Roger D. Knapp

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 alpha-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 alpha-D-(1 leads to 2)-, alpha-D-(1 leads to 3)-, or alpha-D-(1 leads to 4)-linkages. Each chemical shift has been found to be temperature-dependent (deltadelta/deltaT) when referenced to either the deuterium lock or an external standard (tetramethylsilane). All carbohydrate deltadelta values are positive, and range from 0.01 to 0.03 p.p.m./degrees C. These values are considerably larger than analogous deltadelta/deltaT values previously observed for smaller molecules. Larger than average deltadelta/deltaT values are associated with the non-anomeric, sugar-linking carbon atoms.

Structural analysis of dextrans, from strains of leuconostoc and related genera, that contain 3-O-α-d-glucosylated α-d-glucopyranosyl residues at the branch points, or in consecutive, linear positions

Abstract Dextran fractions from NRRL strains Leuconostoc mesenteroides B-523, B-742, B-1149, Betabacterium vermiforme B-1139, and Streptococcus viridans B-1351 were examined by 13 C-n.m.r. spectroscopy. The native, structurally homogeneous dextrans from NRRL strains of L. mesenteroides B-1191, B-1192, and B-1142 were also examined by 13 C-n.m.r. spectroscopy, and the spin-lattice relaxation values of dextran B-1142 were measured. Methylation-fragmentation, structural analyses were performed on dextrans B-1142 and B-1191. Except for the A fractions of dextrans B-523 and B- 1149, all of these dextran fractions differ from linear dextran by branching primarily, if not exclusively, through 3,6-di- O -substituted α- d -glucopyranosyl residues. Dextran B-1149 fraction A is spectroscopically different from the dextrans branching through the 3,6-di- O -substituted residues, and apparently contains significant mole percentages of contiguously linked 3-mono- O -substituted α- d -glucopyranosyl residues.

Structural analysis of leuconostoc dextrans containing 3-O-α-d-glucosylated α-d-glucosyl residues in both linear-chain and branch-point positions, or only in branch-point positions, by methylation and by 13C-N.M.R. spectroscopy

Abstract It had been established by methylation-structural analysis that dextran fraction S from Leuconostoc mesenteroides NRRL B-1355 has two types of α- d -glucopyranosyl residues that are linked through O-3, i.e. , 35% of the residues carry a (1→3)-bond, and ∼10% carry a (1→6)-bond in addition to a (1→3)-bond. Two similarly constituted dextrans have now been identified by methylation-structural analysis, namely, the S-type fractions from L. mesenteroides strains NRRL B-1498 and B-1501. The S-type fractions from L. mesenteroides strains B-1355, B-1498, and B-1501 are structurally differentiated from the α- d -glucans (characteristically insoluble) of certain cariogenic Streptococci which also contain both 3- O - and 3,6-di- O -substituted α- d -glucopyranosyl residues. 13 C-N.m.r. spectra have been recorded at 90° for both the S- and L-type fractions of strains B-1355, b-1498, and B-1501. The L-type fractions have a low degree of branching through 3,6-di- O -substituted α d -glucopyranosyl residues, but no 3-mono- O -substituted residues. (Dextran fraction S of Streptococcus 5000 g.l.c. instrument equipped with hydrogen-flame detectors. On-column injection of glass columns (2 mm i.d. x 1.23 m) was employed for all such chromatography. The 13 C-n.m.r. conditions and methods for preparation of dextran samples have been described(su4). In general, a Varian XL-100-15 spectrometer equipped with a Nicolet TT-100 system was employed in the Fourier-transform mode. Chemical shifts are expressed in p.p.m. relative to external tetramethylsilane, but were actually calculated by reference to the lock signal.

High-temperature enhancement of 13C-N.M.R. chemical-shifts of unusual dextrans, and correlation with methylation structural analysis

Abstract Dextran fractions from NRRL strains Leuconostoc mesenteroides B-742, B-1299, B-1355, and Streptobacterium dextranicum B-1254 were examined by 13 C-n.m.r. spectroscopy at 34 and 90°, and by methylation structural analysis. The native, structurally homogeneous dextran from L. mesenteroides NRRL B-1402 was also examined. The data allow correlations to be made between the structure and physical properties of the S (soluble) and L (less-soluble) fraction pairs of dextrans B-742, B-1254, B-1299, and B-1355. For the dextrans under consideration here, increasing solubility of the dextran (both in water and in aqueous ethanol) was found to correlate with decreasing percentages of α- d -(1→6)-linked d -glucopyranosyl residues. Both the diagnostic nature of the 70–75-p.p.m. spectral region with regard to type of dextran branching, and the increase in resolution of the polysaccharide spectra at higher temperatures, have been further confirmed.

13C-nuclear magnetic resonance spectra of compounds containing β-D-fructofuranosyl groups or residues

Abstract 13 C-N.m.r. spectra have been recorded for sucrose, melezitose, levan, inulin, palatinose, and D -fructose. Except for the last, each compound contains a different O -substituted D -fructofuranose residue or group, or β- D -fructofuranosyl residue or group. On the basis of chemical-shift displacements, resulting from O -substitution at specific carbon atoms, resonances can be assigned to the carbon atoms of the β- D -fructofuranosyl residue. Fortuitously, the α- D -glucopyranosyl group present in some of these compounds exhibits resonances that do not obscure the β- D -fructofuranosyl resonances. O -Substitution of the β- D -fructofuranosyl residue causes a downfield displacement of the corresponding, linked-C resonance; however, the other major resonances of this residue are not affected by bulky substituents. Members of a series of levan fractions, the products of partial, acid hydrolysis of Streptoccoccus salivarius levan, were then examined for changes in relative degree of branching.

Correlation of the structure of dextrans to their 1H-n.m.r. Spectra

Abstract Dextran fractions from NRRL strains Leuconostoc mesenteroides B-742, B-1299, B-1355, and B-1501, Streptobacterium dextranicum B-1254, Streptococcus sp. B-1526, and also the native dextrans from L. mesenteroides B-1142, B-1191, B-1402, and L. dextranicum B-1420 were examined in aqueous solution at 90° by Fourier-transform, 1H-n.m.r. spectroscopy. Branching of dextrans through the 2,6-, 3,6-, and 4,6-di-O-substituted α- d -glucopyranosyl residues was correlated to changes in spectral patterns. The major, spectral differences for these dextrans were in the anomeric (4-6-p.p.m.) region. In general, the degree of branching of a dextran can be determined by comparing anomeric-peak integrals; however, the integrals of the displaced, nonlinear, anomeric resonances are also dependent on the type of branching residues present in the dextran. Although the 1H-n.m.r. resonances for the anomeric region identify the presence of non-6-mono-O-substituted α- d -glucopyranosyl residues, these additional resonances provide little discrimination between other residue types, the exception being the 2,6-di-O-substituted residue, which has a clearly displaced resonance. Techniques for the suppression of the interfering resonance of deuterium hydroxide are also discussed.

Structural analysis of α-d-glucans by 13C-nuclear magnetic resonance, spin-lattice relaxation studies

Abstract Relaxation measurements have been made for the resonances of the 13 C-n.m.r. spectra of the S fractions of dextrans from NRRL strains Leuconostoc mesenteroides B-742, B-1299, B-1355, and B-1498, the S[L] fraction of the dextran from Streptobacterium dextranicum B-1254, pullulan, and a synthetically branched, comb-like amylose. Resonance assignments have been made to carbon-atom positions on the basis that carbon atoms associated with large degrees of segmental motion will have resonances with larger than average dipole-dipole spin-lattice relaxation ( T 1 DD ) values. For a given spectrum, the relative magnitude of T 1 DD values are about the same as the observed spin-lattice relaxation ( T 1 obs ) values for each resonance. Comparisons of the relative magnitudes of relaxation data are most easily made with the resonances of anomeric positions. In general, the relative magnitude of the relaxation values for the spectrum of a given compound did not change between 34° and 90° recording conditions. Relaxation measurements were employed to establish the relative position of O -substituted residues in the average repeating unit of the S fractions of dextrans B-1355 and B-1498. The general parameters controlling the magnitudes of the chemical shifts of the α- d -glucopyranosyl residue were then considered in relation to the nature of the O -substitution for this residue.

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

Abstract 13 C-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 13 C-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.