The Strategy of Peptide

Synthesis
Amino Acids are Structurally Bifunctional

H O
N
H OH H O
H
N
H OH
Glycine

L-Phenylalanine
Possible Products from the Condensation of
Phenylalanine and Glycine

O H O
H2N OH N
N H2N OH
H H
O O
Gly-Gly
Phe-Gly

O O
H H
H
N N
H2N OH H2N OH
H H
O O

Gly-Phe Phe-Phe
 The Challenge of Peptide Synthesis

•Making peptide bonds between amino acids is

not difficult: there are numerous methods to
make amides from amines and carboxylic acids.

•The challenge is connecting amino acids in the

correct sequence.

•Random peptide bond formation in a mixture of

phenylalanine and glycine, for example, will give
four dipeptides.

•Phe—Phe Gly—Gly Phe—Gly Gly—Phe

 Step 1 of Peptide Synthesis: Protection

• 1.Limit the number of possible reactions

by "protecting" the nitrogen of one amino
acid and the carboxyl group of the other.
H O
N
PG OH H O
H PG
N
H O

O-Protected
Glycine
PG
N-Protected N-Nucleophile
L-Phenylalanine
protecting
C-Electrophile group
 Step 2 of Peptide Synthesis: Coupling

• 2. Couple the two protected amino acids.

H O
N
PG OH H O
H PG
N
H O

Peptide
Coupling
H O
N O
PG N PG
H H
O

PG-Phe-Gly-PG
 Peptide Synthesis, Step 3: Global
Deprotection
• 3.Deprotect the amino group at the N-
terminus and the carboxyl group at the C-
terminus. H O
N O
PG N PG
H H
O

Deprotection

O
H3N O
N
H H
O

Phe-Gly
Amino Group Protection
Peptide Synthesis: Amine Protecting Groups

1. Amino groups are normally protected by

converting them to amides. The nitrogen
atom in an amide does not behave as a
nucleophile and will not react with carboxyl
groups.

2. Benzyloxycarbonyl (C6H5CH2O—) is a
common protecting group. It is abbreviated
as Z or Cbz.

3. Cbz-protection is carried out by treating an

amino acid with benzyloxycarbonyl chloride.
 Protecting the Amino Group

Amino groups can behave as nucleophiles and undergo

reaction with carboxylic acid derivatives. The nitrogen atom
in amides is much less nucleophilic. As a result, amide
derivatives of amines can be viewed as protecting groups.

Amine Amide Derivative

O O
H
H2N R2 N
OR OR
O
Nucleophilic
Atom Non-Nucleophilic
Atom
Amine Protecting Groups: Introduction of
Benzyloxycarbonyl Protecting Groups

O
O H3N 1. NaOH
O H2O
O Cl H
2. H3O+

O
H
O N
OH
H
O

(85%)
Benzyloxycarbonyl is Abbreviated to Cbz or Z

O O
H H
O N N
OH Cbz OH
H H
O O
H
N
Z OH
H

Cbz-Phe
 Cleavage of Cbz Groups

• An advantage of the benzyloxycarbonyl protecting

group is that it is easily removed by:

a) catalytic hydrogenation

b) cleavage with HBr in acetic acid

Hydrogenolysis of Cbz Groups

O
H
O N OEt
N
H H
O O

H2, Pd/C
solvent

O
H
HO N OEt
N
H2C H H H
O O

Toluene Carbamic Acid

99 %
(Very Unstable)
(volatile)
Hydrogenolysis of Cbz Groups
O
H
HO N OEt
N
H H
O O

Spontaneous
decarboxylation

O
H2N OEt
N
H H
O
O
C
(100%)
O
Acid-Mediated Cleavage of Cbz Groups

O
H
O N OEt
N
H H
O O

HBr
acetic acid

O
Br H3N OEt
N
H2C Br H H
O
O
Benzyl
bromide C
(volatile) (82%)
O
 Amine Protecting Groups: tert-
Butyloxycarbonyl
O O O O
R
O N O O O O
H
tert-Butyloxycarbonyl Di-tert-butyl dicarbonate
group (Boc 'anhydride')

O Cl
tert-Butyl chloride
(instablity limits use)
tert-Butyloxycarbonyl is Abbreviated to Boc

O O
H H
O N N
OH Boc OH
H H
O

Boc-Phe
 Cleavage of Boc Groups

The tert-butyloxycabonyl protecting group is readily

removed by:

a) Trifluoroacetic acid in methylene chloride

b) cleavage with HBr in acetic acid

Both reagents cleave the quaternary carbon-

oxygen ether bond by an acid-mediated elimination
reaction.
Acid-Mediated Cleavage of Boc Groups
O
H
O N OEt
N
H H
O O
O
trifluoroacetic
acid
F3C OH

O O
H3N OEt
Butene F3C O N
H H
(volatile)
O
H H O
C
(high yield)
H3C CH3 O
Carboxyl Protecting
Group
Peptide Synthesis: Carboxyl Protecting
Groups

-Carboxyl groups are normally activated as

esters.

-Deprotection of methyl and ethyl esters is by

hydrolysis in base.
 Carboxylic acid groups are usually protected by
conversion to anhydride:

ex: Ethyl chloroformate can be used

23
Peptide Bond Formation
 Peptide Coupling is a Condensation
Reaction
• Couple the two protected amino acids.
H O
N
PG OH H O
H PG
N
H O

Peptide
Coupling -H2O
H O
N O
PG N PG
H H
O
 DCC-Mediated Peptide Coupling

• Couple the two protected amino acids.

H O H O
N N Et
Cbz OH H O
H

DCC, CHCl3

H O
N O
Cbz N Et
H H
O

(83%)
 Mechanism of DCC-Promoted
Coupling
H O
N
Cbz OH N C N
H

1,2-Addition

H O N
N
Cbz O N
H H

O-Acylisourea
derivative
 Mechanism of DCC-Promoted
Coupling

H O N
N
Cbz O N
H
H O
N
Attack by the H OEt
amine function of
H OH N
the carboxyl- 1,2-Addition
protected amino then N
Proton Transfer Cbz O N
acid on the N H
carbonyl group H
Unstable
leads to Intermediate
nucleophilic acyl O OEt
substitution.
Mechanism of DCC-Promoted
Coupling

H OH N Unstable
Intermediate
N
Cbz O N
N H
H

Elimination
O OEt
H O
N OEt
Cbz N
H
O H O

N N
H H
Dipeptide
N,N'-dicyclohexylurea