Thermolysin as a catalyst in enzymatic synthesis of asparagine-containing peptides

Abstract

The following di- and tripeptides were synthesized to study the potential utility of thermolysin as a catalyst in reactions of incorporation of N α -acyl- l-asparagine into esters of amino acids and peptides: Boc-Asn-Ile-OBzl, Z-Asn-Ile-OBzl, Moz-Asn-Ile-OBzl, Boc-Asn-Leu-OBzl, Z-Asn-Leu-OBzl, Moz-Asn-Leu-OBzl, Boc-Asn-Phe-OBzl, Z-Asn-Phe-OBzl, Moz-Asn-Phe-OBzl, Z-Asn-Val-OBzl, Moz-Asn-Val-OBzl, Moz-Asn-Ile-Gly-OBzl, Moz-Asn-Ile-Ala-OBzl, Moz-Asn-Ile-Leu-OBzl, and Moz-Asn-Ile-Phe-OBzl. All of these peptides were obtained in pure form in good yield and characterized by thin-layer chromatography, melting point, elemental analysis, amino acid analysis, and proton magnetic resonance. The use of benzyloxycarbonyl (Z) and p-methoxybenzyloxycarbonyl (Moz) as protecting groups for asparagine gave excellent yields of the dipeptides. Relative to the dipeptides, the synthesis of the tripeptides was found to require lower enzyme concentrations and reaction times. Since the yields of the tripeptides failed to exhibit significant differences, it was not possible to establish the existence of a secondary specificity of thermolysin for the residue P′ 2. A methodological study was also performed to determine the optimum conditions for synthesis of Boc-Asn-Ile-OBzl. This study consisted of an analysis of the influence of pH, enzyme concentration, volume and concentration of the solution of sodium acetate, relative proportions of carboxyl and amine components, temperature, and addition of organic solvent to the reaction medium.