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Comparative Study
, 50 (35), 8051-6

Increasing the Efficacy of Bioorthogonal Click Reactions for Bioconjugation: A Comparative Study

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Comparative Study

Increasing the Efficacy of Bioorthogonal Click Reactions for Bioconjugation: A Comparative Study

Christen Besanceney-Webler et al. Angew Chem Int Ed Engl.

Abstract

Raising the bar: the efficacy of bioorthogonal reactions for bioconjugation has been thoroughly evaluated in four different biological settings. Powered by the development of new biocompatible ligands, the copper-catalyzed azide-alkyne cycloaddition has brought about unsurpassed bioconjugation efficiency, and thus it holds great promise as a highly potent and adaptive tool for a broader spectrum of biological applications.

Figures

Figure 1
Figure 1
Comparison of the efficiency of the CuAAC and BARAC-mediated copper-free click chemistry in labeling recombinant proteins and crude cell lysates. a) Western blot analysis of PD1-Fc isolated from HEK cells treated with Ac4ManNAz (top panel). Total protein loading was confirmed by Coomassie staining (bottom panel). b) Western blot analysis of Ac4ManNAz-treated or untreated Jurkat cell lysates.
Figure 2
Figure 2
Relative labeling efficiency of the bioorthogonal click reactions on live cells. a) Schematic representation of metabolic labeling and detection of cell-surface sialic acids using Ac4ManNAz and click chemistry. b) Flow cytometry analysis of cell surface labeling described in a) using Jurkat cells. Error bars represent the standard deviation of two replicate experiments.
Figure 3
Figure 3
Cell proliferation assays indicating that tris(triazolylmethyl)amine-based ligands protect Jurkat cells from Cu(I)-induced long-term perturbation.
Figure 4
Figure 4
Relative efficiencies of CuAAC and copper-free click chemistry in labeling zebrafish glycans. a) One-cell embryos were microinjected with a single dose of Ac4GalNAz. At 24 hpf, the embryos were reacted with biotin-alkyne (50 μM) catalyzed by the BTTAA-Cu(I)-catalysts ([Cu] = 40 μM) or BARAC-biotin (50 μM) for 5 minutes, then probed with streptavidin-Alexa Fluor 488 and imaged using confocal microscopy. b) One-cell embryos were microinjected with a single dose of GDP-FucAl or GDP-Fuc (control) and allowed to develop to 9 hpf. The embryos were then reacted with Alexa Fluor 488-azide (50 μM) catalyzed by BTTAA-Cu(I) or BTTES-Cu(I) ([Cu] = 40 μM) for 3 min and imaged using confocal microscopy. Scale bar: a) 200 μm b) 100 μm. Top: 488 channel; bottom: brightfield.
Scheme 1
Scheme 1
Structural formulae of tris(triazolylmethyl)amine-based ligands and BARAC-biotin. BTTAA=2-[4-({bis[(1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]acetic acid, BTTES=2-[4-({bis[(1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]ethyl hydrogen sulfate, TBTA=tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, THPTA=tris[(1-hydroxypropyl-1H-1,2,3-triazol-4-yl)methyl]amine, BARAC=biarylazacyclooctynone
Scheme 2
Scheme 2
a) A fluorogenic assay for qualitative measurement of CuAAC kinetics. b) Conversion-time profiles of CuAAC in the presence of various ligands. Error bars represent the standard deviation of two replicate experiments.

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