Enzymatic reaction of chlorotrifluoroethene with glutathione: 19F NMR evidence for stereochemical control of the reaction

Chlorotrifluoroethene, a potent nephrotoxin, is a substrate for the glutathione S-transferases present in the cytosolic and microsomal fractions of rat liver. The glutathione conjugate formed by both subcellular fractions has been identified as S-(2-chloro-1,1,2-trifluoroethyl)glutathione by 1H and 19F NMR and by secondary ion mass spectrometry. The conjugate formed by the cytosolic fraction is an equimolar mixture of two diastereomers, whereas the conjugate formed by the microsomal fraction is predominantly one diastereomer, as judged by the 19F NMR spectra. No evidence for the formation of S-(trihalovinyl)glutathione derivatives by an addition/elimination reaction was found. High-performance liquid chromatography was employed to measure the rates of glutathione conjugate formation in vitro. The rates of S-(2-chloro-1,1,2-trifluoroethyl)glutathione formation were 75-107 nmol min-1 (mg of protein)-1 and 151-200 nmol min-1 (mg of protein)-1 catalyzed by the cytosolic and microsomal fractions, respectively (measured at pH 7.4, 37 degrees C, with 5 mM glutathione). These results suggest that glutathione conjugation occurs at high rates in vivo to produce the highly nephrotoxic S-(2-chloro-1,1,2-trifluoroethyl)glutathione.     

Synthesis and capillary electrophoretic analysis of enantiomerically enriched reference standards of MDMA and its main metabolites

Enantiomerically-enriched (S)-3,4-methylenedioxymethamphetamine (MDMA) and its main metabolites (S)-4-hydroxy-3-methoxymethamphetamine (HMMA) and (S)-3,4-dihydroxymethamphetamine (HHMA) were prepared for unequivocal identification of the differential enantioselective metabolism of these compounds as well as for its application in the analysis of biological samples. Capillary electrophoresis with cyclodextrin derivatives and a chemical correlation of (S)-MDMA, (S)-HMMA and (S)-HHMA has been performed to assign the absolute stereochemistry of major isomers in analytical standards enriched with such enantiomers.     

Synthesis and stereochemical characterization of optically active 1,2-diarylethane-1,2-diols: useful chiral controllers in the Ti-mediated enantioselective sulfoxidation

The series of phenylsubstituted 1,2-diphenylethane-1,2-diols 2a-h was prepared in high chemical (70--80%) and optical yields (approximately 90%) by Sharpless syn-dihydroxylation of the corresponding (E)-1,2-diarylethenes, in turn obtained by McMurry or Wittig reactions. The enantiomeric excesses of the samples were determined by HPLC analysis using Chiralcel OD chiral stationary phase (CSP). This CSP was able to resolve all the diols, except for 2g, with alpha values ranging between 1.10--1.64. In all cases the (R,R) antipode was eluted first. (R,R) absolute configuration was assigned to the dextrorotatory (CHCl(3)) diols 2a--h by analyzing the CD spectra of their 2,2-dimethyl-1,3-dioxolanes 3a--h. In fact, the CD spectra of all these dioxolanes present a positive couplet (210--180 nm range) which can be nonempirically related to an (R,R) absolute configuration of the two stereocenters.     

Determination of the enantiomeric composition of chiral delta-2-oxazolines-1,3 by 1H and 19F NMR spectroscopy using chiral solvating agents

Studies of the perturbing effect of chiral solvating agents (CSAs) 5a and mostly of 5c upon the NMR spectra of chiral Delta(2)-oxazoline 1 demonstrated the ability of these fluoroalcohols to afford diastereomeric solvates from these solutes. Thus, for all tested Delta(2)-oxazolines 1Aa-d, 1Ba, and 1e there is at least one possibility to proceed to their enantiomeric discrimination either by (1)H or (19)F NMR using these CSAs (see Fig. 1). NMR results are discussed from substrate and CSA structure standpoints and a solvation model is proposed on the basis of the inequivalence senses generally observed. Then the method was applied to extracts of incubated locust tissues obtained by solid phase extraction (SPE) after a partial unmasking of the substrate 1.     

Comparison of the biological effects of F at different intracellular levels

We herein examined the biological effects of cells treated with ¹⁸F labeled drugs for positron emission tomography (PET). The relationship between the intracellular distribution of ¹⁸F and levels of damaged DNA has yet to be clarified in detail. We used culture cells (Chinese Hamster Ovary cells) treated with two types of ¹⁸F labeled drugs, fluorodeoxyglucose (FDG) and fluorine ion (HF). FDG efficiently accumulated in cells, whereas HF did not. To examine the induction of DNA double strand breaks (DSB), we measured the number of foci for 53BP1 that formed at the site of DNA DSB. The results revealed that although radioactivity levels were the same, the induction of 53BP1 foci was stronger in cells treated with ¹⁸F-FDG than in those treated with ¹⁸F-HF. The clonogenic survival of cells was significantly lower with ¹⁸F-FDG than with ¹⁸F-HF. We concluded that the efficient accumulation of ¹⁸F in cells led to stronger biological effects due to more severe cellular lethality via the induction of DNA DSB.     

Standard reference materials and data quality assurance in the biomedical analysis of trace elements

Accurate and precise analytical data of the concentrations of bioanalytes in bioclinical studies are of fundamental importance. Quality assurance procedures should always be performed to check the overall analytical work. This can be conveniently performed if appropriate standard reference materials with known concentrations of the analyte object of study are available. This paper underlines the key points related to the production and use of biological standard materials for trace element analysis. In particular, the present situation in the field of trace element determination in human biological fluids and the related problems are illustrated. The considerations given in this work may contribute to the preparation of the new biomarker standard materials.     

Evolutionary trace analysis at the ligand binding site of laccase

Laccase belongs to the family of blue multi-copper oxidases and are capable of oxidizing a wide range of aromatic compounds. Laccases have industrial applications in paper pulping or bleaching and hydrocarbon bioremediation as a biocatalyst. We describe the design of a laccase with broader substrate spectrum in bioremediation. The application of evolutionary trace (ET) analysis of laccase at the ligand binding site for optimal design of the enzyme is described. In this attempt, class specific sites from ET analysis were mapped onto known crystal structure of laccase. The analysis revealed 162PHE as a critical residue in structure function relationship studies.     

A micromethod for the stereochemical analysis of fatty acid desaturase-mediated sulfoxidation reactions

The stereochemistry of fatty acid desaturase-mediated sulfoxidation can be evaluated at micromolar concentrations of analyte using (19)F NMR in combination with a chiral shift reagent: (S)-(+)-MPAA.     

Synthesis of a trisaccharide component of the capsular polysaccharide of Streptococcus pneumoniae type 19F

2-O-[4-O-(2-Acetamido-2-deoxy-beta-D-mannopyranosyl)-alpha-D- glucopyranosyl]-alpha,beta-L-rhamnopyranose, a structural component of the capsular polysaccharide of Streptococcus pneumoniae type 19F, has been synthesized by sequential glycosylation reactions using the glycosyl acceptor 2,2,2-trichloroethyl 3,4-di-O-benzyl-alpha-L-rhamnopyranoside (prepared from the known 2-O-acetyl-3,4-di-O-benzyl-alpha-L-rhamnopyranosyl chloride), and the glycosyl donors 4-O-acetyl-2,3,6-tri-O-benzyl-alpha-D-glucopyranosyl chloride and 4,6-di-O-acetyl-2-azido-3-O-benzyl-2-deoxy-alpha-D-mannopyranosyl bromide (prepared in seven steps from the known methyl 2-azido-4,6-O-benzylidene-2-deoxy-alpha-D-altropyranoside). The corresponding 8-(methoxycarbonyl)octyl glycoside has also been synthesized, by coupling of 8-(methoxycarbonyl)octyl trifluoromethanesulfonate and the sodium salt of 2-O-[4-O-(2-acetamido-4,6-di-O-acetyl-3-O-benzyl-2-deoxy-beta-D- mannopyranosyl)-2,3,6-tri-O-benzyl-alpha-D-glucopyranosyl]-3,4-di-O- benzyl-alpha,beta-L-rhamnopyranose.     

Use of ion trap gas chromatography–tandem mass spectrometry for detection and confirmation of anabolic substances at trace levels in doping analysis

A procedure for detecting and confirming 23 anabolic substances and/or metabolites has been developed using a GC–MS–MS ion trap system in full-scan mode. The process used to select the precursor ion, and the optimization of the system parameters used to obtain the daughter ion spectra, are explained. Urine samples were prepared using solid-phase extraction and enzymatic hydrolysis, and after TMS derivatives had been formed, they were injected into the mass spectrometer. This method permits confirmation of the presence of anabolic substances at low ng ml⁻¹ levels without the need of further purification procedures on the samples. This procedure has been used on more than 2000 urine samples collected from sporting competitions and has made it possible to confirm more than 45 true positive cases which could not have been confirmed using routine GC–MS methods.     

Challenges for the modern analytical ultracentrifuge analysis of polysaccharides

This article reviews some of the recent advances in analytical ultracentrifugation and how these advances have impacted--and can impact--on our understanding of the size, shape through conformation modelling, interactions and charge properties of polysaccharides in solution, particularly when used in combination with other solution techniques and also imaging techniques. Specifically we look at (1) polysaccharide polydispersity and simple shape analysis by sedimentation velocity, and in particular using new approaches such as SEDFIT analysis; (2) polysaccharide molecular-weight analysis by sedimentation equilibrium and MSTAR analysis and how this complements analysis of size exclusion chromatography coupled to multi-angle laser light scattering; (3) polysaccharide conformation analysis using traditional procedures such as the Wales-van Holde ratio, power law or 'scaling' relations, more specialised treatments for rigid cylindrical structures, semi-flexible chains and worm-like coils and complications through draining effects; (4) Analysis of polysaccharide interactions and in particular complex formation phenomena, focusing on interesting applications in the areas of mucoadhesion and sedimentation fingerprinting; and (5) the possibilities for macromolecular charge and charge screening measurement.     

NAA methodology for the certification of eight trace elements in a plankton reference material

Biological Trace Element Research - Neutron activation analysis (NAA) methodology was applied for the certification of eight trace elements (As, Co, Cr, Cu, Hg, Mn, Se, and Zn) in a plankton...     

Commerical reference shape standards use in the study of particle shape effect on laser diffraction particle size analysis

The purpose of this paper is to describe the use of LGC Promochem AEA 1001 to AEA 1003 monosized fiberanalog shape standards in the study of the effect of particle shape on laser diffraction (LD) particle size analysis (psa). The psa of the AEA standards was conducted using LD psa systems from Beckman Coulter, Horiba, and Malvern Instruments. Flow speed settings, sample refractive index values, and sample cell types were varied to examine the extent to which the shape effect on LD psa results is modified by these variables. The volume and number probability plots resulting from these measurements were each characterized by a spread in the particle size distribution that roughly extended from the breadth to the longest dimension of the particles. For most of the selected sample refractive index values, the volume probability plots were characterized by apparent bimodal distributions. The results, therefore, provide experimental verification of the conclusions from theoretical studies of LD psa system response to monosized elliptical particles in which this apparent bimodality was the predicted result in the case of flow-oriented particles. The data support the findings from previous studies conducted over the past 10 years that have called into question the verity of the tenets of, and therefore the value of the application of, the equivalent spherical volume diameter theory and the random particle orientation model to the interpretation of LD psa results from measurements made on nonspherical particles.     

Commercial reference shape standards use in the study of particle shape effect on laser diffraction particle size analysis

The purpose of this paper is to describe the use of LGC Promochem AEA 1001 to AEA 1003 monosized fiber-analog shape standards in the study of the effect of particle shape on laser diffraction (LD) particle size analysis (psa). The psa of the AEA standards was conducted using LD psa systems from Beckman Coulter, Horiba, and Malvern Instruments. Flow speed settings, sample refractive index values, and sample cell types were varied to examine the extent to which the shape effect on LD psa results is modified by these variables. The volume and number probability plots resulting from these measurements were each characterized by a spread in the particle size distribution that roughly extended from the breadth to the longest dimension of the particles. For most of the selected sample refractive index values, the volume probability plots were characterized by apparent bimodal distributions. The results, therefore, provide experimental verification of the conclusions from theoretical studies of LD psa system response to monosized elliptical particles in which this apparent bimodality was the predicted result in the case of flow-oriented particles. The data support the findings from previous studies conducted over the past 10 years that have called into question the verity of the tenets of, and therefore the value of the application of, the equivalent spherical volume diameter theory and the random particle orientation model to the interpretation of LD psa results from measurements made on nonspherical particles.     

Evaluation of the methods for enumerating coliform bacteria from water samples using precise reference standards

AIMS: To use BioBall cultures as a precise reference standard to evaluate methods for enumeration of Escherichia coli and other coliform bacteria in water samples. METHODS AND RESULTS: Eight methods were evaluated including membrane filtration, standard plate count (pour and spread plate methods), defined substrate technology methods (Colilert and Colisure), the most probable number method and the Petrifilm disposable plate method. Escherichia coli and Enterobacter aerogenes BioBall cultures containing 30 organisms each were used. All tests were performed using 10 replicates. The mean recovery of both bacteria varied with the different methods employed. CONCLUSIONS: The best and most consistent results were obtained with Petrifilm and the pour plate method. Other methods either yielded a low recovery or showed significantly high variability between replicates. SIGNIFICANCE AND IMPACT OF THE STUDY: The BioBall is a very suitable quality control tool for evaluating the efficiency of methods for bacterial enumeration in water samples.     

Synthesis and analytical characterization of the sodium salt of heptakis(2-O-methyl-3,6-di-O-sulfo)cyclomaltoheptaose, a chiral resolving agent candidate for capillary electrophoresis

A pure, single isomer, strong electrolyte chiral resolving agent candidate for capillary electrophoresis, the sodium salt of heptakis(2-O-methyl-3,6-di-O-sulfo)cyclomaltoheptaose has been synthesized on the 100-g scale, in greater than 97% purity, through heptakis(2,6-di-O-tert-butyldimethylsilyl)cyclomaltoheptaose, heptakis(2-O-methyl-3,6-di-O-tert-butyldimethylsilyl)cyclomaltohep taose and heptakis(2-O-methyl)cyclomaltoheptaose intermediates. The structural identity of each intermediate and the final product was conclusively established by high-resolution MALDI-TOF mass spectrometry, variable-temperature 1H and 13C NMR spectroscopy and X-ray crystallography. The purity of each intermediate and the final product was determined by gradient high-performance liquid chromatography (HPLC) and indirect UV detection capillary electrophoresis.     

A rapid analytical method for monitoring the enantiomeric purity of chiral molecules synthesized in ionic liquid solvents

Ionic liquids (ILs) have been widely used as reaction solvents in asymmetric synthesis due to their interesting physical and chemical properties. However, monitoring reactant-to-product conversion and the enantiopurity of formed stereoisomers often involves a tedious extraction step before chromatographic analysis. In this study, a rapid and sensitive sampling method using headspace solid-phase microextraction (SPME) coupled to chiral gas chromatography was developed for the "on-line" analysis of chiral molecules in the IL solvent. Three different SPME sorbent coatings, namely polydimethylsiloxane, polyacrylate, and a polymeric ionic liquid-based fiber, were examined in this study. The analytical performance of the developed method was evaluated in terms of reproducibility, slope of calibration curve, linear range, calibration linearity, and the determination of detection limits. The SPME method was successfully applied in the determination of enantiomeric excess from selected mixtures of chiral molecules. A preliminary study was performed using an "on-fiber" derivatization approach revealing that the stereoisomers extracted by the SPME fiber can be efficiently derivatized using a short "on-fiber" derivatization step. The developed SPME method eliminates the need of sequestering the reaction, separating the compounds of interest from the IL solvent, and the addition of a derivatizing reagent.