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August 18, 2004
The First Engineering of Cell Surfaces in Living Animals

BERKELEY, CA – Four years ago Carolyn Bertozzi, a member of the Materials Sciences Division at the Department of Energy's Lawrence Berkeley National Laboratory and a professor of chemistry at the University of California at Berkeley, introduced a new way of engineering the surfaces of cells, by arming cell-surface sugars to take part in a modified chemical reaction known as the Staudinger ligation.

The technique allows a variety of markers to be attached to cultured cells without disturbing their biological environment; it has proved valuable in fundamental studies of cell interactions and in the development of new techniques for interfacing cells with synthetic materials and devices.

A sugar on the surface of a mouse cell, right, modified with an azide group, and a probe comprised of a phosphine attached to a peptide (Flag), left, join in the Staudinger ligation. Fluorescence of a Flag antibody reveals the successful ligation.

Now Bertozzi and her colleagues have demonstrated the Staudinger ligation in remodeled cells of living mice. The ability to tag cell surfaces in living beings may someday allow the targeting of specific kinds of cells for noninvasive imaging, for developmental studies, and for treatment of disease.

"There was no precedent for what we were trying to do, which was to modify the cells of a living animal so that they could undergo a chemical reaction without physiological harm," says Bertozzi. "It was a particular challenge because the living animal is such a complex reaction vessel." The researchers report their results in the 19 August 2004 issue of Nature.

Key to their success is what Bertozzi calls the bio-orthogonality of the reaction, literally "at right angles" to biology: chemical reactions artificially induced on the cell surface must be highly selective and able to take place in the warm, watery physiological environment, but at the same time they must have no harmful biological effects.

The Staudinger ligation meets these requirements. It is a reaction between a functional group called an azide (a class of compounds with three nitrogen atoms) and a phosphine (a molecule containing a phosphorus atom). Neither azides nor phosphines have harmful effects — indeed they are useful in some drugs: the A in AZT, azidothymidine, is for azide — but they rapidly react with each other under physiological conditions. Suitably modified by Bertozzi's methods, the ligation product formed from these compounds is stable under physiological conditions.

The trick is to get the azide half of the reaction to show up on cell surfaces. In cell cultures, this is done by diffusing into the cells a precursor, containing the azide group, of a commonly expressed form of oligosaccharide, or sugar, that includes sialic acid. The cells manufacture these azide-bearing sugars and display them on their surfaces.

With mice, Bertozzi's group injected the azide-containing precursor directly into the abdomen, where cells in the spleen and some other organs took it up. Later examination of spleen cells, which are rich in sialic-acid sugars, showed an abundance of cell-surface azides.

Because rodent blood has high levels of esterase, enzymes that might interfere with the conversion of the modified precursor, the first experiments were done with esterase-deficient knockout mice. Later the investigators found that wild-type mice displayed just as many cell-surface azides, so esterase was apparently no hindrance to the uptake of the azide-containing precursor.

The other half of the Staudinger ligation, the phosphine, may be attached to a great variety of markers. To confirm the success of the Staudinger ligation in mice, Bertozzi and her collaborators used a peptide that could be identified by a fluorescing antibody.

"The future holds a range of opportunities for using the Staudinger ligation for noninvasive imaging," says Bertozzi. "Phosphine markers might include radiochemicals for detection by positron emission tomography, or magnetically active molecules for detection by magnetic resonance imaging, or nanocrystals with unique optical properties, or many other methods. These markers could be injected into organs to seek out cells with certain glycosylation patterns" — patterns of cell-surface sugars characteristic of cancerous cells, for example, or sites of inflammation.

Not only does the demonstration of the Staudinger ligation in mice suggest a range of diagnostic and therapeutic possibilities, the Staudinger ligation itself may be only the first of what Bertozzi calls "a future arsenal of chemical reactions used to probe the biology of living animals."

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