H-F cross-polarization magic angle spinning dynamic nuclear polarization NMR investigation of advanced pharmaceutical formulations.

A new 3.2 mm H-F-X magic angle spinning dynamic nuclear polarization NMR (MAS DNP-NMR) probe was developed with a unique coil design with separate radiofrequency channels for H excitation and C or F detection to enable acquisition of H-F cross-polarization (CP) MAS experiments, direct-detected F spectra with proton decoupling, and acquisition on C with simultaneous double decoupling on the H and 19F channels as well as H-F-C double-CP experiments under low temperature MAS DNP conditions. We use these sequences to study AZD2811, which is an active pharmaceutical ingredient (API), in its pure dry state as well as in its corresponding drug delivery formulation consisting of drug-loaded polymeric nanoparticles (PNPs).

Fast magic angle spinning for the characterization of milligram quantities of organic and biological solids at natural isotopic abundance by C-C correlation DNP-enhanced NMR.

We show that multidimensional solid-state NMR C-C correlation spectra of biomolecular assemblies and microcrystalline organic molecules can be acquired at natural isotopic abundance with only milligram quantities of sample. These experiments combine fast Magic Angle Spinning of the sample, low-power dipolar recoupling, and dynamic nuclear polarization performed with AsymPol biradicals, a recently introduced family of polarizing agents. Such experiments are essential for structural characterization as they provide short- and long-range distance information.

Complete resonance assignment of a pharmaceutical drug at natural isotopic abundance from DNP-Enhanced solid-state NMR.

Solid-state dynamic nuclear polarization enhanced magic angle spinning (DNP-MAS) NMR measurements coupled with density functional theory (DFT) calculations enable the full resonance assignment of a complex pharmaceutical drug molecule without the need for isotopic enrichment. DNP dramatically enhances the NMR signals, thereby making possible previously intractable two-dimensional correlation NMR spectra at natural abundance. Using inputs from DFT calculations, herein we describe a significant improvement to the structure elucidation process for complex organic molecules.

Highly Efficient Polarizing Agents for MAS-DNP of Proton-Dense Molecular Solids.

Efficiently hyperpolarizing proton-dense molecular solids through dynamic nuclear polarization (DNP) solid-state NMR is still an unmet challenge. Polarizing agents (PAs) developed so far do not perform well on proton-rich systems, such as organic microcrystals and biomolecular assemblies. Herein we introduce a new PA, cAsymPol-POK, and report outstanding hyperpolarization efficiency on 12.

A novel strategy for site selective spin-labeling to investigate bioactive entities by DNP and EPR spectroscopy.

A novel specific spin-labeling strategy for bioactive molecules is presented for eptifibatide (integrilin) an antiplatelet aggregation inhibitor, which derives from the venom of certain rattlesnakes. By specifically labeling the disulfide bridge this molecule becomes accessible for analytical techniques such as Electron Paramagnetic Resonance (EPR) and solid state Dynamic Nuclear Polarization (DNP). The necessary spin-label was synthesized and inserted into the disulfide bridge of eptifibatide via reductive followed by insertion by a double Michael addition under physiological conditions.

Observing an Antisense Drug Complex in Intact Human Cells by in-Cell NMR Spectroscopy.

Gaining insight into the uptake, trafficking and target engagement of drugs in cells can enhance understanding of a drug's function and efficiency. However, there are currently no reliable methods for studying untagged biomolecules in macromolecular complexes in intact human cells. Here we have studied an antisense oligonucleotide (ASO) drug in HEK 293T and HeLa cells by NMR spectroscopy.

Dynamic Nuclear Polarization / solid-state NMR of membranes. Thermal effects and sample geometry.

Whereas specially designed dinitroxide biradicals, reconstitution protocols, oriented sample geometries and NMR probes have helped to much increase the DNP enhancement factors of membrane samples they still lag considerably behind those obtained from glasses made of protein solutions. Here we show that not only the MAS rotor material but also the distribution of the membrane samples within the NMR rotor have a pronounced effect on the DNP enhancement. These observations are rationalized with the cooling efficiency and the internal properties of the sample, monitored by their T relaxation, microwave ON versus OFF signal intensities and DNP effect.

Efficient 263 GHz magic angle spinning DNP at 100 K using solid-state diode sources.

The development of new, high-frequency solid-state diode sources capable of operating at 263 GHz, together with an optimized stator design for improved millimeter-wave coupling to the NMR sample, have enabled low-power DNP experiments at 263 GHz/400 MHz. With 250 mW output power, signal enhancements as high as 120 are achieved on standard samples - approximately 1/3 of the maximal enhancement available with high-power gyrotrons under similar conditions. Diode-based sources have a number of advantages over vacuum tube devices: they emit a pure mode, can be rapidly frequency-swept over a wide range of frequencies, have reproducible output power over this range, and have excellent output stability.

Improved waveguide coupling for 1.3 mm MAS DNP probes at 263 GHz.

We consider the geometry of a radially irradiated microwave beam in MAS DNP NMR probes and its impact on DNP enhancement. Two related characteristic features are found to be relevant: (i) the focus of the microwave beam on the DNP MAS sample and (ii) the microwave magnetic field magnitude in the sample. We present a waveguide coupler setup that enables us to significantly improve beam focus and field magnitude in 1.

Fast detection and structural identification of carbocations on zeolites by dynamic nuclear polarization enhanced solid-state NMR.

Acidic zeolites are porous aluminosilicates used in a wide range of industrial processes such as adsorption and catalysis. The formation of carbocation intermediates plays a key role in reactivity, selectivity and deactivation in heterogeneous catalytic processes. However, the observation and determination of carbocations remain a significant challenge in heterogeneous catalysis due to the lack of selective techniques of sufficient sensitivity to detect their low concentrations.

Endogenous Dynamic Nuclear Polarization for Natural Abundance O and Lithium NMR in the Bulk of Inorganic Solids.

In recent years magic angle spinning-dynamic nuclear polarization (MAS-DNP) has developed as an excellent approach for boosting the sensitivity of solid-state NMR (ssNMR) spectroscopy, thereby enabling the characterization of challenging systems in biology and chemistry. Most commonly, MAS-DNP is based on the use of nitroxide biradicals as polarizing agents. In materials science, since the use of nitroxides often limits the signal enhancement to the materials' surface and subsurface layers, there is need for hyperpolarization approaches which will provide sensitivity in the bulk of micron sized particles.

Probing the surface of γ-AlO by oxygen-17 dynamic nuclear polarization enhanced solid-state NMR spectroscopy.

γ-Al2O3 is an important catalyst and catalyst support of industrial interest. Its acid/base characteristics are correlated to the surface structure, which has always been an issue of concern. In this work, the complex (sub-)surface oxygen species on surface-selectively labelled γ-Al2O3 were probed by 17O dynamic nuclear polarization surface-enhanced NMR spectroscopy (DNP-SENS).

Dynamic Nuclear Polarization NMR Spectroscopy of Polymeric Carbon Nitride Photocatalysts: Insights into Structural Defects and Reactivity.

Metal-free polymeric carbon nitrides (PCNs) are promising photocatalysts for solar hydrogen production, but their structure-photoactivity relationship remains elusive. Two PCNs were characterized by dynamic-nuclear-polarization-enhanced solid-state NMR spectroscopy, which circumvented the need for specific labeling with either C- or N-enriched precursors. Rapid 1D and 2D data acquisition was possible, providing insights into the structural contrasts between the PCNs.

Analysis of Molecular Orientation in Organic Semiconducting Thin Films Using Static Dynamic Nuclear Polarization Enhanced Solid-State NMR Spectroscopy.

Molecular orientation in amorphous organic semiconducting thin-film devices is an important issue affecting device performance. However, to date it has not been possible to analyze the "distribution" of the orientations. Although solid-state NMR (ssNMR) spectroscopy can provide information on the "distribution" of molecular orientations, the technique is limited because of the small amount of sample in the device and the low sensitivity of ssNMR.

Hydroxyapatites: Key Structural Questions and Answers from Dynamic Nuclear Polarization.

We demonstrate that NMR/DNP (Dynamic Nuclear Polarization) allows an unprecedented description of carbonate substituted hydroxyapatite (CHAp). Key structural questions related to order/disorder and clustering of carbonates are tackled using distance sensitive DNP experiments using C-C recoupling. Such experiments are easily implemented due to unprecedented DNP gain (orders of magnitude).

Dynamic Nuclear Polarization/Solid-State NMR Spectroscopy of Membrane Polypeptides: Free-Radical Optimization for Matrix-Free Lipid Bilayer Samples.

Dynamic nuclear polarization (DNP) boosts the sensitivity of NMR spectroscopy by orders of magnitude and makes investigations previously out of scope possible. For magic-angle-spinning (MAS) solid-state NMR spectroscopy studies, the samples are typically mixed with biradicals dissolved in a glass-forming solvent and are investigated at cryotemperatures. Herein, we present new biradical polarizing agents developed for matrix-free samples such as supported lipid bilayers, which are systems widely used for the investigation of membrane polypeptides of high biomedical importance.

Surface-Sensitive NMR Detection of the Solid Electrolyte Interphase Layer on Reduced Graphene Oxide.

Forming a stable solid electrolyte interphase (SEI) is critical for rechargeable batteries' performance and lifetime. Understanding its formation requires analytical techniques that provide molecular-level insight. Here, dynamic nuclear polarization (DNP) is utilized for the first time to enhance the sensitivity of solid-state NMR (ssNMR) spectroscopy to the SEI.

Temperature dependence of cross-effect dynamic nuclear polarization in rotating solids: advantages of elevated temperatures.

Dynamic nuclear polarization exploits electron spin polarization to boost signal-to-noise in magic-angle-spinning (MAS) NMR, creating new opportunities in materials science, structural biology, and metabolomics studies. Since protein NMR spectra recorded under DNP conditions can show improved spectral resolution at 180-200 K compared to 110 K, we investigate the effects of AMUPol and various deuterated TOTAPOL isotopologues on sensitivity and spectral resolution at these temperatures, using proline and reproducibly prepared SH3 domain samples. The TOTAPOL deuteration pattern is optimized for protein DNP MAS NMR, and signal-to-noise per unit time measurements demonstrate the high value of TOTAPOL isotopologues for Protein DNP MAS NMR at 180-200 K.

On The Potential of Dynamic Nuclear Polarization Enhanced Diamonds in Solid-State and Dissolution (13) C NMR Spectroscopy.

Dynamic nuclear polarization (DNP) is a versatile option to improve the sensitivity of NMR and MRI. This versatility has elicited interest for overcoming potential limitations of these techniques, including the achievement of solid-state polarization enhancement at ambient conditions, and the maximization of (13) C signal lifetimes for performing in vivo MRI scans. This study explores whether diamond's (13) C behavior in nano- and micro-particles could be used to achieve these ends.

Tailoring of Polarizing Agents in the bTurea Series for Cross-Effect Dynamic Nuclear Polarization in Aqueous Media.

A series of 18 nitroxide biradicals derived from bTurea has been prepared, and their enhancement factors ɛ ((1)H) in cross-effect dynamic nuclear polarization (CE DNP) NMR experiments at 9.4 and 14.1 T and 100 K in a DNP-optimized glycerol/water matrix ("DNP juice") have been studied.

Membrane topologies of the PGLa antimicrobial peptide and a transmembrane anchor sequence by Dynamic Nuclear Polarization/solid-state NMR spectroscopy.

Dynamic Nuclear Polarization (DNP) has been introduced to overcome the sensitivity limitations of nuclear magnetic resonance (NMR) spectroscopy also of supported lipid bilayers. When investigated by solid-state NMR techniques the approach typically involves doping the samples with biradicals and their investigation at cryo-temperatures. Here we investigated the effects of temperature and membrane hydration on the topology of amphipathic and hydrophobic membrane polypeptides.

Solid-State NMR/Dynamic Nuclear Polarization of Polypeptides in Planar Supported Lipid Bilayers.

Dynamic nuclear polarization has been developed to overcome the limitations of the inherently low signal intensity of NMR spectroscopy. This technique promises to be particularly useful for solid-state NMR spectroscopy where the signals are broadened over a larger frequency range and most investigations rely on recording low gamma nuclei. To extend the range of possible investigations, a triple-resonance flat-coil solid-state NMR probe is presented with microwave irradiation capacities allowing the investigation of static samples at temperatures of 100 K, including supported lipid bilayers.

Direct Evidence of Imino Acid-Aromatic Interactions in Native Collagen Protein by DNP-Enhanced Solid-State NMR Spectroscopy.

Aromatic amino acids (AAAs) have rare presence (∼1.4% abundance of Phe) inside of collagen protein, which is the most abundant animal protein playing a functional role in skin, bone, and connective tissues. The role of AAAs is very crucial and has been debated.

Up to 100% Improvement in Dynamic Nuclear Polarization Solid-State NMR Sensitivity Enhancement of Polymers by Removing Oxygen.

High-field dynamic nuclear polarization (DNP) has emerged as a powerful technique for improving the sensitivity of solid-state NMR (SSNMR), yielding significant sensitivity enhancements for a variety of samples, including polymers. Overall, depending upon the type of polymer, the molecular weight, and the DNP sample preparation method, sensitivity enhancements between 5 and 40 have been reported. These promising enhancements remain, however, far from the theoretical maximum (>1000).

Observing Apparent Nonuniform Sensitivity Enhancements in Dynamic Nuclear Polarization Solid-State NMR Spectra of Polymers.

High-field dynamic nuclear polarization (DNP) may enhance the sensitivity of solid-state NMR experiments on a wide range of systems, including synthetic polymers, owing to the transfer of electron spin polarization from radicals to nuclei upon microwave irradiation (usually at cryogenic temperatures). Provided that the radicals are homogeneously dispersed in the sample, a uniform DNP enhancement is expected for all the signals in the C cross-polarization magic angle spinning (CPMAS) spectrum. Here, we show that, in the case of methyl group containing polymers, a change in the cross-polarization (CP) dynamics induced by the moderate increase in sample temperature due to microwave irradiation may lead to the observation of apparent nonuniform enhancements in the DNP-enhanced C CPMAS spectra.