The Copper(I)-catalyzed Alkyne-azide Cycloaddition “Click” Reaction in Water Requires the Formation of Two Distinct Mononuclear Copper(I) Complexes

The copper-catalyzed azide-alkyne cycloaddition (CuAAC) has seen widespread adoption amongst numerous fields, but mechanistic details remain elusive and controversial. Using a series of experiments involving fluorogenic coumarin derivatives under pseudo-first order conditions, it was determined that the reaction in conditions close to a physiological environment does not proceed according to the accepted mechanism, but rather requires azide chelation to form a mononuclear copper(I)-azide complex as a preliminary step. Stopped flow studies with a copper-chelating azide further enhanced our understanding of the reaction mechanism, emphasizing the importance of copper-alkyne chelation in which a mononuclear copper(I)-alkyne complex is formed. After both copper(I)-azide and copper(I)-alkyne complexes are formed, they come together to yield the triazole product. This mechanistic proposal of CuAAC in conditions close to a physiological environment is distinct from the currently accepted general model, which proposes dual activation of the alkyne by copper(I) followed by azide interaction. Please note that this manuscript was rejected by several leading chemistry journals for publication in 2015 due to its challenging of existing click chemistry mechanisms. The authors decided to release it to ChemRxiv as a reference for others with a purpose of assisting their experimental designs for bioconjugation.