Category Archives: Radicals

Platforms for Stable Carbon‐Centered Radicals #DNPNMR

Kato, Kenichi, and Atsuhiro Osuka. “Platforms for Stable Carbon‐Centered Radicals.” Angewandte Chemie International Edition 58, no. 27 (July 2019): 8978–86.

Organic radicals can play important roles potentially in diverse functional materials owing to an unpaired electron but are usually highly reactive and difficult to use. Therefore, stabilization of organic radicals is crucially important. Among organic radicals, carbon-centered radicals are promising because of their trivalent nature that enables structural diversity and elaborate designs but they show less stabilities because of reactivities toward carboncarbon bond formation and atmospheric oxygen. Recently, stable carbon-centered radicals have been increasingly explored on the basis of diverse molecular platforms. This minireview highlights these newly explored stable carbon-centered radicals with a particular focus on porphyrinoid-stabilized radicals owing to their remarkable spin delocalization abilities.

Stability of nitroxide biradical TOTAPOL in biological samples

McCoy, Kelsey M., Rivkah Rogawski, Olivia Stovicek, and Ann E. McDermott. “Stability of Nitroxide Biradical TOTAPOL in Biological Samples.” Journal of Magnetic Resonance 303 (June 1, 2019): 115–20.

We characterize chemical reduction of a nitroxide biradical, TOTAPOL, used in dynamic nuclear polarization (DNP) experiments, specifically probing the stability in whole-cell pellets and lysates, and present a few strategies to stabilize the biradicals for DNP studies. DNP solid-state NMR experiments use paramagnetic species such as nitroxide biradicals to dramatically increase NMR signals. Although there is considerable excitement about using nitroxide-based DNP for detecting the NMR spectra of proteins in whole cells, nitroxide radicals are reduced in minutes in bacterial cell pellets, which we confirm and quantify here. We show that addition of the covalent cysteine blocker N-ethylmaleimide to whole cells significantly slows the rate of reduction, suggesting that cysteine thiol radicals are important to in vivo radical reduction. The use of cell lysates rather than whole cells also slows TOTAPOL reduction, which suggests a possible role for the periplasm and oxidative phosphorylation metabolites in radical degradation. Reduced TOTAPOL in lysates can also be efficiently reoxidized with potassium ferricyanide. These results point to a practical and robust set of strategies for DNP of cellular preparations.

Endogenous dynamic nuclear polarization NMR of hydride-terminated silicon nanoparticles #DNPNMR

Ha, Michelle, Alyxandra N. Thiessen, Ivan V. Sergeyev, Jonathan G.C. Veinot, and Vladimir K. Michaelis. “Endogenous Dynamic Nuclear Polarization NMR of Hydride-Terminated Silicon Nanoparticles.” Solid State Nuclear Magnetic Resonance 100 (August 2019): 77–84.

Silicon nanoparticles (SiNPs) are intriguing materials and their properties fascinate the broader scientific community; they are also attractive to the biological and materials science sub-disciplines because of their established biological and environmental compatibility, as well as their far-reaching practical applications. While characterization of the particle nanostructure can be performed using 29Si solid-state nuclear magnetic resonance (NMR) spectroscopy, poor sensitivity due to low Boltzmann population and long acquisition times hinder in-depth studies of these potentially game-changing materials. In this study, we compare two dynamic nuclear polarization (DNP) NMR protocols to boost 29Si sensitivity in hydride-terminated SiNPs. First, we assess a traditional indirect DNP approach, where a nitroxide biradical (AMUPol or bCTbk) is incorporated into a glassing agent and transferred through protons (e− → 1H → 29Si) to enhance the silicon. In this mode, electron paramagnetic resonance (EPR) spectroscopy demonstrated that the hydride-terminated surface was highly reactive with the exogenous biradicals, thus decomposing the radicals within hours and resulting in an enhancement factor, ε, of 3 (TB = 15 s) for the 64 nm SiNP, revealing the surface components. Secondly, direct DNP NMR methods were used to enhance the silicon without the addition of an exogenous radical (i.e., use of dangling bonds as an endogenous radical source). With radical concentrations <1 mM, 29Si enhancements were obtained for the series of SiNPs ranging from 3 to 64 nm. The ability to use direct 29Si DNP transfer (e− → 29Si) shows promise for DNP studies of these inorganic nanomaterials (ε = 6 (TB = 79 min) for 64 nm SiNPs) with highly reactive surfaces, showing the sub-surface and core features. These preliminary findings lay a foundation for future endogenous radical development through tailoring the surface chemistry, targeting further sensitivity gains.

Efficient Hyperpolarization of U- 13C-Glucose Using Narrow-Line UV-Generated Labile Free Radicals #DNPNMR

DNP requires a paramagnetic polarizing agent. This is great for DNP but not so great for the NMR experiment, since the paramagnetic species often causes line broadening due to increase nuclear relaxation. To decrease the unwanted relaxation enhancement some researchers suggested to remove (e.g. filter out) the paramagnetic species after the dissolution step.

The article describes an elegant method, using UV generated radicals for polarization at low temperatures, which recombine once the sample is heated up during the dissolution process, effectively removing the paramagnetic enhanced relaxation process.

Capozzi, Andrea, Saket Patel, Christine Pepke Gunnarsson, Irene Marco-Rius, Arnaud Comment, Magnus Karlsson, Mathilde H. Lerche, Olivier Ouari, and Jan Henrik Ardenkjaer-Larsen. “Efficient Hyperpolarization of U- 13C-Glucose Using Narrow-Line UV-Generated Labile Free Radicals.” Angewandte Chemie, December 20, 2018.

Free radicals generated via irradiation with UV-light of a frozen solution containing a fraction of pyruvic acid (PA), have demonstrated their dissolution Dynamic Nuclear Polarization (dDNP) potential providing up to 30% [1-13C]PA liquid-state polarization. Moreover, their labile nature has proven to pave a way to nuclear polarization storage and transport. Herein, differently from the case of PA, we tackled the issue of providing dDNP UV-radical precursors, trimethylpyruvic acid (TriPA) and its methyl-deuterated form d9-TriPA, not involved in any metabolic pathway. The 13C dDNP performance was evaluated for hyperpolarization of [U-13C6,1,2,3,4,5,6,6-d7]-Dglucose. The generated UV-radical proved to be a versatile and highly efficient polarizing agent providing, after dissolution and transfer (10 s), a 13C liquid-state polarization up to 32%.

BDPA-Nitroxide Biradicals Tailored for Efficient Dynamic Nuclear Polarization Enhanced Solid-State NMR at Magnetic Fields up to 21.1 T #DNPNMR

Wisser, Dorothea, Ganesan Karthikeyan, Alicia Lund, Gilles Casano, Hakim Karoui, Maxim Yulikov, Georges Menzildjian, et al. “BDPA-Nitroxide Biradicals Tailored for Efficient Dynamic Nuclear Polarization Enhanced Solid-State NMR at Magnetic Fields up to 21.1 T.” Journal of the American Chemical Society 140, no. 41 (October 17, 2018): 13340–49.

Dynamic nuclear polarization (DNP) solid-state nuclear magnetic resonance (NMR) has developed into an invaluable tool for the investigation of a wide range of materials. However, the sensitivity gain achieved with many polarizing agents suffers from an unfavorable field and magic angle spinning (MAS) frequency dependence. We present a series of new hybrid biradicals, soluble in organic solvents, that consist of an isotropic narrow electron paramagnetic resonance line radical, α,γ-bisdiphenylene-β-phenylallyl (BDPA), tethered to a broad line nitroxide. By tuning the distance between the two electrons and the substituents at the nitroxide moiety, correlations between the electron–electron interactions and the electron spin relaxation times on one hand and the DNP enhancement factors on the other hand are established. The best radical in this series has a short methylene linker and bears bulky phenyl spirocyclohexyl ligands. In a 1.3 mm prototype DNP probe, it yields enhancements of up to 185 at 18.8 T (800 MHz 1H resonance frequency) and 40 kHz MAS. We show that this radical gives enhancement factors of over 60 in 3.2 mm sapphire rotors at both 18.8 and 21.1 T (900 MHz 1H resonance frequency), the highest magnetic field available today for DNP. The effect of the rotor size and of the microwave irradiation inside the MAS rotor is discussed. Finally, we demonstrate the potential of this new series of polarizing agents by recording high field 27Al and 29Si DNP surface enhanced NMR spectra of amorphous aluminosilicates and 17O NMR on silica nanoparticles.

Computationally Assisted Design of Polarizing Agents for Dynamic Nuclear Polarization Enhanced NMR: The AsymPol Family #DNPNMR

Mentink-Vigier, Frédéric, Ildefonso Marin-Montesinos, Anil P. Jagtap, Thomas Halbritter, Johan van Tol, Sabine Hediger, Daniel Lee, Snorri Th. Sigurdsson, and Gaël De Paëpe. “Computationally Assisted Design of Polarizing Agents for Dynamic Nuclear Polarization Enhanced NMR: The AsymPol Family.” Journal of the American Chemical Society 140, no. 35 (September 5, 2018): 11013–19.

We introduce a new family of highly efficient polarizing agents for dynamic nuclear polarization (DNP)-enhanced nuclear magnetic resonance (NMR) applications, composed of asymmetric bis-nitroxides, in which a piperidine-based radical and a pyrrolinoxyl or a proxyl radical are linked together. The design of the AsymPol family was guided by the use of advanced simulations that allow computation of the impact of the radical structure on DNP efficiency. These simulations suggested the use of a relatively short linker with the intention to generate a sizable intramolecular electron dipolar coupling/J-exchange interaction, while avoiding parallel nitroxide orientations. The characteristics of AsymPol were further tuned, for instance with the addition of a conjugated carbon−carbon double bond in the 5-membered ring to improve the rigidity and provide a favorable relative orientation, the replacement of methyls by spirocyclohexanolyl groups to slow the electron spin relaxation, and the introduction of phosphate groups to yield highly water-soluble dopants. An in-depth experimental and theoretical study for two members of the family, AsymPol and AsymPolPOK, is presented here. We report substantial sensitivity gains at both 9.4 and 18.8 T. The robust efficiency of this new family is further demonstrated through high-resolution surface characterization of an important industrial catalyst using fast sample spinning at 18.8 T. This work highlights a new direction for polarizing agent design and the critical importance of computations in this process.

Photogenerated Radical in Phenylglyoxylic Acid for in Vivo Hyperpolarized 13C MR with Photosensitive Metabolic Substrates #DNPNMR

Marco-Rius, Irene, Tian Cheng, Adam P. Gaunt, Saket Patel, Felix Kreis, Andrea Capozzi, Alan J. Wright, Kevin M. Brindle, Olivier Ouari, and Arnaud Comment. “Photogenerated Radical in Phenylglyoxylic Acid for in Vivo Hyperpolarized 13 C MR with Photosensitive Metabolic Substrates.” Journal of the American Chemical Society 140, no. 43 (October 31, 2018): 14455–63.

Whether for 13C magnetic resonance studies in chemistry, biochemistry or biomedicine, hyperpolarization methods based on dynamic nuclear polarization (DNP) have become ubiquitous. DNP requires a source of unpaired electrons, which are commonly added to the sample to be hyperpolarized in the form of stable free radicals. Once polarized, the presence of these radicals is unwanted. These radicals can be replaced by nonpersistent radicals created by photo-irradiation of pyruvic acid (PA), which are annihilated upon dissolution or thermalization in the solid state. However, since PA is readily metabolized by most cells, its presence may be undesirable for some metabolic studies. In addition, some 13C substrates are photo-sensitive and, therefore, may degrade during photo-generation of PA radical, which requires ultraviolet (UV) light. We show here that photoirradiation of phenylglyoxylic acid (PhGA) using visible light produces a non-persistent radical that, in principle, can be used to hyperpolarize any molecule. We compare radical yields in samples containing PA and PhGA upon photo-irradiation with broadband and narrowband UV-visible light sources. To demonstrate the suitability of PhGA as a radical precursor for DNP, we polarized the gluconeogenic probe 13C-dihydroxyacetone, which is UV-sensitive, using a commercial 3.35 T DNP polarizer and then injected this into a mouse and followed its metabolism in vivo.

Efficiency of Water-Soluble Nitroxide Biradicals for Dynamic Nuclear Polarization in Rotating Solids at 9.4 T: bcTol-M and cyolyl-TOTAPOL as New Polarizing Agents #DNPNMR

Geiger, Michel-Andreas, Anil P. Jagtap, Monu Kaushik, Han Sun, Daniel Stöppler, Snorri T. Sigurdsson, Björn Corzilius, and Hartmut Oschkinat. “Efficiency of Water-Soluble Nitroxide Biradicals for Dynamic Nuclear Polarization in Rotating Solids at 9.4 T: BcTol-M and Cyolyl-TOTAPOL as New Polarizing Agents.” Chemistry – A European Journal 24, no. 51 (September 12, 2018): 13485–94.

Nitroxide biradicals are very efficient polarizing agents in magic angle spinning (MAS) cross effect (CE) dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR). Many recently synthesized, new radicals show superior DNP-efficiency in organic solvents but suffer from insufficient solubility in water or glycerol/water for biological applications. We report DNP efficiencies for two new radicals, the water-soluble bcTol-M and cyolyl-TOTAPOL, and include a comparison with three known biradicals, TOTAPOL, bcTol, and AMUPol. They differ by linker groups, featuring either a 3-aminopropane-1,2-diol or a urea tether, or by the structure of the alkyl substituents that flank the nitroxide groups. For evaluating their performances, we measured both signal enhancements e and DNP-enhanced sensitivity k, and compared the results to electron spin relaxation data recorded at the same magnetic field strength (9.4 T). In our study, differences in DNP efficiency correlate with changes in the nuclear polarization dynamics rather than electron relaxation.

The ratios of their individual e and k differ by up to 20%, which is explained by starkly different nuclear polarization build-up rates. For the radicals compared here empirically, using proline standard solutions, the new radical bcTol-M performs best while being most soluble in water/glycerol mixtures.

Diastereoisomers of L-proline-linked trityl-nitroxide biradicals: synthesis and effect of chiral configurations on exchange interactions #DNPNMR

Zhai, Weixiang, Yalan Feng, Huiqiang Liu, Antal Rockenbauer, Deni Mance, Shaoyong Li, Yuguang Song, Marc Baldus, and Yangping Liu. “Diastereoisomers of L-Proline-Linked Trityl-Nitroxide Biradicals: Synthesis and Effect of Chiral Configurations on Exchange Interactions.” Chemical Science 9, no. 19 (May 16, 2018): 4381–91.

The exchange (J) interaction of organic biradicals is a crucial factor controlling their physiochemical properties and potential applications and can be modulated by changing the nature of the linker. In the present work, we for the first time demonstrate the effect of chiral configurations of radical parts on the J values of trityl-nitroxide (TN) biradicals. Four diastereoisomers (TNT1, TNT2, TNL1 and TNL2) of TN biradicals were synthesized and purified by the conjugation of a racemic (R/S) nitroxide with the racemic (M/P) trityl radical viaL-proline. The absolute configurations of these diastereoisomers were assigned by comparing experimental and calculated electronic circular dichroism (ECD) spectra as (M, S, S) for TNT1, (P, S, S) for TNT2, (M, S, R) for TNL1 and (P, S, R) for TNL2. Electron paramagnetic resonance (EPR) results showed that the configuration of the nitroxide part instead of the trityl part is dominant in controlling the exchange interactions and the order of the J values at room temperature is TNT1 (252 G) > TNT2 (127 G) ≫ TNL2 (33 G) > TNL1 (14 G). Moreover, the J values of TNL1/TNL2 with the S configuration in the nitroxide part vary with temperature and the polarity of solvents due to their flexible linker, whereas the J values of TNT1/TNT2 are almost insensitive to these two factors due to the rigidity of their linkers. The distinct exchange interactions between TNT1,2 and TNL1,2 in the frozen state led to strongly different high-field dynamic nuclear polarization (DNP) enhancements with ε = 7 for TNT1,2 and 40 for TNL1,2 under 800 MHz DNP conditions.

New NMR tools for protein structure and function: Spin tags for dynamic nuclear polarization solid state NMR #DNPNMR

Rogawski, R. and A.E. McDermott, New NMR tools for protein structure and function: Spin tags for dynamic nuclear polarization solid state NMR. Arch. Biochem. Biophys., 2017. 628: p. 102-113.

Magic angle spinning solid state NMR studies of biological macromolecules [1-3] have enabled exciting studies of membrane proteins [4,5], amyloid fibrils [6], viruses, and large macromolecular assemblies [7]. Dynamic nuclear polarization (DNP) provides a means to enhance detection sensitivity for NMR, particularly for solid state NMR, with many recent biological applications and considerable contemporary efforts towards elaboration and optimization of the DNP experiment. This review explores precedents and innovations in biological DNP experiments, especially highlighting novel chemical biology approaches to introduce the radicals that serve as a source of polarization in DNP experiments.

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