Chiral High-pressure Liquid Chromatography Research Articles

Chiral organic stereochemistry: Chiral HPLC, chiral auxiliaries, CD spectroscopy, X-ray crystallography, and light-powered chiral molecular motors.

In the studies of chiral organic stereochemistry, it is important to use enantiopure compounds. For this purpose, the chiral HPLC (High-Pressure Liquid Chromatography) columns containing chiral stationary phases were invented by Y. Okamoto and coworkers for enantio-separating various racemic compounds. In addition, the use of chiral auxiliaries is also useful for preparing enantiopure compounds and also for determining their absolute configurations, where covalent-bonded diastereomers are separated by HPLC on silica gel. In this review article, these HPLC methods will be discussed together with the applications to some interesting organic compounds including light-powered chiral molecular motors.

Enantiomeric chromene derivatives with anticancer effects from Mallotus apelta

Mallotusapelta(Lour.) Müll.Arg has been used in traditional medicine for the treatment of chronic hepatitis. Six new chromene derivatives, malloapeltas C-H (1–6) and one known compound, malloapelta B (7) were isolated and structured from the leaves of M.apelta. Two pairs of enantiomers (1a/1b and 2a/2b) were successfully separated by chiral high-pressure liquid chromatography (HPLC). The structures and absolute configurations of compounds were determined using spectroscopic methods, including 1D, 2D NMR, and MS and quantum chemical calculation methods. All compounds were evaluated for cytotoxic activity using cell counting kit-8 (CCK-8) assay against ovariancancer cell line (TOV-21G). Compounds 1–5 and 7 exhibited significant growth and viability inhibitory effects with GI50 values ranging from 0.06 to 10.39 μM and IC50 values ranging from 1.62 to 10.42 μM on ovarian cancer cell line, TOV-21G. The most cytotoxic compounds 2, 3, and 7 were chosen for studying in apoptosis mechanism. Compounds 2, 3, and 7-induced apoptosis as evidenced by activated caspase 8, caspase 9, and PARP, increased Bak and Bax, and decreased Bcl-xL and survivin. Moreover, compounds 2, 3, and 7 significantly inhibited the NF-κB signaling pathway. Taken together, our findings propose the potential application of compounds 2, 3, and 7 for treating cancer via modulating NF-κB activity.

A Pharmacological Comparison of Two Isomeric Nicotinic Receptor Agonists: The Marine Toxin Isoanatabine and the Tobacco Alkaloid Anatabine.

Many organisms possess “secondary” compounds to avoid consumption or to immobilize prey. While the most abundant or active compounds are initially investigated, more extensive analyses reveal other “minor” compounds with distinctive properties that may also be of biomedical and pharmaceutical significance. Here, we present an initial in vitro investigation of the actions of two isomeric tetrahydropyridyl ring-containing anabasine analogs: isoanatabine, an alkaloid isolated from a marine worm, and anatabine, a relatively abundant minor alkaloid in commercial tobacco plants. Both compounds have a double bond that is distal to the piperidine ring nitrogen of anabasine. Racemic isoanatabine and anatabine were synthesized and their S- and R-enantiomers were isolated by chiral high pressure liquid chromatography (HPLC). Both isoanatabines displayed higher efficacies at α4β2 nicotinic acetylcholine receptors (nAChRs) relative to the anatabines; R-isoanatabine was most potent. Radioligand binding experiments revealed similar α4β2 nAChR binding affinities for the isoanatabines, but R-anatabine affinity was twice that of S-anatabine. While the two anatabines and S-isoanatabine were highly efficacious agonists at α7 nAChRs, R-isoanatabine was only a weak partial agonist. The four compounds share an ability to stimulate both α4β2 and α7 nAChRs, a property that may be useful in developing more efficacious drugs to treat neurodegenerative and other medical disorders.

Enantiomeric Isoflavones with neuroprotective activities from the Fruits of Maclura tricuspidata

Seven pairs of enantiomeric isoflavones (1a/1b–7a/7b) were obtained from the ethyl acetate extract of the fruits of Maclura tricuspidata (syn. Cudrania tricuspidata), and successfully separated by chiral high-pressure liquid chromatography (HPLC). The structures and absolute configurations of the enantiomeric isoflavones were established on the basic of comprehensive spectroscopic analyses and quantum chemical calculation methods. Compounds 1, 1a, and 1b exhibited neuroprotective activities against oxygen-glucose deprivation/reoxygenation (ODG/R)-induced SH-SY5Y cells death with EC50 values of 5.5 µM, 4.0 µM, and 10.0 µM, respectively. Furthermore, 1, 1a, and 1b inhibited OGD/R-induced reactive oxygen species generation in SH-5Y5Y cells with IC50 values of 6.9 µM, 4.5 µM, and 9.5 µM, respectively.

An Integrated Lab‐on‐a‐chip Approach to Study Heterogeneous Enantioselective Catalysts at the Microscale

AbstractAn integrated lab‐on‐a‐chip enables the rapid analysis of heterogenized enantioselective organocatalysis at the microscale. A packed‐bed microreactor was seamlessly integrated with a downstream chiral high pressure liquid chromatography (HPLC) functionality to study enantioselective transformations on a single microfluidic glass chip. Hyphenation to mass spectrometry allows for the rapid investigation of the selectivity and the substrate scope of microgram amounts of catalyst beads. Optimization of reaction conditions is possible with minimal reagent consumption and instant analytical feedback.

Papain Catalyzed Oligomerization in Monophasic Aqueous Organic Media - Synthesis and Characterization of Neutral and Polar Amino Acid Oligomers

Synthesis of oligopeptides is generally carried out in aqueous or bi-phasic reaction media; use of monophasic reaction media is quite limited. In this study, homo-oligopeptides of lysine (Lys), glycine (Gly), methionine (Met) and tyrosine (Tyr) were synthesized through papain-catalyzed reaction in monophasic systems consisting of acetonitrile/ water. Reaction conditions were optimized for peptide bond formation and to minimize enzyme denaturation. Such media are especially attractive because they can minimize the secondary and reverse hydrolysis of the acyl complex and the oligopeptide, respectively. The synthesized oligopeptides were purified and characterized by reversed phase liquid chromatography (RPLC) and Electrospray ionization mass spectrometry (ESI-MS). The yields of oligopeptides were approximately 80% for all amino acids. The stereo specificity of papain in acetonitrile/water media was also investigated. The separated enantiomers of methionine were characterized using Chiral High-Pressure Liquid Chromatography (HPLC). The results indicate the L-specificity of papain is maintained in monophasic media.

Enantioselective pharmacokinetics of doxazosin and pharmacokinetic interaction between the isomers in rats.

In this study, the stereoselective pharmacokinetics of doxazosin enantiomers and their pharmacokinetic interaction were studied in rats. Enantiomer concentrations in plasma were measured using chiral high-pressure liquid chromatography (HPLC) with fluorescence detection after oral or intravenous administration of (-)-(R)-doxazosin 3.0 mg/kg, (+)-(S)-doxazosin 3.0 mg/kg, and rac-doxazosin 6.0 mg/kg. AUC values of (+)-(S)-doxazosin were always larger than those of (-)-(R)-doxazosin, regardless of oral or intravenous administration. The maximum plasma concentration (Cmax ) value of (-)-(R)-doxazosin after oral administration was significantly higher when given alone (110.5 ± 46.4 ng/mL) versus in racemate (53.2 ± 19.7 ng/mL), whereas the Cmax value of (+)-(S)-doxazosin did not change significantly. The area under the curve (AUC) and Cmax values for (+)-(S)-doxazosin after intravenous administration were significantly lower, and its Cl value significantly higher, when given alone versus in racemate. We speculate that (-)-(R)-doxazosin increases (+)-(S)-doxazosin exposure probably by inhibiting the elimination of (+)-(S)-doxazosin, and the enantiomers may be competitively absorbed from the gastrointestinal tract. In conclusion, doxazosin pharmacokinetics are substantially stereospecific and enantiomer-enantiomer interaction occurs after rac-administration.

A Selective, Slow Binding Inhibitor of Factor VIIa Binds to a Nonstandard Active Site Conformation and Attenuates Thrombus Formation in Vivo

The serine protease factor VIIa (FVIIa) in complex with its cellular cofactor tissue factor (TF) initiates the blood coagulation reactions. TF.FVIIa is also implicated in thrombosis-related disorders and constitutes an appealing therapeutic target for treatment of cardiovascular diseases. To this end, we generated the FVIIa active site inhibitor G17905, which displayed great potency toward TF.FVIIa (Ki = 0.35 +/- 0.11 nM). G17905 did not appreciably inhibit 12 of the 14 examined trypsin-like serine proteases, consistent with its TF.FVIIa-specific activity in clotting assays. The crystal structure of the FVIIa.G17905 complex provides insight into the molecular basis of the high selectivity. It shows that, compared with other serine proteases, FVIIa is uniquely equipped to accommodate conformational disturbances in the Gln217-Gly219 region caused by the ortho-hydroxy group of the inhibitor's aminobenzamidine moiety located in the S1 recognition pocket. Moreover, the structure revealed a novel, nonstandard conformation of FVIIa active site in the region of the oxyanion hole, a "flipped" Lys192-Gly193 peptide bond. Macromolecular substrate activation assays demonstrated that G17905 is a noncompetitive, slow-binding inhibitor. Nevertheless, G17905 effectively inhibited thrombus formation in a baboon arterio-venous shunt model, reducing platelet and fibrin deposition by approximately 70% at 0.4 mg/kg + 0.1 mg/kg/min infusion. Therefore, the in vitro potency of G17905, characterized by slow binding kinetics, correlated with efficacious antithrombotic activity in vivo.

Regio- and stereoselective metabolism of 7,12-dimethylbenz[a]anthracene by Mycobacterium vanbaalenii PYR-1.

The degradation of 7,12-dimethylbenz[a]anthracene (DMBA), a carcinogenic polycyclic aromatic hydrocarbon, by cultures of Mycobacterium vanbaalenii PYR-1 was studied. When M. vanbaalenii PYR-1 was grown in the presence of DMBA for 136 h, high-pressure liquid chromatography (HPLC) analysis showed the presence of four ethyl acetate-extractable compounds and unutilized substrate. Characterization of the metabolites by mass and nuclear magnetic resonance spectrometry indicated initial attack at the C-5 and C-6 positions and on the methyl group attached to C-7 of DMBA. The metabolites were identified as cis-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a]anthracene (DMBA cis-5,6-dihydrodiol), trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a]anthracene (DMBA trans-5,6-dihydrodiol), and 7-hydroxymethyl-12-methylbenz[a]anthracene, suggesting dioxygenation and monooxygenation reactions. Chiral stationary-phase HPLC analysis of the dihydrodiols showed that DMBA cis-5,6-dihydrodiol had 95% 5S,6R and 5% 5R,6S absolute stereochemistry. On the other hand, the DMBA trans-5,6-dihydrodiol was a 100% 5S,6S enantiomer. A minor photooxidation product, 7,12-epidioxy-7,12-dimethylbenz[a]anthracene, was also formed. The results demonstrate that M. vanbaalenii PYR-1 is highly regio- and stereoselective in the degradation of DMBA.

Identification of Calcium-independent Phospholipase A2 (iPLA2) β, and Not iPLA2γ, as the Mediator of Arginine Vasopressin-induced Arachidonic Acid Release in A-10 Smooth Muscle Cells: ENANTIOSELECTIVE MECHANISM-BASED DISCRIMINATION OF MAMMALIAN iPLA2s

The agonist-stimulated release of arachidonic acid (AA) from cellular phospholipids in many cell types (e.g. myocytes, beta-cells, and neurons) has been demonstrated to be primarily mediated by calcium-independent phospholipases A(2) (iPLA(2)s) that are inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL). Recently, the family of mammalian iPLA(2)s has been extended to include iPLA(2)gamma, which previously could not be pharmacologically distinguished from iPLA(2)beta. To determine whether iPLA(2)beta or iPLA(2)gamma (or both) were the enzymes responsible for arginine vasopressin (AVP)-induced AA release from A-10 cells, it became necessary to inhibit selectively iPLA(2)beta and iPLA(2)gamma in intact cells. We hypothesized that the R- and S-enantiomers of BEL would possess different inhibitory potencies for iPLA(2)beta and iPLA(2)gamma. Accordingly, racemic BEL was separated into its enantiomeric constituents by chiral high pressure liquid chromatography. Remarkably, (S)-BEL was approximately an order of magnitude more selective for iPLA(2)beta in comparison to iPLA(2)gamma. Conversely, (R)-BEL was approximately an order of magnitude more selective for iPLA(2)gamma than iPLA(2)beta. The AVP-induced liberation of AA from A-10 cells was selectively inhibited by (S)-BEL (IC(50) approximately 2 microm) but not (R)-BEL, demonstrating that the overwhelming majority of AA release is because of iPLA(2)beta and not iPLA(2)gamma activity. Furthermore, pretreatment of A-10 cells with (S)-BEL did not prevent AVP-induced MAPK phosphorylation or protein kinase C translocation. Finally, two different cell-permeable protein kinase C activators (phorbol-12-myristate-13-acetate and 1,2-dioctanoyl-sn-glycerol) could not restore the ability of A-10 cells to release AA after exposure to (S)-BEL, thus supporting the downstream role of iPLA(2)beta in AVP-induced AA release.

In vitro studies on the initial reactions of anaerobic ethylbenzene mineralization.

Anaerobic mineralization of ethylbenzene by the denitrifying bacterium Azoarcus sp. strain EB1 was recently shown to be initiated by dehydrogenation of ethylbenzene to 1-phenylethanol. 1-Phenylethanol is converted to benzoate (benzoyl coenzyme A) via acetophenone as transient intermediate. We developed in vitro assays to examine ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities in cell extracts of this strain. With p-benzoquinone as the electron acceptor, cell extracts of Azoarcus sp. strain EB1 catalyzed ethylbenzene oxidation at a specific rate of 10 nmol min(-1) [mg of protein](-1) and an apparent K(m) for ethylbenzene of approximately 60 microM. The membrane-associated ethylbenzene dehydrogenase activity was found to oxidize 4-fluoroethylbenzene and propylbenzene but was unable to transform 4-chloro-ethylbenzene, the ethyltoluenes, and styrene. Enzymatic ethylbenzene oxidation was stereospecific, with (S)-(-)-1-phenylethanol being the only enantiomer detected by chiral high-pressure liquid chromatography analysis. Moreover, cell extracts catalyzed the oxidation of (S)-(-)-1-phenylethanol but not of (R)-(+)-1-phenylethanol to acetophenone. When cell extracts were dialyzed, (S)-(-)-1-phenylethanol oxidation occurred only in the presence of NAD(+), suggesting that NAD(+) is the physiological electron acceptor of 1-phenylethanol dehydrogenase. Both ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities were present in Azoarcus sp. strain EB1 cells that were grown anaerobically on ethylbenzene, 1-phenylethanol, and acetophenone, but these activities were absent in benzoate-grown cells.

Monohydroxylated fatty acids in mouse epidermis papilloma quantification and stereochemical characterization.

Two-stage chemical carcinogenesis in mouse epidermis by an initiation/promotion protocol based on topical DMBA/TPA application is accompanied by a strong activation of 8(S)- and 12 (S)-lipoxygenase activity (DMBA: dimethylbenzanthracene, TPA: tetradecanoyl-phorbol-lB-acetate) (1–3). Using high pressure liquid chromatography (HPLC), a new method for quantitative profiling of monohydroxylated long-chain fatty acids by gas chromatography-(electron impact) mass spectrometry (GC-(posEI)MS) (4) and chiral phase HPLC (CP-HPLC) (5), we have addressed the question, whether or not this induction of lipoxygenase activity is accompanied by increased epidermal tissue levels of free hydroxylated fatty acids.

Interaction of β2-adrenoceptor agonists with native cyclodextrins: Application to the development of chiral assays for terbutaline

The enantioselectivity of the complexation between β2-adrenoceptor agonists of different structural types and native cyclodextrins (α, β and γ-CDs) has been examined by including CDs as chiral modifiers in high pressure liquid chromatography (HPLC) and in capillary zone electrophoresis (CZE). The best enantiomer separation was obtained for terbutaline with β-CD in both HPLC and CZE systems (resolution (Rs) of 1.0 and 1.1 respectively). The chiral HPLC assay for terbutaline was validated and gave linear standard curves of peak height versus concentration for both enantiomers (r2 > 0.99) with y-intercepts through the origin and intra- and interday coefficients of variation of less than 3% at 100 μg ml−1. Only the enantiomers of the resorcinol type β2-agonists terbutaline and orciprenaline were separated in the CZE and HPLC systems, suggesting the position of substituents in the aromatic ring of β2-agonists has a profound effect on the enantioselectivity of complexation of these drugs with β-CD.

Occurrence and biosynthesis of 11 (R)-hydroxy-eicosatetraenoic acid (11-R-HETE) in the Caribbean soft coralPlexaurella dichotoma

Gorgonian soft corals from the Caribbean Sea are known to contain prostaglandin-like compounds as well as other products of arachidonic acid lipoxygenation, and the formation of the latter has been suggested to represent the first step in prostaglandin biosynthesis. Here we present evidence for the presence of 11-R-hydroxy-5Z, 8Z, 12E, 14Z-eicosatetraenoic acid (11-R-HETE), as well as of the enzyme responsible for its biosynthesis, in the Caribbean gorgonianPlexaurella dichotoma. Lipid extracts fromP. dichotoma were purified by conventional SiO2 column chromatography followed by reversed phase high-pressure liquid chromatography (HPLC). These yielded a component having chromatographic and spectroscopic properties identical to synthetic 11-HETE. Electron impact mass spectrometric analysis of the acetoxy-, methyl ester derivative of the compound confirmed its identity as 11-HETE, while chiral phase HPLC of the methyl ester derivative showed that the stereochemistry of the alcoholic carbon atom wasR. Enzymatically active homogenates fromP. dichotoma were able to convert both unlabelled and [3H] arachidonic acid into 11-HETE. In vitro biosynthesis of the latter metabolite was also observed with homogenates of the Mediterranean gorgonianParamuricea clavata, another non-prostaglandin-containing soft coral, thus suggesting that 11-R-HETE production is not necessarily accompanied by prostaglandin formation in gorgonian corals.

Thioridazine enantiomers in human tissues

Enantiomers of thioridazine (TRZ) were determined in postmortem tissues obtained from a patient on chronic TRZ therapy by sequential achiral and chiral high pressure liquid chromatography. Tissue concentrations of (+)-TRZ found in liver, brain, bile and blood were: 6.46, 0.40, 0.48 and 0.07 mg/l or mg/kg, respectively. Concentrations of (−)-TRZ in liver, brain, bile and blood were: 12.2, 0.81, 1.07 and 0.20 mg/l or mg/kg, respectively. These data demonstrate the stereoselective disposition of TRZ in human tissues.

Chromatographic determination of optical configuration of 3-hydroxy fatty acids composing microbial surfactants

The chromatographic determination of the optical configuration of 3-hydroxy fatty acids of microbial surfactants was achieved in chiral high pressure liquid chromatography (HPLC) by injecting 3,5-dinitroaniline-derivatives of crude hydrolysates (less than 1 mg). Serrawettin W2, a surface-active cyclodepsipeptide of Serratia marcescens, was shown to contain d-3-hydroxydecanoic acid. Rubiwettin R1 and RG1, surface active glycolipid and linked fatty acids of Serratia rubidaea, were shown to contain D-3-hydroxytetradecanoic acid and D-3-hydroxydecanoic acid. The new method does not require purified sample or authentic optical isomers, and could be useful in the structural analysis of microbial lipids.

The stereospecific determination of fluoxetine and norfluoxetine enantiomers in human plasma by high-pressure liquid chromatography (HPLC) with fluorescence detection.

A quantitative method for the simultaneous HPLC resolution and detection of the enantiomers of (R,S) fluoxetine (F) and their metabolites (R,S) norfluoxetine (N) in human plasma has been developed. F is a serotonin uptake inhibitor used in the treatment of depression and is administered as a racemate. After liquid-liquid extraction and derivatization with (R) napthyl ethyl isocyanate (NEI), the separation and detection of the resultant diasteriomers were achieved using normal phase HPLC and fluorescence. The four NEI diastereomers and the internal standard [(-)-N-methyl-gamma-(2-methylphenoxy) benzenepropanamine hydrochloride], representing the enantiomers S-F, R-F, S-N, and R-N were resolved within 15 min. The assay for each analyte was linear using two concentration ranges of 1-10 and 10-500 ng/ml of human plasma. The precision and accuracy are reported as the coefficient of variation (%CV) and relative error (%RE). The sum of the chiral HPLC results from plasma samples were compared to the achiral gas chromatographic/electron capture (GC/EC) results. The correlation between these two methods, for total F and N, resulted in r2 values of 0.98 and 0.89, respectively. The chiral HPLC method is currently being applied to clinical studies for the evaluation of the enantiomeric disposition of F.

PLASMA CONCENTRATIONS OF THE STEREOISOMERS OF PRILOCAINE AFTER ADMINISTRATION OF THE RACEMATE: IMPLICATIONS FOR TOXICITY?

A chiral high pressure liquid chromatography method was developed to measure the separate isomers of prilocaine in plasma after administration of the racemate. The concentrations of the isomers in six patients were similar (S(+)/R(-) = 1.06 (SD 0.06)) after brachial plexus block with 1.5% (RS)-prilocaine hydrochloride 35 ml, suggesting that a higher systemic safety margin may not be achieved by substituting racemic prilocaine by one of its isomers. Much higher plasma concentrations of the S(+)- than the R(-)-form after oral administration of 300 mg of the racemate (n = 4) indicated a large difference in intrinsic metabolic clearance of the isomers on first pass through gut, liver or both organs.

Diethylstilbestrol Metabolites and Analogs: Stereochemical probes for the estrogen receptor binding site

Indenestrol A (IA) and indenestrol B (IB) are analogs and metabolites of diethylstilbestrol (DES). These compounds have high binding affinity with the estrogen receptor (ER) but possess weak uterotropic activity. Due to their chemical structures, IA and IB exist as mixtures of enantiomers. We investigated whether the poor biological activity of these compounds was due to differential activity of the enantiomers. We also utilized these compounds as probes to determine the extent of stereochemical sensitivity in the ER ligand binding site. The IA and IB enantiomers were separated to greater than 98% purity using a chiral high pressure liquid chromatography column. Their enantiomeric nature was confirmed by mass spectrometry and NMR. The purified IA enantiomer peak 1 was derivatized with 4-bromobenzoyl chloride. The resulting di(4-bronobenzoate) IA was analyzed by x-ray crystallography and the absolute enantiomeric conformation assigned is C(3)-R. The IA enantiomers designated IA-R and A-S were assayed by competitive binding to cytosolic ER. The competitive binding index was estradiol, 100; DES, 286; IA-Rac (racemic mixture of IA), 143; IA-R, 3; and IA-S, 285; the index showed that ER demonstrates a stereochemical chiral preference. The IB enantiomers did not show a binding preference: IB, 145; IB-1, 100; and IB-2, 143. The differences in the IA enantiomer binding were shown to be due to competitive interactions by Lineweaver-Burk analysis of saturation binding of estradiol to ER in the presence of 1-, 5-, and 10-fold molar excess of competitor. Differences in binding affinity of the enantiomers could be partially explained by differences in the association rate constant (k+1) determined by association rate inhibition studies in which IA-S was 15 times more active than IA-R. Nuclear estrogen receptor levels were measured 1 h after in vivo treatment with doses of 5-20 micrograms/kg. The IA-Rac produced only 60% of the levels is compared with DES. Nuclear ER levels were checked every 30 min up to 2 h with no apparent difference, indicating that the low early levels were not due to a delayed estrogen receptor retention. When the enantiomers were tested individually only a dose of 10 micrograms/kg IA-S translocated ER to a level comparable to DES, while IA-R showed low levels at several doses. These results suggest that the poor biological activity of IA may be related to the differential ER interaction of its enantiomers.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of the absolute stereochemistry of D- and L-digitoxose using a chiral high-pressure liquid chromatography column

The absolute stereochemistry of D- and L-digitoxose was determined by h.p.l.c., on a chiral column, of the 1-O-(3,5-dinitrophenylcarbamoyl)-3,4-O-isopropylidene-α-D- and -L-digitoxopyranose.