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The main theme of this thesis is to develop a fluorescent probe for imaging the subcellular distribution of kinetically labile copper pools that might play a critical role in copper homeostasis. Various copper-selective sensors were designed by combining 1,3,5-triaryl-2-pyrazoline fluorophores with polythioethers as receptor moieties. A series of donor-substituted 1,3,5-triaryl-2-pyrazoline fluorophores were synthesized and characterized in terms of their photophysical and electrochemical properties. Interestingly, the aryl substituents attached to the 1- and 3-position of the pyrazoline ring influence the photophysical properties of the fluorophore in distinctly different ways. The excited-state equilibrium energy is primarily influenced by changes of the substituent in the 1-position, whereas the reduction potential of the fluorophore is determined by the 3-aryl group. Results from computational analyses agree well with the experimental data. A pyrazoline fluorophore library was synthesized, and their photophysical and electrochemical properties were studied. The compounds cover a broad range of excited state energies and reduction potentials, and allow for selective and differential tuning of these two parameters. A series of thiazacrownethers and tripodal aniline copper(I) receptors were synthesized and their copper binding stoichiometries, stability constants, and copper-self-exchange kinetics were investigated. The measured self-exchange activation parameters revealed for all studied ligands a negative activation entropy, suggesting a predominant associative exchange mechanism. With detailed knowledge of the fluorophore platform and copper receptors, sensor CTAP-1 was designed, synthesized and characterized. The probe shows a 4.6-fold emission enhancement and reaches a quantum yield of 14% upon saturation with Cu(I). The sensor exhibits excellent selectivity towards Cu(I) and is insensitive towards millimolar concentrations of Mg(II) or Ca(II). Mouse fibroblast cells (3T3) incubated with the sensor produced a copper-dependent perinuclear staining pattern, which colocalizes with the subcellular location of the mitochondria and the Golgi apparatus. The subcellular topography of copper was further determined by synchrotron-based x-ray fluorescence (SXRF) microscopy. Furthermore, microprobe x-ray absorption measurements at various subcellular locations showed a near-edge feature that is characteristic for low-coordinate monovalent copper. The data provide a coherent picture with evidence for a kinetically labile copper pool, which is predominantly localized in the mitochondria and the Golgi apparatus.
In this dissertation, we established a new approach assisted by computational chemistry to design fluorescent sensors. The approach is applicable to predict the behavior of a fluorophore-bridge-receptor sensor based on photoinduced electron transfer (PET). Our first designed rhodamine based pH sensor exhibits strong fluorescence under acidic conditions and very weak fluorescence under basic conditions, just as the computations predicted.
Finally, in chapter V, a pyrazoline fluorophore library with varying numbers of fluorine substituents was synthesized. The photophysical and electrochemical properties of these fluorophores were measured in order to determine if careful tuning of the excited state electron transfer thermodynamics is possible. The compounds cover a broad range of excited state energies and reduction potentials, and the data suggest that selective and differential tuning of both the reduction potential of the acceptor as well as the excited state equilibrium energy. These findings show that the individual parameters involved in excited state electron transfer can be tuned by the modular architecture of the pyrazoline fluorophore.
Over the past few decades, there has been significant interest in developing fluorescent probes, because they are useful tools for biological studies. As effective analytical techniques, fluorescent probes utilize distinct advantages offered by fluorescence detection in terms of sensitivity, selectivity, and fast response time. When fluorescent probes interact selectively with target molecules, ions or biological specimens, they can generate large optical responses. Since most ions or molecules, such as Zn2+, Ca2+, or pyrophosphate ion (PPi), are non-fluorescent, chemosensors having analyte binding-triggered fluorescence are appealing in many fields, like analytical chemistry, clinical biochemistry, medicine, and environmental science.This dissertation is devoted to the design, synthesis, and characterization of novel fluorescent sensors for Zn2+ and its associated applications. Chapter II of this dissertation presents several novel terpyridine-based fluorescent sensors with different substituents affecting the electronic and steric nature of the terpyridine (tpy) fluorophore. Sensors are designed to establish the correlation between sensor structure and its photophysical properties. Low temperature fluorescence is used to evaluate the essential role of intramolecular charge transfer (ICT) in zinc binding-induced fluorescence changes. The tpy molecular fragment has a relatively large [pi]-conjugated system which enables the potential [pi-pi] interaction between two tpy platforms and affects the fluorescence of tpy ligands. Chapter III introduces a dimeric tpy ligand containing two tpy fragments connected via a meta-phenylene unit. The detailed spectroscopic study shows that this ligand displays an attractive fluorescence turn-on, in sharp contrast to mono(tpy) ligand that shows fluorescence quenching upon binding Zn2+. The result suggests the existence of delicate structural influences on fluorescence of tpy derivatives.Chapter IV is devoted to 2-(2'-hydroxyphenyl)-1,3-benzoxazole (HBO) and 2-(2'-hydroxyphenyl)-1,3-benzothiazole (HBT) derivatives featured with a structural potential of excited-state intramolecular proton transfer (ESIPT). The study reveals additional information on the binding of HBO or HBT to metal cations, which aids the sensor design for Zn2+ and PPi detection. The molecular design aims to realize ESIPT process control upon complexation with an analyte. Chapter V is devoted to the synthesis of bis(HBO) derivatives which bind Zn2+ selectively and emit near-infrared (NIR) fluorescence as a consequence of metal ion binding-induced ESIPT turn-on. Preliminary cell stain experiment was conducted and indicated the potential biological applications.
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