Category Archives: Trityl

Direct dynamic nuclear polarization of 15N and 13C spins at 14.1 T using a trityl radical and magic angle spinning #DNPNMR

Wang, Xiaoling, Bethany G. Caulkins, Gwladys Riviere, Leonard J. Mueller, Frederic Mentink-Vigier, and Joanna R. Long. “Direct Dynamic Nuclear Polarization of 15N and 13C Spins at 14.1 T Using a Trityl Radical and Magic Angle Spinning.” Solid State Nuclear Magnetic Resonance, April 2019, S0926204019300177.

We investigate solid-state dynamic nuclear polarization of 13C and 15N nuclei using monoradical trityl OX063 as a polarizing agent in a magnetic field of 14.1 T with magic angle spinning at ~100 K. We monitored the field dependence of direct 13C and 15N polarization for frozen [13C, 15N] urea and achieved maximum absolute enhancement factors of 240 and 470, respectively. The field profiles are consistent with polarization of 15N spins via either the solid effect or the cross effect, and polarization of 13C spins via a combination of cross effect and solid effect. For microcrystalline, 15Nenriched tryptophan synthase sample containing trityl radical, a 1500-fold increase in 15N signal was observed under microwave irradiation. These results show the promise of trityl radicals and their derivatives for direct polarization of low gamma, spin-½ nuclei at high magnetic fields and suggest a novel approach for selectively polarizing specific moieties or for polarizing systems which have low levels of protonation.

Influence of 13C Isotopic Labeling Location on Dynamic Nuclear Polarization of Acetate #DNPNMR

Niedbalski, Peter, Christopher Parish, Andhika Kiswandhi, Zoltan Kovacs, and Lloyd Lumata. “Influence of 13C Isotopic Labeling Location on Dynamic Nuclear Polarization of Acetate.” The Journal of Physical Chemistry A 121, no. 17 (May 4, 2017): 3227–33.

Dynamic nuclear polarization (DNP) via the dissolution method has alleviated the insensitivity problem in liquid-state nuclear magnetic resonance (NMR) spectroscopy by amplifying the signals by several thousand-fold. This NMR signal amplification process emanates from the microwavemediated transfer of high electron spin alignment to the nuclear spins at high magnetic field and cryogenic temperature. Since the interplay between the electrons and nuclei is crucial, the chemical composition of a DNP sample such as the type of free radical used, glassing solvents, or the nature of the target nuclei can significantly affect the NMR signal enhancement levels that can be attained with DNP. Herein, we have investigated the influence of 13C isotopic labeling location on the DNP of a model 13C compound, sodium acetate, at 3.35 T and 1.4 K using the narrow electron spin resonance (ESR) line width free radical trityl OX063. Our results show that the carboxyl 13C spins yielded about twice the polarization produced in methyl 13C spins. Deuteration of the methyl 13C group, while proven beneficial in the liquid-state, did not produce an improvement in the 13C polarization level at cryogenic conditions. In fact, a slight reduction of the solid-state 13C polarization was observed when 2H spins are present in the methyl group. Furthermore, our data reveal that there is a close correlation between the solid-state 13C T1 relaxation times of these samples and the relative 13C polarization levels. The overall results suggest the achievable solid-state polarization of 13C acetate is directly affected by the location of the 13C isotopic labeling via the possible interplay of nuclear relaxation leakage factor and cross-talks between nuclear Zeeman reservoirs in DNP.

Electron-Spin Relaxation of Triarylmethyl Radicals in Glassy Trehalose #DNPNMR

Triarylmethyl radicals are commonly used dissolution-DNP experiments (dDNP). This article is a good reference source for the electronic relaxation times T1e and T2e in different solvents.

Kuzhelev, Andrey A., Olesya A. Krumkacheva, Ivan O. Timofeev, Victor M. Tormyshev, Matvey V. Fedin, and Elena G. Bagryanskaya. “Electron-Spin Relaxation of Triarylmethyl Radicals in Glassy Trehalose.” Applied Magnetic Resonance 49, no. 11 (November 2018): 1171–80.

Trehalose was recently proposed as a promising immobilizer of biomolecules for room-temperature electron paramagnetic resonance (EPR) structural studies. The most crucial parameter in these investigations is electron-spin relaxation (namely, phase memory time Tm). Recently, triarylmethyl (TAM) spin labels attached to DNA in trehalose were found to have the longest Tm at room temperature as compared to the existing spin labels and immobilizers. Therefore, in this work, we investigated TAM radicals in trehalose including Finland trityl (H36 form), perdeuterated Finland trityl (D36 form), and a deuterated version of OX063. The temperature dependence of electron-spin relaxation time of these radicals immobilized in trehalose was measured at X-band frequency, and possible mechanisms of relaxation were considered. OX063D in glassy trehalose has longer Tm up to 200 K as compared to Finland trityl, but at higher temperatures, OX063D is inferior in its relaxation properties, and the deuterated form of Finland trityl is preferable for pulse dipolar EPR spectroscopy experiments at 298 K. The influence of various deuterations (TAM or trehalose) on the observed relaxation times was studied, being controlled by the electron-spin-echo envelope modulation at room temperature.

Electronic control of DNA-based nanoswitches and nanodevices #DNPNMR

Ranallo, Simona, Alessia Amodio, Andrea Idili, Alessandro Porchetta, and Francesco Ricci. “Electronic Control of DNA-Based Nanoswitches and Nanodevices.” Chem. Sci. 7, no. 1 (2016): 66–71.

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 conjugation of a racemic (R/S) nitroxide with the racemic (M/P) trityl radical via L-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 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 frozen state led to strongly different high-field dynamic nuclear polarization (DNP) enhancements with ε = 7 for TNT1,2 and 40 for TNL1,2 at 800 MHz DNP conditions.

The effect of Ho3+ doping on 13C dynamic nuclear polarization at 5 T

Sirusi, A.A., et al., The effect of Ho3+ doping on 13C dynamic nuclear polarization at 5 T. PCCP, 2018. 20(2): p. 728-731.

Dissolution dynamic nuclear polarization was introduced in 2003 as a method for producing hyperpolarized 13C solutions suitable for metabolic imaging. The signal to noise ratio for the imaging experiment depends on the maximum polarization achieved in the solid state. Hence, optimization of the DNP conditions is essential. To acquire maximum polarization many parameters related to sample preparation can be modulated. Recently, it was demonstrated that Ho3+, Dy3+, Tb3+, and Gd3+ complexes enhance the polarization at 1.2 K and 3.35 T when using the trityl radical as the primary paramagnetic center. Here, we have investigated the influence of Ho-DOTA on 13C solid state DNP at 1.2 K and 5 T. We have performed 13C DNP on [1-13C] sodium acetate in 1 : 1 (v/v) water/glycerol with 15 mM trityl OX063 radicals in the presence of a series of Ho-DOTA concentrations (0, 0.5, 1, 2, 3, 5 mM). We have found that adding a small amount of Ho-DOTA in the sample preparation not only enhances the 13C polarization but also decreases the buildup time. The optimum Ho-DOTA concentration was 2 mM. In addition, the microwave sweep spectrum changes character in a manner that suggests both the cross effect and thermal mixing are active mechanisms for trityl radical at 5 T and 1.2 K.

EPR Imaging Spin Probe Trityl Radical OX063: A Method for Its Isolation from Animal Effluent, Redox Chemistry of Its Quinone Methide Oxidation Product, and in Vivo Application in a Mouse

Serda, M., et al., EPR Imaging Spin Probe Trityl Radical OX063: A Method for Its Isolation from Animal Effluent, Redox Chemistry of Its Quinone Methide Oxidation Product, and in Vivo Application in a Mouse. Chem Res Toxicol, 2016. 29(12): p. 2153-2156.

We report herein a method for the recovery, purification, and application of OX063, a costly, commercially available nontoxic spin probe widely used for electron paramagnetic resonance (EPR) imaging, as well as its corresponding quinone methide (QM) form. This precious probe can be successfully recovered after use in animal model experiments (25-47% recovery from crude lyophilizate with 98.5% purity), even from samples that are >2 years old. Significantly, the recovered trityl can be reused in further animal model EPR imaging experiments. The work also describes support for the observed formation of an air-sensitive radical derived from the QM under reducing conditions.

The effect of Gd on trityl-based dynamic nuclear polarisation in solids

Ravera, E., et al., The effect of Gd on trityl-based dynamic nuclear polarisation in solids. Phys. Chem. Chem. Phys., 2015. 17(40): p. 26969-78.

In dynamic nuclear polarisation (DNP) experiments performed under static conditions at 1.4 K we show that the presence of 1 mM Gd(iii)-DOTAREM increases the (13)C polarisation and decreases the (13)C polarisation buildup time of (13)C-urea dissolved in samples containing water/DMSO mixtures with trityl radical (OX063) concentrations of 10 mM or higher. To account for these observations further measurements were carried out at 6.5 K, using a combined EPR and NMR spectrometer. At this temperature, frequency swept DNP spectra of samples with 5 or 10 mM OX063 were measured, with and without 1 mM Gd-DOTA, and again a (13)C enhancement gain was observed due to the presence of Gd-DOTA. These measurements were complemented by electron-electron double resonance (ELDOR) measurements to quantitate the effect of electron spectral diffusion (eSD) on the DNP enhancements and lineshapes. Simulations of the ELDOR spectra were done using the following parameters: (i) a parameter defining the rate of the eSD process, (ii) an “effective electron-proton anisotropic hyperfine interaction parameter”, and (iii) the transverse electron spin relaxation time of OX063. These parameters, together with the longitudinal electron spin relaxation time, measured by EPR, were used to calculate the frequency profile of electron polarisation. This, in turn, was used to calculate two basic solid effect (SE) and indirect cross effect (iCE) DNP spectra. A properly weighted combination of these two normalized DNP spectra provided a very good fit of the experimental DNP spectra. The best fit simulation parameters reveal that the addition of Gd(iii)-DOTA causes an increase in both the SE and the iCE contributions by similar amounts, and that the increase in the overall DNP enhancements is a result of narrowing of the ELDOR spectra (increased electron polarisation gradient across the EPR line). These changes in the electron depolarisation profile are a combined result of shortening of the longitudinal and transverse electron spin relaxation times, as well as an increase in the eSD rate and in the effective electron-proton anisotropic hyperfine interaction parameter.

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.

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.

Selective High-Resolution Detection of Membrane Protein-Ligand Interaction in Native Membranes Using Trityl-Nitroxide PELDOR

Joseph, B., et al., Selective High-Resolution Detection of Membrane Protein-Ligand Interaction in Native Membranes Using Trityl-Nitroxide PELDOR. Angew Chem Int Ed Engl, 2016. 55(38): p. 11538-42.

The orchestrated interaction of transmembrane proteins with other molecules mediates several crucial biological processes. Detergent solubilization may significantly alter or even abolish such hetero-oligomeric interactions, which makes observing them at high resolution in their native environment technically challenging. Dipolar electron paramagnetic resonance (EPR) techniques such as pulsed electro-electron double resonance (PELDOR) can provide very precise distances within biomolecules. To concurrently determine the inter-subunit interaction and the intra-subunit conformational changes in hetero-oligomeric complexes, a combination of different spin labels is required. Orthogonal spin labeling using a triarylmethyl (TAM) label in combination with a nitroxide label is used to detect protein-ligand interactions in native lipid bilayers. This approach provides a higher sensitivity and total selectivity and will greatly facilitate the investigation of multimeric transmembrane complexes employing different spin labels in the native lipid environment.

A triarylmethyl spin label for long-range distance measurement at physiological temperatures using T1 relaxation enhancement #DNPNMR

Yang, Z., et al., A triarylmethyl spin label for long-range distance measurement at physiological temperatures using T1 relaxation enhancement. J. Magn. Reson., 2016. 269: p. 50-54.

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy has become an important tool for measuring distances in proteins on the order of a few nm. For this purpose pairs of spin labels, most commonly nitroxides, are site-selectively introduced into the protein. Recent efforts to develop new spin labels are focused on tailoring the intrinsic properties of the label to either extend the upper limit of measurable distances at physiological temperature, or to provide a unique spectral lineshape so that selective pairwise distances can be measured in a protein or complex containing multiple spin label species. Triarylmethyl (TAM) radicals are the foundation for a new class of spin labels that promise to provide both capabilities. Here we report a new methanethiosulfonate derivative of a TAM radical that reacts rapidly and selectively with an engineered cysteine residue to generate a TAM containing side chain (TAM1) in high yield. With a TAM1 residue and Cu2+ bound to an engineered Cu2+ binding site, enhanced T1 relaxation of TAM should enable measurement of interspin distances up to 50 Å at physiological temperature. To achieve favorable TAM1-labeled protein concentrations without aggregation, proteins are tethered to a solid support either site-selectively using an unnatural amino acid or via native lysine residues. The methodology is general and readily extendable to complex systems, including membrane proteins.

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