NMR studies of chiral discrimination relevant to the enantioseparation of N-acylarylalkylamines by an (R)-phenylglycinol-derived chiral selector

Recently, it was reported that the chiral recognition ability of (R)-N-3,5-dinitrobenzoyl phenylglycinol derivative was examined as a new HPLC chiral stationary phase (CSP 1) for the resolution of racemic N-acylnaphthylalkylamines. However, the mechanism of chiral discrimination on the CSP remained elusive until now. In this study, a spectroscopic investigation of the chiral discrimination mechanism of CSP 1 was undertaken using mixtures of (R)-N-3,5-dinitrobenzoyl phenylglycinol-derived chiral selector (2) and each of the enantiomers of N-acylnaphthylalkylamines (3) by NMR study. First, the differences in free energy changes (DeltaDeltaG) upon diastereomeric complexation in solution between the complex of each isomer with chiral selector 2 by NMR titration were calculated. The values were then compared with those estimated by chiral HPLC. The chemical shift changes of each proton on the chiral selector and analytes were also checked and it was found that the chemical shift changes decreased continuously as the acyl group on analytes increased in length. This observation was consistent with the HPLC data. From these experimental results, the interaction mechanism of chiral discrimination between the chiral selector and the analytes is more precisely explained.     

Maltodextrins as Chiral Selectors in CE: Molecular Structure Effect of Basic Chiral Compounds on the Enantioseparation

Prediction of chiral separation for a compound using a chiral selector is an interesting and debatable work. For this purpose, in this study 23 chiral basic drugs with different chemical structures were selected as model solutes and the influence of their chemical structures on the enantioseparation in the presence of maltodextrin (MD) as chiral selector was investigated. For chiral separation, a 100‐mM phosphate buffer solution (pH 3.0) containing 10% (w/v) MD with dextrose equivalent (DE) of 4‐7 as chiral selector at the temperature of 25°C and voltage of 20 kV was used. Under this condition, baseline separation was achieved for nine chiral compounds and partial separation was obtained for another six chiral compounds while no enantioseparation was obtained for the remaining eight compounds. The results showed that the existence of at least two aromatic rings or cycloalkanes and an oxygen or nitrogen atom or –CN group directly bonded to the chiral center are necessary for baseline separation. With the obtained results in this study, chiral separation of a chiral compound can be estimated with MD‐modified capillary electrophoresis before analysis. This prediction will minimize the number of preliminary experiments required to resolve enantiomers and will save time and cost. Chirality 26:620–628, 2014. © 2014 Wiley Periodicals, Inc.     

A versatile chiral selector for determination of enantiomeric composition of fluorescent and nonfluorescent chiral molecules using steady-state fluorescence spectroscopy

A fluorescent chiral molecular micelle (FCMM), poly (sodium N-undecanoyl-L-phenylalaninate) (poly-L-SUF), was developed as a chiral selector for enantiomeric recognition and determination of enantiomeric composition of four fluorescent and four nonfluorescent chiral molecules by use of steady-state fluorescence spectroscopy. The influence of FCMM concentration, buffer pH and complexation medium on FCMM-analyte host-guest complexation, and the emission spectral properties of the resulting complexes were investigated. The chiral interactions of the analytes,1,1'-binaphthyl-2,2'-diamine, 1-(9-anthryl)-2,2,2-trifluoroethanol, propranolol, naproxen, chloromethyl menthyl ether (CME), citramalic acid, tartaric acid, and limonene (LIM), in the presence of poly-L-SUF were based on diastereomeric complex formation. The figures of merit obtained from the partial-least-squares regression modeling of the calibration samples suggested good prediction ability for the validation of six of the eight chiral analytes. Better host-guest complexation of the more hydrophobic molecules, CME and LIM, were obtained in methanol/water mixtures, resulting in better predictability of the regression models. Prediction ability of the models was evaluated by use of the root-mean-square percent relative error (RMS%RE) and was found to range from 1.77 to 15.80% (buffer), 1.26 to 7.95% (25:75 methanol/water), and 1.21 to 4.28% (75:25 methanol/water).     

Chiral separation of unmodified α-hydroxy acids by ligand exchange HPLC using chiral copper(II) complexes of (S)-phenylalaninamide as additives to the eluent

Copper(II) complexes of (S)-phenylalaninamide have been successfully used for the direct enantiomeric separation of unmodified (R,S)-α-hydroxy acids in reversed phase high-performance liquid chromatography (RP-HPLC). The effect of various parameters (pH, eluent polarity, selector concentration) on enantioselectivity is discussed. Evidence is provided that a mechanism of ligand exchange is actually occurring during the chromatographic separation. The method is very convenient and easy to use, and the chiral selector is commercially available and can be recovered at the end of the analysis. A conventional achiral RP-ODS-2 column is used and no pretreatment of the samples is required. This method allows the accurate determination of the enantiomeric excess of α-hydroxy acids in synthetic and biological samples. © 1995 Wiley-Liss, Inc.     

Enantioseparation of citalopram enantiomers by capillary electrophoresis: Method development through experimental design and computational modeling

Citalopram (CIT) is a frequently used modern antidepressant that inhibits selectively serotonin reuptake in the brain. It has a chiral center in its structure and is used in therapy as both racemic mixture and pure enantiomer as its pharmacological effect is almost entirely associated with S-CIT. The aim of this study was the development of a simple and rapid capillary electrophoresis (CE) method for the separation and quantification of CIT enantiomers. To establish the optimum chiral selector, several native and derivatized, neutral, and ionized cyclodextrins (CDs) were examined at different pH levels. An experimental design strategy was adopted for method optimization; a fractional factorial design was applied for screening purposes to identify significant experimental factors followed by a face-centered central composite design used for optimization purposes. Computational modeling was used to obtain information on the interaction energy and the geometry of the complexes to aid in the understanding of chiral separation mechanism. The best results were obtained when using a 25-mM phosphate buffer at pH 7.0, 3-mM CM-β-CD as chiral selector, 17.5°C temperature, 15-kV voltage, and 50 mbar/s hydrodynamic injection. The separation time was fast, below 3 min, and the migration order was S-CIT followed by R-CIT. The analytical performance of the method was verified in terms of precision, linearity, accuracy, sensibility, and robustness, and the method was applied for the determination of CIT enantiomers from pharmaceutical preparations.     

Time-resolved fluorescent chirality sensing and imaging of malate in aqueous solution

Chiral discrimination of malates in aqueous solutions at near-neutral pH is achieved through fluorescence measurement and imaging using the europium-tetracycline complex (EuTc) as a fluorescent probe. The method is based on the significantly different fluorescence properties of the ternary complexes (Eu-Tc-malate) formed between EuTc and the enantiomeric malates. The enantiomeric excess (ee) of chiral malates can be quantified by both steady-state and time-resolved fluorescence, using either a conventional fluorescence microplate reader or fluorescence imaging. It offers a facile and sensitive method for high-throughput chiral discrimination.     

The comparison in enantioseparation ability of the chiral stationary phases with single and mixed selector--the selectors derived from two D-tartrates

(2S,3S)-2,3-Bis(3,5-dimethylphenylcarbonyloxy)-3-(benzyloxycarbonyl)-propanoic acid and (2S,3S)-2,3-bis(1-naphthalenecarbonyloxy)-3-(benzyloxycarbonyl)-propanoic acid were synthesized from D-tartaric acid. These two compounds were chlorinated to afford two chiral selectors for chiral stationary phases (CSPs). The selectors were separately immobilized on aminated silica gel to give two single selector CSPs; and were simultaneously immobilized to obtain a mixed selector CSP. Comparing to the single selector CSPs, the mixed selector CSP bears the enhanced enantioseparation ability, suggesting that the two selectors in the mixed selector CSP are consistent for chiral recognition in most mobile phase conditions.     

Enantioseparation of N-protected α-amino acid derivatives by liquid-liquid extraction technique employing stereoselective ion-pair formation with a carbamoylated quinine derivative

Two-phase liquid-liquid extraction experiments were undertaken to study the enantioselective transport of the chiral N-protected α-amino acid derivatives from an aqueous buffer solution into an organic phase employing highly lipophilic carbamoylated quinine as chiral selector and phase transfer carrier, respectively. The chiral separation, derived from enantioselective ion-pair formation and differential solubility in the aqueous and organic phases of diastereomeric associates thus formed has been shown to be primarily dependent on the structure of the selectand, the nature of the organic solvent, the molar ratio of a given chiral selector to selectand in the two phases, and the pH of the aqueous phase. Extracted enantiomers were recovered by back-extraction using a relatively polar acidic medium in which the selector is barely insoluble. Thus, the enantiomeric purity of N-(3,5-dinitrobenzoyl)-leucine exceeded 95% enantiomeric excess with 70% overall yield with a single extraction and back-extraction step. Chirality 9:268–273, 1997. © 1997 Wiley-Liss, Inc.     

Copper(II) complexes of N2-alkyl-(S)-amino acid amides as chiral selectors for dynamically coated chiral stationary phases in RP-HPLC

Copper(II) complexes of N²-octyl-(S)-phenylalaninamide (Noc-Phe-NH₂), N²-dodecyl-(S)-phenylalaninamide (Ndo-Phe-NH₂), and N²-octyl-(S)-norleucinamide (Noc-NLeu-NH₂), dynamically adsorbed on a reversed-phase C₁₈ column, were able to perform the direct enantiomeric separation of unmodified amino acids, amino acid amides and esters, hydroxy acids, and dipeptides by elution with aqueous or mixed aqueous-organic solutions containing copper(II) sulphate or acetate. The role played by several parameters in the separation procedure was examined with the copper(II) complex of Noc-Phe-NH₂ [concentration of the copper(II) ion in the eluent, pH and eluent polarity, amount of adsorbed selector]. The separation was shown to occur entirely on the stationary phase. The mechanism of chiral discrimination is discussed in terms of the chromatographic parameters and of the structure of the copper(II) complexes in solution and in the solid state. The chiral stationary phase maintained its separation ability for about 3 months. However, the column could be easily restored by recovering the selector with methanol and repeating the loading procedure. © 1996 Wiley-Liss, Inc.     

Immobilization of (1S,2R)-(+)-2-amino-1,2-diphenylethanol derivates on aminated silica gel with different linkages as chiral stationary phases and their enantioseparation evaluation by HPLC

A chiral selector was prepared through the reaction between (1S,2R)-(+)-2-amino-1,2-diphenylethanol and phenyl isocyanate. This selector was immobilized on aminated silica gel, respectively, with bifunctional group linkers of 1,4-phenylene diisocyanate, methylene-di-p-phenyl diisocyanate, and terephthaloyl chloride to produce corresponding three chiral stationary phases. The prepared compounds and chiral stationary phases were characterized by FT-IR, elemental analysis, (1)H NMR, and solid-state (1)H NMR. The enantioseparation ability of these chiral stationary phases was evaluated with structurally various chiral compounds. The chiral stationary phase prepared with 1,4-phenylene diisocyanate as linker showed excellent enantioseparation ability. The influence of different linkages on the enantioseparation was discussed.     

Mechanism of chiral recognition in the enantioseparation of 2-aryloxypropionic acids on new brush-type chiral stationary phases

New brush-type chiral stationary phases (CSP I-IV) comprising N-3,5,6-trichloro-2,4-dicyanophenyl-L-alpha-amino acids (1-4) were prepared by binding of chiral selectors 1-4 to gamma-aminopropyl silica gel. To check the role of excess free aminopropyl groups, CSP V was prepared by binding N-3,5,6-trichloro-2,4-dicyanophenyl-L-alanyl-(3-triethoxysilyl)propylamide to unmodified silica gel. The best separation of racemic 2-aryloxypropionic acids (TR-1-13) was obtained with CSP I; the -(-)-S enantiomer were regularly eluted first, as determined by a CD detector. The mechanism of chiral recognition implies a synergistic interaction of carboxylic acid analyte with the chiral selector and achiral free gamma-aminopropyl units on silica. In fact, CSP V, which is lacking an achiral aminopropyl spacer, shows a lower separation ability for 2-aryloxypropionic acids, but a similar enantioselective discrimination of esters TR-19-20, in comparison with CSP I. CSP I-IV retain unaltered separation ability after a few months of continuous work using a large number of various mobile phases.     

New chiral stationary phases for liquid chromatography based on small molecules: Development,enantioresolution evaluation and chiral recognition mechanisms

Recently, we reported the development of new chiral stationary phases (CSPs) for liquid chromatography (LC) based on chiral derivatives of xanthones (CDXs). Based on the most promising CDX selectors, 12 new CSPs were successfully prepared starting from suitable functionalized small molecules including xanthone and benzophenone derivatives. The chiral selectors comprising one, two, three, or four chiral moieties were covalently bonded to a chromatographic support and further packed into LC stainless-steel columns (150 × 2.1 mm I.D.). The enantioselective performance of the new CSPs was evaluated by LC using different classes of chiral compounds. Specificity for enantioseparation of some CDXs was observed in the evaluation of the new CSPs. Besides, assessment of chiral recognition mechanisms was performed by computational studies using molecular docking approach, which are in accordance with the chromatographic parameters. X-Ray analysis was used to establish a chiral selector 3D structure.     

Probability rule for chiral recognition

Molecular Chirality is of central interest in biological studies because enantiomeric compounds, while indistinguishable by most inanimate systems, show profoundly different properties in biochemical environments. Enantioselective separation methods, based on the differential recognition of two optical isomers by a chiral selector, have been amply documented. Also, great effort has been directed towards a theoretical understanding of the fundamental mechanisms underlying the chiral recognition process. Here we report a comprehensive data examination of enantio separation measurements for over 72000 chiral selector-select and pairs from the chiral selection compendium CHIRBASE. The distribution of alpha = k'(D)/k'(L) values was found to follow a power law, equivalent to an exponential decay for chiral differential free energies. This observation is experimentally relevant in terms of the number of different individual or combinatorial selectors that need to be screened in order to observe alpha values higher than a preset minimum. A string model for enantiorecognition (SMED) formalism is proposed to account for this observation on the basis of an extended Ogston three-point interaction model. Partially overlapping molecular interaction domains are analyzed in terms of a string complementarity model for ligand-receptor complementarity. The results suggest that chiral selection statistics may be interpreted in terms of more general concepts related to biomolecular recognition.     

Synthesis of dendrimer‐type chiral stationary phases based on the selector of (1S,2R)‐(+)‐2‐Amino‐1,2‐diphenylethanol derivate and their enantioseparation evaluation by HPLC

In our recent work, a series of dendritic chiral stationary phases (CSPs) were synthesized, in which the chiral selector was L‐2‐(p‐toluenesulfonamido)‐3‐phenylpropionyl chloride (selector I), and the CSP derived from three‐generation dendrimer showed the best separation ability. To further investigate the influence of the structures of dendrimer and chiral selector on enantioseparation ability, in this work, another series CSPs ( CSPs 1‐4 ) were prepared by immobilizing (1S,2R)‐1,2‐diphenyl‐2‐(3‐phenylureido)ethyl 4‐isocyanatophenylcarbamate (selector II) on one‐ to four‐generation dendrimers that were prepared in previous work. CSPs 1 and 4 demonstrated the equivalent enantioseparation ability. CSPs 2 and 3 showed the best and poorest enantioseparation ability respectively. Basically, these two series of CSPs exhibited the equivalent enantioseparation ability although the chiral selectors were different. Considering the enantioseparation ability of the CSP derived from aminated silica gel and selector II is much better than that of the one derived from aminated silica gel and selector I, it is believed that the dendrimer conformation essentially impacts enantioseparation. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Determination of enantiomeric excess for 2,3-dihydroxy-3-phenylpropionate compounds by capillary electrophoresis using hydroxypropyl-beta-cyclodextrin as chiral selector

A new capillary zone electrophoresis (CZE) method was developed to separate three chiral 2,3-dihydroxy-3-phenylpropionate enantiomers using neutral hydroxypropyl-beta-CD (HP-beta-CD) as chiral selector and borate as background electrolyte. The results showed that HP-beta-CD exhibited good enantioselectivity and high resolution was achieved under the optimum condition of pH 10.3, 200 mM borate buffer containing 6% methanol and 50 mM HP-beta-CD at 15 kV and 20 degrees C within 16 min. The precision of the method was <0.9% for migration time and 4.5% for corrected peak area. In addition, the developed method was successfully applied to the determination of enantiomeric excess (ee) of synthetic 2,3-dihydroxy-3-phenylpropionate samples. With this method, low as 0.2% impurity of the undesirable enantiomer in the presence of high amount of target enantiomer was determined. The results demonstrated that the proposed CZE method is a simple and useful technique and is applicable to ee assay of 2,3-dihydroxy-3-phenylpropionate enantiomers.     

Chiral Discrimination of Ofloxacin Enantiomers Using DNA Double Helix Regulated by Metal Ions

DNA‐based chiral selectors are constructed to discriminate ofloxacin enantiomers through metal‐ion anchoring on a special DNA double helix that contains successive GC pairs. The effects of metal ions involving Mg²⁺, Ni²⁺, Cu²⁺, Ag⁺, and Pt²⁺ were studied on the regulation of DNA chiral discrimination towards ofloxacin enantiomers. It is shown that DNA‐Cu(II) complexes exhibit the highest enantioselectivities at the [Cu²⁺]/base ratio of 0.1. The enantiomeric excess can reach 59% in R‐enantiomer after being adsorbed by the RET‐Cu(II) complex. Stereoselective recognition of ofloxacin enantiomers on the double helix is tunable via external stimulus, providing a programmable desorption process to regenerate DNA. This DNA‐based chiral selector exhibits excellent reusability without apparent loss of enantioselectivity after three cycles of adsorption and desorption. Chirality 26:249–254, 2014. © 2014 Wiley Periodicals, Inc.     

Chiral recognition of binaphthyl derivatives: a chiral recognition model on the basis of chromatography, spectroscopy, and molecular mechanistic calculations for the enantioseparation of 1,1'-binaphthyl derivatives on cholic acid-bonded stationary phases.

In an effort to elucidate the mechanism of chiral discrimination of cholic acid-based stationary phases, the enantiomeric recognition ability of six chiral stationary phases (CSPs), prepared from differently substituted cholic acid derivatives, was evaluated in normal phase high-performance liquid chromatography (HPLC) with a series of 1,1'-binaphthyl compounds. The influence of structural variations of analytes on retention and enantioselectivity was investigated. Particularly high values of enantioselectivity were observed for the binaphthol enantiomers on a CSP prepared from the allyl 7 alpha,12 alpha-dihydroxy-3 alpha-phenylcarbamoyloxy-5 beta-cholan-24-oate. The complexes of this chiral selector with both enantiomers of binaphthol were studied as models for the interactions responsible for the enantioseparation with the cholic acid-based stationary phases. The 1:1 stoichiometry of the complex in solution was determined by UV titration. The chiral selector dissolved in chloroform exhibited a chiral discrimination for the binaphthol in (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopies. Some aromatic proton and carbon resonances of binaphthol were clearly separated into a pair of peaks due to enantiomers in the presence of the chiral selector. Moreover, on the basis of molecular mechanics calculation, a chiral discrimination model was proposed which nicely explains the relevant chromatographic behavior of the 1,1'-binaphthyl derivatives.     

Enantioselective hydrolysis of insoluble (R,S)-ketoprofen ethyl ester in dispersed aqueous reaction system induced by chiral cyclodextrin

The enantioselective hydrolysis of insoluble (R,S)-ketoprofen ethyl ester to the optically active (S)-ketoprofen was carried out in a dispersed aqueous lipase reaction system induced by the inclusion of chiral cyclodextrins for complexation of the substrate. Hydroxypropyl-beta-cyclodextrin was the most effective chiral selector and disperser giving an enantiomeric excess and conversion yield of 0.99 and 0.49, respectively.     

Development of a validated capillary electrophoresis method for enantiomeric purity control and quality control of levocetirizine in a pharmaceutical formulation

A chiral capillary electrophoresis method has been developed for the quantification of 0.1% of the enantiomeric impurity (dextrocetirizine) in levocetirizine and determination of both in pharmaceuticals using sulfated-β-cyclodextrins (CDs) as chiral selector. Several parameters affecting the separation were studied such as the type and concentration of chiral selectors, buffer composition and pH, organic modifier, mixtures of two CDs in a dual system, voltage, and temperature. The optimal separation conditions were obtained using a 50 mM tetraborate buffer (pH 8.2) containing 1% (w/v) sulfated-β-CDs on a fused-silica capillary. Under these conditions, the resolution of two enantiomers was higher than 3. To validate the method, the stability of the solutions, robustness (two level half fraction factorial design for 5 factors using 19 experiments [2(n-1)+3]), precision, linearity (dextrocetirizine 0.25-2.5 μg/ml, R(2) = 0.9994, y = 0.0375x + 0.0008; levocetirizine 15-100 μg/ml, R(2) = 0.9996, y = 0.0213x + 0.0339), limit of detection (0.075 μg/ml, 0.03% m/m), limit of quantification (0.25 μg/ml, 0.1% m/m), accuracy (dextrocetirizine 84-109%, levocetirizine 97.3-103.1%), filter effect, and different CD batches were examined. The validated method was further applied to bulk drug and tablets of levocetirizine.     

Determination of enantiomeric excess of ethyl 3,5-dihydroxy-6-benzyloxy hexanoate by chiral reverse phase high performance liquid chromatography

A simple and reliable chiral HPLC method was developed for the determination of enantiomeric excess of a chiral dihydroxy intermediate for the chemoenzymatic synthesis of side chain of statin drugs. After evaluating different columns and conditions, the four stereoisomers of ethyl 3,5-dihydroxy-6-benzyloxy hexanoate were well resolved by a simple gradient elution on OD-RH column, and the enantiomeric excess of the desired 3R,5S-enantiomer was accurately measured. This study provides a simple, rapid, accurate, and reliable method to assess the enantiomeric quality of such important intermediates.