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

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 ( Rp EH), 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% ee s , a 42.5% yield s , 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 Rp EH plays a crucial role in the enantioselective recognition of bulky 2,2-disubstituted oxirane 1a .