Homonuclear Hartmann-Hahn Spectroscopy Research Articles

Distribution of antigenic oligomannosyl side chains in the cell wall mannans of several strains of Candida tropicalis

In order to clarify the distribution of antigenic oligomannosyl side chains in the cell wall mannans of the pathogenic yeast Candida tropicalis, the chemical structure of mannans isolated from four C. tropicalis strains was investigated using nuclear magnetic resonance, two-dimensional homonuclear Hartmann-Hahn (2D-HOHAHA) spectroscopy. Two-dimensional maps of the 2D-HOHAHA clearly showed the distribution of oligomannosyl side chains in the mannans. The linear side chain Manalpha1-3Manalpha1-(2Manalpha1-)(n)2Man [n> or =2] is present in the mannans from C. tropicalis IFO 0589 and IFO 1400, but not in the mannans from IFO 0199 and IFO 1647. The mannan of IFO 0589 is the only mannan with the branched side chains, Manalpha1-3[Manalpha1-6]Manalpha1-(2Manalpha1-)(n)2Man and Manalpha1-2Manalpha1-3[Manalpha1-6]Manalpha1-(2Manalpha1-)(n)2Man [n> or =2]. However, this mannan lacked the phosphate group and the beta-1,2-linked oligomannosyl side chain which are features of this group. The mannans of the C. tropicalis strains IFO 0589 and IFO 1400 possessed the side chains containing an alpha-1,3-linked mannose residue previously observed in Candida albicans.

Conformational variation of the central CG site in d(ATGACGTCAT)2 and d(GAAAACGTTTTC)2. An NMR, molecular modelling and 3D-homology investigation.

The determination of the solution structure of two self-complementary oligomers d(ATGACGTCAT)2 (CG10) and d(GAAAACGTTTTC)2 (CG12), both containing the 5'-pur-ACGT-pyr-3' sequence, is reported. The impact of the base context on the conformation of the central CpG site has been examined by a combined approach of: (a) 2D 1H-NMR and 31P-NMR; (b) molecular mechanics under experimental constraints; (c) back-calculations of NOESY spectra and iterative refinements of distances; and (d) 3D-homology search of the central tetrad ACGT within the complete oligonucleotides. A full NMR study of each fragment is achieved by means of standard 2D experiments: NOESY, 2D homonuclear Hartmann-Hahn spectroscopy, double-quantum-filtered COSY and heteronuclear 1H-31P correlation. Sugar phase angle, epsilon-zeta difference angle and NOE-derived distances are input as experimental constraints to generate molecular models by energy minimization with the help of jumna. The morass program is used to iteratively refine the structures obtained. The similarity of the two ACGTs within the whole oligonucleotides is investigated. Both the decamer and the dodecamer adopt a B-like DNA conformation. However, the helical parameters within this conformational type are significantly different in CG12 and CG10. The central CpG step conformation is not locked by its nearest environment (5'A and 3'T) as seen from the structural analysis of ACGT in the two molecules. In CG12, despite the presence of runs of A-T pairs, CpG presents a high twist of 43 degrees and a sugar phase at the guanine of about 180 degrees, previously observed in other ACGT-containing-oligomers. Conversely, ACGT in CG10 exhibits strong inclinations, positive rolls, a flat profile of sugar phase, twist and glycosidic angles, as a result of the nucleotide sequence extending beyond the tetrad. The structural specificity of CG10 and its flexibility (as reflected by its energy) are tentatively related to the process of recognition of the cyclic AMP response element by its cognate protein.

Structural study of the LipoMannans from Mycobacterium bovis BCG: characterisation of multiacylated forms of the phosphatidyl-myo-inositol anchor

A biosynthetic filiation is postulated between the mycobacterial phosphatidyl-myo-inositol mannosides (PIMs), the lipomannans (LMs) and the lipoarabinomannans (LAMs), the major antigens of the envelopes. Moreover, as the PI anchor is thought to play a role in the biological functions of the LAMs, we characterized the lipid moiety of the PI anchor from Mycobacterium bovis BCG cellular LMs. Their structure was investigated along with that of a purified tetra-acylated form of PIM2(Ac4PIM2). A two-dimensional 1H-31P heteronuclear multiple quantum correlation homonuclear Hartmann-Hahn spectroscopy study of Ac4PIM2 unambiguously localised a fourth fatty acid on the C3 of the myo-Ins beside the fatty acids already described on the C1 and C2 position of the glycerol and on the C6 position of the mannose. This analytical strategy was extended to the structural study of the cellular LM anchor. Using an appropriate solvent system, the one dimensional 31P NMR spectrum exhibited four major resonances typifying the LM populations. These populations differed in number and location of the fatty acids. For one of these populations, we established the presence of an extra fatty acid on the C3 of the myo-Ins of the LM anchor. The fact that both types of molecules have an elaborated anchor in common, indicates that cellular LMs are multimannosylated forms of PIMs. In addition, the LM mannan core structure was analysed by two-dimensional NMR, pointing to a high level of branching by single α1→2 Man p side-chains.

Demonstration of the presence of alpha-1,6-branched side chains in the mannan of Candida stellatoidea.

A mild acetolysis of the mannans of Candida stellatoidea was performed after acetylation to yielded an alpha-1,6-branched mannohexaose, the presence of which had been predicted from the appearance of a specific H1-H2-correlated cross-peak in two-dimensional homonuclear Hartmann-Hahn spectroscopy. In this study, we found that the de-O-acetylation of a 4-O-acetyl group at the branching point, the 3,6-di-O-substituted mannose unit, of an acetylated oligosaccharide by sodium methoxide is significantly slower than that of other acetyl groups. We could separate the 4-O-acetylated branching oligosaccharide from linear isomer using high-performance liquid chromatography. Before and after the de-O-acetylation of the purified branching oligosaccharide, their 1H-NMR signals were sequentially assigned by means of the nuclear Overhauser effect. In the sequential NMR assignment study, we showed that the alpha-1,6-linked mannose unit is attached to the 3-O-substituted unit based on the presence of NOE cross-peak between H1 of the branching mannose unit and H6 of the 3-O-substituted mannose unit. An enzyme-linked immunosorbent inhibition assay of the reactivity of factor 4 serum to C. stellatoidea mannan by several oligosaccharides indicated that the alpha-1,6-branched oligosaccharide and the beta-1,2 linkage-containing oligosaccharides showed inhibitory activity. This result indicates that factor 4 serum, as well as factor 5 and 6 sera, contains antibodies against beta-1,2-linked mannose units which have been reported to participate in pathogenicity via cytokine production and/or adherence. From the assignment results of H1-H2-correlated cross-peaks of oligosaccharides and mannans, the molar ratio of the mannan side chains was proposed. In this study, we demonstrated that the epitope structure of the C. stellatoidea type I strains was the same as that of the C. albicans NIH B-792 (serotype B) strain.

1H-magnetic resonance spectroscopic observation of cultured malignant cells pharmacologically induced to different phenotypes.

We evaluated the 1H nuclear magnetic resonance spectra of malignant cells after the administration of drugs that cause morphologic changes. 1H spectra of a human lung adenocarcinoma cell line cultured with interferon gamma, dexamethasone, or sodium butyrate were obtained. The peaks were assigned by two-dimensional homonuclear Hartmann-Hahn spectroscopy spectra of the cells and their perchloric acid extracts. Differential spectra were used to evaluate relative changes in the peaks. In the control culture, choline/phosphocholine peaks were increased in the cell-growth phase, and the 1.26-ppm peak was increased in the confluent state. Treatment by interferon gamma and dexamethasone induced reproducible changes in the peaks of differential spectra corresponding to 1.26 ppm, choline/phosphocholine, and glutamate/glutamine. Dexamethasone treatment broadened lipid peaks. Changes after treatment with sodium butyrate were obscure. Microscopically, cells were induced to morphologically different phenotypes by each drug. Cells induced to exhibit morphologically different phenotypes present different 1H spectra.

Characterization of alpha-1,6-mannosyltransferase responsible for the synthesis of branched side chains in Candida albicans mannan.

A particulate insoluble fraction from Candida albicans NIH B-792 (serotype B) strain cells was obtained as the residue after extracting a 105000 x g pellet of cell homogenate with 1% Triton X-100. Incubation of this fraction with a mannopentaose, Man alpha 1-->3Man alpha 1-->2Man alpha 1-->Man alpha 1-->2Man, in the presence of GDP-mannose and Mn2+ at pH 6.0 gave a branched mannohexaose, [sequence: see text] 6 the structure of which was identified by means of sequential off assignment. However, the enzyme fraction obtained from Candida parapsilosis gave Man alpha 1-->2Man alpha 1-->3Man alpha 1-->2Man alpha 1-->2 Man alpha 1-->2Man under the same conditions. These results demonstrate the finding that the structural difference in the mannans of these two species is due to the presence of alpha-1.6-linked branching mannose units in the C. albicans mannan [Shibata, N., Ikuta, K., Imai, T., Satoh, Y., Satoh, R., Suzuki, A., Kojima, C., Kobayashi, H., Hisamichi, K. & Suzuki, S. (1995) J. Biol. Chem. 270, 1113-1122]. The substrate-specificity study of the enzyme indicated that the structural requirement of the alpha-1,6-mannosyltransferase is Man alpha 1-->3Man alpha 1-->. The alpha-1,6-mannosyltransferase also transferred the alpha-1,6-linked branching mannose unit to the mannan of Saccharomyces cerevisiae. The transformation of the mannan was detected by the appearance of antigenic factor 4 using an enzyme-linked immunosorbent assay and two-dimensional homonuclear Hartmann-Hahn spectroscopy.

The effect of conformation on membrane permeability of an acyloxyalkoxy-linked cyclic prodrug of a model hexapeptide.

To determine the different conformations of the acyloxyalkoxy-linked cyclic prodrug 1 of the model hexapeptide 2 in solution and to investigate the relationship between these solution conformations and the cellular permeability characteristics of this prodrug. Two-dimensional Homonuclear Hartmann-Hahn spectroscopy, Rotating-Frame Overhouser effect spectroscopy, circular dichroism and molecular dynamics simulations were used to find the solution conformers of cyclic prodrug 1. Our spectroscopic findings suggest that cyclic prodrug 1 exhibits a major and a minor conformer in solution. The major conformer appears to have a well-defined secondary structure, which involves a beta-turn and 4-->1 intramolecular hydrogen bond, creating a compact structure with a reduced average hydrodynamic radius compared to the model hexapeptide 2. The increased ability of cyclic prodrug 1 to permeate membranes compared to the model hexapeptide 2 could be due to reduction in the average hydrodynamic radius of the molecule facilitating paracellular flux and/or the reduction in the hydrogen bonding potential facilitating transcellular flux.

Existence of Branched Side Chains in the Cell Wall Mannan of Pathogenic Yeast, Candida albicans: STRUCTURE-ANTIGENICITY RELATIONSHIP BETWEEN THE CELL WALL MANNANS OF CANDIDA ALBICANS AND CANDIDA PARAPSILOSIS

Isolation of side chain oligosaccharides from mannans of Candida albicans NIH B-792 (serotype B) and Candida parapsilosis IFO 1396 strains has been conducted by acetolysis under mild conditions. Structural study of these oligosaccharides by 1H and 13C NMR and methylation analyses indicated the presence of novel branched side chains with the following structures in C. albicans mannan. [sequence: see text] It was observed that the H-1 proton chemical shifts of the second and the third mannose units from the reducing terminus in each oligosaccharide are shifted upfield by substitution with an alpha-linked mannose unit at position 6 of the 3-O-substituted mannose unit. An agglutination inhibition assay between factor 4 serum and cells of Candida stellatoidea IFO 1397 lacking the beta-1,2-linked mannose unit, with oligosaccharides obtained from these mannans, indicated that only the branched oligosaccharides were active. This finding suggests that the branched oligosaccharides correspond to the epitope of antigenic factor 4. The presence of the branched structure in other mannans was detected by the characteristic H-1-H-2-correlated cross-peak of the alpha-1,2-linked mannose unit connected with the 3,6-di-O-substituted one by two-dimensional homonuclear Hartmann-Hahn spectroscopy.

Expression of α-1,3 linkage-containing oligomannosyl residues in a cell-wall mannan of Candida tropicalis grown in yeast extract-Sabouraud liquid medium under acidic conditions

We investigated the cell-wall mannan obtained from Candida tropicalis IFO 1647 strain cells grown in yeast extract-Sabouraud medium at pH 3.0 by two-dimensional homonuclear Hartmann-Hahn spectroscopy. The results indicate that the phosphate group and the side chains containing a β-1,2-linked mannopyranose unit decreased compared to those of mannan from cells grown under conventional conditions (pH 5.9) with concomitant expression of α-1,3 linkage-containing oligomannosyl side chains. The results of acetolysis of these mannans indicated that the presence of α-1,3-linked mannopyranose unit existed in side chains corresponding to pentaose and hexaose, Man pα1-3Man pα1-2Man pα1-2Man pα1-2Man, and Man pα1-2Man pα1-3Man pα1-2Man pα1-2Man pα1-2Man, in the mannan from cells grown at pH 3.0.

Triterpenoid saponins from Ardisia crenata and their inhibitory activity on cAMP phosphodiesterase.

Two novel triterpenoid saponins, ardisicrenoside C (1) [3 beta-O-(alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->4)- [beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl)-16 alpha, 28-dihydroxy-olean-12-en-30-oic acid 30-O-beta-D-glucopyranosyl ester] and ardisicrenoside D (2) [3 beta-O-(beta-D-xylopyranosyl-(1-->2)- beta-D-glucopyranosyl-(1-->4)-[beta-D-glucopyranosyl-(1-->2)]-alpha-L- arabinopyranosyl)-16 alpha, 28-dihydroxy-olean-12-en-30-oic acid 30-O-beta-D-glucopyranosyl ester] were isolated from the roots of Ardisia crenata. Structure assignments are based on spectroscopic data including 2D-NMR (correlation spectroscopy (COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA), heteronuclear correlated spectroscopy (HETCOR), heteronuclear multiple bond correlation (HMBC) and rotating frame NOE spectroscopy (ROESY)) experiments and some chemical reactions. In addition, the isolated saponins along with their prosapogenins and sapogenins have been evaluated for their inhibitory activity on cAMP phosphodiesterase as a primary screening test for new medicinal compounds.

Structural study of a cell-wall mannan of Saccharomyces kluyveri IFO 1685 strain. Presence of a branched side chain and beta-1,2-linkage.

Acetolysis of the cell-wall mannan of Saccharomyces kluyveri under mild conditions, gave fragments with 1-6 mannose residues. The structures of mannopentaose and mannohexaose were determined to be [Formula; see text] respectively, by two-dimensional homonuclear Hartmann-Hahn spectroscopy and a sequential NMR assignment method that combines 1H-13C correlated spectroscopy, relayed coherence transfer spectroscopy, 1H-detected heteronuclear multiple-bond connectivity and methylation analysis. The H1 proton chemical shift of a neighboring alpha-1,2-linked mannose unit of the 3-O-substituted structure was shifted upfield by the addition of a mannose unit to the adjacent 3-O-substituted unit by an alpha-1,6 linkage. The characteristic H1--H2-correlated cross-peak of the alpha-1,3-linked mannose unit substituted by a beta-1,2 linkage, beta 1-->2Man alpha 1-->3, in the mannan of S. kluyveri, as also found by two-dimensional homonuclear Hartmann-Hahn spectroscopy in the mannan of Candida guilliermondii, a pathogenic yeast in man.

Heteronuclear nuclear magnetic resonance studies of cobalt corrinoids. 15. The structure of glutathionylcobalamin: a 1H and 13C two-dimensional nuclear magnetic resonance study at 600 MHz.

Glutathionylcobalamin (GSCbl), the complex formed between glutathione (GSH, gamma-glutamylcysteinylglycine) and aquacobalamin (H2OCbl), has been implicated as an intermediate in the pathway for the formation of the cobalamin coenzymes. In chemical model studies, GSCbl has been shown to be a substrate for methylcobalamin formation in the presence of S-adenosylmethionine and a thiol reductant. Although GSCbl was first described in 1964, the structure of this compound, particularly the site of GSH coordination, has been unknown. GSCbl was prepared by reacting GSH (5-fold molar excess) with H2OCbl in 0.1 M sodium phosphate (pH 6.5) and was purified by gel-permeation chromatography on a Bio-Gel P2 polyacrylamide column. By use of a combination of homonuclear [homonuclear J-correlated spectroscopy (COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA), and absorption-mode nuclear Overhauser effect spectroscopy (NOESY)] and inverse detected heteronuclear [1H-detected heteronuclear multiple-quantum coherence (HMQC) and 1H-detected multiple-bond heteronuclear multiple-quantum coherence (HMBC) spectroscopies] two-dimensional NMR methods at 600 MHz, the complete 1H and 13C NMR spectra of GSCbl have now been assigned. Comparison of the 1H and 13C NMR chemical shifts of the GS moiety of GSCbl to those of free GSH and GS- shows that by far the largest differences occur at the cysteine alpha and beta positions. This result strongly suggests that GSH is coordinated to the cobalt atom in GSCbl via the cysteine sulfur atom.

Structure of the O-specific polysaccharide of Salmonella arizonae O45

The O-specific polysaccharide of Salmonella arizonae O45 ( Arizona 11) is acidic and has a branched hexasacharide repeating unit containing two residues of l-fucose, one residue each of d-galactose, d-ribose, d-glucuronic acid, and 2-acetamido-2-deoxy- d-glucose, and an O-acetyl group. It was studied with the help of 1H and 13C NMR spectroscopy, including 1D selective spin-decoupling and homonuclear Hartmann-Hahn spectroscopy, 2D homonuclear and 13C 1H heteronuclear shift-correlated (COSY) and NOE (ROESY) spectroscopy, as well as by methylation analysis, and selective cleavages with anhydrous HF (or dilute HCl) and lithium in ethylenediamine to yield two different tetrasaccharide fragments. As a result, the following structure of the polysaccharide was established: ▪ Anomalous 13C chemical shifts were observed in the spectrum of the trisaccharide fragment α- l-Fuc p-(1 → 2)-β- d-Gal p-(1 → 3)-β- d-Glc pNAc, structurally related to the Le d blood-group determinant, and rationalised by inter-residue proton-proton interactions.

Conformation of deltorphin-II in membrane environment studied by two-dimensional NMR spectroscopy and molecular dynamics calculations.

Two-dimensional homonuclear Hartmann-Hahn spectroscopy and NOESY (nuclear Overhauser effect and exchange spectroscopy) 1H-NMR techniques have been used to obtain complete proton resonance assignments and to perform a conformational investigation of deltorphin-II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2), a naturally occurring delta-selective opioid peptide, in the membrane-mimetic micelles of perdeuterated dodecylphosphocholine. This was done in order to examine conformational characteristics that would be closely related to the selectivity towards the delta-opioid receptor. With the use of the proton-proton distances derived from NOESY measurements, 50 possible three-dimensional structures were generated by means of distance-geometry calculations, and 25 of them were subjected to the molecular-dynamics simulations of 10 ps, which were energetically constrained for the NOE interproton distances. Most of the possible conformers simulated showed a common feature such that the conformation of deltorphin-II is characterized by the S-shaped back-bone structure in which the turn conformation of the Val-Val-Gly-NH2 sequence is located under the helically folded conformation of the N-terminal Tyr-D-Ala-Phe-Glu sequence. The possible relationship between this conformational characteristic and the delta-opioid-receptor selectivity has been discussed.

The utility of one-dimensional homonuclear Hartmann—Hahn spectroscopy (1D HOHAHA) for identifying the suprofen glucuronides fraction from an HPLC separation

Many drug substances that contain carboxylic acid moieties are eliminated from the body via an enzymatic pathway that results in their conjugation with glucuronic acid. Characterization of these acyl glucuronides is complicated because of the ease of acyl migration and hydrolysis. Acyl rearrangements have been well studied and this area has been reviewed by Faed [l]. Liquid chromatography (LC), mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy are the standard analytical methods used for studying glucuronides [2-61. NMR studies are very well suited for identification of positional isomers of acyl glucuronides because of the large chemical shift changes induced by acylation [7]. Additionally, differentiation of the (Yand the P-anomers generally can be obtained from measurement of the proton-proton coupling constant of the anomeric proton [7]. What is needed is a NMR method that aids in the identification of the positional isomers of glucuronides within a mixture. Analysis of this type reduces the chance of hydrolysis or acyl migration as additional purification and separation steps are not required. Homonuclear Hartmann-Hahn (HOHAHA) NMR spectroscopy is well suited for the analysis of mixtures. The technique was first demonstrated by Davis and Bax [8] to assign the proton resonances from the ribose rings in a trinucleotide. They clearly showed the power of the method as a means for spectral editing and thus simplifying complicated spectra. It uses a selective 180 pulse as a means of accessing the spin network followed by a mixing period to label the protons within the coupling network. Onedimensional HOHAHA requires only one resolved proton resonance to be able to identify all the resonances within a sugar ring. For simple sugars this requirement is generally met using the anomeric proton. If needed the sequential assignment of protons can be had by varying the experimental mixing period (spin lock time) because the relay of magnetization occurs sequentially between coupled protons at a rate dependent on their coupling constant [S]. One-dimensional HOHAHA and in particular its 2D analogue [9, lo] has found wide application for structural studies of small proteins, nucleic acids and polysaccharides. For metabolite studies the 2D version may not be practical because of small sample size. Herein is demonstrated the utility of 1D HOHAHA spectroscopy for the identification of acyl glucuronides within a mixture. The glucuronide fraction isolated from an LC column separating the components of human urine from four healthy male subjects given suprofen, (+)-ol-methyl-4-(2-thienyl-

Nondestructive Analyses of Residue Types in β-d-Glucan Derivatives by Two-Dimensional Homonuclear Hartmann-Hahn Spectroscopy

Nondestructive Analyses of Residue Types in β-d-Glucan Derivatives by Two-Dimensional Homonuclear Hartmann-Hahn Spectroscopy

Conformational analysis of the .alpha.-L-arabinofuranosides present in wheat arabinoxylans from proton-proton coupling constants

The conformational behavior of α-L-arabinofuranosides present in wheat endosperm L-arabino-D-xylans has been investigated. To that purpose, oligosaccharides obtained by enzymatic digestion of a wheat endosperm L-arabino-D-xylan were submitted to z-filtered 1D homonuclear Hartmann-Hahn (HOHAHA) spectroscopy, and from the obtained vicinal proton-proton coupling constants, the ring conformation was deduced with the use of appropriate Karplus equations in conjunction with the concept of pseudorotation. The results revealed that α-L-arabinofuranosides linked to 0 3 of an internal O-3-mOnO- or O-2,3-disubstituted (1→4)-linked β-D-XylOpyranOSe residue are present in a conformational equilibrium of equal amounts of 4E-40T (N-type) and 12T-2E (S-type) conformers, whereas the α-L-arabinofuranoside linked to 02 of an internal 02,3-disubstituted (1→4)-linked β-D-xylopyranose residue is present in a conformational equilibrium of equal amounts of 43T-4E (N-type) and 1E-12T (S-type) conformers. Both N- and S-type conformers of the investigated alpha-L-arabinofuranosides contain a rigid C4-04-Cl-01 segment, assuming for 01 a pseudoaxial orientation and for the exocyclic CH20H group a pseudoequatorial orientation. From the proton-proton coupling constants the exocyclic rotamer populations could also be determined showing nearly equal preference for the g- and t rotamers.

Application of two-dimensional homo- and heteronuclear correlation NMR spectroscopy to wood lignin structure determination

A released suspension culture lignin (RSCL) from Norway spruce (Picea abies) was subjected to a detailed two-dimensional NMR study. It was found that the most informative techniques were homonuclear Hartmann-Hahn (HOHAHA) spectroscopy and 13 C-decoupled 1 H-detected multiple quantum 1 H- 13 C-correlation (HMQC) spectroscopy

Reducing the overlap problem in the proton NMR spectra of oligosaccharides by application of pseudo-four-dimensional homonuclear HOHAHA-HOHAHA-COSY.

A new homonuclear NMR experiment is described for the assignment of the proton NMR spectra of oligosaccharides, namely HOHAHA-HOHAHA-COSY (where HOHAHA is homonuclear Hartmann-Hahn spectroscopy and COSY is correlated spectroscopy). While this technique may formally be thought of as a four-dimensional NMR experiment, by use of selective pulses it is demonstrated that the analogous pseudo-four-dimensional experiment is a valuable improvement over conventional three-dimensional HOHAHA-COSY, in that the degree of resonance overlap is markedly reduced by the dispersion of resonances into a fourth effective dimension. The technique is demonstrated by application to the biantennary nonasaccharide Gal beta 1-4-GlcNAc beta 1-2Man alpha 1-6(Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man-beta 1-4GlcNAc beta 1-4GlcNAc.

Proton and carbon-13 NMR studies on xanthan derivatives

High-resolution NMR spectra of a number of xanthan derivatives have been obtained. Enzymic hydrolysis with cellulases was used to produce xanthan fragments as small as the pentasaccharide repeat unit. Spectral resolution improved as the molecular weight of the samples was decreased. Changes in the chemical shift of some anomeric proton and carbon resonances indicated that substantial conformational transitions had taken place on depolymerisation. Partial and complete removal of pyruvyl and acetyl substituents, plus proton-carbon-13 correlation NMR spectroscopy and homonuclear Hartmann-Hahn spectroscopy, were used to assist in the assignment of a number of resonances. Comparison of spectra from samples run at ambient (27°C) and high temperature (90°C) revealed chemical shift changes, particularly of anomeric resonances, which were probably a consequence of conformational transitions induced about the glycosidic linkages by heating or cooling.