Since its discovery in 2002, the copper-catalyzed azide-alkyne cycloaddition (CuAAC)[1] reaction—the most widely recognized example of click chemistry [2]—has been rapidly embraced for applications in myriad fields.[3] The attractiveness of this procedure (and its copper-free strained-alkyne variant [4]) stems from the selective reactivity of azides and alkynes only with each other. Because of the fragile nature and low concentrations at which biomolecules are often manipulated, bioconjugation presents significant challenges for any ligation methodology. Several different CuAAC procedures have been reported to address specific cases involving peptides, proteins, polynucleotides, and fixed cells, often with excellent results,[5] but also occasionally with somewhat less satisfying outcomes.[6] We describe here a generally applicable procedure that solves the most vexing click bioconjugation problems in our laboratory, and therefore should be of use in many other situations.

The CuAAC reaction requires the copper catalyst, usually prepared with an appropriate chelating ligand,[7] to be maintained in the CuI oxidation state. Several years ago we developed a system featuring a sulfonated bathophenanthroline ligand,[8] which was optimized into a useful bioconjugation protocol.[9] A significant drawback was the catalyst’s acute oxygen sensitivity, requiring air-free techniques which can be difficult to execute when an inert-atmosphere glove box is unavailable or when sensitive biomolecules are used in small volumes of aqueous solution. We also introduced an electrochemical method to generate and protect catalytically active CuI–ligand species for CuAAC bioconjugation and synthetic coupling reactions with miminal effort to exclude air.[10] Under these conditions, no hydrogen peroxide was produced in the oxygen-scrubbing process, resulting in protein conjugates that were uncontaminated with oxidative byproducts. However, this solution is also practical only for the specialist with access to the proper equipment. Other protocols have employed copper (I) sources such as CuBr for labeling fixed cells [11] and synthesizing glycoproteins.[12] In these cases, the instability of CuI in air imposes a requirement for large