CAPILLARY ELECTROPHORESIS OF CARBOXYLATED CARBOHYDRATES .1. SELECTIVEPRECOLUMN DERIVATIZATION OF GANGLIOSIDES WITH UV ABSORBING AND FLUORESCENT TAGS

We demonstrate that the precolumn derivatization reaction, recently in troduced by our laboratory for the selective labeling of carboxylated monosaccharides, can be readily transposed to other glycoconjugates co ntaining carboxylated sugar residues, namely sialogangliosides. The se lective derivatization reaction described here involved the attachment of sulfanilic acid (a UV-absorbing tag) or 7-aminonaphthalene-1,3-dis ulfonic acid (a UV-absorbing and also fluorescing tag) to the sialic a cid moiety of the gangliosides via the carboxylic group in the presenc e of water-soluble carbodiimide. This labeling of the sialic acid moie ty of the gangliosides with a chromophore and/or fluorophore leads to the formation of an amide bond between the carboxylic group of the sug ar residue and the amino group of the derivatizing agent, thus replaci ng the weak carboxylic acid group of the carbohydrate species by the s tronger sulfonic acid group which is ionized over the entire pH range. Furthermore, novel electrolyte systems were introduced and evaluated for the separation of the derivatized and underivatized gangliosides. The addition of acetonitrile or alpha-cyclodextrin (alpha-CD) to the r unning electrolyte was necessary to break-up the aggregation of amphip hilic gangliosides and allowed for their efficient separation as monom ers in aqueous media using capillary electrophoresis. Several operatin g parameters were investigated with these electrolyte systems includin g the additive concentration as well as the ionic strength, pH and nat ure of the running electrolyte. Acetonitrile at 50% (v/v) in 5 mM sodi um phosphate at high and low pH or 15 mM alpha-CD in 100 mM sodium ber ate, pH 10.0, proved ideal, in terms of resolution and separation effi ciency, for the group separation of mono-, di- and trisialoganglioside s. On the other hand, the complete resolution of disialoganglioside is omers (e.g., G(D1a) and G(D1b)) necessitated the superimposition of a chromatographic component on the electrophoretic process. This was ach ieved by adding either a hydrophobic (e.g., decanoyl-N-methylglucamide -borate surfactant complex) or hydrophilic [e.g., poly(vinyl alcohol) or hydroxypropyl cellulose] selectors to the running electrolyte.