Subtilisin-catalyzed process

A Subtilisin-catalyzed process was developed for the kilogram-scale continuous production of the drug intermediate (R)-l-aminoindan (R)-19e and of the chiral resolving agent (R)-l-(l-naphthyl)ethylamine (R)-19fin PBR [110]. Note the opposite stereopreference of the protease [110] and Upase-catalysis [111-113]. 

To assess the effect of protein binding on catalysis, we compared the initial rates of [1] oxidation by t-butylhydroperoxide in the presence of selenolsubtilisin and diphenyldiselenide, a nonenzymatic model compound. At low substrate concentrations the enzymatic reaction is roughly 70,000 times faster than the selenocystein catalyzed process. Clearly, the redox activity of the selenium group is considerably increased in the active site of subtilisin. Further characterization of the redox chemistry of selenolsubtilisin is in progress. We believe that the information gained in this study will provide a better understanding of how natural glutathione peroxidase works. 

An efficient enzyme-catalyzed reaction for the large-scale preparation of (R)-2-iso-butyl succinic acid 4-ethyl ester (R)-2a, a key intermediate in the synthesis of collagenase inhibitor R00319790 (1), is described (Fig. 1). The corresponding racemic diethyl ester substrate 9 is applied at 20% concentration and hydrolyzed re-gio- and enantioselectively ( >100) at the sterically more hindered secondary ester group using a cheap commercial subtilisin preparation. The desired (R)-monoacid is separated from the remaining antipodal diester (S)-9 by means of extraction and obtained in > 99% ee and > 43% yield. The development of the reaction from process research to the pilot-scale is described. 

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