The structure of capsule polysaccharide of Klebsiella ozaenae K4 containing 3-deoxynonulosonic acid

3-Deoxy-D-glycero-D-galacto-nonulosonic acid was identified as a component of the Klebsiella ozaenae K4 capsular polysaccharide. On the basis of methylation, complete and partial acid hydrolyses, Smith degradation, and NMR analysis including computer-assisted 13C NMR evaluation, the following structure of the polysaccharide has been established.     

二维核磁共振谱在多糖结构研究中的应用

二维核磁共振谱（2D NMR）是获取多糖结构信息,尤其是在多糖序列分析方面的有力工具。本文重点介绍了在多糖结构解析中常用的几种2D NMR谱以及2D NMR解析多糖结构的方法。     

Structure of the O-specific polysaccharide of Citrobacter braakii O7a,3b,1c.

The following structure of the O-specific polysaccharide of Citrobacter braakii O7a,3b,1c was established using sugar and methylation analyses and NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and 1H, 13C heteronuclear single-quantum coherence (HSQC) experiments: (struture: see text). The main D-mannan chain of the polysaccharide studied has the same structure as the O-specific polysaccharide of Escherichia coli O9, Klebsiella pneumoniae O3, and Hafnia alvei PCM 1223.     

Structure of the exopolysaccharide produced by Streptococcus thermophilus S3

The exopolysaccharide of Streptococcus thermophilus S3, produced in skimmed milk, is composed of D-galactose and L-rhamnose in a molar ratio of 2:1. The polysaccharide contains 0.4 equiv of O-acetyl groups per repeating unit. Linkage analysis and 1D/2D NMR (1H and 13C) studies on native and O-deacetylated EPS together with nanoES-CID tandem mass spectrometry studies on oligosaccharides generated by a periodate oxidation protocol, show the polysaccharide to have the following structure: [structure: see text].     

High-field NMR as a technique for the determination of polysaccharide structures

NMR spectroscopy has played a developing role in the study of polysaccharide structures for over 30 years. Many new bacterial polysaccharide repeat unit structures have recently been published as a result of the application of modern NMR techniques. NMR can also be used to elucidate the structures of both regular and heterogeneous polysaccharides from fungal and plant sources, as well as complex glycosaminoglycans of animal origin. In addition to covalent structure, conformation and dynamics of polysaccharides are susceptible to NMR analysis, both in solution and in the solid state. Improvements in NMR technology with potential applications to polysaccharide studies hold promise for the future.     

Pseudo-3D NMR spectroscopy: Application to oligo- and polysaccharides

Summary Several pseudo-3D NMR experiments are proposed for removal of overlaps in 1D ¹H NMR spectra. A selective pulse and a chemical-shift-selective filter are used for double selection of the magnetization during the course of the pulse sequence. Different polarization transfer mechanisms are combined into pseudo-3D COSY-RELAY, COSY-TOCSY, COSY-NOESY, COSY-ROESY, RELAY-NOESY, RELAY-ROESY, RELAY-RELAY and RELAY-TOCSY experiments. The techniques are illustrated on oligo- and polysaccharide samples.     

黑木耳多糖AAPS-3的化学结构

利用逆流色谱分离技术从黑木耳中分离了黑木耳多糖AAPS-3。通过完全水解、甲基化、一维和二维1H和13C核磁共振(NMR)阐明AAPS-3的化学结构。经完全水解证实AAPS-3主要由Glc单糖组成,用HPLC方法测得其分子量为4.83×105,通过甲基化反应数据和1H和13C NMR等分析发现AAPS-3为由β-D-1,4-Glc,β-D-1,3-Glc和β-D-1,6-Glc残基构成主链的葡聚糖。     

Structure and hydrodynamic properties of the extracellular polysaccharide from a mutant strain (RA3W) of Erwinia chrysanthemi RA3

The structure of the extracellular polysaccharide (EPS) produced by Erwinia chrysanthemi strain RA3W, a mutant strain of E. chrysanthemi RA3, has been determined using low pressure size-exclusion and anion-exchange chromatographies, high pH anion-exchange chromatography, glycosyl linkage analysis, and 1D 1H NMR spectroscopy. The polysaccharide is structurally similar, if not identical, to the family of EPS produced by such as E. chrysanthemi strains Ech9, Ech9Sm6, and SR260. The molecular weight of EPS RA3W by ultracentrifugation (sedimentation equilibrium) and light scattering is compared with those of other E. chrysanthami EPSs, as are the viscometric properties.     

A water-insoluble (1-->3)-beta-D-glucan from the alkaline extract of an edible mushroom Termitomyces eurhizus

A water-insoluble glucan, TEINS has been isolated from the hot alkaline extract of an edible mushroom Termitomyces eurhizus. The total carbohydrate content of the polysaccharide fraction was found to be 98.4%, and it was found to contain only glucose as the monosaccharide constituent. On the basis of total acid hydrolysis, a methylation experiment, periodate oxidation and (13)C NMR experiment, the repeating unit of the polysaccharide was established as: -->3)-beta-D-Glcp-(1-->.     

The structure of a repetitive unit of the glycerolphosphate- containing O-specific polysaccharide chain from Yersinia kristensenii strain 103 (0:12,26) lipopolysaccharide]

Mild acid hydrolysis of the lipopolysaccharide from Yersinia kristensenii strain 103 (0:12.26) afforded teichoic acid-like polysaccharide. From the results of methylation, dephosphorylation, partial Smyth degradation, and 13C and 31P NMR data the structure of the repeating unit of the polysaccharide was deduced as follows: [formula: see text] The structure was confirmed by complete interpretation of polysaccharide 13C NMR spectrum.     

Identification of 3-acetamidino-2-acetamido-2,3-dideoxy-L-guluronic acid in lipopolysaccharide from Pseudomonas aeruginosa immunotype 7

O-Specific polysaccharide chain of Pseudomonas aeruginosa immunotype 7 lipopolysaccharide is composed of 3-acetamidino-2-acetamido-2,3-dideoxy-L-guluronic acid (GulNAcAmA), 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid (ManN2Ac2A), and N-acetyl-D-fucosamine (FucNAc). On solvolysis with anhydrous hydrogen fluoride, the polysaccharide afforded a trisaccharide containing all its components. Borohydride reduction of the trisaccharide in boric acid solution resulted in conversion of reducing fucosamine into fucosaminitol, whereas in water the reduction was accompanied by reductive deamination of acetamidino function into ethylamino group. On hydrolysis with aqueous triethylamine, acetamidino group gave acetamido group. Analysis of the trisaccharides thus obtained by 1H NMR spectroscopy (including nuclear Overhauser effect), 13C NMR spectroscopy, and fast-atom bombardment mass spectrometry allowed the determination of the structure of the unusual uronic acid derivative and the following structure of the polysaccharide repeating unit: -4)-alpha-L-GulNAcAmA-(1-4)-beta-D-ManN2Ac2A-(1-3)-alpha-D-+ ++FucNAc-(1-.     

Structural analysis and chemical depolymerization of the capsular polysaccharide of Streptococcus pneumoniae type 1

NMR spectroscopy can be used to characterize bacterial polysaccharides such as that of Streptococcus pneumoniae type 1 which is a component of the 23-valent pneumococcal vaccine in clinical use. This particular polysaccharide gives NMR spectra with wide lines apparently due to restricted molecular mobility and chain flexibility which leads to rapid dipolar T(2) relaxation limiting the possibility of detailed spectral analysis. Removal of O-acetyl groups found on approximately two thirds of the repeating subunits of pneumococcal type 1 capsule leads to narrower NMR lines facilitating a complete assignment of the 1H and 13C NMR spectra. Degradation of the polysaccharide by periodate oxidation followed by base treatment leads to an oligosaccharide fragment of approximately three repeating trisaccharide units. This oligosaccharide has narrow NMR lines and 1H and 13C assignments very similar to those of the O-deacetylated polysaccharide. In the native polysaccharide, O-acetyl groups are located on the 2- and 3-positions of the 4-linked galacturonic acid residue providing protection against periodate oxidation. Analysis of NOESY spectra combined with molecular modeling of the oligosaccharide shows that flexibility occurs in certain of the saccharide linkages.     

Structure of the O-specific polysaccharide of Providencia alcalifaciens O16 containing N-acetylmuramic acid

The O-specific polysaccharide of Providencia alcalifaciens O16 was obtained by mild-acid degradation of the lipopolysaccharide and studied by chemical methods and NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, NOESY, and 1H,(13)C HSQC experiments. It was found that the polysaccharide contains N-acetylmuramic acid, which was isolated by solvolysis with trifluoromethanesulfonic acid and identified by the specific optical rotation and NMR spectroscopy. The following structure of the trisaccharide repeating-unit of the polysaccharide was established:     

Structural studies of the O-antigen polysaccharide from the enteroinvasive Escherichia coli O164 cross-reacting with Shigella dysenteriae type 3.

The structure of the O-antigen polysaccharide from Escherichia coli O164 has been determined. Nuclear magnetic resonance spectroscopy together with component and methylation analyses of lipid free polysaccharide were the principal methods used. The sequence of the sugar residues could be determined by NOESY and heteronuclear multiple bond connectivity NMR experiments. It is concluded that the polysaccharide is composed of a pentasaccharide repeating unit with the following structure: [structure: see text]. Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) was performed on intact lipopolysaccharide and from the resulting molecular mass, the O-antigen part was estimated to contain approximately 24 repeating units. The nature of the previously reported cross-reactivity of this O-antigen to those of Escherichia coli O124 and Shigella dysenteriae type 3 is discussed.     

Structural studies of an exopolysaccharide produced by Streptococcus thermophilus THS

The structure of an extracellular polysaccharide (EPS) from Streptococcus thermophilus THS has been determined. A combination of component analysis, methylation analysis and NMR spectroscopy shows that the polysaccharide is composed of pentasaccharide repeating units. Sequential information was obtained by two-dimensional (1)H,(1)H-NOESY and (1)H,(13)C-HMBC NMR experiments. NMR data indicate different mobility within the EPS with a stiffer backbone and a more flexible side-chain.     

Structures of two putative O-specific polysaccharides from the Rahnella aquatilis 3-95 lipopolysaccharide

Two polysaccharide preparations (OPSI and OPSII) were obtained by mild acid degradation of the lipopolysaccharide of Rahnella aquatilis 3-95. Studies by chemical methods and 1H and 13C NMR spectroscopy showed that OPSI is a linear alpha-D-mannan having a trisaccharide repeat and OPSII is a approximately 2:1 mixture of the same mannan and an alpha-d-glucan:     

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.     

Structure of an O-specific polysaccharide of Proteus mirabilis 03, containing N-(2-hydroxyethyl)-D-alanine

O-Specific polysaccharide, obtained by mild acid degradation of the Proteus mirabilis 03 lipopolysaccharide, was dephosphorylated with 48% HF to give a linear polysaccharide and an amino acid, N-(2-hydroxyethyl)-D-alanine. The structure of the polysaccharide was determined by methylation, the Smith degradation and computer-assisted analysis of the 13C NMR spectra of original and dephosphorylated polymers and oligomers. The structure of the amino acid was elucidated by using 1H and 13C NMR spectroscopy and mass spectrometry (applied to the acetylated methyl ester derivative), optical rotation and CD spectrum data and comparison with the synthetic sample. The repeating unit of P. mirabilis 03 O-specific polysaccharide is shown to have the following structure: (formula; see text)     

Structural determination and immunochemical characterization of the type V group B Streptococcus capsular polysaccharide

The type V capsular polysaccharide of group B Streptococcus has been isolated and purified, and its repeating unit structure determined. The native type V polysaccharide contains D-glucose, D-galactose, 2-acetamido-2-deoxy-D-glucose, and sialic acid in a molar ratio of 3:2:1:1. Methylation analysis and 1H NMR and 13C NMR analysis of the native type V polysaccharide and of its specifically degraded products permitted the determination of the repeating unit structure of the type V polysaccharide: [formula: see text] The type V polysaccharide has certain structural features in common with other group B streptococcal capsular polysaccharides but is antigenically distinct: no immunologic cross-reactivity was observed between type V and types Ia, Ib, II, III, or IV polysaccharides. Studies of antibody binding to the partially degraded forms of the type V polysaccharide indicated that the native epitope is complex, involving most if not all of the sugar residues of the repeating unit.     

Isolation of oligosaccharides from a partial-acid hydrolysate of pneumococcal type 3 polysaccharide for use in conjugate vaccines

A series of well-defined oligosaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 3 has been generated. Partial-acid hydrolysis of the capsular polysaccharide, followed by fractionation of the oligosaccharide mixture by Sepharose Q ion-exchange chromatography yielded fragments containing one to seven [-->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1-->] repeating units. The isolated fragments were analysed for purity by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using an IonPac AS11 column, and their structures were verified by 1H NMR spectroscopy and nano-electrospray mass spectrometry. The oligosaccharides can be used to produce neoglycoprotein vaccines with a defined carbohydrate part.