Open AccessArticle

Synthesis of a Novel Pyrazine–Pyridone Biheteroaryl-Based Fluorescence Sensor and Detection of Endogenous Labile Zinc Ions in Lung Cancer Cells

Masayori Hagimori

^1,2,*,

Mana Taniura

¹,

Naoko Mizuyama

³,

Yasushi Karimine

²,

Shigeru Kawakami

²,

Hideo Saji

⁴ and

Takahiro Mukai

^1,*

Department of Biophysical Chemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita Machi, Higashinada Ku, Kobe 658-8558, Japan

Graduate School of Biomedical Sciences, Nagasaki University, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan

Clinical Research Center, Nagasaki University Hospital, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan

⁴

Graduate School of Pharmaceutical Sciences, Kyoto University, 46–29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan

Authors to whom correspondence should be addressed.

Sensors 2019, 19(9), 2049; https://doi.org/10.3390/s19092049

Submission received: 7 April 2019 / Revised: 29 April 2019 / Accepted: 1 May 2019 / Published: 2 May 2019

(This article belongs to the Section Chemical Sensors)

Download

Browse Figures

Figure 1
Schematic structure of novel fluorescence Zn2+ sensors. "> Figure 2
Fluorescence spectra of (a) 3a (10−5 M, λex = 381 nm); (b) 3b (10−5 M, λex = 367 nm); (c) 4a (10−5 M, λex = 353 nm); (d) 4b (10−5 M, λex = 360 nm); (e) 5a (10−5 M, λex = 377 nm); (f) 5b (10−5 M, λex = 395 nm) upon addition of Zn2+ in the form of perchlorate salt (0, 1, 2.5, 5, 10, 50, 100 μM) in HEPES buffer (100 mM, 5% DMSO, pH = 7.4). λex = excitation wavelength. The narrow fluorescence band of (a), (b), (d), and (e) were the Raman scattering peak of the solvent. "> Figure 3
Fluorescence responses of (a) 3a (10−5 M, λem = 381 nm); (b) 3b (10−5 M, λem = 367 nm); (c) 4a (10−5 M, λem = 353 nm); (d) 4b (10−5 M, λem = 360 nm); (e) 5a (10−5 M, λem = 377 nm); (f) 5b (10−5 M, λem = 395 nm) upon addition of different metal cations in HEPES buffer (100 mM, 5% DMSO, pH = 7.4). λem = emission wavelength. "> Figure 4
Competitive binding experiments of 5b, in which competing metal ions (10−3 M) and Zn2+ (10−3 M) were added to 5b (10−5 M) in HEPES buffer (100 mM, 5% DMSO, pH = 7.4). "> Figure 5
(a) Effect of pH on the fluorescence properties of 5b in the absence (□) and presence (■) of Zn2+. (b) Enlarged figure of 5b in the absence (□) of Zn2+. "> Figure 6
Fluorescence images of exogenously added Zn2+ in A549 cells: (a) Fluorescence image of cells with 5b (100 μM); (b) fluorescence image incubated with 5b (100 μM) in the presence of Zn2+/pyrithione (100 μM); (c) fluorescence image of (b) after treatment with TPEN (100 μM). Scale bars = 300 μm. "> Figure 7
Fluorescence images of endogenous Zn2+ in A549 cells: (a) Fluorescence image of cells with 5b (100 μM); (b) fluorescence image incubated with 5b (100 μM) after incubation with H2O2 (200 μM) for 24 h; (c) fluorescence image of (b) after treatment with TPEN (100 μM). Scale bars = 300 μm. "> Scheme 1
Synthesis of compounds 3a,b, 4a,b, and 5a,b. ">

Versions Notes

Abstract

A small extent of endogenous labile zinc is involved in many vital physiological roles in living systems. However, its detailed functions have not been fully elucidated. In this study, we developed a novel biheteroaryl-based low molecular weight fluorescent sensor, 3-(phenylsulfonyl)-pyrazine–pyridone (5b), and applied it for the detection of endogenous labile zinc ions from lung cancer cells during apoptosis. The electron-withdrawing property of the sulfonyl group between the phenyl ring as an electron donor and the pyridone ring as a fluorophore inhibited the intramolecular charge transfer state, and the background fluorescence of the sensor was decreased in aqueous media. From the structure–fluorescence relationship analysis of the substituent effects with/without Zn²⁺, compound 5b acting as a sensor possessed favorable properties, including a longer emission wavelength, a large Stokes shift (over 100 nm), a large fluorescence enhancement in response to Zn²⁺ under physical conditions, and good cell membrane permeability in living cells. Fluorescence imaging studies of human lung adenocarcinoma cells (A549) undergoing apoptosis revealed that compound 5b could detect endogenous labile zinc ions. These experiments suggested that the low molecular weight compound 5b is a potential fluorescence sensor for Zn²⁺ toward understanding its functions in living systems.

Keywords:

endogenous Zn²⁺; pyrazine–pyridone biheteroaryl; low background fluorescence; sulfonyl group; cellular imaging

1. Introduction

Essential trace elements are known to play important roles in many biochemical processes, including the construction of biological tissues and the sustaining and adjusting of physiological systems [1]. These elements show positive effects in physiological functions at optimal concentrations, whereas shortage or excessive concentrations affect biochemical processes and may evoke deficiency and toxicity. Because essential trace elements are both directly and indirectly related to diseases, detection with fluorescence imaging has become a useful tool in elucidating their distribution and dynamic states in living organisms, providing extensive information about diseases and facilitating the development of therapeutic strategies [2].

Zinc is the second-most abundant trace element in the living body and is involved in vital physiological roles such as enzyme regulation, gene expression, immune system response, and neurotransmission [2,3,4,5,6]. Most endogenous zinc is tightly bound to proteins for their structural and catalytic functions, whereas a small amount of zinc exists in free or labile forms in several tissues [6,7]. The disruption of zinc homeostasis causes serious damage related to neurodegenerative diseases, indicating that free and labile zinc plays an important role as an intracellular second messenger [7,8,9].

To detect and visualize the complicated physiological and pathological functions of zinc, various fluorescence Zn²⁺ sensors have been developed based on fluorophores such as coumarin, rhodamine, and cyanine [2,10,11,12]. These sensors have unique properties in selectivity and sensitivity. Recently, Zn²⁺ high-specific sensors discriminating Zn²⁺ from Cd²⁺ have been reported [13]. However, the details of zinc homeostatic processes are still unclear. Because the endogenous Zn²⁺ concentration is low (subnanomolar to millimolar range), high background fluorescence in a probe is a significant drawback in imaging small amounts of Zn²⁺ in living cells [12]. We previously reported pyridine–pyridone-based fluorescent Zn²⁺ sensors, in which the pyridine–pyridone core structure acted as both the chelating functionality for Zn²⁺ and the fluorescent moiety, such that the sensor has a low molecular weight (below 500 g mol⁻¹) and has good cell membrane permeability [14,15,16]. The fluorescence mechanism of pyridine–pyridone sensors is the formation of an intramolecular charge transfer (ICT) state, and the separation between the phenyl ring as an electron-donor and the pyridone ring as a fluorophore influenced the push–pull system of the sensors, weakening the ICT and resulting in a decrease in background fluorescence [14]. The sensors could visualize the exogenously-added Zn²⁺ in living cells; however, we realized that further reducing the background fluorescence of the Zn²⁺ sensor was required to detect endogenous Zn²⁺.

Herein, we reported novel biheteroaryl Zn²⁺ fluorescence compounds (3a,b, 4a,b, and 5a,b), which have a sulfonyl group as a strong electron-withdrawing group inhibiting the push–pull system, thus strongly reducing the background fluorescence of the sensors. The introduction of substituents also affected fluorescence properties such as intensity, excitation, and emission wavelength with or without Zn²⁺. From the structure–fluorescence relationship analysis of the substituent effects in the pyridone core structure, 3-(phenylsulfonyl)-pyrazine–pyridone (5b), with pyrazine and tosyl groups, exhibited favorable properties as a Zn²⁺ sensor. In this paper, we described the design and synthesis of fluorescence compounds and tested a biological application that successfully indicated that sensor 5b could detect endogenous Zn²⁺ in lung cancer cells.

2. Materials and Methods

2.1. Materials and Instruments

All chemicals were of the highest purity available. ¹H-NMR was measured on a Varian Mercury-300 (300 MHz) with chemical shifts reported as ppm. Mass spectra (MS) and high-resolution MS (HRMS) were recorded on a Thermo Fisher Scientific Exactive spectrometer. Fluorescence spectra were obtained on a F7000 fluorescence spectrophotometer (Hitachi High-Tech, Tokyo, Japan) and a RF-5300PC fluorescence spectrophotometer (Shimadzu, Kyoto, Japan).

2.2. Synthesis of 4-(Methylsulfanyl)-5-(Phenylsulfonyl)-[2,2’-Bipyridin]-6(1H)-One (3a)

3,3-Bis(methylsulfanyl)-2-(phenylsulfonyl)acrylonitrile (2a; 2.84 g, 10 mmol) [17,18] was slowly added to a solution of 2-acetylpyridine (1a; 1.21 g, 10.0 mmol) and sodium hydroxide (0.56 g, 14 mmol) in DMSO over 30 min, and the mixture was stirred for 2 h at room temperature. After adding sodium hydroxide (0.56 g, 14 mmol), the mixture was stirred for an additional 3 h at room temperature. The reaction mixture was poured into 100 mL of water and adjusted to pH 3–4 with a 10% hydrochloric acid solution. The mixture was extracted with 100 mL of chloroform three times. Organic extracts were combined, washed with water, and dried over anhydrous sodium sulfate. After concentration in vacuo, the residue was refluxed in 1% hydrochloric acid solution for 1 h. The precipitate that appeared was collected by filtration, washed with water, and recrystallized from methanol to give 3a (0.27 g, 5.7 mmol, 7.5%) as brown crystals. Melting point (Mp) 331–332 °C. ¹H-NMR (DMSO-d₆, 300 MHz): δ 2.71 (s, 3H), 7.01 (s, 1H), 7.55–7.64 (m, 3H), 7.90–7.98 (m, 3H), 8.18 (d, J = 8.4 Hz, 2H), 8.74 (d, J = 4.5 Hz, 1H), 11.76 (brs, 1H). MS m/z: 359 [M + H⁺]. HRMS calcd. for C₁₇H₁₅N₂O₃S₂ [M + H⁺]: 359.0524. Found: 359.0518.

2.3. Synthesis of 4-(Methylsulfanyl)-5-Tosyl-[2,2’-Bipyridin]-6(1H)-One (3b)

Compound 3b (0.67 g, 1.8 mmol) was prepared in 39% yield from 0.60 g (5.0 mmol) of 1a and 1.5 g (5.0 mmol) of 3,3-bis(methylsulfanyl)-2-tosylacrylonitrile (2b) in a manner similar to that described for the synthesis of 3a. An analytical sample was recrystallized from methanol to give dark gray crystals. Mp > 400 °C. ¹H-NMR (DMSO-d₆, 300 MHz): δ 2.41 (s, 3H), 2.71 (s, 3H), 6.98 (s, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.54–7.58 (m, 3H), 7.77 (d, J = 8.4 Hz, 2H), 8.71 (d, J = 3.9 Hz, 1H). MS m/z: 371 [M – H⁺]. HRMS calcd. for C₁₈H₁₅N₂O₃S₂ [M – H⁺]: 371.0524. Found: 371.0532.

2.4. Synthesis of 4’-Methyl-4-(Methylsulfanyl)-5-(Phenylsulfonyl)-[2,2’-Bipyridin]-6(1H)-One (4a)

Compound 4a (1.14 g, 3.0 mmol) was prepared in 61% yield from 0.68 g (5.0 mmol) of 2-acetyl-4-methylpyridine (1b) and 1.42 g (5.0 mmol) of 2a in a manner similar to that described for the synthesis of 3a. An analytical sample was recrystallized from methanol to give dark gray crystals. Mp 221–222 °C. ¹H-NMR (DMSO-d₆, 300 MHz): δ 2.51 (s, 3H), 2.70 (s, 3H), 6.91 (s, 1H), 7.25 (d, J = 4.8 Hz, 1H), 7.48 (s, 1H), 7.55–7.62 (m, 3H), 7.75 (d, J = 7.8 Hz, 2H), 8.59 (d, J = 4.8 Hz, 1H). MS m/z: 371 [M – H⁺]. HRMS calcd. for C₁₈H₁₅N₂O₃S₂ [M – H⁺]: 371.0524. Found: 371.0531.

2.5. Synthesis of 4’-Methyl-4-(Methylsulfanyl)-5-Tosyl-[2,2’-Bipyridin]-6(1H)-One (4b)

Compound 4b (0.31 g, 0.8 mmol) was prepared in 27% yield from 0.41 g (3.0 mmol) of 1b and 0.89 g (3.0 mmol) of 2b in a manner similar to that described for the synthesis of 3a. An analytical sample was recrystallized from methanol to give dark gray crystals. Mp > 400 °C. ¹H-NMR (DMSO-d₆, 300 MHz): δ 2.39 (s, 3H), 2.52 (s, 3H), 2.70 (s, 3H), 6.91 (s, 1H), 7.34–7.45 (m, 4H), 7.62 (d, J = 8.4 Hz, 2H), 8.59 (d, J = 4.2 Hz, 1H). MS m/z: 385 [M – H⁺]. HRMS calcd. for C₁₉H₁₇N₂O₃S₂ [M – H⁺]: 385.0681. Found: 385.0689.

2.6. Synthesis of 4-(Methylsulfanyl)-3-(Phenylsulfonyl)-6-(Pyrazin-2-Yl)Pyridin-2(1H)-One (5a)

Compound 5a (0.16 g, 0.5 mmol) was prepared in 9% yield from 0.61 g (5.0 mmol) of 2-acetylpyrazine (1c) and 1.42 g (5.0 mmol) of 2a in a manner similar to that described for the synthesis of 3a. An analytical sample was recrystallized from methanol to give dark gray crystals. Mp 188–189 °C. ¹H-NMR (DMSO-d₆, 300 MHz): δ 2.73 (s, 3H), 7.09 (s, 1H), 7.60 (m, 2H), 7.66 (m, 1H), 7.70 (d, J = 7.2 Hz, 2H), 8.76 (d, J = 1.8 Hz, 1H), 8.83 (d, J = 1.5 Hz, 1H), 9.12 (s, 1H). MS m/z: 358 [M – H⁺]. HRMS calcd. for C₁₆H₁₂N₃O₃S₂ [M – H⁺]: 358.0320. Found: 358.0331.

2.7. Synthesis of 4-(Methylsulfanyl)-6-(Pyrazin-2-Yl)-3-Tosylpyridin-2(1H)-One (5b)

Compound 5b (0.19 g, 0.5 mmol) was prepared in 10% yield from 0.61 g (5.0 mmol) of 1c and 1.50 g (5.0 mmol) of 2b in a manner similar to that described for the synthesis of 3a. An analytical sample was recrystallized from methanol to give dark gray crystals. Mp 232–233 °C. ¹H-NMR (DMSO-d₆, 300 MHz): δ 2.38 (s, 3H), 2.72 (s, 3H), 7.13 (s, 1H), 7.41 (d, J = 8.1 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H), 8.80 (d, J = 0.9 Hz, 1H), 8.83 (d, J = 1.5 Hz, 1H), 9.12 (s, 1H). MS m/z: 372 [M – H⁺]. HRMS calcd. for C₁₇H₁₄N₃O₂S₂ [M – H⁺]: 372.0477. Found: 372.0484.

2.8. Spectral Measurement Studies

A stock solution of each compound (1 mM) was prepared by dissolving in DMSO. Solutions of perchlorate salts of metal ions (Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cu²⁺, Fe²⁺, Fe³⁺, K⁺, Mg²⁺, Mn²⁺, Na⁺, Pb²⁺, and Zn²⁺) were prepared by dissolving in distilled water. The fluorescence of test compounds (10⁻⁵ M) in HEPES buffer (100 mM, 5% DMSO, pH = 7.4) were analyzed in the presence of Zn²⁺. Job’s plot was used to investigate the binding stoichiometries of 3a,b, 4a,b, and 5a,b to Zn²⁺. The dissociation constant (K_d) values were investigated by the following Benesi–Hildebrand plot [19,20].

1/(F – F₀) = 1/{K_a(F_max – F₀)[Zn²⁺]ⁿ} + 1/(F_max – F₀)

where F is the fluorescence intensity, F₀ is the fluorescence intensity without Zn²⁺, and F_max is the fluorescence in addition of excess Zn²⁺. The association constant (K_a) is the inverse of K_d and is determined from the slope of the straight line of the plot of 1/(F – F₀) against 1/[Zn²⁺]. The selectivity of each compound was investigated in HEPES buffer (100 mM, 5% DMSO, pH = 7.4). The fluorescence quantum yields were measured with respect to a quinine sulfate solution (Φ = 0.54) as the standard.

2.9. Cellular Imaging by Fluorescence Microscope

A549 human lung cancer cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) that included 10% fetal bovine serum and 1% penicillin at 37 °C in a humidified atmosphere with 5% CO₂. To investigate the cell-membrane permeability of 5b, the cells were incubated with 100 μM of 1:1 Zn²⁺/pyrithione in the culture media for 30 min at 37 °C. After washing with phosphate-buffered saline (PBS), the treated cells were incubated with 5b (100 μM) in the culture media for 30 min at 37 °C. The incubated cells were imaged with fluorescence microscopy (Nikon Eclipse Ti). For N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) studies, the cells were incubated with 1:1 Zn²⁺/pyrithione (100 μM) in the culture media for 30 min at 37 °C and washed with PBS. After incubating with 5b (100 μM) in the culture media for 30 min at 37 °C and washing with PBS, the cells were incubated with 100 μM of TPEN for an additional 30 min at 37 °C and imaged with fluorescence microscopy (Nikon Eclipse Ti).

To detect endogenous Zn²⁺, the cells were incubated with hydrogen peroxide (200 μM) in DMEM for 24 h at 37 °C. After washing with PBS, the cells were incubated with 5b (100 μM) in the culture media for 30 min at 37 °C. The incubated cells were imaged with fluorescence microscopy (Nikon Eclipse Ti). For TPEN studies, the cells were incubated with hydrogen peroxide (H₂O₂, 200 μM) in the culture media for 24 h at 37 °C and washed with PBS. After incubation with 5b (100 μM) in culture media for 30 min at 37 °C and washing with PBS, the cells were incubated with 100 μM of TPEN for an additional 30 min at 37 °C and imaged with fluorescence microscopy (Nikon Eclipse Ti).

3. Results

Our previous research demonstrated that the NH/OH proton of the pyridone core structure acted as a fluorescence off–on switch and that the weakening of the electron transfer from the electron-donating group to the pyridone core structure reduced the background fluorescence of the Zn²⁺ sensors [14,15]. Then, to develop a lower background fluorescence sensor, we introduced benzensulfonyl or tosyl groups at the two-position of the pyridone ring (Figure 1). In addition, Zn²⁺ was bound to the bipyridyl form. Therefore, we introduced pyridine, 4-methylpyridine, or pyrazine as an electron-withdrawing heteroaryl group at the six-position of the pyridone ring. Pyrazine is a nitrogen-containing, six-membered heterocycle, which has lower basicity and a higher π-acceptor ability than pyridine [21]. Therefore, it was expected that the replacement of pyridine with pyrazine would affect the compound’s coordination ability and fluorescent property towards Zn²⁺. Compound 3a was synthesized from a one-pot reaction of 2-acetylpyridine (1a) with sulfonyl ketene dithioacetal (2a) in the presence of NaOH as a base, followed by treatment with 1% HCl (Scheme 1). In a similar manner, compound 3b, having a tosyl group on the two-position of the pyridone ring, was obtained in 39% yield. Compounds 4a and 4b, having a 4-methyl-2-pyridyl group, were prepared from the reaction of 2-acetyl-4-methylpyridine (1b) and sulfonyl ketene dithioacetal (2a,b). The reaction of 2-acetylpyrazine (1c) with sulfonyl ketene dithioacetals (2a,b) afforded pyrazine–pyridone compounds (5a,b) in 9% and 10% yields, respectively.

Compounds 3a,b, 4a,b, and 5a,b were soluble in aqueous media after dilution of their DMSO stock solutions, and all spectroscopic measurements were performed in HEPES buffer (100 mM, 5% DMSO, pH 7.4). Figure 2 and Table 1 show the fluorescence spectra and corresponding data of 3a,b, 4a,b, and 5a,b. All compounds were excited by 353–395 nm light, and the background fluorescence quantum yields (Φ) of the compounds were in the range of 4.1 × 10⁻⁴–9.8 × 10⁻⁴, which were considerably lower than previously reported pyridine–pyridone type compounds without a sulfonyl group [14,15,16]. As expected, these results suggested that introduction of a sulfonyl group between the pyridone core structure inhibits electron transfer from the electron-donating group and reduces the background fluorescence of 3a,b, 4a,b, and 5a,b. After the addition of Zn²⁺, the fluorescence intensities of all compounds gradually increased in a concentration-dependent manner with Zn²⁺. Compounds 3a,b and 4a,b exhibited blue fluorescence at 430–442 nm, and the emission maximum wavelengths of 3b and 4b exhibited 4–5 nm bathochromic shifts by the introduction of a methyl group at the para position of the phenyl ring. In contrast, the methyl group of the 4-methyl-2-pyrigyl ring of 4a,b induced 7–8 nm hypsochromic shifts in the emission maximum wavelengths. In pyrazine compounds 5a,b, small bathochromic shifts were observed by introduction of the methyl group at the para position of the phenyl ring. On the other hand, the emission maximum wavelengths of compounds 5a,b showed large bathochromic shifts, approximately 60–70 nm, compared to compounds 3a,b and 4a,b with pyridyl moieties, and 5a,b emitted blue–green fluorescence around 500 nm. Since the emission maximum wavelengths of all previously reported pyridine–pyridone-type compounds were near 400–450 nm [14,15,16], it is noteworthy that the emission wavelength could be tuned by the simple replacement of pyridine with pyrazine. In addition, compounds 5a,b showed very large Stokes shifts (over 100 nm), which became an advantage for detecting Zn²⁺ without too much interference from the excitation light. The binding analysis of compounds 3a,b, 4a,b, and 5a,b and Zn²⁺ indicated that the complex formations had 1:1 stoichiometries (Supplementary data). We also calculated the dissociation constants (K_d) of 3a,b, 4a,b, and 5a,b from the Benesi–Hildebrand equation [18,19] using fluorescent titration data (Supplementary data). The K_d values of 5a and 5b were 59 × 10⁻⁵ M and 32 × 10⁻⁵ M, respectively, which were higher than those of 3a,b and 4a,b (1.6 × 10⁻⁵–9.4 × 10⁻⁵ M). These results indicated that the replacement of pyridine with pyrazine obviously influenced the compound’s coordination ability and fluorescent property with Zn²⁺. To investigate the selectivity of these compounds, various cations including Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cu²⁺, Fe²⁺, Fe³⁺, K⁺, Mg²⁺, Mn²⁺, Na⁺, and Zn²⁺ were added to solutions of 3a,b, 4a,b, and 5a,b in HEPES buffer (100 mM, 5% DMSO, pH = 7.4). As shown in Figure 3, all compounds showed selectivity toward Zn²⁺, and especially the pyrazine compound 5b exhibited a large chelation-enhanced fluorescence (CHEF) effect with Zn²⁺ and exhibited an 8.1-fold enhancement over 5b without Zn²⁺ addition. Fluorescence enhancement was also observed upon addition of Cd²⁺. Zn²⁺ and Cd²⁺ belong to the same group of the periodic table, therefore it has been reported that Cd²⁺ interfered with the detection of Zn²⁺ in various types of Zn²⁺ sensors [14,15,16,22,23]. However, Cd²⁺ is not naturally occurring in living systems, so it may have little influence on visualizing cellular Zn²⁺. With addition of other metal ions (Al³⁺, Ca²⁺, Co²⁺, Cu²⁺, Fe²⁺, Fe³⁺, K⁺, Mg²⁺, Mn²⁺, and Na⁺), no large fluorescence changes, including both CHEF effects and chelation-enhanced fluorescence quenching (CHEQ) effects, were observed. We also evaluated the fluorescence change with Pb²⁺ having a wide-spectrum toxic effect in living systems, however both CHEF and CHEQ effects were not observed (Supplementary data).

Among the compounds 3a,b, 4a,b, and 5a,b, pyrazine compound 5b had the most useful properties, namely low background fluorescence, emitted fluorescence at 504 nm, a large Stokes shift (over 100 nm), and an 8.1-fold fluorescence enhancement with Zn²⁺. Next, we performed further experiments, including a competition experiment, a test of pH influence, and cellular fluorescence imaging, to demonstrate the sensing ability of 5b.

Figure 4 shows the results of the competition experiment of 5b between Zn²⁺ and other metal ions. The CHEF effect with Zn²⁺ was quenched by the addition of Co²⁺ or Cu²⁺, whereas it was not affected by addition of alkali-metal ions (Na⁺ and K⁺) or group 2 ions (Ca²⁺ and Mg²⁺), which exist abundantly in millimolar concentrations in the living body. The fluorescence intensity with Cd²⁺ was slightly weakened by the addition of Zn²⁺. Next, we evaluated the fluorescence changes of 5b from pH 4.0 to 10.0 in the absence and presence of Zn²⁺ (Figure 5). The fluorescence intensity of 5b with Zn²⁺ decreased in acidic conditions (pH 4.0 to 6.0). Protons might reflect the complexation Zn²⁺ with 5b. On the other hand, the fluorescence intensity of the emission maximum at 504 nm was stable within the pH range 7.0–8.0. These results indicated that compound 5b could be utilized for cellular experiments under physiological conditions.

To evaluate the efficacy of 5b for living cell Zn²⁺ imaging, we conducted fluorescence microscopy studies in human lung adenocarcinoma cells (A549). The cells incubated with 5b showed a negligible fluorescence due to its low background fluorescence (Figure 6a). After addition of both 5b (100 μM) and Zn²⁺/pyrithione (100 μM), bright intracellular fluorescence was observed (Figure 6b). The addition of TPEN as a Zn²⁺ high-affinity chelator decreased the cellular fluorescence of 5b (Figure 6c) [24]. These results indicated that compound 5b possessed good cell membrane permeability and that the intracellular fluorescence change was due to the selective interaction between compound 5b and Zn²⁺. In these experiments, zinc toxicity was not observed. We further evaluated the ability of 5b to detect endogenous Zn²⁺ in apoptotic cells. It has been reported that Zn²⁺ is released from intracellular zinc stores when the cells are in the apoptosis stage [25]. Fluorescence enhancement was not observed in A549 cells after the induction of apoptosis by incubation with H₂O₂ (200 μM) for 24 h (Figure 7a). On the other hand, bright fluorescence was observed when compound 5b was supplied to the cells and incubated for 30 min at 37 °C (Figure 7b). To investigate fluorescence enhancement due to the interaction between compound 5b and endogenous Zn²⁺, the cells treated with H₂O₂ for 24 h were additionally incubated with TPEN after treatment with 5b. As shown in Figure 7c, the intracellular fluorescence decreased upon the addition of TPEN, suggesting that compound 5b could detect endogenous labile zinc ions.

4. Conclusions

To detect endogenous Zn²⁺, we designed and synthesized low-background-fluorescence sensors 3a,b,4a,b, and 5a,b from one-pot reactions of 2-acetylpyridines, 2-acetyl-4-methylpyridine, or 2-acetylpyrazine with sulfonyl ketene dithioacetals and investigated their fluorescence properties. The sulfonyl group between the phenyl ring and the three-position of the pyridone ring affected the ICT state, and the background fluorescence of 3a,b,4a,b, and 5a,b was considerably decreased. The CHEF effects of 3a,b,4a,b, and 5a,b were observed upon addition of Zn²⁺. Methyl substitution on the R positions of 3a, 4a, and 5a influenced the emission maximum wavelengths, and the replacement of pyridine with pyrazine induced large, approximately 60–70 nm bathochromic shifts. Upon addition of Zn²⁺, the pyrazine compound 5b exhibited favorable properties, including an emission wavelength at 504 nm, a large Stokes shift (over 100 nm), a large fluorescence enhancement, Zn²⁺ selectivity, and stability in physiological pH conditions. Furthermore, compound 5b exhibited favorable cell membrane permeability and selective detection of Zn²⁺ in living human lung adenocarcinoma A549 cells and visualized endogenous labile zinc ions from the cells during apoptosis. We expect that the pyrazine–pyridone biheteroaryl-based compound 5b will contribute toward a better understanding of Zn²⁺ biological functions.

Supplementary Materials

The following are available online at https://www.mdpi.com/1424-8220/19/9/2049/s1, Figure S1: Job’s plot analysis of (a) 3a (10⁻⁵ M, λ_em = 381 nm); ); (b) 3b (10⁻⁵ M, λ_em = 367 nm); (c) 4a (10⁻⁵ M, λ_em = 353 nm); (d) 4b (10⁻⁵ M, λ_em = 360 nm); (e) 5a (10⁻⁵ M, λ_em = 377 nm); (f) 5b (10⁻⁵ M, λ_em = 395 nm). The total concentration of each compound and Zn²⁺ are 10 μM in HEPES buffer (100 mM, 5% DMSO, pH = 7.4). λ_em = emission wavelength. Figure S2: Typical Benesi-Hildebrand analysis of (a) 3a, (b) 3b, (c) 4a, (d) 4b, (e) 5a and (f) 5b. Figure S3: Fluorescence responses of (a) 3a (10⁻⁵ M, λ_em = 381 nm); (b) 3b (10⁻⁵ M, λ_em = 367 nm); (c) 4a (10⁻⁵ M, λ_em = 353 nm); (d) 4b (10⁻⁵ M, λ_em = 360 nm); (e) 5a (10⁻⁵ M, λ_em = 377 nm); (f) 5b (10⁻⁵ M, λ_em = 395 nm) upon addition of Pb²⁺ in HEPES buffer (100 mM, 5% DMSO, pH = 7.4). λ_em = emission wavelength.

Author Contributions

Conceptualization, M.H. and T.M.; investigation, M.T., N.M., and Y.K; writing—original draft preparation, M.H. and T.M.; writing—review and editing, M.H., S.K., H.S., and T.M.; visualization, M.H.; supervision, S.K., H.S., and T.M.; funding acquisition, M.H. and T.M.

Funding

This work was partly supported by the Konica Minolta Imaging Science Encouragement Award of Konica Minolta Science and Technology Foundation.

Conflicts of Interest

The authors declare no conflict of interest.

References

Anastassopoulou, J.; Theophanides, T. The role of metal ions in biological systems and medicine. Bioinorg. Chem. 1995, 459, 209–218. [Google Scholar]
Carter, K.P.; Young, A.M.; Palmer, A.E. Fluorescent sensors for measuring metal ions in living systems. Chem. Rev. 2014, 114, 4564–4601. [Google Scholar] [CrossRef]
Hemmens, B.; Goessler, W.; Schmidt, K.; Mayer, B. Role of bound zinc in dimer stabilization but not enzyme activity of neuronal nitric-oxide synthase. J. Biol. Chem. 2000, 275, 35786–35791. [Google Scholar] [CrossRef] [PubMed]
Choi, S.; Cui, C.; Luo, Y.; Kim, S.H.; Ko, J.K.; Huo, X.; Ma, J.; Fu, L.W.; Souza, R.F.; Korichneva, I.; et al. Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orai1. FASEB J. 2018, 32, 404–416. [Google Scholar] [CrossRef]
Bonaventura, P.; Benedetti, G.; Albarède, F.; Miossec, P. Zinc and its role in immunity and inflammation. Autoimmun. Rev. 2015, 14, 277–285. [Google Scholar] [CrossRef] [PubMed]
Vergnano, A.M.; Rebola, N.; Savtchenko, L.P.; Pinheiro, P.S.; Casado, M.; Kieffer, B.L.; Rusakov, D.A.; Mulle, C.; Paoletti, P. Zinc dynamics and action at excitatory synapses. Neuron 2014, 82, 1101–1104. [Google Scholar] [CrossRef]
Krężel, A.; Maret, W. The biological inorganic chemistry of zinc ions. Arch. Biochem. Biophys. 2016, 611, 3–19. [Google Scholar] [CrossRef]
Kawahara, M.; Mizuno, D.; Koyama, H.; Konoha, K.; Ohkawara, S.; Sadakane, Y. Disruption of zinc homeostasis and the pathogenesis of senile dementia. Metallomics 2014, 6, 209–219. [Google Scholar] [CrossRef] [PubMed]
Li, L.B.; Wang, Z.Y. Disruption of brain zinc homeostasis promotes the pathophysiological progress of Alzheimer’s disease. Histol. Histopathol. 2016, 31, 623–627. [Google Scholar]
Que, E.L.; Domaille, D.W.; Chang, C.J. Metals in neurobiology: Probing their chemistry and biology with molecular imaging. Chem. Rev. 2008, 108, 1517–1549. [Google Scholar] [CrossRef]
Pak, Y.L.; Swamy, K.M.; Yoon, J. Recent progress in fluorescent imaging probes. Sensors 2015, 15, 24374–24396. [Google Scholar] [CrossRef] [PubMed]
Nolan, E.M.; Lippard, S.J. Small-molecule fluorescent sensors for investigating zinc metalloneurochemistry. Acc. Chem. Res. 2009, 42, 193–203. [Google Scholar] [CrossRef] [PubMed]
Dong, W.K.; Akogun, S.F.; Zhang, Y.; Sun, Y.X.; Dong, X.Y. A reversible “turn-on” fluorescent sensor for selective detection of Zn²⁺. Sens. Act. B Chem. 2017, 238, 723–734. [Google Scholar] [CrossRef]
Hagimori, M.; Mizuyama, N.; Tominaga, Y.; Mukai, T.; Saji, H. A low-molecular-weight fluorescent sensor with Zn²⁺ dependent bathochromic shift of mission wavelength and its imaging in living cells. Dyes Pigm. 2015, 113, 205–209. [Google Scholar] [CrossRef]
Hagimori, M.; Mizuyama, N.; Yamaguchi, Y.; Saji, H.; Tominaga, Y. A novel small molecule fluorescent sensor for Zn²⁺ based on pyridine-pyridone scaffold. Talanta 2011, 83, 1730–1735. [Google Scholar] [CrossRef] [PubMed]
Hagimori, M.; Temma, T.; Mizuyama, N.; Uto, T.; Yamaguchi, Y.; Tominaga, Y.; Mukai, T.; Saji, H. A high-affinity fluorescent Zn²⁺ sensor improved by the suppression of pyridine-pyridone tautomerism and its application in living cells. Sens. Act. B Chem. 2015, 213, 45–52. [Google Scholar] [CrossRef]
Tominaga, Y.; Hidaki, S.; Matsuda, Y.; Kobayashi, G.; Sakemi, K. Sulfonyl ketenethioacetal. II. Reactions of pyridinium and isoquinolinium N-ylides with phynylsulfonyl ketenethioacetal. Yakugaku Zasshi 1979, 99, 540–542. [Google Scholar] [CrossRef]
Hagimori, M.; Matsui, S.; Mizuyama, N.; Yokota, K.; Nagaoka, J.; Tomionaga, Y. Novel synthesis of 4H-quinolizine derivatives using sulfonyl ketene dithioacetals. Eur. J. Org. Chem. 2009, 2009, 5847–5853. [Google Scholar] [CrossRef]
Benesi, H.A.; Hildebrand, J.H. A spectrophotometric investigation of the interaction of iodine with aromatic hydrocarbons. J. Am. Chem. Soc. 1949, 71, 2703–2707. [Google Scholar] [CrossRef]
Kumawat, L.M.; Asif, M.; Gupta, V.K. Dual ion selective fluorescence sensor with potential applications in sample monitoring and membrane sensing. Sens. Act. B Chem. 2017, 241, 1090–1098. [Google Scholar] [CrossRef]
Olguín, J.; Brooker, S. Spin crossover active iron(II) complexes of selected pyrazole-pyridine/pyrazine ligands. Cood. Chem. Rev. 2011, 255, 203–240. [Google Scholar] [CrossRef]
Weng, Y.; Chen, Z.; Wang, F.; Xue, L.; Jiang, H.. High sensitive determination of zinc with novel water-soluble small molecular fluorescent sensor. Anal. Chem. Acta 2009, 647, 215–218. [Google Scholar] [CrossRef] [PubMed]
Ghosh, K.; Saha, I. Selective sensing of Zn(II) ion by a simple anthracene-based tripodal chemosensor. Tetrahedron Lett. 2010, 51, 4995–4999. [Google Scholar] [CrossRef]
Golovine, K.; Uzzo, R.G.; Makhov, P.; Crispen, P.L.; Kunkle, D.; Kolenko, V.M. Depletion of intracellular zinc increases expression of tumorigenic cytokines VEGF, IL-6 and IL-8 in prostate cancer cells via NF-κB dependent pathway. Prostate 2008, 68, 1443–1449. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Zalewski, P.D.; Forbes, I.J.; Seamark, R.F.; Borlinghaus, R.; Betts, W.H.; Lincoln, S.F.; Ward, A.D. Flux of intracellular labile zinc during apoptosis (gene-directed cell death) revealed by a specific chemical probe. Chem. Biol. 1994, 1, 53–61. [Google Scholar] [CrossRef]

Figure 1. Schematic structure of novel fluorescence Zn²⁺ sensors.

Scheme 1. Synthesis of compounds 3a,b, 4a,b, and 5a,b.

Figure 2. Fluorescence spectra of (a) 3a (10⁻⁵ M, λ_ex = 381 nm); (b) 3b (10⁻⁵ M, λ_ex = 367 nm); (c) 4a (10⁻⁵ M, λ_ex = 353 nm); (d) 4b (10⁻⁵ M, λ_ex = 360 nm); (e) 5a (10⁻⁵ M, λ_ex = 377 nm); (f) 5b (10⁻⁵ M, λ_ex = 395 nm) upon addition of Zn²⁺ in the form of perchlorate salt (0, 1, 2.5, 5, 10, 50, 100 μM) in HEPES buffer (100 mM, 5% DMSO, pH = 7.4). λ_ex = excitation wavelength. The narrow fluorescence band of (a), (b), (d), and (e) were the Raman scattering peak of the solvent.

Figure 3. Fluorescence responses of (a) 3a (10⁻⁵ M, λ_em = 381 nm); (b) 3b (10⁻⁵ M, λ_em = 367 nm); (c) 4a (10⁻⁵ M, λ_em = 353 nm); (d) 4b (10⁻⁵ M, λ_em = 360 nm); (e) 5a (10⁻⁵ M, λ_em = 377 nm); (f) 5b (10⁻⁵ M, λ_em = 395 nm) upon addition of different metal cations in HEPES buffer (100 mM, 5% DMSO, pH = 7.4). λ_em = emission wavelength.

Figure 4. Competitive binding experiments of 5b, in which competing metal ions (10⁻³ M) and Zn²⁺ (10⁻³ M) were added to 5b (10⁻⁵ M) in HEPES buffer (100 mM, 5% DMSO, pH = 7.4).

Figure 5. (a) Effect of pH on the fluorescence properties of 5b in the absence (□) and presence (■) of Zn²⁺. (b) Enlarged figure of 5b in the absence (□) of Zn²⁺.

Figure 6. Fluorescence images of exogenously added Zn²⁺ in A549 cells: (a) Fluorescence image of cells with 5b (100 μM); (b) fluorescence image incubated with 5b (100 μM) in the presence of Zn²⁺/pyrithione (100 μM); (c) fluorescence image of (b) after treatment with TPEN (100 μM). Scale bars = 300 μm.

Figure 7. Fluorescence images of endogenous Zn²⁺ in A549 cells: (a) Fluorescence image of cells with 5b (100 μM); (b) fluorescence image incubated with 5b (100 μM) after incubation with H₂O₂ (200 μM) for 24 h; (c) fluorescence image of (b) after treatment with TPEN (100 μM). Scale bars = 300 μm.

Table 1. Fluorescence data for compounds 3a,b, 4a,b, and 5a,b.

Compounds	E_x max (nm)	E_m max (nm)	SS^a (nm)	Ф^b Zn²⁺ free (×10^–4)	Ф^b Zn²⁺ complex (×10⁻⁴)	K_d (×10⁻⁵ M)
3a	381	438	57	9.8 ± 3.0	35 ± 3	8.0 ± 7.1
3b	367	442	75	9.2 ± 0.9	21 ± 7	5.5 ± 3.3
4a	353	430	77	4.1 ± 1.0	10 ± 4	9.4 ± 3.4
4b	360	435	75	5.1 ± 1.7	11 ± 1	1.6 ± 1.4
5a	377	502	125	4.5 ± 1.1	10 ± 4	59 ± 24
5b	395	504	109	5.4 ± 1.6	40 ± 13	32 ± 25

^a Stokes shift. ^b The fluorescence quantum yield values were measured with respect to quinine sulfate solution as standard.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Share and Cite

MDPI and ACS Style

Hagimori, M.; Taniura, M.; Mizuyama, N.; Karimine, Y.; Kawakami, S.; Saji, H.; Mukai, T. Synthesis of a Novel Pyrazine–Pyridone Biheteroaryl-Based Fluorescence Sensor and Detection of Endogenous Labile Zinc Ions in Lung Cancer Cells. Sensors 2019, 19, 2049. https://doi.org/10.3390/s19092049

AMA Style

Hagimori M, Taniura M, Mizuyama N, Karimine Y, Kawakami S, Saji H, Mukai T. Synthesis of a Novel Pyrazine–Pyridone Biheteroaryl-Based Fluorescence Sensor and Detection of Endogenous Labile Zinc Ions in Lung Cancer Cells. Sensors. 2019; 19(9):2049. https://doi.org/10.3390/s19092049

Chicago/Turabian Style

Hagimori, Masayori, Mana Taniura, Naoko Mizuyama, Yasushi Karimine, Shigeru Kawakami, Hideo Saji, and Takahiro Mukai. 2019. "Synthesis of a Novel Pyrazine–Pyridone Biheteroaryl-Based Fluorescence Sensor and Detection of Endogenous Labile Zinc Ions in Lung Cancer Cells" Sensors 19, no. 9: 2049. https://doi.org/10.3390/s19092049

APA Style

Hagimori, M., Taniura, M., Mizuyama, N., Karimine, Y., Kawakami, S., Saji, H., & Mukai, T. (2019). Synthesis of a Novel Pyrazine–Pyridone Biheteroaryl-Based Fluorescence Sensor and Detection of Endogenous Labile Zinc Ions in Lung Cancer Cells. Sensors, 19(9), 2049. https://doi.org/10.3390/s19092049

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Menu