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Rhodamine based guanidinobenzimidazole functionalized fluorescent probe for tetravalent tin and its application in living cells imaging
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Rhodamine based guanidinobenzimidazole functionalized fluorescent probe for tetravalent tin and its application in living cells imaging Zheng Yang a,b , Mengyao She b , Siyue Ma b , Bing Yin b , Ping Liu b , Xiangrong Liu a , Shunsheng Zhao a , Jianli Li b,∗ a
School of Chemistry & Chemical Engineering, Xi’an University of Science and Technology, Xi’an, 710054, China Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, College of Chemistry & Materials Science, Northwest University, Xi’an, 710069, China b
a r t i c l e
i n f o
Article history: Received 22 March 2016 Received in revised form 26 September 2016 Accepted 28 September 2016 Available online xxx Keywords: Fluorescence Probe Tetravalent tin DFT calculation Bio-imaging
a b s t r a c t A novel rhodamine based 2-guanidinobenzimidazole functionalized probe RhoG was rationally designed and synthesized for the detection of tetravalent tin in environmental, geological, and biological samples with excellent selectivity, sensitivity, photostability and anti-interference performance. The mechanism investigation confirmed that guanidinobenzimidazole provided binding sites for Sn4+ and facilitate the formation of the complex. Successfully fluorescent imaging experiments in living L929 cells and MG-63 cells by confocal laser scanning microscopy presented a promising candidate for tracking tetravalent tin in biological organisms and processes thus contribute to significant breakthroughs in understanding the critically important functions of tetravalent tin. © 2016 Published by Elsevier B.V.
1. Introduction The design and development of fluorescent chemosensors for sensitive and selective detection of fluorescent cellular and subcellular imaging chemically, environmentally and biologically important heavy and transition-metal ions in combination with confocal microscopy has evolved into an emerging attractive area of particular interest in chemical biology because of the attractive properties including simplicity, high sensitivity, high spatial resolution, non-destructive and real time monitoring [1–5]. Among which, rhodamine derivatives appear to be the ideal model for the construction of the “off-on” type chemosensors, owing to their excellent spectroscopic properties, such as long wavelength excitation and emission profiles, great photostability, high extinction coefficient and excellent fluorescence quantum yields [6–8]. Recently, on the basis of spirolactam (colorless, non-fluorescent) to ring-open amide (pink, fluorescent) equilibrium of rhodamine, a large number of promising selective fluorescence sensors for the detection of biological relevant metal ions that are known to have harmful effects on the environment and living organisms including
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (J. Li).
Cu2+ [9–12], Cr3+ [13], Fe3+ [14–19], Hg2+ [20–23], Zn2+ [24,25] and other metal ions [26–29] have been preferentially concerned and fruitful developed at a high speed with worldwide emerging. Amongst a variety of the attractive metal ions, tetravalent tin, which was known as the widely used in chemical reagent, agriculture, chemical weapon, industrial activities. It is also an important trace mineral for human that stored in the adrenal glands, liver, brain, spleen and thyroid gland, is of indispensable importance that involved significant roles in our daily life, industry, environmental science, medicine as well as several crucial biochemical processes at the cellular level [30]. The importance of tetravalent tin is reflected by the fact that abnormal levels of tetravalent tin have been implicated in a series of human diseases, such as increase the risk factors associated with poor growth, hearing loss, and cancer prevention caused by the deficiency of tetravalent tin as well as eye and skin irritation, headaches and dizziness, gastrointestinal effects and stomachaches, breathlessness, liver damage, immune systems damage and even chromosomal damage caused by the excess accumulation of tetravalent tin [31–33]. However, we have little knowledge of the mechanisms of how tetravalent tin affects our metabolism and body health due to the lack of effective tools for the recognition and sensing of tetravalent tin in biological samples. In recent years, significant emphasis has been placed on the exploitation of facile, highly selective fluorescent probes for
http://dx.doi.org/10.1016/j.snb.2016.09.170 0925-4005/© 2016 Published by Elsevier B.V.
Please cite this article in press as: Z. Yang, et al., Rhodamine based guanidinobenzimidazole functionalized fluorescent probe for tetravalent tin and its application in living cells imaging, Sens. Actuators B: Chem. (2016), http://dx.doi.org/10.1016/j.snb.2016.09.170
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tetravalent tin and several progress has been made [34–39]. Wang et al. [40] reported a naphthalimide–rhodamine B derivative that could be used as a fluorescent probe for Sn4+ in biological systems. Mahapatra et al. [41] also presented two probes and investigated the recognition mechanisms through DFT computational studies as well as put them into the use of biological imaging of Sn4+ in living cells. However, the reported probes not only influenced by other metal ions such as Cu2+ and Cr3+ but also suffered the shortcomings of poor water solubility, fluorescence stability and fluorescent sustainability. As a result, selection of adequate probes with excellent fluorescent properties for tetravalent tin investigations in biological, geological, and environmental sciences remains to be of great significance. With the above mentioned factors in mind, we herein present a wonderful tetravalent tin probe RhoG (Scheme 1) in which rhodamine was used as the chromophore in considering the wonderful photophysical properties and 2-guanidinobenzimidazole was chosen as the recognition functional group for the certain consideration of the potential excellent bio-medical and optical properties that the amino group in this group can not only improve the solubility, selectivity, biocompatibility and cellular affinity but also can provide more binding sites along with significantly promotion of the fluorescent stability and sustainability. Strong fluorescence emission was observed upon addition of tetravalent tin due to the spirolacton ring-opening power of tetravalent tin caused by the metal-to-ligand chelation charge transfer, which was reasonably proved by combined experimental and computational investigation. The fluorescent imaging experiments in living cells demonstrated that the probe could be successfully applied for mapping tetravalent tin concentration and distribution in living biological samples, thus contributed to significant breakthroughs in understanding the correlation and functional mechanism between tetravalent tin and related cells and organic substances.
2. Material and methods 2.1. General methods Elemental analyses were measured on a Vario EL III analyzer. IR spectra were recorded on a Bruker Tensor 27 spectrometer. NMR spectra were performed on a Varian Inova-400 MHz spectrometer. Mass spectra were performed with Bruker micrOTOF-Q II ESI-QTOF LC/MS/MS Spectroscopy. X-ray crystal data were collected on the Bruker Smart APEX II CCD diffractometer. The absorbance spectra were performed on a Shimadzu UV-1700 spectrophotometer. Fluorescent spectra measurements measured were on a Hitachi F4500 fluorescence spectrophotometer. Bioimaging of the sensors were performed on an Olympus FV1000 confocal microscopn and the excitation wavelength was set as 543 nm. The cell viability was determined at 490 nm absorbance using an ELx800 Absorbance Reader (BioTek Instruments, Inc.).
2.1.1. Synthesis The chemicals and reagents were all obtained from SigmaAldrich Co. LLC. Analytical thin layer chromatography was performed using Merck 60 GF254 silica gel (precoated sheets,
0.25 mm thick). Silica gel (0.200–0.500 mm, 60A, J&K Scientific Ltd.) was used for column chromatography. 2.1.2. Synthesis of the probe RhoG In brief, to a stirred solution of rhodamine B hydrochloride (0.4780 g, 0.001 mol) and 2-dichloroethane (5 mL), 1 mL of phosphorus oxychloride was added. The solution was refluxed for 6 h and concentrated by evaporation. The obtained crude acid chloride was dissolved in acetonitrile (2 mL). Then, a solution of the 2-guanidinobenzimidazole (0.001 mol) and triethylamine (1 mL) in acetonitrile (50 mL) was added dropwise in 30 min. After refluxing for 4 h, the solvent was removed under reduced pressure to give a violet oil. Water was then added to the mixture, and the aqueous phase was extracted with dichloromethane (5 mL × 3). The organic layer was washed with water, dried over anhydrous MgSO4 , and filtered. Purification of the products was by column chromatography on silica gel (eluant: CH2 Cl2 :CH3 OH = 60:1, v:v) to get the probe as a light yellow crystal (0.4833 g, yield: 80.7%), Mp.: 272–273 ◦ C. Anal. calcd. for C36 H37 N7 O2 : H, 6.22; C, 72.10; N, 16.35. Found: H, 6.18; C, 72.30; N, 16.29. IR (KBr, /cm−1 ): 3433.2, 3377.3, 3064.8, 3016.6, 2966.4, 2927.9, 1689.6, 1633.7, 1614.4, 1517.9, 1452.4, 1319.3, 1269.1, 1220.9, 1151.5, 1118.7, 815.8, 785.0, 758.0. 1 H NMR (400 MHz, CDCl ) ı 9.46 (s, 1H), 8.94 (s, 1H), 8.14 (s, 1H), 3 7.99 (d, J = 7.4 Hz, 1H), 7.56 (dt, J = 23.2, 7.0 Hz, 2H), 7.41 (d, J = 6.2 Hz, 1H), 7.20 (dd, J = 18.8, 7.0 Hz, 2H), 7.11–6.98 (m, 2H), 6.40 (dd, J = 23.4, 5.5 Hz, 4H), 6.17 (dd, J = 8.8, 2.3 Hz, 2H), 3.30 (dd, J = 13.9, 6.9 Hz, 8H), 1.12 (t, J = 7.0 Hz, 12H). 13 C NMR (101 MHz, CDCl3 ) ı 164.69, 150.24, 148.57, 147.46, 145.11, 143.06, 137.05, 129.00, 125.75, 124.16, 122.97, 122.31, 119.25, 117.74, 115.39, 115.16, 111.57, 103.23, 102.66, 101.36, 91.33, 61.30, 38.69, 7.01. Calc. for [M + H]+ = 600.3089, found MS (ESI) m/z = 600.3187. Crystal data and structure refinement for RhoG: [C37 H39 Cl2 N7 O2 ]; Mr = 684.65; size: 0.25 × 0.24 × 0.23 mm3 ; block; yellow; Orthorhombic; T = 296(2) K; space group Pbca; a = 11.8726(17) Å, b = 23.890(3) Å, c = 25.006(3) Å; ␣ = 90.00◦ , ˇ = 90.00◦ , = 90.00◦ ; V = 7092.6(17) Å3 ; Z = 8; Dc = 1.282 Mg m−3 , = 0.226 mm−1 ; F(000) = 288; range for data collection = 2.36 to 23.18; reflections collected = 4750; independent reflections = 8085 (Rint = 0.0436); full-matrix least-squares refinement on F2 ; goodness-of-fit on F2 = 1.047; final R1 = 0.0636, wR2 = 0.1818; R indices (all data) R1 = 0.1091, wR2 = 0.2145; Largest diff. peak and hole = 0.529 and −0.549 eÅ−3 . 2.2. General procedure Stock solutions of the probe (500 mol L−1 ), metal ions (500 mol L−1 ), other organotin compounds (500 mol L−1 , including butyltin trichloride, dibutyltin dichloride, tributyl tin chloride and tetrabutyltin) were all prepared in EtOH-H2 O (5:5, v/v) solution. The solutions of metal ions were prepared with hydrochloride salts of Li+ , Na+ , K+ , Cu+ , Ba2+ , Ca2+ , Cd2+ , Mg2+ , Co2+ , Mn2+ , Zn2+ , Pb2+ , Ni2+ , Cu2+ , Sn2+ , Hg2+ , Fe2+ , Cr2+ , Fe3+ , Al3+ , Au3+ , Cr3+ and Sn4+ and the nitrate salt of Ag+ . Double distilled water was used throughout the experiment. To a 25 mL volumetric tube, 5.0 mL 0.2 mol L−1 PBS, 0.50 mL of 500 mol L−1 probe and different concentration of Sn4+ were added. The mixture was diluted to the mark with EtOH-H2 O (5:5, v/v) mixed solution. The Absorptions were recorded at 559 nm and the fluorescence intensities were recorded at 581 nm. The excita-
Scheme 1. The synthesis of the probe.
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tion and emission wavelength bandpasses were both set as 5.0 nm and the excitation wavelength was set at 540 nm. 2.3. Crystal growth and conditions White single crystal of the probe was obtained at room temperature from the mixed solvents of CH2 Cl2 –CH3 CN solution by slow evaporation and then mounted on the goniometer of single crystal diffractometer. The crystal data have been collected at 296 K by using Mo K␣ radiation by using ϕ/ω scan mode and collected for Lorentz and polarization effect (SADABS). The structure was solved using the direct method and refined by full-matrix least-squares fitting on F2 by SHELX-97. 2.4. Computation details The calculations of all the structures were performed with PBE1PBE function [42] via Gaussian 09 Program [43]. The probe was optimized with a combination of basis of double- quality consisting of 6-31G** for C, H elements, 6-31 + G* for N, O elements [44]. The possible complexes were optimized with a combination of basis of double- quality consisting of 3-21G** for C, H elements, 3-21 + G* for Cl, N, O, and SVP for Sn element [44]. The environmental effect was included via PCM model with ethanol as the solvent molecule. 2.5. Fluorescent imaging in living cells Fluorescent imaging in living mouse fibroblast cells L929 cells and Human Osteosarcoma MG-63 cells were performed in the similar procedures [45,46]. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS, at 37 ◦ C in the humidified atmosphere with 5% CO2 and 95% air. The cells were then cultured for 2 h until they plated on glass-bottomed dishes. The growth medium was then removed and the cells were washed with DMEM without FBS and incubated with 10 mol L−1 of the probe for 30 min at 37 ◦ C, washed three times with PBS and imaged. Then the cells were supplemented with 50 mol L−1 Sn4+ in the growth medium for 30 min at 37 ◦ C and imaged. 2.6. In vitro cell viability assay MCF-7 (1 × 106 to 5 × 106 cells) were seeded in 96-well plates and incubated in 150 L of medium (RPMI Medium Modified, HyClone with 10% gibco FBS (Art. No. 10270) and 1% hydrostreptomycin (P/S), HyClone) added into each plate at 37 ◦ C in the atmosphere of 5% CO2 to allow the cells to attach. After incubated for 24 h, the medium was sucked out and 100 L of fresh medium was added. A stock solution of 5 mM of the probe was prepared in DMSO (Sigma, Biological reagent level) and stored at −20 ◦ C. The stock solution was diluted to the appropriate concentrations with cultured medium. The final concentration of DMSO was 2% (v/v). The same amount of DMSO was used as the vehicle control throughout this study. The solution of the probe in the cultured medium were serial diluted with final concentration of 100, 50, 25, 12.5, 6.25, 3.125 mol/L and added to the microtiter plate and incubated for 24 h in the atmosphere of 5% CO2 at 37 ◦ C and then was sucked out the medium. MTT (3-(4,5-dimethy-1-thiazol-2-yl)-2,5diphenyltetrazolium bromide) with 1 × PBS (HEART) was filtered with water-based 0.22 m filter membrane to get the MTT solution in the concentration of 5 mg/mL. This solution was added to the previous medium in 10% proportion to form the MTT medium. 150 L of fresh MTT medium was then added into each plate. After placed for 4 h in the atmosphere of 5% CO2 at 37 ◦ C, the MTT medium was sucked out and 150 L of DMSO was added and placed on the shaking table for 10 min and then determined at 490 nm absorbance
Fig. 1. Molecular structure of the probe.
using an ELx800 Absorbance Reader. Experiments were performed in triplicate. 3. Results and discussion The probe was readily synthesized according to the reported synthetic procedure using rhodamine B acyl chloride with 2guanidinobenzimidazole as reactants and characterized by X-ray crystallography, elemental analysis, FTIR, 1 H NMR, 13 C NMR spectra and HRMS. The X-ray crystallography structural investigation of the probe (Fig. 1) clearly revealed the unique spirolactam ringclosed formation with the C4 N spiro ring (Marked with red circle in Fig. 1) in a solid state which was also supported by a distinctive spirocycle carbon shift at 61.3 ppm in 13 C NMR, thus agreed well with the colorless and non-fluorescence status of the probe in the metal-free condition. Fig. 1. The solution investigation was firstly performed in EtOH-H2 O MeOH-H2 O, (CH3 )2 CHOH-H2 O, (CH3 )2 CO-H2 O, THFH2 O, CH3 CN-H2 O, DMSO-H2 O, and DMF-H2 O mixed solutions (Fig. 2). The result suggested that other solvent systems seemed all to be inferior to EtOH-H2 O since the fluorescence intensities and absorptions of the probe in these solvents were much lower than that in EtOH-H2 O mixed solution upon addition of Sn4+ except for MeOH-H2 O, which was quite close to EtOH-H2 O mixed solution. As methanol are more harmful to the biological samples than ethanol in cell imaging experiments, EtOH-H2 O mixed solution was chosen. We then evaluated the fluorescence intensity and absorption of the probe with Sn4+ in the solutions with the containing water varies from 0 to 100% (Figs. S4 and S5). It is clear that the pure solution of water caused negligible signal changing. The fluorescence intensity and absorption increased with the increasing of ethanol in the mixed solvent. As the probe was designed for detection of tetravalent tin in environmental and biological samples, the containing of organic solvent should be as little as possible, thus EtOH-H2 O (5:5, v/v) was chosen for photo-property studies and EtOH-H2 O (2:8, v/v) was chosen for bio-imaging. Originally, the hydrolytic process of Sn4+ can be happen in aqueous media and results in the decrease of pH and induces the protonation-induced ring-opening process. Therefore, the pH responses of the probes in EtOH-H2 O (5:5, v/v) were then evaluated using solutions with pH values from 1.7 to 13.0 (Figs. S6 and S7). As expected, the fluorescence intensity and absorption increased when the pH value is lower than 5, which was corresponding to the protonation-induced ring-opening process. Delightfully, both the fluorescence intensity and the absorption exhibited unconspicuous changes in the pH range of 5.0–8.0, suggesting that the probes were insensitive to pH at neutral range and could work in
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Fig. 2. Fluorescence intensity (a) and absorption (b) changes of the probe (10 M) in the presence of 5.0 equiv. of Sn4+ in different solutions with PBS buffer (0.2 M, pH 7.4), ex = 540 nm.
approximate physiological condition with negligible background fluorescence. Thus, further investigations for the ultraviolet and fluorescent properties of the probes were carried out in the nearly neutral EtOH-H2 O (5:5, v/v, PBS, pH 7.4) mixed system to insure that the signal changings were caused by the coordination of Sn4+ to the probe instead of the decrease of pH induced by the hydrolytic process of Sn4+ . When related heavy, transition and main group metal ions including Li+ , Na+ , K+ , Ag+ , Cu+ , Ca2+ , Ba2+ , Cd2+ , Mg2+ , Co2+ , Mn2+ , Zn2+ , Pb2+ , Ni2+ , Cu2+ , Sn2+ , Hg2+ , Fe2+ , Cr2+ , Fe3+ , Al3+ , Cr3+ , Au3+ and Sn4+ were used to evaluate the selective property of probes in EtOHH2 O (5/5, v/v, PBS, pH 7.4) solution (Fig. 3). As expect, only the introduction of Sn4+ give rise to remarkable absorbance and fluorescence enhancement while Sn2+ did not cause any absorbance and fluorescence changes. Although Pb2+ , Fe3+ , and Cr3+ also had slight responses, they were significantly inferior and easily quenched compared with Sn4+ induced spectroscopic changes (Figs. S10 and S11). The results obviously implied the significant selectivity of the probe to Sn4+ . Time dependent study showed that the coordination of the probe with Sn4+ completed rapidly within 10 s (Figs. S16 and S17) and showed prolonged stability since the fluorescence intensity showed unconspicuous decrease for several hours, indicating a significant resistance to irreversible photo bleaching. In addition, other organic tetravalent tin compounds including butyltin trichloride, dibutyltin dichloride, tributyl tin chloride and tetrabutyltin
could also gave rise to distinct spectroscopic changes in a short time and exhibited long time stability (Figs. S12 and S13). The competition experiments were then performed by adding the competing ion to the solutions which contained the probe and Sn4+ . The results indicated that the selectivity of the probe did not significantly experience interference from the commonly coexistent ions, except for the slight increasing and quenching caused by Fe3+ , Cr3+ and Cu2+ , respectively. The spectroscopic titrations were then performed to demonstrate the efficiency of quantitative signaling behavior of the probe (Fig. 4). Both the absorption band centered at 559 nm and the fluorescence intensity around 581 nm increased accompanied by a brilliant pink coloration and a strong orange fluorescent emission with the increasing concentration of Sn4+ in high fluorescence quantum yield when the concentration varied in the range of 0 mol L−1 –50 mol L−1 (Fig. 4, inset). The fluorescence intensity and absorption both saturated with 5.0 equiv. of Sn4+ and higher concentration of Sn4+ would cause signal quenching. Good linear relationships were observed between the relative absorbance as well as fluorescence intensities and the concentration of Sn4+ in the 0.06 mol L−1 –50 mol L−1 range with a detection limit of 0.03 mol L−1 (Figs. S8 and S9) in EtOH-H2 O (5/5, v/v, PBS, pH 7.4) mixed solution when 10 mol L−1 of probe was used, indicating the highly suitability for quantitative determination of Sn4+ through this probe.
Fig. 3. Histogram showing of the fluorescence intensity and absorption for the selectivity and competition investigation of the probe RhoG (10 mol L−1 ) in EtOH-H2 O (5:5, v/v, PBS, pH 7.4) mixed solution, ex = 540 nm. The pillars in the front row represent fluorescence response of the probe to the metal ions of interest. The pillars in the back row represent the subsequent addition 5.0 equiv. Sn4+ to the solution containing RhoG and the metal ions, respectively.
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Fig. 4. Fluorescence intensity (a) and absorption (b) changes of the probe (10 mol L−1 ) upon addition of Sn4+ (0 − 5.0 equiv.) in EtOH-H2 O (5/5, v/v, PBS, pH 7.4) mixed solution. Inset: Changes of emission intensity at 581 nm (a) and absorption at 559 nm (b), ex = 540 nm.
In order to gain an insight of the ring-opening mechanism, intensive investigation has been performed. The stoichiometric ratio investigation through job’s plot established the formation of the 1:1 stoichiometry between the probe and Sn4+ with high association constant of 2.99 × 106 M−1 (Figs. S14 and S15). The research of addition ethylenediamine to the solution of the complex indicated the reversible nature of the binding process since the fluorescence intensity gradually decreased with the increasing concentration of ethylenediamine and completed quenched when excess ethylenediamine was added, coupled with the changing of pink color back to colorless (Figs. S18 and S19). On the hand of structural investigation, the mass spectrum manifested the peak at m/z 790.5088 (Fig. S24), which was assigned as [RhoG + Sn4+ + 2Cl− + H]+ , providing a powerful evidence for the structure of the binding complex. This result also coincided perfectly with the job’s plot investigation which confirmed the 1:1 binding stoichiometry. For better understand, DFT-NBO analysis focusing on the electron lone pairs (LP) was performed for the probe [47,48]. The LP of the N atoms on the guanidine group and benzimidazole moiety were computed to be the most possible ones to coordinate with Sn4+ . Thus, several possible structures of the complex were reasonably proposed and theoretically optimized at DFT level with PBE1PBE functional (Fig. S25). The optimized geometries of the probe and the complex were shown in Fig. 5, of which the local minimum character was confirmed by the nonexistence of imaginary frequency. The most probable structure which was proposed and validated to have the lowest energy compared to others clearly indicated that Sn4+ coordinate with the NH moiety of guanidine
Scheme 2. Proposed mechanism of the probe upon addition of Sn4+ .
group and the N atom of benzimidazole functional group inducing the metal-to-ligand chelation charge transfer ring-opening process inducing the color and fluorescent changes (Scheme 2). With the formation of the complex, the bond length of N1C1 and C4-C7 decreased from 1.387 Å to 1.359 Å and 1.516 Å (S1) to 1.409 Å, respectively, indicating the transformation of the phenol structure to the quinoid structure (Fig. 6). The investigation of spatial distributions and orbital energies of HOMO and LUMO (Fig. 6) demonstrated that the HOMO distribution of the probe was located essentially over the xnathene moiety, while the LUMO was mainly distributed over the volution neighboring benzene ring. In contrast, both HOMO and LUMO of the complex were mostly positioned at the rhodamine framework. In addition, although the HOMO-LUMO energy gap of complex decreased by 0.74 eV relative to that of probe, the large energy gap of 3.10 eV between HOMO and LUMO in the complex still provide powerful evidence for the strong interaction and excellent stability of the complex, which was well consistent with the designing demand.
Fig. 5. PBE1PBE optimized structure of the probe (a) and the complex (b). Carbon atoms are yellow, oxygen atoms are red, nitrogen atoms are purple, hydrogen atoms are blue, chlorine atoms are gray, and tetravalent tin atom is dark yellow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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fluorescence inside the cells could be monitored. However, after three times washing with PBS buffer and supplemented with 5.0 equiv. of Sn4+ in the same growth medium for another 30 min at 37 ◦ C, prominent fluorescent increases were observed in the intracellular area. Moreover, bright-field transmission images of cells suggested that the cell morphology remained in good condition throughout the imaging experiments and established the wonderful cell-membrane permeability of the probe. In addition, the overlay of fluorescent and bright-field images confirmed that the fluorescent signals were localized in the perinuclear area of the cytosol, demonstrating the subcellular distribution of Sn4+ . The preliminary experiments in living L929 cells provided an suitable candidate for in vivo tracking and imaging of Sn4+ . Similarity, as another proof-of-concept, the biological imaging of Sn4+ in Human Osteosarcoma MG-63 cells (Fig. 7) was also successfully performed with the same procedure, thus contributed to significant breakthroughs in understanding the importance and functional mechanism of tetravalent tin in environmental samples as well as related cells and organs.
4. Conclusion Fig. 6. HOMO and LUMO distributions of the probe and the complex. Carbon atoms are yellow, oxygen atoms are red, nitrogen atoms are purple, hydrogen atoms are blue, chlorine atoms are gray, and tetravalent tin atom is dark yellow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
The MTT assay that uses mitochondrial metabolic enzyme activity as an indicator of cell viability was conducted following the protocol described previously [49–51] to identify the toxicity of the probe. The MCF-7 cell was treated with different concentrations of RhoG. The fifty percent (IC50 ) value was calculated as the ratio of the mean of OD obtained for the treated cell to that of the vehicle control. As shown in Table S2, the IC50 value was more than 100, indicating the strong cell viability and low toxicity of RhoG. To further evaluate the practical bio-imaging applications of the probe for quantifying Sn4+ in biological systems, cultured Mouse fibroblast cells L929 were incubated with the probes in culture medium for 30 min at 37 ◦ C (Fig. 7). Fluorescence laser scanning confocal microscopy images demonstrated that no intracellular
In conclusion, the design and synthesis of the novel 2guanidinobenzimidazole functionalized rhodamine based fluorescent probe RhoG for specific detection of tetravalent tin with special selectivity and sensitivity were presented. The recognition behavior was investigated both experimentally and computationally. The results suggested that the Sn4+ induced metal-to-ligand chelation charge transfer in complexation with the NH moiety of guanidine group and the N atom of benzimidazole functional group reasonably promoted the ring-opening process, indicating that the introduction of guanidinobenzimidazole functional group provided binding sites for Sn4+ and facilitate the formation of the complex along with improving the spectral properties and biocompatibility of the probe. Preliminary MTT assay and bio-imaging experiments in living L929 cells and MG-63 cells confirmed the membrane permeability of the probe and the appropriateness of using the probe as a promising candidate for intracellular identifying of Sn4+ , which was supposed to provide a better understanding of the deleterious effects and biological functions of Sn4+ in related environmental, geological, and biological samples.
Fig. 7. Fluorescent images of Mouse fibroblast cells L929 and human Osteosarcoma MG-63 cells incubated with 10 mol L−1 RhoG for 30 min at 37 ◦ C (a) and then further incubated with 50 mol L−1 SnCl4 for 30 min at 37 ◦ C (b). (c). Bright-field transmission image of cell treated with the probe (10 mol L−1 ) and SnCl4 (50 mol L−1 ). (d). The overlay images of (b) and (c).
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Acknowledgements We are grateful for the support from the National Natural Science Foundation of China (NSFC 21572177; 21301139; 21272184; 21103137; 21103135; 21073139 and J1210057), the Shaanxi Provincial Natural Science Fund Project (No. 2015JZ003), the Shaanxi Provincial Higher Education Teaching Reform Project (No. 15BY47), the Xi’an City Science and Technology Project (Nos. CXY1511(3)), Scientific Research Cultivating Fund of Xi’an University of Science and Technology (No. 201619), the Xi’an University of Science and Technology Teaching Reform Project (No. JG14052), the Chinese National Innovation Experiment Program for University Students (No. 201510697004) and the Northwest University Science Foundation for Postgraduate Students (No. YZZ14052). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2016.09.170. References [1] M.H. Lee, J.S. Kim, J.L. Sessler, Small molecule- based ratiometric fluorescence probes for cations, anions, and biomolecules, Chem. Soc. Rev. 44 (2015) 4185–4191. [2] H. Qian, Z.C. Xu, Fluorescence imaging of metal ions implicated in diseases, Chem. Soc. Rev. 44 (2015) 4487–4493. [3] Y.H. Tang, D. Lee, J.L. Wang, G.H. Li, J.H. Yu, W.Y. Lin, J. Yoon, Development of fluorescent probes based on protection- deprotection of the key functional groups for biological imaging, Chem. Soc. Rev. 44 (2015) 5003–5015. [4] H. Li, X.H. Gao, W. Shi, H.M. Ma, Design strategies for water-soluble small molecular chromogenic and fluorogenic probes, Chem. Rev. 114 (2014) 590–659. [5] Y.Y. Huang, M.J. Wang, M.Y. She, Z. Yang, P. Liu, J.L. Li, Z. Shi, Recent progress in the fluorescent probe based on spiro ring opening of xanthenes and related derivatives, Chin. J. Org. Chem. 34 (2014) 1–25. [6] Q. Chen, T. Pradhan, F. Wang, J.S. Kim, J. Yoon, Fluorescent chemosensors based on spiroring-opening of xanthenes and related derivatives, Chem. Rev. 112 (2012) 1910–1956. [7] R.W. Ramette, E.B. Sandell, Rhodamine B equilibriums, J. Am. Chem. Soc. 78 (1956) 4872–4878. [8] M. Beija, C.A. Afonso, J.M. Martinho, Synthesis and applications of rhodamine derivatives as fluorescent probes, Chem. Soc. Rev. 38 (2009) 2410–2433. [9] Y. Wang, H.Q. Chang, W.N. Wu, W.B. Peng, Y.F. Yan, C.M. He, T.T. Chen, X.L. Zhao, Z.Q. Xu, Rhodamine 6G hydrazone bearing pyrrole unit: ratiometric and selective fluorescent sensor for Cu2+ based on two different approaches, Sens. Actuators B: Chem. 228 (2016) 395–400. [10] J.L. Fan, P. Zhan, M.M. Hu, W. Sun, J.Z. Tang, J.Y. Wang, S.G. Sun, F.L. Song, X.J. Peng, A fluorescent ratiometric chemodosimeter for Cu2+ based on TBET and its application in living cells, Org. Lett. 15 (2013) 492–495. [11] L. Yuan, W.Y. Lin, B. Chen, Y.N. Xie, Development of FRET- based ratiometric fluorescent Cu2+ chemodosimeters and the applications for living cell imaging, Org. Lett. 14 (2012) 432–435. [12] J. Tang, S.G. Ma, D. Zhang, Y.Q. Liu, Y.F. Zhao, Y. Ye, Highly sensitive and fast responsive ratiometric fluorescent probe for Cu2+ based on a naphthalimiderhodamine dyad and its application in living cell imaging, Sens. Actuators B: Chem. 236 (2016) 109–115. [13] K. Gupta, N. Mergu, A.K. Singh, Rhodamine- derived highly sensitive and selective colorimetric and off- on optical chemosensors for Cr3+ , Sens. Actuators B: Chem. 220 (2015) 420–432. [14] S. Li, D. Zhang, X.Y. Xie, S.G. Ma, Y. Liu, Z.H. Xu, Y.F. Gao, Y. Ye, A novel solvent- dependently bifunctional NIR absorptive and fluorescent ratiometric probe for detecting Fe3+ /Cu2+ and its application in bioimaging, Sens Actuators B: Chem 224 (2016) 661–667. [15] S.Y. Ma, Z. Yang, M.Y. She, W. Sun, B. Yin, P. Liu, S.Y. Zhang, J.L. Li, Design and synthesis of functionalized rhodamine based probes for specific intracellular fluorescence imaging of Fe3+ , Dyes Pigm. 115 (2015) 120–126. [16] Z. Yang, M.Y. She, B. Yin, J.H. Cui, Y.Z. Zhang, W. Sun, J.L. Li, Z. Shi, Three rhodamine- based off- on chemosensors with high selectivity and sensitivity for Fe3+ imaging in living cells, J. Org. Chem. 77 (2012) 1143–1147. [17] M.Y. She, Z. Yang, B. Yin, J. Zhang, J. Gu, W.T. Yin, J.L. Li, G.F. Zhao, Z. Shi, A novel rhodamine- based fluorescent and colorimetric off- on chemosensor and investigation of the recognizing behavior towards Fe3+ , Dyes Pigm. 92 (2012) 1337–1343. [18] W.T. Yin, H. Cui, Z. Yang, C. Li, M.Y. She, B. Yin, J.L. Li, G.F. Zhao, Z. Shi, Facile synthesis and characterization of rhodamine- based colorimetric and off- on fluorescent chemosensor for Fe3+ , Sens. Actuators B: Chem. 157 (2011) 675–680.
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Northwest University. His research interests also focus on developing fluorescent functional chemosensors. Siyue Ma received her BS degree from Heilongjiang University, PR China, in 2014. She is currently a Ph.D candidate at College of Chemistry and Materials Science, Northwest University. Her research interests also focus on developing fluorescent functional chemosensors. Dr. Bing Yin was born in 1981, and is now working in Northwest University, PR China. He received his Ph.D. in physical chemistry from Beijing Normal University in 2009. His current research interests include theoretical investigation of molecular magnetic and theoretically model of functional organic molecules. Peof. Ping Liu was born in 1974, and is a professor at Northwest University, PR China. She received her Ph.D. in inorganic chemistry from Northwest University in 2005. Her current research interests include synthesis and study of new ligand and their functional complexes. Prof. Xiangrong Liu was born in 1967, and is a professor at Xi’an University of Science and Technology, PR China. She received his Ph.D. in Physical chemistry of materials from Northwestern Polytechnical University in 2004. Her current research interests include synthesis and research methods for functional molecular, the thermal kinetics of microbial interactions, and bioconversion of coal.
Biographies
Dr. Shunsheng Zhao was born in 1981, and is now working in Xi’an University of Science and Technology, PR China. He received his Ph.D. in inorganic chemistry from Wuhan University in 2010. His current research interests include biological imaging of water-soluble luminescence of rare earth complexes, transition metal complexes and their catalysis in the polymerization reaction and the thermodynamics and thermal kinetics of complexes.
Dr. Zheng Yang was born in 1988, and is now working in Xi’an University of Science and Technology, PR China. He received his Ph.D. in organic chemistry from Northwest University in 2015. His research interests focus on developing fluorescent functional chemosensors.
Prof. Jianli Li was born in 1973, and is a professor at Northwest University, PR China. He received his Ph.D. in organic chemistry from Northwest University in 2007. His current research interests include synthesis and research methods for organic substance, fluorescent molecular devices, chemical and biological sensors and molecular recognition.
MengyaoShe received his BS degree from Northwest University, PR China, in 2012. He is currently a Ph.D candidate at College of Chemistry and Materials Science,
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Rhodamine based guanidinobenzimidazole functionalized fluorescent probe for tetravalent tin and its application in living cells imaging Zheng Yang a,b , Mengyao She b , Siyue Ma b , Bing Yin b , Ping Liu b , Xiangrong Liu a , Shunsheng Zhao a , Jianli Li b,∗ a
School of Chemistry & Chemical Engineering, Xi’an University of Science and Technology, Xi’an, 710054, China Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, College of Chemistry & Materials Science, Northwest University, Xi’an, 710069, China b
a r t i c l e
i n f o
Article history: Received 22 March 2016 Received in revised form 26 September 2016 Accepted 28 September 2016 Available online xxx Keywords: Fluorescence Probe Tetravalent tin DFT calculation Bio-imaging
a b s t r a c t A novel rhodamine based 2-guanidinobenzimidazole functionalized probe RhoG was rationally designed and synthesized for the detection of tetravalent tin in environmental, geological, and biological samples with excellent selectivity, sensitivity, photostability and anti-interference performance. The mechanism investigation confirmed that guanidinobenzimidazole provided binding sites for Sn4+ and facilitate the formation of the complex. Successfully fluorescent imaging experiments in living L929 cells and MG-63 cells by confocal laser scanning microscopy presented a promising candidate for tracking tetravalent tin in biological organisms and processes thus contribute to significant breakthroughs in understanding the critically important functions of tetravalent tin. © 2016 Published by Elsevier B.V.
1. Introduction The design and development of fluorescent chemosensors for sensitive and selective detection of fluorescent cellular and subcellular imaging chemically, environmentally and biologically important heavy and transition-metal ions in combination with confocal microscopy has evolved into an emerging attractive area of particular interest in chemical biology because of the attractive properties including simplicity, high sensitivity, high spatial resolution, non-destructive and real time monitoring [1–5]. Among which, rhodamine derivatives appear to be the ideal model for the construction of the “off-on” type chemosensors, owing to their excellent spectroscopic properties, such as long wavelength excitation and emission profiles, great photostability, high extinction coefficient and excellent fluorescence quantum yields [6–8]. Recently, on the basis of spirolactam (colorless, non-fluorescent) to ring-open amide (pink, fluorescent) equilibrium of rhodamine, a large number of promising selective fluorescence sensors for the detection of biological relevant metal ions that are known to have harmful effects on the environment and living organisms including
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (J. Li).
Cu2+ [9–12], Cr3+ [13], Fe3+ [14–19], Hg2+ [20–23], Zn2+ [24,25] and other metal ions [26–29] have been preferentially concerned and fruitful developed at a high speed with worldwide emerging. Amongst a variety of the attractive metal ions, tetravalent tin, which was known as the widely used in chemical reagent, agriculture, chemical weapon, industrial activities. It is also an important trace mineral for human that stored in the adrenal glands, liver, brain, spleen and thyroid gland, is of indispensable importance that involved significant roles in our daily life, industry, environmental science, medicine as well as several crucial biochemical processes at the cellular level [30]. The importance of tetravalent tin is reflected by the fact that abnormal levels of tetravalent tin have been implicated in a series of human diseases, such as increase the risk factors associated with poor growth, hearing loss, and cancer prevention caused by the deficiency of tetravalent tin as well as eye and skin irritation, headaches and dizziness, gastrointestinal effects and stomachaches, breathlessness, liver damage, immune systems damage and even chromosomal damage caused by the excess accumulation of tetravalent tin [31–33]. However, we have little knowledge of the mechanisms of how tetravalent tin affects our metabolism and body health due to the lack of effective tools for the recognition and sensing of tetravalent tin in biological samples. In recent years, significant emphasis has been placed on the exploitation of facile, highly selective fluorescent probes for
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tetravalent tin and several progress has been made [34–39]. Wang et al. [40] reported a naphthalimide–rhodamine B derivative that could be used as a fluorescent probe for Sn4+ in biological systems. Mahapatra et al. [41] also presented two probes and investigated the recognition mechanisms through DFT computational studies as well as put them into the use of biological imaging of Sn4+ in living cells. However, the reported probes not only influenced by other metal ions such as Cu2+ and Cr3+ but also suffered the shortcomings of poor water solubility, fluorescence stability and fluorescent sustainability. As a result, selection of adequate probes with excellent fluorescent properties for tetravalent tin investigations in biological, geological, and environmental sciences remains to be of great significance. With the above mentioned factors in mind, we herein present a wonderful tetravalent tin probe RhoG (Scheme 1) in which rhodamine was used as the chromophore in considering the wonderful photophysical properties and 2-guanidinobenzimidazole was chosen as the recognition functional group for the certain consideration of the potential excellent bio-medical and optical properties that the amino group in this group can not only improve the solubility, selectivity, biocompatibility and cellular affinity but also can provide more binding sites along with significantly promotion of the fluorescent stability and sustainability. Strong fluorescence emission was observed upon addition of tetravalent tin due to the spirolacton ring-opening power of tetravalent tin caused by the metal-to-ligand chelation charge transfer, which was reasonably proved by combined experimental and computational investigation. The fluorescent imaging experiments in living cells demonstrated that the probe could be successfully applied for mapping tetravalent tin concentration and distribution in living biological samples, thus contributed to significant breakthroughs in understanding the correlation and functional mechanism between tetravalent tin and related cells and organic substances.
2. Material and methods 2.1. General methods Elemental analyses were measured on a Vario EL III analyzer. IR spectra were recorded on a Bruker Tensor 27 spectrometer. NMR spectra were performed on a Varian Inova-400 MHz spectrometer. Mass spectra were performed with Bruker micrOTOF-Q II ESI-QTOF LC/MS/MS Spectroscopy. X-ray crystal data were collected on the Bruker Smart APEX II CCD diffractometer. The absorbance spectra were performed on a Shimadzu UV-1700 spectrophotometer. Fluorescent spectra measurements measured were on a Hitachi F4500 fluorescence spectrophotometer. Bioimaging of the sensors were performed on an Olympus FV1000 confocal microscopn and the excitation wavelength was set as 543 nm. The cell viability was determined at 490 nm absorbance using an ELx800 Absorbance Reader (BioTek Instruments, Inc.).
2.1.1. Synthesis The chemicals and reagents were all obtained from SigmaAldrich Co. LLC. Analytical thin layer chromatography was performed using Merck 60 GF254 silica gel (precoated sheets,
0.25 mm thick). Silica gel (0.200–0.500 mm, 60A, J&K Scientific Ltd.) was used for column chromatography. 2.1.2. Synthesis of the probe RhoG In brief, to a stirred solution of rhodamine B hydrochloride (0.4780 g, 0.001 mol) and 2-dichloroethane (5 mL), 1 mL of phosphorus oxychloride was added. The solution was refluxed for 6 h and concentrated by evaporation. The obtained crude acid chloride was dissolved in acetonitrile (2 mL). Then, a solution of the 2-guanidinobenzimidazole (0.001 mol) and triethylamine (1 mL) in acetonitrile (50 mL) was added dropwise in 30 min. After refluxing for 4 h, the solvent was removed under reduced pressure to give a violet oil. Water was then added to the mixture, and the aqueous phase was extracted with dichloromethane (5 mL × 3). The organic layer was washed with water, dried over anhydrous MgSO4 , and filtered. Purification of the products was by column chromatography on silica gel (eluant: CH2 Cl2 :CH3 OH = 60:1, v:v) to get the probe as a light yellow crystal (0.4833 g, yield: 80.7%), Mp.: 272–273 ◦ C. Anal. calcd. for C36 H37 N7 O2 : H, 6.22; C, 72.10; N, 16.35. Found: H, 6.18; C, 72.30; N, 16.29. IR (KBr, /cm−1 ): 3433.2, 3377.3, 3064.8, 3016.6, 2966.4, 2927.9, 1689.6, 1633.7, 1614.4, 1517.9, 1452.4, 1319.3, 1269.1, 1220.9, 1151.5, 1118.7, 815.8, 785.0, 758.0. 1 H NMR (400 MHz, CDCl ) ı 9.46 (s, 1H), 8.94 (s, 1H), 8.14 (s, 1H), 3 7.99 (d, J = 7.4 Hz, 1H), 7.56 (dt, J = 23.2, 7.0 Hz, 2H), 7.41 (d, J = 6.2 Hz, 1H), 7.20 (dd, J = 18.8, 7.0 Hz, 2H), 7.11–6.98 (m, 2H), 6.40 (dd, J = 23.4, 5.5 Hz, 4H), 6.17 (dd, J = 8.8, 2.3 Hz, 2H), 3.30 (dd, J = 13.9, 6.9 Hz, 8H), 1.12 (t, J = 7.0 Hz, 12H). 13 C NMR (101 MHz, CDCl3 ) ı 164.69, 150.24, 148.57, 147.46, 145.11, 143.06, 137.05, 129.00, 125.75, 124.16, 122.97, 122.31, 119.25, 117.74, 115.39, 115.16, 111.57, 103.23, 102.66, 101.36, 91.33, 61.30, 38.69, 7.01. Calc. for [M + H]+ = 600.3089, found MS (ESI) m/z = 600.3187. Crystal data and structure refinement for RhoG: [C37 H39 Cl2 N7 O2 ]; Mr = 684.65; size: 0.25 × 0.24 × 0.23 mm3 ; block; yellow; Orthorhombic; T = 296(2) K; space group Pbca; a = 11.8726(17) Å, b = 23.890(3) Å, c = 25.006(3) Å; ␣ = 90.00◦ , ˇ = 90.00◦ , = 90.00◦ ; V = 7092.6(17) Å3 ; Z = 8; Dc = 1.282 Mg m−3 , = 0.226 mm−1 ; F(000) = 288; range for data collection = 2.36 to 23.18; reflections collected = 4750; independent reflections = 8085 (Rint = 0.0436); full-matrix least-squares refinement on F2 ; goodness-of-fit on F2 = 1.047; final R1 = 0.0636, wR2 = 0.1818; R indices (all data) R1 = 0.1091, wR2 = 0.2145; Largest diff. peak and hole = 0.529 and −0.549 eÅ−3 . 2.2. General procedure Stock solutions of the probe (500 mol L−1 ), metal ions (500 mol L−1 ), other organotin compounds (500 mol L−1 , including butyltin trichloride, dibutyltin dichloride, tributyl tin chloride and tetrabutyltin) were all prepared in EtOH-H2 O (5:5, v/v) solution. The solutions of metal ions were prepared with hydrochloride salts of Li+ , Na+ , K+ , Cu+ , Ba2+ , Ca2+ , Cd2+ , Mg2+ , Co2+ , Mn2+ , Zn2+ , Pb2+ , Ni2+ , Cu2+ , Sn2+ , Hg2+ , Fe2+ , Cr2+ , Fe3+ , Al3+ , Au3+ , Cr3+ and Sn4+ and the nitrate salt of Ag+ . Double distilled water was used throughout the experiment. To a 25 mL volumetric tube, 5.0 mL 0.2 mol L−1 PBS, 0.50 mL of 500 mol L−1 probe and different concentration of Sn4+ were added. The mixture was diluted to the mark with EtOH-H2 O (5:5, v/v) mixed solution. The Absorptions were recorded at 559 nm and the fluorescence intensities were recorded at 581 nm. The excita-
Scheme 1. The synthesis of the probe.
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tion and emission wavelength bandpasses were both set as 5.0 nm and the excitation wavelength was set at 540 nm. 2.3. Crystal growth and conditions White single crystal of the probe was obtained at room temperature from the mixed solvents of CH2 Cl2 –CH3 CN solution by slow evaporation and then mounted on the goniometer of single crystal diffractometer. The crystal data have been collected at 296 K by using Mo K␣ radiation by using ϕ/ω scan mode and collected for Lorentz and polarization effect (SADABS). The structure was solved using the direct method and refined by full-matrix least-squares fitting on F2 by SHELX-97. 2.4. Computation details The calculations of all the structures were performed with PBE1PBE function [42] via Gaussian 09 Program [43]. The probe was optimized with a combination of basis of double- quality consisting of 6-31G** for C, H elements, 6-31 + G* for N, O elements [44]. The possible complexes were optimized with a combination of basis of double- quality consisting of 3-21G** for C, H elements, 3-21 + G* for Cl, N, O, and SVP for Sn element [44]. The environmental effect was included via PCM model with ethanol as the solvent molecule. 2.5. Fluorescent imaging in living cells Fluorescent imaging in living mouse fibroblast cells L929 cells and Human Osteosarcoma MG-63 cells were performed in the similar procedures [45,46]. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS, at 37 ◦ C in the humidified atmosphere with 5% CO2 and 95% air. The cells were then cultured for 2 h until they plated on glass-bottomed dishes. The growth medium was then removed and the cells were washed with DMEM without FBS and incubated with 10 mol L−1 of the probe for 30 min at 37 ◦ C, washed three times with PBS and imaged. Then the cells were supplemented with 50 mol L−1 Sn4+ in the growth medium for 30 min at 37 ◦ C and imaged. 2.6. In vitro cell viability assay MCF-7 (1 × 106 to 5 × 106 cells) were seeded in 96-well plates and incubated in 150 L of medium (RPMI Medium Modified, HyClone with 10% gibco FBS (Art. No. 10270) and 1% hydrostreptomycin (P/S), HyClone) added into each plate at 37 ◦ C in the atmosphere of 5% CO2 to allow the cells to attach. After incubated for 24 h, the medium was sucked out and 100 L of fresh medium was added. A stock solution of 5 mM of the probe was prepared in DMSO (Sigma, Biological reagent level) and stored at −20 ◦ C. The stock solution was diluted to the appropriate concentrations with cultured medium. The final concentration of DMSO was 2% (v/v). The same amount of DMSO was used as the vehicle control throughout this study. The solution of the probe in the cultured medium were serial diluted with final concentration of 100, 50, 25, 12.5, 6.25, 3.125 mol/L and added to the microtiter plate and incubated for 24 h in the atmosphere of 5% CO2 at 37 ◦ C and then was sucked out the medium. MTT (3-(4,5-dimethy-1-thiazol-2-yl)-2,5diphenyltetrazolium bromide) with 1 × PBS (HEART) was filtered with water-based 0.22 m filter membrane to get the MTT solution in the concentration of 5 mg/mL. This solution was added to the previous medium in 10% proportion to form the MTT medium. 150 L of fresh MTT medium was then added into each plate. After placed for 4 h in the atmosphere of 5% CO2 at 37 ◦ C, the MTT medium was sucked out and 150 L of DMSO was added and placed on the shaking table for 10 min and then determined at 490 nm absorbance
Fig. 1. Molecular structure of the probe.
using an ELx800 Absorbance Reader. Experiments were performed in triplicate. 3. Results and discussion The probe was readily synthesized according to the reported synthetic procedure using rhodamine B acyl chloride with 2guanidinobenzimidazole as reactants and characterized by X-ray crystallography, elemental analysis, FTIR, 1 H NMR, 13 C NMR spectra and HRMS. The X-ray crystallography structural investigation of the probe (Fig. 1) clearly revealed the unique spirolactam ringclosed formation with the C4 N spiro ring (Marked with red circle in Fig. 1) in a solid state which was also supported by a distinctive spirocycle carbon shift at 61.3 ppm in 13 C NMR, thus agreed well with the colorless and non-fluorescence status of the probe in the metal-free condition. Fig. 1. The solution investigation was firstly performed in EtOH-H2 O MeOH-H2 O, (CH3 )2 CHOH-H2 O, (CH3 )2 CO-H2 O, THFH2 O, CH3 CN-H2 O, DMSO-H2 O, and DMF-H2 O mixed solutions (Fig. 2). The result suggested that other solvent systems seemed all to be inferior to EtOH-H2 O since the fluorescence intensities and absorptions of the probe in these solvents were much lower than that in EtOH-H2 O mixed solution upon addition of Sn4+ except for MeOH-H2 O, which was quite close to EtOH-H2 O mixed solution. As methanol are more harmful to the biological samples than ethanol in cell imaging experiments, EtOH-H2 O mixed solution was chosen. We then evaluated the fluorescence intensity and absorption of the probe with Sn4+ in the solutions with the containing water varies from 0 to 100% (Figs. S4 and S5). It is clear that the pure solution of water caused negligible signal changing. The fluorescence intensity and absorption increased with the increasing of ethanol in the mixed solvent. As the probe was designed for detection of tetravalent tin in environmental and biological samples, the containing of organic solvent should be as little as possible, thus EtOH-H2 O (5:5, v/v) was chosen for photo-property studies and EtOH-H2 O (2:8, v/v) was chosen for bio-imaging. Originally, the hydrolytic process of Sn4+ can be happen in aqueous media and results in the decrease of pH and induces the protonation-induced ring-opening process. Therefore, the pH responses of the probes in EtOH-H2 O (5:5, v/v) were then evaluated using solutions with pH values from 1.7 to 13.0 (Figs. S6 and S7). As expected, the fluorescence intensity and absorption increased when the pH value is lower than 5, which was corresponding to the protonation-induced ring-opening process. Delightfully, both the fluorescence intensity and the absorption exhibited unconspicuous changes in the pH range of 5.0–8.0, suggesting that the probes were insensitive to pH at neutral range and could work in
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Fig. 2. Fluorescence intensity (a) and absorption (b) changes of the probe (10 M) in the presence of 5.0 equiv. of Sn4+ in different solutions with PBS buffer (0.2 M, pH 7.4), ex = 540 nm.
approximate physiological condition with negligible background fluorescence. Thus, further investigations for the ultraviolet and fluorescent properties of the probes were carried out in the nearly neutral EtOH-H2 O (5:5, v/v, PBS, pH 7.4) mixed system to insure that the signal changings were caused by the coordination of Sn4+ to the probe instead of the decrease of pH induced by the hydrolytic process of Sn4+ . When related heavy, transition and main group metal ions including Li+ , Na+ , K+ , Ag+ , Cu+ , Ca2+ , Ba2+ , Cd2+ , Mg2+ , Co2+ , Mn2+ , Zn2+ , Pb2+ , Ni2+ , Cu2+ , Sn2+ , Hg2+ , Fe2+ , Cr2+ , Fe3+ , Al3+ , Cr3+ , Au3+ and Sn4+ were used to evaluate the selective property of probes in EtOHH2 O (5/5, v/v, PBS, pH 7.4) solution (Fig. 3). As expect, only the introduction of Sn4+ give rise to remarkable absorbance and fluorescence enhancement while Sn2+ did not cause any absorbance and fluorescence changes. Although Pb2+ , Fe3+ , and Cr3+ also had slight responses, they were significantly inferior and easily quenched compared with Sn4+ induced spectroscopic changes (Figs. S10 and S11). The results obviously implied the significant selectivity of the probe to Sn4+ . Time dependent study showed that the coordination of the probe with Sn4+ completed rapidly within 10 s (Figs. S16 and S17) and showed prolonged stability since the fluorescence intensity showed unconspicuous decrease for several hours, indicating a significant resistance to irreversible photo bleaching. In addition, other organic tetravalent tin compounds including butyltin trichloride, dibutyltin dichloride, tributyl tin chloride and tetrabutyltin
could also gave rise to distinct spectroscopic changes in a short time and exhibited long time stability (Figs. S12 and S13). The competition experiments were then performed by adding the competing ion to the solutions which contained the probe and Sn4+ . The results indicated that the selectivity of the probe did not significantly experience interference from the commonly coexistent ions, except for the slight increasing and quenching caused by Fe3+ , Cr3+ and Cu2+ , respectively. The spectroscopic titrations were then performed to demonstrate the efficiency of quantitative signaling behavior of the probe (Fig. 4). Both the absorption band centered at 559 nm and the fluorescence intensity around 581 nm increased accompanied by a brilliant pink coloration and a strong orange fluorescent emission with the increasing concentration of Sn4+ in high fluorescence quantum yield when the concentration varied in the range of 0 mol L−1 –50 mol L−1 (Fig. 4, inset). The fluorescence intensity and absorption both saturated with 5.0 equiv. of Sn4+ and higher concentration of Sn4+ would cause signal quenching. Good linear relationships were observed between the relative absorbance as well as fluorescence intensities and the concentration of Sn4+ in the 0.06 mol L−1 –50 mol L−1 range with a detection limit of 0.03 mol L−1 (Figs. S8 and S9) in EtOH-H2 O (5/5, v/v, PBS, pH 7.4) mixed solution when 10 mol L−1 of probe was used, indicating the highly suitability for quantitative determination of Sn4+ through this probe.
Fig. 3. Histogram showing of the fluorescence intensity and absorption for the selectivity and competition investigation of the probe RhoG (10 mol L−1 ) in EtOH-H2 O (5:5, v/v, PBS, pH 7.4) mixed solution, ex = 540 nm. The pillars in the front row represent fluorescence response of the probe to the metal ions of interest. The pillars in the back row represent the subsequent addition 5.0 equiv. Sn4+ to the solution containing RhoG and the metal ions, respectively.
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Fig. 4. Fluorescence intensity (a) and absorption (b) changes of the probe (10 mol L−1 ) upon addition of Sn4+ (0 − 5.0 equiv.) in EtOH-H2 O (5/5, v/v, PBS, pH 7.4) mixed solution. Inset: Changes of emission intensity at 581 nm (a) and absorption at 559 nm (b), ex = 540 nm.
In order to gain an insight of the ring-opening mechanism, intensive investigation has been performed. The stoichiometric ratio investigation through job’s plot established the formation of the 1:1 stoichiometry between the probe and Sn4+ with high association constant of 2.99 × 106 M−1 (Figs. S14 and S15). The research of addition ethylenediamine to the solution of the complex indicated the reversible nature of the binding process since the fluorescence intensity gradually decreased with the increasing concentration of ethylenediamine and completed quenched when excess ethylenediamine was added, coupled with the changing of pink color back to colorless (Figs. S18 and S19). On the hand of structural investigation, the mass spectrum manifested the peak at m/z 790.5088 (Fig. S24), which was assigned as [RhoG + Sn4+ + 2Cl− + H]+ , providing a powerful evidence for the structure of the binding complex. This result also coincided perfectly with the job’s plot investigation which confirmed the 1:1 binding stoichiometry. For better understand, DFT-NBO analysis focusing on the electron lone pairs (LP) was performed for the probe [47,48]. The LP of the N atoms on the guanidine group and benzimidazole moiety were computed to be the most possible ones to coordinate with Sn4+ . Thus, several possible structures of the complex were reasonably proposed and theoretically optimized at DFT level with PBE1PBE functional (Fig. S25). The optimized geometries of the probe and the complex were shown in Fig. 5, of which the local minimum character was confirmed by the nonexistence of imaginary frequency. The most probable structure which was proposed and validated to have the lowest energy compared to others clearly indicated that Sn4+ coordinate with the NH moiety of guanidine
Scheme 2. Proposed mechanism of the probe upon addition of Sn4+ .
group and the N atom of benzimidazole functional group inducing the metal-to-ligand chelation charge transfer ring-opening process inducing the color and fluorescent changes (Scheme 2). With the formation of the complex, the bond length of N1C1 and C4-C7 decreased from 1.387 Å to 1.359 Å and 1.516 Å (S1) to 1.409 Å, respectively, indicating the transformation of the phenol structure to the quinoid structure (Fig. 6). The investigation of spatial distributions and orbital energies of HOMO and LUMO (Fig. 6) demonstrated that the HOMO distribution of the probe was located essentially over the xnathene moiety, while the LUMO was mainly distributed over the volution neighboring benzene ring. In contrast, both HOMO and LUMO of the complex were mostly positioned at the rhodamine framework. In addition, although the HOMO-LUMO energy gap of complex decreased by 0.74 eV relative to that of probe, the large energy gap of 3.10 eV between HOMO and LUMO in the complex still provide powerful evidence for the strong interaction and excellent stability of the complex, which was well consistent with the designing demand.
Fig. 5. PBE1PBE optimized structure of the probe (a) and the complex (b). Carbon atoms are yellow, oxygen atoms are red, nitrogen atoms are purple, hydrogen atoms are blue, chlorine atoms are gray, and tetravalent tin atom is dark yellow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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fluorescence inside the cells could be monitored. However, after three times washing with PBS buffer and supplemented with 5.0 equiv. of Sn4+ in the same growth medium for another 30 min at 37 ◦ C, prominent fluorescent increases were observed in the intracellular area. Moreover, bright-field transmission images of cells suggested that the cell morphology remained in good condition throughout the imaging experiments and established the wonderful cell-membrane permeability of the probe. In addition, the overlay of fluorescent and bright-field images confirmed that the fluorescent signals were localized in the perinuclear area of the cytosol, demonstrating the subcellular distribution of Sn4+ . The preliminary experiments in living L929 cells provided an suitable candidate for in vivo tracking and imaging of Sn4+ . Similarity, as another proof-of-concept, the biological imaging of Sn4+ in Human Osteosarcoma MG-63 cells (Fig. 7) was also successfully performed with the same procedure, thus contributed to significant breakthroughs in understanding the importance and functional mechanism of tetravalent tin in environmental samples as well as related cells and organs.
4. Conclusion Fig. 6. HOMO and LUMO distributions of the probe and the complex. Carbon atoms are yellow, oxygen atoms are red, nitrogen atoms are purple, hydrogen atoms are blue, chlorine atoms are gray, and tetravalent tin atom is dark yellow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
The MTT assay that uses mitochondrial metabolic enzyme activity as an indicator of cell viability was conducted following the protocol described previously [49–51] to identify the toxicity of the probe. The MCF-7 cell was treated with different concentrations of RhoG. The fifty percent (IC50 ) value was calculated as the ratio of the mean of OD obtained for the treated cell to that of the vehicle control. As shown in Table S2, the IC50 value was more than 100, indicating the strong cell viability and low toxicity of RhoG. To further evaluate the practical bio-imaging applications of the probe for quantifying Sn4+ in biological systems, cultured Mouse fibroblast cells L929 were incubated with the probes in culture medium for 30 min at 37 ◦ C (Fig. 7). Fluorescence laser scanning confocal microscopy images demonstrated that no intracellular
In conclusion, the design and synthesis of the novel 2guanidinobenzimidazole functionalized rhodamine based fluorescent probe RhoG for specific detection of tetravalent tin with special selectivity and sensitivity were presented. The recognition behavior was investigated both experimentally and computationally. The results suggested that the Sn4+ induced metal-to-ligand chelation charge transfer in complexation with the NH moiety of guanidine group and the N atom of benzimidazole functional group reasonably promoted the ring-opening process, indicating that the introduction of guanidinobenzimidazole functional group provided binding sites for Sn4+ and facilitate the formation of the complex along with improving the spectral properties and biocompatibility of the probe. Preliminary MTT assay and bio-imaging experiments in living L929 cells and MG-63 cells confirmed the membrane permeability of the probe and the appropriateness of using the probe as a promising candidate for intracellular identifying of Sn4+ , which was supposed to provide a better understanding of the deleterious effects and biological functions of Sn4+ in related environmental, geological, and biological samples.
Fig. 7. Fluorescent images of Mouse fibroblast cells L929 and human Osteosarcoma MG-63 cells incubated with 10 mol L−1 RhoG for 30 min at 37 ◦ C (a) and then further incubated with 50 mol L−1 SnCl4 for 30 min at 37 ◦ C (b). (c). Bright-field transmission image of cell treated with the probe (10 mol L−1 ) and SnCl4 (50 mol L−1 ). (d). The overlay images of (b) and (c).
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Acknowledgements We are grateful for the support from the National Natural Science Foundation of China (NSFC 21572177; 21301139; 21272184; 21103137; 21103135; 21073139 and J1210057), the Shaanxi Provincial Natural Science Fund Project (No. 2015JZ003), the Shaanxi Provincial Higher Education Teaching Reform Project (No. 15BY47), the Xi’an City Science and Technology Project (Nos. CXY1511(3)), Scientific Research Cultivating Fund of Xi’an University of Science and Technology (No. 201619), the Xi’an University of Science and Technology Teaching Reform Project (No. JG14052), the Chinese National Innovation Experiment Program for University Students (No. 201510697004) and the Northwest University Science Foundation for Postgraduate Students (No. YZZ14052). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2016.09.170. References [1] M.H. Lee, J.S. Kim, J.L. Sessler, Small molecule- based ratiometric fluorescence probes for cations, anions, and biomolecules, Chem. Soc. Rev. 44 (2015) 4185–4191. [2] H. Qian, Z.C. Xu, Fluorescence imaging of metal ions implicated in diseases, Chem. Soc. Rev. 44 (2015) 4487–4493. [3] Y.H. Tang, D. Lee, J.L. Wang, G.H. Li, J.H. Yu, W.Y. Lin, J. Yoon, Development of fluorescent probes based on protection- deprotection of the key functional groups for biological imaging, Chem. Soc. Rev. 44 (2015) 5003–5015. [4] H. Li, X.H. Gao, W. Shi, H.M. Ma, Design strategies for water-soluble small molecular chromogenic and fluorogenic probes, Chem. Rev. 114 (2014) 590–659. [5] Y.Y. Huang, M.J. Wang, M.Y. She, Z. Yang, P. Liu, J.L. Li, Z. Shi, Recent progress in the fluorescent probe based on spiro ring opening of xanthenes and related derivatives, Chin. J. Org. Chem. 34 (2014) 1–25. [6] Q. Chen, T. Pradhan, F. Wang, J.S. Kim, J. Yoon, Fluorescent chemosensors based on spiroring-opening of xanthenes and related derivatives, Chem. Rev. 112 (2012) 1910–1956. [7] R.W. Ramette, E.B. Sandell, Rhodamine B equilibriums, J. Am. Chem. Soc. 78 (1956) 4872–4878. [8] M. Beija, C.A. Afonso, J.M. Martinho, Synthesis and applications of rhodamine derivatives as fluorescent probes, Chem. Soc. Rev. 38 (2009) 2410–2433. [9] Y. Wang, H.Q. Chang, W.N. Wu, W.B. Peng, Y.F. Yan, C.M. He, T.T. Chen, X.L. Zhao, Z.Q. Xu, Rhodamine 6G hydrazone bearing pyrrole unit: ratiometric and selective fluorescent sensor for Cu2+ based on two different approaches, Sens. Actuators B: Chem. 228 (2016) 395–400. [10] J.L. Fan, P. Zhan, M.M. Hu, W. Sun, J.Z. Tang, J.Y. Wang, S.G. Sun, F.L. Song, X.J. Peng, A fluorescent ratiometric chemodosimeter for Cu2+ based on TBET and its application in living cells, Org. Lett. 15 (2013) 492–495. [11] L. Yuan, W.Y. Lin, B. Chen, Y.N. Xie, Development of FRET- based ratiometric fluorescent Cu2+ chemodosimeters and the applications for living cell imaging, Org. Lett. 14 (2012) 432–435. [12] J. Tang, S.G. Ma, D. Zhang, Y.Q. Liu, Y.F. Zhao, Y. Ye, Highly sensitive and fast responsive ratiometric fluorescent probe for Cu2+ based on a naphthalimiderhodamine dyad and its application in living cell imaging, Sens. Actuators B: Chem. 236 (2016) 109–115. [13] K. Gupta, N. Mergu, A.K. Singh, Rhodamine- derived highly sensitive and selective colorimetric and off- on optical chemosensors for Cr3+ , Sens. Actuators B: Chem. 220 (2015) 420–432. [14] S. Li, D. Zhang, X.Y. Xie, S.G. Ma, Y. Liu, Z.H. Xu, Y.F. Gao, Y. Ye, A novel solvent- dependently bifunctional NIR absorptive and fluorescent ratiometric probe for detecting Fe3+ /Cu2+ and its application in bioimaging, Sens Actuators B: Chem 224 (2016) 661–667. [15] S.Y. Ma, Z. Yang, M.Y. She, W. Sun, B. Yin, P. Liu, S.Y. Zhang, J.L. Li, Design and synthesis of functionalized rhodamine based probes for specific intracellular fluorescence imaging of Fe3+ , Dyes Pigm. 115 (2015) 120–126. [16] Z. Yang, M.Y. She, B. Yin, J.H. Cui, Y.Z. Zhang, W. Sun, J.L. Li, Z. Shi, Three rhodamine- based off- on chemosensors with high selectivity and sensitivity for Fe3+ imaging in living cells, J. Org. Chem. 77 (2012) 1143–1147. [17] M.Y. She, Z. Yang, B. Yin, J. Zhang, J. Gu, W.T. Yin, J.L. Li, G.F. Zhao, Z. Shi, A novel rhodamine- based fluorescent and colorimetric off- on chemosensor and investigation of the recognizing behavior towards Fe3+ , Dyes Pigm. 92 (2012) 1337–1343. [18] W.T. Yin, H. Cui, Z. Yang, C. Li, M.Y. She, B. Yin, J.L. Li, G.F. Zhao, Z. Shi, Facile synthesis and characterization of rhodamine- based colorimetric and off- on fluorescent chemosensor for Fe3+ , Sens. Actuators B: Chem. 157 (2011) 675–680.
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Northwest University. His research interests also focus on developing fluorescent functional chemosensors. Siyue Ma received her BS degree from Heilongjiang University, PR China, in 2014. She is currently a Ph.D candidate at College of Chemistry and Materials Science, Northwest University. Her research interests also focus on developing fluorescent functional chemosensors. Dr. Bing Yin was born in 1981, and is now working in Northwest University, PR China. He received his Ph.D. in physical chemistry from Beijing Normal University in 2009. His current research interests include theoretical investigation of molecular magnetic and theoretically model of functional organic molecules. Peof. Ping Liu was born in 1974, and is a professor at Northwest University, PR China. She received her Ph.D. in inorganic chemistry from Northwest University in 2005. Her current research interests include synthesis and study of new ligand and their functional complexes. Prof. Xiangrong Liu was born in 1967, and is a professor at Xi’an University of Science and Technology, PR China. She received his Ph.D. in Physical chemistry of materials from Northwestern Polytechnical University in 2004. Her current research interests include synthesis and research methods for functional molecular, the thermal kinetics of microbial interactions, and bioconversion of coal.
Biographies
Dr. Shunsheng Zhao was born in 1981, and is now working in Xi’an University of Science and Technology, PR China. He received his Ph.D. in inorganic chemistry from Wuhan University in 2010. His current research interests include biological imaging of water-soluble luminescence of rare earth complexes, transition metal complexes and their catalysis in the polymerization reaction and the thermodynamics and thermal kinetics of complexes.
Dr. Zheng Yang was born in 1988, and is now working in Xi’an University of Science and Technology, PR China. He received his Ph.D. in organic chemistry from Northwest University in 2015. His research interests focus on developing fluorescent functional chemosensors.
Prof. Jianli Li was born in 1973, and is a professor at Northwest University, PR China. He received his Ph.D. in organic chemistry from Northwest University in 2007. His current research interests include synthesis and research methods for organic substance, fluorescent molecular devices, chemical and biological sensors and molecular recognition.
MengyaoShe received his BS degree from Northwest University, PR China, in 2012. He is currently a Ph.D candidate at College of Chemistry and Materials Science,
Please cite this article in press as: Z. Yang, et al., Rhodamine based guanidinobenzimidazole functionalized fluorescent probe for tetravalent tin and its application in living cells imaging, Sens. Actuators B: Chem. (2016), http://dx.doi.org/10.1016/j.snb.2016.09.170