Tetrazole-Based Probe: A New Frontier for Scalable, Selective Peroxynitrite Sensing in Cells

Peroxynitrite (ONOO−), a highly reactive oxygen and nitrogen species produced during cellular oxidative stress, has emerged as a key tar-get for responsive fluorescent probes. The existing toolbox of responsive chemistries, which predominantly rely on oxidative bond cleav-age and fluorogenic activation, remains constrained by complex syntheses and irreversible detection mechanisms. Herein, we present a streamlined, efficient strategy for synthesizing a tetrazole-based peroxynitrite-responsive fluorescent probe (Tet-Probe: TP), that exhibits reversible behavior. Our strategy relies on a three-step, two-pot process: (1) synthesis of tetrazole carboxylic acid, (2) simple esterification of tetrazole carboxylic acid with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), and (3) light activation to transform our non-fluorescent pre-probe into a fluorescent peroxynitrite-responsive chemistry. This new class of tetrazole-based probe demonstrates ratiometric sensing, reversibility, and high selectivity for peroxynitrite over other biologically relevant redox active species. Additionally, when incubated with CHO cells treated with SIN-1, TP-2 achieves efficient cellular internalization and peroxynitrite-induced fluores-cence enhancement with minimal toxicity. This seminal study introduces a new type of scalable (gram-scale) tetrazole-based probe for reversible peroxynitrite sensing, making it a promising molecular tool for real-time analysis of biological processes in cell.