Useful Methods in Enzymatic Synthesis of Peptides: A Comparative Study Focussing on Kinetically Controlled Synthesis of Ac-Phe-Ala-NH2 Catalyzed by α-Chymotrypsin

Abstract

The usefulness of different reaction systems for enzymatic peptide synthesis was evaluated by studying the formation of a dipeptide from equimolar amounts of acyl-donor and acyl-acceptor. α-Chymotrypsin (EC 3.4.21.1.), dissolved or deposited on celite, was used for the synthesis of Ac-Phe-Ala-NH2 from Ac-Phe-OEt and H-Ala-NH2. Reactions were carried out at room temperature (≈25°C), if not otherwise stated, in aqueous solutions (<21% yield), frozen solutions at -30°C (<60% yield), suspensions in aqueous media with and without 0.5% (v/v) of Triton X-100 (<82% yield), suspensions in hydrophobic organic media with Na2CO3 10H2O as water source (<90% yield), eutectic mixtures at 37°C (<97% yield), arid acetonitrile containing 1-5% (v/v) of aqueous buffer (<99% yield). Initial rates of synthesis were in the range of 5.9 × 10-2- 7.1 × 10-2μmol min-1 (mg of enzyme)-1. The lowest one was obtained with dissolved reactants in acetonitrile containing 1 % (v/v) of aqueous buffer, and the highest one was obtained with suspended acyl-donor in aqueous medium containing 0.5% (v/v) of Triton X-100.