Continuous Production of N-(Benzyloxycarbonyl)-L-Glycyl-L-Phenylalanine Methyl Ester Utilizing Extractive Reaction in Aqueous/Organic Biphasic Medium
ABSTRACT N-(Benzyloxycarbonyl)-L-glycyl-L-phenylalanine methyl ester was continuously synthesized, enzymatically, utilizing an extractive reaction in an aqueous/organic biphasic system. The extremely high yield, ca.100%, was obtained continuously in a water/butyl acetate biphasic medium.
Continuous enzymatic synthesis of aspartame precursor at low pH using an extractive reaction
Peptides are synthesized in reactions catalyzed by the protease at the optimal pH, although the enzymatic synthesis of peptides at low pH has many advantages from the view point of an industrial-scale production, such as, suppression of nonenzymatic decomposition of amino acid ester substrates and a lower requirement of NaOH for pH adjustment. We developed an extractive synthesis of peptides in an aqueous/organic biphasic system at low pH and applied it to the protease-catalyzed synthesis of N-(benzyloxycarbonyl)-l-aspartyl-l-phenylalanine methyl ester (Z-APM), the precursor of an artificial sweetener, aspartame. The Z-APM yield in a pure aqueous monophasic system is less than 10%, however, it was over 96% at low pH using our extractive synthesis in an aqueous/organic biphasic system although the enzyme activity was very low. The extractive synthesis at low pH has advantages, such as, suppression of substrate inhibition by decreasing the concentration of N-(benzyloxycarbonyl)-l-aspartic acid (Z-l-Asp) in the aqueous phase, and shifting of the chemical equilibrium towards peptide bond formation because of a high distribution of Z-l-Asp to the organic phase. We expect that our method of extractive synthesis at low pH can be applied to other reaction systems.
Protease‐catalyzed synthesis of oligopeptides in heterogenous substrate mixtures
A systematic study of enzymatic peptide synthesis in heterogeneous substrate mixtures was carried out, with the aim of establishing the preparative scope of this methodology. Semiliquid eutectics were obtained with various combinations of neutral, acidic, and basic amino acid derivatives, in the presence or absence of adjuvants. A range of serine cysteine, and metalloproteases readily catalyzed the formation of the required dipeptides under these conditions. The synthetic usefulness of the approach was demonstrated by the sequential and convergent synthesis of derivatives of a number of bioactive di‐, tri‐, and pentapeptides, including aspartame, sweet lysine peptide, kyotorphin amide, ACE‐inhibiting and ‐immunoactive tripeptides, and Leu‐enkephalin amide, with overallyields of 21% to 84% and productivities of 0.13 to 0.75 g/g being obtained. © 1994 John Wiley & Sons, Inc.