Structure of an abequose-containing O-polysaccharide from Citrobacter freundii O22 strain PCM 1555

The lipopolysaccharide of Citrobacter freundii O22 (strain PCM 1555) was degraded under mild acidic conditions and the O-polysaccharide released was isolated by gel chromatography. Sugar and methylation analyses along with ¹H and ¹³C NMR spectroscopy, including two-dimensional ¹H,¹H ROESY and ¹H,¹³C HMBC experiments, showed that the repeating unit of the O-polysaccharide has the following structure:

Full-size image (4K)

where Abe is abequose (3,6-dideoxy-d-xylo-hexose). SDS–PAGE and immunoblotting revealed that the O-antigen of C. freundii O22 is serologically indistinguishable from those of Salmonella group B serovars (Typhimurium, Brandenburg, Sandiego, Paratyphi B) but not related to other abequose-containing O-antigens tested (Citrobacter werkmanii O38 and Salmonella Kentucky) or colitose (l enantiomer of abequose)-containing O-antigen of Escherichia coli O111.     

Chemical analysis of a polysaccharide of unripe (green) tomato (Lycopersicon esculentum)

A polysaccharide (PS-I) isolated from the aqueous extract of the unripe (green) tomatoes (Lycopersicon esculentum) consists of d-galactose, d-methyl galacturonate, d-arabinose, l-arabinose, and l-rhamnose. Structural investigation of the polysaccharide was carried out using total acid hydrolysis, methylation analysis, periodate oxidation study, and NMR studies (¹H, ¹³C, DQF-COSY, TOCSY, NOESY, ROESY, HMQC, and HMBC). On the basis of above-mentioned experiments the structure of the repeating unit of the polysaccharide (PS-I) was established as:

Full-size image (8K)

     

Structural studies of the O-antigen polysaccharide from the enteroinvasive Escherichia coli O173

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O173 has been investigated. Sugar and methylation analyses, electrospray ionisation mass spectrometry together with ¹H, ³¹P and ¹³C NMR spectroscopy were the main methods used. The structure of the pentasaccharide repeating unit of the PS was found to be:

By treatment with 48% HF the phosphoric diester linkage was cleaved together with the glycosidic linkage of the fucosyl group, rendering a tetrasaccharide with the structure:

     

New cell wall glycopolymers of the representatives of the genus Kribbella

Each of the cell walls of four representatives of the genus Kribbella (order Actinomycetales; suborder Propionibacterineae; family Nocardioidaceae) contains a neutral polysaccharide and an acidic polysaccharide with unusual structures. Common to all four strains studied is a mannan with the following repeating unit: In the cell wall of the strain VKM Ac-2541, a teichulosonic acid was identified with a monosaccharide component that has not hitherto been found in Gram-positive bacteria, viz., pseudaminic acid, and an unusual linkage type in the polymeric chain,

Full-size image (1K)

where R = Н (45%), α-d-Galp3OMe (37%) or α-d-Galp2,3OMe (18%).The anionic cell wall components of three other strains are represented by teichuronic acids with a rare constituent, viz., a diaminosugar, 2,3-diacetamido-2,3-dideoxyglucopyranose. The structures of their repeating units differ in the nature of the acidic components:→4)-β-d-Manp2,3NAcA-(1→6)-α-d-Glcp2,3NAc-(1→ (VKM Ас-2538 and VKM Ас-2540) and →4)-β-d-ManpNAcA-(1→6)-α-d-Glcp2,3NAc-(1→ (VKM Ас-2539).The structures of all the glycopolymers were established by chemical and NMR spectroscopic methods; they are identified in Gram-positive bacteria for the first time.     

Structural studies of the O-antigenic polysaccharide from Escherichia coli O177

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O177 has been determined. Component analysis together with ¹H and ¹³C NMR spectroscopy experiments was used to determine the structure. Inter-residue correlations were determined by ¹H,¹³C-heteronuclear multiple-bond correlation and ¹H,¹H-NOESY experiments. PS is composed of tetrasaccharide repeating units with the following structure:→2)-α-l-Rhap-(1→3)-α-l-FucpNAc-(1→3)-α-l-FucpNAc-(1→3)-β-d-GlcpNAc-(1→An α-l-Rhap residue is suggested to be present at the terminal part of the polysaccharide, which on average is composed of ∼20 repeating units, since the ¹H and ¹³C chemical shifts of an α-linked rhamnopyranosyl group could be assigned by a combination of 2D NMR spectra. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. The repeating unit of the E. coli O177 O-antigen shares the →3)-α-l-FucpNAc-(1→3)-β-d-GlcpNAc-(1→ structural element with the O-antigen from E. coli O15 and this identity may then explain the reported cross-reactivity between the strains.     

Structural elucidation of the O-antigenic polysaccharide from Escherichia coli O175

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O175 has been elucidated. Component analysis together with ¹H and ¹³C NMR spectroscopy experiments were used to determine the structure. Inter-residue correlations were determined by ¹H,¹H-NOESY, and ¹H,¹³C-heteronuclear multiple-bond correlation experiments. The PS is composed of pentasaccharide repeating units with the following structure:→2)-α-d-Glcp-(1→4)-α-d-GlcpA-(1→3)-α-d-Manp-(1→2)-α-d-Manp-(1→3)-β-d-GalpNAc-(1→Cross-peaks of low intensity from an α-linked glucopyranosyl residue were present in the ¹H,¹H-TOCSY NMR spectra. The α-d-Glcp residue is suggested to originate from the terminal part of the polysaccharide and consequently the biological repeating unit has a 3-substituted N-acetyl-d-galactosamine residue at its reducing end. The repeating unit of the E. coli O175 O-antigen is similar to those from E. coli O22 and O83, both of which carry an α-d-Glcp-(1→4)-d-GlcpA structural element, thereby explaining the reported cross-reactivities between the strains.     

Structural elucidation of an exopolysaccharide from mycelial fermentation of a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis

A novel polysaccharide designated EPS-1A with an average molecular weight around 40 kDa was fractionated and purified by anion-exchange and gel-filtration chromatography from the crude exopolysaccharide (EPS) isolated from fermentation broth of Cs-HK1, a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis. The structural characteristics of EPS-1A were determined with various methods (e.g. GC, GC–MS, FT-IR, ¹H NMR and ¹³C NMR) and through acid hydrolysis, methylation, periodate-oxidation and Smith degradation. The results suggested that EPS-1A was composed of glucose, mannose and galactose at 15.2:3.6:1.0 M ratio. EPS-1A was a slightly branched polysaccharide and its backbone was composed of (1 → 6)-α-d-glucose residues (77%) and (1 → 6)-α-d-mannose residues (23%). Branching occurred at O-3 position of (1 → 6)-α-d-mannose residues of the backbone with (1 → 6)-α-d-mannose residues and (1 → 6)-α-d-glucose residues, and terminated with β-d-galactose residues.     

A new ethanolamine phosphate-containing variant of the O-antigen of Shigella flexneri type 4a

The O-specific polysaccharide (O-antigen) structure of a Shigella flexneri type 4a strain from the Dysentery Reference Laboratory (London, UK) was elucidated in 1978 and its characteristic feature was found to be α-d-glucosylation of GlcNAc at position 6, which defines O-factor IV. Our NMR spectroscopic studies of the O-specific polysaccharides of two other strains belonging to S. flexneri type 4a (G1668 from Adelaide, Australia, and 1359 from Moscow, Russia) confirmed the carbohydrate backbone structure but revealed in both strains an additional component, ethanolamine phosphate (EtnP), attached at position 3 of one of the rhamnose residues:

Full-size image (5K)

Phosphorylation has not been hitherto reported in any S. flexneri O-antigen. Reinvestigation of the O-specific polysaccharide of S. flexneri type 4b showed that it is not phosphorylated and confirmed its structure established earlier.     

Structure of the oligosaccharides isolated from Dalbergia sissoo Roxb. leaf polysaccharide

A water-soluble polysaccharide isolated from Dalbergia sissoo Roxb. leaves was purified and major homogeneous fraction obtained by GPC. Complete hydrolysis of the polysaccharide followed by paper chromatography and GLC analysis indicated the presence of l-rhamnose, d-glucuronic acid, d-galactose and d-glucose in molar ratio of 1:1:2:2.33, respectively. Partial hydrolysis of the polysaccharide furnished one tri-[I], one hepta-[II] and one nona-[III] saccharides. Hydrolysis of the oligosaccharide I, II and III followed by GLC analysis furnished d-glucose and l-rhamnose (2:1); l-rhamnose, d-galactose and d-glucuronic acid (1:3:3); and l-rhamnose, d-galactose and d-glucose (1:3:5), respectively. Methylation analysis and periodate oxidation of the oligosaccharide I indicated the presence of two non reducing glucose units linked to rhamnose by 1→2 and 1→4 linkages, respectively. Oligosaccharide II is a branched molecule with a main chain consisting of 1,3-linked β-d-galactopyranosyl (2 mol), 1,3,4 linked α-l-rhamnopyranosyl (1 mol) and 1,4,6 linked β-d-galactopyranosyl unit (1 mol) and non reducing β-d-glucuronic acid at the end along with side chains of β-d-glucouronopyranosyl units (2 mol). Oligosaccharide III is also a branched molecule with a main chain consisting of 1,3,4 linked α-l-rhamnopyranosyl (1 mol), 1,2,4 linked β-d-glucopyranosyl (1 mol), 1,3 and 1,4 linked β-d-galactopyranosyl (2 and 1 mol, respectively) having β-d-glucopyranosyl as a non reducing end.     

Structural elucidation of the O-antigen of the Shigella flexneri provisional serotype 88-893: structural and serological similarities with S. flexneri provisional serotype Y394 (1c)

The structure of the repeating unit of the O-antigen polysaccharide from Shigella flexneri provisional serotype 88-893 has been determined. ¹H and ¹³C NMR spectroscopy as well as 2D NMR experiments were employed to elucidate the structure. The carbohydrate part of the hexasaccharide repeating unit is identical to the previously elucidated structure of the O-polysaccharide from S. flexneri prov. serotype Y394. The O-antigen of S. flexneri prov. serotype 88-893 carries 0.7 mol O-acetyl group per repeating unit located at O-2 of the 3-substituted rhamnosyl residue, as identified by H2BC and BS-CT-HMBC NMR experiments. The O-antigen polysaccharide is composed of hexasaccharide repeating units with the following structure: →2)-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-α-l-Rhap2Ac-(1→3)[α-d-Glcp-(1→2)-α-d-Glcp-(1→4)]-β-d-GlcpNAc-(1→. Serological studies showed that type antigens for the two provisional serotypes are identical; in addition 88-893 expresses S. flexneri group factor 6 antigen. We propose that provisional serotypes Y394 and 88-893 be designated as two new serotypes 7a and 7b, respectively, in the S. flexneri typing scheme.     

Structural characterization of an acidic exoheteropolysaccharide produced by the nitrogen-fixing bacterium Burkholderia tropica

An acidic exopolysaccharide (EPS) produced by the diazotrophic bacterium Burkholderia tropica, strain Ppe8, was isolated from the culture supernatant of bacteria grown in a synthetic liquid medium containing mannitol and glutamate. Monosaccharide composition showed Rha, Glc and GlcA in a 2.0:2.0:1.0 molar ratio, respectively. Further structural characterization was performed by a combination of NMR, mass spectrometry and chemical methods. Partial acid hydrolysis of EPS provided a mixture of acidic oligosaccharides that were characterized by ESI-MS, giving rise to ions with m/z 193 (GlcA-H)⁻, 339 (GlcA,Rha-H)⁻, 501 (GlcA,Rha,Glc-H)⁻, 647 (GlcA,Rha₂,Glc,-H)⁻, 809 (GlcA,Rha₂,Glc₂,-H)⁻ and 851 (GlcA,Rha₂,Glc₂,OAc-H)⁻. Carboxyreduced EPS (EPS-CR) had Glc and Rha in a 3:2 ratio, present as d- and l-enantiomers, respectively. Methylation and NMR analysis of EPS and EPS-CR showed a main chain containing 2,4-di-O-Rhap, 3-O-Rhap and 4-O-Glcp. A GlcA side chain unit was found in the acidic EPS, substituting O-4 of α-l-Rhap units. This was observed as a non-reducing end unit of glucopyranose in the EPS-CR. Acetyl esters occured at O-2 of β-l-Rhap units. From the combined results herein, we determined the structure of the exocellular polysaccharide produced by B. tropica, Ppe8, as being a pentasaccharide repeating unit as shown:

Full-size image (8K)

     

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 94/D4 and the international type strain Escherichia coli O82

The structure of the O-antigen polysaccharides (PS) from the enteroaggregative Escherichia coli strain 94/D4 and the international type strain E. coli O82 have been determined. Component analysis and ¹H, ¹³C, and ³¹P NMR spectroscopy experiments were employed to elucidate the structure. Inter-residue correlations were determined by ¹H, ¹³C-heteronuclear multiple-bond correlation, and ¹H, ¹H-NOESY experiments. d-GroA as a substituent is linked via its O-2 in a phosphodiester-linkage to O-6 of the α-d-Glcp residue. The PS is composed of tetrasaccharide repeating units with the following structure:→4)-α-d-Glcp6-(P-2-d-GroA)-(1→4)-β-d-Galp-(1→4)-β-d-Glcp-(1→3)-β-d-GlcpNAc-(1→Cross-peaks of low intensity from an α-d-Glcp residue were present in the NMR spectra and spectral analysis indicates that they originate from the terminal residue of the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. Enzyme immunoassay using specific anti-E. coli O82 rabbit sera showed identical reactivity to the LPS of the two strains, in agreement with the structural analysis of their O-antigen polysaccharides.     

Structure of the O-specific polysaccharide of Hafnia alvei PCM 1196

An acidic O-specific polysaccharide was isolated from Hafnia alvei PCM 1196 lipopolysaccharide and studied by sugar and methylation analyses along with one- and two-dimensional ¹H and ¹³C NMR spectroscopy, including NOESY and HMBC experiments. The following structure of the pentasaccharide repeating unit was established:

     

Morphological and structural characterization of a polysaccharide from Gynostemma pentaphyllum Makino and its anti-exercise fatigue activity

The crude polysaccharide was obtained from Gynostemma pentaphyllum Makino by water extraction followed by ethanol precipitation. The polysaccharide was successively purified by chromatography on DEAE-52 and SephadexG-150 column, and three polysaccharide fractions were obtained and termed GPP1-a, GPP2-b, and GPP3-a, respectively. The administration with GPP1-a markedly prolonged exhaustive exercise time of the mice. Structural features of GPP1-a were investigated by a combination of instrumental and chemical analyses, including atomic force microscope (AFM), scanning electron microscope (SEM), partial acid hydrolysis, periodate oxidation, Smith degradation, methylation analysis, gas chromatography–mass spectrometry (GC–MS) analysis and NMR spectroscopy. The results indicate that GPP1-a has a backbone of (1 → 4)-linked α-d-Glucose residues, which occasionally branches at O-6. The branches are mainly composed of (1 → 6)-linked α-d-Glucose, (1 → 3)-linked β-d-Galactose and (1 → 6)-linked α-d-Galactose residues, and terminated with β-d-Galactose residues and β-l-Arabinose residues.     

E versus Z geometry in β-d-arabino-hexopyranosidulose oximes

Koenigs–Knorr-type glycosidations of peracylated 2Z-benzoyloxyimino-glycopyranosyl bromides invariably proceed with retention of the Z-geometry. Accordingly, the many β-d-hexosidulose oximes in literature which were prepared in this way and for which the oxime geometry has not been addressed explicitly, are the Z-oximes throughout. By contrast, oximation of β-d-hexopyranosid-2-uloses leads to mixtures of E and Z oximes readily separable and structurally verifiable by ¹H and ¹³C NMR. Configurational assignments rested on comparative evaluation of NMR data of E and Z isomers, and, most notably on an X-ray structural analysis of the pivaloylated isopropyl 2E-benzoyloxyimino-2-deoxy-β-d-arabino-hexopyranoside revealing the unusual ¹S₅^1,4B conformation for the pyranoid ring.     

Glycosylated nervogenic acid derivatives from Liparis condylobulbon (Reichb.f.) leaves

Three new nervogenic acid glycosides, 1-O-α-l-rhamnopyranosyl 3,5-bis(3-methyl-but-2-enyl)-4-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]-benzoate, 3,5-bis(3-methyl-but-2-enyl)-4-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]-benzoic acid, and bis{3,5-bis(3-methyl-but-2-enyl)-4-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]-benzoyl} 1,2-O-β-d-glucopyranose, which we named condobulbosides A–C, were isolated from a methanol extract of the leaves of Liparis condylobulbon together with an apigenin C-glycoside, schaftoside. Their structures were established on the basis of spectral techniques, namely, UV, IR, HR-MS spectroscopy, both 1D and 2D NMR experiments, and chemical reactions.     

Gene cloning, purification, and characterization of α-methylserine aldolase from Bosea sp. AJ110407 and its applicability for the enzymatic synthesis of α-methyl-l-serine and α-ethyl-l-serine

The gene encoding α-methylserine aldolase was isolated from Bosea sp. AJ110407. Sequence analysis revealed that the predicted amino acid sequence encoded by the 1320-bp open reading frame was 65.0% similar to the corresponding sequence of the enzyme isolated from Ralstonia sp. AJ110405. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. Gel filtration revealed the molecular mass of the purified enzyme to be approximately 78 kDa, suggesting that the enzyme is a homodimer. The enzyme exhibited a specific peak at 429 nm in the spectrum and contained 1 mol pyridoxal 5′-phosphate per mole of the subunit. The V_max value was 1.40 μmol min⁻¹ mg⁻¹, and the K_m value was 1.5 mM for the reaction wherein formaldehyde was released from α-methyl-l-serine. This enzyme could also catalyze the reverse reaction, i.e., the synthesis of α-methyl-l-serine from l-alanine and formaldehyde. This activity was inhibited in the excess of formaldehyde; however, α-methyl-l-serine was efficiently produced from l-alanine in the presence of formaldehyde. This method was also applicable for producing α-ethyl-l-serine from l-2-aminobutyric acid.     

Structural characterization of an active polysaccharide from Phellinus ribis

A water-soluble polysaccharide named as PRP was isolated from the fruiting bodies of Phellinus ribis by hot water extraction, DEAE-cellulose and Superdex 30 column chromatography. Its structural characteristics were investigated by FT-IR, NMR spectroscopy, GLC-MS, methylation analysis, periodate oxidation and Smith degradation. Based on the data obtained, PRP was found to be a β-d-glucan containing a (1 → 4), (1 → 6)-linked backbone, with a single β-d-glucose at the C-3 position of (1 → 6)-linked glucosyl residue every eight residues, along the main chain. The glucan has a weight-average molecular weight of about 8.59 kDa by HPGPC determination using dextran samples as the standards. Preliminary activity tests in vitro revealed that PRP could stimulate the proliferation of spleen lymphocyte.     

Structural characterization of an O-linked tetrasaccharide from Pseudomonas syringae pv. tabaci flagellin

The flagellin of Pseudomonas syringae pv. tabaci is a glycoprotein that contains O-linked oligosaccharides composed of rhamnosyl and 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methylglucosyl residues. These O-linked glycans are released by hydrazinolysis and then labeled at their reducing ends with 2-aminopyridine (PA). A PA-labeled trisaccharide and a PA-labeled tetrasaccharide are isolated by normal-phase high-performance liquid chromatography. These oligosaccharides are structurally characterized using mass spectrometry and NMR spectroscopy. Our data show that P. syringae pv. tabaci flagellin is glycosylated with a tetrasaccharide, 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methyl-Glcp-(1→3)-α-l-Rhap-(1→2)-α-l-Rhap-(1→2)-α-l-Rha-(1→, as well a trisaccharide, 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methyl-Glcp-(1→3)-α-l-Rhap-(1→2)-α-l-Rha-(1→, which was identified in a previous study.     

N-Acetylhexosamine triad in one molecule: Chemoenzymatic introduction of 2-acetamido-2-deoxy-β-d-galactopyranosyluronic acid residue into a complex oligosaccharide

A complex trisaccharide β-d-GalpNAcA-(1 → 4)-β-d-GlcpNAc-(1 → 4)-d-ManpNAc (3) was prepared in a good yield (35%) in a transglycosylation reaction catalyzed by β-N-acetylhexosaminidase from Talaromyces flavus using p-nitrophenyl 2-acetamido-2-deoxy-β-d-galacto-hexodialdo-1,5-pyranoside (1) as a donor followed by the in situ oxidation of the aldehyde functionality by NaClO₂. The disaccharide β-d-GlcpNAc-(1 → 4)-d-ManpNAc (2) was used as galactosyl acceptor. A disaccharide β-d-GalpNAcA-(1 → 4)-d-GlcpNAc (4; 39%) originated as a by-product in the reaction. Oligosaccharides comprising a carboxy moiety at C-6 are shown to be very efficient ligands to natural killer cell activation receptors, particularly to human receptor CD69. Thus, oxidized trisaccharide 3 is the best-known oligosaccharidic ligand to this receptor, with IC₅₀ = 2.5 × 10⁻⁹ M. The presented method of introducing a β-d-GalpNAcA moiety into carbohydrate structures is versatile and can be applied in the synthesis of other complex oligosaccharides.