MitoNeoD: A Mitochondria-Targeted Superoxide Probe

Summary Mitochondrial superoxide (O 2 ⋅− ) underlies much oxidative damage and redox signaling. Fluorescent probes can detect O 2 ⋅− , 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 2 ⋅− probe, MitoNeoD, which can assess O 2 ⋅− changes in vivo by mass spectrometry and in vitro by fluorescence. MitoNeoD comprises a O 2 ⋅− -sensitive reduced phenanthridinium moiety modified to prevent DNA intercalation, as well as a carbon-deuterium bond to enhance its selectivity for O 2 ⋅− 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 2 ⋅− from isolated mitochondria to animal models, thus offering a way to examine the many roles of mitochondrial O 2 ⋅− production in health and disease.