Amino acid methyl esters as amine components in CPD-Y catalyzed peptide synthesis: Control of side reactions

Abstract

Carboxypeptidase Y has previously been shown to catalyze the formation of peptide bonds from N-protected amino acid methyl esters (acyl components) and amino acid methyl esters (amine components) (Carlsberg Res. Commun. 45, 453–463, 1980). However, the reaction course was difficult to control and often resulted in oligomerization. It is here demonstrated that knowledge of the kinetic constants for the CPD-Y catalyzed hydrolysis of ester substrates can be exploited in such a way that amino acid esters may be used as amine components without oligomerization. This is achieved by utilizing the fact that N-blocked amino acid benzyl esters are much better substrates of carboxypeptidase Y than the corresponding methyl esters. Thus, by using N-blocked amino acid benzyl esters as acyl components and amino acid methyl esters as amine components, peptide methyl esters are formed as the major product because they are turned over by the enzyme with a lower rate than the initial benzyl ester acyl component. It is furthermore demonstrated that carboxypeptidase Y modified with phenylmercuric chloride or mercuric chloride can be used for the incorporation of amino acid methyl esters without oligomerization in cases where the unmodified enzyme yields several products.