Synthesis Of Chiral Alcohols Research Articles

Pickering Emulsion Promoted Interfacial Sequential Chemo-Biocatalytic Reaction for the Synthesis of Chiral Alcohols from Styrene.

Chemo-biocatalytic cascades have emerged as a promising approach in the realm of advanced synthesis. However, reconciling the incompatible reaction conditions among distinct catalytic species presents a significant challenge. Herein, we introduce an innovative solution using an emulsion system stabilized by Janus silica nanoparticles, which serve as a bridge for both chemo-catalysts and biocatalysts at the interface. The chemo-catalyst is securely anchored within a hydrophobic polymer matrix, ensuring its residence in an organic environment. Meanwhile, the negatively charged E. coli cells containing enzymes are attracted to the aqueous phase at the interface, facilitating their optimal positioning. We demonstrate the efficacy of this system through a two-step cascade reaction. Initially, the oxidation of styrene to acetophenone using palladium as a chemocatalyst achieves a 6-fold increase in yield compared to the control system. Subsequently, the reduction of achiral acetophenone to its chiral alcohol derivative presents a 17-fold yield enhancement relative to that of the control reaction. Importantly, our system exhibits versatility, accommodating a wide range of substrates for both individual and sequential reactions. This work not only validates the concept but also paves the way for the integration of chemo- and biocatalysts in the synthesis of a broader array of high-value chemical compounds.

Chemoenzymatic Sequential Catalysis with Carbonic Anhydrase for the Synthesis of Chiral Alcohols from Alkanes, Alkenes, and Alkynes

Chemoenzymatic Sequential Catalysis with Carbonic Anhydrase for the Synthesis of Chiral Alcohols from Alkanes, Alkenes, and Alkynes

Engineering diaryl alcohol dehydrogenase KpADH reveals importance of retaining hydration shell in organic solvent tolerance.

Alcohol dehydrogenases (ADHs) are synthetically important biocatalysts for the asymmetric synthesis of chiral alcohols. The catalytic performance of ADHs in the presence of organic solvents is often important since most prochiral ketones are highly hydrophobic. Here, the organic solvent tolerance of KpADH from Kluyveromyces polyspora was semi-rationally evolved. Using tolerant variants obtained, meticulous experiments and computational studies were conducted to explore properties including stability, activity and kinetics in the presence of various organic solvents. Compared with WT, variant V231D exhibited 1.9-fold improvement in ethanol tolerance, while S237G showed a 6-fold increase in catalytic efficiency, a higher , as well as 15% higher tolerance in 7.5% (v/v) ethanol. Based on 3 × 100 ns MD simulations, the increased tolerance of V231D and S237G against ethanol may be ascribed to their enhanced ability in retaining water molecules and repelling ethanol molecules. Moreover, 6.3-fold decreased KM value of V231D toward hydrophilic ketone substrate confirmed its capability of retaining hydration shell. Our results suggest that retaining hydration shell surrounding KpADH is critical for its tolerance to organic solvents, as well as catalytic performance. This study provides useful guidance for engineering organic solvent tolerance of KpADH and other ADHs.

Cordyceps fungus-mediated enantioselective synthesis of chiral alcohol and enhanced biocatalytic efficiency in cyclodextrin/surfactant-buffer system

Asymmetric synthesis of chiral alcohols by biocatalysts holds promising prospect. In this study, a medicinal Cordyceps fungus, namely Tolypocladium inflatum, was adopted as a novel biocatalyst for the synthesis of chiral alcohols. Among the assessed prochiral ketone substrates, T. inflatum exhibited the best catalytic activity towards 1-(4-bromophenyl)−2,2,2-trifluoroethanone (BPFO), yielding a valuable pharmaceutical intermediate (S)−1-(4-bromophenyl)−2,2,2-trifluoroethanol ((S)-BPFL) with >99.9 % enantiomeric excesses (ee). For more efficient biosynthesis of (S)-BPFL, cyclodextrins or surfactants were introduced to the reaction system as additives to strengthen this bioprocess. Among which methyl β-cyclodextrin (MCD) improved cell membrane permeability and alleviated product inhibition, while Triton X-100 enhanced BPFO solubility. Furthermore, synergistic promotion for BPFO bioreduction by MCD and Triton X-100 was observed. Under the optimal reaction conditions, 50 mM BPFO was converted into (S)-BPFL with 89.1 % yield and >99.9 % ee in 12 h, showing 16-fold increase of space-time yield in the developed MCD/Triton X-100-buffer system compared to neat buffer solution. This study illustrated the potential of the enantioselective preparation of chiral alcohols mediated by Cordyceps fungus. Additionally, the biocatalytic efficiency was significantly improved in the constructed synergistic reaction system, and the underlying mechanism was also explored.

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.

Rhodium‐Catalyzed Asymmetric Transfer Hydrogenation of Heterocyclic Diaryl Ketones: Facile Access to Key Intermediate of Baloxavir†

Comprehensive SummaryTransition metal‐catalyzed asymmetric transfer hydrogenation has been proven to be a powerful approach for the synthesis of chiral alcohols. Herein, a highly efficient and enantioselective transfer hydrogenation of dibenzoheptaheterocyclic ketones catalyzed by an arene‐tethered TsDPEN‐based Rh(III) catalyst has been successfully developed, and a variety of dibenzoheptaheterocyclic ketones were reduced by a 1/1 mixture of formic acid and DBU (1,8‐diazabicyclo[5.4.0]undec‐7‐ene) with high yields and enantioselectivities. With this method, the asymmetric reduction of 7,8‐difluorodibenzo[b,e]thiepin‐11(6H)‐one has been realized, providing the key intermediate of baloxavir marboxil with >99% yield and >99% ee at a substrate/catalyst molar ratio of 1000.

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.

Light Controlled Nanobiohybrids for Modulating Chiral Alcohol Synthesis.

The modulation of whole-cell activity presents a considerable challenge in biocatalysis. Conventional approaches to whole-cell catalysis, while having their strengths, often rely on complex and deliberate enzyme designs, which could result in difficulties in activity modulation and prolonged response times. Additionally, the activity of intracellular enzymes in whole-cell catalysis is influenced by temperature. To address these limitations, we introduced a relationally designed nanobiohybrid system that utilized light to modulate whole-cell catalysis for chiral alcohol production. By incorporating platinum nanoparticles onto Rhodotorula sp. cell surfaces, the nanobiohybrid capitalized on the photothermal properties of the nanoparticles to regulate the overall cell activity. When exposed to light, the Pt nanoparticles generate heat through the photothermal effect, consequently leading to an increase in the catalytic activity of the whole cells. This innovative approach facilitates control over whole-cell production and provides an efficient method for regulating biocatalytic processes. The findings of this study demonstrate the significant potential of switchable control strategies in biomanufacturing across a wide range of industries.

Biocatalytic Foams from Microdroplet-Formulated Self-Assembling Enzymes.

Industrial biocatalysis plays an important role in the development of a sustainable economy, as enzymes can be used to synthesize an enormous range of complex molecules under environmentally friendly conditions. To further develop the field, intensive research is being conducted on process technologies for continuous flow biocatalysis in order to immobilize large quantities of enzyme biocatalysts in microstructured flow reactors under conditions that are as gentle as possible in order to realize efficient material conversions. We report here on monodisperse foams consisting almost entirely of enzymes covalently linked via SpyCatcher/SpyTag conjugation. The biocatalytic foams are readily available from recombinant enzymes via microfluidic air-in-water droplet formation, can be directly integrated into micro reactors, and can be used for biocatalytic conversions after drying. Reactors prepared by this method show surprisingly high stability and biocatalytic activity. We describe the physicochemical characterization of the new materials and show exemplary applications in biocatalysis using two-enzyme cascades for the stereoselective synthesis of chiral alcohols and the rare sugar tagatose. This article is protected by copyright. All rights reserved.

A curated list of targeted optimized promiscuous ketoreductases (TOP-K).

Enzymes are either specific or promiscuous catalysts in nature. The latter is portrayed by protein families like CYP450Es, Aldo-ketoreductases and short/medium-chain dehydrogenases which participate in detoxification or secondary metabolite production. However, enzymes are evolutionarily 'blind' to an ever-increasing synthetic substrate library. Industries and laboratories have circumvented this by high-throughput screening or site-specific engineering to synthesize the product of interest. However, this paradigm entails cost and time-intensive one-enzyme, one-substrate catalysis model. One of the superfamilies regularly used for chiral alcohol synthesis are short-chain dehydrogenases/reductases (SDRs). Our objective is to determine a superset of promiscuous SDRs that can catalyze multiple ketones. They are typically classified into shorter 'Classical' and longer 'Extended' type ketoreductases. However, current analysis of modelled SDRs reveals a length-independent conserved N-terminus Rossmann-fold and a variable substrate-binding C-terminus substrate-binding region for both categories. The latter is recognized to influence the enzyme's flexibility and substrate promiscuity and we hypothesize these properties are directly linked with each other. We tested this by catalyzing ketone intermediates with the essential and specific enzyme: FabG_E, as well as non-essential SDRs such as UcpA and IdnO. The experimental results confirmed this biochemical-biophysical association, making it an interesting filter for ascertaining promiscuous enzymes. Hence, we created a dataset of physicochemical properties derived from the protein sequences and employed machine learning algorithms to examine potential candidates. This resulted in 24 targeted optimized ketoreductases (TOP-K) from 81 014 members. The experimental validation of select TOP-Ks demonstrated the correlation between the C-terminal lid-loop structure, enzyme flexibility and turnover rate on pro-pharmaceutical substrates.

Structural Features of Diacyldodecaheterocycles with Pseudo-C2-Symmetry: Promising Stereoinductors for Divergent Synthesis of Chiral Alcohols.

Pseudo-C2-symmetric dodecaheterocyclic structures, which possess two acyl/aroyl groups disposed on either a cis- or trans-relative configuration, were prepared from the naturally occurring (-)-(1R)-myrtenal. Addition of Grignard reagents (RMgX) to the diastereoisomeric mixture of these compounds unexpectedly showed that nucleophilic additions to the two prochiral carbonyl centers gave the same stereochemical result in both cis/trans diastereoisomers, making unnecessary the separation of this mixture. Noticeably, both carbonyl groups showed different reactivity because one of them is attached to an acetalic carbon and the other to a thioacetalic carbon. Furthermore, addition of RMgX to the carbonyl attached to the former carbon takes place through the re face, while addition to the second one proceeds through the si face, thus affording the corresponding carbinols in a highly diastereoselective process. This structural feature allowed the sequential hydrolysis of both carbinols, yielding separately (R)- and (S)-1,2-diols after reduction with NaBH4. The mechanism of the asymmetric Grignard addition was explained by density functional theory calculations. This approach contributes to the development of the divergent synthesis of structurally and/or configurationally different chiral molecules.

Magnetic wrinkled organosilica-based metal-enzyme integrated catalysts for enhanced chemoenzymatic catalysis

Core-shell structured magnetic wrinkled organosilica-based metal-enzyme integrated catalysts were synthesized, and their catalytic performances were studied in the chemoenzymatic dynamic kinetic resolution of chiral amines in an organic solvent, as well as in the chemoenzymatic synthesis of chiral alcohols in water. Structure-performance studies revealed the important influence of their tunable structure and composition on the optimization of activity, stability, and recyclability in chemoenzymatic catalysis.

Development of an enzymatic process for the synthesis of (1S)-2-chloro-1-(3, 4-difluorophenyl) ethanol, the key intermediate of ticagrelor

Asymmetric bioreductions catalyzed by ketoreductases have shown considerable potential in the synthesis of chiral alcohols. However, the synthesis routes of (1S)-2-chloro-1-(3, 4-difluorophenyl) ethanol ((S)-CFPL), a key chiral intermediate in the synthesis of ticagrelor, still pose significant challenges in term of unsatisfactory substrate loading and production cycle. In this study, a structure-guided rational design was applied to a ketoreductase from Candida glabrata (CgKR1) for the asymmetric reduction of the ketone precursor 2-chloro-1-(3, 4-difluorophenyl) ethanone (CFPO). A number of mutants were obtained with remarkably improved activity and inversed stereoselectivity compared with CgKR1-WT. In particular, F92V/Y208T mutant and F92T/Y208V mutant exhibited strict S-stereoselectivity and extremely high catalytic activity for CFPO. In addition, CFPO with the concentration of 200 g/L can be completely converted into (S)-CFPL on a preparative scale (5.0 g, 92.8% isolated yield) in 4 h through optimizing some parameters in the reduction process, delivering a high space-time yield (STY) of 1200 g L−1 day−1. This study not only provides insight into the relationship between the structure and function of CgKR1, but also establishes an efficient enzymatic process for the biocatalytic synthesis of crucial chiral intermediate (S)-CFPL of ticagrelor.

Chiral Alcohols from Alkenes and Water: Directed Evolution of a Styrene Hydratase.

Enantioselective synthesis of chiral alcohols through asymmetric addition of water across an unactivated alkene is a highly sought-after transformation and a big challenge in catalysis. Herein we report the identification and directed evolution of a fatty acid hydratase from Marinitoga hydrogenitolerans for the highly enantioselective hydration of styrenes to yield chiral 1-arylethanols. While directed evolution for styrene hydration was performed in the presence of heptanoic acid to mimic fatty acid binding, the engineered enzyme displayed remarkable asymmetric styrene hydration activity in the absence of the small molecule activator. The evolved styrene hydratase provided access to chiral alcohols from simple alkenes and water with high enantioselectivity (>99 : 1 e.r.) and could be applied on a preparative scale.

Chiral Alcohols from Alkenes and Water: Directed Evolution of a Styrene Hydratase

AbstractEnantioselective synthesis of chiral alcohols through asymmetric addition of water across an unactivated alkene is a highly sought‐after transformation and a big challenge in catalysis. Herein we report the identification and directed evolution of a fatty acid hydratase from Marinitoga hydrogenitolerans for the highly enantioselective hydration of styrenes to yield chiral 1‐arylethanols. While directed evolution for styrene hydration was performed in the presence of heptanoic acid to mimic fatty acid binding, the engineered enzyme displayed remarkable asymmetric styrene hydration activity in the absence of the small molecule activator. The evolved styrene hydratase provided access to chiral alcohols from simple alkenes and water with high enantioselectivity (>99 : 1 e.r.) and could be applied on a preparative scale.

Protein engineering of multi-enzyme virus-like particle nanoreactors for enhanced chiral alcohol synthesis.

In the past decade, virus-like particles (VLPs) that can encapsulate single or multiple enzymes have been studied extensively as typical nanoreactors for biocatalysis in vitro, yet their catalytic efficiencies are usually inadequate for real applications. These biocatalytic nanoreactors should be engineered like their free-enzyme counterparts to improve their catalytic performance for potential applications. Herein we engineer biocatalytic VLPs for the enhanced synthesis of chiral alcohols. Different methods including directed evolution were applied to the entire bacteriophage P22 VLPs (except the coat protein), which encapsulated a carbonyl reductase from Scheffersomyces stipitis (SsCR) and a glucose dehydrogenase from Bacillus megaterium (BmGDH) in their capsids. The best variant, namely M5, showed an enhanced turnover frequency (TOF, min-1) up to 15-fold toward the majority of tested aromatic prochiral ketones, and gave up to 99% enantiomeric excess in the synthesis of chiral alcohol pharmaceutical intermediates. A comparison with the mutations of the free-enzyme counterparts showed that the same amino acid mutations led to different changes in the catalytic efficiencies of free and confined enzymes. Finally, the engineered M5 nanoreactor showed improved efficiency in the scale-up synthesis of chiral alcohols. The conversions of three substrates catalyzed by M5 were all higher than those catalyzed by the wild-type nanoreactor, demonstrating that enzyme-encapsulating VLPs can evolve to enhance their catalytic performance for potential applications.

Engineering ketoreductases for the enantioselective synthesis of chiral alcohols.

The use of engineered ketoreductases (KREDS), both as whole microbial cells and isolated enzymes, in the highly enantiospecific reduction of prochiral ketones is reviewed. The homochiral alcohol products are key intermediates in, for example, pharmaceuticals synthesis. The application of sophisticated protein engineering and enzyme immobilisation techniques to increase industrial viability are discussed.

Directed reconstruction of a novel ancestral alcohol dehydrogenase featuring shifted pH-profile, enhanced thermostability and expanded substrate spectrum

Ancestral enzymes are promising for industrial biotechnology due to high stability and catalytic promiscuity. An effective protocol was developed for the directed resurrection of ancestral enzymes. Employing genome mining with diaryl alcohol dehydrogenase KpADH as the probe, descendant enzymes D10 and D11 were firstly identified. Then through ancestral sequence reconstruction, A64 was resurrected with a specific activity of 4.3 U·mg−1. The optimum pH of A64 was 7.5, distinct from 5.5 of D10. The T15 50 and Tm values of A64 were 57.5 °C and 61.7 °C, significantly higher than those of the descendant counterpart. Substrate spectrum of A64 was quantitively characterized with a Shannon-Wiener index of 2.38, more expanded than D10, especially, towards bulky ketones in Group A and B. A64 also exhibited higher enantioselectivity. This study provides an effective protocol for constructing of ancestral enzymes and an efficient ancestral enzyme of industrial relevance for asymmetric synthesis of chiral alcohols.

Substrate selectivity and kinetic studies of (S)-specific alcohol dehydrogenase purified from Candida parapsilosis ATCC 7330

Biocatalytic reduction catalysed by alcohol dehydrogenases is a valuable tool for asymmetric synthesis of chiral alcohols. This study reports the broad substrate specificity of NADH dependent (S) - specific alcohol dehydrogenase (S-ADH) purified from Candida parapsilosis ATCC 7330. The substrates for this enzyme include aliphatic and aromatic ketones, cyclic and diketones, aldehydes, ketoesters, primary and secondary alcohols. The kinetic studies of different substrates indicate that ketones and secondary alcohols are the most preferred substrates of S-ADH with highest catalytic efficiencies reported for reduction of acetone (4153 s−1mM−1) and oxidation of 2-propanol (1358 s−1mM−1). The double reciprocal plots obtained for varied concentrations of acetophenone (0.2–16 mM) at a fixed concentration of NADH (0.05, 0.1 and 0.2 mM) and with varied concentrations of NADH (0.01–0.2 mM) at a fixed concentration of acetophenone (1, 4, 8 and 16 mM) showed intersecting lines indicating sequential kinetic mechanism. S-ADH follows Prelog's stereopreference in reducing prochiral carbonyl substrates to yield (S)-alcohols with >99% enantiomeric excess using a simple coupled substrate approach for cofactor recycling.

Enantioselective Nickel-Catalyzed Si–C(sp2) Bond Activation and Migratory Insertion to Aldehydes: Reaction Scope and Mechanism

Transition-metal-catalyzed silicon–carbon bond activation is one of the most important processes in both organosilicon chemistry and homogeneous catalysis that is still rarely reported in the past decades, and the enantioselective versions based on transition-metal-catalyzed Si–C bond activation remain an ongoing challenge in asymmetric catalysis. Herein, we report a convenient and enantioselective Si–C bond cleavage-initiated [4 + 2] annulation of benzosilacyclobutenes with aldehydes, which provides an access to the direct synthesis of chiral six-membered oxasilacycles and their derivatives with high yields and enantioselectivities (up to 97% ee). The catalytic asymmetric reaction proceeds smoothly with the aid of a chiral TADDOL-derived phosphoramidite ligand and its chiral Ni complex with a suitable cavity. By switching the work-up of the reaction involved, the present strategy may be extended to subsequent downstream transformations of silyl ether-containing oxasilacycles to give chiral o-tolyl arylmethanols with high ees and quantitative conversions. A series of experimental results support that the strategy of silicon-mediated organic synthesis controlled by nickel catalysis demonstrates a powerful potential for the facile synthesis of chiral alcohols and its drug-like derivatives. Finally, mechanistic and computational investigations of the nickel-catalyzed Si–C bond activation offer insights into the origin of the observed stereoselective outcome, and the density functional theory calculation shows that the nickel-controlled Si–C(sp2) bond activation enables the controllable migratory insertion of benzaldehyde into the Ni–Si bond, which is recognized as the enantioselectivity-determining step.