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Stereochemical structures of synthesized and natural plasmalogalactosylceramides from equine brain

1998; Elsevier BV; Volume: 39; Issue: 5 Linguagem: Inglês

10.1016/s0022-2275(20)33872-4

1539-7262

Youichi Yachida, Motoi Kashiwagi, Takeshi Mikami, Keiko Tsuchihashi, Takumi Daino, Toyoaki Akino, Shinsei Gasa,

Lipid Membrane Structure and Behavior

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-O-acetal 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.—Yachida, Y., M. Kashiwagi, T. Mikami, K. Tsuchihashi, T. Daino, T. Akino, and S. Gasa. Stereochemical structures of synthesized and natural plasmalogalactosylceramides from equine brain. J. Lipid Res. 1998. 39: 1039–1045. 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-O-acetal 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.—Yachida, Y., M. Kashiwagi, T. Mikami, K. Tsuchihashi, T. Daino, T. Akino, and S. Gasa. Stereochemical structures of synthesized and natural plasmalogalactosylceramides from equine brain. J. Lipid Res. 1998. 39: 1039–1045. Glycosphingolipids (GSLs) play roles in intercellular recognition and transmembrane signaling (1Hannun Y.A. Bell R.M. Functions of sphingolipids and sphingolipid breakdown products in cellular regulation.Science. 1989; 243: 500-507Google Scholar, 2Hakomori S. Bifunctional role of glycosphingolipids. Modulators for transmembrane signaling and mediators for cellular interactions.J. Biol. Chem. 1990; 265: 18713-18716Google Scholar). Some of these GSLs are modified in normal and tumor tissues, such as O-fatty acylated galactosylceramide (GalCer) in several mammalian (3Klenk E. Lohr J.P. On the ester cerebrosides of brain.Hoppe-Seylers Z. Physiol. Chem. 1967; 348: 1712-1714Google Scholar, 4Kishimoto Y. Wajda M. Radin N.S. 6-acyl galactosyl ceramides of pig brain: structure and fatty acid composition.J. Lipid Res. 1968; 9: 27-33Google Scholar, 5Tamai Y. Further study on the faster running glycolipid in brain.Jpn. J. Exp. Med. 1968; 38: 65-73Google Scholar) and fish (6Tamai Y. Nakamura K. Takayama-Abe K. Uchida K. Kasama T. Kobatake H. Less polar glycolipids in Alaskan pollack brain: isolation and characterization of acyl galactosyl diacylglycerol, acyl galactosyl ceramide, and acyl glucosyl ceramide.J. Lipid Res. 1993; 34: 601-608Google Scholar) brains, O-fatty acylated glucosylceramide in mammalian epidermis (7Gray G.M. White R.J. Majer J.R. 1-(3′-O-acyl)-beta-glucosyl-N-dihydroxypentatriacontadienoylsphingosine, a major component of the glucosylceramides of pig and human epidermis.Biochim. Biophys. Acta. 1978; 528: 127-137Google Scholar), O-acetyl GD3 in melanoma cells (8Cheresh D.A. Reisfeld R.A. Varki A.P. O-acetylation of disialoganglioside GD3 by human melanoma cells creates a unique antigenic determinant.Science. 1984; 225: 844-846Google Scholar, 9Thurin J. Herlyn M. Hindsgaul O. Stromberg N. Karlsson K.A. Elder D. Steplewski Z. Koprowski H. Proton NMR and fast-atom bombardment mass spectrometry analysis of the melanoma-associated ganglioside 9-O-acetyl-GD3.J. Biol. Chem. 1985; 260: 14556-14563Google Scholar), O-acetyl GM3 in horse erythrocytes (10Hakomori S. Saito T. Isolation and characterization of a glycosphingolipid having a new sialic acid.Biochemistry. 1969; 8: 5082-5088Google Scholar, 11Gasa S. Makita A. Kinoshita Y. Further study of the chemical structure of the equine erythrocyte hematoside containing O-acetyl ester.J. Biol. Chem. 1983; 258: 876-881Google Scholar, 12Gasa S. Makita A. Yanagisawa K. Nakamura M. Glycosphingolipids of equine erythrocytes membranes: complete characterization of a fucoganglioside.Adv. Exp. Med. Biol. 1984; 174: 111-117Google Scholar, 13Yachida Y. Tsuchihashi K. Gasa S. Characterization of novel mono-O-acetylated GM3s containing 9-O-acetyl sialic acid and 6-O-acetyl galactose in equine erythrocytes.Glycoconj. J. 1996; 13: 225-233Google Scholar, 14Yachida Y. Tsuchihashi K. Gasa S. Novel di-O-acetylated GM3s from equine erythrocytes one containing 4,9-di-O-acetyl N-glycolyl neuraminic acid and another containing 4-O-acetyl N-glycolyl neuraminic acid and 6-O-acetyl galactose.Carbohydr. Res. 1997; 298: 201-212Google Scholar), and O-acetylated GM3 at the Cer moiety in glioma (15Suetake K. Tsuchihashi K. Inaba K. Chiba M. Ibayashi Y. Hashi K. Gasa S. Novel modification of ceramide: rat glioma ganglioside GM3 having 3-O-acetylated sphingenine.FEBS Lett. 1995; 361: 201-205Google Scholar). PlasmaloGSLs, which are conjugated with a long-chain fatty aldehyde at the sugar moiety and reported to form a cyclic acetal linkage exclusively on galactose, have recently been isolated from normal human brain as novel modified GSLs (16Levery S.B. Nudelman E.D. Hakomori S. Novel modification of glycosphingolipids by long-chain cyclic acetals: isolation and characterization of plasmalocerebroside from human brain.Biochemistry. 1992; 31: 5335-5340Google Scholar, 17Nudelman E.D. Levery S.B. Igarashi Y. Hakomori S. Plasmalopsychosine, a novel plasmal (fatty aldehyde) conjugate of psychosine with cyclic acetal linkage. Isolation and characterization from human brain white matter.J. Biol. Chem. 1992; 267: 11007-11016Google Scholar). Modification with fatty acetal was reported to occur at 3,4-O and 4,6-O on Gal of GalCer (16Levery S.B. Nudelman E.D. Hakomori S. Novel modification of glycosphingolipids by long-chain cyclic acetals: isolation and characterization of plasmalocerebroside from human brain.Biochemistry. 1992; 31: 5335-5340Google Scholar) and Gal sphingosine (psychosine) (17Nudelman E.D. Levery S.B. Igarashi Y. Hakomori S. Plasmalopsychosine, a novel plasmal (fatty aldehyde) conjugate of psychosine with cyclic acetal linkage. Isolation and characterization from human brain white matter.J. Biol. Chem. 1992; 267: 11007-11016Google Scholar), and their chemical structures were characterized mainly with mass spectra using fast-atom bombardment–mass spectrometry, and by methylation analysis. The presence and chain length of fatty aldehyde have also been determined by gas chromatography–mass spectrometry (GC–MS) as an enol methyl ether derivative. In particular, plasmalopsychosines have been chemically synthesized from psychosine with acetalization, giving acetal structures identical to naturally occurring plasmalolipid (18Sadozai K.K. Anand J.K. Nudelman E.D. Hakomori S. Synthesis of plasmalopsychosines A and B two novel lysosphingolipids found in human brain.Carbohydr. Res. 1993; 241: 301-307Google Scholar). However, the stereostructure of natural plasmaloGalCer has not yet been characterized. In the present paper, we describe isolation of plasmaloGalCer from equine brain, its synthesis and identification of its stereochemical structure. DEAE-Sephadex, A-25 and LH-20 were purchased from Pharmacia-LKB (Sweden). Iatrobeads (8060) were from Iatron (Tokyo). Precoated thin-layer chromatography (TLC)-plates (Silica gel 60) and pyridine-d5 were obtained from Merck (Germany). Other reagents were of analytical grade. The ratio of solvent mixtures is expressed by volume. Whole horse brain (457 g wet weight) was homogenized with acetone (1 g/9 ml) to yield an acetone powder with a dry weight of 104 g. The glycolipids were extracted three times from the powder with chloroform–methanol–water (CMW, 4:8:3) at room temperature. The neutral glycolipid fraction was isolated from the combined and concentrated extracts with a DEAE-Sephadex, A-25 (acetate form) column (2.5 × 30 cm) by elution with CMW (40:60:10). The total neutral glycolipids were chromatographed on a silica-gel (Iatrobeads) column (2.5 × 50 cm) by stepwise elution with CM from 95:5 to 90:10, 85:15, 80:2, 75:25, and 70:30 each with 1,000 ml. The fraction that consisted of several less polar glycolipids was eluted with CM, 95:5 and 90:10, and the combined eluates were further chromatographed on a silica-gel column with a smaller column size (1.2 × 40 cm) by stepwise elution with the above CM, and chromatography was repeated to obtain homogenous glycolipid. The purified glycolipids were chromatographed on a TLC plate, developed with CMW (90: 10:0.5), and visualized by spraying with orcinol–sulfuric acid reagent, for estimation of the purity. The chemical acetalization of GalCer was performed according to the method of Sadozai et al., with a slight modification, utilizing synthesis of plasmalo-psychosine (18Sadozai K.K. Anand J.K. Nudelman E.D. Hakomori S. Synthesis of plasmalopsychosines A and B two novel lysosphingolipids found in human brain.Carbohydr. Res. 1993; 241: 301-307Google Scholar) and -methyl galactoside (19Sadozai K.K. Levery S.B. Anand J.K. Hakomori S. Model compounds from plasmaloglycolipids: preparation of long chain cyclic acetals of methyl beta-d-galactopyranoside and determination of their regio- and stereochemistry by proton NMR.J. Carbohydr. Chem. 1996; 15: 715-725Google Scholar). Briefly, equine brain GalCer (30 mg containing non-hydroxy fatty acid, co-purified as described above) in dimethylformamide (2 ml) was incubated with p-toluene sulfonic acid (5 mg) and 1,1-dimethoxyhexadecane (30 mg). The reaction mixture was applied to an LH-20 column (1 × 50 cm) with chloroform to remove dimethylformamide and p-toluene sulfonic acid. The acetalized products were further purified on a silica-gel column (1 × 40 cm) by stepwise elution with CM from 98:2 to 96:4, 94:6, 92:8 and 90:10 each with 300 ml. An aliquot of the fraction was chromatographed by TLC as above. The fatty acid and fatty aldehyde of synthesized and natural plasmaloGalCer were analyzed from the methanolyzates of the purified GSL using a gas–liquid chromatography (GLC) apparatus (GC-14A, Shimadzu) equipped with a capillary column (0.25 mm × 50 m) coated with 1% of DB-5, with programmed temperatures from 150 to 250°C at 5°C per min. The methanolysis of the glycolipid (0.1 mg) was carried out with 1 ml of 1 N HCl in anhydrous methanol at 80°C for 16 h, followed by extraction three times with 1 ml of n-hexane. After concentration of the extracts under N2 gas to approximately 20 μl, an aliquot of the extracts was subjected to GLC. The GLC peaks were characterized using a GC–MS (JEOL JMS-OISG-2) with electron impact ionization, equipped with a capillary column (0.25 mm × 50 m) coated with 1% of OV-1 and the same programmed temperatures as the above GLC, at the NMR–MS Laboratory of the Faculty of Agriculture in Hokkaido University. The sugar species and the substituted sites of the synthesized and natural plasmaloGalCers were determined by GC–MS as above with partially methylated alditol acetate derived from permethylated GSL in basic conditions following acetolysis/hydrolysis, reduction with NaBH4, and peracetylation as reported previously (20Sako F. Gasa S. Makita A. Hayashi A. Nozawa S. Human blood group glycosphingolipids of porcine erythrocytes.Arch. Biochem. Biophys. 1990; 278: 228-237Google Scholar). One (1-D)- and two-dimensional (2-D) proton nuclear magnetic resonance spectroscopy (NMR) spectra of the glycolipids (approximately 1 mg) in 0.3 ml of pyridine-d5 containing 2% D2O were obtained at 90°C in a Fourier-transform mode on a Bruker AMX-500 spectrometer at the above laboratory, as described previously (13Yachida Y. Tsuchihashi K. Gasa S. Characterization of novel mono-O-acetylated GM3s containing 9-O-acetyl sialic acid and 6-O-acetyl galactose in equine erythrocytes.Glycoconj. J. 1996; 13: 225-233Google Scholar, 15Suetake K. Tsuchihashi K. Inaba K. Chiba M. Ibayashi Y. Hashi K. Gasa S. Novel modification of ceramide: rat glioma ganglioside GM3 having 3-O-acetylated sphingenine.FEBS Lett. 1995; 361: 201-205Google Scholar). Chemical shifts (δ, ppm) were measured using tetramethylsilane as an internal standard. The TLC profiles of the GSLs purified from equine brain are presented in Fig. 1, together with O-acylated- and unacylated-GalCers. Of these less-polar GSLs, the lipid abbreviated as N-1 was a plasmaloGalCer (see below) with a yield of 2.3 mg, and co-purified with 6-O-fatty acyl GalCer (12 mg yield) and 2-O-acyl GalCer (8 mg) as well as unmodified GalCer. The mobility of N-1 did not change on TLC analysis after saponification with 0.2 N sodium methoxide in methanol (data not shown), by which an O-acyl group was released from the O-acylated GalCers, indicating no modification with an alkali-labile group as in O-acylation. The structures of N-1 and O-acylated GalCers were determined by NMR, MS, and GLC as described below (MS and GLC data not shown for the N-1). To identify the structure of naturally acetalized GalCer (N-1) from equine brain, GalCer from the brain was chemically acetalized with a 1,1-dimethoxy derivative of hexadecanal. Three products, abbreviated as S-1, -2, and -3, were obtained from the reaction followed by purification. S-1 (0.9 mg, 3% yield) eluted faster with CM, 98:2; S-2 (22.5 mg, 75%) eluted later with 98:2; and S-3 (2.4 mg, 7%) eluted with 96:4 from the silica-gel column. Of these chemically acetalized GalCers, S-2 showed an Rf identical to N-1 as demonstrated in Fig. 1. The lipid moieties of the above plasmaloGalCers were analyzed by GLC and GC–MS after methanolysis with anhydrous methanolic HCl, which converted the fatty acid to the methyl ester; the fatty aldehyde was derivatized to dimethyl acetal as a major component and an enol methyl ether as a minor one. Such conversions of fatty aldehydes under an anhydrous acidic condition were confirmed by analysis of the methanolyzates of authentic hexadecanal. The starting fatty aldehyde itself was detected at a level of less than 0.1%. The lipid data are summarized in Tables 1, 2, 3, 4. From the sum of the respective areas with aldehyde and fatty acid derivatives in the gas chromatograms, synthesized and natural GalCers were estimated to have both lipid components with approximately similar ratios. The saturated fatty aldehydes were detected in N-1 with hexadecanal and octadecanal at 33% and 44%, respectively, together with their monounsaturated aldehydes as a minor component. The composition and ratio of the lipid moiety released from S-1 and S-3 by methanolysis were similar in these lipids, indicating that they were stereoisomers of the acetal ring and/or positional isomers of S-2. The lipid compositions of S-1, -2, -3, and N-1 were alternatively confirmed by positive ion fast-atom bombardment–mass spectrometry (data not shown).TABLE 1Composition of fatty acids aaMeasured as methyl ester by gas–liquid chromatography. Trace, <0.4%.Carbon Chain LengthS-2bbLipid composition was identical to those of S-1 and S-3.N-1SaturatedMonounsaturatedSaturatedMonounsaturated%%163tracetracetrace1811trace34219tracetracetracetrace2012trace622112trace1trace22tracetrace7123tracetrace8trace2444724trace258trace51261trace42Unknown23a aMeasured as methyl ester by gas–liquid chromatography. Trace, <0.4%.b bLipid composition was identical to those of S-1 and S-3. Open table in a new tab TABLE 2.Composition of fatty aldehydesCarbon Chain LengthS-2N-1%%16:0Aldehydetrace1Enol ether41Acetal963216:1Aldehyde—traceEnol ether—3Acetal—818:0Aldehyde—traceEnol ether—7Acetal—3718:1Aldehyde—traceEnol ether—traceAcetal—11Trace, <0.4%. Open table in a new tab TABLE 3.Composition of long chain baseLong Chain BaseS-2N-1%%Sphingenined18:19795Sphinganined18:0trace1Unknown34Trace, <0.4%. Open table in a new tab TABLE 4.Composition of saccharidesSaccharidePartially Methylated Alditol AcetateaaMeasured as methyl ester by gas–liquid chromatography. Trace, <0.4%.S-11,4,5,6-tetra-O-acetyl 2,3-di-O-methyl galactitolS-21,4,5,6-tetra-O-acetyl 2,3-di-O-methyl galactitolS-31,3,4,5-tetra-O-acetyl 2,6-di-O-methyl galactitolN-11,4,5,6-tetra-O-acetyl 2,3-di-O-methyl galactitolaAnalyzed by gas chromatography–mass spectrometry. Open table in a new tab Trace, <0.4%. Trace, <0.4%. aAnalyzed by gas chromatography–mass spectrometry. The substituted sites of Gal on S-1, -2, -3 and N-1 with fatty acetal were further analyzed by GC–MS of the partially methylated alditol acetates, in which acetalized positions in the intact sugar moiety were replaced with O-acetates. S-1, S-2, and N-1 gave only 2,3-di-O-methyl-1, 4, 5, 6-tetra-O-acetyl galactitol, indicating these GSLs to be substituted at C-4,6-O on the Gal, whereas S-3 gave 2, 6-di-O-methyl-1, 3, 4, 5-tetra-O-acetyl galactitol, substituted at C-3,4-O on Gal (Table 4). Combined data from this methylation analysis and GLC analysis of the lipid moiety suggested that S-1 and S-2 were diastereomeric isomers of each other for the cyclic acetal ring and that S-3 was the positional isomer of S-1 and S-2. The partial 1-D proton NMR spectra of S-1, -2, -3 and N-1 are shown in Fig. 2, together with that of GalCer as a reference. The assignment of proton signals inserted in the figure was performed with the 2-D chemical shift-correlated spectroscopy spectra (data not shown), and the data are summarized in Table 5. The triplet signal H-1′′ (peak 1′′ in Fig. 2; the carbon number in each group is shown in Fig. 4) at δ5.291 ppm with one proton from the relative integration newly appeared in the 1-D spectrum of S-1 in comparison with the spectrum of GalCer, and the H-1′′ was coupled to an upper field signal H-2′′ (1.75 ppm) having two-proton intensity, which was further coupled to H-3′′ (1.48 ppm) with two protons in the 2-D spectrum. The chemical shifts, splitting pattern, and integrations of these proton signals suggested the presence of a sequence of dioxomethine–methylene–methylene, and H-1′′ was consequently identified as a methine proton of the acetal group.TABLE 5.Chemical shifts and coupling constants (Hz) of ring proton on galactoside and acetal proton of plasmaloGalCers and reference GalCerChemical Shift (∂, ppm)GalactosideAcetalPlasmaloGalCerH-1′H-2′H-3′H-4′H-5′H-6′aH-6′bH-1′′H-2′′H-3′′S-14.6444.2033.914.3303.8184.0084.2305.2911.751.48S-24.5894.1463.8754.0943.4133.874.1874.6651.741.48S-34.6023.8844.3254.2194.1664.1484.175.0661.741.48N-14.6204.1833.954.1173.4303.8574.2034.6821.791.62GalCer4.7054.2403.9514.3413.8824.2264.238Coupling Constant (Hz)J1′,2′J2′,3′J3′,4′J4′,5′J5′,6′aJ5′,6′bJ6′a,6′bJ1′′,2′′S-17.47.93.5<1.5<1.54.911.85.4S-27.98.04.0<1.5<1.5<1.512.34.9S-37.96.66.4<1.54.03.510.34.5N-17.97.94.0<1.5ND<1.512.34.9GalCer7.410.03.5<1.56.95.9eqND, not determined; eq, equivalent. Open table in a new tab Fig. 4.Stereostructure of “exo”- and “endo”-type plasmaloGalCers. The NOE peak was observed in the 2-D spectra (Fig. 3) between protons indicated by arrow.View Large Image Figure ViewerDownload (PPT) ND, not determined; eq, equivalent. The 1-D spectrum of S-2 also showed a new triplet H-1′′ signal at δ4.665 ppm (Fig. 2), and the proton was coupled with H-2′′ (1.74 ppm) and the H-2′′ further coupled with H-3′′ (1.48 ppm) in the 2-D spectrum, indicating H-1′′ to be an H-1 of the acetal as in the case of S-1. The S-1 and S-2 were, therefore, each configurational isomers at C-1′′ of the acetal with an asymmetric center having C-4-O, C-6-O, H-1′′ of the acetal and a hydrocarbon chain. These isomers were each characterized by 2-D homonuclear Overhauser effect spectroscopy (NOESY). The H-1′′ of the acetal of S-1 was positioned close to H-2′ on Gal based on the appearance of an NOE peak (1′′–2′), and acetal H-2′′ nearby H-4' and H-6'a on Gal from peaks 2′′–4′ and 2′′–6′a, respectively, as shown in Fig. 3A. These NOE peaks involved a stereostructure of a cyclic acetal ring having an 'exo'-type hydrocarbon chain and consequently an equatorial orientation of H-1′′ of the acetal on a six-membered ring (1,3-dioxane ring) as illustrated by structure 1 in Fig. 4. On the other hand, H-1′′ of the acetal of S-2 had NOE peaks toward H-4′ and –6′a (Fig. 3B), indicating proximity of acetal H-1′′ and Gal H-4′ and H-6′a, and consequently S-2 was an “endo”-type stereoisomer of the hydrocarbon chain having an axial H-1′′ of the acetal (structure 2 in Fig. 4). Of the two stereoisomers, S-1 and S-2, the 1-D and 2-D spectra of S-2 were most similar to those of N-1, indicating that N-1 and S-2 had isomeric structures identical to that of structure 2. The 1-D spectrum of S-3 demonstrated the presence of H-1′′ of the acetal (Fig. 2), coupled with H-2′′ and the H-2′′ sequentially with H-3′′ as observed in the 2-D spectrum (data not shown), just as in the cases of S-1 and S-2. The stereochemical assignment of the “endo”- or “exo”-type hydrocarbon chain, of S-3 with a cyclic acetal ring having a five-membered ring was, however, unsuccessful, as the NOE spectrum was very complex. In the present paper, plasmaloGalCer having 4,6-O-hexadecylidene and -octadecylidene and their unsaturated derivatives was isolated from equine brain, and the stereoisomeric structure was characterized by NMR study as structure 2 with the “endo”-type in comparison to the stereoisomeric structures of chemically synthesized plasmaloGalCers. The synthesized 4,6-O-hexadecylidene derivative was chromatographically separated into two diastereomeric isomers of the structures 1 and 2, S-1 and S-2, respectively, with long hydrocarbon chains of fatty aldehyde, forming the 1,3-dioxane structure of the cyclic acetal ring. Though the two stereoisomers of 3,4-O-hexadecylidene derivatives should also occur, one of these isomers was barely detected on TLC analysis, probably because of the much lower yield of the isomer. The configuration at acetal carbon, the asymmetric center of the synthesized 4,6-plasmaloGalCers, was effectively characterized by the NOESY spectrum as above, revealing the acetal proton (H-1′′) to be equatorial in S-1 and axial in S-2 in the 1,3-dioxane ring (see Fig. 4). These assignments were supported by previous data with different chemical shifts of H-1 of the acetal between equatorial and axial protons on a 1,3-dioxane ring, in which the equatorial proton resonated in a field lower than δ4.8 ppm whereas the chemical shift of the axial proton was δ4.5 to 4.7 ppm (22Jackman L.M. Sternhell S. Applications of nuclear magnetic resonance spectroscopy in organic chemistry. Pergamon Press, London1969Google Scholar). The chemical shifts of the axial H-1 of the acetal of other plasmalo derivatives have also been observed comparably in the upper field with δ4.582 ppm (observed in CDCl3–CD3OD) in synthesized plasmalopsychosine (18Sadozai K.K. Anand J.K. Nudelman E.D. Hakomori S. Synthesis of plasmalopsychosines A and B two novel lysosphingolipids found in human brain.Carbohydr. Res. 1993; 241: 301-307Google Scholar) and with δ4.500 ppm (CDCl3) of per-O-acetyl methyl β-galactopyranoside 4,6-O-hexadecylidene (21Budzikiewicz H. Brauman J.I. Djerassi C. Massenspektrometrie und ihre anwendung auf strukturelle und stereochemische probleme-LXVII.Tetrahedron. 1965; 21: 1855-1879Google Scholar), in accordance with the above observations. With respect to biological activity of plasmaloGSL, plasmalopsychosine has been reported to have a weak inhibitory effect on protein kinase C activity, and an enhancements of p140trk phosphorylation, mimicking activity of nerve growth factor on rat pheochromocytoma cells (23Sakakura C. Igarashi Y. Anand J.K. Sadozai K.K. Hakomori S. Plasmalopsychosine of human brain mimics the effect of nerve growth factor by activating its receptor kinase and mitogen-activated protein kinase in PC12 cells. Induction of neurite outgrowth and prevention of apoptosis.J. Biol. Chem. 1996; 271: 946-952Google Scholar). These phenomena indicate that plasmalopsychosine has important biological roles in the nervous system, though the biosynthetic pathway of the plasmalolipids is not yet known. We are deeply indebted to Mr. Kim Barrymore for his help in the preparation of this article, Dr. Yoshihiro Maeda for his technical help in the preparation of glycolipids, and the Hokkaido Ebetsu Meat Inspection Office for donation of equine brain. This work was supported in part by Grant-in-Aid for Scientific Research on Priority Areas No. 10142712 from the Ministry of Education, Science and Culture, Japan. chloroform–methanol–water galactosylceramide gas chromatography–mass spectrometry gas–liquid chromatography glycosphingolipid nuclear magnetic resonance spectroscopy homonuclear Overhauser effect spectroscopy thin-layer chromatography.