Investigators have increased the shelf life and detection ability of fluorescent probes that are necessary for studying a variety of inflammatory diseases, including cancer and atherosclerosis, by trading one specific hydrogen atom for an isotope that is twice as heavy. The probes detect and measure reactive oxygen species, which play a role in disease processes. “By replacing a hydrogen atom with a deuterium atom during the synthesis of several fluorescent probes, we increased the stability and shelf life of the dyes and also improved their ability to detect smaller concentrations of reactive oxygen species,” said Niren Murthy, associate professor of biomedical engineering at Georgia Tech and Emory University. The atomic weight of deuterium, an isotope of hydrogen with a single proton and single neutron in its nucleus, is twice that of the common hydrogen atom, which lacks a neutron. When Murthy and postdoctoral fellow Kousik Kundu designed and produced a range of fluorescent probes with deuterium instead of hydrogen, the dyes were not as likely as their hydrogen counterparts to degrade spontaneously from contact with air and light, which made them significantly more accurate at detecting reactive oxygen species in cells and animals. The researchers studied probes such as dihydroethidium (DHE), the current gold standard for imaging reactive oxygen species, and hydrocyanines. Associate professor of biomedical engineering Niren Murthy (left, standing) and postdoctoral fellows Seungjun Lee and Kousik Kundu (seated) display fluorescence images showing how well deuterium-containing DHE probes detect reactive oxygen species. Courtesy of Gary Meek. The study, sponsored by the National Institutes of Health and the National Science Foundation, was published in the journal Angewandte Chemie International. According to the research, after 10 days in storage, the standard fluorescent probe DHE had been 60 percent oxidized by air and light, whereas the deuterium equivalent had been only 20 percent oxidized. These findings could have significance for companies that make fluorescent probes and other compounds, according to Murthy, because commercializing and shipping the modified probes will be easier. “One of the major limitations of the radical oxidant probes is their susceptibility to aerial oxidation. Therefore, it is challenging to store/ship the probes without them getting oxidized. The deuterated probes are stabilized to aerial oxidation and therefore have improved stability and storage shelf life, making their shipping/ handling easier,” he explained. He added that another benefit of using deuterium-containing fluorescent probes is that they produce the same fluorescent dye as their hydrogen counterparts after reacting with reactive oxygen species. “This is important from a practical standpoint because scientists have developed protocols with DHE and other fluorescent probes that they will be able to continue using by simply substituting the more stable and accurate deuterated version into the assay,” Murthy said. To detect reactive oxygen species, fluorescent probes undergo a chemical process called amine oxidation. The mechanism of amine oxidation for reactions involving reactive oxygen species differs significantly from results involving air and light. Additionally, because deuterium is a heavier atom, reactions with deuterium-containing probes occur much more slowly than with hydrogen probes. Murthy and Kundu used these mechanistic and kinetic differences to selectively slow the oxidation of the fluorescent probes by air and light while maintaining their reactivity with cellular reactive oxygen species. To test the selective suppression of oxidation, they examined the kinetic isotope effect – a value that measures the ratio of the rate of a chemical reaction with hydrogen compared to the same reaction with deuterium to air and radical oxidation. They investigated the ability of the deuterium-containing probes to compete with a common enzyme for superoxide, a reactive oxygen species that is a form of molecular oxygen with one extra electron. The researchers found that the probes’ oxidation mechanism with superoxide differed from that for spontaneous oxidation because the two reactions exhibited dissimilar values for the kinetic isotope effect. For spontaneous oxidation, the values ranged from 3.7 to 4.7, whereas for superoxide oxidation, they were between 2.5 and 2.8 for various types of deuterium-containing fluorescent dyes, including DHE. “This was the key experiment that demonstrated that there was a much larger difference in the way the hydrogen and deuterium compounds reacted to spontaneous oxidation than how they dealt with oxidation by a reactive oxygen species,” Murthy explained. W. Robert Taylor and Sarah Knight, Murthy’s collaborators, tested the ability of both types of dyes to detect reactive oxygen species inside cells. Because the deuterium-containing probes were less affected by air and light, and background fluorescence was suppressed, the researchers found that the dyes more accurately detected small amounts of reactive oxygen species. Knight is a postdoctoral fellow at Emory University, and Taylor is the director of Emory’s cardiology division. After the cellular experiments, Knight and postdoctoral fellow Seungjun Lee investigated whether the kinetic isotope effect would similarly improve the ability of H-Cy7 – a hydrocyanine dye developed by Murthy – to detect radical oxidants in vivo. They found that the version of the dye with deuterium generated a fluorescence intensity that was 10 times that of the control probe, whereas the hydrogen probe’s intensity was five times that of the control. “This new process of replacing hydrogen with deuterium is potentially valuable because the positive results are universal among many different types and classes of probes,” Murthy explained. “All of the modified probes generated less background fluorescence, while maintaining high reactivity with reactive oxygen species and generating similar levels of fluorescence in cells and animals stimulated to produce them.” Murthy indicated that the kinetic isotope effect has been used to improve drug stability but that it has never been used to improve probe development. “Several drugs are metabolized into toxic byproducts via an amine oxidation reaction, which involves the abstraction of a hydrogen atom. A few pharmaceutical companies have therefore replaced the labile hydrogen atom with deuterium to create safer drugs, and several clinical trials are currently under way (Concert Pharmaceuticals, Lexington, Mass.). However, utilization of the kinetic isotope effect (by replacing hydrogen by a deuterium) has never been used in improving the efficacy of a fluorescent probe.” “Based on our results, we anticipate numerous applications of deuterated radical oxidant probes in biology and an increased application of the kinetic isotope effect in biological probe development,” Murthy added. “The higher stability of the deuterated probes will allow the researchers to study more complex oxidative processes, where longer incubation times are necessary.”