A Time-Honored Chemical Reaction Generates an Unexpected Product

By Jason Socrates Bardi

Call it the mystery of Chemistry 101.

If you take hydrogen peroxide, the familiar antiseptic and bleaching agent, mix it with water, and bubble it through ozone, the atmospheric gas and hazardous component of smog, you will produce a powerful and extremely microbicidal chemical reaction that can kill just about any kind of bacteria and render harmful chemicals benign.

Chemists have applied this bubbling brew industrially for many years—using it for processing wastewater and treating soil and groundwater contaminated with PCBs, MTBE, or other organic pollutants. The reaction is an attractive decontamination process because of it is potent and because ozone and hydrogen peroxide naturally break down into water, hydrogen, and other harmless waste products.

But why is it such a potent reaction?

“That’s the question,” says Paul Wentworth, Jr., who is a professor in the Department of Chemistry at The Scripps Research Institute. “This reaction [between ozone and hydrogen peroxide] has been studied for over 100 years, and there are still big questions as to what intermediates are generated.”

Now Wentworth and his colleagues at Scripps Research are suggesting that one reason why ozone and hydrogen peroxide make such a potent brew is that they create a short-lived and powerful intermediate molecule called “dihydrogen trioxide” or H2O3, which is itself a powerful oxidant.

In the latest issue of the journal Angewandte Chemie, Wentworth and his colleagues at Scripps Research report that they have detected the formation of the elusive dihydrogen trioxide in a reaction between ozone and hydrogen peroxide.

This is especially significant, says Wentworth, because ozone and hydrogen peroxide may both be found in vivo as part of an immune process whereby the body produces oxidants in order to kill invading microorganisms. Since ozone and hydrogen peroxide mixed together can form dihydrogen trioxide, and since ozone and hydrogen peroxide can be found in living systems, dihydrogen trioxide may also be a part of biology.

The research was led by Wentworth and Scripps Research President Richard A. Lerner, who is the Lita Annenberg Hazen Professor of Immunochemistry, a member of the Skaggs Institute for Chemical Biology, and Cecil H. and Ida M. Green Chair in Chemistry.

Antibodies, Ozone, and Oxidative Killing

This research by Wentworth, Lerner, and their colleagues supports a hypothesis they first arrived at a few years ago when they reported that all antibodies have a novel catalytic ability that is unique among proteins.

Antibodies are produced by the immune system’s B cells, which recognize a wide range of foreign viruses and bacteria. B cells produce specific antibodies that circulate through the blood, track down, bind to, and help eliminate these microbial invaders.

Wentworth, Lerner, and their colleagues discovered that in the test tube, antibodies can take singlet oxygen, a highly-reactive, electronically-excited form of oxygen that forms spontaneously during normal metabolic processes, and turn it into hydrogen peroxide. They also speculated about what this might mean for immunology—perhaps antibodies do more than was previously thought.

For the last 100 years, immunologists have firmly believed that the sole purpose of antibodies was to recognize pathogens and trigger cells in the immune system to kill the pathogen. But the ability of antibodies to convert singlet oxygen into hydrogen peroxide could mean that antibodies have a previously unrecognized killing mechanism that would enhance their defensive role by allowing them to participate directly in killing pathogens with hydrogen peroxide.

This previously unrecognized ability offers exciting possibilities for new antibody-mediated therapies for conditions ranging from bacterial and viral infection to cancer. Furthermore, this process could be linked to a number of other diseases.

But it also raises more questions.

The antibacterial activity of the antibodies was more intense than what the researchers would have expected if the antibodies were just producing hydrogen peroxide, says Wentworth, because the amount of hydrogen peroxide the antibodies generated was not enough to kill the number of bacteria they observed dying. So perhaps the antibodies were producing other oxidative species as well.

Then they made the startling discovery that antibodies generated a product with the chemical signature of ozone, a particularly reactive form of oxygen that exists naturally as a trace gas in the atmosphere, constituting on average fewer than one part per million air molecules. The gas plays a crucial role in protecting life on earth from damaging solar radiation by concentrating in the upper reaches of Earth's stratosphere—about 25 kilometers above the surface—and absorbing ultraviolet radiation. Ozone is also a familiar component of air in industrial and urban settings where the highly reactive gas is a hazardous component of smog in the summer months.

Ozone had never been considered a part of biology before, but they had detected it to be produced by antibodies, implicating ozone in the killing of the bacteria.

Perhaps ozone together with hydrogen peroxide are unleashed on the bacteria, where they can either react with the surface proteins of the bacteria or trigger a cascade of reactions on the surface of the bacterial membrane that destroy the bacteria by poking holes in their cell walls.

And dihydrogen trioxide, they proposed, was being produced as a transient intermediary molecule in this process. Dihydrogen trioxide had also never before been observed in biological systems.

Perhaps for good reason. The molecule is extremely reactive and very short-lived—it has a lifetime of a few thousandths of a second or less before its inherent instability causes it to react with other molecules and be converted into something else. To be able to detect dihydrogen trioxide in the body, you would need it to either outlive its natural lifetime or to be present in unnaturally high concentrations.

So the scientists asked a simpler question: Can dihydrogen trioxide be produced in the test tube, in a reaction involving hydrogen peroxide and ozone? Now the team is reporting, in an article published by the journal Angewandte Chemie, that the answer is yes.

They placed hydrogen peroxide and ozone in an organic solution designed to prolong the lifetime of the dihydrogen trioxide, and they were able to detect the presence of dihydrogen trioxide in the vessel through nuclear magnetic resonance.

In order to verify that the chemical signal they detected was indeed dihydrogen trioxide, they created a new system for generating high concentrations of pure dihydrogen trioxide for the sake of comparison.

Since ozone and hydrogen peroxide are both part of biological systems, and since dihydrogen trioxide has now been detected in the reaction of hydrogen peroxide and ozone, it stands to reason, says Wentworth, that the powerful oxidant dihydrogen trioxide may be a biologically relevant molecule present in biological systems and acting as an oxidant in the immune system’s reaction against bacteria.

To read the article, " Dihydrogen Trioxide (HOOOH) is Generated during the Thermochemical Reaction between Hydrogen Peroxide and Ozone " by Paul T. Nyffeler, Laxman Eltepu, Nicholas A. Boyle, Chi-Huey Wong, Albert Eschenmoser, Richard A. Lerner, and Paul Wentworth, Jr., see the September 6, 2004 issue of the journal Angewandte Chemie. The article is accessible to journal subscribers online at: http://dx.doi.org/10.1002/ange.200460457.

The research was funded in part by the National Institutes of Health and The Skaggs Institute for Research.

Send comments to: jasonb@scripps.edu

"This reaction [between ozone and hydrogen peroxide] has been studied for over 100 years and there are still big questions as to what intermediates are generated."

-Professor Paul Wentworth, Jr.