A Spectrometer for Dynamic Nuclear Polarization and Electron Paramagnetic Resonance at High Frequencies

Solid-state MAS NMR at ultra low temperature of hydrated alanine doped with DNP radicals

The membrane-bound heterotrimeric nitrate reductase A (NarGHI) catalyzes the oxidation of quinols in the cytoplasmic membrane of Escherichia coli and reduces nitrate to nitrite in the cytoplasm. The enzyme strongly stabilizes a menasemiquinone intermediate at a quinol oxidation site (Q(D)) located in the vicinity of the distal heme b(D). Here molecular details of the interaction between the semiquinone radical and the protein environment have been provided using advanced multifrequency pulsed EPR methods. (14)N and (15)N ESEEM and HYSCORE measurements carried out at X-band ( approximately 9.7 GHz) on the wild-type enzyme or the enzyme uniformly labeled with (15)N nuclei reveal an interaction between the semiquinone and a single nitrogen nucleus. The isotropic hyperfine coupling constant A(iso)((14)N) approximately 0.8 MHz shows that it occurs via an H-bond to one of the quinone carbonyl group. Using (14)N ESEEM and HYSCORE spectroscopies at a lower frequency (S-band, approximately 3.4 GHz), the (14)N nuclear quadrupolar parameters of the interacting nitrogen nucleus (kappa = 0.49, eta = 0.50) were determined and correspond to those of a histidine N(delta), assigned to the heme b(D) ligand His-66 residue. Moreover S-band (15)N ESEEM spectra enabled us to directly measure the anisotropic part of the nitrogen hyperfine interaction (T((15)N) = 0.16 MHz). A distance of approximately 2.2 Abetween the carbonyl oxygen and the nitrogen could then be calculated. Mechanistic implications of these results are discussed in the context of the peculiar properties of the menasemiquinone intermediate stabilized at the Q(D) site of NarGHI.

Dynamic nuclear polarization (DNP) is a nuclear magnetic resonance (NMR) hyperpolarization technique that mediates polarization transfer from unpaired electrons to nuclear spins. DNP performance can vary significantly depending on the types of polarizing agents employed, and the criteria for optimum DNP efficiency are not fully understood. Thus, a better understanding of the structure, electron paramagnetic resonance (EPR) line widths, and relaxation properties would aid in designing more efficient DNP polarizing agents. However, EPR characterizations of the polarizing agents are typically performed in different environments (e.g., strength of magnetic field and microwave power) than typical DNP experiments. Here, we demonstrate a low-cost and home-built setup that enables in situ EPR detection in a dual resonance DNP-NMR/EPR probe using an optical chopper. The optical chopper modulates the microwave irradiation, thereby modulating the longitudinal magnetization (Mz) of the electron spins, which results in a LOngitudinal-Detected (LOD) EPR spectrum. The EPR spectrum acquired for TEMPOL at 6.7 T/188 GHz using a mechanical modulation showed a good agreement with the spectrum acquired using an electrical modulation. Hence, our work provides a proof-of-principle setup that enables an optical chopper EPR spectrometer.

The relationship between structure and nonlinear optical properties in LiCsB6O10 is characterized using single-crystal nuclear magnetic resonance (NMR) and magic-angle spinning (MAS) NMR. Although the quadrupole parameters for B(1) and B(2) sites were obtained using single-crystal NMR, the T 1 values for these atomic sites could not be distinguished in this way. Thus, the structural nature of lithium and boron sites in LiCsB6O10 was investigated using MAS NMR. B(1) and B(2) sites could be distinguished based on the spectrum and T 1ρ obtained from 11B MAS NMR. In addition, the T 1 and T 1ρ values and activation energies for 7Li and 11B are compared. No significant changes were seen in the T 1ρ at the lithium and boron nuclei in LiCsB6O10.

Nuclear magnetic resonance (NMR) provides unparalleled information on molecular structure and intermolecular interactions with atomic-level resolution, but it nevertheless lacks sensitivity in comparison to other forms of spectroscopy. This problem is more acute in cases where multidimensional spectra are required, such as in protein NMR structural studies and in metabolomic experiments where biomarkers of interest may be present in low concentrations. An approach to addressing the issue of sensitivity is dynamic nuclear polarization (DNP) in which NMR signal intensities are enhanced by transferring the large Boltzmann polarization of electrons to nuclear spins via microwave irradiation at the electron Larmor frequency. This article describes some recent technological innovations—in particular, the development of high-frequency gyrotron oscillators and amplifiers and novel polarizing agents—that enable applications of DNP at the 10–20 T fields where contemporary NMR experiments are performed. In addition, we discuss potential and current applications which are wide ranging, and include magic angle spinning (MAS) NMR studies of amyloid fibrils and membrane proteins, and melting and dissolution experiments applicable to solution NMR that could serve as a tool for high throughput small molecule analysis such as metabolic screening.

Solid state nuclear magnetic resonance (NMR) enables atomic-resolution characterization of the molecular structure and dynamics within complex heterogeneous samples, but it is typically insensitive. Dynamic nuclear polarization (DNP) increases the NMR signal intensity by orders of magnitude and can be performed in combination with magic angle spinning (MAS) for sensitive, high-resolution spectroscopy. Here we report MAS DNP experiments, for the first time, within intact human cells with >40-fold DNP enhancement and a sample temperature of <6 K. In addition to cryogenic MAS results at <6 K, we also show in-cell DNP enhancements of 57-fold at 90 K. In-cell DNP is demonstrated using biradicals and sterically shielded monoradicals as polarizing agents. A novel trimodal polarizing agent is introduced for DNP, which contains a nitroxide biradical, a targeting peptide for cell penetration, and a fluorophore for subcellular localization with confocal microscopy. The fluorescent polarizing agent provides in-cell DNP enhancements of 63-fold at a concentration of 2.7 mM. These experiments pave the way for structural characterization of biomolecules in an endogenous cellular context.

Static NMR experiments on insensitive nuclei (e.g., (13)C) have been generally difficult because of low magnetogyric ratios and/or poor natural abundance, even at the highest magnetic fields available. One method of overcoming these sensitivity limitations is dynamic nuclear polarization (DNP), which can be utilized to enhance NMR signals relative to those obtained with static NMR detection. In this study, we report a recycled-flow DNP apparatus capable of monitoring (13)C scalar-dominated enhancements 1-2 orders of magnitude greater than those obtainable using the conventional recycled-flow (13)C NMR experiment. Specifically, a mixture of benzene and several chlorocarbons were continuously "recycled" through a (13)C DNP spectrometer. Results indicate successful detection with the (13)C DNP approach in scalar-dominated cases, where analytes could not be observed with conventional static or flow NMR detection in a reasonable length of time.

Spectral diffusion and dynamic nuclear polarization: Beyond the high temperature approximation

Magic angle spinning (MAS) NMR studies of amyloid and membrane proteins and large macromolecular complexes are an important new approach to structural biology. However, the applicability of these experiments, which are based on (13)C- and (15)N-detected spectra, would be enhanced if the sensitivity were improved. Here we discuss two advances that address this problem: high-frequency dynamic nuclear polarization (DNP) and (1)H-detected MAS techniques. DNP is a sensitivity enhancement technique that transfers the high polarization of exogenous unpaired electrons to nuclear spins via microwave irradiation of electron-nuclear transitions. DNP boosts NMR signal intensities by factors of 10(2) to 10(3), thereby overcoming NMR's inherent low sensitivity. Alternatively, it permits structural investigations at the nanomolar scale. In addition, (1)H detection is feasible primarily because of the development of MAS rotors that spin at frequencies of 40 to 60 kHz or higher and the preparation of extensively (2)H-labeled proteins.

The inherently low sensitivity of nuclear magnetic resonance(NMR)spectroscopy is the major limiting factor for its application to elucidatestructure and dynamics in solids. In the solid state, nuclear spinhyperpolarization methods based on microwave-induced dynamic nuclearpolarization (DNP) provide a versatile platform to enhance the bulkNMR signal of many different sample formulations, leading to significantsensitivity improvements. Here we show that 1H NMR hyperpolarizationcan also be generated in solids at high magnetic fields by opticalirradiation of the sample. We achieved this by exploiting a donor–chromophore–acceptormolecule with an excited state electron–electron interactionsimilar to the nuclear Larmor frequency, enabling solid-state 1H photochemically induced DNP (photo-CIDNP) at high magneticfields. Through hyperpolarization relay, we obtained bulk NMR signalenhancements εH by factors of ∼100 at both9.4 and 21.1 T for the 1H signal of o-terphenylin magic angle spinning (MAS) NMR experiments at 100 K. These findingsopen a pathway toward a general light-induced hyperpolarization approachfor dye-sensitized high-field NMR in solids.

Solid-state nuclear magnetic resonance (NMR) spectroscopy is an atomic-level method used to determine the chemical structure, three-dimensional structure, and dynamics of solids and semi-solids. This Primer summarizes the basic principles of NMR as applied to the wide range of solid systems. The fundamental nuclear spin interactions and the effects of magnetic fields and radiofrequency pulses on nuclear spins are the same as in liquid-state NMR. However, because of the anisotropy of the interactions in the solid state, the majority of high-resolution solid-state NMR spectra is measured under magic-angle spinning (MAS), which has profound effects on the types of radiofrequency pulse sequences required to extract structural and dynamical information. We describe the most common MAS NMR experiments and data analysis approaches for investigating biological macromolecules, organic materials, and inorganic solids. Continuing development of sensitivity-enhancement approaches, including 1H-detected fast MAS experiments, dynamic nuclear polarization, and experiments tailored to ultrahigh magnetic fields, is described. We highlight recent applications of solid-state NMR to biological and materials chemistry. The Primer ends with a discussion of current limitations of NMR to study solids, and points to future avenues of development to further enhance the capabilities of this sophisticated spectroscopy for new applications.

The compositional dependence of water incorporation mechanisms in melts and glasses belonging to the quartz (Qz)-albite (Ab) join was studied with nuclear magnetic resonance (NMR) spectroscopy. Dry and hydrous glasses (containing 3.8 ± 0.1 wt% water) ranging in composition from Qz 90Ab 10 to Qz 28Ab 72 (in wt%) were synthesised and studied with 1H, 23Na, 27Al and 29Si magic angle spinning (MAS) NMR and 1H- 29Si cross polarisation (CP) MAS NMR at magnetic fields of 8.45 and 14.1 T. The results show that both molecular H 2O and OH groups are present in the hydrous glasses, represented by a broad and a narrow component in the static 1H NMR spectra. The changes in the 23Na MAS NMR spectra of the hydrated glasses indicate that sodium associates with water in all compositions studied. In contrast to the Ab-rich glasses, the 29Si MAS spectra of Qz-rich glasses (Qz 73Ab 27 - Qz 90Ab 10) change upon water incorporation, indicating the presence of Si-OH groups at least in the Qz-richest sample. The 27Al MAS data demonstrate that Al is only present in tetrahedral coordination for all glasses studied and that unless δ iso for Al Q 4(4Si) and Al Q 3(3Si)-OH are identical, Al-OH groups cannot be present in significant concentrations. Thus, in hydrous Ab-rich glasses there is probably no significant depolymerisation as suggested by Kohn et al. (1989a). However, more 27Al data for appropriate model compounds or reliable calculations of 27Al shifts and quadrupolar coupling constants are required before this conclusion can be considered to be definitive. For Qz-rich glass compositions a second solubility mechanism involving the formation of Si-OH and depolymerisation of the silicate network is inferred. The data suggest that only in Qz-rich glasses do both mechanisms coexist.

Investigation of a naphthalene pitch by high-resolution solid-state dynamic nuclear polarization

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 polarisation (DNP) is a process that transfers electron spin polarisation to nuclei by applying resonant microwave radiation, and has been widely used to improve the sensitivity of nuclear magnetic resonance (NMR). Here we demonstrate new levels of performance for static cross-effect proton DNP using high peak power chirped inversion pulses at 94 GHz to create a strong polarisation gradient across the inhomogeneously broadened line of the mono-radical 4-amino TEMPO. Enhancements of up to 340 are achieved at an average power of a few hundred mW, with fast build-up times (3 s). Experiments are performed using a home-built wideband kW pulsed electron paramagnetic resonance (EPR) spectrometer operating at 94 GHz, integrated with an NMR detection system. Simultaneous DNP and EPR characterisation of other mono-radicals and biradicals, as a function of temperature, leads to additional insights into limiting relaxation mechanisms and give further motivation for the development of wideband pulsed amplifiers for DNP at higher frequencies.