Developing Photoenzymes for Selective Chemical Synthesis

Photoenzymes are biological catalysts that use photonic energy to drive chemical reactions. The key intermediates in these enzymes are organic free radicals, highly reactive intermediates that can be challenging to control using small molecule catalysts. In this proposal, we will use naturally occurring dark enzymes as photoenzymes to address enantio-, regio-, and chemoselectivity challenges associated with radical organic reactions. These non-natural functions will be optimized to directed evolution to enable access to chemical structures that would be challenging to access using existing synthetic methods. In this application, we will use previously identified radical initiation methods to solve new synthetic challenges. To this end, we propose to merge ‘ene’-reductase catalyzed hydroalkylations with imine reductases to prepare saturated heterocycles bearing multiple stereocenters with excellent control over the stereochemistry of the product. In a related area of research, we will develop regioselective difluoro- and trifluoromethylation of arenes to access substitution patterns that are elusive using existing C–H functionalization strategies. We will also use Baeyer-Villager Monooxygenases as potent photoreductants to convert styrenyl alkenes to the corresponding benzylic radical for various cyclization reactions to access N-heterocyclic motifs found in biologically active molecules. Concurrently, we will develop new initiation mechanisms that will expand the types of reactivity available to photoenzymes. In this context, we will use lactate monooxygenase’s ability to form alkylated flavin adducts to facilitate Csp3-Csp3 cross-coupling reactions. This work will enable asymmetric cross-coupling and strategies for preparing congested carbons. Additionally, we will engineer ‘ene’-reductases to use oxidative mechanisms for radical initiation–a challenge because of the oxidative sensitivity of these proteins. This new initiation mechanism can interface with the existing radical termination mechanism to unlock new photoenzymatic chemical transformations. Finally, we will develop energy transfer mechanisms with pyridoxal-dependent enzymes to unlock new biocatalytic transformations. Together, these methods and the goals have the potential to streamline the synthesis of biological probes and drug targets, creating a significant benefit to human health and associated biomedical sciences. Project Number: 1R35GM161429-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Todd Hyster | Institution: PRINCETON UNIVERSITY, Princeton, NJ | Award Amount: $165,366 | Activity Code: R35 | Study Section: Maximizing Investigators' Research Award - E Study Section[MRAE] View on NIH RePORTER: https://reporter.nih.gov/project-details/11259880