Short Peptide Synthesis

Keith Proinsias
8
th
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
H
2
N R'
+
R COOH
R
O
H
N
R
-H
2
0
Basic amide synthesis is the reaction of a carboxylic
acid and an amine with the loss of water.
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
N
H
O
N
H
Basic Synthesis
At the beginning acid chlorides where used.
Not good for peptide synthesis mainly due to
racemisation occurring.
R OH
O
SO
2
Cl
R Cl
O
+ H
2
N R'
R N
H
O
R'
+ HCl
Advanced Organic Chemistry- Reaction Mechanisms by Brukner, R., 2002, 216
base
Solution Phase Peptide Synthesis
H
2
N COOH
H
2
N COOPG
R
R
PGHN COOH
R'
PGHN
R'
O
H
N COOPG
R
H
2
N
R'
O
H
N COOH
R
H
2
N
R'
O
H
N COOPG
R
PGHN COOH
R'
N
H
R'
O
H
N COOPG
R
O
PGHN
R
N
H
R'
O
H
N COOH
R
O
H
2
N
R
Coupling
Deprotection
Dimer
Triemer
Acid Protection
Coupling
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
H
2
N
O
OH
R
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.
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.
Alanine methyl ester
H
2
N
O
O
H
2
N
O
O
Alanine allyl ester
Protecting Groups
Amine Protection
H
2
N
O
OH
R
Protecting Groups
N-Boc cleavage
H
2
N COOH
BocHN COOH
Glycine
50% TFA in DCM
N-Fmoc cleavage
H
2
N COOH
FmocHN COOH
Glycine
20% Piperidine in DMF
There are two standard types of N-protecting groups used, the
Boc and Fmoc group.
O
O
C H
3
CH
3
CH
3
O
O
Protecting Groups
Other protecting groups that can be used are Cbz and more recently
the Nosyl group.
Leggio, A.; Gioia, M.L.D.; Perri, F.; Liguori, A. Tetrahedron, 2007, 63, 8164-8173
O
O
CBZ
Protecting Groups
Protection of the R-group
H
2
N
O
OH
R
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).
HO
NH
2
COOH
HS
NH
2
COOH
NH
2
COOH
HOOC
NH
2
COOH H
2
N
Serine (Ser) Cysteine (Cys)
Lysine (Lys) Aspartic Acid (Asp)
HO
NH
2
COOH
HS
NH
2
COOH
NH
2
COOH
HOOC
NH
2
COOH H
2
N
Serine (Ser) Cysteine (Cys)
Lysine (Lys) Aspartic Acid (Asp)
Protecting Groups
Base Sensitive Protecting Groups
Used in N-Boc protected peptide synthesis.
Cleavage : 2M NaOH (aq) EtOH (1:1)
Cleavage : 20% Piperidine in DMF
O
NH
2
COOH
Si
Ph
Ph
TBDPS
S
NH
2
COOH
Fm
NH
2
COOH
H
N
O
O
Fmoc
NH
2
COOH O
O
Fm
Cleavage : 20% Piperidine in DMF
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.
O
NH
2
COOH
Ph
Ph
Ph
Trityl
NH
2
COOH
H
N
Boc
O
O
S
NH
2
COOH
t-Butyl
NH
2
COOH O
O
t-Butyl
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
O
Alloc
S
O
O
NO
2
Nosyl
Allyl
O
PMB
Cleavage: Pd(PPh
3
)
4
Cleavage: Thiophenol 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-(3-Dimethylaminopropyl)-N-ethylcarbonate (EDC)
N C N
N C N N N C N N
N,N-Dicyclohexylcarbodimide (DCC)
N
N
DMAP
Coupling Reagents
Organic Chemistry by Bruce, 977-978
Coupling Reagents
PG
O
N
X O
R
H
O
N
O
PG
R
O
N
O
PG
R
base
O
N
O
PG
R
O
N
O
PG
R
O
N
O
PG
R
Racemisation of an activated amino acid
X= Activator
Formation of oxazolone intermediates
Coupling Reagents
N
N
N
O
Me
2
N NMe
2
PF
6
(BF
4
)
N
N
N
N
O
Me
2
N NMe
2
HBTU (TBTU)
HATU
PF
6
N
N
N
OH
N
N
N
N
OH
HOBt
HOAt
N
N
N
O
P N N
N
PF
6
N
N
N
O
P N N
N
PF
6
BOP
PyBOP
P N N
N
PF
6
PyBrOP
Br
Coupling Reagents
R O
O
N
N
N
O
N
Me
2
N
+
R O
O N
N
N
O
N
NMe
2
R O
O
N
N
N
O
NMe
2
NMe
2
R O
O
N
N
N
Me
2
N NMe
2
O
+
R'-NH
R
HO
O
N
N
N
N
H
R' +
Mechanismof HBTU Coupling
Coupling reagents
Brink, H.T.; Rijkers, D.T.S.; J. Org. Chem., 2006, 71, 1824
BOP coupling reagent
Coupling reagents
Anderson, R.J.; Coleman, J.E.; Tetrahedron Lett., 1997, 38, 317-320
PyBrOP coupling reagent
Coupling reagents
R. Wischnat, Tetrahedron Lett., 2003, 44, 4393-4394
Synthesis of new coupling reagents
Coupling reagents
El-Faham, A.; Albericio, F., Org. Lett., 2007, 9, 4475-4477
Synthesis of new coupling reagents
Solid Phase Peptide Synthesis
Solid Phase Vs Solution Phase
Advantages
Disadvantages
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.
Expensive resin and can require
specialised equipment.
Limited scale-up.
More difficult to purify.
Longer reaction time
Solid Phase Peptide Synthesis
Chan, W.; White, P. Fmoc Solid Phase Peptide Synthesis, Oxford, New York, 2000.
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.
It can then be coupled to another N-
protected amino acid.
The first step is to deprotect the amino group
to produce a free amine.
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
Nitrogen
Vacuum
Resin - Amino acids - Coupling reagents - Base - Solevnt
Sinter
Merrifield Bubbler
1. Add resin to column.
2. Swell resin using DMF and bubbling with N
2
.
3. Remove solvent using vacuum.
4. To cleave the first N-protecting group the appropriate deprotecting
reagent is added.
5. Bubble N
2
and then remove solvent under vacuum and wash with DCM.
6. Add DMF plus coupling reagent, base and N-protected amino acid.
7. Bubble N
2
until reaction is complete.
8. 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?