Synthesis of Glycan Molecular Probes for Easy Detection of Endoglycosidase Activities

Endo-β-N-acetylglucosaminidases (ENGases) are endoglycosidases that hydrolyze the glycosidic bond between two N-acetylglucosamine residues in asparagine-linked glycans. ENGases are crucial tools for the structural analysis and glycan remodeling of glycoproteins. However, current ENGase activity assays are often complex and unsuitable for high-throughput analysis. To address this, we developed Förster resonance energy transfer (FRET)-based glycan molecular probes for the real-time detection of ENGase activity. We synthesized di-, tri-, and pentasaccharide probes bearing a fluorophore at the non-reducing end and a quencher at the reducing end, and evaluated their quenching efficiencies for activity detection. The pentasaccharide probe, MM3D, was efficiently cleaved by Endo-M, resulting in a significant increase in fluorescence. These results successfully demonstrate that our FRET-based glycan probes enable straightforward, real-time ENGase activity detection. Furthermore, we constructed a library of probes with diverse glycan structures. Using this library, we evaluated the activities of six commercially available ENGases and observed their distinct substrate specificities. This FRET probe library represents a valuable tool for detecting ENGase activity and will significantly contribute to advances in glycobiology research.