摘要：Yong-ZeXie,# Dong-Sheng Yang,# Li-Gang Bai, Jin-Wei Yang, BoGu, Meng-Ting Liu, Qi-Yue Wang, Wang Wang, Wen-Bo Liu,* and Heng Song*

转自：康龙化成

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

Yong-Ze Xie,# Dong-Sheng Yang,# Li-Gang Bai, Jin-Wei Yang, Bo Gu, Meng-Ting Liu, Qi-Yue Wang, Wang Wang, Wen-Bo Liu,* and Heng Song*

College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P.R. China

—J. Am. Chem. Soc. 2025, DOI: 10.1021/jacs.5c09258

Recommended by Yuquan Liu_PT

KEY WORDS:Biocatalysis, enzyme catalysis, desymmetrizing hydrolysis (反应类型),Malononitriles (原料), α-cyanocarboxylic acids (产物)

ABSTRACT: 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.

Selected substrate scope

Prof. Heng Song et alhave established a robust enzymatic platform that achieves divergent desymmetrizing hydrolysis of disubstituted malononitriles, enabling the construction of both (R)-and (S)-enantiomers of the acyclic quaternary carbon stereocenters. By employing a structure-guided rational engineering strategy, they successfully achieved bidirectional stereocontrol of nitrilase SsNIT, thereby generating a complementary pair of enantioselective variants: (R)-enantioselective mutant SsNIT-(M1) and (S)-enantioselective mutant SsNIT(M3). The efficacy of the aforementioned approachrelied on a comprehensive strategy combining covalent docking analysis with systematic alanine scanning mutagenesis to identify key residues governing stereoselectivity within the substrate binding pocket, followed by precise refinement and reshaping of substrate pockets of the SsNITarchitecture through Focused Rational Iterative Site-directed Mutagenesis (FRISM). This integrated methodology provides a generalizable framework for engineering stereoselective enzymes.

来源：新浪财经