Isolation and identification of oligomeric procyanidins from Crataegus leaves and flowers

Oligomeric procyanidins were isolated from the leaves and flowers of hawthorn (Crataegus laevigata). A trimer, epicatechin-(4β→8)-epicatechin-(4β→6)-epicatechin, and a pentamer consisting of (−)-epicatechin units linked through C-4β/C-8 bonds have been isolated from hawthorn for the first time, in addition to known procyanidins including dimers B-2, B-4 and B-5, trimers C-1 and epicatechin-(4β→6)-epicatechin-(4β→8)-epicatechin, and tetramer D-1. A fraction containing a hexamer was also found.     

Structure of the serine-containing capsular poly-saccharide K40 antigen from Escherichia coli O8:K40:H9

The structure of the K40 antigenic capsular polysaccharide (K40 antigen) of E. coli O8:K40:H9 was elucidated by determination of the composition, ¹H- and ¹³C-n.m.r. spectroscopy, periodate oxidation and Smith degradation, and methylation analysis. The K40 polysaccharide consists of [(O-β- -glucopyranosyluronic acid)-(1→4)-O-(2-acetamido-2-deoxy-- -glucopyranosyl)-(1→6)-O-(2-acetamido-2-deoxy-- -glucopyranosyl)-(1→4)] repeating units. All of the glucuronic acid residues are substituted amidically with -serine.     

Structure elucidation of the core octasaccharide from Citrobacter PCM 1487 with the aid of 500-MHz, two-dimensional phase-sensitive correlated, relayed-coherence transfer, double-quantum, triple-quantum filtered, and N.O.E. H-n.m.r. spectra

The main features of the primary structure of the octasaccharide, - -Glcp-(1→2)-- -Glcp-(1→2)-[- -GalpNAc- (1→3)]-- -Galp-(1→3)-- -Glcp-(1→3)-[- -Hepp-(1→7)]-- -Hepp-(1→3)-- -Hep, have been determined in the ab initio manner by ¹H-n.m.r. spectroscopy without resorting to biochemical methods of analysis. Several nontypical interresidue n.O.e. values point to a preferred solution conformation of the molecule.     

Structure of hardwood glucuronoxylans: modifications and impact on pulp retention during wood kraft pulping

The behaviour of different hardwood glucuronoxylans during the kraft pulping process was investigated. Woods and pulps xylans were isolated and characterized by size exclusion chromatography, methylation (linkage) analysis and ¹H NMR. Eucalyptus globulus and Eucalyptus urograndis showed xylan retention significantly higher than that of Betula pendula. The higher retention of Eucalyptus xylans was assigned to (i) their higher average molecular weight (31 against 24 KDa in B. pendula) and to (ii) the presence of O-2 substituted 4-O-methyl--d-glucuronic acid groups ([→2)-GlcpA-(1→]) with galactopyranosyl/glucopyranosyl residues belonging to fragments of galactan/glucan chains that were absent in B. pendula xylans. A significant part of uronic acids, particularly [→2)-GlcpA-(1→] units, remain in fibres until the end of pulping. The acetylation degree and distribution of acetyl groups between Xylp units, in general terms, was similar in the three types of xylans. Unexpectedly, about 20% of the acetyl groups persisted in pulps xylans till the end of pulping.     

Structural characterization of cold extracted fraction of soluble sulfated polysaccharide from red seaweed Gracilaria birdiae

Water soluble polysaccharide from Gracilaria birdiae cultivated along the northeast coast of Brazil was characterized by infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy. The composition of the polysaccharide in wt% was determined as: β-d-galp (50.3%), 3,6-anhydro--l-galp (40.5%) and --l-galp-6 sulfate (9.2%). The ratio of l/d units (β-d-galp units and 3,6-anhydro--l-galp + -l-galp-6 sulfate) is that of an ideal agarose. The sulfate content calculated by S% accounts for 6.4%. 1D and 2D NMR techniques were employed in order to assign the spin system of polysaccharide without partial degradation. The structure is composed of → 4-3,6-anhydro--l-galp (1 → 3)β-d-galp 1 → segments, with the possibility of a -l-galp unit substituted at the 6-position by sulfate ester.     

Pustulan and branched β-galactofuranan from the phytopathogenic fungus Guignardia citricarpa, excreted from media containing glucose and sucrose

Guignardia citricarpa is a phytopathogenic fungus and the causal agent of citrus black spot. Incubation in a semi-defined media resulted in formation of exopolysaccharides [EPS(s)]. A medium containing glucose gave rise to a (1→6)-linked β-glucan (200 kD), pustulan, which was characterized by NMR and methylation analysis. A sucrose-containing medium provided a homogalactan (376 kD) and methylation analysis showed nonreducing end- (20%), 6-O- (53%) and 5,6-di-O-substituted Galf units (27%). An HMQC spectrum of the homogalactan showed C-1/H-1 signals at δ 108.2/4.820, 108.3/4.820 and 107.1/5.079, corresponding to three types of β- -Galf units. A DEPT analysis showed inverted signals (CH₂) at δ 67.8 and 67.2, corresponding to 6-O-substituted β- -Galf units, whereas a C-5 signal at δ 77.0 suggests 5-O-substitution, confirming a novel structure for a β-galactofuranan.     

An unusual juxtaposition of polysaccharide components of Collema leptosporum

The lichenised ascomycete, Collema leptosporum Malme, was extracted with aqueous methanol to give traces of mannitol and 3-O-β- -glucopyranosyl- -mannitol (2.7% yield). The residue was consecutively extracted with hot water to give a complex uronic acid-containing polysaccharide, and then with hot aqueous alkali which provided a mixture of polysaccharides. This was fractionated with Cetavlon to give a branched galactomannan, which had the lowest content of galactose yet reported for such a lichen polysaccharide. It has a main chain of (1→6)-linked -Manp units partly substituted at O-2,4 by non-reducing end-units of Manp and Galp, shown by NMR spectroscopy to have - and β-configurations, respectively. The other polysaccharide component was unexpectedly a branched (1→3), (1→6)-linked β-glucan, which is typical of a basidiomycete, whereas those of ascomycetes contain similar linkages but in linear glucans.     

Kinetics and substrate specificity of the glycanase activity associated with particles of Klebsiella bacteriophage No. 13

There is a glycanase activity associated with the particles of Klebsiella bacteriophage No. 13 which catalyzes hydrolysis of O-β-d-glucopyranosyl-(1 → 4)-β-d-mannopyranose linkages in Klebsiella serotype 13 capsular polysaccharide. The initial glycanase reaction velocity (at 37° and the optimum pH of 7–8) exhibits hyperbolical dependence on low concentrations of substrate, with K_m = 170 μm (in repeating units), and V_max = 140 nmol/min (glucose equivalents, for 10¹⁰ plaque-forming units). The reaction is inhibited by reaction products (type-13 penta- and decasaccharides, one and two repeating-units), and also by the substrate at concentrations above 1mm. Of seventy-four heterologous Klebsiella, and two other bacterial capsular heteropolysaccharides, as well as two substrate derivatives tested, only the Klebsiella serotype-2, -22, and -37 glycans were also depolymerized by the phage-13 enzyme. The reacting polysaccharides consist of repeating units, characteristically containing (a) chain 3eq,1eq → 4eq,1eq dihexopyranosyl sequences around the susceptible linkages, with equatorial hydroxyl and hydroxymethyl groups at C-2 and C′-5 next to them, and (b) branch carboxyl groups that may have to be located within a certain distance from these linkages.     

Structures of the galactomannans from seeds of Annona muricata, Arenga saccharifera, Cocos nucifera, Convolvulus tricolor, and Sophora japonica

Galactomannans were isolated from ripe seeds of Annona muricata (Annonaceae), Convolvulus tricolor (Convolvulaceae), Sophora japonica (Leguminosae), and from immature seeds of Arenga saccharifera and Cocos nucifera (both Palmae). Their sugar compositions were determined and their structures studied by the methylation and periodate-oxidation techniques. All the galactomannans studied are of the leguminous type, the molecules having main chains consisting of (1→4)-linked β- -mannose residues, with differing proportions of side chains consisting of single - -galactose residues linked to the main chains by (1→6)-bonds. The molecular weights were found to vary from 6,000 (Sophora) to 17,000 (Arenga). The isolation of the galactomannan of Annona is the first recorded occurrence of this type of polysaccharide in the family Annonaceae, whereas there has been a previous report of the occurrence of a galactomannan in the Convolvulaceae; the study of the structure of the Sophora galactomannan is the first one in the tribe Sophoreae of the Leguminosae.     

The structure of a polysaccharide isolated from Inonotus levis P. Karst. mushroom (Heterobasidiomycetes)

Inonotus levis biomass was extracted with 5% NaOH containing NaBH₄, the insoluble material was discarded and the solution dialyzed. It was further treated with proteinase and the polymeric fraction isolated by gel chromatography. It contained mostly a polysaccharide of the following structure:

β-GlcA-(1→2)--Gal-(1→6)--Gal3OMe-(1→[→6)--Gal-(1→6)--Gal3OMe-(1→]_5–10

where a non-reducing terminal glucuronic acid residue was present in about half of the molecules, making thus some of the chains acidic and others neutral. Methyl groups were present at about half of the galactose residues, however, no direct proof of the presence of defined repeating units was obtained due to NMR signals overlap. We believe that these short polymeric chains might be originally attached to a protein via serine or threonine residues and were cleaved off due to the alkaline conditions of extraction. Another polymer, co-extracted with this galactan, was a branched phosphorylated mannan with a structure similar to that of the mannan from Saccharomyces cerevisiae yeast.     

Structure and physicochemical properties of barley non-starch polysaccharides—II. Alkaliextractable β-glucans and arabinoxylans

Three fractions containing hemicellulosic material were obtained by sequential extraction of barley residue (left after removal of water-extractable polysaccharides) with saturated barium hydroxide [Ba(OH)₂ fraction], distilled water [Ba(OH)₂/H₂O fraction], and 1 m sodium hydroxide [NaOH fraction]. The yields of the fractions were 1.6, 1.7, and 2.6% (w/w), respectively, of the dry barley grist. The Ba(OH)₂ fraction contained mainly arabinose and xylose, 35.8% and 60.9%, respectively. The Ba(OH)₂/H₂O fraction in addition to 26.7% Ara and 36.6% Xyl contained also 34.8% Glc. The NaOH fraction was composed of 14.2% Ara, 44.0% Xyl, and 40.9% Glc. The Ba(OH)₂/H₂O and NaOH extracts were further fractionated by stepwise (NH₄)₂SO₄ precipitation into several subfractions with varying amounts of β-glucans and arabinoxylans. β-Glucans in Ba(OH)₂/H₂O and NaOH fractions were characterized by high ratios of β-(1→4)/β-(1→3) linkages, large amounts of contiguously linked β-(1→4) segments, and high ratios of cellotriosyl/cellotetraosyl units. The alkali-extractable arabinoxylans, especially those NaOH-extractable, were characterized by a very low degree of substitution, high xylose/arabinose ratio, and a small content of doubly substituted xylose residues. Some populations of arabinoxylans displayed structural features that would enable them to self-associate or to interact with β-glucans.     

Uronic acid-containing glycopeptides from Fusarium oxysporum: Possible significance as chemotypes

Hot aqueous extraction of mycelia of Fusarium oxysporum, followed by fractionation on an anionic resin column gave glycopeptides FL-2 and FL-3. Methylation analysis and 1D and 2D NMR data demonstrated β-d-Manp units and partial hydrolysis gave -d-GlcpA(1→2)-d-Gal, arising from β-d-Galf-containing groups. Both are chemotaxonomic markers of Fusarium spp. FL-3 contained 2,6-di-O-substituted Manp, as well as 2,6-di-O-substituted Galf units, raising the possibility that the former are main-chain constituents, as well as the expected latter structure. The carbohydrate structures of FL-2 and FL-3 differ from those of previously examined polysaccharides of Fusarium spp., which are in turn all different from each other, so that they can serve as fingerprints. Possible variations in their main-chain structures can occur as well as those of their side-chains.     

Structural characterization and antioxidant activity of a polysaccharide from the fruiting bodies of cultured Cordyceps militaris

The water-soluble crude polysaccharides were obtained from the fruiting bodies of cultured Cordyceps militaris by hot water extraction followed by ethanol precipitation. The polysaccharides were successively purified by chromatography on DEAE–cellulose-52 and Sephacryl S-100 HR columns, giving main three polysaccharide fractions termed P50-1, P70-1, and P70-2. Structural features of P70-1 were investigated by a combination of chemical and instrumental analysis, such as partial acid hydrolysis, methylation analysis, periodate oxidation – Smith degradation, GC–MS, ¹³C NMR, HPAEC-PAD, and FT-IR. The results indicated that P70-1 has a backbone of (1 → 6)-linked β-d-mannopyranosyl residues, which occasionally branches at O-3. The branches were mainly composed of (1 → 4)-linked -d-glucopyranosyl and (1 → 6)-linked β-d-galactopyranosyl residues, and terminated with β-d-galactopyranosyl residues and -d-glucopyranosyl residues. In the in vitro antioxidant assay, P70-1 was found to possess hydroxyl radical-scavenging activity with an IC₅₀ value of 0.548 mg/ml.     

Enzymatic transglycosylation of xylose using a glycosynthase

The application of the hyperactive glycosynthase derived from Agrobacterium sp. β-glucosidase (AbgE358G-2F6) to the synthesis of xylo-oligosaccharides by using -d-xylopyranosyl fluoride as donor represents the first successful use of glycosynthase technology for xylosyl transfer. Transfer to p-nitrophenyl β-d-glucopyranoside yields di- and trisaccharide products with β-(1→4) linkages in 63% and 35% yields, respectively. By contrast, transfer to p-nitrophenyl β-d-xylopyranoside yielded the β-(1→3) linked disaccharide and β-d-Xyl-(1→4)-β-d-Xyl-(1→3)-β-d-Xyl-pNP as major products in 42% and 30% yields, respectively. Transfer of xylose to β-d-Xyl-(1→4)-β-d-Xyl-pNP yielded the β-(1→4) linked trisaccharide in 98% yield, thereby indicating that transfers to xylo-disaccharides occur with formation of β-(1→4) bonds. Xylosylation of carbamate-protected deoxyxylonojirimycin produced a mixture of di- and tri-‘saccharide’ products in modest yields.     

Polysaccharides from the green seaweeds Codium fragile and C. vermilara with controversial effects on hemostasis

Codium fragile and Codium vermilara biosynthesize water-soluble sulfated arabinans and galactans (and/or sulfated arabinogalactans), (1 → 4)-d-glucans and β(1 → 4)-d-mannans. The former polysaccharides are composed by 3-linked β-d-galactopyranose and β-l-arabinopyranose residues, they are highly sulfated and substituted with pyruvic acid ketals. For both seaweeds, they have the same main structural units, but in different percentages. All the room-temperature water extracts from both seaweeds showed a dual haemostatic effect: they prevented coagulation, but they induced platelet aggregation. Anticoagulant activity and platelet aggregation were higher in the samples with polysaccharides richer in sulfate, mainly in those from C. vermilara, which have a higher degree of sulfation and arabinose content.     

Crystallinity and polysaccharide chains of beta-glucan in white sorghum, SK(5912).

The polysaccharide chains and the crystallinity of β-glucan in a white sorghum variety, SK₅₉₁₂ were investigated using chemical and enzymic studies. Mild periodate oxidation and methylation, coupled to descending paper chromatography of products revealed the presence of unresolved non-carbohydrate moiety, 2, 4-and 2, 3-di-O-methyl -glucose residues (molar ratio; 18:3) and 2, 4, 6-and 2, 3, 6-tri-O-methyl -glucose residues (molar ratio; 1:14). Paper chromatography of the total acid hydrolysate also revealed a non-carbohydrate spot, identified as protein on the basis of positive Biuret and ninhydrin tests. The O-methyl -glucose residues suggest two polysaccharide chains designated X and Y. Chain X is formed through linking of β- -glucopyranosyl residues by (1→3) linkages with 85–86% (1→6) bonds at branch points and constitute about 6–7% of the β-glucan sample. Chain Y, which is 93–94% of the β-glucan polysaccharide chains, constitutes β- -glucopyranosyl residues in (1→4) linkages and 4–5% (1→6) bonds at branch points. Of the 18 branch points on the X-chains in a given β-glucan sample, about 15 are the Y chains interlinked to the X-chains through their (Y-chains) reducing ends. Both acid and enzyme hydrolyses of the β-glucan suggest two structural organizations, a crystalline and less crystalline granules, based on two first order kinetics. This was correlated by the progress curves obtained during hydrolysis with two purified isoforms of β-glucanases from the sorghum malt. The short and highly branched polysaccharide chains, and longer but less branched polysaccharide chains found in this β-glucan are reminiscent of the structures of amylopectin and amylose, respectively. The K_ms of 0.30–0.32 and 0.42–0.50 mg β-glucan/ml for the β-glucanase isoforms also lay credence to both the crystalline forms and the highly polymerised nature of the β-glucan in white sorghum.     

Assignment of the H-N.M.R. spectra of heparin and heparan sulphate

Resonances from the main repeating unit of heparan, →4)-β- -GlcA-(1→4)-- -GlcNAc-(1→, have been assigned by using a sample of the capsular polysaccharide of E. coli K5. Comparison of the spectra of heparan sulphate samples before and after O- and/or N-desulphation, with re-N-acetylation or re-N-sulphation, allowed assignment of some of the H-1 doublets in terms of sequence effects. Chemical shifts for H-1 of unsulphated uronic acid residues are influenced by 6-sulphation of the nearest neighbor GlcN on the reducing side; those of GlcN residues vary according to whether they have IdoA or GlcA as the nearest neighbour on the reducing side. The H-1 doublets due to residues in the binding sequence for antithrombin have been assigned by comparison of the spectra of heparins having high and low affinities for immobilised antithrombin.     

Capsular and extracellular polysaccharides from Rhizobium microsymbionts of Acacia decurrens

The capsular polysaccharide produced by a Rhizobium isolated from a root nodule of Acacia decurrens is composed of 3-O-methyl- -rhamnose: -rhamnose: - mannose: -glucose: -galacturonic acid in the molar ratios of 1:2:2:4:1. The extracellular polysaccharide is similarly constituted. Structural analyses indicate a decasaccharide repeating-unit in which the -rhamnosyl groups occur as single-unit side-chains. The 3-O-methyl- -rhamnosyl and one of the α- -rhamnosyl groups are (1→6)-linked to two of the -glucosyl residues. The other α- -rhamnosyl group is (1→4)-linked to the -galacturonic acid residue. The main-chain residues are all (1→3)-linked, and are partially identified as -(1→3)-α- -GalpA-(1→3)-α- -Manp- (1→3)-α- -Glcp-(1→3)-.     

Further structural studies of an anti-complementary acidic heteroglycan from the leaves of Artemisia princeps

Partial acid hydrolysis of the anti-complementary acidic heteroglycan, AAFIIb-3, isolated from the leaves of Artemisia princeps PAMP gave the oligosaccharides Gal-(1→6)-Gal, Gal-(1→6)-Gal-(1→6)-Gal, GalA-(1→4)-Rha, GalA-(1→2)-Rha, GlcA-(1→4)-Gal, GlcA-(1→4)-Rha, GlcA-(1→6)-Gal, and GlcA-(1→4)-Xyl. On methylation of AAFIIb-3 without de-esterification, 4-linked and 3,6-disubstituted galactan, 3-linked galactan, 4-linked galactan, and branched arabinan-rich fragments were obtained. The results of base-catalysed β-elimination indicated that AAFIIb-3 has a backbone consisting of 4-linked GalA and 2-linked Rha to which a highly branched arabino-3,6-galactan and arabino-4-galactan are linked at positions 4 of some 2-linked Rha units. Xyl-(1→4)-GalA, GlcA-(1→4)-Xyl-GalA, and →3)-Gal-(1→4)-GalA might also be joined to other 2-linked Rha at the same position. Some 6-linked and 4-linked Gal were terminated by GlcA.     

Structural studies of gum arabic, the exudate polysaccharide from Acacia senegal

A 25.182-MHz ¹³C-n.m.r. spectrum of gum arabic allows unambiguous characterisation of all the C-1 resonances. These assignments have been confirmed by correlation of the modification of the intensities of these signals after controlled acid hydrolysis and characterisation of the released fragments. The resonances of the other carbons have been assigned through partial relaxed T₁ spectra of the polysaccharides obtained by graded degradation of the gum. These results indicate gum arabic to consist mainly of a (1→3)-β- -galactan core with (1→6)-β- -galacto-pyranosyl branches and with - -arabinofuranosyl-(1→3)-- -arabinofuranosyl and - -rhamnopyranosyl-(1→4)-β- -glucopyranosyluronic acid groups attached to positions 3 and 6, respectively, of the branch units.