Computer analysis of the structure of branched O-specific polysaccharide of Salmonella arizonae O63 (Arizona 08) from 13C-NMR data

A computerised approach to the structural analysis of branched regular polysaccharides on the basis of the 13C NMR spectra is described. Deviations from additivity of the glycosidation effects for the monosaccharides arranged in branch points are taken into account. The approach has been verified by using data on bacterial polysaccharide of S. arizonae 063.     

Sequence determination of oligosaccharides and regular polysaccharides using NMR spectroscopy and a novel Web-based version of the computer program CASPER

A WWW-interface to a program for structure elucidation of oligo- and polysaccharides using NMR data, CASPER, is presented. The interface and the underlying program have been extensively tested using published data and it was able to simulate 13C NMR spectra of >200 structures with an average error of about 0.3 ppm/resonance. When applied to the repeating units of Escherichia coli O-antigens the published structures were found among the five highest ranked structures in 75% of the cases. The average deviation between calculated and experimental 13C chemical shifts was 0.45 ppm. Oligosaccharide spectra were calculated with even better accuracy (0.23 ppm/resonance) and the correct structure was ranked 1st or 2nd in all the cases examined. Additional NMR experiments that may be required to distinguish between candidate structures are aided by the assignments provided by the program. This computational approach is also suitable for use in structural confirmation of chemically or enzymatically synthesized oligosaccharides. The program is found at http://www.casper.organ.su.se/casper.     

Antigenic polysaccharides of bacteria. 33. The structure of O-specific polysaccharide chain of lipopolysaccharide from Pseudomonas cepacia serotype 6

On mild acid degradation of the Pseudomonas cepacia serotype 6 lipopolysaccharide, the O-specific polysaccharide was obtained, which contains D-mannose and D-galactose residues in the ratio approximately 1:1, as well as O-acetyl groups. On the basis of 1H and 13C NMR analysis, calculation of specific optical rotation, and methylation, it was concluded that the polysaccharide possesses the following structure: (formula; see text) Regularities in glycosidation effects in 13C NMR spectra of 1,3-linked disaccharides containing furanoside residues are discussed.     

Somatic antigens of Pseudomonas aeruginosa. The structure of the O-specific polysaccharide chains of lipopolysaccharides of P. aeruginosa serogroup O4 (Lányi) and related serotype O6 (Habs) and immunotype 1 (Fisher)

Acidic O-specific polysaccharides were isolated on mild acidic degradation of lipopolysaccharides of Pseudomonas aeruginosa serotypes O4a,b, O4a,c, O4a,d (Lányi classification) and serologically related to them serotype O6 (Habs classification) and immunotype 1 (Fisher classification). The polysaccharides had identical monosaccharide composition and were built up of L-rhamnose, 2-acetamido-2,6-dideoxy-D-glucose,2-formamido-2-deoxy-D-galacturonic acid and 2-acetamido-2-deoxy-D-galactouronamide residues. The latter two derivatives of D-galactosaminuronic acid were found in nature for the first time. All the polysaccharides, but Lányi serotype O4a,c, contained O-acetyl groups. The polysaccharides were readily de-O-acetylated with aqueous triethylamine and de-N-formylated with dilute hydrochloric acid. De-N-formylated polysaccharide of serotype O4a,c was selectively cleaved with nitrous acid upon 2-amino-2-deoxygalacturonic acid residues to form a tetrasaccharide with a 2,5-anhydrotaluronic acid residue on the reducing end. The tetrasaccharide represented a modified repeating unit of the polysaccharide. Solvolysis of all intact polysaccharides with hydrogen fluoride selectively split the glycosidic linkages of 6-deoxy sugars to give the same trisaccharide, including both derivatives of galactosaminuronic acid and having 2-acetamido-2,6-dideoxyglucose on the reducing end. Structural investigation of the oligosaccharides obtained together with methylation analysis and 13C nuclear magnetic resonance data revealed the following structures of the O-specific polysaccharides: (Formula: see text) An independent confirmation of the structures of the repeating units was obtained as the result of full interpretation of the 13C nuclear magnetic resonance spectra of the intact and modified polymers. Spectral data analysis revealed a number of regularities in the effects of glycosidation connecting their values with the anomeric and absolute configuration of pyranose residues. The data on the structures of the O-specific polysaccharides indicated that each of the five P. aeruginosa strains under study should be considered as an individual O-serotype within one O-serogroup.     

Structure of the O-specific polysaccharides of the lipopolysaccharides of Xanthomonas campestris pv. vignicola GSPB 2795 and GSPB 2796

The O-specific polysaccharides of Xanthomonas campestris pv. vignicola GSPB 2795 and GSPB 2796 were studied by sugar and methylation analyses, Smith degradation, ID, 2D 1H and 13C NMR spectroscopy. It was found that the polysaccharides are similar branched D-rhamnans lacking strict regularity, and their structures can be described as follows: [carbohydrate equation: see text] where Rha(v) is present in a non-stoichiometric amount, which varies from strain to strain.     

Antigenic polysaccharides of bacteria: 41. Structures of the O-specific polysaccharides of Shigella dysenteriae types 4 and 5 revised by NMR spectroscopy

The earlier established structures of the acidic O-specific polysaccharides from two typical strains of the Shigella dysenteriae bacterium were revised using modern NMR spectroscopy techniques. In particular, the configurations of the glycosidic linkages of GlcNAc (S. dysenteriae type 4) and mannose (S. dysenteriae type 5) residues were corrected. In addition, the location of the sites of nonstoichiometric O-acetylation in S. dysenteriae type 4 was determined: the lateral fucose residue was shown to be occasionally O-acetylated; also, the position of the O-acetyl group present at the stoichiometric quantity in S. dysenteriae type 5 was corrected. The revised structures of the polysaccharides studied are shown below. The known identity of the O-specific polysaccharide structures of S. dysenteriae type 5 and Escherichia coli O58 was confirmed by 13C NMR spectroscopy and, hence, the structure of the E. coli O58 polysaccharide should be revised in the same manner. [Formula: see text].     

Antigenic polysaccharides of bacteria. 32. The structure of O-specific polysaccharide chains of Pseudomonas cepacia serotype B and E lipopolysaccharides containing D-fucose

On the basis of acid hydrolysis, methylation, 1H and 13C NMR analysis, and calculation of specific optical rotation, the following structures were established for O-specific polysaccharides of Pseudomonas cepacia serotypes B and E: ----3)-beta-D-Galf-(1----3)-alpha-D-Fucp-(1----serotype B ----3)-beta-D-GlcpNAc-(1----3)-alpha-D-Fucp-(1----serotype E A characteristic feature of the polysaccharides is the presence of D-fucose, rather rare for bacterial antigens.     

Structural investigation of the O-specific polysaccharides of Morganella morganii consisting of two higher sugars

The lipopolysaccharide of the bacterium Morganella morganii (strain KF 1676, RK 4222) yielded two polysaccharides, PS1 and PS2, when subjected to mild acid degradation followed by GPC. The polysaccharides were studied by 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, 1H,(13)C HMQC, and HMBC experiments. Each polysaccharide was found to contain a disaccharide repeating unit consisting of two higher sugars, 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-L-glycero-D-galacto-non-2-ulosonic acid (a derivative of 8-epilegionaminic acid, 8eLeg5Am7Ac) and 2-acetamido-4-C-(3'-carboxamide-2',2'-dihydroxypropyl)-2,6-dideoxy-D-galactose (shewanellose, She). The two polysaccharides differ only in the ring size of shewanellose and have the following structures:Shewanellose has been previously identified in a phenol-soluble polysaccharide from Shewanella putrefaciens A6, which shows a close structural similarity to PS2.     

Computer-assisted structural analysis of regular glycopolymers on the basis of 13C NMR data

A computer-assisted approach to the prediction of the primary structures of regular glycopolymers is described. The analysis is based on comparing the calculated 13C NMR spectra of all the possible structures of the repeating unit (for the given monomeric composition) to an experimental 13C NMR spectrum. The spectra generation is based on the spectral database containing information on the 13C chemical shifts of monomers, di- and trimeric fragments. If the required data are missing from this database, the special database for average glycosylation effects is used. The analysis reveals those structures with the calculated 13C NMR spectrum most close to observed. The structures of repeating units of any topology containing up to six residues linked by glycosidic, amidic or phospho-diester bridges can be predicted. Unambiguous selection of the proper structure from the output list of possible structures may require additional experimental data. Testing the created program and databases on bacterial polysaccharides and their derivatives containing up to three non-sugar residues (alditols, amino acids, phosphate groups etc.) per repeating unit revealed the good convergence of prediction with independently obtained structural data.     

Complete (1)H and (13)C NMR chemical shift assignments of mono-, di-, and trisaccharides as basis for NMR chemical shift predictions of polysaccharides using the computer program casper

The computer program casper uses (1)H and (13)C NMR chemical shift data of mono- to trisaccharides for the prediction of chemical shifts of oligo- and polysaccharides. In order to improve the quality of these predictions the (1)H and (13)C, as well as (31)P when applicable, NMR chemical shifts of 30 mono-, di-, and trisaccharides were assigned. The reducing sugars gave two distinct sets of NMR resonances due to the α- and β-anomeric forms. In total 35 (1)H and (13)C NMR chemical shift data sets were obtained from the oligosaccharides. One- and two-dimensional NMR experiments were used for the chemical shift assignments and special techniques were employed in some cases such as 2D (1)H,(13)C-HSQC Hadamard Transform methodology which was acquired approximately 45 times faster than a regular t(1) incremented (1)H,(13)C-HSQC experiment and a 1D (1)H,(1)H-CSSF-TOCSY experiment which was able to distinguish spin-systems in which the target protons were only 3.3Hz apart. The (1)H NMR chemical shifts were subsequently refined using total line-shape analysis with the PERCH NMR software. The acquired NMR data were then utilized in the casper program (http://www.casper.organ.su.se/casper/) for NMR chemical shift predictions of the O-antigen polysaccharides from Klebsiella O5, Shigella flexneri serotype X, and Salmonella arizonae O62. The data were compared to experimental data of the polysaccharides from the two former strains and the lipopolysaccharide of the latter strain showing excellent agreement between predicted and experimental (1)H and (13)C NMR chemical shifts.     

Structure of O-specific polysaccharide chains of Pseudomonas aeruginosa II (Sandwick) and V (Verder-Evans) lipopolysaccharides containing N-acetyl-D-galactosaminuronic acid

Acidic polysaccharides were isolated from the Pseudomonas aeruginosa II (Sandvik) and V (Verder-Evans) lipopolysaccharides on mild acid hydrolysis followed by gel filtration on Sephadex G-50. The Sandvik II polysaccharide consists of 2-acetamido-2-deoxy-D-galacturonic acid, 2-acetamido-2,6-dideoxy-D-glucose, and L-rhamnose in the ratio 1:1:2. The Verder-Evans V polysaccharide contained the same monosaccharides and, in addition, a D-glucose residue. On the basis of 13C NMR data, methylation analysis, Smith degradation and solvolysis with hydrogen fluoride, the following structures were determined for the repeating units of the polysaccharides: (Formula: see text).     

Structural analysis of the O-polysaccharide from the lipopolysaccharide of Azospirillum brasilense S17

A mixture of two structurally distinct neutral O-polysaccharides was obtained by mild acid degradation of the lipopolysaccharide isolated by the phenol/water extraction from the asymbiotic diazotrophic rhizobacterium Azospirillum brasilense S17. The following structures of the O-polysaccharides were established by composition and methylation analyses, Smith degradation, and 1H and 13C NMR spectroscopy, including a 2D NOESY experiment: [Formula: see text] where L-Rha2Me stands for 2-O-methyl-L-rhamnose and SHb for the (S)-3-hydroxybutanoyl group. The occurrence of two distinct polysaccharides is reported for the first time in Azospirillum spp.     

The structure of O-specific polysaccharide of Proteus mirabilis 027 containing amino acids and phosphoethanolamine

The following structure of the repeating unit of the Proteus mirabilis O27 O-specific polysaccharide was established: (formula; see text) where (formula; see text) is N-glucopyranuronoyl-L-lysine, (formula; see text) is N-galactopyranuronoyl-L-alanine. The polysaccharide was parially solvolysed with anhydrous HF and the resulting dephosphorylated tri- and tetrasaccharide with N-acetylglucosamine at the reducing end were studied by means of 1H and 13C NMR spectroscopy and (for methylated derivative of trisaccharide) mass-spectrometry. Smith degradation of the polysaccharide afforded linear polymer, and its structure was investigated by 13C NMR spectroscopy. The position of the ethanolamine phosphate group was determined by means of the analysis of the phosphorylation effects in the 13C NMR spectra of the linear and branched polysaccharides.     

Structural characterization by (13)C-NMR spectroscopy of products synthesized in vitro by polysaccharide synthases using (13)C-enriched glycosyl donors: application to a UDP-glucose:(1-->3)-beta-D-glucan synthase from blackberry (Rubus fruticosus)

A simple and sensitive method for the characterization of products synthesized in vitro by polysaccharide synthases is described. It relies on the use of (13)C-enriched nucleotide sugars as substrates and on the analysis of the newly synthesized polysaccharides by (13)C-nuclear magnetic resonance (NMR) spectroscopy. The method was validated with a (1-->3)-beta-d-glucan synthase from blackberry, but it may be applied to the study of any glycosyltransferase. The chemical synthesis of UDP-d-[U-(13)C]glucose was achieved in a classical procedure with an overall yield of 50%. A uniformly labeled (1-->3)-beta-d-glucan was synthesized from this substrate, using detergent extracts of blackberry cell membranes as a source of synthase. One hundred micrograms of product was sufficient for liquid and solid-state (13)C-NMR spectroscopy analyses. The method is at least 100 times more sensitive than in the case of nonenriched polysaccharides. It allows the unequivocal identification and direct structural characterization of the products synthesized in vitro, as opposed to conventional methods that rely on the use of radioactive substrates and enzymatic hydrolysis of the polysaccharides with specific glycoside hydrolases. The method proves that the glycan analyzed was synthesized de novo because the final product is enriched in (13)C. Information on the 3D organization of the polymer may also be obtained by solid-state NMR spectroscopy.     

Detection of conserved N-linked glycans and phase-variable lipooligosaccharides and capsules from campylobacter cells by mass spectrometry and high resolution magic angle spinning NMR spectroscopy

Glycomics, the study of microbial polysaccharides and genes responsible for their formation, requires the continuous development of rapid and sensitive methods for the identification of glycan structures. In this study, methods for the direct analysis of sugars from 108 to 1010 cells are outlined using the human gastrointestinal pathogen, Campylobacter jejuni. Using capillary-electrophoresis coupled with sensitive electrospray mass spectrometry, we demonstrate variability in the lipid A component of C. jejuni lipooligosaccharides (LOSs). In addition, these sensitive methods have permitted the detection of phase-variable LOS core structures that were not observed previously. High resolution magic angle spinning (HR-MAS) NMR was used to examine capsular polysaccharides directly from campylobacter cells and showed profiles similar to those observed for purified polysaccharides analyzed by solution NMR. This method also exhibited the feasibility of campylobacter serotyping, mutant verification, and preliminary sugar analysis. HR-MAS NMR examination of growth from individual colonies of C. jejuni NCTC11168 indicated that the capsular glycan modifications are also phase-variable. These variants show different staining patterns on deoxycholate-PAGE and reactivity with immune sera. One of the identified modifications was a novel -OP=O(NH2)OMe phosphoramide, not observed previously in nature. In addition, HR-MAS NMR detected the N-linked glycan, GalNAc-alpha1,4-GalNAc-alpha1,4-[Glc-beta1,3-]GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac, where Bac is 2,4-diacetamido-2,4,6-trideoxy-d-glucopyranose, in C. jejuni and Campylobacter coli. The presence of this common heptasaccharide in multiple campylobacter isolates demonstrates the conservation of the N-linked protein glycosylation pathway in this organism and describes the first report of HR-MAS NMR detection of N-linked glycans on glycoproteins from intact bacterial cells.     

轻度降解核磁共振法研究多糖链结构

 建立了轻度降解~(13)C核磁共振法研究多糖结构的方法。美国瓜胶和国产胶1在70℃下用90％甲酸降解3小时。用Bio-Gel p-4的凝胶色谱分离。含20个以上糖残基的片断为主要降解成份。用~(13)C核磁共振法研究,确定它们都是具有以β1-4甘露糖为骨架,其中某些甘露糖的6位碳有α-半乳糖侧链的结构。这些结果与组成分析和甲基化分析结果一致。     

Structural studies of water-soluble polysaccharides of an edible mushroom, Termitomyces eurhizus. A reinvestigation

The structure of two polysaccharides isolated from the hot aqueous extract of fruiting bodies of the mushroom, Termitomyces eurhizus, have been reinvestigated. These consist of two homogeneous fractions PS-I and PS-II. PS-I contains only D-glucose as the monosaccharide constituent. From methylation analysis and periodate oxidation studies, followed by GLC-MS analysis the linkages, the sugar units in PS-I were identified as (1-->3)-D-Glcp and (1-->6)-D-Glcp. PS-II contains D-glucose, and the mode of linkage of d-glucose was identified as (1-->6)-D-Glcp. Finally, the following possible structures of the polysaccharides were assigned using 1H, 2D-COSY, TOCSY, NOESY and 13C NMR spectral analysis: [carbohydrate structure: see text].     

Structures of the O-polysaccharide chains of the lipopolysaccharides of Xanthomonas campestris pv phaseoli var fuscans GSPB 271 and X campestris pv malvacearum GSPB 1386 and GSPB 2388

O-polysaccharides of phytopathogenic bacteria Xanthomonas campestris were isolated by mild acid degradation of the lipopolysaccharides and studied by sugar and methylation analysis, along with 1H and 13C NMR spectroscopy. The following structures of the repeating units of the polysaccharides of X. campestris pv. phaseoli var. fuscans GSPB 271 (1). and X. campestris pv. malvacearum GSPB 1386 and GSPB 2388 (2). were established:The O-polysaccharides of X. campestris are structurally similar to those of some Pseudomonas syringae strains.     

Structure elucidation and sulfated derivatives preparation of two alpha-D-glucans from Gastrodia elata Bl. and their anti-dengue virus bioactivities

The structures of two glucans, WGEW and AGEW, isolated from Gastrodia elata Bl. were elucidated using monosaccharide composition analysis by gas chromatography (GC), methylation analysis by gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy. Their structures were deduced as an alpha-D-(1-->4)-glucan with an alpha-(1-->4) linked branch attached to O-6 branch points with different branch degrees. Their sulfate derivatives with distinct degrees of substitution (DS) were prepared. The substitution position was assigned to O-6 according to the (13)C NMR spectra. All sulfated derivatives showed strong anti-dengue virus bioactivities. The structure-activity relationships (SAR) between the polysaccharides and their sulfated derivatives were also investigated. Results showed that the higher the DS is, the more potent the impact on the dengue virus infection would be.     

Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspen employing 1H and 13C NMR spectroscopy

Water-soluble hemicelluloses were extracted from milled aspen wood (Populus tremula) employing microwave oven treatment at 180 degrees C for 10 min. The final pH of this extract was 3.5. From this extract oligo- and polysaccharides were isolated and subsequently fractionated by size-exclusion chromatography. The structures of the saccharides in three of the fractions obtained were determined by 1H and 13C NMR spectroscopy, using homonuclear and heteronuclear two-dimensional techniques. The polysaccharides present in the two fractions eluted first were O-acetyl-(4-O-methylglucurono)xylans. The average degree of acetylation of the xylose residues in these compounds was 0.6. The structural element -->4)[4-O-Me-alpha-D-GlcpA-(1-->2)][3-O-Ac]-beta-D-Xylp-(1 --> could also be identified. On the average, these two xylans were composed of the following (1-->4)-linked beta-D-xylopyranosyl structural elements: unsubstituted (50 mol%), 2-O-acetylated (13 mol%), 3-O-acetylated (21 mol%), 2,3-di-O-acetylated (6 mol%) and [MeGlcA alpha-(1-->2)][3-O-acetylated] (10 mol%). Most of the 4-O-methylglucuronyl and acetyl substituents in the isolated polysaccharides survived the microwave oven treatment. The third fraction, eluted last, contained acetylated xylo-oligosaccharides, with minor contamination by an acetylated mannan. In the case of these xylo-oligosaccharides, the average degree of acetylation was 0.3.