On the Meaning of Affinity:  Cluster Glycoside Effects and Concanavalin A

Abstract

The inhibition of protein−carbohydrate interaction provides a powerful therapeutic strategy for the treatment of myriad human diseases. To date, application of such approaches have been frustrated by the inherent low affinity of carbohydrate ligands for their protein receptors. Because lectins typically exist in multimeric assemblies, a variety of polyvalent saccharide ligands have been prepared in the search for high affinity. The cluster glycoside effect, or the observation of high affinity derived from multivalency in oligosaccharide ligands, apparently represents the best strategy for overcoming the “weak binding” problem. Here we report the synthesis of a series of multivalent dendritic saccharides and a biophysical evaluation of their interaction with the plant lectin concanavalin A. Although a 30-fold enhancement in affinity on a valence-corrected basis is observed by agglutination assay, calorimetric titration of soluble protein with a range of multivalent ligands reveals no enhancement in binding free energies. Rather, IC50 values from agglutination measurements correlate well with entropies of binding. This observation suggests that hemagglutination measures a phenomenon distinct from binding that is typified by a large favorable entropy and an unfavorable enthalpy: this process is almost certainly aggregation. Supporting this assertion, we report crystal structures of multivalent ligands cross-linking concanavalin A dimers. To the best of our knowledge, these structures are the first reported of their kind. Our results indicate that hemagglutination assays evaluate the ability of ligands to inhibit the formation of cross-linked lattices, a process only tangentially related to reversible ligand binding. Cluster glycoside effects observed in agglutination assays must, therefore, be viewed with caution. Such effects may or may not be relevant to the design of therapeutically useful saccharides.

Lectins: Tools for the Molecular Understanding of the Glycocode

“…The diminution of −DH was therefore considered the thermodynamic signature for the intermolecular aggregation process. 91, 97 Studies previously carried out by Toone and co-workers employing another series of dendritic multivalent ligands, 96 where crystals of both bi-and trivalent ligands were grown in the presence of succinylated Con A, showed that the carbohydrate orientation within the binding site was identical to that of the corresponding monovalent ligand. Interestingly, ligands with valencies greater than three showed affinity enhancements in agglutination assay but not by ITC.…”

Development of Bis(2‐picolyl)amine–Zinc Chelates for Imidazole Receptors

“…The formyl substituents were reduced with NaBH 4 [25] and the primary alcohols were selectively converted into chlorides with SOCl 2 . [26] Final substitution with Dpa was performed as described above.…”

Responsive Glyco-poly(2-oxazoline)s: Synthesis, Cloud Point Tuning, and Lectin Binding

“…In this respect, it was possible to control the hydrophilic/hydrophobic ratio of the copolymer by increasing the amount of sugar moieties or by attaching long aliphatic chains as well as to generate a pH-sensitive system using 3-mercaptopropionic acid as thiol component (Scheme 3). The reaction was performed according to the method reported by Hawker et al 39 The thiols were added in slight excess (2 equiv of Ac 4 GlcSH with respect to the amount of double bonds) up to a large excess (10 equiv of 3-mercaptopropionic acid as well as dodecanethiol with respect to the number of double bonds) to the polymer solution. A radical photoinitiator (DMPA) was employed to accelerate the reaction.…”

“…The rate constant of EtOx at 100°C was determined to be 6.5 Â 10 À3 L 3 mol À1 3 s À1 , which is one order of magnitude slower than a polymerization at 140°C in the microwave. The disappearance of the monomer signal in the SEC, as depicted in Figure 2 (left), allowed a rough estimation of the required time for a full conversion of Ac 4 GlcYnOx. The exact calculation of the polymerization rate was not possible with the common methods because Ac 4 GlcYnOx could not be detected in GC.…”

“…The disappearance of the monomer signal in the SEC, as depicted in Figure 2 (left), allowed a rough estimation of the required time for a full conversion of Ac 4 GlcYnOx. The exact calculation of the polymerization rate was not possible with the common methods because Ac 4 GlcYnOx could not be detected in GC. Also, the sugar signals were overlapping with the oxazoline ring signals in the 1 H NMR spectrum.…”