In vivo imaging of hydrogen peroxide production in a murine tumor model with a chemoselective bioluminescent reporter

Living organisms produce hydrogen peroxide ([H.sub.2][O.sub.2]) to kill invading pathogens and for cellular signaling, but aberrant generation of this reactive oxygen species is a hallmark of oxidative stress and inflammation in aging, injury, and disease, The effects of [H.sub.2][O.sub.2] on the overall health of living animals remain elusive, in part owing to a dearth of methods for studying this transient small molecule in vivo. Here we report the design, synthesis, and in vivo applications of Peroxy Caged Luciferin-1 (PCL-1), a chemoselective bioluminescent probe for the real-time detection of [H.sub.2][O.sub.2] within living animals. PCL-1 is a boronic acid-caged firefly luciferin molecule that selectively reacts with [H.sub.2][O.sub.2] to release firefly luciferin, which triggers a bioluminescent response in the presence of firefly luciferase. The high sensitivity and selectivity of PCL-1 for [H.sub.2][O.sub.2], combined with the favorable properties of bioluminescence for in vivo imaging, afford a unique technology for real-time detection of basal levels of [H.sub.2][O.sub.2] generated in healthy, living mice. Moreover, we demonstrate the efficacy of PCL-1 for monitoring physiological fluctuations in [H.sub.2][O.sub.2] levels by directly imaging elevations in [H.sub.2][O.sub.2] within testosterone-stimulated tumor xenografts in vivo. The ability to chemoselectively monitor [H.sub.2][O.sub.2] fluxes in real time in living animals offers opportunities to dissect [H.sub.2][O.sub.2]'s disparate contributions to health, aging, and disease. cancer | molecular imaging | redox biology doi/ 10.1073/pnas.1012864107