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Content archived on 2024-05-29

Applications of NMR spectroscopy to the study of the interactions of glyconanoparticles with other biomolecules as models of multivalent biological molecular recognition processes

Final Activity Report Summary - GLYCONANONMR (Aplications of NMR spectroscopy to the study of the interactions of glyconanoparticles with other biomolecules as models of ... recognition processes)

Understanding molecular recognition of carbohydrates by other biomolecules at atomic level not only increases knowledge about the molecular recognition phenomena, but also allows gaining key structural information for the design of specific carbohydrates-binding agents (for diagnostics, drugs, vaccines,...). NMR spectroscopy is a very useful tool for the study of biomolecular interactions in solution, with a variety of experiments particularly suited for small ligands and large receptors.

In these experiments (STD NMR, tr-NOESY, WaterLOGSY,...) normally the carbohydrate molecules constitute the small partner of the interacting pair, in the form of mono- or oligosaccharides. In this project the work has been focused on the application of NMR techniques to study 'small molecule'-'small molecule' binding processes in solution, with particular emphasis put on molecular recognition processes involving small carbohydrates. Recent advances in the field of glyconanotechnology have allowed getting large 3D polyvalent clusters in the form of gold nanoparticles functionalised with monolayers of synthetic oligosaccharides (glyconanoparticles, GNPs). These particles have diameters above 1-2 nm with molecular weights well above 50 kDa, and we wanted to consider them as potential large 'receptors' in NMR ligand-based binding experiments.

To this aim we have explored the applicability of NMR experiments to study the interactions of the sugars present on the surfaces of these glyconanoclusters with other small biomolecules. The first studies focused on the biologically relevant carbohydrate-carbohydrate interactions and different systems were explored. The results indicated that, unfortunately, the affinity of these interactions is likely to be at the lower limit for the quantitation by these techniques (STD NMR and transferred-NOE), and the detection of signals hence requires very long measuring times with results more prone to be contaminated by experimental artefacts.

Work is still in progress for these systems. Nevertheless, the proof of principle for the proposed concept was finally successfully obtained through the unambiguous detection by STD NMR of the interactions between some D-hexopyranoses immobilised on GNPs and the amino acids L-tryptophane and L-phenylalanine, which model the well-known aromatic-carbohydrate interactions that play an important role in a number of molecular recognition processes of biologically interesting carbohydrates by some proteins (e.g. lectins). STD NMR experiments allowed not only to detect the binding in solution, but the quantitation of the signals led to structural information about which parts of the amino acid make most intimate contacts with the sugar rings in the bound state (binding epitope).

The results clearly indicate that the aromatic side-chains of the amino acids are the moieties making closest contacts with the sugars on the surfaces of the glyconanoparticles, in perfect agreement with the expected CH-p stacking interactions between the biomolecules, as it is observed in different bound states of carbohydrates in the binding site of some proteins. The versatility of the approach is actually being tested by analysing other models of molecular recognition processes that involve weak 'small-molecule'-'small-molecule' interactions in solution.