Recognition in the Domain of Molecular Chirality: From Noncovalent Interactions to Separation of Enantiomers.

It is not a coincidence that both chirality and noncovalent interactions are ubiquitous in nature and synthetic molecular systems. Noncovalent interactivity between chiral molecules underlies enantioselective recognition as a fundamental phenomenon regulating life and human activities. Thus, noncovalent interactions represent the narrative thread of a fascinating story which goes across several disciplines of medical, chemical, physical, biological, and other natural sciences. This review has been conceived with the awareness that a modern attitude toward molecular chirality and its consequences needs to be founded on multidisciplinary approaches to disclose the molecular basis of essential enantioselective phenomena in the domain of chemical, physical, and life sciences. With the primary aim of discussing this topic in an integrated way, a comprehensive pool of rational and systematic multidisciplinary information is provided, which concerns the fundamentals of chirality, a description of noncovalent interactions, and their implications in enantioselective processes occurring in different contexts. A specific focus is devoted to enantioselection in chromatography and electromigration techniques because of their unique feature as "multistep" processes. A second motivation for writing this review is to make a clear statement about the state of the art, the tools we have at our disposal, and what is still missing to fully understand the mechanisms underlying enantioselective recognition.

A liquid chromatography-mass spectrometry method for the enantioselective multiresidue determination of nine chiral agrochemicals in urine using an enrichment procedure based on graphitized carbon black.

Many agrochemicals are chiral molecules, and most of them are marketed as racemates or diastereomeric mixtures. Stereoisomers that are not the active enantiomer have little or no pesticidal activity and can exert serious toxic effects towards non-target organisms. Thus, investigating the possible exposure to different isomers of chiral pesticides is an urgent need. The present work was aimed at developing a new enantioselective high-performance liquid chromatography-mass spectrometry method for the simultaneous determination of nine chiral pesticides in urine. Two solid-phase extraction (SPE) procedures, based on different carbon-based sorbents (graphitized carbon black (GCB) and buckypaper (BP)), were developed and compared. By using GCB, all analytes were recovered with yields ranging from 60 to 97%, while BP allowed recoveries greater than 54% for all pesticides except those with acid characteristics. Baseline separation was achieved for the enantiomers of all target agrochemicals on a Lux Cellulose-2 column within 24 min under reversed-phase mode. The developed method was then validated according to the FDA guidelines for bioanalytical methods. Besides recovery, the other evaluated parameters were precision (7-15%), limits of detection (0.26-2.21 µg/L), lower limits of quantitation (0.43-3.68 µg/L), linear dynamic range, and sensitivity. Finally, the validated method was applied to verify the occurrence of the pesticide enantiomers in urine samples from occupationally exposed workers.

Enantioseparation of planar chiral ferrocenes on cellulose-based chiral stationary phases: Benzoate versus carbamate pendant groups.

In this study, the enantioseparation of 14 planar chiral ferrocenes containing halogen atoms, and methyl, iodoethynyl, phenyl, and 2-naphthyl groups, as substituents, was explored with a cellulose tris(4-methylbenzoate) (CMB)-based chiral column under multimodal elution conditions. n-Hexane/2-propanol (2-PrOH) 95:5 v/v, pure methanol (MeOH), and MeOH/water 90:10 v/v were used as mobile phases (MPs). With CMB, baseline enantioseparations were achieved for nine analytes with separation factors (α) ranging from 1.24 to 1.77, whereas only three analytes could be enantioseparated with 1.14 ≤ α ≤ 1.51 on a cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC)-based column, used as a reference for comparison, under the same elution conditions. Pendant group-dependent reversal of the enantiomer elution order was observed in several cases by changing CMB to CDMPC. The impact of analyte and chiral stationary phase (CSP) structure, and MP polarity on the enantioseparation, was evaluated. The two cellulose-based CSPs featured by different pendant groups were also compared in terms of thermodynamics. For this purpose, enthalpy (ΔΔH°), entropy (ΔΔS°) and free energy (ΔΔG°) differences, isoenantioselective temperatures (Tiso ), and enthalpy/entropy ratios (Q), associated with the enantioseparations, were derived from van 't Hoff plots by using n-hexane/2-PrOH 95:5 v/v and methanol/water 90:10 v/v as MPs. With the aim to disclose the functions of the different substituents in mechanisms and noncovalent interactions underlying analyte-selector complex formation at molecular level, electrostatic potential (V) analysis and molecular dynamics simulations were used as computational techniques. On this basis, enantioseparations and related mechanisms were investigated by integrating theoretical and experimental data.

Our research cooperation with Professor Yoshio Okamoto.

This article summarizes our cooperation with the research group of Prof. Yoshio Okamoto at Nagoya University during the period of time between 1992 and 2005. Although the text deals entirely with enantioseparations in high-performance liquid chromatography, capillary electrophoresis, and capillary electrochromatography, this is not a detailed review in any of these areas. The text highlights selected aspects of these techniques, which have been the subject of our joint research and in part their reflection in follow-up research by our and other research groups. Together with more systematically studied topics, aspects such as ultrafast separation of enantiomers, uncommonly high separation factor of enantiomers and other related issues are also addressed.

Comparative enantioseparation of planar chiral ferrocenes on polysaccharide-based chiral stationary phases.

Planar chiral ferrocenes are well-known compounds that have attracted interest for application in synthesis, catalysis, material science, and medicinal chemistry for several decades. In spite of the fact that asymmetric synthesis procedures for obtaining enantiomerically enriched ferrocenes are available, sometimes, the accessible enantiomeric excess of the chiral products is unsatisfactory. In such cases and for resolution of racemic planar chiral ferrocenes, enantioselective high-performance liquid chromatography (HPLC) on polysaccharide-based chiral stationary phases (CSPs) has been used in quite a few literature articles. However, although moderate/high enantioselectivities have been obtained for planar chiral ferrocenes bearing polar substituents, the enantioseparation of derivatives containing halogens, or exclusively alkyl groups, remains rather challenging. In this study, the enantioseparation of ten planar chiral 1,2- and 1,3-disubstituted ferrocenes was explored by using five polysaccharide-based CSPs under multimodal elution conditions. Baseline enantioseparations were achieved for nine analytes with separation factors (α) ranging from 1.20 to 2.92. The presence of π-extended systems in the analyte structure was shown to impact affinity of the most retained enantiomer toward amylose-based selectors, observing retention times higher than 80 min with methanol-containing mobile phases (MPs). Electrostatic potential (V) analysis and molecular dynamics (MD) simulations were used in order to study interaction modes at the molecular level.

Native and substituted cyclodextrins as chiral selectors for capillary electrophoresis enantioseparations: Structures, features, application, and molecular modeling.

CDs are cyclic oligosaccharides consisting of α-d-glucopyranosyl units linked through 1,4-linkages, which are obtained from enzymatic degradation of starch. The coexistence of hydrophilic and hydrophobic regions in the same structure makes these macrocycles extremely versatile as complexing host with application in food, cosmetics, environmental, agriculture, textile, pharmaceutical, and chemical industries. Due to their inherent chirality, CDs have been also successfully used as chiral selectors in enantioseparation science, in particular, for CE enantioseparations. In the last decades, multidisciplinary approaches based on CE, NMR spectroscopy, X-ray crystallography, microcalorimetry, and molecular modeling have shed light on some aspects of recognition mechanisms underlying enantiodiscrimination. With the ever growing improvement of computer facilities, hardware and software, computational techniques have become a useful tool to model at molecular level the dynamics of diastereomeric associate formation to sample low-energy conformations, the binding energies between the enantiomer and the CD, and to profile noncovalent interactions contributing to the stability of CD/enantiomer association. On this basis, the aim of this review is to provide the reader with a critical overview on the applications of CDs in CE. In particular, the contemporary theory of the electrophoretic technique and the main structural features of CDs are described, with a specific focus on techniques, methods, and approaches to model CE enantioseparations promoted by native and substituted CDs. A systematic compilation of all published literature has not been attempted.

Enantioseparations of polyhalogenated 4,4'-bipyridines on polysaccharide-based chiral stationary phases and molecular dynamics simulations of selector-selectand interactions.

2'-(4-Pyridyl)- and 2'-(4-hydroxyphenyl)-TCIBPs (TCIBP = 3,3',5,5'-tetrachloro-2-iodo-4,4'-bipyridyl) are chiral compounds that showed interesting inhibition activity against transthyretin fibrillation in vitro. We became interested in their enantioseparation since we noticed that the M-stereoisomer is more effective than the P-enantiomer. Based thereon, we recently reported the enantioseparation of 2'-substituted TCIBP derivatives with amylose-based chiral columns. Following this study, herein we describe the comparative enantioseparation of both 2'-(4-pyridyl)- and 2'-(4-hydroxyphenyl)-TCIBPs on four cellulose phenylcarbamate-based chiral columns aiming to explore the effect of the polymer backbone, as well as the nature and position of substituents on the side groups on the enantioseparability of these compounds. In the frame of this project, the impact of subtle variations of analyte and polysaccharide structures, and mobile phase (MP) polarity on retention and selectivity was evaluated. The effect of temperature on retention and selectivity was also considered, and overall thermodynamic parameters associated with the analyte adsorption onto the CSP surface were derived from van 't Hoff plots. Interesting cases of enantiomer elution order (EEO) reversal were observed. In particular, the EEO was shown to be dependent on polysaccharide backbone, the elution sequence of the two analytes being P-M and M-P on cellulose and amylose tris(3,5-dimethylphenylcarbamate), respectively. In this regard, a theoretical investigation based on molecular dynamics (MD) simulations was performed by using amylose and cellulose tris(3,5-dimethylphenylcarbamate) nonamers as virtual models of the polysaccharide-based selectors. This exploration at the molecular level shed light on the origin of the enantiodiscrimination processes.

Enantioseparation of 5,5'-Dibromo-2,2'-Dichloro-3-Selanyl-4,4'-Bipyridines on Polysaccharide-Based Chiral Stationary Phases: Exploring Chalcogen Bonds in Liquid-Phase Chromatography.

The chalcogen bond (ChB) is a noncovalent interaction based on electrophilic features of regions of electron charge density depletion (σ-holes) located on bound atoms of group VI. The σ-holes of sulfur and heavy chalcogen atoms (Se, Te) (donors) can interact through their positive electrostatic potential (V) with nucleophilic partners such as lone pairs, π-clouds, and anions (acceptors). In the last few years, promising applications of ChBs in catalysis, crystal engineering, molecular biology, and supramolecular chemistry have been reported. Recently, we explored the high-performance liquid chromatography (HPLC) enantioseparation of fluorinated 3-arylthio-4,4'-bipyridines containing sulfur atoms as ChB donors. Following this study, herein we describe the comparative enantioseparation of three 5,5'-dibromo-2,2'-dichloro-3-selanyl-4,4'-bipyridines on polysaccharide-based chiral stationary phases (CSPs) aiming to understand function and potentialities of selenium σ-holes in the enantiodiscrimination process. The impact of the chalcogen substituent on enantioseparation was explored by using sulfur and non-chalcogen derivatives as reference substances for comparison. Our investigation also focused on the function of the perfluorinated aromatic ring as a π-hole donor recognition site. Thermodynamic quantities associated with the enantioseparation were derived from van't Hoff plots and local electron charge density of specific molecular regions of the interacting partners were inspected in terms of calculated V. On this basis, by correlating theoretical data and experimental results, the participation of ChBs and π-hole bonds in the enantiodiscrimination process was reasonably confirmed.

Separation of terbutaline enantiomers in capillary electrophoresis with neutral cyclodextrin-type chiral selectors and investigation of the structure of selector-selectand complexes using nuclear magnetic resonance spectroscopy.

The major goal of this study was to determine the affinity pattern of the terbutaline (TB) enantiomers toward α-, ß-, γ-, and heptakis(2,3-di-O-acetyl)-ß-cyclodextrins and using NMR spectroscopy for the understanding of the fine mechanisms of interaction between the cyclodextrins (CD) and TB enantiomers. It was shown once again that CE in combination with NMR spectroscopy represents a sensitive tool to study the affinity patterns and structure of CD complexes with chiral guests. Opposite affinity patterns of TB enantiomers toward native α- and ß-CDs were associated with significant differences between the structure of the related complexes in solution. In particular, the complex between TB enantiomers and α-CD was of the external type, whereas an inclusion complex was formed between TB enantiomers and ß-CD. One of the possible structures of the complex between TB and heptakis(2,3-di-O-acetyl)-ß-CD (HDA-ß-CD) was quite similar to that of TB and ß-CD, although the chiral recognition pattern and enantioselectivity of TB complexation with these two CDs were very different.

The Effect of Enantiomer Elution Order on the Determination of Minor Enantiomeric Impurity in Ketoprofen and Enantiomeric Purity Evaluation of Commercially Available Dexketoprofen Formulations.

In a recent study, opposite enantiomer elution order was observed for ketoprofen enantiomers on two amylose-phenylcarbamate-based chiral columns with the same chemical composition of the chiral selector but in one case with coated while in the other with an immobilized chiral selector. In the present study, the influence of this uncommon effect on method validation parameters for the determination of minor enantiomeric impurity in dexketoprofen was studied. The validated methods with two alternative elution orders for enantiomers were applied for the evaluation of enantiomeric impurity in six marketed dexketoprofen formulations from various vendors. In most of these formulations except one the content of enantiomeric impurity exceeded 0.1% (w/w).

Some thoughts about enantioseparations in capillary electrophoresis.

In this overview the goal of the authors was to analyze from the historical perspective the reasons of success and failure of chiral capillary electrophoresis. In addition, the current trends are analyzed, unique advantages of capillary electrophoresis are highlighted and some future directions are discussed.

Separation of brombuterol enantiomers in capillary electrophoresis with cyclodextrin-type chiral selectors and investigation of structure of selector-selectand complexes using nuclear magnetic resonance spectroscopy.

The major goal of this study was to determine the affinity pattern of brombuterol (BB) enantiomers toward various cyclodextrins (CD) and to evaluate the potential of NMR spectroscopy for understanding fine mechanisms of interactions between CDs and BB enantiomers. Separation of BB enantiomers was performed in a fused-silica capillary using a phosphate buffer, pH 2.5, at the room temperature in the normal polarity mode. It was shown once again that CE in combination with NMR spectroscopy represents a very sensitive tool for studies of affinity patterns and structure of CD complexes with chiral guests. Although opposite affinity patterns of BB enantiomers were observed toward native ß- and γ-CDs, no significant differences between the structures of the complexes of these two CDs with BB were detected by NMR spectroscopy. In contrary to this, the opposite affinity pattern of BB enantiomers toward ß-CD and its two sulfated derivatives, heptakis (2,3-O-diacetyl-6-sulfo)-ß-CD (HDAS-ß-CD) and heptakis (2-O-methyl-3,6-di-O-sulfo)-ß-CD (HMDS-ß-CD) was associated with major differences in the structure of the complexes. In addition, it was shown again that HMDS-ß-CD provides separation of enantiomers without formation of inclusion-type complex with the chiral analyte.

An attempt for fast separation of enantiomers in nano-liquid chromatography and capillary electrochromatography.

In the present study, an attempt was made to achieve separation of enantiomers within a minute in nano-LC and CEC. In order to achieve this goal several parameters were optimized from the viewpoint of the property of chiral analytes, concentration of the chiral selector in the packing material, capillary dimensions, and separation mode. The enantiomers of several of the applied chiral sulfoxides could be resolved with the analysis time <1 min. Some instrumental obstacles hindering further reduction of analysis time are also highlighted.

Separation of enilconazole enantiomers in capillary electrophoresis with cyclodextrin-type chiral selectors and investigation of structure of selector-selectand complexes by using nuclear magnetic resonance spectroscopy.

In the present study, the enantiomer migration order (EMO) of enilconazole in the presence of various cyclodextrins (CDs) was investigated by capillary electrophoresis (CE). Opposite EMO of enilconazole were observed when ß-CD or the sulfated heptakis(2-O-methyl-3,6-di-O-sulfo)-ß-CD (HMDS-ß-CD) was used as the chiral selectors. Nuclear magnetic resonance (NMR) spectroscopy was used to study the mechanism of chiral recognition between enilconazole enantiomers and those two cyclodextrins. On the basis of rotating frame nuclear Overhauser (ROESY) experiments, the structure of an inclusion complex between enilconazole and ß-CD was derived, in which (+)-enilconazole seemed to form a tighter complex than the (-)-enantiomer. This correlates well with the migration order of enilconazole enantiomers observed in CE. No evidence of complexation between enilconazole and HMDS-ß-CD could be gathered due to lack of intermolecular nuclear Overhauser effect (NOE). Most likely the interaction between enilconazole and HMDS-ß-CD leads to formation of a shallow external complex that is sufficient for separation of enantiomers in CE but cannot be evidenced based on ROESY experiment. Thus, in this particular case CE documents the presence of intermolecular interactions which are at least very difficult to be evidenced by other instrumental techniques.

Structural rationale for the chiral separation and migration order reversal of clenpenterol enantiomers in capillary electrophoresis using two different ß-cyclodextrins.

NMR spectroscopy experiments, molecular dynamics simulations, and theoretical chemistry calculations provide insight into the structural and energetic determinants of the distinct binding of clenpenterol enantiomers to two cyclodextrins and the migration order reversal of their respective inclusion complexes in capillary electrophoresis.

Chip-based high-performance liquid chromatography for high-speed enantioseparations.

In this work, the first high-performance chiral liquid chromatography in packed microfluidic chips is presented. The chromatographic separation was performed on a column integrated into the microfluidic glass chip and packed with the particulate chiral stationary phase. Cellulose tris(3,5-dimethylphenylcarbamate) coated on 5-µm fully porous silica was used as chiral stationary phase material. Several racemic analytes including pharmaceutical products were baseline separated into their corresponding enantiomers under reversed-phase, polar organic and normal-phase conditions, demonstrating the versatility of the glass chip in the field of chiral separations. Van Deemter plots revealed a reduced plate height of 2.2 and a trend to enhanced mass transfer processes for solutes under low retention conditions. The utilization of very short column lengths of down to 12 mm led to ultrafast separations of enantiomers within 5 s.

Comparative enantiomer affinity pattern of ß-blockers in aqueous and nonaqueous CE using single-component anionic cyclodextrins.

A series of eight chiral ß-blocker drugs, acebutolol, atenolol, carazolol, carteolol, carvedilol, propranolol, sotalol, and talinolol, have been enantioseparated using two single-component anionic ß-CD derivatives, namely heptakis (2,3-di-O-methyl-6-sulfo)-ß-CD (HDMS-ß-CD) and heptakis (2,3-di-O-acetyl-6-sulfo)-ß-CD (HDAS-ß-CD), in aqueous CE and NACE. The influence of the nature of substituents (methyl or acetyl) in positions 2 and 3 on the CD derivatives and of the electrophoretic medium (water or methanol) on the enantioselectivity and enantiomer affinity pattern (EAP) of these structurally related compounds was systematically studied. All eight ß-blockers could be enantioseparated at least partially in the four CE systems, except sotalol with HDMS-ß-CD in NACE. In general, lower affinity and enantioselectivity were obtained in the presence of HDMS-ß-CD compared to HDAS-ß-CD. Reversals of EAPs were observed for all compounds. EAPs toward these two CDs were found to be opposite to each other in NACE for all compounds except carvedilol and in aqueous CE for atenolol, carteolol, talinolol, and sotalol. It is particularly noteworthy that opposite EAPs were also observed using the same CD derivative when the aqueous BGE was replaced with the methanolic one: for carazolol, carvedilol, and propranolol in the presence of HDMS-ß-CD and for acebutolol and carvedilol with HDAS-ß-CD.

Effect of basic and acidic additives on the separation of some basic drug enantiomers on polysaccharide-based chiral columns with acetonitrile as mobile phase.

The separation of enantiomers of 16 basic drugs was studied using polysaccharide-based chiral selectors and acetonitrile as mobile phase with emphasis on the role of basic and acidic additives on the separation and elution order of enantiomers. Out of the studied chiral selectors, amylose phenylcarbamate-based ones more often showed a chiral recognition ability compared to cellulose phenylcarbamate derivatives. An interesting effect was observed with formic acid as additive on enantiomer resolution and enantiomer elution order for some basic drugs. Thus, for instance, the enantioseparation of several ß-blockers (atenolol, sotalol, toliprolol) improved not only by the addition of a more conventional basic additive to the mobile phase, but also by the addition of an acidic additive. Moreover, an opposite elution order of enantiomers was observed depending on the nature of the additive (basic or acidic) in the mobile phase.

Stopped-Flow Enantioselective HPLC-CD Analysis and TD-DFT Stereochemical Characterization of Methyl Trans-3-(3,4-Dimethoxyphenyl)Glycidate.

Caffeic acid-derived polyethers are a class of natural products isolated from the root extracts of comfrey and bugloss, which are endowed with intriguing pharmacological properties as anticancer agents. The synthesis of new polyether derivatives is achieved through ring-opening polymerization of chiral 2,3-disubstituted oxiranes, whose absolute configurations define the overall stereochemistry of the produced polymer. The absolute stereochemistry of one of these building blocks, methyl trans-3-(3,4-dimethoxy-phenyl)glycidate (3), was therefore characterized by the combination of enantioselective high-performance liquid chromatography (HPLC), electronic circular dichroism (ECD) spectroscopy, and time-dependent density functional theory (TD-DFT) calculations. Initial efforts aiming at the isolation of enantiomers by means of a standard preparative HPLC protocol followed by offline ECD analysis failed due to unexpected degradation of the samples after collection. The stopped-flow HPLC-CD approach, by which the ECD spectra of enantiomers are measured online with the HPLC system, was applied to overcome this issue and allowed a fast, reliable, and chemical-saving analysis, while avoiding the risks of sample degradation during the collection and processing of enantiomeric fractions. Subsequent TD-DFT calculations identified ( as the first eluted enantiomeric fraction on the Lux Cellulose-2 column, therefore achieving a full stereochemical characterization of the chiral oxirane under investigation.