Alcohol dehydrogenases (ADHs) have become important catalysts for stereoselective oxidation and reduction reactions of alcohols, aldehydes and ketones. The aim of this contribution is to provide the reader with a timely update on the state-of-the-art of ADH-catalysis. Mechanistic basics are presented together with practical information about the use of ADHs. Current concepts of ADH engineering and ADH reactions are critically discussed. Finally, this contribution highlights some prominent examples and future-pointing concepts.

/pdf/alcohol-dehydrogenases-as-catalysts-in-organic-synthesis-1wwcp4iz.pdf

Alcohol Dehydrogenases as Catalysts in Organic Synthesis

• para -position of diaryl ketones is critical in manipulating stereoselectivity of Kp ADH. • Two stereocomplementary mutants were obtained by stereodivergent evolution. • Side-chain collision and electrostatic interactions are vital for stereoselectivity. Kp ADH is a promising biocatalyst for the synthesis of chiral bulky-bulky alcohols. Substrate specificity toward diaryl ketone substrates with different substituents at phenyl group was investigated. The results reveal that the para -position of diaryl ketones plays important role in manipulating the stereoselectivity of Kp ADH. Two vital residues, E214 and T215, were identified through single-point mutagenesis, and saturation mutagenesis was performed to evaluate their contributions to the stereoselectivity. Furthermore, stereodivergent evolution of Kp ADH was achieved through combinatorial mutagenesis. Among them, E214I/T215S/S237A (ISA) and F161V/S196G/E214G (VGG) exhibited e.e. values of >99% ( S ) and 99% ( R ) toward [4-(trifluoromethyl)phenyl]-2-pyridinylmethanone ( 5a ), and e.e. values of >99% ( S ) and 98% ( R ) toward (4-methylphenyl)-2-pyridinylmethanone ( 6a ), respectively. Kinetic characterization and interaction analysis further prove that the eliminated side-chain collision and enhanced electrostatic interactions are mainly responsible for the increased catalytic efficiency and stereoselectivity. This study provides beneficial mutants with complementary stereoselectivity toward diaryl ketones with para -substituents and also provides guidance for the engineering of homologous alcohol dehydrogenases toward bulky-bulky ketones. 

Stereodivergent evolution of KpADH for the asymmetric reduction of diaryl ketones with para-substituents

cis-4-Propylcyclohexanol is an important intermediate for synthesizing trans-2-(4-propylcyclohexyl)-1,3-propanediol, which is widely used in the manufacture of liquid crystal displays. In this study, cis-4-propylcyclohexanol was prepared using a mutant alcohol dehydrogenase from Lactobacillus kefir (LK-TADH, A94T/F147L/L199H/A202L) coupled with glucose dehydrogenase. Using the optimal catalytic conditions, 125 g/L (250 g) of 4-propylcyclohexanone was completely transformed after 5 h, and 225.8 g of cis-4-propylcyclohexanol (cis/trans ratio of 99.5:0.5) was obtained through extraction and rotary evaporation at a yield of 90.32%. This study reports a potential method for the green production of cis-4-propylcyclohexanol as the key intermediate of trans-2-(4-propylcyclohexyl)-1,3-propanediol at an industrial level.

https://www.mdpi.com/2073-4344/12/4/406/pdf?version=1649297703

Efficient Synthesis of cis-4-Propylcyclohexanol Using a Mutant Alcohol Dehydrogenase Coupled with Glucose Dehydrogenase

A Novel Organic Solvent Tolerant NAD+-dependent Formate Dehydrogenase from Halophilic Yeast Candida diddensiae

Control of the activity and enantioselectivity in biocatalyzed procedures: immobilization, medium engineering, and protein engineering

(S)-4-Chlorophenylpyridylmethanol and (R)-4-chlorobenzhydrol are key pharmaceutical intermediates for the synthesis of bepotastine and cloperastine, respectively. However, the biocatalytic approach to prepare these bulky diaryl ketones remains challenging because of the low activity of naturally occurring alcohol dehydrogenases (ADH). In the present study, ADH seq5, which has an adequate binding pocket volume and accepts bulky diaryl ketones, was further engineered with a binding pocket of increased hydrophobicity. Based on molecular simulation and binding free energy analyses, a small mutation library was constructed, and mutant seq5-D150I with a threefold increase in kcat and a low Km was obtained successfully. The comparison of kinetic parameters, binding free energy, docking conformation, and critical catalytic distances calculated by molecular dynamic simulations revealed the source of increased activity. To develop a practical approach with seq5-D150I, reaction conditions including pH, temperature, buffer, and metal ions were optimised and applied to synthesise (S)-4-chlorophenylpyridylmethanol and (R)-4-chlorobenzhydrol with high enantiomeric excess. The space-time yields for (S)-4-chlorophenylpyridylmethanol and (R)-4-chlorobenzhydrol increased dramatically to as high as 263.4 g∙L-1 day-1 and 150 g∙L-1 day-1, respectively, which, to our knowledge, is the highest reported yield to date. These results show that the biocatalytic approach with seq5-D150I may be practical for future industrial applications.Key points An alcohol dehydrogenase was engineered based on binding free energy analysis. The mutant seq5-D150I obtained a threefold increase in kcat and a low Km. Two important pharmaceutical intermediates were obtained with high space-time yield.

Efficient synthesis of bepotastine and cloperastine intermediates using engineered alcohol dehydrogenase with a hydrophobic pocket.

Biocatalytic preparation of a key intermediate of antifungal drugs using an alcohol dehydrogenase with high organic tolerance

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Efficient synthesis of bepotastine and cloperastine intermediates using engineered alcohol dehydrogenase with a hydrophobic pocket.

Biocatalytic preparation of a key intermediate of antifungal drugs using an alcohol dehydrogenase with high organic tolerance