Abstract
It is well known that acid hydrolysis of natural sphingomyelin in aqueous methanol or 1-butanol at refluxing temperature is accompanied by epimerization at the C-3 position of the long-chain base. An improved procedure for the hydrolysis of commercially available, naturally occurring sphingomyelin is described. Prolonged exposure (3;-4 days) of sphingomyelin to freshly prepared 0.5 M anhydrous methanolic hydrogen chloride (generated by trapping the gas evolved from the reaction of concentrated sulfuric acid with solid sodium chloride in anhydrous methanol) at 50 degrees C resulted in cleavage of the amide side chain. The extent of epimerization of the allylic alcohol stereocenter was quantified by integration of the C-5 signal of the (13)C nuclear magnetic resonance spectrum of lysosphingomyelin. The method described here is superior to the traditional acid hydrolysis methods because it provides the product as a approximately 10:1 ratio of d-erythro/l-threo epimers; in contrast, a ratio of approximately 1. 3:1 was obtained by the previous methods. We also report that use of dichloromethane as a cosolvent with N,N-dimethylformamide in the reaction of lysosphingomyelin with an activated fatty acid reduced the time required for completion of the N-acylation reaction.
Highlights
It is well known that acid hydrolysis of natural sphingomyelin in aqueous methanol or 1-butanol at refluxing temperature is accompanied by epimerization at the C-3 position of the long-chain base
We illustrate here the first use of 13C nuclear magnetic resonance (NMR) spectroscopy to evaluate the degree of epimerization with the known strategies for acid hydrolysis of sphingomyelin, and we describe a new method for preparing lysosphingomyelin with a low extent of C-3 epimerization
Conditions under which a variety of amides are known to undergo hydrolysis [25] were applied to egg-yolk sphingomyelin, and the reactions were analyzed by Thin-layer chromatography (TLC) (Table 1)
Summary
It is well known that acid hydrolysis of natural sphingomyelin in aqueous methanol or 1-butanol at refluxing temperature is accompanied by epimerization at the C-3 position of the long-chain base. The impracticality associated with the use (and subsequent removal) of high-boiling solvents prompted Gaver and Sweeley [14] to report a second strategy in which naturally occurring sphingomyelin is heated in 1 M methanolic HCl at reflux. Each of these methods causes a significant loss of configuration at the C-3 (allylic) stereocenter. Commercially available semisynthetic sphingomyelins that are prepared by N-acylation of lysosphingomyelin (obtained by acid hydrolysis of sphingomyelin) are mixtures of d-erythro (2S,3R) and l-threo (2S,3S) diastereomers [17]. The presence of silica gel in the product lysosphingomyelin causes an inaccurate determination of the yields of both the hydrolysis and reacylation reactions
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