Low-Affinity Zinc Sensor Showing Fluorescence Responses with

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Low-Affinity Zinc Sensor Showing Fluorescence Responses with Minimal Artifacts Xinhao Yan,†,⊥ Jin Ju Kim,‡,⊥ Hye Sun Jeong,§,⊥ Yu Kyung Moon,‡ Yoon Kyung Cho,§ Soyeon Ahn,† Sang Beom Jun,*,§,∥ Hakwon Kim,*,† and Youngmin You*,‡ †

Department of Applied Chemistry, Kyung Hee University, Yongin, Gyeonggi-do 17104, Korea Division of Chemical Engineering and Materials Science, §Department of Electronics Engineering, and ∥Department of Brain and Cognitive Sciences, Ewha Womans University, Seoul 03760, Korea

‡

S Supporting Information *

ABSTRACT: The study of the zinc biology requires molecular probes with proper zinc affinity. We developed a low-affinity zinc probe (HBO−ACR) based on an azacrown ether (ACR) and an 2-(2hydroxyphenyl)benzoxazole (HBO) fluorophore. This probe design imposed positive charge in the vicinity of a zinc coordination center, which enabled fluorescence turn-on responses to high levels of zinc without being affected by the pH and the presence of other transitionmetal ions. Steady-state and transient photophysical investigations suggested that such a high tolerance benefits from orchestrated actions of proton-induced nonradiative and zinc-induced radiative control. The zinc bioimaging utility of HBO−ACR has been fully demonstrated with the use of human pancreas epidermoid carcinoma, PANC-1 cells, and rodent hippocampal neurons from cultures and acute brain slices. The results obtained through our studies established the validity of incorporating positively charged ionophores for the creation of low-affinity probes for the visualization of biometals.

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INTRODUCTION Labile zinc is ubiquitous in the human body, and its homeostasis is governed precisely by the action of several intracellular machineries.1−5 Growing studies evidence that basal levels of labile zinc vary considerably, depending on the types of organs, cells and subcellular organelles.6 For instance, synaptic vesicles at glutamatergic nerve terminals contain several millimolar zinc,7 whereas cytosolic zinc is regulated at as low as ∼10 nM.8,9 This implies that studies on the zinc biology demand a library of sensing tools that can respond to different zinc levels. This is particularly the case for zinc-rich specimens. High-affinity zinc sensors will be saturated in such specimens and, thus, cannot retain the capacity to report zinc fluctuation. Therefore, it is of considerable importance to create sensors with the zinc affinity properly tailored for the locations of interest.10−13 While great advances have been made in the development of fluorescence zinc sensors,14−24 low-affinity zinc probes remain relatively underdeveloped. Several weakly zinc-binding motifs have been utilized for the creation of low-affinity probes, including N,N-di-2-picolylamino derivatives,25−36 N-(2-picolyl)-N-alkylamine,33 N-[ω-(picolylamino)ethyl]aniline,30 N-(2picolyl)-N-(2-thienylmethyl)amine, 37 N-alkyl-N-carboxylamine, 38 N,N-dicarboxylaniline, 39−41 5-hydroxy-6-[8(quinolylamino)methyl]xanthene,42 N-arylsulfonamide,43,44 2picolinamine,45 benzoylhydrizine,46 2,2′-bypyridine,47 1,4,7,10tetraazacyclododecane,48 bipyricorrole,49 and dipyrrin.10 These synthetic modifications were intended to reduce the zinc© XXXX American Chemical Society

binding strength by providing steric hindrance around a zinc coordination center or by substituting hard nitrogen atoms with soft sulfur atoms. It is, however, noted that these approaches inevitably led to poor zinc selectivity27−30,34−36,41,47,48,50−81 and proton-induced false signaling.33,34,36,48,54,56,67,75 We reasoned that the incorporation of a positively charged ionophore vicinal to a zinc receptor could serve as an alternative strategy to creating a low-affinity zinc sensor. To examine this hypothesis, we chose a 2-(2-hydroxyphenyl)benzoxazole (HBO) fluorophore as the sensory unit. The emission properties of HBO have been thoroughly investigated, and previous studies demonstrated that the fluorescence of HBO is highly sensitive to external stimuli.82−93 Merit of the HBO fluorophore is the capability for direct coordination with zinc ions.44,71,89,92,94−100 18-Azacrown-6 ether (ACR) was introduced at the phenyl ring ortho to the hydroxyl group in HBO (Scheme 1). The potassium dissociation constant (Kd) value of ACR was previously determined to be 0.53 mM in methanolic aqueous solutions [CH3OH:water = 1:1 (v/v)].101 It was, thus, envisioned that the ACR unit would readily scavenge alkaline metal ions in the biological milieu, being positively charged.102 Such K+ chelation would impose throughspace Coulombic repulsion on the Zn2+-binding moiety in HBO, potentially leading to low zinc affinity. This synthetic approach is advantageous in that the electronic structure of the Received: November 21, 2016

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DOI: 10.1021/acs.inorgchem.6b02786 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

Scheme 1. Chemical Structures of the Low-Affinity Zinc Sensor (HBO−ACR) and Its Control Compounds (HBO, HBO−ACR′, and HBO−ACR′′)a

a

Colored structures indicate the zinc- (red), potassium- (blue), and sodium-binding (orange) moieties.

the control compounds, HBO−ACR′ and HBO−ACR′′, are described in the Experimental Details. HBO−ACR, HBO− ACR′, HBO−ACR′′, and their precursors were structurally characterized with standard spectroscopic identification techniques, including 1H and 13C NMR and high-resolution mass spectrometry (HR MS). The characterization data were fully consistent with the proposed structures. Spectroscopic data and copies of the NMR spectra are summarized in the Supporting Information (SI), Figures S1−S20. Fluorescence Zinc-Sensing Behaviors. The zinc-sensing properties of HBO−ACR were evaluated in buffered aqueous solutions that were supplemented with 100 mM KCl in order to simulate the biological milieu [pH 7.4, 25 mM piperazineN,N′-bis(2-ethanesulfonic acid) (PIPES), and 100 mM KCl]. HBO−ACR exhibited high solubility in the buffered solutions. Five micromolar HBO−ACR displayed weak green fluorescence emission in the absence of zinc ions [λems = 473 nm and fluorescence quantum yield (Φ) = 0.023]. The emission originated from a phenolate form because the emission spectrum was identical with that of 5 μM HBO−ACR in the presence of a base (200 equiv of Bu4NOH in CH3CN; SI, Figure S21). In addition, the spectrum of HBO−ACR resembled that of 5 μM HBO (i.e., no ACR unit; SI, Figure S22). Henary and Fahrni established that photoexcited HBO becomes more acidic than the ground state, generating phenolate fluorescence.89 A Stokes shift as large as 9300 cm−1 indicates the intramolecular charge-transfer (ICT) nature due to the negative charge. The addition of ZnCl2 provoked prompt fluorescence turn-on responses with a hypsochromic shift (λems = 443 nm; Figure 1a). The fluorescence zinc response entailed a large bathochromic shift of 3300 cm−1 in the UV−vis absorption spectra (SI, Figure S23). The chromic shifts in the fluorescence and UV−vis absorption spectra point to zinc-induced deprotonation of HBO [see SI, Figure S24 and

HBO motif is not altered. In addition, the ACR group can significantly enhance the solubility in aqueous solutions. Herein, we report the design, synthesis, and zinc-sensing properties of a novel fluorescence zinc sensor, HBO−ACR. Our studies revealed that HBO−ACR exhibits low zinc affinity (Kd = 60 μM), while maintaining fluorescence properties favorable for zinc bioimaging: (1) a 10-fold fluorescence turnon, (2) no proton-induced background emission in the range pH 5−11, and (3) zinc responses selective over a competing cadmium ion. Control molecules having the ACR unit at a different position (HBO−ACR′; see Scheme 1 for the structure) or having the 15-azacrown-5 ether,103,104 instead of the 18-azacrown-6 ether (HBO−ACR′′; see Scheme 1 for the structure), were also prepared to support the validity of our strategy. More importantly, we successfully demonstrated the zinc bioimaging utility of HBO−ACR by estimating the basal levels of endogenous zinc within PANC-1, human pancreas epidermoid carcinoma, and also by visualizing neuronal zinc in rodent hippocampal slices and cultured neurons.

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RESULTS AND DISCUSSION Synthesis of HBO−ACR. The low-affinity zinc sensor, HBO−ACR, has been synthesized through a six-step procedure (Scheme 2). Briefly, 2-(2-hydroxy-5-methylphenyl)-1,3-benzoxazole was formylated to 2-(3-formyl-2-hydroxy-5methylphenyl)benzoxazole (1 in Scheme 2) with hexamethylenetetramine in a mixture of toluene and acetic acid. After protection of the hydroxyl group to benzyl ether, the formyl unit was reduced to hydroxymethyl by employing NaBH4. Subsequent chlorination of the terminal hydroxyl group furnished compound 4. The chloride was substituted with 18azacrown-6 ether in the presence of Cs2CO3. Finally, deprotection of the benzyl group with trifluoroacetic acid furnished HBO−ACR in an overall 45% yield. The syntheses of B


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Inorganic Chemistry Scheme 2. Synthetic Routes to HBO−ACR and Its Control Compounds (HBO−ACR′ and HBO−ACR′′)

ions. UV−vis absorption titration results were also consistent with the fluorescence responses (SI, Figure S23). These titration behaviors are indicative of loose zinc binding, while fluorescence Job analysis and an appearance of an isosbestic point at λabs = 347 nm in the UV−vis absorption titration data (SI, Figures S23 and S25) indicated a 1:1 zinc-binding stoichiometry. Nonlinear least-squares fitting of the fluorescence zinc titration data using a 1:1 binding model yielded a Kd value of 60 μM (Figure 1b). This large Kd value can be ascribed to the presence of the positively charged ionophore near the zinc center because control zinc titration experiments performed in KCl-free buffered aqueous solutions (25 mM PIPES, pH 7.4) revealed a 1 order of magnitude decrease in Kd (7.8 μM; SI, Figure S26). MS spectra [electrospray ionization

Table S1, for the time-dependent density functional theory (TD-DFT) calculations]. Although repeated attempts to obtain a crystal structure were unsuccessful, it is reasonable that zinc coordination involves Zn−O of the phenolate and Zn−N of the imino nitrogen atom in HBO. This binding motif is reminiscent of the zinc complex of our previous probe based on 2-(2′hydroxy-3′-naphthyl)benzoxazol.96 The zinc binding led to a 10-fold enhancement in the fluorescence intensity (Φ = 0.23), upon photoexcitation at the isosbestic point observed in the zinc-responsive UV−vis absorption spectra (347 nm). Table 1 lists the photophysical and analytical parameters of HBO−ACR. The fluorescence intensities kept increasing even after the addition of 1 equiv of ZnCl2, and no saturation was observed until 200 equiv of zinc C


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Inorganic Chemistry

Figure S27a]. MS spectra acquired after the subsequent addition of 5 mM ZnCl2 revealed the presence of a heterodinuclear complex of K+ and Zn2+ [m/z values: 301.07 ([ZnK(HBO−ACR−H)]2+); 333.01 ([ZnK(HBO−ACR−H)(CH3OH)2]2+); 337.60 ([ZnK(HBO−ACR−H)(CH3CN)(CH3OH)]2+); SI, Figure S27c]. These results unambiguously point to the presence of a repulsive interaction between K+- and Zn2+-binding moieties in HBO−ACR. Kd values of HBO (the reference compound without the ACR unit) and HBO−ACR′ (the control compound having the ACR unit) were determined to be 5.7 and 59 μM, respectively, which further validated our hypothesis (SI, Figure S28). In addition, the potassium dissociation constants for HBO−ACR and compound 5 (Scheme 2) were determined to be 6.4 and 2.1 mM, respectively (SI, Figure S29). These values are 2 orders of magnitude smaller than the concentration of potassium ion present in our buffer solutions (100 mM), which implies that the major species of our zinc probe is the K+-bound form. As a final proof of our hypothesis, we determined the Kd value of HBO−ACR′′ in buffered aqueous solutions containing 25 mM PIPES and 100 mM NaCl. It was envisaged that the 15azacrown-5 ether unit in HBO−ACR′′ would chelate Na+, exerting the Coulombic repulsion to the zinc coordination center, an effect identical with the K+ binding in HBO−ACR and HBO−ACR′. As expected, HBO−ACR′′ displayed a gradual increase in the fluorescence intensity due to loose zinc binding (SI, Figure S30). A nonlinear least-squares fit to the zinc titration isotherm returned a Kd value of 260 μM. This large Kd value originated from preoccupation of the 15azacrown-5 ether unit by a sodium ion. Taken together, the zinc titration experiments revealed antagonistic effects of the Na+- or K+-coordinated azacrown ether units upon zinc binding. The zinc responses were fully reversible, as demonstrated by the complete recovery of the fluorescence and UV−vis absorption spectra after the subsequent addition of a strong zinc chelator, N,N,N′,N′-tetrakis(2-picolyl)ethylenediamine (TPEN; SI, Figure S31). HBO−ACR displayed fluorescence turn-on responses exclusively to zinc ions. As shown in Figure 2, the zinc-sensing capability was not influenced by the presence of biologically abundant metal ions, including sodium (1.0 mM), magnesium (100 μM), potassium (100 mM), and calcium (1.0 mM). Biologically important transition-metal ions, such as chromium, manganese, iron, cobalt, and nickel, did not intervene the zinc responses, while cupric ions abrogated the zinc-induced fluorescence turn-on signaling. Silencing by cupric ions is commonly observed for zinc sensors and is consistent with the Irving−Williams series. Nevertheless, this cupric interference will not interfere with the zinc-sensing capability because the intracellular levels of free cupric ions are extremely low.105 Of particular advantage of HBO−ACR is the zinc selectivity over Cd2+ and Hg2+. These metal ions did not affect the fluorescence emission of HBO−ACR, but the subsequent addition of ZnCl2 to the mixture solution immediately turned on fluorescence. The absence of fluorescence responses to Cd2+ and Hg2+ indicates that these metal ions are not bound to HBO−ACR in the PIPES buffer solution. The high selectivity toward zinc binding was retained in aqueous buffers devoid of KCl (SI, Figure S32), which indicated an insignificant role of the [K(ACR)] ionophore in the zinc selectivity. Similar fluorescence zinc selectivity was observed for HBO and HBO− ACR′ in buffers containing 100 mM KCl, further supporting this notion (SI, Figure S33). The capability of discriminating

Figure 1. Fluorescence zinc titration of HBO−ACR. (a) Fluorescence spectra (λex = 347 nm) of 5.0 μM HBO−ACR with an increase in the concentration of ZnCl2 (0−1.0 mM). (b) Corresponding fluorescence zinc titration isotherm (black squares) and a nonlinear least-squares fit (red curve) to a 1:1 binding model (top panel) and the residuals (bottom panel). Note that the isotherm is a function of the calculated concentrations of free zinc ions ([Zn2+]free). Refer to the Experimental Details for the mathematical equations and details of the analysis. The error bars are obtained after three independent measurements. Conditions: air-equilibrated buffer solutions containing 25 mM PIPES and 100 mM KCl (pH 7.4).

Table 1. Photophysical Data for HBO−ACRa

zinc-free +50 equiv ZnCl2

λabs,b nm (log ε)

λems,c nm

Φd

τobs,e ns

kr (106 s−1)f

knr (108 s−1)g

328 (2.98) 368 (2.88)

473 443

0.023 0.23

4.7 4.9

4.9 48

2.1 1.6

5 μM in air-equilibrated aqueous buffer solutions containing 25 mM PIPES and 100 mM KCl (pH 7.4). bAbsorption peak wavelength. c Fluorescence peak wavelength. dFluorescence quantum yield determined relatively by using 9,10-diphenylanthracene as a standard. e Fluorescence decay lifetimes. fRadiative rate constant. gNonradiative rate constant. a

(ESI), positive mode] taken for aqueous solutions of 100 μM HBO−ACR and 1.0 mM KCl indicated the presence of a K+bound form [m/z values: 539.22 ([K(HBO−ACR)]+; SI, D


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Inorganic Chemistry

fluorescence intensity decreased with decreasing pH, a behavior opposite to typical zinc sensors. Actually, the Φ value (0.0096) measured at pH 4.1 was about half that (0.023) obtained at pH 7.4. The significant attenuation of the fluorescence intensities offers great advantages in detecting labile zinc in the acidic milieu. To investigate the photophysical mechanism, we determined the fluorescence lifetime (τobs) of HBO−ACR by employing time-correlated single-photon-counting (TCSPC) techniques after picosecond-pulsed laser excitation at 345 nm. τobs was 0.59 ns in the absence of zinc at pH 4.1, being 1 order of magnitude shorter than the τobs value (4.7 ns) at pH 7.4 (SI, Figure S35 and Table S2). A comparison of the nonradiative decay rate constants (knr), which were calculated by the relationship knr = (1 − Φ)/τobs, revealed a 1 order of magnitude increase in knr at low pH (knr = 1.7 × 109 s−1 at pH 4.1 vs 2.1 × 108 s−1 at pH 7.4; SI, Table S3). The results suggest that nonradiative processes would become predominant at low pH values. To support this hypothesis, we compared the fluorescence intensities of HBO−ACR in acidic solutions of different viscosities at 25 °C (5.0 μM HBO−ACR and 0.1 M HCl). Methanol, ethylene glycol, and glycerol with viscosities of 0.593, 16.9, and 1070 cP, respectively, were employed for this purpose. As anticipated, the fluorescence intensities increased linearly with the viscosity (SI, Figure S36). On the contrary, such fluorescence increases were not observed in the absence of HCl or in the presence of 1.0 mM Bu4NOH. This pH-gated rigidochromic behavior indicates that protonation facilitates nonemissive motional relaxation, leading to fluorescence turnoff. Zinc-induced (50 equiv) fluorescence turn-on properties were maintained over a broad pH range (Figure 3). Actually, the fluorescence turn-on at pH 4.7 was still ∼3-fold. The pKa value was determined to be 5.9 in the presence of zinc ions. This value is smaller than that for the zinc-free form, indicating that the zinc-bound form is less basic due to zinc coordination. Contrary to the nonradiative control by protonation, zincinduced fluorescence turn-on results from radiative control. The τobs values were unaffected by zinc coordination at pH 7.4 (4.7 and 4.9 ns for the zinc-free and zinc-bound forms, respectively), but the increase in Φ led to 1 order of magnitude elongation of the radiative rate constant (kr) from 4.9 × 106 s−1 (zinc-free form) to 4.8 × 107 s−1 (zinc-bound form). One possible mechanism for the increase in kr is zinc-induced rigidification of the HBO scaffold. On the contrary, knr remained almost the same (1.6 × 108 s−1 for the zinc-bound form vs 2.1 × 108 s−1 for the zinc-free form). Taking the photophysical results into account, it is concluded that the high proton tolerance of the fluorescence zinc responses of HBO− ACR is due to the synergy of protonation-induced nonradiative and zinc-coordiation-induced radiative control. Scheme 3 depicts the plausible mechanism for the photophysical processes of HBO−ACR in aqueous solutions. In the absence of zinc ions, there are three proton-equilibrated species of the potassium-bound form of HBO−ACR: [K(HBO−ACR−H)], [K(HBO−ACR)], and [KH(HBO−ACR)]. [K(HBO−ACR− H)] and [KH(HBO−ACR)] are weakly emissive. Excited-state intramolecular proton transfer does not occur in aqueous solutions because of excited-state deprotonation. Nonradiative deactivation by twisted ICT cannot be ruled out for the deprotonated form.109−111 Zinc binding of [K(HBO−ACR− H)] or [K(HBO−ACR)] generates the highly fluorescent [ZnK(HBO−ACR−H)]. Zinc coordination at HBO is stronger

Figure 2. Fluorescent zinc selectivity of 5 μM HBO−ACR: blue bars, metal-free state; red bars, in the presence of metal ions; black bars, after the subsequent addition of 50 μM ZnCl2. Na+ and Ca2+ ions are 1.0 mM. The Mg2+ ion is 100 μM. Other divalent metal ions are 10 μM. Chloride salts were used. Conditions: air-equilibrated, buffered aqueous solutions containing 25 mM PIPES and 100 mM KCl (pH 7.4), with λex = 350 nm. Fluorescence spectra are integrated over the range λems = 380−620 nm.

zinc over cadmium is particularly appealing and has rarely been observed among biologically applicable zinc sensors.26,42,45,46,106−108 Another advantage of HBO−ACR is proton tolerance of the fluorescence zinc responses. Figure 3 depicts the fluorescence

Figure 3. pH titration isotherm of 50 μM HBO−ACR (airequilibrated Milli-Q water containing 100 mM KCl), which plots the integrated photoluminescence intensities in the absence (empty triangles) and presence (filled triangles) of 2.5 mM ZnCl2 as a function of the pH values. The curves are nonlinear least-squares fits to the relationship photoluminescence intensity = [HBO−ACR]total(A·Ka + B[H+])/([H+] + Ka), where [HBO−ACR]total is the total concentration of HBO−ACR and A and B are proportionality constants. See SI, Figure S34, for the full spectral evolution.

titration isotherms obtained for 50 μM HBO−ACR with decreasing pH from 10.5 to 3.1 in the absence and presence of 50 equiv of ZnCl2 (air-equilibrated Milli-Q water containing 100 mM KCl; see SI, Figure S34, for the spectra). The fluorescence titration isotherm for the zinc-free form displays a typical sigmoidal behavior with a pKa value of 8.1. This pKa value is identical with that obtained from the UV−vis absorption data (SI, Figure S34), which corresponds to protonation of the phenolate in HBO. It is notable that the E


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Inorganic Chemistry Scheme 3. Plausible Mechanism for the Photophysical Processes of HBO−ACR in Aqueous Solutionsa

a

Equilibria with potassium-free forms are not included.

Colocalization experiments with the use of organelle-specific fluorescence stains, including DRAQ5 (nucleus stain), ER− Tracker (ER stain), MitoTracker (mitochondrion stain), and LysoTracker (lysosome stain), revealed localization of HBO− ACR within lysosomes (overlap coefficient = 0.69; SI, Figure S39). Such lysosomal enrichment was likely due to the presence of the basic diethylamino moiety in ACR.108,113−119 Because lysosomes maintain pH values as low as ∼5, the lysosomal localization may provide unique opportunities to examine the capability of our probe for fluorescence zinc imaging. In order to estimate the basal levels of labile zinc in PANC-1 cells, we treated the cells with HBO−ACR and a high-affinity zinc sensor, HNBO−DPA, which was established by us.96 It is notable that zinc concentrations in PANC-1 cells have not been determined to date. The Kd value of HNBO−DPA is 12 pM, being 6 orders of magnitude smaller than that of HBO−ACR. The fluorescence emissions of the two sensors could be spectrally well resolved. Figure 4a depicts fluorescence micrographs recorded through 450 and 581 nm channels, which correspond to the HBO−ACR and HNBO−DPA emission regions, respectively. The HBO−ACR emission is considerably weaker than the HNBO−DPA emission. Such weak fluorescence intensities of HBO−ACR may be due to decreased zinc levels by preferential zinc scavenging of HNBO−DPA. Subsequent treatment of the cells with 100 μM zinc pyrithione (ZnPT) provoked enhancement in the HBO−ACR fluorescence, while that of HNBO−DPA was relatively intact. These fluorescence responses can be

than protonation at the benzoxazole moiety, as evidenced by the smaller pKd(Zn) (4.2) than pKa (8.1). This tighter interaction with zinc ions over protons enables fluorescence zinc detection, without suffering from protonation over the broader pH range. Zinc Bioimaging. Finally, the zinc bioimaging utility of our probe has been demonstrated by the visualization of zinc in human pancreatic cells and rodent hippocampal slices and primary cultured neurons. PANC-1 cells, human pancreas epidermoid carcinoma, were chosen for zinc bioimaging because the pancreas is known to contain high levels of labile zinc.112 The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C under a 5% CO2 atmosphere. Ten micromolar HBO−ACR (dimethyl sulfoxide, DMSO) was added into the medium, and the cells were incubated for 15 min. Fluorescence micrographs (λex = 405 nm and λems = 400−700 nm) revealed that HBO−ACR readily permeated PANC-1 cells. Subsequent treatment of the cells with 100 μM ZnCl2/sodium pyrithione promptly turned on the fluorescence intensity (SI, Figure S37). Finally, a huge decrease in the fluorescence emission was observed upon the addition of 100 μM TPEN, confirming that fluorescence signaling was due to zinc binding. The treated cells were viable throughout the microscopic experiments, suggesting that the phototoxicity was minimal. MTT assays indicated that 0.1−50 μM HBO−ACR did not influence the viability of the PANC-1 cells (2 h of incubation at 37 °C and 5% CO2; SI, Figure S38). F


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Figure 4. Intracellular zinc imaging of PANC-1 cells coincubated with the low-affinity zinc sensor, HBO−ACR, and the high-affinity zinc sensor, HNBO−DPA. (a) Fluorescence microscope images detected through a 450 nm emission channel that recorded the HBO−ACR emission (excitation at 405 nm; top panels) and a 581 nm emission channel that recorded the HNBO−DPA emission (excitation at 405 nm; middle panels) before (left panels) and after (middle panels) treatment with 100 μM ZnPT (10 min), followed by the subsequent addition of 100 μM TPEN (right panels; 5 min). The bottom panels are ratio images that were constructed by dividing the HBO−ACR (top) images with the HNBO−DPA (middle) images. Scale bar = 100 μm. (b) Corresponding changes in the fluorescence intensity ratios (i.e., 450 nm/581 nm) of the PANC-1 cells. (c) Photoluminescence spectra reconstructed from the fluorescence emissions of PANC-1 cells treated with 10 μM HBO−ACR (red) or 10 μM HNBO−DPA (blue).

Figure 5. Zinc imaging of hippocampal cultured neurons treated with 10 μM HBO−ACR. (a) Wide-field image. Fluorescence micrographs (λex = 350 nm and λems > 400 nm) before (b) and after (c) the addition of 50 μM ZnPT. Scale bar = 100 μm.

rationalized by the different Kd values; the high-affinity zinc probe HNBO−DPA was already saturated because of the basal level of zinc ions in PANC-1. Thus, the supply of exogenous zinc could not produce additional fluorescence enhancement. On the contrary, the low-affinity zinc probe HBO−ACR remained as the zinc-free form in the cells, and the escalation of intracellular zinc concentrations by the ZnPT treatment led to fluorescence turn-on. Further treatment of the cells with 100 μM TPEN reduced the fluorescence intensities of both HBO− ACR and HNBO−DPA, with the former being more affected. On the basis of the results, the basal zinc level of PANC-1 can be estimated to be within the range 12 pM to 60 μM. As a final demonstration of our study, we attempted fluorescence visualization of neuronal zinc in rodent hippocampal neurons and slices. Among a variety of neurotransmitters, glutamate is the major excitatory neurotransmitter in the central nervous system. It has been known that labile zinc exists at high levels in the glutamatergic synaptic vesicles.

However, the role of synaptic zinc has remained unclear until recent studies revealed that endogenous synaptic zinc is crucial for proper neuronal signaling.120 Primary hippocampal neurons harvested from the brain of an embryonic 18-day-gestation Sprague−Dawley (SD) rat were grown in serum-free media for 2 weeks. The cultured neurons were incubated in artificial cerebrospinal fluid (ACSF) with 10 μM HBO−ACR for 30 min at DIV 14, after which fluorescence imaging experiments were performed (Figure 5). The acquired fluorescence micrographs (λex = 350 nm and λems > 400 nm) revealed that HBO−ACR permeated into the cultured hippocampal neurons. An exogenous supply of zinc in the form of ZnPT provoked prompt enhancement in the fluorescence intensity of the neurons. On the basis of the results, the basal zinc level in the cultured hippocampal neurons can be estimated as much lower than 60 μM. This estimation was consistent with the previous research, which predicted ∼10 nM resting zinc levels in neurons.121 G


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Figure 6. Fluorescence zinc imaging of the CA1, CA3, and DG regions of hippocampal slices coincubated with the low-affinity zinc sensor, HBO− ACR, and the high-affinity zinc sensor, HNBO−DPA. Fluorescence microscope images were obtained through a 450 nm emission channel that recorded the HBO−ACR emission (λex = 405 nm; top green panels) and a 581 nm emission channel that recorded the HNBO−DPA emission (λex = 405 nm; middle orange panels) before (left panels) and after (right panels) treatment with 100 μM TPEN (10 min). Bottom panels are ratio images that were constructed by dividing the intensity of the 581 nm images with the intensity of the 450 nm images. Scale bar = 200 μm.

It is well-known that, among the hippocampal subregions, the CA1 and CA3 regions are densely populated with glutamatergic neurons. Therefore, there is an area-dependent zinc level gradient in the hippocampal area.122,123 In order to estimate the basal zinc levels in the hippocampal regions, we treated hippocampal slices from adult C57BL/6 mice (male, 9 weeks old) with 10 μM HBO−ACR and 10 μM HNBO−DPA. Fluorescence micrographs were recorded through 450 and 581 nm channels, which correspond to HBO−ACR and HNBO− DPA fluorescence, respectively. As shown in Figure 6, the HNBO−DPA emission intensity is considerably stronger than the HBO−ACR emission. In analogy with the PANC-1 cell results (Figure 4), such fluorescence responses can be rationalized by the different Kd values; the high-affinity zinc probe HNBO−DPA was already saturated by the basal level of zinc ions in CA1, CA3, and DG. Moderate fluorescence of the low-affinity zinc probe HBO−ACR was clearly observed in the CA3 and DG subregions but not in the CA1 area. The results reveal that CA3 and DG contain higher levels of zinc than CA1. This result is consistent with the previous study, which reported that the mossy fibers between the DG and CA3 regions contain distinctive amounts of zinc.124 Subsequent treatment with 100 μM TPEN reduced the fluorescence intensities of both HBO− ACR and HNBO−DPA, confirming that the fluorescence responses were due to zinc binding. Although the results successfully demonstrated the feasibility of the developed zinc sensors for neuronal cells, the exact intracellular distribution of the fluorescence signals have not been identified yet. Therefore, in order to investigate the origin of zinc sensor fluorescence signals, fluorescence images with a higher magnification were obtained. According to the previous literature, it is known that the concentration of labile zinc in the intracellular and extracellular fluids of neurons are below the detection limits of conventional analytical techniques.125

Rather, several studies revealed the existence of zinc ions in presynaptic vesicles.126−128 The imaging with a higher magnification showed that the zinc fluorescence signals from both HBO−ACR and HNBO−DPA have the punctated forms in the proximity of neuronal cell bodies, as shown in Figure 7. This indicates that the sensors permeated both the neuronal and vesicular membranes and detected the zinc ions existing presumably in the presynaptic vesicles inside the presynaptic boutons. This observation is consistent with the previous study, which directly showed concentrated zinc ions in a single presynaptic bouton.127

Figure 7. High-magnification fluorescence micrographs of the CA region of a rat brain slice treated with HBO−ACR, HNBO−DPA, and DRAQ5: (a) HBO−ACR, (b) HNBO−DPA, (c) DRAQ5, and (d) an overlay image. Scale bar = 10 μm. H


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Inorganic Chemistry

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135.8, 134.1, 132.8, 130.4, 128.4, 128.3, 128.1, 125.0, 124.4, 120.2, 119.9, 110.4, 76.8, 60.3, 20.6. MS (ESI). Calcd for C22H20NO3 ([M + H]+): m/z 346.39. Found: m/z 346.2. 2-[2-(Benzyloxy)-3-(chloromethyl)-5-methylphenyl]benzo[d]oxazole (4). To a stirred CH2Cl2 solution (2 mL) of 3 (100 mg, 0.29 mmol) was added thionyl chloride (0.3 mL, 4.6 mmol) at 0 °C. The reaction mixture was stirred at 70 °C for 1 h and cooled to room temperature. Concentration in vacuo afforded compound 4 (98 mg, 93%) as a white solid, which was used in the next reaction without additional purification. Mp: 127−128 °C. Rf: 0.62 (EtOAc:hexane = 1:6). 1H NMR (300 MHz, CDCl3): δ 8.03 (d, 1H, J = 1.6 Hz), 7.84− 7.81 (m, 1H), 7.56−7.53 (m, 3H), 7.44 (d, 1H, J = 1.6 Hz), 7.42−7.35 (m, 5H), 5.07 (s, 2H), 4.69 (s, 2H), 2.43 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 160.9, 153.8, 150.5, 141.8, 136.7, 134.8, 134.5, 132.7, 132.1, 128.6, 128.4, 128.3, 125.3, 124.5, 121.1, 120.1, 110.5, 76.6, 40.8, 20.6. MS (ESI). Calcd for C22H19ClNO2 ([M + H]+): m/z 364.84. Found: m/z 364.1. 2-[3-[(1,4,7,10,13-Pentaoxa-16-azacyclooctadecan-16-yl)methyl]-2-(benzyloxy)-5-methylphenyl]benzo[d]oxazole (5). To a stirred solution of 1-aza-18-crown-6-ether (80 mg, 0.3 mmol) and anhydrous Cs2CO3 (180 mg, excess) in dry CH3CN (10 mL) was added 4 (109 mg, 0.3 mmol). The reaction mixture was refluxed for 10 h. After cooling to room temperature, the residual cesium salts were filtered off. Column chromatography (CH2Cl2:EtOH = 10:1) on silica gel afforded the desired product 5 (117 mg, 66%) as a yellow oil. Rf: 0.42 (CH2Cl2:CH3OH = 10:1). 1H NMR (300 MHz, CDCl3): δ 7.89 (s, 1H), 7.78−7.75 (m, 1H), 7.54 (s, 1H), 7.47−7.45 (m, 3H), 7.35− 7.31 (m, 5H), 4.94 (s, 2H), 3.74 (s, 2H), 3.66−3.63 (m, 13H), 3.60− 3.59 (m, 7H), 2.80 (t, 4H, J = 5.6 Hz), 2.40 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 161.67, 154.03, 150.49, 141.80, 137.13, 134.65, 134.58, 133.78, 130.06, 128.36, 128.20, 127.89, 124.88, 124.27, 120.48, 119.90, 110.40, 76.79, 70.76, 70.65, 70.24, 69.74, 53.85, 53.66, 20.76. MS (ESI). Calcd for C34H43N2O7 ([M + H]+): m/z 591.71. Found: m/z 592.0. HBO−ACR. A solution of trifluoroacetic acid (5 mL) containing 5 (90 mg, 0.15 mmol) was refluxed for 18 h. The reaction mixture was concentrated in vacuo and subjected to silica gel column chromatography (CH2Cl2:EtOH = 10:1). HBO−ACR was obtained as a solid (64 mg, 85%). Mp: 75−76 °C. Rf: 0.40 (CH2Cl2:CH3OH = 10:1). 1H NMR (300 MHz, CDCl3): δ 7.78 (s, 1H), 7.75−7.72 (m, 1H), 7.61−7.58 (m, 1H), 7.39−7.34 (m, 2H), 7.27 (s, 1H), 3.88 (s, 2H), 3.74−3.64 (m, 20H), 2.92 (t, 4H, J = 5.2 Hz), 2.35 (s, 3H). 13C NMR (75 MHz, (CD3)2CO): δ 162.28, 161.83, 149.92, 140.25, 130.29, 129.68, 127.07, 126.35, 119.88, 115.94, 111.73, 111.43, 70.89, 70.13, 69.89, 66.50, 53.18, 20.21. HR MS (ESI). Calcd for C27H37N2O7 ([M + H]+): m/z 501.2599. Found: m/z 501.2599. 2-[3-[(1,4,7,10-Tetraoxa-13-azacyclopentadecan-13-yl)methyl]2-(benzyloxy)-5-methylphenyl]benzo[d]oxazole (6). To a stirred dry CH3CN solution (6.0 mL) containing 1-aza-15-crown-5-ether (30 mg, 0.14 mmol) and anhydrous Cs2CO3 (90 mg, excess) was added 4 (50 mg, 0.14 mmol). The reaction mixture was refluxed for 10 h. The solution was cooled to room temperature, and then residual salts were filtered off. Column chromatography (CH2Cl2:EtOH = 10:1) on silica gel afforded the desired product 6 (50 mg, 66%) as a colorless oil. Rf: 0.40 (CH2Cl2:CH3OH = 10:1). 1H NMR (300 MHz, CDCl3): δ 7.98 (s, 1H), 7.86−7.83 (m, 1H), 7.64−7.60 (m, 1H), 7.51−7.49 (m, 2H), 7.48−7.34 (m, 6H), 4.89 (s, 2H), 3.75−3.73 (m, 6H), 3.59−3.39 (m, 12H), 2.70 (s, 4H,), 2.45 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 160.68, 153.76, 150.42, 141.77, 141.74, 136.45, 135.01, 131.60, 129.36, 129.07, 128.89, 125.77, 124.88, 120.91, 120.87, 120.11, 110.61, 77.59, 77.58, 77.16, 76.74, 68.73, 68.36, 67.35, 55.60, 54.25, 20.64. HR MS (ESI). Calcd for C32H39N2O6 ([M + H]+): m/z 546.2733. Found: m/z 546.2733. HBO−ACR″. A trifluoroacetic acid (5.0 mL) containing 6 (50 mg, 0.09 mmol) was refluxed for 18 h. The reaction mixture was concentrated in vacuo and subjected to silica gel column chromatography (CH2Cl2:EtOH = 5:1). HBO−ACR″ was obtained as a colorless oil (34 mg, 83%). Rf: 0.26 (CH2Cl2:CH3OH = 10:1). 1H NMR (300 MHz, (CD3)2CO): δ 7.84−7.77 (m, 3H), 7.52−7.47 (m, 3H), 3.93 (s, 2H), 3.74−3.66 (m, 16H), 2.93 (s, 4H), 2.39 (s, 3H).

SUMMARY AND CONCLUSIONS We developed a fluorescence low-affinity zinc probe, HBO− ACR. HBO−ACR was created by introducing an azacrown ether ionophore into an HBO fluorophore. The azacrown ether unit harvested potassium ions, being positively charged. This modification resulted in several advantages in fluorescence zinc signaling; the ionophore imposed Coulombic repulsion on the zinc-binding unit, which led to low zinc affinity (Kd = 60 μM). Steady-state and transient photophysical studies revealed that the high proton tolerance was due to a synergy of nonradiative and radiative controls. Finally, the capability of HBO−ACR for fluorescence zinc visualization has been successfully demonstrated for human pancreas cells and rodent hippocampal slices and cultured neurons. Despite the utility, our probe carries a disadvantage due to near-UV excitation (∼350 nm). This drawback can be overcome by utilization of low-band-gap fluorophores.

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EXPERIMENTAL DETAILS

Materials and Synthesis. Commercially available chemicals were used as received. All glassware and magnetic stir bars were thoroughly dried in a convection oven. Reactions were monitored using thin-layer chromatography (TLC). Commercial TLC plates (silica gel 254, Merck Co.) were developed, and the spots were visualized under UV light at 254 or 365 nm. Silica gel column chromatography was performed with silica gel 60 G (particle size 0.063−0.200 mm, Merck Co.). 1H and 13C NMR spectra were collected with a JEOL ECX-400 (300 MHz for 1H NMR and 75 MHz for 13C NMR) spectrometer, and the chemical shifts were recorded with respect to tetramethylsilane [Si(CH3)4] as an internal reference or referenced to residual proton peaks of the deuterated solvent. Mass spectrometry (MS) spectra were recorded using an Agilent Technologies 6120 quadrupole liquid chromatograph/mass spectrometer. 3-(Benzo[d]oxazol-2-yl)-2-(benzyloxy)-5-methylbenzaldehyde (2). Compound 1 was prepared following the procedure reported previously.129 1 (500 mg, 1.98 mmol), benzyl bromide (507 mg, 2.97 mmol), and Cs2CO3 (965 mg, 2.97 mmol) were dissolved in CH3CN (8 mL). The mixture was stirred at 80 °C for 5 h. The residual cesium salts were filtered off, and the solution was concentrated under reduced pressure. The crude product was extracted with EtOAc (×3), and the combined organic extracts were washed with brine (×2), dried over anhydrous MgSO4, and concentrated in vacuo. The crude product was purified by column chromatography (EtOAc:hexane = 1:6) on silica gel to afford 2 (598 mg, 88%) as a white solid. Mp: 136− 138 °C. Rf: 0.56 (EtOAc:hexane = 1:6). 1H NMR (300 MHz, CDCl3): δ 10.27 (s, 1H), 8.53 (s, 1H), 7.85−7.81 (m, 2H), 7.59−7.56 (m, 1H), 7.42−7.35 (m, 7H), 5.11 (s, 2H), 2.46 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 189.38, 160.14, 158.47, 150.58, 141.75, 137.48, 135.49, 134.79, 131.61, 130.89, 128.92, 128.75, 128.61, 125.60, 124.76, 121.98, 120.35, 110.65, 79.49, 20.65. MS (ESI). Calcd for C22H18NO3 ([M + H]+): m/z 344.38. Found: m/z 344.1. [3-(Benzo[d]oxazol-2-yl)-2-(benzyloxy)-5-methylphenyl]methanol (3). To a solution of 2 (100 mg, 0.29 mmol) in CH3OH (4 mL) was added NaBH4 (23 mg, 0.58 mmol) portionwise over 10 min at 0 °C. After gas evolution ceased, the reaction mixture was warmed to room temperature and stirred for an additional 30 min. The reaction mixture was concentrated, and the residue was partitioned between H2O (10 mL) and EtOAc (50 mL). The organic layer was recovered, and the crude product in the aqueous layer was extracted with EtOAc (×2). The combined organic extracts were dried over anhydrous MgSO4, filtered, and concentrated to afford 3 (97 mg, 97%) as a white solid. 3 was used for the next reaction without further purification. Mp: 129−131 °C. Rf: 0.13 (EtOAc:hexane = 1:6). 1H NMR (300 MHz, CDCl3): δ 7.98 (d, 1H, J = 1.6 Hz), 7.83−7.80 (m, 1H), 7.58−7.54 (m, 1H), 7.47−7.45 (m, 2H), 7.40−7.35 (m, 6H), 5.00 (s, 2H), 4.67 (d, 2H, J = 6.2 Hz), 2.43 (s, 3H), 2.02 (t, 1H, J = 6.3 Hz). 13C NMR (75 MHz, CDCl3): δ 161.2, 153.2, 150.3, 141.6, 136.6, I


Article

Inorganic Chemistry

(99.99%, Aldrich), CaCl2 (99.99%, Aldrich), CrCl3·6H2O (98%, Aldrich), MnCl2 (99.99%, Aldrich), FeCl2 (99.99%, Aldrich), CoCl2 (99.9%, Aldrich), NiCl2 (99.99%, Aldrich), CdCl2 (99.99%, Aldrich), and ZnCl2 (99.999%, Aldrich). A TPEN solution was prepared by dissolving TPEN (≥99%, Sigma) in DMSO (99.9%, Aldrich). Zn(ClO4)2·6H2O (Aldrich) was dissolved in CH3CN (spectrophotometric grade, Aldrich) to 1.0, 10, and 100 mM concentrations. HBO− ACR solutions were prepared by dissolution in DMSO to a concentration of 10 mM and partitioned into Eppendorf tubes. The sensor stock solution was stored in a refrigerator at −10 °C and thawed before measurement. A total of 3.0 mL of the buffer and 3 μL of the sensor stock solution (10 mM in DMSO) were mixed to give a 10 μM solution. A 1 cm × 1 cm fluorimeter cell (Hellma) was used for the steady-state photophysical measurements. UV−vis absorption spectra were collected on a Varian Cary 300 spectrophotometer at room temperature. Photoluminescence spectra were obtained using a Quanta Master 400 scanning spectrofluorimeter at room temperature. The photoluminescence quantum yields (Φ) were relatively determined according to the following standard equation: Φ = Φref(I/Iref)(Aref/A)(n/nref)2, where A, I, and n are the absorbance at the excitation wavelength, integrated photoluminescence intensity, and refractive index of the solvent, respectively. 9,10-Diphenylanthracene in a deaerated toluene solution was used as the external reference (Φref = 1.00). Photoluminescence decay traces were acquired based on TCSPC techniques using a FluoTime 200 instrument (PicoQuant, Germany). A 377 nm diode laser (PicoQuant, Germany; pulse energy = 35 pJ) with a repetition rate of 125 kHz was used as the excitation source. The signals at 456 and 493 nm were obtained using an automated motorized monochromator and recorded with a NanoHarp unit. The decay profiles were analyzed (OriginPro 8.0, OriginLab) using a single- or double-exponential decay model. Determination of Kd. An approach previously reported by us was used to determine the Kd values for zinc binding to HBO−ACR, HBO−ACR′, and HBO−ACR′′. An equilibrium model for the formation of a 1:1 ligand/metal complex yields the following equations for the photoluminescence intensity (eq 1) and the free zinc concentration (eq 2):

C NMR (75 MHz, (CD3)2CO): δ 164.13, 156.13, 150.27, 140.97, 137.35, 129.87, 129.54, 127.48, 126.70, 126.21, 120.08, 111.73, 111.15, 71.43, 70.83, 70.53, 70.36, 69.20, 68.98, 54.80, 53.99, 20.48. HR MS (ESI). Calcd for C25H33N2O6 ([M + H]+): m/z 456.2260. Found: m/z 456.2262. 2-(Benzo[d]oxazol-2-yl)-4-methylphenyl acetate (7). Compound 1′ was prepared according to the previous method.129 1′ (160 mg, 0.46 mmol) was dissolved in 2 mL of acetic anhydride. A total of 1 mL of pyridine was added, and the mixture was stirred at room temperature for 2 h. The crude solution was diluted in 30 mL of dichloromethane and washed with 0.1 N HCl(aq) and water. The organic layer was recovered, dried over anhydrous MgSO4, and concentrated in vacuo to yield compound 7 quantitatively. Mp: 139−141 °C. Rf: 0.26 (EtOAc:hexane = 1:10). 1H NMR (300 MHz, CDCl3): δ 8.11 (d, 1H, J = 1.8 Hz), 7.76−7.73 (m, 1H), 7.56−7.53 (m, 1H), 7.38−7.34 (m, 3H), 7.11 (d, 1H, J = 8.3 Hz), 2.47 (s, 3H), 2.45 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 169.86, 159.75, 149.88, 146.86, 141.63, 135.98, 132.88, 130.24, 125.09, 124.29, 123.56, 119.98, 119.58, 110.13, 21.04, 20.54. MS (ESI). Calcd for C16H14NO3 ([M + H]+): m/z 267.28. Found: m/z 268.1. 2-(Benzo[d]oxazol-2-yl)-4-(bromomethyl)phenyl acetate (8). NBromosuccinimide (120 mg, 0.67 mmol) and benzoyl peroxide (32 mg, 3.30 mmol) were added into a 5 mL CCl4 solution containing 7 (120 mg, 0.44 mmol). The reaction mixture was refluxed for 24 h and then cooled to room temperature. After filtration, the filtrate was concentrated in vacuo. The concentration solution was redissolved in CH2Cl2, washed with water, dried over anhydrous MgSO4, and concentrated through vacuum. Column chromatography on silica gel (EtOAc:hexane = 1:30) yielded compound 8 as a white solid (61 mg, 40%). Mp: 146−147 °C. Rf: 0.28 (EtOAc:hexane = 1:6). 1H NMR (300 MHz, CDCl3): δ 8.33 (d, 1H, J = 2.0 Hz), 7.77−7.74 (m, 1H), 7.60−7.55 (m, 2H), 7.41−7.34 (m, 2H), 7.21 (d, 1H, J = 8.3 Hz), 4.56 (s, 2H), 2.49 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 169.65, 159.07, 149.97, 148.89, 141.59, 136.10, 132.84, 130.59, 125.54, 124.59, 124.56, 120.51, 120.24, 110.37, 31.68, 21.19. MS (ESI). Calcd for C16H13BrNO3 ([M + H]+): m/z 346.18. Found: m/z 348.0. HBO−ACR′. To a stirred solution of dry CH3CN (10 mL) containing 1-aza-18-crown-6-ether (50 mg, 0.18 mmol) and anhydrous Cs2CO3 (110 mg, 0.35 mmol) was added 8 (61 mg, 0.18 mmol). The reaction mixture was refluxed for 24 h. After cooling, the reaction mixture was filtered to remove residual cesium salts and concentrated in vacuo. The crude product 9 was dissolved in methanol, to which hydrazine hydrate (0.1 mL, excess) was added. The reaction mixture was stirred for 30 min at room temperature. Neutralization with 1 N HCl(aq) was performed, and the solution was concentrated. The crude product was extracted with EtOAc (×3), and the combined organic extracts were washed with brine (×2), dried over anhydrous MgSO4, and concentrated in vacuo. Silica gel column chromatography (CH2Cl2:EtOH = 10:1) was subsequently carried out to afford HBO− ACR′ (24 mg, 27%) as a sticky oil. Rf: 0.40 (CH2Cl2:CH3OH = 10:1). 1 H NMR (300 MHz, CDCl3): δ 7.96 (s, 1H), 7.73−7.70 (m, 1H), 7.63−7.60 (m, 1H), 7.43−7.36 (m, 3H), 7.06 (d, 1H, J = 8.4 Hz), 3.70−3.64 (m, 22H), 2.83 (t, 4H, J = 5.7 Hz). 13C NMR (75 MHz, CDCl3): δ 162.9, 157.7, 149.1, 140.0, 134.4, 130.6, 127.2, 125.3, 124.9, 119.1, 117.2, 110.6, 110.0, 77.2, 70.7, 70.6, 70.2, 69.8, 53.6. HR MS (ESI). Calcd for C26H35N2O7 ([M + H]+): m/z 478.2446. Found: m/z 478.2446. Spectroscopic Measurements. Milli-Q-grade water (18.2 MΩ· cm) was used to prepare solutions for spectroscopic measurements. Piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES; ≥99%) was purchased from Aldrich. A pH 7.4 buffer solution was prepared by dissolving PIPES (25 mM) and KCl (100 mM) in Milli-Q water and adjusting the pH with a standard KOH solution (45 wt %, Aldrich) or concentrated HCl (Aldrich). The buffer solution was further treated with Chelex 100 resin (BIO-RAD) to remove trace metal ions and filtered through a membrane (pore size = 0.45 μm), and its pH was reexamined prior to use. Fresh metal stock solutions (typically, 0.10 or 0.010 M except for CrCl3·6H2O) were prepared in Milli-Q water using the corresponding chloride salts: CuCl2 (99.999%, Aldrich), NaCl (≥99.5%, Aldrich), KCl (puratonic grade, Calbiochem), MgCl2 13

photoluminescence intensity (PI) = [α1 + α2([Zn 2 +]free /Kd)]/[([Zn 2 +]free /Kd) + 1]

(1)

2

[Zn 2 +]free /Kd + [([sensor]total / Kd) − ([Zn 2 +]total /Kd) + 1] [Zn 2 +]free − [Zn 2 +]total (2)

=0 −1

In eq 1, α1 = PI/[sensor]total = 2.38 × 10 M (without zinc ions) and α2 = PI/[sensor]total = 1.81 × 107 M−1 (in the presence of an excess amount of zinc ions) were employed. [sensor]total was 5 μM. A nonlinear least-squares method was applied to fit the metal-ion titration data to eq 1 for the determination of Kd, which subsequently gave a set of free-zinc-ion concentrations ([Zn2+]free) by applying eq 2. The free-zinc-ion concentration was used to revise Kd. This process was iterated (i.e., Kd and [Zn2+]free) until the r2 value of the nonlinear least-squares fit could not be improved. A nonlinear curve fitting module embedded in OriginPro 8.5 (OriginLab) was used for this purpose. Cell Culture. PANC-1 cells were obtained from Korean Cell Line Bank and cultured in DMEM, supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in a humidified incubator under 5% CO2. Two days before imaging, the cells were passed and plated onto poly(D-lysine)-coated glass-bottom culture dishes. Preparation of Rat Hippocampal Neurons. Primary hippocampal neurons were obtained from the brain of embryonic 18-daygestation SD rats. Hippocampi were dissociated and seeded through one of the wells to achieve a cell density of 200 cells mm−2. Neuronal cells were seeded at a density of about 200 cells mm−2 on the 12 wells in a minimal essential mediumsupplemented with 10% horse serum and 0.1% pyruvic acid (Invitrogen). After 3 h of incubation, the medium was replaced with serum-free neurobasal media (Invitrogen) 6

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Article

Inorganic Chemistry supplemented with 1% Anti−Anti (Gibco), 2% B27 (Gibco), and 1% Gluta-MAX (Gibco). Cultured hippocampal neurons were maintained at 37 °C in a 5% CO2/95% air humidified atmosphere. Preparation of Rat Hippocampal Slices. A hippocampal slice was prepared from adult C57BL/6 mice (male, 9 weeks old). The animals were anesthetized with isoflurane before decapitation, and the brain was rapidly removed and sliced in ice-cold solution containing 86 mM NaCl, 3.0 mM KCl, 1.0 mM NaH2PO4, 20 mM NaHCO3, 1.0 mM CaCl2, 4.0 mM MgCl2, 25 mM glucose, and 75 mM sucrose (saturated with 95% O2 and 5% CO2, pH 7.3). Slices were cut at 300 μm coronal sections using a vibratome (Vibratome 1000plus). The slices were then incubated for >30 min at 35−37 °C in ACSF containing 124 mM NaCl, 4.5 mM KCl, 1.2 mM NaH2PO4, 26 mM NaHCO3, 2.0 mM CaCl2, 1.0 mM MgCl2, and 10 mM glucose (saturated with 95% O2 and 5% CO2, pH 7.3). After primary incubation, slices were loaded with both zinc probes and incubated at 37 °C in a humidified atmosphere (5% CO2 and 95% air) for 30 min. Confocal Laser Scanning Microscopy. A 10 μM sensor solution was prepared by dissolving a 10 mM stock solution (DMSO) in serum-free DMEM. A 100 mM ZnPT solution prepared by mixing 200 mM ZnCl2(aq) and 400 mM sodium pyrithione was diluted in serumfree DMEM to 100 μM, prior to the microscopic experiments. Serumfree DMEM solutions containing 100 μM TPEN were also prepared similarly. After the cells were washed with PBS, the medium containing a 10 μM sensor was added to the culture dish. The cells were incubated for 10 min at 37 °C. The incubated cells were washed with PBS and fresh DMEM (serum-free), and photoluminescence micrographs were taken using a Carl Zeiss LSM 510 META confocal laser scanning microscope equipped with a Newport MaiTai eHP DeepSee multiphoton excitation system at Korea Basic Science Institute, Chuncheon Center. An excitation beam (405 nm) was focused on the dish, and the signals were acquired through 32 emission channels covering the range of 415−686 nm. After removal of the cell medium, the cells were washed with fresh DMEM and incubated in DMEM containing 100 μM ZnPT for 10 min. The cells were imaged after washing with FBS and supplemented with fresh DMEM. Finally, the cells were treated with 100 μM TPEN (DMEM) for 5 min, and microscopic visualization was performed. Photoluminescence images and mean intensities were processed using ZEN2000 and ImageJ software, respectively. The sensor stock solution was added to the hippocampal slice with ACSF and incubated for 30 min in the 37 °C and 5% CO2/95% air humidified atmosphere. Additionally, for nuclear staining, 10 μM DRAQ5 (Thermo Fisher Scientific, USA) was added to the slice for 3 min. For DRAQ5, an excitation beam at 633 nm was used. The incubated hippocampal slice was washed with fresh ACSF and treated with 100 μM TPEN for 10 min. Photoluminescence images and mean intensities were processed using ZEN2000 software.

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determined in KCl-free buffers, zinc selectivity results of HBO and HBO−ACR′, pH titration results, photoluminescence decay traces of HBO−ACR in the absence and presence of zinc ions at various pH values, photoluminescence spectra of HBO−ACR in solvents of different viscosities, MTT cell proliferation assays, intracellular localization of HBO−ACR, and additional fluorescence cell images and Tables S1−S3 listing a summary of the TD-DFT calculation results, a summary of the nonlinear least-squares fits of the fluorescence zinc titration data, and the photophysical data for HBO−ACR determined at various pH values (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (S.B.J.). *E-mail: [email protected] (H.K.). *E-mail: [email protected] (Y.Y.). ORCID

Youngmin You: 0000-0001-5633-6599 Author Contributions ⊥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the MSIP of Korea through the GFP (Grant CISS-2012M3A6A6054204 to S.B.J. and Y.Y.) and NRF (Grant 2012M2B2B1055502 to H.K. and 2014R1A1A1A05003770 to S.B.J.). The authors acknowledge Dr. Seung-Hae Kwon and So-Young Kim at Korea Basic Science Institute, Chuncheon Center, for microscopic experiments.

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REFERENCES

(1) Beyersmann, D.; Haase, H. Functions of zinc in signaling, proliferation and differentiation of mammalian cells. BioMetals 2001, 14, 331−341. (2) King, J. C.; Shames, D. M.; Woodhouse, L. R. Zinc homeostasis in humans. J. Nutr. 2000, 130, 1360S−1366S. (3) Maret, W. Molecular aspects of human cellular zinc homeostasis: redox control of zinc potentials and zinc signals. BioMetals 2009, 22, 149−157. (4) Chasapis, C. T.; Loutsidou, A. C.; Spiliopoulou, C. A.; Stefanidou, M. E. Zinc and human health: an update. Arch. Toxicol. 2012, 86, 521− 534. (5) Fukada, T.; Yamasaki, S.; Nishida, K.; Murakami, M.; Hirano, T. Zinc homeostasis and signaling in health and diseases-zinc signaling. JBIC, J. Biol. Inorg. Chem. 2011, 16, 1123−1134. (6) Kelleher, S. L.; McCormick, N. H.; Velasquez, V.; Lopez, V. Zinc in specialized secretory tissues: roles in the pancreas, prostate, and mammary gland. Adv. Nutr. 2011, 2, 101−111. (7) Frederickson, C. J.; Giblin, L. J.; Rengarajan, B.; Masalha, R.; Frederickson, C. J.; Zeng, Y.; Lopez, E. V.; Koh, J.-Y.; Chorin, U.; Besser, L.; Hershfinkel, M.; Li, Y.; Thompson, R. B.; Krezel, A. Synaptic release of zinc from brain slices: factors governing release, imaging, and accurate calculation of concentration. J. Neurosci. Methods 2006, 154, 19−29. (8) Sensi, S. L.; Paoletti, P.; Bush, A. I.; Sekler, I. Zinc in the physiology and pathology of the CNS. Nat. Rev. Neurosci. 2009, 10, 780−791. (9) Finney, L. A.; O’Halloran, T. V. Transition metal speciation in the cell: insights from the chemistry of metal ion receptors. Science 2003, 300, 931−936.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b02786. Figures S1−S39 displaying copies of 1H and 13C NMR spectra, UV−vis absorption spectra obtained in basic conditions, photoluminescence spectra of HBO and HBO−ACR in buffered solutions, UV−vis absorption changes in response to zinc additions, TD-DFT calculation results for the model structures of the zincfree and zinc-bound forms of HBO−ACR, a fluorescence Job plot, fluorescence zinc titration results obtained in KCl-free aqueous buffers, ESI-MS results, fluorescence zinc titration of HBO, HBO−ACR′, and HBO−ACR′′, fluorescence potassium titration results of HBO−ACR and compound 5, reversible zinc responses of HBO− ACR, fluorescence zinc selectivity of HBO−ACR K


Article

Inorganic Chemistry

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