Methoxyneuraminic acid

Methoxyneuraminic Acid (crystalline) By acid methanolysis of brain glyco-lipids (and later BSM)... 

Nonulosaminic acids are found as constituents of certain water-soluble, lipid fractions (glycolipids) of animal tissues, being particularly associated with gangliosides as well as with more complex lipopolysaccharides. The detection and characterization of the nonulosaminic acid component in these mucolipids has been based upon one or more of the color reactions (see below) applied to the macromolecular material, followed by isolation therefrom of methoxyneuraminic acid after methanolysis. The question of whether or not one or more acylated forms of neuraminic acid are actually present in the mucolipid has still to be resolved. Svennerholm has reported the isolation of A-acetylneuraminic acid from brain gangliosides. 

Qualitatively, the presence of the nonulosaminic acids is best indicated by the brilliant-red coloration formed on addition of an acidic solution of p-dimethylaminobenzaldehyde (the so-called direct Ehrlich reaction ), and by the bright-purple coloration which develops upon boiling with Bial s reagent for several minutes at 100°. In addition, the nonulosaminic acids give a blue-violet coloration with Dische s diphenylamine reagent for deoxypentoses and a positive reaction in the tryptophan-perchloric acid test. Since no single one of these color reactions is absolutely specific for a nonulosaminic acid, it is advisable to carry out at least two of these for a qualitative analysis. For quantitative determinations, all four reactions have been employed, using either A/ -acetylneuraminic acid (m. p., 183-185° [a] —32.0°) or methoxyneuraminic acid [m. p., 200° (dec.) [a]o —55.0°] as colorimetric standards. 

For their detection, the papers may be sprayed with reagents commonly used for the detection of keto and aminodeoxy sugars. Those of particular value include the direct Ehrlich reaction, the orcinol-trichloroacetic acid reagent, the chlorine-benzidine reagent for the detection of —NH—CO— groupings, and the Bial reaction as modified by Bohm und Baumeister. The limit of detection with the orcinol-trichloroacetic acid reagent is reported to be 5 ixg. of V-acetylneuraminic acid. For the detection of methoxyneuraminic acid, ninhydrin may also be used. 

A further modification of the method employs aqueous ammonia instead of the strong-alkali treatment, thus ensuring a minimal decomposition of the methanolysis products. These latter include both the methyl glycoside (methoxyneuraminic acid) and its methyl ester. After separation on a cation exchanger (Dowex 50), the crystalline material is obtained in yields of 60 % or higher. 

A comparison between certain chemical characteristics of the siaUc, iV-acetylneuraminic, and methoxyneuraminic acids, especially their functional groups and the color reactions afforded with Ehrlich s and Bial s reagents, suggested a close relationship between these nonulosaminic acids and indicated that methoxyneuraminic acid might be the methyl glycoside of a completely deacylated sialic acid. This concept was substantiated with the conversion of A-acetylneuraminic acid into methoxyneuraminic... 

This was clearly at variance with Gottschalk s former hypothesis and led him to propose the following structures for bovine sialic acid, iV-acetylneuraminic acid, and methoxyneuraminic acid. 

FIGURE 3. Foimation of methoxyneuraminic acid (V) from ketosidically bound NANA (IV), shown as Fischer projection formulas. Configuration of C2-ketoside changes from a (IV)toi8(V). 

FIGURE 14. Gas-liquid chromatograms of sialic acid liberated by mild methanolysis from purified ovine adrenal medulla and converted to trimethylsilyl ether derivatives. Left, 3% OV-1, 225°C right, 3% OV-225, 225°C. Peak identification A, methoxyneuraminic acid methyl ester (de-N-acylated product) B, methyl a-ketoside methyl ester of NANA C, methyl ketoside methyl ester of NANA D, phenyl N-acetyl-a-D-glucosaminide (internal standard) E, methyl a-ketoside methyl ester of NONA F, methyl /8-ketoside methyl ester of NONA. (Price and Yu, 1976). 

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