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Imaging Molecules On Living Cells

Click-chemistry technique provides a nontoxic way to visualize biomolecules on cell surfaces in vivo

by Stu Borman
November 29, 2010 | A version of this story appeared in Volume 88, Issue 48

A new biocompatible molecular visualization technique based on click chemistry offers a potentially advantageous alternative to previous methods for imaging specific biomolecules on the surfaces of living cells.

Click chemistry is a method in which heteroatom links are used to join together small molecular units in a modular fashion. A click-chemistry reaction called copper-catalyzed azide-alkyne cycloaddition (CuAAC), developed in 2002, has in the past been used to add fluorescent labels to cell-surface biomolecules, enabling scientists to image them. But the copper catalyst causes impairment or death of cells and organisms exposed to it, and the slow kinetics of the reaction have hindered broader use of the approach.

A copper-free cycloaddition developed in 2004 by chemical biologist Carolyn R. Bertozzi of the University of California, Berkeley, and coworkers is much more biocompatible and nontoxic than the CuAAC method.

In this confocal image, fluorescently labeled glycans light up the boundaries of cell surfaces in a zebrafish embryo.
In this confocal image, fluorescently labeled glycans light up the boundaries of cell surfaces in a zebrafish embryo.

Now, synthetic chemist and glycobiologist Peng Wu of Albert Einstein College of Medicine, in the Bronx, N.Y., and coworkers have developed an alternative in vivo labeling and imaging procedure that is easy to carry out and, like Bertozzi’s technique, seems to minimize cytotoxicity problems (J. Am. Chem. Soc., DOI: 10.1021/ja106553e).

The researchers accomplished this feat by screening a compound library for ligands that could sequester copper in such a way that it can still catalyze CuAAC but is rendered nontoxic. In the screen, they found BTTES, a tris(triazolylmethyl)amine-based ligand that coordinates with copper, accelerates CuAAC dramatically, and is easily prepared on the gram scale from commercially available materials in five steps. Tests on four different cell lines and in live zebra­fish embryos suggest that it’s nontoxic.

“One question the field will have to address is how much exposure to these new CuAAC conditions cells and organisms can take,” Bertozzi comments. “It seems that any more than 15 minutes leads to toxicity.” But so far, Wu and coworkers find that the procedure requires organisms to be exposed to reagents for only one to three minutes, and Bertozzi agrees that a 15-minute toxicity limit “may be plenty long for many applications.”

The researchers used the BTTES-based approach to detect sialylated glycans on surfaces of live mammalian cells and to image fucosylated glycans in live zebrafish embryos.

Wu and coworkers “have identified ligand systems that both increase the rate of CuAAC substantially and largely obviate toxicity issues, at least for systems involving cell-surface modifications,” says Stephen G. Withers, a glycoenzyme expert at the University of British Columbia. The work could “greatly open up the areas of application of click reagents,” he adds.

The approach “represents a significant advance for the use of CuAAC in living systems,” says James C. Paulson, a glycobiologist at Scripps Research Institute. “The reduced toxicity of the ligand-coordinated copper will make it possible to explore limitless applications of bioorthogonal azide-alkyne cycloaddition reactions in living systems.”

Credit: J. Am. Chem. Soc. (Both)


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