SimLabel: a graphical user interface to simulate continuous wave EPR spectra from site‐directed spin labeling experiments

Site‐directed spin labeling (SDSL) combined with continuous wave electron paramagnetic resonance (cw EPR) spectroscopy is a powerful technique to reveal, at the residue level, structural transitions in proteins. SDSL‐EPR is based on the selective grafting of a paramagnetic label on the protein under study, followed by cw EPR analysis. To extract valuable quantitative information from SDSL‐EPR spectra and thus give reliable interpretation on biological system dynamics, numerical simulations of the spectra are required. Such spectral simulations can be carried out by coding in MATLAB using functions from the EasySpin toolbox. For non‐expert users of MATLAB, this could be a complex task or even impede the use of such simulation tool. We developed a graphical user interface called SimLabel dedicated to run cw EPR spectra simulations particularly coming from SDSL‐EPR experiments. Simlabel provides an intuitive way to visualize, simulate, and fit such cw EPR spectra. An example of SDSL‐EPR spectra simulation concerning the study of an intrinsically disordered region undergoing a local induced folding is described and discussed. We believe that this new tool will help the users to rapidly obtain reliable simulated spectra and hence facilitate the interpretation of their results. Copyright © 2017 John Wiley & Sons, Ltd.     

Interaction between Molecular Oxygen and Nitroxide Radicals: A Search for a Reversible Complex

At high concentrations of oxygen, the EPR spectrum of the nitroxide radical 4‐oxo‐TEMPO (= 4‐oxo‐2,2,6,6‐tetramethylpiperidin‐1‐yloxy) is found to broaden significantly. In addition to the expected broadening, double integration of the EPR signals indicates that a significant fraction of the nitroxide spins has ‘disappeared’. In perfluoro(2‐butyltetrahydrofuran) at 273 K, the extent of diminution of the EPR signal intensity is ca. 20%. The results are analyzed in terms of collision and supramolecular complexes between oxygen and 4‐oxo‐TEMPO. It is concluded that a supramolecular complex is responsible for the observed phenomenon.     

Surface‐directed morphology evolution in ternary blends of polyethylene,polypropylene, and ethylene–propylene copolymer: A study by laser scanning confocal fluorescence microscopy

With laser scanning confocal fluorescence microscopy, we demonstrate a novel type of morphology evolution in moderately thick films (70–100 μm) of ternary blends of polypropylene (PP), polyethylene (PE), and ethylene–propylene rubber (EPR), in which EPR is labeled with a benzothioxanthene dye (HY‐EPR). The blends are prepared by solution blending, and the phase morphology evolves during the annealing of the blend films in a stainless steel mold. Our results indicate that wetting of the mold surface is a driving force in morphology evolution for the two blend compositions investigated. For 81/14/5 PP/PE/HY‐EPR, phase evolution within the mold results in a laminar structure and hydrodynamic channels, features which have previously been found in thin films of polymer blends as a result of surface‐directed spinodal decomposition. In a blend with a lower weight fraction of the dispersed phase (92/7/1 PP/PE/HY‐EPR), we find that the PE/HY‐EPR domains are larger and more polydisperse closer to the surface because of wetting of the mold wall. We also show that the phase morphology in these films can be controlled by the nature of one or both of the surfaces being varied. When one of the mold surfaces is replaced with a thin film of PP homopolymer, we observe draining of PE/HY‐EPR from the PP to the mold surface, which results in a bilayer structure. A trilayer morphology is likewise obtained by the replacement of both mold surfaces with PP. We also carry out three‐dimensional image reconstruction on a single PE/HY‐EPR particle within the 81/14/5 PP/PE/HY‐EPR blend to obtain detailed information on the interphase structure. We find that HY‐EPR of this composition (30/70 ethylene/propylene) fully coats the PE dispersed phase and partially penetrates the PE droplets. This result falls between the interphase structures found for previously investigated EPR compositions (40/60 and 80/20 ethylene/propylene). © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 637–654, 2003     

Evidence of a Photo‐Radical Pathway Responsible of the Rearrangement of a Manganese(III)‐Schiff Base Complex in Solution

A new Mn^III‐Schiff base complex, [MnL(OH₂)](ClO₄) ( 1 ) (H₂L = N, N′‐bis‐(3‐Br‐5‐Cl‐salicylidene)‐1, 2‐diimino‐2‐methylethane), an inorganic model of the catalytic center (OEC, Oxygen Evolving Complex) in photosystem II (PSII), has been synthesized and characterized by elemental analysis, IR and EPR spectroscopy, mass spectrometry, magnetic susceptibility measurement and the study of its redox properties by cyclic and normal pulse voltammetry. This complex mimics reactivity (showing a relevant photolytic activity), and also some structural characteristics (parallel‐mode Mn^III EPR signal from partially assembled OEC cluster) of the natural OEC. The complex 1 was found to rearrange in solution into a crystallographically solved square‐pyramidal complex, [MnLL′] ( 2 ) (HL′ = 6‐bromo‐4‐chloro‐2‐cyanophenol), through a process, which probably liberates radical species (detected by EPR), and provokes a C—N bond cleavage in the ligand. A photo‐radical mechanism is discussed to explain this rearrangement.     

Characterizing Active Pharmaceutical Ingredient Binding to Human Serum Albumin by Spin‐Labeling and EPR Spectroscopy

Drug binding to human serum albumin (HSA) has been characterized by a spin‐labeling and continuous‐wave (CW) EPR spectroscopic approach. Specifically, the contribution of functional groups (FGs) in a compound on its albumin‐binding capabilities is quantitatively described. Molecules from different drug classes are labeled with EPR‐active nitroxide radicals (spin‐labeled pharmaceuticals (SLPs)) and in a screening approach CW‐EPR spectroscopy is used to investigate HSA binding under physiological conditions and at varying ratios of SLP to protein. Spectral simulations of the CW‐EPR spectra allow extraction of association constants (K_A) and the maximum number (n) of binding sites per protein. By comparison of data from 23 SLPs, the mechanisms of drug–protein association and the impact of chemical modifications at individual positions on drug uptake can be rationalized. Furthermore, new drug modifications with predictable protein binding tendency may be envisaged.     

Role of radicals in UV‐initiated postplasma grafting of poly‐ε‐caprolactone: An electron paramagnetic resonance study

Electron paramagnetic resonance (EPR) measurements were performed on poly‐ε‐caprolactone (PCL) films at different stages of the postplasma‐grafting process. PCL films prepared by solvent casting (SC) or electrospinning (ESP) yield very similar EPR spectra after Ar‐plasma treatment and subsequent exposure to air, but the EPR signal is much stronger in the PCL‐ESP films. The free radicals appear to be mainly, and possibly exclusively, oxygen centered. The radicals generated by UV irradiation in PCL‐ESP films were studied in situ with EPR, using a UV‐LED (λ = (285 ± 5) nm). Their EPR spectrum is distinctly different from the plasma‐induced signal, indicative of carbon‐centered radicals, and appears to be independent of the plasma pretreatment. UV‐induced homolytic splitting of (hydro)peroxide bonds was not observed. Both the plasma‐ and UV‐induced radicals decay at room temperature (RT), even in an inert atmosphere. This study demonstrates the potential of electrospun films and UV‐LEDs for the study of plasma‐ and UV‐generated free radicals with EPR in polyesters, and raises questions with respect to the validity of some generally accepted molecular mechanisms underpinning the postplasma grafting technique for polyesters. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Polyamide 66/EPR‐g‐MA blends: mechanical modeling and kinetic analysis of thermal degradation

The present investigation deals with the mechanical, thermal, and morphological properties of binary nylon 66/maleic anhydride grafted ethylene propylene rubber (EPR‐g‐MA) blends at different dispersed phase (EPR‐g‐MA) concentrations. The effects of EPR‐g‐MA concentration and dispersed particle size on the mechanical properties of the blends were studied. Analysis of the tensile data in terms of various theoretical models revealed the variation of stress concentration effect with blend composition and the improvement of interfacial adhesion between dispersed rubber phase and nylon 66 matrix. The thermal degradation of the blends was analyzed by nonisothermal thermogravimetric analysis (TGA). It was found that the activation energy (E_a) and overall reaction order of thermal degradation decreased with increasing EPR‐g‐MA content. The scanning electron microscopic (SEM) analysis showed a significant decrease in dispersed particle size with increasing EPR‐g‐MA content, which was explained on the basis of the level of chemical interaction (in situ compatibilization) between nylon 66 and EPR‐g‐MA. The surface morphology of the nylon 66/EPR‐g‐MA blends was illustrated by the roughness of atomic force microscopy (AFM) images. Copyright © 2008 John Wiley & Sons, Ltd.     

Syntheses of graft and star copolymers possessing polyolefin branches by using polyolefin macromonomer

Graft and star copolymers having poly(methacrylate) backbone and ethylene–propylene random copolymer (EPR) branches were successfully synthesized by radical copolymerization of an EPR macromonomer with methyl methacrylate (MMA). EPR macromonomers were prepared by sequential functionalization of vinylidene chain‐end group in EPR via hydroalumination, oxidation, and esterification reactions. Their copolymerizations with MMA were carried out with monofunctional and tetrafunctional initiators by atom transfer radical polymerization (ATRP). Gel‐permeation chromatography and NMR analyses confirmed that poly(methyl methacrylate) (PMMA)‐g‐EPR graft copolymers and four‐arm (PMMA‐g‐EPR) star copolymers could be synthesized by controlling EPR contents in a range of 8.6–38.1 wt % and EPR branch numbers in a range of 1–14 branches. Transmission electron microscopy of these copolymers demonstrated well‐dispersed morphologies between PMMA and EPR, which could be controlled by the dispersion of both segments in the range between 10 nm and less than 1 nm. Moreover, the differentiated thermal properties of these copolymers were demonstrated by differential scanning calorimetry analysis. On the other hand, the copolymerization of EPR macromonomer with MMA by conventional free radical polymerization with 2,2′‐azobis(isobutyronitrile) also gave PMMA‐g‐EPR graft copolymers. However, their morphology and thermal property remarkably differed from those of the graft copolymers obtained by ATRP. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 5103–5118, 2005     

The nature of Cu(II) species in ATRP: New insights via EPR

Identification of the paramagnetic species present in the Cu(I)Br‐catalyzed atom transfer radical polymerization (ATRP) of a model monomer (isobornyl acrylate) has been carried out by electron paramagnetic resonance (EPR) in the continuous wave mode at 90 K. Up to five different species—four copper‐based species and one organic radical—were detected with this technique. The EPR parameters of the copper‐based species are found to differ strongly, and originate from diverse isolated Cu(II) complexes, as well as dipolarly interacting and even exchange‐coupled Cu(II) species. The work highlights the complexity of the copper‐based EPR signal observed in copper‐mediated ATRP reactions. Analysis of the time evolution of the individual EPR contributions reveals the disadvantages of quantitative kinetics studies based on the summed EPR intensity of all copper‐based species, as is commonly used in literature for this type of reactions. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 1493–1501, 2010     

Reactivity of functional SAN toward coreactive EPR‐g‐MA at planar interface

The grafting kinetics of reactive poly(styrene‐co‐acrylonitrile) (SAN) onto EPR‐g‐MA was studied under isothermal conditions, at the planar interface of an SAN/ethylene‐propylene rubber (EPR) bilayer film in relation to the type of reactive groups, NH₂ versus carbamate (which is an amine precursor), attached to SAN. The amount of SAN chemically bound to EPR chains at the interface was estimated by selectively washing off the unreacted SAN chains before X‐ray photon spectroscopic analysis of the released surface. It is clear that the mutual reactivity of the reactive groups, i.e., the NH₂–MA pair versus the carbamate–MA pair, has a decisive effect on the amount of SAN that reacts with EPR‐g‐MA at the interface. In case of SAN‐carb, the grafting reaction is controlled by the thermolysis of the carbamate groups into primary amines. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 3682–3689, 2000     

Discriminative Detection of Biothiols by Electron Paramagnetic Resonance Spectroscopy using a Methanethiosulfonate Trityl Probe

Biothiols, such as glutathione (GSH), homocysteine (Hcy), and cysteine (Cys), coexist in biological systems with diverse biological roles. Thus, analytical techniques that can detect, quantify, and distinguish between multiple biothiols are desirable but challenging. Herein, we demonstrate the simultaneous detection and quantitation of multiple biothiols, including up to three different biothiols in a single sample, using electron paramagnetic resonance (EPR) spectroscopy and a trityl‐radical‐based probe (MTST). We term this technique EPR thiol‐trapping. MTST could trap thiols through its methanethiosulfonate group to form the corresponding disulfide conjugate with an EPR spectrum characteristic of the trapped thiol. MTST was used to investigate effects of l ‐buthionine sulfoximine (BSO) and pyrrolidine dithiocarbamate (PDTC) on the efflux of GSH and Cys from HepG2 cells.     

Radiochemical aging of ethylene–propylene–diene monomer elastomers. I. Mechanism of degradation under inert atmosphere

This article is devoted to the study of electron‐beam‐induced degradation under argon atmosphere of an ethylene–propylene–diene monomer (EPDM, based on 5‐ethylidene 2‐norbornene) and an ethylene–propylene rubber (EPR) containing the same molar ratio of ethylene/propylene. The chemical structure modifications of polymeric samples were analyzed by ultraviolet–visible and IR spectroscopies. Crosslinking reactions were deduced by measuring the changes in gel fraction and the degree of swelling in n‐heptane. Irradiation of EPDM and EPR created trans‐vinylene, vinyl, vinylidene, and dienic‐type unsaturations. The radiochemical yields for unsaturation formations in EPDM and EPR were similar. Degradation also involved crosslinking and the production of molecular hydrogen. The comparison between EPDM and EPR showed that the diene (in which a double bond is consumed with a high radiochemical yield) contributes to the increase in rate and intermolecular bridges density. Mechanisms are proposed to account for the main routes of EPDM degradation. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 1239–1248, 2004     

Molecular‐Weight‐Dependence on Domain Formation of Grafted Poly(ethylene‐co‐propylene) in a Poly(propylene) Matrix

Several novel poly(propylene)‐graft‐poly(ethylene‐co‐propylene) copolymers with isotactic poly(propylene) (PP) backbones and ethylene/propylene rubber (EPR) branches were synthesized. The thermomechanical properties of these samples were investigated using a dynamic mechanical analyzer. There appeared to be a critical EPR molecular weight above which a two‐phase system developed with EPR domains dispersed in a PP matrix. This domain formation gave an enhanced loss modulus compared to a commercial high impact PP product below 40°C.     

Spectroscopic Observation of the Triplet Diradical State of a Cyclobutadiene

Tetrakis(trimethylsilyl)cyclobuta‐1,3‐diene ( 1 ) was subjected to a temperature‐dependent EPR study to allow the first spectroscopic observation of a triplet diradical state of a cyclobutadiene ( 2 ). From the temperature dependent EPR absorption area we derive a singlet→triplet ( 1 → 2 ) energy gap, E _ST, of 13.9 kcal mol⁻¹, in agreement with calculated values. The zero‐field splitting parameters D =0.171 cm⁻¹, E =0 cm⁻¹ are accurately reproduced by DFT calculations. The triplet diradical 2 is thermally accessible at moderate temperatures. It is not an intermediate in the thermal cycloreversion of cyclobutadiene to two acetylene molecules.     

Improved Sensitivity for Imaging Spin Trapped Hydroxyl Radical at 250 MHz

Radicals, including hydroxyl, superoxide, and nitric oxide, play key signaling roles in vivo. Reaction of these free radicals with a spin trap affords more stable paramagnetic nitroxides, but concentrations in vivo still are so low that detection by electron paramagnetic resonance (EPR) is challenging. Three innovative enabling technologies have been combined to substantially improve sensitivity for imaging spin‐trapped radicals at 250 MHz. 1) Spin‐trapped adducts of BMPO have lifetimes that are long enough to make imaging by EPR at 250 MHz feasible. 2) The signal‐to‐noise ratio of rapid‐scan EPR is substantially higher than for conventional continuous‐wave EPR. 3) An improved algorithm permits image reconstruction with a spectral dimension that encompasses the full 50 G spectrum of the BMPO–OH spin adduct without requiring the wide sweeps that would be needed for filtered backprojection. A 2D spectral–spatial image is shown for a phantom containing ca. 5 μM BMPO–OH.     

Phase Separation of Poly(N‐isopropylacrylamide) in Mixtures of Water and Methanol: A Spectroscopic Study of the Phase‐Transition Process with a Polymer Tagged with a Fluorescent Dye and a Spin Label

The thermoreversible phase transition of poly(N‐isopropylacrylamide) randomly labeled with a spin label, 4‐amino‐2,2′,6,6′‐tetramethylpiperidine 1‐oxide (TEMPO), and a fluorescent dye, 4‐(pyren‐1‐yl)butyl (PNIPAM‐Py‐T), in different H₂O/MeOH mixtures was studied by turbidimetry, continuous‐wave electron paramagnetic resonance spectroscopy (CW‐EPR), and fluorescence spectroscopy. The macroscopic phase diagram of PNIPAM‐Py‐T in H₂O/MeOH measured by turbidimetry was identical to those of poly(N‐isopropylacrylamide) (PNIPAM) and of TEMPO‐labeled PNIPAM (PNIPAM‐T) in H₂O/MeOH mixtures. However, distinct differences among the three polymers were detected in their solvent‐dependent EPR and fluorescence‐spectroscopic properties. The EPR spectra were analyzed in terms of the isotropic hyperfine coupling constants, which monitor the variation in environmental polarity of the radical labels occurring for the conformational transitions of the polymer as a function of temperature, as well as the correlation time for reorientation motion, the increase of which is indicative of the increased viscosity of the radical environment and interactions occurring between the radical and other surface groups of the precipitated polymer, if compared to the soluble polymer. The fluorescence of Py in PNIPAM‐Py‐T displayed contributions from isolated excited pyrenes (monomer emission) and from preformed pyrene ground‐state aggregates (excimer emission). The quantum efficiencies of monomer and excimer emission were monitored as a function of solvent composition. By the two experimental approaches, we demonstrate the profound influence of the PNIPAM‐attached pyrene units in increasing the hydrophobicity of the nanodomains formed upon heat‐induced precipitation of PNIPAM‐Py‐T.     

Bioconjugation of Proteins with a Paramagnetic NMR and Fluorescent Tag

Site‐specific labeling of proteins with lanthanide ions offers great opportunities for investigating the structure, function, and dynamics of proteins by virtue of the unique properties of lanthanides. Lanthanide‐tagged proteins can be studied by NMR, X‐ray, fluorescence, and EPR spectroscopy. However, the rigidity of a lanthanide tag in labeling of proteins plays a key role in the determination of protein structures and interactions. Pseudocontact shift (PCS) and paramagnetic relaxation enhancement (PRE) are valuable long‐range structure restraints in structural‐biology NMR spectroscopy. Generation of these paramagnetic restraints generally relies on site‐specific tagging of the target proteins with paramagnetic species. To avoid nonspecific interaction between the target protein and paramagnetic tag and achieve reliable paramagnetic effects, the rigidity, stability, and size of lanthanide tag is highly important in paramagnetic labeling of proteins. Here 4′‐mercapto‐2,2′: 6′,2′′‐terpyridine‐6,6′′‐dicarboxylic acid (4MTDA) is introduced as a a rigid paramagnetic and fluorescent tag which can be site‐specifically attached to a protein by formation of a disulfide bond. 4MTDA can be readily immobilized by coordination of the protein side chain to the lanthanide ion. Large PCSs and RDCs were observed for 4MTDA‐tagged proteins in complexes with paramagnetic lanthanide ions. At an excitation wavelength of 340 nm, the complex formed by protein–4MTDA and Tb³⁺ produces high fluorescence with the main emission at 545 nm. These interesting features of 4MTDA make it a very promising tag that can be exploited in NMR, fluorescence, and EPR spectroscopic studies on protein structure, interaction, and dynamics.     

Fourier Transform EPR Spectroscopy of Trityl Radicals for Multifunctional Assessment of Chemical Microenvironment

Pulse techniques in electron paramagnetic resonance (EPR) allow for a reduction in measurement times and increase in sensitivity but require the synthesis of paramagnetic probes with long relaxation times. Here it is shown that the recently synthesized phosphonated trityl radical possesses long relaxation times that are sensitive to probe the microenvironment, such as oxygenation and acidity of an aqueous solution. In principle, application of Fourier transform EPR (FT‐EPR) spectroscopy makes it possible to acquire the entire EPR spectrum of the trityl probe and assess these microenvironmental parameters within a few microseconds. The performed analysis of the FT‐EPR spectra takes into consideration oxygen‐, proton‐, buffer‐, and concentration‐induced contributions to the spectral shape, therefore enabling quantitative and discriminative assessment of pH, pO₂, and concentrations of the probe and inorganic phosphate.     

Pulse EPR Methods for Studying Chemical and Biological Samples Containing Transition Metals

This review discusses the application of pulse EPR to the characterization of disordered systems, with an emphasis on samples containing transition metals. Electron nuclear double‐resonance (ENDOR), electron‐spin‐echo envelope‐modulation (ESEEM), and double electron–electron resonance (DEER) methodologies are outlined. The theory of field modulation is outlined, and its application is illustrated with DEER experiments. The simulation of powder spectra in EPR is discussed, and strategies for optimization are given. The implementation of this armory of techniques is demonstrated on a rich variety of chemical systems: several porphyrin derivatives that are found in proteins and used as model systems, otherwise highly reactive aminyl radicals stabilized with electron‐rich transition metals, and nitroxide–copper–nitroxide clusters. These examples show that multi‐frequency continuous‐wave (CW) and pulse EPR provides detailed information about disordered systems.     

Eine neuartige Methode zur Analyse kombinatorischer Substanzbibliotheken mittels paramagnetischer Reporter

A Novel Method to Identify Chemical Compounds of Combinatorial Libraries by the Use of Paramagnetic Tags An EPR method to identify non‐destructively chemical compounds bound to a single solid‐phase‐synthesis bead for combinatorial chemistry applications is discribed. During synthesis chemical inert paramagnetic substances can be attached in small amounts to a solid‐phase‐synthesis resin for tagging of organic compounds or even reaction steps. The identification of single members of a combinatorial library in short time and high sensitivity can be carried out by using an EPR‐spectrometer.