Preparation of deacetyl-, lyso-, and deacetyl-lyso-GM(3) by selective alkaline hydrolysis of GM(3) ganglioside

Three methods (using GM(3) quantities ranging from a few milligrams to grams) have been developed to prepare, in high yield, the three derivatives of ganglioside GM(3) [alpha -Neu5A-c(2-3)-beta -Gal-(1-4)-beta -Glc-(1-1)-ceramide]: deacetyl-GM(3) [alpha -Neu-(2-3)-beta -Gal-(1-4)-beta -Glc-(1-1)-ceramide], lyso-GM(3) [alpha -Neu5Ac-(2-3)-beta -Gal-(1-4)-beta -Glc-(1-1)-sphingosine], and deacetyl-lyso-GM(3) [alpha -Neu-(2-3)-beta -Gal-(1-4)-beta -Glc-(1-1)-sphingosine]. This is the first report of the preparation of lyso-GM(3) by a one-pot reaction. We can now define the optimal conditions for the different preparations. Preparation of deacetyl-GM(3): alkaline reagent, 2 M KOH in water; GM(3) concentration, 33 mg/nl; reaction temperature, 90 degreesC; reaction time, 3.5 h; nitrogen atmosphere. Preparation of deacetyl-lyso-GM(3): alkaline reagent, 8 M KOH in water; GM(3) concentration, 10 mg/ml; reaction temperature, 90 degreesC; reaction time, 18 h; nitrogen atmosphere. Preparation of lyso-GM(3): alkaline reagent, 1 M sodium tertbutoxide in methanol; GM(3) concentration, 10 mg/ml; reaction temperature, 80 degreesC; reaction time, 18 h; anhydrous conditions. The percentage yield of deacetyl-GM(3) was 70-75%, that of deacetyl-lyso-GM(3) 100%, and of lyso-GM(3) 36-40%. 12 Deacetyl-GM(3), deacetyl-lyso-GM(3), and lyso-GM(3) were purified by column chromatography, and chemical structures were confirmed by electron spray-mass spectrometry.