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

Abstract

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 tert-butyl group makes it a great challenge to synthesize tebuconazole enantiomers. Here, we designed a simple chemoenzymatic approach for the asymmetric synthesis of (R)-tebuconazole, which includes the biocatalytic resolution of racemic epoxy-precursor (2-tert-butyl-2-[2-(4-chlorophenyl)ethyl] oxirane, rac-1a) by Escherichia coli/Rpeh whole cells expressed epoxide hydrolase from Rhodotorula paludigensis (RpEH), followed by a one-step chemocatalytic synthesis of (R)-tebuconazole. It was observed that (S)-1a was preferentially hydrolyzed by E. coli/Rpeh, whereas (R)-1a was retained with a specific activity of 103.8 U/g wet cells and a moderate enantiomeric ratio (E value) of 13.4, which was remarkably improved to 43.8 after optimizing the reaction conditions. Additionally, a gram-scale resolution of 200 mM rac-1a was performed using 150 mg/mL E. coli/Rpeh wet cells, resulting in the retention of (R)-1a in a 97.0% ees, a 42.5% yields, and a 40.5 g/L/d space-time yield. Subsequently, the synthesis of highly optical purity (R)-tebuconazole (>99% ee) was easily achieved through the chemocatalytic ring-opening of the epoxy-precursor (R)-1a with 1,2,4-triazole. To elucidate insight into the enantioselectivity, molecular docking simulations revealed that the unique L-shaped substrate-binding pocket of RpEH plays a crucial role in the enantioselective recognition of bulky 2,2-disubstituted oxirane 1a.