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Benzothiazole-based fluorescent sensor for ratiometric detection of Zn(II) ion and secondary sensing PPi and its applications for biological imaging and PPase catalysis assays Chengcheng Chang, Fang Wang, Tingwen Wei, and Xiaoqiang Chen Ind. Eng. Chem. Res., Just Accepted Manuscript • Publication Date (Web): 09 Jun 2017 Downloaded from http://pubs.acs.org on June 11, 2017

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Industrial & Engineering Chemistry Research

Benzothiazole-based fluorescent sensor for ratiometric detection of Zn(II) ion and secondary sensing PPi and its applications for biological imaging and PPase catalysis assays

Chengcheng Chang, Fang Wang, Tingwen Wei, Xiaoqiang Chen*

State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemical Engineering, Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University, Nanjing 210009, P. R. China.

* Corresponding author Tel. (Fax): +86 025 83587856 E-mail address: [email protected] (X. Chen)

ABSTRACT In this paper, we designed and synthesized three benzothiazole-based fluorescent probes L1, L2 and L3 for zinc ion detection. Among various metal ions, only the zinc ion exhibited fluorescence enhancement at 475 nm accompanied with the blue-shift emission wavelength in HEPES buffer solution containing probes. Through titration experiment, the detection limit of L1 for zinc ion sensing was calculated to be as low as 7 nM, which showed a high sensitivity. Furthermore, the confocal laser scanning micrographs of HeLa cells demonstrate good cell permeability of probe L1 and selective detection of zinc ion in living cells. The L1-Zn2+ complex was further used for pyrophosphate (PPi) sensing in HEPES buffer solution, the limit of detection was calculated to be

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as low as 60 nM. The L1-Zn2+ can monitor enzyme catalyzed degradation process of PPi, thus providing a meaningful way for tracking of zinc ion and pyrophosphate in biological system.

1. INTRODUCTION Zinc(II) ion plays an important role in the physiological process as the second most abundant transition metal ion in human body after iron ion. It is believed to be related to many essential physiological activities, such as brain function and pathology, DNA synthesis, immune function, enzyme activity and neurotransmission.1-5 Therefore, developing effective methods for monitoring Zn2+ in living systems is very important for understanding the functions of zinc ion in physiological and pathological processes. In addition, pyrophosphate (PPi) plays a crucial role in various biological processes, such as metabolic enzymatic reactions and physiological energetics transduction.6 PPi is relevant to many diseases, such as calcium pyrophosphate dehydrate crystals and chondrocalcinosis.7,8 Thus, selectively recognition of PPi is also very important during various biological processes. Although conventional methods for anion sensing such as ion selective electrodes have been found, there is still a need for an alternative detection method, including the use of selective fluorescent chemosensors.9-12 In general, anions sensing in aqueous solution is a much more challenging mission than the detection of cations due to the strong hydration effects of anions. Biologically significant anion pyrophosphate (PPi) sensor particularly requires an understanding of the molecular recognition between PPi and the binding sites, the communicating and signaling mechanism, the desired solubility in an aqueous solution, and most importantly, the selectivity for PPi over other related anions. Based on the above considerations, metal ion complexes are ideal binding sites for PPi recognition in aqueous solutions.


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Fluorescence methods provided preferable approaches for the measurement of Zn2+ with high sensitivity, simplicity and real-time detection.13-16 A number of fluorescent sensors based on various fluorophores including quinoline,17-19 coumarin,20-22 fluorescein,23-25 naphthalimide,26-28 BODIPY29,30 and others31-34 were developed for detection of Zn2+, most of them show excellent sensitivity and high selectivity toward Zn2+ based on an “enhanced photon-induced electron transfer (PET)” mechanism. However, the probes based PET effects are prone to be disturbed by illumination intensity and optical path length, leading to the inaccuracy in quantitative detection. A ratiometric approach is preferable because it can eliminate the disturbing effects of these factors to realize quantitative detection by measuring the ratio of fluorescence intensities at two different emissions. 2-(2’-hydroxyphenyl)benzothiazole and its derivatives have gained wide attention in recent years because they can undergo an excited-state intramolecular photon transfer (ESIPT) process upon photoexcitation.35,36 Here we designed and synthesized three compounds L1, L2 and L3 (Figure 1) by introducing a Zn-binding moiety to 2-(2’-hydroxyphenyl)benzothiazole fluorophore, which may realize ratiometric detection of Zn(II) ion. All the three probes L1, L2 and L3 produced yellow fluorescence in the absence of Zn2+ and generated blue-shifted fluorescence after the addition of zinc ion. Among three probes, L1 showed a most remarkable fluorescence response to Zn2+, which made it a much more preferable option for Zn2+ sensing. The bio-imaging experiment was also carried out to demonstrate the application of probe L1 as zinc ion imaging agent in living cells. In addition, the probe of metal ion complexes has become increasingly popular for the detection of PPi in recent years.37-40 Therefore the L1-Zn2+ complex-type probe was applied to sensing PPi, and exhibited good selectivity among various phosphorous-containing



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anions. It is further used to monitor the degradation of PPi in the pyrophosphatase (PPase)-catalyzed reaction.

N

OH

S

HO

OH N

N

L1

OH

OH

S

HO N N

OH

HO

OH

L2

S

N

L3

Figure 1. The structures of probes L1, L2 and L3 2. EXPERIMENTAL 2.1. Materials and equipments Unless specially noted, all reagents and solvents were purchased from commercial suppliers and used without any purification. Column chromatography was performed on silica gel. 1H NMR and 13

C NMR spectra were collected on Bruker AV II-400. Mass spectra were obtained from Q-Tof

mass spectrometer (Agilent 6530). Fluorescent spectra were measured on RF-5301/PC spectrofluorophotometer. The cell images were collected using a confocal scanning microscopy (Leica, TCS sp5II).

2.2. The preparation of stock solutions for fluorescent analysis Stock solutions of various metal cations (1 mM) were prepared from NaClO4, KClO4, Cs(ClO4)2, EuCl3, Co(ClO4)2, Zn(ClO4)2, Cu(ClO4)2, Ni(ClO4)2, Zr(ClO4)4, Cd(ClO4)2, AgClO4, Cr(ClO4)3, Fe(ClO4)2, Fe(ClO4)3, LiClO4, Mg(ClO4)2, Hg(ClO4)2, Ca(ClO4)2, Al(ClO4)3 and Mn(ClO4)2. For the selective investigation for probe L1-Zn2+ toward anions, stock solutions of various anions (1 mM) were prepared from Na2HPO4, CH3COONH4, MgSO4, K2CO3, NaF, NaCl, NaBr, KI, Na3PO4, Na2H2P2O7 and NaH2PO4. All stock solutions of analytes were dissolved in deionized


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water. The probes L1, L2 and L3 (1 mM) were dissolved in DMSO. All the fluorescence and absorbance measurments were studied in HEPES buffer solution (10 mM, pH = 7.4).

2.3. Synthesis The synthesis routes of probes are showed in Scheme 1. 2.3.1. Synthesis of 3 2-hydroxy-5-methyl-benzaldehyde (2.5 g, 18.35 mmol), 2-amino-benzenethiol (2.3 g, 18.35 mmol), Na2S2O5 (3 g, 15.8 mmol) was dissolved in DMF (60 mL) and refluxed at 110 oC for overnight, the reaction progress was monitored by thin-layer chromatography (TLC). Deionized water (100 mL) was added after the reaction was completed, while a white solid was precipitated. After filtration, washing with water, drying to give the crude product benzothiazole derivatives which was further purified by silica gel column chromatography using dichloromethane as the eluent to obtain pure compound 3 (4 g, 90%). 1H NMR (400 MHz, CDCl3) δ: 12.24 (s, 1H), 7.92 (d, J =8.1Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.45 (t, J = 8.4 Hz, 1H), 7.41 (s, 1H), 7.35 (t, J = 7.6 Hz, 1H), 7.13 (d, J = 8.3Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 2.28 (s, 3H).

13

C NMR (100 MHz,

CDCl3) δ: 168.37, 154.75, 150.85, 132.72, 131.56，127.66, 127.29, 125.61, 124.40, 121.09, 120.46, 116.62, 115.29, 19.45. TOF-MS-ESI m/z found for C14H12NOS ([M] + H)+ 242.0594, calculated 242.0640 (SI Figures S1-S3). 2.3.2. Synthesis of 2 Compound 3 (2 g, 8.3 mmol) and methenamine (2.25 g, 16.07 mmol) were refluxed in trifluoroacetic acid (40 mL) at 100 oC for 5 h. After the completion of the reaction, HCl (100 mL, 4 M) was added, stirring for another 2 h. The reaction mixture was extracted by using dichloromethane. The organic phase was separated and dried over anhydrous sodium sulfate,



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filtered, and evaporated under reduced pressure and the crude product was further purified by silica gel column chromatography using dichloromethane as the eluent to give pure compound 2 (2.25 g, 80%). 1H NMR (400 MHz, CDCl3) δ: 12.99(s, 1H), 10.42 (s, 1H, ), 7.98 (d, J = 8.1 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.86 (s, 1H), 7.64 (s, 1H), 7.49 (t, J = 7.6 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 2.34 (s, 3H). 13C NMR (100 MHz, CDCl3) δ: 189.84, 165.91, 157.49, 150.35, 134.16， 132.08, 131.58, 127.91, 125.85, 124.81, 122.66, 121.32, 120.56, 117.60, 19.30. TOF-MS-ESI m/z found for C15H10NO2S ([M] - H)- 268.0463, calculated 268.0432 (SI Figures S4-S6).

2.3.3. Synthesis of L1 Compound 2 (1 g, 3.72 mmol) and tris(hydroxymethyl)aminomethane (0.45 g, 3.72 mmol) were refluxed in ethanol (40 mL) at 80 oC for 3 h. After the completion of the reaction, cooled to room temperature, the mixture was filtered and dried under vacuum. Recrystallization from CH3OH and dried under vacuum gave the target product L1 (1.05 g, 76%). 1H NMR (400 MHz, DMSO) δ: 14.70 (s, 1H), 8.54 (d, J = 12.9 Hz, 1H), 8.41 (s, 1H), 8.06 (d, J = 7.8 Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.41 (s, 1H), 7.34 (t, J = 7.5 Hz, 1H), 5.25 (s, 3H), 3.70 (s, 6H), 2.30 (s, 3H). 13C NMR (100 MHz, DMSO) δ: 172.50, 165.34, 163.35, 151.90, 137.80，136.20, 136.17, 126.16, 124.16, 123.98, 122.18, 121.91, 120.62, 116.09, 66.36, 60.88, 20.31. TOF-MS-ESI m/z found for C19H20N2O4S ([M] + H)+ 373.1153, calculated 373.1144 (SI Figures S7-S9).

2.3.4. Synthesis of L2 Compound 2 (0.2 g, 0.74 mmol) and 2-amino-1,3-dihydroxypropane (0.068 g, 0.74 mmol) were dissolved in ethanol (15 mL) and refluxed at 80 oC for 3 h. After the completion of the reaction,


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cooled the reaction liquid to room temperature, the mixture was filtered and dried under vacuum. Recrystallization from CH3OH and dried under vacuum gave the target product L2 (0.177 g, 70%). 1

H NMR (400 MHz, DMSO) δ: 14.82 (s, 1H), 8.57 (s, 1H), 8.40 (s, 1H), 8.06 (d, J = 7.4 Hz, 1H),

7.98 (d, J = 8.0 Hz, 1H), 7.50 (t, J = 7.0 Hz, 1H), 7.39 (s, 1H), 7.38 (t, J = 7.5 Hz, 1H), 5.19 (t, J = 4.6 Hz, 2H), 3.72 (m, 5H), 2.31 (s, 3H). 13C NMR (100 MHz, DMSO) δ: 170.36, 166.94, 163.09, 151.86, 136.93, 136.08, 135.64, 126.28, 124.39, 123.41, 122.18, 122.07, 121.94, 116.43, 66.62, 61.01, 20.33.TOF-MS-ESI m/z found for C18H18N2O3S ([M] + Na)+ 365.0920, calculated 365.0936 (SI Figures S10-S12).

2.3.5. Synthesis of L3 Compound 2 (0.2 g, 0.74 mmol) and ethanolamine (0.046 g, 0.74 mmol) were refluxed in ethanol (15 mL) at 80 oC for 3 h. After the completion of the reaction, cooled the reaction liquid to room temperature, the mixture was filtered then dried under vacuum. Recrystallization from CH3OH and dried under vacuum gave the target product L3 (0.18 g, 78%). 1H NMR (400 MHz, DMSO) δ: 14.61 (s, 1H), 8.55 (s, 1H), 8.41 (s, 1H), 8.06 (d, J = 7.5 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H) 7.50 (t, J = 7.6 Hz, 1H), 7.38 (t, J = 7.0 Hz, 1H), 7.34 (s, 1H), 5.15 (t, J = 4.9 Hz, 1H), 3.73 (m, 4H), 2.30 (s, 3H).

13

C NMR (100 MHz, DMSO) δ: 170.88, 167.53, 163.12, 151.86, 136.92,

136.06, 135.89, 126.27, 124.38, 123.58, 122.16, 122.06, 121.63, 116.25, 60.10, 54.63, 20.31.TOF-MS-ESI m/z found for C17H16N2O2S ([M] + Na)+ 335.0817, calculated 335.0830 (SI Figures S13-S15).

2.4 Calculation of detection limits and determination of binding constants



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Fluorescence titration was carried out in HEPES buffer solution (10 mM, pH = 7.4) to determine the detection limit. The detection limit is calculated using the following equation：

Detection of limit = 3σbi/m where σbi is the standard deviation of blank measurements, and m is the slope between the ration of fluorescent intensity (I475nm/I543nm) and sample concentration. The binding constant Ka was calculated from the emission intensity–titration curve according to the following equation41: 1 = ∆F

1 + (1 / K [C ]n )(1 / ∆F max ) ∆F max

Here ∆F = Fx - F0 and ∆Fmax = F∞ - F0 , where F0, Fx, and F∞ are the emission intensities of probe, in the absence of Zn2+, at an intermediate Zn2+ concentration, and at a concentration of complete interaction, respectively. K is the binding constant, [C] is [Zn2+], and n is the number of Zn2+ bound per probe (here n = 1).

2.5 PPase assays The stock solutions of L1-Zn2+ and PPi was added to the HEPES buffer (10 mM, pH 7.4) to make a final concentration 20 µM and 200 µM, respectively. PPase was pre-incubated for 5 min at 25 oC. The fluorescent responses of L1-Zn2+ to PPi were recorded under three catalytic conditions: (a) only PPase (20 units); (b) only Mg2+ (40 µM); (c) PPase (20 units) and Mg2+ (40 µM).

2.6 Preparation of paper test strips loaded with probes L1 The filter paper with a diameter of 1 cm was soaked in probe 10 mM of L1 solution for about 2 mins, then removed the filter paper and dried them, after that, the dried filter papers were immersed in different concentration of zinc ion solution (1 mM, 10 mM, 50 mM and 100 mM) for


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2 mins, we removed the filter paper and dried them, then we observed the fluorescence changes under the portable UV lamp. Next, filter papers were soaked in different concentrations of PPi solutions (10 mM, 20 mM, 60 mM and 150 mM) for 2 min, then were removed and dried. The changes of fluorescence with zinc ion and PPi were photographed with irradiation by the portable UV lamp.

2.7 Cell culture and fluorescence imaging experiments HeLa cells were purchased from Nanjing Cobioer Biosciences company, incubated in dulbeccos modified eagles medium (DMEM) supplement with 10% (V/V) fetal fovine serum(FBS, Gibco), 100U/mL penicillin, and 100 µg/mL streptomycin at 37 oC with 5% CO2 in appropriate humidity. Cells were pre-transferred to culture dishes and then incubated for 20 hours. Three groups were studied as follows: (I) HeLa cells were incubated with the probe L1 (10 µM) for 10 min. (II) HeLa cells were treated with probe L1 (10 µM) for 10 min and then were exposed to Zn(ClO4)2 (100 µM) for 10 min. (III) HeLa cells were pre-incubated with probe L1 (10 µM) and Zn(ClO4)2 (100 µM) for 10 min and then were exposed to PPi (100 µM) for 10 min. Cell imaging experiments were carried out after washing the cells with PBS buffer (pH = 7.4). All of the cell images were collected on a confocal scanning microscopy (Leica, TCS sp5II).

3. RESULT AND DISCUSSION 3.1 Synthesis of compound L1, L2 and L3 The synthetic routes of sensor L1, L2 and L3 were shown in Scheme 1, which were started with the reaction of 2-aminobenzenethiol and 2-hydroxy-5-methyl-benzaldehyde to form 3, after



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formulation,

reacting

respectively

with

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tris(hydroxymethyl)aminomethane,

2-amino-1,3-dihydro-xypropane and ethanolamine to obtain the final compound L1, L2 and L3 respectively. The products and intermediates were characterized by 1H NMR,

13

C NMR and

HR-Mass (SI Figures S1-S15).

N O

OH

NH2

N Na2S2O 5

OH

N

N

TFA， 100

oC，

S

2

HO

OH O

N OH

Ethanol

OH

oC，

80

N Ethanol

H2N OH

OH

S

HO N

OH

80 oC， Reflux

2

L2

N

OH OH

S

OH

Reflux

HO O

N

OH

L1

OH

S

HO N

2

N

OH

S

H2N

O

Reflux

3

N

OH

S

S

DMF， 110 oC， Reflux

SH

N N

O

Ethanol

H2N 80

oC，

OH

S

HO N

Reflux L3

2

Scheme 1. The routes of synthesis of compounds L1, L2 and L3

3.2 X-ray structure determination The single crystal structures of L1, L2 and L3 were confirmed by X-ray diffraction analyses. Crystals of L1, L2 and L3 were obtained by slow evaporation in methanol solvent respectively. The single-crystal structures of L1, L2 and L3 were shown in SI Figure S16. SI Table S1 showed the crystallographic data of L1, L2 and L3. Compound L1 was crystallized in the Monoclinic space


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group P21/n, with a = 6.757(5), b = 12.885(9), c = 20.418(14), β = 95.296(8). Compound L2 was crystallized in the Monoclinic space group P21/n, with a = 12.812 (16), b = 9.347 (12), c = 15.73 (2), β = 91.689 (15). Compound L3 was crystallized in the Monoclinic space group P21/n, with a = 8.991(3), b = 8.989(3), c = 18.683(7), β = 93.581(4). In the molecular configuration of L1, the benzothiazole group and the phenol group are in the same plane, and there existed an intramolecular H-bond between the proton of phenolic hydroxyl group and the nitrogen atom.

3.3 Fluorescent and colorimetric responses of probes to Zn2+ In order to investigate whether probes can selectively respond to zinc ion, the fluorescence responses of probe L1, L2 and L3 to various metal ions were performed in HEPES buffer solution (10 mM, pH = 7.4). As shown in Figure 2, the probes themselves showed weak fluorescence emission at 543 nm, after the addition of Zn2+, emission peak at 475 nm significantly enhanced when the excitation wavelength at 417 nm. The marked fluorescence change from light yellow to blue when mixed L1 and zinc ion (SI Figure S17). In contrast, other metal ions including Na+, K+, Cs2+, Zr4+, Co2+, Ni2+, Hg2+, Cd2+, Ag+, Cr3+, Fe3+, Eu3+, Li+, Mn2+, Mg2+, Ca2+ and Al3+ did not show obvious fluorescence response while the addition of Cu2+ and Fe2+ lead to the fluorescence quenched. Obviously, among the three probes, probe L1 exhibited the most changes in fluorescence in the presence of zinc ion (Figure 2 and SI Figure S18).



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Figure 2. a) Fluorescence responses of L1 (10 µM), L2 (10 µM) and L3 (10 µM) in the presence of 10 equiv. of various metal ions in HEPES buffer solution (10 mM, pH = 7.4). b) The histogram of selectivity for various metal ions. (λex= 417 nm)

In order to further study the responsive nature of probe L1 to Zn2+, we have carried out the fluorescence and UV-Vis spectra in HEPES buffer solution (10 mM, pH = 7.4) (Figure 3 and SI Figure S19). As shown in Figure 3, once zinc ion was added, a new emission peak at 475 nm appeared, and gradually increased with the increase of the concentration of zinc ion. There is a linear relationship between the fluorescence intensity and the concentration of Zn2+ ranging from 0 to 10 µM. Through the fluorescence intensity ratio (I475nm/I543nm) versus the concentration of Zn2+, the detection limit was calculated to be 7 nM, means that the probe can be used for the quantitative detection of zinc ion at low concentration. The UV-Vis absorption spectra of probe L1 with the addition of Zn2+ have also been investigated, and the results were shown in SI Figure S19. The initial probe showed an absorption band centered at 445 nm in the absence of Zn2+. With the addition of Zn2+, the absorption peak gradually declined and accompanied by changes in the absorption peak blue shift from 445 nm to 433 nm. Furthermore, the response reversibility of probe to Zn2+ was further studied. The probe could be reversed back by the addition of Na2EDTA


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in HEPES buffer solution (10 mM, pH = 7.4) (SI Figure S20), which is quite useful for developing the reusable fluorescent probe for Zn2+ sensing.

Figure 3. a) The changes of the fluorescence spectra of L1 with different equivalent Zn2+ (0 - 1 equiv.) in HEPES buffer solution (10 mM, pH = 7.4). b) A linear calibration curve between the ratio of fluorescence intensity of L1 at 475 nm to 543 nm (I475nm/I543nm) and the concentration of Zn2+. (λex= 417 nm)

The responses of fluorescence under different pH values were investigated. As show in SI Figure S21, the fluorescence change of probe itself is negligible in the range of pH 5.5-8. Once the zinc ion was added, obvious changes were observed under the neutral pH condition. Therefore, the probe can be well applied to detection of Zn2+ under physiological conditions. Furthermore, the binding stoichiometry of probe L1 and zinc ion was also investigated. We carried out the job’s plot experiments in which the total concentration of L1 and Zn2+ was 30 µM. As show in SI Figure S22, the plot of fluorescence intensity versus XZn (XZn= [Zn2+]/([Zn2+] + [L1])) showed the maximum fluorescence value at XZn = 0.5, which indicated that it was a 1:1 stoichiometry of the binding mode of L1 and Zn2+ in HEPES buffer solution. Through the fluorescence titration



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experiments, the binding constants between probes L1 , L2, L3 and zinc ion were calculated to be 4.53 × 104 M−1 , 4.19 × 104 M−1 and 4.12 × 104 M−1 , respectively. (SI Figure S23) The result showed the binding affinity between L1 and Zn2+ is stronger than the other two probes. In view of the fact that the Schiff base is easy to be hydrolyzed in aqueous solution, we carried out the curve of the fluorescence intensity of L1 at 543 nm along with time. As show in SI Figure S24, the fluorescence intensity of the probe remained almost stable within the first four hours. After four hours, the fluorescence intensity decreased gradually, indicating that the probe remained almost stable in aqueous solution for four hours and would not decompose.

3.6 Anion sensing properties Given that copper or zinc-based complexes have become increasingly popular for sensing PPi in recent years,42-44 we further investigated the application of complex L1-Zn2+ in the recognition of PPi. The complex of L1-Zn2+ was prepared by mixing L1 with 2 equiv. of Zn(ClO4)2. As shown in Figure 4, the fluorescence response of L1-Zn2+ to various anions were determined in HEPES buffer solution (10 mM, pH = 7.4). With the addition of PPi, the fluorescence of the complex L1-Zn2+ between L1 and zinc was significantly reduced. Finally, the fluorescence intensity remained stable, and the fluorescent intensity is almost the same as the probe L1. Moreover, the release of L1 when added PPi to the solution containing the complex L1-Zn2+ was demonstrated by the ESI-Mass spectra, in which a peak at m/z 373.1170 assigned to [L1 + H+]+ was observed (SI Figure S25). We proposed that the fluorescent intensity enhancement was due to the dissociation of L1-Zn2+ complex in the presence of PPi. In contrast, other anions including HPO42-, H2PO4-, PO43-, CH3COO-, SO42-, CO32-, F-, Cl-, Br- and I- did not show obvious fluorescence response. Therefore, L1-Zn2+ showed excellent selectivity for the detection of PPi over other anions.


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Figure 4. a) Fluorescence response of L1-Zn2+ (20 µM) in the presence of different anions (40 µM) in HEPES buffer solution (10 mM, pH = 7.4). b) The histogram of selectivity for L1-Zn2+ toward various anions. (λex= 417 nm) Furthermore, we also carried out the fluorescence titration and UV-Vis titration experiments of L1-Zn2+ with increasing concentration of PPi in HEPES buffer solution (10 mM, pH = 7.4). As shown in Figure 5, the emission peak of L1-Zn2+ at 475 nm gradually decreased with the increase of the concentration of the PPi. When the concentration of PPi increase to 60 µM, the fluorescence intensity tends to saturation and is almost the same as that of L1. Through the ratio of fluorescence intensity at 543 nm to 475 nm(I543nm/I475nm) versus the concentration of PPi, the linear relationship between I543nm/I475nm and the concentration of PPi ranging from 0 to 60 µM, and the detection limit is calculated to be 60 nM. In the absence of PPi, the complex of L1-Zn2+ showed an absorption band centered at 433 nm. With the addition of PPi, the absorption peak gradual increase and red shift to 433 nm from 445 nm (SI Figure S19), which exhibited the release of L1 from the complex L1-Zn2+ in the presence of PPi.



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Figure 5. a) The fluorescence spectra of 20 µM of L1-Zn2+ complex upon addition of 0 - 60 µM of PPi in HEPES buffer solution (10 mM, pH = 7.4). b) A linear calibration curve between the ratio of fluorescence intensity at 543 nm to 475 nm (I543nm/I475nm) and the concentration of PPi. (λex= 417 nm)

3.7 PPase catalysis assays As we known, PPase is highly active in catalyzing the cleavage reaction from PPi to HPO42- in the presence of Mg2+.45,46 Thus, we further explored whether the L1-Zn2+ can be used for detect enzyme-catalyzed degradation of PPi. The solutions of L1-Zn2+ (20 µM) and PPi (200 µM) were added to the HEPES buffer (10 mM, pH = 7.4). PPase was pre-incubated for 5 min at 25 oC, after fluorescent response process of L1-Zn2+ to PPi in PPase/Mg2+ catalytic system was monitored. As shown in Figure 6, only upon the addition of both PPase and Mg2+, the ratio of fluorescence intensity at 475 nm to 543 nm (I475nm /I543nm) was increased after 5 min, and the ratio of intensity was stable about 40 min. However, there was no obvious intensity change in the presence of only PPase or Mg2+, which demonstrated that the complex of L1-Zn2+ can monitor the hydrolysis procedure of PPi catalyzed by PPase-Mg2+ system.


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Figure 6. Fluorescent response of L1-Zn2+ to PPi generated in PPase/Mg2+ catalytic system in HEPES buffer (10 mM, pH 7.4): ▲only Mg2+ (40 µM); ● only PPase (20 units); ■PPase (20 units) + Mg2+ (40 µM).

3.8 Test paper-based application Considering the good selectivity of the probe L1 to Zn2+, we decided to put the probe for practical application by making it test papers. Firstly, the test papers loaded with probe were treated with different concentrations of Zn2+, and then fluorescence changes were observed directly using 365 nm UV lamp. As shown in Figure 7, yellow fluorescence was observed with L1 loaded on test strips. With increasing Zn2+ concentrations, the fluorescence gradually changed from yellow to cyan color. Subsequently, the papers were further exposed to different concentrations of PPi, the fluorescence gradually returned from cyan to yellow. Therefore, L1 can be successfully used as handy fluorescent probes for zinc ion and PPi.



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Figure 7. The pictures of probe L1 loaded test strips, treated with different concentrations of Zn2+, then further exposed to different concentrations of PPi. The photographs were taken with the irradiation of 365 nm UV light.

3.9 Proposed mechanism We had calculated that the binding stoichiometry of probe 1 and zinc ion was 1:1 by Job’s plot and further confirmed by MS-ESI (SI Figure S25) (a peak at m/z 435.0341 was assigned to [L1 – H+ + Zn2+]+). In order to further determine the binding mode of L1 and zinc ions, 1H NMR titration spectrum of L1 with Zn2+ was tested. SI Figure S26 showed the 1H NMR spectroscopy of L1 in the absence and presence of Zn2+. As shown in SI Figure S26, the coordination of sensor L1 with Zn2+ could induce the changes and reorganization of certain H-chemical shifts from sensor L1. The relative height of 1H NMR peak at 5.23 ppm (-OH from tris moiety) and 14.72 ppm (-OH from phenolic) reduced obviously with addition of Zn2+. By the information from H-chemical shifts, we could confirm that the coordinative cites of L1 for Zn2+ was from hydroxyl groups of tris moiety and benzothiazole. In addition, the supposed fluorescence emission mechanism was listed in Figure 8.


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Figure 8. Binding between probe L1 with Zn2+ and dissociation of complex L1-Zn2+ in the presence of PPi.

3.10 Cell cytotoxicity of L1 and cellular imaging To ascertain the cytotoxic effect of L1, the MTT assay was performed according to the reported method.47 HeLa cells were treated with 0, 10, 20, 30, 40, and 50 µM L1 for 24 h (SI Figure S27). The cell viability remained 85% under the treatment of 50 µM L1, which indicated that the probe is of low toxicity towards the HeLa cells under the experimental conditions. Confocal microscopy experiments were carried out to verify the permeability and real-time monitoring of zinc ion and PPi. As shown in Figure 9, When HeLa cells were incubated with probe L1 for 10 min (10 µM), the yellow fluorescence was observed from the green channel (Figure 9c) while blue channel showed negligible fluorescence (Figure 9b). After 10 equiv. of Zn2+ was added and incubated for 10 min, the strong blue fluorescence appeared from the blue channel (Figure 9g) though the yellow fluorescence still exist from the green channel(Figure 9h). Finally, addition of the PPi faded the blue fluorescence from the blue channel (Figure 9l). Thus, the probe can be applied to the detection of zinc ion and PPi in living cells.



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Figure 9. Confocal fluorescence images of HeLa cells. (a-k) Bright-field image; (b-l) blue channel fluorescence image (λex= 405 nm, λem= 425-490 nm); (c-m) green channel fluorescence image (λex= 480 nm, λem= 500-600 nm); (d-n) fluorescence merged image; (e-o) blue/green channel fluorescence image. (a-e) HeLa cells were incubated with the probe L1 (10 µM) for 10 min. (f-j) HeLa cells treated with probe L1 (10 µM) for 10 min and then exposed to Zn(ClO4)2 (100 µM) for 10 min. (k-o) HeLa cells were pre-incubated with probe L1 (10 µM) and Zn(ClO4)2 (100 µM) for 10 min and then exposed to PPi (100 µM) for 10 min.

4. CONCLUSIONS In conclusion, we have successfully developed benzothiazole-based fluorescent probes L1, L2 and L3 for specific detection of zinc ion. All the three probes L1, L2 and L3 produced yellow fluorescence in the absence of Zn2+ and generated blue-shifted fluorescence after the addition of zinc ion. Because L1 showed a most remarkable fluorescence response to Zn2+, which made it a much more preferable option for Zn2+ sensing. Furthermore, the L1-Zn2+ complex could also be


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used for further sensing of pyrophosphate (PPi) and monitoring the PPase catalysis process. The imaging experiments demonstrated that the probe L1 can be applied to detection of zinc ion and PPi in living cells.

ACKNOWLEDGMENTS X. Chen acknowledges funding from the National Natural Science Foundation of China (21376117), the Jiangsu Natural Science Funds for Distinguished Young Scholars (BK20140043), and the Natural Science Foundation of Jiangsu Higher Education Institutions of China (14KJA150005), the Qing Lan Project and the Project of Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version. Crystallographic data for the structures reported in this article have been deposited with the Cambr idge Crystallographic Data Center as supplementary publication Nos.

CCDC

1538581

(L1),

1538582 (L2), 1538580 (L3). Copies of the data can be obtained free of charge via www.ccdc.cam.ac.uk (or from the Cambridge Crystallographic Centre, 12 Union Road, Cambridge CB21EZ, UK; Fax: +44 1223 336033; e-mail: [email protected]).

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The benzothiazole-based fluorescent probe for detection of zinc ion was synthesized for ratiometric detection of Zn(II) ion and secondary sensing PPi. 254x190mm (96 x 96 DPI)


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