MitoNeoD: A Mitochondria-Targeted Superoxide Probe

Maria M Shchepinova,Victoria R Pell,Sara Vidoni,Thomas Krieg,Tracy A Prime,Stuart T Caldwell,Robert F Anderson,Pooja Yadav,Andrew M James,James N Cobley,Thomas P Bright,Hiran A Prag,Andrew G Cairns,Richard C Hartley,Angela Logan,Sabine Arndt,Michael P Murphy,Andrew Hall ,Hans Martin Senn ,John Mulvey

doi:10.1016/j.chembiol.2017.08.003

Abstract

SummaryMitochondrial superoxide (O2⋅−) underlies much oxidative damage and redox signaling. Fluorescent probes can detect O2⋅−, 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 O2⋅− probe, MitoNeoD, which can assess O2⋅− changes in vivo by mass spectrometry and in vitro by fluorescence. MitoNeoD comprises a O2⋅−-sensitive reduced phenanthridinium moiety modified to prevent DNA intercalation, as well as a carbon-deuterium bond to enhance its selectivity for O2⋅− 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 O2⋅− from isolated mitochondria to animal models, thus offering a way to examine the many roles of mitochondrial O2⋅− production in health and disease.

Highlights

The production of O2,À within the mitochondrial matrix varies under different conditions (Murphy, 2009; Winterbourn, 2008)
It was thought that HE was oxidized by O2,À to the fluorescent product ethidium (E+), but Kalyanaraman and colleagues showed that HE reacts with O2,À to form 2-hydroxyethidium (2-OH-E+) (Zhao et al, 2003), while the production of E+ from
The neopentylamino groups are more acidstable than tertiary butyl derivatives and do not sterically impede the reaction with O2,À. These electronic and steric properties played a significant role in the chemical synthesis of MitoNeoD (Figure 1C)

Summary

Introduction

The production of O2,À within the mitochondrial matrix varies under different conditions (Murphy, 2009; Winterbourn, 2008). To assess O2,À production reliably the E+ and 2-OH-E+ products of HE and its derivatives have to be separated by high-pressure liquid chromatography (HPLC) followed by detection by fluorescence or mass spectrometry (Kalyanaraman et al, 2014; Maghzal and Stocker, 2007; Michalski et al, 2014; Zielonka and Kalyanaraman, 2010). Another factor affecting the fluorescence of E+ and 2-OH-E+ is that both intercalate into DNA and double-stranded RNA (Horobin et al, 2013), thereby increasing the fluorescence quantum yield by 10- to 40-fold (Zhao et al, 2005; Zielonka et al, 2008)

Methods

Results

Discussion

Conclusion