105 - Development and characterization of a mitochondria-targeted boronate-based fluorescent probe for peroxynitrite detection

Abstract

The unambiguous detection, formation rates and steady-state concentrations of peroxynitrite (ONOO‒/ONOOH) in biological systems is a great challenge due to its high reactivity towards several biomolecules. The accurate cellular detection of peroxynitrite in different cellular compartments and the estimation of its formation rates, represent fundamental steps to understand how nitric oxide-derived oxidants affect biological processes, including mitochondrial dysfunction and cell death. Aryl-boronates react rapidly and stoichiometrically with ONOO− to yield the corresponding hydroxylated compound as the major reaction product. It has been demonstrated by others and us, that in cellular systems where several reactive oxygen and nitrogen species coexist, boronates are kinetically selective for peroxynitrite. We have recently reported a boronate-based fluorescent probe for the direct and sensitive detection and quantitation of endogenously-produced peroxynitrite [1]. Furthermore, those data allowed us to tailor boronate-derivatives targeted to identify subcellular sites and rates of peroxynitrite generation. Herein, we communicate the design, synthesis and characterization of new organelle-targeted boronate-based fluorescent probe MitoCT. MitoCT chemical structure consist of a boronic ester attached to a modified coumarin in order to enhance spectroscopic properties, and supporting a triphenylphosphonium cation to target and concentrate in mitochondria matrix. The synthetic route involves a convergent route and results in reasonable product yields. Kinetic and mechanistic characterization of the reaction of MitoCT with peroxynitrite and other oxidants, including a side-by-side comparison with the commercially-available and putative H2O2 mitochondrial probe MitoPy1, are underway.