Project Summary The rapid and modular generation of molecular diversity is key to the search for new chemical functions. One particularly useful functional group is the halogen (X = Cl, Br, I), which enables many selective and effective downstream strategies for creating structural complexity. In this regard, halogenase enzymes have provided an important and complementary approach to synthetic catalysts for regio- and stereoselective introduction of a halogen substituent on a complex scaffold. While many families of halogenases exist, the radical halogenases provide the greatest potential for reaction diversity, as they are competent to replace unactivated C-H bonds with a halogen unlike those that operate by electrophilic or nucleophilic mechanisms. However, the substrate scope of these enzymes has been limited to date to either protein- bound substrates or large late-stage natural product intermediates. Our group has discovered a new clade of radical halogenases capable of reacting with small molecule substrates. We now seek to take advantage of this discovery to develop new tools for in vitro and in vivo synthesis. Specific aims of this proposal include: (i) elucidating the structure and mechanism of these new radical halogenases, which will provide important insight into their engineering, (ii) investigating and engineering selectivity in halogenases.