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Small molecule drug discovery is strongly supported by biophysical data. In the reach of this thesis, cell free protein expression was used to produce human target proteins for ligand binding assays using Surface Plasmon Resonance spectroscopy (SPR). In the second step the binding and interaction characteristics of small molecules and fragments were analyzed using Nuclear Magnetic Resonance spectroscopy (NMR).
The first target protein was the human acid sensing channel 1 (ASIC1a). ASIC1a was expressed in a cell free expression system based on E.coli lysate. To optimize the expression, several parameters including fusion tags, ion concentrations and different hydrophobic environments were tested.
The adaption of the folding environment for ASIC1a needed more optimization, because it is a very challenging target to express in an in vitro system. Three different expression modes were employed to find a suitable folding environment.
SPR binding studies with ASIC1a were performed with chicken ASIC1a expressed in insect cells. The immobilization of cASIC1a and the used buffer conditions were tested using Psalmotoxin 1, a naturally occurring peptide venom which binds strong to the trimeric form of ASIC1a. Compound characterization experiments were performed with a variety of different ligands including amiloride, a general blocker of the whole ENaC protein family. None of the used ligands showed titration curves that would match a simple 1:1 binding model. The experiments either show no binding signal or signal that could be interpreted as unspecific binding. Even amiloride that should be binding the protein shows no signals that fit a simple binding model.
Another target protein that was investigated is the soluble prolyl cis/trans isomerase Cyclophilin D (or peptidyl prolyl isomerase F – PPIF). This protein is involved in the regulation of the mitochondrial permeability transition pore and therefore a potential drug target to treat neurodegenerative diseases. Small molecule binding was tested with CypD using SPR. Following the kinetic analysis of small molecule ligands, the binding position of different binding fragments was analyzed. These fragments originated from a SPR based fragment screen and gave no co-crystal structures with CypD. Therefore NMR was used to investigate the binding position of these fragments. An analysis of the chemical shift perturbations upon ligand addition revealed that the NMR analysis was in line with the results gathered by x-ray crystallography. The fragments with unknown binding position however, all bind to a specific patch slightly outside the binding pocket.
The ligand CL1 showed a special behavior in the NMR experiments. Upon addition to CypD, it produced large shifts on many signals of the protein, accompanied by a severe line broadening. The shift perturbations were so numerous and large that the spectrum had to be reassigned in complex with the ligand. Triple selective labeling was applied to allow a fast and nearly complete signal assignment. The possibility to use highly sophisticated labeling schemes, is one of the advantages of cell free protein expression. After the assignment of the complex spectrum, the chemical shift perturbations were analyzed and quantified. The residues showing the strongest CSPs are also identified in the crystal structure to be involved in the binding of CL1, giving a consistent picture. The numerous and large shift perturbations, produced by CL1 led to the assumption, that the ligand induces a conformational change in CypD, which is not represented in the co-crystal structure. This conformational change was characterized by a NMR based structure determination. CypD apo yielded a defined bundle, whose folded regions overlap well with the corresponding crystal structure.
For the calculation of the CypD-CL1 complex structure, the sidechain resonances were assigned using an automated assignment approach with the software FLYA. The calculation of the CypD-CL1 complex structure did not result in a defined bundle. While parts of the protein converge in a well folded state, the region around the active site shows no defined folding. Careful analysis of the structure calculation suggests that the problems during structure calculation did not originate from an incorrect resonance assignment, but rather from a lack of NOE crosspeaks. This might be due to a broadening of the corresponding NOE crosspeaks or the coexistence of many different conformations. This leads to the conclusion, that the protein conformation is not defined by the NMR data and could be in a dynamic interchange between multiple structures.
This hypothesis is supported by other observations. The line broadening of the signals in the complex is pronounced in the area around the active site and the substrate binding pocket, hinting to a connection between catalytic activity and protein dynamics. In addition many NMR signals are sensitive to changes in the measurement field strength and the temperature. This field dependent signal splitting suggests dynamic conformational changes in the protein between at least two different conformations on a millisecond timescale.
The current working model is that CL1 binds to CypD and induces the catalytic cycle and the connected conformational changes in CypD. As a result the proline like moiety in CL1 is constantly switching between the cis and the trans conformation. Due to the high affinity of CL1, the inhibitor does not leave the binding pocket after successful catalysis, but stays bound in the pocket stimulating further catalytic cycles. These findings as well as the working model are well in line with data published for Cyclophilin A, another member of the cyclophilin family, thereby supporting the model.
During the last decade of the 20th century, the field of mass spectrometry has seen a revolutionary change in its application and scope. The introduction of soft ionization methods for the analysis of biological molecules has expanded the area of mass spectrometry from its early roots in the analysis of inorganic and organic species into the fields of biology and medicine.
Today, the use of the mass spectrometry is extended to a wide range of applications in biotechnology and pharmaceutical industry, in geological, environmental and clinical research. In biochemistry, the principles of mass spectrometry are, however, broadly applicable in accurate molecular weight determination, reaction monitoring, amino acid sequencing, oligonucleotide sequencing and protein structure.
In order to carry out their biological activities, proteins interact most often to each other and form transient or stable complexes. In addition, some proteins specifically interact also with other proteins or with non-protein molecules, such as DNA, RNA or metabolites, these interactions being critical for their function. Hence, defining the composition of protein complexes, as well as understanding how protein complexes are assembled and regulated yield invaluable insights into protein function. Coupled with an isolation technique to purify a specific protein complex of interest, mass spectrometry can rapidly and reliably identify the components of complexes. In addition, quantitative MS techniques offer the possibility of studying dynamically regulated interactions....
Die Biosynthese der Fettsäuren (FS) ist in Eukaryoten und Bakterien ein hochkonserviert zentraler Stoffwechselweg, der in zwei strukturell verschiedenen Systemen ausgeführt wird. Die meisten Bakterien, Parasiten, Pflanzen und Mitochondrien nutzen ein Fettsäuresesynthase Typ-II (FAS-II) System. Bei FAS II Systemen sind alle katalytischen Domänen separate lösliche Proteine. In Eukaryoten wie auch den Bakterien Corynebakteria, Mycobakteria, Nocardia (Klasse der CMN Bakterien) liegen die katalytischen Domänen fusioniert auf einer Polypeptidkette vor, die zu einem Multienzymkomplex der Fettsäuresynthase Typ I (FAS-I) assemblieren. Die Architektur der FAS-I zeigt große Unterschiede; die X förmige Säuger-FAS-I (Maier et al., 2006), sowie die fassartigen Enzyme der Pilz FAS-I (Jenni et al., 2007; Leibundgut et al., 2007; Lomakin et al., 2007; Johansson et al., 2008) und der bakteriellen FAS-I (Boehringer et al., 2013; Ciccarelli et al., 2013). Zwischen Pilz- und bakterieller FAS-I gibt es trotz des ähnlichen Aufbaus bedeutende Unterschiede. Mycobakterium tuberculosis, der Auslöser von Tuberkulose (TB), an der jährlich über eine Million Menschen weltweit sterben (WHO, 2014), synthetisiert durch eine Symbiose von FAS-I, FAS-II und der Polyketidsynthase-13 Mykolsäuren. Durch die Mykolsäuren ist M. tuberculosis resistent gegen äußere Einflüsse. FAS-I ist in die Synthese der Vorstufen der Mykolsäuren involviert. Sie stellt im Kampf gegen TB ein potentielles Inhibierungstarget dar.
Strukturell war die bakterielle FAS-I beim Beginn der vorliegenden Arbeit, nur durch negative-stain-Elektronenmikroskopie (EM) Aufnahmen aus dem Jahr 1982 charakterisiert (Morishima et al., 1982). In dieser Arbeit konnte die bakteriellen FAS I aus M. tuberculosis (MtFAS), sowie Corynebacterium ammoniagenes (CaFAS) und Corynebacterium efficiens (CeFAS) strukturell untersucht werden. Dies geschah mit den Methoden negative-stain-EM, Einzelmolekül-Cryo-EM (Cryo-EM), Cryo EM Tomographie (CET) und Röntgenkristallographie.
Anhand von CeFAS-Kristallen konnte erstmals durch Röntgenkristallographie die Struktur einer bakteriellen FAS-I bestimmt werden. Zudem wurde die hohe konformationelle Flexibilität der bakteriellen FAS-I mit mehreren Methoden gezeigt. Für die CaFAS konnte mit Cryo-EM initiale Prozesse der Proteinkristallbildung abgebildet werden.
A novel series of ribonucleosides of 1,2,3-triazolylbenzyl-aminophosphonates was synthesized through the Kabachnik–Fields reaction using I2 as catalyst followed by copper-catalyzed cycloaddition of the azide–alkyne reaction (CuAAC). All structures of the newly prepared compounds were characterized by 1H NMR, 13C NMR, and HRMS spectra. The structures of 2e, 2f, 3d, and 3g were further confirmed by X-ray diffraction analysis. These compounds were tested against various strains of DNA and RNA viruses; compounds 4b and 4c showed a modest inhibitory activity against respiratory syncytial virus (RSV) and compound 4h displayed modest inhibitory activity against Coxsackie virus B4.
Rotary adenosine triphosphate (ATP)ases are ubiquitous, membrane-bound enzyme complexes involved in biological energy conversion. The first subtype, the so-called F1Fo ATP synthase, predominantly functions as an ATP synthesizing machinery in most bacteria, mitochondria and chloroplasts. The vacuolar subtype of enzyme, the V1Vo ATPase, operates as an ATP driven ion pump in eukaryotic membranes. The subtype found in archaea and some bacteria is called A1Ao ATP (synth)ase and is capable of working in both directions either to synthesize ATP or to generate an ion motive force by consuming the same.
All the three above-mentioned subtypes of rotary ATPases work as nanomolecular machines sharing a conserved mechanism to perform the energy conservation process. The simplest form of these enzymes is the bacterial F1Fo ATP synthase. Here, ions are channelled via the membrane stator subunit a to the rotor ring of the enzyme. After almost a complete rotation of the ring the ions are released again on the other side of the membrane. This rotation is further transmitted via the central stalk to the soluble part of the enzyme, the F1-complex, where conformational changes within the nucleotide binding sites result in the synthesis of ATP from ADP and Pi.
The rotor or c-ring of the enzyme is the key protein complex in mediating transmembrane ion translocation. Several structural and biochemical methods have been applied in the past years to study the rotor rings from many different organisms. The results revealed that the stoichiometry of a c-ring of a given species is constant while it can vary between different species within a range of 8 to 15 c subunits. The c-ring stoichiometry determines directly the number of ions transported through Fo per rotation whereby three molecules of ATP are concurrently synthesized in the water-soluble F1 headgroup. Hence the number of c subunits has an important influence on the bioenergetics of the corresponding enzyme and thus the entire organism.
The c-ring of a rotary ATPase is able to specifically bind either protons (H+) or sodium ions (Na+) as the coupling ion for the enzyme. Several structures are already available revealing the coordination network of both types of rotor rings. In each case ion binding includes a highly-conserved carboxylic acid residue (glutamate or aspartate), in addition to a more varying combination of amino acid residues, whereby Na+ coordination is structurally more demanding than H+ binding.
In the first part of my PhD thesis, I aimed to characterize the F1Fo ATP synthase rotor ring of the opportunistic pathogenic bacterium Fusobacterium nucleatum on a functional and structural level. F. nucleatum is an anaerobic bacterium which uses peptides and amino acids as a primary energy source. It is one of the most frequently occuring bacteria in human body infections and involved in human periodontal diseases.
The protein complex was heterologously expressed within a hybrid ATP synthase in Escherichia coli and purified without an affinity tag for further analysis. Two high resolution X-ray structures of the c-ring were solved at low (5.3) and high (8.7) pH to 2.2 and 2.64 Å, respectively. In both structures, the conserved glutamate is in an ion-locked conformation, revealing that the conformational state of the ion binding carboxylate is not depending on the pH of the crystallization condition, which is in good agreement with previous structural and biochemical studies of other c-rings.
A Na+ ion is present within the c-ring binding site and directly coordinated by four amino acid residues and a structural water molecule. Remarkably, the Na+ is bound by two glutamate residues instead of one as is the case in the I. tartaricus Na+ binding c-ring, of which the first high resolution X-ray structure of a c-ring has been solved in 2005. Thus, a new type of Na+ coordination in an ATP synthase rotor ring with a two-carboxylate ion binding motif is described here, which also occurs in other bacteria, including several pathogens. Na+ specificity of the investigated c-ring was further confirmed by a competitive biochemical labeling reaction performed with a fluorescent ATP synthase inhibitor molecule (N-cyclohexyl-N`-[4(dimethylamino)-α-naphtyl] carbodiimide, NCD-4).
We furthermore complemented our functional and structural data of the F. nucleatum c-ring by computational studies to explore the ion translocation mechanism of this enzyme in more details. We therefore analyzed the protonation state of the second, additional glutamate in the ion binding site. Molecular dynamics (MD) simulations and free-energy calculations indicated that this glutamate is constitutively protonated, in the ion-locked as well as in a simulated, more hydrated open-conformation of the ion binding glutamate as when it is travelling through the a/c-ring interface upon c-ring rotation.
During my thesis, I worked on two different membrane proteins. One is a bacterial secondary transporter and the second is a human mitochondrial calcium channel.
The first part of my thesis involves the structural and biochemical characterization of an L-carnitine/ γ-butyrobetaine antiporter from bacteria called CaiT. The aim of the project was to understand the Na+ independence of CaiT and to determine the crystal structures of CaiT in different conformations to expand the mechanistic understanding of substrate/ product antiport in CaiT.
The study revealed how a positively charged amino acid side chain (arginine 262) in CaiT could structurally and functionally mimic a sodium ion. Additionally, various crystal structures of CaiT obtained in this study demonstrate that the central substrate-binding site is highly dynamic and can accommodate the substrate in various orientations.
In the second part of my thesis, I was able to optimize the expression and purification conditions for the human mitochondrial calcium uniporter or the MCU. Understanding how this channel functions can help us unravel the mechanism of calcium uptake by mitochondria. Secondary structure prediction analysis in combination with mass spectrometry of degraded MCU products obtained during the purification of the full-length protein led to the identification of a stable MCU construct. This study resulted in the successful purification of milligram quantities of stable MCU protein for the first time. Further optimization may be required to obtain more homogenous protein that is amenable for crystallization.
Protein folding in cells is regulated by networks of chaperones, including the heat shock protein 70 (Hsp70) system, which consists of the Hsp40 cochaperone and a nucleotide exchange factor. Hsp40 mediates complex formation between Hsp70 and client proteins prior to interaction with Hsp90. We used mass spectrometry (MS) to monitor assemblies formed between eukaryotic Hsp90/Hsp70/Hsp40, Hop, p23, and a client protein, a fragment of the glucocorticoid receptor (GR). We found that Hsp40 promotes interactions between the client and Hsp70, and facilitates dimerization of monomeric Hsp70. This dimerization is antiparallel, stabilized by post-translational modifications (PTMs), and maintained in the stable heterohexameric client-loading complex Hsp902Hsp702HopGR identified here. Addition of p23 to this client-loading complex induces transfer of GR onto Hsp90 and leads to expulsion of Hop and Hsp70. Based on these results, we propose that Hsp70 antiparallel dimerization, stabilized by PTMs, positions the client for transfer from Hsp70 to Hsp90.
Habituation ist eine der einfachsten Formen des Gedächtnisses. Hierbei handelt es sich um die erlerne Gewöhnung an einen harmlosen Reiz. Dies bedeutet, dass nach mehrfacher wiederholter Repräsentation eines harmlosen Reizes die Reaktion darauf stetig abnimmt, bis sie völlig zum erliegen kommt. Je nach Trainingsprotokoll kann diese Gewöhnung bis zu mehren Tagen andauern. Habituation ist hoch konserviert und ein Verhaltensmuster, dass auch bei sehr einfachen vielzelligen Organismen zu finden ist und untersucht werden kann. Zur Untersuchung des Zusammenspiels innerhalb eines neuronalen Netzwerkes, welches für die Habituation des Rückzugsreflexes (Ausweichreaktion nach Berührung) verantwortlich ist wurde hier der Fadenwurm Caenohabditis elegans (C. elegans) als Modell Organismus verwendet. Aufgrund seines einfachen, nur 302 Zellen umfassenden, Nervensystems eignet sich C. elegans sehr gut für Grundlagenforschung in diesem Bereich. Das neuronale Netzwerk, das verantwortlich ist für den Rückzugsreflex ist in drei Ebenen organisiert. Wahrgenommen wird der Reiz von sensorischen Neuronen (ASH, ALM, AVM, PLM, PVM). Die Weiterleitung erfolgt über verschiedene Interneuronen (AVA, AVB, AD, AVE, PVC) hin zu den Motorneuronen, welche die Muskeln enervieren und somit die Reaktion auf den in erster Ebenen wahrgenommen Reiz auslösen.
Mit Hilfe von optogenetischen Werkzeugen wurde hier Untersucht welche Rolle einzelne Zellen innerhalb dieses Netzwerkes innehaben und an welcher Stelle innerhalb des Netzwerkes die kurzzeitige Habituation des Reizes, nach einem Einfachen Lernprotokoll stattfindet. Zuerst musste eine Möglichkeit gefunden werden die zur Verfügung stehenden optogenetischen Werkzeuge zellspezifisch zu exprimieren. In dieser Arbeit wurden hierfür Rekombinasesysteme verwendet, die es ermöglichten zur Expression eine Kombination aus 2 verschiedenen Promotoren zu verwenden. Beide Promotoren dürfen hierbei nur in einer Zelle, der Zielzelle, überlappen. Es konnte zellspezifische Expression des Kationenkanals Chanelrhodopsin 2 (ChR2) in den beiden Zellparen AVAL/R und ASHL/R (nimmt aversive Reize wahr) erreicht werden.
Zur Untersuchung der Habituation wurde zusätzlich noch ein Wurmstamm verwendet, welcher ChR2 unter dem mec-4 Promotor exprimiert. ChR2 ist hier in den Mechanorezeptorneuronen (MRN) ALM, AVM, PLM und PVM exprimiert. Die hier durchgeführten Experimente deuten darauf hin das den MRNs die Größte Rolle bei der Ausbildung einer Habituation zukommt. Es gibt jedoch auch Hinweise darauf, dass AVA zusätzlich eine Rolle spielt.
Im weiteren Verlauf der Arbeit wurde die Rolle von AVA genauer untersucht. AVA gilt als der Hauptsignalgeber für eine Rückwärtsbewegung (spontan und nach Reizempfang). Es konnte gezeigt werden dass eine Unterbrechung der ’Gap Junktionen’ zwischen AVA und PVC eine stärkere Reaktion zur Folge haben. AVA scheint also durch PVC inhibiert zu werden. Ebenfalls mit AVA direkt interagierende Neuronen sind AVD und AVE. Mit den hier zur Verfügung stehenden Mitteln konnte die genaue Modulation von AVA durch diese Zellen jedoch nicht gezeigt werden.
In dieser Arbeit konnte der Grundstein für eine funktionale Aufklärung des Nervensystems von C. elegans gelegt werden. Vor allem durch die Möglichkeit der zellspezifischen Expression kann es zukünftig gelingen das Zusammenspiel der einzelnen Nervenzellen und ihren Anteil an einem bestimmtem Verhalten zu Untersuchen.
Pflanzen, aber auch einige Bakterien und Archäen verfügen über hocheffiziente Mechanismen, Licht in Energie umzuwandeln. Photovoltaik-Zellen reichen an die Perfektion dieser natürlichen Systeme noch lange nicht heran. Deshalb versuchen Forscher, mit ultraschnellen spektroskopischen Methoden der Natur in die Karten zu schauen und von ihr zu lernen.
Die Glühbirne hat ausgedient. Auch Energiesparlampen sind nur eine Übergangslösung. Große Hoffnungen richten sich auf organische Leuchtdioden, zumal man daraus auch großflächige und biegsame Displays und Flachbildschirme herstellen kann. Für eines der größten Probleme, das Ausbleichen der blauen Leuchtstoffe, findet man immer bessere Lösungen. Anwendungen, die heute noch wie Science-Fiction klingen, rücken damit in erreichbare Nähe.
Der Auflösung mikroskopischer Verfahren ist durch die Beugungsgrenze eine natürliche Schranke gesetzt. Strukturen, die näher als die halbe Wellenlänge des verwendeten Lichts zusammenliegen, können nicht aufgelöst werden. Doch Forscher haben einen Weg gefunden, diese Grenze zu umgehen. Die entstehenden Bilder ähneln dem Pointillismus in der Malerei.
Introduction: Interferon alpha (IFNα) is routinely used in the clinical practice for adjuvant systemic melanoma therapy. Understanding the molecular mechanism of IFNα effects and prediction of response in the IFNα therapy regime allows initiation and continuation of IFNα treatment for responder and exclusion of non-responder to avoid therapy inefficacy and side-effects. The transporter protein associated with antigen processing-1 (TAP1) is part of the MHC class I peptide-loading complex, and important for antigen presentation in tumor and antigen presenting cells. In the context of personalized medicine, we address this potential biomarker TAP1 as a target of IFNα signalling.
Results: We could show that IFNα upregulates TAP1 expression in peripheral blood mononuclear cells (PBMCs) of patients with malignant melanoma receiving adjuvant high-dose immunotherapy. IFNα also induced expression of TAP1 in mouse blood and tumor tissue and suppressed the formation of melanoma metastasis in an in vivo B16 tumor model. Besides its expression, TAP binding affinity and transport activity is induced by IFNα in human monocytic THP1 cells. Furthermore, our data revealed that IFNα clearly activates phosphorylation of STAT1 and STAT3 in THP1 and A375 melanoma cells. Inhibition of Janus kinases abrogates the IFNα-induced TAP1 expression. These results suggest that the JAK/STAT pathway is a crucial mediator for TAP1 expression elicited by IFNα treatment.
Conclusion: We suppose that silencing of TAP1 expression provides tumor cells with a mechanism to escape cytotoxic T-lymphocyte recognition. The observed benefit of IFNα treatment could be mediated by the shown dual effect of TAP1 upregulation in antigen presenting cells on the one hand, and of TAP1 upregulation in ‘silent’ metastatic melanoma cells on the other hand. In conclusion, this work contributes to a better understanding of the mode of action of IFNα which is essential to identify markers to predict, assess and monitor therapeutic response of IFNα treatment in the future.
Recent studies indicate that the abnormal microenvironment of tumors may play a critical role in carcinogenesis, including lung cancer. We comprehensively assessed the number of stromal cells, especially immune/inflammatory cells, in lung cancer and evaluated their infiltration in cancers of different stages, types and metastatic characteristics potential. Immunohistochemical analysis of lung cancer tissue arrays containing normal and lung cancer sections was performed. This analysis was combined with cyto-/histomorphological assessment and quantification of cells to classify/subclassify tumors accurately and to perform a high throughput analysis of stromal cell composition in different types of lung cancer. In human lung cancer sections we observed a significant elevation/infiltration of total-T lymphocytes (CD3+), cytotoxic-T cells (CD8+), T-helper cells (CD4+), B cells (CD20+), macrophages (CD68+), mast cells (CD117+), mononuclear cells (CD11c+), plasma cells, activated-T cells (MUM1+), B cells, myeloid cells (PD1+) and neutrophilic granulocytes (myeloperoxidase+) compared with healthy donor specimens. We observed all of these immune cell markers in different types of lung cancers including squamous cell carcinoma, adenocarcinoma, adenosquamous cell carcinoma, small cell carcinoma, papillary adenocarcinoma, metastatic adenocarcinoma, and bronchioloalveolar carcinoma. The numbers of all tumor-associated immune cells (except MUM1+ cells) in stage III cancer specimens was significantly greater than those in stage I samples. We observed substantial stage-dependent immune cell infiltration in human lung tumors suggesting that the tumor microenvironment plays a critical role during lung carcinogenesis. Strategies for therapeutic interference with lung cancer microenvironment should consider the complexity of its immune cell composition.
The title compound, [Li2(C25H23BN4OP)2], features a centrosymmetric dimeric complex. The four-memberered Li2O2 ring is exactly planar due to symmetry. The Li atom is four-coordinated by two O atoms and by two N atoms of two different pyrazole rings. The dihedral angle between two pyrazole rings bonded to the same B atom is 45.66 (9)°. The B—N—N—Li—N—N metalla ring adopts a boat conformation. The crystal packing is stabilized by van der Waals interactions only.
The structure of the title compound, C8H16N4, which consists of four fused seven-membered rings, has been redetermined at 173 K. This redetermination corrects the orientation of two H atoms, which were located at unrealistic positions in the original room-temperature study [Murray-Rust (1974[Murray-Rust, P. (1974). J. Chem. Soc. Perkin Trans. 2, pp. 1136-1141.]). J. Chem. Soc. Perkin Trans. 2, pp. 1136–1141]. The complete molecule is generated by -42m symmetry, with one quarter of a molecule [one N atom (site symmetry m), two C atoms (one with site symmetry m and the other with site symmetry 2) and two H atoms] in the asymmetric unit. No directional interactions beyond van der Waals contacts are apparent in the crystal structure.
Single crystals of the title compound, C10H11NO4, an intermediate in the industrial synthesis of yellow azo pigments, were obtained from the industrial production. The molecules crystallize as centrosymmetic dimers connected by two symmetry-related N—H⋯O=C hydrogen bonds. Each molecule also contains an intramolecular N—H⋯O=C hydrogen bond. The dimers form stacks along the a-axis direction. Neighbouring stacks are arranged into a herringbone structure.
In the title compound, C40H76Si, the Si atom is located on a special position of site symmetry -4. Thus, there is just a quarter of a molecule in the asymmetric unit. The C=C double bonds exhibit a trans configuration. The Si atom and the tert-butyl group are located on the same side of the plane formed by the C=C double bond and its four substituents. The crystal packing shows no short contacts between the molecules and despite the low crystal density (0.980 Mg m−3), there are no significant voids in the structure.
In the title compound, C19H24N2O2, a di-Mannich base derived from 2-methylphenol and 1,3,6,8-tetraazatricyclo[4.4.1.13,8]dodecane, the imidazolidine ring adopts a twist conformation, with a twist about the ring N—C bond [C—N—C—C torsion angle = −44.34 (14)°]. The two 2-hydroxy-3-methylbenzyl groups are located in trans positions with respect to the imidazolidine fragment. The structure displays two intramolecular O—H⋯N hydrogen bonds, which each form an S(6) ring motif. In the crystal, the molecules are linked by weak C—H⋯O interactions with a bifurcated acceptor, forming a three-dimensional network.
The title compound, C12H20N4O, undergoes a phase transition on cooling. The room-temperature structure is tetragonal (P43212, Z′ = 1), with the methoxybornyl group being extremely disordered. Below 213 K the structure is orthorhombic (P212121, Z′ = 2), with ordered molecules. The two independent molecules (A and B) have very similar conformations; significant differences only occur for the torsion angles about the Cbornyl—Ctetrazole bonds. The independent molecules are approximately related by the pseudo-symmetry relation: xB = −1/4 + yA, yB = 3/4 - xA and zB = 1/4 + zA. In the crystal, molecules are connected by N—H⋯N hydrogen bonds between the tetrazole groups, forming a pseudo-43 helix parallel to the c-axis direction. The crystal studied was a merohedral twin with a refined twin fraction value of 0.231 (2).
The title molecule, C34H28I4·4C6H6, has crystallographic 4 symmetry and crystallizes with four symmetry-related benzene solvent molecules. The phenyl group is eclipsed with one of the adamantane C—C bonds. The tetraphenyladamantane units and the benzene solvent molecules are connected by weak intermolecular phenyl–benzene C—H⋯π and benzene–benzene C—H⋯π interactions. In the crystal, molecules are linked along the c-axis direction via the iodophenyl groups by a combination of weak intermolecular I⋯I [3.944 (1) Å] and I⋯π(phenyl) [3.608 (6) and 3.692 (5) Å] interactions.