PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
ACS Sens. Author manuscript; available in PMC 2017 December 23.
Published in final edited form as:
PMCID: PMC5569883
NIHMSID: NIHMS858724

Near-Infrared Fluorescent Probes with Large Stokes Shifts for Sensing Zn(II) Ions in Living Cells

Abstract

We report two new near-infrared fluorescent probes based on Rhodol counterpart fluorophore platforms functionalized with dipicolylamine Zn(II)-binding groups. The combinations of the pendant amines and fluorophores provide the probes with an effective three-nitrogen-atom and one-oxygen-atom binding motif. The fluorescent probes with large Stokes shifts offer sensitive and selective florescent responses to Zn(II) ions over other metal ions, allowing a reversible monitoring of Zn(II) concentration changes in living cells, and detecting intracellular Zn(II) ions released from intracellular metalloproteins.

Keywords: fluorescent probe, near-infrared, Rhodol counterpart, Zn(II) ions, photoinduced electron transfer

Graphical abstract

An external file that holds a picture, illustration, etc.
Object name is nihms858724u1.jpg

Zinc (II), the second most abundant transition metal ion in the human body after iron, is often present in a tightly bound form in proteins. It plays several vital roles in a variety of physiological and pathological processes including cellular metabolism, gene expression, cell apoptosis, regulation of metalloenzymes, neural signal transmission and mammalian reproduction.13 The disruption of Zn(II) homeostasis may contribute to Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, cerebral ischemia (ischemic stroke), epilepsy, prostate cancer, diabetes, immune dysfunction, and infantile diarrhea.1,46 Therefore, the development of fluorescent probes to effectively quantify and visualize Zn(II) concentration in biological systems is key to understanding of Zn(II) important roles in biological processes. Furthermore, the fluorescent probes with high sensitivity and spatial resolution of fluorescence would assist in identifying the molecular mechanisms that link zinc homeostasis and human pathophysiology. Several fluorescent probes for Zn(II) sensing have been developed by using different fluorophores such as 2-aryl benzimidazoles,7,8 benzoxazoles,9,10 indoles,11 benzofurans, quinoline,12,13 dansyl,14 anthracene,15 coumarin,16,17 naphtha-limide,18 fluorescein,1925 Rhodamine,24,26,27 cyanine dye,2831 BODIPY dyes,32 and porphyrins.33 They can detect Zn(II) concentration in the range of 10−10 M in the cytoplasm to 10−4 M in some vesicles.1,6,34 Most of these probes still suffer from some significant drawbacks such as autofluorescence and high light scattering. Near-infrared fluorescent probes have been developed to overcome these limitations by reducing background signals and photoinduced damages to biological samples. However, the near-infrared (NIR) fluorescent probes for Zn(II) ions are still less common, as only a very few of them could be used to monitor the levels of endogenous Zn(II) ions in living cells and organisms.28,33,35 The search for readily accessible NIR fluorescent probes with excellent hydrophilic properties, high specificity, large Stokes shifts and dynamic responsive range is still a challenging task for imaging of Zn(II) concentration changes inside living cells. Fluorescent probes with large Stokes shifts possess advantages as spectral overlap between absorption and emission spectra can be eliminated. It allows for detection of fluorescence by avoiding measurement errors by excitation and scattered lights.

In this paper, we report a rational design and synthesis of near-infrared fluorescent probes (A and B) with large Stokes shifts to image the changes in Zn(II) concentration in living cells by incorporating the di-2-picolylamine as a Zn-specific receptor into the near-infrared emissive Rhodol counterpart fluorophores (Figure 1). Fluorescent probes A and B display absorption peaks at 576 and 586 nm and weak fluorescence peaks at 701 and 702 nm with large Stokes shifts of 125 and 116 nm in 10 mM HEPES buffer (pH 7.0), respectively. Near-infrared fluorescent probes A and B display sensitive and selective fluorescent responses to Zn(II) over most other metal ions with detection limits of 0.19 and 0.086 μM, allow for reversibly monitoring Zn(II) concentration in living cells, and sensitively detect intracellular levels of free Zn(II) ions which are released from intracellular metalloproteins when the cells were treated with 2,2′-dithiodipyridine. First-principles density functional calculations are carried out to gain electronic structure level understanding of the fluorescence behavior of probes A and B.

Figure 1
Chemical structures of near-infrared fluorescent probes for Zn(II) ions.

Experimental Section

Instrumentation

1H NMR and 13C NMR spectra were collected via a 400 MHz Varian Unity Inova NMR spectrophotometer. 1H NMR and 13C NMR spectra were recorded in CDCl3 solutions. Chemical shifts (δ) were given in ppm relative to solvent residual peaks (1H, δ 7.26 for CDCl3; 13C, δ 77.3 for CDCl3) as internal standards. High-resolution mass spectrometry data (HRMS) of the intermediates and fluorescent probes were measured with fast atom bombardment (FAB) ionization mass spectrometer, double focusing magnetic mass spectrometer or matrix assisted laser desorption/ionization time-of-flight mass spectrometer. Absorption spectra were taken on a Perkin Elmer Lambda 35 UV/vis spectrometer. Fluorescence spectra were recorded on a Jobin Yvon Fluoromax-4 spectrofluorometer.

Cell Culture and Fluorescence Imaging

Breast cancer (MDA-MB-231) and normal endothelial (HUVEC-C) cell lines obtained from ATCC were cultured according to the published protocols.36 Cells were plated on 12-well culture plates at a density of 1 × 105 cells/mL for live cell imaging. After 24 h incubation at 37 °C with a 5% CO2 incubator, the media was removed and cells were rinsed three times with PBS. Fresh serum free media with 2, 5, or 10 μM of probe A or B was added, and then cells were further incubated for 1 h at 37 °C with 5% CO2. After the cells were rinsed three times with PBS, fresh serum-free media with either 100 μM Zn(II) or 100 μM Zn(II) plus sodium pyrithione (Pyr) was added. Cells were then incubated for 30 min at 37 °C with 5% CO2. At this point, the cells were rinsed three times with PBS, and fresh serum free media was added before acquiring images using an inverted fluorescence microscope (model AMF-4306; EVOS, AMG). Fluorescence images were obtained at either 20× or 60× magnification and the exposure times were kept constant for each image series. 100 μM of TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine) was added to the wells containing 100 μM Zn(II) + 100 μM sodium pyrithione, and then cells were incubated for 10 min at room temperature. Fluorescence images were acquired at 20× magnification using the inverted fluorescence microscope as described previously. Similar experiments with TPEN were also conducted by using 5 μM of each probe at either 10 or 30 μM Zn(II) plus sodium pyrithione. The ability to detect intracellular levels of Zn(II) ions in HUVEC-C cells was evaluated using 2,2′-dithiodipyridine (DTDP) using a modified protocol.37 Cells were serum starved for 3 h at 37 °C with 5% CO2 and then incubated with 2, 5, or 10 μM of probe A or B for 1 h at 37 °C with 5% CO2. To corresponding wells, 100 μM DTDP was added and cells were further incubated for 30 min at 37 °C with 5% CO2. Finally, cells were imaged at 20× or 60× using the inverted fluorescence microscope (model AMF-4306; EVOS, AMG).

Materials

Unless specifically indicated, all reagents and solvents were obtained from commercial suppliers and used without further purification. 3-((Bis(pyridin-2-ylmethyl)amino)methyl)-4-hydroxy-benzaldehyde (1),38 9-(2-carboxyphenyl)-6-(diethylamino)-1,2,3,4-tetrahydroxanthylium perchlorate (2),39,40 and 4-(2-carboxyphenyl)-7-(diethylamino)-2-methylchromenylium perchlorate (3)41 were prepared according to reported procedures.

Synthesis of Fluorescent Probes A and B

Fluorescent Probe A

3-((Bis(pyridin-2-ylmethyl)amino)methyl)-4-hydroxybenzaldehyde (1) (0.30 g, 0.90 mmol) and 9-(2-carboxyphenyl)-6-(diethylamino)-1,2,3,4-tetrahydroxanthylium perchlorate (2) (0.43 g, 0.90 mmol) were dissolved in acetic acid (10 mL) in a 25 mL round-bottom flask. The reaction mixture was heated at 100 °C and stirred for 3 h. When the solvent was evaporated under reduced pressure, the crude product was purified by silica gel chromatography using CH2Cl2/CH3OH/ CH3COOC2H5/CH3COOH (20/2/10/0.32, v/v/v/v) as eluent to obtain fluorescent probe A as deep blue solid (0.12 g, 16.8%). 1H NMR (400 MHz, CDCl3) δ 8.51 (d, J = 4.0 Hz, 2H), 8.04 (d, J = 8.0 Hz, 1H), 7.62–7.58 (m, 3H), 7.54–7.51 (m, 2H), 7.31–7.26 (m, 3H), 7.23 (s, 1H), 7.15–7.11 (m, 3H), 6.86 (d, J = 8.0 Hz, 1H), 6.65–6.62 (m, 2H), 6.50–6.47 (m, 1H), 3.85–3.78 (m, 6H), 3.42–3.37 (m, 4H), 2.78–2.64 (m, 2H), 2.15–2.11 (m, 1H), 1.83–1.79 (m, 1H), 1.63–1.59 (m, 2H), 1.17 (t, J = 4.0 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 169.9, 158.3, 158.1, 154.7, 151.6, 148.8, 137.4, 133.6, 133.3, 131.4, 129.6, 129.4, 127.7, 127.3, 125.3, 123.5, 123.3, 122.6, 116.9, 111.7, 97.0, 59.0, 57.0, 45.2, 27.5, 24.3, 22.3, 12.8. HRMS (ESI): calculated for C44H43N4O4+ [M-ClO4]+, 691.3279; found, 691.3275.

Fluorescent Probe B

3-((Bis(pyridin-2-ylmethyl)amino)methyl)-4-hydroxybenzaldehyde (1) (0.12 g, 0.26 mmol) and 4-(2-carboxyphenyl)-7-(diethylamino)-methylchromenylium perchlorate (3) (0.09 g, 0.26 mmol) were dissolved in acetic acid (4 mL) in a 10 mL round-bottom flask. The reaction mixture was heated at 100 °C and stirred for 2 h. After the solvent was evaporated under reduced pressure, the crude product was purified by silica gel chromatography using CH2Cl2/CH3OH/CH3COOC2H5/ CH3COOH (30/3/10/0.4, v/v/v/v) as eluent to obtain fluorescent probe B as deep blue solid (0.04 g, 19.3%). 1H NMR (400 MHz, CDCl3) δ 8.57 (d, J = 4.0 Hz, 2H), 7.93 (d, J = 4.0 Hz, 1H), 7.65–7.61 (m, 3H), 7.56–7.52 (m, 2H), 7.37–7.30 (m, 3H), 7.26–7,24 (m, 2H), 7.19–7.16 (m, 3H), 6.91 (d, J = 8.0 Hz, 1H), 6.55 (d, J = 8.0 Hz, 1H), 6.47–6.45 (m, 1H), 6.41–6.37 (m, 1H), 3.89 (s, 4H), 3.81 (s, 2H), 3.40–3.34 (m, 4H), 1.18 (t, J = 4.0 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 170.2, 158.8, 158.3, 153.3, 152.7, 149.8, 148.9, 137.3, 134.4, 132.3, 129.7, 129.5, 128.8, 128.3, 127.3, 127.2, 124.2, 123.5, 122.6, 118.3, 117.4, 109.5,105.9, 100.5, 97.9, 94.6, 59.1, 57.2, 44.7, 30.0, 12.8. HRMS (ESI): calculated for C41H39N4O4+ [M-ClO4]+, 651.2966; found, 651.2966.

Results and Discussion

Synthetic Approach to Fluorescent Probes A and B

Dipicolylamine (DPA), a zinc binding ligand with three nitrogen atoms for Zn(II) chelation, was incorporated into near-infrared emissive Rhodol counterpart fluorophores where an oxygen atom from a hydroxyl group can involve in Zn(II) chelation to enhance Zn(II) binding strengths. We conducted a condensation reaction of 3-((bis(pyridin-2-ylmethyl)amino)-methyl)-4-hydroxybenzaldehyde with 9-(2-carboxyphenyl)-6-(diethylamino)-1,2,3,4-tetrahydroxanthylium perchlorate (2), and 4-(2-carboxyphenyl)-7-(diethylamino)-methylchromenylium perchlorate (3) in acetic acid solution at 100 °C, respectively, affording fluorescent probes for Zn(II) (A and B) (Scheme 1). We intentionally controlled one carbon chain length between zinc-binding ligand and the fluorophores in order to manipulate the sensitive fluorescence responses of the probes to Zn(II) ions via effective photoinduced electron transfer effect of a tertiary amine from zinc-binding ligand as an electron donor to the fluorophores. The fluorescent probes were characterized by 1H NMR, 13C NMR, and high-resolution mass spectrometry.

Scheme 1
Synthetic Route to Fluorescent Probes A and B

Optical Properties Of Fluorescent Probes A And B In Different Solvents

All optical data of fluorescent probes were obtained with 10 μM concentration of fluorescent probe A or B in different solvents. Fluorescent probes A and B were very stable in 10 mM HEPES buffer (pH 7.0) containing 1% EtOH solution. These fluorescent probes possess advantageous photophysical properties including a large absorption extinction coefficient, excellent photostability, and large Stokes shifts with near-infrared emission (Table 1 and Figure S7). Probes A and B display large Stokes shifts of 111 and 101 nm with absorption and emission peaks at 581, 594 nm and 692, 695 nm in ethanol solution, respectively. However, the fluorescent probes exhibit smaller Stoke shifts in less polar solvents such as dichloromethane and toluene. The fluorescent probes possess low fluorescence quantum yields in different solvents (Table 1). Probe B shows slightly longer absorption and emission wavelengths in all solvents because of its slightly better π-conjugation than probe A although they have very similar structures (Table 1). The slightly better π-conjugation of the fluorescent probe B is due to less steric hindrance between two bulky groups in a trans configuration in a double bond (Scheme 1).

Table 1
Optical Properties of Fluorescent Probes A and B

Optical Responses of Fluorescent Probes A and B to Zn(II) Ions

The optical responses of the fluorescent probes to Zn(II) ions were investigated in aqueous HEPES buffer (pH 7.0) containing 1% EtOH solution. Probe A displays a main absorption peak at 576 nm and a shoulder peak at 430 nm. Upon gradual addition of Zn(II) to 10 μM solution of fluorescent probe A, the main and shoulder absorption peaks of the probe A undergo red shifts by 26 and 20 nm, respectively, and absorbance of the probe A increases as the molar absorptivity of probe A increases from 1.77 × 104 M−1cm−1 in the absence of Zn(II) ion and to 2.06 × 104 M−1cm−1 in the presence of 10 μM Zn(II) ions (Figure 2). Fluorescent probe B exhibits a main broad absorption peak at 586 nm and a shoulder peak at 435 nm in the same buffer solution. Gradual addition of Zn(II) to 10 μM fluorescent probe B solution triggers red shifts of the main and shoulder absorption peaks by 38 and 21 nm, respectively, and results in enhancement of the absorbance as the molar absorptivity of probe B increases from 1.80 × 104 M−1cm−1 in the absence of Zn(II) ion and to 2.10 × 104 M−1cm−1 in the presence of 10 μM Zn(II) ions (Figure 2).

Figure 2
UV/vis absorption spectra of 10 μM probes A (a) and B (b) upon gradual addition of Zn(II) from 1 μM to 20 μM in 10 mM HEPES buffer solutions (pH 7.0).

Fluorescent probe A displays a very weak fluorescence peak at 701 nm with a fluorescence quantum yield of 0.06% in HEPES buffer (pH 7.0) containing 1% EtOH solution in the absence of Zn(II) ions. This is due to fluorescence quenching effect via photoinduced electron transfer from the tertiary amine of a Zn(II)-binding dipicolylamine ligand as an electron donor to the Rhodol counterpart fluorophore (Table 1). Gradual addition of Zn(II) to a 10 μM solution of fluorescent probe A causes significant fluorescence increases because binding of Zn(II) to the dipicolylamine ligand effectively suppresses the fluorescence quenching effect and recovers the probe fluorescence (Figure 3). Probe A shows a linear fluorescence response to Zn(II) from 0.3 to 4.0 μM with a detection limit of 0.19 μM (Figure S8). The 1:1 stoichiometry was further confirmed by a nonlinear fitting of the titration data at 701 nm by assuming a 1:1 association between fluorescent probe A and Zn(II) (Figure S14). Fluorescent probe B displays a slight higher fluorescence background with fluorescence quantum yield of 0.10% in HEPES buffer than the probe A (Figure 3), and possesses a linear fluorescence response to Zn(II) from 0.3 μM to 4.0 μM with a lower detection limit of 0.086 μM when compared to the probe A (Figure S13). Probe B displays similar fluorescent responses to zinc(II) ions with 1:1 stoichiometry which is similar as probe A. This 1:1 binding stoichiometry between probe B and Zn(II) was verified by Job's plot that is based on nonlinear fitting of the titration curve using 1:1 binding model (Figure S15).

Figure 3
Fluorescence spectra of 10 μM fluorescent probes A (a) and B (b) upon gradual addition of Zn(II) from 1 to 20 μM in 10 mM HEPES buffer solution (pH 7.0) with excitation at 595 nm. Fluorescence responsive curves of fluorescent probes A ...

Theoretical Modeling

We performed first-principles quantum mechanical calculations to gain an electronic structure level understanding of the fluorescence behavior of fluorescent probes A and B acting as multidentate chelators for Zn(II) ions. The stable conformations of the aforementioned probes and their corresponding Zn(II)/probe complexes were determined through optimization of their geometries (Figure 4). A posteriori hybrid density functional method (B3LYP) that includes a portion of the exact Hartree–Fock exchange, and a 6-311+G(d,p) Gaussian basis set42,43 were used for our calculation. We used an implicit solvent model (taking water as the solvent) to mimic the solvent effect.

Figure 4
Optimized conformations of fluorescent probes. Fluorescent probe A (a) and Zn(II)/probe A complex (b). Fluorescent probe B (c) and Zn(II)/probe B complex (d), respectively.

The tetra dentate ligands undergoing the Zn(II) chelation involve three nitrogen atoms of the dipicolylamine (DPA) and an oxygen atom from a nearby hydroxyl group. The hydroxyl group is found to be deprotonated upon binding to Zn(II) for both the probes A and B. The dihedral angle between the two pyridinal planes of DPA in probes A and B are calculated to be 115.12° and 102.12° respectively. Upon the addition of Zn(II) ion, the dihedral angle changes to 132.90° and 129.90° in the so formed Zn(II)/probe A and B complexes, respectively, suggesting a significant conformational change upon binding. The calculated Zn–N and Zn–O bonding distances in Zn(II)/probes A and B complexes are explicitly shown above in Figure 4b and d. Further analysis reveals that the addition of Zn(II) ion in the probes A and B is also accompanied by the redistribution of charge between Zn(II) ions and the associated ligands. The Mulliken charges on three nitrogen atoms an oxygen atom in probes A and B before binding to Zn(II) are found 0.57 e, 0.08 e, −0.07 e, −0.20 e and 0.68 e, −0.07 e, −0.09 e, −0.21 e, respectively. Upon the addition of Zn(II), the charge configuration in these species are found 0.43 e, 0.21 e, 0.03 e, −0.34 and 0.44 e, 0.18 e, 0.04 e, −0.32 e, respectively. On the other hand, the charge on Zn ion in fluorescent complex A and B after binding are found to be 1.21 and 1.12 e, respectively.

Next, to understand the observed fluorescence phenomenon, we performed the excitonic calculations using time-dependent density functional methods (TD-DFT)44,45 for all of the above-mentioned stabilized conformations of the probes. The Lorentzian fitted plot of the absorption spectrum with a half width maximum value of 5 nm were shown in the Figure 5. The variation of oscillator strength (f) with wavelength is presented (Figure 5).The oscillator strength (f) defines the probability of absorption of electromagnetic radiation for transitions between energy levels of the molecule. The strength of maximum absorption is found to be higher in both the Zn(II)/probe A and B complexes than that in fluorescent probes A and B, respectively. The maximum f-values for probes A and B are found to be 0.89 and 1.13, while in the case of Zn(II)/probes A and B complexes, it is found to be 0.98 and 1.28, respectively. This clearly explains the observed sensitivity of Zn(II) ions toward fluorescence behavior due to a much higher absorption observed in both the complexes, which match experimental results of absorbance enhancement when Zn(II) ions bind to fluorescent probes (Figure 2).

Figure 5
Absorption spectra of probe A (a), Zn(II)/probe A complex (b), probe B (c), and Zn(II)/probe B complex (d).

For the fluorescent probes A and B, the calculated dominant absorption are found at wavelengths 547 and 540 nm, respectively. Upon the addition of Zn(II), the maximum absorption in Zn(II)/probe A complex shifts by 34 nm toward the red end to lie at 581 nm. A similar shift of 28 nm in the maximum absorption is seen in the case Zn(II)/probe B complex which is found to lie at 568 nm. Thus, in both cases of probes A and B, our results agree well with the experimentally reported findings of absorption spectra. Further analysis also reveals that in all of these four cases, the dominant absorption peak, without any ambiguity, does correspond to the HOMO–LUMO excitation. It is important to point out that, the experimentally observed higher values of the emission wavelength (λem) of 701 and 702 nm for the Zn(II)/probes A and B complexes may be attributed to the transition of electron from an excited state to a higher energy metastable structure with different conformation than the true ground state. It should also be noted that the calculation presented here is carried out for a single molecule in the presence of an implicit solvent unlike that in the experiment. Nevertheless, the red shifts observed upon binding with Zn(II) to probe A or B is in good agreement with our experimental results.

Selectivity of Fluorescent Probes to Zn(II) Ions over Other Metal Ions

Selectivity of fluorescent probes A and B to Zn(II) over other metal ions was investigated in 10 mM HEPES buffer (Figure 6). No significant responses of fluorescent probes A and B were observed to 20 μM alkali, and alkaline-earth metal ions such as Na+, K+, Ca2+, Mg2+, and Al3+ ions, and some transitional metallic ions such as Mn2+, Fe3+, Ni2+, Co2+, Hg2+, Ag+, and Cr3+ ions. However, the presence of Cu2+ ions causes fluorescence quenching of the fluorescent probe A or B as the fluoroescence quenching may be due to metal-to-ligand electron transfer upon excitation for paramagnetic ions such as Cu2+ for fluorescence quenching which was encountered in fluorescein-based probes for Zn(II) ions.1924 The fluorescent probes can be used to detect Zn(II) with minimum interference from other competing metal ions except Cd2+ ions because Cu2+ ions are rarely present as free ions in the living cells.

Figure 6
Fluorescence spectra of 10 μM probes A (a) and B (b) to 20 μM of various metal anions in 10 mM HEPES buffer solutions (pH 7.0).

In Vitro Imaging of Zn(II) in Living Cells

After we established the fluorescence responses of the florescent probes to Zn(II) ions, we investigated whether the fluorescent probes A and B are cell permeable and stable, and respond to Zn(II) ions reversibly in living cells. We used mixtures of Zn(II) and pyrithione, a cell-permeable ionophore, to deliver exogenous Zn(II) to the cells, and employed N,N,N′,N′-tetrakis(2-picolyl)ethylenediamine (TPEN), a cell-permeable metal ion chelator to test fluorescently responsive reversibility of the probes to Zn(II) ions in living cells according to our reported procedures.46 Fluorescent probe A at 5 μM concentration displayed extremely weak fluorescence background signals in HUVEC normal cells and MDA-MB231 breast cancer cells before the addition of exogenous Zn(II) ions (Figures 7 and and8).8). This is because of the low level of endogenous fluorescence from the cells in near-infrared region, and the low fluorescence quantum yield of the probe. No significant fluorescence change was observed when the cells treated with fluorescent probe A were supplemented by Zn(II) ions alone. However, fluorescence turn-on was observed when sodium pyrithione was added to the cells treated with fluorescent probe A and supplemented by Zn(II) ions (Figures 7 and and8).8). Increase of fluorescent probe A concentration from 2 to 10 μM in living cells significantly enhances turn-on fluorescence intensity of the probe (Figures S22–S29). These results demonstrated that the fluorescent probe A is cell-permeable and intact in living cells, and can be effectively applied to image intracellular Zn(II) ions in living cells. In contrast, fluorescent probe B showed weaker fluorescence turn-on in both HUVEC and MDA-MB231 cells (Figures 7, ,8,8, and S22–S29).

Figure 7
Fluorescence images of 5 μM fluorescent probes A and B in HUVEC-C cells. Cells were then supplemented with either 100 μM zinc(II) chloride or 100 μM each of zinc(II) chloride plus sodium pyrithione (Pyr) for 30 min before acquiring ...
Figure 8
Fluorescence images of 5 μM probes A and B in MDA-MB231 cells. Cells were supplemented with 100 μM zinc(II) chloride or 100 μM each of zinc(II) chloride and sodium pyrithione (Pyr) for 30 min before acquiring images.

We tested whether the fluorescent probes can respond reversibly to Zn(II) ions in living cells by using N,N,N′,N′-tetrakis(2-picolyl)ethylenediamine (TPEN), a cell-permeable Zn(II) ion chelator.46 Upon addition of TPEN to the cells, fluorescence intensity of fluorescent probe-labeled cells decreased dramatically by more than 90% (Figure 9), indicating that the fluorescent probes respond to Zn(II) reversibly since TPEN effectively removes Zn(II) ions from the fluorescent probes (Figures S30 and S31).

Figure 9
Fluorescence images of MDA-MB231 cells incubated with fluorescent probe A. 100 μM each of zinc(II) chloride and sodium pyrithione (Pyr) were supplemented to cells for 30 min before acquiring images. Then 100 μM TPEN (zinc chelator) was ...

We also investigated whether the fluorescent probes could be used to detect intracellular Zn(II) concentration changes in living cells by using 2,2′-dithiodipyridine (DTDP) to promote the release of Zn(II) ions from intracellular metalloproteins.47 When DTDP is used alone, only modest increases (nM) in intracellular Zn(II) ions have been reported.48,45 The HUVECC cells treated with fluorescent probe A showed very weak fluorescence without DTDP treatment. However, the cells displayed accumulation of endogenous Zn(II) ions because addition of DTDP to the HUVEC-C cells treated with fluorescent probe A significantly enhances the fluorescence intensity of fluorescent probe A even at 2 μM concentration in living cells without the addition of external ionophores (Figures 10 and S32). Further, the cells treated with TPEN (zinc chelator) showed the decrease in fluorescence intensity of fluorescent probe-labeled cells (Figure 10). These results unambiguously demonstrated that fluorescent probe A can be effectively used to detect intracellular Zn(II) ion. Fluorescent probe B displays much weaker fluorescence responses to intracellular Zn(II) changes in living cells when compared with fluorescent probe A (Figure S32).

Figure 10
Fluorescence images of HUVEC-C cells in the presence of 5 μM each of probes A and B. Cells were supplemented with 100 μM 2,2′-dithiodipyridine (DTDP) for 30 min before acquiring images. Then 100 μM TPEN (zinc chelator) ...

Conclusion

In this study, we report synthesis and characterization of novel near-infrared fluorescent probes for Zn(II) based on Rhodol counterpart fluorophores functionalized with dipicolylamine Zn(II)-binding groups. The fluorescent probes display selective and sensitive responses to Zn(II) ions over most other metal ions. The binding of Zn(II) ions to fluorescent probes effectively triggers fluorescence turn-on of the fluorophores by suppressing fluorescence quenching effect via photoinduced electron transfer from tertiary amine of dipicolylamine residues to Rhodol counterpart fluorophores. The fluorescent probes offer the reversible visualization of exogenously supplemented Zn(II) ions in living normal and cancer cells. In addition, these probes effectively detect intracellular Zn(II) ions which are released from intracellular metalloproteins in the cells treated with 2, 2′-dithiodipyridine.

Supplementary Material

Supporting information

Acknowledgments

The first-principles results reported here were obtained using RAMA and Superior, the high performance computing cluster at Michigan Technological University. This research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM114751 (to H.Y.L. and A.T.). The research was also partially supported by the National Science Foundation (award number 1048655) (to H.Y.L.), Michigan Technological University faculty start-up fund, Linda J. Horton Laboratory Research Fund, and Research Excellence Fund (to A.T.).

Footnotes

Supporting Information: The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.6b00490.

Detailed synthetic procedures, 1H and 13C NMR spectra of fluorescent probes A and B, detailed supplemental optical spectra of fluorescent probes A and B in the absence and presence of different metal ions, ESI-MS spectra for probes A and B in the absence and presence of Zn(II) ions, photostability, binding constants, and detection limits of probes A and B, Job's plot, selectivity of probes A and B to Zn(II) ions over hydrogen peroxide, in vitro cell imaging, and intracellular detection of Zn(II) by using probes A and B (PDF)

Notes: The authors declare no competing financial interest.

References

1. Pluth MD, Tomat E, Lippard SJ. Biochemistry of Mobile Zinc and Nitric Oxide Revealed by Fluorescent Sensors. Annu Rev Biochem. 2011;80:333–355. [PMC free article] [PubMed]
2. Kay AR, Toth K. Is Zinc a Neuromodulator? Sci Signaling. 2008;1(19):re3–re3. [PMC free article] [PubMed]
3. Sensi SL, Paoletti P, Bush AI, Sekler I. Zinc in the physiology and pathology of the CNS. Nat Rev Neurosci. 2009;10(11):780–U38. [PubMed]
4. Frederickson CJ, Koh JY, Bush AI. The neurobiology of zinc in health and disease. Nat Rev Neurosci. 2005;6(6):449–462. [PubMed]
5. Takeda A, Tamano H. Insight into zinc signaling from dietary zinc deficiency. Brain Res Rev. 2009;62(1):33–44. [PubMed]
6. Pluth MD, Tomat E, Lippard SJ. Biochemistry of Mobile Zinc and Nitric Oxide Revealed by Fluorescent Sensors. Annu Rev Biochem. 2011;80:333–355. [PMC free article] [PubMed]
7. Li PX, Zhou XY, Huang RY, Yang LZ, Tang XL, Dou W, Zhao QQ, Liu WS. A highly fluorescent chemosensor for Zn2+ and the recognition research on distinguishing Zn2+ from Cd2+ Dalton Transactions. 2014;43(2):706–713. [PubMed]
8. Xu YQ, Xiao LL, Zhang YF, Sun SG, Pang Y. Substituent effect on fluorophores instead of ionophores: its implication in highly selective fluorescent probes for Zn2+ over Cd2+ RSC Adv. 2014;4(10):4827–4830.
9. Jiang W, Fu Q, Fan H, Wang W. An NBD fluorophore-based sensitive and selective fluorescent probe for zinc ion. Chem Commun. 2008;2:259–261. [PubMed]
10. Liu ZP, Zhang CL, Chen YC, Qian F, Bai Y, He WJ, Guo ZJ. In vivo ratiometric Zn2+ imaging in zebrafish larvae using a new visible light excitable fluorescent sensor. Chem Commun. 2014;50(10):1253–1255. [PubMed]
11. Taki M, Watanabe Y, Yamamoto Y. Development of ratiometric fluorescent probe for zinc ion based on indole fluorophore. Tetrahedron Lett. 2009;50(12):1345–1347.
12. Zhang Y, Guo X, Si W, Jia L, Qian X. Ratiometric and water-soluble fluorescent zinc sensor of carboxamidoquinoline with an alkoxyethylamino chain as receptor. Org Lett. 2008;10(3):473–476. [PubMed]
13. Ma Y, Wang F, Kambam S, Chen XQ. A quinoline-based fluorescent chemosensor for distinguishing cadmium from zinc ions using cysteine as an auxiliary reagent. Sens. Actuators, B. 2013;188:1116–1122.
14. Jiang PJ, Chen LZ, Lin J, Liu Q, Ding J, Gao X, Guo ZJ. Novel zinc fluorescent probe bearing dansyl and aminoquinoline groups. Chem Commun. 2002;13:1424–1425. [PubMed]
15. Hennrich G, Sonnenschein H, Resch-Genger U. Redox switchable fluorescent probe selective for either Hg(II) or Cd(II) and Zn(II) J Am Chem Soc. 1999;121(21):5073–5074.
16. Komatsu K, Urano Y, Kojima H, Nagano T. Development of an iminocoumarin-based zinc sensor suitable for ratiometric fluorescence imaging of neuronal zinc. J Am Chem Soc. 2007;129(44):13447–13454. [PubMed]
17. Mizukami S, Okada S, Kimura S, Kikuchi K. Design and Synthesis of Coumarin-Based Zn(2+) Probes for Ratiometric Fluorescence Imaging. Inorg Chem. 2009;48(16):7630–7638. [PubMed]
18. Xu Z, Qian X, Cui J, Zhang R. Exploiting the deprotonation mechanism for the design of ratiometric and colorimetric Zn(2+) fluorescent chemosensor with a large red-shift in emission. Tetrahedron. 2006;62(43):10117–10122.
19. Nolan EM, Burdette SC, Harvey JH, Hilderbrand SA, Lippard SJ. Synthesis and characterization of zinc sensors based on a monosubstituted fluorescein platform. Inorg Chem. 2004;43(8):2624–2635. [PubMed]
20. Buccella D, Horowitz JA, Lippard SJ. Understanding Zinc Quantification with Existing and Advanced Ditopic Fluorescent Zinpyr Sensors. J Am Chem Soc. 2011;133(11):4101–4114. [PMC free article] [PubMed]
21. Nolan EM, Jaworski J, Okamoto KI, Hayashi Y, Sheng M, Lippard SJ. QZ1 and QZ2: Rapid, reversible quinoline-derivatized fluoresceins for sensing biological Zn(II) J Am Chem Soc. 2005;127(48):16812–16823. [PMC free article] [PubMed]
22. Nolan EM, Jaworski J, Racine ME, Sheng M, Lippard SJ. Midrange affinity fluorescent Zn(II) sensors of the Zinpyr family: Syntheses, characterization, and biological imaging applications. Inorg Chem. 2006;45(24):9748–9757. [PMC free article] [PubMed]
23. Nolan EM, Ryu JW, Jaworski J, Feazell RP, Sheng M, Lippard SJ. Zinspy sensors with enhanced dynamic range for imaging neuronal cell zinc uptake and mobilization. J Am Chem Soc. 2006;128(48):15517–15528. [PMC free article] [PubMed]
24. Du P, Lippard SJ. A Highly Selective Turn-On Colorimetric, Red Fluorescent Sensor for Detecting Mobile Zinc in Living Cells. Inorg Chem. 2010;49(23):10753–10755. [PMC free article] [PubMed]
25. Chyan W, Zhang DY, Lippard SJ, Radford RJ. Reaction-based fluorescent sensor for investigating mobile Zn2+ in mitochondria of healthy versus cancerous prostate cells. Proc Natl Acad Sci U S A. 2014;111(1):143–148. [PubMed]
26. Han ZX, Zhang XB, Zhuo L, Gong YJ, Wu XY, Zhen J, He CM, Jian LX, Jing Z, Shen GL, Yu RQ. Efficient Fluorescence Resonance Energy Transfer-Based Ratiometric Fluorescent Cellular Imaging Probe for Zn(2+) Using a Rhodamine Spirolactam as a Trigger. Anal Chem. 2010;82(8):3108–3113. [PubMed]
27. Tomat E, Lippard SJ. Ratiometric and Intensity-Based Zinc Sensors Built on Rhodol and Rhodamine Platforms. Inorg Chem. 2010;49(20):9113–9115. [PMC free article] [PubMed]
28. Komatsu K, Kikuchi K, Kojima H, Urano Y, Nagano T. Selective zinc sensor molecules with various affinities for Zn2+, revealing dynamics and regional distribution of synaptically released Zn2+ in hippocampal slices. J Am Chem Soc. 2005;127(29):10197–10204. [PubMed]
29. Menendez GO, Lopez CS, Jares-Erijman EA, Spagnuolo CC. A Versatile Near-Infrared Asymmetric Tricarbocyanine for Zinc Ion Sensing in Water. Photochem Photobiol. 2013;89(6):1354–1361. [PubMed]
30. Rivera-Fuentes P, Lippard SJ. SpiroZin1: A Reversible and pH-Insensitive, Reaction-Based, Red-Fluorescent Probe for Imaging Biological Mobile Zinc. ChemMedChem. 2014;9(6):1238–1243. [PMC free article] [PubMed]
31. Guo ZQ, Kim GH, Yoon J, Shin I. Synthesis of a highly Zn2+-selective cyanine-based probe and its use for tracing endogenous zinc ions in cells and organisms. Nat Protoc. 2014;9(6):1245–1254. [PubMed]
32. Ojida A, Sakamoto T, Inoue Ma, Fujishima Sh, Lippens G, Hamachi I. Fluorescent BODIPY-Based Zn(II) Complex as a Molecular Probe for Selective Detection of Neurofibrillary Tangles in the Brains of Alzheimer's Disease Patients. J Am Chem Soc. 2009;131(18):6543–6548. [PubMed]
33. Zhang XA, Lovejoy KS, Jasanoff A, Lippard SJ. Water-soluble porphyrins as a dual-function molecular imaging platform for MRI and fluorescence zinc sensing. Proc Natl Acad Sci U S A. 2007;104(26):10780–10785. [PubMed]
34. Nolan EM, Lippard SJ. Small-Molecule Fluorescent Sensors for Investigating Zinc Metalloneurochemistry. Acc Chem Res. 2009;42(1):193–203. [PMC free article] [PubMed]
35. Haas KL, Franz KJ. Application of Metal Coordination Chemistry To Explore and Manipulate Cell Biology. Chem Rev. 2009;109(10):4921–4960. [PMC free article] [PubMed]
36. Zhu S, Zhang J, Janjanam J, Bi J, Vegesna G, Tiwari A, Luo FT, Wei J, Liu H. Highly water-soluble, near-infrared emissive BODIPY polymeric dye bearing RGD peptide residues for cancer imaging. Anal Chim Acta. 2013;758:138–44. [PMC free article] [PubMed]
37. Baek NY, Heo CH, Lim CS, Masanta G, Cho BR, Kim HM. A highly sensitive two-photon fluorescent probe for mitochondrial zinc ions in living tissue. Chem Commun (Cambridge, U K) 2012;48(38):4546–4548. [PubMed]
38. Murale DP, Singh AP, Lavoie J, Liew H, Cho J, Lee HI, Suh YH, Churchill DG. Novel molecular tools to discriminate Fe3+ and Fe2+ by fluorescence via ″turn-on″ responses within neuronal cells. Sens Actuators, B. 2013;185:755–761.
39. Yuan L, Lin WY, Yang YT, Chen H. A Unique Class of Near-Infrared Functional Fluorescent Dyes with Carboxylic-Acid-Modulated Fluorescence ON/OFF Switching: Rational Design, Synthesis, Optical Properties, Theoretical Calculations, and Applications for Fluorescence Imaging in Living Animals. J Am Chem Soc. 2012;134(2):1200–1211. [PubMed]
40. Vegesna GK, Janjanam J, Bi JH, Luo FT, Zhang JT, Olds C, Tiwari A, Liu HY. pH-activatable near-infrared fluorescent probes for detection of lysosomal pH inside living cells. J Mater Chem B. 2014;2(28):4500–4508.
41. Zheng KB, Lin WY, Huang WM, Guan XY, Cheng D, Wang JY. Facile synthesis of a class of aminochromene-aniliniumion conjugated far-red to near-infrared fluorescent dyes for bioimaging. J Mater Chem B. 2015;3(5):871–877.
42. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V, Mennucci B, Petersson GA, Nakatsuji H, Caricato M, Li X, Hratchian HP, Izmaylov AF, Bloino J, Zheng G, Sonnenberg JL, Hada M, Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H, Vreven T, Montgomery JA, Jr, Peralta JE, Ogliaro F, Bearpark M, Heyd JJ, Brothers E, Kudin KN, Staroverov VN, Kobayashi R, Normand J, Raghavachari K, Rendell A, Burant JC, Iyengar SS, Tomasi J, Cossi M, Rega N, Millam JM, Klene M, Knox JE, Cross JB, Bakken V, Adamo C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev O, Austin AJ, Cammi R, Pomelli C, Ochterski JW, Martin RL, Morokuma K, Zakrzewski VG, Voth GA, Salvador P, Dannenberg JJ, Dapprich S, Daniels AD, Farkas O, Foresman JB, Ortiz JV, Cioslowski J, Fox DJ. Gaussian 09. Gaussian, Inc.; Wallingford, CT: 2009. Revision E.01.
43. Mandal S, Pati R. Mechanism behind the switching of current induced by a gate field in a semiconducting nanowire junction. Phys Rev B: Condens Matter Mater Phys. 2011;84(11):115306.
44. Bartolotti LJ. Time-Dependent Kohn-Sham Density-Functional Theory. Phys Rev A: At, Mol, Opt Phys. 1982;26(4):2243–2244.
45. Appel H, Gross EKU, Burke K. Excitations in time-dependent density-functional theory. Phys Rev Lett. 2003;90(4):043005. [PubMed]
46. Zhu SL, Zhang JT, Janjanam J, Vegesna G, Luo FT, Tiwari A, Liu HY. Highly water-soluble BODIPY-based fluorescent probes for sensitive fluorescent sensing of zinc(II) J Mater Chem B. 2013;1(12):1722–1728.
47. Aizenman E, Stout AK, Hartnett KA, Dineley KE, McLaughlin B, Reynolds IJ. Induction of Neuronal Apoptosis by Thiol Oxidation. J Neurochem. 2000;75(5):1878–1888. [PubMed]
48. Sensi SL, Ton-That D, Sullivan PG, Jonas EA, Gee KR, Kaczmarek LK, Weiss JH. Modulation of mitochondrial function by endogenous Zn2+ pools. Proc Natl Acad Sci U S A. 2003;100(10):6157–6162. [PubMed]