Directed Evolution of Copper-Substituted Nonheme Enzymes for Enantioselective Alkene Oxytrifluoromethylation

Trifluoromethylation is a coveted transformation due to the unique properties of the trifluoromethyl (−CF₃) group and the importance of organofluorine compounds. Enzymes that can catalyze the formation of C−CF₃ bonds would therefore be highly desirable. However, such “trifluoromethylases” are rare. Here, we report a biocatalytic platform for constructing CF₃-substituted lactones via intramolecular alkene oxytrifluoromethylation based on hydroxymandelate synthase from Amycolatopsis orientalis (AoHMS), a nonheme iron enzyme. The key feature that enabled the development of this enzymatic system was the substitution of the native catalytic iron center with copper. This modification retained the ability of the AoHMS protein scaffold to facilitate CF₃ radical generation while harnessing the exceptional catalytic activity of copper for alkene oxyfunctionalizations. Directed evolution of copper-substituted AoHMS resulted in an engineered variant capable of producing β-, γ-, and δ-lactones bearing quaternary stereocenters with high efficiency and enantiocontrol (up to 99% yield and 98.5:1.5 e.r.). This work not only expands the biocatalytic toolbox for organofluorine synthesis but also highlights the immense potential of metal-substituted nonheme iron enzymes for evolving new-to-nature transformations.