Stereoselective photoenzymatic o- and n-centered radical transformations mediated by the pyridoxal 5'-phosphate cofactor

The National Institute of General Medical Sciences, part of the National Institutes of Health within the Department of Health and Human Services, awarded $227,652 to The Trustees of Princeton University on June 26, 2026, under the Biomedical Research and Research Training program (CFDA 93.859) to develop stereoselective photoenzymatic methods for oxygen- and nitrogen-centered radical transformations mediated by pyridoxal 5'-phosphate cofactors. The project will leverage pyridoxal 5'-phosphate-dependent enzymes and triplet energy transfer catalysis as a platform for generating and controlling oxyl-imidyl and amidyl-imidyl biradical pairs to enable oxyamination and diamination of alkenes, with the goal of providing a stereocontrolled route to chiral oxygen- and nitrogen-containing heterocycles relevant to pharmaceutical development and synthesis of natural products. Performance occurs at Princeton, New Jersey, with a period of performance extending through March 14, 2029.

Abstract

Chiral oxygen and nitrogen containing heterocycles are prolific in pharmaceutical agents, natural products, and biologically relevant molecules. The cyclization of heteroatom-centered radicals with alkenes provides a direct and atom-efficient route towards the synthesis of chiral heterocycles. However, this seemingly simple transformation presents substantial challenges due to limited avenues of generating heteroatom-centered radicals and controlling their resultant stereochemistry. Triplet energy transfer (ENT) from an excited state photocatalyst is a growing conceptual strategy for the mild generation of o- and n-centered radicals by direct bond homolysis of oximes and hydrazones but still suffers from a lack of stereocontrol. Photoenzymatic catalysis is an emerging strategy for initiating and controlling radical intermediates with high stereoselectivity. The highly ordered active site of enzymes overcomes selectivity issues by maintaining a strong association between radical intermediates. Non-natural mechanisms of initiating radical generation have mainly taken advantage of the excited state redox properties of the flavin and nadp cofactors, outer sphere electron transfer from exogenous photocatalysts, and metallocofactor-to-ligand charge transfer. Herein, we propose leveraging pyridoxal 5'-phosphate (PLP) dependent enzymes and ENT catalysis as a platform for generating and controlling oxyl-imidyl and amidyl-imidyl biradical pairs for the intramolecular OXY- amination or diamination of alkenes. By taking advantage of a substantial body of known hydroxylamine and hydrazine based PLP inhibitors, we hypothesize the well characterized PLP oxime and hydrazone intermediates are primed for ENT-mediated homolysis for the generation of o- and n-centered radicals. Substrate pre- organization within the PLP bound active site will exert stereocontrol and the rapid rates of heteroatom radical- ENE cyclization will limit deleterious side reactions. By developing the stereoselective radical-ENE cyclization of o- and n-centered radicals, we would address one of chemistry's grand challenges and broadly benefit the synthetic community. Thus, our following specific aims are: (aim 1) identify PLP-dependent enzymes for oxyl radical cyclization and imidyl recombination for the synthesis of chiral tetrahydrofuryl amines. PLP dependent enzymes are an extremely versatile class of enzymes that facilitate a plethora of biochemical pathways, and we anticipate this will promote the promiscuous reactivity we seek. We will use the oxyl-ENE cyclization as a testbed for the development of this novel mechanism within enzymes for the synthesis of valuable alpha-stereogenic tetrahydrofurans. (aim 2) by expanding reactivity to o-acyl hydroxylamines, hydrazines, and hydrazides we seek to access the stereoselective synthesis of c-c and c-n unsaturated cyclic species which constitute the two most frequently used unsaturated ring systems in FDA approved drugs. Concurrent directed evolution and protein engineering will allow for the optimization of reaction yield, selectivity, and expansion of scope.