A novel small molecule fluorescent sensor for Zn 2+ based on pyridine–pyridone scaffold

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A novel small molecule fluorescent sensor for Zn 2+ based on pyridine–pyridone scaffold

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   1 A Novel Small Molecule Fluorescent Sensor for Zn 2+  Based on Pyridine-Pyridone Scaffold  Masayori Hagimori, a  Naoko Mizuyama, b Yasuchika Yamaguchi, a  Hideo Saji, c and Yoshinori Tominaga d  * a Faculty of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo 859-3298, Japan, b  Department of Hospital Pharmacy, Saga University Hospital, 5-1-1  Nabeshima, Saga 849-8521, Japan, c Graduate School of Pharmaceutical Sciences, Kyoto University, 46–29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto 606–8501, Japan, d  Faculty of Environmental Studies,  Nagasaki University,1-14 Bunkyo-machi, Nagasaki 852-8521, Japan Abstract The development of a water-soluble and small molecular weight fluorescent probe, 3-(4-methoxyphenyl)-4-(methylsulfanyl)-6-(pyridin-2-yl)pyridin-2(1H)-one ( 3 ), for detecting Zn 2+  based on  pyridine-pyridone skeleton is reported. We observed a clear chelation enhanced fluorescence effect of 3  in the presence of Zn 2+ . Other fluorescent properties of 3  are discussed. Keywords Zn 2+ , Fluorescent sensor, Pyridine-pyridone, Small molecule, Water-soluble 1. Introduction Zinc is an essential element for life and is known to play important roles in biological processes including gene expression, apoptosis, enzyme regulation, immune system and neurotransmission [1–6]. Most of Zn 2+  in living cells are bound to the proteins, which are called as metalloprotein [7–9]. Zinc metalloproteases employ Zn 2+  as the catalysis center and a most well-known example in this class is angiotensin converting enzyme (ACE) [10–12]. Zinc chelation also contributes to the structure stabilization of proteins. For example, the zinc finger proteins form a stable three dimensional structure to interact a specific DNA sequence for controlling gene functions [13].     2There also are free or chelatable Zn 2+  in living cells, which may be involved in both physiology and disease states. In pancreatic β -cells, Zn 2+  participates in controlling synthesis, storage and secretion of insulin. When Zn 2+  is short, blood sugar level rises by a secretion delay of the insulin [14,15]. In central nervous system, free or chelatable Zn 2+  are co-localized with glutamate in presynaptic vesicles of the mammalian hippocampus [16,17]. To investigate physiological roles of free or chelatable Zn 2+  in living cells, we should know their concentration in each tissue and in different (patho-) physiological states. In this context, a number of fluorescent sensors have recently been developed  based on elegant ideas [18–20]. Although they contributed great extends to understand Zn 2+  roles in physiology and particularly in the field of neurochemistry [16,17], its mechanisms of action is not well understood in comparison with other cations such as Na + , K  + , Ca 2 +, etc. We realized that Zn 2+  selective fluorescent probes have mainly used three core structures, namely quinoline, BF 2  chelated dipyrromethene and fluorescein [21–30]. After modification of the core structure in getting selectivity toward Zn 2+ , the molecule obviously became bigger and more hydrophobic. Therefore most of the fluorescent probes prepared so far have undesirable characteristics in terms of aqueous solubility. This problem should be overcome somehow to develop a new simple fluorescent probe based upon different chemical backbone structures. We thought that if we could find a new and small molecular weight (MW = ca. 300) fluorescent core structure having certain selectivity toward Zn 2+ , such molecules would become an advantageous starting point for designing a new   fluorescent sensor. If the initial core structure is small enough, the fluorescent probes may still be molecular weight below 500 with desirable physico-chemical properties, even after the modifications [31]. We have recently published a new and simple synthetic method for preparing pyridine-pyridone derivative 3  [32]. Since then we have been interested in the molecule characters of this class of heterocyclic compounds. In this paper, we report the fluorescent characteristics of pyridine-pyridone derivative 3 , MW = 324, that can potentially be used as a lead structure for finding a new fluorescent sensor for Zn 2+ . 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, BaCl 2 , MgCl 2 ·6H 2 O, CaCl 2 , FeCl 3 ·6H 2 O, CoCl 2 ·6H 2 O, NiCl 2 ·6H 2 O, ZnCl 2 , CdCl 2 ·2.5H 2 O, CuCl 2 , MnCl 2 ·4H 2 O, AlCl 3 ·6H 2 O, respectively, and were dissolved in distilled water. 1 H NMR was measured on a JEOL-GX-400 (400MHz) with chemical shifts reported as ppm (in   3DMSO- d  6 ). HRMS was measured on a JMS-T100LP mass spectrometer. Mass spectra (MS) were recorded on a JEOL-DX-303 mass spectrometer. Fluorescence spectra were determined on a Jasco FP-6200 spectrofluorometer. Ultraviolets (UVs) absorption spectra were determined in 95% ethanol on a Hitachi 323 spectrometer. Infrared (IR) spectra were recorded in potassium bromide pellets on JASCO 810. 2.2. Synthesis of 3-(4-methoxyphenyl)-4-(methylsulfanyl)-6-(pyridin-2-yl)pyridin-2(1H)-one (  3 ) Powdered sodium hydroxide (0.40 g, 10.0 mmol) was added to a solution of 1.13 g (5.0 mmol) of 3,3- bis-methylsulfanyl-1-pyridin-2-yl-propenone   ( 1 ) [32] and 0.88 g (6.0 mmol) of 2 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 3  (1.01 g, 62%) as yellow leaflets. Mp: 260-261 °C. IR (KBr, cm -1 ): 3456, 1605, 1510, 1275, 1175. 1 H NMR (DMSO- d  6 , 400 MHz) δ  2.48 (s, 3H), 3.80 (s, 3H), 6.96 (d,  J   = 8.8 Hz, 2H), 7.20 (d,  J   = 8.8 Hz, 2H), 7.50 (ddd,  J   = 2.9, 4.9, 7.8 Hz, 1H), 7.97 (ddd,  J = 1.5, 2.0, 7.8 Hz, 1H), 8.25 (d,  J   = 7.8 Hz, 1H), 8.25 (d,  J = 7.8 Hz, 1H), 8.72 (ddd,  J   = 1.0, 3.9, 4.9 Hz, 1H), 11.09 (brs, 1H). 13 C  NMR (DMSO- d  6 , 100 MHz) δ  14.46, 55.06, 113.41, 120.93, 124.73, 127.09, 131.35, 137.63, 149.41, 151.54, 158.62, 159.71. Ms m/z: 325 (M + +1, 12), 324 (M + , 36), 310 (11), 309 (42), 294 (7), 278 (10), 149 (11), 84 (19), 66 (21), 57 (11), 44 (100). Anal. Calcd. for C 18 H 16  N 2 O 2 S: C, 66.64; H, 4.97; N, 8.64%. Found: C, 66.51, H, 5.07; N, 8.51%. HRMS calcd. for C 18 H 16  N 2 O 2 S: 324.0932; found: 324.0949. 2.3. Synthesis of 6-methoxy-5-(4-methoxyphenyl)-4-(methylsulfanyl)-2,2'-bipyridine (  4 ) Powdered sodium hydroxide (0.20 g, 5.0 mmol) was added to a solution of 0.56 g (2.5 mmol) of 1  and 0.37 g (2.5 mmol) of 2 in 25 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 in 100 mL of methanol was refluxed for 6 h. After evaporation, the residual solid was recrystallized from methanol to give 4  (0.38 g, 45%) as yellow leaflets. Mp: 149-150 °C. IR (KBr, cm -1 ): 3425, 2940, 1610, 1570, 1540, 1510, 1450, 1430, 1350, 1270, 1250, 1170, 1105, 1035. 1 H NMR (DMSO- d  6 , 400 MHz) δ  2.47 (s, 3H), 3.81 (s, 3H), 3.90 (s, 3H), 7.00 (d,  J   = 7.8 Hz, 2H), 7.20 (d,  J   = 8.3 Hz, 2H), 7.45 (dd,  J   = 5.4, 6.3 Hz, 1H), 7.95 (dd,  J   = 7.3, 8.3 Hz, 2H), 8.41 (d,  J   = 7.8 Hz, 1H), 8.71 (d,  J   = 4.4 Hz, 1H). 13 C NMR (DMSO- d  6 , 100 MHz) δ  14.18, 53.35,   455.07, 108.54, 113.77, 120.57, 120.67, 124.18, 126.03, 131.14, 137.32, 149.22, 151.00, 152.19, 154.74, 158.85, 159.90. Ms m/z : 339 (M +  + 1, 24), 338 (M + , 100), 337 (29), 323 (19), 293 (8), 291 (7), 161 (6), 121 (5), 105 (5), 44 (4). Anal. Calcd. for C 19 H 18  N 2 SO 2 : C, 67.43; H, 5.36; N, 8.28%. Found: C, 67.61; H, 5.50; N, 8.10%. HRMS calcd. for C 19 H 18  N 2 O 2 S: 338.1089; found: 338.1098. 2.4. Synthesis of 3-(4-methoxyphenyl)-4-(methylsulfanyl)-6-phenylpyridin-2(1H)-one ( 6  ) Powdered sodium hydroxide (0.40 g, 10.0 mmol) was added to a solution of 1.13 g (5.0 mmol) of 3,3- bis-methylsulfanyl-1-phenyl-propenone ( 5 ) and 0.88 g (6.0 mmol) of 2 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 6  (0.38 g, 24%) as yellow leaflets. Mp: 238-240 °C. IR (KBr, cm -1 ): 3056, 1568, 1527, 1285, 1176. 1 H NMR (DMSO- d  6 , 400 MHz) δ  2.45 (s, 3H), 3.79 (s, 3H), 6.56 (s, 1H), 6.95 (d,  J   = 8.8 Hz, 2H), 7.18 (d,  J   = 8.8 Hz, 2H), 7.49 (m, 3H), 7.82 (dd,  J   = 3.9, 7.8 Hz, 2H), 11.75 (brs, 1H). 13 C  NMR (DMSO- d  6 , 100 MHz) δ  14.31, 55.04, 101.25, 113.37, 123.56, 127.04, 127.34, 128.74, 129.72, 131.44, 133.78, 151.43, 158.50, 160.63. HRMS calcd. for C 19 H 17  NO 2 S: 323.0980; found: 323.0940.  2.5. Spectral measurement The compounds 3 ,  4 and 6  stock solution (1×10 -2 M) was prepared by directly dissolving in DMSO. The UV/vis spectrum of 3 (10 -4  M) was measured in HEPES buffer (100 mM, 5% DMSO, pH = 7.4), as shown in Fig.1. For the fluorescence analysis, 3  (10 -6  M) upon addition of Zn 2+  in the form of  perchlorate salt was measured in HEPES buffer (100 mM, 5% DMSO, pH 7.4). The binding stoichiometry of 3  to Zn 2+  was investigated by Job’s plot. We measured the fluorescence intensity of 3  in the following buffers: 100 mM glycine - HCl buffer (pH 2.0), 100 mM citrate 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 ( K  d  ) of 3  in HEPES buffer was determined by plotting the fluorescence intensity to free Zn 2+  concentration. The metal selectivity of 3  was investigated in HEPES buffer (100 mM, 5% DMSO, pH 7.4) and that of 4  and 6  were investigated in aqueous EtOH solution (1:1 EtOH/water (v/v)). The cational solutions were prepared from NaCl, KCl, BaCl 2 , MgCl 2 ·6H 2 O, CaCl 2 , FeCl 3 ·6H 2 O, CoCl 2 ·6H 2 O, NiCl 2 ·6H 2 O, ZnCl 2 , CdCl 2 ·2.5H 2 O, CuCl 2 , MnCl 2 ·4H 2 O, AlCl 3 ·6H 2 O   (10 -3  M), respectively. The measurements were carried out at 298 K. The
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