title: Copper-Catalyzed Click Reaction on/in Live Cells.
abstract: We demonstrated that 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 atris(triazolylmethyl)amine CuIligand 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% cells remained viable. By reducing the biothiols in the system by scraping or treatment withN-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 CuAAC reaction in the cytosol. Overall, we have improved the efficiency for CuAAC reaction on live cells by 3-fold. Despite the low yielding inside live cells, the products that strongly bind to the intracellular targets can be detected by mass spectrometry. Hence, thein situCuAAC reaction can be potentially used for screening of cell-specific enzyme inhibitors or biomarkers containing 1,4-substituted 1,2,3-triazoles.
authors.affiliation: Department of Chemistry, University of Houston, 4800 Calhoun Rd., Houston, TX 77204, USA.
authors.forename: Siheng
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Chemistry, University of Houston, 4800 Calhoun Rd., Houston, TX 77204, USA; College of Materials Science and Engineering, South China University of Technology, Guangzhou, 510640, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of Houston, 4800 Calhoun Rd., Houston, TX 77204, USA.
authors.forename: Fei
authors.initials: F
authors.lastname: Yu
authors.affiliation: Department of Chemistry, University of Houston, 4800 Calhoun Rd., Houston, TX 77204, USA.
authors.forename: Zhiling
authors.initials: Z
authors.lastname: Zhu
authors.affiliation: Department of Chemistry, University of Houston, 4800 Calhoun Rd., Houston, TX 77204, USA.
authors.forename: Dema
authors.initials: D
authors.lastname: Shobaki
authors.affiliation: Department of Chemistry, University of Houston, 4800 Calhoun Rd., Houston, TX 77204, USA.
authors.forename: Haoqing
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Chemistry, University of Houston, 4800 Calhoun Rd., Houston, TX 77204, USA.
authors.forename: Mu
authors.initials: M
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of Houston, 4800 Calhoun Rd., Houston, TX 77204, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of Houston, 4800 Calhoun Rd., Houston, TX 77204, USA.
authors.forename: Guoting
authors.initials: G
authors.lastname: Qin
authors.affiliation: Department of Chemistry, University of Houston, 4800 Calhoun Rd., Houston, TX 77204, USA.
authors.forename: Uriel J
authors.initials: UJ
authors.lastname: Erasquin
authors.affiliation: College of Materials Science and Engineering, South China University of Technology, Guangzhou, 510640, China.
authors.forename: Li
authors.initials: L
authors.lastname: Ren
authors.affiliation: College of Materials Science and Engineering, South China University of Technology, Guangzhou, 510640, China.
authors.forename: Yingjun
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of Houston, 4800 Calhoun Rd., Houston, TX 77204, USA.
authors.forename: Chengzhi
authors.initials: C
authors.lastname: Cai