An enantioselective asymmetric reduction of phenyl ring-containing prochiral ketones to yield the corresponding optically active secondary alcohols was achieved with W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (W110A TESADH) in Tris buffer using 2-propanol (30%, v/v) as cosolvent and cosubstrate. This concentration of 2-propanol was crucial not only to enhance the solubility of hydrophobic phenyl ring-containing substrates in the aqueous reaction medium, but also to shift the equilibrium in the reduction direction. The resulting alcohols have S-configuration, in agreement with Prelog's rule, in which the nicotinamide-adenine dinucleotide phosphate (NADPH) cofactor transfers its pro-R hydride to the re face of the ketone. A series of phenyl ring-containing ketones, such as 4-phenyl-2-butanone (1a) and 1-phenyl-1,3-butadione (2a), were reduced with good to excellent yields and high enantioselectivities. On the other hand, 1-phenyl-2-propanone (7a) was reduced with lower ee than 2-butanone derivatives. (R)-Alcohols, the anti-Prelog products, were obtained by enantiospecific oxidation of (S)-alcohols through oxidative kinetic resolution of the rac-alcohols using W110A TESADH in Tris buffer/acetone (90:10, v/v).

The secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus 39E (TeSADH) is highly thermostable and solvent-stable, and it is active on a broad range of substrates. These properties make TeSADH an excellent template to engineer an industrial catalyst for chiral chemical synthesis. (S)-1-Phenyl-2-propanol was our target product because it is a precursor to major pharmaceuticals containing secondary alcohol groups. TeSADH has no detectable activity on this alcohol, but it is highly active on 2-butanol. The structural model we used to plan our mutagenesis strategy was based on the substrate's orientation in a horse liver alcohol dehydrogenase*p-bromobenzyl alcohol*NAD(+) ternary complex (PDB entry 1HLD). The W110A TeSADH mutant now uses (S)-1-phenyl-2-propanol, (S)-4-phenyl-2-butanol and the corresponding ketones as substrates. W110A TeSADH's kinetic parameters on these substrates are in the same range as those of TeSADH on 2-butanol, making W110A TeSADH an excellent catalyst. In particular, W110A TeSADH is twice as efficient on benzylacetone as TeSADH is on 2-butanol, and it produces (S)-4-phenyl-2-butanol from benzylacetone with an enantiomeric excess above 99%. W110A TeSADH is optimally active at 87.5 degrees C and remains highly thermostable. W110A TeSADH is active on aryl derivatives of phenylacetone and benzylacetone, making this enzyme a potentially useful catalyst for the chiral synthesis of aryl derivatives of alcohols. As a control in our engineering approach, we used the TbSADH*(S)-2-butanol binary complex (PDB entry 1BXZ) as the template to model a mutation that would make TeSADH active on (S)-1-phenyl-2-propanol. Mutant Y267G TeSADH did not have the substrate specificity predicted in this modeling study. Our results suggest that (S)-2-butanol's orientation in the TbSADH*(S)-2-butanol binary complex does not reflect its orientation in the ternary enzyme-substrate-cofactor complex.

To gel well: The asymmetric reduction of hydrophobic ketones by xerogel‐immobilized W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (TeSADH) in organic solvents affords their S‐configured alcohols in yields comparable with those achieved by using the free enzyme, and, in some cases, with higher enantioselectivities. R=phenyl‐ring‐containing substituent.

The asymmetric reduction of hydrophobic phenyl-ring-containing ketones and the enantiospecific kinetic resolution of the corresponding racemic alcohols catalyzed by Thermoanaerobacter ethanolicus W110A secondary alcohol dehydrogenase were performed in mono- and biphasic systems containing either organic solvents or ionic liquids. Both yield and enantioselectivity for these transformations can be controlled by changing the reaction medium. The enzyme showed high tolerance to both water-miscible and -immiscible solvents, which allows biotransformations to be conducted at high substrate concentrations.