Issue 36, 2023

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

Abstract

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.

Graphical abstract: SNAP-tagging live cells via chelation-assisted copper-catalyzed azide–alkyne cycloaddition

Supplementary files

Article information

Article type
Paper
Submitted
23 Jun 2023
Accepted
26 Aug 2023
First published
29 Aug 2023

Org. Biomol. Chem., 2023,21, 7419-7436

Author version available

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

D. J. Stone, M. Macias-Contreras, S. M. Crist, C. F. T. Bucag, G. Seo and L. Zhu, Org. Biomol. Chem., 2023, 21, 7419 DOI: 10.1039/D3OB01003A

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