Condensation without NProtection

Various strategies have been used in the attempt to improve the APM production process. The possibility of reducing enzyme consumption was indicated by mutant thermolysins and by adjusting of the concentration of substrates. On the other hand, an alternative production system is desired to take long strides in reducing costs. Much of this research has been focused on avoiding N-protection of aspartic acid. If APM can be produced without N-protection, it will not only reduce raw materials costs but will also simplify the production process by eliminating the protection and de-protection steps. Although low yields have prevented industry adoption of a new method, the production costs of APM would be further reduced by N-protection-free methods if productivity could be improved. Hereafter, I summarize such attempts as appeared in patent applications.

Direct production of APM by condensation of aspartic acid and PheOMe was proposed by TOSOH and Ajinomoto, respectively, where both starting materials are condensed by microbial cells belonging to Pseudomonas, Alkaligenes, and so forth (36,37), as shown in Scheme 4. In this process, the reaction seems to be catalyzed by aminopeptidases in the bacterial cells. Although this is a very simple production method, its yield was very low. According to the TOSOH patent application (36), only 0.06 g of APM was produced from 1.0 g of aspartic acid after 16 h of reaction. Because APM is not removed from the reaction mixture as a precipitate under this condition, it seems to be hydro-lyzed back by the aminopeptidases. If the product could be separated from the reaction mixture in situ, the production yield will be increased. It is, however, difficult to remove APM from the reaction mixture as a precipitate, because APM is not only highly soluble (similar to the substrates) but also does not form an insoluble addition compound with remaining substrate. No other additive that would form an insoluble addition compound with APM has yet been found. Although separation of APM by electrodialysis was attempted by Snedecor and Hsu (38), sufficient separation yields do not seem to be obtained. Development of an effective method to separate APM from its starting materials and enzymes will be of importance in establishing a feasible production process based on those patent applications.

Another APM production method without N-protection is the coupling of a-AspOR (R indicates a lower alkyl group) and PheOMe by an ester-amino exchange reaction catalyzed by a protease from Staphylococcus aureus strain

V8 (V8 protease) (39) as shown in Scheme 5. Because this reaction is a dealcoholation reaction, the reaction rate is higher than that of a dehydration reaction, such as the reaction shown in Scheme 4. Additionally, by-products due to misconfiguration, such as PheAspOR or PhePheOMe, are not produced because V8 protease is specific for a pep-tide bond or ester bond that involves the carbonyl group of amino acids with an acidic side chain (40), for example, aspartic acid at the P1 position (Table 1). However, removal of APM from the reaction system encounters the same difficulties as in the first example of the APM direct production; therefore, a higher production yield cannot be expected. It will also be difficult to produce a-AspOR industrially without formation of/¡-AspOR and Asp(OR)2, because the reactivities of the two carboxyl group of aspartic acid are not so different. Thus, various barriers exist to establishing a production process based on this method.

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