Enzymatic Stereodivergent Access to Fluorinated β-Lactam Pharmacophores via Triple-Parameter Engineered Ketoreductases

Abstract

Enzyme-catalyzed stereodivergent synthesis to access all possible stereoisomers of organofluorine compounds bearing multiple stereogenic centers remains an important and challenging subject. By integrative data-driven mining and mechanism-guided engineering of ketoreductases, we identified a stereodivergent biocatalytic platform to produce four stereoisomeric fluoroalkyl amino acid esters bearing two vicinal stereocenters. Fast triple-parameter coevolution via a semirational CAST/ISM strategy provided the quadruple mutant M5 (A140K/L203T/G92A/V84I) of ketoreductase BgADH not only displayed high stereoselectivity toward the target stereoisomers (99:1 dr, 99% ee) but also observed with enhanced activity (kcat/Km, 6.3 folds) and improved thermostability (T5015, 4 °C). Crystal structural analysis and molecular dynamics (MD) simulation studies unveil two residues (A140 and F148) of BgADH to be the key sites that are responsible for the control of the stereoselectivity. The L203T/G92A mutation enhanced activity by affecting the conformational distribution of the α-helix within the active-site region, and V84I improved thermal stability by strengthening the hydrogen bonding network with neighboring residues. The synthetic utility was further demonstrated by fluoroalkyl substrate scope expansion, gram-scale reactions (648 g L–1 day–1), and synthetic transformations to chiral fluorinated β-lactams that are the antibiotic carbapenem cores.