Biocatalytic Synthesis of Ruxolitinib Intermediate via Engineered Imine Reductase

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.