The separation of cyclic diadenosine diphosphorothioate and the diastereomers of its difluorinated derivative and the estimation of the binding constants and ionic mobilities of their complexes with 2-hydroxypropyl-ß-cyclodextrin by affinity capillary electrophoresis.

The incorporation of phosphorothioate linkages has recently been extensively employed in therapeutic oligonucleotides. For their separation and quality control, new high-efficient and high-sensitive analytical methods are needed. In this work, a new affinity capillary electrophoresis method has been developed and applied for the separation of a potential anticancer drug, 2',3'-cyclic diadenosine diphosphorothioate (Rp, Rp) (ADU-S100), and three recently newly synthesized diastereomers of its difluorinated derivative, 3',3'-cyclic di(2'-fluoro, 2'-deoxyadenosine phosphorothioate). The separation was performed in the various background electrolytes (BGEs) within a pH range 5-9 using several native and derivatized cyclodextrins (CDs) as chiral additives of the BGE. Relatively good separations were obtained with ß-, γ-, and 2-hydroxypropyl-γ-CDs in some of the BGEs tested. However, the best separation was achieved using the 2-hydroxypropyl-ß-CD chiral selector at 43.5 mM average concentration in the BGE composed of 40 mM Tris, 40 mM tricine, pH 8.1. Under these conditions, all the previous four cyclic dinucleotides (CDNs) were baseline separated within 4 min. Additionally, the average apparent binding constants and the average actual ionic mobilities of the complexes of all four CDNs with 2-hydroxypropyl-ß-CD in the above BGE were determined. The formed complexes were found to be relatively weak, with the average apparent binding constants in the range of 12.2-94.1 L mol-1 and with the actual ionic mobilities spanning the interval (-7.8 to -12.7) × 10-9 m2 V-1 s-1. The developed method can be applied for the separation, analysis, and characterization of the above and similar CDNs.

Single ion mobility monitoring (SIM2) stitching method for high-throughput and high ion mobility resolution chiral analysis.

Chiral analysis is of high interest in many fields such as chemistry, pharmaceuticals and metabolomics. Mass spectrometry and ion mobility spectrometry are useful analytical tools, although they cannot be used as stand-alone methods. Here, we propose an efficient strategy for the enantiomer characterization of amino acids (AAs) using non-covalent copper complexes. A single ion mobility monitoring (SIM2) method was applied on a TIMS-ToF mass spectrometer to maximize the detection and mobility separation of isomers. Almost all of the 19 pairs of proteinogenic AA enantiomers could be separated with at least one combination with the chiral references L-Phe and L-Pro. Furthermore, we extended the targeted SIM2 method by stitching several mobility ranges, in order to be able to analyze complex mixtures in a single acquisition while maintaining high mobility resolution. Most of the enantiomeric pairs of AAs separated with the SIM2 method were also detected with this approach. The SIM2 stitching method thus opens the way to a more comprehensive chiral analysis with TIMS-ToF instruments.

A new HPLC method with multiple detection systems for impurity analysis and discrimination of natural versus synthetic cannabidiol.

Cannabidiol (CBD) is the main non-psychoactive phytocannabinoid derived from Cannabis sativa L. It is now an active pharmaceutical ingredient (API), given its usage in treating some types of pediatric epilepsy. For this reason, this compound requires a deep characterization in terms of purity and origin. Previous research work has shown two impurities in CBD samples from hemp inflorescences, namely, cannabidivarin (CBDV) and cannabidibutol (CBDB), while abnormal-cannabidiol (abn-CBD) has been described as the primary by-product that is generated from CBD synthesis. Both natural and synthetic CBD samples exhibit the presence of Δ9-tetrahydrocannabinol (Δ9-THC) and Δ8-THC. This study aimed to develop a new analytical method based on high-performance liquid chromatography (HPLC) with different detection systems to study the purity of CBD and to define its origin based on the impurity profile. In addition to the above-mentioned cannabinoids, other compounds, such as cannabigerovarin (CBGV), cannabigerol (CBG), cannabichromevarin (CBCV), and cannabichromene (CBC), were examined as potential discriminating impurities. Qualitative and quantitative analyses were carried out by UHPLC-HRMS and HPLC-UV/Vis, respectively. Principal component analysis was applied for statistical exploration. Natural CBD samples exhibited purities ranging between 97.5 and 99.7%, while synthetic samples were generally pure, except for three initially labeled as synthetic, revealing natural-derived impurities. To further confirm the origin of CBD samples, the presence of other two minor impurities, namely cannabidihexol (CBDH) and cannabidiphorol (CBDP), was assessed as unequivocal for a natural origin. Finally, an enantioselective HPLC analysis was carried out and the results confirmed the presence of the (-)-trans enantiomer in all CBD samples. In conclusion, the HPLC method developed represents a reliable tool for detecting CBD impurities, thus providing a clear discrimination of the compound origin.

Enantiomeric Analysis of Chiral Drugs Using Mass Spectrometric Methods: A Comprehensive Review.

Chirality plays a crucial role in the drug development process, influencing fundamental chemical and biochemical processes and significantly affecting our daily lives. This review provides a comprehensive examination of mass spectrometric (MS) methods for the enantiomeric analysis of chiral drugs. It thoroughly investigates MS-hyphenated techniques, emphasizing their critical role in achieving enantioselective analysis. Furthermore, it delves into the intricate chiral recognition mechanisms inherent in MS, elucidating the fundamental principles that govern successful chiral separations. By critically assessing the obstacles and potential benefits associated with each MS-based method, this review offers valuable insights for researchers navigating the complexities of chiral analysis. Both qualitative and quantitative approaches are explored, presenting a comparative analysis of their strengths and limitations. This review is aimed at significantly enhancing the understanding of chiral MS methods, serving as a crucial resource for researchers and practitioners engaged in enantioselective studies.

A Squaramide-Based Organocatalyst as a Novel Versatile Chiral Solvating Agent for Carboxylic Acids.

A squaramide-based organocatalyst for asymmetric Michael reactions has been tested as a chiral solvating agent (CSA) for 26 carboxylic acids and camphorsulfonic acid, encompassing amino acid derivatives, mandelic acid, as well as some of its analogs, propionic acids like profens (ketoprofen and ibuprofen), butanoic acids and others. In many cases remarkably high enantiodifferentiations at 1H, 13C and 19F nuclei were observed. The interaction likely involves a proton transfer from the acidic substrates to the tertiary amine sites of the organocatalyst, thus allowing for pre-solubilization of the organocatalyst (when a chloroform solution of the substrate is employed) or the simultaneous solubilization of both the catalyst and the substrate. DOSY experiments were employed to evaluate whether the catalyst-substrate ionic adduct was a tight one or not. ROESY experiments were employed to investigate the role of the squaramide unit in the adduct formation. A mechanism of interaction was proposed in accordance with the literature data.

Stereoselective study of fluoxetine and norfluoxetine across the blood-brain barrier mediated by organic cation transporter 1/3 in rats using an enantioselective UPLC-MS/MS method.

Fluoxetine (FLT) is a widely used antidepressant in clinical practice, which can be metabolized into active norfluoxetine (NFLT) in vivo. The stereoselectivity of FLT and NFLT enantiomers across the blood-brain barrier (BBB) is still to be clarified. In this study, accurate and reliable UPLC-MS/MS enantioselective analysis was established in rat plasma and brain. The characteristics of FLT and NFLT enantiomers across the BBB were studied by chemical knockout of rat transporters. We found that the dominant enantiomers of FLT and NFLT were S-FLT and R-NFLT, respectively, both in plasma and in brain. The FLT and NFLT enantiomers showed significant stereoselectivity across the BBB, and S-FLT and S-NFLT were the dominant configurations across the BBB. Chemical knockout of organic cation transporter 1 (OCT1) and OCT3 can affect the ratio of plasma FLT and NFLT enantiomers into the brain, suggesting that OCT1/3 is stereoselective for FLT and NFLT transport across the BBB.

Assignment of the absolute configuration of molecules that are chiral by virtue of deuterium substitution using chiral tag molecular rotational resonance spectroscopy.

Chiral tag molecular rotational resonance (MRR) spectroscopy is used to assign the absolute configuration of molecules that are chiral by virtue of deuterium substitution. Interest in the improved performance of deuterated active pharmaceutical ingredients has led to the development of precision deuteration reactions. These reactions often generate enantioisotopomer reaction products that pose challenges for chiral analysis. Chiral tag rotational spectroscopy uses noncovalent derivatization of the enantioisotopomer to create the diastereomers of the 1:1 molecular complexes of the analyte and a small, chiral molecule. Assignment of the absolute configuration requires high-confidence determinations of the structures of these weakly bound complexes. A general search method, CREST, is used to identify candidate geometries. Subsequent geometry optimization using dispersion corrected density functional theory gives equilibrium geometries with sufficient accuracy to identify the isomers of the chiral tag complexes produced in the pulsed jet expansion used to introduce the sample into the MRR spectrometer. Rotational constant scaling based on the fact that the diastereomers have the same equilibrium geometry gives accurate predictions allowing identification of the homochiral and heterochiral tag complexes and, therefore, assignment of absolute configuration. The method is successfully applied to three oxygenated substrates from enantioselective Cu-catalyzed alkene transfer hydrodeuteration reaction chemistry.

The separation of the enantiomers of diquats by capillary electrophoresis using randomly sulfated cyclodextrins as chiral selectors.

Diquats, derivatives of the widely used herbicide diquat, represent a new class of functional organic molecules. A combination of their special electrochemical properties and axial chirality could potentially result in their important applications in supramolecular chemistry, chiral catalysis, and chiral analysis. However, prior to their practical applications, the diquats have to be prepared in enantiomerically pure forms and the enantiomeric purity of their P- and M-isomers has to be checked. Hence, a chiral capillary electrophoresis (CE) method has been developed and applied for separation of P- and M-enantiomers of 11 new diquats. Fast and better than baseline CE separations of enantiomers of all 11 diquats within a short time 5-7 min were achieved using acidic buffer, 22 mM NaOH, 35 mM H3 PO4 , pH 2.5, as a background electrolyte, and 6 mM randomly sulfated α-, ß-, and γ-cyclodextrins as chiral selectors. The most successful selector was sulfated γ-cyclodextrin, which baseline separated the enantiomers of all 11 diquats, followed by sulfated ß-cyclodextrin and sulfated α-cyclodextrin, which baseline separated enantiomers of 10 and nine diquats, respectively. Using this method, a high enantiopurity degree of the isolated P- and M-enantiomers of three diquats with a defined absolute configuration was confirmed and their migration order was identified.

The Phenomenon of Self-Induced Diastereomeric Anisochrony and Its Implications in NMR Spectroscopy.

Nuclear magnetic resonance (NMR) spectroscopy is an analytical technique largely applied in the analysis of discrimination processes involving enantiomeric substrates and chiral agents, which can interact with the analyte either via covalent bonding or via formation of diastereomeric solvates. However, enantiodiscrimination has been observed, in some cases, even in the absence of any additional chiral selector. The reasons behind this phenomenon must be found in the capability of some chiral substrates to interact with themselves by forming diastereomeric solvates in solution that can generate nonequivalences in the NMR spectra of enantiomerically enriched mixtures. As a result, differentiation of enantiomers is observed, thus allowing the quantification of the enantiomeric composition of the mixture under investigation. The tendency of certain substrates to self-aggregate and to generate diastereomeric adducts in solution can be defined as Self-Induced Diastereomeric Anisochrony (SIDA), but other acronyms have been used to refer to this phenomenon. In the present work, an overview of SIDA processes investigated via NMR spectroscopy will be provided, with a particular emphasis on the nature of the substrates involved, on the interaction mechanisms at the basis of the phenomenon, and on theoretical treatments proposed in the literature to explain them.

Chiral secondary amino acids, their importance, and methods of analysis.

Naturally occurring secondary amino acids, with proline as the main representative, contain an alpha-imino group in a cycle that is typically four-, five-, and six-membered. The unique ring structure exhibits exceptional properties-conformational rigidity, chemical stability, and specific roles in protein structure and folding. Many proline analogues have been used as valuable compounds for the study of metabolism of both prokaryotic and eukaryotic cells and for the synthesis of compounds with desired biological, pharmaceutical, or industrial properties. The D-forms of secondary amino acids play different roles in living organisms than the L-forms. They have different metabolic pathways, biological, physiological, and pharmacological effects, they can be indicators of changes and also serve as biomarkers of diseases. In the scientific literature, the number of articles examining D-amino acids in biological samples is increasing. The review summarises information on the occurrence and importance of D- and L-secondary amino acids-azetidic acid, proline, hydroxyprolines, pipecolic, nipecotic, hydroxypipecolic acids and related peptides containing these D-AAs, as well as the main analytical methods (mostly chromatographic) used for their enantiomeric determination in different matrices (biological samples, plants, food, water, and soil).

Separation and identification of the DL-forms of short-chain peptides using a new chiral resolution labeling reagent.

There are several reports of D-amino acids being the causative molecules of serious diseases, resulting in the formation of, for example, prion protein and amyloid ß. D-Amino acids in peptides and proteins are typically identified by sequencing each residue by Edman degradation or by hydrolysis with hydrochloric acid for amino acid analysis. However, these approaches can result in racemization of the L-form to the D-form by hydrolysis and long pre-treatment for hydrolysis. To address these problems, we aimed to identify the DL-forms of amino acids in peptides without hydrolysis. Here, we showed that the DL-forms in peptides which are difficult to separate on a chiral column can be precisely separated by labeling with 1-fluoro-2,4-dinitrophenyl-5-D-leucine-N,N-dimethylethylenediamine-amide (D-FDLDA). Additionally, the peptides could be quantitatively analyzed using the same labeling method as for amino acids. Furthermore, the detection sensitivity of a sample labeled with D-FDLDA was higher than that of the conventional reagents Nα-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide (L-FDAA) and Nα-(5-fluoro-2,4-dinitrophenyl)-L-leucinamide (L-FDLA) used in Marfey's method. The proposed method for identifying DL-forms of amino acids in peptides is a powerful tool for use in organic chemistry, biochemistry, and medical science.

Impact of cyclofructan derivatives as efficient chiral selector in chiral analysis: An overview.

The development of chiral selectors for the separation and analysis of chiral molecules has been an evolving process happening over three decades, since the introduction of the first chiral stationary phase (CSP) in 1938. The main impetus for designing new chiral selectors is to get to most promising one which has a broad chiral recognition property, separation capability for a wide range of chiral analytes, and the cost-effective CSP, which is also a major concern. Today, we have more than 100 commercially available CSPs, and these are prepared by coating or immobilizing the classical chiral selectors on to the chromatographic support, normally, silica gel. The purpose of this review is to look at progress and the impact of cyclofructan derivatives, a novel chiral selector introduced recently, for performing chiral analysis.

Enantioselective analyses of chloroquine and hydroxychloroquine in rat liver microsomes through chiral liquid chromatography-tandem mass spectrometry.

An efficient, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) chiral analysis method was established for determination of chloroquine and hydroxychloroquine enantiomers in rat liver microsomes. Effects of polysaccharide chiral stationary phases and basic additives on chiral separations of two analytes were discussed in detail. Amylose tris(3, 5-dimethylphenylcarbamate)-coated chiral stationary phase showed the best separation performance for them with acetonitrile-diethylamine-ethanol-diethylamine mixture (90:0.1:10:0.1, v/v/v/v) among four chiral stationary phases. Then, multiple reaction monitoring mode was selected as the data acquisition for determination of two pairs of enantiomers. The proposed LC-MS/MS chiral analysis method was validated in terms of linearity, accuracy, precision, and specificity. Good linearity with correlation coefficient over 0.998 was obtained in the concentration range of 0.05-5 µM. Limits of quantification for chloroquine and hydroxychloroquine enantiomers were 5.0 and 1.0 nM, respectively. The recoveries ranged from 81.14% to 111.09%. The intra-day and inter-day relative standard deviation were less than 6.5%. Moreover, concentrations of chloroquine and hydroxychloroquine enantiomers in rat liver microsomes were determined through the proposed LC-MS/MS analysis method. After incubated with rat liver microsomes for 10 min, the enantiomeric factor of hydroxychloroquine decreased from 0.50 to 0.45 (p < 0.001). In brief, our developed determination method for chloroquine and hydroxychloroquine enantiomers through LC-MS/MS spectrometry showed the characteristics of high-efficiency, fast speed, and very low detection limit, and would be greatly beneficial for screening and quantitation of them in biological matrices.

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.

Self-Propelled Multifunctional Microrobots Harboring Chiral Supramolecular Selectors for "Enantiorecognition-on-the-Fly".

Herein, a general procedure for the synthesis of multifunctional MRs, which simultaneously exhibit i) chiral, ii) magnetic, and iii) fluorescent properties in combination with iv) self-propulsion, is reported. Self-propelled Ni@Pt superparamagnetic microrockets have been functionalized with fluorescent CdS quantum dots carrying a chiral host biomolecule as ß-cyclodextrin (ß-CD). The "on-the-fly" chiral recognition potential of MRs has been interrogated by taking advantage of the ß-CD affinity to supramolecularly accommodate different chiral biomolecules (i.e., amino acids). As a proof-of-concept, tryptophan enantiomers have been discriminated with a dual-mode (optical and electrochemical) readout. This approach paves the way to devise intelligent cargo micromachines with "built-in" chiral supramolecular recognition capabilities to elucidate the concept of "enantiorecognition-on-the-fly", which might be facilely customized by tailoring the supramolecular host-guest encapsulation.

Past, present, and future developments in enantioselective analysis using capillary electromigration techniques.

Enantioseparation of chiral products has become increasingly important in a large diversity of academic and industrial applications. The separation of chiral compounds is inherently challenging and thus requires a suitable analytical technique that can achieve high resolution and sensitivity. In this context, CE has shown remarkable results so far. Chiral CE offers an orthogonal enantioselectivity and is typically considered less costly than chromatographic techniques, since only minute amounts of chiral selectors are needed. Several CE approaches have been developed for chiral analysis, including chiral EKC and chiral CEC. Enantioseparations by EKC benefit from the wide variety of possible pseudostationary phases that can be employed. Chiral CEC, on the other hand, combines chromatographic separation principles with the bulk fluid movement of CE, benefitting from reduced band broadening as compared to pressure-driven systems. Although UV detection is conventionally used for these approaches, MS can also be considered. CE-MS represents a promising alternative due to the increased sensitivity and selectivity, enabling the chiral analysis of complex samples. The potential contamination of the MS ion source in EKC-MS can be overcome using partial-filling and counter-migration techniques. However, chiral analysis using monolithic and open-tubular CEC-MS awaits additional method validation and a dedicated commercial interface. Further efforts in chiral CE are expected toward the improvement of existing techniques, the development of novel pseudostationary phases, and establishing the use of chiral ionic liquids, molecular imprinted polymers, and metal-organic frameworks. These developments will certainly foster the adoption of CE(-MS) as a well-established technique in routine chiral analysis.

A sensitive HPLC-MS/MS method for the detection, resolution and quantitation of cathinone enantiomers in horse blood plasma and urine.

Resolution of cathinone enantiomers in equine anti-doping analysis is becoming more important to distinguish the inadvertent ingestion of plant-based products from those of deliberate administration of designer synthetic analogs. With this in mind, a rapid and sensitive method was developed and validated for the detection, resolution and quantitative determination of cathinone enantiomers in horse blood plasma and urine. The analytes were recovered from the blood plasma and urine matrices by using a liquid-liquid extraction after adjusting the pH to 9. The recovered analytes were derivatized with Nα-(2,4-dinitro-5-fluorophenyl)-L-valinamide, a chiral derivatizing agent analogous to Marfey's reagent. The resulting diastereoisomers were baseline resolved under a reversed-phase liquid chromatographic condition. Derivatization of the analytes not only allowed the separation of the enantiomers using cost-effective traditional liquid chromatography conditions and reversed-phase columns but also increased the sensitivity, at least to an order of magnitude, when tandem mass spectrometry is used for the detection. A limit of detection of 0.05 ng/mL was achieved for cathinone enantiomers for both matrices. Acceptable intraday and interday precision and accuracy along with satisfactory dilution accuracy and precision were observed during the method validation. The method suitability was tested using the post administration urine samples collected after single doses of cathinone and ephedrine as single-enantiomeric form and methcathinone as racemic form. Finally, a proof of concept of the isomeric ratio in urine samples to distinguish the presence of cathinone as a result of accidental ingestion of plant-based product from that of an illicit use of a designer product is demonstrated. To the best of our knowledge, this is the first such work where cathinone enantiomers were resolved and quantified in horse blood plasma and urine at sub nanogram levels.

Adulteration of Essential Oils: A Multitask Issue for Quality Control. Three Case Studies: Lavandula angustifolia Mill., Citrus limon (L.) Osbeck and Melaleuca alternifolia (Maiden & Betche) Cheel.

The quality control of essential oils (EO) principally aims at revealing the presence of adulterations and at quantifying compounds that are limited by law by evaluating EO chemical compositions, usually in terms of the normalised relative abundance of selected markers, for comparison to reference values reported in pharmacopoeias and/or international norms. Common adulterations of EO consist of the addition of cheaper EO or synthetic materials. This adulteration can be detected by calculating the percent normalised areas of selected markers or the enantiomeric composition of chiral components. The dilution of the EO with vegetable oils is another type of adulteration. This adulteration is quite devious, as it modifies neither the qualitative composition of the resulting EO nor the marker's normalised percentage abundance, which is no longer diagnostic, and an absolute quantitative analysis is required. This study aims at verifying the application of the two above approaches (i.e., normalised relative abundance and absolute quantitation) to detect EO adulterations, with examples involving selected commercial EO (lavender, bergamot and tea tree) adulterated with synthetic components, EO of different origin and lower economical values and heavy vegetable oils. The results show that absolute quantitation is necessary to highlight adulteration with heavy vegetable oils, providing that a reference quantitative profile is available.

Improving enantiomeric resolutions by avoiding peak distortion effects in on-line coupled liquid chromatography to gas chromatography.

In this work, we study the effect of different variables affecting elution profile distortion on the enantiomeric resolution eventually achievable when working with on-line coupled liquid chromatography to gas chromatography (LC-GC). Specifically, the proposed configuration combines achiral reversed-phase liquid chromatography (RPLC) and chiral gas chromatography (enantio-GC), with heptakis-(2,3,6-tri-O-methyl)-ß-cyclodextrin as enantioselective stationary phase to analyse target fractions transferred (from LC to GC) via the through oven transfer adsorption desorption (TOTAD) interface. The high degree of orthogonality resulting from the combination of two chromatographic columns having very different separation mechanisms (and also requiring mobile phases in distinct physical states), as well as integration of the sample preparation step in the first dimension of the system, significantly contributed to exploit the performance of the proposed two-dimensional approach. Occasional adverse effects, which may result in severe peak distortions during LC-GC analysis and could be explained by flow instabilities due to viscous fingering, are circumvented by using the outstanding capacity of the TOTAD interface for achieving effective elimination of the eluent arriving from the LC preseparation.

The determination of enantiomer composition of 1-((3-chlorophenyl)-(phenyl)methyl) amine and 1-((3-chlorophenyl)(phenyl)-methyl) urea (Galodif) by NMR spectroscopy, chiral HPLC, and polarimetry.

For the first time, a method for enantiomer resolution of the anticonvulsant Galodif (1-((3-chlorophenyl)(phenyl)methyl) urea) by chiral HPLC was developed, whereas the enantiomeric composition of 1-((3-chlorophenyl)(phenyl)methyl) amine-precursor in Galodif synthesis-cannot be resolved by this method. However, starting 1-((3-chlorophenyl)(phenyl)methyl) amine quantitatively forms diastereomeric N-((3-chlorophenyl)(phenyl)methyl)-1-camphorsulfonamides in reaction with chiral (1R)-(+)- or (1S)-(-)-camphor-10-sulfonyl chlorides. The diastereomeric ratio of obtained camphorsulfonamides can be easily determined by NMR 1 H and 13 C spectroscopy. The DFT calculations of specific rotation of Galodif enantiomers showed good agreement with experimental data. The absolute configuration of enantiomers was proposed for the first time.