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.     

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.     

Recent progresses in protein-based chiral stationary phases for enantioseparations in liquid chromatography

Chiral stationary phases (CSPs) based on proteins orglycoproteins have been developed for the enantioseparations of various compounds. In 2001, a review article [J. Haginaka, J. Chromatogr. A, 906 (2001) 253] dealing with CSPs based on proteins and glycoproteins was published. After that serum albumin from other species, penicillin G-acylase, antibodies, fatty acid binding protein and streptavidin were newly introduced as the chiral selectors in CSPs. This review article deals with recent progresses in CSPs based on protein or glycoproteins in LC after 2001, focusing on their enantioselective properties and chiral recognition mechanisms.     

Combining the enantioselectivity of a cyclodextrin and a diamide selector in a mixed binary gas‐chromatographic chiral stationary phase

The present work reports on the investigation of a mixed binary chiral stationary phase (CSP) prepared by simultaneous attachment of permethylated‐β‐cyclodextrin ( D selector) and resorcinarene with pendant l ‐ or d ‐valine diamide groups ( L′ and D′ selectors, respectively) to a polysiloxane matrix via platinum‐catalyzed hydrosilylation. The gas‐chromatographic investigation of a number of racemates on the four different CSPs ( D, D′, DD′, and DL′ ) showed that the enantioselectivity of the individual chiral selectors was retained in the mixed binary CSPs. As a consequence, hydrocarbons, underivatized alcohols, ketones, and almost all proteinogenic amino acid derivatives could be separated simultaneously on each of the mixed CSPs. Matched and mismatched cases of enantioseparation on the mixed binary CSPs were observed but turned out to be of minor importance for enantiomeric separation. In general, more racemates were separated with α ≥ 1.02 on the mixed phases as compared to the single phases. In order to analyze the influence of the presence of the diamide selector on the enantioselectivity of the cyclodextrin selector, a mixed ternary CSP containing the selector D and a racemic mixture of the selectors D′ and L′ [ D ( D′L′ )] was prepared and investigated. Merits and limitations of the approach of mixed binary CSPs are discussed. © 2005 Wiley‐Liss, Inc. Chirality     

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.     

Enantioselective anion exchangers based on cinchona alkaloid‐derived carbamates: Influence of C8/C9 stereochemistry on chiral recognition

Four diastereomeric chiral stationary phases (CSPs) based on quinine, quinidine, epiquinine, and epiquinidine tert‐butyl carbamate selectors were synthesized and evaluated under ion exchange HPLC conditions with a set of racemic N‐acylated and N‐oxycarbonylated α‐amino acids as selectands. The enantioseparation potential of quinine‐ and quinidine‐derived CSPs proved to be far superior to that of their C₉‐epimeric congeners. The absolute configuration of C₉ stereogenic center of the cinchonan backbone of these selectors was identified as the structural feature controlling the elution order. Guided by an X‐ray structure of a most favorable selector–selectand complex and the observed chromatographic enantioseparation data, a chiral recognition model was advanced. The contributions of ion‐pairing, π–π donor–acceptor, hydrogen bonding and steric interactions were established as crucial factors. Chirality 11:522–528, 1999. © 1999 Wiley‐Liss, Inc.     

Chiral recognition mechanisms with macrocyclic glycopeptide selectors

Macrocyclic glycopeptide selectors are naturally occurring antibiotics produced by microorganisms. They were found to be excellent chiral selectors for a wide range of enantiomers, including amino acids. Four selectors are commercialized as chiral stationary phases (CSP) for chromatography. They are ristocetin, teicoplanin, vancomycin, and the teicoplanin aglycone (TAG). The key docking interaction for amino acid recognition was established to be a charge-charge interaction between the anionic carboxylate group of the amino acid and a cationic amine group of the macrocyclic peptidic selector basket. The carbohydrate units are responsible for secondary interactions. However, they hinder somewhat the charge-charge docking interaction. The TAG selector is more effective for amino acid enantioseparations than the other CSPs. The "sugar" units are however useful allowing for chiral recognitions of other analytes, e.g., beta-blockers, not possible with the aglycone. Thermodynamic studies established that normal phase and reversed phase enantioseparations were enthalpy-driven. With polar waterless mobile phases used in the polar ionic mode, some separations were enthalpy-driven and others were entropy-driven. The linear solvation energy method was tentatively used to gain knowledge about the chiral recognition mechanism. It appeared to be a viable approach with neutral molecules but it failed with ionizable solutes. With molecular solutes and the teicoplanin CSP, the study showed a significant role of the surface charge-induced dipole interaction and steric effects. The remarkable complementary enantioselectivity effect observed with the four CSPs is discussed.     

Separation of drug enantiomers by liquid chromatography and capillary electrophoresis, using immobilized proteins as chiral selectors

Proteins display interesting chiral discrimination properties owing to multiple possibilities of intermolecular interactions with chiral compounds. This review deals with proteins which have been used as immobilized chiral selectors for the enantioseparation of drugs in liquid chromatography and capillary electrophoresis. The main procedures allowing the immobilization of proteins onto matrices, such as silica and zirconia particles, membranes and capillaries are first presented. Then the factors affecting the enantioseparation of drugs in liquid chromatography, using various protein-based chiral stationary phases (CSPs), are reviewed and discussed. Last, chiral separations already achieved using immobilized protein selectors in affinity capillary electrochromatography (ACEC) are presented and compared in terms of efficiency, stability and reproducibility.     

Enantiomeric Separations of Ruthenium (II) Polypyridyl Complexes Using HPLC With Cyclofructan Chiral Stationary Phases

The enantiomeric separation of 21 ruthenium (II) polypyridyl complexes was achieved with a novel class of cyclofructan‐based chiral stationary phases (CSPs) in the polar organic mode. Aromatic derivatives on the chiral selectors proved to be essential for enantioselectivity. The R‐napthylethyl carbamate functionalized cyclofructan 6 (LARIHC CF6‐RN) column proved to be the most effective overall, while the dimethylphenyl carbamate cyclofructan 7 (LARIHC CF7‐DMP) showed complementary selectivity. A combination of acid and base additives was necessary for optimal separations. The retention factor vs. acetonitrile/methanol ratio plot showed a U‐shaped retention curve, indicating that different interactions take place at different polar organic solvent compositions. The separation results indicated that π–π interactions, steric effects, and hydrogen bonding contribute to the enantiomeric separation of ruthenium (II) polypyridyl complexes with cyclofructan chiral stationary phases in the polar organic mode. Chirality 27:64–70, 2015. © 2014 Wiley Periodicals, Inc.     

Novel chiral stationary phases based on 3,5-dimethyl phenylcarbamoylated β-cyclodextrin combining cinchona alkaloid moiety

Novel chiral selectors based on 3,5-dimethyl phenylcarbamoylated β-cyclodextrin connecting quinine (QN) or quinidine (QD) moiety were synthesized and immobilized on silica gel. Their chromatographic performances were investigated by comparing to the 3,5-dimethyl phenylcarbamoylated β-cyclodextrin (β-CD) chiral stationary phase (CSP) and 9-O-(tert-butylcarbamoyl)-QN-based CSP (QN-AX). Fmoc-protected amino acids, chiral drug cloprostenol (which has been successfully employed in veterinary medicine), and neutral chiral analytes were evaluated on CSPs, and the results showed that the novel CSPs characterized as both enantioseparation capabilities of CD-based CSP and QN/QD-based CSPs have broader application range than β-CD-based CSP or QN/QD-based CSPs. It was found that QN/QD moieties play a dominant role in the overall enantioseparation process of Fmoc-amino acids accompanied by the synergistic effect of β-CD moiety, which lead to the different enantioseparation of β-CD-QN-based CSP and β-CD-QD-based CSP. Furthermore, new CSPs retain extraordinary enantioseparation of cyclodextrin-based CSP for some neutral analytes on normal phase and even exhibit better enantioseparation than the corresponding β-CD-based CSP for certain samples.     

Structure-activity relationships of carboxymethylpullulan-peptide-doxorubicin conjugates--systematic modification of peptide spacers

A series of carboxymethylpullulan (CMPul)-doxorubicin (DXR) conjugates bound by peptide spacers of different compositions and lengths were prepared and evaluated for their in vivo antitumor effects. Systematic study of the peptide spacers indicated that CMPul-DXR conjugates bound via appropriate dipeptide spacers were more potent than DXR.     

Enantiomeric Separation of Bicyclo[2.2.2]octane‐Based 2‐Amino‐3‐Carboxylic Acids on Macrocyclic Glycopeptide Chiral Stationary Phases

Direct high‐performance liquid chromatographic (HPLC) separation of four bicyclo[2.2.2]octane based 2‐amino‐3‐carboxylic acid enantiomers were developed on chiral stationary phases (CSPs) containing different macrocyclic glycopeptide antibiotic selectors. The analyses were performed under reversed‐phase, polar organic and polar ionic mode on macrocyclic‐glycopeptide‐based Chirobiotic T, T2, TAG, and R columns. The effects of the mobile phase composition including the acid and base modifier, the structure of the analytes, and the temperature on the separations were investigated. Experiments were achieved at constant mobile phase compositions on different stationary phases in the temperature range 5–40°C. Thermodynamic parameters were calculated from plots of ln k or ln α versus 1/T. It was recognized that the enantioseparations in reversed‐phase and polar organic mode were enthalpically driven, but under polar‐ionic conditions entropically driven enantioseparation was observed as well. Baseline separation and determination of elution sequence were achieved in all cases. Chirality 26:200–208, 2014. © 2014 Wiley Periodicals, Inc.     

Evaluation of dalbavancin as chiral selector for HPLC and comparison with teicoplanin‐based chiral stationary phases

Dalbavancin is a new compound of the macrocyclic glycopeptide family. It was covalently linked to 5 μm silica particles using two different binding chemistries. Approximately 250 racemates including (a) heterocyclic compounds, (b) chiral acids, (c) chiral amines, (d) chiral alcohols, (e) chiral sulfoxides and sulfilimines, (f) amino acids and amino acid derivatives, and (g) other chiral compounds were tested on the two new chiral stationary phases (CSPs) using three different mobile phases. As dalbavancin is structurally related to teicoplanin, the same set of chiral compounds was screened on two commercially available teicoplanin CSPs for comparison. The dalbavancin CSPs were able to separate some enantiomers that were not separated by the teicoplanin CSPs and also showed improved separations for many racemates. However, there were other compounds only separated or better separated on teicoplanin CSPs. Therefore, the dalbavancin CSPs are complementary to the teicoplanin CSPs. Chirality, 2010. © 2009 Wiley‐Liss, 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.     

Comparative HPLC Enantioseparation of Thirty‐Six Aromatic Compounds on Four Columns of the Lux® Series: Impact of Substituents,Shapes and Electronic Properties

With the aim to define a combined computational/chromatographic empirical approach useful for the high‐performance liquid chromatography (HPLC) method development of new chiral compounds, 36 racemic aromatic compounds with different chemical structures were used as test probes on four polysaccharide‐based chiral stationary phases (CSPs) of the Lux series, namely Lux Cellulose‐1, Lux Cellulose‐2, Lux Cellulose‐4, and Lux Amylose‐2, using classical n‐hexane/2‐propanol mixtures as mobile phase. Electrostatic potential surfaces (EPSs) determined using Density Functional Theory (DFT) calculations were used to derive size, shape, and electronic properties of each analyte. Then a comparative HPLC screening was carried out in order to evaluate the impact of substituents, shapes, and electronic properties of the analytes on the chromatographic behavior as the column changes. The four CSPs showed good complementary recognition ability. The elution sequence was determined in 30 cases out of 36. The success rate to afford baseline separations (R_s ≥ 1.5) was estimated: 29 compounds out of 36 showed baseline enantioseparation on at least one of the four selected CSPs. The combined computational‐chromatographic screening furnished useful collective structure‐chromatographic behavior relationships and a map of the chiral discrimination abilities of the considered CSPs towards the analytes. On this basis, the chromatographic behavior of new analytes on a set of polysaccharide‐based CSPs can be mapped through the qualitative correlation of chromatographic parameters (k, α, R_s) to computed molecular properties of the analytes. Chirality 25:709–718, 2013. © 2013 Wiley Periodicals, Inc.     

Solid-phase synthesis of chiral stationary phases based on 2,4,5,6-tetrachloro-1,3-dicyanobenzene derivatives spaced from N-3,5-dinitrobenzoyl alpha-amino acids: comparative study of their resolution efficacy

Two new chiral stationary phases, 3-[5-chloro-1,3-dicyano-2,4-[2'-(N'-1,3-dinitrobenzoyl-D-phenylglycinyl) aminoethyl]aminophen-1-yl] aminopropyl silica (CSP-1) and 3-[5-chloro-1,3-dicyano-2,4-[2'-(N'-1,3-dinitrobenzoyl-L-leucinyl) aminoethyl] aminophen-1-yl] aminopropyl silica (CSP-2), were prepared by solid-phase synthesis. They comprise chiral unit, 3,5-dinitrobenzoyl derivative of the amino acid, D-PhGly or L-Leu, bound via spacer 1,2-diaminoethane to 2,4-positions of the persubstituted benzene ring, derived from compound 1, and possess pseudo-C2 symmetry. Preparation of model compounds 6 and 7 confirmed the structure of chiral selectors, which comprise pi-donor persubsituted aromatic ring and two strong pi-acceptor 3,5-dinitrobenzoyl amido units. CD spectra of model selectors 6 and 7, run in DMSO above 250 nm, exhibit negative exciton coupling (EC) between pi-acceptor and pi-donor chromophores, C(1) symmetric model compound 8 exhibited much weaker EC and 9, devoid of pi-donor unit, does not exhibit any significant CD. Combined pi-donor and pi-acceptor properties enable the new CSPs to separate a broad range of racemates. The columns with CSP-1 and CSP-2 were tested for the separation of 22 racemates by HPLC with two different mobile phase systems and the results are compared with those obtained by using a structurally related commercial column.     

Chromatographic optical resolution on trans-1,2-diaminocyclohexane derivatives: Theory and applications

An overall view on some new chiral stationary phases based on (trans)-1,2-diaminocyclohexane is illustrated. The selected chiral moiety, derivatized with different aroyl groups, has been linked to a silica matrix in order to give chiral stationary phases (CSPs) enabling them to be used efficiently in the normal and reverse phase, both for analytical and preparative purposes. In addition new polymeric CSPs have been prepared by using the same selector, suitably modified, as monomer. The new chiral stationary phases have been characterised by physicochemical methods and used for the resolution of various racemic compounds classes such as α-aryloxyacetic acids, alcohols, sulfoxides, selenoxides, phosphinates, tertiaryphosphine oxides, benzodiazepines etc. without prederivatization or as amines, amino acids, amino alcohols, nonsteroidal antiinflammatory agents in a derivatized form. The separated solutes structural variety suggests that multiple interaction sites are involved in the recognition process: some thermodynamic data relative to the CSPs—selectands interactions are also illustrated. © 1992 Wiley-Liss, Inc.     

Force measurement for antigen-antibody interaction by atomic force microscopy using a photograft-polymer spacer

To determine the intermolecular force on protein-protein interaction (PPI) by atomic force microscopy (AFM), a photograft-polymer spacer for protein molecules on both surfaces of the substrate and AFM probe tip was developed, and its effectiveness was assessed in a PPI model of a pair of human serum albumin (HSA) and its monoclonal antibody (anti-HSA). A carboxylated photoiniferter, N-(dithiocarboxy)sarcosine, was derivatized on both surfaces of the glass substrate and AFM probe tip, and subsequently water-soluble nonionic vinyl monomers, N,N-dimethylacrylamide (DMAAm), were graft-polymerized on them upon ultraviolet light irradiation. DMAAm-photograft-polymerized spacers with carboxyl groups at the growing chain end but with different chain lengths on both surfaces were prepared. The proteins were covalently bound to the carboxyl terminus of the photograft-polymer chain using a water-soluble condensation agent. The effects of the graft-spacer length on the profile of the force-distance curves and on the unbinding characteristics (unbinding force and unbinding distance) were examined in comparison with those in the case of the commercially available poly(ethylene glycol) (PEG) spacer. The frequency of the nonspecific adhesion force profile was markedly decreased with the use of the photograft spacers. Among the force curves detected, a high frequency of single-peak curves indicating the unbinding process of a single pair of proteins and a very low frequency of multiple-peak profiles were observed for the photograft spacers, regardless of the graft chain length, whereas a high frequency of no-force peaks was noted. These observations were in marked contrast with those for the PEG spacer. The force peak values determined ranged from 88 to 94 pN, irrespective of the type of spacer, while the standard deviation of force distribution observed for the photograft spacer was lower than that for the PEG spacer, indicating that the photograft spacers provide a higher accuracy of force determination.     

Chromatographic properties of composite chiral stationary phases based on cellulose derivatives

Chiral stationary phases (CSPs) prepared by mixing together two different cellulose derivatives, before or after being coated on macroporous silica gel, were developed in order to determine the mutual influence of two different polymers on global chiral recognition capacity. The chromatographic properties of these CSPs were evaluated using a wide range of racemic test solutes. The mixing method does not significantly affect the enantioselectivities. The composite CSPs obtained by cocoating of two different cellulose derivatives on silica generally exhibit chiral recognition capacities intermediate between those of the two individual phases, and thus broadening the application range of a single column. These results indicate that the simultaneous coating of two different cellulose derivatives does not significantly alter the optical resolution power of each chiral material and are discussed in relationship with the supramolecular structure of the polymeric stationary phases. © 1995 Wiley-Liss, Inc.     

Enantiomeric separation of substituted 2-arylindoles on derivatised polysaccharide chiral stationary phases

The enantiomeric separation of a series of 2-arylindoles, developed as 5HT(2A) receptor antagonists for the treatment of schizophrenia, was investigated. Evaluation of a number of chiral stationary phases (CSPs) suggested that Chiralcel OD-H and Chiralpak AD were the most versatile for these compounds, and were employed for more detailed studies. A degree of complementarity between the CSPs was observed, such that Chiralcel OD-H was more effective for piperidine-containing molecules and Chiralpak AD for piperazine- and morpholine-containing molecules. The presence of a basic secondary amine was detrimental chromatographically, but resolution was improved substantially by employing diethylamine (DEA) in the mobile phase. All separations were either enthalpy-controlled or showed no temperature dependence. Differential temperature effects between series highlighted the possibility of multiple binding modes on these CSPs. Based on this study, it is possible to make a more rational selection of chromatographic conditions for future novel analogues.