Tuning the Spectroscopic Properties of Ratiometric Fluorescent Metal Indicators: Experimental and Computational Studies on Mag-fura‑2 and Analogues

In this joint theoretical and experimental work, we investigate the properties of Mag-fura-2 and seven structurally related fluorescent sensors designed for the ratiometric detection of Mg²⁺ cations. The synthesis of three new compounds is described, and the absorption and emission spectra of all of the sensors in both their free and metal-bound forms are reported. A time-dependent density functional theory approach accounting for hydration effects using a hybrid implicit/explicit model is employed to calculate the absorption and fluorescence emission wavelengths, study the origins of the hypsochromic shift caused by metal binding for all of the sensors in this family, and investigate the auxochromic effects of various modifications of the “fura” core. The metal-free forms of the sensors are shown to undergo a strong intramolecular charge transfer upon light absorption, which is largely suppressed by metal complexation, resulting in predominantly locally excited states upon excitation of the metal complexes. Our computational protocol might aid in the design of new generations of fluorescent sensors with low-energy excitation and enhanced properties for ratiometric imaging of metal cations in biological samples

Visualizing Compartmentalized Cellular Mg²⁺ on Demand with Small-Molecule Fluorescent Sensors

The study of intracellular metal ion compartmentalization and trafficking involved in cellular processes demands sensors with controllable localization for the measurement of organelle-specific levels of cations with subcellular resolution. We introduce herein a new two-step strategy for in situ anchoring and activation of a fluorescent Mg²⁺ sensor within an organelle of choice, using a fast fluorogenic reaction between a tetrazine-functionalized pro-sensor, Mag-<i>S</i>-Tz, and a strained bicyclononyne conjugated to a genetically encoded HaloTag fusion protein of known cellular localization. Protein conjugation does not affect the metal-binding properties of the <i>o</i>-aminophenol-<i>N</i>,<i>N</i>,<i>O</i>-triacetic acid (APTRA)-based fluorescent indicator, which displays a dissociation constant <i>K</i>_d = 3.1 mM suitable for the detection of low millimolar concentrations of chelatable Mg²⁺ typical of the intracellular environment. We demonstrate the application of our sensing system for the ratiometric detection of Mg²⁺ in target organelles in HEK 293T cells, providing the first direct comparison of subcellular pools of the metal without interfering signal from other compartments. Activation of the fluorescence in situ through a fluorogenic conjugation step effectively constrains the fluorescence signal to the locale of interest, thus improving the spatial resolution in imaging applications and eliminating the need for washout of mislocalized sensor. The labeling strategy is fully compatible with live cell imaging, and provides a valuable tool for tracking changes in metal distribution that to date have been an unsolved mystery in magnesium biology

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 <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

Formation of Ternary Complexes with MgATP: Effects on the Detection of Mg²⁺ in Biological Samples by Bidentate Fluorescent Sensors

Fluorescent indicators based on β-keto-acid bidentate coordination motifs display superior metal selectivity profiles compared to current <i>o</i>-aminophenol-<i>N</i>,<i>N</i>,<i>O</i>-triacetic acid (APTRA) based chelators for the study of biological magnesium. These low denticity chelators, however, may allow for the formation of ternary complexes with Mg²⁺ and common ligands present in the cellular milieu. In this work, absorption, fluorescence, and NMR spectroscopy were employed to study the interaction of turn-on and ratiometric fluorescent indicators based on 4-oxo-4H-quinolizine-3-carboxylic acid with Mg²⁺ and ATP, the most abundant chelator of biological magnesium, thus revealing the formation of ternary complexes under conditions relevant to fluorescence imaging. The formation of ternary species elicits comparable or greater optical changes than those attributed to the formation of binary complexes alone. Dissociation of the fluorescent indicators from both ternary and binary species have apparent equilibrium constants in the low millimolar range at pH 7 and 25 °C. These results suggest that these bidentate sensors are incapable of distinguishing between free Mg²⁺ and MgATP based on ratio or intensity-based steady-state fluorescence measurements, thus posing challenges in the interpretation of results from fluorescence imaging of magnesium in nucleotide-rich biological samples