While the enzymatic oxidative cleavage of C–F bonds at achiral centers catalyzed by P450 monooxygenases has been studied extensively, less is known about the oxidative defluorination mechanism of chiral substrates with fluorine being at chirality centers. Here, we report that the use of ethyl 2-fluoro-2-phenylacetate as a stereochemical probe enabled us to discover two oxidative defluorination paths catalyzed by P450-BM3: α-site hydroxylation and hydroxylation at the remote phenyl group. By means of intermediate identification and capture, chemical mimics, key intermediate derivatization, and deuterium insertion experiments as well as QM theoretical calculations, we succeeded in demonstrating an unusual mechanism in which these two oxidative defluorination routes occur in a P450 monooxygenase. This work presents valuable insights into the oxidative degradation of organofluorines and provides a convenient method for remote C–F bond activation.

中文翻译：

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发现 P450 催化的手性有机氟氧化脱氟机制：揭示隐藏的途径

虽然已经广泛研究了由 P450 单加氧酶催化的非手性中心 C-F 键的酶促氧化裂解，但对氟位于手性中心的手性底物的氧化脱氟机制知之甚少。在这里，我们报告了使用 2-氟-2-苯基乙酸乙酯作为立体化学探针使我们能够发现 P450-BM3 催化的两种氧化脱氟途径：α-位点羟基化和远程苯基羟基化。通过中间体识别和捕获、化学模拟、关键中间体衍生化和氘插入实验以及 QM 理论计算，我们成功地证明了这两种氧化脱氟途径在 P450 单加氧酶中发生的不同寻常的机制。