Magdalena Zdrowowicz

Fluorescent Probes Used for Detection of Hydrogen Peroxide under Biological Conditions.

ABSTRACT Hydrogen peroxide is a well-established precursor of reactive oxygen and nitrogen species that are known to contribute to oxidative stress—the crucial factor responsible for the course of a wide range of phy-sicochemical processes as well as the genesis of various diseases, such as cancer and neurodegenerative disorders. Thus, the development of sensitive and selective methods for the detection and quantitative determination of hydrogen peroxide is of great importance in monitoring the in vivo production of that species and elucidating its biological functions. This review highlights the progress that has been made in the development of fluorescent and luminescent probes (excluding nanoparticles) employed to monitor hydrogen peroxide under biological conditions. Attention was focused on probes developed in the past 10 years.

5-Selenocyanatouracil: A Potential Hypoxic Radiosensitizer. Electron Attachment Induced Formation of Selenium Centered Radical

The propensity of 5-selenocyanatouracil (SeCNU) to decomposition induced by attachment of electron was scrutinized with the G3B3 composite quantum-chemical method and radiolytic studies. Favorable thermodynamic (Gibbs free reaction energy of -13.65 kcal/mol) and kinetic (Gibbs free activation energy of 1.22 kcal/mol) characteristics revealed by the G3B3 free energy profile suggest SeCNU to be sensitive to electron attachment. The title compound was synthesized in the reaction between uracil and selenocyanogen chloride in acetic acid. Then, an aqueous and deoxygenated solution of the HPLC purified compound containing tert-butanol as a hydroxyl radical scavenger was irradiated with X-rays. SeCNU radio-degradation results in two major products: the U-Se-Se-U dimer and the adduct of the ●OtBu radical to the U-Se● radical, U-Se-OtBu. The effects of radiolysis as well as the results of G3B3 calculations point to U-Se● as the primary product of dissociative electron attachment to SeCNU. The MTT test shows that SeCNU is nontoxic in vitro in concentrations equal to or lower than 10-6 M. Ionizing radiation will probably induce cytotoxic intra- and interstrand DNA cross-links as well as protein-DNA cross-links in the genomic DNA labeled with SeCNU.

Dihydroxycoumarins as highly selective fluorescent probes for the fast detection of 4-hydroxy-TEMPO in aqueous solution

Due to the biological significance of 4-hydroxy-TEMPO, its detection in biological systems such as blood serum is of great importance in both research and clinical applications. The following paper reports a novel fluorimetric method for the detection and quantitative determination of 4-hydroxy-TEMPO radicals in an aqueous solution using dihydroxycoumarins as fluorescent probes. Among the 17 coumarin derivatives studied, only some dihydroxycoumarins show high sensitivity, specificity and selectivity for 4-hydroxy-TEMPO. Among them, 6,7-dihydroxycoumarin (esculetin) exhibits the strongest fluorescence enhancement under the action of 4-hydroxy-TEMPO. In this assay, esculetin reacts with 4-hydroxy-TEMPO to exclusively yield a dimer. 6,7-Dihydroxycoumarin responds to 4-hydroxy-TEMPO quickly and shows a 2.5-fold fluorescence enhancement with an estimated detection limit of 285 μM.

Fluorescent and Luminescent Probes for Monitoring Hydroxyl Radical under Biological Conditions

ABSTRACT Detection and quantitative determination in biological media of the hydroxyl radical are of great importance due to the role this radical plays in many physiological and pathological processes. This review focuses on the progress that has been made in recent years in the development of fluorescent and luminescent probes employed to monitor hydroxyl radical concentrations under biological conditions.

5-Selenocyanato and 5-trifluoromethanesulfonyl derivatives of 2′-deoxyuridine: synthesis, radiation and computational chemistry as well as cytotoxicity

5-Selenocyanato-2′-deoxyuridine (SeCNdU) and 5-trifluoromethanesulfonyl-2′-deoxyuridine (OTfdU) have been synthesized and their structures have been confirmed with NMR and MS methods. Both compounds undergo dissociative electron attachment (DEA) when irradiated with X-rays in an aqueous solution containing a hydroxyl radical scavenger. The DEA yield of SeCNdU significantly exceeds that of 5-bromo-2′-deoxyuridine (BrdU), remaining in good agreement with the computationally revealed profile of electron-induced degradation. The radiolysis products indicate, in line with theoretical predictions, Se–CN bond dissociation as the main reaction channel. On the other hand, the DEA yield for OTfdU is slightly lower than the degradation yield measured for BrdU, despite the fact that the calculated driving force for the electron-induced OTfdU dissociation substantially overpasses the thermodynamic stimulus for BrdU degradation. Moreover, the calculated DEA profile suggests that the electron attachment induced formation of 5-hydroxy-2′-deoxyuridine (OHdU) from OTfdU, while 2′-deoxyuridine (dU) is mainly observed experimentally. We explained this discrepancy in terms of the increased acidity of OTfdU resulting in efficient deprotonation of the N3 atom, which brings about the domination of the OTfdU(N3–H)− anion in the equilibrium mixture. As a consequence, electron addition chiefly leads to the radical dianion, OTfdU(N3–H)˙2−, which easily protonates at the C5 site. As a result, the C5–O rather than O–S bond undergoes dissociation, leading to dU, observed experimentally. A negligible cytotoxicity of the studied compounds toward the MCF-7 cell line at the concentrations used for cell labelling calls for further studies aiming at the clinical use of the proposed derivatives.

UV-induced electron transfer between triethylamine and 5-bromo-2′-deoxyuridine. A puzzle concerning the photochemical debromination of labeled DNA

Graphical abstract Figure. No caption available. HighlightsTriethylamine (TEA) is an impurity of synthetic oligonucleotides not reported by oligo vendors.In commercial samples, TEA content may exceed by orders of magnitude that of synthetic oligo.TEA completely changes the photochemistry of duplex DNA labeled with 5‐bromo‐2′‐deoxyurdine.The electron‐donor properties of TEA leads to photoinduced debromination of labeled DNA. Abstract 5‐Bromo‐2′‐deoxyuridine (BrdU) photosensitizes DNA to strand break formation. However, this type of photodamage is completely quenched by the presence of triethylamine (TEA) which originates from RP‐HPLC purification commonly employed by oligonucleotide providers. While the presence of TEA in oligonucleotide samples does not interfere with PCR or other molecular biology applications, the mechanism of photochemical reaction proceeding in the labeled DNA is dramatically changed due to the photoinduced electron transfer (PET) between the photoexcited BrdU and the ground state TEA. For the first time, we demonstrated that the latter process produces 2′‐deoxyuridne2′‐deoxyuridine (debromination) in the labeled DNA instead of the expected strand break. PET between TEA and BrdU was additionally confirmed by the UV irradiations of aqueous solutions containing both species. Indeed, the efficient formation of 2′‐deoxyuridine was observed in the studied photolytes. Moreover, we showed the formation of an additional product in these binary mixtures, i.e. imidazole derivative, that is not formed in DNA and was reported in the literature in the context of dark rather than photochemical processes. Using mass spectrometry we demonstrated that the amount of TEA impurity in the commercial samples of oligos exceeds up to 3 orders of magnitude that of the purchased DNA.

The development of 1,3-diphenylisobenzofuran as a highly selective probe for the detection and quantitative determination of hydrogen peroxide

Abstract 1,3-Diphenylisobenzofuran (DPBF) has been developed as a selective probe for the detection and quantitative determination of hydrogen peroxide in samples containing different reactive nitrogen and oxygen species (RNOS). DPBF is a fluorescent probe which, for almost 20 years, was believed to react in a highly specific manner toward some reactive oxygen species (ROS) such as singlet oxygen and hydroxy, alkyloxy or alkylperoxy radicals. Under the action of these individuals DPBF has been rapidly transformed to 1,2-dibenzoylbenzene (DBB). In order to check if DPBF can act as a unique indicator of the total amount of different RNOS, as well as oxidative stress caused by an overproduction of these individuals, a series of experiments was carried out, in which DPBF reacted with peroxynitrite anion, superoxide anion, hydrogen peroxide, hypochlorite anion, and anions commonly present under biological conditions, namely nitrite and nitrate. In all cases, except for hydrogen peroxide, the product of the reaction is DBB. Only under the action of H2O2 9-hydroxyanthracen-10(9H)-one (oxanthrone) is formed. This product has been identified with the use of fluorescence spectroscopy, NMR spectroscopy, high performance liquid chromatography coupled with mass spectrometry, infrared spectroscopy, elemental analysis, and cyclic voltammetry (CV). A linear relationship was found between a decrease in the fluorescence intensity of DPBF and the concentration of hydrogen peroxide in the range of concentrations of 0.196–3.941 mM. DPBF responds to hydrogen peroxide in a very specific way with the limits of detection and quantitation of 88 and 122.8 μM, respectively. The kinetics of the reaction between DBBF and H2O2 was also studied.