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Alcohol dehydrogenases (ADHs) catalyze the reversible reduction of a carbonyl group to its corresponding alcohol. ADHs are widely employed for organic synthesis due to their lack of harm to the environment, broad substrate acceptance, and high enantioselectivity. This review focuses on the impact and relevance of ADH enantioselectivities on their biotechnological application. Stereoselective ADHs are beneficial to reduce challenging ketones such as ketones owning two bulky substituents or similar-sized substituents to the carbonyl carbon. Meanwhile, in cascade reactions, non-stereoselective ADHs can be utilized for the quantitative oxidation of racemic alcohol to ketone and dynamic kinetic resolution.

Biocatalysis is always considered as an efficient alternative for preparing a wide range of compounds with pharmaceutical/industrial importance. The immense popularity of biocatalysts is due to its striking features of being an environmentally benign, renewable and sustainable.In this present review, different methods of preparation of alcohols from their respective carbonyl compounds using different plant cells as biocatalysts are discussed. In addition, the importance of these alcohols in pharmaceutical industries and their methods of preparation in optically as well as chemically pure form are also discussed.

Biocatalytic approaches have gained increasing attention as sustainable alternatives to metal-catalyzed asymmetric reductions of ketones to obtain enantiopure alcohols, important intermediates for pharmaceutical synthesis. For example, enzyme-catalyzed reduction of substituted benzophenone analogs to produce chiral diaryl methanols has attracted interest, as they are the key intermediates in the synthesis of antihistamines. However, benzophenone analogs are difficult to be reduced by enzymes due to steric hindrance. Moreover, the similarities between the two groups adjacent to the carbonyl group make achieving high enantioselectivity in reduction challenging. In this study, we examined the reduction of benzophenone and its analogs by Geotrichum candidum acetophenone reductase ( Gc APRD). However, the wild type did not exhibit activity toward benzophenone due to the substrate’s bulkiness. Then, two mutants of Gc APRD (Trp288Ala and Phe56Ile/Trp288Ala) were applied to catalyze the reduction of benzophenone, resulting in high reduction yield (≥ 80%). In addition, both mutants exhibited catalytic activity toward methyl- and halogen-substituted benzophenones, especially toward 3- and 4-substituted substrates. Regarding enantioselectivity, Trp288Ala generally reduced both 3- and 4-substituted substrates to ( R )-alcohols with up to 97% ee . In contrast, Phe56Ile/Trp288Ala reduced 3-substituted substrates to ( R )-alcohols with up to 89% ee but reduced 4-substituted substrates to ( S )-alcohols with up to 92% ee . At last, the reduction mechanism was investigated using molecular docking simulations. Key points • GcAPRD mutants exhibited catalytic performance toward benzophenone analogs. • GcAPRD Phe56Ile/Trp288Ala exhibited substituent-dependent enantioselectivity. • Introducing Phe56Ile into GcAPRD Trp288Ala resulted in a clear enantiopreference. Graphical Abstract

Old yellow enzymes (OYEs) have been proven as powerful biocatalysts for the asymmetric reduction of activated alkenes. Fungi appear to be valuable sources of OYEs, but most of the fungal OYEs are unexplored. To expand the OYEs toolbox, a new thermophilic-like OYE ( Af OYE1) was identified from Aspergillus flavus strain NRRL3357. The thermal stability analysis showed that the T 1/2 of Af OYE1 was 60 °C, and it had the optimal temperature at 45 °C. Moreover, Af OYE1 exhibited high reduction activity in a wide pH range (pH 5.5–8.0). Af OYE1 could accept cyclic enones, acrylamide, nitroalkenes, and α, β-unsaturated aldehydes as substrates and had excellent enantioselectivity toward prochiral alkenes (> 99% ee). Interestingly, an unexpected ( S )-stereoselectivity bioreduction toward 2-methylcyclohexenone was observed. The further crystal structure of Af OYE1 revealed that the “cap” region from Ala132 to Thr182, the loop of Ser316 to Gly325, α short helix of Arg371 to Gln375, and the C-terminal “finger” structure endow the catalytic cavity of Af OYE1 quite deep and narrow, and flavin mononucleotide (FMN) heavily buried at the bottom of the active site tunnel. Furthermore, the catalytic mechanism of Af OYE1 was also investigated, and the results confirmed that the residues His211, His214, and Tyr216 compose its catalytic triad. This newly identified thermophilic-like OYE would thus be valuable for asymmetric alkene hydrogenation in industrial processes. Key points A new thermophilic-like OYE AfOYE1 was identified from Aspergillus flavus, and the T 1/2 of AfOYE1 was 60 °C AfOYE1 catalyzed the reduction of 2-methylcyclohexenone with (S)-stereoselectivity The crystal structure of AfOYE1 was revealedv

A convenient protocol for the synthesis of 25,26,27-tribenzoyl-28-[((S)-1-diphenylphos- phanyl-propan-2-yl)oxy]-calix[4]arene via stereospecific methylation on Evans’ oxazolidinone moiety was reported. According to the 13C NMR analysis of this phosphine, the calix[4]arene skeleton adopted a 1,3-alternate conformation. The latter conformation of the macrocycle and the (S)-chirality of the carbon atom bearing the methyl substituent were confirmed by a single-crystal X-ray diffraction study. After coordination of the phosphinated ligand to the dimeric [RuCl2(p-cymene)]2 organometallic precursor, the resulting arene–ruthenium complex was tested in the asymmetric reduction of acetophenone and alcohol was obtained with modest enantiomeric excess.

Aliphatic chiral secondary alcohols are important chiral intermediates widely used in the synthesis of drugs and fine chemicals. The whole cells of recombinant E. coli co-expressing alcohol dehydrogenase and glucose dehydrogenase were used to catalyze the asymmetric reduction of three fatty ketones without the addition of exogenous cofactors. The reaction medium and reaction conditions were systematically studied. As expected, the kinds of co-solvents, the molar ratio of glucose/substrate, temperature, pH and cell amount have important influence on the reaction. Under the optimal condition (glucose at 1.5 times substrate concentration, isopropanol at 2%, PBS (0.1 M, pH 8.0), and 45℃), the cells co-expressing both enzymes efficiently catalyzed the asymmetric reduction of three fatty ketones to the corresponding ( S )-fatty alcohols. The conversion and ee value of the products were both greater than 99% after 8 h reaction at 1 M of substrate concentration, and the space-time yield was between 346 ~ 470 g/(L·d). All the products can be obtained on gram scale. The results indicated that this method has potential application value in the efficient and green synthesis of aliphatic chiral secondary alcohols. Graphical Abstract

The importance of yeast old yellow enzymes is increasingly recognized for direct asymmetric reduction of (E/Z)-citral to (R)-citronellal. As one of the most performing old yellow enzymes, the enzyme OYE3 from Saccharomyces cerevisiae S288C exhibited complementary enantioselectivity for the reduction of (E)-citral and (Z)-citral, resulting in lower e.e. value of (R)-citronellal in the reduction of (E/Z)-citral. To develop a novel approach for the direct synthesis of enantio-pure (R)-citronellal from the reduction of (E/Z)-citral, the enzyme OYE3 was firstly modified by semi-rational design to improve its (R)-enantioselectivity. The OYE3 variants W116A and S296F showed strict (R)-enantioselectivity in the reduction of (E)-citral, and significantly reversed the (S)-enantioselectivity in the reduction of (Z)-citral. Next, the double substitution of OYE3 led to the unique variant S296F/W116G, which exhibited strict (R)-enantioselectivity in the reduction of (E)-citral and (E/Z)-citral, but was not active on (Z)-citral. Relying on its capability discriminating (E)-citral and (Z)-citral, a new cascade reaction catalyzed by the OYE3 variant S296F/W116G and glucose dehydrogenase was developed, providing the enantio-pure (R)-citronellal and the retained (Z)-citral after complete reduction of (E)-citral.

Chiral diaryl alcohols, such as (4-chlorophenyl)(pyridin-2-yl)methanol, are important intermediates for pharmaceutical synthesis. However, using alcohol dehydrogenases (ADHs) in the asymmetric reduction of diaryl ketones to produce the corresponding alcohols is challenging due to steric hindrance in the substrate binding pockets of the enzymes. In this study, the steric hindrance of the ADH from Geotrichum candidum NBRC 4597 ( G. candidum acetophenone reductase, Gc APRD) was eliminated by simultaneous site-directed mutagenesis of Phe56 (in the large pocket) and Trp288 (in the small pocket). As a result, two double mutants, Phe56Ile/Trp288Ala, and Phe56Ala/Trp288Ala, exhibited much higher specific activities towards 2-(4′-chlorobenzoyl)pyridine (4.5 μmol/min/mg and 3.4 μmol/min/mg, respectively) than the wild type (< 0.2 μmol/min/mg). In whole-cell-catalyzed asymmetric reductions of diaryl ketones, Phe56Ile/Trp288Ala significantly increased the isolated yields, which were over 90% for the reactions of most of the tested substrates. Regarding enantioselectivity, Phe56Ile/Trp288Ala and Phe56Ala/Trp288Ala, and Trp288Ala generally exhibited similar selectivity to produce ( R )-alcohols with up to 97% ee . Key points • Phe56 in Geotrichum reductase (GcAPRD) was mutated to eliminate steric hindrance. • Mutation at Phe56 increased enzymatic activity and expanded substrate specificity. • Phe56Ile/Trp288Ala showed high activity and (R)-selectivity towards diaryl ketones. Graphical Abstract

A series of squaric acid amides (synthesized in 66–99% isolated yields) and a set of chiral aminoalcohols were comparatively studied as ligands in a model reaction of reduction of α-chloroacetophenone with BH3•SMe2. In all cases, the aminoalcohols demonstrated better efficiency (up to 94% ee), while only poor asymmetric induction was achieved with the corresponding squaramides. A mechanistic insight on the in situ formation and stability at room temperature of intermediates generated from ligands and borane as possible precursors of the oxazaborolidine-based catalytic system has been obtained by 1H DOSY and multinuclear 1D and 2D (1H, 10/11B, 13C, 15N) NMR spectroscopy of equimolar mixtures of borane and selected ligands. These results contribute to better understanding the complexity of the processes occurring in the reaction mixture prior to the possible oxazaborolidine formation, which play a crucial role on the degree of enantioselectivity achieved in the borane reduction of α-chloroacetophenone.

The intracellular NADPH insufficient supply is the main bottleneck to the synthesis of chiral alcohols by asymmetric reduction with whole-cell catalysis. Herein, we provide a novel strategy to strengthen intracellular NADPH supply through introducing an NADP + -dependent glyceraldehyde 3-phosphate dehydrogenase ( gap B from Bacillus subtilis 168) into the Embden-Meyerhof pathway and a NAD kinase ( yfj B from E. coli MG1655) to further enhance the NADP(H) pool. A recombinant E. coli ( E. coli BL21 (DE3)/pETDuet-1- gap B- yue D&pET28a- yfj B) was constructed to co-express gap B and yfj B with a carbonyl reductase gene yue D together. The result showed that the intracellular NADPH amount increased by 134.4% with the strategy. To the model reaction (asymmetric reduction of acetophenone to S -phenyl ethanol), the yield was 3.7-fold with this strategy compared to the control. This provides a technological route for strengthening the intracellular NADPH supply in E. coli for biocatalysis and biosynthesis. Graphic Abstract