Chiral Metabolites Research Articles

Advances in Chiral Metabolomic Profiling and Biomarker Discovery.

Chiral metabolomics entails the enantioselective measurement of the metabolome present in a biological system. Over recent years, it has garnered significant interest for its potential in discovering disease biomarkers and aiding clinical diagnostics. D-Amino acids and D-hydroxy acids, traditionally overlooked as unnatural, are now emerging as novel signaling molecules and potential biomarkers for a range of metabolic disorders, brain diseases, kidney disease, diabetes, and cancer. Despite their significance, simultaneous measurements of multiple classes of chiral metabolites in a biological system remain challenging. Hence, limited information is available regarding the metabolic pathways responsible for synthesizing D-amino/hydroxy acid and their associated pathophysiological mechanisms in various diseases. Capitalizing on recent advancements in sensitive analytical techniques, researchers have developed various targeted chiral metabolomic methods for the analysis of chiral biomarkers. Here, we highlight the pivotal role of chiral metabolic profiling studies in disease diagnosis, prognosis, and therapeutic interventions. Furthermore, we describe cutting-edge chromatographic and mass spectrometry methods that enable enantioselective analysis of chiral metabolites. These advanced techniques are instrumental in unraveling the complexities of disease biomarkers, contributing to the ongoing efforts in disease biomarker discovery.

A dried sweat spot paper (DSSP) method based on novel mass spectrometry probe labeling for detection and resolution of DL-lactate enantiomers as potential biomarkers for diabetes mellitus

BackgroundHuman sweat can be collected non-invasively with low infectivity; however, its application as a determination method has been challenged due to the presence of trace amounts of chiral metabolites. Moreover, its application as a biological fluid for disease diagnosis has not been previously reported. In this study, the human dried sweat spot paper (DSSP) method was proposed for the derivatization of a novel mass spectrometric chiral probe, N-[1-Oxo-5-(triphenylphosphonium) pentyl]-(S)-3-aminopyrrolidine (OTPA), determination and resolution of DL-lactic acid (DL-LA) enantiomers in human elbow sweat. ResultsThe methodological validation revealed the resolution (Rs) as 1.78, the limit of detection (S/N = 3) as 20.83 fmol, good linearity (R2 ≥ 0.9996), and the intra-day and intra-day stability with RSD ranging from 0.53 to 10.85 %, while the average recovery rate of D-LA and L-LA were 104.00 % ± 4.68 % and 107.41 % ± 8.34 %, respectively, with high accuracy. In addition, the method was applied for the determination of DL-LA in the sweat on elbow of 10 healthy volunteers and 30 diabetic patients. The results demonstrated that the D/L ratio and L/D ratio were significantly different (p < 0.0001). In addition, a moderate positive linear correlation between the D/L-LA ratio in human sweat and fasting blood glucose level (r = 0.7744, p < 0.0001) was observed, thereby suggesting that the D/L ratio of lactate in human sweat correlate the glucose level in human fasting blood. Significance and noveltyThe D/L lactate ratio in human sweat could be used as a potential biomarker for diabetes screening. The method can be used to screen for diabetes by providing a dry sweat paper to test equipment and has the potential to be a non-invasive early-warning diagnostic tool for diabetes.

Enantiomeric profile of promethazine in metabolic studies in liver microsomes

Promethazine (PMZ) is metabolized in the liver giving rise to chiral metabolites, including promethazine sulfoxide (PMZSO) and desmonomethyl promethazine (DMPMZ). Other metabolites, such as dioxopromethazine (DOPMZ), hydroxy promethazine (PMZOH), N-desmethyl-promethazine sulfoxide, and promethazine N-oxide (PMZNO) can also be formed, but information about them is limited. In this work, the enantiomeric metabolic profile of PMZ in ultra-pool human liver microsomes (HLM) was evaluated. For that, a novel enantioselective analytical method, by liquid chromatography coupled with high-resolution mass spectrometry was established and employed to monitor the enantiomers of PMZ and the formation of its main metabolites PMZSO and DMPMZ in two-hours assay with HLM. The enantioseparation optimized conditions were achieved with two immobilized carbamate amylose-based (3‑chloro-5-methylphenylcarbamate) columns: Lux® 3 µm i-Amylose-3 and Chiralpak® IG-U 1.6 µm. The optimized mobile phase for enantioseparation used buffer and ethanol, a green organic solvent, in a low flow rate. The sample preparation was based only in liquid-liquid extraction. (R)-PMZ consistently exhibited higher concentrations than (S)-PMZ, indicating less extension in metabolization. Regarding the metabolites, PMZSO exhibited higher concentrations compared to DMPMZ. A higher concentration for (S)-PMZSO was found when compared to the (R)-PMZSO and (R)-DMPMZ showing higher concentration compared to (S)-DMPMZ, indicating enantioselectivity in the metabolization process. The metabolites, PMZOH or PMZNO and DOPMZ were also identified. The second enantiomer of DOPMZ also showed a higher proportion than the first eluted enantiomer. These results demonstrated enantioselectivity of liver metabolism of PMZ, as well as confirmed PMZOH, PMZNO, and DOPMZ metabolites formation.

Chiral Hydroxy Metabolite of Mebendazole: Analytical and Semi-Preparative High-Performance Liquid Chromatography Resolution and Chiroptical Properties.

Mebendazole (MBZ) is a benzimidazole carbamate anthelmintic used worldwide for the treatment and prevention of parasitic disorders in animals and humans. A large number of in vivo and in vitro studies have demonstrated that MBZ also has anticancer activity in multiple types of cancers. After oral administration, the phenylketone moiety of MBZ is rapidly reduced to the hydroxyl group to form the chiral hydroxy metabolite (MBZ-OH). To the best of our knowledge, there is no information in the literature on the stereochemical course of transformation and the anthelmintic and antitumor activity of individual enantiomers of MBZ-OH. In the present study, we describe in detail the direct HPLC resolution of MBZ-OH on a 100 mm × 4.6 mm Chirapak IG-3 column packed with 3 μm silica particles containing amylose (3-chloro-5-methylphenylcarbamate) as a selector. At 25 °C and using pure methanol as the mobile phase, the enantioseparation and resolution factors were 2.38 and 6.13, respectively. These conditions were scaled up at a semi-preparative scale using a 250 mm × 10 mm Chiralpak IG column to isolate multi-milligram amounts of both enantiomeric forms of the chiral metabolite. The chiroptical properties of the collected enantiomers were determined and, through a theoretical study, were related to the more stable conformations of MBZ-OH. The first and second eluted enantiomers were dextrorotatory and levorotatory, respectively, in dimethylformamide solution. Finally, by recording the retention factors of the enantiomers as the water content in the water-acetonitrile mobile phases was progressively varied, U-shaped retention maps were generated, indicating a dual and competitive hydrophilic interaction liquid chromatography and reversed-phase liquid chromatography retention mechanism on the Chirapak IG-3 chiral stationary phase.

Chemo- and enantioselective analysis of high hydrophilic organophosphate pesticides by liquid chromatography under different elution modes

Acephate and malathion are prevalent chiral organophosphate insecticides, giving rise to more toxic chiral metabolites such as methamidophos and malaoxon, respectively. Enantioselective separation of chiral pesticides is crucial due to the differing pharmacological, environmental, and toxicological profiles of enantiomers. This study systematically evaluates an enantio‑ and chemoselective performance of eight polysaccharide-based chiral columns (Chiralcel OB-H, Chiralcel OD-H, Chiralcel OJ-R, Chiralpak AD-H, Chiralpak AS-H, Chiralpak IG-3, Chiralpak IK-3, and Lux-Amylose-2) and ten mobile phases across multimodal elution (normal-phase, polar organic, and reversed-phase conditions). Within the 80 combinations per analyte, the chiral screening highlights specific enantioselectivity trends concerning hydrophilic and hydrophobic compounds when utilizing amylose- or cellulose-based polymers under different conditions. Notably, a set of columns including IK-3, OJ-R, AS-H, and AD-H demonstrated superior coverage, achieving at least a 62 % success rate for enantioseparation. For the resolution between the pairs acephate/methamidophos and malathion/malaoxon, where no further optimizations were undertaken, the success rate for enantio‑ and chemoselectivity was 10 % and 30 %, respectively.

Synergetic Multichiral Covalent Organic Framework for Enantioselective Recognition and Separation.

In enantiomer recognition and separation, a highly enantioselective approach with universal applicability is urgently desired but hard to realize, especially in the case of chiral molecules. To resolve the trade-off between enantioselectivity and universality, a glutathione (GSH) and methylated cyclodextrins (MCD)-functionalized covalent organic framework (GSH-MCD COF) with porosity and abundant chiral surfaces is presented that was designed and synthesized for recognition and separation of various enantiomers. As expected, the GSH-MCD COF can be used as chiral stationary phases for the separation of various enantiomers, including aromatic alcohols, aromatic acids, amides, amino acids, and organic acids, with performance and versatility even superior to some widely used commercial chiral chromatographic columns. Furthermore, the synthesized GSH-MCD COF shows high enantioselectivity for the rapid recognition and identification of enantiomers and chiral metabolites when coupling to Raman spectroscopy. Molecular simulations suggest that the COF provides a confined microenvironment for cyclodextrins and peptides that dictates the separation and recognition capability. This work provides a strategy to synthesize synergetic multichiral COF and achieve separations and recognitions of enantiomers in complex samples.

Determination of chiral prothioconazole and its chiral metabolite in water, juice, tea, and vinegar using emulsive liquid–liquid microextraction combined with ultra-high performance liquid chromatography

Emulsive liquid–liquid microextraction (ELLME), a simple, rapid, and environmentally friendly technique, was established to identify chiral prothioconazole and its chiral metabolite in water, juice, tea, and vinegar using ultra-high-performance liquid chromatography (UPLC). Environmentally friendly extractant was mixed with pure water to prepare a high-concentration emulsion, which was added to samples to complete the emulsification and extraction in 1 s. Afterward, an electrolyte solution was added to complete the demulsification without centrifugation. ELLME did not use dispersants compared to the familiar dispersive liquid–liquid microextraction (DLLME), thus reducing the use of toxic solvents and avoiding the effect of dispersants on the partition coefficient. The linear range was from 0.01 to 1 mg/L. The limit of detection was 0.003 mg/L. The extraction recoveries ranged from 82.4 % to 101.6 %, with relative standard deviations of 0.7–5.2 %. The ELLME method developed has the potential to serve as an alternative to DLLME.

Residue Behavior of Chiral Fungicide Prothioconazole and Its Major Chiral Metabolite in Flour Product Processing.

This study systematically investigates the stereoselective metabolism and residue behavior of chiral pesticide prothioconazole enantiomers during the steaming, baking, and frying of steamed buns, bread, and deep-fried dough sticks. The results show that steaming, baking, and frying can significantly promote the degradation of the prothioconazole enantiomers. In low- and high-concentration treatments, the degradation rates of prothioconazole enantiomers were over 96.0% and 45.4%, respectively, and the residual concentration of prothioconazole-desthio enantiomers was less than 32.7 μg/kg (excluding fried processing). During the processing of steamed buns, bread, and deep-fried dough sticks, the enantiomer fraction (EF) value of the prothioconazole enantiomer was close to 0.5, and the stereoselectivity was not significant. During the processing of steamed buns (low concentration), bread (low and high concentrations), and deep-fried dough sticks (low concentration), the stereoselectivity of prothioconazole-desthio was significant, and preferential enantiomer degradation occurred. Following the analysis of 120 flour product samples, the residual risk.

Metabolic Transformation of Gentiopicrin, a Liver Protective Active Ingredient, Based on Intestinal Bacteria.

Gentiopicrin, the main component of the famous Chinese patent medicine Long Dan Xie Gan Wan, has the characteristics of fast absorption in vivo and low bioavailability. Intestinal bacteria play an important role in the absorption and pharmacokinetics of oral drugs. In this study, the metabolic transformation of gentiopicrin by intestinal bacteria was examined. High-performance liquid chromatography coupled with ion trap time-of-flight mass spectrometry (LC/MSn-IT-TOF) and nuclear magnetic resonance (NMR) were used, and six metabolites were identified, including reduction products (G-M1, G-M2, G-M4, and G-M6), a hydrolytic product (G-M3), and a dehydration product (G-M5) of gentiopicrin aglycone after hydrolysis, reduction, and dehydration reactions were performed by the intestinal flora. This is the first time that chiral metabolites of gentiopicrin (G-M1 and G-M2) were found in this study. In addition, the precursors of glucuronic acid conjugates previously reported in vivo may have come from the intestinal bacterial metabolites G-M1, G-M2, and G-M3. In addition, the metabolic transformation of gentiopicrin in liver microsomes was studied in vitro, and it was found that gentiopicrin did not undergo metabolic transformation under the action of liver microsomes. It is suggested that gentiopicroside may be metabolized in the intestine. This study provides both new insight regarding the investigation of effective substances and an exploration of the pharmacodynamic and toxicological properties of gentiopicrin.

The essential oil from leaves of Mauria heterophylla Kunth (Anacardiaceae): chemical and enantioselective analyses

ABSTRACT The first chemical and enantioselective analyses of the essential oil from the leaves of Mauria heterophylla Kunth were carried out. The dry plant materialproduced an essential oil with a 0.09 ± 0.05% yield by weight. The volatile fraction was submitted to GC-MS (qualitative) and GC-FID (quantitative) analyses, on two different stationary phases. A total of forty-four constituents were detected and quantified with at least one column, corresponding to about 85.5% of the whole oil mass. Limonene (15.2%), (E)-β-caryophyllene (11.1%), α-phellandrene (10.9%), germacrene D (6.6%), α-pinene (6.4%), myrcene (3.9%), β-phellandrene (2.8%), bicyclogermacrene (2.8%), (E)-β-farnesene (2.2%) and δ-cadinene (1.2%) were major components. Furthermore, the study was complemented with the enantioselective analysis of eleven common chiral terpenes, carried out on two β-cyclodextrin-based chiral selectors. Among the chiral metabolites, (1R,4S)-(+)-camphene, (1R,5R)-(+)-β-pinene, (S)-(+)-α-phellandrene and (S)-(+)-β-phellandrene were enantiomerically pure, whereas α-pinene, sabinene, limonene, linalool, terpinen-4-ol, α-terpineol and germacrene D were present as scalemic mixtures.

Enantioselective metabolism of novel chiral insecticide Paichongding by human cytochrome P450 3A4: A computational insight

As a novel chiral neonicotinoid insecticide, Paichongding (IPP) has been widely applied in agriculture due to its excellent insecticidal activity. However, the enantioselective metabolism of IPP stereoisomers (5R7R-IPP, 5S7S-IPP, 5R7S-IPP, and 5S7R-IPP) mediated by enzymes in non-target organisms, especially the cytochrome P450s (CYPs), remains unknown. To address this knowledge gap, we developed an integrated computational framework to elucidate the binding interactions and enantioselective metabolism of IPP stereoisomers in human CYP3A4. The results reveal that 5R7R-IPP shows much stronger binding affinity to CYP3A4 than 5S7S-IPP, while enantiomers 5R7S-IPP and 5S7R-IPP have no essential difference in their binding potential, owing to their specific interactions with key CYP3A4 residues. Although enantiomers 5R7R-IPP and 5S7S-IPP feature distinct binding modes resulting from the chiral differences, their transformation activities are slightly different, with C5 and C13 being the primary metabolic sites, respectively. In contrast, CYP3A4 preferably metabolizes 5R7S-IPP over 5S7R-IPP. The metabolism of epimers 5R7R-IPP and 5R7S-IPP share C5-hydroxylation routes due to the conserved 5R-conformaitons, but differ with the transformation routes at C11/C13 and C3 sites. The 7R-chirality of 5S7R-IPP significantly reduces the metabolic potency compared to 5S7S-IPP. CYP3A4-catalyzed hydroxylation and desaturation of IPP stereoisomers generate various chiral metabolites, with C5- and C13-hydroxyIPPs further transforming into depropylated products. Furthermore, the toxicity assessment reveals that IPP, along with the majority of its hydroxylated, desaturated, and depropylated metabolites, can potentially induce adverse effects on human health, specifically hepatotoxicity, respiratory toxicity, and carcinogenicity. This study provides valuable insights into the enantioselective fate of chiral IPP metabolism by CYP3A4, and the identified metabolites can serve as potential biomarkers for monitoring IPP exposure and associated health risk in human body.

Ecological threat caused by malathion and its chiral metabolite in a honey bee-rape system: Stereoselective exposure risk and the mechanism revealed by proteome

Honey bees play an important role in the ecological environment. Regrettably, a decline in honey bee colonies caused by chemical insecticides has occurred throughout the world. Potential stereoselective toxicity of chiral insecticides may be a hidden source of danger to bee colonies. In this study, the stereoselective exposure risk and mechanism of malathion and its chiral metabolite malaoxon were investigated. The absolute configurations were identified using an electron circular dichroism (ECD) model. Ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for chiral separation. In pollen, the initial residues of malathion and malaoxon enantiomers were 3571–3619 and 397–402 μg/kg, respectively, and R-malathion degraded relatively slowly. The oral LD50 values of R-malathion and S-malathion were 0.187 and 0.912 μg/bee with 5 times difference, respectively, and the malaoxon values were 0.633 and 0.766 μg/bee. The Pollen Hazard Quotient (PHQ) was used to evaluate exposure risk. R-malathion showed a higher risk. An analysis of the proteome, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and subcellular localization, indicated that energy metabolism and neurotransmitter transport were the main affected pathways. Our results provide a new scheme for the evaluation of the stereoselective exposure risk of chiral pesticides to honey bees.

Chiral separation and quantitation of methylphenidate, ethylphenidate, and ritalinic acid in blood using supercritical fluid chromatography.

Supercritical fluid chromatography (SFC)is a technique that analyzes compounds that are temperature-labile, have moderately low weight, or are chiral compounds. Methylphenidate (MPH) is a chiral compound with two chiral centers. MPH has two chiral metabolites, ethylphenidate (EPH) and ritalinic acid (RA). MPH is sold as a racemic mixture. The d-enantiomer of threo-MPH is responsible for medicinal effects. Due to the differing effects of the enantiomers, it is important to analyze the enantiomers individually to better understand their effects. This method utilizes SFCand solid-phase extraction (SPE) to separate and analyze the enantiomers of MPH, EPH, and RA in postmortem blood. The objective of this method was to assess a unique approach with SFC for enantiomeric separation of MPH, EPH, and RA. A SPE method was developed and optimized to isolate the analytes in blood and validated as fit-for-purpose following international guidelines. The linear range for MPH and EPH was 0.25-25 and 10-1000 ng/mL for RA in blood. Bias was -8.6% to 0.8%, and precision was within 15.4% for all analytes. Following method validation, this technique was applied to the analysis of 49 authentic samples previously analyzed with an achiral method. Quantitative results for RA were comparable to achiral technique, whereas there was loss of MPH and EPH over time. The l:d enantiomer ratio was calculated, and MPH demonstrated greater abundance of the d-enantiomer. This is the first known method to separate and quantify the enantiomers of all three analytes utilizing SFC and SPE.

Production of novel Rieske dioxygenase metabolites enabled by enzyme engineering

Rational engineering of toluene dioxygenase expands the substrate scope of this enzyme, enabling the production of new, amide-functionalized chiral metabolites.

Environmental behavior of the chiral fungicide epoxiconazole in earthworm-soil system: Enantioselective enrichment, degradation kinetics, chiral metabolite identification, and biotransformation mechanism.

The environmental impact of the chiral fungicide epoxiconazole and its chiral transformation products (TPs) on non-target organisms and the environment has become a significant concern due to its widespread use in agricultural practice. Enantioselectivity studies of parent contaminants cannot adequately assess the complexity of its chiral TPs in the environment. This study aimed to investigate the environmental behavior of epoxiconazole in an earthworm-soil system. 2S,3R-(-)-epoxiconazole was preferentially enriched in earthworms during the accumulation phase (p<0.05), but no enantioselectivity was observed during the elimination phase. One methoxylated and four hydroxylated chiral TPs were identified in soil, earthworm, and excrement. The epoxy ring hydroxylated TP and methoxylated TP of epoxiconazole were discovered for the first time in the environment. The chemically specific enantioselectivity with enantiomer fraction (EF)>0.8 was observed for the TPs in different matrices. The CYP450 monooxygenase of earthworm was significant activated. In vitro enzyme metabolism experiments (earthworm microsomes and recombinant CYP450 enzymes CYP2A6, CYP 2C9, and CYP 3A4) were carried out to further explain the biotransformation mechanism of epoxiconazole in earthworm. This study provides new evidence of enantiomeric biotransformation of chiral fungicide epoxiconazole in the earthworm-soil system and could provide valuable insights into their environmental risk assessment.

Indirect Enantioseparations: Recent Advances in Chiral Metabolomics for Biomedical Research.

Chiral metabolomics is starting to become a well-defined research field, powered by the recent advances in separation techniques. This review aimed to cover the most relevant advances in indirect enantioseparations of endogenous metabolites that were published over the last 10 years, including improvements and development of new chiral derivatizing agents, along with advances in separation methodologies. Moreover, special emphasis is put on exciting advances in separation techniques combined with mass spectrometry, such as chiral discrimination by ion-mobility mass spectrometry together with untargeted strategies for profiling of chiral metabolites in complex matrices. These advances signify a leap in chiral metabolomics technologies that will surely offer a solid base to better understand the specific roles of enantiomeric metabolites in systems biology.

Chiral Posttranslational Modification to Lysine ε-Amino Groups.

The sophistication of proteomic analysis has revealed that protein lysine residues are posttranslationally modified by a variety of acyl groups. Protein lysine acetylation regulates metabolism, gene expression, and microtubule formation and has been extensively studied; however, the understanding of the biological significance of other acyl posttranslational modifications (PTMs) is still in its infancy. The acylation of lysine residues is mediated either by acyltransferase "writer" enzymes or by nonenzymatic mechanisms and hydrolase enzymes, termed "erasers", that cleave various acyl PTMs to reverse the modified state. We have studied the human lysine deacylase enzymes, comprising the 11 Zn2+-dependent histone deacetylases (HDACs) and the 7 NAD+-consuming sirtuins (SIRTs), over the past decade. We have thus developed selective inhibitors and molecular probes and have studied the acyl substrate scope of each enzyme using chemically synthesized peptide substrates and photo-cross-linking probes. Recently, we have turned our attention to PTMs containing a stereogenic center, such as ε-N-β-hydroxybutyryllysine (Kbhb) and ε-N-lactyllysine (Kla), that each comprise a pair of mirror image stereoisomers as modifications. Both modifications are found on histones, where they affect gene transcription in response to specific metabolic states, and they are found on cytosolic and mitochondrial enzymes involved in fatty acid oxidation (Kbhb) and glycolysis (Kla), respectively. Thus, chiral modifications to lysine side chains give rise to two distinct diastereomeric products, with separate metabolic origins and potentially different activities exhibited by writer and eraser enzymes. Lysine l-lactylation originates from l-lactate, a major energy carrier produced from pyruvate after glycolysis, and it is highly induced by metabolic states such as the Warburg effect. l-Lactate can possibly be activated by acyl-coenzyme A (CoA) synthetases and transferred to lysine residues by histone acetyltransferases such as p300. d-Lactylation, on the other hand, arises primarily from a nonenzymatic reaction with d-lactylglutathione, an intermediate in the glyoxalase pathway. In addition to their distinct origin, we found that both K(l-la) and K(d-la) modifications are erased by HDACs with different catalytic efficiencies. Also, K(l-bhb) and K(d-bhb) arise from different metabolites but depend on interconnected metabolic pathways, and the two stereoisomers of ε-N-3-hydroxy-3-methylglutaryllysine (Khmg) originate from a single precursor that may then be regulated differently by eraser enzymes. Distinguishing between the individual stereoisomers of PTMs is therefore of crucial importance. In the present Account, we will (1) revisit the long-standing evidence for the distinct production and dynamics of enantiomeric forms of chiral metabolites that serve as ε-N-acyllysine PTMs and (2) highlight the outstanding questions that arise from the recent literature on chiral lysine PTMs resulting from these metabolites.

A New Essential Oil from the Leaves of the Endemic Andean Species Gynoxys miniphylla Cuatrec. (Asteraceae): Chemical and Enantioselective Analyses

A previously uninvestigated essential oil (EO) was distilled from Gynoxys miniphylla Cuatrec. (Asteraceae) and submitted to chemical and enantioselective analyses. The qualitative and quantitative analyses were conducted by GC-MS and GC-FID, over two orthogonal columns (5%-phenyl-methylpolysiloxane and polyethylene glycol stationary phases). Major constituents (≥2%) were, on both columns, respectively, as follows: α-phellandrene (16.1–17.2%), α-pinene (14.0–15.0%), germacrene D (13.3–14.8%), trans-myrtanol acetate (8.80%), δ-cadinene (4.2–4.6%), β-phellandrene (3.3–2.8%), (E)-β-caryophyllene (3.1–2.0%), o-cymene (2.4%), α-cadinol (2.3–2.6%), and α-humulene (1.7–2.0%). All the quantified compounds corresponded to 93.5–97.3% by weight of the whole essential oil, with monoterpenes counting for 53.8–55.6% of the total, and sesquiterpenes for 38.5–41.4%. For what concerns the enantioselective analyses, the chiral components were investigated through a β-cyclodextrin-based enantioselective column (2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin). A total of six chiral metabolites were analysed and the respective enantiomeric excess calculated as follows: (1S,5S)-(−)-α-pinene (98.2%), (1S,5S)-(−)-β-pinene (11.9%), (1R,5R)-(+)-sabinene (14.0%), (R)-(−)-α-phellandrene (100.0%), (R)-(−)-β-phellandrene (100.0%), and (S)-(−)-germacrene D (95.5%). According to the chemical composition and enantiomeric distribution of major compounds, this EO can be considered promising as a cholinergic, antiviral and, probably, analgesic product.

Monooxygenase- and Dioxygenase-Catalyzed Oxidative Dearomatization of Thiophenes by Sulfoxidation, cis-Dihydroxylation and Epoxidation.

Enzymatic oxidations of thiophenes, including thiophene-containing drugs, are important for biodesulfurization of crude oil and drug metabolism of mono- and poly-cyclic thiophenes. Thiophene oxidative dearomatization pathways involve reactive metabolites, whose detection is important in the pharmaceutical industry, and are catalyzed by monooxygenase (sulfoxidation, epoxidation) and dioxygenase (sulfoxidation, dihydroxylation) enzymes. Sulfoxide and epoxide metabolites of thiophene substrates are often unstable, and, while cis-dihydrodiol metabolites are more stable, significant challenges are presented by both types of metabolite. Prediction of the structure, relative and absolute configuration, and enantiopurity of chiral metabolites obtained from thiophene enzymatic oxidation depends on the substrate, type of oxygenase selected, and molecular docking results. The racemization and dimerization of sulfoxides, cis/trans epimerization of dihydrodiol metabolites, and aromatization of epoxides are all factors associated with the mono- and di-oxygenase-catalyzed metabolism of thiophenes and thiophene-containing drugs and their applications in chemoenzymatic synthesis and medicine.

Dimethylcysteine (DiCys)/o-Phthalaldehyde Derivatization for Chiral Metabolite Analyses: Cross-Comparison of Six Chiral Thiols.

Metabolomics profiling using liquid chromatography-mass spectrometry (LC-MS) has become an important tool in biomedical research. However, resolving enantiomers still represents a significant challenge in the metabolomics study of complex samples. Here, we introduced N,N-dimethyl-l-cysteine (dimethylcysteine, DiCys), a chiral thiol, for the o-phthalaldehyde (OPA) derivatization of enantiomeric amine metabolites. We took interest in DiCys because of its potential for multiplex isotope-tagged quantification. Here, we characterized the usefulness of DiCys in reversed-phase LC-MS analyses of chiral metabolites, compared against five commonly used chiral thiols: N-acetyl-l-cysteine (NAC); N-acetyl-d-penicillamine (NAP); isobutyryl-l-cysteine (IBLC); N-(tert-butoxycarbonyl)-l-cysteine methyl ester (NBC); and N-(tert-butylthiocarbamoyl)-l-cysteine ethyl ester (BTCC). DiCys and IBLC showed the best overall performance in terms of chiral separation, fluorescence intensity, and ionization efficiency. For chiral separation of amino acids, DiCys/OPA also outperformed Marfey’s reagents: 1-fluoro-2-4-dinitrophenyl-5-l-valine amide (FDVA) and 1-fluoro-2-4-dinitrophenyl-5-l-alanine amide (FDAA). As proof of principle, we compared DiCys and IBLC for detecting chiral metabolites in aqueous extracts of rice. By LC–MS analyses, both methods detected twenty proteinogenic l-amino acids and seven d-amino acids (Ala, Arg, Lys, Phe, Ser, Tyr, and Val), but DiCys showed better analyte separation. We conclude that DiCys/OPA is an excellent amine-derivatization method for enantiomeric metabolite detection in LC-MS analyses.