Pullulans produced by strains of Cryphonectria parasitica—II. Nuclear magnetic resonance evidence

The structure of pullullan-like polysaccharides produced as exocellular material by different strains of Cryphonectria parasitica, the fungus responsible for chestnut tree cankers, was investigated with nuclear magnetic resonance (NMR) techniques. ¹³C, mono- and bidimensional ¹H, and ¹H–¹³C heteronuclear correlated NMR spectra (HSQC and HMBC) were recorded. Advanced analysis of the NMR spectra allowed the main resonance of the atoms in the maltotriose and in the maltotetraose repeat units of pullulan-like polysaccharides from C. parasitica to be recognised with confidence. In all cases investigated, the presence of large amounts of -(1→6) maltotetraose subunits was evidenced, in addition to the -(1→6) maltotriose subunits, corresponding to the repeating unit of pullulan produced by Aureobasidium pullulans and other fungi. The results were in agreement with other data from this laboratory, obtained with independent techniques. The belief that in ‘pullulans’ the maximum amount of -(1→6) maltotetraose subunits is about 7% can thus be considered as definitely outdated.     

Chemical structure and conformational features of cell-wall polysaccharides isolated from Aphanoascus mephitalus and related species

The structures of cell-wall mannans isolated from Aphamoascus mephitalus, A. Fulvescens, A. verrucosus, and A. reticulisporus have been investigated by chemical analyses and 1D and 2D ¹H and ¹³C NMR techniques. It was found that all of them consists of a relatively simple comb-like structure of the disaccharide repeating block {→ 6)-[-Man p-(1 → 2)]--Man p-(1 →}. The conformations around the -(1 → 2) and -(1 → 6) linkages in thes kinds of polymers were also studied by using molecular mechanics and dynamics calculations, together with NOE data. The results are similar to those found within the oligosaccharide chains of glycoproteins, with a well-defined conformation for the -(1 → 2) linkage and a certain restriction around the -(1 → 6) bonding imposed by the 2-substitution.     

Saponins from Albizzia lucida.

Three main saponins were isolated from the seeds of Albizzia lucida. Their structures were established by spectral analyses and chemical and enzymatic transformations as 3-O-[β- -xylopyranosyl(1→2)-- -arabinopyranosyl (1→6)] [β- -glucopyranosyl (1→2)] β- -glucopyranosyl echinocystic acid; 3-O-[- -arabinopyranosyl (1→6)][β- -glucopyranosyl (1→2)]-β- -glucopyranosyl echinocystic acid and 3-O-[β- -xylopyranosyl (1→2)-β- -fucopyranosyl (1→6)-2-acetamido-2-deoxy-β- -glucopyranosyl echinocystic acid, characterized as its methyl ester.     

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.     

Structural investigation of Ceratozamia spinosa mucilage

The polysaccharide fraction from Ceratozamia spinosa appears to be made up mainly by a chemically homogeneous polysaccharide but with a wide range of molecular weight. By NMR and chemical degradative methods, it is shown to consist essentially of a backbone of alternate → 4)-β- -GlcpA-(1 → and → 2)-- -Manp-(1 → units. On the 4 position of the latter, β- -GlcpA residues are linked. End units of - -Ara f, β- -Xylp, - -Rhap, and - -3-OMe-Rhap are linked to C-3 and/or C-4 positions of β- -Glc pA residues.     

Observations on the structure of pullulan

An extracellular α-glucan, pullulan, elaborated by a strain of Pullularia pullulans, contains 0.6% of a maltotetraose subunit, as well as the already known major component, maltotriose. The majority, at least, of the maltotetraose is contained within the polymeric chains and is linked, through its terminal glucose units, by α-1→6— bonds.     

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.     

Structure of the phosphopeptidomannans from flocculent and non-flocculent yeast Kluyveromyces lactis

After extraction from whole cells, and purification by gel filtration, the chemical composition and molecular mass estimation of the cell-wall phosphopeptidomannan (PPM) showed no significant difference respectively between flocculent, weakly, very weakly and non-flocculent Kluyveromyces lactis yeast strains. However, when PPMs were tested as ligands of a lectin, extracted from the flocculent strain, the PPM isolated from the flocculent and weakly flocculent strain were recognized to a higher degree than those isolated from the non and very weakly flocculent strains. Acetolysis of PPM extracted from the four strains produced five oligosaccharide fractions corresponding to mono-, di-, tri-, penta-and hexa-saccharides. The flocculent strain was characterised by a high content of di-and penta-saccharides. The ¹H NMR analysis of the oligosaccharides demonstrated that the flocculent strain contained equivalent levels of the two mannobioses: Man( 1 → 2)Man and Man( 1 → 3)Man and of the two mannotrioses Man( 1 → 2)Man( 1 → 2)Man and Man( 1 → 3)Man( 1 → 2)Man. In contrast, the non-flocculent and the very weakly flocculent strains contained a single type of mannobiose Man( 1 → 2)Man and one type of mannotriose Man( 1 → 2)Man( 1 → 2)Man.     

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.     

Cyclic (1 → 2)-β-d-glucans excreted by the glucuronan-producing strain Rhizobium meffloti M5N1CS CS (NCIM B 40472) and by the succinoglycan-producing strain Rhizobium meliloti M5N1

During fermentation, the mutant strain Rhizobium mefliloti M5N1 CS, which induces nodule formation on alfalfa roots, produces a partially acetylated (1 → 4)-β-d-glucuronan. In addition to this exopolysaccharide of high molecular weight, the mutant strain produces oligoglucoronates and cyclic (1 → 2)-β-d-glucans with degrees of polymerization from 17 to 30. Under the conditions applied, magnesium has no effect on cyclic glucan production by the mutant strain, but the succinoglycan production by the wild-type strain Rhizobium meliloti M5N1 increases.     

Purification and determination of the action pattern of Haliotis tuberculata laminarinase

The major laminarinase activity (EC 3.2.1.39) from the gastropodean marine mollusc Haliotis tuberculata was purified to homogeneity by cation exchange chromatography and its action pattern was investigated by HPAEC-PAD analysis of the degradation of various laminarin samples. It consists of a 60 kDa protein capable of depolymerizing the unbranched portions of the β-(1→3), β-(1→6)-glucan, down to laminaritriose. The enzyme operates via a molecular mechanism retaining the anomeric configuration. As the purified protein does not cleave the β-(1→6) linkages, it can be used for the structural analysis of laminarins.     

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.     

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 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.     

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.     

Triterpenoidal saponins from Dumasia Truncata

Extraction of the aerial parts of Dumasia truncata Sieb et Zucc. afforded two new triterpenoidal saponins, together with four known ones. The structures of the new compounds were elucidated by spectral analysis as 3-O--l-rhamnopyranosyl-(1 → 3)-β-d-glucuronopyranosy-28-O-β-d-glucopyransoyl hederagenin and 3-O-β-d-xylopyranosyl-(1 → 2)-[-l-rhamnopyranosyl(1 → 3)]-β-d-glucuronopyranosyl oleanic acid.     

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.     

A spirostane hexaglycoside from Agave cantala fruits.

A new steroidal glycoside, agaveside D, isolated from the fruits of Agave cantata was characterized as 3β-{- -rhamnopyranosyl-(1→2), β- -glycopyranosyl-(1→3)-β- -glucopyranosyl[β- -xylopyransoyl-(1→4)-- -rhamnopyranosyl-(1→2)]-β- -glucopyranosyl}-25R-5- spirostane on the basis of chemical degradation and spectrometry.     

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.     

Cycloartane triterpene glycosides from Astragalus trigonus

Three new cycloartane glycosides, trigonoside I, II and III, and the known astragalosides I and II were isolated from the roots of Astragalus trigonus. The structures of the new glycosides were totally elucidated by high field (600 MHz) NMR analyses as cycloastragenol-6-O-β-xylopyranoside, cycloastragenol-3-O-[-l-arabinopyranosyl(1 → 2)-β-d-xylopyranosyl]-6-O-β- d-xylopyranoside and cycloastragenol-3-O-[-l-arabinopyranosyl(1 → 2)-β-d-(3-O-acetyl)-xylopyranosyl]-6-O-β-d-xylopyranoside.