MitoNeoD: A Mitochondria-Targeted Superoxide Probe.

Mitochondrial superoxide (O) underlies much oxidative damage and redox signaling. Fluorescent probes can detect O, but are of limited applicability in vivo, while in cells their usefulness is constrained by side reactions and DNA intercalation. To overcome these limitations, we developed a dual-purpose mitochondrial O probe, MitoNeoD, which can assess O changes in vivo by mass spectrometry and in vitro by fluorescence. MitoNeoD comprises a O-sensitive reduced phenanthridinium moiety modified to prevent DNA intercalation, as well as a carbon-deuterium bond to enhance its selectivity for O over non-specific oxidation, and a triphenylphosphonium lipophilic cation moiety leading to the rapid accumulation within mitochondria. We demonstrated that MitoNeoD was a versatile and robust probe to assess changes in mitochondrial O from isolated mitochondria to animal models, thus offering a way to examine the many roles of mitochondrial O production in health and disease.