The present PhD thesis entitled “Design, Synthesis and Evaluation of Chromofluorogenic Probes for Contaminating Species” is focused on the development of new chromo-fluorogenic sensors based on the principles of molecular recognition. The first part of this thesis is focused on the design and synthesis of suitable organic compounds as sensors for metal cations. The selected sensing paradigm was the binding site-signalling subunit approach. The synthetized receptors employs a chromophore (fluorescein or BODIPY) skeleton as signalling subunit and it is functionalized with aminoethoxy moieties as binging site; the metal coordination reduces the electron-donating ability of the nitrogen atom conjugated to the chromophore resulting in optical changes noticeable to the naked-eye. The sensing behavior is highly selective to trivalent cations (Fe3+, Al3+ and Cr3+) with remarkable limits of detection. The receptors based on BODIPYdyes retain the sensing abilities in mixed aqueous solutions. The remaining chapters of the thesis are centered in the detection and removal of nerve agents surrogates. The design, synthesis, characterization and application of new BODIPY chemosensors were studied. These chemosensors were designed containing different reactive sites in order to avoid interferences produced by acids or hydrolysis products, and also be able to distinguish between the different G-nerve agent mimics (DCNP and DFP). The BODIPY-probes allows screening of nerve agent surrogates with remarkable limits of detection and optical changes noticeable to the naked-eye. The sensing abilities are retained in solid support, allowing practical application in real-time monitoring by simple colorimetric tests. The displacement assay approach has been used to develop a selective sensor for V-nerve agent surrogates versus G-type. For this purpose, two Eu3+ and Au3+ BODIPY-complexes were prepared. In this case, V-surrogate is capable of coordinate the metallic center, releasing the BODIPY ligand. This causes a change in the optical properties visible to the naked-eye. Finally, the use of supramolecular-based organocatalyst for destruction of OP nerve agent surrogates was studied. Hydrolysis studies were performed in presence of 1,3-diindolylureas and thioureas, amines, aminoalcohol and glycols. Addition of catalyst enhances the electrophilic character of the P atom, and the final nucleophilic attack of water that results in the formation of the corresponding less toxic organophosphate derivatives, thus higher hydrolysis rates are obtained. / Barba Bon, A. (2014). Design, Synthesis and Evaluation of Chromo-fluorogenic Probes for Contaminating Species [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48521 / TESIS

Xanthene dyes are common fluorophores which have been widely used as molecular probes. The xanthene fluorophores can be used as highly selective optical sensors to detect disease biomarkers. A new fluorogenic dye containing an alpha, beta-unsaturated aldehyde moiety exhibits selective fluorescent signal enhancement in the presence of cysteine or peptides containing N-terminal cysteine residues. The mechanism is based on synergistic covalent and supramolecular interactions. A unique rhodamine boronic acid indicator is used in an optimized data collection protocol for wavelength- and time-dependent selectivity of various saccharides and nucleosides. One indicator is thereby capable of selectively distinguishing structurally related analytes in mixtures. Moreover, the rhodamine-based boronic acid responds linearly to increasing riboside concentrations in urine samples, potentially enabling the screening for inborn purine metabolism disorders.

[Truncated abstract] The scientific endeavours described within this thesis attempt to create novel solutions to current scientific, commercial and industrial downfalls, and contribute to the advancement of technologies in these areas. This has been achieved through the application of theoretical and experimental principles, entrenched in the domains of chemistry and physics, which have been harnessed to assist in the transformation from nanoscience to nanotechnology. These solutions range from unique supramolecular systems capable of selective-diameter enrichment of single-walled carbon nanotubes (SWCNTs), to the fabrication of low-cost, potentially remote deployable carbon nanotube-based gas and vapour sensors, and expand right through to the development of water-soluble fluoroionophoric sensors and manipulations of a molecular form of carbon in constructing all-carbon nano-architectures. For the advancement and successful integration of carbon nanotubes (CNTs) into commercial processes, the advent of scalable separation protocols based on their electronic properties is required. SWCNTs have been successfully solubilised using water-soluble p-phosphonated calix[n]arenes (n = 4, 6, 8) and 'extended arm' upper rim functionalised (benzyl, phenyl) p-sulfonated calix[8]arenes. Selective SWCNT diameter solubilisation has been demonstrated and subsequent preferential enrichment of SWCNTs with semiconducting or metallic electronic properties has been achieved. In addition, semiconducting nanotube-enriched supernatants (liquid) have been utilised to fabricate on/off field effect transistors (FET). These water-soluble supramolecular systems can be incorporated into post-growth purification protocols, with direct implications in areas such as carbon nano-electronics and device fabrication. In the current global environment there is a heightened level of public and governmental disquiet due to the reality of impending terrorist attacks. This is compounded by the inherent ease of manufacture and effectiveness of specific chemical warfare agents (CWAs) used in small-scale terrorist operations. ... Additional all-carbon structures are described with the formation of rings of helical SWCNT bundles through post-growth SWCNT modifications, and a variety of fibrous all-carbon structures, most notably novel square-geometry carbon nano-fibres (CNFs), through catalytic-chemical vapour deposition (C-CVD) synthesis strategies. The current requirement for entirely water-soluble fluorescent sensors is routinely documented in the literature. The autofluorescence properties of p-phenyl-sulfonated calix[8]arene are characterised and this water-soluble cavitand is surveyed as a metal cation sensor candidate. This particular system was found to exhibit a change in fluorescence response when exposed to divalent metal cations, and interactions with [UO2]2+, Pb2+, Co2+, and Cu2+ ions are discussed in detail. The system is characterised through a variety of analytical techniques to yield sensor calibration data, degradation characteristics, pH sensitivity and suitability as a 'small molecule' drug-carrier.

Un (bio)capteur classique est principalement composé de deux éléments essentiels : une couche réceptrice sensible à l’analyte à laquelle on s’intéresse et un transducteur qui permet de convertir une stimulation chimique / biologique en un signal physique mesurable. Dans le cas idéal, un capteur ne doit pas nécessiter de marquage de la cible, doit posséder de très grandes sensibilité et sélectivité envers elle, ne requiert qu’une faible quantité de cette dernière et doit présenter un temps de réponse très court. Au vu de ces critères, les microsystèmes électromécaniques (MEMS) sont des candidats très prometteurs dans le développement de capteurs. Les polymères fonctionnels, tels que les polymères à empreinte moléculaire (MIPs), sont une approche très intéressante dans l’utilisation des MEMS car ils peuvent être intégrés dans des technologies existantes de MEMS à base de silicium ou complètement remplacer ces technologies. Le but de cette thèse porte sur le développement d’un capteur MEMS composé de polymères (fonctionnels). Un chapitre initial (chapitre 2) introduit des nouveaux systèmes de fabrication de polymères fonctionnels. Des biopuces composées de MIPs imprimés par jet d’encre sont présentées ainsi qu’une technique basée sur la polymérisation radicale contrôlée qui permet le dépôt d’un fin enrobage de MIPs sur des microstructures. La deuxième partie de ce chapitre présente la fabrication de polymères à empreinte moléculaire par stéréolithographie deux-photons, qui peut être vue comme une extension de l’impression 3D. Afin d’illustrer cette technologie de prototypage rapide, deux capteurs composés de MIPs sont présentés : un capteur à grille de diffraction et un capteur en microlevier. Les deux principaux chapitres de ce manuscrit (chapitre 3 et 4) se focalisent sur le développement d’un nouveau concept de fabrication pour les capteurs MEMS. Ce concept est basé sur la polymérisation d’une poutre à fort ratio de forme à l’extrémité d’une fibre optique de télécommunication. Cette poutre a été mise en vibration à sa résonnance et a ainsi pu être utilisée comme un capteur à base de levier. Le capteur en polymère a permis l’intégration de MIPs comme élément récepteur et la reconnaissance sélective de l’antibiotique enrofloxacine. De plus, un nouveau système de mesure intégré est présenté dans le chapitre 4. Ce système de mesure intègre la fibre optique en guidant un rayon laser à travers elle ainsi qu’à travers le levier qui y est attaché.Le rayon lumineux sortant est ensuite focalisé sur une photodiode sensible à la position du rayon lumineux, permettant ainsi la mesure du spectre de résonance de la poutre en polymère. Ce système de mesure est caractérisé et ses performances sont présentées au travers de la détection de masse du levier en polymère et de mesures faites en milieu liquide. / A classical (bio)sensor consists of two key components: A receptor layer that detects the analyte of interest and the transducer which converts the chemical / biological stimuli into a physical measurable signal. Ideally a sensor is label-free, highly sensitive and selective towards the target, requires low sample amount and shows a fast response time. Regarding these criteria microelectromechanical systems (MEMS) offer great potential for the sensor development. One interesting approach for this development are functional polymer materials, such as molecularly imprinted polymers (MIPs), that can be either integrated to existing MEMS based on silicon or completely replace the silicon technology. The emphasis of this thesis is focused on the development of a MEMS sensor based on (functional) polymers. In an initial chapter (chapter 2) new fabrication schemes for functional polymers are introduced. Inkjet-printed biochips based on MIPs are presented and a technique based on controlled radical polymerization is shown that allows the deposition of thin MIP shells on a microfabricated pattern. In the second part of this chapter the fabrication of molecularly imprinted polymers by two-photon stereolithography is shown which can be seen as an extension of 3dimensional printing. As possible application of this rapid prototyping technology two sensors based on MIPs are introduced a diffraction grating sensor and a microcantilever sensor. The two main chapters of this manuscript (chapter 3 and chapter 4) report the development of a new fabrication concept for MEMS sensors. It is based on the polymerization of a high aspect ratio beam on the extremity of an optical telecommunication fiber which was actuated at resonance and thus could be used as a cantilever sensor. The polymer sensor allowed the integration of MIPs as sensing element and the selective recognition of the antibiotic enrofloxacin. Furthermore, is a new, integrated read-out scheme presented in chapter 4. This read-out scheme integrates the optical fiber, by guiding a probe laser beam through it and attached cantilever beam. The output light beam is then focused on a position sensitive photodiode and thus enabled to monitor the resonance spectra of the polymer beam. The read-out scheme is characterized and its performance is shown by demonstrating the mass sensitivity of the polymeric cantilever beam and by measurements in liquid environments.

A ce jour, le développement de textiles intelligents, de peaux artificielles, de capteurs de paramètres environnementaux et de composés optoélectroniques souples ; qui nécessite des innovations à la fois dans la synthèse des matériaux, leur conception mécanique mais aussi, à l’échelle industrielle, en stratégie de production ; présente un intérêt majeur dans le domaine du prêt-à-porter connecté. D’un point de vue mécanique, l’obtention des propriétés de flexibilité et d’étirabilité à faible coût, via un procédé simple, au sein d’un matériau léger et capable de s’expandre sur de grandes surfaces est un prérequis essentiel pour incorporer des dispositifs optoélectroniques au sein des objets connectés portables. Parmi les différents procédés couramment utilisés, l’electrospinning est une technique simple, facilement adaptable et peu onéreuse qui permet un ajustement fin et flexible des morphologies de fibres, l’assemblage de plusieurs nanofibres fonctionnelles et une production en continue à haut débit. Ces multiples avantages sont à l’origine des nombreux travaux concernant l’utilisation de l’electrospinning dans le domaine du prêt-à-porter électronique et/ou connecté. Cependant, il est nécessaire de développer des projets innovants pour ce secteur qui incluent des capteurs détectant des paramètres environnementaux (pH, température), des chemo-capteurs colorimétriques à large spectre (full-color), des composantes électroniques étirables et capteurs tactiles. / To date, the development of smart textiles, artificial skins, environmental sensory devices, and flexible/stretchable optoelectronics involve the innovation of material synthesis, mechanical design, and fabrication strategies have attracted considerable attention in wearable displays. The mechanically flexible and stretchable functions with cost-effective, facile, lightweight, and large-area expandability are essential modules to fabricate the optoelectronic devices in various wearable display applications. Among them, electrospinning is an easy, versatile, and inexpensive technique enables flexible morphology tuning, assembling various functional nanofibers, and high-throughput continuous production has motivated extensive studies on wearable electronics applications. Therefore, it is necessary to develop innovative projects including the environment-sensing elements with pH-sensing dependency, temperature-sensitive, full-color switchable chemosensors, stretchable electronics, and tactile sensors for various wearable electronics applications.

Ohne zuverlässige Chemo- und Biosensoren wären beispielsweise die Überwachung von Prozessparametern in der chemischen und biotechnologischen Industrie, die Detektion von geringsten Analytkonzentrationen in der biomedizinischen Analytik oder die Spurenanalyse von Schadstoffen undenkbar. Neue Sensormaterialien wie stimuliresponsive Hydrogele spielen bei der Entwicklung neuer chemischer und biochemischer Sensoren eine immer größere Rolle. Hydrogele sind „intelligente“ hydrophile Polymernetzwerke, die in Abhängigkeit von spezifischen Stimuli quellen und entquellen können. In Kombination mit piezoresistiven Drucksensoren wird dann der resultierende Quelldruck in eine messbare Ausgangsspannung umgewandelt. In dieser Arbeit werden sowohl neuartige stimuliresponsive Hydrogele für die Detektion von Ethanol in alkoholischen Getränken als auch zum Nachweis von gelöstem Ammoniak und Harnstoff für biotechnologische Prozesse vorgestellt. Nach der gezielten Synthese und Funktionalisierung der Gele werden zunächst die Quelleigenschaften in freier Quellung untersucht. Im Mittelpunkt der Charakterisierung stehen dabei sensorisch relevante Eigenschaften wie beispielsweise das Quellverhalten in Abhängigkeit vom jeweiligen Stimulus. Im nächsten Schritt werden piezoresistive Hydrogelsensoren aufgebaut und vermessen. Dabei werden wichtige Sensoreigenschaften wie der sensitive Messbereich, Nachweisgrenzen oder Querempfindlichkeiten detailliert untersucht und die Sensorkonzepte hinsichtlich ihres Anwendungspotenzials bewertet.

Pyrazoloanthrone and its analogues form the central core of the thesis and the work is focused on the evaluation of chemical and biological applications of pyrazoloanthrones. Selective and sensitive detection of biologically, environmentally and industrially important molecular species such as fluoride, cyanide and picric acid by using pyrazoloanthrones as sensors form the first part while the second part deals with selective and specific kinase inhibition by pyrazoloanthrones to moderate inflammation associated disorders like septic shock. All the investigations are based on extensive crystallographic studies of the participating molecules. Chapter 1 provides a brief review on the history and biological importance of 1,9-pyrazoloanthrones. The potential of these molecules as probes in sensor chemistry and protein kinase inhibition is envisaged. A short account of the techniques employed for the investigations along with a preamble is presented. Chapter 2 is divided into two parts. Part A deals with the design of a colorimetric and “turn-on” fluorescent chemosensor based on 1,9-pyrazoloanthrone specifically for cyanide and fluoride ion detection. A remarkable solid state reaction indicated by the development of intense red color occurs when crystals of tetrabutylammonium cyanide/fluoride are brought in physical contact with 1,9¬pyrazoloanthrone resulting in corresponding molecular complexes (Figure 1). X-ray crystal structures of these complexes and also of 1,9-pyrazoloanthrone have been determined and the ion sensing activity has been substantiated on the basis of spectroscopic (absorption, fluorescence and NMR) and structural analyses. The crystal structure of the parent compound exhibits a disorder as a consequence of tautomerism and the disorder gets carried on to the complexes as well with even the cyanide and the fluoride ions showing partial occupancy sites. The presence of the –NH group and associated intramolecular charge transfer upon complex formation is attributed to the extreme sensitivity of 1,9-pyrazoloanthrone for cyanide and fluoride (detection limits of 0.2 ppb and 2 ppb) ions respectively. Figure 1. Development of intense red color during the solid state reaction (shown on left) and the turn on fluorescence behavior (shown to the right) Part B demonstrates the utilization of electron rich N-alkyl substituted pyrazoloanthrones to design sensors for detecting explosive and electron deficient nitro aromatics such as picric acid (PA). The N-alkyl derivative of 1,9-pyrazoloanthrone has been synthesized, characterized by single crystal X-ray diffraction studies and evaluated as a potent sensor for picric acid. NMR and fluorescence lifetime measurements validate that the fluorescence quenching of sensor compound by PA (Figure 2) as due to the formation of excited state charge-transfer complex resulting in dynamic quenching. Figure 2. Fluorescence quenching measurements demonstrating the dynamic quenching in the charge transfer complex. Chapter 3 deals with the biological evaluation of 1,9-pyrazoloanthrone and its alkyl derivatives towards the inhibition of a decisive protein kinase called c-Jun N-terminal Kinase (JNK), an important member of MAP kinase family. JNK controls crucial cellular processes like apoptosis and cell proliferation and is implicated in disorders associated with inflammation such as septic shock, arthritis, inflammatory bowel disease, etc. Therapeutic inhibition of JNK activity by small molecules has proven to be advantageous in the treatment of diseases coupled with derailed inflammation. In this context, it is already established that 1,9-pyrazoloanthrone (SP600125) effectively and selectively inhibits JNK at concentrations beyond 10 M. A series of alkyl isomers of pyrazoloanthrone derivatives have been synthesized to evaluate the structural implications of inhibition and to elevate both selectivity and sensitivity at lower concentrations. The crystal structures of these isomers have been characterized and their utility as inhibitors has been tested for their in vitro inhibitory activity over c-Jun N-terminal kinase (JNK). The minimum inhibitory concentrations required by these molecules to inhibit JNK was found to be lesser as compared to 1,9-pyrazoloanthrone (<5 µM; Figure 3). Critically, it turns out that among the various inhibitors synthesized, the lead candidates SPP1 and SPB1 display specific inhibition of JNK among other LPS activated MAP kinases like ERK1/2 and p38. These results suggest that N-alkyl (propyl and butyl) bearing pyrazoloanthrone scaffolds provide promising therapeutic inhibitors for JNK in regulating inflammation associated disorders. Figure 3. Inhibition of JNK in macrophages by the SPP1 and SPB1 compared to the known SP600125. Inspired by the results reported in the previous chapter, Chapter 4 is devoted to the generation of a library of compounds based on SPP1 and SPB1 with a purpose to design inhibitors of JNK which perform at the lowest possible concentrations and the consequent evaluation of their potential on endotoxin induced septic shock. Severe sepsis or septic shock is one of the rising causes for mortality worldwide representing nearly 10% of intensive care unit admissions. Susceptibility to sepsis is identified to be mediated by innate pattern recognition receptors and responsive signaling pathways of the host. The c-Jun N-terminal Kinase (JNK)-mediated signaling events play critical role in bacterial infection triggered multi-organ failure, cardiac dysfunction and mortality. Figure 4. Two selected molecules for specific inhibition studies of JNK at lower concentrations. It is demonstrated that alkyl and halogen substitution on the periphery of anthrapyrazolone increases the binding potency of the inhibitors specifically towards JNK. Based on the results from both in vitro with macrophages and in vivo with the mouse model of septicemia, the potential role of two selected molecules D1 and D2 (Figure 4) in regulating endotoxin induced inflammation is firmly established. Further, it is demonstrated that hydrophobic and hydrophilic interactions generated by these small molecules effectively block endotoxin-induced inflammatory genes expression in in vitro and septic shock in vivo, in a mouse model, with remarkable efficacies. Altogether, the in vitro as well as the in vivo data clearly potentiates the selective inhibitory capacity of small molecule inhibitors like D1 and D2 which can facilitate the treatment of current inflammatory disorders when used in combination with the available drugs having varied efficacies. The results rationalize the significance of the diversity oriented synthesis of small molecules for selective inhibition of JNK and their potential in the treatment of severe sepsis.