Engineering an\nEpoxide Hydrolase for Chemoenzymatic\nAsymmetric Synthesis of Chiral Triazole Fungicide (<i>S</i>)- and (<i>R</i>)‑Flutriafol

Flutriafol, a globally utilized triazole fungicide in\nagriculture,\nis typically applied as a racemic mixture, but its enantiomers differ\nin bioactivity and environmental impact. The synthesis of flutriafol\nenantiomers is critically dependent on chiral precursors: 2,2-bisaryl-substituted\noxirane [(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><sup>H336W/L360F</sup> with a 6.4-fold enhanced enantiomeric ratio (<i>E</i>)\nfrom 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><sup>H336W/L360F</sup>, producing (<i>S</i>)-<b>1a</b> (98.2% <i>ee</i><sub>s</sub>) and (<i>R</i>)-<b>1b</b> (75.0% <i>ee</i><sub>p</sub>) with 44.3 and 55.7% analytical yields, respectively. As follows,\nchiral (<i>S</i>)-flutriafol (98.2% <i>ee</i>)\nand (<i>R</i>)-flutriafol (75.0% <i>ee</i>) were\neasily synthesized by a one-step chemocatalytic process from (<i>S</i>)-<b>1a</b> and a two-step chemocatalytic process\nfrom (<i>R</i>)-<b>1b</b>, respectively. This chemoenzymatic\napproach offers a superior alternative for the asymmetric synthesis\nof flutriafol enantiomers. Furthermore, molecular dynamics simulations\nrevealed insight into the enantioselectivity improvement of <i>Rp</i>EH toward bulky 2,2-bisaryl-substituted oxirane <b>1a</b>.