AContinuous-Flow Cascade Reactor System for Subtilisin A-Catalyzed Dynamic Kinetic Resolution of N-tert-Butyloxy-carbonylphenylalanine Ethyl Thioester with Benzylamine

摘要

Alcalase O (Subtilisin A) was immobilized by simple hydrophobic adsorption onto various surface-grafted macroporous silica gels resulting in easy-to-prepare and stable biocatalysts enabling the efficient kinetic resolution (KR) and dynamic kinetic resolution (DKR) of racemic N-Boc-phenylalanine ethyl thioester with benzylamine. The immobilized Alcalase biocatalysts, which retained their activity and selectivity when stored at 4 degrees C for more than a year, were tested in enzymatic aminolysis in batch and continuous-flow KRs resulting in (S)-N-Boc-phenylalanine benzylamide in high enantiomeric purity. In KR of the racemic thioester by Alcalase-catalyzed aminolysis in a continuous-flow reactor, the productivity (specific reaction rate, r(flow)) and enantiomeric ratio (E) were studied in the 0-100 degrees C range. The effect of the temperature on base-catalyzed racemization of the non-transformed (R)-thio-ester in a continuous-flow reactor was also investigated in the 0-150 degrees C range. The continuous-mode DKR of the racemic thioester in a serial cascade system of six biocatalyst-filled columns at 50 8C for KR and five grafted silica gel-filled columns at 150 8C for racemization resulted in the formation of the (S)-benzylamide in 79% conversion, 8.17 gL(-1)h(-1) volumetric productivity and 98% ee. This is the first example of a dynamic kinetic resolution of an amino acid derivative in continuous-flow mode using an alternating cascade of packed-bed enzyme reactors and racemization reactors kept at different temperatures.