Increasing the efficacy of bioorthogonal click reactions for bioconjugation: a comparative study

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.

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

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

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

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

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

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.