Enantiodivergent Access to Acyclic Quaternary Stereocenters by Nitrilase-Catalyzed Desymmetrizing Hydrolysis of Malononitriles.

The desymmetrization of fully substituted carbons bearing a pair of enantiotopic functional groups represents a pragmatic approach to the synthesis of quaternary stereocenters. Prochiral α,α-disubstituted malononitriles are of particular interest due to the simplicity of their preparation, the programmable introduction of functional substituents, and the high synthetic value of the resulting enantioenriched nitriles. Nevertheless, the asymmetric catalytic transformation of these malononitriles to all-carbon quaternary stereocenters represents a significant challenge. Here, we present a nitrilase (SsNIT)-catalyzed desymmetrizing hydrolysis of α,α-disubstituted malononitriles, resulting in the production of enantioenriched α-cyanocarboxylic acids containing acyclic all-carbon quaternary stereocenters in good yields (up to 99% yield) with excellent enantioselectivities (up to 99% ee). By fine-tuning and reshaping the substrate pockets of the enzyme through Focused Rational Iterative Site-directed Mutagenesis (FRISM), we successfully engineered a pair of enantiocomplementary nitrilases (M1 and M3), thereby providing a divergent synthetic route to assemble both R and S configurations of the quaternary stereocenters. The synthetic robustness and scalability of this strategy were further validated through gram-scale reactions using whole-cell biocatalysts, followed by derivatization into diverse enantioenriched noncanonical amino acid derivatives bearing quaternary stereocenters. This study highlights the potential of biocatalysis in facilitating the efficient synthesis of quaternary centers and their enantiomers through the rational engineering of enzymes.