Preparation and application of biologically active fluorescent hyaluronan oligosaccharides

We report the production of biologically active hyaluronan (HA) oligosaccharides labeled with the fluorophore 2-aminobenzoic acid (2AA). Oligosaccharides from 4 to 40 residues in length were purified to homogeneity. by ion exchange chromatography using a logarithmic gradient. Molecular weight and purity characterization of HA oligosaccharides is' facilitated by 2AA derivatization because it enhance's signals in MALDI-TOF MS and improves FACE (fluorophore-assisted carbohydrate electrophoresis) - analysis by avoiding the inverted parabolic migration characteristic of 2-aminoacridone (AMAC)-labeled sugars., The small size and shape, of the fluorophore maintains. the. biological activity of the. derivatized oligosaccharides,. as demonstrated, by their ability to compete for polymeric HA binding,to, the G1-domain of human recombinant versican (VG1). An electrophoretic mobility. shift assay was used to. study VG1 binding to labeled HA 8-, 10-, 20-, 30-,, and 40-mers; and although no stable VG1 binding was observed to labeled 8-mers, the equilibrium dissociation - constant. (100 nM) for VG1 with HA(10) was estimated from densitometry analysis of the free oligosaccharide., Interactions involving HA 20-, 30-, and. 40-mers (proposed to be multivalent), could also be studied using this protocol. Oligosaccharides labeled with 2AA therefore show excellent potential as probes in fluorescence-based assays that investigate protein-carbohydrate, interactions.