High-Field DNP and ENDOR with a Novel Multiple-Frequency Resonance Structure

In a previous communication [Hu et al., J. Am. Chem. Soc. 126, 10844 (2004)], an approach was demonstrated that improves the efficiency of the cross-effect polarization mechanism employed in high field dynamic nuclear polarization (DNP) experiments. Specifically, it was shown that tethering two TEMPO (2,2,6,6-tetramethylpiperidin-1-oxyl) radicals increases the electron-electron dipole coupling from approximately 1 MHz in solutions of monomeric TEMPO to approximately 25 MHz in a tethered biradical. The larger coupling resulted in an increase in the DNP enhancements by a factor of approximately 3-4, from 45-50 to approximately 165. Here, a second approach to improving the efficiency of the polarization process is described that involves approximately satisfying the matching condition |omega(2e)-omega(1e)|=omega(n), where omega(2e) and omega(1e) are two frequencies in the electron paramagnetic resonance (EPR) spectrum and omega(n) is the Larmor frequency of the nuclear spins being polarized. Specifically, in a mixture of TEMPO and trityl [tris (8-carboxy-2,2,6,6-tetramethyl(-d3)-benzo[1,2d:4,5-d']bis(1,3)dithiol-4-yl) methyl] radicals, the intensity maxima in the EPR spectra of these two species are approximately separated by the (1)H NMR frequency. In this case the frequency difference between the g(yy) value of TEMPO and the narrow pseudo-isotropic g-value of trityl is approximately 224 MHz and the (1)H Larmor frequency is 211 MHz. The optimal magnetic field for DNP using the mixtures was found to coincide with the trityl EPR resonance. At 90 K and 5 T, a mixture of 20 mM TEMPO and 20 mM trityl enhanced the (1)H polarization by a factor of approximately 160, an improvement over the enhancement of approximately 50 with 40 mM TEMPO. The reasons for the improvement are discussed and evidence is presented suggesting that DNP enhancement can be improved further by tethering TEMPO and trityl or two similar radicals.

Proton Dynamic Nuclear Polarization (DNP) experiments were conducted on a 3.4 T homebuilt hybrid pulsed-EPR-NMR spectrometer, on static samples containing 10 mM or 40 mM TEMPOL in frozen glassy solutions of DMSO/water. During DNP experiments proton-NMR signals are enhanced with the help of microwave (MW) irradiation on or close to the Electron Paramagnetic Resonance (EPR) spectrum of the free radicals in the sample, transferring polarization from the free electrons to the nuclei. In the solid state a distinction is made between three DNP enhancement mechanisms: the Solid Effect (SE), the Cross Effect (CE) and Thermal Mixing (TM). In an effort to determine the dominant DNP mechanisms responsible for the enhancement of the nuclear signals, electron and nuclear spin-lattice relaxation rates, enhancement buildup times and microwave (MW) swept DNP spectra were measured as a function of temperature and MW irradiation strength. We observed lineshape variations of the DNP spectra that indicated changes in the relative contributions of SE-DNP and CE-DNP with temperature and MW power. Using a theoretical model describing the SE-DNP and CE-DNP the DNP spectra could be analyzed without involving the TM-DNP mechanism and the relative SE-DNP and CE-DNP contributions to the nuclear enhancement could be determined. From this analysis it follows that lowering the temperature beyond 20 K increases the SE-DNP and decreases the CE-DNP contributions. Possible explanations for this behavior are suggested.

Impact of non-polar solvents in dynamic nuclear polarization at high magnetic fields.

Paramagnet induced signal quenching in MAS–DNP experiments in frozen homogeneous solutions

To investigate the experimental technique and the high magnetic field effect in the dynamic nuclear polarization (DNP) experiments, X-band 1H-DNP and high-power subterahertz wave irradiation experiments have been performed on bisdiphenylene-phenylallyl-doped toluene solution at room temperature. We have established a sample-shuttle X-band DNP experimental system, and investigated the external magnetic field, the microwave power and 1H T 1 −1 dependences of the Overhauser enhancement factor (e). A maximal signal enhancement of approximately −19 has been observed. As for the subterahertz wave experiments, we have constructed a nuclear magnetic resonance and electron spin resonance measurement system for the experiments under the magnetic field of about 10.7 T, with a high-power light source gyrotron CW-I (TE22,8 mode, f ∼ 299.23 GHz). when the high-power subterahertz wave of a gyrotron was irradiated on the toluene solution in order to confirm the evidence of DNP, the sample has evaporated due to the electromagnetic wave heating.

Breaking the maximum enhancement barrier of 660 at room temperature in a conventional dynamic nuclear polarization (DNP) experiment has the immense potential of practical applications. Optical DNP experiments with radical-chromophore (RC) adducts, which harnesses hyperpolarized radicals, instead of thermalized radicals, offers a powerful way to achieve this. Typical DNP and NMR experiments, however, are carried out at high magnetic fields of about 5-10 T, whereas the large electron spin hyperpolarization (ESP) demonstrated in the RC adducts so far are at a much low field of 0.3 T. Thus, in order to realize a successful optical DNP experiment, it is imperative to ask whether the RC adducts, which are currently available, can achieve a large ESP even at high fields. The present work poses this question and shows that the current RC adducts would not generate a large ESP at high fields unless the separation between the chromophore and nitroxyl moiety is reduced to less than four bonds. Two serious bottlenecks in this direction are the near impossibility of synthesizing such RC adducts using the common nitroxyl radicals and the absence of any photophysical studies on RC adducts with such short spacer groups. In this regard, the present work exploits the spin trapping methodology to synthesize one- and two-atom separated naphthalene-nitroxyl RC adducts. Good yields and excellent stability of the adducts have been demonstrated. Furthermore, the present work presents their detailed photophysical and photochemical studies by transient optical and time-resolved EPR studies. On the basis of the present results, a potential RC adduct is proposed for the high field optical DNP experiments. Finally, the prospect of exploiting the large EPR signal enhancement due to ESP in the field of spin trapping studies has been discussed.

Dynamic nuclear polarization (DNP) is a nuclear magnetic resonance (NMR) hyperpolarization technique that mediates polarization transfer from unpaired electrons with large thermal polarization to NMR-active nuclei via microwave (mw) irradiation. The ability to generate arbitrarily shaped mw pulses using arbitrary waveform generators allows for remarkable improvement of the robustness and versatility of DNP. We present here novel design principles based on single-spin vector effective Hamiltonian theory to develop new broadband DNP pulse sequences, namely, an adiabatic version of XiX (X–inverse X)–DNP and a broadband excitation by amplitude modulation (BEAM)–DNP experiment. We demonstrate that the adiabatic BEAM-DNP pulse sequence may achieve a 1H enhancement factor of ∼360, which is better than ramped-amplitude NOVEL (nuclear spin orientation via electron spin locking) at ∼0.35 T and 80 K in static solids doped with trityl radicals. In addition, the bandwidth of the BEAM-DNP experiments (~50 MHz) is about three times the 1H Larmor frequency. The generality of our theoretical approach will be helpful in the development of new pulsed DNP sequences.

Dynamic nuclear polarization (DNP) combined with high magnetic fields and fast magic angle spinning (MAS) has opened up a new avenue for the application of exceptionally sensitive 1H NMR detection schemes to study protonated solids. Recently, it has been shown that DNP experiments at fast MAS rates lead to slower spin diffusion and hence reduced DNP enhancements for impregnated materials. However, DNP enhancements alone do not determine the overall sensitivity of a NMR experiment. Here we measure the overall sensitivity of one-dimensional 1H detected relayed DNP experiments as a function of the MAS rate in the 20–60 kHz regime using 0.7 mm diameter rotors at 21.2 T. Although faster MAS rates are detrimental for the DNP enhancement on the target material, due to slower spin diffusion, we find that with increasing spinning rates the gain in sensitivity due to 1H line-narrowing and the folding-in of sideband intensity compensates a large part of the loss of overall hyperpolarization. We find that sensitivity depends on the atomic site in the molecule, and is maximised at between 40 and 50 kHz MAS for the sample of L-histidine.HCl·H2O studied here. There is a 10–20 % difference in sensitivity between the optimum MAS rate and the fastest rate currently accessible (60 kHz).

Dynamic nuclear polarization experiments were performed to study the solutions of the stable free radical α,γ‐Bisdiphenylene‐β‐phenyl allyl complex with benzene (1∶1) in some highly fluorinated aliphatic and aromatic solvents. In solutions examined in this study, the Overhauser effect, which normally arises due to both dipolar and scalar interactions between the unpaired electrons of the free radical molecules and fluorine nuclei of solvent molecules occurs mainly. 1‐Iodotridecafluorohexane, 2,2,3,4,4,4‐Hexafluoro‐1‐butanol, N‐methyl‐bis‐trifluoroacetamide, hexafluoroacetylacetone, octafluorotoluene, and hexafluorobenzene were used as the solvents. The experiments were performed at a low field double resonance NMR spectrometer, which operates at 1.53 mT. The NMR enhancements depend on competition between intermolecular magnetic interactions. The measurements were performed at four different temperatures to test the dipolar and the scalar part of the coupling between the fluorine nucleus (19F) and the unpaired electron. It was found that the dipolar interactions are more effective for the aliphatic solvents, while the scalar interactions are more effective for the aromatics. The nuclear‐electron coupling parameter varies between 0.018 and 0.157 in all aliphatic solvents and between −0.063 and −0.035 in aromatic solvents.

Dynamic nuclear polarization of nitrogen-15 in benzamide

The Overhauser effect type dynamic nuclear polarization was used to study some colloidal suspensions with benzene, toluene, ethylbenzene, mesitylene, tetralin, and chlorobenzene solvent media of asphaltene obtained from MC-30 liquid asphalt in a weak magnetic field of 1.437 mT at room temperature. For each sample of solvent medium, the samples were prepared in three different asphaltene concentrations. The samples were held for ten years and then dynamic nuclear polarization parameters were found via dynamic nuclear polarization experiments that were performed again. Thus, the effect of sample age to dynamic nuclear polarization parameters was investigated. The results were discussed.

An apparatus for pulsed ESR and DNP experiments using optically excited triplet states down to liquid helium temperatures

Two different approaches for assigning electron nuclear double resonance (ENDOR) signals to their respectiveM s manifolds by a controlled generation of asymmetric ENDOR spectra, are described and applied to a number of systems. This assignment then allows a straightforward determination of the sign of the hyperfine coupling. Both approaches rely on a high thermal polarization that is easily achieved at high fields and low temperatures. For high-spin systems, such asS = 5/2 the assignment is afforded by the selective inversion of the | −3/2〉 → | −1/2〉 electron paramagnetic resonance (EPR) transition which is highly populated as compared to its symmetric counterpart, the |1/2〉 → |3/2〉 EPR transition, and therefore is easily identified. ForS = 1/2 the determination of the sign of the hyperfine coupling becomes possible when the cross- and nuclear-spin relaxation rates are much slower than the electron-spin relaxation rate and variable mixing time pulse ENDOR is used to measure the spectrum. Under these conditions the signals of theM s = 1/2 (α) manifold become negative when the mixing time is on order of the electron-spin relaxation time, whereas those of theM s =−1/2 (β) manifold remain positive. Under partial saturation of the nuclear transitions and short mixing time the opposite behavior is observed. Pulse W-band1H ENDOR experiments demonstrating these approaches were applied and the signs of the hyperfine couplings of the water ligands in Mn(H2O) 6 2+ , the Hα and Hβ histidine protons in the Cu(histidine)2 complex, the imidazole protons in Cu(imidazole) 4 2+ and the cysteine β-protons in nitrous oxide reductase were determined.

Thermosetting polymer for dynamic nuclear polarization: Solidification of an epoxy resin mixture including TEMPO

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms, thereby providing the essential precursors for DNA synthesis and replication. The E.coli RNR is composed of two homodimeric subunits: alpha2 and beta2. In alpha2 nucleotides are converted to deoxynucleotides and beta2 contains a stable di-iron tyrosyl radical (Y122 ) cofactor. A proton-coupled electron transfer (PCET) mechanism is proposed to occur over a distance of more than 35 Å originating from Y122 and ending up in the generation of a C439 in alpha2 which in turn initiates the nucleotide reduction process. Three conserved redox-active tyrosines (beta2-Y356, alpha2-Y730 and alpha2-Y731) are essential for the radical transfer to Cys439 and are proposed to be connected through hydrogen- bonds during reversible charge migration over both subunits. In order to probe for structural and mechanistic requirements of a PCET event in alpha2, NH2Y has been incorporated through the suppressor tRNA/aminoacyl-tRNAsynthetase methodology at positions 730 and 731, respectively. NH2Y functions hereby as a radical spin probe on the PCET pathway and allows the elucidation of chemical environments through high-field EPR and electron-nuclear double resonance (ENDOR) spectroscopy. In order to characterize the generated 3-aminotyrosyl radical (NH2Y ) in D2O and H2O buffer, multifrequency EPR spectroscopy was first applied on NH2Y730 . The characterization of the electronic and molecular structure of a NH2Y was a crucial step in order to deconvolute the hyperfine couplings of internal protons from external protons which build up a hydrogen-bond network around alpha-NH2Y730 . High-field ENDOR spectroscopy detected two hydrogen-bonds which point into the direction of the next PCET pathway residues and additionally a water molecule which is placed near alpha-NH2Y730 and could have mechanistic functions during the proposed H-atom transfer process. A similar hydrogen-bond network was detected for alpha-NH2Y731 with a water molecule placed near the alpha/beta interface. Recent DFT calculations on the PCET mechanism in alpha2 are hereby in agreement with structural insights obtained from high-field EPR and ENDOR studies.