RESUMO
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 (â¼ 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 ß-D-galactose units partially substituted on C-6 with sulfate or single stubs of ß-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.
Assuntos
Carragenina/química , Galactanos/química , Rodófitas/química , Alga Marinha/química , Carragenina/isolamento & purificação , Galactanos/isolamento & purificação , Temperatura Alta , Água/químicaRESUMO
A highly sulfated 3-linked ß-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.
Assuntos
Anticoagulantes/farmacologia , Polissacarídeos/química , Piranos/química , Trombina/química , Animais , Anticoagulantes/química , Biofísica/métodos , Coagulação Sanguínea , Configuração de Carboidratos , Bovinos , Parede Celular/metabolismo , Dicroísmo Circular , Eletroforese , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Cinética , Espectroscopia de Ressonância Magnética/métodos , Metilação , Modelos Químicos , Conformação Molecular , Ligação Proteica , Alga Marinha , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Trombina/antagonistas & inibidoresRESUMO
Xylans from five seaweeds belonging to the order Nemaliales (Galaxaura marginata, Galaxaura obtusata, Tricleocarpacylindrica, 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 ß-D-(1â4), ß-D-(1â3) 'mixed linkage' structures were determined using methylation analysis and 1D and 2D NMR spectroscopy. The presence of large sequences of ß-(1â4)-linked units suggests transient aggregates of ribbon- or helical-ordered structures that would explain the low optical rotations.
Assuntos
Rodófitas/química , Xilanos/química , Configuração de Carboidratos , Espectroscopia de Ressonância Magnética , Metilação , Fenômenos Ópticos , Estereoisomerismo , Xilanos/isolamento & purificaçãoRESUMO
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.
Assuntos
Galactanos/química , Rodófitas/química , Ressonância Magnética Nuclear Biomolecular , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
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 ß-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.
RESUMO
The polysaccharide extracted from cystocarpic Callophyllis variegata was fractionated with potassium chloride yielding three small fractions that precipitated in the ranges of 0-0.05 M KCl, 1.20-1.25 M KCl, and 1.80-2.00 M KCl, and a main product soluble in 2.00 M KCl. These fractions were analyzed, and as the first one contained very high amounts of protein, it was not studied further. Structural analysis of the rest of the fractions (F1-F3) was carried out by methylation, desulfation-methylation, IR, and 13C NMR spectroscopy. The results are consistent for F1 with a carrageenan-type backbone mainly constituted by beta-D-galactose 2-sulfate linked to alpha-D-galactose 2,3,6-trisulfate and beta-D-galactose 2,4-disulfate linked to 3,6-anhydro-D-galactose 2-sulfate as dominant diads. In F2 these diads are present together with low amounts of beta-D-galactose 2-sulfate linked to 3,6-anhydro-D-galactose 2-sulfate, whose contribution becomes higher in F3. In addition, minor but significant amounts of L-galactose were detected. F1-F3 showed potent antiviral activity against herpes simplex types 1 and 2 and dengue type 2.
Assuntos
Antivirais , Galactanos , Alga Marinha/química , Antivirais/síntese química , Antivirais/química , Antivirais/farmacologia , Isótopos de Carbono , Galactanos/síntese química , Galactanos/química , Galactanos/farmacologia , Espectroscopia de Ressonância Magnética , Extratos Vegetais/química , Hidróxido de Sódio/química , Espectrofotometria InfravermelhoRESUMO
Cryptonemia crenulata biosynthesizes a family of dl-hybrid galactans that are based on the classical 3-linked beta-d-galactopyranosyl-->4-linked alpha-galactopyranosyl alternating sequence (A-units-->B-units). The dispersion of structures in these galactans is based on four factors, namely: (a) the amount and position of substituent groups as sulfate (major), pyruvic acid ketals, methoxyl and side substituents of beta-D-xylose and/or beta-D-galactose; (b) the ratio galactose/3,6-anhydrogalactose in the B-units; (c) the ratio D-/L-galactoses and 3,6-anhydrogalactoses also in the B-units and (d) the sequence of the diads in the linear backbone. Alkali treatment carried out on the major fraction produced a nearly quantitative formation of 3,6-anhydrogalactose units from precursor units (alpha-galactose 6-sulfate (major) and alpha-galactose 2,6-sulfate, minor). Kinetic studies show a rate constant, for the diad G2S-D(L) 6-S, of 1.7 x 10(4)s(-1) indicating a reaction faster than in lambda-carrageenans but slower than in porphyrans.
Assuntos
Galactanos/química , Galactanos/isolamento & purificação , Hidróxidos/química , Rodófitas/química , Cromatografia por Troca Iônica , Ciclização , Cinética , Metilação , Ressonância Magnética Nuclear Biomolecular , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
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.
Assuntos
Galactanos/isolamento & purificação , Alga Marinha/química , Cálcio/análise , Carragenina/química , Cromatografia por Troca Iônica , Galactanos/química , Magnésio/análise , Monossacarídeos/análise , Ressonância Magnética Nuclear Biomolecular , SulfatosRESUMO
Several sulfated seaweed polysaccharides show high antiviral activity against enveloped viruses, including important human pathogens such as human immunodeficiency virus, herpes simplex virus, human cytomegalovirus, dengue virus and respiratory syncytial virus. They can be obtained in major amounts and at low costs, have low toxicity and in some cases, lack anticoagulant effects. Even if the systemic applications have many drawbacks, their structure and mode of action indicate potential for topical uses to prevent virus infection. The herpes simplex viruses attach to cells by an interaction between the envelope glycoprotein C and the cell surface heparan sulfate (HS). The virus-cell complex is formed by ionic interactions between the anionic (mainly sulfate) groups in the polysaccharide and basic amino acids of the glycoprotein, and non-ionic ones depending on hydrophobic amino acids interspersed between the basic ones in the glycoprotein-binding zone. Hypothesis are advanced of the corresponding hydrophobic structures in the polysaccharides. The antiviral activity of the sulfated seaweed polysaccharides is based on the formation of formally similar complexes that block the interaction of the viruses with the cells. Correlations are established between different structural parameters and antiviral activity. The minimal, ionic and hydrophobic, structures in the seaweed polysaccharides were hypothesized by comparison of the polysaccharides with the known minimal binding structure in HS/heparin, together with a correlation between those structures of the polysaccharides and their antiviral activity.
Assuntos
Antivirais/química , Polissacarídeos/química , Alga Marinha/química , Sulfatos/química , Antivirais/isolamento & purificação , Antivirais/farmacologia , Sequência de Carboidratos , Humanos , Dados de Sequência Molecular , Polissacarídeos/isolamento & purificação , Polissacarídeos/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
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.