Development of a synthetic strategy for Water soluble tripodal receptors: Two novel fluorescent receptors for highly selective and sensitive detections of Fe3+ and Cu2+ ions and biological evaluation

Development of a synthetic strategy for Water soluble tripodal receptors: Two novel fluorescent receptors for highly selective and sensitive detections of Fe3+ and Cu2+ ions and biological evaluation

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Journal Pre-proof Development of a Synthetic Strategy for Water Soluble Tripodal Receptors: Two Novel Fluorescent Receptors for Highly Selective and Sensitive Detections of Fe3+ and Cu2+ Ions and Biological Evaluation ¨ ˘ Tumay, Aylin Uslu, Elif Ozcan, Sureyya ¨ Oguz ¨ Hasan Huseyin ¨ Kazan, Serkan Yes¸ilot

PII:

S1010-6030(19)31162-1

DOI:

https://doi.org/10.1016/j.jphotochem.2020.112411

Reference:

JPC 112411

To appear in:

Journal of Photochemistry & Photobiology, A: Chemistry

Received Date:

9 July 2019

Revised Date:

24 January 2020

Accepted Date:

24 January 2020

¨ Please cite this article as: Uslu A, Ozcan E, Tumay ¨ SO, Kazan HH, Yes¸ilot S, Development of a Synthetic Strategy for Water Soluble Tripodal Receptors: Two Novel Fluorescent Receptors for Highly Selective and Sensitive Detections of Fe3+ and Cu2+ Ions and Biological Evaluation, Journal of Photochemistry and amp; Photobiology, A: Chemistry (2020), doi: https://doi.org/10.1016/j.jphotochem.2020.112411

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Development of a Synthetic Strategy for Water Soluble Tripodal Receptors: Two Novel Fluorescent Receptors for Highly Selective and Sensitive Detections of Fe3+ and Cu2+ Ions and Biological Evaluation

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Aylin Uslua*, Elif Özcana, Süreyya Oğuz Tümaya, Hasan Hüseyin Kazanb, Serkan Yeşilota*

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Department of Chemistry, Gebze Technical University, 41400 Gebze Kocaeli Turkey.

Department of Biological Sciences, Middle East Technical University, Ankara, Turkey.

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∗ Corresponding authors. Tel.: +90 262 6053009 and +90 262 6053014

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E-mail addresses: [email protected] and [email protected]

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Graphical abstarct

Research Highlights

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General synthetic strategy for water soluble tripodal system based on a cyclotriphosphaze platform was developed. Presented receptors demonstrated high selectivity towards Fe3+ and Cu2+ over other metal ions with the low detection limits. 3 is able bind Fe3+ and the fluorescent signal obtained via 3 is able to be quenched by the presence of Fe3+ in live cells, in vitro. The proposed synthetic strategy could be applied to develop water soluble receptors for other metal ions

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ABSTRACT A general synthetic strategy is developed to synthesize water soluble receptors by employing tripodal system based on a cyclotriphosphaze platform. The developed model is successfully synthesized and characterized by using elemental analysis, FT-IR, MALDI-TOF, 1H NMR, 13C 31

P NMR techniques. The fluorescence sensing performance of prepared water

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NMR and

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soluble tripodal systems were evaluated by UV/Vis and fluorescence spectroscopies. According to obtained results, two novel water-soluble sensing platforms were selective and

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sensitive fluorescence receptors for detections of Fe3+ and Cu2+ ions in both the absence and presence of competitive ions. In addition, the iron receptor also displays biological function,

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therefore, the cytotoxicity and fluorescence microscopy experiments were applied and it was demonstrated that compound 3 was non-cytotoxic, and can be used as fluorescence

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imaging sensors for Fe3+ in living cells. According to obtained results, proposed synthetic strategy could be applied to develop water soluble receptors for not only iron and/or copper

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ions but also many different metal ions of interest using a variety of fluoroionophores.

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keywords: Fluorescence receptors, water-soluble, phosphazene, live-cell imaging 1. Introduction

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Selective and sensitive detection of chemical species is very important in solving physiological and environmental problems [1-4]. Fluorescence sensor technology has been widely used for determination of these systems, compared to conventional analytical methods,

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due to their many distinct advantages such as facile detection, high sensitivity, and easy tenability [3, 4]. Therefore, one of the most challenging tasks facing synthetic chemists is the

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design and synthesis of these artificial receptors for detection of chemical species, especially

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metals used in many fundamental biological and environmental processes [3, 4]. The analyte, such as a metal ion, can react with the receptor unit through complexation, hydrolysis, substitution or oxidation which would lead to a signal change in fluorescence measurements [3,

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4]. However, prediction of receptor affinities to different analyte is very difficult because the receptor affinities cannot be anticipated in a straightforward manner [3, 4]. Heterocyclic

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chromophores are one of the most investigated classes of optical sensing molecules due to

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excellent spectral properties and their ability to detect diverse analytes. Amongst them, benzimidazole and thiazole units often captures the attention of scientists [5, 6]. Their derivatives are important candidates for development of novel optical chemical sensors [5, 6]. Their electron accepting and π-bridging properties combined with chromogenic pH sensitivity/switching, metal-ion chelating properties and compatibility with biomolecules makes them an especially attractive building block in molecular systems for different

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applications such as optoelectronics and non-linear optics (NLO), sensing and bioimaging [7, 8]. Fluorescent sensing in 100% aqueous medium is also another obstacle because for practical applications of the probes in biochemical studies such as living cell imaging good water solubility is expected [9]. The development of suitable water-soluble receptors for the selective recognition of analyte remains a great challenge and a lot of effort has been spent on the design and synthesis of tailored receptors with different chemical structures [10]. Generally, in order to prepare water-soluble fluorescent receptors, hydrophilic groups such as quaternary

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ammonium salts [11], sulfonates [12], phosphonates [13] and oligo-ethyleneglycol [14] groups need to be introduced into the chemical structures. However, neutral water-soluble receptors such as containing oligo-ethyleneglycol groups are superior as to ionic receptors because they

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can avoid potential nonspecific interactions through electrostatic interactions between probes and physical or biological medium in various applications. It is also advantage of

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oligoethyleneglycol groups that they can increase the cell permeability in biological systems

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[14]. Recently, considering several advantages including remarkable synthetic versatility, specific functionalization and optical inertness, the using of cyclotriphosphazene (N3P3Cl6) has been reported in the field of fluorescent sensors for detection of various metal ions [15-17].

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Although these materials exhibited highly sensitive and selective sensing properties for the detection of metal ions, they were not soluble in 100% aqueous media. Another concept is that

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N3P3Cl6 scaffold directs the six chlorines three by three to the opposite sides of the phosphazene

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ring. Thus regio- and stereo-chemically control of the nucleophilic substitutions of chlorine is possible. Phosphazene ring greatly benefits as a core when three hydrophilic groups were substituted in lower or upper side opposite to remaining side [18, 19]. These all results prompted us to build up a template compound by combining the two synthetic strategies described above [16, 18-21]. Considering these all requirements, we have chosen to investigate the design and synthetic strategies of water soluble tripodal receptors regarding their

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ability to chelate toward the desired metal ion in aqueous environments, which can provide more choices and options in the development of such receptor systems. To test the versatility and usefulness of this template compound (Fig. S1), we designed and synthesized two novel receptors by different combinations from chelating group and we examined their binding behavior toward selected metal ions. All synthesized compounds were characterized by the usual spectroscopic techniques. Binding modes and sensing mechanisms of compounds were proposed and investigated by Job plots and

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non-linear curve fitting analysis. We also show the results of imaging and cytotoxicity potential on cancer cells by using one of the receptors. We also show the results of imaging and cytotoxicity potentials of one of the receptors on cancer cells since the biological compatibility

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underlying cell imaging for detection of such compounds in living organisms, pointing possible biosensing systems, and evaluation of the cytotoxicity as a route to discover novel therapeutic

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compounds has been one of the fundamental approaches to address the novel compounds an

Experimental

2.1. Materials

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2.

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application area.

Hexachlorocyclotriphosphazene, (NPCl2)3 from Aldrich and was purified by fractional

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crystallization from hexane. The deuterated solvents (CDCl3 for NMR spectroscopy and

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Sodium hydride (60.0%) were obtained from Merck; The following chemicals were obtained from Sigma Aldrich; Methoxy Polyethylene glycol 350 (99.0%), tetrahydrofuran (THF) (≥99.9%). 4-(2-Thiazolyl)phenol (97%) was obtained from Alfa Aesar. Column chromatography was performed on silica gel (Merck, Kieselgel 60, 230–400 mesh). All reactions were carried out under a dry argon atmosphere.

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2.2. Equipment Elemental analyses were obtained using an Ele-mentarVario MICRO Cube Instrument. Mass spectra were recorded on a Bruker MicrOTOF MALDITOF–MS spectrometer with the Matrix-assisted Laser Desorption/Ionization Time of Flight method.

31

P, 1H and

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C NMR

spectra were recorded in CDCl3 solution on a Varian INOVA 500 MHz spectrometer using TMS as an internal reference for 1H and 13C NMR and 85% H3PO4 as an external reference for 31

P. Electronic absorption spectra were recorded with a Shimadzu 2101 UV spectrophotometer

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in the UV-visible region. Fluorescence excitation and emission spectra were recorded on a Varian Eclipse spectrofluorometer using 1 cm pathlength cuvettes at room temperature. The fluorescence lifetimes were obtained using a Horiba-Jobin-Yvon-SPEX Fluorolog 3-2iHR

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instrument with a Fluoro Hub-B Single Photon Counting Controller at an excitation wavelength of 390 nm. Signal acquisition was performed using a TCSPC module. Fluorescence titration

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data was fitted with the appropriate non-linear regression analyses using Sigma-Plot 14.0

modifications [22-24].

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(Systat Software Inc., Point Richmond, CA) via reported equation with appropriate

Human cervical cancer cell line, HeLa was used. Cells were grown in RPMI 1640 medium

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(Lonza, Switzerland) supplemented with 10% FBS (Biochrom, Germany) and gentamycin (Biological Industries, USA) at 37°C in a humidified atmosphere with 5% CO2 for used for cell

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lines and cell culture. To perform live cell imaging, 4x105 cells/well were seeded into 6-well

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plates and incubated for 24 h. Next, cells were washed with PBS twice, and medium was renewed. Cells were treated with 500 μM of FeCl3 for 1 h at 37oC. Then, cells were washed with PBS twice and compound at certain concentrations were added onto cells for 30 min in PBS. Images were taken by FLoid Cell Imaging Station (Thermo Fischer Scientific, USA) under white light and by DAPI filter (Ex:390/40; Em:446/33). Only FeCl3+- and only compound treated-cells were used as control. Cell viabilities were determined by 3-(4,5-dimethylthiazol-

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2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay (SERVA Electrophoresis GmbH, Germany) as described previously [25]. 1x104 cells/well were seeded into 96-well microplates and incubated for 48 h. Next, cells were treated with compound with increasing concentrations for 24 h. Water-treated cells were used as control group. Cells were washed with PBS twice and medium was renewed. 10 μl of MTT solution (5 mg/ml) was added onto cells and incubated for 4 h. Finally, cells were disrupted with SDS-HCl solution (1 g SDS and 0.01 M HCI in 10 ml final volume) overnight at 37oC and the microplates were read by microplate

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spectrophotometer (Multiskan GO; Thermo Fisher Scientific, USA) at wavelength 570 nm. Optical densities were converted to % viability by using water-treated cells as reference (100%). All data were representative of three independent experiments with each set containing

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triplicates of the same sample (n=9). Results were analyzed by GraphPad Prism 6 (GraphPad Software, Inc., USA) with One-Way ANOVA and post-hoc Tukey’s test, and significant when

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p<0.05.

2.3. Synthesis

2.3.1. Synthesis of compound 2.

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Hexachlorocyclotriphosphazene (NPCl2)3 (Compound 1) was reacted with methoxy

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polyethtlene glycol-350 to obtained compounds 2 according to the literature procedure [26].

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2.3.2. Synthesis of compound 3. Compound 2 (0.5 g, 0.39 mmol) was dissolved in dry THF (25 mL) in a 100 mL three-

necked round-bottomed flask under an argon atmosphere. Dry THF solution (10 mL) of 4-(2thiazolyl)phenol (0.21 g, 1.17 mmol) was added to the reaction mixture dropwise under an argon atmosphere and NaH (0.05 g, 1.17 mmol, 60%) was added to the reaction mixture. The reaction mixture was stirred for five days in room temperature and followed by TLC, which

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indicated product formation. After the solution was filtered to remove sodium chloride salt, the solvent was removed under reduced pressure, using a rotary evaporator. The oily residue was purified using a column chromatography using THF as the mobile phase. The compound 3 was isolated (0.20 g, ~30%). Found: C, 53.26; H, 7.14; N, 4.23 %; MS: m/z = 1.691,66-1.722,15. Calculated: C66-78H99-123N6O24-30P3S3; C, 51.16-51.65; H, 6.44-6.84; N, 4.63-5.42 %; MS: m/z = 1549.64-1813.95.

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2.3.3. Synthesis of compound 4.

Compound 2 (1.00 g, ~0.77 mmol) was dissolved in dry THF in a 100 mL three-necked round-bottomed flask under an argon atmosphere. Triethylamine (0.35 ml, 2.32 mmol) was

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added to the reaction mixture. Dry THF solution (10 mL) of benzimidazole (0.27 g, 2.32 mmol) was added to the reaction mixture. The reaction mixture was refluxed for three days. After the

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solution was filtered to remove sodium chloride salt, the solvent was removed under reduced

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pressure, using a rotary evaporator. The oily residue was purified using a column chromatography using THF as the mobile phase. The compound 4 was isolated (0.33 g, ~30%).

129N6O21-27P3;

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Found: C, 52.72; H, 7.35; N, 8.42 %; MS: m/z = 1.131,13-1.650,63. Calculated: C75-87H105C, 55.94-56.17; H, 6.60-6.96; N, 9.00-10.08 %; MS: m/z = 1603.63-1867.95.

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The compounds 3 and 4 contain polymer units named methoxypolyethyleneglycol-350 as formula CH3O(CH2CH2O)nH and n about ⁓ 6.8-6.5 average molecular weight 350 and between

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335-365. For this reason, Compound 3 and 4 have no sharp single molecular ion peak in their mass spectra. In the mass spectrum of compound 3, there are multiple peaks between 1600 and 1800 m/z areas. These values similar and within range as calculated molecular weight of compound 3 and arises from polymer units on compound 3. Mass spectrum of compound 4 has serial peaks between 1100 and 1600 m/z areas. Between consecutive peaks differences

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generally corresponds to the same values. These results may arise partially breakage of polymer units from the molecule.

3.

Results and discussions

3.1. Synthesis/structural characterization

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Water soluble fluorescent efficient molecules (3,4) containing thiazole or benzimidazole side groups and polyethylene glycol cosubstitutent groups were designed and synthesized using cyclotriphosphazene (1) as a core according to Scheme 1. After prepared water–soluble

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cyclotriphosphazene core, to obtain tripodal structured molecular receptors which have different metal binding properties, thiazole and benzimidazole reagents were selected as a

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ionofluorophore due to their well-known fluorescence and metal binding properties [5, 6, 27].

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In addition, considering the effect of the tripodal structure formed by the binding of thiazole and benzimidazole to the cyclotriphosphazene core, the metal binding process such as selectivity, sensitivity and complex stability through rigidity of arms and their cavity may also

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be controlled [28]. Synthesized compounds were characterized using mass,

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P, 1H and

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C

NMR spectroscopies and elemental analysis. All the results were consistent with the predicted

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structures as given in the Supp. Inf (Fig.S2-Fig.S8).

Scheme 1.

3.2. Spectral studies Photophysical properties of 3 and 4 were investigated by UV/Vis and fluorescence spectrophotometers at 25oC with spectroscopic cuvette. Different solvents were used to

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evaluate electronic absorption and fluorescence spectra of 3 and 4 such as cyclohexane, dichloromethane, ethanol (EtOH) and water. As can be seen at Fig.S9, electronic properties of 3 and 4 are not changed by solvent system except for absorbance which is probably due to dissolution differences of compounds in different solvents. Electronic absorption maxima of 3 and 4 were observed at 290 nm and 265-280 nm, which were same with thiazole and benzimidazole moieties and they were attributed to π--π* transitions [27, 29]. This situation was expected because cyclotriphosphazenes does not have any optical properties in UV/Vis region

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as we mentioned previously published papers [15-17]. In addition, fluorescence emission properties of 3 and 4 were investigated at same solvent systems (Fig.S10) and fluorescence maxima were observed at 296 nm and 378 nm for 3 and 4 respectively, which were also same

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with fluorescence emissions of thiazole and benzimidazole [27, 29]. According to obtained results (Fig.S9-S10), optic properties of 3 and 4 were consistent and did not affected by the

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change of solvent systems. Stokes shifts of 3 and 4 in water were calculated as 88 nm and 23

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nm, respectively (Fig.S11).

The synthesized compounds (3 and 4) were found to be highly soluble in water as expected due to glycol groups hence their receptor properties were investigated in deionized water. To

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determine appropriate concentration for emission properties, different concentration from 100 mg.L-1 to 6.25 mg.L-1 of 3 and 4 were prepared in water from stock solution (250 mg.L-1). In

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Fig. S9, the fluorescence response of 25 mg.L-1 3 was more intense than 100 mg.L-1 and then

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fluorescence signal decreased proportional after dilution to 6.25 mg.L-1. This behavior was probably due to intermolecular self-quenching of thiazole groups because of high concentration (100 mg.L-1) which was previously worked for many fluorophores [30]. On the other hand, fluorescence response of 4 was decreased with diluted concentration which showed that interor intramolecular interactions were not effective in compound 4. When consider the adequacy

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of the analytical signal intensity, 6.25 mg.L-1 and 25 mg.L-1 were chosen as a valid concentration for 3 and 4, respectively. The chemosensor properties of 6.25 mg.L-1 of 3 and 25 mg.L-1 of 4 were evaluated with 500 µM different competitive metal ions (Al3+, Li+, Na+, K+, Cs+ , Mg2+, Ca2+, Ba2+, Hg2+ , Pb2+, Mn2+, Cd2+, Ag+, Ni2+, Cu2+, Zn2+, Co2+, Cr3+, Fe2+ and Fe3+). As can be seen Fig. 1a-b, the UVVis spectra of 3 and 4 were unchanged upon the addition of 500 µM of various metal ions except for Fe2+ and Fe3+ (for 3) and Cu2+ (for 4). Upon addition 500 µM of Fe2+ and Fe3+ ions

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to 3, which its absorbance (at 290 nm) was significantly change when other metal ions cause meaningless change (Fig. 1a). This changes in absorption band was 69.30% and 24.39% for Fe3+ and Fe2+, respectively. Also, upon addition 500 µM of Cu2+ ions to 4, the absorbance band

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between 265-280 nm which can be attributed to benzimidazole moieties [31] were changed and new absorption band at 305 nm was observed which can be attributed to charge transfer between

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metal and 4 [27]. These changes in UV/Vis electronic spectra of target compound can be attributed to electron reorganization by formation of compound-metal complexes with addition

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of Fe3+ and Cu2+ [17, 32]. After evaluated the UV-Vis responses of 3 and 4 to metal ions, fluorescence responses of 3 and 4 were investigated under the same conditions. According to

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Fig. 2, when fluorescence signal of 3 completely quenched after addition of 500 µM of Fe3+ in water and Fe2+ ion was quenched 45.4%, fluorescence signal of 4 was nearly completely

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quenched after addition of 500 µM of Cu2+ and other competitive metal ions did not affect the

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florescence signals of 3 and 4 (Fig. 2). This differences between quenching efficiency of Fe3+ and Fe2+ might be useful for speciation of iron ions in fluorescence sensor application of 3, additionally, adjustable selectivity of 3 and 4 might be led to the development of new strategies for molecular receptor applications. Moreover, after addition of Fe3+ and Cu2+ to aqueous solution of 3 and 4 caused important color change while other various metal ions induced no significant solution color change at daylight and under fluorescence excitation (Fig. 2c and d).

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Fig. 1.

Fig. 2.

The selectivity of 3 and 4 towards different competitive ions (Ag+, Al3+, Ba2+, Cr3+, Co2+, Cs2+, Ca2+, Cd2+, Cu2+, Hg2+, K+, Li+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Zn2+, Fe2+, Fe3+, NO3-, CO32-, Cl-

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, F-) were evaluated by the change of fluorescence signal and showed at Fig. 3. It was obviously seen from Fig. 3, the competitive ions which could be found in biological sample did not effect to fluorescence signal of 3 and 4. The influence of the pH of 3, 4 and their metal complexes

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(Fe3+-3 and Cu2+-4) were studied by using 6.25 mg.L-1 of 3, 25 mg.L-1 of 4 and 500 µM of metal ions which were changed from 3.5 to 9.0 with Britton-Robinson buffer system when other

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parameters kept constant. 0.1 mL of Britton-Robinson buffer systems were used to adjust the

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pH of the solvents. According to Fig.S10, the highest fluorescence intensity of 3 and 4 were obtained at 6.09 and relatively decreased above and below 6.09 when their metal complexes nearly did not affect by pH. Considered sensitivity and stability of 3 and 4 and their metal

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Fig. 3.

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sensor application.

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complexes, Britton-Robinson buffer system and pH 6.09 was chosen for rest of fluorescence

To understand binding mode of Fe3+-3 and Cu2+-4, fluorescence titration experiments were carried out with 6.25 mg.L-1 of 3, 25 mg.L-1 of 4 and gradually increased concentration of metal ions from 0 to 500 µM in water. According to Fig. 4, fluorescence intensity of 3 and 4 decreased gradually and proportional after addition increased amount of Fe3+ and Cu2+. The dynamic

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ranges for detection of Fe3+ and Cu2+ obtained according to fluorescence titration experiment which are linear up to 500 µM Fe3+ and Cu2+.

Fig. 4.

This quenching of fluorescence for paramagnetic guest such as (Fe3+ (d5), Fe2+(d6), Cu2+(d9)) was generally explained by chelation enhanced fluorescent quenching (CHEQ) mechanism via

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coordination of host [33, 34]. As can be seen in Fig.4 inset, linear regression equations were calculated as F= -1.0059[Fe3+] + 500.02 and F= -1.6045[Cu2+] + 938.28 when correlation coefficients were 0.997 and 0.999 (n=3) for Fe3+ and Cu2+, respectively. The limit of detections

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(LODs) were calculated as 8.04 µM (0.511 mg.L-1) and 9.68 µM (0.615 mg.L-1) for Fe3+ and Cu2+, respectively, according to 3σ /K [15-17]. The detection limit is superior than previously

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reported sensor application for Fe3+ and Cu2+ therefore these results pointing high selectivity and sensitivity for Fe3+ and Cu2+ detection [35-38]. According to analytical parameters, 3 and

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4 are convenient candidate for selective and sensitive detection of Fe3+ and Cu2+ in 100% aqueous media.

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The continuous variation method (Job’s plot) and non-linear curve fitting analyses were carried out for understand the stoichiometry of Fe3+-3 and Cu2+-4 complex (Fig.S11). As can be seen

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from Fig.S11, when the mole fraction of Fe3+ and Cu2+ were 0.33, signal of complex reached

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turning point which means stoichiometry of Fe3+-3 and Cu2+-4 complex were 1:2 (metal:ligand). In addition, plots of decreasing fluorescence intensity with increasing amount of Fe3+/Cu2+ to 3 and 4 demonstrated an inflection point where the stoichiometric ratio of Fe3+/Cu2+ to 3 and 4 were determined to be 2:1 (ligand:metal) according to non-linear curve fitting analyses (Fig. S12). Based on obtained results from continuous variation method (Job’s plot) and non-linear

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curve fitting analyses, proposed binding mechanism for 3 and 4 with metal ions were presented at Fig. 5.

Fig. 5.

Reproducibly of chemical sensors is very important for detection and monitoring process. Therefore, precision of 3 and 4 were determined via ten measurements of model working with

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500 µM of Fe3+ and Cu2+ under the same conditions. The relative standard deviation (RSD %) was calculated as 2.48% and 3.01% for 3 and 4 which pointing high reproducibility. The photostability of 3, 4 and their metal complexes (Fe3+-3 and Cu2+-4) against daylight were

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investigated between 0 and 60 minutes. As can be seen Fig.S13, the fluorescence signal of 3, 4 and their metal complexes (Fe3+-3 and Cu2+-4) nearly unchanged until 60 minutes. Therefore,

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these results indicated that 3, 4 and their metal complexes (Fe3+-3 and Cu2+-4) have high photostability which is important for real-time detection analysis. In addition, fluorescence

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quenching mechanism of 3, 4 with Fe3+ and Cu2+ were investigated by using Stern-Volmer equation [15, 17]. As can be seen from Fig.S14, Stern-Volmer graph of 3, 4 with Fe3+ and Cu2+

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are linear in the range of 5x10-5 M – 2.5x10-4 M but when the concentration of Fe3+ and Cu2+ increased a positive deviation is observed which pointed out that both dynamic and static

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quenching are effective in the systems [15, 17].

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We also performed preliminary investigation of the Fe3+ quenching of compound 3 in live cells by using human cervical cancer cell line, HeLa. For this purpose, cells were treated with either Fe3+ alone, 3 alone or both. According to results, Fe3+-treated cells were not fluorescent, as expected. Treatment of cells with 3 resulted in a detectable fluorescent signal. However, the signal obtained from the cells which were at first treated with Fe3+ and then 3 was lost, pointing availability of excess Fe3+ resulted in quenching (Fig. 6). This result is critical in terms of

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illustrating that 3 is able bind Fe3+ and the fluorescent signal obtained via 3 is able to be quenched by the presence of Fe3+ in live cells, in vitro. Still, the intensity of the signal was low since the iron is a supplement of the growth medium and cells stores the iron as iron pool inside the cells. Beside ability to sense iron inside the live cells, this and similar fluorescent compounds are regarded as iron chelators [39, 40]. Iron is a critical inorganic element that is inevitably used for cell survival and proliferation. Thus, iron chelators, alone or as co-therapy, are used as anti-cancer agents [41-43]. To see the anti-proliferative effect of the compound, we

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checked the cell viabilities by MTT assay.

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Fig. 6.

According to results, the compound significantly reduced the live cell ratio with an inhibitory

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concentration 50 (IC50) value of 51.3 ± 5.19 μg/ml after 24 h treatment (Fig. S15). All these

4. Conclusion

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agent by chelating iron.

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results suggest that the novel compound is a good candidate as both iron sensor and anti-cancer

In summary, a template compound by the development of step by step the design

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strategies for the preparation of water soluble tripodal receptors is presented. Two novel water-

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soluble fluorescence receptors (3,4) are synthesized by the proposed synthetic strategy and, they showed a unique selectivity and sensitivity for Fe3+ and Cu2+ ions, respectively. The fluorescence microscopy experiments also demonstrate that receptor (3) is non-cytotoxic, and can be used as a fluorescent imaging receptor for Fe3+ in living cells. We believe that the strategies can provide more choices and options in the development of such systems for further studies based on both increasing sensitivity and controlling. The significance of this model is

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that metal-chelation could be manipulated depending on the chelating functional group, not only iron and/or copper ions but also many different analytes of interest using a variety of tripodal systems. In addition, proposed model can be also used in the preparation of watersoluble materials for other potential applications such as photodynamic therapy, cell imaging and pH indicators.

Authors Statements

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Aylin USLU analysed the synthesis data, checked the manuscript and supervised the project. Elif ÖZCAN synthesized compound 3 and 4, performed its characterization and wrote the manuscript. Süreyya Oğuz TÜMAY carried out the UV-Vis/fluorescence measurements for photophysical properties and fluorescence sensor experiment, analysed the data and wrote the manuscript. Hasan Hüseyin KAZAN carried out live cell imaging and cytotoxicity experiments, analysed the data and wrote the manuscript. Serkan YEŞİLOT checked the manuscript, conceived the original idea and provided theoretical guidance during the research process. All co-authors reviewed and approved the manuscript.

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Declaration of interests

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Acknowledgements

This work was partially supported (synthesis of compound 3) by the Scientific and

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Technological Research Council of Turkey (TÜBİTAK), (Project number: 115Z897) and partially supported (synthesis of compound 4) by Gebze Technical University (Project number: GTU-BAP-2017-A-13).

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References [1] P.D. Beer, P.A. Gale, Anion Recognition and Sensing: The State of the Art and Future Perspectives, Angewandte Chemie International Edition, 40 (2001) 486-516. [2] W.-W. Zhao, J.-J. Xu, H.-Y. Chen, Photoelectrochemical detection of metal ions, Analyst, 141 (2016) 4262-4271. [3] K.P. Carter, A.M. Young, A.E. Palmer, Fluorescent Sensors for Measuring Metal Ions in Living Systems, Chemical Reviews, 114 (2014) 4564-4601. [4] J.F. Zhang, Y. Zhou, J. Yoon, J.S. Kim, Recent progress in fluorescent and colorimetric

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chemosensors for detection of precious metal ions (silver, gold and platinum ions), Chemical Society Reviews, 40 (2011) 3416-3429.

[5] A. Maji, S. Pal, S. Lohar, S.K. Mukhopadhyay, P. Chattopadhyay, A new turn-on benzimidazole-based greenish-yellow fluorescent sensor for Zn2+ ions at biological pH

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applicable in cell imaging, New Journal of Chemistry, 41 (2017) 7583-7590.

[6] K. Mahesh, S. Karpagam, Thiophene-thiazole functionalized oligomers-excellent

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fluorescent sensing and selective probe for copper and iron ion, Sensors and Actuators B: Chemical, 251 (2017) 9-20.

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[7] N. Perin, M. Hranjec, G. Pavlović, G. Karminski-Zamola, Novel aminated benzimidazo[1,2-a]quinolines as potential fluorescent probes for DNA detection: Microwaveassisted synthesis, spectroscopic characterization and crystal structure determination, Dyes and

na

Pigments, 91 (2011) 79-88.

[8] M.-Y. Lai, C.-H. Chen, W.-S. Huang, J.T. Lin, T.-H. Ke, L.-Y. Chen, M.-H. Tsai, C.-C. Wu, Benzimidazole/Amine-Based Compounds Capable of Ambipolar Transport for

ur

Application in Single-Layer Blue-Emitting OLEDs and as Hosts for Phosphorescent Emitters, Angewandte Chemie International Edition, 47 (2008) 581-585.

Jo

[9] L. Xue, C. Liu, H. Jiang, A ratiometric fluorescent sensor with a large Stokes shift for imaging zinc ions in living cells, Chemical Communications, (2009) 1061-1063. [10] X. Li, X. Gao, W. Shi, H. Ma, Design Strategies for Water-Soluble Small Molecular Chromogenic and Fluorogenic Probes, Chemical Reviews, 114 (2014) 590-659. [11] L. Li, Y. Ji, X. Tang, Quaternary Ammonium Promoted Ultra Selective and Sensitive Fluorescence Detection of Fluoride Ion in Water and Living Cells, Analytical Chemistry, 86 (2014) 10006-10009.

17

[12] W. Wu, A. Chen, L. Tong, Z. Qing, K.P. Langone, W.E. Bernier, W.E. Jones, Facile Synthesis of Fluorescent Conjugated Polyelectrolytes Using Polydentate Sulfonate as Highly Selective and Sensitive Copper(II) Sensors, ACS Sensors, 2 (2017) 1337-1344. [13] G.G. Vinoth Kumar, R.S. Kannan, T. Chung-Kuang Yang, J. Rajesh, G. Sivaraman, An efficient “Ratiometric” fluorescent chemosensor for the selective detection of Hg2+ ions based on phosphonates: its live cell imaging and molecular keypad lock applications, Analytical Methods, 11 (2019) 901-916. [14] J. Madsen, R.E. Ducker, O. Al Jaf, M.L. Cartron, A.M. Alswieleh, C.H. Smith, C.N. Hunter, S.P. Armes, G.J. Leggett, Fabrication of microstructured binary polymer brush

Chemical Science, 9 (2018) 2238-2251.

ro of

“corrals” with integral pH sensing for studies of proton transport in model membrane systems, [15] S.O. Tümay, S.Y. Sarıkaya, S. Yeşilot, Novel iron(III) selective fluorescent probe based on synergistic effect of pyrene-triazole units on a cyclotriphosphazene scaffold and its utility in real samples, Journal of Luminescence, 196 (2018) 126-135.

-p

[16] S.O. Tümay, A. Uslu, H. Ardıç Alidağı, H.H. Kazan, C. Bayraktar, T. Yolaçan, M. Durmuş, S. Yeşilot, A systematic series of fluorescence chemosensors with multiple binding

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sites for Hg(ii) based on pyrenyl-functionalized cyclotriphosphazenes and their application in live cell imaging, New Journal of Chemistry, 42 (2018) 14219-14228.

lP

[17] S.O. Tümay, S. Yeşilot, Tripodal synthetic receptors based on cyclotriphosphazene scaffold for highly selective and sensitive spectrofluorimetric determination of iron(III) in water samples, Journal of Photochemistry and Photobiology A: Chemistry, 372 (2019) 156-167.

na

[18] A. Uslu, Ş. Güvenaltın, The investigation of structural and thermosensitive properties of new phosphazene derivatives bearing glycol and amino acid, Dalton Transactions, 39 (2010) 10685-10691.

ur

[19] A. Uslu, E. Özcan, Synthesis of water soluble cyclotriphosphazenes with thiazolecontaining side groups: Amphiphilic and hydrolytic degradable, Polyhedron, 148 (2018) 49-54.

Jo

[20] A. Uslu, S. Yeşilot, Chiral configurations in cyclophosphazene chemistry, Coordination Chemistry Reviews, 291 (2015) 28-67. [21] W. Wei, R. Lu, S. Tang, X. Liu, Highly cross-linked fluorescent poly(cyclotriphosphazeneco-curcumin) microspheres for the selective detection of picric acid in solution phase, Journal of Materials Chemistry A, 3 (2015) 4604-4611. [22] A.E. Brummett, N.J. Schnicker, A. Crider, J.D. Todd, M. Dey, Biochemical, Kinetic, and Spectroscopic Characterization of Ruegeria pomeroyi DddW—A Mononuclear IronDependent DMSP Lyase, PLOS ONE, 10 (2015) e0127288. 18

[23] S.C. Chai, Q.-Z. Ye, Analysis of the stoichiometric metal activation of methionine aminopeptidase, BMC Biochemistry, 10 (2009) 32. [24] S.C. Chai, J.-P. Lu, Q.-Z. Ye, Determination of binding affinity of metal cofactor to the active site of methionine aminopeptidase based on quantitation of functional enzyme, Analytical Biochemistry, 395 (2009) 263-264. [25] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays, Journal of Immunological Methods, 65 (1983) 55-63. [26] S.-C. Song, S.B. Lee, J.-I. Jin, Y.S. Sohn, A New Class of Biodegradable Thermosensitive Polymers. I. Synthesis and Characterization of Poly(organophosphazenes) with Methoxy-

2188-2193. [27] A.

Uslu,

S.O.

Tümay,

A.

Şenocak,

F.

ro of

Poly(ethylene glycol) and Amino Acid Esters as Side Groups, Macromolecules, 32 (1999) Yuksel,

E. Özcan,

S.

Yeşilot,

Imidazole/benzimidazole-modified cyclotriphosphazenes as highly selective fluorescent probes for Cu2+: synthesis, configurational isomers, and crystal structures, Dalton Transactions, 46

-p

(2017) 9140-9156.

[28] M. Shahid, L.S. Arora, B. Uttam, Synthesis and evaluation of a tri-armed molecular

re

receptor for recognition of mercury and cyanide toxicants AU - Chawla, Har Mohindra, Supramolecular Chemistry, 29 (2017) 111-119.

lP

[29] A. Helal, H.G. Kim, M.K. Ghosh, C.-H. Choi, S.-H. Kim, H.-S. Kim, New regioisomeric naphthol–thiazole based ‘turn-on’ fluorescent chemosensor for Al3+, Tetrahedron, 69 (2013) 9600-9608.

na

[30] E. Morgounova, Q. Shao, B.J. Hackel, D.D. Thomas, S. Ashkenazi, Photoacoustic lifetime contrast between methylene blue monomers and self-quenched dimers as a model for duallabeled activatable probes, Journal of Biomedical Optics, 18 (2013) 056004.

ur

[31] X.Z. Kai Liu, Qingxiang Liu, Jianzhong Huo, Bolin Zhu, Shihua Diao, Beilstein J. Org. Chem., 11 (2015) 563–567.

Jo

[32] T. Senthilkumar, N. Parekh, S.B. Nikam, S.K. Asha, Orientation effect induced selective chelation of Fe2+ to a glutamic acid appended conjugated polymer for sensing and live cell imaging, Journal of Materials Chemistry B, 4 (2016) 299-308. [33] J.H. Chang, Y.M. Choi, S. Young-Kook, Bull. Korean Chem. Soc., 22 (2001) 527-530. [34] Y. Zheng, J. Orbulescu, X. Ji, F.M. Andreopoulos, S.M. Pham, R.M. Leblanc, Development of Fluorescent Film Sensors for the Detection of Divalent Copper, Journal of the American Chemical Society, 125 (2003) 2680-2686.

19

[35] S. Joshi, S. Kumari, R. Bhattacharjee, A. Sarmah, R. Sakhuja, D.D. Pant, Experimental and theoretical study: Determination of dipole moment of synthesized coumarin–triazole derivatives and application as turn off fluorescence sensor: High sensitivity for iron(III) ions, Sensors and Actuators B: Chemical, 220 (2015) 1266-1278. [36] D. Shi, M. Ni, J. Luo, M. Akashi, X. Liu, M. Chen, Fabrication of novel chemosensors composed of rhodamine derivative for the detection of ferric ion and mechanism studies on the interaction between sensor and ferric ion, Analyst, 140 (2015) 1306-1313. [37] A.A. Bhatti, M. Oguz, S. Memon, M. Yilmaz, Dual Fluorescence Response of Newly Synthesized Naphthalene Appended Calix[4]arene Derivative towards Cu2+ and I−, Journal of

ro of

Fluorescence, 27 (2017) 263-270. [38] A.K. Manna, J. Mondal, R. Chandra, K. Rout, G.K. Patra, A thio-urea based chromogenic and fluorogenic chemosensor for expeditious detection of Cu2+, Hg2+ and Ag+ ions in aqueous medium, Journal of Photochemistry and Photobiology A: Chemistry, 356 (2018) 477-488.

[39] Y. Ma, W. Luo, P.J. Quinn, Z. Liu, R.C. Hider, Design, Synthesis, Physicochemical

-p

Properties, and Evaluation of Novel Iron Chelators with Fluorescent Sensors, Journal of Medicinal Chemistry, 47 (2004) 6349-6362.

re

[40] S. Fakih, M. Podinovskaia, X. Kong, H.L. Collins, U.E. Schaible, R.C. Hider, Targeting the Lysosome: Fluorescent Iron(III) Chelators To Selectively Monitor Endosomal/Lysosomal

lP

Labile Iron Pools, Journal of Medicinal Chemistry, 51 (2008) 4539-4552. [41] D.R. Richardson, Iron chelators as therapeutic agents for the treatment of cancer, Critical Reviews in Oncology/Hematology, 42 (2002) 267-281.

na

[42] D.R. Richardson, D.S. Kalinowski, S. Lau, P.J. Jansson, D.B. Lovejoy, Cancer cell iron metabolism and the development of potent iron chelators as anti-tumour agents, Biochimica et Biophysica Acta (BBA) - General Subjects, 1790 (2009) 702-717.

ur

[43] H.H. Kazan, C. Urfali-Mamatoglu, U. Gunduz, Iron metabolism and drug resistance in

Jo

cancer, BioMetals, 30 (2017) 629-641.

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Figure and Scheme Caption:

Scheme 1. Synthesis of receptors 3 and 4. Fig. 1. UV-Vis absorption spectra of a) compound 3 (6.25 mg.L-1) and b) compound 4 (25 mg.L-1) in water upon the addition of 500 µM various metal ions (pH 6.09). Fig. 2. Fluorescence responses of a) 3 (6.25 mg.L-1, λex= 300 nm), b) 4 (25 mg.L-1, λex= 250 nm) and naked-eye (up )/fluorescence color changes (down) of c) 3 (6.25 mg.L-1) and d) 4 (25 mg.L-1) in water upon the addition of 500 µM various metal ions (pH 6.09).

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Fig. 3. Selectivities of a) 3 (6.25 mg.L-1, λex= 300 nm) and b) 4 (25 mg.L-1, λex= 250 nm) in water. The red bars represent the fluorescent intensity of 3 and 4. The dark blue bars represent the fluorescent intensity of 3 and 4 + competitive ions (500 µM, pH 6.09). Fig. 4. Fluorescence signal changes of a) 3 (6.25 mg.L-1, λex= 300 nm) and b) 4 (25 mg.L-1, λex= 250 nm) upon the addition of gradually increased concentration of Fe3+ and Cu2+. Insets: Fluorescence titration curves of 3 versus Fe3+ ions and 4 versus Cu2+ ions (in water, pH 6.09).

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Fig. 5. Proposed binding mechanism for compound 3-Fe3+ and compound 4-Cu2+.

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Fig. 6. Live cell imaging study in HeLa cell lines. Cells were treated with either Fe3+ alone (500 μM), Fe3+ (500 μM; 1 h) plus 3 (100 μg/ml; 30 min in PBS) or 3 alone (100 μg/ml); then, images were obtained (Ex:390/40; Em:446/33). The presence of Fe3+ quenched the signal coming from 3.

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Scheme 1. Synthesis of receptors 3 and 4.

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Fig. 1. UV-Vis absorption spectra of a) compound 3 (6.25 mg.L-1) and b) compound 4 (25 mg.L-1) in water upon the addition of 500 µM various metal ions (pH 6.09).

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Fig. 2. Fluorescence responses of a) 3 (6.25 mg.L-1, λex= 300 nm), b) 4 (25 mg.L-1, λex= 250 nm) and naked-eye (up )/fluorescence color changes (down) of c) 3 (6.25 mg.L-1) and d) 4 (25 mg.L-1) in water upon the addition of 500 µM various metal ions (pH 6.09).

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Fig. 3. Selectivities of a) 3 (6.25 mg.L-1, λex= 300 nm) and b) 4 (25 mg.L-1, λex= 250 nm) in water. The red bars represent the fluorescent intensity of 3 and 4. The dark blue bars represent the fluorescent intensity of 3 and 4 + competitive ions (500 µM, pH 6.09).

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Fig. 4. Fluorescence signal changes of a) 3 (6.25 mg.L-1, λex= 300 nm) and b) 4 (25 mg.L-1, λex= 250 nm) upon the addition of gradually increased concentration of Fe3+ and Cu2+. Insets: Fluorescence titration curves of 3 versus Fe3+ ions and 4 versus Cu2+ ions (in water, pH 6.09).

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Fig. 5. Proposed binding mechanism for compound 3-Fe3+ and compound 4-Cu2+.

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Fig. 6. Live cell imaging study in HeLa cell lines. Cells were treated with either Fe3+ alone (500 μM), Fe3+ (500 μM; 1 h) plus 3 (100 μg/ml; 30 min in PBS) or 3 alone (100 μg/ml); then, images were obtained (Ex:390/40; Em:446/33). The presence of Fe3+ quenched the signal coming from 3.

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