Ketoreductase Engineering for a Chemoenzymatic Fluorination and Dynamic Kinetic Reduction Cascade

We report the engineering of a highly active and solvent-stable ketoreductase (KRED) for a sequential chemoenzymatic fluorination and dynamic kinetic reduction (DKR) cascade en route to the active pharmaceutical ingredient belzutifan. The protein engineering strategy used a tiered approach to identify beneficial mutational diversity across protein structural regions, simultaneously targeting multiple specific attributes. By recombining these beneficial mutations over 14 rounds of evolution, the KRED was significantly improved in activity, stereoselectivity, and stability, particularly in the presence of up to 50 vol % mixed organic solvents. Structural analysis revealed that the monomeric parent enzyme became homodimeric via introduction of a hydrophobic region, providing a probable mechanistic rationale for the observed improvement in solvent stability. Defining performance targets at the onset of the engineering campaign guided the multidimensional screening approach and resulted in an efficient evolution to the desired KRED, replacing a through-process employing a rare metal catalyst. Our chemoenzymatic fluorination and DKR cascade process will facilitate broader access to belzutifan for a larger patient population.