Characterization of the reactivity of luciferin boronate - A probe for inflammatory oxidants with improved stability

• Published in: Dyes and pigments 2020-12, Vol.183, p.108693, Article 108693
• Main Authors: Szala, Marcin, Grzelakowska, Aleksandra, Modrzejewska, Julia, Siarkiewicz, Przemysław, Słowiński, Daniel, Świerczyńska, Małgorzata, Zielonka, Jacek, Podsiadły, Radosław
• Format: Article
• Language: English
• Subjects: Firefly luciferin-based probe; Luminogenic probes; Peroxynitrite-specific product
• ISSN: 0143-7208; 1873-3743
• DOI: 10.1016/j.dyepig.2020.108693

Boronate derivatives of luciferin, containing oxidant-activated self-immolative moieties, recently have been developed for bioluminescent detection of hydrogen peroxide in animal models. Here, we report the synthesis and characterization of luciferin boronic acid pinacol ester (LBE) as a probe for detection of hydrogen peroxide, hypochlorous acid, and peroxynitrite, with improved stability and response time. HPLC analyses showed that LBE quickly hydrolyzes in phosphate buffer to luciferin boronic acid (LBA). Hydrogen peroxide oxidizes LBA slowly, with the formation of luciferase substrate, luciferin (Luc-OH), as the only product. Hypochlorite also oxidizes LBA to luciferin, but the subsequent reaction of Luc-OH with hypochlorite gives a chlorinated luciferin Luc–OH–Cl, which has a higher fluorescence quantum yield than luciferin at pH 7.4 and is also a substrate for luciferase (Takakura H, et. all. ChemBioChem 2012; 13:1424). Similar to other boronate probes, LBA is oxidized by peroxynitrite in two pathways. Luc-OH is the product of the major pathway, common for all the oxidants tested, whereas the non-fluorescent nitrated derivative, Luc-NO2, is formed in the minor pathway, specific for peroxynitrite. Formation of luciferin radical intermediate in the minor pathway has been confirmed by EPR spin trapping and mass spectrometric analyses of the spin adducts. We conclude that LBE shows potential as an improved probe for the detection of inflammatory oxidants in biological settings. Complementation of the bioluminescence measurements by HPLC or LC-MS-based identification of chlorinated and nitrated luciferin(s) will help identify the oxidants detected. [Display omitted] •Firefly luciferin-based probe for detection of peroxynitrite.•Luciferin boronic acid (LBA) is oxidized to luciferin by H2O2, HOCl, and peroxynitrite.•7′-Chloroluciferin is a product specific for HOCl.•6′-Nitroluciferin serves as peroxynitrite-specific product.•LBA enables real-time monitoring of the ONOO− formation in activated RAW 264.7 macrophages.