Biocatalytic Synthesis of Ruxolitinib Intermediate via Engineered Imine Reductase

• Published in: Journal of organic chemistry Vol. 89; no. 16; pp. 11446 - 11454
• Main Authors: Huang, Aiping, Zhang, Xuewen, Yang, Yiming, Shi, Chengcheng, Zhang, Bifei, Tuo, Xinkun, Shen, Peili, Jiao, Xuecheng, Zhang, Na
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 16.08.2024
• ISSN: 0022-3263; 1520-6904; 1520-6904
• DOI: 10.1021/acs.joc.4c01119

An enzyme catalyzed strategy for the synthesis of a chiral hydrazine from 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 is presented. An imine reductase (IRED) from Streptosporangium roseum was identified to catalyze the reaction between 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 to produce trace amounts of (R)-3-cyclopentyl-3-hydrazineylpropanenitrile 4. We employed a 2-fold approach to optimize the catalytic performance of this enzyme. First, a transition state analogue (TSA) model was constructed to illuminate the enzyme–substrate interactions. Subsequently, the Enzyme_design and Funclib methods were utilized to predict mutants for experimental evaluation. Through three rounds of site-directed mutagenesis, site saturation mutagenesis, and combinatorial mutagenesis, we obtained mutant M6 with a yield of 98% and an enantiomeric excess (ee) of 99%. This study presents an effective method for constructing a hydrazine derivative via IRED-catalyzed reductive amination of ketone and hydrazine. Furthermore, it provides a general approach for constructing suitable enzymes, starting from nonreactive enzymes and gradually enhancing their catalytic activity through active site modifications.