Manipulating the stereoselectivity of a thermostable alcohol dehydrogenase by directed evolution for efficient asymmetric synthesis of arylpropanols

Chiral arylpropanols are valuable components in important pharmaceuticals and fragrances, which is the motivation for previous attempts to prepare these building blocks enantioselectively in asymmetric processes using either enzymes or transition metal catalysts. Thus far, enzymes used in kinetic resolution proved to be best, but several problems prevented ecologically and economically viable processes from being developed. In the present study, directed evolution was applied to the thermostable alcohol dehydrogenase TbSADH in the successful quest to obtain mutants that are effective in the dynamic reductive kinetic resolution (DYRKR) of racemic arylpropanals. Using rac-2-phenyl-1-propanal in a model reaction, (S)- and (R)-selective mutants were evolved which catalyzed DYRKR of this racemic substrate with formation of the respective (S)- and (R)-alcohols in essentially enantiomerically pure form. This was achieved on the basis of an unconventional form of iterative saturation mutagenesis (ISM) at randomization sites lining the binding pocket using a reduced amino acid alphabet. The best mutants were also effective in the DYRKR of several other structurally related racemic aldehydes.