Influence of ionic liquids on enzymatic asymmetric carboligations

The asymmetric mixed carboligation of aldehydes catalyzed by thiamine diphosphate (ThDP)-dependent enzymes provides a sensitive system for monitoring changes in activity, chemo-, and enantioselectivity. While previous studies have shown that organic cosolvents influence these parameters, we now demonstrate that similar effects occur upon addition of water-miscible ionic liquids (ILs). In this study, six ThDP-dependent enzymes were analyzed in the presence of 14 ILs under comparable conditions to assess their influence on enzymatic carboligation reactions yielding 2-hydroxy ketones. ILs exerted a moderate to strong influence on activity and, more notably, altered enantioselectivity. (<i>R</i>)-selective reactions were generally stable upon IL addition, while (<i>S</i>)-selective reactions frequently showed reduced selectivity or even inversion to the (<i>R</i>)-enantiomer. The most significant change was observed for the <i>Ap</i>PDC_E469G variant of pyruvate decarboxylase from <i>Acetobacter pasteurianus</i>, where the enantiomeric excess shifted from 86 % (<i>S</i>) to 60 % (<i>R</i>) in the presence of 9 % (w/v) Ammoeng 102. Control experiments indicated that this shift was primarily due to the Ammoeng cation rather than the anion. To explore the molecular basis of this phenomenon, all-atom molecular dynamics (MD) simulations were performed on wild-type <i>Ap</i>PDC and the E469G variant in Ammoeng 101 and Ammoeng 102. The simulations revealed that hydrophobic and hydrophilic regions of the Ammoeng cations interact with the (<i>S</i>)-selective binding pocket, thereby favoring formation of the (<i>R</i>)-product. These results highlight the potential of solvent engineering for modulating enzyme selectivity and demonstrate that MD simulations can capture functionally relevant enzyme-solvent interactions at the atomic level.