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Trehalose has been recently promoted as efficient immobilizer of biomolecules for room-temperature EPR studies, including distance measurements between attached nitroxide spin labels. Generally, the structure of nitroxide influences the electron spin relaxation times, being crucial parameters for room-temperature pulse EPR measurements. Therefore, in this work we investigated a series of nitroxides with different substituents adjacent to NO-moiety including spirocyclohexane, spirocyclopentane, tetraethyl and tetramethyl groups. Electron spin relaxation times (T1, Tm) of these radicals immobilized in trehalose were measured at room temperature at X- and Q-bands (9/34GHz). In addition, a comparison was made with the corresponding relaxation times in nitroxide-labeled DNA immobilized in trehalose. In all cases phase memory times Tm were close to 700ns and did not essentially depend on structure of substituents. Comparison of temperature dependences of Tm at T=80-300K shows that the benefit of spirocyclohexane substituents well-known at medium temperatures (∼100-180K) becomes negligible at 300K. Therefore, unless there are specific interactions between spin labels and biomolecules, the room-temperature value of Tm in trehalose is weakly dependent on the structure of substituents adjacent to NO-moiety of nitroxide. The issues of specific interactions and stability of nitroxide labels in biological media might be more important for room temperature pulsed dipolar EPR than differences in intrinsic spin relaxation of radicals., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The influence of bovine serum albumin (BSA) on collapsing poly(N-isopropylacrylamide) (PNIPAM) chains was studied with turbidimetry and spin probe and spin label electron paramagnetic resonance spectroscopy. An increased ratio of collapsed chains in aqueous solutions in the narrow temperature region near the LCST appeared in the presence of 2.5–10 wt% BSA. The spin probe EPR data indicate that the inner cavities of the BSA dimers are probably responsive to the capture of small hydrophobic or amphiphilic molecules, such as TEMPO nitroxyl radical. The observed features of the structure and dynamics of inhomogeneities of aqueous PNIPAM-BSA solutions, including their mutual influence on the behavior of the polymer and protein below the LCST, should be considered when developing and investigating PNIPAM-based drug delivery systems. [ABSTRACT FROM AUTHOR]

The flavoprotein Cytochrome P450 reductase (CPR) is the unique electron pathway from NADPH to Cytochrome P450 (CYPs). The conformational dynamics of human CPR in solution, which involves transitions from a "locked/closed" to an "unlocked/open" state, is crucial for electron transfer. To date, however, the factors guiding these changes remain unknown. By Site‐Directed Spin Labelling coupled to Electron Paramagnetic Resonance spectroscopy, we have incorporated a non‐canonical amino acid onto the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) domains of soluble human CPR, and labelled it with a specific nitroxide spin probe. Taking advantage of the endogenous FMN cofactor, we successfully measured for the first time, the distance distribution by DEER between the semiquinone state FMNH• and the nitroxide. The DEER data revealed a salt concentration‐dependent distance distribution, evidence of an "open" CPR conformation at high salt concentrations exceeding previous reports. We also conducted molecular dynamics simulations which unveiled a diverse ensemble of conformations for the "open" semiquinone state of the CPR at high salt concentration. This study unravels the conformational landscape of the one electron reduced state of CPR, which had never been studied before. [ABSTRACT FROM AUTHOR]

Highly resistant to reduction nitroxides open new opportunities for structural studies of biological macromolecules in their native environment inside living cells and for functional imaging of pH and thiols, enzymatic activity and redox status in living animals. 3,4-Disubstituted nitroxides of 2,2,5,5-tetraethylpyrrolidine and pyrroline series with a functional group for binding to biomolecules and a polar moiety for higher solubility in water and for more rigid attachment via additional coordination to polar sites were designed and synthesized. The EPR spectra, lipophilicities, kinetics of the reduction in ascorbate-containing systems and the decay rates in liver homogenates were measured. The EPR spectra of all 3,4-disubstituted pyrrolidine nitroxides showed additional large splitting on methylene hydrogens of the ethyl groups, while the spectra of similar pyrroline nitroxides were represented with a simple triplet with narrow lines and hyperfine structure of the nitrogen manifolds resolved in oxygen-free conditions. Both pyrrolidine and pyrroline nitroxides demonstrated low rates of reduction with ascorbate, pyrrolidines being a bit more stable than similar pyrrolines. The decay of positively charged nitroxides in the rat liver homogenate was faster than that of neutral and negatively charged radicals, with lipophilicity, rate of reduction with ascorbate and the ring type playing minor role. The EPR spectra of N,N-dimethyl-3,4-bis-(aminomethyl)-2,2,5,5-tetraethylpyrrolidine-1-oxyl showed dependence on pH with pKa = 3, ΔaN = 0.055 mT and ΔaH = 0.075 mT. [ABSTRACT FROM AUTHOR]

This work aims at developing a diagnostic method based on Electron Paramagnetic Resonance (EPR) measurements of stable nitroxide radicals released from "EPR silent" liposomes. The liposome destabilisation and consequent radical release is enzymatically triggered by the action of phospholipase A2 (PLA2) present in the biological sample of interest. PLA2 are involved in a broad range of processes, and changes in their activity may be considered as a unique valuable biomarker for early diagnoses. The minimum amount of PLA2 measured "in vitro" was 0.09 U/mL. Moreover, the liposomes were successfully used to perform Overhauser-enhanced Magnetic Resonance Imaging (OMRI) in vitro at 0.2 T. The amount of radicals released by PLA2 driven liposome destabilization was sufficient to generate a well detectable contrast enhancement in the corresponding OMRI image. [ABSTRACT FROM AUTHOR]

Nitroxide (NO) spin radicals are effective in characterizing structures, interactions and dynamics of biomolecules. The EPR applications in cell lysates or intracellular milieu require stable spin labels, but NO radicals are unstable in such conditions. We showed that the destabilization of NO radicals in cell lysates or even in cells is caused by NADPH/NADH related enzymes, but not by the commonly believed reducing reagents such as GSH. Maleimide stabilizes the NO radicals in the cell lysates by consumption of the NADPH/NADH that are essential for the enzymes involved in destabilizing NO radicals, instead of serving as the solo thiol scavenger. The maleimide treatment retains the crowding properties of the intracellular components and allows to perform long‐time EPR measurements of NO labeled biomolecules close to the intracellular conditions. The strategy of maleimide treatment on cell lysates for the EPR applications has been demonstrated on double electron‐electron resonance (DEER) measurements on a number of NO labeled protein samples. The method opens a broad application range for the NO labeled biomolecules by EPR in conditions that resemble the intracellular milieu. [ABSTRACT FROM AUTHOR]

Water-soluble spin probes were obtained from reduction-resistant sterically shielded pyrrolidine nitroxides by forming a 4,5-dicarboxy-1H-1,2,3-triazole fragment in the side chain by the Huisgen reaction of the azido group with acetylenedicarboxylic acid ester followed by hydrolysis. For the synthesized radicals, the reduction rate constants by ascorbate and the partition coefficient in an octanol—water system were determined. [ABSTRACT FROM AUTHOR]

Nitroxides are widely used as probes and polarization transfer agents in spectroscopy and imaging. These applications require high stability towards reducing biological environments, as well as beneficial relaxation properties. While the latter is provided by spirocyclic groups on the nitroxide scaffold, such systems are not in themselves robust under reducing conditions. In this work, we introduce a strategy for stability enhancement through conformational tuning, where incorporating additional substituents on the nitroxide ring effects a shift towards highly stable closed spirocyclic conformations, as indicated by X-ray crystallography and density functional theory (DFT) calculations. Closed spirocyclohexyl nitroxides exhibit dramatically improved stability towards reduction by ascorbate, while maintaining long relaxation times in electron paramagnetic resonance (EPR) spectroscopy. These findings have important implications for the future design of new nitroxide-based spin labels and imaging agents. Nitroxides are common probes and polarization transfer agents used in spectroscopy and imaging, however, reducing conditions limit their robustness. Here, the authors tune the conformation of the spirocyclic nitroxide ring by incorporating additional substituents, which enhances their stability under reducing conditions, and maintains long relaxation times in electron paramagnetic resonance spectroscopy. [ABSTRACT FROM AUTHOR]

The cationic charged chitosan and the weak polyacid poly(ethylene‐alt‐maleic acid) are used to build polyelectrolyte multilayers (PEMs). To study the rotational dynamics of the polymer backbone in the swollen state of the PEMs using electron paramagnetic resonance spectroscopy, a nitroxide spin‐label is covalently attached to the polyacid. It is found that spectra of PEMs with even number of layers show a superposition of two spectral components: a major, slow‐motion component, which arises from chain segments intrinsically compensated by protonated amino groups of the chitosan, and a minor, fast‐motion component, which is assumed to originate from loops and tails of the polyacid chain located at the surface of the PEMs. The dissociated acid groups in the loops and tails are extrinsically compensated by small counterions and the rotational mobility of these chain segments is consequently much higher compared to chain segments interacting with the oppositely charged chitosan. [ABSTRACT FROM AUTHOR]

Site-directed spin labeling (SDSL) combined with continuous wave electron paramagnetic resonance (cw EPR) spectroscopy is a powerful technique to reveal, at the local level, the dynamics of structural transitions in proteins. Here, we consider SDSL-EPR based on the selective grafting of a nitroxide on the protein under study, followed by X-band cw EPR analysis. To extract valuable quantitative information from SDSL-EPR spectra and thus give a reliable interpretation on biological system dynamics, a numerical simulation of the spectra is required. However, regardless of the numerical tool chosen to perform such simulations, the number of parameters is often too high to provide unambiguous results. In this study, we have chosen SimLabel to perform such simulations. SimLabel is a graphical user interface (GUI) of Matlab, using some functions of Easyspin. An exhaustive review of the parameters used in this GUI has enabled to define the adjustable parameters during the simulation fitting and to fix the others prior to the simulation fitting. Among them, some are set once and for all (gy, gz) and others are determined (Az, gx) thanks to a supplementary X-band spectrum recorded on a frozen solution. Finally, we propose guidelines to perform the simulation of X-band cw-EPR spectra of nitroxide labeled proteins at room temperature, with no need of uncommon higher frequency spectrometry and with the minimal number of variable parameters. [ABSTRACT FROM AUTHOR]

Sterically shielded nitroxides, which demonstrate high resistance to bioreduction, are the spin labels of choice for structural studies inside living cells using pulsed EPR and functional MRI and EPRI in vivo. To prepare new sterically shielded nitroxides, a reaction of cyclic nitrones, including various 1-pyrroline-1-oxides, 2,5-dihydroimidazole-3-oxide and 4H-imidazole-3-oxide with alkynylmagnesium bromide wereused. The reaction gave corresponding nitroxides with an alkynyl group adjacent to the N-O moiety. The hydrogenation of resulting 2-ethynyl-substituted nitroxides with subsequent re-oxidation of the N-OH group produced the corresponding sterically shielded tetraalkylnitroxides of pyrrolidine, imidazolidine and 2,5-dihydroimidazole series. EPR studies revealed large additional couplings up to 4 G in the spectra of pyrrolidine and imidazolidine nitroxides with substituents in 3- and/or 4-positions of the ring. [ABSTRACT FROM AUTHOR]

The nitroxide TPA (2,2,5,5‐tetramethyl‐pyrrolin‐1‐oxyl‐3‐acetylene) is an excellent spin label for EPR studies of RNA. Previous synthetic methods, however, are complicated and require special equipment. Herein, we describe a uridine derived phosphoramidite with a photocaged TPA unit attached. The light sensitive 2‐nitrobenzyloxymethyl group can be removed in high yield by short irradiation at 365 nm. Based on this approach, a doubly spin‐labeled 27mer neomycin sensing riboswitch was synthesized and studied by PELDOR. The overall thermal stability of the fold is not much reduced by TPA. In‐line probing nevertheless detected changes in local mobility. [ABSTRACT FROM AUTHOR]

[Display omitted] • Spin-labeled pH-responsive diblock copolymers could be potentially used for controlled drug delivery applications. • Microfluidic technique offers a powerful and versatile platform for the precise synthesis of nanoparticles. • Electron paramagnetic resonance is a valuable technique for the molecular-level analysis of colloidal polymer systems. This research emphasizes the crucial role of electron paramagnetic resonance (EPR) spectroscopy in nanoparticle analysis, showcasing its distinctive ability to probe molecular-level details. We demonstrate the synthesis and self-assembly of a new class of pH-responsive amphiphilic diblock copolymers, specifically poly[ N -(2-hydroxypropyl)-methacrylamide]- block -poly[2-(diisopropylamino)ethyl methacrylate] (PHPMA- b -PDPA), incorporating 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) radicals covalently bound to the hydrophilic PHPMA segment. The copolymer synthesis involved a three-step process, combining reversible addition − fragmentation chain transfer (RAFT) polymerization with carbodiimide (DCC) chemistry. TEMPO radical-containing nanoparticles (RNPs) were created using microfluidic (MF) nanoprecipitation, a technique essential for generating uniform nanoparticles with predictable biodistribution and cellular uptake. Adjusting MF protocol parameters allowed fine-tuning of RNP sizes. EPR spectroscopy confirmed the formation of core–shell RNPs, with features aligning closely with those obtained from scattering techniques like dynamic light scattering (DLS), static light scattering (SLS), small-angle X-ray scattering (SAXS), and cryo-transmission electron microscopy (cryo-TEM). EPR spectroscopy emerges as a potent tool for molecular-level analysis of colloidal polymer systems. This study highlights the EPR-spin label method's efficacy in probing the internal structural features and dynamics of core–shell nanoparticles, especially their response to pH-triggered disassembly. The findings open new avenues for the use of pH-responsive TEMPO-labeled diblock copolymers in controlled drug delivery applications. [ABSTRACT FROM AUTHOR]

The possibility of the application of nitronyl-nitroxide ligand (NIT) as a spin label for the study of the composition of transition metals complexes with M-NIT bonds by EPR spectroscopy was explored using PCP-pincer complexes of Pd and Ni as examples. [ABSTRACT FROM AUTHOR]

The recent finding that hydropersulfides (RSSH) are biologically prevalent in mammalian systems has prompted further investigation of their chemical properties in order to provide a basis for understanding their potential functions, if any. Hydropersulfides have been touted as hyper-reactive thiol-like species that possess increased nucleophilicity and reducing capabilities compared to their thiol counterparts. Herein, using persulfide generating model systems, the ability of RSSH species to act as one-electron reductants has been examined. Not unexpectedly, RSSH is relatively easily oxidized, compared to thiols, by weak oxidants to generate the perthiyl radical (RSS·). Somewhat surprisingly, however, RSS· was found to be stable in the presence of both O2 and NO and only appears to dimerize. Thus, the RSSH/RSS· redox couple is readily accessible under biological conditions and since dimerization of RSS· may be a rare event due to low concentrations and/or sequestration within a protein, it is speculated that the general lack of reactivity of individual RSS· species may allow this couple to be utilized as a redox component in biological systems.

Four glycolipids carrying different glycans and a nitroxide free radical spin at the glycan non-reducing end were designed and synthesized from free glucose and maltooligosaccharides by an efficient and streamlined synthetic strategy. The main features of this synthetic strategy include regioselective functionalization of the free carbohydrates and coupling of the radical spin label with functionalized free glycans as the last synthetic step. These glycolipids are useful probes for the study of cell surface glycans by electron paramagnetic resonance spectroscopy. Moreover, the key synthetic intermediates, free glycolipids carrying a flexible azido group at the glycan non-reducing end, are widely useful platforms for accessing glycolipids with other molecular labels. [ABSTRACT FROM AUTHOR]

A method, based on the doubly rotating frame (DRF) technique to calculate the basis DEER (Double Electron–Electron Resonance) signals [Physica B: Condensed Matter, 625, 413,511 (2022)] accurately by numerical techniques over a range of r values, where r is the distance between the two nitroxides in a biradical in a biological system, has been exploited to calculate the probabilities of distance distribution, P r , by the use of Tikhonov regularization. It is demonstrated here by applying it to the data reported by Lovett et al. [J. Magn. Reson., 223, 98–106 (2012)] on a sample of bis-nitroxide nanowire, P1, in deuterated ortho-terphenyl solvent with 5% BnPy (d14-oTP/BnPy) in semi-rigid state. An improvement in the agreement of the calculated signal with respect to the experimental signal and thus in the probabilities of the distance distribution, P r , so obtained, is found, as compared to that obtained using the kernel signals based on analytical expressions. [ABSTRACT FROM AUTHOR]

Nitroxide radicals are characterized by a long‐lived spin‐unpaired electronic ground state and are strongly sensitive to their chemical surroundings. Combined with electron paramagnetic resonance spectroscopy, these electronic features have led to the widespread application of nitroxide derivatives as spin labels for use in studying protein structure and dynamics. Site‐directed spin labelling requires the incorporation of nitroxides into the protein structure, leading to a new protein–ligand molecular model. However, in protein crystallographic refinement nitroxides are highly unusual molecules with an atypical chemical composition. Because macromolecular crystallography is almost entirely agnostic to chemical radicals, their structural information is generally less accurate or even erroneous. In this work, proteins that contain an example of a radical compound (Chemical Component Dictionary ID MTN) from the nitroxide family were re‐refined by defining its ideal structural parameters based on quantum‐chemical calculations. The refinement results show that this procedure improves the MTN ligand geometries, while at the same time retaining higher agreement with experimental data. [ABSTRACT FROM AUTHOR]

• Modification of DSSC architecture by incorporating microscopic reference electrodes. • Separation of contributions of photoanode and counter electrode to EIS. • Identification of recombination and mediator regeneration rates in DSSCs. • In-depth analysis of the rate-limiting steps of aqueous TEMPO-based electrolytes. For new components in dye-sensitized solar cells (DSSCs), identification and quantification of rate-limiting steps is needed to evaluate their applicability. This is particularly important if fundamental changes are studied. In this work, the use of a micro-reference electrode in DSSCs is proposed to increase the significance of electrochemical impedance spectroscopy. The recombination of charge carriers at the photoanode and the regeneration of redox mediators at the counter electrode, which typically occur on similar timescales, could be studied separately but simultaneously in cells under operating conditions. This is particularly useful in the study of water-based DSSCs. Here, cells with 2,2,6,6-Tetramethylpiperidinoxyl (TEMPO) or 4-Hydroxy-TEMPO (OH-TEMPO) as redox mediators using additives like 1-Methylbenzimidazole (MBI) are discussed. It was revealed that the charge transfer resistance (R CE) for the reduction of the oxidized redox mediator at the counter electrode (CE) limits the fill factor (FF) of such DSSCs. TEMPO/MBI electrolytes yielded low R CE and high FF , whereas OH-TEMPO in otherwise identical cells resulted in large R CE , low FF , and low conversion efficiencies. This indicates that the interface between the CE and the electrolyte significantly influences the DSSC performance of these cells and strongly depends on the electrolyte composition. Important optimization strategies could be discussed based on the present results. [Display omitted] [ABSTRACT FROM AUTHOR]

Herein, we report an EPR-based method for protease enzymatic characterization and inhibitor screening. This method utilizes dual paramagnetically-labeled probes consisting of a nitroxide spin probe and a Gd3+ ion flanking a peptide that could be specifically cleaved by protease caspase-3. Distance-dependent dipolar coupling between the two paramagnetic centers can be modulated by the protease cleavage activity, thus providing a straightforward and convenient method for protease activity detection using EPR spectroscopy under ambient conditions. Moreover, time-course monitoring of the protease-catalyzed cleavage reaction demonstrated that this EPR-based method could not only allow a direct quantitative enzymatic kinetic assessment, but also could be used for protease inhibitor screening, thus holding great potential in drug discovery studies. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease with a poor prognosis and limited treatment options. Oxidative and nitrosative stress is implicated as one of the main pathogenic pathways in IPF. The rationale for the use of antioxidants to treat lung fibrosis is appealing, however to date a consistent beneficial effect for such an approach has not been observed. We have recently demonstrated that nitroxides, particularly 3-carbamoyl-proxyl (3-CP), markedly reduce airway inflammation, airway hyper-responsiveness, and protein nitration of the lung tissue in a mouse model of ovalbumin-induced acute asthma, thus prompting its use for the treatment of IPF. The present study investigates the effect of 3-CP on the development of lung fibrosis using the murine intratracheal bleomycin model. 3-CP was administered either intranasally or orally during the entire experiment or starting 7 days after induction of the lung injury. 3-CP was found to be both a preventive and a therapeutic drug reducing the lung fibrosis (histological score), the increase in collagen content, protein nitration, TGF-β levels, the degree of weight loss as well as inhibiting the impairment of lung function. Nitroxides are catalytic antioxidants that preferentially detoxify radicals, and therefore the effect of 3-CP on the severity of the disease supports the involvement of reactive oxygen and nitrogen species in the disease pathology. [Display omitted] • Idiopathic pulmonary fibrosis is a fatal disease with limited treatment options. • Nitroxides, such as 3-carbamoyl-proxyl (3-CP), are powerful antioxidants. • 3-CP reduces the severity of bleomycin-induced lung fibrosis in mice. • 3-CP provided protection when administered either before or after lung injury. • The results support the role of oxidative and nitrosative stress in the disease pathology. [ABSTRACT FROM AUTHOR]

This study focuses on analyzing the noncovalent interaction patterns between three lipid‐nanodisc‐forming polymers and nitroxide radicals which are used as small organic tracer molecules. Besides the negatively charged polymers diisobutylene/maleic acid (DIBMA) and styrene/maleic acid (SMA) (2:1), the solvation behavior of a newly synthesized zwitterionic styrene/maleic amide sulfobetaine copolymer named SMA‐sulfobetaine (SB) is characterized. The applied spin probes vary in their respective chemical structure, allowing the report of different local micropolarities and nanoscopic regions by recording temperature‐dependent continuous‐wave electron paramagnetic resonance (CW EPR) spectra. In combination with light scattering experiments, a nanoscopic interpretation of the dominant polymer/guest molecule interaction patterns is provided. The results indicate that in SMA and DIBMA, ionic interactions dominate the interaction patterns with other molecules. In SMA‐SB, the zwitterionic side chains mainly induce a dynamic assembly with guest molecules due to weaker noncovalent interactions. Depending on the applied spin probe, temperature‐dependent CW EPR measurements reveal nanoscopic cloud points depending on the interaction patterns with SMA‐SB which can occur more than 20 °C below its macroscopically observed upper critical solution temperature. Finally, the detailed dissection of interaction patterns may provide a better understanding that may even allow tuning the polymers' properties for use in lipid nanodisc formation. [ABSTRACT FROM AUTHOR]

Aminoxyl radicals (nitroxides) are a class of compounds with important biomedical applications, serving as antioxidants, spin labels for proteins, spin probes of oximetry, pH, or redox status in electron paramagnetic resonance (EPR), or as contrast agents in magnetic resonance imaging (MRI). However, the fast reduction of the radical moiety in common tetramethyl-substituted cyclic nitroxides within cells, yielding diamagnetic hydroxylamines, limits their use in spectroscopic and imaging studies. In vivo half-lives of commonly used tetramethyl-substituted nitroxides span no more than a few minutes. Therefore, synthetic efforts have focused on enhancing the nitroxide stability towards reduction by varying the electronic and steric environment of the radical. Tetraethyl-substitution at alpha position to the aminoxyl function proved efficient in vitro against reduction by ascorbate or cytosolic extracts. Moreover, 2,2,6,6-tetraethyl-4-oxo(piperidin-1-yloxyl) radical (TEEPONE) was used successfully for tridimensional EPR and MRI in vivo imaging of mouse head, with a reported half-life of over 80 min. We decided to investigate the stability of tetraethyl-substituted piperidine nitroxides in the presence of hepatic microsomal fractions, since no detailed study of their "metabolic stability" at the molecular level had been reported despite examples of the use of these nitroxides in vivo. In this context, the rapid aerobic transformation of TEEPONE observed in the presence of rat liver microsomal fractions and NADPH was unexpected. Combining EPR, HPLC-HRMS, and DFT studies on a series of piperidine nitroxides — TEEPONE, 4-oxo-2,2,6,6-tetramethyl(piperidin-1-yloxyl) (TEMPONE), and 2,2,6,6-tetraethyl-4-hydroxy(piperidin-1-yloxyl) (TEEPOL), we propose that the rapid loss in paramagnetic character of TEEPONE is not due to reduction to hydroxylamine but is a consequence of carbon backbone modification initiated by hydrogen radical abstraction in alpha position to the carbonyl by the P450-Fe(V)=O species. Besides, hydrogen radical abstraction by P450 on ethyl substituents, leading to dehydrogenation or hydroxylation products, leaves the aminoxyl function intact but could alter the linewidth of the EPR signal and thus interfere with methods relying on measurement of this parameter (EPR oximetry). Image 1 • Tetraethyl-substituted aminoxyl radicals are valuable probes for biological EPR. • They are indeed more resistant to reduction than their tetramethyl analogs. • Here, the metabolic stability of three piperidine nitroxides was studied in vitro. • Surprisingly TEEPONE rapidly decayed in contrast to TEMPONE or TEEPOL. • The trigger is hydrogen abstraction by P450-FeV=O on the lipophilic probe. [ABSTRACT FROM AUTHOR]

Understanding the segmental dynamics of polymer chains is cardinal to decipher the microscopic behaviour in order to modulate the bulk properties of polymers. The study of electron spin resonance (ESR) spectroscopy of spin-labelled polymers is useful to understand the segmental dynamics of polymer chains in solution. In this paper, poly(acrylic acid)s (PAAs) were spin labelled with 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl radicals. Spin-labelled PAAs (SL-PAAs) were characterised by Fourier-transform infrared spectroscopy, ultraviolet–visible spectroscopy, thermogravimetric analysis, differential scanning calorimetry, cyclic voltammetry (CV), and ESR analyses. The polyelectrolyte complexes of SL-PAAs were prepared by employing poly(diallyldimethylammonium chloride) (PDADMAC) as the polycation and analysed by transmission electron microscopy, dynamic light scattering (DLS), and ESR spectroscopies. The effect of molar mass on the segmental dynamics of SL-PAAs in pristine as well as in the form of polyelectrolyte complexes (PECs) was studied. The results indicated that SL-PAAs show a differential complexation behaviour with PDADMAC in the PECs depending on their molar mass. [ABSTRACT FROM AUTHOR]

The synthesis of a novel nitronylnitroxyl radical 2-(2,5-di(pyridin-4-yl)phenyl)-4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazol-3-oxide-1-oxyl (L) and the first Mn(II) complex with it [Mn(NO3)2(L)(H2O)× ×(i-PrOH)] is elaborated and performed. It is found that the complex is formed by polymeric chains in which (NO3)2(H2O)(i-PrOH) fragmentы are linked by L coordinated by N atoms of pyridine rings. The chains are combined into layers by hydrogen bonds between the coordinated water molecules, i-PrOH, and nitrate anions. The layers are joined into a framework by weak C-H⋯O contacts between O atoms of the nitronylnitroxyl fragment and the pyridine C-H group. [ABSTRACT FROM AUTHOR]

Pulsed electron-electron double resonance (PELDOR) has proven to be a valuable tool to measure the distribution of long range distances in noncrystalline macromolecules. These experiments commonly use nitroxide spin labels as paramagnetic markers that are covalently attached to the macromolecule at specific positions. Unless these spin labels are flexible in such a manner that they exhibit an almost random orientation, the PELDOR signals will—apart from the interspin distance—also depend on the orientation of the spin labels. This effect needs to be considered in the analysis of PELDOR signals and can, moreover, be used to obtain additional information on the structure of the molecule under investigation. In this work, we demonstrate that the PELDOR signal can be represented as a convolution of a kernel function containing the distance distribution function and an orientation intensity function. The following strategy is proposed to obtain both functions from the experimental data. In a first step, the distance distribution function is estimated by the Tikhonov regularization, using the average over all PELDOR time traces with different frequency offsets and neglecting angular correlations of the spin labels. Second, the convolution relation is employed to determine the orientation intensity function, using again the Tikhonov regularization. Adopting small nitroxide biradical molecules as simple examples, it is shown that the approach works well and is internally consistent. Furthermore, independent molecular dynamics simulations are performed and used to calculate PELDOR signals, distance distributions, and orientational intensity functions. The calculated and experimental results are found to be in excellent overall agreement. [ABSTRACT FROM AUTHOR]

Spectroscopic and biophysical methods for structural determination at atomic resolution are fundamental in studies of biological function. Here we introduce an approach to measure molecular distances in bio‐macromolecules using 19F nuclear spins and nitroxide radicals in combination with high‐frequency (94 GHz/3.4 T) electron–nuclear double resonance (ENDOR). The small size and large gyromagnetic ratio of the 19F label enables to access distances up to about 1.5 nm with an accuracy of 0.1–1 Å. The experiment is not limited by the size of the bio‐macromolecule. Performance is illustrated on synthesized fluorinated model compounds as well as spin‐labelled RNA duplexes. The results demonstrate that our simple but strategic spin‐labelling procedure combined with state‐of‐the‐art spectroscopy accesses a distance range crucial to elucidate active sites of nucleic acids or proteins in the solution state. [ABSTRACT FROM AUTHOR]

The availability of bioresistant spin labels is crucial for the optimization of site‐directed spin labeling protocols for EPR structural studies of biomolecules in a cellular context. As labeling can affect proteins' fold and/or function, having the possibility to choose between different spin labels will increase the probability to produce spin‐labeled functional proteins. Here, we report the synthesis and characterization of iodoacetamide‐ and maleimide‐functionalized spin labels based on the gem‐diethyl pyrroline structure. The two nitroxide labels are compared to conventional gem‐dimethyl analogs by site‐directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy, using two water soluble proteins: T4 lysozyme and Bid. To foster their use for structural studies, we also present rotamer libraries for these labels, compatible with the MMM software. Finally, we investigate the "true" biocompatibility of the gem‐diethyl probes comparing the resistance towards chemical reduction of the NO group in ascorbate solutions and E. coli cytosol at different spin concentrations. [ABSTRACT FROM AUTHOR]

Oligonucleotides containing nitroxide spin labels, used in biophysical studies of nucleic acids, are frequently prepared by chemical synthesis. However, during the synthesis of spin‐labeled oligonucleotides, the nitroxides are partially reduced to the corresponding amines. Here we report that a benzoylated hydroxylamine can be used as a protected form of the nitroxide to avoid this reduction. The benzoyl group is stable through the oligonucleotide synthesis and is readily removed under standard oligonucleotide deprotection conditions, yielding a hydroxylamine that is oxidized in situ to the nitroxide. This method was used to incorporate the rigid spin labels Ç and Çm into DNA and RNA oligonucleotides, respectively, including a doubly labeled 36‐nucleotide long DNAzyme. Enzymatic digestion of the spin‐labeled oligonucleotides and subsequent HPLC analysis showed that the nitroxides were intact. This protecting group strategy facilitates the high‐yielding synthesis of spin‐labeled DNA and RNA oligonucleotides using the phosphoramidite method. [ABSTRACT FROM AUTHOR]

• Oxidation of nitroxides in water examined by theoretical calculations. • Inaccuracy in thermodynamic cycle approach revealed. • Structure of nitroxide adducts with chitosan and fullerene C60 explored. • Oxidation of nitroxide adducts examined. Density functional theory and continuum solvent model have been used to determine standard redox potentials of oxidation of cyclic nitroxide radicals and the nitroxide derivatives of chitosan and fullerene C 60 in water. Inclusion of structural relaxation of solute in water improves correlation between the measured and calculated oxidation potentials. The underlying limitations of the thermodynamic cycle model have been discussed. The computed oxidation potential of the piperidine–N–oxide derivative of chitosan is several tens mV higher than of the separate radical while the opposite trend was found for the pyrroline–N–oxide derivative of chitosan. The calculations of fullerene C 60 –TEMPO have shown that the 6,6–methanofullerene is more stable that the 5,6–fulleroid structure. The determined redox potential of oxidation of fullerene C 60 –TEMPO is significantly higher than of TEMPO in water. [ABSTRACT FROM AUTHOR]

A method is developed for the direct simulation of continuous wave electron paramagentic resonance (CW-EPR) line shapes from molecular trajectories. The method is applied to the cases of an axially symmetric nitroxide spin label undergoing two different types of motion (i) two-site jumping motion and (ii) isotropic rotational Brownian motion. Probabilistic models are developed that generate trajectories stable for large numbers of evolution time steps. Spectra at both 10 (X band) and 35 (Q band) GHz of satisfactory quality are generated from 40 trajectories of 214 steps sampled at 0.05 ns intervals. The resulting calculations illustrate that this method can be used as a basis of CW-EPR spectral simulation for systems undergoing arbitrarily complex dynamics using trajectories generated by classical Brownian dynamics. [ABSTRACT FROM AUTHOR]

β‐Peptides are an interesting new class of transmembrane model peptides based on their conformationally stable and well‐defined secondary structures. Herein, we present the synthesis of the paramagnetic β‐amino acid β3‐hTOPP (4‐(3,3,5,5‐tetramethyl‐2,6‐dioxo‐4‐oxylpiperazin‐1‐yl)‐d‐β3‐homophenylglycine) that enables investigations of β‐peptides by EPR spectroscopy. This amino acid adds to the, to date, sparse number of β‐peptide spin labels. Its performance was evaluated by investigating the helical turn of a 314‐helical transmembrane model β‐peptide. Nanometer distances between two incorporated β3‐hTOPP labels in different environments were measured by using pulsed electron/electron double resonance (PELDOR/DEER) spectroscopy. Due to the semi‐rigid conformational design, the label delivers reliable distances and sharp (one‐peak) distance distributions even in the lipid bilayer. The results indicate that the investigated β‐peptide folds into a 3.2514 helix and maintains this conformation in the lipid bilayer. Nitroxide radicals: New semi‐rigid spin label β3‐hTOPP has allowed the investigation of a β‐peptide structure by EPR spectroscopy. Well‐defined inter‐spin distances between two β3‐hTOPP labels in a model β‐peptide have been measured in lipid bilayers using pulsed electron/electron double resonance (PELDOR) spectroscopy. The labelling strategy has permitted to determine the peptide helical structure with resolution at molecular‐length scale (see figure). [ABSTRACT FROM AUTHOR]

A large number (63) of well‐defined nitroxide radicals, all phenol‐based TEMPO and PROXYL esters, were synthesized using different strategies based on well‐established Steglich esterifications. All of these radicals can be used as spin probes (SPs) and spin labels (SLs) for electron paramagnetic resonance (EPR) spectroscopy of supramolecular systems. Depending on the nature of the functional group(s) on each SP/SL, the synthesized nitroxide radicals serve as polyphilic molecular "toolbox" for the EPR‐spectroscopic detection and characterization of specific types of interactions, e. g. π–π interactions, sulfur‐sulfur interactions, hydrogen bonding, electrostatic and dipole‐dipole interactions, and van der Waals and hydrophobic interactions, in the presence of the selected supramolecular systems of interest (e. g. proteins, peptides). For each synthesized SP/SL the water solubility was gravimetrically determined for use in aqueous solution at pH 7. Mix or click! A large toolbox of 59 spin probes for detection of supramolecular interactions and four spin labels for "click" chemistry is presented. All nitroxide radicals are phenol‐based and accessible in high yields through a versatile synthetic approach. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • "2+1" train ESE artifact is present in conventional 4-pulse DEER. • AWG-created Gaussian pulses elegantly remove this artifact. • GaussDEER increases accuracy of the obtained distance distributions. Abstract The introduction of arbitrary waveform generator (AWG) technology and the availability of high power microwave amplifiers mark a "new era" in pulse EPR due to significant sensitivity improvements and the possibility to perform novel types of experiments. We present an optimized 4-pulse DEER setup that uses Gaussian observer pulses (GaussDEER) in connection with a Gaussian/shaped pump pulse. Gaussian pulses allow to experimentally remove the "2+1" pulse train ESE signal which is intrinsically present in any DEER experiment performed with rectangular pulses. Further signal improvements are obtained with shaped pump pulses, which can significantly increase the modulation depth of the DEER experiment due to their tailored excitation bandwidth. Although sequences like CP (Carr-Purcell) DEER offer advantages such as a prolongation of the dipolar evolution time, they suffer from post-processing of the time-domain data to remove artifacts. Therefore, it is worth having a 4-pulse DEER experiment free of residual "2+1" signal since this is still the main dipolar spectroscopic technique used in structural biology. In this work we focus on nitroxides, which are the spin probes primarily used in site-directed spin labeling studies of biomolecules, however, the advantages introduced by Gaussian pulses can be extended to any spin type. [ABSTRACT FROM AUTHOR]

Abstract: The investigation of the structure and conformational dynamics of biomolecules under physiological conditions is challenging for structural biology. Although pulsed electron paramagnetic resonance (like PELDOR) techniques provide long‐range distance and orientation information with high accuracy, such studies are usually performed at cryogenic temperatures. At room temperature (RT) PELDOR studies are seemingly impossible due to short electronic relaxation times and loss of dipolar interactions through rotational averaging. We incorporated the rigid nitroxide spin label Ç into a DNA duplex and immobilized the sample on a solid support to overcome this limitation. This enabled orientation‐selective PELDOR measurements at RT. A comparison with data recorded at 50 K revealed averaging of internal dynamics, which occur on the ns time range at RT. Thus, our approach adds a new method to study structural and dynamical processes at physiological temperature in the <10 μs time range with atomistic resolution. [ABSTRACT FROM AUTHOR]

Abstract: Nitronyl nitroxide (NN)‐substituted conjugated oligomers, which were expected to self‐associate in biradical cation states, were designed to analyze the capability of π‐dimers as molecular wires. The oligomer moieties were composed of dithienyl‐N‐methylpyrrole with methoxy substituents at the inner β‐position of thiophene rings (DTP‐NN.) and its propylenedioxythiophene (ProDOT) inserted derivative (DTP‐P‐NN.), or two ethylenedioxythiophene (EDOT) and two ProDOT units (E2P2‐NN.). Among them, chemical one‐electron oxidation gave biradical cations (DTP‐P).+‐NN. and (E2P2).+‐NN. that formed π‐dimers (DTP‐P‐NN.)22+ and (E2P2‐NN.)22+ in dichloromethane at low temperatures. ESR studies of (DTP‐P‐NN.)22+ and (E2P2‐NN.)22+ showed the presence of a relatively strong exchange interaction between two NN radicals through the radical cation π‐dimer moieties. DFT calculations supported these experimental results and predicted that exchange interactions between two NN radicals were comparable or stronger than those through covalently linked quaterthiophene. Thus, the conjugated oligomer radical cation π‐dimers acted as efficient molecular wires for electronic communication. [ABSTRACT FROM AUTHOR]

Electron spin echo envelope modulation (ESEEM) spectroscopy in combination with site-directed spin labeling (SDSL) has been established as a valuable biophysical technique to provide site-specific local secondary structure of membrane proteins. This pulsed electron paramagnetic resonance (EPR) method can successfully distinguish between α-helices, β-sheets, and 3 10 -helices by strategically using 2 H-labeled amino acids and SDSL. In this study, we have explored the use of 13 C-labeled residues as the NMR active nuclei for this approach for the first time. 13 C-labeled d 5 -valine (Val) or 13 C-labeled d 6 -leucine (Leu) were substituted at a specific Val or Leu residue ( i ), and a nitroxide spin label was positioned 2 or 3 residues away (denoted i -2 and i -3) on the acetylcholine receptor M2δ (AChR M2δ) in a lipid bilayer. The 13 C ESEEM peaks in the FT frequency domain data were observed for the i -3 samples, and no 13 C peaks were observed in the i -2 samples. The resulting spectra were indicative of the α-helical local secondary structure of AChR M2δ in bicelles. This study provides more versatility and alternative options when using this ESEEM approach to study the more challenging recombinant membrane protein secondary structures. [ABSTRACT FROM AUTHOR]

Abstract: EPR studies on RNA are complicated by three major obstacles related to the chemical nature of nitroxide spin labels: Decomposition while oligonucleotides are chemically synthesized, further decay during enzymatic strand ligation, and undetected changes in conformational equilibria due to the steric demand of the label. Herein possible solutions for all three problems are presented: A 2‐nitrobenzyloxymethyl protective group for nitroxides that is stable under all conditions of chemical RNA synthesis and can be removed photochemically. By careful selection of ligation sites and splint oligonucleotides, high yields were achieved in the assembly of a full‐length HIV‐1 TAR RNA labeled with two protected nitroxide groups. PELDOR measurements on spin‐labeled TAR in the absence and presence of arginine amide indicated arrest of interhelical motions on ligand binding. Finally, even minor changes in conformation due to the presence of spin labels are detected with high sensitivity by in‐line probing. [ABSTRACT FROM AUTHOR]

EPR spectroscopy is an increasingly useful analytical tool to probe biomolecule structure, dynamic behaviour, and interactions. Nitroxide radicals are the most commonly used radical probe in EPR experiments, and many methods have been developed for their synthesis, as well as incorporation into biomolecules using site-directed spin labelling. In this Tutorial Review, we discuss the most practical methods for the synthesis of nitroxides, focusing on the tunability of their structures, the manipulation of their sidechains into spin labelling handles, and their installation into biomolecules. [ABSTRACT FROM AUTHOR]

Abstract: Approaching protein structural dynamics and protein–protein interactions in the cellular environment is a fundamental challenge. Owing to its absolute sensitivity and to its selectivity to paramagnetic species, site‐directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR) has the potential to evolve into an efficient method to follow conformational changes in proteins directly inside cells. Until now, the use of nitroxide‐based spin labels for in‐cell studies has represented a major hurdle because of their short persistence in the cellular context. The design and synthesis of the first maleimido‐proxyl‐based spin label (M‐TETPO) resistant towards reduction and being efficient to probe protein dynamics by continuous wave and pulsed EPR is presented. In particular, the extended lifetime of M‐TETPO enabled the study of structural features of a chaperone in the absence and presence of its binding partner at endogenous concentration directly inside cells. [ABSTRACT FROM AUTHOR]

In situ monitoring of biomolecular recognition, especially at surfaces, still presents a significant technical challenge. Electron paramagnetic resonance (EPR) of biomolecules spin-labeled with nitroxides can offer uniquely sensitive and selective insights into these processes, but new spin-labeling strategies are needed. The synthesis and study of a bromoacrylaldehyde spin label (BASL), which features two attachment points with orthogonal reactivity is reported. The first examples of mannose and biotin ligands coupled to aqueous carboxy-functionalized gold nanoparticles through a spin label are presented. EPR spectra were obtained for the spin-labeled ligands both free in solution and attached to nanoparticles. The labels were recognized by the mannose-binding lectin, Con A, and the biotin-binding protein avidin-peroxidase. Binding gave quantifiable changes in the EPR spectra from which binding profiles could be obtained that reflect the strength of binding in each case. [ABSTRACT FROM AUTHOR]

The synthesis of a spin label based on PD168393, a covalent inhibitor of a major anticancer drug target, the epidermal growth factor receptor (EGFR), is reported. The label facilitates the analysis of the EGFR structure in solution by pulsed electron paramagnetic resonance (EPR) spectroscopy. For various EGFR constructs, including near-full-length EGFR, we determined defined distance distributions between the two spin labels bound to the ATP binding sites of the EGFR dimer. The distances are in excellent agreement with an asymmetric dimer of the EGFR. Based on crystal structures, this dimer had previously been proposed to reflect the active conformation of the receptor but structural data demonstrating its existence in solution have been lacking. More generally, our study provides proof-of-concept that inhibitor-based spin labeling enables the convenient introduction of site-specific spin labels into kinases for which covalent or tight-binding small-molecule modulators are available. [ABSTRACT FROM AUTHOR]

We address the interpretation, via an integrated computational approach, of the experimental continuous-wave electron paramagnetic resonance (cw-EPR) spectra of a complete set of conformationally highly restricted, stable 310-helical peptides from hexa- to nonamers, each bis-labeled with nitroxide radical-containing TOAC (4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-4-carboxylic acid) residues. The usefulness of TOAC for this type of analysis has been shown already to be due to its cyclic piperidine side chain, which is rigidly connected to the peptide backbone α-carbon. The TOAC α-amino acids are separated by two, three, four, and five intervening residues. This set of compounds has allowed us to modulate both the radical⋯radical distance and the relative orientation parameters. To further validate our conclusion, a comparative analysis has been carried out on three singly TOAC-labeled peptides of similar main-chain length. [ABSTRACT FROM AUTHOR]

[Display omitted] Sterically shielded nitroxides were shown to demonstrate resistance to bioreduction. For this reason, they are attracting much interest as spin labels and probes for in-cell and in vivo EPR and NMR applications. Formal [3 + 2] cycloaddition reaction of donor–acceptor cyclopropanes with propionitrile provided 1-pyrrolines which were converted to the new sterically shielded achiral pyrrolidine nitroxides via several steps. [ABSTRACT FROM AUTHOR]

Structure determination of biomacromolecules under in-cell conditions is a relevant yet challenging task. Electron paramagnetic resonance (EPR) distance measurements in combination with site-directed spin labeling (SDSL) are a valuable tool in this endeavor but the usually used nitroxide spin labels are not well-suited for in-cell measurements. In contrast, triarylmethyl (trityl) radicals are highly persistent, exhibit a long relaxation time and a narrow spectral width. Here, the synthesis of a versatile collection of trityl spin labels and their application in in vitro and in-cell trityl-iron distance measurements on a cytochrome P450 protein are described. The trityl labels show similar labeling efficiencies and better signal-to-noise ratios (SNR) as compared to the popular methanethiosulfonate spin label (MTSSL) and enabled a successful in-cell measurement. [ABSTRACT FROM AUTHOR]

Nuclear relaxation is a sensitive monitor of rotational dynamics in spin-label EPR. It also contributes competing saturation transfer pathways in T 1 -exchange spectroscopy, and the determination of paramagnetic relaxation enhancement in site-directed spin labelling. A survey shows that the definition of nitrogen nuclear relaxation rate W n commonly used in the CW-EPR literature for 14 N-nitroxyl spin labels is inconsistent with that currently adopted in time-resolved EPR measurements of saturation recovery. Redefinition of the normalised 14 N spin-lattice relaxation rate, b = W n /(2 W e ), preserves the expressions used for CW-EPR, whilst rendering them consistent with expressions for saturation recovery rates in pulsed EPR. Furthermore, values routinely quoted for nuclear relaxation times that are deduced from EPR spectral diffusion rates in 14 N-nitroxyl spin labels do not accord with conventional analysis of spin-lattice relaxation in this three-level system. Expressions for CW-saturation EPR with the revised definitions are summarised. Data on nitrogen nuclear spin-lattice relaxation times are compiled according to the three-level scheme for 14 N-relaxation: T 1 n = 1/ W n . Results are compared and contrasted with those for the two-level 15 N-nitroxide system. [ABSTRACT FROM AUTHOR]

Cross relaxation, and m I -dependence of the intrinsic electron spin-lattice relaxation rate W e , are incorporated explicitly into the rate equations for the electron-spin population differences that govern the saturation behaviour of 14 N- and 15 N-nitroxide spin labels. Both prove important in spin-label EPR and ELDOR, particularly for saturation recovery studies. Neither for saturation recovery, nor for CW-saturation EPR and CW-ELDOR, can cross relaxation be described simply by increasing the value of W e , the intrinsic spin-lattice relaxation rate. Independence of the saturation recovery rates from the hyperfine line pumped or observed follows directly from solution of the rate equations including cross relaxation, even when the intrinsic spin-lattice relaxation rate W e is m I -dependent. [ABSTRACT FROM AUTHOR]

A quantum mechanical (QM) method rooted on density functional theory (DFT) has been employed to determine conformations of the methane-thiosulfonate spin label (MTSL) attached to a fragment extracted from the activation loop of Aurora-A kinase. The features of the calculated energy surface revealed low energy barriers between isoenergetic minima, and the system could be described in a population of 76 rotamers that can be also considered for other systems since it was found that the χ 3 , χ 4 and χ 5 do not depend on the previous two dihedral angles. Conformational states obtained were seen to be comparable to those obtained in the α-helix systems studied previously, indicating that the protein backbone does not affect the torsional profiles significantly and suggesting the possibility to use determined conformations for other protein systems for further modelling studies. [ABSTRACT FROM AUTHOR]