Coupling of polysaccharides activated by means of chloroacetaldehyde dimethyl acetal to amines or proteins by reductive amination

A novel method has been developed for the coupling of modified polysaccharides to proteins or other amines. Chloroacetaldehyde dimethyl acetal has been used for the introduction of O-(2,2-dimethoxyethyl) groups into amylose, dextran, and a linear (1→3)-β-d-glucan. In amylose and the (1→3)-β-d-glucan, these groups were attached preponderantly at O-6 and in dextran at O-2. Mild treatment with acid then gave polysaccharide derivatives substituted with aldehyde groups which were coupled in good yields to proteins and other amines by reductive amination with sodium cyanoborohydride in aqueous solution at pH 7. An aminated (1→3)-β-d-glucan derivative that induced antitumor activity in mouse macrophages in vitro is reported.     

Structural features of immunologically active polysaccharides from Ganoderma lucidum.

Three polysaccharides, two heteroglycans (PL-1 and PL-4) and one glucan (PL-3), were solubilized from the fruit bodies of Ganoderma lucidum and isolated by anion-exchange and gel-filtration chromatography. Their structural features were elucidated by glycosyl residue and glycosyl linkage composition analyses, partial acid hydrolysis, acetolysis, periodate oxidation, 1D and 2D NMR spectroscopy, and ESI-MS experiments. The data obtained indicated that PL-1 had a backbone consisting of 1,4-linked alpha-D-glucopyranosyl residues and 1,6-linked beta-D-galactopyranosyl residues with branches at O-6 of glucose residues and O-2 of galactose residues, composed of terminal glucose, 1,6-linked glucosyl residues and terminal rhamnose. PL-3 was a highly branched glucan composed of 1,3-linked beta-D-glucopyranosyl residues substituted at O-6 with 1,6-linked glucosyl residues. PL-4 was comprised of 1,3-, 1,4-, 1,6-linked beta-D-glucopyranosyl residues and 1,6-linked beta-D-mannopyranosyl residues. These polysaccharides enhanced the proliferation of T- and B-lymphocytes in vitro to varying contents and PL-1 exhibited an immune-stimulating activity in mice.     

The system of xylogalactans from the red seaweed Jania rubens (Corallinales, Rhodophyta)

The main acidic polysaccharides from the red seaweed Jania rubens share the general characteristics of corallinans (agar-like xylogalactans). After fractionation by ion-exchange chromatography, ten fractions were separated and characterized by sugar composition, other components, methylation, ethylation, desulfation-methylation, and NMR analyses. The main group of fractions carry the agaran disaccharidic repeating unit [-->3)-beta-d-Gal-(1-->4)-alpha-l-Gal-(1-->] substituted mainly on O-6 of the beta-d-Gal unit by beta-xylosyl side stubs, and less with sulfate or methoxyl groups, and also on O-2 of the alpha-l-Gal unit with methoxyl or sulfate, or less on O-3 of the same unit with methoxyl groups. These features are somehow common to the four members of the order already studied. However, a sugar uncommon to the order appears in moderate proportions for all the fractions: it is 3,6-anhydro-l-galactose (partly sulfated or methoxylated on O-2) replacing the l-Gal unit. Besides, several other structural features never found in the order (and uncommon in any polysaccharide) appear in some minor fractions: the presence of side stubs of 2,3-di- and 3-O-methyl-d-galactose, and also part of the 3-O-methyl-l-galactose acting as side stubs. These results show that, although the main features of the corallinean xylogalactans are common to all the species studied, each one has minor characteristics of its own.     

Three exopolysaccharides of the beta-(1-->6)-D-glucan type and a beta-(1-->3;1-->6)-D-glucan produced by strains of Botryosphaeria rhodina isolated from rotting tropical fruit

Four exopolysaccharides (EPS) obtained from Botryosphaeria rhodina strains isolated from rotting tropical fruit (graviola, mango, pinha, and orange) grown on sucrose were purified on Sepharose CL-4B. Total acid hydrolysis of each EPS yielded only glucose. Data from methylation analysis and (13)C NMR spectroscopy indicated that the EPS from the graviola isolate consisted of a main chain of glucopyranosyl (1-->3) linkages substituted at O-6 as shown in the putative structure below: [carbohydrate structure: see text]. The EPS of the other fungal isolates consisted of a linear chain of (1-->6)-linked glucopyranosyl residues of the following structure: [carbohydrate structure: see text]. FTIR spectra showed one band at 891 cm(-1), and (13)C NMR spectroscopy showed that all glucosidic linkages were of the beta-configuration. Dye-inclusion studies with Congo Red indicated that each EPS existed in a triple-helix conformational state. beta-(1-->6)-d-Glucans produced as exocellular polysaccharides by fungi are uncommon.     

Evidence of the presence of 2-O-beta-D-xylopyranosyl-alpha-l-arabinofuranose side chains in barley husk arabinoxylan

(Glucurono)arabinoxylans were extracted from barley husks and degraded with endo-beta-xylanase or subjected to periodate oxidation. The released oligosaccharide fragments were separated and isolated on Biogel-P2, and their structures were determined by NMR spectroscopy. The oligosaccharides identified consisted of beta-d-(1-->4)-linked xylopyranosyl residues, of which some were substituted at O-3 with alpha-l-arabinofuranosyl groups or at O-2 with 4-O-methylglucuronic acid. In addition to these substituents, a disaccharide side chain, 2-O-beta-d-xylopyranosyl-alpha-l-arabinofuranose, attached at position O-3 of the main chain, was proved to exist in arabinoxylan from barley husks. The compound was fully characterized with NMR, and all (1)H and (13)C NMR signals were assigned. The arabinose to xylose ratio was low (approximately 0.2) and no 2,3-disubstitution existed. No blocks of substituted xylose residues could be observed along the main chain.     

Structural characterization of a 2-O-acetylglucomannan from Dendrobium officinale stem

A heteropolysaccharide obtained from an aqueous extract of dried stem of Dendrobium officinale Kimura and Migo by anion-exchange chromatography and gel-permeation chromatography, was investigated by chemical techniques and NMR spectroscopy, and is demonstrated to be a 2-O-acetylglucomannan, composed of mannose, glucose, and arabinose in 40.2:8.4:1 molar ratios. It has a backbone of (1-->4)-linked beta-d-mannopyranosyl residues and beta-d-glucopyranosyl residues, with branches at O-6 consisting of terminal and (1-->3)-linked Manp, (1-->3)-linked Glcp, and a small proportion of arabinofuranosyl residues at the terminal position. The acetyl groups are substituted at O-2 of (1-->4)-linked Manp and Glcp. The main repeating unit of the polysaccharides is reported.     

漆斑霉属(丝孢纲):3新种、1新变种暨中国土壤中已知漆斑霉属真菌分种(变种)检索表

报道分离自中国土壤的漆斑霉属Myrothecium的3个新种:杆状漆斑霉M.bacilliforme、二形孢漆斑霉M.biforme、大孢漆斑霉M.macrosporum,和1个新变种:外来漆斑霉土栖变种M.advena var.terricola,对它们作了较详细的形态描述、图解和讨论。文后列出了中国土壤中12个已知漆斑霉分种(变种)检索表。模式种和所有研究过的标本(干制培养物)及活菌种保存在山东农业大学植物病理学标本室(HSAUP)。等模式标本(干制培养物)存放在中国科学院菌物标本馆(HMAS)。     

The O18 antigens (lipopolysaccharides) of Escherichia coli. Structural characterization of the O18A, O18A1, O18B and O18B1-specific polysaccharides.

The O-specific polysaccharide moieties (PS) of the O18A, O18A1, O18B, and O18B1 antigens (lipopolysaccharides, LPS) consist of L-rhamnose (Rha), N-acetyl-D-glucosamine, D-galactose, and D-glucose in different molar ratios. By using chemical fragmentation, methylation, as well as one- and two-dimensional NMR spectroscopy, the structures of these polysaccharides were found to be [formula: see text] In O18A-PS and O18A1-PS x = 2, whereas in O18B-PS and in O18B11-PS x = 3. In all four polysaccharides alpha-D-Galp (residue D) is substituted at O-3. This substituent L (residue E) is beta-D-GlcpNAc-(1 in O18A-PS and O18A1-PS and it is alpha-D-Glcp-(1 in O18B-PS and O18B1-PS. Whereas there is no further substituent on the main chain of the O18A and O18B polysaccharides, in O18A1-PS and O18B1-PS the alpha-D-GlcpNAc residue A is substituted with alpha-Glcp-(1 (residue F), which is linked to O-6 in O18A1-PS and to O-4 in O18B1-PS. These results show that the O18 antigen comprises a group of four related LPS (O18A and O18B, with their glucosylated forms O18A1 and O18B1). The results are discussed with respect to epitope definition and biochemical implications.     

Configuration of the acetal carbon atom of pyruvic acid acetals in some bacterial polysaccharides

The configuration at the acetal carbon atom of pyruvic acid acetals present in some extracellular bacterial polysaccharides has been investigated. Assignment of the absolute configuration was made by comparing signals in the ¹³C- and ¹H-n.m.r. spectra of the polysaccharides with those of model substances. The S-configuration was demonstrated in eight polysaccharides in which pyruvic acid is linked to O-4 and O-6 of D-glucopyranosyl or D-mannopyranosyl residues. The R-configuration was demonstrated in four polysaccharides in which pyruvic acid is linked to O-4 and O-6 of D-galactopyranosyl residues. Consequently, in each of these acetals, which form 1,3-dioxane rings, the methyl group is equatorial and the carboxyl group axial. The S-form was further demonstrated in four polysaccharides in which the pyruvic acid is linked to O-3 and O-4 of D-galactopyranosyl groups.     

Structural study of the extracellular polysaccharide gum from Rhizobium strain CB744

The structure of the extracellular polysaccharide gum from nitrogen-fixing Rhizobium sp. strain CB744 (a member of the slow-growing Cowpea group) has been investigated. Gas-chromatographic analysis of the alditol acetates of the acid hydrolysate showed the gum to be composed of galactose, 4-O-methylgalactose, mannose, and glucose in the molar ratio of 1:2.5:3.5:7.0. The polysaccharide is unusual in that it contains no carbonyl substituent, although such substituents are common amongst polysaccharides produced by the slow-growing group. The native and de-branched polysaccharides were examined by methylation analysis. The anomeric configurations were determined by ¹³C-n.m.r. and oxidation by chromium trioxide. It is concluded that there are two β-(1→4)-linked glycopyranosyl residues for each α-(1→4)-linked mannopyranosyl residue, and that each mannose is substituted at O-6 by a β-galactopyranosyl residue, with 71% of the galactose groups being present as 4-O-methylgalactose.     

The structure of the neutral polysaccharide gum secreted by Rhizobium strain cb744

A previous investigation of the structure of the extracellular polysaccharide gum from the nitrogen-fixing Rhizobium strain cb744 (a member of the slow-growing Cowpea group) indicated that there were two β-(1→4)-linked d-glucopyranosyl residues for each α-(1→4)-linked d-mannopyranosyl residue, and that each mannose was substituted at O-6 by a β-d-galactopyranosyl residue having 71% of the galactose present as 4-O-methylgalactose. The present study shows that, although the gum appeared to have a simple tetrasaccharide repeating unit, it is composed of two closely associated components. One is a (1→4)-linked α-d-mannan substituted at each O-6 by a β-d-galactopyranosyl residue (71% 4-O-methylated). The second component is a (1→4)-linked β-d-glucan. The existence of the two polysaccharides was established by separation of the β-d-galactosidase-treated gum on a column of concanavalin A-Sepharose 4B. The d configurations were determined and the anomeric attribution of the linkages confirmed by the use of enzymes. The interaction between the two gum components is discussed.     

Structural data for the carbohydrate of ispaghula husk ex Plantago ovata forsk

The husk from the seeds of Plantago ovata Forsk yielded two fractions when exposed to mild aikali, namely, the mucilage polysaccharide (85%, apparently a single species) and the non-polysaccharide component (15%). Methylation analysis and partial hydrolysis with acid showed the mucilage polysaccharide to be a highly branched, acidic arabinoxylan, the xylan backbone having both (1→4) and (1→3) linkages. The majority of the residues in the xylan backbone are variously substituted at O-2 and O-3 with arabinose, xylose, and an aldobiouronic acid identified as 2-O-(galactopyranosyluronic acid)-rhamnose. A structure incorporating these features for the husk polysaccharide is proposed.     

Xyloglucan from the leaves of Hymenaea courbaril

A fucosylated xyloglucan was isolated from the leaves of Hymenaea courbaril by alkaline extraction, followed by ethanol precipitation and ion-exchange chromatography. The isolated polysaccharide showed Glc:Xyl:Gal:Fuc in molar ratio of 8:5:2.5:1 and (D)(25) +40.5 degrees. Composition and linkage analyses, supported by NMR spectroscopic measurements, showed that the polysaccharide has a glucan backbone which is highly substituted at O-6 with D-xylopyranose residues, about a half of which are substituted at O-2 by D-galactopyranosyl units. Some of the galactose residues are further substituted by L-fucopyranose at O-2. The M(r), as determined by HPSEC, was 49,500.     

Polysaccharides from the fruit bodies of the basidiomycete Laetiporus sulphureus (Bull.: Fr.) Murr

The two main polysaccharides from the basidiomycetous fungus Laetiporus sulphureus were isolated, purified and characterized. The structural assignments were carried out using (13)C, (1)H, and (1)H,(13) HSQC nuclear magnetic resonance spectroscopy, methylation analysis, and Smith degradation. One was a linear beta-glucan having a (1-->3)-linked main chain, namely laminaran. The other was a fucomannogalactan, which consisted of a main chain of (1-->6)-linked alpha-D-galactopyranosyl residues, a part of them being substituted at O-2 by 3-O-D-mannopyranosyl-L-fucopyranosyl, alpha-D-mannopyranosyl and in a minor proportion, alpha-L-fucopyranosyl groups. This heteropolysaccharide is related to those of other Basidiomycetes heterogalactans, although it differs distinctly in its side-chain structures. Whereas part of the single-unit L-fucopyranosyl and/or 3-O-alpha-mannopyranosyl-L-fucopyranosyl residues are present as side chains of the other heterogalactans, additional alpha-D-mannopyranosyl units are present in our fucomannogalactan of L. sulphureus.     

Comparative studies of the polysaccharides from species of the genus Ramalina-lichenized fungi-of three distinct habitats

Several structurally different glucans (alpha- and beta-) and galactomannans were characterized as components of four species of the genus Ramalina, namely R. dendriscoides, R. fraxinea, R. gracilis and R. peruviana. Freeze-thawing treatment of hot aqueous extracts furnished as precipitates (PW) linear alpha-D-glucans of the nigeran type, with regularly distributed (1-->3)- and (1-->4)-linkages in a 1:1 ratio. The supernatants (SW) contained alpha-D-glucans with (1-->3)- and (1-->4)-linkages in a molar ratio of 3:1. The lichen residues were then extracted with 2% aq. KOH, and the resulting extracts submitted to the freeze-thawing treatment, giving rise to precipitates (PK2) of a mixture of alpha-glucan (nigeran) and beta-glucan, which were suspended in aqueous 0.5% NaOH at 50 degrees C, dissolving preferentially the beta-glucan. These were linear with (1-->3)-linkages (laminaran). The mother liquor of the KOH extractions (2% and 10% aq. KOH) was treated with Fehling's solution to give precipitates (galactomannans). The galactomannans are related, having (1-->6)-linked alpha-D-mannopyranosyl main chains, substituted at O-4 and in a small proportion at O-2,4 by beta-D-galactopyranosyl units. Despite the different habitats of these lichenized fungi, all species studied in this investigation have a similar pool of polysaccharides.     

Structure of a highly substituted beta-xylan of the gum exudate of the palm Livistona chinensis (Chinese fan)

Structural features of the acidic, highly substituted glycanoxylan (LCP; 87% yield) from the gum exudate of the palm, Livistona chinensis, family Arecaceae, were determined. It had [alpha]D -30 degrees, Mw 1.9x10(5) and a polydispersity ratio Mw/Mn of approximately 1.0. Acid hydrolysis gave rise to Rha, Fuc, Ara, Xyl, and Gal, in a 1:6:46:44:3 molar ratio, and 12% of uronic acid was present. LCP had a highly branched structure with side-chains containing nonreducing end-units (% values are approximate) of Araf (15%), Fucp (4%), Xylp (7%), GlcpA, and 4-Me-GlcpA, and internal 2-O- (5%) and 3-O-substituted Araf (8%), and 2-O-substituted Xylp (14%) units. The (1-->4)-linked beta-Xylp main-chain units of LCP were substituted at O-3 (4%), O-2 (17%), and O-2,3 (16%). Partial acid hydrolysis gave 4-Me-alpha-GlcpA-(1-->2)-[beta-Xylp-(1-->4)](0-2)-Xyl, identified by showing that the uronic acids were single-unit side-chain substituents on O-2. Milder hydrolysis conditions removed from O-3 other side-chains containing Fucp and Araf nonreducing end-units and internal Arap, and 2-O- and 3-O-substituted Araf units. Carboxyl-reduced LCP contained 4-O-methylglucose and glucose in a 3.2:1 molar ratio, arising from GlcpA and 4-OMe-GlcpA nonreducing end-units, respectively. The gum contained small amounts of free alpha-Fucp-(1-->2)-Ara, which corresponds to structures in the polysaccharide. Free myo- and D- or L-chiro-inositol were present in a 9:1 ratio.     

Structural studies on water-soluble arabinoxylans in rye grain using enzymatic hydrolysis

The major water-soluble arabinoxylan fraction from rye grain, containing 4-linked β- -xylopyranosyl residues of which about 43% were substituted solely at O-3 and 7% at both O-2 and O-3 with terminal - -arabinofuranosyl units, was hydrolysed to different extents using semi-purified xylanase from Trichoderma reesei. Products were fractionated on Biogel P-2 and structurally elucidated by sugar, methylation and high-field ¹H-NMR analysis. Moderate hydrolysis released arabinose, xylose, xylobiose, xylotriose and xylotetraose together with xylo-oligosaccharides (DP ≥ 4) in which one or more of the residues were substituted at O-3 with a terminal arabinose unit. The xylose residues substituted with arabinose units at both O-2 and O-3 became enriched in the remaining polymeric fraction. Extensive hydrolysis with the enzyme released arabinose, xylose and xylobiose as major products together with small amounts of two oligosaccharides and a polymeric fraction. One of the oligosaccharides was identified as xylotriose in which the non-reducing end was substituted at O-2 and O-3 with terminal arabinose units and the other as xylotetraose in which one of the interjacent residues was substituted with arabinose units in the same way. The polymeric fraction contained a main chain of 4-linked xylose residues in which 60–70% of the residues were substituted at both O-2 and O-3 with arabinose units.

The semi-purified enzyme contained xylanase and arabinosidase activities which rapidly degraded un- and mono-substituted xylose residues while the degradation of double-substituted xylose residues was much slower. The results show that the mono- and double-substituted xylose residues were present in different polymers or different regions of the same polymer.     

Fragmentation analysis of extracellular acid polysaccharides from seven Rhizobium strains. Part I. D-glucuronic acid-containing oligosaccharides.

The extracellular, bacterial polysaccharides from seven Rhizobium strains have been submitted to partial hydrolysis with acid. Several neutral oligosaccharides, some containing pyruvic acid, were isolated together with D-glucuronic acid-containing oligosaccharides. The polysaccharide from Rh. meliloti did not contain glucuronic acid. For the other six strains, the following components were characterized: 4-O-(beta-D-glucopyranosyluronic acid)-D-glucuronic acid, 4-O-(beta-D-glucopyranosyluronic acid)-D-glucose, and O-(beta-D-glucopyranosyluronic acid)-(1leads to4)-O-(beta-D-glucopyranosyluronic acid)-(1leads to4)-D-glucose. These results indicate the presence of chains containing two beta-(1leads to4)-linked D-glucuronic acid residues, beta-linked to D-glucose at position 4.     

The structures of the cell wall teichoic acids from the thermophilic microorganism Geobacillus thermoleovorans strain Fango

The structures of two teichoic acid fractions (TA1 and TA2) isolated from the thermophilic gram-positive bacterium Geobacillus thermoleovorans strain Fango were investigated by means of chemical and NMR spectroscopic methods. The most abundant species (TA1) exhibited a rather regular structure comprising two different repeating units of 1,3-glycerol phosphate nonstoichiometrically substituted by terminal-alpha-D-Gal p (t-alpha-D-Gal p). The second molecular species (TA2) presented a higher structural variability and t-alpha-D-Glc p and the disaccharides t-alpha-D-Glc pNAc-(1-->2)-alpha-D-Glc p and t-alpha-D-Glc pNAc-(1-->3)-alpha-D-Glc p were also present as minor substituents at O-2 of the glycerol phosphate residues. Minor substitution by alanine could also be detected.     

Structure of a highly pyruvylated galactan sulfate from the Pacific green alga Codium yezoense (Bryopsidales, Chlorophyta)

A polysaccharide fraction consisting of d-galactose, sulfate, and pyruvate in a molar proportion of 4:2:1 was isolated from the green seaweed Codium yezoense by water extraction followed by ion-exchange chromatography. To elucidate its structure, modified polysaccharides were prepared by desulfation, depyruvylation, and by total removal of non-carbohydrate substituents. Structures of the native polysaccharide and of the products of its chemical modifications were investigated by methylation analysis as well as by 1D and 2D (1)H and (13)C NMR spectroscopy. The polysaccharide devoid of sulfate and pyruvate was subjected to two subsequent Smith degradations to afford a rather low-molecular and essentially linear (1-->3)-beta-d-galactan. A highly ramified structure was suggested for the native polysaccharide, which contains linear backbone segments of 3-linked beta-d-galactopyranose residues connected by (1-->6) linkages, about 40% of 3-linked residues being additionally substituted at C-6, probably by short oligosaccharide residues also containing (1-->3) and (1-->6) linkages. Sulfate groups were found mainly at C-4 and in minor amounts at C-6. Pyruvate was found to form mainly five-membered cyclic ketals with O-3 and O-4 of the non-reducing terminal galactose residues. The minor part of pyruvate forms six-membered cyclic ketals with O-4 and O-6. The absolute configurations of ketals (R for six-membered ketals and S for five-membered ones) were established using NMR spectral data.