Harnessing Alcohol Dehydrogenases in Organic Reaction Cascades: A Strategy for Enhanced Efficiency in Synthetic Organic Chemistry

Abstract

Alcohol dehydrogenases (ADHs) are versatile enzymes that enable the reversible reduction of aldehydes and ketones to their corresponding alcohols. The exceptional chemo-, regio-, and stereoselectivity of ADHs position them as attractive catalysts for generating enantiopure alcohols, whether through deracemization of racemates or asymmetric reduction of prochiral ketones. The emergence of robust ADHs capable of functioning effectively at elevated temperatures and in high concentrations of non-aqueous media has stimulated interest in integrating ADH-catalyzed asymmetric transformations with other chemical processes in a single pot, either in a stepwise mode or simultaneously. This review presents an overview of one-pot organic transformations that combine ADH-catalyzed asymmetric reductions with additional nonenzymatic chemical reactions, demonstrating the potential for enhanced efficiency and sustainability in synthetic organic chemistry.

1. Introduction

One-pot stepwise chemical synthesis, wherein multiple reactions occur sequentially within the same vessel without the need to isolate intermediates, represents a promising strategy in chemical synthesis [1,2,3]. This approach aligns with key principles of green chemistry, particularly in minimizing waste and reducing environmental impacts of chemical processes [4,5]. However, the compatibility of the reactions involved poses significant challenges in the design of effective one-pot processes.

Biocatalysis, which utilizes enzymes to facilitate chemical reactions, offers an environmentally friendly alternative for traditional chemical synthesis [6,7]. Alcohol dehydrogenases (ADHs) are pivotal enzymes that catalyze the reversible interconversion of alcohols and their corresponding aldehydes or ketones [8,9,10,11,12]. ADHs are nicotinamide–adenine–dinucleotide (phosphate) [NAD(P)⁺]-dependent enzymes that require either an enzyme-coupled or substrate-coupled approach to regenerate the cofactor [13], thereby rendering the process catalytic. The stereopreference of ADH-catalyzed asymmetric reductions of prochiral ketones is controlled by the orientation of the prochiral ketone substrate within the active site of the enzyme. This orientation allows NAD(P)H to deliver its hydride from either the re or si face of the ketone, resulting in the formation of one enantiomer of the corresponding alcohols (Figure 1). Most ADHs conform to Prelog’s rule [14], delivering the hydride from the re face of prochiral ketones. This leads to the production of (S)-alcohols, assuming that the larger alkyl group of the ketone exhibits a higher Kahn–Ingold–Prelog priority than that of the smaller alkyl group.

Alcohol dehydrogenases (ADHs) have been established as robust catalysts in organic synthesis [15] due to their remarkable tolerance to non-aqueous media [16] and elevated temperatures. This resilience enables their incorporation into various organic reactions under extreme conditions of pH, temperature, and solvent. Moreover, recent advancements in biotechnology have enhanced the applicability of ADHs in organic transformations, particularly by broadening their substrate scope [17] and tuning their stereopreference and stereoselectivity [18].

The ability to perform multiple steps in a single pot, whether sequentially or concurrently, without isolating intermediates is crucial for streamlining organic synthesis, as it eliminates the need for extensive workup procedures and reduces solvent use. Recent reviews have explored one-pot, two-step reactions with a particular emphasis on enzymatic cascades [19]. Additionally, another review discussed the integration of chemo- and biocatalysis within one-pot systems [20]. In contrast, the present review specifically focuses on the synergy between ADH-catalyzed reactions and other chemically catalyzed processes within a one-pot framework. It is worth noting that the current review will not focus on deracemization strategies for obtaining enantiopure alcohols, as this topic has been covered in other review articles [21,22].

2. One-Pot, Two-Step Reactions Utilizing Alcohol Dehydrogenases

2.1. Two-Step Sequential Reactions in a One-Pot Setup

This section highlights examples of two-step transformations conducted sequentially in a single pot, eliminating the need to isolate the product from the first reaction. These one-pot, two-step sequential transformations are typically enabled by the compatibility of both reactions with the reaction medium. Gröger and coworkers developed a one-pot, two-step method that combines the Wittig reaction with enzymatic reduction to produce optically active allylic alcohols [23]. This approach commenced with the formation of α, β-unsaturated ketones via the Wittig reaction in aqueous media containing 2-propanol, followed by ADH-catalyzed reduction of the ketone in the same vessel. The same reaction medium was utilized throughout both steps. ADHs from Lactobacillus kefir and Rhodococcus sp. were used to independently synthesize (R)- and (S)-alcohols, respectively. These reactions achieved moderate conversion rates and very high enantioselectivities (Scheme 1). Although 2-propanol did not participate in the Wittig reaction, it performed as a cosolvent to enhance the solubility of the selected hydrophobic aryl-ring-containing ketones in the reaction mixture, and it was subsequently used in the regeneration of the cofactor in the ADH-catalyzed step [23]. This one-pot, two-step reaction was facilitated by the ability to perform Wittig reaction in aqueous media [24].

Cacchi and coworkers reported two-step sequential reactions involving Heck coupling followed by ADH-catalyzed asymmetric reduction to synthesize optically active allylic alcohols [25]. In this process, aryl iodide derivatives were coupled with butenone using palladium catalyst [Pd(OAc)₂] at 80 °C, resulting in the formation of α,β-unsaturated ketones. This reaction was followed by an ADH-catalyzed symmetric reduction in the same pot, eliminating the need for isolation of the intermediate ketone. The enantiopure form of each enantiomer was obtained by using enantiocomplementary ADHs, from Lactobacillus brevis (LbADH) and from Thermoanaerobacter sp. (TADH). A variety of aryl iodides produced enantiomerically pure allylic alcohols in excellent yields and high enantioselectivity (Scheme 2).

Inspired by the capability of palladium-based catalysts to facilitate Suzuki cross-coupling reactions in aqueous media [26,27], Gröger and coworkers pioneered the first one-pot palladium-catalyzed Suzuki cross-coupling followed by asymmetric enzymatic reduction in an aqueous medium. In the cross-coupling step, they used Pd(PPh₃)₂Cl₂ as a catalyst without the addition of phosphane additives, conducting the reaction at 70 °C in aqueous media. This Suzuki cross-coupling reaction produced the corresponding biaryl ketone in high conversion (Scheme 3). Subsequently, the biaryl ketone was reduced using ADH from Rhodococcus sp., leading to the formation of the desired (S)-configured biaryl-containing alcohols in good conversions and high enantioselectivity without the need to isolate the ketone intermediate. This reaction was carried out by in situ recycling of the cofactor using 2-propanol as a cosubstrate [28]. The same strategy was used to synthesize (S,S)-4,4′-bis(1-hydroxyethyl)biphenyl, a C2-symmetric biphenyl diol, in excellent enantio- and diastereoselectivities starting from a halogenated ketone and a phenylboronic substituted ketone (Scheme 4) [29].

Following this, the same research group coupled 4′-iodoacetophenone with phenylboronic acid in the presence of a water-soluble palladium catalyst. This catalyst was prepared by mixing palladium chloride and tris(3-sulfonatophenyl)phosphine hydrate, sodium salt (TPPTS), at room temperature in aqueous media containing 2-propanol [30]. Following a pH adjustment to 7.0, the resulting biaryl ketone was enzymatically reduced to yield the corresponding optically active biaryl alcohol, achieving high conversions and excellent enantioselectivities. Both enantiomers of the biaryl alcohol were independently synthesized using the enantiocomplementary ADHs from Lactobacillus kefir (LkADH) and from Rhodococcus sp. (Scheme 5).

The integration of palladium-based cross-coupling and ADH-catalyzed reduction in one pot was also exemplified by Gotor-Fernández and coworkers in their chemoenzymatic synthesis (R)-2,2,2-trifluoro-1-[4′-(methylsulfonyl)-(1,1′-biphenyl)-4-yl]ethanol, a precursor for Odanacatib (a Cathepsin K inhibitor) [31]. More specifically, they reported a sequential chemoenzymatic palladium-catalyzed Suzuki–Miyaura cross-coupling reaction between 1-(4′-bromophenyl)-2,2,2-trifluoroethanone and 4-(methylsulfonyl)phenylboronic acid, followed by asymmetric reduction of the resulting 2,2,2-trifluoro-1-[4′-(methylsulfonyl)-(1,1′-biphenyl)-4-yl]ethenone using the ADH from Ralstonia sp. (RasADH) without isolation of this ketone intermediate. This one-pot, two-step reaction yielded (R)-2,2,2-trifluoro-1-[4′-(methylsulfonyl)-(1,1′-biphenyl)-4-yl]ethanol in a good isolated yield and high enantioselectivity (Scheme 6).

Lui, Ren, and their coworkers developed a one-pot, two-step synthetic strategy for diarylmethanols that combines a Suzuki cross-coupling reaction of acyl halides with boronic acids using resin-immobilized palladium acetate, followed by the asymmetric reduction of the resulting diarylketones using ADH from Kluyveromyces polyspora (KpADH), also immobilized on amino resin [32]. After the first step, solvent evaporation allowed for the direct introduction of the immobilized KpADH to facilitate the asymmetric reduction of the diarylketones in phosphate buffer solution. This approach yielded diarylmethanols with good to excellent yields and enantioselectivities, highlighting the effectiveness of employing immobilized catalysts in one-pot, multi-step transformations (Scheme 7).

Gröger and coworkers reported a chemoenzymatic catalytic method that enables highly enantioselective hydration of styrene analogs. More specifically, this approach combines palladium-catalyzed Wacker-Tsuji oxidation with LkADH-catalyzed asymmetric reduction of the resulting ketone in one pot and without isolation of the acetophenone intermediate [33]. The authors noted that the activity of LkADH was significantly inhibited by the palladium species generated during the Wacker–Tsuji oxidation, leading to extremely low yields of the desired chiral alcohol. To mitigate the inhibitory effect of Pd(II) species, various Pd(II) complexing agents, such as thiourea, EDTA, or 2,2-bipyridine, were introduced following the oxidation stage. This optimized procedure resulted in good yields of the (R)-1-phenylethanols from their styrene analogs (Scheme 8).

Mihovilovic and coworkers developed a one-pot, two-step reaction sequence for synthesizing optically active 1-phenylethanol analogs from terminal alkynes [34]. The process was initiated with gold(III)-catalyzed hydration of terminal alkynes, which is followed by an ADH-catalyzed reduction conducted in Tris-HCl buffer solution adjusted to pH 8. Interestingly, 2-propanol played a dual role as both a cosubstrate and a cofactor regeneration agent in the ADH-catalyzed reduction during the second step, while also being utilized as the solvent in the first step. The enantiocomplementary ADH from Rhodococcus ruber (ADH-A) and LkADH were successfully employed to produce (S)- and (R)-alcohols, respectively, demonstrating the effectiveness of this method (Scheme 9).

Lipshutz and coworkers developed a one-pot sequential transformation that combines the Pd-catalyzed Sonogashira cross-coupling reaction with an ADH-catalyzed enantioselective reduction of the ketone intermediates produced from the first step (Scheme 10A) [35]. Moreover, they reported enhanced catalytic efficiency of this method by encapsulating ADH101 within a micelle, which serves as a reservoir for both the product and the catalyst. This sequential reaction yielded (R)-configured alcohols with high enantiopurity (>99% ee). Notably, the overall yields from this two-step process were comparable to those of the first step alone, highlighting the remarkable efficiency of the ADH-catalyzed reaction. Additionally, this methodology was expanded to combine the Heck reaction with an ADH-catalyzed asymmetric reduction (Scheme 10B). Excellent performance was achieved with the (R)- and (S)-selective ADHs, ADH101 and ADH112, respectively. Remarkably, these ADHs demonstrated impressive tolerance to high concentrations of Pd catalysts, up to 20,000 ppm (2 mol%).

Gotor-Fernández, Lavandera, and their coworkers reported a stereoselective method for synthesizing β-hydroxy sulfones via a one-pot chemoenzymatic process that combines oxosulfonylation with bioreduction [36]. The method involves treating terminal acetylene derivatives with sodium sulfinates under aerobic conditions using FeCl₃·6H₂O, leading to the formation of 1-phenyl-2-(phenylsulfonyl)ethan-1-one. After cooling the reaction mixture, they employed an ADH to perform the asymmetric reduction of the β-keto sulfone intermediate. This approach successfully produced optically active β-hydroxy sulfones with varying stereopreference, achieving high conversion rates with high enantioselectivities. Specifically, ADH from Ralstonia species (RasADH) and KRED-P1-B02 generated (R)-β-hydroxy sulfones, and KRED-119 produced (S)-β-hydroxy sulfones. However, a concurrent cascade approach was not feasible due to the high temperature required for the oxosulfonylation reaction, which would inhibit the enzyme used in the subsequent reduction step (Scheme 11).

The ability to conduct organocatalytic aldol reaction under neat conditions [37] allowed Gröger and his coworkers to effectively construct both stereogenic centers of aryl-ring-containing 1,3-diols through a one-pot, two-step process involving an aldol reaction followed by ADH-catalyzed reduction [38]. Initially, they employed an organocatalytic aldol reaction to enantioselectively introduce the first stereogenic center. Subsequently, the asymmetric reduction of the β-hydroxy ketone using the (S)-selective ADH from Rhodococcus sp. yielded the corresponding (1R,3S)-1,3-diol with high yield, excellent enantioselectivity, and favorable diastereoselectivity. Their method demonstrated the versatility to generate all four possible stereoisomers of the 1,3-diol by carefully selecting the organocatalyst and the ADH. For instance, utilizing the (R, R)-organocatalyst in the initial step, coupled with the (R)-selective ADH from Lactobacillus kefir, produced the (1S,3R)-1,3-diol (Scheme 12). The enantioselectivity of the first step was further tuned by the use of less organocatalyst loading [39].

Berkessel, Gröger, and their coworkers reported the bioreduction of 1-bromo-2-octanone to its corresponding (S)-1-bromo-2-octanol by recombinant whole cells containing the (S)-selective LkADH, and glucose dehydrogenase from Thermoplasma acidophilum (TaGDH), the latter used for enzyme-based regeneration of the cofactor (Scheme 13) [40]. Following the asymmetric reduction, pH adjustment to 11 was performed, accompanied by stirring for 2 h, without the need to isolate the bromohydrin. This led to the formation of the corresponding epoxide with an isolated yield of 37%. The lower yield was attributed to the lower efficiency of extraction at high pH compared to that at low pH. Subsequently, Wei, Wang, and their coworkers employed a similar strategy to synthesize styrene oxide and phenyl glycidyl ether from α-chloroacetophenone and chloro-1-phenoxy-2-propanone, respectively, using ADH from Kuraishia capsulata CBS1993 (KcADH), followed by the addition of excess NaOH, achieving excellent enantiomeric excess and good yield [41]. They also demonstrated the applicability of this method in large-scale reactions with substrate loading of up to 738 mg (Scheme 14).

2.2. Concurrent Reaction Cascades Involving Alcohol Dehydrogenases

Rudrof, Mihovilovic, and their coworkers developed a concurrent chemoenzymatic reaction cascade that combines metal-catalyzed Liebeskind–Srogl coupling, using Pd/Cu catalysts, with an enantioselective biocatalytic reduction [42]. The coupling reaction combines aryl boronic acids with a thioester in the presence of copper salts, while a biphasic compartmentalized reactor facilitates compatibility by isolating the deactivating components in separate chambers (Scheme 15). This innovative approach features a concurrent C-C cross-coupling reaction together with an enzymatic reduction using either of the enantiocomplementary LkADH, an (R)-selective ADH, or ADH-A, an (S)-selective ADH, to independently produce the optically active form of both enantiomers of 1-arylethanols with high enantiomeric purity. Notably, a high substrate loading of up to 100 mM was achieved through the use of a polydimethylsiloxane (PDMS) membrane reactor.

Gonzalez-Sabin, Rebolledo, Crochet, and their coworkers reported a method for simultaneous asymmetric bioreduction and metal-catalyzed nitrile hydrogenation of β-ketonitriles to β-hydroxyamides [43]. This innovative approach involved a ruthenium (IV) catalyst for nitrile hydration while employing LkADH or a commercially available ketoreductase for the chemo- and enantioselective reduction of the resultant ketone moiety. This concurrent method yielded enantiopure β-hydroxyamides from β-ketonitriles, achieving high enantioselectivities and conversion rates (Scheme 16).

Lavandera, Gotor-Fernández, and their coworkers demonstrated a concurrent Meyer–Schuster rearrangement followed by a stereoselective reduction catalyzed by an ADH [44]. This approach was achieved using active N-heterocyclic carbene gold (I) catalysts in an aqueous medium, in combination with various ADHs, including KRED-P3-H12, KRED-P2-H07, LbADH, and ADH-A. The gold catalysts facilitated the formation of α,β-unsaturated ketones from propargylic alcohols, which were subsequently asymmetrically reduced by an ADH in situ, to produce the corresponding β,β-disubstituted allylic alcohols. Notably, the gold catalyst exhibited varying selectivity towards the tested substrates, resulting in 14 out of 24 substrates yielding E-isomers. Consequently, the substrate scope for this cascade reaction was limited to E-allylic isomers, as the ADHs were only capable of reducing these ketones (Scheme 17).

Gröger and coworkers reported a tandem chemoenzymatic transformation that integrates high-pressure syngas metal catalysis with ADH-catalyzed reduction for the synthesis of alcohols from 1-octene [45]. This innovative one-pot process utilized rhodium-catalyzed hydroformylation using 6-diphenylphosphanyl-2-pyridone (6-DPPon) ligand and Triton X-100 as a surfactant to enhance the solubility of the alkene in aqueous media containing phosphate buffer. The resulting aldehyde was concurrently reduced to the corresponding alcohol using LbADH. Remarkably, both catalytic systems demonstrated remarkable efficiency, with no significant deactivation of either the biocatalyst or the chemo-catalyst, achieving >99% conversion and an isolated yield of 80% (Scheme 18). Furthermore, the methodology was successfully extended to the enantioselective synthesis of (S)-2-phenylpropanol through hydroformylation of styrene in tandem with a dynamic kinetic resolution (DKR) of the resulting aldehyde facilitated by ADH from Thermoanaerobacter brockii (TbADH) yielding the (S)-alcohol with an enantiomeric ratio of 91:9. This study demonstrates the feasibility of combining disparate catalytic systems in a sustainable aqueous environment, paving the way for scalable and stereoselective chemoenzymatic syntheses.

Kroutil and coworkers reported a novel one-pot, one-step strategy for synthesizing enantiopure epoxides from α-chloro-ketones [46]. This approach operates at pH levels exceeding 12 and combines biocatalytic asymmetric reduction using whole cells containing ADH from Rhodococcus ruber with in situ base-catalyzed epoxide formation. The protection of the enzyme within the shielding environment of whole microbial cells enabled effective biocatalytic reduction under extreme pH conditions, allowing both reactions to concurrently occur in a single pot. These findings demonstrate the efficiency and effectiveness of this method, facilitating the direct conversion of halo-ketones into valuable enantiopure epoxide products while ensuring high enantiomeric purity (Scheme 19).

Concurrent chemoenzymatic transformations are relatively rare, primarily due to the challenges in ensuring compatibility between chemical and enzyme-based processes. However, recent advancements in biotechnology will lead to the development of more robust enzymes that demonstrate improved stability. These are expected to enhance the compatibility of enzymatic reactions with chemical transformations, potentially increasing the viability and efficiency of concurrent chemoenzymatic processes in organic synthesis.

3. One-Pot, Three-Step Reaction Cascades Utilizing Alcohol Dehydrogenases

Gröger, Berkessel, and their coworkers reported a chemoenzymatic synthesis of (R)-pantolactone, a key intermediate in the production of vitamin B5 [47]. This method integrates an L-histidine-catalyzed asymmetric aldol reaction of isobutanal and ethyl glyoxylate with an ADH-catalyzed reduction in a “one-pot-like” setup, leading to the formation of a chiral aldehyde intermediate. Following the evaporation of volatile components under vacuum, the intermediate was subsequently reduced using ADH-200, yielding a hydroxyester that spontaneously cyclizes to form (R)-pantolactone (Scheme 20). Interestingly, a molecular weight cut-off membrane was used to recycle enzyme, significantly reducing the enzyme loading to a minimal level. The combined process achieved a total yield of 55% with an enantiomeric excess of 95%.

Surfactants, such as TPGS-750-M, have demonstrated promising attributes for performing multi-step reactions in the same pot [48]. Lipshutz and his coworkers developed a one-pot method that involves a sequential 1,4-addition, reduction of the nitro group using carbonyl iron powder (CIP), and ADH101-catalyzed reduction to synthesize (R)-4-(3′-aminophenyl)butan-2-ol, achieving an overall yield of 75% with an ee exceeding 99% (Scheme 21) [35]. Notably, the enzyme was able to perform asymmetric reduction within two hours, despite the presence of Rh, Fe, and other metal salts. This finding highlights the resilience of ADHs to residual catalysts from other steps, which enables a prolonged sequence of reactions.

Aue, Lipshutz, and their coworkers developed a one-pot three-step sequence that integrates compatible chemo- and biocatalytic reactions in aqueous media [49]. The process began with an ADH-catalyzed reduction of 4-bromoacetophenone in an aqueous buffer solution. The product from this initial step was not isolated; instead, a Suzuki–Miyaura coupling catalyst was directly added to the reaction mixture. Subsequently, the resulting biaryl intermediate was treated with CIP, yielding the corresponding non-racemic amino alcohol with a yield exceeding 80% (Scheme 22). The compatibility of reaction conditions was achieved by the design of a biaryl phosphine-containing ligand (N₂Phos), which facilitated the Suzuki–Miyaura reaction at low palladium concentrations within an aqueous micellar reaction media.

Recent advancements in biotechnology, including directed evolution [50,51] and the innovative use of computational protein design [52], are anticipated to yield a greater variety of ADHs with broad substrate specificity and the desired stereopreference. These developments are expected to enhance the employability of ADHs in chemoenzymatic transformations, facilitating more versatile and efficient applications in synthetic organic chemistry.

4. Conclusions

In this review, we highlighted the significant enhancement in chemoenzymatic cascade reactions that integrate chemical transformations with ADH-catalyzed asymmetric reductions, enabling the production of enantiopure alcohols in a single pot, either concurrently or sequentially. These methodologies streamline synthesis, improve reaction yields, and minimize the use of organic solvents, aligning with environmentally friendly practices. Despite these benefits, challenges remain, particularly in achieving optimal compatibility between chemical and enzymatic processes and expanding the substrate scope of ADHs to accommodate diverse substrates. To address these limitations, future research should focus on the development of more robust enzymes through protein engineering and directed evolution. This would allow organic chemists to explore a broader range of chemoenzymatic transformations, ultimately enhancing the versatility and applicability of these innovative methodologies in synthetic chemistry.