The Strategy of Peptide Synthesis 4

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

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

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

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

Amino Group Protection 4

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.

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

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

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

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

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

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

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

Carboxyl Group Protection 4

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.

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

Peptide Bond Formation 4

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.

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

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

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

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