Biocatalytic enantioselective C(sp)-H fluorination enabled by directed evolution of non-haem iron enzymes.

Due to the scarcity of C-F bond forming enzymatic activities in nature and the contrasting ubiquity of organofluorine moieties in bioactive compounds, developing new biocatalytic fluorination reactions represents a preeminent challenge in enzymology, biocatalysis, and synthetic biology. Additionally, catalytic asymmetric C(sp)-H fluorination remains a challenging problem facing synthetic chemists. Although many nonheme Fe halogenases have been discovered to promote C(sp)-H halogenation reactions, to date, efforts to convert these Fe halogenases to fluorinases have remained unsuccessful. We repurposed a plant-derived natural nonheme enzyme 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO) to catalyze unnatural enantioselective C(sp)-H fluorination via a radical rebound mechanism. Directed evolution afforded C-H fluorinating enzyme ACCO displaying 200-fold higher activity, substantially improved chemoselectivity and excellent enantioselectivity, converting a range of substrates into enantioenriched organofluorine products. Notably, almost all the beneficial mutations were found to be distal to the Fe centre, underscoring the importance of substrate tunnel engineering in nonheme Fe biocatalysis. Computational studies revealed that the radical rebound step with the Fe(III)-F intermediate has an exceedingly low activation barrier of 3.4 kcal/mol, highlighting a new avenue to expand the catalytic repertoire of enzymes to encompass asymmetric C-F bond formation.