High-throughput chemoenzymatic synthesis of bioactive compounds

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Proposal Summary With drug-resistant infections and cancers becoming more prominent, it is crucial to investigate new classes of small molecules to combat these growing health crises. Azaphilones, an underexplored class of fungal natural products, have been shown to exhibit diverse biological properties, making them candidates as a novel class of therapeutics. Although preliminary studies demonstrate their utility in this regard, further investigation of their activities has been hindered due to the challenges of constructing their densely functionalized core and congested stereocenter. Furthermore, selectively accessing either C7 configuration of these scaffolds presents an addition hurdle to exploring azaphilone structure-activity relationships. The most concise approach toward this class of molecules relies on the oxidative dearomatization of prefunctionalized arenes. Current state-of-the-art methods in asymmetric oxidative dearomatization require superstoichiometric quantities of both an oxidant and expensive chiral ligand. These methods have also only been demonstrated on a limited substrate scope, can exhibit poor site-selectivity, and require forcing reaction conditions. Fortunately, Nature has evolved superior catalysts to perform oxidative dearomatization with greater site- and stereoselectivity than these established chemical methods. Promisingly, we have identified two flavin- dependent monooxygenase homologs, AzaH and AfoD, which can perform an oxidative dearomatization on aromatic substrates with the same site-selectivity, but provide the opposite stereochemical configuration in the subsequent azaphilone product. This pivotal discovery enables their use in the synthesis of various natural products and synthetic building blocks, providing orthogonal site- and stereoselectivity to more readily access greater chemical space in an environmentally-benign manner. Furthermore, these catalysts can operate mild reaction conditions, making them compatible with high-throughput compound generation platforms. This proposal describes strategies for the use of these biocatalysts in stereodivergent, chemoenzymatic syntheses for the rapid generation of azaphilone analogs. In summary, this work aims to investigate azaphilone structure-activity relationships through high-throughput and strategic diversification of these scaffolds, directed by a fluorescence polarization assay and a cell painting screen. The methods established herein will provide a means to efficiently develop increasingly potent azaphilone-based drugs.