The document describes the strategy and key steps of solid phase peptide synthesis using the Merrifield method. In this method, the C-terminus of the first amino acid is attached to an insoluble polystyrene resin support. Subsequent amino acids are then added step-wise through repetition of deprotecting the N-terminus, coupling with the next protected amino acid, and further deprotection until the full peptide is synthesized while still attached to the resin. The final peptide is then cleaved off the resin support to obtain the pure peptide product.

1. The Strategy of Peptide Synthesis
Rajendra A Sonawane

2. 27.14
The Strategy of Peptide
Synthesis

3. •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
The Challenge of Peptide Synthesis

4. Amino Acids are Structurally Bifunctional
L-Phenylalanine
O
OH
N
H
H O
OH
N
H
H
H
Glycine

5. Possible Products from the Condensation of
Phenylalanine and Glycine
Phe-Phe
O
N
H
H2N
O
OH
N
H
H
Gly-Gly
OH
O
O
OH
H
N
H
O
H2N
O
H2N
O
OH
H
N
H
O
H2N
H
Phe-Gly
Gly-Phe

6. • 1.Limit the number of possible reactions by
"protecting" the nitrogen of one amino acid
and the carboxyl group of the other.
Step 1 of Peptide Synthesis: Protection
N-Protected
L-Phenylalanine
C-Electrophile
O
OH
N
H
O
O
N
H
H
H PG
PG
O-Protected
Glycine
N-Nucleophile
PG
protecting
group

7. • 2. Couple the two protected amino acids.
Step 2 of Peptide Synthesis: Coupling
O
OH
N
H
O
O
N
H
H
H PG
PG
O
N
H
N
H
H
PGPG
O
O
Peptide
Coupling
PG-Phe-Gly-PG

8. • 3.Deprotect the amino group at the N-terminus
and the carboxyl group at the C-terminus.
Peptide Synthesis, Step 3: Global Deprotection
Phe-Gly
O
N
H
N
H
H
PGPG
O
O
Deprotection
O
N
H
H3N
H
O
O

9. Does the Requirement for Three Extra Steps
Outweigh the Need to Purify a Mixture?
Phe-Phe
O
N
H
H2N
O
OH
N
H
H
Gly-Gly
OH
O
O
OH
H
N
H
O
H2N
O
H2N
O
OH
H
N
H
O
H2N
H
Phe-Gly
Gly-Phe
Yes - synthesis is easier than purification

10. 27.15
Amino Group Protection

11. What are we Trying to Achieve by 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.
O
OR
H2N
O
OR
H
NR2
O
Nucleophilic
Atom Non-Nucleophilic
Atom
Amine Amide Derivative

12. 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.

13. Amine Protecting Groups: Benzyloxycarbonyl
O
O Cl
Benzyloxycarbonyl
chloride
(Cbz-Cl)
O
O N
H
R
Benzyloxycarbonyl
group

14. Amine Protecting Groups: Introduction of
Benzyloxycarbonyl Protecting Groups
O
O
H3N
H
O
O Cl
1. NaOH
H2O
2. H3O+
O
OH
H
N
H
O
O
(85%)

15. Benzyloxycarbonyl is Abbreviated to Cbz or Z
O
OH
H
N
H
O
O
O
OH
H
N
H
Cbz
O
OH
H
N
H
Z
Cbz-Phe

16. •An advantage of the benzyloxycarbonyl
protecting group is that it is easily removed by:
•a)catalytic hydrogenolysis under extremely mild
conditions
•b) cleavage with HBr in acetic acid
•Both reagents cleave the relatively weak
benzylic carbon-oxygen ether bond, albeit by
different mechanisms
Cleavage of Cbz Groups

17. Hydrogenolysis of Cbz Groups
O
N
H
H
N
H
O
O OEt
O
H2, Pd/C
solvent
O
N
H
H
N
H
O
HO OEt
O
Carbamic Acid
(Very Unstable)
H2C H
Toluene
(volatile)
ethyl ester is
stable to
hydrogenolysis

18. Hydrogenolysis of Cbz Groups
O
N
H
H
N
H
O
HO OEt
O
Spontaneous
decarboxylation
O
N
H
H2N
H
OEt
O
C
O
O
(100%)

19. Acid-Mediated Cleavage of Cbz Groups
O
N
H
H
N
H
O
O OEt
O
HBr
acetic acid
O
N
H
H3N
H
OEt
O
H2C Br
Benzyl
bromide
(volatile)
Br
C
O
O
(82%)

20. Amine Protecting Groups: tert-Butyloxycarbonyl
O
O O
Di-tert-butyl dicarbonate
(Boc 'anhydride')
O
O N
H
R
tert-Butyloxycarbonyl
group
O
O O
O
O
O Cl
tert-Butyl chloride
(instablity limits use)

21. O
OH
H
N
H
O
O
O
OH
H
N
H
Boc
Boc-Phe
tert-Butyloxycarbonyl is Abbreviated to Boc

22. Cleavage of Boc Groups
The tert-butyloxycabonyl protecting group is readily
removed by treatment wit strong, anhydrous BrØnsted
acids:
a) cleavage with 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.

23. Acid-Mediated Cleavage of Boc Groups
O
N
H
H
N
H
O
O OEt
O
O
N
H
H3N
H
OEt
O
Butene
(volatile)
C
O
O
(high yield)
CH3H3C
H H
O
OHF3C
trifluoroacetic
acid
O
OF3C

24. 27.16
Carboxyl Group Protection

25. Peptide Synthesis: Carboxyl Protecting Groups
Carboxyl groups are normally protected as
esters.
Deprotection of methyl and ethyl esters is by
hydrolysis in base.
Benzyl esters can be cleaved by
hydrogenolysis.

26. Simultaneous Hydrogenolysis of Cbz
Group and Benzyl Ester
O
N
H
H
N
H
O
O O
O
H2, Pd/C
solvent
O
N
H
H
N
H
O
HO OH
O
Carbamic Acid
(Very Unstable)
H2C H
Toluene
(volatile)

27. Simultaneous Hydrogenolysis of Cbz
Group and Benzyl Ester
O
N
H
H
N
H
O
HO OEt
O
Spontaneous
decarboxylation
O
N
H
H2N
H
OEt
O
C
O
O
(87%)

28. 27.17
Peptide Bond Formation

29. Peptide Synthesis: Forming Peptide Bonds
The two major methods are:
1. coupling of suitably protected amino acids
using N,N'-dicyclohexylcarbodiimide (DCC)
2. via an active ester of the N-terminal amino
acid.

30. N,N'-Dicyclohexylcarbodiimide (DCC) is a
Powerful Dehydrating Agent
N C N
N,N'-dicyclohexylcarbodiimide
O
N
H
N
H
N,N'-dicyclohexylurea
'H2O'
O
OR1
H
H
N
R2H
O
NR1
R2
H
Amide
O
OR1
H
N
R2H
H
-H2OVery high
temps

31. • 2. Couple the two protected amino acids.
Peptide Coupling is a Condensation Reaction
O
OH
N
H
O
O
N
H
H
H PG
PG
O
N
H
N
H
H
PGPG
O
O
Peptide
Coupling -H2O

32. • 2. Couple the two protected amino acids.
DCC-Mediated Peptide Coupling
O
OH
N
H O
O
N
H
H
H
Et
O
N
H
N
H
Cbz
H
O
O
Et
DCC, CHCl3
(83%)
Cbz

33. Mechanism of DCC-Promoted Coupling
O
OH
N
H
H
Cbz N C N
O
O
N
H
H
Cbz N
H
N
1,2-Addition
O-Acylisourea
derivative

34. O-Acylisoureas are Powerful
Acylating Agents
The O-acylisourea
intermediate formed by
addition of the Cbz-
protected amino acid to
DCCI is similar in
structure to an acid
anhydride and acts as an
acylating agent.
O
O
N
H
H
Cbz N
H
N
O-Acylisourea
Derivative
H3C O
O
CH3
O
Acid
Anhydride

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

36. Attack by the
amine function of
the carboxyl-
protected amino
acid on the
carbonyl group
leads to
nucleophilic acyl
substitution.
Mechanism of DCC-Promoted Coupling
OH
O
N
H
Cbz N
H
N
N
H
OEtO
Unstable
Intermediate
O
N
H
N
H
Cbz
OEt
OHO
N
H
N
H
N,N'-dicyclohexylurea
Dipeptide
Elimination

37. Peptide Synthesis: Forming Peptide Bonds
The two major methods are:
1. coupling of suitably protected amino acids
using N,N'-dicyclohexylcarbodiimide (DCCI)
2. via an active ester of the N-terminal amino
acid.

38. Peptide Synthesis: Active Ester Method
A p-nitrophenyl ester is an example of an "active
ester.”
p-Nitrophenyl is a better leaving group than methyl
or ethyl, and p-nitrophenyl esters are more reactive
in nucleophilic acyl substitution.
O
O
Alkyl
O
O
N+
O–
O
4-Nitrophenyl
(PNP) Ester
Alkyl Ester
is a more powerful
acylating agent than......

39. Peptide Synthesis: Active Ester Method
O
O
N
H
Cbz
O
OEt
N
H
H
OH
O
N
H
Cbz
N
H
OEtO
1,2-Addition
then
Proton Transfer
Unstable
Intermediate
NO2
NO2

40. Peptide Synthesis: Active Ester Method
Unstable
Intermediate
O
N
H
N
H
Cbz
OEt
OH
para-Nitrophenol Dipeptide
Elimination
OH
O
N
H
Cbz
N
H
OEtO
NO2
O2N OH

41. 27.18
Solid-Phase Peptide Synthesis:
The Merrifield Method

42. 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.

43. •The solid support is a copolymer of styrene and
divinylbenzene. It is represented above as if it
were polystyrene. Cross-linking with
divinylbenzene simply provides a more rigid
polymer.
Polystyrene is the Basis for the Solid Support
C
C
C
C
C
C
C
C
C
C
H H H H H H H H
HHHH
H H

44. Functionalization of Polystyrene
•Treating the polymeric support with
chloromethyl methyl ether (ClCH2OCH3) and
SnCl4 places ClCH2 side chains on some of
the benzene rings.

45. Chloromethylation of Polystyrene
C
H2
O
Me
Cl
SnCl4
CH2 CH2
ClCl

46. Solid Phase Peptide Synthesis
CH2 CH2
ClCl
The side chain chloromethyl group is a benzylic
halide, reactive toward nucleophilic substitution
(SN2).

47. 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.
Solid Phase Peptide Synthesis
CH2 CH2
ClCl

48. CHCH22 CHCH22 CHCH22 CHCH22
CHCH CHCH CHCH CHCH
CHCH22ClCl
BocBocNNHHCHCOCHCO
RR
OO
––
Merrifield Procedure

49. BocBocNNHHCHCOCHCO
RR
OO
CHCH22 CHCH22 CHCH22 CHCH22
CHCH CHCH CHCH CHCH
CHCH22
Merrifield Procedure
Next, the Boc
protecting group is
removed with HCl.

50. HH22NNCHCOCHCO
RR
CHCH22 CHCH22 CHCH22 CHCH22
CHCH CHCH CHCH CHCH
CHCH22
OO
Merrifield Procedure
DCCI-promoted
coupling adds the
second amino acid

51. NNHCHCOHCHCO
RR
OO
CHCH22 CHCH22 CHCH22 CHCH22
CHCH CHCH CHCH CHCH
CHCH22
BocBocNNHCHCHCHC
R'R'
OO
Merrifield Procedure
Remove the Boc
protecting group.

52. CHCH22 CHCH22 CHCH22 CHCH22
CHCH CHCH CHCH CHCH
CHCH22
NNHCHCOHCHCO
RR
OO
HH22NNCHCCHC
R'R'
OO
Merrifield Procedure
Add the next
amino acid and
repeat.

53. • Remove the
peptide from the
resin with HBr in
CHCH22 CHCH22 CHCH22 CHCH22
CHCH CHCH CHCH CHCH
CHCH22
NNHCHCOHCHCO
RR
OO
NNHCHCHCHC
R'R'
OO
CC
OO
++
HH33NN peptide
Merrifield Procedure

54. CHCH22 CHCH22 CHCH22 CHCH22
CHCH CHCH CHCH CHCH
CHCH22BrBr
NNHCHCOHCHCO
RR
OO
NNHCHCHCHC
R'R'
OO
CC
OO
++
HH33NN peptide
––
Merrifield Procedure

55. •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
Merrifield Procedure