Correlation Nuclear Magnetic Resonance Spectroscopy Research Articles

Automated screening for metabolites in complex mixtures using 2D COSY NMR spectroscopy

One of the greatest challenges in metabolomics is the rapid and unambiguous identification and quantification of metabolites in a biological sample. Although one-dimensional (1D) proton nuclear magnetic resonance (NMR) spectra can be acquired rapidly, they are complicated by severe peak overlap that can significantly hinder the automated identification and quantification of metabolites. Furthermore, it is currently not reasonable to assume that NMR spectra of pure metabolites are available a priori for every metabolite in a biological sample. In this paper we develop and report on tests of methods that assist in the automatic identification of metabolites using proton two-dimensional (2D) correlation spectroscopy (COSY) NMR. Given a database of 2D COSY spectra for the metabolites of interest, our methods provide a list sorted by a heuristic likelihood of the metabolites present in a sample that has been analyzed using 2D COSY NMR. Our models attempt to correct the displacement of the peaks that can occur from one sample to the next, due to pH, temperature and matrix effects, using a statistical and chemical model. The correction of one peak can result in an implied correction of others due to spin–spin coupling. Furthermore, these displacements are not independent: they depend on the relative position of functional groups in the molecule. We report experimental results using defined mixtures of amino acids as well as real complex biological samples that demonstrate that our methods can be very effective at automatically and rapidly identifying metabolites.

New modulated metallic macrocycles: Electrochemistry and their interaction with calf thymus DNA

New modulated pentacoordinate complexes [C 17H 34N 7O 2Cu]ClO 4 ( 5), [C 17H 34N 7O 2Co]ClO 4 ( 6), [C 17H 34N 7O 2Ni] ClO 4 ( 7) have been synthesized by the interaction of 1,8-dihydro-1,3,6,8,10,13 Cu(II) ( 2), Co(II) ( 3), Ni(II) ( 4) hexaazacyclotetradecane complexes and Hsalea N-(2-hydroxy benzyl)-2-amino-1-ethanol ligand ( 1). All the complexes have been characterized by infrared, electron paramagnetic resonance, 1H and 13C nuclear magnetic resonance (NMR), 2D correlation spectroscopy NMR and ultraviolet–visible (UV–vis) spectroscopy. In all the complexes, the metal center is encapsulated by the Hsalea ligand in a pentacoordinate environment. Conductance measurements show that the complexes are ionic in nature. UV–vis absorption titration and viscometric studies have been carried out to ascertain the interaction of complexes 5 and 6 with calf thymus DNA (CT DNA). The experimental results suggest that complex 5 binds to CT DNA through partial intercalation of the aromatic ring into the base pair of DNA while complex 6 binds to CT DNA by electrostatic mode. The intrinsic binding constants K b of complex 5 and 6 were found to be 6.8 × 10 −5 M −1 and 1.8 × 10 −4 M −1, respectively. The binding of complexes 5 and 6 with CT DNA has also been investigated by cyclic voltammetry.

Rapid identification of Candida species by using nuclear magnetic resonance spectroscopy and a statistical classification strategy.

Nuclear magnetic resonance (NMR) spectra were acquired from suspensions of clinically important yeast species of the genus Candida to characterize the relationship between metabolite profiles and species identification. Major metabolites were identified by using two-dimensional correlation NMR spectroscopy. One-dimensional proton NMR spectra were analyzed by using a staged statistical classification strategy. Analysis of NMR spectra from 442 isolates of Candida albicans, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis resulted in rapid, accurate identification when compared with conventional and DNA-based identification. Spectral regions used for the classification of the five yeast species revealed species-specific differences in relative amounts of lipids, trehalose, polyols, and other metabolites. Isolates of C. parapsilosis and C. glabrata with unusual PCR fingerprinting patterns also generated atypical NMR spectra, suggesting the possibility of intraspecies discontinuity. We conclude that NMR spectroscopy combined with a statistical classification strategy is a rapid, nondestructive, and potentially valuable method for identification and chemotaxonomic characterization that may be broadly applicable to fungi and other microorganisms.

Synthesis and [formula omitted] NMR study of R F-oleic acid-F 13

A seven-step synthesis of Z-13,13,14,14,15,15,16,16,17,17,18,18,18-tridecafluoro-octadec-9-enoic acid (oleic acid-F 13) is reported. The key step is a Wittig reaction to form the C9–C10 double bond with a Z: E isomeric ratio greater than 20:1. The 19 F nuclear magnetic resonance (NMR) spectrum is included and unambiguous assignments have been made with the aid of 19 F – 19 F correlation NMR spectroscopy (COSY spectra).

The phage T4 restriction endoribonuclease RegB: a cyclizing enzyme that requires two histidines to be fully active.

The regB gene, from the bacteriophage T4, codes for an endoribonuclease that controls the expression of a number of phage early genes. The RegB protein cleaves its mRNA substrates with an almost absolute specificity in the middle of the tertranucleotide GGAG, making it a unique well-defined restriction endoribonuclease. This striking protein has no homology to any known RNase and its catalytic mechanism has never been investigated. Here, we show, using 31P nuclear magnetic resonance (NMR), that RegB produces a cyclic 2',3'-phosphodiester product. In order to determine the residues crucial for its activity, we prepared all the histidine-to- alanine point mutants of RegB. The activity of these mutants was characterized both in vivo and in vitro. In addition, their binding capability was quantified by surface plasmon resonance and their structural integrity was probed by 1H/15N NMR correlation spectroscopy. The results obtained show that only the H48A and the H68A substitutions significantly reduce RegB activity without changing its ability to bind the substrate or affecting its overall structure. Altogether, our results define RegB as a new cyclizing RNase and present His48 and His68 as potent catalytic residues. The effect of the in vivo selected R52L mutation is also described and discussed.

Identification of metabolites of importance in the pathogenesis of pulmonary cryptococcoma using nuclear magnetic resonance spectroscopy

Primary lung infection with Cryptococcus neoformans is characterised by circumscribed lesions (cryptococcomas). To identify cryptococcal and/or host products of importance in pathogenesis, we applied proton nuclear magnetic resonance (NMR) spectroscopy, which identifies mobile compounds present in complex mixtures, to experimental pulmonary cryptococcomas from rats. Magnetic resonance experiments were performed on cryptococcomas ( n = 10) and healthy lungs ( n = 8). Signal assignment to key metabolites was confirmed by homo-nuclear and hetero-nuclear NMR correlation spectroscopy. Cryptococcal metabolites, dominating spectra from cryptococcomas included the stress protectants, trehalose and mannitol, acetate, and in some animals, ethanol. Glycerophosphorylcholine was also abundant in cryptococcomas, consistent with hydrolysis of phospholipids in vivo by the cryptococcal enzyme, phospholipase B (PLB). PLB has been identified by molecular studies as a cryptococcal virulence determinant. We propose that PLB secreted by cryptococci promotes tissue invasion by hydrolysing host phospholipids, such as dipalmitoyl phosphatidyl choline, which is abundant in pulmonary surfactant, and lung cell membrane phospholipids. Our results confirm the utility of NMR spectroscopy in studies of microbial pathogenesis.

Quantitative Analysis of Intracellular Metabolic Fluxes Using GC-MS and Two-Dimensional NMR Spectroscopy.

A method for metabolic flux determination by carbon labeling experiments is described. The cells were grown on 13C-labeled substrate, and the labeling patterns of the amino acids in biomass hydrolysate were measured using two dimensional 1H-13C correlation nuclear magnetic resonance spectroscopy (2D 1H-13C COSY NMR) and gas chromatography/mass spectrometry (GC-MS) technique. The estimation of intracellular flux distribution from the measured labeling data and extracellular fluxes was achieved by a nonlinear least squares fitting approach, since a framework was developed that allows mathematical description relating the measured quantities and the intracellular fluxes. The described method was used to investigate the central carbon metabolism of the mixotrophically cultivated Synechocystis sp. PCC6803. The statistical quality of the estimated fluxes was also evaluated.

Quantitative analysis of intracellular metabolic fluxes using GC-MS and two-dimensional NMR spectroscopy

A method for metabolic flux determination by carbon labeling experiments is described. The cells were grown on 13C-labeled substrate, and the labeling patterns of the amino acids in biomass hydrolysate were measured using two dimensional 1H- 13C correlation nuclear magnetic resonance spectroscopy (2D 1H- 13C COSY NMR) and gas chromatography/mass spectrometry (GC-MS) technique. The estimation of intracellular flux distribution from the measured labeling data and extracellular fluxes was achieved by a nonlinear least squares fitting approach, since a framework was developed that allows mathematical description relating the measured quantities and the intracellular fluxes. The described method was used to investigate the central carbon metabolism of the mixotrophically cultivated Synechocystis sp. PCC6803. The statistical quality of the estimated fluxes was also evaluated.

Lysophosphatidylcholine derived from deer antler extract suppresses hyphal transition in Candida albicans through MAP kinase pathway

A family of 2-lysophosphatidylcholines (lyso-PCs) was isolated from deer antler extract, guided exclusively by hyphal transition inhibitory activity in Candida albicans. Structural determination of the isolated lyso-PCs by spectroscopic methods, including infrared spectroscopy, 1H nuclear magnetic resonance (NMR), 13C NMR, 2D correlation spectroscopy NMR, fast atom bombardment mass spectrometry and tandem mass spectrometry, confirmed that the natural products were composed of at least four different lyso-PCs varying in fatty acid moiety at the sn-1 position of the glycerol backbone. The major lyso-PCs were confirmed as 1-stearoyl-, 1-oleoyl-, 1-linoleoyl- and 1-palmitoyl-2-lyso- sn-glycero-3-phosphatidylcholines. Lyso-PC specifically suppressed the morphogenic transition from yeast to hyphae in C. albicans, without affecting the growth of either yeast or hyphae. Lyso-PC exerted hyphal transition that suppressed activity in the broad spectrum of the Candida species, such as C. albicans, Candida krusei, Candida guilliermondii and Candida parapsilosis. Northern analysis indicated that the suppression was mediated through the mitogen-activated protein kinase pathway.

Metabolic flux ratio analysis of genetic and environmental modulations of Escherichia coli central carbon metabolism.

The response of Escherichia coli central carbon metabolism to genetic and environmental manipulation has been studied by use of a recently developed methodology for metabolic flux ratio (METAFoR) analysis; this methodology can also directly reveal active metabolic pathways. Generation of fluxome data arrays by use of the METAFoR approach is based on two-dimensional (13)C-(1)H correlation nuclear magnetic resonance spectroscopy with fractionally labeled biomass and, in contrast to metabolic flux analysis, does not require measurements of extracellular substrate and metabolite concentrations. METAFoR analyses of E. coli strains that moderately overexpress phosphofructokinase, pyruvate kinase, pyruvate decarboxylase, or alcohol dehydrogenase revealed that only a few flux ratios change in concert with the overexpression of these enzymes. Disruption of both pyruvate kinase isoenzymes resulted in altered flux ratios for reactions connecting the phosphoenolpyruvate (PEP) and pyruvate pools but did not significantly alter central metabolism. These data indicate remarkable robustness and rigidity in central carbon metabolism in the presence of genetic variation. More significant physiological changes and flux ratio differences were seen in response to altered environmental conditions. For example, in ammonia-limited chemostat cultures, compared to glucose-limited chemostat cultures, a reduced fraction of PEP molecules was derived through at least one transketolase reaction, and there was a higher relative contribution of anaplerotic PEP carboxylation than of the tricarboxylic acid (TCA) cycle for oxaloacetate synthesis. These two parameters also showed significant variation between aerobic and anaerobic batch cultures. Finally, two reactions catalyzed by PEP carboxykinase and malic enzyme were identified by METAFoR analysis; these had previously been considered absent in E. coli cells grown in glucose-containing media. Backward flux from the TCA cycle to glycolysis, as indicated by significant activity of PEP carboxykinase, was found only in glucose-limited chemostat culture, demonstrating that control of this futile cycle activity is relaxed under severe glucose limitation.

Two-dimensional nuclear magnetic resonance correlation spectroscopy at zero field

Three methods for two-dimensional correlation nuclear magnetic resonance spectroscopy at zero field are discussed. All three involve coherence transfer via longitudinal polarization, double quantum coherence, or both in parallel. The double quantum pulse sequences exploit the spinor property of spin states. These sequences have been applied to connected Δm=1 transitions, as well as for the indirect detection of forbidden or nearly forbidden Δm>1 transitions.

Bioreaction Network Topology and Metabolic Flux Ratio Analysis by Biosynthetic Fractional 13C Labeling and Two-Dimensional NMR Spectroscopy

Biosynthetically directed fractional 13C labeling of the proteinogenic amino acids is achieved by feeding a mixture of uniformly 13C-labeled and unlabeled carbon source compounds into a bioreaction network. Analysis of the resulting labeling pattern enables both a comprehensive characterization of the network topology and the determination of metabolic flux ratios. Attractive features with regard to routine applications are (i) an inherently small demand for 13C-labeled source compounds and (ii) the high sensitivity of two-dimensional [13C,1H]-correlation nuclear magnetic resonance spectroscopy for analysis of 13C-labeling patterns. A user-friendly program, FCAL, is available to allow rapid data analysis. This novel approach, which recently also has been employed in conjunction with metabolic flux balancing to obtain reliable estimates of in vivo fluxes, enables efficient support of metabolic engineering and biotechnology process design.

Molecular Motion in the Two Amorphous Phases of Triphenyl Phosphite

Molecular reorientation in the two amorphous phases of triphenyl phosphite, namely the supercooled liquid (phase aI) and the newly discovered second amorphous phase (phase aII), was investigated by dielectric relaxation and by two-dimensional (2D) nuclear magnetic resonance spectroscopy (NMR) in the time and frequency domain. Whereas phase aI exhibits the relaxational features typical of supercooled liquids, the molecular motion in phase aII is characterized by an extremely broad dielectric loss and by a pronounced nonexponential reorientational correlation function. Using a Gaussian distribution of correlation times, both dielectric and NMR data reveal consistently correlation times on the order of seconds. The quantitative analysis of the 2D spectra favors the interpretation that molecular motion in phase aII leads to an isotropic distribution of molecular orientation on the surface of a sphere. In addition, we find a secondary relaxation process that shows basically the same features in both phases. We conclude that the newly discovered phase is a second liquid phase with a very unusual reorientational correlation function. However, a nematic liquid crystal cannot completely be ruled out.

Use of rapid scan correlation nuclear magnetic resonance spectroscopy as a quantitative analytical method

Rapid scan correlation (RSC) nuclear magnetic resonance (NMR) spectroscopy combines the advantages of rapidity of Fourier transform methods with advantages in dynamic range of continuous wave methods. A systematic investigation of the technique for quantitative analysis has been carried out. It is concluded, provided suitable precautions are taken, that RSCNMR can be useful as a quantitative method. Application is illustrated by the determination of ethanol in aqueous media and wines over the range 0.01–15% v/v.

High-resolution two-dimensional in-phase multiplets in nuclear magnetic resonance correlation spectroscopy

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTHigh-resolution two-dimensional in-phase multiplets in nuclear magnetic resonance correlation spectroscopySebastien J. F. Vincent, Catherine Zwahlen, and Geoffrey BodenhausenCite this: J. Am. Chem. Soc. 1992, 114, 27, 10989–10990Publication Date (Print):December 1, 1992Publication History Published online1 May 2002Published inissue 1 December 1992https://doi.org/10.1021/ja00053a061RIGHTS & PERMISSIONSArticle Views41Altmetric-Citations6LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (270 KB) Get e-Alerts Get e-Alerts

Two new 9,11-secosterols from the marine sponge Spongia officinalis. Synthesis of 9,11-seco-3β,6α,11-trihydroxy-5α-cholest-7-en-9-one

Two new 9,11-secosterol, 9,11-seco-3β,6α,11-trihydroxy-5α-cholest-7-en-9-one (2) and 9,11-seco-3β,6α,11-trihydroxy-24-methylene-5α-cholest-7-en-9-one (3), have been isolated from the marine sponge Spongia officinalis and their structures elucidated by analysis of spectral data including 1H nuclear magnetic resonance correlation spectroscopy (COSY) experiments. Partial synthesis of 2 starting from 3β,6α-dihydroxy-9-oxo-9,11-seco-5α-cholest-7-en-11-al (1) confirmed the structure assignment .

S-(2-chloroacetyl)glutathione, a reactive glutathione thiol ester and a putative metabolite of 1,1-dichloroethylene.

Conversion of the toxic vinyl halide 1,1-dichloroethylene (DCE) to S-(2-S-glutathionyl-acetyl)glutathione (GSCH2COSG) involves sequential acylation and alkylation of two glutathione (GSH) molecules by the microsomal DCE metabolite ClCH2COCl. To examine its possible role in DCE biotransformation, we synthesized the putative intermediate S-(2-chloroacetyl)glutathione (ClCH2COSG). In aqueous buffer, ClCH2COSG did not hydrolyze to release GSH, but instead underwent a two-step rearrangement to yield a cyclic product. Product analyses by liquid secondary ion mass spectrometry and 1H-13C heteronuclear correlation nuclear magnetic resonance spectroscopy indicated that rearrangement involved initial transfer of the chloroacetyl moiety from the cysteinyl thiol to the gamma-glutamyl alpha-amine. The cysteinyl thiol then displaced chloride from the 2-chloroacetyl methylene carbon to yield the cyclic product. Incubation of 2 mM ClCH2COSG with 20 mM GSH yielded approximately 4.5-fold more cyclic product than GSCH2COSG. ClCH2COSG alkylated oxytocindithiol and N-acetyl-L-cysteine to yield S-[2-(alkylthio)acetyl]glutathione adducts analogous to GSCH2COSG. S-2-Chloroacetylation products were absent. In reacting with thiols by alkylation and in decomposing by rearrangement, ClCH2COSG displayed properties strikingly different from those of ClCH2COCl. Although much less reactive than its acyl halide precursor, ClCH2COSG may display greater selectivity in covalent modification of cellular targets in DCE intoxication.

Ryanoid insecticides: structural examination by fully coupled two-dimensional1H–13C shift correlation nuclear magnetic resonance spectroscopy

The two-dimensional pulse sequence, (π/2, 1H)–Δt1–(π/2, 1H; π/2, 13C)–acquire, for ‘fully coupled’1H–13C shift correlation n.m.r. has been applied to structural analysis of ryanodine (1) and 9,21-didehydroryanodine (2)(a new botanical insecticide from Ryania speciosa). This procedure allowed total assignment of the 13C n.m.r. spectra using one-, two-, and three-bond 1H–13C couplings, stereochemical assignments of protons, and some conformational data via relative 3JcH couplings. The structure (2) defined by n.m.r. was confirmed by reductive conversion into ryanodine (1) and 9-epi-ryanodine (3).

Proton nuclear magnetic resonance and biochemical studies of oxygenation of human adult hemoglobin in deuterium oxide.

The proton nuclear magnetic resonance spectrum of human adult deoxyhemoglobin in D2O in the region from 6 to 20 ppm downfield from the proton resonance of residual water shows a number of hyperfine shifted proton resonances that are due to groups on or near the alpha and beta hemes. The sensitivity of these resonances to the ligation of the heme groups and the assignment of these resonances to the alpha and beta chains provide an opportunity to investigate the cooperative oxygenation of an intact hemoglobin molecule in solution. By use of the nuclear magnetic resonance correlation spectroscopy technique, at least two resonances, one at approximately 18 ppm downfield from HDO due to the beta chain and the other at approximately 12 ppm due to the alpha chain, can be used to study the binding of oxygen to the alpha and beta chains of hemoglobin. The present results using approximately 12% hemoglobin concentration in 0.1 M Bistris buffer at pD 7 and 27 degrees C with and without organic phosphate show that there is no significant line broadening on oxygenation (from 0 to 50% saturation) to affect the determination of the intensities or areas of these resonances. It is found that the ratio of the intensity of the alpha-heme resonance at 12 ppm to that of the beta-heme resonance at 18 ppm is constant on oxygenation in the absence of organic phosphate but decreases in the presence of 2,3-diphosphoglycerate or inositol hexaphosphate, with the effect of the latter being the stronger. On oxygenation, the intensities of the alpha-heme resonance at 12 ppm and of the beta-heme resonance at 18 ppm decreases more than the total number of deoxy chains available as measured by the degree of O2 saturation of hemoglobin. This shows the sensitivity of these resonances to structural changes which are believed to occur in the unligated subunits upon the ligation of their neighbors in an intact tetrameric hemoglobin molecule. A comparison of the nuclear magnetic resonance data with the populations of the partially saturated hemoglobin tetramers (i.e., hemoglobin with one, two, or three oxygen molecules bound) leads to the conclusion that in the presence of organic phosphate the hemoglobin molecule with one oxygen bound maintains the beta-heme resonance at 18 ppm but not the alpha-heme resonance at 12 ppm. These resluts suggest that some cooperativity must exist in the deoxy quaternary structure of the hemoglobin molecule during the oxygenation process. Hence, these results are not consistent with the requirements of two-state concerted models for the oxygenation of hemoglobin. In addition, we have investigated the effect of D2O on the oxygenation of hemoglobin by measuring the oxygen dissociation curves of normal adult hemoglobin as a function of pH in D2O andH2O media. We have found that (1) the pH dependence of the oxygen equilibrium of hemoglobin (the Bohr effect) in higher pH in comparison to that in H2O medium and (2) the Hill coefficients are essentially the same in D2O and H2O media over the pH range from 6.0 to 8.2...

Comparable investigations on the ultrasonic pulse-echo equipment for use as measuring instruments

A seven-step synthesis of Z-13,13,14,14,15,15,16,16,17,17,18,18,18-tridecafluoro-octadec-9-enoic acid (oleic acid-F13) is reported. The key step is a Wittig reaction to form the C9–C10 double bond with a Z:E isomeric ratio greater than 20:1. The 19F nuclear magnetic resonance (NMR) spectrum is included and unambiguous assignments have been made with the aid of 19F–19F correlation NMR spectroscopy (COSY spectra).