The structure of a sulfated galactan from Porphyra haitanensis and its in vivo antioxidant activity

The sulfated galactan fraction F1 isolated from the red seaweed, Porphyra haitanensis, showed typical porphyran structure. It has a linear backbone of alternating 3-linked beta-D-galactosyl units and 4-linked alpha-L-galactosyl 6-sulfate and 3,6-anhydro-alpha-L-galactosyl units. The L-residues are mainly composed of alpha-L-galactosyl 6-sulfate units, and the 3,6-anhydrogalactosyl units are minor. Partial methylation occurred at the C-6 position of the D-galactosyl units and at the C-2 position of the 3,6-anhydro-alpha-L-galactosyl units. Intraperitoneal administration of F1 significantly decreased the lipid peroxidation in aging mice. F1 treatment increased the total antioxidant capacity and the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in aging mice. The results indicated that F1 had significant in vivo antioxidant activity.     

Further studies on the composition and structure of a fucoidan preparation from the brown alga Saccharina latissima

The polysaccharide composition of a fucoidan preparation isolated from the brown alga Saccharina latissima (formerly Laminaria saccharina) was reinvestigated. The preparation was fractionated by anion-exchange chromatography, and the fractions obtained were analyzed by chemical methods combined with NMR spectroscopy. Several 2D procedures, including HSQC, HMQC-TOCSY, and HMQC-NOESY, were used to obtain reliable structural information from the complex spectra, and the signal assignments were additionally confirmed by comparison with the literature spectra of the related polysaccharides and synthetic oligosaccharides. In accordance with the previous data, the main polysaccharide component was shown to be a fucan sulfate containing a backbone of 3-linked α-l-fucopyranose residues sulfated at C-4 and/or at C-2 and branched at C-2 by single sulfated α-l-fucopyranose residues. In addition, three other types of sulfated polysaccharide molecules were detected in the total fucoidan preparation: (i) a fucogalactan having a backbone of 6-linked β-d-galactopyranose residues branched mainly at C-4 and containing both terminal galactose and fucose residues; (ii) a fucoglucuronomannan having a backbone of alternating 4-linked β-d-glucopyranosyluronic acid and 2-linked α-d-mannopyranose residues with α-l-fucopyranose residues as single branches at C-3 of α-d-Manp; and (iii) a fucoglucuronan having a backbone of 3-linked β-d-glucopyranosyluronic acid residues with α-l-fucopyranose residues as single branches at C-4. Hence, even a single algal species may contain, at least in minor amounts, several sulfated polysaccharides differing in molecular structure. Partial resolution of these polysaccharides has been accomplished, but unambiguous evidence on their presence as separate entities was not obtained.     

The system of galactans of the red seaweed, Kappaphycus alvarezii, with emphasis on its minor constituents

The galactans extracted with hot water from Kappaphycus alvarezii, after previous extraction at room temperature, are mainly composed of kappa-carrageenans (approximately 74%) and micro-carrageenans (approximately 3%). However, a significant percentage of these galactans (at least 14%) is composed of sulfated agarans and, possibly, agaran-type sulfated DL-hybrid galactans. These agarans are partially substituted on C-2 or C-4 or disubstituted on both positions of the beta-D-galactose units and on C-3 or C-2 and C-3 of the alpha-L-galactose residues with sulfate groups or single stubs of beta-D-xylopyranose, D-glucopyranose, and galactose or with D-glucopyranosyl-(1-->4)-D-glucopyranose side chains. Significant quantities of 2-O-methyl- and 3-O-methyl-L-galactose units are also present. A great tendency to retain Ca2+ and Mg2+, in spite of massive treatments with Na+ and K+ salts, was observed. The complexation between agarans and agarans-kappa-carrageenans through divalent cations and the possible zipper-type carbohydrate-carbohydrate interactions would be two complementary mechanisms of interactions.     

Chemical and in situ characterization of macromolecular components of the cell walls from the green seaweed Codium fragile

A comprehensive analysis of the carbohydrate-containing macromolecules from the coencocytic green seaweed Codium fragile and their arrangement in the cell wall was carried out. Cell walls in this seaweed are highly complex structures composed of 31% (w/w) of linear (1-->4)-beta-D-mannans, 9% (w/w) of pyruvylated arabinogalactan sulfates (pAGS), and low amounts of hydroxyproline rich-glycoprotein epitopes (HRGP). In situ chemical imaging by synchrotron radiation Fourier transform infrared (SR-FTIR) microspectroscopy and by immunolabeling using antibodies against specific cell wall carbohydrate epitopes revealed that beta-d-mannans and pAGS are placed in the middle part of the cell wall, whereas HRGP epitopes (arabinogalactan proteins (AGPs) and extensins) are located on the wall boundaries, especially in the utricle apical zone. pAGS are sulfated at C-2 and/or C-4 of the 3-linked beta-L-arabinopyranose units and at C-4 and/or C-6 of the 3-linked beta-D-galactopyranose residues. In addition, high levels of ketals of pyruvic acid were found mainly at 3,4- of some terminal beta-D-Galp units forming a five-membered ring. Ramification was found at some C-6 of the 3-linked beta-D-Galp units. In agreement with the immunolabeled AGP epitopes, a nonsulfated branched furanosidic arabinan with 5-linked alpha-L-Araf, 3,5-linked alpha-L-Araf, and terminal alpha-L-Araf units and a nonsulfated galactan structure composed of 3-(3,6)-linked beta-D-Galp residues, both typical of type-II AG glycans were found, suggesting that AGP structures are present at low levels in the cell walls of this seaweed. Based on this study, it is starting to emerge that Codium has developed unique cell wall architecture, when compared, not only with that of vascular plants, but also with other related green seaweeds and algae.     

Fucoidans from the brown seaweed Adenocystis utricularis: extraction methods,antiviral activity and structural studies

The brown seaweed Adenocystis utricularis (family Adenocystaceae, order Ectocarpales sensu lato) was extracted in parallel with three solvents usually utilized for obtaining fucoidans: distilled water, 2% calcium chloride solution and diluted hydrochloric acid (pH 2) solution. In each case, the extraction was effected at room temperature and then at 70 degrees C. The extraction yields and characteristics of the products were similar in the three cases, with only minor differences. The analytical features of the products indicate that two different types of fucoidans are present in this seaweed. One of them, mostly extracted at room temperature, is composed mainly of L-fucose, D-galactose and ester sulfate (the 'galactofucan'). The other product (the 'uronofucoidan') is the major component of the extracts obtained at 70 degrees C. It is composed mainly of fucose, accompanied by other monosaccharides (mostly Man, but also Glc, Xyl, Rha and Gal), significant amounts of uronic acids and low proportions of sulfate ester. Fractionation with the cationic detergent cetrimide has allowed achieving a better separation of the galactofucan and uronofucoidan components. The galactofucans show a high inhibitory activity against herpes simplex virus 1 and 2, with no cytotoxicity, whereas the uronofucoidans carry no antiviral activity. Structural studies on the galactofucan fractions were carried out by methylation analysis, desulfation and NMR spectroscopy. The fucan constituent is mainly composed of 3-linked alpha-L-fucopyranosyl backbone, mostly sulfated at C-4, and branched at C-2 with non-sulfated fucofuranosyl and fucopyranosyl units, and 2-sulfated fucopyranosyl units. The galactan moiety is more heterogeneous, with predominant D-galactopyranose units linked on C-3 and C-6, and sulfation mostly on C-4, even in terminal non-reducing units. It may be inferred that at least some of these galactose units carry the alpha-configuration.     

Structural analysis and antiviral activity of a sulfated galactan from the red seaweed Schizymenia binderi (Gigartinales, Rhodophyta)

Aqueous extraction of gametophytic Schizymenia binderi afforded a polysaccharide composed of galactose and sulfate groups in a molar ratio of 1.0:0.89 together with uronic acids (6.8 wt%) and minor amounts of other neutral sugars. Alkali-treatment of the polysaccharide afforded a polysaccharide devoid of 3,6-anhydrogalactose. 13C NMR spectroscopy of the desulfated alkali-treated polysaccharide showed a backbone structure of alternating 3-linked beta-D-galactopyranosyl and 4-linked alpha-galactopyranosyl units that are predominantly of the D-configuration and partly of the L-configuration. Methylation, ethylation and NMR spectroscopic studies of the alkali-treated polysaccharide indicated that the sulfate groups are located mainly at positions O-2 of 3-linked beta-D-galactopyranosyl residue and at position O-3 of 4-linked-alpha-galactopyranosyl residues, the latter is partially glycosylated at position O-2. The sulfated galactan from S. binderi exhibited highly selective antiviral activity against Herpes simplex virus types 1 and 2, with selectivity indices (ratio cytotoxicity/antiviral activity) >1000 for all assayed virus strains. This compound was shown to interfere with the initial adsorption of viruses to cells.     

Structural investigation of a fucoidan containing a fucose-free core from the brown seaweed, Hizikia fusiforme

A fucoidan, obtained from the hot-water extract of the brown seaweed, Hizikia fusiforme, was separated into five fractions by DEAE Sepharose CL-6B and Sepharose CL-6B column chromatography. All five fractions contained predominantly fucose, mannose and galactose and also contained sulfate groups and uronic acid. The fucoidans had MWs from 25 to 950 kDa. The structure of fraction F32 was investigated by desulfation, carboxyl-group reduction, partial hydrolysis, methylation analysis and NMR spectroscopy. The results showed that the sugar composition of F32 was mainly fucose, galactose, mannose, xylose and glucuronic acid; sulfate was 21.8%, and the MW was 92.7 kDa. The core of F32 was mainly composed of alternating units of -->2)-alpha-D-Man(1--> and -->4)-beta-D-GlcA(1-->, with a minor portion of -->4)-beta-D-Gal(1--> units. The branch points were at C-3 of -->2)-Man-(1-->, C-2 of -->4)-Gal-(1--> and C-2 of -->6)-Gal-(1-->. About two-thirds of the fucose units were at the nonreducing ends, and the remainder were (1-->4)-, (1-->3)- and (1-->2)-linked. About two-thirds of xylose units were at the nonreducing ends, and the remainder were (1-->4)-linked. Most of the mannose units were (1-->2)-linked, and two-thirds of them had a branch at C-3. Galactose was mainly (1-->6)-linked. The absolute configurations of the sugar residues were alpha-D-Manp, alpha-L-Fucp, alpha-D-Xylp, beta-D-Galp and beta-D-GlcpA. Sulfate groups in F32 were at C-6 of -->2,3)-Man-(1-->, C-4 and C-6 of -->2)-Man-(1-->, C-3 of -->6)-Gal-(1-->, C-2, C-3 or C-4 of fucose, while some fucose had two sulfate groups. There were no sulfate groups in either the GlcA or xylose residues.     

A polysaccharide fraction from the red seaweed Champia novae-zealandiae Rhodymeniales,Rhodophyta

The polysaccharide recovered after extraction of Champia novae-zealandiae is a galactan with alternating 3-linked d-galactopyranosyl units sulfated at the 2-position, and 4-linked galactopyranosyl units sulfated at both the 2- and 3-positions that are predominantly of the l- and partly of the d-configuration. Other minor substitution includes 6-O-methyl ether or 4,6-pyruvate acetal on the 3-linked residues. Techniques used in determining the structure include infrared and ¹³C-NMR spectroscopy, and GC-MS analysis of alditol acetate derivatives produced by reductive hydrolysis/acetylation of native, methylated, and/or desulfated samples. These results are of particular interest because 4-linked 2,3-desulfated galactosyl residues have not been encountered as major constituents of red algal polysaccharides.     

NMR structural determination of dissolved O-acetylated galactoglucomannan isolated from spruce thermomechanical pulp

Water-soluble O-acetylated galactoglucomannan (GGM) isolated from spruce thermomechanical pulp (TMP) by hot-water extraction was characterized by 1D and 2D (homo- and heteronuclear) NMR analysis. The backbone was found to consist of (1-->4)-linked mannopyranosyl and glucopyranosyl units in a ratio of 10:1.9-2.6. The mannopyranosyl units were acetylated at C-2 and C-3 with a degree of acetylation around 0.28-0.37 as determined by NMR. A slightly larger amount of 2-O-acetylated mannopyranosyl was detected when compared to the 3-O-acetylated component. Approximately every 10th mannopyranosyl unit was substituted at C-6 by a single alpha-galactopyranosyl unit. Fine structure determination based on sequence-specific chemical shift variations showed that the distribution of glycosyl residues is random. Small amounts of other minor polysaccharide species including xylans and galactans could also be identified by NMR.     

Chemical structure and antiviral activity of the sulfated heterorhamnan isolated from the green seaweed Gayralia oxysperma

A homogeneous sulfated heterorhamnan was obtained by aqueous extraction, then by ultrafiltration from the green seaweed Gayralia oxysperma. Besides α-l-rhamnose it contains glucuronic and galacturonic acids, xylose and glucose. The structure was established by methylation analyses of the carboxyl-reduced, carboxyl-reduced/desulfated, carboxyl-reduced/Smith-degraded, and carboxyl-reduced/Smith-degraded/desulfated products and 1D, 2D NMR spectroscopy analyses. The heterorhamnan backbone is constituted by 3- and 2-linked rhamnosyl units (1.00:0.80), the latter being ∼50% substituted at C-3 by side chains containing 2-sulfated glucuronic and galacturonic acids and xylosyl units. The 3- and 2-linked rhamnosyl units are unsulfated (20%), disulfated (16%), and mostly monosulfated at C-2 (27%) and C-4 (37%). The branched and sulfated heterorhamnan had high and specific activity against herpes simplex virus.     

Production and properties of agar from the invasive marine alga, Gracilaria vermiculophylla (Gracilariales, Rhodophyta)

The utilization potential, in terms of agar production, of the invasive alga, Gracilaria vermiculophylla, collected at Ria de Aveiro, northwestern Portugal was investigated. The agar yield ranged from 15% to 33%, with pre-extraction treatment with alkali generally increasing the yield. The gel quality (gel strength and apparent Young’s modulus) was best (>600 g cm^?2 and >1,000 kPa, respectively) when alkali treatment with 6% NaOH for 3.5 h was performed. At these pretreatment conditions, the effect of extraction time was also investigated and highest yield and best gel quality were obtained with a 2 h extraction time. By employing these extraction conditions, G. vermiculophylla can be a source of industrial food-grade agar. The structure of agar from G. vermiculophylla was determined through chemical techniques and FTIR and NMR spectrometry. It is mainly composed of alternating 3-linked d-galactose and 4-linked 3,6-anhydro-l-galactose, with methyl substitution occurring at 16–19 mol% of C-6 in 3-linked units and 2–3 mol% of C-2 in 4-linked units. A minor sulfation on C-4 of 3-linked units was also detected; while precursor units (6-sulfated 4-linked galactosyl moieties) were found in the native extract.     

Isolation,chemical investigation and antiviral activity of polysaccharides from Gracilaria corticata (Gracilariaceae,Rhodophyta)

Polysaccharides were sequentially extracted from the agarophyte Gracilaria corticata. Chemical analysis combined with infrared spectroscopy showed that the cold water extracted material consists mainly of a high molecular weight sulfated galactan. Most of the sulfate groups are alkali labile and are located at C-4 of the 1,3-linked D-galactose units and C-6 of the 1,4-linked L-galactose residues. The autoclaved extracts contain agar type polysaccharide having a high pyruvate content and a variable degree of methylation, but were contaminated with floridean starch. 1H-NMR studies indicate that methoxyl groups, when present, occur at C-6 of the 1,3-linked D-galactose units and C-2 of the 1,4-linked L-galactose residues of agar polymer. Bioassays showed that a high molecular weight galactan sulfate, exhibited selective antiviral activity against herpes simplex virus types 1 and 2, likely due to an inhibition of the initial virus attachment to the host cell.     

Chemical characteristics of a polysaccharide from Porphyra capensis (Rhodophyta)

The structure of a polysaccharide from the red seaweed, Porphyra capensis, growing along the coast of Namibia and South Africa was investigated. Algae growing at different sites and collected at different times gave a polysaccharide extract with similar chemical components. FTIR and NMR spectral analysis showed that the polysaccharide from P. capensis had a typical porphyran structure. It has the linear backbone of alternating 3-linked beta-D-galactose and 4-linked alpha-L-galactose-6-sulfate or 3,6-anhydro-alpha-L-galactose units. The ratio of alpha-L-galactose-6-sulfate and the 3,6-anhydrogalactose is 1.2:1, as reflected by a 1H NMR spectrum. A high degree of methylation occurred at the C-6 position of the D-galactose units. The degree of methylation was 0.64 for the D-galactose residues.     

The galactomannan-like oligosaccharides from the endosperm of the seed of Gleditsia triacanthos

The endosperm of the seed of Gleditsia triacanthos contains 4.8% of 85% ethanol-soluble, galactomannan-like oligosaccharides having Man:Gal ratios of 1.5–2.6:1 and an average degree of polymerization of 15. They have a narrow distribution of molecular weights and of ratio of components. The oligosaccharides have the gross structure accepted for the galactomannans, namely, a β-(1→4)-linked d-mannopyranosyl backbone having single stubs of α-(1→6)-linked d-galactopyranosyl groups. Some of the lateral chains contain more than one unit, and a minor proportion of the branches are ended by arabinofuranose or fucopyranose residues. Unusual branching points formed by 3,4-linked d-mannosyl, or 3,6-linked d-galactosyl units, or both, were also found. Despite their low molecular weight, the oligosaccharides form aggregates with a structure similar to that of the aggregates of the related galactomannans, but having a lower association energy. This fact, together with the difficulty of combining with more than one partner (due to the short, central chain), results in an increased solubility and in nonviscous solutions. The ¹³C-n.m.r. spectrum differentiated clearly the five structural units of the oligosaccharides, namely, the reducing and nonreducing end-chains of the d-mannosyl backbone; substituted and nonsubstitued, internal β-(1→4)-linked mannopyranosyl units of the backbone; and the galactosyl nonreducing end-chain of the lateral chains. The C-4 signal of the (1→4)-linked d-mannose and the C-6 signal of the same, but substituted, units showed splitting into three lines. The first has been attributed to sequence-related heterogeneity, whereas the latter is tentatively explained by assuming that this resonance is sensitive to whether the mannosyl units linked to that residue are also branched, or not.     

The structure of the agaran sulfate from Acanthophora spicifera (Rhodomelaceae, Ceramiales) and its antiviral activity. Relation between structure and antiviral activity in agarans

The sulfated agaran isolated by water extraction from the red seaweed, Acanthophora spicifera (Rhodomelaceae, Ceramiales), is made up of A-units highly substituted with sulfate groups on C-2 (28-30%), sulfates on C-2 and 4,6-O-(1'-carboxyethylidene) groups (9-15%), and only the C-2 sulfate groups (5-8%) with small amounts of C-6 sulfate, 6-O-methyl, and nonsubstituted residues. B-units are formed mainly by 3,6-anhydro-alpha-L-galactose (15-16%) and its precursor, alpha-L-galactose 6-sulfate (10-17%), together with lesser amounts of 3,6-anhydro-alpha-L-galactose 2-sulfate, alpha-L-galactose 2,6-disulfate, alpha-L-galactose 2,3,6-tri-sulfate, alpha-L-galactose 2,6-disulfate 3-xylose, 2-O-methyl-alpha-L-galactose, and unsubstituted alpha-L-galactose. Small, but significant quantities of beta-D-xylose were found in all the fractions, together with small amounts to traces of D-glucose. Some of the fractions have high antiviral activity. Attempts to correlate structure and antiviral activity in agarans are presented.     

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.     

A preponderantly 4-sulfated, 3-linked galactan from the green alga Codium isthmocladum

The green algae of the genus Codium have recently been demonstrated to be an important source of sulfated galactans from the marine environment. Here, a sulfated galactan was isolated from the species Codium isthmocladum and its structure was studied by a combination of chemical analyses and NMR spectroscopy. Two fractions (SG 1, approximately 14 kDa, and SG 2, approximately 20 kDa) were derived from this highly polydisperse and heterogeneous polysaccharide. Both exhibited similar structures in (1)H 1D NMR spectra. The structural features of SG 2 and its desulfated derivative were analyzed by COSY, TOCSY, DEPT-HSQC, HSQC, and HMBC. This sulfated galactan is composed preponderantly of 4-sulfated, 3-linked beta-D-galactopyranosyl units. In minor amounts, it is sulfated and glycosylated at C-6. Pyruvate groups are also found, forming five-membered cyclic ketals as 3,4-O-(1'carboxy)-ethylidene-beta-D-galactose residues. A comparison of sulfated galactans from different marine taxonomic groups revealed similar backbones of 3-beta-D-Galp-1.     

ESIMS analysis of fucoidan preparations from Costaria costata, extracted from alga at different life-stages

Four fucoidan fractions from brown alga Costaria costata, collected at different life-stages: vegetative, May (5F2 and 5F3) and generative, July (7F1 and 7F2) collections were characterized. It was found that seaweed synthesizes different set of fucoidans - one with high fucose content and substantial percentage of hexoses and uronic acid and lower sulfate content (7F1, 5F2 and 5F3) and other - highly sulfated galactofucan (7F2). Structural features of fractions 7F2 and 5F3 were predominantly determined by mass spectrometric analysis of low-molecular-weight (LMW) oligosaccharide fragments, obtained by autohydrolysis of 7F2 and mild acid hydrolysis of 5F3 fucoidans. It was found that oligosaccharides from 7F2 fractions were mainly built up of sulfated at C-2 and/or at C-2/C-4 (1→3)-linked α-l-fucopyranose residues. d-Galactose residues, sulfated either at C-2 or C-6, were found as parts of mixed di- and trisaccharides at both termini and, probably, internal. Fucose residues in 5F3 fucoidan fragments were sulfated at C-2 and sometimes at C-4. Galactose residues were sulfated at C-4 and less frequently at C-2. Resistant to hydrolysis fraction was probably a core, built up with fucose, mannose and glucuronic acid. Presumably, oligosaccharide fragments were branches at C-4 of GlcA. They were sulfated at C-2 and sometimes at C-4 (1→3)- and/or (1→4)-linked fucooligosaccharides (sometimes terminated with (1→3)-linked galactose) and sulfated at C-4 or C-2 (1→4)- or, probably, (1→6)-linked galactooligosaccharides, probably, with own branches, formed by (1→2)-linked galactose residues. Unsulfated xylose residues were probably terminal in chains built up of fucose. It was confirmed, that monosaccharide content and structure of fucoidans of vegetative algae changed following its life stage. Generative alga in general produced highly sulfated galactofucan having lower MW along with less sulfated mannoglucuronofucan with higher MW, which was extensively synthesized by vegetative algae.     

Chemical structure of the complex pyruvylated and sulfated agaran from the red seaweed Palisada flagellifera (Ceramiales, Rhodophyta)

A homogeneous agaran fraction from Palisada flagellifera (Laurencia complex, Rhodomelaceae, Ceramiales) was obtained by aqueous room-temperature extraction, followed by ion-exchange chromatography. This galactan presents a highly complex structure with at least 18 different types of derivatives. The A units were found mostly pyruvylated, 2-sulfated (～34%), and 6-methylated (～34%), with the latter partially 2- and 2,4-sulfated. Minor amounts of β-D-galactopyranosyl units 2-, 6- and 2,6-sulfated, 6-glycosylated, and non-substituted are also present. The B-units are L-sugars composed predominantly of their cyclized derivatives, 3,6-anhydrogalactose and 3,6-anhydro-2-O-methylgalactose (～56%). The former are linked to β-D-galactosyl (6-methyl) (6-glycosylated) units, as well as to 4,6-O-(1-carboxyethylidene)-β-D-galactose 2-sulfate in the proportion of 3:1.8, respectively. A significant amount (～18%) of the α-L-galactopyranosyl units are linked to pyruvylated β-D-galactose 2-sulfate residues. An important part of the B-units (20%) is represented by α-L-galactose 6-sulfate substituted on C-3 by xylosyl, galactosyl and/or 2,3-di-O-methylgalactose units or sulfate groups that preclude their cyclization to 3,6-anhydrogalactosyl derivative. The precursor units are present in relatively low percentages. Kinetic studies suggest that in P. flagellifera agaran the cyclizable units are linked to 6-O-methyl-β-D-galactosyl and/or β-D-galactosyl units (6-glycosylated). The structural complexity of this polysaccharide is increased by the presence of 2- and 3,6-sulfated α-L-galactoses, with the latter additionally 2-O-methylated. Therefore, the major subfraction obtained from the cold extract contains structurally complex sulfated, methylated, and pyruvylated agaran.     

A sulfated galactan from the mucilaginous sheath of the red filamentous alga Chroodactylon ornatum (Stylonematophyceae,Rhodophyta)

The pseudofilamentous red alga Chroodactylon ornatum was grown in f/2 culture medium with the addition of 10, 34, and 100 μM (nominal concentration) cupric sulfate. The bioassays were terminated at two selected end points (days 4 and 12). Growth inhibition, changes in pigment composition, and oxidative stress indicators such as phenolic compounds and lipid peroxidation (dosed as thiobarbituric reactive substances) were observed in cultures with 34 and 100-μM cupric sulfate. Quinacrine (Atebrin) and chlorotetracycline fluorochromes showed abundant vacuoles of acidic content, related with mucilage secretion. Structural analyses by methylation, desulfation-methylation, alkaline treatment, and NMR spectroscopy revealed that the mucilaginous sheath of C. ornatum contains a sulfated galactan with a backbone of alternating 3-linked β-d-galactopyranose and 4-linked α-l-galactopyranose moieties, i.e., an agaran. The absence of 3,6-anhydrogalactose and of its precursor unit (α-galactose 6-sulfate) were confirmed. The highly sulfated polysaccharide contained these ester groups on the C-2 and C-4 hydroxyl groups of the 3-linked unit and on C-3 of the 4-linked units. The large proportion of sulfate esterification in this polysaccharide can be related to the extracellular biosorption of copper divalent cation (2.5?±?0.4 mg per gram of dry weight) and to copper tolerance in bioassays.