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Melaccio F.,University of Siena | Melaccio F.,Bowling Green State University | Ferre N.,CNRS Institute of Radical Chemistry | Olivucci M.,University of Siena | Olivucci M.,Bowling Green State University
Physical Chemistry Chemical Physics | Year: 2012

We look at the possibility to compute and understand the color change occurring upon mutation of a photochromic protein. Accordingly, ab initio multiconfigurational quantum chemical methods are used to construct basic quantum-mechanics/molecular-mechanics (QM/MM) models for a small mutant library of the sensory rhodopsin of Anabaena (Nostoc) sp. PCC7120 cyanobacterium. Together with the wild-type forms, a set of 26 absorption maxima spanning a ca. 80 nm range is obtained. We show that these models can be used to capture the electrostatic change controlling the computed color variation and the change in the ionization of specific side chains. This journal is © the Owner Societies 2012. Source

Michalski R.,Medical College of Wisconsin | Michalski R.,University of Lodz | Zielonka J.,Medical College of Wisconsin | Hardy M.,CNRS Institute of Radical Chemistry | And 2 more authors.
Free Radical Biology and Medicine | Year: 2013

Here we report the synthesis and characterization of a membrane-impermeant fluorogenic probe, hydropropidine (HPr+), the reduction product of propidium iodide, for detecting extracellular superoxide (O2 •-). HPr+ is a positively charged water-soluble analog of hydroethidine (HE), a fluorogenic probe commonly used for monitoring intracellular O2 •-. We hypothesized that the presence of a highly localized positive charge on the nitrogen atom would impede cellular uptake of HPr+ and allow for exclusive detection of extracellular O2 •-. Our results indicate that O 2 •- reacts with HPr+ (k=1.2×10 4 M-1 s-1) to form exclusively 2-hydroxypropidium (2-OH-Pr2+) in cell-free and cell-based systems. This reaction is analogous to the reaction between HE and O2 •- (Zhao et al., Free Radic. Biol. Med. 34:1359-1368; 2003). During the course of this investigation, we also reassessed the rate constants for the reactions of O2 •- with HE and its mitochondria targeted analog (Mito-HE or MitoSOX Red) and addressed the discrepancies between the present values and those reported previously by us. Our results indicate that the rate constant between O2 •- and HPr+ is slightly higher than that of HE and O2 •- and is closer to that of Mito-HE and O 2 •-. Similar to HE, HPr+ undergoes oxidation in the presence of various oxidants (peroxynitrite-derived radicals, Fenton's reagent, and ferricytochrome c) forming the corresponding propidium dication (Pr2+) and the dimeric products (e.g., Pr 2+-Pr2+). In contrast to HE, there was very little intracellular uptake of HPr+. We conclude that HPr+ is a useful probe for detecting O2 •- and other one-electron oxidizing species in an extracellular milieu. Source

Kalyanaraman B.,Medical College of Wisconsin | Dranka B.P.,Medical College of Wisconsin | Hardy M.,Medical College of Wisconsin | Hardy M.,CNRS Institute of Radical Chemistry | And 3 more authors.
Biochimica et Biophysica Acta - General Subjects | Year: 2014

Background: Nearly ten years ago, we demonstrated that superoxide radical anion (O2×̄) reacts with the hydroethidine dye (HE, also known as dihydroethidium, DHE) to form a diagnostic marker product, 2-hydroxyethidium (2-OH-E+). This particular product is not derived from reacting HE with other biologically relevant oxidants (hydrogen peroxide, hydroxyl radical, or peroxynitrite). This discovery negated the longstanding view thatO2×̄ reacts with HE to form the other oxidation product, ethidium (E+). It became clear that due to the overlapping fluorescence spectra of E+ and 2-OH-E+, fluorescence-based techniques using the "red fluorescence" are not suitable for detecting and measuring O2×̄ in cells using HE or other structurally analogous fluorogenic probes (MitoSOXTM Red or hydropropidine). However, using HPLC-based assays, 2-OH-E+ and analogous hydroxylated products can be easily detected and quickly separated from other oxidation products. Scope of review: The principles discussed in this chapter are generally applicable in free radical biology and medicine, redox biology, and clinical and translational research. The assays developed here could be used to discover new and targeted inhibitors for various superoxide-producing enzymes, including NADPH oxidase (NOX) isoforms. Major conclusions: HPLC-based approaches using site-specific HE-based fluorogenic probes are eminently suitable for monitoring O2×̄ in intra- and extracellular compartments and in mitochondria. The use of fluorescence-microscopic methods should be avoided because of spectral overlapping characteristics of O2×̄-derived marker product and other, non-specific oxidized fluorescent products formed from these probes. General significance: Methodologies and site-specific fluorescent probes described in this review can be suitably employed to delineate oxy radical dependent mechanisms in cells under physiological and pathological conditions. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. © 2013 Elsevier B.V. All rights reserved. Source

Yue L.,Beijing Normal University | Roca-Sanjuan D.,Uppsala University | Lindh R.,Uppsala University | Ferre N.,CNRS Institute of Radical Chemistry | Liu Y.-J.,Beijing Normal University
Journal of Chemical Theory and Computation | Year: 2012

The chemiluminescent decomposition of 1,2-dioxetanone has in the past been studied by state-of-the-art multireference quantum chemical calculations, and a stepwise biradical mechanism was established. Recently, this decomposition has been reinvestigated, and a concerted mechanism has been proposed based on calculations performed at the closed-shell density functional theory (DFT) level of theory. In order to solve this apparent mechanistic contradiction, the present paper presents restricted and unrestricted DFT results obtained using functionals including different amounts of Hartree-Fock (HF) exchange, repeating and complementing the above-mentioned DFT calculations. The calculated results clearly indicate that the closed-shell DFT methods cannot correctly describe the thermolysis of 1,2-dioxetanone. It is found that unrestricted Kohn-Sham reaction energies and barriers are always lower than the ones obtained using a restricted formalism. Hence, from energy principles, the biradical mechanism is found to be prevailing in the understanding of the 1,2-dioxetanone thermolysis. © 2012 American Chemical Society. Source

Al Ouahabi A.,Charles Sadron Institute | Charles L.,CNRS Institute of Radical Chemistry | Lutz J.-F.,Charles Sadron Institute
Journal of the American Chemical Society | Year: 2015

Sequence-defined non-natural polyphosphates were prepared using iterative phosphoramidite protocols on a polystyrene solid support. Three monomers were used in this work: 2-cyanoethyl (3-dimethoxytrityloxy-propyl) diisopropylphosphoramidite (0), 2-cyanoethyl (3-dimethoxytrityloxy-2,2-dimethyl-propyl) diisopropylphosphoramidite (1), and 2-cyanoethyl (3-dimethoxytrityloxy-2,2-dipropargyl-propyl) diisopropylphosphoramidite (1′). Phosphoramidite coupling steps allowed rapid synthesis of homopolymers and copolymers. In particular, the comonomers (0, 1), (0, 1′), and (1, 1′) were used to synthesize sequence-encoded copolymers. It was found that long encoded sequences could be easily built using phosphoramidite chemistry. ESI-HRMS, MALDI-HRMS, NMR, and size exclusion chromatography analyses indicated the formation of monodisperse polymers with controlled comonomer sequences. The polymers obtained with the comonomers (0, 1′) and (1, 1′) were also modified by copper-catalyzed azide-alkyne cycloaddition with a model azide compound, namely 11-azido-3,6,9-trioxaundecan-1-amine. 1H and 13C NMR analysis evidenced quantitative modification of the alkyne side-chains of the monodisperse copolymers. Thus, the molecular structure of the coding monomer units can be easily varied after polymerization. Altogether, the present results open up interesting avenues for the design of information-containing macromolecules. © 2015 American Chemical Society. Source

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