Isatin-Schiff base copper(II) complexes-A DFT study of the metal-ligand bonding situation

Herein, we report results of calculations based on density functional theory (BP86/TZVP) of a set of isatin-Schiff base copper(II) and related complexes, 1-12, that have shown significant pro-apoptotic activity toward diverse tumor cells. The interaction of the copper(II) cation with different ligands has been investigated at the same level of theory. The strength and character of the Cu(II)-L bonding was characterized by metal-ligand bond lengths, vibrational frequencies, binding energies, ligand deformation energies, and natural population analysis. The metal-ligand bonding situation was also characterized by using two complementary topological approaches, the quantum theory of atoms-in-molecules (QTAIM) and the electron localization function (ELF). The calculated electronic g-tensor and hyperfine coupling constants present significant agreement with the EPR experimental data. The calculated parameters pointed to complex 10 as the most stable among the isatin-Schiff base copper(II) species, in good agreement with experimental data that indicate this complex as the most reactive in the series. (C) 2011 Wiley Periodicals, Inc. Int J Quantum Chem, 2012

Two structurally discrete GH7-cellobiohydrolases compete for the same cellulosic substrate fiber

Background: Cellulose consisting of arrays of linear beta-1,4 linked glucans, is the most abundant carbon-containing polymer present in biomass. Recalcitrance of crystalline cellulose towards enzymatic degradation is widely reported and is the result of intra-and inter-molecular hydrogen bonds within and among the linear glucans. Cellobiohydrolases are enzymes that attack crystalline cellulose. Here we report on two forms of glycosyl hydrolase family 7 cellobiohydrolases common to all Aspergillii that attack Avicel, cotton cellulose and other forms of crystalline cellulose. Results: Cellobiohydrolases Cbh1 and CelD have similar catalytic domains but only Cbh1 contains a carbohydrate-binding domain (CBD) that binds to cellulose. Structural superpositioning of Cbh1 and CelD on the Talaromyces emersonii Cel7A 3-dimensional structure, identifies the typical tunnel-like catalytic active site while Cbh1 shows an additional loop that partially obstructs the substrate-fitting channel. CelD does not have a CBD and shows a four amino acid residue deletion on the tunnel-obstructing loop providing a continuous opening in the absence of a CBD. Cbh1 and CelD are catalytically functional and while specific activity against Avicel is 7.7 and 0.5 U. mg prot-1, respectively specific activity on pNPC is virtually identical. Cbh1 is slightly more stable to thermal inactivation compared to CelD and is much less sensitive to glucose inhibition suggesting that an open tunnel configuration, or absence of a CBD, alters the way the catalytic domain interacts with the substrate. Cbh1 and CelD enzyme mixtures on crystalline cellulosic substrates show a strong combinatorial effort response for mixtures where Cbh1 is present in 2: 1 or 4: 1 molar excess. When CelD was overrepresented the combinatorial effort could only be partially overcome. CelD appears to bind and hydrolyze only loose cellulosic chains while Cbh1 is capable of opening new cellulosic substrate molecules away from the cellulosic fiber. Conclusion: Cellobiohydrolases both with and without a CBD occur in most fungal genomes where both enzymes are secreted, and likely participate in cellulose degradation. The fact that only Cbh1 binds to the substrate and in combination with CelD exhibits strong synergy only when Cbh1 is present in excess, suggests that Cbh1 unties enough chains from cellulose fibers, thus enabling processive access of CelD.

Salts of the anti-HIV drug lamivudine with phthalic and salicylic acids

Salts of the anti-HIV drug lamivudine, with phthalic acid and salicylic acid as counterions, were investigated in this study. Neither the packing of the (lamivudine)(+)(phthalic acid)(-) ion pairs nor the conformation of the lamivudine moiety itself were similar to those found in other multicomponent molecular salts of the drug, such as hydrogen maleate and saccharinate ones, even though all three salts crystallize in the same P2(1)2(1)2(1) orthorhombic space group with similar unit cell metrics. Lamivudine salicylate assumes a different crystal structure to those of the hydrogen maleate and saccharinate salts, crystallizing in the P2(1) monoclinic space group as a monohydrate whose (lamivudine)(+)(salicylic acid)(-) ion pair is assembled through two hydrogen bonds with cytosine as a dual donor to both oxygens of the carboxylate, such as in the pairing of lamivudine with a phthalic acid counterion. In lamivudine salicylate monohydrate, the drug conformation is related to the hydrogen maleate and saccharinate salts. However, such a conformational similarity is not related to the intermolecular interaction patterns. Lamivudine and water molecules alternate into helical chains in the salicylate salt monohydrate.

Probing the relationships between molecular conformation and intermolecular contacts in N,N-dibenzyl-N '-(furan-2-carbonyl)thiourea

In the crystal structure of the title compound, C20H18N2O2S, molecules are linked by bifurcated C-H center dot center dot center dot O hydrogen-bond interactions, giving rise to chains whose links are composed of alternating centrosymmetrically disposed pairs of molecules and characterized by R-2(2)(10) and R-2(2)(20) hydrogen-bonding motifs. Also, N-H center dot center dot center dot S hydrogen bonds form infinite zigzag chains along the [010] direction, which exhibit the C(4) motif. Hirshfeld surface and fingerprint plots were used to explore the intermolecular interactions in the crystal structure. This analysis confirms the important role of C-H center dot center dot center dot O hydrogen bonds in the molecular conformation and in the crystal structure, providing a potentially useful tool for a full understanding of the intermolecular interactions in acylthiourea derivatives.

Atomistic Study of Water Confined in Silica.

In this work, we have used a combined of atomistic simulation methods to explore the effects of confinement of water molecules between silica surfaces. Firstly, the mechanical properties of water severe confined (~3A) between two silica alpha-quartz was determined based on first principles calculations within the density functional theory (DFT). Simulated annealing methods were employed due to the complex potential energry surface, and the difficulties to avoid local minima. Our results suggest that much of the stiffness of the material (46%) remains, even after the insertion of a water monolayer in the silica. Secondly, in order to access typical time scales for confined systems, classical molecular dynamics was used to determine the dynamical properties of water confined in silica cylindrical pores, with diameters varying from 10 to 40A. in this case we have varied the passivation of the silica surface, from 13% to 100% of SiOH, and the other terminations being SiOH2 and SiOH3, the distribution of the different terminations was obtained with a Monte Carlo simulation. The simulations indicates a lowering of the diffusion coefficientes as the diameter decreases, due to the structuration of hydrogen bonds of water molecules; we have also obtained the density profiles of the confined water and the interfacial tension.

Studio di biomateriali usati come scaffold per Tissue Engineering e loro caratterizzazione con tecniche spettroscopiche vibrazionali e di analisi termica

This research investigated someone of the main problems connected to the application of Tissue Engineering in the prosthetic field, in particular about the characterization of the scaffolding materials and biomimetic strategies adopted in order to promote the implant integration. The spectroscopic and thermal analysis techniques were usefully applied to characterize the chemico-physical properties of the materials such as – crystallinity; – relative composition in case of composite materials; – Structure and conformation of polymeric and peptidic chains; – mechanism and degradation rate; – Intramolecular and intermolecular interactions (hydrogen bonds, aliphatic interactions). This kind of information are of great importance in the comprehension of the interactions that scaffold undergoes when it is in contact with biological tissues; this information are fundamental to predict biodegradation mechanisms and to understand how chemico-physical properties change during the degradation process. In order to fully characterize biomaterials, this findings must be integrated by information relative to mechanical aspects and in vitro and in vivo behavior thanks to collaborations with biomedical engineers and biologists. This study was focussed on three different systems that correspond to three different strategies adopted in Tissue Engineering: biomimetic replica of fibrous 3-D structure of extracellular matrix (PCL-PLLA), incorporation of an apatitic phase similar to bone inorganic phase to promote biomineralization (PCL-HA), surface modification with synthetic oligopeptides that elicit the interaction with osteoblasts. The characterization of the PCL-PLLA composite underlined that the degradation started along PLLA fibres, which are more hydrophylic, and they serve as a guide for tissue regeneration. Moreover it was found that some cellular lines are more active in the colonization of the scaffold. In the PCL-HA composite, the weight ratio between the polymeric and the inorganic phase plays an essential role both in the degradation process and in the biomineralization of the material. The study of self-assembling peptides allowed to clarify the influence of primary structure on intermolecular and intermolecular interactions, that lead to the formation of the secondary structure and it was possible to find a new class of oligopeptides useful to functionalize materials surface. Among the analytical techniques used in this study, Raman vibrational spectroscopy played a major role, being non-destructive and non-invasive, two properties that make it suitable to degradation studies and to morphological characterization. Also micro-IR spectroscopy was useful in the comprehension of peptide structure on oxidized titanium: up to date this study was one of the first to employ this relatively new technique in the biomedical field.

De Novo Design of secondary structures: Synthesis and conformational studies

Chemists have long sought to extrapolate the power of biological catalysis and recognition to synthetic systems. These efforts have focused largely on low molecular weight catalysts and receptors; however, biological systems themselves rely almost exclusively on polymers, proteins and RNA, to perform complex chemical functions. Proteins and RNA are unique in their ability to adopt compact, well-ordered conformations, and specific folding provides precise spatial orientation of the functional groups that comprise the “active site”. These features suggest that identification of new polymer backbones with discrete and predictable folding propensities (“foldamers”) will provide a basis for design of molecular machines with unique capabilities. The foldamer approach complements current efforts to design unnatural properties into polypeptides and polynucleotides. The aim of this thesis is the synthesis and conformational studies of new classes of foldamers, using a peptidomimetic approach. Moreover their attitude to be utilized as ionophores, catalysts, and nanobiomaterials were analyzed in solution and in the solid state. This thesis is divided in thematically chapters that are reported below. It begins with a very general introduction (page 4) which is useful, but not strictly necessary, to the expert reader. It is worth mentioning that paragraph I.3 (page 22) is the starting point of this work and paragraph I.5 (page 32) isrequired to better understand the results of chapters 4 and 5. In chapter 1 (page 39) is reported the synthesis and conformational analysis of a novel class of foldamers containing (S)-β3-homophenylglycine [(S)-β3-hPhg] and D- 4-carboxy-oxazolidin-2-one (D-Oxd) residues in alternate order is reported. The experimental conformational analysis performed in solution by IR, 1HNMR, and CD spectroscopy unambiguously proved that these oligomers fold into ordered structures with increasing sequence length. Theoretical calculations employing ab initio MO theory suggest a helix with 11-membered hydrogenbonded rings as the preferred secondary structure type. The novel structures enrich the field of peptidic foldamers and might be useful in the mimicry of native peptides. In chapter 2 cyclo-(L-Ala-D-Oxd)3 and cyclo-(L-Ala-DOxd) 4 were prepared in the liquid phase with good overall yields and were utilized for bivalent ions chelation (Ca2+, Mg2+, Cu2+, Zn2+ and Hg2+); their chelation skill was analyzed with ESI-MS, CD and 1HNMR techniques and the best results were obtained with cyclo-(L-Ala-D-Oxd)3 and Mg2+ or Ca2+. Chapter 3 describes an application of oligopeptides as catalysts for aldol reactions. Paragraph 3.1 concerns the use of prolinamides as catalysts of the cross aldol addition of hydroxyacetone to aromatic aldeydes, whereas paragraphs 3.2 and 3.3 are about the catalyzed aldol addition of acetone to isatins. By means of DFT and AIM calculations, the steric and stereoelectronic effects that control the enantioselectivity in the cross-aldol addition of acetone to isatin catalysed by L-proline have been studied, also in the presence of small quantities of water. In chapter 4 is reported the synthesis and the analysis of a new fiber-like material, obtained from the selfaggregation of the dipeptide Boc-L-Phe-D-Oxd-OBn, which spontaneously forms uniform fibers consisting of parallel infinite linear chains arising from singleintermolecular N-H···O=C hydrogen bonds. This is the absolute borderline case of a parallel β-sheet structure. Longer oligomers of the same series with general formula Boc-(L-Phe-D-Oxd)n-OBn (where n = 2-5), are described in chapter 5. Their properties in solution and in the solid state were analyzed, in correlation with their attitude to form intramolecular hydrogen bond. In chapter 6 is reported the synthesis of imidazolidin-2- one-4-carboxylate and (tetrahydro)-pyrimidin-2-one-5- carboxylate, via an efficient modification of the Hofmann rearrangement. The reaction affords the desired compounds from protected asparagine or glutamine in good to high yield, using PhI(OAc)2 as source of iodine(III).

Wasserstoffbrücken-gebundene Netzwerke

Die supramolekularen Organisationen von 5-n-Alkoxyisophthaläuren und 2,5-Di-n-alkoxyterephthalsäuren bilden durch sekundäre Wechselwirkungen in Form van der Waals-Kräften und Wasserstoffbrückenbindungen hochgeordnete lamellare Strukturen, die sowohl mittels Röntgen-kristallgraphie als auch durch Rastertunnelmikroskopie (STM) visualisierbar sind. Diese Art der Aggregationsmuster ermöglicht den potentiell reaktiven Gruppen (Diacetylene, Zimtsäuren) in der Alkoxykette, einen optimalen Abstand und geometrische Orientierungen im Kristall einzunehmen, um lichtinduzierte Reaktionen im Festkörper durchzuführen. Einführung einer Amidgruppe an die Alkoxykette erweitert dieses Konzept, durch Erhalt einer zusätzlichen Wasserstoffbrückenbindung im hydrophoben Bereich der Organisation.Die Stabilität solche supramolekularer Aggregate kann durch Einführung semifluorierter Alkoxyketten stark beeinflußt werden, da die Inkompatibilität der pefluorierten Gruppen durch viele organische Verbindungen das Gleichgewicht nicht-kovalenter Wechselwirkung drastisch verändert. Diese Eigenschaft der semifluorierten 5-n-Alkoxyisopthalsäuren und 2,5-Di-n-alkoxyterephthalsäuren zeigt sich erstaunlicherweise jedoch nur bei einem großen Anteil des perfluorierten Alkylteils als der des nicht-perfluorierten. Da sich dann die perfluorierten Alkylteile untereinander organisieren können, erscheint die Änderung der Assoziationsstruktur von interdigitiert-lamellar zu nicht-interdigitiert-lamellar plausibel.Das gezielte Design eines neuen Organisationsmusters der 5-n-Alkoxyisophthalsäuren gelang durch Brechung der Symmetrie dieses Bausteins. Die Einführung zweier Alkoxyketten ließ die zweidimensionale Anordnung der wasserstoffbrückengebundenen Aromate unverändert, resultierte aber mit Ausbruch einer Alkoxykette aus dieser Ebene.

Complexes of polyelectrolytes with defined charge distance and different dendrimer counterions

Complexes of polyelectrolytes with defined charge distance and different dendrimer counterions Magdalena Chelmecka Max Planck Institute for Polymer Research; Ackermannweg 10; D-55128 Mainz ; Tel.: (+49) 06131- 379 – 226 A study of complexes in solution is of interest to investigate whether the formation of well-defined assemblies like in classical surfactant systems is possible. Aim of this thesis is to investigate the electrostatic self-assembly of linear polycations of varying charge distance with “large” counterions of varying architecture. We especially investigate the morphology of objects formed, but also their stability under salt free condition and after low molecular mass salt addition. As polycations, Poly(dialkylimino)-alkylene salts (Ionenes) I65MeBr and I25MeBr were chosen. Ionenes are synthesized via Menschutkin reaction and characterized by standard methods. Counterions are Polyamidoamine (PAMAM) dendrimers of generations G2.5, G5.5, G7.5 with -COONa surface groups and shape-persistent, Polyphenylene dendrimers of generation G1 with surface -COOH groups. A complex interplay of interactions is expected to direct the self assembly via electrostatic interaction, geometric factors, hydrophobic interaction or hydrogen bonds. Methods used for the investigation of complexes are: UV-spectroscopy, pH-metric techniques, dynamic and static light scattering, small angle neutron scattering,  potential measurements and potentiometric titration. Under certain conditions, (i.e. charge ratio of compounds, charge density of ionene and dendrimer also concentration of sample) polyelectrolyte systems composed of ionenes and dendrimers build complexes in solution. System compounds are typical polyelectrolytes, but structures which they build behave not usual for typical polyelectrolytes. In a one diffusion mode regime aggregates of about 100 nm hydrodynamic radius have been found. Such aggregates are core-shell or anisotropic core shell structures in the case of ionenes/PAMAM dendrimers complexes. These complexes are stable even at high ionic strength. In case of ionenes with poly(phenylene) dendrimers, hard sphere-like objects or spherical objects with hairy-like surface have been found in a one diffusion mode regime. Their stability at high ionic strength is lower. For the ionenes/poly(phenylene) dendrimers systems one transition point has been found from one to two diffusion processes, towards increasing ionene concentration, i.e. for the samples with fixed dendrimer concentration towards increasing ionic strength. For the diffusion profile of ionene/PAMAM dendrimers in most cases two transition regimes are observed. One at very low ionene concentration, the second one at high ionene concentrations, which again means for the samples with fixed dendrimer concentration, also at higher ionic strength. Both two mode regimes are separated by the one mode regime. As was confirmed experimentally, the one diffusion mode regime is caused by the motion of well defined assemblies. The two diffusion mode regimes are caused by the movement of different sized species in solution, large aggregates and middle-size aggregates (oligoaggregates). The location and also the number of transition points in the diffusion profiles is dependent on the ionene to dendrimer charge ratio, charge density of the compounds and concentration. No influence of the molecular mass of the ionene has been found. The aggregates are found to be charged on the surface, however this surface charge does not significantly influence the diffusion properties of the system.

Characterisation of supramolecular structures by novel recoupling methods in solid-state NMR

In this work, solid-state NMR methods suitable for the investigation of supramolecular systems were developed and improved. In this context, special interest was focussed on non-covalent interactions responsible for the formation of supramolecular structures, such as pi-pi interacions and hydrogen-bonds. In the first part of this work, solid-state NMR methods were presented that provide information on molecular structure and motion via the investigation of anisotropic interactions, namely quadrupole and dipole-dipole couplings, under magic-angle spinning conditions. A two-dimensional 2H double quantum experiment was developed, which is performed under off magic-angle conditions and correlates 2H isotropic chemical shifts with quasistatic DQ-filtered line shapes. From the latter, the quadrupole coupling parameters of samples deuterated at multiple sites can be extracted in a site-selective fashion. Furthermore, 7Li quadrupole parameters of lithium intercalated into TiO2 were determined by NMR experiments performed under static and MAS conditions, and could provide information on the crystal geometry. For the determination of 7Li-7Li dipole-dipole couplings, multiple-quantum NMR experiments were performed. The 1H-13C REREDOR experiment was found to be capable of determining strong proton-carbon dipole-dipole couplings with an accuracy of 500~Hz, corresponding to a determination of proton-carbon chemical-bond lengths with picometer accuracy In the second part of this work, solid-state NMR experiments were combined with quantum-chemical calculations in order to aid and optimise the interpretation of experimental results. The investigations on Calix[4]hydroquinone nanotubes have shown that this combined approach can provide information on the presence of disordered and/or mobile species in supramolecular structures. As a second example, C3-symmetric discs arranging in helical columnar stacks were investigated. In these systems, 1H chemical shifts experience large pi-shifts due to packing effects, which were found to be long-ranged. Moreover, quantum-chemical calculations revealed that helicity in these systems is induced by the propeller-like conformation of the core of the molecules.

Thermodynamics of aqueous biopolymer solutions and fractionation of Dextran

Flory-Huggins interaction parameters and thermal diffusion coefficients were measured for aqueous biopolymer solutions. Dextran (a water soluble polysaccharide) and bovine serum albumin (BSA, a water soluble protein) were used for this study. The former polymer is representative for chain macromolecules and the latter is for globular macromolecules. The interaction parameters for the systems water/dextran and water/BSA were determined as a function of composition by means of vapor pressure measurements, using a combination of headspace sampling and gas chromatography (HS-GC). A new theoretical approach, accounting for chain connectivity and conformational variability, describes the observed dependencies quantitatively for the system water/dextran and qualitatively for the system water/BSA. The phase diagrams of the ternary systems water/methanol/dextran and water/dextran/BSA were determined via cloud point measurements and modeled by means of the direct minimization of the Gibbs energy using the information on the binary subsystems as input parameters. The thermal diffusion of dextran was studied for aqueous solutions in the temperature range 15 < T < 55 oC. The effects of the addition of urea were also studied. In the absence of urea, the Soret coefficient ST changes its sign as T is varied; it is positive for T > 45.0 oC, but negative for T < 45.0 oC. The positive sign of ST means that the dextran molecules migrate towards the cold side of the fluid; this behavior is typical for polymer solutions. While a negative sign indicates the macromolecules move toward the hot side; this behavior has so far not been observed with any other binary aqueous polymer solutions. The addition of urea to the aqueous solution of dextran increases ST and reduces the inversion temperature. For 2 M urea, the change in the sign of ST is observed at T = 29.7 oC. At higher temperature ST is always positive in the studied temperature range. To rationalize these observations it is assumed that the addition of urea opens hydrogen bonds, similar to that induced by an increase in temperature. For a future extension of the thermodynamic studies to the effects of poly-dispersity, dextran was fractionated by means of a recently developed technique called Continuous Spin Fractionation (CSF). The solvent/precipitant/polymer system used for the thermodynamic studies served as the basis for the fractionation of dextran The starting polymer had a weight average molar mass Mw = 11.1 kg/mol and a molecular non-uniformity U= Mw / Mn -1= 1.0. Seventy grams of dextran were fractionated using water as the solvent and methanol as the precipitant. Five fractionation steps yielded four samples with Mw values between 4.36 and 18.2 kg/mol and U values ranging from 0.28 to 0.48.

Synthetical engineering of supramolecular properties of large polycyclic aromatic hydrocarbons

An important property for devices is the charge-carrier mobility values for discotic organic materials like hexa-peri-hexabenzocoronenes. A close relation exists between the degree of their columnar self-arrangement of the molecules and their mobilities. Within this first step an induction of a higher order via hydrogen-bonding was considered, which mainly pointed towards the improvement of the intracolumnar stacking of the materials. For the analytics a broad range of methods was used including differential scanning calorimetry (DSC), wide-angle X-ray diffractometry (WAXS), solid-state NMR spectroscopy and scanning tunneling microscopy (STM). Indeed, a specific influence of the hydrogen-bonds could be identified, although in several cases by the cost of a severe reduction of solubility and processability. This effect was dampened by the addition of a long alkyl chain next to the hydrogen-bond exerting functional group, which resulted in an improved columnar arrangement by retention of processability. In contrast to the before mentioned example of inducing a higher intracolumnar order by hydrogen-bonding, the focus was also be set upon larger aromatic systems. The charge-carrier mobility is also in close relation to the size of the aromatic core and larger π-areas are expected to lead to improved mobilities. For photovoltaic applications a high extinction coefficient over a broad range of the spectrum is favorable, which can also be achieved by enlarging the aromatic core component. In addition the stronger π-interactions between the aromatic core components should yield an improved columnar stability and order. However the strengthening of the π-interactions between the aromatic core components led to a reduction of the solubility and the processability due to the stronger aggregation of the molecules. This required the introduction of efficiently solubilizing features in terms of long alkyl chains in the corona of the aromatic entity, in combination of a distortion of the aromatic core moiety by bulky tert-butyl groups. By this approach not only the processing and cleaning of the materials with standard laboratory techniques became possible, but moreover the first structure-rich UV/vis and a resolved 1H-NMR spectra for an aromatic system two times larger than hexa-peri-hexabenzocoronene were recorded. The bulk properties in an extruded fiber as well as on the surface showed a columnar self-assembly including a phase in which a homeotropic alignment on a substrate was observed, which turns the material into an interesting candidate for future applications in electronic devices.

Selektiv deblockierbare 2,6-Diamino-2,6-didesoxy-D-glucose-Scaffolds für die kombinatorische Synthese potentieller RNA-Liganden

Aminoglydosid-Antibiotika wie Neomycin B oder Cyclopeptid-Antibiotike wie Viaomycin sind dafür bekannt, daß sie selektiv an RNA binden können. Diese Interaktionen beruhen sowohl auf elektrostatischen Wechselwirkungen als auch auf H-Brücken-Bindungen. Des weiteren ist die definierte räumliche Anordnung von Donor- und Akzeptor-Resten in den Strukturen der RNA-Liganden wichtig für die Affinität. Eine Möglichkeit natürliche RNA-Liganden zu imitieren ist der Einsatz polyfunktioneller Template wie zum Beispiel das 2,6-Diamino-2,6-didesoxy-D-glucose-Scaffold. Mit Hilfe dieser Scaffolds können dann verschiedene positv geladene Reste und Donatoren sowie Akzeptoren für H-Brücken-Bindungen oder auch Interkalatoren räumlich definiert präsentiert werden. Für die unabhängige Funktionalisierung einer jeden Position ist ein Satz orthogonal stabiler Schutzgruppen nötig, wobei eine Hydroxylguppe durch einen Anker ersetzt wird, der eine Anbindung des Scaffolds an einen polymeren Träger ermöglicht. Das neu entwickelte 2,6-Diamino-2,6-didesoxy-D-glucose-Scaffold ist das erste Monosaccharid-Templat, das in allen fünf Positionen mit orthogonal stabilen Schutzgruppen blockiert ist. Alle Positionen könne in beliebiger Reihenfolge selektiv deblockiert und anschließend derivatisiert werden. Das Scaffold kann mit Aminosäuren, Guanidinen oder Interkalatoren umgesetzt werden, um so natürlich vorkommende RNA-bindende Aminoglycoside oder Peptide zu imitieren. Aufbauend auf diesem Monosaccharid-Templat wurde eine Bibliothek von über 100 potentiellen RNA-Liganden synthetisiert, die im Rahmen des Sonderforschungsbereichs 579 (RNA-Liganden-Wechselwirkungen) in Zellassays auf ihre Fähigkeit zur Hemmung der Tat/TAR-Wechselwirkung untersucht wurden, wobei bis jetzt 9 Verbindungen mit einer hemmenden Wirkung im micromolaren Bereich gefunden wurden.

Untersuchung der Bindung des humanen Papillomvirus HPV16 an Heparansulfate der Zelloberfläche

In 99,7% aller Zervixkarzinome kann die DNA humaner Papillomviren (HPV) nachgewiesen werden, die somit den Hauptauslöser für eine der häufigsten Krebserkrankungen bei Frauen weltweit darstellen. HPV16 ist verantwortlich für etwa 50% aller Zervixkarzinome. Für die Infektion von Zellen mit HPV16 ist die Interaktion mit Heparansulfatproteoglykanen der Zelloberfläche essentiell. Um Aminosäuren auf der Oberfläche des majoren Kapsidproteins L1 von HPV16 zu identifizieren, die zu dieser Interaktion beitragen, wurden im Rahmen dieser Arbeit zahlreiche Punktmutanten hergestellt und analysiert. Der Austausch der drei Lysine K278, K356 und K361 zu Alaninen führte zu signifikant verminderter Zell-, Heparin- und Heparansulfatbindung, die noch weiter reduziert wurde, wenn zwei oder drei der Lysine gleichzeitig mutiert waren. Auch die Infektiosität der mutanten Pseudovirionen war stark beeinträchtigt, die Trippelmutante zeigte nur noch 5% Infektiosität. Diese Ergebnisse demonstrieren, dass die drei Lysine gemeinsam die Bindestelle für Heparansulfate bilden. Ihr Austausch zu Argininen beeinflusste die Infektiosität der Partikel hingegen nicht, was bestätigt, dass die Interaktion mit Heparansulfaten von der positiven Ladungsdichte abhängt und nicht sequenzspezifisch ist. Die drei Lysine befinden sich auf der Spitze des HPV16-Kapsomers in einer flachen Tasche, die aufgrund ihrer Struktur bereits früher als potentielle Rezeptorbindestelle vorgeschlagen wurde. Fab-Fragmente des bindungsneutralisierenden Antikörpers H16.56E, dessen Epitop in direkter Nachbarschaft der Lysine liegt, inhibierten die heparansulfatvermittelte Zellbindung viraler Partikel. Auch Epitope anderer bindungsneutralisierender Antikörper befinden sich in der Nähe. Dies untermauert die Hypothese, dass die Lysine K278, K356 und K361 die Heparansulfatbindestelle von HPV16 bilden. Der Austausch von Threoninen, die genau zwischen den Lysinen liegen, hatte keine Auswirkung auf Bindung der Partikel und Infektiosität. Sie könnten jedoch durch die Bildung von Wasserstoffbrücken die Bindung an Heparansulfat stabilisieren. Die Bedeutung der Lysine K278, K356 und K361 bei der primären Interaktion von HPV16 mit Heparansulfaten konnte durch die Computersimulation der Interaktion der Virusoberfläche mit einem Heparinmolekül bestätigt werden. Des Weiteren konnten Anforderungen ermittelt werden, die eine solche Interaktion an das Heparinmolekül stellt. Weiterhin zeigten die Ergebnisse dieser Arbeit, dass basische Aminosäuren in der interkapsomeren Grube nicht an der primären Zellbindung an Heparansulfate beteiligt zu sein scheinen, aber eine Rolle bei sekundären Interaktionen mit der Zelloberfläche spielen könnten.

Struktur-Dynamik-Beziehungen in amorphen festen Lösungen

Feste Lösungen homogen dispergierter Wirkstoffmoleküle in amorphen Polymermatrizen sind wichtige Materialien in vielen pharmazeutischen Anwendungen, bei denen eine kontrollierte Abgabe wasserunlöslicher Wirkstoffe in wässrige Systeme eine Rolle spielt. Die intermolekulare Bindungs-stärke zwischen Polymer- und Wirkstoffmolekülgruppen bestimmt die Stabilität der festen Lösung und steuert somit die biologische Aktivität der Wirkstoffmoleküle. In festen Lösungen, die aus acryl-säurehaltigen Copolymeren (Protonendonoren) und basischen Wirkstoffmolekülen (Protonenakzepto-ren) hergestellt werden, sind intermolekulare Wasserstoffbrücken zwischen den Systemkomponenten Triebkraft für die Bildung einer stabilen homogenen Dispersion und für die Entstehung struktureller Merkmale zwischen den Molekülgruppen der Systemkomponenten. Zudem ist die Bindungsstärke der Wasserstoffbrücken im Hinblick auf die kontrollierte Abgabe der Wirkstoffe von Bedeutung. Da dynamische chemische Gleichgewichte bei der Bildung der Wasserstoffbrücken eine wichtige Rolle spielen müssen neben strukturellen Parametern auch dynamische Faktoren beleuchtet werden. Ziel dieser Arbeit ist neben der Ermittlung von intermolekularen Bindungsstärken vor allem die Identifika-tion struktureller Verhältnisse zwischen den Systemkomponenten auf molekularer Ebene. Die Be-stimmung der Abhängigkeit dieser Parameter von der Struktur der verwendeten Polymere und einer Vielzahl weiterer Einflüsse wie z.B. Feuchtigkeit, Lagerdauer oder Wirkstoffkonzentration soll ein kontrolliertes Design fester Lösungen mit definierten anwendungsspezifischen Eigenschaften ermögli-chen. Temperaturabhängige 1H-Festkörper-MAS-NMR (Magic Angle Spinning Nuclear Magnetic Resonance) Experimente an festen Lösungen mit unterschiedlichen Copolymer-Zusammensetzungen weisen die Existenz dynamischer chemischer Gleichgewichte in den komplexen Wasserstoffbrücken-netzwerken nach. Veränderungen in der chemischen Verschiebung und in der Linienform der Reso-nanzlinien acider Protonen erlauben einen tiefen Einblick in die Architektur dieser Netzwerke und legen die Bindungsverhältnisse unter Berücksichtigung der Polymerchemie und der Mobilität der Systemkomponenten dar, wobei die Befunde mithilfe quantenchemischer Rechnungen untermauert werden können. Die Gegenwart acider Protonen ermöglicht einen einfachen 1H-2H-Austausch, wor-aufhin mithilfe rotorsynchronisierter temperaturabhängiger 2H-MAS-NMR Experimente die Wasser-stoffbrückenbindungsstärke bestimmt werden kann. Mit 1H-1H-Korrelationsexperimenten (Doppelquantenspektroskopie) stehen Methoden für die Bestimmung homonuklearer dipolarer 1H-1H-Kopplungen zur Verfügung, die strukturelle Aussagen aufgrund von bevorzugten räumlichen Kontak-ten bestimmter Molekülgruppen ermöglichen. Weiterhin können diese Experimente verwendet werden, um Wasserstoffbrücken zwischen Polymergruppen von Polymer-Wirkstoff-Wasserstoffbrücken zu unterscheiden, wodurch eine quantitative Beschreibung des Bindungsnetzwerks und der Konkurrenz-prozesse zwischen den einzelnen wasserstoffverbrückten Spezies ermöglicht wird. Eine Kristallisation der Wirkstoffmoleküle ist in vielen Anwendungen unerwünscht, da sie die biologische Verfügbarkeit des Wirkstoffs reduzieren. Mit 1H-Festkörper-MAS-NMR Experimenten können kristalline von amorph dispergierten Wirkstoffmolekülen unterschieden werden, wodurch eine Quantifizierung der Destabilisierungsprozesse ermöglicht wird, die durch Exposition der festen Lösungen mit Wasserdampf ausgelöst werden können. Die Zeit- und Konzentrationsabhängigkeit der Wasseraufnahme kann mit NMR-Experimenten verfolgt werden, wobei unterschiedlich mobile Was-serspezies an unterschiedlichen Bindungsorten identifiziert werden können, was zum molekularen Verständnis der Destabilisierungsprozesse beiträgt. Zusätzlich wird die Mobilität der Wirkstoffmole-küle bestimmt, die sich – wie auch die Wirkstoffkonzentration - als wichtige Größe in der Beschrei-bung der Destabilisierung erweist. Aufbauend auf den Beobachtungen wird ein Zusammenhang zwischen der Copolymerzusammensetzung und einer kritischen Wirkstoffkonzentration hergestellt, der für die Anwendungen amorpher fester Lösungen in biologischen Systemen von großer Bedeutung ist.