Engineering an Epoxide Hydrolase for Chemoenzymatic Asymmetric Synthesis of Chiral Triazole Fungicide (<i>S</i>)- and (<i>R</i>)-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, <b>1a</b>] and 1,2-diol [1-(2-fluorophenyl)-1-(4-fluorophenyl)ethane-1,2-diol, <b>1b</b>]. Here, we engineered a <i>Rhodotorula paludigensis</i> epoxide hydrolase (<i>Rp</i>EH), obtaining mutant <i>Escherichia coli</i>/<i>Rpeh</i>^H336W/L360F with a 6.4-fold enhanced enantiomeric ratio (<i>E</i>) from 5.5 to 35.4. This enabled a gram-scale resolution of <i>rac</i>-<b>1a</b> by <i>E. coli</i>/<i>Rpeh</i>^H336W/L360F, producing (<i>S</i>)-<b>1a</b> (98.2% <i>ee</i>_s) and (<i>R</i>)-<b>1b</b> (75.0% <i>ee</i>_p) with 44.3 and 55.7% analytical yields, respectively. As follows, chiral (<i>S</i>)-flutriafol (98.2% <i>ee</i>) and (<i>R</i>)-flutriafol (75.0% <i>ee</i>) were easily synthesized by a one-step chemocatalytic process from (<i>S</i>)-<b>1a</b> and a two-step chemocatalytic process from (<i>R</i>)-<b>1b</b>, 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 <i>Rp</i>EH toward bulky 2,2-bisaryl-substituted oxirane <b>1a</b>.