Stereodivergent protein engineering of alcohol dehydrogenase for the asymmetric synthesis of 2-arylidenequinuclidin-3-ols

The enzymatic synthesis of chiral 2-arylidenequinuclidin-3-ols remains unexplored, despite their importance as pharmaceutical intermediates. In this study, an alcohol dehydrogenase from Lactobacillus brevis ( Lb ADH) was rationally engineered using the FRISM (focused rational iterative site-specific mutagenesis) strategy and applied to the stereodivergent reduction of a series of ( Z )-2-arylidenequinuclidin-3-ones. Two enantiocomplementary variants were identified, affording a range of quinuclidinol products (13 examples) with satisfactory conversions and enantioselectivities (up to 99%). Gram-scale reactions demonstrated the practical applicability of this biotransformation. Molecular dynamics simulations elucidated the structural basis for the enhanced and inverted stereoselectivity. Overall, this work significantly expanded the application of alcohol dehydrogenases in the precision synthesis of key pharmaceutical intermediates. This study addressed the previously unreported enzymatic synthesis of chiral 2-arylidenequinuclidin-3-ols by developing enantiocomplementary variants of Lactobacillus brevis alcohol dehydrogenase ( Lb ADH). These engineered catalysts enabled stereodivergent reductions to access valuable 3-quinuclidinol products with high enantioselectivity (up to > 99%). Molecular dynamics simulations elucidated the structural basis for selectivity, offering a sustainable and practical biocatalytic strategy.