Stressing the differences in alizarin and purpurin dyes through UV-visible light absorption and 1H-NMR spectroscopies.

Three anthraquinone-based chromophores (9,10-anthraquinone, alizarin, purpurin) are compared from the point of view of their experimental and computed NMR and UV-visible light absorption spectra. Using a hybrid (explicit/implicit) solvent model, each proton chemical shift can be reproduced with an error of less than 7%, even when such protons are engaged in inter-molecular hydrogen bonds with the solvent or when the analyzed sample contains a significant amount of impurities, for instance, 9,10-anthraquinone in purpurin. All the steady-state UV-visible absorption spectra feature a significant vibrational progression in the first absorption band. The shape of the corresponding computed spectra, including vibronic couplings obtained with the adiabatic Hessian approach and the Franck-Condon and Herzberg-Teller approximation of the transition dipole, are in excellent agreement with the experimental ones. The importance and the nature of the vibronic couplings are different for the three molecules, even if they only differ by the number of hydroxyl groups.

Investigating the efficiency of silica materials with wall-embedded nitroxide radicals for dynamic nuclear polarisation NMR.

Dynamic nuclear polarisation (DNP) can significantly enhance the sensitivity of solid-state nuclear magnetic resonance (SSNMR) experiments by transferring the electron spin polarisation of paramagnetic species to nuclei through microwave irradiation of the sample at cryogenic temperatures. Paramagnetic species required for DNP can be provided in the form of mesoporous silica materials containing nitroxide radicals either located on the porous surface or embedded in the pore walls. The present study focuses specifically on porous materials with wall-embedded radicals that were synthesised using conventional molecular imprinting protocols. More remarkably, by changing the molecular structure of the TEMPO precursor, the theoretical distance between the oxygen atoms in a pair of wall-embedded face-to-face TEMPO radicals was increased stepwise (0.7, 0.9, 1.1, 1.3 and 1.5 nm). The thermal activation of these five series of materials led to 37 TEMPO-functionalised silica materials with different radical concentrations. Their efficiency as DNP polarising agents was subsequently investigated at 9.4 T and ∼110 K under magic-angle spinning conditions (10 kHz) after impregnating them at room temperature with an aqueous solution of isotopically enriched proline. Our results show that the highest DNP efficiency was obtained for the silica materials that exhibited the shortest theoretical oxygen-oxygen distance between the TEMPO rings, suggesting that the design rules accepted for soluble DNP polarising agents may not be transposed to these materials with wall-embedded pairs of nitroxides.

Monitoring the influence of additives on the crystallization processes of glycine with dynamic nuclear polarization solid-state NMR.

Crystallization is fundamental in many domains, and the investigation of the sequence of solid phases produced as a function of crystallization time is thus key to understand and control crystallization processes. Here, we used a solid-state nuclear magnetic resonance strategy to monitor the crystallization process of glycine, which is a model compound in polymorphism, under the influence of crystallizing additives, such as methanol or sodium chloride. More specifically, our strategy is based on a combination of low-temperatures and dynamic nuclear polarization (DNP) to trap and detect transient crystallizing forms, which may be present only in low quantities. Interestingly, our results show that these additives yield valuable DNP signal enhancements even in the absence of glycerol within the crystallizing solution.

Supercritical CO2 impregnation process applied to polymer samples preparation for dynamic nuclear polarization solid-state NMR.

In this study, supercritical CO2 (scCO2 ) was used to impregnate polymers with paramagnetic polarizing agents to prepare samples for dynamic nuclear polarization (DNP) solid-state NMR (ssNMR) experiments. As a proof of concept, we impregnated polystyrene samples with bTbK, which stands for bis-TEMPO-bisketal where TEMPO is 2,2,6,6-tetra-methylpiperindin-1-oxyl. Substantial DNP signal enhancements could be measured on DNP-enhanced 1 H → 13 C cross-polarization (CP) magic-angle spinning (MAS) spectra recorded at 9.4 T and ~100 K, reaching a maximum value of 8 in the most favorable case, which appeared comparable or even higher than what is typically obtained on similar systems for former sample preparation methods. These results highlight the potential of scCO2 impregnation as an efficient and possibly versatile methodology to prepare polymer samples for DNP ssNMR investigations.

Monitoring Crystallization Processes in Confined Porous Materials by Dynamic Nuclear Polarization Solid-State Nuclear Magnetic Resonance.

Establishing mechanistic understanding of crystallization processes at the molecular level is challenging, as it requires both the detection of transient solid phases and monitoring the evolution of both liquid and solid phases as a function of time. Here, we demonstrate the application of dynamic nuclear polarization (DNP) enhanced NMR spectroscopy to study crystallization under nanoscopic confinement, revealing a viable approach to interrogate different stages of crystallization processes. We focus on crystallization of glycine within the nanometric pores (7-8 nm) of a tailored mesoporous SBA-15 silica material with wall-embedded TEMPO radicals. The results show that the early stages of crystallization, characterized by the transition from the solution phase to the first crystalline phase, are straightforwardly observed using this experimental strategy. Importantly, the NMR sensitivity enhancement provided by DNP allows the detection of intermediate phases that would not be observable using standard solid-state NMR experiments. Our results also show that the metastable ß polymorph of glycine, which has only transient existence under bulk crystallization conditions, remains trapped within the pores of the mesoporous SBA-15 silica material for more than 200 days.

Solvent suppression in solid-state DNP NMR using Electronic Mixing-Mediated Annihilation (EMMA).

We show here that the Electronic Mixing-Mediated Annihilation (EMMA) method, previously reported for the suppression of background signals in solid-state nuclear magnetic resonance spectra, can be successfully applied to remove the solvent signals observed in the case of nuclear magnetic resonance spectra obtained with dynamic nuclear polarization. The methodology presented here is applied to two standard sample preparation methods for dynamic nuclear polarization, namely, glass forming and incipient wetness impregnation. It is demonstrated that the Electronic Mixing-Mediated Annihilation method is complementary to the different methods for solvent suppression based on relaxation filters and that it can be used to preserve the quantitative information that might be present in the pristine spectra.

Brute-force solvent suppression for DNP studies of powders at natural isotopic abundance.

A method based on highly concentrated radical solutions is investigated for the suppression of the NMR signals arising from solvents that are usually used for dynamic nuclear polarization experiments. The presented method is suitable in the case of powders, which are impregnated with a radical-containing solution. It is also demonstrated that the intensity and the resolution of the signals due to the sample of interest is not affected by the high concentration of radicals. The method proposed here is therefore valuable when sensitivity is of the utmost importance, namely samples at natural isotopic abundance.

Reducing the computational cost of NMR crystallography of organic powders at natural isotopic abundance with the help of 13 C-13 C dipolar couplings.

Structure determination of functional organic compounds remains a formidable challenge when the sample exists as a powder. Nuclear magnetic resonance crystallography approaches based on the comparison of experimental and Density Functional Theory (DFT)-computed 1 H chemical shifts have already demonstrated great potential for structure determination of organic powders, but limitations still persist. In this study, we discuss the possibility of using 13 C-13 C dipolar couplings quantified on powdered theophylline at natural isotopic abundance with the help of dynamic nuclear polarization, to realize a DFT-free, rapid screening of a pool of structures predicted by ab initio random structure search. We show that although 13 C-13 C dipolar couplings can identify structures possessing long range structural motifs and unit cell parameters close to those of the true structure, it must be complemented with other data to recover information about the presence and the chemical nature of the supramolecular interactions.

A Karplus Equation for the Conformational Analysis of Organic Molecular Crystals.

Vicinal scalar couplings (3 J) are extensively used for the conformational analysis of organic compounds in the liquid state through empirical Karplus equations. In contrast, there are no examples of such use for the structural investigation of solids. With the support of first principles calculations, we demonstrate here that 13 C-13 C 3 J coupling constants (3 JCC ) measured on a series of isotopically enriched solid amino acids and sugars can be related to dihedral angles by a simple Karplus-like relationship, and we provide a parameterized Karplus function for the conformational analysis of organic molecular crystals. Under the experimental conditions discussed, torsional angles can be estimated from the experimental 3 JCC values with an accuracy of 10° using this function. These results open new perspectives towards the use of 3 JCC as a new analytical tool that could considerably simplify structure determination of functional organic solids.

Insights into the Crystallization and Structural Evolution of Glycine Dihydrate by In†Situ Solid-State NMR Spectroscopy.

In situ solid-state NMR spectroscopy is exploited to monitor the structural evolution of a glycine/water glass phase formed on flash cooling an aqueous solution of glycine, with a range of modern solid-state NMR methods applied to elucidate structural properties of the solid phases present. The glycine/water glass is shown to crystallize into an intermediate phase, which then transforms to the ß polymorph of glycine. Our in situ NMR results fully corroborate the identity of the intermediate crystalline phase as glycine dihydrate, which was first proposed only very recently.

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). Crucial to the success of DNP SSNMR is the DNP signal enhancement (εDNP ), which is the ratio of the NMR signal intensities with and without DNP. It is shown here that, for polymers exhibiting high affinity toward molecular oxygen (e.g., polystyrene), removing part of the absorbed (paramagnetic) oxygen from the solid-state samples available as powders (instead of dissolved or dispersed in a solvent) increases proton nuclear relaxation times and εDNP, hereby providing up to a two-fold sensitivity increase (i.e., a four-fold reduction in experimental time).

Probing crystal packing of uniformly (13)C-enriched powder samples using homonuclear dipolar coupling measurements.

The relationship between the crystal packing of powder samples and long-range (13)C-(13)C homonuclear dipolar couplings is presented and illustrated for the case of uniformly (13)C-enriched L-alanine and L-histidine·HCl·H2O. Dipolar coupling measurement is based on the partial reintroduction of dipolar interactions by spinning the sample slightly off-magic-angle, while the coupling of interest for a given spin pair is isolated with a frequency-selective pulse. A cost function is used to correlate the so-derived dipolar couplings to trial crystal structures of the samples under study. This procedure allowed for the investigation of the l-alanine space group and L-histidine·HCl·H2O space group and unit-cell parameters.

Quantitative structural constraints for organic powders at natural isotopic abundance using dynamic nuclear polarization solid-state NMR spectroscopy.

A straightforward method is reported to quantitatively relate structural constraints based on (13)C-(13)C double-quantum build-up curves obtained by dynamic nuclear polarization (DNP) solid-state NMR to the crystal structure of organic powders at natural isotopic abundance. This method relies on the significant gain in NMR sensitivity provided by DNP (approximately 50-fold, lowering the experimental time from a few years to a few days), and is sensitive to the molecular conformation and crystal packing of the studied powder sample (in this case theophylline). This method allows trial crystal structures to be rapidly and effectively discriminated, and paves the way to three-dimensional structure elucidation of powders through combination with powder X-ray diffraction, crystal-structure prediction, and density functional theory computation of NMR chemical shifts.

A facile approach for estimating radio-frequency field strength of low-receptivity nuclei.

Radio-frequency (RF) field calibration is essential in NMR spectroscopy. A common practice is to collect a nutation curve by varying the pulse length in a direct single-pulse excitation experiment or in a cross-polarization magic-angle spinning with a flip-back pulse experiment. From the null points on this curve, one can calculate the RF field strength. Nevertheless, the practical implementation is not always straightforward or can even be unrealizable, especially for low-receptivity nuclei owing to their associated low sensitivity. Several researchers used an approach that involves utilizing other nuclei with more sensitivity but nearly identical Larmor frequencies to that of the nucleus of interest. However, such an approach has not been a common practice so far. In this work, we have systematically revisited this approach using 3.2 mm rotors on different sets of nuclei covering a Larmor frequency range up to 80 MHz. The effect of solid- and solution-states on RF field strength measurements has been investigated. The detection of each set of nuclei is then carried out with a resonant circuit in the NMR probe consisting of identical coils and capacitors. Our methodology is illustrated by recording 135/137Ba NMR spectra of BaTiO3 without prior 135/137Ba RF field calibration.

43Ca MAS-DNP NMR of Frozen Solutions for the Investigation of Calcium Ion Complexation.

Calcium ion complexation in aqueous solutions is of paramount importance in biology as it is related to cell signaling, muscle contraction, or biomineralization. However, Ca2+-complexes are dynamic soluble entities challenging to describe at the molecular level. Nuclear magnetic resonance appears as a method of choice to probe Ca2+-complexes. However, 43Ca NMR exhibits severe limitations arising from the low natural abundance coupled to the low gyromagnetic ratio and the quadrupolar nature of 43Ca, which overall make it a very unreceptive nucleus. Here, we show that 43Ca dynamic nuclear polarization (DNP) NMR of 43Ca-labeled frozen solutions is an efficient approach to enhance the NMR receptivity of 43Ca and to obtain structural insights about calcium ions complexed with representative ligands including water molecules, ethylenediaminetetraacetic acid (EDTA), and l-aspartic acid (l-Asp). In these conditions and in combination with numerical simulations and calculations, we show that 43Ca nuclei belonging to Ca2+ complexed to the investigated ligands exhibit rather low quadrupolar couplings (with CQ typically ranging from 0.6 to 1 MHz) due to high symmetrical environments and potential residual dynamics in vitrified solutions at a temperature of 100 K. As a consequence, when 1H→43Ca cross-polarization (CP) is used to observe 43Ca central transition, "high-power" νRF(43Ca) conditions, typically used to detect spin 1/2 nuclei, provide ∼120 times larger sensitivity than "low-power" conditions usually employed for detection of quadrupolar nuclei. These "high-power" CPMAS conditions allow two-dimensional (2D) 1H-43Ca HetCor spectra to be readily recorded, highlighting various Ca2+-ligand interactions in solution. This significant increase in 43Ca NMR sensitivity results from the combination of distinct advantages: (i) an efficient 1H-mediated polarization transfer from DNP, resembling the case of low-natural-abundance spin 1/2 nuclei, (ii) a reduced dynamics, allowing the use of CP as a sensitivity enhancement technique, and (iii) the presence of a relatively highly symmetrical Ca environment, which, combined to residual dynamics, leads to the averaging of the quadrupolar interaction and hence to efficient high-power CP conditions. Interestingly, these results indicate that the use of high-power CP conditions is an effective way of selecting symmetrical and/or dynamic 43Ca environments of calcium-containing frozen solution, capable of filtering out more rigid and/or anisotropic 43Ca sites characterized by larger quadrupolar constants. This approach could open the way to the atomic-level investigation of calcium environments in more complex, heterogeneous frozen solutions, such as those encountered at the early stages of calcium phosphate or calcium carbonate biomineralization events.

Polarization Amplification in Dynamic Nuclear Polarization Magic-Angle Spinning Solid-State Nuclear Magnetic Resonance by Solubilizing Traditional Ionic Salts.

Dynamic nuclear polarization can improve the sensitivity of magic-angle spinning solid-state NMR experiments by 1-2 orders of magnitude. In aqueous media, experiments are usually performed using the so-called DNP juice, a glycerol-d8/D2O/H2O mixture (60/30/10, v/v/v) that can form a homogeneous glass at cryogenic temperatures. This acts as a cryoprotectant and prevents phase separation of the paramagnetic polarizing agents (PAs) that are added to the mixture to provide the source of electron spin polarization required for DNP. Here, we show that relatively high 1H DNP enhancements (∼60) can also be obtained in water without glycerol (or other glass forming agents) simply by dissolving high concentrations of electrolytes (such as NaCl or LiCl), which perturb the otherwise unavoidable ice crystallization observed upon cooling, thereby reducing PA phase separation and restoring DNP efficiency.

Exploiting solid-state dynamic nuclear polarization NMR spectroscopy to establish the spatial distribution of polymorphic phases in a solid material.

Solid-state DNP NMR can enhance the ability to detect minor amounts of solid phases within heterogenous materials. Here we demonstrate that NMR contrast based on the transport of DNP-enhanced polarization can be exploited in the challenging case of early detection of a small amount of a minor polymorphic phase within a major polymorph, and we show that this approach can yield quantitative information on the spatial distribution of the two polymorphs. We focus on the detection of a minor amount (<4%) of polymorph III of m-aminobenzoic acid within a powder sample of polymorph I at natural isotopic abundance. Based on proposed models of the spatial distribution of the two polymorphs, simulations of 1H spin diffusion allow NMR data to be calculated for each model as a function of particle size and the relative amounts of the polymorphs. A comparison between simulated and experimental NMR data allows the model(s) best representing the spatial distribution of the polymorphs in the system to be established.

Probing the motional behavior of eumelanin and pheomelanin with solid-state NMR spectroscopy: new insights into the pigment properties.

Melanin is the most widespread pigment in the animal kingdom. Despite its importance, its detailed structure and overall molecular architecture remain elusive. Both eumelanin (black) and pheomelanin (red) occur in the human body. These two melanin compounds show very different responses to UV-radiation exposure, which could relate to their microscopic features. Herein, the structural properties and motional behavior of natural eu- and pheomelanin extracted from black and red human hair are investigated by means of solid-state NMR spectroscopy. Several 1D and 2D NMR spectroscopic techniques were combined to highlight the differences between the two forms of the pigment. The quantitative analysis of the (1) H NMR wide-line spectra extracted from 2D (1) H-(13) C LG-WISE experiments revealed the presence of two dynamically distinguishable components in both forms. Remarkably, the more mobile fraction of the pigment showed a higher mobility with respect to the proteinaceous components that coexist in the melanosome, which is particularly evident for the red pigment. An explanation of the observed effects takes into account the different architecture of the proteinaceous matrix that constitutes the physical substrate onto which melanin polymerizes within the eu- and pheomelanosomes. Further insight into the molecular structure of the more mobile fraction of pheomelanin was also obtained by means of the analysis of 2D (1) H-(13) C INEPT experiments. Our view is that not only structural features inherent in the pure pigment, but also the role of the matrix structure in defining the overall melanin supramolecular arrangement and the resulting dynamic behavior of the two melanin compounds should be taken into account to explain their functions. The reported results could pave a new way toward the explanation of the molecular origin of the differences in the photoprotection activity displayed by black and red melanin pigments.

Towards measurement of homonuclear dipolar couplings in 1H solid-state NMR: recoupling with a rotor-synchronized decoupling scheme.

We describe a magic-angle spinning NMR experiment for (1)H-(1)H homonuclear dipole-dipole coupling estimations in organic solids. The methodology involves reintroducing dipolar interactions with rotor-synchronized homonuclear decoupling pulse sequences. Frequency-selective DANTE pulses are used to isolate a specific spin pair from a natural isotopic abundance sample. The coupling of interest, between the selected spin pair, may be extracted by a non linear least-squares fit of the experimentally observed modulation of the signal intensity to an exact analytical formula. The experiment is demonstrated on natural isotopic abundance glycine and alanine powder samples.