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.     

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.     

Assignment of structures to oligosaccharides produced by enzymic degradation of a beta-D-glucan from barley by 1H- and 13C-n.m.r. spectroscopy.

The structures of one tri-(1), two tetra-(2 and 3), and one hexa-saccharide (4) produced by treatment of barley flour, after removal of the starch components, with a fungal beta-D-glucanase (Finizyme) have been assigned on the basis of 1H- and 13C-n.m.r. data as follows: beta-D-Glcp-(1----3)-beta-D-Glcp-(1----4)-D-Glcp (1), beta-D-Glcp-(1----4)-beta-D-Glcp-(1----3)-beta-D-Glcp-(1----4)-D-Glcp (2), beta-D-Glcp-(1----3)-beta-D-Glcp-(1----4)-beta-D-Glcp-(1----4)-D-Glcp (3), and beta-D-Xylp-(1----4)-[alpha-L-Araf-(1----3)]-[alpha-L-Ara f-(1----2)-beta-D-Xylp-(1----4)-beta-D-Xylp- (1----4)-D-Xylp (4).     

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.     

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.     

Assignments of the 1H- and 13C-n.m.r. spectra of tobramycin at low and high pH

The 1H-n.m.r. spectra (200 MHz) of protonated (pD 3.9) and non-protonated (pD 11.9) tobramycin have been analysed completely by 2D methods. The 3JH,H values are consistent with an essentially undistorted 4C1 conformation for each of the three moieties and are practically independent of the state of protonation. The resonances in the 13C-n.m.r. spectrum (50 MHz) have been reassigned at both pD values on the basis of 2D1H-13C chemical-shift correlation and 1D selective INEPT measurements.     

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.     

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.     

1H- and 13C-n.m.r. studies of the antitumour antibiotic luzopeptin. Resonance assignments, conformation and flexibility in solution.

The depsipeptide DNA-intercalating antibiotic luzopeptin was studied in solution by n.m.r. methods. Two-dimensional 1H double-quantum-filtered correlation spectroscopy (DQF-COSY) and nuclear-Overhauser-effect spectroscopy (NOESY) confirm the primary structure and twofold symmetry of luzopeptin and provide details of its three-dimensional conformation in solution. Trans-annular hydrogen bonds between the glycine NH groups and carbonyl oxygen atoms have been identified in the crystalline state [Arnold & Clardy (1981) J. Am. Chem. Soc. 103, 1243-1244], and are important in maintaining an antiparallel beta-sheet conformation. The n.m.r. data indicate that the glycine NH protons are appreciably shielded from the solvent molecules, which suggests that these hydrogen bonds are maintained in solution. The orientation of the quinoline chromophores is defined by two-dimensional NOE cross-peaks that position the N-methyl group of the L-beta-hydroxyvaline residue close in space to both the quinoline H-8 and serine NH proton. This pattern of NOEs is in accord both with the chromophore configuration found in the crystal and one where the quinoline rings are aligned in a parallel manner at right-angles to the depsipeptide ring. The n.m.r. data are consistent with a hydrogen bond between the quinoline hydroxy groups and the quinoline carbonyl oxygen atoms. The pyridazine acetylmethyl groups give NOEs to the C(alpha)H groups of the beta-hydroxy-N-methylvaline residues, showing that the acetyl groups, for at least some of the time, stretch over the depsipeptide ring, occluding one face of the molecule. Both of the latter features are also found in the crystal structure. Resonances in the 13C-n.m.r. spectrum of luzopeptin have been assigned by transferring 1H assignments to their covalently bonded carbon atoms via a heteronuclear shift-correlation experiment (HETCOR). The measurement of spin-lattice relaxation times and 1H-13C NOEs at specific sites in the molecule has led us to conclude that segmental motions within the depsipeptide ring are restricted and that the 13C relaxation data for luzopeptin's protonated carbon atoms are adequately described by isotropic tumbling in solution. Furthermore, relaxation data for the carbon atoms of the quinoline chromophores show that these rings exhibit similar motion to the depsipeptide ring and are not rotating rapidly with respect to it. Taken together all the data imply that luzopeptin is fairly rigid in solution, on the time scale of molecular tumbling, and has, or can readily attain, a staple-like structure suitable for bisintercalation.(ABSTRACT TRUNCATED AT 400 WORDS)     

A 13C-n.m.r. study of intermediates in the L-type pentose phosphate cycle

The structures in aqueous solution of all major contributing forms of D-altro-heptulose 1,7-diphosphate, and D-glycero-D-altro and D-glycero-D-ido-octulose 1,8-diphosphates have been established by 13C-n.m.r. spectroscopy. Assignments to individual carbon atoms were made with the aid of isotopically enriched analogues and by comparison with related sugars.     

High-resolution H- and C-n.m.r. spectra of the group A-variant streptococcal polysaccharide

The molecular weight of the water-soluble polysaccharide of Phellodendron amurense Ruprecht was found to differ with the sample used. The difference is considered to be due to different degrees of degradation of the polysaccharide chains, together with oxidation of galactose to galacturonic acid residues.     

13C-n.m.r. study of citrate metabolism in rabbit renal proximal-tubule cells.

U.v.-visible-absorption and e.p.r. spectroscopy were used to study the type 2 and type 3 copper centres in the mercury derivative of laccase. After treatment with peroxide the mercury derivative of laccase exhibits a fully developed absorption band at 330 nm (delta epsilon = 2900 +/- 100 M-1.cm-1, which is characteristic of type 3 copper in the oxidized state. In addition, there is a weak ligand-field absorption at 740 nm (epsilon = 380 +/- 30 M-1.cm-1), which can be assigned to the type 3 pair. Because the e.p.r. spectrum of the type 2 copper is well resolved in the case of the mercury derivative of laccase, for the first time we have been able to observe spectroscopic evidence for a pH-dependent structural transition that has been invoked to explain the kinetics of enzyme reduction [Andréasson & Reinhammar (1979) Biochim. Biophys. Acta 568, 145-156]. According to the e.p.r. data the pKa lies in the range 6-7, and comparisons with a model compound show that the spectral changes can plausibly be interpreted in terms of the deprotonation of a water molecule in the co-ordination sphere of the type 2 copper.     

C-n.m.r. spectroscopic investigation of methylated and charged agarose oligosaccharides and polysaccharides

The ¹³C-n.m.r. signals of agarose oligomers with various substituted repeating units have been assigned. Enzymic hydrolysis of agaroses gave 2¹-O-methylagarobiose, 6²-O-methylagarobiose, the agarobiose biological precursor, agarobiose 4²-sulfate, 2¹-O-methylagarobiose 4²-sulfate, and pyruvylated agarobiose. The chemical shift data of the oligomers and the parent polymers were compared, and indicated the distribution of the substituents in hybrid polymers.