Rational engineering of an imine reductase unlocks stereochemical gatekeeping for efficient synthesis of Evocalcet chiral intermediate
Chiral amines serve as vital building blocks in pharmaceutical synthesis, yet their stereoselective production remains a significant challenge. Biocatalytic approaches harness the inherent stereospecificity of enzymes to enable precise, sustainable, and highly selective construction of stereocenters. Here, we engineer an imine reductase (IRED) to catalyze the asymmetric synthesis of (3 S )-3-{[(1 R )-1-(naphthalen-1-yl)ethyl]amino}-1-[(2-methylpropan-2-yl)oxycarbonyl]pyrrolidine, the key chiral intermediate of Evocalcet . Computational enzyme design of PcIRED-M3 identified the W219L variant, which enhanced catalytic efficiency 8.9-fold and achieved >99 % diastereomeric excess ( d.e. ) versus 63.15 % in wild-type. The W219L substitution presumably reduces steric hindrance by extending the residue-substrate naphthyl distance from 2.1 Å to 4.7 Å while concurrently improving hydride transfer efficiency. This improvement is also owing to the optimized π-σ orbital alignment with the decreased spacing (from 6.2 Å to 4.9 Å) between the imine carbon and the C-4 position of NADPH’s nicotinamide ring. Reaction parameters optimization (30 % DMSO, K 2 HPO 4 -KH 2 PO 4 buffer pH 7.5, 35 °C) achieved 62.7 % conversion in 48 h. This work establishes the first enzymatic route to Evocalcet intermediates and identifies W219 as a stereochemical gatekeeper residue that allosterically coordinates substrate-cofactor geometry.
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