epr测自由基

We explored the capability of low-frequency Electron Paramagnetic Resonance (EPR) to noninvasively detect melanin (a stable semiquinone free radical) in the human skin. As previous in vitro studies on biopsies suggested that the EPR signal from melanin was different when measured in skin melanomas or benign nevi, we conducted a prospective first-in-man clinical EPR study in patients with skin lesions suspicious of melanoma. EPR spectra were obtained using a spectrometer operating at 1 GHz, with a surface coil placed over the area of interest. Two clinical studies were carried out: 1) healthy volunteers (n = 45) presenting different skin phototypes; 2) patients (n = 88) with skin lesions suspicious of melanoma (n = 100) requiring surgical resection. EPR data obtained before surgery were compared with histopathology results. The method was not sensitive enough to measure differences in melanin content due to changes in skin pigmentation. In patients, 92% of the spectra were analyzable. The EPR signal of melanin was significantly higher (p < 0.0001) in melanoma lesions (n = 26) than that in benign atypical nevi (n = 62). A trend toward a higher signal intensity (though not significant) was observed in high Breslow depth melanomas (a marker of skin invasion) than in low Breslow lesions. To date, no naturally occurring free radicals have been detected by low-frequency EPR systems adapted for clinical studies. Here, we demonstrated for the first time the ability of this technology to detect an endogenous free radical, opening new avenues for evaluating clinical EPR as a potential aid in the diagnosis of pigmented skin lesions.

The purpose of this study was to examine the free radical scavenging and antioxidant activities of ellagic acid (EA) and ellagic acid peracetate (EAPA) by measuring their reactions with the radicals, 2,2-diphenyl-1-picrylhydrazyl and galvinoxyl using EPR spectroscopy. We have also evaluated the influence of EA and EAPA on the ROS production in L-6 myoblasts and rat liver microsomal lipid peroxidation catalyzed by NADPH. The results obtained clearly indicated that EA has tremendous ability to scavenge free radicals, even at concentration of 1 µM. Interestingly even in the absence of esterase, EAPA, the acetylated product of EA, was also found to be a good scavenger but at a relatively slower rate. Kinetic studies revealed that both EA and EAPA have ability to scavenge free radicals at the concentrations of 1 µM over extended periods of time. In cellular systems, EA and EAPA were found to have similar potentials for the inhibition of ROS production in L-6 myoblasts and NADPH-dependent catalyzed microsomal lipid peroxidation.

The quantitative measurement of free radicals in liquid using an X-band electron paramagnetic resonance (EPR) was systematized. Quantification of free radicals by EPR requires a standard sample that contains a known spin amount/concentration. When satisfactory reproducibility of the sample material, volume, shape, and positioning in the cavity for EPR measurements can be guaranteed, a sample tested and a standard can be directly compared and the process of quantification can be simplified. The purpose of this study was to simplify manual quantitative EPR measurement. A suitable sample volume for achieving a stable EPR intensity was estimated. The effects of different solvents on the EPR sensitivity were compared. The stability and reproducibility of the EPR intensity of standard nitroxyl radical solutions were compared among different types of sample tubes. When the sample tubes, sample volumes, and/or solvents were the same, the EPR intensity was reproduced with an error of 2% or less for μM samples. The quantified sample and the standard sample in the same solvent and the same volume drawn into the same sample tube was able to be directly compared. The standard sample for quantification should be measured just before or after every daily experiment.

Peroxymonosulfate (PMS)-based advanced oxidation processes (AOPs) represent an effective method for the remediation of antibiotic-contaminated soils. In this study, a natural pyrite-biochar composite material (FBCx) was developed, demonstrating superior activation performance and achieving a 76% removal rate of SMX from soil within 120 min. There existed different degradation mechanisms for SMX in aqueous and soil solutions, respectively. The production of 1O2 and inherent active species produced by soil slurry played an important role in the degradation process. The combination of electron paramagnetic resonance (EPR) and free radical probe experiments confirmed the presence of free radical transformation processes in soil. Wherein, the·OH and SO4·- generated in soil slurry did not directly involve in the degradation process, but rather preferentially reacted with soil organic matter (SOM) to form alkyl-like radicals (R·), thereby maintaining a high concentration of reactive species in the system. Furthermore, germination and growth promotion of mung bean seeds observed in the toxicity test indicated the environmental compatibility of this remediation method. This study revealed the influence mechanism of SOM in the remediation process of contaminated soil comprehensively, which possessed enormous potential for application in practical environments.

The influence of heating at a temperature of 50 °C and UV-irradiation of propolis drops and spray on their free radical scavenging activity was determined. The kinetics of interactions of the propolis samples with DPPH free radicals was analyzed. Interactions of propolis drops and propolis spray with free radicals were examined by electron paramagnetic resonance spectroscopy. A spectrometer generating microwaves of 9.3 GHz frequency was used. The EPR spectra of the model DPPH free radicals were compared with the EPR spectra of DPPH in contact with the tested propolis samples. The antioxidative activity of propolis drops and propolis spray decreased after heating at the temperature of 50 °C. A UV-irradiated sample of propolis drops more weakly scavenged free radicals than an untreated sample. The antioxidative activity of propolis spray increased after UV-irradiation. The sample of propolis drops heated at the temperature of 50 °C quenched free radicals faster than the unheated sample. UV-irradiation weakly changed the kinetics of propolis drops or spray interactions with free radicals. EPR analysis indicated that propolis drops and spray should not be stored at a temperature of 50 °C. Propolis drops should not be exposed to UV-irradiation.

Nitrogen fixation is essential for the sustainable development of both human society and the environment. Due to the chemical inertness of the N≡N bond, the traditional Haber-Bosch process operates under extreme conditions, making nitrogen fixation under ambient conditions highly desirable but challenging. In this study, we present an ultrasonic atomizing microdroplet method that achieves nitrogen fixation using water and air under ambient conditions in a rationally designed sealed device, without the need for any catalyst. The total nitrogen fixation rate achieved is 6.99 μmol/h, yielding ammonium as the reduction product and nitrite and nitrate as the oxidation products, with hydrogen peroxide produced as a byproduct at a rate of 4.29 μmol/h. Using electron paramagnetic resonance (EPR) spectroscopy, we captured reactive species, including hydrogen, hydroxyl, singlet oxygen, superoxide anion, and NO radicals. In conjunction with in situ mass spectrometry (MS) and isotope labeling, we confirmed the presence of nitrogen-containing intermediates, such as HN═NOH+•, H2N-N(OH)2+•, HNO+, and NH2OH+•. Supported by these findings and theoretical calculations, we propose a radical-mediated nitrogen disproportionation mechanism. Simulations of naturally occurring condensed microdroplets also demonstrated nitrogen redox fixation. This microdroplet-based method not only offers a potential pathway for nitrogen fixation in practical applications and sustainable development but also deepens our understanding of the natural nitrogen cycle.

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The purpose of this study was to estimate the effect of chlorpromazine on free radical concentration in HEMn-DP melanocytes using electron paramagnetic resonance (EPR) spectroscopy. It was found that chlorpromazine at concentrations of 1 × 10−7 and 1 × 10−6 M contributed to the formation of free radicals (g values ~2) in a dose-dependent manner. The increase in free radical formation was accompanied by an increase in cytotoxicity, as shown by a tetrazolium assay. Homogeneous broadening of EPR lines, slow spin–lattice relaxation processes, and strong dipolar interactions characterized all the tested cellular samples. The performed examination of free radical formation in cells exposed to different chlorpromazine concentrations confirmed the usefulness of electron paramagnetic resonance spectroscopy to determine the effect of a drug on free radical production in a cellular model system in vitro.

In peaches, phenolic compounds are the major sources of antioxidants, and cyanidin-3-O-glucoside is the main anthocyanin present, above all in the skin. Anthocyanin content has been shown to increase after UV-B irradiation, which may be very harmful for all biological organisms due to the induction of the generation of reactive oxygen species (ROS). Peach fruits (cv. 'Suncrest') were exposed during post-harvest to supplemental ultraviolet-B radiation. A spin-trapping technique was used to monitor the generation of free radicals under UV-B, and 5-(diethoxy-phosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) was used as the spin trap. The flesh of peaches was essentially unaffected by the treatment, whereas the skin was responsive at the end of the treatment, accumulating ascorbate, flavonoids, cyanidin-3-O-glucoside, and showing a higher antioxidant activity. The levels of stable free radicals were also lower at the end of treatment. Carbon-centred radicals contributed the most to the total amounts of free radicals, whereas hydroxyl radicals and oxygen-centred free radicals contributed minimally. The carbon-centred free radical identified was the same as the one obtained after irradiation of authentic cyanidin-3-O-glucoside. During UV-B treatment cyanidin-3-O-glucoside increased and was capable of radicalization protecting the other organic molecules of the cell from oxidation. ROS, among which hydroxyl radicals, were thus maintained to minimal levels. This ability of cyanidin-3-O-glucoside displayed the mechanism underlined the tolerance to UV-B irradiation indicating that shelf life can be prolonged by the presence of anthocyanins. Thus, UV-B technique results a good approach to induce antioxidant production in peach fruits increasing their nutraceutical properties.

The interaction between CuII, FeIII and MnII complexes, derived from the ligands 1-[bis(pyridine-2-ylmethyl)amino]-3-chloropropan-2-ol (hpclnol) and bis(pyridine-2-ylmethyl)amine (bpma), and the free radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH) and reactive oxygen species (ROS), was investigated by colorimetric and EPR (Electron Paramagnetic Resonance) techniques. A comparison between these results and those reported to [Mn(salen)Cl] or EUK-8 was also addressed. EPR studies allowed us the identification of intermediates species such as superoxide‑copper(I) and superoxide‑copper(II), a mixed-valence FeIIIFeII species and a 16-line feature attributed to MnIII-oxo-MnIV species. The biomarker malondialdehyde (MDA) was determined by TBARS assay in S. cerevisiae cells, and the determination of the IC50 indicate that the antioxidant activity shown dependence on the metal center (CuII ≈ FeIII > MnII ≈ [Mn(salen)Cl]. The lipid peroxidation attenuation was also investigated in liver homogenates obtained from Swiss mice and the IC50 values were in the nanomolar concentrations. We demonstrated here that all the complexes interact with the free radical DPPH and with ROS (H2O2, O2•- and hydroxyl radical), enhancing the cellular protection against oxidative stress generated by hydroxyl radical, employing two experimental model systems, S. cerevisiae (in vivo) and mouse liver (ex vivo).

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With conventional cigarettes, the burning cone reaches temperatures of >900°C resulting in the production of numerous toxicants and significant levels of highly reactive free radicals. In attempts to eliminate combustion while still delivering nicotine and flavorings, a newer alternative tobacco product has emerged known as "heat-not-burn" (HnB). These products heat tobacco to temperatures of 250-350°C depending on the device allowing for the volatilization of nicotine and flavorants while potentially limiting the production of combustion related toxicants. To better understand how the designs of these new products compare to conventional cigarettes and different styles of electronic cigarettes (e-cigs), we measured and partially characterized their production of free radicals. Smoke or aerosols were trapped by a spin trap phenyl-N-tert-butylnitrone (PBN) and analyzed for free radicals using electron paramagnetic resonance (EPR). Free radical polarity was assessed by passing the aerosol or smoke through either a polar or non-polar trap prior to being spin trapped with PBN. Particulate-phase radicals were detected only for conventional cigarettes. Gas-phase free radicals were detected in smoke/aerosol from all products with levels for HnB (IQOS, Glo) (12 pmol/puff) being similar to e-cigs (Juul, SREC, box mod e-cig) and hybrid devices (Ploom) (5-40 pmol/puff) but 50-fold lower than conventional cigarettes (1R6F). Gas phase radicals differed in polarity with HnB products and conventional cigarettes producing more polar radicals compared to those produced from e-cigs. Free radical production should be considered in evaluating the toxicological profile of nicotine delivery products and identification of the radicals is of paramount importance.

The aim of this study was to investigate the inhibitory effect of non-precursors amino acids (histidine, leucine, proline and methionine) which have advantages of safety, inexpensiveness and high standardization on the formation of β-carbolines in roast beef patties and glucose/creatine/creatinine/tryptophan model system, and the possible pathway of inhibition by monitoring the scavenging of free radicals by electron paramagnetic resonance (EPR) spectroscopy and the consumption of tryptophan by HPLC in a glucose/tryptophan model system. Almost all amino acids can inhibit β-carbolines in roast beef patties (up to 80.62%) and model system (up to 67.01%). Histidine showed an excellent alkyl radical scavenging ability (up to 82.59%) and a highly competitive inhibition ability (up to 65.60%) against β-carbolines generation. The corresponding abilities of leucine and methionine were less remarkable. Proline could only suppress β-carbolines through competitive inhibition. The results could shed light on the reduction of β-carbolines during meat processing.

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Aerosols from electronic cigarettes and heat-not-burn tobacco products have been found to contain lower levels of almost all compounds from the list of Harmful and Potentially Harmful Constituents known to be present in tobacco products and tobacco smoke than smoke from conventional cigarettes. Free radicals, which also pose potential health risks, are not considered in this list, and their levels in the different product types have not yet been compared under standardized conditions. We compared the type and quantity of free radicals in mainstream aerosol of 3R4F research cigarettes, two types of electronic cigarettes, and a heat-not-burn tobacco product. Free radicals and NO in the gas phases were separately spin trapped and quantified by electron paramagnetic resonance (EPR) spectroscopy by using a smoking machine for aerosol generation and a flow-through cell to enhance reproducibility of the quantification. Particulate matter was separated by a Cambridge filter and extracted, and persistent radicals were quantified by EPR spectroscopy. Levels of organic radicals for electronic cigarettes and the heat-not-burn product, as measured with the PBN spin trap, did not exceed 1% of the level observed for conventional cigarettes and were close to the radical level observed in air blanks. The radicals found in the smoke of conventional cigarettes were oxygen centered, most probably alkoxy radicals, whereas a signal for carbon-centered radicals near the detection limit was observed in aerosol from the heat-not-burn product and electronic cigarettes. The NO level in aerosol produced by electronic cigarettes was below our detection limit, whereas for the heat-not-burn product, it reached about 7% of the level observed for whole smoke from 3R4F cigarettes. Persistent radicals in particulate matter could be quantified only for 3R4F cigarettes. Aerosols from vaping and heat-not-burn tobacco products have much lower free radical levels than cigarette smoke, however, the toxicological implications of this finding are as yet unknown.

Persulfate-based in situ chemical oxidation (ISCO) for soil remediation has received great attention in recent years. However, the mechanisms of interaction between persulfate (PS) and soil constituents are not fully understood. In this study, PS decomposition, activation, free radical formation and conversion processes in 10 different soils were examined. The results showed that soil organic matter (SOM) was the dominant factor affecting PS decomposition in soil, but Fe/Mn-oxides were mainly responsible for PS decomposition when SOM was removed. Electron paramagnetic resonance (EPR) spectroscopy analysis showed that sulfate radicals (SO4•-) and hydroxyl radicals (•OH) generated from PS decomposition subsequently react with SOM to produce alkyl-like radicals (R•), and this process is dependent on SOM content. R• and SO4•-/•OH radicals predominated in soil with high and low SOM, respectively, and all three radicals coexist in soil with medium SOM. Chemical probe analysis further identified the types of radicals, and R• can reductively degrade hexachloroethane in high SOM soil, while SO4•- and •OH oxidatively degrade phenol in low SOM soil. These findings provide valuable information for PS-ISCO, and new insight into the role of SOM in the remediation of contaminated soil.

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Dye decolouring peroxidases (DyPs) are the most recent class of heme peroxidase to be discovered. On reacting with H2 O2 , DyPs form a high-valent iron(IV)-oxo species and a porphyrin radical (Compound I) followed by stepwise oxidation of an organic substrate. In the absence of substrate, the ferryl species decays to form transient protein-bound radicals on redox active amino acids. Identification of radical sites in DyPs has implications for their oxidative mechanism with substrate. Using a DyP from Streptomyces lividans, referred to as DtpA, which displays low reactivity towards synthetic dyes, activation with H2 O2 was explored. A Compound I EPR spectrum was detected, which in the absence of substrate decays to a protein-bound radical EPR signal. Using a newly developed version of the Tyrosyl Radical Spectra Simulation Algorithm, the radical EPR signal was shown to arise from a pristine tyrosyl radical and not a mixed Trp/Tyr radical that has been widely reported in DyP members exhibiting high activity with synthetic dyes. The radical site was identified as Tyr374, with kinetic studies inferring that although Tyr374 is not on the electron-transfer pathway from the dye RB19, its replacement with a Phe does severely compromise activity with other organic substrates. These findings hint at the possibility that alternative electron-transfer pathways for substrate oxidation are operative within the DyP family. In this context, a role for a highly conserved aromatic dyad motif is discussed.

ABSTRACT Sun radiation is indispensable to our health, however, a long term and high exposure could lead to erythema, premature skin aging and promotion of skin tumors. An underlying pathomechanism is the formation of free radicals. First, reactive oxygen species (*OH, *O2‐) and then, secondary lipid oxygen species (C centered radicals, CCR) are formed. A high amount of free radicals results in oxidative stress with subsequent cell damage. In dermatological research different skin models are used, however, comparative data about the cutaneous radical formation are missing. In this study, the radical formation in porcine‐, (SKH‐1) murine‐, human‐ ex vivo skin and reconstructed human skin (RHS) were investigated during simulated sun irradiation (305–2200 nm), with X‐band EPR spectroscopy. The amount of radical formation was investigated with the spin probe PCA exposed to a moderate sun dose below one minimal erythema dose (MED, ˜25 mJ/cm2 UVB) in all skin models. Furthermore, the *OH and *CCR radical concentrations were measured with the spin trap DMPO within 0–4 MED (porcine‐, human skin and RHS). The highest amount of radicals was found in RHS followed by murine and porcine, and the lowest amount in human ex vivo skin. In all skin models, more *OH than CCR radicals were found at 0–4 MED. Additionally, this work addresses the limitations in the characterization with the spin trap DMPO. The measurements have shown that the most comparable skin model to in vivo human skin could differ depending on the focus of the investigation. If the amount of radial production is regarded, RHS seems to be in a similar range like in vivo human skin. If the investigation is focused on the radical type, porcine skin is most comparable to ex vivo human skin, at an irradiation dose not exceeding 1 MED. Here, no comparison to in vivo human skin is possible. Graphical abstract Figure. No Caption available. HighlightsRadical quantification and characterization in skin models is measurable by EPR.RHS shows the highest cumulative radical production, comparable to human skin in vivo.All investigated skin models showed more *OH radicals than *CCR during irradiation.Porcine skin is comparable to ex vivo human skin concerning the ratio of radical types.

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ABSTRACT Molecular oxygen, reactive oxygen species and free radicals derived from oxygen play important roles in a broad spectrum of physiological and pathological processes. The quantitative measurement of molecular oxygen in tissues by electron paramagnetic resonance (EPR) has great potential for understanding and diagnosing a number of diseases, and for developing and guiding therapies. This requires improvements in the free radical probe systems that sense and report molecular oxygen levels in vivo. We report on the encapsulation of existing free radical probes in lipophilic gel implants: an in‐situ‐oleogel and an emulgel, based only on well‐known, safe excipients for the incorporation of lipophilic and hydrophilic radicals, respectively. The EPR signals of encapsulated radicals were not altered compared to dissolved radicals. The high solubility of oxygen in lipophilic solvents enhanced oxygen sensitivity. The gels extended the lifetime of the radicals in tissues from tens of minutes to many days, simplifying studies with extended series of measurements. The encapsulated radicals showed a good in vivo response to changes in oxygen supply and seem to circumvent concerns from toxicity of the radical probes. These gels simplify the development of new oxygen‐sensitive free radical probes for EPR oximetry by making their in vivo stability, persistence and toxicity a function of the encapsulating gel and not a set of additional requirements for the free radical probe. HighlightsHydrophilic and lipophilic trityl radicals were encapsulated in gels.The gel constituents were known, safe pharmaceutical excipients.The encapsulation of radicals did not alter their linewidths.The gel formulations were shown to be suitable for local oxygen measurements in vivo.EPR signals were detectable up to three weeks after injection. Graphical abstract Figure. No Caption available.

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Microbubbles are very fine bubbles that shrink and collapse when underwater within several minutes, leading to the generation of free radicals. Electron spin resonance spectroscopy (ESR) confirmed the generation of hydroxyl radicals under strongly acidic conditions. The drastic environmental change caused by the collapse of the microbubbles may trigger radical generation via the dispersion of the elevated chemical potential that had accumulated around the gas-water interface. The present study also confirmed the generation of ESR signals from the microbubble-treated waters even after several months had elapsed following the dispersion of the microbubbles. Bulk nanobubbles were expected to be the source of the spin-adducts of hydroxyl radicals. Such microbubble stabilization and conversion might be caused by the formation of solid microbubble shells generated by iron ions in the condensed ionic cloud around the microbubble. Therefore, the addition of a strong acid might cause drastic changes in the environment and destroy the stabilized condition. This would restart the collapsing process, leading to hydroxyl radical generation.

In this study, 8% hydrogen (H2) in argon (Ar) and carbon dioxide (CO2) gas nanobubbles was produced at 10, 30, and 50 vol.% of ethanol aqueous solution by the high-speed agitation method with gas. They became stable for a long period (for instance, 20 days), having a high negative zeta potential (−40 to −50 mV) at alkaline near pH 9, especially for 10 vol.% of ethanol aqueous solution. The extended Derjaguin, Landau, Verwey, and Overbeek (DLVO) theory was used to evaluate the nanobubble stability. When the nanobubble in ethanol alkaline aqueous solution changed to an acidic pH of around 5, the zeta potential of nanobubbles was almost zero and the decrease in the number of nanobubbles was identified by the particle trajectory method (Nano site). The collapsed nanobubbles at zero charge were detected thanks to the presence of few free radicals using G-CYPMPO spin trap reagent in electron spin resonance (ESR) spectroscopy. The free radicals produced were superoxide anions at collapsed 8%H2 in Ar nanobubbles and hydroxyl radicals at collapsed CO2 nanobubbles. On the other hand, the collapse of mixed CO2 and H2 in Ar nanobubble showed no free radicals. The possible presence of long-term stable nanobubbles and the absence of free radicals for mixed H2 and CO2 nanobubble would be useful to understand the beverage quality.

The main findings are the hydroxyl radical scavenging and the superoxide anion diminishing by mixing the carbon dioxide (CO2) nanobubbles after hydrogen nanobubble blowing in water and alcohol aqueous solution. The nanobubbles produce the hydroxyl radical by ultrasonic waves, changing the pH and catalyst and so on, while the nanobubble is very reactive to scavenge free radicals. In this research especially hydrogen (4% H2 in argon) and CO2 nanobubbles have been blown into hydrogen peroxide (H2O2) added pure water, ethanol, and ethylene glycol aqueous solution through a porous ceramic sparger from the gas cylinder. The aqueous solutions with H2O2 are irradiated by ultraviolet (UV) light and the produced hydroxyl radical amount is measured with spin trapping reagent and electron spin resonance (ESR). The CO2 nanobubble blowing extremely has reduced the hydroxyl radical in water, ethanol, and ethylene glycol aqueous solution. On the other hand, when H2 nanobubbles are brown after CO2 nanobubble blowing, the hydroxyl radical amount has increased. For the disinfection test, the increase of hydroxyl radicals is useful to reduce the bacteria by the observation in the agar medium. Next, when the superoxide anion solution is mixed with nanobubble containing water, ethanol, and ethylene glycol aqueous solution, H2 nanobubble has reduced the superoxide anion slightly. The water containing both CO2 and H2 nanobubble reduces the superoxide anion. The less than 20% ethanol and the 30% ethylene glycol aqueous solution containing CO2 nanobubbles generated after H2 nanobubble blowing can diminish the superoxide anion much more. While the H2 nanobubble blowing after CO2 nanobubble blowing scavenges the superoxide anion slightly. The experimental results have been considered using a chemical reaction formula.

Although metal-free electrodes in molecular oxygen-activated Fenton-like wastewater treatment technologies have been developed, the reactive oxygen species (ROS) generation mechanisms are still not sufficiently clear. As a typical example of refractory phenolic wastewater, p-nitrophenol (PNP) has been widely studied. This study demonstrated the critical role of superoxide radicals (O2•-) in PNP degradation by metal-free electrodes through electron spin resonance (ESR), ROS quenching, and density functional theory (DFT) tests. The most superior metal-free electrode exhibited a mass activity of approximately 133.5 h-1 gcatalyst-1. Experimental and theoretical studies revealed the mechanism of O2•- generation via oxygen activation, including one- and three-electron transfer pathways, and found that O2•- mainly attacked the nitro group of PNP to degrade and transform the pollutant. This study enhances the mechanistic understanding of metal-free materials in the electrochemical degradation of refractory pollutants.

Secondary plant metabolites, e.g., polyphenols, are widely known as health-improving compounds that occur in natural functional foods such as pomegranates. While extracts generated from these fruits inhibit oxidative stress, the allocation of these effects to the different subgroups of substances, e.g., anthocyanins, “copigments” (polyphenols without anthocyanins), or polymeric compounds, is still unknown. Therefore, in the present study, polyphenols from pomegranate juice were extracted and separated into an anthocyanin and copigment fraction using adsorptive membrane chromatography. Phenolic compounds were determined by high performance liquid chromatography with photodiode array (HPLC–PDA) detection and HPLC-PDA electrospray ionization tandem mass spectrometry (HPLC–PDA–ESI–MS/MS), while the free radical scavenging activity of the pomegranate XAD‑7 extract and its fractions was evaluated by the Trolox equivalent antioxidant capacity (TEAC) assay and electron spin resonance (ESR) spectroscopy. Compared to juice, the total phenolic content and free radical scavenging potential was significantly higher in the pomegranate XAD-7 extract and its fractions. In comparison to the anthocyanin and copigment fraction, pomegranate XAD-7 extract showed the highest radical scavenging activity against galvinoxyl and DPPH radicals. Moreover, the enriched XAD-7 extract and its fractions were able to protect human hepatocellular HepG2 cells against oxidative stress induced by hydrogen peroxide. Overall, these results indicated that anthocyanins and copigments act together in reducing oxidative stress.

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Seahorses, Hippocampus abdominalis, have a long history in traditional Chinese medicine as an important healthy ingredient in foods. This study evaluated the antioxidant activity of an enzymatic hydrolysate prepared from a seahorse bred in Jeju, South Korea. Experiments were performed in vitro using electron spin resonance spectrometry (ESR) to determine the free radical scavenging activity and in vivo using a zebrafish model to determine the protective effects against 2,2-azobis hydrochloride (AAPH)-induced oxidative damage. H. abdominalis protein hydrolysate (HPH) exhibited peroxyl radical scavenging activity (IC50  = 0.58 mg/ml) generated by the water-soluble AAPH (azo initiator of peroxyl radicals). HPH reduced dose-dependently both intracellular reactive oxygen species (ROS) levels in AAPH-induced cells and cell death in AAPH-induced zebrafish embryos. The antioxidant peptide purified from HPH was identified as a tripeptide (alanine-glycine-aspartic acid) using Q-TOF ESI mass spectroscopy. Thus, this study demonstrated that HPH contains antioxidant peptides that exhibit a strong antioxidant activity. PRACTICAL APPLICATIONS: Hippocampus abdominalis is one of the largest seahorse species and cultivated in many countries. Because of its large body size compared to other seahorse species, H. abdominalis has acquired considerable consumer attraction in the global market. Owing to its biologically useful properties, it recently gained attention as the natural products obtained from H. abdominalis have varied applications in the field of medicine, health care products, and functional foods. Thus, commercial products of this particular seahorse species are popular among customers, especially in China. The purpose of this study was to evaluate the antioxidant property of H. abdominalism, cultured in a commercial seahorse farm in Jeju Island. Owing to its prominent antioxidant activity, it could be used as an ingredient in medicinal preparations, nutraceuticals, and functional foods.

The only general technique that allows the unambiguous detection of free radicals is electron spin resonance (ESR). However, ESR spin trapping has severe limitations especially in biological systems. The greatest limitation of ESR is poor sensitivity relative to the low steady-state concentration of free radical adducts, which in cells and in vivo is much lower than the best sensitivity of ESR. Limitations of ESR have led to an almost desperate search for alternatives to investigate free radicals in biological systems. Here we explore the use of the immuno-spin trapping technique, which combine the specificity of the spin trapping to the high sensitivity and universal use of immunological techniques. All of the immunological techniques based on antibody binding have become available for free radical detection in a wide variety of biological systems.

Starting from dansyl-chloride, in reaction with 1,1-diphenylhydrazine and methoxyamine, two new fluorescent derivatives 1 and 2 were obtained and characterized by NMR, IR, UV-Vis, HR-MS, and fluorescence spectroscopy. The single-crystal X-ray structure was obtained for compound 2. Both compounds generate free radicals by oxidation, as demonstrated by ESR spectroscopy. Compound 1 generates the corresponding hydrazyl-persistent free radical, evidenced directly by ESR spectroscopy, while compound 2 generates in the first instance the methoxyaminyl short-lived free radical, which decomposes rapidly with the formation of the methoxy radical, evidenced by the ESR spin-trapping technique. By oxidation of compounds 1 and 2, their fluorescence is quenched.

To achieve sustainable metal-free electron-Fenton, N self-doped biochar air-cathode (BCAC) was prepared by pyrolyzing coffee residues. During the pyrolysis process, the endogenous N transformed from edge-doping to graphite-doping. Particularly, N vacancies started to evolve when the peak temperature exceeded 700 °C. A high Tetracycline removal rate of 70.42% was obtained on the BCAC at the current density of 4 mA cm-2. Quenching tests incorporated with ESR spectroscopy were adopted to identify the specific oxidants produced on the cathode. The results showed that •OH (37.36%), •O2- (29.67%) and 1O2 (24.17%) played comparable role in the tetracycline removal, suggesting the coexist of radical and non-radical oxidants in our electro-Fenton system. According to the structure characterization and the DFT calculation, graphitic N was suggested as the critical site for H2O2 generation, and both graphitic N and pyridinic N were electroactive sites for H2O2 activation to •OH. Graphitic N and N vacancies with stronger capabilities in O2 adsorption and electron-trapping were proposed as the electroactive sites for 1O2 and •O2- formation. This work predicts a novel electro-Fenton process with cooperative radical and non-radical degradation on N self-doped carbonaceous catalysts at a mild condition, which is extremely meaningful for boosting sustainable electro-Fenton technology.

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Electron paramagnetic resonance (EPR) spectroscopy using sterically hindered amine is extensively applied to detect singlet oxygen (1O2) possibly generated in advanced oxidation processes. However, EPR-detectable 1O2 signals were observed in not only the 1O2-dominated hydrogen peroxide (H2O2)/hypochlorite (NaClO) reaction but surprisingly also the 1O2-absent Fe(II)/H2O2, UV/H2O2, and ferrate [Fe(VI)] process with even stronger intensities. By taking advantage of the characteristic reaction between 1O2 and 9,10-diphenyl-anthracene and near-infrared phosphorescent emission of 1O2, 1O2 was excluded in the Fe(II)/H2O2, UV/H2O2, and Fe(VI) process. The false detection of 1O2 was ascribed to the direct oxidation of hindered amine to piperidyl radical by reactive species [e.g., •OH and Fe(VI)/Fe(V)/Fe(IV)] via hydrogen transfer, followed by molecular oxygen addition (forming a piperidylperoxyl radical) and back reaction with piperidyl radical to generate a nitroxide radical, as evidenced by the successful identification of a piperidyl radical intermediate at 100 K and theoretical calculations. Moreover, compared to the highly oxidative species (e.g., •OH and high-valence Fe), the much lower reactivity of 1O2 and the profound nonradiative relaxation of 1O2 in H2O resulted it too selective and inefficient in organic contaminant destruction. This study demonstrated that EPR-based 1O2 detection could be remarkably misled by common oxidative species and thereby jeopardize the understandings on 1O2.

Depicting how biomolecules move and interact within their physiological environment is one of the hottest topics of structural biology. This Feature Article gives an overview of the most recent advances in Site-directed Spin Labeling coupled to Electron Paramagnetic Resonance spectroscopy (SDSL–EPR) to study biomolecules in living cells. The high sensitivity, the virtual absence of background, and the versatility of spin-labeling strategies make this approach one of the most promising techniques for the study of biomolecules in physiologically relevant environments. After presenting the milestones achieved in this field, we present a summary of the future goals and ambitions of this community.

Abstract Dipolar electron paramagnetic resonance (EPR) spectroscopy (DEER and other techniques) enables the structural characterization of macromolecular and biological systems by measurement of distance distributions between unpaired electrons on a nanometer scale. The inference of these distributions from the measured signals is challenging due to the ill-posed nature of the inverse problem. Existing analysis tools are scattered over several applications with specialized graphical user interfaces. This renders comparison, reproducibility, and method development difficult. To remedy this situation, we present DeerLab, an open-source software package for analyzing dipolar EPR data that is modular and implements a wide range of methods. We show that DeerLab can perform one-step analysis based on separable non-linear least squares, fit dipolar multi-pathway models to multi-pulse DEER data, run global analysis with non-parametric distributions, and use a bootstrapping approach to fully quantify the uncertainty in the analysis.

The exploration of heavy main-group radicals is rapidly expanding, for which electron paramagnetic resonance (EPR) spectroscopic characterisation plays a key role. EPR spectroscopy has the capacity to deliver information of the radical's electronic, geometric and bonding structure. Herein, foundations of electron-nuclear hyperfine analysis are detailed before reviewing more recent applications of EPR spectroscopy to As, Sb, and Bi centred radicals. Additional diverse examples of the application of EPR spectroscopy to other heavy main group radicals are highlighted.

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Energy-transducing respiratory complex I (NADH:ubiquinone oxidoreductase) is one of the largest and most complicated enzymes in mammalian cells. Here, we used hyperfine electron paramagnetic resonance (EPR) spectroscopic methods, combined with site-directed mutagenesis, to determine the mechanism of a single proton-coupled electron transfer reaction at one of eight iron-sulfur clusters in complex I, [4Fe-4S] cluster N2. N2 is the terminal cluster of the enzyme's intramolecular electron-transfer chain and the electron donor to ubiquinone. Because of its position and pH-dependent reduction potential, N2 has long been considered a candidate for the elusive "energy-coupling" site in complex I at which energy generated by the redox reaction is used to initiate proton translocation. Here, we used hyperfine sublevel correlation (HYSCORE) spectroscopy, including relaxation-filtered hyperfine and single-matched resonance transfer (SMART) HYSCORE, to detect two weakly coupled exchangeable protons near N2. We assign the larger coupling with A(1H) = [-3.0, -3.0, 8.7] MHz to the exchangeable proton of a conserved histidine and conclude that the histidine is hydrogen-bonded to N2, tuning its reduction potential. The histidine protonation state responds to the cluster oxidation state, but the two are not coupled sufficiently strongly to catalyze a stoichiometric and efficient energy transduction reaction. We thus exclude cluster N2, despite its proton-coupled electron transfer chemistry, as the energy-coupling site in complex I. Our work demonstrates the capability of pulse EPR methods for providing detailed information on the properties of individual protons in even the most challenging of energy-converting enzymes.

The contribution of copper complexes of salen-based Schiff bases N, N'-bis(salicylidene)ethylenediamine (C1), N, N'-bis(4-hydroxysalicylidene)ethylenediamine (C2), and N, N'-bis(5-hydroxysalicylidene)ethylenediamine (C3) to the flame retardancy of thermoplastic polyurethane (TPU) is investigated in the context of minimizing the inherent flammability of TPU. Thermal and fire properties of TPU are evaluated. It is observed that fire performances vary depending upon the substitution of the salen framework. Cone calorimetry [mass loss calorimetry (MLC)] results show that, in TPU at 10 wt % loading, C2 and C3 reduce the peak of heat release rate by 46 and 50%, respectively. At high temperature, these copper complexes undergo polycondensation leading to resorcinol-type resin in the condensed phase and thus acting as intumescence reinforcing agents. C3 in TPU is particularly interesting because it delays significantly the time to ignition (MLC experiment). In addition, pyrolysis combustion flow calorimetry shows reduction in the heat release rate curve, suggesting its involvement in gas-phase action. Structural changes of copper complexes and radical formation during thermal treatment as well as their influence on fire retardancy of TPU in the condensed phase are investigated by spectroscopic studies supported by microscopic and powder diffraction studies. Electron paramagnetic resonance (EPR) spectroscopy was fully used to follow the redox changes of Cu(II) ions as well as radical formation of copper complexes/TPU formulations in their degradation pathways. Pulsed EPR technique of hyperfine sublevel correlation spectroscopy reveals evolution of the local surrounding of copper and radicals with a strong contribution of nitrogen fragments in the degradation products. Further, the spin state of radicals was investigated by the two-dimensional technique of phase-inverted echo-amplitude detected nutation experiment. Two different radicals were detected, that is, one monocarbon radical and an oxygen biradical. Thus, the EPR study permits to deeply investigate the mode of action of copper salen complexes in TPU.

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The structures of the final products of peroxidatic oxidations (la, 2), the stoichiometries and relative rates of reduction of peroxidase Compounds I and II (3, 4), and the redox prop-erties of substrate intermediates (5) make it likely that the intermediates are free radicals. We have now obtained direct physical evidence, with electron paramagnetic resonance spectroscopy, that free radicals are formed from substrates in this enzymic oxidation-reduction.

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Nitric oxide, NO, exerts numerous important regulatory functions in biological tissues and has been hypothesized to have a role in the pathogenesis of cellular injury in a number of diseases. It has been suggested that alterations in NO generation are a critical cause of injury in the ischemic heart. However, the precise alterations in NO generation which occur are not known, and there is considerable controversy regarding whether myocardial ischemia results in increased or decreased NO formation. Therefore, electron paramagnetic resonance studies were performed to directly measure NO in isolated rat hearts subjected to global ischemia, using the direct NO trap Fe-N-methyl-D-glucamine dithiocarbamate, which specifically binds NO giving rise to a characteristic triplet EPR spectrum with g = 2.04 and aN = 13.2 G. While only a small triplet signal was observed in normally perfused hearts, a 10-fold increase in this triplet EPR spectrum was observed after 30 min of ischemia indicating a marked increase in NO formation and trapping. Measurements were performed as a function of the duration of ischemia, and it was determined that with increased duration of ischemia NO formation and trapping was also increased. NO generation was inhibited by the nitric oxide synthase blocker, N-nitro-L-arginine methyl ester (L-NAME), suggesting that NO was generated via nitric oxide synthase. Blockade of NO generation with L-NAME resulted in more than a 2-fold increase in the recovery of contractile function in hearts reperfused after 30 min of global ischemia. Thus, ischemia causes a marked duration-dependent increase of NO in the heart which may in turn mediate postischemic injury.

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Electron spin resonance (ESR) spectroscopy is a crucial tool, through spin labelling, in investigations of the chemical structure of materials and of the electronic structure of materials associated with unpaired spins. ESR spectra measured in molecular systems, however, are established on large ensembles of spins and usually require a complicated structural analysis. Recently, the combination of scanning tunnelling microscopy with ESR has proved to be a powerful tool to image and coherently control individual atomic spins on surfaces. Here we extend this technique to single coordination complexes—iron phthalocyanines (FePc)—and investigate the magnetic interactions between their molecular spin with either another molecular spin (in FePc–FePc dimers) or an atomic spin (in FePc–Ti pairs). We show that the molecular spin density of FePc is both localized at the central Fe atom and also distributed to the ligands (Pc), which yields a strongly molecular-geometry-dependent exchange coupling. Electron spin resonance spectroscopy has traditionally been used to study large ensembles of spins, but its combination with scanning tunnelling microscopy recently enabled measurements on single adatoms. Now, individual iron phthalocyanine complexes adsorbed on a surface have been probed. Their spin distribution partially extends on the phthalocyanine, leading to a strong geometry-dependent exchange coupling interaction.

Abstract Flavoproteins are a versatile class of proteins involved in numerous biological processes, including redox reactions, electron transfer, and signal transduction, often relying on their ability to stabilize different oxidation states of their flavin cofactor. A critical feature of flavin cofactors is their capacity to achieve, within particular protein environments, a semiquinone state that plays a pivotal role in mediating single-electron transfer events and that is key to understanding flavoprotein reactivity. Hyperfine interactions between the unpaired electron and magnetic nuclei in the isoalloxazine ring provide valuable insights into the semiquinone state and its mechanistic roles. This study investigates the hyperfine interactions of isotopically labeled flavodoxin (Fld) with 13C and 15N in specific positions of the flavin mononucleotide (FMN) ring using advanced electron paramagnetic resonance (EPR) techniques. The combination of continuous-wave (CW) EPR at the X-band and ELDOR-detected NMR and HYSCORE at the Q-band revealed a strong and anisotropic hyperfine interaction with the nucleus of 13C at 4a and yielded principal tensor values of 40, -13.5 , and -9 MHz, the first of which is associated with the axis perpendicular to the flavin plane. On the other hand, as predicted, the hyperfine interaction with the 13C nucleus in position 2 was minimal. Additionally, HYSCORE experiments on 15N -FMN-labeled Fld provided precise axial hyperfine parameters, i.e., (74, 5.6, 5.6) MHz for 15N (5) and (38, 3.2, 3.2) MHz for 15N (10). These were used to refine quadrupole tensor values for 14N nuclei through isotope-dependent scaling. These results showcase the potential of combining CW EPR, ELDOR-detected NMR, and HYSCORE with isotopic labeling to probe electronic and nuclear interactions in flavoproteins. The new data complete and refine the existing experimental map for the electronic structure of the flavin cofactor and expose systematic divergences between the calculated and experimental values of hyperfine couplings of the atoms that contribute most to the semi-occupied orbital (SOMO). This could indicate a slight but significant shift in the unpaired electron density from position 4a towards the central nitrogens of the pyrazine ring as compared with the calculations. These results highlight the importance of integrating computational and experimental approaches to refine our understanding of flavin cofactor reactivity.

Abstract Electron spin resonance (ESR) is a widely employed spectroscopic technique for studying systems with unpaired electron spins, such as molecular radicals. Typically, many billions of spins are required to get a detectable ESR signal, which is subject to extensive ensemble averaging. Downscaling ESR to a single molecule allows studying the signatures of each individual molecule separately, applicable to biomolecules in their native environment, for example. Single‐molecule ESR offers several novel research avenues, such as in quantum sensing with a single molecule. Over the last decades, four different single‐molecule ESR approaches have been developed, which rely on either optically detected magnetic resonance or scanning‐probe microscopy. An introduction into these four approaches including their deployment in pioneering works will be provided.

Electron Spin Resonance (ESR), also known as Electron Paramagnetic Resonance (EPR), is a sophisticated spectroscopic technique that provides critical insights into the electronic structure and dynamics of materials with unpaired electrons. This experiment focuses on the application of ESR to study the stable free radical Diphenyl-Picryl-Hydrazyl (DPPH), a compound widely utilized in various scientific fields due to its well-defined resonance characteristics. The primary objectives of this study were to observe the resonance curve of DPPH, determine the resonant frequency as a function of the applied magnetic field, and calculate the Landé g-factor for free electrons subjected to an external alternating magnetic field. The experimental setup involved the use of an ESR spectrometer, a microwave source, and a magnetic field source, with DPPH dissolved in a suitable solvent to create a homogeneous sample. Data collection was performed by varying the magnetic field while monitoring the intensity of the resonance signal, allowing for the construction of a resonance curve. The results indicated a clear peak in signal intensity at a specific magnetic field strength, corresponding to the resonance condition where the energy differ- ence between the electron spin states matched the energy of the microwave radiation. The analysis of the resonance curve revealed a linear relationship between the magnetic field strength and the resonant microwave frequency, consistent with theoretical predictions. The calculated Landé g-factor for the DPPH radical was found to be approximately 1.99, closely aligning with the expected value for free electrons, thus confirming the reliability of the experimental methodology. This study highlights the significance of ESR as a powerful tool for investigating paramagnetic species, providing valuable information about their electronic properties and behavior. The findings not only reinforce the fundamental principles of electron spin resonance but also pave the way for future research into the dynamics of free radicals and their implications in various scientific domains, including chemistry, biology, and materials science. Overall, the successful execution of this experiment underscores the versatility and importance of ESR in advancing our understanding of electron spin phenomena and their applications in real-world scenarios.

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EPR spectroscopy, in a constant-frequency field-swept experiment, was used to determine the resonant magnetic field, g-factor, line width, and hyperfine constant for diphenyl picrylhydrazyl at room temperature and manganese (II) chloride at cryogenic temperatures (using liquid nitrogen). Diphenyl picrylhydrazyl was determined to be a free radical, indicated by a g-factor of 2.022, with a resonant magnetic field of 3.23 kG at a microwave frequency of 9.14 GHz. The manganese (II) chloride was found to have six signal peaks, indicating hyperfine interaction of a nucleus with non-zero spin with unpaired electrons (spin was determined to be 5/2).

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Dissolution dynamic nuclear polarization (dDNP) increases the sensitivity of magnetic resonance experiments by >104-fold, permitting isotopically labeled molecules to be transiently visible in magnetic resonance imaging scans. dDNP mechanistically takes place at ~1 K and requires unpaired electrons and microwaves. These electrons are usually chemical radicals, requiring removal by filtration prior to injection into humans. Alternative sources, such as ultraviolet irradiation, generate lower polarization and require cryogenic transport. We present ultrahigh–dose rate electron irradiation as an alternative for generating nonpersistent radicals in alanine/glycerol mixtures. These are stable for months at room temperature, quench spontaneously upon dissolution, are present in dose-dependent concentrations, and generate comparable nuclear polarization (17%) to trityl radicals used clinically (19%) through a previously unknown mechanism we believe to involve partial ordering and electron-electron interactions. Owing to the large radiation doses required, this process is sterilizing, permits imaging of alanine metabolism in vivo in the rat kidney, and may aid clinically translating dDNP.

Dynamic nuclear polarization (DNP) has emerged as a powerful technique to overcome the sensitivity limit of solid-state nuclear magnetic resonance (ssNMR) spectroscopy through polarizing agents (PAs) carrying unpaired electrons. Nitroxide biradical-based PAs have garnered great attention due to their superior DNP performance. Here, we report TJPols─water-soluble acrylamide-linked nitroxide biradicals featuring facile large-scale synthesis. These PAs yield fast polarization build-up and reduced depolarization effect in magic-angle spinning (MAS) DNP experiments at 14.1 T, compensating for the moderate DNP enhancements and thus achieving higher overall NMR sensitivity gains than AMUPol, the most widely utilized binitroxide PA. Electron paramagnetic resonance (EPR) spectroscopy and molecular dynamics (MD) simulations reveal strong electron-electron dipolar and exchange couplings, validating the strategy of introducing a double bond in the linker region to tune the DNP behaviors. Our work presents a new synthetically feasible and functionalizable scaffold for developing new generations of PAs for DNP ssNMR at medium-to-high magnetic fields.

The presence of unpaired electrons, i.e., radicals, equips organic molecules with unique magnetic and reactivity properties. However, due to the reactive nature of radicals and the nontrivial chemistry required for their preparation, strict structural and electronic limitations are imposed on the available systems, limiting their potential applications. Thus, developing mechanisms that enable facile radical formation in simple reaction conditions, employing available and inexpensive reactants and applicable to general types of molecules, holds the key to capitalize on the extraordinary properties that radicals have to offer. Here, combining electron paramagnetic resonance spectroscopy and ab initio calculations, we uncover an unprecedented single electron transfer from multiple anionic organic bases (B–X+) to nitrobenzene [1], leading to the formation of stable nitrobenzenide radical ion-pair [1 •– ][X + ] (X = Li, Na, K) and transient oxidized, radical bases B•. Our results establish nitroarenes as versatile radical precursors, providing a broadly applicable protocol for generating heteroatom-centered radicals and enabling radical transformations under mild conditions from inexpensive and readily available starting materials. Finally, we propose the [1]–[B–X+] couple as an unexplored platform with the potential to advance the field of frustrated radical pairs.

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Abstract Polarisation transfer schemes and indirect detection are central to magnetic resonance. Using the trityl radical OX063 and a pulse electron paramagnetic resonance spectrometer operating in the Q-band (35 GHz, 1.2 T), we show here that it is possible to use pulsed dynamic nuclear polarisation (DNP) to transfer polarisation from electrons to protons and back. The latter is achieved by first saturating the electrons and then simply using a reverse DNP step. A variable mixing time between DNP and reverse DNP allows us to investigate the decay of polarisation on protons in the vicinity of the electrons. We qualitatively investigate the influence of solvent deuteration, temperature, and electron concentration. We expect reverse DNP to be useful in the investigation of nuclear spin diffusion and envisage its use in electron–nuclear double-resonance (ENDOR) experiments.

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The development of open‐shell organic molecules that magnetically order at room temperature,which can be practically applied, remains a grand challenge in chemistry, physics, and materials science. Despite the exploration of vast chemical space, design paradigms for organic paramagnetic centers generally result in unpaired electron spins that are unstable or isotropic. Here, a high‐spin conjugated polymer is demonstrated, which is composed of alternating cyclopentadithiophene and benzo[1,2‐c;4,5‐c′]bis[1,2,5]thiadiazole heterocycles, in which macromolecular structure and topology coalesce to promote the spin center generation and intermolecular exchange coupling. Electron paramagnetic resonance (EPR) spectroscopy is consistent with spatially localized spins, while magnetic susceptibility measurements show clear anisotropic spin ordering and exchange interactions that persist at room temperature. The application of long‐range π‐correlations for spin center generation promotes remarkable stability. This work offers a fundamentally new approach to the implementation of this long‐sought‐after physical phenomenon within organic materials and the integration of manifold properties within emerging technologies.

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Flow-injection spin-trapping electron paramagnetic resonance (FI-EPR) methods that involve the use of 5,5-dimethyl-pyrroline-N-oxide (DMPO) as a spin-trapping reagent have been developed for the kinetic study of the O2•- radical scavenging reactions occurring in the presence of various plant-derived and synthetic phenolic antioxidants (Aox), such as flavonoid, pyrogallol, catechol, hydroquinone, resorcinol, and phenol derivatives in aqueous media (pH 7.4 at 25 °C). The systematically estimated second-order rate constants (ks) of these phenolic compounds span a wide range (from 4.5 × 10 to 1.0 × 106 M-1 s-1). The semilogarithm plots presenting the relationship between ks values and oxidation peak potential (Ep) values of phenolic Aox are divided into three groups (A1, A2, and B). The ks-Ep plots of phenolic Aox bearing two or three OH moieties, such as pyrogallol, catechol, and hydroquinone derivatives, belonged to Groups A1 and A2. These molecules are potent O2•- radical scavengers with ks values above 3.8 × 104 (M-1 s-1). The ks-Ep plots of all phenol and resorcinol derivatives, and a few catechol and hydroquinone derivatives containing carboxyl groups adjacent to the OH groups, were categorized into the group poor scavengers (ks < 1.6 × 103 M-1 s-1). The ks values of each group correlated negatively with Ep values, supporting the hypothesis that the O2•- radical scavenging reaction proceeds via one-electron and two-proton processes. The processes were accompanied by the production of hydrogen peroxide at pH 7.4. Furthermore, the correlation between the plots of ks and the OH proton dissociation constant (pKa•) of the intermediate aroxyl radicals (ks-pKa• plots) revealed that the second proton transfer process could potentially be the rate-determining step of the O2•- radical scavenging reaction of phenolic compounds. The ks-Ep plots provide practical information to predict the O2•- radical scavenging activity of plant-derived phenolic compounds based on those molecular structures.

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A central goal of photoprotective energy dissipation processes is the regulation of singlet oxygen (1O2*) and reactive oxygen species in the photosynthetic apparatus. Despite the involvement of 1O2* in photodamage and cell signaling, few studies directly correlate 1O2* formation to nonphotochemical quenching (NPQ) or lack thereof. Here, we combine spin-trapping electron paramagnetic resonance (EPR) and time-resolved fluorescence spectroscopies to track in real time the involvement of 1O2* during photoprotection in plant thylakoid membranes. The EPR spin-trapping method for detection of 1O2* was first optimized for photosensitization in dye-based chemical systems and then used to establish methods for monitoring the temporal dynamics of 1O2* in chlorophyll-containing photosynthetic membranes. We find that the apparent 1O2* concentration in membranes changes throughout a 1 h period of continuous illumination. During an initial response to high light intensity, the concentration of 1O2* decreased in parallel with a decrease in the chlorophyll fluorescence lifetime via NPQ. Treatment of membranes with nigericin, an uncoupler of the transmembrane proton gradient, delayed the activation of NPQ and the associated quenching of 1O2* during high light. Upon saturation of NPQ, the concentration of 1O2* increased in both untreated and nigericin-treated membranes, reflecting the utility of excess energy dissipation in mitigating photooxidative stress in the short term (i.e., the initial ∼10 min of high light).

Significance 1O2 is a vital species for the selective oxidations of chemicals. However, detecting its production and understanding the underlying mechanisms in complex systems remains challenging. The spin-trapping method based on EPR (electron paramagnetic resonance) analysis has emerged as an indispensable tool for identifying the generation of 1O2. Here, we applied EPR analysis to track the fates of 1O2 in catalytic oxidation processes, offering time-dependent profiles of trapping products. These detailed examinations can unveil the molecular mechanism of direct 2,2,6,6-tetramethylpiperidine oxidation and mitigate the risk of false positives. This study paves the way for exploring 1O2 generation in aqueous solutions and catalytic oxidations governed by other oxidative species and also offers thorough clarification from the mechanism of spin-trapping to its application scenarios.

Ultrasound coupled with activated persulfate can synergistically degrade aqueous organic contaminants. Here, in situ electron paramagnetic resonance spin trapping was used to compare radicals produced by ultrasonically activated persulfate (US-PS) and its individual technologies, ultrasound alone (US) and heat-activated persulfate (PS), with respect to temperature. Radicals were trapped using 5,5-dimethyl-1-pyrroline-N-oxide, DMPO, to form detectable nitroxide adducts. Using initial rates of radical adduct formation, and compared to US and PS, US-PS at 40 and 50 °C resulted in the largest synergistic production of radicals. Radicals generated from US were reasonably consistent from 40 to 70 °C, indicating that temperature had little effect on cavitational bubble collapse over this range. However, synergy indexes calculated from initial rates showed that ultrasonic activation of persulfate at the bubble interface changes with temperature. From these results, we speculate that higher temperatures enhance persulfate uptake into cavitation bubbles via nanodroplet injection. DMPO-OH was the predominant adduct detected for all conditions. However, competition modeling and spin trapping in the presence of nitrobenzene and atrazine probes showed that SO4•- predominated. Therefore, the DMPO-OH signal is derived from SO4•- trapping with subsequent DMPO-SO4- hydrolysis to DMPO-OH. Spin trapping is effective in quantifying total radical adduct formation but limited in measuring primary radical speciation in this case.

Both hydroxyl radical (•OH) and chlorine radical (Cl•) are recognized as significant reactive species in aquatic and atmospheric sciences. While •OH can be readily detected with the conventional spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), Cl• is often considered difficult to detect by electron paramagnetic resonance (EPR) technique and has therefore been largely underexploited. In this work, we clarify the complicated reactions of Cl• with DMPO, which pose challenges for the direct detection of Cl•. To overcome this limitation, we take advantage of the differences in the products formed when methanol reacts with Cl• (i.e. •OCH3) compared to its reaction with •OH (i.e. •CH2OH), thereby developing a novel approach for detecting the elusive Cl•. The reliability and adaptability of this solvent-independent method are validated across various natural and engineered scenarios. Therefore, this work unlocks opportunities for identifying Cl• in the saline environments and further elucidating its roles in biogeochemical cycles of elements and environmental remediation.

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Lipid hydroperoxides play an important role in various pathophysiological processes. Therefore, a simple model for organic hydroperoxides could be helpful to monitor the biologic effects of endogenous and exogenous compounds. The electron paramagnetic resonance (EPR) spin-trapping technique is a useful method to study superoxide (O2•−) and hydroxyl radicals. The aim of our work was to use EPR with the spin trap 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO), which, by trapping O2•− produces relatively stable •BMPO-OOH spin-adduct, a valuable model for organic hydroperoxides. We used this experimental setup to investigate the effects of selected sulfur/selenium compounds on •BMPO-OOH and to evaluate the antioxidant potential of these compounds. Second, using the simulation of time-dependent individual BMPO adducts in the experimental EPR spectra, the ratio of •BMPO-OH/•BMPO-OOH—which is proportional to the transformation/decomposition of •BMPO-OOH—was evaluated. The order of potency of the studied compounds to alter •BMPO-OOH concentration estimated from the time-dependent •BMPO-OH/•BMPO-OOH ratio was as follows: Na2S4 > Na2S4/SeO32− > H2S/SeO32− > Na2S2 ~Na2S2/SeO32− ~H2S > SeO32− ~SeO42− ~control. In conclusion, the presented approach of the EPR measurement of the time-dependent ratio of •BMPO-OH/•BMPO-OOH could be useful to study the impact of compounds to influence the transformation of •BMPO-OOH.

The oxidative stability of myrtle hydroalcoholic extracts was measured, over storage time, with the EPR spin trapping method under forced ageing conditions. The extracts were prepared with 150 and 300 g l-1 of berries and extraction media with ethanol ranging from 60 to 90%. Two radicals were detected: the PBN-1-hydroxyethyl adduct and the tert-butyl aminoxyl radical. A dimensionless parameter (Ω) calculated on the basis of the lag time, the rate of formation and concentration of the radical species was used to estimate the extracts' oxidative stability. Ω was strongly influenced by the extraction medium, being lower in extracts with ethanol 60%, and by the time of storage. An inverse correlation was calculated between Ω and ellagic acid concentration, thus suggesting the role of this phenolic acid in the antioxidant properties of the extracts. The radical scavenging activity of the extracts against the hydroxyl radical was also measured.

The generation of superoxide radical anion in biological systems is one of the major initiating events in the redox biology of NADPH oxidases and mitochondrial redox signalling. However, the pallette of chemical tools for superoxide detection is very limited, hampering progress in understanding the chemical biology of superoxide. Although EPR spin trapping is regarded as the most rigorous technique for superoxide detection, rapid reduction of the EPR-active superoxide spin adducts to EPR-silent hydroxylamines, or to hydroxyl radical adducts by bioreductants, significantly limits the applicability of this technique in biological systems. To overcome these limitations, in this work, we report the synthesis and characterization of a new mesoporous silica functionalized with a phosphorylated cyclic spin trap (DIPPMPO nitrone). The DIPPMPO-grafted silica is a versatile spin-trap agent enabling the identification of a wide range of carbon or oxygen-centered transient radicals in organic and in aqueous media. Moreover, superoxide was efficiently trapped under in vitro conditions in both cell-free and cellular systems. The generated superoxide adduct exhibited an exceptional half-life of 3.5 h and a resistance toward bioreductant agents such as glutathione for several hours.

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The electron spin resonance (EPR) spin-trapping technique allows detection of radical species with nanosecond half-lives. This technique is based on the high rates of addition of radicals to nitrones or nitroso compounds (spin traps; STs). The paramagnetic nitroxides (spin-adducts) formed as a result of reactions between STs and radical species are relatively stable compounds whose EPR spectra represent “structural fingerprints” of the parent radical species. Herein we report a novel protocol for the synthesis of N-tert-butylmethanimine N-oxide (EBN), which is the simplest nitrone containing an α-H and a tertiary α′-C atom. We present EPR spin-trapping proof that: (i) EBN is an efficient probe for the analysis of glutathione thiyl radical (GS•); (ii) β-cyclodextrins increase the kinetic stability of the spin-adduct EBN/•SG; and (iii) in aqueous solutions, EBN does not react with superoxide anion radical (O2−•) to form EBN/•OOH to any significant extent. The data presented complement previous studies within the context of synthetic accessibility to EBN and efficient spin-trapping analysis of GS•.

Electron paramagnetic resonance (EPR)-spin trapping and flow cytometry were used to identify free radicals generated using argon-cold atmospheric plasma (Ar-CAP) in aqueous solutions and intracellularly in comparison with those generated by X-irradiation. Ar-CAP was generated using a high-voltage power supply unit with low-frequency excitation. The characteristics of Ar-CAP were estimated by vacuum UV absorption and emission spectra measurements. Hydroxyl (·OH) radicals and hydrogen (H) atoms in aqueous solutions were identified with the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO), and phenyl N-t-butylnitrone (PBN). The occurrence of Ar-CAP-induced pyrolysis was evaluated using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) in aqueous solutions of DNA constituents, sodium acetate, and L-alanine. Human lymphoma U937 cells were used to study intracellular oxidative stress using five fluorescent probes with different affinities to a number of reactive species. The analysis and quantification of EPR spectra revealed the formation of enormous amounts of ·OH radicals using Ar-CAP compared with that by X-irradiation. Very small amounts of H atoms were detected whereas nitric oxide was not found. The formation of ·OH radicals depended on the type of rare gas used and the yield correlated inversely with ionization energy in the order of krypton > argon = neon > helium. No pyrolysis radicals were detected in aqueous solutions exposed to Ar-CAP. Intracellularly, ·OH, H2O2, which is the recombination product of ·OH, and OCl- were the most likely formed reactive oxygen species after exposure to Ar-CAP. Intracellularly, there was no practical evidence for the formation of NO whereas very small amounts of superoxides were formed. Despite the superiority of Ar-CAP in forming ·OH radicals, the exposure to X-rays proved more lethal. The mechanism of free radical formation in aqueous solutions and an intracellular milieu is discussed.

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Electron paramagnetic resonance (EPR) has been extensively used for the identification of free radicals that are generated from advanced oxidation processes (AOPs) so as to establish the reaction mechanism. However, some misinterpretations or controversies on the identity of detected EPR signals remain in the literature. This study, with Cu(II)-based AOPs as examples, comprehensively investigated the origin of 5,5-dimethyl-l-pyrroline N-oxide (DMPO) adducts in Cu(II) alone, Cu(II)/H2O2, Cu(II)/peroxymonosulfate (PMS), and Cu(II)/peroxydisulfate (PDS) systems. In most Cu(II) systems, DMPO-OH signals can be detected even without any peroxygens, indicating the presence of other origins of this adduct in addition to the genuine spin trapping of •OH by DMPO. According to the formed secondary radical adducts (DMPO-OCH3 from a nonradical process or DMPO-CH2OH from a radical oxidation) derived from methanol quenching, we propose that CuO+, instead of free radicals, is involved in the Cu(II)/PMS system, while •OH is indeed generated in the Cu(II)/H2O2 and Cu(II)/PDS systems under neutral conditions. Notably, 17O-incorporation experiments demonstrate that -OH in the detected DMPO-OH adduct originates 100% from water in the Cu(II) alone system but the amount of -OH is over 99.8% from the oxidant while peroxygens are added. In addition, DMPO-O2- appears only in the Cu(II)/PDS system under highly alkaline conditions and H2O is not involved in superoxide formation.

The radical intermediates formed upon UVA irradiation of titanium dioxide suspensions in aqueous and non-aqueous environments were investigated applying the EPR spin trapping technique. The results showed that the generation of reactive species and their consecutive reactions are influenced by the solvent properties (e.g., polarity, solubility of molecular oxygen, rate constant for the reaction of hydroxyl radicals with the solvent). The formation of hydroxyl radicals, evidenced as the corresponding spin-adducts, dominated in the irradiated TiO2 aqueous suspensions. The addition of 17O-enriched water caused changes in the EPR spectra reflecting the interaction of an unpaired electron with the 17O nucleus. The photoexcitation of TiO2 in non-aqueous solvents (dimethylsulfoxide, acetonitrile, methanol and ethanol) in the presence of 5,5-dimethyl-1-pyrroline N-oxide spin trap displayed a stabilization of the superoxide radical anions generated via electron transfer reaction to molecular oxygen, and various oxygen- and carbon-centered radicals from the solvents were generated. The character and origin of the carbon-centered spin-adducts was confirmed using nitroso spin trapping agents.

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When photosystem II (PSII) is exposed to excess light, singlet oxygen (1O2) formed by the interaction of molecular oxygen with triplet chlorophyll. Triplet chlorophyll is formed by the charge recombination of triplet radical pair 3[P680•+Pheo•−] in the acceptor-side photoinhibition of PSII. Here, we provide evidence on the formation of 1O2 in the donor side photoinhibition of PSII. Light-induced 1O2 production in Tris-treated PSII membranes was studied by electron paramagnetic resonance (EPR) spin-trapping spectroscopy, as monitored by TEMPONE EPR signal. Light-induced formation of carbon-centered radicals (R•) was observed by POBN-R adduct EPR signal. Increased oxidation of organic molecules at high pH enhanced the formation of TEMPONE and POBN-R adduct EPR signals in Tris-treated PSII membranes. Interestingly, the scavenging of R• by propyl gallate significantly suppressed 1O2. Based on our results, it is concluded that 1O2 formation correlates with R• formation on the donor side of PSII due to oxidation of organic molecules (lipids and proteins) by long-lived P680•+/TyrZ•. It is proposed here that the Russell mechanism for the recombination of two peroxyl radicals formed by the interaction of R• with molecular oxygen is a plausible mechanism for 1O2 formation in the donor side photoinhibition of PSII.

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Site-directed spin labeling (SDSL) ESR is a valuable tool to probe protein systems that are not amenable to characterization by x-ray crystallography, NMR or EM. While general principles that govern the shape of SDSL ESR spectra are known, its precise relationship with protein structure and dynamics is still not fully understood. To address this problem, we designed seven variants of GB1 domain bearing R1 spin label and recorded the corresponding MD trajectories (combined length 180 μs). The MD data were subsequently used to calculate time evolution of the relevant spin density matrix and thus predict the ESR spectra. The simulated spectra proved to be in good agreement with the experiment. Further analysis confirmed that the spectral shape primarily reflects the degree of steric confinement of the R1 tag and, for the well-folded protein such as GB1, offers little information on local backbone dynamics. The rotameric preferences of R1 side chain are determined by the type of the secondary structure at the attachment site. The rotameric jumps involving dihedral angles χ1 and χ2 are sufficiently fast to directly influence the ESR lineshapes. However, the jumps involving multiple dihedral angles tend to occur in (anti)correlated manner, causing smaller-than-expected movements of the R1 proxyl ring. Of interest, ESR spectra of GB1 domain with solvent-exposed spin label can be accurately reproduced by means of Redfield theory. In particular, the asymmetric character of the spectra is attributable to Redfield-type cross-correlations. We envisage that the current MD-based, experimentally validated approach should lead to a more definitive, accurate picture of SDSL ESR experiments.

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ESR spectra of erythrocyte membranes labeled with a maleimide spin label (MSL) show two types of label environment: a weakly immobilized component and a strongly immobilized component. Chlorpromazine (CPZ) markedly altered the spectra: at pH 8.0, 3 mM CPZ reduced the amplitude of the spectrum by 40%, and the weakly immobilized component was almost completely removed. In order to clarify the mechanisms of these spectral changes the protein release from erythrocyte membranes induced by CPZ has been followed. CPZ had a weak solubilizing effect on erythrocyte membranes: less than 1% of the membrane protein was released, mainly Band 6. By comparison with the protein release induced by low-salt treatment it was found that the "detergent-like" property of CPZ cannot explain the alterations in the ESR spectra. The nature of the spectral changes induced by CPZ was different from that of changes induced by lowering the pH to 4.5; correlated with other data this shows that changes in organization or conformation of membrane protein cannot explain the CPZ-induced alterations in the ESR spectra. These spectral changes appeared to be due to the reduction by CPZ of the nitroxide free radical. This was documented by the marked reduction of spin concentration of the labeled ghosts in the presence of CPZ resulting in a decrease in amplitude of the ESR spectrum of MSL-labeled erythrocyte ghosts induced by CPZ. The reduction by CPZ of the nitroxide free radical was compared with that induced by ascorbate. It was found that CPZ preferentially reduces the mobile component of the ESR spectrum of MSL-labeled ghosts. The action of CPZ in reducing free radicals may have consequences for patients receiving long-term treatment with phenothiazine derivatives.

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The advanced electron paramagnetic resonance (EPR) techniques, electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopies, provide unique insights into the structure, coordination chemistry, and biochemical mechanism of nature's widely distributed iron-sulfur cluster (FeS) proteins. This review describes the ENDOR and ESEEM techniques and then provides a series of case studies on their application to a wide variety of FeS proteins including ferredoxins, nitrogenase, and radical SAM enzymes. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

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Lytic Polysaccharide Monooxygenases (LPMOs) catalyze the oxidative depolymerization of polysaccharides at a monocopper active site, that is coordinated by the so-called histidine brace. In the past, this motif has sparked considerable interest, mostly due to its ability to generate and stabilize highly oxidizing intermediates during catalysis. We used a variety of advanced EPR techniques, including Electron Nuclear Double Resonance (ENDOR), Electron Spin Echo Envelope Modulation (ESEEM) and Hyperfine Sublevel Correlation (HYSCORE) spectroscopy in combination with isotopic labelling (15N, 2H) to characterize the active site of the bacterial LPMO SmAA10A over a wide pH range (pH 4.0–pH 12.5). At elevated pH values, several ligand modifications are observed, including changes in the HxO ligand coordination, but also regarding the protonation state of the histidine brace. At pH > 11.5, the deprotonation of the two remote nitrogen nuclei of the imidazole moieties and of the terminal amine is observed. These deprotonations are associated with major electronic changes, including increased σ-donor capabilities of the imidazolates and an overall reduced interaction of the deprotonated amine function. This observation highlights a potentially more significant role of the imidazole ligands, particularly for the stabilization of potent oxidants during turnover. The presented study demonstrates the application of advanced EPR techniques for a thorough characterization of the active site in LPMOs, which ultimately sets a foundation for and affords an outlook on future applications characterizing reaction intermediates.

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In addition to the commonly used electron–electron double resonance (ELDOR) technique, there are several other electron paramagnetic resonance (EPR) methods by which structure information can be obtained by exploiting the dipolar coupling between two radicals based on its characteristic r −3 dependence. In this contribution, we explore the potential of out-of-phase-electron-spin echo envelope modulation (OOP-ESEEM) spectroscopy to collect accurate distance information in photo-sensitive (bio) molecules. Although the method has already been applied to spin-correlated radical pairs in several classes of light-active proteins, the accuracy of the information obtained has not yet been extensively evaluated. To do this in a system-independent fashion, OOP-ESEEM time traces simulated with different values of the dipolar and exchange couplings were generated and analyzed in a best-possible way. Excellent agreement between calculated and numerically fitted values over a wide range of distances (between 15 and 45 Å) was obtained. Furthermore, the limitations of the method and the dependence on various experimental parameters could be evaluated.

Light-driven formation of radical ion pairs that occurs much faster than their electron spin dynamics results in correlated spins whose coherence properties can be used as a quantum-based electric field sensor. This results from the radical ion pair having charge and spin distributions that track one another. Thus, electric field induced changes in the distance between the two charges are reflected in the spin-spin distance that can be measured directly using out-of-phase electron spin echo envelope modulation (OOP-ESEEM), a pulse-EPR technique. OOP-ESEEM produces oscillations whose frequency depends on the spin-spin exchange (J) and dipolar (D) interactions as well as the electron-nuclear hyperfine couplings of the radicals. To date, analyses of OOP-ESEEM data have focused predominately on determination of J and D, largely because D serves to measure the spin-spin distance. In this study we compare the OOP-ESEEM signals obtained from radical ion pairs that are fully deuterated with those that contain hydrogen. The results show that low modulation amplitudes occur in the OOP-ESEEM data using deuterated radicals, making it difficult to accurately measure radical ion pair distances. Further, the use of hydrogen-containing radicals provides the modulation amplitude enhancement necessary to measure small distances accurately using OOP-ESEEM data despite the somewhat shorter electron spin coherence times of the hydrogen-containing radical ion pairs. This is an important consideration when designing photogenerated radical ion pairs to serve as quantum sensors of electric fields.

The magnetic compass sensor in night-migratory songbirds is thought to be a flavin-tryptophan radical pair formed by blue-light excitation of the protein cryptochrome-4a (Cry4a) localized in photoreceptor cells in the birds’ retinas. The effects of applied magnetic fields on the photochemistry of purified Cry4a from the migratory European robin are well characterized, but it is less clear what, if anything, distinguishes the magnetic responses of the Cry4a proteins from migratory and nonmigratory species. We present here a detailed study of the magnetic sensitivity of Cry4a from the nonmigratory chicken. The wild-type protein is compared with two mutants in which either Arg317 or Glu320, both close to the tryptophan radical, were replaced by the amino acids Cys and Lys, respectively, found in Cry4a from robins and other night-migratory passerines. These sites had previously been identified as probably facilitating the evolution of an optimized magnetic sensor for nocturnal orientation in songbirds. Neither of these mutations was found to affect the reaction kinetics or magnetic sensitivity of the radical pairs, suggesting that any differences in Cry4a between robin and chicken must stem from their ability to transmit magnetic information, for example via protein–protein interactions. In contrast, a Trp → Phe mutation at the end of the tryptophan-tetrad electron transfer chain in both cryptochromes led to a large increase in magnetic sensitivity, suggesting different sensing and signaling roles for the third and fourth tryptophans.

Recently, Marina Bennati and coworkers (M. Bennati et al., Angew. Chem., Int. Ed., 2020, 59, 373-379., M. Bennati et al., J. Magn. Reson., 2021, 333, 107091) proposed to use electron nuclear double resonance (ENDOR) spectroscopy in the W-band for a pair of labels, nitroxide and 19F, for measurements of short (0.5-1.0 nm) distances in biomolecules. In our paper, we investigated the suitability of high-field ENDOR spectroscopy in the W-band for pairs of triarylmethyl and fluorine labels using five newly synthesized model compounds. It is shown that the application of strong magnetic fields allows distinguishing nuclear frequencies of 19F and protons with sufficient resolution. On the one hand, in contrast to nitroxides, for triarylmethyl radicals, it is not necessary to obtain spectra in different orientations owing to low g-factor anisotropic and long electron spin relaxation times of triarylmethyls. On the other hand, the size of the triarylmethyl radical is substantially larger than that of nitroxide and comparable with measured distances. We theoretically analyzed the suitability of the dipole-dipole approach for triarylmethyl to be used in a 19F ENDOR experiment and determined limitations of this approach. Finally, for comparison, we performed paramagnetic relaxation enhancement (PRE) NMR on the same compounds. In addition, we applied this approach to study the process of a thiol exchange between molecules of triarylmethyl-labeled and 19F-labeled human serum albumin (HSA).

Radical enzymes orchestrate challenging chemical transformations by devising strategies to tame the highly reactive radical intermediates. Electron paramagnetic resonance (EPR) spectroscopy is the most suitable technique to study various aspects of the radical enzymes. Lysine 5,6-aminomutase (5,6-LAM) is one such radical enzyme and employs coenzyme B12 and pyridoxal 5'-phosphate (PLP) to catalyze the 1,2-amino shift reaction through a radical mechanism. 5,6-LAM accepts either d-lysine or l-β-lysine as the substrate. EPR and electron nuclear double resonance (ENDOR) spectroscopies have played major roles in deciphering the mechanism of action of 5,6-LAM, while density functional theoretical (DFT) computation and synthetic isotopologues have played supporting roles. This comprehensive toolkit has revealed that 5,6-LAM undergoes large-scale conformational movement to bring PLP and coenzyme B12 close together, which allows the reaction to progress. The conformational change also closes the active site, which protects the radical intermediates and enables their transformation to product without unwanted side reactions. The substrate-related radical (S•), which is spin-coupled with Co2+ generated from homolysis of the CoC bond in coenzyme B12, was unequivocally characterized when a substrate analog, 4-thia-l-lysine, and isotopologues of it were reacted with 5,6-LAM. Studies with substrate analogs revealed a unique "odd-even" correlation with opening of the closed state. Moreover, mutagenesis studies identified the contributions that conserved residues in 5,6-LAM make toward binding of the substrate. Further studies with a cofactor analog, PLP-N-oxide, have shed light on various aspects of the mechanism of action of 5,6-LAM.

Carotenoids are indispensable molecules for life. They are present everywhere in plants, algae, bacteria whom they protect against free radicals and oxidative stress. Through the consumption of fruits and vegetables and some carotenoid-containing fish, they are introduced into the human body and, similarly, protect it. There are numerous health benefits associated with the consumption of carotenoids. Carotenoids are antioxidants but at the same time they are prone to oxidation themselves. Electron loss from the carotenoid forms a radical cation. Furthermore, proton loss from a radical cation forms a neutral radical. In this mini-review, we discuss carotenoid radicals studied in our groups by various physicochemical methods, namely the radical cations formed by electron transfer and neutral radicals formed by proton loss from the radical cations. They contain many similar hyperfine couplings due to interactions between the electron spin and numerous protons in the carotenoid. Different EPR and ENDOR methods in combination with DFT calculations have been used to distinguish the two independent carotenoid radical species. DFT predicted larger coupling constants for the neutral radical compared to the radical cation. Previously, INDO calculations miss assigned the large couplings to the radical cation. EPR and ENDOR have aided in elucidating the physisorb, electron and proton transfer processes that occur when carotenoids are adsorbed on solid artificial matrices, and predicted similar reactions in aqueous solution or in plants. After years of study of the physicochemical properties of carotenoid radicals, the different published results start to merge together for a better understanding of carotenoid radical species and their implication in biological systems. Up until 2008, the radical chemistry in artificial systems was elucidated but the correlation between quenching ability and neutral radical formation was an inspiration to look for these radical species in vivo. In addition, the EPR spin-trapping technique has been applied to study inclusion complexes of carotenoids with different delivery systems.

Mo-dependent nitrogenase is a major contributor to global biological N2 reduction, which sustains life on Earth. Its multi-metallic active-site FeMo-cofactor (Fe7MoS9C-homocitrate) contains a carbide (C4-) centered within a trigonal prismatic CFe6 core resembling the structural motif of the iron carbide, cementite. The role of the carbide in FeMo-cofactor binding and activation of substrates and inhibitors is unknown. To explore this role, the carbide has been in effect selectively enriched with 13C, which enables its detailed examination by ENDOR/ESEEM spectroscopies. 13C-carbide ENDOR of the S = 3/2 resting state (E0) is remarkable, with an extremely small isotropic hyperfine coupling constant, Ca = +0.86 MHz. Turnover under high CO partial pressure generates the S = 1/2 hi-CO state, with two CO molecules bound to FeMo-cofactor. This conversion surprisingly leaves the small magnitude of the 13C carbide isotropic hyperfine-coupling constant essentially unchanged, Ca = -1.30 MHz. This indicates that both the E0 and hi-CO states exhibit an exchange-coupling scheme with nearly cancelling contributions to Ca from three spin-up and three spin-down carbide-bound Fe ions. In contrast, the anisotropic hyperfine coupling constant undergoes a symmetry change upon conversion of E0 to hi-CO that may be associated with bonding and coordination changes at Fe ions. In combination with the negligible difference between CFe6 core structures of E0 and hi-CO, these results suggest that in CO-inhibited hi-CO the dominant role of the FeMo-cofactor carbide is to maintain the core structure, rather than to facilitate inhibitor binding through changes in Fe-carbide covalency or stretching/breaking of carbide-Fe bonds.

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