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Synthesis

Copper-Free Click Chemistry

Reagent for labeling biomolecules eliminates need for toxic metal catalyst

by Celia Henry Arnaud
October 18, 2007

DIFLUORINATED CYCLOOCTYNE
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A new reagent developed by chemistry professor Carolyn R. Bertozzi and coworkers at the University of California, Berkeley, eliminates the toxicity usually associated with a rapid and irreversible reaction strategy commonly known as "click chemistry" (Proc. Natl. Acad. Sci. USA, DOI: 10.1072/pnas.0707090104). This tweak to remove copper catalysts makes the reaction, azide-alkyne cycloaddition, biologically friendly and thus useful for labeling biomolecules in cells.

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Credit: Proc. Natl. Acad. Sci. USA © 2007
Copper-free click chemistry labels cell surface carbohydrates (green), which then move inside the cell.
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Credit: Proc. Natl. Acad. Sci. USA © 2007
Copper-free click chemistry labels cell surface carbohydrates (green), which then move inside the cell.

The reagent helps Bertozzi and her team study dynamic biochemical processes that are otherwise difficult to follow in real time. Bertozzi is particularly interested in studying glycosylation, the addition of sugar molecules to proteins. The reaction is tough to track because the sugar molecules, or glycans, are continuously recycled. "Most imaging of carbohydrates uses fixed systems," says Jeremy M. Baskin, a grad student in Bertozzi's lab and lead author of the report. "You can take a snapshot, but you can???t make the equivalent of a movie."

Azides make a handy tag for labeling biomolecules. They don't react with other molecules in the system, and they can be added to a range of biomolecules, including sugars, lipids, and proteins.

Unfortunately, the two reactions most commonly used to affix fluorescent or other labels to azide-tagged biomolecules have limitations. The Staudinger ligation, which forms an amide bond between the azide and an ester-derivatized phosphine, is too slow. Azide-alkyne cycloaddition is much faster, but the conventional copper catalysts required are toxic to living systems.

Bertozzi's team has designed a reagent that eliminates those copper catalysts. "This copper-free chemistry is really designed for applications that require this extra burden of nontoxicity," Baskin says. "I see it as one reaction in a family of click chemistries."

The UC Berkeley researchers speed up the reaction even without a catalyst by using a difluorinated cyclooctyne instead of the usual terminal alkyne. The ring strain and the electron-withdrawing difluoro group activate the alkyne for copper-free click chemistry.

Bertozzi and coworkers use the reagent to attach fluorescent labels to cells with azide-containing sialic acid in their surface glycans. The team studied the dynamics of glycan trafficking over the course of 24 hours with no indication that the reaction or the labels perturb the process.

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