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

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. ¹³C-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 αd-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 ¹³C-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.     

13C-N.M.R.-spectroscopic investigation of agarose oligomers

A complete, unambiguous assignment of all of the ¹³C-n.m.r.-spectral signals of agarose oligomers produced by enzymic hydrolysis has been achieved. The ¹J¹³C-H coupling constants are reported, and the chemical shifts and coupling constants of both the agarose polymer and oligomers are compared.     

High resolution 13C-N.M.R. spectroscopy of legume-seed galactomannans

Five galactomannans obtained by aqueous extraction at different temperatures from the endosperm of the seed of Gleditsia triacanthos, and having different Gal:Man ratios, were submitted to a preliminary degradation, and the products analyzed by high resolution ¹³C-n.m.r. spectroscopy. In these spectra all carbon atoms yield several lines. C-6 (substituted Man) shows, for the first time, a clear splitting, which is explained by considering that this carbon atom is sensitive to whether or not its neighbors are branched; this provides a basis for determining the next-nearest-neighbor probabilities in the galactomannans (“triad frequencies”). Although diad frequencies are roughly consistent with a random arrangement, the values obtained for the triad frequencies indicate a more complex kind of arrangement of the lateral chains.     

13C-N.M.R. spectra of cyclomalto-oligosaccharides (cyclodextrins), their derivatives, and complexes with azo dyes

The nature of the inclusion complexes of several cyclomalto-oligosaccharides (cyclodextrins, CDs) with azo dyes has been studied on the basis of 13C-n.m.r. chemical shifts, relaxation times, correlation times, and broadening and doubling of the n.m.r. signals. All CDs show the azo dye-induced shifts at the narrow-rim side of the CD, indicating that the azo dyes protrude from the cavity. CD-induced shifts of azo dyes depend on the hydrophobic nature of the cavity, van der Waals forces, as well as ring-current and deformation effects, and suggest inclusion essentially from the hydrophobic site. The broadening and the doubling of the 13C-n.m.r. signals, the altered relaxation and correlation times, as well as the temperature dependence for these phenomena, also provide particular information about the characteristic host-guest interactions.     

1H- and 13C-N.M.R. spectra of eledoisin and intermediate oligopeptides.

The 1H- and 13C-n.m.r. spectra of eledoisin and minor oligopeptides were measured and assigned. The proton spectra were interpreted on the basis of homonuclear decoupling, chemical shift criteria and spectra simulation. The information obtained was used in the assignment of the 13C spectrum via heteronuclear 1H-13C. The steric arrangement of proline residue was deduced from the 13C spectrum. Moreover the similarity of the 13C spectrum of eledoisin with that of component oligopeptides suggests that no considerable conformational change occurs in the undecapeptide relative to the component fragments.     

Six unusual dextrans: methylation structural analysis by combined g.l.c.—m.s. of per-O-acetyl-aldononitriles

Six bacterial dextrans from NRRL strains Leuconostoc mesenteroides B-1299, B-1303. B-1355, and B-1399; Streptobacterium dextranicum B-1254; andA g.l.c. procedure permitted, for the first time. separation of the 2,3,4- from the 2.3.6-tri-O-methyl derivative of d-glucose. Deuteriomethyla     

Methylation structural analysis of unusual dextrans by combined gas-liquid chromatography-mass spectrometry

Eight bacterial dextrans from NRRL strainsLeuconostoc mesenteroides B-742, B-1299, B-1355, B-1399, and B-1402, and from Streptobacterium dextranicum B-1254 were examined by methylation structural analysis. Methyl ethers of d-glucose that were present in hydrolyzates of permethylated dextrans were analyzed by combined g.l.c.?m.s. as the peracetylated aldononitriles. The various dextrans differed significantly in frequency and type of chain branching.     

Effects of carbohydrate-structure changes on induced shifts in differential isotope-shift 13C-N.M.R.

Deuterium-induced, ¹³C-isotope shifts are shown to vary considerably from the initially predicted values calculated for ordinary pyranose and furanose sugars, when minor structural changes are introduced into the carbohydrate ring. Both substitution of C-OH groups or reduction of C-OH to CH₂ permitted the evaluation of γ effects of OD without the contribution of β-OD-induced shifting. The observed γ-shift values for these modified structures were twice as large as those previously noted. This difference is most probably due to favored salvation. Substitution of OH at C-6 led to the predicted loss of differential isotope-shift (d.i.s.) at C-6 because of its isolation from all β and γ OD groups. The ³¹P resonances of d-glucose 6-phosphate show downfield deuterium shifts. Based on d.i.s. values, new ¹³C-shift assignments are proposed for isomaltose and 2-amino-2-deoxy-α-d-glucose. A study of acidic carbohydrates has demonstrated that isotope shifts are somewhat larger for sp²-hybridized carbon atoms whose OH groups are acidic. Relaxation times for sp² carbon atoms isolated from dipolar interaction with protons were very long in D₂O relative to their relaxation time in the H₂O environment.     

The 13C-N.M.R. spectra of disaccharides of D-glucose, D-galactose, and L-rhamnose as models for immunological polysaccharides.

Complete assignments of the 13C-n.m.r. spectra of disaccharides having beta-glycosidic linkages are presented and discussed. The disaccharides of D-glucose, D-galactose, L-rhamnose, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-D-galactose are model compounds for 13C-n.m.r. studies of immunological polysaccharides. Changing the nature of the reducing glucopyranose rings (D-glucose to L-rhamnose) has no important influence on the chemical shifts of the carbons of the non-reducing glucopyranose ring (D-glucose). The converse is also true: the chemical shifts of the carbons of the reducing glucopyranose ring (L-rhamnose) are not noticeably affected by a change of the non-reducing unit (D-glucose to D-galactose or 2-acetamido-2-deoxy-D-glucose).     

13C-N.M.R. study of the conformation of helical complexes of amylodextrin and of amylose in solution

Amylose (average d.p. 1000) and amylodextrin (average d.p. 25) have identical 13C-n.m.r. spectra, except for some minor signals from the small amount of alpha-1----6 branch linkages present in amylodextrin. Amylodextrin can be obtained as stable solutions in much higher concentrations than amylose and so requires only 1/100th as many scans to obtain a spectrum comparable to that of amylose. 13C-N.m.r. spectroscopy has been used to study the formation of amylodextrin complexes with organic complexing agents in aqueous solution. A control study using dextran, which does not form helical complexes, showed that, when complexing agents are added, the signals from all of the carbons show a slight downfield shift due to a general solvent effect. In the case of amylodextrin, the addition of increasing concentrations of complexing agent also produced a downfield shift of the signals of all the carbons, but there was a greater shift of the signals for carbons 1 and 4 than for carbons 2, 3, and 6, indicating that something more than a solvent effect was occurring. The cycloamyloses (cyclic alpha-1----4 linked D-glucose oligosaccharides which may be considered as model for an amylose helix) in water have chemical shifts for carbons 1 and 4 that are comparable to those shown by the amylodextrin complexes. It is thus proposed that the formation of a helical complex with amylodextrin results in a change in the conformation of the glycosidic linkage, which is reflected by greater downfield shifts of the signals for carbons 1 and 4, relative to those for carbons 2, 3, and 6. It was observed that differences in the ratio of the downfield shifts of C-1 and C-4 of the different amylodextrin complexes indicate differences in the degree of compactness of the helical structures. A comparison of the 13C chemical shifts of methyl alpha-D-glucoside and methyl alpha-maltoside showed that, for a molecule as small as a disaccharide, there is a conformational change about the glycosidic linkage when complexing agents are added.     

Fourier-transform,infrared difference-spectrometry for structural analysis of dextrans

The Fourier-transform (F.t.), infrared (i.r.) spectra of a series of branched dextrans were examined. The dextrans studied were those from the N R R L collection designated Leuconostoc mesenteroides B-1142, B-1191, B-1299 fraction S, B-1355 fraction S, B-1402, and B-1422, and Streptobacterium dextranicum B-1254 fractions S[L] and L[S]. The spectrum of a levan, N R R L L. mesenteroides B-523 fraction M, was also examined, for comparison with the spectra of the dextrans. Meaningful results were obtained by “weight-normalizing” the spectral absorbance to that of the dextran of very low degree of branching (dextran B-1254 fraction L[S]), and then subtracting this spectrum of linear dextran from each of the other polysaccharide spectra. The resulting i.r.-absorbance difference-spectra were plotted, at uniform scale-expansion across the 1800-400-cm^?1 region, resulting in difference-absorbance features at ≈ 1100 and ≈ 800 cm^?1 for all branched dextrans. These absorbance differences could be correlated to the type and degree of dextran branching, which had previously been established by permethylation analysis. It was concluded that such F.t.-i.r. difference-spectra have general application for the structural analysis of polysaccharides.     

Distribution of substituents in O-(2-hydroxyethyl)cellulose: A 13C-N.M.R. approach

O-(2-Hydroxyethyl)cellulose was converted into a mixture of the corresponding d-glucitol derivatives by hydrolysis followed by reduction of the sugars with NaBH₄. On the basis of the spectra of individual O-(2-hydroxyethyl)-d-glucitols, the ¹³C-n.m.r. spectrum of this mixture was assigned to the extent that permitted quantitative analysis in terms of monomer composition of the polymer. The monomer mole-fractions conform to a statistical, kinetic model that assumes that the reactivity of the 3-hydroxyl group of the d-glucosyl residues of cellulose depends on the state of substitution at O-2. The relative rate-constants of the hydroxyl groups in the (hydroxyethyl)ation reaction are k₂:k₃:k₃′:k₆:k_x = 6.0:1.0:4.0:11.1:34.6, indicating that the reactivity of OH-3 increases fourfold upon (hydroxyethyl)ation of OH-2.     

High resolution1H- and13C-N.M.R spectra ofD-glucopyranose, 2-acetamido-2-deoxy-D-glucopyranose,and related compounds in aqueous media

The¹H- and¹³C-n.m.r spectra ofD-glucopyranose and 2-acetamido-2-deoxy-D-glucopyranose and its derivatives in D₂O at 25° have been completely interpreted. Iterative analysis allowed accurate determination of the chemical shifts and coupling constants in the 270-MHz¹H-spectra, and these are used to correlate the chemical shift changes with substitution patterns. The implications of the systematic errors from assuming first-order conditions for the p.m.r spectra of sugars are discussed in relation to measuring shift changes of sugar-enzyme complexes.     

Benzylidene acetal structural elucidation by N.M.R. Spectroscopy: Application of carbon-13. N.M.R.-Spectral parameters

The ₁₃C-n.m.r. spectra of 19 2-phenyl-1,3-dioxolane, -1,3-dioxane and -1,3-dioxopane derivatives were examined and it was found that both the ₁₃C-n.m.r. chemical shift for the acetal carbon atom and the one-bond coupling constant between the acetal carbon atom and the acetal proton had values that could be used to distinguish between acetals having different ring sizes. In addition, the presence of axial substituents at positions 4 or 6 in substituted 2-phenyl-1,3-dioxane rings and 4 or 7 in substituted 2-phenyl-1,3-dioxepane rings could be readily detected. The structures of a number of carbohydrate examples were determined by using these two parameters and also the chemical shift of the acetal proton from ₁H-n.m.r. spectra. The use of all three parameters made assignment of benzylidene acetal ring-size unambiguous.     

C.P.-M.A.S. 13C-N.M.R. study of some solid-state inclusion complexes of cyclomalto-oligosaccharides with para-substituted benzenes

Cross polarisation—magic-angle sample spinning ¹³C-n.m.r. spectral have been measured in the solid state for p-nitrophenol, p-iodophenol, and their inclusion complexes with cyclomaltohexaose, cyclomaltoheptaose, and methylated cyclomaltohexaose. Analysis of the line-shapes of the resonances and the dipolar-dephasing experiments indicate that the guest molecules undergo motion in the host cavities, whereas the host molecules are almost static. The mode and rate of guest motion depend on the size of the cavity.     

The α-d-glucopyranosidic linkages of dextrans: comparison of percentages from structural analysis by periodate oxidation and by methylation

The analyses of 25 dextrans by g.l.c.-m.s., methylation-fragmentation, and periodate-oxidation techniques have been compared. Although in general agreement for slightly branched dextrans, the two techniques yield divergent analyses for highly branched dextrans. Employing the methylation-fragmentation data as the standard, the periodate-oxidation data were examined to establish the extent of deviation for the types of α-d-glucopyranosidic linkages that occur in dextrans, that is, the (1→6)-like, the (1→4)-like, and the (1→3)-like. The unexpected behavior of the dextrans was correlated with whether branching occurs through either C-2 or C-4, or C-3 or both of these types. Possible causes for the effects observed are discussed.     

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.     

C.P.-D.D.-M.A.S. 13C-N.M.R. investigations of anhydrous and hydrated cyclomalto-oligosaccharides: The role of water of hydration

Hydrated and anhydrous cyclomaltohexaose, cyclomaltoheptaose, and cyclomalto-octaose (cyclodextrins) have been investigated by the c.p.-d.d.-m.a.s. ¹³C-n.m.r. technique. The chemical shifts of the signals of C-1 and C-6 provide information about conformation and the results agree fairly well with the earlier scattering data on hydrated systems, but some discrepancies have been found for cyclomaltohexaose. Th conformation of the macro-rings seems to be determined by the hydration water. The unique role of water in forming crystals of cyclomalto-oligosaccharides is demonstrated.     

Loss of R Factors Promoted by Bacteriophage M13 and the M13 Carrier State

The infection of different Hfr strains of Escherichia coli bearing derepressed R factors of the fi(+) or fi(-) type can result in the loss of the R factor and the conversion of the infected cells to the R(-) state. This extends earlier observations on the elimination of F' factors by bacteriophage M13 infection. Variability in the efficiency of this conversion can arise because of genetic factors independent of the R factor being eliminated. A fraction of the infected but unconverted R(+) cells were M13 carrier strains. The carrier state had an intracellular basis, and single R(+) cells could maintain the carrier state.     

High-field, C-N.M.R. spectroscopy of β-d-glucans, amylopectin, and glycogen

¹³C-N.m.r. spectra of several d-glucans, recorded at 100 MHz, have afforded information about structural detail not previously accessible at lower frequencies. Spectra of (1→4)- and (1→3)-linked β-d-glucans of oats, barley, and lichenan of Iceland moss demonstrate the presence, in each, of three, non-equivalent, 4-O-substituted residues, that the ratio of these to 3-O-substituted residues averages 2.4–2.5, and hence that the patterns of repeating sequences in the three polymers are essentially the same. A comparison of wheat amylopectin with a minor, amylopectin-like fraction of wheat starch indicates that they are strictly analogous in basic structure, and differ only in that the average length of branches in the minor fraction is 20–25% shorter. By combining the advantages of high-field operation with the use of dimethyl sulfoxide as solvent, a large number of distinctive resonances have been observed, representing end-units, branch-points, and residues adjacent to branch-points. Accordingly, these signals are even more prominent in the spectrum of glycogen, reflecting the higher incidence of branching in this polymer. At 100 MHz, the excellent resolution and sensitivity afforded constitute a potent basis for assessing the purity of polysaccharide preparations, as illustrated with wheat amylose and barley β-d-glucans.