Non-Covalent N-Glycan-Directed Paramagnetic Tagging: Structural Analysis of Glycoproteins by NMR

Paramagnetic probes have become powerful tools in structural biology, providing access to long-range structural information in biomolecular systems1. However, the success of these approaches relies on the precise positioning of the paramagnetic centre relative to the biomolecular surface, which is commonly achieved through covalent protein modification. Although effective, this strategy often requires extensive protein engineering and may perturb the native structure of the system2. Consequently, there remains a need for alternative non-covalent anchoring methods. Supramolecular chemistry offers an attractive opportunity in this context, particularly for glycoprotein targeting3. Significant progress has recently been achieved through the development of aminocarbazole-based molecular tweezers able to selectively recognize, in water, the conserved GlcNAc2 disaccharide motif within the Man3GlcNAc2 core of N-linked glycans4. We developed a paramagnetic aminocarbazole-based synthetic receptor that combines selective recognition of the same N-glycan core with the coordination of paramagnetic metal ions. The potential of this supramolecular probe was evaluated using the sialylglycopeptide SGP, a ubiquitous sialylated N-glycan in mammals, and ovalbumin, a well-characterized glycoprotein. Paramagnetic relaxation enhancement (PRE) nuclear magnetic resonance (NMR) experiments were used to demonstrate the effective generation of long-range restraints without the need for covalent protein tagging. Our data, supported by molecular dynamics simulations, confirmed selective recognition of the GlcNAc₂ motif and provided insight into the structure and dynamics of the glycosylation site and nearby residues of the investigated systems. Overall, this work provides a proof-of-concept demonstration that N-glycan-directed supramolecular recognition can serve as a modular and non-covalent strategy for the paramagnetic investigation of native glycoproteins.