Visualization of Peroxynitrite-Induced Changes of Labile Zn²⁺ in the Endoplasmic Reticulum with Benzoresorufin-Based Fluorescent Probes

Zn2+ plays essential roles in biology, and the homeostasis of Zn2+ is tightly regulated in all cells. Subcellular distribution and trafficking of labile Zn2+, 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 Zn2+, 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 Zn2+ 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 Zn2+ thus providing evidence for a direct connection between ER stress and ER Zn2+ homeostasis.锌离子（Zn²+）在生物学中发挥着不可或缺的作用，且所有细胞内的锌离子稳态均受到严格调控。由于缺乏合适的成像工具，人们对游离锌离子（labile Zn²+）的亚细胞分布、转运过程及其与活性氮物种（reactive nitrogen species）的相互关联尚缺乏深入了解。本研究报道了一类基于苯并试卤灵（benzoresorufin）平台、修饰有二吡啶甲基胺（dipicolylamine）或吡啶甲基胺（picolylamine）衍生金属结合基团的新型游离锌离子红色发射型荧光传感器家族ZBR1~3。该传感器的悬垂胺基与荧光团共同构成了[N3O]结合基序，与此前报道的Zinpyr家族荧光素基荧光传感器的结合基序高度相似，不仅很好地复刻了其金属结合能力，还在亚纳摩尔至皮摩尔范围内获得了相当的解离常数（Kd）值。ZBR系列传感器在比色皿中与锌离子结合后，荧光发射强度最高可提升8.4倍；在活细胞中采用标准宽场荧光显微镜成像时，也能获得类似的响应效果。这类新型传感器可自发定位于多种受试细胞系的内质网（ER），从而实现活细胞内细胞器特异性的锌水平监测。通过使用过氧亚硝酸盐发生器（peroxynitrite generator）Sin-1处理神经干细胞并检测其内质网锌水平，研究发现游离锌离子水平迅速下降，这为内质网应激（ER stress）与内质网锌离子稳态之间的直接关联提供了实验证据。