RATIOMETRIC NEAR-INFRARED FLUORESCENT PROBES FOR THE SENSITIVE DETECTION OF INTRACELLULAR pH AND BIO-THIOLS IN LIVE CELLS

In the past twenty years, fluorescence sensing and imaging based on fluorescent probes has been developed as an imperative technique due to the merits including excellent sensitivity, operational simplicity, instant time effectiveness and outstanding selectivity in the research areas such as mineralogy, gemology, biological medicine, materials and environmental engineering. Protons act as a significant role in a variety of pathological and physiological processes, and there are obvious differences in the pH among organelles: the pH in lysosomes is acid within the range of 4.5–5.5, whereas mitochondrial pH is basic that can be as high as 8.0. Abnormal intracellular pH is always an indication of a disrupted pH homeostasis in the whole cell. Furthermore, intracellular bio-thiols are vital to cell metabolism, which by either elevated or deficiency levels of bio-thiols will lead to some diseases. Possessing the advantages of avoiding systematic errors and undesirable photophysical properties of certain fluorophores, novel near-infrared ratiometric fluorescent sensors for the accurately monitoring intracellular pH and biothiols have become the spotlight in research topics. Throughout this dissertation, we firstly have designed and synthesized two novel rhodamine-based dyes with high fluorescence quantum yield, good pH stability large Stokes shifts and excellent photostability by introducing an additional amino residue with fused rings into a classic rhodamine skeleton. We also have constructed a fluorescent sensor by incorporating a receptor to one of these dyes and applied it as an effective sensor for the quick and sensitive monitoring of lysosomal pH fluctuations. Then, we have prepared two sets of ratiometric fluorescent probes for the sensitive detection of lysosomal pH values. The former series were based on π-conjugation modulation strategy, which was accomplished by conjugating a visible coumarin motif to a classic near-infrared hemicyanine skeleton via a vinyl linker. The lysosome-targeting goal was reached by introducing a morpholine ligand or a o-phenylenediamine group to the hemicyanine acceptor. For the latter series, we have obtained three near-infrared ratiometric fluorescent sensors containing a TPE as a donor and a rhodamine as an acceptor for the quantitative, sensitive and comparative analysis of lysosomal pH alterations through FRET and TBET approaches. Furthermore, we have prepared two near-infrared hybrid rhodol dyes for the ratiometric and sensitive visualization of pH value alterations in mitochondria taking advantage of conjugating typical hemicyanine fluorophores into a classic rhodol motif. Upon pH changes, a rhodol hydroxyl group in the probe acts as a spiropyran switch, resulting in the change of π-conjugation and the appearance of a new fluorescent peak. Due to the positive charge, these two novel rhodol dyes possessed the mitochondria-targeting property. In the end, besides the ratiometric fluorescent pH probes, we have reported a FRET-based fluorescent sensor for the ratiometric, selective and accurate detection of cysteine (Cys), which was achieved by linking a visible coumarin skeleton and a near-infrared rhodamine motif through a piperazine spacer. This probe could be used to monitor the intracellular cysteine concentration ratiometrically and be further applied for imaging of Drosophila melanogaster larvae to detect cysteine concentration alterations in vivo

Novel systems for bioimaging and photodynamic therapy: BODIPY dyes and silica-based nanocarriers.

235 p.En este trabajo se han propuesto nuevos colorantes basados principalmente en la estructura del cromóforo BODIPY, así como nuevos nanosistemas híbridos fotoactivos para su potencial implementación en el campo biomedicina, en particular para bioimagen y terapia fotodinámica del cáncer.Primero, el desarrollo de nuevos biomarcadores fluorescentes se basó en la modificación del cromóforo BODIPY para mejorar su solubiliad en medios acuosos y su selectividad hacia orgánulos específicos de la célula i.e. mitocondria. Para ellos, se añadieron grupos hidrofílicos y biomoleculas especificas, respectivamente. Posteriormente, se estudió la funcionalización del cromóforo BODIPY con el objetivo de lograr fotosensibilizadores con alta producción de oxigeno singlete por un lado mediante la introducción de átomos halogenados y además sistemas ¿-conjugados que desplazan la absorción hacia la región roja dentro de la ventana clínica, o por otro lado anclando grupos dadores de electrones que activen un estado de transferencia de carga intramolecular capaz de modular la capacidad fluorescente junto con la generación de oxígeno singlete, ideal para aplicaciones teranosticas.Además, se sintetizaron tres tipos de nanopartículas esféricas de sílice (no porosas, mesoporosas y ormosil) de entorno a 50 nm de tamaño, que junto con una nanoarcilla sintética (laponita) de unos 100 nm, se utilizaron como trasportadores de colorantes: fluoroforos (principalmente ocluidos en el core) o fotosensibilizadores (unidos covalentemente en la superficie externa). Estos nanosistemas se funcionalizaron además con polietilen glicol y ácido fólico para mejorar su estabilidad en agua y su selectividad hacia células tumorales

Synthesis and Supramolecular Functional Assemblies of Ratiometric pH Probes

Tracking the pH with spatiotemporal resolution is a critical challenge for synthetic chemistry, chemical biology and beyond. Over the last decade different small probes and supramolecular systems have emerged for in celluloor in vivo pH tracking. However, pH reporting still presents critical limitations such as background reduction, sensor improved stability, cell targeting, endosomal escape, near and far infrared ratiometric pH tracking, adaptation to the new imaging techniques (i.e. super‐resolution), etc. These challenges will demand the combined efforts of synthetic and supramolecular chemistry working together to develop a next generation of smart materials that will resolve the current limitations. In this review we describe the recent advances in the synthesis of small fluorescent probes together with new supramolecular functional systems employed for pH tracking with emphasis in ratiometric probes. The combination of organic synthesis and stimuli‐responsive supramolecular functional materials will be essential to solve future challenges of pH tracking such as the improved signal to noise ratio, on target activation and microenvironment reportingThis work was partially supported by the Spanish Agencia Estatal de Investigación (AEI) [SAF2017-89890-R], the Xunta de Galicia (ED431C 2017/25, 2016-AD031 and Centro Singular de Investigación de Galicia accreditation 2016–2019, ED431G/09), the ISCIII (RD16/0008/003), and the European Union (European Regional Development Fund –ERDF). A.M. received a Marie Curie fellowship (GLYCONANOPEP-750248). J.M. received a Ramón y Cajal (RYC-2013-13784), an ERC Starting Investigator Grant (DYNAP-677786) and a Young Investigator Grant from the Human Frontier Science Research Program (RGY0066/2017)S

SYNTHESIS OF DYE LIBRARIES FOR NEURODEGENERATIVE DISEASE AND UNIQUE FRET STUDY

Ph.DDOCTOR OF PHILOSOPH

Dynamics and Design of Green Fluorescent Protein-Inspired Superphotoacids and Red-Emitting Fluorophores with Transient Absorption and Femtosecond Stimulated Raman Spectroscopy

In the past decades, femtosecond stimulated Raman spectroscopy (FSRS) has been gaining tremendous popularity in fundamental sciences stemming from chemistry to biology. It is capable of capturing both equilibrium and non-equilibrium structural information across a broad range of timescales with simultaneously high temporal and spectral resolutions. Femtosecond transient absorption spectroscopy (fs-TA) acts as a powerful partner technique that complements the dynamics analysis and guides the resonance selection for FSRS pulses. In this dissertation, two bodies of work on the excited-state dynamics and design strategies of the green fluorescent protein (GFP) chromophore-based superphotoacids and red-emitting fluorophores studied by FSRS and TA are presented. First, FSRS and fs-TA were implemented to dissect the ultrafast excited-state proton transfer (ESPT) dynamics of superphotoacids in aqueous and nonaqueous solutions. The superphotoacids were derived from the synthetic GFP chromophore, namely, p-hydroxybenzylidiene-dimethylimidazolinone or p-HBDI. By strategic conformational locking and multi-site halogenation, p-HBDI was transformed from a dim weak photoacid to a bright superphotoacid. The locking enables a three-order increase of magnitude in fluorescence quantum yield from 10−4 to 10−1 while the halogenation remarkably elevates the photoacidity characterized by the excited-state pKa or pKa* (asterisk denotes excited state) value which is lowered from ~2.1 of p-HBDI to negative values of the derived superphotoacids. FSRS and fs-TA results showed that the ESPT dynamics of these superphotoacids in protic organic solvents such as methanol (an inert proton acceptor) are inhomogeneous. Three pathways including direct, solvent reorientation-controlled, and rotational diffusion-controlled ESPT are coexisting within the excited-state lifetime. Through a systematic comparison of different photoacids, new design principles of tuning photoacidity was proposed. Besides, a comparison between locked and unlocked halogenated superphotoacids of similar strengths was made. They exhibit drastically different ESPT dynamics and thus highlight the importance of structure-activity relationship of functional molecules. Second, ground- and excited-state FSRS in conjunction with quantum calculations allows a deep understanding of the emission mechanism in GFP chromophore and its derivatives. A unique “double-donor-one-acceptor” red-shifting strategy thereby was proposed based on the GFP chromophore and we showed that this strategy can be generalized to other fluorophore scaffolds. Excited-state intramolecular charge transfer (ICT) was extensively proven to be able to red-shift emission wavelength by stabilizing the excited state. The presence of excited-state ICT in GFP chromophore and its derivatives were both experimentally and theoretically demonstrated with site precision by FSRS and quantum calculations, respectively. Moreover, a “double-donor” structure that incorporates another electron-donating group (EDG) to the ortho site of the −OH/O− group of p-HBDI was discovered to destabilize the ground state. In synergy with the “one-acceptor” that lowers excited-state energy through ICT, the emission energy gap could be lowered to a remarkable extent. The red emission wavelength is highly desired in biological imaging due to the resultant small background noise and has drawn lots of attention in the design of imaging probes. The “double-donor-one-acceptor” strategy is advantageous in that it does not require large π-conjugation extension and hence provides space for further chemical engineering for other wanted imaging properties such as high FQY

Detail Synthetic Study of Infrared Fluorescent Dyes: Design, Synthesis and Chemical Properties of their Photodynamic Therapy Probes

Dipyrromethene boron difluoride (BODIPY) derivatives can be used as effective photosensitizers (PS’s) to eradicate a broad spectrum of microbes that threaten the global population health. Moreover, these compounds could be used in diagnostic or therapy, controlling the balance between the fluorescence emission and the photodynamic activity. There is still much work to be done in the search for ideal PS’s with applications in photodynamic therapy (PDT). To effectively use near infrared region BODIPY dyes for labelling during biological analyses, or as biomarkers in biomedical applications such as imaging diagnosis, a hydrophilic character is usually required. It was found that the introduction of the strong electron-withdrawing group at the meso position in the BODIPY skeleton was responsible for the drastic bathochromic shift in the absorption spectrum. Several studies on the development of small molecule fluorescent probes have been performed with short wavelengths and with poor water solubility. There should be new investigations to obtain more information on the mechanisms of photodynamic action relating to cell damage and experiments in vivo infection models. In order to understand the effect of the substituents, a predictive quantitative structure-activity relationship (QSAR) regression model, based on theoretical holistic molecular descriptors as developed. An even better fluorescent probe would combine the photostability of the BODIPY group with a chromophore that absorbs at longer wavelength that makes for better light penetration in cells and tissues. In this review, we will summarize ideas on different wavelengths and hydroelectric abilities through modifications of molecular structures of the biological probe molecule. BODIPY’s materials and chemical modification methods for modulating the optical properties presented here could be versatile for developing efficient photo-responsive bio-related materials to control the biological activities and efficient quenchers on the biotechnological assays with labelled biomolecules

Shifting Gears Towards the Red: Novel Boron-Based Fluorophores for Bioimaging Applications

Advancements in near-infrared (NIR) fluorescence imaging have enabled greater tissue penetration depths, high spatial resolution, reduced photon scattering, and minimal interference from tissue autofluorescence. Hence, NIR fluorophores exist as viable candidates for biological imaging applications, as well as providing unique insights into complex biological processes to better understand disease etiology. In this work, a series of novel boron-based coumarin and rhodamine-like fluorophores were developed and tested. Initially, modifications to the coumarin scaffold were investigated to develop more red-shifted dyes, whereby incorporation of a p-conjugated bridge was determined to be a critical component. Confocal microscopy studies with A549 lung cancer cells showed clear differences in the intra-cellular distributions of the fluorophores. The lipophilic carborane coumarin derivatives exhibited superior selectively within lipid droplets. In contrast, the polar boronic acid hydrazone-coumarins displayed intracellular localisation within the endoplasmic reticulum. A library of boron-containing rhodamine-like probes were also synthesised. All compounds exhibited near-infrared emission wavelengths with large Stokes shifts. Furthermore, modifications to the terminal boron moiety were not found to impact the overall red-shift of the molecules, although increasing the donor group strength favourably enhanced this shift. The low brightness of some of the probes, related to rhodamine spirocyclisation, meant that conclusive intracellular localisations could not be confirmed. The near-IR emitting nature of the rhodamine-like probes was recognised as a highly advantageous tool for bioimaging applications. Two fluorescent-labelled ligands with varying linker lengths, of the allosteric adamantyl benzamide P2X7R antagonist were prepared, based on the para-MIDA ester rhodamine-like fluorophore. Both probes were utilised in preliminary fluorescence studies, whereby the near-IR emitting nature of the fluorophore was retained despite conjugation to biomolecules. As well, a longer emission wavelength was observed with a shorter linker length. This research ultimately highlights the versatility of boron as a unique element in fluorescence and biological imaging applications

Spectroscopic Characterization and Bioanalytical Applications of Benzophenoxazine Derivatives and the Use of Dyes and Dye-Encapsulated Silica Nanoparticles for Fingerprint Detection

The use of benzophenoxazine dyes such as Nile red and Nile blue in various applications has received increasing attention in recent years. Due to the limitations of using the two dyes in aqueous media because of their poor solubility, extensive efforts have been made to synthesize new benzophenoxazine analogues. Therefore, the first part of the work aims to characterize modified structures of benzophenoxazine derivatives as well as Nile red and Nile blue using spectroscopic techniques. The optical properties of the dyes involve the determination of molar absorptivity and quantum yield values as well as photostability studies. The absorbance and emission wavelengths are in the visible and near-infrared region of the electromagnetic spectrum which is the most useful region for bioanalytical applications due to the reduced autofluorescence from biomolecules. The first part also includes the interactions between benzophenoxazine derivatives and human serum albumin. The affinity of the dye to the protein is hydrophobicity-dependent, but other parameters such as steric hindrance and electrostatic interaction play a role too as confirmed by binding constant values. Detection of fingerprints is considered one of the most valuable pieces of evidence in forensic investigations. The second part of the work aims to use benzophenoxazine derivatives for fingerprint detection on porous surfaces. The factors affecting the ability of Nile red and Nile blue derivatives to develop luminescent and visible fingerprints with good background contrast are discussed. The second part also contains the application of silica nanoparticles encapsulating some benzophenoxazine derivatives as well as fluorescein isothiocyanate as fingerprint reagents. The efficiency to develop fingerprints is governed by the nature of the encapsulated dye and organosilicate precursors

Exclusive Feature Papers in Colorants

“Exclusive Feature Papers in Colorants” is a collection of important high-quality papers (original research articles or comprehensive review papers) published in open access. This Special Issue aims to discuss new knowledge or new cutting-edge developments in the colorants research field through selected works, in the hope of making a great contribution to the community. We intend for this issue to be the best forum for disseminating excellent research findings as well as sharing innovative ideas in the field

A photophysical study of substituted arylethynylenes

Molecules based on the 1,4-bis(phenylethynyl)benzene (BPEB) architecture are an important class of compounds because of their luminescent properties. The rigid rod-like backbone and extended π-conjugation serves as the base upon which a wide variety of substituted species can be synthesised with a variety of photophysical properties. This thesis represents a detailed study of the photophysical properties of a series of compounds based on BPEB using a combination of steady-state and time- resolved spectroscopic methods. A comparative study of the photophysical properties of BPEB, 1,2,4,5-tetramethyl- 3,6-bis(phenylethynyl)benzene, l,2,4,5-tetramethyl-3,6-bis-(2,4,6-trimethylphenyl- ethynyl)benzene, 1,4-bis(2-(2-tórř-butylphenyl)ethynyl)benzene, 1,4-bis(2-(2-tert- butylphenyl)ethynyl)-2,3,5,6-tetramethylbenzene, 1,4-dibromo-2,5-bis(phenylethy- nyl)benzene and l,2,4,5-tetrafluoro-3,6-bis(phenylethynyl)benzene is reported. These compounds exist as a series of geometric conformers, in solution, in the ground state due the low barrier to rotation about the carbon-carbon triple bond (C≡C). The absorption and emission dipoles of BPEB are found to be almost linear and lie along the long axis of the molecule. The substituents red shift the absorption and emission spectra of BPEB. l,4-Bis(2-(2-tert-butylphenyl)ethynyl)-2,3,5,6- tetramethylbenzene, exhibits fluorescence from both high and low energy conformations at 77 K. Only l,4-dibromo-2,5-bis(phenylethynyl)benzene has a long lived phosphorescence emission detectable at 77 K. Time-resolved Raman spectroscopy reveals that the C≡C stretching vibrations of BPEB, and its substituted derivatives in the excited singlet and triplet states, which shows evidence of only minor structural changes upon excitation. The photophysical properties, and solvatochromism, of a series of compounds having donor and acceptor groups bridged by BPEB are reported. They are methyl- 4-(4-(4-dimethylaminophenylethynyl)phenylethynyl)benzoate, 4-(4-(4-dimethyl- aminophenylethynyl)phenylethynyl)benzonitrile, 4-(4-(4-dimethylammo-3,5-dimeth- ylphenylethynyl)phenylethynyl)benzonitrile, 4-(4-(4-dimethylaminophenylethynyl)- phenylethynyl)-N,N-dimethylbenzenamine, 4-(4-(4-cyanophenyIethynyl)phenylethy֊nyl)benzonitrile, 4-(4-(4-dimethylaminophenylethynyl)phenylethynyl)benzene, 4-(4- (4-cyanophenylethynyl)phenylethynyl)benzene and methyl-4-(4-phenylethynyl)- phenylethynylbenzoate. The donor-acceptor systems exhibit intramolecular charge transfer (ICT) and twisted internal charge transfer in the excited state, when in polar solvents. In non-polar solvents only the locally excited state is observed. The largest change in dipole moment in the excited state occurred when the dimethylamino group was fixed perpendicular to the adjacent phenyl ring. In protic solvents, hydrogen bonds to the amino group in the ICT state to form a hydrogen- bonded ICT state. The photophysical properties of 9,10-bis(phenylethynyl)anthracene (BPEA) and a series of nine of its donor and acceptor substituted derivatives are reported. Compounds with the strongest donor (dimethylamino) and acceptor (nitro) groups exhibit ICT. All substituted BPEA compounds had lower fluorescence quantum yields and lifetimes, and higher non-radiative decay constants than BPEA. The molecular and photophysical properties of 2,5-bis(phenylethynyl)thiophene, 1,4-bis(2-thienylethynyl)benzene and 2,5-bis(2-thienylethynyl)thiophene were investigated with specific emphasis on the bonding character of their triplet excited states. Like the parent, BPEB, these compounds exhibit evidence of triple bond character in the excited singlet and triplet states