Stable Nitroxide Research Articles

Keep Your TEMPO Up: Nitroxide Radicals as Sensors of Intermolecular Interactions.

This study examines experimental data on the influence of the surrounding medium and non-covalent interactions on the isotropic hyperfine coupling constant, Aiso(14N), of the stable nitroxide radical 2,2,6,6-Tetramethylpiperidin-1-yl)oxyl (TEMPO) in solution. The data were used to identify a density functional theory functional/basis set combination that accurately reproduces the experimental Aiso(14N) values. The variations in Aiso(14N) due to external factors are two orders of magnitude greater than the accuracy of its experimental measurements, making Aiso(14N) a highly sensitive experimental probe for quantifying these effects. Additionally, it was found that the proton-accepting ability of the N-O• moiety in TEMPO resembles that of the P=O moiety, enabling the simultaneous formation of two equally strong hydrogen bonds.

Combine calculation and experiment to study the undesired polymerization of styrene for screening of effective inhibitors

Regarding undeniable formation of the undesired polymer (UP) in the styrene purification process, it is of vital importance to inhibit its production. To address the mentioned issue, effects of injecting antipolymers (e.g., stable nitroxide radicals (i.e., SNRs) and antioxidants (e.g., alkylhydroxylamines (AHA)) on the controlling the UP formation have been studied through density functional theory (DFT) calculations as well as experimental method in this study. In fact, electrophilicity and growth percentage were evaluated by DFT calculations and experimental approach, correspondingly. Accordingly, it was revealed that the best antipolymers in terms of inhibiting performance were 4‑hydroxy-2,2,6,6-tetramethyl piperidine 1-Oxyl (4‑hydroxy-TEMPO) and 4-oxo-2,2,6,6-tetramethylpiperidine 1-Oxyl (4-oxo-TEMPO), and from the antioxidants, N-Ethyl-N-phenyl hydroxylamine (EPHA) and N-Benzyl-N-phenyl hydroxylamine (BPHA) exhibited the best performance. In addition, the growth percentage of 18.70, 20.55, 24.85 and 46.8, were obtained for the EPHA, BPHA, 4‑hydroxy-TEMPO, and 4-oxo-TEMPO after 4 hrs of operation, correspondingly. Besides, the synergetic effects of the used inhibitors were determined experimentally. Among the evaluated inhibitors, EPHA/4‑hydroxy-TEMPO demonstrated the best synergetic effects over the control of UP formation. Finally, the blend of 4‑hydroxy-TEMPO (40 wt.%) and EPHA (60 wt.%) was obtained to be the optimum (best) inhibitor with 7.2 % polymer growth after 4 hrs of operation.

Intrinsic Proteolytic Activities from Cancer Cells Are Sufficient to Activate Alkoxyamine Prodrugs and Induce Cell Death.

In search of better specificity and lower chances of resistance, protease-activatable alkoxyamine prodrugs to fight cancer have been proposed. These molecules are made of a peptide linked to an alkoxyamine. Proteolysis of the peptide converts the stable prodrug at 37 °C to a metastable alkoxyamine that spontaneously homolyzes into two free radicals: a stable nitroxide and a very reactive alkyl radical. The alkyl radical induces apoptosis in the surrounding cells by inducing random chemical alterations. Here, we show that varying the peptide moiety from succinyl-Ala-Ala-Pro-Val- to PyroGlu-Gly-Arg- or PyroGlu-Gly-Lys- is effective in switching the activating enzyme from elastase to urokinase. Furthermore, these prodrugs induce the death of HT-1080 cells, a cell line that secretes several active proteases in culture. This cytotoxic activity can be suppressed by protease inhibitors and does not affect cell lines devoid of active urokinase. We thus provide examples of alkoxyamine prodrugs that are efficiently activated by the limited intrinsic protease activity and that succeed in the destruction of cancer cell lines and cancer cells from tumor explants.

Competition between n → π* interactions and H-bonding: a way to stabilize aminoxyl anions and to improve electron transfer kinetics

A new approach to stabilization of the reduced state of nitroxides in solution is suggested. Thermodynamic stabilization of the strongly basic aminoxyl anions toward protonation and hydrogen bond formation with the solvent was achieved by intramolecular n → π* interactions. A new stable nitroxide radical bearing ortho-acyl group was synthesized to demonstrate this effect by means of cyclic voltammetry.

Synthesis of Nitronyl Nitroxide Radical-Modified Multi-Walled Carbon Nanotubes and Oxidative Desulfurization in Fuel.

Novel and highly stable nitronyl nitroxide radical (NIT) derivatives were synthesized and coated on the surface of multi-walled carbon nanotubes (MWCNTs) to improve their desulfurization performance. They were characterized by FTIR, UV-vis, SEM, XRD, Raman spectroscopy and ESR. Thiophene in fuel was desulfurized by molecular O2, and the oxidation activity of these compounds was evaluated. At a normal temperature and pressure, the degradation rates of thiophene by four compounds in 4 h can reach 92.66%, 96.38%, 93.25% and 89.49%, respectively. The MWCNTs/NIT-F have a high special activity for the degradation of thiophene, and their desulfurization activity can be recycled for five times without a significant reduction. The mechanistic studies of MWCNTs/NIT composites show that the ammonium oxide ion is the key active intermediate in catalytic oxidative desulfurization, which provides a new choice for fuel oxidative desulfurization. The results show that NIT significantly improves the photocatalytic performance of MWCNTs.

Stable Nitroxide as Diagnostic Tools for Monitoring of Oxidative Stress and Hypoalbuminemia in the Context of COVID-19.

Oxidative stress is a major source of ROS-mediated damage to macromolecules, tissues, and the whole body. It is an important marker in the severe picture of pathological conditions. The discovery of free radicals in biological systems gives a "start" to studying various pathological processes related to the development and progression of many diseases. From this moment on, the enrichment of knowledge about the participation of free radicals and free-radical processes in the pathogenesis of cardiovascular, neurodegenerative, and endocrine diseases, inflammatory conditions, and infections, including COVID-19, is increasing exponentially. Excessive inflammatory responses and abnormal reactive oxygen species (ROS) levels may disrupt mitochondrial dynamics, increasing the risk of cell damage. In addition, low serum albumin levels and changes in the normal physiological balance between reduced and oxidized albumin can be a serious prerequisite for impaired antioxidant capacity of the body, worsening the condition in patients. This review presents the interrelationship between oxidative stress, inflammation, and low albumin levels, which are hallmarks of COVID-19.

Two-electron conversion nitroxide radicals-based electrode synergistically enhancing charge storage in water-in-salt electrolyte

Water-in-salt electrolytes (WISEs) are attractive in aqueous batteries due to their non-flammability, environmental friendliness, and wide electrochemical stability window. Stable nitroxide radicals with fast electron transfer rate and good cycling stability are promising electrode materials applied for WISEs, while their limited conductivity and high solubility in electrolytes are still challenges. Here, porous carbon with high specific area is used as conductive substrate for the deposition of 4-amino-TEMPO (TEMPO = 2,2,6,6-tetramethylpiperidine-1-oxyl), and MXene nanosheets are employed as their conductive binder to construct freestanding electrode with “hyacinth bean-like” structure. Nitroxide radicals undergo a two-step two-electron reversible redox reaction and MXene exhibits pseudocapacitive ion intercalation, leading to synergistically enhanced discharge capacity in WISE (19.8 m LiCl). The incorporated redox active nitroxide radicals in MXene/porous carbon-TEMPO electrode boost the surface pseudocapacity and the rationally designed three-dimensional structure further facilitates the ions diffusion and electrons transfer, endowing the composite electrode with fast charge storage kinetic. This work provides a promising potential for the design and fabrication of high-performance aqueous batteries.

Towards liquid EPR dosimetry using nitroxides in aqueous solution.

Objective. Water-equivalent dosimeters are desirable for dosimetry in radiotherapy. The present work investigates basic characteristics of novel aqueous detector materials and presents a signal loss approach for electron paramagnetic resonance (EPR) dosimetry.Approach. The proposed principle is based on the radiation dose dependent annihilation of EPR active nitroxides (NO·) in aqueous solutions. Stable nitroxide radicals (3-Maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (MmP), 3-Carbamoyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (CmP)) in aqueous solutions containing dimethyl sulfoxide (DMSO) as an additive were filled in glass capillaries for irradiation and EPR readout. Radiation doses ranging from 1 to 64 Gy were applied with a clinical 6 MV flattening filter free photon beam. EPR readout was then performed with a X-band benchtop spectrometer. The dose response, temporal stability and reproducibility of the samples' EPR signal amplitudes as well as the influence of the nitroxide concentration between 10 and 160μM on the absolute signal loss were investigated using MmP. CmP was used to examine the dependence of the dose response on DMSO concentration between 0 and 10 vol%. An indirect effect model was fitted to the experimental data assuming irradiation induced radical reactions as the underlying mechanism.Main results. For an initial MmP concentration of 20μM, absolute EPR signal loss is linear up to a dose of 16 Gy with a yield G(-NO·) of approximately 0.4μmol J-1. Within five weeks upon sample irradiation to doses between 0 and 32 Gy relative EPR signal fluctuations were on average (126 readouts) below 1% (1σ). For c(MmP) ≥ 20μM, absolute signal loss is only weakly dependent on c(MmP), whereas it increases strongly with increasing c(DMSO) in the range 0-5 vol%. An indirect effect model is applicable to describe the reaction mechanism resulting in the observed dose response curve.Significance. Liquids consisting of nitroxides in aqueous solution and small amounts of DMSO (2 vol%) show promising basic characteristics for application as water-equivalent EPR dosimeter materials in radiotherapy. The EPR signal loss is based on an indirect effect mediated by diffusing radicals originating from the radiolysis of the water/DMSO mixture.

Hair Follicle Damage after 100 mGy Low-Dose Fractionated X-Ray Irradiation and the Protective Effects of TEMPOL, a Stable Nitroxide Radical, against Radiation.

The effects of long-term low-dose X-ray irradiation on the outer root sheath (ORS) cells of C3H/He mice were investigated. Mice were irradiated with a regime of 100 mGy/day, 5 days/week, for 12 weeks (Group X) and the results obtained were compared to those in a non-irradiated control (Group C). Potential protection against ORS cells damage induced by this exposure was investigated by adding the stable nitroxide radical 4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) at 1 mM to the drinking water of mice (Group X + TEMPOL). The results obtained were compared with Group C and a non-irradiated group treated with TEMPOL (Group C + TEMPOL). After fractionated X-ray irradiation, skin was removed and ORS cells were examined by hematoxylin and eosin staining and electron microscopy for an abnormal nuclear morphology and nuclear condensation changes. Fractionated X-irradiated mice had an increased number of ORS cells with an abnormal nuclear morphology as well as nuclear condensation changes. Sections were also immunohistochemically examined for the presence of TdT-mediated dUTP nick-end labeling (TUNEL), 8-hydroxy-2'-deoxyguanosine (8-OHdG), 4-hydroxynonenal (4-HNE), vascular endothelial growth factor (VEGF), nitrotyrosine, heme oxygenase 1 (HO-1), and protein gene product 9.5 (PGP 9.5). Significant increases were observed in TUNEL, 8-OHdG, and 4-HNE levels in ORS cells from mice in Group X. Electron microscopy also showed irregular shrunken ORS cells in Group X. These changes were prevented by the presence of TEMPOL in the drinking water of the irradiated mice. TEMPOL alone had no significant effects. These results suggest that fractionated doses of radiation induced oxidative damage in ORS cells; however, TEMPOL provided protection against this damage, possibly as a result of the rapid reaction of this nitroxide radical with the reactive oxidants generated by fractionated X-ray irradiation.

A water-soluble aza-adamantyl nitroxide radical and its complexes with β-cyclodextrin derivatives

A water-soluble, stable aza-adamantyl nitroxide radical has been synthesized. Complexation with selected β-cyclodextrin derivatives increases its water-solubility and maintains well the stability of the radical.

Enhancing Coherence Times of Chromophore-Radical Molecular Qubits and Qudits by Rational Design.

An important criterion for quantum operations is long qubit coherence times. To elucidate the influence of molecular structure on the coherence times of molecular spin qubits and qudits, a series of molecules featuring perylenediimide (PDI) chromophores covalently linked to stable nitroxide radicals were synthesized and investigated by pulse electron paramagnetic resonance spectroscopy. Photoexcitation of PDI in these systems creates an excited quartet state (Q) followed by a spin-polarized doublet ground state (D0), which hold promise as spin qudits and qubits, respectively. By tailoring the molecular structure of these spin qudit/qubit candidates by selective deuteration and eliminating intramolecular motion, coherence times of Tm = 9.1 ± 0.3 and 4.2 ± 0.3 μs at 85 K for D0 and Q, respectively, are achieved. These coherence times represent a nearly 3-fold enhancement compared to those of the initial molecular design. This approach offers a rational structural design protocol for effectively extending coherence times in molecular spin qudits/qubits.

4-Amino-TEMPO loaded liposomes as sensitive EPR and OMRI probes for the detection of phospholipase A2 activity

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.

An iron–sulfur cluster in the zinc-binding domain of the SARS-CoV-2 helicase modulates its RNA-binding and -unwinding activities

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, uses an RNA-dependent RNA polymerase along with several accessory factors to replicate its genome and transcribe its genes. Nonstructural protein (nsp) 13 is a helicase required for viral replication. Here, we found that nsp13 ligates iron, in addition to zinc, when purified anoxically. Using inductively coupled plasma mass spectrometry, UV-visible absorption, EPR, and Mössbauer spectroscopies, we characterized nsp13 as an iron-sulfur (Fe-S) protein that ligates an Fe4S4 cluster in the treble-clef metal-binding site of its zinc-binding domain. The Fe-S cluster in nsp13 modulates both its binding to the template RNA and its unwinding activity. Exposure of the protein to the stable nitroxide TEMPOL oxidizes and degrades the cluster and drastically diminishes unwinding activity. Thus, optimal function of nsp13 depends on a labile Fe-S cluster that is potentially targetable for COVID-19 treatment.

Efficacy of the Radical Scavenger, Tempol, to Reduce Inflammation and Oxidative Stress in a Murine Model of Atopic Dermatitis.

Atopic dermatitis (AD) is the most common chronically relapsing inflammatory skin disease, predominantly common in children; it is characterized by an eczematous pattern generally referable to skin dryness and itchy papules that become excoriated and lichenified in the more advanced stages of the disease. Although the pathophysiology of AD is not completely understood, numerous studies have demonstrated the complex interaction between genetic, immunological, and environmental factors, which acts to disrupt skin barrier function. Free radicals play a key role by directly damaging skin structure, inducing inflammation and weakening of the skin barrier. Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) is a membrane-permeable radical scavenger, known to be a stable nitroxide, which exhibits excellent antioxidant effects in several human disorders, such as osteoarthritis and inflammatory bowel diseases. Considering the few existing studies on dermatological pathologies, this study aimed to evaluate tempol, in a cream formulation, in a murine model of AD. Dermatitis was induced in mice via dorsal skin application of 0.5% Oxazolone, three times a week for two weeks. After induction, mice were treated with tempol-based cream for another two weeks at three different doses of 0.5%, 1% and 2%. Our results demonstrated the ability of tempol, at the highest percentages, to counteract AD by reducing the histological damage, decreasing mast cell infiltration, and improving the skin barrier properties, by restoring the tight junction (TJs) and filaggrin. Moreover, tempol, at 1% and 2%, was able to modulate inflammation by reducing the nuclear factor kappa-light-chain-enhancer of the activated B cell (NF-κB) pathway, as well as tumor necrosis factor (TNF)-α and interleukin (IL)-1β expression. Topical treatment also attenuated oxidative stress by modulating nuclear factor erythroid 2-related factor 2 (Nrf2), manganese superoxide dismutase (MnSOD), and heme oxygenase I (HO-1) expression levels. The obtained results demonstrate the numerous advantages provided by the topical administration of a tempol-based cream formulation, in reducing inflammation and oxidative stress through modulation of the NF-κB/Nrf2 signaling pathways. Therefore, tempol could represent an alternative anti-atopic approach to treating AD, thereby improving skin barrier function.

Magnetically Cured Macroradical Epoxy as Antimicrobial Coating.

The radical-bearing epoxy monomer could be the ideal embodiment of multifunctionality in epoxy-based materials. This study demonstrates the potential of macroradical epoxies as surface coating materials. A diepoxide monomer derivatized with a stable nitroxide radical is polymerized with a diamine hardener under the influence of a magnetic field. The magnetically oriented and stable radicals in the polymer backbone render the coatings antimicrobial. The unconventional use of magnets during polymerization proved crucial in correlating the structure-property relationships with antimicrobial performance inferred from oscillatory rheological technique, polarized macro-attenuated total reflectance - infrared (macro-ATR-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The magnetic thermal curing influenced the surface morphology, resulting in a synergy of the coating's radical nature with microbiostatic performance assessed using the Kirby-Bauer test and liquid chromatography - mass spectroscopy (LC-MS). Further, the magnetic curing of blends with a traditional epoxy monomer demonstrates that radical alignment is more critical than radical density in imparting biocidal behavior. This study shows how the systematic use of magnets during polymerization could pave for probing more significant insights into the mechanism of antimicrobial action in radical-bearing polymers.

Direct Application of 3-Maleimido-PROXYL for Proving Hypoalbuminemia in Cases of SARS-CoV-2 Infection: The Potential Diagnostic Method of Determining Albumin Instability and Oxidized Protein Level in Severe COVID-19.

Oxidative stress and the albumin oxidized form can lead to hypoalbuminemia, which is a predisposing factor for reduced treatment effectiveness and an increased mortality rate in severe COVID-19 patients. The aim of the study is to evaluate the application of free radical 3-Maleimido-PROXYL and SDSL-EPR spectroscopy in the in vitro determination of ox/red HSA in serum samples from patients with SARS-CoV-2 infection. Venous blood was collected from patients intubated (pO2 < 90%) with a positive PCR test for SARS-CoV-2 and controls. At the 120th minute after the incubation of the serum samples from both groups with the 3-Maleimido-PROXYL, the EPR measurement was started. The high levels of free radicals were determined through the nitroxide radical TEMPOL, which probably led to increased oxidation of HSA and hypoalbuminemia in severe COVID-19. The double-integrated spectra of 3-Maleimido-PROXYL radical showed a low degree of connectivity due to high levels of oxidized albumin in COVID-19 patients. The low concentrations of reduced albumin in serum samples partially inhibit spin-label rotation, with Amax values and ΔH0 spectral parameters comparable to those of 3-Maleimido-PROXYL/DMSO. Based on the obtained results, we suggest that the stable nitroxide radical 3-Maleimido-PROXYL can be successfully used as a marker to study oxidized albumin levels in COVID-19.

A system for in vivo on-demand ultra-low field Overhauser-enhanced 3D-Magnetic resonance imaging

Development of very-low field MRI is an active area of research. It aims at reducing operating costs and improve portability. However, the signal-to-noise issue becomes prominent at ultra-low field (<1 mT), especially for molecular imaging purposes that addresses specific biochemical events. In the context of preclinical molecular MRI of abnormal proteolysis the paper describes a MRI system able to produce Overhauser-enhanced MR images in living rats through in situ Dynamic Nuclear Polarization at 206 µT using stable and non-toxic nitroxides. In parallel conventional images are generated at 206 µT following pre-polarization at 20 mT. Results show that nitroxides are visualized in 3D within a few minutes in the lungs, kidneys and bladder post-administration. This system will be used for molecular imaging of inflammation using protease-specific nitroxide probes.

Novel Orientation-Sensitive Spin Probes for Graphene Oxide Membranes Study.

Spin probe EPR spectroscopy is currently the only method to quantitatively report on the orientational ordering of graphene oxide membranes. This technique is based on the analysis of EPR spectra of a membrane containing stable radicals sorbed on oxidized graphene planes. The efficiency of the method depends on the spin probe structure; therefore, it is important to find stable paramagnetic substances that are most sensitive to the alignment of graphene oxide membranes. In the present work, three novel stable nitroxide radicals containing aromatic fragments with two nitrogen atoms were tested as spin probes to study graphene oxide membranes. The spin-Hamiltonian parameters of the radicals in graphite oxide powder and orientational order parameters of the probes inside graphene oxide membrane were determined. The sensitivity of one of these radicals to membrane orientational ordering was found to be higher than for any of spin probes used previously. A likely reason for this higher sensitivity is the presence of heteroatoms which can facilitate interaction between paramagnetic molecules and oxygen-containing groups on the inner surface of the membrane. The new high-sensitivity spin probe may significantly increase the potential of EPR spectroscopy for studying the internal structure of graphene oxide membranes.

Detection of molecular transitions with nitrogen-vacancy centers and electron-spin labels

We present a protocol that detects molecular conformational changes with two nitroxide electron-spin labels and a nitrogen-vacancy (NV) center in diamond. More specifically, we demonstrate that the NV can detect energy shifts induced by the coupling between electron-spin labels. The protocol relies on the judicious application of microwave and radiofrequency pulses in a range of parameters that ensures stable nitroxide resonances. Furthermore, we demonstrate that our scheme is optimized by using nitroxides with distinct nitrogen isotopes. We develop a simple theoretical model that we combine with Bayesian inference techniques to demonstrate that our method enables the detection of conformational changes in ambient conditions including strong NV dephasing rates as a consequence of the diamond surface proximity and nitroxide thermalization mechanisms. Finally, we counter-intuitively show that with our method the small residual effect of random molecular tumbling becomes a resource that can be exploited to extract inter-label distances.

Robust compound elastomers of integrated polymer cathode and electrolyte for high-performance all-solid-state lithium battery

A novel type of all-solid-state lithium-organic batteries based on redox active multicomponent polyethers comprising stable nitroxide radicals, ethylene oxide groups and cross-linkable moieties has been demonstrated. The radical copolymer with a low glass transition temperature exhibits enhanced mixed conductivities of electron and ion. Furthermore, the in-situ cross-linking of the radical copolymer cathode and a comb-branched polyether electrolyte results in a flexible yet strong compound elastomer, which demonstrates a low interfacial resistance and fast ion transport. The fabricated lithium cell shows a high cycling stability with a low polarization and durable rate capability at room temperature, benefiting from the fast redox reactions in the organic cathode and the integrated structure. This work is expected to advance the development of flexible solid batteries with high electrochemical performance.