Development of zinc detection system with novel zinc-fluorophores

用选择性分析试剂定量分析痕量锌离子在环境和生物领域的应用已变得极为重要。虽然对其他具有重要生物学意义的二价金属离子，特别是钙（II）离子有了一些选择性荧光团，如Quin-2和Fura-2，取得了显著的发展，但很少有锌（II）选择性分析试剂可用于锌（II）离子的生物学研究。目前，用于检测细胞内锌（II）离子最广泛的探针是8-氨基喹啉磺酰胺衍生物，如Zinquine。最近，荧光素附加的锌（II）螯合物如氮杂碳荧光酮被开发出来，作为理解生物系统中锌（II）化学的改进证明。在利用大环多胺配合物（如锌（II）与1,5,9-三氮杂环十二烷（[12]aneN_3）和1,4,7,10-四氮杂环十二烷（cyclen）配合物）对锌酶催化位点锌（II）离子的内在性质进行研究时，我们发现了锌（II）离子独特的酸性质。锌（II）最突出的特性之一是对芳香磺胺具有很强的亲和力，在生理ph值下，大环多胺中的去质子化磺胺N^-阴离子与锌（II）之间形成了强键。我们设计了5-二甲氨基萘-1-磺酸[2-（1,4,7,10-四氮杂环十二-1-基）-乙基]-酰胺（dansylamidoethyl-cyclen）作为一种新型的锌(II)-荧光基团，在生理ph下与锌（II）离子形成高荧光1:1配合物。锌（II）解离常数远小于细胞内锌（II）浓度，这是细胞内锌（II）离子动态分析的一个缺点。为了将分析范围转移到较高浓度（即生物条件），我们设计了一些具有较大K_d值的锌荧光团（例如，dansylamidoethyl-[12]aneN_3）。本研究确定了这些新型荧光团和锌（II）配合物的合成方法和表征。少

Quantitative analysis of trace zinc(II) ion with a selective analytical reagent has become extremely important for environmental and biological applications. While remarkable development has been made for other biologically important divalent metal ions, in particular calcium(II) ion, with several selective fluorophores such as Quin-2 and Fura-2, there have been few zinc(II)-selective analytical reagents available for biological research on zinc(II) ion. Currently, the most widely used probes for detecting intracellular zinc(II) ion are 8-aminoquinoline sulfonamide derivatives such as Zinquine. Recently, fluorescein-appended zinc(II) chelates such as azacrownfluorone have been developed as improved proves for understanding the zinc(II) chemistry in biological systems. While working on elucidation of intrinsic properties of zinc(II) ion in the catalytic sites of zinc enzymes by use of macrocyclic polyamine complexes (e.g., zinc(II) complexes with 1,5,9-triazacyclododecane ([12]aneN_3) a … More nd 1,4,7,10-tetraazacyclododecane (cyclen)), we have discovered unique acid properties of zinc(II) ion. One of the most outstanding zinc(II) properties is a strong affinity to aromatic sulfonamides, as illustrated by formation of the strong bonds between the deprotonated sulfonamide N^- anions and zinc(II) in the macrocyclic polyamines at physiological pH. On the basis of these findings, we have designed 5-dimethylamino-naphthalene-1-sulfonic acid [2-(1,4,7,10-tetraazacyclododec-1-yl)-ethyl]-amide (dansylamidoethyl-cyclen) that serves as a novel type of zinc(II)-fluorophore by forming a highly fluorescent 1:1 complex with zinc(II) ion at physiological pH. The zinc(II) dissociation constant is much smaller than intracellular zinc(II) concentrations, which is a drawback on the dynamic analysis of intracellular zinc(II) ion. In order to shift the analytical range to the higher concentrations (I.e., biological conditions), we have designed some zinc-fluorophores (e.g., dansylamidoethyl-[12]aneN_3) having a larger K_d value. In this study we deterimined the synthetic method and characterization of those novel fluorophores and those zinc(II) complexes. Less