Influence of Organic Solvents on Enzymatic Asymmetric Carboligations

The asymmetric mixed carboligation of aldehydes with thiamine diphosphate (ThDP)-dependent enzymes is an excellent example where activity as well as changes in chemo- and stereoselectivity can be followed sensitively. To elucidate the influence of organic additives in enzymatic carboligation reactions of mixed 2-hydroxy ketones, we present a comparative study of six ThDP-dependent enzymes in 13 water-miscible organic solvents under equivalent reaction conditions. The influence of the additives on the stereoselectivity is most pronounced and follows a general trend. If the enzyme stereoselectivity in aqueous buffer is already >99.9% ee, none of the solvents reduces this high selectivity. In contrast, both stereoselectivity and chemoselectivity are strongly influenced if the enzyme is rather unselective in aqueous buffer. For the S-selective enzyme with the largest active site, we were able to prove a general correlation of the solvent-excluded volume of the additives with the effect on selectivity changes: the smaller the organic solvent molecule, the higher the impact of this additive. Further, a correlation to log?P of the additives on selectivity was detected if two additives have almost the same solvent-excluded volume. The observed results are discussed in terms of structural, biochemical and energetic effects. This work demonstrates the potential of medium engineering as a powerful additional tool for varying enzyme selectivity and thus engineering the product range of biotransformations. It further demonstrates that the use of cosolvents should be carefully planned, as the solvents may compete with the substrate(s) for binding sites in the enzyme active site. Abbreviations: ApPDC: pyruvate decarboxylase from Acetobacter pasteurianus; DCM: dichloromethane; DIPE: diisopropyl ether; DMSO: dimethyl sulfoxide; ee: enantiomeric excess; EtOAc: ethyl acetate; EtOH: ethanol; GC: gas chromatography; HPLC: high-performance liquid chromatography; HPP: 2-hydroxy-1-phenylpropan-1-one; iProp: isopropyl alcohol; LlKdcA: branched-chain keto acid decarboxylase from Lactococcus lactis; MIBK: methyl isobutyl ketone; MTBE: methyl tert-butyl ether; MTHF: 2-methyltetrahydrofuran; PAC: 1-hydroxy-1-phenylpropan-2-one; PfBAL: benzaldehyde lyase from Pseudomonas fluorescens; PpBFD: benzoylformate decarboxylase from Pseudomonas putida; rpd: relative product distribution; TCM: trichloromethane; THF; tetrahydrofuran