Abstract
As described by Carter et al. (J. Biol. Chem. 1951. 192: 197-207), O-methyl derivatives of sphingosine are formed upon acid hydrolysis of sphingolipids in the presence of methanol. In the present study, we have isolated four O-methyl ethers of C18-sphingosine by medium pressure liquid chromatography of their diacetate derivatives, i.e., (2S,3R,4E)-1-acetoxy-2-acetamido-3-methoxy-4-octadecene, its (2S,3S) epimer, (2R,3E,5R)-1-acetoxy-2-acetamido-5-methoxy-3-octadecene, and its (2R,5S) epimer. Structures were determined by physical, chromatographic, and spectral properties. The 5-O-methyl ethers, which were the predominant byproducts of sphingolipid hydrolysis, were easily distinguished from the 3-O-methyl ethers by chromatography, and all four isomers could be differentiated by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. NMR analysis of the original N-acetate and diacetate samples of O-methylsphingosines I and II of Carter et al. demonstrated that they correspond to the 5-O-methyl ethers (2R,5R and 2R,5S, respectively), with purities of approximately 90-99%. Resolution enhancement of the 126-MHz 13C NMR spectra of the O-methyl ethers and D-erythro-C18-sphingosine (Ia) afforded distinct signals for nearly all carbon atoms. 13C NMR assignments of carbons 7-15 were made from their lanthanide-induced shifts, and revised assignments for olefinic carbons of Ia were established based upon 1H-13C shift correlation experiments.
Highlights
Considerable attention has recently been directed towards free sphingolipid bases and Abbreviations: COSY, IH-IH correlation spectroscopy; COSYDEC, f,decoupled COSY; Gas chromatography (GC), gas chromatography; HMBC, heteronuclear multiple bond correlation; High performance liquid chromatography (HPLC), high performance liquid chromatography; HSQC, 1H-'9C heteronuclear single quantum coherence; IR, infrared; Lanthanide-induced shifts (LIS), lanthanide-induced shift; mp, melting point; MPLC, medium pressure liquid chromatography; Mass spectra (MS), mass spectrometry or mass spectrum; NMR, nuclear magnetic resonance; Pr(tfc)J, tris[3-(trifluoromethylhydroxymethylene)-(+)-camphorato]praseodymium(II1); Thin-layer chromatography (TLC), thin-layer chromatography; trimethylsilyl. IPresent addless (TMS), trimethylsilyl
Brain and spinal cord represent rich sources of sphingolipids from which enantiomerically pure Ia can be prepared in quantity [15].,hydrolysis of sphingolipids results in the formation of byproducts that complicate analyses of sphingolipid base composition and present problems in the isolation of pure D-erythm-sphingosine from natural sources [16,17,18]. 0-Methyl ethers have long beenrecognized as one type of byproduct formed during acid hydrolysis of sphingolipids in the presence of methanol [16, 19]
In the present study we describe the first isolation of each epimer of the 3-0-methyl and 5-0-methyl derivatives of C18-sphingosineby medium pressure liquid chromatography (MPLC) of their diacetates
Summary
As described by Carter et al (J Bid. C h . 1951. 192: 197-207), 0-methyl derivatives of sphingosine are formed upon acid hydrolysis of sphingolipids in the presence of methanol.
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