Bright Fluorescent Chemosensor Platforms for Imaging Endogenous Pools of Neuronal Zinc

AbstractA series of new fluorescent Zinpyr (ZP) chemosensors based on the fluorescein platform have been prepared and evaluated for imaging neuronal Zn2+. A systematic synthetic survey of electronegative substitution patterns on a homologous ZP scaffold provides a basis for tuning the fluorescence responses of “off-on” photoinduced electron transfer (PET) probes by controlling fluorophore pKa values and attendant proton-induced interfering fluorescence of the metal-free (apo) probes at physiological pH. We further establish the value of these improved optical tools for interrogating the metalloneurochemistry of Zn2+; the novel ZP3 fluorophore images endogenous stores of Zn2+ in live hippocampal neurons and slices, including the first fluorescence detection of Zn2+ in isolated dentate gyrus cultures. Our findings reveal that careful control of fluorophore pKa can minimize proton-induced fluorescence of the apo probes and that electronegative substitution offers a general strategy for tuning PET chemosensors for cellular studies. In addition to providing improved optical tools for Zn2+ in the neurosciences, these results afford a rational starting point for creating superior fluorescent probes for biological applications

Investigation of zinc metalloneurochemistry with fluorescent sensors based on fluorescein platforms

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

Increasing the Dynamic Range of Metal Ion Affinity Changes in Zn²⁺ Photocages Using Multiple Nitrobenzyl Groups

Two generations of DiCast photocages that exhibit light-induced decreases in metal ion affinity have been prepared and characterized. Expansion of the common Zn²⁺ chelator of <i>N</i>,<i>N</i>-dipicolylaniline (DPA) to include additional aniline ligand provides <i>N</i>,<i>N</i>′<i>-</i>diphenyl-<i>N</i>,<i>N</i>′<i>-</i>bis­(pyridin-2-ylmethyl)­ethane-1,2-diamine, a tetradentate ligand that was functionalized with two photolabile groups to afford DiCast-1. Uncaging of the nitrobenzhydrol reduces the electron density on two metal-bound aniline ligands, which decreases the Zn²⁺ affinity 190-fold. The analogous MonoCast photocage with a single nitrobenzhydrol group only undergoes a 14-fold reduction in affinity after an identical photochemical transformation. A second series of DiCast photocages based on a <i>N</i>,<i>N</i>′-(pyridine-2,6-diylbis­(methylene))­dianiline scaffold, which allows the introduction of two additional Zn²⁺-binding ligands into a preorganized chelator, expand on the multi-photolabile group strategy. DiCast-2 includes two pyridine ligands while DiCast-3 adds two carboxylate groups. Addition of bridging pyridine to the second generation photocages leads to more stable Zn²⁺ complexes, and photolysis of two photolabile groups increases the Zn²⁺ affinity changes to 480-fold. The Zn²⁺, Cu²⁺, and Cd²⁺ binding properties were examined in all the DiCast photocages and the corresponding photoproducts using UV–vis spectroscopy. Further insight into the photocage Zn²⁺-binding motifs was obtained by X-ray analysis of DiCast-2 and DiCast-3 model ligands

Increasing the Dynamic Range of Metal Ion Affinity Changes in Zn²⁺ Photocages Using Multiple Nitrobenzyl Groups

Two generations of DiCast photocages that exhibit light-induced decreases in metal ion affinity have been prepared and characterized. Expansion of the common Zn²⁺ chelator of <i>N</i>,<i>N</i>-dipicolylaniline (DPA) to include additional aniline ligand provides <i>N</i>,<i>N</i>′<i>-</i>diphenyl-<i>N</i>,<i>N</i>′<i>-</i>bis­(pyridin-2-ylmethyl)­ethane-1,2-diamine, a tetradentate ligand that was functionalized with two photolabile groups to afford DiCast-1. Uncaging of the nitrobenzhydrol reduces the electron density on two metal-bound aniline ligands, which decreases the Zn²⁺ affinity 190-fold. The analogous MonoCast photocage with a single nitrobenzhydrol group only undergoes a 14-fold reduction in affinity after an identical photochemical transformation. A second series of DiCast photocages based on a <i>N</i>,<i>N</i>′-(pyridine-2,6-diylbis­(methylene))­dianiline scaffold, which allows the introduction of two additional Zn²⁺-binding ligands into a preorganized chelator, expand on the multi-photolabile group strategy. DiCast-2 includes two pyridine ligands while DiCast-3 adds two carboxylate groups. Addition of bridging pyridine to the second generation photocages leads to more stable Zn²⁺ complexes, and photolysis of two photolabile groups increases the Zn²⁺ affinity changes to 480-fold. The Zn²⁺, Cu²⁺, and Cd²⁺ binding properties were examined in all the DiCast photocages and the corresponding photoproducts using UV–vis spectroscopy. Further insight into the photocage Zn²⁺-binding motifs was obtained by X-ray analysis of DiCast-2 and DiCast-3 model ligands

Improved Photodecarboxylation Properties in Zinc Photocages Constructed using M‐nitrophenylacetic Acid Variants

The methoxy- and fluoro-derivatives of meta-nitrophenylacetic acid (mNPA) chromophores undergo photodecarboxylation with comparable quantum yields to unsubstituted mNPA, but uncage at red-shifted excitation wavelengths. This observation prompted us to investigate DPAdeCageOMe (2-[bis(pyridin-2-ylmethyl)amino]-2-(4-methoxy-3-nitrophenyl)acetic acid) and DPAdeCageF (2-[bis(pyridin-2-ylmethyl)amino]-2-(4-fluoro-3-nitrophenyl)acetic acid) as Zn2+ photocages. DPAdeCageOMe has a high quantum yield and exhibits other photophysical properties comparable to XDPAdeCage ({bis[(2-pyridyl)methyl]amino}(9-oxo-2-xanthenyl) acetic acid), the best perforiming Zn2+ photocage reported to date. Since the synthesis of DPAdeCageOMe is more straightforward than XDPACage, the new photocage will be a highly competitive tool for biological applications