Design and Synthesis of Mg2+-Selective Fluoroionophores Based on

Koji Suzuki*,†,‡. Collaboration of Regional Entities for the Advancement of Technological Excellence (CREATE), Kanagawa Academy of. Science and Te...
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Anal. Chem. 2002, 74, 1423-1428

Design and Synthesis of Mg2+-Selective Fluoroionophores Based on a Coumarin Derivative and Application for Mg2+ Measurement in a Living Cell Yoshio Suzuki,† Hirokazu Komatsu,‡ Takafumi Ikeda,‡ Naohiko Saito,‡ Sawa Araki,‡,¶ Daniel Citterio,‡ Hideaki Hisamoto,,§ Yoshiichiro Kitamura,| Takeshi Kubota,⊥ Jun Nakagawa,# Kotaro Oka,⊥,# and Koji Suzuki*,†,‡

Collaboration of Regional Entities for the Advancement of Technological Excellence (CREATE), Kanagawa Academy of Science and Technology, 3-2-1 Sakato, Takatsu-ku, Kawasaki, Kanagawa 213-0012, Japan, Department of Applied Chemistry, Faculty of Science and Technology, Institute of Biomedical Engineering, Graduate School of Science and Technology, Center for Life Science and Technology, School of Fundamental Science and Technology, and Department of System Design Engineering, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan

Novel Mg2+ fluorescent molecular probes (KMG-20-AM and KMG-27-AM; where AM is an acetoxymethyl group) based on a coumarin possessing a charged β-diketone structure were designed and synthesized. These fluorescent probes produced a red shift from 425 to 445 nm in the absorption spectra after formation of a complex with Mg2+. The fluorescence spectra of these probes also showed a red shift from 485 to 495 nm and an increasing fluorescence intensity after formation of a complex with Mg2+. The optimum experimental conditions were excitation wavelength of 445 nm and a monitored wavelength of 500 nm, where these probes functioned as an indicator showing an image of increasing fluorescence in the presence of Mg2+. These probes showed a “seesaw-type” fluorescent spectral change with the isosbestic point at 480 nm due to the light excitation at 445 nm, which indicates that ratiometry can be used for the measurement. The molecular probes formed a 1:1 complex with Mg2+ and the dissociation constant (Kd) was 10.0 mM for KMG-20. The association constants of the probes with Mg2+ were ∼3 times higher than that with Ca2+, which showed that the selectivity of Mg2+ versus Ca2+ for these probes was over 200 times higher than that for commercially available Mg2+ fluorescent molecular probes such as mag-fura-2, Magnesium Green. As an application of these probes, intracellular fluorescent imaging of Mg2+ was demonstrated using a fluorescent microscope. After the addition of KMG-20-AM and KMG-27-AM into PC12 cells, a strong fluorescence was observed in the cytoplasm and a weak fluorescence in the nuclei region. After treatment with a high-K+ medium, the fluorescence intensity increased due to increasing intracellular Mg2+. The real image of Mg2+ release from the magnesium store was successfully observed with these Mg2+ fluorescent probes. Fluorescent probes that indicate a spectral response upon binding ions or neutral organic or inorganic molecules have enabled researchers to investigate the changes in intracellular free 10.1021/ac010914j CCC: $22.00 Published on Web 02/09/2002

© 2002 American Chemical Society

guest ions or concentrations of molecules by means of fluorescent microscopy, flow cytometry, and fluorescent spectroscopy. Recently, a fluorescent imaging technique has been developed coupled with the advances in various optical instruments; fluorescent imaging is one of the most powerful techniques available for determining dynamic behaviors and for observing inside a single living cell.1 The technique of intracellular fluorescent bioimaging started with the success of intracellular Ca2+ measurement using a Ca2+ fluorescent molecular probe. Then various types of Ca2+ fluorescent molecular probes, such as Fura, Indo, CalciumGreen, and Cameleon, were designed and synthesized that contributed to the understanding of Ca2+ dynamics in biology and physiology2-6 These Ca2+ probes have had a significant impact on the field of biological science. Various types of molecular probes targeting not only for ions but also for molecules (for example, cyclic AMP,7,8 cyclic GMP,9,10 nitric oxide,11,12 and inositol †

Kanagawa Academy of Science and Technology. Department of Applied Chemistry, Faculty of Science and Technology, Keio University. § Present address: Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. | Institute of Biomedical Engineering, Graduate School of Science and Technology, Keio University. ⊥ Center for Life Science and Technology, School of Fundamental Science and Technology, Keio University. # Department of System Design Engineering, Faculty of Science and Technology, Keio University. ¶ Present address: Nano-Material Division, Nissan Arc, Ltd., 1 Natsushimacho, Yokosuka, Kanagawa 237-0061, Japan. (1) Ishikawa, H.; Suzuki, K.; Nakanishi, M.; Inokai, A. Bioimaging for Molecular Dynamics and Cellular Functions; Kyoritsushuppan Co., Ltd.: Tokyo, 1998. (2) Tsien, R. Y. Biochemistry 1980, 19, 2396-2404. (3) Grynkiewicz, G.; Poenie. M.; Tsien, R. Y. J. Biol. Chem. 1985, 260, 34403450. (4) Minta, A.; Kao, J. P.; Tsien, R. Y. J. Biol. Chem. 1989, 264, 8171-8178. (5) Takesako, K.; Sasamoto, K.; Ohkura, Y.; Hirose, K.; Iino, M. Anal. Commun. 1997, 391-392. (6) Miyawaki, J.; Llopis, R.; Heim, J.; McCaffey, M.; Adams, J. A.; Ikura, M.; Tsien, R. Y. Nature 1997, 388, 882-887. (7) Adams, S. R.; Harootunian, A. T.; Buechler, Y. J.; Taylor, S. S.; Tsien, R. Y. Nature 1991, 349, 694-697. (8) Sammak, P. J.; Adams, S. R.; Harootunian, A. T.; Schliwa, M.; Tsien, R. Y. J. Cell. Biol. 1992, 117, 57-72. ‡

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Figure 1. Chemical structures of fluorescent Mg2+ indicator probes, KMG-20-AM and KMG-27-AM.

1,4,5-triphosphate13) and macromolecules (protein) have been designed and actively investigated. Mg2+ is one of most important divalent cations in the cell, whose many intracellular processes, such as those known for Ca2+,14 need to be understood. There are several hundreds of enzymatic reactions intermediated by Mg2+ in cells, especially, excessive ATP hydrolysis causes the intracellular free Mg2+ concentration to increase. It has been also known that Mg2+ concentration changes respond to chemical stimulation like other ions.15-17 Therefore, it can be expected that the change of Mg2+ concentration has physiological importance (e.g., photosynthesis, oxidative phosphorylation, and muscle contraction are also modulated by Mg2+).18-20 Although Mg2+ plays such important roles, the number of studies is less than that for Ca2+. There are several methods to determine the amount of Mg2+ in cells such as atomic absorbance, Mg2+-selective electrodes, NMR, and fluorescent dyes.21-23 Only the imaging method using fluorescent dyes is able to investigate the spatial-temporal patterns of Mg2+ mobilization. To understand the intracellular Mg2+ concentration and distribution that plays an important role in the body, fluorescent magnesium indicators (for example, MagFura, Magnesium Green, Magnesium Orange, etc.) have been designed and synthesized.24-27 Using Mag-Fura-2, intracellular Mg2+ concentration has already been studied in cells from the liver,28 heart,29 muscle,30 and nervous system.31 Likewise, glutamate(9) Sato, M.; Hida, N.; Ozawa, T.; Umezawa, Y. Anal. Chem. 2000, 72, 59185924. (10) Wolfe, L.; Francis, S. H.; Corbin, J. D. J. Biol. Chem. 1989, 264, 41574162. (11) Kojima, H.; Nakatsubo, N.; Kikuchi, K.; Kawahara, S.; Kirino, Y.; Nagoshi, H.; Hirata, Y.; Nagano, T. Anal. Chem. 1998, 70, 2446-2453. (12) Kojima, H.; Nakatsubo, N.; Kikuchi, K.; Tanaka, J.; Kubo, Y.; Nagano, T. Neuroreport 1998, 9, 3345-3348. (13) Rhee, S. G.; Choi, K. D. J. Biol. Chem. 1992, 267, 12393-12396. (14) Romani, A.; Scarpa, A. Arch. Biochem. Biophys. 1992, 298, 1-12. (15) Fatholahi, M.; LaNoue, K.; Romani, A.; Scarpa, A. Arch. Biochem. Biophys. 2000, 374, 395-401. (16) Gaussin, V.; Gailly, P.; Gillis, J. M.; Hue, L. Biochem. J. 1997, 326, 823827. (17) Zhang, A.; Altura, B. T.; Altura, B. M. Front. Biosci. 1997, 2, A13-17. (18) Romani, A. M.; Scarpa, A. Arch. Biochem. Biophys. 1992, 298, 1-12. (19) Romani, A. M.; Scarpa, A. Front. Biosci. 2000, 5, D720-D734. (20) Garfinkel, L.; Garfinkel, D. Magnesium 1985, 4, 60-72. (21) Yago, M. D.; Manas, M.; Singh, J. Front. Biosci. 2000, 5, D602-D618. (22) Romani, A.; Marfella, C.; Scarpa, A. Circ. Res. 1993, 72, 1139-1148. (23) Kennedy, H. J. Exp. Physiol. 1998, 83, 449-460. (24) London, R. E. Annu. Rev. Physiol. 1991, 53, 241-258. (25) Murphy, E.; Freudenrich, C. C.; Lieberman, M. Annu. Rev. Physiol. 1991, 53, 273-287. (26) Jung, D. W.; Chapman, C. J.; Baysal, K.; Pfeiffer, D. R.; Brierley, G. P. Arch. Biochem. Biophys. 1996, 332, 19-29. (27) van der Wolk, J. P. W.; Klose, M.; de Wit, J. G.; den Blaauwen, T.; Freudl, R.; Driessen, A. J. M. J. Biol. Chem. 1995, 270, 18975-18982. (28) Raju, B.; Murphy, E.; Levy, L. A.; Hall. R. D.; London, R. E. Am. J. Physiol. 1989, 256, C540-C548.

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stimulated Mg2+ concentration change,32 or temporal analysis of Ca2+-induced Mg2+ mobilization in neurons,33 was examined using Mag-Indo-1 and Magnesium Green, respectively. However, these fluorescent indicators bind Ca2+ more tightly than Mg2+ (for example, the ratio between KMg and KCa was 0.013 in Mag-Fura2, 0.013 in Mag-Indo-1, and 0.003 in Magnesium Green),34 which interferes with the correct [Mg2+] measurement that limits the real information about how Mg2+ is regulated and contributes in intracellular roles. Therefore, a novel Mg2+-specific indicator that is not disturbed by any other ions, especially Ca2+, is much needed. We considered several requirements when designing the fluorescent Mg2+ molecular probe: (1) efficient excitation with most laser-based instrumentation, (2) reduced interference from sample autofluorescence, (3) less cellular photodamage and scatter, (4) higher molar extinction coefficient and quantum yield, which may guarantee the use of lower dye concentrations and prevent toxicity in the living cell, and (5) introduction of the β-diketone group that selectively complexes with Mg2+.35-39 For synthesizing an excellent Mg2+ fluorescent probe for realtime cell imaging, we chose a coumarin possessing a charged or uncharged β-diketone moiety as the fluorophore ligand for Mg2+ based on the above requirements.40-43 In these studies, new fluorescent reagents, which have one or two coumarin 343 groups at the terminals of the alkyl or oxyethylene chain, were synthesized and characterized. All the reagents produced large absorption and fluorescence spectral changes after the addition of Mg2+. These reagents formed a 1:1 complex with Mg2+; the order of (29) Murphy, E.; Freudenrich, C. C.; Levy, L. A.; London. R. E.; Lieberman, M. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 2981-2984. (30) Touyz, R. M.; Schiffrin, E. L. J. Biol. Chem. 1996, 271, 24353-24358. (31) Brocard, J. B.; Rajdev, S.; Reynolds, I. J. Neuron 1993, 11, 751-757. (32) Cheng, C.; Reynolds, I. J. Neurosciences 2000, 95, 973-979. (33) Gotoh, H.; Kajikawa, M.; Kato, H.; Suto, K. Brain Res. 1999, 828, 163168. (34) Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals, 6th ed.; Molecular Probes Inc.: Leiden, 1996. (35) Nagashima, H.; Tohda, K.; Matsunari, Y.; Tsunakawa, Y.; Watanabe, K.; Inoue, H.; Suzuki, K. Anal. Lett. 1990, 23, 1993-2004. (36) Suzuki, K.; Watanabe, K.; Matsumoto, Y.; Kobayashi, M.; Sato, S.; Siswanta, D.; Hisamoto, H. Anal. Chem. 1995, 67, 324-334. (37) Hu, Z.; Buhrer, T.; Muller, M.; Rusterholz, B.; Rouilly, M.; Simon, W. Anal. Chem. 1989, 61, 574. (38) Forgues, S. F.; Lavabre, D.; Rochal, A. D. New J. Chem. 1998, 1531-1538. (39) Otten, P. A.; London, R. E.; Levy, L. A. Bioconjugate Chem. 2001, 12, 203212. (40) Hisamoto, H.; Araki, S.; Kawasaki, N.; Kosugi, M.; Suzuki, K. Pittsburgh Conf. Abstr. 1999, 586. (41) Hisamoto, H.; Kosaka, K.; Araki, S.; Kosugi, M.; Citterio, D.; Suzuki, K. 76th CSJ National Meeting Abstracts; Kanagawa, Japan, 1999; 1E214. (42) Suzuki, S.; Ikeda, T.; Saito, N.; Citterio, D.; Sasaki, S.; Suzuki K. 78th CSJ National Meeting Abstracts; Funabashi, Japan, 2000; 3C207. (43) Suzuki, Y.; Saito, N.; Komatsu, H.; Citterio, D.; Kubota, T.; Kitamura, Y.; Oka, K.; Suzuki, K. Anal. Sci., in press.

Figure 2. Absorption spectra (a) and fluorescence spectra (b) of 10.0 µM KMG-20 before and after the addition of MgCl2 at 37 °C in 10.0 mM HEPES, 120.0 mM KCl, 20.0 mM NaCl (pH 7.2). [MgCl2] ) 0, 0.1, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500 mM. Excitation at 445 nm for the fluorescence measurements.

the complex formation constant with Mg2+ depended on the kind of spacer part connecting the coumarin moieties and the number of β-diketone groups. Based on our previous investigations and our Mg2+ ionophore knowledge,35,36,40-43 we have obtained two typical excellent Mg2+ indicators, KMG-20-AM and KMG-27-AM, as shown in Figure 1. These indicators have a β-hydroxycarboxylic acid group and an aromatic amino group connected by a conjugated π-electron system which enables bright fluorescence detection without the influence of protons under neutral conditions and brings about a large fluorescence spectral change after formation of the Mg2+ complexes. In addition, the acetoxymethyl group is introduced on the residual carboxyl group, which enables its easy passage through a cell membrane and produces easy hydrolysis (decomposition) of the acetoxymethyl group in a cell. In this paper, the fluorescent responses of KMG-20 and KMG27 against Mg2+ and other alkali and alkaline earth metal cations were examined using absorption and fluorescence spectroscopies. In addition, the measurement of intracellular magnesium ion concentration imaging was demonstrated using KMG-20-AM and KMG-27-AM with fluorescent microscopy with PC12 cells. The experimental results clearly showed that KMG-20-AM and KMG27-AM are good Mg2+ fluorescent probes. EXPERIMENTAL SECTION Synthesis of Fluoromagnesium Probes (KMG-20-AM and KMG-27-AM). KMG-20-AM ((13-aza-3-oxa-4-oxotetracyclo[7.7.1.0〈2,7〉‚0〈13,17〉]heptadeca-1(17),2(7),5,8-tetraen-5-ylcarbonyloxy)methyl acetate) was synthesized by the reaction of 13-aza-3-oxa4-oxotetracyclo[7.7.1.0〈2,7〉‚0〈13,17〉]heptadeca-1(17),2(7),5,8-tetraene-5-carboxylic acid (coumarin 343)44 and bromomethyl acetate in THF at room temperature in the presence of triethylamine. KMG-27-AM (methyl-13-aza-10,10,16,16-tetramethyl-3-oxa4oxotetracyclo[7.7.1.0〈2,7〉‚0〈13,17〉]heptadeca-1(17),2(7),5,8-tetraene5-carboxylate) was synthesized using 8-hydroxy-1,1,7,7-tetramethyljulolidine-9-carboxaldehyde as the starting material, similar to the synthetic method for KMG-20-AM. All the compounds were identified by means of 1H NMR, elemental analysis, and ESI-TOF mass spectroscopy. The detailed synthetic procedures can be obtained from the Supporting Information. Absorption and Fluorescence Spectrometry. Fluorescent reagents were dissolved in buffer solution ([HEPES] ) 10.0 mM, (44) Gomplel, J. V.; Schuster, G. B. J. Org. Chem. 1987, 52, 1465-1468.

[KCl] ) 130.0 mM, [NaCl] ) 20.0 mM, [EGTA] ) 2.0 mM, pH 7.0) at 10.0 µM. Alkali and alkaline earth metal cations were added to the solution of the fluorescent reagent as chloride salts. KMG-20-AM and KMG-27-AM Loading. KMG-20-AM and KMG-27-AM were stored as 10 mM stock solutions in DMSO. PC12 cells were incubated with 10 µM KMG-20-AM and KMG27-AM in the culture medium for 30 min at 37 °C. The cells were then washed twice with a recording solution containing (in mM) the following: NaCl, 125; KCl, 5; MgSO4, 1.2; CaCl2, 2; KH2PO4, 1.2; glucose, 6; and HEPES, 25 (pH 7.4). The cells were incubated for a further 15 min for complete hydrolysis of the acetoxymethyl ester of KMG-20-AM and KMG-27-AM. Recording of Intracellular Mg2+. PC12 cells loaded with the indicators were set in an experimental chamber. This chamber was mounted on the stage of an inverted fluorescent microscope, Axiovert S 100 (Zeiss, Jena, Germany). The light through a 440nm wavelength filter from a 75-W Xe lamp was used for excitation. Fluorescence through a 500-530 band-pass filter was acquired with a CCD camera system (T.I.L.L. Photonics, Planegg, Germany) and analyzed with Scion Image (Scion Corp.). Instruments for Characterization of the Dyes. The 1H NMR spectra were recorded on a JEOL JSM-GSX270 or a JEOL JNMLA300 instrument. The 1H chemical shifts are reported in ppm relative to tetramethylsilane as the internal reference. Mass spectra were run on an Applied Biosystems Mariner ESI-TOFMS instrument. Absorption spectra were recorded at 25 °C on a Hitachi U-2000 UV/visible spectrophotometer and a Hitachi U-3000 UV/ visible spectrophotometer. Fluorescence spectra were recorded at 25 °C on a Hitachi F-2000 fluorescence spectrophotometer or a Hitachi F-4500 fluorescence spectrophotometer. RESULTS AND DISCUSSION To study the in vitro photophysical properties of the Mg2+ indicators, KMG-20 and KMG-27, the absorption and fluorescence spectral measurements were obtained in HEPES buffer solution of pH 7.0 at 25 °C. Figure 2a) indicates the typical absorption spectra of KMG-20 before and after the additions of MgCl2. KMG20 itself showed an absorption maximum at 425 nm, while the KMG-20‚Mg2+ complex showed an absorption maximum at 445 nm; the isosbestic point was observed at 423 nm. On the other hand, no spectral change was observed after the addition of the Na+ and K+ alkali metal cations at 100 mM. Similar results were Analytical Chemistry, Vol. 74, No. 6, March 15, 2002

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Table 1. Complex Formation Constants of KMG-20, KMG-27, and Other Magnesium Indicators with Mg2+ and Ca2+ indicators

Ks(Mg) (M-1)

Ks(Mg) (mM)

KCa (M-1)

KMg/KCa

KMG-20 KMG-27 Mag-Fura-2 Mag-Indo-1 Mag-Fura-5 Magnesium Green

100.0 102.0 526.3a 370.4a 434.8a 500.0a

10.0 9.80 1.90a 2.69a 2.29a 2.00a

33.3 33.0 40000a 28570a 35710a 166660a

3.0 3.09 0.013 0.013 0.012 0.003

a

These values were cited in ref 34.

Figure 3. Ratio of fluorescence intensity excitation at 400 nm and at 460 nm as a function of Mg2+ concentration. Monitored wavelength, 500 nm.

Figure 4. Benesi-Hildebrand plots for the complexation of KMG20 with Mg2+ and Ca2+.

obtained in the absorption spectra of KMG-27 (absorption maximums of free KMG-27, 425 nm; of the Mg2+ complex, 445 nm). Figure 2b shows the fluorescence spectral changes of KMG20 before and after the addition of MgCl2. KMG-20 itself had an emission maximum at 485 nm, whereas the emission maximum of KMG-20‚Mg2+ shifted to 495 nm with increasing fluorescence intensity (1300 before complexation, 1880 after complexation at emission maximum wavelength). In the presence of other metal cations such as Na+, K+, and Ca2+, the emission maxium of KMG20 did not shift. Similar results were obtained in the fluorescence spectra of KMG-27 (Emission maximums of the free form, 483 nm; of the complexed form, 494 nm. Fluorescence intensities were 1850 before complexation and 2500 after complexation at emission maximum wavelength.). These spectral changes were caused by complex formation, in which the Mg2+ strongly bound at the β-diketone positions of the coumarin moiety supported by the carbonyl group, attracting an electron (negative charge) and increasing the polarity of the coumarin group. This interaction involves the electron lone pair of the oxygen atoms at the carboxyl group. The stability of the complex increased with the basicity of the oxygen atom that depended on the capacity of the ligand molecule to delocalize the charge borne by the metal atom. The fluorescence ratio of KMG-20 monitored at 500 nm between the excitation at 400 nm and at 460 nm was plotted as a function of the Mg2+ concentration; the result is shown in Figure 3. This plot provided a good calibration curve with ratiometry measurements to obtain the precise Mg2+ concentration. 1426 Analytical Chemistry, Vol. 74, No. 6, March 15, 2002

Figure 5. Responses of fluorescence intensity of KMG-20 and Magnesium Green for Ca2+. Arrow indicates the timing of 10 µM CaCl2 addition ([Ca2+] increased from 140 to 850 nM).

To investigate the detailed complexation properties, the ground-state association constants of the complexes between each fluorescent indicator and the metal cations were determined from absorption and fluorometric titrations as a function of the concentration of the Mg2+ or Ca2+/metal cation. The plots of the emission maximum of KMG-20 versus the Mg2+ concentration showed a sigmoid curve. The complex formation constants of the Mg2+ probes with Mg2+ and Ca2+ (KMg and KCa) were calculated using the Benesi-Hildebrand plot method45 shown in Figure 4 with the values summarized in Table 1. The association constants of the alkali metal ion complexes (Li+, Na+, K+) could not be calculated because no spectral changes were observed. Both probes formed a 1:1 complex with Mg2+ and Ca2+. The complex formation constants for Mg2+ and Ca2+ (KMg and KCa) of commercially available magnesium indicators of Mag-Fura-2, MagIndo-1, and Magnesium Green34 are summarized in Table 1 along with the values for KMG-20 and KMG-27. Though the KMg values of KMG-20 and KMG-27 was slightly lower than that of Mag-Fura2, Mag-Indo-1, and Magnesium Green, the KCa of the magnesium indicators for the products were about 2000-5000 times stronger than those of KMG-20 and KMG-27. From these results, KMG20 and KMG-27 had much higher selectivity values for Mg2+ versus Ca2+ (KMg/KCa) than those of commercially available Mg2+ fluorescent probes. The intracellular Mg2+ concentration typically ranges from about 0.1 to 6.0 mM, whereas the typical physiological Ca2+ concentration is 10 nM-1 µM.46,47 Therefore, the influence of Ca2+ on KMG-20 and KMG-27 was negligible in the physiological range of Ca2+ mobilization. (45) Benesi, H. A.; Hildebrand, J. H. J. Am. Chem. Soc. 1949, 71, 2703-2707.

Figure 6. DIC and fluorescent images of PC12 cells loaded with KMG-20-AM (10 µM) and KMG-27-AM (10 µM). The outline of the nucleus is evident, and some brighter spots were observed in the cytoplasm and growth cone.

To compare the Mg2+/Ca2+ selectivity of KMG-20 with that of commercially available fluorescent probe in detail, the fluorescence intensity changes of KMG-20 and Magnesium Green were monitored as a function of time after acute Ca2+ increase and the results are shown in Figure 5. At 1 min, CaCl2 and EGTA were added ([Ca2+] increased from 140 to 850 nM) into the solution containing 1 mM MgCl2 and fluorescent dyes ([dye] ) 10 µM, respectively). Compared with Magnesium Green, the response of KMG-20 to Ca2+ was very small. This also indicated that the sensitivity of KMG-20 for Ca2+ was relatively low, which was consistent with the data of KMg/KCa values in Table 1. KMG-27 indicated similar absorption and fluorescence spectral changes after the formation of a complex with Mg2+ to that of KMG-20 under the same experimental conditions (HEPESbuffered solution at pH 7.0 with 500 mM Mg2+ at 25 °C). Also, the KMg value of KMG-27 was the same as that of KMG-20. From this result, introducing functional groups into this cyclohexyl ring moiety in KMG-20 to create a modified KMG-20 may be effective without the loss of the optical and complexation properties of KMG-20. To investigate photodamage, a Xe lamp was used to irradiate a solution of KMG-20; fluorescence intensity was monitored with time. As a result, fluorescence intensity and spectra had essentially no change after several hours. To demonstrate the application of the new Mg2+ fluorescent probes in living cell experiments, PC12 cells were incubated in medium containing KMG-20-AM and KMG-27-AM in order to determine the appropriate indicator-loading concentration for Mg2+ distribution imaging. Differential interference contrast (DIC) and fluorescent images of the PC12 cells loaded with KMG-20-AM and KMG-27-AM are shown in Figure 6. Fluorescence was mainly observed in the cytoplasm and growth cone and not in the nuclei. This result means that intracellular free Mg2+ distributes in the (46) Kelepouris, E.; Kasama, R.; Agus, Z. S. Miner. Electrolyte Metab. 1993, 19, 277-281. (47) Ebel, H.; Gunther, T. J. Clin. Chem. Clin. Biochem. 1980, 18, 257-270.

cytoplasm area rather than in the nucleus area. The cells were cultured intact for several days after the KMG-20-AM and KMG27-AM loading. The concentration of Mg2+ in the PC12 cells ([Mg2+]) was calculated using the method developed for [Ca2+] measurement.48 The maximum (Fmax) and the minimum (Fmin) fluorescence intensities of KMG-20 by external concentrations of 0 or 100 mM in Mg2+-permeated PC12 cells with ionomycin (10 µM) were monitored and [Mg2+] was calculated according to the following equation: [Mg2+] ) Kd(F - Fmin)/(Fmax - F), where Kd is the dissociation constant and F is fluorescence intensity. The [Mg2+] of PC12 cells at rest was 0.68 ( 0.05 mM (n ) 10). This value was consistent with previous data.46,47 The change in the [Mg2+] in KMG-20-AM-loaded PC12 cells was observed after the addition of K+ and this result is shown in Figure 7. It is known that high K+ depolarizes the cell membrane and opens several ion channels. The fluorescent intensity increased after the administration of K+ (120 mM). A few seconds after the onset of the K+ application, the fluorescent intensity increased in the cytoplasm and growth cone. The [Mg2+] abruptly increased to the highest level (F/F0 ) 1.5) in the cytoplasm (F0 is fluorescence intensity of KMG-20 itself at the emission maximum wavelength. F is that of KMG-20 after administration of K+ at the emission maximum wavelength.). After 1 min, [Mg2+] decreased but maintained a slightly higher level (F/F0 ) 1.0) for several minutes. The [Mg2+] increase started just after the K+ administration and then gradually decreased in the cytoplasm and growth cone for 1 min. The Mg2+ concentration in the cytoplasm, growth cone, and nuclei reached the same level (F/F0 ) 1.0) after ∼4 min. This phenomenon was caused by the fact that K+ depolarized the cell membrane and Ca2+ was introduced into the cell via Ca2+ ion channels. The high intracellular Ca2+ concentration triggered Ca2+-induced Mg2+ release from the organelle (mitochondria and endoplasmic reticulum) due to signal trans(48) Brocard, J. B.; Rajdev, S.; Reynolds, I. J. Neuron 1993, 11, 751-757.

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Figure 7. Fluorescent image of PC12 cells before (A) and after (B) the administration of K+ and time course of the change of [Mg2+] in response to K+ (C). Fluorescence acquired from PC12 cells loaded with KMG-20-AM (10 µM). At 30 s, KCl solution (120 mM) was applied. In cytoplasm and growth cone, [Mg2+] transiently increased, and decreased to the level that was slightly higher than the initial level.

duction.31,33 Consequently, the intracellular [Mg2+] increased and KMG-20 recognized the free Mg2+ and produced the increasing fluorescence intensity. CONCLUSIONS The present study demonstrated the highly selective detection of Mg2+ versus Ca2+ by means of monitoring the absorption and fluorescence spectral change of two novel fluorescent molecular probes, KMG-20 and KMG-27, in water. In addition, the successful demonstration of intracellular fluorescent imaging of Mg2+ was performed using the fluorescent imaging probe KMG-20. The new molecular probes were able to be loaded easily to the cells by path application, and their fluorescence was bright. The selectivity values of KMg/KCa for KMG-20 and KMG-27 was over 200 times higher than that of the commercially available Mg2+ fluorescent molecular probes, which means that KMG-20 and KMG-27 are (49) Kubota, T.; Nakagawa, J.; Kitamura, Y.; Suzuki Y.; Suzuki K.; Oka, K. Neurosci. Res. Suppl. 2000, 24, s66. (50) Kitamura Y.; Suzuki Y.; Suzuki K.; Oka, K. Soc. Neurosci. Abstr. 2000, 26, 875. (51) Suzuki Y.; Ikeda T.; Saito N.; Komatsu H.; Kitamura Y.; Oka K.; Suzuki K. Bioimages 2001, 9, 29.

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the most appropriate Mg2+ molecular probes at the present time. In the 1980s and 1990s, the design and synthesis of Ca2+ fluorescent molecular probes produced a large amount of information about inter- and intracellular Ca2+ dynamics. These new Mg2+ fluorescent molecular probes, therefore, will be used to investigate Mg2+ dynamics. Now, we expect to clarify the role of intracellular Mg2+ using KMG-20 and KMG-27. Moreover, we are currently designing other Mg2+ indicators which have absorption and emission wavelengths higher than those of KMG-20 and KMG27.49-51 SUPPORTING INFORMATION AVAILABLE Detailed synthetic procedures and absorption and fluorescence spectra of KMG-20 and KMG-27. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review August 14, 2001. Accepted November 19, 2001. AC010914J