Lin Wei, Buccella Daniela, Lippard Stephen J
Department of Chemistry, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
J Am Chem Soc. 2013 Sep 11;135(36):13512-20. doi: 10.1021/ja4059487. Epub 2013 Sep 3.
Zn(2+) plays essential roles in biology, and the homeostasis of Zn(2+) is tightly regulated in all cells. Subcellular distribution and trafficking of labile Zn(2+), and its inter-relation with reactive nitrogen species, are poorly understood due to the scarcity of appropriate imaging tools. We report a new family of red-emitting fluorescent sensors for labile Zn(2+), ZBR1-3, based on a benzoresorufin platform functionalized with dipicolylamine or picolylamine-derived metal binding groups. In combination, the pendant amines and fluorophore afford an [N3O] binding motif that resembles that of previously reported fluorescein-based sensors of the Zinpyr family, reproducing well their binding capabilities and yielding comparable Kd values in the sub-nanomolar and picomolar ranges. The ZBR sensors display up to 8.4-fold emission fluorescence enhancement upon Zn(2+) binding in the cuvette, with similar responses obtained in live cells using standard wide-field fluorescence microscopy imaging. The new sensors localize spontaneously in the endoplasmic reticulum (ER) of various tested cell lines, allowing for organelle-specific monitoring of zinc levels in live cells. Study of ER zinc levels in neural stem cells treated with a peroxynitrite generator, Sin-1, revealed an immediate decrease in labile Zn(2+) thus providing evidence for a direct connection between ER stress and ER Zn(2+) homeostasis.
锌离子（Zn(2+)）在生物学中发挥着重要作用，并且在所有细胞中锌离子的稳态都受到严格调控。由于缺乏合适的成像工具，不稳定锌离子（Zn(2+)）的亚细胞分布和转运及其与活性氮物种的相互关系尚不清楚。我们报道了基于用二吡啶基胺或吡啶基胺衍生的金属结合基团功能化的苯并试卤灵平台的用于不稳定锌离子（Zn(2+)）的新型红色发射荧光传感器ZBR1 - 3。 pendant胺和荧光团共同提供了一个[N3O]结合基序，类似于先前报道的基于荧光素的锌吡啶家族传感器的结合基序，很好地再现了它们的结合能力，并在亚纳摩尔和皮摩尔范围内产生了相当的解离常数（Kd）值。在比色皿中，ZBR传感器在结合锌离子（Zn(2+)）时显示出高达8.4倍的发射荧光增强，使用标准宽场荧光显微镜成像在活细胞中也获得了类似的响应。新传感器自发地定位于各种测试细胞系的内质网（ER）中，从而能够对活细胞中的锌水平进行细胞器特异性监测。在用过氧亚硝酸盐发生器Sin - 1处理的神经干细胞中对内质网锌水平的研究表明，不稳定锌离子（Zn(2+)）立即减少，从而为内质网应激与内质网锌离子（Zn(2+)）稳态之间的直接联系提供了证据。
