SNAP-tagging live cells via chelation-assisted copper-catalyzed azide-alkyne cycloaddition

SNAP-tag is a single-turnover enzyme that has become a powerful tool, hence a popular choice, of targeted cellular protein labeling. Three SNAP-tag substrates that carry the copper-chelating 2-picolyl azide moiety are prepared, one of which has an unconventional 5-pyridylmethyl-substituted guanine structure, rather than the usual benzylguanine that is optimized to be accepted by SNAP-tag. All three substrates are effective in transferring a 2-picolyl azide moiety to SNAP-tag in live cells under conventional labeling conditions (30-minute incubation of cells with labeling reagents at 37 °C under 5% CO₂). Live cells that are decorated with chelating azido groups on the extracellular side of membranes undergo copper-catalyzed azide-alkyne cycloaddition (CuAAC) with an ethynyl-functionalized fluorophore to accomplish membrane protein labeling by a fluorescent dye. The chelation-assisted CuAAC labeling step is rapid (<1 minute) with a relatively low dose of the copper catalyst (20 μM), and consequently exerts no ill effect on the labeled cells. A SNAP-tag substrate that carries a non-chelating azide moiety, on the other hand, fails to produce satisfactory labeling under the same constraints. The rapid, live cell-compatible SNAP-tag/chelation-assisted CuAAC two-step method expands the utility of SNAP-tag in protein labeling applications.