin hormone (ACTH) and testing their ability to bind the recepors for ACTH (Bost and Blalock, 1986). It was found that these antibodies, being an image of an image, behaved like ACTH in the receptor-binding assays and were successfully used to purify an ACTH-binding protein or receptor. This concept of receptor purificaion has since been applied to the purification of binding proteins for ACTH (Bost and Blalock, 1986), Fibronectin (Brentani et al., 1988), vasopressin receptor (Abood et al., 1989), y-endorphin (Carr et al., 1986), luteinizing hormone releasing hormone, LHRH (Mulchahey et al., 1986), and angiotensin-II (Elton et al., 1988a). It also has been shown that antibodies against a pair of complementary peptides exhibit idiotypic-anti-idiotypic relationships (Smith et al., 1987). The antibodies against synthetic ACTH(1-13) respectively bound the antibodies against "HTCA," the peptide antisense to ACTH. The selective interactions discussed above gave rise to a suggestion that the DNA strand complementary to the strand coding a peptide hormone might be coding for its receptor. Sequence search analysis performed by Bost et al. (1985a) revealed that regions of complementarity exist between mRNAs for epidermal growth factor (EGF), transferrin (TF), interleukin-2 (IL-2) and their receptors, but that these complementary sequences were small (six amino acids) and were obtained upon 3' -> 5' reading of the nucleotide sequence which s not the usual transcriptional or translational direction during protein synthesis. Though other similar reports have appeared in the iterature, the occurrence of such small complementary fragments in nature appears not to be common. Thus the significance of these observations is unclear. There are many instances reported (and perhaps many others unreported) where no measurable interactions between antisense-sense peptide pairs have been observed (e.g. Rasmussen and Hesch 1987; Eberle and Huber 1991; De Gasparo et al., 1989; Guillemette et al. 1989). For example, Eberle and coworkers failed to raise antibodies against a peptide antisense to human ACTH. Such suggestions of he nonuniversality of this phenomenon, weak unmeasurable interactions, or difficulties in generating antiself antibodies by an animal
Sense peptide SYSMEHFRWGKPVGKKRRPVKVYP (ACTH) YGGFMTSEKSQTPLVTL (y-Endorphin) QHWSYGLRPG (LI1RH) RPKPQQFFGLM ^Substance PI KETAAAKFERQHMDSSTSAA (Bovine pancreatic RNase-S-peptide)
Antisense peptide GVHLHRAPLLAHRLAPAEVFHGVR (HTCA) RMRYLVKATPFGHPFFAAGHKHMG QRDKGRLALLGGHEPAV SRAQSIGPVL
Reading direction 5'->3' 3'->5' 5'->3' 5'-3'
Dissociation constant (M) 0.3 X 1<T 1.9X10* 0.3 X l<r' 0.2 X 10J
-1 X 10"1
Assail SPBA" SPBA SPBA SPBA Inhibition of a biological assay SPBA
Reference Blalock et al., 1986 Bostetal., 1985 Blalock et al.. 1986 Carretal., 1986 Mulchahey et al., 1986 Also see Bost & Blalock, 1989 Bost & Blalock, 1989 Shaietal., 1987 Shai et al., 1989
AGFGVVKKPNY RGGSRSVHVLPLKLSRRSFL SRSVHVLPLKLSRRSFL RGGSRSVHVLPLK VHVLPLKLSRRSFL PLKLSRRSFL RSVHVLPLK VHVLPLKLSRRSFL LFSRRSLKLPLVHVSRSGGR SRRSLKLPLVHVSRSGGR RSLKLPLVHVSRSGGR KLPLVHVSRSGGR LVHVSRSGGR KESRRSLKLPLVHVSRSGGR EKSRRSLKLPLVHVSRSGGR EESRRSLKLPLVHVSRSGGR LFPRRPLKLPLVPVPRSGGR
3'-5' 5'->3'
6 X 10* 4.0 x 10' 3.5 x 10* l.8xlO J 8.3 x 10* 2.3 x 10-5 >5.9 x Iff5 1.2 xlO' 5 8.0 x lO'7 1.0 x 10"4 6.5 x 10* 2.3 x 10'5 >5.9xlO' 5 1.5 x 10* 1.5x10* 3.7 x 10* 1.2 xlO J
7
ZE-HPAC"
N-C Terminal inversion of the above Further substitution
of
underlined amino acids 5'-3' B.G.S.' 5'->3' B.G.S.Rd Computer optimized sequences
FNLDAEAPAVLSG (Rat liver glycoprotein fragment)
AAQHGGGLGVQVE AAQHGRGFGIQIE TAKDSGSLSVKVE AGKDSGSFSVKVE PAKDSGSFSVKVK TAKDSWSFSVKVK AAKDSRGLGIEIE AGKYSWRLRIK1K
920 x 10* 460 x 10* 460 x 10* 160 x 10* 59x10* 22x10* 40 x 10* 0.7 x 10*
ZE-HPAC
Omichinski et al., 1989
(continued)

Sense peptide GSGSFGTVYKGKWHGDVAVK (c-RAFjM.j7i sequence) Antisense peptide LYSNISMPLALVHSAKGARA SAKGARA All D-isomeis of the above: lysnismplalvhsakgara sakgara FHGHISMPFAFVYSSKAAAA Reading direction 5'-3' Dissociation constant (M) 380x10-* 1500 x lO*
380 x W* 1500 x lO"6 SxlO" 4 3.5 x \(f3 5.9 x 10*' 7.3 x lO'3 >30xlO' 3
Assay ZE-HPAC
Reference Fassinaetal., 1989a
CYFQNCPRGGKR (Arg8-Vasopressin-GIy-Lys-Arg)
SQLQVGHGPLAAPWAVLEVA PLAAPWAVLEVA SQLQVGHG
Computer optimized sequence 5'->3'
ZE-HPAC
Fassinaetal., 1989b
* SPBA = solid phase binding assay. b ZE-HPAC = zonal clutionhigh-performance affinity chromatography in which sense peptide has been immobilized on the column matrix. c B.G.S. = best guess sequence. d B.G.S.R. = best guess sequence for rabbit. e Binding constant for soluble sense and immobilized antisense peptide.
351
(antisense- peptide-making antireceptor antibodies) have been made to explain these cases. It can, therefore, be concluded that at present the concept of antisense-sense peptides is unclear. The biological relevance, and the mechanistic aspects of the interactions exhibited by these peptide pairs remain the main issues to be understood. Approaches for the Generation of Antisense Peptides The generation of an antisense sequence is straightforward in cases where DNA sequence information on a sense peptide is available. A sequence of complementary bases is written opposite the nucleotide sequence of the sense peptide. The entire complementary or antisense base sequence is then read in both 5' -> 3' and 3' -> 5' direction and translated to the peptide sequence according to the genetic code. It has been observed that antisense peptides generated in both the natural 5' -> 3' reading of the nucleotide sequence frame as well as opposite 3' -* 5' direction exhibit the ability to selectively bind the sense peptide. As discussed elsewhere in this section, Table 5-8 describes the amino acids and their corresponding complementary or antisense amino acids when translated in both 5' -> 3' and 3' -> 5' direction. As is evident from table 5-8, in the absence of knowledge of the nucleotide sequence of a sense peptide, one would be required to test all the various combinations of antisense amino acid sequences resulting due to the degeneracy of the genetic code. None the less, few approaches to generating antisense peptides in such instances have been proposed. One such approach makes a series of educated guesses based on the use of preferred codon usage tables (Hartfield and Rice, 1986; Granthan et al., 1980; Hartfield et al., 1979; Aota et al., 1988) which allow one to assess the probability of a particular codon to be used for each amino acid for a given peptide. In certain instances the frequency of encoding a particular antisense amino acid among a combination of antisense amino acids may also help in making a choice. For example, in 3'-> 5' reading, the arginine codon may give rise to six antisense codons, two of which code for serine and the remaining four for alanine. Alanine may, therefore, be preferred in designating amino acid antisense to arginine. Similarly, in the 5' > 3' reading frame, arginine may serve as an antisense replacement for proline. The combined use of these approaches may help one to obtain a "best guess" or "consensus" antisense peptide sequence. Examples of this approach have been discussed in the generation of correct antisense peptides to Substance-P read in the 3' - 5' direction (Bost and Blalock, 1989), and design of a 13-mer peptide antisense to a rat glycoprotein sequence (Omichinski et al., 1989). As is evident from table 5-8, certain anticodons either in 5' - 3' or 3' -> 5' direction are stop codons. To overcome this problem in generating antisense
352
Synthetic Peptides
peptides, Bost and Blalock (1989) have proposed substituting this codon by an amino acid that is suggested by the best-guess or consensus amino acid for that position. Similarly, Shai et al. (1989) replaced the half-Cys residues in a deduced antisense peptide by Ser residues. Omichinski and coworkers (1989) and Fassina and coworkers (1989a) have successfully demonstrated another approach to the design of antisense peptides. Their approach stems from the observed hydropathy anticomplementarity in the sense-antisense amino acid pairs and consequent suggestion that a hydropathic complementarity may be responsible for the selective interactions between a pair of sense-antisense peptides. As described in table 5-8, hydrophobic and hydrophilic amino acids are, respectively, complemented by hydrophilic and hydrophobic amino acids. Uncharged (slightly hydrophilic) amino acids are complemented by uncharged amino acids. Under this approach the consensus or best-guess sequence obtained earlier or the actual antisense peptide sequence, if known, is subjected to further refinements in order to maximize the existing hydropathic complementarity. The amino acid substitution is made in the antisense peptide so as to maximize a defined "moving average hydropathic score" for it with respect to the sense peptide. In one example, these authors (Omichinski et al., 1989) demonstrated the use of this approach in refining a best-guess antisense sequence for a rat glycoprotein fragment (table 5-9). One of the optimized sequences, AGKYSWRLRIKIK, exhibited much higher binding affinity for the 13-mer sense peptide, FNLDAEAPAVLSG, than the previous bestguess sequence AAQHGGGLGVQVE. In the other example, Fassina and co workers (1989a) have shown that an optimized sequence antisense to 356-375 peptide sequence of c-raf protein exhibited 47-fold higher binding affinity to immobilized c-raf^56_^5 peptide than the actual c-raf25(,~3i5 antisense peptide (table 5-9). Although not applied yet, these examples strongly suggest this approach, in principle, may allow a de novo design of antisense peptides that are highly selective to a given sense peptide.
Assays for Studying the Interactions Between an Antisense Peptide and Its Sense Counterpart In general, two types of assays, solid-phase-binding assay and high performance affinity chromatography (HPAC), have been used to study antisense-sense peptide interactions. Both these assays are solid-phase assays designed to overcome the main difficulty one would face in applying the standard techniques of receptor-ligand interactions of high affinity ligands, where the unbound ligand is
removed either by filtration, or centrifugation, or selective adsorption on an inert insoluble additive like charcoal.
Solid-Phase Binding Assay Well-established techniques of enzyme-linked immunosorbent assay (ELISA) were used by Blalock and coworkers (Bost et al., 1985) to develop the binding assay for studying the interactions between a sense and antisense peptide. Typically, multi-well microtiter plates are coated with varying amounts of an antisense peptide (10-50 fig) or another peptide or protein serving as control for 12-18 hrs. The wells are then washed and incubated with a fixed concentration of the radiolabeled sense peptide for 1-2 h. The plates are then washed with appropriate buffer to remove the unbound radiolabeled sense peptide and the amount of bound radiolabeled sense peptide measured. In case antibodies against the sense peptide are available, radiolabeled sense peptide may be substituted by "cold" sense peptide and the amounts of the bound sense peptide estimated by the standard techniques of ELISA using antibodies.
High-Performance Affinity Chromatography (HPAC) Method Chaiken and coworkers (Shai et al., 1987) have developed the methods of high-performance affinity chromatography (HPAC) as a direct powerful tool to study the interactions between immobilized-sense peptide and a mobile-antisense peptide. The affinity matrix is prepared by the standard techniques for reacting the sense peptide with the activated matrix. Chaiken and coworkers (Shai et al., 1987, 1989) have employed Accell 78 (Waters Chromatography Division, Mileford, Mass.) as matrix. This is a silica-based support containing a six-carbon spacer with terminal jV-hydroxysuccinimide active ester for immobilizing molecules through their amino functionalities. The matrix is reacted with the peptide, then end-capped by aminoethanol and packed in a glass column. An identical column containing blank end capped matrix also is prepared. Alternatively, Sharma and coworkers (S. D. Sharma, F. Al-Obeidi, M. E. Hadley and V. J. Hruby, unpublished data) have used a prepacked activated tresyl column (SelectiSphere 10, Pierce Chemical Company, Rockford, Illinois) for immobilizing [Nle4]-a-melanotropin (a-MSH) a tridecapeptide analogue of a-MSH. This was achieved by pumping a solution containing a determined amount of a-melanotropin through the column at pH 7.2. Spectrophotometric evaluations of the eluant indicated the immobilization of the peptide on the column. The
354
Synthetic Peptides
column was subsequently end capped with aminoethanol to block all the remaining reactive tresyl groups. The affinity column thus prepared is then used to study the interactions of antisense peptide with the immobilized sense peptide according to experimental and theoretical techniques. Typically, the extent of retardation of different concentrations of a mobile antisense peptide under binding conditions on this column can then be used to calculate equilibrium binding constant for mobile-antisense-immobile sense-peptide complex according to equation 1.
where V experimental elution volume of mobile-antisense peptide, F0 = unretarded elution volume, Fm = mobile-phase volume, [M]T = total concentration of matrix-bound sense peptide, [P] = concentration of mobile-antisense peptide in the chromatographed zone, and A^M/p = dissociation constant of the complex of mobile-antisense peptide (P) and matrix-bound sense peptide (M). Alternatively to the above, a fixed concentration of antisense peptide can be made to compete on this column with different concentrations of soluble sense peptide. This affords the binding constants for mobile-antisense-mobile-sense peptide complex (equation 2). In both these methods, called "zonal elution methods," the antisense peptide is applied to the column as a fixed zone of different concentrations.
where [L]T = total concentration of competing mobile-sense peptide, KL/P = dissociation constant between mobile-antisense peptide and mobile-competing-sense peptide, and the rest of the parameters are as above. In yet another method, referred to as the "continuous elution method," the solutions containing different concentrations of an antisense peptide are passed through the affinity column and the plateau of maximum absorbance (UV) is observed. The volume of the eluant at which the affinity matrix is half saturated is used to calculate the binding constant of the mobile-antisense-matrix-bound-sense peptide complex according to equation 3.
355
where V = volume at which the affinity matrix is half saturated, FO = void volume, A^M/P = dissociation constant of complex of the immobilized-sense peptide and antisense peptide, MT = total amount of immobilized-sense peptide, and [P0] = initial concentration of mobile antisense peptide. Biological Activity of Antisense Peptides In some cases, the antisense peptides have been found to exert biological responses through the biological receptors for the sense peptides. Al-Obeidi and coworkers (1990) assayed a series of antisense peptides corresponding to the /3-melanotropin (/3-MSH) sequence for melanotropic (MSH-like) activity in frog skin bioassay, and for melanin concentrating hormone (MCH-like) activity in frog skin and fish skin bioassays, respectively. Surprisingly, it was found that some of the antisense peptides exhibited either agonistic or antagonistic activity in these in vitro bioassays suggesting the interaction of these peptides with MSH or MCH receptors. Jones (1972) has reported that after administration of a peptide antisense to C-terminal tetrapeptide of gastrins in conscious dogs some inhibition of gastric juice volume and pepsin output was observed. Bost and Blalock (1989) have also observed a partial inhibition of stress-induced steroid production after in vivo injection of a peptide antisense to ACTH. It could be argued that these inhibitions may result either from the antagonistic action of the antisense peptide on the sense peptide receptors or as a result of interaction between administered antisense peptide and endogenous sense peptide, thereby leaving low levels of sense peptide to elicit its normal biological response. Antibodies against antisense peptides also may exert biological responses when administered in whole animals. As shown by Gores and co workers (1986), intravenous administration of antiserum against a peptide antisense to human placental progonadotropin- releasing hormone, when injected in ovariectomized rats, caused a significant decrease in plasma levels of luteinizing hormone. Since the antibodies against an antisense peptide will behave as the sense peptide in recognition aspects (being an image of an image), this effect has been interpreted in terms of an interference of this antiserum with the actions of endogenous gonadotropin-releasing hormone. Immunocytochemical staining of receptors for gonadotropin-releasing hormone on female rat pituitary cells by this antiserum further supported this inference. Potential Applications of Antisense Peptides Though experiments with antisense peptides have shown interesting, even provocative, results from time to time, the early promise has not
356
Synthetic Peptides
lead to a systematically useful application of peptide science to problems in biology and medicine, or to develop useful leads for drug candidates in medicinal chemistry. Furthermore, to our knowledge, there is no direct evidence that biological systems synthesize or use antisense peptides, and their applications to peptide hormones, neurotransmitters, enzyme substrates, immunology have not met with any dramatic success. Though this is an interesting area of research, its significance is still not established. It will be interesting to see what further developments may occur in this area.
Summary and Future Perspectives In this overview of some possible applications of synthetic peptides to modern biology and medicine, we have tried to emphasize the need for both physical/chemical and biological considerations in peptide ligand design, and the application of modern synthetic chemistry including asymmetric syntheses and macrocyclic design and synthesis. Because macromolecular recognition and selectivity are essential features of most biological systems, it is critical that highly purified peptide ligands be utilized to examine these systems, whether the examination is in the form of a binding, in vitro, or in vivo bioassay, or whether it is in the form of a biophysical or biochemical measurement. It is also clear that peptides and their analogues, pseudo-peptides, and peptidomimetics are likely to be the major drugs of the future. These findings raise many issues in need of further study. As already outlined herein, the discovery process for obtaining peptide ligands that are more or less selective for a particular receptor (or acceptor) type or subtype, a specific antibody or antigen, a specific enzyme, and so on, has been greatly accelerated in recent years by the tremendous advances in synthetic peptide chemistry, combinatorial chemistry which peptide chemists pioneered, and the development of multiple, high-throughput binding and bioassay systems. The following factors all provide powerful tools for future developments: The availability by synthesis of large (billions) peptide libraries that can be used to screen for binding and biologically relevant activities; the ability to design peptide derivatives and analogues with constrained conformations and topographies, and specific two- and three-dimensional properties; the utilization of modern synthetic chemistry to prepare novel amino acids, and designed peptides, pseudo-peptides, and peptide mimetics; the ability to design peptide analogues that are stable to biodegradation and have good bioavailability; the developments of computational methods using modern fast computers to examine the conformations, topographies, and dynamics of peptides. All will be critically important to future developments. Further work is needed
357
in these areas, as well as greatly accelerated efforts to improve methods of peptide delivery in biological systems, to find methods for passing peptides and peptide mimetics through various membrane barriers, to develop further rapid-throughput multiple-assay systems, and to obtain more reliable methods for evaluating peptide conformations and dynamic properties. Most important, there is a need to find ways to facilitate the proper collaboration between chemists, physicists, biologists, and physicians that will be necessary to optimize progress in this area. The prospects for important scientific advances and for their application to the treatment of disease are exceptionally good. The extent to which these prospects will be realized depends increasingly on comprehensive collaborations to solve these problems. This will require a considerable culture change in the way science is practiced and rewarded, especially in the interactions between chemists and biologists. We hope that this chapter will help facilitate this process.
List of Abbreviations
or-MSH: a-melanotropin, amelanocyte stimulating hormone ACTH: adrenocorticotropin hormone CCK: cholecystokinin Cp: /J-cyclopentamethylene-/3mercaptopropionic acid CTOP: D-Phe-Cys-Tyr-D-Trp-OrnThr-Pen-Thr-NH2 DADLE: [o-Ala2, DLeu5]enkephalin Dep: ft, ft-diethyl-ftmercaptopropanoic acid Dmp: ft, /3-dimethyl-/3mercaptopropanoic acid DPDPE: c[D-Pen2, DPen5]enkephalin DSLET: Tyr-D-Ser-Gly-Phe-Leu-Thr DTT: dithiothreitol EGF: epidermal growth factor ELISA: enzyme-linked immunosorbant assay FMDV: foot and mouth disease virus GPI: guinea pig ileum HP AC: high-performance affinity chromatography IL-2: interleukin-2 MAP: multiple antigenic peptide MCH: melanocyte concentrating hormone Mpa: /3-mercaptoacetic acid MVD: mouse vas deference NMR: nuclear magnetic NOESY: nuclear Overhauser enhancement spectroscopy OXT: oxytocin Pap: />-azidophenylalanine Pen: penicillamine, ft, /J-dimethyl cysteine Pip: L-pipecolic acid TASP: template assembled synthetic protein TASP: template assembled synthetic protein TCTAP: D-Tic-Cys-Tyr-o-Trp-ArgThr-Pen-Thr-NH2 TF: transferrin Thr-ol: Threonal, the reduced form of threonine, 2-amino-l,3-butanedio Tic: 1,2,3,4-tetrahydroisoquinoline carboxylate
358
Synthetic Peptides
ACKNOWLEDGMENTS This work was supported by grants from the US Public Health Service and the National Institute of Drug Abuse. We thank Cheryl McKinley and especially Margie Colie for extraordinary help in typing and editing this manuscript. We especially thank Drs. Shubh D. Sharma, Nathan Collins and K. C. Russell who helped to write the earlier version of this chapter, much of which still remains in modified form in this chapter.
References Abbenante, G., March, D. R., Bergman, D. A., Hunt, P. A., Garnham, B., Dancer, R. J., Martin, J. L., and Fairlie, D. P. (1995). Regioselective structural and functional mimicry of peptides. Design of hydrolyticallystable cyclic peptidomimetic inhibitors of HIV-1 protease. J. Am. Chem. Soc. 117:10220-10226. Abood, L. G., Michael, G. J., Xin, L., and Knigge, K. M. (1989). Interaction of putative vasopressin receptor proteins of rat brain and bovine pituitary gland with an antibody against a nanopeptide encoded by the reverse message of the complementary mRNA to vasopressin. J. Receptor Res. 9:19-25. Al-Obeidi, F., Hadley, M. E., Pettitt, M. B., and Hruby V. J. (1989a). Design of a new class of superpotent cyclic a-melanotropins based on quenched dynamic simulations. J. Am. Chem. Soc. 111:3413-3416. Al-Obeidi, F. de L., Castrucci, A. M., Hadley, M. E., and Hruby, V. J. (1989b). Potent and prolonged acting cyclic lactam analogues of a-melanotropin: design based on molecular dynamics. J. Med. Chem. 32:25552561. Al-Obeidi, F. A., Hruby, V. J., Sharma, S. D., Hadley, M. E., and Castrucci, A. M. (1990). Antisense peptides of melanocyte stimulating hormone (MSH): Surprising results. In Peptides: Chemistry, Structure and Biology, J. E. Rivier and G. R. Marshall, eds., Leiden, Escom, pp. 530-532. Al-Obeidi, F., Hruby, V. J., and Sawyer, T. K. (1998). Peptide and peptidomimetic libraries: Molecular diversity and drug design. Mol. Biotech. 9:205-223. Amit, A. G., Mariuzza, R. A., Phillips, S. E. V., and Paljak, R. J. (1986). Three-dimensional structure of an antigen-antibody complex at 2.8 A resolution. Science 233:747-753. Aota, S., Gojobori, T., Ishibashi, F., Marvyama, T., and Ikamvea, T. (1988). Codon usage tabulated from the gene bank genetic sequence data. Nucleic Acids Res. 16: Suppl. R315-391. Appella, E., Loftus, D. J., Sakaguchi, K., Sette, A., and Celis, E. (1995). Synthetic antigenic peptides as a new strategy for immunotherapy of cancer. Biomed. Peptides, Proteins, Nucleic Acids 1(3): 177-184. Bankowski, K., Manning, M., Haider, J., and Sawyer, W. H. (1978). Design of potent antagonist of the vasopressor response to arginine-vasopressin. J. Med. Chem. 21:850-853. Barlow, D. J., Edwards, M. S., and Thornton, J. M. (1986). Continuous and discontinuous protein antigenic determinants. Nature 322:744751.
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Bates, R. B., Gin, S. L., Hassen, H. A., Hruby, V. J., Janda, K. M., Kriek, G. R., Michaud, J-P., and Vine, D. B. (1984). Synthesis of cyclo-W-methyl-L-TyrAr-Methyl-L-Tyr-D-Ala-L-Ala-O,A'-diinethyl-L-Tyr-L-Ala, a cyclic hexapeptide related to the antitumor agent deoxybouvardin. Hetero-cycles 22:785-790. Bauer, W., Boiner, U., Doepfner, W., Haller, R., Hugeunin, R., Marbach, P., Petcher, T. J., and Pless, J. (1983) Structure-activity relationships of highly potent octapeptide analogues of somatostatin. In Peptides 1982, K. Blaha and P. Malon, eds., Berlin, de Gruyter. Bayley, H. (1983). Photogenerated Reagents in Biochemistry and Molecular Biology, Amsterdam, Elsevier. Bayley, H., and Knowles, J. R. (1978a). Photogenerated reagents for membrane labeling. 1. Phenylnitrene formed within the lipid bilayer. Biochemistry 17: 2414-2419. Bayley, H., and Knowles, J. R. (1978b). Photogenerated reagents for membrane labeling. 2. Phenylcarbene and adamentylidene formed within the lipid bilayer. Biochemistry 17:2420-2423. Berzofsky, J. A. (1993). Epitope selection and design of synthetic vaccines: Molecular approaches to enhancing immunogenicity and cross-reactivity of engineered vaccines. Ann. N.Y. Acad. Sci. 690:256264. Berzofsky, J. A. (1995). Designing peptide vaccines to broaden recognition and enhance potency. Ann. N.Y. Acad. Sci. 574:161-168. Bilsky, E. J., Qian, X., Hruby, V. J., and Porreca, F. (2000). Antinociceptive activities of [/?-methyl-2',6'-dimethyltyrosinel]-substituted cyclic-[oPen2, DPen5]enkephalin and [o-Ala2, Asp4]deltorphin analogues. J. Pharmacol. Exp. Ther. 293:151-158. Biro, J. (1981a). Comparative analysis of specificity in protein-protein interactions. Part I: A theoretical and mathematical approach to specificity in protein-protein interactions. Med. Hypotheses 7:969-979. Biro, J. (1981b). Comparative analysis of specificity in protein-protein interactions. Part II: The complementary coding of some proteins as the possible source of specificity in protein-protein interactions. Med. Hypotheses 7:981-993. Biro, J. (1981c). Comparative analysis of specificity in protein-protein interactions. Part III: Models of the gene expression based on the sequential complementary coding of some pituitary proteins. Med. Hypotheses 7:995-1007. Blalock, J. E., and Smith, E. M. (1984). Hydropathic anti-complementarity of amino acids based on the genetic code. Biochem. Biophys. Res. Commun. 121:203-207. Blalock, J. E., and Bost, K. L. (1986). Binding of peptides that are specified by complementary RNAs. Biochem. J. 234:679-683. Bodanszky, M., and du Vingeaud, V. (1959). Synthesis of a biologically active analog of oxytocin, with phenylalanine replacing tyrosine. J. Am. Chem. Soc. 81:6072-6075. Boehncke, W.-H., Takeshita, T., Pendleton, C. D., Sadegh-Nasseri, S., Racioppi, L., Houghten, R. A., Berzofsky, J. A., and Germain, R. N. (1993). The importance of dominant negative effects of amino acids side
360
Synthetic Peptides
chain substitution in peptide-MHC molecule-restricted murine cytotoxic T lymphocytes. J. Immunol. 150:331-341. Bohm, H.-J., and Klebe, G. (1996). What can we learn from molecular recognition in protein-ligand complexes for the design of new drugs. Angew. Chem. Int. Ed. Engl. 35:2589-2614. Bost, K. L., and Blalock, J. E. (1986). Molecular characterization of a corticotropin (ACTH) receptor. Mol. Cell. Endocrinol. 44:1-9. Bost, K. L., and Blalock, J. E. (1989). Preparation and use of complementary peptides. Methods Enzymol. 168:16-28. Bost, K. L., Smith, E. M., and Blalock, J. E. (1985a). Regions of complementarity between the messenger RNAs for epidermal growth factor, tranferrin, interleukin-2 and their respective receptors. Biochem. Biophys. Res. Commun. 128:1373-1380. Bost, K. L., Smith, F. M., and Blalock, J. E. (1985b). Similarity between the corticotropin (ACTH) receptor and a peptide encoded by an RNA that is complementary to ACTH mRNA. Proc. Natl. Acad. Sci. USA 82:13721375. Brentani, R. R., Ribeiro, S. F., Potocnjak, P., Pasqualini, R., Lopes, J. D., and Nakaie, C. R. (1988). Characterization of the cellular receptor for fibronectin through a hydropathic complementarity approach. Proc. Natl. Acad. Sci. USA 85:364-367. Brunner, J., and Richards, F. M. (1980). Analysis of membranes photolabeled with lipid analogues. Reaction of phospholipids containing a disulfide group and a nitrene or carbene precursor with lipids and with gramicidin A. J. Biol. Chem. 255:3319-3329. Brunner, J., Senn, H., and Richards, F. M. (1980). 3-Trifluoromethyl-3-phenyl diazirine. A new carbene containing group for photolabeling reagents. J. Biol. Chem. 255:3313-3318. Budker, V. G., Knorre, D. G., Kravchenko, V. V., Lavrik, O. I., Nevinsky, G. A., and Teplova, M. (1974). Photoaffinity reagents for modifications of amino acyl-tRNA synthetases. FEBS Lett. 49:159-162. Bueno, E. C., Vaz, A. J. Machado, L. D., Livramento, J. A., and Mielle, S. R. (2000). Specific Taenia crassiceps and Taenia solium antigenic peptides for neurocysticercosis immunodiagnosis using serum samples. J. Clin. Microbiol. 38(1): 146-151. Burgermeister, W., Nassal, M., Wieland, T., and Helmreich, E. J. M. (1983). A carbene-generating photoaffinity probe for beta-adrenergic receptors. Biochem. Biophys. Acta 729:219-228. Carr, D. J. J., Bost, K. L., and Blalock, J. E. (1986). An antibody to a peptide specified by an RNA that is complementary to y-endorphin mRNA recognizes an opiate receptor. J. Neuroimmunol. 12:329-337. Cease, K. B., Margalit, H., Cornette, J. L., Putney, S. D., Robey, W. G., Ouyang, C., Streicher, H. Z., Fischinger, P. J., Gallo, R. C., Delisi, C., and Berzofsky, J. A. (1987). Helper T cell antigenic site identification in the AIDS virus gp!20 envelope protein and induction of immunity in mice to the native protein using a 16-residue synthetic peptide. Proc. Natl. Acad. Sci. USA 84:4249^253. Chan, W. Y., and Kelly, N. (1969). A pharmacologic analysis on the significance of the chemical functional groups of oxytocin on its oxytocic
activity and on the effect of magnesium on the in vitro and in vivo oxytocic activity of neurohypophyseal hormones. J. Pharmacol. Exp. Ther. 156:150-158. Charpentier, B., Durieux, C., Menant, E., and Roques, B. P. (1987). Investigation of peripheral cholecystokinin receptor homogeneity by cyclic and related linear analogues of CCK26_33: Synthesis and biological properties. J. Med. Chem. 30:962-968. Chowdhry, V., and Westheimer, F. H. (1978). /?-Toluenesulfonyl diazoacetates: Reagents for photoaffinity labeling. Bioorg. Chem. 7:189-205. Chowdhry, V., and Westheimer, F. H. (1979). Photoaffinity labeling of biological systems. Annu. Rev. Biochem. 48:293-325. Chowdhry, V., Vaughan, R., and Westheimer, F. H. (1976). 2-Diazo-3,3,3, trifluoropropionyl chloride: Reagents for photoaffinity labeling. Proc. Natl. Acad. Sci. USA 73:1406-1408. Collins, N., Flippen-Anderson, J. L., Haaseth, R. C., Deschamps, J. R., George, C., Kover, K., and Hruby, V. J. (1996). Conformational determinants of agonist versus antagonist properties of [o-Pen2,DPen5]enkephalin (DPDPE) analogs at opioid receptors. Comparison of X-ray crystallographic structure, solution 1H NMR data, and molecular dynamic simulations of [L-Ala2]DPDPE and [o-Ala3]DPDPE. J. Am. Chem. Soc. 118:2143-2152. Coy, D. H., Horvath, A., Nekola, M. V., Coy, F. J., Erchogyi, J., and Schally, A. V. (1982). Peptide antagonists of LH-RH: Large increases in antiovulatory activities produced by basic D-amino acids in the six position. Endocrinology 110:1445-1447. Cruz, L. J., Quintana, D., Iglesias, E., Garcia, Y., Huerta, V., Garay, H. E., Duarte, C., and Reges, O. (2000). Immunogenicity comparison of a multiantigenic peptide bearing V3 sequences of the human immunodeficiency virus type 1 with TAB9 protein in mice. J. Peptide Sci. 6(5):217-224. Cwirla, S. E., Peters, E. A., Barrett, R. W., and Dower, W. J. (1990). Peptides on phage: A vast library of peptides for identifying ligands. Proc. Natl. Acad. Sci. USA 87:6378-6382. Dakappagari, N. K., Douglas, D. B., Triozzi, P. L., Stevens, V. C., and Kaumaya, T. P. (2000). Prevention of mammary tumors with a chimeric Her-2 B-cell epitope peptide vaccine. Cancer Res. 60(14):3782-3789. Das, M., and Fox, C. F. (1978). Molecular mechanism of nitrogen action: Processing of receptor induced by epidermal growth factor. Proc. Natl. Acad. Sci. USA 75:2644-2648. Das, M., Miyakawa, T., Fox, C. F., Pruss, R. M., Aharonov, A., and Herschman, H. R. (1977). Specific radiolabeling of a cell surface receptor for epidermal growth factor. Proc. Natl. Acad. Sci. USA 74: 2790-2794. De Gasparo, M., Whitebread, S., Einsle, K., and Heussen, C. (1989). Are the antibodies to a peptide complementary to angiotensin II useful to isolate the angiotensin II receptor? Biochem. J. 261:310-311. De Grado, W. F. (1988). Design of peptides and proteins. Adv. Protein Chem. 39:51-124. De Lisi, C., and Berzofsky, J. A. (1986). T-cell antigenic sites tend to be amphipathic structures. Proc. Natl. Acad. Sci. USA 82:7048-7059.
362
Synthetic Peptides
Delay-Goyet, P., Sequin, C, Gacel, G., and Roques, B. P. (1988). [3H][o-Ser2 (0-terf-butyl), Leu5]enkephalyl-Thr6 and [o-Ser2 (O-tert-butyl), Leu5]enkephalyl-Thr6 (O-tert-butyY). Two new enkephalin analogs with both a good selectivity and a high affinity toward 6-opioid binding sites. J. Biol. Chem. 263:4124-^130. Demoliou, C. D., and Epand, R. M. (1980). Synthesis and characterisation of a heterobifunctional photoaffmity reagent for modification of tryptophan residues and its application to the preparation of a photoreactive glucagon derivative. Biochemistry 19:539. Dennis, S., Wallace, A., Hofsteenge, J., and Stone, S. R. (1990). Use of fragments of hirudin to investigate thrombin-hirudin interactions. Eur. J. Biochem. 188:61-66. Devlin, J. J., Panganiban, L. C., and Devlin, P. E. (1990). Random peptide libraries: A source of specific protein binding molecules. Science 249:404406. Dooley, C. T., and Houghten, R. A. (2000). New opioid peptides, peptidomimetics, and heterocyclic compounds from combinatorial libraries. Biopolymers: Peptide Sci. 51(6):91-110. Dutta, A. D. (1991). Design and therapeutic potential of peptides. Adv. Drug Res. 21:145-286. Dyckes, D. F., Nestor, Jr., J. J., Jr., Ferger, M. F., and du Vigneaud, V. (1974). [l-/3-Mercapto-/8, /5-diethylpropanoic acid]-8-lysine-vasopressin, A potent inhibitor of 8-lysine-vasopressin and oxytocin. J. Med. Chem. 17:250-252. Dyson, H. J., Ranee, M., Houghten, R. A., Lerner, R. A., and Wright, P. E. (1988). Folding of immunogenic peptide fragments of proteins in water solution. J. Mol. Biol. 201:161-201. Eberle, A. N. (1983). Photoaffinity labeling of peptide hormone receptors. J. Receptor Res. 3:313-316. Eberle, A. N. (1984). Photoaffinity labeling of MSH receptors in Anolis melanophores: Irradiation techniques and MSH photolabels for irreversible stimulation. J. Receptor Res. 4:315-329. Eberle, A. N., and de Graan, P. N. E. (1985). General principles for photoaffinity labeling of peptide hormone receptors. Methods Enzymol. 109:129-156. Eberle, A. N., and Huber, M. (1991). Antisense peptides: Tools for receptor isolation? Lack of antisense MSH and ACTH to interact with their sense peptides and to induce receptor-specific antibodies. J. Receptor Res. 11:13-43. Eberle, A. N., and Schwyzer, R. (1976). Syntheses von [o-alanin1, 4'-azidoS'jS'-ditritio-L-phenylalanine , norvalin4]-a-melanotropins als "Photoaffinits-probe" fur Hormon-Rezeptor-Wechselwirkungen. Helv. Chim. Acta 59:2421-2431. Eberle, A. N., Hhbscher W., and Schwyzer, R. (1977). Synthesis von radioaktiv markierten Bromacetyl- und Diazoacetyl-or-Melanotropin-Derivaten zum Studium von kovalenten Hormone-Makromolekhl-Komplexen. Helv. Chim. Acta 60:2895-2910. Eisenbach, L., Bar-Haim, E., and El-Shami, K. (2000). Antitumor vaccination using peptide based vaccines. Immunol. Lett. 74(l):27-34.
363
Elton, T. S., Dion, L. D., Host, K. L., Oparil, S., and Blalock, J. E. (1988a). Purification of an angiotensin II binding protein by using antibodies to a peptide encoded by angiotensin II complementary RNA. Proc. Natl. Acad. Sci. USA 85:2518-2522. Elton, T. S., Oparil, S., and Blalock, J. E. (1988b). The use of complementary peptides in the purification of an angiotensin II binding proteins. J. Hypertension 6:S404-S407. Emmett, J., ed. (1990). Comprehensive Medicinal Chemistry, Vol. 3, Oxford, Pergamon Press. Escher, E., and Guillemette, G. (1978). Photoaffinity labeling of the angiotensin II receptor. 3. Receptor inactivation with photolabile hormone analogue. J. Med. Chem. 22:1047-1050. Escher, E., and Schwyzer, R. (1974). /?-Nitrophenylalanine, /?-azidophenylalanine, m-azidophenylalanine, and o-nitro-p-azido-phenylalanine as photoaffinity labels. FEES Lett. 46:347-350. Escher, E. H. F., Robert, H., and Guillemette, G. (1979). 4-Azidoaniline, a versatile protein and peptide modifying agent for photo affinity labeling. Helv. Chim. Acta 62:1217-1222. Escher, E., Laczko, E., Guillemette, G., and Regoli, D. (1981). Biological activities of photoaffinity labeling analogues of kinins and their irreversible effects on kinin receptors. J. Med. Chem. 24:1409-1413. Fanning, D. W., Smith, J. A., Rose, G. D. (1986). Molecular cartography of globular proteins with application to antigenic sites. Biopolymers 25:863882. Farlie, D. P., Abbenante, G., and March, D. R. (1995). Macrocyclic peptidomimetics-forcing peptides into bioactive conformations. Curr. Med. Chem. 2:654-686. Fassina, G., Roller, P. P., Olson, A. D., Thorgeirsson, S. S., and Omichinski, J. G. (1989a). Recognition properties of peptides hydropathically complementary to residues 356-375 of the c-raf protein. J. Biol. Chem. 264:11252-11257. Fassina, G., Zamai, M., Brigham-Burke, M., and Chaiken, L M. (1989b). Recognition properties of antisense peptides to Arg8-vasopressin/bovine neurophysin II biosynthetic precursor sequence. Biochemistry 28:88118818. Feng, Y., Wang, Z., Jin, S., and Burgess, K. (1998). SNAr cyclizations to form cyclic peptidomimetics of ^-turns. J. Am. Chem. Soc. 120:10768-10769. Ferrier, B. M., Jarvis, D., and du Vigneaud, V. (1965). Deamino-oxytocin. Its isolation by partition chromatography on sephadex and crystallization from water and its biological activities. J. Biol. Chem. 240:4264-4266. Filatova, M. P., Kri, N. A., Komarova, O. M., Orfkchovich, V. N., Reiss, S., Liepinya, I. T., and Nikiforovich, G. V. (1986). Synthesis and studies of conformationally restricted analogues of peptide inhibitors of the angiotensin-converting enzyme. Bioorg. Khim. 12:59-70. Finnegan, A., Smith, M. A., Smith, J. A., Berzofsky, J., Sachs, D. H., and Hades, R. J. (1986). The T-cell repertoire for recognition of a phylogenetically distant protein antigen: Peptide specificity and MHC restriction of staphylococcal nuclease-specific T cell clones. J. Exp. Med. 164:897-903.
364
Synthetic Peptides
Fleet, G. W. J., Porter, R. R., and Knowles, J. R. (1969). Affinity labeling of antibodies with aryl nitrene as reactive group. Nature 224:511-512. Flippen-Anderson, J, L., Hruby, V. J., Collins, N., George, C., and Cudney, B. (1994). X-ray structure of [D-Pen2,D-Pen5]enkephalin, A highly potent, delta opioid receptor selective compound: Comparisons with proposed solution conformations. J. Am. Chem. Soc. 116:7523-7531. Fodor, S. P., Read J. L., Pirrung, U. C., Stryer, 1., Lu, A. T., Salas, D. (1991). Light directed, spatially addressable parallel chemical synthesis. Science 251:767-771. Fournie-Zaluski, M.-C., Gacel, G. W., Maigret, B., Premilat, S., and Roques, B. P. (1981). Structural requirements for specific recognition of a or ft opiate receptors. Mol. Pharmacol. 20:484-491. Freidinger, R. M., and Veber D. F. (1984). Design of novel cyclic hexapeptide somatostatin analogs from a model of the bioactive conformation. In Confor nationally Directed Drug Design: Peptides and Nucleic Acids as Templates and Targets, J. A. Vida and M. Gordon, eds., ACS Symposium Series 251, Washington, D.C., American Chemical Society. Freidinger, R. M., Colton, C. D., Randall, W. C., Pitzenberger, S. M., Veber, D. F., Saperstein, R., Brady, E. J., and Arison, B. H. (1985). A cyclic hexapeptide LH-RH antagonist. In Peptides: Structure and Function, C. M. Deber, V. J. Hruby, and K. D. Kopple, eds. Rockford, 111., Pierce Chemical Co. Furka, A., Sebestyen, F., Asgedom, M., and Debo, G. (1991). General method for rapid synthesis of multicomponent peptide mixtures. Int. J. Peptide Protein Res. 37:487^193. Gacel, G. W., Fournie-Zaluski, M.-C., and Roques, B. P. (1980). Tyr-o-SerGly-Phe-Leu-Thr, a highly preferential ligand for S opiate receptors. FEBS Lett. 18(2):245-147. Gacel, G. W., Dauge, V., Breuze, P., Delay-Goyet, P., and Roques, B. P. (1988b). Development of conformationally constrained linear peptides exhibiting a high affinity and pronounced selectivity for 8 opioid receptors. J. Med. Chem. 31:1891-1897. Gacel, G. W., Zajac, J. M., Delay-Goyet, P., Dauge, V., and Roques, B. P. (1988a). Investigation of the structural parameters involved in the fj, and S opioid receptor discrimination of linear enkephalin-related peptides. J. Med. Chem. 31:374-383. Galardy, R. E., Craig, L. C., Jamieson, J. D., and Printz, M. P. (1974). Photoaffinity labeling of peptide hormone binding sites. J. Biol. Chem. 249:3510-3518. Geyer, A., Muller, G., and Kessler, H. (1994). Conformational analysis of a cyclic RGD peptide containing a PSI[CH2-NH] bond: A positional shift in backbone structure caused by a single dipeptide mimetic. J. Am. Chem. Soc. 116:7735-7743. Geysen, H. M., Meloen, R. H., and Bartelling S. J. (1984). Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci. USA 81:3998^002. Geysen, H. M., Rodda, S. J., and Mason, T. J. (1986). A priori delineation of a peptide which mimics a discontinuous antigenic determinant. Mol. Immunol. 23:709-715.
Gierasch, L. M., Deber, C. M., Madison, V., Niu, C. H., and Blout, E. R. (1981). Conformations of (X-L-Pro-Y)2 cyclic hexapeptides. Prefered fiturn conformers and implications for /Rurns in proteins. Biochemistry 20:4730-4736. Gomara, M. J., Riedemann, S., Vega, I., Ibarra, H., Ercilla, G., and Haro, I. (2000). Use of linear and multiple antigenic peptides in the immunodiagnosis of acute Hepatitis A virus infection. J. Immunol. Methods 234(l-2):23-34. Goodman, M., and Ro, S. (1994). Peptidomimetics for drug design. In Burger's Medicinal Chemistry and Drug Discovery, Vol. I, M. E. Wolff, ed., New York, Wiley-Interscience, pp. 803-861. Goudsmit, J., Debouck, C., Meloen, R. H., Smit, L., Bakker, M., Asher, D. M., Wolff, A. V., Gibbs, C. J., and Gajdusek, D. C. (1988). Human immunodeficiency virus type 1 neutralization epitope with conserved architecture elicits early type-specific antibodies in experimentally infected chimpanzees. Proc. Natl. Acad. Sci. USA 85:4478-4482. Gores, T. J., Gottschall, P. E., Coy, D. H., and Arimura, A. (1986). Possible recognition of the GnRH receptor by an antiserum against a peptide encoded by nucleotide sequence complementary to mRNA of a GnRH precursor peptide. Peptides 7:1137-1145. Granthan, R., Gautier, C., and Gouy, M. (1980). Codon frequencies in 119 individual genes confirm consistent choices of degenerate bases according to genome type. Nucleic Acids Res. 8:1893-1912. Grieco, P., Balse, P. M., Weinberg, D., MacNeil, T., and Hruby, V. J. (2000). D-Amino acid scan of y-melanocyte-stimulating hormone: Importance of Trp8 on human MC3 receptor selectivity. J. Med. Chem. 43:4998-5002. Guillemette, G., Boulay, G., Gaynon, S., Bosse, R., and Escher, E. (1989). The peptide encoded by angiotensin II complementary RNA does not interfere with antiotensin II action. Biochem. J. 261:309. Guire, P., Fligen, D., and Hodgson, J. (1977). Photochemical coupling of enzymes to mammalian cells. Pharmacol. Res. Commun. 9:131-141. Hartfield, D., and Rice, M. (1986). Amino acyl-/RNA (anticodon): Codon adaptation in human and rabbit reticulocytes. Biochem. Int. 13:835-842. Hartfield, D., Mathew, C. R., and Rice, M. (1979). Aminoacyl-transfer RNA populations in mammalian cells: Chromatographic profiles and patterns of codon recognition. Biochim. Biophys. Acta 564:414-423. Hawiger, J. (1995). Platelets in thrombosis and rethrombosis. In Molecular Cardiovascular Medicine, E. Haber, ed., New York, Scientific American, Inc., pp. 157-175. Henkin, J. (1977). Photolabeling reagents for thiol enzymes. Studies on rabbit muscle creatine kinase. J. Biol. Chem. 252:4293-4297. Hill, P. S., Smith, D. D., Slaninova, J., and Hruby, V. J. (1990). Bicyclization of a weak oxytocin agonist produces a highly potent oxytocin antagonist. J. Am. Chem. Soc. 112:3110-3113. Hinds, M. G., Welsh, J. H., Biennoud, D. M., Fisher, J., Glennie, M. G., Richards. N. J. R., Turner, D. L., and Robinson, J. A. (1991). Synthesis, conformational properties and antibody recognition of peptides containing /6-turn mimetics based on a-alkyl proline derivatives. J. Med. Chem. 34:1777-1789.
Synthetic Peptides
rschmann, R. (1991). Medicinal chemistry in the golden age of biology Lessons from steroid and peptide research. Angew. Chem. Int. Ed. Engl. 30:1278-1301. xson, S. H., and Hixson, S. S. (1975). /?-Azidophenacyl bromide, a versatile photolabile bifunctional reagent. Reaction with glyceraldehyde-3-phosphate dehyrogenase. Biochemistry 14:4251^254. opp, T. P. (1986). Protein surface analysis. Methods for identifying antigenic determinants and other interaction sites. J. Immunol. Methods 88:1-10. opp, T. P., and Woods, K. R. (1981). Prediction of protein antigenic determinants from amino acid sequences. Proc. Natl. Acad. Sci. USA 78:3824-3839. oughten, R. A., Pinilla, C., Blondelle, S. E., Appel, J. R., Dooley, C. T., and Cuervo, J. H. (1991). Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery. Nature 354:84-86. uby, V. J. (198la). Structure and conformation related to the activity of peptide hormones. In Perspectives in Peptide Chemistry, A. Eberle, R. Geiger and T. Wieland, eds., Basel, S. Karger. uby, V. J. (1981b). Relation of conformation to biological activity in oxytocin, vasopressin, and their analogues. In Topics in Molecular Pharmacology, Vol. 1, A.S.V. Burgen and G.C.K. Roberts, eds., Amsterdam, Elsevier/North-Holland Biomedical Press. ruby, V. J. (1982). Conformational restrictions of biologically active peptides via amino acid side chain groups. Life Sciences 31:189-199. ruby, V. J. (1996). Synthesis of peptide libraries for lead structure screening. In The Practice of Medicinal Chemistry, C. G. Wermuth, ed., London, Academic Press, pp. 135-151. uby, V. J., and Balse, P. M. (2000). Conformational and topographical considerations in designing agonist peptidomimetics from peptide leads. Curr. Med. Chem. 7:945-970. ruby, V. J., and Boteju, L. W. (1997). Design of conformationally constrained peptides and mimics. In Molecular Biology and Technology, R. A. Meyers, ed., New York, VCH Publishers Inc., pp. 371-382. ruby, V. J., and Smith, C. W. (1987). Structure-function studies of neurohypophyseal hormones. In The Peptides: Analysis, Synthesis, Biology, Vol. 8: Chemistry, Biology and Medicine of Neurohypophyseal Hormones and Their Analogs, C. W. Smith, ed., New York, Academic Press, pp. 77207. ruby, V. J., Sawyer, T. K., Yang, Y. C. S., Bregman, M. D., Hadley, M. E., and Heward C. B. (1980). Synthesis and structure-function studies of melanocyte stimulating hormone (MSH) analogues modified in the 2 and 4(7) position: Comparison of activities on frog skin melanophores and melanoma adenylate cyclase. J. Med. Chem. 23:1432-1437. uby, V. J., Kao, L.-F., Pettitt, B. M., and Karplus, M. (1988). The conformational properties of the delta opioid peptide [D-Pen2, D-Pen5]-enkephalin in aqueous solution determined by NMR and energy minimization calculations. J. Am. Chem. Soc. 110:3351-3359. uby, V. J., Al-Obeidi, F., and Kazmierski, W. (1990a). Emerging approaches in the molecular design of receptor selective peptide ligands:
367
Conformational, topographical and dynamic considerations. Biochem. J. 268:249-262. Hruby, V. J., Fang, S. Knapp, R., Kazmierski, W. M., Lui, G. K., and Yamamura, H. I. (1990b). Cholecystokinin analogues with high affinity and selectivity for brain membrane receptors. Int. J. Peptide Protein Res. 35:566-573. Hruby, V. J., Kazmierski, W., Kawasaki, A. M., and Matsunaga, T. (1991a). Synthetic peptide chemistry and the design of peptide based drugs. In Peptide Pharmaceuticals: Approaches to the Design of Novel Drugs, D. J. Ward, ed., London, Open University Press, pp. 135-184. Hruby, V. J., Toth, G., Gehrig, C. A., Kao, L.-F., Knapp, R., Lui, G. K., Yamamura, H. I., Kramer, T. H., Davis, P, and Burks T. F. (1991b). Topographically designed analogues of [o-Pen2,D-Pen5]enkephalin. J. Med. Chem. 34:1823-1830. Hruby, V. J., Li, G., Haskell-Luevano, C., and Shenderovich, M. D. (1997). Design of peptides, proteins and peptidomimetics in chi space. Biopolymers (Peptide Sci.) 43:219-266. Huang, C. K., and Richards, F. M. (1977). Reaction of a lipid soluble, unsymmetrical, cleavable cross-linking reagent with muscle aldolase and erythrocyte membrane protein. J. Biol. Chem. 252:5514-5521. Hughes, J., Kosterlitz, H. W., and Leslie, F. M. (1975). Effect of morphine on adrenergic transmission in the mouse vas deferens. Assessment of agonist and antagonist potencies of narcotic analgesics. Br. J. Pharmacol. 53:371381. Jaffe, C. L., Lis, H., and Sharon, N. (1979). Identification of peanut agglutinin receptors on human erythrocyte ghosts by affinity crosslinking using a cleavable heterobifunctional reagent. Biochem. Biophys. Res. Commun. 91:402-109. Jaffe, C. L., Lis, H., and Sharon, N. (1980). New cleavable photoreactive heterobifunctional crosslinking reagent for studying membrane organization. Biochemistry 19:4423^429. Ji, T. H. (1977). A novel approach to the identification of surface receptors. The use of photosensitive hetero-bifunctional cross-linking reagent. J. Biol. Chem. 252:1566-1570. Ji, T. H. (1979). The application of chemical crosslinking for studies on cell membranes and the identification of surface receptors. Biochem. Biophys. Acta 559:39-69. Ji, T. H., and Ji, I. (1982). Macromolecular photoaffinity labeling with radioactive photoactivable heterobifunctional reagents. Anal. Biochem. 121: 286-289. Jones, D. S. (1972). Polypeptides. Part XIII. Peptides related to the C-terminal tertapeptide sequence of gastrins by complementary reading of the genetic message. J. Chem. Soc. Perkin Trans. 1:1407-1415. Jost, K. (1977). Handbook of Neurohypophyseal Hormone Analogues, Vol. 1, Part 2, K. Jost, M. Lebl, and F. Brtnik, eds. Boca Raton, Fla., CRC Press. Kahn, M. (1993). Peptide secondary structure mimetics: Recent advances and future challenges. SYNLETT 11:821-826. Karplus, P. A., and Schulz, G. E. (1985). Prediction of chain flexibility in proteins. Naturwissenschaften 72:212-222.
368
Synthetic Peptides
Kaurov, O. A., Martynov, V. F., Milhailov, Y. D., and Auna, Z. P. (1972). Synthesis of the new analogues of oxytocin, modified in position 2 [in Russian]. Zh. Obshch. Khim. 42:1654. Kawasaki, A. M., Knapp, R. J., Kramer, T. H., Wire, W. S., Vasquez, O. S., Yamamura, H. I., Burks, F., and Hruby, V. J. (1990). Design and synthesis of highly potent and selective cyclic dynorphin A analogues. J. Med. Chem. 33:1874-1879. Kayser, S. (1983). Thrombosis. In Applied Therapeutics: The Clinical Use of Drugs, B. S. Katcher, L. Y. Young, and M. A. Koda-Kimble, eds., San Francisco, Applied Therapeutics Inc., pp. 333-360. Kazmierski, W. M., and Hruby, V. J. (1988). A new approach to receptor ligand design: synthesis and conformation of a new class of potent and highly selective ^ opioid antagonists utilizing tetrahydroisoquinoline carboxylic acid. Tetrahedron 44:697-710. Kazmierski, W. M., Wire, W. S., Lui, G. K., Knapp, R. J., Shook, J. E., Burks, T. F., Yamamura H. I., and Hruby, V. J. (1988). Design and synthesis of somatostatin analogues with topographical properties that lead to highly potent and specific \JL opioid receptor antagonists with greatly reduced binding at somatostatin receptors. J. Med. Chem. 31:2170-2177. Kessler, H. (1982). Conformation and biological activity of cyclic peptides. Angew. Chem. Int. Ed. Engl. 21:512-523. Kitagawa, T., and Aikawa, T. (1976). Enzyme coupled immunoassay of insulin using a novel coupling reagent. J. Biochem. 79:233-236. Kosterlitz, H. W., Lord, J. A. H, Paterson, S. J., and Waterfield, A. A. (1980). Effects of changes in the structure of enkephalins and of narcotic analgesic drugs on their interactions with \L and 8 receptors. Br. J. Pharmacol. 68:333-342. Krstenansky, J. L., and Mao, S. J. T. (1987). Antithrombin properites of Cterminus of hirudin using synthetic unsulfated Ara-acetyl-hirudin45_65. FEBSLett. 211(1): 10-16. Kruszynski, M., Lammek, B., Manning, M., Seto, J., Haldar, J., and Sawyer, W. H. (1980). [l-(/J-Mercapto-/3, /J-cyclopentamethylenepropanoic acid), 2(O-methyl)tyrosine]arginine-vasopressin and [ 1 -(/3-mercapto-/J,j8-cyclopentamethylenepropanoic acid)]arginine-vasopressin, two highly potent antagonists of the vasopressor response to arginine-vasopressin. J. Med. Chem. 23:364-368. Kyte, J., and Doolittle, R. F. (1982). A simple method for displaying hydropatic character of a protein. J. Mol. Biol. 157:105-132. Lam, K. S., Salmon, S. E., Hersh, E. M. Hruby, V. J., Kazmierski, W. M., and Knapp, R. J. (1991). A new type of synthetic peptide library for identifying ligand-binding activities. Nature 354:82-84. Lam, K. S., Salmon, S. E., Hersh, E. M., Al-Obeidi, F., and Hruby, V. J. (1992). The selectide process: Rapid generation of large synthetic peptide libraries linked to identification and structure determination of acceptor, binding ligands. In Peptides: Chemistry and Biology, J. A. Smith and J. E. Rivier, eds., Leiden, Escom, pp. 492-495. Lebl, M., Barth, T., Servitova, L., Slaninova, J., and Jost, K. (1985). Oxytocin analogues with inhibitory properties, containing in position 2 A hydro-
369
phobic amino acid of o-configuration. Collec. Czech. Chem. Commun. 50:132-145. Leslie, F. M., Chavkin, C., and Cox, B.M (1980). Opioid binding properties of brain and periperal tissues: Evidence for heterogeneity in opioid ligand binding sites. J. Pharm. Exp. Ther. 214:395-402. Levy, D. (1973). Preparation of photo-affinity probes for the insulin receptor site in adipose and liver cell membranes. Biochem. Biophys. Acta 322:329-336. Liao, M., Lu, Y., Xiao, Y., Dierich, M. P., and Chen, Y.-H. (2000). Induction of high level of specific antibody response to the neutralizaing epitope ELDKWA on HIV-1 gp41 by peptide-vaccine. Peptides (N.Y.) 21(4):463468. Liao, S., Shenderovich, M. D., Zhang, Z., Maletinska, L., Slaninova, J., and Hruby, V. J. (1998) Substitution of the side-chain constrained amino acids /3-methyl-2',6'-dimethyl-4'-methoxytyrosine in position 2 of a bicyclic oxytocin analogue provides unique insights into the bioactive topography of oxytocin antagonists. J. Am. Chem. Soc. 120:7393-7394. Lin, Y., Trivedi, D., Siegel, M., and Hruby, V. J. (1992). Conformationally constrained glucagon analogues: New evidence for the conformational features important to glucagon-receptor interactions. In Peptides: Chemistry and Biology. J. A. Smith and J. E. Rivier, eds., Leiden, Escom, pp. 439-440. Lord, I. A. H., Waterfield, A. A., Hughes, J., and Kosterlitz, H. W. (1977). Endogenous opioid peptides: multiple agonists and receptors. Nature 267:495^99. Lowbridge, J., Manning, M., Seto, J., Haldar, J., and Sawyer, W. H. (1979). Synthetic Antagonists of in vivo responses by the rat uterus to oxytocin. J. Med. Chem. 22:565-569. Ma, S., and Spatola, A. F. (1993). Conformations of ^[CH2NH]pseudopeptide-cyclo[Gly-Pro^[CH2NH]Gly-D-Phe-Pro]-TFA and cyclo[Gly-Pro^]CH2NH]Gly-D-Phe-Pro]. Int. J. Peptide Protein Res. 41:204-206. Mann, K. G. (1987). The assembly of blood clotting complexes on membranes. Trends Biochem. Sci. 12:229-233. Mao, S. J. T., Yates, M. T., Owen, T. J., and Krstenansky, J. L. (1988). Interaction of hirudin with thrombin: Identification of a minimal binding domain of hirudin that inhibits clotting activity. Biochemistry 27:81708173. Maraganore, J. M. (1993b). Thrombotic process and emerging drugs for its control. Tex. Heart Inst. J. 20:43^7. Marshall, G. R. (1993). A hierarchical approach to peptidomimetic design. Tetrahedron 49:3547-3558. Martin, W. R., Eades, C. G., Thompson, J. A., Huppler, R. E., and Gilbert, P. E. (1976). The effects of morphine and nalorphine-like drugs in the nondependent and morphine-dependent chronic spinal dog. J. Pharmacol. Exp. Ther. 197:517-532. Martin, F. J., and Papahadjopoulos, D. (1982). Irreversible coupling of immunoglobulin fragments to preformed vesicles. An improved method for liposome targeting. J. Biol. Chem. 257:286-288.
370
Synthetic Peptides
Matsunaga, T. O., de Lauro Castrucci, A. M., Hadley, M. E., and Hruby, V. J. (1989). Melanin concentrating hormone (MCH): Synthesis and bioactivity studies of MCH fragment analogues. Peptides 10:349-354. McRobbie, M., Meth-Cohn, O., and Suschitzky, H. (1976a). Competitive cyclizations of singlet and triplet nitrenes. Part I. Cyclization of l-(2nitrophenyl)pyrazoles. Tetrahedron Lett. 925-928. McRobbie, M., Meth-Cohn, O., and Suschitzky, H. (1976b). Competitive cyclizations of singlet and triplet nitrenes. Part II. Cyclization of 2-nitrophenyl-, thiophens-, benzo thiazoles-, and benzimidazoles. Tetrahedron Lett. 929-932. Mekler, L. B. (1969). On specific selective interactions between amino acid residues of poly peptide chain. Biophys. USSR (Engl. Trans.) 14: 613-617. Melin, P., Vilhardt, H., and Akerlund, M. (1983). Uteronic oxytocin and vasopressin antagonists with minimal structure modifications. In Peptides: Structure and Function, V. J. Hruby and D. H. Rich, eds., Rockford, 111., Pierce Chemical Co. Meraldi, J. P., Yamamoto, D., Hruby, V. J., and Brewster, A. I. R. (1975). Conformational studies of neurohypophyseal hormones and inhibitors using high resolution NMR spectroscopy. In Peptides: Chemistry, Structure and Biology. R. Walter and J. Meinhofer, eds., Ann Arbor, Mich., Ann Arbor Sci. Publ., pp. 803-814. Meraldi, J. P., Hruby, V. J., and Brewster, A. I. R. (1977). Relative conformational rigidity in oxytocin and [l-penacillamine]-oxytocin: A proposal for the relationship of conformational flexibility to peptide hormone agonism and antagonism. Proc. Natl. Acad. Sci. USA 74:1373-1377. Merrifield, R. B. (1963). Solid phase peptide synthesis. I. The synthesis of a tetrapeptide J. Am. Chem. Soc. 85:2149-2152. Moreland, R. B., Smith, P. K., Fujimoto, E. K., and Dockter, M. E. (1982). Synthesis and characterisation of 7V-[4-azido phenylthio] phaliimidate: a cleavable photoactivable crosslinking reagent that reacts with sulfhydryl groups. Anal. Biochem. 121:321-326. Morgan, B. A., Bowers, J. D., Guest, K. P., Handa, B. K., Metcalf, G., and Smith, C. F. C. (1977). Structure-activity relationships of enkephalin analogues. In Peptides, M. Goodman and J. Meienhofer, eds., New York, Wiley. Mosberg, H. I., Hurst, R., Hruby, V. J., Galligan, J. J., Burks, T. F., Gee, K., and Yamamura, H. I. (1983a). Conformationally constrained cyclic enkephalin analogs with pronounced delta opioid receptor agonist selectivity. Life Sciences 32:2565-2569. Mosberg, H. I., Hurst, R., Hruby, V. J., Gee, K., Yamamura, H. I., Galligan, J. J., and Burks. T. F. (1983b). Bis-penicillamine enkephalins possess highly improved sensitivity toward delta opioid receptors. Proc. Natl. Acad. Sci. USA 80:5871-5874. Mulchahey, J. J., Neill, J. D., Dion, L. D., Bost, K. L., and Blalock, J. E. (1986). Antibodies to the binding site of the receptor for luteinizing hormone-releasing hormone (LHRH): generation with a synthetic decapeptide encoded by an RNA complementary to LHRH mRNA. Proc. Natl. Acad. Sci. USA 83:9714-9718.
371
Muller, S. and Briand, J. P. (1998). On the potential of retro-inverso peptides in vaccine design. Res. Immunol. 149:55-57. Muramoto, K., and Ramachandran, J. (1980). Photoreactive derivative of corticotropin 2. Preparation and characterisation of 2-nitro-4(5)-azido phenylsulfenyl derivatives of corticotropin. Biochemistry 19:3280-3286. Nassal, M. (1983). 4(l-Azi-2,2,2,trifluoroethyl)benzoic acid, a highly photolabile carbene generating label readily fixable to biochemical agents. Liebigs Ann. Chem. 1510-1523. Nikiforovich, G. V., Prakash, O., Gehrig, C. A., and Hruby, V. J. (1993). Solution conformations of the peptide backbone for DPDPE and its fiMe-Phe4-substituted analogs. Int. J. Peptide Protein Res. 41:347-361. Ngo, T. T., Yam, C. F., Lenhoff, H. M., and Ivy, I. I. (1981). pAzidophenylglyoxal. A heterobifunctional photoactivable cross-linking reagent selective for arginyl residues. J. Biol. Chem. 256:11313-11318. Ogasawara, K. (1999). Synthetic peptide vaccines effective in preventing virus infection. Microbiol. Immunol. 43(10):915-923. Omichinski, J. G., Olson, A. D., Thorgeirsson, S. S., and Fassina, G. (1989). Computer assisted design of recognition peptides. In Techniques in Protein Chemistry, T. E. Hugh, ed., San Diego, Calif., Academic Press, pp. 430^38. Palker, T. J., Clark, M. E., Langlois, A. J., Matthews, T. J., Weinhold, K. J., Randall, R. R., Bolognesi, D. P., and Haynes, B. F. (1988). Type-specific neutralization of the human immunodeficiency virus with antibodies to ev-encoded synthetic peptides. Proc. Natl. Acad. Sci. USA 85:19321936. Pelton, J. T., Gulya, K., Hruby, V. J., Duckies, S. P., and Yamamura, H. I. (1985). Conformationally restricted analogues of somatostatin with high //.-opiate receptor specificity. Proc. Natl. Acad. Sci. USA 82:236-239. Pettitt, B. M., Matsunaga, T. O., Al-Obeidi, F., Gehrig, C. A., Hruby, V. J., and Karplus, M. (1991). Dynamic search for bis-penicillamine enkephalin conformations. Biophys. J. 60:1540-1544. Pfaff, E., Mussgay, M., Bohm, H. O., Schulz, G. E., and Scalier, H. (1982). Antibodies against a preselected peptide recognize and neutralize foot and mouth disease virus. EMBO J. 1:869-874. Piercy, M. F., Schroeder, A., Einspahr, F. J., Folkers, K., Xu, J.-C., and Horig, J. (1981). Behavioral evidence that substance P may be a spinal cord nociceptor neurotransmitter. In Peptides: Synthesis-StructureFunction, D. H. Rich and E. Gross, eds., Rockford, 111., Pierce Chemical Co. Pierschbacher, M. D., and Ruoslahti, E. (1987). Influence of stereochemistry of the sequence Arg-Gly-Asp-Xaa on binding specificity in cell adhesion. J. Biol. Chem. 262:17294-17298. Qian, X., Kover, K. E., Shenderovich, M. D., Lou, B-S., Misicka, A., Zalewska, T., Horvath, R., Davis, P., Bilsky, E. J., Porreca, F., Yamamura, H. I. and Hruby, V. J. (1994). Newly discovered stereochemical requirements in side chain conformation of 8 opioid agonists for recognizing opioid 8 receptors. J. Med. Chem. 37:1746-1757. Qian, X., Russell, K. C., Boteju, L. W., and Hruby, V. J. (1995). Stereoselective total synthesis of topographically constrained designer
372
Synthetic Peptides
amino acids: 2', 6'-dimethyl-/3-methyltyrosines. Tetrahedron 51:10331054. Qian, X., Shenderovich, M. D., Kover, K. E., Davis, P., Horvath, R., Zalewska, T., Yamamura, H. I., Porreca F., and Hruby, V. J. (1996). Probing the stereochemical requirements for receptor recognition of 8 opioid agonists through topographic modifications in position 1. J. Am. Chem. Soc. 118:7280-7290. Rasmussen, U. B., and Hesch, R.-D. (1987). On antisense peptides: The parathyroid hormone as an experimental example and a critical theoretical view. Biochem. Biophys. Res. Commun. 149:930-938. Rinke, J., Meinke, M., Brimacombe, R., Fink, G., Rommel, W., and Fasold, H. (1980). The use of azidoarylimidoesters in RNA-protein cross-linking studies with Escherichia coli ribosomes. J. Mol. Biol. 137:301-314. Ripka, W. C., De Lucca, G. V., Bach II, A. C, Pottorf, R. S., and Blaney, J. M. (1993a). Protein /3-turn mimetics I. Design, synthesis, and evaluation in model cyclic peptides. Tetrahedron 49:3593-3608. Ripka, W. C., De Lucca, G. V., Bach II, A. C., Pottorf, R. S., and Blaney, J. M. (1993b). Protein ^-turn mimetics II. Design, synthesis, and evaluation in the cyclic peptide gramicidin S. Tetrahedron 49:3609-3628. Rizo, J., and Gierasch, L. M. (1992). Constrained peptides: Models of bioactive-peptides and protein structures. Annu. Rev. Biochem. 61:387^18. Rodda, S. J., Geysen, H. M., Manson, I.J, and Schaafs, P. G. (1986). The antibody response to myoglobin-I. Systematic synthesis of myoglobin peptides reveals location and substructure of species dependent continuous antigenic determinants. Mol. Immunol. 23:603-610. Rodriguez, M., Lignon, M.-F., Galas, M.-C, and Martinez, J. (1990). Highly selective cyclic cholecystokinin analogs for central receptors. In Peptides: Chemistry, Structure, and Biology. J. E. Rivier and G. R. Marshall, eds., Leiden, Escom. Rothbard, J. B., and Taylor, W. R. (1988). A sequence pattern common to Tcell epitopes. EMBO J. 7:93-101. Rudinger, J., Pliska, V., and Krejci, I. (1972). Oxytocin analogues in the analysis of some phases of hormone action. Rec. Prog. Hormone Res. 28:131-172. Ruoslahti, E. (1991) Integrins. J. Clin. Invest. 87:1-5. Rusche, J. R., Javaherian, K., McDanal, C., Petro, J., Lynn, D. L., Grimaila, R., Langlois, A., Gallo, R. C., Arthur, L. O., Fischinger, P. J., Bolognesi, D. P., Putney, S. D., and Matthews, T. J. (1988). Antibodies that inhibit fusion of HIV infected cells bind a 24 amino acid sequence of the viral envelope, gp!20. Proc. Natl. Acad. Sci. USA 85:3198-3202. Salamon, Z., Cowell, S., Varga, E., Yamamura, H. I., Hruby, V. J., and Tollin, G. (2000). Plasmon resonance studies of agonist/antagonist binding to the human 5-opioid receptor: New structural insights into receptor-ligand interactions. Biophys. J. 79:2463-2474. Sawyer, T. K. (1995). Peptidomimetic design and chemical approaches to peptide metabolism. In Peptide-Based Drug Design: Controlling Transport and Metabolism, M. D. Taylor and G. L. Amidon, eds., Washington, D.C., American Chemical Society, pp. 387^22. Sawyer, T. K. (1977). Peptidomimetic and nonpeptide drug discovery: Impact of structure-based drug design. In Structural-Based Drug Design:
373
Diseases, Targets, Techniques and Developments, P. Veerapandian, ed., New York, Marcel Dekker, pp. 559-634. Sawyer, T. K., Sanfilippo, P. J. Hruby, V. J., Engel, M. H., Heward, C. B., Burnett, C. B., and Hadley, M. E. (1980). [Nle4, o-Phe7]-a-Melanocyte stimulating hormone: A highly potent a-melanotropin with ultralong biological activity. Proc. Natl. Acad. Sci. USA 77:5754-5758. Sawyer, T. K., Hruby, V. J., Darman, P. S., and Hadley, M. E. (1982). [halfCys4, half-Cys10]-o;-melanocyte stimulating hormone: A cyclic cc-melanotropin exhibiting superagonist biological activity. Proc. Natl. Acad. Sci. USA 79:1751-1755. Scarborough, R. M., Naughton, M. A., Teng, W., Rose, J. W., Philips, D. R., Nannizzi, L., Arfsten, A., Campbell, A. M., and Charo, I. F. (1993). Design of potent and specific integrin antagonists. J. Biol. Chem. 268(2): 1066-1073. Schiller, P. W., Eggimann, B., DiMaio, J., Lemieux, C., and Nguyen, T. M.-D. (1981). Cyclic enkephalin analogs containing a cystine bridge. Biochem. Biophys. Res. Commun. 101:337-343. Schiller, P. W., Nguyen, T. M.-D., Maziak, L. A., and Lemieux C. (1985). Novel cyclic opioid peptide analog showing high preference for /z-receptors. Biochem. Biophys. Res. Commun. 127:558-564. Schneider, J. P. and Kelly, J. W. (1995). Templates that induce a-helical, /3-sheet, and loop conformations. Chem. Rev. 95:2169-2187. Schulz, H., and du Vigneaud, V. (1966). Synthesis of 1-L-penacillamineoxytocin, 1-D-penacillamine-oxytocin, and 1 -deamino-penacillamine-oxytocin, potent inhibitors of oxytocic response to oxytocin. J. Med. Chem. 9:647-650. Schwartz, I., and Ofengand, J. (1974). Photoaffmity labeling of tRNA binding site in macromolecules. I. Linking of the phenacyl-p-azide of 4-thiouridine in (Escherichia coli) valyWRNA to 16S RNA at the ribosomal P site. Proc. Natl. Acad. Sci. USA 71:3951-3955. Schwyzer, R. (1977). ATCH: A short introductory review. Ann. N.Y. Acad. Sci. 297:3-26. Scott, J. K., and Smith, G. P. (1990). Searching for peptide ligands with an epitope library. Science 249:386-390. Seela, F., and Rosemeyer, H. (1977). 5-Azido-x-bromo-2-nitroacetophenone. A cross-linking reagent with groups of selective reactivity. Hoppe-Seyler's Z. Physiol. Chem. 358:129-131. Sette, A., Doria, G., and Adauin, L. (1986). A microcomputer program for hydrophilicity and amphipathicity analysis of protein antigens. Mol. Immunol. 23:807-815. Shai, Y., Flashner, M., and Chaiken, I. M. (1987). Anti-sense peptide recognition of sense peptide: Direct quantitative characterization with the ribonuclease S-peptide system using analytical high-performance affinity chromatography. Biochemistry 26:669-675. Shai, Y., Brunck, T. K., and Chaiden, I. M. (1989). Antisense peptide recognition of sense peptides: Sequence simplification and evaluation of forces underlying the interaction. Biochemistry 28:8804-8811. Sharma, S. D., Nikiforovich, G. V., Jiang, J., de Lauro Castrucci, A.-M., Hadley, M. E., and Hruby, V. J. (1993). A new class of positively charged
374
Synthetic Peptides
melanotropin analogs: A new concept in peptide design. In Peptides 1992, C. H. Schneider and A. N. Eberle, eds., Leiden, Escom, pp. 95-96. Shenderovich, M. D., Liao, S., Qian, X., and Hruby, V. J. 2000. A three dimensional model of the 8 opioid pharmacophore: Comparative molecular modeling of peptide and non-peptide ligands. Biopolymers 53:565580. Siemion, I. Z., Szewezuk, Z., Herman, Z. S., and Stachura, Z. (1980). To the problem of the biologically active conformation of enkephalin. Mol. Cell. Biochem. 34:23-29. Sigrist, H., and Zahler, P. (1980). Heterobifunctional crosslinking of bacteriorhodopsin by azidophenylisothiocanate. FEES Lett. 113:307-311. Singh, A., Thornton, E. R., and Westheimer, F. H. (1962). The photolysis of diazoacetyl chymotrypsin. J. Biol. Chem. 237:PC3006-PC3008. Slomczynska, U., Chalmers, D. K., Cornille, F., Smythe, M. L., Beusen, D. D., Moeller, K. D., and Marshall, G. R. (1996). Electrochemical cyclization of dipeptides to form novel bicyclic, reverse-turn peptidomimetics. 2. Synthesis and conformational analysis of 6,5-bicyclic systems. J. Org. Chem. 61:1198-1204. Smith, J. A. (1989). Synthetic peptides: Tools for elucidating mechanism of protein antigen processing and presentation. In Synthetic Peptides: Approaches to Biological Problems, J. P. Tarn and E. T. Kaiser, eds., New York, Alan R. Liss, pp. 31-42. Smith, J. A., Hunnell J. G. R., and Leach, S. J. (1977). A novel method for delineating antigenic determinants: Peptide synthesis and radioimmunoassay using the same solid support. Immunochemistry 14:565572. Smith, L. R., Bost, K. L., and Blalock, J. E. (1987). Generation of idiotypic and anti-idiotypic antibodies by immunnization with peptides encoded by complementary RNA: A possible molecular basis for the network theory. J. Immunol. 138:7-9. Smith, R. A. G., and Knowles, J. R. (1974). The utility of photoaffmity labels as mapping reagents. Biochem. J. 141:51-56. Sonder, S. A., and Fenton, J. W. (1984). Proflavin binding within the fibrinopeptide groove adjacent to the catalytic site of human a-thrombin. Biochemistry 23:1818. Stone, S. R., and Hofsteenge, J. (1986). Kinetics of the inhibition of thrombin by hirudin. Biochemistry 25:4622. Strand, F. L. (1999). Neuropeptides: Regulators of Physiological Processes, Cambridge, Mass., MIT Press. Struthers, R. S., Rivier, J., and Hagler, A. (1984). Molecular dynamics and minimum energy of GnRH and analogs: A methodology for computeraided drug design. Ann. N.Y. Acad. Sci. 439:81-96. Stryer, L. (1975). Biochemistry, San Francisco, W. H. Freeman & Co. Takahashi, H., Merli, S., Putney, S. D., Houghten, R., Moss, B., Germain, R. N., and Berzofsky, J. A. (1989). A single amino acid interchange yields reciprocal CTL specificities for HIV gp!60. Science 246:118-121. Thornton, J. M., Edward, M. S., Taylor, W. R., and Barlow, D. J. (1986). Location of "continuous" antigenic determinants in the protruding regions of proteins. EMBO J. 5:409-415.
375
Toniolo, C. (1990). Conformationally restricted peptides through short-range cyclizations. Int. J. Peptide Protein Res. 35:287-300. Torchiana, M. L., Cook, P. G., Weise, S. R., Saperstein, R., and Veber, D. F. (1978). Subcutaneous administration of somatostatin analogs as a major factor in the enhancement of drug duration of action. Arch. Int. Pharmacol. Ther. 235:170-176. Toth, G., Russell, K. C., Landis, G., Kramer, T. H., Fang, L., Knapp, R., Davis, P., Burks, T. F., Yamamura, H. L, and Hruby, V. J. (1992). Ring substituted and other conformationally constrained tyrosine analogues of [D-Pen2,o-Pen5]enkephalin with 8 opioid receptor selectivity. J. Med. Chem. 35:2384-2391. Trivedi, D., Lin, Y., Ahn, J-M., Siegel, M., Mollova, N. N., Schram, K. H., and Hruby, V. J. (2000). Design and synthesis of conformationally constrained glucagon analogues. J. Med. Chem. 43:1714-1722. Trommer, W. E., and Hendrick, M. (1973). The formation of maleimides by a new mild cyclization process. Synthesis 484-^485. Turro, N. J. (1965). Molecular Photochemistry, New York, Benjamin. Turro, N. J. (1980). Structure and dynamics of important reactive intermediates involved in photobiological systems. Ann. N.Y. Acad. Sci. 346:1-17. Unanue, E. R., and Allen, P. M. (1987). The basis for the immunoregulatory role of macrophages and other accesory cells. Nature 236:551-557. van Regenmortel, M. H. V., Briand, J. P., Muller, S., and Plause, S. (1988). Synthetic polypeptides as antigens. In Laboratory Techniques in Biochemistry and Molecular Biology, R. H. Burden and P. H. von Knippenberg, eds., Amsterdam, Elsevier. Vanin, E. F., and Ji, T. H. (1981). Synthesis and applications of cleavable photoactivable heterbifunctional reagents. Biochemistry 20:6754-6760. Vanin, E. F., Burkhard, S. J., and Kaiser, I. I. (1981). ^-Azidophenylglyoxal: A heterobifunctional photosensitive reagent. FEBS Lett. 124:89-92. Vaughan, R. J., and Westheimer, F. H. (1969). A method for marking the hydrophobic binding sites of enzymes. An insertion into the methyl group of an alanine residue of trypsin. J. Am. Chem. Soc. 91:217-218. Vavrek, R. J., Ferger, M. F., Allen, G. A., Rich, D. H., Blomquist, A. T., and du Vigneaud, V. (1972). Synthesis of three oxytocin analogs related to [1deaminopenacillaminejoxytocin possessing antioxytocic activity. J. Med. Chem. 15:123-126. Veber, D. F., Freidinger, R. M., Prelow, D. S., Poleveda, W. J., Holly, F. W., Strachan, R. G., Nutt, R. F., Arison, B. H., Homnick, C., Randall, W. C., Glitzer, M. S., Saperstein, R., and Hirchmann, R. (1981). A potent cyclic hexapeptide of somatostatin. Nature (London) 292:55-57. Virgilio, A. A., and Ellman, J. A. (1994). Simultaneous solid-phase synthesis of /}-turn mimetics incorporating side-chain functionality. J. Am. Chem. Soc. 116:11580-11581. Virgilio, A. A., Schurer, S. C., and Ellman, J. A. (1997a). Expedient solidphase synthesis of putative /?-turn mimetics incorporating the i+\,i + 2, and i + 3 sidechains. Tetrahedron Lett. 37:6961-6964. Virgilio, A. A., Bray, A. A., Zhang, W., Trinh, L., Snyder, M., Morrissey, M. M., and Ellman, J. A. (1997b). Synthesis and evaluation of a
376
Synthetic Peptides
library of peptidomimetics based upon the /3-turn. Tetrahedron 53:6635-6644. Walker, B., Wikstrom, P., and Shaw, E. (1985). Evaluation of inhibitor constants and alkylation rates for a series of thrombin affinity labels. Biochem J. 230:245. Walter, R. (1977). Identification of sites in oxytocin involved in uterine receptor recognition and activation. Fed. Proc. 36:1872-1878. Wang, W., Borchardt, R. T., and Wang, B. (2000). Orally active peptidomimetic RGD analogs that are glycoprotein Ilb/IIIa antagonists. Curr. Med. Chem. 7:437^53. Ward, D. J., ed. (1990). Peptide Pharmaceuticals: Approaches to the Design of Novel Drugs, London, Open University Press. Whittle, P. J., and Blundell, T. L. (1994). Protein/structure-based drug design. Ann. Rev. Biophys. Biomol. Struct. 23:349-375. Wiley, R. A., and Rich, D. H. (1993). Peptidomimetics derived from natural products. Med. Res. Rev. 13:327-384. Wunderlin R., Minakakis P., Tun-Kyi, A., Sharma, S. D., and Schwyzer, R. (1985a). Melanotropin receptors. I. Synthesis and biological activity of ^"-(S-bromovaleryO-A^-desacetyl-a-melanotropin. Helv. Chim. Acta 68:1-11. Wunderlin R., Sharma, S. D., Minakakis P., and Schwyzer, R. (1985b). Melanotropin receptors. II. Syntheisis and biological activity of a-melanotropin/tobacco mosaic virus disulfide conjugates. Helv. Chim. Acta 68:12-22. Yip, C. C, Yeung, C. W. T., and Moule, M. L. (1980). Photoaffinity labeling of insulin receptor proteins of liver plasma membrane preparations. Biochemistry 19:70-76. Yoshitake, S., Yamada Y., Ishikawa, E., and Masseyeff, R. (1979). Conjugation of glucose oxidase from Aspergillus niger and rabbit antibodies using TV-hydroxysuccinimide ester of jV-(4-carboxy cyclohexylmethyl)-maleimide. Eur. J. Biochem. 101:395-399.
Index
acetic acid 232 acetic anhydride 226 acetonitrile 232 W-acetyl 50 acetylation 226 acid halides 140 acid-sensitive side chains and bonds 164 acidolysis 119 acids, use in cleavage 148-149 ACTH. See adrenocorticotropic hormone active esters 137-139 activity 49-50 biological 35, 52 antisense peptides 355 argininc vasopressin analogs 69 catalytic 51 conformation-biological studies 300 See also structure-activity relationships acyl carrier protein 11 acyl hydrazide linkage 72 acyl transferases 12 adrenocorticotropic hormone (ACTH) 11,93, 348
affinity labeling of receptors 318-319 aggregation 165-166 agonists, design 49 AIDS 341 alanine 12 alkaline hydrolysis 266 alkane chains 239 5-alkylating reactions 325 alkylation 275 amide bonds 23-24, 31, 72 characteristics 24 formation 301 polarity 24 replacement 71-72 amide geometry 72 amide torsional angle 25 amine-reacting derivatives 326 amino acid(s) 11, 38, 39, 58, 61, 342 Q-alkylated 298 amide nitrogen, reversible protection 166 analysis 57, 222, 223, 262-268, 277 antisense 346-347 chemistry 12 coded 345
377
378
Index
amino acid(s) (continued) equimolar distribution 5960 hydrophilic 345 hydrophobic 345 and î-turn indices 41 internal reference (IRAA) 143 nonprotein 16-23 novel 302 phenylthiohydantoins 143 post-translational modification 15 protein 12-16 replacement 297 residues 26, 40, 227, 255 in helices 55 mass 257 sequence 25, 35 side-chain protection 96, 102-111 specific derivation 323 structures and properties 13, 25 systematic substitution 295 uncharged 345 amino functionalities 326 aminoacyl resin 52 aminoacyl transferases 10 3-(2-aminoethyl) indole 266 aminoisobutyric acid 77 aminomethyl groups 96 aminomethyl-polystyrene resin 128 aminopeptidase 67, 68 degradation 52 p-aminophenylalanine-substituted peptide 329 ammo-terminal glutamine 227 ammonium bicarbonate 232 amphipathic (amphiphilic) helix 34, 36, 46, 74 amphipathic secondary structures 43 amphipathic (amphiphilic) structures 34-35 analogues antigenic peptides 338-340 bombesin 50 enkephalin 299, 301 with modified peptide bonds 71 fragment 296 Hirulog 308 oxytocin 299 somatostatin 297, 299 tyrosine 299
analysis of synthetic product 232-233 See aho specific methods anchoring linkages 97, 99, 122, 129 benzyl ester 151 Androctonus australis 167 angiotensin converting enzyme (ACE) 11, 68,93 angiotensin II 328, 329 anhydrides, preformed symmetrical 139 140 antagonists, design 49 antibodies 49 against antisense peptides 355 binding 52 equilibrium binding to 339 protein-specific 54-55 See also antigens antibody/antigen recognition 343 anticomplementarity 348 antigenic peptides 340 analogues of 338-340 applications of 341-344 sequences 332-334 antigenic uses of synthetic peptides 331-332 antigenicity, prediction of 335-338 antigens 160-161 protein 332 T-cell 334 antisense peptides 344-345 and sense counterpart interactions 352-353 antibodies against 355 as internal images of sense peptides 345-351 biological activity 355 generation of 351-352 potential applications 355-356 antisense-sense peptide interactions 349-350 antithrombotic agents 80 apolipoprotein A 35 applications of antigenic peptides 341-344 of synthetic peptides 292-376 aqueous formic acid 232 arginine 307 arginine vasopressin 10, 68-69
Index
379
analogs, biological activity 69 ArgoGel 122 aryl azide 326-328 aryl diazirine 327 asparagine 12, 265, 266 aspartic acid 39, 227, 267 aspartic acid-X sequences 229-231 aspartic protease 37 ASPECT 122 Aspergillus tereus 307 Association of Biomolecular Resource Facilities (ABRF) 233 ATP 62 32 P-labeled 64 ATZ-amino acid 275 autoimmune diseases 343-345 automated synthesizers 144 automation, of solid-phase peptide synthesis 96, 143-144 p-azido benzoic acid 330 /i-azidophenyl 328 p-azidophenyl glyoxal 331 /)-azidophenylacetic acid 330 ;?-azidophenylalanine (Pap) residue 329 substituted peptides 330 p-azidophenylisothiocyanates 330
BHA-resin 170 Bibapcitide 310 binding affinity 80 binding constants 349-350 biological activity 52 BLAST 37, 38 blocking groups 50 blood clots 79 Boc chemistry 3, 98-100, 113, 151, 162, 170 peptide acids 128 standard Merrifield system 98-99 Boc-His 116 bombesin 49 analogues 50 bonded phase 239-240 bonds, acid-sensitive 164 BOP 115, 135, 136 BOP/DIEA 133 bovardin 301 Bpoc 119 bradykinin 329 bromophenol blue 142 BUBU 14 buffer modulation 240 buffer system 239 buried helices 43
backbone alteration 304 amide linkage (BAL) 134, 160 strategy 170 anchoring 134 structure. See helix, helices torsional angles 26 bead libraries 66 beads labeled 64 substrate-bearing 64 benzyl ester anchoring linkages 151 ,6-sheet 42 potential 40 structure 31, 43 /3-structure 29-30, 40, 48 side chains 35 torsional angles for 31 /3-turns. See turns
C-terminal carbonyl 161 residue 272 thioacid 170 Cahn-Ingold-Prelog convention 16 calcitonin 11, 35, 93 capillary electrophoresis (CE) 222-224, 227, 232-235, 284 capping 163, 226 carbodiimide activation 114 carbodiimide acylations 115 /^-carbon 16 carbon load 239 carboxamide 50 side chains 115 y-carboxyglutamate (Gla) 153 X-carboxyglutamic acid (Gla) 15 carboxypeptidase Y ladder sequencing 260 carboxypeptidases 67
380
Index
cardiovascular agents 309 catalytic activity 51 catalytic efficiency 50 catalytic rate constant 50 cation exchange chromatography 238 CCK-8 301 CCK-A 298, 301 CCK-B 298, 301 C-H bonds 327 OH hydrogens 328 characterization 96, 340 charged moiety 51 chemical-affinity labeling 318-323 chemical tags 66 chemoselective ligation 3, 168-170 chloranil 262 chloroform-acetic acid-NMM 160 cholecystokinin 155 chromatography 66, 171, 233-241, 262 cation exchange 238 chromophores 140 chymotrypsin 68 a-chymotrypsin 328 cis-trans ratio 76 CLEAR 122 cleavage 67-68, 96, 97, 99, 129, 146, 233, 304 alternative strong acids 148-149 C1D 256 common reagents and proteases for 274 hydrogen fluoride 52 nonacidolytic methods 151-152 photolytic 152 TFA 143, 150 cloning methods 312 clotting formation 307 prevention of 310 coagulation 308 collision-induced decomposition (CID) 253-258 cleavage 256 column chromatography 262 combinatorial chemistry 58 combinatorial libraries 58-67 combinatorial techniques 57
complement protein C5a 46-47 complementary peptide 344 computer-assisted deconvolution of electrospray data 252-253 conformation-biological activity studies 300 conformational constraints 73, 80-81, 297, 312 local 76-77 regional 77-79 conformational design 72-76 conformational features 300 CONH peptide bond 72 consensus libraries 62 conservative substitution 39 constraint(s) 297-300, 315 conformational 73, 76-81, 297, 312 cyclic 300-301 geometric 161 topographical 297, 312 continuous elution method 354 controlled-pore silica glass 121 corticotropin releasing factor 35 coupling constants 305 coupling efficiency 261 coupling methods 137 covalent structure 226-227 detailed characterization 243-244 CPK models 316 4-cresol 146 CTOP 312 cyclic j6-turn peptidomimetics, onresin formation of 305 cyclic constraints 300-301 cyclic imide formation 229-230 cyclic peptides 68, 78 cyclization(s) 79-81, 301, 304, 312 electrochemical 306 Cys protecting groups 117, 119 Cys racemization 118, 119 Cys residues 117-119, 161 deprotection/oxidation 158 Cys side chain 118 cysteine 12, 80, 227, 265-266, 298, 299 formation 161 residues 21, 56, 228 cytokines 10
Index
381
DADLE 313 314 DAVP 70 DBU 100, 156 DBU-piperidine 167 DCBC 130 DCC 130, 132, 134, 136 DCC/HOBt 156 DCM. See dichloromethane dDAVP 70 Ddz 119 DEAD 130 deconvolution of spectra 249, 252-253 deletion peptides 163-164, 295 deletion sequences 226, 276, 277 <5-selective opioid receptor peptides 313-318 de novo synthesis of photoaffinity peptides 329 deprotection 233, 304 procedure 228 reactions 148 deprotection/coupling 94, 140 deprotection/oxidation, Cys residues 158 design of synthetic peptides 10-92 based on sequence 49-52 See also synthesis desolvation 18 diazirines 326, 328 or-diazocarbonyl 327 a-diazoketones 326-328 dichloromethane (DCM) 98, 100, 115, 116, 121, 122, 132, 139, 141, 152 DIEA98, 115, 129, 130, 132, 135,
160
5,5-dimethylt hiazolidine-4-carboxylic acid 76 DIPCDI 130, 132, 134, 136, 137 dipeptidyl carboxypeptidases 67-68 dissociation constant 339 disulfide bonds 21-23 formation 157-159 disulfide bridge forming reactions 325-326 disulfide-containing peptides 22 disulfides 68 dithiothreitol (DTT) 228, 273, 325 1,3-divinyl benzene 168 DMA 101, 130 DMAP 130, 132 DMF. See dimethylformamide DMSO. See dimethylsulfoxide DNA 12, 20 binding proteins 20 coding sequence 25 sequence information 351 Dnp removal 115 dodecanethiol 266 doubly charged peptide 249 DPDCE 314 DPDPE 312, 314, 316-317 DPLCE 314 DSTBULET 314 Dtz 119 Edman degradation analysis 269-276. See also amino acid sequence Edman sequential degradation 143 electrochemical cyclizations 306 electron capture gas chromatography 66 electron density 47 electrophoresis 171, 233-241 electrospray ionization (ESI) 243-244, 249, 250, 253-259 electrospray-ionization mass spectrometry (ESMS) 248 ELISA 53, 332, 333, 353 endopeptidases 67-68 ^-endorphin 35 enkephalin analogues 299, 301
,a-diethylglycine 76 dihydrofolate reductase 306 diketopiperazine formation 161-162 &j8-dimethyl cysteine 80 dimethylformamide (DMF) 21, 100, 101, 112, 114-116, 121, 132, 139, 152, 165, 232 dimethylsulfide 146 dimethylsulfoxide (DMSO) 21, 157, 165,232 dimethylthiazolidine carboxylic acid 77
382
Index
enkephalin analogues (continued) with modified peptide bonds 71 enkephalins 296, 297, 313 enzymatic hydrolysis 68 enzymatic stability 67-72 enzyme inhibitors 306-309 enzyme specificity 64 enzymes, proteolytic 67 epidermal growth factor (EGF) 348 epitope-binding sites 334 epitope mapping 52-54, 66 procedures 332 epitopes 81 equilibrium binding to antibody 339 erythrocytes 74 ester bonds 131 esters, active 137-139 1,2-ethanedithiol (EDT) 117,149 ethanol 21 ethylene 70 ethylenediamine 128 European Molecular Biology Laboratory Protein and Peptide Group 37 evaluation of synthetic peptides 220-291 advantages and limitations of analytical techniques 223 battery of analytical tests 220-291 cumulative 6-year study 284 general considerations 221 how much needs to be done? 284-285 initial criteria 233 residues presenting potential problems 227-231 strategy for 221-225 exopeptidases 67
fibronectin 80 filtration 95 fluorescamine 262 fluorescence-quenched substrates 64 fiuoroarylazides 330 fluorodinitrobenzene 269 cc-fluoroketones 325 fMLF 305 Fmoc chemistry 3, 99-100, 161, 170, 232, 278 peptide acids 129 Fmoc synthesis 273 Fmoc-amino acid resins 133 Fmoc-amino acids 99, 100, 130, 138-140 Fmoc-His 116 Fmoc-OSu 99, 100 folding 37 foot and mouth disease 332, 333, 337 formaldehyde 232 formic acid, aqueous 232 ,/V-formyl tryptophan 266 fraction-by-fraction analysis 243 fragmentation 256 Friedel-Crafts catalyst 164 Friedel-Crafts reaction 96 function 37
FAB 244 FASTA 38 fibrin clot formation 307 fibrin monomers 307 fibrinogen 80 cleaving 307 fibrinogen receptor antagonists 49, 79 binding peptides 80
gas-phase sequencer 270, 271 gastrin 49, 155 gastrin-releasing peptide (GRP) 49, 50 gel-phase NMR spectroscopy 142 gene products, isolation and evaluation 340 geometric constraints 161 globally optimized secondary structure 74-76 glucagon 300 glutamine 12, 265, 266 as problem residue 229 glutathione 68 glycine 12 glycoprotein Ilb/IIa receptor 309-310 glycosyl transferases 10 glycosylation 156
Index
383
site motifs 56 gonadotropin releasing hormone (GnRH) 93 G-protein coupled receptors (GPCRs) 65, 318 Grarnacidin S 302 growth factors 10 growth hormone 300 guanidine 297, 306 guanidino group 114 guinea pig ileum (GPI) 313
HAL 129 halides, acid 140 a-haloketones 325 handles, preformed 128 handling of synthetic peptides 278-283 HATU 135, 136, 156 HBTU 135, 136, 137 helical axis 30 helical structures 41 helical wheel diagram 335 projection 36, 75 helix, helices 26-29 310, backbone structure 29 a-, backbone structure 28 amino acid residues in 55 amphipathic (amphiphilic) 34, 36, 46, 74 boundaries 40 buried 43 membrane-spanning 43 prediction 40 structures 27 torsional angles for 27 helix-breaking residues 40 hepatitis 332, 341 heterobifunctional reagents 320-322, 330, 331 cleavable 324 heterogeneity 233, 267 hexapeptide library, iterative deconvolution 61 high-performance affinity chromatography. See HPAC analysis
high-performance liquid chromatography. See HPLC analysis high-resolution spectra 251 hirudin 307, 308 recombinant analogue 309 Hirudo medicinalix 307 Hirulog analogues 308 His(Dnp)-containing peptideresins 115 HIV 332, 341 envelope protein gp!20 54 protease 11, 93, 303 HIV-1 166 protease 37,47,48, 50, 51, 168 HMFA 128 HMP/PAB linkage 99, 129 HO Ac 101 HOAt 135, 136, 156 HOBt 114-116, 128, 130, 132, 135-137 homogeneity 225-226 checking 57 homologous proteins 38 homology 37 HOPip 128 hormones 10, 295, 302, 329 HPAC analysis 345, 352, 353-355 HPLC analysis 57, 66, 171, 222, 224, 227, 232, 233, 236-237, 275, 276, 278, 284 reverse-phase 223, 225, 228, 229, 232-233, 237-241, 269, 277 HTCA 348 HYCRAM 129, 151, 156 hydrogen bonds 18-19, 25, 27, 28, 30, 31 hydrogen fluoride 98, 146-148 cleavage 52 hydrolysis procedures 265-268 hydropathy profile 53 hydrophilic peaks 43 hydrophilic residues 12 hydrophilic segments 43 hydrophilic side chains 36, 43 hydrophilicity 42, 55, 335, 336, 337 analysis 42 index of 55 hydrophobic interactions 19-21
384
Index
hydrophobic moment 43, 46 hydrophobic peptides 52 hydrophobic propensity values 42 hydrophobic residues 12, 36, 231 hydrophobic side chains 20 hydrophobicity 41-42, 44-45, 55, 335 measurements 42^6 3-hydroxy-3 methyl-glutaryl Co-A 307 hydroxylated residues 153 hydroxylysine (Hyl) 153 hydroxyproline (Hyp) 153 A'-hydroxysuccinimide ester 330 imidazole ring 115 imide formation 164-165 immobilized-sense peptides 355 immunogen 55 design 56-57 immunogenic uses of synthetic peptides 331-332 immunological agents 309 incomplete coupling 226 infrared spectroscopy 224 in situ coupling reagents and additives 134, 136 insulin 300 integrase 47 integrins 309 interleukin-2 (IL-2) 348 internal reference amino acids (IRAA) 143 ion-exchange chromatography 262 ion-exchange HPLC 235, 237 Irori Quantum Microchemistry 67 isatin test 141 isoleucine 12, 16, 39 isopropylidene 156 isosteric substitutions 297 iterative deconvolution 60-62 hexapeptide library 61 Kel-F particles 121 KGD 310 kinases 10, 62, 64 kinetic fine-tuning 97 Kyte-Doolittle analysis 42
labeled beads 64 X-lactam 78-79 5-lacta.m formation 114 lactams 159-160 head-to-tail 159 intrachain 159 side-chain-to-head 159 side-chain-to-tail 159 leucine 12 leucine enkephalin 70 ligand-receptor/acceptor interactions 294 linkages, anchoring 97, 99, 122, 129, 151 linkers 65, 129 LKTEAE 333 long chain syntheses 166-167 loop structures 55 lovastatin 306 luteinizing-hormone-releasing hormone (LHRH) 10-11, 78-79, 296 lysozyme 334 major histocompatibility complex (MHC) 333, 341-344 polymorphism 342 maleimido groups 319, 325 mass differences relevant to synthetic peptides 245-248 mass evaluation 244-253 mass spectrometry 3, 57, 143, 222-224,253-261,331 mass spectroscopy 243 matrix-assisted laser desorption ionization (MALDI) 143, 243-244, 259 mass spectrometry 248, 253 MBHA 98, 132, 146 melanin concentrating hormone (MCH) 295, 355 fragment analogues, relative potencies 296 melanophores 65 mellitin 35, 74, 75 membrane-spanning helices 43 mercaptoethanesulfonic acid 266 Merrifield protection scheme 98-99 Merrifield solid-phase
Index
385
chemistry 333 procedure 332 methanesulfonic acid 266 methionine 12, 227, 265, 297 as problem residue 228 TV-methyl amino acids 76, 298 N-methyl-alanine 76, 77 JV-methylmercaptoacetamide 273 mevolonate 307 Michaelis binding constant 50 microsequencing 65 mimotopes 340, 342 MM A 117 mobile-antisense-matrix-boundsense peptide complex 354 molecular biology 312 molecular genetics 53 monitoring 140-143 continuous-flow 142 invasive 143 peptide purification 224 qualitative 141 quantitative 142 mouse vas deferens (MVD) 313 Moz 119 Mpc 121 Msc 121 a-MSH 328, 329, 353, 355 superagonists 300 MSNT 130, 132 multiple antigenic peptide (MAP) 55-57, 161 myoglobin 336 N" amino-protecting groups 98-100, 116, 119, 120, 161 N01 protected amino acid ODhbt esters 138 N" protected amino acids 131 National Biomedical Research Foundation (NBRF) 37 National Center for Biotechnology Information 37, 38 native-like conformation 161 NCAs 130, 139 neurotransmitters 10, 302 Newman projections of torsional angles 26 NHCO linkage 72
ninhydrin test 141 nisin 23 nitrenes 326-327 2-nitro-4-azido phenylsulfenyl chloride 331 />-nitrophenyI 326, 328 p-nitrophenylalanine derivatives 328 residue 329 NMM 98, 130, 135 NMP 100, 101, 165 NMP DMSO 170 nonenzymatic transformation 15 nonionic polar residues 18 nonpeptide peptide mimetics 302 nonpolar residues 20 nonprotein amino acids 16-23 norleucine (Nle) 297 normal-phase chromatography 237 norpholine 156 NPE 128 Npys 119 nuclear magnetic resonance (NMR) 23, 171, 315, 318 analysis 305 constraints 304 spectroscopy 224 techniques 47 nuclear Overhauser enhancement (NOE) spectroscopy 304, 315 nucleophilicity effect 324 Nvoc 119
OBt 137 ODhbt 137 esters 138 O-H hydrogens 328 on-bead analysis 64-66 on-resin formation of cyclic /3-turn peptidomimetics 305 on-resin sequencing 276-278 one-bead, one-compound screening 64-66 ONo 137 ONp 137 OPfp 137 opioid receptor peptides, ^-selective 313-318 opioid receptors 312
386
Index
/u-opioid selective peptides 297 Orn formation 114 orthogonal protection schemes 98,99 orthogonal side-chain protection 159-160 OSBA 163 OTDO 130 overlapping peptides 334 oxidative reagents 119 oxytocin 10, 93, 297, 298, 301 analogues 299 antagonists 296
PAL 133, 134 palladium-catalyzed peptide-resin cleavage 151 palladium-sensitive allyl (OA1) group 112 PAM 98, 99, 128, 146 parallel synthesis 66-67 particle-induced desorption techniques 244 particle size 240-241 PE Biosystems Model 431 A/ 4334A 144 PEG-polyacrylamide (PEGA) 58, 64, 122, 170 PEG-polystyrene 58, 64, 122, 159 penicillamine 80, 298 penicillins 306 pentapeptide sequence, scanning libraries 63 Pepsyn 121 PepsynK 122 peptidase ladder sequencing 259 peptide(s) biological activity 35 complementary 344 cyclic 68, 78 de novo synthesis of photoaffinity 329 deletion 163-164, 295 (^-selective opioid receptor 313-318 development prospects 4 doubly charged 249 fibrinogen receptor binding 80 hydrophobic 52 immobilized-sense 355
medical and biological applications 292-376 overlapping 334 purified 225-226 retroinverso 342 roles in biochemistry 93 sequence 11 singly charged 249 structure 11 prediction 35 unpurified 225-226 peptide acids 128-132 Boc chemistry 128 Fmoc chemistry 129 linkers 129 peptide amides 132-134 peptide antigens 160-161 peptide backbone, conformation 25,26 peptide bonds formation 134 hydrolysis 266 replacements 72 peptide-hormone receptor systems 318 peptide hormones 68, 78 peptide libraries 340, 343 design and use 57 peptide ligand design 298 peptide-resins treatment following synthesis 152 peptide sequences 295, 310 antigenic 332-334 as potential vaccines 341-344 peptide sequencing 253, 259 Peptide Synthesis Database 101 peptide therapy 292 peptidomimetics 302-306 peptidyl resins 60 performic acid oxidation 266 permanent protecting groups 97, 101-121 pH effects 231, 239, 249, 322, 331 phenylalanine 12 phosphatases 10 phospholipid bilayers and membranes 74 phosphorus-containing thermolysin inhibitors 19
Index
387
phosphorylation 153-155 phosphoserine 265 phosphothreonine 265 phosphotyrosine 265 photoaffinity labels 318, 326-327, 330 photoaffinity peptides 320-322, 330 de novo synthesis 329 photolabile groups 329 photolysis 119 ofarylazides 328 photolytic cleavage 152 picric acid 141 piperidine 114, 156 piperidine-DMF 112, 118, 133, 162 3-( 1 -piperidinyl)alanine 119 plasma-desorption mass spectrometry (PDMS) 248 platelets 79, 80, 303, 309-310 polar side chains 73 polyamide 121, 168 cylinder 27 gel beads 121 supports 128 polyethylene glycol (PEG)polystyrene 58 polyethylene sheets 121 polymerase chain reaction (PCR) 66 polymeric support 94, 96, 121-122 polynucleotide tags 66 polypeptide chain 49, 271 polyprotein precursors 48 polystyrene 121, 168 pore size 240-241 porous graphitized carbon (PGC) 163 post-column ninhydrin detection 263 post-column o-phthalaldehyde detection 263 post-source decay 259-261 post-translational modifications 3, 152-153 potassium ferricyanide 157 preformed handles 128 preformed symmetrical anhydrides (PSAs) 114, 116, 139-140 preview analysis 143
production stages 222 proline 12, 77 proteases 10, 64 protected segments, solid-phase assembly 167 protecting groups permanent 97, 101-121 temporary 97 protection chemistry 97 protection schemes 94, 97-98 orthogonal 98, 99 protein(s) 11, 12 amphipathic structures 35 homologous 38 information content 35 primary structure 25 quaternary structure 25 receptor/acceptor proteins 302 secondary structure 25-33 tertiary structure 25 protein amino acids 12-16 protein antigenic regions, prediction of 334 protein antigens, identification of 332 protein biochemistry 53 protein biosynthesis 37 protein databases 37, 38 protein folding 37 protein secondary structure 38 protein sequence(s) 37, 38, 41-42, 44-45 analysis software 42 homology 35-39 protein-specific antibodies 54-55 protein structure 25 proteolysis 70, 71 proteolytic enzymes 67 protonation 255 pseudo-dilution 158 pseudo-isosteric replacements 297 pseudo-pentapeptide 304 pseudo-peptides 302 PTH-amino acids 268-270, 275, 278 relative elution positions 279-282 purification 96, 224-226, 241-243, 340 purified peptides 225-226 PVDF (polyvinylidene difluoride) 272
388 Index PyAOP 137 PyBOP 136, 137 PyBroP 137 3-(2-pyridyldithio)propionate (POP) 325 S-pyridylethyl derivative 275 pyroglutamate formation 116 quinoline yellow 142 salt bridges 18-19 SASRIN 129 scanning libraries 62 63 pentapeptide sequence 63 Schiffs base 163-164, 326 scoring table 38-39 secondary structure 12, 55 globally optimized 74-76 prediction 39 42 segment condensation 167-168 segmental flexibility 12 segmental mobility 55 sense peptide 344-351 Sequelon 272 sequence analysis 222, 223, 269-276 application to modeling and design 46^18 limitations 269-271 sequence homology 39 searching 38 sequence selection, general procedure 55-56 sequence variability 55 serine 12, 265 side chain(s) acid-sensitive 164 arrangement of 35 carboxamide 115 Cys 118 hydrophilic 36, 43 hydrophobic 20 polar 73 pseudo-axial 32 pseudo-equatorial 32 reactive amino acid 101-121 side-chain accessibility 55 side-chain carboxyls 112 side-chain conformations 302 side-chain cyclization 301 side-chain hydroxyls 112 side-chain interactions 17-23 side-chain phosphorylated Tyr 154 side-chain protected amino acids 96, 102-111 side-chain protecting groups 95, 98, 99, 227 side-chain protection orthogonal 159-160 Trt 117
racemization 114, 132 radioactive heterobifunctional reagents 323 Raman spectroscopy 331 random adducts 227 random peptide synthesis 344 reactive amino acid side chains 101-121 reagent K 164 real-time feedback 140 receptor/acceptor protein(s) 302 receptor studies 311-313 receptors affinity labeling 318-319 multiple opioid 312 steric and stereoelectronic nature of 298 residue mass 255 residue side chains 30 resin beads 65 linkers and handles 122-128 sampling 233, 261-262 resin-bound jV-amino groups 141 retroinverso peptides 342 retroviruses 48 reverse-phase HPLC 223, 225, 228, 229, 232-233, 237-241, 269, 277 reverse transcriptase 47 reversible labeling 324 ROD 303, 309, 310 ribonuclease A l l ring size 300 Rink amide resin 305, 306 RNase H 47 Ruhemann's purple 141
Index
389
side-chain SjVAr reaction 305 side-chain-to-head cyclizations 160 1-3 simultaneous syntheses 144 singly charged peptide 249 sodium thiosulfate 326 solid-phase binding assay 353 solid-phase peptide synthesis (SPPS) 11, 52, 58 attachment to support 122-128 automation 96, 143-144 auxiliary issues 161 170 batchwise 122 Boc 146 chemistry of 171 classical methods 94 continuous-flow 122, 162 development 4 Fmoc 146, 162 general Boc chemistry 134 general Fmoc chemistry 135 overview 94-97 potential side reactions 161 principles and practice 93-219 stepwise 95,96, 114, 140 solubility, properties of synthetic peptides 231-232 solvation 18, 165-166 somatostatin 296 analogues 297, 299 spectroscopic measurements 171 spinning cup sequencer 269, 270 y-spirolactam 339 split-and-mix technique 58-60, 67 SPPS. See solid-phase peptide synthesis stepwise SPPS 95, 96, 114, 140 stereochemical substitutions 295-297 storage of synthetic peptides 278-283 structural information, tertiary 334-335 ai-structure 29-30 structure-activity relationships (SAR) 57, 311 313 structure/function studies 294, 301, 309 structures, unnatural 152-153 substance P antagonists 296 substrate-bearing beads 64
subtilin 23 succinimide formation 164 succinyl groups 52 sulfation 155 Synbranchus marmoratus 296 synthesis early 4 methods 58-67 1-3 simultaneous 144 steps involved 5-6 See also design of synthetic peptides; solid-phase peptide synthesis synthesizers automated 144 automated and semi-automated multiple peptide 144-145 automated solid-phase organic 145-146 systematic modifications 294-295 T-bags 52, 67 t-Boc chemistry 277 T-cell antigens 334 T-cell epitopes 333 T-helper cells 333 tagging 65-66 TBDMS 153, 156 TBDPS 156 TBTU 135, 137 TCTAP 312 temporary protecting groups 97 temporary protection of W*-amino groups 98-100 TentaGel 64, 122 tertiary structural information 334-335 TFA. See trifluoroacetic acid TFA/scavenger mixture 164 TFE 158, 165 TFE-DCM 165-166 TFFH 140 TFMSA 98, 115, 128, 148 therapeutic agents 309-311 THF-NMP 165-166 thioamide 70, 72 thioesters 21-23, 170 thioethers 21-23 thiol reacting derivatives 323-324
390
Index
thiolysis 119 thiomethyl ether 70, 72 thioproline 76 thiopropionic acid 170 three-dimensional orthogonal protection scheme 160 threonine 12, 16, 265 thrombin, inhibition of 307-308 thrombin-hirudin complex 308 thrombosis, prevention of 310 thymidylate synthetase 306 TMSBr 117 TNBS 262 toluenesulfonic acid 266 topographical constraints 297, 312 TOPPipU 135 torsional angles 25, 26 /3-structure 31 helices 27 Newman projections of 26 turns 32 transferrin (TF) 348 translocation 37 trifluoroacetic acid (TFA) 65, 98, 99, 114, 117, 119, 129, 132, 133, 149-151, 155, 162,232, 238, 239 cleavage 143, 150 tripeptide RGD sequence 302 Trt group 115 Trt side-chain protection 117 trypsin 68 tryptophan 12, 227, 265, 266, 295 as problem residue 229 tryptophan-19 75 tryptophan-containing peptide 267 tumor cells 303 turn formation 339 turn mimetics 303 libraries 303 turn structures 41 turns 55, 77-78 3- 30-32, 33, 40, 44-45, 76, 78, 298, 305 conformations 305 8-1 340 fl-ll 33, 304, 305
/3-II1 32 /?-IV 315 conformational aspects 302 7- 31, 34, 76 peptide backbone 32 reverse 340 role in peptide secondary structure 30-33 structural features 32 topographical aspects 302 torsional angles for 32 type I and II 340 type III 32 types 315 Tyr 155 Tyr sulfate-containing peptides 155 tyrosine 12, 265 analogues 299 unnatural structures 152-153 unpurified peptides 225-226 a, /?-unsaturated ketones 326, 328 UV spectroscopy 222-224, 267 vaccines, development of 341-344 valine 12 van der Waals (or London) interactions 20 vascular agents 309 diagnostic 309-311 4-vinylpyridine 275 viral diseases 343-345 washing 95 washout 272 Water's Accu-Tag system 264 xanthenyl 118, 133 xanthenylamide 155 X-ray crystal structure 18, 37, 39 YPGDV 338 YPYDV 338, 339 YPYDVPDYA 338 Zinsser Analytic Sophas M 145

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SYNTHETIC PEPTIDES

SYNTHETIC PEPTIDES A User's Guide

Copyright 2002 by Oxford University Press, Inc.

987654321 Printed in the United States of America on acid free paper

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Victor J. Hruby Department of Chemistry University of Arizona Tucson, Arizona 85721

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Figure 1 -1. Diagram of the steps involved in obtaining a synthetic peptide.

Peptide Design Considerations

Peptide Design Considerations

rotein and Amino Acid Chemistry

rotein Amino Acids

Alanine Ala A Neutral, hydrophobic, aliphatic

Leucine Leu L Neutral, hydrophobic, aliphatic

Tyrosine Tyr Y Neutral, hydrophobic, aromatic

Table 2-1 (Continued)

Threonine Thr T Neutral, hydrophilic

Glutamine Gin Q Neutral, hydrophilic

Aspartic Acid Asp D

Glutamic Acid Glu E

Lysine Lys K Basic, hydrophilic

Cystetne Cys C Thiol-containing

Proline Pro P Imino acid

Peptide Design Considerations

Figure 2-1. Some amino acids formed by post-translational modification.

Peptide Design Considerations

Figure 2-2. Some nonprotein amino acids.

Peptide Design Considerations

Table 2-2 Phosphorus-containing thermolysin inhibitors

Peptide Design Considerations 21

Figure 2-3. Disulfide, thioester, and thioether linkages.

Figure 2-4. Disulfide-containing peptides.

Peptide Design Considerations 23

The Amide Bond

Figure 2-5. Characteristics of amide bonds.

Peptide Design Considerations 25

Protein Secondary Structure

Peptide Design Considerations 27 Table 2-3 Torsional angles for helices

Angles taken from IUPAC-IUB Commission on Biochemical Nomenclature (1970).

Peptide Design Considerations

Peptide Design Considerations 31

Angles taken from IUPAC-IUB Commission Biochemical Nomenclature (1970)

Table 2-5 Torsional angles for turns

Angles taken from Smith and Pease (1980).

Peptide Design Considerations

Peptide Design Considerations

Peptide Design Considerations

Peptide Design Considerations

Secondary Structure Prediction

'Kyte and Doolittle (1982). 'Chou and Fasman (1978).

Peptide Design Considerations

Application of Sequence Analysis to Modeling and Design

Yamashiro & Li, 1973- Tarn & Merrifield, 1987*

Chan etal, 1995-