Design and synthesis of optical sensors for the detection of molecules with biological activity

A la present tesi doctoral, titulada “Disseny i síntesi de sensors òptics per a la detecció de molècules amb activitat biològica”, s’han utilitzat diferents conceptes de la química supramolecular per a preparar compostos capaços de senyalitzar macroscópicament la presència de molècules tòxiques o actives biològicament. Així, s’han preparat quimio-sensors i quimiodosímetres per a la detecció d’anions, sals, zwitterions i agents de guerra química tal i com agents nerviosos i agents sanguinis. Addicionalment, es descriu el disseny i la construcció d’un aparell electrònic per a la monitorització automàtica d’aquests compostos. La tesi té un capítol introductori on s'expliquen els conceptes bàsics de la química supramolecular i especialment, sensors moleculars i quimiodosímetres. El Capítol 2 descriu l’ús de derivats de calix[4]pirrol com a quimiosensors de tipus pinça per a la detecció de dianions de alfa,omega-dicarboxilats, i l’ús de calix[4]pirrols bloquetjats conformacionalment com a receptor per a anions sulfonat. El Capítol 3 descriu el disseny i síntesi d’ un quimio-sensor heteroditòpic per a la detecció i sensat de cations, anions, o parells iònics, així com espècies zwitteriòniques com aminoàcids. Aquest sensor heteroditòpic, que està basat a una estructura de BODIPY connectat a dos diferents receptors per cations i anions (una 18-azacorona-6 i un calix[4]pirrol respectivament) també és capaç de solubilitzar parells iònics i espècies zwitteriòniques en acetonitril. Addicionalment, aquest compost pot realitzar totes les operacions lògiques bàsiques quan s’utilitza K+ i F- com entrades, emetent fotons com a eixida. Al Capítol 4, es descriu una biblioteca de quimiodosímetres òptics per a la detecció selectiva d'agents nerviosos. La biblioteca té dos grups principals: indicadors cromogènics i indicadors fluorogènics. El primer grup està basat en derivats de triarilmetanol. L’ alcohol terciari d’ aquestos compostos reacciona amb els compostos organofosforats tòxics i després d’ una reacció de desfosforilació, l’ espècie catiònica resultant adquireix color mitjançant una deslocalització de la càrrega sobre els tres anells aromàtics. Per altra banda, elsindicadors fluoro-cromogènics estan basats en l'ús de fluoròfors de BODIPY o trans-estilbe com a unitats senyalitzadores i 2-(2-dimetilamino)feniletanol com a unitat sensora. Amb la presència d’agents nerviosos, aquests indicadors es fosforilen al residu d'etanol, que activa una ciclació intramolecular duta a terme pel grup dimetilamino. Aquesta ciclació canvia les propietats electròniques del sistema, canviant així el seu espectre d'absorció i emissió. Tots els indicadors d'aquesta biblioteca han demostrat la seua capacitat en la detecció de compostos organofosforats en dissolucions orgàniques, aquoses o a la fase gas. El Capítol 5 descriu la síntesi i avaluació de dos quimiodosímetres colorimètrics per a la detecció de l’agent sanguini HCN. Aquests indicadors estan basats en derivats altament colorejats de p-quinometà capaços de reaccionar amb l’anió CN- en una reacció d’ addició de Michael, produint un derivat de leucocianur no acolorit. Aquests quimiosensors operen en dissolució aquosa, amb límits de detecció entre 165 - 300 ppb. També s'han realitzat experiments de detecció a la fase gas mitjançant l'exposició dels indicadors a una atmosfera contaminada amb HCN (g), amb resultats positius. Amb aquest simple procediment s'han aconseguit límits de detecció de HCN (g) de 2 ppm. Finalment, al capítol 6 es descriu el disseny, construcció i avaluació d'un aparell electrònic per a la monitorització automàtica de l'estat òptic de quimio-sensors i quimiodosímetres. El dispositiu està pensat per a treballar com a sensor de gasos. L’aparell, basat en una placa d'Arduino, utilitza software i hardware de codi lliure. Un parell de sensors de color basats al xip TCS230 monitoritzen continuament el color (com a valors RGB) d’una referència i d’un indicador químic immobilitzat mentre que el gas passa sobre ell. La capacitat del prototip ha sigut demostrada a la detecció de HCN(g)

Synthesis and applications of novel fluorescent and colorimetric coumarin-based sensors towards analyte sensing in aqueous systems

The continuous growth of mankind has not been considerate to the environment. The release of millions of tonnes of toxic heavy metal cations and anionic species through industrial, mining, agricultural, and electronic dumping has led to disease and, in many instances, death. This is usually suffered by low-income informal populations residing in third world countries. Moreover, many unnecessary deaths of children are becoming more prevalent because of consumption and contact with contaminated water, agricultural, and animal sources. Bioaccumulation of these toxic species in fish, plants, and animals, inevitably make their way back to the unaware general population. As growth by mining, agriculture, and electronics are indeed vital aspects of human development, the negative side effects of these activities usually continue unregulated. Therefore, as these processes are set to continue until more stringent regulatory processes are put into legislature; low-cost, sensitive, selective organic based sensors are a step in the right direction towards highlighting the need for environmental restoration and remediation; whilst also aiming to preventing unnecessary disease and death in the process. Herein, coumarin derived small-molecule fluorescent and colorimetric sensors for the quantitative and qualitative assessment of cationic and anionic species in aqueous and organic media are described. Ten fluorescent sensors supporting 1,4-disubstituted triazolyl moieties were synthesized according to Cu(I)-catalyzed azide-alkyne cycloaddition “click” reactions. These sensors were screened for their cationic and anionic affinities in a variety of solvent systems. Majority of the sensors responded well towards Fe3+, characterized by a strong fluorescent quenching response with a good degree of sensitivity and selectivity. Selected sensors were further investigated for their affinities towards anionic species; however, they did not display the same degree of selectivity or sensitivity towards these chosen anions. Titration studies of selected sensors with Fe3+ were able to be used towards determining the modes of fluorescent quenching; the photophysical mechanisms by which quenching occurs; stoichiometric binding ratios, association constants, and the number of coordination sites present between the sensors and Fe3+. Reversibility studies of the sensor-metal complex was investigated with EDTA. Partial reversibility was achieved for the chosen sensors with Fe3+. Hydrogen potential studies further described the application of these sensors over a good pH range. The binding site between the sensors and Fe3+ was investigated by NMR studies.Thesis (PhD) -- Faculty of Science, School of Biomecular and Chemical Sciences, 202

Synthesis and applications of novel fluorescent and colorimetric coumarin-based sensors towards analyte sensing in aqueous systems

The continuous growth of mankind has not been considerate to the environment. The release of millions of tonnes of toxic heavy metal cations and anionic species through industrial, mining, agricultural, and electronic dumping has led to disease and, in many instances, death. This is usually suffered by low-income informal populations residing in third world countries. Moreover, many unnecessary deaths of children are becoming more prevalent because of consumption and contact with contaminated water, agricultural, and animal sources. Bioaccumulation of these toxic species in fish, plants, and animals, inevitably make their way back to the unaware general population. As growth by mining, agriculture, and electronics are indeed vital aspects of human development, the negative side effects of these activities usually continue unregulated. Therefore, as these processes are set to continue until more stringent regulatory processes are put into legislature; low-cost, sensitive, selective organic based sensors are a step in the right direction towards highlighting the need for environmental restoration and remediation; whilst also aiming to preventing unnecessary disease and death in the process. Herein, coumarin derived small-molecule fluorescent and colorimetric sensors for the quantitative and qualitative assessment of cationic and anionic species in aqueous and organic media are described. Ten fluorescent sensors supporting 1,4-disubstituted triazolyl moieties were synthesized according to Cu(I)-catalyzed azide-alkyne cycloaddition “click” reactions. These sensors were screened for their cationic and anionic affinities in a variety of solvent systems. Majority of the sensors responded well towards Fe3+, characterized by a strong fluorescent quenching response with a good degree of sensitivity and selectivity. Selected sensors were further investigated for their affinities towards anionic species; however, they did not display the same degree of selectivity or sensitivity towards these chosen anions. Titration studies of selected sensors with Fe3+ were able to be used towards determining the modes of fluorescent quenching; the photophysical mechanisms by which quenching occurs; stoichiometric binding ratios, association constants, and the number of coordination sites present between the sensors and Fe3+. Reversibility studies of the sensor-metal complex was investigated with EDTA. Partial reversibility was achieved for the chosen sensors with Fe3+. Hydrogen potential studies further described the application of these sensors over a good pH range. The binding site between the sensors and Fe3+ was investigated by NMR studies.Thesis (PhD) -- Faculty of Science, School of Biomecular and Chemical Sciences, 202

Supramolecular and heterosupramolecar chemistry in controlled release and molecular recognition processes

La presente tesis doctoral titulada ¿Supramolecular and heterosupramolecular chemistry in controlled release and molecular recognition processes¿ está centrada en los dos aspectos principales de la química supramolecular que han experimentado un gran auge en los últimos años: el reconocimiento molecular y los procesos de liberación controlada. En particular la primera parte de la tesis se focaliza en el diseño y síntesis de moléculas orgánicas que pueden ser empleados cómo sensores para especies aniónicas y neutras. El paradigma seleccionado para los procesos de reconocimiento molecular fue la aproximación del dosimetro químico. Esta aproximación presenta ventajas con respecto a los otros dos métodos de determinación de aniones (desplazamiento y unidad coordinanteunidad indicadora), cómo, por ejemplo, la posibilidad de determinar los analitos en disolución acuosa. Así se sintetizaron dos sensores selectivos, uno para el anión fluoruro (F-) y el otro para glutatión (GSH). El sensor selectivo para la determinación de F- está basado en un colorante azoico funcionalizado, en su ¿OH fenólico, cómo silileter. Esta molécula presenta una banda de absroción muy intensa centrada a 350 nm que, después de la adición de F- , sufre un efecto hipocrómico significativo y un desplazamiento batocromico ligero (de ca. 10 nm), mientras aparece una nueva banda a 470 nm, determinando un cambio de incoloro a amarillorojo. Para obtener un sensor selectivo para GSH se sintetizó una sonda químico basado en una sal de 2,6-difenilpirilio. Sucesivamente se preparó una disolución de este compuesto en agua/CTAB, que se caracterizaba por un intenso color azul. En este caso, la adición de GSH produce una disminución significativa de la banda del visible, acompañada por la consecuente decoloración. Además la adicón de GSH induce la aparición de Resumen vi una intensa banda de emisión centrada a 485 nm (después de la irradiación a 350 nm). La segunda parte de esta tesis doctoral se basa en el diseño y síntesis de nuevos sistemas híbridos orgánicos-inorgánicos para procesos de liberación controlada en ambiente celular. Estos materiales híbridos se componen en general, de dos unidades: una matriz inorgánica mesoporosa de base silícea, capaz de almacenar moléculas orgánicas (colorantes, farmacos...) y un compuesto orgánico anclado covalentemente a la superficie externa del soporte inorgánico mesoporoso, que actúa cómo puerta molecular. La aplicación de un estímulo externo puede modificar la conformación de la puerta molecular permitiendo o bien impidiendo la difusión de la carga almacenada en los mesoporos hacía el exterior (disolución o citoplasma). El primer sistema sintetizado y estudiado se compone de una matriz inorgánica mesoporosa (MCM-41), cargada con el colorante Ru(bipy)3 2+ y funcionalizada en la superficie con un oligoetilen glicol mediante un grupo ester. La adición de la enzima esterasa determinaba la hidrólisis del grupo ester y la consecuente reducción del tamaño de la puerta molecular, acompañada por la liberación del colorante previamente cargado. Otro sistema de liberación preparado consiste en el uso de la misma matriz MCM-41 nanoscópica y el mismo colorante Ru(bipy)3 2+, pero se funcionalizó la superficie con una puerta molecular fotolabil. La irradiación en el maximo de absorción de la puerta molecular inducía la fotodegradación de la misma y la consecuente liberación del colorante. Un tercer ejemplo de sistema de liberación consiste en una puerta molecular caracterizada por la presencia de dos grupos funcionales hidrolizables con enzimas diferentes: grupos urea y amida. vii El material final, caracterizado por la presencia del mismo esqueleto inorgánico, y cargado con Ru(bipy)3 2+, era capaz de liberar selectivamente cantidades distintas de colorante, dependiendo del enzima empleado. Así se podían conseguir dos tipos de perfiles de liberación: uno muy rápido y poco intenso y otro más lento pero mucho mas intenso. Finalmente se sintetizó un material híbrido siempre basado en la misma matriz de MCM- 41, cargado con rodamina-B y funcionalizado en la superficie con galactooligosacáridos. Con este material se podía conseguir una liberación controlada del colorante selectivamente en células senescentes, debido a que estas sobreexpresan el enzima ß-galactosidasa que es capaz de hidrolizar los galactooligosacáridos.Agostini, A. (2013). Supramolecular and heterosupramolecar chemistry in controlled release and molecular recognition processes [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/29397TESI

Developing fluorescent probes for cellular imaging of the environment

Cellular pH environment is a crucial for healthy physiological process, and abnormal pH regulation can inflict stress to cells and drive disease. Fluorescence imaging with selective sensors is an effective method for examining living cells, but current fluorescent pH probes have a limited sensing range. New tools are needed to cover the full pH range to advance health and disease research. Chapter 2 details the development of novel small molecule fluorescent pH sensor with ratiometric output that can be further functionalised for specific biological studies in Chapter 3 and 4. Chapter 3 focuses on functionalising a nanoparticle platform, carbon dot, with an small organic based ratiometric pH sensor (NpRho1). The resultant nanoprobe NpRhoDot has a fluorescence response to a widened pH range from pH 4.5 – 7.5 compared to NpRho1 that was developed in Chapter 2 (pH 4.0 – 6.0). The nanoprobe demonstrated excellent selectivity and sensitivity to pH, and was applied in different cell lines in 2D and 3D cell cultures in one- and two-excitation microscope system to visualise intracellular pH through a ratiometric response. Chapter 4 explores a multivariate approach using multiple fluorescent pH probes. Two probes (Rho1 and Fl) were selected to best discriminate pH 4 to 8 using machine learning algorithms and co-conjugated to plasma-polymerised nanoparticle for simple, reagent-free conjugation process. Their fluorescence to pH was predicted using Gaussian process regression and could determine the pH of unknown samples. Preliminary cell experiment demonstrated internalisation of PPN-Rho1-Fl into A549 cells, showing potential for multivariate analysis of pH in microscope experiments. The pH sensing systems developed in this thesis expanded the fluorescent tools that can be used to probe intracellular pH to better understand the roles of pH in human health and diseases

Coherent Dynamics in the Ultrafast Relaxation of BODIPY Dyes: 2D Electronic Spectroscopic Characterization

The work presented in this thesis deals with the investigation of the relaxation dynamics of molecules occurring after excitation, a process whereby the excess of energy provided to the molecule by the interaction with light is released. To this aim, we applied 2D electronic spectroscopy, a recently developed advanced optical technique now recognized as one of the best tool to capture the ultrafast relaxation dynamics occurring in the first picosecond after excitation. Indeed, its capability to reveal quantum coherent mechanisms acting during relaxation dynamics makes it one of the most powerful and informative spectroscopic techniques currently available. Recent experimental data obtained on biological light harvesting complexes suggested the presence of coherent effects during the electronic energy transport, surviving for times longer than expected even at physiological conditions. The understanding of this phenomenon in biological complexes and the possibility of control and engineer these effects in ‘smart’ artificial devices is one of the hottest topics across different communities. In this context, it is now agreed that environment, including surroundings and vibrations intrinsic to the molecular structure, plays a crucial role in the dynamics of coherences, being responsible of their manifestation as long-lived oscillations. Despite this, a systematic study on the role of the molecular structure, the nature of the vibrational modes and solvent involved in the coherent dynamics of the chromophores is still missing. The research activity described in this thesis aims to fill this gap. Indeed, we applied 2D spectroscopy to study the coherent relaxation dynamics of single chromophores as a function of their structure (i.e. mono or multi-branching, role of substituents, etc.), solvent and exciting bandwidth with the objective of identify possible ‘structure-to-properties’ relationships to be used in the design of multi-chromophoric systems with artificial lightharvesting capabilities. The attention has been focused in particular on organic dyes known as BODIPYs, structurally analogue to half-porphyrins, and thus possibly envisioned in the preparation of multi-chromophoric bio-mimetic systems. Particular attention was spent in understanding the effect of the introduction of specified substituents in significant positions of the BODIPY core and on the role of solvent in the ultrafast relaxation of these chromophores. The background of information acquired on these simplified systems is of pivotal importance for the characterization of quantum effects in energy transfer processes in dimers of BODIPY dyes, planned in the near future. Il lavoro presentato in questa tesi si occupa di investigare dinamiche di rilassamento di molecole a seguito di fotoeccitazione, un processo in cui la molecola rilascia l’eccesso di energia acquisito tramite l’interazione con la luce. A questo scopo, abbiamo applicato la spettroscopia elettronica 2D, una tecnica avanzata di spettroscopia ottica recentemente sviluppata, oggi riconosciuta come uno degli strumenti migliori per catturare la dinamica di rilassamento ultraveloce che ha luogo nel primo picosecondo a seguito dell’eccitazione. Infatti, la sua abilità nel rivelare meccanismi coerenti quantistici attivi durante il trasporto di energia elettronica, la rendono una delle più efficaci e informative tecniche di spettroscopia ad oggi disponibili. Recenti dati sperimentali ottenuti su complessi antenna biologici hanno suggerito la presenza di effetti coerenti durante il trasporto di energia elettronica, in grado di persistere per tempi maggiori di quelli attesi, persino in condizioni fisiologiche. La comprensione di questo fenomeno in complessi biologici e la possibilità di controllare e ingegnerizzare questi effetti in dispositivi artificiali, rappresenta una delle tematiche più di picco in diversi ambiti di ricerca. In questo contesto, ad oggi si è raggiunto un certo consenso sul fatto che l’ambiente, con cui si indica in modo generico sia l’intorno che le vibrazioni intrinseche alla struttura molecolare, rivesta un ruolo cruciale nella dinamica delle coerenze; si pensa infatti che l’interazione con l’ambiente sia responsabile del loro manifestarsi come oscillazioni di lunga durata. Nonostante ciò, uno studio sistematico sul ruolo della struttura molecolare, sulla natura dei modi vibrazionali e sul solvente, fattori coinvolti nella dinamica coerente dei cromofori, non è ancora ad oggi stato effettuato. L’attività di ricerca descritta in questa tesi ha lo scopo di colmare questa mancanza. Infatti, si è applicata la spettroscopia 2D allo studio della dinamica di rilassamento coerente di singoli cromofori in funzione della loro struttura (per esempio mono o multi ramificato, ruolo dei sostituenti, ecc.), del solvente e della banda di eccitazione con l’obiettivo di identificare possibili relazioni tra struttura e proprietà, da poter eventualmente utilizzare nel design di sistemi multicromoforici artificiali per light-harvesting artificiale. L’attenzione è stata focalizzata in particolare su coloranti organici noti come BODIPY, strutturalmente analoghi a metà porfirine, dunque potenzialmente utilizzabili nella preparazione di sistemi multicromoforici biomimetici. Particolare attenzione è stata inoltre rivolta alla comprensione dell’effetto del solvente e di specifici sostituenti introdotti in posizioni significative del BODIPY, sul rilassamento ultraveloce di questi cromofori. Il background di informazioni acquisite su questi sistemi semplificati è di centrale importanza per la caratterizzazione di effetti quantistici nei processi di trasferimento di energia in dimeri di BODIPY, pianificata per il futuro

Development of Fluorescein-Based Fluorescent Chemosensors and Convergent Approaches in Solid-Phase Organic Synthesis

Palladium and platinum are widely used precious metals that play a pivotal role in energy, materials and drugs. Using a common principle of fluorescent sensor design, a highly sensitive fluorescent detection method for these metals has been developed. A key aspect of this method is the amplification of fluorescence signal directly through a catalytic process, which is conceptually new for metal sensors. Using this method, sub-ppb levels of these metals can be detected with potential applications in the pharmaceutical industry, hospitals and pharmacies. As an extensive of this palladium- and platinum-specific fluorescent sensor, determination of the oxidation state of palladium and platinum based on the aromatic Claisen rearrangement is also demonstrated. Using this mechanism, the probe can fluorescently distinguish Pd0 from PdII/IV and Pt0 from PtIV in both organic and aqueous solvents. Ozone exposure is a significant global health problem, especially in urban areas. While ozone in the stratosphere protects the earth from damaging ultraviolet light, tropospheric is toxic resulting in damage to the respiratory tract. In addition, tt has recently been shown that ozone may be produced endogenously in inflammation and antibacterial responses of the immune system; however, these results have sparked controversy due to the use of a non-specific colorimetric probe. A fluorescent sensor capable of unambiguously detecting ozone has been developed. This is the first sensor that is specific for ozone and can be used in both biological and atmospheric samples. Solid-phase organic synthesis is an enabling technology and its automation has profoundly impacted the scientific community and society in general. However, to date, the structural diversity of the molecules that can be synthesized by this technology is limited partly because current solid-phase organic synthesis relies on linear approaches. Described here are efforts toward the development of an enabling resin-to-resin olefin cross-metathesis technology that unites two functionalized alkene fragments to form highly complex small molecules in a convergent manner. It is also demonstrated that olefin cross-metathesis is a synthetically viable method particularly when a traceless longer linker is inserted between solid support and reacting olefins

Estudio analítico de la contaminación metálica en aguas afectadas por actividades antropogénicas crónicas. Métodos de control de la biodisponibilidad y toxicidad metálica.

Los principales objetivos perseguidos en estos trabajos de investigación han sido abordados desde dos puntos de vista: el desarrollo de nuevos métodos de análisis químico que sean fiables para evaluar los niveles subtrazas y ultratrazas de metales pesados y la aplicación de éstas y otras metodologías al estudio de la contaminación metálica de origen antropogénico en aguas costeras andaluzas, así como la evaluación de las formas químicas más biodisponibles, que pueden ser relacionadas con la toxicidad ambiental. Así, los estudios descritos en la presente memoria de investigación, se encuentran distribuidos de la siguiente forma: - En primer lugar se presenta el Capítulo I, el cual consta de una introducción general acerca los metales pesados en los ecosistemas acuáticos. En los dos siguientes capítulos se han aplicado ligandos orgánicos con altas propiedades quelatantes, caracterizados por la presencia del grupo imino, en la determinación de trazas metálicas en muestras acuosas. - En el Capítulo II se desarrolla un nuevo método para la preconcentración y análisis de tazas de plomo basado en el empleo de una membrana líquida de volumen, mediante el uso del ligando orgánico 2-hidroxibenzaldehido N-etiltiosemicarbazona (2-HBET) como agente transportador en la membrana. Cabe destacar la aplicación de este método a muestras naturales, especialmente muestras salinas. - El Capítulo III, el cual fue llevado a cabo durante una estancia de investigación en la University of Ulster (Coleraine, UK) bajo la dirección del Profesor D. John Callan, donde se estudiaron dos bases de Schiff como moléculas sensoras fluorescentes para hierro, proponiéndose dichas moléculas como potenciales indicadores para futuros estudios en el campo de los sensores ópticos. - Por último, en el Capítulo IV se muestra el estudio sobre la influencia global de la actividad antropogénica en un medio costero singular caracterizado por un impacto crónico persistente y de gran interés como es la Bahía de Algeciras, donde: Se establecen los niveles de concentración metálica total de Zn, Cd, Pb y Cu, presentes en las aguas y sedimentos tomados de la zona, donde además se evalúa su distribución en función de las distintas fracciones químicas (estudios de especiación) en ambas muestras medioambientales. Se analizan y cuantifican los niveles de concentración metálica total de estos metales en tejidos biológicos de branquias, hígado y músculo en peces de interés comercial. Así se evalúa el nivel de contaminación química por metales pesados que presentan estas especies biomonitoras de contaminantes inorgánicos. Se estudia la correlación de los niveles metálicos en el medio biótico con el medio abiótico, identificando las formas químicas más biodisponibles y, en consecuencia con mayor potencial tóxico