Proteins are the primary functional manifestation of the information in

genomes

atgcaaactctttctgaacgcctcaagaagaggcgaattgcgttaaaaatg
DNA sequence acgcaaaccgaactggcaaccaaagccggtgttaaacagcaatcaattca
actgattgaagctggagtaaccaagcgaccgcgcttcttgtttgagattgct
transcription atggcgcttaactgtgatccggtttggttacagtacggaactaaacgcggta
aagccgcttaa
augcaaacucuuucugaacgccucaagaagaggcgaauugcguuaaaaaugacgca
RNA sequence aaccgaacuggcaaccaaagccgguguuaaacagcaaucaauucaacugauugaagc
uggaguaaccaagcgaccgcgcuucuuguuugagauugcuauggcgcuuaacugug
auccgguuugguuacaguacggaacuaaacgcgguaaagccgcuuaa
translation
MQTLSERLKKRRIALKMTQTELATKAGVKQQSIQLIEA
protein GVTKRPRFLFEIAMALNCDPVWLQYGTKRGKAA
sequence

protein protein
structure function
 Immune peptides: Hormones: Neuropeptides:
synthetic antigens; oxytocin substance P
vaccines vasopressin cholecystokinin
diagnostic tools insulin neurotensin
immunostimulator peptides; somatostatin
muramyl dipeptide GnRH
etc. Antibiotics:
tuftsin derivatives
tachikinin
gramicidine S

Transporter peptides: Applications of

penetratin synthetic peptides Toxins:
oligoarginine conoAtoxins
HIV-Tat protein spider toxins
snake toxins
Carriers: ionchanel blockers
templates
Peptides
for structural studies: Enzymes and
turn mimicking cyclic peptides enzyme inhibitors:
miniproteins
Ribonuclease A
 alpha carbon
O O

H2N CH C OH H3N CH C O

R R

amino group carboxylic acid

group The zwitterionic form is
the predominant form at
neutral pH
side chain

C O
H3N C

H R

The alpha carbon is

a chiral center--natural
proteins are made of
L amino acids (shown
above) as opposed to
D
 The protein alphabet--the 20 amino acid R groups
NH3 

2 1 CH2 
2
O O 1 HN
C CH2  1 H3C CH3
O O
2 N 1 CH3 
2 1 CH 
SH  C  CH2    CH2 
2 H3C CH2 1

CH2  CH2  CH2  CH2  H CH2  CH  CH2  CH2 

CH3 

A C D E F G H I K L

2
 H2N NH2   
2 1 OH
C  NH

CH3  3
 2
NH2 2
NH  1
1 O 2
S NH2 2 3 1
O 1 HO CH3  
C CH2 
 CH  2 1
CH2  C  CH2 
H2 
 C CH2  CH2  OH 
1 H3C CH3 
CH2  CH2  H2C  CH2 
CH2 CH2  CH2  CH2  CH 
HC N

V W Y
M N Q R S T
P
 Proteins are made by controlled polymerization of amino acids

water is eliminated

O O

two amino acids H2N CH C OH H2N CH C OH

condense to form...
R1 R2

N or amino C or carboxy
terminus O O terminus

...a dipeptide. If H2N CH C NH CH C OH + HOH

there are more it
becomes a polypeptide. R1 R2
Short polypeptide chains
are usually called peptides
while longer ones are called peptide bond is formed
proteins.
residue 1 residue 2
Peptide synthesis – Production of peptides, in which
multiple amino acids are linked via amide bonds which are
also known as peptide bonds

Objectives

 Structure verifications
 Structure activity relations
 Synthesis of medicinally important peptides
 New peptide based immunogens
Peptide synthesis is the stepwise condensation based on the
repetitive addition of single N protected amino groups to a
growing amino component

Chemical peptide synthesis starts at the C-terminal end of the

peptide and ends at the N-terminus

C N
 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

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

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
 Step 1 of Peptide Synthesis: Protection

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

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

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
 Solid phase peptide synthesis (SPPS)

P2
P1
O
O

Fmoc HN AA2 Fmoc HN AA1 Resin

OH

activation deblocking
Fmoc

P2 P1
O O

Fmoc HN AA2 H2N AA1 Resin

Resin A
solid support

Fmoc coupling repeat steps

fmoc protecting group A for each amino
acid in peptide,
then deblock,
P1 P2 protecting groups deprotect,
for side chains P2 P1 cleave off resin
O O
AA1 AA2 1st and 2nd
amino acids Fmoc HN AA2 NH AA1 Resin
A carbonyl activating
group
 Solid-phase peptide synthesis (SPPS), pioneered by Robert
Bruce Merrifield

 It is now the accepted method for creating Peptides

and proteins in the lab in a synthetic manner

 SPPS allows the synthesis of natural peptides which are

difficult to express in bacteria, the incorporation of
unnatural amino acids, peptide/protein backbone
modification, and the synthesis of D-proteins, which consist
of D-amino acids
 Solid phase peptide synthesis involves three steps:

 Immobilization – The C-terminal amino acid of the growing peptide

chain is anchored covalently to a solid support or resin during
synthesis
 Chain assembly – Each amino acid is then added to the peptide
chain one at a time by a cyclic process involving three chemical
reactions
 activation
 coupling
 deprotection
 Cleavage – After the synthesis is completed, the peptide must be
removed from the resin and the protecting groups must be
removed from the side chains.
Activation
Coupling
Deprotection
Cleavage
Amino acids have reactive moieties at the N- and C-termini,
which facilitates amino acid coupling during synthesis

Many amino acids also have reactive side chain functional

groups, which can interact with free termini or other side chain
groups during synthesis and peptide elongation and negatively
influence yield and purity

To facilitate proper amino acid synthesis with minimal side

chain reactivity, chemical groups have been developed to bind
to specific amino acid functional groups and block, or protect,
the functional group from nonspecific reactions
 Reactive groups
Amino
Carboxyl
Amide
Alcoholic
Phenolic
Hydroxyl
Thiol
Indole
Imidazole
 Step 1 of Peptide Synthesis: Protection

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
Functions of a protective group
Selective introduction
Stable
Selective removable
 These protecting groups, while vast in nature, can be
separated into three groups, as follows:

 N-terminal protecting groups

 C-terminal protecting groups (mostly used in liquid-

phase synthesis)

 Side chain protecting groups

 Purified, individual amino acids are reacted with these

protecting groups prior to synthesis and then selectively
removed during specific steps of peptide synthesis
For amines:

 Boc / t-Boc (Butoxy carbonyl , ter Butoxy carbonyl)

 Fmoc (Fluorenylmethyloxycarbonyl)
 Tmsec (Tri methyl silyl ethyl chloroformate)

For Carboxylic acids:

 Ter-Bu-ester
 Fm ester (fluro methyl esters)
 Tmse ester (Tri methyl silyl ethyl ester)
Acid labile protecting groups: Boc and tert – Bu ester
Base labile protecting groups: Fmoc and Fm ester
Fluoride labile protecting groups: Tmsec and Tmse ester
Amino Group Protection
Amino groups can behave as nucleophiles and undergo reaction
with carboxylic acid derivatives

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

Amine Amide Derivative

O O
H
H2N R2 N
OR OR
O
Nucleophilic
Atom Non-Nucleophilic
Atom
 Peptide Synthesis: Amine Protecting Groups

 Benzyloxycarbonyl (C6H5CH2O—) is a common protecting

group. It is abbreviated as Z or Cbz

 Cbz-protection is carried out by treating an amino acid with

benzyloxycarbonyl chloride

O O
R
O Cl O N
H

Benzyloxycarbonyl Benzyloxycarbonyl
chloride group
(Cbz-Cl)
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%)
 Cleavage of Cbz Groups

 An advantage of the benzyloxycarbonyl protecting group is

that it is easily removed by:

 Catalytic hydrogenolysis under extremely mild

conditions

 Cleavage with HBr in acetic acid

 Both reagents cleave the relatively weak benzylic carbon-

oxygen ether bond, albeit by different mechanisms
 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

(volatile) (Very Unstable)
 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
(2 molecules of HBr reacts)

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 as 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 treatment with strong, anhydrous BrØnsted
acids:

 cleavage with trifluoroacetic acid in methylene chloride

 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 Group Protection
 Carboxyl groups are normally protected as esters – methyl,
ethyl and benzyl esters

 Deprotection of methyl and ethyl esters is by hydrolysis in

base

 Benzyl esters can be cleaved by hydrogenolysis (benzyl esters)

or by base hydrolysis (methyl and ethyl esters)
Simultaneous Hydrogenolysis of Cbz Group and Benzyl Ester

O
H
O N O
N
H H
O O

H2, Pd/C
solvent

O
H
HO N OH
N
H2C H H H
O O

Toluene Carbamic Acid

(volatile) (Very Unstable)
Simultaneous Hydrogenolysis of Cbz Group and Benzyl Ester

O
H
HO N OEt
N
H H
O O

Spontaneous
decarboxylation

O
H2N OEt
N
H H
O
O
C
(87%)
O
Peptide Bond Formation
 Peptide Synthesis: Forming Peptide Bonds

The two major methods are:

 coupling of suitably protected amino acids using N,N'-

dicyclohexylcarbodiimide (DCC)

 via an active ester of the N-terminal amino acid

 Step 2 of Peptide Synthesis: Coupling

 Peptide Coupling is a Condensation Reaction

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

Peptide
-
CouplingH2O
H O
N O
PG N PG
H H
O

PG-Phe-Gly-PG
Why are we using coupling agents ?

These coupling agents activates either the carboxyl (more

electrophile) or amino termial groups (more nucleophile)

The transient intermediates form a better leaving group

Activated groups are

 CO – N3
 CO – Cl
 – CO – O – CO – R
 – CO – OR’
N,N'-Dicyclohexylcarbodiimide (DCC) is a Powerful Dehydrating Agent

 First generation agents

 Forms symmetrical anhydrides which are highly reactive species

O H

H N
R1 O H R2 N C N

N,N'-dicyclohexylcarbodiimide
O H
H
N
R1 O H R2
'H2O'
Very high -H2O
temps O

O N N
H H
R2 N,N'-dicyclohexylurea
R1 N Amide

H
 DCC-Mediated Peptide Coupling

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 – Nucleophilic acyl
substitution reaction
H O
N
Cbz OH N C N
H

1,2-Addition

H O N
N
Cbz O N
H H

O-Acylisourea
derivative

Promotes dehydration between the free carboxyl group of an N

protected amino acid and the free amino group of the C
protected amino acid.
 H O N
N
Cbz O N
H
H O
N
H OEt

H OH N
1,2-Addition
then N
Proton Transfer Cbz O N
N H
H
Unstable
Intermediate
O OEt
 Peptide Synthesis: Active Ester Method

p-nitrophenyl ester

Is an example of an "active ester.”

Better leaving group than methyl or ethyl

More reactive in nucleophilic acyl substitution

O
N+ O
O O– is a more powerful Alkyl
acylating agent than...... O
O
Alkyl Ester
4-Nitrophenyl
(PNP) Ester
 NO2
H O
N
Cbz O
H O
N
H OEt
NO2
H OH
1,2-Addition
then N
Proton Transfer Cbz O
N
H Unstable
Intermediate

O OEt
 NO2
H OH
N Unstable
Cbz O Intermediate
N
H

Elimination
O OEt
H O
N OEt
Cbz N
H
H O
O2N OH

para-Nitrophenol Dipeptide
 Blocking side chains of amino acids

The side chain functional groups are masked with semi

permanent PG
 Peptide Synthesis, Step 3: Deprotection

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
Solid-Phase Peptide Synthesis:
The Merrifield Method
 Solid Phase Peptide Synthesis

 In solid-phase synthesis, the starting material is bonded to

an inert solid support.

 Reactants are added in solution.

 Reaction occurs at the interface between the solid and the

solution. Because the starting material is bonded to the
solid, any product from the starting material remains bonded
as well.

 Purification involves simply washing the byproducts from

the solid support.
 Solid Phase Peptide Synthesis - Overview
 In solid-phase synthesis, the starting material is bonded to an inert
solid support
 Reactants are added in solution
 Reaction occurs at the interface between the solid and the solution
 Because the starting material is bonded to the solid, any product from
the starting material remains bonded as well
 Purification involves simply washing the byproducts from the solid
support
 Synthetic intermediates don’t have to be isolated
 Excess reagents are used to drive reactions to completion
 Reagents simply washed away each step
 Overall quicker
 Can be automated with robots
 Solid Support

The characteristics of an efficient solid support include:

It must be physically stable and permit the rapid filtration of

liquids, such as excess reagents
It must be inert to all reagents and solvents used during SPPS
It must swell extensively in the solvents used to allow for
penetration of the reagents
It must allow for the attachment of the first amino acid
1. Gel-type supports
 Polystyrene: Styrene cross-linked with 1-2%
divinylbenzene
 Polyacrylamide: A hydrophilic alternative to polystyrene
 Polyethylene glycol (PEG)
 PEG-based supports: Composed of a PEG-polypropylene
glycol network or PEG with polyamide or polystyrene
2. Surface-type supports: controlled pore glass, cellulose fibers,
and highly cross-linked polystyrene
3. Composites: Gel-type polymers supported by rigid matrices.
 RESINS
 Can be functionalised
 Chemical stability (it must be inert to all applied chemicals)
 Mechanical stability (it shouldn’t brake under stirring)
 It must swell extensively in the solvents used for the
synthesis
 Peptide-resin bond should be stable during the synthesis
 Peptide-resin bond can be cleaved effectively at the end of
the synthesis
The basic of the most common used resins: polystyrene-1,4-
divinylbenzene (1-2%) copolymer

polymerisation
+
 Polystyrene is the Basis for the Solid Support

The solid support is a copolymer of styrene and

divinylbenzene – Merrifield resin

H H H H H H H H H H
C C C C C
C C C C C
H H H H
 Polystyrene resin is a versatile resin

Minimal swelling in dichloromethane

New resins have been developed with the following
advantages:
 Enhanced swelling or rigidity (a property of
mechanical strength)
 Chemical inertness
Highly cross-linked (50%) polystyrene has been developed
that possesses the features of increased mechanical stability,
better filtration of reagents and solvents, and rapid reaction
kinetics.
 Functionalization of Polystyrene – Chloromethylation of
Polystyrene

 Treating the polymeric support with chloromethyl methyl

ether (ClCH2OCH3) and SnCl4 places ClCH2 side chains on
some of the benzene rings
 Cl O
C Me
H2
SnCl4

CH2 CH2
Cl Cl
 CH2 CH2
Cl Cl

The side chain chloromethyl group is a benzylic halide, reactive

toward nucleophilic substitution (SN2)
 CH2 CH2
Cl Cl

 The chloromethylated resin is treated with the Boc-protected

C-terminal amino acid
 Nucleophilic substitution occurs, and the Boc-protected
amino acid is bound to the resin as an ester
 Merrifield Procedure

CH2 CH2 CH2 CH2

CH CH CH CH

O CH2Cl
–
BocNHCHCO
R
 CH2 CH2 CH2 CH2
CH CH CH CH

CH2
O
BocNHCHCO
R
Next, the Boc protecting group is removed with HCl
 CH2 CH2 CH2 CH2
CH CH CH CH

O
CH2
H2NCHCO
R

DCCI-promoted coupling adds the second amino acid

 CH2 CH2 CH2 CH2
CH CH CH CH

O O
CH2
BocNHCHC NHCHCO
R' R

Remove the Boc protecting group

 CH2 CH2 CH2 CH2
CH CH CH CH

O O
CH2
H2NCHC NHCHCO
R' R

Add the next amino acid and repeat

 CH2 CH2 CH2 CH2
CH CH CH CH

O O O
CH2
+
H3N peptide C NHCHC NHCHCO
R' R

Remove the peptide from the resin with HBr in CF3CO2H

 CH2 CH2 CH2 CH2
CH CH CH CH

CH2Br

O O O
+ –
H3N peptide C NHCHC NHCHCO
R' R
 Merrifield automated his solid-phase method

 Synthesized a nonapeptide (bradykinin) in 1962 in 8 days in

68% yield

 Synthesized ribonuclease (124 amino acids) in 1969,

369 reactions; 11,391 steps

 Nobel Prize in chemistry: 1984

 Liquid-phase peptide synthesis

 step-by-step peptide synthesis with subsequent adding of

one amino acid at ones from C-terminal to N-terminal and
block-synthesis with coupling of polypeptide fragments

 used in large-scale peptide production for industry