A Continuous-Flow Cascade Reactor System for Subtilisin A- Catalyzed Dynamic Kinetic Resolution of N-tert-Butyloxycarbonylphenylalanine Ethyl Thioester with Benzylamine

Alcalase® (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 °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, rflow) and enantiomeric ratio (E) were studied in the 0–100 °C range. The effect of the temperature on base‐catalyzed racemization of the non‐transformed (R)‐thioester in a continuous‐flow reactor was also investigated in the 0–150 °C range. The continuous‐mode DKR of the racemic thioester in a serial cascade system of six biocatalyst‐filled columns at 50 °C for KR and five grafted silica gel‐filled columns at 150 °C for racemization resulted in the formation of the (S)‐benzylamide in 79% conversion, 8.17 g L−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.