Asymmetric Hydrolysis Research Articles

Actinobacteria as Microbial Cell Factories and Biocatalysts in The Synthesis of Chiral Intermediates and Bioactive Molecules; Insights and Applications.

Actinobacteria are one of the most intriguing bacterial phyla in terms of chemical diversity and bioactivities of their reported biomolecules and natural products, including various types of chiral molecules. Actinobacterial genera such as Detzia, Mycobacterium, and Streptomyces are among the microbial sources targeted for selective reactions such as asymmetric biocatalysis catalyzed by whole cells or enzymes induced in their cell niche. Remarkably, stereoselective reactions catalyzed by actinobacterial whole cells or their enzymes include stereoselective oxidation, stereoselective reduction, kinetic resolution, asymmetric hydrolysis, and selective transamination, among others. Species of actinobacteria function with high chemo-, regio-, and enantio-selectivity under benign conditions, which could help current industrial processing. Numerous selective enzymes were either isolated from actinobacteria or expressed from actinobacteria in other microbes and hence exploited in the production of pure organic compounds difficult to obtain chemically. In addition, different species of actinobacteria, especially Streptomyces species, function as natural producers of chiral molecules of therapeutic importance. Herein, we discuss some of the most outstanding contributions of actinobacteria to asymmetric biocatalysis, which are important in the organic and/or pharmaceutical industries. In addition, we highlight the role of actinobacteria as microbial cell factories for chiral natural products with insights into their various biological potentialities.

Chemoenzymatic Synthesis of Chiral Building Blocks Based on the Kinetic Resolution of Glycerol‐Derived Cyclic Carbonates

AbstractThe biocatalytic kinetic resolution of cyclic carbonates derived from glycerol is reported. A selection of 26 esterases and lipases was tested for the asymmetric hydrolysis of the model substrate (epichlorohydrin carbonate) in aqueous medium. Among them, Pig Liver Esterase and Novozym® 435 showed the best selectivity with E=38 and 49, respectively. Both enzymes were employed for the conversion of 12 glycerol derivatives under optimized conditions. The resolution of halogenated carbonates afforded the unconverted enantiomer in up to >99 : 1 er. Furthermore, Novozym® 435 was successfully recycled 10 times without significant loss of activity. Upscaling and isolation of the chiral carbonate was also demonstrated. Subsequent conversion of this chiral building block allowed the direct one‐pot synthesis of (S)‐Guaifenesin, (S)‐Mephenesin and (S)‐Chlorphenesin in up to 89 % yield and 94 : 6 er.

Chiral Phosphoric Acid Catalyzed Asymmetric Hydrolysis of Biaryl Oxazepines for the Synthesis of Axially Chiral Biaryl Amino Phenol Derivatives

AbstractThe development of catalytic asymmetric reaction with water as the reactant is challenging due to the reactivity‐ and stereoselectivity‐control issues resulted from the low nucleophilicity and the small size of water. We disclose herein a chiral phosphoric acid (CPA) catalyzed atroposelective ring‐opening reaction of biaryl oxazepines with water. A series of biaryl oxazepines undergo the CPA catalyzed asymmetric hydrolysis in a highly enantioselective manner. The key for the success of this reaction is the use of a new SPINOL‐derived CPA catalyst and the high reactivity of biaryl oxazepine substrates towards water under acidic conditions. Density functional theory calculations suggest that the reaction proceeds via a dynamic kinetic resolution pathway and the CPA catalyzed addition of water to the imine group is both enantio‐ and rate‐determining.

Chiral Phosphoric Acid Catalyzed Asymmetric Hydrolysis of Biaryl Oxazepines for the Synthesis of Axially Chiral Biaryl Amino Phenol Derivatives.

The development of catalytic asymmetric reaction with water as the reactant is challenging due to the reactivity- and stereoselectivity-control issues resulted from the low nucleophilicity and the small size of water. We disclose herein a chiral phosphoric acid (CPA) catalyzed atroposelective ring-opening reaction of biaryl oxazepines with water. A series of biaryl oxazepines undergo the CPA catalyzed asymmetric hydrolysis in a highly enantioselective manner. The key for the success of this reaction is the use of a new SPINOL-derived CPA catalyst and the high reactivity of biaryl oxazepine substrates towards water under acidic conditions. Density functional theory calculations suggest that the reaction proceeds via a dynamic kinetic resolution pathway and the CPA catalyzed addition of water to the imine group is both enantio- and rate-determining.

Facile Synthesis of Both Enantiomers of (Z)-1,5-Octadien-3-ol (Oyster Alcohol) and Its Identification from the Pacific Oyster Crassostrea gigas.

The aim of this study was to present a facile protocol for preparation of both enantiomerically pure forms of (Z)-1,5-octadien-3-ol with lipases and to identify the stereochemistry of oyster alcohol from Crassostrea gigas. The asymmetric hydrolysis of (±)-(Z)-1,5-octadien-3-yl acetate with CHIRAZYME L-2 afforded the (R)-alcohol with ≧99% ee in 37.8% conversion. On the other hand, the first asymmetric acylation of the alkadienol with lipase PS recovered the (S)-alcohol with 79.5% ee in 47.8% conversion. Then, the second asymmetric acylation of the recovered (S)-alcohol with lipase PS gave the remaining (S)alcohol with ≧99% ee in 14.1% conversion. Thus, we have successfully prepared both enantiomerically pure forms of (Z)-1,5-octadien-3-ol with high ee (≧99%) separately. On the other hand, oyster alcohol in the extract from C. gigas was purified by silica gel column chromatography and the structure was confirmed by 1H- and 13C-nuclear magnetic resonance spectra. Furthermore, the stereochemistry of oyster alcohol was decided as the (R)-form from the specific rotation and its optical purities were determined as 20.45 ± 0.2% ee by a chiral gas chromatograph/mass spectrometry for the first time.

A new highly enantioselective stable epoxide hydrolase from Hypsibius dujardini: Expression in Pichia pastoris and immobilization in ZIF-8 for asymmetric hydrolysis of racemic styrene oxide

In this study, an epoxide hydrolase gene (hdeh) from extremophile Hypsibius dujardini was expressed heterologously in Pichia pastoris under the optimized conditions. The purified HdEH was immobilized in a metal-organic framework (HdEH@ZIF-8) using zinc nitrate and 2-methylimidazole. The HdEH@ZIF-8 was further treated with different concentration of glutaraldehyde (HdEH@Glu/ZIF-8) to investigate the effect of glutaraldehyde concentration on the activity and stability. The cross-linking of HdEH@ZIF-8 with glutaraldehyde resulted in decreasing the activity, while increasing the thermal stability. The optimum pH of free and immobilized HdEH samples was all determined as 8.0. The free and both immobilized HdEHs exhibit its optimal activity at 20 °C and 30 °C respectively. The HdEH@ZIF-8 and HdEH@Glu/ZIF-8 showed around 16- and 18-fold higher stability than free HdEH at 30 °C. All the HdEHs were used in the asymmetric hydrolysis of racemic styrene oxide (R/S-SO) and the results showed that R-enantiomer of epoxide was preferentially hydrolyzed by free HdEH, HdEH@ZIF-8 and HdEH@Glu/ZIF-8. After 360 min reaction time, (S)-styrene oxide was obtained with 99% enantiopurity for the all the HdEH preparations. HdEH@ZIF-8 and HdEH@Glu/ZIF-8 showed 1.9- and 1.4-fold higher enantioselectivity than that of the free HdEH. HdEH@ZIF-8 and HdEH@Glu/ZIF-8 were remained 91% and 93% of its initial activity after 5 reuses. The results show that HdEH@ZIF-8 and HdEH@Glu/ZIF-8 are a promising stable and enantioselective EH preparations for the asymmetric hydrolysis of racemic styrene oxide.

Informing our understanding of the copper-cuprizone reaction with computational chemistry.

The reaction of copper with bis(cyclohexanone)oxaldihydrazone (cuprizone) is a challenging coordination chemistry problem that has confounded attempts at elucidation for the past 70 years. The product of the reaction, a blue copper complex, wherein the cuprizone ligand is hydrolyzed, has been the primary focus during its history. We have recently characterized an additional green multi-copper product which contains unhydrolyzed cuprizone, which only added to the mystery. Using density functional structure models and thermodynamic calculations we address several of the long-standing questions surrounding the copper-cuprizone reaction, as well as identify the likely reaction pathway that gives rise to the blue and green products. Cu(II)-induced asymmetric hydrolysis of the cuprizone ligand is essential for formation of the blue product, followed by a series of Cu(II)-induced deprotonation and coordination events, with complex formation terminating with hydrolyzed cuprizone tautomerization and intramolecular electron transfer, generating a pseudo-macrocyclic Cu(III) species. Alternatively, in the presence of excess Cu(II), or in non-aqueous solvents, a green multi-Cu(II) complex forms comprised of alternating Cu(II)-cuprizone units. Structure calculations are supported by experimental data and represent the most rigorous approach to-date toward understanding the complex solution chemistry of copper with cuprizone.

A integrated process for nitrilase-catalyzed asymmetric hydrolysis and easy biocatalyst recycling by introducing biocompatible biphasic system

The whole-cell nitrilase-catalyzed asymmetric hydrolysis of nitriles is a green and efficient preparation approach for chiral carboxylic acids, but often suffers from toxicity and cell lysis from organic substrates. In this work, a novel integrated process for whole-cell nitrilase-catalyzed asymmetric hydrolysis was developed for the first time by introducing a biocompatible ionic liquid (IL)-based biphasic system. The whole-cell nitrilases displayed an outstanding stability and recyclability in the biphasic system and still retained > 85% activity even after 7 cycles reaction. A preparative-scale fed-batch hydrolysis of o-chloromandelonitrile to (R)-o-chloromandelic acid (R-CMA) was performed using the integrated process. The results revealed a yield of 91.3% and a space–time yield of 746.4 g·L−1·d−1, which are currently the highest reported values for R-CMA biosynthesis. The proposed integrated process avoids substrate inhibition, facilitates the reusability of whole-cell nitrilases, and thus shows great potential for the sustainable production of chiral carboxylic acids.

Kinetic Resolution of α-Nitrolactones by Catalytic Asymmetric Hydrolysis or Ester–Amide Exchange Reaction

Abstract C 1-Symmetric chiral ammonium salt catalysts induced a kinetic resolution of racemic α-nitrolactones through an asymmetric ester–amide exchange reaction. The corresponding amides were obtained with high enantioselectivities and high S (= k fast/k slow) values. This reaction system is a useful approach for obtaining carbocyclic quaternary α-nitroamides as chiral building blocks.

Isolation of Penicillium expansum WH-3 for the production of L(+)-tartaric acid.

The L(+)-form of tartaric acid (L(+)-TA) exists extensively in nature, and is widely used in the food, chemical, textile, building, and pharmaceutical industries (Su et al., 2001). The main method for L(+)-TA production is microbial transformation by cis-epoxysuccinate hydrolase (CESH), which can catalyze the asymmetric hydrolysis of cis-epoxysuccinic acid or its salts to TA or tartrate (Bao et al., 2019). Seventeen species containing CESH have been isolated so far. However, most species for L(+)-TA production have been reported from bacteria (Xuan and Feng, 2019). The only fungus isolated from soil by our lab recently, that could be used as catalyst for the process under acidic condition, is Aspergillus niger WH-2 (Bao et al., 2020). In order to find strains with new characteristics, this study attempted to isolate a new CESH source from fungi and investigate its application value.

Highly regio- and enantio-selective hydrolysis of two racemic epoxides by GmEH3, a novel epoxide hydrolase from Glycine max

A novel epoxide hydrolase from Glycine max, designated GmEH3, was excavated based on the computer-aided analysis. Then, gmeh3, a GmEH3-encoding gene, was cloned and successfully expressed in E. coli Rosetta(DE3). Among the ten investigated rac-epoxides, GmEH3 possessed the highest and best complementary regioselectivities (regioselectivity coefficients, αS = 93.7% and βR = 97.2%) in the asymmetric hydrolysis of rac-m-chlorostyrene oxide (5a), and the highest enantioselectivity (enantiomeric ratio, E = 55.6) towards rac-phenyl glycidyl ether (7a). The catalytic efficiency (kcatS/KmS = 2.50 mM−1 s−1) of purified GmEH3 for (S)-5a was slightly higher than that (kcatR/KmR = 1.52 mM−1 s−1) for (R)-5a, whereas the kcat/Km (5.16 mM−1 s−1) for (S)-7a was much higher than that (0.09 mM−1 s−1) for (R)-7a. Using 200 mg/mL wet cells of E. coli/gmeh3 as the biocatalyst, the scale-up enantioconvergent hydrolysis of 150 mM rac-5a at 25 °C for 1.5 h afforded (R)-5b with 90.2% eep and 95.4% yieldp, while the kinetic resolution of 500 mM rac-7a for 2.5 h retained (R)-7a with over 99% ees and 43.2% yields. Furthermore, the sources of high regiocomplementarity of GmEH3 for (S)- and (R)-5a as well as high enantioselectivity towards rac-7a were analyzed via molecular docking (MD) simulation.

Improved enantioselectivity of E. coli BioH in kinetic resolution of methyl (S)-3-cyclohexene-1-carboxylate by combinatorial modulation of steric and aromatic interactions.

As a chiral precursor for the important anticoagulant Edoxaban, enantioselective synthesis of (S)-3-cyclohexene-1-carboxylic acid is of great significance. The complicated procedures and generation of massive solid waste discourage its chemical synthesis, and the alternative biocatalysis route calls for an enzyme capable of asymmetric hydrolysis of racemic methyl-3-cyclohexene-1-carboxylate. To this end, we engineered the E. coli esterase BioH for improved S-enantioselectivity via rational design. By combinatorial modulation of steric and aromatic interactions, a positive mutant Mu3 (L24A/W81A/L209A) with relatively high S-selectivity in hydrolyzing racemic methyl-3-cyclohexene-1-carboxylate was obtained, improving the enantiomeric excess from 32.3% (the wild type) to 70.9%. Molecular dynamics simulation was conducted for both (R)- or (S)- complexes of the wild type and Mu3 to provide hints for the mechanism behind the increased S-selectivity. Moreover, the reaction conditions of Mu3 in methyl-3-cyclohexene-1-carboxylate hydrolysis was optimized to improve the conversion rate to 2 folds.

Biphasic Enantioselective Fluorescent Recognition of Amino Acids by a Fluorophilic Probe

A fluorophilic fluorescent probe based on a perfluoroalkyl-substituted bis(binaphthyl) compound was designed and synthesized. It displayed a highly enantioselective fluorescence response toward structurally diverse amino acids in a biphasic fluorous/aqueous system with enantiomeric fluorescent enhancement ratio (ef; ΔID /ΔIL ) values up to 45.2 (histidine). It can be used to determine the enantiomeric compositions of amino acids and also allows the amino acid enantiomers to be visually discriminated. NMR and mass-spectroscopic investigations provided insights into the observed high enantioselectivity. This biphasic fluorescent recognition was used to determine the enantiomeric composition of the crude phenylalanine products generated by an enzyme-catalyzed asymmetric hydrolysis under various reaction conditions. The fluorous-phase-based fluorescence measurement under the biphasic conditions was able to minimize the interference of other reaction components and thus has potential in asymmetric reaction screening.

Increased Selectivity of Novozym 435 in the Asymmetric Hydrolysis of a Substrate with High Hydrophobicity Through the Use of Deep Eutectic Solvents and High Substrate Concentrations.

The effects of the reaction medium and substrate concentration were studied on the selectivity of Novozym 435 using the asymmetric hydrolysis of dimethyl-3-phenylglutarate as a model reaction. Results show that the use of choline chloride ChCl:urea/phosphate buffer 50% (v/v) as a reaction medium increased the selectivity of Novozym 435 by 16% (e.e = 88%) with respect to the one in 100% phosphate buffer (e.e = 76%). Best results were obtained when high substrate concentrations (well above the solubility limit, 27-fold) and ChCl:urea/phosphate buffer 50% (v/v) as reaction medium at pH 7 and 30 °C were used. Under such conditions, the R-monoester was produced with an enantiomeric purity of 99%. Novozym 435 was more stable in ChCl:urea/phosphate buffer 50% (v/v) than in phosphate buffer, retaining a 50% of its initial activity after 27 h of incubation at pH 7 and 40 °C. Results suggest that the use of deep eutectic solvents (ChCl:urea/phosphate buffer) in an heterogeneous reaction system (high substrate concentration) is a viable and promising strategy for the synthesis of chiral drugs from highly hydrophobic substrates.

Stereoselective ring-opening of styrene oxide at elevated concentration by Phaseolus vulgaris epoxide hydrolase, PvEH2, in the organic/aqueous biphasic system

To elevate the concentration of racemic styrene oxide (rac-SO), its asymmetric hydrolysis in organic/aqueous biphasic system was researched. Firstly, n-octane/phosphate buffer reaction system was constructed, where PvEH2 retained 75.9% activity at 25 °C for 8 h. Secondly, the optimal volume ratio of two phases and weight ratio of rac-SO to E. coli/pveh2 were 4:6 and 0.8. Then, the maximum allowable rac-SO concentration was assayed as 200 mM, 10-fold that in aqueous phase. Finally, the hydrolysis of rac-SO was performed, affording (R)-SO with over 99.5% ees and 47.8% yield, and (R)-phenyl-1,2-ethanediol with 93.6% eep and 50.4% yield in 12 h.

Efficient asymmetric hydrolysis of styrene oxide by Mung bean epoxide hydrolases in a supercritical CO2/buffer biphasic system

The asymmetric hydrolysis of styrene oxide (SO) by Mung bean epoxide hydrolases in a supercritical carbon dioxide (scCO2)/buffer biphasic system was investigated. With the addition of 0.80 M sodium bicarbonate, the yield of (R)-1-phenyl-1,2-ethanediol ((R)-PED) improved from 20.8% to 47.1%. Compared to the case of the aqueous monophasic system, the enantiomer excess (ee) of (R)-PED improved from 81.8% to 91.5%. Under the optimized conditions for the enzymatic reaction, i.e., at a pressure, temperature, SO concentration, and water content of 12 MPa, 40 °C, 80 mM, and 6.0% (v/v), respectively, an (R)-PED yield of 49.2% and ee of 94.1% were achieved. As the water content had a significant effect on the (R)-PED yield and ee, a stepwise reaction method was adopted. When the initial water content was 8.0% (v/v) and 1% (v/v) water was added at intervals of 1 h, the (R)-PED yield and ee reached 49.1% and 97.2%, respectively.

Structural investigation of the enantioselectivity and thermostability mechanisms of esterase RhEst1

The esterase RhEst1 can catalyze the asymmetric hydrolysis of ethyl (±)-2,2-dimethylcyclopropane carboxylate (DmCpCe), yielding a pharmaceutically relevant (S)-carboxylic acid. A triple mutant RhEst1A147I/V148F/G254A showed a 5-fold increase in the catalytic activity but a significant decrease in the enantioselectivity. Further optimization studies led to a new enzyme with an additional A143T mutation, which showed both increased catalytic activity and recovered enantioselectivity as well as improved thermostability. To reveal the detailed structural mechanisms for these improved properties, we performed all-atom molecular dynamics simulations on the wild type and two mutants A147I/V148F/G254A and A143T/A147I/V148F/G254A RhEst1, in complex with R-DmCpCe and S-DmCpCe substrates, respectively. The structural stability of the enzyme variants was investigated with the residue interaction network analysis. In RhEst1M2, S-DmCpCe was observed to adopt a more “activated” conformation than R-DmCpCe, with the active site residues better prearranged for the reaction, leading to the improved enantioselectivity towards S-DmCpCe. The mutations in the two mutants, especially A143T, could lead to different motion patterns in the cap domain, thus affecting the structure of the substrate entrance tunnel. The residue interaction networks analysis showed an increased number of interactions in RhEst1M1 and RhEst1M2 as compared to the wild type enzyme, especially the π-π stacking interactions between Phe148 and the nearby residues, providing an explanation for the increased thermostability of the two mutant enzymes observed experimentally. Our work provides essential molecular insights into the substrate binding, enantioselectivity and structural stability of esterase RhEst1, which will facilitate the development of more efficient RhEst1 variants for pharmaceutical applications.

Utilization of one novel deep-sea microbial protease sin3406-1 in the preparation of ethyl (S)-3-hydroxybutyrate through kinetic resolution.

One novel protease sin3406-1 was identified from Streptomyces niveus SCSIO 3406, which was isolated from the deep sea of the South China Sea, and heterologously expressed in E. coli BL21(DE3). Protease sin3406-1 was further used as a green biocatalyst in the kinetic resolution of racemic ethyl-3-hydroxybutyrate. After careful process optimization, chiral product ethyl (S)-3-hydroxybutyrate was generated with an enantiomeric excess of over 99% and a conversion rate of up to 50% through direct hydrolysis of inexpensive racemic ethyl-3-hydroxybutyrate catalyzed by sin3406-1. Interestingly, protease sin3406-1 exhibited the same enantio-preference as that of esterase PHE21 during the asymmetric hydrolysis of the ester bonds of racemic ethyl-3-hydroxybutyrate. Through mutation studies and molecular docking, we also demonstrated that the four residues close to the catalytic center, S85, A86, Q87 and Y254, played key roles in both the hydrolytic activity and the enantioselectivity of protease sin3406-1, possibly through forming hydrogen bonds between the enzyme and the substrates. Deep-sea microbial proteases represented by sin3406-1 are new contributions to the biocatalyst library for the preparation of valuable chiral drug intermediates and chemicals through enzymatic kinetic resolution.

The effect of deep eutectic solvents on the asymmetric hydrolysis of styrene oxide by mung bean epoxide hydrolases

BackgroundDeep eutectic solvents have attracted considerable attention in numerous fields. There is little information on mung bean epoxide hydrolase-catalyzed epoxides in deep eutectic solvent-containing system.ResultsAdding deep eutectic solvents with hydrogen bond donor of acids to phosphate buffer resulted in an obvious decrease in the optical purity of product; nevertheless, the relative slight change was observed by the addition of the deep eutectic solvents with hydrogen bond donor of alcohols and urea. Of the tested deep eutectic solvents, 10% additional amount of choline chloride/trithylene glycol can cause a significant improvement in the enantiopurity of product, from 83.2 ± 1.3% to 87.9 ± 0.3%. Moreover, with the increase in the addition amount of choline chloride/triethylene glycol from 10 to 30%, the enantiomeric excess of product enhanced from 87.9 to 94%, but a decline of product yield was observed. Finally, the evaluation of enzyme stability showed that the additional amount from 10% to 30% was not beneficial to the activity recovery.ConclusionIn short, adding choline chloride/triethylene glycol contributes to the improvement in the optical purity of (R)-1-phenyl-1, 2-ethanediol in the catalysis of styrene oxide by mung bean epoxide hydrolases; meanwhile, the enzyme immobilization could be essential.

Functional Characterization of a New Antarctic Microbial Esterase EST112-2 and Its Use in the Preparation of Chiral Tertiary Alcohol (S)-Linalool

Chiral tertiary alcohols (TAs) are key building blocks for the synthesis of many crucial flavor compounds and pharmaceuticals. The two enantiomers of tertiary alcohol, linalool, differ in odor. So, sustainable strategies for the manufacture of optically pure TAs represented by linalool, are highly desirable. But the enzymatic synthesis of chiral tertian' alcohols through kinetic resolution was not easily achieved, possibly because of the steric hindrance from the chemical structures of tertiary alcohols. Herein, we identified and functionally characterized a new microbial esterase EST112-2 from the antarctic sediments and utilized esterase EST112-2 as a green biocatalyst in the synthesis of chiral tertiary alcohol (S)-linalool through asymmetric hydrolysis of racemic linalyl acetate. Parameters such as pH, temperature, co-solvents, substrate concentrations, enzyme loading and reaction time were optimized for the kinetic resolutions. Desired chiral product (S)-linalool was finally obtained with an enantiomeric excess of over 66% and a yield of over 72% after process optimization. The enantiomeric excess of (S)-linalool prepared by esterase EST112-2 was much higher than that from previous reports.