PROJECT SUMMARY & ABSTRACT Nitrogen heterocycles are ubiquitous pharmacophores found in nearly 60% of all pharmaceuticals. Despite the prevalence of these moieties there are no catalyst classes that form C–N bonds enantioselectively to produce diverse nitrogen-containing heterocycles (i.e. cyclic amines and lactams) in an industrially viable fashion. Therefore, developing catalysts for direct and expedient access to nitrogen heterocycles is of great synthetic and medicinal interest. A desirable method for nitrogen heterocycle synthesis is using intramolecular nitrene insertion reactions to form a new C–N bond. The laboratories of Breslow, Du Bois, Betley, and Zhang have laid the instrumental groundwork for developing these nitrene insertion reactions. But, current precious metal catalysts are limited and display little to no enantioselectivity. Engineered enzyme catalysts can solve this longstanding synthetic challenge as they have exquisite regio-, chemo-, and stereo-selectivity in mild conditions with fast kinetics and are biosynthesized from renewable materials. Ongoing research on engineered hemoproteins shows that they catalyze an ever increasing number of asymmetric reactions of carbenoids and nitrenoids. I propose to engineer hemoproteins to catalyze C–N bond formation by nitrene insertion reactions to directly form important nitrogen-containing heterocycles (i.e. cyclic amines and lactams). Such reactions are unknown in Nature and grant ready access to numerous bioactive molecules. The specific aims are: (1) to develop hemoproteins for nitrene C–H insertion reactions to form cyclic amines; (2) to develop hemoproteins for nitrene C–H insertion reactions to form lactams; (3) rationalize the origins of selectivity in developed hemoproteins for engineering new reactivity. I will begin by screening compilation plates containing hundreds of the Arnold laboratory’s hemoproteins that catalyze various carbene and nitrene transfer and insertion reactions against five safe and easy-to-synthesize nitrene sources. This process will identify an enzyme-nitrene-source pair to optimize with directed evolution. After multiple rounds of evolution, I will then analyze the biocatalyst evolutionary trajectory with computational models to guide future evolutionary campaigns and streamline choosing mutational sites for evolving enzymes to catalyze new-to-Nature reactions. In total, development and implementation of such biocatalysts will allow for sustainable and asymmetric syntheses of highly valuable commodity chemicals, pharmacophores, natural products and pharmaceuticals.