With her student Eliana Saxon, Bertozzi, a member of Berkeley Lab's Materials Sciences and Physical Biosciences Divisions and an associate professor of chemistry at the University of California at Berkeley, describes the Staudinger ligation in the March 17 issue of Science magazine.

This gives us a new avenue for engineering cell chemistry. The cell is now less of a black box.

Bertozzi and her colleagues have engineered human cells to display markers on oligosaccharides, the complex natural sugars that crowd cell surfaces. Through ligation with specific reagents, the markers provide new ways of labeling cells as targets for cancer therapy; adhering cells to nonbiological materials such as medical implants; forming new receptors for viral-mediated gene transfer; and other functions.

Unlike the ketone markers Bertozzi developed earlier, which may encounter interference from natural ketone metabolites inside the cell, the new Staudinger ligation yields azide markers that react exclusively with nonbiological groups. Since azides (a class of compounds with three nitrogen atoms) do not react with any other biological molecules outside or inside the cell, they hold promise for engineering the cell's interior as well as its surface.

"In all cellular engineering, the nature of the chemistry is key," Bertozzi says. "We need reactions that are stable in a watery environment at body temperature, that are highly selective, and that won't interfere with normal cell processes or with each other -- that are what we call 'orthogonal' to cellular components. That means we have to create new reactions."

Casting about for a reaction that was suitably orthogonal and harmless to cells, Bertozzi remembered "one of my favorite reactions from undergraduate chemistry class, the Staudinger reaction -- it's stunningly selective."

Named for the German synthetic-organic chemist Hermann Staudinger, who won the Nobel Prize in 1953 for his pioneering work in polymer chemistry, the Staudinger reaction occurs between an azide and a phosphine, a molecule containing a phosphorus atom. The azide sheds two nitrogen atoms, and a compound called an aza-ylide is formed.

At first glance, this reaction seems ideal for cell engineering: neither phosphines nor azides react with biological molecules, but they react rapidly and with high efficiency with each other, in water and at room temperature. Unfortunately, the resulting aza-ylides fall apart in water almost as easily as they form.

"We asked ourselves if we could create a different kind of aza-ylide that transformed into a stable adduct. Staudinger would have loved the challenge!" Bertozzi and Saxon added an electron-hungry carbohydrate trap to the phosphine, which attaches to the electron-rich aza-ylide and prevents it from falling apart in water, subsequently yielding a stable amide bond.

The technique worked well on the lab bench, but, says Bertozzi, "the cell surface is a lot more demanding than the test tube. Now we had to find a way to install azides on cells."

Partly because azides are small functional groups, they are readily incorporated into sialic-acid residues, sugars that are components of some cell-surface oligosaccharides. When Bertozzi and Saxon fed cultured cells with a sialic-acid precursor containing the azide group, the cells manufactured azide-containing oligosaccharides in abundance and displayed them on their surfaces.

Next a water-soluble phosphine was used to label fluorescent probes to seek out the azide markers. When these chemical probes were allowed to interact with the treated cells, the cells became intensely fluorescent -- the phosphines had found the azides.

"We have developed a useful reaction based on a biological mandate: that the new markers and reagents must be abiotic and harmless to the cells," Bertozzi says. Treated cells cultured for several days showed no change in growth rate, indicating that neither the artificial sugars nor the attachment of the probes affected cell viability.

Because the "Staudinger ligation," named by Bertozzi after the classic reaction that inspired it, is potentially useful for labeling components of the cell interior -- such as amino acids from which proteins are assembled -- previously unobservable interactions in the cell may become accessible. "This gives us a new avenue for engineering cell chemistry. The cell is now less of a black box."

"Cell Surface Engineering By a Modified Staudinger Reaction," by Eliana Saxon and Carolyn R. Bertozzi, appears in the 17 March 2000 issue of the journal Science.

The Berkeley Lab is a U.S. Department of Energy national laboratory located in Berkeley, California. It conducts unclassified scientific research and is managed by the University of California.

USING THE STAUDINGER LIGATION, HUMAN CELLS HAVE BEEN ENGINEERED TO DISPLAY AZIDE-CONTAINING OLIGOSACCHARIDES ON THEIR SURFACES IN ABUNDANCE. WATER-SOLUBLE PROBES SEEK OUT AND REACT WITH THE AZIDE MARKERS

Additional Information:

• Cellular Engineers Design Custom Cell Surfaces Able to Adhere to Synthetic Materials
• Bertozzi Lab Web site
• Carolyn Bertozzi Named Macarthur Fellow for cell surface research