Development of a C5-Azidoacetamide-modified 4-Amino-2,3-difluorosialic Acid Activity-based Probe for Labelling of Influenza A Neuraminidases

Influenza A virus neuraminidases play a crucial role in the viral life cycle, making them important targets for research into their interactions with sialoside receptors and their effects on viral uptake, tropism, and pathogenesis. In this work, we describe the design, synthesis, and evaluation of a novel 2,3-difluorosialic acid-based probe for applications in activity-based protein profiling of influenza A neuraminidases. The probe features a C4-amino group to facilitate active-site binding and achieve a stabilized covalent intermediate. Crucially, a C5-modified azidoacetamide group enables subsequent bioorthogonal conjugation for detection or, when pre-clicked with a biotin tag, one-step labeling. Both azido- and biotin pre-clicked versions of the probe were synthesized, and their inhibitory activity against recombinant influenza A viral neuraminidases was assessed. Our findings show that these probes exhibit a prolonged reactivation half-life compared to their 2,3-difluoro counterparts, as determined by a reactivation assay. However, compared to 4-amino-2,3-difluoro-Neu5Ac, the NA affinity and half-life were diminished for C5-modified probes. Labeling studies with both probes demonstrated their ability to label various recombinant viral neuraminidases, as well as neuraminidases in a virus sample. During these evaluations, we observed that the 9-azido-2,3-difluoro-Neu5Ac and its biotin pre-clicked version were potent activity-based probes for viral neuraminidases. Together, these four activity-based probes represent useful lead structures for further development into molecular tools for applications such as cellular profiling, detection of viral neuraminidase activity, and diagnostics.