The Structure of the Necrosome RIPK1-RIPK3 Core, a Human Hetero-Amyloid Signaling Complex.

The RIPK1-RIPK3 necrosome is an amyloid signaling complex that initiates TNF-induced necroptosis, serving in human immune defense, cancer, and neurodegenerative diseases. RIPK1 and RIPK3 associate through their RIP homotypic interaction motifs with consensus sequences IQIG (RIPK1) and VQVG (RIPK3). Using solid-state nuclear magnetic resonance, we determined the high-resolution structure of the RIPK1-RIPK3 core. RIPK1 and RIPK3 alternately stack (RIPK1, RIPK3, RIPK1, RIPK3, etc.) to form heterotypic ß sheets. Two such ß sheets bind together along a compact hydrophobic interface featuring an unusual ladder of alternating Ser (from RIPK1) and Cys (from RIPK3). The crystal structure of a four-residue RIPK3 consensus sequence is consistent with the architecture determined by NMR. The RIPK1-RIPK3 core is the first detailed structure of a hetero-amyloid and provides a potential explanation for the specificity of hetero- over homo-amyloid formation and a structural basis for understanding the mechanisms of signal transduction.

Structural Studies of Amyloid Proteins at the Molecular Level.

Dozens of proteins are known to convert to the aggregated amyloid state. These include fibrils associated with systemic and neurodegenerative diseases and cancer, functional amyloid fibrils in microorganisms and animals, and many denatured proteins. Amyloid fibrils can be much more stable than other protein assemblies. In contrast to globular proteins, a single protein sequence can aggregate into several distinctly different amyloid structures, termed polymorphs, and a given polymorph can reproduce itself by seeding. Amyloid polymorphs may be the molecular basis of prion strains. Whereas the Protein Data Bank contains some 100,000 globular protein and 3,000 membrane protein structures, only a few dozen amyloid protein structures have been determined, and most of these are short segments of full amyloid-forming proteins. Regardless, these amyloid structures illuminate the architecture of the amyloid state, including its stability and its capacity for formation of polymorphs.

Direct Synthesis of N-formamides by Integrating Reductive Amination of Ketones and Aldehydes with CO2 Fixation in a Metal-Organic Framework.

Formamides are important feedstocks for the manufacture of many fine chemicals. State-of-the-art synthesis of formamides relies on the use of an excess amount of reagents, giving copious waste and thus poor atom-economy. Here, we report the first example of direct synthesis of N-formamides by coupling two challenging reactions, namely reductive amination of carbonyl compounds, particularly biomass-derived aldehydes and ketones, and fixation of CO2 in the presence of H2 over a metal-organic framework supported ruthenium catalyst, Ru/MFM-300(Cr). Highly selective production of N-formamides has been observed for a wide range of carbonyl compounds. Synchrotron X-ray powder diffraction reveals the presence of strong host-guest binding interactions via hydrogen bonding and parallel-displaced π⋅⋅⋅π interactions between the catalyst and adsorbed substrates facilitating the activation of substrates and promoting selectivity to formamides. The use of multifunctional porous catalysts to integrate CO2 utilisation in the synthesis of formamide products will have a significant impact in the sustainable synthesis of feedstock chemicals.

Electron and Spin Delocalization in [Co6 Se8 (PEt3 )6 ]0/+1 Superatoms.

Molecular clusters can function as nanoscale atoms/superatoms, assembling into superatomic solids, a new class of solid-state materials with designable properties through modifications on superatoms. To explore possibilities on diversifying building blocks, here we thoroughly studied one representative superatom, Co6 Se8 (PEt3 )6 . We probed its structural, electronic, and magnetic properties and revealed its detailed electronic structure as valence electrons delocalize over inorganic [Co6 Se8 ] core while ligands function as an insulated shell. 59 Co SSNMR measurements on the core and 31 P, 13 C on the ligands show that the neutral Co6 Se8 (PEt3 )6 is diamagnetic and symmetric, with all ligands magnetically equivalent. Quantum computations cross-validate NMR results and reveal degenerate delocalized HOMO orbitals, indicating aromaticity. Ligand substitution keeps the inorganic core nearly intact. After losing one electron, the unpaired electron in [Co6 Se8 (PEt3 )6 ]+1 is delocalized, causing paramagnetism and a delocalized electron spin. Notably, this feature of electron/spin delocalization over a large cluster is attractive for special single-electron devices.

The structure of a minimum amyloid fibril core formed by necroptosis-mediating RHIM of human RIPK3.

Receptor-interacting protein kinases 3 (RIPK3), a central node in necroptosis, polymerizes in response to the upstream signals and then activates its downstream mediator to induce cell death. The active polymeric form of RIPK3 has been indicated as the form of amyloid fibrils assembled via its RIP homotypic interaction motif (RHIM). In this study, we combine cryogenic electron microscopy and solid-state NMR to determine the amyloid fibril structure of RIPK3 RHIM-containing C-terminal domain (CTD). The structure reveals a single protofilament composed of the RHIM domain. RHIM forms three ß-strands (referred to as strands 1 through 3) folding into an S shape, a distinct fold from that in complex with RIPK1. The consensus tetrapeptide VQVG of RHIM forms strand 2, which zips up strands 1 and 3 via heterozipper-like interfaces. Notably, the RIPK3-CTD fibril, as a physiological fibril, exhibits distinctive assembly compared with pathological fibrils. It has an exceptionally small fibril core and twists in both handedness with the smallest pitch known so far. These traits may contribute to a favorable spatial arrangement of RIPK3 kinase domain for efficient phosphorylation.

1 H-13 C cross-polarization kinetics to probe hydration-dependent organic components of bone extracellular matrix.

Bone is a living tissue made up of organic proteins, inorganic minerals, and water. The organic component of bone (mainly made up of Type-I collagen) provides flexibility and tensile strength. Solid-state nuclear magnetic resonance (ssNMR) is one of the few techniques that can provide atomic-level structural insights of such biomaterials in their native state. In the present article, we employed the variable contact time cross-polarization (1 H-13 C CP) kinetics experiments to study the hydration-dependent atomic-level structural changes in the bone extracellular matrix (ECM). The natural abundant 13 C CP intensity of the bone ECM is measured by varying CP contact time and best fitted to the nonclassical kinetic model. Different relaxation parameters were measured by the best-fit equation corresponding to the different hydration conditions of the bone ECM. The associated changes in the measured parameters due to varying levels of hydration observed at different sites of collagen protein have provided its structural arrangements and interaction with water molecules in bone ECM. Overall, the present study reveals a better understanding of the kinetics of the organic part inside the bone ECM that will help in comprehending the disease-associated pathways.

A Review of Applications of Solid-State Nuclear Magnetic Resonance (ssNMR) for the Analysis of Cyclodextrin-Including Systems.

Cyclodextrins, cyclic oligosaccharides composed of five or more α-D-glucopyranoside units linked by α-1,4 glycosidic bonds, are widely used both in their native forms as well as the components of more sophisticated materials. Over the last 30 years, solid-state nuclear magnetic resonance (ssNMR) has been used to characterize cyclodextrins (CDs) and CD-including systems, such as host-guest complexes or even more sophisticated macromolecules. In this review, the examples of such studies have been gathered and discussed. Due to the variety of possible ssNMR experiments, the most common approaches have been presented to provide the overview of the strategies employed to characterize those useful materials.

Density Functional Theory and Density Functional Tight Binding Studies of Thiamine Hydrochloride Hydrates.

Thiamine hydrochloride (THCL), also known as vitamin B1, is an active pharmaceutical ingredient (API), present on the list of essential medicines developed by the WHO, which proves its importance for public health. THCL is highly hygroscopic and can occur in the form of hydrates with varying degrees of hydration, depending on the air humidity. Although experimental characterization of the THCL hydrates has been described in the literature, the questions raised in previously published works suggest that additional research and in-depth analysis of THCL dehydration behavior are still needed. Therefore, the main aim of this study was to characterize, by means of quantum chemical calculations, the behavior of thiamine hydrates and explain the previously obtained results, including changes in the NMR spectra, at the molecular level. To achieve this goal, a series of DFT (CASTEP) and DFTB (DFTB+) calculations under periodic boundary conditions have been performed, including molecular dynamics simulations and GIPAW NMR calculations. The obtained results explain the differences in the relative stability of the studied forms and changes in the spectra observed for the samples of various degrees of hydration. This work highlights the application of periodic DFT calculations in the analysis of various solid forms of APIs.

Dual Functionality of Bile Acid: Physical Stabilization of Drugs in the Amorphous Form and Solubility Enhancement in Solution.

Drugs containing an amino aromatic nitrogen moiety were stabilized in the amorphous form by the surfactant cholic acid (CA). Coamorphous systems of lamotrigine (LAM), pyrimethamine (PYR), and trimethoprim (TRI) were each prepared with CA. Drug-CA interactions, investigated by IR and solid-sate NMR spectroscopy, revealed deprotonation of the carboxylic acid group in CA and the protonation of the most basic nitrogen of the drug. The coamorphous systems exhibited exceptional physical stability and resisted crystallization at (i) elevated temperatures (>100 °C) and (ii) accelerated storage conditions, 40 °C/75% relative humidity for 15 months. The dissolution performance of each coamorphous system was compared with the respective crystalline drug based on the area under the curve (AUC) of the concentration-time profiles. A 25-fold increase in AUC was observed in the PYR-CA coamorphous system. The solubility enhancement is attributed not only due to drug amorphization but also due to solubilization by CA. The supramolecular synthon approach, through a drug-CA interaction, yielded physically stable coamorphous systems with enhanced aqueous drug solubility.

Characterization of crystalline and amorphous forms of irbesartan by multi-nuclear solid-state NMR.

Irbesartan (IRB) is an antihypertensive drug which exhibits the rare phenomenon of desmotropy; its 1H- and 2H- tetrazole tautomers can be isolated as distinct crystalline forms. The crystalline forms of IRB are poorly soluble, hence the amorphous form is potentially of interest for its faster dissolution rate. The tautomeric form and the nature of hydrogen bonding in amorphous IRB are unknown. In this study, crystalline form A and amorphous form of irbesartan were studied using 13C, 15N and 1H solid-state NMR. Variable-temperature 13C SSMNR studies showed alkyl chain disorder in the crystalline form of IRB, which may explain the conflicting literature crystal structures of form A (the marketed form). 15N NMR indicates that the amorphous material contains an approximately 2:1 ratio of 1H- and 2H-tetrazole tautomers. Static 1H SSNMR and relaxation time measurements confirmed different molecular mobilities of the samples and provided molecular-level insight into the nature of the glass transition. SSNMR is shown to be a powerful technique to investigate the solid state of disordered active pharmaceutical ingredients.

Separated quadrupole and shift interactions of 2H NMR spectra in paramagnetic solids by asymmetric pulse sequences.

Separated pure-quadrupole (PQ) and -shift (PS) spectra of 2H nuclear magnetic resonance (NMR) of paramagnetic solids are obtained and correlated by simple pulse sequences that can acquire the full magnetization under ideal conditions. Two-dimensional NMR signals obtained using an asymmetric π-pulse-inserted quadrupole-echo (APIQE) sequence yielded separated spectra through the skew operation of an affine transform (AT) before a Fourier transform. Modified APIQE sequences that acquire whole echo signals were fabricated, and separated PQ and PS spectra were obtained by applying a combination of AT, such as rotation and skew operations, to the signal data. These methods were demonstrated for diamagnetic Zn(CD3CO2)2⋅2H2O and paramagnetic Nd(CD3CO2)3⋅1.5H2O. Further, the dynamics of the D2O molecule and [Co(D2O)6]2+ ion in paramagnetic CoSiF6⋅6D2O was analyzed based on the temperature dependence of the separated spectra.

A structural analysis of 2,5-diaryl-4H-2,4-dihydro-3H-1,2,4-triazol-3-ones: NMR in the solid state, X-ray crystallography, and GIPAW calculations.

The 1 H, 13 C, 15 N, and 19 F nuclear magnetic resonance (NMR) spectra of 11 2,5-diaryl-2,4-dihydro-3H-1,2,4-triazol-3-ones have been acquired in DMSO-d6 solution and the 13 C, 15 N, and 19 F NMR spectra have also been acquired in the solid state (solid-state nuclear magnetic resonance [SSNMR] and magic angle spinning [MAS]). The X-ray structures of Compounds 3, 5, and 6 have been determined by X-ray diffraction. Theoretical calculations at the gauge-independent atomic orbital (GIAO)/B3LYP/6-311++G(d,p) level have provided a set of 321 chemical shifts that were compared with 310 experimental values in DMSO-d6 . To obtain good agreements, some effects need to be included. The SSNMR chemical shifts have been compared with gauge-including projector-augmented wave (GIPAW) calculations and with the heavy atom-light atom (HALA) effects.

Reconstitution of Caveolin-1 into Artificial Lipid Membrane: Characterization by Transmission Electron Microscopy and Solid-State Nuclear Magnetic Resonance.

Caveolin-1 (CAV1), a membrane protein that is necessary for the formation and maintenance of caveolae, is a promising drug target for the therapy of various diseases, such as cancer, diabetes, and liver fibrosis. The biology and pathology of caveolae have been widely investigated; however, very little information about the structural features of full-length CAV1 is available, as well as its biophysical role in reshaping the cellular membrane. Here, we established a method, with high reliability and reproducibility, for the expression and purification of CAV1. Amyloid-like properties of CAV1 and its C-terminal peptide CAV1(168-178) suggest a structural basis for the short linear CAV1 assemblies that have been recently observed in caveolin polyhedral cages in Escherichia coli (E. coli). Reconstitution of CAV1 into artificial lipid membranes induces a caveolae-like membrane curvature. Structural characterization of CAV1 in the membrane by solid-state nuclear magnetic resonance (ssNMR) indicate that it is largely α-helical, with very little ß-sheet content. Its scaffolding domain adopts a α-helical structure as identified by chemical shift analysis of threonine (Thr). Taken together, an in vitro model was developed for the CAV1 structural study, which will further provide meaningful evidences for the design and screening of bioactive compounds targeting CAV1.

New Pharmaceutical Salts of Trazodone.

New pharmaceutically acceptable salts of trazodone (trazodone hydrogen bromide and trazodone 1-hydroxy-2-naphthonic acid) for the treatment of central nervous system disorders are synthesized and described. Although trazodone salts are poorly crystalline, single-crystal X-ray diffraction data for trazodone 1-hydroxy-2-naphthonic acid were collected and analyzed as well as compared to the previously described crystal structure of commercially available trazodone hydrochloride. The powder samples of all new salts were characterized by Fourier transform infrared spectroscopy, X-ray diffraction and 13C solid-state nuclear magnetic resonance spectroscopy. Spectroscopic studies were supported by gauge including projector augmented wave (GIPAW) calculations of carbon chemical shielding constants. The main goal of our research was to find salts with better physicochemical properties and to make an attempt to associate them with both the anion structure and the most prominent interactions exhibited by the protonated trazodone cation. The dissolution profiles of trazodone from tablets prepared from various salts with lactose monohydrate were investigated. The studies revealed that salts with simple anions show a fast release of the drug while the presence of more complex anion, more strongly interacting with the cation, effects a slow-release profile of the active substance and can be used for the preparation of the tables with a delay or prolonged mode of action.

In Situ Observation of Non-Classical 2-Norbornyl Cation in Confined Zeolites at Ambient Temperature.

Carbonium ions are an important class of reaction intermediates, but their dynamic evolution is difficult to be monitored by in situ techniques under experimental conditions because of their extremely short lifetime. Probably the most famous case is 2-norbornyl cation (2NB+ ): its existing form (classical or non-classical) had been debated for decades, until the concrete proof of non-classical geometry was achieved by X-ray crystallographic characterization at ultra-low temperature (40 K) and super acidic environment. However, we lack the understanding about 2NB+ at ambient conditions. Herein, by taking advantage of the confinement effect and delocalized acidic environment of zeolites, we successfully stabilized 2NB+ and unequivocally confirmed its "non-classical" structure inside the ZSM-5 zeolite by ab initio molecular dynamics simulations and 13 C solid-state nuclear magnetic resonance experiments. It is the first time to in situ observe the non-classical 2NB+ without the super acidic environment at ambient temperature, which provides a new strategy to expand the carbocation chemistry.

Controls of nature: Secondary, tertiary, and quaternary structure of the enamel protein amelogenin in solution and on hydroxyapatite.

Amelogenin, a protein critical to enamel formation, is presented as a model for understanding how the structure of biomineralization proteins orchestrate biomineral formation. Amelogenin is the predominant biomineralization protein in the early stages of enamel formation and contributes to the controlled formation of hydroxyapatite (HAP) enamel crystals. The resulting enamel mineral is one of the hardest tissues in the human body and one of the hardest biominerals in nature. Structural studies have been hindered by the lack of techniques to evaluate surface adsorbed proteins and by amelogenin's disposition to self-assemble. Recent advancements in solution and solid state nuclear magnetic resonance (NMR) spectroscopy, atomic force microscopy (AFM), and recombinant isotope labeling strategies are now enabling detailed structural studies. These recent studies, coupled with insights from techniques such as CD and IR spectroscopy and computational methodologies, are contributing to important advancements in our structural understanding of amelogenesis. In this review we focus on recent advances in solution and solid state NMR spectroscopy and in situ AFM that reveal new insights into the secondary, tertiary, and quaternary structure of amelogenin by itself and in contact with HAP. These studies have increased our understanding of the interface between amelogenin and HAP and how amelogenin controls enamel formation.

Iterative baseline correction algorithm for dead time truncated one-dimensional solid-state MAS NMR spectra.

We present an algorithm suitable for automatically correcting rolling baseline coming from time-domain truncation induced by the dead time in pulse-acquire one-dimensional MAS NMR spectra. It relies on an iterative estimation of the baseline restricted in the time-domain by the dead time duration combined with a histogram filter allowing adaptive selection of the baseline points. This method does not make any assumption regarding the NMR resonances line shapes or widths and does not modify the acquired free induction decay points. This makes it suitable for accurate deconvolution and quantification of single-pulse MAS NMR spectra. The baseline correction accuracy is evaluated on synthetic solid-state spectra of 19F, 71Ga, and 23Na by comparing the fitted baseline to the theoretical one. The versatility of the algorithm is also exemplified on three additional solid-state spectra of 23Na and 71Ga. The algorithm is made available to the community through a user-friendly standalone Matlab® application.

Efficient assignment and NMR analysis of an intact virus using sequential side-chain correlations and DNP sensitization.

An experimental strategy has been developed to increase the efficiency of dynamic nuclear polarization (DNP) in solid-state NMR studies. The method makes assignments simpler, faster, and more reliable via sequential correlations of both side-chain and Cα resonances. The approach is particularly suited to complex biomolecules and systems with significant chemical-shift degeneracy. It was designed to overcome the spectral congestion and line broadening that occur due to sample freezing at the cryogenic temperatures required for DNP. Nonuniform sampling (NUS) is incorporated to achieve time-efficient collection of multidimensional data. Additionally, fast (25 kHz) magic-angle spinning (MAS) provides optimal sensitivity and resolution. Data collected in <1 wk produced a virtually complete de novo assignment of the coat protein of Pf1 virus. The peak positions and linewidths for samples near 100 K are perturbed relative to those near 273 K. These temperature-induced perturbations are strongly correlated with hydration surfaces.

Quantification of Monomer Units in Insoluble Polymeric Active Pharmaceutical Ingredients Using Solid-State NMR Spectroscopy I: Patiromer.

Although extensive precautions are taken to limit batch-to-batch variation in pharmaceutical manufacturing, differences between lots may still exist, particularly in complex formulations. When polymerization is used in the production process, the potential for varying chain lengths and incorporation of different monomers increases the likelihood of batch-to-batch variation. This poses a significant challenge for demonstrating active pharmaceutical ingredient (API) sameness between the innovator and generic drug under development. Therefore, the ability to accurately analyze and quantify the relative amounts of active ingredients present in a formulated product is critically important. Solid-state nuclear magnetic resonance (SSNMR) spectroscopy was used to identify, quantify, and compare the relative amounts of the three polymer groups in the amorphous block copolymer drug, patiromer (Veltassa®). Techniques such as cross polarization (CP) and magic angle spinning were used to quantify each polymer group while the importance of understanding CP dynamics to obtain quantitative data was also addressed. It was found that the magnetization transfer rate and chemical shift anisotropy for different functional groups present in patiromer play a large role when optimizing parameters for spectral acquisition. Once accounted for, the average patiromer lot contained 90.9%, 7.6%, and 1.5% carboxylate, aromatic, and aliphatic blocks, respectively, with little lot-to-lot variation between different dosage strengths and expiration dates. SSNMR proved to be a sensitive analytical technique for evaluating and quantifying different monomer groups present in patiromer. This procedure may serve as a guide for similar quantitation studies on complex drug products and for demonstrating API sameness during generic drug development.

A comprehensive and comparative study of the internal structure and dynamics of natural ß-keratin and regeneratedß-keratin by solid state NMR spectroscopy.

Structure and dynamics of natural and regenerated chicken feather ß-keratin were investigated by 13C cross-polarization (CP) magic angle spinning (MAS) solid state nuclear magnetic resonance (SSNMR) spectral analysis, 13C and 1H spin-lattice relaxation time measurements, and 13C two dimensional phase adjusted spinning sidebands (2DPASS) MAS SSNMR measurements. Chemical shift anisotropy (CSA) parameters of both natural and regenerated chicken feather ß-keratin were extracted by using 2DPASS MAS SSNMR experiment. The beauty of 2DPASS MAS SSNMR experiment is it can correlate the isotropic and anisotropic dimension with the help of shearing transformation and two dimensional Fourier Transformation. Molecular correlation time at each and every magnetically inequivalent carbon site of both natural and regenerated chicken feather ß-keratin were also determined. The change in molecular dynamics of structural protein after pretreatment was monitored by 2DPASS MAS SSNMR and 13C relaxation measurement. This type of comprehensive study will provide the information about the interrelation between the structure and dynamics of structural protein and will also shed light in the way of developing methods for conversion of animal by-products to novel product.