High-field Dynamic Nuclear Polarization Research Articles

Proton hyperfine couplings and Overhauser DNP.

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.

Nitrogen Substitutions Aggregation and Clustering in Diamonds as Revealed by High-Field Electron Paramagnetic Resonance.

Diamonds have been shown to be an excellent platform for quantum computing and quantum sensing applications. These applications are enabled by the presence of defects in the lattice, which are also known as color centers. The most common nitrogen-based defect in synthetic diamonds is the paramagnetic nitrogen substitution (P1) center. While the majority of quantum applications rely on nitrogen-vacancy (NV) centers, the properties of the latter are heavily influenced by the presence and the spatial distribution of the P1 centers. Hence, understanding the spatial distribution and mutual interactions of P1 centers is crucial for the successful development of diamond-based quantum devices. Unlike NV centers, P1 centers do not have a spin-dependent optical signature, and their spin-related properties, therefore, have to be detected and characterized using magnetic resonance methods. We show that using high-field (6.9 and 13.8 T) pulsed electron paramagnetic resonance (EPR) and dynamic nuclear polarization (DNP) experiments, we can distinguish and quantify three distinct populations of P1 centers: isolated P1 centers, weakly interacting ones, and exchange-coupled ones that are clustered together. While such clustering was suggested before, these clusters were never detected directly and unambiguously. Moreover, by using electron-electron double resonance (ELDOR) pump-probe experiments, we demonstrate that the latter clustered population does not exist in isolation but coexists with the more weakly interacting P1 centers throughout the diamond lattice. Its presence thus strongly affects the quantum properties of the diamond. We also show that the existence of this population can explain recent hyperpolarization results in type Ib high-pressure, high-temperature (HPHT) diamonds. We propose a combination of high-field pulsed EPR, ELDOR, and DNP as a tool for probing the aggregation state and interactions among different populations of nitrogen substitution centers.

The solid effect of dynamic nuclear polarization in liquids - accounting for g-tensor anisotropy at high magnetic fields.

In spite of its name, the solid effect of dynamic nuclear polarization (DNP) is also operative in viscous liquids, where the dipolar interaction between the polarized nuclear spins and the polarizing electrons is not completely averaged out by molecular diffusion on the timescale of the electronic spin-spin relaxation time. Under such slow-motional conditions, it is likely that the tumbling of the polarizing agent is similarly too slow to efficiently average the anisotropies of its magnetic tensors on the timescale of the electronic . Here we extend our previous analysis of the solid effect in liquids to account for the effect of -tensor anisotropy at high magnetic fields. Building directly on the mathematical treatment of slow tumbling in electron spin resonance , we calculate solid-effect DNP enhancements in the presence of both translational diffusion of the liquid molecules and rotational diffusion of the polarizing agent. To illustrate the formalism, we analyze high-field (9.4 T) DNP enhancement profiles from nitroxide-labeled lipids in fluid lipid bilayers. By properly accounting for power broadening and motional broadening, we successfully decompose the measured DNP enhancements into their separate contributions from the solid and Overhauser effects.

13C Hyperpolarization of Viscous Liquids by Transfer of Solid-Effect 1H Dynamic Nuclear Polarization at High Magnetic Field.

Dynamic nuclear polarization (DNP) is routinely used as a method for increasing the sensitivity to nuclear magnetic resonance (NMR). Recently, high-field solid-effect DNP in viscous liquids on 1H nuclei was demonstrated using narrow-line polarizing agents. Here we expand the applicability of DNP in viscous media to 13C nuclei. To hyperpolarize 13C nuclei, we combined solid-effect 1H DNP with a subsequent transfer of the 1H polarization to 13C via insensitive nuclei enhanced by polarization transfer (INEPT). We demonstrate this approach using a triarylmethyl radical as a polarizing agent and glycerol-13C3 as an analyte. We achieved 13C enhancement factors of up to 45 at a magnetic field of 9.4 T and room temperature.

Efficient 18.8T MAS-DNP NMR reveals hidden side chains in amyloid fibrils.

Amyloid fibrils are large and insoluble protein assemblies composed of a rigid core associated with a cross-β arrangement rich in β-sheet structural elements. It has been widely observed in solid-state NMR experiments that semi-rigid protein segments or side chains do not yield easily observable NMR signals at room temperature. The reasons for the missing peaks may be due to the presence of unfavorable dynamics that interfere with NMR experiments, which result in very weak or unobservable NMR signals. Therefore, for amyloid fibrils, semi-rigid and dynamically disordered segments flanking the amyloid core are very challenging to study. Here, we show that high-field dynamic nuclear polarization (DNP), an NMR hyperpolarization technique typically performed at low temperatures, can circumvent this issue because (i) the low-temperature environment (~ 100K) slows down the protein dynamics to escape unfavorable detection regime, (ii) DNP improves the overall NMR sensitivity including those offlexible side chains, and (iii) efficient cross-effect DNP biradicals (SNAPol-1) optimized for high-field DNP (≥ 18.8T) are employed to offer high sensitivity and resolution suitable for biomolecular NMR applications. By combining these factors, we have successfully established an impressive enhancement factor of ε ~ 50 on amyloid fibrils using an 18.8T/ 800MHz magnet. We have compared the DNP efficiencies of M-TinyPol, NATriPol-3, and SNAPol-1 biradicals on amyloid fibrils. We found that SNAPol-1 (with ε ~ 50) outperformed the other two radicals. The MAS DNP experiments revealed signals of flexible side chains previously inaccessible at conventional room-temperature experiments. These results demonstrate the potential of MAS-DNP NMR as a valuable tool for structural investigations of amyloid fibrils, particularly for side chains and dynamically disordered segments otherwise hidden at room temperature.

Mechanistic origins of methyl-driven Overhauser DNP.

The Overhauser effect in the dynamic nuclear polarization (DNP) of non-conducting solids has drawn much attention due to the potential for efficient high-field DNP as well as a general interest in the underlying principles that enable the Overhauser effect in small molecules. We recently reported the observation of 1H and 2H Overhauser effects in H3C- or D3C-functionalized Blatter radical analogs, which we presumed to be caused by methyl rotation. In this work, we look at the mechanism for methyl-driven Overhauser DNP in greater detail, considering methyl librations and tunneling in addition to classical rotation. We predict the temperature dependence of these mechanisms using density functional theory and spin dynamics simulations. Comparisons with results from ultralow-temperature magic angle spinning-DNP experiments revealed that cross-relaxation at temperatures above 60K originates from both libration and rotation, while librations dominate at lower temperatures. Due to the zero-point vibrational nature of these motions, they are not quenched by very low temperatures, and methyl-driven Overhauser DNP is expected to increase in efficiency down to 0K, predominantly due to increases in nuclear relaxation times.

Triangulating Dopant-Level Mn(II) Insertion in a Cs2NaBiCl6Double Perovskite Using Magnetic Resonance Spectroscopy

Lead-free metal halide double perovskites are gaining increasing attention for optoelectronic applications. Specifically, doping metal halide double perovskites using transition metals enables broadband tailorability of the optical bandgap for these emerging semiconducting materials. One candidate material is Mn(II)-doped Cs2NaBiCl6, but the nature of Mn(II) insertion on chemical structure is poorly understood due to low Mn loading. It is critical to determine the atomic-level structure at the site of Mn(II) incorporation in doped perovskites to better understand the structure-property relationships in these materials and thus to advance their applicability to optoelectronic applications. Magnetic resonance spectroscopy is uniquely qualified to address this, and thus a comprehensive three-pronged strategy, involving solid-state nuclear magnetic resonance (NMR), high-field dynamic nuclear polarization (DNP), and electron paramagnetic resonance (EPR) spectroscopies, is used to identify the location of Mn(II) insertion in Cs2NaBiCl6. Multinuclear (23Na, 35Cl, 133Cs, and 209Bi) one-dimensional (1D) magnetic resonance spectra reveal a low level of Mn(II) incorporation, with select spins affected by paramagnetic relaxation enhancement (PRE) induced by Mn(II) neighbors. EPR measurements confirm the oxidation state, octahedral symmetry, and low doping levels of the Mn(II) centers. Complementary EPR and NMR measurements confirm that the cubic structure is maintained with Mn(II) incorporation at room temperature, but the structure deviates slightly from cubic symmetry at low temperatures (<30 K). HYperfine Sublevel CORrelation (HYSCORE) EPR spectroscopy explores the electron-nuclear correlations of Mn(II) with 23Na, 133Cs, and 35Cl. The absence of 209Bi correlations suggests that Bi centers are replaced by Mn(II). Endogenous DNP NMR measurements from Mn(II) → 133Cs (<30 K) reveal that the solid effect is the dominant mechanism for DNP transfer and supports that Mn(II) is homogeneously distributed within the double-perovskite structure.

PyrroTriPol: a semi-rigid trityl-nitroxide for high field dynamic nuclear polarization.

Magic angle spinning (MAS) dynamic nuclear polarization (DNP) has significantly broadened the scope of solid-state NMR to study biomolecular systems and materials. In recent years, the advent of very high field DNP combined with fast MAS has brought new challenges in the design of polarizing agents (PA) used to enhance nuclear spin polarization. Here, we present a trityl-nitroxide PA family based on a piperazine linker, named PyrroTriPol, for both aqueous and organic solutions. These new radicals have similar properties to that of TEMTriPol-I and can be readily synthesized, and purified in large quantities thereby ensuring widespread application. The family relies on a rigid bridge connecting the trityl and the nitroxide offering a better control of the electron spin-spin interactions thus providing improved performance across a broad range of magnetic fields and MAS frequencies while requiring reduced microwave power compared to bis-nitroxides. We demonstrate the efficiency of the PyrroTriPol family under a magnetic field of 9.4, 14.1 and 18.8 T with respect to TEMTriPol-I. In particular, the superiority of PyrroTriPol was demonstrated on γ-Al2O3 nanoparticles which enabled the acquisition of a high signal-to-noise surface-selective 27Al multiple-quantum MAS experiment at 18.8 T and 40 kHz MAS frequency.

Polarizing agents for efficient high field DNP solid-state NMR spectroscopy under magic-angle spinning: from design principles to formulation strategies.

Dynamic Nuclear Polarization (DNP) has recently emerged as a cornerstone approach to enhance the sensitivity of solid-state NMR spectroscopy under Magic Angle Spinning (MAS), opening unprecedented analytical opportunities in chemistry and biology. DNP relies on a polarization transfer from unpaired electrons (present in endogenous or exogenous polarizing agents) to nearby nuclei. Developing and designing new polarizing sources for DNP solid-state NMR spectroscopy is currently an extremely active research field per se, that has recently led to significant breakthroughs and key achievements, in particular at high magnetic fields. This review describes recent developments in this area, highlighting key design principles that have been established over time and led to the introduction of increasingly more efficient polarizing sources. After a short introduction, Section 2 presents a brief history of solid-state DNP, highlighting the main polarization transfer schemes. The third section is devoted to the development of dinitroxide radicals, discussing the guidelines that were progressively established to design the fine-tuned molecular structures in use today. In Section 4, we describe recent efforts in developing hybrid radicals composed of a narrow EPR line radical covalently linked to a nitroxide, highlighting the parameters that modulate the DNP efficiency of these mixed structures. Section 5 reviews recent advances in the design of metal complexes suitable for DNP MAS NMR as exogenous electron sources. In parallel, current strategies that exploit metal ions as endogenous polarization sources are discussed. Section 6 briefly describes the recent introduction of mixed-valence radicals. In the last part, experimental aspects regarding sample formulation are reviewed to make best use of these polarizing agents in a broad panel of application fields.

Design of a cryogen-free high field dual EPR and DNP probe

We present the design and construction of a cryogen free, dual electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) probe for novel dynamic nuclear polarization (DNP) experiments and concurrent “in situ” analysis of DNP mechanisms. We focus on the probe design that meets the balance between EPR, NMR, and low temperature performance, while maintaining a high degree of versatility: allowing multi-nuclear NMR detection as well as broadband DNP/EPR excitation/detection. To accomplish high NMR/EPR performance, we implement a novel inductively coupled double resonance NMR circuit (1H-13C) in a solid state probe operating at cryogenic temperatures. The components of the circuit were custom built to provide maximum NMR performance, and the physical layout of this circuit was numerically optimized via magnetic field simulations to allow maximum microwave transmission to the sample for optimal EPR performance. Furthermore this probe is based around a cryogen free gas exchange cryostat and has been designed to allow unlimited experiment times down to 8.5 Kelvin with minimal cost. The affordability of EPR/DNP experiment is an extremely important aspect for broader impact with magnetic resonance measurements. The purpose of this article is to provide as complete information as we have available for others with interest in building a dual DNP/EPR instrument based around a cryogen-free cryostat.

Information-Guided Dynamic Nuclear Polarization

A spin ensemble in thermal equilibrium continuously undergoes random fluctuations analogous to those observed in Brownian motion, a process known as spin noise. Here, we investigate the dynamics of a system comprising a spin-1 paramagnetic center and a hyperfine-coupled spin-1/2 nucleus in the vicinity of a level crossing. We theoretically show that nuclear spins polarize efficiently under the combined action of thermal fluctuations and a closed-loop feedback protocol. The latter articulates periodic observations of the electronic magnetization and radio-frequency pulses connecting hybrid states with opposite nuclear spin alignment. Since nuclear polarization emerges from electronic spin fluctuations, not spin order, this microwave-free technique generically benefits from warmer, not colder, operation temperatures. Furthermore, because the spin dynamics at play near a level crossing is rather insensitive to the absolute value of the magnetic field, our work promises opportunities for high-field dynamic nuclear polarization, which is difficult to attain through present methods.

Highly bioresistant, hydrophilic and rigidly linked trityl-nitroxide biradicals for cellular high-field dynamic nuclear polarization.

Cellular dynamic nuclear polarization (DNP) has been an effective means of overcoming the intrinsic sensitivity limitations of solid-state nuclear magnetic resonance (ssNMR) spectroscopy, thus enabling atomic-level biomolecular characterization in native environments. Achieving DNP signal enhancement relies on doping biological preparations with biradical polarizing agents (PAs). Unfortunately, PA performance within cells is often limited by their sensitivity to the reductive nature of the cellular lumen. Herein, we report the synthesis and characterization of a highly bioresistant and hydrophilic PA (StaPol-1) comprising the trityl radical OX063 ligated to a gem-diethyl pyrroline nitroxide via a rigid piperazine linker. EPR experiments in the presence of reducing agents such as ascorbate and in HeLa cell lysates demonstrate the reduction resistance of StaPol-1. High DNP enhancements seen in small molecules, proteins and cell lysates at 18.8 T confirm that StaPol-1 is an excellent PA for DNP ssNMR investigations of biomolecular systems at high magnetic fields in reductive environments.

Racing toward Fast and Effective 17O Isotopic Labeling and Nuclear Magnetic Resonance Spectroscopy of N-Formyl-MLF-OH and Associated Building Blocks.

Solid-state 1H, 13C, and 15N nuclear magnetic resonance (NMR) spectroscopy has been an essential analytical method in studying complex molecules and biomolecules for decades. While oxygen-17 (17O) NMR is an ideal and robust candidate to study hydrogen bonding within secondary and tertiary protein structures for example, it continues to elude many. We discuss an improved multiple-turnover labeling procedure to develop a fast and cost-effective method to 17O label fluoroenylmethyloxycarbonyl (Fmoc)-protected amino acid building blocks. This approach allows for inexpensive ($0.25 USD/mg) insertion of 17O labels, an important barrier to overcome for future biomolecular studies. The 17O NMR results of these building blocks and a site-specific strategy for labeled N-acetyl-MLF-OH and N-formyl-MLF-OH tripeptides are presented. We showcase growth in NMR development for maximizing sensitivity gains using emerging sensitivity enhancement techniques including population transfer, high-field dynamic nuclear polarization, and cross-polarization magic-angle spinning cryoprobes.

Structural Analysis of an Antigen Chemically Coupled on Virus-Like Particles in Vaccine Formulation.

Structure determination of adjuvant-coupled antigens is essential for rational vaccine development but has so far been hampered by the relatively low antigen content in vaccine formulations and by their heterogeneous composition. Here we show that magic-angle spinning (MAS) solid-state NMR can be used to assess the structure of the influenza virus hemagglutinin stalk long alpha helix antigen, both in its free, unformulated form and once chemically coupled to the surface of large virus-like particles (VLPs). The sensitivity boost provided by high-field dynamic nuclear polarization (DNP) and proton detection at fast MAS rates allows to overcome the penalty associated with the antigen dilution. Comparison of the MAS NMR fingerprints between the free and VLP-coupled forms of the antigen provides structural evidence of the conservation of its native fold upon bioconjugation. This work demonstrates that high-sensitivity MAS NMR is ripe to play a major role in vaccine design, formulation studies, and manufacturing process development.

Solvent-free dynamic nuclear polarization enhancements in organically modified mesoporous silica.

High-field dynamic nuclear polarization is a powerful tool for the structural characterization of species on the surface of porous materials or nanoparticles. For these studies the main source of polarization are radical-containing solutions which are added by post-synthesis impregnation of the sample. Although this strategy is very efficient for a wide variety of materials, the presence of the solvent may influence the chemistry of functional species of interest. Here we address the development of a comprehensive strategy for solvent-free DNP enhanced NMR characterization of functional (target) species on the surface of mesoporous silica (SBA-15). The strategy includes the partial functionalization of the silica surface with Carboxy-Proxyl nitroxide radicals and target Fmoc-Glycine functional groups. As a proof of principle, we have observed for the first time DNP signal enhancements, using the solvent-free approach, for 13C{1H} CPMAS signals corresponding to organic functionalities on the silica surface. DNP enhancements of up to 3.4 were observed for 13C{1H} CPMAS, corresponding to an experimental time save of about 12 times. This observation opens the possibility for the DNP-NMR study of surface functional groups without the need of a solvent, allowing, for example, the characterization of catalytic reactions occurring on the surface of mesoporous systems of interest. For 29Si with direct polarization NMR, up to 8-fold DNP enhancements were obtained. This 29Si signal enhancement is considerably higher than the obtained with similar approaches reported in literature. Finally, from DNP enhancement profiles we conclude that cross-effect is probably the dominant polarization transfer mechanism.

Dynamic Nuclear Polarization with Vanadium(IV) Metal Centers

Summary Dynamic nuclear polarization (DNP) harnesses the large polarization of electron spins to dramatically increase nuclear magnetic resonance (NMR) sensitivity. This study expands the scope of DNP beyond its traditional focus on hyperpolarizing the solvent network using exogenous polarizing agents (PAs). We introduce 1H DNP with endogenous V4+ centers positioned in a set of vanadyl complexes with tunable V4+-1H distances. We traced the polarization transfer from V4+ to 1H spins, specifically differentiating between direct V4+-1Hs polarization transfer and the 1H spin-diffusion-mediated bulk solvent 1H polarization buildup and illuminated the effect of the V4+-1H distance on these processes. These results deepen our understanding of polarization pathways and expand the catalog of PAs to broad-line transition metals. This study establishes crucial first steps toward employing strategically positioned endogenous paramagnetic metal centers for DNP and the conceptual framework of hyperfine DNP spectroscopy that merges both spatial and chemical diagnosis of target nuclear spins.

A Factor Two Improvement in High-Field Dynamic Nuclear Polarization from Gd(III) Complexes by Design

Gadolinium(III) complexes have recently been demonstrated to have potential as polarizing agents for high-field dynamic nuclear polarization (DNP) NMR spectroscopy. By tailoring the ligand design to reduce the zero-field splitting (ZFS), we demonstrate a quadratic improvement in DNP through the investigation of a stable, water-soluble, narrow-line Gd(III) complex, [Gd(tpatcn)], doubling the magic-angle-spinning DNP enhancement of the previous state-of-the-art [Gd(dota)(H2O)]- at 9.4 T and 100 K.

A Factor Two Improvement in High-Field Dynamic Nuclear Polarization from Gd(III) Complexes by Design

A Factor Two Improvement in High-Field Dynamic Nuclear Polarization from Gd(III) Complexes by Design

Compact radiation module for THz spectroscopy using 300 GHz continuous-wave clinotron.

The results of the development of compact radiation module based on a 300 GHz continuous-wave (CW) clinotron are presented. The clinotron oscillator is proposed as a part of the module designated for high-field dynamic nuclear polarization (DNP) systems for applications in nuclear magnetic resonance (NMR) spectroscopy. The simulation results of clinotron radiation spectra considering the influence of accelerating voltage pulsations are compared with the requirements for THz radiation linewidth for efficient NMR signal enhancement. Based on the simulations, the 300 GHz CW clinotron oscillator was developed and tested together with the high-voltage (HV) power supply, providing the output voltage stability better than 20 ppm. The frequency stability of 33 ppm was observed during the clinotron operation within several hours. The spectral linewidth is better than 8 MHz at 300 GHz that satisfies the requirements for DNP-NMR spectroscopy.

Absolute 1H polarization measurement with a spin-correlated component of magnetization by hyperpolarized MAS-DNP solid-state NMR

Sensitivity of magic-angle spinning (MAS) NMR spectroscopy has been dramatically improved by the advent of high-field dynamic nuclear polarization (DNP) technique and its rapid advances over the past decades. In this course, discussions on ways to improve the DNP enhancement factor or the overall sensitivity gain have been numerous, and led to a number of methodological and instrumental breakthroughs. Beyond the sensitivity gain, however, discussions on accurate quantification of the 1H polarization amplitude achievable in a sample with DNP have been relatively rare. Here, we propose a new method for quantifying the local 1H hyperpolarization amplitude, which is applicable to un-oriented/powdered solid samples under MAS NMR conditions. The method is based on the ability to observe the high-order spin-correlated term (2IzSz) intrinsic to a hyperpolarized IS two-spin state, separately from the lowest-order Zeeman term (Sz) in quasi-equilibrium magnetization. The quantification procedure does not require evaluation of signal amplitudes for a “microwave-off” condition and for an un-doped reference sample, and thus enables quick and accurate quantification unaffected by the effects of the paramagnetic quenching and the MAS-induced depolarization. The method is also shown to elucidate spatial polarization distribution through the 2IzSz term prepared domain-selectively. As a potential application, we also demonstrate 2D DQ-SQ spectroscopy utilizing the 2IzSz term that is generated in a spatially selective manner without using IS dipolar or J coupling. These salient features may be evolved into a way for characterizing mesoscopic molecular assemblies of medical/biological importance.