Semisynthesis of new sulfated heterorhamnan derivatives obtained from green seaweed Gayralia brasiliensis and evaluation of their anticoagulant activity.

Marine green algae produce sulfated polysaccharides with diverse structures and a wide range of biological activities. This study aimed to enhance the biotechnological potential of sulfated heterorhamnan (Gb1) from Gayralia brasiliensis by chemically modifying it for improved or new biological functions. Using controlled Smith Degradation (GBS) and O-alkylation with 3-chloropropylamine, we synthesized partially water-soluble amine derivatives. GBS modification increase sulfate groups (29.3 to 37.5 %) and α-l-rhamnose units (69.9 to 81.2 mol%), reducing xylose and glucose, compared to Gb1. The backbone featured predominantly 3- and 2-linked α-l-rhamnosyl and 2,3- linked α-l-rhamnosyl units as branching points. Infrared and NMR analyses confirmed the substitution of hydroxyl groups with aminoalkyl groups. The modified compounds, GBS-AHCs and GBS-AHK, exhibited altered anticoagulant properties. GBS-AHCs showed reduced effectiveness in the APTT assay, while GBS-AHK maintained a similar anticoagulant activity level to Gb1 and GBS. Increased nitrogen content and N-alkylation in GBS-AHCs compared to GBS-AHK may explain their structural differences. The chemical modification proposed did not enhance its anticoagulant activity, possibly due to the introduction of amino groups and a positive charge to the polymer. This characteristic presents new opportunities for investigating the potential of these polysaccharides in various biological applications, such as antimicrobial and antitumoral activities.

Semisynthesis and characterization of versatile azide intermediates using sodium alginate and its homopolymeric derivatives as starting material.

Alginate, a polyuronic biopolymer composed of mannuronic and guluronic acid units, contain hydroxyl and carboxyl groups as targeting modification sites to obtain structures with new and/or improved biological properties. The copper-catalyzed azide-alkyne cycloaddition (CuAAC) is a versatile click reaction for polymer functionalization, but it typically requires a "pre-click" modification to introduce azide or alkyne groups. Here, we described a straightforward chemical path to selectively modify alginate carboxyl groups producing versatile azido derivatives through N-acylation using 3-azydopropylamine. The resulting azide-functionalized polysaccharides underwent click chemistry to yield amino derivatives, confirmed by NMR and FTIR analyses. The 1H NMR spectrum reveals a characteristic triazole group signal at 8.15 ppm. The absence of the azide FTIR band for all amino derivatives, previously observed for the N-acylation products, indicated reaction success. Antibacterial and antioxidant assessments revealed that the initial polysaccharide lacks E. coli inhibition, while the click chemistry-derived amine products exhibit growth inhibition at 5.0 mg/mL. Lower molecular weight derivatives demonstrate superior DPPH scavenging ability, particularly amino-derivatives (24-33 % at 1.2 mg/mL). This innovative chemical pathway offers a promising strategy for developing polysaccharide structures with enhanced properties, demonstrating potential applications in various fields.

Synthesis of C6-amino agarose and evaluation of its antibacterial activity.

In this paper, the biologically inert agarose was selectively modified at C6 of ß-d-Galp units to produce an amino derivative with antibacterial property. The synthetic route involved the preparation of tosyl and azido agarose intermediates. All the polysaccharide derivatives were characterized by mono- and bidimensional 1H and 13C NMR and FT-IR analysis. A water-soluble amino polymer (Mw = 39,000 g mol-1, DSamino = 0.50) was produced by partial acid hydrolysis showing bactericidal and bacteriostatic activity against P. aeruginosa (ATCC 9027), S. aureus (ATCC 6538), and E. coli (ATCC 25922), with MIC values lower than 2.5 mg mL-1 and MBC values ranging from 2.5 to 5.0 mg mL-1.

Semi-synthesis of hybrid ulvan-kappa-carrabiose polysaccharides and evaluation of their cytotoxic and anticoagulant effects.

In this study we described the synthesis of a hybrid polysaccharide harboring moieties of ulvan and kappa-carrabiose. Alkylamines (1,3-diaminopropane and 1,6-diaminohexane) were selectively inserted into ß-D-GlcAp and α-L-IdoAp units in the ulvan structure via an amide bond formation producing ulvan-amide derivatives F-DAP (N% = 1.77; Mw = 208 kg mol-1) and F-DAH (N% = 1.77; Mw = 202 kg mol-1), which were reacted with kappa-carrabiose via reductive amination to produce hybrid ulvan-kappa-carrabiose polysaccharides F-DAP-Kb (N% = 1.56; Mw = 206 kg mol-1) and F-DAH-Kb (N% = 1.16; Mw = 200 kg mol-1). All the ulvan derivatives were characterized by 1H and 13C NMR spectroscopy and did not show cytotoxicity against human dermal fibroblasts (HDFa) at the concentrations of 25, 100, and 500 µg mL-1, neither anticoagulant properties at the range of 10-150 µg mL-1. Therefore, the ulvan-amide derivatives and the hybrid ulvan-kappa-carrabiose polysaccharides showed good biocompatibility in vitro, presenting as worthy candidates for tailoring scaffolds for biomedical applications.

Semi-synthesis of N-alkyl-kappa-carrageenan derivatives and evaluation of their antibacterial activity.

In this article, we describe the semi-synthesis of N-alkyl-kappa-carrageenan derivatives and their antibacterial activity against Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 8739), and Pseudomonas aeruginosa (ATCC 9027). Kappa-carrageenan was submitted to partial acid hydrolysis promoting the selective cleavage of α-glycosidic bonds involving 3,6-anhydro-α-D-Galp units, giving rise to reducing low-molecular weight polysaccharide fragments, which were reacted with alkylamines of varying chain lengths by reductive amination. The carrageenan derivatives were characterized by HPSEC-MALLS-RID and 1D and 2D 1H and 13C NMR spectroscopy. The antibacterial activity of N-alkyl-kappa-carrageenan derivatives was compared with N-alkyl-(1-deoxylactitol-1-yl)-amines using a microdilution test, which indicated that inhibitory activity was dependent on the degree of substitution by hydrophobic groups at the polysaccharide structure. Comparing the effect of different N-alkyl chains, those with longer chains showed higher activity.

Conformational analysis of ulvans from Ulva fasciata and their anticoagulant polycarboxylic derivatives.

This study investigates conformational aspects of ulvans (F2) and their polycarboxyl derivatives obtained through periodate-chlorite oxidation (C3) followed by DEAE-Sephacel fractioning (C3b and C3c). Size exclusion chromatography coupled with laser light scattering and viscometric detection, in addition to circular dichroism (CD) and molecular modeling analyses, suggested that F2 had a compact sphere conformation with a helical motif as secondary structure. In contrast, all the polycarboxyl ulvans showed a random coil conformation, although C3c (NaSO3- 21.0%; COO- 1.81 mmol·g-1; Mw 18 kg·mol-1) had a more rigid and constrained backbone than C3 (NaSO3- 21.0%; COO- 1.81 mmol·g-1; Mw 49 kg·mol-1), largely due to its higher sulfate and carboxyl content. Despite the higher ionic character of C3c, its anticoagulant activity (ACA), determined by activated partial thromboplastin time (APTT) assay, was not improved compared to that of C3. Moreover, C3b (NaSO3- 14.1%; COO- 1.23 mmol·g-1; Mw 8.1 kg·mol-1) had higher activity than F2 (NaSO3- 20.6.%; COO- 0.36 mmol·g-1; Mw 123 kg·mol-1), even with its lower sulfate content and molar mass. These results suggest that the ACA of polycarboxyl ulvans relies on carboxyl and sulfate content and may depend, in addition, on a proper flexible conformation.

Chemical structure and snake antivenom properties of sulfated agarans obtained from Laurencia dendroidea (Ceramiales, Rhodophyta).

Aqueous and KCl-soluble polysaccharides were extracted from Laurencia dendroidea (Rhodomelaceae, Ceramiales) and their chemical profile was accessed by anion-exchange chromatography, chemical and spectroscopic analyses. The homogeneous agaran DHS-4 (181.3 × 103 g. mol-1, 21.3% of NaSO3) presents A units mostly 2-sulfated (18.9 mol%), nonsubstituted (15.3 mol%) and 6-O-methylated (10.1 mol%), while B units are l-sugars composed predominantly by galactose 6-sulfate precursor units (19.2 mol%) and 3,6-anhydrogalactose (13.8 mol%), besides non-precursor galactose 6-sulfate units bearing d-xylose substituents on C-3 (8.1 mol%). The crude KCl-soluble DHS agaran (20.5% of NaSO3) inhibited proteolysis and hemolysis induced by Lachesis muta and Bothrops jararaca venoms. DHS was able to inhibit up to 75% the L. muta venom hemorrhagic effect and to reduce the lethality displayed by B. jararaca venom, increasing the mice survival time up to 3 times. Therefore, this agaran has high potential to be used as an additional tool to treat snakebite envenomation.

Effects of carboxyl group on the anticoagulant activity of oxidized carrageenans.

In this paper, carrageenans having distinct sulfation patterns (κ-, ι-, ι/ν-, θ- and λ-carrageenans), were fully or partially oxidized at C-6 of the ß-d-Galp units using 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and trichloroisocyanuric acid (TCCA) in bicarbonate buffer. The modified carrageenans were characterized by mono- and bidimensional 1H and 13C NMR spectroscopy. The influence of the sulfate and carboxyl groups onto anticoagulant activity was evaluated using Activated Partial Thromboplastin Time (aPTT) in vitro assay. The results showed a synergic effect of the carboxyl groups on the anticoagulant activity, which was dependent on the regiochemistry of the sulfate groups in the polysaccharide backbone. Sulfate groups at C2 of the ß-d-GalAp units appeared to positively influence the anticoagulant effect in comparison to C4-sulfate samples. Also, the partially oxidized κ-carrageenan derivative (κLO) showed better anticoagulant effect than the fully oxidized carrageenan (κHO).

Modification of ulvans via periodate-chlorite oxidation: Chemical characterization and anticoagulant activity.

Native (F2) and carboxyl-reduced (R) ulvans from Ulva fasciata were sequentially oxidized with periodate-chlorite affording the polycarboxyl ulvans C1, C2 and C3 (1.20, 1.41 and 1.81 mmol g-1 of COOH, respectively; 19.7, 21.3 and 21.0% of NaSO3, respectively) and R-C3 (1.86 mmol g-1 of COOH; NaSO3 = 22.7%), respectively. APTT assay (polysaccharide fractions at 150 µg mL-1) showed clotting time of 45.6 s for F2 fraction. For polycarboxyl ulvans C1, C2, C3 and R-C3 the clotting times were 101.0, 122.2, 222.0 and 227.0 s, respectively. Comparison of the APTT assay results using ulvans chemically modified by carboxyl-reduction, desulfation, periodate oxidation and/or chlorite oxidation showed the anticoagulant activity of polycarboxyl ulvans is dependent of the sulfate groups present in the native polymer. In addition, the increase of the anticoagulant activity was accompanied by the increasing of the carboxyl groups and the content of this acidic substituent seems to be more important than its positioning.

Aqueous semisynthesis of C-glycoside glycamines from agarose.

Agarose was herein employed as starting material to produce primary, secondary and tertiary C-glycoside glycamines, including mono- and disaccharide structures. The semisynthetic approach utilized was generally based on polysaccharide-controlled hydrolysis followed by reductive amination. All reactions were conducted in aqueous media and without the need of hydroxyl group protection. We were able to identify optimal conditions for the reductive amination of agar hydrolysis products and to overcome the major difficulties related to this kind of reaction, also extending it to reducing anhydrosugars. The excess of ammonium acetate, methyl- or dimethylamine, and the use of a diluted basic (pH 11) reaction media were identified as important aspects to achieve improved yields, as well as to decrease the amount of byproducts commonly related to reductive amination of carbohydrates. This strategy allowed the transposition of the 3,6-anhydro-α-L-galactopyranose unit (naturally present in the agarose structure) to all glycamines synthesized, constituting an amino-substituted C-threofuranoside moiety, which is closely related to (+)-muscarine.

Ulvans induce resistance against plant pathogenic fungi independently of their sulfation degree.

The present work aimed to evaluate the defense responses induced by chemically sulfated ulvans in Arabidopsis thaliana plants against the phytopathogenic fungi Alternaria brassicicola and Colletotrichum higginsianum. Derivatives with growing sulfate content (from 20.9 to 36.6%) were prepared with SO3-pyridine complex in formamide. NMR and FTIR spectroscopic analyses confirmed the increase of sulfate groups after the chemical sulfation process. The native sulfated polysaccharide (18.9% of sulfate) and its chemically sulfated derivatives similarly reduced the severity of both pathogenic fungi infections. Collectively, our results suggest that ulvans induce resistance against both fungal pathogens independently of its sulfation degree.

Selective sulfation of carrageenans and the influence of sulfate regiochemistry on anticoagulant properties.

Sulfated polysaccharides are recognized for their broad range of biological activities, including anticoagulant properties. The positions occupied by the sulfate groups are often related to the level of the inherent biological activity. Herein the naturally sulfated galactans, kappa-, iota- and theta-carrageenan, were additionally sulfated by regioselective means. The anticoagulant activity of the resulting samples was then studied using the aPTT in vitro assay. The influence of sulfate regiochemistry on the anticoagulant activity was evaluated. From kappa-carrageenan three rare polysaccharides were synthesized, one of them involved a synthetic route with an amphiphilic polysaccharide intermediate containing pivaloyl groups. Iota- and theta-carrageenan were utilized in a selective C6 sulfation at ß-D-Galp units to produce different structures comprising trisulfated diads. All the samples were characterized by NMR (1D and 2D). The resulting aPPT measurements suggested that sulfation at C2 of 3,6-anhydro-α-D-Galp and C6 of ß-D-Galp increased the anticoagulant activity.

ß-D-(1→4), ß-D-(1→3) 'mixed linkage' xylans from red seaweeds of the order Nemaliales and Palmariales.

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-(1→4), ß-D-(1→3) 'mixed linkage' structures were determined using methylation analysis and 1D and 2D NMR spectroscopy. The presence of large sequences of ß-(1→4)-linked units suggests transient aggregates of ribbon- or helical-ordered structures that would explain the low optical rotations.

Brown algae overproduce cell wall polysaccharides as a protection mechanism against the heavy metal toxicity.

Brown algae are often used as heavy metal biomonitors and biosorbents because they can accumulate high concentrations of metals. Cation-exchange performed by cell wall polysaccharides is pointed out as the main chemical mechanism for the metal sequestration. Here, we biochemically investigated if the brown alga Padina gymnospora living in a heavy metal contaminated area would modify their polysaccharidic content. We exposed non-living biomass to Cd and Pb and studied the metals adsorption and localization. We found that raw dried polysaccharides, sulfate groups, uronic acids, fucose, mannose, and galactose were significantly higher in contaminated algae compared with the control ones. Metal concentrations adsorbed by non-living biomass were rising comparatively to the tested concentrations. Electron microscopy showed numerous granules in the cell walls and X-ray microanalysis revealed Cd as the main element. We concluded that P. gymnospora overproduces cell wall polysaccharides when exposed to high metal concentrations as a defense mechanism.

Dihydropyridine C-glycoconjugates by organocatalytic Hantzsch cyclocondensation. Stereoselective synthesis of alpha-threofuranose C-nucleoside enantiomers.

The Hantzsch reaction of C-glycosyl aldehyde/enamino ester/beta-ketoester systems under l-proline catalysis to give dihydropyridine C-glycoconjugates is reported. Asymmetric cyclocondensations of differentially substituted enamine and beta-dicarbonyl components with formyl alpha-L-C-threofuranoside and with the alpha-D-isomer were also carried out. Each reaction occurred with high yet opposite stereoselectivity (de >95%) so that the pair of alpha-threofuranose C-nucleoside enantiomers was prepared.

Production of carbohydrate building blocks from red seaweed polysaccharides. Efficient conversion of galactans into C-glycosyl aldehydes.

Agarans and carrageenans are abundant natural polysaccharides which are obtained on a large scale by water extraction from a variety of red seaweeds. These galactans, in addition to being valuable products for the pharmaceutical and food industries, are low cost starting materials for the preparation of useful and rare carbohydrate-based building blocks whose access by total synthesis is difficult and expensive. The semisynthesis of two sets of C-glycosyl aldehydes with l- and d-configuration from agarose and kappa-carrageenan respectively is described. Succinctly, the partial acid-catalyzed mercaptolysis of the two galactans under mild conditions afforded agarobiose and carrabiose (beta-d-Galp-(1-->4)-3,6-anhydro-aldehydo-l- and d-galactose, respectively) derivatives. Complete depolymerization of agarose and kappa-carrageenan under harsher conditions produced 3,6-anhydro l- and d-galactose aldehyde derivatives. Chain shortening of these products via alditol formation and oxidative carbon-carbon bond cleavage furnished C-formyl alpha-l- and alpha-d-threofuranosides. The above C-glycosyl aldehydes were all prepared on a meaningful preparative scale starting from gram quantities of galactans. Finally, a new procedure for the preparation of the 2,3-O-benzyl l-threofuranose was established by Baeyer-Villiger oxidation of the benzylated C-formyl alpha-l-threofuranoside here prepared from agarose.

Complexation of vanadium(V) oxyanions with hexopyranose- and mannopyranoseuronic acid-containing polysaccharides: stereochemical considerations.

Carbohydrates containing galactopyranosyl and mannopyranosyl units with vicinal cis-diols were treated with NaVO(3) in D(2)O, and complexation was determined by (51)V NMR spectroscopy. Me alpha-Galp, Me beta-Galp (3,4-cis-diols), and Me alpha-Manp (2,3-cis-diol) complexed, but Me beta-Manp barely did so. This low degree of complexation also occurred with a beta-mannan containing alternate (1-->3)- and (1-->4)-linkages and an alginate having beta-ManpA blocks. In contrast, branched alpha-mannans complexed readily, although the (51)V resonances for one with side chains terminated with alpha-Manp-(1-->3)-alpha-Manp-(1--> differed from another with only alpha-Manp-(1-->2)-alpha-Manp-(1--> groups. The anomeric configuration of Me alpha-Galp and Me beta-Galp, each with 3,4-cis-diols remote from C-1, gave rise to three (51)V signals of complexes with similar shifts and proportions. The shifts of a galactomannan with terminal alpha-Galp-(1-->2)-alpha-Manp- were the same as those with alpha-Galp-(1-->6)-beta-Manp- groups, but fewer complexes were formed with the former structure, probably due to greater steric crowding of the vanadate esters. Most of the complexes gave rise to a signal in the delta515 region, consistent with the dimeric trigonal-bipyramidal structure.

Avaliaçäo das atividades hemaglutinantes e de interaçäo com linfócitos T humanos nas fraçöes parcialmente purificadas da lectina de massas de ovas de Biomphalaria Glabrata (variedade albina) / Evaluation of hemagglutination activity and linfocyte T interaction in the fraction parcially purified from apawn lectin of Biomphalaria glabrata

O extrato bruto de massas de ovas (MO) de Biomphalaria glabrata (variedade albina), após precipitaçäo fracionada com sulfato de amônia, forneceu uma fraçäo protéica de 30-70 por cento de saturaçäo, que além da atividade lectínica hemaglutinante, também foi capaz de interagir com linfócitos T humanos. Tal fraçäo foi submetida à cromatografia em coluna de DEAE Sephadex A-50-120, e três fraçöes com atividades biológicas distintas foram obtidas: F-I) fraçäo hemaglutinante; F-III) fraçäo de interaçäo com linfócitos, e F-II) contendo as duas atividades. A fraçäo de interaçäo com linfócitos (FIL) se mostrou um potente bloqueador da formaçäo de rosáceas espontâneas entre linfócitos T e eritrócitos de carneiro. Essa inibiçäo näo foi afetada por íons Ca++ e Mg++ e näo foi revertida na presença dos inibidores preferenciais de hemaglutinaçäo (cetohexoses) ou de outros açúcares. Além disso, o fenômeno de rosáceas näo foi inibido quando os eritrócitos foram préviamente incubados com FIL. Ensaios de imunofluorescência indireta e citometria de fluxo demonstraram que FIL bloqueou a ligaçäo do anticorpo monoclonal anti-CD2 com o receptor de membrana de linfócitos T, reforçando os dados preliminares obtidos com a inibiçäo de rosáceas