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

Living organisms produce hydrogen peroxide (H 2 O 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 2 O 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 2 O 2 within living animals. PCL-1 is a boronic acid-caged firefly luciferin molecule that selectively reacts with H 2 O 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 2 O 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 2 O 2 generated in healthy, living mice. Moreover, we demonstrate the efficacy of PCL-1 for monitoring physiological fluctuations in H 2 O 2 levels by directly imaging elevations in H 2 O 2 within testosterone-stimulated tumor xenografts in vivo. The ability to chemoselectively monitor H 2 O 2 fluxes in real time in living animals offers opportunities to dissect H 2 O 2 ’s disparate contributions to health, aging, and disease.