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

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.