Short Peptide Synthesis

Keith Proinsias
8th February 2010

Introduction
Amide bond and basic amide synthesis
Solution phase peptide synthesis
Protecting groups required for peptide synthesis
Coupling reagents used in peptide synthesis
Solid phase peptide synthesis

The Amide Bond

Some of the main properties of the amide bond is its
low basicity, which is useful in purification, and its
stability, due to resonance.
O

O
N
H

N
H

Basic amide synthesis is the reaction of a carboxylic

acid and an amine with the loss of water.
R

COOH

H2N

R'

-H20

H
N
R

R
O

Basic Synthesis
At the beginning acid chlorides where used.
O
R

OH

SO2Cl

+ H2N
R

Cl

R'
base

N
H

R'

+ HCl

Not good for peptide synthesis mainly due to

racemisation occurring.

Advanced Organic Chemistry- Reaction Mechanisms by Brukner, R., 2002, 216

Solution Phase Peptide Synthesis

R
H2N

COOH
Acid Protection

R
H2N

COOPG

R'
Coupling
PGHN

COOH
R'

R'

H
N

PGHN

COOPG

H
N

H2N

O
Dimer

COOH
R

Deprotection
R'

H
N

H2N
O

COOPG
R

R'
Coupling
PGHN

COOH
O

PGHN
R

R'
N
H

H
N
O

COOPG
R

H2N
R

R'
N
H

O
Triemer

H
N

COOH
R

Planning For Solution Phase Peptide Synthesis

Before starting.

Choose the C-terminal protecting group

Choose the N-terminal protecting group

Choose protecting groups for any other R-group on the

amino acids

Choose the coupling reagent

Protecting Groups
Acid Protection
R
OH
H 2N
O

Protecting Groups
The most common acid protecting group used is the methyl ester.
It is stable in most coupling reaction and deprotection reaction
conditions.
Difficult to selectively remove.

H2N

Alanine methyl ester

O

Depending on what type of coupling reaction and with what amino acids
will be used other acid protecting groups can be used, such as the allyl
ester.
O

H2N
O

Alanine allyl ester

Protecting Groups
Amine Protection
R
OH
H 2N
O

Protecting Groups
There are two standard types of N-protecting groups used, the
Boc and Fmoc group.
O H C
3
O

CH3

N-Boc cleavage

CH3

50% TFA in DCM

BocHN

H 2N

COOH

COOH
Glycine

N-Fmoc cleavage

O
O

20% Piperidine in DMF

FmocHN

COOH

H2N

COOH
Glycine

Protecting Groups
Other protecting groups that can be used are Cbz and more recently
the Nosyl group.

O
O
CBZ

Leggio, A.; Gioia, M.L.D.; Perri, F.; Liguori, A. Tetrahedron, 2007, 63, 8164-8173

Protecting Groups
Protection of the R-group

R
OH
H 2N
O

Protecting Groups
Some of the different R-groups that must be protected before coupling
are hydroxyl groups (Ser), thiol groups (Cys), amines (Lys) and
carboxylic acids (Asp).

COOH

HO

COOH

HS
NH2

NH2

Serine (Ser)
H2N

Cysteine (Cys)
COOH

COOH

HOOC
NH2

Lysine (Lys)

NH2

Aspartic Acid (Asp)

Protecting Groups
Base Sensitive Protecting Groups
Used in N-Boc protected peptide synthesis.
Ph
COOH
Si

Ph
TBDPS

COOH

NH2

NH2

Cleavage : 2M NaOH (aq) EtOH (1:1)

Fm

Cleavage : 20% Piperidine in DMF

H
N

O
O

Fmoc

Cleavage : 20% Piperidine in DMF

COOH

NH2

COOH
O

NH2

Fm

Cleavage : 20% Piperidine in DMF

Greene, T.W.; Wuts, P.G.M. Protecting groups in organic synthesis, Fourth edition, Wiley-interscience, New York, 2006.

Protecting Groups
Acid Sensitive Protecting Groups
Used in N-Fmoc protected peptide synthesis.
Normally the t-Butyl, Boc or Trityl group is used.
Cleaved using 5-50% TFA in DCM.
Ph

COOH

COOH
Ph

O
Ph

t-Butyl

Trityl

COOH

O
t-Butyl

NH2

NH2

H
N

NH2

COOH

NH2

Boc

Greene, T.W.; Wuts, P.G.M. Protecting groups in organic synthesis, Fourth edition, Wiley-interscience, New York, 2006.

Protecting Groups
Other Protecting Groups
O

Allyl

Alloc

Cleavage: Pd(PPh3)4
O

NO2
O

S
O

Nosyl

Cleavage: Thiophenol

PMB

Cleavage: Ceric Ammonium Nitrate (CAN)

Greene, T.W.; Wuts, P.G.M. Protecting groups in organic synthesis, Fourth edition, Wiley-interscience, New York, 2006.

Protecting Groups
There are some exception were an unprotected amino
acid, such as serine, can be used without being
protected.

Rothman, D.M.; Vazquez, M.E.; Vogel, E.M.; Imperiali, B., Org. Lett., 2002, 4, 2865-2868

Coupling Reagents
N

N
N,N-Dicyclohexylcarbodimide (DCC)

N
N

N-(3-Dimethylaminopropyl)-N-ethylcarbonate (EDC)

DMAP

Coupling Reagents

Organic Chemistry by Bruce, 977-978

Coupling Reagents
Racemisation of an activated amino acid
H
PG

PG

PG

N
R

O
X

O
O
O

base

X= Activator
PG

PG

PG

N
R

O
O

N
R

O
O

O
O

Formation of oxazolone intermediates

Coupling Reagents
N

N
N

N
N

OH

HOBt

HOAt

PF6 (BF4 )

OH
Me2N

NMe2

N
PF6

BOP

N P

PF6
Br
N P

O
N

PF6

N
PyBrOP

PyBOP

PF6

NMe2
Me2N
HATU

HBTU (TBTU)

N P

Coupling Reagents
Mechanism of HBTU Coupling
N

N
N

O
N
R

NMe2

Me2N

NMe2

NMe2

N
N
N

O
N
H

R' +
HO

N
N

R'-NH

N
N

+
Me2N

NMe2

Coupling reagents
BOP coupling reagent

Brink, H.T.; Rijkers, D.T.S.; J. Org. Chem., 2006, 71, 1824

Coupling reagents
PyBrOP coupling reagent

Anderson, R.J.; Coleman, J.E.; Tetrahedron Lett., 1997, 38, 317-320

Coupling reagents
Synthesis of new coupling reagents

R. Wischnat, Tetrahedron Lett., 2003, 44, 4393-4394

Coupling reagents
Synthesis of new coupling reagents

El-Faham, A.; Albericio, F., Org. Lett., 2007, 9, 4475-4477

Solid Phase Peptide Synthesis

Solid Phase

Vs

Solution Phase

Advantages
Fast production of long peptides by
increasing the amount of reactant.
Quick purification by filtration.
Automated or manual option.

Easily scaled up from mg to kg.

No need for excess reactants or
expensive machinery.

Disadvantages
Expensive resin and can require
specialised equipment.
Limited scale-up.

More difficult to purify.

Longer reaction time

Solid Phase Peptide Synthesis

The first step is to deprotect the amino group
to produce a free amine.

It can then be coupled to another Nprotected amino acid.

By deprotecting the final -amino group

and cleaving the peptide from the resin
after peptide chain elongation, using the
appropriate cleavage conditions, the
peptide is isolated.

Chan, W.; White, P. Fmoc Solid Phase Peptide Synthesis, Oxford, New York, 2000.

Solid Phase Peptide Synthesis

The resin is not completely spherical, with the reaction only occur on
the surface of the resin.
There are cavities were the coupling takes place and is the reason
why swelling is very important before coupling can occur.

Types of Resin.
You buy commercially available pre-loaded resins.
Different resins can be cleaved under basic (N-Boc) or acidic
(N-Fmoc) conditions.
Depending on the resin the final peptide can have an amide or
acid C-terminal.

Solid Phase Peptide Synthesis

Merrifield Bubbler

Resin - Amino acids - Coupling reagents - Base - Solevnt

Sinter
Nitrogen

Vacuum
1.
2.
3.
4.
5.
6.
7.
8.

Add resin to column.

Swell resin using DMF and bubbling with N2.
Remove solvent using vacuum.
To cleave the first N-protecting group the appropriate deprotecting
reagent is added.
Bubble N2 and then remove solvent under vacuum and wash with DCM.
Add DMF plus coupling reagent, base and N-protected amino acid.
Bubble N2 until reaction is complete.
Remove solvent and wash with DCM.

Chan, W.; White, P. Fmoc Solid Phase Peptide Synthesis, Oxford, New York, 2000.

Thank You For

Your
Attention
Any Questions?