13C-n.m.r. spectroscopy of cellulose ethers

Characteristics of the ¹³C-n.m.r. spectra of cellulose ethers (methyl, carboxymethyl, and hydroxyethyl) have been examined at 22.6 MHz. Partial depolymerization with acid or cellulase proved to be a requisite preliminary step. Strong deshielding of ¹³C nuclei bearing alkoxyl groups was clearly evident in these spectra, which permitted an assessment of the degree of substitution at individual positions of the d-glucose residues. Better resolved spectra, and more-detailed structural analyses, were afforded by complete hydrolysates of the polymers. The findings are wholly consistent with data obtained for these derivatives by other methods, showing that the reactivities of the hydroxyl groups of cellulose are OH-2>OH-6 ? OH-3. It is also shown that reducing-end residues liberated during enzymic hydrolysis of the cellulose derivatives are not substituted at the 2-position.     

Analysis of bromine-oxidised dextran by 13C-n.m.r. spectroscopy

Dextran T 10, elaborated by Leuconostoc mesenteroides NRRL B-512, was oxidised with aqueous bromine at pH 7.0. The resulting oxodextran and its methoximated derivative were analysed by ¹³C-n.m.r. spectroscopy. The total amount of keto groups and their positions were established. Assignments of the ¹³C signals were made by referring to spectra of the corresponding methyl glucosiduloses and an oxodextran having most of the carbonyl groups at position 3 of the glycopyranosyl residues. In accordance with the mechanism for bromine oxidation of mono- and di-saccharides, the glucopyranosyl residues of dextran were oxidised mainly at C-2 and C-4. Over-oxidation resulted in a small proportion of acidic, ring-cleavage products.     

13C-n.m.r. spectra of some ribitol teichoic acids

Proton-decoupled ¹³C-n.m.r. spectra were determined for D₂O solutions of several wall teichoic acids containing glycosylated ribitol 1,5-diphosphate residues and for their dephosphorylated repeating-units. Assignments were made by correlating the chemical shift values observed with those reported for isolated constituents, allowing for perturbations of the latter resonances because of the presence of O-glycosyl or phosphodiester bonds. Anomeric configurations of hexopyranosyl residues and their position of substitution on ribitol were indicated from the distinctive chemical shifts of the carbons concerned. Three-bond ¹³C³¹P couplings (6–8 Hz) were observed, and two-bond ¹³C³¹P couplings were indicated by broadened signals. The lack of resolution for the latter resonances is probably due to the heterogeneous nature of the polymers.     

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.     

13C-n.m.r. studies of monomeric composition and sequence in alginate

High-resolution, ¹³C-n.m.r. spectra of slightly depolymerised alginates have been interpreted. The sequence of monomer units, l-guluronate (G) and d-mannuronate (M), markedly influenced the chemical shifts. At 50 MHz, some of the individual carbon resonances of both units were resolved into four lines, in evident dependence upon the identities of the units immediately preceding and following them in the chains. The relative intensifies of the signals permitted rapid computation of (1) monomeric composition (M/G ratio), (2) monomeric sequence in terms of a complete set of four diad and eight triad frequencies, and (3) the composition (M/G ratio) of end units and of the units adjacent to M-residues at the non-reducing end. The diad frequencies indicated that alginate was a block co-polymer containing number-average, co-monomer block-lengths of ～2?8. The triad frequencies indicated average lengths of ～4?8 for blocks containing two or more units, these being somewhat longer for G- than for M-blocks. Regions of the chains having a strictly alternating sequence of M- and G-residues were short. The relative occurrence of G-centred triads deviated significantly from those predicted by first-order Markovian statistics.     

Chemical and 13C-n.m.r. studies of an arabinogalactan from Larix sibirica L.

An arabinogalactan isolated from the wood of Larix sibirica L. was investigated by methylation analysis, partial hydrolysis, enzymic oxidation, and ¹³C-n.m.r. spectroscopy. The structural conclusions arrived at by ¹³C-n.m.r. spectroscopy were consistent with the data from methylation analysis. The polysaccharide is highly branched and similar in structure to those of arabino-3,6-galactans isolated from other Larix species.     

15N n.m.r. spectroscopy: 36. 1H n.m.r. and 15N n.m.r.spectroscopic investigation on the protonation of polypeptides

¹⁵N n.m.r. (9.12 MHz) spectra of acetamide, polyglycine, poly([l-alanine) and poly(l-leucine) were measured in various acidic solvents. These solvents include dichloroacetic acid (DCA), trifluoroacetic acid (TFA), methane sulphonic acid (MSA) and fluorosulphonic acid (FSA). Full protonation of both amides and polypeptides causes downfield shifts of 17–20 ppm. Furthermore, the concentration dependence of the chemical shift was measured. In solvents which cause partial protonation, decreasing concentration of amide groups may cause downfield shifts up to 8.5 ppm, while in the case of full protonation or in the absence of protonation no concentration dependence is observable. The protonation of peptide groups induces H/D-exchange of the αC proton which was monitored by ¹H n.m.r. spectroscopy. The mechanism of this H/D-exchange is discussed.     

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.     

Determination of the structure of dextran by 13C-nuclear magnetic resonance spectroscopy

The ¹³C-n.m.r. spectra have been recorded for a series of dextrans whose structures, in terms of degree and type of branching, had previously been determined by methylation analysis. The spectra established that all observable linkages in these dextrans are α-linked. Correlation of the spectra with methylation data indicated that the 75–85-p.p.m. spectral region is diagnostic for establishing the presence of α-D-(1→2)-, α-D-(1→3)-, or α-D-(1→4)-linkages. Each chemical shift has been found to be temperature-dependent (Δδ/ΔT) when referenced to either the deuterium lock or an external standard (tetramethylsilane). All carbohydrate Δδ/ΔT values are positive, and range from 0.01 to 0.03 p.p.m./°C. These values are considerably larger than analogous Δδ/ΔT values previously observed for smaller molecules. Larger than average Δδ/ΔT values are associated with the non-anomeric, sugar-linking carbon atoms.     

13C-NMR spectroscopy of biflavanoids

The ¹³C-NMR spectra of nine naturally occurring CC linked biflavanoids have been assigned. The signals for the carbon atoms I-6, I-8, II-6, and II-8 appear in the region 90.0 ppm to 105.0 ppm. On the basis of the chemical shifts of these signals and their multiplicities in the off-resonance spectra, it is possible to determine the interflavonoid linkage in biflavanoids, provided that the A ring is involved. The level of oxidation of the ring C can be readily determined by a consideration of the chemical shift value of the carbonyl resonances. The position of the methoxyl substitution can also be inferred.     

Two-dimensional J-resolved 1H n.m.r. spectroscopy for studies of biological macromolecules

The technique of two-dimensional J-resolved ¹H n.m.r. spectroscopy has been extended to handle the very wide spectra of proteins and other macromolecules at 360 MHz. The potential of the method to resolve and assign individual spin multiplets in the complex spectra encountered in structural studies of biopolymers is illustrated with some experiments with amino acids and with a protein, the basic pancreatic trypsin inhibitor.     

A high-resolution c.p.-m.a.s. 13C-n.m.r. study of solid-state cyclomaltohexaose inclusion-complexes: Chemical shifts and structure of the host cyclomaltohexaose

High-resolution, solid-state ¹³C-n.m.r. spectra were obtained for several crystalline cyclomaltohexaose inclusion-complexes. The resonances of C-1, C-4, and C-6 of the host were dispersed. The averaged ¹³C shifts of these resonances were in good agreement with the ¹³C shifts observed in solution, where the dispersion due to conformational diversity is expected to be averaged by rapid interconversion of the conformers. This result indicates that the most plausible source of the solid-state ¹³C-shift dispersions of the resonances of C-1 and C-4 is the diversity of conformations about the glycosidic linkage. The molecular origins of conformation-dependent ¹³C shifts are discussed.     

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.     

13C NMR spectroscopy of Pyrrolizidine alkaloids

The ¹³C NMR spectra of nine pyrrolizidine alkaloids of the macrocyclic diester type, seven of the corresponding N-oxides and of the parent base retronecine have been recorded and the signals assigned. The 13C NMR signals were found to be sensitive to structural variation in both the diester moiety and the heterocyclic ring system, providing useful information for structural elucidation, particularly when the ¹H NMR spectra may be difficult to interpret.     

Structural analysis of comb-like,amylose derivatives by 13C-N.M.R. spectroscopy

¹³C-N.m.r. spectra have been recorded for previously reported, comb-like derivatives of amylose produced by orthoester and Helferich condensation of D-glucose to amylose. As known from monomeric studies, the Helferich condensation conditions (the presence of mercury salts) favor α-D-glucosylation, and orthoester condensation conditions favor β-D-glycosylation. It was anticipated that, for these polymer condensations, the Helferich and orthoester condensations would also favor α- or β-D-glycosylation, respectively. The ¹³C-n.m.r. spectra of representative products of the Helferich and orthoester condensations confirmed the presence of 4,6-di-O-substituted α-D-glucopyranosyl residues, and also the degree of polymer linearity derived from independent, analytical data. However, these spectra indicate extensive, if not exclusive, β-D-glycosylation for both the helferich and the orthoester conditions. These results were obtained by using the product from an enzymically synthesized, strictly linear amylose in the Helferich condensation reaction.