The Strategy of Peptide Synthesis

1. The Strategy of Peptide Synthesis

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—PheGly—GlyPhe—Gly Gly—Phe

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

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

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

6. Amino Group Protection

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

9. O O CH2OC NHCHCOH CH2C6H5 O ZNHCHCOH CH2C6H5 Protect Amino Groups as Amides is abbreviated as: 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. O O CH2OC NHCHCNHCH2CO2CH2CH3 CH2C6H5 O CH3 H2NCHCNHCH2CO2CH2CH3 CH2C6H5 Hydrogenolysis of Z-Protecting Group H2, Pd CO2 (100%)

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

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

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

15. Carboxyl Group Protection

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

18. Peptide Bond Formation

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

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

22. H O C6H11N C OCCHNHZ CH2C6H5 C6H11N 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.

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