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A mixture of oligosaccharides produced by beta-galactosidase using lactose as a substrate was fractionated according to degree of polymerization using gel filtration, followed by high-pH anion-exchange chromatography. The fractions obtained were analyzed using monosaccharide analysis, methylation analysis, mass spectrometry, and NMR spectroscopy. Twelve novel non-reducing oligosaccharides were characterized, namely, [beta-D-Galp-(1-->4)]n-alpha-D-Glcp- (1<-->1)-beta-D-Galp[-(4<--1)-beta-D-Galp]m, with n, m = (1, 2, 3, or 4) and beta-D-Galp-(1-->2)-alpha-D-Glcp- (1<-->1)-beta-D-Galp.

In the framework of studies towards oligosaccharide-conjugate-based vaccines against Streptococcus pneumoniae, the synthesis is reported of several spacer-containing oligosaccharides that represent parts of the capsular polysaccharide of S. pneumoniae serotype 6B, namely alpha-L-rhamnopyranosyl-(1-->4)-5-O-(3-aminopropyl hydrogen phosphate)-D-ribitol, 3-aminopropyl D-ribitol-(5-->hydrogen phosphate-->2)-alpha-D-galactopyranoside, 3-aminopropyl alpha-L-rhamnopyranosyl-(1-->4)-D-ribityl-(5-->hydrogen phosphate-->2) -alpha-D-galactopyranoside, and 3-aminopropyl D-ribityl-(5-->hydrogen phosphate-->2) -alpha-D-galactopyranosyl-(1-->3)-alpha-D-glucopyranoside. Phosphorylations were carried out using the H-phosphonate method.

In the framework of studies towards oligosaccharide-conjugate based vaccines against Streptococcus pneumoniae, the synthesis is reported of four spacer-containing tetrasaccharides that each can be conceived as representing a repeating unit of the capsular polysaccharide of S. pneumoniae serotype 6B, namely, 3-aminopropyl D-ribityl-(5-->hydrogen phosphate-->2)-alpha-D-galactopyranosyl-(1-->3) -alpha-D-glucopyranosyl-(1-->3)-alpha-L-rhamnopyranoside, 3-aminopropyl alpha-L-rhamnopyranosyl-(1-->4)-D-ribityl-5(-->hydrogen phosphate-->2)-alpha-D-galactopyranosyl-(1-->3)-alpha-D-glucopyranoside, 3-aminopropyl alpha-D-glucopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl-(1-->4) -D-ribityl-(5-->hydrogen phosphate-->2) -alpha-D-galactopyranoside, and alpha-D-galactopyranosyl-(1-->3)-alpha-D-glucopyranosyl-(1-->3)-alpha-L -rhamnopyranosyl-(1-->4)-5-O-(3-aminopropyl hydrogen phosphate)-D-ribitol. Phosphorylations were carried out using the H-phosphonate method.

Apple fruit xyloglucan is predominantly built up from XXXG, XXFG, and XLFG units (G = beta-D-Glcp-, X = alpha-D-Xylp-(1-->6)-beta-D-Glcp-, L = beta-D-Galp-(1-->2)-alpha-D-Xylp-(1-->6)-beta-D-Glcp-, F = alpha-L-Fucp-(1-->2)-beta-D-Galp-(1-->2)-alpha-D-Xyl p-(1-->6)-beta-D-Glcp-). However, small amounts of oligosaccharides with a less heavily branched glucan backbone also occur. Structural analysis of two such oligosaccharides, isolated from a xyloglucan preparation digested with endoglucanase i.v., using a combination of FAB mass spectrometry and 1H NMR spectroscopy, afforded the identification of GFG and a dimer of GFG. The finding of the dodecasaccharide GFGGFG as a structural element of apple fruit xyloglucan is most unusual.

The exopolysaccharide produced by Lactobacillus acidophilus LMG9433 in a semi-defined medium was found to be a charged heteropolymer, with a composition of D-glucose, D-galactose, D-glucuronic acid, and 2-acetamido-2-deoxy-D-glucose in molar ratios of 2:1:1:1. By means of methylation analysis, uronic acid degradation, de-N-acetylation/deamination, partial acid hydrolysis, and 1D/2D NMR studies the polysaccharide was demonstrated to consist of repeating units with the following structure: [Table: see text]

The synthesis is reported of methyl 2-acetamido-4-O-[2-acetamido-2-deoxy-O-(3,6-di-O-alpha-D- mannopyranosyl-2-O-beta-D-xylopyranosyl-beta-D-mannopyranosyl)-beta-D- glucopyranosyl]-2-deoxy-beta-D-glucopyranoside (4) and methyl 2-acetamido-4-O-[2-acetamido-2-deoxy-4-O- (3,6-di-O-alpha-D- mannopyranosyl-2-O-beta-D-xylopyranosyl-beta-D-mannopyranosyl)-beta-D- glucopyranosyl]-2-deoxy-6- O-alpha-L-fucopyranosyl-beta-D-glucopyranoside (5), which represent the invariant hexasaccharide core structure of the xylose-containing glycans of N-glycoproteins and its 6-O- fucosylated derivative. Ethyl 4-O-[3-O-allyl-4-O-benzoyl-6-O-tert-butyldimethylsilyl-2-O- (2,3,4-tri-O-acetyl-beta-D-xylopyranosyl)- beta-D-mannopyranosyl]-3,6-di-O-benzyl-2-deoxy-2-phthalimido-1- thio-beta-D-glucopyranoside (9) was coupled with methyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D- glucopyranoside (11). Desilylation of the resulting tetrasaccharide derivative, followed by condensation with 2,3,4,6- tetra-O-acetyl-alpha-D-mannopyranosyl trichloroacetimidate (7), gave methyl 4-O-(4-O-[3-O-allyl-4- O-benzoyl-6-O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)-2-O-(2,3,4 -tri-O- acetyl-beta-D-xylopyranosyl)- beta-D-mannopyranosyl]-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D- glucopyranosyl)- 3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranoside (14). Deallylation of 14, followed by condensation with 7 and deprotection, gave hexasaccharide 4. Ethyl 3,6-di-O- benzyl-2-deoxy-4-O- [4,6-di-O-acetyl-3-O-allyl-2-O-(2,3,4-tri-O-acetyl-beta-D-xylopyranosyl) - beta-D-mannopyranosyl]-2- phthalimido-1-thio-beta-D-glucopyranoside (17) was coupled with methyl 3-O- benzyl-2-deoxy-6-O- (4-methoxybenzyl)-2-phthalimido-beta-D-glucopyranoside. Demethoxybenzylation of the tetrasaccharide derivative thus obtained, followed by fucosylation using ethyl 2,3,4-tri-O- benzyl-1-thio- beta-L-fucopyranoside, gave methyl 3-O-benzyl-2-deoxy-4-O-[3,6-di-O-benzyl-2- deoxy-4-O-[4,6- di-O-acetyl-3-O-allyl-2-O-(2,3,4-tri-O-acetyl-beta-D-xylopyranosyl)-beta -D- mannopyranosyl]-2-phthalimido- beta-D-glucopyranosyl)-2-phthalimido-6-O-(2,3,4-tri-O-benzyl-alpha-L- fucopyranosyl)-beta-D-glucopyranoside (23). O-Deacetylation followed by tert-butyldimethylsilylation, benzoylation, and desilylation gave methyl 4-O-(4-O-[3-O-allyl-4-O-benzoyl-2-O-(2,3,4-tri-O-benzoyl-beta-D- xylopyranosyl)- beta-D-mannopyranosyl]-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta- D-glucopyranosyl)-3- O-benzyl-2-deoxy-2-phthalimido-6-O-(2,3,4-tri-O-benzyl-alpha-L-fucopy ran osyl)- beta-D-glucopyranoside (24). Mannosylation of 24 using 7, followed by deallylation, further mannosylation with 7, and deprotection, gave the heptasaccharide 5.

The synthesis is reported of 4-methoxyphenyl O-(beta-D-glucopyranosyluronic acid)-(1-->3)-2-acetamido-2-deoxy-beta-D-glucopyranoside (1), 4-methoxyphenyl O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1-->4)-O-(beta-D- glucopyranosyluronic acid)-(1-->3)-2-acetamido-2-deoxy-beta-D-glucopyranoside (5), and 4-methoxyphenyl O-(beta-D-glucopyranosyluronic acid)-(1-->3)-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1-->4)-O-(b eta-D- glucopyranosyluronic acid)-(1-->3)-2-acetamido-2-deoxy-beta-D-glucopyranoside (10), which are structural elements of the extracellular polysaccharide hyaluronic acid. 6-O-Levulinoyl-2,3,4-tri-O-p-toluoyl-alpha-D-glucopyranosyl trichloroacetimidate (3) was condensed with 4-methoxyphenyl 2-deoxy-4,6-O-isopropylidene-2-phthalimido-beta-D-glucopyranoside (4). De-isopropylidenation and acetylation of the obtained disaccharide derivative yielded 4-methoxyphenyl O-(6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-beta-D-glucopyranosyl)-(1-->3)-4 ,6-di-O - acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranoside, and subsequent delevulinoylation, oxidation, complete deprotection, and N-acetylation gave 1. Coupling of 4-O-allyloxycarbonyl-6-O-levulinoyl-2,3-di-O-p-toluoyl-alpha-D-glu copyranosyl trichloroacetamidiate with 4 followed by de-isopropylidenation, acetylation, and deallyloxycarbonylation of the obtained disaccharide derivative gave 8. Condensation of 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl trichloroacetimidate with 8 afforded trisaccharide derivative 4-methoxyphenyl O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1--> 4)-O-(6 - O-levulinoyl-2,3-di-O-p-toluoyl-beta-D-glucopyranosyl)-(1-->3)-4,6-di-O- acetyl- 2-deoxy-2-phthalimido-beta-D-glucopyranoside, and subsequent delevulinoylation, oxidation, complete deprotection, and N-acetylation gave 5. 3-O-Allyloxycarbonyl-2-deoxy-4,6-O-isopropylidene-2-phthalimido-be ta-D- glucopyranosyl trichloroacetimidate was coupled with disaccharide acceptor 8, and deallyloxycarbonylation of the obtained trisaccharide derivative yielded 12. Condensation of 3 with 12 followed by de-isopropylidenation and acetylation of the obtained tetrasaccharide derivative gave 4-methoxyphenyl O-(6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-beta-D-glucopyranosyl)-(1-->3)-O -(4,6- di-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1-->4)-O-(6-O- levulinoyl-2,3-di-O-p-toluoyl-beta-D-glucopyranosyl)-(1-->3)-4,6-di-O-ac etyl-2- deoxy-2-phthalimido-beta-D-glucopyranoside, and delevulinoylation, oxidation, complete deprotection, and N-acetylation yielded 10.

The synthesis is reported of methyl 3,6-di-O-(3-O-methyl-alpha-D-mannopyranosyl)-2-O-beta-D-xylopyranosyl-be ta-D- mannopyranoside (2), methyl 6-O-alpha-D-mannopyranosyl-3-O-(3-O-methyl-alpha-D-mannopyranosyl)-2-O-b eta-D- xylopyranosyl-beta-D-mannopyranoside (3), and methyl 3-O-alpha-D-mannopyranosyl-6-O-(3-O-methyl-alpha-D-mannopyranosyl)-2-O-b eta-D- xylopyranosyl-beta-D-mannopyranoside (4). The various methyl beta-D-Manp acceptor derivatives were prepared from the corresponding methyl beta-D-Glcp derivatives via oxidation-reduction. All glycosyl donors were coupled using the trichloroacetimidate method at -40 degrees C in dichloromethane with trimethylsilyl triflate as a catalyst. Methyl-3-O-benzyl-4,6-O-benzylidene-beta-D-mannopyranoside (7) was condensed with 2,3,4-tri-O-acetyl-alpha-D-xylopyranosyl trichloroacetimidate (8). Regioselective reductive 4,6-O-benzylidene ring-opening on the resulting disaccharide derivative, followed by acetylation, and hydrogenation gave methyl 4-O-acetyl-2-O-(2,3,4-tri-O-acetyl-beta-D-xylopyranosyl)-beta-D-mannopyr anoside (12). Coupling of 12 with 2,4,6-tri-O-acetyl-3-O-methyl-alpha-D-mannopyranosyl trichloroacetimidate (18) afforded tetrasaccharide derivative 19, and subsequent O-deacetylation gave 2. Methyl 3-O-benzyl-4,6-O-prop-2-enylidene-beta-D-mannopyranoside (22) was condensed with 2,3,4-tri-O-acetyl-alpha-D-xylopyranosyl trichloroacetimidate (8). Regioselective reductive 4,6-O-prop-2-enylidene ring-opening on the resulting disaccharide derivative, followed by acetylation, and deallylation at O-6 gave methyl 4-O-acetyl-3-O-benzyl-2-O-(2,3,4-tri-O-acetyl-beta-D-xylopyranosyl)-beta -D- mannopyranoside (26-a), which was either condensed with 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl trichloroacetimidate (27) or 18, to give trisaccharide derivatives 28 or 31, respectively. Debenzylation of 28 followed by condensation with 18 gave, after O-deacetylation, 3, whereas debenzylation of 31 followed by condensation with 27 gave, after O-deacetylation, 4.

The exopolysaccharide of Lactobacillus delbrückii subsp. bulgaricus rr, isolated from skimmed milk, is a heteropolymer of D-galactopyranosyl, D-glucopyranosyl, and L-rhamnopyranosyl residues in the molar ratio 5:1:1. The structure was established by linkage analysis and 1D and 2D NMR spectroscopy of the native polysaccharide, in combination with characterisation of oligosaccharide fragments, obtained by Smith degradation and partial acid hydrolysis, using methylation analysis, EIMS, and 1D and 2D 1H NMR spectroscopy. The polysaccharide has a branched heptasaccharide repeating unit with the following structure: -->2)-[beta-D-Galp-(1-->3)]-alpha-D-Galp-(1-->3)- beta-D-Glcp-(1-->3)-[beta-D-Galp-(1-->4)]-beta-D-Galp-(1-->4)-[alpha-L- Rhap-(1-->3)]-alpha-D-Galp-(1-->.

A 1H NMR database computer program has been developed to determine the primary structure of complex carbohydrates. The database contains carbohydrate structures, their corresponding 1H NMR data, and literature references. From an input list of chemical shift values, the program generates an output list of partially or completely matching carbohydrate structures. In order to facilitate the recognition of the matching part of the selected carbohydrate structures, these structures are displayed with the matching structural elements highlighted. This new 1H NMR database, together with the search program described, now provides a fast access to the published 1H NMR data of complex carbohydrates and furnishes easy links to carbohydrate structures. The performance of the program is demonstrated by the analysis of five carbohydrate fractions prepared from a pool of horse serum glycoproteins.

The structure of the exopolysaccharide of Lactococcus lactis subsp. cremoris H414, isolated from a defined medium or skimmed milk, was established by linkage analysis on the native polysaccharide, and by characterisation of oligosaccharide fragments, obtained by Smith degradation and partial acid hydrolysis, using methylation analysis, FABMS, EIMS, and 1H-NMR spectroscopy. The polysaccharide has the branched-pentasaccharide repeating unit: [formula: see text]

The existence of an HO-3...O-5' intramolecular hydrogen bond in methyl beta-cellobioside in solution in Me2SO-d6 and H2O-CD3OD (4:1 w/w) was studied by 500-MHz 1H-NMR spectroscopy and MD simulations. Temperature coefficients for the chemical shift of the hydroxyl resonances in these solvents were determined and the rates of proton exchange in the latter solvent were obtained from NOE data. With H2O-CD3OD as the solvent, the HO-3...O-5' hydrogen bond was insignificant, but its presence in Me2SO-d6 was confirmed.