# Development of a C5‐Azidoacetamide‐Modified 4‐Amino‐2,3‐Difluorosialic Acid Activity‐Based Probe for Labeling of Influenza A Neuraminidases

## TL;DR

Scientists developed new probes to detect and study influenza A virus neuraminidase, a key protein in the virus's life cycle.

## Contribution

The paper introduces novel activity-based probes with a stabilized covalent intermediate for improved detection of neuraminidase activity.

## Key findings

- The probes effectively label multiple neuraminidases and intact virions with high sensitivity.
- The C5 modification allows bioorthogonal tagging via CuAAC or one-step biotin labeling.
- The probes show hour-scale reactivation and can detect nanogram levels of neuraminidase.

## Abstract

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.

We report DFSA5Az/DFSA5bio, C5‐azidoacetamide 4‐amino‐2,3‐difluorosialic acid activity‐based probes that trap influenza A viral (IAV) neuraminidases (NA) via a stabilized covalent Tyr‐sialosyl intermediate with hour‐scale reactivation. A bioorthogonal azide enables CuAAC (or one‐step biotin) tagging for activity‐dependent Western‐blot detection at nanogram levels, labeling recombinant N1/N2, paramyxovirus HN, and neuraminidase on intact virions.© 2026 WILEY‐VCH GmbH