Atomic-Scale Description of Interfaces between Antigen and Aluminum-Based Adjuvants Used in Vaccines by Dynamic Nuclear Polarization (DNP) Enhanced NMR Spectroscopy.

The addition of aluminum-based adjuvants in vaccines enhances the immune response to antigens. The strength of antigen adsorption on adjuvant gels is known to modulate vaccine efficacy. However, a detailed understanding of the mechanisms of interaction between aluminum gels and antigens is still missing. Herein, a new analytical approach based on dynamic nuclear polarization (DNP) enhanced NMR spectroscopy under magic angle spinning (MAS) is implemented to provide a molecular description of the antigen-adjuvant interface. This approach is demonstrated on hepatitis B surface antigen particles in combination with three aluminum gels obtained from different suppliers. Both noncovalent and covalent interactions between the phospholipids of the antigen particles and the surface of the aluminum gels are identified by using MAS DNP NMR 27 Al and 31 P correlation experiments. Although covalent interactions were detected for only one of the formulations, dipolar recoupling rotational echo adiabatic passage double resonance (REAPDOR) experiments reveal significant differences in the strength of weak interactions.

Homonuclear Decoupling in 1 H†NMR of Solids by Remote Correlation.

The typical linewidths of 1 H NMR spectra of powdered organic solids at 111 kHz magic-angle spinning (MAS) are of the order of a few hundred Hz. While this is remarkable in comparison to the tens of kHz observed in spectra of static samples, it is still the key limit to the use of 1 H in solid-state NMR, especially for complex systems. Here, we demonstrate a novel strategy to further improve the spectral resolution. We show that the anti-z-COSY experiment can be used to reduce the residual line broadening of 1 H NMR spectra of powdered organic solids. Results obtained with the anti-z-COSY sequence at 100 kHz MAS on thymol, ß-AspAla, and strychnine show an improvement in resolution of up to a factor of two compared to conventional spectra acquired at the same spinning rate.

Rapid Structure Determination of Molecular Solids Using Chemical Shifts Directed by Unambiguous Prior Constraints.

NMR-based crystallography approaches involving the combination of crystal structure prediction methods, ab initio calculated chemical shifts and solid-state NMR experiments are powerful methods for crystal structure determination of microcrystalline powders. However, currently structural information obtained from solid-state NMR is usually included only after a set of candidate crystal structures has already been independently generated, starting from a set of single-molecule conformations. Here, we show with the case of ampicillin that this can lead to failure of structure determination. We propose a crystal structure determination method that includes experimental constraints during conformer selection. In order to overcome the problem that experimental measurements on the crystalline samples are not obviously translatable to restrict the single-molecule conformational space, we propose constraints based on the analysis of absent cross-peaks in solid-state NMR correlation experiments. We show that these absences provide unambiguous structural constraints on both the crystal structure and the gas-phase conformations, and therefore can be used for unambiguous selection. The approach is parametrized on the crystal structure determination of flutamide, flufenamic acid, and cocaine, where we reduce the computational cost by around 50%. Most importantly, the method is then shown to correctly determine the crystal structure of ampicillin, which would have failed using current methods because it adopts a high-energy conformer in its crystal structure. The average positional RMSE on the NMR powder structure is ⟨rav⟩ = 0.176 Å, which corresponds to an average equivalent displacement parameter Ueq = 0.0103 Å2.

High-resolution 1H NMR of powdered solids by homonuclear dipolar decoupling.

The development of homonuclear dipolar decoupling sequences to obtain high-resolution 1H NMR spectra from solids has recently celebrated its 50th birthday. Over the years, a series of different decoupling schemes have been developed, starting with the pioneering Lee-Goldburg and WAHUHA sequences up to the most recent generation of experimentally optimized phase-modulated schemes such as eDUMBO-122 and LG4. These schemes can all yield over an order of magnitude reduction in 1H NMR linewidths in solids. Here we provide an overview and a broad experimental comparison of the performance of the main sequences, which has so far been absent in the literature, especially between the newest and the oldest decoupling schemes. We compare experimental results obtained using eight different decoupling schemes (LG, WHH-4, MREV-8, BR-24, FSLG/PMLG, DUMBO-1, eDUMBO-122 and LG4) on three different microcrystalline powdered samples (alanine, glycine and ß-AspAla) and at three different MAS rates (3.0, 12.5 and 22.0 kHz). Finally, since these sequences can be technically demanding, we describe the experimental protocol we have used to optimize these schemes with the aim to provide simple guidelines for the optimization of CRAMPS experiments for all NMR users.

Line narrowing in 1H NMR of powdered organic solids with TOP-CT-MAS experiments at ultra-fast MAS.

The residual broadening observed in 1H spectra of rigid organic solids at natural abundance under 111 kHz magic angle spinning (MAS) is typically a few hundred Hertz. Here we show that refocusable and non-refocusable interactions contribute roughly equally to this residual at high-fields (21.14 T), and suggest that the removal of the non-refocusable part will produce significant increase in spectral resolution. To this end, we demonstrate an experiment for the indirect acquisition of constant-time experiments at ultra-fast MAS (CT-MAS) which verifies this hypothesis. The combination of this experiment with the two-dimensional one pulse (TOP) transformation reduces the experimental time to a fraction of the original cost while retaining the narrowing effects. Results obtained with TOP-CT-MAS at 111 kHz MAS on a sample of ß-AspAla yield up to 30% higher resolution spectra than the equivalent one-pulse experiment, in less than 10 min.

Measurement of Proton Spin Diffusivity in Hydrated Cementitious Solids.

The study of hydration and crystallization processes involving inorganic oxides is often complicated by poor long-range order and the formation of heterogeneous domains or surface layers. In solid-state NMR, 1H-1H spin diffusion analyses can provide information on spatial composition distributions, domain sizes, or miscibility in both ordered and disordered solids. Such analyses have been implemented in organic solids but crucially rely on separate measurements of the 1H spin diffusion coefficients in closely related systems. We demonstrate that an experimental NMR method, in which "holes" of well-defined dimensions are created in proton magnetization, can be applied to determine spin diffusion coefficients in cementitious solids hydrated with 17O-enriched water. We determine proton spin diffusion coefficients of 240 ± 40 nm2/s for hydrated tricalcium aluminate and 140 ± 20 nm2/s for hydrated tricalcium silicate under quasistatic conditions.

Chemical shifts in molecular solids by machine learning.

Due to their strong dependence on local atonic environments, NMR chemical shifts are among the most powerful tools for strucutre elucidation of powdered solids or amorphous materials. Unfortunately, using them for structure determination depends on the ability to calculate them, which  comes at the cost of high accuracy first-principles calculations. Machine learning has recently emerged as a way to overcome the need for quantum chemical calculations, but for chemical shifts in solids it is hindered by the chemical and combinatorial space spanned by molecular solids, the strong dependency of chemical shifts on their environment, and the lack of an experimental database of shifts. We propose a machine learning method based on local environments to accurately predict chemical shifts of molecular solids and their polymorphs to within DFT accuracy. We also demonstrate that the trained model is able to determine, based on the match between experimentally measured and ML-predicted shifts, the structures of cocaine and the drug 4-[4-(2-adamantylcarbamoyl)-5-tert-butylpyrazol-1-yl]benzoic acid.

Refocused linewidths less than 10 Hz in 1H solid-state NMR.

Coherence lifetimes in homonuclear dipolar decoupled 1H solid-state NMR experiments are usually on the order of a few ms. We discover an oscillation that limits the lifetime of the coherences by recording spin-echo dephasing curves. We find that this oscillation can be removed by the application of a double spin-echo experiment, leading to coherence lifetimes of more than 45 ms in adamantane and more that 22 ms in ß-AspAla, corresponding to refocused linewidths of less than 7 and 14 Hz respectively.

Structure elucidation of a complex CO2-based organic framework material by NMR crystallography.

A three-dimensional structural model of a complex CO2-based organic framework made from high molecular weight, self-assembled, flexible and multi-functional oligomeric constituents has been determined de novo by solid-state NMR including DNP-enhanced experiments. The complete assignment of the 15N, 13C and 1H resonances was obtained from a series of two-dimensional through space and through bond correlation experiments. MM-QM calculations were used to generate different model structures for the material which were then evaluated by comparing multiple experimental and calculated NMR parameters. Both NMR and powder X-ray diffraction were evaluated as tools to determine the packing by crystal modelling, and at the level of structural modelling used here PXRD was found not to be a useful complement. The structure determined reveals a highly optimised H-bonding network that explains the unusual selectivity of the self-assembly process which generates the material. The NMR crystallography approach used here should be applicable for the structure determination of other complex solid materials.