13C Dynamic Nuclear Polarization Research Articles

Sedimentation of large, soluble proteins up to 140kDa for 1H-detected MAS NMR and 13C DNP NMR - practical aspects.

Solution NMR is typically applied to biological systems with molecular weights < 40kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40-140kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SaETF), tryptophan synthases from Salmonella typhimurium (StTS) and their dimeric β-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20-30Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1.

P1 Center Electron Spin Clusters Are Prevalent in Type Ib Diamonds.

Understanding the spatial distribution of the P1 centers is crucial for diamond-based sensors and quantum devices. P1 centers serve as polarization sources for dynamic nuclear polarization (DNP) quantum sensing and play a significant role in the relaxation of nitrogen vacancy (NV) centers. Additionally, the distribution of NV centers correlates with the distribution of P1 centers, as NV centers are formed through the conversion of P1 centers. We utilized DNP and pulsed electron paramagnetic resonance (EPR) techniques that revealed strong clustering of a significant population of P1 centers that exhibit exchange coupling and produce asymmetric line shapes. The 13C DNP frequency profile at a high magnetic field revealed a pattern that requires an asymmetric EPR line shape of the P1 clusters with electron-electron (e-e) coupling strengths exceeding the 13C nuclear Larmor frequency. EPR and DNP characterization at high magnetic fields was necessary to resolve energy contributions from different e-e couplings. We employed a two-frequency pump-probe pulsed electron double resonance technique to show cross-talk between the isolated and clustered P1 centers. This finding implies that the clustered P1 centers affect all of the P1 populations. Direct observation of clustered P1 centers and their asymmetric line shape offers a novel and crucial insight into understanding magnetic noise sources for quantum information applications of diamonds and for designing diamond-based polarizing agents with optimized DNP efficiency for 13C and other nuclear spins of analytes. We propose that room temperature 13C DNP at a high field, achievable through straightforward modifications to existing solution-state NMR systems, is a potent tool for evaluating and controlling diamond defects.

THz-enhanced dynamic nuclear polarized liquid spectrometer

Dynamic nuclear polarization (DNP) technology can be utilized to dramatically enhance NMR signal. In this paper, we report on the development of a self-constructed 5 T DNP spectrometer for liquid samples and the 13C DNP enhancement achieved with this spectrometer. The DNP spectrometer is comprised of a wide-bore superconducting magnet, a home-made console, a dual resonance probe and a self-built 140 GHz microwave source for the spectrometer. Specifically, a microwave source of traveling wave tube (TWT) amplifier has been developed, which can provide a maximum power output of 4.4 W and a wide frequency tuning range of 1 GHz. The excellent performance of our built liquid-state DNP spectrometer is verified by the observation of more than 100-fold DNP enhancement of the 13C NMR signal for liquid 13CCl4 sample. Our result shows the superiority of DNP technology in the liquid-state high-field NMR spectrometer.

Dynamic Nuclear Polarization of Silicon Carbide Micro- and Nanoparticles.

Two dominant crystalline phases of silicon carbide (SiC): α-SiC and β-SiC, differing in size and chemical composition, were investigated regarding their potential for dynamic nuclear polarization (DNP). 29Si nuclei in α-SiC micro- and nanoparticles with sizes ranging from 650 nm to 2.2 μm and minimal oxidation were successfully hyperpolarized without the use of free radicals, while β-SiC samples did not display appreciable degrees of polarization under the same polarization conditions. Long T1 relaxation times in α-SiC of up to 1600 s (∼27 min) were recorded for the 29Si nuclei after 1 h of polarization at a temperature of 4 K. Interestingly, these promising α-SiC particles allowed for direct hyperpolarization of both 29Si and 13C nuclei, resulting in comparably strong signal amplifications. Moreover, the T1 relaxation time of 13C nuclei in 750 nm-sized α-SiC particles was over 33 min, which far exceeds T1 times of conventional 13C DNP probes with values in the order of 1–2 min. The present work demonstrates the feasibility of DNP on SiC micro- and nanoparticles and highlights their potential as hyperpolarized magnetic resonance imaging agents.

13C Dynamic Nuclear Polarization using SA-BDPA at 6.7 T and 1.1 K: Coexistence of Pure Thermal Mixing and Well-Resolved Solid Effect.

SA-BDPA is a water-soluble, narrow-line width radical previously used for dynamic nuclear polarization (DNP) signal enhancement in solid-state magic angle spinning NMR spectroscopy. Here, we report the first study using SA-BDPA under dissolution DNP conditions (6.7 T and 1.15 K). Longitudinal-detected (LOD)-electron spin resonance (ESR) and 13C DNP measurements were performed on samples containing 8.4 M [13C]urea dissolved in 50:50 water:glycerol (v/v) doped with either 60 or 120 mM SA-BDPA. Two distinct DNP mechanisms, both "pure" thermal mixing and a well-resolved solid effect could clearly be identified. The radical's ESR line width (30-40 MHz), broadened predominantly by dipolar coupling, excluded any contribution from the cross effect. Microwave frequency modulation increased the enhancement by DNP at the lower radical concentration but not at the higher radical concentration. These results are compared to data acquired with trityl radical AH111501, highlighting the unusual 13C DNP properties of SA-BDPA.

Carbon-13 dynamic nuclear polarization in diamond via a microwave-free integrated cross effect

Color-center-hosting semiconductors are emerging as promising source materials for low-field dynamic nuclear polarization (DNP) at or near room temperature, but hyperfine broadening, susceptibility to magnetic field heterogeneity, and nuclear spin relaxation induced by other paramagnetic defects set practical constraints difficult to circumvent. Here, we explore an alternate route to color-center-assisted DNP using nitrogen-vacancy (NV) centers in diamond coupled to substitutional nitrogen impurities, the so-called P1 centers. Working near the level anticrossing condition-where the P1 Zeeman splitting matches one of the NV spin transitions-we demonstrate efficient microwave-free 13C DNP through the use of consecutive magnetic field sweeps and continuous optical excitation. The amplitude and sign of the polarization can be controlled by adjusting the low-to-high and high-to-low magnetic field sweep rates in each cycle so that one is much faster than the other. By comparing the 13C DNP response for different crystal orientations, we show that the process is robust to magnetic field/NV misalignment, a feature that makes the present technique suitable to diamond powders and settings where the field is heterogeneous. Applications to shallow NVs could capitalize on the greater physical proximity between surface paramagnetic defects and outer nuclei to efficiently polarize target samples in contact with the diamond crystal.

Effects of glassing matrix deuteration on the relaxation properties of hyperpolarized 13C spins and free radical electrons at cryogenic temperatures.

Glassing matrix deuteration could be a beneficial sample preparation method for 13C dynamic nuclear polarization (DNP) when large electron paramagnetic resonance (EPR) width free radicals are used. However, it could yield the opposite DNP effect when samples are doped with small EPR width free radicals. Herein, we have investigated the influence of solvent deuteration on the 13C nuclear and electron relaxation that go along with the effects on 13C DNP intensities at 3.35 T and 1.2 K. For 13C DNP samples doped with trityl OX063, the 13C DNP signals decreased significantly when the protons are replaced by deuterons in glycerol:water or DMSO:water solvents. Meanwhile, the corresponding solid-state 13C T1 relaxation times of trityl OX063-doped samples generally increased upon solvent deuteration. On the other hand, 13C DNP signals improved by a factor of ∼1.5 to 2 upon solvent deuteration of samples doped with 4-oxo-TEMPO. Despite this 13C DNP increase, there were no significant differences recorded in 13C T1 values of TEMPO-doped samples with nondeuterated or fully deuterated glassing matrices. While solvent deuteration appears to have a negligible effect on the electron T1 relaxation of both free radicals, the electron T2 relaxation times of these two free radicals generally increased upon solvent deuteration. These overall results suggest that while the solid-phase 13C DNP signals are dependent upon the changes in total nuclear Zeeman heat capacity, the 13C relaxation effects are related to 2H/1H nuclear spin diffusion-assisted 13C polarization leakage in addition to the dominant paramagnetic relaxation contribution of free radical centers.

The influence of Ho3+ doping on 13C DNP in the presence of BDPA.

Polarization transfer from unpaired electron radicals to nuclear spins at low-temperature is achieved using microwave irradiation by a process broadly termed dynamic nuclear polarization (DNP). The resulting signal enhancement can easily exceed factors of 104 when paired with cryogenic cooling of the sample. Dissolution-DNP couples low temperature polarization methods with a rapid dissolution step, resulting in a highly polarized solution that can be used for metabolically sensitive magnetic resonance imaging (MRI). Hyperpolarized [1-13C]pyruvate is a powerful metabolic imaging agent for investigation of in vitro and in vivo cellular metabolism by means of NMR spectroscopy and MRI. Radicals (trityl OX063 and BDPA) with narrower EPR linewidths typically produce higher nuclear polarizations when carbon-13 is the target nucleus. Increased solid-state polarization is observed when narrow line radicals are doped with lanthanide ions such as Gd3+, Ho3+, Dy3+, and Tb3+. Earlier results have demonstrated an incongruence between DNP experiments with trityl and BDPA, where the optimal concentrations for polarization transfer are disparate despite similar electron spin resonance linewidths. Here, the effects of Ho-DOTA on the solid-state polarization of [1-13C]pyruvic acid were compared for 3.35 T (1.4 K) and 5 T (1.2 K) systems using BDPA as a radical. Multiple concentrations of BDPA were doped with variable concentrations of Ho-DOTA (0, 0.2, 0.5, 1, and 2 mM), and dissolved in 1 : 1 (v/v) of [1-13C] pyruvic acid/sulfolane mixture. Our results reveal that addition of small amounts of Ho-DOTA in the sample preparation increases the solid-state polarization for [1-13C] pyruvic acid, with the optimum Ho-DOTA concentration of 0.2 mM. Without Ho-DOTA doping, the optimum BDPA concentration found for 3.35 T (1.4 K) is 40 mM, and for 5 T (1.2 K) system it is about 60 mM. In both systems, inclusion of Ho-DOTA in the 13C DNP sample leads to a change in the breadth (ΔDNP) of the extrema between the P(+) and P(-) frequencies in microwave spectra. At no combination of BDPA and Ho3+ did polarizations reach those achievable with trityl. Simplified analysis of increased polarization as a function of decreased electron T1e used to explain results in trityl are insufficient to describe DNP with BDPA.

Multi-resonant photonic band-gap/saddle coil DNP probehead for static solid state NMR of microliter volume samples.

The most critical condition for performing Dynamic Nuclear Polarization (DNP) NMR experiments is achieving sufficiently high electronic B1e fields over the sample at the matched EPR frequencies, which for modern high-resolution NMR instruments fall into the millimeter wave (mmW) range. Typically, mmWs are generated by powerful gyrotrons and/or extended interaction klystrons (EIKs) sources and then focused onto the sample by dielectric lenses. However, further development of DNP methods including new DNP pulse sequences may require B1e fields higher than one could achieve with the current mmW technology. In order to address the challenge of significantly enhancing the mmW field at the sample, we have constructed and tested one-dimensional photonic band-gap (PBG) mmW resonator that was incorporated inside a double-tuned radiofrequency (rf) NMR saddle coil. The photonic crystal is formed by stacking ceramic discs with alternating high and low dielectric constants and thicknesses of λ/4 or 3λ/4, where λ is the wavelength of the incident mmW field in the corresponding dielectric material. When the mmW frequency is within the band gap of the photonic crystal, a defect created in the middle of the crystal confines the mmW energy, thus forming a resonant structure. An aluminum mirror in the middle of the defect has been used to substitute one-half of the structure with its mirror image in order to reduce the resonator size and simplify its tuning. The latter is achieved by adjusting the width of the defect by moving the aluminum mirror with respect to the dielectric stack using a gear mechanism. The 1D PBG resonator was the key element for constructing a multi-resonant integrated DNP/NMR probehead operating at 190-199 GHz EPR/300 MHz 1H/75.5 MHz 13C NMR frequencies. Initial tests of the multi-resonant DNP/NMR probehead were carried out using a quasioptical mmW bridge and a Bruker Biospin Avance II spectrometer equipped with a standard Bruker 7 T wide-bore 89 mm magnet parked at 300.13 MHz 1H NMR frequency. The mmW bridge built with all solid-state active components allows for the frequency tuning between ca. 190 and ca. 199 GHz with the output power up to 27 dBm (0.5 W) at 192 GHz and up to 23 dBm (0.2 W) at 197.5 GHz. Room temperature DNP experiments with a synthetic single crystal high-pressure high-temperature (HPHT) diamond (0.3 × 0.3 × 3.0 mm3) demonstrated dramatic 1500-fold enhancement of 13C natural abundance NMR signal at full incident mmW power. Significant 13C DNP enhancement (of about 90) have been obtained at incident mmW powers of as low as <100 μW. Further tests of the resonator performance have been carried out with a thin (ca. 100 μm thickness) composite polystyrene-microdiamond film by controlling the average mmW power at the optimal DNP conditions via a gated mode of operation. From these experiments, the PBG resonator with loaded Q ≃ 250 and finesse F≈75 provides up to 12-fold or 11 db gain in the average mmW power vs. the non-resonant probehead configuration employing only a reflective mirror.

Transition Metal Doping Reveals Link between Electron T1 Reduction and 13C Dynamic Nuclear Polarization Efficiency.

Optimal efficiency of dissolution dynamic nuclear polarization (DNP) is essential to provide the required high sensitivity enhancements for in vitro and in vivo hyperpolarized 13C nuclear magnetic resonance (NMR) spectroscopy and imaging (MRI). At the nexus of the DNP process are the free electrons, which provide the high spin alignment that is transferred to the nuclear spins. Without changing DNP instrumental conditions, one way to improve 13C DNP efficiency is by adding trace amounts of paramagnetic additives such as lanthanide (e.g., Gd3+, Ho3+, Dy3+, Tb3+) complexes to the DNP sample, which has been observed to increase solid-state 13C DNP signals by 100-250%. Herein, we have investigated the effects of paramagnetic transition metal complex R-NOTA (R = Mn2+, Cu2+, Co2+) doping on the efficiency of 13C DNP using trityl OX063 as the polarizing agent. Our DNP results at 3.35 T and 1.2 K show that doping the 13C sample with 3 mM Mn2+-NOTA led to a substantial improvement of the solid-state 13C DNP signal by a factor of nearly 3. However, the other transition metal complexes Cu2+-NOTA and Co2+-NOTA complexes, despite their paramagnetic nature, had essentially no impact on solid-state 13C DNP enhancement. W-band electron paramagnetic resonance (EPR) measurements reveal that the trityl OX063 electron T1 was significantly reduced in Mn2+-doped samples but not in Cu2+- and Co2+-doped DNP samples. This work demonstrates, for the first time, that not all paramagnetic additives are beneficial to DNP. In particular, our work provides a direct evidence that electron T1 reduction of the polarizing agent by a paramagnetic additive is an essential requirement for the improvement seen in solid-state 13C DNP signal.

13C Dynamic Nuclear Polarization Using Derivatives of TEMPO Free Radical

The nitroxide-based 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) free radical is widely used in 13C dynamic nuclear polarization (DNP) due to its relatively low cost, commercial availability, and effectiveness as polarizing agent. While a large number of TEMPO derivatives are available commercially, so far, only few have been tested for use in 13C DNP. In this study, we have tested and evaluated the 13C hyperpolarization efficiency of eight derivatives of TEMPO free radical with different side arms in the 4-position. In general, these TEMPO derivatives were found to have slight variations in efficiency as polarizing agents for DNP of 3 M [1-13C] acetate in 1:1 v/v ethanol:water at 3.35 T and 1.2 K. X-band electron paramagnetic resonance (EPR) spectroscopy revealed no significant differences in the spectral features among these TEMPO derivatives. 2H enrichment of the ethanol:water glassing matrix resulted in further improvement of the solid-state 13C DNP signals by factor of 2 to 2.5-fold with respect to the 13C DNP signal of non-deuterated DNP samples. These results suggest an interaction between the nuclear Zeeman reservoirs and the electron dipolar system via the thermal mixing mechanism.

Assembly and performance of a 6.4T cryogen-free dynamic nuclear polarization system.

We report on the assembly and performance evaluation of a 180-GHz/6.4T dynamic nuclear polarization (DNP) system based on a cryogen-free superconducting magnet. The DNP system utilizes a variable-field superconducting magnet that can be ramped up to 9T and equipped with cryocoolers that can cool the sample space with the DNP assembly down to 1.8K via the Joule-Thomson effect. A homebuilt DNP probe insert with top-tuned nuclear magnetic resonance coil and microwave port was incorporated into the sample space in which the effective sample temperature is approximately 1.9K when a 180-GHz microwave source is on during DNP operation. 13 C DNP of [1-13 C] acetate samples doped with trityl OX063 and 4-oxo-TEMPO in this system have resulted in solid-state 13 C polarization levels of 58±3% and 18±2%, respectively. The relatively high 13 C polarization levels achieved in this work have demonstrated that the use of a cryogen-free superconducting magnet for 13 C DNP is feasible and in fact, relatively efficient-a major leap to offset the high cost of liquid helium consumption in DNP experiments.

Directly vs Indirectly Enhanced 13C in Dynamic Nuclear Polarization Magic Angle Spinning NMR Experiments of Nonionic Surfactant Systems

A study of dynamic nuclear polarization (DNP) in polyethylene glycol and related nonionic surfactants is presented. In these experiments, we found the surprising result that DNP enhanced 13C magic angle spinning (MAS) spectra display two sets of resonances, one with broad and one with sharp spectral features that are 180° opposite in phase. These two sets indicate the presence of a direct polarization transfer channel as expected for 13C MAS experiments, and a second unexpected indirect polarization transfer channel. Plots of DNP enhancements as a function of applied magnetic field for the two resonances show a superposition of two DNP enhancement profiles for AMUpol in the nonionic surfactant C10E6. The indirect polarization channel can be suppressed by application of a string of 1H 180° pulses during 13C DNP buildup. The presence of direct and indirect polarization channels is observed in a total of four different nonionic surfactants and with three different radicals, showing that these concurring pola...

Influence of Dy3+ and Tb3+ doping on 13C dynamic nuclear polarization.

Dynamic nuclear polarization (DNP) is a technique that uses a microwave-driven transfer of high spin alignment from electrons to nuclear spins. This is most effective at low temperature and high magnetic field, and with the invention of the dissolution method, the amplified nuclear magnetic resonance (NMR) signals in the frozen state in DNP can be harnessed in the liquid-state at physiologically acceptable temperature for in vitro and in vivo metabolic studies. A current optimization practice in dissolution DNP is to dope the sample with trace amounts of lanthanides such as Gd3+ or Ho3+, which further improves the polarization. While Gd3+ and Ho3+ have been optimized for use in dissolution DNP, other lanthanides have not been exhaustively studied for use in C13 DNP applications. In this work, two additional lanthanides with relatively high magnetic moments, Dy3+ and Tb3+, were extensively optimized and tested as doping additives for C13 DNP at 3.35 T and 1.2 K. We have found that both of these lanthanides are also beneficial additives, to a varying degree, for C13 DNP. The optimal concentrations of Dy3+ (1.5 mM) and Tb3+ (0.25 mM) for C13 DNP were found to be less than that of Gd3+ (2 mM). W-band electron paramagnetic resonance shows that these enhancements due to Dy3+ and Tb3+ doping are accompanied by shortening of electron T1 of trityl OX063 free radical. Furthermore, when dissolution was employed, Tb3+-doped samples were found to have similar liquid-state C13 NMR signal enhancements compared to samples doped with Gd3+, and both Tb3+ and Dy3+ had a negligible liquid-state nuclear T1 shortening effect which contrasts with the significant reduction in T1 when using Gd3+. Our results show that Dy3+ doping and Tb3+ doping have a beneficial impact on C13 DNP both in the solid and liquid states, and that Tb3+ in particular could be used as a potential alternative to Gd3+ in C13 dissolution DNP experiments.

13C dynamic nuclear polarization for measuring metabolic flux in endothelial progenitor cells

Endothelial progenitor cells (EPCs) represent a heterogeneous cell population that is believed to be involved in vasculogenesis. With the purpose of enhancing endothelial repair, EPCs could have a potential for future cell therapies. Due to the low amount of EPCs in the peripheral circulating blood, in vitro expansion is needed before administration to recipients and the effects of in vitro culturing is still an under-evaluated field with little knowledge of how the cells change over time in culture. The aim of this study was to use hyperpolarised carbon-13 magnetic resonance spectroscopy to profile important metabolic pathways in a population of progenitor cells and to show that cell culturing in 3D scaffolds seem to block the metabolic processes that leads to cell senescence.The metabolic breakdown of hyperpolarized [1-13C]pyruvate was followed after injection of the substrate to a bioreactor system with EPCs either adhered to 3D printed scaffolds or kept in cell suspension. The pyruvate-to-lactate conversion was elevated in suspension of EPCs compared to the EPCs adhered to scaffolds. Furthermore in the setup with EPCs in suspension, an increase in lactate production was seen over time indicating that the older the cultures of EPCs was before using the cells for cell suspension experiments, the more lactate they produce, compared to a constant lactate level in the cells adhered to scaffolds. It could therefore be stated that cells grown first in 2D culture and subsequent prepared for cell suspension show a metabolism with higher lactate production consistent with cells senescence processes compared to cells grown first at 2D culture and subsequent in the 3D printed scaffolds, where metabolism shows no sign of metabolic shifting during the monitored period.

The effect of glassing solvent deuteration and Gd3+ doping on 13C DNP at 5 T

We report the influence of glassing solvent deuteration and Gd3+ doping on 13C dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR) performed on [1-13C] sodium acetate at B0 = 5 T and 1.2 K.

Optimization and prediction of the electron-nuclear dipolar and scalar interaction in 1H and 13C liquid state dynamic nuclear polarization.

During the last 10-15 years, dynamic nuclear polarization (DNP) has evolved as a powerful tool for hyperpolarization of NMR and MRI nuclides. However, it is not as well appreciated that solution-state dynamic nuclear polarization is a powerful approach to study intermolecular interactions in solution. For solutions and fluids, the 1H nuclide is usually dominated by an Overhauser dipolar enhancement and can be significantly increased by decreasing the correlation time (τc) of the substrate/nitroxide interaction by utilizing supercritical fluids (SF CO2). For molecules containing the ubiquitous 13C nuclide, the Overhauser enhancement is usually a profile of both scalar and dipolar interactions. For carbon atoms without an attached hydrogen, a dipolar enhancement usually dominates as we illustrate for sp2 hybridized carbons in the fullerenes, C60 and C70. However, the scalar interaction is dependent on a Fermi contact interaction which does not have the magnetic field dependence inherent in the dipolar interaction. For a comprehensive range of molecular systems we show that molecules that exhibit weakly acidic complexation interaction(s) with nitroxides provide corresponding large scalar enhancements. For the first time, we report that sp hybridized (H-C) alkyne systems, for example, the phenylacetylene-nitroxide system exhibit very large scalar dominated enhancements. Finally, we demonstrate for a wide range of molecular systems that the Fermi contact interaction can be computationally predicted via electron-nuclear hyperfine coupling and correlated with experimental 13C DNP enhancements.

The interplay between the solid effect and the cross effect mechanisms in solid state 13C DNP at 95 GHz using trityl radicals

The 13C solid state Dynamic Nuclear Polarization (DNP) mechanism using trityl radicals (OX63) as polarizers was investigated in the temperature range of 10–60K. The solutions used were 6M 13C urea in DMSO/H2O (50% v/v) with 15mM and 30mM OX63. The measurements were carried out at ∼3.5T, which corresponds to Larmor frequencies of 95GHz and 36MHz for the OX63 and the 13C nuclei, respectively. Measurements of the 13C signal intensity as a function of the microwave (MW) irradiation frequency yielded 13C DNP spectra with temperature dependent lineshapes for both samples. The maximum enhancement for the 30mM sample was reached at 40K, while that of the 15mM sample at 20–30K. Furthermore, the lineshapes observed showed that both the cross effect (CE) and the solid effect (SE) DNP mechanisms are active in this temperature range and that their relative contribution is temperature dependent. Simulations of the spectra with the relative contributions of the CE and SE mechanisms as a fit parameter revealed that for both samples the CE contribution decreases with decreasing temperature while the SE contribution increases. In addition, for the 15mM sample the contributions of the two mechanisms are comparable from 20K to 60K while for the 30mM the CE dominates in this range, as expected from the higher concentration. The steep decrease of the CE contribution towards low temperatures is however unexpected. The temperature dependence of the OX63 longitudinal relaxation, DNP buildup times and 13C spin lattice relaxation times did not reveal any obvious correlation with the DNP temperature dependence. A similar behavior of the CE and SE mechanism was observed for 1H DNP with the nitroxide radical TEMPOL as a polarizer. This suggests that this effect is a general phenomenon involving a temperature dependent competition between the CE and SE mechanisms, the source of which is, however, still unknown.

Temperature dependence of high field 13C dynamic nuclear polarization processes with trityl radicals below 35 Kelvin

In order to facilitate versatile applications with high field dynamic nuclear polarization (DNP), it is important to be able to optimize the DNP performance, i.e. reach high nuclear hyperpolarization within a short signal build up time. Given that the solid-state DNP process is strongly temperature-dependent, it is important to benchmark the temperature dependence of various DNP and electron paramagnetic resonance (EPR) parameters that can then be used to test and develop theories and models for high field DNP mechanisms. However, DNP and EPR experiments at high fields and cryogenic temperatures below 20 Kelvin usually require home built instrumentation, and therefore even basic experimental observations are lacking in the literature. DNP and EPR experiments at 7 T (197 GHz) and 8.5 T (240 GHz), respectively, were conducted at temperatures between 35 K and 3.7 K where the electron thermal polarization changes from 13.4% to 85.6%, respectively. The samples are frozen solutions of 15 mM OX063Me trityl radicals in various mixtures of [1-(13)C]pyruvic acid, glycerol, and Gd(3+)-chelates. For all sample mixtures, the trityl EPR lines are found to be inhomogeneously broadened and the dominant DNP mechanism is shown to be the cross effect (CE). A 20%, 11%, and 6.77% (13)C polarization is achieved at 3.7 K with a [1-(13)C]pyruvic-glycerol-H2O sample, the addition of 2 mM of Gd(3+)-chelates, and pure [1-(13)C]pyruvic acid, respectively. When T1n is sufficiently long, our results seem to suggest T1e is a key variable in the DNP process, where longer T1e values correlate with larger DNP enhancements (εDNP). The experimental data reported here on the temperature dependence of T1n, T1e, Tm (electron phase memory time), the EPR linewidth, TDNP and ε(DNP) at high fields will be helpful for testing the mechanism and theory of DNP processes.

Influence of deuteration in the glassing matrix on 13C dynamic nuclear polarization

Replacement of protons by deuterons in the glassing solvents led to 2-3-fold improvement of the (13)C dynamic nuclear polarization (DNP) solid-state NMR signal for samples doped with large electron spin resonance (ESR) linewidth free radicals galvinoxyl, DPPH, and 4-oxo-TEMPO. Meanwhile, the reverse effect is observed for (13)C DNP using small ESR linewidth free radicals BDPA and trityl OX063.