Very-high-field n.m.r. studies of bovine lung heparan sulphate tetrasaccharides produced by nitrous acid deaminative cleavage. Determination of saccharide sequence, uronate composition and degrees of sulphation.

Tetrasaccharides with the general structure UA-GlcNAc-GlcUA-aManOH (where UA represents uronate, GlcNAc N-acetylglucosamine, GlcUA glucuronate and aManOH anhydromannitol) were prepared from low-sulphated heparan sulphates of bovine lung origin by complete nitrous acid deaminative cleavage followed by reduction and fractionated by gel filtration. Ion-exchange chromatography of the tetrasaccharides yielded three major fractions in approximate yields of 37%, 45% and 14%. These were shown to be non-, mono- and di-sulphated respectively. Complete structural characterization of the tetrasaccharide fractions by quantitative high-field n.m.r. spectroscopy showed that each fraction contained only two discrete species and led to the following observations. (1) All of the uronate residues in the tetrasaccharides (and in larger oligosaccharides) are unsulphated, and hence sulphated iduronate [IdUA(2SO3)] must occur exclusively within -GlcNSO3-IdUA(2SO3)-GlcNSO3- sequences (where GlcNSO3 represents N-sulpho-glucosamine) in the parent polymers. (2) The GlcNAc residues in the tetrasaccharides are more highly C-6-O-sulphated than are the aManOH residues, and furthermore sulphation on the aManOH appears to occur only where the GlcNAc is also sulphated. (3) Where the GlcNAc is C-6-O-sulphated, iduronate is the major non-reducing terminal residue, whereas glucuronate predominates in this position if the GlcNAc is unsulphated. The quantitative data obtained are used to determine the degree of C-6-O-sulphation of glucosamine residues in specific sequences within the parent heparan sulphates.     

13C-n.m.r. analysis of some sulphate derivatives of chitosan.

Positions of substitution with sulphate in three water-soluble sulphated derivatives of chitosan were analysed by 13C n.m.r. The structures of N-acetylchitosan 3,6-O-disulphate, sodium chitosan N-, 6-O-disulphate, and sodium chitosan 6-O-monosulphate were confirmed.     

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).     

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.     

13C-n.m.r. studies of the conformational changes in proline oligomers brought about by lithium and calcium salts

A detailed ¹³C-n.m.r. investigation has been carried out on the conformational changes in proline oligomers brought about by interaction with lithium and calcium perchlorates. Interaction of lithium and calcium salts with Piv-(Pro)_n-OMe, n = 2, 4 and 5 results in trans-cis isomerization. In the case of pentaproline, metal salts also give rise to other trans-isomers caused by the rotation about the C^αC(O) bond (Ψ, cis). Calcium salts seem to stabilize cis'-isomers and produce effects somewhat different from those of lithium salts.     

Reassignment of the methine resonances of D-fructose based on the carbon-13 n.m.r. spectrum of 3-O-methyl-D-fructose

Several disagreements in the ¹³C n.m.r. assignments of the methine carbons of D-fructose exist in the literature. In order to settle these inconsistencies, we examined the ¹³C n.m.r. spectrum of 3-O-methyl-D-fructose. By following the methyl induced shift in this spectrum, as compared to the parent sugar, we identified the alkylated C-3 resonance of all four tautomeric forms of D-fructose. This information, together with our previous identification of the C-5 resonances of the α- and β-forms of D-fructofuranose 6-phosphate, allow the unambiguous identification of all methine carbons of D-fructose in its ¹³C n.m.r. spectrum. The tautomeric composition of 3-O-methyl-D-fructose at 16.5°, in aqueous solution, was found to be as follows: α-pyranose 18%, β-pyranose 37%, α-furanose 11% and β-furanose 34%.     

A two-dimensional 1H-n.m.r. (500 MHz) and 13C-n.m.r. (125 MHz) study of N-linked glycopeptides derived from calf fetuin

The oligosaccharide part of an N-linked triantennary glycopeptide from calf fetuin with fourteen carbohydrate residues and its smaller derivatives obtained by successive enzymic cleavage of the terminal residues were investigated using 2D 1H-n.m.r. (500 MHz) and 13C-n.m.r. (125 MHz) spectroscopy. Assignments have been made of the resonances of almost all the protons of the constituent carbohydrate residues in these glycopeptides. A comparison of the 1H chemical shifts and coupling constants, as determined from the cross-peaks, has shown the dependence of these parameters on the interactions of spatially related neighbouring carbohydrates. Small conformational changes take place upon elongation of the oligosaccharide side-chains.     

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)     

H and 13C n.m.r. studies of serum from normal and Echinococcus multilocularis infected jirds

The major components of the 13C and high field region of the 1H nuclear magnetic resonance (n.m.r.) spectra of normal and Echinococcus multilocularis infected jirds were identified and compared. Substantial depletion of the glucose and fatty acid chains from lower density lipoproteins was detected in sera from infected animals. In addition, this proliferating metacestode markedly changed the appearance of the spectral region recently assigned to N-acetyl protons of carbohydrate side chains of N-acetylated glycoproteins.     

1H and 13C n.m.r. studies of pseudo-peptide analogues of the C-terminal tetrapeptide of gastrin

1H and 13C n.m.r. study of pseudo-peptide analogues of the C-terminal tetrapeptide of gastrin, obtained by replacing each peptide bond by a "reduced peptide bond", one at a time, e.g. Boc-Trp psi (CH2NH)Leu-Asp-Phe-NH2 2, Boc-Trp-Leu psi (CH2NH) Asp-Phe-NH2 3, Boc-Trp-Leu-Asp psi (CH2NH)Phe-NH2 4, were reported. The CH2NH bond was completely characterized. 1H and 13C spectroscopic data were reported. It appeared from the present work that the modifications produced by the replacement of a peptide bond by a CH2NH bond were localized around the CH2NH.     

Biosynthesis and n.m.r. studies of deuterated poly(3-hydroxybutyrate) produced by Alcaligenes eutrophus H16.

Alcaligenes eutrophus H16 was grown on mixtures of 1H- and 2H-acetate as carbon sources. The accumulation of deuterated poly(3-hydroxybutyrate) (P(3HB)) was observed. The deuterium distributions in the isolated P(3HB)s were determined from 1H and 2H-n.m.r. spectra and confirmed by 13C-n.m.r. spectra. Although one would expect to synthesize P([2,2,4,4,4-2H5]3HB) when the cells were grown on 2H-acetate as the sole carbon source, the methyl, methylene and methine groups of the P(3HB) contained both deuterium and proton. This observation indicates some substitution from 2H to 1H during the P(3HB) synthesis. The 2H content in the methyl groups was larger than that in the methylene groups, which suggests a kinetic isotope effect in the P(3HB) synthesizing process. The deuterium distributions in the two magnetically non-equivalent methylene protons were determined to be different, which indicates stereoselectivity at the C2 site.     

Analysis of conformationally restricted models for the (1----6)-branch of asparagine-linked oligosaccharides by n.m.r.-spectroscopy and HSEA calculation.

The conformational preferences of the trisaccharide, beta-D-GlcpNAc-(1----2)-alpha-D-Manp-(1----6)-beta-D-GlcpOR (1) have been investigated by n.m.r.-spectroscopy and HSEA calculation. The fixed omega-angle bicyclic analogs 2 and 3, models for the gt and gg rotamers, respectively, of 1, were furthermore examined with the same techniques in an attempt to deduce which of the conformations accessible to 1 was recognized and glycosylated by the enzyme GlcNAc-transferase-V, which acts on a component of the (1----6)-arm of glycoproteins. Only the gg bicyclic 3 was found to be reactive with the enzyme and this study concludes, based on conformational analysis, that 1 as well as the natural Asn-linked oligosaccharide are recognized by GlcNAc-transferase-V in only one of the two local minimum energy conformations energetically accessible to these molecules in their gg rotamer.     

X-ray structural and n.m.r.-spectral studies of methyl alpha-L-evalopyranoside: reassignment of anomeric configuration for the methanolysis product of methyl 6-deoxy-3-C-methyl-alpha-L-mannofuranoside

The methanolysis product of methyl 6-deoxy-3-C-methyl-alpha-L-mannofuranoside has been reassigned as methyl 6-deoxy-3-C-methyl-alpha-L-mannopyranoside by X-ray crystallographic and n.m.r.-spectral analyses. The crystals of methyl alpha-L-evalopyranoside are monoclinic, space group C2, with cell dimensions: a = 12.913(2), b = 8.052(1), c = 9.766(2) A, B = 105.13(2) degrees. The pyranoside ring exists in the 1C4 conformation, with the methoxyl and 3-C-methyl groups axial. Nuclear Overhauser effects were measured for selected proton resonances in the 1H-n.m.r. spectrum. Irradiation of the 3-C-methyl and 5-C-methyl group proton signals resulted in enhancements for H-2, H-4, H-5, and the methoxyl group hydrogen atoms, but not for H-1.     

1H n.m.r. studies of a neurotoxin and a cardiotoxin from Naja mossambica mossambica: Amide proton resonances

Proton n.m.r. spectra at 360 MHz of neurotoxin II and cardiotoxin V^II4 from the venom of $\text{Naja mossambica mossambica}$ are reported. From the n.m.r. spectra the solution conformations of the two proteins seem to be quite closely related. However, the exchange rates of the n.m.r. observable labile protons with deuterium of the solvent were markedly different, showing that the molecular structure of the cardiotoxin must be more flexible than that of the neurotoxin and suggesting that the different functional properties of the two toxins might be related to the different molecular dynamics.     

Synthesis of methyl O-(3-deoxy-3-fluoro-β--galactopyranosyl)-(1→6)-O-β--galactopyranosyl-(1→6)-3-deoxy-3-fluoro -β--galactopyranoside and related n.m.r. studies

Sequential tritylation, benzoylation, and detritylation of methyl 3-deoxy-3-fluoro-β--galactopyranoside gave crystalline methyl 2,4-di-O-benzoyl-3-deoxy-3-fluoro-β--galactopyranoside (9), which was used as the initial nucleophile in the synthesis of the target oligosaccharide (16). Treatment of 9 with 2,3,4-tri-O-benzoyl-6-O-bromoacetyl-α--galactopyranosyl bromide gave the corresponding disaccharide derivative 13, having a selectively removable blocking group at O-6′. Debromoacetylation of 13 afforded the disaccharide nucleophile 14 which, when treated with 2,4,6-tri-O-benzoyl-3-deoxy-3-fluoro-α--galactopyranosyl bromide, gave the fully protected trisaccharide 15. Debenzoylation of 15 gave the title glycoside 16. Condensation reactions were performed with silver trifluoromethane-sulfonate as a promoter in the presence of sym-collidine under base-deficient conditions, and gave excellent yields of the desired β-(trans)-products. Analyses of the ¹H- and ¹³C-n.m.r. spectra, as well as determination of the J_CF and J_HF coupling constants, were made by using various one- and two-dimensional n.m.r. techniques.