Diego A. Navarro

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.

Determination of the configuration of 3,6-anhydrogalactose and cyclizable α-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.

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.

DIFFERENCES IN POLYSACCHARIDE STRUCTURE BETWEEN CALCIFIED AND UNCALCIFIED SEGMENTS IN THE CORALLINE CALLIARTHRON CHEILOSPORIOIDES (CORALLINALES, RHODOPHYTA)1

The articulated coralline Calliarthron cheilosporioides Manza produces segmented fronds composed of calcified segments (intergenicula) separated by uncalcified joints (genicula), which allow fronds to bend and reorient under breaking waves in the wave‐swept intertidal zone. Genicula are formed when calcified cells decalcify and restructure to create flexible tissue. The present study has identified important differences in the main agaran disaccharidic repeating units [→3)‐β‐d‐Galp (1→ 4)‐α‐l‐Galp(1→] synthesized by genicular and intergenicular segments. Based on chemical and spectroscopical analyses, we report that genicular cells from C. cheilosporioides biosynthesize a highly methoxylated galactan at C‐6 position with low levels of branching with xylose side stubs on C‐6 of the [→3)‐β‐d‐Galp (1→] units, whereas intergenicular segments produce xylogalactans with high levels of xylose and low levels of 6‐O‐methyl β‐d‐Gal units. These data suggest that, during genicular development, xylosyl branched, 3‐linked β‐d‐Galp units present in the xylogalactan backbones from intergenicular walls are mostly replaced by 6‐O‐methyl‐d‐galactose units. We speculate that this structural shift is a consequence of a putative and specific methoxyl transferase that blocks the xylosylation on C‐6 of the 3‐linked β‐d‐Galp units. Changes in galactan substitutions may contribute to the distinct mechanical properties of genicula and may lend insight into the calcification process in coralline algae.

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.

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.

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.

Isolation of xylogalactans from the Corallinales: influence of the extraction method on yields and compositions

Abstract An already studied seaweed, Corallina officinalis and two other species of the red seaweed order Corallinales, i.e. Bossiella orbigniana and Jania rubens, were extracted using three different standard procedures: the method of Usov, which destroys the calcium carbonate cover by the addition of concentrated acid, the method of Cases, which applies controlled acid degradation of this cover, and the method of Takano, which avoids an acid medium, and uses hot water. The three methods extract similar amounts of products, but those using acid give better yields of xylogalactans, without evidence for degradation. However, subsequent hot water extraction shows that the traces of acid present when applying the method of Usov yield partially degraded products at this stage. The characteristics of the xylogalactans of the newly studied seaweeds are similar to those previously studied. However, in Jania rubens significant amounts of 3,6-anhydrogalactose, and in Bossiella orbigniana small amounts of the d -enantiomer of 2-O-methylgalactose were found.

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 β-furanosic form, was found linked to C-6 of β-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.