Chiral Amine Synthesis Using Engineered Pyridoxal-Dependent Enzymes

Researchers from Princeton University are developing new enzyme catalysts that transform simple amines into complex, chiral molecules used in medicines and agrochemicals. Chiral amines are found in many societally valuable molecules, yet their synthesis often requires multiple chemical steps and expensive catalysts because existing methods separate carbon–carbon bond formation from the creation of molecular chirality. Pyridoxal phosphate (PLP)-dependent enzymes offer exceptional stereochemical control but are naturally limited to a narrow range of amino acid substrates and native reaction pathways. The project seeks to overcome these limitations by expanding the substrate scope and catalytic mechanisms of these enzymes. The research team will engineer them to accept broadly available benzylic, allylic, and propargylic amines while introducing new radical reaction pathways that enable carbon–carbon bond formation inaccessible to natural enzymes. Their discoveries could establish a fundamentally new platform for asymmetric synthesis, reveal how protein environments control radical and photochemical reactivity, and broaden the use of biocatalysis in chemical synthesis. The project will also train undergraduate, graduate, and postdoctoral researchers, expand biocatalysis-focused laboratory experiences, and engage K–12 students through hands-on STEM outreach activities that highlight the role of enzymes in sustainable chemistry. This project will develop pyridoxal phosphate (PLP)-dependent enzymes as versatile catalysts for the stereoselective synthesis of chiral amines by engineering for expanded amine scope and non-natural reaction mechanisms. Although PLP-dependent enzymes catalyze a broad range of transformations in nature, their synthetic applications remain limited by a narrow substrate scope and reliance on closed-shell catalytic pathways. This research will overcome these limitations by engineering threonine aldolases and related PLP-dependent enzymes to accept benzylic, allylic, and propargylic amines while introducing radical mechanisms for carbon–carbon bond formation. Directed evolution, structural biology, spectroscopy, and mechanistic studies will be integrated to define how protein active sites control substrate recognition, radical generation, and stereoselectivity. The project will establish strategies to expand enzyme substrate scope, exploit pyridoxal-redox chemistry to enable new catalytic functions. Beyond providing efficient routes to valuable amino alcohols, amino acids, and other nitrogen-containing building blocks, the work will generate fundamental insights into the design principles governing enzyme-controlled radical and excited-state reactivity. These advances are expected to establish broadly applicable approaches for engineering PLP-dependent enzymes and expand the synthetic capabilities of biocatalysis for asymmetric chemical synthesis. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.