Category Archives: Nitroxides

Continuous wave electron paramagnetic resonance of nitroxide biradicals in fluid solution

Eaton, Sandra S., Lukas B. Woodcock, and Gareth R. Eaton. “Continuous Wave Electron Paramagnetic Resonance of Nitroxide Biradicals in Fluid Solution.” Concepts in Magnetic Resonance Part A 47A, no. 2 (March 2018): e21426.

https://doi.org/10.1002/cmr.a.21426

Nitroxide biradicals have been prepared with electron-electron spin-spin exchange interaction, J, ranging from weak to very strong. EPR spectra of these biradicals in fluid solution depend on the ratio of J to the nitrogen hyperfine coupling, AN, and the rates of interconversion between conformations with different values of J. For relatively rigid biradicals EPR spectra can be simulated as the superposition of AB splitting patterns arising from different combinations of nitrogen nuclear spin states. For more flexible biradicals spectra can be simulated with a Liouville representation of the dynamics that interconvert conformations with different values of J on the EPR timescale. Analysis of spectra, factors that impact J, and examples of applications to chemical and biophysical problems are discussed.

Succinyl-DOTOPA: An effective triradical dopant for low-temperature dynamic nuclear polarization with high solubility in aqueous solvent mixtures at neutral pH

Yau, Wai-Ming, Jaekyun Jeon, and Robert Tycko. “Succinyl-DOTOPA: An Effective Triradical Dopant for Low-Temperature Dynamic Nuclear Polarization with High Solubility in Aqueous Solvent Mixtures at Neutral PH.” Journal of Magnetic Resonance 311 (February 2020): 106672.

https://doi.org/10.1016/j.jmr.2019.106672

We report the synthesis of the nitroxide-based triradical compound succinyl-DOTOPA and the characterization of its performance as a dopant for dynamic nuclear polarization (DNP) experiments in frozen solutions at low temperatures. Compared with previously described DOTOPA derivatives, succinyl-DOTOPA has substantially greater solubility in glycerol/water mixtures with pH > 4 and therefore has wider applicability. Solid state nuclear magnetic resonance (ssNMR) measurements at 9.39 T and 25 K, with magic-angle spinning at 7.00 kHz, show that build-up times of DNP-enhanced, cross-polarized 13C ssNMR signals are shorter and that signal amplitudes are larger for glycerol/water solutions of L-proline containing succinyl-DOTOPA than for solutions containing the biradical AMUPol, with electron spin concentrations of 15 mM or 30 mM, resulting in greater net sensitivity gains from DNP. In similar measurements at 90 K, AMUPol yields greater net sensitivity, apparently due to its longer electron spin-lattice and spin-spin relaxation times. One- and two-dimensional 13C ssNMR measurements at 25 K on the complex of the 27-residue peptide M13 with the calcium-sensing protein calmodulin, in glycerol/water with 10 mM succinyl-DOTOPA, demonstrate the utility of this compound in DNP-enhanced ssNMR studies of biomolecular systems.

Optimizing nitroxide biradicals for cross-effect MAS-DNP: the role of g-tensors’ distance #DNPNMR

Mentink-Vigier, Frédéric. “Optimizing Nitroxide Biradicals for Cross-Effect MAS-DNP: The Role of g-Tensors’ Distance.” Physical Chemistry Chemical Physics 22, no. 6 (2020): 3643–52.

https://doi.org/10.1039/C9CP06201G

Nitroxide biradicals are common polarizing agents used to enhance the sensitivity of solid-state NMR experiments via Magic Angle Spinning Dynamic Nuclear Polarization (MAS-DNP). These biradicals are used to increase the polarization of protons through the cross-effect mechanism, which requires two unpaired electrons with a Larmor frequency difference greater than that of the protons. From their early conception, the relative orientation of the nitroxide rings has been identified as a critical factor determining their MAS-DNP performance. However, the MAS leads to a complex DNP mechanism with time dependent energy level anti-crossings making it difficult to pinpoint the role of relative g-tensor orientation. In this article, a single parameter called “g-tensors’ distance” is introduced to characterize the relative orientation’s impact on the MAS-DNP field profiles. It is demonstrated for the first time how the g-tensors’ distance determines the nuclear hyperpolarization and depolarization properties of a given biradical. This provides a new critical parameter that paves the way for more efficient bis-nitroxides for MAS-DNP.

Nitroxides: Brief History, Fundamentals, and Recent Developments #DNPNMR

Nitroxide-based radicals are essential to many DNP-NMR experiments and a profound understanding of their chemistry is essential to synthesize new polarizing agents with increased DNP performance. This book gives a comprehensive overview of nitroxides – a good introduction for the new comer but also a reference guide for the expert.

Likhtenshtein, Gertz I. Nitroxides: Brief History, Fundamentals, and Recent Developments. Springer Series in Materials Science. Springer International Publishing, 2020.

https://doi.org/10.1007/978-3-030-34822-9

Written by a pioneer in the development of spin labeling in biophysics, this expert book covers the fundamentals of nitroxide spin labeling through cutting-edge applications in chemistry, physics, materials science, molecular biology, and biomedicine. Nitroxides have earned their place as one of the most popular organic paramagnets due to their suitability as inhibitors of oxidative processes, as a means to polarize magnetic nuclei, and, in molecular biology, as probes and labels to understand molecular structures and dynamics AS DRAGS FOR CANCER AND OTHER DISEASES. Beginning with an overview of the basic methodology and nitroxides’ 145-year history, this book equips students with necessary background and techniques to undertake original research and industry work in this growing field.

Persistence of Nitroxide Radicals in Solution #EPR #DNPNMR

Elajaili, Hanan, Jessica Sedhom, Sandra S. Eaton, and Gareth R. Eaton. “Persistence of Nitroxide Radicals in Solution.” Applied Magnetic Resonance 50, no. 10 (October 2019): 1177–81.

https://doi.org/10.1007/s00723-019-01135-7

Data on long-term persistence of nitroxide radicals typically are focused on solid samples. Less information is available for nitroxides in fluid solution. Sealed deoxygenated solutions of a doxyl nitroxide in tetrahydrofuran and a piperidinyl nitroxide in toluene in 4 mm EPR tubes were kept in a laboratory environment at ambient temperature and without protection from light. After more than 40 years, the concentrations of the solutions had decreased by about factors of 12 and 6, respectively. The longevity in solution probably depends strongly on the purity of the solvent, but these results indicate remarkable persistence.

Anisotropic longitudinal electronic relaxation affects DNP at cryogenic temperatures #DNPNMR

Anisotropic relaxation effects are well know and understood in EPR spectroscopy and have long served as measures to understand the motion (libration) of paramagnetic co-factors (quinones, nitroxide radicals etc.) in biological system. In this study the authors investigate the effect of anisotropic relaxation effects in DNP experiments.

To find more about anisotropic relaxation effects studied by EPR take a look at the work by Sergei Dzuba or the Eatons:

Weber, E.M.M., et al., Anisotropic longitudinal electronic relaxation affects DNP at cryogenic temperatures. Phys. Chem. Chem. Phys., 2017. 19(24): p. 16087-16094.

http://dx.doi.org/10.1039/C7CP03242K

We report the observation of anisotropic longitudinal electronic relaxation in nitroxide radicals under typical dynamic nuclear polarization conditions. This anisotropy affects the efficiency of dynamic nuclear polarization at cryogenic temperatures of 4 K and high magnetic fields of 6.7 T. Under our experimental conditions, the electron paramagnetic resonance spectrum of nitroxides such as TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl) is only partly averaged by electronic spectral diffusion, so that the relaxation times T1e([small omega]) vary across the spectrum. We demonstrate how the anisotropy of T1e([small omega]) can be taken into account in simple DNP models.

EPR Spectroscopy of Nitroxide Spin Probes #EPR #DNPNMR

Nitroxide spin labels are extensively used in EPR for distance measurements and many polarizing agents are based on nitroxides. More recently they are also used in Overhauser DNP measurements (ODNP) to study surface hydration dynamics of larger (membrane) proteins. Although the article is already a bit older, it is a nice review of spin labels and their use in EPR spectroscopy.

Bordignon, E., EPR Spectroscopy of Nitroxide Spin Probes, in eMagRes. 2017, John Wiley & Sons, Ltd. p. 235-254.

http://dx.doi.org/10.1002/9780470034590.emrstm1513

In this article, we will introduce the main chemical and spectroscopic properties of nitroxides. These paramagnetic non-endogenous probes have been widely used in EPR spectroscopy in the last decade due to their high stability and simple spectral fingerprint, which provides a wealth of qualitative and quantitative information about their microscopic environment under almost unrestricted experimental conditions. Nitroxides can be covalently or noncovalently introduced into a variety of different materials to monitor viscosity, local dynamics, pH, polarity, H-bond networks, transition temperatures, and distances toward other nitroxide probes. In general, these small probes minimally perturb the system under investigation, and being the unique paramagnetic centers in an otherwise diamagnetic sample, they provide unequivocal information. Here we will focus on their exquisite sensitivity to report molecular motions within defined ‘EPR timescales’ and spin-spin interactions via changes in their spectral lineshape. Additionally, we will discuss some methods to monitor polarity and formation of H-bonds in their microenvironment.

A combined EPR and MD simulation study of a nitroxyl spin label with restricted internal mobility sensitive to protein dynamics

Oganesyan, V.S., et al., A combined EPR and MD simulation study of a nitroxyl spin label with restricted internal mobility sensitive to protein dynamics. J. Magn. Reson., 2017. 274: p. 24-35.

www.sciencedirect.com/science/article/pii/S1090780716302270

EPR studies combined with fully atomistic Molecular Dynamics (MD) simulations and an MD-EPR simulation method provide evidence for intrinsic low rotameric mobility of a nitroxyl spin label, Rn, compared to the more widely employed label MTSL (R1). Both experimental and modelling results using two structurally different sites of attachment to Myoglobin show that the EPR spectra of Rn are more sensitive to the local protein environment than that of MTSL. This study reveals the potential of using the Rn spin label as a reporter of protein motions.

Trityl-based alkoxyamines as NMP controllers and spin-labels

Audran, G., et al., Trityl-based alkoxyamines as NMP controllers and spin-labels. Polym. Chem., 2016. 7(42): p. 6490-6499.

http://dx.doi.org/10.1039/C6PY01303A

Recently, new applications of trityl-nitroxide biradicals were proposed. In the present study, attachment of a trityl radical to alkoxyamines was performed for the first time. The rate constants kd of C-ON bond homolysis in these alkoxyamines were measured and found to be similar to those for alkoxyamines without a trityl moiety. The electron paramagnetic resonance (EPR) spectra of the products of alkoxyamine homolysis (trityl-TEMPO and trityl-SG1 biradicals) were recorded, and the corresponding exchange interactions were estimated. The decomposition of trityl-alkoxyamines showed more than an 80% yield of biradicals, meaning that the C-ON bond homolysis is the main reaction. The suitability of these labelled initiators/controllers for polymerisation was exemplified by means of a successful nitroxide-mediated polymerisation (NMP) of styrene. Thus, this is the first report of a spin-labelled alkoxyamine suitable for NMP.

Nuclear spin-lattice relaxation in nitroxide spin-label EPR

Marsh, D., Nuclear spin-lattice relaxation in nitroxide spin-label EPR. J Magn Reson, 2016. 272: p. 166-171.

https://www.ncbi.nlm.nih.gov/pubmed/27712989

Nuclear relaxation is a sensitive monitor of rotational dynamics in spin-label EPR. It also contributes competing saturation transfer pathways in T1-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 Wn commonly used in the CW-EPR literature for 14N-nitroxyl spin labels is inconsistent with that currently adopted in time-resolved EPR measurements of saturation recovery. Redefinition of the normalised 14N spin-lattice relaxation rate, b=Wn/(2We), 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 14N-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 14N-relaxation: T1n=1/Wn. Results are compared and contrasted with those for the two-level 15N-nitroxide system.

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