Preparation of deacetyl-, lyso-, and deacetyl-lyso-GM3 by selective alkaline hydrolysis of GM3 ganglioside

Abstract

Three methods (using GM3 quantities ranging from a few milligrams to grams) have been developed to prepare, in high yield, the three derivatives of ganglioside GM3 [α-Neu5Ac-(2-3)-β-Gal-(1-4)-β-Glc-(1-1)-ceramide]: deacetyl-GM3 [α-Neu-(2-3)-β-Gal-(1-4)-β-Glc-(1-1)-ceramide], lyso-GM3 [α-Neu5Ac-(2-3)-β-Gal-(1-4)-β-Glc-(1-1)-sphingosine], and deacetyl-lyso-GM3 [α-Neu-(2-3)-β-Gal-(1-4)-β-Glc-(1-1)-sphingosine]. This is the first report of the preparation of lyso-GM3 by a one-pot reaction. We can now define the optimal conditions for the different preparations. Preparation of deacetyl-GM3: alkaline reagent, 2 M KOH in water; GM3 concentration, 33 mg/ml; reaction temperature, 90 °C; reaction time, 3.5 h; nitrogen atmosphere. Preparation of deacetyl-lyso-GM3: alkaline reagent, 8 M KOH in water; GM3 concentration, 10 mg/ml; reaction temperature, 90 °C; reaction time, 18 h; nitrogen atmosphere. Preparation of lyso-GM3: alkaline reagent, 1 M sodium tert-butoxide in methanol; GM3 concentration, 10 mg/ml; reaction temperature, 80 °C; reaction time, 18 h; anhydrous conditions. The percentage yield of deacetyl-GM3 was 70–75%, that of deacetyl-lyso-GM3 100%, and of lyso-GM3 36–40%. Deacetyl-GM3, deacetyl-lyso-GM3, and lyso-GM3 were purified by column chromatography, and chemical structures were confirmed by electron spray-mass spectrometry. —Valiente, O., L. Mauri, R. Casellato, L. E. Fernandez, and S. Sonnino. Preparation of deacetyl-, lyso-, and deacetyl-lyso-GM3 by selective alkaline hydrolysis of GM3 ganglioside.

Keywords

• TLC staining
• tritium
• tert-butoxide
• ESI-MS