When is a mouse like a test tube?

By Robert Sanders, Media Relations

All cells decorate their surfaces with a variety of sugars and sugar polymers, called oligosaccharides. Carolyn Bertozzi attaches unnatural chemicals to simple sugars and feeds them to cells in order to get these chemicals onto the cell surface as part of the sugary landscape. (Bertozzi lab/UC Berkeley)

Carolyn Bertozzi has put a new twist on the standard chemistry experiment: Instead of using a test tube or flask, she mixes and reacts chemicals in living organisms.

Bertozzi's innovative approach involves chemicals that don't interact with the molecules in the body, only with each other. But her in vivo chemistry has great potential for studying cells in living organisms and creating new diagnostics, and perhaps treatments, for disease.

"We're using the mouse as a reaction vessel, designing chemical reactions that will teach us about biology and disease without causing physiological harm," said Bertozzi, a professor of chemistry and a faculty scientist at Lawrence Berkeley National Laboratory. "It's a really powerful technique, with the ability to change how we think about applying chemical processes in biology."

She and her colleagues report their novel chemical experiments - the first reaction in a living organism between two chemicals that don't react with the organism - in the Aug. 19 issue of Nature.

In this particular experiment, they showed that they could use this type of chemical reaction to tag cells in live mice, specifically, to attach tracer molecules to sugars on the surface of cells. The sugars Bertozzi targeted are produced abundantly by inflamed cells and by cancer cells, which means her technique could be used to attach medical tracers to such cells to allow doctors to pinpoint them in the body.

"The fact that this works is remarkable," wrote David A. Tirrell of the California Institute of Technology in an accompanying News & Views article in Nature. "The labeling strategy described by Bertozzi and colleagues allows one to probe the set of sugars arrayed by the cell, to explore biosynthetic pathways and to examine the functional consequences of modifying the complement of cell-surface sugars."

Bertozzi, an investigator in the Howard Hughes Medical Institute at UC Berkeley and a member of the California Institute for Quantitative Biomedical Research (QB3), is a sugar chemist who studies the sugars and sugar polymers (oligosaccharides and carbohydrates) with which cells decorate themselves. Such sugars play major roles in cell-cell interactions, and often provide entrée to bacteria and viruses that cause disease.

More than five years ago, she conceived of the idea of instigating reactions in the body between unnatural chemicals - ones created by humans and never before seen by living organisms. The unnatural chemicals she chose do not react with any biological molecules, so that the only reaction is between them, as if the body were simply a flask of water.

Her idea runs counter to conventional techniques, where chemists try to control the environment of a reaction by removing as many other chemicals as possible.

"Synthetic chemists try to eliminate all interfering chemical groups, which can be difficult in a biological system," Bertozzi said. "We design functional groups to be specific, so they don't react with the environment in the body. Ours is an elegant system, highly targeted and very selective."

The reaction she focused on was a simple one between azides and phosphines, described last century by German synthetic-organic chemist Hermann Staudinger, a pioneering polymer chemist who won the Nobel Prize in 1953. Azides are simple, three-nitrogen molecules that can be added to biological molecules, in particular sugars, without really being noticed by the body. Phosphines are phosphorous-containing molecules that react with azides to form a stable phosphorous-nitrogen compound. Both types of molecules have shown no harmful effects in the body, based on use of the AIDS drug AZT (azidothymidine) and phosphine-gold compounds for treating arthritis.

Bertozzi figured that if she could attach an azide to a natural sugar and feed it to cells, she could insinuate the sugar into the sugar polymers (oligosaccharides) that decorate the exterior of cells, then use the Staudinger ligation to attach phosphines to these azides. If a label, such as a fluorescent dye or a contrast agent, were attached to the phosphine, she would be able to label cells for diagnostic purposes.

In early cell culture experiments reported in 2000, Bertozzi showed that an azide attached to the sugar mannose is taken up by cells in a test tube and converted to another type of sugar, sialic acid, which in turn is incorporated into sugar polymers on the cell surface. More importantly, the more azide-sugar she fed cells, the more appeared on the surface.

In the current experiment, her research group extended this to living mice. Graduate students Jennifer Prescher and Danielle Dube injected the mice with an azide-mannose compound for seven days, then harvested spleens from the mice to look for the azide. As predicted, it appeared on the surface of spleen cells attached to sialic acid, with more azide on the surface associated with higher doses of the azide-mannose sugar. Azide-labeled cells also were found in the heart, kidney and liver, though not the brain, thymus or gut.

They then repeated the experiment, but on the eighth day injected each mouse with a phosphine attached to a fluorescent molecule called FLAG. After only 90 minutes, the FLAG tag appeared on spleen cells, proving that the Staudinger ligation had occurred in living mice.

"We successfully marked cells as a function of the robustness of a metabolic pathway, the sialic acid biosynthetic pathway," Bertozzi said. "This offers an alternative way to visualize cells that are undergoing a change in metabolism, such as happens with cancer cells and inflamed cells that over-produce sialic acid. The technique opens the door to non-invasive imaging of sugars as markers of disease."

"Irrespective of its biological relevance, the method introduced by Bertozzi and colleagues is remarkable as a chemical process," Tirrell added in his commentary. "The fact that specific chemical transformations can now be accomplished with spatial and temporal control in live animals is a major step forward for chemistry."

She and colleagues are now developing radio-labeled azide compounds that could be detected by PET (positron emission spectroscopy) or SPECT (single photon emission computed tomography), fluorescent compounds that could be detected visually, and magnetic compounds that could be detected by MRI (magnetic resonance imaging). She also is investigating another type of probe that is not a phosphine but will also react with azides in a highly selective manner.

The work was supported by the Department of Energy.

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