Research Projects

The proposal originates from a growing demand for sustainable, stereoselective, and environmentally benign synthetic methodologies to access chiral amines—key intermediates in the pharmaceutical, agrochemical, and fine chemical industries. Traditional synthetic routes to β-nitroamines, particularly via aza-Michael reaction and aza-Henry reaction, often require harsh conditions, heavy metal catalysts, and deliver suboptimal stereoselectivity.1-2 Biocatalysis offers an elegant alternative, with enzymes enabling highly chemo-, regio-, and stereoselective transformations under mild aqueous conditions.3 Despite the remarkable progress in the field of enzyme catalysis, no naturally occurring enzyme is known to catalyze the direct, enantioselective addition of ammonia to α-nitro olefins. This gap represents an opportunity to explore and engineer enzyme scaffolds for ‘new-to-nature’ reactivities. Aspartate ammonia lyase (AAL), a PLP-independent lyase known for its reversible addition of ammonia to fumarate to yield L-aspartate, has shown promiscuous activity toward structurally related electrophilic alkenes, especially α,β-unsaturated carboxylic acids.4 Computational design-based protein engineering of AAL from Bacillus sp. has highlighted the potential plasticity of the enzyme and broad substrate tolerance, suggesting that AALs can be engineered for novel C–N bond-forming activities.5 Recently, we have successfully engineered Ureibacillus thermophilus AAL (UtAAL) towards hydroamination of crotonic acid to (R)-3-amino butyric acid, which makes this enzyme a promising scaffold to engineer towards asymmetric β-nitroamine synthesis by altering its substrate selectivity while retaining the promiscuous regio (β-amination) and enantioselectivity (R-selectivity).6 Directed evolution on different ammonia lyases (e.g., methylaspartate ammonia lyase, phenylalanine ammonia lyase) has shown that relatively few mutations can expand substrate scope toward non-natural substrates.7-8 We hypothesize that, based on our understanding of UtAAL catalytic mechanism, the role of binding pocket residues interacting with the proposed substrate, the enzyme can be engineered to generate variants capable of catalyzing enantioselective ammonia addition to α-nitro olefins, thereby affording access to chiral β-nitroamines with high stereocontrol. To test this hypothesis, the project aims to: (i) rationally redesign and engineer UtAAL to accommodate structurally diverse α-nitro olefins; (ii) screening of the variant libraries to identify biocatalysts with the new activity and enantioselectivity; (iii) kinetic study of the successful evolved UtAAL variants, mechanistic investigation on the molecular basis of new activity, and evaluate the synthetic scope in the synthesis of a repertoire of (R)-β-nitroamines and (iv) preparative-scale synthesis of chiral β-nitroamines to validate synthetic applicability and demonstrate the practical value of the engineered enzyme. The novelty of the proposed work lies in repurposing an existing lyase enzyme toward a completely new-to-nature transformation, combining principles of enzyme promiscuity, directed evolution, and mechanistic enzymology. This proposal integrates modern enzyme engineering, synthetic biology, and sustainable organic synthesis, offering a biocatalytic route to access functionally and stereochemically rich, diverse nitrogen-containing molecules. It would expand the biocatalytic toolbox for asymmetric synthesis, provide a green route to valuable β-nitroamine building blocks, and open new avenues in enzyme catalysis for non-natural reactions. It could lead to cost-effective and scalable biocatalysts for industrial asymmetric amination processes, supporting the shift toward greener chemical manufacturing.