13C-n.m.r. spectra of xylo-oligosaccharides and their application to the elucidation of xylan structures

¹³C-N.m.r. spectra of thirteen xylo-oligosaccharides [a complete series of α- and β-d-xylopyranosyl derivatives of methyl α-d-xylopyranoside, β-d-xylopyranosyl derivatives of methyl 4-O-β-d-xylopyranosyl-d-xylopyranoside, methyl O-α-d-xylopyranosyl-(1→3)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranoside, and a branched methyl β-xylotetraoside] have been interpreted. The data obtained have been used for the carbon signal assignment in the spectra of a number of red-algal xylans. ¹³C-N.m.r. spectroscopy is shown to be a rapid and convenient method for the structural analysis of xylose-rich polysaccharides.     

A 13c-n.m.r. study of a di- and a tri-saccharide containing the α-d-galactopyranosyl group

The ¹³C-n.m.r. spectra of methyl 4-O-α-d-galactopyranosyl-α-d-galactopyranoside (1) and methyl 4-O-[4-O-(α-d-galactopyranosyl)-β-d-galactopyranosyl]-β-d-glucopyranoside (2) in D₂O were recorded. Comparison of these spectra with the spectra of methyl α-d-galactopyranoside (4) and methyl β-lactoside (5) provided substantial confirmation of the structures of 1 and 2.     

Untersuchung der 13c-kernresonanz-spektren der acht isomeren 1,6-anhydro-β-D-hexopyranosen

The signals of the ¹³C-n.m.r. spectra of the eight isomeric 1,6-anhydro-β-D-hexopyranoses having the ¹C₄ conformation were assigned by comparison with the spectra of selectively deuterated derivatives and by observation of the substituent effect of the O-isopropylidene derivatives. Of the two substituted C atoms in the O-isopropylidene derivatives, the signal of the equatorially substituted C atom was shifted to a lower field more strongly than that of the carbon atom bearing an axial substituent. The chemical shifts and their calculation with empirical parameters are discussed.     

Multi-nuclear (119Sn, 13C, 1H), fourier-transform,N.M.R. studies of di- and tri-butylstannyl ethers of carbohydrates

¹¹⁹Sn-N.m.r. spectra are reported for toluene solutions of the tributylstannyl ethers of 2,3,4,6-tetra-O-methyl-d)-glucose, 1,2:3,4-di-O-isopropylidene-α-d-galactopyranose, methyl 2,3-di-O-methyl-α-d-glucopyranoside, and methyl 4,6-O-benzylidene-α-d-glucopyranoside, and the dibutylstannyl ether of the last sugar. In the reaction of bis(tributyltin) oxide with methyl 4,6-O-benzylidene-α-d-glucopyranoside in toluene, HO-3 is much more reactive than HO-2. The presence of the various tin-containing species is readily apparent from the ¹¹⁹Sn-spectra. The importance of suppressing the nuclear Overhauser effect is demonstrated. The appearance of ¹¹⁹Sn satellites in the ¹³C-n.m.r. spectra demonstrates couplings of the types, ²J(¹¹⁹Sn-O-¹³C) and ³J(¹¹⁹Sn-O-C-¹³C), forthe first time, and, together with the ¹³C-chemical shifts, facilitates the determination of the site of substitution. The ¹¹⁹Sn-chemical shifts show that different states of coordination may be recognised. However, although different sites of substitution produce separate resonances, no simple relationship between shift and position is found. ¹³C-Chemical shifts are reported for methyl 4,6-O-benzylidene-α-d-glucopyranoside and its tributylstannyl ethers, and substituent effects are discussed.     

Proton and carbon-13 nuclear magnetic resonance studies on methyl (methyl d-galactosid)uronates and their per-O-acetyl derivatives

The ¹H- and ¹³C-n.m.r. spectra of the anomeric methyl (methyl d-galactosid)uronates, as well as the ¹H-n.m.r. spectra of their acetyl derivatives, were analyzed. The spectra of the unacetylated d-galactopyranosiduronates showed good correlation with those of the corresponding anomeric d-galactopyranuronic acids and their methyl esters, and with those of the anomeric methyl d-galactopyranosides. From the values of the chemical shifts and coupling constants, it was concluded that the anomeric methyl (methyl d-galactopyranosid)uronates and their corresponding peracetates are in the ⁴C₁(d) conformation. The chemical shifts in the ¹³C-n.m.r. spectra show good correlation with those of the methyl d-galactosides. The signals of the furanose derivatives appear at fields lower than those of the corresponding pyranose compounds.     

Synthesis of 5-thio-D-galactose

A synthesis of 5-thio-D-galactose, in the form of its crystalline, anomeric methyl glycopyranosides, is described. Compounds prepared as intermediates included ethyl 2,3-di-O-(tert-butyldimethylsilyl)-5,6-O-carbonyl-β-D-galactofuranoside, the corresponding 5,6-dideoxy-5,6-epithio derivative, and ethyl 2,3,6-tri-O-acetyl-5-S-acetyl-5-thio-β-D-galactofuranoside. On methanolysis, the latter afforded methyl 5-thio-α-D-galactopyranoside which, in turn, was transformed into methyl 5-thio-β-D-galactopyranoside. Acetolysis proved to be less satisfactory for incorporation of the sulfur atom into a pyranose ring-form. Characteristics of the ¹³C-n.m.r. spectra of derivatives of 5-thio-D-galactose are described, including the fact that ¹J_C,H values for the anomeric pyranosides differ by only 1–3 Hz, as compared with ≈ 10 Hz for their oxygen analogs.     

Carbon-13 nuclear magnetic resonance spectra of carbohydrate oxirane derivatives

The ¹³C-chemical shifts and ¹J_C,H values of two series of carbohydrate oxirane derivatives, namely methyl 2,3-anhydro-ribo- and -lyxofuranosides and methyl 2,3-anhydro-4,6-O-benzylidene-manno- and -allopyranosides have been determined. The assignment of ¹³C resonances has been established mainly by the examination of the proton-coupled and the selective proton-decoupled spectra. The effect of the oxirane rings on the chemical shifts of β and γ carbon atoms (from the oxirane ring oxygen atom) has been observed. Large ¹J_C,H values associated with cis CH bonds adjacent to the oxirane rings relative to those of trans counterparts have been found.     

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

Sequential tritylation, benzoylation, and detritylation of methyl 3-deoxy-3-fluoro-β-d-galactopyranoside gave crystalline methyl 2,4-di-O-benzoyl-3-deoxy-3-fluoro-β-d-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-d-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-d-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.     

1H-N.M.R. spectra of glycosaminoglycan monomers and dimers in solution in methyl sulphoxide and water

¹H-N.m.r. spectra of glycosaminoglycuronan monomers and dimers in solution in methyl sulphoxide-d₆ have been investigated; N-H and O-H resonances were observed and partially assigned. Their temperature-dependence suggests hydrogen-bonding to the solvent, with the notable exception of that of HO-4 Of sodium D-gluctironate, which was consistently downfield and relatively temperature-insensitive. The concentration-dependence ofthis signal indicates that the corresponding hydroxyl group is involved in the formation of a dimer. Signals for N-H and O-H were observed for aqueous solutions, especially at subzero temperatures.     

Assignment of signals of the carbon-13 magnetic resonance spectrum of a selected polysaccharide: Comments on methodology

The mannan from Rhodotorula glutinis contains alternate (1→3)- and (1→4)- linked β-D-mannopyranose residues (1) and its carbon-13 magnetic resonance spectrum displays 12 signals. These were assigned in terms of the positions of their parent nuclei in the sugar rings [but not whether the signals arose from a (1→3)- or (1→4)-linked residue] by preparation of D-mannans from specifically deuterated D-glucoses and observation of α- and β-deuterium isotope-effects. Individual assignments could then be made for carbon atoms of each unit by using the spectra of known oligo- and polysaccharides. The signal displacements of certain ¹³C nuclei observed on O-methylation were compared with those obtained on O-mannosylation in order to determine whether methyl ethers could be used as model compounds for signal assignments in spectra of mannose-containing polysaccharides. The displacements observed were in the same direction and of a similar order of magnitude. An assessment is made of the use of the various techniques in assigning signals of polysaccharides and their possible interpretation in terms of chemical structure.     

Studies of metal-sugar complexes in the solid state by the 13C-n.m.r. c.p.-m.a.s. method

Several metal-sugar conjugates have been prepared. Chelate coordination complexes of copper(II) and zinc(II) were prepared from salicylaldimine ligands derived from combinations of methyl 3,4,6-tri-O-acetyl-2-amino-2-deoxy-β-d-glucopyranoside or 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-β-d-glucopyranose and salicylaldehyde. Similar Schiff's bases were prepared from chitosan and complexed to copper(II). Cross-polarisation-magic angle spinning ¹³C-n.m.r. spectroscopy showed that paramagnetic ions have profound effects on the resolution obtainable and, at high concentration, result in broad, featureless spectra. In contrast, diamagnetic ions have little effect on the isotropic chemical shifts of the sugar chelates.     

Sequential synthesis and 13C-N.m.r. spectra of methyl β-glycosides of (1→4)-β-d-xylo-oligosaccharides

The reaction of 2,3-di-O-acetyl-4-O-benzyl-α,β-d-xylopyranosyl bromide (2) with methyl 2,3-di-O-acetyl-β-d-xylopyranoside gave methyl O-(2,3-di-O-acetyl-4-O-benzyl-β-d-xylopyranosyl)-(1→4)-2,3-di-O-acetyl-β-d-xylopyranoside (22). Catalytic hydrogenolysis of 22 exposed HO-4′ which was then condensed with 2. This sequence of reactions was repeated three more times to afford, after complete removal of protecting groups, a homologous series of methyl β-glycosides of (1→4)-β-d-xylo-oligosaccharides. ¹³C-N.m.r. spectra of the synthetic methyl β-glycosides (di- to hexa-saccharide) are presented together with data for six other, variously substituted, homologous series of (1→4)-d-xylo-oligosaccharides.     

Hydrogenolysis of the isomeric 1,2:4,6-di-O-benzylidene-α-d-glucopyranose derivatives with the LiAlH4AlCl3 reagent

Both isomers of 1,2:4,6-di-O-benzylidene-α-d-glucopyranose (and their 3-O-acetyl and 3-O-benzyl derivatives) have been prepared and their ¹H- and ¹³C-n.m.r. spectra assigned. The mode of hydrogenolysis of the dioxolane ring in these isomers by the LiAlH₄AlCl₃ reagent is determined by the configuration at the acetal carbon and is independent of the electronic character of the two oxygen atoms.     

Conformational changes in erythrocyte membranes by prostaglandins as measured by circular dichroism

Membranes were prepared from fresh, washed human erythrocytes by hemolysis and washing with 5 mm sodium phosphate buffer (pH 7.4). The mean residue ellipticity, [θ], of erythrocyte membrane circular dichroism was altered by prostaglandin E₁ or prostaglandin F_2α at 37 °C when observed from 250 nm to 190 nm. The decrease in negativity of [θ] with 10^?6m prostaglandin E₁ was 12.7% at 222 nm and 17.7% at 208 nm, and with 10^?6m prostaglandin F_2α 22.5% and 34.2%, respectively (P < 0.01). Similar changes in [θ] were observed at lower concentrations of prostaglandins. No strict relationship between amount of change of [θ] and prostaglandin concentrations of 3 × 10^?5m to 3 × 10^?12m was evident. A persistent alteration of [θ] with prostaglandin was observed at 37 °C. Transient change of [θ] occurred at 25 °C with prostaglandin. No change of [θ] was observed at 15 or 20 °C. Buffer or palmitic acid were without effect on membrane [θ]. Phosphatidyl inositol or methyl arachidonate caused an increase in negativity of membrane spectra. The observed alterations of membrane [θ] did not arise from changes in light scattering as the OD₇₀₀–OD₂₀₀ of membranes was not changed by prostaglandin. Effects of prostaglandin were not dependent on light path length. The prostaglandin E₁ antagonist, 7-oxa-13-prostynoic acid, at 10^?7m produced no change of [θ] of membrane spectra and prevented the otherwise demonstrable effects of 10^?10m prostaglandin E₁ on [θ]. The decrease in negativity of [θ] at 222 nm is indicative of a decrease in ellipticity of membrane protein. These studies suggest that prostaglandins may act by inducing a conformational change in membrane protein.     

Synthesis of 4,5-dideoxy-4-C-[(R,S)-phenylphosphinyl]-d-ribo- and l-lyxo-furanose and their 1,2,3-triacetates

2,3-O-Isopropylidene-d-ribose diethyl dithioacetal, prepared from d-ribose, was converted in three steps into the corresponding dimethyl acetal, which was monotosylated at O-5, and the ester oxidized at C-4 with pyridinium chlorochromate; addition of methyl phenylphosphinate to the resulting pentos-4-ulose derivative then provided (4R,S)-4,5-anhydro-2,3-O-isopropylidene-4-C-[(R,S)-(methoxy)phenylphosphinyl]-d-erythro-pentose dimethyl acetal. Hydrogenation of this compound in the presence of Raney Ni, followed by reduction with SDMA, hydrolysis, and acetylation, yielded the title compounds (seven kinds), the structures of which were established on the basis of their 400-MHz, ¹H-n.m.r. and mass spectra. A general dependence of the ²J_PH and ³J_PH values on the OPCH and PCCH dihedral angles provided an effective method for the assignment of the configurations and conformations of these 4-deoxy-4-phosphinyl-pentofuranoses.     

The synthesis and the 1h- and 13c-nuclear magnetic resonance spectroscopy of the cyclic sulfites of some sugars

Treatment of methyl β-D-ribofuranoside with thionyl chloride in hexamethyl-phosphoric triamide gives two diastereoisomeric methyl 5-chloro-5-deoxy-β-D-ribo-furanoside 2,3-cyclic sulfites. Similar cyclic sulfites are formed from benzyl β-D-ribofuranoside and 1,4-anhydro-DL-ribitol. If acetonitrile is substituted for hexa-methylphosphoric triamide, the cyclic sulfites are the main products, and only traces of the chlorinated sugars are formed. ¹H- and ¹³C-n.m.r.-spectral analysis of these reactions demonstrated that one of the diastereomers preponderates. The structure of these cyclic sulfites was established by comparison of the ¹H-n.m.r. spectra with those of the propylene sulfites. Treatment of 1,2-O-isopropylidene-α-D-glucofuranose (14) with thionyl chloride in hexamethylphosphoric triamide yields 3-chloro-3-deoxy-1,2-O-isopropylidene-α-D-allofuranose 5,6-cyclic suffite. In contrast to the 2,3-cyclic suffites, which are stable, the cyclic sulfites derived from 14 slowly decompose at room temperature.     

A 13C-n.m.r. spectral study of chondroitin sulfates A,B, and C: evidence of heterogeneity

Chondroitin sulfates A, B, and C produce well-resolved ¹³C-n.m.r. spectra which allow for a more complete characterization than that available from their p.m.r. spectra. The ¹³C data fully support earlier evidence as to the main structural features of these glycosaminoglycans, but they also show that many chondroitin preparations are substantially heterogeneous in composition. Thus, spectra of chondroitin A and C have the appearance of composites representative of both types of polymer: specimens of A may contain 25% of the C-type of structural sequence, and C, 30% of the A-type of sequence; 10–20% of unidentified constituents, including a residue bearing a 6-sulfate group, are present in the specimens of chondroitin B. Chemical-shift and ¹J_C-H values found for the L-iduronic acid residues of chondroitin B, as well as the effect of gadolinium nitrate on the relaxation properties of its ¹³C nuclei, indicate that this moiety possesses the α configuration and favors the ¹C₄(L) conformation. Corresponding data for the acetamidodeoxy-D-galactose and D-glucuronic acid residues of the chondroitins are consistent with the β-anomeric configuration and ⁴C₁(D) conformation in all instances.     

Synthesis and spectra of some octa-O-benzoylaldo-biononitriles

2,3,5,6,2′,3′,4′,6′-Octa-O-benzoyl-cellobiononitrile, -lactobiononitrile, -maltobiononitrile, and 2,3,4,5,2′,3′,4′,6′-octa-O-benzoyl-melibiononitrile were prepared by benzoylation and dehydration of the corresponding disaccharide oximes, and their ¹H- and ¹³C-n.m.r., and e.i.m.s. spectra are described.     

13C/1H high power double magnetic resonance investigation of collagen backbone motion in fibrils and in solution

${}^{13}\text{C}{}^1\text{H}$ high power double magnetic resonance spectroscopy was used to investigate the mobility of the collagen peptide backbone. [1-¹³C]- and [2-¹³C]-glycine-labeled collagen samples (with >50% enrichment in ¹³C) were prepared via chick calvaria culture. ¹³C n.m.r.² spectra of labeled reconstituted collagen fibrils, of labeled helical collagen in solution, and of unlabeled bovine Achilles tendon collagen were obtained with scalar decoupling and with dipolar decoupling of protons. Proton-enhanced spectra were also obtained using cross-polarization techniques. n.m.r. parameters (linewidths, lineshapes, T₁ values, nuclear Overhauser enhancements, and cross polarization enhancements) were measured for the labeled samples and for collagen in natural abundance. Comparison of ¹³C n.m.r. parameters for bovine Achilles tendon fibrils and for reconstituted chick calvaria collagen fibrils established that chick calvaria collagen is a good model for the molecular dynamics of collagen in vivo.Spin-lattice relaxation times and nuclear Overhauser enhancements for [1-¹³C]- and [2-¹³C]glycine-labeled collagen indicated that R₁ ~2 × 10⁷s^?1 in solution, where R₁ is the diffusion constant for reorientation about the long axis of the molecule. A substantially smaller value for R₁ (2.6 × 10⁶s^?1) was calculated for an axially symmetric ellipsoid of revolution having dimensions appropriate to the collagen helix. The discrepancy between the rigid ellipsoid and n.m.r. values of R₁ suggests that the collagen molecule undergoes torsional reorientation, as well as rod-like reorientation, about its long axis.The T₁ and NOE values measured in the glycine-labeled fibrils show that rapid axial motion (R₁ ~ 10⁷s^?1) persists in the fibrillar state. In the collagen fibril the full width of the glycyl carbonyl powder pattern is 103 p.p.m. This value is substantially smaller than the rigid lattice value, 144 p.p.m., which provides further evidence for motion in the fibril. The observed powder pattern is axially asymmetric, which shows that certain azimuthal orientations are energetically preferred in the fibril. Taken together, the n.m.r. data provide strong evidence that rapid reorientation of the helix backbone occurs in the fibrils. This result shows that formation of a fibrillar structure does not require the existence of a unique set of intermolecular interactions at the helical surfaces.     

Analysis of some partly and fully esterified oligogalactopyranuronic acids by p.m.r. spectrometry at 220 MHz

The p.m.r. spectra of mono-, di-, tri-, tetra-, and penta-galactopyranuronic acids (1–5), the corresponding fully esterified methyl esters (6–10), the partly esterified di- (11) and tri-galactopyranuronic acids (12, 13), and the unsaturated di-, tri-, and tetra-galactopyranuronic acids (14–16) were measured on solutions in D₂O at 220 MHz at a pH of 1 and 6. Observation of doublets (J 4 Hz) in the range δ 4.90–5.05 p.p.m. indicates the site of esterification in the non-reducing or reducing sugar residue. Esterification of the sugar residue at the non-reducing end can be deduced from both the presence of a methyl resonance peak at δ 3.80 and the indifference of the signal at δ 4.35 (H-4) to the change in pH. The δ values and coupling constants confirm that all the d-galacturonic acid residues have the CI conformation and are α-(1→4)-linked. In the unsaturated oligogalactopyranuronic acids, the double bond is located between C-4 and C-5 of the sugar unit at the non-reducing end. The 4-deoxyhex-4-enopyranosyluronic acid residue occurs in the ²H₁(d) conformation. Compound 11 was identified as O-(α-d-galactopyranosyluronic acid)-(1→4)-(methyl α,β-d-galactopyranuronate). Compounds 12 and 13 each consisted of a mixture of the three possible isomers; preference for the site of esterification decreases in the order reducing sugar unit, non-reducing sugar unit, sugar unit at the non-reducing end.