From Oxygen to Sulfur: Rhodamine Hydrazide Probes for Peroxynitrite Detection from the Perspective of Theoretical...

The hydrazide functional group is known for its specific recognition of peroxynitrite. Upon incorporation into rhodamine fluorophores, the resulting fluorescent probes have been widely used for the real-time tracking of peroxynitrite in biological systems. However, the lack of in-depth research on the fundamental reaction mechanism in peroxynitrite detection has limited the optimization of these probes. In this study, we developed two hydrazide-based peroxynitrite probes by linking hydrazine moiety to rhodamine and thio-rhodamine. The responsiveness of these probes toward peroxynitrite was also systematically investigated. Theoretical calculations indicate that the key mechanism of hydrazide-based probes in peroxynitrite detection lies in the reduced Gibbs free energy difference between the ring-open and ring-closed isomers of the oxidized intermediate, which thereby facilitates the ring-opening process. Overall, this study elucidates the reaction mechanism of hydrazide-based peroxynitrite probes from the perspective of Gibbs free energy, providing valuable insights for the rational design and optimization of rhodamine ring-opening probes.