With the support of the Chemistry of Life Processes (CLP) program in the Division of Chemistry, John Jewett from the University of Arizona is studying releasable reactive species to probe biological systems. Everything within biology can be analyzed based on its chemical composition. Reactive chemical probes form chemical bonds with molecules, both small and macromolecular-such as proteins or DNA, and these reactions are important because they can report back on the state of the system (healthy versus diseased, growing versus quiescent). Aryl diazonium ions (ADI) are a unique class of highly reactive molecules that the Jewett lab has learned to harness by rendering them deliverable using masking functional groups. These ADI could be leveraged to report on biological systems using fluorescence as an output to detect the chemical environment or enzymatic activity. The Jewett research team will also investigate the multifaceted fate of ADI once they enter a complex biological environment. In addition to their utility as biochemical tools, the study and synthesis of these compounds will serve to train undergraduate and graduate students. Moreover, this class of compounds generally exhibits fascinating color changes, fluorescence, solubility changes, and other properties that make them well suited to integrate with outreach goals. Dr. Jewett will develop an outreach program for a local school district with the goal of empowering elementary school students with safe experiments that they can carry out at home. Aryl diazonium ions (ADI) are powerful hypothesis-generating biochemical tools. A better understanding of ADIs is required to optimize their utility. One piece of this proposal seeks to develop a class of fluorogenic molecules with multiple mechanisms and inputs for activation. Multiple inputs could imbue these molecules with power to provide advanced spatiotemporal probing of the environment or of enzymatic status within a biochemical system. Additionally, the proposed research seeks to understand the nature of the reactivity and selectivity of ADI in living systems. While ADI have been used for decades for bioconjugation of proteins, or to degrade DNA, a holistic view of these disparate reactivities has not been undertaken. Following the synthesis of a small library of ADI, competition studies between disparate classes of biomolecules will be analyzed by NMR, mass spectrometry and gel electrophoresis. These studies could culminate in intracellular delivery of certain ADI using a triazabutadiene strategy. The findings of these studies may potentially impact the future of how this class of reactive probes is used for biological systems, from modifying purified proteins to probing whole organisms. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.