Scheme 2.

Although the j-carboxylic group of aspartic acid does not need to be protected, the a-amino group of aspartic acid and the carboxylic group of phenylalanine must be protected. This plays an important role not only for the selective reaction of the a-carboxylic group of L-aspartic acid and the amino group of L-phenylalanine, but also for being suitable to be used by thermolysin as substrates. Ther-molysin can not catalyze the condensation reaction without such protections (9,10). The methyl ester group is used preferably for the protection of the carboxyl group of phe-nylalanine because this is an essential group expressing the sweetness of APM. The amino group of aspartic acid is protected by the Z-group because it is easily removed by hydrogenolysis without removal of the methyl ester group at the C-terminal of APM. Additionally, a higher reaction rate is obtained when a hydrophobic and bulky group exists at this position. Although formyl or acetyl groups can also be used for the protection, the Z-group, which is hy-drophobic and bulky, is more suitable for the reaction rate of the condensation.

Thermolysin and similar microbial metalloproteases (thermolysin-like metalloproteases) were selected from among a number of industrially available proteases. Although other serine or cysteine proteases, such as subtili-sin or papain, have been more widely used in various industrial fields, they are not very suitable for APM production because they tend to hydrolyze lower alkyl esters at the C-terminal of peptides (11-13) and also the methyl ester group of L-PheOMe and Z-APM. The cleaving sites of proteases in Z-APM are summarized in Table 1, where residues of Z-APM are numbered as P1, P2, and P3, respectively, for those toward the N-terminal from the cleavage site, and as P'1 and P'2, respectively, for those toward the C-terminal from the cleavage site. Specificity of thermo-lysin and thermolysin-like metalloproteases is restricted to hydrophobic and bulky amino acids, such as phenylal-anine or leucine (14) at the P'1 position. The metallopro-teases also have esterase activity, but this activity is restricted in peptide mimic esters like Bz-Gly-OPhe-Ala or furylacryloyl-Gly-OLeu-NH2 (15). The methyl ester of L-PheOMe or Z-APM is not hydrolyzed by them at all, because the methyl ester group is too small at P'1 position. Z-APM is, therefore, produced by thermolysin or thermo-lysin-like metalloproteases without by-products.

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