Optimizing the selectivity of DIFO-based reagents for intracellular bioorthogonal applications

- We optimized the selectivity of DIFO-based reagents for examining intracellular glycans.
- We illustrated the potential non-specific labeling with difluorinated cyclooctynes.
- Biotin- and TAMRA-DIFO are highly reactive toward a reactive free peptidylcysteine.
- Biotin- and TAMRA-DIFO are almost inert to nonpeptidyl thiols (DTT, glutathione, etc.).
- Blocking of the free-thiols prior to SPAAC reaction restores its bioorthogonal nature.