Enzymatic peptide synthesis in frozen aqueous solution: use of N alpha-unprotected peptide esters as acyl donors

The ability of the endopeptidase alpha-chymotrypsin (EC 3.4.21.1) to catalyse the reaction of various N alpha-unprotected di- and tripeptide ester derivatives with H-Leu-NH2, and with a series of C-terminal free di- and tripeptides at -15 degrees C in frozen aqueous solution was investigated. The enzyme is able to synthesize N- and C-terminal unprotected penta- and hexapeptides in up to 92% yield, depending on the amino component used, in a single-step segment-condensation reaction. Freezing the reaction mixture resulted in significantly increased peptide yields compared with the reaction at room temperature. The enzyme shows a modified nucleophilic specificity in frozen solution compared with room temperature. Nucleophilic amino components with positively charged amino acids in P2'-position are accepted.