Selective zinc sensor molecules with various affinities for Zn2+, revealing dynamics and regional distribution of synaptically released Zn2+ in hippocampal slices

We have developed a series of fluorescent Zn(2+) sensor molecules with distinct affinities for Zn(2+), because biological Zn(2+) concentrations vary over a wide range from sub-nanomolar to millimolar. The new sensors have K(d) values in the range of 10(-8)-10(-4) M, compared with 2.7 nM for ZnAF-2. They do not fluoresce in the presence of other biologically important metal ions such as calcium or magnesium, and they can detect Zn(2+) within 100 ms. In cultured cells, the fluorescence intensity of ZnAF-2 was saturated at low Zn(2+) concentration, while that of ZnAF-3 (K(d) = 0.79 muM) was not saturated even at relatively high Zn(2+) concentrations. In hippocampal slices, we measured synaptic release of Zn(2+) in response to high-potassium-induced depolarization. ZnAF-2 showed similar levels of fluorescence increase in dentate gyrus (DG), CA3 and CA1, which were indistinguishable. However, ZnAF-3 showed a fluorescence increase only in DG. Thus, by using a combination of sensor molecules, it was demonstrated for the first time that a higher Zn(2+) concentration is released in DG than in CA3 or CA1 and that we can easily visualize Zn(2+) concentration over a wide range. We believe that the use of various combinations of ZnAF family members will offer unprecedented versatility for fluorescence-microscopic imaging of Zn(2+) in biological applications.     

Modulating affinities of di-2-picolylamine (DPA)-substituted quinoline sensors for zinc ions by varying pendant ligands

We have developed a series of di-2-picolylamine (DPA)-substituted quinoline sensors, HQ1- 4, bearing a pendant ligand at the 8 position of quinoline. UV-vis spectra of HQ1- 4 showed similar variations to that of HQ5 but with different varying extents upon the titration of zinc ions. Fluorescence intensities of HQ1, HQ3, and HQ4 were enhanced 4-6 times upon the addition of 1 equiv of zinc ions under an aqueous buffer. Somewhat unexpectedly, HQ2 is nonfluorescent in the presence of metal ions, including zinc ions. The affinities of HQ sensors are distributed in a broad range from nanomolarity to femtomolarity by varying the pendant ligands near the coordination unit. More importantly, these new sensors exhibited very high selectivity for Zn(2+) over Na(+), K(+), Mg(2+), and Ca(2+) at the millimolar level and over other transition metal ions at the micromolar level, except for Cd(2+). These findings indicated that the incorporations of the pendant groups exerted no effect on the spectroscopic properties and selectivity of the parent fluorescent sensor, with the exception of HQ2. Finally, X-ray crystal structures of ZnHQ's revealed that the auxiliary pendant groups at the 8 position participated in zinc coordination and were able to tune the affinities of HQ sensors.     

A minimal core based fluorophore for selective detection of Zn(II) ions in aqueous solution and living cells

A minimal core based fluorophore was introduced as a selectively fluorescent "turn on" sensor for Zn(2+) ions in aqueous solution. Addition of Zn(2+) ions to the fluorophore generates a significant emission through a 1:1 ligand-to-metal complex. The fluorescence titration experiment of the minimal core based fluorophore with various metal ions shows that the pyromellitic diimide derivative also has the advantage of a high selectivity to Zn(2+) ions over other metals such as Ni(2+), or Co(2+), Cu(2+), Fe(3+), Fe(2+). More than 8 fold increase in the intensity of fluorescence was observed for the Zn(2+)-bound fluorophore compared to Zn-free fluorophore. Due to its small molecular size, the fluorophore was cell-permeable and successfully applied to the detection of Zn(2+) in living cells. With its relatively high sensitivity to Zn(2+) in living cells, the synthesized new fluorophore will be very useful in the studies on various biological functions of Zn(2+).     

Zn2+-triggered excited-state intramolecular proton transfer: a sensitive probe with near-infrared emission from bis(benzoxazole) derivative

Near-infrared (NIR) emission can offer distinct advantages for biological applications. A fluorescent sensor, Zinhbo-1, based on bis(benzoxazole) ligand with 2,2'-dipicolylamine (DPA) as receptor, was synthesized. In aqueous solution, Zinhbo-1 demonstrates high sensitivity and selectivity for sensing Zn(2+) with about 10-fold enhancement and nanomolar sensitivity (K(d) = 0.29 nM). Moreover, sensor Zinhbo-1 can detect Zn(2+) in near-infrared region (over 700 nm) with large Stokes shift (ca. 230 nm) attributing to the Zn(2+)-induced excited state intramolecular proton transfer (ESIPT).     

An efficient sensor for Zn2+ and Cu2+ based on different binding modes

An efficient sensor for Zn(2+) and Cu(2+) was designed based on different binding modes. The sensor displays ratiometric signals for Zn(2+), due to the Zn(2+)-triggered amide tautomerization; while dual-mode selective behaviors for Cu(2+) result from the deprotonation of the amide tautomer.     

1,3-Di-amidoquinoline conjugate of calix[4]arene (L) as a ratiometric and colorimetric sensor for Zn2+: spectroscopy, microscopy and computational studies

Carboxamidoquinoline appended calix[4]arene-1,3-di-conjugate (L) has been synthesized and characterized and its single crystal XRD structure has been established. L has been shown to act as selective ratiometric turn-on fluorescence sensor for Zn(2+) up to a lowest concentration of 183 ± 18 ppb (2.82 μM) with a nine-fold enhancement by exhibiting blue-green emission. The coordination features of the species of recognition have been computationally evaluated by DFT methods and found to have distorted tetrahedral Zn(2+) center in an N(4) core. The spherical nano-structural features observed for L in TEM are being transformed into the Koosh nano-flower like structure when complexed with Zn(2+) and hence these two can be easily differentiated. Even the features observed in AFM can distinctly differentiate L from its Zn(2+) complex.     

Calix[4]arene-based 1,3-diconjugate of salicylyl imine having dibenzyl amine moiety (L): synthesis, characterization, receptor properties toward Fe2+, Cu2+, and Zn2+, crystal structures of its Zn2+ and Cu2+ complexes, and selective phosphate sensing by the [ZnL

A calix[4]arene conjugate bearing salicylyl imine having dibenzyl moiety (L) has been synthesized and characterized, and its ability to recognize three most important essential elements of human system, viz., iron, copper, and zinc, has been addressed by colorimetry and fluorescence techniques. L acts as a sensor for Cu(2+) and Fe(2+) by exhibiting visual color change and for Zn(2+) based on fluorescence spectroscopy. L shows a minimum detection limit of 3.96 ± 0.42 and 4.51 ± 0.53 ppm and 45 ± 4 ppb, respectively, toward Fe(2+), Cu(2+), and Zn(2+). The in situ prepared [ZnL] exhibits phosphate sensing among 14 anions studied with a detection limit of 247 ± 25 ppb. The complexes of Zn(2+), Cu(2+), and Fe(2+) of L have been synthesized and characterized by different techniques. The crystalline nature of the zinc and copper complexes and the noncrystalline nature of simple L and its iron complex have been demonstrated by powder XRD. The structures of Cu(2+) and Zn(2+) complexes have been established by single crystal XRD wherein these were found to be 1:1 monomeric and 2:2 dimeric, respectively, using N(2)O(2) as binding core. The geometries exhibited by the Zn(2+) and the Cu(2+) complexes were found to be distorted tetrahedral and distorted square planar, respectively. The iron complex of L exists in 1:1 stoichiometry as evident from the mass spectrometry and elemental analysis.     

ZP4, an improved neuronal Zn2+ sensor of the Zinpyr family

A second-generation fluorescent sensor for Zn(2+) from the Zinpyr family, ZP4, has been synthesized and characterized. ZP4 (Zinpyr-4, 9-(o-carboxyphenyl)-2-chloro-5-[2-(bis(2-pyridylmethyl)aminomethyl)-N-methylaniline]-6-hydroxy-3-xanthanone) is prepared via a convergent synthetic strategy developed from previous studies with these compounds. ZP4, like its predecessors, has excitation and emission wavelengths in the visible range ( approximately 500 nm), a dissociation constant (K(d)) for Zn(2+) of less than 1 nM and a high quantum yields (Phi = approximately 0.4), making it well suited for biological applications. A 5-fold fluorescent enhancement is observed under simulated physiological conditions corresponding to the binding of the Zn(2+) cation to the sensor, which inhibits a photoinduced electron transfer (PET) quenching pathway. The metal-binding stereochemistry of ZP4 was evaluated through the synthesis and X-ray structural characterization of [M(BPAMP)(H(2)O)(n)](+) complexes, where BPAMP is [2-(bis(2-pyridylmethyl)aminomethyl)-N-methylaniline]-phenol and M = Mn(2+), Zn(2+) (n = 1) or Cu(2+) (n = 0).     

An excitation ratiometric Zn2+ sensor with mitochondria-targetability for monitoring of mitochondrial Zn2+ release upon different stimulations

A mitochondria-targeted fluorescent sensor (Mito-ST), constructed by integrating a sulfamoylbenzoxadiazole fluorophore with a phosphonium group, displays the specific Zn(2+)-induced hypsochromic shifts of both excitation (69 nm) and emission (35 nm) maxima. Its ratiometric Zn(2+) imaging ability via dual excitation mode has been applied in MCF-7 cells to clarify the different behaviours of mitochondrial Zn(2+) release stimulated by H(2)O(2) and SNOC.     

Design and synthesis of a caged Zn2+ probe, 8-benzenesulfonyloxy-5-N,N-dimethylaminosulfonylquinolin-2-ylmethyl-pendant 1,4,7,10-tetraazacyclododecane, and its hydrolytic uncaging upon complexation with Zn2+

8-Benzenesulfonyloxy-5- N,N-dimethylaminosulfonylquinolin-2-ylmethyl-pendant cyclen (BS-caged-L(4), BS = benzenesulfonyl) was designed and synthesized as a "caged" derivative of a previously described Zn(2+) fluorophore, 8-hydroxy-5- N,N-dimethylaminosulfonylquinolin-2-ylmethyl-pendant cyclen (L(4)) (cyclen = 1,4,7,10-tetraazacyclododecane). In the absence of metal ions and in the dark, BS-caged-L(4) (10 microM) showed negligible fluorescence emission at pH 7.4 (10 mM HEPES with I = 0.1 (NaNO3)) and 25 degrees C (excitation at 328 nm). Addition of Zn(2+) induced an increase in the UV/vis absorption of BS-caged-L(4) (10 microM) at 258 nm and a significant increase in fluorescence emission at 512 nm. These responses are results from the formation of Zn(H-1L(4)) by the hydrolysis of the sulfonyl ester at the 8-position of the quinoline unit promoted by the Zn(2+)-bound HO(-). Improvement of cell membrane permeation in comparison with L(4) is also described.     

A Zn2+-specific turn-on fluorescent probe for ratiometric sensing of pyrophosphate in both water and blood serum

A novel fluorescent sensor composed of a naphthalene functionalized tetraazamacrocycle ligand 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3-methyl naphthalene (1) and Zn(2+) has been designed and prepared, which can be utilized for selective and ratiometric sensing of pyrophosphate (PPi) over other phosphate-containing anions in aqueous solution at physiological pH. Notably, the water soluble 1 itself also exhibits a selective enhanced fluorescent response to Zn(2+), and the complex 1-Zn(2+) thus formed eventually fulfils the synergic Zn(2+) coordination-altered strategy with PPi. Furthermore, the ratiometric sensing of 1-Zn(2+) towards PPi performed well even in blood serum milieu. Finally, the sensor 1-Zn(2+) was successfully employed to monitor a real-time assay of inorganic pyrophosphatase (PPase) by means of ratiometric fluorescent measurements for the first time.     

EPR spectra of Cu(2+) doped [Zn(sac)2(dmen)] and [Zn(sac)2(paen)] single crystals

Cu(2+) doped single crystals of [Zn(sac)2(dmen)] (sac: saccharinate, dmen: N,N'-dimethylethylendiamine) and [Zn(sac)2(paen)], (paen: N,N'-bis(3-propylamine)ethylendiamine) complexes have been investigated by electron paramagnetic resonance (EPR) technique. Detailed investigations of the EPR spectra indicate that Cu(2+) ion substitute with Zn(2+) ion and forms tetrahedral complex in [Zn(sac)2(dmen)] and octahedral complex in [Zn(sac)2(paen)] hosts. Principal values of the g and hyperfine tensors are determined and the ground state wave functions of Cu(2+) ions are obtained using EPR parameters.     

A turn-on and reversible fluorescence sensor for zinc ion

A simple Schiff base type fluorescent receptor was prepared and evaluated for its fluorescence response to heavy metal ions. Receptor exhibits an "off-on-type" mode with high selectivity in the presence of Zn(2+) ion. The addition of EDTA quenches the fluorescence of receptor -Zn(2+) complex, making receptor a reversible chemosensor. The selectivity of for Zn(2+) is the consequence of combined effects of CHEF, C[double bond, length as m-dash]N isomerization and inhibition of ESIPT.     

A differentially selective sensor with fluorescence turn-on response to Zn2+ and dual-mode ratiometric response to Al3+ in aqueous media

A novel quinoline-coumarin (QC) fluoroionophore conjugated by means of a triazolyl-pyrrolidinyl linker exhibits differential dual selectivity for Zn(2+) and Al(3+) in mixed media. QC acts as a turn on fluorescence sensor for Zn(2+) while exhibiting overall ratiometric selectivity for Al(3+) in aqueous media. Moreover, QC exhibited preferential second mode of selectivity for Al(3+) as it ratiometrically displaces Zn(2+) from the [QC + Zn(2+)] complex.     

A highly selective fluorescent sensor for distinguishing cadmium from zinc ions based on a quinoline platform

A fluorescent sensor, N-(quinolin-8-yl)-2-(quinolin-8-yloxy)acetamide (HL), based on 8-aminoquinoline and 8-hydroxyquinoline platforms has been synthesized. This sensor displays high selectivity and sensitive fluorescence enhancement to Cd(2+) in ethanol. Moreover, sensor HL can distinguish Cd(2+) from Zn(2+) via two different sensing mechanisms (photoinduced electron transfer for Cd(2+); internal charge transfer for Zn(2+)). The composition of the complex Cd(2+)/HL or Zn(2+)/L(-) has been found to be 1:1, based on the fluorescence/absorption titration and further confirmed by X-ray crystallography.     

Rhodamine-based highly sensitive colorimetric off-on fluorescent chemosensor for Hg2+ in aqueous solution and for live cell imaging

A novel rhodamine-based highly sensitive and selective colorimetric off-on fluorescent chemosensor for Hg(2+) ions is designed and prepared by using the well-known thiospirolactam rhodamine chromophore and furfural hydrazone as signal-reporting groups. The photophysical characterization and Hg(2+)-binding properties of sensor RS1 in neutral N, N-dimethylformamide (DMF) aqueous solution are also investigated. The signal change of the chemosensor is based on a specific metal ion induced reversible ring-opening mechanism of the rhodamine spirolactam. The response of the chemosensor for Hg(2+) ions is instantaneous and reversible. And it successfully exhibits a remarkably "turn on" response toward Hg(2+) over other metal ions (even those that exist in high concentration). Moreover, this sensor is applied for in vivo imaging in Rat Schwann cells to confirm that RS1 can be used as a fluorescent probe for monitoring Hg(2+) in living cells with satisfying results, which further demonstrates its value of practical applications in environmental and biological systems.     

Development and applications of fluorescent indicators for Mg2+ and Zn2+

In a study of the spectroscopic behavior of two Schiff base derivatives, salicylaldehyde salicylhydrazone (1) and salicylaldehyde benzoylhydrazone (2), Schiff base 1 has high selectivity for Zn(2+) ion not only in abiotic systems but also in living cells. The ion selectivity of 1 for Zn(2+) can be switched for Mg(2+) by swapping the solvent from ethanol-water to DMF (N,N-dimethylformamide)-water mixtures. Imine 2 is a good fluorescent probe for Zn(2+) in ethanol-water media. Many other ions tested, such as Li(+), Na(+), Al(3+), K(+), Ca(2+), Cr(3+), Mn(2+), Fe(3+), Co(2+), Ni(2+), Cu(2+), Ag(+), Cd(2+), Sn(2+), Ba(2+), Hg(2+), and Pb(2+), failed to induce any spectral change in various solvents. The selectivity mechanism of 1 and 2 for metal ions is based on a combinational effect of proton transfer (ESPT), C═N isomerization, and chelation-enhanced fluorescence (CHEF). The coordination modes of the complexes were investigated.     

Coumarin-derived transformable fluorescent sensor for Zn2+

We report a coumarin-derived fluorescent sensor for Zn(2+) termed CTS. CTS shows excellent binding selectivity for Zn(2+) over competing metal ions due to the transformable ability of CTS, that is the displacement of other metal ions by Zn(2+), which induces transformation of chelation from an amide to an imidic acid tautomeric form.     

Development of a cholesterol-conjugated fluorescent sensor for site-specific detection of zinc ion at the plasma membrane

A cholesterol-conjugated fluorescence Zn(2+) sensor based on the fluorescein platform was designed and synthesized. The cholesterol moiety is essential for localizing the Zn(2+) sensor to the cell membrane, allowing the sensor to probe changes in the Zn(2+) concentration in a localized area of the cell.     

Aqueous fluorescence turn-on sensor for Zn2+ with a tetraphenylethylene compound

A new sensitive and selective fluorescence turn-on sensor for Zn(2+) (1) was developed by taking advantage of the aggregation-induced emission of the tetraphenylethylene framework. In addition, the corresponding ester precursor of 1 was successfully used for intracellular Zn(2+) imaging.