Controllable Multihalogenation of a Non-native Substrate by the SyrB2 Iron Halogenase

Geminal, multihalogenated functional groups are widespread in natural products and pharmaceuticals, yet no synthetic methodologies exist that enable selective multihalogenation of unactivated C–H bonds. Biocatalysts are powerful tools for late-stage C–H functionalization as they operate with high degrees of regio-, chemo-, and stereoselectivity. 2-Oxoglutarate (2OG)-dependent nonheme iron halogenases chlorinate and brominate aliphatic C–H bonds and offer a solution for achieving these challenging transformations. Here, we describe the ability of a nonheme iron halogenase, SyrB2, to controllably halogenate a non-native substrate α-aminobutyric acid (Aba) to yield monochlorinated, dichlorinated, and trichlorinated products. These chemoselective outcomes are achieved by controlling the loading of the 2OG cofactor and SyrB2 biocatalyst. Furthermore, by using a ferredoxin-based biological reductant for electron transfer to the catalytic center of SyrB2, we demonstrate order-of-magnitude enhancement in the yield of trichlorinated products that were previously inaccessible using any single halogenase enzyme. We also apply these strategies to broaden SyrB2's reactivity scope to include multibromination and demonstrate chemoenzymatic conversion of the ethyl side chain in Aba to an ethynyl functional group. We show how steric hindrance induced by the successive addition of halogen atoms on Aba's C4 carbon dictates the degree of multihalogenation by hampering C3–C4 bond rotation within SyrB2's catalytic pocket. Overall, our work showcases the potential of iron halogenases to facilitate multi–C–H functionalization chemistry.