An Improved Understanding of Fluorescent Zn(II) Sensors and Their Uses in Biological Settings

Chapter 1. An Introduction to Fluorescent Zn(II) Sensors and Their Applications in Biological Systems This chapter opens with an overview of the numerous roles of zinc in biology, with an emphasis on labile Zn(II), that is, loosely bound or kinetically available stores of the ion. The synthesis, characterization, and application of fluorescent sensors for Zn(II) detection constitute an active field of research. A survey of the history and development of these sensors is presented, along with a sample of the knowledge acquired through their use in biological systems. An examination of methods for the subcellular localization of fluorescent probes is included. Chapter 2. Solution and Fluorescence Properties of Symmetric DipicolylamineContaining Dichlorofluorescein-Based Zn2+ Sensors The syntheses of five new analogues of the 2',7'-dichlorofluorescein-based Zn sensor Zinpyr-1 (ZP1) are reported. Analysis of these fluorophores by absorption and emission spectroscopy, in combination with potentiometric titrations, produced a detailed molecular picture of the Zn 2+ and H+ binding properties of the ZP1 family of sensors. The two separate N 30 donor atom sets on ZP1 converge to form binding pockets in which all four heteroatoms participate in coordination to either Zn or protons. The position of the pyridyl group nitrogen atom, 2-pyridyl or 4-pyridyl, has a large impact on the fluorescence response of the dyes to protons in spite of relatively small changes in pKa values. Despite the structural complexity of ZP1, we provide evidence that the pyridyl arms of the DPA appendages participate in the quenching process, in addition to the contribution from the tertiary nitrogen amine atom. Potentiometric titrations reveal ZP1 dissociation constants (Kd) for Zn2 + of 0.04 pM and 1.2 nM for binding to the first and second binding pockets of the ligand, respectively, the second of which correlates with the value observed by fluorescence titration. Chapter 3. Subtle Modification of 2,2-Dipicolylamine Lowers the Affinity and Improves the Turn-On of Zn(II)-Selective Fluorescent Sensors The spectroscopic and protonand Zn(II)-binding properties of two new members of the Zinpyr family of fluorescent sensors are reported. In ZP1B and ZP3B, a (2-picolyl)(4picolyl)amine (2,4-DPA) moiety is installed in place of the di(2-picolyl)amine (2,2-DPA) ligand used in the parent sensors ZP1 and ZP3. This modification has the benefit of both lowering the proton-induced turn-on at physiological pH levels and altering the Zn(II) affinity so as to detect only the most concentrated stores of this ion in biological samples. Comparison of the proton affinities of all four sensors, as determined by potentiometric titrations, contributes to our understanding of the solution properties of this family of