Synthesis of a highly HOCl-selective fluorescent probe and its use for imaging HOCl in cells and organisms

During infection, nicotinamide adenine dinucleotide phosphate-oxidase of innate immune cells generates important microbicidal reactive oxygen species (ROSROSROS) such as hypochlorous acid (HOCOCl) to kill the invading pathogens. However, excess amounts of HOCOCl induce oxidative damage of functional biomolecules such as DNANA and proteins, which may cause chronic inflammatory diseases. Herein, we outline protocols for the preparation of a rhodamine-based HOCOCl probe, as well as applications thereof, with which to detect HOCOCl in living cells and organisms. The probe (R19S) can be prepared from a commercially available rhodamine, rhodamine 6G, in two steps. When R19S is treated with HOCOCl, the sulfur atom is replaced by an oxygen atom, resulting in opening of the lactone ring; thus, nonfluorescent R19S is converted to highly fluorescent rhodamine 19 (R19). R19S exhibits high selectivity for HOCOCl over other ROSROSROS and high sensitivity in a weakly acidic environment. In addition, we describe fluorescence imaging assays of HOCOCl in mouse neutrophils and Drosophila targeted using this probe. The approximate amount of time required to synthesize the probe is 2-3 d, after which it can be used for up to 5 h in the bioimaging of living cells.