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Triple Quad LC-MS for detection of luciferin-6'-p-nitrobenzyl ether - a footprint of peroxynitrite

Radoslaw Podsiadly, Aleksandra Grzelakowska, Lodz University of Technology - Institute of Polymer and Dye Technology

Jacek Zielonka, Medical College of Wisconsin


Peroxynitrite (ONOO-), a highly reactive oxidizing and nitrating agent is generated via a spontaneous, diffusion-controlled reaction between nitric oxide (·NO) and superoxide radical anion (O2·-). Peroxynitrite has been implicated as a key pathophysiological intermediate in various diseases, including acute and chronic inflammatory processes, diabetes, sepsis, ischemia-reperfusion, atherosclerosis and neurodegenerative disorders as well as chemotherapy-induced nephrotoxicity.

Low steady state concentrations and unfavorable spectroscopic properties make direct detection of peroxynitrite in cells practically impossible. One of the methods to detect ONOO- is to use a chemical probe which is selective and/or forms a footprint product after reaction with this oxidant.

Oxidation of arylboronic acid

Over the last decade, it has been demonstrated that several oxidants such as hydrogen peroxide, hypochlorous acid, protein hydroperoxides and peroxynitrite convert arylboronic acids (ArB(OH)2) to phenolic products ArOH. However, the reactivity of boronates with peroxynitrite (ONOO-) is of special interest. Not only the rate constant of the reaction of boronate probes with ONOO- is the highest of all tested biological oxidants, but the reaction also typically leads to the formation of minor but ONOO--specific products (ArNO2, ArH) in addition to the major phenolic product (ArOH). Since the minor pathway is specific only for the reaction of boronates with peroxynitrite, formation of these products (ArNO2, ArH) may serve as a unique footprint providing a diagnostic marker for peroxynitrite formation in cells-free and cellular systems.

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