Issue 3, 2017

Copper-catalyzed click reaction on/in live cells

Abstract

We demonstrated that the copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction could be performed inside live mammalian cells without using a chelating azide. Under optimized conditions, the reaction was performed in human ovary cancer cell line OVCAR5 in which newly synthesized proteins were metabolically modified with homopropargylglycine (HPG). This model system allowed us to estimate the efficiency of the reaction on the cell membranes and in the cytosol using mass spectrometry. We found that the reaction was greatly promoted by a tris(triazolylmethyl)amine CuI ligand tethering a cell-penetrating peptide. Uptake of the ligand, copper, and a biotin-tagged azide in the cells was determined to be 69 ± 2, 163 ± 3 and 1.3 ± 0.1 μM, respectively. After 10 minutes of reaction, the product yields on the membrane and cytosolic proteins were higher than 18% and 0.8%, respectively, while 75% of cells remained viable. By reducing the biothiols in the system by scraping or treatment with N-ethylmalemide, the reaction yield on the cytosolic proteins was greatly improved to ∼9% and ∼14%, respectively, while the yield on the membrane proteins remained unchanged. The results indicate that out of many possibilities, deactivation of the current copper catalysts by biothiols is the major reason for the low yield of the CuAAC reaction in the cytosol. Overall, we have improved the efficiency for the CuAAC reaction in live cells by 3-fold. Despite the low yield inside live cells, products that strongly bind to the intracellular targets can be detected by mass spectrometry. Hence, the in situ CuAAC reaction can be potentially used for screening of cell-specific enzyme inhibitors or biomarkers containing 1,4-substituted 1,2,3-triazoles.

Graphical abstract: Copper-catalyzed click reaction on/in live cells

Supplementary files

Article information

Article type
Edge Article
Submitted
24 May 2016
Accepted
21 Nov 2016
First published
25 Nov 2016
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2017,8, 2107-2114

Copper-catalyzed click reaction on/in live cells

S. Li, L. Wang, F. Yu, Z. Zhu, D. Shobaki, H. Chen, M. Wang, J. Wang, G. Qin, U. J. Erasquin, L. Ren, Y. Wang and C. Cai, Chem. Sci., 2017, 8, 2107 DOI: 10.1039/C6SC02297A

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