Fluorescent chemosensors for exploring zinc metalloneurochemistry and detecting mercury in aqueous solution

Abstract

Chapter 1. An Introduction to Zinc Metalloneurochemistry and Zinc Detection in Biology. This chapter presents an overview of zinc neurophysiology and pathology, which provides motivation for the design of new tools and tactics for zinc detection in vivo. A historical account of biological zinc detection is also given, followed by a summary of recent progress in the development and use of fluorescent Zn(II) sensors for in vivo studies. A summary of project goals and thesis organization is also included. Chapter 2. Zinc Sensors Based on Monosubstituted Fluorescein Platforms I: Routes to Electronic Variation, Syntheses and Spectroscopic Characterization. In this work, a convergent synthetic approach for the assembly of fluorescent zinc sensors from aniline-derivatized ligands and a fluroescein carboxaldehyde platform is presented. These sensors are based on the previously reported ZP4 motif and incorporate a di(2-picolyl)amine moiety in the aniline-based ligand framework. The effects of electronic variation, achieved by halogenation of either the zinc-binding unit or the fluorophore platform, on the fluorescence properties and aniline nitrogen atom pKa values are considered.

(cont.) Chapter 3. Spectroscopic Characterization and Biological Applications of Halogenated Zinpyr Sensors. In this chapter, the effects of fluorescein halogenation on the photophysical properties and protonation equilbria of symmetrical Zinpyr derivatives, which contain two di(2-picolyl)amine-based ligand appendages, are considered. These sensors have sub-nM affinity for Zn(II) and are selective for Zn(II) over biologically relevant alkali and alkaline earth metals. Fluorescein halogenation influences background fluorescence, dynamic range, tertiary amine pKa, and both excitation and emission wavelengths. Extensive biological work, including cytotoxicity assays and confocal imaging, are also presented. Studies in a number of cell lines, including neurons, show that ZP3 is a versatile Zn(II) imaging tool. Chapter 4. Zinc Sensors Based on Monosubstituted Fluorescein Platforms II: Modulation of Zinc Affinity and Biological Applications. To access ZP sensors with lower Zn(II) affinity, pyrrole moieties were incorporated into an aniline-based ligand unit to give sensors ZP9 and ZP10. The photophysical characterization and metal binding properties of these sensors are described.

(cont.) The pyrrole-for-pyridyl substitution affords Zn(II) sensors with improved Zn(II) selectivity and sub-[M dissociation constants. Biological imaging studies revealed that asymmetrical ZP probes, including ZP4, are cell permeable and Zn(II) responsive in vivo. Both ZP4 and ZP9 detect endogenous Zn(II) in acute hippocampal slices from the adult rat. Chapter 5. The Zinspy Family of Fluorescent Zinc Sensors: Syntheses and Spectroscopic Investigations. Four fluorescent sensors designed for Zn(II) detection and which contain a fluorescein reporting group and a pyridyl-amine-thioether derivatized ligand moiety were prepared and their photophyiscal properties characterized. These "Zinspy" sensors are water soluble and generally display -1.4 to -4.5-fold fluorescence enhancement upon Zn(II) coordination, depending upon fluorescein halogenation and the number and nature of the Zn(II)-binding appendages. The Zinspy sensors exhibit improved selectivity and lower affinity for Zn(II) compared to the di(2-picolyl)amine-based Zinpyr family members. Chapter 6. Zinspy Sensors with Enhanced Dynamic Range: Imaging Zinc Uptake and Mobilization with a Low Affinity Probe.

(cont.) This chapter describes the preparation and characterization of Zinspy sensors containing non-coordinating thiophene heterocycles in the metal-binding unit. These probes show improved dynamic range relative to thioether-containing ZS sensors, low M dissociation constants for Zn(II) and improved Zn(II) selectivity. Stopped-flow kinetics investigations indicate fast association rates and reversible Zn(II) coordination with kon > 1.8 x 106 M'-s- and koff > 3 s-1 at 25 C. ZS5 is cell permeable, Zn(II)-responsive in vivo and localizes to the mitochondria of certain cell types. ZS5 can detect Zn(II) released from neurons following nitrosative stress. Chapter 7. QZ1 and QZ2, Rapid Reversible Quinoline Derivatized Fluoresceins for Sensing Biological Zinc. Two fluorescein-based dyes derivatized with 8-aminoquinoline were prepared and their photophysical, thermodynamic and zinc-binding kinetic properties determined. Because of their low background fluorescence and highly emissive Zn(II) complexes, QZ1 and QZ2 display a large dynamic range, with -42- and -150-fold fluorescence enhancements upon Zn(II) coordination, respectively.

(cont.) These sensors have micromolar dissociation constants for Zn(II), improved selectivity and bind Zn(II) rapidly and reversibly with kon values >106 M-'s-1 and koffvalues of ~150 s-1. Biological imaging studies with ZP3 and QZ2 show that binding affinity is an important parameter for metal ion detection in vivo. QZ1 and QZ2 also respond to two-photon excitation and two-photon microscopy was used to visualize Zn(II) with QZ2 in live HeLa cells. Chapter 8. A "Turn-On" Fluorescent Sensor for the Selective Detection of Mercuric Ion in Aqueous Media. This chapter describes the synthesis, photophysical characterization and metal-binding properties of mercury sensor 1 (MS1). This sensor is based on a fluorescein platform and has a thioether-rich metal-binding unit, which conveys high selectivity for Hg(II). To the best of our knowledge, MS1 was the first reversible fluorescent Hg(II) sensor to give fluorescence turn-on in water. MS1 can detect low ppb levels of Hg(II) in aqueous solution at neutral pH. Chapter 9. Selective Hg(II) Detection in Aqueous Solution with Thiol Derivatized Fluoresceins. The syntheses and photophysical properties of MS2 and MS3, two asymmetrically derivatized fluorescein-based dyes designed for Hg(II) detection, are described.

(cont.) These sensors each contain a single pyridyl-amine-thiol metal-binding moiety, form 1:1 complexes with Hg(II) and exhibit selectivity for Hg(II) over other Group 12 metals, alkali and alkaline earth metals, and most divalent first-row transition metals. Both dyes display superior brightness ( x ) and fluorescence enhancement following Hg(II) coordination in aqueous solution. At neutral pH, electron transfer (PET) quenching of the free dye is enhanced, and the Hg(II)-induced turn-on also benefits from alleviation of this pathway. MS2 can detect ppb levels of Hg(II) in aqueous solution, demonstrating its ability to identify environmentally relevant concentrations of Hg(II). Chapter 10. MS4, A Seminaphthofluorescein-Based Chemosensor for the Ratiometric Detection of Hg(II). The synthesis and photophysical characterization of MS4, an aniline-derivatized seminaphthofluorescein-based dye that contains a pyridyl-amine-thioether ligand analogous to that employed in the Zinspy Zn(II) sensor family (Chapter 5) are reported. Sensor MS4 provides single-excitation, dual-emission ratiometric detection of Hg(II) in aqueous solution. An -4-fold ratiometric change (624/524) is observed upon introduction of Hg(II) to an aqueous chloride-containing solution of MS4 at pH 8.

(cont.) In this milieu, MS4 shows selectivity for Hg(II) over a background of alkali and alkaline earth metals, a number of divalent first-row transition metals and its Groupl2 congeners Zn(II) and Cd(II). Chapter 11. Turn-On and Ratiometric Mercury Sensing in Water with a Seminaphthofluorescein-Based Probe. The synthesis and characterization of MS5 are presented in this chapter. This sensor incorporates the aniline-derivatized thioether ligand used in the preparation of MS1 and the seminaphthofluorescein platform described in the design of MS4. MS5 gives selective fluorescence turn-on for Hg(II) at pH > 7. At pH > 8, single-excitation dual-emission ratiometric Hg(II) detection is possible by comparison of the (624 / 524) ratio before and after Hg(II) coordination. Studies of the pH dependence suggest that the seminaphthofluorescein dianion is critical for generating the ratiometric response. X-ray crystallographic studies with a salicylaldehyde-based model complex are presented to help elucidate the nature of Hg(II) coordination to MS1 and MS5. MS5 can respond to Hg(II) added to natural water samples, which points to its potential utility in the field.

(cont.) Appendix 1. Miscellenous Fluorescein-Based Ligands. This appendix details the preparation and, in some cases, characterization of potential fluorescein based sensors for either Zn(II) or Hg(II) detection that were not described in earlier chapters. Many of these compounds give fluorescence turn-off or no fluorescence change with analyte binding. Sensors ZP11 and MS6 are lower-affinity probes that give fluorescence turn-on for Zn(II) and Hg(II), respectively. Appendix 2. Theoretical Investigations of Fluorescein Derivatives. In this chapter, we present the results from DFT and TDDFT calculations on fluorescein and its derivatives. These studies include establishing protocols for fluorescein pKa determination and TDDFT analysis. The absorption spectra of the fluorescein dianion and monoanion were assigned and a detailed molecular orbital analysis for a fluorescein dianion analog was conducted. These studies indicate that oxygen atoms in the xanthenone moiety influence the amount of C1 character in the donor and acceptor molecular orbitals responsible for fluorescein absorption.