Investigation of zinc metalloneurochemistry with fluorescent sensors based on fluorescein platforms

• Main Author: Burdette, Shawn C. (Shawn Christopher), 1975-
• Other Authors: Stephen J. Lippard.
• Format: Others
• Language: English
• Published: Massachusetts Institute of Technology 2005
• Subjects: Chemistry.
• Online Access: http://hdl.handle.net/1721.1/17567

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, February 2003. === Vita. === Includes bibliographical references. === Chapter 1. CoordinationChemistry for the Neurosciences Metal ions are integral components of numerous enzymes and proteins. Although the field of bioinorganic chemistry has not focused on the brain and central nervous system, metal ions are vital to many neurological functions and are implicated in several neurological disorders. In this chapter, I present a brief overview of the functions of metal ions in neurobiology and highlight recent advances in the use of fluorescent sensors to study the neurotransmitters zinc and nitric oxide. Chapter 2. Fluorescent Sensors for Zn2' Based on a Fluorescein Platform: Synthesis, Properties and Intracellular Distribution Two new fluorescent sensors for Zn2' that utilize fluorescein as a reporting group, Zinpyr-1 (ZP1) and Zinpyr-2 (ZP2), have been synthesized and characterized. ZP1 is prepared in one step via a Mannich reaction, and ZP2 is obtained in a multi-step synthesis that utilizes 4',5'-fluorescein dicarboxaldehyde as a key intermediate. Both ZP sensors have excitation and emission wavelengths in the visible range (500 nm), dissociation constants (Kdl) for Zn2* of less than 1 nM, quantum yields approaching unity ... and cell permeability, making them well suited for intracellular applications. A 3- to 5-fold fluorescent enhancement occures under simulated physiological conditions corresponding to the binding of the Zn2+ cation to the sensor, which inhibits a photo-induced electron transfer (PET) quenching pathway. The X-ray crystal structure of a 2:1 Zn2/ZP1 complex has been solved. It is the first structurally characterized example of a complex of fluorescein substituted with metal binding ligands. === (cont.) Chapter 3 Improved Synthetic Methods for Preparing Fluorescein-Based Sensors and Application to the Preparation of ZP3 The synthetic precursor to ZP2 is a fluorescein dialdehyde prepared by a low yielding oxidation reaction. Several pathways for accessing versatile fluorescein scaffolds for Zn2* sensors have been explored. Although attempts to convert 4',5'- bis(bromomethyl)fluorescein dibenzoate to a diol were unsuccessful, substitution of the benzoate ester protecting groups of silyl ethers permitted the bromo groups to be activated toward nucleophilic substitution upon treatment with AgNO3. This method has been applied to the synthesis of ZP3 (Zinpyr-3, 9-(o-carboxyphenyl)-2-chloro-5-[2-[bis(2-pyridylmethyl)aminomethyl]-N-(p-anisidine)]-6-hydroxy-3-xanthanone). ZP3 binds Zn2*, but exhibits only a modest enhancement of the quantum yield from 0.04 to 0.05. Chapter 4 ZP4, an Improved Neuronal Zn2+ Sensor of the Zinpyr FamilyA second-generation fluorescent sensor for Zn2+ from the Zinpyr family, ZP4, has been synthesized and characterized. ZP4 (Zinpyr-4, 9-(o-carboxyphenyl)-2-chloro-5-[2-[bis(2-pyridylmethyl)aminomethyl]-N-methylaniline]-6-hydroxy-3-xan-thanone) is prepared by a convergent synthetic strategy developed from previous studies with these compounds. ZP4, like its predecessors, has excitation and emission wavelengths in the visible range (500 nm), a dissociation constant (Kd) for Zn2+ of less than 1 nM and a high quantum yields ... === by Shawn C. Burdette. === Ph.D.