Preparation and application of HPLC chiral stationary phases based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid

Preparation of liquid chromatographic chiral stationary phases (CSPs) based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid and their application are reviewed. The various methods of connecting (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid to silica gel covalently or dynamically are demonstrated. The CSPs based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid have been very successful for the resolution of various primary amino compounds with the use of an aqueous mobile phase containing organic and acidic modifiers. In addition, the resolution of secondary amino compounds including beta-blockers and N-(3,5-dinitrobenzoyl)-alpha-amino acids has been demonstrated on a CSP based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid with a non-aqueous mobile phase.     

Chiral discrimination studies of (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid by high-performance liquid chromatography and NMR spectroscopy

Chiral discrimination studies using (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18-C-6-TA) as a chiral selector were performed by high-performance liquid chromatography (HPLC) and NMR spectroscopy. The enantiomers of alanine (Ala) or alanine methyl ester (Ala-ME) were well separated on the chiral stationary phases (CSPs) derived from (+)-18-C-6-TA by HPLC. The chiral selector, (+)-18-C-6-TA, used in the CSP was also applied for the chiral discrimination of the Ala and Ala-ME enantiomers, and it discriminated these enantiomers successfully by NMR spectroscopy. The chemical shift differences (Delta Delta delta) of the alpha-proton of these enantiomers in the presence of an equimolecular solution of 18-C-6-TA were observed to be 0.10 ppm for Ala in methanol-d4 containing 10 mM H2SO4 and 0.11 ppm for Ala-ME in methanol-d4. The observed NMR results agreed with the chromatographic data on the (+)-18-C-6-TA-derived CSP by HPLC in terms of both the elution order and solvents effects.     

非甾体抗炎药对映体在冠醚手性固定相上的拆分

利用高效液相色谱法,采用从(18-冠-6)-2,3,11,12-四羧((18-crown-6)-2,3,11,12-tetracarboxylic acid,18-C-6-TA)衍生的冠醚类型手性固定相(CSPs),对非甾体抗炎药酰肼衍生物进行手性分离研究.为了手性酸类非甾体抗炎药在冠醚手性固定相上进行手性拆分,采用与肼合成方法导入氨基基团,合成了其酰肼衍生物.色谱条件为:流动相:80%甲醇/水(V/V)含10 mmol/L H2SO4;流速:1.0 mL/min;紫外检测波长:210 nm.结果表明,除酮洛芬之外,其它非甾体抗炎药的酰肼衍生物拆分效果较好(α=1.14～1.26,Rs=0.88～1.43).而且非甾体抗炎药酰肼衍生物在(+)18-C-6-TA衍生的CSP 1和(-)18-C-6-TA衍生的CSP 2上的洗脱顺序得到了相反结果.     

Comparison of enantiomer separation on two chiral stationary phases derived from (+)-18-crown-6-2,3,11,12-tetracarboxylic acid of the same chiral selector

Liquid chromatographic comparisons for enantiomer resolution of α-amino acids and chiral primary amino compounds were made using chiral stationary phases (CSPs) prepared by covalently bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18-C-6-TA) of the same chiral selector. The resolution of all α-amino acids on CSP 1 developed in our group was found to be better than that on CSP 2 reported by Machida et al. All α-amino acids examined in this study were well enantioseparated on CSP 1 (α=1.22–2.47), while four analytes were not resolved or all the other analytes were poorly resolved on CSP 2 than on CSP 1. However, in resolving the primary amino compounds without a carbonyl group, CSP 1 was comparable with CSP 2. Although (+)-18-C-6-TA of the same chiral selector was used to prepare CSP 1 and CSP 2, this study showed that different connecting methods for the CSPs might influence their ability to resolve the analytes depending on their structures related to the chiral recognition mechanism.     

High-performance liquid chromatographic enantioseparation of beta-amino acid stereoisomers on a (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid-based chiral stationary phase

Direct reversed-phase high-performance liquid chromatographic methods were developed for the separation of enantiomers of 14 unnatural beta-amino acids, including several beta-3-homo-amino acids on a chiral stationary phase containing (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid bonded to 3-aminopropyl silica gel as chiral selector. The effects of the organic and the acidic modifiers and the mobile phase composition on the separation were investigated. The natures and positions of the substituents on the aromatic ring substantially influenced the retention and enantioseparation. The elution sequence in most cases was determined and the R enantiomers were eluteted before the S enantiomers.     

Enantiomeric separation of underivatized small amines in conventional and on-chip capillary electrophoresis with contactless conductivity detection

The determination of the enantiomers of small non-UV-absorbing amines which otherwise can only be achieved with difficulty was possible by using a combination of the chiral crown ether (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18C6H4) and dimethyl-beta-CD as selectors in CE and contactless conductivity measurement for detection. Alkylamines without any other functional group, amino alcohols, species with ether or ester groups and with a cyclic moiety were investigated. The detection limits were found to be about 1.0 microM and the determination is possible up to at least 1.0 mM. The determination of enantiomeric ratios of up to 99.5:0.5 was also found feasible.     

Chiral resolution of diphenylalanine by high-performance liquid chromatography on a crown-ether-based chiral stationary phase and by NMR spectroscopy

Summary The enantiomers of diphenylalanine (DPA) were well separated by chiral HPLC and NMR spectroscopy on the chiral stationary phase (CSP) derived from (18-crown-6)-2,3,11,12-tetracarboxylic acid (18-C-6-TA). The chromatographic parameters such as separation factors and retention times were greatly influenced by the mobile phase conditions. The (+)-18-C-6-TA used in the CSP was also employed as a chiral solvating agent for the enantiodiscrimination of the DPA enantiomers by NMR spectroscopy. The proton of the DPA analyte showing the chemical shift nonequivalences was used in determining the enantiomeric composition of the analyte.     

Separation of the stereoisomers of racemic fluoroquinolone antibacterial agents on a crown-ether-based chiral HPLC stationary phase

Summary A chiral stationary phase derived from (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid has been successfully used for the direct separation of the enantiomers of recemic fluoroquinolines containing a primary amino group being investigated as antibacterial agents. The chromatographic resolution behavior was found to depend on the content and the type of acidic and organic modifiers in the mobile phase and on the column temperature.     

Stereoisomeric separation of pharmaceutical compounds using CE with a chiral crown ether

Optical pure (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid, a chiral crown ether, was successfully used as a chiral selector for the stereoisomeric separation of numerous real pharmaceutical compounds. Both practical and mechanistic aspects were described. Effects of chiral selector concentration under different pH values of BGE were discussed. Chiral recognition for the enantiomeric compounds with (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was investigated through model compounds using CE and infrared spectroscopic techniques. Relations between the enantioselectivity of the chiral crown ether and the structural features of the studied compounds were also investigated. Unusual resolutions of compound-p and its enantiomer as well as compound-o and its 2b epimer were described. These compounds contained only tertiary amine, believed to be nonbinding with crown ethers in general. The possible mechanisms for the interaction between compound-o and the chiral crown ether were investigated using CE, electrospray MS (ESI-MS), and proton ((1)H) NMR spectroscopy. All experiments provided clear evidence that binding between compound-o and the chiral crown ether had occurred. ESI-MS spectra indicated that the complexes had a 1:1 stoichiometric ratio. The advantages and disadvantages of using chiral crown ether for stereoisomeric separations were compared with those using sulfated CDs.     

Chiral counter-current chromatography of gemifloxacin guided by capillary electrophoresis using (+)-(18-crown-6)-tetracarboxylic acid as a chiral selector

(+)-(18-crown-6)-tetracarboxylic acid (18C6H4) has been known as a highly efficient chiral selector for resolving primary amine enantiomers in capillary electrophoresis (CE). We investigated the chiral separation of gemifloxacin using 18C6H4 in analytical counter-current chromatography (CCC). The separation conditions for CE, including the binding constant, pH, and run buffer constituents, provided a helpful guideline for chiral CCC. A successful separation of gemifloxacin enantiomers could be achieved using a two-phase solvent system composed of 1-butanol-ethyl-acetate-bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane acetate buffer with a small amount of 18C6H4. The hydrophobicity of the solvent system and the 18C6H4 concentration were varied to optimize the chiral separation.     

High-performance liquid chromatographic enantioseparation of beta-3-homo-amino acid stereoisomers on a (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid-based chiral stationary phase

High-performance liquid chromatographic methods were developed for the separation of the enantiomers of thirteen unusual beta-3-homo-amino acids and three of its ethyl esters on a chiral stationary phase containing (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid as chiral selector. The effects of the mobile phase composition and the acidic modifiers on the separation were investigated. The structures of the substituents in beta-position substantially influenced the retention and enantioseparation. The influence of ionic strength on the enantioseparation was established experimentally. The elution sequence was determined in all cases.     

High performance liquid chromatographic separation of dipeptide and tripeptide enantiomers using a chiral crown ether stationary phase

A chiral stationary phase (CSP) based on (-)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was evaluated for the direct resolution of the enantiomers of dipeptides and tripeptides. The type and concentration of the acid and the methanol content were optimized with regard to retention time and resolution using Ala-Phe as model peptide. A mobile phase consisting of 10 mM sulfuric acid in 70% aqueous methanol was applied to the separation of a set of 16 structurally diverse dipeptides and tripeptides. Generally, the configuration of the amino acid at the N-terminus determined the enantiomer elution order. With a few exceptions the LL- and LD-enantiomers interacted stronger with the CSP compared to the corresponding DD- or DL-enantiomers. The experimental conditions also allowed the simultaneous separation of all four stereoisomers of Ala-Phe. Addition of ammonium sulfate generally reduced retention times and enantiomer resolution. Addition of triethylamine as modifier led to an overall increase of the retention times while the resolution did not show a general trend, increasing in the case of Ala-Ala but decreasing in the case of Ala-Phe.     

Determination of chromatographic separation parameters of tryptophan enantiomers on a Chirosil-SCA chiral stationary phase by using the inverse method based on the initial guesses estimated from elution by characteristic point method

An effective chiral stationary phase (CSP) for enantioseparation of amino acids was established previously by bonding (18-crown-6)-2, 3, 11, 12-tetracarboxylic acid to silica gel. This CSP has recently been commercialized under the name of Chirosil-SCA. As a first step for developing a Chirosil-SCA simulated moving bed chromatographic process for separation of tryptophan enantiomers, the adsorption isotherm and mass-transfer parameters of each tryptophan enantiomer on the Chirosil-SCA CSP were determined in this study while using only water as a mobile phase. For this task, inverse method (IM) was applied on the basis of the initial guesses estimated from elution by characteristic point (ECP) method, which was found to be more advantageous in the aspects of both accuracy and computational efficiency than the case of utilizing individually only IM or ECP method. The results revealed that the adsorption behavior of each tryptophan enantiomer on the Chirosil-SCA could be well described by the Langmuir-Freundlich isotherm. The model predictions based on the determined parameter values were in close agreement with the experimental chromatograms from a series of single-component or mixture pulse tests that were performed under various feed concentrations and flow rates. It was also found that the Langmuir-Freundlich isotherm parameters of each enantiomer were largely affected by temperature. Such a marked dependence of the parameters on temperature was investigated quantitatively. The results of such an investigation indicated that as the temperature decreases, the adsorption affinities of both enantiomers become higher and the heterogeneity of the Chirosil-SCA becomes more pronounced.     

Chiral separation of gemifloxacin in sodium-containing media using chiral crown ether as a chiral selector by capillary and microchip electrophoresis

Chiral crown ether, (+)-(18-crown-6)-tetracarboxylic acid (18C6H(4)), is an effective chiral selector for resolving enantiomeric primary amines owing to the difference in affinities between 18C6H(4) and each of the amine enantiomers. In addition to the destacking effect of sodium ion in the sample solution, the strong affinity of sodium ion to the polyether ring of crown ether is unfavorable to chiral capillary electrophoresis using 18C6H(4) as a chiral selector. In this report, the chiral separation of gemifloxacin dissolved in a saline sample matrix using 18C6H(4) was investigated. Adding a chelating agent, ethylenediaminetetraacetic acid (EDTA), to the run buffer greatly improved the separation efficiencies and peak shapes. The successful chiral separation of gemifloxacin in a urinary solution was demonstrated for both capillary and microchip electrophoresis.     

Structural scaffold of 18-crown-6 tetracarboxylic acid for optical resolution of chiral amino acid: X-ray crystal analyses and energy calculations of complexes of D- and L-isomers of tyrosine, isoleucine, methionine and phenylglycine

To clarify the structural scaffold of (+)-18-crown-6 tetracarboxylic acid ((+)-18C6H4) for the optical resolution of a chiral amino acid, the crystal structures of its equimolar complexes with L- and D-isomers of tyrosine (Tyr), isoleucine (Ile), methionine (Met) and phenylglycine (PheG) were analysed by X-ray diffraction methods. (+)-18C6H4 took very similar conformations for all complexes. Although the chemical structure of (+)-18C6H4 is C2-symmetric, it took a similar asymmetric ring conformation of radius ca. 6.0 A. In all complexes, the amino group of chiral amino acids was located near the center of the ring and formed three hydrogen bonds and five electrostatic interactions with eight oxygen atoms of the ether ring and carboxyl groups. Also, the Calpha atom of chiral amino acids participated in Calpha-H...O interaction with the oxygen atom of (+)-18C6H4. In contrast, the carboxyl group of chiral amino acids did not directly interact with (+)-18C6H4. These results indicate that the structural scaffold of (+)-18C6H4 for the optical resolution of chiral amino acids is mainly based on the mode of interaction of (+)-18C6H4 with the amino and Calpha-H groups of chiral amino acids. The differences in interaction pattern and binding energy between the L- and D-isomers of each amino acid are discussed in relation to the chiral recognition of (+)-18C6H4.     

Simultaneous analysis of underivatized chiral amino acids by liquid chromatography-ionspray tandem mass spectrometry using a teicoplanin chiral stationary phase

Simultaneous chiral separations of underivatized amino acids have been performed using a teicoplanin-based chiral stationary phase and ionspray tandem mass spectrometry for their ionisation and detection. Different amino acid enantiomer pairs were separated simultaneously, including those of positional isomeric amino acids (e.g., L,D-Leu/Ile, or L,D-Val/Iva). Due to the specificity of tandem mass spectrometry, co-eluting enantiomers of different amino acids could also be determined. Fifteen chiral underivatized proteinogenic and non-proteinogenic amino acids were analysed simultaneously under isocratic conditions (acetonitrile-water, 75:25) in less than 25 min. For maximum sensitivity, post-column addition of 500 mM aqueous HCOOH was necessary. Detection limits varied from 2.5 to 50 microg l(-1) depending on the amino acid. The signal vs. concentration relationship was linear for all D- and L-amino acids (0.9995 < or = r2 < or = 1) for three orders of magnitude.     

Noncovalent interactions between ([18]crown-6)-tetracarboxylic acid and amino acids: electrospray-ionization mass spectrometry investigation of the chiral-recognition processes

Chiral recognition of enantiomers by host compounds is one of the most challenging topics in modern host-guest chemistry. Amongst the well-established methods, mass spectrometry (MS) is increasingly used nowadays, due to its low detection limit, short analysis time, and suitability for analyzing mixtures and for studying chiral effects in the gas phase. The development of electrospray-ionization (ESI) techniques provides an invaluable tool to study, in the gas phase, diastereoisomeric complex ions prepared from enantiomer ions and a chiral selector. This paper reports on an ESIMS and ESIMSMS study of the molecular mechanisms that intervene in the chiral-recognition phenomena observed between amino acids and a chiral crown ether. The modified crown ether, namely (+)-([18]crown-6)-2,3,11,12-tetracarboxylic acid, is used as the chiral selector when covalently bound on a stationary phase in liquid chromatography. This study was stimulated by the fact that, except with threonine and proline, consistent elution orders were observed, which indicates that the D enantiomers interact more strongly with the chiral selector than the L enantiomers. For proline, the lack of a primary amino group is likely to be responsible for the nonresolution of the two forms, whereas the second stereogenic center on threonine could explain the reversed elution order. In light of those observations, we performed mass spectrometry experiments to understand more deeply the enantiomeric recognition phenomena, both in solution by the enantiomer-labeled guest method and in the gas phase by gas-phase ligand-exchange ion/molecule reactions. The results have been further supported by quantum chemical calculations. One of the most interesting features of this work is the identification of a nonspecific interaction between proline and the crown ether upon ESIMS analysis.     

Detection and Separation of Free Amino Acid Enantiomers by Capillary Electrophoresis with a Chiral Crown Ether and Indirect Photometric Detection

18-Crown-6 tetracarboxylic acid (18C6H₄) has been successfully used as a chiral selector for capillary electrophoretic (CE), high-performance liquid chromatographic (HPLC), and gas chromatographic (GC) separation of the enantiomers of DL-amino compounds. We have previously used X-ray crystallographic analysis and HPLC with an immobilized 18C6H₄ chiral stationary phase to study chiral recognition by 18C6H₄ of several DL amino acids (DL-AA). In this study CE was used for chiral recognition of several DL-AA in electrolyte solution containing 18C6H₄, in which the analyte (D or L amino acid) interacts freely. Among 14 DL-AA investigated, the enantiomers of nine (Glu, Ile, Met, PheG, Phe, Ser, Tyr, Val, and Thr) were successfully recognized in 4-15 mM 18C6H₄. Indirect photometric detection with a cationic dye, chrysoidine, was used to monitor non-chromophoric DL-AA. Among nine successfully recognized DL-AA, the D forms of Ser, Thr and Met migrated faster than the corresponding L forms. The strengths of interactions predicted from the order of migration of each enantiomer in CE were different from those in HPLC analysis. The different enantiomer recognition probably can be ascribed to the difference between CE in which the selector is not immobilized and HPLC in which the selector is immobilized by means of a spacer.     

Liquid chromatographic resolution of gemifloxacin mesylate on a chiral stationary phase derived from crown ether

A new racemic fluoroquinolone antibacterial agent, gemifloxacin mesylate, has been successfully resolved on a chiral stationary phase (CSP) derived from (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid. Compared to the Crownpak CR(+) column, the CSP used in this study was more effective for the resolution of racemic gemifloxacin mesylate, especially in terms of analytical time. The resolution of gemifloxacin mesylate enantiomers on the CSP was found to be dependent on the type and content of organic and acidic modifiers in the aqueous mobile phase and the column temperature.     

(+)-(18-Crown-6)-2,3,11,12-tetracarboxylic acid and its Ytterbium(III) complex as chiral NMR discriminating agents

The compound (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (I) and its ytterbium(III) complex are evaluated as chiral NMR discriminating agents. The crown ether is a useful chiral discriminating agent for protonated amino acid esters, amines, and amino alcohols. The crown can also be used with neutral primary amines since amines are protonated through a neutralization reaction with a carboxylic acid moiety of the crown. Enantiodiscrimination with the crown is observed in methanol and acetonitrile. Addition of ytterbium(III) nitrate to crown-substrate mixtures causes upfield shifts in the NMR spectrum of the substrate and often enhances the enantiomeric discrimination. Evidence indicates that the ytterbium(III) bonds to the carboxylic acid moieties of the crown, but enhancements in enantiomeric discrimination result from either the different association constants of the enantiomers with the crown or diastereomeric nature of the resulting crown-substrate complexes. The ytterbium complex with the crown is suitable for use in methanol but precipitates in acetonitrile.