Kinetics of enzymatic synthesis of peptides in aqueous/organic biphasic systems. Thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-phenylalanine methyl ester.

Abstract

We studied kinetics of thermolysin-catalyzed peptide synthesis in an aqueous/organic biphasic system theoretically and experimentally. As a model reaction producing a condensation product having no dissociating groups, we used the synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-phenylalanine methyl ester (Z-Phe2OMe) from N-(benzyloxycarbonyl)-L-phenylalanine (Z-Phe) and L-phenylalanine methyl ester (PheOMe). Usually, ethyl acetate was used as the organic solvent. First we studied the kinetics of the synthesis of Z-Phe2OMe in a buffer solution saturated with ethyl acetate. Then, factors that may affect the kinetics in the biphasic system were examined. The course of Z-Phe2OMe synthesis in the biphasic system was explained by the rate equations obtained, using the partitions of substrate and product and non-enzymatic decomposition of PheOMe. In the biphasic reaction system, the rate of synthesis was lower for a wide range of pH due to the unfavorable partition of PheOMe in the aqueous phase, but yields were higher than in the buffer solution. The effects of the organic solvents on the rate of synthesis could also be explained by variations in the partition coefficient of PheOMe. Finally, we gave a way to predict the aqueous-phase pH change caused by partitioning of the substrate. The significance of the pH change was shown in connection with the reaction using the immobilized enzyme in an organic solvent.