Category Archives: Polarizing Agents

Polarizing Agents: Evolution and Outlook in Free Radical Development for DNP #DNPNMR

Casano, Gilles, Hakim Karoui, and Olivier Ouari. “Polarizing Agents: Evolution and Outlook in Free Radical Development for DNP” 7 (2018): 14.

https://doi.org/10.1002/9780470034590.emrstm1547.

In this article, we describe an in-depth overview of the development of paramagnetic polarizing agents for dynamic nuclear polarization (DNP)-enhanced solid-state magic angle spinning (MAS) nuclear magnetic resonance (NMR). In DNP experiments, the large polarization of unpaired electrons is transferred to surrounding nuclei, which provides a maximum theoretical DNP enhancement of 660 for 1H NMR. The article includes a description of the different polarizing mechanisms and outlines key structural and magnetic parameters that contributed to the rational design of improved polarizing sources. The application of (di)nitroxides, heterobiradicals, narrow-line radicals, paramagnetic metal ions as well as site-specific polarizing agents is discussed. With the best polarizing agents, ssNMR MAS NMR/DNP enhances sensitivity by a factor of up to 200, providing decreased experiment time by five orders of magnitude and opening new avenues for NMR.

Dipolar Order Mediated 1H -> 13C Cross-Polarization for Dissolution-Dynamic Nuclear Polarization #DNPNMR

Elliott, Stuart J., Samuel F. Cousin, Quentin Chappuis, Olivier Cala, Morgan Ceillier, Aurélien Bornet, and Sami Jannin. “Dipolar Order Mediated 1H -> 13C Cross-Polarization for Dissolution-Dynamic Nuclear Polarization.” Preprint. Hyperpolarization/Pulse-sequence development, February 20, 2020.

https://doi.org/10.5194/mr-2020-4

Magnetic resonance imaging and spectroscopy often suffer from a low intrinsic sensitivity, which can in some cases be circumvented by the use of hyperpolarization techniques. Dissolution-dynamic nuclear polarization offers a way of hyperpolarizing 13C spins in small molecules, enhancing their sensitivity by up to four orders of magnitude. This is usually performed by direct 13C polarization, which is straightforward but often takes more than an hour. Alternatively, indirect 1H polarization followed by 1H®13C polarization transfer can be implemented, which is more efficient and faster but is technically very challenging and hardly implemented in practice. Here we propose to remove the main roadblocks of the 1H®13C polarization transfer process by using alternative schemes with: (i) less rf-power; (ii) less overall rf-energy; (iii) simple rf-pulse shapes; and (iv) no synchronized 1H and 13C rf-irradiation. An experimental demonstration of such a simple 1H®13C polarization transfer technique is presented for the case of [1-13C]sodium acetate, and is compared with the most sophisticated cross-polarization schemes. A polarization transfer efficiency of ~0.43 with respect to cross-polarization was realized, which resulted in a 13C polarization of ~8.7% after ~10 minutes of microwave irradiation and a single polarization transfer step.

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.

Targetable Tetrazine‐Based Dynamic Nuclear Polarization Agents for Biological Systems #DNPNMR

Lim, Byung Joon, Bryce E. Ackermann, and Galia T Debelouchina. “Targetable Tetrazine‐Based Dynamic Nuclear Polarization Agents for Biological Systems.” ChemBioChem, November 19, 2019, cbic.201900609.

https://doi.org/10.1002/cbic.201900609.

Dynamic nuclear polarization (DNP) has shown great promise as a tool to enhance the nuclear magnetic resonance (NMR) signals of proteins in the cellular environment. As the sensitivity increases, the ability to select and efficiently polarize a specific macromolecule over the cellular background has become desirable. Here, we address this need and present a tetrazine-based DNP polarization agent that can be targeted selectively to proteins containing the unnatural amino acid (UAA) norbornene-lysine. The UAA can be introduced efficiently by genetic means in the cellular milieu. Our approach is bio-orthogonal and easily adaptable to any protein of interest. We illustrate the scope of our methodology and investigate the DNP polarization transfer mechanisms in several biological systems. Our results shed light on the complex polarization transfer pathways in targeted DNP and ultimately pave the way to selective DNPenhanced NMR spectroscopy in both bacterial and mammalian cells.

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.

Sensitivity analysis of magic angle spinning dynamic nuclear polarization below 6 K #DNPNMR

Judge, Patrick T., Erika L. Sesti, Edward P. Saliba, Nicholas Alaniva, Thomas Halbritter, Snorri Th. Sigurdsson, and Alexander B. Barnes. “Sensitivity Analysis of Magic Angle Spinning Dynamic Nuclear Polarization below 6 K.” Journal of Magnetic Resonance 305 (August 2019): 51–57.

https://doi.org/10.1016/j.jmr.2019.05.011

Dynamic nuclear polarization (DNP) improves signal-to-noise in nuclear magnetic resonance (NMR) spectroscopy. Signal-to-noise in NMR can be further improved with cryogenic sample cooling. Whereas MAS DNP is commonly performed between 25 and 110 K, sample temperatures below 6 K lead to further improvements in sensitivity. Here, we demonstrate that solid effect MAS DNP experiments at 6 K, using trityl, yield 3.2Â more sensitivity compared to 90 K. Trityl with solid effect DNP at 6 K yields substantially more signal to noise than biradicals and cross effect DNP. We also characterize cross effect DNP with AMUPol and TEMTriPol-1 biradicals for DNP magic angle spinning at temperatures below 6 K and 7 Tesla. DNP enhancements determined from microwave on/off intensities are 253 from AMUPol and 49 from TEMTriPol-1. The higher thermal Boltzmann polarization at 6 K compared to 298 K, combined with these enhancements, should result in 10,000Â signal gain for AMUPol and 2000Â gain for TEMTriPol-1. However, we show that AMUPol reduces signal in the absence of microwaves by 90% compared to 41% by TEMTriPol-1 at 7 Tesla as the result of depolarization and other detrimental paramagnetic effects. AMUPol still yields the highest signal-to-noise improvement per unit time between the cross effect radicals due to faster polarization buildup (T1DNP = 4.3 s and 36 s for AMUPol and TEMTriPol-1, respectively). Overall, AMUPol results in 2.5Â better sensitivity compared to TEMTriPol-1 in MAS DNP experiments performed below 6 K at 7 T. Trityl provides 6.0Â more sensitivity than TEMTriPol-1 and 1.9Â more than AMUPol at 6 K, thus yielding the greatest signal-to-noise per unit time among all three radicals. A DNP enhancement profile of TEMTriPol-1 recorded with a frequency-tunable custom-built gyrotron oscillator operating at 198 GHz is also included. It is determined that at 7 T below 6 K a microwave power level of 0.6 W incident on the sample is sufficient to saturate the cross effect mechanism using TEMTriPol-1, yet increasing the power level up to 5 W results in higher improvements in DNP sensitivity with AMUPol. These results indicate MAS DNP below 6 K will play a prominent role in ultra-sensitive NMR spectroscopy in the future.

Biradical rotamer states tune electron J coupling and MAS dynamic nuclear polarization enhancement #DNPNMR

Tagami, Kan, Asif Equbal, Ilia Kaminker, Bernard Kirtman, and Songi Han. “Biradical Rotamer States Tune Electron J Coupling and MAS Dynamic Nuclear Polarization Enhancement.” Solid State Nuclear Magnetic Resonance 101 (September 2019): 12–20.

https://doi.org/10.1016/j.ssnmr.2019.04.002

Cross Effect (CE) Dynamic Nuclear Polarization (DNP) relies on the dipolar (D) and exchange (J) coupling interaction between two electron spins. Until recently only the electron spin D coupling was explicitly included in quantifying the DNP mechanism. Recent literature discusses the potential role of J coupling in DNP, but does not provide an account of the distribution and source of electron spin J coupling of commonly used biradicals in DNP. In this study, we quantified the distribution of electron spin J coupling in AMUPol and TOTAPol biradicals using a combination of continuous wave (CW) X-band electron paramagnetic resonance (EPR) lineshape analysis in a series of solvents and at variable temperatures in solution – a state to be vitrified for DNP. We found that both radicals show a temperature dependent distribution of J couplings, and the source of this distribution to be conformational dynamics. To qualify this conformational dependence of J coupling in both molecules we carry out “Broken Symmetry” DFT calculations which show that the biradical rotamer distribution can account for a large distribution of J couplings, with the magnitude of J coupling directly depending on the relative orientation of the electron spin pair. We demonstrate that the electron spin J couplings in both AMUPol and TOTAPol span a much wider distribution than suggested in the literature. We affirm the importance of electron spin J coupling for DNP with density matrix simulations of DNP in Liouville space and under magic angle spinning, showcasing that a rotamer with high J coupling and “optimum” relative g-tensor orientation can significantly boost the DNP performance compared to random orientations of the electron spin pair. We conclude that moderate electron spin J coupling above a threshold value can facilitate DNP enhancements.

Dynamic Nuclear Polarization / solid-state NMR of membranes. Thermal effects and sample geometry #DNPNMR

Salnikov, Evgeniy Sergeevich, Fabien Aussenac, Sebastian Abel, Armin Purea, Paul Tordo, Olivier Ouari, and Burkhard Bechinger. “Dynamic Nuclear Polarization / Solid-State NMR of Membranes. Thermal Effects and Sample Geometry.” Solid State Nuclear Magnetic Resonance 100 (August 2019): 70–76.

https://doi.org/10.1016/j.ssnmr.2019.03.004.

Whereas specially designed dinitroxide biradicals, reconstitution protocols, oriented sample geometries and NMR probes have helped to much increase the DNP enhancement factors of membrane samples they still lag considerably behind those obtained from glasses made of protein solutions. Here we show that not only the MAS rotor material but also the distribution of the membrane samples within the NMR rotor have a pronounced effect on the DNP efficiency. These observations are rationalized with the cooling efficiency and the internal properties of the sample, monitored by their T1 relaxation, microwave on versus off signal intensities and DNP enhancement. The data are suggestive that for membranes the speed of cooling has a pronounced effect on membrane phase transitions and concomitantly the distribution of biradicals within the sample.

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