Probing the Nature of the Cluster Effect Observed with Synthetic Multivalent Galactosides and Peanut Agglutinin Lectin

Abstract

We designed a set of multi‐galactosides with valencies ranging from one to seven and different spacer‐arm lengths. The compounds display a high structural homology for a strict assessment of multivalent phenomena. The multimers were first evaluated by an enzyme‐linked lectin assay (ELLA) toward the peanut agglutinin (PNA). The binding affinity was shown to be dependent on the spacer‐arm length, and cluster effects were observed for the galactosides bearing the shortest and the longest linkers. The latter compounds were shown to be much more potent PNA cross‐linkers in a “sandwich assay”. Dynamic light scattering (DLS) experiments also revealed the formation of soluble aggregates between heptavalent derivatives with medium or long linkers and the labeled PNA. ELLA experiments performed with valency‐controlled clusters and labeled lectins are therefore not always devoid from aggregative processes. The precise nature of the multivalent interaction observed by ELLA for the compounds bearing the shortest linkers, which are unable to form PNA aggregates, was further investigated by atomic force microscopy (AFM). The galactosides were grafted onto the tip of a cantilever and the PNA lectin onto a gold surface. Similar unbinding forces were registered when the valency of the ligands was increased, thus showing that the multimers cannot interact more strongly with PNA. Multiple binding events to the PNA were also never observed, thus confirming that a chelate binding mode does not operate with the multivalent galactosides, probably because the linkers are too short. Altogether, these results suggest that the cluster effect that operates in ELLA with the multimers is not related to additional PNA stabilizations and can be ascribed to local concentration effects that favor a dynamic turnover of the tethered galactosides in the PNA binding sites.

The Impact of Heteromultivalency in Lectin Recognition and Glycosidase Inhibition: An Integrated Mechanistic Study

“…The concentrations needed to achieve 50 % inhibition of the reference ligand–lectin association (IC 50 values; assumed to be proportional to the corresponding binding energies) are collected in Table . The IC 50 values obtained for methyl α‐ d ‐mannopyranoside (1050±50 μ m ) and methyl α‐ d ‐glucopyranoside (11 000±100 μ m ) against ConA and for lactose against PNA (1350±10 μ m ) under the same experimental settings were consistent with values reported in the literature, validating the method and providing a reference for the analysis of the impact of structure modifications in the stability of the ligand‐lectin complex.…”

Synthesis of β-galactosylamides as ligands of the peanut lectin. Insights into the recognition process

“…On the other hand, the affinity of D-galactopyranosides for the PNA lectin may be enhanced by means of their multivalent presentation onto a suitable platform. The mechanisms governing the cluster effect in the binding of both D-galactose and lactose glycotopes to PNA have been previously studied [22]. With respect to multivalency, the glycoside cluster effect clearly depends on the number of copies (valency) of the carbohydrate residues but their spatial disposition is also a determinant feature [23].…”

“…As azide counterparts for click reactions we selected a monoazide carbohyhdrate platform as a precursor of monovalent species, and three diazide scaffolds that would give rise to divalent compounds, having similar intersaccharide distances. We and others demonstrated the suitability of sugars as scaffolds for multivalent ligands [7][8][9]22,[34][35][36][37]. Methyl 6-azido-2,3,4-tri-O-acetyl-β-D-glucopyranoside (7) and 6,6'-diazido-2,3,4,2′,3′,4'-hexa-O-acetyl-α,α'-trehalose (8), were prepared as previously described [37].…”

Potent Glycosidase Inhibition with Heterovalent Fullerenes: Unveiling the Binding Modes Triggering Multivalent Inhibition

“…[53] Similarly to ConA-HRP, HRP-labeled PNA (PNA-HRP) is essentially not cross-linkable and suitable for ELLA as well as for two-site sandwich ELLA in combination with the unlabeled immobilizedl ectin. [54] Thec orresponding plots forc ompounds 10, 12, 14 and 16 are depicted in Figures S1 and S2 (in the Supporting Information). All four galactose-bearing fullerenes behaved as potent PNA ligands,w ith IC 50 values in the 2.2-15.5 mm range for the inhibition of PNA binding to the reference galatosylated polymer in the microtiter well ( Figure S1).…”