Monitoring of κ-carrageenan depolymerization by capillary electrophoresis and semisynthesis of oligosaccharide alditols.

Different hydrolysis conditions to produce κ-carrageenan oligosaccharide alditols were studied and the depolymerization process monitored by capillary electrophoresis (CE). Semisynthesis, ion-exchange and exclusion chromatography were used to obtain and isolate sulfated di-, tetra- and hexasaccharide alditols, the last being fully characterized for the first time. Those derivatives were used as standards to validate a new quantitative CE analytical method which was used to compare two different partial hydrolysis methodologies: an acid hydrolysis followed by reduction and a one-pot reductive hydrolysis using 4-methylmorpholine borane. The resulting depolymerization profiles were quite different from each other. Optimal hydrolysis conditions to produce high yields of specific sulfated oligosaccharides as well as particular mixtures of oligosaccharide alditols were determined. Moreover, using the novel CE method, we were able to distinguish up to eight different oligosaccharides in the hydrolysate mixtures.

Photodynamic effect of meso-(aryl)porphyrins and meso-(1-methyl-4-pyridinium)porphyrins on HaCaT keratinocytes.

Sixteen porphyrins, including neutral, anionic and cationic meso-(aryl)porphyrins and meso-(1-methyl-4-pyridinium)porphyrins were herein evaluated in terms of their photosensitizing properties against HaCaT keratinocytes. After an initial screening, the cationic porphyrins were studied in more details, by both determining their log POW and performing PDT assays in lower porphyrin concentrations. Porphyrins presenting two or more adjacent positively charged groups, directly linked to the macrocycle meso positions, appeared to be the most effective photosensitizers. The present study also included the dicationic 5,10-diphenyl-15,20-di(1-methylpyridinium-4-yl)porphyrin (14b), which has previously shown promising results on a psoriasis-like in vivo model. Overall results indicated that the beneficial effect related to porphyrins on psoriasis can be related to the decreasing of keratinocyte viability. Furthermore, some of the cationic porphyrins studied appeared as candidates to be utilized as photosensitizers for psoriasis treatment.

Production of agaro- and carra-oligosaccharides by partial acid hydrolysis of galactans

Agaro- and carra-oligosaccharides were produced by partial acid hydrolysis of commercial agarose and kappa-carrageenan. Di- and tetrasaccharides were purified by gel filtration chromatography and characterized by NMR (1D and 2D) spectroscopy and ESIMS. The following oligosaccharides were obtained: agarobiose, agarotetraose, kappa-carrabiose and kappa-carratetraose. Agarobiose and agarotetraose were used as standards to develop a high performance size exclusion chromatography (HPSEC) method which was utilized to study the hydrolysis rate of agarose and oligosaccharide production. Six hours of hydrolysis (0.1 M TFA, 65 ºC) produced mainly di- and tetrasaccharides. The methodology for oligosaccharide production and evaluation developed in the present work shows good potential for the production of bioactive oligosaccharides.

ESI-MS differential fragmentation of positional isomers of sulfated oligosaccharides derived from carrageenans and agarans.

We have prepared a number of isomeric red seaweed galactan-derivative sulfated oligosaccharides to determine whether there were diagnostic differences among the isomeric mass spectra obtained using ESI CID MS/MS (triple quadrupole instrument). Fragmentation of the single or multicharged molecular ions from di-, tetra-, and hexasaccharides indicated that the relative positioning of the sulfate groups and type of monosaccharide unit affect the rate of cleavage of the glycosidic bonds. We also performed a comparative [M-Na](-) fragmentation study of positional isomers of sulfated disaccharides that present all four monosulfation possibilities on the galactopyranosidic ring. In this case, negative-ion ESI CID MS/MS approach gave diagnostic product ions from cross-ring cleavages along with the same main B(1) ion (from sulfated Galp), at m/z 241, for all isomers. The isomeric disaccharides were also submitted to increased spray energy conditions inducing in-source fragmentation; preformed B(1) ions were then fragmented to give similar product ions as those found in [M-Na](-) analysis. Evaluation of the relative abundances mainly for cross-ring fragment ions at m/z 138, 139, 151, 153 allowed clear distinction among the members of the disaccharide series. The different ratios for m/z 151/153 ions were consistent with the predominance of m/z 153 being related to the cases when the bond involved in the cleavage process links a sulfated carbon. A quadrupole ion trap instrument (MS(n) analysis) was also utilized to compare the results obtained with the triple quadrupole instrument.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of oligosaccharides and oligosaccharide alditols obtained by hydrolysis of agaroses and carrageenans, two important types of red seaweed polysaccharides.

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.

Semi-synthesis of a 3-O-sulfated red seaweed galactan-derived disaccharide alditol.

Beta-D-Galp3-SO3-(1-->4)-3,6-anhydro-L-GalOH (agarobiitol 3(2)-sulfate, 4) was semi-synthetically prepared as follows: production of agarobiitol (1) from agarose by partial reductive hydrolysis, protection of the primary hydroxyl groups of 1 with trityl groups to produce the 1(1),6(2)-di-O-tritylated derivative (2), regioselective dibutylstannylene-mediated sulfation of 2 to give the 3(2)-O-sulfated-1(1),6(2)-di-O-tritylated compound (3), and detritylation of compound 3 to give the final product (4). This semi-synthetic route allowed the preparation of a red seaweed galactan-derived disaccharide alditol with sulfate group located at C-3 of the galactopyranosidic ring. Because red seaweed galactans are glycosidically linked at C-3 of the beta-D-Galp unit, a sulfated derivative with this structure could not be obtained by partial reductive hydrolysis of sulfated red seaweed galactans.

Complete 1H and 13C NMR assignment of digeneaside, a low-molecular-mass carbohydrate produced by red seaweeds.

Digeneaside (alpha-D-mannopyranosyl-(1-->2)-D-glycerate) was extracted from the red algae, Bostrychia binderii, and purified by adsorption and gel-filtration chromatography. HPLC and ESI-MS techniques were used to follow purification steps and characterize digeneaside. NMR spectroscopy experiments (1D 1H, 13C, DEPT and 2D HMQC, COSY and TOCSY) were used to fully assign the 1H and 13C spectra.

Sulfated and pyruvylated disaccharide alditols obtained from a red seaweed galactan: ESIMS and NMR approaches.

The water-soluble acid agaran isolated from Acanthophora spicifera (Rhodophyta) was submitted to alkaline treatment for the complete cyclization of alpha-L-Galp 6-sulfate to 3,6-An-alpha-L-Galp units. The modified agaran was then partially depolymerized using partial reductive hydrolysis. The resulting oligosaccharide mixture was fractionated by adsorption and ion-exchange chromatography. Fractions were purified by gel-filtration chromatography and studied by ESIMS and NMR spectroscopy, including 1D 1H, 13C, DEPT and 2D 1H, 1H COSY, TOCSY and 1H, 13C HMQC procedures. The following neutral, pyruvylated, sulfated and sulfated/pyruvylated disaccharide alditols were obtained: beta-D-Galp-(1-->4)-3,6-An-L-GalOH; 4,6-O-(1-carboxyethylidene)-beta-D-Galp-(1-->4)-3,6-An-L-GalOH; beta-D-Galp 2-sulfate-(1-->4)-3,6-An-L-GalOH and 4,6-O-(1-carboxyethylidene)-beta-D-Galp 2-sulfate-(1-->4)-3,6-An-L-GalOH.