Chemoenzymatic Asymmetric Synthesis of Chiral Triazole Fungicide ()-Tebuconazole in High Optical Purity Mediated by an Epoxide Hydrolase from .

Tebuconazole is a chiral triazole fungicide used globally in agriculture as a racemic mixture, but its enantiomers exhibit significant enantioselective dissimilarities in bioactivity and environmental behaviors. The steric hindrance caused by the -butyl group makes it a great challenge to synthesize tebuconazole enantiomers. Here, we designed a simple chemoenzymatic approach for the asymmetric synthesis of ()-tebuconazole, which includes the biocatalytic resolution of racemic epoxy-precursor (2--butyl-2-[2-(4-chlorophenyl)ethyl] oxirane, -) by / whole cells expressed epoxide hydrolase from (EH), followed by a one-step chemocatalytic synthesis of ()-tebuconazole. It was observed that ()- was preferentially hydrolyzed by /, whereas ()- was retained with a specific activity of 103.8 U/g wet cells and a moderate enantiomeric ratio ( value) of 13.4, which was remarkably improved to 43.8 after optimizing the reaction conditions. Additionally, a gram-scale resolution of 200 mM - was performed using 150 mg/mL wet cells, resulting in the retention of ()- in a 97.0% , a 42.5% yield, and a 40.5 g/L/d space-time yield. Subsequently, the synthesis of highly optical purity ()-tebuconazole (>99% ) was easily achieved through the chemocatalytic ring-opening of the epoxy-precursor ()- with 1,2,4-triazole. To elucidate insight into the enantioselectivity, molecular docking simulations revealed that the unique L-shaped substrate-binding pocket of EH plays a crucial role in the enantioselective recognition of bulky 2,2-disubstituted oxirane .