Sulfated Ligands for the Copper(I)‐Catalyzed Azide–Alkyne Cycloaddition

Abstract

The copper(I)-catalyzed azide-alkyne cycloaddition, the most widely recognized reaction of click chemistry, is accelerated by tris(triazolylmethyl)amine-based ligands. Here, we compared two new ligands in this family, BTTP and the corresponding sulfated ligand BTTPS, for three bioconjugation applications: (1) labeling of alkyne-tagged glycoproteins in crude cell lysates, (2) labeling of alkyne/azide-tagged glycoproteins on the surface of live mammalian cells, and (3) labeling of azides in surface proteins of live Escherichia coli. Though BTTPS exhibits faster kinetics than BTTP in accelerating the CuAAC in in vitro kinetic measurements, its labeling efficiency is slightly lower than BTTP in conjugating biomolecules bearing a significant amount of negative charges due to electrostatic repulsion. Nevertheless, the negative charge conferred by the sulfate at physiological conditions significantly reduced the cellular internalization of the coordinated Cu(I), thus making BTTPS-Cu(I) a better choice for live cell labeling.

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

“…tris(triazolylmethyl)amines) were developed. 146,[151][152][153] When these ligand accelerated CuAAC reactions were used in vitro, live cells showed normal functions after the treatment evidencing their applicability in living systems. 146,[151][152][153] The reaction time of CuAAC reaction with these accelerator ligands was optimized for 5-10 min, because in longer reaction time significant cell death was observed.…”

“…146,[151][152][153] When these ligand accelerated CuAAC reactions were used in vitro, live cells showed normal functions after the treatment evidencing their applicability in living systems. 146,[151][152][153] The reaction time of CuAAC reaction with these accelerator ligands was optimized for 5-10 min, because in longer reaction time significant cell death was observed. 146,147,151,154 In Kennedy's work L-histidine (His) was used as accelerator ligand that resulted in a two-to threefold slower reaction as compared to other ligands (e.g.…”

“…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,[151][152][153] The reaction time of CuAAC reaction with these accelerator ligands was optimized for 5-10 min, because in longer reaction time significant cell death was observed. 146,147,151,154 In Kennedy's work L-histidine (His) was used as accelerator ligand that resulted in a two-to threefold slower reaction as compared to other ligands (e.g. BTTAA, BTTP), but the cytotoxicity was lower that makes the application of longer reaction time possible.…”

“…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.…”