The galactan system biosynthesized by the red seaweed Gymnogongrus tenuis (Phyllophoraceae) is constituted by major amounts of κ/ι-carrageenans, with predominance of ι-structures, which were isolated by extraction with hot water in high yield (∼ 45%). A small amount of non-cyclized carrageenans mostly of the ν-type was also obtained. Besides, 12% of these galactans are agaran structures, which were present in major quantities in the room temperature water extracts, but they were also found in the hot water extract. They are constituted by 3-linked β-D-galactose units partially substituted on C-6 with sulfate or single stubs of β-D-xylose and 4-linked residues that comprise α-L-galactose units partially sulfated or methoxylated on C-3 or sulfated on C-3 and C-6 and 3,6-anhydro-α-L-galactose. Related structural patterns were previously found for agarans synthesized by other carrageenophytes. Results presented here show that these agarans are low molecular weight molecules independent of the carrageenan structures, with strong interactions between them.

A highly sulfated 3-linked β-arabinan (Ab1) with arabinose in the pyranose form was obtained from green seaweed Codium vermilara (Bryopsidales). It comprised major amounts of units sulfated on C-2 and C-4 and constitutes the first polysaccharide of this type isolated in the pure form and fully characterized. Ab1 showed anticoagulant activity by global coagulation tests. Less sulfated arabinans obtained from the same seaweed have less or no activity. Ab1 exerts its activity through direct and indirect (antithrombin- and heparin cofactor II-mediated) inhibition of thrombin. Direct thrombin inhibition was studied in detail. By native PAGE, it was possible to detect formation of a complex between Ab1 and human thrombin (HT). Ab1 binding to HT was measured by fluorescence spectroscopy. CD spectra of the Ab1 complex suggested that ligand binding induced a small conformational change on HT. Ab1-thrombin interactions were studied by molecular dynamic simulations using the persulfated octasaccharide as model compound. Most carbohydrate-protein contacts would occur by interaction of sulfate groups with basic amino acid residues on the surface of the enzyme, more than 60% of them being performed by the exosite 2-composing residues. In these interactions, the sulfate groups on C-2 were shown to interact more intensely with the thrombin structure. In contrast, the disulfated oligosaccharide does not promote major conformational modifications at the catalytic site when complexed to exosite 1. These results show that this novel pyranosic sulfated arabinan Ab1 exerts its anticoagulant activity by a mechanism different from those found previously for other sulfated polysaccharides and glycosaminoglycans.

Xylans from five seaweeds belonging to the order Nemaliales (Galaxaura marginata, Galaxaura obtusata, Tricleocarpa cylindrica, Tricleocarpa fragilis, and Scinaia halliae) and one of the order Palmariales (Palmaria palmata) collected on the Brazilian coasts were extracted with hot water and purified from acid xylomannans and/or xylogalactans through Cetavlon precipitation of the acid polysaccharides. The β-d-(1→4), β-d-(1→3) 'mixed linkage' structures were determined using methylation analysis and 1D and 2D NMR spectroscopy. The presence of large sequences of β-(1→4)-linked units suggests transient aggregates of ribbon- or helical-ordered structures that would explain the low optical rotations.

β-(1→4)-d-Mannans constitute the major component of the cell wall of seaweeds of the genus Codium and replace cellulose as the major fibrillar component. They were found as major constituents of the hot water extracts of green seaweed Codium vermilara. By anion exchange chromatography of the first hot water extract, a pure sulfated mannan with a molar ratio carbohydrates:sulfate of 2.7:1 was isolated. The sulfate groups are linked to C-2 of 23% of the mannose units, while most of these units are not substituted. This degree of sulfation would explain the higher solubility of the polymer, compared to that of the non-sulfated fibrillar mannan. Taking into account that the fibrillar polysaccharides form two external layers in the cell wall, while the sulfated polymers are forming an amorphous central layer, it is postulated that these sulfated mannans could act as an interphase region between the neutral and acidic layers.

The anticoagulant behavior of sulfated polysaccharides from seaweeds is reviewed based on their chemical structures. Analysis of the literature suggested that the driving force for the formation of the sulfated polysaccharide/protein complex is the non-specific polar interaction between the negatively and positively charged groups in the polysaccharide and protein, respectively and that the complex is further stabilized by short-range interactions. The polysaccharide binding site should be able to go through the following conformational steps in the formation of the complex: random coil-->ordered conformation--> low distortion of this conformation to form a complementary fitting structure with the protein backbone. The sulfated monosaccharide units with the highest potential for anticoagulant activity should have two sulfate groups and a glycosidic linkage on the pyranose ring with C-2, C-3 and C-4 in 2S, 3R, 4R or 2R, 3S, 4S configurations for galactose, fucose and arabinose and 2S, 3S, 4R, for rhamnose. Three distributions of these substituents appear: 3-linked 2,4-disulfated units, 4-linked 2,3-disulfated units and 2-linked 3,4-disulfated residues. These types of units have the possibility, through the equilibrium of the chair conformations, to place their sulfate groups in adequate special positions to interact with basic groups of the protein. The anticoagulant activity is mainly attributed to thrombin inhibition mediated by antithrombin and/or heparin cofactor II, with different effectivenesses depending of the compound. Other mechanisms are also proposed and these differences could be attributed to the diversity of structures of the polysaccharides evaluated and to the fact that one compound may have more than one target protease.

MALDI-TOF mass spectrometry analyses of several oligosaccharides (aldoses) and oligosaccharide alditols derived from agaroses, kappa- and iota-carrageenans using different matrices (2,5-dihydroxybenzoic acid, nor-harmane, ferulic acid, and the ionic liquid matrices 2,5-dihydroxybenzoic acid–n-butylamine and ferulic acid–n-butylamine) were conducted. These carbohydrates were selected as model compounds to study the MALDI prompt and post-source decay (PSD) fragmentation processes of both families of oligosaccharides. Sulfated alditols showed in the negative-ion mode the molecular ion as [M−Na]− together with the species yielded by their prompt fragmentation (mainly desulfation) while the sulfated oligosaccharides (aldoses) showed mainly glycosidic prompt fragmentation (glycosidic C-cleavages and desulfation). Non-sulfated aldoses and alditols, which could only be analyzed in positive-ion mode ([M+Na]+), did not suffer any prompt fragmentation. The former yielded cross-ring fragmentation in the PSD mode. Best results were obtained by using 2,5-dihydroxybenzoic acid and/or nor-harmane as matrices for all the compounds studied.

The anticoagulant behavior of sulfated polysaccharides from seaweeds is reviewed based on their chemical structures. Analysis of the literature suggested that the driving force for the formation of the sulfated polysaccharide/protein complex is the non-specific polar interaction between the negatively and positively charged groups in the polysaccharide and protein, respectively and that the complex is further stabilized by short-range interactions. The polysaccharide binding site should be able to go through the following conformational steps in the formation of the complex: random coil-->ordered conformation--> low distortion of this conformation to form a complementary fitting structure with the protein backbone. The sulfated monosaccharide units with the highest potential for anticoagulant activity should have two sulfate groups and a glycosidic linkage on the pyranose ring with C-2, C-3 and C-4 in 2S, 3R, 4R or 2R, 3S, 4S configurations for galactose, fucose and arabinose and 2S, 3S, 4R, for rhamnose. Three distributions of these substituents appear: 3-linked 2,4-disulfated units, 4-linked 2,3-disulfated units and 2-linked 3,4-disulfated residues. These types of units have the possibility, through the equilibrium of the chair conformations, to place their sulfate groups in adequate special positions to interact with basic groups of the protein. The anticoagulant activity is mainly attributed to thrombin inhibition mediated by antithrombin and/or heparin cofactor II, with different effectivenesses depending of the compound. Other mechanisms are also proposed and these differences could be attributed to the diversity of structures of the polysaccharides evaluated and to the fact that one compound may have more than one target protease.

Several points concerning the presence in red seaweeds of galactans with cooccurrence of carrageenan and agaran structures (DL-hybrid galactans) are addressed:a. Analysis of D- and L-galactose enantiomers and their methylated and cyclic derivatives. Spectroscopic, enzymatic and chemical methodologies.b. Systems of galactans, in which the presence of D- and L-galactose has been determined (DL-galactan systems).c. The possible deviant structures of carrageenans and agarans in DL-hybrid galactans.d. The isolation of "pure" carrageenans and agarans from DL-galactan systems.e. The formation of molecular complexes between carrageenans, agarans and carrageenans/agarans.f. DL-hybrid galactans or molecular complexes: Evidences in favor of each hypothesis.

Cryptonemia seminervis biosynthesizes a family of D,L-hybrid galactans based on the classical 3-linked beta-D-galactopyranosyl-->4-linked alpha-D- and alpha-L-galactopyranosyl alternating sequence (A-units-->B-units) with major amounts of alpha-D- and alpha-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 beta-D-galactopyranosyl 2-sulfate (20 and 11.9 mol%), beta-d-galactopyranosyl 2-sulfate 4,6-O-(1'-carboxyethylidene) (8.9 and 6.0 mol%) and beta-D-galactopyranosyl 2,6-sulfate (5.4 and 18.6 mol%), together with 3,6-anhydro-alpha-l-galactopyranosyl (11.4 and 7.3 mol%) and 3,6-anhydro-alpha-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 alpha-L-galactopyranosyl units from precursor units (alpha-L-galactose 6-sulfate and alpha-L-galactose 2,6-sulfate). Kinetic studies on this 3,6-anhydro cyclization show a rate constant of 5.2 x 10(4)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 approximately 2:1.

Sulfated polysaccharides were localized in the cuticle, cortex and medulla of the gametophyte thallus, being more concentrated in the intercellular matrix than in the cell walls. During the water extraction sequence, a small percentage of galactan sulfates (5.1% of dry seaweed) with average low Mr (6-11.4kDa) were extracted at room temperature without disturbing the cellular arrangement, while sulfated galactans of average medium Mr (18-45kDa) were obtained by further hot-water extractions (52.4% of dry seaweed), with diorganization of the tissue. The residue (40.0% of dry seaweed) still contained carrageenan-type (major) and agaran-type (minor) galactans. Part of these galactans was extracted with 8.4% LiCl solution in DMSO, from which "pure" κ/ι-carrageenans were isolated. Carrageenans and agarans were extracted in a ratio 1:0.5, showing the highest amount of agaran-structures for a carrageenophyte. The galactans comprise alternating 4-sulfated (major) and non-sulfated (minor) 3-linked β-d-galactopyranose units, and 4-linked α-galactopyranose units with the following substitutions: (i) non-sulfated and 2-sulfated 3,6-anhydro-α-d-galactopyranose residues in the carrageenan-structures, which belong to the κ-family (κ/ι-carrageenans); (ii) 3-sulfated α-l-galactopyranose units and 2-sulfated 3,6-anhydro-α-l-galactopyranose residues in the agaran-structures. Alkaline treatment and alkaline dialysis of the main extracts gave "pure" κ/ι-carrageenans, showing that carrageenan molecules are extracted together with low Mr agarans or agaran-dl-hybrids.

Codium fragile and Codium vermilara biosynthesize water-soluble sulfated arabinans and galactans (and/or sulfated arabinogalactans), alpha(1-->4)-D-glucans and beta(1-->4)-D-mannans. The former polysaccharides are composed by 3-linked beta-D-galactopyranose and beta-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.

Matrix-assisted ultraviolet laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOF-MS) has shown to be a very useful technique for the study of the non-volatile and thermally non-stable N-acylated glycopyranosyl- and glycofuranosyl-amines. Of the several matrices tested, 2,5-dihydroxybenzoic acid (DHB) was the most effective giving good spectra in the positive-ion mode. In the linear and reflectron modes, the [M+Na](+) ions appeared with high intensity. Their fragmentation patterns were investigated by post-source decay (PSD) UV-MALDI-TOF-MS showing mainly cross-ring cleavages. In addition, N,O-acylated glycopyranosyl- and glycofuranosyl-amines were also analyzed by this technique. PSD UV-MALDI-TOF-MS gave significant signals for several primary fragment ions, which were proposed but not detected, or observed with very low abundance, in electron ionization mass spectrometry (EI-MS) experiments.

Bovine seminal plasma was submitted to chromatography on Con A-Sepharose. The “non-interacting”, “weakly-interacting” and “strongly-interacting” fractions were analyzed through UV-MALDI-TOF MS together with a subfraction of the “non-interacting” material, using sinapinic acid (SA) as matrix. The spectra were obtained in linear positive mode in the 4.0-90.0 kDa mass/charge range showing peaks in well defined zones, namely: 5.5-8.0 kDa, 10.0-12.0 kDa, 12.5-14.0 kDa (major), 23.2-23.7 kDa, 26.1-27.5 kDa and 38.0-40.0 kDa. High sensitivity spectra showed some very small peaks until 90 kDa. Bovine seminal protein (BSP-A3), acidic seminal fluid protein (αSFP) and PDC-109 glycoproteins (BSP-A1 and BSP-A2) were identified. Caltrin, the human epididymis-specific glycoprotein (HE4), the calcium transport inhibitor protein, the inhibitor of metalloprotease 2 (TIMP-2), osteopontin (OPN) and the prostatic acid protease (PAP) were tentatively identified. The molecular weight of some peaks can be arranged in a sequence from that of BSP-A3 going through the molecular weights of glycoforms (including the known BSP-A1 and BSP-A2) which differ in the amounts of neutral hexoses and sialic acids, composing a BSP-family more extended than previously reported. Another two families could be builded up from proteins of molecular weight of about 12730 and 12750 Da and glycoforms which differ from them also by hexoses and sialic acids. The structures of the deduced O-linked oligosaccharides of the glycoforms are in complete agreement to that determined for the BSP-A1 oligosaccharide. Small differences in the m. w. of some (glyco)proteins were attributed to genetic polymorphysm. The identification of proteins and O-linked glycoproteins in the “interacting” fractions of the chromatography suggests that the fractionation was not due to specific affinity interactions but to non-specific hydrophobic interactions of the proteins with the hydrophobic pocked of the Con A.

The partially cyclized mu/nu-carrageenan 1C3, isolated from the red seaweed Gigartina skottsbergii, was previously shown to be a potent inhibitor of the in vitro replication of Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). Here the protective effect of 1C3 in a murine model of intraperitoneal ( i. p.) HSV-1 infection was evaluated. OF1 mice were i. p. infected with 5 x 10 (5) PFU of HSV-1 KOS strain, and the effects of different treatments with 1C3 were studied. When 30 mg/kg of body weight of 1C3 was administered by the i. p. route immediately after HSV-1 infection, 87.5 % survival of the animals was achieved (p < 0.005), associated with a delay in the mean day of death in 1C3-treated non-surviving mice. Animal survival was not improved when multiple doses of 1C3 were also given in the period 1 - 48 h post-infection, and no protection was afforded when treatment was started after 24 h of infection. When virus and compound were injected by different routes, i. p. and intravenous ( i. v.), respectively, a still significant protection was achieved (40 % survival, p < 0.05). No toxicity of 1C3 for the animals was recorded. The pharmacokinetic properties were analyzed after injection of 1C3 into the tail vein by monitoring of [ (3)H]-1C3 in plasma and organs and by a bioassay of the anti-HSV-1 activity remaining in serum after non-radioactive 1C3 inoculation. A very rapid disappearance of the compound from the blood was observed since only 5.9 - 0.9 % of the radioactivity of the initially administered [ (3)H]-1C3 appeared in the plasma between 5-300 minutes after administration. A transient peak of radioactivity was detected in the kidney 15 minutes after inoculation. The bioassay confirms the presence of the compound circulating in a biologically active form up to 1 hour after injection.

A partially cyclized mu/nu-carrageenan from Gigartina skottsbergii was submitted to autohydrolysis and the resulting mixture of oligosaccharides was fractionated using sequential dialysis through tubings of molecular weight cutoff 12000, 3500 and 1000. In this way four fractions were separated and characterized. Structural analysis of the major fraction, obtained from the solution inside the dialysis tubing of molecular weight cutoff 12000 (D120), was carried out. A significant difference was found by 13 C NMR spectroscopy and UV-MALDI-TOF mass spectrometry: in the 13 C NMR spectrum the reducing end-chain unit was the hydrated aldehyde of 3,6-anhydrogalactose while the UV-MALDI-TOF mass spectrum showed an unimodal distribution of even and odd peaks, suggesting fragmentation of glycosidic linkages.

The conformational maps of eight derivatives of the disaccharide alpha-D-Galp-(1-->3)-beta-D-Galp sulfated in different positions were obtained using the MM3 force-field specially parameterized for sulfate ester groups. As occurred with MM2, the conformational flexibility of the glycosidic linkage is only slightly hindered by sulfation. A substantial effect of sulfation of the beta-D-galactose unit on position 4 shifts the global minimum to positive psi(H) (C1'-O3-C3-H3) angles, while sulfation at position 2 of the same unit deepens the well at negative psi(H) angles. On the other hand, sulfation on the alpha-D-galactose unit has a lesser effect, which in any case tends to stabilize the minimum at negative psi(H) angles.

Three xylan fractions isolated from the red seaweed Nothogenia fastigiata (Nemaliales) were analyzed by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOFMS). UV-MALDI-TOFMS was carried out in the linear and reflectron modes, and as routine in the positive and negative ion modes. Of the several matrices tested, nor-harmane was the only effective one giving good spectra in the positive ion mode. The number-average molar masses of two of the fractions, calculated from the distribution profiles, were lower than those determined previously by (1)H NMR analysis, suggesting a decrease in the ionization efficiency with increasing molecular weight; weight-average molar mass and polydispersity index were also determined. As the xylans retained small but significant quantities of calcium salts, the influence of added Ca(2+) as CaCl(2) on UV-MALDI-MS was investigated. The simultaneous addition of sodium chloride and calcium chloride was also analyzed. Addition of sodium chloride did not change the distribution profile of the native sample showing that the inhibitory effect is due to Ca(2+) and not to Cl(-). Addition of calcium chloride with 1:1 analyte/salt molar ratio gave spectra with less efficient desorption/ionization of oligomers; the signals of these oligomers were completely suppressed when the addition of the salt became massive (1:100 analyte/salt molar ratio).

The whole system of low molecular weight/highly hydrophilic weak-bond linked components of the endosperm of the legume seed of Gleditsia triacanthos was studied on the basis of previous and new data. This reserve incrustant/fibrillar cell wall system may be considered a continuous phase formed by three types of compounds, namely: a) galactomannans/galactomannan-like oligosaccharides highly substituted (reserve) and with low branching (near mannans, fibrillar); b) "mixed- linkage"-glucans (reserve) and cellulose (fibrillar), and c) arabinans (reserve) and low-substituted arabinogalactans (fibrillar). The biological role of these "weak bond-linked" components is not fully understood but they may be multifunctional molecules, as they provide not only the weak bonds necessary for the swelling properties of the endosperm, but also they may contribute to the stabilization of enzymes in the dry endosperm by preventing dehydration.