p‐Azido‐L‐Phenylalanine Peptides. I: Synthesis of Peptide Ligands for Chymotrypsin and Aminopeptidases

Abstract p ‐Azido‐L‐phenylalanine peptides as ligands for α‐chymotrypsin and aminopeptidases were synthesized in two ways: (i) by direct incorporation of f‐butoxycarbonyl‐ p ‐azido‐L‐phenylalanine or its active esters, or (ii) by conversion of the nitro groups of the corresponding p ‐nitrophenylalanine‐containing peptides into azido functions. Both procedures yielded O‐ t ‐butylthreonyl‐ p ‐azidophenylalanylproline (L, L, L) which, like its parent tripeptide O‐ t ‐butylthreonyl‐phenylalanylproline (L, L, L), acted as a competitive inhibitor for leucine aminopeptidase (K i ≈ 7 to 8 × 10 −6 M). The two peptides p ‐azidophenylalanylphenylalanine (L, L) and p ‐azido‐phenylalanylphenylalanylproline (L, L, L) proved to be substrates for leucine aminopeptidase and for both aminopeptidases I and II from Bacillus stearothermophilus . Acetyl‐ p ‐azidophenylalanine ethyl ester (L) is a substrate of α‐chymotrypsin with K m ≈ 6 × 10 −4 M, closely resembling that of N‐acetyltyrosine ethyl ester (L), K m ≈ 5.4 × 10 −4 M, but with an approximately 100‐fold reduced maximum velocity of hydrolysis (V m ≈ 1.3 s −1 versus 211 s −1 ). The results obtained with photo‐affinity labelling will be described in a forthcoming paper.