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.

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.

Cell proliferation assays indicating that tris(triazolylmethyl)amine-based ligands protect Jurkat cells from Cu(I)-induced long-term perturbation.

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.

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

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.