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

Abstract 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 Acl HMT. QCMT can cleave F‐SAM to produce the 5'‐dA radical. The significant reaction intermediate CH 2 FCbI was characterized by HR‐MS, 19 F 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 CH 2 FCbI with CH 2 FI and transfer the CH 2 F 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.