Exploring the origins of L-selectin specificity: Synthesis and biological evaluation of mono- and multivalent L-selectin ligands

To identify the molecular factors responsible for the specificity of carbohydrate-binding proteins, we have synthesized mono- and multivalent inhibitors of the cell adhesion molecule L-selectin. We have developed methods for the stereoselective synthesis of analogs of the Lewis x trisaccharide (Le{dollar}sp{lcub}rm x{rcub}{dollar}: Gal{dollar}beta 1 to 4{dollar}(Fuc{dollar}alpha 1 to{dollar} 3)GlcNAc) and for the regioselective introduction of sulfate esters in polyhydroxylated systems, and have identified conditions for the polymerization of sulfated saccharide-derived norbornene monomers. The ability of the synthetic saccharide ligands to block binding of L-selectin has been assessed as has the ability of these agents to interfere with L-selectin mediated cell rolling.; Six sulfated derivatives of the Lewis x trisaccharide were synthesized and the inhibitory potencies of these molecules demonstrate that 3{dollar}spprime{dollar},6-disulfo Lewis x is approximately three times more effective at inhibiting the L-selectin binding than is the sialyl Lewis x analog 3{dollar}spprime{dollar}-sulfo Lewis x, while 3{dollar}spprime,6spprime{dollar}-disulfo Lewis x is no more effective than 3{dollar}spprime{dollar}-sulfo Lewis x. These results suggest that only sulfation of sialyl Lewis x at the 6-position of glucosamine is a favorable modification for binding to L-selectin.; In order to determine the effects of multivalent ligand display on L-selectin recognition, multivalent derivatives of the L-selectin ligands 3{dollar}spprime{dollar}-sulfo Lewis x, 3{dollar}spprime,6spprime{dollar}-disulfo Lewis x and 3{dollar}spprime{dollar},6-disulfo Lewis x were synthesized and tested in three different assays. Static assays demonstrate that multivalent ligands are of higher potency than their monovalent counterparts. In addition, static inhibition assays reveal no difference in inhibitory potency between multivalent 3{dollar}spprime{dollar},6- and 3{dollar}spprime,6spprime{dollar}-disulfo Lewis x. Flow assays, however, demonstrate that there are marked differences in the abilities of the multivalent ligands to inhibit L-selectin mediated rolling. Although both 3{dollar}spprime{dollar},6- and 3{dollar}spprime,6spprime{dollar}-disulfo Lewis x-containing polymers inhibit static binding of L-selectin with similar efficacies, only the 3{dollar}spprime{dollar},6-disulfo derivative inhibits rolling. Finally, shedding experiments demonstrate that substrates which inhibit L-selectin mediated rolling also promote the proteolytic cleavage of L-selectin from the cell surface.