LemengChao, Erianna I.Alvarado-Melendez, Katherine. Development of a C5-Azidoacetamide-Modified 4-Amino-2,3-Difluorosialic Acid Activity-Based Probe for Labeling of Influenza A Neuraminidases. ChemBioChem2026, 27, e70288
Influenza A virus neuraminidase (NA) is central to the viral life cycle and a key target for studying sialoside recognition and hydrolysis and its impact on viral uptake, tropism, and pathogenesis. Here, we report the design, synthesis, and evaluation of 2,3-difluorosialic acid-based activity-based probes for NA profiling. The probes carry a C4-amino substituent to promote active-site engagement and stabilize covalent trapping through a tyrosine-sialosyl intermediate. A C5 azidoacetamide handle enables bioorthogonal tagging by CuAAC for detection, while a preclicked biotin variant supports one-step labeling. We synthesized both the azide and biotin formats and assessed their inhibitory activity against recombinant influenza A NAs. A reactivation assay showed prolonged, hour-scale recovery relative to related 2,3-difluoro analogs, although the C5 modification reduced NA affinity and covalent half-life compared with 4-amino-2,3-difluoro-Neu5Ac. In labeling experiments, the probes tagged multiple recombinant viral neuraminidases and NA present in virus samples. In addition, 9-azido-2,3-difluoro-Neu5Ac and its biotin preclicked counterpart proved potent activity-based probes for NAs. Together, these four probes provide lead structures for further development of molecular tools for cellular profiling, viral NA activity detection, and diagnostics.
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