Aspartam enzymatic coupling

Since the reaction (Fig. 19-34) is limited by the equilibrium the products have to be removed from the reaction mixture to reach high yields. Therefore an excess of racemic phenylalanine methylester (which is inert to the reaction) is added. The carboxylic anion of the protected aspartame forms a poorly soluble adduct with d-Phe-OCH3 that precipitates from the reaction mixture. The precipitate can be removed easily by filtration. Final steps of the process are the separation of D-Phe-ester, removal of protecting groups and racemi2ation of the formed L-amino acid, a-Aspartame is produced with > 99.9% and a worldwide capacity of -10,000 t a 1, - 2,5001 a 1 by enzymatic coupling. 

Proteases have been much less studied than lipases in ionic liquid media and generally require the presence of water for activity. We note that the thermolysin-catalyzed amide coupling of benzoxycarbonyl-L-aspartate and L-phenylalanine methyl ester into Z-aspartame in [BMIm][PF6] was an early example of an enzymatic reaction in an ionic liquid medium [8]. 

Enzymatic methods are now preferred for the commercial production of aspartame from aspartate and phenylalanine. Typically, these use thermolysin (EC 3.4.24.4 derived from Bacillus spp.) to couple benzyloxycarbonyl-protected aspartate (Z-asp) with phenylalanine methyl ester. The resulting Z-APM is then deprotected using standard methods [14-18]. Numerous enzymatic methods have been reported [10]. 

A prominent example of peptide coupling is used for the production of the synthetic dipeptide sweetener aspartame (a-APM) (28) (Chapter 31). The chemical coupling method yields approximately an 80 20 mixture of the a- and P-isomers, whereas the regioselectivity of the enzymatic... 

Aspartame is produced by coupling together L-phenylalanine (or L-phenylalanine methyl ester) and L-aspartic acid, either chemically or enzymatically. The former procedure yields both the sweet a-aspartame and nonsweet P-aspartame from which the a-aspartame has to be separated and purified. The enzymatic process yields only a-aspartame. 

The enzymatic aspartame process of Tosoh can also be regarded as a reactive deracemization of D,L-phenylalanine methyl ester (D.L-Phe-OCHa). In the key step the metalloproteinase thermolysin couples only the L-isomer with Z-aspartic acid in a thermodynamically controlled peptide synthesis the remaining D-Phe-OCH3 is racemized and reused [107]. 

The enzyme has also been used in the production of several natural amino acids such as L-serine from glycine and formaldehyde and L-tryptophan from glycine, formaldehyde, and indole [77-79], In addition, SHMT has also been used for the production of a precursor, 20, to the artificial sweetener aspartame (21) through a non-phenylalanine-requiring route (Scheme 14) [80-83]. Glycine methyl ester (22) is condensed with benzaldehyde under kinetically controlled conditions to form L-enY/ ra-p-phenylserine (23). This is then coupled enzymatically using thermolysin with Z-aspartic acid (24) to form A -carbobcnzyloxy-L-a-aspartyl-L-eryt/zro-p-phenylserine (20). and affords aspartame upon catalytic hydrogenation. 

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