The interaction of multi-LacNAc (Galβ1-4GlcNAc)-containing N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers with human galectin-1 (Gal-1) and the carbohydrate recognition domain (CRD) of human galectin-3 (Gal-3) was analyzed using NMR methods in addition to cryo-electron-microscopy and dynamic light scattering (DLS) experiments. The interaction with individual LacNAc-containing components of the polymer was studied for comparison purposes. For Gal-3 CRD, the NMR data suggest a canonical interaction of the individual small-molecule bi- and trivalent ligands with the lectin binding site and better affinity for the trivalent arrangement due to statistical effects. For the glycopolymers, the interaction was stronger, although no evidence for forming a large supramolecule was obtained. In contrast, for Gal-1, the results indicate the formation of large cross-linked supramolecules in the presence of multivalent LacNAc entities for both the individual building blocks and the polymers. Interestingly, the bivalent and trivalent presentation of LacNAc in the polymer did not produce such an increase, indicating that the multivalency provided by the polymer is sufficient for triggering an efficient binding between the glycopolymer and Gal-1. This hypothesis was further demonstrated by electron microscopy and DLS methods.

BioOrgNMR Lab Department of Biotechnology and Biosciences University of Milano Bicocca Piazza della Scienza 2 20126 Milano Italy

CIC bioGUNE Basque Research and Technology Alliance BRTA Bizkaia Technology Park 48162 Derio Bizkaia Spain

Department of Health Care Disciplines and Population Protection Faculty of Biomedical Engineering Czech Technical University Prague Nám Sítná 27201 Kladno Czech Republic

Department of Organic Chemistry 2 University of the Basque Country UPV EHU 48940 Leioa Bizkaia Spain

Ikerbasque Basque Foundation for Science 48013 Bilbao Bizkaia Spain

Institute of Macromolecular Chemistry of the Czech Academy of Sciences Heyrovského Nám 2 16206 Prague Czech Republic

Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 14220 Prague Czech Republic

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