Reprogramming ω -Transaminase for Enantioselective Assembly of Axially Chiral Spiro[3.3]heptane Building Blocks

Abstract Axially chiral amines embedded in rigid spirocyclic frameworks are valuable motifs in medicinal chemistry, yet their synthesis by direct asymmetric amination of sterically congested ketones remains highly challenging. Here, we report an engineered ω-transaminase variant for the direct asymmetric amination of 2,6-disubstituted spiro[3.3]heptanones, providing access to enantioenriched axially chiral spiro amines under mild aqueous conditions. Starting from a weakly active wild-type ω-transaminase from Arthrobacter sp. KNK168 (ArTA), semi-rational engineering identified a double variant, ArTA-M2 (G136M/S223P), that substantially improved both catalytic activity and stereocontrol. This variant enables the asymmetric amination of a broad range of 2,6-disubstituted spiro[3.3]heptanone ketones with high conversions (up to 91%) and enantioselectivities (up to 99% ee). The preparative utility of ArTA-M2 was further demonstrated through the synthesis of pharmaceutically relevant building blocks and gram-scale whole-cell biotransformations. Molecular docking and molecular dynamics simulations suggest that the beneficial mutations reshape the binding pocket to promote a productive and stereodiscriminating binding mode. This work establishes a streamlined biocatalytic pathway to a challenging class of axially chiral building blocks.