Highly Regioselective Trifluoromethylation of Unprotected Phenols and Anilines Enabled by an Engineered P450 Peroxizyme System.

The trifluoromethyl (CF3) group is a pivotal motif in pharmaceuticals, agrochemicals, and functional materials, yet achieving direct and regioselective aromatic C─H trifluoromethylation-especially for phenols and anilines-remains a formidable challenge. Here, we transform a dual-functional small molecule (DFSM)-facilitated P450BM3 peroxizyme into a versatile biocatalytic platform that enables highly ortho-selective trifluoromethylation of diverse phenols and anilines. Through protein engineering, we achieve up to 98% ortho-selectivity for phenol-the first highly selective enzymatic trifluoromethylation reported. The system exhibits broad substrate scope and functional-group tolerance, delivering >99% regioselectivity and over 6700 turnovers for leading substrates. Mechanistic studies confirm a radical pathway and reveal that selectivity is engineered via synergistic mutations-DFSM interactions that pre-organize substrates in a confined active site. This work establishes a programmable strategy to bridge biocatalysis and organofluorine chemistry, offering a sustainable route to valuable fluorinated building blocks.