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.
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Abstract |
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Key words: Dictyonema glabratum/fatty acids/sulfonoglycolipids/NMR/ESI-MS
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Introduction |
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Results |
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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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NMR examination confirmed the structure determined for PF1a, namely, an -quinovosyl-6-sulfono unit, linked (1'
1) to D-glycerol. Its 13C NMR spectrum showed lipid signals at
14.4 (CH3) and from
23.3 to 35.0 (CH2), the largest being at
30.3 An ester carbonyl signal at
174.6 was also present. Double bonds were characterized by signals at
126.4 to 131.5. The anomeric 1H- and 13C NMR regions of the monosaccharide unit gave rise to a signal at
4.70/99.8, corresponding to an
-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
-Quip in the high field region at
55.8 (3.04, 2.69) and signals at
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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Discussion |
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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) 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., 1995) and green marine algae (Fusetani and Hashimoto, 1975
), the 2,3-di-O-dipalmitoyl derivative of SQDC was the most abundant component (Araki et al., 1989
; Son, 1990
; Siddhanta et al., 1991
), as in the cyanobacterial, blue-green algae (Gustafson et al., 1989
), 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, 1963; Güler et al., 2000
), which are associated with the galactolipids in the photosynthetic membranes, especially that of the tylakoides (Chapman and Barber, 1987
).
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) 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 -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
-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
-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., 2000) 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.
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Materials and methods |
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General procedures
Extracts of lipid-containing compounds, obtained from D. glabratum (see below), were evaporated at <40°C under reduced pressure (Sassaki et al., 1999). 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, 1985
).
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 min1) 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., 1950). 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, 1982) 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 min1, 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, 1983
), CHCl3-MeOH-HOAc-H2O (85:15:10:3 v/v/v/v; Roughan and Batt, 1968
), 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, 1986). 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 1001000. 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 ( = 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.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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