Alexander S. Shashkov

A computer-assisted structural analysis of regular polysaccharides on the basis of 13C-n.m.r. data.

A computerised approach to the structural analysis of unbranched regular polysaccharides is described, which is based on an evaluation of the 13C-n.m.r. spectra for all possible primary structures within the additive scheme starting from the chemical shifts of the 13C resonances of the constituent monosaccharides and the average values of the glycosylation effects. The analysis reveals a structure (or structures), the evaluated spectrum of which resembles most closely that observed. The approach has been verified by using a series of bacterial polysaccharides of known structure and, in combination with methylation analysis data, for the determination of the presently unknown structures of the O-specific polysaccharides from Salmonella arizonae O59 and O63, and Proteus hauseri O19.

Structure of a fucoidan from the brown seaweed Fucus evanescens C.Ag.

A fucoidan consisting of L-fucose, sulfate and acetate in a molar proportion of 1:1.23:0.36 was isolated from the Pacific brown seaweed Fucus evanescens. The structures of its desulfated and de-O-acetylated derivatives were investigated by 1D and 2D (1)H and (13)C NMR spectroscopy, and the data obtained were confirmed by methylation analysis of the native and desulfated polysaccharides. The fucoidan was shown to contain a linear backbone of alternating 3- and 4-linked alpha-L-fucopyranose 2-sulfate residues: -->3)-alpha-L-Fucp(2SO(3)(-))-(1-->4)-alpha-L-Fucp(2SO(3)(-))-(1-->. Additional sulfate occupies position 4 in a part of 3-linked fucose residues, whereas a part of the remaining hydroxyl groups is randomly acetylated.

A study of fucoidan from the brown seaweed Chorda filum

Fucoidan fractions from the brown seaweed Chorda filum were studied using solvolytic desulfation. Methylation analysis and NMR spectroscopy were applied for native and desulfated polysaccharides. Homofucan sulfate from C. filum was shown to contain poly-alpha-(1-->3)-fucopyranoside backbone with a high degree of branching, mainly of alpha-(1-->2)-linked single units. Some fucopyranose residues are sulfated at O-4 (mainly) and O-2 positions. Some alpha-(1-->3)-linked fucose residues were shown by NMR to be 2-O-acetylated. The 1H and 13C NMR spectra of desulfated, deacetylated fucan were completely assigned. The spectral data obtained correspond to a quasiregular polysaccharide structure with a branched hexasaccharide repeating unit. Other fucoidan fractions from C. filum have more complex carbohydrate composition and give rather complex methylation patterns. [formula: see text]

Structure of the polysaccharide chains of Pseudomonas pseudomallei lipopolysaccharides

The pathogenic bacterium Pseudomonas pseudomallei strain 57576 produces two partially O-acetylated O-antigenic polysaccharides (PS-I and PS-II). Methylation analysis and 1H and 13C NMR spectroscopy, including NOE experiments, showed PS-I to have the structure [formula: see text] and PS-II to have the structure [formula: see text] where 6dmanHep is the unusual higher sugar 6-deoxy-D-manno-heptose. PS-II is produced also by P. pseudomallei strains 100 and 110, and PS-I and O-deacetylated PS-II by strain 97.

The nuclear overhauser effect and structural factors determining the conformations of disaccharide glycosides

Abstract Conformations of (1→2)-, (1→3)-, and (1→4)-linked disaccharide methyl glycosides involving aldohexopyranose residues have been studied on the basis of the inter-unit n.O.e. and theoretical calculations using atom-atom potential functions. The preferred conformations and the n.O.e. values are determined by the absolute configurations of constituent residues, the configuration and position of the glycosidic linkage, and the orientation of the protons at the aglycon carbon associated with the glycosidic linkage and the adjacent carbons.

Further studies on the composition and structure of a fucoidan preparation from the brown alga Saccharina latissima

The polysaccharide composition of a fucoidan preparation isolated from the brown alga Saccharina latissima (formerly Laminaria saccharina) was reinvestigated. The preparation was fractionated by anion-exchange chromatography, and the fractions obtained were analyzed by chemical methods combined with NMR spectroscopy. Several 2D procedures, including HSQC, HMQC-TOCSY, and HMQC-NOESY, were used to obtain reliable structural information from the complex spectra, and the signal assignments were additionally confirmed by comparison with the literature spectra of the related polysaccharides and synthetic oligosaccharides. In accordance with the previous data, the main polysaccharide component was shown to be a fucan sulfate containing a backbone of 3-linked alpha-l-fucopyranose residues sulfated at C-4 and/or at C-2 and branched at C-2 by single sulfated alpha-l-fucopyranose residues. In addition, three other types of sulfated polysaccharide molecules were detected in the total fucoidan preparation: (i) a fucogalactan having a backbone of 6-linked beta-d-galactopyranose residues branched mainly at C-4 and containing both terminal galactose and fucose residues; (ii) a fucoglucuronomannan having a backbone of alternating 4-linked beta-d-glucopyranosyluronic acid and 2-linked alpha-d-mannopyranose residues with alpha-l-fucopyranose residues as single branches at C-3 of alpha-d-Manp; and (iii) a fucoglucuronan having a backbone of 3-linked beta-d-glucopyranosyluronic acid residues with alpha-l-fucopyranose residues as single branches at C-4. Hence, even a single algal species may contain, at least in minor amounts, several sulfated polysaccharides differing in molecular structure. Partial resolution of these polysaccharides has been accomplished, but unambiguous evidence on their presence as separate entities was not obtained.

5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids in bacterial glycopolymers: chemistry and biochemistry.

Publisher Summary This chapter provides an overview of the chemistry of 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids in bacterial glycopolymers. Ald-2-ulosonic acids are the important components of natural glycoconjugates. Sialic acids—namely, N - and O -acyl derivatives of 5-amino-3,5-dideoxy- D -glycero- D -galacto-non-2-ulosonic acid (neuraminic acid, Neu), generally occur in glycoconjugates of vertebrates and play a significant role in their recognition, regulation, and protection. A deamino analogue of neuraminic acid—3-deoxy- D -glycero- D -galacto-non-2-ulosonic acid (Kdn)—has also been found in a variety of animal tissues. 3-Deoxy- D - manno -oct-2-ulosonic acid (Kdo) is an essential component of lipopolysaccharides (LPSs) of Gram-negative bacteria that functions to link the carbohydrate portion to the lipid moiety. In rare cases, Kdo in LPS is replaced with a 3-hydroxylated analogue— D -glycero- D -talo-oct-2-ulosonic acid. The chapter focuses on the occurrence and characterization of derivatives of 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids and presents experimental approaches that are used to identify them and to elucidate the structures of the bacterial polysaccharides that contain the nonulosonic acids. It also presents the recent data on the biosynthesis of these sugars and discusses their role in immune recognition.

Structural and Genetic Characterization of the Shigella boydii Type 13 O Antigen

Shigella is an important human pathogen. It is generally agreed that Shigella and Escherichia coli constitute a single species; the only exception is Shigella boydii type 13, which is more distantly related to E. coli and other Shigella forms and seems to represent another species. This gives S. boydii type 13 an important status in evolution. O antigen is the polysaccharide part of the lipopolysaccharide in the outer membrane of gram-negative bacteria and plays an important role in pathogenicity. The chemical structure and genetic organization of the S. boydii type 13 O antigen were investigated. The O polysaccharide was found to be acid labile owing to the presence of a glycosyl phosphate linkage in the main chain. The structure of the linear pentasaccharide phosphate repeating unit (O unit) was established by nuclear magnetic resonance spectroscopy, including two-dimensional COSY, TOCSY, ROESY, and H-detected 1H, 13C and 1H, 31P HMQC experiments, along with chemical methods. The O antigen gene cluster of S. boydii type 13 was located and sequenced. Genes for synthesis of UDP-2-acetamido-2,6-dideoxy-L-glucose and genes that encode putative sugar transferases, O unit flippase, and O antigen polymerase were identified. Seven genes were found to be specific to S. boydii type 13. The S. boydii type 13 O antigen gene cluster has higher levels of sequence similarity with Vibrio cholerae gene clusters and may be evolutionarily related to these gene clusters.

Polysaccharides of Algae XXXIII: Isolation and 13C-NMR Spectral Study of Some New Gel-forming Polysaccharides from Japan Sea Red Seaweeds

Fractional extraction of eight red seaweed species from Japan Sea was carried out and the yields and monosaccharide composition of polysaccharide fractions were determined. Six species were showa to contain gelforming polysaccharides which were characterized by chemical composition, gel strength measurements and by change of these parameters after alkali treatment. Carbon-13 NMR spectroscopy was used for the structural analysis of gel-forming polysaccharides; a table of resonance assignments for agarand porphyran-like polysaccharides is given in revised form. It was stated from the chemical and spectral data, that the gel-forming polysaccharide from Campylaephora hypnaeoides is a porphyran analogue, and the polysaccharides from Gracilaria cuneifolia and Polysiphonia morrowü are modified agaroses having the sulfate and O-methyl groups mainly at position 6 of the 3-O-linked ß-D-galactopyranose residues.

Structure of the O-antigen of Francisella tularensis strain 15

The O-specific polysaccharide, obtained by mild acid degradation of the lipopolysaccharide of Francisella tularensis strain 15, contained 2-acetamido-2,6-dideoxy-D-glucose (D-QuiNAc), 4,6-dideoxy-4-formamido-D-glucose (D-Qui4NFm), and 2-acetamido-2-deoxy-D-galacturonamide (D-GalNAcAN) in the ratios 1:1:2. Tri- and tetra-saccharide fragments were obtained on treatment of the polysaccharide with anhydrous hydrogen fluoride and partial hydrolysis with 0.1 M hydrochloric acid, respectively. On the basis of 1H- and 13C-n.m.r. spectroscopy of the polysaccharide and the saccharides, it was concluded that the O-antigen had the structure: ----4)-alpha-D-GalpNAcAN-(1----4)-alpha-D-GalpNAcAN-(1----3) -beta-D-QuipNAc-(1----2)-beta-D-Quip4NFm-(1----. This O-antigen is related in structure to those of Pseudomonas aeruginosa O6, immunotype 1, and IID 1008, and Shigella dysenteriae type 7.