Engineering an Epoxide Hydrolase for Chemoenzymatic Asymmetric Synthesis of Chiral Triazole Fungicide ( S )- and ( R )-Flutriafol.

Flutriafol, a globally utilized triazole fungicide in agriculture, is typically applied as a racemic mixture, but its enantiomers differ in bioactivity and environmental impact. The synthesis of flutriafol enantiomers is critically dependent on chiral precursors: 2,2-bisaryl-substituted oxirane [(2-fluorophenyl)-2-(4-fluorophenyl)oxirane, 1a ] and 1,2-diol [1-(2-fluorophenyl)-1-(4-fluorophenyl)ethane-1,2-diol, 1b ]. Here, we engineered a Rhodotorula paludigensis epoxide hydrolase ( Rp EH), obtaining mutant Escherichia coli / Rpeh H336W/L360F with a 6.4-fold enhanced enantiomeric ratio ( E ) from 5.5 to 35.4. This enabled a gram-scale resolution of rac - 1a by E. coli / Rpeh H336W/L360F , producing ( S )- 1a (98.2% ee s ) and ( R )- 1b (75.0% ee p ) with 44.3 and 55.7% analytical yields, respectively. As follows, chiral ( S )-flutriafol (98.2% ee ) and ( R )-flutriafol (75.0% ee ) were easily synthesized by a one-step chemocatalytic process from ( S )- 1a and a two-step chemocatalytic process from ( R )- 1b , respectively. This chemoenzymatic approach offers a superior alternative for the asymmetric synthesis of flutriafol enantiomers. Furthermore, molecular dynamics simulations revealed insight into the enantioselectivity improvement of Rp EH toward bulky 2,2-bisaryl-substituted oxirane 1a .