A Bright Spiropyran-Based Zinc Sensor for Live-Cell Imaging

Abstract

Pools of labile bound zinc ions are essential for signal transduction in the human body. At the cellular level, such pools occur in the cytosol, discrete organelles, and secretory vesicles. These zinc-containing vesicles are found in distinct regions of the central nervous system, modulating calcium ion channels that play an essential role in olfaction, audition, and somatosensory perception. Dysregulation of these receptors is associated with a number of neurodegenerative diseases. To understand the underlying mechanisms at the molecular level, zinc fluorescence sensors are versatile tools. In this report, a new member of the spiropyran-based sensor family SpiroZin, which has proven useful for the investigation of zinc in living cells, is presented: SpiroZin2-COOH. This sensor can be synthesized in a 5-step synthesis and shows superior zinc-sensing properties in cuvette as well as live cell studies. The quantum yield is approximately seven times higher than that of the parent zinc sensor, which also results in an approximately 6-fold higher brightness and a turn-on of 30 at pH 7 in cuvette studies. Another advantage is a significant red-shift of 30 nm in comparison to the parent sensor SpiroZin2. Other basic properties of the SpiroZin family are retained, as revealed by a similar binding constant and negligible pH dependence in zinc sensing. Similar to other members of the SpiroZin family, SpiroZin2-COOH images intracellular zinc pools in living cells. Lysotracker costaining reveals lysosomal localization of SpiroZin2-COOH. The turn-on is determined to be 14.6, which is the highest turn-on within the SpiroZin family reported so far in live-cell studies.

1. Zinc Sensing Mechanism of SpiroZin Sensors (L = H₂O and/or Anion

2. Synthetic Route to SpiroZin2-COOH

1

Absorption (black lines) and fluorescence (red lines) spectra of 5 μM SpiroZin2-COOH in aqueous buffer (50 mM PIPES, 100 mM KCl, pH 7) before (solid lines) and after (dotted lines) the addition of 100 equiv of ZnSO₄. Photophysical properties: λ_abs = 526 nm (ε₅₂₆ = 2,6(8) × 10⁴ cm^–1 M^–1); λ_em = 675 nm (ϕ = 0.0065(6)).

2

Influence of chosen metal ions on the fluorescence intensity of SpiroZin2-COOH. Intensities are normalized to that of the SpiroZin2-COOH zinc complex.

3

A. Confocal fluorescence microscopy of living HeLa cells pretreated with 5 μM SpiroZin2-COOH before and after subsequent addition of 50 μM zinc pyrithione in water (10 equiv) and addition of 50 μM TPEN (20 equiv). Nuclei were counterstained with Hoechst 33342. Representative images are depicted (n = 3). B. Fluorescence normalized to the background fluorescence of the cells after the addition of SpiroZin2-COOH. Data are shown as mean + SEM (n = 3). **p < 0.01.

4

A. Confocal fluorescence microscopy images of living HeLa cells pretreated with 20 μM Hoechst 33342, 5 μM SpiroZin2-COOH, 50 μM zinc pyrithione, and 33 nM LysoTracker Green DND-26. Representative images are depicted (n = 3). B. Determination of Pearson’s correlation coefficient. Data are shown as mean + SEM (n = 3).