Biocatalytic Generation\nof Trifluoromethyl Radicals\nby Nonheme Iron Enzymes for Enantioselective Alkene Difunctionalization

The trifluoromethyl (−CF<sub>3</sub>) group represents\na\nhighly prevalent functionality in pharmaceuticals. Over the past few\ndecades, significant advances have been made in the development of\nsynthetic methods for trifluoromethylation. In contrast, there are\ncurrently no metalloenzymes known to catalyze the formation of C(sp<sup>3</sup>)–CF<sub>3</sub> bonds. In this work, we demonstrate\nthat a nonheme iron enzyme, hydroxymandelate synthase from Amycolatopsis orientalis (<i>Ao</i>HMS),\nis capable of generating CF<sub>3</sub> radicals from hypervalent\niodine(III) reagents and directing them for enantioselective alkene\ntrifluoromethyl azidation. A high-throughput screening (HTS) platform\nbased on Staudinger ligation was established, enabling the rapid evaluation\nof <i>Ao</i>HMS variants for this abiological transformation.\nThe final optimized variant accepts a range of alkene substrates,\nproducing the trifluoromethyl azidation products in up to 73% yield\nand 96:4 enantiomeric ratio (e.r.). The biocatalytic platform can\nbe further extended to alkene pentafluoroethyl azidation and diazidation\nby altering the iodine(III) reagent. In addition, anion competition\nexperiments provide insights into the radical rebound process for\nthis abiological transformation. This study not only expands the catalytic\nrepertoire of metalloenzymes for radical transformations but also\ncreates a new enzymatic space for organofluorine synthesis.