La liaison Bore-Fluor (B-F) a une longue et riche histoire dans le domaine des transformations chimiques. De plus, cette liaison B-F est utilisée dans une grande diversité de domaines tel que la médecine nucléaire avec l'imagerie de Tomographie par Émission de Positrons (TEP) ou l'imagerie de fluorescence. Dans le cadre de cette thèse, nous nous intéressons à l'utilisation de la liaison B-F pour la formation nucléophile de liaison Carbone-Fluor (C-F) et le développement de nouveaux fluorophores ayant des applications potentielles dans les domaines de l'imagerie bimodale TEP et l'imagerie de fluorescence. Les organotrifluoroborates sont connus pour être des outils de synthèse d'importance majeure pour la conception de molécules organiques. Cependant, le groupement trifluoroborate n'est généralement utilisé qu'en tant que groupement partant, c'est à dire par rupture de la liaison C-B. Ainsi, son potentiel en tant que source de fluorure nucléophile (F-) n'est pas étudié. C'est pourquoi, nous nous sommes intéressé à l'aptitude des organotrifluoroborates à servir de source de fluorure. En effet, si historiquement la réaction de Balz-Schiemann, utilise le BF4- comme source de F-, nous avons démontré que les organotrifluoroborates sont capables de réaliser la réaction de fluoro-dédiazotation de sels d'aryldiazonium générés in situ dans des conditions réactionnelles douces. De plus, dans des réactions avec une classe de substrats analogues, des sels dissymétriques de diaryliodonium pourraient être fluorés par des phényltrifluoroborates par décomposition thermique de la paire d'ion. Finalement, l'aptitude du groupement trifluoroborate à promouvoir des réactions de fluoration intramoléculaires a été étudiée par la synthèse de divers motifs contenant ce groupement. Nous avons également étudié de nouveaux complexes fluoroborates (B-Fn) dérivés du motif 2-aryl-benzothiazole comme sondes fluorescentes. Trois complexes ont été isolés, caractérisés, et leurs propriétés photo-physiques rapportées. Un complexe monofluoroborate de 2,4-diaryl-benzothiazole présentant les propriétés photo-physiques les plus prometteuses s'est révélé stable en conditions aqueuses. De plus, l'abstraction de fluorure de ce complexe a été démontrée, fournissant ainsi des résultats préliminaires prometteurs vers la viabilité du radiomarquage par échange isotopiques 19F-18F. Ce complexe monofluoroborate a également montré des propriétés structurales intéressantes par la présence d'un bore stéréogène. Les énantiomères de ce complexe ont été dédoublés par chromatographie chirale à partir du mélange racémique puis caractérisés par dichroïsme circulaire. Enfin, la stabilité configurationnelle du bore a été étudiée. / The B-F bond has an expansive and rich history in chemical transformations and the versatility of the B-F bond has also shown immense utility in fields as far reaching as Positron Emission Tomography (PET) and fluorescence imaging. The application of B-F bonds for nucleophilic Carbon-Fluorine (C-F) bond formation and the development of novel fluorophores with potential applications in PET/fluorescence bi-modal imaging is reported in this document. Organotrifluoroborates are well known as indispensable synthetic tools for the elaboration of organic molecules. However, the trifluoroborate functionality is generally regarded as an auxiliary group, and its potential as a nucleophilic fluoride (F-) source has remained untested. In this context, the ability of organotrifluoroborates to serve as competent sources of F- was investigated. In an update to the historic Balz-Schiemann reaction, that traditionally uses BF4- as a source of F-, organotrifluoroborates were shown to mediate the fluorodediazoniation of in situ generated aryl diazonium salts under mild reaction conditions. In reactions with an analogous substrate class, unsymmetrical diaryliodonium salts could also be fluorinated using phenyltrifluoroborate via thermal decomposition of the ion pair. Finally, the ability of the trifluoroborate moiety to mediate intramolecular fluorination reactions was investigated through the synthesis of various trifluoroborate-containing molecular scaffolds. We also investigated novel fluoroborate (B-Fn) complexes of the 2-aryl-benzothiazole scaffold, known for its interaction with Aß aggregates, as fluorescent probes. Three complexes were isolated, characterized and their photophysical properties are reported. A 2,4-diaryl-benzothiazole monofluoroborate complex displaying the most promising photophysical properties, was shown to be stable to aqueous conditions. Fluoride abstraction from this complex was also demonstrated, providing promising preliminary results towards the viability of radiolabelling via 19F-18F isotope exchange. The monofluoroborate complex also showed interesting structural properties given the asymmetric boron centre. Enantiomers of the complex were successfully resolved by chiral chromatography from the racemic mixture, characterized by Circular Dichroism and their stability to inversion investigated.

The development of single-molecule and super-resolution fluorescence techniques has revolutionised biological imaging. Nano-scale cellular structures and heterogeneous dynamic processes are now able to be visualised with unprecedented resolution in both time and space. The achievable localisation precision and therefore the resolution is fundamentally limited by the number of photons a single-fluorophore can emit. The ideal super-resolution dye would emit a large number of photons over a short period of time. On the contrary, an optimal single-molecule tracking probe would be highly photostable and undergo no transient dark-state transitions. Single-molecule instrument development is beginning to reach technological saturation and as the frontiers of bioimaging expand, exorbitant demands are placed on the gamut of available probes that often cannot be met. Thus, the next key challenge in the field is the development of the better fluorophores that underlie these techniques; this includes both the synthesis of new chemical derivatives and alternative novel strategies to augment existing technologies. The results of this thesis are divided into two distinct parts; Project One details the development of new synthetic fluorescent probes for the study of amyloid protein aggregates implicated in neurodegenerative diseases. This includes a study of the photophysical and binding properties of a novel fluorophore library based on the amyloid dye Thioflavin-T. Following on from this, is the presentation of novel bifunctional dyes capable of simultaneously identifying hydrogen peroxide and amyloid aggregates by combining existing tools for the independent detection of these species. The sensing capabilities of these dyes are explored at the bulk and single-molecule levels. Project Two describes a new photo-modulatable fluorescent-protein fusion construct that can undergo Förster resonance energy transfer (FRET) to an organic dye molecule. This FRET cassette is comprised of a photoconvertible fluorescent protein donor, mEos3.2 and acceptor fluorophore, JF646. This strategy imparts a strong photostabilising effect on the fluorescent protein and a resistance to photobleaching. The functionality of this approach is demonstrated with in vitro single-molecule fluorescence studies and its biological applicability shown by tracking single proteins in the nuclei of live embryonic stem cells. Furthermore, initial characterisations of the excited state dynamics in effect are presented through the systematic modification of parameters.

<P> A great number of cellular processes can lead to local changes in proton concentration and temperature. So, it is desirable to be able to measure pH and temperature with non-invasive and spatially resolved methods. In this thesis, I describe two unique methods to measure pH and temperature using the environmental sensitivity of the proton transfer of fluorescent molecules. The first method is based on the detection of the temperature and pH dependent parameters associated with the blinking of fluorophores by fluorescence correlation spectroscopy (FCS). Employing EGFP as the probe, I used this method to characterize temperature increase at a laser focus due to light absorption in a thin liquid sample. Using pyranine as a probe, I extended the applicability of this method to a range of pH including physiological pH. Also, I investigated in details the effect of buffer composition on the blinking of the fluorophores. Then, I concluded that one limitation of this method is its strong dependence on buffer conditions, which are not well characterized in vivo. The second approach is a pH measurement method based on ratiometric imaging. This method is not as dependent on buffer condition. We improved on current ratiometric imaging techniques by demonstrating the possibility of using two-photon excitation. This method was used to measure pH in pyranine loaded vesicles created during receptor-mediated endocytosis of µ-Opioid receptors expressed in HEK 293 cells. Preliminary results showed that the pH in the endocytic vesicles dropped to a value similar to those measurements in late endosomes roughly~ 10 minutes after triggering the endocytosis, and eventually, the pH reading reached a value similar to that of the pH oflysosomes. </p> / Thesis / Doctor of Philosophy (PhD)

L’utilisation de nano-objets luminescents en milieu biologique est devenue très répandue, notamment en vue d’applications biomédical est elles que l’imagerie, la thérapie et le diagnostic. Jusqu’à récemment, les principaux travaux réalisés dans ce domaine concernaient les nanoparticules de silice dopées ou fonctionnalisées avec des molécules organiques, les nanoparticules d’or et les nanoparticules semi-conductrices (quantum dots, i.e., QDs). Toutefois, un certain nombre de limitations demeurent pour les applications dans le domaine du vivant, en lien notamment avec des problèmes de stabilité, de biocompatibilité et de toxicité ou encore de biodégradabilité. En parallèle,un certain nombre de molécules organiques fluorescentes non-toxiques ont été utilisées comme sondes fluorescentes en milieu biologique, mais leur brillance demeure limitée. L’idée directrice de la thèse est de concevoir et synthétiser de nouveaux chromophores organiques présentant une émission modulable (du visible au proche infrarouge) et adaptés à la préparation de nanoparticules organiques fluorescentes (FONs) combinant à la fois une brillance extrêmement élevée, une excellente stabilité colloïdale et une photostabilité adaptée à leur utilisation en imagerie in vitro et in vivo. De tels nanoobjets ultra-brillants pourraient alors représenter une alternative très intéressante aux nanoparticules actuellement les plus utilisées en imagerie de fluorescence du vivant (QDs). Le manuscrit décrit la synthèse et les propriétés de plusieurs classes de molécules fluorescentes spécifiquement conçues pour former des telles FONS par auto-assemblage dans l’eau. La préparation de ces FONs est présentée et leurs propriétés étudiées et discutées. Enfin des applications concrètes en bio-imagerie sont présentées. / Nowadays the use of bright luminescent nano-objects in biological environment is a topic that is gaining more and more importance, especially for biomedical applications such as imaging, the rapyand diagnostic. So far, numerous studies have been conducted with gold nanoparticles, silica nanoparticles (doped or functionalized with organic molecules), as well as semiconductor nanoparticles (quantum dots, i.e., QDs). However, most of these nanoparticles suffer from drawbacks (in terms of stability, biocompatibility, eco-toxicity or degradability). On the other hand, several nontoxic fluorescent molecular probes have been widely used, but most of the time their brightness remain modest in biological environments compared to QDs. Our idea is to engineer new organicchromophores with tunable emission wavelength (from visible to near infrared) for further preparation of organic fluorescent nanoparticles (so called FONs) that display giant one-photon and two-photonbrightness, as well as good colloidal and chemical stability, and suitable photostability for in vitro andin vivo imaging. As such, these FONs would represent interesting alternatives to QDs for use in bioimaging. This manuscript describes the synthesis and characterization of new classes of fluorescent molecules specifically engineered as building blocks for the fast preparation of such nanoparticles byself-aggregation in water. The FONs were fully characterized from both morphological and photophysical points of view and further used in bioimaging.

Face à la recrudescence de l'utilisation des nanotubes de carbone (NTCs) pour des applications de pointe, leur impact potentiel sur la santé et sur l'environnement est devenu une question centrale. Afin d'évaluer les risques liés à l'exposition accidentelle des êtres-vivants à ces nanoparticules de nombreuses études de toxicité ont été réalisées. Pour certaines, il est important de pouvoir déterminer précisément où et comment les NTCs s'accumulent dans les organismes aquatiques ou dans les cellules. Un des moyens habituel pour réaliser de telles expériences consiste à fonctionnaliser les NTCs avec des molécules fluorescentes, qui seront par la suite mises en évidence sous un rayonnement de longueur d'onde appropriée, permettant ainsi de localiser les nanotubes. Malgré le fait que cette technique soit bon marché et facilement mise en œuvre, elle souffre d'un défaut majeur: elle présuppose en effet que les molécules fluorescentes resteront attachées aux nanotubes de façon définitive. Cependant, cette supposition peut être sérieusement questionnée car les fluorophores possèdent généralement un ou plusieurs cycles aromatiques pouvant facilement conduire à l'adsorption de ces derniers sur les parois des NTCs. Dès lors, une fois que les NTCs atteignent l'environnement chimique complexe d'une cellule, on peut s'attendre à une désorption des molécules fluorescentes, pouvant de fait conduire à de mauvaises interprétations des résultats expérimentaux. Il est par conséquent essentiel d'évaluer l'efficacité du protocole de greffage et de déterminer les différentes proportions de molécules greffées de façon covalente et non-covalente (adsorbées). Bien entendu, la fonctionnalisation des NTCs étant une problématique clef dans le développement de leurs applications en science des matériaux, les questions soulevées ici dépassent le simple cadre de la santé et de l'environnement. Pour y répondre, nous avons décidé de nous intéresser à la fonctionnalisation de nanotubes de carbone double-parois (DWNTs) par deux molécules fluorescentes, l'Isothiocyanate de Fluorescéine (FITC) et la cyanine 5Me(Net2)2. Nous avons, pour ce faire, réaliser le greffage des fluorophores sur des DWNTs oxydés purifiés à l'aide d'un procédé de fonctionnalisation en 3 étapes. Enfin, nous avons caractérisé nos échantillons au moyen de deux techniques différentes de spectroscopie, la spectroscopie de photoélectron X (XPS) et la spectroscopie de diffusion inélastique de neutrons (INS). En complément de la spectroscopie de neutrons, nous nous sommes appuyés sur des simulations numériques, telle que la théorie de la fonctionnelle de la densité (DFT) pour les analyses de nos données expérimentales. Les résultats ainsi obtenus, ont permis de montrer que, contrairement à ce qui était communément attendu lors d'un greffage covalent, une part non négligeable des marqueurs fluorescent restait adsorbée à la surface des NTCs, et ce, en dépit de lavages consciencieux par des solvants appropriés. La présence de ces marqueurs fluorescents adsorbés montre, dans le cadre des études de toxicité, qu'il existe une certaine probabilité que ces derniers se détachent des NTCs lors de leur voyage dans un organisme ou dans une cellule, ce qui conduirait l'établissement de conclusions potentiellement erronées quant au destin de ces nanoparticules. / Facing the growing use of carbon nanotubes (CNTs) in state-of-the-art applications, the question of their potential impact on health and environment became a central one. In order to evaluate the risks related to living being exposure to those nanoparticles, several toxicity studies have been performed aiming at knowing the exact location of the CNTs accumulating inside organisms or cells. A very common way to track them in such conditions is to functionalize the CNTs with fluorescent molecules which would be highlighted afterwards under a light with appropriate exciting wavelength. Despite the fact that these fluorescence techniques are very cheap and easy to operate, they suffer from a major drawback: they assumes that the fluorescent molecule is permanently linked to the CNTs. It is however reasonable to question this assumption as the fluorescent molecules are usually constituted by one or more 6-carbon rings that can easily also simply adsorb on the surface of the CNTs. This non-covalent binding could lead to the desorption of the fluorophore once the CNTs reach the complex chemical environment of a living cell or organism. Therefore, the fluorescence data could lead to wrong information about the CNTs location. Therefore, it is fundamental to understand the grafting mechanisms in order to estimate the efficiency of the covalent functionalization of the CNTs as well as the amount of simply adsorbed fluorophores. Of course, the impact of such a question clearly exceeds the field of the health and the environment, because the functionalization of CNTs is a key for their application is Materials Science in general. In order to answer to these questions, we chose to study the functionalization of Double-Walled carbon Nanotubes (DWNTs) with two different fluorophores, the Fluorescein Isothiocyanate (FITC) and the STREPTOcyanine 5Me(Net2)2. We used a three-step functionalization process to graft the fluorophores on highly-purified oxidized DWNTs. Finally, both the surface and the bulk of the sample have been investigated using two different spectroscopic techniques, the X-ray photoelectron spectroscopy (XPS) and the inelastic neutron scattering spectroscopy (INS). In addition to neutron techniques we also used computational techniques such as Density Functional Theory (DFT) calculations for a better analysis of our results. The results obtained by means of this two powerful techniques highlighted that, although the fluorescent markers is always considered to be strongly bonded onto carbon nanotubes when using a covalent strategy, a non-negligible part may in fact be only adsorbed, even after thorough washings in appropriate solvents. This is likely, in the particular field of toxicology, to lead to a release of the fluorescent marker at some point along the journey of the nanoparticle throughout the cells or the whole organism, and thus to partially wrong conclusions in terms of their fate in terms of biodistribution, accumulation or excretion.

The use of fluorescent imaging probes for the real time detection of cellular malfunctions, such as enzyme over expression has shown promise. Fluorescent dyes with absorption and emission values below 600 nm are limited in their in vivo applications due to high background auto-fluorescence and low resolution images. Employing near infrared (NIR) fluorophores such as cyanine dyes can overcome this disadvantage. Cyanine dyes can be synthesised using solution or solid-phase techniques with the use of solution phase chemistry allowing for larger scale and higher yielding reactions. Utilising a selection of functional groups and varying polymethine chain lengths a cyanine dye library with tuneable absorption and emission wavelengths was synthesised. This thesis gives the first detailed examples of how modifications on heptamethine cyanine dyes alter their cellular uptake and cellular toxicity. Furthermore, a NIR fluorescent microsphere is reported as well as NIR functionalised microspheres with the ability to be tracked within cells. Additional lines of work involved the synthesis of a fluorescent sensor for the visualisation of bacteria. Aminopeptidases are present within the peptidoglycan cell wall of Gram negative bacteria and therefore can be targeted for real time detection of bacteria to aid in the detection of infectious diseases. A coumarin based probe is reported which detects aminopeptidase in gram negative bacteria in vitro.

Thesis advisor: Abhishek Chatterjee / Förster Resonance Energy Transfer (FRET) offers a powerful approach to study biomolecular dynamics in vitro as well as in vivo. The ability to apply FRET imaging to proteins in living cells provides an excellent tool to monitor important dynamic events such as protein conformational changes, protein-protein interactions, and proteolysis reactions. However, selectively incorporating two distinct fluorophores into the target protein(s) that are capable of FRET interaction within the complex cellular milieu is challenging. Consequently, terminal fusion to genetically encoded fluorescent proteins has emerged as the predominant labeling strategy for FRET studies in vivo. However, a major limitation of this strategy stems from the large size of the fluorescent proteins, which may perturb the native properties of the target, and restricted attachment only to the termini of the target. We reasoned that using genetically encoded fluorescent unnatural amino acids would overcome several of these challenges associated with currently available labeling strategies owing to their small size and the ability to introduce them site- specifically and co-translationally. Here, we report the use of the fluorescent unnatural amino acid “Anap” as a FRET donor with green and yellow fluorescent protein acceptors. We demonstrate the utility of this labeling strategy using proteolysis and conformational change models, and step towards in vivo studies by further developing a proteolysis system in cell lysates. / Thesis (MS) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

BODIPYs são compostos fluorescentes que possuem uma ampla gama de aplicações tecnológicas em diversas áreas do conhecimento, recebendo considerável destaque na literatura, tanto do ponto de vista fotoquímico, quanto sintético. Entretanto, a reatividade química desses fluorórofos ainda não é totalmente compreendida. Neste sentido, neste trabalho utilizou-se a química de sais de diazônio aplicada aos BODIPYs, explorando uma série de perspectivas para diversificação estrutural desses fluoróforos. Como estratégias para a obtenção do BODIPY funcionalizado com o grupo diazo foram testados três diferentes métodos, que envolvem a utilização de NOBF4, NaNO2/HCl e NaNO2/HBF4. O método que envolve a utilização de NOBF4 não levou a obtenção do composto diazotado, no entanto, resultou na obtenção de compostos nitrosilados. Quando utilizado NaNO2/HCl, o composto diazotado foi obtido in situ, seguido pela reação de acoplamento diazóico. Com o método que envolve o uso de NaNO2/HBF4 foi possível obter melhores rendimentos para as reações de acoplamento diazóico, e abriu a possibilidade de novas explorações química dos sais de diazônio-BODIPY / BODIPYs are fluorescent compounds which have a wide range of technological applications in different areas of knowledge. They have remarkable presence in the literature because of their synthetic and photochemical properties. However, the chemical reactivity of these fluorophores are not fully known. Considering this, our study applied the diazonium salts chemistry to BODIPYs aiming to explore the structure diversification of these fluorophores. Three different methods were used to obtain the diazo derivatives of BODIPYs: NOBF4, NaNO2/HCl and NaNO2/HBF4. The method using the NOBF4 instead of afford the diazotized compound as expected, a nitrosylated compound was obtained. When NaNO2/HCl was used, the diazotized compound was obtained in situ, followed by diazo coupling reaction. The best yields diazo coupling reactions were obtained when NaNO2/HBF4 was used. The reactions described in our work showed new possibilities of chemical tractability of BODIPY compounds.

xx, 361 p. A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / Highly conjugated carbon-rich molecules have attracted interest in recent years due to unique electronic, optical and materials properties. Carbon networks based upon the phenylacetylene subunit are increasingly recognized as building blocks for a host of sensing and electronics components due to the rigidity and linearity of carbon-carbon triple bonds. Further extending this motif into a macrocycle, generating a dehydrobenzoannulene (DBA), also confers planarity, increasing the à -conjugation and giving rise to enhanced materials behavior. Functionalization of arylacetylenes and DBAs with electron donating and accepting groups manipulates the energetics such that finely-tuned optoetectronic properties can be devised for customized applications, including fluorescent sensor arrays, organic light-emitting diodes, and nonlinear optical materials. Fundamental structure-property relationship studies into certain physical modifications of molecular architecture effects on the photophysics, intramolecular charge transfer (ICT), or complexation properties are of importance in the rational design of the next generation of organic electronics. Chapter I provides a review of recent advances in the field of annulene chemistry. It is organized by cycle type, size, and application within each category. Chapter II describes syntheses and ion responses of an array of donor/acceptor-functionalized arylacetylenes. The independent manipulation of frontier molecular orbital (FMO) energy levels is discussed in relation to a fluorescent switching phenomenon. Chapter III expands this effect to include [15]DBAs. The consequences of incorporating protonatable donor/acceptor groups into a macrocycle, as well as placement of the acceptor nitrogen are examined, and comparison of calculations to experimental results imply generation of transient ICT species with induced FMO localizations. Chapter IV describes the syntheses of acyclic tetrakis(phenylethynyl)benzene (TPEB) and [14]- and [15]DBA systems utilizing fluorinated acceptor groups. Comparisons between these inductive acceptors and earlier resonance acceptors are made, and imply greater stability and processing potential for optoelectronic applications. Chapter V describes a series of bis[18]DBAs functionalized with dibutylamino groups as donors and nitro groups as acceptors. The effects of 2-donor/2-acceptor versus 4-donor/4-acceptor motifs are explored, and trends are identified in the systematic adjustment of the optical band gap that will have important implications for the design of two-photon absorbing materials. This dissertation includes my previously published and co-authored material. / Adviser: Michael M. Haley

Conjugated macrocycles have emerged as novel structural motifs that modulate the electronic properties of organic molecules because of their strained and contorted structures. Cycloparaphenylenes, known as nanohoops, are a particularly attractive scaffold for the design of new types of carbon nanomaterials because of their size-selective synthesis, radially oriented π-systems and tunable electronic properties. The development of modular syntheses of nanohoops over the past decade should enable the preparation of substituted derivatives that can be tuned for applications in biology and materials science. Chapter I provides a brief overview of conjugated macrocycles recently reported in the literature with a discussion of the structural effects that are responsible for the remarkable properties of this class of molecules. Chapter II highlights a scalable and mild synthetic approach developed in our lab to prepare nanohoop conjugated macrocycles and expands the generality of this methodology with the formal synthesis of natural product Acerogenin E. Chapter III describes the synthesis of cycloparaphenylenes with versatile functional handles and uncovers the reactivity of the strain nanohoop backbone under reaction conditions that promote the formation of radical cations. Chapter IV takes advantage of the functional groups described in chapter III to develop the first example of nanohoops as a new class of biocompatible fluorophores. Chapter V details a novel synthetic approach that enables the incorporation of the linear acene pentacene into the nanohoop backbone and reports our findings on the impact that the macrocyclic structure has on the properties of this organic semiconductor. In summary, the findings discussed in this dissertation provide synthetic strategies for the selective functionalization of nanohoops and highlight this class of molecules as a novel scaffold for the design of new types of carbon nanomaterials. This dissertation includes previously published and unpublished co-authored material.