UNIT 18

POLYPEPTIDES AND PROTEINS

1. GENERAL FEATURES

2. STRUCTURE

3. DETERMINATION OF THE PRIMARY STRUCTURE

4. SYNTHESIS OF PEPTIDES

1
 Unit 18

Literature:

• Units 2-5 Foundations of Chemical Biology, C. M. Dobson, J. A.

Gerrard, A. J. Pratt, pp. 7-50

• Tema 6 Química Orgánica, Parte 4, J. M. Tedder, A. Nechvatal, A.

W. Murray, J. Carnduff, pp. 305-328

2
 UNIT 18
POLYPEPTIDES AND PROTEINS

1. GENERAL FEATURES

2. STRUCTURE

3. DETERMINATION OF THE PRIMARY STRUCTURE

4. SYNTHESIS OF PEPTIDES

3
1. GENERAL FEATURES Unit 18

The Peptide Bond

_
O O
+
N N
H H

The peptide bond is very strong. It has some carácter It is a very stable bond. Unreactive
of doublé bond with Erot = 20 Kcal/mol Hydrolysis, alkylation, etc.. 4
 UNIT 18
POLYPEPTIDES AND PROTEINS

1. GENERAL FEATURES

2. STRUCTURE

3. DETERMINATION OF THE PRIMARY STRUCTURE

4. SYNTHESIS OF PEPTIDES

5
2. STRUCTURE Unit 18

Composition and Sequence

Polar chains are linked by hydrogen bonds

O
N H O
N H

Disulfide bridges are formed between two molecules of cysteine. This weak bond can be easily
broken by thyolates (RS-Na), bases, reductants (NaBH4 ) or oxidants (KMnO4)

O
H
O
H N H [O] H N S
SH + HS S N H
H N H [H] H
O O

Cys Cys 6
2. STRUCTURE Unit 18

Primary Structure

The amino acid sequence in a polypeptide chain (proteins), including the disulfide bridges.
The number of possible combinations of natural aas is enormous.

7
2. STRUCTURE Unit 18

Secondary Structure Well defined tridimensional arrangement for each chain.

Repetitive patterns with hydrogen bonds in specific positions.

β- Turn
-Helix Two types of β-sheets

Antiparallel
β- sheets

Parallel β- sheets
(Main chains in the
same direction)

8
2. STRUCTURE Unit 18

Ternary Structure Quaternary Structure

Association of separate protein chains to

Overall tridimensional organization
form a cohesive structure.
of a single chain (intramolecular interactions)
(Oligomers, dimers, tetramers, etc)

Classification Fibrous proteins (fibers)

Globular proteins (compact)

e.g. collagen enzymes, etc.

Dodecamer of aspartate
transcarbamilase

9
 UNIT 18
POLYPEPTIDES AND PROTEINS

1. GENERAL FEATURES

2. STRUCTURE

3. DETERMINATION OF THE PRIMARY STRUCTURE

4. SYNTHESIS OF PEPTIDES

10
3. DETERMINATION OF THE PRIMARY STRUCTURE Unit 18

STEPS

I. Purification
Usually contaminated by other peptides

Electrophoresis
Separation Column Chromatography
Techniques Dialysis
Preparative HPLC

II. Protein degradation into amino acids

Common process: Trp, Asn and Gln decompose under these conditions.
Complete hydrolysis with 6 M HCl at 110 ºC Asn and Gln give Asp and Glu, respectively.

Before hydrolysis, Cys (-CH2SH) is quantitatively oxidized to cysteic acid (CH2SO3H) which
is more robust.
After hydrolylis, the free amino acids are isolated by ion-exchange chromatography and
the TLC is stained with ninhydrin.

11
3. DETERMINATION OF THE PRIMARY STRUCTURE Unit 18

III. Determination of the sequence of aa

Sanger’s reagent (DNFB)

Identification of the N-terminal aa

The peptide is labelled by the reaction shown below and, after hydrolysis,
the N-terminus is determined by comparison with all DNP-aa by
chromatography. The intensity of the yellow color its proportional to [aa].

The Dansyl reagent

(similar method)

12
3. DETERMINATION OF THE PRIMARY STRUCTURE Unit 18

Edman degradation
The reaction with phenyl isothiocyanate gives a thiazolinone with the N-terminal aa. Under acidic
conditions, this intermediate rearrange to form a more stable phenylthiohydantoin, which is
identified by chromatography. The process is repeated to determine the new N-terminal residue.

13
3. DETERMINATION OF THE PRIMARY STRUCTURE Unit 18

Hydrazinolysis

Might help to identify the C-terminus of the sequence ( free CO2H)

14
3. DETERMINATION OF THE PRIMARY STRUCTURE Unit 18

Enzymes that selectively cleave amino acids from the end of the
Exopeptidases
polypeptide chain

Aminopeptidases
Carboxypeptidases

15
3. DETERMINATION OF THE PRIMARY STRUCTURE Unit 18

Sequencing using
Useful for small peptides. Fragmentations occur at the peptide
Mass Spectrometry bond.

442 475 362 249

Ph
H O H O H O
( )18 N N N
N N OMe (OH)
O H O H O Me
Ph
16
102
3. DETERMINATION OF THE PRIMARY STRUCTURE Unit 18

IV. Relate all information

Estructura elucidada por Frederick Sanger (1954, Universidad de Cambridge)

Insulin
It was the first protein elucidated by Frederick Sanger in 1954 (Cambridge University).
F. S. was awarded the Nobel Prize for Chemistry for this work in 1958.

a) The Sanger’s reagent as a label for N-terminal aa

Tools used:
b) Fractional hydrolysis
c) TLC for the analysis of aa

Two peptide chains of 30 and 21 amino acids link by two disulfide bridges.

H2N Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Tyr Thr Pro Lys Thr

S S
S S
H2N Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn

S S
17
 UNIT 18
POLYPEPTIDES AND PROTEINS

1. GENERAL FEATURES

2. STRUCTURE

3. DETERMINATION OF THE PRIMARY STRUCTURE

4. SYNTHESIS OF PEPTIDES

18
4. SYNTHESIS OF PEPTIDES Unit 18

Steps

19
4. SYNTHESIS OF PEPTIDES Unit 18

Protecting groups of -NH2

Benzyloxycarbonyl (Z or Cbz)

tert-Butoxycarbonyl (Boc)

20
30
4. SYNTHESIS OF PEPTIDES Unit 18

9-Fluorenylmethoxycarbonyl (Fmoc)

Protecting groups of –CO2H

Esters

21
4. SYNTHESIS OF PEPTIDES Unit 18

Activation of –CO2H to form the peptide bond

Dicyclohexilcarbodiimide (DCC)

R'
O
R Cy H2N P' R O
Cy R H
P O N O P N P'
N C N + P N OH N C N O
Cy H H H
O HN O R'
O Cy O
Cy Cy
N N
H H

Mixed Anhydride methods

R'
O
H2N P'
R O R R O
O H
P OH + P O X P N P'
N X Cl N N O
H H H
O HCl O O O R'
O
[X = t-Bu, OEt] (base)
HO X

22
4. SYNTHESIS OF PEPTIDES Unit 18

Synthesis of Ala-Val-Phe

Ph Ph
MeOH
OH OMe
H2N H+ H2N
O O
Phe

O
BnOCOCl H
OH OH N
H2N CbzN DCC CbzN OMe
H H
O O O
Ph
Val
H2 / Pd

O O O
H H DCC H
CbzN N N
N OMe H2N OMe
H
O O
Ph Ph
1) H2 / Pd
2) HCl / H2O

OH BnOCOCl OH
O O CbzN H2N
H H
H2N N O O
N OH
H Ala
O 23
Ph
4. SYNTHESIS OF PEPTIDES Unit 18

Solid phase peptide synthesis

R
_
O K+
BocN
ClCH2OMe H
O
SnCl4 SN
Cl

R R
O O
NBoc HCl NH2
H
O O

R'
DCC OH
BocN
H
O
Cl
+ R O
H
1) CF3CO2H O NBoc
N
R O H
2) HBr O R'
HO NH2
N
H 24
O R'