Enantioselective Biotransformation Research Articles

Enzymatic Kinetic Resolution of Racemic 1-(Isopropylamine)-3-phenoxy-2-propanol: A Building Block for β-Blockers.

This study aimed to optimize the kinetic resolution of building blocks for the synthesis of β-blockers using Candida rugosa lipases, which could be potentially used to synthesize enantiomerically pure β-blockers further. Reaction mixtures were incubated in a thermostated shaker. Qualitative and quantitative analyses of the reaction mixtures were performed using chiral stationary phases and the UPLC-IT-TOF system. Of the 24 catalytic systems prepared, a system containing lipase from Candida rugosa MY, [EMIM][BF4] and toluene as a two-phase reaction medium and isopropenyl acetate as an acetylating agent was optimal. This resulted in a product with high enantiomeric purity produced via biotransformation, whose enantioselectivity was E = 67.5. Using lipases from Candida rugosa enables the enantioselective biotransformation of the β-blockers building block. The biocatalyst used, the reaction environment, and the acetylating agent significantly influence the efficiency of performer kinetic resolutions. The studies made it possible to select an optimum system, a prerequisite for obtaining a product of high enantiomeric purity. As a result of the performed biotransformation, the (S)-enantiomer of the β-blocker derivative was obtained, which can be used to further synthesize enantiomerically pure β-blockers.

Development of enantioselective high-performance liquid chromatography-tandem mass spectrometry method for quantitative determination of methylone and some of its metabolites in oral fluid

In the present study an enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the first time for quantitative determination of the recreational drug of abuse methylone and its major metabolites in oral fluid. The simultaneous chemo- and enantioseparation of methylone and its major metabolites was performed on a polysaccharide-based chiral column based on amylose tris(5-chloro-3-methylphenylcarbamate) as chiral selector (Lux i-Amylose-3) with methanol containing 0.4 % (v/v) aqueous ammonium hydroxide as mobile phase. The time required for enantioselective analysis of methylone and its 2 major metabolites was 15 min. This method was fully validated following the Organization of Scientific Area Committees (OSAC) for Forensic Science guidelines.This method was applied for the enantioselective determination of methylone and its metabolites in oral fluid and enantioselectivity in metabolism and pharmacokinetic of the parent compound and metabolites was observed.While the first enantiomer of methylone was found at higher concentration, both metabolites shown greater concentration for the second enantiomer. The results revealed that MET undergoes an enantioselective biotransformation to its metabolites HMMC and MDC, with S-(−)-MET more rapidly metabolized and eliminated from the body.

Bio-Approach for Obtaining Enantiomerically Pure Clopidogrel with the Use of Ionic Liquids.

Clopidogrel is a chiral compound widely used as an antiplatelet medication that lowers the risk of blood clots, strokes, and heart attacks. The main aim of the study presented herein was to obtain (S)-clopidogrel, which is commercially available in treatments, via the kinetic resolution of racemic clopidogrel carboxylic acid with the use of lipase from Candida rugosa and a two-phase reaction medium containing an ionic liquid. For this purpose, the enantioselective biotransformation of clopidogrel carboxylic acid and chiral chromatographic separation with the use of a UPLC-MS/MS system were optimized. The best kinetic resolution parameters were obtained by using a catalytic system containing lipase from Candida rugosa OF as a biocatalyst, cyclohexane and [EMIM][BF4] as a two-phase reaction medium, and methanol as an acyl acceptor. The enantiomeric excess of the product was eep = 94.21% ± 1.07 and the conversion was c = 49.60% ± 0.57%, whereas the enantioselectivity was E = 113.40 ± 1.29. The performed study proved the possibility of obtaining (S)-clopidogrel with the use of lipase as a biocatalyst and a two-phase reaction medium containing an ionic liquid, which is in parallel with green chemistry methodology and does not require environmentally harmful conditions.

The Utilization of Two-Phase Catalytic System in Enantioselective Biotransformation of Racemic Atenolol

There are several methods that allow enantiomerically pure compounds to be obtained. In the study presented herein, the enantioselective biotransformations of (R,S)-atenolol were performed with the use of various catalytic systems containing ionic liquids and toluene as a reaction medium, vinyl acetate as an acetylating agent as well as lipases from Candida rugosa. The conducted studies profs that, the use of the two-phase reaction system enables the reuse of the biocatalyst in another cycle and allows to achieve satisfactory kinetic resolution parameters.

Deracemization of 1-phenylethanols in a one-pot process combining Mn-driven oxidation with enzymatic reduction utilizing a compartmentalization technique.

Racemic 1-phenylethanols were converted into enantiopure (R)-1-phenylethanols via a chemoenzymatic process in which manganese oxide driven oxidation was coupled with enzymatic biotransformation by compartmentalization of the reactions, although the two reactions conducted under mixed conditions are not compatible due to enzyme deactivation by Mn ions. Achiral 1-phenylethanol is oxidized to produce acetophenone in the interior chamber of a polydimethylsiloxane thimble. The acetophenone passes through the membrane into the exterior chamber where enantioselective biotransformation takes place to produce (R)-1-phenylethanol with an enantioselectivity of >99% ee and with 96% yield. The developed sequential reaction could be applied to the deracemization of a wide range of methyl- and chloro-substituted 1-phenylethanols (up to 93%, >99% ee). In addition, this method was applied to the selective hydroxylation of ethylbenzene to afford chiral 1-phenylethanol.

Newly elected Chinese Academy of Sciences academicians in chemistry division in 2021

Newly elected Chinese Academy of Sciences academicians in chemistry division in 2021

Significance of biotransformation and excretion on the enantioselective bioaccumulation of hexabromocyclododecane (HBCDD) in laying hens and developing chicken embryos

Although (–)-α-hexabromocyclododecane (HBCDD) and (+)-γ-HBCDD are preferentially enriched in chickens, the key factors contributing to their selective bioaccumulation in hens and their potential biotransformation in developing chicken embryos remain unclear. Herein, in vivo and in ovo exposure experiments using hens and fertilized eggs were conducted to investigate the absorption, excretion, and biotransformation of HBCDDs in chickens. γ-HBCDD (76%) exhibited a higher absorption efficiency than α- (22%) and β- (69%) HBCDDs. However, α-HBCDD was dominant in hen tissues, although γ-HBCDD accounted for >75% in the spiked feed. Moreover, chicken embryos biotransformed approximately 9.5% and 11.7% of absorbed α- and γ-HBCDDs, respectively, implying that diastereomer-selective elimination causes the predominance of α-HBCDD in hens. The concentration and enantiomer fraction (EF) of α-HBCDD in laid eggs were significantly positively correlated, suggesting enantioselective elimination. The EFs of α- and γ-HBCDDs varied between feces from the exposure and depuration periods, indicating the preferred excretion of (+)-α- and (–)-γ-HBCDDs. Furthermore, the enantioselective biotransformation of (–)-γ-HBCDD was confirmed in developing chicken embryos. These results show that excretion and biotransformation contribute to the diastereomer- and enantiomer-selective bioaccumulation of HBCDDs in chickens; The results may improve our understanding of the environmental fate and ecological risks of HBCDDs in biota.

Overview: Enzyme-catalyzed Enantioselective Biotransformation of Chiral Active Compounds Used in Hypertension Treatment

Enzymatic kinetic resolution is one of the methods which allows for the synthesis of enantiomerically pure various active pharmaceutical ingredients. In contrast to chemical routes, enzymatic reactions have characteristics, including mild reaction conditions, a few byproducts, and relatively high activity of the used enzymes. β-adrenolytic drugs are widely used in the treatment of hypertension and cardiovascular disorders. Due to the fact that β- blockers possess an asymmetric carbon atom in their structure, they are presented in two enantiomeric forms. It was reported by many studies that only the (S)-enantiomers of these drugs possess the desired therapeutic effect, whereas the administration of the racemate may cause dangerous side effects, such as bronchoconstriction or diabetes. Nevertheless, β- blockers are still commercially available drugs mainly used in medicine as racemates, whereas there are several methods that are widely used in order to obtain enantiomerically pure compounds.

Application of the redox system of Nocardia corallina B-276 in the enantioselective biotransformation of ketones and alcohols

The aim of this research was to evaluate the redox system of Nocardia corallina B-276 in the biotransformation of 1-phenyl-1-propanone (1a), 2-hydroxy-1-phenylethanone (2a) and methyl (2-chlorophenyl)(oxo)acetate (3a) into 1-phenylpropan-1-ol (1b), 1-phenyl-1,2-ethanediol (2b) and methyl (2-chlorophenyl)(hydroxy)acetate (3b). The biomass of N. corallina was obtained in a liquid medium with an initial pH of 8.50, but the pH changed during the 96 h of the culture media, the final pH was between 4.74 and 7.62. The N. corallina biomass biocatalyzed the enantioselective reduction of 1a?3a to the corresponding alcohols. Whereas, during the process of oxidation of the rac-alcohols 1b?3b, 1b was oxidized in enantioselective way, the oxidation of 2b was not selective, but 3b was biotransformed mainly to (R)-3b. These results are indicative that N. corallina produced reductases and oxidases, whereby the biocatalytic activity was influenced by the final pH of the culture media, the reaction time and structure of the substrate.

Use of Candida rugosa lipase as a biocatalyst for L-lactide ring-opening polymerization and polylactic acid production

Abstract Candida rugosa lipase (CRL, EC Number: 232–619-9) is a stable enzyme, and its use is relatively widespread in the field of biotechnology, especially in hydrolysis reactions, esterification, transesterification and enantioselective biotransformation. This study examined the role of CRL as a biocatalyst for L-lactide ring-opening polymerization in the production of metal-free polylactic acid (PLA). The ring-opening polymerization reaction was conducted at various temperatures (70, 90, 110 and 130 °C) and CRL concentrations (1, 2, 3 (%w/w)). The results revealed a strong relationship between PLA formation and CRL activity. The highest CRL activity was obtained in ring-opening polymerization of L-lactide at a temperature of 90 °C and concentration of 2% (w/w). Under these conditions, the weight-average molecular weight (Mw) and number-average molecular weight (Mn) of PLA analyzed by gel permeation chromatography was highest at 5428 and 2854 g/mol respectively, with a yield of 93% and the enzyme activity 0.39 U. The polymerization of lactide at 90 °C occurred only in the presence of the catalyst. The crystallinity and melting point of PLA were 31% and 120 °C, respectively. Scanning electron microscopy (SEM) analysis of the morphology of PLA revealed smooth and uniform pores in each region, with mass percentage of the elements carbon (C) and oxygen (O) of 52% and 48%, respectively.

Diastereoisomer-Specific Biotransformation of Hexabromocyclododecanes by a Mixed Culture Containing Dehalococcoides mccartyi Strain 195.

Hexabromocyclododecane (HBCD) stereoisomers may exhibit substantial differences in physicochemical, biological, and toxicological properties. However, there remains a lack of knowledge about stereoisomer-specific toxicity, metabolism, and environmental fate of HBCD. In this study, the biotransformation of (±)α-, (±)β-, and (±)γ-HBCD contained in technical HBCD by a mixed culture containing the organohalide-respiring bacterium Dehalococcoides mccartyi strain 195 was investigated. Results showed that the mixed culture was able to efficiently biotransform the technical HBCD mixture, with 75% of the initial HBCD (∼12 μM) in the growth medium being removed within 42 days. Based on the metabolites analysis, HBCD might be sequentially debrominated via dibromo elimination reaction to form tetrabromocyclododecene, dibromocyclododecadiene, and 1,5,9-cyclododecatriene. The biotransformation of the technical HBCD was likely diastereoisomer-specific. The transformation rates of α-, β-, and γ-HBCD were in the following order: α-HBCD > β-HBCD > γ-HBCD. The enantiomer fractions of (±)α-, (±)β-, and (±)γ-HBCD were maintained at about 0.5 during the 28 days of incubation, indicating a lack of enantioselective biotransformation of these diastereoisomers. Additionally, the amendment of another halogenated substrate tetrachloroethene (PCE), which supports the growth of strain 195, had a negligible impact on the transformation patterns of HBCD diastereoisomers and enantiomers. This study provided new insights into the stereoisomer-specific transformation patterns of HBCD by anaerobic microbes and has important implications for microbial remediation of anoxic environments contaminated by HBCD using the mixed culture containing Dehalococcoides.

Kinetic resolution of a b-adrenolytic drug with the use of lipases as enantioselective biocatalysts

The study presented herein is focused on optimisation of the kinetic resolution of racemic propranolol by screening acetylating agent, reaction medium, and enantioselective biocatalysts. The effects of acetylating agent and reaction medium on the efficiency of performed enantioselective biotransformation were inves-tigated. Furthermore, the catalytic activities on nine commercially available lipases were tested. Finally, the composition of reaction medium was selected, based on previously obtained results, providing the most efficient process of kinetic resolution of racemic propranolol.

Enantioselective biotransformation of sterically hindered amine substrates by the fungus Stemphylium lycopersici.

To screen 20 micro-organisms for ω-transaminase (ω-TA) activity by the kinetic resolution of rac-1-phenylethylamine, followed by testing rac-amines of pharmaceutical interest with bulky substituents and to conduct the asymmetric synthesis of a chiral amine. Stemphylium lycopersici was selected as the best biocatalyst. By the central composite rotatable design (CCRD), it was found that, at lower pH (5·5 and 6·5), the lyophilized micro-organism biocatalysed the kinetic resolution of rac-1-phenylethylamine with 99% enantiomeric excess (e.e.) ((R)-enantiomer) with acetophenone conversions ranged from 41 to 45%. Interestingly, the lyophilized crude enzymatic extract lead to better results at pH from 7·0 to 9·0, with conversions up to 47% and about 99% e.e. We also attested that as much as higher is the pyruvate (amino acceptor) concentration, higher is the acetophenone conversion, corroborating the presence of ω-TA-type enzymes. Among different sterically hindered racemic amines tested, rac-1,2,3,4-tetrahydro-1-naphthylamine and rac-phenylbutylamine were satisfactorily kinetically resolved in up to 91% e.e. (R). The results for the asymmetric synthesis showed excellent conversion (>85%) for the S-1-phenylethylamine, indicating (S)-stereopreference. Stemphylium lycopersici showed to be an important tool for broader substrate scope transaminases and a relevant player on the development of new biocatalysts with ability in asymmetric synthesis reactions. Here in, we contribute to the improvement of the biocatalytic toolbox for chiral amines synthesis. Interestingly, we have found that the crude enzymatic extract of the endophytic fungus S. lycopersici could accept bulky substrates with reasonable activity, compared to the wild-type transaminase already published over literature, and with high enantioselectivity.

Microbiological bio-reduction of prochiral carbonyl compounds by antimycotic agent Boni Protect

The selective properties of the fungus Aureobasidium pullulans, in the antifungal agent Boni Protect, were investigated in the fermentative bioreduction of selected carbonyl compounds. Catalyzed by oxidoreductases contained in the microorganism Aureobasidium pullulans highly enantioselective biotransformation of prochiral ketones provides the secondary alcohols when the reaction is done in the presence of specific additives. Aureobasidium pullulans has also proved to be an effective bioreagent in the reduction of α- and β-keto esters. Optically pure hydroxy esters were obtained under fermentation conditions without the use of additives.

Enantioselective Biotransformation of Chiral Persistent Organic Pollutants.

Enantiomers of chiral compounds commonly undergo enantioselective transformation in most biologically mediated processes. As chiral persistent organic pollutants (POPs) are extensively distributed in the environment, differences between enantiomers in biotransformation should be carefully considered to obtain exact enrichment and specific health risks. This review provides an overview of in vivo biotransformation of chiral POPs currently indicated in the Stockholm Convention and their chiral metabolites. Peer-reviewed journal articles focused on the research question were thoroughly searched. A set of inclusion and exclusion criteria were developed to identify relevant studies. We mainly compared the results from different animal models under controlled laboratory conditions to show the difference between enantiomers in terms of distinct transformation potential. Interactions with enzymes involved in enantioselective biotransformation, especially cytochrome P450 (CYP), were discussed. Further research areas regarding this issue were proposed. Limited evidence for a few POPs has been found in 30 studies. Enantioselective biotransformation of α-hexachlorocyclohexane (α-HCH), chlordane, dichlorodiphenyltrichloroethane (DDT), heptachlor, hexabromocyclododecane (HBCD), polychlorinated biphenyls (PCBs), and toxaphene, has been investigated using laboratory mammal, fish, bird, and worm models. Tissue and excreta distributions, as well as bioaccumulation and elimination kinetics after administration of racemate and pure enantiomers, have been analyzed in these studies. Changes in enantiomeric fractions have been considered as an indicator of enantioselective biotransformation of chiral POPs in most studies. Results of different laboratory animal models revealed that chiral POP biotransformation is seriously affected by chirality. Pronounced results of species-, tissue-, gender-, and individual-dependent differences are observed in in vivo biotransformation of chiral POPs. Enantioselective biotransformation of chiral POPs is dependent on enzyme amounts and activities. However, the role of cytochrome P450 in enantioselective biotransformation has not yet been confirmed. Currently available data on biotransformation of chiral POPs provide a preliminary understanding of the fate of chiral compounds in organisms. Further detailed studies of species-dependent biotransformation pathway and molecular mechanism in various animal models should be performed to comprehensively understand chiral POP biotransformation.

Efficient synthesis of (S)-epichlorohydrin in high yield by cascade biocatalysis with halohydrin dehalogenase and epoxide hydrolase mutants

Enantioselective biotransformation of prochiral 1,3-DCP by halohydrin dehalogenases (HHDHs) is particularly attractive since 100% yield of chiral epichlorohydrin (ECH) may be obtained. HheC mutant (P175S/W249P) displayed greatly improved enantiomeric excess (ee) of (S)-ECH from 5% to 95.3% in the catalyzed dehalogenation of 1,3-DCP at pH8.0. (S)-ECH was enantioselectively biotransformed from 40mM 1,3-DCP with 92.3% ee and 93.2% yield at pH10.0. To increase the ee of (S)-ECH, the catalysis was carried out using HheC mutant coupled with epoxide hydrolase mutant and the maximum yield and ee of (S)-ECH reached 91.2% and >99%.

Polychlorinated biphenyls (PCBs) and polybrominated biphenyl ethers (PBDEs) in environmental samples from Ny-Ålesund and London Island, Svalbard, the Arctic

Polychlorinated biphenyls (PCBs) and polybrominated biphenyl ethers (PBDEs) were determined in environmental samples collected from Ny-Ålesund and London Island, Svalbard, the Arctic. Total PCB concentrations (∑25PCBs) varied from 0.57 to 2.52ngg−1 dry weight (dw) in soil, 0.30 to 1.16ngg−1dw in plants and 0.56 to 0.98ngg−1dw in reindeer dung. The non-Aroclor congener of CB-11 was predominant in most samples compared to other congeners, accounting for 16.0±9.8% to the ∑25PCBs. The ∑13PBDEs concentrations were 1.7–416, 36.7–495 and 28.1–104pgg−1dw in soil, plants and reindeer dung, respectively. The signature of enantioselective biotransformation was observed in all samples for chiral CB-95, whereas in parts of samples for other chiral PCBs. Bioaccumulation factors (BAFs) in six plant species varied within individual contaminant congeners and plant species, with BAFs less than 1 for ∑PCBs and higher than 1 for ∑PBDEs. BAF values decreased with increasing soil concentrations, suggesting that high background levels in soil restricted the accumulation of these contaminants by plants.

A new enantioselective CE method for determination of oxcarbazepine and licarbazepine after fungal biotransformation.

The present work describes, for the first time, the simultaneous separation of oxcarbazepine (OXC) and its active metabolite 10-hydroxy-10,11-dihydrocarbamazepine (licarbazepine, Lic) by chiral CE. The developed method was employed to monitor the enantioselective biotransformation of OXC into its active metabolite by fungi. The electrophoretic separations were performed using 10 mmol/L of a Tris-phosphate buffer solution (pH 2.5) containing 1% w/v of β-CD phosphate sodium salt (P-β-CD) as running electrolyte, -20 kV of applied voltage and a 15°C capillary temperature. The method was linear over the concentration range of 1000-30 000 ng/mL for OXC and 75-900 ng/mL for each Lic enantiomer (r ≥ 0.9952). Within-day precision and accuracy evaluated by RSD and relative errors, respectively, were lower than 15% for all analytes. The validated method was used to evaluate the enantioselective biotransformation of OXC, mediated by fungi, into its active metabolite Lic. This study showed that the fungi Glomerella cingulata (VA1) and Beuveria bassiana were able to enantioselectively metabolize the OXC into Lic after 360 h of incubation. Biotransformation by the fungus Beuveria bassiana showed 79% enantiomeric excess for (S)-(+)-Lic, while VA1 gave an enantiomeric excess of 100% for (S)-(+)-Lic. This study opens a new route to the drug (S)-(+)-licarbazepine.

Elevated Levels of Polychlorinated Biphenyls in Plants, Air, and Soils at an E-Waste Site in Southern China and Enantioselective Biotransformation of Chiral PCBs in Plants

E-waste that contains polychlorinated biphenyls (PCBs) is moved across national boundaries, often from industrialized countries in the northern hemisphere, where the items were formerly used, to subtropical and tropical regions in southeastern Asia and Africa. As a result, there is a high likelihood that PCBs will be released into the environment from a primary source due to the elevated temperatures encountered in these low-latitude regions. In the present study, PCBs and enantiomer fractions (EFs) of chiral PCBs (PCB 84, 95, 132, 136, 149, and 183) were analyzed in air, eucalyptus leaves, pine needles, and soil at an e-waste site and a rural site in southern China. The concentrations of PCBs at the e-waste site ranged from 7825 to 76330 pg/m(3), 27.5 to 1993 ng/g, and 24.2 to 12045 ng/g in the air (gas plus particle), plant leaves, and soils, respectively. The atmospheric PCB composition profiles in the present study indicated relatively high abundances of penta- and hexa-PCBs, which were different from those previously observed in the air across China. The Clausius-Clapeyron regression analysis indicated that evaporation from local contaminated surfaces constitutes a primary emission source of PCBs in the air at the e-waste site. The chiral signatures of PCBs in the air at the e-waste site were essentially racemic (mean EFs = (0.484 ± 0.022)-(0.499 ± 0.004) in the gaseous phase) except for PCB 84 (0.420 ± 0.050), indicating that racemic sources dominate the PCB emission in the air. PCB chiral signatures in the soils ((0.422 ± 0.038)-(0.515 ± 0.016)) were similar to those in the air except for PCB 95. However, the chiral PCBs in the plants (especially the eucalyptus leaves) had significantly nonracemic residues ((0.368 ± 0.075)-(0.561 ± 0.045)) compared to those in the air and soil. This finding suggests that enantioselective biotransformation of these atropisomeric PCBs was very likely to occur in the plant leaves, possibly due to metabolism by cytochrome P-450 enzymes in leaves. To our knowledge, this is the first report on the enantioselective metabolism of chiral PCBs in plants under field conditions.

Enantioselective Biotransformation of Hexabromocyclododecane by in Vitro Rat and Trout Hepatic Sub-Cellular Fractions

α-, β-, and γ-Hexabromocyclododecanes (HBCDs) were subjected to in vitro biotransformation experiments with rat and trout liver S9 fractions for different incubation times (10, 30, and 60 min) at 2 concentration levels (1 and 10 μM). The metabolic degradation of target HBCDs followed first order kinetics. Whereas β-HBCD undergoes rapid biotransformation (t0.5 = 6.4 and 38.1 min in rat and trout, respectively), α-HBCD appears the most resistant to metabolic degradation (t0.5 = 17.1 and 134.9 min). The biotransformation rate in trout was slower than in rat. Investigation of HBCD degradation profiles revealed the presence of at least 3 pentabromocyclododecene (PBCD) and 2 tetrabromocyclododecadiene (TBCD) isomers indicating reductive debromination as a metabolic pathway for HBCDs. Both mono- and di- hydroxyl metabolites were identified for parent HBCDs, while only mono hydroxyl metabolites were detected for PBCDs and TBCDs. Interestingly, δ-HBCD was detected only in trout S9 fraction assays indicating metabolic interconversion of test HBCD diastereomers during biotransformation in trout. Finally, enantioselective analysis showed significant enrichment of the (-)-α-HBCD enantiomer (EF = 0.321 and 0.419 after 60 min incubation in rat and trout, respectively). The greater enrichment of (-)-α-HBCD in rat than in trout underlines the species-specific differences in HBCD metabolism and the need for caution when extending similar results from animal studies to humans.