¹H NMR study of the diastereomeric forms of the protease inhibitor antipain

Antipain [(1‐carboxy‐2‐phenylethyl)carbamoyl‐ L ‐arginyl‐ L ‐valyl‐ DL ‐arginal] is a potent inhibitor of papain and related proteases. The assignment of 1 H NMR signals of the interconverting forms in equilibrium in aqueous solution at pH 5.8 were investigated using two‐dimensional DQF‐COSY, DOUBLE‐RELAYED‐COSY, HOHAHA and ROESY nuclear magnetic resonance techniques. The assignments of protons of several forms in equilibrium of the D ‐ and L ‐arginal antipain isomers were determined in aqueous solution at 300 K, and their corresponding stereochemistry is tentatively proposed. Addition of equimolar amounts of papain resulted in spectral changes and chemical shifts which are compatible with enzyme‐ L ‐carbinolamine interactions.