Synthesis and identification in bacterial lipopolysaccharides of 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto- and -D-glycero-D-talo-non-2-ulosonic acids

5,7-Diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto- and -D-glycero-D-talo-non-2-ulosonic acids were synthesized by condensation of 2,4-diacetamido-2,4,6-trideoxy-D-mannose with oxalacetic acid. Comparison of the 1H and 13C NMR data and the specific optical rotation values of these monosaccharides and the corresponding L-glycero-D-galacto and L-glycero-D-talo isomers synthesized earlier [Tsvetkov, Y. E.; Shashkov, A. S.; Knirel, Y. A.; Backinowsky, L. V.; Z?hringer, U. Mendeleev Commun. 2000, 90-92] with data of the natural compounds enabled the identification in bacterial lipopolysaccharides of derivatives of 5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic (legionaminic) acid and epimers of legionaminic acid at C-4 and C-8.     

A pseudoaminic acid-containing O-specific polysaccharide from a marine bacterium Cellulophaga fucicola

The O-polysaccharide was isolated from the lipopolysaccharide of Cellulophaga fucicola and studied by chemical analyses along with (1)H and (13)C NMR spectroscopy. The following new structure of the O-polysaccharide of C. fucicola containing 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid residue (pseudoaminic acid, Psep) was elucidated as the following:     

Structure and serological characterization of 5,7-diamino-3,5,7,9-tetradeoxy-non-2-ulosonic acid isolated from lipopolysaccharides of Vibrio parahaemolyticus O2 and O-untypable strain KX-V212

Lipopolysaccharides (LPS) of Vibrio parahaemolyticus O2 and O-untypable (OUT) strain (KX-V212) isolated from an individual patient were shown to contain 5,7-diamino-3,5,7,9-tetradeoxy-non-2-ulosonic acid (NonlA), which was readily released from LPS by mild acid hydrolysis. In the present study, we investigated the chemical and serological properties of NonlA isolated from LPS of V. parahaemolyticus O2 and OUT KX-V212. GC-MS and NMR analysis identified the NonlA from LPS of O2 to be 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (5NAc7NAcNonlA) and that from LPS of KX-V212 to be 5-acetamido-7-(N-acetyl-D-alanyl)amido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (5NAc7NAlaNAcNonlA). In ELISA inhibition analysis, 5NAc7NAcNonlA inhibited the O2 LPS/anti-O2 antiserum system, whereas, 5NAc7NAlaNAcNonlA did not show any inhibitory activity. However, after N-deacylation of 5NAc7NAlaNAcNonlA followed by N-acetylation, the product (5NAc7NAcNonlA) inhibited the O2 LPS/anti-O2 antiserum system to the same extent as that of 5NAc7NAcNonlA obtained from O2 LPS. These results suggest that 5NAc7NAcNonlA might be related to the serological specificity of O2 LPS as one of main epitope(s) involved in O2 LPS.     

Structure of an acidic polysaccharide from the agar-decomposing marine bacterium Pseudoalteromonas atlantica strain IAM 14165 containing 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid

The structure of an acidic polysaccharide from Pseudoalteromonas atlantica strain 14165 containing 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid (di-N-acetylpseudaminic acid, Pse5Ac7Ac) has been elucidated. The polysaccharide was studied by 1H and 13C NMR spectroscopy, including 2D experiments, along with sugar and methylation analyses. After a selective hydrolysis a modified polysaccharide devoid of its side chain could be isolated. It was found that the polysaccharide has pentasaccharide repeating units with following structure: [structure: see text].     

Structure of the O-antigen of Providencia stuartii O20, a new polysaccharide containing 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic acid

The O-polysaccharide chain of the lipopolysaccharide (LPS) of Providencia stuartii O20 was found to contain d-glucuronic acid, N-acetyl-d-glucosamine, and a rarely occurring higher sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic acid (di-N-acetyl-8-epilegionaminic acid, 8eLeg5Ac7Ac). Degradation of the LPS with dilute acetic acid caused depolymerization of the polysaccharide chain by the ketosidic linkage to give a tetrasaccharide corresponding to the repeating unit of the polysaccharide. Based on sugar and methylation analyses of the tetrasaccharide and O-deacylated LPS as well as ESIMS, (1)H and (13)C NMR spectroscopy data, the structure of the O-polysaccharide of P. stuartii O20 was established.     

Structure of the acidic O-specific polysaccharide from Proteus vulgaris O39 containing 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid.

The O-specific polysaccharide of Proteus vulgaris O39 was found to contain a new acidic component of Proteus lipopolysaccharides, 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid (di-N-acetylpseudaminic acid, Pse5Ac7Ac). The following structure of the polysaccharide was determined by NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, ROESY, and 1H,(13)C HMQC experiments, along with selective cleavage of the polysaccharide by solvolysis with anhydrous trifluoromethanesulfonic (triflic) acid: -->8)-beta-Psep5Ac7Ac-(2-->3)-alpha-L-FucpNAc-(1-->3)-alpha-D-GlcpNAc-(1--> The structure established is unique among the O-specific polysaccharides, which is in accordance with classification of the strain studied into a separate Proteus serogroup.     

Teichuronic and teichulosonic acids of actinomycetes

The subject of the present review is the structural diversity and abundance of cell wall teichuronic and teichulosonic acids of representatives of the order Actinomycetales. Recently found teichulosonic acids are a new class of natural glycopolymers with ald-2-ulosonic acid residues: Kdn (3-deoxy-D-glycero-D-galacto-non-2-ulosonic acid) or di-N-acyl derivatives of Pse (5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic or pseudaminic acid) as the obligatory component. The structures of teichuronic and teichulosonic acids are presented. Data are summarized on the occurrence of the glycopolymers of different nature in the cell wall of the studied actinomycetes. The biological role of the glycopolymers and their possible taxonomic implication are discussed. The comprehensive tables given in the Supplement show ¹³C NMR spectroscopic data of teichuronic and teichulosonic acids obtained by the authors.     

Characterization of the lipopolysaccharide O-antigen of Cronobacter turicensis HPB3287 as a polysaccharide containing a 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (legionaminic acid) residue

Cronobacter turicensis, previously known as Enterobacter sakazakii, is a Gram-negative opportunistic food-borne pathogen that has been reported as a cause of life-threatening neonatal infections. From chemical and physical analyses involving composition analysis, methylation, two-dimensional high-resolution nuclear magnetic resonance, and mass spectrometry methods, the antigenic O-polysaccharide in the smooth-type lipopolysaccharide of C. turicensis (strain HPB 3287) was determined to be a high molecular mass polymer of a repeating pentasaccharide unit composed of D-galactose, D-glucose, 2-acetamido-2-deoxy-D-galactose, and 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (legionaminic acid), in a molar ratio 2:1:1:1, and having the structure: [see formula in text].     

Structure of the O-antigen and characterization of the O-antigen gene cluster of Escherichia coli O108 containing 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic (8-epilegionaminic) acid

On mild acid degradation of the lipopolysaccharide of Escherichia coli O108, the O-polysaccharide was isolated and studied by sugar analysis and one- and two-dimensional ¹H- and ¹³C-NMR spectroscopy. The polysaccharide was found to contain an unusual higher sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic acid (di-N-acetyl-8-epilegionaminic acid, 8eLeg5Ac7Ac). The following structure of the tetrasaccharide repeating unit of the polysac-charide was established: →4)-α-8eLegp5Ac7Ac-(2→6)-α-D-Galp-(1→3)-α-L-FucpNAc-(1→3)-α-D-GlcpNAc-(1→. Functions of the E. coli O108 antigen biosynthetic genes, including seven putative genes for synthesis of 8eLeg5Ac7Ac, were assigned by sequencing the O-antigen gene cluster along with comparison with gene databases and known biosynthetic pathways for related nonulosonic acids.     

Structure of the O-Specific polysaccharide from Shewanella japonica KMM 3601 containing 5,7-Diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulosonic acid

Structure of the O-specific polysaccharide chain of the lipopolysaccharide (LPS) of Shewanella japonica KMM 3601 was elucidated. The initial and O-deacylated LPS as well as a trisaccharide representing the O-deacetylated repeating unit of the O-specific polysaccharide were studied by sugar analysis along with ¹H and ¹³C NMR spectroscopy. The polysaccharide was found to contain a rare higher sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-d-talo-non-2-ulosonic acid (a derivative of 4-epilegionaminic acid, 4eLeg). The following structure of the trisaccharide repeating unit was established: →4)-α-4eLegp5Ac7Ac-(2→4)-β-d-GlcpA3Ac-(1→3)-β-d-GalpNAc-(1→.     

Characterization of the lipopolysaccharide O-antigen of Cronobacter turicensis HPB3287 as a polysaccharide containing a 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulosonic acid (legionaminic acid) residue

Cronobacter turicensis, previously known as Enterobacter sakazakii, is a Gram-negative opportunistic food-borne pathogen that has been reported as a cause of life-threatening neonatal infections. From chemical and physical analyses involving composition analysis, methylation, two-dimensional high-resolution nuclear magnetic resonance, and mass spectrometry methods, the antigenic O-polysaccharide in the smooth-type lipopolysaccharide of C. turicensis (strain HPB 3287) was determined to be a high molecular mass polymer of a repeating pentasaccharide unit composed of d-galactose, d-glucose, 2-acetamido-2-deoxy-d-galactose, and 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulosonic acid (legionaminic acid), in a molar ratio 2:1:1:1, and having the structure:     

The structure of O-specific polysaccharide from Pseudomonas aeruginosa 013 containing 5,7-diacetoamido-3,5,7,9-tetradeoxy-D- glycero-L-galacto-nonulosonic acid and 2-acetamidino-2,6-dideoxy-L-galactose

O-Specific polysaccharide chain of P. aeruginosa 013 (Lányi) lipopolysaccharide is composed of N-acetyl-D-quinovosamine (QuiNAc), acetamidino derivative of L-fucosamine (FucNAm), and 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-L-galacto-nonuloso nic acid (Sug). On solvolysis with HF in methanol, the polysaccharide afforded methylglycosides of a disaccharide and a trisaccharide both containing fucosamine and ulosonic acid derivatives. Chemical transformations (alkaline hydrolysis, reductive deamination, acetylation accompanied by intramolecular acylation of acetamidino group by ulosonic acid), 1H and 13C NMR analysis and mass spectral data proved the following structure of the trisaccharide unit of the polysaccharide: -8)-beta-Sug-(1-3)-alpha-L-FucNAm-(1-3)-alpha-D-QuiNAc -(1-     

Identification of a novel sugar 5,7-diacetamido-8-amino-3,5,7,8,9-pentadeoxy-D-glycero-D-galacto-non-2-ulosonic acid present in the lipooligosaccharide of Vibrio parahaemolyticus O3:K6

A novel sugar, 5,7-diacetamido-8-amino-3,5,7,8,9-pentadeoxy-d-glycero-d-galacto-non-2-ulosonic acid (NonlA), has been identified as a component of the oligosaccharide (OS) isolated from the lipooligosaccharide (LOS) of the emerging strain of Vibrio parahaemolyticus O3:K6 associated with a global pandemic. In the present study we report the identification and characterization of this novel sugar present in the OS of V. parahaemolyticus O3:K6, using chemical analysis, NMR spectroscopy and mass spectrometry.     

Structure of the phenol-soluble polysaccharide from Shewanella putrefaciens strain A6

The structure of the phenol-soluble polysaccharide from Shewanella putrefaciens strain A6 has been elucidated. Chemical modifications of the polymer in conjunction with 1H and 13C NMR spectroscopy, including 2D techniques, were employed in the analysis. It is concluded that the repeating unit is composed of two nine-carbon sugars as follows: -->4)-alpha-NonpA-(2-->3)-beta-Sugp-(1--> where alpha-NonpA is 5-acetamido-7-acetamidino-8-O-acetyl-3,5,7,9-tetradeoxy-L-glycero-alpha-D-galacto-non-2-ulosonic acid (8eLeg) and beta-Sugp is 2-acetamido-2,6-dideoxy-4-C-(3'-carboxamide-2',2'-dihydroxypropyl)-beta-D-galactopyranose, with the proposed name Shewanellose (She).     

Structural analysis of the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides

A structural investigation has been carried out on the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides (LPS) isolated by dephosphorylation, O-deacylation and N-deacylation. The carbohydrate backbone is a short-chain saccharide consisting of nine monosaccharide units i.e., 1 mol each of D-galactose (Gal), D-glucose (Glc), D-glucuronic acid (GlcA), L-glycero-D-manno-heptose (L,D-Hep), D-glycero-D-manno-heptose (D,D-Hep), 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (NonlA), and 2 mol of 2-amino-2-deoxy-D-glucose (D-glucosamine, GlcN). Based on the data obtained by NMR spectroscopy, fast-atom bombardment mass spectrometry (FABMS) and methylation analysis, a structure was elucidated for the carbohydrate backbone of O2 LPS. In the native O2 LPS, the 2-amino-2-deoxy-D-glucitol (GlcN-ol) at the reducing end of the nonasaccharide is present as GlcN. The lipid A backbone is a beta-D-GlcN-(1-->6)-D-GlcN disaccharide as is the case for many Gram-negative bacterial LPS. The lipid A proximal Kdo is substituted by the distal part of the carbohydrate chain at position-5. In the native O2 LPS, D-galacturonic acid, which is liberated from LPS by mild acid treatment or by dephosphorylation in hydrofluoric acid, is present although its binding position is unknown at present.     

Teichulosonic acid,an anionic polymer of a new class from the cell wall of <Emphasis Type="Italic">Actinoplanes utahensis</Emphasis> VKM Ac-674<Superscript>T</Superscript>

The cell wall of Actinoplanes utahensis VKM Ac-674^T contains two anionic polymers: teichoic acid 1,3-poly(glycerol phosphate) that is widespread in cell walls of Gram-positive bacteria; and a unique teichulosonic acid belonging to a new class of bioglycans described only in microorganisms of the Actinomycetales order. The latter polymer contains residues of di-N-acyl derivative of sialic acid-like monosaccharide — 5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-β-L-manno-non-2-ulosonic or pseudaminic acid (Pse) which bears the N-(3,4-dihydroxybutanoyl) group (Dhb) at C7. This polymer has irregular structure and consists of fragments of two types, which differ in substitution of the Dhb residues at O4 either with β-D-glucopyranose or with β-Pse residues. Most of the β-Pse residues (∼80%) are glycosylated at position 4 with α-D-galactopyranose residues in both types of fragments. The glucose, galactose, and Dhb residues are partly O-acetylated. The structures of the polymers were established by chemical and NMR spectroscopy methods.     

Structural and genetic characterization of the O-antigen of Escherichia coli O161 containing a derivative of a higher acidic diamino sugar, legionaminic acid

The O-antigen is an essential component of lipopolysaccharide on the surface of Gram-negative bacteria and plays an important role in its pathogenicity. Composition and structure of the O-antigens of Escherichia coli are highly diverse mainly due to genetic variations in the O-antigen gene cluster. In this work, the chemical structure and the gene cluster of the O-antigen of E. coli O161 were studied. Chemical degradations, sugar analyses, and NMR spectroscopy showed that the O161 antigen possesses a trisaccharide O-repeating unit containing a 5-N-acetyl-7-N-(d-alanyl) derivative of 5,7-diamino-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulosonic (legionaminic) acid (Leg5Ac7Ala) and having the following structure:

→8)-α-Legp5Ac7Ala-(2→4)-β-d-GlcpA-(1→3)-β-d-GlcpNAc-(1→     

Protein Glycosylation in Helicobacter pylori: Beyond the Flagellins?

Glycosylation of flagellins by pseudaminic acid is required for virulence in Helicobacter pylori. We demonstrate that, in H. pylori, glycosylation extends to proteins other than flagellins and to sugars other than pseudaminic acid. Several candidate glycoproteins distinct from the flagellins were detected via ProQ-emerald staining and DIG- or biotin- hydrazide labeling of the soluble and outer membrane fractions of wild-type H. pylori, suggesting that protein glycosylation is not limited to the flagellins. DIG-hydrazide labeling of proteins from pseudaminic acid biosynthesis pathway mutants showed that the glycosylation of some glycoproteins is not dependent on the pseudaminic acid glycosylation pathway, indicating the existence of a novel glycosylation pathway. Fractions enriched in glycoprotein candidates by ion exchange chromatography were used to extract the sugars by acid hydrolysis. High performance anion exchange chromatography with pulsed amperometric detection revealed characteristic monosaccharide peaks in these extracts. The monosaccharides were then identified by LC-ESI-MS/MS. The spectra are consistent with sugars such as 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (Pse5Ac7Ac) previously described on flagellins, 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (Pse5Am7Ac), bacillosamine derivatives and a potential legionaminic acid derivative (Leg5AmNMe7Ac) which were not previously identified in H. pylori. These data open the way to the study of the mechanism and role of protein glycosylation on protein function and virulence in H. pylori.     

Efficient preparation of steroidal 5,7-dienes of high purity.

Protected forms of dehydroepiandrosterone, delta 5 cholenic acid, (25R)-26-hydroxycholesterol and diosgenin were converted to the corresponding delta 5,7 dienes by successive treatment with 1,3-dibromo-5,5-dimethylhydantoin (dibromantin), tetrabutylammonium bromide and tetrabutylammonium fluoride. The crude products, which contained the delta 5,7 species contaminated by minor amounts of the delta 5 and delta 4,6 steroids, were purified by silica gel-AgNO3 chromatography to give the following steroids in approximately 99% purity and at least 50% yield: 3 beta-acetoxyandrosta-5,7-dien-17-one, methyl 3 beta-acetoxychola-5,7-dien-24-oate, (25R)-3 beta,26-diacetoxycholesta-5,7-diene and (25R)-3 beta-acetoxyspirosta-5,7-diene. Analogous treatment of acetate derivatives of pregnenolone and stigmasterol gave 3 beta-acetoxypregna-5,7-dien-20-one and 3 beta-acetoxystigmasta-5,7,22-triene in approximately 50% yield but of lower purity. Full 1H and 13C NMR assignments are given for seven delta 5,7 steroid acetates and the corresponding delta 5 starting materials. Coupling constants for rings A, B and C of delta 5,7 steroids are presented and stereochemical assignments have been made for the following 1H NMR signals: the C-11 protons of delta 5,7 steroids, the C-16 protons of sterols and bile acids, the C-22 and C-23 protons of bile acid esters and the C-28 protons of stigmasterol derivatives.     

5,7-Diamino-5,7,9-trideoxynon-2-ulosonic acid: a novel sugar from a phytopathogenic Pseudomonas lipopolysaccharide

Preliminary results on the structure of a novel sugar from a lipopolysaccharide from Pseudomonas corrugata, a plant pathogenic bacterium whose several aspects of phytopathogenic mechanism are under investigation, are described. This is a 5,7-diamino-5,7,9-trideoxynon-2-ulosonic acid, isolated as an O-glycoside from the Smith degradation of the O-chain. The structure was obtained both with NMR and MS methodologies. To the best of our knowledge, this is the first example of 3-hydroxylated non-2-ulosonic acid.