Mechanisms of retention of pyrrolidinyl norephedrine on immobilized alpha(1)-acid glycoprotein

The HPLC separation of the R,S and S,R enantiomers of pyrrolidinyl norephedrine on immobilized alpha-1 glycoprotein (AGP) was investigated. Conditions for the separation were varied using a premixed mobile phase containing an ammonium phosphate buffer and an organic modifier. The influence of mobile phase pH, ionic strength, organic modifier composition, modifier type, and temperature on the chiral selectivity and retention were investigated. The presented data demonstrate that independent phenomena govern the enantioselectivity and retention. Retention is a function of both ion exchange equilibria and hydrophobic adsorption. Thermodynamic data derived from van't Hoff plots illustrates that while enantioselectivity is also enthalpically driven, the magnitude of the enthalpy term is governed by pH. Enantioselectivity has little dependence on ionic strength. Hydrophobic interactions appear to foster hydrogen bonding interactions; the two appear to be mutually responsible for chiral selectivity. The chiral selectivity decreases as the pH is decreased and increases with mobile phase buffer strength.     

Retentivity and enantioselectivity of uniformly sized molecularly imprinted polymers for d-chlorpheniramine and -brompheniramine in hydro-organic mobile phases

Uniformly sized molecularly imprinted polymers (MIPs) for d-chlorpheniramine (CP) and -brompheniramine (BP) have been prepared by a multi-step swelling and polymerization method using methacrylic acid (MAA) or 2-(trifluoromethyl)acrylic acid (TFMAA) and ethylene glycol dimethacrylate (EDMA) as a functional monomer and cross-linker, respectively. The retentive and enantioselective properties of CP, BP and their structurally related compounds on the MIPs were evaluated using hydro-organic mobile phases. CP and BP enantiomers were retained the most as a monovalent cation on MAA-co-EDMA polymers and a divalent cation on TFMAA-co-EDMA polymers. Ion exchange and hydrophobic interactions could mainly work for the retention and enantioseparation of CP and BP on both MAA-co-EDMA and TFMAA-co-EDMA polymers in hydro-organic mobile phases. Though the respective MIPs gave the highest enantioselectivity for the template molecule, cross-reactivity for CP and BP was observed with them.     

Comparative study of coated and immobilized polysaccharide-based chiral stationary phases and their applicability in the resolution of enantiomers

The enantioselective and chromatographic properties of Chiralpak AD and Chiralpak IA as well as those of Chiralcel OD and Chiralpak IB have been evaluated using a set of 48 compounds that differ in their physical and chemical properties. The impact of the different immobilisation methodologies of the chiral polysaccharide, i.e., coated or immobilized on retention and enantioselectivity was studied. The study on immobilized chiral stationary phases (CSPs) was expanded to also include mobile phases containing mixtures of alkanes and more non-conventional solvents such as ethyl acetate, ethers, acetone and dichloromethane. In this paper we report some of the general trends observed for the 48 racemic compounds with respect to retention, alpha and Rs. Further, the impact of the immobilisation methodology and the choice of the mobile phase on the elution order of the enantiomers is also discussed.     

Some mechanistic aspects on chiral discrimination of organic acids by immobilized bovine serum albumin (BSA)

Although chiral anionic compounds, notably a large number of organic acids, have been found to be readily separated into enantiomers on BSA-based columns, the structural requirements for an efficient enantiomer discrimination by the protein is still not very well known. Since it is often observed that very hydrophobic acids, like many of the antiinflammatory “profens,” can be resolved with large separation factors for the enantiomers, a systematic study of a series of racemic α-substituted alkanoic acids was made. The series of analytes was prepared from α-amino acids, RCH(NH₂)CO₂H (where R = C₁-C₆), by reaction with N-(chloroformyl)-carbazole. A rapid increase in the capacity ratios of both enantiomers was found with increasing length of R. The effect, however, was larger for the last eluted enantiomer, leading to a substantial increase in the separation factor; this being 7.3 for R = C₆ in 20 mM phosphate buffer (pH 8.0) with 30% of acetonitrile. Further, the separation factor also increased with decreasing organic modifier content. Thus when the R = C₆-analyte was run at a mobile phase concentration of 20% acetonitrile and a flow rate of 1.5 ml/min, the time difference between the two eluted enantiomers exceeded 20 hr. A reasonable interpretation of our results seems to be that enantioselectivity is promoted by increased hydrophobic interaction. Since the anionic charge of the analyte is also taking part in the retention mechanism, a tight binding of the analyte will result from simultaneous electrostatic and hydrophobic interaction. When the latter is increased, less conformational freedom will be left for the analyte and the steric configuration at the α-carbon atom will become more and more important. Steric hindrance by the α-substituent in the first eluted enantiomer will counteract the tight binding caused by the combined binding interactions and lead to a smaller increase in the capacity ratio.     

Investigation of a tartaric acid-based linear polyamide and dimer as chiral selectors in liquid chromatography

A twin selector for enantioselective liquid chromatography based on O,O'-bis(dimethyl)benzoyl tartaric diamide was synthesized and compared to commercially available Kromasil CHI-DMB (O,O'-bis(dimethyl)benzoyl tartaric diamide). A linear polyamide based on the same tartaric acid derivative was also synthesized, immobilized on silica, and evaluated as stationary phase. The twin selector was immobilized as a brush-type phase, with similar bonding chemistry as in Kromasil CHI-DMB. It was shown to exhibit lower resolution power than Kromasil CHI-DMB. However, retention and separation factors obtained on the respective sorbents were shown to exhibit interdependence. CD spectra of the twin selector give no indication that the respective branches interact in the solvent mixtures employed for chromatography. The linear polyamide showed lower enantioselectivity and higher retention than Kromasil CHI-DMB.     

Influence of endotoxin on the stereoselective pharmacokinetics of oxprenolol,propranolol, and verapamil in the rat

The influence of endotoxin-induced inflammation on the enantioselective pharmacokinetics of propranolol, oxprenolol, and verapamil, which bind to α₁-acid glycoprotein, was studied in the rat. The racemic mixtures were given orally. In the control animals, for propranolol and oxprenolol, the plasma concentrations of the (R)-enantiomer were higher than those of the (S)-enantiomer, while for verapamil the reverse was true. Protein binding and intrinsic clearance are the main factors responsible for this enantioselectivity. After endotoxin treatment, for the three drugs tested the plasma concentrations and the plasma binding of both enantiomers were significantly increased. This effect was more pronounced for (R)-propranolol, (R)-oxprenolol, and (S)-verapamil than for their respective antipodes. The enantioselective effect of endotoxin on the plasma concentrations of the drugs studied seems mainly due to the enantioselective increase in binding to α₁-acid glycoprotein. © 1994 Wiley-Liss, Inc.     

Structure‐retention relationship for enantioseparation of selected fluoroquinolones

Fluoroquinolones are popular class of antibiotics with distinct chemical functionality. Most of them are ampholytes with one chiral center. Stereogeneic center is located either in the side ring of Gatifloxacin (GFLX) or in the quinolone core of Ofloxacin (OFLX). These two amphoteric fluoroquinolones have terminal amino groups in common. The unusual Nadifloxacin (NFLX) is an acidic fluoroquinolone with a core chiral center. Owing to chirality and functionality differences among GFLX, OFLX, and NFLX, we mapped these enantiomers onto structure‐retention relationship. Amount of acetic acid modifier was studied in screened mobile phase and cellulose tris(3‐chloro‐4‐methyl phenyl carbamate) (Lux cellulose‐2) stationary phase. Experimental design of acetic acid% along with column temperature have been applied. Resolution and enantioselectivity have been related to structural features of the studied enantiomers. High amount of acid (0.4%) was optimum for the separation of either side chirality with a proximate amino group (GFLX) or core chirality without basic functionality (NFLX), while low amount (0.2%) is optimum for core chiral center with distal amino group (OFLX). Temperature has no significant effect on resolution and retention of enantiomers except for OFLX. Enantio‐retention explains possible chiral selective and nonselective interactions. The proposed methods have been validated for pharmaceutical analyses.     

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.     

Isolation of enantioselective α-hydroxyacid dehydrogenases based on a high-throughput screening method

To isolate enantioselective α-hydroxyacid dehydrogenases (α-HADHs), a high-throughput screening method was established. 2,4-Dinitrophenylhydrazine solution forms a red-brown complex with ketoacid produced during the α-HADH-mediated oxidation of α-hydroxyacid. The complex can be easily quantified by spectrophotometric measurement at 458?nm. The enantioselectivity of α-HADH in each strain can be measured with this colorimetric method using (R)- and (S)-α-hydroxyacid concurrently as substrates to evaluate the apparent enantioselectivity (E _app). The E _app closely matches the value of true enantioselectivity (E _true) determined by HPLC analysis. With this method, a total of 34 stains harboring enantioselective α-HADHs were selected from 526 potential α-HADH-producing microorganisms. Pseudomonas aeruginosa displayed the highest (S)-enantioselective α-HADH activity. This strain appears promising for potential application in industry to produce (R)-α-hydroxyacids. The method described herein represents a useful tool for the high-throughput isolation of enantioselective α-HADHs.     

Enantioselective phenylacetylene addition to aldehydes and ketones catalyzed by recyclable polymeric Zn(salen) complex

New polymeric Zn(salen) complex was employed in the enantioselective phenylacetylene addition to aldehydes and ketones to produce corresponding chiral secondary propargylic alcohols with yields (up to 96%) and enantioselectivity (up to 72%) and tertiary propargylic alcohols with yields (up to 79%) and enantioselectivity (up to 68%) at room temperature, with added advantage of four times reuse with retention of enantioselectivity.     

The stereochemical resolution of the enantiomers of aspartame on an immobilized alpha-chymotrypsin HPLC chiral stationary phase: the effect of mobile-phase composition and enzyme activity

The enantioselective and diastereoselective resolutions of the stereoisomers of N alpha-aspartyl-phenylalanine 1-methyl ester (APME) have been accomplished on an HPLC chiral stationary phase based upon alpha-chymotrypsin (the ACHT-CSP) with observed enantioselectivities (alpha 1) for the DL-/LD-enantiomer of as high as 29.17 and for the DD-/LL-enantiomers of as high as 28.97. In addition, the effect on the chromatographic retention of the APME stereoisomers of the activity of the ACHT and the composition of the mobile phase--structure of the anionic component, molarity, and pH--have been studied. The results of this study suggest that the aspartyl moiety and/or the aspartyl-phenylalanine amide linkage play key roles in the observed enantioselectivity; the APME stereoisomers containing L-phenylalanine, i.e., DL- and LL-APME, bind at a different site in the ACHT molecule (the L-Phe site) than the APME stereoisomers containing D-phenylalanine (the D-Phe site); and the observed enantioselectivity is a measure of the difference in the binding affinities at the two sites rather than the consequence of differential affinities at a single site.     

Elucidation of the enantioselective recognition mechanism of a penicillin G acylase-based chiral stationary phase towards a series of 2-aryloxy-2-arylacetic acids

A series of structurally related 2-aryloxy-2-arylacetic acids (1-3, 5-16) together with a thioisostere derivative (4) have been synthesized and characterized by GC-MS and 1H NMR. The designed compounds were analyzed on a Penicillin G Acylase chiral stationary phase (PGA-CSP) and the influence of the structure variations on retention and enantioselectivity was investigated. The chromatographic study includes the direct separation of the enantiomers of the synthesized compounds and the determination of the elution order of selected racemic mixtures. 10 out of 16 racemates were separated; high chromatographic enantioseparation factors (alpha > 2) were achieved for some compounds. For the enantiomers of four compounds whose absolute configuration was known (1, 3, 12, 16), the elution order was R:S with the exception of 2-(4-chloro-phenoxy)phenylacetic acid (1), for which the elution order was reversed. Preliminary molecular modeling studies suggest that both polar and charge-transfer interactions as well as steric effects play an important role in determining the retention factors and the enantioselectivities observed.     

Exploring enantiospecific ligand-protein interactions using cellular membrane affinity chromatography: chiral recognition as a dynamic process

The chiral recognition mechanisms responsible for the enantioselective binding on the alpha3beta4 nicotinic acetylcholine receptor (alpha3 beta4 nAChR) and human organic cation transporter 1 (hOCT1) have been reviewed. The results indicate that chiral recognition on the alpha3beta4 nAChR is a process involving initial tethering of dextromethorphan and levomethorphan at hydrophobic pockets within the central lumen followed by hydrogen bonding interactions favoring dextromethorphan. The second step is the defining enantioselective step. Studies with the hOCT1 indentified four binding sites within the transporter that participated in chiral recognition. Each of the enantiomers of the compounds used in the study interacted with three of these sites, while (R)-verapamil interacted with all four. Chiral recognition arose from the conformational adjustments required to produce optimum interactions. With respect to the prevailing interaction-based models, the data suggest that chiral recognition is a dynamic process and that the static point-based models should be amended to reflect this.     

Paralogous gene analysis reveals a highly enantioselective 1,2-O-isopropylideneglycerol caprylate esterase of Bacillus subtilis.

Carboxylesterase NP of Bacillus subtilis Thai I-8, characterized in 1992 as a very enantioselective (S)-naproxen esterase, was found to show no enantiopreference towards (S)-1,2-O-isopropylideneglycerol (IPG) esters. The ybfK gene was identified by the B. subtilis genome project as an unknown gene with homology to carboxylesterase NP. The purpose of the present study was to characterize the ybfK gene product in order to determine whether this paralogue of carboxylesterase NP had an altered or enhanced stereospecificity. The ybfK gene was cloned and expressed in B. subtilis using a combination of two strong promoters in a multicopy vector. The enzyme was purified from the cytoplasm of B. subtilis by means of anion exchange and hydrophobic interaction chromatography. The purified YbfK is an enzyme of 296 amino acids and shows an apparent molecular mass of 32 kDa (SDS/PAGE). Comparison of the activities of YbfK and carboxylesterase NP towards caprylate esters of IPG revealed that YbfK produces (S)-IPG with 99.9% enantioselectivity. Therefore, we conclude that we have isolated a paralogue of carboxylesterase NP that can be used for the enantioselective production of (S)-IPG.     

Penicillin G acylase as chiral selector in LC and CE: exploring the origins of enantioselectivity

The review examines the most recent achievement of immobilized Penicillin G acylase (PGA) as chiral stationary phases (PGA-CSP) for the separation of acidic enantiomers. Particular attention is paid to the influence of structural variations of a large number of analytes on retention and enantioselectivity with a special emphasis on advances in the elucidation of the chiral recognition mechanism. Docking calculations and molecular dynamic simulations are discussed to rationalize the origins of enantioselective behaviour. The review also touches the chiral behaviour of PGA in partial filling CE. The CE results, obtained using PGA in free solution, suggest that the immobilization procedure used in the development of PGA-CSPs did not alter the enantioselective properties of the enzyme.     

Elucidation of vancomycin's enantioselective binding site using its copper complex

Vancomycin forms a stable complex with Cu²⁻ in neutral aqueous solutions. The enantioselectivity of native vancomycin was compared to that of the copper-vancomycin complex using capillary electrophoresis (CE). There were significant differences in their enantioselectivities. This can be attributed to the fact that copper ion coordinates with some of the same functional groups in vancomycin that are essential for chiral recognition and enantioresolution. An amine moiety that provides one of the more important enantioselective interactions was identified. This chiral interaction site was illustrated using a color-coded, space-filling model of the X-ray crystal structure of the copper-vancomycin complex. Successful enantioselective interactions at lower pHs were attributed to the partial dissociation of the copper-vancomycin complex. Chirality 8:590–595, 1996. © 1997 Wiley-Liss, Inc.     

High performance liquid chromatography of leukotriene C4 on the aminopropyl sorbent

Chromatographic behaviour of leukotriene C4, its methyl ester and hydrophobic side products of their synthesis on an aminopropyl sorbent has been investigated, the retention time values being determined by both hydrophobic and ionic interaction of the substances with the sorbent. Optimal conditions of the separation are determined.     

Chaperone-like functions of high-mannose type and complex-type N-glycans and their molecular basis

It has recently become apparent that high-mannose type N-glycans directly promote protein folding, whereas complex-type ones play a crucial role in the stabilization of protein functional conformations through hydrophobic interactions with the hydrophobic protein surfaces. Here an attempt was made to understand more deeply the molecular basis of these chaperone-like functions with the aid of information obtained from spacefill models of N-glycans. The promotion of protein folding by high-mannose N-glycans seemed to be based on their unique structure, which includes a hydrophobic region similar to the cyclodextrin cavity. The promotive features of high-mannose N-glycans newly observed under various conditions furnished strong support for the view that both intra- and extramolecular high-mannose N-glycans are directly involved in the promotion of protein folding in the endoplasmic reticulum. Further, it was revealed that the N-acetyllactosamine units in complex-type N-glycans have an amphiphilic structure and greatly contribute to the formation of extensive hydrophobic surfaces and, consequently, to the N-glycan-protein hydrophobic interactions. The processing of high-mannose type N-glycans to complex-type ones seems to be an ingenious device to enable the N-glycans to perform these two chaperone-like functions.     

Simultaneous enantioselective separation of azelastine and three of its metabolites for the investigation of the enantiomeric metabolism in rats. I. Liquid chromatography-ionspray tandem mass spectrometry and electrokinetic capillary chromatography

Enantioselective separation methods and the enantioselective determination of the anti-allergic drug azelastine and of three of its main phase I metabolites in a biological matrix underwent chromatographic and electrophoretic investigations. An enantioselective assay of a coupling of HPLC using a beta-cyclodextrin chiral stationary phase to ionspray tandem mass spectrometry is presented. Additionally, this assay is compared to another enantioselective assay using electrokinetic capillary chromatography with beta-cyclodextrin and carboxymethyl-beta-cyclodextrin in polyacrylamide-coated capillaries. For capillary electrophoresis (CE) the importance of polyacrylamide coating for the validation of this separation method is highlighted. Extracted rat plasma samples of enantioselective metabolism studies were measured by both validated assays. Differences in the pharmacokinetics and pharmacodynamics were evaluated for the main substance azelastine and its main metabolite demethylazelastine. So, a first hint about the enantioselectivity of biotransformation of azelastine in rats was seen after oral application of either enantiomer or the racemate to rats.