A novel 3-Hydroxychromone fluorescence sensor for intracellular Zn2+ and its application in the recognition of prostate cancer cells

Accepted Manuscript Title: A novel 3-Hydroxychromone fluorescence sensor for intracellular Zn2+ and its application in the recognition of prostate cancer cells Authors: Xiang Li, Jie Li, Xiongwei Dong, Xiang Gao, Dan Zhang, Changlin Liu PII: DOI: Reference:

S0925-4005(17)30177-6 http://dx.doi.org/doi:10.1016/j.snb.2017.01.170 SNB 21695

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

13-12-2016 21-1-2017 25-1-2017

Please cite this article as: Xiang Li, Jie Li, Xiongwei Dong, Xiang Gao, Dan Zhang, Changlin Liu, A novel 3-Hydroxychromone fluorescence sensor for intracellular Zn2+ and its application in the recognition of prostate cancer cells, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2017.01.170 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A novel 3-Hydroxychromone fluorescence sensor for intracellular Zn2+ and its application in the recognition of prostate cancer cells Xiang Li, Jie Li, Xiongwei Dong, Xiang Gao, Dan Zhang*, and Changlin Liu*

Key Laboratory of Pesticide and Chemical Biology, Ministry of Education and School of Chemistry, Institute of Public Health and Molecular Medicine Analysis, Central China Normal University, Wuhan 430079, P. R. China.

* Corresponding author. Tel.: 86 027-67867232. E-mail: [email protected] (Dan Zhang); [email protected] (Changlin Liu).

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Graphical abstract

Highlights: 1: 3HC-DPA can chelate Zn2+ in a dimeric form (2:2), emitting strong fluorescence sensitively and selectively. 2: A novel fluorescent ESIPT-blocking zinc sensor 3HC−DPA was reported, which provided an opportunity to further development of ESIPT-based metal sensors in bio-imaging. 3: 3HC-DPA possessed the ability to monitor intracellular free zinc ions in live cells, and had an excellent mean cell retention time. 4: 3HC-DPA has a promising application in the recognition of cancerous prostate cells from normal prostate cells. Abstract: A novel fluorescent zinc sensor 3HC−DPA, containing a 3-hydroxychromone (3HC) chromophore and a zinc-selective metal chelator di(2-picolyl)amine (DPA), was prepared and found to chelate Zn2+ in a dimeric form. Due to the deprotonation of 3HC and occupation of the lone electron pairs on the nitrogen atoms of DPA, the complex [Zn2(3HC-DPA)2]2+ emitted sensitively and selectively strong fluorescence in solution. Moreover, cell experiments, including flow cytometry and two-photo excitation fluorescence microscopy, as well as inductively coupled plasma-atomic emission spectrometry (ICP-AES), demonstrated that 3HC-DPA possessed the ability to monitor intracellular free zinc ions in live cells. A promising use was developed for the ability of 3HC-DPA in the recognition of cancerous prostate cells from normal prostate cells, based on the added Zn2+ detection by flow cytometry and cell imaging. Keywords: Zinc-selective metal chelator; Biosensors; Recognition; Prostate cancer cells; Bioimaging

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1. Introduction Zinc ion plays important roles in both physiological and pathological processes [1-5]. The failures in homeostasis of free zinc ions are closely associated with many pathological states [6-9], therefore, the development of new sensors for intracellular free zinc ions is of critical importance. Recently, it has been widely accepted that mobile zinc in cells could be taken as an excellent biomarker candidate for prostate cancer progression, as the level of free zinc ions decreases dramatically during the development of prostate cancer, in agreement with the downregulation of ZIP1 transporter or the inability to accumulate zinc in cancer cells [9-11]. Lippard and co-workers have first used zinc as an imaging biomarker and successfully employed a Zinpyr family zinc probe ZPP1 for early detection of prostate cancer in a transgenic mouse model [9,12-14]. Although many zinc sensors have been reported in recent years [14-27], just few of them can be used in the recognition of prostate cancer cells except Zinpyr family. Actually, fluorescent dyes with lower fluorescence background, higher sensitivity and selectivity, better cell-permeable properties and longer mean cell retention time are still in high demand, which would be more suitable for the bioimaging of intracellular free zinc ions. ESIPT (excited-state intramolecular proton transfer) is emerging as a new design principle for fluorescence probes and attracts extensive attention for their unique photophysical properties [28,29]. ESIPT molecules possess a protondonor moiety (mainly OH) and a protonacceptor moiety (mainly N=, =O) with a suitable distance, which could form an intra-molecular hydrogen bond in a five- or six-membered ring. Upon photoexcitation, one part of molecules emits fluorescence with short wavelength in the process of returning to the ground state, the other part of molecules undergoes ESIPT, in which the proton transfers from its H−donor moiety to the H−acceptor moiety, resulting in distinct emission with a typical large Stokes shift (about 150−200 nm) [30,31]. When the proton undergoing ESIPT is lost in the presence of competing Lewis acids, high pH, or a solvent with high polarity and Hbond acceptor ability, ESIPT process is suppressed and then only one emission with short wavelength can be observed, which could be called as ESIPT-blocking [32-37]. Despite the limited studies in the research of ESIPT probes, ESIPT-based metal sensors (ESIPT-blocking system) are attractive for their facile controls in fluorescence properties, which could be attributed to the direct interaction between metal ions and the key points (such as −OH) of ESIPT chromophores [20,21,37]. As an important member of ESIPT-based molecules, 3-hydroxychromone (3HC) derivatives have been reported for their excellent sensitivity to the polarity of microenvironment [38-41], which were used to investigate the organized ensembles such as micelles [42,43], phosphor lipid vesicles [44,45], proteins [46-50], nucleic acids [51-53] and polymers [54], and to obtain insights into some biological processes [55]. Here, a novel 3HC fluorescent zinc probe 3HC−DPA, which is consisted of 3HC chromophore and a zinc-selective chelator di(2-picolyl)amine (DPA), was prepared (Scheme 1) and applied in the recognition of cancerous prostate cells from normal prostate cells by flow cytometry and two-photo excitation fluorescence microscopy. We expected that the zinc-based fluorescence properties of 3HC-DPA and its promising application in the recognition of prostate cancer cells could be an effective complement to the investigation of the fluorescence

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response of ESIPT suppression mediated by zinc-coordination, and provide opportunities to further develop 3hydroxychromone sensors for bioimaging application, as well as zinc-based cancerous prostate diagnostics. 2. Material and methods 2.1. Materials and instrumentation Unless otherwise noted, materials were purchased from commercial suppliers and used without further purification. All the solvents were treated according to standard methods. 1H NMR spectra were recorded on 400 or 600 MHz spectrophotometers (Vadan Mercury Plus-400/600 MHz spectrometer). Chemical shifts are reported in delta (δ) units in parts per million (ppm) relative to the singlet for tetramethylsilane (TMS). 13C NMR spectra were recorded on 100 or 150 MHz with complete proton decoupling spectrophotometers. Chemical shifts are reported in ppm relative to the central line of the heptalet at 77.0 ppm for CDCl3 or 39.6.0 ppm for DMSO-d6. The HRMS were obtained on Agilent 6460 Triple Quadrupole mass spectrometer. The concentrations of metal ions were performed on ICP-AES (Thermo scientific 6300, USA). The fluorescence imaging experiments were carried out using Carl Zeiss LSM710 laser scanning confocal microscopy (Carl Zeiss, Germany). X-ray single crystal diffraction analysis was carried out on Bruker APEX DUO and Brucker AXS SMART 4000. 2.2. General spectroscopic methods All solutions were prepared with spectrophotometric grade solvents. All of the UV/Vis and fluorescence spectroscopy were working in deionized water solutions. Absorption spectra were recorded on a SPECORD 210 Ultra-visible spectrophotometer (Analytic Jena AG) using 1 cm path length quartz cuvette with a total volume of 200 µL (at 25 °C in 50 mM Tris-buffer, pH 7.4). Fluorescence spectra were recorded on a Cary Eclipse fluorescence spectrophotometer (Varian, USA). Fluorescence spectra were routinely acquired at 25 °C in a 1 cm quartz cuvette with a total volume of 400 μL, unless otherwise stated, 5 nm slit widths and a photomultiplier tube power of 700 V were used. The fluorescence emission spectra of 3HC-DPA or [Zn2(3HC-DPA)2]2+ were collected between 400 and 600 nm, and the excitation wavelength of them was set at 382 nm (in 50 mM Tris-buffer, pH 7.4). 2.3. Confocal imaging The cells were seeded in a 6-well plate at a density of 104 cells per mL in culture media. After 24 h, the cells were treated without or with Zn2+ of different concentrations (0, 5, 10, 20, 50 μM) in culture media for 4 h at 37 oC. After washing with D-PBS (RWPE-1) or PBS (DU145 and Hela) to remove the remaining Zn2+, the cells were further incubated with 20 μM of 3HC-DPA in culture media for 2 h at 37 oC. Then, cells were washed by D-PBS or PBS to remove the remaining 3HC-DPA. Finally, cells were imaged by confocal microscopy with two-photon excitation at 780 nm for 3HC-DPA and the fluorescence signals for 3HC-DPA were acquired for imaging with a×63 (Plan-Apo, NA 1.4, oil immersion) objective. 2.4. Zinc detection in prostate cells by flow cytometry The cells were seeded in a 6-well plate at a density of 104 cells per mL in culture media. After 24 h, the cells were treated without or with Zinc ions of different concentrations (0, 5, 10, 20, 50 μM) in culture media for 4 h at 37 oC. After washing with D-PBS (RWPE-1) or PBS (DU145) to remove the remaining Zn2+, the cells were further

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incubated with 20 μM of 3HC-DPA in culture media for 2 h at 37 oC. Then, cells were washed three times with DPBS (PRWE-1) or PBS (DU145) to remove the remaining 3HC-DPA. Finally, cells were dissociated by 0.25% trypsin-EDTA and resuspended in D-PBS (PRWE-1) or PBS (DU145). Fluorescence data was collected and analyzed on the BD FACSAria III in FL1 using a 520/30 BP filter. 3. Results and discussion 3.1. Design and Synthesis of 3HC-DPA An ESIPT-blocking zinc sensor HNBO−DPA, consisting of 2-(2’-hydroxy-3’-naphthyl)benzoxazole (HNBO) chromophore and DPA, previously reported by Nam and co-workers [21], showed a strong fluorescence turn-on response toward Zn2+. In their study, the mechanism was considered to suppress of photoinduced electron transfer (PeT) exerted by deprotonation of ESIPT chromophore HNBO as well as occupation of the electron pair of DPA. We thus designed another ESIPT-based fluorescent sensor to detect intracellular zinc ions and further recognize prostate cancer cells. In addition, as a widely known 3HC derivative fluorescent probe, FA, 2-(6diethylaminobenzo[b]furan-2-yl)-3-hydroxychromone, has been reported to bind to bovine serum albumin (BSA) with a very high binding constant [40], which may be helpful to increase the mean cell retention time when 3HC derivatives are used in cell imaging, and consequently improve the sensitivity of detection. Therefore, a new ESIPTblocking fluorescent zinc sensor, 3HC−DPA that is consisted of an ESIPT chromophore moiety 3HC and a zincselective metal chelator moiety DPA, was designed and prepared in this study. As shown in Scheme 1, 3HC-DPA was synthesized in four steps. Acid-catalyzed condensation between ophthalaldehyde and ethylene glycol produced the intermediate product 1, in which one aldehyde group of ophthalaldehyde was protected. Next, DPA reacted with another aldehyde group based on aldehyde-ammonia condensation, and then yielded the zinc-selective chelating unit (2) [14]. After deprotection in the presence of HCl, the obtained compound 3 subsequently underwent an oxidative cyclization to form the ESIPT unit flavonol (3HCDPA) [56]. All novel compounds were fully characterized by NMR (1H and 13C) and HRMS, and 3HC-DPA was additionally characterized by X-ray single crystal diffraction analysis (ESI, detailed crystallographic data were summarized in Table S1). 3.2. Fluorescence Properties of 3HC-DPA in the presence of Zn2+ To demonstrate the fluorescent response and sensing property of 3HC-DPA toward zinc ions, we first evaluated the interaction between 3HC-DPA and Zn2+ in 50 mM Tris-buffer, pH 7.4. As shown in Fig. S1a, upon titration of Zn2+, the intense absorption band at 331 nm decreased gradually along with the increase of a new band at 382 nm, and a well-defined isosbestic point at 350 nm distinguished free 3HC-DPA and the corresponding zinc complex clearly. In addition, through plotting the absorbance ratio (λ382 nm /λ331 nm) against the concentration ratio r ([Zn2+]/[3HC-DPA]) (Fig. S1b), we found that the absorbance intensity of the two species almost maintained the same when r was equal or greater than 1, which indicated that the coordination between 3HC-DPA and Zn2+ in solution has a 1:1 binding stoichiometry. Moreover, a Job’s plot also confirmed the 1:1 complexation (Fig. S2a). Based on the results of spectrophotometric titrations, we further measured the excitation and emission spectra of 3HC-DPA as well as the complex, respectively (Fig. S3a and Fig. S3b). The results displayed that the

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excitation/emission maximum pair of 3HC-DPA was 331 nm/512 nm, upon addition of two equivalents of Zn2+, the complex was formed and its excitation/emission maximum pair was 382 nm/466 nm. Then, the Zn2+-induced fluorescence response of 3HC−DPA was investigated by fluorescence titration in the same buffer. As shown in Fig. 1a, the background fluorescence of 3HC-DPA (10 μM) was very weak when excited at 382 nm, upon titration with Zn2+, 3HC−DPA underwent a significant increase in fluorescence, and almost kept unchanged after one equal Zn2+ was added (Fig. 1b). Plotting the relative fluorescence intensity F/F0 (F and F0, the emission intensity of 3HC-DPA at 466 nm in the presence and in the absence of Zn2+, respectively) against the concentration of Zn2+, a linear response of F/F0 versus zinc concentration could be observed over the range of 0∼10 μM with a correlation coefficient (R) of 0.99 (Fig. 1b, insert). As expected for a zinc-responsive sensor with bioimaging application, a cell membrane-permeable Zn2+ chelator, TPEN (N,N,N',N'-tetrakis(2pyridylmethyl)ethylenediamine) [57], was subsequently added into the mixture of 3HC-DPA and Zn2+, the results (Fig. S2b) indicated that changes in the emission spectra of 3HC-DPA observed upon zinc coordination can be easily reversed by the treatment with TPEN. In addition, we further evaluate the stability of 3HC-DPA through examining its ability of resistance to acid and alkali as well as the anti-photobleaching. The results showed that the intensity of the emission maximum of the complex at 466 nm almost maintained in pH=5-10 buffer solutions, but slightly lower in pH 4.0 buffer solution (Fig. S4a). Therefore, it’s reasonable to conclude that 3HC-DPA is stable enough to be applied in bio-imaging on physiological condition (pH=7.4). Irradiation under UV light for a long time would photobleach the fluorescence intensity of the complex to some degree (Fig. S4b), however, it would have little effect on the results of imaging if we finished the imaging process within a few minutes. Furthermore, the selectivity of Zn2+-induced fluorescence response of 3HC-DPA was determined by recording the emission spectra of 3HC-DPA (10 μM), respectively, before and after the treatment with a variety of biologically relevant metal ions, such as K+, Li+, Na+, Mg2+, Mn2+, Hg2+ (100 equiv to 3HC-DPA), Cr3+, Cd2+ (10 equiv) and Fe3+, Fe2+, Co2+, Ni2+, Cu2+ (1 equiv), followed by subsequent addition of Zn2+ (10 μM). As shown in Fig. 2a, the presence of the tested metal ions (except Cu2+) had no obvious effect on the fluorescent zinc response, indicating the high selectivity of 3HC-DPA toward Zn2+. As to Cu2+, it could be explained by competitive binding for the interference of Cu2+ with zinc detection, which made it hard for Zn2+ to reverse the fluorescence. On the other hand, the fluorescence quenching effect of Cu2+ assured that no “false positive” turn-on response would be induced by Cu2+ in the analysis of biological samples [12]. In addition, it is noted that both Hg2+ (100 equiv) and Cd2+ (10 equiv) had little effect on the fluorescence response of 3HC-DPA to Zn2+, although the three elements have similar physical characteristics and chemical behavior in solutions, and Co2+ is more interferential than Hg2+ and Cd2+. Here, the interference of Hg2+, Cd2+ and Co2+ can be explained by Lewis acid-Lewis base interactions and the principle of hard and soft acids and bases (HASB). Zn2+ is hard metal ion, Co2+ is intermediate metal ion, and both Hg2+ and Cd2+ are soft metal ions, [58] therefore, when reacting with the hard N- and O-donor atoms of 3HC-DPA, the order of stabilities for the complexes between 3HC-DPA and Zn2+, Co2+, Hg2+ and Cd2+ is as following: Zn2+ > Co2+ > Hg2+ and Cd2+. 3.3. The Binding between 3HC-DPA and Zn2+

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In order to elucidate the zinc binding mode of 3HC−DPA, crystals were grown in CH3OH solution consisting of 0.04 mM Zn(NO3)2 and 0.04 mM 3HC−DPA. The X-ray single crystal diffraction analysis exhibited that 3HC-DPA chelated Zn2+ in a penta-coordinate manner, and formed a zinc complex [Zn2(3HC-DPA)2]2+ with a 2:2 zinc-toligand ratio (Fig. 2b and Scheme 1, detailed crystallographic data were summarized in Table S3, S4). The dimeric crystal structure provided convincing evidence that one Zn2+ coordinates to DPA “N” atoms in one 3HC-DPA and to two 3HC “O” atoms in another 3HC-DPA, thereby prohibited ESIPT process through displacing the ESIPT hydroxyl proton of 3HC. A similar fluorescence response was previously reported by Nam and co-workers for HNBO−DPA [21], accordingly, the fluorescence turn-on mechanism of 3HC-DPA could be analogous to HNBO−DPA, that is, in the zinc-free form, DPA transferred its electron to the adjacent fluorophores 3HC in the excited state, quenching the fluorescence emission of 3HC-DPA to a great extent. However, when the “N” atoms of DPA and the “O” atoms of 3HC coordinate with zinc ions, the nonradiative electron transfer was suppressed and thus resulted in the fluorescence emission at 466 nm. Therefore, the fluorescence turn-on response for Zn2+ could be attributed to the suppression of photoinduced electron transfer (PeT) exerted by deprotonation of 3HC and occupation of the lone electron pairs of DPA. We further verified the existence of the dimeric form in solution through NMR titration and HRMS. As shown in Fig. S5, it was suggested that the 1H NMR chemical shifts of the ortho and para positions of pyridyls in 3HC-DPA were δ 8.31-8.33 ppm and δ 7.12-7.16 ppm, respectively. When Zn2+ was added gradually (from 0.5 equiv to 2 equiv) to the solution and the complex was formed, the 1H chemical shifts in the ortho-position of pyridyl might change from δ 8.31-8.33 ppm to δ 8.51 ppm, and the peak at δ 7.12-7.16 ppm disappeared. Besides, the peaks of six hydrogen-atoms in -CH2- distributed in three regions (4.08, 3.91, 3.72 ppm) after mixing Zn2+ with 3HC-DPA, which declared that the nitrogen-atom bonding with -CH2- coordinated with Zn2+. In this respect, the results of NMR titrations suggested that all of the three nitrogen-atoms of 3HC-DPA coordinated with Zn2+, which was accorded with the results of crystal structure. Moreover, the dimeric form of [Zn2(3HC-DPA)2]2+ was also confirmed by HRMS (HRMS (EI): m/z [M+K]+ calcd for C56H44N6O6Zn2K: 1063.1520, found: 1063.1564. the data was shown in the SI). Based on the binding mode between 3HC-DPA and Zn2+, we further detected the stability constant logK of the zinc complex of 3HC-DPA by potentiometric titrations, the detail data were shown in Fig. S6 and the result was 16.82. In addition, the molar extinction coefficient of the zinc complex of 3HC-DPA was observed to be 18440 L∙mol-1∙cm-1 (Fig. S7), and its fluorescence quantum yield was 0.432 (Table S2) [59]. 3.4. Application in the Recognition of Prostate Cancer Cells The above-mentioned fluorescence response study of 3HC-DPA to zinc ions encouraged us to further investigate its application in intracellular zinc sensing in cell culture. First, we examined the response of 3HC-DPA to Zn2+ within HeLa cells. As shown in Fig. 3a-d, when incubated with 3HC-DPA (20 μM) alone, the HeLa cells displayed a very faint background by two-photo excitation in laser-scanning microscopy (Fig. 3b). However, bright green fluorescence images could be observed easily when incubating Hela cells with Zn2+ (50 μM) for 30 min first, and reincubating with 3HC-DPA (20 μM) for another 2 h after washing the extracellular Zn2+ (Fig. 3c). In order to prove that the fluorescence signal was a consequence of the response to intracellular zinc ions, TPEN (100 μM) was

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subsequently added, and the result showed that the bright fluorescence was quenched and almost disappeared (Fig. 3d), which confirmed the reversibility of Zn2+-induced fluorescence response. Then, we examined the biocompatibility and cytotoxicity of 3HC-DPA for DU145 and RWPE-1 through cell proliferation and MTT assay. Thereinto, cell counting assay was used to determine a cell proliferation [60]. The results showed that, little cytotoxicity could be observed by 3HC-DPA against DU145 or RWPE-1 cell lines when the concentration of 3HC-DPA reached 40 μM (Fig. 3e). In addition, little effect on the cell proliferation of DU145 or RWPE-1 could be observed by incubating 3HC-DPA with cells for 24 h at 37 oC (Fig. S8a). Finally, we evaluated the mean cell retention time of 3HC-DPA within human normal prostate cells (RWPE-1). In this experiment, 100 μM Zn2+ was incubated with RWPE-1 cells for 4 h in advance, after washing extracellular Zn2+, 3HC-DPA (20 μM) was added into the plates, and continued incubation for 0.5, 2, 4, 6 h, respectively, before measurements by flow cytometry. The results displayed that the fluorescence intensity of 3HC−DPA did not significantly decrease in this incubation period, and maintained 80% after incubation for 6 h compared with that of 0.5 h (Fig. 3f). The longer mean cell retention time might be related to the high binding affinity between 3HC and proteins in cells [40], which was beneficial to improve the sensitivity of detection. To confirm the selectivity of 3HC-DPA to free zinc ions but not to zinc in proteins, 3HC-DPA was incubated for 30 min with free zinc ions, SOD1 (copper-zinc superoxide dismutase 1), proteomes extracted from Hela, RWPE-1 (normal prostate cell lines) and DU145 (cancerous prostate cell lines) cells respectively, and then measured by fluorescence spectrophotometer. The results shown in Fig. S8b indicated that 3HC-DPA only reacted with free zinc ions and gave rise to strong emission, compared with SOD1 or the proteomes extracted from cells. As the above-described results of cell experiments displayed better cell-permeable properties, longer mean cell retention time and promising bioimaging application of 3HC−DPA for intracellular free zinc ions, we further explored the utility of zinc sensing using 3HC-DPA in DU145 and RWPE-1 cell lines, based on the reported research results that the concentration of zinc ions decreased dramatically during the development of prostate cancer [10, 61-63]. As shown in Fig. S9 and Fig. 4, when the added concentrations of zinc ions were 0, 5, 10, 20, 50 μM, respectively, the zinc concentration-dependent fluorescence images could be observed in the normal prostate cells RWPE-1 after the treatment with 3HC−DPA (20 μM). However, almost no fluorescence signals in the cancerous prostate cells DU145 could be found even when the added concentration of Zn2+ was 50 μM. The significant difference in the fluorescence imaging between the two cell lines was consistent with the uptake of extracellular zinc by the cells through zinc transporters such as ZIP1 [10], that is, compared with normal RWPE-1 cells, zinc transporters are down-regulated in cancerous DU145 cells, which leads to overall reduced levels of zinc uptake. This conclusion was also supported by the result of inductively coupled plasma-atomic emission spectrometry (ICP-AES). In these measurements, RWPE-1 and DU145 cells were first incubated with 0, 1, 10 and 50 μM zinc ions for 4 h, respectively. Then, we took the supernatant of each cell lysate sample after washing extracellular Zn2+ of the cells and detected the concentration of protein through BCA Protein Assay Kit. Finally, the contents of Zn2+ and Cu2+ in the supernatants were identified by ICP-AES following diluting to maintain the protein concentration same in each sample. It was easy to find that the intracellular zinc levels detected by ICP-AES kept consistent with the added Zn2+ concentration in RWPE-1 cells, while only the little change could be observed in DU145 cells when the added zinc

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ions increased from 0 to 50 μM (Fig. 5a). The concentration of endogenic Cu2+ inside the cancerous cells and normal cells maintained at very low levels, which were obviously less than Zn2+ (Fig. 5a). In this respect, the interference from Cu2+ in vivo could be negligible, as the endogenic Cu2+ inside the cancerous cells is about one-fifth of Zn2+. In order to exclude the possibility that 3HC-DPA might act as an ionphore (i.e. carrying the metal across the membrane) when incubation with RWPE-1 cells, we did a flow cytometry experiment (Fig. S10), the result displayed that the relative fluorescence intensity of per DU145 or RWPE-1 cell was almost the same when zinc ions and 3HC-DPA were incubated with DU145 or RWPE-1 cells simultaneously, which indicated that 3HC-DPA didn’t carry more zinc ions into RWPE-1 cells compared with that in DU145 cells. The turn-on fluorescence response of 3HC-DPA both in RWPE-1 and DU145 cell lines were further detected by flow cytometry measurements. On the one hand, we maintained the concentration of 3HC-DPA at 20 μM and altered the concentration of added Zn2+ (0, 1, 10, 30 and 50 μM, respectively), the result shown in Fig. 5b displayed that the mean fluorescence intensity per cell in RWPE-1 cells increased gradually with the increase of added Zn2+, but the fluorescence intensity was not obvious in DU145 cells. Significantly, the difference in 3HC-DPA fluorescence was also visible between the two cell lines when no zinc ions was added, which was consistent with that the level of endogenous free zinc ions decreased dramatically during the development of prostate cancer. On the other hand, we kept the concentration of added Zn2+ at 80 μM and changed the concentration of 3HC-DPA (0, 20, 40, 60 and 80 μM, respectively). As shown in Fig. S11, the mean fluorescence intensity per cell of RWPE-1 increased dramatically upon the addition of 20 μM and 40 μM 3HC-DPA, and then maintained when 60 μM or 80 μM 3HCDPA was added, while almost no change in the fluorescence intensity could be found in DU145 cells when different concentrations of 3HC-DPA were added. Therefore, we concluded that 3HC-DPA was able to recognize cancerous prostate cells from normal prostate cells based on Zn2+ sensing. It’s reasonable to attribute the fluorescence properties and the successful application of 3HC-DPA in the recognition of prostate cancer cells to the advantageous characteristics of 3HC chromophore as well as the special chelating manner between 3HC-DPA and zinc ions. In the zinc complex [Zn2(3HC-DPA)2]2+, Zn2+ coordinates with three “N” and two “O” atoms of 3HC-DPA in a penta-coordinate manner. On the one hand, the coordination of 3HC−DPA with Zn2+ exerted deprotonation of 3HC and occupation of the electron pair of DPA, thus suppressed PeT process and then emitted fluorescence sensitively, on the other hand, the ligating “N” and “O” atoms was helpful to improve the selectivity of 3HC−DPA to zinc ions in detection among biologically relevant metal ions. Moreover, the high binding affinity between 3HC derivatives and proteins (mainly BSA) was benefit to enhance the cell-permeable properties, mean cell retention time and the detection sensitivity of 3HC-DPA in cell experiments, thus realizing a promising application in the recognition of cancerous prostate cells. 4. Conclusions In this study, we reported the synthesis and photophysical evaluation of a novel ESIPT-blocking zinc sensor 3HC−DPA, containing the ESIPT chromophore moiety 3HC and a zinc-selective metal chelator moiety DPA. The results of crystal structure, NMR titration and HRMS indicated that 3HC−DPA chelated zinc ions in a pentacoordinate manner, and the resulting zinc complex was found to have a dimeric structure with a 2:2 zinc-to-ligand ratio. Cell experiments demonstrated that 3HC-DPA possessed the ability to monitor intracellular free zinc ions in

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live mammalian cells with longer mean cell retention time, therefore, was able to distinguish cancerous prostate cells from normal prostate cells, based on the added free zinc ions sensing. Overall, the fluorescence property study of 3HC-DPA here provided an opportunity to further development of ESIPT-based metal sensors for bioimaging application, and also clearly illustrated the value of zinc-based cancerous prostate diagnostics. Acknowledgments This work was financially supported by National Natural Science Foundation of China [21302059, 21271079]; Natural Science Foundation of Hubei Province of China [2014CFB654]; Self-determined research funds of CCNU from the colleges’ basic research and operation of MOE [CCNU14A05004]. Appendix A. Supporting information Supplementary data associated with this article can be found, in the online version, at doi:XXXXXXXXX.

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Biographies: Xiang Li is studying for PhD degree at Central China Normal University. His research focuses on the biochemistry and the interactions between proteins and DNA. Jie Li is a MS student graduated from Central China Normal University. Her research focuses on the development of new fluorescent sensors. Xiongwei Dong is a PhD student graduated from Central China Normal University. His research focuses on the regulation of ROS in cells by inorganic methods.

Xiang Gao is studying for MS degree at Central China Normal University. Her research focuses on the development of new fluorescent sensors of GSH and H2O2. Dan Zhang is an associate professor of the College of Chemistry, Central China Normal University (CCNU). She received her PhD in 2003 from Wuhan University. She joined the faculty at Central China Normal University in 2009. Her recent research focuses on molecular recognition, prostate cancer and G-quadruplex. Changlin Liu is a professor of the College of Chemistry, Central China Normal University (CCNU). He received her PhD in 1998 from Huazhong University of Science and Technology. He worked in Huazhong University of Science and Technology from 1986 to 2006. He joined the faculty at Central China Normal University in 2006. His recent research focuses on the regulation of ROS and the interactions of proteins and DNA.

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List of Figures: Fig. 1. (a) Fluorescence spectra of 3HC-DPA (10 μM) in titrations with Zn2+ (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200 μM). The arrow indicates the development of bands with increasing Zn2+ concentration. (b) Plot of the relative fluorescence intensity of 3HC-DPA at 466 nm with Zn2+ (F) and without Zn2+ (F0) against the concentration of Zn2+. Insert: A linear response of emission intensity ratio F/F0 versus Zn2+ concentration (R2 = 0.99). Fig. 2. (a) Metal ion selectivity of 3HC−DPA (10 μM): White bar, fluorescence intensity of the zinc-free form; Black bar, fluorescence intensity in the presence of metal ion (K+, Li+, Na+, Mg2+, Mn2+, Hg2+ ions, 100 equiv; Cr3+, Cd2+,10 equiv; Fe3+, Fe2+, Co2+, Ni2+, Cu2+, 1 equiv to 3HC−DPA); Red bar, fluorescence intensity after subsequent addition of zinc ion (1 equiv) to the mixture. λex = 382 nm. (b) X-ray crystal structure of zinc complex [Zn2(3HCDPA)2]2+ (CCDC: 1414677). ‡ Fig. 3. Fluorescence microscope images of HeLa cells (a), incubation with 20 μM 3HC-DPA (b), in the presence of 50 μM added Zn2+ and 20 μM 3HC-DPA (c), and incubation with 50 μM Zn2+, 20 μM 3HC-DPA, and 100 μM membrane-permeable zinc ion chelator TPEN (d). Scale bar = 20 μm. λex = 780 nm. (e) MTT assay of the cytotoxicity for 3HC-DPA (0, 0.5, 5, 10, 20, 30, 40 μM) in DU145 and RWPE-1. The results were representative of at least five independent experiments. (f) 3HC−DPA in RWPE-1 during different retention time. The concentration of Zn2+ added in advance was 100 μM, and incubation time of 3HC−DPA (20 μM) in RWPE-1 cells was 0.5 h. The mean cell retention times were 0.5, 2, 4, 6 h, respectively. The results were representative of at least three independent experiments. Fig. 4. Fluorescence microscope images in human cancerous prostate cell line DU145 and human normal prostate cell line RWPE-1 cell line with 3HC−DPA, respectively. The concentration of 3HC-DPA was 20 μM, and the concentration of added Zn2+ was 50 μM. Scale bar = 50 μm. λex = 780 nm. Fig. 5. (a) Detection of Zn2+ and Cu2+ in human cancerous prostate cell line DU145 and human normal prostate cell line RWPE-1 with ICP-AES (*P<0.001, Student’s t-test). 0, 1, 10, and 50 μM Zn2+ was added in those cell lines, respectively. (b) Both DU145 and RWPE-1 cell lines were incubated with 80 μM Zn2+ for 4 h. After washing extracellular Zn2+, relative fluorescence intensity of per RWPE-1 or DU145 cell was measured by flow cytometry after incubation with 20 μM 3HC−DPA for 1 h, respectively. The results were representative of at least three independent experiments (*P<0.001, Student’s t-test).

List of Scheme: Scheme 1. Synthesis of the fluorescent Zn2+ sensor 3HC-DPA.

16

a)

30

Zn2+

b)

200 M

30

600

20 400 0

F/F0

F/F0

Fluorescence Intensity (a.u.)

800

10

200

20

R2=0.99

10 0 0

0

0 400

450

500 550 Wavelength (nm)

600

0

3

6

2+

9

[Zn ] (M)

50 100 150 2+ Concentration of Zn (M)

12 200

Fig. 1. (a) Fluorescence spectra of 3HC-DPA (10 μM) in titrations with Zn2+ (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200 μM). The arrow indicates the development of bands with increasing Zn2+ concentration. (b) Plot of the relative fluorescence intensity of 3HC-DPA at 466 nm with Zn2+ (F) and without Zn2+ (F0) against the concentration of Zn2+. Insert: A linear response of emission intensity ratio F/F0 versus Zn2+ concentration (R2 = 0.99).

800

metal-free +metal +zinc

a)

F466nm

600

400

200

0 +

+

+

2+

2+

2+

3+

2+

3+

2+

2+

2+

2+

K Li Na g n Hg Cr Cd Fe Fe Co Ni Cu M M

Fig. 2. (a) Metal ion selectivity of 3HC−DPA (10 μM): White bar, fluorescence intensity of the zinc-free form; Black bar, fluorescence intensity in the presence of metal ion (K+, Li+, Na+, Mg2+, Mn2+, Hg2+ ions, 100 equiv; Cr3+, Cd2+,10 equiv; Fe3+, Fe2+, Co2+, Ni2+, Cu2+, 1 equiv to 3HC−DPA); Red bar, fluorescence intensity after subsequent addition of zinc ion (1 equiv) to the mixture. λex = 382 nm. (b) X-ray crystal structure of zinc complex [Zn2(3HCDPA)2]2+ (CCDC: 1414677). ‡

17

DU145 cell viability RWPE-1 cell viability

Cell Viability (%)

100 80 60 40 20 0

0 10 0.5 5 20 30 40 Concentration of 3HC-DPA/M

Relative Mean F per Cell

10

e)

f)

8 6 4 2 0

0.5

2 4 6 Incubation time (h)

Control

Fig. 3. Fluorescence microscope images of HeLa cells (a), incubation with 20 μM 3HC-DPA (b), in the presence of 50 μM added Zn2+ and 20 μM 3HC-DPA (c), and incubation with 50 μM Zn2+, 20 μM 3HC-DPA, and 100 μM membrane-permeable zinc ion chelator TPEN (d). Scale bar = 20 μm. λex = 780 nm. (e) MTT assay of the cytotoxicity for 3HC-DPA (0, 0.5, 5, 10, 20, 30, 40 μM) in DU145 and RWPE-1. The results were representative of at least five independent experiments. (f) 3HC−DPA in RWPE-1 during different retention time. The concentration of Zn2+ added in advance was 100 μM, and incubation time of 3HC−DPA (20 μM) in RWPE-1 cells was 0.5 h. The mean cell retention times were 0.5, 2, 4, 6 h, respectively. The results were representative of at least three independent experiments.

18

Fig. 4. Fluorescence microscope images in human cancerous prostate cell line DU145 and human normal prostate cell line RWPE-1 cell line with 3HC−DPA, respectively. The concentration of 3HC-DPA was 20 μM, and the concentration of added Zn2+ was 50 μM. Scale bar = 50 μm. λex = 780 nm.

19

*

b)16000

a) Cu2+ of DU145 Cu2+ of RWPE-1

5

2 1 0

*

DU145 RWPE-1

* *

*

Mean F per cell

ppm/(mg protein)

Zn2+ of DU145 Zn2+ of RWPE-1

12000

* *

8000

*

*

4000 0 μM 1 μM 10 μM 50 μM Concentration of zinc ions

0

30 1 10 Concentration of Zn2+ (M)

50

Fig. 5. (a) Detection of Zn2+ and Cu2+ in human cancerous prostate cell line DU145 and human normal prostate cell line RWPE-1 with ICP-AES (*P<0.001, Student’s t-test). 0, 1, 10, and 50 μM Zn2+ was added in those cell lines, respectively. (b) Both DU145 and RWPE-1 cell lines were incubated with 80 μM Zn2+ for 4 h. After washing extracellular Zn2+, relative fluorescence intensity of per RWPE-1 or DU145 cell was measured by flow cytometry after incubation with 20 μM 3HC−DPA for another 1 h, respectively. The results were representative of at least three independent experiments (*P<0.001, Student’s t-test).

Scheme 1. Synthesis of the fluorescent Zn2+ sensor 3HC-DPA.

20