Project Summary The ability to access complex target molecules with sustainable methodology is imperative to the development of novel drugs, which will be necessary to treat antimicrobial resistance and emerging infectious diseases.1 Site- selective oxidation reactions are fundamental to the current synthetic logic towards complex scaffolds and the decoration of these cores, which is key to the development of novel drugs.2,3 Nature can use enzymes to impart exquisite site-selectivity in oxidations based on the three-dimensional control exerted by the active site while bringing a substrate and oxidant together. Taking inspiration from Nature, I plan to utilize flavin-dependent monooxygenases to site-selectively oxidize a variety of non-native arenes and heteroarenes substrates (Aim 1), which will allow access to novel building blocks, pharmaceuticals, and natural products and their derivatives. The sequence space of flavin-dependent monooxygenases is vast, therefore, logically picking biocatalysts that have improved, site-selectivity, yield, or substrate scope, requires a strategy. I plan to use sequence similarity networks, (to visualize how similar sequences are), sequence alignments, (to probe amino acid changes proximal to active sites), three-dimensional active site analysis, (to identify key amino acids and size of active site), and docking studies, (to understand how a substrate interacts with an active site) (Aim 2). These techniques can be used together to explain and predict trends in substrate scope, and site-selectivity. Ultimately, these techniques will help me to logically choose biocatalysts that have improved properties such as expanded substrate scope, divergent site-selectivity, or improved yield to strengthen the proposed oxidative method development. Together these aims will develop site-selective biocatalytic oxidation methods applicable to a variety of arene and heteroarene substrates. This research will broadly impact biocatalysis investigations by developing a high throughput platform for nonnative substrate screening across a family of enzymes, and informing what tools are important for rationally choosing existing biocatalysts. Finally, the proposed site-selective oxidative method will improve the ease and efficiency of synthesis and derivatization of molecules that are important to human health.