Abstract
Herein, we report the first application of grating-coupled interferometry (GCI) to quantify the binding affinity of synthetically defined mannose-based neo-glycoproteins to Concanavalin A (ConA), a model lectin for carbohydrate recognition. Mono-, di-, and tri-saccharides were conjugated to carrier proteins to create multivalent ligands in a structurally defined yet heterogeneous format. Unlike free sugars, which can interact with lectins in multiple orientations, conjugation constrains sugar presentation and provides a more realistic proxy of how glycans are encountered in glycoconjugates. This approach enabled a systematic comparison of sugar epitopes, confirming that Man(α1,2)Man motif shows stronger binding than Man(α1,6), and that the trisaccharide displays the highest affinity. Notably, the multivalent display of these relatively simple sugar motifs resulted in affinities comparable to or higher than those of natural glycoproteins, highlighting the contribution of valency and presentation rather than glycan complexity. Dissociation constants measured by GCI are consistent with literature values obtained by surface plasmon resonance (SPR) and other methods, confirming the reliability of this platform. This work establishes GCI as a reliable tool for affinity profiling of glycoconjugates and highlights the value of a simple and reproducible screening strategy for evaluating structure-affinity relationships of glycans in a presentation format closer to their intended applications.