Protease-catalyzed synthesis of Leu-enkephalin in a solvent-free system.

The total enzymatic synthesis of a model peptide Leu-enkephalin on a preparative scale was accomplished in the so-called solvent-free system. The syntheses were carried out in a rotary glass homogenizer by admixing solid reactants with native proteases and Na2CO3.10H2O. The most feasible way leading to biologically active Leu-enkephalin, was based on the strategy of 2 + (1 + 2) condensation catalyzed by alpha-chymotrypsin, thermolysin and papain for the final segment coupling. Subtilisin was used for the ester hydrolysis of peptide intermediates. Alternative strategies as well as the influence of several reaction conditions on the yield of the protease-catalyzed synthesis of Leu-enkephalin or Leu-enkephalin amide were also investigated.