On the phosphate linkages and the structure of a disaccharide unit of the type-specific polysaccharide of pneumococcus type XIX

The structure of the capsular polysaccharide (S-XIX) of Pneumococcus Type XIX, which contains residues of d-glucose, l-rhamnose, 2-acetamido-2-deoxy- d-mannose, and phosphate, has been investigated by acid hydrolysis, treatment with acid phosphatase, mass spectrometry, and ¹³C-n.m.r. spectroscopy. Phosphoric esters in S-XIX were largely resistant to hydrolysis (4M HCl, 100°, 3 h). With M or 2M HCl at 100° for 3 h, 4-O-(2-amino-2-deoxy-β-d-mannopyranosyl)-d-glucose 4′-phosphate was liberated. More-drastic hydrolysis of S-XIX gave 2-amino-2-deoxy-d-mannose 3-, 4-, and 6-phosphates, and 4-O-(2-amino-2-deoxy-d-mannopyranosyl)-d-glucose and its 4′-phosphate.     

Synthese der “repeating unit” der O-spezifischen kette des lipopolysaccharides aus Aeromonas salmonicida

8-Methoxycarbonyloctyl 2-azido-4,6-O-benzylidene-2-deoxy-β-d-mannopyranoside reacted with 2,3,4-tri-O-acetyl-α-l-rhamnopyranosyl bromide to give a disaccharide from the which the glycosyl-acceptor 8-methoxycarbonyloctyl 2-azido-4,6-O-benzylidene-2-deoxy-3-O-(2,4,-di-O-acetyl-α-l-rhamnopyranosyl)-β-d-manno pyranoside (19) was obtained. This glycosyl-acceptor with 2,3,4,6-tetra-O-benzyl-α-d-glucopyranosyl chloride to give trisaccharide derivative 22 and with 2,3,6-tri-O-(α-²H₂)benzyl-4-O-(2,3,4,6-tetra-O-(α-²H₂)benzyl-α-d-glucopyranosyl)-α-d-glucopyranosyl chloride to give tetrasaccharide derivative 29. Deblocking of 22 yielded 8-methoxycarbonyloctyl O-(α-d-glucopyranosyl)-(1→3)-O-α-l-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-d-mannopyranoside and deblocking of 29 8-methoxycarbonyloctyle O-α-d-glucopyranosyl-(1→4)-O-α-d-glucopyranosyl-(1→3)-O-α-l-rhamnopyranosyl- (1→3)-2-acetamido-2-deoxy-β-d-mannopyranoside. Both oligosaccharides represent the “repeating unit” of the O-specific chain of the lipopolysaccharide from Aeromonas salmonicida.     

An extracellular fungal polysaccharide composed of 2-acetamido-2-deoxy-d-glucuronic acid residues

The black yeast-like fungus NRRL YB-4163, now tentatively identified as Rhinocladiella elatior Mangenot, has been found to produce an extracellular microbial polysaccharide composed mainly of 2-acetamido-2-deoxy-d-glucuronic acid residues. Polysaccharide (PS) YB-4163, when isolated in good yield as the neutral potassium salt, dissolves readily in water to produce extremely viscous solutions, which form stable foams and emulsions. By depolymerizing PS YB-4163 with [¹⁴C]methanol—HCl, the polysaccharide can be both identified and quantitated radiochemically by determining the individual [¹⁴C]methyl glycosides after their separation by paper chromatography. When the methyl glycosides of PS YB-4163 were reduced with NaB³H₄, only the methyl glycosides of 2-acetamido-2-deoxy-d-[6-³H]glucose were found. Analysis of the monosaccharide released from carboxyl-reduced PS YB-4163 by acid hydrolysis or methanolysis also showed 2-acetamido-2-deoxy-d-glucuronic acid to be the main constituent. Previously, the only polysaccharides known to be composed entirely or hexosaminuronic acid have been cellular products from pathogens. Of these, the antigenic polysaccharide (SPSA) from Staphylococcus aureus is composed entirely of 2-amino-2-deoxy-d-glucuronic acid, but its amino groups are substituted equally with acetyl and N-acetylalanyl groups. The specific optical rotation of PS YB-4163,

75° (c 0.5, water), is similar to that of SPSA (?91°), and suggests β-d-linkages that must be either (1→3) or (1→4).     

Structure of the O-polysaccharides of the lipopolysaccharides of Mesorhizobium loti HAMBI 1148 and Mesorhizobium amorphae ATCC 19655 containing two O-methylated monosaccharides

The O-polysaccharide of Mesorhizobium loti HAMBI 1148 was obtained by mild acid degradation of the lipopolysaccharide and studied by sugar and methylation analyses, Smith degradation, and ¹H and ¹³C NMR spectroscopies, including 2D ¹H/¹H COSY, TOCSY, ROESY, and H-detected ¹H/¹³C HSQC experiments. The O-polysaccharide was found to have a branched hexasaccharide-repeating unit of the following structure:where 2-acetamido-2-deoxy-4-O-methyl-d-glucose (d-GlcNAc4Me) and methyl group on 2-substituted d-rhamnose (Me) shown in italics are present in ∼80% and ∼40% repeating units, respectively. Similar studies of the O-polysaccharide from Mesorhizobium amorphae ATCC 19655 by sugar analysis and NMR spectroscopy revealed essentially the same structure but a higher content of 3-O-methyl-d-rhamnose (∼70%).     

Synthesis and immunoadjuvant activities of the repeating,disaccharide-dipeptide unit of the bacterial,cell-wall peptidoglycan and of some carbohydrate analogs

The repeating disaccharide-dipeptide units of the bacterial, cell-wall peptidoglycan, one being O-(N-acetyl-β-muramoyl-l-alanyl-d-isoglutamine)-(1→4)-2-acetamido-2-deoxy-d-glucose, and the other, O-(2-acetamido-2-deoxy-β-d-glucosyl)-(1→4)-N-acetyl-muramoyl-l-alanyl-d-isoglutamine, have been synthesized. Some carbohydrate analogs, such as O-(N-acetyl-β-muramoyl-l-alanyl-d-isoglutamine)- (1→4)-N-acetylmuramoyl-l-alanyl-d-isoglutamine, O-β-d-glucosyl-(1→4)-N-acetylmuramoyl-l-alanyl-d-isoglutamine, and O-(6-acetamido-6-deoxy-β-d-glucosyl)-(1→4)-N-acetylmuramoyl-l-alanyl-d-isoglutamine, were also synthesized. Their immunoadjuvant activities were examined in guinea-pigs.     

Structure of the extracellular polysaccharide produced by the bacterium Alcaligenes (ATCC 31555) species

The extracellular anionic polysaccharide produced by the bacterium Alcaligenes (ATCC 31555) contains l-mannose, l-rhamnose, d-glucose, and d-glucuronic acid in the molar ratios 1.0:4.5:3.1:2.3. Analysis of the methylated and methylated, carboxyl-reduced polysaccharide indicated terminal non-reducing rhamnose and mannose, (1→4)-linked rhamnose, (1→3)- and (1→3,1→4)-linked glucose, and (1→4)-linked glucuronic acid to be present in the ratios 1.0:0.8:2.1:2.2:2.0:2.2. Partial acid hydrolysis and base-catalysed β-elimination gave a series of oligosaccharides that were isolated as their alkylated alditol derivatives by reverse-phase h.p.l.c. and characterised by f.a.b.-m.s., e.i.-m.s., and ¹H-n.m.r. spectroscopy. The repeating unit 1, excluding O-acyl groups, is proposed.

     

Artificial carbohydrate antigens: the synthesis of a tetrasaccharide hapten,a Shigella flexneri O-antigen repeating unit

The 8-methoxycarbonyloctyl glycoside of the tetrasaccharide hapten, O-α-l-rhamnopyranosyl-(1→2)-O-α-l-rhamnopyranosyl-(1→3)-O-α-l-rhamnopyranosyl-(1→ 3)-2-acetamido-2-deoxy-β-d-glucopyranoside and the trisaccharide glycoside 8-methoxycarbonyloctyl O-α-l-rhamnopyranosyl-(1→3)-O-α-l-rhamnopyr-anosyl-(1→3)-2-acetamido-2-deoxy-β-d-glucopyranoside were synthesized by sequential Koenigs-Knorr reactions from monosaccharide units. The tetrasaccharide represents the complete skeletal repeating unit of Shigella flexneri serogroup Y lipopolysaccharide. Both oligosaccharide haptens are functionalized for covalent attachment to proteins, cell surfaces, and solid supports. ¹H-N.m.r. evidence for the conformations of these oligosaccharides in solution is presented and shown to be consistent with predictions based on the exo-anomeric effect     

Cell wall glycopolymers of type strains from three species of the genus <Emphasis Type="Italic">Actinoplanes</Emphasis>

The structures of cell wall glycopolymers from the type strains of three Actinoplanes species were investigated using chemical methods, NMR spectroscopy, and mass spectrometry. Actinoplanes digitatis VKM Ac-649^T contains two phosphate-containing glycopolymers: poly(diglycosyl-1-phosphate) →6)-α-D-GlcpNAc-(1-P-6)-α-D-GlcpN-(1→ and teichoic acid →1)-sn-Gro-(3-P-3)-β-[β-D-GlcpNAc-(1→2]-D-Galp-(1→. Two glycopolymers were identified in A. auranticolor VKM Ac-648^T and A. cyaneus VKM Ac-1095^T: minor polymer–unsubstituted 2,3-poly(glycerol phosphate), widely abundant in actinobacteria (Ac-648^T), and mannan with trisaccharide repeating unit →2)-α-D-Manp-(1→2)-α-D-Manp(1→6)-α-D-Manp-(1→(Ac-1095^T). In addition, both microorganisms contain a teichuronic acid of unique structure containing a pentasaccharide repeating unit with two residues of glucopyranose and three residues of diaminouronic acids in D-manno- and/or D-gluco-configuration. Each of the strains demonstrates peculiarities in the structure of teichuronic acid with respect to the ratio of diaminouronic acids and availability and location of O-methyl groups in glucopyranose residues. All investigated strains contain a unique set of glycopolymers in their cell walls with structures not described earlier for prokaryotes.     

Covalent structure of the extracellular polysaccharide from Xanthomonas campestris: evidence from partial hydrolysis studies.

Partial, acid hydrolysis of the extracellular polysaccharide from Xanthomonas campestris gave products that were identified as cellobiose, 2-O-(β-d-glucopyranosyluronic acid)-d-mannose, O(β-d-glucopyranosyluronic acid)-(1→2)-O-α-d-mannopyranosyl-(1→3)-d-glucose, O-(β-d-glucopyranosyluronic acid)-(1→2)-O-α-d-mannopyranosyl-(1→3)-[O-β-d-glucopyranosyl-(1→4)]-d-glucose, and O-(β-d-glucopyranosyluronic acid)-(1→2)-O-α-d-mannopyranosyl-(1→3)-[O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→4)-d-glucose. This and other evidence supports the following polysaccharide structure (1) which has been proposed independently by Jansson, Kenne, and Lindberg:

     

The structure of the O-antigen in the endotoxin of the emerging food pathogen Cronobacter (Enterobacter) muytjensii strain 3270

Strains of the Gram-negative bacterium Cronobacter (formerly known as Enterobacter) sakazakii have been identified as emerging opportunistic pathogens that can cause enterocolitis, bacteraemia, meningitis, and brain abscess, and they have been particularly associated with meningitis in neonates where infant milk formulae have been epidemiologically linked to the disease. A study of the lipopolysaccharides produced by clinical isolates using chemical, 2D ¹H and ¹³C NMR, and MS methods revealed that the O-polysaccharide produced by Cronobactermuytjensii strain 3270, isolated from powdered infant formula from Denmark, was a linear unbranched polymer of a repeating pentasaccharide unit composed of 2-acetamido-2-deoxy-d-galactose (d-GalNAc), 2-acetamido-2-deoxy-d-glucose (d-GlcNAc), 3-acetamido-3-deoxy-d-quinovose (d-Qui3NAc), l-rhamnose (l-Rha), and d-glucuronic acid (d-GlcA) in equimolar ratio, and has the structureThe specific structural characteristics of the O-polysaccharides of C.muytjensii may be of value in the identification and tracking of the bacterial pathogen.     

Bacterial antigen polysaccharides. 43. Structure for the O-specific polysaccharide of <Emphasis Type="Italic">Providencia alcalifaciens</Emphasis> O46

Acidic O-specific polysaccharide containing D-glucose, D-glucuronic acid, L-fucose, and 2-acetamido-2-deoxy-D-glucose was obtained by mild acid degradation of lipopolysaccharide from Providencia alcalifaciens O46. The following structure of the hexasaccharide repeating unit of the O-specific polysaccharide was established using methylation analysis along with ¹H and ¹³C NMR spectroscopy, including 2D ¹H, ¹H-COSY, TOCSY, ROESY, ¹H, ¹³C-HSQC, and HMQC-TOCSY experiments:

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New versatile phosphorus ligands containing SPNSO skeleton - synthesis and characterization of (SPPh2)(O2SR)NH acids (R=Me, C6H4Me-4) and their alkaline metal salts. Crystal and molecular structures of (SPPh2)(O2SR)NH

New phosphorus ligands of the type (SPPh₂)(O₂SR)NH [R=Me (1), C₆H₄Me-4 (2)] were prepared as white crystalline solids using the reactions between Li[HN(S)PPh₂] and RSO₂Cl. They were easily converted into their alkali metal salts, M[(SPPh₂)(O₂SR)N] (M=Li, Na, K). Both the free acids and their alkali metal salts were characterised by multinuclear (¹H, ¹³C, ³¹P) NMR spectroscopy. The molecular structures of the free acids were established by single crystal X-ray diffraction. They crystallize in the tetragonal space group I4₁/a (1) and the triclinic space group P−1 (2), respectively. In both compounds the acidic proton is attached to nitrogen and the molecular units are associated through SO?H-N intermolecular hydrogen bonding [H?O 2.216 in 1 and 2.029 Å in 2]. A supramolecular chain-like structure is formed in 1 and dimeric units are built in 2. For both compounds a conformation close to syn-syn can be considered for the SP(C)₂-N-SC(O)₂ fragment.     

Disaccharide 1-phosphate polymers of some representatives of the Bacillus subtilis group

Disaccharide 1-phosphate polymers as well as teichoic acids of various structures have been found in the cell walls of the representatives of the Bacillus subtilis group, namely Bacillus subtilis subsp. spizizenii VKM B-720 and VKM B-916, B. subtilis VKM B-517, and Bacillus vallismortis VKM B-2653^T. Disaccharide 1-phosphate polymers are composed of repeating units of the following structure: -P-4)-β-D-GlcpNAc-(1→6)-α-D-Galp-(1-, the N-acetylglucosamine residues are partially acetylated at positions O3 and O6 (VKM B-720 and VKM B-916); -P-4)-β-D-Glcp-(1→6)-α-D-GlcpNAc-(1-, the glucopyranose residues are partially acetylated at positions O2 or O3 (VKM B-517); -P-6)-α-D-GlcpNH ₃ ⁺ /α-D-GlcpNAc-(1→2)-α-D-Glcp-(1-, the N-acetylglucosamine residues are partially deacetylated (VKM B-2653^T). The structures of the two last disaccharide 1-phosphate polymers have not been reported so far for Gram-positive bacteria. The teichoic acids in the studied strains are O-D-alanyl-1,5-poly(ribitol phosphates) substituted with β-D-glucopyranose (VKM B-517, VKM B-720, VKM B-916) or 2-acetamido-2-deoxy-β-D-glucopyranose (VKM B-2653^T). The structures of the phosphate-containing polymers have been studied by chemical methods and by NMR spectroscopy.     

Entada saponin-III,a saponin isolated from the bark of Entada phaseoloides

The structure of Entada saponin (ES)-III, one of the main saponins of Entada phaseoloides bark, was established to be 3-O-[β-d-xylopyranosyl (1 → 2)-α-l-arabinopyranosyl (1 → 6)] [β-l-glucopyranosyl (1 → 4)]-2-acetamido-2-deoxy-β-l-glucopyranosyl-28-O-[β-l-apiofuranosyl (1 → 3)-β-d-xylopyranosyl (1 → 2)] [(2-O-acetoxyl)-β-d-glucopyranosyl-(1 → 4)] (6 − O(R) (−)2,6-dimethyl-2-trans-2,7-octadienoyl)-β-d-glucopyranosyl echinocystic acid.     

Isolation and characterization of a heteropolysaccharide from the corm of Amorphophallus campanulatus

A water-soluble polysaccharide isolated from the aqueous extract of the corm of Amorphophallus campanulatus was found to contain d-galactose, d-glucose, 4-O-acyl-d-methyl galacturonate, and l-arabinose in a molar ratio 2:1:1:1. Structural investigation of the polysaccharide was carried out using acid hydrolysis, methylation analysis, periodate oxidation study, and NMR studies (¹H, ¹³C, DQF-COSY, TOCSY, NOESY, ROESY, HMQC, and HMBC). On the basis of the above-mentioned experiments the structure of the repeating unit of the polysaccharide was established as:This molecule showed splenocyte activation.     

Receptors for glucocorticoids in lung tissue

Gas chromatography-mass spectrometric identification of partially methylated aminosugars has been developed: (a) various kinds of O-methylated 2-deoxy-2-(N-methyl)-acetamidohexitols were prepared from partially O-(1-methoxy)-ethylated 2-deoxy-2-acetamidohexoses, and their gas chromatography-mass spectrometric patterns were determined; (b) permethylated glycolipids gave a satisfactory yield of 2-deoxy-2-N-methyl-amidohexoses by acetolysis with 0.5 n sulfuric acid in 95% acetic acid, followed by aqueous hydrolysis; (c) the resulting partially methylated aminosugars and neutral sugars were analyzed after borohydride reduction and acetylation according to the procedure of Lindberg and associates (Björndal, Lindberg and Svennson, 1967; Björndal, Hellerqvist, Lindberg and Svensson, 1970).This method was successfully applied to analysis of aminosugar linkages in blood group B-active ceramide pentasaccharide from rabbit erythrocytes and in Forssman antigen of equine spleen. The structure of blood group B-active glycolipid of rabbit erythrocyte was found to be Galα1 → 3Galβ1 → 4G1cNAcβ1 → 3Ga11 → 4Glc → Cer, and that of Forssman antigen to be GaNAcα1 → 3GalNAcβ1 → 3Galα1 → 4Ga11 → 4Glc → Cer.     

Anionic polymers of the cell wall of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> subsp. <Emphasis Type="Italic">subtilis</Emphasis> VKM B-501<Superscript>T</Superscript>

Teichoic acid and disaccharide-1-phosphate polymer were identified in the cell walls of Bacillus subtilis subsp. subtilis VKM B-501^T. The teichoic acid represents 1,3-poly(glycerol phosphate) 80% substituted by α-D-glucopyranose residues at O-2 of glycerol. The linear repeating unit of disaccharide-1-phosphate polymer contains the residues of β-D-glucopyranose, N-acetyl-α-D-galactosamine, and phosphate and has the following structure: -6)-β-D-Glcp-(1→3)-α-D-GalpNAc-(1-P-. The structures of two anionic polymers were determined by chemical and NMR-spectroscopic methods. The ¹H- and ¹³C-NMR spectral data on disaccharide-1-phosphate polymer are presented for the first time.     

Isolation and characterization of membrane-associated proteoglycans from normal and malignant human mammary epithelial cells

The plasma membrane-associated proteoglycans of a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal cell line (HBL-100). The labeled proteoglycans were isolated from the plasma membranes of cells grown in the presence of [³H]glucosamine and [³⁵S]Na₂SO₄ by extraction with guanidine hydrochloride and subsequently purified by DEAE-ion exchange chromatography. Their structural properties were established by treatment with nitrous acid, heparitinase and chondroitinase ABC, and by gel filtration before and after alkaline -elimination. About 18% of the proteoglycans synthesized by these cell lines were associated with the plasma membranes. The HBL plasma membranes contained 80% heparan sulfate and 20% chondroitin sulfate proteoglycans whereas MDA plasma membranes had 50% heparan sulfate and 50% chondroitin sulfate proteoglycans. The MDA plasma membrane contained two heparan sulfate proteoglycans, both having nearly the same molecular size as the two species secreted into the medium by these cells. The HBL plasma membrane also contained two hydrodynamic size heparan sulfate proteoglycans. The larger hydrodynamic size species has a slightly lower molecular size than that secreted into the medium, and the smaller hydrodynamic size species was not detectable in the medium. Even though the major chondroitin sulfate proteoglycans from MDA plasma membranes were smaller in size than those from HBL plasma membrane, a larger proportion of the glycosaminoglycan chains of the former were bigger than those from the latter.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate - Di-OS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-d-galactose - Di-4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-4-O-sulfo-d-galactose - Di-6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-6-O-sulfo-d-galactose - Gdn-HCl guanidine hydrochloride - WGA wheat germ agglutinin