Fast, Cell‐Compatible Click Chemistry with Copper‐Chelating Azides for Biomolecular Labeling

Abstract

We report that azides capable of copper-chelation undergo much faster “Click chemistry” (copper-accelerated azide-alkyne cycloaddition, or CuAAC) than nonchelating azides under a variety of biocompatible conditions. This kinetic enhancement allowed us to perform site-specific protein labeling on the surface of living cells with only 10–40 µM CuI/II and much higher signal than could be obtained using the best previously-reported live-cell compatible CuAAC labeling conditions. Detection sensitivity was also increased for CuAAC detection of alkyne-modified proteins and RNA labeled by metabolic feeding.

Metal-catalyzed azide-alkyne “click” reactions: Mechanistic overview and recent trends

“…This showed that the triazoles linker between rGO and AuNPs were very efficient for charge separation and electron transfer [203]. In the field of biology, click chemistry has also been extensively used with various successful biological, chemical and medicinal applications [204] including nucleic acid ligation [205], bioconjugation for protein modification [206], drug design [207], gel syntheses for biomedical applications [208], chemical modification of biological molecules [209], and biomolecular labeling [210] activity-based protein profiling (ABPP) [211].…”

Development of fluorescent cholesterol derivatives for the exogenous introduction of proteins to the plasma membrane

“…This chemical transformation is characterized by high chemoselectivity and broad tolerance to diverse reaction condition and functional groups. 23,24,[126][127][128][129] In addition, as neither azide nor alkyne is generally present in biomolecules, they are bioorthogonal functional groups.…”

“…135,[137][138][139] In order to facilitate this CuAAC reaction for biomolecules chelating ligands were developed to stabilize the Cu(I)-ion (Figure 8). [127][128][129][140][141][142][143][144][145][146][147] The application of these Cu(I)-ligands decreased the effective concentration of reducing agent and copper-salts, and also accelerated the bioconjugation reaction and protected the biomolecules from the oxidation by ROS. 127,129,[148][149][150] Aminoguanidine, a carbonyl-capturing reagent is frequently used because it stabilizes proteins during CuAAC by avoiding the adverse effects of ascorbate and its byproducts.…”

“…146,147,151,154 This latter, the so-called copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) was developed by the Bertozzi group for live cell application. 128,132,152,[155][156][157][158][159][160][161][162][163][164][165] It excludes the application of the cytotoxic copper salts, but the rate of the SPAAC is lower than that of the CuAAC. 152,155 Even the most reactive strained alkyne biarylazacyclooctynone reacted significantly slower with azides than alkynes in the ligand-accelerated CuAAC reaction.…”

“…152,155 Even the most reactive strained alkyne biarylazacyclooctynone reacted significantly slower with azides than alkynes in the ligand-accelerated CuAAC reaction. 128 Furthermore, the nonspecific conjugation of cyclooctynes to the thiols of Cys containing proteins can be significant. 152,[155][156][157][158][159][160][161][162][163][164][166][167][168][169] The inverse electron-demand Diels-Alder reaction between a strained alkene like norbornene or trans-cyclooctyne and a tetrazine is also an alternative for bioconjugations.…”