Oxygen is an integral component of proteins and nucleic acids, but remains sparsely studied in such samples because its only NMR active isotope, 17O, is a low sensitivity and resolution species. These properties are a consequence of its low natural abundance (0.039%) and the fact that 17O is a S = 5/2 nuclide with large quadrupolar couplings (6-11 MHz). In this work, we address these issues with efficient isotopic labeling, high magnetic fields, fast magic-angle spinning and indirect 1H detection. This combination of refinements, in conjunction with multidimensional heteronuclear correlation experiments, improves sensitivity and permits observation of oxygen sites specific to each amino acid residue in a model dipeptide sample in a manner consistent with the goal of high resolution. In particular, double-quantum cross-polarization at high sample spinning frequencies is found to provide efficient polarization transfer between 13C and 17O nuclei. Notably, the use of 17O as the initial source of polarization for experiments, as opposed to 1H, is found to be advantageous in terms of sensitivity per unit time due to the short 17O T1 relaxation. Additionally, the second-order quadrupolar broadening in the 17O dimension is averaged by incorporation of a low-power multiple-quantum sequence to yield sharp isotropic peaks. Comparison of isotropic and anisotropic 17O spectra allows extraction of quadrupolar parameters for each oxygen site. Finally, the high 17O resolution obtained is used in 3D experiments in combination with 13C polarization transfers and subsequent 1H detection to demonstrate the potential to determine sequential assignments and long range distance restraints. Collectively, these results suggest that 17O correlation spectroscopy can become an essential tool in the repertoire of techniques for biomolecular structure determination with the backbone 17O that has not yet been fully utilized.

Solid-state NMR methods with high resolution and sensitivity are presented for identification and charaterization of hydrogen-bonded 15N/17O atomic pairs in peptide samples. Indirect 1H detection under fast magic-angle spinning, and the stronger 1H-15N and 1H-17O couplings are leveraged to significantly improve sensitivity over previous methods that use direct 15N-17O interactions.

The synthesis and redox transformations of 6,12-diboraindeno[1,2-b]fluorene (DBIF)─a pentacyclic π-system with diboron incorporation─are reported. In notable contrast to the all-hydrocarbon indenofluorenes, a ligand coordination and reduction strategy allows tuning of the electronic structure across four redox states. Accordingly, we introduce an 18π e- neutral DBIF, a 20π e- diradical, a 21π e- radical anion, and a 22π e- dianion, all of which have been isolated and structurally authenticated. The diradicals exhibit diradical character of up to 77% and possess open-shell singlet ground states with thermally accessible triplet states.

Magic Angle Spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy is limited in spectral resolution by the spinning frequency of rotors that hold the analyte. Traditional yttria-stabilized zirconia (YSZ) rotors have mechanical constraints that typically limit spinning frequencies of 0.7 mm rotors to ωr/2π ∼ 110 kHz. These frequencies are not sufficient to achieve resolution comparable to that of solution NMR, which theoretically requires ωr/2π > 300 kHz. Building upon prior work that utilized rotary-assisted drilling, we present significant advances in diamond rotor fabrication using a high precision lathe and a centerless laser machining fixture that achieves improved concentricity of the rotor outer and inner diameters and spinning stability. The new crop of diamond rotors, which interface with the Bruker MAS 3 spinning system equivalently or better than commercial rotors, were spun using automatic 0.7 mm profiles. Furthermore, diamond rotors can be emptied and repacked, and we describe a set of 3D-printed centrifuge tools for efficient execution of this process. We evaluate chemical vapor deposition (CVD) versus high-pressure high-temperature (HPHT) diamonds as rotor material and find HPHT preferable. Extended spin stability tests and multidimensional NMR spectra of Aβ1-40 demonstrate the robustness and usability of these rotors. These advances pave the way for higher frequency spinning with helium gas in the future, enabling transformative improvements in MAS NMR for biological and material sciences.

Amyloid fibrils have been implicated in the pathogenesis of several neurodegenerative diseases, the most prevalent example being Alzheimer's disease (AD). Despite the prevalence of AD, relatively little is known about the structure of the associated amyloid fibrils. This has motivated our studies of fibril structures, extended here to the familial Arctic mutant of A beta(1-42), E22G-A beta(1-42). We found E22G-A beta(M0,1-42) is toxic to Escherichia coli, thus we expressed E22G-A beta(1-42) fused to the self-cleavable tag N-Pro in the form of its EDDIE mutant. Since the high surface activity of E22G-A beta(1-42) makes it difficult to obtain more than sparse quantities of fibrils, we employed H-1 detected magic angle spinning (MAS) nuclear magnetic resonance (NMR) experiments to characterize the protein. The H-1 detected C-13-C-13 methods were first validated by application to fully protonated amyloidogenic nanocrystals of GNNQQNY, and then applied to fibrils of the Arctic mutant of A beta, E22G-A beta(1-42). The MAS NMR spectra indicate that the biosynthetic samples of E22G-A beta(1-42) fibrils comprise a single conformation with C-13 chemical shifts extracted from hCH, hNH, and hCCH spectra that are very similar to those of wild type A beta(1-42) fibrils. These results suggest that E22G-A beta(1-42) fibrils have a structure similar to that of wild type A beta(1-42).

Collagen-mimetic peptides (CMP) have been invaluable tools for understanding the structure and function of collagen, which is the most abundant protein in animals. CMPs have also been developed as probes that detect damaged collagen because of the specificity required to form a collagen triple helix. These probes are not, however, ratiometric. Here, we used EPR spectroscopy to determine the end-to-end distances of CMPs that do not form stable homotrimeric helices. We found that those distances are shorter than the distances in the context of a collagen triple helix, suggesting their potential utility as a "molecular beacon" and guiding the choice and location of a pendant fluorophore-quencher pair. We then showed that a molecular beacon based on a glycine-(2S,4S)-4-fluoroproline-(2S,4R)-4-hydroxyproline tripeptide repeat and EDANS-DABCYL pair enabled the ratiometric detection of its binding to both other CMPs and natural mammalian collagen. These results provide guidance for the development of a new modality for detecting damaged collagen in physiological settings.

We present 3D electromagnetic simulations of the coupling of a 250 GHz beam to the sample in a 380 MHz DNP NMR spectrometer. To obtain accurate results for magic angle spinning (MAS) geometries, we first measured the complex dielectric constants of zirconia, sapphire, and the sample matrix material (DNP juice) from room temperature down to cryogenic temperatures and from 220 to 325 GHz with a VNA and up to 1 THz with a THz TDS system. Simulations of the coupling to the sample were carried out with the ANSYS HFSS code as a function of the rotor wall material (zirconia or sapphire), the rotor wall thickness, and the THz beam focusing (lens or no lens). For a zirconia rotor, the B 1 field in the sample was found to be strongly dependent on the rotor wall thickness, which is attributed to the high refractive index of zirconia. The optimum thickness of the wall is likely due to a transmission maximum but is offset from the thickness predicted by a simple calculation for a flat slab of the wall material. The B 1 value was found to be larger for a sapphire rotor than for a zirconia rotor for all cases studied. The results found in this work provide new insights into the coupling of THz radiation to the sample and should lead to improved designs of future DNP NMR instrumentation.

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We have prepared trityl radicals with protons at the positions of the -COOH group in the phenyl rings and examined their EPR spectra, which show large - hyperfine couplings, and their dynamic nuclear polarization (DNP) Zeeman field profiles . By assessing these polarizing agents for high-field and Overhauser effect DNP, we gain insight into the roles that these hyperfine couplings and other molecular properties play in the DNP performance of these radicals. Interestingly, we do not observe OE DNP in any of the three molecules we examined. This suggests that hyperfine couplings by themselves are not sufficient to support OE DNP. In this case the electron spin density is ∼75 % localized on the central carbon atom rather than being distributed uniformly over the aromatic rings. This is in contrast to BDPA where the distribution is delocalized. Our findings do not suggest that any of these radicals are particularly well-suited to high-field DNP. Furthermore, we emphasize that polarizing agents can be extremely sensitive to their solvent environment, even obscuring the intrinsic magnetic properties of the radical.

Magic angle spinning (MAS) nuclear magnetic resonance (NMR) has evolved significantly over the past three decades and established itself as a vital tool for the structural analysis of biological macromolecules and materials. This review delves into the development and application of dipolar recoupling techniques in MAS NMR, which are crucial for obtaining detailed structural and dynamic information. We discuss a variety of homonuclear and heteronuclear recoupling methods which are essential for measuring spatial restraints and explain in detail the spin dynamics that these sequences generate. We also explore recent developments in high spinning frequency MAS, proton detection, and dynamic nuclear polarization, underscoring their importance in advancing biomolecular NMR. Our aim is to provide a comprehensive account of contemporary dipolar recoupling methods, their principles, and their application to structural biology and materials, highlighting significant contributions to the field and emerging techniques that enhance resolution and sensitivity in MAS NMR spectroscopy.

Substitutional nitrogen impurities within the diamond lattice, known as P1 centers, have unpaired electrons that can mediate microwave driven dynamic nuclear polarization (DNP). In this paper we explore DNP of the bulk 13C spins in micrometer-sized P1 diamond particles and demonstrate a 550-fold DNP enhancement of the bulk 13C spins at room temperature in a 9 T magnetic field or 250 GHz for g approximate to 2 electrons. We study the DNP mechanisms, exploring their dependence on sample spinning frequency and microwave irradiation frequency using both continuous wave and frequency swept microwave irradiation, and discuss the results alongside recent DNP studies in the literature. Even with a modest microwave irradiation power of 160 mW from our frequency swept solid-state microwave source, we achieve a significant 13C signal enhancement, epsilon = 270 at room temperature. The enhancements were found to increase with the magic angle spinning (MAS) frequency, omega r /2 pi, and the results provide mechanistic insights into how different electron populations contribute to the observed DNP efficiency. These findings are inherently interesting and of practical importance in view of the recently reported diamond rotors fabricated from P1 high-pressure, high-temperature (HPHT) diamond.

In magic angle spinning (MAS) experiments involving uniformly C-13/N-15 labeled proteins, C-13-C-13 and C-13-N-15 dipolar recoupling experiments are now routinely used to measure direct dipole-dipole couplings that constrain distances and torsion angles and determine molecular structures. When the distances are short (<4 & Aring;), the direct couplings dominate the evolution of the spin system, and the C-13-C-13 and C-13-N-15 J-couplings (scalar couplings) are ignored. However, for structurally interesting >4 & Aring; distances, the dipolar and J-couplings are generally of comparable magnitude, and the variation in J must be included in order to optimize the precision of the experiment. This problem is circumvented in cases with well resolved spectra by using frequency-selective dipolar recoupling methods where the effects of J-couplings are refocused. However, for larger molecules with more spectral crowding, the requisite pulse length to achieve selectivity becomes long and leads to unacceptable sensitivity losses during the pulse or the spectral overlap precludes selective excitation. In this paper, we address this problem with two approaches aimed at facilitating higher precision internuclear distance measurements in systems that are not fully resolved. Namely, (1) we describe an approach for high precision measurements of specific J-couplings using the in-phase anti-phase (IPAP) sequence which is integrated into a nonselective dipolar recoupling technique and (2) we utilize the measured J-couplings to implement a double quantum filter experiment capable of providing the resolution necessary for frequency selective dipolar recoupling techniques without resorting to multidimensional spectroscopy. We illustrate these methods using a 7-peptide segment from the amyloidogenic Sup-35p protein, U-13C/15N-GNNQQNY, where we have measured 25 of the 27 possible one bond 13C-13C J-couplings.

Dynamic nuclear polarization (DNP) improves the sensitivity of NMR spectroscopy by the transfer of electron polarization to nuclei via irradiation of electron-nuclear transitions with microwaves at the appropriate frequency. For fields > 5 T and using g ∼ 2 electrons as polarizing agents, this requires the availability of microwave sources operating at >140 GHz. Therefore, microwave sources for DNP have generally been continuous-wave (CW) gyrotrons, and more recently solid state, oscillators operating at a fixed frequency and power. This constraint has limited the DNP mechanisms which can be exploited, and stymied the development of new time domain mechanisms. We report here the incorporation of a microwave source enabling facile modulation of frequency, amplitude, and phase at 9 T (250 GHz microwave frequency), and we have used the source for magic-angle spinning (MAS) NMR experiments. The experiments include investigations of CW DNP mechanisms, the advantage of frequency-chirped irradiation, and a demonstration of an Overhauser enhancement of ∼25 with a recently reported water-soluble BDPA radical, highlighting the potential for affordable and compact microwave sources to achieve significant enhancement in aqueous samples, including biological macromolecules. With the development of suitable microwave amplifiers, it should permit exploration of multiple new avenues involving time domain experiments.

Pancreatic cancer remains a pre-eminent cause of cancer-related deaths with late-stage diagnoses leading to an 11% five-year survival rate. Moreover, perineural invasion (PNI), in which cancer cells migrate into adjacent nerves, occurs in an overwhelming majority of patients, further enhancing tumor metastasis. PNI has only recently been recognized as a key contributor to cancer progression; thus, there are insufficient treatment options for the disease. Attention has been focused on glial Schwann cells (SC) for their mediation of pancreatic PNI. Under stress, SCs dedifferentiate from their mature state to facilitate the repair of peripheral nerves; however, this signaling can also re-direct cancer cells to accelerate PNI. Limited research has explored the mechanism that causes this shift in SC phenotype in cancer. Tumor-derived extracellular vesicles (TEV) have been implicated in other avenues of cancer development, such as pre-metastatic niche formation in secondary locations, yet how TEVs contribute to PNI has not been fully explored. In this study, we highlight TEVs as initiators of SC activation into a PNI-associated phenotype. Proteomic and pathway assessments of TEVs revealed an elevation in interleukin-8 (IL-8) signaling and nuclear factor kappa B (NFκB) over healthy cell-derived EVs. TEV-treated SCs exhibited higher levels of activation markers, which were successfully neutralized with IL-8 inhibition. Additionally, TEVs increased NFκB subunit p65 nuclear translocation, which may lead to increased secretion of cytokines and proteases indicative of SC activation and PNI. These findings present a novel mechanism that may be targeted for the treatment of pancreatic cancer PNI. Statement of Significance Identifying pancreatic tumor extracellular vesicles as key players in Schwann cell activation and perineural invasion by way of IL-8 will educate for more specialized and effective targets for an under-valued disease.

The resolution of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra remains bounded by the spinning frequency, which is limited by the material strength of MAS rotors. Since dia-mond is capable of withstanding 1.5-2.5x greater MAS frequencies, compared to state-of-the art zirconia, we fabricated rotors from single crystal diamond. When combined with bearings optimized for spinning with helium gas, diamond rotors could achieve the highest MAS frequencies to date. Furthermore, the excellent microwave transmission properties and thermal conductivity of diamond could improve sensi-tivity enhancements in dynamic nuclear polarization (DNP) experiments. The fabrication protocol we report involves novel laser micromachining and produced rotors that presently spin at xr=2p= 111.000 & PLUSMN; 0.004 kHz, with stable spinning up to 124 kHz, using N2 gas as the driving fluid. We present the first proton-detected 13C/15N MAS spectra recorded using diamond rotors, a critical step towards studying currently inaccessible ex-vivo protein samples with MAS NMR. Previously, the high aspect ratio of MAS rotors (-10:1) precluded fabrication of MAS rotors from diamond.& COPY; 2023 Elsevier Inc. All rights reserved.

The application of radio frequency (RF) vacuum electronics for the betterment of the human condition began soon after the invention of the first vacuum tubes in the 1920s and has not stopped since. Today, microwave vacuum devices are powering important applications in health treatment, material and biological science, wireless communication-terrestrial and space, Earth environment remote sensing, and the promise of safe, reliable, and inexhaustible energy. This article highlights some of the exciting application frontiers of vacuum electronics.

We recently used selective 2H labeling of BDPA to investigate the Overhauser Effect (OE) dynamic nuclear polarization (DNP) mechanism in insulating solids doped with 1,3-bis(diphenylene)-2-phenylallyl (BDPA), and established that the α and γ 1H spins on the fluorene rings are responsible for generating a zero quantum (ZQ) mediated positive bulk polarization. Here, we establish that the phenyl 1H spins relax via double-quantum (DQ) processes and therefore contribute negative enhancements which attenuate the OE-DNP. With measurements at different magnetic field strengths, we show that phenyl-d5-BDPA offers >50% improvement in OE-DNP enhancement compared to h21-BDPA attaining a maximum of ∼90 at 14.1 T and 5 kHz MAS, the highest observed OE-DNP enhancement to date under these conditions. The approach may be utilized to optimize other polarizing agents exhibiting an OE, an important DNP mechanism with a favorable field and spinning frequency dependence.

Despite the expanding applications of dynamic nuclear polarization (DNP) to problems in biological and materials science, there remain unresolved questions concerning DNP mechanisms. In this paper, we investigate the Zeeman DNP frequency profiles obtained with trityl radicals, OX063 and its partially deuterated analog OX071, in two commonly used glassing matrices based on glycerol and dimethyl sulfoxide (DMSO). When microwave irradiation is applied in the neighborhood of the narrow EPR transition, we observe a dispersive shape in the 1H Zeeman field and the effects are larger in DMSO than in glycerol. With the help of direct DNP observations on 13C and 2H nuclei, we investigate the origin of this dispersive field profile. In particular, we observe a weak nuclear Overhauser effect between 1H and 13C in the sample, which, when irradiating at the positive 1H solid effect (SE) condition, results in a negative enhancement of 13C spins. This observation is not consistent with thermal mixing (TM) being the mechanism responsible for the dispersive shape in the 1H DNP Zeeman frequency profile. Instead, we propose a new mechanism, resonant mixing, involving mixing of nuclear and electron spin states in a simple two-spin system without invoking electron-electron dipolar interactions.

We propose a mechanism for dynamic nuclear polarization that is different from the well-known Overhauser effect, solid effect, cross effect, and thermal mixing processes. We term it Resonant Mixing (RM), and we show that it arises from the evolution of the density matrix for a simple electron-nucleus coupled spin pair subject to weak microwave irradiation, the same interactions as the solid effect. However, the SE is optimal when the microwave field is off-resonance, whereas RM is optimal when the microwave field is on-resonance and involves the mixing of states by the microwave field together with the electron-nuclear coupling. Finally, we argue that this mechanism is responsible for the observed dispersive-shaped DNP field profile for trityl samples near the electron paramagnetic resonance center.