Electron Paramagnetic Resonance Spin Research Articles

Preliminary analyses of radiophotoluminescence centers in a recently developed Ag-doped alkali-phosphate glass (SAPANS)

The mechanism behind radiophotoluminescence (RPL) emission in Ag-doped alkali-phosphate glass remains unclear. To contribute to resolving this issue, the authors measured and analyzed the temporal change of electron spin/paramagnetic resonance (ESR) spectrum of a recently developed, weather-resistant RPL glass, named “SAPANS,” after irradiation with X-rays. As a result, a continuous gradual increase in Ag2+-derived signals was observed even at four weeks post-irradiation, which corresponded well with the time build-up pattern of the RPL of SAPANS. In contrast, the signals derived from phosphorous-oxygen-hole centers (POHC) rapidly decreased over a few days. The peaks of Ag0 and Ag2+ were not successfully separated from the ESR spectra because of the possible overlap of unidentified peaks, which implies a complex mechanism of RPL-center formation in Ag-doped alkali-phosphate glass that requires further investigation.

Metal-free catalysts with local fluorination regulation for peroxymonosulfate activation: Nearly 100 % singlet oxygen production for selective degradation of aqueous organic pollutants

Generating singlet oxygen (1O2) via activating peroxymonosulfate (PMS) is crucial for the selective removal of organic pollutants in aquatic environments. However, achieving efficient and selective conversion of PMS into 1O2 without consuming energy or metals remains a significant challenge. In this study, graphite phase carbon nitride (g-C3N4) was locally fluorinated via a simple polymerization method. The local fluorinated g-C3N4 (FCN) was used as a metal-free catalyst to activate PMS, enabling highly efficient degradation of 4-chlorophenol (100 %) and detoxification. The FCN/PMS system demonstrates exceptional selectivity for electron-rich pollutants, functions effectively across a broad pH range (2–10), and exhibits remarkable interference resistances to background scavenging ions and natural organic compounds. Furthermore, quenching experiments, probe tests, electron paramagnetic spin resonance measurements, electrochemical characterizations, and theoretical calculations demonstrated that 1O2 was the only reactive species in the catalytic process. Local fluorination regulated the electronic structure of g-C3N4, juxtaposing electron-rich fluorine with electron-deficient carbon dual active sites. This modification alters the electron transfer direction between PMS and g-C3N4, allowing for nearly 100 % selective generation of 1O2via dual electron transfer channels without the need for energy or metals. This study provides a new strategy for designing metal-free heterogeneous catalysts to selectively generate 1O2 in a green way and can facilitate the wider applications of advanced oxidation processes for water purification.

The oxidation of fenamic acid NSAIDs by neutrophil myeloperoxidase produces toxic reactive metabolites that induce leukemic cell death

Fenamic acids are a group of non-steroidal anti-inflammatory drugs (NSAIDs) that are among the most common drugs prescribed globally. However, they have been associated with many adverse effects, such as agranulocytosis, neutropenia, hepatotoxicity, and nephrotoxicity. The interactions between peroxidase enzymes and fenamic acid-like NSAIDs cause the formation of reactive species, potentially involved in side effects. The aim of this study was to investigate the neutrophil myeloperoxidase (MPO)-mediated bioactivation of fenamic acids based on N-phenylanthranilic acid (NPA) and its four drug analogues: flufenamic acid (FFA), mefenamic acid (MFA), meclofenamic acid (MCFA), and tolfenamic acid (TFA). We hypothesized that the enzymatic oxidation of fenamic acids by MPO/hydrogen peroxide (H2O2) would produce reactive metabolites, cause oxidative damage and induce cytotoxicity. We utilized UV–Vis spectrophotometry, liquid chromatography-mass spectrometry (LC-MS), and electron paramagnetic spin resonance (EPR) spectroscopy using purified MPO from human neutrophils. In addition, in vitro studies were performed with MPO-containing human promyelocytic leukemia (HL-60) cells for cytotoxicity and immuno-spin trapping to detect protein-free radicals. UV–Vis spectrophotometry revealed that MPO oxidized the fenamic acids. LC-MS analyses revealed the formation of dimers, hydroxylated, and quinoneimine species, and glutathione (GSH) conjugates. EPR spin trapping with DMPO using GSH revealed that fenamic acids produced glutathionyl radicals in a concentration-dependent manner. We also detected the formation of protein-free radicals in HL-60 cells, which correlated with cytotoxicity. Despite the minor structural differences between the fenamic acids, there were variations in their oxidation potential. These findings revealed a correlation between pro-oxidant metabolite reactivity and cytotoxicity caused by fenamic acid NSAIDs.

Use of Electron Paramagnetic Resonance (EPR) to Evaluate Redox Status in a Preclinical Model of Acute Lung Injury.

Patients with hyper- vs. hypo-inflammatory subphenotypes of acute respiratory distress syndrome (ARDS) exhibit different clinical outcomes. Inflammation increases the production of reactive oxygen species (ROS) and increased ROS contributes to the severity of illness. Our long-term goal is to develop electron paramagnetic resonance (EPR) imaging of lungs in vivo to precisely measure superoxide production in ARDS in real time. As a first step, this requires the development of in vivo EPR methods for quantifying superoxide generation in the lung during injury, and testing if such superoxide measurements can differentiate between susceptible and protected mouse strains. In WT mice, mice lacking total body extracellular superoxide dismutase (EC-SOD) (KO), or mice overexpressing lung EC-SOD (Tg), lung injury was induced with intraperitoneal (IP) lipopolysaccharide (LPS) (10mg/kg). At 24h after LPS treatment, mice were injected with the cyclic hydroxylamines 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride (CPH) or 4-acetoxymethoxycarbonyl-1-hydroxy-2,2,5,5-tetramethylpyrrolidine-3-carboxylic acid (DCP-AM-H) probes to detect, respectively, cellular and mitochondrial ROS - specifically superoxide. Several probe delivery strategies were tested. Lung tissue was collected up to one hour after probe administration and assayed by EPR. As measured by X-band EPR, cellular and mitochondrial superoxide increased in the lungs of LPS-treated mice compared to control. Lung cellular superoxide was increased in EC-SOD KO mice and decreased in EC-SOD Tg mice compared to WT. We also validated an intratracheal (IT) delivery method, which enhanced the lung signal for both spin probes compared to IP administration. We have developed protocols for delivering EPR spin probes in vivo, allowing detection of cellular and mitochondrial superoxide in lung injury by EPR. Superoxide measurements by EPR could differentiate mice with and without lung injury, as well as mouse strains with different disease susceptibilities. We expect these protocols to capture real-time superoxide production and enable evaluation of lung EPR imaging as a potential clinical tool for subphenotyping ARDS patients based on redox status.

The Effects of Mono- and Bivalent Linear Alkyl Interlayer Spacers on the Photobehavior of Mn(II)-Based Perovskites.

Mn(II)-based perovskite materials are being intensively explored for lighting applications; understanding the role of ligands regarding their photobehavior is fundamental for their development. Herein, we report on two Mn (II) bromide perovskites using monovalent (perovskite 1, P1) and bivalent (perovskite 2, P2) alkyl interlayer spacers. The perovskites were characterized with powder X-ray diffraction (PXRD), electron spin paramagnetic resonance (EPR), steady-state, and time-resolved emission spectroscopy. The EPR experiments suggest octahedral coordination in P1 and tetrahedral coordination for P2, while the PXRD results demonstrate the presence of a hydrated phase in P2 when exposed to ambient conditions. P1 exhibits an orange-red emission, while P2 shows a green photoluminescence, as a result of the different types of coordination of Mn(II) ions. Furthermore, the P2 photoluminescence quantum yield (26%) is significantly higher than that of P1 (3.6 %), which we explain in terms of different electron-phonon couplings and Mn-Mn interactions. The encapsulation of both perovskites into a PMMA film largely increases their stability against moisture, being more than 1000 h for P2. Upon increasing the temperature, the emission intensity of both perovskites decreases without a significant shift in the emission spectrum, which is explained in terms of an increase in the electron-phonon interactions. The photoluminescence decays fit two components in the microsecond regime-the shortest lifetime for hydrated phases and the longest one for non-hydrated phases. Our findings provide insights into the effects of linear mono- and bivalent organic interlayer spacer cations on the photophysics of these kinds of Mn (II)-based perovskites. The results will help in better designs of Mn(II)-perovskites, to increase their lighting performance.

(Invited) Operando EPR Study on Radicals in Anion-Exchange Membrane Fuel Cells

In the rapidly developing modern society, there is an urgent need for the wide-ranging availability of advanced and eco-friendly energy sources. One of the possible alternatives is the application of anion exchange membrane fuel cells (AEMFCs) with a catalyst reducing dioxygen efficiently. These promising devices can revolutionize the energy sector since they practically produce no pollution. However, to make them more widely used, several obstacles must be overcome. As a result, vast-ranging investigations focus on improving the properties of conductive polymer membranes [1] and catalysts for the oxygen reduction reaction (ORR) [2]. The durability of the membrane electrode assembly (MEA) is one of the critical requirements for the successful commercialization of anion exchange membrane fuel cells (AEMFCs). Despite significant impacts of nucleophilic degradation on ion-exchange capability and the anionic conductivity of investigated membranes, it is believed to affect only cationic sites of membrane polymers and thus cannot explain the reported loss in the mechanical strength of degraded AEMs. Such a phenomenon might be related to polymer backbone degradation caused by free radicals. This was widely described in the literature in the case of fuel cells using proton-conducting membranes [3] but barely mentioned for AEMFCs [4]. Since the oxidative degradation of hydrocarbon polymers is very well known, we aimed to comprehensively investigate the formation of the short-lived species generated during the operation of AEMFCs as well as stable radicals present in the polymer membranes.We investigated the LDPE-base membranes with Pt black, Pd black, PdAg, and Ag as the ORR catalysts, whereas for HOR the Pt black, Pd black, and NiFe catalysts were used. The in-situ measurements are performed with a micro-AEMFC inserted into a resonator of an electron paramagnetic resonance (EPR) spectrometer, which enables separate monitoring of radicals formed on the anode and cathode sides. The creation of radicals was monitored by the EPR spin trapping technique. In Figure 1 the EPR spectra of DMPO spin adducts trapped during operation of micro-fuel cell placed in EPR spectrometer cavity are presented. In this experiment, the LDPE-base membrane with platinum catalysts on both sides was used. The main detected adducts during the operation of the micro-AEMFC were DMPO-OOH and DMPO-OH on the cathode side and DMPO-H on the anode side. Additionally, we clearly show the formation and presence of stable radicals in AEMs during and after long-term AEMFC operation [5]. Preliminary results suggest that the creation of the short-living radicals during AEMFCs operation is independent of the used membrane. However, the applied catalysts determine the number of detected radicals. The EPR investigations indicate that, in addition to the known chemical degradation mechanisms of the cationic ammonium groups of the membrane, oxidative degradation, including radical reactions, has to be taken into account when the stability of an anion conductive polymer for AEMFCs is investigated. The formation of stable radicals in AEMs was proven for the first time in this study. All short-living radicals formed during the AEMFC operation were fully identified. The presence of radicals in the AEM after AEMFC testing indicates that reactive oxygen species may play a very important role in the degradation mechanism of the anion conducting polymers. Results from this study shed light on the understanding of radical formation and presence in the membranes during AEMFC tests, which in turn may help to solve the challenge of anion exchange membrane stability. Acknowledgments. This work was supported by the Polish National Science Centre (NCN) project OPUS-14, No. 2017/27/B/ST5/01004.

Electrochemical-enhanced Fe3O4/biochar activates peroxymonosulfate (E/nano-Fe3O4/BC/PMS) for degradation of oxytetracycline

To find cost-effective and environmentally friendly free radical activators to stimulate peroxymonosulfate (PMS) oxidative degradation of organic pollutants, nano-Fe3O4/biochar (BC) composites were prepared and characterized in this work to examine their effectiveness in stimulating PMS oxidative degradation of Oxytetracycline (OTC) in water enhanced with electrochemical degradation. When the mass ratio of nano-Fe3O4 to BC is 1:1, the catalyst nano-Fe3O4/BC exhibits the most obvious degradation effect on OTC. After 4 h of degradation, the OTC concentrations were reduced from 20 to 2.65 mg L−1, while treated with a single nano-Fe3O4 and a single BC are reduced by only 67.7% and 61.8%. Anions HCO3− and H2PO4− significantly inhibit OTC degradation, and HCO3− has a stronger inhibitory effect than H2PO4−, while Cl− and NO3− can promote OTC degradation. Quenching test and electron spin paramagnetic resonance (EPR) detection showed that singlet oxygen (1O2) was the main active species in the degradation process, followed by hydroxyl radical (·OH). When reused for the third time, the removal rate of OTC by nano-Fe3O4/BC composites with mass ratios of 1:4, 1:2 and 1:1 was still more than 70%. Therefore, the nano-Fe3O4/BC composite is a promising PMS activator, which can realize the rapid oxidative degradation of OTC.

Protonation and Non-Innocent Ligand Behavior in Pyranopterin Dithiolene Molybdenum Complexes.

The complex [TEA][Tp*MoIV(O)(S2BMOPP)] (1) [TEA = tetraethylammonium, Tp* = tris(3,5-dimethylpyrazolyl)hydroborate, and BMOPP = 6-(3-butynyl-2-methyl-2-ol)-2-pivaloyl pterin] is a structural analogue of the molybdenum cofactor common to all pyranopterin molybdenum enzymes because it possesses a pyranopterin-ene-1,2-dithiolate ligand (S2BMOPP) that exists primarily in the ring-closed pyrano structure as a resonance hybrid of ene-dithiolate and thione-thiolate forms. Compound 1, the protonated [Tp*MoIV(O)(S2BMOPP-H)] (1-H) and one-electron-oxidized [Tp*MoV(O)(S2BMOPP)] [1-Mo(5+)] species have been studied using a combination of electrochemistry, electronic absorption, and electron paramagnetic resonance (EPR) spectroscopy. Additional insight into the nature of these molecules has been derived from electronic structure computations. Differences in dithiolene C-S bond lengths correlate with relative contributions from both ene-dithiolate and thione-thiolate resonance structures. Upon protonation of 1 to form 1-H, large spectroscopic changes are observed with transitions assigned as Mo(xy) → pyranopterin metal-to-ligand charge transfer and dithiolene → pyranopterin intraligand charge transfer, respectively, and this underscores a dramatic change in electronic structure between 1 and 1-H. The changes in electronic structure that occur upon protonation of 1 are also reflected in a large >300 mV increase in the Mo(V/IV) redox potential for 1-H, resulting from the greater thione-thiolate resonance contribution and decreased charge donation that stabilize the Mo(IV) state in 1-H with respect to one-electron oxidation. EPR spin Hamiltonian parameters for one-electron-oxidized 1-Mo(5+) and uncyclized [Tp*MoV(O)(S2BDMPP)] [3-Mo(5+)] [BDMPP = 6-(3-butynyl-2,2-dimethyl)-2-pivaloyl pterin] are very similar to each other and to those of [Tp*MoVO(bdt)] (bdt = 1,2-ene-dithiolate). This indicates that the dithiolate form of the ligand dominates at the Mo(V) level, consistent with the demand for greater S → Mo charge donation and a corresponding increase in Mo-S covalency as the oxidation state of the metal is increased. Protonation of 1 represents a simple reaction that models how the transfer of a proton from neighboring acidic amino acid residues to the Mo cofactor at a nitrogen atom within the pyranopterin dithiolene (PDT) ligand in pyranopterin molybdenum enzymes can impact the electronic structure of the Mo-PDT unit. This work also illustrates how pyran ring-chain tautomerization drives changes in resonance contributions to the dithiolene chelate and may adjust the reduction potential of the Mo ion.

Enhanced photocatalytic performance and persulfate activation properties by BiOBr supported waste rock wool fibers under LED blue light

An advanced oxidation process was developed under LED blue light (BL) photocatalysis in the presence of ammonium persulfate (APS) for effective degradation of tetracycline (TC) from contaminated water. Waste rock wool (RW) fibers modified with BiOBr nano-sheet (BiOBr/RW) using impregnation combined with hydrolysis process, was employed to activate APS to produce reactive radicals under LED BL irradiation (BiOBr/RW+APS+BL system). The influence factors on TC degradation were investigated. Besides, the radical scavengers such as methanol (MA), phenol (PN), tertbutanoland (TBA) and ethylene diamine tetraacetate (EDTA-2Na) combined electron spin paramagnetic resonance (EPR) analysis were used to confirm the reactive radical species. The developed BiOBr/RW+APS+BL system exhibited much higher TC removal efficiency compared with other reported oxidation systems. In addition, the BiOBr/RW could directly activate the APS leading to degrade TC in the darkness. A maximum TC removal of 84.3% was achieved under the optimal conditions such as initial concentration of APS 100 mg/L, BiOBr/RW dosage 0.1 g/L and pH 7 in 80 min. Results also ascertained that h+, •OH and •SO4- over surface of the BiOBr/RW were the primary active chemical species in the BiOBr/RW+APS+BL system. It was found that the •OH and •SO4- in solution play critical roles for degradation of TC on the BiOBr/RW+APS system in dark. Finally, the repeated experiments indicated the excellent stability, photocatalytic ability and cycling property of BiOBr/RW.

Neutrophil Myeloperoxidase-Mediated N-Demethylation of Quetiapine Leads to N-Desalkylquetiapine, a Pharmacologically Active Cytochrome P450 Metabolite

The atypical antipsychotic drugs, quetiapine and clozapine, are associated with idiosyncratic drug reactions (such as agranulocytosis or neutropenia) that are thought to involve reactive metabolites. Neutrophil myeloperoxidase (MPO) metabolism of quetiapine is not well-studied, but is metabolized by cytochrome P450. Based on structural similarity to clozapine, we hypothesized that quetiapine can be metabolized by MPO and that there is overlap between cytochrome P450 and MPO metabolism of quetiapine. The interaction of quetiapine and clozapine with MPO and MPO chlorination activity was studied using UV-vis spectrophotometry. The metabolites were characterized using liquid chromatography-mass spectrometry (LC-MS), and electron paramagnetic resonance (EPR) spectroscopy was used for detecting drug-catalyzed glutathione oxidation. In the presence of quetiapine, MPO compound II accumulated for about 7.5 min, whereas in the presence of clozapine, MPO compound II was not observed as it was rapidly reduced back to the resting state. Increasing quetiapine concentrations resulted in a decrease in MPO chlorination activity, while the opposite result was found in the case of clozapine. UV-vis spectral studies showed no change when quetiapine was oxidized in the absence and presence of chloride anion (Cl-, to catalyze chlorination reactions). Significant changes, however, were observed in the same assay with clozapine, where Cl- appeared to hinder the rate of clozapine metabolism. The MPO-catalyzed hydroxylated and dealkylated metabolites of quetiapine and hydroxylated metabolites of clozapine were observed from the LC-MS analyses, particularly when Cl- was included in the reaction. In addition, hydroxylated, dealkylated, and a proposed sulfoxide metabolite of quetiapine were also observed in the reaction catalyzed by human microsomes/NADPH. Lastly, compared to quetiapine, clozapine metabolism by MPO/H2O2 and glutathione produced more glutathionyl radicals using EPR spin trapping. In conclusion, MPO/H2O2/Cl- was shown to metabolize quetiapine to S-oxidation and P450-like dealkylation products, and quetiapine metabolites were generally less reactive than clozapine.

Z-scheme bismuth-rich bismuth oxide iodide/bismuth oxide bromide hybrids with novel spatial structure: Efficient photocatalytic degradation of phenolic contaminants accelerated by in situ generated redox mediators

Z-scheme photocatalysts commonly possess both high charge separation efficiency and strong redox ability. In this paper, novel 3-dimensional/2-dimensional (3D/2D) structured bismuth-rich bismuth oxide iodide/bismuth oxide bromide (Bi4O5I2/BiOBr) hybrids with Z-scheme heterojunctions were first prepared. The in situ generated I3−/I− and Bi5+/Bi3+ redox mediators in Bi4O5I2/BiOBr hybrids greatly improve their photocatalytic activity toward phenolic contaminants. Their structure, morphology, optical properties, and electrochemical properties were characterized. Scanning electron microscopy images demonstrated that the 2D BiOBr nanoplates were evenly and tightly anchored on the surface of the 3D Bi4O5I2 microspheres. This novel 3D/2D spatial structure was beneficial for the formation of heterojunctions between BiOBr and Bi4O5I2, which improved the quantum efficiency through interfacial charge transfer. The Bi4O5I2/BiOBr hybrids exhibited excellent photocatalytic activities toward o-phenylphenol (OPP), p-tert-butylphenol, 4-chlorophenol, and p-nitrophenol. Bi4O5I2/BiOBr-5 possessed the best activity in decomposing OPP, which was approximately 3.43 times higher than that of pure Bi4O5I2. The well-matched energy bands of components in the hybrids facilitated the interfacial charge separation through an effective Z-scheme transfer direction guided by I3−/I− and Bi5+/Bi3+ redox mediators. Based on the results of electron spin paramagnetic resonance and trapping experiments, a mechanism was proposed for the degradation of pollutants using the Bi4O5I2/BiOBr hybrids.

Nanosized Zn-In spinel-type sulfides loaded on facet-oriented CeO2 nanorods heterostructures as Z-scheme photocatalysts for efficient elemental mercury removal

Developing of effective photocatalysts is of great significance for realizing photocatalytic environment purification. Herein, an interfacial bent bands and internal electric field modulated CeO2/ZnIn2S4 Z-scheme heterojunction for photocatalytic Hg0 oxidation. It is found that the charge transfer mechanism of Z-scheme was driven by the interfacial bent bands and internal electric field, which was confirmed by electrochemical measurements, electron spin paramagnetic resonance spectroscopy and density functional theory calculations. Moreover, the (110) dominant CeO2 nanorods partially converted Ce4+ to Ce3+ and formed oxygen vacancies, and as an electron mediator in Z-scheme systems to further facilitate charge transfer process and molecular oxygen activation. Under the strong synergistic effect between the large specific surface area, Z-scheme heterojunction and oxygen vacancies, the optimized photocatalyst exhibits 86.7% of photocatalytic removal efficiency. This work provides Z-scheme heterojunction photocatalyst design perspective for photocatalytic air purification.

EPR Study of KO2 as a Source of Superoxide and •BMPO-OH/OOH Radical That Cleaves Plasmid DNA and Detects Radical Interaction with H2S and Se-Derivatives.

Superoxide radical anion (O2•−) and its derivatives regulate numerous physiological and pathological processes, which are extensively studied. The aim of our work was to utilize KO2 as a source of O2•− and the electron paramagnetic resonance (EPR) spin trapping 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) technique for the preparation of •BMPO-OOH and/or •BMPO-OH radicals in water solution without DMSO. The method distinguishes the interactions of various compounds with •BMPO-OOH and/or •BMPO-OH radicals over time. Here, we show that the addition of a buffered BMPO-HCl mixture to powdered KO2 formed relatively stable •BMPO-OOH and •BMPO-OH radicals and H2O2, where the •BMPO-OOH/OH ratio depended on the pH. At a final pH of ~6.5–8.0, the concentration of •BMPO-OOH radicals was ≥20 times higher than that of •BMPO-OH, whereas at pH 9.0–10.0, the •BMPO-OH radicals prevailed. The •BMPO-OOH/OH radicals effectively cleaved the plasmid DNA. H2S decreased the concentration of •BMPO-OOH/OH radicals, whereas the selenium derivatives 1-methyl-4-(3-(phenylselanyl) propyl) piperazine and 1-methyl-4-(4-(phenylselanyl) butyl) piperazine increased the proportion of •BMPO-OH over the •BMPO-OOH radicals. In conclusion, the presented approach of using KO2 as a source of O2•−/H2O2 and EPR spin trap BMPO for the preparation of •BMPO-OOH/OH radicals in a physiological solution could be useful to study the biological effects of radicals and their interactions with compounds.

EPR Spin-Trapping Study of Free Radical Intermediates in Polyalphaolefin Base Oil Autoxidation

Autoxidation is a set of spontaneous oxygen-mediated radical reactions common to degradation processes of all hydrocarbon-based materials. In this work, EPR (electron paramagnetic resonance) spin-trapping technique was used to study radical species involved in the autoxidation of a sample of synthetic base oil. A sample of polyalphaolefin base oil was aged at 140°C in the presence of oxygen and the difference in the tendency to generate radical species between the aged and non-aged samples of the same base oil was investigated using DMPO (5,5-dimethyl-1-pyrroline N-oxide) spin-trap. The ageing was performed in such a way that a small amount of hydroperoxides, the primary and key intermediate in autoxidation reactions, accumulated in the sample. Subsequently, the degradation of hydroperoxides was triggered by three different means: thermal treatment, introduction of soluble transition metal ions, and UV irradiation. Radical intermediates were monitored by EPR spin trapping; mixtures of peroxyl, alkoxyl, and carbon-centered radicals were detected and their origin was discussed.

Perchlorinated Triarylmethyl Radical 99% Enriched 13C at the Central Carbon as EPR Spin Probe Highly Sensitive to Molecular Tumbling.

Soluble stable radicals are used as spin probes and spin labels for in vitro and in vivo electron paramagnetic resonance (EPR) spectroscopy and imaging applications. We report the synthesis and characterization of a perchlorinated triarylmethyl radical enriched 99% at the central carbon, 13C1-PTMTC. The anisotropy of the hyperfine splitting with the 13C1 (Ax = 26, Ay = 25, Az = 199.5 MHz) and the g (gx = 2.0015, gy = 2.0015, gz = 2.0040) are responsible for a strong effect of the radical tumbling rate on the EPR spectrum. The rotational correlation time can be determined by spectral simulation or via the line width or the apparent Az after calibration, so the spin probe 13C1-PTMTC can be used to measure media microviscosity with high sensitivity.

A novel strategy for site selective spin-labeling to investigate bioactive entities by DNP and EPR spectroscopy

A novel specific spin-labeling strategy for bioactive molecules is presented for eptifibatide (integrilin) an antiplatelet aggregation inhibitor, which derives from the venom of certain rattlesnakes. By specifically labeling the disulfide bridge this molecule becomes accessible for analytical techniques such as Electron Paramagnetic Resonance (EPR) and solid state Dynamic Nuclear Polarization (DNP). The necessary spin-label was synthesized and inserted into the disulfide bridge of eptifibatide via reductive followed by insertion by a double Michael addition under physiological conditions. This procedure is universally applicable for disulfide containing biomolecules and is expected to preserve their tertiary structure with minimal change due to the small size of the label and restoring of the previous disulfide connection. HPLC and MS analysis show the successful introduction of the spin label and EPR spectroscopy confirms its activity. DNP-enhanced solid state NMR experiments show signal enhancement factors of up to 19 in 13C CP MAS experiments which corresponds to time saving factors of up to 361. This clearly shows the high potential of our new spin labeling strategy for the introduction of site selective radical spin labels into biomolecules and biosolids without compromising its conformational integrity for structural investigations employing solid-state DNP or advanced EPR techniques.

A Dual Fluorescence–Spin Label Probe for Visualization and Quantification of Target Molecules in Tissue by Multiplexed FLIM–EPR Spectroscopy

AbstractSimultaneous visualization and concentration quantification of molecules in biological tissue is an important though challenging goal. The advantages of fluorescence lifetime imaging microscopy (FLIM) for visualization, and electron paramagnetic resonance (EPR) spectroscopy for quantification are complementary. Their combination in a multiplexed approach promises a successful but ambitious strategy because of spin label‐mediated fluorescence quenching. Here, we solved this problem and present the molecular design of a dual label (DL) compound comprising a highly fluorescent dye together with an EPR spin probe, which also renders the fluorescence lifetime to be concentration sensitive. The DL can easily be coupled to the biomolecule of choice, enabling in vivo and in vitro applications. This novel approach paves the way for elegant studies ranging from fundamental biological investigations to preclinical drug research, as shown in proof‐of‐principle penetration experiments in human skin ex vivo.

Coupling of ethanolamine ammonia-lyase protein and solvent dynamics characterized by the temperature-dependence of EPR spin probe mobility and dielectric permittivity.

Electron paramagnetic resonance (EPR) spectroscopy is used to address the remarkable persistence of the native Arrhenius dependence of the 2-aminopropanol substrate radical rearrangement reaction in B12-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium from physiological to cryogenic (220 K) temperatures. Two-component TEMPOL spin probe mobility in the presence of 10 mM (0.08% v/v) 2-aminopropanol over 200-265 K demonstrates characteristic concentric aqueous-cosolvent mesodomain and protein-associated domain (PAD, hydration layer) solvent phases around EAL in the frozen solution. The mesodomain formed by the relatively small amount of 2-aminopropanol is highly confined, as shown by an elevated temperature for the order-disorder transition (ODT) in the PAD (230-235 K) and large activation energy for TEMPOL rotation. Addition of 2% v/v dimethylsulfoxide expands the mesodomain, partially relieves PAD confinement, and leads to an ODT at 205-210 K. The ODT is also manifested as a deviation of the temperature-dependence of the EPR amplitude of cob(II)alamin and the substrate radical, bound in the enzyme active site, from Curie law behavior. This is attributed to an increase in sample dielectric permittivity above the ODT at the microwave frequency of 9.5 GHz. The relatively high frequency dielectric response indicates an origin in coupled protein surface group-water fluctuations of the Johari-Goldstein β type that span spatial scales of ∼0.1-10 Å on temporal scales of 10-10-10-7 s. The orthogonal EPR spin probe rotational mobility and solvent dielectric measurements characterize features of EAL protein-solvent dynamical coupling and reveal that excess substrate acts as a fluidizing cryosolvent to enable native enzyme reactivity at cryogenic temperatures.

Photoreactivity of Hair Melanin from Different Skin Phototypes-Contribution of Melanin Subunits to the Pigments Photoreactive Properties.

Photoreactivity of melanin has become a major focus of research due to the postulated involvement of the pigment in UVA-induced melanoma. However, most of the hitherto studies were carried out using synthetic melanin models. Thus, photoreactivity of natural melanins is yet to be systematically analyzed. Here, we examined the photoreactive properties of natural melanins isolated from hair samples obtained from donors of different skin phototypes (I, II, III, and V). X-band and W-band electron paramagnetic resonance (EPR) spectroscopy was used to examine the paramagnetic properties of the pigments. Alkaline hydrogen peroxide degradation and hydroiodic acid hydrolysis were used to determine the chemical composition of the melanins. EPR oximetry and spin trapping were used to examine the oxygen photoconsumption and photo-induced formation of superoxide anion, and time-resolved near infrared phosphorescence was employed to determine the singlet oxygen photogeneration by the melanins. The efficiency of superoxide and singlet oxygen photogeneration was related to the chemical composition of the studied melanins. Melanins from blond and chestnut hair (phototypes II and III) exhibited highest photoreactivity of all examined pigments. Moreover, melanins of these phototypes showed highest quantum efficiency of singlet oxygen photogeneration at 332 nm and 365 nm supporting the postulate of the pigment contribution in UVA-induced melanoma.

Site-directed spin labeling-electron paramagnetic resonance spectroscopy in biocatalysis: Enzyme orientation and dynamics in nanoscale confinement

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy probes the otherwise inaccessible structural information in complex biological systems. We recently extended SDSL-EPR to reveal the relative orientation and backbone dynamics of enzymes upon encapsulation in mesoporous nanostructures, which set the structural basis underlying the observed biocatalytic activity. Our strategy had generated interest in the biocatalysis community, and thus in this resource article, we contribute an introduction to the principles and experimental procedure that generalize SDSL-EPR to heterogeneous biocatalysis. We will focus on enzymes in mesoporous materials with examples demonstrating the methods and cautions of potential pitfalls. The ultimate goal is to provide the biocatalysis community with a powerful resource to fill in a long-standing knowledge gap in heterogeneous biocatalysis and the structure-function relationship of enzymes at the interface of enzyme-mesoporous materials and utilize the structural insights to guide the rational design of porous platforms for enzyme immobilization. • Enzyme orientation is determined by using EPR to probe its contact with nanoscale pores • Enzyme dynamics in nanoscale pores and associated catalytic activities are revealed • Protocols and procedures are given for experimental data acquisition, analysis, and interpretation • Various EPR techniques and spin labels can be extended to heterogeneous biocatalysis Enzyme immobilization in nanoscale confinement offers enhanced enzyme protection and reusability, yet the structure-function relationship of the entrapped enzymes remains elusive. Site-directed spin labeling in combination with electron paramagnetic resonance (EPR) spectroscopy has primarily been applied in determining protein structural information in complex biological systems. In this resource article, we introduce the extension of the X-band continuous-wave EPR with a methanethiosulfonate spin label to reveal enzyme structural basis upon immobilization into nanoscale confinement. Using our recent works as examples, we demonstrate the method for determining enzyme orientation and backbone dynamics upon immobilization with a special focus on generalizing the method to heterogeneous biocatalysis and cautions in each step. Furthermore, we summarize the recent findings in other EPR techniques and spin labels so that the whole EPR toolbox can be extended to heterogeneous biocatalysis. In this resource article, Yang and colleagues introduce a general procedure that can reveal the orientation and backbone dynamics of enzymes entrapped in mesoporous materials based on metal-organic materials (MOMs) by using X-band SDSL-EPR. They demonstrate the method by using recently published works focusing on enzyme-MOM interfaces, as well as highlight cautions and potential solutions at each step. They then summarize a variety of other EPR techniques and spin labels in order to pinpoint their potential applications in heterogeneous biocatalysis in the near future.