Controllable Multihalogenation\nof a Non-native Substrate\nby the SyrB2 Iron Halogenase

Geminal, multihalogenated functional groups are widespread\nin natural\nproducts and pharmaceuticals, yet no synthetic methodologies exist\nthat enable selective multihalogenation of unactivated C–H\nbonds. Biocatalysts are powerful tools for late-stage C–H functionalization\nas they operate with high degrees of regio-, chemo-, and stereoselectivity.\n2-Oxoglutarate (2OG)-dependent nonheme iron halogenases chlorinate\nand brominate aliphatic C–H bonds and offer a solution for\nachieving these challenging transformations. Here, we describe the\nability of a nonheme iron halogenase, SyrB2, to controllably halogenate\na non-native substrate α-aminobutyric acid (Aba) to yield monochlorinated,\ndichlorinated, and trichlorinated products. These chemoselective outcomes\nare achieved by controlling the loading of the 2OG cofactor and SyrB2\nbiocatalyst. Furthermore, by using a ferredoxin-based biological reductant\nfor electron transfer to the catalytic center of SyrB2, we demonstrate\norder-of-magnitude enhancement in the yield of trichlorinated products\nthat were previously inaccessible using any single halogenase enzyme.\nWe also apply these strategies to broaden SyrB2’s reactivity\nscope to include multibromination and demonstrate chemoenzymatic conversion\nof the ethyl side chain in Aba to an ethynyl functional group. We\nshow how steric hindrance induced by the successive addition of halogen\natoms on Aba’s C<sub>4</sub> carbon dictates the degree of\nmultihalogenation by hampering C<sub>3</sub>–C<sub>4</sub> bond\nrotation within SyrB2’s catalytic pocket. Overall, our work\nshowcases the potential of iron halogenases to facilitate multi–C–H\nfunctionalization chemistry.