Isolation and characterization of poly- and oligosaccharides from the red microalga Porphyridium sp.

The current study forms part of an ongoing research effort focusing on the elucidation of the chemical structure of the sulfated extracellular polysaccharide of the red microalga Porphyridium sp. (UTEX 637). We report here on the chemical structure of a fraction separated from an acidic crude extract of the polysaccharide, as investigated by methylation analysis, carboxyl reduction-methylation analysis, desulfation-methylation analysis, partial acid hydrolysis, Smith degradation, together with 1D and 2D ¹H and ¹³C NMR spectroscopy. This fraction with a molar mass of 2.39 × 10⁵ g mol⁻¹ comprised d- and l-Gal, d-Glc, d-Xyl, d-GlcA, and sulfate groups in a molar ratio of 1.0:1.1:2.1:0.2:0.7. The almost linear backbone of the fraction is composed of (1→2)- or (1→4)-linked d-xylopyranosyl, (1→3)-linked l-galactopyranosyl, (1→3)-linked d-glucopyranosyl, and (1→3)-linked d-glucopyranosyluronic acid and comprises a possible acidic building unit:

[(2 or 4)-β-d-Xylp-(l→3)]_m-α-d-Glcp-(1→3)-α-d-GlcpA-(1→3)-l-Galp(l→

Attached to the backbone are sulfate groups and nonreducing terminal d-xylopyranosyl and galactopyranosyl residues, which occur at the O-6 positions of Glc-derived moieties in the main chain.     

Structural elucidation of the O-antigenic polysaccharide from Escherichia coli O175

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O175 has been elucidated. Component analysis together with ¹H and ¹³C NMR spectroscopy experiments were used to determine the structure. Inter-residue correlations were determined by ¹H,¹H-NOESY, and ¹H,¹³C-heteronuclear multiple-bond correlation experiments. The PS is composed of pentasaccharide repeating units with the following structure:→2)-α-d-Glcp-(1→4)-α-d-GlcpA-(1→3)-α-d-Manp-(1→2)-α-d-Manp-(1→3)-β-d-GalpNAc-(1→Cross-peaks of low intensity from an α-linked glucopyranosyl residue were present in the ¹H,¹H-TOCSY NMR spectra. The α-d-Glcp residue is suggested to originate from the terminal part of the polysaccharide and consequently the biological repeating unit has a 3-substituted N-acetyl-d-galactosamine residue at its reducing end. The repeating unit of the E. coli O175 O-antigen is similar to those from E. coli O22 and O83, both of which carry an α-d-Glcp-(1→4)-d-GlcpA structural element, thereby explaining the reported cross-reactivities between the strains.     

Presence of alternating glucosaminoglucan in the sheath of Thiothrix nivea

A sheath-forming sulfa oxidizer, Thiothrix nivea, was mixotrophically cultured in a medium supplemented with acetic acid and sodium disulfide. Its sheath, a microtube-like extracellular supermolecule, was prepared by selectively removing the cells with lysozyme, sodium dodecyl sulfate, and sodium hydroxide. The sheath was not visibly affected by hydrazine treatment, suggesting that it is not a proteinous supermolecule. From the acid hydrolysate of the sheath, glucose and glucosamine were detected in an approximate molar ratio of 1:1. Three other saccharic compounds were detected and recovered by HPLC as fluorescent derivatives prepared by reaction with 4-aminobenzoic acid ethyl ester. Nuclear magnetic resonance (NMR) analysis suggested that one of the derivatives was derived from an unidentified deoxypentose. NMR analysis for the other 2 derivatives showed that they were derived from β-1,4-linked disaccharides and tetrasaccharides, which were composed of glucose and glucosamine. The sheath was readily broken down by weak HCl treatment, releasing an unidentified deoxypentose and polymer. Chemical analysis showed the presence of β-1,4-linked d-Glcp and d-GlcNp in the polymer. NMR analysis revealed that the polymer had a repeating unit of →4)-d-Glcp-(β1→4)-d-GlcNp-(β1→. The solid-state 1D-¹³C NMR spectrum of the polymer in N-acetylated form supported this result. The molecular weight of the polymer was estimated to be 8.2 × 10⁴ by size exclusion chromatography. Based on these results, the sheath of T. nivea is hypothesized to be assembled from alternately β-1,4-linked glucosaminoglucan grafted with unidentified deoxypentose.     

Structure of the O-polysaccharide of Salmonella enterica O41

An O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Salmonella enterica O41, and the following structure of the O-unit was determined by chemical analyses along with 1D and 2D ¹H and ¹³C NMR spectroscopy:→2)-β-d-Manp-(1→4)-α-d-Glcp-(1→3)-α-l-QuipNAc-(1→3)-α-d-GlcpNAc-(1→where QuiNAc stands for 2-acetamido-2,6-dideoxyglucose. The structure established is in agreement with the O-antigen gene cluster of S. enterica O41 and tentative assignment of the gene functions reported earlier.     

Synthesis of two isomeric methyl β-D-xylotriosides containing a (1→2)-β-linkage

Partial hydrolysis with acid, methylation analysis (including uronic acid degradation), Smith degradation, and p.m.r. spectroscopy have been used to determine the primary structure of the capsular polysaccharide of Klebsiella serotype k64. The hexasaccharide repeating-unit, which also contains one O-acetyl substituent, comprises a 4)-α-d-GlcpA-(1 → 3)-α-d-Manp-(1 → 3)-β-d-Glcp-(1 → 4)-α-d-Manp-(1 → chain with a 4,6-O-(l-carboxyethylidene)-β-d-glucopyranosyl and an l-rhamnosyl group attached to the 4-linked d-mannosyl residue at O-2 and O-3, respectively.     

Isolation of phosphorylated polysaccharides from algae: the immunostimulatory principle of Chlorella pyrenoidosa

The major immunostimulatory principle in the hot aqueous extract of Chlorella pyrenoidosa has been isolated by a sequence of ethanol precipitation, precipitation with a cationic surfactant (CTAB), size exclusion chromatography, and anion exchange chromatography. A series of phosphorylated polysaccharides were obtained having different molecular masses but with similar structures. The higher molecular mass fractions showed considerable activity in the stimulation of mouse peritoneal macrophages to synthesize nitric oxide. The structure of the major polysaccharide was established by sugar analysis, configurational analysis, and 1D and 2D NMR experiments at 500 and 800 MHz on the parent polysaccharide, the de-O-acetylated polysaccharide, and on the components obtained after hydrolysis of the phosphate diesters. It had a β-d-Galp-(1→3)-β-d-Galp-(1→3)-backbone with half of the Galp units substituted at O-6 by terminal β-d-Glcp units. The remaining Galp units were substituted on O-6 by about equal amounts of α-d-Manp-1-phosphate and 3-O-Me-α-Manp-1-phosphate diesters. The substituents were not located in a regularly alternating fashion on the backbone. The O-acetyl groups were largely located on O-2 and O-4 of Galp and 35% of the Galp residues were O-acetylated. This is the second observation of a phosphorylated polysaccharide in an alga and the first where it is present to a significant extent.     

A dual role for the lex2 locus: identification of galactosyltransferase activity in non-typeable Haemophilus influenzae strains 1124 and 2019

Lipopolysaccharide (LPS) of Haemophilus influenzae comprises a conserved tri-l-glycero-d-manno-heptosyl inner-core moiety (l-α-d-Hepp-(1→2)-[PEtn→6]-l-α-d-Hepp-(1→3)-[β-d-GlcIp-(1→4)]-l-α-d-Hepp-(1→5)-α-Kdop) to which addition of β-d-Glcp to O-4 of GlcI in serotype b strains is controlled by the gene lex2B. In non-typeable H. influenzae strains 1124 and 2019, however, a β-d-Galp is linked to O-4 of GlcI. In order to test the hypothesis that the lex2 locus is involved in the expression of β-d-Galp-(1→4-β-d-Glcp-(1→ from HepI, lex2B was inactivated in strains 1124 and 2019, and LPS glycoform populations from the resulting mutant strains were investigated. Detailed structural analyses using NMR techniques and electrospray-ionisation mass spectrometry (ESIMS) on O-deacylated LPS and core oligosaccharide material (OS), as well as ESIMSⁿ on permethylated dephosphorylated OS, indicated both lex2B mutant strains to express only β-d-Glcp extensions from HepI. This provides strong evidence that Lex2B functions as a galactosyltransferase adding a β-d-Galp to O-4 of GlcI in these strains, indicating that allelic polymorphisms in the lex2B sequence direct alternative functions of the gene product.     

Glycan flexibility: insights into nanosecond dynamics from a microsecond molecular dynamics simulation explaining an unusual nuclear Overhauser effect

An atomistic all-atom molecular dynamics simulation of the trisaccharide β-d-ManpNAc-(1→4)[α-d-Glcp-(1→3)]-α-l-Rhap-OMe with explicit solvent molecules has been carried out. The trisaccharide represents a model for the branching region of the O-chain polysaccharide of a strain from Aeromonas salmonicida. The extensive MD simulations having a 1-μs duration revealed a conformational dynamics process on the nanosecond time scale, that is, a ‘time window’ not extensively investigated for carbohydrates to date. The results obtained from the MD simulation underscore the predictive power of molecular simulations in studies of biomolecular systems and also explain an unusual nuclear Overhauser effect originating from conformational exchange.     

Improved procedures for the selective chemical fragmentation of rhamnogalacturonans

The structural characterization of branched rhamnogalacturonans (RGs) requires the availability of methods that selectively cleave the Rhap-(1→4)-α-GalAp linkage and thereby generate oligosaccharide fragments that are suitable for mass spectrometric and NMR spectroscopic analyses. Enzymic cleavage of this linkage is often ineffective, especially in highly branched RGs. Therefore, we have developed an improved chemical fragmentation method based on β-elimination of esterified 4-linked GalpA residues. At least 85% of the carboxyl groups of the GalA residues in Arabidopsis thaliana seed mucilage RG is esterified using methyl iodide or 3-iodopropanol in Me₂SO containing 8% water and 1% tetrabutylammonium fluoride. However, β-elimination fragmentation at pH 7.3 and 120 °C is far more extensive with hydroxypropyl-esterified RG than with methyl-esterified RG. The non-reducing 4-deoxy-β-l-threo-hex-4-enepyranosyluronic acid residue formed by the β-elimination reaction is completely removed by treatment with aqueous N-bromosuccinimide, thereby simplifying the structural characterization of the chemically generated oligoglycosyl fragments. This newly developed procedure was used to selectively fragment the branched RG from peppergrass seed mucilage. The products were characterized using MALDI-TOF mass spectrometry, glycosyl residue composition analysis, and 1 and 2D NMR spectroscopy. Our data show that the most abundant low-molecular weight fragments contained a backbone rhamnose residue substituted at O-4 with a single sidechain, and suggest that peppergrass seed mucilage RG is composed mainly of the repeating unit 4-O-methyl-α-d-GlcpA-(1→4)-β-d-Galp-(1→4)-[→4)-α-d-GalpA-(1→2)-]-α-l-Rhap-(1→.     

Structural characterization of a highly branched polysaccharide from the seeds of Plantago asiatica L.

In this paper, polysaccharides were extracted from the seeds of Plantago asiatica L. with hot water and separated into three fractions PLP-1 (18.9%), PLP-2 (52.6%) and PLP-3 (28.5%) by Sephacryl™ S-400 HR column chomatography. The main fraction PLP-2's structure was elucidated using oxalic acid hydrolysis, partial acid hydrolysis, methylation, GC, GC-MS, 1D and 2D NMR. PLP-2 was composed of Rha, Ara, Xyl, Man, Glc and Gal, in a molar ratio of 0.05:1.00:1.90:0.05:0.06:0.10. Its uronic acid was GlcA. PLP-2 was highly branched heteroxylan which consisted of a β-1,4-linked Xylp backbone with side chains attached to O-2 or O-3. The side chains consisted of β-T-linked Xylp, α-T-linked Araf, α-T-linked GlcAp, β-Xylp-(1 → 3)-α-Araf and α-Araf-(1 → 3)-β-Xylp, etc. Based on these results, the structure of PLP-2 was proposed.     

Double diastereoselection explains limitations in synthesizing mannose-containing β-(1,3)-glucans

It is known that 3-O-glycosylation of glucosidic acceptors bearing acyl groups in the 4 and 6 positions instead of a 4,6-O-benzylidene ring mainly affords α-glycosides. Described here is an unexpected stereochemical outcome for elongation at glucose O-3 of a β-d-Glcp-(1→3)-α-d-Manp disaccharide using peracetylated ethyl thioglucoside as a donor. This unexpected reaction was correlated with match-mismatch effects, as shown by efficient coupling of the same acceptor by a donor of l-configuration.     

Structure of the high-molecular weight exopolysaccharide isolated from Lactobacillus pentosus LPS26

The strain Lactobacillus pentosus LPS26 produces a capsular polymer composed of a high- (2.0 × 10⁶ Da) (EPS A) and a low-molecular mass (2.4 × 10⁴ Da) (EPS B) polysaccharide when grown on semi-defined medium containing glucose as the carbon source. The structure of EPS A and its deacetylated form has been determined by monosaccharide and methylation analysis as well as by 1D/2D NMR studies (¹H and ¹³C). We conclude that EPS A is a charged heteropolymer, with a composition of d-glucose, d-glucuronic acid and l-rhamnose in a molar ratio 1:2:2. The repeating unit is a pentasaccharide with two O-acetyl groups at O-4 of the 3-substituted α-d-glucuronic acid and at O-2 of the 3-substituted β-l-rhamnose, respectively.→4)-α-d-Glcp-(1→3)-α-d-GlcpA4Ac-(1→3)-α-l-Rhap-(1→4)-α-d-GlcpA-(1→3)-β-l-Rhap2Ac-(1→This unbranched structure is not common in EPSs produced by Lactobacilli. Moreover, the presence of acetyl groups in the structure is an unusual feature which has only been reported in L. sake 0-1 [Robijn et al. Carbohydr. Res., 1995, 276, 117-136].     

Studies on the chemical structure and antitumor activity of an exopolysaccharide from Rhizobium sp. N613

The molecular structure of the rhizobium exopolysaccharide (REPS) was analyzed by enzymolysis, periodate oxidation, and Smith degradation, and by IR and NMR spectroscopy. The results indicated that REPS was a β-glucan with a backbone of β-d-(1→4)-linked glucose residues and branches of β-d-(1→6)-linked glucose residues. The branch was attached to the main chain at the 6-O-position. The molar ratio of 1→4 and 1→6 was 2:1. The terminal C3 of the (1→6)-Glc branch had an O-acetyl group. The molecular weight was estimated to be 35 kDa by Sephadex G-100 column chromatography. The antitumor activity of REPS was evaluated in mice bearing sarcoma 180, hepatoma 22, and Ehrlich ascites carcinoma tumor, respectively. At doses of 10-60 mg/kg, it was observed that tumor formation decreased significantly (P <0.01), but the relative spleen and thymus weight, the phagocytic function of monocytes, lymphocyte proliferation, and serum hemolysis antibody increased significantly (P <0.05). Results of these studies demonstrated that the REPS polysaccharide possessed antitumor activity.     

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 94/D4 and the international type strain Escherichia coli O82

The structure of the O-antigen polysaccharides (PS) from the enteroaggregative Escherichia coli strain 94/D4 and the international type strain E. coli O82 have been determined. Component analysis and ¹H, ¹³C, and ³¹P NMR spectroscopy experiments were employed to elucidate the structure. Inter-residue correlations were determined by ¹H, ¹³C-heteronuclear multiple-bond correlation, and ¹H, ¹H-NOESY experiments. d-GroA as a substituent is linked via its O-2 in a phosphodiester-linkage to O-6 of the α-d-Glcp residue. The PS is composed of tetrasaccharide repeating units with the following structure:→4)-α-d-Glcp6-(P-2-d-GroA)-(1→4)-β-d-Galp-(1→4)-β-d-Glcp-(1→3)-β-d-GlcpNAc-(1→Cross-peaks of low intensity from an α-d-Glcp residue were present in the NMR spectra and spectral analysis indicates that they originate from the terminal residue of the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. Enzyme immunoassay using specific anti-E. coli O82 rabbit sera showed identical reactivity to the LPS of the two strains, in agreement with the structural analysis of their O-antigen polysaccharides.     

Galactans from Cryptonemia species. Part II: Studies on the system of galactans of Cryptonemia seminervis (Halymeniales) and on the structure of major fractions

Cryptonemia seminervis biosynthesizes a family of d,l-hybrid galactans based on the classical 3-linked β-d-galactopyranosyl→4-linked α-d- and α-l-galactopyranosyl alternating sequence (A-units→B-units) with major amounts of α-d- and α-l-galactose and 3,6-anhydro-d- and l-galactose and lesser percentages of 3,6-anhydro-2-O-methyl-l-galactose, 2-O-methyl-, 4-O-methyl- and 6-O-methylgalactoses. The dispersion of structures in this family is based on five structural factors, namely: (a) the amount and position of substituent groups as sulfate (major), pyruvic acid ketals, methoxyl and glycosyl side-chain (4-O-methyl galactopyranosyl and/or xylosyl); (b) the ratio galactose/3,6-anhydrogalactose in the B-units; (c) the ratio d,l-galactoses and d,l-3,6-anhydrogalactoses also in the B-units, (d) the formation of diads and (e) the sequence of the diads in the linear backbone. Considering these variables it is not unexpected to find in the fractions studied at least 18 structural units producing highly complex structures. Structural studies carried out in two major fractions (S2S-3 and S2S-4) showed that these galactans were formed mainly by β-d-galactopyranosyl 2-sulfate (20 and 11.9 mol %), β-d-galactopyranosyl 2-sulfate 4,6-O-(1′-carboxyethylidene) (8.9 and 6.0 mol %) and β-d-galactopyranosyl 2,6-sulfate (5.4 and 18.6 mol %), together with 3,6-anhydro-α-l-galactopyranosyl (11.4 and 7.3 mol %) and 3,6-anhydro-α-l-galactopyranosyl 2-sulfate (4.9 and 15.4 mol %) and minor quantities of 12-15 other structural units.Preparative alkaline treatment carried out on fraction (S2S-3) produced a quantitative formation of 3,6-anhydro α-l-galactopyranosyl units from precursor units (α-l-galactose 6-sulfate and α-l-galactose 2,6-sulfate). Kinetic studies on this 3,6-anhydro cyclization show a rate constant of 5.2 × 10⁴ s⁻¹ indicating diads of the type G→L6S/2,6S. Data from chemical, spectroscopic and kinetic studies suggest that, in S2S-3, the agaran block in the d,l-hybrid galactan is composed of the following diads: G(6R)→L6S/2,6S and G2S(P)(2,6S)→LA(2S)(2R)(2M) and the carrageenan block of G2S(P)→D(2S)(2,3S)(3S)(3,6S) in a molar ratio of agaran to carrageenan structures of ∼2:1.     

O-Methylated mannogalactan from the microalga Coccomyxa mucigena, symbiotic partner of the lichenized fungus Peltigera aphthosa

A structural study of the carbohydrates from Coccomyxa mucigena, the symbiotic algal partner of the lichenized fungus Peltigera aphthosa, was carried out. It produced an O-methylated mannogalactan, with a (1 → 6)-linked β-galactopyranose main-chain partially substituted at O-3 by β-Galp, 3-OMe-α-Manp or α-Manp units. There were no similarities with polysaccharides previously found in the lichen thallus of P. aphthosa. Moreover, the influence of lichenization in polysaccharide production by symbiotic microalgae and the nature of the photobiont in carbohydrate production in lichen symbiosis are also discussed.     

NMR study on the hydroxy protons of the Lewis X and Lewis Y oligosaccharides

The ¹H NMR chemical shifts and NOEs of hydroxy protons in Lewis X trisaccharide, β-d-Galp-(1 → 4)[α-l-Fucp-(1 → 3)]-β-d-GlcpNAc, and Lewis Y tetrasaccharide, α-l-Fucp-(1 → 2)-β-d-Galp-(1 → 4)[α-l-Fucp-(1 → 3)]-β-d-GlcpNAc, were obtained for 85% H₂O/15% (CD₃)₂CO solutions. The OH-4 signal of Galp in Lewis X and OH-3, OH-4 signals of Galp, and OH-2 signal of Fucp linked to Galp in Lewis Y had chemical shifts which indicate reduced hydration due to their proximity to the hydrophobic face of the Fucp unit linked to GlcpNAc. The inter-residue NOEs involving the exchangeable NH and OH protons confirmed the stacking interaction between the Fucp linked to the GlcpNAc and the Galp residues in Lewis X and Lewis Y.     

The synthesis of antigenic determinants for yeast d-mannan,and a linear (1→6)-α-d-gluco-d-mannan,and their protein conjugates

2-O-Benzoyl-3,4,6-tri-O-benzyl-1-O-tosyl-d-mannopyranose and 2,3,4-tri-O- benzyl-6-O-(N-phenylcarbamoyl)-1-O-tosyl-d-glucopyranose were allowed to react with partially blocked 2-[4-(p-toluenesulfonamido)phenyl]ethyl α-d-manno- and -gluco-pyranosides. Disaccharides having α-d-Manp-(1→2)-α-D-Manp, α-d-manp-(1→6)-α-d-Manp, α-d-Manp-(1→6)-α-d-Manp, and α-d-Glcp-(1→6)-α-d-Manp structures, and a branched trisaccharide having the structure α-d-Manp-(1→2)-[α-d-Manp-(1→6)]-α-d-Manp were synthesized. The oligosaccharides were deblocked with sodium in liquid ammonia to give glycopyranosides having a free primary aromatic amine which were converted into isothiocyanate derivatives with thiophosgene. The functionalized oligosaccharides were then coupled to bovine serum albumin to give protein conjugates.     

Acylated flavonol tetraglycosides from Delphinium gracile

An ethanol extract of the aerial parts of Delphinium gracile DC. yielded five flavonol glycosides quercetin-3-O-{[β-d-xylopyranosyl (1 → 3)-4-O-(E-p-caffeoyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranosyl (1 → 2)]}-β-d-glucopyranoside (1), quercetin-3-O-{[β-d-xylopyranosyl (1 → 3)-4-O-(E-p-coumaroyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranosyl (1 → 2)]}-β-d-glucopyranoside (2), quercetin-3-O-{[β-d-xylopyranosyl (1 → 3)-4-O-(Z-p-coumaroyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranosyl (1 → 2)]}-β-d-glucopyranoside (3), kaempferol-3-O-{[β-d-glucopyranosyl (1 → 3)-4-O-(E-p-coumaroyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranoside-7-O-(4-O-acetyl)-α-l-rhamnopyranoside (4) kaempferol-3-O-{[β-d-glucopyranosyl (1 → 3)-4-O-(E-p-coumaroyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranoside-7-O-(4-O-acetyl)-α-l-rhamnopyranoside (5) in addition to 4-(β-d-glucopyranosyloxy)-6-methyl-2H-pyran-2-one (6) and rutin. Structures were elucidated by spectroscopic methods.     

Isolation and structural characterization of a polysaccharide FCAP1 from the fruit of Cornus officinalis

A water-soluble polysaccharide, FCAP1, was isolated from an alkaline extract from the fruits of Cornus officinalis. Its molecular weight was 34.5 kDa. Monosaccharide composition analysis revealed that it was composed of fucose, arabinose, xylose, mannose, glucose, and galactose in a molar ratio of 0.29:0.19:1.74:1:3.30:1.10. On the basis of partial acid hydrolysis and methylation analysis, FCAP1 was shown to be a highly branched polysaccharide with a backbone of β-(1→4)-linked-glucose partially substituted at the O-6 position with xylopyranose residues. The branches were composed of (1→3)-linked-Ara, (1→4)-linked-Man, (1→4,6)-linked-Man, (1→4)-linked-Glc, and (1→2)-linked-Gal. Arabinose, fucose, and galactose were located at the terminal of the branches. The structure was further elucidated by a specific enzymatic degradation with an endo-β-(1→4)-glucanase and MALDI-TOF-MS analysis. Oligosaccharides generated from FCAP1 indicated that FCAP1 contained XXXG-type and XXG-type xyloglucan fragments.