AsymPol-TEKs as efficient polarizing agents for MAS-DNP in glass matrices of non-aqueous solvents.

Two polarizing agents from the AsymPol family, AsymPol-TEK and cAsymPol-TEK (methyl-free version) are introduced for MAS-DNP applications in non-aqueous solvents. The performance of these new biradicals is rationalized in detail using a combination of electron paramagnetic resonance spectroscopy, density functional theory, molecular dynamics and quantitative MAS-DNP spin dynamics simulations. By slightly modifying the experimental protocol to keep the sample temperature low at insertion, we are able to obtain reproducable DNP-NMR data with 1,1,2,2-tetrachloroethane (TCE) at 100 K, which facilitates optimization and comparison of different polarizing agents. At intermediate magnetic fields, AsymPol-TEK and cAsymPol-TEK provide 1.5 to 3-fold improvement in sensitivity compared to TEKPol, one of the most widely used polarizing agents for organic solvents, with significantly shorter DNP build-up times of ∼1 s and ∼2 s at 9.4 and 14.1 T respectively. In the course of the work, we also isolated and characterized two diastereoisomers that can form during the synthesis of AsymPol-TEK; their difference in performance is described and discussed. Finally, the advantages of the AsymPol-TEKs are demonstrated by recording 2D 13C-13C correlation experiments at natural 13C-abundance of proton-dense microcrystals and by polarizing the surface of ZnO nanocrystals (NCs) coated with diphenyl phosphate ligands. For those experiments, cAsymPol-TEK yielded a three-fold increase in sensitivity compared to TEKPol, corresponding to a nine-fold time saving.

Sustainable and cost-effective MAS DNP-NMR at 30 K with cryogenic sample exchange.

We report here instrumental developments to achieve sustainable, cost-effective cryogenic Helium sample spinning in order to conduct dynamic nuclear polarisation (DNP) and solid-state NMR (ssNMR) at ultra-low temperatures (<30 K). More specifically, we describe an efficient closed-loop helium system composed of a powerful heat exchanger (95% efficient), a single cryocooler, and a single helium compressor to power the sample spinning and cooling. The system is integrated with a newly designed triple-channel NMR probe that minimizes thermal losses without compromising the radio frequency (RF) performance and spinning stability (±0.05%). The probe is equipped with an innovative cryogenic sample exchange system that allows swapping samples in minutes without introducing impurities in the closeloop system. We report that significant gain in sensitivity can be obtained at 30-40 K on large micro-crystalline molecules with unfavorable relaxation timescales, making them difficult or impossible to polarize at 100 K. We also report rotor-synchronized 2D experiments to demonstrate the stability of the system.

Solvent-derived defects suppress adsorption in MOF-74.

Defects in metal-organic frameworks (MOFs) have great impact on their nano-scale structure and physiochemical properties. However, isolated defects are easily concealed when the frameworks are interrogated by typical characterization methods. In this work, we unveil the presence of solvent-derived formate defects in MOF-74, an important class of MOFs with open metal sites. With multi-dimensional solid-state nuclear magnetic resonance (NMR) investigations, we uncover the ligand substitution role of formate and its chemical origin from decomposed N,N-dimethylformamide (DMF) solvent. The placement and coordination structure of formate defects are determined by 13C NMR and density functional theory (DFT) calculations. The extra metal-oxygen bonds with formates partially eliminate open metal sites and lead to a quantitative decrease of N2 and CO2 adsorption with respect to the defect concentration. In-situ NMR analysis and molecular simulations of CO2 dynamics elaborate the adsorption mechanisms in defective MOF-74. Our study establishes comprehensive strategies to search, elucidate and manipulate defects in MOFs.

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.

Optimizing chemistry at the surface of prodrug-loaded cellulose nanofibrils with MAS-DNP.

Studying the surface chemistry of functionalized cellulose nanofibrils at atomic scale is an ongoing challenge, mainly because FT-IR, NMR, XPS and RAMAN spectroscopy are limited in sensitivity or resolution. Herein, we show that dynamic nuclear polarization (DNP) enhanced 13C and 15N solid-state NMR is a uniquely suited technique to optimize the drug loading on nanocellulose using aqueous heterogenous chemistry. We compare the efficiency of two conventional coupling agents (DMTMM vs EDC/NHS) to bind a complex prodrug of ciprofloxacin designed for controlled drug release. Besides quantifying the drug grafting, we also evidence the challenge to control the concurrent prodrug adsorption and to optimize washing procedures. We notably highlight the presence of an unexpected prodrug cleavage mechanism triggered by carboxylates at the surface of the cellulose nanofibrils.

Fast magic angle spinning for the characterization of milligram quantities of organic and biological solids at natural isotopic abundance by 13C-13C correlation DNP-enhanced NMR.

We show that multidimensional solid-state NMR 13C-13C 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. This approach is demonstrated on diverse sample types, including polyglutamine fibrils implicated in Huntington's disease and microcrystalline ampicillin, a small antibiotic molecule.

Biomolecular and Biological Applications of Solid-State NMR with Dynamic Nuclear Polarization Enhancement.

Solid-state NMR spectroscopy (ssNMR) with magic-angle spinning (MAS) enables the investigation of biological systems within their native context, such as lipid membranes, viral capsid assemblies, and cells. However, such ambitious investigations often suffer from low sensitivity due to the presence of significant amounts of other molecular species, which reduces the effective concentration of the biomolecule or interaction of interest. Certain investigations requiring the detection of very low concentration species remain unfeasible even with increasing experimental time for signal averaging. By applying dynamic nuclear polarization (DNP) to overcome the sensitivity challenge, the experimental time required can be reduced by orders of magnitude, broadening the feasible scope of applications for biological solid-state NMR. In this review, we outline strategies commonly adopted for biological applications of DNP, indicate ongoing challenges, and present a comprehensive overview of biological investigations where MAS-DNP has led to unique insights.

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 12.76 kDa U-13 C,15 N-labeled LecA protein and pharmaceutical drugs at high magnetic fields (up to 18.8 T) and fast magic angle spinning (MAS) frequencies (up to 40 kHz). The performance of cAsymPol-POK is rationalized by MAS-DNP simulations combined with electron paramagnetic resonance (EPR), density functional theory (DFT) and molecular dynamics (MD). This work shows that this new biradical is compatible with challenging biomolecular applications and unlocks the rapid acquisition of 13 C-13 C and 15 N-13 C correlations of pharmaceutical drugs at natural isotopic abundance, which are key experiments for structure determination.

Targeted DNP for biomolecular solid-state NMR.

High-field dynamic nuclear polarization is revolutionizing the scope of solid-state NMR with new applications in surface chemistry, materials science and structural biology. In this perspective article, we focus on a specific DNP approach, called targeted DNP, in which the paramagnets introduced to polarize are not uniformly distributed in the sample but site-specifically located on the biomolecular system. After reviewing the various targeting strategies reported to date, including a bio-orthogonal chemistry-based approach, we discuss the potential of targeted DNP to improve the overall NMR sensitivity while avoiding the use of glass-forming DNP matrix. This is especially relevant to the study of diluted biomolecular systems such as, for instance, membrane proteins within their lipidic environment. We also discuss routes towards extracting structural information from paramagnetic relaxation enhancement (PRE) induced by targeted DNP at cryogenic temperature, and the possibility to recover site-specific information in the vicinity of the paramagnetic moieties using high-resolution selective DNP spectra. Finally, we review the potential of targeted DNP for in-cell NMR studies and how it can be used to extract a given protein NMR signal from a complex cellular background.

Correction: De novo prediction of cross-effect efficiency for magic angle spinning dynamic nuclear polarization.

Correction for 'De novo prediction of cross-effect efficiency for magic angle spinning dynamic nuclear polarization' by Frédéric Mentink-Vigier et al., Phys. Chem. Chem. Phys., 2019, 21, 2166-2176, DOI: 10.1039/C8CP06819D.

On the use of radio-frequency offsets for improving double-quantum homonuclear dipolar recoupling of half-integer-spin quadrupolar nuclei.

Detecting proximities between nuclei is crucial for atomic-scale structure determination with nuclear magnetic resonance (NMR) spectroscopy. Different from spin-1/2 nuclei, the methodology for quadrupolar nuclei is limited for solids due to the complex spin dynamics under simultaneous magic-angle spinning (MAS) and radio-frequency irradiation. Herein, the performances of several homonuclear rotary recoupling (HORROR)-based homonuclear dipolar recoupling sequences are evaluated for 27 Al (spin-5/2). It is shown numerically and experimentally on mesoporous alumina that BR 2 2 1 outperforms the supercycled S3 sequence and its pure double-quantum (DQ) (bracketed) version, [S3 ], both in terms of DQ transfer efficiency and bandwidth. This result is surprising since the S3 sequence is among the best low-power recoupling schemes for spin-1/2. The superiority of BR 2 2 1 is thoroughly explained, and the crucial role of radio-frequency offsets during its spin dynamics is highlighted. The analytical approximation of BR 2 2 1 , derived in an offset-toggling frame, clarifies the interplay between offset and DQ efficiency, namely, the benefits of off-resonance irradiation and the trough in DQ efficiency for BR 2 2 1 when the irradiation is central between two resonances, both for spin-1/2 and half-integer-spin quadrupolar nuclei. Additionally, density matrix propagations show that the BR 2 2 1 sequence, applied to quadrupolar nuclei subject to quadrupolar interaction much larger than radio-frequency frequency field, can create single- and multiple-quantum coherences for near on-resonance irradiation. This significantly perturbs the creation of DQ coherences between central transitions of neighboring quadrupolar nuclei. This effect explains the DQ efficiency trough for near on-resonance irradiation, in the case of both cross-correlation and autocorrelation peaks. Overall, this work aids experimental acquisition of homonuclear dipolar correlation spectra of half-integer-spin quadrupolar nuclei and provides theoretical insights towards improving recoupling schemes at high magnetic field and fast MAS.

The surface chemistry of a nanocellulose drug carrier unravelled by MAS-DNP.

Cellulose nanofibrils (CNF) are renewable bio-based materials with high specific area, which makes them ideal candidates for multiple emerging applications including for instance on-demand drug release. However, in-depth chemical and structural characterization of the CNF surface chemistry is still an open challenge, especially for low weight percentage of functionalization. This currently prevents the development of efficient, cost-effective and reproducible green synthetic routes and thus the widespread development of targeted and responsive drug-delivery CNF carriers. We show in this work how we use dynamic nuclear polarization (DNP) to overcome the sensitivity limitation of conventional solid-state NMR and gain insight into the surface chemistry of drug-functionalized TEMPO-oxidized cellulose nanofibrils. The DNP enhanced-NMR data can report unambiguously on the presence of trace amounts of TEMPO moieties and depolymerized cellulosic units in the starting material, as well as coupling agents on the CNFs surface (used in the heterogeneous reaction). This enables a precise estimation of the drug loading while differentiating adsorption from covalent bonding (∼1 wt% in our case) as opposed to other analytical techniques such as elemental analysis and conductometric titration that can neither detect the presence of coupling agents, nor differentiate unambiguously between adsorption and grafting. The approach, which does not rely on the use of 13C/15N enriched compounds, will be key to further develop efficient surface chemistry routes and has direct implication for the development of drug delivery applications both in terms of safety and dosage.

Disclosing Interfaces of ZnO Nanocrystals Using Dynamic Nuclear Polarization: Sol-Gel versus Organometallic Approach.

The unambiguous characterization of the coordination chemistry of nanocrystal surfaces produced by wet-chemical synthesis presently remains highly challenging. Here, zinc oxide nanocrystals (ZnO NCs) coated by monoanionic diphenyl phosphate (DPP) ligands were derived by a sol-gel process and a one-pot self-supporting organometallic (OSSOM) procedure. Atomic-scale characterization through dynamic nuclear polarization (DNP-)enhanced solid-state NMR (ssNMR) spectroscopy has notably enabled resolving their vastly different surface-ligand interfaces. For the OSSOM-derived NCs, DPP moieties form stable and strongly-anchored µ2 - and µ3 -bridging-ligand pairs that are resistant to competitive ligand exchange. The sol-gel-derived NCs contain a wide variety of coordination modes of DPP ligands and a ligand exchange process takes place between DPP and glycerol molecules. This highlights the power of DNP-enhanced ssNMR for detailed NC surface analysis and of the OSSOM approach for the preparation of ZnO NCs.

Natural Isotopic Abundance 13C and 15N Multidimensional Solid-State NMR Enabled by Dynamic Nuclear Polarization.

Dynamic nuclear polarization (DNP) has made feasible solid-state NMR experiments that were previously thought impractical due to sensitivity limitations. One such class of experiments is the structural characterization of organic and biological samples at natural isotopic abundance (NA). Herein, we describe the many advantages of DNP-enabled ssNMR at NA, including the extraction of long-range distance constraints using dipolar recoupling pulse sequences without the deleterious effects of dipolar truncation. In addition to the theoretical underpinnings in the analysis of these types of experiments, numerous applications of DNP-enabled ssNMR at NA are discussed.

Detection of the Surface of Crystalline Y2O3 Using Direct 89Y Dynamic Nuclear Polarization.

Nuclei with low gyromagnetic ratio (γ) present a serious sensitivity challenge for nulear magnetic resonance (NMR) spectroscopy. Recently, dynamic nuclear polarization (DNP) has shown great promise in overcoming this hurdle by indirect hyperpolarization (via 1H) of these low-γ nuclei. Here we show that at a magnetic field of 9.4 T and cryogenic temperature of about 110 K direct DNP of 89Y in a frozen solution of Y(NO3)3 can offer signal enhancements greater than 80 times using exogeneous trityl OX063 monoradical by satisfying the cross effect magic angle spinning (MAS) DNP mechanism. The large signal enhancement achieved permits 89Y NMR spectra of Y2O3 and Gd2O3-added Y2O3 materials to be obtained quickly (∼30 min), revealing a range of surface yttrium hydroxyl groups in addition to the two octahedral yttrium signals of the core. The results open up promises for the observation of low gyromagnetic ratio nuclei and the detection of corresponding surface and (sub-)surface sites.

Selective high-resolution DNP-enhanced NMR of biomolecular binding sites.

Locating binding sites in biomolecular assemblies and solving their structures are of the utmost importance to unravel functional aspects of the system and provide experimental data that can be used for structure-based drug design. This often still remains a challenge, both in terms of selectivity and sensitivity for X-ray crystallography, cryo-electron microscopy and NMR. In this work, we introduce a novel method called Selective Dynamic Nuclear Polarization (Sel-DNP) that allows selective highlighting and identification of residues present in the binding site. This powerful site-directed approach relies on the use of localized paramagnetic relaxation enhancement induced by a ligand-functionalized paramagnetic construct combined with difference spectroscopy to recover high-resolution and high-sensitivity information from binding sites. The identification of residues involved in the binding is performed using spectral fingerprints obtained from a set of high-resolution multidimensional spectra with varying selectivities. The methodology is demonstrated on the galactophilic lectin LecA, for which we report well-resolved DNP-enhanced spectra with linewidths between 0.5 and 1 ppm, which enable the de novo assignment of the binding interface residues, without using previous knowledge of the binding site location. Since this approach produces clean and resolved difference spectra containing a limited number of residues, resonance assignment can be performed without any limitation with respect to the size of the biomolecular system and only requires the production of one protein sample (e.g. 13C,15N-labeled protein).

De novo prediction of cross-effect efficiency for magic angle spinning dynamic nuclear polarization.

Magic angle spinning dynamic nuclear polarization (MAS-DNP) has become a key approach to boost the intrinsic low sensitivity of NMR in solids. This method relies on the use of both stable radicals as polarizing agents (PAs) and suitable high frequency microwave irradiation to hyperpolarize nuclei of interest. Relating PA chemical structure to DNP efficiency has been, and is still, a long-standing problem. The complexity of the polarization transfer mechanism has so far limited the impact of analytical derivation. However, recent numerical approaches have profoundly improved the basic understanding of the phenomenon and have now evolved to a point where they can be used to help design new PAs. In this work, the potential of advanced MAS-DNP simulations combined with DFT calculations and high-field EPR to qualitatively and quantitatively predict hyperpolarization efficiency of particular PAs is analyzed. This approach is demonstrated on AMUPol and TEKPol, two widely-used bis-nitroxide PAs. The results notably highlight how the PA structure and EPR characteristics affect the detailed shape of the DNP field profile. We also show that refined simulations of this profile using the orientation dependency of the electron spin-lattice relaxation times can be used to estimate the microwave B1 field experienced by the sample. Finally, we show how modelling the nuclear spin-lattice relaxation times of close and bulk nuclei while accounting for PA concentration allows for a prediction of DNP enhancement factors and hyperpolarization build-up times.

Sparsely Pillared Graphene Materials for High-Performance Supercapacitors: Improving Ion Transport and Storage Capacity.

Graphene-based materials are extensively studied as promising candidates for supercapacitors (SCs) owing to the high surface area, electrical conductivity, and mechanical flexibility of graphene. Reduced graphene oxide (RGO), a close graphene-like material studied for SCs, offers limited specific capacitances (100 F·g-1) as the reduced graphene sheets partially restack through π-π interactions. This paper presents pillared graphene materials designed to minimize such graphitic restacking by cross-linking the graphene sheets with a bifunctional pillar molecule. Solid-state NMR, X-ray diffraction, and electrochemical analyses reveal that the synthesized materials possess covalently cross-linked graphene galleries that offer additional sites for ion sorption in SCs. Indeed, high specific capacitances in SCs are observed for the graphene materials synthesized with an optimized number of pillars. Specifically, the straightforward synthesis of a graphene hydrogel containing pillared structures and an interconnected porous network delivered a material with gravimetric capacitances two times greater than that of RGO (200 F·g-1 vs 107 F·g-1) and volumetric capacitances that are nearly four times larger (210 F·cm-3 vs 54 F·cm-3). Additionally, despite the presence of pillars inside the graphene galleries, the optimized materials show efficient ion transport characteristics. This work therefore brings perspectives for the next generation of high-performance SCs.

Structural Fingerprinting of Protein Aggregates by Dynamic Nuclear Polarization-Enhanced Solid-State NMR at Natural Isotopic Abundance.

A pathological hallmark of Huntington's disease (HD) is the formation of neuronal protein deposits containing mutant huntingtin fragments with expanded polyglutamine (polyQ) domains. Prior studies have shown the strengths of solid-state NMR (ssNMR) to probe the atomic structure of such aggregates, but have required in vitro isotopic labeling. Herein, we present an approach for the structural fingerprinting of fibrils through ssNMR at natural isotopic abundance (NA). These methods will enable the spectroscopic fingerprinting of unlabeled (e.g., ex vivo) protein aggregates and the extraction of valuable new long-range 13C-13C distance constraints.

Computationally Assisted Design of Polarizing Agents for Dynamic Nuclear Polarization Enhanced NMR: The AsymPol Family.

We introduce a new family of highly efficient polarizing agents for dynamic nuclear polarization (DNP)-enhanced nuclear magnetic resonance (NMR) applications, composed of asymmetric bis-nitroxides, in which a piperidine-based radical and a pyrrolinoxyl or a proxyl radical are linked together. The design of the AsymPol family was guided by the use of advanced simulations that allow computation of the impact of the radical structure on DNP efficiency. These simulations suggested the use of a relatively short linker with the intention to generate a sizable intramolecular electron dipolar coupling/ J-exchange interaction, while avoiding parallel nitroxide orientations. The characteristics of AsymPol were further tuned, for instance with the addition of a conjugated carbon-carbon double bond in the 5-membered ring to improve the rigidity and provide a favorable relative orientation, the replacement of methyls by spirocyclohexanolyl groups to slow the electron spin relaxation, and the introduction of phosphate groups to yield highly water-soluble dopants. An in-depth experimental and theoretical study for two members of the family, AsymPol and AsymPolPOK, is presented here. We report substantial sensitivity gains at both 9.4 and 18.8 T. The robust efficiency of this new family is further demonstrated through high-resolution surface characterization of an important industrial catalyst using fast sample spinning at 18.8 T. This work highlights a new direction for polarizing agent design and the critical importance of computations in this process.