Autohydrolysis of a partially cyclized mu/nu-carrageenan and structural elucidation of the oligosaccharides by chemical analysis, NMR spectroscopy and UV-MALDI mass spectrometry

A partially cyclized mu/nu-carrageenan from Gigartina skottsbergii was submitted to autohydrolysis and the resulting mixture of oligosaccharides was fractionated using sequential dialysis through tubings of molecular weight cutoff 12000, 3500 and 1000. In this way four fractions were separated and characterized. Structural analysis of the major fraction, obtained from the solution inside the dialysis tubing of molecular weight cutoff 12000 (D120), was carried out. A significant difference was found by 13 C NMR spectroscopy and UV-MALDI-TOF mass spectrometry: in the 13 C NMR spectrum the reducing end-chain unit was the hydrated aldehyde of 3,6-anhydrogalactose while the UV-MALDI-TOF mass spectrum showed an unimodal distribution of even and odd peaks, suggesting fragmentation of glycosidic linkages


Introduction
Red seaweeds of the family Gigartinaceae are a rich source of sulfated galactans known as carrageenans which are widely used in food industry as thickening and gelling agents. 1 Carrageenans consist of an alternating structure of 3-linked β-D-galactoyranose and 4-linked 3,6anhydro-α-D-galactopyranose or α-D-galactopyranose.The different hydroxyl groups present in these units can be substituted, mainly with sulfate, but also with single stubs of xylose or methyl groups. 1 Usually, carrageenans have structures that are a mixture of two or more idealized repeating units (e.g.kappa/iota-carrageenan, mu/nu-carrageenan or a partially cyclized mu/nucarrageenan) and are called hybrid carrageenans. 2The repeating units of kappa-, iota-, mu-and nu-carrageenans are shown in Figure 1.In a previous publication 3 , the feasibility of matrix-assisted ultraviolet laser-desorption ionization time-of-flight mass spectrometry (UV-MALDI-TOF-MS) of carrageenans was studied.Several commercial sulfated neocarrabiose oligosaccharides were analyzed in the positive-and negative-ion modes using 2,5-dihydroxybenzoic acid and nor-harmane as matrices.Post-source decay (PSD) experiments were also carried out, and structures of the fragment ions were assigned.
5][6][7] This reaction, applied to carrageenans, splits glycosidic linkages between 3,6-anhydro-α-galactose 2-sulfate and β-galactose 4-sulfate or between α-galactose 2,6-disulfate and β-galactose 4-sulfate; as soon as these linkages are cleaved, there is a loss of sulfate groups on C-2.][6] A partially cyclized mu/nu-carrageenan from Gigartina skottsbergii, whose structure was previously determined by methylation analysis and NMR spectroscopy, [8][9][10] was subjected to autohydrolysis and the mixture of oligosaccharides fractionated.Herein we report the characterization of the fractions and structural analysis of the major one by the combined use of methylation analysis, 1 H and 13 C NMR spectroscopy and UV-MALDI-TOF-MS.
1C 3 (molecular weight 198000) was submitted to autohydrolysis and the rate constant of the reaction, calculated from internal 3,6-anhydrogalactose was 6.04 x 10 -2 h -1 ; a value of the same order was previously determined 6 for a mu/nu-carrageenan from Sarcothalia crispata (Iridaea undulosa).After 11 h at 60 °C, the acid solution was neutralized and the mixture of oligosaccharides was fractionated using dialysis tubing of molecular weight cutoff 12000.The dialysis tubing content was concentrated and freeze-dried to give D120 (56.6% of the recovered) and the dialyzate was subjected to further fractionation with dialysis tubing of molecular weight cutoff 3500 and 1000.Table 1 shows the yields and analyses of the fractions recovered from the dialysis tubings (D120, D35 and D10) and the fraction DW constituted by the oligosaccharides that passed through the dialysis tubing of molecular weight cutoff 1000.In order to investigate the homogeneity of the fractions, these were submitted to GPC on Sephadex G-25 using 0.1 M NaCl as eluant for D35, D10 and DW and 1 M NaCl for D120; chromatography of all the fractions was also carried on Sephadex G-75 with 0.1 M NaCl.Each  Structural analysis of the major fraction D120 was carried out by 1 H and 13 C NMR spectroscopy, methylation analysis and UV-MALDI-TOF-MS.
Figure 2 shows the 1 H-decoupled 13 C NMR spectrum of D120 and Table 3 indicates the assignment of the signals.In this spectrum, the diads corresponding to kappa-, iota-, mu-and nu-structures were clearly observed, 10,12 with prevalence of the mu-and kappa-diads.Besides, peaks of carrabiose 4´sulfate and oligosaccharides containing carrabiose units were also important, 13 and no anomeric resonances due to reducing end-chain galactose were detected.This result is in agreement with the 1 H NMR spectrum of this fraction where only weak signals at 5.53 and 5.30 ppm, due to the H-1 of α-D-galactose 2,6-disulfate and 3,6-anhydrogalactose 2-sulfate, both linked to β-Dgalactose 4-sulfate, were found. 10educed D120 was converted into the triethylammonium salt and methylated according the method of Hakomori. 14,15Table 4 shows the composition of the permethylated product.The galactose:3,6-ahydrogalactose molar ratio, calculated from Table 4 is 1.00:0.34,very similar to that of the non-methylated fraction (1.00:0.39),and if it is considered that the 2,3,6-tri-O-methylgalactose derives from non-reducing end-chain β-galactose 4-sulfate units, the molecular weight is ~ 3000, slightly higher to that obtained by GPC.In addition, the lower galactose:3,6-ahydrogalactose molar ratio calculated from Table 4 was consistent with the loss of permethylated small carrabiose oligosaccharides (see later the analysis of the UV-MALDI-TOF mass spectrum) during the dialysis of the methylation procedure.It should be noted that no methylated derivatives corresponding to the 4-linked reducing end-chain 3,6-anhydrogalactose or galactose 6-sulfate residues were detected by gas-chromatography analysis of the alditol acetates.D120 was also analyzed by UV-MALDI-TOF-MS using 2,5-dihydroxybenzoic acid and norharmane as matrices.Experiments were carried out in the linear and reflectron modes and, as routine, in the positive-and negative-ion modes; good spectra were only obtained with norharmane in the negative-ion mode.Table 5 shows the m/z values and the assignment of the main peaks observed in the linear mode spectrum of this fraction (Figure 3).For the assignment, the information acquired by methylation analysis and 13 C NMR spectroscopy was essential.Thus, it was assumed that: a) all the 3-linked units were sulfated in the 4-position (G4S); b) the major kappa-(G4S→DA) and mu-diads (G4S→D6S) were the only observed; and c) the 4-linked reducing end-chain residues should be, according to previous reports [4][5][6] on the autohydrolysis reaction, the hydrated aldehyde of 3,6-anhydrogalactose (reducing end-chain DA) or galactose 6sulfate (reducing end-chain D6S), with high prevalence of the former according to the 13 C NMR spectrum.

Table 5. UV-MALDI-TOF-MS of D120 a m/z
Assignment consistent again with a maximum loss of (x -1) sulfate groups, where x is the number of sulfates in the analyte, suggesting that the non-ionized sulfate groups are more easily lost than the stabilized sulfate anion or that the monocharged anion with only one sulfate group is the most stable in the UV-MALDI process. 3For each peak of the asymmetrical curve shown in Figure 3, a minor signal at m/z -18 Da was observed.This difference could be either attributed to: a) the replacement in the oligosaccharide structure of a galactose by a 3,6-anhydrogalactose unit and/or b) the loss of water from the reducing end-chain hydrated aldehyde of 3,6-anhydrogalactose.It is noteworthy that no loss of water was previously observed in the mass spectra of the sulfated neocarrabiose oligosaccharides determined in the same conditions. 3This asymmetrical profile suggested a mass-dependent discrimination 17 for m/z higher than 2349.6Da.Some of the peaks also showed a minor signal at m/z + 18 Da, which could derive from the replacement of a 3,6anhydrogalactose unit by a non-sulfated galactose in the oligosaccharide structure.
Besides, it is important to note that there is an alternant sequence of signals corresponding to oligosaccharides with even (m/z 727.6, 1052.0,1376.5, 1700.5, 2024.7 and 2349.6 Da) and odd (m/z 889.8, 1214.3,1538.5, 1862.6 and 2186.9Da) number of residues.However, the most intense peaks of the spectrum correspond to oligosaccharides with even number of residues.
The presence of fragments with an even and odd number of residues could indicate that, during the autohydrolysis reaction, some β-galactosidic linkages were cleaved, or that a significant amount of 3,6-anhydrogalactose residues was degraded.However, this result is not in agreement with the 13 C NMR spectrum where it is clearly observed that the reducing end-chain unit is the hydrated aldehyde of 3,6-anhydrogalactose. Therefore, the presence of oligosaccharides with an odd number of residues would arise in the UV-MALDI process 17 probably due to glycosidic C-cleavages 18 which involve breaking the bond between the reducing end-chain hydrated aldehyde of 3,6-anhdrogalactose and the adjacent galactose 4-sulfate unit.In the negative-ion mode UV-MALDI-TOF reflectron mass spectrum of neocarrahexaose 4 1 ,4 3 ,4 5 trisulfate, a C 5 -ion was detected; 3 however, this fragmentation involved the cleavage between the reducing end-chain galactose 4-sulfate and the adjacent 3,6-anhydrogalactose residue.
In conclusion, though the oligosaccharides present in D120 are of low molecular weight, their regular structure would promote aggregation precluding dialysis through tubing of molecular weight cutoff 12000.In addition this is the first report on UV-MALDI-TOF-MS of sulfated oligosaccharides with reducing end-chain 3,6-anhydrogalactose units.Furthermore, norharmane showed again to be a high efficient UV-MALDI matrix for desorption/ionization of sulfated oligosaccharides in negative-ion mode.

Analytical autohydrolysis of 1C 3
The sample (214 mg) was dissolved in water (21 mL) and was passed through an Amberlite IR-120 (H + ) column (10 x 1 cm i.d.); the final volume was 60 mL.The solution was shaken at 60 °C and aliquots (0.5 mL) were taken at different times, neutralized with calcium carbonate and reduced with sodium borohydride.The 3,6-anhydrogalactose content was measured on the reduced solutions and the values were used to determine the rate constant.After 11 h, the remaining solution was allowed to reach room temperature and neutralized with sodium carbonate.The solution gave an absorbance of 0.191 at 280 nm, indicating than less than 0.5 % of the total 3,6-anhydrogalactose was degraded to hydroxymethylfurfural.The solution was concentrated and freeze-dried.

Preparative autohydrolysis of 1C 3
The sample (2.5 g) was dissolved in water (250 mL) and was passed throght an Amberlite IR-120 (H + ) column (25 x 2 cm i.d.); the final volume was 500 mL.The solution was shaked at 60 °C for 11 h, then it was allowed to reach room temperature, neutralized with sodium carbonate, and concentrated to give a volume of 125 mL.

Fractionation using dialysis tubing of different molecular weight cutoff
The concentrated solution was dialyzed (Spectra/Por molecular weight cutoff 12000-14000) against distilled water (2.5 L) with constant agitation during 18 h.The procedure was repeated once more with a new batch of distilled water (1 L), and the dialysis tubing content was concentrated and freeze-dried to give D120 (1.08 g).The two dialyzates were pooled and the resulting solution (3.5 L) was concentrated to 80 mL and dialyzed (Spectra/Por molecular weight cutoff 3500) against distilled water (700 mL).The same procedure described before led to the isolation of D35 (135 mg).The dialyzates obtained in this step were submitted to a similar procedure (Spectra/Por molecular cutoff 1000).The solution inside the dialysis tubing was concentrated and freeze-dried to give D10 (640 mg) and the dialyzates gave DW (59 mg) after carrying out the same procedure.

Reduction of the samples for methylation analysis and GPC
The sample was dissolved in a minimum amount of water and sodium borohydride was added.The solution was left overnight at room temperature and the excess of reductant was quenched with acetic acid.The mixture was heated to 40 °C and evaporated to dryness with a stream of dry air.Methanol (4 x 0.5 mL) was added and the solution was evaporated.

Methylation analysis
Reduced D120 (6 mg) was converted into the corresponding triethylammonium salt and methylated according to Hakomori, as described elsewhere. 14,15The methylated derivative was recovered by dialysis (Spectra/Por molecular weight cutoff 1000) and freeze-drying.

GPC on Sephadex G-25 and G-75
The sample (10 mg) was chromatographed on a Sephadex G-25 column (74 x 1 cm i.d.) using (0.1 M, 1 M, or 2 M) sodium chloride or 0.1 M LiCl as eluants; with Sephadex G-75 the eluant was 0.1 M NaCl.Fractions of 1.2 mL were collected and aliquots were assayed by the phenolsulfuric acid method.The dextran sulfates and the sulfated oligosaccharides described in Materials were cromatographed in the same conditions as the samples.
1 H NMR and 13 C NMR spectroscopy 50-MHz 13 C NMR 1 H-decoupled spectrum of D120 (30 mg) was recorded at room temperature on a Bruker AC 200 spectrometer, in H 2 O-D 2 O 1:1 solution (0.5 mL), using a 5-mm NMR tube and with external reference to TMS. Specific parameters included a pulse angle of 90°, an acquisition time of 0.74 s, no pulse delay, a spectral width of 11 KHz and 150000 scans.
For the 1 H NMR spectrum, the sample (10 mg) was dissolved in D 2 O (0.5 mL) and a 5-mm NMR tube was used.The spectrum was recorded on a Bruker AM 500 spectrometer, at room temperature using a spectral width of 6.4 kHz, 90° pulse, an acquisition time of 5.1 s, for 240 scans.Acetone was used as internal standard, at 2.20 ppm.
199-Welled gold sample plates (P/N V700401) were used in the PerSeptive Biosystems Voyager DE-STR mass spectrometer; samples were placed at locations which were mirrorpolished.Sample preparation.Matrix stock solutions were made by dissolving 2 mg of the selected compound in 0.2 mL of MeOH−H2O (1:1, v/v) or in 0.2 mL of MeCN−H2O (2:3, v/v).Analyte solutions were freshly prepared by dissolving the carbohydrates (1 mg) in pure water (0.5 mL).Best results were afforded when the matrix solutions were prepared in MeOH-H2O.
To prepare the analyte-matrix deposit two methods were used: Method A (thin-film layer method) and Method B. 3,[24][25][26] Best results were obtained using nor-harmane as matrix and Method B. In this method, the analyte stock solution was mixed with the matrix solution in 1:4 v/v ratio.A 0.5 µL aliquot of this analyte-matrix solution was deposited onto the stainless steel probe tip and dried with a stream of forced room temperature air.Then, an additional portion of 0.5 µL was applied to the dried solid layer on the probe, causing it to re-dissolve partially, and the solvent was removed by blowing air.Spectrum calibration.Spectra were calibrated by use of external calibration reagents.In the linear and reflectron modes: a) commercial proteins (neurotensin; insulin) with SA as matrix in positive-and negative-ion mode, and b) α-, β-and γ-cyclodextrins with nor-harmane as matrix in positive-and in negative-ion modes.The Kratos Kompact calibration program and the Voyager DE-STR calibration program were respectively used.

550 1590 a
Determined by the method of Park and Johnson (see Experimental). b On Sephadex G-25 and 0.1 M NaCl as eluant for D35, D10 and DW; for D120 the eluant was 1 M NaCl.c n.d.= not determined.

Figure 3 .
Figure 3. Negative-ion mode UV-MALDI-TOF mass spectrum of D120 carried out in the linear mode using nor-harmane as matrix.Peaks at 413.1 and 565.2 Da were not assigned.

Table 1 .
Yields, analyses and monosaccharide composition of the fractions isolated after autohydrolysis of 1C 3 and further fractionation of the oligosaccharide mixture with dialysis tubings of different molecular weight cutoff

Table 2 .
Molecular weight of the different fractions obtained by end-group analysis and GPC

Table 4 .
Composition (mol%) of monosaccharides produced by permethylation and hydrolysis of D120