Vinylboronic Acids as Efficient Bioorthogonal Reactants for Tetrazine Labeling in Living Cells

[Image: see text] Bioorthogonal chemistry can be used for the selective modification of biomolecules without interfering with any other functionality present in the cell. The tetrazine ligation is very suitable as a bioorthogonal reaction because of its selectivity and high reaction rates with several alkenes and alkynes. Recently, we described vinylboronic acids (VBAs) as novel hydrophilic bioorthogonal moieties that react efficiently with dipyridyl-s-tetrazines and used them for protein modification in cell lysate. It is not clear, however, whether VBAs are suitable for labeling experiments in living cells because of the possible coordination with, for example, vicinal carbohydrate diols. Here, we evaluated VBAs as bioorthogonal reactants for labeling of proteins in living cells using an irreversible inhibitor of the proteasome and compared the reactivity to that of an inhibitor containing norbornene, a widely used reactant for the tetrazine ligation. No large differences were observed between the VBA and norbornene probes in a two-step labeling approach with a cell-penetrable fluorescent tetrazine, indicating that the VBA gives little or no side reactions with diols and can be used efficiently for protein labeling in living cells.