Asymmetric Reduction Of Ketones Research Articles

Co‐Immobilization of ADH and GDH on Metal‐organic‐framework: An Effective Biocatalyst for Asymmetric Reduction of Ketones

Co‐Immobilization of ADH and GDH on Metal‐organic‐framework: An Effective Biocatalyst for Asymmetric Reduction of Ketones

Near-infrared light-driven asymmetric photolytic reduction of ketone using inorganic-enzyme hybrid biocatalyst

Effective photolytic regeneration of the NAD(P)H cofactor in enzymatic reductions is an important and elusive goal in biocatalysis. It can, in principle, be achieved using a near-infrared light (NIR) driven artificial photosynthesis system employing H2O as the sacrificial reductant. To this end we utilized TiO2/reduced graphene quantum dots (r-GQDs), combined with a novel rhodium electron mediator, to continuously supply NADPH in situ for aldo-keto reductase (AKR) mediated asymmetric reductions under NIR irradiation. This upconversion system, in which the Ti-O-C bonds formed between r-GQDs and TiO2 enabled efficient interfacial charge transfer, was able to regenerate NADPH efficiently in 64 % yield in 105 min. Based on this, the pharmaceutical intermediate (R)-1-(3,5-bis(trifluoromethyl)phenyl)ethan-1-ol was obtained, in 84 % yield and 99.98 % ee, by reduction of the corresponding ketone. The photo-enzymatic system is recyclable with a polymeric electron mediator, which maintained 66 % of its original catalytic efficiency and excellent enantioselectivity (99.9 % ee) after 6 cycles.

Vigna radiata (mung) as superior natural biobased catalyst for asymmetric reduction of prochiral acetophenone

Asymmetric reduction of prochiral ketone via biocatalysis is one of the most promising and straight forward routes to prepare optically active alcohols. In this work, whole cells of Vigna radiata (also called mung) catalyzed enantioselective reduction of prochiral aromatic ketones was investigated. Effects of various parameters such as co-substrate, temperature, cell loading, substrate concentration on conversion and enantioselectivity were studied in a systematic manner by using acetophenone as the model compound. Glucose was found to be the most effective co-substrate for co-factor regeneration among different co-substrates employed. Whole cells of mung have shown excellent thermostability and high substrate tolerance (up to 200 mM). Under optimum conditions, maximum conversion of 93.44% and enantioselectivity of 98.92% were obtained in 9 h. Good conversion and enantioselectivity were observed with various substituted aromatic ketones. This is a cheap, clean, and eco-friendly process for preparation of chiral secondary alcohols compared to chemical processes.

Two anti‐Prelog NAD‐Dependent Alcohol Dehydrogenases with Broad Substrate Scope and Excellent Enantioselectivity

AbstractEnantiomerically pure alcohols are important to produce active pharmaceutical ingredients, agrochemicals and fine chemicals. Herein, we explored the substrate scope and chemo‐ and enantioselectivity of two NAD‐dependent anti‐Prelog alcohol dehydrogenases from Candida maris (Cm‐ADH) and Pichia finlandica (Pf‐ADH) in the asymmetric reduction of ketones. The ADHs were tested for the reduction of acetophenone with NADH that was recycled using a formate dehydrogenase and sodium formate. Cm‐ADH and Pf‐ADH performed best at 30 °C and at around pH 7 and pH 6, respectively. Pf‐ADH operated well at 50 mM acetophenone concentration, while Cm‐ADH was limited to 10 mM. Regarding the substrate scope, linear‐chain alkyl ketones were efficiently reduced (up to 98 % conversion), while branched and cyclic ketones gave lower conversions (up to 60 %). Aryl‐aliphatic ketones showed variable levels of conversion (<1–79 %), while α,β‐unsaturated and heteroaromatic ketones exhibited good to excellent conversions. In most of the cases, the enantiomeric excess was >99 %. Aliphatic and aryl‐aliphatic aldehydes were converted with up to >99 % conversion. A scale‐up experiment with Pf‐ADH using acetophenone as substrate led to 73 % isolated yield and >99 % ee (R).This work contributes to filling the gap in biocatalytic asymmetric synthesis of chiral alcohols by introducing two NAD‐dependent ADHs with anti‐Prelog selectivity.

Enantioselective Regulation of Short‐Chain Dehydrogenases via Key Sites in its Loop and Adjacent Regions for the Enantiodivergent Reduction of Difficult‐To‐Reduce Ketones

AbstractShort‐chain dehydrogenases (SDRs) are powerful catalysts for the asymmetric reduction of prochiral ketones in pharmaceutical products. Herein, through gene mining and evolutionary analysis, we obtained two major types of SDRs (SDR‐1 and SDR‐2) for the enantioselective complementary reduction of N‐Boc‐piperidone, which gives the corresponding product (R)‐ or (S)‐N‐Boc‐piperidol, an intermediate of the interleukin inhibitor and lymphoma treatment drug (imbruvica), respectively. By integrating multiple sequence alignment, site‐directed mutagenesis and computational modeling, we proposed a “loop regulation” mechanism for the enantioselective control of SDRs, through which residues in the loop region could potentially fine‐tune their enantioselectivity. Further, site‐directed mutagenesis assays showed that two key residues (L201 and F205 for SDR‐1, F92 and H93 for SDR‐2) in the loop and its adjacent region played critical roles in fine‐tuning the enantioselectivity of SDRs. Understanding this mechanism of SDR stereo preference in catalyzing asymmetric reduction, we further switched the enantioselectivity of the homologous enzymes. The obtained enzymes catalyzed the enantiodivergent synthesis of chiral heterocyclic alcohols with different ring sizes and substituents (25–99% conversion and 25–99% ee (R/S)), including piperidols, 4‐hydroxy azepanes, 3‐hydroxy azepanes and pyrrolidinols. These findings could potentially guide future attempts at protein engineering of stereoselective SDRs.

Development of a novel magnetic metal-organic framework for the immobilization of short-chain dehydrogenase for the asymmetric reduction of pro-chiral ketone

Short-chain dehydrogenase/reductase (SDR) acts as a biocatalyst in the synthesis of chiral alcohols with high optical purity. Herein, we achieved immobilization via crosslinking on novel magnetic metal–organic framework nanoparticles with a three-layer shell structure (Fe3O4@PDA@Cu (PABA)). The results of scanning electron microscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy, and energy dispersive X-ray spectroscopy confirmed the morphology and cross-linking property of immobilized SDR, which was more durable, stable, and reusable and exhibited better kinetic performance than free enzyme. The SDR and glucose dehydrogenase (GDH) were co-immobilized and then used for the asymmetric reduction of COBE and ethyl 2-oxo-4-phenylbutanoate (OPBE). These finding suggest that enzymes immobilized on novel MOF nanoparticles can serve as promising biocatalysts for asymmetric reduction prochiral ketones into chiral alcohols.

Modified distance optimization method for the asymmetric bioreduction conditions of phenyl(thiophen-2-yl)methanone by Weissella paramesenteroides N7

Chirality plays a significant part in life since it is closely linked to carrying out the many metabolic processes that make up a living being. Chiral secondary alcohols such as diaryl-, aryl heteroaryl-, and diheteroaryl are employed in pharmaceuticals as drug intermediates. Although biocatalytic asymmetric reduction of prochiral ketones containing aromatic and heteroaromatic groups is widely known, biocatalytic reduction of heteroaromatic prochiral ketones containing sulfur heteroatoms is rarely known. Very few studies exist in the literature, including the biocatalytic reduction of phenyl(thiophen-2-yl)methanone (1). Moreover, the biocatalytic reduction of 1 with a mathematical modelling and optimization technique is unknown until now. In this study, Weissella paramesenteroides N7 biocatalyst for the asymmetric bioreduction of 1 using a novel modified distance optimization method. Optimization conditions were found as pH = 6.46, temperature = 26 °C, incubation period = 71 h, agitation speed = 200 rpm by the modified distance optimization method, and it was determined that the conversion and enantiomeric excess (ee) under these conditions could be 98.7% and 98%, respectively. Under these proposed optimization conditions, (S)-phenyl(thiophen-2-yl)methanol ((S)-2) was obtained with >99% ee, >99% conversion, and 97% yield. In addition, 11.29 g of 1 was completely converted into (S)-2 (11.07 g, 97% isolated yield) under optimized conditions. This is the first report about the fabrication of enantiopure (S)-2 in high gram scale using a biocatalyst and a novel modified distance optimization technique. In this study, the successful applicability of the new modified distance optimization method in biocatalytic asymmetric reduction reactions has been successfully demonstrated.

Optimization of asymmetric bioreduction conditions of 1-indanone by Leuconostoc mesenteroides N6 using a face-centered design-based multi-objective optimization model

There has been an increasing interest in biocatalysts over the past few decades in order to obtain high efficiency, high yield, and environmentally benign procedures aiming at the manufacture of pharmacologically relevant chemicals. Lactic Acid Bacteria (LAB), a microbial group, can be employed as biocatalysts while performing asymmetric reduction of prochiral ketones. In this study, Leuconostoc mesenteroides N6 was used for the asymmetric bioreduction 1-indanone. And then, a novel and innovative face-centered design-based multi-objective optimization model was used to optimize experimental conditions. Also, the experimental design factors were defined as agitation speed, incubation period, pH, and temperature for optimization to acquire the maximum enantiomeric excess (ee) and conversion rate (cr) values. When using the face-centered design-based multi-objective optimization model, the optimum culture conditions corresponded to 96.34 and 99.42%, ee and cr responses, respectively, were pH = 5.87, incubation temperature = 35 °C, incubation period = 50.88 h, and agitation speed = 152.60 rpm. Notably, the validation experiment under the optimum model conditions confirmed the model results. This study demonstrated the importance of the optimization and the efficiency of the face-centered design-based multi-objective model.

Computationally Supported Inversion of Ketoreductase Stereoselectivity.

Whereas directed evolution and rational design by structural inspection are established tools for enzyme redesign, computational methods are less mature but have the potential to predict small sets of mutants with desired properties without laboratory screening of large libraries. We have explored the use of computational enzyme redesign to change the enantioselectivity of a highly thermostable alcohol dehydrogenase from Thermus thermophilus in the asymmetric reduction of ketones. The enzyme reduces acetophenone to (S)-1-phenylethanol. To invert the enantioselectivity, we used an adapted CASCO workflow which included Rosetta for enzyme design and molecular dynamics simulations for ranking. To correct for unrealistic binding modes, we used Boltzmann weighing of binding energies computed by a linear interaction energy approach. This computationally cheap method predicted four variants with inverted enantioselectivity, each with 6-8 mutations around the substrate-binding site, causing only modest reduction (2- to 7-fold) of kcat /KM values. Laboratory testing showed that three variants indeed had inverted enantioselectivity, producing (R)-alcohols with up to 99 % enantiomeric excess. The broad substrate range allowed reduction of acetophenone derivatives with full conversion to highly enantioenriched alcohols. The results demonstrate the use of computational methods to control ketoreductase stereoselectivity in asymmetric transformations with minimal experimental screening.

High-throughput ab initio reaction mechanism exploration in the cloud with automated multi-reference validation.

Quantum chemical calculations on atomistic systems have evolved into a standard approach to studying molecular matter. These calculations often involve a significant amount of manual input and expertise, although most of this effort could be automated, which would alleviate the need for expertise in software and hardware accessibility. Here, we present the AutoRXN workflow, an automated workflow for exploratory high-throughput electronic structure calculations of molecular systems, in which (i) density functional theory methods are exploited to deliver minimum and transition-state structures and corresponding energies and properties, (ii) coupled cluster calculations are then launched for optimized structures to provide more accurate energy and property estimates, and (iii) multi-reference diagnostics are evaluated to back check the coupled cluster results and subject them to automated multi-configurational calculations for potential multi-configurational cases. All calculations are carried out in a cloud environment and support massive computational campaigns. Key features of all components of the AutoRXN workflow are autonomy, stability, and minimum operator interference. We highlight the AutoRXN workflow with the example of an autonomous reaction mechanism exploration of the mode of action of a homogeneous catalyst for the asymmetric reduction of ketones.

New Anti-Prelog Stereospecific Whole-Cell Biocatalyst for Asymmetric Reduction of Prochiral Ketones.

The biocatalytic asymmetric reduction of prochiral ketones for the production of enantiopure alcohols is highly desirable due to its inherent advantages over chemical methods. In this study, a new bacterial strain capable of transforming ketones to corresponding alcohols with high activity and excellent enantioselectivity was discovered in a soil sample. The strain was subsequently identified as Bacillus cereus TQ-2 based on its physiological characteristics and 16S rDNA sequence analysis. Under optimized reaction conditions, the resting cells of B. cereus TQ-2 converted acetophenone to enantioenriched (R)-1-phenylethanol with 99% enantiometric excess following anti-Prelog's rule, which is scarce in biocatalytic ketone reduction. The optimum temperature for the cells was 30 °C, and considerable catalytic activity was observed over a broad pH range from 5.0 to 9.0. The cells showed enhanced catalytic activity in the presence of 15% (v/v) glycerol as a co-substrate. The catalytic activity can also be substantially improved by adding Ca2+ or K+ ions. Moreover, the B. cereus TQ-2 cell was highly active in reducing several structurally diverse ketones and aldehydes to form corresponding alcohols with good to excellent conversion. Our study provides a versatile whole-cell biocatalyst that can be used in the asymmetric reduction of ketones for the production of chiral alcohol, thereby expanding the biocatalytic toolbox for potential practical applications.

Cover Feature: Recycling of Cofactors in Crude Enzyme Hydrogels as Co‐immobilized Heterogeneous Biocatalysts for Continuous‐Flow Asymmetric Reduction of Ketones (ChemSusChem 3/2023)

The Cover Feature shows a river in which blue enzyme molecules are induced to form porous enzyme-rich hydrogels in the flow channel and the stable hydrogels have a good tethering effect on the flowing orange cofactor molecules. The orange molecules are trapped in the hydrogel networks, thus forming co-immobilized heterogeneous biocatalysts with self-sufficient cofactors. More information can be found in the Research Article by C. Chen et al.

Structural analysis of an anthrol reductase inspires enantioselective synthesis of enantiopure hydroxycycloketones and β-halohydrins

Asymmetric reduction of prochiral ketones, particularly, reductive desymmetrization of 2,2-disubstituted prochiral 1,3-cyclodiketones to produce enantiopure chiral alcohols is challenging. Herein, an anthrol reductase CbAR with the ability to accommodate diverse bulky substrates, like emodin, for asymmetric reduction is identified. We firstly solve crystal structures of CbAR and CbAR-Emodin complex. It reveals that Tyr210 is critical for emodin recognition and binding, as it forms a hydrogen-bond interaction with His162 and π-π stacking interactions with emodin. This ensures the correct orientation for the stereoselectivity. Then, through structure-guided engineering, variant CbAR-H162F can convert various 2,2-disubstituted 1,3-cyclodiketones and α-haloacetophenones to optically pure (2S, 3S)-ketols and (R)-β-halohydrins, respectively. More importantly, their stereoselectivity mechanisms are also well explained by the respective crystal structures of CbAR-H162F-substrate complex. Therefore, this study demonstrates that an in-depth understanding of catalytic mechanism is valuable for exploiting the promiscuity of anthrol reductases to prepare diverse enantiopure chiral alcohols.

Recycling of Cofactors in Crude Enzyme Hydrogels as Co-immobilized Heterogeneous Biocatalysts for Continuous-Flow Asymmetric Reduction of Ketones.

Flow biocatalysis involving oxidoreductase is limited by the difficulty in recycling expensive cofactors. In this study, an enzyme-rich hydrogel monolithic microreactor was developed via in situ microfluidic assembly of inexpensive crude enzymes. This porous gel biocatalyst exhibited good tethering functions to nicotinamide cofactors; thus, they were retained by the hydrogel to controllably form a novel heterogeneous biocatalyst with self-sufficient cofactors. The flow asymmetric production of a chiral alcohol in this cofactor-entrapped gel microreactor achieved >99 % enantioselectivity and a high space-time yield of 46.3 g L-1 h-1 at 94.8 % conversion. Moreover, the turnover number of cofactors reached as high as 4800 after continuous operation of 160 reactor volumes, realizing significantly higher utilization of the cofactors compared with many reported strategies. Furthermore, this engineered heterogeneous biocatalyst exhibited improved performance in terms of product tolerance and storage stability, paving the way for a green, cost-effective, and sustainable continuous-flow production of enantiopure alcohols.

Synthesis and Biological Evaluation of Bicyclo[1.1.1]pentane-Containing Aromatic Lipoxin A4 Analogues.

Lipoxins are important drivers of inflammation resolution, suggesting a potential therapeutic benefit. Bicyclo[1.1.1]pentanes (BCPs) are potential isosteric replacements for arenes and/or alkyl groups within drug candidates. We carried out an asymmetric synthesis of four BCP-containing synthetic lipoxin A4 mimetics (BCP-sLXms) in which the key steps were a Suzuki coupling, an asymmetric ketone reduction, and a triethylborane-initiated radical bicyclopentylation. These mimetics were screened for their impact on inflammatory responses, and one imidazolo-BCP-sLXm (6a) was found to possess high anti-inflammatory activity.

Ketoreductase Catalyzed (Dynamic) Kinetic Resolution for Biomanufacturing of Chiral Chemicals.

Biocatalyzed asymmetric reduction of ketones is an environmentally friendly approach and one of the most cost-effective routes for producing chiral alcohols. In comparison with the well-studied reduction of prochiral ketones to generate chiral alcohols with one chiral center, resolution of racemates by ketoreductases (KREDs) to produce chiral compounds with at least two chiral centers is also an important strategy in asymmetric synthesis. The development of protein engineering and the combination with chemo-catalysts further enhanced the application of KREDs in the efficient production of chiral alcohols with high stereoselectivity. This review discusses the advances in the research area of KRED catalyzed asymmetric synthesis for biomanufacturing of chiral chemicals with at least two chiral centers through the kinetic resolution (KR) approach and the dynamic kinetic resolution (DKR) approach.

Advanced Insights into Catalytic and Structural Features of the Zinc‐Dependent Alcohol Dehydrogenase from Thauera aromatica

The asymmetric reduction of ketones to chiral hydroxyl compounds by alcohol dehydrogenases (ADHs) is an established strategy for the provision of valuable precursors for fine chemicals and pharmaceutics. However, most ADHs favor linear aliphatic and aromatic carbonyl compounds, and suitable biocatalysts with preference for cyclic ketones and diketones are still scarce. Among the few candidates, the alcohol dehydrogenase from Thauera aromatica (ThaADH) stands out with a high activity for the reduction of the cyclic α‐diketone 1,2‐cyclohexanedione to the corresponding α‐hydroxy ketone. This study elucidates catalytic and structural features of the enzyme. ThaADH showed a remarkable thermal and pH stability as well as stability in the presence of polar solvents. A thorough description of the substrate scope combined with the resolution and description of the crystal structure, demonstrated a strong preference of ThaADH for cyclic α‐substituted cyclohexanones, and indicated structural determinants responsible for the unique substrate acceptance.

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

KpADH 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 KpADH. 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 KpADH 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.

A new facile synthesis of (2S,5S)-5-hydroxypipecolic acid hydrochloride

A simple and efficient synthesis of (2S,5S)-5-Hydroxypipecolic acid hydrochloride is reported. The key features of the synthesis involve the asymmetric reduction of ketone using (S)-CBS oxazaborolidine and the use of commercially available methyl pyroglutamate as a starting material.

Recent Advances in Organocatalyzed Asymmetric Reduction of Prochiral Ketones: An Update

AbstractChiral alcohols are important synthetic intermediates and building blocks for the synthesis of drugs, agrochemicals, and natural products. Asymmetric reduction of prochiral ketones has been the most investigated method for accessing chiral alcohols. In this regard, organocatalyzed asymmetric reduction, as a complementary method to transition-metal- and enzyme-catalyzed reactions, has attracted tremendous interest in the past decades due to the reactions with such catalysts being metal-free and easy to operate, and principally, the ease of recovery and the ability to reuse the catalysts. Following up on a comprehensive overview on organocatalyzed asymmetric reductions of prochiral ketones in early 2018, this short review is intended to summarize the recent progress in this area from the beginning of 2018 until the end of August 2021.1 Introduction2 Boron-Based Chiral Organocatalysts2.1 Boron-Containing Chiral Schiff Base Catalysts2.2 Chiral Alpine-Borane Catalysts2.3 Boron-Containing Chiral Frustrated Lewis Pair Catalysts2.4 Chiral Borate Ester–Amine Complex Catalysts3 Phosphorus-Based Chiral Organocatalysts3.1 Chiral Phosphoric Acid Organocatalysts3.2 Chiral Phosphinamide and Phosphoramide Organocatalysts4 Chiral Ionic Liquid Organocatalysts5 Chiral-Oxazoline-Based Organocatalysts6 Conclusion and Outlook