Chirality Flipping: Engineering <i>Lf</i> SDR1 via Opposing Residue Substitutions to Expand the Substrate Scope for Chiral Alcohol Synthesis from Halogen-Substituted Acetophenone Derivatives

Enzymatic enantioselective reduction is an excellent technique in pharmaceutical manufacturing, enabling elaborate chirality tuning. KREDs have been implemented into practice for producing many chiral drug intermediates, while multihalogen-substituted acetophenones persist as a monolith in the path toward on-demand inversion of the chirality. (<i>S</i>)-2-Chloro-1-(2,4-difluorophenyl) ethanols constitute the key chiral fragment of voriconazole, of which the <i>S</i>-enantiomer is more rarely achieved than its counterpart through industrial reductases, i.e., <i>Lf</i>SDR1s. In this regard, a selection strategy based on residues with opposing physicochemical properties was developed to construct a minimal but highly effective library of <i>Lf</i>SDR1, where five amino acids (namely, A, W, T, S, and L) were classified and selected by hydrophobicity and steric hindrance. <i>Lf</i>SDR1-E141T-G92S-V186W (denoted as TSW, <i>S</i>-preferred) and <i>Lf</i>SDR1-E141L-G92W-V186A (denoted as LWA, <i>R</i>-preferred) were screened out from a 17-membered library, achieving <i>S</i>-specific reduction with 168 mM <b>1a</b> in 20 min. Furthermore, various halogenated 2-phenylethanol derivatives that could constitute diverse drug intermediates were stereoselectively prepared with optimized <i>Lf</i>SDR1 variants. This success highlights the advantages of devising a labor-saving yet effective approach toward asymmetric reduction of target substrates, balancing activities and enantioselectivities. Our findings offer a powerful choice for shortening the timelines of enzyme engineering works.