Biocatalytic Olefin Difunctionalization for Synthesis of Chiral 2‐Azidoamines Using Nonheme Iron Enzymes

Abstract Alkene difunctionalization represents an important category of reactions in organic synthesis, with a diverse array of transformations developed over the past decades for various synthetic applications. Nevertheless, the scope and diversity of biocatalytic alkene difunctionalization have been limited, constraining its synthetic utility. In this study, we repurposed nonheme iron enzymes to generate iron nitrene intermediates for alkene difunctionalization. 4‐hydroxymandelate synthase from Amycolatopsis orientalis ( Ao HMS) was successfully engineered for direct alkene aminoazidation to produce chiral 2‐azidoamines. Directed evolution was performed on Ao HMS to provide evolved variants that could utilize O ‐pivaloylhydroxylamine triflic acid as the nitrene precursor and produced various primary aminoazidation products with up to 44% yield, 44 total turnover number (TTN), and 98.5:1.5 enantiomeric ratio (e.r.). Mechanistic studies indicated that this new biocatalytic transformation proceeds through a stepwise radical addition and azide recombination pathway. This work expands the catalytic toolbox of metalloenzymes and opens up new opportunities for biosynthesis by introducing nonnatural olefin difunctionalization reactions into biocatalysis.