Chiral Stationary Phase HPLC Research Articles

Assignment of absolute configuration to an improved enantioselective naproxen selector

AbstractAssignment of absolute configuration to a recently developed chiral selector useful in the separation of the underivatized enantiomers of naproxen and other nonsteroidal anti‐inflammatory drugs (NSAIDs) is described. Circular dichroism, 1H NMR, and X‐ray diffraction have been used to confirm the original assignment which was based solely upon elution orders from HPLC chiral stationary phases. All of these techniques agree in the assignment of the (S,S) absolute configuration to the enantiomer of the chiral selector which associates preferentially with (S)‐naproxen. © 1994 Wiley‐Liss, Inc.

Self-Normalized Analysis of Lipase-Catalyzed Conversion of Naproxen Enantiomers

Abstract Improved equations were derived to determine the conversion of Naproxen enantiomers or racemate by lipase-catalyzed esterification in an organic solvent. From the resultant ratio of peak areas for Naproxen and Naproxen ester, an accurate quantification of the conversion was obtained by a commercially available HPLC chiral stationary phase (CSP).

On-line determination of lipase activity and enantioselectivity using an immobilized enzyme reactor coupled to a chiral stationary phase

Lipase has been immobilized on an artificial immobilized membrane HPLC phase and the enzyme reactor coupled to a HPLC chiral stationary phase. The resulting system was used for on-line determination of the enantioselectivity of the immobilized enzyme. Enantioselective hydrolyses using lipase from Candida cylindracea immobilized on an IAM HPLC support.

An amylopectin tris(phenylcarbamate) chiral stationary phase for high performance liquid chromatography

AbstractAmylopectin tris(phenylcarbamate) has been evaluated as a chiral stationary phase for HPLC; the influence on its cptical resolving capabilities of mobile phase composition and nature of the alcohol used as modifier has been studied. Separation and resolution of twelve arylalcohol racemates were examined. In most instances, the stationary phase exhibited high optical resolving capacity.

Comparison of Avidin and Ovomucoid as Chiral Selectors for the Resolution of Drug Enantiomers by High-Performance Liquid Chromatography

The effects of the pH, organic modifiers and buffer salt of the mobile phase upon the retention and separation characteristics of avidin- and ovomucoid-bonded silica columns as proteinous chiral stationary phases for HPLC were investigated. The enantioselectivity was greatly affected by changing the mobile-phase composition, possibly because of induced alterations in the protein conformation. Hydrophobic and Coulombic interactions play major roles in the retention, and there are some similarities between the avidin column and the ovomucoid column. However, the stereoselectivity does not necessarily reflect the retention processes on these columns. No significant decrease in the retention or enantioselectivity was observed, even after the injection of 200 samples.

Evaluation of 3,5-Dimethylphenyl Carbamoylated α-, β-, and γ-Cyclodextrins as Chiral Stationary Phases for HPLC

Abstract Optical resolving power of chiral stationary phases (CSPs) containing 3,5-dimethylphenyl carbamoylated β-cyclodextrin chemically bonded to silica was compared with those of two commercial stationary phases containing carbamoylated β-cyclodextrin (β-CD), Cyclobond I SN and Cyclobond I DMP. For most of examined 14 racemates, higher selectivities were obtained on our CSPs than on the commercial CSPs. This may be ascribed to the higher degree of substitution of carbamate groups on β-CD and to the opposite orientation of the immobilized β-CD on our materials. The present CSPs were evaluated under reverse, normal and supercritical fluid chromatographic conditions. The highest selectivities were usually obtained under normal phase chromatographic conditions. The influence of cyclodextrins, α-, β- and γ-CD, on the enantioselectivity was compared. Each CSP containing different cyclodextrin showed the highest enantioselectivity for different racemates. By using silica particles with different average pore ...

The Use of Displacement Chromatography to Alter Retention and Enantioselectivity on a Human Serum Albumin-Based HPLC Chiral Stationary Phase: A Mini-Review

Abstract The chromatographic properties of protein-based HPLC chiral stationary phases (CSPs) have been manipulated using a variety of neutral and charged mobile phase modifiers. These compounds have usually been alkyl alcohols and amines, acetonitrile and octanoic acid. This mini-review discusses another approach to the control of chromatographic retention (k′) and enantioselectivity (α) on a human serum albumin based-CSP, HSA-CSP, which utilizes the binding characteristics of the protein. The addition to the mobile phase of compounds known to bind to HSA such as warfarin, ibuprofen and tryptophan altered k′ and α for a variety of solutes. In some instances, direct competition reduced these variables, but the opposite result was also obtained due to allosteric interactions. These interactions, their chromatographic applications and pharmacological implications are examined in this presentation.

Identification of xyloside conjugates formed from anthracene by Rhizoctonia solani

Biotransformation experiments showed that a strain of Rhizoctonia solani was able to metabolize anthracene, a tricyclic aromatic hydrocarbon. The fungus was grown in a complex liquid medium containing [9-14C]anthracene; after 6 d, 98·8% of the anthracene had been converted to ethyl acetate-extractable metabolites. These compounds were separated by high-performance liquid chromatography (hplc) and detected by ultraviolet (uv) absorbance and liquid scintillation counting. The major metabolites were identified by their uv, mass, and nuclear magnetic resonance spectra. One of the principal metabolites was identified as trans-1,2-dihydroxy-1,2-dihydroanthracene (anthracene trans-1,2-dihydrodiol), which was shown by circular dichroism spectroscopy to be a mixture of two enantiomers. Chiral stationary phase hplc was used to resolve the trans-1,2-dihydrodiol into a (−)-1S,2S enantiomer (60%) and a (+)-1R,2R enantiomer (40%). The other principal metabolites were novel xyloside conjugates of anthracene: 1-O-(2-hydroxy-trans-1,2-dihydroanthryl)-β- d -xylopyranoside, 2-O-(1-hydroxy-trans-1,2-dihydroanthryl)-β- d -xylopyranoside, and 1-O-anthryl-β- d -xylopyranoside. Anthraquinone was found in the culture media but was also present in non-inoculated controls.

Quantitative structure-enationselective retention relationships for the chromatography of 1,4-benzodiazepines on a human serum albumin based HPLC chiral stationary phase: An approach to the computational prediction of retention and enantioselectivity

Quantitative structure-enantiospecific retention relation-ships (QSERR) have been derived for a series of 1,4-benzodiazepines. The compounds were chromatographed on a human serum albumin based HPLC chiral stationary phase (HSA-CSP). Molecular modeling of the solutes allowed the determiantion of various structural descriptors. Among these descriptors, a submolecular polarity parameter, PSM, was identified which was able to characterize enantiospecific interactions of benzodiazepines with the HSA-CSP. Combining PSM with the retention parameter of the less retained enantiomer permitted precise prediction of the retention of the second eluting enantiomer. Highly statistically significant regression equations were also derived which described the retentions of both enantiomers in terms of nonempirical molecular descriptors. The calculated enantioselectivity correlated well with the experimentally observed values. A significant conclusion from this study is support for the existence of two different binding sites for chiral benzodiazepines on HSA.

A simple asymmetric synthesis of 2-substituted pyrrolidines and 5-substituted pyrrolidinones

An efficient procedure for the preparation of the title compounds in high enantiomeric purity has been realized starting from 3-acylpropionic acids. Stereoselective reduction of chiral bicyclic lactams 2a-h, prepared from the corresponding γ-keto acid and (R)-phenylglycinol, using alane or triethylsilane with titanium tetrachloride provided the N-substituted pyrrolidines and pyrrolidinones, respectively. Subsequent cleavage of the phenylglycinol returned the desired amines and lactams. The enantiomeric purity of these compounds was determined to be >98% by chiral stationary-phase HPLC

Synthesis of Optically Active Polymethacrylates Bearing Axially Dissymmetric 1,1′-Binaphthalene Skeleton as a Pendant Group and Their Optical Resolution Ability as Chiral Adsorbent for HPLC

Abstract Optically active polymethacrylates 6 and 8, which have pendant axially dissymmetric 1,1′-binaphthalene moiety, were synthesized by radical polymerization of (S)-2-methacryloyloxy- and (S)-2-(3-methacryloyloxypropoxy)-2′-methoxy-1,1′-binaphthalene, respectively. Specific rotation and CD spectrum of 6 are rather different from those of the model compound 2-(2,2-dimethylpropanoyloxy)-2′-methoxy-1,1′-binaphthalene (7), while the chiroptical properties of 8 are almost the same as those of the corresponding model compound. The difference of the chiroptical properties between 6 and 7 was attributed to the difference of the dihedral angles between the naphthalene planes of the 1,1′-binaphthalene skeleton. The polymethacrylates 6 and 8 were coated on a spherical silica gel to give HPLC chiral stationary phases (CSP-6 and CSP-8). These CSPs differentiated several enantiomeric 1-aryl-1-alkanols and 1,2-diols as the 3,5-dinitrophenylcarbamates, CSP-8 showing better resolution than CSP-6. Furthermore, (S)-2-(5-carboxypentyloxy)-2′-methoxy-1,1′-binaphthalene, a model compound of 8, was prepared and covalently bonded to an aminopropylsilanized silica gel to afford CSP-16, which also resolved these racemates. Thus, the chiral discriminating ability of the polymethacrylate 8 would be based not on the secondary and/or higher-ordered structure, but mainly on the interaction between the individual 1,1′-binaphthalene units of 8 and the racemates.

Synthesis, separation and absolute configuration assignment to enantiomers of 1,3-benzodithiole 1-oxide and 2-alkyl-1,3-benzodithiole 1-oxides

1,3-Benzodithiole 1-oxide, 5, has been resolved into enantiomers using chiral stationary phase high performance liquid chromatography. Alkylation of an excess (25%) of the late eluting (+)-(1R) enantiomer of the sulfoxide 5 under basic conditions yielded both cis and trans isomers of 2-methyl (6cis-1R,2S′,6trans-1R,2R), 2-ethyl (7cis-1R,2S′, 7trans-1R,2R) and 2-isopropyl (8cis-1R,2S′, 8trans-1R,2R)-benzodithiole 1-oxide (25% e.e.) and allowed a stereochemical correlation of absolute configuration between the sulfoxides 5–8.Treatment of racemic 1,3-benzodithiole 1-oxide (51S/51R) with potassium bis(trimethylsilyl)amide and (S)-(+)-1-iodo-2-methylbutane yielded four diastereoisomers of 2-(2′-methylbutyl)-1,3-benzodithiole 1-oxide (9cis-1R,2S,2′S′, 9cis-1S,2R,2′S′, 9trans-1S,2S,2′S, 9trans-1R,2R,2′S) which were separated by chiral stationary phase HPLC (CSP-HPLC). The most strongly retained diastereoisomer was analysed by X-ray crystallography and was assigned the (1S:2S:2′S) absolute configuration. Further alkylation of 9trans-1S,2S,2′S under similar conditions yielded 2-(2′S-methylbutyl)-2-(2″S-methylbutyl)-1,3·benzodithiole 1-oxide 10 of (1S,2′S,2″S) configuration exclusively. The same stereoisomer of the sulfoxide 10 was also obtained by dialkylation of the early eluting (–)-enantiomer of 1,3-benzodithiole 1-oxide (51S) using (+)-1-iodo-2-methylbutane, thus enabling the unequivocal establishment of the absolute configurations of the enantiomers of 1,3-benzodithiole 1-oxides listed in Table 1. A comparison of circular dichroism (CD) spectra for sulfoxides 5–9 indicates that this method may also be used to correlate absolute configurations of 2-alkyl substituted 1,3-benzodithiole 1-oxides.

Chromatographic Separation of Underivatized Naproxen Enantiomers

Abstract The enantiomers of underivatized Naproxen are resolved on a previously described commercially available HPLC chiral stationary phase (CSP). This same CSP also separates the enantiomers of a variety of ester and amide derivatives of Naproxen.

Polysaccharide derivatives as chiral stationary phases in HPLC

AbstractTwenty different tris(phenylcarbamate)s of cellulose were synthesized and evaluated as chiral stationary phases for HPLC. Optical resolving power of the tris(phenylcarbamate)s depends on the substituents introduced on the phenyl groups. Optical resolving abilities of amylose tris(phenylcarbamate)s were also evaluated. In most cases, either cellulose tris(3,5‐dimethylphenylcarbamate) or amylose tris(3,5‐dimethylphenylcarbamate) exhibited the highest optical resolving ability. Aralkylcarba‐mates such as benzyl‐ and 1‐phenylethylcarbamates of cellulose and amylose were also tested as chiral stationary phases. (S)‐1‐Phenylethylcarbamate of amylose showed a high optical resolving power.

Restrictive cardiomyopathy.

Rates of hydrolysis of racemic and enantiomeric lorazepam 3-acetates (LZA) by esterases in human and rat liver microsomes and rat brain S9 fraction were compared. LZA and its hydrolysis product were analyzed by chiral stationary phase HPLC. When rac-LZA was the substrate, the (R)-LZA was hydrolyzed 2.7-fold and 6.8-fold faster than the (S)-LZA by esterases in rat and human liver microsomes, respectively. In contrast, esterases in rat brain S9 fraction were enantioselective toward the (S)-LZA. The specific activities (nmol of LZA hydrolyzed/mg protein/min) of liver microsomes in the hydrolysis of enantiomerically pure (R)-LZA were approximately 210 (rat) and 1330 (human), and in the hydrolysis of enantiomerically pure (S)-LZA were 25 (rat) and 8 (human). The specific activities of rat brain S9 fraction in the hydrolysis of enantiomerically pure (R)-LZA and (S)-LZA were approximately 3 and 6 nmol/mg protein/min, respectively. Results also indicated an enantiomeric interaction in the hydrolysis of rac-LZA; the presence of (R)-LZA stimulated the hydrolysis of (S)-LZA by all esterase preparations, whereas the presence of (S)-LZA stimulated the hydrolysis of (R)-LZA in rat brain S9 fraction and inhibited the hydrolysis of (R)-LZA in rat and human liver microsomes.

Synthesis and chromatographic properties of an HPLC chiral stationary phase based upon human serum albumin

A new HPLC stationary phase was synthesized by thein situ covalent immobilization of human serum albumin (HSA). The protein was immobilized on a commerically available diol column which had been activated with 1,1-carbonyldiimidazole. Initial chromatographic studies show that this phase can be used for chiral separations of enantiomeric solutes and that these separations may reflectin vitro binding to the HSA. The effects of mobile phase composition and temperature on the stereochemical resolutions are reported.

The stereochemical resolution of the enantiomers of aspartame on an immobilized α‐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.

1-Hydroxy- and 2-hydroxy-3-methylcholanthrene: regioselective and stereoselective formations in the metabolism of 3-methylcholanthrene and enantioselective disposition in rat liver microsomes

Absolute configurations of enantiomeric 1-hydroxy-3-methylcholanthrene (1-OH-3MC) and 2-hydroxy-3-methyl-cholanthrene (2-OH-3MC) were determined by the exciton chirality circular dichroism (CD) method as their p-nitrobenzoate derivatives. Enantiomers of 1-OH-3MC were resolved by HPLC using a column packed with chiral stationary phase (CSP) (R)-N-(3,5-dinitrobenzoyl)phenylglycine covalently bonded to gamma-aminopropylsilanized silica. Enantiomers of 2-OH-3MC were resolved as diastereomeric (-)-methoxyacetates by normal-phase HPLC. 1-OH-3MC and 2-OH-3MC, formed in the metabolism of 3MC by liver microsomes from untreated, phenobarbital (PB)-treated and 3MC-treated male Sprague-Dawley rats, were first isolated as a mixture by reversed-phase HPLC and subsequently separated by normal-phase HPLC. Concentration ratios of [1-OH-3MC]:[2-OH-3MC] formed in the metabolism of 3MC by three rat liver microsomal preparations (at 0.5 mg protein per ml of incubation mixture and an incubation time of 10 min) were found to be: 30:70 (control), 21:79 (PB treated) and 10:90 (3MC treated) respectively. R/S enantiomer ratios of 1-OH-3MC formed in the metabolism of 3MC by three rat liver microsomal preparations were determined by CSP HPLC: 35:65 (control), 39:61 (PB treated) and 46:54 (3MC treated) respectively. R/S enantiomer ratios of 2-OH-3MC formed in the metabolism of 3MC by three rat liver microsomal preparations were determined by CD spectral data: 14:86 (control), 6:94 (PB treated) and 6:94 (3MC treated) respectively. Metabolism of racemic 1-OH-3MC and 2-OH-3MC by all three rat liver microsomal preparations was found to be substrate enantioselective; the rate of 1S-OH-3MC metabolism was faster than that of 1R-OH-3MC, whereas the rate of 2R-OH-3MC metabolism was faster than that of 2S-OH-3MC.

Absolute configuration ofcis‐5,6‐dihydrodiol enantiomers derived from helical conformers of 1,12‐dimethylbenz[a]anthracene

1,12-Dimethylbenz[a]anthracene (1,12-DMBA) cis-5,6-dihydrodiol was synthesized by oxidation of 1,12-DMBA with osmium tetroxide in pyridine in low yield (less than or equal to 3%) and was purified by sequential use of reversed-phase and normal-phase HPLC. Two pairs of 1,12-DMBA cis-5,6-dihydrodiol enantiomers, derived from P (right-handed helix) and M (left-handed helix) conformers, were eluted as a single chromatographic peak on both reversed-phase and normal-phase HPLC. However, these four enantiomers were resolved by sequential use of two chiral stationary phase (CSP) HPLC columns. CSP (Pirkle type I) columns were packed with either (R)-N-(3,5-dinitrobenzoyl)phenylglycine or (S)-N-(3,5-dinitrobenzoyl)leucine, which is ionically bonded to gamma-aminopropylsilanized silica. Absolute configurations of enantiomers were determined by comparing their circular dichroism spectra with those of conformationally similar cis-5,6-dihydrodiol enantiomers of 4-methylbenz[a]anthracene and 7,12-dimethylbenz[a]anthracene with known absolute stereochemistry.

Immobilized serum albumin: Rapid HPLC probe of stereoselective protein‐binding interactions

A human serum albumin-based HPLC chiral stationary phase (HSA-CSP) has been examined as a tool to investigate binding of chiral drugs to HSA and drug-drug protein-binding interactions. Rac-oxazepam hemisuccinate (OXH) was used as a model compound and the chromatographic retention (k') of its enantiomers was determined after addition of displacers to the mobile phase. Compounds known to bind at the same site as OXH and at different sites were tested for their displacing capacities. Competitive binding interactions between the OXH enantiomers and displacers in the mobile phase were reflected by decreases in the k's of (R)- and (S)-OXH. The results indicate that retention on the HSA-CSP accurately reflects binding to native HSA and the technique can determine enantioselective and competitive binding interactions at specific sites on HSA. The HSA-CSP was also able to recognize separate binding areas for (S)- and (R)-OXH.