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.     

Chiral separation and CD characterisation of enantiomeric cyclotriphosphazene derivatives

The gem-disubstituted cyclotriphosphazene 1 reacted with piperazine (pip) to give the piperazine-bridged derivative 2, which is expected to exist in meso and racemic forms because the two PCl (pip) groups are stereogenic. The proton-decoupled (31)P NMR spectrum of 2 gave rise to two similar sets of ABX signals in a 1:1 ratio, consistent with formation of diastereoisomers. The meso and racemic forms of compound 2 were separated by column chromatography on silica gel and characterised by elemental analysis, mass spectrometry, (31)P NMR spectroscopy, and X-ray crystallography. Using HPLC with a chiral stationary phase, the racemic form of compound 2 was further separated into enantiomers, which were characterised by circular dichroism (CD) spectroscopy. This is the first report of the separation of enantiomers in the field of cyclophosphazene chemistry and hence the first CD spectra of derivatives in which the cyclophosphazene ring is at the chiral centre.     

A surface molecularly imprinted polymer as chiral stationary phase for chiral separation of 1,1′‐binaphthalene‐2‐naphthol racemates

Acrylamide (AM) was copolymerized with ethylene glycol dimethacrylate (EGDMA) in the presence of (R )‐1,1′‐binaphthalene‐2‐naphthol (BINOL) as the template molecules on the surface of silica gel by a free radical polymerization to produce a chiral stationary phase based on the surface molecularly imprinted polymer (SMIP‐CSP). The SMIP‐CSP showed a much better separation factor (α = 4.28) than the CSP based on the molecularly imprinted polymer (MIP‐CSP) without coating on the silica gel (α = 1.96) during the chiral separation of BINOL enantiomers by high‐performance liquid chromatography. The influence of the pretreatment temperature and the content of the template molecule ((R )‐BINOL) of the SMIP‐CSP, and the mobile phase composition on the separation of the racemic BINOL were systematically investigated.     

Practical enantioresolution of alcohols with 2-methoxy-2-(1-naphthyl)propionic acid and determination of their absolute configurations by the (1)H NMR anisotropy method.

The enantioresolution of racemic alcohols as esters of 2-methoxy-2-(1-naphthyl)propionic acid (MalphaNP acid 1) and the determination of their absolute configurations on the basis of (1)H NMR anisotropy effect are described. The enantiopure MalphaNP acid (S)-(+)-1 was allowed to react with racemic 2-alkanols and 1-octyn-3-ol, yielding diastereomeric mixtures of esters, which were easily separated by HPLC on silica gel. To determine the absolute configurations of the first-eluted diastereomeric esters by the (1)H NMR anisotropy method, the general scheme was proposed. Separated esters were reduced with LiAlH(4) or hydrolyzed with KOH/EtOH to recover enantiopure alcohols.     

Enantioresolution by the chiral phthalic acid method: absolute configurations of substituted benzylic alcohols

Substituted benzylic alcohols were enantioresolved by the chiral phthalic acid method as follows; 1) esterification of racemic alcohols with chiral phthalic acid, 2) separation of a diastereomeric mixture of the esters formed by HPLC on silica gel, and 3) recovery of enantiopure alcohols from the separated esters. The absolute configurations of chiral phthalic acid esters of benzylic alcohols were unambiguously determined by the X-ray crystallography using the campharsultam moiety as the internal standard of absolute configuration.     

Investigation of the stereoselective in vitro metabolism of the chiral antiasthmatic/antiallergic drug flezelastine by high-performance liquid chromatography and capillary zone electrophoresis

An achiral HPLC method using a silica gel column as well as two independent chiral analytical methods by HPLC and capillary zone electrophoresis (CZE) were developed in order to investigate the in vitro metabolism of the racemic antiasthmatic/antiallergic drug flezelastine. The chiral HPLC analysis was performed on a Chiralpak AD column, which also allowed the simultaneous separation of the N-dephenethyl metabolite. The chiral separation by CZE was achieved by the addition of β-cyclodextrin to the run buffer. The stereoselectivity of the in vitro biotransformation of flezelastine was investigated using liver homogenates of different species. Depending on the species, diverse stereoselective aspects were demonstrated. The determination of the enantiomeric ratios of flezelastine and of N-dephenethylflezelastine after incubations of racemic flezelastine with liver microsomes revealed that porcine liver microsomes showed the greatest enantioselectivity of the biotransformation. (−)-Flezelastine was preferentially metabolized. After incubations with bovine liver microsomes the enantiomer of N-dephenethylflezelastine formed from (+)-flezelastine dominated. Incubations of the pure enantiomers of flezelastine with induced rat liver microsomes resulted in the stereoselective formation of a hitherto unknown metabolite, which was only detected in samples of (+)-flezelastine. Initial structure elucidation of the compound indicated that the new     

MalphaNP acid, a powerful chiral molecular tool for preparation of enantiopure alcohols by resolution and determination of their absolute configurations by the (1)H NMR anisotropy method

A novel methodology using a chiral molecular tool of MalphaNP acid (1), 2-methoxy-2-(1-naphthyl)propionic acid, useful for preparation of enantiopure secondary alcohols and determination of their absolute configurations by the (1)H NMR anisotropy method was developed; racemic MalphaNP acid (1) was enantioresolved with (-)-menthol, and the enantiopure MalphaNP acid (S)-(+)-(1) obtained was allowed to react with racemic alcohol, yielding a mixture of diastereomeric esters, which was clearly separated by HPLC on silica gel. By applying the sector rule of (1)H NMR anisotropy effect, the absolute configuration of the first-eluted MalphaNP ester was unambiguously determined. Solvolysis or reduction of the first-eluted MalphaNP esters yielded enantiopure alcohols.     

Synthesis of dendritic stationary phases with surface-bonded L-phenylalanine derivate as chiral selector and their evaluation in HPLC resolution of racemic compounds

Four dendrimers were synthesized on aminopropyl-modified silica gel using methyl acrylate and ethylene diamine as building blocks by divergent method. Four generations of chiral stationary phases (CSPs) were prepared by coupling of L-2-(p-toluenesulfonamido)-3-phenylpropionyl chloride to corresponding dendrimers. The derivatives prepared on silica gel were characterized by FT-IR, (1)H NMR, and elemental analysis. The selector loadings of these four generations of CSPs generally showed a decrease tendency with the increase of generation numbers of dendrimers. The enantioseparation properties of these CSPs were preliminarily investigated by high-performance liquid chromatography. The CSP derived from the three-generation dendrimer exhibited the best enantioseparation capability. Effects of the mobile phase composition and molecular structures of racemic mixtures on enantioseparation were further studied.     

Capillary zone electrophoresis in normal or reverse polarity separation modes for the analysis of hydroxy acid oligomers in neutral phosphate buffer

Capillary zone electrophoresis (CZE) with neutral phosphate buffer as the background electrolyte was used to analyse water-soluble oligomers obtained by polycondensation of racemic lactic acid. Two CZE separation modes were tested. The first mode was based on normal separation (injection at the anodic side) using a fused-silica capillary. Eight peaks were observed within a 60-min migration time range. They were ascribed to dimer and higher water-soluble oligomers. Peaks from dimer to tetramer were split due to sensitivity for the fine structures at the level of the distribution of chiral lactic acid moieties in oligomer chains. The second mode was based on reverse separation (injection at the cathodic side) using a fused-silica capillary modified by adsorption of a polycation on its inner wall. Under these conditions, oligomers were rapidly separated without peak splitting. Considering the forces which are involved in CZE, data were plotted as a function of 1/t scale, according to the equation [signal]=f((−1)^k/t) where k=0 and k=1 for normal and reverse separation modes, respectively. Such a plot allowed direct comparison between the various runs after a simple translation along the 1/t axis, regardless of the separation mode and the variation of electroosmotic flow. The second separation mode allowed separation of 3-hydroxybutyric acid and 6-hydroxyhexanoic acid oligomers. For the former series of oligomers, a side reaction generating crotyl bonds was observed due to the high sensitivity of CZE. It was shown that separation was governed by the ratio charge/mass of the oligoesters whatever their structure.     

Preparation of enantiopure Wieland-Miescher ketone and derivatives by the MalphaNP acid method: substituent effect on the HPLC separation

Enantiopure Wieland-Miescher ketone (4, W-M ketone) and derivatives were prepared by the enantioresolution with 2-methoxy-2-(1-naphthyl)propionic acid (MalphaNP acid 1). Various racemic derivatives of 4 were esterified with acid (S)-(+)-1 yielding diastereomeric MalphaNP esters, which were separated by HPLC on silica gel. It was clarified that the HPLC separation of diastereomers depended on the substituent of the derivatives, leading to the working hypothesis that MalphaNP acid esters of alcohols with less polar and more bulky aliphatic substituents are more effectively separated. The best separation was obtained in the case of tert-butyldimethylsilyl (TBDMS) ether derivative (12a/12b): separation factor alpha=1.80, and resolution factor, Rs=1.30. The (1)H NMR spectra of separated MalphaNP esters showed anomalously large magnetic anisotropy effects, from which their absolute configurations were determined. Solvolysis or reduction of the separated MalphaNP esters yielded alcohols, which were converted to enantiopure W-M ketones 4. The results thus provided another route for preparation of enantiopure ketones (8aR)-(-)-4 and (8aS)-(+)-4.     

Impact of substituents on the enantioseparation of racemic 2-amidotetralins on polysaccharide stationary phases. I. chiralcel OD

The direct enantiomeric separation of 32 racemic 2-amidotetralins on the commercially available tris-(3,5-dimethylphenylcarbamate) derivative of cellulose, coated on silica gel (Chiralcel OD), is presented. To date, the selection of a column for the chiral separation of a racemic mixture is done empirically. Studying the impact of small changes in the chemical structure of a series of amidotetralins on the separation behavior may help to give an insight in the chiral recognition mechanism. The amidotetralins differed structurally in three of their substituents, which were never directly located on the chiral carbon atom. The enantiomers of 24 out of 32 amidotetralins could be resolved with a resolution >1.5. Hydrogen bonding and π-π interactions are supposed to be the major analyte-chiral stationary phase (CSP) interactions. However, the spatial arrangement of the enantiomers may play an important role too. Increasing the bulkiness of the acyl substituent led to an increase in the resolution (R_S), whereas a more bulky substituent on the aromatic ring resulted in a very low resolution. The introduction of a chlorine atom into the acyl substituent additionally increased the resolving power. Chirality 8:574–578, 1996. © 1997 Wiley-Liss, Inc.     

Preparation and application of a new doubly tethered chiral stationary phase containing N-CH3 amide linkage based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid

A new doubly tethered chiral stationary phase (CSP 5) based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was developed by attaching the second tethering group to silica gel through a carbon atom of the first tethering group of the corresponding singly tethered CSP (CSP 2) containing an N-CH3 tertiary amide linkage, which was previously developed in our laboratory, in order to enhance the CSP stability without the loss of chiral recognition efficiency. The new CSP was quite effective in the resolution of various racemic alpha-amino acids, amines, and amino alcohols, and the chiral recognition efficiency of the new CSP was even greater than that of the corresponding singly tethered CSP especially in terms of the resolution factors (RS). The stability of the new CSP was greater than that of the corresponding singly tethered CSP. The chromatographic resolution behaviors of the new CSP were generally consistent with those of the corresponding singly tethered CSP.     

Versatile methods for the synthesis of mixed-acid 1,2-diacylglycerols

Two methods for synthesizing mixed-acid 1,2-diacylglycerols starting from 2,3-epoxy-1-propanol (glycidol) or 3-chloro-1,2-propanediol have been described. This first method involves fatty acid addition to a protected glycidol derivative and solid-state isomerization. The second approach exploits the specificity of the trityl group for primary alcohols and the nucleophilic replacement of chlorine by a carboxylate ion in an aprotic solvent. The second method proves to be more general: with 3-chloro-1-O-trityl-1,2-propanediol as an intermediate compound apparently all types of mixed-acid, saturated and unsaturated, chiral and racemic 1,2-diacylglycerols can be prepared in good yields. In the first method tritylglycidol is a good starting compound. The use of this method, however, is restricted because only the 2-position of glycerol can be occupied with an unsaturated fatty acid. For de-blocking protected 1,2-diacylglycerols, the trityl group and other protecting groups were exchanged for the trifluoroacetyl group, which group could then be removed without any detectable acyl migration (< 1%). To this end, the 1,2-diacyl-3-trifluoroacetyl-glycerols were dissolved at room temperature in methanol containing pyridine, whereby the trifluoroacetyl group was split off, giving the 1,2-diacylglycerol.     

Separation of major polar lipids in Pecten maximus by high-performance liquid chromatography and subsequent determination of their fatty acids using gas chromatography

An easy method for the separation of major polar lipid classes by HPLC is described. Maximum resolution was achieved by an automated combination of a silica gel column and a diol column. Polar lipid analysis of the larvae and gonads of Pecten maximus showed the presence of a particular glycolipid especially rich in 22:6(n-3) and the predominance of 20:4(n−6) in the phosphatidylinositol. The phosphatidylcholine and phosphatidylethanolamine (diacyl form + alkenylacyl) were the major fractions. The plasmalogen form (25% in larvae, 34% in gonads) was essentially composed of polyunsaturated fatty acids of 20 and 22 carbons in the sn-2 position.     

Separation of neutral lipids and free fatty acids by high-performance liquid chromatography using low wavelength ultraviolet detection

Normal phase, isocratic high-performance liquid chromatography methods are described for the separation of neutral lipid and fatty acid classes using low wavelength detection. Prior to high-performance liquid chromatography, methods were developed and are described for the separation of phospholipids from neutral lipids and fatty acids using small (600 mg) silica Sep-PaksTM. Recoveries of cholesteryl esters, triglycerides, fatty acids, and phospholipids from the silica columns were greater than 95%. Two mobile phases are described for lipid class separation by high-performance liquid chromatography. The first mobile phase, hexane-2-propanol-acetic acid 100:0.5:01, resulted in incomplete separation of cholesteryl ester and triglyceride but excellent separations of fatty acids and cholesterol. The second mobile phase, hexane-n-butyl chloride-acetonitrile-acetic acid 90:10:1.5:0.01, resulted in complete separation of the four lipid classes. This mobile phase also separated individual triglycerides and fatty acids based on the number of double bonds. Recoveries of radiolabeled lipids for the four lipid classes from high-performance liquid chromatography was greater than 95% with both mobile phases.     

Evaluation of comprehensive multidimensional separations using reversed-phase, reversed-phase liquid chromatography/mass spectrometry for shotgun proteomics

Two-dimensional liquid-chromatographic (LC) separation followed by mass spectrometric (MS) analysis was examined for the identification of peptides in complex mixtures as an alternative to widely used two-dimensional gel electrophoresis followed by MS analysis for use in proteomics. The present method involves the off-line coupling of a narrow-bore, polymer-based, reversed-phase column using an acetonitrile gradient in an alkaline mobile phase in the first dimension with octadecylsilanized silica (ODS)-based nano-LC/MS in the second dimension. After the first separation, successive fractions were acidified and dried off-line, then loaded on the second dimension column. Both columns separate peptides according to hydrophobicity under different pH conditions, but more peptides were identified than with the conventional technique for shotgun proteomics, that is, the combination of a strong cation exchange column with an ODS column, and the system was robust because no salts were included in the mobile phases. The suitability of the method for proteomics measurements was evaluated.     

Separation of glycoprotein-derived oligosaccharides by thin-layer chromatography.

A thin-layer chromatography (tlc) system has been developed for the separation of glycoprotein-derivedoligosaccharides. The method involves chromatography on silica gel using n-propanol/acetic acid/water (3:3:2 v/v) as the solvent. This tlc method was used to separate pathological oligosaccharides isolated from individuals with G_M1 gangliosidosis and with neuraminidase deficiency. The results indicate the potential usefulness of the system in the analysis of complex carbohydrates.     

Separation of [1-3H]cellooligosaccharides by thin-layer chromatography: assay for cellulolytic enzymes

A thin-layer chromatographic method for the separation with high resolution of [1-3H]cellooligosaccharides on silica gel plates has been developed. Reducing end-labeled glucose through cellohexaose were separated on silica gel plates with three ascents of ethyl acetate:water: methanol (40:15:20; v:v) and each was extracted with an efficiency of 88 +/- 3%. Separations of cellooligosaccharides using other adsorbents, solvents, and impregnants are also described. This thin-layer chromatographic method facilitated analysis of the activity of cellulolytic enzymes.     

Determination of ((R)-(+)- and (S)-(−)-isomers of thiopentone in plasma by chiral high-performance liquid chromatography

A method for the determination of (R)-(+)- and (S)-(−)-isomers of thiopentone in plasma was developed. Following liquid-liquid extraction, the separation of enantiomers of thiopentone and the internal standard (racemic ketamine) was achieved by high-performance liquid chromatography on an α₁-acid glycoprotein (AGP) column with ultraviolet detection at 280 nm. The mobile phase consisted of 20 mM KH₂PO₄ buffer-propanol-methanol (93.5:5.0:1.5) at pH 5.0. The flow-rate was 0.9 ml/min. The limit of quantification for earch isomer was approximately 10 ng/ml. The assay is suitable for pharmacokinetic studies of (R)-(+)- and (S)-(−)-isomers of thiopentone, following usual bolus intravenous clinical doses of the racemic drug.     

Determination of some pyridinium aldoxime compounds by means of ion-pair reversed-phase high-performance liquid chromatography: Application in biological material

Two reversed-phase high-performance liquid chromatographic systems are presented for the separation and assay of the pyridinium aldoximes benzyl-P2A, HI-6 and obidoxime in aqueous solutions and biological samples. The systems involve a 5-μm C₁₈ silica gel stationary phase. The eluent consists of methanol, acetic acid buffer (pH 4.80), a counter ion (perchlorate or n-octanesulphonate) and a surfactant. The compounds were detected spectrophotometrically at 304 nm. In the concentration range used, linear plots of concentration versus extinction were obtained, both in blood and in water. Detection limits, even in blood, are satisfactory (0.5–1 μM).Evidence is presented that, at least for HI-6, the addition of counter ions to the system does not lead to the formation of ion pairs to be retained by partition, but rather to a mechanism based on adsorption chromatography.