New synthetic strategies to tethered bilayer lipid membranes

Tethered bilayer lipid membranes provide an efficient, stable and versatile platform for the investigation of integrated membrane proteins. However, the incorporation of large proteins, as well as of proteins with a large submembrane part is still a very critical issue and therefore, further optimisation of the system is necessary. The central element of a tBLM is a lipid bilayer. Its proximal leaflet is, at least to some extend, covalently attached to a solid support via a spacer group. The anchor lipid consists of three distinct parts, a lipid headgroup, a spacer group and an anchor. All parts together influence the final bilayer properties. In the frame of this work, the synthesis of new thiolipids for tBLMs on gold has been investigated. The aim was to obtain molecules with longer spacers in order to increase the submembrane space. The systems obtained have been characterized using SPR and EIS. The results obtained during this study are multiple. First, the synthesis of a previously synthesized architecture was successfully scaled up in an industrial lab using a new synthetic approach. The synthesis of large amounts is now feasible. Then, the synthesis of the new thiolipids was carried out taking into account the following requirements: the increase of the submembrane space by having longer ethyleneglycol spacers, the attachment of the molecules to a gold substrate via a thiol bond, and the tunability of the lateral mobility by changing the lipid headgroup. Three different synthetic strategies have been investigated. The polymeric approach did not prove to be successful, merely because of the broad molecular weight distribution. The synthesis of heterofunctionally protected oligoethyleneglycols allowed to obtain ethyleneglycol moieties with 6 and 8 units, but the tedious purification steps gave very low yields. Finally, the block by block synthesis using ethyleneglycol precursors proved to be an efficient and fast method to synthesize the target molecules. Indeed, these were obtained with very high yields, and the separation was very efficient. A whole family of new compounds was obtained, having 6, 8 and 14 ethyleneglycol units and with mono- or diphytanyl lipid headgroups. This new pathway is a very promising synthetic strategy that can be used further in the development of new compounds of the tether system. The formation of bilayers was investigated for the different thiolipids mainly by using EIS. The electrical properties of a bilayer define the quality of the membrane and allow the study of the functionality of proteins embedded in such a system. Despite multiple trials to improve the system using self assembly, Langmuir Blodgett transfer, and detergent mixed vesicles, the new polymer thiolipids did not show as high electrical properties as tBLMs reported in the literature. Nevertheless, it was possible to show that a bilayer could be obtained for the different spacer lengths. These bilayers could be formed using self assembly for the first monolayer, and two different methods for bilayer formation, namely vesicle fusion and solvent exchange. We could furthermore show functional incorporation of the ion carrier valinomycin: the selective transport of K+ ions could be demonstrated. For DPHL, it was even possible to show the functional incorporation of the ion channel gramicidin. The influence of the spacer length is translated into an increase of the spacer capacitance, which could correspond to an increase in the capacity of charge accumulation in the submembrane space. The different systems need to be further optimised to improve the electrical properties of the bilayer. Moreover, the incorporation of larger proteins, and proteins bearing submembrane parts needs to be investigated.

Synthetic strategies of new molecular paramagnetic mechanically-interlocked complexes

Supramolecular chemistry is a multidisciplinary field which impinges on other disciplines, focusing on the systems made up of a discrete number of assembled molecular subunits. The forces responsible for the spatial organization are intermolecular reversible interactions. The supramolecular architectures I was interested in are Rotaxanes, mechanically-interlocked architectures consisting of a "dumbbell shaped molecule", threaded through a "macrocycle" where the stoppers at the end of the dumbbell prevent disassociation of components and catenanes, two or more interlocked macrocycles which cannot be separated without breaking the covalent bonds. The aim is to introduce one or more paramagnetic units to use the ESR spectroscopy to investigate complexation properties of these systems cause this technique works in the same time scale of supramolecular assemblies. Chapter 1 underlines the main concepts upon which supramolecular chemistry is based, clarifying the nature of supramolecular interactions and the principles of host-guest chemistry. In chapter 2 it is pointed out the use of ESR spectroscopy to investigate the properties of organic non-covalent assemblies in liquid solution by spin labels and spin probes. The chapter 3 deals with the synthesis of a new class of p-electron-deficient tetracationic cyclophane ring, carrying one or two paramagnetic side-arms based on 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) moiety. In the chapter 4, the Huisgen 1,3-dipolar cycloaddition is exploited to synthesize rotaxanes having paramagnetic cyclodextrins as wheels. In the chapter 5, the catalysis of Huisgen’s cycloaddition by CB[6] is exploited to synthesize paramagnetic CB[6]-based [3]-rotaxanes. In the chapter 6 I reported the first preliminary studies of Actinoid series as a new class of templates in catenanes’ synthesis. Being f-block elements, so having the property of expanding the valence state, they constitute promising candidates as chemical templates offering the possibility to create a complex with coordination number beyond 6.

Modeling and simulation of a synthetic genetic circuit that implements a multicelled behavior in a growing microcolony of E. coli

Synthetic Biology is a relatively new discipline, born at the beginning of the New Millennium, that brings the typical engineering approach (abstraction, modularity and standardization) to biotechnology. These principles aim to tame the extreme complexity of the various components and aid the construction of artificial biological systems with specific functions, usually by means of synthetic genetic circuits implemented in bacteria or simple eukaryotes like yeast. The cell becomes a programmable machine and its low-level programming language is made of strings of DNA. This work was performed in collaboration with researchers of the Department of Electrical Engineering of the University of Washington in Seattle and also with a student of the Corso di Laurea Magistrale in Ingegneria Biomedica at the University of Bologna: Marilisa Cortesi. During the collaboration I contributed to a Synthetic Biology project already started in the Klavins Laboratory. In particular, I modeled and subsequently simulated a synthetic genetic circuit that was ideated for the implementation of a multicelled behavior in a growing bacterial microcolony. In the first chapter the foundations of molecular biology are introduced: structure of the nucleic acids, transcription, translation and methods to regulate gene expression. An introduction to Synthetic Biology completes the section. In the second chapter is described the synthetic genetic circuit that was conceived to make spontaneously emerge, from an isogenic microcolony of bacteria, two different groups of cells, termed leaders and followers. The circuit exploits the intrinsic stochasticity of gene expression and intercellular communication via small molecules to break the symmetry in the phenotype of the microcolony. The four modules of the circuit (coin flipper, sender, receiver and follower) and their interactions are then illustrated. In the third chapter is derived the mathematical representation of the various components of the circuit and the several simplifying assumptions are made explicit. Transcription and translation are modeled as a single step and gene expression is function of the intracellular concentration of the various transcription factors that act on the different promoters of the circuit. A list of the various parameters and a justification for their value closes the chapter. In the fourth chapter are described the main characteristics of the gro simulation environment, developed by the Self Organizing Systems Laboratory of the University of Washington. Then, a sensitivity analysis performed to pinpoint the desirable characteristics of the various genetic components is detailed. The sensitivity analysis makes use of a cost function that is based on the fraction of cells in each one of the different possible states at the end of the simulation and the wanted outcome. Thanks to a particular kind of scatter plot, the parameters are ranked. Starting from an initial condition in which all the parameters assume their nominal value, the ranking suggest which parameter to tune in order to reach the goal. Obtaining a microcolony in which almost all the cells are in the follower state and only a few in the leader state seems to be the most difficult task. A small number of leader cells struggle to produce enough signal to turn the rest of the microcolony in the follower state. It is possible to obtain a microcolony in which the majority of cells are followers by increasing as much as possible the production of signal. Reaching the goal of a microcolony that is split in half between leaders and followers is comparatively easy. The best strategy seems to be increasing slightly the production of the enzyme. To end up with a majority of leaders, instead, it is advisable to increase the basal expression of the coin flipper module. At the end of the chapter, a possible future application of the leader election circuit, the spontaneous formation of spatial patterns in a microcolony, is modeled with the finite state machine formalism. The gro simulations provide insights into the genetic components that are needed to implement the behavior. In particular, since both the examples of pattern formation rely on a local version of Leader Election, a short-range communication system is essential. Moreover, new synthetic components that allow to reliably downregulate the growth rate in specific cells without side effects need to be developed. In the appendix are listed the gro code utilized to simulate the model of the circuit, a script in the Python programming language that was used to split the simulations on a Linux cluster and the Matlab code developed to analyze the data.

Development of new therapeutic approaches in the treatments of Inflammatory Bowel Diseases: functional food and nutraceuticals vs synthetic peptides based vaccines

Inflammatory Bowel Diseases (IBD) are intestinal chronic relapsing diseases which ethiopathogenesis remains uncertain. Several group have attempted to study the role of factors involved such as genetic susceptibility, environmental factors such as smoke, diet, sex, immunological factors as well as the microbioma. None of the treatments available satisfy several criteria at the same time such as safety, long-term remission, histopatological healing, and specificity. We used two different approaches for the development of new therapeutic treatment for Inflammatory Bowel Disease. The first is focused on the understanding of the potential role of functional food and nutraceuticals nutrients in the treatment of IBD. To do so, we investigated the role of Curcuma longa in the treatment of chemical induced colitis in mice model. Since Curcma Longa has been investigated for its antinflammatory role related to the TNFα pathway as well investigators have reported few cases of patients with ulcerative colites treated with this herbs, we harbored the hypothesis of a role of Curcuma Longa in the treatment f IBD as well as we decided to assess its role in intestinal motility. The second part is based on an immunological approach to develop new drugs to induce suppression in Crohn’s disease or to induce mucosa immunity such as in colonrectal tumor. The main idea behind this approach is that we could manipulate relevant cell-cell interactions using synthetic peptides. We demonstrated the role of the unique interaction between molecules expressed on intestinal epithelial cells such as CD1d and CEACAM5 and on CD8+ T cells. In normal condition this interaction has a role for the expansion of the suppressor CD8+ T cells. Here, we characterized this interaction, we defined which are the epitope involved in the binding and we attempted to develop synthetic peptides from the N domain of CEACAM5 in order to manipulate it.

New synthetic bile acid analogue agonists of FXR and TGR5 receptors: Analytical methodologies for the study of their physico-chemical properties, pharmacokinetic activity and metabolism.

This thesis reports an integrated analytical approach for the study of physicochemical and biological properties of new synthetic bile acid (BA) analogues agonists of FXR and TGR5 receptors. Structure-activity data were compared with those previous obtained using the same experimental protocols on synthetic and natural occurring BA. The new synthetic BA analogues are classified in different groups according also to their potency as a FXR and TGR5 agonists: unconjugated and steroid modified BA and side chain modified BA including taurine or glycine conjugates and pseudo-conjugates (sulphonate and sulphate analogues). In order to investigate the relationship between structure and activity the synthetic analogues where admitted to a physicochemical characterization and to a preliminary screening for their pharmacokinetic and metabolism using a bile fistula rat model. Sensitive and accurate analytical methods have been developed for the quali-quantitative analysis of BA in biological fluids and sample used for physicochemical studies. Combined High Performance Liquid Chromatography Electrospray tandem mass spectrometry with efficient chromatographic separation of all studied BA and their metabolites have been optimized and validated. Analytical strategies for the identification of the BA and their minor metabolites have been developed. Taurine and glycine conjugates were identified in MS/MS by monitoring the specific ion transitions in multiple reaction monitoring (MRM) mode while all other metabolites (sulphate, glucuronic acid, dehydroxylated, decarboxylated or oxo) were monitored in a selected-ion reaction (SIR) mode with a negative ESI interface by the following ions. Accurate and precise data where achieved regarding the main physicochemical properties including solubility, detergency, lipophilicity and albumin binding . These studies have shown that minor structural modification greatly affect the pharmacokinetics and metabolism of the new analogues in respect to the natural BA and on turn their site of action, particularly where their receptor are located in the enterohepatic circulation.

Novel Synthetic Procedures in Organocatalysis

The main aim of my PhD project was the design and the synthesis of new pyrrolidine organocatalysts. New effective ferrocenyl pyrrolidine catalysts, active in benchmark organocatalytic reactions, has been developed. The ferrocenyl moiety, in combination with simple ethyl chains, is capable of fixing the enamine conformation addressing the approach trajectory of the nucleophile in the reaction. The results obtained represent an interesting proof-of-concept, showing for the first time the remarkable effectiveness of the ferrocenyl moiety in providing enantioselectivity through conformational selection. This approach could be viably employed in the rational design of ligands for metal or organocatalysts. Other hindered secondary amines has been prepared from alkylation of acyclic chiral nitroderivatives with alcohols in a highly diastereoselective fashion, giving access to functionalized, useful organocatalytic chiral pyrrolidines. A family of new pyrrolidines bearing sterogenic centers and functional groups can be readily accessible by this methodology. The second purpose of the project was to study in deep the reactivity of stabilized carbocations in new metal-free and organocatalytic reactions. By taking advantage of the results from the kinetic studies described by Mayr, a simple and effective procedure for the direct formylation of aryltetrafluoroborate salts, has been development. The coupling of a range of aryl- and heteroaryl- trifluoroborate salts with 1,3-benzodithiolylium tetrafluoroborate, has been attempted in moderate to good yields. Finally, a simple and general methodology for the enamine-mediated enantioselective α-alkylation of α-substituted aldehydes with 1,3-benzodithiolylium tetrafluoroborate has been reported. The introduction of the benzodithiole moiety permit the installation of different functional groups due to its chameleonic behaviour.

Bioconjugation and synthetic approach towards enantioenriched gem-difluoromethylene compounds through carbenium ions

Bioconjugation of peptides and asymmetric synthesis of gem-difluoromethylene compounds are areas of the modern organic chemistry for which mild and selective methods continue to be developed. This thesis reports new methodologies for these two areas based on the use of stabilized carbenium ions. The reaction that makes the bioconjugation of peptides possible takes place via the direct nucleophilic substitution of alcohols and is driven by the spontaneous formation of stabilized carbenium ions in water. By reacting with the thiol group of cysteine in very mild conditions and with a high selectivity, these carbenium ions allow the site-specific ligation of polypeptides containing cysteine and their covalent derivatization with functionalized probes. The ligation of the indole ring of tryptophan, an emerging target in bioconjugation, is also shown and takes place in the same conditions. The second area investigated is the challenging access to optically active gem-difluoromethylene compounds. We describe a methodology relying on the synthesis of enantioenriched 1,3-benzodithioles intermediates that are shown to be precursors of the corresponding gem-difluoromethylene analogues by oxidative desulfurization-fluorination. This synthesis takes advantage of the highly enantioselective organocatalytic α-alkylation of aldehydes with the benzodithiolylium ion and of the wide possibilities of synthetic transformations offered by the 1,3-benzodithiole group. This approach allows the asymmetric access to complex gem-difluoromethylene compounds through a late-stage fluorination step, thus avoiding the use of fluorinated building blocks.

Fast and accurate numerical solutions in some problems of particle and radiation transport: synthetic acceleration for the method of short characteristics, Doppler-broadened scattering kernel, remote sensing of the cryosphere

The aim of this work is to present various aspects of numerical simulation of particle and radiation transport for industrial and environmental protection applications, to enable the analysis of complex physical processes in a fast, reliable, and efficient way. In the first part we deal with speed-up of numerical simulation of neutron transport for nuclear reactor core analysis. The convergence properties of the source iteration scheme of the Method of Characteristics applied to be heterogeneous structured geometries has been enhanced by means of Boundary Projection Acceleration, enabling the study of 2D and 3D geometries with transport theory without spatial homogenization. The computational performances have been verified with the C5G7 2D and 3D benchmarks, showing a sensible reduction of iterations and CPU time. The second part is devoted to the study of temperature-dependent elastic scattering of neutrons for heavy isotopes near to the thermal zone. A numerical computation of the Doppler convolution of the elastic scattering kernel based on the gas model is presented, for a general energy dependent cross section and scattering law in the center of mass system. The range of integration has been optimized employing a numerical cutoff, allowing a faster numerical evaluation of the convolution integral. Legendre moments of the transfer kernel are subsequently obtained by direct quadrature and a numerical analysis of the convergence is presented. In the third part we focus our attention to remote sensing applications of radiative transfer employed to investigate the Earth's cryosphere. The photon transport equation is applied to simulate reflectivity of glaciers varying the age of the layer of snow or ice, its thickness, the presence or not other underlying layers, the degree of dust included in the snow, creating a framework able to decipher spectral signals collected by orbiting detectors.

Semi-synthetic bile acids as novel drug candidate in liver diseases: physico-chemical characterization and HPLC-ES-MS/MS methods for their quali-quantitative analysis in different experimental animal models

The physico-chemical characterization, structure-pharmacokinetic and metabolism studies of new semi synthetic analogues of natural bile acids (BAs) drug candidates have been performed. Recent studies discovered a role of BAs as agonists of FXR and TGR5 receptor, thus opening new therapeutic target for the treatment of liver diseases or metabolic disorders. Up to twenty new semisynthetic analogues have been synthesized and studied in order to find promising novel drugs candidates. In order to define the BAs structure-activity relationship, their main physico-chemical properties (solubility, detergency, lipophilicity and affinity with serum albumin) have been measured with validated analytical methodologies. Their metabolism and biodistribution has been studied in “bile fistula rat”, model where each BA is acutely administered through duodenal and femoral infusion and bile collected at different time interval allowing to define the relationship between structure and intestinal absorption and hepatic uptake ,metabolism and systemic spill-over. One of the studied analogues, 6α-ethyl-3α7α-dihydroxy-5β-cholanic acid, analogue of CDCA (INT 747, Obeticholic Acid (OCA)), recently under approval for the treatment of cholestatic liver diseases, requires additional studies to ensure its safety and lack of toxicity when administered to patients with a strong liver impairment. For this purpose, CCl4 inhalation to rat causing hepatic decompensation (cirrhosis) animal model has been developed and used to define the difference of OCA biodistribution in respect to control animals trying to define whether peripheral tissues might be also exposed as a result of toxic plasma levels of OCA, evaluating also the endogenous BAs biodistribution. An accurate and sensitive HPLC-ES-MS/MS method is developed to identify and quantify all BAs in biological matrices (bile, plasma, urine, liver, kidney, intestinal content and tissue) for which a sample pretreatment have been optimized.

Synthetic strategies of new spin-labelled supramolecular systems

Molecular recognition and self-assembly represent fundamental issues for the construction of supramolecular systems, structures in which the components are held together through non-covalent interactions. The study of host-guest complexes and mechanical interlocked molecules, important examples in this field, is necessary in order to characterize self-assembly processes, achieve more control over the molecular organization and develop sophisticated structures by using properly designed building blocks. The introduction of paramagnetic species, or spin labelling, represents an attractive opportunity that allows their detection and characterization by the Electron Spin Resonance spectroscopy, a valuable technique that provides additional information to those obtained by traditional methods. In this Thesis, recent progresses in the design and the synthesis of new paramagnetic host-guest complexes and rotaxanes characterized by the presence of nitroxide radicals and their investigation by ESR spectroscopy are reported. In Chapter 1 a brief overview of the principal concepts of supramolecular chemistry, the spin labelling approach and the development of ESR methods applied to paramagnetic systems are described. Chapter 2 and 3 are focused on the introduction of radicals in macrocycles as Cucurbiturils and Pillar[n]arenes, due to the interesting binding properties and the potential employment in rotaxanes, in order to investigate their structures and recognition properties. Chapter 4 deals with one of the most studied mechanical interlocked molecules, the bistable [2]rotaxane reported by Stoddart and Heath based on the ciclobis (paraquat-p-phenylene) CBPQT4+, that represents a well known example of molecular switch driven by external stimuli. The spin labelling of analogous architectures allows the monitoring by ESR spectroscopy of the switch mechanism involving the ring compound by tuning the spin exchange interaction. Finally, Chapter 5 contains the experimental procedures used for the synthesis of some of the compounds described in Chapter 2-4.

Synthetic routes toward functional block copolymers and bioconjugates via RAFT polymerization

Synthetic Routes toward Functional Block Copolymers and Bioconjugates via RAFT PolymerizationrnSynthesewege für funktionelle Blockcopolymere und Biohybride über RAFT PolymerisationrnDissertation von Dipl.-Chem. Kerstin T. WissrnIm Rahmen dieser Arbeit wurden effiziente Methoden für die Funktionalisierung beider Polymerkettenenden für Polymer- und Bioanbindung von Polymeren entwickelt, die mittels „Reversible Addition-Fragmentation Chain Transfer“ (RAFT) Polymerisation hergestellt wurden. Zu diesem Zweck wurde ein Dithioester-basiertes Kettentransferagens (CTA) mit einer Aktivestereinheit in der R-Gruppe (Pentafluorphenyl-4-phenylthiocarbonylthio-4-cyanovaleriansäureester, kurz PFP-CTA) synthetisiert und seine Anwendung als universelles Werkzeug für die Funktionalisierung der -Endgruppe demonstriert. Zum Einen wurde gezeigt, wie dieser PFP-CTA als Vorläufer für die Synthese anderer funktioneller CTAs durch einfache Aminolyse des Aktivesters genutzt werden kann und somit den synthetischen Aufwand, der üblicherweise mit der Entwicklung neuer CTAs verbunden ist, reduzieren kann. Zum Anderen konnte der PFP-CTA für die Synthese verschiedener Poly(methacrylate) mit enger Molekulargewichtsverteilung und wohl definierter reaktiver -Endgruppe verwendet werden. Dieses Kettenende konnte dann erfolgreich mit verschiedenen primären Aminen wie Propargylamin, 1-Azido-3-aminopropan und Ethylendiamin oder direkt mit den Amin-Endgruppen verschiedener Peptide umgesetzt werden.rnAus der Reaktion des PFP-CTAs mit Propargylamin wurde ein Alkin-CTA erhalten, der sich als effizientes Werkzeug für die RAFT Polymerisation verschiedener Methacrylate erwiesen hat. Der Einbau der Alkin-Funktion am -Kettenende wurde mittels 1H und 13C NMR Spektroskopie sowie MALDI TOF Massenspektroskopie bestätigt. Als Modelreaktion wurde die Kopplung eines solchen alkin-terminierten Poly(di(ethylenglykol)methylethermethacrylates) (PDEGMEMA) mit azid-terminiertem Poly(tert-butylmethacrylat), das mittels Umsetzung einer Aktivester-Endgruppe erhalten wurde, als kupferkatalysierte Azid-Alkin-Cycloaddition (CuAAC) durchgeführt. Die Aufarbeitung des resultierenden Diblockcopolymers durch Fällen ermöglichte die vollständige Abtrennung des Polymerblocks 1, der im Überschuss eingesetzt wurde. Darüber hinaus blieb nur ein sehr kleiner Anteil (< 2 Gew.-%) nicht umgesetzten Polymerblocks 2, was eine erfolgreiche Polymeranbindung und die Effizienz der Endgruppen-Funktionalisierung ausgehend von der Aktivester--Endgruppe belegt.rnDie direkte Reaktion von stimuli-responsiven Polymeren mit Pentafluorphenyl(PFP)ester-Endgruppen, namentlich PDEGMEMA und Poly(oligo(ethylenglykol)methylethermethacrylat), mit kollagen-ähnlichen Peptiden ergab wohl definierte Polymer-Peptid-Diblockcopolymere und Polymer-Peptid-Polymer-Triblockcopolymer unter nahezu quantitativer Umsetzung der Endgruppen. Alle Produkte konnten vollständig von nicht umgesetztem Überschuss des Homopolymers befreit werden. In Analogie zu natürlichem Kollagen und dem nicht funktionalisierten kollagen-ähnlichen Peptid bilden die PDEGMEMA-basierten, entschützten Hybridcopolymere Trimere mit kollagen-ähnlichen Triple-Helices in kalter wässriger Lösung, was mittels Zirkular-Dichroismus-Spektroskopie (CD) nachgewiesen werden konnte. Temperaturabhängige CD-Spektroskopie, Trübungsmessungen und dynamische Lichtstreuung deuteten darauf hin, dass sie bei höheren Temperaturen doppelt stimuli-responsive Überstrukturen bilden, die mindestens zwei konformative Übergänge beim Aufheizen durchlaufen. Einer dieser Übergänge wird durch den hydrophoben Kollaps des Polymerblocks induziert, der andere durch Entfalten der kollagen-ähnlichen Triple-Helices.rnAls Ausweitung dieser synthetischen Strategie wurde homotelecheles PDEGMEMA mit zwei PFP-Esterendgruppen dargestellt, wozu der PFP-CTA für die Funktionalisierung der -Endgruppe und die radikalische Substitution des Dithioesters durch Behandlung mit einem Überschuss eines funktionellen AIBN-Derivates für die Funktionalisierung der -Endgruppe ausgenutzt wurde. Die Umsetzung der beiden reaktiven Kettenenden mit dem N-Terminus eines Peptidblocks ergab ein Peptid-Polymer-Peptid Triblockcopolymer.rnSchließlich konnten die anorganisch-organischen Hybridmaterialien PMSSQ-Poly(2,2-diethoxyethylacrylat) (PMSSQ-PDEEA) und PMSSQ-Poly(1,3-dioxolan-2-ylmethylacrylat) (PMSSQ-PDMA) für die Herstellung robuster, peptid-reaktiver Oberflächen durch Spin Coaten und thermisch induziertes Vernetzen angewendet werden. Nach saurem Entschützen der Acetalgruppen in diesen Filmen konnten die resultierenden Aldehydgruppen durch einfaches Eintauchen in eine Lösung mit einer Auswahl von Aminen und Hydroxylaminen umgesetzt werden, wodurch die Oberflächenhydrophilie modifiziert werden konnte. Darüber hinaus konnten auf Basis der unterschiedlichen Stabilität der zwei hier verglichenen Acetalgruppen Entschützungsprotokolle für die exklusive Entschützung der Diethylacetale in PMSSQ-PDEEA und deren Umsetzung ohne Entschützung der zyklischen Ethylenacetale in PMSSQ-PDMA entwickelt werden, die die Herstellung multifunktioneller Oberflächenbeschichtungen z.B. für die Proteinimmobilisierung ermöglichen.

Development of synthetic methods of silicon nanocrystals functionalized with photoactive molecules

The thesis is focused on the development of a method for the synthesis of silicon nanocrystals with different sizes, narrow size distribution, good optical properties and stability in air. The resulting silicon nanocrystals have been covalently functionalized with different chromophores with the aim to exploit the new electronic and chemical properties that emerge from the interaction between silicon nanocrystal surface and ligands. The purpose is to use these chromophores as light harvesting antennae, increasing the optical absorption of silicon nanocrystals. Functionalized silicon nanocrystals have been characterized with different analytical techniques leading to a good knowledge of optical properties of semiconductor quantum dots.

Surface-enhanced vibrational techniques for the detection of synthetic organic colorants

The research project object of this thesis is focused on the development of an advanced analytical system based on the combination of an improved thin layer chromatography (TLC) plate coupled with infrared (FTIR) and Raman microscopies for the detection of synthetic dyes. Indeed, the characterization of organic colorants, which are commonly present in mixtures with other components and in a very limited amount, still represents a challenging task in scientific analyses of cultural heritage materials. The approach provides selective spectral fingerprints for each compound, foreseeing the complementary information obtained by micro ATR-RAIRS-FTIR and SERS-Raman analyses, which can be performed on the same separated spot. In particular, silver iodide (AgI) applied on a gold coated slide is proposed as an efficient stationary phase for the discrimination of complex analyte mixtures, such as dyes present in samples of art-historical interest. The gold-AgI-TLC plate shows high performances related both to the chromatographic separation of analytes and to the spectroscopic detection of components. The use of a mid-IR transparent inorganic salt as the stationary phase avoids interferences of the background absorption in FTIR investigations. Moreover, by ATR microscopy measurements performed on the gold-AgI surface, a considerable enhancement in the intensity of spectra is observed. Complementary information can be obtained by Raman analyses, foreseeing a SERS activity of the AgI substrate. The method has been tested for the characterization of a mixture of three synthetic organic colorants widely used in dyeing processes: Brilliant Green (BG1), Rhodamine B (BV10) and Methylene Blue (BB9).

New synthetic strategies towards indolizines and pyrroles

Indolizines and pyrroles are considered as “privileged” structures since their skeletons were found in many biologically active natural products and they possess a wide range of pharmaceutical properties. Syntheses of these small drug-like molecules are very important in medicinal chemistry. However, most existent methodologies are usually limited to specific substitution patterns or require impractical starting materials or expensive catalysts. Therefore, developing new methodologies for the synthesis of indolizines and pyrroles from commercially available or readily accessible sources is highly desirable.rnIn this PhD thesis, several methods has been described for the synthesis of indolizines and pyrroles. In the first part, indolizines carrying substituents in positions 1-3 were synthesized via a formal [3+2]-cycloaddition of pyridinium ylides and nitroalkenes. Pyridinium salts were prepared by N-alkylation of pyridines with cyanohydrin triflates which could be prepared from corresponding aldehydes via a Strecker reaction followed by O-triflylation. Nitroalkenes were simply prepared from the corresponding aldehydes and nitroalkanes in a nitroaldol condensation. Overall, this modular approach allows to construct the indolizine framework with various substitution patterns starting from a pyridine, two different aldehydes and a nitroalkane. In contrast to reported methods, the produced indolizines do not have to contain an electron-withdrawing group.rnIt has also been found that nitrile-stabilized 2-alkylpyridinium ylides cyclize to unstable 2-aminoindolizines via an intramolecular 5-exo-dig cyclization. Using an in situ acetylation of the amino group, N-protected 2-aminoindolizines could be synthesized. As a less common substitution pattern, indolizines carrying substituents in positions 5–8 were synthesized from enones and 2-(1H-pyrrol-1-yl)nitriles obtained from α-aminonitriles using a modified Paal-Knorr pyrrole synthesis. The decoration of the pyridine unit in the indolizine skeleton has been achieved by a one-pot conjugate addition/cycloaromatization sequence.rnIn the second part of the thesis, the diversity-oriented synthesis of pyrroles from 3,5-diaryl substituted 2H-pyrrole-2-carbonitriles (cyanopyrrolines) obtained in a cyclocondensation of enones with aminoacetonitrile hydrochloride is being discussed. 2,4-Di-, 2,3,5-trisubstituted pyrroles, pyrrole-2-carbonitriles and 2,2’-bipyrroles were synthesized in a one- or two-step protocol. While the microwave-assisted thermal elimination of HCN from cyanopyrrolines gave 2,4-disubstituted pyrroles, DDQ-oxidation of the same intermediates furnished pyrrole-2-carbonitriles. Furthermore, 2,3,5-trisubstituted pyrroles were obtained via a C-2-alkylation of the deprotonated cyanopyrrolines followed by the elimination of HCN. Finally, it has also been found that tetraaryl substituted 2,2’-bipyrroles could be synthesized by the oxidative dimerization of cyanopyrrolines using copper (II) acetate at 100 °C.rn

Synthetic studies towards a new fulvestrant analogue

A study towards the synthesis of a new fulvestrant analogue with improved bioavailability was carried out. In this work a twelve-step synthetic route starting from β-estradiol was optimized and a palladium (Pd)-catalyzed endo-selective Heck reaction for the functionalization of an advanced intermediate was investigated.