Time domain DNP at 1.2 T.

We present the results of an experimental pulsed DNP study at 1.2 T (33.5 GHz/51 MHz electron and 1H Larmor frequencies, respectively). The results include a comparison of constant-amplitude NOVEL (CA-NOVEL), ramped-amplitude NOVEL (RA-NOVEL) and the frequency-swept integrated solid effect (FS-ISE) experiments all of which were performed at the NOVEL matching condition, ω1S=ω0I, where ω1S is the electron Rabi frequency andω0I the proton Larmor frequency. To the best of our knowledge, this is the first pulsed DNP study carried out at field higher than X-band (0.35 T) using the NOVEL condition. A combination of high microwave power (∼150 W) and a microwave cavity with a high Q (∼500) allowed us to satisfy the NOVEL matching condition. We also observed stretched solid effect (S2E) contributions in the Zeeman field profiles when chirped pulses are applied. Furthermore, the high quality factor of the cavity limits the concentration of the radical to ∼5 mM and generates a hysteresis in the FS-ISE experiments. Nevertheless, we observe very high DNP enhancements that are comparable to the results at X-band. These promising outcomes suggest the importance of further studies at even higher fields that delineate the instrumentation and methods required for time domain DNP.

Diverse approaches to predicting drug-induced liver injury using gene-expression profiles.

BACKGROUND: Drug-induced liver injury (DILI) is a serious concern during drug development and the treatment of human disease. The ability to accurately predict DILI risk could yield significant improvements in drug attrition rates during drug development, in drug withdrawal rates, and in treatment outcomes. In this paper, we outline our approach to predicting DILI risk using gene-expression data from Build 02 of the Connectivity Map (CMap) as part of the 2018 Critical Assessment of Massive Data Analysis CMap Drug Safety Challenge. RESULTS: First, we used seven classification algorithms independently to predict DILI based on gene-expression values for two cell lines. Similar to what other challenge participants observed, none of these algorithms predicted liver injury on a consistent basis with high accuracy. In an attempt to improve accuracy, we aggregated predictions for six of the algorithms (excluding one that had performed exceptionally poorly) using a soft-voting method. This approach also failed to generalize well to the test set. We investigated alternative approaches-including a multi-sample normalization method, dimensionality-reduction techniques, a class-weighting scheme, and expanding the number of hyperparameter combinations used as inputs to the soft-voting method. We met limited success with each of these solutions. CONCLUSIONS: We conclude that alternative methods and/or datasets will be necessary to effectively predict DILI in patients based on RNA expression levels in cell lines. REVIEWERS: This article was reviewed by Pawel P Labaj and Aleksandra Gruca (both nominated by David P Kreil).

Frequency-Swept Integrated and Stretched Solid Effect Dynamic Nuclear Polarization.

We investigate a new time domain approach to dynamic nuclear polarization (DNP), the frequency-swept integrated solid effect (FS-ISE), utilizing a high power, broadband 94 GHz (3.35 T) pulse EPR spectrometer. The bandwidth of the spectrometer enabled measurement of the DNP Zeeman frequency/field profile that revealed two dominant polarization mechanisms, the expected ISE, and a recently observed mechanism, the stretched solid effect (S2E). At 94 GHz, despite the limitations in the microwave chirp pulse length (10 µs) and the repetition rate (2 kHz), we obtained signal enhancements up to ∼70 for the S2E and ∼50 for the ISE. The results successfully demonstrate the viability of the FS-ISE and S2E DNP at a frequency 10 times higher than previous studies. Our results also suggest that these approaches are candidates for implementation at higher magnetic fields.

High-precision measurement of the electron spin g factor of trapped atomic nitrogen in the endohedral fullerene N@C60.

The electronic g factor carries highly useful information about the electronic structure of a paramagnetic species, such as spin-orbit coupling and dia- or paramagnetic (de-)shielding due to local fields of surrounding electron pairs. However, in many cases, a near "spin-only" case is observed, in particular for light elements, necessitating accurate and precise measurement of the g factors. Such measurement is typically impeded by a "chicken and egg situation": internal or external reference standards are used for relative comparison of electron paramagnetic resonance (EPR) Larmor frequencies. However, the g factor of the standard itself usually is subject to a significant uncertainty which directly limits the precision and/or accuracy of the sought after sample g factor. Here, we apply an EPR reference-free approach for determining the g factor of atomic nitrogen trapped within the endohedral fullerene C60:N@C60 in its polycrystalline state by measuring the 1H NMR resonance frequency of dispersing toluene at room temperature. We found a value of g=2.00204(4) with a finally reached relative precision of ∼20 ppm. This accurate measurement allows us to directly compare the electronic properties of N@C60 to those found in atomic nitrogen in the gas phase or trapped in other solid matrices at liquid helium temperature. We conclude that spin-orbit coupling in N@C60 at room temperature is very similar in magnitude and of same sign as found in other inert solid matrices and that interactions between the quartet spin system and the C60 molecular orbitals are thus negligible.

Overhauser effects in non-conducting solids at 1.2 K.

Recently, it was observed that protons in non-conducting solids doped with 1,3-bisdiphenylene-2-phenylallyl (BDPA) or its sulfonated derivative (SA-BDPA) can be polarized through Overhauser effects via resonant microwave irradiation. These effects were present under magic angle spinning conditions in magnetic fields between 5 and 18.8 T and at temperatures near 100 K. This communication reports similar effects in static samples at 6.7 T and, more importantly, at temperatures as low as 1.2 K, in a different dynamic regime than in the previous study. Our results provide new information towards understanding the mechanism of the Overhauser effect in non-conducting solids. We discuss possible origins of the fluctuations that can give rise to an Overhauser effect at such low temperatures.

Reprint of: Localization of Cl-35 Nuclei in Biological Solids using Rotational-Echo Double-Resonance Experiments.

Chloride ions play important roles in many chemical and biological processes. This paper investigates the possibility of localizing 35Cl nuclei using solid-state NMR. It demonstrates that distances shorter than 3.8Å, between 13C atoms and 35Cl atoms in 10% uniformly labeled 13C L-tyrosine·HCl and natural abundance Glycine·HCl can be measured using rotational-echo (adiabatic passage) double-resonance (RE(AP)DOR). Furthermore the effect of quadrupolar interaction on the REDOR/REAPDOR experiment is quantified. The dephasing curve is plotted in a three dimensional chart as a function of the dephasing time and of the strength of quadrupolar interaction felt by each orientation. During spinning each orientation feels a quadrupolar interaction that varies in time, and therefore at each moment in time we reorder the crystallite orientations as a function of their contribution to the dephasing curve. In this way the effect of quadrupolar interaction on the dipolar dephasing curve can be fitted with a polynomial function. The numerical investigation performed allows us to generate REDOR/REAPDOR curves which are then used to simulate the experimental data.

Ramped-amplitude NOVEL.

We present a pulsed dynamic nuclear polarization (DNP) study using a ramped-amplitude nuclear orientation via electron spin locking (RA-NOVEL) sequence that utilizes a fast arbitrary waveform generator (AWG) to modulate the microwave pulses together with samples doped with narrow-line radicals such as 1,3-bisdiphenylene-2-phenylallyl (BDPA), sulfonated-BDPA (SA-BDPA), and trityl-OX063. Similar to ramped-amplitude cross polarization in solid-state nuclear magnetic resonance, RA-NOVEL improves the DNP efficiency by a factor of up to 1.6 compared to constant-amplitude NOVEL (CA-NOVEL) but requires a longer mixing time. For example, at τmix = 8 µs, the DNP efficiency reaches a plateau at a ramp amplitude of ∼20 MHz for both SA-BDPA and trityl-OX063, regardless of the ramp profile (linear vs. tangent). At shorter mixing times (τmix = 0.8 µs), we found that the tangent ramp is superior to its linear counterpart and in both cases there exists an optimum ramp size and therefore ramp rate. Our results suggest that RA-NOVEL should be used instead of CA-NOVEL as long as the electronic spin lattice relaxation T1e is sufficiently long and/or the duty cycle of the microwave amplifier is not exceeded. To the best of our knowledge, this is the first example of a time domain DNP experiment that utilizes modulated microwave pulses. Our results also suggest that a precise modulation of the microwave pulses can play an important role in optimizing the efficiency of pulsed DNP experiments and an AWG is an elegant instrumental solution for this purpose.

Localization of Cl-35 nuclei in biological solids using rotational-echo double-resonance experiments.

Chloride ions play important roles in many chemical and biological processes. This paper investigates the possibility of localizing 35Cl nuclei using solid-state NMR. It demonstrates that distances shorter than 3.8Å, between 13C atoms and 35Cl atoms in 10% uniformly labeled 13C L-tyrosine·HCl and natural abundance Glycine·HCl can be measured using rotational-echo (adiabatic passage) double-resonance (RE(AP)DOR). Furthermore the effect of quadrupolar interaction on the REDOR/REAPDOR experiment is quantified. The dephasing curve is plotted in a three dimensional chart as a function of the dephasing time and of the strength of quadrupolar interaction felt by each orientation. During spinning each orientation feels a quadrupolar interaction that varies in time, and therefore at each moment in time we reorder the crystallite orientations as a function of their contribution to the dephasing curve. In this way the effect of quadrupolar interaction on the dipolar dephasing curve can be fitted with a polynomial function. The numerical investigation performed allows us to generate REDOR/REAPDOR curves which are then used to simulate the experimental data.

Three pulse recoupling and phase jump matching.

The paper describes a family of novel recoupling pulse sequences, called three pulse recoupling. These pulse sequences can be employed for both homonuclear and heteronuclear recoupling experiments and are robust to dispersion in chemical shifts and rf-inhomogeneity. These recoupling pulse sequences can be used in design of two-dimensional solid state NMR experiments that use powdered dephased antiphase coherence (γ preparation) to encode chemical shifts in the indirect dimension. Both components of this chemical shift encoded gamma-prepared states can be refocused into inphase coherence by a recoupling element. This helps to achieve sensitivity enhancement in 2D NMR experiments by quadrature detection.

Time domain DNP with the NOVEL sequence.

We present results of a pulsed dynamic nuclear polarization (DNP) study at 0.35 T (9.7 GHz/14.7 MHz for electron/(1)H Larmor frequency) using a lab frame-rotating frame cross polarization experiment that employs electron spin locking fields that match the (1)H nuclear Larmor frequency, the so called NOVEL (nuclear orientation via electron spin locking) condition. We apply the method to a series of DNP samples including a single crystal of diphenyl nitroxide (DPNO) doped benzophenone (BzP), 1,3-bisdiphenylene-2-phenylallyl (BDPA) doped polystyrene (PS), and sulfonated-BDPA (SA-BDPA) doped glycerol/water glassy matrices. The optimal Hartman-Hahn matching condition is achieved when the nutation frequency of the electron matches the Larmor frequency of the proton, ω(1S) = ω(0I), together with possible higher order matching conditions at lower efficiencies. The magnetization transfer from electron to protons occurs on the time scale of ∼100 ns, consistent with the electron-proton couplings on the order of 1-10 MHz in these samples. In a fully protonated single crystal DPNO/BzP, at 270 K, we obtained a maximum signal enhancement of ε = 165 and the corresponding gain in sensitivity of ε(T1/T(B))(1/2)=230 due to the reduction in the buildup time under DNP. In a sample of partially deuterated PS doped with BDPA, we obtained an enhancement of 323 which is a factor of ∼3.2 higher compared to the protonated version of the same sample and accounts for 49% of the theoretical limit. For the SA-BDPA doped glycerol/water glassy matrix at 80 K, the sample condition used in most applications of DNP in nuclear magnetic resonance, we also observed a significant enhancement. Our findings demonstrate that pulsed DNP via the NOVEL sequence is highly efficient and can potentially surpass continuous wave DNP mechanisms such as the solid effect and cross effect which scale unfavorably with increasing magnetic field. Furthermore, pulsed DNP is also a promising avenue for DNP at high temperature.

Mechanisms of dynamic nuclear polarization in insulating solids.

Dynamic nuclear polarization (DNP) is a technique used to enhance signal intensities in NMR experiments by transferring the high polarization of electrons to their surrounding nuclei. The past decade has witnessed a renaissance in the development of DNP, especially at high magnetic fields, and its application in several areas including biophysics, chemistry, structural biology and materials science. Recent technical and theoretical advances have expanded our understanding of established experiments: for example, the cross effect DNP in samples spinning at the magic angle. Furthermore, new experiments suggest that our understanding of the Overhauser effect and its applicability to insulating solids needs to be re-examined. In this article, we summarize important results of the past few years and provide quantum mechanical explanations underlying these results. We also discuss future directions of DNP and current limitations, including the problem of resolution in protein spectra recorded at 80-100 K.

Confined crystallization of fenofibrate in nanoporous silica.

Producing stable nanocrystals confined to porous excipient media is a desirable way to increase the dissolution rate and improve the bioavailability of poorly water soluble pharmaceuticals. The poorly soluble pharmaceutical fenofibrate was crystallized in controlled pore glass (CPG) of 10 different pore sizes between 12 nm and 300 nm. High drug loadings of greater than 20 wt% were achieved across all pore sizes greater than 20 nm. Nanocrystalline fenofibrate was formed in pore sizes greater than 20 nm and showed characteristic melting point depressions following a Gibbs-Thomson relationship as well as enhanced dissolution rates. Solid-state Nuclear Magnetic Resonance (NMR) was employed to characterize the crystallinity of the confined molecules. These results help to advance the fundamental understanding of nanocrystallization in confined pores.

Formation of Organic Molecular Nanocrystals under Rigid Confinement with Analysis by Solid State NMR.

Crystallization in rigid confinement is a promising method to obtain organic molecular nanocrystals. However, the crystallization behavior and the related characterization methods are not well studied. Here we present a systematic study of the nucleation of organic molecular nanocrystals in rigid pores. Four different compounds were studied, ibuprofen, fenofibrate, griseofulvin, and indomethacin, which range from simple to complex molecules. Solid-state Nuclear Magnetic Resonance (NMR) was employed to analyse the structure of these compounds inside pores which are difficult to characterize by other analytical methods. We successfully demonstrated the production of nano-crystalline ibuprofen, fenofibrate and griseofulvin in porous silica particles with ~ 40 nm pores. These nanocrystals showed significant enhancement in dissolution rates. These results help advance the fundamental understanding of nucleation under rigid confinement and may lead to potential applications in developing new formulations in the pharmaceutical industry.

Overhauser effects in insulating solids.

We report magic angle spinning, dynamic nuclear polarization (DNP) experiments at magnetic fields of 9.4 T, 14.1 T, and 18.8 T using the narrow line polarizing agents 1,3-bisdiphenylene-2-phenylallyl (BDPA) dispersed in polystyrene, and sulfonated-BDPA (SA-BDPA) and trityl OX063 in glassy glycerol/water matrices. The (1)H DNP enhancement field profiles of the BDPA radicals exhibit a significant DNP Overhauser effect (OE) as well as a solid effect (SE) despite the fact that these samples are insulating solids. In contrast, trityl exhibits only a SE enhancement. Data suggest that the appearance of the OE is due to rather strong electron-nuclear hyperfine couplings present in BDPA and SA-BDPA, which are absent in trityl and perdeuterated BDPA (d21-BDPA). In addition, and in contrast to other DNP mechanisms such as the solid effect or cross effect, the experimental data suggest that the OE in non-conducting solids scales favorably with magnetic field, increasing in magnitude in going from 5 T, to 9.4 T, to 14.1 T, and to 18.8 T. Simulations using a model two spin system consisting of an electron hyperfine coupled to a (1)H reproduce the essential features of the field profiles and indicate that the OE in these samples originates from the zero and double quantum cross relaxation induced by fluctuating hyperfine interactions between the intramolecular delocalized unpaired electrons and their neighboring nuclei, and that the size of these hyperfine couplings is crucial to the magnitude of the enhancements. Microwave power dependent studies show that the OE saturates at considerably lower power levels than the solid effect in the same samples. Our results provide new insights into the mechanism of the Overhauser effect, and also provide a new approach to perform DNP experiments in chemical, biophysical, and physical systems at high magnetic fields.

Photonic-band-gap traveling-wave gyrotron amplifier.

We report the experimental demonstration of a gyrotron traveling-wave-tube amplifier at 250 GHz that uses a photonic band gap (PBG) interaction circuit. The gyrotron amplifier achieved a peak small signal gain of 38 dB and 45 W output power at 247.7 GHz with an instantaneous -3 dB bandwidth of 0.4 GHz. The amplifier can be tuned for operation from 245-256 GHz. The widest instantaneous -3 dB bandwidth of 4.5 GHz centered at 253.25 GHz was observed with a gain of 24 dB. The PBG circuit provides stability from oscillations by supporting the propagation of transverse electric (TE) modes in a narrow range of frequencies, allowing for the confinement of the operating TE03-like mode while rejecting the excitation of oscillations at nearby frequencies. This experiment achieved the highest frequency of operation for a gyrotron amplifier; at present, there are no other amplifiers in this frequency range that are capable of producing either high gain or high output power. This result represents the highest gain observed above 94 GHz and the highest output power achieved above 140 GHz by any conventional-voltage vacuum electron device based amplifier.

Recoupling in solid state NMR using γ prepared states and phase matching.

The paper describes two-dimensional solid state NMR experiments that use powdered dephased antiphase coherence (γ preparation) to encode chemical shifts in the indirect dimension. Both components of this chemical shift encoded gamma-prepared states can be refocused into inphase coherence by a recoupling element. This helps to achieve sensitivity enhancement in 2D NMR experiments by quadrature detection. The powder dependence of the gamma-prepared states allows for manipulating them by suitable insertion of delays in the recoupling periods. This helps to design experiments that suppress diagonal peaks in 2D spectra, leading to improved resolution. We describe some new phase modulated heteronuclear and homonuclear recoupling pulse sequences that simplify the implementation of the described experiments based on γ prepared states. Recoupling in the heteronuclear spin system is achieved by matching the difference in the amplitude of the sine/cosine modulated phase on the two rf-channels to the spinning frequency while maintaining the same power on the two rf-channels.

Dipolar recoupling in solid state NMR by phase alternating pulse sequences.

We describe some new developments in the methodology of making heteronuclear and homonuclear recoupling experiments in solid state NMR insensitive to rf-inhomogeneity by phase alternating the irradiation on the spin system every rotor period. By incorporating delays of half rotor periods in the pulse sequences, these phase alternating experiments can be made gamma encoded. The proposed methodology is conceptually different from the standard methods of making recoupling experiments robust by the use of ramps and adiabatic pulses in the recoupling periods. We show how the concept of phase alternation can be incorporated in the design of homonuclear recoupling experiments that are both insensitive to chemical shift dispersion and rf-inhomogeneity.

High-Field Dynamic Nuclear Polarization for Solid and Solution Biological NMR.

Dynamic nuclear polarization (DNP) results in a substantial nuclear polarization enhancement through a transfer of the magnetization from electrons to nuclei. Recent years have seen considerable progress in the development of DNP experiments directed towards enhancing sensitivity in biological nuclear magnetic resonance (NMR). This review covers the applications, hardware, polarizing agents, and theoretical descriptions that were developed at the Francis Bitter Magnet Laboratory at Massachusetts Institute of Technology for high-field DNP experiments. In frozen dielectrics, the enhanced nuclear polarization developed in the vicinity of the polarizing agent can be efficiently dispersed to the bulk of the sample via (1)H spin diffusion. This strategy has been proven effective in polarizing biologically interesting systems, such as nanocrystalline peptides and membrane proteins, without leading to paramagnetic broadening of the NMR signals. Gyrotrons have been used as a source of high-power (5-10 W) microwaves up to 460 GHz as required for the DNP experiments. Other hardware has also been developed allowing in situ microwave irradiation integrated with cryogenic magic-angle-spinning solid-state NMR. Advances in the quantum mechanical treatment are successful in describing the mechanism by which new biradical polarizing agents yield larger enhancements at higher magnetic fields. Finally, pulsed methods and solution experiments should play a prominent role in the future of DNP.