Reprogramming nonheme iron enzymes for abiotic alkene trifluoromethylazidation

In nature, nonheme iron enzymes catalyze a variety of chemical transformations, such as hydroxylation, halogenation, desaturation, and oxidative cyclizations, through the activation of dioxygen. Beyond their native functions, these enzymes can also serve as versatile biocatalysts for new-to-nature metal-catalyzed reactions. In particular, we evolved the nonheme iron oxygenase, hydroxymandelate synthase from Amycolatopsis orientalis (AoHMS), to catalyze an enantioselective alkene trifluoromethylazidation reaction using hypervalent iodine(III) reagents as a source of trifluoromethyl (-CF₃) radicals. In this chapter, we describe experimental protocols for the directed evolution of nonheme iron trifluoromethylases using a Staudinger ligation-based high-throughput screening (HTS) platform. We also include detailed procedures for the setup of analytical scale biotransformations, generation of gas chromatography-mass spectrometry (GC-MS) calibration curves, and protein purification. We hope these methods will prove useful for the engineering of nonheme iron enzymes for future biocatalytic applications.