Solvent-free enzymatic synthesis of alitame precursor using eutectic substrate mixtures

N-benzyloxycarbonyl-l-aspartic acid ethyl ester-D-alanine amide, a derivative of alitame, was synthesized from a eutectic mixture of the substrates N-benzyloxycarbonyl-l-aspartic acid diethyl ester and D-alanine amide using α-chymotrypsin. The hydrophilic solvents DMSO and MEA were found to be the best adjuvants for formation of a eutectic substrate mixture. A low eutectic temperature of 27°C was obtained for the substrate mixture containing 9% DMSO, 18% MEA, and 12% water. Under these conditions a conversion yield of 70.3% (mol/mol) was obtained at 37°C. The optimum molar ratio of the acyl acceptor D-alanine amide and the acyl donor N-benzyloxycarbonyl-l-aspartic acid diethyl ester was 1:1.