Enzymatic Synthesis and Chemical Properties of Sweet Aminomalonyl (Ama) Dipeptide Esters (R)-Ama-(S)-Phe-OMe and (R)-Ama-(S)-Phe-OEt

Abstract

Proteases catalyze the coupling of N-protected L-amino acids and C-protected L-amino acids, but the enzymatic synthesis of aminomalonic acid (Ama)-containing peptides has not been reported. We undertook the synthesis of sweet (R)-Ama-(S)-Phe-OMe (800 times sweeter than sucrose) and (R)-Ama-(S)-Phe-OEt (50 times sweeter than sucrose) by coupling Z-(RS)-Ama(OBzl)-OH and H-L-Phe-OR (R, Me or Et) with thermolysin (EC 3.4.24.4) and subsequent hydrogenolysis. Thermolysin catalyzed the synthesis of Z-(R)-Ama(OBzl)-(S)-Phe-OR (R, Me or Et), which is the precursor of sweet (R)-Ama-(S)-Phe-OR (R, Me or Et). The Ama residues of Z-(R or S)-Ama(OBzl)-(S)-Phe-OR (R, Me or Et) and (R or S)-Ama-(S)-Phe-OR (R, Me or Et) were susceptible to facile racemization. Both Z-(R)-Ama(OBzl)-(S)-Phe-OMe and Z-(S)-Ama(OBzl)-(S)-Phe-OMe were sparingly soluble in water. The desired Z-(R)-Ama(OBzl)-(S)-Phe-OMe was more soluble than Z-(S)-Ama(OBzl)-(S)-Phe-OMe; consequently, the precipitated Z-(R)-Ama(OBzl)-(S)-Phe-OMe gradually dissolved in water, and changed to Z-(R, S)-Ama(OBzl)-(S)-Phe-OMe, because of racemization of the Ama residue. The less-soluble Z-(S)-Ama(OBzl)-(S)-Phe-OMe, which is the precursor of non-sweet (S)-Ama-(S)-Phe-OMe, remained after 24 h. On the other hand, Z-(R)-Ama(OBzl)-(S)-Phe-OEt, which is the precursor of sweet (R)-Ama-(S)-Phe-OEt, was less soluble than Z-(S)-Ama(OBzl)-(S)-Phe-OEt, and therefore remained throughout the reaction.