Stable Organic Free Radical Research Articles

Unusual reactivity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) with Fe3+ controlled by competing reactions.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) is a stable organic free radical widely used in various fields as a model free radical. There is scarce information about the stability of this compound in the chemical environments in which it is used. Side reactions between DPPH and other species can alter the precision of experiments that use DPPH, such as the evaluation of antioxidant properties amongst others. Following recent investigations highlighting reactions between DPPH and metal cations or Lewis acids, a quantitative reaction between DPPH and Fe3+ in acetonitrile was studied in the present work. UV-Vis spectroscopy and electrochemistry were used to monitor the reaction. The results obtained indicate a fast and multistep reaction between Fe3+ and DPPH that can be simplified as a simple redox reaction with the formation of Fe2+ and DPPH+. The reaction mechanism proposed follows complex steps involving two competing phenomena: a disproportionation which accelerates the reaction and an oxidation process that slows it down through DPPH regeneration.

EPR Spectroscopy: A Powerful Tool to Analyze Supramolecular Host•Guest Complexes of Stable Radicals with Cucurbiturils.

Stable organic free radicals are increasingly studied compounds due to the multiple and unusual properties imparted by the single electron(s). However, being paramagnetic, classical methods such as NMR spectroscopy can hardly be used due to relaxation and line broadening effects. EPR spectroscopy is thus better suited to get information about the immediate surroundings of the single electrons. EPR has enabled obtaining useful data in the context of host•guest chemistry, and a classical example is reported here for the stable (2,2,6,6-tetramethyl-4-oxo-piperidin-1-yl)oxyl or 4-oxo-TEMPO nitroxide (TEMPONE) inside the macrocycle host cucurbit[7]uril (CB[7]). Generally and also observed here, a contraction of the spectrum is observed as a result of the reduced nitrogen coupling constant due to inclusion complexation in the hydrophobic cavity of the host. Simulations of EPR spectra allowed determining the corresponding binding constant pointing to a weaker affinity for CB[7], compared to TEMPO with CB[7]. We complement this work by the results of EPR spectroscopy of a biradical: bis-TEMPO-bis-ketal (bTbk) with cucurbit[8]uril (CB[8]). Initial investigations pointed to very weak effects on the spectrum of the guest and incorrectly led us to conclude an absence of binding. However, simulations of EPR spectra combined with NMR data of reduced bTbk allowed showing inclusion complexation. EPR titrations were performed, and the corresponding binding constant was determined. 1H NMR spectra with reduced bTbk suggested a shuttle mechanism, at nearly one equivalent of CB[8], for which the host moves rapidly between two stations.

Proton polarization enhancement of up to 150 with dynamic nuclear polarization of plasma-treated glucose powder

Dynamic nuclear polarization (DNP) for the enhancement of the NMR signals of specific metabolites has recently found applications in the context of magnetic resonance imaging (MRI). Currently, DNP signal enhancement is implemented in clinical systems through the use of exogenous stable organic free radicals, known as polarization agents (PAs), mixed in a solution with the metabolite of interest. These PAs are medically undesirable and thus must be filtered out prior to patient injection - a task that involves considerable technical complexity and consumes valuable time during which the polarization decays. Here, we aim to demonstrate DNP enhancements large enough for clinical relevance using a process free of exogenous PAs. This is achieved by processing (soft grinding) the metabolite in its solid form and subsequently exposing it to plasma in a dilute atmosphere to produce chemically-unstable free radicals (herein referred to as electrical-discharge-induced radicals - EDIRs) within the powder. These samples are then subjected to the normal DNP procedure of microwave irradiation while placed under a high static magnetic field, and their NMR signal is measured to quantify the enhancement of the protons’ signal in the solid. Proton signal enhancements (measured as the ratio of the NMR signal with microwave irradiation to the NMR signal without microwave irradiation) of up to 150 are demonstrated in glucose. Upon fast dissolution, the free radicals are annihilated, leaving the sample in its original chemical composition (which is safe for clinical use) without any need for filtration and cumbersome quality control procedures. We thus conclude that EDIRs are found to be highly efficient in providing DNP enhancement levels that are on par with those achieved with the exogenous PAs, while being safe for clinical use. This opens up the possibility of applying our method to clinical scenarios with minimal risks and lower costs per procedure.

Electron-Spin-Resonance Dipstick.

Electron spin resonance (ESR) is a powerful analytical technique used for the detection, quantification, and characterization of paramagnetic species ranging from stable organic free radicals and defects in crystals to gaseous oxygen. Traditionally, ESR requires the use of complex instrumentation, including a large magnet and a microwave resonator in which the sample is placed. Here, we present an alternative to the existing approach by inverting the typical measurement topology, namely placing the ESR magnet and resonator inside the sample rather than the other way around. This new development relies on a novel self-contained ESR sensor with a diameter of just 2 mm and length of 3.6 mm, which includes both a small permanent magnet assembly and a tiny (∼1 mm in size) resonator for spin excitation and detection at a frequency of ∼2.6 GHz. The spin sensitivity of the sensor has been measured to be ∼1011 spins/√Hz, and its concentration sensitivity is ∼0.1 mM, using reference samples with a measured volume of just ∼10 nL. Our new approach can be applied for monitoring the partial pressure of oxygen in vitro and in vivo through its paramagnetic interaction with another stable radical, as well as for simple online quantitative inspection of free radicals generated in reaction vessels and electrochemical cells via chemical processes.

Covalent Modification of Highly Ordered Pyrolytic Graphite with a Stable Organic Free Radical by Using Diazonium Chemistry.

A novel, persistent, electrochemically active perchlorinated triphenylmethyl (PTM) radical with a diazonium functionality has been covalently attached to highly ordered pyrolytic graphite (HOPG) by electrografting in a single-step process. Electrochemical scanning tunneling microscopy (EC-STM) and Raman spectroscopy measurements revealed that PTM molecules had a higher tendency to covalently react at the HOPG step edges. The cross-section profiles from EC-STM images showed that there was current enhancement at the functionalized areas, which could be explained by redox-mediated electron tunneling through surface-confined redox-active molecules. Cyclic voltammetry clearly demonstrated that the intrinsic properties of the organic radical were preserved upon grafting and DFT calculations also revealed that the magnetic character of the PTM radical was preserved.

Pharmacological effects of the phytochemicals of Anethum sowa L. root extracts.

Background Anethum sowa L. is widely used as an important spice and traditional medicinal plants to treat various ailments. On the basis of scientific ethnobotanical information, this study was undertaken to evaluate the antioxidant, antimicrobial and cytotoxic activity of the crude extracts of Anethum sowa L. roots as well as to identify the classes of phytochemicals by chemical tests.MethodsThe antioxidant potential of the extracts was ascertained with the stable organic free radical (2, 2-diphenyl-1-picryl-hydrazyl). The agar well diffusion method was used to determine the susceptibility of bacterial and fungal strains of the crude extracts. The minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) were determined by the microdilution test. Cytotoxic activities were screened using brine shrimps (Artemia salina) lethality assay. Finally, phytochemicals were profiled using standard procedures.ResultsA preliminary phytochemical screening of the different crude extracts by methanol, ethyl acetate and chloroform showed the presence of secondary metabolites such as flavonoids, alkaloids, saponin, cardiac glycosides and tannins while cyanogenetic glycosides were not detected. The methanol, ethyl acetate and chloroform extracts displayed high antioxidant activity (IC50 = 13.08 ± 0.03, 33.48 ± 0.16 and 36.42 ± 0.41 μg/mL, respectively) in the DPPH assay comparable to that of the standard ascorbic acid and BHT (IC50 = 3.74 ± 0.05 and 11.84 ± 0.29 μg/mL). The cytotoxic activity of the crude ethyl acetate and chloroform extracts possessed excellent activity (LC50 = 5.03 ± 0.08, 5.23 ± 0.11 and 17.22 ± 0.14 μg/mL, respectively) against brine shrimp larvae after 24 h of treatment and compared with standard vincristine sulphate (LC50 = 0.46 ± 0.05 μg/mL). The extracts also showed good antimicrobial activity against both Gram-positive and Gram-negative bacteria when compared with two standard antibiotics ciprofloxacin and tetracycline.ConclusionThese results showed that the Anethum sowa root extracts are the important source of the antioxidant, antimicrobial and cytotoxic agent. So, further research is necessary to isolate and characterize of different phytoconstituents for pharmaceutical drug lead molecules and also to verify its traditional uses.

Catechol degradation on hematite/silica-gas interface as affected by gas composition and the formation of environmentally persistent free radicals.

Environmentally persistent free radicals (EPFRs) formed on a solid particle surface have received increasing attention because of their toxic effects. However, organic chemical fate regulated by EPFRs has rarely been investigated, and this information may provide the missing link in understanding their environmental behavior. Previous studies have suggested that the reduction of transition metals is involved in EPFRs formation. We thus hypothesize that an oxidative environment may inhibit EPFRs formation in particle-gas interface, which will consequently release free radicals and accelerate organic chemical degradation. Our result indicates that a 1% hematite coating on a silica surface inhibited catechol degradation in N2, especially at low catechol loadings on solid particles (SCT). However, under an O2 environment, catechol degradation decreased when SCT was <1 μg/mg but increased when SCT was >1 μg/mg. Stable organic free radicals were observed in the N2 system with g factors in the 2.0035–2.0050 range, suggesting the dominance of oxygen-centered free radicals. The introduction of O2 into the catechol degradation system substantially decreased the free radical signals and decreased the Fe(II) content. These results were observed in both dark and light irradiation systems, indicating the ubiquitous presence of EPFRs in regulating the fate of organic chemicals.

Phytochemicals, antioxidant, antinociceptive and anti-inflammatory potential of the aqueous extract of Teucrium stocksianum bioss

BackgroundDespite availability of a substantial number of potent synthetic drugs, medicinal plants are still playing a key role in the discovery of novel and effective drug molecules. Numerous researchers are focusing on the plant based medicines due to its strong safety profiles. Teucrium species exhibit profound antidiabetic, analgesic and spasmolytic activities. The methanolic extract and essential oil of Teucrium stocksianum possess strong antinociceptive activity. The aim of the current research study was to determine the phytochemicals, antioxidant, analgesic and anti-inflammatory potential of the aqueous extract of Teucrium stocksianum Bioss (AETS).MethodPhytochemical screening was carried out according to standard procedures. The antioxidant potential of the extract was ascertained with the stable organic free radical (2, 2-diphenyl-1-picryl-hydrazyl). Three different pain models, including acetic acid induced writhing, formalin induced paw licking and tail immersion tests were carried out for the determination of antinociceptive potential, while the anti-inflammatory activity was evaluated through carrageenan induced paw edema test in mice. The antinociceptive and anti-inflammatory potentials of AETS were assessed at 100, 200 and 300 mg/kg body weight, while acute toxicity were observed at 1500 mg/kg body weight in various groups of mice.ResultsPhytochemical screening has shown the occurrence of flavonoids saponins, reducing sugars, terpenoids and tannins. AETS exhibited profound antioxidant activity and has shown maximum activity (60.06 ± 0.846) at 250 μg/ml. In the three pain models AETS displayed marked dose dependent antinociceptive potential. AETS exhibited 63.5, 67.61 and 64 % activity in acetic acid induced, formalin induced paw licking and tail immersion tests respectively. The antinociceptive effect of AETS and reference standard drug TramadolR was significantly reversed by Naloxone, endorsed the central analgesic potential of AETS. Similarly the extract also reversed the paw edema in dose dependent manner. AETS displayed significant (53.81 %) anti-inflammatory effects at a dose of 300 mg/kg that persisted till 5th h. In acute toxicity test AETS was found safe at 1500 mg/kg body weight.ConclusionsAETS exhibited profound antioxidant activity. The test sample displayed marked antinociceptive potential in all the test procedures, indicating the peripheral and central analgesic effects of AETS. The plant extract also displayed marked anti-inflammatory activity at all test doses.

Comparison of magnetic properties of DNA-cetyltrimethyl ammonium complex with those of natural DNA

We prepared the DNA-cetyltrimethyl ammonium complex, as well as the same complex intercalated with stable organic free radicals, and studied their magnetic properties by electron magnetic resonance (EMR) spectroscopy and by measuring the magnetization on a superconducting quantum interference device (SQUID). The UV-vis and CD spectra of DNA-quaternary alkyl ammonium complex (DNA−-Q+) in organic solvent clearly demonstrated that it retained the double helical B-form conformation. The interhelical spacing of double strand DNA (dsDNA) increased when the counter ions (Na+) of phosphate groups of the natural DNA were replaced with the long alkyl quaternary ammonium groups. The inter-helical distance of DNA-cetyltrimethyl ammonium (CTMA) was 39.1 A as confirmed by X-ray diffractometry. In general, the magnetization of the DNA-CTMA complex solid was found to be significantly lower than that of natural DNA. Moreover, intercalation of the complex with stable organic free radicals did not improve magnetization, which again was in marked contrast to natural DNA. EMR spectroscopic behavior of the complex in the solid state also was quite different from that of natural DNA: The unique broad EMR signal of natural DNA in the low field region with g-value greater than 10 disappeared in the DNA-CTMA complex.

Synthesis of a new stable and water-soluble tris(4-hydroxysulfonyltetrachlorophenyl)methyl radical with selective oxidative capacity

A new stable organic free radical of the PTM (perchlorotriphenylmethyl) series very soluble in water is reported. This free radical is sensitive to electron transfer processes, and the selectivity of these reactions in the presence of ascorbic acid, pyrogallol, and catechol as reducing species is described. The electron paramagnetic resonance spectrum and the electrochemical behavior are also presented.

Oxidant Activity of Tris(2,4,6-trichloro-3,5-dinitrophenyl)methyl Radical with Catechol and Pyrogallol. Mechanistic Considerations

The reducing activity of simple polyphenols (PhOH), catechol and pyrogallol, is tested in different solvents in front of tris(2,4,6-trichloro-3,5-dinitrophenyl)methyl (HNTTM) radical, a stable organic free radical of the TTM series. HNTTM radical is very active in electron-transfer reactions to give a very stable anion. The standard potential for the reduction of HNTTM radical by cyclic voltammetry in different solvents is E(o) = 0.60 +/- 5 V vs SCE. In hydroxylic solvents, the electron transfer is a very rapid process and the electron-donating species is the ionized PhO(-), whereas in nonpolar solvents, it is suggested that the electron transfer is facilitated by the formation of an intermediate complex between HNTTM and PhOH.

Encapsulation of a Highly Sensitive EPR Active Oxygen Probe into Sonochemically Prepared Microspheres

High-power ultrasound (20 kHz) was used to encapsulate a solution of perchlorotriphenylmethyl triester (PTM-TE, a stable organic free radical) dissolved in hexamethyldisiloxane (HMDS) into a polymerized shell of bovine serum albumin (BSA). The size distribution of the microspheres was between 0.5 and 3 microm with a maximum at approximately 1.2 microm. The electron paramagnetic resonance spectrum of PTM-TE consists of a single, sharp line which is sensitive to the surrounding concentration of oxygen. It was found that the technique of encapsulating a solution of PTM-TE dissolved in HMDS into the BSA microspheres resulted in an overall loss of EPR signal intensity from the washed suspension of microspheres. However, the encapsulated PTM-TE/HMDS solution remained sensitive to the partial pressure of oxygen in the surrounding environment. The microspheres were found to be useful for determining the partial pressure of oxygen in the muscle and tumor tissue of mice.

Solid-state magnetic resonance studies of polyaniline as a radical scavenger

The first detailed study of polyaniline (PANI) and reduced PANI (R-PANI) before and after reaction with the stable organic free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) by using solidstate13C CP MAS (cross-polarization magic-angle spinning), NQS (nonquaternary suppressed) and CPPI (cross-polarization phase inversion) nuclear magnetic resonance techniques is reported. These techniques reveal relatively subtle structural changes that occur upon reaction with DPPH free radicals and confirm the radical scavenging ability of PANI. The integrated intensity of the PANI CP MAS spectrum after reaction with DPPH is about 8% larger than that of PANI and this cannot be explained by an oxidation process alone. The increase is attributed to a decrease in the polaron concentration in the polymer, which is consistent with electron paramagnetic resonance (EPR) data that show a decrease of the EPR signal intensity after reaction of chemically synthesized PANI with DPPH.

Use of Nitroxide Spin Probes and Electron Paramagnetic Resonance for Assessing Reducing Power of Beer. Role of SH Groups

Intensity of EPR spectra of stable organic free radicals, nitroxides, is decreasing with time if the radicals are dissolved in beer. The process is determined by a chemical reaction of nitroxide reduction by components naturally present in beer. Kinetics can be described as a simple irreversible first order with respect to both nitroxide and one reducing agent. Effective concentration of the reducing agent and the corresponding reaction rate constant has been determined. It is demonstrated that the nitroxide reduction is sensitive to the presence of solvent-accessible SH groups of proteins present in beer. It is proposed that quantitative analysis of reduction kinetics of small water-soluble nitroxide radicals such as TEMPO and TEMPOL can be used to assess the reducing power of beer. The effect of accelerated aging of beer achieved at elevated temperatures on nitroxide reduction kinetics is demonstrated.

Reducing Activity of Polyphenols with Stable Radicals of the TTM Series. Electron Transfer versus H-Abstraction Reactions in Flavan-3-ols

A new method to test the antioxidant activity of polyphenols by electron transfer reactions to a stable organic free radical, tris(2,4,6-trichloro-3,5-dinitrophenyl)methyl radical (HNTTM), is reported. Therefore, the activity of the natural flavanols, (-)-epicatechin, and two synthetic derivatives, 4beta-(S-cysteinyl)epicatechin and 4beta-(2-aminoethylthio)epicatechin, can be differentiated by their capacity to transfer hydrogen atoms to 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and to transfer electrons to HNTTM. [structure: see text]

A Stable Organic Free Radical in Anaerobic Benzylsuccinate Synthase of Azoarcus sp. Strain T

The novel enzyme benzylsuccinate synthase initiates anaerobic toluene metabolism by catalyzing the addition of toluene to fumarate, forming benzylsuccinate. Based primarily on its sequence similarity to the glycyl radical enzymes, pyruvate formate-lyase and anaerobic ribonucleotide reductase, benzylsuccinate synthase was speculated to be a glycyl radical enzyme. In this report we use EPR spectroscopy to demonstrate for the first time that active benzylsuccinate synthase from the denitrifying bacterium Azoarcus sp. strain T harbors an oxygen-sensitive stable organic free radical. The EPR signal of the radical was centered at g = 2.0021 and was characterized by a major 2-fold splitting of about 1.5 millitesla. The strong similarities between the EPR signal of the benzylsuccinate synthase radical and that of the glycyl radicals of pyruvate formate-lyase and anaerobic ribonucleotide reductase provide evidence that the benzylsuccinate synthase radical is located on a glycine residue, presumably glycine 828 in Azoarcus sp. strain T benzylsuccinate synthase.

Magneto-Structural Correlation in Two Isomeric Series of Nitronyl Nitroxide Molecular Magnets: Intermolecular Interactions Relevant to Ferromagnetic Exchange in Naphthyl and Quinolyl Derivatives

Two isomeric series of stable organic free radicals, 1- and 2-naphthyl nitronyl nitroxide (1- and 2-NAPNN) and 2-, 3-, and 4-quinolyl nitronyl nitroxide (2-, 3-, and 4-QNNN), were synthesized and their crystal structures were determined by X-ray diffraction. The crystal structure of 1-NAPNN consists of a three-dimensional (3D) molecular network formed by several atomic contacts between the O atom of the ONCNO group and the aromatic ring as well as between the O atom and the methyl groups. Since 1-NAPNN shows intra- and interlayer ferromagnetic (FM) intermolecular interactions with exchange coupling constants J/k=0.09 K and J′/k=0.03 K (H=−2J∑SiSj), respectively, these contacts are relavant to the FM intermolecular interaction. In contrast, 4-QNNN, having a molecular structure close to that of 1-NAPNN, contains molecular chains with alternating antiferromagnetic (AFM) intermolecular interaction with J/k=−7.8 K and α=0.5 [H=−2J∑(S2i−1S2i+ αS2iS2i+1)]. The crystal structures of 2-NAPNN, 2-QNNN, and 3-QNNN consist of 2D molecular layers formed through contacts similar to those observed in 1-NAPNN. The radical molecules are oriented in the same direction forming a herringbone-like arrangement in the layers. 2-NAPNN exhibits interlayer AFM interaction with J′/k=−0.31 K in addition to FM interaction with J/k=0.14 K in the layer, whereas 2- and 3-QNNN show 3D FM intermolecular interactions with J/k=0.018 K, J′/k=0.01 K (2-QNNN) and J/k=0.12 K, J′/k=0.03 K (3-QNNN). Each layer in 2-NAPNN is related to adjacent layers by twofold screw symmetry to yield atomic contacts between the naphthyl rings standing opposite across the interface of the layers. Since the contacts between the aromatic rings of these nitronyl nitroxides favor AFM intermolecular interaction while the contacts within the 2D layer favor FM interaction, 2-NAPNN shows overall AFM behavior. In contrast, the crystals of 2- and 3-QNNN have no such twofold axis and hence the 2D FM layers are connected through interlayer contacts, which result overall in FM behavior. Possible origins of the characteristic molecular arrangements that give rise to FM intermolecular interaction in the 2D layer are discussed in terms of hydrogen-bond formation between the O atom of the ONCNO group and the methyl groups of the nitronyl nitroxide moieties.

Single-Crystal Multifrequency EPR Evidence for a Quasi-Low-Dimensional Spin Exchange in 3-n-Butyl-2,4,6-Triphenylverdazyl

Verdazyls, 1,2,3,4-tetrahydro-s-tetrazin-1-(2H-yls), are a series of stable organic free radicals some of which in solid form exhibit ferromagnetism and spin-Peierls transitions. Molecular mechanisms responsible for these properties remain somewhat unclear. This study reports on the growth of single crystals of several substituted verdazyls and multifrequency (1.4−94.3 GHz) EPR measurements on single crystals of 3-n-butyl-2,4,6-triphenylverdazyl, a model compound in the series. The angular, temperature, and magnetic field dependence of the EPR line width and the temperature dependence of the g-factor anisotropy provide direct evidence of a pseudo-low-dimensional spin diffusion in the crystal lattice. Analysis of the EPR line width for the crystal, oriented with the c-axis at the magic angle, θ ≈ 54.7° with respect to the external magnetic field, indicates a weak logarithmic dependence upon the frequency of the EPR experiment typical for a quasi-two-dimensional spin system.

The occurrence of a stable quinone radical accumulating in vivo during natural and induced senescence in a range of plants

SynopsisA correlation has been firmly established, in a wide range of plants, between environmental stress, the onset of senescence, loss of viability in seeds and the development and accumulation of a stable organic free radical. On the basis of the EPR response obtained at 95 GHz (W-band) and ENDOR spectra, and comparisons with quinone radical anions, we present evidence from contrasted plant species, plant tissues and sub-cellular fractions that this stable radical originates from one or more quinones possibly, though perhaps not exclusively, associated with stressed or age-impaired photosynthetic and respiratory electron transport chains. The radical appears to be ubiquitously associated with sub-lethal stress-induced damage and with senescence and arises during the sub-cellular structural and biochemical processes associated with the final phases of metabolism prior to death. As the free radical persists for some considerable time after death, it may have value in long-term studies of seed viability and in broader areas of plant pathology and stress physiology.

Thermal decomposition of Athabasca tar sand and bitumen

A study has been made of the thermal cracking of Athabasca tar sand and bitumen in an inert atmosphere of He at temperatures between 350°C and 550°C. The products were collected in a cold water trap and analysed by gas chromatography (G.C.). ESR studies were also made of the residue. The G.C. analysis showed that n-paraffinic hydrocarbons from C 5 to at least C 21 were present in the liquid product. At temperatures greater than 400°C, the concentration of n-paraffins (C 10C 21) decreased with the increase in their molecular weight. No significant effect of the inorganic matrix of tar sand was noticed on the products obtained when compared with the products obtained from bitumen. The number of spins/g of the stable organic free radical in the residue was directly related to the formation of n-paraffins. The maximum amount of n-paraffins was formed when tar sand and bitumen were cracked at 450°C. The residue obtained from tar sand at temperatures greater than 450°C gave a broad resonance at g ≅ 2.267 and width, ΔH pp = 1400 G. The physical structure of the residue obtained on the thermal cracking of tar sand and bitumen was found by reflected light microscopy and found to depend on the cracking temperature.