Maria Baias

Powder crystallography of pharmaceutical materials by combined crystal structure prediction and solid-state 1H NMR spectroscopy

A protocol for the ab initio crystal structure determination of powdered solids at natural isotopic abundance by combining solid-state NMR spectroscopy, crystal structure prediction, and DFT chemical shift calculations was evaluated to determine the crystal structures of four small drug molecules: cocaine, flutamide, flufenamic acid, and theophylline. For cocaine, flutamide and flufenamic acid, we find that the assigned (1)H isotropic chemical shifts provide sufficient discrimination to determine the correct structures from a set of predicted structures using the root-mean-square deviation (rmsd) between experimentally determined and calculated chemical shifts. In most cases unassigned shifts could not be used to determine the structures. This method requires no prior knowledge of the crystal structure, and was used to determine the correct crystal structure to within an atomic rmsd of less than 0.12 Å with respect to the known reference structure. For theophylline, the NMR spectra are too simple to allow for unambiguous structure selection.

De novo determination of the crystal structure of a large drug molecule by crystal structure prediction based powder NMR crystallography

The crystal structure of form 4 of the drug 4-[4-(2-adamantylcarbamoyl)-5-tert-butyl-pyrazol-1-yl]benzoic acid is determined using a protocol for NMR powder crystallography at natural isotopic abundance combining solid-state (1)H NMR spectroscopy, crystal structure prediction, and density functional theory chemical shift calculations. This is the first example of NMR crystal structure determination for a molecular compound of previously unknown structure, and at 422 g/mol this is the largest compound to which this method has been applied so far.

Structure and dynamics of water in native and tanned collagen fibers: Effect of crosslinking

The influence of crosslinking on the hydration structure of collagen has been investigated. Nuclear magnetic resonance, dielectric relaxation and thermoporometry were used to investigate water structure in native and crosslinked collagen fibers on both wet and dried specimen. Measurements reveal the influence of different chemical treatments on the transverse relaxation time and polarization of the collagen fibers. The frequency dependence of dielectric constant of collagen fibers displays an induction behavior on low frequencies. Bound water constrained in collagen fibers seems to provide signatures for changes induced by crosslinking agents on the pore diameter and distribution in collagen fibers. A correlation of transverse relaxation time of water in dry and wet states presented in this study presents an experimental tool for examining the differences in efficacy of crosslinking agents. Changes in the dielectric relaxation, dynamics of water structure and hydroporometric structure of collagen are dependent on the nature of crosslinking material.

Atomic-Resolution Structural Dynamics in Crystalline Proteins from NMR and Molecular Simulation

Solid-state NMR can provide atomic-resolution information about protein motions occurring on a vast range of time scales under similar conditions to those of X-ray diffraction studies and therefore offers a highly complementary approach to characterizing the dynamic fluctuations occurring in the crystal. We compare experimentally determined dynamic parameters, spin relaxation, chemical shifts, and dipolar couplings, to values calculated from a 200 ns MD simulation of protein GB1 in its crystalline form, providing insight into the nature of structural dynamics occurring within the crystalline lattice. This simulation allows us to test the accuracy of commonly applied procedures for the interpretation of experimental solid-state relaxation data in terms of dynamic modes and time scales. We discover that the potential complexity of relaxation-active motion can lead to significant under- or overestimation of dynamic amplitudes if different components are not taken into consideration.

Superstructure of a Substituted Zeolitic Imidazolate Metal–Organic Framework Determined by Combining Proton Solid‐State NMR Spectroscopy and DFT Calculations

We report the supercell crystal structure of a ZIF-8 analog substituted imidazolate metal-organic framework (SIM-1) obtained by combining solid-state nuclear magnetic resonance and powder X-ray diffraction experiments with density functional theory calculations.

Thermal denaturation of hydrated wool keratin by 1H solid-state NMR.

Thermal denaturation of hydrated keratin in wool was investigated by NMR using 1H wide-line spectra to obtain the phase composition and 1H spin-diffusion experiments using a double-quantum filter to obtain the domain sizes for the wool fibers. The denaturation process detected by DSC takes place for wool fibers in deuterated water in the temperature range 140-144 degreeC. The phase composition measured by 1H wide line NMR spectra reveals a rigid, semirigid and an amorphous phase for temperatures in the range 25-160 degreeC. A dramatic change in the phase composition was detected around 142 degreeC, corresponding to the denaturation temperature. The morphological domain sizes measured by 1H spin-diffusion NMR experiments were obtain from the solutions of the spin-diffusion equations for two-dimensional rectangular and cylindrical morphologies. The keratin mobility gradient in the interfacial region at different denaturation temperatures was measured from the 1H spin-diffusion data. A qualitative model describing the denaturation process of hydrated keratin protein was developed that explains the changes in domain thickness, spin diffusivities, phase composition, and thermodynamic parameters.

Ultrafast NMR T1 Relaxation Measurements: Probing Molecular Properties in Real Time

The longitudinal relaxation properties of NMR active nuclei carry useful information about the site-specific chemical environments and about the mobility of molecular fragments. Molecular mobility is in turn a key parameter reporting both on stable properties, such as size, as well as on dynamic ones, such as transient interactions and irreversible aggregation. In order to fully investigate the latter, a fast sampling of the relaxation parameters of transiently formed molecular species may be needed. Nevertheless, the acquisition of longitudinal relaxation data is typically slow, being limited by the requirement that the time for which the nucleus relaxes be varied incrementally until a complete build-up curve is generated. Recently, a number of single-shot-inversion-recovery methods have been developed capable of alleviating this need; still, these may be challenged by either spectral resolution restrictions or when coping with very fast relaxing nuclei. Here, we present a new experiment to measure the T1s of multiple nuclear spins that experience fast longitudinal relaxation, while retaining full high-resolution chemical shift information. Good agreement is observed between T1s measured with conventional means and T1s measured using the new technique. The method is applied to the real-time investigation of the reaction between D-xylose and sodium borate, which is in turn elucidated with the aid of ancillary ultrafast and conventional 2D TOCSY measurements.

Structure and Dynamics of the Huntingtin Exon-1 N-Terminus: A Solution NMR Perspective

Many neurodegenerative diseases are characterized by misfolding and aggregation of an expanded polyglutamine tract (polyQ). Huntingtons Disease, caused by expansion of the polyQ tract in exon 1 of the Huntingtin protein (Htt), is associated with aggregation and neuronal toxicity. Despite recent structural progress in understanding the structures of amyloid fibrils, little is known about the solution states of Htt in general, and about molecular details of their transition from soluble to aggregation-prone conformations in particular. This is an important question, given the increasing realization that toxicity may reside in soluble conformers. This study presents an approach that combines NMR with computational methods to elucidate the structural conformations of Htt Exon 1 in solution. Of particular focus was Htts N17 domain sited N-terminal to the polyQ tract, which is key to enhancing aggregation and modulate Htt toxicity. Such in-depth structural study of Htt presents a number of unique challenges: the long homopolymeric polyQ tract contains nearly identical residues, exon 1 displays a high degree of conformational flexibility leading to a scaling of the NMR chemical shift dispersion, and a large portion of the backbone amide groups are solvent-exposed leading to fast hydrogen exchange and causing extensive line broadening. To deal with these problems, NMR assignment was achieved on a minimal Htt exon 1, comprising the N17 domain, a polyQ tract of 17 glutamines, and a short hexameric polyProline region that does not contribute to the spectrum. A pH titration method enhanced this polypeptides solubility and, with the aid of ≤5D NMR, permitted the full assignment of N17 and the entire polyQ tract. Structural predictions were then derived using the experimental chemical shifts of the Htt peptide at low and neutral pH, together with various different computational approaches. All these methods concurred in indicating that low-pH protonation stabilizes a soluble conformation where a helical region of N17 propagates into the polyQ region, while at neutral pH both N17 and the polyQ become largely unstructured-thereby suggesting a mechanism for how N17 regulates Htt aggregation.

Morphology and Molecular Mobility of Fibrous Hard α-Keratins by 1H, 13C, and 129Xe NMR

The morphology and molecular mobility changes of the side chains for hard alpha-keratin due to oxidative and reductive/oxidative treatments for temperatures around the DSC denaturation peak were investigated by (1)H, (13)C, and (129)Xe NMR spectroscopy and (1)H spin diffusion. Proton wide-line spectra were used to obtain the phase composition (rigid, interface, and amorphous fractions) and molecular dynamics of each phase. Proton spin diffusion experiments using a double-quantum filter and initial rate approximation were employed to obtain the dependence of the rigid domain sizes on chemical treatments and denaturation temperatures. A drastic reduction in the rigid domain thickness takes place for the reductive/oxidative treatment. The keratin mobility gradient in the interfacial region at different denaturation temperatures was measured for hard alpha-keratin from (1)H spin diffusion data. (13)C CPMAS spectra were used to provide a detailed examination of the effects of the chemical treatments especially on the disulfide bonds. Thermally polarized (129)Xe spectra suggest the existence of voids in the hard alpha-keratin induced by the reductive and oxidative treatment. The surface of the hard alpha-keratin fiber surface is probed by the laser hyperpolarized (129)Xe. A qualitative model describing the changes induced in hard alpha-keratin protein by chemical transformation was developed and could be correlated with the changes in domain thickness, phase composition, and molecular dynamics.

Nanostructure of Materials Determined by Relayed Paramagnetic Relaxation Enhancement

Particle and domain sizes strongly influence the properties of materials. Here we present an NMR approach based on paramagnetic relaxation enhancement (PRE) relayed by spin diffusion (SD), which allows us to determine lengths in the nm−μm range. We demonstrate the method on multicomponent organic polymer mixtures by selectively doping one component with a paramagnetic center in order to measure the domain size in a second component. Using this approach we determine domain sizes in ethyl cellulose/hydroxypropyl cellulose film coatings in pharmaceutical controlled release formulations. Here we measure particle sizes ranging from around 50 to 200 nm.