The influence of water on protease‐catalyzed peptide synthesis in acetonitrile/water mixtures

Protease‐catalyzed peptide synthesis in acetonitrile water mixtures, containing 0–90% water, was investigated. α‐Chymotrypsin, as well as thermolysin, were deposited on solid supports, prior to exposure to the reaction media. Peptide syntheses were performed using both a kinetically controlled process (chymotrypsin) and an equilibrium‐controlled synthesis (thermolysin). The activity of chymotrypsin decreased at low water contents. However, at low water contents (1–10%) hydrolytic side reactions were suppressed and high yields of dipeptides were obtained. Optimal water content for the thermolysin‐catalyzed reaction was 4–8%. The dipeptides produced were fully soluble in the reaction mixtures. High operational stability for α‐chymotrypsin was obtained during 216 h of reaction