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

Abstract

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 <i>K</i>_d 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