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 (k cat /K m , 6.3 folds) and improved thermostability (T 50 15 , 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, gramscale reactions (648 g L −1 day −1 ), and synthetic transformations to chiral fluorinated β-lactams that are the antibiotic carbapenem cores.

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“…Data-focused mining and triple parameter engineering of ketoreductases have generated stereocomplementary biocatalysts that allow access to the four stereoisomers of fluoroalkyl β-hydroxyl-β′-amino esters from racemic ketoesters (Scheme 1). 29 Directed evolution generated additional mutants that displayed enhanced activity and thermostability and was used for gram-scale transformations. Two regioselective and diastereoselective enzymatic C–H oxidations of l -isoleucine have been used for the synthesis of 4,5-dihydroxy- l -isoleucine, an important constituent of the RNA polymerase II inhibitor, α-amanitin.…”