Project Summary This project aims to develop chemical tools to precisely measure the concentration of reactive sulfur, oxygen, and nitrogen (RSON) species in living systems. These endogenous small molecule species are critical to healthy function of the heart, lungs, and brain, but if concentrations of RSON species become too high or too low, then this can lead to heart disease, cancer, and neurodegeneration. There is significant clinical interest in targeting RSON signaling for therapeutic benefit and examples included organic nitrates for treating angina, direct inhalation of nitric oxide to treat infants with respiratory failure, and hydrogen sulfide releasing non- steroidal anti-inflammatory drugs for long-term pain management. Despite the increasing importance of these therapeutic approaches, clinical translation and a robust understanding of biological mechanisms are hindered by a lack of methods to precisely measure the concentration of RSON species in real-time. This project aims to use an innovative kinetics-based approach and newly developed chemiluminescent reagents for quantifying the concentrations of peroxynitrite, nitroxyl, hydrogen sulfide, and other RSON species. Specifically, we aim to: 1. Develop chemical probes for real-time quantification of peroxynitrite (ONOO?). ONOO? is an oxidative product of nitric oxide and is a key species that leads to disease when nitric oxide signaling is misregulated. ONOO? production can lead to decoupling of iNOS in response to organic nitrate treatment, contributing to the rapid tolerance of these important drugs. This study will develop methods to quantify the concentration of ONOO? generated by various chemical donors, providing deep clinical insight into next generation therapies. 2. Develop chemical probes for real-time quantification of nitroxyl (HNO). HNO is related to nitric oxide and has similar vasorelaxation properties, with the additional ability to act as an inotropic factor. Importantly, HNO donors are not vulnerable to the same type of tolerance that is observed with organic nitrates. A number of HNO donor compounds have entered into clinical trials. This study will develop methods for measuring the concentration of HNO released from donor molecules in real-time and is expected to accelerate the development and translation of new HNO-based therapies. 3. Develop chemical probes for real-time quantification of hydrogen sulfide (H2S). H2S is a reactive sulfur species that is toxic at high concentrations, but vital for healthy physiology at low concentrations. The body produces H2S enzymatically as a signaling molecule and as a protective agent. The transition from benign to toxic is critically dependent on the concentration and this study will develop new chemical probes to quantify H2S concentration generated from donor compounds and naturally produced by mammalian cells, increasing the understanding of the physiological roles of this molecule and how it could be used in a therapeutic context.