Synthesis of α-hydroxy ketones through novel one-pot multi-enzyme cascade system from racemic epoxides

Abstract

α-Hydroxy ketones, highly versatile building blocks, present challenges for their green and efficient synthesis. Based on thermodynamic analysis, a novel in vitro one-pot multi-enzyme cascade system was designed for synthesizing 2-hydroxyacetophenone (1c) from racemic styrene oxide (1a). This system incorporated a Rhodotorula paludigena epoxide hydrolase (RpEHL360V) for the enantioconvergent hydrolysis of rac-1a to (R)-phenyl-1,2-ethanediol (1b), a Bacillus subtilis (2,3)-butanediol dehydrogenase (BsBDHAI49L/V266L/G292A) for the subsequent oxidation to 1c, and a Lactobacillus casei lactate dehydrogenase (LcLDH1) for the reduction of pyruvate to lactate, thereby facilitating coenzyme recycling and shifting the reaction's thermodynamic equilibrium. After optimizing the reaction conditions, the system reached its highest efficiency using cell-free extracts of RpEHL360V (4.0 U/mL), BsBDHAI49L/V266L/G292A (0.3 U/mL), and LcLDH1 (0.1 U/mL), along with 4 mM NADH and 200 mM sodium pyruvate as cofactor and cosubstrate, respectively, in a Na2HPO4-NaH2PO4 buffer (100 mM, pH 8.0) at 30 °C. Under these conditions, the system catalyzed the conversion of 50 mM rac-1a to 1c with a total yield of 73.1 % and a maximum space-time yield of 10.7 mmol/L/h within 24 h. Furthermore, this one-pot system was applied to catalyze 20 mM of rac-4-chlorostyrene oxide and 1,2-epoxyhexane, successfully synthesizing two corresponding α‑hydroxy ketones, each with a space-time yield of 0.7 mmol/L/h, demonstrating the system's efficacy for the production of α‑hydroxy ketones.