The structures of APM and its regioisomer are shown in Figure 1. In APM production by conventional organic synthesis, the side-chain carboxylic group (S-carboxylic group) of aspartic acid must be protected to avoid the formation of unwanted /-APM; additionally, optically pure substrates are required to avoid the production of stereoisomers of APM. These APM-related compounds, such as /-APM and stereoisomers, exhibit a bitter taste (1). Use of a protease as catalyst does not give formation of these undesirable compounds, even if racemic substrates are used as starting materials and without protection of the /-carboxylic group of aspartic acid. The use of racemic phenylalanine in the enzyme process is very advantageous, because L-phenyl-alanine is more expensive than racemic phenylalanine.
Proteases such as thermolysin, subtilisin, or papain were found to be useful tools for the peptide synthesis because of their high stereo and regio selectivity, and they have been applied in the synthesis of various biologically active peptides, such as peptide hormones, neuropeptides, and insulin (3). APM synthesis catalyzed by a protease was also investigated by Isowa et al. (4). They reported that N-protected aspartic acid and Phe-alkyl ester are condensed to N-protected L-a-Asp-L-Phe-alkyl ester by thermolysin in high yield (83-96%) as a precipitate consisting of the addition compound of the condensation product and one molecule of Phe-alkyl ester, as shown in Scheme 1.
In this scheme, R indicates N-protective group, such as Z or PMZ, and R1 indicates lower alkyl ester, such as methyl or ethyl ester. Proteases usually catalyze the hydrolysis of peptides in water as solvent; the condensation reaction is, in other words, the reverse reaction. Therefore, it is usually difficult to obtain products in high yield because of the occurrence of hydrolysis of the products by the proteases. The synthesis of APM-related peptides can, however, be performed in high yield when the product is removed from the reaction system as a precipitate and is not hydrolyzed back by the protease, for example, by ther-molysin.
Although this method requires an expensive catalyst, such as thermolysin, the high stereo and regio selectivity is a great advantage in APM production. The industrial APM production process based on the proposal of Isowa et al. was developed by Toyo Soda Manufacturing and was adopted for APM production by Holland Sweetener Company.
In contrast to the enzymatic method, the chemical method produces an APM precursor where formyl-APM (f-APM) is derived from A-f-Asp anhydride and L-PheOMe, (5) as is shown in Scheme 2.
Although /-APM is produced as by-product in this method, a-selectivity of the reaction can be enhanced by selection of the optimal solvent for the reaction, such as alkaline aqueous medium (6) or acetic acid partially replaced with an alkyl ester with a secondary or tertiary alcohol (7). It was reported that the production of /-APM was reduced to 20% of total peptide by selection of a proper solvent. The /-APM reportedly can be converted to APM by alkaline treatment in alcohol in the presence of metal ions such as zinc or copper as catalysts (8). Productivity of APM seems to be enhanced by these methods in the chemical procedure.
As described earlier, the enzymatic and chemical methods both have individual advantages. An advantage of the enzymatic method is the high selectivity of the reaction, where APM is produced from cheap raw materials by a simple procedure in high yield. Not requiring expensive catalysts, such as enzymes, in the condensation step is an advantage of the chemical method; however, expensive starting materials must be used and by-product is produced. Attempting cost reduction in APM production and improving the condensation step are of great importance for APM production by Holland Sweetener Company, and many efforts have been made by both TOSOH and DSM. Hereafter, I describe the enzymatic APM production process and the efforts to improve the condensation reaction.
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