Andrzej Marcinek

Reaction between Peroxynitrite and Boronates: EPR Spin-Trapping, HPLC Analyses, and Quantum Mechanical Study of the Free Radical Pathway

Recently, we showed that peroxynitrite (ONOO(-)) reacts directly and rapidly with aromatic and aliphatic boronic acids (k ≈ 10(6) M(-1)s(-1)). Product analyses and substrate consumption data indicated that ONOO(-) reacts stoichiometrically with boronates, yielding the corresponding phenols as the major product (∼85-90%), and the remaining products (10-15%) were proposed to originate from free radical intermediates (phenyl and phenoxyl radicals). Here, we investigated in detail the minor, free radical pathway of boronate reaction with ONOO(-). The electron paramagnetic resonance (EPR) spin-trapping technique was used to characterize the free radical intermediates formed from the reaction between boronates and ONOO(-). Using 2-methyl-2-nitrosopropane (MNP) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) spin traps, phenyl radicals were trapped and detected. Although phenoxyl radicals were not detected, the positive effects of molecular oxygen, and inhibitory effects of hydrogen atom donors (acetonitrile, and 2-propanol) and general radical scavengers (GSH, NADH, ascorbic acid, and tyrosine) on the formation of phenoxyl radical-derived nitrated product, suggest that the phenoxyl radical was formed as the secondary species. We propose that the initial step of the reaction involves the addition of ONOO(-) to the boron atom in boronates. The anionic intermediate undergoes both heterolytic (major pathway) and homolytic (minor pathway) cleavage of the peroxy (O-O) bond to form phenol and nitrite as a major product (via a nonradical mechanism), or a radical pair PhB(OH)(2)O(•-)···(•)NO(2) as a minor product. It is conceivable that phenyl radicals are formed by the fragmentation of the PhB(OH)(2)O(•-) radical anion. According to the DFT quantum mechanical calculations, the energy barrier for the dissociation of PhB(OH)(2)O(•-) radical anion to form phenyl radical is only a few kcal/mol, suggesting rapid and spontaneous fragmentation of the PhB(OH)(2)O(•-) radical anion in aqueous media. Biological implications of the minor free radical pathway are discussed in the context of ONOO(-) detection, using the boronate probes.

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

Background: Several amino acids in proteins form hydroperoxides during oxidative stress. Results: A fluorometric assay for real-time analyses of amino acid and protein hydroperoxides is developed. Conclusion: The boronate-based assay is a convenient method for detection and absolute quantitation of amino acid/protein hydroperoxides. Significance: The proposed strategy will enhance understanding of the mechanism(s) of oxidative and post-translational modification of proteins. 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.

Nitroxyl (HNO) Reacts with Molecular Oxygen and Forms Peroxynitrite at Physiological pH BIOLOGICAL IMPLICATIONS

Background: Nitroxyl (HNO) is a reactive nitrogen species implicated in cardioprotection. Results: Nitroxyl reacts with oxygen to form an oxidizing and nitrating species, peroxynitrite. Conclusion: In the presence of oxygen, HNO donors may be a source of peroxynitrite. Significance: Peroxynitrite formation should be taken into account in the extracellular milieu when exposing cells to HNO donor under aerobic conditions. Nitroxyl (HNO), the protonated one-electron reduction product of NO, remains an enigmatic reactive nitrogen species. Its chemical reactivity and biological activity are still not completely understood. HNO donors show biological effects different from NO donors. Although HNO reactivity with molecular oxygen is described in the literature, the product of this reaction has not yet been unambiguously identified. Here we report that the decomposition of HNO donors under aerobic conditions in aqueous solutions at physiological pH leads to the formation of peroxynitrite (ONOO−) as a major intermediate. We have specifically detected and quantified ONOO− with the aid of boronate probes, e.g. coumarin-7-boronic acid or 4-boronobenzyl derivative of fluorescein methyl ester. In addition to the major phenolic products, peroxynitrite-specific minor products of oxidation of boronate probes were detected under these conditions. Using the competition kinetics method and a set of HNO scavengers, the value of the second order rate constant of the HNO reaction with oxygen (k = 1.8 × 104 m−1 s−1) was determined. The rate constant (k = 2 × 104 m−1 s−1) was also determined using kinetic simulations. The kinetic parameters of the reactions of HNO with selected thiols, including cysteine, dithiothreitol, N-acetylcysteine, captopril, bovine and human serum albumins, and hydrogen sulfide, are reported. Biological and cardiovascular implications of nitroxyl reactions are discussed.

Toward selective detection of reactive oxygen and nitrogen species with the use of fluorogenic probes – Limitations, progress, and perspectives

Over the last 40 years, there has been tremendous progress in understanding the biological reactions of reactive oxygen species (ROS) and reactive nitrogen species (RNS). It is widely accepted that the generation of ROS and RNS is involved in physiological and pathophysiological processes. To understand the role of ROS and RNS in a variety of pathologies, the specific detection of ROS and RNS is fundamental. Unfortunately, the intracellular detection and quantitation of ROS and RNS remains a challenge. In this short review, we have focused on the mechanistic and quantitative aspects of their detection with the use of selected fluorogenic probes. The challenges, limitations and perspectives of these methods are discussed.

Mechanism of oxidative conversion of Amplex® Red to resorufin: Pulse radiolysis and enzymatic studies

Amplex® Red (10-acetyl-3,7-dihydroxyphenoxazine) is a fluorogenic probe widely used to detect and quantify hydrogen peroxide in biological systems. Detection of hydrogen peroxide is based on peroxidase-catalyzed oxidation of Amplex® Red to resorufin. In this study we investigated the mechanism of one-electron oxidation of Amplex® Red and we present the spectroscopic characterization of transient species formed upon the oxidation. Oxidation process has been studied by a pulse radiolysis technique with one-electron oxidants (N3(•), CO3(•-),(•)NO2 and GS(•)). The rate constants for the Amplex® Red oxidation by N3(•) ((2)k=2.1·10(9)M(-1)s(-1), at pH=7.2) and CO3(•-) ((2)k=7.6·10(8)M(-1)s(-1), at pH=10.3) were determined. Two intermediates formed during the conversion of Amplex® Red into resorufin have been characterized. Based on the results obtained, the mechanism of transformation of Amplex® Red into resorufin, involving disproportionation of the Amplex® Red-derived radical species, has been proposed. The results indicate that peroxynitrite-derived radicals, but not peroxynitrite itself, are capable to oxidize Amplex® Red to resorufin. We also demonstrate that horseradish peroxidase can catalyze oxidation of Amplex® Red not only by hydrogen peroxide, but also by peroxynitrite, which needs to be considered when employing the probe for hydrogen peroxide detection.

Antithrombotic Effects of Pyridinium Compounds Formed from Trigonelline upon Coffee Roasting

Coffee may exert a preventive effect on arterial thrombosis. Trigonelline is one of the most abundant compounds in coffee that undergoes pyrolysis upon roasting of coffee beans. The aim of the present study was to identify pyridinium compounds formed upon trigonelline pyrolysis and coffee roasting and to investigate the effect of three of them, i.e., 1-methylpyridine and 1,3- and 1,4-dimethylpyridine, on experimentally induced arterial thrombosis in rats. 1,3- and 1,4-dimethylpyridine but not 1-methylpyridine inhibited arterial thrombus formation. 1,3-Dimethylpyridine inhibited platelet aggregation and reduced fibrin formation in platelet-rich plasma, whereas 1,4-dimethylpyridine increased the plasma level of 6-keto-PGF1α. 1,4-Dimethylpyridine slightly increased rat tissue plasminogen activator plasma activity. In summary, we demonstrated that pyridinium compounds display mild antithrombotic properties due to stimulation by prostacyclin release (1,4-dimethylpyridine) and inhibition of platelet aggregation (1,3-dimethylpyridine). Those pyridinium compounds may, to some extent, be responsible for the beneficial effects of coffee drinking.

Characterization of Fluorescein-Based Monoboronate Probe and Its Application to the Detection of Peroxynitrite in Endothelial Cells Treated with Doxorubicin

Boronate probes have emerged recently as a versatile tool for the detection of reactive oxygen and nitrogen species. Here, we present the characterization of a fluorescein-based monoboronate probe, a 4-(pinacol boronate)benzyl derivative of fluorescein methyl ester (FBBE), that proved to be useful to detect peroxynitrite in cell culture experiments. The reactivity of FBBE toward peroxynitrite as well hypochlorite, hydrogen peroxide, and tyrosyl hydroperoxide was determined. Second-order rate constants of the reactions of FBBE with peroxynitrite, HOCl, and H2O2 at pH 7.4 were equal to (2.8 ± 0.2) × 10(5) M(-1) s(-1), (8.6 ± 0.5) × 10(3) M(-1) s(-1), and (0.96 ± 0.03) M(-1) s(-1), respectively. The presence of glutathione completely blocked the oxidation of the probe by HOCl and significantly inhibited its oxidation by H2O2 and tyrosyl hydroperoxide but not by peroxynitrite. The oxidative conversion of the probe was also studied in the systems generating singlet oxygen, superoxide radical anion, and nitric oxide in the presence and absence of glutathione. Spectroscopic characterization of FBBE and its oxidation product has been also performed. The differences in the reactivity pattern were supported by DFT quantum mechanical calculations. Finally, the FBBE probe was used to study the oxidative stress in endothelial cells (Ea.hy926) incubated with doxorubicin, a quinone anthracycline antibiotic. In endothelial cells pretreated with doxorubicin, FBBE was oxidized, and this effect was reversed by PEG-SOD and L-NAME but not by catalase.

Dimer Radical Cations of Indole and Indole-3-carbinol: Localized and Delocalized Radical Cations of Diindolylmethane

Extending our previous study on the title species (J. Phys. Chem. A2010, 114, 6787), we investigated the dimer cations that are formed on oxidation of the glucobrassin derivatives indole-3-carbinol (I3C) and diindolylmethane (DIM) and of parent indole (I). Radiolysis in ionic liquid and Ar matrices shows that, at sufficiently high concentrations and/or on annealing the solid glasses, intense intermolecular charge-resonance (CR) absorption bands in the NIR herald the formation of sandwich-type dimer cations. The molecular and electronic structure of these species is modeled by calculations with the double-hybrid B2-PLYP-D density functional method which yields predictions in good accord with experiment. The radical cation of DIM also shows a CR band, but unlike in the case of I and I3C, its occurrence is not dependent on the concentration but instead on the solvent: in ionic liquid the CR band is initially absent and arises only on annealing, whereas in Ar matrices it is present from the outset and undergoes blue shifting and sharpening on annealing. These puzzling findings are rationalized on the basis of B2-PLYP-D calculations which predict that neutral DIM exists in the form of two conformers, present in different relative amounts in the two experiments, which on vertical ionization form distinct radical cations, a nonsymmetric one where the odd electron is largely localized on one of the two indole moieties and one with C(2) symmetry where charge and spin are completely delocalized over both halves of the molecule, thus giving rise to an intramolecular CR transition. On annealing, the nonsymmetric cation relaxes to a similarly delocalized structure with C(s) symmetry, thus explaining the observed increase and the shift of the CR band. We believe that DIM(•+) represents the first example of a radical cation which can exist under the same conditions as a localized and a delocalized complex cation.

Radical scavenging and NO-releasing properties of selected β-adrenoreceptor antagonists

It is claimed that novel β-adrenolytic drugs possess superior antioxidant properties as compared to classical selective or non-selective β-adrenoceptor antagonists. Here we tested this notion by analyzing radical scavenging properties of selected β-adrenolytic drugs and their ability to release nitric oxide in biological preparations. Selective β1-adrenolytics such as nebivolol, atenolol, metoprolol and non-selective β-adrenolytics with α1-receptor blocking properties such as carvedilol and labetalol were chosen for analysis. NO-releasing properties of nebivolol and carvedilol distinguished third generation β-adrenolytics from their older counterparts while the reactivity towards hydroxyl and peroxyl radicals discerns only carvedilol but not nebivolol. Thus, superior clinical efficacy of third generation β-adrenolytics may be related to their ability to release NO rather then to their direct antioxidant properties.

The radical cation of syn-tricyclooctadiene and its rearrangement products

The syn dimer of cyclobutadiene (tricyclo[4.2.0.0(2.5)]octa-3,7-diene, TOD) is subjected to ionization under different conditions and the resulting species are probed by optical and ESR spectroscopy. By means of quantum chemical modelling of the potential energy surfaces and the optical spectra, it is possible to assign the different products that arise spontaneously after ionization or after subsequent warming or illumination of the samples. Based on these findings, we propose a mechanistic scheme which involves a partitioning of the incipient radical cation of TOD between two electronic states. These two states engage in (near) activation-less decay to the more stable valence isomers, cyclooctatetraene (COT*+) and a bis-cyclobutenylium radical cation BCB*+. The latter product undergoes further rearrangement, first to tetracyclo[4.2.0.0(2,4).0(3,5]oct-7-ene (TCO*+) and eventually to bicyclo[4.2.0]octa-2,4,7-triene (BOT*+) which can also be generated photochemically from BCB*+ or TCO*+. The surprising departure of syn-TOD*+ from the least-motion reaction path leading to BOT*+ can be traced to strong vibronic interactions (second-order Jahn-Teller effects) which prevail in both possible ground states of syn-TOD*+. Such effects seem to be more important in determining the intramolecular reactivity of radical cations than orbital or state symmetry rules.