solid-state NMR spectroscopy
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NMR spectroscopy is the key analytical method for determination of chemical structure and investigation of physico-chemical properties, such as non-covalent interac-tions, tautomeric equilibria or polymorphism. In this work, selected examples of using advanced NMR methods in structural analysis are discussed. (1) NMR spectroscopy with in situirradiation useful to monitor photochemical processes, (2) 31P NMR spectroscopy for reaction monitor-ing and probing stereochemistry and (3) solid-state NMR spectroscopy to investigate polymorphism.
Changes in the protonation state of lyophilized proteins can impact structural integrity, chemical stability, and propensity to aggregate upon reconstitution. When a buffer is chosen, the freezing/drying process may result in dramatic changes in the protonation state of the protein due to ionization shift of the buffer. In order to determine whether protonation shifts are occurring, ionizable probes can be added to the formulation. Optical probes (dyes) have shown dramatic ionization changes in lyophilized products, but it is unclear whether the pH indicator is uniform throughout the matrix and whether the change in the pH indicator actually mirrors drug ionization changes. In solid-state NMR (SSNMR) spectroscopy, the chemical shift of the carbonyl carbon in carboxylic acids is very sensitive to the ionization state of the acid. Therefore, SSNMR can be used to measure ionization changes in a lyophilized matrix by employing a small quantity of an isotopically-labeled carboxylic acid species in the formulation. This paper compares the apparent pH of six trehalose-containing lyophilized buffer systems using SSNMR and UV-Vis diffuse reflectance spectroscopy (UVDRS). Both SSNMR and UVDRS results using two different ionization probes (butyric acid and bromocresol purple, respectively) showed little change in apparent acidity compared to the pre-lyophilized solution in a sodium citrate buffer, but a greater change was observed in potassium phosphate, sodium phosphate, and histidine buffers. While the trends between the two methods were similar, there were differences in the numerical values of equivalent pH (pHeq) observed between the two methods. The potential causes contributing to the differences are discussed.
- MeSH
- fosfáty * chemie MeSH
- histidin * chemie MeSH
- koncentrace vodíkových iontů MeSH
- kyselina citronová chemie MeSH
- lyofilizace * metody MeSH
- magnetická rezonanční spektroskopie * metody MeSH
- pufry MeSH
- spektrofotometrie ultrafialová metody MeSH
- trehalosa * chemie MeSH
- Publikační typ
- časopisecké články MeSH
Solid dispersions of active pharmaceutical ingredients are of increasing interest due to their versatile use. In the present study polyvinylpyrrolidone (PVP), poly[N-(2-hydroxypropyl)-metacrylamide] (pHPMA), poly(2-ethyl-2-oxazoline) (PEOx), and polyethylene glycol (PEG), each in three Mw, were used to demonstrate structural diversity of solid dispersions. Acetylsalicylic acid (ASA) was used as a model drug. Four distinct types of the solid dispersions of ASA were created using a freeze-drying method: (i) crystalline solid dispersions containing nanocrystalline ASA in a crystalline PEG matrix; (ii) amorphous glass suspensions with large ASA crystallites embedded in amorphous pHPMA; (iii) solid solutions with molecularly dispersed ASA in rigid amorphous PVP; and (iv) nanoheterogeneous solid solutions/suspensions containing nanosized ASA clusters dispersed in a semiflexible matrix of PEOx. The obtained structural data confirmed that the type of solid dispersion can be primarily controlled by the chemical constitutions of the applied polymers, while the molecular weight of the polymers had no detectable impact. The molecular structure of the prepared dispersions was characterized using solid-state NMR, wide-angle X-ray scattering (WAXS), and differential scanning calorimetry (DSC). By applying various (1)H-(13)C and (1)H-(1)H correlation experiments combined with T1((1)H) and T1ρ((1)H) relaxation data, the extent of the molecular mixing was determined over a wide range of distances, from intimate intermolecular contacts (0.1-0.5 nm) up to the phase-separated nanodomains reaching ca. 500 nm. Hydrogen-bond interactions between ASA and polymers were probed by the analysis of (13)C and (15)N CP/MAS NMR spectra combined with the measurements of (1)H-(15)N dipolar profiles. Overall potentialities and limitations of individual experimental techniques were thoroughly evaluated.
In this work, we studied model stratum corneum lipid mixtures composed of the hydroxylated skin ceramides N-lignoceroyl 6-hydroxysphingosine (Cer[NH]) and α-hydroxylignoceroyl phytosphingosine (Cer[AP]). Two model skin lipid mixtures of the composition Cer[NH] or Cer[AP], N-lignoceroyl sphingosine (Cer[NS]), lignoceric acid (C24:0) and cholesterol in a 0.5:0.5:1:1 molar ratio were compared. Model membranes were investigated by differential scanning calorimetry and 2H solid-state NMR spectroscopy at temperatures from 25 °C to 80 °C. Each component of the model mixture was specifically deuterated for selective detection by 2H NMR. Thus, the exact phase composition of the mixture at varying temperatures could be quantified. Moreover, using X-ray powder diffraction we investigated the lamellar phase formation. From the solid-state NMR and DSC studies, we found that both hydroxylated Cer[NH] and Cer[AP] exhibit a similar phase behavior. At physiological skin temperature of 32 °C, the lipids form a crystalline (orthorhombic) phase. With increasing temperature, most of the lipids become fluid and form a liquid-crystalline phase, which converts to the isotropic phase at higher temperatures (65-80 °C). Interestingly, lignoceric acid in the Cer[NH]-containing mixture has a tendency to form two types of fluid phases at 65 °C. This tendency was also observed in Cer[AP]-containing membranes at 80 °C. While Cer[AP]-containing lipid models formed a short periodicity phase featuring a repeat spacing of d = 5.4 nm, in the Cer[NH]-based model skin lipid membranes, the formation of unusual long periodicity phase with a repeat spacing of d = 10.7 nm was observed.
- MeSH
- biologické modely MeSH
- ceramidy chemie metabolismus MeSH
- cholesterol chemie MeSH
- deuterium chemie MeSH
- hydroxylace fyziologie MeSH
- kůže chemie metabolismus MeSH
- lidé MeSH
- lipidové dvojvrstvy chemie metabolismus MeSH
- magnetická rezonanční spektroskopie metody MeSH
- permeabilita buněčné membrány MeSH
- prášková difrakce metody MeSH
- rentgenové záření MeSH
- teplota kůže fyziologie MeSH
- teplota MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Sensitivity and resolution together determine the quality of NMR spectra in biological solids. For high-resolution structure determination with solid-state NMR, proton-detection emerged as an attractive strategy in the last few years. Recent progress in probe technology has extended the range of available MAS frequencies up to above 100 kHz, enabling the detection of resolved resonances from sidechain protons, which are important reporters of structure. Here we characterise the interplay between MAS frequency in the newly available range of 70-110 kHz and proton content on the spectral quality obtainable on a 1 GHz spectrometer for methyl resonances. Variable degrees of proton densities are tested on microcrystalline samples of the α-spectrin SH3 domain with selectively protonated methyl isotopomers (CH3, CH2D, CHD2) in a perdeuterated matrix. The experimental results are supported by simulations that allow the prediction of the sensitivity outside this experimental frequency window. Our results facilitate the selection of the appropriate labelling scheme at a given MAS rotation frequency.
New drug formulations are sought for poorly water-soluble substances because there is a risk of compromised bioavailability if such substances are administered orally. Such active pharmaceutical ingredients can be reformulated as solid dispersions with suitable water-soluble polymers. In this contribution, formulation of a novel and physically stable dispersion of Simvastatin in poly(2-hydroxypropyl) methacrylamide (pHPMA) is demonstrated. Due to the limited water sorption of pHPMA and a high Tg, the prepared dispersion is more suited for oral administration and storage compared with neat amorphous Simvastatin. Surprisingly, the rate of global reorientation and the internal motion of Simvastatin molecules were enhanced and exhibited dynamical heterogeneities when incorporated into the pHPMA matrix. As revealed by solid-state nuclear magnetic resonance combined with Raman spectroscopy exploiting the fluorescence phenomenon the mobility of the ester and lactone components increased considerably, whereas the naphthalene ring remained rigid. Furthermore, the solid dispersion was found to be nano-heterogeneous with nanometer-sized Simvastatin domains. The presence of these clusters had no impact on the dynamics of the rigid pHPMA chains. Thus, the diffusion of Simvastatin molecules through the glassy pHPMA walls and the subsequent transformation of the clusters into larger crystallites were prevented. No crystallization was detected for more than two years.
- MeSH
- adsorpce MeSH
- diferenciální skenovací kalorimetrie MeSH
- kyseliny polymethakrylové chemie MeSH
- magnetická rezonanční spektroskopie MeSH
- molekulární struktura MeSH
- Ramanova spektroskopie MeSH
- simvastatin chemie MeSH
- stabilita léku MeSH
- voda chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The solid-state NMR measurements play an indispensable role in studies of interactions between biological membranes and peptaibols, which are amphipathic oligopeptides with a high abundance of alpha-aminobutyric acid (Aib). The solid-state NMR investigations are important in establishing the molecular models of the pore forming and antimicrobial properties of peptaibols, but rely on certain simplifications. Some of the underlying assumptions concern the parameters describing the 15N NMR chemical shielding tensor (CST) of the amide nitrogens in Aib and in conventional amino acids. Here the density functional theory (DFT) based calculations were applied to the known crystal structure of one of peptaibols, Ampullosporin A, in order to explicitly describe the variation of the 15N NMR parameters within its backbone. Based on the DFT computational data it was possible to verify the validity of the assumptions previously made about the differences between Aib and other amino acids in the isotropic part of the CST. Also the trends in the magnitudes and orientations of the anisotropic components of the CST, as revealed by the DFT calculations of the full periodic structure of Ampullosporin A, were thoroughly analyzed, and may be employed in future studies of peptaibols.
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Reliable values of the solid-state NMR (SSNMR) parameters together with precise structural data specific for a given amino acid site in an oligopeptide are needed for the proper interpretation of measurements aiming at an understanding of oligopeptides' function. The periodic density functional theory (DFT)-based computations of geometries and SSNMR chemical shielding tensors (CSTs) of solids are shown to be accurate enough to support the SSNMR investigations of suitably chosen models of oriented samples of oligopeptides. This finding is based on a thorough comparison between the DFT and experimental data for a set of tripeptides with both 13Cα and 15Namid CSTs available from the single-crystal SSNMR measurements and covering the three most common secondary structural elements of polypeptides. Thus, the ground is laid for a quantitative description of local spectral parameters of crystalline oligopeptides, as demonstrated for the backbone 15Namid nuclei of samarosporin I, which is a pentadecapeptide (composed of five classical and ten nonproteinogenic amino acids) featuring a strong antimicrobial activity.
Alginate gels are an outstanding biomaterial widely applicable in tissue engineering, medicine, and pharmacy for cell transplantation, wound healing and efficient bioactive agent delivery, respectively. This contribution provides new and comprehensive insight into the atomic-resolution structure and dynamics of polyvalent ion-cross-linked alginate gels in microbead formulations. By applying various advanced solid-state NMR (ssNMR) spectroscopy techniques, we verified the homogeneous distribution of the cross-linking ions in the alginate gels and the high degree of ion exchange. We also established that the two-component character of the alginate gels arises from the concentration fluctuations of residual water molecules that are preferentially localized along polymer chains containing abundant mannuronic acid (M) residues. These hydrated M-rich blocks tend to self-aggregate into subnanometer domains. The resulting coexistence of two types of alginate chains differing in segmental dynamics was revealed by 1H-13C dipolar profile analysis, which indicated that the average fluctuation angles of the stiff and mobile alginate segments were about 5-9° or 30°, respectively. Next, the 13C CP/MAS NMR spectra indicated that the alginate polymer microstructure was strongly dependent on the type of cross-linking ion. The polymer chain regularity was determined to systematically decrease as the cross-linking ion radius decreased. Consistent with the 1H-1H correlation spectra, regular structures were found for the gels cross-linked by relatively large alkaline earth cations (Ba2+, Sr2+, or Ca2+), whereas the alginate chains cross-linked by bivalent transition metal ions (Zn2+) and trivalent metal cations (Al3+) exhibited significant irregularities. Notably, however, the observed disordering of the alginate chains was exclusively attributed to the M residues, whereas the structurally well-defined gels all contained guluronic acid (G) residues. Therefore, a key role of the units in M-rich blocks as mediators promoting the self-assembly of alginate chains was experimentally confirmed. Finally, combining 2D 27Al 3Q/MAS NMR spectroscopy with density functional theory (DFT) calculations provided previously unreported insight into the structure of the Al3+ cross-linking centers. Notably, even with a low residual amount of water, these cross-linking units adopt exclusively 6-fold octahedral coordination and exhibit significant motion, which considerably reduces quadrupolar coupling constants. Thus, the experimental strategy presented in this study provides a new perspective on cross-linked alginate structure and dynamics for which high-quality diffraction data at the atomic resolution level are inherently unavailable.
