Sulfonoglycolipids from the lichenized basidiomycete Dictyonema glabratum: isolation, NMR, and ESI-MS approaches

Guilherme L. Sassaki, Philip A.J. Gorin, Cesar A. Tischer and Marcello Iacomini1

Departamento de Bioquímica, Universidade Federal do Paraná, Curitiba-PR, Cx.P. 19046, 81.531-990, Brazil

Received on August 24, 2000; revised on October 31, 2000; accepted on November 3, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Two distinct sulfonoglycolipid fractions were isolated from the basidiolichen Dictyonema glabratum by chromatography on Diethylaminoethyl (DEAE)-Sepharose, which resulted in rapid elution, followed by partition between aqueous sulfuric acid and an ethyl acetate-methanol-chloroform mixture and the content of the organic layer chromatographed of a column of silicic acid. The products were examined by nuclear magnetic resonance spectroscopy in their native rather than their acetylated forms, as in previous investigations. Each was methanolyzed to give the same methyl glycoside, Me-G. On electrospray ionization mass spectrometry (ESI-MS), it provided a pseudomolecular ion at m/z 303 in the positive-ion mode and a molecular ion at m/z 257 with a daughter ion at m/z 146 in the negative-ion mode, showing the presence of a sulfonate group S-linked to a hexosyl ring. An exclusively {alpha}-glucopyranosyl configuration was indicated by 1H, 1H correlation spectroscopy (COSY) and 1H, 1H total correlation spectroscopy (TOCSY). S-substitution occurred at CH2-6, because a high-field 13C signal at {delta} 52.6 gave an inverted distortionless enhancement by polarization transfer (DEPT) signal and 1H, 3C heteronuclear multiple quantum coherence (HMQC) showed correlation with two H-6 signals. This 6-sulfono-{alpha}-quinovopyranoside group was present in the glycolipid fractions, O-{alpha}-D-Quip-6-sulfono-(1'{leftrightarrow}1)-2,3-diacyl-D-glycerol (polar fraction 1a; PF1a) and O-{alpha}-D-Quip-6-sulfono-(1'{leftrightarrow}1)-2- or -3-monoacyl-D-glycerol (polar fraction 1b; PF1b), the monoacyl derivatives not having been previously completely characterized in other systems. All components are typical of plant glycolipids. The most abundant fatty acid ester in PF1a and PF1b was C16:0. Other esters present in PF1a were C14:0, C17:0, C18:0, C18:1 (oleic), C20:0, C21:0, and C24:0, in contrast with C14:0, C17:0, C18:0, and C20:0 in PF1b. HMQC and TOCSY data can be used as fingerprints for detection of glycosylacylglycerides and sulfonoglycolipids and the positive ESI-MS ions at m/z 329 and 271 for identification of the latter.

Key words: Dictyonema glabratum/fatty acids/sulfonoglycolipids/NMR/ESI-MS


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The first glycolipids to be reported in lichens were glycosyldiacylglycerides, based on thin-layer chromatrography (TLC) evidence (Dertien et al., 1977Go). Such structures were completely characterized in the ascolichen Ramalina celastri (Machado et al., 1994Go) and the basidiolichen Dictyonema glabratum (Sassaki et al., 1999Go) using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, complemented by gas chromatography–mass spectrometry (GC-MS) and field desorption MS. In the latter, three galactolipids, namely, mono-, di-, and trigalactosydiacylglyceride were found, presumably arising from photosynthetic tissues (Nichols, 1974Go). A galactosphingolipid was also found in the ascolichen (Machado et al., 1997Go), with a structure similar to those occurring in fungi. Another possible glycolipid candidate in lichens is the sulfonoglycolipid, the most common of which is sulfoquinovosyldiacylglyceride (SQDG), commonly referred to as the "plant sulfolipid" (Benson, 1963Go), with acyl groups on both hydroxyl groups of the glycerol moiety. The related lysosulfolipid only has acyl groups on its primary hydroxyl group (Benson, 1963Go). In contrast with sulfate esters, the sulfur is linked directly to the carbon atom, in this case, C-6. These sulfonate-glycolipids appear to be involved with proteins to maintain their catalytic activities (Gournais and Barber, 1985Go). This association probably occurs by electrostatic interactions between the negative portion of the glycolipid (sulfonate), and positive charges of the proteins. Another property attributed to SQDG is a potential effect against the AIDS virus (Gustafson et al., 1989Go). If SQDG has an effect on cell-surface receptors that interfere in viral recognition characteristics, determination of its conformation is essential (Howard and Prestergard, 1996Go). We now report the determination of the structures of two sulfonoglycolipids obtained from the basidiolichen Dictyonema glabratum (Sprengel) D. Hawks (Hawksworth and Hill, 1988Go), family Dictyonematacea.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Extraction and fractionation of glycolipids using relatively nontoxic solvents
A lipid extract from D. glabratum was obtained by successive homogenization of a cleaned sample with phosphate buffer, followed by extraction with refluxing 90% aqueous EtOH (6.1% yield, w/w, based on lichen). This was extracted using solvent B and the product was chromatographed on a column of fast-flow DEAE-Sepharose (OAc form) with eluants, solvents A, B, and C (for composition, see Materials and methods). Based on the weight of the lipid extract, solvent A gave rise to a fractions 2a (13%) and 2b (14%), solvent B to fraction 1 (57%), and solvent C to fraction 3 (5.7%). The resulting fractions were examined by TLC, and orcinol-positive spots were detected in 2a and 2b; these were combined (polar fraction, PF), evaporated, and dissolved in CHCl3-MeOH-H2O (60:40:9 v/v/v) and partitioned between an EtOAc-MeOH-CHCl3 mixture and 0.5% v/v aqueous H2SO4, following the steps described in Materials and methods (also see Scheme 1), giving a polar lipid fraction from the organic phase (PF1 yield 0.53%, based on lichen). TLC examination showed two orcinol-positive spots with Rfs of 0.26 and 0.46 in CHCl3-MeOH-H2O (65:25:4 v/v/v). PF1 was chromatographed on a column of silicic acid, which was eluted with CHCl3 and then CHCl3-MeOH mixtures with increasing concentrations of MeOH. Further chromatography on the partially purified fractions gave small amounts of pure PF1a (Rf 0.46) and PF1b (Rf 0.26).



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Scheme 1. Sequential organic extractions, isolation, and purification of the polar glycolipids.

 
Structural characterization of the purified glycolipids
Fractions PF1a and PFb were characterized as follows. The methyl glycoside, Me-G, obtained from both fractions via methanolysis was examined by electrospray ionization mass spectrometry (ESI-MS) in the positive-ion mode. It gave a pseudomolecular ion with m/z 303, corresponding to the sodium salt, Na+ form, of a methyl glycoside of a methylpentose directly linked to the sulfur atom of a sulfonate group. This was confirmed in the negative-ion mode, which gave a molecular ion with m/z 257, accompanied by a daughter ion at m/z 146, corresponding to removal of sulfono and methoxy groups (Table I; Figure 1A, 1B).


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Table I. Molecular and daughter ions formed in ESI-MS spectra of glycosyldiacylglycerolpids (PF1a and PF1b) and its methyl glycoside derivative (Me-G)
 


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Fig. 1. ESI-MS spectra of Me-G (A) positive-ion mode: m/z 303 (pseudomolecular ion), m/z 271 [Z+2Na]+; (B) negative-ion mode: m/z 257 molecular ion, daughter ion: m/z 146 [M-H-Z-W].

 
13C and 1H NMR examination showed Me-G to be homogeneous with respective anomeric signals at {delta} 99.7 and 4.78 (J = 3.4 Hz) and a 13C,1H coupled spectrum showed JC-1,H-1 = 174.4 Hz (Perlin and Casu, 1969Go), these data indicating an {alpha}-glycosidic configuration (no ß-glycoside was detected). COSY and TOCSY examination of the glycoside allowed assignment of all the protons of the methylpentosyl unit, and HMQC was used to assign correlated 13C signals (Table II). An inverted DEPT signal at {delta} 52.9 corresponded to CH2-6 linked directly to a sulfur atom, and which correlated (HMQC) with two H-6 signals at {delta} 3.06 and 3.42.


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Table II. 1H- and 13C NMR spectroscopic data of glycolipids (PF1a PF1b) and methyl glycoside sulfonate derivative, Me-G ({delta} values, p.p.m., and coupling constants J = Hz)
 
The vicinal coupling constants of each proton, with the exception of JH-1,H-2, which was 3.4 Hz, varied from 6.0 to 10.1 Hz, showing that all the other protons were trans-diaxial. This corresponds to an {alpha}-D-glucopyranosyl configuration, identical to that of the quinovose structure present in SQDG.

The fatty acid composition of the purified glycolipids, PF1a and PF1b, was determined by GC-MS of methyl esters derived on methanolysis (Table III). In each fraction, the most abundant fatty acid by far was palmitic acid C16:0. Qualitative and quantitative differences were found between the fractions, for example much more stearic acid ester was found in PF1a than PF1b. Also, PF1a contained smaller amounts of C14:0, C17:0, C18:0, C18:1, C20:0, C21:0, and C24:0 esters, but those of C18:1, C21:0, and C24:0 were absent from PF1b.


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Table III . Fatty acid compositions of purified glycolipids.
 
ESI-MS examination of PF1a in the positive-ion mode gave a major pseudomolecular ion of the Na salt (Na+ form) at m/z 839 (Table I). Based on the presence of the sulfonoquinovosyl residue, this disubstitution is by esters of palmitic acid. That this occurred at C-2 and C-3 of the glycerol moiety is shown by the presence of pseudomolecular daughter ions with m/z 584 and 329, due to the removal of one and two palmitic ester groups, respectively (Figure 2A). Other pseudomolecular ion peaks at m/z 853, 867, 879, and 895 showed respective disubstitutions of combinations of C16:0 and C17:0, C17:0 and C17:0, C17:0 and C18:1, C18:0 and C18:0, and/or C16:0 and C20:0.



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Fig. 2. ESI-MS spectra, positive-ion mode of glycolipids PF1a sulfoquinovosyldiacylglycerol (A) pseudomolecular ions: m/z 839, 853, 867, 879, and 895 (varying according to the fatty acid composition, see Table I). PF1b sulfoquinovosymonoacylglycerol (B) pseudomolecular ions: m/z 601, 615, 629, and 657 (varying according to the fatty acid composition, see Table I).

 
Fraction PF1b differed because its ESI-MS spectrum contained a major pseudomolecular ion at m/z 601, derived from palmitic ester at C-2 or C-3 of the glycerol moiety, which was confirmed by the presence of the pseudomolecular daughter ion with m/z 329. Smaller peaks were at m/z 615, 629, and 657, arising from substitution at C-2 or C-3 by C17:0, C18:0, and C20:0 esters, respectively (Table I).

NMR examination confirmed the structure determined for PF1a, namely, an {alpha}-quinovosyl-6-sulfono unit, linked (1'{leftrightarrow}1) to D-glycerol. Its 13C NMR spectrum showed lipid signals at {delta} 14.4 (CH3) and from {delta} 23.3 to 35.0 (CH2), the largest being at {delta} 30.3 An ester carbonyl signal at {delta} 174.6 was also present. Double bonds were characterized by signals at {delta} 126.4 to 131.5. The anomeric 1H- and 13C NMR regions of the monosaccharide unit gave rise to a signal at {delta} 4.70/99.8, corresponding to an {alpha}-Quip-6-sulfonate unit. HMQC examination indicated 1H- and 13C NMR signals belonging to the glycerol moiety and these were confirmed by its TOCSY spectrum (Figure 3). A DEPT experiment showed inverted CH2 signals of S-substituted C-6', corresponding to units of {alpha}-Quip in the high field region at {delta} 55.8 (3.04, 2.69) and signals at {delta} 66.0 (4.00, 3.52), 72.1/5.24, and 63.7 (4.47, 4.25), which arose from C-1, C-2, and C-3 and adjacent protons, respectively, of the glycerol moiety.



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Fig. 3. Partial 2D NMR spectra (HMQC and TOCSY experiments) of Me-G and glycolipids PF1a and PF1b. (A) Partial HMQC spectrum of PF1a (monosaccharide and glycerol regions), the proton signals at {delta} 5.27 (H-2), 4.00 and 3.52 (H-1a/H-1b) and 4.47 and 4.25 (H-3a/H-3b), as fingerprint of glycosylglycerolipids. Partial TOCSY spectra of the glycolipids PF1a (B), PF1b (C), and Me-G (D). The monosaccharide ring and glycerol moiety are represented in this sequence: a fingerprint for sulfonoglycolipids and their derivatives.

 
A similar approach was adopted for PF1b. Its 13C NMR spectrum had similarities to the spectrum of PF1a. It contained lipid signals of at {delta} 13.9 (CH3), from {delta} 22.6 to 34.1 (CH2), with a predominant one at {delta} 29.8, along with a carbonyl signal of an ester group at {delta} 173.5. However, as expected from GC-MS data (Table III), signals of double bonds were not detected. Anomeric carbohydrate signals were found in the 1H- and 13C NMR spectra, with one signal at {delta} 4.59/98.7 that belongs to an {alpha}-Quip-6-sulfonate unit linked (1'{leftrightarrow}1) to D-glycerol. As with PF1a, the DEPT, TOCSY, and HMQC spectra were similar. Assigned were C-6' and H-6' signals of the monosaccharide ring [{delta} 54.3 (2.94, 2.56)], and C-1/H-1 [{delta} 69.0 (3.71, 3.38)], C-3/H-3 [{delta} 65.4 (4.01, 3.92)], and C-2/H-2 ({delta} 68.4/5.13) of the glycerol moiety.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Two sulfonoglycolipids have been isolated for the first time from a lichen, using an improved method of extraction. This was carried out on the lichenenized basidiomycete D. glabratum, unusual because the mycobiont is a basidio- and not an ascomycete. In it, the presence of other galactosylacylglycerides with one, two, and three units of galactose has been reported (Sassaki et al., 1999Go). Now, isolated from the lichen and characterized by 1D and 2D NMR spectroscopy and ESI-MS, were two derivatives of 6-sulfono-{alpha}-quinovosyl-(1'{leftrightarrow}1)-D-glycerol, one with the glycerol moiety disubstituted with long-chain fatty esters (PF1a) and the other monosubstituted (PF1b). Methanolysis of the glycolipids gave rise to the methyl glycoside of 6-sulfono-{alpha}-quinovopyranoside (Me-G) and no ß-anomer, based on NMR and ESI-MS data. Previous articles only described NMR assignments of acetylated derivatives of the glycolipids (Tulloch et al., 1973Go; Siddhanta et al., 1995Go).

Fatty acid esters of PF1a and PF1b were characterized as their methyl esters (GC-MS), following acid methanolysis and the principal component of each was palmitic ester. There were minor components, the most unusual being the odd-number C17:0 esters in both glycolipids and that of C21:0 in PF1a (Table III). The C17:0 ester has already been described as a component of D. glabratum, but not that of C21:0 (Sassaki et al., in preparation).

It is of interest to compare the fatty acids obtained from our lichen with those previously found in sulfonoglycolipids of plants. Tulloch et al. (1973)Go studied six plant species and found a great variation in their composition. In contrast to our data, their principal compound in most plants was the diacylated 2-O-palmitoyl-3-O-linolenoyl derivative, sometimes accompanied by a combination of palmitic, oleic, and linolenic esters of SQDG. In lyso-SQG, the fatty acids could be linolenic, palmitic, and linolenic, or mainly palmitic. The results are in contrast with ours, which showed mainly palmitic ester at C-2 and C-3 in PF1a, or at C-2 or C-3 in PF1b. In their investigation of minor fatty acid esters, they did not find those of C17:0 and C21:0.

In red (Siddhanta et al., 1995Go) and green marine algae (Fusetani and Hashimoto, 1975Go), the 2,3-di-O-dipalmitoyl derivative of SQDC was the most abundant component (Araki et al., 1989Go; Son, 1990Go; Siddhanta et al., 1991Go), as in the cyanobacterial, blue-green algae (Gustafson et al., 1989Go), which had lower levels and a variation of unsaturated fatty acids, when compared with those of higher plants.

Sulfonoglycolipids most likely arise from the D. glabratum photobiont, which is a cyanobacterium: Stigonema or Scytonema sp. (M. Marcelli, personal communication). This is by analogy with photosynthetic plants, algae, and bacteria, including cyanobacteria (Benson, 1963Go; Güler et al., 2000Go), which are associated with the galactolipids in the photosynthetic membranes, especially that of the tylakoides (Chapman and Barber, 1987Go).

In terms of our isolation methodology, the use of 90% aqueous ethanol under reflux gave lipids in a high yield; this procedure was described by Ledeen and Yu (1982)Go to extract gangliosides and more recently by Sassaki et al. (in preparation), who showed that this solvent is ideal for extraction of medium chain length fatty acids, the most common of which are C16:0 and C18:0 with the others varying from C14:0 to C24:0. The most useful modification of the method described by Ledeen and Yu was the substitution of the solvent system CHCl3-MeOH-0.8M NaOAc (60:40:8 v/v/v) by the EtOH-MeOH-0.8M NaOAc (89:11:9 v/v/v), which eluted polar lipids from DEAE-Sepharose, cheaper and less toxic. Fast-flow DEAE-Sepharose, instead of DEAE-Sephadex, gave rise to a more homogeneous flux and faster elution. This could be predicted by the low gel compaction of DEAE-Sepharose when eluted with organic solvents.

ESI-MS was useful to characterize molecular ions and the sequence of groups in the studied molecules and to distinguish lipid structures that gave similar NMR spectra. The positively charged fragment at m/z 329 arose from both glycolipids and was formed by removal of two O-acyl groups from PF1a and an O-acyl and a hydroxyl groups from PF1b, followed by a rearrangement in the glycerol moiety to form a double bond between C-2 and C-3 (Table I, Figure 2A, 2). The negative-ion mode spectrum showed that carbon covalently linked to the high oxidized sulfur (sulfonate group) can be cleaved (Figure 1), as confirmed by the fragment at m/z 146.

The pseudomolecular ions obtained in the positive-ion mode showed different types of O-substituted fatty acids. These observations confirmed the GC-MS data of derived methyl esters, although in some of the -MS spectra the presence of ion peaks were not as evident. The utilization of ESI-MS spectra (Table I, Figure 2A, 2B) can provide useful information to detect and identify fatty acids.

The NMR spectra showed that methanolysis gave exclusively the {alpha}-anomer of the methyl glycoside, although the presence of the ß-anomer might be expected. The 13C-NMR spectra of the sulfonoglycolipids contain complex CH2 and CH signals, although HMQC readily differentiated CH groups of the carbohydrate ring from that of the glycerol moiety, the latter having a distinctive {alpha}-shift to lower field for 13C and 1H nuclei when substituted by an O-acyl group. The O-acylated CH2 group gave rise to a doublet, which has an {alpha}-downfield shift of only 3 to 5 p.p.m., less than those of O-methyl and O-glycosyl substitutions. This characteristic makes HMQC an ideal experiment for analyzing glycosylglycerides, because it can serve as a fingerprint for these types of structure (Figure 3A). The TOCSY experiment, which shows relative configurations of protons present in the carbohydrate ring, indicates the structure of the possible monosaccharide units. Also, a 2-O-acyl substituent on the 1-O-linked D-glycerol moiety can be detected because H-2 would appear as a combination of two doublets (Figure 3B, 3C, 3D). These NMR data were characteristic to glycosylglycerides, distinguishing them from glycosphingolipids.

The lysosulfonolipids were completely characterised for the first time using spectroscopic data and revealed the presence of O-acyl groups at C-2 or C-3 of the D-glycerol moiety. This suggests that the lysosulfonolipids were formed by hydrolytic processes, although recent observations (Mongrand et al., 2000Go) showed that the galactolipids were acylated via assimilation of lysophosphatidylcholine (lyso-PC), which acts as a precursor for the synthesis of eukaryotic plastid lipids. Because sulfonoglycolipids are involved in photosynthesis as well as galactolipids, they should be associated with the cyanobacerial photobiont.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Lichen material
Dictyonema glabratum was collected in March 1999 from an embankment near to the 47 km sign of the National Highway (BR) 277, at an altitude of 900 m, in the proximity of Curitiba, State of Paraná, Brazil.

General procedures
Extracts of lipid-containing compounds, obtained from D. glabratum (see below), were evaporated at <40°C under reduced pressure (Sassaki et al., 1999Go). TLC was performed on silica gel plates (Merck), and the presence of glycolipids was detected by spraying with carbohydrate-specific orcinol-H2SO4, followed by heating at 100°C (Skipki, 1985Go).

Fatty acids methyl esters were obtained from the glycolipids by methanolysis (see below) and were analyzed by GC-MS on a DB-23 capillary column (30 m x 0.25 mm i.d.), programmed from 50°C to 220°C (40°C min–1) and then held, and characterized by their electron impact MS spectra and retention times compared with those of standards (Sigma products for lipids).

For detection of monosaccharides, descending PC was carried out on Whatman no. 1 filter paper (solvent: n-BuOH-pyridine-H2O, 5:3:3 v/v/v), developed with the acetone-AgNO3-NaOH dip reagent (Trevelyan et al., 1950Go). Isolated fractions were stored in sealed tubes at <10°C.

Extraction, fractionation, and isolation of glycolipids
Lichen (200 g) was cleaned and homogenized in a blender with a 0.1 M phosphate buffer at pH 7.2 to extract most of the proteins. The residue (145 g) was extracted 3x with 10 v/w of 90% aqueous EtOH (Ledeen and Yu, 1982Go) under reflux for 2 h. The resulting lipid-containing extracts were chromatographed on TLC and then combined and evaporated. Prior to fractionation of the extract on a column (3.9 x 16 cm) of fast-flow DEAE-Sepharose, it was eluted with 200 ml of EtOH-MeOH-0.8M NaOAc (80:11:9 v/v/v; solvent A) to give rise to the OAc form, and then 200 ml of EtOH-MeOH-H2O (89:11:9 v/v/v; solvent B) to remove excess NaOAc. The lipid extract (12.3 g) was applied to the column in 200 ml of solvent B and was successively eluted with solvent B (400 ml) and solvent A (300 ml) to give fractions 1 and 2a and 2b, respectively, and then with of EtOH-MeOH-1.2M NaOAc (80:11:9 v/v/v; 200 ml; solvent C) at a flow rate of 1 ml x min–1, the last step being repeated to give an eluate, which was negative to PhOH-H2SO4, giving Fraction 3. The three fractions obtained were dissolved in CHCl3-MeOH (2:1 v/v) and examined on TLC (solvent: CHCl3-MeOH-H2O, 65:25:4 v/v/v; spray: orcinol-H2SO4). Fractions 2a, 2b, and 3 (polar fraction: PF) were combined and to remove residual NaOAc; it was dissolved in 50 ml CHCl3-MeOH-H2O (60:40:9 v/v/v), to which was added 0.5% v/v aqueous H2SO4 (20 ml) and shaken for 2 min with EtOAc (90 ml). The upper phase was washed with H2O (120 ml) and evaporated to give fraction PF1, which was then dissolved in CHCl3 and chromatographed on a column of silicic acid (5.6 x 15 cm, particle size 0.1 mm). Elution was carried out with CHCl3 (500 ml) and then with increasing concentrations of MeOH in CHCl3 (500 ml each), with successive concentrations of 5%, 10%, 15%, 20%, 25%, 30%, 40%, and 50%. Isolated glycolipids were chromatographed by TLC using successively as solvents: CHCl3-MeOH-HOAc-H2O (100:20:12:5 v/v/v/v; Kataoka and Misaki, 1983Go), CHCl3-MeOH-HOAc-H2O (85:15:10:3 v/v/v/v; Roughan and Batt, 1968Go), and CHCl3-MeOH-H2O (65:25:4 v/v/v). Selected for their chromatographic purity were the fractions eluted from the column with 25% (PF1a) and 30% MeOH in CHCl3 w/v (PF1b).

Analysis of fatty acid components of glycolipids
Purified glycolipids (1 mg) were methanolyzed by refluxing in 3% MeOH-HCl for 2 h (Morrison, 1986Go). The resulting fatty acid methyl esters were extracted from excess. We added water with CHCl3 and analyzed the fatty acid methyl esters by GC-MS, as described above.

Isolation of methyl sulfonoglycoside from the glycolipids
Methanolysis of each sulfonoglycolipid fraction (20 mg) was performed refluxing in 3% w/v MeOH-HCl (1.0 ml) for 20 min. After neutralization (AgCO3), the filtered solution was evaporated and the residue fractionated on Whatman No. 1 filter paper (solvent: n-BuOH-pyridine-H2O, 5:3:3 v/v/v). The methyl glycoside, designated Me-G, which barely moved from the origin, was separated from the faster glycerol. The yield was 9 mg from each glycolipid.

ESI-MS
ESI-MS experiments were carried out using a Micromass Quattro LC spectrometer. The samples (10 µg) were dissolved in MeOH containing 0.01% NH4OAc, which was introduced by direct infusion. Analysis incorporated single quadrupole scanning, searching for a mass range of m/z 100–1000. The tuning parameters in the positive-ion mode were: capillary (4.75 kV), cone (192 V), and ion energy (0.6 V), which gave rise principally to pseudomolecular [M-H+2Na]+ ions. To give negative ions, the parameters were 5.00 kV, 200 V, and 1.9 V, respectively, to provide molecular ions [M].

1H and 13C NMR spectroscopy
For NMR experiments the purified samples were prepared following these steps, PF1a (13.6 mg) and PF1b (17.1 mg) were deuterium exchanged by repeated evaporation in CDCl3-CD3OD-D2O (1:1:0.5 v/v/v). Me-G (9 mg) was deuterium exchanged by successive lyophilizations in D2O. The NMR spectra were determined measured in DMSO-d6 and D2O at 30°C, using MS as internal standard ({delta} = 0). All spectra were obtained with a Bruker 400 MHz NMR spectrometer with a 5-mm inverse probe. Signal assignments in the 1D 1H- and 13C-NMR (decoupled and coupled) spectra of the glycolipids and the methyl glycoside were carried out using COSY, TOCSY, HMQC, ROESY, and DEPT programs.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank the Brazilian agencies FINEP (PRONEX-CARBOIDRATOS, PADCT II/SBIO) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
COSY, 1H, 1H correlation spectroscopy; DEAE, diethylaminoethyl; DEPT, distortionless enhancement by polarization transfer; GC-MS, gas chromatography–mass spectroscopy; ESI-MS, electrospray ionization mass spectrometry; HMQC, 1H, 13C heteronuclear multiple quantum coherence; ROESY, rotating frame Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy; TLC, thin-layer chromatography; PC, paper chromatography; NMR, nuclear magnetic resonance; PF, polar fraction; PF1a, polar fraction 1a; PF1b, polar fraction 1b; Me-G, methyl glycoside; SQDG, sulphoquinovosyldiacylglycerol; lyso-SQDG, sulphoquinovosylmonoacylglycerol.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Araki, S., Sakurai, T., Oohusa, T., Kayama, M., and Sato, M. (1989) Characterisation of sulphonoquinovosyl diacylglycerol from marine red alga. Plant Cell Physiol., 30, 775–781.[ISI]

Benson, A.A. (1963) The plant sulpholipid. Adv. Lipid Res., 1, 387–394.[ISI]

Chapman, D. and Barber, J. (1987) Polar lipids of chloroplast membranes. Methods Enzymol., 148, 294–319.[ISI]

Dertien, R.D., Kok, L.J., and Kuiper, P. (1977) Lipid and fatty acid composition of tree-growing and terrestrial lichens. Physiol. Plant., 40, 175–180.[ISI]

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