Ic103: Materials Chemistry-I: Institute Core Course For Btech Program

Institute Core Course for BTech Program
IC103: Materials Chemistry-I
Dr. Sanjib Banerjee

Assistant Professor
Department of Chemistry
IIT Bhilai
Aug 21, 2018 Materials Chemistry-I, IC103, 2018-19 M Semester 1

Lecture 7: Peptides/Proteins

Peptides/Proteins
Proteins and peptides are polymers made up of amino acid units (residues)
that are linked together through the formation of amide bonds (peptide bonds)
from the amino group of one residue and the carboxylate of a second residue
Peptide
By convention, peptide sequences

are written left to right from the
N-terminus to the C-terminus
Protein

Amide bond
The amide (peptide) bond has C=N double bond character due to resonance
resulting in a planar geometry
restricts rotations
resistant to hydrolysis
The N-H bond of one amide linkage can form a hydrogen bond with the C=O of another.
N-O distance 2.85 - 3.20 Å
optimal N-H-O angle is 180 °
Disulfide bonds: the thiol groups of cysteine can be oxidized to form disulfides (Cys-S-S-Cys)
NaOH/Br2
Or Zn/HCl enzymetic catalyst
RSH
The Strategy for Peptide Synthesis
The need for protecting groups
Orthogonal protecting group strategy: the carboxylate protecting group must be stable to the
reaction conditions for the removal of the -amino protecting group and ( vice versa)

Amino Group Protection
The -amino group is protected as a carbamate.

Carboxyl Group Protection
Protected as a benzyl ester; removed by hydrogenolysis

Peptide Bond Formation
Amide formation from the reaction of an amine with a carboxylic acid is slow. Amide bond
formation (peptide coupling) can be accelerated if the carboxylic
acid is activated. Reagent: dicyclohexylcarbodiimide (DCC)
O O C6H11 C6H11
O O
H NH NH
R O R O R O C R O C
+ +N
N R' H N
C6H11 N C N C6H11 + H C6H11
C6H11 N C N C6H11 C6H11
(DCC) H ••
R'-NH2 "activated acid"
C6H11 O O
O
NH R' + C6H11 C6H11
R O C R N N N
NH H H H
R' HN
+ C6H11 Amide DCU

Peptide Synthesis
• In order to practically synthesize peptides and proteins, time

consuming purifications steps must be avoided until the very
end of the synthesis.
• Large excesses of reagents are used to drive reactions forward
and accelerate the rate of reactions.
• How are the excess reagents and by-products from the reaction,
which will interfere with subsequent coupling steps, removed
without a purification step?

Solid-Phase Peptide Synthesis
The Merrifield Method.

Peptides and proteins up to ~ 100 residues long are synthesized on a solid,
insoluble, polymer support. Purification is conveniently accomplished after
each step by a simple wash and filtration.


Ribonuclease A- 124 amino acids, catalyzes the hydrolysis of RNA

Solid-phase synthesis of RNase A:
Synthetic RNase A: 78 % activity

0.4 mg was synthesized
2.9 % overall yield
average yield ~ 97% per coupling step His-119 A His-12 A
LYS GLU THR ALA ALA ALA LYS PHE GLU ARG
GLN HIS MET ASP SER SER THR SER ALA ALA
SER SER SER ASN TYR CYS ASN GLN MET MET
LYS SER ARG ASN LEU THR LYS ASP ARG CYS
LYS PRO VAL ASN THR PHE VAL HIS GLU SER
LEU ALA ASP VAL GLN ALA VAL CYS SER GLN His-12 B
LYS ASN VAL ALA CYS LYS ASN GLY GLN THR His-119 B
ASN CYS TYR GLN SER TYR SER THR MET SER
ILE THR ASP CYS ARG GLU THR GLY SER SER
LYS TYR PRO ASN CYS ALA TYR LYS THR THR
GLN ALA ASN LYS HIS ILE ILE VAL ALA CYS
GLU GLY ASN PRO TYR VAL PRO VAL HIS PHE pdb code: 1AFL
ASP ALA SER VAL
R. Bruce Merrifield, Rockefeller University, 1984 Nobel Prize in Chemistry:

“for his development of methodology for chemical synthesis on a solid matrix.”
Importance of Disulfide Linkage
Epidermal Growth Factor (EGF): the miracle of mother’s spit

53 amino acid, 3 disulfide linkages
1986 Nobel Prize in Medicine or Physiology :

Stanley Cohen
Rita Levi-Montalcini
Protein Structure
primary (1°) : the amino acid sequence (peptide bonds)

secondary (2°): frequently occurring substructures or folds
(H-bonding of the backbone)
tertiary (3°): three-dimensional arrangement of all atoms in a
single polypeptide chain
quaternary (4°): overall organization of non-covalently linked
subunits of a functional protein
Protein Structure
NC
anti-parallel loop
or
turn
NC
CN
CN
parallel
NC
NC
crossover
NC
NC
Protein Structure
-helix: 3.6 amino acids per coil, 5.4 Å
C 3.6 AA
5.4 Å

Protein Structure
myoglobin
pdb code: 1WLA
Bacteriorhodopsin
pdb code: 1AP9
Parallel -sheets Anti-parallel

carbonic anhydrase -sheets
pdb code: 1QRM
of lectin
pdb code: 2LAL
Protein Structure
Tertiary Structure of polypeptides and Proteins.
Fibrous. Polypeptides strands that “bundle” to form elongated
fibrous assemblies; insoluble.
Globular. Proteins that fold into a “spherical” conformation.
Hydrophobic effect. Proteins will fold so that hydrophobic amino

acids are on the inside (shielded from water) and hydrophilic
amino acids are on the outside (exposed to water)
Pro • Ile • Lys • Tyr • Leu • Glu • Phe • Ile • Ser • Asp • Ala • Ile • Ile • His •Val • His • Ser • Lys

Protein Structure Determination
1. Determine the amino acids present and their relative ratios

2. Cleave the peptide or protein into smaller peptide fragments
and determine their sequences
3. Cleave the peptide or protein by another method and
determine their sequences. Align the sequences of the
peptide fragments from the two methods

Amino Acid Analysis
Automated method to determine the amino acid content of a peptide or protein
Reaction of primary amines with ninhydrin
• Amine functional group of α-amino acids reacts with ninhydrin to form purple-
colored compounds.
• Ninhydrin is used to detect fingerprints because it reacts with amino acids
from the proteins in skin cells transferred to the surface by the individual
leaving the fingerprint.

Amino Acid Analysis
Enzymatic
peptide [H] reduce any digestion
-or- disulfide individual
-or- amino acids
protein bonds
H 3O + , 
liquid derivatize w/ Detected w/
chromatography ninhydrin UV-vis
Different amino
acids have different 1972 Nobel Prize in Chemistry
chromatographic William Stein
mobilities (retention Stanford Moore
times)

Partial Hydrolysis of Peptides
Acidic hydrolysis of peptides cleave the amide bonds indiscriminately.
Proteases (peptidases): Enzymes that catalyzed the hydrolysis of the
amide bonds of peptides and proteins.
Enzymatic cleavage of peptides and proteins at defined sites:
• trypsin: cleaves at the C-terminal side of basic residues,
Arg, Lys but not His
• chymotrypsin: cleaves at the C-terminal side of aromatic residues

Phe, Tyr, Trp
• Trypsin and chymotrypsin are endopeptidases

• Carboxypeptidase: Cleaves the amide bond of the C-terminal amino acid (exopeptidase)
End Group Analysis
• The C-terminal AA is identified by treating with peptide with

carboxypeptidase, then analyzing by liquid chromatography
(AA Analysis).
• N-labeling: The peptide is first treated with 1-fluoro-2,4-
dinitro benzene (Sanger’s reagent), which selectively reacts
with the N-terminal amino group. The peptide is then
hydrolyzed to their amino acids and the N-terminal amino
acid identified as its N-(2,4-dinitrophenyl) derivative (DNP).

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Institute Core Course for BTech Program

IC103: Materials Chemistry-I

Dr. Sanjib Banerjee

Aug 21, 2018 Materials Chemistry-I, IC103, 2018-19 M Semester 1

Aug 21, 2018 Materials Chemistry-I, IC103, 2018-19 M Semester 2

By convention, peptide sequences

Aug 21, 2018 Materials Chemistry-I, IC103, 2018-19 M Semester 3

N-O distance 2.85 - 3.20 Å

optimal N-H-O angle is 180 °

The need for protecting groups

Aug 21, 2018 Materials Chemistry-I, IC103, 2018-19 M Semester 5

The -amino group is protected as a carbamate.

Aug 21, 2018 Materials Chemistry-I, IC103, 2018-19 M Semester 6

Protected as a benzyl ester; removed by hydrogenolysis

Aug 21, 2018 Materials Chemistry-I, IC103, 2018-19 M Semester 7

Aug 21, 2018 Materials Chemistry-I, IC103, 2018-19 M Semester 8

• In order to practically synthesize peptides and proteins, time

Aug 21, 2018 Materials Chemistry-I, IC103, 2018-19 M Semester 9

The Merrifield Method.

Aug 21, 2018 Materials Chemistry-I, IC103, 2018-19 M Semester 10

Aug 21, 2018 Materials Chemistry-I, IC103, 2018-19 M Semester 11

Ribonuclease A- 124 amino acids, catalyzes the hydrolysis of RNA

Synthetic RNase A: 78 % activity

R. Bruce Merrifield, Rockefeller University, 1984 Nobel Prize in Chemistry:

Epidermal Growth Factor (EGF): the miracle of mother’s spit

1986 Nobel Prize in Medicine or Physiology :

primary (1°) : the amino acid sequence (peptide bonds)

-helix: 3.6 amino acids per coil, 5.4 Å

Aug 21, 2018 Materials Chemistry-I, IC103, 2018-19 M Semester 16

Parallel -sheets Anti-parallel

Globular. Proteins that fold into a “spherical” conformation.

Hydrophobic effect. Proteins will fold so that hydrophobic amino

Aug 21, 2018 Materials Chemistry-I, IC103, 2018-19 M Semester 18

1. Determine the amino acids present and their relative ratios

Aug 21, 2018 Materials Chemistry-I, IC103, 2018-19 M Semester 19

Aug 21, 2018 Materials Chemistry-I, IC103, 2018-19 M Semester 20

Aug 21, 2018 Materials Chemistry-I, IC103, 2018-19 M Semester 21

• chymotrypsin: cleaves at the C-terminal side of aromatic residues

• Trypsin and chymotrypsin are endopeptidases

• The C-terminal AA is identified by treating with peptide with

Aug 21, 2018 Materials Chemistry-I, IC103, 2018-19 M Semester 23

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