Cell-permeable probe for identification and imaging of sialidases

Abstract

Alkyne-hinged 3-fluorosialyl fluoride (DFSA) containing an alkyne group was shown to be a mechanism-based target-specific irreversible inhibitor of sialidases. The ester-protected analog DFSA (PDFSA) is a membrane-permeable precursor of DFSA designed to be used in living cells, and it was shown to form covalent adducts with virus, bacteria, and human sialidases. The fluorosialyl–enzyme adduct can be ligated with an azide-annexed biotin via click reaction and detected by the streptavidin-specific reporting signals. Liquid chromatography-mass spectrometry/mass spectrometry analysis on the tryptic peptide fragments indicates that the 3-fluorosialyl moiety modifies tyrosine residues of the sialidases. DFSA was used to demonstrate influenza infection and the diagnosis of the viral susceptibility to the anti-influenza drug oseltamivir acid, whereas PDFSA was used for in situ imaging of the changes of sialidase activity in live cells.

Covalent protein modification: the current landscape of residue-specific electrophiles

“…Similarly, sulfonylfluorides ( Figure 4b) react with highly conserved tyrosines in multiple GSTs, but also target the activated serine in serine proteases [19]. Recently, 3-fluorosialyl fluoride (DFSA; Figure 4c) was shown to modify a tyrosine within sialidase active sites [58]. However, this electrophile is specific to the sialic-acid scaffold, and is therefore not amenable as a general tool for profiling tyrosine reactivity.…”

Convenient analysis of protein modification by chemical blotting with fluorogenic “click” reagents

“…Perhaps surprisingly, we are not aware of this technique being used elsewhere, apart from an example of on-membrane antigen attachment for western blotting. 11 …”

Chemical Methods for Probing Virus–Host Proteomic Interactions

“…ABPP imaging has also been applied by Tsai et al through the developed of a target-specific irreversible inhibitor of sialidases to be used for the study of influenza virus infection. 59 They synthesized an alkyne- proprotein processing is required for the propagation of many non-enveloped or lipid-enveloped viral pathogens and mediates viral invasion into host cells. 101 The probe that was used was designed by flanking a furin-cleavable peptide with FITC and a quencher, Dabcyl.…”