OFF switching Zn2+ sensor based on pyridine–pyridone scaffold

Sensors and Actuators B 181 (2013) 823–828

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Fluorescence ON/OFF switching Zn2+ sensor based on pyridine–pyridone scaffold Masayori Hagimori a,∗ , Takuhiro Uto b , Naoko Mizuyama c , Takashi Temma d , Yasuchika Yamaguchi b , Yoshinori Tominaga e , Hideo Saji d,∗∗ a

Faculty of Pharmaceutical Sciences, Kobe Pharmaceutical University, 4-19-1 Motoyamakita Machi, Higashinada Ku, Kobe 658-8558, Japan Faculty of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo 859-3298, Japan Department of Hospital Pharmacy, Saga University Hospital, 5-1-1 Nabeshima, Saga 849-8521, Japan d Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan e Faculty of Environmental Studies, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan b c

a r t i c l e

i n f o

Article history: Received 27 November 2012 Received in revised form 4 February 2013 Accepted 7 February 2013 Available online 27 February 2013 Keywords: Zn2+ Fluorescent ON/OFF switching Pyridine–pyridone Small molecule Water-soluble Large Stokes shift

a b s t r a c t We report a novel series of fluorescent Zn2+ sensors based on a pyridine–pyridone core structure, which functions as both the chelating part for Zn2+ and as the fluorophore. Pyridine–pyridone derivatives obtained by replacement of an electron-donating group with an electronic-withdrawing group at the 3-position of the pyridone ring showed almost no background fluorescence because electron transfer to the fluorophore was inhibited. However, these derivatives showed strong fluorescence with Zn2+ . Coordination with Zn2+ greatly influenced the electron transfer of the fluorophore, and, as a result, enabled fluorescence ON/OFF switching. In these sensors, 4-[4-(methylsulfanyl)-2-oxo-6-(pyridin-2yl)-1,2-dihydropyridin-3-yl]benzoic acid (5) displayed excellent properties: small molecular weight (MW = 338), good water solubility, large Stokes shift (132 nm), and fluorescence ON/OFF switching with Zn2+ . In addition, fluorescence images of Zn2+ with 5 in living cells show that the new fluorescent sensor is useful for biological applications. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Zinc is the second most abundant metal, after iron, and is an essential element for supporting life. It plays an important role in many biochemical processes, including gene expression, apoptosis, enzyme regulation, immune responses, and neurotransmission [1–6]. Zn2+ is present in living cells over a wide concentration range (nanomolar to millimolar). There are two forms of Zn2+ in living systems. The first is a form that is tightly chelated with proteins, which is known as a metalloprotein. The other is the free or chelatable form. Although most Zn2+ exists in chelated forms, the latter form is also believed to play an important role in biological systems. It is known that free or chelatable Zn2+ is released in response to cellular signaling [7–9]. In the central nervous system, free or chelatable Zn2+ ions are co-localized with glutamate in the presynaptic vesicles of the mammalian hippocampus [10,11]. Although a large amount of work has contributed to the understanding of the roles played by Zn2+ in physiology, and particularly in the field of

∗ Corresponding author. Tel.: +81 784417540; fax: +81 784417541. ∗∗ Corresponding author. Tel.: +81 757534556. E-mail addresses: [email protected] (M. Hagimori), [email protected] (H. Saji). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.02.033

neurochemistry, the actions of chelatable or free Zn2+ in terms of human health and diseases remain largely unexplored. To address the basic questions in zinc biology, we need to know the concentration of free or chelatable Zn2+ in each tissue and in different (patho-) physiological states. For this purpose, however, we cannot use conventional analytical methods such as ultraviolet/visible (UV/vis), nuclear magnetic resonance (NMR), or electron paramagnetic resonance (EPR) spectroscopies because of the 3d10 4s0 electronic configuration of Zn2+ . Much attention has therefore been paid to fluorescence-based detection methods, because of their high optical sensitivity. Indeed, a variety of elegant fluorescent sensors have been developed recently [12–19]. In principle, such tools can be used to monitor Zn2+ in biological samples and to provide useful information about zinc biology. However, most of the fluorescent sensors presented so far possess a fluorescent core and a separate part for binding to Zn2+ within the molecule, so the molecular weight is usually large, and the molecules are hydrophobic. As a result, the applications of such molecules in biological systems are often difficult because of their low solubility in aqueous media. We clearly need to develop a new class of fluorescent sensors for Zn2+ with improved molecular characteristics. Recently, we reported the small molecular weight fluorescent sensor 1 (Fig. 1) for Zn2+ , based on pyridine–pyridine [20]. The pyridine–pyridone sensor, as the chelating functionality for

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2.2. Synthesis of 4-(methylsulfanyl)-3-(naphthalen-1-yl)-6(pyrid-2-yl)pyridin-2(1H)-one (4a)

Fig. 1. Structure of compound 1.

Zn2+ , also nicely provides the fluorescent part within this single molecule, so the molecular weight can be minimized (MW = 324). A small molecular weight is a primary requisite for aqueous solubility and cell permeability [21]. In addition, this sensor exhibited a very large Stokes shift (>100 nm), enabling researchers to detect emitted light without too much interference from the excitation light, which is a potential advantage for biological applications. However, we need to address two issues in order to realize a new fluorescent Zn2+ -selective sensor for biological investigations. A selective turn-on response to Zn2+ was seen with 1, but the fluorescence enhancement with Zn2+ was only 4.4-fold because of the relatively strong background fluorescence. Moreover, the aqueous solubility of 1 was still low for the analysis of biological samples. To improve these properties, we investigated a series of pyridine–pyridone compounds with different substituents at the 3-position of the pyridone ring. The mechanism of the fluorescence signal transduction of 1 is internal charge transfer (ICT), which has been widely used in metal-detecting sensors [22–24]. A fluorophore with both an electron-acceptor and an electron-donor can form an ICT state and show strong fluorescence on excitation with light. Compound 1 therefore showed relatively strong background fluorescence. In this paper, we report a new water-soluble fluorescent sensor for Zn2+ , based on a pyridine–pyridone core structure. Pyridine–pyridone derivatives obtained by replacement of an electron-donating group with an electronic-withdrawing group at the 3-position of the pyridone ring showed almost no background fluorescence because electron transfer to the fluorophore was inhibited. However, these derivatives showed strong fluorescence with Zn2+ . Coordination with Zn2+ greatly influenced the electron transfer of the fluorophore, and, as a result, enabled fluorescence ON/OFF switching. We describe the synthesis and fluorescence properties of the new compounds, and the evaluation of 4-[4-(methylsulfanyl)-2oxo-6-(pyridin-2-yl)-1,2-dihydropyridin-3-yl]benzoic acid (5) for biological investigations.

2. Experimental 2.1. Materials and instruments All the solvents were of analytic grade and used as received. The solutions of metal ions were prepared from NaCl, KCl, MgCl2 ·6H2 O, CaCl2 , FeCl2 ·4H2 O, FeCl3 ·6H2 O, CoCl2 ·6H2 O, NiCl2 ·6H2 O, ZnCl2 , CdCl2 ·2.5H2 O, CuCl2 , MnCl2 ·4H2 O, AlCl3 ·6H2 O, respectively, and were dissolved in distilled water. 1 H and 13 C NMR were measured on a JEOL-GX-400 (400 MHz) and a Varian Mercury-300 (300 MHz) with chemical shifts reported as ppm (in DMSO-d6 and CDCl3 ). HRMS were measured on a JMS-T100LP mass spectrometer. Mass spectra (MS) were recorded on a JEOL-DX-303 mass spectrometer and a JMS-T100LP mass spectrometer. Microanalyses were measured on a Perkin-Elmer instrument. Fluorescence spectra were determined on a Jasco FP-6200 spectrofluorometer. Infrared (IR) spectra were recorded in potassium bromide pellets on JASCO 810.

Powdered sodium hydroxide (0.40 g, 10.0 mmol) was added to a solution of 0.56 g (2.49 mmol) of 3,3-bis-methylsulfanyl-1-pyridin2-yl-propenone (2) and 0.50 g (2.99 mmol) of 3a in 50 mL of DMSO, and the mixture was stirred for 2 h at room temperature. The reaction was poured into 300 mL of ice water and neutralized with a 10% hydrochloric acid solution. The mixture was extracted with 100 mL of dichloromethane three times. The combined organic extracts were washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. A mixture of the residue and a 1% hydrochloric acid solution was refluxed for 1 h. After evaporation, the residual solid was recrystallized from methanol to give 4a (0.26 g, 31%) as pale yellow crystals. mp 231–232 ◦ C. IR (KBr, cm−1 ): 3450, 1580, 1540, 1515, 1350, 1250, 1210, 1170, 1140, 1030. 1 H NMR (CDCl3 , 400 MHz) ı 2.44 (s, 3H), 7.31 (d, J = 7.3 Hz, 1H), 7.41 (d, J = 6.9 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.53 (m, 3H), 7.94 (m, 3H), 8.32 (d, J = 8.3 Hz, 1H), 8.75 (d, J = 5.4 Hz, 1H), 11.23 (bs, 1H). 13 C NMR (CDCl3 , 75 MHz) ı 14.17, 101.70, 121.07, 124.78, 124.83, 125.02, 125.61, 125.67, 125.83, 126.02, 127.99, 128.07, 128.25, 131.18, 133.22, 133.26, 133.42, 137.65, 149.44, 149.71, 153.40, 159.75. MS (ESI) m/z: 345 (M+ +1). HRMS calcd for C21 H16 N2 OS: 344.0983; found: 344.0988. 2.3. Synthesis of 4-(methylsulfanyl)-3-phenyl-6-(pyridin-2-yl) pyridin-2(1H)-one (4b) This compound 4b (0.68 g, 2.31 mmol) was prepared in 46% yield from 1.13 g (5.02 mmol) of 2 and 0.70 g (5.98 mmol) of 3b in a manner similar to that described for the synthesis of 4a. An analytical sample was recrystallized from methanol to give pale yellow crystals, mp 281–283 ◦ C. IR (KBr, cm−1 ): 3370, 1695, 1610, 1550, 1510, 1460, 1420, 1065. 1 H NMR (CDCl3 , 400 MHz) ı 2.46 (s, 3H), 6.85 (s, 1H), 7.34–7.50 (m, 6H), 7.83 (ddd, J = 1.7, 8.0, 8.0 Hz, 1H), 7.90 (d, J = 7.8 Hz, 1H), 8.68 (d, J = 3.9 Hz, 1H), 10.6 (brs, 1H). 13 C NMR (CDCl , 100 MHz) ı 15.40, 99.81, 119.48, 124.62, 128.17, 3 128.42, 130.06, 134.67, 137.28, 139.80, 147.85, 149.42, 151.87, 160.13. Ms m/z: 295 (M+ +1, 3), 294 (M+ , 13), 248 (20), 247 (100), 140 (8), 106 (10), 79 (8), 78 (43), 51 (7), 44 (6). Anal. calcd for C17 H14 N2 OS: C, 69.36; H, 4.79; N, 9.52%. Found: C, 69.37; H, 4.29; N, 9.35%. 2.4. Synthesis of 3-(4-chlorophenyl)-4-(methylsulfanyl)-6(pyridin-2-yl)pyridin-2(1H)-one (4c) This compound 4c (0.64 g, 1.95 mmol) was prepared in 39% yield from 1.13 g (5.02 mmol) of 2 and 0.91 g (6.03 mmol) of 3c in a manner similar to that described for the synthesis of 4a. An analytical sample was recrystallized from methanol to give pale yellow crystals, mp 195–196 ◦ C. IR (KBr, cm−1 ): 3317, 1620, 1592, 1561, 1517, 1455, 1422, 1090. 1 H NMR (DMSO-d6 , 400 MHz) ı 2.50 (s, 3H), 7.18 (s, 1H), 7.30 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.3 Hz, 2H), 7.51 (dd, J = 5.3, 6.8 Hz, 1H), 7.98 (dd, J = 6.8, 7.8 Hz, 1H), 8.26 (d, J = 7.8 Hz, 1H), 8.72 (d, J = 4.3 Hz, 1H), 11.3 (1H, brs, NH). 13 C NMR (DMSO-d6 , 100 MHz) ı 14.40, 121.13, 124.92, 128.13, 128.90, 130.05, 131.95, 132.13, 132.25, 133.98, 137.70, 149.48, 151.91, 159.44. Ms m/z: 328 (M+ ), 330 (M+ +2). HRMS calcd for C17 H13 ClN2 OS: 328.0437; found: 328.0460. 2.5. Synthesis of 4-(methylsulfanyl)-6-(pyrid-2-yl)-3[4-(trifluoromethyl)phenyl]pyridin-2(1H)-one (4d) This compound 4d (0.56 g, 1.55 mmol) was prepared in 31% yield from 1.13 g (5.02 mmol) of 2 and 1.11 g (6.00 mmol) of 3d in a manner similar to that described for the synthesis of 4a. An

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analytical sample was recrystallized from methanol to give pale yellow crystals, mp 263–264 ◦ C. IR (KBr, cm−1 ): 3330, 3051, 1617, 1599, 1577, 1581, 1439, 1021. 1 H NMR (DMSO-d6 ) ı: 2.51 (s, 3H), 4.90 (brs, 1H), 7.33 (s, 1H), 7.53 (d, J = 8.3 Hz, 2H), 7.61 (dd, J = 4.8, 6.8 Hz, 1H), 7.79 (d, J = 7.8 Hz, 2H), 8.09 (dd, J = 5.8, 7.6, 1H), 8.34 (d, J = 7.8 Hz, 1H), 8.76 (d, J = 4.8 Hz, 1H). 13 C NMR (DMSO-d6 ) ı: 14.49, 102.98, 121.77, 122.96, 123.78, 125.02, 125.29, 125.66, 127.95, 128.27, 131.16, 138.89, 139.47, 143.29, 148.59, 149.06, 152.32, 159.46. MS (ESI) m/z: 363 (M+ +1). HRMS calcd for C18 H13 F3 N2 OS: 362.0701; found: 362.0657. 2.6. Synthesis of methyl 4-(4-(methylsulfanyl)-2-oxo-6-(pyridin2-yl)-1,2-dihydropyridin-3-yl)benzoate (4e)

825

buffer (pH 3.0), 100 mM acetate buffer (pH 4.0–5.0), 100 mM phosphate buffer (pH 6.0), 100 mM HEPES buffer (pH 7.0–8.0), 100 mM tris–HCl buffer (pH 9.0). The dissociation constant (Kd ) of 5 in HEPES buffer was determined by plotting the fluorescence intensity to free Zn2+ concentration. The metal selectivity of 5 was investigated in HEPES buffer (100 mM, 5% DMSO, pH = 7.4). The cational solutions were prepared from NaCl, KCl, MgCl2 ·6H2 O, CaCl2 , FeCl2 ·4H2 O, FeCl3 ·6H2 O, CoCl2 ·6H2 O, NiCl2 ·6H2 O, ZnCl2 , CdCl2 ·2.5H2 O, CuCl2 , MnCl2 ·4H2 O, AlCl3 ·6H2 O (10−3 M), respectively. The measurements were carried out at 298 K. The fluorescence quantum yield values were measured with respect to quinine sulfate solution (˚ = 0.54) as standard. 2.9. Fluorescence microscope images in cells

This compound 4e (0.93 g, 2.64 mmol) was prepared in 53% yield from 1.13 g (5.02 mmol) of 2 and 1.05 g (6.00 mmol) of 3e in a manner similar to that described for the synthesis of 4a. An analytical sample was recrystallized from methanol to give pale yellow leaflets, mp 225–226 ◦ C. IR (KBr, cm−1 ): 3099, 2813, 1612, 1599, 1324, 1275, 1113. 1 H NMR (DMSO-d6 , 400 MHz) ı 2.58 (s, 3H), 3.88 (s, 3H), 7.22 (s, 1H), 7.45 (d, J = 7.3 Hz, 2H), 7.52 (ddd, J = 3.4, 3.9, 4.8 Hz, 1H), 8.02–8.05 (m, 3H), 8.27 (d, J = 8.2 Hz, 1H), 8.73 (d, J = 4.4 Hz, 1H), 11.3 (brs, 1H). 13 C NMR (DMSO-d6 , 100 MHz) ı 14.43, 52.16, 100.20, 118.50, 121.15, 125.75, 128.84, 129.13, 130.69, 137.68, 138.64, 140.31, 149.46, 151.93, 157.15, 159.27, 166.07. Ms m/z: 353 (M+ +1), 322 (M+ ). HRMS calcd for C19 H16 N2 O3 S: 352.0882; found: 352.0897. 2.7. Synthesis of 4-(4-(methylsulfanyl)-2-oxo-6-(pyridin-2-yl) -1,2-dihydropyridin-3-yl)benzoic acid (5) The compound 4e (0.20 g, 0.57 mmol) was stirred in 30 mL of 1 N NaOH for 2.5 h at room temperature. The reaction was neutralize with 1 N HCl. The precipitate that appeared was collected by filtration, washed with water, and dried by air to give 0.18 g (0.53 mmol, 95% yield) as colorless leaflets, mp 287–288 ◦ C. IR (KBr, cm−1 ): 3460, 3052, 2824, 1710, 1584, 1464, 1235, 1175, 1101, 1003. 1 H NMR (DMSO-d6 , 400 MHz) ı 2.50 (s, 3H), 7.22 (s, 1H), 7.40 (d, J = 7.8 Hz, 2H), 7.53 (ddd, J = 2.4, 4.9, 7.3 Hz, 1H), 7.90 (d, J = 8.3 Hz, 2H), 8.01 (dd, J = 1.5, 7.8 Hz, 1H), 8.27 (d, J = 7.8 Hz, 1H), 8.73 (d, J = 2.9 Hz, 1H). 13 C NMR (DMSO-d6 , 100 MHz) ı 14.34, 121.04, 124.84, 128.89, 130.33, 137.59, 139.60, 149.37, 151.80, 159.25, 167.13. Ms m/z: 339 (M+ +1). HRMS calcd for C18 H14 N2 O3 S: 338.0725; found: 338.0676. 2.8. Spectral measurement The compound stock solution (1 × 10−2 M) was prepared by directly dissolving in DMSO. For the fluorescence analysis, 5 (10−6 M) upon addition of Zn2+ in the form of perchlorate salt was measured in HEPES buffer (100 mM, 5% DMSO, pH = 7.4). The binding stoichiometry of 5 to Zn2+ was investigated by Job’s plot. We measured the fluorescence intensity of 5 in the following buffers: 100 mM glycine–HCl buffer (pH 2.0), 100 mM citrate

RAW264 macrophage cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FBS and 1% penicillin, at 37 ◦ C in a humidified atmosphere of 5% CO2 . The cells were incubated with 5 (30 ␮M) in culture media for 30 min at 37 ◦ C. After washing with phosphate-buffered saline (PBS), the treated cells were incubated with Zn2+ /pyrithione (50, 100, 150 ␮M) in culture media for 15 min at 37 ◦ C. The incubated cells were imaged by fluorescence microscopy (Nikon Eclipse E600). 3. Results and discussion We previously revealed that the pyridine–pyridone structure of 1 interacts with Zn2+ in the bipyridyl form, and the NH/OH proton of the pyridine–pyridone structure is needed for chelation-enhanced fluorescence (CHEF) effects with Zn2+ [20]. We therefore studied the substituent effects of different electron-withdrawing/donating groups on the aryl ring that did not participate in coordination with Zn2+ . Compounds 4a–e were synthesized by a one-pot reaction of 3,3-bis(methylsulfanyl-1-pyridin-2-yl)propenone (2) with 2-(4-substituted-phenyl)acetonitrile (3a–e) in the presence of powdered sodium hydroxide in dimethyl sulfoxide (DMSO), followed by treatment with 1% HCl (Scheme 1). We analyzed the fluorescence properties of 4a–e and compared them with those of 1; the fluorescence data are listed in Table 1. The fluorescence maxima of 4a–e ranged from 468 to 480 nm, and all the compounds exhibited significantly large Stokes shifts (>100 nm) in HEPES buffer (100 mM, 5% DMSO, pH = 7.4). Without Zn2+ , compound 4a, with a 1-naphthyl group at the 3position of the pyridone ring as an electron-donating group and a pyridine group at the 6-position of the pyridone ring as an electronwithdrawing group, exhibited higher fluorescence quantum yields (˚) than that of 1 (˚ = 0.25). This result was in accordance with the ICT mechanism; the introduction of electron-donating and electron-withdrawing groups has a great influence on the fluorescence intensity. Compound 4b, with an aryl group at the 3-position of the pyridone ring as a weak electron-donating group, exhibited weak fluorescence. Compounds 4c–e with electron-withdrawing groups exhibited almost no fluorescence. On addition of Zn2+ , all

Scheme 1. Synthesis of compounds 4a–e.

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Table 1 Fluorescence data for 4a–e. Compounds

Ex max (nm)

Em max (nm)

SSa (nm)

˚free b

˚Zn(II) c

˚Zn(II) /˚free d

4a 4b 4c 4d 4e 1

362 362 360 350 346 366

470 478 470 468 480 490

108 116 110 118 134 124

0.34 0.1 0.03 0.02 0.02 0.25

0.61 0.79 0.44 0.46 0.35 0.73

1.6 7.9 14.7 23 17.5 2.9

a b c d

Stokes shift. The fluorescence quantum yield values without Zn2+ were measured with respect to quinine sulfate solution as standard. The fluorescence quantum yield values with Zn2+ were measured with respect to quinine sulfate solution as standard. The ratio of ˚ with Zn2+ to ˚ without Zn2+ .

Scheme 2. Synthesis of compound 5.

350

Fluorescence Intensity

300

250

200

Fig. 4. Effect of pH on the fluorescence properties of 5 in the absence () and presence () of Zn2+ .

Zn2+

150

100

50

0

360

400

450

500

550

600

650

Wavelength (nm) Fig. 2. Fluorescence response of 5 (10−6 M) on addition of Zn2+ in the form of perchlorate salt (0, 0.25, 0.5, 1, 2.5, 5, 10, 50, and 100 ␮M) in HEPES buffer (100 mM, 5% DMSO, pH = 7.4; excitation wavelength: 350 nm).

Fluorescence Intensity at 472 nm

the compounds showed CHEF effects. Although 4a showed strong fluorescence, the ˚ value ratio in the presence/absence of Zn2+ was 1.6 because of its high background fluorescence. In contrast, the ˚ value ratios of 4b–e were higher than those of 4a and 1. In particular, 4c–e, with electron-withdrawing groups, showed high ˚ value

ratios. The electron density of the aryl ring at the 3-position of the pyridone ring weakened in the presence of electron-withdrawing groups, and electrons were pushed from the fluorophore to the aryl ring. As a result, the background fluorescences of 4c–e were almost zero. However, in coordination with Zn2+ , it was speculated that the electron density of the fluorophore greatly decreased because the fluorophore and the coordination site were almost the same. Electron transfer from the fluorophore to the aryl ring was therefore inhibited, resulting in the exhibited fluorescence. These results indicate that simple para modification of the phenyl ring strongly influences the fluorescence properties and improves the turn-on response to Zn2+ . Compounds 4c–e showed good fluorescence ON/OFF switching properties with Zn2+ ; however, high solubility to aqueous solutions was more necessary. We selected a compound with a carboxyl derivative at the para position of the aryl ring. The hydrolysis of 4e using 1 N NaOH solution gave the carboxylic derivative 5 in 95%

350 300 250 200 150 100 50 0

no

Al

Ba

Ca

Cd

Co

Cu

Fe

Fe

K

Mg

Mn

Na

Ni

Zn

Cations Fig. 3. Job’s plot of compound 5 and Zn2+ . The total concentrations of compound 5 and Zn2+ are 1 ␮M in HEPES buffer (100 mM, 5% DMSO, pH = 7.4).

Fig. 5. Fluorescence intensities of 5 (1 ␮M) at 472 nm with addition of metal cations (100 ␮M) in HEPES buffer (100 mM, 5% DMSO, pH = 7.4).

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Fig. 6. Fluorescence images in macrophages (RAW 264). (a) Images of cells incubated with only 5 (30 ␮M) and (b) images of cells incubated with 5 (30 ␮M) and Zn2+ /pyrithione (100 ␮M). Pyrithione is a zinc-selective ionophore.

yield, as established by 1 H and 13 C NMR spectroscopies and mass spectrometry (MS) (Scheme 2). Fig. 2 shows the emission spectra of 5 excited at 350 nm for various Zn2+ concentrations. Compound 5 also showed almost no fluorescence without Zn2+ (˚ = 0.02). On addition of Zn2+ , the emission intensity increased significantly (˚ = 0.32 at 100 ␮M ZnCl2 ) and a significantly large Stokes shift (132 nm) was observed. The detection limit was 0.5 ␮M ZnCl2 . In the spectral measurements, we confirmed that components of HEPES buffer solution did not affect the emission intensity and wavelength. Binding analysis (using a Job’s plot) between 5 and Zn2+ indicated that the complex formation has 1:1 stoichiometry (Fig. 3), and the dissociation constant Kd was calculated to be 3 × 10−5 M from the resulting titration data. Next, we investigated the effect of pH on the fluorescence properties of 5 in the absence/presence of Zn2+ (Fig. 4). When the solution pH decreased from pH 9.0 to pH 2.0, the fluorescence intensity of 5 remained constant for pH > 6, suggesting that this compound can be used to monitor intracellular Zn2+ under physiological conditions. We then examined the selectivity of 5 toward other cations (Al3+ , Ca2+ , Cd2+ , Co2+ , Cu2+ , Fe2+ , Fe3+ , K+ , Mg2+ , Mn2+ , Na+ , and Ni2+ ) in HEPES buffer (100 mM, 5% DMSO, pH = 7.4); the results are shown in Fig. 5. The results show that only Zn2+ and Cd2+ induce a large CHEF effect of 5 (24-fold enhancement for Zn2+ and 22-fold for Cd2+ ). There is very little Cd2+ in living systems, so we believe that this will have little influence on intracellular imaging of Zn2+ . Transition-metal ions (Cu2+ , Co2+ , Fe2+ , and Ni2+ ), except for Mn2+ , quenched the fluorescence of 5. CHEF effects in 5 were not observed for group III ions (Al3+ and Fe3+ ). In addition, alkali-metal ions (Na+ and K+ ) and alkaline-earth metal ions (Ca2+ and Mg2+ ) did not induce CHEF effects in 5. These ions are present in high concentrations in cells. These results therefore indicate that 5 might be used under a wide range of biological conditions. To evaluate the use of 5 in biological applications, we obtained fluorescence microscope images in living cells (mouse macrophage-like cells; RAW264). The cell membrane acts as a barrier to some chemicals; in particular, water-soluble (hydrophilic) substances cannot penetrate the cell membrane. Compound 5 has a low molecular weight, so it should be able to penetrate into the cell without further modification. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), and then incubated with 5 (30 ␮M) for 30 min at 37 ◦ C in a humidified atmosphere of 5% CO2 ; they showed very weak fluorescence [Fig. 6(a), n = 3]. After incubation with several concentrations of Zn2+ /pyrithione (50, 100, 150 ␮M) for 15 min, very strong intracellular fluorescence was observed in more than 100 ␮M [Fig. 6(b), n = 3]. These results indicate that 5 can be used as the fluorescence ON/OFF switching sensor for the detection of intracellular Zn2+ .

4. Conclusion We have described the synthesis, spectroscopy, and applications of a novel and simple fluorescent sensor, based on a pyridine–pyridone scaffold, for the detection of Zn2+ in biological applications. Compound 5 is a unique Zn2+ -responsive smallmolecule sensor that displays a selective turn-on response to Zn2+ by replacement of an electron-donating group with an electronicwithdrawing group at the 3-position of the pyridone ring. The molecular weight (MW = 338) is small enough for 5 to exhibit good cell permeability. The fluorescence microscopy experiments showed that 5 may be applicable to the detection of Zn2+ within living cells. We consider that this compound will be useful for studying the biological functions of Zn2+ . References [1] K.H. Falchuk, The molecular basis for the role of zinc in developmental biology, Molecular and Cellular Biochemistry 188 (1998) 41–48. [2] D.K. Perry, M.J. Smyth, H.R. Stennicke, G.S. Salvesen, P. Duriez, G.G. Poirier, Y.A. Hannun, Zinc is a potent inhibitor of the apoptotic protease, caspase-3. A novel target for zinc in the inhibition of apoptosis, Journal of Biological Chemistry 272 (1997) 18530–18533. [3] P.D. Zalewski, I.J. Forbes, W.H. Betts, Correlation of apoptosis with change in intracellular labile Zn(II) using zinquin [(2-methyl-8-p-toluenesulphonamido6-quinolyloxy)acetic acid], a new specific fluorescent probe for Zn(II), Biochemical Journal 296 (1993) 403–408. [4] W. Maret, Y. Li, Coordination dynamics of zinc in proteins, Chemical Reviews 109 (2009) 4682–4707. [5] H. Kitamura, H. Morikawa, H. Kamon, M. Iguchi, S. Hojyo, T. Fukada, S. Yamashita, T. Kaisho, S. Akira, M. Murakami, T. Hirano, Toll-like receptormediated regulation of zinc homeostasis influences dendritic cell function, Nature Immunology 7 (2006) 971–977. [6] M. Hershfinkel, K. Kandler, M.E. Knoch, M. Dagan-Rabin, M.A. Aras, C. Abramovitch-Dahan, I. Sekler, E. Aizenman, Intracellular zinc inhibits KCC2 transporter activity, Nature Neuroscience 12 (2009) 725–727. [7] C.J. Frederickson, J.Y. Koh, A.I. Bush, The neurobiology of zinc in health and disease, Nature Reviews Neuroscience 6 (2005) 449–462. [8] A.I. Bush, Metals and neuroscience, Current Opinion in Chemical Biology 4 (2000) 184–191. [9] M. Paker, F.L. Humoller, D.J. Mahler, Determination of copper and zinc in biological material, Clinical Chemistry 13 (1967) 40–48. [10] S.C. Burdette, S.J. Lippard, Meeting of the minds: metalloneurochemistry, Proceedings of the National Academy of Sciences of the United States of America 100 (2003) 3605–3610. [11] E.M. Nolan, S.J. Lippard, Small-molecule fluorescent sensors for investigating zinc metalloneurochemistry, Accounts of Chemical Research 42 (2009) 193–203. [12] K.L. Haas, K.J. Franz, Application of metal coordination chemistry to explore and manipulate cell biology, Chemical Reviews 109 (2009) 4921–4960. [13] E.L. Que, D.W. Domaille, C.J. Chang, Metals in neurobiology: probing their chemistry and biology with molecular imaging, Chemical Reviews 108 (2008) 1517–1549. [14] K. Kikuchi, K. Komatsu, T. Nagano, Zinc sensing for cellular application, Current Opinion in Chemical Biology 8 (2004) 182–191. [15] G.K. Walkup, S.C. Burdette, S.J. Lippard, R.Y. Tsien, A new cell-permeable fluorescent probe for Zn2+ , Journal of the American Chemical Society 122 (2000) 5644–5645.

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Biographies Masayori Hagimori received his BS in Pharmaceutical Sciences in 2001 from Nagasaki University, and his PhD in Pharmaceutical Sciences in 2012 from Kyoto University. After working on drug metabolism at Tukuba Research Institute of Banyu pharmaceutical CO., Ltd., he moved to Nagasaki International University, where he was an assistant professor in pharmaceutical science, and currently, he is a lecturer at Kobe Pharmaceutical University. He is currently working in the area of biophysical chemistry and focuses on development of optical and nuclear probes for biological analysis. Takuhiro Uto received his BS and MS in Biochemical Sciences and Technology from Kagoshima University in 2001 and 2003, respectively. He received his PhD in Biochemical Science and Technology from the United Graduate School of Agricultural Sciences, Kagoshima University in 2006. Following his degree, he was a post-doctoral fellow in the Department of Pediatrics at Medical University of South

Carolina (MUSC) from 2006 to 2007. Now he is working as an assistant professor at Nagasaki International University. His current research interests are target molecules of phytochemicals. Naoko Mizuyama received her BS in Pharmaceutical Science in 2000 and her PhD in Environmental Science in 2007 from Nagasaki University. She spent some years as a pharmacist at University of Occupational and Environmental Health, and then she moved to the department of hospital pharmacy of Saga University. Her research focuses on synthesis of fluorescent heterocycles from ketene-dithioacetals and chemical luminescence. Takashi Temma received his BS in Pharmaceutical Sciences in 2000 and his PhD in Pharmaceutical Sciences in 2008 from Kyoto University. Currently, he is an assistant professor in the Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University. He is currently working in the area of in vivo molecular imaging using optical and nuclear medical techniques, and focuses on development of molecular probes and in vivo functional analysis on cancers, atherosclerosis, and cerebral diseases. Yasuchika Yamaguchi was born in 1960 in Sasebo, Japan. He graduated from Kyushu University (1983) and received his PhD (1988) in Pharmaceutical Sciences from the same university (Thesis Director: late Prof. Ken Kanematsu). After postdoctoral work under the supervision of Sir Jack E. Baldwin at Oxford University, he joined CIBA-GEIGY AG in Basel (now Novartis Pharma AG) in 1990. He moved to Neurogen (CT, US) (2002–2005) and he currently is a professor of medicinal chemistry in Faculty of Pharmaceutical Sciences at Nagasaki International University since 2006. He was a co-recipient of Ciba Pharma Forschungspris (Ciba Pharmaceutical Research Prize) in 1996 and ACS Heroes of Chemistry Award in 2009. His current research interests include medicinal chemistry of ATP related compounds and phosphorylated amino acid mimics, and organic chemistry of cationic reactions. Yoshinori Tominaga received his BS in Pharmaceutical Sciences from Nagasaki University in 1969 and then received his PhD from Kyusyu University in 1974, working with the late Professor Goro Kobayashi. In 1971–1975 he was a Special Fellow of Japan Society for the Promotion of Science. In 1975 he joined the faculty of Nagasaki University in Nagasaki, Japan. In 1981–1982 and 1991–1992 he was postdoctoral fellow under later Professor Raymon N. Castle at Bring Young University and South Flroride University. His research interests have been in developing new synthetic reactions, use of ketene dithioacetals and carbon disulfide in heterocyclic chemistry, and synthesis of fluorescent materials. Hideo Saji received his BS in Pharmaceutical Sciences in 1974 and his PhD in Pharmaceutical Sciences from Kyoto University at Kyoto in 1980. He was an assistant professor of Nuclear Medicine at Kyoto University from 1977 to 1988, an associate professor of Pharmaceutical Sciences at Kyoto University from 1988 to 1996, and currently, he is a professor of Pharmaceutical Sciences at Kyoto University (1996–present). He is currently working in the area of molecular imaging and focuses on development of molecular probe and its application for basic life sciences, pharmaceutical development and medical application.