FLUORESCENCE ANISOTROPY ASSAYS REVEAL AFFINITIES OF C-GLYCOSIDES AND O-GLYCOSIDES FOR CONCANAVALIN-A

The free energies of binding of various C- and O-glycosides to the lec tin concanavalin A were measured using fluorescence anisotropy. Fluore scein derivatives of mannose and glucose were synthesized and were sho wn to bind to concanavalin A with free energies of -5.1 and -4.3 kcal mol(-1), respectively. Competition experiments were performed to deter mine the binding energies of different nonfluorescent carbohydrates, a nd the results were in excellent agreement with the binding energies d etermined by microcalorimetry. The minimum carbohydrate epitope that f ills the lectin carbohydrate binding site, methyl di-O-(alpha-mannopyr anosyl)-alpha-mannopyranoside, competes directly for the site with the fluorescent ligands, indicating that the fluorescent ligands bind spe cifically. The binding affinities of C-glycosides to concanavalin A we re compared with those of O-glycosides. The free energies of binding f or corresponding C- and O-glycosides differed by less than 0.5 kcal mo l(-1), indicating that recognition properties of C- and O-glycosides a re very similar. It was found that for some ligands the use of a carbo n linkage rather than an oxygen linkage caused the specificity of bind ing to decrease slightly.