Project Abstract Halogenated organic compounds are used extensively as building blocks, synthetic intermediates, and end use products for pharmaceutical and agrochemical applications. The utility of these compounds, including their biological activity, arises from the reactivity and physical properties uniquely conferred to them by halogen substitution. The importance of halogenation and limitations associated with current halogenation methods prompted us to develop enzymes for biocatalytic halogenation. This proposal outlines the evolution of flavin dependent halogenases (FDHs) and Fe(II)/α-ketoglutarate dependent halogenases (FeDHs) for a range selective halogenation and related (i.e. pseudohalogen) atom transfer reactions. Specifically, we will build on our extensive experience with FDH engineering to expand the range of substrates and sites on those substrates that can be halogenated. We will focus on enabling halogenation of electron deficient aromatic substrates with high site- and enantioselectivity. These efforts will benefit from recently characterized single component flavin reductase/FDH enzymes, and we will optimize conditions for large scale halogenation using these simplified biocatalysts. Finally, we recently reported that FDHs catalyze enantioselective halocyclization, and we will expand the substrate scope of these reactions to include systems that cannot be achieved using small molecule catalysts. In the FeDH space, we will engineer enzymes with expanded substrate scope to enable site-selective C-H azidation of natural products and pharmaceuticals. This capability will be used for chemoenzymatic synthesis via fragment coupling reactions and other processes that leverage the unique reactivity of azides for more extensive remodeling of substrates to generate natural product-like heterocycles. Finally, we will use both computational design and directed evolution to enable site-selective fluorination using FeDHs. In addition to addressing a major synthetic challenge, this effort will improve our understanding of non- native rebound in FeDHs to improve these enzymes for different C-H functionalization processes.