Modified galactosylceramide with a long-chain cyclic acetal at the sugar moiety, plasmalogalactosylceramide, was isolated from equine brain. To identify the isomeric stereostructure of the natural product, the plasmalo derivative was chemically synthesized from galactosylceramide through acetalization. The presence of cyclic acetal linkage, the linked position and length of the acetal chain of the synthesized and natural products were determined by proton nuclear magnetic resonance spectroscopy and fast-atom bombardment-mass spectrometry, as well as gas chromatography-mass spectrometry and gas-liquid chromatography. The orientation of the acetal chain linked to galactoside was characterized by connectivity between the cyclic acetal proton and ring proton(s) on the sugar moiety using the homonuclear Overhauser effect. This revealed that, of the two positional isomers of the acetal linkage with 4,6-Oacetal and 3,4-O -acetal derivatives obtained from the acetalization reaction, the former positional isomer, separated into two spots, was identified to 'endo'-and 'exo'-type acetal chains. In comparison to the NMR data of the synthesized derivative, equine brain acetalized lipid was found to be an 'endo'-type 4,6-O -acetal derivative.-
Novel mono-O-acetylated GM3s, one containing 9-O-acetyl N-glycolyl neuraminic acid and another containing 6'-O-acetyl galactose, were isolated as a mixture from equine erythrocytes, and the structures were characterized by one- and two-dimensional proton nuclear magnetic resonance (NMR) and fast atom bombardment-mass spectrometry (FAB-MS). The position of the O-acetyl residue was identified by the downfield shift of the methylene protons at C-9 of N-glycolyl neuraminic acid (9-O-Ac GM3) and C-6 of galactose (6'-O-Ac GM3) in the NMR spectrum, in comparison to the respective non-acetylated counterparts. To confirm the presence of 6'-O-Ac GM3, the O-acetylated GM3 mixture was desialylated with Arthrobacter neuraminidase, giving 6-O-acetyl galactosyl glucosylceramide, the structure of which was estimated by NMR and FAB-MS, together with non-acetylated lactosylceramide with a ratio of 1:1.
A novel galactosylalkylglycerol modified with a long-chain cyclic acetal at the sugar moiety, 3-O -(4 6 -plasmalogalactosyl) 1-O -alkylglycerol, was isolated from equine brain. The presence of cyclic acetal linkage, its linked position, and the length of the acetal chain of the natural plasmalo lipid were determined by proton NMR spectroscopy and fast-atom bombardment-mass spectrometry, as well as gas chromatography-mass spectrometry and gas-liquid chromatography. To identify the isomeric stereostructure of the natural product, the plasmalo derivative was chemically synthesized from 3-O -galactosyl 2-O -acyl 1-O -alkyl glyceride through acetalization after deacylation. As a result, the direction and position of the acetal chain of the natural plasmalo lipid were characterized as an " endo "-type 4 ,6 -Oacetal derivative linked to galactoside by comparison with the NMR data of the synthesized product. The chain lengths of alkyl and acetal groups were C 14 for the former and C 16 and C 18 for the latter, and those for the latter group were mostly similar to those of plasmalogalactosyl ceramide, which was previously isolated from equine brain. -Yachida,
A sulfatide, O-fatty-acylated 3-sulfogalactosylceramide at C6-O on galactoside, was isolated from equine brain and the chemical structure was characterized by proton NMR and MS. The O-acylation site of the acylated sulfatide was determined by the down-field shift of protons attached to a carbon having an O-acyl group in the NMR spectrum and by analysis of a partially methylated derivative before and after acetalization of the intact sulfatide using GC-MS. The O-acyl chain length was determined by GLC, revealing that it exclusively had palmitoyl and stearoyl residues as the major fatty acids. The enzymatic conversion to the O-acyl sulfatide was further examined using equine brain microsomes as an enzyme source and different lipid substrates, resulting in O-acylation of 3-sulfogalactosylceramide from stearoyl CoA, while 6-O-acyl galactosylceramide was not O-sulfated from phosphoadenosine phosphosulfate. The results were supported by the comparably different N-linked fatty acid components between two lipid substrates, in which the component of 6-O-acyl sulfatide was mostly similar to that of sulfatide, but not to 6-O-acyl galactosylceramide. Keywords : O-acyl sulfatide; O-acylation; esterified sulfatide ; NMR.Of the many glycolipids, sulfated glycosphingolipids (sulfa-MATERIALS AND METHODS tides) are distributed in a wide variety of mammalian tissues and Chemicals. DEAE-Sephadex, A-25 and Sephadex, LH-20 fluids, and have been reported to have several roles in binding were purchased from Pharmacia-LKB, silica beads (Iatrobeads) to some biologically active proteins such as laminin, thrombo-from Iatron Laboratories, TLC-plates (silica-gel 60) and spondin, von Willebrand factor and antistasin [1Ϫ6]. 3-O-hexadeuterodimethylsulfoxide ([ 2 H6]Me2SO) from Merck. SteaSulfogalactosylceramide (Gal3S-Cer) is a major glycolipid of royl CoA was from Sigma (MO). 3′-[ 35 S]Phosphoadenosine 5′-the sulfatides and localizes mainly in the central nervous system phosphosulfate (2 Ci/mmol) and [1-14 C]stearic acid (55 mCi/ and kidney, and to a minor extent in other tissues and fluids mmol) were obtained from NEN Bioproducts. All other reagents [7, 8]. The Gal3S-Cer is biosynthesized from the precursor were of analytical grade. galactosylceramide (GalCer) with glycolipid sulfotransferase loIsolation of O-acylated sulfatide. The ratio of solvent mixcalizing in the Golgi membrane [9,10].tures is expressed by volume. An acetone powder (104 g dry Esterified glycolipids with long-chain fatty acids have al-mass) was obtained from whole equine brain (457 g wet mass) ready been isolated from mammalian brain, spleen and epider-by homogenization with acetone (1 g/9 ml). The glycolipids mis [11Ϫ17]. However, these modified glycolipids had an ex-were extracted three times from the powder with chloroform/ clusively non-acidic glycolipid skeleton such as GalCer and glu-methanol/water, 4:8:3, at room temperature. The acidic glycocosylCer; where the O-acylation occurred at C2-, C3-, C4-, C6-lipid fraction was isolated from the combined and concentrated ...
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