Copper-free click chemistry

Copper-free click chemistry is a bioorthogonal reactionas a variant of an azide alkyne Huisgen cycloaddition. By eliminating cytotoxic copper catalysts, the reaction proceeds without live cell toxicity. It was developed as a faster alternative to the Staudinger ligation, with the first generation producing rate constants over 63 times faster.

Although the reaction produces a regioisomeric mixture of triazoles, the lack of regioselectivity in the reaction is not a major concern for its applications in bioorthogonal chemistry. More regiospecific and less bioorthogonal requirements are best served by the traditional Huisgen cycloaddition, especially given the low yield and synthetic difficulty (compared to the addition of a terminal alkyne) of synthesizing a strained cyclooctyne.

The bioorthogonality of the reaction has allowed the Cu-free click reaction to be applied within cultured cells, live zebrafish, and mice.

OCT was the first of all cyclooctynes developed for Cu-free click chemistry; it had only ring strain to drive the reaction forward, and the kinetics were barely improved over the Staudinger ligation. After OCT and MOFO (monofluorinated cyclooctyne), the difluorinated cyclooctyne (DIFO) was developed. An improved synthetic approach to a monofluorosubstituted cyclooctyne (MFCO) was introduced that could be easily converted to a useful reactive intermediate for bioconjugation applications, although the reactivity was somewhat slower than the DIFO. The MFCO demonstrated excellent stability characteristics for long term storage.

The substituted cyclooctyne is activated for a 1,3-dipolar cycloaddition by its ring strain and electron-withdrawing fluorine substituents, which allows the reaction to take place with kinetics comparable to the Cu-catalyzed Huisgen cycloaddition. Ring strain (~18 kcal/mol) arises from the deviation of the bond angles from the ideal 180 in order to form an eight-membered ring, the smallest of all cycloalkynes. The electron-withdrawing fluorine substituents were chosen due to synthetic ease and compatibility with living biological systems. Additionally, the group cannot produce cross-reacting Michael acceptors that could act as alkylating agents towards nucleophilic species within cells.

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