Structural studies of capsular polysaccharide of type XII Diplococcus pneumoniae

The capsular polysaccharide of Diplococcus pneumoniae Type XII contains residues of d-glucose and d-galactose in a molar ratio of 2:1. The methylated polysaccharide yielded upon hydrolysis 2,3,4,6-tetra- and 3,4,6-tri-O-methyl-d-glucose and 2,3,4,6-tetra-O-methyl-d-galactose as the only neutral methyl sugars. Periodate oxidation of the polysaccharide resulted in destruction of all neutral sugars and immunochemical activity against rabbit antisera. Periodate oxidation of the methyl O-methylglycosides obtained after hydrolysis of the methylated polysaccharide indicated that at least 30% of the l-fucosamine residues are substituted at C-4 in the polysaccharide. It is concluded that the polysaccharide consists of a hexosamine backbone that is substituted by d-galactosyl and kojibiosyl side-chains. The proposed terminal d-galactosyl residues apparently are sterically hindered from interacting with several d-galactose-binding proteins.     

Structural and immunochemical characterization of the acidic arabinomannan of mycobacterium smegmatis

A serologically active, acidic arabinomannan has been isolated from Mycobacterium smegmatis. The polysaccharide contains approximately 56 arabinosyl and 11 mannosyl residues, and 2 phosphate, 6 monoesterified succinate, and 4 ether-linked lactate groups. After saponification to remove succinyl groups, the polysaccharide can be separated into phosphorylated (55%) and nonphosphorylated (45%) forms, the former containing a little more arabinose and a little less mannose than the latter. The structures of these polysaccharides were investigated by ¹H- and ¹³C-n.m.r. spectroscopy and methylation analysis, before and after selective cleavage of furanosyl linkages. The phosphorylated and nonphosphorylated forms of the polysaccharide were found to have similar, if not identical, structures. The main structural feature of the polysaccharides is the presence of chains of contiguous arabinofuranosyl residues linked α-(1→5). These chains are attached at O-4 of arabinopyranosyl residues that are present in a core region of the polysaccharide that also contains mannopyranosyl residues. Immunochemical studies demonstrated that the polysaccharide is an effective, precipitating antigen with antisera from rabbits immunized with cell walls or heat-killed cells of M. smegmatis. The polysaccharide is, however, more effective as a precipitating antigen after removal of the succinate groups, and completely ineffective after removal of arabinofuranosyl residues. The polysaccharide therefore contains an important antigen in common with the arabinogalactan lipopolysaccharide of the cell wall of the bacterium, i.e., chains of contiguous α-(1→5)-linked arabinofuranosyl residues.     

Structural studies of the Rhizobium trifolii extracellular polysaccharide

The structure of the extracellular polysaccharide of Rhizobium trifolii has been investigated. Methylation analysis, sequential degradations by oxidation and elimination of oxidized residues, uronic acid degradation, and degradation by oxidation of the acetylated polysaccharide with chromium trioxide in acetic acid were the main methods used. It is proposed that the polysaccharide is composed of heptasaccharide repeating-units having the following structure:

An unusual feature is that some of the repeating units are incomplete and lack the terminal β-d-galactopyranosyl group. The polysaccharide contains O-acetyl groups (somewhat more than 1 mol. per unit), linked to O-2 and O-3 of 4-O-substituted d-glucopyranosyl chain-residues. A previous finding that the polysaccharide contains 2-deoxy-d-arabino-hexose (2-deoxy-d-glucose) residues is erroneous.     

Modification of polysaccharides containing uronic acid residues

Klebsiella Type 47 capsular polysaccharide has side chains attached to the main chain viaD-glucuronic acid residues. The side chains have been removed to yield an essentially linear polysaccharide by the following sequence of reactions: (1) substitution of hydroxyl and car?yl groups with methyl vinyl ether; (2) β-elimination by treatment with base; (3) removal of modified uronic acid residues and protecting groups by mild acid hydrolysis. The possibility of modifying other uronic acid-containing polysaccharides by this method is discussed.     

Structure and biodegradation of the polysaccharide components of egg masses isolated from snails of an Ampullarius species

A polysaccharide preparation, isolated from egg masses deposited by snails of an Ampullarius species, was purified via precipitation with Cetavlon in the presence of sodium borate, and found to contain d-galactose and a smaller proportion of d-glucose, and to have two components with sedimentation coefficients of 10S and 40S. A polysaccharide, isolated from freshly laid egg masses, was highly branched and was shown to contain nonreducing α-d-glucopyranosyl and β-d-galactopyranosyl end-groups, and 3,6-di-O-substituted β-d-galactopyranosyl residues. One or more of the polysaccharide components was a d-glucopyrano-d-galactopyranan with non-reducing α-d-glucopyranosyl end-groups (1→4)-linked to β-d-galactopyranosyl residues. The polysaccharide preparations, obtained from freshly laid egg masses and from those that were left for 10 and 15 days after being laid, were structurally different from each other. With the passage of time, progressive diminution of the 10S component and the proportion of d-glucose in the polysaccharide took place, suggesting that each constituent was consumed preferentially by the snail embryos as an energy source.     

Structural investigation of the acidic polysaccharide secreted by Zoogloea ramigera 115

Batch-culture growth of Zoogloea ramigera 115 in a defined medium produced a weakly acidic polysaccharide containing glucose and galactose residues, and (S)-pyruvic acetal groups. Analytical results indicated that the polysaccharide does not have a simple repeating-unit. Mainly with the aid of Smith degradation of the native polysaccharide and oxidation and subsequent β-elimination of the methylated and then depyruvylated polysaccharide, some structural features of the polysaccharide were identified.     

Strucrural investigation of Klebsiella serotype K7 polysaccharide

Methylation analysis of and partial hydrolysis studies on the Klebsiella K7 capsular polysaccharide and its carboxyl-reduced derivative indicated the recurrence of D-glucopyranuronic acid, D-mannopyranose, and D-glucopyranose residues, linearly linked in a specific manner, in the molecular structure. D-Galactopyranose and pyruvic acid residues are linked to the main chain on the D-mannose residues (at O-3) and the D-glucose residues (at O-4 and O-6), respectively; the simplest interpretation of this evidence is that nine sugar residues and pyruvic acid constitute a repeating unit. The sequence →3)-β-D-GlcAp-(1→2)-α-D-Manp-(1→2)-α-D-Manp-(1→3)-D-Glcp→ was demonstrated by the isolation from the polysaccharide of an aldotetraouronic acid of this structure.     

Structural studies of a specific polysaccharide isolated from non-agglutinable Vibrio

The purified, specific polysaccharide from Vibrio cholera type NAG, NV 384, O-antigen, 2A, 2Bhuman, contains glucose (5.14%), galactose (4.21%), mannose (64.8%), xylose (3.16%), arabinose (1.98%), fucose (1.50%), mannuronic acid (14.3%), phosphate (0.32%), 2-amino-2-deoxy-D-glucose (2.9%), and 2-amino-2-deoxy-D-galactose (1.0%). Various reactions have shown that the material comprises a phosphoric diester-linked polysaccharide containing mainly (1→2)-linked mannopyranose residues that are highly branched with other sugar residues.     

Structural features of a sulphated,fucose-containing polysaccharide from the brown seaweed dictyota dichotoma

A sulphated heteropolysaccharide (～15% of the acid-extractable material) isolated from the brown alga Dictyota dichotoma contains residues of D-glucuronic acid, D-galactose, D-mannose, D-xylose, and L-fucose¹. Partial hydrolysis of the polysaccharide with acid gave one neutral and two acidic oligosaccharides. The behaviour towards periodate of the polysaccharide before and after partial hydrolysis, alkali-treatment, and methanolysis has been studied. Evidence is thereby provided that the polysaccharide is partially sulphated and composed of (1→4)-linked residues of D-glucuronic acid, D-galactose, D-mannose, and D-xylose, and (1→2)-linked L-fucose.     

Structural studies on an antitumor polysaccharide from Microellobosporia grisea

The structure of the antitumor polysaccharide from the actinomycete Microellobosporia grisea has been investigated. By methylation and periodate-oxidation studies, the polysaccharide was shown to consist of (nonreducing)d-mannosyl groups, (1→4)-linkedd-glucosyl residues, and 3,6-branched, (1→4)-linkedd-glucosyl residues in the approximate molar ratios of 2:1:1. Periodate oxidation of the polysaccharide, followed by borohydride reduction and mild hydrolysis with acid yielded glycerol, erythritol, 2-O-β-d-glucopyranosyl-d-erythritol, and 5-O-β-d-glucopyranosyl-2,4-bis(hydroxymethyl)-1,3-dioxane, which were isolated in the molar ratios of 2.0:0.14:0.74:0.35. Partial hydrolysis of the polysaccharide gave α-d-Man p-(1→6)-d-Glcp, β-d-Glcp-(1→4)-d-Glcp, α-d-Man p-(1→3)-d-Glcp, and β-d-Glcp-(1→4)-[α-d-Man p-(1→3)-]-d-Glcp. From these results, it is proposed that the polysaccharide is mainly composed of tetrasaccharide repeating-units having the following structure.     

Selective cleavage of glycosidic linkages: Studies with the O-specific polysaccharide from Shigella dysenteriae type 3

Treatment of the O-specific polysaccharide from Shigella dysenteriae Type 3 with hydrazine in the presence of hydrazine sulphate resulted in quantitative N-deacetylation with the formation of a modified polysaccharide containing free amino groups. Oxidation of the modified polysaccharide with periodate did not destroy the 2-amino-2-deoxygalactose residues, thus indicating that they were substituted at position 3. Acid hydrolysis of the modified polysaccharide afforded 3-O-(2-amino-2-deoxy-β-D-galactopyranosyl)-D-galactose, which was identified as the N-acetyl derivative. Deamination of the modified polysaccharide with nitrous acid cleaved the 2-amino-2-deoxy-D-galactopyranosyl linkages to give a pentasaccharide as the major product, which appeared to be the modified chemical repeating unit of the O-specific polysaccharide.     

Structural studies of the capsular polysaccharide from streptococcus pneumoniae type 1

The capsular polysaccharide from Streptococcus pneumoniae type 1 is composed of D-galactopyranosyluronic acid residues and 2-acetamido-4-amino-2,4,6-trideoxy-D-galactopyranosyl residues. The latter sugar, previously unknown in Nature, was not isolated but was identified from the products obtained on deamination of the polymer. Using n.m.r. spectroscopy, methylation analysis, and Smith degradation as the principal methods of structural investigation, it is concluded that the polysaccharide is composed of trisaccharide repeating-units having the structure: →3)-α-Sugp-(1→)-α-D-GalpA-(1→3)-α-D-GalpA-(1→, in which Sug denotes the new sugar.     

Structural investigations of the extracellular polysaccharides elaborated by Beijerinckia mobilis

The extracellular mucilage from Beijerinckia mobilis, a member of the Azotobacteriaceae, after removal of contaminating protein, was separated into a neutral polysaccharide (N-2, 10%); a neutral, dialysable fraction (N-1, 5%), consisting of glucose and oligosaccharides containing glucose, arabinose, and rhamnose; and an acidic polysaccharide (85%). N-2 (mol. wt, 1900) was highly branched and comprised glucopyranose, mannopyranose, and arabinofuranose residues (1:1:1). The various linkages were determined. The acid fraction was a polymer of high molecular weight composed of L-guluronic acid (65%), D-glucose (15%), and D-glycero-D-mannoheptose (20%), together with acetic and pyruvic acids. From the results of methylation, periodate oxidation, and partial hydrolysis, a branched molecule with a backbone of guluronic acid and heptose, and side chains of glucose and guluronic acid is proposed. Pyruvic acid was found to be acetal-linked to 2?5% of the heptose residues. The similarities between this polysaccharide and that from the related species Azotobacter indicum are discussed.     

High-resolution H- and C-n.m.r. spectra of the group A-variant streptococcal polysaccharide

The molecular weight of the water-soluble polysaccharide of Phellodendron amurense Ruprecht was found to differ with the sample used. The difference is considered to be due to different degrees of degradation of the polysaccharide chains, together with oxidation of galactose to galacturonic acid residues.     

Bis(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)amine obtained by a fusion reaction

A unique, alkali-soluble polysaccharide has been isolated from the cell walls of the basidiomycete Coprinus macrorhizus microsporus. The polysaccharide, which is primarily a glucan, contains a large proportion of α-(1→4)-linked d-glucose residues and a smaller amount of β-(1→3) and (1→6) linkages, as suggested by methylation, partial acid hydrolysis, periodate oxidation, and enzymic studies. Hydrolysis of the methylated polysaccharide gave equimolar amounts of 2,4-di- and 2,3-di-O-methyl-d-glucose; no 2,6-di-O-methyl-d-glucose was identified, indicating the absence of branch points joined through O-1, O-3, and O-4. The isolation and identification of 2-O-α- glucopyranosylerythritol from the periodate-oxidized polysaccharide suggests that segments of the a-(1→4)-linked d-glucose residues are joined by single (1→3)-linkages. An extracellular enzyme-preparation from Sporotrichum dimorphosporum (QM 806) containing both β-(1→3)- and α-(1→4)-d-glucanohydrolase activity released 76% of the reducing groups from the polysaccharide. The polysaccharide also contains minor proportions of xylose, mannose, 2-amino-2-deoxyglucose, and amino acids.     

Mucilage from a fresh-water red alga of the genus Batrachospermum

The gelatinous polysaccharides of a Batrachospermum species have been extracted from the alga. The major polysaccharide is acidic and has been separated from neutral polysaccharides by chromatography on DEAE-cellulose. The constituent sugars of the acidic polysaccharide include d- and l-galactose, d-mannose, d-xylose, l-rhamnose, d-glucuronic acid, and two O-methyl sugars, which have been characterized as 3-O-methyl-l-rhamnose (l-acofriose and 3-O-methyl-d-galactose. Partial acid hydrolysis of this polysaccharide has given a complex mixture of neutral and acidic oligosaccharides. The two preponderant acidic oligosaccharides contained galactose and glucuronic acid in 1:1 ratio, suggesting the presence of a repeating sequence of these two residues as a major structural feature of the polysaccharide.     

Acylated oligosaccharides from Klebsiella K63 capsular polysaccharide: Depolymerization by partial hydrolysis and by bacteriophage-borne enzymes

The extracellular polysaccharide from Klebsiella K63 is unique in having acetic and formic ester groups attached to the d-galactopyranosyluronic residues in the trisaccharide repeating-sequence. These O-acyl substituents are shown to be some what resistant to mild hydrolysis by both acid and alkali. Bacteriophage-induced depolymerization of the polysaccharide generated a series of acylated oligosaccharides comprising one, or more, repeating unit(s). By mild hydrolysis with acid, the same series of oligomers was released from the polysaccharide, together with the corresponding non-acylated compounds and the expected acylated and non-acylated aldobiouronic acids. A study of these oligosaccharides, as well as of a number of their related compounds, is described, with particular emphasis on the methods used to locate the formic and acetic ester groups. The location of the O-acyl substituents on the galactosyluronic residues was further supported by the results obtained from the high-resolution, 400-MHz, p.m.r. spectra and ¹³C-n.m.r. spectra of a number of the oligosaccharides.     

Methylation studies of the polysaccharides resulting from sequential Smith-degradations of the galactan from the snail strophocheilus oblongus

When the galactan from the albumen glands of the snail Strophocheilus oblongus was submitted to three Smith-degradations, the degraded polysaccharide, isolated in 6% yield, was much more linear. Methylation analysis showed that the Smith-degraded polysaccharide gave an increased percentage of 2,4,6-tri-, decreased percentages of 2,3,4,6-tetra- and 2,4-di-, and a large variation in the amount of 2,3,4-tri-O-methyl-d-galactose. The sugars in the polysaccharide which result in the formation of 2,3,4,6-tetra- and 2,3,4-tri-O-methyl-d-galactose are destroyed in subsequent degradation procedures. The above observations suggest that the degradation by periodate oxidation proceeds via non-reducing end-groups and through some internal residues that are exposed as the degradation proceeds. As a result of the overall process, new non-reducing end-groups are formed and new (1 → 6)-linked d-galactose residues are then exposed. The isolation of glycosides of low molecular weight supports the suggestion that the molecule, in the main, is sequentially degraded from the external layers.     

Host-Pathogen Interactions: VIII. Isolation of a Pathogen-synthesized Fraction Rich in Glucan That Elicits a Defense Response in the Pathogen's Host

A polysaccharide from the fungal pathogen Colletotrichum lindemuthianum causes browning and phytoalexin production when applied to the cut surfaces of bean (Phaseolus vulgaris) cotyledons and hypocotyls. The application of an amount of polysaccharide equivalent to less than 100 ng of glucose will elicit this response in the bean tissues. The polysaccharide has been isolated both from culture filtrates and from the mycelial walls of the fungus. Purification of the polysaccharide involved anion and cation exchange chromatography and gel filtration. The polysaccharide has an apparent molecular weight between 1,000,000 and 5,000,000 daltons, and consists predominantly of 3- and 4-linked glucosyl residues.     

Glycosylation by d-fructofuranose thio-orthoesters

The capsular polysaccharide from Klebsiella type K54, containing both O-formyl and O-acetyl groups, has been investigated by using the techniques of methylation analysis (by gas-liquid chromatography), periodate oxidation-Smith degradation, and both ¹H- and ¹³C-n.m.r. spectroscopy. Degradation of the native polysaccharide with a bacteriophage-induced glucosidase generated a formylated, as well as a formylated and acetylated, tetrasaccharide, whereas similar depolymerization of the deacetylated polysaccharide yielded a single tetrasaccharide; the corresponding, O-acylated octasaccharides were also isolated and characterized. These oligosaccharides, utilized in chemical and spectroscopic studies in order to determine the location of the O-acyl substituents in the repeating sequence, indicated formylation at O-4 of each lateral d-glucosyl group and acetylation at O-2 of alternate l-fucosyl residues. A new structure for the repeating unit in the polysaccharide is proposed.