Monitoring of Protease-Catalyzed Peptide Synthesis by High Performance Liquid Chromatography

Abstract

High performance liquid chromatography (HPLC) on prepacked silica gel columns has been applied to the separation of closely related protected peptides and amino acids. In the course of a protease-catalyzed synthesis of Leu-enkephalin this chromatogra-phic technique was found to be a valuable tool to rapidly and reliably characterize the outcome of enzymatic reactions, the nature of which was often difficult to be predicted. Stepwise gradient elution was employed to enable fractionation of mixture components, which covered only a short polarity range. The solvent systems composed of dichloromethane, anhydrous ethanol and acetic acid were mixed in such ratios so as to provide completely resolved peaks for the sample problems studied so far. Enzymatically prepared compounds and their chemically synthesized, authentic analogues were cochromatographed to enable the assignment of the eluted peaks. The provisional identification thus obtained could finally be established for all compounds under study by standard methods for chemical analysis.