Development and application of procedures for the highly sensitive quantification of cyclosarin enantiomers in hemolysed swine blood samples

The present study was initiated to develop a sensitive method for the analysis of cyclosarin (O-cyclohexyl methylphosphonofluoridate, GF) enantiomers in biological samples utilizing classical configurations of GC-MS and automated solid phase extraction. To achieve this goal, a specific procedure had to be developed to extract cyclosarin from swine blood samples thereby stabilising and minimising the racemisation/deracemisation of its enantiomers. The chiral stationary phase was GAMMA DEX (gamma cyclodextrin), on which GF and deuterated GF enantiomers were baseline-resolved. The limit of detection was 1 pg for (-)-GF with GC-EI-MS and 5 pg for (+)-GF with GC-NCI-MS. The absolute recovery of the overall procedure for sample preparation was 85%. After an intravenous infusion of a supralethal dose of GF in anaesthetised swine only (-)-GF could be quantified, (+)-GF was not detected.     

The dirhodium-method in the determination of absolute configurations of phospholene chalcogenides

The 1H, 13C, and 31P NMR signals of six chiral phospholene chalcogenides (X = O, S, Se) are duplicated in the presence of one mole equivalent of the chiral auxiliary Rh2[(R)-MTPA]4 (diastereomeric dispersion Deltanu; in Hz). The samples were investigated as nonracemic mixtures of enantiomers with known absolute configurations so that signs can be attributed to the Deltanu-values and each signal set can be assigned to the respective enantiomer. The signs are uniform--in particular those of 1H nuclei--and nearly independent of the nature of the chalcogen atom. Thus, if the absolute configuration of one compound is known, it is possible to derive absolute configurations in the whole series (correlation method).     

Separation of enantiomers by open capillary electrochromatography on polysiloxane-bonded permethyl-beta-cyclodextrin

The separation of enantiomers by open capillary electrochromatography (o-CEC) using Chirasil-Dex as chiral stationary phase (CSP) is reviewed. In Chirasil-Dex, permethylated beta-cyclodextrin is linked via a single octamethylene spacer to polydimethylsiloxane. The CSP is coated and thermally immobilized onto the internal surface of a fused-silica column (i.d. 50 microm). Employing a single open-tubular column coated with Chirasil-Dex, a unified enantioselective approach can be realized using the four common chromatographic techniques: o-GC, o-SFC, o-LC and o-CEC. The chiral stationary phase Chirasil-Dex can be combined with a charged cyclodextrin derivative, which is added into the mobile phase. In the resulting dual chiral recognition system, enhancement of enantioselectivity (matched case) or compensation of enantioselectivity (mismatched case) are observed. The overall enantioselectivity is dependent on the sense of enantioselectivity of the selectors chosen and their influence on the electrophoretic and electroosmotic migration of the enantiomers of a selectand. The feasibility to couple chiral o-CEC and ESI/MS is demonstrated for trace analysis of enantiomeric drugs in body fluids.     

Resolution and absolute configuration of 7,12-dimethylbenz[a]anthracene 5,6-epoxide enantiomers

The enantiomers of 7,12-dimethylbenz[a]anthracene (DMBA) 5,6-epoxide were directly resolved by normal-phase high-performance liquid chromatography with an ionically bonded chiral stationary phase. The absolute configurations of the resolved enantiomers were determined by comparison of circular dichroism spectra of the methanolysis products formed from the epoxide enantiomers with that of a DMBA trans-5,6-dihydrodiol enantiomer of known absolute stereochemistry. DMBA 5R,6S-epoxide is hydrated by rat liver microsomal epoxide hydrolase predominantly (95%) to a 5S,6S-dihydrodiol. The results indicate that the 5S,6S-dihydrodiol formed from the metabolism of DMBA by microsomes prepared from the livers of 3-methylcholanthrene-treated rats is predominantly derived from a 5R,6S-epoxide intermediate.     

Synthesis, chiral separation, and absolute configuration of bis-(N-aryl) atropisomeric triads: 1,2-bis-[4-methyl-2-(thi)oxo-2,3-dihydrothiazol-3-yl]-benzene

In this work, a series of 1,2-bis-[4-methyl-2-(thi)oxo-2,3-dihydrothiazol-3-yl]-benzene has been prepared. These atropisomeric molecular triads were exclusively found to exist in the anti-form. They were separated into enantiomers by liquid chromatography on a chiral support. The absolute configurations of the enantiomers were determined using a chemical correlation method together with chiral chromatography. The barriers to interconversion of the enantiomers were determined.     

Enantiomers of a nonylphenol isomer: absolute configurations and estrogenic potencies

Enantiomers of 4-(1,1,2-trimethylhexyl)phenol, a chiral isomer of the endocrine disrupting chemical nonylphenol, have been resolved and isolated by preparative chiral HPLC. The absolute configurations of the enantiomers were then determined by an X-ray crystallographic study of the (-)-camphanoyl derivative of the first eluted enantiomer NP(35)E1. The first enantiomer (NP(35)E1) and the second enantiomer (NP(35)E2) eluted were found to have the S and R absolute configurations, respectively. The estrogenic potencies of the S and R enantiomers were tested by the E-screen assay. A slight difference was observed in the relative proliferative effect between the S enantiomer and R enantiomer in the E-screen assay.     

Synthesis, resolution, and investigation of the rotational interconversion process of atropisomeric 1,n-diaza[n]paracyclophanes using cyclodextrin-mediated capillary zone electrophoresis.

A number of variously monosubstituted 1,n-diaza[n]paracyclophanes (n = 10-12), which show planar chirality and atropisomerism due to hindered rotation about single bonds, were synthesized via a classical route to analyze their stereodynamic properties. Racemic analytes with 10- and 11-membered bridges were resolved by capillary zone electrophoresis (CZE) in acidic phosphate buffers (pH 2.5-4.5) employing permethylated beta- and gamma-cyclodextrin as chiral additives. Moreover, cyclodextrin mediated CZE was used in a discontinuously driven mode for investigations of the rotational interconversion process of conformationally labile homologues (n = 11). In stopped-flow experiments, after baseline separation enantiomers were partially enantiomerized in situ inside the capillary by heating. The rate constants (k(enan) = 1/2 k(rac)) and rotational energy barriers (Delta G(++)) were determined from the resulting enantiomeric ratios. Energy barriers between 113-126 kJ/mol were found depending on the substituent of the benzene ring and the degree of ionization of the amino groups in bridgehead positions. The energy barriers increased in order of the substituents: NO(2) > CF(3) > Br > Cl > CH(3) approximately F. In addition, the rotational energy barriers were decreased by approximately 6-8 kJ/mol in the presence of the chiral selector.     

Absolute configuration of a diaryldiacyloxy spiroselenurane and resolution of a carbon analog

The enantiomers of 3,3'-spirobi(3-selenaphthalide) (2) were obtained via direct separation by liquid chromatography on a chiral stationary phase. Determination of the absolute configuration was made by X-ray crystallography with the use of the anomalous dispersion technique. The first eluted (+)-form (lambda = 302 and 365 nm) of 2 was found to have (S)-configuration. By a comparison of CD-spectra, the same (S)-configuration could be assigned to the (+)-forms of the sulfur (1) and tellurium (7) analogs of 2. An asymmetric dichloro-substituted spirobilactone (4) was also synthesized and separated into its enantiomers. Relative configurations between 4 and its parent compound (3) were established from the corresponding chiroptical data obtained. Chirality 12: 71-75, 2000.     

Resolution of racemic mixtures of carbocyclic analogues of nucleosides and assignment of their absolute configuration

Racemic trans-6-chloro-9-[2-(hydroxymethyl)cyclopentyl]purine was resolved using HPLC with a chiral column. The absolute configurations of the enantiomers were determined by NMR studies of their (R)- and (S)-methoxyphenylacetates.     

NMR determination of the absolute configuration of an alpha-exo-methylene-gamma-lactone

Both the enantiomers of the axially chiral reagent, 2'-methoxy-1,1'-binaphthalene-2-carbohydroximoyl chloride (MBCC), were used to convert igalan, an alpha-exo-methylene-gamma-lactone, to yield 4,5-dihydroisoxazoles. The absolute configuration of igalan was determined to be (3aR,5R,6R,7aR)-6-ethenylhexahydro-6-methyl-3-methylen e-5-(1-methylethenyl)-2(3H)-benzofuranone (IUPAC name) on the basis of NOE correlations in these derivatives. The absolute configurations of other alpha-exo-methylene-gamma-lactones can be determined by the same method.     

RESOLUTION OF RACEMIC MIXTURES OF CARBOCYCLIC ANALOGUES OF NUCLEOSIDES AND ASSIGNMENT OF THEIR ABSOLUTE CONFIGURATION

Racemic trans-6-chloro-9-[2-(hydroxymethyl)cyclopentyl]purine was resolved using HPLC with a chiral column. The absolute configurations of the enantiomers were determined by NMR studies of their (R)- and (S)-methoxy-phenylacetates.     

New insights into the ligninolytic capability of a wood decay ascomycete

Wood-grown cultures of Daldinia concentrica oxidized a permethylated beta-(14)C-labeled synthetic lignin to (14)CO(2) and also cleaved a permethylated alpha-(13)C-labeled synthetic lignin to give C(alpha)-C(beta) cleavage products that were detected by (13)C nuclear magnetic resonance spectrometry. Therefore, this ascomycete resembles white-rot basidiomycetes in attacking the recalcitrant nonphenolic structures that predominate in lignin.     

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.     

Explanation of enantioseparation of amino acid derivatives in gas chromatography

The enantioseparation of amino acid derivatives by gas chromatography was investigated through molecular dynamics simulation. The chiral stationary phase was based on permethylated β-cyclodextrin (PM-β-CD). The model enantiomers were four amino acid derivatives. For the inclusion complexes of PM-β-CD with the enantiomers, we studied the binding energy. The competitive binding of l- or d-enantiomers to PM-β-CD was simulated. The interaction energy of the enantiomers with PM-β-CD and the appearing frequency of l- and d-enantiomers around a certain distance from the centre of mass of PM-β-CD were obtained. It was found that the appearing frequency is an important parameter to explain the enantioseparation of the amino acid derivatives in gas chromatography. The appearing frequency of the enantiomer together with the binding and interaction energy can be used to predict the elution orders of the enantiomers in gas chromatography.     

Preparative chiral chromatography and chiroptical characterization of enantiomers of omeprazole and related benzimidazoles

To chiroptically characterize the enantiomers of omeprazole and some structurally related benzimidazoles with circular dichroism (CD), preparative chiral liquid chromatography was utilized for the isolation of the pure enantiomers. A limited analytical column screen was performed identifying Kromasil-CHI-TBB and the amylose-based phases Chiralpak AD and AS as possible chiral stationary phases (CSPs) for the preparative scale separation of the enantiomers of the different benzimidazoles. Optimization of the chromatographic conditions with respect to retention, enantioseparation, and resolution was achieved by variation of the mobile phase constituents as well as of temperature. Because of the lability of the compound in slightly acidic media, supercritical fluid chromatography (SFC) could not be applied for a preparative scale separation of the enantiomers. The separation of omeprazole was optimized to give high throughput (2.6 kg racemate/kg CSP/day) and high enantiomeric excess of the obtained isomers. The absolute configurations of the pure enantiomers of rabeprazole, lansoprazole, and pantoprazole were determined from the strong correlation to the CD spectrum of (+)-(R)-omeprazole. For all the compounds, the (+)-enantiomers displayed similar chiroptical features as (+)-(R)-omeprazole and were thus assigned the (R)- configuration. Elution order of the optical isomers was monitored by injecting racemic solutions spiked with one of the isomers and also by an on-line laser polarimeter. Both the type of CSP and also the mobile phase constituents had a strong effect on elution order of the enantiomers.     

High-performance liquid chromatographic enantioseparation of 1-(aminoalkyl)-2-naphthol analogs on polysaccharide-based chiral stationary phases

The enantiomers of various 1-(alpha-aminobenzyl)-2-naphthol and 1-(aminoalkyl)-2-naphthol analogs were separated on cellulose-tris-3,5-dimethylphenyl carbamate-based chiral stationary phases (Chiralcel OD-H and Chiralcel OD-RH), using n-hexane/2-propanol/diethylamine or phosphate buffer/organic modifier mobile phases. The 3,5-dimethylphenyl carbamoylated cellulose columns were effective in both normal and rev ersed-phase modes. The effects of the mobile phase composition, the pH, the buffer concentration, and the structures of the substituents on the 2-naphthol on the enantioseparations were studied. The absolute configuration and elution sequence were determined for 1-(1-amino-2-methylpropyl)-2-naphthol: the elution sequence was S < R.     

Enantiomeric Polyketides from the Starfish‐Derived Symbiotic Fungus Penicillium sp. GGF16‐1‐2

One new racemic mixture, penicilliode A ( 1 ) and four pairs of enantiomeric polyketides, penicilliode B and C ( 2 and 3 ) and coniochaetone B and C ( 4 and 5 ), were obtained from the starfish‐derived symbiotic fungus Penicillium sp. GGF16‐1‐2. Interestingly, the strain GGF16‐1‐2 can produce enantiomers. The absolute configuration of 1 was determined by X‐ray diffraction (XRD) analysis, and the absolute configurations of 2 – 4 were determined by the optical rotation (OR) values and electronic circular dichroism (ECD) calculations. Compounds 1 – 5 were firstly isolated from the marine‐derived fungus Penicillium as racemates, and 2 – 5 were separated by HPLC with a chiral stationary phase. All the compounds were evaluated for their antibacterial, cytotoxic and inhibitory activities against PDE4D2.     

The role of pi-acidic and pi-basic chiral stationary phases in the high-performance liquid chromatographic enantioseparation of unusual beta-amino acids

The application of 3,5-dimethylphenyl-carbamoylated-beta-cyclodextrin (Cyclobond I 2000 DMP) and 2,6-dinitro-4-trifluoromethylphenyl-ether-beta-cyclodextrin-based (Cyclobond DNP) chiral stationary phases for the high-performance liquid chromatographic enantioseparation of unusual beta-amino acids is reported. The investigated amino acids were saturated or unsaturated alicyclic beta-3-homo-amino acids and bicyclic beta-amino acids. Prior to chromatographic analyses, all amino acids were transformed to N-3,5-dinitrobenzoyl- or N-3,5-dimethylbenzoyl form to ensure a pi-acidic or pi-basic function and to enhance the pi-acidic-pi-basic interactions between analytes and chiral selectors. Chromatographic results are given as retention, separation and resolution factors. The chromatographic conditions were varied to achieve optimal separation. The sequence of elution of the enantiomers was determined in some cases.     

Separation and absolute configuration of the enantiomers of a degradation product of the new inhalation anesthetic sevoflurane

In a rebreathing anesthesia circuit, the inhaled anesthetic sevoflurane degrades into at least two products, termed "compound A" and "compound B." The enantiomer separation of the chiral compound B (1,1,1,3,3-pentafluoro-2-(fluoromethoxy)-3-methoxypropane ) by capillary gas chromatography (cGC) using heptakis (2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl)-beta-cyclodextrin as chiral selector was studied. With this cyclodextrin derivative diluted in the polysiloxane PS 86, an unprecedented high separation factor alpha of 4.1 (at 30 degrees C) was found. Consequently, the enantiomers of compound B were isolated by preparative GC and their specific rotations were measured. In addition, their absolute configurations were determined by X-ray crystallography. To collect the X-ray data, single crystals of both enantiomers were grown in situ on the diffractometer. The levorotatory enantiomer B(-) has the R-configuration while the dextrorotatory enantiomer B(+) has the S-configuration. The elution order of the compound B enantiomers on heptakis (2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl)-beta-cyclodextrin is R before S.     

Chromatographic resolution, characterisation and quantification of VX enantiomers in hemolysed swine blood samples

The present study was initiated to develop a sensitive and highly selective method for the analysis of the enantiomers of the nerve agent VX (O-ethyl S-[2(diisopropylamino)ethyl] methylphosphonothioate) in blood samples for toxicokinetic and therapeutic research. To achieve this goal, analytical and semi-preparative enantioseparation of VX were carried out with gas and liquid chromatography. The GC chiral stationary phase was HYDRODEX-beta-TBDAc (beta cyclodextrin), on which VX was baseline-resolved. On the chiral HPLC phase CHIRALCEL OD-H the enantiomers of VX were isolated with enantiomeric excess >99.99%. They were characterised by specific optical rotation (+/-25.8degmldm(-1)g(-1) at 20 degrees C and 589nm) and by determination of cholinesterase inhibition rate constants. For the quantitative chiral detection of VX the enantioresolution was realized on the HPLC chiral phase CHIRAL AGP. A specific procedure was developed to isolate VX from swine blood samples thereby stabilising its enantiomers. The limit of detection was 200fg per enantiomer on column. The absolute recovery of the overall sample preparation procedure was 75%. After an intravenous and percutaneous administration of a supralethal dose of VX in anesthetised swine (+)-VX and (-)-VX could be quantified up to 720min.