Controllable multi-halogenation of a non-native substrate by SyrB2 iron halogenase

Geminal, multi-halogenated functional groups are widespread in natural products and pharmaceuticals, yet no synthetic methodologies exist that enable selective multi-halogenation 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 non-heme iron halogenases chlorinate and brominate aliphatic C-H bonds offering a solution for achieving these challenging transformations. Here, we describe the ability of a non-heme iron halogenase, SyrB2, to controllably halogenate non-native substrate alpha-aminobutyric acid (Aba) to yield mono-chlorinated, di-chlorinated, and tri-chlorinated products. These chemoselective outcomes are achieved by controlling the loading of 2OG cofactor and SyrB2 biocatalyst. 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 tri-chlorinated product that were previously inaccessible using any single halogenase enzyme. We also apply these strategies to broaden SyrB2's reactivity scope to include multi-bromination and demonstrate chemoenzymatic conversion of the ethyl side chain in Aba to an ethylyne functional group. We show how steric hindrance induced by the successive addition of halogen atoms on Aba's C₄ carbon dictates the degree of multi-halogenation by hampering C₃-C₄ bond rotation within SyrB2's catalytic pocket. Overall, our work showcases the synthetic potential of iron halogenases to facilitate multi-C-H functionalization chemistry.