13C-n.m.r. study of C hordein.

Insoluble xylan was prepared from ground birch (Betula pubescens) pulp by alkali extraction and precipitation with ethanol. The only sugar detected after acid hydrolysis of the preparation was xylose. The insoluble xylan was used as substrate in a nephelometric assay to determine the xylanase (EC 3.2.1.8, 1,4-beta-D-xylan xylanohydrolase and EC 3.2.1.37, 1,4-beta-D-xylan xylohydrolase) activities of Aspergillus and Trichoderma enzymes. The nephelometric method is reliable in evaluating xylanase hydrolysis of insoluble xylan.     

The 13C-n.m.r. spectra of peracetylated cello-oligosaccharides

The reaction of 1,4-anhydro-2-deoxy-5,6-O-isopropylidene-d-arabino-hex-1-enitol (1) with m-chloroperbenzoic acid in ethanol gives 2,3-unsaturated ethyl glycosides together with saturated ethyl glycosides formed by trans-ring opening of 1,2-epoxide intermediates. Similar results are obtained on peroxidation of 1,4-anhydro-2-deoxy-3-O-(2,3:5,6-di-O-isopropylidene-α-d-mannofuranosyl)-5,6-O-isopropylidene-d-arabino-hex-1-enitol (2). Products resulting from osmylation of 1 and 2 and cleavage of the osmate esters are also described. 2-Deoxy derivatives are prepared from 1 and 2 by methoxymercuration-demercuration and also by reduction of 2-bromo-2-deoxy derivatives obtained by ethoxybromination.     

High resolution 13C-n.m.r. spectroscopy of 'mixed linkage' xylans.

Three xylan fractions, obtained by stepwise precipitation with ethanol, were analysed by 75-MHz 13C-n.m.r. spectroscopy. Diad frequencies, determined from the C-2 resonances, show that the (1----3)-linkages are interspersed throughout the chain rather than grouped contiguously. This type of distribution is in agreement with a random coil conformation and with the constancy of the optical rotation in solvents of different ionic strength and chaotropic power. These diad frequencies were compared with the theoretical values calculated for a random distribution from the ratio of (1----4)-:(1----3)-linkages in the 1H-n.m.r. spectra, and from the methylation analysis for one of the fractions.     

13C-substituted pentos-2-uloses: synthesis and analysis by 1H- and 13C-n.m.r. spectroscopy

D-erythro-Pentos-2-ulose and D-threo-pentos-2-ulose and their 1-13C- and 2-13C-substituted derivatives have been prepared by oxidizing the corresponding natural and 13C-substituted D-aldopentoses (D-arabinose, D-xylose) with cupric acetate, and purifying the products by chromatography on a cation-exchange resin in the calcium or barium form. The equilibrium compositions of the pentos-2-uloses in 2H2O were determined by 13C-n.m.r. spectroscopy (75 MHz) at 25 degrees and 80 degrees. Among the eighteen possible monomeric acyclic, cyclic, and bicyclic forms, the anomeric pairs of the unhydrated aldopyranoses, aldopyranose endocyclic hydrates, aldofuranose endocyclic hydrates, and ketofuranose exocyclic hydrates were identified on the basis of 13C chemical shifts and 13C-1H and 13C-13C spin-coupling constants. 1H-N.m.r. (300, 500, and 620 MHz) and 13C-n.m.r. (75 MHz) spectroscopic data in one and two dimensions (DQF-COSY, homonuclear 2D-J) were used to evaluate the conformational properties of the cyclic structures. The unhydrated pyranoses are highly conformationally homogeneous; the erythro and threo isomers prefer 1C4 and 4C1 conformations, respectively. D-threo-Pentos-2-ulopyranose hydrate prefers the 4C1 conformation whereas the erythro isomers exists in both the 4C1 and 1C4 conformations. The furanoid forms favor structures having quasi-axial anomeric hydroxyl groups and quasi-equatorial exocyclic hydroxymethyl or dihydroxymethyl groups.     

1H- and 13C-n.m.r.-spectral study of synthetic methyl d-manno-oligosaccharides

¹H- and ¹³C-n.m.r. spectra for 16 synthetic methyl manno-oligosaccharides were recorded, and the signals for the anomeric protons and anomeric carbon atoms in branched manno-pentaosides and -hexaosides were assigned, based on the data for methyl manno-biosides and -triosides. These n.m.r. data identified the branching pattern of high-mannose types of glycans of glycopeptides with those of unambiguously synthesized manno-oligosaccharides, and confirmed the structures proposed for such glycans.     

The application of 13C-n.m.r. spectroscopy to products derived from sucrose

Glycogen has been isolated from the livers of rats which had been fasted and then intubated with d-glucose. The structure of the glycogen, as determined by iodine staining and enzymic methods, was shown to be very similar to that from control animals. There were slight differences in the iodine-staining properties, but not as marked as that previously reported in the literature.     

Room temperature, low-field 13C-n.m.r. spectra of degraded kappa/iota carrageenans

Using samples previously degraded by autohydrolysis, it is possible to obtain valuable low-field 13C-n.m.r. spectra of samples of kappa/iota carrageenans at room temperature. Spectroscopy of samples extracted at different stages of autohydrolysis helps to determine the mechanism of the reaction and the presence of low proportion kinks and other irregularities in the molecules.     

Very-high-field n.m.r. studies of bovine lung heparan sulphate oligosaccharides produced by nitrous acid deaminative cleavage. 13C-n.m.r. study of methylene resonances: degree and positions of C-6 sulphation.

Oligosaccharides with the general structure UA-(GlcNAc-GlcUA-)m-aManOH (m = 1-5) (where UA represents uronic acid, GlcNAc N-acetylglucosamine, GlcUA glucuronic acid and aManOH anhydromannitol) were prepared from low-sulphated heparan sulphates of bovine lung origin by nitrous acid deaminative cleavage followed by reduction. Analysis of the methylene signals in the 100 MHz 13C-n.m.r. spectrum of the tetrasaccharide (m = 1) shows that, whereas the extent of C-6 O-sulphation in the GlcNAc is approx. 65%, in the aManOH [formerly a GlcNSO3 (N-sulphoglucosamine) residue in the parent heparan sulphate] it is only approx. 10%. In the higher oligosaccharides (m = 2-5) the gross extent of C-6 O-sulphation of GlcNAc residues falls systematically with increasing oligosaccharide size, whereas that in the aManOH residues remains below 10%. There is also evidence that the C-6 O-sulphation of the GlcNAc residues is confined to the GlcNAc residue adjacent to the non-reducing terminal uronic acid residue. It is therefore tentatively proposed that the GlcNAc in the sequence -GlcNSO3-UA-GlcNAc- might be a favoured substrate for the 6-O-sulphotransferase. It is concluded that in the low-sulphated heparan sulphates GlcNSO3 residues that do not occur in (GlcNSO3-UA-)n blocks tend to have a significantly smaller extent of C-6 O-sulphation than do GlcNAc residues that occur in -GlcNSO3-UA-GlcNAc-GlcUA-GlcNSO3-sequences.     

Determination of arrangement of isoprene units in pig liver dolichol by 13C-n.m.r. spectroscopy.

The arrangement of isoprene units in pig liver dolichol-18, -19 and -20 was determined by 1H- and 13C-n.m.r. spectroscopies. The alignment of trans and cis isoprene units was found to be in the order: dimethylallyl unit, two trans units, a sequence of 14-16 cis units, and a saturated isoprene unit terminated with a hydroxyl group, which verified the presumed chemical structure of dolichol. The absence of geometric isomers was confirmed. A slight amount of impurity was detected in each reversed-phase h.p.l.c. fraction of dolichol obtained by a conventional method. Detailed assignments of the 13C-n.m.r. spectrum were given for these dolichols by using model compounds and INEPT (insensitive nuclei enhanced by polarization transfer) measurement. The chemical structure of synthetic dolichol-19, which was prepared by the addition of a saturated isoprene unit to the polyprenol-18 isolated from Ginkgo biloba, was confirmed to be identical with that of pig liver dolichol-19.     

Complete 1H- and 13C-n.m.r. assignments for two sulphated oligosaccharide alditols of hen ovomucin

The complete 1H- and 13C-n.m.r. assignments for beta-D-Galp-(1----4)-beta-D-GlcpNAc-6-SO3H-(1----6)-[beta-D-Galp-(1----3 )]- D-GalNAcol and alpha-NeuAcp-(2----3)-beta-D-Galp-(1----3)-[beta-D-Galp-(1----4)-b eta-D- GlcpNAc-6-SO3H-(1----6)]-D-GalNAcol were made by a combination of 2-D correlation experiments (Relayed-Cosy; and 13C,1H Correlation-shift n.m.r. spectroscopy), and 1-D n.m.r. spectroscopy. The results illustrate the ability of these methods to locate sulphate and NeuAc groups in anionic mucinous glycoproteins.     

13C-n.m.r.-spectral study of galactosyltransferase specificity with regard to the carbohydrate side-chain of native ovalbumin

As a prelude to studies using bovine N-acetylglucosaminide-β-(1→4)-galactosyltransferase to label membrane-surface glycoproteins with isotopically enriched d-galactose, the structural specificity of the enzymic reaction with water-soluble, hen ovalbumin has been examined. The enzyme-catalyzed transfer of d-galactose from UDP-d-galactose requires a (nonreducing) terminal 2-acetamido-2-deoxy-d-glucosyl group and exhibits selectivity towards saccharide chains containing d-mannose. This study considers the structural specificity of the enzyme with regard to the anomeric linkage between 2-acetamido-2-deoxy-d-glucose and d-mannose in the carbohydrate chains of hen ovalbumin. Uniformly ¹³C-enriched d-galactose was enzymically attached to the ovalbumin carbohydrate chain (which exhibits microheterogeneity in its structure), the protein was hydrolyzed, and separate glycopeptide fractions were chromatographically isolated. The ¹³C-n.m.r. spectra (60.5 MHz) of the fractions revealed two peaks for the anomeric carbon atom of d-galactose. The two peaks, at 104.20 and 104.39 p.p.m., were ascribed to d-galactosyl groups attached to 2-acetamido-2-deoxy-d-glucose respectively linked β-(1→4) and β-(1→2), to d-mannose in the glycopeptide chains. Quantifying of the spectral data revealed no specificity of d-galactosyltransferase towards the linkage from the terminal 2-acetamido-2-deoxy-d-glucosyl group to the penultimate d-mannosyl residue.     

Furanose ring anomerization: kinetic and thermodynamic studies of the D-2-pentuloses by 13C-n.m.r. spectroscopy

The tautomeric compositions of D-erythro-2-pentulose (D-ribulose) and D-threo-2-pentulose (D-xylulose) in aqueous solution have been studied by 13C-n.m.r. spectroscopy at various temperatures using 2-13C-substituted compounds. The alpha-furanose, beta-furanose, and acyclic carbonyl (keto) forms were detected at all temperatures, whereas the acyclic hydrate (gem-diol) form was not observed. The percentage of keto form increased with increasing temperature, at the expense of the furanose forms. Thermodynamic (delta G0, delta H0, delta S0) and kinetic parameters for the interconversion of alpha- and beta-furanoses with the acyclic carbonyl form were determined and compared with those determined under similar conditions for the structurally-related aldotetrofuranoses. The ring-opening rate constant (kopen) measured by 13C saturation-transfer n.m.r. spectroscopy in 50mM sodium acetate (pH 4.0) at 55 degrees were as follows: beta-threofuranose (0.65 s-1) greater than alpha-erythrofuranose (0.51 s-1) greater than beta-erythrofuranose (0.37 s-1) approximately beta-threo-2-pentulofuranose (0.35 s-1) greater than alpha-threofuranose (0.25 s-1) greater than alpha-threo-2-pentulofuranose (0.20 s-1) approximately alpha-erythro-2-pentulofuranose (0.18 s-1) approximately beta-erythro-2-pentulofuranose (0.18 s-1). Within each structural type the pentulofuranose anomer having O-2 and O-3 cis (O-1 and O-2 cis in aldotetrofuranoses) opens faster than, or at a similar rate to, the alternative anomer having these oxygen atoms trans. Ring-closing rate constants (kclose), calculated from kopen and Keq, decrease in the order beta-erythrofuranose (15 s-1) greater than beta-threofuranose (12 s-1) greater than alpha-erythrofuranose (9.9 s-1) greater than alpha-threofuranose (6.2 s-1) greater than beta-threo-2-pentulofuranose (0.71 s-1) greater than alpha-erythro-2-pentulofuranose (0.38 s-1) greater than alpha-threo-2-pentulofuranose (0.13 s-1) approximately beta-erythro-2-pentulofuranose (0.13 s-1). Replacement of H-1 in aldotetrofuranoses by a hydroxymethyl group (i.e., conversion to 2-pentuloses) significantly decreases the ring-opening and ring-closing rate constants of furanose anomerization.