Amidation of arabinoglucuronoxylans to modulate their flow behavior.

Arabinoglucuronoxylans obtained from the exudate of Cercidium praecox (Brea gum) were subjected to an amidation reaction to modulate their flow behavior to obtain a product with similar behavior to gum Arabic. The amidation reaction of the uronic acids present in this exudate was studied using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) system with the aim of maximizing product yield and minimizing by-product. An analysis of the significant factors involved in the reaction was carried out and a response surface methodology was conducted to optimize the stoichiometry of the reagents used. It was possible to obtain models for predicting the degree of amidation (DA) of arabinoglucuronoxylans and the formation of by-products. The formation of a secondary product derived from the amino acid ß-alanine which has not been reported previously in the reaction with polysaccharides, was described. The flow behavior of an amidated product (DA = 52 %) was determined, showing a pseudoplastic behavior and a decreased Newtonian viscosity (η0 = 36.2 Pa s) at the lowest shear rate range with respect to native product solution (η0 = 115 Pa s). Amidated arabinoglucuronoxylans had a flow behavior more similar to that of gum Arabic.

Perennial halophyte Salicornia neei Lag.: Cell wall composition and functional properties of its biopolymers.

Salicornia neei halophyte extends in Argentina seashores. To envisage potential applications, cell wall sequential extraction performed on dry plant yielded 1.1, 2.4, 0.3 and 0.9% of pectin fractions respectively extracted by room temperature water, 90 °C-water, CDTA and Na2CO3. They contained 21-33% uronic acids (UA) with low degree of methylation and 0.5-1.2 M ratios of neutral sugars to UA. High arabinose level suggests that long arabinan side-chains maintain cell wall flexibility in water deficit. Fractions also contained 10-36% of proteins. The KOH-soluble fractions (4.3%) were mainly arabinoxylans. At 2.0% w/v, pectin fractions developed "weak gel"-type networks with Ca2+, while arabinoxylans generated "dilute solutions". Cellulose (28%) and lignin (45.1%) were the main biopolymers in the final residue, which showed low water swelling capacity (3.6 mL/g) due to lignin, increasing when arabinoxylans were also present. Phenolics (9.8%) were mainly water-extractable. Salicornia is a source of biopolymers and antioxidants potentially useful for food applications.

Rheology of partially and totally oxidized red seaweed galactans.

Agarose and κ-carrageenan were oxidized using (2,2,6,6-tetramethylpiperidinyl)oxy (TEMPO) in the presence of NaOCl and NaBr. Products with several degrees of oxidation were structurally characterized. The mechanical spectra were determined: derivatives with a medium to high degree of oxidation give rise to polysaccharides that behave like dilute solutions in water, whereas those with a degree of oxidation close to 20 % keep the gelling properties with a different thermo-rheological response towards pH (6.5 or 4.0) and counterions (K+ or Ca2+) in comparison with the native polysaccharides. For instance, they showed a marked dependence on the presence of calcium ions, observed in the increase of thermal stability and dynamic elastic component (G') value, due to the known interaction of this divalent cation with the carboxylate groups. In this sense, these derivatives with low oxidation degrees have proven to be not only thermosensitive, like the native polysaccharide, but also pH- and calcium-sensitive.

Chemical structure and rheological studies of arabinoglucuronoxylans from the Cercidium praecox exudate brea gum.

The structure of the arabinoglucuronoxylans from brea gum was elucidated through an chemical and NMR spectroscopical analysis. They are composed of xylose, arabinose, glucuronic acid and 4-O-methylglucuronic acid in a molar ratio 1:0.44:0.16:0.22. The structure consists of a central chain of (1→4)-ß-d-xylopyranose of which ca.70% are susbstituted in C2 with single stubs of others sugars (ß-d-Xylp, α-d-GlcpA and 4-O-Me-α-d-GlcpA), with disaccharides (α-l-Arap-(1→2)-4-O-Me-α-d-GlcpA-(1→, α-l-Arap-(1→2)-α-d-GlcpA-(1→, ß-l-Araf-(1→3)-α-l-Araf-(1→ and α-l-Araf-(1→3)-α-l-Araf-(1→5), and possibly with trisaccharides of xylose. The determination of the location of the acetyl groups and their quantification in these arabinoglucuronoxylans has been achieved for the first time. Brea gum presents a higher thickening effect than gum arabic in 5% aqueous solution, demonstrating its potential usefulness for food and pharmaceutical applications.

Disaccharides obtained from carrageenans as potential antitumor agents.

Carrageenans are sulfated galactans found in certain red seaweeds with proven biological activities. In this work, we have prepared purified native and degraded κ-, ι-; and λ-carrageenans, including the disaccharides (carrabioses) and disaccharide-alditols (carrabiitols) from seaweed extracts as potential antitumor compounds and identified the active principle of the cytotoxic and potential antitumor properties of these compounds. Both κ and ι-carrageenan, as well as carrageenan oligosaccharides showed cytotoxic effect over LM2 tumor cells. Characterized disaccharides (carrabioses) and the reduced product carrabiitols, were also tested. Only carrabioses were cytotoxic, and among them, κ-carrabiose was the most effective, showing high cytotoxic properties, killing the cells through an apoptotic pathway. In addition, the cells surviving treatment with κ-carrabiose, showed a decreased metastatic ability in vitro, together with a decreased cell-cell and cell-matrix interactions, thus suggesting possible antitumor potential. Overall, our results indicate that most cytotoxic compounds derived from carrageenans have lower molecular weights and sulfate content. Potential applications of the results emerging from the present work include the use of disaccharide units such as carrabioses coupled to antineoplasics in order to improve its cytotoxicity and antimetastatic properties, and the use of ι-carrageenan as adjuvant or carrier in anticancer treatments.

Fucoidans from the phaeophyta Scytosiphon lomentaria: Chemical analysis and antiviral activity of the galactofucan component.

The brown seaweed Scytosiphon lomentaria produces moderate amounts of fucoidans. By cetrimide fractionation, typical heavily sulfated galactofucans are obtained, with no major signs of chemical heterogeneity, together with fractions with higher proportions of xylose, mannose and uronic acids. Anyway, fucose is the most important monosaccharide in most of the subfractions of the subsequent extracts. The fucan moieties appear to be mostly as 3-linked α-l-fucopyranosyl units, with several patterns of sulfate and branching. Galactose is mostly 6-linked, whereas mannose appears to be 2-linked, and xylose appears mostly as terminal stubs. Small amounts of 2-O-acetylated fucose units appear. A high and selective antiviral activity against HSV-1 and HSV-2 was determined for the galactofucan fractions whereas the uronofucoidans were inactive.

Minor polysaccharidic constituents from the red seaweed Hypnea musciformis. Appearance of a novel branched uronic acid.

Two polysaccharide fractions isolated from Hypnea musciformis after room temperature- and hot water extraction, soluble after KCl precipitation of the more abundant carrageenans, were subfractionated by ion-exchange chromatography eluting with increasing concentrations of NaCl. The lowest NaCl concentration (0.2M) eluted agarans. The dl-hybrids (or mixtures) eluted at intermediate concentrations of NaCl. The d/l-galactose ratio and the sulfate proportion increased with the NaCl concentration. Different types of substitution were present, mainly at C-3 with sulfate, Xyl and methylated Gal stubs, as well as low amounts of 3,6-AnGal. A novel constituent, identified as 3-C-carboxy-d-erythrose1 in its ß-furanosic form, was found linked to C-6 of ß-Gal units. A search carried out in other species like Iridaea undulosa and Kappaphycus alvarezii also revealed the same constituent. Finally, the late-eluting fractions were mostly carrageenans, with a structure consistent with that of a κ/ι/ν-carrageenan hybrid.

DFT/PCM theoretical study of the conversion of methyl 4-O-methyl-α-d-galactopyranoside 6-sulfate and its 2-sulfated derivative into their 3,6-anhydro counterparts.

Modeling of the conversion of methyl 4-O-methyl-α-d-galactopyranoside 6-sulfate (2) and 2,6-disulfate (1) into methyl 3,6-anhydro-4-O-methyl-α-d-galactopyranoside (4) and its 2-sulfate (3), respectively (Scheme 1) has been carried out using DFT at the M06-2X/6-311 + G(d,p)//M06-2X/6-31 + G(d,p) level with the polarizable continuum model (PCM) in water. The three steps necessary for the alkaline transformation of 6-sulfated (and 2,6-disulfated) galactose units into 3,6-anhydro derivatives were evaluated. The final substitution step appears to be the rate limiting, involving an activation energy of ca. 23 kcal/mol. The other two steps (deprotonation and chair inversion) combined involve lower activation energies (9-12 kcal/mol). Comparison of the thermodynamics and kinetics of the reactions suggest that if the deprotonation step precedes the chair inversion, the reaction should be faster for both compounds. No major differences in reaction rate can be theoretically predicted to be caused by the presence of sulfate on O-2, although one experimental result suggested that O-2 sulfation should increase the reaction rate. The conformational pathways are complex, given the large number of rotamers available for each compound, and the way that some of these rotamers combine into some of the pathways. In any case, the conformation (O)S2 appears as a common intermediate for the chair inversion processes.

Partial and total C-6 oxidation of gelling carrageenans. Modulation of the antiviral activity with the anionic character.

The optimal conditions for the full C-6 oxidation of κ- and ι-carrageenans using (2,2,6,6-tetramethylpiperidinyl)oxy (TEMPO) in the presence of sodium hypochlorite and sodium bromide were assessed. The fully oxidized products were characterized by NMR spectroscopy. Partially oxidized products were also obtained and analyzed by chemical and spectroscopical methods. The antiviral activity of carrageenans against herpes simplex virus HSV-1 and HSV-2 determined by plaque reduction assay, was not largely affected by full oxidation of the polysaccharides, but an increase in activity was detected by partial oxidation. A specific overoxidation on C-2 of the 3,6-anhydrogalactose moiety of κ-carrageenan was identified, solved experimentally and rationalized through the application of molecular modeling.

Chemical and rheological characterization of the carrageenans from Hypnea musciformis (Wulfen) Lamoroux.

This work reports a chemical and rheological study of the carrageenans from Hypnea musciformis, a red seaweed commercially known for its production of κ-carrageenan. The polysaccharides were extracted with water both at room temperature and at 90 °C: the yield of the latter was about six times larger than the former. Fractionation with KCl yielded a large proportion (50-67%) of a precipitate with 0.125 M of this salt for both extracts, with characteristics of a nearly pure κ-carrageenan, as determined by methylation analysis and NMR spectroscopy. Smaller amounts of fractions precipitating at higher concentrations showed a basic κ-carrageenan structure, but included some ι-carrageenan diads. The KCl-soluble polysaccharides showed a larger complexity, containing d- and l-galactans or d/l-hybrids. Some differences in the rheological properties of these carrageenans have been found. Although all KCl-precipitating polysaccharides form true gels at 10 °C in presence of KCl, those extracted with hot water form stronger gels than those extracted at room temperature. Both purified κ-carrageenans show lower gelling and melting temperature than the whole polysaccharides from which they were originated.

The system of fucoidans from the brown seaweed Dictyota dichotoma: chemical analysis and antiviral activity.

Room-temperature acid (pH 2) extraction of Dictyota dichotoma thalli yielded 2.2% of sulfated polysaccharides. Further extraction with the same solvent at 70°C was conducted sequentially for nine times, with a total yield of 7.2%. Fucose was the main monosaccharide only in the room-temperature extract (EAR) and in the first 70°C extract (EAH1). The remaining fractions showed increasing amounts of mannose (the main neutral monosaccharide), xylose and uronic acids. Fractionation by means of cetrimide precipitation and redissolution in increasing sodium chloride solutions has allowed obtaining several subfractions from each extract. The fractions redissolved at lower NaCl concentrations have large amounts of uronic acids and lesser sulfate contents, whereas those redissolved at higher NaCl concentrations are heavily sulfated and have low uronic acid contents. For the fucose-rich extracts (EAR and EAH1), fractionation leads to uronoxylomannofucan-rich and galactofucan-rich fractions. The remaining extracts gave rise to complex mixtures, with mannose and uronic acid-rich polysaccharides. Moderate inhibitory effect against herpes virus (HSV-1) and Coxsackie virus (CVB3) were found for the galactofucan-rich fractions. Most of the other fractions were inactive against both viruses, although some xylomannan-rich fractions were also active against HSV-1.

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.

DFT/MM modeling of the five-membered ring in 3,6-anhydrogalactose derivatives and its influence on disaccharide adiabatic maps.

Different conformations of methyl 3,6-anhydro-4-O-methyl-alpha-d-galactoside (1) and 3,6-anhydro-4-O-methylgalactitol (2) were studied by molecular mechanics (using the program mm3) and by quantum mechanical (QM) methods at the B3LYP/6-31+G( * *) and MP2/6-311++G( * *) levels, with and without solvent emulation. In 2, where the five-membered ring is free to move, two main stable conformations of this ring were found, identified as North (N) and South (S). The latter appears to be more stable, by either calculation, though the energy difference is reduced when emulating solution behavior. In order to find out the possible influence of a glycosidic bond over its shape, and to explain the marked NMR chemical shift displacements observed by opening of the ring, the adiabatic maps of two disaccharides carrying an analog of beta-galactoside linked to O-4 of 1 and 2 were generated. It was shown that the characteristics of the 3,6-AnGal terminal influence the characteristics of the map, especially at lower dielectric constants. On the other hand, different glycosidic angles also promote distinct stable conformations of the five-membered ring, changing from N to S, or even variants. Comparison with experimental results leads to the idea of highly flexible disaccharides, with variable values for both the five-membered ring and the glycosidic angles.

Modeling ring puckering in strained systems: application to 3,6-anhydroglycosides.

Different conformations of methyl 3,6-anhydroglycosides with the beta-D-galacto, alpha-D-galacto, and beta-D-gluco configurations were studied by molecular mechanics (using the program mm3) and by quantum mechanical (QM) methods at the HF/- and B3LYP/6-31+G** levels, with and without solvent emulation. Using molecular mechanics, the energies were plotted against the phi, theta puckering coordinates of Cremer and Pople. In such strained systems, only two extreme conformations of the six-membered ring are likely: (1)C(4) and B(1,4), or any one close to either of them. Results show the preponderance of a distorted chair conformation over that of the distorted boat, though the energy difference is lower and the distortions are larger for the compound with the beta-D-galacto configuration. For derivatives of this compound, experimental data in solution indicate both chair and boat forms, depending on the compound and the solvent, whereas for the remaining compounds, experimental data always show the preponderance of the chair conformation. The more accurate DFT calculations lead to the lower energy differences, suggesting that HF and MM3 underestimate the stability of the boat-like conformations. Similar studies on model compounds depict the importance of the anomeric effect in the conformational preferences.

Determination of the configuration of 3,6-anhydrogalactose and cyclizable alpha-galactose 6-sulfate units in red seaweed galactans.

A combination of two reported procedures was used in order to determine the configuration of the 3,6-anhydrogalactose present in red seaweed polysaccharides. A mild hydrolysis (to cleave only 3,6-anhydrogalactosyl linkages) was followed by a reductive amination with a chiral amine. Then, the total hydrolysis proceeded, followed by a new step of reductive amination. In this way, using (S)-alpha-methylbenzylamine as the chiral amine, it was possible to separate and quantitate both enantiomers of 3,6-AnGal and its 2-O-methyl ether as their diastereomeric acetylated aminoalditols. On the other hand, using (S)-1-amino-2-propanol, even though the derivatives of both enantiomers of 3,6-AnGal are not separated, the mixture can be safely quantitated with respect to galactose. Furthermore, a one-pot technique was developed to carry out an alkaline treatment of the polysaccharides, followed by the double hydrolysis-reductive amination procedure, which is useful to determine the proportions of both enantiomers of 6-sulfated 4-linked galactose units in the native polysaccharides. The unexpected presence of small amounts of units of this type belonging to the D-series in a porphyran sample is revealed by this novel procedure.

NMR spectroscopy and chemical studies of an arabinan-rich system from the endosperm of the seed of Gleditsia triacanthos.

Exhaustive extraction of the endosperm from the seed of Gleditsia triacanthos using water at room temperature and 50 degrees C left a residue, which was further extracted at 95 degrees C. Precipitation of this extract with 2-propanol yielded major amounts of galactomannan components, while the supernatant was mainly composed of arabinose-rich constituents. Two fractions were obtained by anion-exchange chromatography. The fraction that eluted with water is an arabinan with (1-->5) alpha-L linkages and branching mainly on C-2, accompanied with equal amounts of a low-galactose galactomannan oligosaccharide, and a small proportion of a beta-(1-->4)-galactan. The fraction eluted with an increased ionic strength consists mainly of a similar arabinan, and lower proportions of a high-galactose galactomannan, galactan, and protein. The arabinan moiety in both fractions was characterized by chemical analysis and 1D and 2D NMR spectroscopic techniques.