Synthesis, Optical Resolution, and Configurational Assignment of Novel Axially Chiral Quateraryls

A one-pot, general synthesis of highly functionalized quateraryls through carbanion-induced, base-catalyzed ring transformation of 5,6-diaryl-2H-pyran-2-ones and core-substituted phenylacetones is delineated. These conversions were found to give diversely functionalized benzenes bearing peripheral aryl rings, some of which possess inherent atropisomerism. Exemplarily for one of the quateraryls, the optical resolution of the respective atropo-enantiomers by HPLC on a chiral phase and the assignment of their absolute axial configurations succeeded by LC-CD coupling in combination with semiempirical CNDO/S and TDDFT CD calculations. This synthetic approach offers – in a transition metal-free environment – high flexibility in the construction of quateraryls with the desired conformational freedom along the molecular axis, which may help in exploring and developing new potential ligands for asymmetric synthesis.

Combinatorial synthesis and biological evaluation of isoxazole-based libraries as antithrombotic agents

The 3-substitutedphenyl-5-isoxazolecarboxaldehydes have been identified as activated aldehydes for the generation of isoxazole-based combinatorial libraries on solid phase through automation. Three highly functionalized isoxazole-based libraries comprising of 32, 96 and 45 compounds each have been synthesized in parallel format using Baylis Hillman reaction, Michael addition, reductive amination and alkylation reactions. With an objective of lead generation all the three libraries were evaluated for their antithrombin activity in vivo.

An in-situ surface electrochemistry approach toward whole-cell studies: Charge transfer between Geobacter sulfurreducens and electrified metal/electrolyte interfaces through linker molecules

Electrochemical reactivity and structure properties of electrogenic bacteria, Geobacter sulfurreducens (Gs) were studied to explore the heterogeneous electron transfer at the bacteria/electrode interface using electrochemical and in-situ spectroscopic techniques. The redox behavior of Gs adsorbed on a gold electrode, which is modified with a ω-functionalized self-assembled monolayer (SAM) of alkanethiols, depends strongly on the terminal group. The latter interacts directly with outermost cytochromes embedded into the outer membrane of the Gs cells. The redox potential of bacterial cells bound electrostatically to a carboxyl-terminated SAM is close to that observed for bacteria attached to a bare gold electrode, revealing a high electronic coupling at the cell/SAM interface. The redox potentials of bacterial cells adsorbed on amino- and pyridyl-terminated SAMs are significantly different suggesting that the outermost cytochromes changes their conformation upon adsorption on these SAMs. No redox activity of Gs was found with CH3-, N(CH3)3+- and OH-terminated SAMs. Complementary in-situ spectroscopic studies on bacteria/SAMs/Au electrode assemblies were carried out to monitor structure changes of the bacterial cells upon polarization. Spectro-electrochemical techniques revealed the electrochemical turnover of the oxidized and reduced states of outer membrane cytochromes (OMCs) in Gs, providing evidence that the OMCs are responsible for the direct electron transfer to metal electrodes, such as gold or silver, during the electricity production. Furthermore, we observed spectroscopic signatures of the native structure of the OMCs and no conformational change during the oxidation/reduction process of the microorganisms. These findings indicate that the carboxyl-anchoring group provides biocompatible conditions for the outermost cytochromes of the Gs, which facilitate the heterogeneous electron transfer at the microorganism/electrode interface.

Oligo(aryleneethynylene)s with Terminal Pyridyl Groups: Synthesis and Length Dependence of the Tunneling-to-Hopping Transition of Single-Molecule Conductances

The synthesis is reported of a new series of oligo(aryleneethynylene) (OAE) derivatives of up to ca. 6 nm in molecular length (OAE9) using iterative Pd-mediated Sonogashira cross-coupling methodology. The oligo-p-phenyleneethynylene cores of the molecular wires are functionalized at both termini with pyridyl units for attachment to gold leads. The molecular structures determined by single-crystal X-ray analysis are reported for OAE4, OAE5, OAE7, and OAE8a. The charge transport characteristics of derivatives OAE3–OAE9 in single-molecular junctions have been studied using the mechanically controlled break junction technique. The data demonstrate that the junction conductance decreases with increasing molecular length. A transition from coherent transport via tunneling to a hopping mechanism is found for OAE wires longer than ca. 3 nm.

Promises of cyclotron-produced 44Sc as a diagnostic match for trivalent β--emitters: in vitro and in vivo study of a 44Sc-DOTA-folate conjugate

In recent years, implementation of 68Ga-radiometalated peptides for PET imaging of cancer has attracted the attention of clinicians. Herein, we propose the use of 44Sc (half-life = 3.97 h, average β+ energy [Eβ+av] = 632 keV) as a valuable alternative to 68Ga (half-life = 68 min, Eβ+av = 830 keV) for imaging and dosimetry before 177Lu-based radionuclide therapy. The aim of the study was the preclinical evaluation of a folate conjugate labeled with cyclotron-produced 44Sc and its in vitro and in vivo comparison with the 177Lu-labeled pendant. Methods: 44Sc was produced via the 44Ca(p,n)44Sc nuclear reaction at a cyclotron (17.6 ± 1.8 MeV, 50 μA, 30 min) using an enriched 44Ca target (10 mg 44CaCO3, 97.00%). Separation from the target material was performed by a semiautomated process using extraction chromatography and cation exchange chromatography. Radiolabeling of a DOTA-folate conjugate (cm09) was performed at 95°C within 10 min. The stability of 44Sc-cm09 was tested in human plasma. 44Sc-cm09 was investigated in vitro using folate receptor–positive KB tumor cells and in vivo by PET/CT imaging of tumor-bearing mice Results: Under the given irradiation conditions, 44Sc was obtained in a maximum yield of 350 MBq at high radionuclide purity (>99%). Semiautomated isolation of 44Sc from 44Ca targets allowed formulation of up to 300 MBq of 44Sc in a volume of 200–400 μL of ammonium acetate/HCl solution (1 M, pH 3.5–4.0) within 10 min. Radiolabeling of cm09 was achieved with a radiochemical yield of greater than 96% at a specific activity of 5.2 MBq/nmol. In vitro, 44Sc-cm09 was stable in human plasma over the whole time of investigation and showed folate receptor–specific binding to KB tumor cells. PET/CT images of mice injected with 44Sc-cm09 allowed excellent visualization of tumor xenografts. Comparison of cm09 labeled with 44Sc and 177Lu revealed almost identical pharmacokinetics. Conclusion: This study presents a high-yield production and efficient separation method of 44Sc at a quality suitable for radiolabeling of DOTA-functionalized biomolecules. An in vivo proof-of-concept study using a DOTA-folate conjugate demonstrated the excellent features of 44Sc for PET imaging. Thus, 44Sc is a valid alternative to 68Ga for imaging and dosimetry before 177Lu-radionuclide tumor therapy.

Anti-PSMA antibody-coupled gold nanorods detection by optical and electron microscopies

While cancer is one of the greatest challenges to public health care, prostate cancer was chosen as cancer model to develop a more accurate imaging assessment than those currently available. Indeed, an efficient imaging technique which considerably improves the sensitivity and specificity of the diagnostic and predicting the cancer behavior would be extremely valuable. The concept of optoacoustic imaging using home-made functionalized gold nanoparticles coupled to an antibody targeting PSMA (prostate specific membrane antigen) was evaluated on different cancer cell lines to demonstrate the specificity of the designed platform. Two commonly used microscopy techniques (indirect fluorescence and scanning electron microscopy) showed their straightforwardness and versatility for the nanoparticle binding investigations regardless the composition of the investigated nanoobjects. Moreover most of the research laboratories and centers are equipped with fluorescence microscopes, so indirect fluorescence using Quantum dots can be used for any active targeting nanocarriers (polymers, ceramics, metals, etc.). The second technique based on backscattered electron is not only limited to gold nanoparticles but also suits for any study of metallic nanoparticles as the electronic density difference between the nanoparticles and binding surface stays high enough. Optoacoustic imaging was finally performed on a 3D cellular model to assess and prove the concept of the developed platform.

Fluorescence-encoded gold nanoparticles: library design and modulation of cellular uptake into dendritic cells.

In order to harness the unique properties of nanoparticles for novel clinical applications and to modulate their uptake into specific immune cells we designed a new library of homo- and hetero-functional fluorescence-encoded gold nanoparticles (Au-NPs) using different poly(vinyl alcohol) and poly(ethylene glycol)-based polymers for particle coating and stabilization. The encoded particles were fully characterized by UV-Vis and fluorescence spectroscopy, zeta potential and dynamic light scattering. The uptake by human monocyte derived dendritic cells in vitro was studied by confocal laser scanning microscopy and quantified by fluorescence-activated cell sorting and inductively coupled plasma atomic emission spectroscopy. We show how the chemical modification of particle surfaces, for instance by attaching fluorescent dyes, can conceal fundamental particle properties and modulate cellular uptake. In order to mask the influence of fluorescent dyes on cellular uptake while still exploiting its fluorescence for detection, we have created hetero-functionalized Au-NPs, which again show typical particle dependent cellular interactions. Our study clearly prove that the thorough characterization of nanoparticles at each modification step in the engineering process is absolutely essential and that it can be necessary to make substantial adjustments of the particles in order to obtain reliable cellular uptake data, which truly reflects particle properties.

Functionalization of PAMAM dendrimers with nitronyl nitroxide radicals as models for the outer-sphere relaxation in dentritic potential MRI contrast agents

PAMAM dendrimers functionalized with nitronyl nitroxide radicals were characterized. Quantitative determination of substitution with radicals was performed using EPR and electrochemical methods. The study of the 1H NMR relaxation of the surrounding water showed how the outer-sphere contribution to the relaxivity may be limited by the presence of the dendrimer core.

Excited state interactions between the chiral Au 38 L 24 cluster and covalently attached porphyrin

A protected S-acetylthio porphyrin was synthesized and attached to the Au38(2-phenylethanethiolate)24 cluster in a ligand exchange reaction. Chiral high performance liquid chromatography of the functionalized cluster yielded enantiomeric pairs of clusters probably differing in the binding site of the porphyrin. As proven by circular dichroism, the chirality was maintained. Exciton coupling between the cluster and the chromophore is observed. Zinc can be incorporated into the porphyrin attached to the cluster, as evidenced by absorption and fluorescence spectroscopy, however, the reaction is slow. Quenching of the chromophore fluorescence is observed, which can be explained by energy transfer from the porphyrin to the cluster. Transient absorption spectra of Au38(2-phenylethanethiolate)24 and the functionalized cluster probe the bleach of the gold cluster due to ground state absorption and the characteristic excited state absorption signals. Zinc incorporation does not have a pronounced effect on the photophysical behaviour. Decay times are typical for the molecular behaviour of small monolayer protected gold clusters.

C1-C8 hydrocarbons in DSDP Leg 64 Holes sediments

Sediments from the Baja California Continental Margin Transect - Sites 474 and 476 - showed small amounts of C2-C8 hydrocarbons and functionalized compounds (alkenes) typical of organic-rich, Recent, cold (<30°C) marine sediments. In contrast, some samples from Sites 477, 478, 479, and Hole 481A in the Guaymas Basin, an active spreading center, showed the characteristics of thermally generated hydrocarbons. These include an increase (sometimes exponential) in amount and diversity of C2-C8 hydrocarbons and a decrease in alkenes in more thermally mature sediments. The results indicate that the injection of basaltic sills has minimal effect on C2-C8 hydrocarbon generation except in the immediate vicinity of the sill. The absence of light hydrocarbons close to the hottest sills suggests that the compounds distill away as they are formed in these areas of very active hydrothermal circulation. A sample of young sediment exposed to very high temperatures (>300°C) from deeper thermal sources at the hottest site, 477, showed a very limited hydrocarbon distribution, including primarily ethane, benzene, and toluene, together with smaller amounts of propane and butane.

Topographical and mechanical characterization of living eukaryotic cells on opaque substrates: development of a general procedure and its application to the study of non-adherent lymphocytes

The mechanical behavior of living murine T-lymphocytes was assessed by atomic force microscopy (AFM). A robust experimental procedure was developed to overcome some features of lymphocytes, in particular their spherical shape and non-adherent character. The procedure included the immobilization of the lymphocytes on amine-functionalized substrates, the use of hydrodynamic effects on the deflection of the AFM cantilever to monitor the approaching, and the use of the jumping mode for obtaining the images. Indentation curves were analyzed according to Hertz's model for contact mechanics. The calculated values of the elastic modulus are consistent both when considering the results obtained from a single lymphocyte and when comparing the curves recorded from cells of different specimens

Nanoscale imaging of chemical interactions: Fluorine on graphite

Using C60-functionalized scanning tunneling microscope tips, we have investigated the adsorption of fluorine on graphite. Based on characteristics of the accompanying electron standing waves, we are able to distinguish the fluorine adatoms that have bonded ionically to the graphite surface from those that have formed covalent bonds with the surface. This result permits determination of the ratio of ionic to covalent C–F bonds on graphite obtained by gas phase fluorination, which seems to be temperatureindependent between 200 and 300°C under the reaction conditions used.

Measuring the forces involved in polyvalent adhesion of uropathogenic Escherichia coli to mannose-presenting surfaces

Mechanisms of bacterial pathogenesis have become an increasingly important subject as pathogens have become increasingly resistant to current antibiotics. The adhesion of microorganisms to the surface of host tissue is often a first step in pathogenesis and is a plausible target for new antiinfective agents. Examination of bacterial adhesion has been difficult both because it is polyvalent and because bacterial adhesins often recognize more than one type of cell-surface molecule. This paper describes an experimental procedure that measures the forces of adhesion resulting from the interaction of uropathogenic Escherichia coli to molecularly well defined models of cellular surfaces. This procedure uses self-assembled monolayers (SAMs) to model the surface of epithelial cells and optical tweezers to manipulate the bacteria. Optical tweezers orient the bacteria relative to the surface and, thus, limit the number of points of attachment (that is, the valency of attachment). Using this combination, it was possible to quantify the force required to break a single interaction between pilus and mannose groups linked to the SAM. These results demonstrate the deconvolution and characterization of complicated events in microbial adhesion in terms of specific molecular interactions. They also suggest that the combination of optical tweezers and appropriately functionalized SAMs is a uniquely synergistic system with which to study polyvalent adhesion of bacteria to biologically relevant surfaces and with which to screen for inhibitors of this adhesion.

Controlling cell attachment on contoured surfaces with self-assembled monolayers of alkanethiolates on gold.

This paper describes a method based on experimentally simple techniques--microcontact printing and micromolding in capillaries--to prepare tissue culture substrates in which both the topology and molecular structure of the interface can be controlled. The method combines optically transparent contoured surfaces with self-assembled monolayers (SAMs) of alkanethiolates on gold to control interfacial characteristics; these tailored interfaces, in turn, control the adsorption of proteins and the attachment of cells. The technique uses replica molding in poly(dimethylsiloxane) molds having micrometer-scale relief patterns on their surfaces to form a contoured film of polyurethane supported on a glass slide. Evaporation of a thin (< 12 nm) film of gold on this surface-contoured polyurethane provides an optically transparent substrate, on which SAMs of terminally functionalized alkanethiolates can be formed. In one procedure, a flat poly(dimethylsiloxane) stamp was used to form a SAM of hexadecanethiolate on the raised plateaus of the contoured surface by contact printing hexadecanethiol [HS(CH2)15CH3]; a SAM terminated in tri(ethylene glycol) groups was subsequently formed on the bare gold remaining in the grooves by immersing the substrate in a solution of a second alkanethiol [HS(CH2)11(OCH2CH2)3OH]. Then this patterned substrate was immersed in a solution of fibronectin, the protein adsorbed only on the methyl-terminated plateau regions of the substrate [the tri(ethylene glycol)-terminated regions resisted the adsorption of protein]; bovine capillary endothelial cells attached only on the regions that adsorbed fibronectin. A complementary procedure confined protein adsorption and cell attachment to the grooves in this substrate.

Innovative analytical strategies for the development of sensor devices and mass spectrometry methods

Il lavoro presentato in questa tesi di Dottorato è incentrato sullo sviluppo di strategie analitiche innovative basate sulla sensoristica e su tecniche di spettrometria di massa in ambito biologico e della sicurezza alimentare. Il primo capitolo tratta lo studio di aspetti metodologici ed applicativi di procedure sensoristiche per l’identificazione e la determinazione di biomarkers associati alla malattia celiaca. In tale ambito, sono stati sviluppati due immunosensori, uno a trasduzione piezoelettrica e uno a trasduzione amperometrica, per la rivelazione di anticorpi anti-transglutaminasi tissutale associati a questa malattia. L’innovazione di questi dispositivi riguarda l’immobilizzazione dell’enzima tTG nella conformazione aperta (Open-tTG), che è stato dimostrato essere quella principalmente coinvolta nella patogenesi. Sulla base dei risultati ottenuti, entrambi i sistemi sviluppati si sono dimostrati una valida alternativa ai test di screening attualmente in uso per la diagnosi della celiachia. Rimanendo sempre nel contesto della malattia celiaca, ulteriore ricerca oggetto di questa tesi di Dottorato, ha riguardato lo sviluppo di metodi affidabili per il controllo di prodotti “gluten-free”. Il secondo capitolo tratta lo sviluppo di un metodo di spettrometria di massa e di un immunosensore competitivo per la rivelazione di prolammine in alimenti “gluten-free”. E’ stato sviluppato un metodo LC-ESI-MS/MS basato su un’analisi target con modalità di acquisizione del segnale selected reaction monitoring per l’identificazione di glutine in diversi cereali potenzialmente tossici per i celiaci. Inoltre ci si è focalizzati su un immunosensore competitivo per la rivelazione di gliadina, come metodo di screening rapido di farine. Entrambi i sistemi sono stati ottimizzati impiegando miscele di farina di riso addizionata di gliadina, avenine, ordeine e secaline nel caso del sistema LC-MS/MS e con sola gliadina nel caso del sensore. Infine i sistemi analitici sono stati validati analizzando sia materie prime (farine) che alimenti (biscotti, pasta, pane, etc.). L’approccio sviluppato in spettrometria di massa apre la strada alla possibilità di sviluppare un test di screening multiplo per la valutazione della sicurezza di prodotti dichiarati “gluten-free”, mentre ulteriori studi dovranno essere svolti per ricercare condizioni di estrazione compatibili con l’immunosaggio competitivo, per ora applicabile solo all’analisi di farine estratte con etanolo. Terzo capitolo di questa tesi riguarda lo sviluppo di nuovi metodi per la rivelazione di HPV, Chlamydia e Gonorrhoeae in fluidi biologici. Si è scelto un substrato costituito da strips di carta in quanto possono costituire una valida piattaforma di rivelazione, offrendo vantaggi grazie al basso costo, alla possibilità di generare dispositivi portatili e di poter visualizzare il risultato visivamente senza la necessità di strumentazioni. La metodologia sviluppata è molto semplice, non prevede l’uso di strumentazione complessa e si basa sull’uso della isothermal rolling-circle amplification per l’amplificazione del target. Inoltre, di fondamentale importanza, è l’utilizzo di nanoparticelle colorate che, essendo state funzionalizzate con una sequenza di DNA complementare al target amplificato derivante dalla RCA, ne permettono la rivelazione a occhio nudo mediante l’uso di filtri di carta. Queste strips sono state testate su campioni reali permettendo una discriminazione tra campioni positivi e negativi in tempi rapidi (10-15 minuti), aprendo una nuova via verso nuovi test altamente competitivi con quelli attualmente sul mercato.