ICPBCZin: A red emitting ratiometric fluorescent indicator with nanomolar affinity for Zn2+ ions

Cell Calcium (2008) 44, 270—275

journal homepage: www.elsevier.com/locate/ceca

ICPBCZin: A red emitting ratiometric fluorescent indicator with nanomolar affinity for Zn2+ ions Emmanuel Roussakis a, Styliani Voutsadaki a, Eftychia Pinakoulaki a, Dionisia P. Sideris b,c, Kostas Tokatlidis b,d, Haralambos E. Katerinopoulos a,∗ a

Department of Chemistry, University of Crete, Heraklion 71003, Crete, Greece Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas (IMBB-FORTH), Heraklion 71110, Crete, Greece c Department of Biology, University of Crete, Heraklion 71409, Crete, Greece d Department of Materials Science and Technology, University of Crete, Heraklion 710 03, Crete, Greece b

Received 17 May 2007; received in revised form 26 November 2007; accepted 10 December 2007 Available online 19 February 2008

KEYWORDS Intacellular zinc; Zinc ion sensor; Ratiometric probe; Fluorescent dye

Summary A new fluorescent Zn2+ indicator, namely, ICPBCZin was synthesized and the spectral profile of its free and Zn2+ bound forms was studied. The newly synthesized zinc indicator incorporates as chromophore the chromeno [3 ,2 :3,4]pyrido[1,2a] [1,3]benzimidazole moiety and belongs to the dicarboxylate-type of zinc probes. The compound is excited with visible light, exhibits high selectivity for zinc in the presence of calcium and other common biological ions, and its Zn2+ dissociation constant is 4.0 nM. Fluorescence spectra studies of ICPBCZin indicated a clear shift in its emission wavelength maxima upon Zn2+ binding, as it belongs to the class of Photoinduced Charge Transfer (PCT) indicators, along with changes in fluorescence intensity that enable the compound to be used as a ratiometric, visible-excitable Zn2+ probe. © 2007 Elsevier Ltd. All rights reserved.

Introduction Zn2+ is a key element for sustaining life and the second most abundant transition metal, after iron, in biological systems [1]. Since the 1940s there has been a steady stream of data implicating Zn2+ in a number of biological processes.

∗ Corresponding author. Tel.: +30 2810 545 026; fax: +30 2810 545 001. E-mail address: [email protected] (H.E. Katerinopoulos).

Zinc plays a central role in cellular metabolism regulation [2,3] may serve as a structural element in enzymes [2,4] and transcription factors, as well as at the catalytic site of a number of enzymes. The interest in Zn2+ is compounded by its involvement in a number of neuropathologies such as Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson’s disease, hypoxia-ischemia and epilepsy [5—14]. Zn2+ is often referred to as the ‘‘silent ion’’ since, unlike other biological transition metal ions (such as Fe2+ , Mn2+ or Cu2+ ), it does not have an intrinsic spectroscopic or magnetic signal because of its 3d10 4s0 electronic configuration. How-

0143-4160/$ — see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2007.12.008

ICPBCZin: A red emitting ratiometric fluorescent indicator with nanomolar affinity for Zn2+ ions ever, its ability to form tetra-coordinated complexes with a variety of organic moieties that can be connected to a fluorophore, can be used as the basis for synthesizing fluorescent probes that may be useful in biological systems [15]. A number of studies analyzing the reasoning behind the design of such molecules have been published and, as a result, a host of fluorescent zinc probe syntheses have been reported and reviewed in recent articles [16—22]. Gee et al. published the synthesis of a series of dicarboxylate zinc sensors, namely FuraZin, IndoZin, FluoZin-1, X-RhodZin and FluoZin-2 the prefixes chosen from the well known chomophores in the corresponding calcium indicators [23]. The above compounds exhibit a micromolar affinity for zinc. As expected, the first two congeners may be used as ratiometric dyes where the rest maintain the same excitation and emission maxima in their free and zinc-bound forms. In common with the corresponding calcium probes, none of the aforementioned compounds may be used as a visible-excited ratiometric probe, a set of highly desirable properties. We were therefore tempted to extend this new generation of zinc sensors with ratiometric dyes incorporating visible-excited chromophores while preserving the dicarboxylate nature of the zinc chelator. Based on our experience in the chromeno [3 ,2 :3,4]pyrido[1,2a] [1,3]benzimidazole chemistry [24], we synthesized ICPBCZin and we explored the ability of the dye on [Zn2+ ] detection.

Materials and methods General NMR spectra were taken on an AMX500 Bruker FT-NMR spectrometer; proton chemical shifts are reported in relative to tetramethylsilane. Mass spectra were recorded on a Shimadzu ESI—MS and a Finnigan LCQAD-30000 mass spectrometers. Fluorescence spectra were recorded on an Aminco Bowman spectrofluorimeter (Spectronics Co., USA). Optical absorption spectra were recorded with a PerkinElmer Lamda 20 UV—Vis spectrophotometer. The FTIR spectra were recorded at 4 cm−1 resolution with a BRUKER Equinox 55 FTIR spectrometer equipped with a liquid nitrogen cooled mercury cadmium telluride detector. Standard free Zn2+ concentration buffers were prepared according to a published procedure [23]. EGTA, KCl, and CaCO3 were purchased from Sigma (St. Louis, MO, USA).

Preparation of methyl 2-((3-(1H-1,3-benzimidazol2-yl)-2-imino-6-methoxy-2H-chromen-7-yl) (2-methoxy-2-oxoethyl)amino)acetate (2) To a solution of methyl 2-(4-formyl-5-hydroxy-2methoxy(2-methoxy-2-oxoethyl)anilino)acetate (1) (350 mg, 1.12 mmol) in 10 ml MeOH was added piperidine (954 mg, 1.12 mmol) and the solution was stirred at room temperature for 1.0 min. To this system was added a solution of 2-benzimidazolyl acetonitrile (176 mg, 1.12 mmol) in 3.0 ml dry MeOH. The reaction was completed after 2 h stirring at room temperature. The yellow

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precipitate was filtered, washed with dry ether and dried in high vacuum. Flash chromatography using a gradient of 10—30% acetone/toluene as eluent yielded 375 mg (75%). 1 H NMR (CDCl3 , 500 MHz): 12.62 (br s., 1H), 8.57 (s, 1H), 7.75 (m, 2H), 7.45—7.49 (m, 2H), 6.83 (s, 1H), 6.48 (s, 1H), 4.18 (s, 4H), 3.78 (s, 3H), 3.77 (s, 6H).

Preparation of methyl 2-((6-cyano-7-imino-2methoxy-7H-chromeno[3 ,2 :3,4]pyrido[1,2-a] [1,3]benzimidazol-3-yl) (2-methoxy-2-oxoethyl) amino)acetate (3) To a solution of compound 2 (70 mg, 0.16 mmol) in 0.5 ml dry 2-methoxyethanol was added malononitrile (11 mg, 0.17 mmol) and the solution was stirred at 90 ◦ C for 0.5 h and then the system was cooled to room temperature. Upon addition of 2 ml ether a precipitate was formed, which was centrifuged, the solvents were decanted and the solid was resuspended in ether and centrifuged repeatedly. The yellow precipitate was heated at 110 ◦ C for 2.0 h. The yellow solid turned deep red indicating the formation of the product. The compound was purified by flash chromatography using a gradient of 5—20% acetone/toluene as eluent. The product yield was 60 mg (75%). 1 H NMR (500 MHz, CDCl3 ): 8.71—8.69 (m, 1H), 8.44 (s, 1H), 7.79—7.76 (m, 1H), 7.43—7.38 (m, 2H), 7.23—7.12 (m, 1H), 6.80 (s, 1H), 6.68 (s, 1H), 4.25 (s, 4H), 3.83 (s, 6H), 3.78 (s, 3H).

Preparation of 2-((carboxymethyl)(6-cyano-7imino-2-methoxy-7H-chromeno[3 ,2 :3,4] pyrido[1,2-a] [1,3]benzimidazol-3-yl)amino)acetic acid disodium salt (4) In a flame dried round bottomed flask, equipped with a magnetic stirrer, were placed 0.4 ml DMSO and compound 3 (9.0 mg, 0.018 mmol). To this suspension was added potassium tert-butoxide (8.0 mg, 0.07 mmol) in one portion at room temperature and under vigorous stirring. After 30 min all the solids were dissolved and TLC analysis (1-propanol:water 3:1) indicated the consumption of the starting material and the formation of a single, soluble component. The product precipitated upon addition of 1 ml ether. The precipitate formed was centrifuged, the solvents were decanted, and the product was dissolved in 0.5 ml of methanol, precipitated by addition of 1 ml ether, centrifuged, resuspended in acetonitrile, centrifuged repeatedly and dried in high vacuum to give 8 mg (81%) of pure dye. 1 H NMR (500 MHz, DMSO-d6): 8.79 (d, J = 7.5 Hz, 1H), 7.42 (d, J = 8.5 Hz, 1H), 7.16 (dd, J1 = J2 = 7.5 Hz, 1H), 7.00 (dd, J1 = 8 Hz, J2 = 7.5 Hz, 1H), 6.76 (s, 1H), 6.52 (br s., 1H), 3.79—3.76 (m, 6H), 3.19—3.16 (m, 2H). IR (cm−1 ): (free dye) 830, 910, 976, 1080, 1147, 1193, 1246, 1312, 1400, 1448, 1520, 1590, 1642, 2180, 2214. IR (cm−1 ): (zinc-bound dye) 1182, 1200, 1240, 1258, 1321, 1418, 1449, 1512, 1590, 1632, 2216. UV—Vis (cm−1 ): (free dye) 223, 274, 328, 424, 539, 580. UV—Vis (cm−1 ): (zinc-bound dye) 394, 539, 574, 674.

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Preparation of indicator solutions containing adjusted Zn2+ concentrations A buffer solution of 50 mM HEPES (2-[4-(2-hydroxyethyl)-1piperazinyl]ethanesulfonic acid), containing 0.1 M KNO3 and 10 mM EGTA (ethylenebis(oxyethylenenitrilo) tetraacetic acid) was prepared. The pH was adjusted to 7.2 via addition of KOH solution. In 2 ml samples of the above solution were added the appropriate aliquots of 0.02 M or 1 M ZnSO4 solutions to give a series of solutions with known concentrations of free Zn2+ according to a published procedure [23]. To each of the above solutions were added 5 ␮l aliquots of a 0.2 mM dye solution to make a final indicator concentration of 0.5 ␮M. All solutions were prepared using nanopure water. Calculated values of free zinc resulting from the addition of total zinc quantities to the buffer are given in the following chart. [Zn2+ ]total (mM) [Zn2+ ]free (nM)

— 0

1 0.29

5 2.6

6 4

7 6.1

8 11

Cell staining visualization with confocal microscopy The yeast wild type strain FT5 (˛ ura3-52 trp1-63 his3leu2::PET56) was used for this study. YPD (yeast extract 1%,

Figure 1

peptone 2%, glucose 2%) was used for the growth of the yeast cells. Staining of yeast cells was performed according to the protocol available at http://www.bioprotocol.com. The zinc probe was added as its diacetoxymethyl ester [27] at 50 ␮M concentration for 30 min after growth to midlog phase before the fixation step. To check nuclei staining DAPI (4 ,6-diamidino-2-phenylindole, dihydrochloride) (Molecular Probes, OR, USA) was added at the mounting solution before applying the coverslip. Cells were viewed with appropriate filters in a Bio-Rad Radiance 2100 microscope and analyzed with the software AxioVision 3.1 from Carl Zeiss.

Results and discussion Chemistry Salicylaldehyde 1 [25] was synthesized from hydroquinone in 8 steps in 35% overall yield. Reactions for the formation of the iminocoumarin intermediate 2 proceeded smoothly and in relatively high yield as shown in Fig. 1. The key step for the synthesis of the dye appeared to be the construction of the polycyclic chromophore of compound 3. Literature data indicated that cyclization can be induced by reaction of benzimidazole-substituted iminocoumarins with malononitrile in neutral as well as

Synthesis of ICPBCZin.

ICPBCZin: A red emitting ratiometric fluorescent indicator with nanomolar affinity for Zn2+ ions

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in basic or acidic conditions [26]. Thus, compound 2 was heated with malononitrile in methoxyethanol to give a yellow precipitate, which upon dissolution in ethyl acetate and treatment with NaOH solution gave the desired product. Product formation was also observed while heating a thin film of this intermediate at 110 ◦ C for 2 h, a procedure that furnished the desired compound in practically the same yield. Deprotection of the methyl esters took place smoothly and in high yield via treatment of 3 with t-BuOK/DMSO.

Spectral properties The fluorescence spectra of ICPBCZin (4) are shown in Fig. 2A and B. The free probe exhibits an excitation maximum wavelength at 553 nm, which shifts to 543 nm upon zinc binding. When excited at its free form the molecule shows an emission maximum at 590 nm, which shifts to 564 nm upon Zn2+ binding with an isosbestic point at 572 nm and a 50% decrease in flu-

Figure 2 Excitation (upper) and emission (lower) spectra of ICPBCZin in solutions of increasing Zn2+ concentrations.

Figure 3 Ion competition study. First set of measurements: increase of fluorescence ratio (Iisosb /I590 ) in the presence of 50 M zinc (black bar), as compared to that of the free dye (grey bar). The rest of the measurement sets indicate a fluorescence ratio of the dye in the presence of 50 M of metal (grey bars) vs. ratio fluorescence after addition of one equivalent of zinc to the sample (black bars).

orescence intensity of the free vs. the zinc-saturated dye. The dissociation constant of the new probe was calculated according to Tsien’s algorithm [27] to be 4.0 nM. The potential use of this compound as a ratiometric dye in biological systems is shown in an ion competition study of a range of metals binding to the sensor. Samples were excited at exc (553 nm) and the fluorescence intensity ratio was calculated (Fig. 3). Given that addition of zinc is decreasing the fluorescence of the dye at 590 nm, fluorescence changes were expressed in positive numbers as the ratio Iisosb /I590 . Solutions were made in the HEPES buffer mentioned above. In the first set of measurements the fluorescence ratio after addition of 50 ␮M zinc is shown (dark blue bar), as compared to that of the free dye (light blue bar). The rest of the measurement sets indicate a fluorescence ratio of the dye in the presence of one equivalent of metal (light blue bars) vs. ratio fluorescence after addition of one equivalent of zinc to the sample (dark blue bars). Binding by these metal ions appears to be weak since (a) the presence of metals does not increase the fluorescence ratio Iisosb /I590 of the free dye and (b) subsequent addition of Zn2+ to these solutions increases the fluorescence ratio of the dye-metal solution to the same extent as in the case of the first measurement (competitive ion-free solution). Alterations in the structural and electronic characteristics of ion indicators frequently result in changes in affinity of the sensors for the target ion or even in changes in ion selectivity. Thus, modification of the tetracarboxylic system of well known calcium indicator Fura-2 [27] yielded magnesium indicator Mag-Fura-2 [28,29] whereas further reduction of the number of carboxylates gave bis-carboxylate zinc indicator FuraZin [23]. In this example, ion selectivity depends on the participation of the various functional groups present in the molecule for an optimum coordination with the target ion. In order to explore the mode of bind-

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E. Roussakis et al. ing of Zn2+ to ICPBCZin we recorded the FTIR and UV—Vis spectra of both, free and zinc-bound dyes (Fig. 4). In the FTIR spectra zinc binding caused a distinct reduction of intensity at 1080 cm−1 , an absorption band corresponding to the symmetric C—O—C streching vibration of aryl—alkyl ethers [30,31]. A similar reduction in intensity appears at 1312 cm−1 , a band characteristic of C—N streching of tertiary amines. Participation of the two carboxylate moieties was indicated by a shift of a band at 1642—1632 cm−1 . These findings were further supported by UV—Vis spectroscopy data indicating a 424—394 cm−1 bathochromic shift for the carboxylate [32] and a 580—574 cm−1 shift for the alkyl—aryl ether absorption, respectively. These results provide evidence that the zinc ion is coordinated to all four moieties of the ionophore a fact that could rationalize the selectivity of the dye for zinc ions vs. other cations included in the above mentioned ion-competition study. Finally, the cell permeability of the probe was tested by treating yeast cells with the diacetoxymethyl ester of ICPBCZin. The yeast FT5 strain (˛ ura3-52 trp163 his3-leu2::PET56) was used for this study. The zinc probe was added in its diacetoxymethyl ester form [27] at 50 ␮M concentration for 30 min after growth of the cells for 2—3 doublings before the fixation step. Nuclei staining was performed by adding DAPI at the mounting solution before applying the coverslip [33]. As shown in Fig. 5 both intact yeast cells and yeast spheroplasts are stained by DAPI as well as ICPBCZinAM suggesting that the probe may be used as indicator in intracellular zinc concentration measurement studies.

Figure 4 IR (upper) and UV—Vis (lower) spectra of the free and the zinc-bound forms of ICPBCZin.

Figure 5 Staining of WT cells grown in glucose. The upper panel shows stained yeast cells and the lower panel yeast spheroplasts. The samples were excited at exc = 365 nm to observe nuclei by DAPI (em = 420 nm, left panel) and at exc = 543—553 nm to check ICPBCZin-AM internalisation (em = 564—590 nm, middle panel).

ICPBCZin: A red emitting ratiometric fluorescent indicator with nanomolar affinity for Zn2+ ions

Conclusions ICPBCZin (4) has been synthesized from salicylaldehyde 1 in three steps and 42% overall yield. This dye fulfills all the basic requirements for an efficient fluorescent zinc probe since it: (a) absorbs and emits in the visible region, an advantage in intracellular zinc measurements (b) exhibits a 26 nm emission maximum shift between its free and zinc-bound form as well as an isosbestic point, a fact that makes it usable as a ratiometric dye (c) has a Kd value of 4.0 nM and is able to respond to changes of free cytocolic zinc concentrations, and (d) shows considerable selectivity for zinc ions vs. other common biological ions. Comparison of the above properties to those of other dyes, as summarized by recent reviews [16], may rank ICPBCZin as one of the most promising fluorescent zinc sensors to be used in a wide range of biological studies. The study of such applications is currently under investigation in our laboratory.

Acknowledgements This work was supported by an EPEAEK grant by the Greek Ministry of Education (Herakleitos). DPS was supported by a PENED grant from the Greek Secretariat of Research and Technology.

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