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In cancer diagnostics, magnetic resonance imaging (MRI) uses contrast agents to enhance the distinction between the target tissue and background. Several promising approaches have been developed to increase MRI sensitivity, one of which is Overhauser dynamic nuclear polarization (ODNP)-enhanced MRI (OMRI). In this study, a macromolecular construct based on human serum albumin and nitroxyl radicals (HSA-NIT) was developed using a new synthesis method that significantly increased the modification to 21 nitroxide residues per protein. This was confirmed by electron paramagnetic resonance (EPR) spectroscopy and matrix-assisted laser desorption/ionization time-of-flight (MALDI ToF) mass spectrometry. Gel electrophoresis and circular dichroism showed no significant changes in the structure of HSA-NITs, and no oligomers were formed during modification. The cytotoxicity of HSA-NITs was comparable to that of native albumin. HSA-NITs were evaluated as potential "metal-free" organic radical relaxation-based contrast agents for 1H-MRI and as hyperpolarizing contrast agents for OMRI. Relaxivities (longitudinal and transversal relaxation rates r1 and r2) for HSA-NITs were measured at different magnetic field strengths (1.88, 3, 7, and 14 T). Phantoms were used to demonstrate the potential use of HSA-NIT as a T1- and T2-weighted relaxation-based contrast agent at 3 T and 14 T. The efficacy of 1H Overhauser dynamic nuclear polarization (ODNP) in liquids at an ultralow magnetic field (ULF, B0 = 92 ± 0.8 µT) was investigated for HSA-NIT conjugates. The HSA-NITs themselves did not show ODNP enhancement; however, under the proteolysis conditions simulating cancer tissue, HSA-NIT conjugates were cleaved into lower-molecular-weight (MW) protein fragments that activate ODNP capabilities, resulting in a maximum achievable enhancement |Emax| of 40-50 and a radiofrequency power required to achieve half of Emax, P1/2, of 21-27 W. The HSA-NIT with a higher degree of modification released increased the number of spin probes upon biodegradation, which significantly enhanced the Overhauser effect. Thus, HSA-NITs may represent a new class of MRI relaxation-based contrast agents as well as novel cleavable conjugates for use as hyperpolarizing contrast agents (HCAs) in OMRI.
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Neoplasias , Óxidos de Nitrogênio , Albumina Sérica Humana , Humanos , Meios de Contraste , Imageamento por Ressonância MagnéticaRESUMO
Photo-excited triplet states represent a new class of spin labels in pulse electron paramagnetic resonance (EPR), attracting increasing attention because of their unique spectroscopic properties. Despite certain advantages, the use of photo-labels has also some challenges, e.g. low repetition rates due to technical laser-related limitations and intrinsic properties of the labels. The application of additional pulse trains for multiple refocusing of the electron spin echo and integration of all observed echoes can significantly enhance sensitivity at a given repetition rate. In this work, we demonstrate that the use of Carr-Parcel-Meiboom-Gill (CPMG) blocks followed by multiple echo integration is a promising route for sensitivity gain in pulsed EPR utilizing photo-excited triplet states, including light-induced pulsed dipolar spectroscopy (LiPDS). The reduction of accumulation time by a factor of 5.3 has been achieved using a commercial pulsed EPR spectrometer with the implementation of a CPMG block and an external digitizer. The methodology of using CPMG refocusing with multiple echo integration in light-induced pulsed EPR experiments is discussed, aiding future applications of this approach in LiPDS experiments.
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The binding of G-quadruplex structures (G4s) with photosensitizers is of considerable importance in medicinal chemistry and drug discovery due to their promising potential in photodynamic therapy applications. G4s can experience structural changes as a result of ligand interactions and light exposure. Understanding these modifications is essential to uncover the fundamental biological roles of the complexes and optimize their therapeutic potential. The structural diversity of G4s makes it challenging to study their complexes with ligands, necessitating the use of various complementary methods to fully understand these interactions. In this study, we introduce, for the first time, the application of laser-induced dipolar EPR as a method to characterize G-quadruplex DNA complexes containing photosensitizers and to investigate light-induced structural modifications in these systems. To demonstrate the feasibility of this approach, we studied complexes of the human telomeric G-quadruplex (HTel-22) with cationic 5,10,15,20-tetrakis(1-methyl-4-pyridinio) porphyrin tetra(p-toluenesulfonate) (TMPyP4). In addition to showcasing a new methodology, we also aimed to provide insights into the mechanisms underlying photoinduced HTel-22/TMPyP4 structural changes, thereby aiding in the advancement of approaches targeting G4s in photodynamic therapy. EPR revealed G-quadruplex unfolding and dimer formation upon light exposure. Our findings demonstrate the potential of EPR spectroscopy for examining G4 complexes with photosensitizers and contribute to a better understanding of G4s' interactions with ligands under light.
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Quadruplex G , Porfirinas , Humanos , Fármacos Fotossensibilizantes , Ligantes , Porfirinas/química , DNA/químicaRESUMO
Triplet states of photoexcited organic molecules are promising spin labels with advanced spectroscopic properties for pulsed dipolar electron paramagnetic resonance (PD EPR) spectroscopy. Recently proposed triplet fullerene labels have shown great potential for double electron-electron resonance (DEER) distance measurements as "observer spins" due to a high quantum yield of the triplet state, hyperpolarization and relatively narrow EPR spectra. Here, we demonstrate the applicability of fullerene labels to other PD EPR techniques, such as relaxation induced dipolar modulation enhancement (RIDME) and laser induced magnetic dipolar spectroscopy (LaserIMD). In particular, a specific contaminating signal in LaserIMD experiments was observed, explained and mitigated. Comparative analyses of the signal-to-noise (SNR) ratios were performed for all employed methods. DEER on the fullerene-triarylmethyl pair shows the best performance, which allows state-of-the-art DEER acquisition at 100 nM with a SNR of â¼35 within reasonable 42 hours.
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Fulerenos , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Marcadores de SpinRESUMO
Zeolite imidazolate framework-8 (ZIF-8) is a promising platform for drug delivery, and information regarding the stability of ZIF-8 nanoparticles in cell culture media is essential for proper interpretation of in vitro experimental results. In this work, we report a quantitative investigation of the ZIF-8 nanoparticle's stability in most common cell culture media. To this purpose, ZIF-8 nanoparticles containing sterically shielded nitroxide probes with high resistance to reduction were synthesized and studied using electron paramagnetic resonance (EPR). The degradation of ZIF-8 in cell media was monitored by tracking the cargo leakage. It was shown that nanoparticles degrade at least partially in all studied media, although the degree of cargo leakage varies widely. We found a strong correlation between the amount of escaped cargo and total concentration of amino acids in the environment. We also established the role of individual amino acids in ZIF-8 degradation. Finally, 2-methylimidazole preliminary dissolved in cell culture media partially inhibits the degradation of ZIF-8 nanoparticles. The guidelines for choosing the proper cell culture medium for the in vitro study of ZIF-8 nanoparticles have been formulated.
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Nanopartículas , Zeolitas , Aminoácidos , Técnicas de Cultura de Células , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Zeolitas/químicaRESUMO
The features of previously unexplored labile complexes of human 40S ribosomal subunits with RNAs, whose formation is manifested in the cross-linking of aldehyde derivatives of RNAs to the ribosomal protein uS3 through its peptide 55-64 located outside the mRNA channel, were studied by EPR spectroscopy methods. Analysis of subatomic 40S subunit models showed that a likely site for labile RNA binding is a cluster of positively charged amino acid residues between the mRNA entry site and uS3 peptide 55-64. This is consistent with our finding that the 3'-terminal mRNA fragment hanging outside the 40S subunit prevents the cross-linking of an RNA derivative to this peptide. To detect labile complexes of 40S subunits with RNA by DEER/PELDOR spectroscopy, an undecaribonucleotide derivative with nitroxide spin labels at terminal nucleotides was utilized. We demonstrated that the 40S subunit channel occupancy with mRNA does not affect the RNA derivative binding and that uS3 peptide 55-64 is not involved in binding interactions. Replacing the RNA derivative with a DNA one revealed the importance of ribose 2'-OH groups for the complex formation. Using the single-label RNA derivatives, the distance between the mRNA entry site and the loosely bound RNA site on the 40S subunit was estimated.
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Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Ligação Proteica , RNA Mensageiro/química , RNA de Transferência/química , RNA de Transferência/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/químicaRESUMO
A DNA molecule is under continuous influence of endogenous and exogenous damaging factors, which produce a variety of DNA lesions. Apurinic/apyrimidinic sites (abasic or AP sites) are among the most common DNA lesions. In this work, we applied pulse dipolar electron paramagnetic resonance (EPR) spectroscopy in combination with molecular dynamics (MD) simulations to investigate in-depth conformational changes in DNA containing an AP site and in a complex of this DNA with AP endonuclease 1 (APE1). For this purpose, triarylmethyl (TAM)-based spin labels were attached to the 5' ends of an oligonucleotide duplex, and nitroxide spin labels were introduced into APE1. In this way, we created a system that enabled monitoring the conformational changes of the main APE1 substrate by EPR. In addition, we were able to trace substrate-to-product transformation in this system. The use of different (orthogonal) spin labels in the enzyme and in the DNA substrate has a crucial advantage allowing for detailed investigation of local damage and conformational changes in AP-DNA alone and in its complex with APE1.
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DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA/química , Oligonucleotídeos/química , Marcadores de Spin/síntese química , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , DNA/genética , DNA/metabolismo , Dano ao DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Oligonucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
RL2 is a recombinant analogue of a human κ-casein fragment, capable of penetrating cells and inducing apoptosis of cancer cells with no toxicity to normal cells. The exact mechanism of RL2 penetration into cells remains unknown. In this study, we investigated the mechanism of RL2 penetration into human lung cancer A549 cells by a combination of electron paramagnetic resonance (EPR) spectroscopy and confocal laser scanning microscopy. EPR spectra of A549 cells incubated with RL2 (sRL2) spin-labeled by a highly stable 3-carboxy-2,2,5,5-tetraethylpyrrolidine-1-oxyl radical were found to contain three components, with their contributions changing with time. The combined EPR and confocal-microscopy data allowed us to assign these three forms of sRL2 to the spin-labeled protein sticking to the membrane of the cell and endosomes, to the spin-labeled protein in the cell interior, and to spin labeled short peptides formed in the cell because of protein digestion. EPR spectroscopy enabled us to follow the kinetics of transformations between different forms of the spin-labeled protein at a minimal spin concentration (3-16 µM) in the cell. The prospects of applications of spin-labeled cell-penetrating peptides to EPR imaging, DNP, and magnetic resonance imaging are discussed, as is possible research on an intrinsically disordered protein in the cell by pulsed dipolar EPR spectroscopy.
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Peptídeos Penetradores de Células , Proteínas Intrinsicamente Desordenadas , Neoplasias Pulmonares , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , CinéticaRESUMO
Trityl radicals (TAMs) have recently appeared as an alternative source of spin labels for measuring long distances in biological systems. Finland trityl radical (FTAM) served as the basis for this new generation of spin labels, but FTAM is rather lipophilic and susceptible to self-aggregation, noncovalent binding with lipophilic sites of proteins, and noncovalent docking at the termini of duplex DNA. In this paper the very hydrophilic OX063 TAM with very low toxicity and little tendency for aggregation is used as the basis for a spin label. Human serum albumin (HSA) labeled with OX063 has an intense narrow line typical of TAM radicals in solution, whereas HSA labeled with FTAM shows broad lines and extensive aggregation. In pulse EPR measurements, the measured phase memory time TM for HSA labeled with OX063 is 6.3â µs at 50â K, the longest yet obtained with a TAM-based spin label. The lowered lipophilicity also decreases side products in the labeling reaction.
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Indicadores e Reagentes/química , Mesilatos/química , Albumina Sérica Humana/química , Compostos de Sulfidrila/química , Espectroscopia de Ressonância de Spin Eletrônica , Interações Hidrofóbicas e Hidrofílicas , Estrutura Molecular , Marcadores de Spin , Relação Estrutura-Atividade , TemperaturaRESUMO
The model mRNA (MR), 11-mer RNA containing two nitroxide spin labels at the 5'- and 3'-terminal nucleotides and prone to form a stable homodimer (MR)2, was used for Electron Paramagnetic Resonance study of structural rearrangements in mRNA occurring upon its binding to human 80S ribosomes. The formation of two different types of ribosomal complexes with MR was observed. First, there were stable complexes where MR was fixed in the ribosomal mRNA-binding channel by the codon-anticodon interaction(s) with cognate tRNA(s). Second, we for the first time detected complexes assembled without tRNA due to the binding of MR most likely to an exposed peptide of ribosomal protein uS3 away from the mRNA channel. The analysis of interspin distances allowed the conclusion that 80S ribosomes facilitate dissociation of the duplex (MR)2: the equilibrium between the duplex and the single-stranded MR shifts to MR due to its efficient binding with ribosomes. Furthermore, we observed a significant influence of tRNA bound at the ribosomal exit (E) and/or aminoacyl (A) sites on the stability of ribosomal complexes. Our findings showed that a part of mRNA bound in the ribosome channel, which is not involved in codon-anticodon interactions, has more degrees of freedom than that interacting with tRNAs.
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Espectroscopia de Ressonância de Spin Eletrônica/métodos , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Anticódon/metabolismo , Sítios de Ligação , Códon/metabolismo , Humanos , Conformação de Ácido Nucleico , Ligação Proteica , RNA Mensageiro/química , RNA de Transferência/química , RNA de Transferência/metabolismo , Marcadores de SpinRESUMO
Recently, the photoexcited triplet state of porphyrin was proposed as a promising spin-label for pulsed dipolar electron paramagnetic resonance (EPR). Herein, we report the factors that determine the electron spin echo dephasing of the photoexcited porphyrin in a water-glycerol matrix. The electron spin relaxation of a water-soluble porphyrin was measured by Q-band EPR, and the temperature dependence and the effect of solvent deuteration on the relaxation times were studied. The phase memory relaxation rate (1/Tm) is noticeably affected by solvent nuclei and is substantially faster in protonated solvents than in deuterated solvents. The Tm is as large as 13-17 µs in deuterated solvent, potentially expanding the range of distances available for measurement by dipole spectroscopy with photoexcited porphyrin. The 1/Tm depends linearly on the degree of solvent deuteration and can be used to probe the environment of a porphyrin in or near a biopolymer, including the solvent accessibility of porphyrins used in photodynamic therapy. We characterized the noncovalent binding of porphyrin to human serum albumin (HSA) from 1/Tm and electron spin echo envelope modulation (ESEEM) and found that porphyrin is quite exposed to solvent on the surface of HSA. The 1/Tm and ESEEM are equally effective and provide complementary methods to determine the solvent accessibility of a porphyrin bound to protein or to determine the location of the porphyrin.
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Vidro/química , Glicerol/química , Porfirinas/química , Albumina Sérica Humana/química , Água/química , Elétrons , Humanos , Porfirinas/metabolismo , Albumina Sérica Humana/metabolismo , Marcadores de SpinRESUMO
Pulsed Dipolar Spectroscopy (PDS) methods of Electron Paramagnetic Resonance (EPR) were used to detect and characterize reversible non-covalent dimers of Human Serum Albumin (HSA), the most abundant protein in human plasma. The spin labels, MTSL and OX063, were attached to Cys-34 and these chemical modifications of Cys-34 did affect the dimerization of HSA, indicating that other post-translational modifications can modulate dimer formation. At physiologically relevant concentrations, HSA does form weak, non-covalent dimers with a well-defined structure. Dimer formation is readily reversible into monomers. Dimerization is very relevant to the role of HSA in the transport, binding, and other physiological processes.
Assuntos
Albumina Sérica Humana/química , Cisteína/química , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Multimerização Proteica , Marcadores de SpinRESUMO
Four albumin-nitroxide conjugates were prepared and tested as metal-free organic radical contrast agents (ORCAs) for magnetic resonance imaging (MRI). Each human serum albumin (HSA) carrier bears multiple nitroxides conjugated via homocysteine thiolactones. These molecular conjugates retain important physical and biological properties of their HSA component, and the resistance of their nitroxide groups to bioreduction was retained or enhanced. The relaxivities are similar for these four conjugates and are much greater than those of their individual components: the HSA or the small nitroxide molecules. This new family of conjugates has excellent prospects for optimization as ORCAs.
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Meios de Contraste/química , Imageamento por Ressonância Magnética , Óxidos de Nitrogênio/química , Albumina Sérica Humana/química , Coloração e Rotulagem , Ácidos Carboxílicos/química , Morte Celular , Espectroscopia de Ressonância de Spin Eletrônica , Homocisteína/análogos & derivados , Homocisteína/química , Humanos , Cinética , Óxidos de Nitrogênio/síntese química , Imagens de Fantasmas , Estrutura Secundária de ProteínaRESUMO
Trityl and nitroxide radicals are connected by π-topologically controlled aryl linkers, generating genuinely g-engineered biradicals. They serve as a typical model for biradicals in which the exchange (J) and hyperfine interactions compete with the g-difference electronic Zeeman interactions. The magnetic properties underlying the biradical spin Hamiltonian for solution, including J's, have been determined by multifrequency CW-ESR and 1H ENDOR spectroscopy and compared with those obtained by quantum chemical calculations. The experimental J values were in good agreement with the quantum chemical calculations. The g-engineered biradicals have been tested as a prototype for AWG (Arbitrary Wave Generator)-based spin manipulation techniques, which enable GRAPE (GRAdient Pulse Engineering) microwave control of spins in molecular magnetic resonance spectroscopy for use in molecular spin quantum computers, demonstrating efficient signal enhancement of specific weakened hyperfine signals. Dynamic nuclear polarization (DNP) effects of the biradicals for 400 MHz nuclear magnetic resonance signal enhancement have been examined, giving efficiency factors of 30 for 1H and 27.8 for 13C nuclei. The marked DNP results show the feasibility of these biradicals for hyperpolarization.
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Precise nanoscale distance measurements by pulsed electron paramagnetic resonance (EPR) spectroscopy play a crucial role in structural studies of biomolecules. The properties of the spin labels used in this approach determine the sensitivity limits, attainable distances, and proximity to biological conditions. Herein, we propose and validate the use of photoexcited fullerenes as spin labels for pulsed dipolar (PD) EPR distance measurements. Hyperpolarization and the narrower spectrum of fullerenes compared to other triplets (e.g., porphyrins) boost the sensitivity, and superior relaxation properties allow PD EPR measurements up to a near-room temperature. This approach is demonstrated using fullerene-nitroxide and fullerene-triarylmethyl pairs, as well as a supramolecular complex of fullerene with nitroxide-labeled protein. Photoexcited triplet fullerenes can be considered as new spin labels with outstanding spectroscopic properties for future structural studies of biomolecules.
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The unexpected formation of a highly strained polycyclic amine was observed in a one-pot synthesis from cyclopentanone, dimethyl fumarate and ammonium acetate. This multistep reaction includes 1,3-dipolar cycloaddition of dimethyl fumarate to the cyclic azomethine ylide formed in situ from cyclopentanone and ammonia. The polycyclic amine product was easily converted into a sterically shielded polycyclic nitroxide. The EPR spectra and spin relaxation behavior of the nitroxide were studied in solution. The spin relaxation seems well suited for the use as a biological spin label and are comparable with those of cyclic nitroxides with two spirocyclic moieties adjacent to the N-O · group.
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Electron paramagnetic resonance (EPR) based nanometer distance measurements at ambient temperatures are of particular interest for structural biology applications. The nitroxide spin labels commonly used in EPR reveal relatively short transverse relaxation under these conditions, which limits their use for detecting static dipolar interactions. At the same time, the longitudinal relaxation of nitroxide spin labels is still long enough to allow using them as 'pumped' species in the relaxation induced dipolar modulation enhancement (RIDME) experiment where the detection is carried out on the slower relaxing triarylmethyl (TAM) spin labels. In the present study, we report the first demonstration of room-temperature RIDME distance measurements in nucleic acids using TAM as the slow-relaxing detected species and traditional nitroxide as the fast-relaxing partner spin. Two types of immobilizers, glassy trehalose and the modified silica gel Nucleosil, were used for immobilization of the spin-labeled biomolecules. The room-temperature RIDME-based distance distributions are in good agreement with those measured at 80 K by other techniques. Room-temperature RIDME on the spin pairs trityl/nitroxide may become a useful method for the structural characterization of biomacromolecules and biomolecular complexes at near physiological temperatures.
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Nanoscale distance measurements by pulse dipolar Electron paramagnetic resonance (EPR) spectroscopy allow new insights into the structure and dynamics of complex biopolymers. EPR detection requires site directed spin labeling (SDSL) of biomolecule(s), which remained challenging for long RNAs up-to-date. Here, we demonstrate that novel complementary-addressed SDSL approach allows efficient spin labeling and following structural EPR studies of long RNAs. We succeeded to spin-label Hepatitis C Virus RNA internal ribosome entry site consisting of ≈330 nucleotides and having a complicated spatial structure. Application of pulsed double electron-electron resonance provided spin-spin distance distribution, which agrees well with the results of molecular dynamics (MD) calculations. Thus, novel SDSL approach in conjunction with EPR and MD allows structural studies of long natural RNAs with nanometer resolution and can be applied to systems of biological and biomedical significance.
Assuntos
Hepacivirus/genética , RNA Viral/metabolismo , Marcadores de Spin , Alquilação , Sequência de Bases , Espectroscopia de Ressonância de Spin Eletrônica , Sítios Internos de Entrada Ribossomal , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/metabolismo , RNA Viral/química , RNA Viral/genética , TemperaturaRESUMO
Microscopic molecular organization and heterogeneities in ionic liquids (ILs) are of significant fundamental and applied interest. Although many theoretical studies have been dedicated to this topic, the development of experimental methods for studying such heterogeneities is still in demand. In this work we propose a new approach for the characterization of microscopic rigidity and heterogeneities in ILs using stochastic librations (small angle motions) of the nitroxide radicals as a probe. Stable nitroxides are dissolved in ILs, which are then shock-frozen and investigated using pulse Electron Paramagnetic Resonance (EPR) at variable temperatures. Stochastic molecular librations of nitroxides depend on local rigidity of a medium and manifest themselves in different electron dephasing times across the EPR spectrum. The use of advanced spiro-cyclohexane-substituted nitroxides allows implementation of this approach at temperatures up to â¼150 K and above. Remarkably, we have found that librational motions in ILs arise at much lower temperatures (â¼75 K for [Bmim]BF4, [Bmim]PF6, [C10mim]BF4) compared to common organic solvents. This can be rationalized by smaller local rigidity of the medium in ILs, most likely due to localization of nitroxides in low-density heterogeneities formed by non-polar alkyl chains. The structure, size, solubility and other properties of nitroxides are adjustable for particular tasks, therefore the proposed approach can potentially be implemented to probe the rigidity and heterogeneities of any ILs, thus providing vital insights into their molecular-scale self-organization.
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Triarylmethyl (TAM) radicals are widely used in Electron Paramagnetic Resonance (EPR) spectroscopy as spin labels and in EPR imaging as spin probes for in vivo oxymetry. One of the key advantages of TAMs is extremely narrow EPR line, especially in case of deuterated analogues (~5 µT). Another advantage is their slow spin relaxation even at physiological temperatures allowing, in particular, application of pulsed dipolar EPR methods for distance measurements in biomolecules. In this paper a large series of TAM radicals and their deuterated analogues is synthesized, and corresponding spectroscopic parameters including 13C hyperfine constants are obtained for the first time. The negligible dependence of 13C hyperfine constants on solvent, as well as on structure and number of substituents at para-C atoms of aromatic rings, has been found. In addition, we have demonstrated that 13C signals at natural abundance can be employed for successful room-temperature distance measurements using Pulsed Electron Double Resonance (PELDOR or DEER).