1. The Strategy of Peptide Synthesis4

2. General Considerations
Making peptide bonds between amino acids is not difficult.
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

3. N-Protected phenylalanine
General Strategy
1. Limit the number of possibilities by "protecting" the nitrogen of one amino acid and the carboxyl group of the other.
N-Protected phenylalanine
C-Protected glycine
NHCHCOH
CH2C6H5 O X
H2NCH2C O Y

4. General Strategy
2. Couple the two protected amino acids.
NHCH2C O Y
NHCHC
CH2C6H5 X
NHCHCOH
CH2C6H5 O X
H2NCH2C O Y

5. General Strategy
3. Deprotect the amino group at the N-terminus and the carboxyl group at the C-terminus.
NHCH2C O Y
NHCHC
CH2C6H5 X
NHCH2CO O
H3NCHC
CH2C6H5 + –
Phe-Gly

6. Amino Group Protection4

7. Protect Amino Groups as Amides
Amino groups are normally protected by converting them to amides.
Benzyloxycarbonyl (C6H5CH2O—) is a common protecting group. It is abbreviated as Z.
Z-protection is carried out by treating an amino acid with benzyloxycarbonyl chloride.

8. Protect Amino Groups as Amides
CH2OCCl O
H3NCHCO
CH2C6H5 O – + +
1. NaOH, H2O
2. H+
NHCHCOH
CH2C6H5 O
CH2OC
(82-87%)

9. Protect Amino Groups as Amides
NHCHCOH
CH2C6H5 O
CH2OC
is abbreviated as:
ZNHCHCOH
CH2C6H5 O
or Z-Phe

10. Removing Z-Protection
An advantage of the benzyloxycarbonyl protecting group is that it is easily removed by:
a) hydrogenolysis
b) cleavage with HBr in acetic acid

11. Hydrogenolysis of Z-Protecting Group
NHCHCNHCH2CO2CH2CH3
CH2C6H5 O
CH2OC
H2, Pd
H2NCHCNHCH2CO2CH2CH3
CH2C6H5 O
CH3
CO2
(100%)

12. HBr Cleavage of Z-Protecting Group
NHCHCNHCH2CO2CH2CH3
CH2C6H5 O
CH2OC
HBr
H3NCHCNHCH2CO2CH2CH3
CH2C6H5 O
CH2Br +
CO2 Br –
(82%)

13. The tert-Butoxycarbonyl Protecting Group
NHCHCOH
CH2C6H5 O
(CH3)3COC
is abbreviated as:
BocNHCHCOH
CH2C6H5 O
or Boc-Phe

14. HBr Cleavage of Boc-Protecting Group
(CH3)3COC
NHCHCNHCH2CO2CH2CH3
CH2C6H5
HBr
H3NCHCNHCH2CO2CH2CH3
CH2C6H5 O
CH2 C
H3C +
CO2 Br –
(86%)

15. Carboxyl Group Protection4

16. Protect Carboxyl Groups as Esters
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.

17. Hydrogenolysis of Benzyl Esters
NHCHCNHCH2COCH2C6H5
CH2C6H5 O
C6H5CH2OC
H2, Pd
H3NCHCNHCH2CO
CH2C6H5 O + –
C6H5CH3
CO2
CH3C6H5
(87%)

18. Peptide Bond Formation4

19. 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.

20. DCCI-Promoted Coupling
ZNHCHCOH
CH2C6H5 O
H2NCH2COCH2CH3 O +
DCCI, chloroform
ZNHCHC
CH2C6H5 O
NHCH2COCH2CH3
(83%)

21. Mechanism of DCCI-Promoted Coupling
ZNHCHCOH
CH2C6H5 O +
C6H11N C
NC6H11
CH2C6H5 O
C6H11N C H
OCCHNHZ

22. Mechanism of DCCI-Promoted Coupling
The species formed by addition of the Z-protected amino acid to DCCI is similar in structure to an acid anhydride and acts as an acylating agent.
Attack by the amine function of the carboxyl-protected amino acid on the carbonyl group leads to nucleophilic acyl substitution.
CH2C6H5 O
C6H11N C H
OCCHNHZ

23. Mechanism of DCCI-Promoted Coupling
C6H11N C
C6H11NH H O
ZNHCHC
CH2C6H5 O
NHCH2COCH2CH3 +
H2NCH2COCH2CH3 O
CH2C6H5 O
C6H11N C H
OCCHNHZ

24. The 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.

25. The Active Ester Method
ZNHCHCO
CH2C6H5 O
H2NCH2COCH2CH3 O
NO2 +

26. The Active Ester Method
ZNHCHCO
CH2C6H5 O
H2NCH2COCH2CH3 O
NO2 +
chloroform
ZNHCHC
CH2C6H5 O
NHCH2COCH2CH3 HO
NO2 +
(78%)

27. Solid-Phase Peptide Synthesis: The Merrifield Method4

28. 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.

29. The Solid Support
CH2 CH
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.

30. The Solid Support
CH2 CH
Treating the polymeric support with chloromethyl methyl ether (ClCH2OCH3) and SnCl4 places ClCH2 side chains on some of the benzene rings.

31. The Solid Support
CH2 CH
CH2Cl
The side chain chloromethyl group is a benzylic halide, reactive toward nucleophilic substitution (SN2).

32. The Solid Support
CH2 CH
CH2Cl
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.

33. The Merrifield Procedure
CH2 CH
CH2Cl
BocNHCHCO R O –

34. The Merrifield Procedure
CH2 CH
BocNHCHCO R O
Next, the Boc protecting group is removed with HCl.

35. The Merrifield Procedure
H2NCHCO R
CH2 CH O
DCCI-promoted coupling adds the second amino acid

36. The Merrifield Procedure
NHCHCO R O
CH2 CH
BocNHCHC R'
Remove the Boc protecting group.

37. The Merrifield Procedure
CH2 CH
NHCHCO R O
H2NCHC R'
Add the next amino acid and repeat.

38. The Merrifield Procedure
CH2 CH
NHCHCO R O
NHCHC R' C +
H3N
peptide
Remove the peptide from the resin with HBr in CF3CO2H

39. The Merrifield Procedure
CH2 CH
CH2Br
NHCHCO R O
NHCHC R' C +
H3N
peptide –

40. Peptide Synthesis

41. The Merrifield Method
Merrifield also automated his solid-phase method.
Synthesized a nonapeptide (bradykinin) in 1962 in 8 days in 68% yield.
Synthesized ribonuclease (124 amino acids) in reactions; 11,391 steps
Nobel Prize in chemistry: 1984