Bioconjugate Chemistry

Objective: Biodegradable polycaprolactone/starch composites can be used for bone tissue engineering applications. The effect of the ratio of components on composite properties is of tremendous importance. Methods: Polycaprolactone/starch composite of 80/20 and 70/30 ratios were fabricated by dissolving them in chloroform followed by evaporation of the solvent. Results: The composites were characterized by fourier transform infrared spectroscopy. Their bioactivity was evaluated by studying the apatite formation ability after immersing the specimens in simulated body fluid. The results of compressive test on samples showed that the composite's modulus and strength approximated that of human trabecular bone. Mass loss in distilled water and starch degradation rate in PBS was evaluated, which showed that the starch ratio was effective in composite degradation. MTT analysis and alkaline phosphatase levels showed that this composite had no toxicity and could increase G-299 cell line osteoblastic activities. Conclusion: The increase in cellular osteoblastic activities and the ability for apatite formation on the composite surface, in addition to the polycaprolactone/starch samples' mechanical properties shows their capability to be used as substitutes for bone. Because this composite degradation rate is controlled by changing the starch ratio, it has the potential for use in bone tissue engineering applications. Samples that have a 70/30 ratio are considered optimal due to their enhanced cellular response and better mechanical properties.

Supporting information AFM Studies. Alkanethiol materials. 1-adamantanethiol (AD) of 95% purity was purchased from Sigma-Aldrich (Missouri, USA) and was used as received. Ethanol solvent of 99.99% purity was purchased from Gold Shield Chemical Co. (California, USA) and served as the solvent for AD. Preparation of 1-adamantanethiol Self-Assembled Monolayers (SAM). The SAM preparation used in this study follows established procedures. 1, 2 Gold with 99,999% purity was purchased from Alfa Aesar (Massachusetts, USA) was deposited in a high-vacuum evaporator (Model DV502-A, Denton Vacuum, New Jersey, USA) at a base pressure below 2 x 10-7 Torr onto freshly cleaved mica substrates

Background: 1E10 monoclonal antibody is a murine anti-idiotypic antibody that mimics N-glycolyl-GM3 gangliosides. This antibody has been tested as an anti-idiotypic cancer vaccine, adjuvated in Al(OH) 3 , in several clinical trials for melanoma, breast, and lung cancer. During early clinical development this mAb was obtained in vivo from mice ascites fluid. Currently, the production process of 1E10 is being transferred from the in vivo to a bioreactor-based method.

Recently, a bivalent recombinant anti-human CD3 diphtheria toxin (DT) based immunotoxin derived from the scFv of UCHT1 antibody has been made that shows enhanced bioactivity and is free from the side effects of Fc receptor interaction. In this case, the diminution of CD3 binding due to the placement of the scFv domain at the C-terminus of the truncated DT in single scFv immunotoxins was compensated by adding an additional scFv domain. However, this strategy was less successful for constructing an anti-rhesus recombinant immunotoxin derived from the scFv of FN18 antibody due to poor binding of the anti-rhesus bivalent immunotoxin. We report here that, by increasing the FN18 scFv affinity through random mutagenesis and selection with a dye-labeled monkey CD3 γ recombinant heterodimer, we greatly improved the bioactivity of FN18 derived immunotoxin. The best mutant, C207, contained nine mutations, two of which were located in CDRs that changed the charge from negative to positive. Binding affinity of the C207 scFv to the monkey T cell line HSC-F increased 9.8-fold. The potency of the C207 bivalent immunotoxin assayed by inhibition of protein synthesis increased by 238-fold.

Extremely stable, peptide-capped gold nanoparticles with two different biomolecular recognition motifs expressed on their surface have been prepared, and their specific and selective binding to artificial, DNA-modified target particles and to DNA and protein microarrays has been demonstrated. Stabilization and biofunctionalization has been achieved in a single preparative step starting with citrate-stabilized gold hydrosols and a derivatization cocktail of peptide-capping ligands, which carry the functionalities of choice.

A series of human insulin maleimido derivatives with short and long linkers was synthesized by exploiting the variations in the pK a values and environment of the three amino groups present in the protein. The syntheses were accomplished in organic solvent because of maleimide's instability in basic aqueous media. The derivatives thus obtained were conjugated to the free thiol on Cys34 of human serum albumin (HSA) and purified. A structure-activity relationship based on in vitro receptor binding and activation results for this series of insulin-HSA conjugates showed that the best compounds were attached at the B1 position of insulin with either short or long linkers. Two conjugates were administered subcutaneously to streptozotocin-induced diabetic rats and found to possess blood glucose normalizing activity up to 8 h postadministration. The return to diabetic plasma glucose levels was not observed within the time frame of the experiment (48 h). In comparison, the insulin-treated group's normalization activity lasted 2 h and returned to a diabetic level at 8 h. The onset of the conjugate activities were delayed by 1 h when compared to the activity of human insulin. The study results led to the identification of CJC-1575 as a potent and long lasting human insulin analogue.

Posttranslational modification catalyzed by phosphopantetheinyl transferases (PPTases) has previously been used to site-specifically label proteins with structurally diverse molecules. PPTase catalysis results in covalent modification of a serine residue in acyl/peptidyl carrier proteins and their surrogate substrates which are typically fused to the N- or C-terminus. To test the utility of PPTases for preparing antibody-drug conjugates (ADCs), we inserted 11 and 12-mer PPTase substrate sequences at 110 constant region loop positions of trastuzumab. Using Sfp-PPTase, 63 sites could be efficiently labeled with an auristatin toxin, resulting in 95 homogeneous ADCs. ADCs labeled in the CH1 domain dis-played in general excellent pharmacokinetic profiles and negligible drug loss. A subset of CH2 domain conjugates underwent rapid clearance in mouse pharmacokinetic studies. Rapid clearance correlated with lower thermal stability of the particular antibodies. Independent of conjugation site...

In this paper are reported the synthesis and characterization of three LSD derivatives. On the basis of several analytical characterization studies, the most stable derivative has been selected and a procedure to covalently link the derivative to polystyrene microparticles through a carrier protein has been developed. In addition, two new LSD immunogens have been synthesized and characterized, and from these immunogens antibodies that recognize not only LSD but also several major LSD metabolites have been generated. Using the selected derivative and antibody, a homogeneous microparticle-based immunoassay has been developed for the detection of LSD in human urine with the required sensitivity and specificity for an effective screening assay. The performance of this LSD OnLine assay has been evaluated using the criteria of precision, cross-reactivity, correlation to the Abuscreen LSD RIA and GC/MS/MS, assay specificity, and limit of detection.

During protein synthesis the nascent polypeptide chain (NC) extends through the ribosomal exit tunnel (NPET). Also, the large group of macrolide antibiotics binds in the nascent peptide exit tunnel. In some cases interaction of NC with NPET leads to the ribosome stalling, a significant event in regulation of translation. In other cases NC-ribosome interactions lead to pauses in translation that play an important role in cotranslational folding of polypeptides emerging from the ribosome. The precise mechanism of NC recognition in NPET as well as factors that determine NC conformation in the ribosomal tunnel are unknown. A number of derivatives of the macrolide antibiotic 5-O-mycaminosyltylonolide (OMT) containing N-acylated amino acid or peptide residues were synthesized in order to study potential sites of NC-NPET interactions. The target compounds were prepared by conjugation of protected amino acids and peptides with the C23 hydroxyl group of the macrolide. These OMT derivatives showed high although varying abilities to inhibit the firefly luciferase synthesis in vitro. Three glycil-containing derivatives appeared to be strong inhibitors of translation, more potent than parental OMT. Molecular dynamics (MD) simulation of complexes of tylosin, OMT, and some of OMT derivatives with the large ribosomal subunit of E. coli illuminated a plausible reason for the high inhibitory activity of Boc-Gly-OMT. In addition, the MD study detected a new putative site of interaction of the nascent polypeptide chain with the NPET walls.

R-Melanocyte-stimulating hormone (R-MSH) is known to bind to the melanocortin receptor 1 (MC1R) which is overexpressed on melanotic and amelanotic melanoma cells. R-MSH analogues are potential candidates for specific targeting of melanoma metastases. Several linear and cyclic radiolabeled MSH peptides have been designed and tested in the past, showing both high affinity for the MC1R in vitro and good incorporation in tumor xenografts in vivo. However, considerable kidney reabsorption of the radiopeptides could not be avoided. With the aim to increase the tumor-to-kidney ratio, we synthesized six glycosylated derivatives of NAPamide, an R-MSH octapeptide analogue with high tumor selectivity and coupled them to the chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). The peptides were evaluated in vitro for MC1R binding and bioactivity and, after labeling with 111 In, for in vitro cellular uptake and in vivo tissue distribution in mice carrying B16F1 melanoma tumors. The glycopeptides showed excellent binding affinities in the low nanomolar to subnanomolar range using both murine and human melanoma cell lines. However, five glycopeptides displayed lower selectivity in vivo than the parent DOTA-NAPamide, because of either a lower tumor uptake or a higher kidney uptake. In particular C-terminal extension of the amide group by a galactosyl moiety increased the kidney retention dramatically. By contrast, an N-terminally positioned galactose residue in DOTA-Gal-NAPamide improved the tumor-to-kidney ratio (4-48 h AUC of 1.34) by a factor of about 1.2 as compared to the parent DOTA-NAPamide (4-48 h AUC of 1.11), thus serving as new lead compound for MC1R-targeting molecules.

Nine complexes of the type [Ru(N-N) 2 (BPG)]Cl 2 1-4, [Ru(N-N)(BPG) 2 ]Cl 2 5-8, and [Ru(BPG) 3 ]Cl 2 9 where N-N is 2,2′-bipyridine (bpy), 1,10-phenanthroline (phen), dipyrido [3,2-d:2′,3′-f]quinoxaline (dpq), dipyrido[3,2a:2′,3′-c]phenazine (dppz), which incorporates bipyridine-glycoluril 5,[1,10]phenanthroline-6,13-dione) as the ancillary ligand, have been synthesized and characterized. These complexes with the peripheral polypyridyl ligands have the ability to form conjugates with DNA. The DNA binding (absorption spectroscopy, steady-state and time-resolved emission measurements, steady-state emission quenching measurements) and cleavage (under dark and irradiated conditions) by these complexes has been studied to investigate the influence of the ancillary ligand. The binding ability of these complexes to DNA is dependent on the planarity of the intercalative polypyridyl ligand, which is further affected by the ancillary bipyridine-glycoluril ligand. The complexes 3, 4, 7, and 8 bind to CT-DNA with binding constants on the order of 10 4 M -1 . Time-resolved emission measurements on the DNA-bound complexes 1, 3, 5-7, and 9 show monoexponential decay of the excited states, whereas complexes 2, 4, and 8 show biexponential decay with short-and long-lived components. Interaction of complexes 2-9 with plasmid pBR322 DNA studied by gel electrophoresis experiments reveals that all complexes cleave DNA efficiently at micromolar concentrations under dark and anaerobic conditions probably by a hydrolytic mechanism. Complexes 3, 4, 7, 8, and [Ru(bpy) 2 (dppz)] 2+ show extensive DNA cleavage in the presence of light with a shift in mobility of form I of DNA probably due to the high molecular weight of DNA-complex conjugates. However, the extent of the cleavage is augmented on irradiation in the case of complexes 3, 4, 7, and 8, which include the planar dpq and dppz ligands, suggesting a combination of hydrolytic and oxidative mechanism for the DNA scission. Molecular mechanics calculations of these systems corroborate the DNA binding and cleavage mechanisms. *

Bioconjugates can be synthesized by reaction of synthetic peptides and polyelectrolytes. They are biologicallay important molecules. Their application on biology and nanotechnology fields has great potential. There are lots of researchs related to functional biopolymer systems in last years. These researchs are about synthesis, characterization and application properties of biologically important polyelectrolytes. This work is aimed to giving information about polyelectrolytes, bioconjugation, microwave energy, solid phase peptide synthesis

The gastrin-releasing peptide receptor (GRPR) is overexpressed on a number of human tumors and has been targeted with radiolabeled bombesin analogues for the diagnosis and therapy of these cancers. Seven bombesin analogues containing various linkers and peptide sequences were designed, synthesized, radiolabeled with 18 F, and characterized in Vitro and in ViVo as potential PET imaging agents. Binding studies displayed nanomolar binding affinities toward human GRPR for all synthesized bombesin analogues. Two high-affinity peptide candidates 6b (K i ) 0.7 nM) and 7b (K i ) 0.1 nM) were chosen for further in ViVo evaluation. Both tracers revealed specific uptake in GRPR-expressing PC-3 tumors and the pancreas. Compared to [ 18 F]6b, compound [ 18 F]7b was characterized by superior tumor uptake, higher specificity of tracer uptake, and more favorable tumor-to-nontarget ratios. In ViVo PET imaging allowed for the visualization of PC-3 tumor in nude mice suggesting that [ 18 F]7b is a promising PET tracer candidate for the diagnosis of GRPR-positive tumors in humans.

Today in the world of pharmacy, evaluation parameters play a vital and critical role in deciding the best method for the preparation of various dosages forms. The R & D department is responsible for carrying out various researches and later deciding the most appropriate among the all. The process should be economical and at the most rationalized to be considered as the process of choice.

Liposomes are of interest as drug delivery tools for therapy of cancer and infectious diseases. We investigated conjugation of epidermal growth factor, EGF, to liposomes using the micelle-transfer method. EGF was conjugated to the distal end of PEG-DSPE lipid molecules in a micellar solution and the EGF-PEG-DSPE lipids were then transferred to preformed liposomes, either empty or containing the DNA-binding compound, water soluble acridine, WSA. We found that the optimal transfer conditions were a 1-h incubation at 60°C. The final conjugate, 125 I-EGF-liposome-WSA, contained approximately 5 mol % PEG, 10-15 EGF molecules at the liposome surface, and 10 4 to 10 5 encapsulated WSA molecules could be loaded. The conjugate was shown to have EGF-receptor-specific cellular binding in cultured human glioma cells.

The P2Y 1 receptor is a prothrombotic G protein-coupled receptor (GPCR) activated by ADP. Preference for the North (N) ring conformation of the ribose moiety of adenine nucleotide 3′,5′bisphosphate antagonists of the P2Y 1 receptor was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute. A series of covalently linkable N 6 -methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphates containing extended 2alkynyl chains was designed and binding affinity at the human (h) P2Y 1 receptor determined. The chain of these functionalized congeners contained hydrophilic moieties, a reactive substituent, or biotin, linked via an amide. Variation of the chain length and position of an intermediate amide group revealed high affinity of carboxylic congener 8 (K i 23 nM) and extended amine congener 15 (K i 132 nM), both having a 2-(1-pentynoyl) group. A biotin conjugate 18 containing an extended ε-aminocaproyl spacer chain exhibited higher affinity than a shorter biotinylated analogue. Alternatively, click coupling of terminal alkynes of homologous 2-dialkynyl nucleotide derivatives to alkyl azido groups produced triazole derivatives that bound to the P2Y 1 receptor following deprotection of the bisphosphate groups. The preservation of receptor affinity of the functionalized congeners was consistent with new P2Y 1 receptor modeling and ligand docking. Attempted P2Y 1 antagonist conjugation to PAMAM dendrimer carriers by amide formation or palladium-catalyzed reaction between an alkyne on the dendrimer and a 2-iodopurine-derivatized nucleotide was unsuccessful. A dialkynyl intermediate containing the chain length favored in

N,N,N-Trimethylated chitosan (TMC) is a biodegradable polymer emerging as a promising nonviral vector for nucleic acid and protein delivery. In the present study, we investigated whether the introduction of thiol groups in TMC enhances the extracellular stability of the complexes based on this polymer and promotes the intracellular release of siRNA. The gene silencing activity and the cellular cytotoxicity of polyplexes based on thiolated TMC were compared with those based on the nonthiolated counterpart and the regularly used lipidic transfection agent Lipofectamine. Incubation of H1299 human lung cancer cells expressing firefly luciferase with siRNA/thiolated TMC polyplexes resulted in 60-80% gene silencing activity, whereas complexes based on nonthiolated TMC showed less silencing (40%). The silencing activity of the complexes based on Lipofectamine 2000 was about 60-70%. Importantly, the TMC-SH polyplexes retained their silencing activity in the presence of hyaluronic acid, while nonthiolated TMC polyplexes hardly showed any silencing activity, demonstrating their stability against competing anionic macromolecules. Under the experimental conditions tested, the cytotoxicity of the thiolated and nonthiolated siRNA complexes was lower than those based on Lipofectamine. Given the good extracellular stability and good silencing activity, it is concluded that polyplexes based on TMC-SH are attractive systems for further in vivo evaluations.

Science in a Global Community T hings have continued to progress and evolve at Bioconjugate Chemistry in 2016. Scientifically, we are continuing to expand the scope of the journal into new areas, while building on our strength in delivery and imaging. While expanding the scope of BC, we have maintained our ∼40 day submission to online publication average. Along with our publishing efforts, we presented the inaugural Bioconjugate Chemistry Lecturer award to Shawn Chen. We have also had dramatic growth in the BC social media community, with over 1000 Twitter followers and 1500 likes on Facebook. We have a busy year ahead in 2017. In January we published a special issue on "Interfacing Inorganic Nanoparticles with Biology" and we will be putting out an issue on "Peptide Conjugates for Biological Applications" in March. We will also be presenting the next BC lecturer award in August at the National ACS meeting in Washington, DC. And, as you can see below, we at BC are working to expand the global presence of both the journal and the scientific community. As you can see from the messages from the BC team, we are happy to reach out to researchers around the world to help you publish your work. We also want to go beyond the walls of the journal. Our goal is to do everything we can as a journal and as scientists to knit together the global community of researchers working at the interface between biology and chemistry. Best wishes for the coming year,

This review outlines the synthesis of covalent conjugates of oligonucleotides and their analogues that are obtained by reactions of carbonyl compounds with various nucleophiles such as primary amines, N-alkoxyamines, hydrazines, and hydrazides. The products linked by imino, oxime, hydrazone, or thiazolidine groups are shown to be useful intermediates for a wide range of chemical biology applications. Methods for their preparation, isolation, purification, and analysis are highlighted, and the comparative stabilities of the respective linkages are evaluated. The relative merits of incorporation of a carbonyl group, particularly an aldehyde group, into either the oligonucleotide or the ligand parts are considered. Examples of harnessing of aldehyde-nucleophile coupling for the labeling of nucleic acids are given, as well as their conjugation to various biomolecules (e.g. peptides and small molecule ligands), site-specific cross-linking of oligonucleotides to nucleic acid-binding proteins, assembly of multibranched supramolecular structures, and immobilization on functionalized surfaces. Future perspectives of bioconjugation and complex molecular engineering via carbonyl group additionelimination reactions in nucleic acids chemistry are discussed. Scheme 23. Aldehyde Oligonucleotides Used for Oxime Formation Scheme 24. Aminooxy Compounds Used for Oxime Formation Scheme 25. Double Conjugation to 3′,5′-Bis-Modified Oligonucleotides Reviews

Interest in taking advantage of the unique spectral properties of semiconductor quantum dots (QDs) has driven their widespread use in biological applications such as in vitro cellular labeling/imaging and sensing. Despite their demonstrated utility, concerns over the potential toxic effects of QD core materials on cellular proliferation and homeostasis have persisted, leaving in question the suitability of QDs as alternatives for more traditional fluorescent materials (e.g., organic dyes, fluorescent proteins) for in vitro cellular applications. Surprisingly, direct comparative studies examining the cytotoxic potential of QDs versus these more traditional cellular labeling fluorophores remain limited. Here, using CdSe/ZnS (core/shell) QDs as a prototypical assay material, we present a comprehensive study in which we characterize the influence of QD dose (concentration and incubation time), QD surface capping ligand and delivery modality (peptide or cationic amphiphile transfection r...

A new amino acid derivative, N~-(tert-butoxycarbonyl)-N~-[N-(bromoacetyl)-@-alanyl]-~-lysine (BBAL), has been synthesized as a reagent to be used in solid-phase peptide synthesis for introducing a sidechain bromoacetyl group at any desired position in a peptide sequence. The bromoacetyl group subsequently serves as a sulfhydryl-selective cross-linking function for the preparation of cyclic peptides, peptide conjugates, and polymers. BBAL is synthesized by condensation of N-bromoacetyl-@alanine with Nu-Boc-L-lysine and is a white powder which is readily stored, weighed, and used with a peptide synthesizer, programmed for Na-Boc amino acid derivatives. BBAL residues are stable to final HF deprotection/cleavage. BBAL peptides can be directly coupled to other molecules or surfaces which possess free sulfhydryl groups by forming stable thioether linkages. Peptides containing both BBAL and cysteine residues can be self-coupled to produce either cyclic molecules or linear peptide polymers, alsolinked through thioether bonds. Products made with BBAL peptides may be characterized by amino acid analysis of acid hydrolyzates by quantification of @-alanine, which separates from natural amino acids in suitable analytical systems. Where sulfhydryl groups on coupling partners arise from cysteine residues, S-(carboxymethy1)cysteine in acid hydrolyzates may also be assayed for this purpose. Examples are given of the use of BBAL in preparing peptide polymers and a peptide conjugate with bovine albumin to serve as immunogens or model vaccine components.

Affibody molecules present a new class of affinity proteins, which utilizes a scaffold based on a 58-amino acid domain derived from protein A. The small (7 kDa) Affibody molecule can be selected to bind to cell-surface targets with high affinity. An Affibody molecule (Z ...

Conjugates of a dicarba analogue of octreotide, a potent somatostatin agonist whose receptors are overexpressed on tumor cells, with [PtCl 2 (dap)] (dap = 1-(carboxylic acid)-1,2-diaminoethane) (3), [(η 6bip)Os(4-CO 2 -pico)Cl] (bip = biphenyl, pico = picolinate) (4), [(η 6 -p-cym)RuCl(dap)] + (p-cym = pcymene) (5), and [(η 6 -p-cym)RuCl(imidazole-CO 2 H)(PPh 3 )] + (6), were synthesized by using a solidphase approach. Conjugates 3-5 readily underwent hydrolysis and DNA binding whereas conjugate 6 was inert to ligand substitution. NMR spectroscopy and molecular dynamics calculations showed that conjugate formation does not perturb the overall peptide structure. Only 6 exhibited antiproliferative activity in human tumor cells (IC 50 = 63 + 2 µM in MCF-7 cells and IC 50 = 26 + 3 µM in DU-145 cells)

A new biomolecule labeling method that utilizes the [ 99m Tc(N)(PNP)] 2+ metal fragment is presented. Thus, a series of nitrido mixed-ligand M(V) complexes (M ) 99m Tc, 99g Tc, Re), [M(N)(Ln)(PNP)], where Ln is the dianionic form of a dithiolate or substituted-dithiolate ligand and PNP is an aminodiphosphine, is described. 99m Tc complexes can be prepared using either a two-step or a three-step procedure starting from generator-eluted pertechnetate through a prereduced mixture of [ 99m Tc(N)]-containing species, followed by sequential or contemporary addition of the relevant dithiolate and aminodiphosphine. The reactions of 2,3-dimercaptopropionic acid (H 2 L1) with [Tc(N)(PNP)] 2+ were investigated in detail. It was found that this bidentate ligand coordinated the metal fragment through the [S -,S -] donor atom pair, to yield neutral mixed-ligand complexes [ 99m Tc(N)(L1)(PNP)] in high specific activity. The additional carboxylic functional group was not involved in metal coordination, thus remaining available for conjugation to target-specific molecules. Dithiolates incorporating pendant functional group(s) gave rise to a 1:1

Proteins are hetero-polymers made-up of single building blocks (aminoacids) whose composition determines folding and final architecture. Some proteins are able to undergo self-assembly process
enabling the formation of ordered molecular aggregates that in some cases assume conformations particularly suitable to nanotechnological applications. In this work we describe the properties of a ringlike decameric protein, Peroxiredoxin (Prx), to build composite materials interacting with or catalyzing the formation of selectively metal nanoparticles that can be trapped over the surface of nanostructured
graphene oxide (GO) sheets. We demonstrate furthermore the ability of Prx to guide the formation of 3D layers of GO embedding metal nanoparticles in the composite material. These composites are discussed as possible precursors to electronic and chemical devices.

Despite the improvements in cancer therapy during the past years, high-grade gliomas and many other types of cancer are still extremely resistant to current forms of therapy. Boron neutron capture therapy (BNCT) provides a promising way to destroy cancer cells without damaging healthy tissue. However, BNCT in practice is still limited due to the lack of boron-containing compounds that selectively deliver boron to cancer cells. Since many neuroendocrine tumors show an overexpression of the somatostatin receptor, it was our aim to synthesize compounds that contain a large number of boron atoms and still show high affinity toward this transmembrane receptor. The synthetic peptide Tyr 3 -octreotate (TATE) was chosen as a high-affinity and internalizing tumor targeting vector (TTV). Novel boron cluster compounds, containing 10 or 20 boron atoms, were coupled to the N-terminus of TATE. The obtained affinity data demonstrate that the use of a spacer between TATE and the closo-borane moiety is the option to avoid a loss of biological affinity of closo-borane conjugated TATE. For the first time, it was shown that closo-borane conjugated regulatory peptides retain high biological affinity and selectivity toward their transmembrane tumor receptors. The results obtained and the improvement of spacer and boron building block chemistry may stimulate new directions for BNCT. * Figure 1. Simplified scheme of the BNCT reaction inside a cell.

A new class of vesicular acetylcholine transporter inhibitor that incorporates a carbonyl group into the benzovesamicol structure was synthesized and analogs were evaluated in vitro. (±)-trans-2-Hydroxy-3-(4-(4-[ 18 F]fluorobenzoyl)piperidino)tetralin (9e) has K i values of 2.70 nM for VAChT, 191 nM for σ 1 and 251 nM for σ 2 . The racemic precursor (9d) was resolved via chiral HPLC and (±)-[ 18 F]9e, (-)-[ 18 F]9e, and (+)-[ 18 F]9e were respectively radiolabeled via microwave irradiation of the appropriate precursors with [ 18 F]/F-and Kryptofix/K 2 CO 3 in DMSO with radiochemical yields ∼50-60% and specific activities >2000 mCi/μmol. (-)-[ 18 F]9e uptake in rat brain was consistent with in vivo selectivity for the VAChT with an initial uptake of 0.911 %ID/g in rat striatum and a striatum: cerebellum ratio of 1.88 by 30 min p.i.. MicroPET imaging of macaques demonstrated a 2.1 ratio of (-)-[ 18 F]9e in putamen versus cerebellum at 2 h. p.i. (-)-[ 18 F]9e has potential to be a PET tracer for clinical imaging of the VAChT. . Supporting Information Available: (1) Crystal structure data for (-)9e: final residual values, structure refinement parameters, complete listings of positional and isotropic displacement coefficients for hydrogen atoms, anisotropic displacement coefficients for the nonhydrogen atoms.

A new ligand derivative of 1,4,7-triazacyclononane (TACN), 2-[4,7-bis(2-pyridylmethyl)-1,4,7-triazacyclononan-1-yl]acetic acid (6), has been synthesized and its complexation behavior toward Cu 2+ ions investigated. The ligand 6 has been characterized by spectroscopic methods, and a molecular structure of a corresponding Cu(II) complex has been elucidated by single-crystal X-ray analysis. The suitability of 6 for conjugation to peptide substrates has been shown by amide coupling of 6 to the stabilized derivative of bombesin (BN), Ala-Ala-[Cha 13 , Nle 14 ]BN(7-14), to give the conjugate 8. The free ligand 6 and the bioconjugate 8 were labeled with 64 Cu 2+ , and the resulting complexes, 64 Cu⊂6 and 64 Cu⊂8, were found to be stable in the presence of a large excess of a competing ligand (cyclam) or copper-seeking superoxide dismutase (SOD), as well as in rat plasma. Biodistribution studies of 64 Cu⊂8 in Wistar rats showed a high activity uptake into the pancreas (5.76 ( 0.25 SUV, 5 min p.i.; 3.93 ( 0.25 SUV, 1 h p.i.), which is the organ with high levels of gastrin-releasing peptide receptor (GRPR). This receptor is overexpressed in a large number of breast and prostate carcinomas. The novel 64 Cu⊂6 complex had a dominating influence on the nonspecific activity biodistribution of its BN conjugate, since the distribution data of 64 Cu⊂6 are similar to those of 64 Cu⊂8. The 64 Cu complexes exhibited a low activity accumulation in the liver tissue and an extensive renal clearance, which was distinctively different to the biodistribution of 64 CuCl 2 , suggesting that 64 Cu⊂6 does not undergo significant demetalation, but rather exhibits high in ViVo stability. * Corresponding authors.

Arginine-rich cell penetrating peptides are short cationic peptides able to cross biological membranes despite their peptidic character. In order to optimize their penetration properties and further elucidate their mechanisms of cellular entry, these peptides have been intensively studied for the last two decades. Although several parameters are simultaneously involved in the internalization mechanism, recent studies suggest that structural modifications influence cellular internalization. Particularly, backbone rigidification, including macrocyclization, was found to enhance proteolytic stability and cellular uptake. In the present work, we describe the synthesis of macrocyclic arginine-rich cell penetrating peptides and study their cellular uptake properties using a combination of flow cytometry and confocal microscopy. By varying ring size, site of cyclization, and stereochemistry of the arginine residues, we studied their structure−uptake relationship and showed that the mode and site of cyclization as well as the stereochemistry influence cellular uptake. This study led to the identification of a hepta-arginine macrocycle as efficient as its linear nona-arginine congener to enter cells.

Successful imaging of somatostatin receptor-positive tumors with 111 In-DTPA-D-Phe 1 -octreotide has stimulated development of peptide radiopharmaceuticals using DTPA as the chelating agent. However, use of cyclic DTPA dianhydride (cDTPA) resulted in low synthetic yields of DTPA-peptide by either solution or solid-phase syntheses. This paper reports a novel high-yield synthetic procedure for DTPA-D-Phe 1 -octreotide that is applicable to other peptides of interest using a monoreactive DTPA derivative. A monoreactive DTPA that possesses one free terminal carboxylic acid along with four carboxylates protected with tert-butyl ester (mDTPA) was synthesized. Fmoc-Thr( t Bu)-ol, prepared from Fmoc-Thr( t Bu)-OH, was loaded onto 2-chlorotrityl chloride resin. After construction of the peptide chains by Fmoc chemistry, mDTPA was coupled to the R amine group of the peptide on the resin in the presence of 1,3-diisopropylcarbodiimide and 1-hydroxybenzotriazole. Treatment of the mDTPApeptide-resin with trifluoroacetic acid-thioanisole removed the protecting groups and liberated [Cys(Acm) 2,7 ]-octreotide-D-Phe 1 -DTPA from the resin. Iodine oxidation of the DTPA-peptide, followed by the reversed-phase HPLC purification, produced DTPA-D-Phe 1 -octreotide in overall 31.8% yield based on the starting Fmoc-Thr( t Bu)-ol-resin. The final product gave a single peak on analytical HPLC, and amino acid analysis and mass spectrometry confirmed the integrity of the product. 111 In radiolabeling of the product provided 111 In-DTPA-D-Phe 1 -octreotide with >95% radiochemical yield, as confirmed by analytical reversed-phase HPLC, TLC, and CAE. These findings indicated that use of mDTPA during solid-phase peptide synthesis greatly increased the synthetic yield of DTPA-D-Phe 1 -octreotide, due to the absence of nonselective reactions that are unavoidable when cDTPA is used. These results also suggested that mDTPA would be a versatile reagent to introduce DTPA with high yield into peptides of interest.

ImmunoPET with 89Zr-labeled antibodies has shown great potential in cancer imaging. It can provide important information about the pharmacokinetics and tumor targeting properties of monoclonal antibodies and may help in anticipating on toxicity. Furthermore, it allows accurate dose-planning for individualized radioimmunotherapy and may aid in patient selection and early response monitoring for targeted therapies. The most commonly used chelator for 89Zr is desferrioxamine (DFO). Preclinical studies have shown that DFO is a not an ideal chelator, since the 89Zr-DFO complex is partly unstable in vivo, which results in release of 89Zr from the chelator, and subsequent accumulation of 89Zr in bone. This bone accumulation interferes with accurate interpretation and quantification of bone uptake on PET images. Therefore, there is a need for novel chelators that allow more stable complexation of 89Zr. In this review we will describe the most recent developments in 89Zr-radiochemistry, incl...

Optimal PET imaging of tumors with radiolabeled engineered antibodies requires, among other parameters, matching blood clearance and tumor uptake with the half-life of the engineered antibody. Although diabodies have favorable molecular sizes (50 kDa) for rapid blood clearance (t 1/2 = 30-60 min) and are bivalent, thereby increasing tumor uptake, they exhibit substantial kidney uptake as their major route of clearance, which is especially evident when they are labeled with the PET isotope 64 Cu (t 1/2 = 12 hr). To overcome this drawback, diabodies may be conjugated to PEG, a modification that increases the apparent molecular size of the diabody and reduces kidney uptake without adversely affecting tumor uptake or the tumor to blood ratio. We show here that site specific attachment of monodispersed PEGn of increasing molecular size (n= 12, 24, and 48) can uniformly increase the apparent molecular size of the PEG-diabody conjugate, decrease kidney uptake and increase tumor uptake, the latter due to the increased residence time of the conjugate in the blood. Since the monodispersed PEGs were pre-conjugated to the chelator DOTA, the conjugates were able to bind radiometals such as 111 In and 64 Cu that can be used for SPECT and PET imaging, respectively. To allow conjugation of the DOTA-PEG to the diabody, the DOTA-PEG incorporated a terminal Cysteine conjugated to a vinyl sulfone moiety. In order to control the conjugation chemistry, we have engineered a surface thiolated diabody that incorporates two cysteines per monomer (four per diabody). The thiolated diabody was expressed and purified from bacterial fermentation and only needs to be reduced prior to conjugation to the DOTA-PEGn-Cys-VS. This novel imaging agent (a diabody with DOTA-PEG48-Cys-VS attached to introduced thiols) gave up to 80 %ID/g of tumor uptake with a tumor to blood ratio (T/B) of 8 at 24h when radiolabeled with 111 In and 37.9% ID/g of tumor uptake (T/B= 8) at 44h when radiolabeled with 64 Cu in PET imaging in an animal model. Tumor uptake was significantly improved from the 50% ID/g at 24 hours observed with diabodies that were pegylated on surface Lysine residues. Importantly, there was no loss of immunoreactivity of the site-specific Cysconjugated diabody to its antigen (TAG-72) compared to the parent, unconjugated diabody. We propose that thiolated diabodies conjugated to DOTAylated monodisperse PEGs have the potential for superior SPECT and PET imaging in a clinical setting.

Microarray technology has become an important tool for detection and analysis of nucleic acid targets. Immobilization of modified and unmodified oligonucleotides on epoxy-functionalized glass surfaces is often used in microarray fabrication. Here, we demonstrate a protocol that employs coating of SU-8 (glycidyl ether of bisphenol A) onto glass microslides to obtain high density of epoxy functions for efficient immobilization of aminoalkyl-, thiophosphoryl-, and phosphorylated oligonucleotides with uniform spot morphology. The resulting microarrays exhibited high immobilization (∼65%) and hybridization efficiency (30-36%) and were sufficiently stable over a range of temperature and pH conditions. The prominent feature of the protocol is that spots can be visualized distinctly at 0.05 µM probe (a 20-mer oligonucleotide) concentration. The constructed microarrays were subsequently used for detection of base mismatches and bacterial diseases (meningitis and typhoid).

The 5 0 -γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates (3 and 4), containing the poly(ethylene glycol) spacers, were synthesized and compared to a hydrophobic analogue as co-substrates for the following protein kinases: sarcoma related kinase (Src), cyclin-dependent kinase (CDK), casein kinase II (CK2R), and protein kinase A (PKA). Electrochemical kinase assays indicate that the hydrophobic Fc-ATP analogue was an optimal co-substrate for which K M values were determined to be in the 30À200 μM range, depending on the particular protein kinase. The luminescence kinase assay demonstrated the kinase utility for all Fc-ATP conjugates, which is in line with the electrochemical data. Moreover, Fc-ATP bioconjugates exhibit competitive behavior with respect to ATP. Relatively poor performance of the polar Fc-ATP bioconjugates as co-substrates for protein kinases was presumably due to the additional H-bonding and electrostatic interactions of the poly(ethylene glycol) linkers of Fc-ATP with the kinase catalytic site and the target peptides. Phosphorylation of the full-length protein, His-tagged pro-caspase-3, was demonstrated through Fc-phosphoamide transfer to the Ser residues of the surface-bound protein by electrochemical means. These results suggest that electrochemical detection of the peptide and protein Fc-phosphorylation via tailored Fc-ATP co-substrates may be useful for probing proteinÀprotein interactions. ARTICLE similar spacer length. 14 Here, we explore the kinase promiscuity, only with respect to Fc-ATP, and investigate the usefulness of hydrophobic and hydrophilic Fc-ATP co-substrates for peptide phosphorylations by multiple protein kinases. We probe the effects of the polar versus nonpolar Fc-ATP conjugates on four protein kinases and their ability to drive peptide phosphorylation. By electrochemical means, we demonstrate the usefulness of the analytical method for detection of protein phosphorylation as well, as presented in Scheme 1B. The kinase reaction parameters were estimated electrochemically, and the electrochemical trends were compared to the fluorescence kinase assay for the first time (Scheme 1C).

Radiolabeling of liposomes with 64 Cu (t 1/2 = 12.7 h) is attractive for molecular imaging and monitoring drug delivery. A simple chelation procedure, performed at a low temperature and under mild conditions, is required to radiolabel pre-loaded liposomes without lipid hydrolysis or the release of the encapsulated contents. Here we report a 64 Cu post-labeling method for liposomes. A 64 Cuspecific chelator, 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N',N",N"'tetraacetic acid (BAT), was conjugated with an artificial lipid to form a BAT-PEG-lipid. After incorporation of 0.5% (mol/mol) BAT-PEG-lipid during the liposome formulation, liposomes were successfully labeled with 64 Cu in 0.1 M NH 4 OAc pH 5 buffer, at 35 °C for 30~40 min with an incorporation yield as high as 95%. After 48 hour incubation of 64 Cu-liposomes in 50/50 serum/PBS solution, more than 88% of the 64 Cu label was still associated with liposomes. After injection of liposomal 64 Cu in a mouse model, 44 ± 6.9, 21 ± 2.7, 15 ± 2.5, and 7.4 ± 1.1 (n = 4) % of the injected dose per cubic centimeter remained within the blood pool at 30 min, 18, 28, and 48 hours, respectively. The biodistribution at 48 hours after injection verified that 7.0 ± 0.47 (n = 4), and 1.4 ± 0.58 (n = 3) % of the injected dose per gram of liposomal 64 Cu and free 64 Cu remained in the blood pool, respectively. Our results suggest that this fast and easy 64 Cu labeling of liposomes could be exploited in tracking liposomes in vivo for medical imaging and targeted delivery.

Targeted delivery of therapeutics possesses the potential to localize therapeutic agents to a specific tissue as a mechanism to enhance treatment efficacy and abrogate side effects. Antibodies have been used clinically as therapeutic agents and are currently being explored for targeting drug-loaded nanoparticles. Peptides such as RGD peptides are also being developed as an inexpensive and stable alternative to antibodies. In this study, cyclo(1,12)PenITDGEATDSGC (cLABL) peptide was used to target nanoparticles to human umbilical cord vascular endothelial cell (HUVEC) monolayers that have upregulated intercellular cell-adhesion molecule-1 (ICAM-1) expression. The cLABL peptide has been previously demonstrated to possess high avidity for ICAM-1 receptors on the cell surface. Poly(DL-lactic-coglycolic acid) nanoparticles conjugated with polyethylene glycol and cLABL demonstrated rapid binding to HUVEC with upregulated ICAM-1, which was induced by treating cells with the proinflammatory cytokine, interferon-γ. Binding of the nanoparticles could be efficiently blocked by preincubating cells with free peptide suggesting that the binding of the nanoparticles is specifically mediated by surface peptide binding to ICAM-1 on HUVEC. The targeted nanoparticles were rapidly endocytosed and trafficked to lysosomes to a greater extent than the untargeted PLGA-PEG nanoparticles. Verification of peptide-mediated nanoparticle targeting to ICAM-1 may ultimately lead to targeting therapeutic agents to inflammatory sites expressing upregulated ICAM-1.