USE OF DIETHYL SQUARATE FOR THE COUPLING OF OLIGOSACCHARIDE AMINES TOCARRIER PROTEINS AND CHARACTERIZATION OF THE RESULTING NEOGLYCOPROTEINS BY MALDI-TOF MASS-SPECTROMETRY

The 8-methoxycarbonyloctyl glycosides of GlcNAc beta, Gal beta 1-4Glc beta, Fuc alpha 1-2Fuc alpha 1-3GalNAc beta and Fuc alpha 1-2Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta were converted to primary amines by reac tion with neat ethylenediamine and then coupled to bovine serum albumi n (BSA) using diethyl squarate as the connector. The average degree of incorporation of the sugar onto the protein, as well as the molecular weight distribution, could be conveniently determined using matrix as sisted laser desorption ionization/time of flight (MALDI-TOF) mass spe ctrometry thus avoiding cumbersome structure-dependent colour-tests or analysis of cleaved ligand. The present coupling method has the advan tages of proceeding under very mild conditions, yielding controlled in corporation values and can reliably be used for the coupling of very s mall amounts (mg) of oligosaccharide.