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.     

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.     

ZP8, a neuronal zinc sensor with improved dynamic range; imaging zinc in hippocampal slices with two-photon microscopy

The synthesis of a difluorofluorescein monocarboxaldehyde platform and its use for preparing ZP8, a new member of the Zinpyr family of neuronal Zn(2+) sensors, are described. By combining an aniline photoinduced electron transfer (PET) switch and an electron-withdrawing fluorescein scaffold, ZP8 displays reduced background fluorescence and improved dynamic range compared to previous ZP probes. The bright sensor undergoes an 11-fold increase in fluorescence intensity upon Zn(2+) complexation (Phi = 0.03-0.35) with high selectivity over cellular concentrations of Ca(2+) and Mg(2+). In addition, sensors in the ZP family have been utilized for optical imaging in biological samples using two-photon microscopy (TPM). The cell-permeable ZP3 probe is capable of identifying natural pools of labile Zn(2+) within the mossy fiber synapses of live hippocampal slices using TPM, establishing the application of this technique for monitoring endogenous Zn(2+) stores.     

A novel,cell-permeable,fluorescent probe for ratiometric imaging of zinc ion

Zn(2+) plays important roles in various biological systems; as a result, the development of tools that can visualize chelatable Zn(2+) has attracted much attention recently. We report here newly synthesized fluorescent sensors for Zn(2+), ZnAF-Rs, whose excitation maximum is shifted by Zn(2+) under physiological conditions. Thus, these sensors enable ratiometric imaging, which is a technique to reduce artifacts by minimizing the influence of extraneous factors on the fluorescence of a probe. Ratiometric measurement can provide precise data, and some probes allow quantitative detection. ZnAF-Rs are the first ratiometric fluorescent sensors for Zn(2+) that enable quantitative analysis under physiological conditions. ZnAF-Rs also possess suitable K(d) for applications, and high selectivity against other biologically relevant cations, especially Ca(2+). Using these probes, changes of intracellular Zn(2+) concentration in cultured cells were monitored successfully. We believe that these probes will be extremely useful in studies on the biological functions of Zn(2+).     

Ratiometric and water-soluble fluorescent zinc sensor of carboxamidoquinoline with an alkoxyethylamino chain as receptor

A novel "naked-eye" and ratiometric fluorescent zinc sensor (AQZ) of carboxamidoquinoline with an alkoxyethylamino chain as receptor was designed and synthesized. AQZ shows good water solubility and high selectivity for sensing; about an 8-fold increase in fluorescence quantum yield and a 75 nm red-shift of fluorescence emission upon binding Zn2+ in buffer aqueous solution are observed. Moreover, AQZ can enter yeast cells and signal the presence of Zn2+.     

Quantifying factors that influence metal ion release in photocaged complexes using ZinCast derivatives

Two generations of nitrobenzhydrol-based photocages for Zn(2+) have been prepared and characterized. The first series includes the tridentate ZinCast-1 utilizes a bis-pyridin-2-ylmethyl-aniline ligand that forms a 5,5-chelate ring upon metal binding. The related photocages ZinCast-2 with a N-[2-(pyridine-2-yl)ethyl]-N-(pyridine-2-ylmethyl)aniline (5,6-chelate ring) and ZinCast-3 with a N,N-bis[2-(pyridine-2-yl)ethyl]aniline (6,6-chelate ring) were synthesized for comparative studies. The complexes formed by the ions Cu(2+), Zn(2+) and Cd(2+) with three ZinCast and their photoproducts (ZinUnc) were interrogated by UV-Vis spectroscopy. The studies indicate that ZinCast-1 forms complexes of the highest stability and ZinCast-3 exhibits the most significant changes in metal affinity upon uncaging. These results suggest that the changes in nitrogen atom donor ability as well as the initial complex stability must be considered to design a photocage with the desired properties. The composite results were used to design ZinCast-4 and ZinCast-5, the second generation photocages that incorporate an additional adjacent ether ligand into the Zn(2+) chelator.     

Design and synthesis of a novel magnetic resonance imaging contrast agent for selective sensing of zinc ion

A series of new diethylenetriaminepentaacetic acid (DTPA)-bisamide chelators has been prepared and characterized for application as zinc sensors. We have designed and synthesized (GdL(a))(2-), which contains a DTPA-bisamide moiety. The R(1) relaxivity of (GdL(a))(2-) solution decreased monotonically on the addition of Zn(2+). Moreover, (GdL(a))(2-) showed high selectivity for Zn(2+) against Ca(2+) and Mg(2+). We also measured the UV-visible spectra and the coldspray ionization (CSI) MS spectra and concluded that the 1-to-1 Zn(2+) complex of (GdL(a))(2-) is stable at higher concentrations of Zn(2+). These complexes should provide the basis for creating a superior Zn(2+)-sensitive MRI contrast agent and are excellent candidates for incorporation into sensors designed for selective detection of Zn(2+) in biological applications.     

The rhodafluor family. An initial study of potential ratiometric fluorescent sensors for Zn2+

A new class of ratiometric Zn(2+) sensors that employ a hybrid fluorescein and rhodamine fluorophore has been designed, and two members of the rhodafluor family of sensors, RF1 and RF2, have been synthesized. The preparation of RF1 (9-(o-carboxyphenyl)-2-chloro-6-[bis(2-pyridylmethyl)amino]-3-xanthanone, Rhodafluor-1), uses conventional synthetic methods. Elaboration of the RF1 synthesis in an effort to enhance the Zn(2+) affinity was unsuccessful, so palladium-catalyzed aryl amination was applied to prepare RF2 (1-[9'-(o-carboxyphenyl)-6'-amino-2'-chloro-3'-xanthanone]-4,10-(diethyl)-7-(2-pyridylmethyl)-1,4,7,10-tetraazacyclododecane, Rhodafluor-2). The key step in the synthesis of RF2 is coupling of a triprotected tetraazamacrocycle (cyclen) to 3-bromoanisidine. RF2 binds Zn(2+) with a dissociation constant of 13.5 microM accompanied by an approximately 50% increase in quantum yield. Although only small shifts in absorption wavelength were observed, because protonation of the amino nitrogen atoms of the macrocycle prevents the uncomplexed sensor from adopting the desired mesomer, the intensity doubling makes the probe of value for immediate application in situations where our previous tight binding (<1 nM) sensors are inadequate.     

Recognition of Hg2+ and Cr3+ in physiological conditions by a rhodamine derivative and its application as a reagent for cell-imaging studies

A new rhodamine-based receptor, derivatized with an additional fluorophore (quinoline), was synthesized for selective recognition of Hg(2+) and Cr(3+) in an acetonitrile/HEPES buffer medium of pH 7.3. This reagent could be used as a dual probe and allowed detection of these two ions by monitoring changes in absorption and the fluorescence spectral pattern. In both instances, the extent of the changes was significant enough to allow visual detection. More importantly, the receptor molecule could be used as an imaging reagent for detection of Hg(2+) and Cr(3+) uptake in live human cancer cells (MCF7) using laser confocal microscopic studies. Unlike Hg(ClO(4))(2) or Hg(NO(3))(2) salts, HgCl(2) or HgI(2) failed to induce any visually detectable change in color or fluorescence upon interaction with L(1) under identical experimental conditions. Presumably, the higher covalent nature of Hg(II) in HgCl(2) or HgI(2) accounts for its lower acidity and its inability to open up the spirolactam ring of the reagent L(1). The issue has been addressed on the basis of the single-crystal X-ray structures of L(1)·HgX(2) (X(-) = Cl(-) or I(-)) and results from other spectral studies.     

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.     

Tetrahydroindazolone substituted 2-aminobenzamides as fluorescent probes: switching metal ion selectivity from zinc to cadmium by interchanging the amino and carbamoyl groups on the fluorophore

Three fluorescent probes CdABA', CdABA and ZnABA', which are structural isomers of ZnABA, have been designed with N,N-bis(2-pyridylmethyl) ethylenediamine (BPEA) as chelator and 2-aminobenzamide as fluorophore. These probes can be divided into two groups: CdABA, CdABA' for Cd(2+) and ZnABA, ZnABA' for Zn(2+). Although there is little difference in their chemical structures, the two groups of probes exhibit totally different fluorescence properties for preference of Zn(2+) or Cd(2+). In the group of Zn(2+) probes, ZnABA/ZnABA' distinguish Zn(2+) from Cd(2+) with F(Zn)(2+)-F(Cd)(2+) = 1.87-2.00. Upon interchanging the BPEA and carbamoyl groups on the aromatic ring of the fluorophore, the structures of ZnABA/ZnABA' are converted into CdABA/CdABA'. Interestingly, the metal ions selectivity of CdABA/CdABA' was switched to discriminate Cd(2+) from Zn(2+) with F(Cd)(2+)-F(Zn)(2+) = 2.27-2.36, indicating that a small structural modification could lead to a remarkable change of the metal ion selectivity. (1)H NMR titration and ESI mass experiments demonstrated that these fluorescent probers exhibited different coordination modes for Zn(2+) and Cd(2+). With CdABA' as an example, generally, upon addition of Cd(2+), the fluorescence response possesses PET pathway to display no obvious shift of maximum λ(em) in the absence or presence of Cd(2+). However, an ICT pathway could be employed after adding Zn(2+) into the CdABA' solution, resulting in a distinct red-shift of maximal λ(em).     

High-throughput examination of fluorescence resonance energy transfer-detected metal-ion response in mammalian cells

Fluorescence resonance energy transfer (FRET)-based genetically encoded metal-ion sensors are important tools for studying metal-ion dynamics in live cells. We present a time-resolved microfluidic flow cytometer capable of characterizing the FRET-based dynamic response of metal-ion sensors in mammalian cells at a throughput of 15 cells/s with a time window encompassing a few milliseconds to a few seconds after mixing of cells with exogenous ligands. We have used the instrument to examine the cellular heterogeneity of Zn(2+) and Ca(2+) sensor FRET response amplitudes and demonstrated that the cluster maps of the Zn(2+) sensor FRET changes resolve multiple subpopulations. We have also measured the in vivo sensor response kinetics induced by changes in Zn(2+) and Ca(2+) concentrations. We observed an ～30 fold difference between the extracellular and intracellular sensors.     

Methoxy-substituted TQEN family of fluorescent zinc sensors

Two methoxy-substituted TQEN (N,N,N',N'-tetrakis(2-quinolylmethyl)ethylenediamine) derivatives, T(MQ)EN (N,N,N',N'-tetrakis(6-methoxy-2-quinolylmethyl)ethylenediamine) and T(TMQ)EN (N,N,N',N'-tetrakis(5,6,7-trimethoxy-2-quinolylmethyl)ethylenediamine), have been prepared, and their fluorescence properties with respect to Zn2+ coordination were investigated. Introduction of a methoxy substituent at 6-position of the quinoline ring enhances the fluorescence intensity by 10-fold, and the three methoxy substituents in the 5,6,7-positions afford significant enhancement of the long-wavelength component of the fluorescence of zinc complex. The substituents did not alter the binding affinity of these compounds toward zinc ion significantly. T(MQ)EN was proved to be effective in detection of zinc ion in cells by fluorescent microscopy.     

Infrared absorbing croconaine dyes: synthesis and metal ion binding properties

Quinaldine-based croconaine dyes synthesized by the condensation reaction between croconic acid and the respective quinaldinium salts are described. These dyes exhibit absorption maximum in the infrared region (840-870 nm) with high molar extinction coefficients (1-5 x 10(5) M(-1) cm(-1)) and have very low fluorescence quantum yields. Upon binding to divalent metal ions, these dyes were found to form complexes with a 2:1 stoichiometry having high association constants of the order of 10(11)-10(14) M(-2), while the monovalent metal ions showed negligible affinity. The binding of the croconaine dye 3d with divalent metal ions especially Zn(2+), Pb(2+), and Cd(2+) led to significant chelation-enhanced fluorescence emission. The broadening of the aromatic signals, vinylic and N-methyl protons and the negligible changes at the aliphatic region of the dye 3d in the (1)H NMR spectrum in the presence of Zn(2+), indicate that the binding occurs at the carbonyl groups of the croconyl ring. The shift in the croconyl carbonyl stretching frequency in the [3d-Zn(2+)] complex analyzed through FT-IR analysis further confirms the involvement of two electron-rich carbonyl groups of the croconyl moiety in the complexation. These results demonstrate that the binding of the divalent metal ions at the carbonyl oxygens of these infrared absorbing dyes can be favorably utilized for the development of potential sensors for the detection of metal ions and further can be exploited as sensitizers for photodynamic therapeutic applications.     

Modular and tunable chemosensor scaffold for divalent zinc

A modular peptide scaffold has been developed for fluorescent sensing of divalent zinc. The signaling component of the chemosensor is the chelation-sensitive fluorophore 8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline, which is prepared as the protected amino acid derivative Fmoc-Sox-OH and integrated into peptide sequences. Nineteen synthetic peptides incorporating the signaling element exhibit a range of affinities for Zn(2+) through variation of the type and number of Zn(2+) ligands, ligand arrangement and the beta-turn sequence that acts as a preorganization element between the ligands. The stoichiometry of the peptide-Zn(2+) complexes is evaluated by several criteria. The fluorescence response of these peptides to pH and various important metal ions is reported. Eleven of these sequences form only 1:1 complexes with Zn(2+) and their affinities range from 10 nM to nearly 1 microM. When used in concert, these sensors can provide Zn(2+) concentration information in a valuable range.     

A novel quinoline-based two-photon fluorescent probe for detecting Cd2+ in vitro and in vivo

A new two-photon fluorescent Cd(2+) probe APQ is developed by introducing a N(1),N(1)-dimethyl-N(2)-(pyridin-2-ylmethyl)ethane-1,2-diamine binding group and a 4-methoxyphenylvinyl conjugation-enhancing group to the 2- and 6-positions of quinoline. This probe shows a large red shift and good emission enhancement under Cd(2+) binding. It also exhibits a high ion selectivity for Cd(2+) (especially over Zn(2+)) and a large two-photon absorption cross section at 710 nm. Two-photon microscopy imaging studies reveal that the new probe is non-toxic and cell-permeable and can be used to detect intracellular Cd(2+) under two-photon excitation.     

Polymethylated DOTA ligands. 2. Synthesis of rigidified lanthanide chelates and studies on the effect of alkyl substitution on conformational mobility and relaxivity

M4DOTA, [(2S,5S,8S,11S)-4,7,10-tris-carboxymethyl-2,5,8,11-tetramethyl-1,4,7,10-tetraazacyclododecan-1-yl]acetic acid (2e), and M4DOTMA, (R)-2-[(2S,5S,8S,11S)-4,7,10-tris-((R)-1-carboxyethyl)-2,5,8,11-tetramethyl-1,4,7,10-tetraazacyclododecan-1-yl]propionic acid (3e), are derivatives of ligand DOTA (1e) that form sterically crowded lanthanide chelates. M4DOTMA forms highly symmetric and totally rigid single Y(3+) and Yb(3+) species in which the ring substituents occupy corner positions in a square antiprismatic arrangement as shown by molecular mechanics calculations and by a quantitative interpretation of the relative magnitudes of the paramagnetic (1)H NMR shifts of dipolar origin. The NMR spectrum of YbM4DOTMA(-) displays two intense methyl peaks outside the 0-10 ppm range whose shift difference is strongly temperature dependent. YbM4DOTMA(-) (3d) could be a useful probe in magnetic resonance thermometric imaging. With only four methyl substituents on the tetraaza ring, M4DOTA forms three Yb(3+) species in solution. The methyl substituents prevent the inversion of configuration of the ethylenic groups but not of the acetate arms. Although the methyl groups are likely to preferably occupy ring corner positions, the dipolar equations do not allow one to distinguish with certainty between the two available corner (equatorial) orientations. Reliably applying the dipolar equations is less obvious than usually assumed. A single methyl substituent as in ligand MDOTA (5e) suffices to rigidify the tetraaza cycle but not the acetate arms. Racemic YbMDOTA(-) (5d) is present in solution as four totally asymmetric topomers with the methyl groups occupying either one of the two equatorial positions. A complete assignment of the solution structures on the basis of the dipolar equations is again uncertain. The nuclear magnetic relaxation dispersion curves of the Gd(3+) chelates of all the methylated DOTA ligands including DOTMA, (R)-2-[4,7,10-tris-((R)-carboxyethyl)- 1,4,7,10- tetraazacyclododecan-1-yl]propionic acid, are very similar, and intermolecular conformational processes appear to have no influence on the relaxivity of these small complexes for which the relaxation T(1) is mainly determined by the rotational correlation time (tau(r)). The hydration number of the Tb(3+) chelates measured by fluorescence decreases from DOTMA to M4DOTMA presumably because steric crowding leads to an increase of the metal-water distance.     

Ribonucleic acid as a novel ionophore for potentiometric membrane sensors of some transition metal ions

Ribonucleic acid (RNA) is used as a novel ionophore in plasticized poly(vinyl chloride) matrix membrane sensors for some transition metal ions. Membranes incorporating RNA and doped in Cu(2+), Cd(2+) and Fe(2+) display fast near-Nernstian and stable responses for these ions with cationic slopes of 31.1, 31.3 and 35.5 mV per decade, respectively, over the concentration range 10(-6)-10(-2) M and pH range 4-6.5. The cadmium RNA-based sensor shows no interference by Cu(2+), Fe(2+) Hg(2+) and Ag(+), which are known to interfere significantly with the solid-state CdS/Ag(2)S membrane electrode. The copper RNA-based sensor displays general potentiometric characteristics similar to those based on macrocyclic ionophores and organic ion exchangers and has the advantage of a better selectivity for Cu(2+) over some alkaline earth, divalent and transition metal ions. The iron RNA-based membrane sensor exhibits no interference by Hg(2+) and Zn(2+), which are known to interfere with other previously suggested sensors. The nature and composition of the RNA ionophore and its cadmium complex are examined using electrophoresis, Fourier-transform infrared analysis, elemental analysis and X-ray fluorescence techniques.     

表面活性剂增溶比率荧光Zn2+检测方法研究

合成了一种新的Zn2+荧光检测试剂8-(2-(十八氨基)乙酰氨基)喹啉(AQZ-18)。通过自发荧光的非离子表面活性剂OP-10增溶AQZ-18,获得了一个与Zn2+结合后在320 nm和505 nm分别有两个荧光发射峰的溶液体系。短波长荧光峰来自OP-10,荧光峰强度不随Zn2+浓度变化;长波长荧光峰来自AQZ-18,荧光峰强度随Zn2+浓度增加而增强。利用上述两个荧光峰强度随Zn2+浓度变化时的比值变化建立了一种新的比率荧光Zn2+检测方法。研究表明,Zn2+与AQZ-18形成1∶1型基态配合物,其表观结合常数为1.1×106L/mol。常见金属离子对Zn2+荧光检测无干扰,Zn2+浓度在0～1.1×10-5mol/L范围内与荧光强度变化的比值呈良好的线性关系,相关系数(r2)为0.996 2,检出限为55 nmol/L。该方法可用于水样中Zn2+的检测。