Probing reversible covalent ligand–lectin interactions via NMR spectroscopy

Covalent ligands have long been used in drug development for their ability to form reversible or irreversible bonds with specific protein residue, offering high potency, selectivity, and prolonged action. A range of complementary analytical techniques has been developed to determine the mode of action of a covalent ligand and confirm its covalent interaction with the target protein, validating the formation and specificity of the covalent adduct. The most widely used are mass spectrometry (MS) and X-ray crystallography, even though they both require large amounts of protein and complex workflows. Here, we present a workflow for characterising reversible covalent glycomimetic ligands that target the fucose-specific lectin domain BC2L-C-Nt from Burkholderia cenocepacia, a multidrug-resistant pathogen associated with severe respiratory infections in patients with cystic fibrosis. The designed ligands feature an L-fucose core linked to a salicylaldehyde warhead, which selectively forms a reversible Schiff base with Lys108 near the fucose-binding pocket. Covalent interaction was confirmed by MALDI-MS and LC-MS/MS peptide analysis following NaBH₄ reduction, together with negative control studies. Validating covalent interactions proved challenging due to the significant amounts of purified soluble protein required for mass spectrometry experiments, and complex data analysis. We therefore focused on developing an alternative approach utilising NMR techniques to evaluate the formation of covalent lectin-ligand adducts. This approach employed both well-established NMR techniques, such as Saturation Transfer Difference (STD)-NMR and ¹H-¹³C Heteronuclear Single Quantum Coherence (HSQC), as well as a less conventional yet efficient method using Diffusion-Ordered Spectroscopy (DOSY). This approach offers a generalisable and accessible method to validate covalent binding, particularly for reversible systems and in contexts where protein quantity is limited.