Elisa Tomat

Organelle-Specific Zinc Detection Using Zinpyr-Labeled Fusion Proteins in Live Cells

A protein labeling approach is employed for the localization of a zinc-responsive fluorescent probe in the mitochondria and in the Golgi apparatus of living cells. ZP1, a zinc sensor of the Zinpyr family, was functionalized with a benzylguanine moiety and thus converted into a substrate (ZP1BG) for the human DNA repair enzyme alkylguaninetransferase (AGT or SNAP-Tag). The labeling reaction of purified glutathione S-transferase tagged AGT with ZP1BG and the zinc response of the resulting protein-bound sensor were confirmed in vitro. The new detection system, which combines a protein labeling methodology with a zinc fluorescent sensor, was tested in live HeLa cells expressing AGT in specific locations. The enzyme was genetically fused to site-directing proteins that anchor the probe onto targeted organelles. Localization of the zinc sensors in the Golgi apparatus and in the mitochondria was demonstrated by fluorescence microscopy. The protein-bound fluorescence detection system is zinc-responsive in living cells.

Biochemistry of Mobile Zinc and Nitric Oxide Revealed by Fluorescent Sensors

Biological mobile zinc and nitric oxide (NO) are two prominent examples of inorganic compounds involved in numerous signaling pathways in living systems. In the past decade, a synergy of regulation, signaling, and translocation of these two species has emerged in several areas of human physiology, providing additional incentive for developing adequate detection systems for Zn(II) ions and NO in biological specimens. Fluorescent probes for both of these bioinorganic analytes provide excellent tools for their detection, with high spatial and temporal resolution. We review the most widely used fluorescent sensors for biological zinc and nitric oxide, together with promising new developments and unmet needs of contemporary Zn(II) and NO biological imaging. The interplay between zinc and nitric oxide in the nervous, cardiovascular, and immune systems is highlighted to illustrate the contributions of selective fluorescent probes to the study of these two important bioinorganic analytes.

Ratiometric and Intensity-Based Zinc Sensors Built on Rhodol and Rhodamine Platforms

A xanthene-forming condensation reaction yields rhodol and rhodamine dyes carrying a zinc-binding ligand that includes the aniline-type nitrogen donor of the fluorophores. Upon zinc coordination in neutral aqueous solution, rhodol RF3 behaves as a ratiometric sensor, and rhodamine RA1 acts as a turn-off intensity-based indicator. Both fluorescent compounds bind the divalent zinc cation with micromolar affinity.

Binuclear organometallic ruthenium complexes of a Schiff base expanded porphyrin

The synthesis of binuclear organometallic ruthenium complexes of an expanded porphyrin-type macrocycle is reported; pyrrolic hydrogen bonding donors were found to interact with ancillary ligands in the primary coordination sphere and to stabilize coordinated dioxygen in an eta(2)-fashion.

Tripyrrindione as a Redox‐Active Ligand: Palladium(II) Coordination in Three Redox States

The tripyrrin-1,14-dione scaffold of urinary pigment uroerythrin coordinates divalent palladium as a planar tridentate ligand. Spectroscopic, structural and computational investigations reveal that the tripyrrindione ligand binds as a dianionic radical, and the resulting complex is stable at room temperature. One-electron oxidation and reduction reactions do not alter the planar coordination sphere of palladium(II) and lead to the isolation of two additional complexes presenting different redox states of the ligand framework. Unaffected by stability problems common to tripyrrolic fragments, the tripyrrindione ligand offers a robust platform for ligand-based redox chemistry.

Intracellular iron chelation modulates the macrophage iron phenotype with consequences on tumor progression

A growing body of evidence suggests that macrophage polarization dictates the expression of iron-regulated genes. Polarization towards iron sequestration depletes the microenvironment, whereby extracellular pathogen growth is limited and inflammation is fostered. In contrast, iron release contributes to cell proliferation, which is important for tissue regeneration. Moreover, macrophages constitute a major component of the infiltrates in most solid tumors. Considering the pivotal role of macrophages for iron homeostasis and their presence in association with poor clinical prognosis in tumors, we approached the possibility to target macrophages with intracellular iron chelators. Analyzing the expression of iron-regulated genes at mRNA and protein level in primary human macrophages, we found that the iron-release phenotype is a characteristic of polarized macrophages that, in turn, stimulate tumor cell growth and progression. The application of the intracellular iron chelator (TC3-S)2 shifted the macrophage phenotype from iron release towards sequestration, as determined by the iron-gene profile and atomic absorption spectroscopy (AAS). Moreover, whereas the addition of macrophage supernatants to tumor cells induced tumor growth and metastatic behavior, the supernatant of chelator-treated macrophages reversed this effect. Iron chelators demonstrated potent anti-neoplastic properties in a number of cancers, both in cell culture and in clinical trials. Our results suggest that iron chelation could affect not only cancer cells but also the tumor microenvironment by altering the iron-release phenotype of tumor-associated macrophages (TAMs). The study of iron chelators in conjunction with the effect of TAMs on tumor growth could lead to an improved understanding of the role of iron in cancer biology and to novel therapeutic avenues for iron chelation approaches.

Prodigiosin analogue designed for metal coordination: stable zinc and copper pyrrolyldipyrrins.

The pyrrolyldipyrrin motif is found in several naturally occurring prodigiosin pigments. The potential roles of the interactions of prodigiosins with transition metals and the properties of metal-bound pyrrolyldipyrrins, however, have been difficult to assess because of the very limited number of well-characterized stable complexes. Here, we show that the introduction of a meso-aryl substituent and an ethyl ester group during the sequential assembly of the three heterocycles affords a pyrrolyldipyrrin of enhanced coordinating abilities when compared to that of natural prodigiosins. UV–visible absorption studies indicate that this ligand promptly binds Zn(II) ions with 2:1 ligand-to-metal stoichiometry and Cu(II) ions with 1:1 stoichiometry. Notably, no addition of base is required for the formation of the resulting stable complexes. The crystal structures reveal that whereas the tetrahedral zinc center engages two nitrogen donors on each ligand, the pseudosquare planar copper complex features coordination of all three pyrrolic nitrogen atoms and employs the ester group as a neutral ligand. This first example of coordination of a redox-active transition metal within a fully conjugated pyrrolyldipyrrin framework was investigated spectroscopically by electron paramagnetic resonance to show that the 1:1 metal-to-ligand ratio found in the crystal structure is also maintained in solution.

Disulfide/thiol switches in thiosemicarbazone ligands for redox-directed iron chelation

A disulfide bond is incorporated in the scaffold of thiosemicarbazone iron chelators as a reduction/activation switch. Following reduction, thiol-containing ligands stabilize iron ions in their trivalent oxidation state. The antiproliferative activity of the new chelating systems is assessed in human cancer cell lines and in normal tissue.

Coordination Chemistry of Linear Tripyrroles: Promises and Perils

GRAPHICAL ABSTRACT Presenting an electron-rich π system and an array of three pyrrolic nitrogen donors, linear tripyrrolic ligands are attractive platforms for metal coordination. While retaining several advantageous features of porphyrin-type ligands (e.g., tunable electronic structure, ligand-based one-electron redox chemistry), tripyrroles permit in-plane access to the metal center. The metal-binding properties of both synthetic and naturally occurring tripyrroles have been documented; however, this chemistry is still in its early stages and the full potential of this class of ligands has not yet come to fruition. Through a selection of examples, several areas of current and potential growth are described along with known hurdles that have emerged thus far.

Intracellular reduction/activation of a disulfide switch in thiosemicarbazone iron chelators

Iron scavengers (chelators) offer therapeutic opportunities in anticancer drug design by targeting the increased demand for iron in cancer cells as compared to normal cells. Prochelation approaches are expected to avoid systemic iron depletion as chelators are liberated under specific intracellular conditions. In the strategy described herein, a disulfide linkage is employed as a redox-directed switch within the binding unit of an antiproliferative thiosemicarbazone prochelator, which is activated for iron coordination following reduction to the thiolate chelator. In glutathione redox buffer, this reduction event occurs at physiological concentrations and half-cell potentials. Consistent with concurrent reduction and activation, higher intracellular thiol concentrations increase cell susceptibility to prochelator toxicity in cultured cancer cells. The reduction of the disulfide switch and intracellular iron chelation are confirmed in cell-based assays using calcein as a fluorescent probe for paramagnetic ions. The resulting low-spin Fe(III) complex is identified in intact Jurkat cells by EPR spectroscopy measurements, which also document a decreased concentration of active ribonucleotide reductase following exposure to the prochelator. Cell viability and fluorescence-based assays show that the iron complex presents low cytotoxicity and does not participate in intracellular redox chemistry, indicating that this antiproliferative chelation strategy does not rely on the generation of reactive oxygen species.