Solid-Phase Synthesis for the Identification of High-Affinity Bivalent Lectin Ligands

Abstract

The development of carbohydrate-based therapeutics has been frustrated by the low affinities that characterize protein-carbohydrate complexation. Because of the oligomeric nature of most lectins, the use of multivalency may offer a successful strategy for the creation of high-affinity ligands. The solid-phase evaluation of libraries of peptide-linked multivalent ligands facilitates rapid examination of a large fraction of linker structure space. If such solid-phase assays are to replicate solution binding behavior, the potential for intermolecular bivalent binding on bead surfaces must be eliminated. Here we report the solid-phase synthesis and analysis of peptide-linked, spatially segregated mono- and bivalent ligands for the legume lectin concanavalin A. Bead shaving protocols were used for the creation of beads displaying spatially segregated binding sequences on the surface of Tentagel resins. The same ligands were also synthesized on PEGA resin to determine the effect of ligand presentation on solid-phase binding. While we set out to determine the lower limit of assay sensitivity, the unexpected observation that intermolecular bivalent ligand binding is enhanced for bivalent ligands relative to monovalent ligands allowed direct observation of the level of surface blocking required to prevent intermolecular bivalent ligand binding. For a protein with binding sites separated by 65 A, approximately 99.9% of Tentagel(1) surface sites and 99.99% of the total sites on a PEGA bead must be blocked to prevent intermolecular bivalent binding. We also report agglutination and calorimetric solution-phase binding studies of mono- and bivalent peptide-linked ligands.

On-Bead Screening of Combinatorial Libraries: Reduction of Nonspecific Binding by Decreasing Surface Ligand Density

“…The problem can be largely overcome by synthesizing OBOC libraries on spatially segregated beads that carry a reduced ligand loading on the bead surface. Reduction of ligand loading can also increase the screening stringency and facilitate the identification of the most potent ligands 25. Although only demonstrated with peptide libraries in this work, the general principle should be applicable to other types of OBOC libraries and any studies involving the interaction between a macromolecule and surface-immobilized ligands.…”

Helical Disposition of Proteins and Lipopolysaccharide in the Outer Membrane ofEscherichia coli

“…LPS might simply be an inert, randomly diffusing matrix in which the immobile protein helices were embedded, or else a portion of the LPS might also be stable and immobile. To distinguish between these possibilities, we labeled the O-antigen side chain of E. coli 2443 with ConA-AF488, a fluorescent lectin that binds specifically to ␣-glucose and ␣-mannose residues of the O8 antigen present in this strain (5,8).…”

Combinatorial/Library Peptide Synthesis

“…This allows for encoding nonsequencable parts of the peptide or nonpeptide structure on the bead surface, or for increasing the efficiency of Edman degradation by not including conserved parts of the peptide structure (if any) in the bead interior. Debenham et al [267] used decreased substitution of the surface for the screening of high-affinity lectin ligands in an attempt to mimic the solution assay and avoid bivalent interaction. They found that actually 99.9% of the ligands had to be eliminated from the surface to prevent bidentate binding.…”