Dynamic Nuclear Polarization Nuclear Magnetic Resonance in Human Cells Using Fluorescent Polarizing Agents

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).

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

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.

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

Nuclear Magnetic Resonance (NMR) spectroscopy would be a method of choice to follow biochemical events in cells because it can analyze molecules in complex environments. However, the intrinsically low sensitivity of NMR makes in-cell measurements challenging. Dynamic Nuclear Polarization (DNP) has emerged as a method to circumvent this limitation, but most polarizing agents developed for DNP are unstable in reducing cellular environments. Here, we introduce the use of Gd(III)-based DNP polarizing agents for in-cell NMR spectroscopy. Specifically, we show their persistent stability in cellular formulations, and we investigate the DNP performance of the Gd(III)-based complex [Gd(tpatcn)] in human embryonic kidney cell lysates and intact cells. For cell lysates, DNP enhancements up to -27 are obtained on the cellular signals, reproducible even after storage at room temperature for days. Mixing the [Gd(tpatcn)] solution with intact cells enables the observation of cellular signals with DNP, and DNP enhancement factors of about -40 are achieved.

Solid Effect DNP in a Rapid-melt setup

Dynamic nuclear polarization on a hybridized hammerhead ribozyme: An explorative study of RNA folding and direct DNP with a paramagnetic metal ion cofactor

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

Dynamic nuclear polarization (DNP) employs paramagnetic species to increase the NMR signal of nuclear spins. In liquids, the efficiency of the effect depends on the strength of the interaction between the electron and nuclear spins and the time scales on which this interaction is modulated by the physical motion of the spin-bearing molecules. An approach to quantitatively predict the contribution of molecular motions to the DNP enhancement using molecular dynamics (MD) simulations is developed and illustrated for the nitroxide radical TEMPOL in liquid toluene. A multi-resolution strategy that combines explicit treatment of the solvent at short distances from the free radical with implicit description at large intermolecular distances is adopted. Novel analytical expressions are obtained to correct for the finite spatial extent of the MD simulations. The atomistic and analytical descriptions are sewn seamlessly together by ensuring that for molecular trajectories that start in the near (explicit) region and end in the distant (implicit) region the analytical dipolar spectral densities reproduce the MD estimates. The spectral densities obtained from the developed approach are used to calculate DNP coupling factors separately for the ring and methyl protons of toluene. The agreement with previously reported experimental DNP data at a magnetic field of 3.4 T is noteworthy and encouraging. Maximum obtainable DNP enhancements at other magnetic fields are predicted.

Stability of the nitroxide biradical AMUPol in intact and lysed mammalian cells

Perspectives on microwave coupling into cylindrical and spherical rotors with dielectric lenses for magic angle spinning dynamic nuclear polarization.

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

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.

We use microwave-induced dynamic nuclear polarization(DNP) ofthe substitutional nitrogen defects (P1 centers) in diamond to hyperpolarizebulk 13C nuclei in both single crystal and powder samplesat room temperature at 3.34 T. The large (>100-fold) enhancementsdemonstrated correspond to a greater than 10 000-fold improvementin terms of signal averaging of the 1% abundant 13C spins.The DNP was performed using low-power solid state sources under static(nonspinning) conditions. The DNP spectrum (DNP enhancement as a functionof microwave frequency) of diamond powder shows features that broadlycorrelate with the EPR spectrum. A well-defined negative Overhauserpeak and two solid effect peaks are observed for the central (mI = 0) manifold of the 14N spins. Previous low temperature measurements in diamondhad measured a positive Overhauser enhancement in this manifold. Frequency-chirpedmillimeter-wave excitation of the electron spins is seen to significantlyimprove the enhancements for the two outer nuclear spin manifolds(mI = ±1) and to blur some of thesharper features associated with the central manifold. The outer linesare best fit using a combination of the cross effect and the truncatedcross effect, which is known to mimic features of an Overhauser effect.Similar features are also observed in experiments on single crystalsamples. The observation of all of these mechanisms in a single materialsystem under the same experimental conditions is likely due to thesignificant heterogeneity of the high pressure, high temperature (HPHT)type Ib diamond samples used. Large room temperature DNP enhancementsat fields above a few tesla enable spectroscopic studies with betterchemical shift resolution under ambient conditions.

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.