B12‐Dependent Radical SAM Enzymes Catalyze C‐Fluoromethylation via a CH₂F‐Cobalamin Intermediate

Fluorine and fluorine-containing functional groups play important roles in drugs and agrochemicals. Recently, SAM-dependent methyltransferases and several SAM analogues have been reported for fluoromethyl transfer through a nucleophilic mechanism. However, fluoromethylation of unactivated carbon centers is very challenging, and their substitution usually involves a radical mechanism. To date, no biocatalysts have been developed for fluoromethylation of unactivated carbon centers. In this study, we found that the B12-dependent radical SAM methyltransferase (B12-RSMT) QCMT can fluoromethylate the glutamine Cα position of peptides with fluorinated SAM (F-SAM) generated in situ by the enzyme AclHMT. QCMT can cleave F-SAM to produce the 5'-dA radical. The significant reaction intermediate CH₂FCbI was characterized by HR-MS, ¹⁹F NMR spectroscopy and X-ray crystallography. In addition, B12-RSMTs CysS and GenD1 can also transfer fluoromethyl groups onto natural products. We also found that F-SAM is not compulsory. The reduced B12-RSMTs can directly generate CH₂FCbI with CH₂FI and transfer the CH₂F group when SAM is used as the radical initiator. Our results demonstrate a radical-mediated enzymatic strategy for fluoromethylation with abiological cofactors and expand radical SAM enzymes to the field of fluorine chemistry.