Nitroxide Free Radicals Research Articles

The semisynthetic spin-labelled derivatives of 3-hydroxybutanolide as potential oxidative stress inhibitors

To obtain more accessible oxidative stress inhibitors, a series of novel spin-labelled derivatives of 3-hydroxybutanolide (2a–d, 3a–d) with the natural active compound (kinsenoside) as the lead compound were designed, synthesised from the nitroxide free radical piperidine (pyrroline) and the main structural unit of kinsenoside: 3-hydroxybutanolide. Antioxidant activity screening of these derivatives was performed using MTT method on rat pheochromocytoma PC12 cells. The antioxidative stress effect was further investigated on the changes of the important antioxidant enzyme activities and intracellular reactive oxygen species production. Among the derivatives, 2b–d, 3a and 3c showed comparable or superior antioxidative stress activity to kinsenoside. Also, most of the tested derivatives displayed obvious antioxidative ability in concentrations. Cytotoxic assay simultaneously indicated that all compounds had very low toxicity to normal cells. Based on the observed results, the structure–activity relationship of these derivatives was discussed.

Photolysis of endoperoxides in the presence of nitroxides: a laser flash photolysis study with optical and ESR detection

Time-resolved electron paramagnetic resonance spectroscopy, transient absorption, and phosphorescence spectroscopy were used to investigate the spin polarization of a nitroxide free radical induced by interaction with singlet oxygen ((1)O2). The latter was generated by photolysis of endoperoxides of two anthracene derivatives. Although both anthracene endoperoxides are structurally similar, opposite spin polarization of the nitroxide was observed. Photolysis of one endoperoxide leads to absorptive nitroxide spin polarization due to interaction with the generated (1)O2. Photolysis of the other endoperoxide generated emissive nitroxide spin polarization, probably due to interaction of the endoperoxide triplet states with nitroxides.

Nitronyl and imino nitroxide free radicals as precursors of magnetic phthalocyanine and porphyrin building blocks

Phthalonitrile and benzaldehyde bearing α-nitronyl and α-imino nitroxide free radicals have been synthesized as precursors of nitroxide substituted phthalocyanine and porphyrin macrocycle.

Modulation of CEST Images in Vivo by T1 Relaxation: A New Approach in the Design of Responsive PARACEST Agents

A novel approach for the design of responsive paramagnetic chemical exchange saturation transfer (PARACEST) magnetic resonance imaging (MRI) agents has been developed where the signal is "turned on" by altering the longitudinal relaxation time (T1) of bulk water protons. To demonstrate this approach, a model Eu(DOTA-tetraamide) complex (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) containing two nitroxide free radical units was synthesized. The nitroxide groups substantially shortened the T1 of the bulk water protons which, in turn, resulted in quenching of the CEST signal. Reduction of paramagnetic nitroxide moieties to a diamagnetic species resulted in the appearance of CEST. The modulation of CEST by T1 relaxation provides a new platform for designing biologically responsive MRI agents.

Modulating Crystal Packing and Magnetic Properties of Nitroxide Free Radicals by Halogen Bonding

By combining a halogen bonding donor of 1,4-diiodotetrafluorobenzene (1,4-DITFB) or 1,2-diiodotetrafluorobenzene (1,2-DITFB) with 4-benzoyloxy-2,2,6,6-tetramethyl-piperidine-1-oxy free radical (BTEMPO, 1), two novel cocrystals, BTEMPO·1,4-DITFB (2) and BTEMPO·2(1,2-DITFB) (3), featuring a one-dimensional (1D) zigzag chain and a cyclic tetrameric structure, respectively, were constructed via halogen bonding. Furthermore, by tuning the halogen bonding between BTEMPO and halogen bonding donors, the packing modes of organic free radicals in both 2 and 3 are successfully modulated to result in side-by-side 1D mode in 2 and head-to-head dimer manner in 3. Magnetic studies reveal that in all compounds the organic radicals are antiferromagnetically coupled; however, the magnetic coupling in 2 and 3 is stronger than that in 1, indicating the modulating effect of halogen bonding on magnetic properties that are further confirmed by quantum chemistry calculations.

Nitroxide TEMPO: A genotoxic and oxidative stress inducer in cultured cells

2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) is a low molecular weight nitroxide and stable free radical. In this study, we investigated the cytotoxicity and genotoxicity of TEMPO in mammalian cells using the mouse lymphoma assay (MLA) and in vitro micronucleus assay. In the absence of metabolic activation (S9), 3mM TEMPO produced significant cytotoxicity and marginal mutagenicity in the MLA; in the presence of S9, treatment of mouse lymphoma cells with 1–2mM TEMPO resulted in dose-dependent decreases of the relative total growth and increases in mutant frequency. Treatment of TK6 human lymphoblastoid cells with 0.9–2.3mM TEMPO increased the frequency of both micronuclei (a marker for clastogenicity) and hypodiploid nuclei (a marker of aneugenicity) in a dose-dependent manner; greater responses were produced in the presence of S9. Within the dose range tested, TEMPO induced reactive oxygen species and decreased glutathione levels in mouse lymphoma cells. In addition, the majority of TEMPO-induced mutants had loss of heterozygosity at the Tk locus, with allele loss of ⩽34Mbp. These results indicate that TEMPO is mutagenic in the MLA and induces micronuclei and hypodiploid nuclei in TK6 cells. Oxidative stress may account for part of the genotoxicity induced by TEMPO in both cell lines.

Overhauser-Enhanced MRI of Elastase Activity from In Vitro Human Neutrophil Degranulation

BackgroundMagnetic resonance imaging can reveal exquisite anatomical details. However several diseases would benefit from an imaging technique able to specifically detect biochemical alterations. In this context protease activity imaging is one of the most promising areas of research.Methodology/Principal FindingsWe designed an elastase substrate by grafting stable nitroxide free radicals on soluble elastin. This substrate generates a high Overhauser magnetic resonance imaging (OMRI) contrast upon digestion by the target proteases through the modulation of its rotational correlation time. The sensitivity is sufficient to generate contrasted images of the degranulation of neutrophils induced by a calcium ionophore from 2×104 cells per milliliter, well under the physiological neutrophils concentrations.Conclusions/SignificanceThese ex-vivo experiments give evidence that OMRI is suitable for imaging elastase activity from neutrophil degranulation. Provided that a fast protease-substrate is used these results open the door to better diagnoses of a number of important pathologies (cystic fibrosis, inflammation, pancreatitis) by OMRI or Electron Paramagnetic Resonance Imaging in vivo. It also provides a long-expected method to monitor anti-protease treatments efficiency and help pharmaceutical research.

Spectroscopic studies on the photochemical decarboxylation mechanisms of synthetic pyrethroids

A novel radical trapping technique combined with a fluorescence spectroscopic analysis has been employed to investigate the radical intermediates produced by photodecarboxylation of four synthetic pyrethroids: fenvalerate (SMD), fenpropathrin (DTL), cyphenothrin (GKL), and cypermethrin (AGT). Under photoirradiation at >290 nm, all pyrethroids underwent direct photolysis via homolytic cleavage of the carbon-oxygen bonds in the ester groups. The consumed amount of a nitroxide free radical, as a trapping agent for the intermediate radical of a pyrethroid, was determined by ESR, which was the measure of the reaction yield of a photochemically generated α-cyano-3-phenoxybenzyl radical common to all pyrethroids. The reactivities of the pyrethroids studied was in the sequence of SMD >> DTL > GKL > AGT. Furthermore, nanosecond transient absorption spectroscopy demonstrated that geminate recombination of the radical pair within a solvent cage is the main deactivation route of the photochemically generated α-cyano-3-phenoxybenzyl radical common for all pyrethroids studied.

Comproportionation versus Disproportionation in the Initiation Step of Cu(0)-Mediated Living Radical Polymerization

We present a study of the initiation step in copper(0)-mediated atom transfer radical polymerization (also known as SET-LRP or SARA ATRP), in which a nitroxide free radical, N-tert-butyl-N-[1-diethylphosphono-(2,2-dimethylpropyl)] nitroxide (SG1), is used to trap radicals derived from ethyl 2-bromoisobutyrate (EBiB), copper metal and pentamethyl diethylene triamine (PMDETA) ligand. A kinetic model was developed relating the rate of conversion of EBiB to the rate constants of activation by copper metal (ka0) and comproportionation of Cu0 and CuII (kcomp). This allowed the determination of these parameters in several solvents, including dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ethanol (EtOH), and acetonitrile (MeCN), in which significant levels of comproportionation were observed, leading to autoacceleration of the reaction. A similar treatment of copper-mediated polymerization kinetics predicts 1/2 order dependence on initiator concentration and copper surface area, in accordance with previously...

Fluorescent polyolefins by free radical post-reactor modification with functional nitroxides

The 4-(1-naphthoate)-2,2,6,6-tetramethylpiperidine-1-oxyl (NfO-TEMPO) has been synthesized and successfully grafted in the melt onto a random poly(ethylene-co-1-ottene) copolymer. Functionalized polyolefins have been prepared by coupling reaction between NfO-TEMPO free radicals and macroradicals which have been formed by H-abstraction induced by the presence of a peroxide. In order to deepen insight into the functionalization mechanism, the reaction has been investigated by Electron Paramagnetic Resonance (EPR). EPR spectra collected during the reaction run, have evidenced the decrease of TEMPO signal as a consequence of temperature increasing. This decrease has been attributed to the formation of a covalent bond between macroradicals and nitroxide free radical. The resulting functionalized polyolefins, PO-g-(NfO-TEMPO), have been characterized by FT-IR and 1H NMR which has allowed to evaluate their functionalization degree, whereas UV–Vis and fluorescence spectroscopy have been used to investigate their optical properties. The comparison with low molecular weight model compounds, has allowed to state that our methodology can be conveniently adopted to prepare fluorescent polyolefins where the optical properties of the chromophore has been completely transferred to polymer backbone both in solution and in the condensed phase.

Characterization of Hindered Amine Light Stabilizers in Polymer Matrix Using Terahertz Time‐Domain Spectroscopy

AbstractTerahertz time‐domain spectroscopy (THz‐TDS) technique allows us to analyze plastic additives. A series of hindered amine light stabilizers (HALSs), including TMP, PMP, BB‐PMP, Chimassorb 944 and 119, and a nitroxide free radical of 4‐hydroxy‐2,2,6,6‐tetramethyl‐1‐piperidinyloxy (HTEMPO) in the matrix of high‐density polyethylene are characterized in the range of 0.2–2.6 THz. TMP, PMP, and BB‐PMP exhibit individual well‐resolved terahertz (THz) absorption spectra at room temperature. Furthermore, a linear relationship of the content of TMP versus the intensity of it first THz absorption peak are demonstrated with the detection limit of about 1.6% (w/w). In addition, PMP exhibit water‐dependent characteristic THz absorption spectra. THz‐TDS will afford a potential tool to investigate the stabilizing mechanism of HALS and monitor their evolution.

Electron Paramagnetic Resonance Line Shifts and Line Shape Changes Due to Heisenberg Spin Exchange and Dipole–Dipole Interactions of Nitroxide Free Radicals in Liquids 8. Further Experimental and Theoretical Efforts to Separate the Effects of the Two Interactions

The work in part 6 of this series (J. Phys. Chem. A 2009, 113, 4930), addressing the task of separating the effects of Heisenberg spin exchange (HSE) and dipole-dipole interactions (DD) on electron paramagnetic resonance (EPR) spectra of nitroxide spin probes in solution, is extended experimentally and theoretically. Comprehensive measurements of perdeuterated 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl (pDT) in squalane, a viscous alkane, paying special attention to lower temperatures and lower concentrations, were carried out in an attempt to focus on DD, the lesser understood of the two interactions. Theoretically, the analysis has been extended to include the recent comprehensive treatment by Salikhov (Appl. Magn. Reson. 2010, 38, 237). In dilute solutions, both interactions (1) introduce a dispersion component, (2) broaden the lines, and (3) shift the lines. DD introduces a dispersion component proportional to the concentration and of opposite sign to that of HSE. Equations relating the EPR spectral parameters to the rate constants due to HSE and DD have been derived. By employing nonlinear least-squares fitting of theoretical spectra to a simple analytical function and the proposed equations, the contributions of the two interactions to items 1-3 may be quantified and compared with the same parameters obtained by fitting experimental spectra. This comparison supports the theory in its broad predictions; however, at low temperatures, the DD contribution to the experimental dispersion amplitude does not increase linearly with concentration. We are unable to deduce whether this discrepancy is due to inadequate analysis of the experimental data or an incomplete theory. A new key aspect of the more comprehensive theory is that there is enough information in the experimental spectra to find items 1-3 due to both interactions; however, in principle, appeal must be made to a model of molecular diffusion to separate the two. The permanent diffusion model is used to illustrate the separation in this work. In practice, because the effects of DD are dominated by HSE, negligible error is incurred by using the model-independent extreme DD limit of the spectral density functions, which means that DD and HSE may be separated without appealing to a particular model.

Two-photon fluorescence microscopy imaging of cellular oxidative stress using profluorescent nitroxides.

A range of varying chromophore nitroxide free radicals and their nonradical methoxyamine analogues were synthesized and their linear photophysical properties examined. The presence of the proximate free radical masks the chromophore’s usual fluorescence emission, and these species are described as profluorescent. Two nitroxides incorporating anthracene and fluorescein chromophores (compounds 7 and 19, respectively) exhibited two-photon absorption (2PA) cross sections of approximately 400 G.M. when excited at wavelengths greater than 800 nm. Both of these profluorescent nitroxides demonstrated low cytotoxicity toward Chinese hamster ovary (CHO) cells. Imaging colocalization experiments with the commercially available CellROX Deep Red oxidative stress monitor demonstrated good cellular uptake of the nitroxide probes. Sensitivity of the nitroxide probes to H2O2-induced damage was also demonstrated by both one- and two-photon fluorescence microscopy. These profluorescent nitroxide probes are potentially powerful tools for imaging oxidative stress in biological systems, and they essentially “light up” in the presence of certain species generated from oxidative stress. The high ratio of the fluorescence quantum yield between the profluorescent nitroxide species and their nonradical adducts provides the sensitivity required for measuring a range of cellular redox environments. Furthermore, their reasonable 2PA cross sections provide for the option of using two-photon fluorescence microscopy, which circumvents commonly encountered disadvantages associated with one-photon imaging such as photobleaching and poor tissue penetration.

Low Operational Stability of Enzymes in Dry Organic Solvents: Changes in the Active Site Might Affect Catalysis

The potential of enzyme catalysis in organic solvents for synthetic applications has been overshadowed by the fact that their catalytic properties are affected by organic solvents. In addition, it has recently been shown that an enzyme’s initial activity diminishes considerably after prolonged exposure to organic media. Studies geared towards understanding this last drawback have yielded unclear results. In the present work we decided to use electron paramagnetic resonance spectroscopy (EPR) to study the motion of an active site spin label (a nitroxide free radical) during 96 h of exposure of the serine protease subtilisin Carlsberg to four different organic solvents. Our EPR data shows a typical two component spectra that was quantified by the ratio of the anisotropic and isotropic signals. The isotropic component, associated with a mobile nitroxide free radical, increases during prolonged exposure to all solvents used in the study. The maximum increase (of 43%) was observed in 1,4-dioxane. Based on these and previous studies we suggest that prolonged exposure of the enzyme to these solvents provokes a cascade of events that could induce substrates to adopt different binding conformations. This is the first EPR study of the motion of an active-site spin label during prolonged exposure of an enzyme to organic solvents ever reported.

Reversibility of solid state radical reactions in thermally remendable polymers with C–ON bonds

The C–ON covalent bond with thermally responsive function opens a new avenue to prepare remendable polymers. Carbon-centered radicals and oxygen-centered nitroxide free radicals produced by homolytic cleavage of C–ON bonds at healing temperature synchronously reorganize to form cross-linked networks, offering effective damage repairing characteristics without losing integrity and load bearing ability of the material even above Tg. To gain a deeper understanding of the mechanisms, thermally remendable two-component polymer blends carrying two types of C–ON bonds in the side chains were synthesized. With the aid of small molecule mimetics, intermolecular reactions involved in thermal reversibility of the polymer blends and self-healing capability are analyzed. It was found that crossover reaction between carbon-centered radicals and oxygen-centered nitroxide radicals is necessary, but the possibility of the irreversible combination of carbon-centered radicals should be eliminated. Polymer solid hinders diffusion of carbon-centered radicals so that the unwanted secondary reaction is depressed even though complete recombination of the cleft C–ON bond is also somewhat blocked. Basically, reversibility and multiple self-healing behaviors of the synthesized polymer blends are satisfactory in addition to the expanded healing temperature range. These results are believed to favor materials design and improve crack healing performance based on efficient bond fission and radical recombination.

Synthesis of Deprenyl-like nitroxide free radicals and their diamagnetic derivatives

Synthesis of paramagnetically modified deprenyl and oxotremorine is reported. Starting from 5- and 6-membered 2,5-disubstituted nitrones (1, 6) or 4-phenyl-2,5,5-trimethyl-1H-pyrroline 1-oxide (11) deprenyl or oxotremorine like nitroxides were synthesized via Grignard reactions. The corresponding pre-nitroxides with propargylamine structure were achieved by reduction of nitroxides followed by methylation.

ChemInform Abstract: Profluorescent Nitroxides as Sensitive Probes of Oxidative Change and Free Radical Reactions

AbstractReview: 126 refs.

Preparation and characterization of novel spin labeled magnetic nanoparticles

Novel spin-labeled magnetic nanoparticles (MNPs) were prepared through the reaction between carboxylic acids modified Fe3O4 particles and 4-amino-2,2,6,6-tetramethylpiperidin-1-oxyl (4-NH2-TEMPO). And X-ray diffractometer (XRD), FT-IR spectra, thermogravimetric analysis (TGA), transmission electronic microscopy (TEM) and room temperature electron paramagnetic resonance (EPR) were employed to characterize the prepared materials. The results of EPR measurements indicated that the spin-labeled MNPs exhibited both the paramagnetism of nitroxide free radicals and superparamagnetism of ferrimagnetic nanoparticles could act as a bio-probe or potential drug delivery vehicles tracking by EPR technique.

Exploring phases of a physiologically-active nitronyl nitroxide free radical

Exploring phases of a physiologically-active nitronyl nitroxide free radical

Single-Molecule Magnetic Behavior in a Neutral Terbium(III) Complex of a Picolinate-Based Nitronyl Nitroxide Free Radical

The terdentate anionic picolinate-based nitronyl nitroxide (picNN) free radical forms neutral and robust homoleptic complexes with rare earth-metal ions. The nonacoordinated Tb(3+) complex Tb(picNN)(3)·6H(2)O is a single-molecule magnet with an activation energy barrier Δ = 22.8 ± 0.5 K and preexponential factor τ(0) = (5.5 ± 1.1) × 10(-9) s. It shows magnetic hysteresis below 1 K.