Enzymic synthesis of aspartame precursors from eutectic substrate mixtures

Abstract

The precursor of aspartame N-benzyloxycarbonyl- l-aspartic acid- l-phenylalanine methyl ester ( N-CBZ- l-Asp- l-PheOMe) was synthesized using thermolysin from eutectic mixtures of the substrates N-benzyloxycarbonyl- l-aspartic acid ( N-CBZ- l-Asp) and l-phenylalanine methyl ester ( l-PheOMe). Eutectic mixtures were easily formed by mixing the substrates in the presence of water and/or organic solvents as adjuvants. Among the tested solvents the hydrophilic solvents dimethyl sulfoxide (DMSO) and 2-methoxy ethyl acetate (MEA) were the best adjuvants for enzymic reactions. A maximum conversion yield of 73.8% was obtained for a combination of 6% DMSO and 6% MEA. In addition to benzyloxycarbonyl (CBZ), l-Asp derivatives with other protecting groups were used. Reactions with N-CBZ- l-Asp or N- tert-butyloxycarbonyl (Boc)- l-Asp formed eutectic mixtures at approximately 0 °C, whereas reactions with hydrophobic N-Ac- l-Asp and N-9-fluorenylmethyloxycabronyl (Fmoc)- l-Asp formed mixtures at 25 and 40 °C, respectively. High conversion yields of 74 and 78% were achieved for N-CBZ- l-Asp and N-Boc- l-Asp but low yields of 56 and 35% were obtained for N-Ac- l-Asp and N-Fmoc- l-Asp. Thermolysin was most effective with a yield of 72% compared to fungal protease, subtilsin, α-chymotrypsin, papain, and pepsin with yields of 50% or less.