Real-time Measurements of Amino Acid and Protein Hydroperoxides Using Coumarin Boronic Acid

Hydroperoxides of amino acid and amino acid residues (tyrosine, cysteine, tryptophan, and histidine) in proteins are formed during oxidative modification induced by reactive oxygen species. Amino acid hydroperoxides are unstable intermediates that can further propagate oxidative damage in proteins. The existing assays (oxidation of ferrous cation and iodometric assays) cannot be used in real-time measurements. In this study, we show that the profluorescent coumarin boronic acid (CBA) probe reacts with amino acid and protein hydroperoxides to form the corresponding fluorescent product, 7-hydroxycoumarin. 7-Hydroxycoumarin formation was catalase-independent. Based on this observation, we have developed a fluorometric, real-time assay that is adapted to a multiwell plate format. This is the first report showing real-time monitoring of amino acid and protein hydroperoxides using the CBA-based assay. This approach was used to detect protein hydroperoxides in cell lysates obtained from macrophages exposed to visible light and photosensitizer (rose bengal). We also measured the rate constants for the reaction between amino acid hydroperoxides (tyrosyl, tryptophan, and histidine hydroperoxides) and CBA, and these values (7–23 m⁻¹ s⁻¹) were significantly higher than that measured for H₂O₂ (1.5 m⁻¹ s⁻¹). Using the CBA-based competition kinetics approach, the rate constants for amino acid hydroperoxides with ebselen, a glutathione peroxidase mimic, were also determined, and the values were within the range of 1.1–1.5 × 10³ m⁻¹ s⁻¹. Both ebselen and boronates may be used as small molecule scavengers of amino acid and protein hydroperoxides. Here we also show formation of tryptophan hydroperoxide from tryptophan exposed to co-generated fluxes of nitric oxide and superoxide. This observation reveals a new mechanism for amino acid and protein hydroperoxide formation in biological systems.     

Ferrocenylboronates: Crystal structures and electrochemical properties

The electrochemical behavior of a series of eight previously reported monoferrocenyl- and diferrocenyl-boronates derived from tridentate ligands has been studied. Even if most mononuclear and all the dinuclear complexes examined showed to undergo slow decomposition in nonaqueous solution (releasing free ferrocenyl boronic acid), their redox activity has been investigated. It has been proved that the oxidation of the two ferrocenyl subunits in the dimeric species proceeds simultaneously, indicating that no mutual electronic interaction exists between them. Additionally, the solid-state molecular structures of two diferrocenyl complexes (2a and 2b) were studied by X-ray diffraction. The analysis confirms the formation of a [5.4.0] heterobicycle with the presence of two different boron atoms, one in a tetrahedral geometry and the other one in a trigonal geometry.     

Direct oxidation of boronates by peroxynitrite: Mechanism and implications in fluorescence imaging of peroxynitrite

In this study, we show that boronates, a class of synthetic organic compounds, react rapidly and stoichiometrically with peroxynitrite (ONOO⁻) to form stable hydroxy derivatives as major products. Using a stopped-flow kinetic technique, we measured the second-order rate constants for the reaction with ONOO⁻, hypochlorous acid (HOCl), and hydrogen peroxide (H₂O₂) and found that ONOO⁻ reacts with 4-acetylphenylboronic acid nearly a million times (k = 1.6 × 10⁶ M^− 1 s^− 1) faster than does H₂O₂ (k = 2.2 M^− 1 s^− 1) and over 200 times faster than does HOCl (k = 6.2 × 10³ M^− 1 s^− 1). Nitric oxide and superoxide together, but not alone, oxidized boronates to the same phenolic products. Similar reaction profiles were obtained with other boronates. Results from this study may be helpful in developing a novel class of fluorescent probes for the detection and imaging of ONOO⁻ formed in cellular and cell-free systems.