DOI: 10.1002/chem.201404224
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& Fluorescent Probes
A Highly Selective Two-Photon Fluorescent Probe for Detection of Cadmium(II) Based on Intramolecular Electron Transfer and its Imaging in Living Cells Zhaohua Shi,[a] Qingxin Han,[a] Lizi Yang,[a] Huan Yang,[a] Xiaoliang Tang,[a] Wei Dou,[a] Zhiqi Li,[a] Yange Zhang,[a] Yongliang Shao,[a] Liping Guan,[b] and Weisheng Liu*[a]
Abstract: A new quinoline-based probe was designed that shows one-photon ratiometric and two-photon off–on changes upon detecting Cd2 + . It exhibits fluorescence emission at 407 nm originating from quinoline groups in Tris-HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v). Coordination with Cd2 + causes quenching of the emission at 407 nm and simultaneously yields a remarkable redshift of the emission maximum to 500 nm with an isoemissive point at 439 nm owing to an intramolecular charge-transfer mechanism. Thus, dual-emission ratiometric measurement with a large redshift (Dl = 93 nm) and significant changes in the ratio (F500/F439) of the emission intensity (R/R0 up to 27) is estab-
lished. Moreover, the sensor H2L displays excellent selectivity response, high sensitive fluorescence enhancement, and strong binding ability to Cd2 + . Coordination properties of H2L towards Cd2 + were fully investigated by absorption/fluorescence spectroscopy, which indicated the formation of a 2:1 H2L/Cd2 + complex. All complexes were characterized by X-ray crystallography, and TD-DFT calculations were performed to understand the origin of optical selectivity shown by H2L. Two-photon fluorescence microscopy experiments have demonstrated that H2L could be used in live cells for the detection of Cd2 + .
Introduction
Therefore, it is of great significance to develop excellent methods for the quantification of Cd2 + in environment or living cells. As one of powerful means and techniques for detecting metal ions, fluorescent sensors have attracted increasing attention in recent years because of simplicity, high sensitivity and selectivity, instantaneous response, and in vivo applications, which convert interaction of the target species with the receptor unit into an optical signal changing.[4] The main challenge of fluorescent sensors for Cd2 + is to discriminate it from Zn2 + . As a result of being in the same group of the periodic table and closed-shell d10 electronic configuration, they usually possess similar chemical behaviors, including similar spectral changes after interactions with fluorescent sensors.[5] So far, a few fluorescent Cd2 + probes have been reported, and most of them are intensity-based. As is well-known, the emission intensity is dependent on many other factors, such as emission collection efficiency, sample environment, sensor concentration, bleaching, optical path length, and illumination intensity.[5, 6] In contrast, a ratiometric sensor exhibits spectral shift in absorption and emission spectra upon binding to the analyte of interest, and the ratio of emission intensities at two different wavelengths can be used to evaluate the analyte concentration, thus reducing the interference induced by the unknown local sensor concentration and the deviations in detecting conditions.[7] Moreover, two-photon (TP) probes that can selectively detect Cd2 + are very rare, which utilize two photons of lower energy for the excitation and have evolved into a widely used method in biomedical research.[8] To date, sever-
Cadmium, an important heavy and transition metal, is widely used in many industrial and agricultural processes, thus resulting in serious pollution to air, water, and soil. It is one of the highly toxic metals and listed seventh on the Top 20 Hazardous Substances Priority List by the Agency for Toxic Substances and Disease Registry and US Environmental Protection Agency (EPA).[1] Cd2 + can accumulate in ecosystems and humans owing to its long biological half-life (15–20 years for humans) through the soil-plant-animal-human food chain and inhaling of cigarette smoke.[2] Either short-term or long-term exposure to Cd2 + can lead to mutations and cancer, such as renal dysfunction, calcium metabolism disorders, and pulmonary, prostatic, and renal cancer.[3] Nevertheless, cellular Cd2 + uptake and carcinogenic mechanisms are still poorly understood. [a] Dr. Z. Shi, Q. Han, Dr. L. Yang, H. Yang, Dr. X. Tang, Dr. W. Dou, Z. Li, Y. Zhang, Y. Shao, Prof. Dr. W. Liu Key Laboratory of Nonferrous Metals Chemistry and Resources Utilization of Gansu Province and State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering Lanzhou University, Lanzhou 730000 (P. R. China) Fax: (+ 86) 931-8912582 E-mail: [email protected] [b] L. Guan School of Life Sciences Lanzhou University, Lanzhou 730000 (P. R. China) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201404224. Chem. Eur. J. 2015, 21, 290 – 297
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Full Paper al ratiometric Cd2 + sensors have been developed and successfully used in biological applications recently.[9] Chemists still need to design novel methods that can quantify Cd2 + and overcome limitations, including poor selectivity and water solubility, small spectral shift, or operation at non-physiological pH. In our previous research, we have successfully designed and synthesized HL, a combination of 8-HQ or 8-AQ, which showed a bright “switch-on” optical response based on the photoinduced electron-transfer (PET) mechanism upon binding Cd2 + .[6d] However, recognition of Cd2 + using HL must be performed in absolute ethanol and easily suffers from interference from anions. These disadvantages encouraged us to improve sensing properties by incorporating with auxiliary receptors. According to the ionic radius and the limitation of coordination numbers, we hope that introducing a hydroxymethyl at the 2-position of 8-HQ would generate a novel sensor with high selectivity and sensitivity for Cd2 + . Herein, we present our design and synthesis of sensor H2L, featuring a modified 8-HQ. As expected, metal-ion complexation and accompanying selectivity of H2L is enhanced. H2L showed redshifts in both emission and absorption wavelength upon binding Cd2 + that is based on the ICT mechanism in aqueous media. Furthermore, we carefully studied its complexation with Cd2 + by fluorescence/absorption titration, X-ray crystallography, and TD-DFT calculations. Above all, the complex of H2L with Cd2 + exhibits a large two-photon absorption cross section at 700 nm and TP fluorescence imaging have demonstrated that H2L is membrane-permeable and can detect intracellular Cd2 + .
X-ray crystallography was obtained by slow evaporation from a CH3CN/CH3OH solution. All of the complexes were fully characterized by 1H NMR, 13C NMR, ESI-MS, FTIR, and elemental analysis or X-ray diffraction measurement. Spectroscopic measurements of H2L were performed under physiological pH conditions (Tris-HCl, 25 mm, pH 7.40, H2O/ EtOH 8:2, v/v). The UV/Vis spectrum of H2L exhibits an absorption maximum at 302 nm at room temperature, which can be assigned to the p–p* transitions of the quinoline groups (Figure 1).[6d] When titrated by Cd(ClO4)2 (0–2.0 equiv), the absorb-
Figure 1. UV/Vis spectral changes of 0.10 mm H2L upon addition of Cd2 + (0– 0.3 mm) in Tris-HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v) at room temperature. Inset: Absorbance changes at * 302 and * 342 nm as a function of the Cd2 + concentration, indicating the 2:1 stoichiometry for H2L-Cd2 + .
Results and Discussion ance of H2L at 302 nm gradually decreased with an increasing concentration of Cd2 + (Figure 1). Moreover, a new absorption band appeared at 342 nm with a distinct isosbestic point at 325 nm, suggesting chelation-elicited effective ICT. The absorbance at 342 and 302 nm changed linearly with the concentration of Cd2 + up to a molar ratio (H2L/Cd2 + ) of 2:1, and there was saturation at even higher [Cd2 + ] (Figure 1, inset). Upon addition of Zn2 + , similar changes in the absorption spectrum were observed (Supporting Information, Figure S1), indicating the similar coordination pattern between H2L-Cd2 + and H2LZn2 + . These indicate the 2:1 binding stoichiometry between H2L and Cd2 + , Zn2 + , which was further confirmed by Job’s plots (Supporting Information, Figures S2 and S3), fluorescence titrations, and crystal structures. The optical properties of H2L are mainly dominated by the quinoline platforms. We investigated its fluorescence properties in the presence of common metal ions such as Li + , Na + , K + , Ca2 + , Mg2 + , Al3 + , Cr3 + , Mn2 + , Fe3 + , Co2 + , Ni2 + , Cu2 + , Ag + , Zn2 + , Cd2 + , and Hg2 + in Tris—HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v). As shown in Figure 2, the free sensor H2L displayed an emission maximum at around 407 nm upon excitation at 325 nm. Physiologically important metal ions and most heavy and transition-metal ions, including Li + , Na + , K + , Ca2 + , Mg2 + , Cr3 + , Ni2 + , Mn2 + , Hg2 + , Ag + , and Al3 + , induced negligible changes in the emission profiles. Fe3 + , Co2 + , and Cu2 +
The synthesis of H2L is shown in Scheme 1. Compound 2 was synthesized by the reaction of 8-hydroxyquinaldine with 1 using anhydrous potassium carbonate in refluxing CH3CN. Oxidation of active methyl group of 2 to formyl with SeO2 followed by reduction using NaBH4 afforded H2L. Significantly, the metal complex of [Cd(H2L)2(ClO4)2]H2O was obtained through the reaction of H2L with Cd(ClO4)2. A suitable single crystal for
Scheme 1. Synthesis of H2L. Chem. Eur. J. 2015, 21, 290 – 297
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Full Paper arly with the total concentration of Cd2 + up to a mole ratio (H2L/Cd2 + ) of 2:1 (Figure 3, inset). Job’s plot and ESI-MS (m/z [Cd(H2L)2(ClO4)] + 930.9) also confirmed the binding stoichiometry of 2:1 between H2L and Cd2 + . Moreover, introduction of Cd2 + turned the visual emission of the sensor from blue to green (Supporting Information, Figure S4), which further supports the ratiometric fluorescence response. Based on the fluorescence titration studies, the association constant (log K) of H2L for Cd2 + was calculated to be 10.86 using Thordarson’s fitting program,[10] suggesting the strong binding ability of H2L with Cd2 + . The method detection limits (MDL) was measured to be 23.63 nm,[11] which is lower than the maximum limit (45 nm) for drinking water emphasized by The US Environmental Protection Agency (EPA).[12] Furthermore, the direct proof experiment for the trace Cd2 + determination was performed (Supporting Information, Figure S5). To establish the practical application of H2L, we carried out an experiment to monitor residual Cd2 + in a reactor using H2L. A CH3CN (10 mL) solution of Cd(ClO4)2·6H2O (4.2 mg) was stirred in three flasks for two days at room temperature. After the solution was poured out, the three flasks were treated with different washing procedures respectively (washing with H2O (1 10 mL); washing with H2O (3 10 mL); and washing with H2O (3 10 mL) and CH3OH (3 10 mL)). Subsequently, H2L solution (Tris-HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v)) was added into the three flasks and R was determined to be 3.23, 1.68, and 1.06 respectively. The results demonstrate that our detection system could function well in Cd2 + analysis in environmental samples. The sensing process of Cd2 + by H2L was found to be reversible. This was demonstrated by the addition of an ethylenediaminetetraacetic acid (EDTA) aqueous solution to the glowing H2L-Cd2 + solution, which can almost recover the original emission of H2L (Supporting Information, Figure S6). These indicate that H2L is an excellent ratiometric fluorescent sensor with high sensitivity and reversibility. In the selectivity experiment, Zn2 + also showed some ratiometric changes as similar as that of Cd2 + (Figure 2). To understand the distinction between the interaction of H2L with Cd2 + and Zn2 + , we also investigated the fluorescence titration spectra of Zn2 + (0–1.2 equiv) to H2L (Figure 4). Similarly, the emission spectrum showed a redshift to 495 nm with an isoemissive point at 465 nm. Therefore, it may be difficult to avoid the interference of Zn2 + for detecting trace Cd2 + as there are some obvious overlap in the fluorescent spectra for the Cd2 + and Zn2 + determinations as shown in Figure 3 and Figure 4. Efforts are under way to design and synthesize analogues, which can be better used for detecting trace Cd2 + in the water media. Job’s plot (Supporting Information, Figure S3) and crystal structure (see below) confirmed the binding stoichiometry of 2:1 between H2L and Zn2 + . The binding constant (log K) of H2L for Zn2 + was determined to be 6.98 using a fitting program, which was much less than that for Cd2 + (10.86).[10] These results imply that highly selective fluorescent response may arise from selective combination. The structures of the Cd2 + and Zn2 + complexes with H2L are shown in Figure 5 and the Supporting Information, Figure S8, respectively. Selected bond lengths and bond angles are given
Figure 2. Fluorescence emission spectra of 50 mm H2L in the presence of different metal ions (1 mm for s-block and 0.1 mm for d-block metal ions) in Tris-HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v). lex = 325 nm. Inset: Visual fluorescent photographs of H2L-Zn2 + , H2L, and H2L-Cd2 + in Tris-HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v). The photographs were taken under a handheld UV/Vis (365 nm) lamp.
quenched the fluorescence in varied degrees, which always meet in the other metal ion sensors. Interestingly, on addition of Cd2 + , the emission band at 407 nm had disappeared with the appearance of a new striking increased band at 500 nm. Although Zn2 + could induce similar redshifts of fluorescence spectra, the enhancements in fluorescence intensity at 495 nm are negligible compared with that induced by Cd2 + . These results indicate that H2L shows excellent selectivity for Cd2 + in aqueous media. The fluorescence titration spectra of Cd2 + to H2L (50 mm) are displayed in Figure 3. Addition of gradually increasing concentrations of Cd2 + caused a significant effect on the emission profile: the emission intensity at 407 nm gradually decreased, and simultaneously, a new peak appeared at 500 nm with a large redshift of 93 nm and dramatically enhanced intensity, which can be attributed to the ICT structure in the excited state of the H2L-Cd2 + complex.[9a] A well-defined isoemissive point at 439 nm was noted. The emission ratio at 500 and 439 nm (F500/F439) increased by about 27-fold and changed line-
Figure 3. Fluorescence emission spectra of 50 mm H2L upon addition of Cd2 + (0–0.2 mm) in Tris-HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v) at room temperature. lex = 325 nm. Inset: Ratio (F500/F439) changes as a function of the Cd2 + concentration, indicating the 2:1 stoichiometry for H2L-Cd2 + . Chem. Eur. J. 2015, 21, 290 – 297
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Full Paper square-bipyramidal coordination geometry. These results imply that H2L would grasp Cd2 + better than Zn2 + . The larger Cd2 + (ionic radii r + = 0.96 for Cd2 + and r + = 0.74 for Zn2 + ) may be more suitable for the binding pocket of H2L, resulting in a high affinity for Cd2 + .[9b, 15] It has been noted that five-membered chelate rings favor the complexation of larger metal ions, whereas six-membered chelate rings favor the complexation of smaller metal ions.[15] The new emission band (500 nm) of H2L-Cd2 + may arise from two effects. First, after binding Cd2 + , the intramolecular hydrogen bond (N···H···O) is broken, and the radiationless relaxation is forbidden.[16] Second, the coordination of Cd2 + to 8-HQ induces an intramolecular electron transfer process (ICT) from the nitrogen atom of the heterocycle to the metal ion. Consequently, an obvious redshift in both emission and absorption wavelength could be observed. Time-dependent density functional theory (TD-DFT) calculations were performed to clarify the Zn2 + /Cd2 + optical discrimination by H2L. As illustrated in Figure 6 a, the absorption band at about 302 nm of H2L was calculated at 324 nm, which was assigned to the S0!S2 monoelectronic vertical transition. This transition corresponds to HOMO!LUMO + 1 (f = 0.1202).
Figure 4. Fluorescence emission spectra of 50 mm H2L upon addition of Zn2 + in Tris-HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v) at room temperature. lex = 325 nm. Inset: Ratio (F500/F439) changes as a function of the Zn2 + concentration.
Figure 5. ORTEP (ellipsoids set at 30 % probability) of [Cd(H2L)2(ClO4)2]H2O. Hydrogen atoms, disordered perchlorate, and solvent molecules are omitted for clarity.
in the Supporting Information, Table S2 and S3. They crystallize in the monoclinic system from methanol/acetonitrile solvent, with space group P2/n and P2/c, respectively. We can see that H2L chelates Cd2 + with 2:1 stoichiometry, and the asymmetric unit consists of H2L and half of Cd2 + . Cd2 + is eight-coordinate and surrounded by two carbonyl oxygen atoms, two 8-HQ oxygen atoms, two 8-HQ nitrogen atoms, and two hydroxymethyl oxygen atoms from two H2L ligands, which forms three five-membered chelate rings with each one H2L ligand. The bond length of Cd1N3 is 2.322 (4) , and the Cd1O distance ranges from 2.367 (3) to 2.498 (3) (Supporting Information, Table S2), which are within the reported extent (2.26–2.52 for CdN and 2.20–2.59 for CdO).[13] In Zn(H2L)2(ClO4)2 as demonstrated in the Supporting Information, Figure S8, although Zn2 + adopts coordination conformation similar to Cd2 + in Cd(H2L)2(ClO4)2, Zn1O2 distance is 2.464(3) and it is beyond the reported bond length range (1.95–2.37 for ZnO).[14] Consequently, Zn2 + is six-coordinate and forms a distorted Chem. Eur. J. 2015, 21, 290 – 297
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Figure 6. Interface plots of the molecular orbitals calculated for respective compounds in the gas phase involved in the principal singlet vertical electronic transitions evaluated by TD-DFT: a) H2L, b) H2L-Cd2 + complex, c) H2LZn2 + complex. Black and gray parts on molecular orbitals refer to the different phases of the molecular wave functions, where the isovalue is 0.02 au.
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Full Paper For [Cd(H2L)2]2 + (Figure 6 b), the two experimental absorption bands at about 342 nm and 302 nm were calculated at 334 nm (HOMO1!LUMO, f = 0.0772, p–p*) and 309 nm (HOMO!LUMO + 1, f = 0.3106), respectively; these transitions involve ICT processes within the two ligands. While in the case of [Zn(H2L)2]2 + (Figure 6 c), the two experimental absorption bands were calculated at 319 nm (HOMO1!LUMO + 1, f = 0.0943) and 308 nm (HOMO!LUMO + 1, f = 0.3189, p–p*), respectively. Some of differences between experimental and theoretical absorption bands are perhaps caused by the solvent effects. These results strongly imply that the optical selectivity shown by H2L arises from different types of transition process among the respective compounds of H2L with metal ions.[17] To further understand the nature of interactions between sensor H2L and Cd2 + , 1H NMR titration was carried out (Figure 7). It can be seen that the NMR signals of methylene (CH2(a) and CH2(b)) and hydroxy protons of H2L are significant-
Figure 8. Selectivity of H2L for Cd2 + in the presence of other metal ions in Tris-HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v). lex = 325 nm. Black bars represent the addition of an excess of the appropriate metal ion (1 mm for Li + , Na + , K + , Ca2 + , Mg2 + ; 0.1 mm for all other metal ions) to a 50 mm solution of H2L. White bars represent the subsequent addition of 0.1 mm Cd(ClO4)2(H2O)6 to the solution.
ure S11, the fluorescence intensity of H2L-Cd2 + is hardly interfered with by these anions. Along with excellent selectivity for practical applications, it is essential that the probe can be operated in the physiological pH range. Therefore, we investigated the pH effect on the fluorescence response of H2L in the absence and presence of Cd2 + . The emission peak of H2L is observed at approximately 406 nm at neutral pH, and no dramatic change is expected within a broad pH range of 4.0–12.0.[18] At pH 13, owing to the deprotonation of the NH moiety of the aminoquinoline, a new redshifted emission band around 486 nm with slight enhancements could be observed (Supporting Information, Figure S12). The complexation of Cd2 + induces a fluorescence-observable increase of H2L at 500 nm and decrease at 407 nm within a pH range of 5–13. As can be seen from the Supporting Information, Figure S13, the fluorescence intensity ratio (F500/F439) of Cd2 + -H2L continues increasing in the pH 4.0–6.0 range, which may be due to the competition of H + . Moreover, H2L and the Cd2 + -H2L complex showed a stable F500/ F439 ratio under neutral and alkaline conditions. These results demonstrated that H2L can be used in complex system (environment samples or living systems) as a ratiometric fluorescent probe for Cd2 + without interference from other cations, anions, and pH effects. Having established the utility of H2L by one-photon spectroscopy, we have investigated the two-photon properties of H2L and its complexes using the TP induced fluorescence measurement technique. As shown in Figure 9, H2L shows no obvious two-photon action cross-section, and Zn2 + can induce weak enhancement in TP action cross-section around 700 nm. However, its Cd2 + complex has satisfactory efficiency in TP sensitization at 700 nm. This indicates the capability of H2L for detecting Cd2 + by the TP fluorescence. Figure 10 shows that changes in the TP excited fluorescence spectra (lex = 700 nm) of H2L titration with Cd2 + are qualitatively similar to those of corresponding one-photon excited fluorescence spectra. The fluorescence intensity at 515 nm dramatically enhanced with increasing concentrations of Cd2 + and increases linearly with
Figure 7. 1H NMR spectra (400 MHz) of H2L and H2L-Cd2 + in CD3CN.
ly shifted to the down-field direction upon titration with Cd2 + ion, indicating that 8-HQ is metal ion receptor to effectively interact with Cd2 + . However, addition of Cd2 + induces a marked up-field shift of the NH signal from 11.14 to 10.68, suggesting the increase of electron density of 8-AQ in the H2L-Cd2 + system. This is due to the effective ICT process upon binding Cd2 + . Owing to similar coordination patterns of H2L-Zn2 + and H2L-Cd2 + , 1H NMR signal changes of H2L upon addition of Zn2 + ion are similar to that of Cd2 + (Supporting Information, Figure S9). The emission response of H2L with different metal ions revealed a remarkable selectivity for Cd2 + (Figure 2). However, the most important criterion for a selective sensor is the ability to detect a specific species in a complex system. Therefore, we investigated the stability of the H2L-Cd2 + complex in the presence of other metal ions in Tris—HCl (25 mm, pH 7.20), H2O/ EtOH (8:2, v/v; Figure 8; Supporting Information; Figure S10). Most representative metal ions have little effect on the fluorescence of H2L for Cd2 + detection. Owing to competition coordination, Fe3 + slightly quenched the fluorescence of H2L-Cd2 + but clearly detectable (Supporting Information, Figure S10). The anion responses to the detection systems were further investigated. As shown in the Supporting Information, FigChem. Eur. J. 2015, 21, 290 – 297
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Figure 9. Two-photon excitation spectra of H2L with and without Cd2 + /Zn2 + in Tris-HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v).
Figure 11. TP image of SMMC-7721 cells. lex = 900 nm. a) Bright-field transmission image of cells incubated with 100 mm H2L for 2 h at 37 8C; b) TP image of the cells shown in (a); c) an overlay image of (a) and (b); d) brightfield transmission image of cells supplemented with 100 mm H2L for 2 h and then further incubated with 100 mm Cd(NO3)2 for 1 h at 37 8C; e) TP image of the cells shown in (d); f) an overlay image of (d) and (e); g) bright-field transmission image of cells that after treatment with H2L and Cd(NO3)2 and subsequent treatment with 200 mm EDTA for 30 min at 37 8C. h) TP image of the cells shown in (g); i) an overlay image of (g) and (h). Scale bars: 10 mm.
Cd2 + were further treated with EDTA (200 mm for 30 min) that decreases the level of Cd2 + , it showed a weaker fluorescent signal (Figure 11 g–i). These cell experiments show that H2L is cell-membrane-permeable and can be used to monitor Cd2 + reversibly in vivo.
Figure 10. Two-photon emission spectra of 50 mm H2L upon addition of Cd2 + (0–0.2 mm) in Tris-HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v) at room temperature. lex = 700 nm. Inset: 1) Enlarged figure from 385 nm to 415 nm of two-photon emission spectra; 2) emission intensity (515 nm) changes as a function of the Cd2 + concentration.
Conclusion
the total concentration of Cd2 + up to a mol ratio (H2L/Cd2 + ) of 2:1 (Figure 10, inset), indicating the capability of H2L for quantitatively detecting Cd2 + by the TP fluorescence. Therefore, on the basis of 2:1 stoichiometry and TP fluorescence titration data, the association constant (log K) of with Cd2 + was calculated to be 10.69, which is comparable to that based on onephoton excited fluorescence titration spectra. Because of extremely weak two-photon action cross section, H2L shows no obvious TP fluorescence, so it showed two-photon off–on changes for Cd2 + . In vitro studies demonstrated the ability of H2L to detect Cd2 + with excellent properties. We next sought to investigate the utility of H2L for monitoring the intracellular (living SMMC7721 cells) exogenous Cd2 + under two-photon excitation. SMMC-7721 cells treated with 100 mm H2L alone in the growth medium for 2 h at 37 8C gave a very weak fluorescence (Figure 11 a–c). However, the cells were subsequently incubated with Cd2 + (100 mm) for another 1 h and it displayed green fluorescence (Figure 11 d–f). When the cells exposed to H2L and Chem. Eur. J. 2015, 21, 290 – 297
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We have successfully designed and synthesized a ratiometric fluorescent sensor (H2L) for Cd2 + , and evaluated the Cd2 + fluorescence sensing and binding properties of H2L in aqueous media. It shows high selectivity and sensitivity for Cd2 + , reversible fluorescent response, as well as two-photon absorption upon binding with Cd2 + . The sensor provides dual-emission ratiometric detection of Cd2 + with a significant red-shift in emission and remarkable changes in the ratio (F500/F439) of the emission intensity. Two-photon imaging demonstrates that H2L is membrane-permeable and can monitor the Cd2 + in living cells. The Cd2 + selective fluorescence response is attributed to the enhanced selective binding ability of H2L by auxiliary receptor. Introducing of auxiliary binding site can prevent anion from coordinating and enhance the binding ability and selectivity of ligands toward analytes. The special size selectivity may give some insight into how to construct fluorescent sensors with special properties, which is very useful for probe design in the future. 295
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Full Paper Experimental Section
(w), 1539 (vs), 1485 (s), 1428 (m), 1321 (w), 1254 (m), 1114 (m), 1064 (m); ESI-MS: m/z [(M + 1) + ]: 360.3.
Synthesis of H2L and complexes
Synthesis of [Cd(H2L)2(ClO4)2]H2O: A methanol solution (8 mL) of Cd(ClO4)2·6H2O (0.0210 g, 0.05 mmol) was added to a magnetically stirred acetonitrile solution (5 mL) of H2L (0.0179 g, 0.05 mmol). The mixture was stirred in air for 30 min. It was filtered and kept in air. The transparent colorless single crystals suitable for X-ray crystallography were obtained on slow evaporation of the filtrate within 2 days. Anal. calcd for [Cd(H2L)2(ClO4)2]H2O: C 48.13, H 3.46, N 8.02; found: C 48,03, H 3.27, N 7.97; FTIR (KBr pellet, cm1): 3375 (br), 1663 (s), 1550 (vs), 1484 (m), 1434 (m), 1325 (m), 1265 (m), 1126 (s), 1095 (vs). ESI-MS m/z [Cd(H2L)2(ClO4)] + : 930.9. Synthesis of [Zn(H2L)2(ClO4)2]H2O: A methanol solution (8 mL) of Zn(ClO4)2·6H2O (0.0286 g, 0.05 mmol) was added to a magnetically stirred acetonitrile solution (3 mL) of H2L (0.0179 g, 0.05 mmol). The mixture was stirred in air for 30 min. It was filtered and kept in air. The transparent colorless single crystals suitable for X-ray crystallography were obtained on slow evaporation of the filtrate within 2 days. Anal. calcd for [Zn(H2L)2(ClO4)2]H2O: C 50.39, H 3.62, N 8.40; found: C 50.27, H 3.43, N 8.29; FTIR (KBr pellet, cm1): 3421 (br), 1655 (s), 1554 (s), 1513 (m), 1488 (m), 1325 (m), 1268 (m), 1121 (s), 1096 (vs). ESI-MS m/z [Zn(H2L)(ClO4)] + : 522.0.
Preparation of 2-((2-methylquinolin-8-yl)oxy)-N-(quinolin-8-yl)acetamide (2): Compound 1 (1.10 g, 5.00 mmol) and K2CO3 (1.38 g, 10 mmol) were added to a solution of 8-hydroxyquinadine (0.80 g, 5.00 mmol) in degassed CH3CN (100 mL). The reaction mixture was refluxed overnight. After removal of the insoluble materials by filtration, the filtrate was evaporated. The residue was dissolved in EtOAc (100 mL), and the organic layer was washed with water (2 100 mL) and brine (100 mL), dried over MgSO4, and concentrated. The crude residue was purified by chromatography (SiO2, EtOAc/ petroleum ether = 1:2) to give 2 as a white solid (1.55 g, 90 %). Mp: 160.1 161.5 8C. Anal. calcd for C20H17N3O2 : C 73.45, H 4.99, N 12.24; found: C 73.03, H 4.81, N 11.96; 1H NMR (400 MHz, CDCl3) d = 2.86 (s, 3 H), 5.03(s, 2 H), 7.19(d, J=7.6 Hz, 1 H), 7.32–7.43 (m, 4 H), 7.49–7.55 (m, 2 H), 8.02(d, J=8.0 Hz, 1 H), 8.10 (d, J = 8.0 Hz, 1 H), 8.78(d, J=2.4 Hz, 1 H), 8.87 (d, J = 7.2 Hz, 1 H), 11.25(s, 1 H); 13 C NMR (100 MHz, CDCl3) d = 25.67, 69.44, 111.20, 116.96, 121.29, 121.44, 122.10, 122.56, 125.41, 127.02, 127.72, 127.87, 133.92, 135.93, 138.85, 139.93, 148.35, 152.90, 158.21, 167.18; FT-IR (KBr pellet, cm1): 3315 (br), 1686 (s), 1534 (vs), 1505 (m), 1488 (m), 1426 (m),1384 (w), 1326 (m), 1236 (m), 1116 (m); ESI-MS m/z [(M + 1) + ]: 344.3.
Crystallographic studies
Preparation of 2-((2-formylquinolin-8-yl)oxy)-N-(quinolin-8-yl)acetamide (3): A solution of 2 (0.65 g, 1.88 mmol) in 1,4-dioxane (70 mL) was added in several portions to a vigorously stirred hot suspension of SeO2 (0.24 g, 2.1 mmol) in 1,4-dioxane (50 mL). The mixture was stirred for 10 h at approximately 80 8C, after which deposited selenium was filtered off and the solution was evaporated under reduced pressure. The crude residue was purified by chromatography (SiO2, EtOAc/petroleum ether = 1:1) to give 3 as a white solid (0.60 g, 90 %). Mp: 211.2–211.5 8C. Anal. calcd for C21H15N3O3 : C 70.58, H 4.23, N 11.76; found: C 70.72, H 4.09, N 11.54. 1H NMR (400 MHz, CDCl3) d = 5.07 (s, 2 H), 7.30 (dd, J = 0.8, 7.2 Hz, 1 H), 7.46 (dd, J = 4.4, 8.4 Hz, 1 H), 7.57–7.60 (m, 3 H), 7.64 (t, J = 8.4 Hz, 1 H), 8.13 (d, J = 8.4 Hz, 1 H), 8.17 (dd, J = 1.6, 8.4 Hz, 1 H), 8.34 (d, J = 8.4 Hz, 1 H), 8.75 (dd, J = 1.6, 4.0 Hz, 1 H), 8.89 (dd, J = 3.6, 5.6 Hz, 1 H), 10.41 (d, J = 0.8 Hz, 1 H), 11.29 (s, 1 H); 13C NMR (100 MHz, CDCl3) d = 69.09, 111.45, 117.22, 118.01, 121.32, 121.74, 122.44, 127.19, 128.05, 129.60, 131.39, 133.87, 136.23, 137.29, 138.91, 140.18, 148.48, 151.76, 154.02, 166.42, 193.95; FT-IR (KBr pellet, cm1): 3321 (m), 1707 (s), 1679 (s), 1615 (w), 1545 (vs), 1491 (m), 1426 (m), 1382 (m), 1322 (s), 1239 (m), 1112 (m); ESI-MS m/z [(M + 1) + ]: 358.3.
Single-crystal X-ray diffraction measurements were collected with an Agilent Technologies SuperNova diffractometer operating at 50 kV and 30 mA using Mo Ka radiation (l = 0.71073 ). Data collection and reduction were performed using CrysAlis PRO, Agilent Technologies, version 1.171.36.28. Empirical absorption correction using spherical harmonics, implemented in SCALE3 ABSPACK scaling algorithm. The structure was solved by direct methods and refined by full-matrix least-squares on F 2 using the SHELXL97 program package. Crystal data and details of the structure determination for H2L, [Cd(H2L)2(ClO4)2]H2O and [Zn(H2L)2(ClO4)2]H2O are summarized in the Supporting Information, Table S1. CCDC 998925, 956497, and 973060 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data request/cif.
Computational methods Theoretical calculations were carried out by using Gaussian 09 (Rev. A.02)[19] programs at Density Functional Theory (DFT) and TD-DFT levels. A double-& quality basis set consisting of Hay and Wadt’s effective core potentials (LANL2DZ)[20] was employed for the Zn and Cd atom[21] and a 6–31G* basis set[22] for the H, C, N, and O atoms.[23] TD-DFT calculation for the S0 !Sn transitions using the same functional and basis set were then performed based on the single-crystal structures at ground states. Typically, the lowest 100 singlet roots of the nonhermitian eigenvalue equations were obtained to determine the vertical excitation energies. Oscillator strengths were deduced from the dipole transition matrix elements (for singlet states only).
Preparation of 2-((2-(hydroxymethyl)quinolin-8-yl)oxy)-N-(quinolin-8-yl)acetamide (H2L): Excess solid NaBH4 (0.076 g, 2.0 mmol) was added to the methanol solution of 3 (0.60 g, 1.69 mmol) in portions and the mixture was stirred for 30 min at room temperature. After removal of the solvent, the residue was partitioned between CH2Cl2 and 0.1 m HCl. The organic layer was dried (Na2SO4) and concentrated. The crude product was purified by chromatography (SiO2, petroleum ether/EtOAc 1:5, v/v) to afford H2L as a white solid (0.43 g, 71 %). Mp: 167.3–167.9 8C. Anal. calcd. for C21H17N3O3 : C 70.18, H 4.77, N 11.69; found: C 69.12, H 4.69, N 11.36; 1H NMR (400 MHz, CDCl3) d = 4.89 (s, 2 H), 5.02 (s, 2 H), 7.12 (dd, J = 2.0, 6.4 Hz, 1 H), 7.31 (d, J = 8.4 Hz, 1 H), 7.42–7.49 (m, 3 H), 7.54 (d, J = 4.4 Hz, 2 H), 8.10 (d, J = 8.4 Hz, 1 H), 8.13 (dd, J = 1.6, 8.0 Hz, 1 H), 8.91 (t, J = 4.4 Hz, 1 H), 9.15 (dd, J = 1.6, 4.4 Hz, 1 H), 11.22 (s, 1 H);13C NMR (100 MHz, CDCl3) d = 64.25, 68.05, 110.16, 117.11, 119.03, 120.98, 121.68, 122.45, 126.19, 126.82, 127.94, 128.77, 133.63, 136.06, 136.63, 138.38, 138.79, 149.76, 152.33, 158.33, 166.36; FT-IR (KBr pellet, cm1): 3397 (br), 1672 (s), 1609 Chem. Eur. J. 2015, 21, 290 – 297
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Cell culture and two-photon fluorescence microscopy imaging SMMC-7721 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum in an atmosphere of 5 % CO2 and 95 % air at 37 8C humidified air for
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Full Paper 24 h. One day before imaging, the cells were passaged and plated in phenol red-free medium on glass coverslips in 24-well plates. H2L in the culture media (a solution of H2L in DMSO (0.01 m) was diluted into DMEM at 100 mm) was added to the cells, and the cells were incubated for 2 h at 37 8C. After washing twice with PBS buffer, the cells were further incubated with 100 mm Cd2 + for 1 h. For EDTA experiment, the cells incubated with H2L and Cd2 + were further treated with 200 mm EDTA for 30 min. The treated cells were imaged by a two-photon fluorescence microscopy (Olympus FV 1000 MPE) with a mode-locked laser source set at wavelength 900 nm.
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Acknowledgements This work was supported by the NSFC (Grant 20931003, 91122007, 21301080) and the Specialized Research Fund for the Doctoral Program of Higher Education (Grant No. 20110211130002). Keywords: cadmium · fluorescence · intramolecular electron transfer · ratiometry · semirigid structures [1] Agency for Toxic Substances and Disease Registry, 4770 Buford Hwy NE, Atlanta, GA 30341. http://www.atsdr.cdc.gov/cercla/07 List.html. [2] a) T. Jin, J. Lu, M. Nordberg, Neurotoxicology 1998, 19, 529 – 535; b) M. P. Waalkes, Mutat. Res. 2003, 533, 107 – 120; c) M. Waisberg, P. Joseph, B. Hale, D. Beyersmann, Toxicology 2003, 192, 95 – 117. [3] R. Peng, F. Wang, Y. Sha, Molecules 2007, 12, 1191 – 1201. [4] a) H. N. Kim, W. X. Ren, J. S. Kim, J. Yoon, Chem. Soc. Rev. 2012, 41, 3210 – 3244; b) M. Vendrell, D. Zhai, J. C. Er, Y.-T. Chang, Chem. Rev. 2012, 112, 4391 – 4420; c) M. C. Aragoni, M. Arca, A. Bencini, C. Caltagirone, A. Garau, F. Isaia, M. E. Light, V. Lippolis, C. Lodeiro, M. Mameli, R. Montis, M. C. Mostallino, A. Pintus, S. Puccioni, Dalton Trans. 2013, 42, 14516 – 14530. [5] M. Mameli, M. C. Aragoni, M. Arca, C. Caltagirone, F. Demartin, G. Farruggia, G. De Filippo, F. A. Devillanova, A. Garau, F. Isaia, V. Lippolis, S. Murgia, L. Prodi, A. Pintus, N. Zaccheroni, Chem. Eur. J. 2010, 16, 919 – 930. [6] a) Y. Pourghaz, P. Dongare, D. W. Thompson, Y. Zhao, Chem. Commun. 2011, 47, 11014 – 11016; b) H. Tian, B. Li, H. Wang, Y. Li, J. Wang, S. Zhao, J. Zhu, Q. Wang, W. Liu, X. Yao, Y. Tang, J. Mater. Chem. 2011, 21, 10298 – 10303; c) W. Wang, Q. Wen, Y. Zhang, X. Fei, Y. Li, Q. Yang, X. Xu, Dalton Trans. 2012, 41, 1827 – 1833; d) X. Zhou, P. Li, Z. Shi, X. Tang, C. Chen, W. Liu, Inorg. Chem. 2012, 51, 9226 – 9231; e) Q. Zhao, R.-F. Li, S.-K. Xing, X.-M. Liu, T.-L. Hu, X.-H. Bu, Inorg. Chem. 2011, 50, 10041 – 10046; f) T. Cheng, Y. Xu, S. Zhang, W. Zhu, X. Qian, L. Duan, J. Am. Chem. Soc. 2008, 130, 16160 – 16161; g) T. Gunnlaugsson, T. C. Lee, R. Parkesh, Org. Lett. 2003, 5, 4065 – 4068; h) Y. Yang, T. Cheng, W. Zhu, Y. Xu, X. Qian, Org. Lett. 2011, 13, 264 – 267; i) W. Liu, L. Xu, R. Sheng, P. Wang, H. Li, S. Wu, Org. Lett. 2007, 9, 3829 – 3832; j) X.-L. Tang, X.-H. Peng, W. Dou, J. Mao, J.-R. Zheng, W.-W. Qin, W.-S. Liu, J. Chang, X.-J. Yao, Org. Lett. 2008, 10, 3653 – 3656; k) L. Xue, C. Liu, H. Jiang, Org. Lett. 2009, 11, 1655 – 1658. [7] a) X. Zhou, B. Yu, Y. Guo, X. Tang, H. Zhang, W. Liu, Inorg. Chem. 2010, 49, 4002 – 4007; b) Q. Zhao, F. Li, C. Huang, Chem. Soc. Rev. 2010, 39, 3007 – 3030. [8] a) Y. Wang, Y. Cui, R. Liu, Y. Wei, X. Jiang, H. Zhu, L. Gao, Y. Zhao, Z. Chai, X. Gao, Chem. Commun. 2013, 49, 10724 – 10726; b) J. F. Zhang, C. S. Lim, S. Bhuniya, B. R. Cho, J. S. Kim, Org. Lett. 2011, 13, 1190 – 1193.
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Received: July 2, 2014 Published online on October 24, 2014
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& Fluorescent Probes
A Highly Selective Two-Photon Fluorescent Probe for Detection of Cadmium(II) Based on Intramolecular Electron Transfer and its Imaging in Living Cells Zhaohua Shi,[a] Qingxin Han,[a] Lizi Yang,[a] Huan Yang,[a] Xiaoliang Tang,[a] Wei Dou,[a] Zhiqi Li,[a] Yange Zhang,[a] Yongliang Shao,[a] Liping Guan,[b] and Weisheng Liu*[a]
Abstract: A new quinoline-based probe was designed that shows one-photon ratiometric and two-photon off–on changes upon detecting Cd2 + . It exhibits fluorescence emission at 407 nm originating from quinoline groups in Tris-HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v). Coordination with Cd2 + causes quenching of the emission at 407 nm and simultaneously yields a remarkable redshift of the emission maximum to 500 nm with an isoemissive point at 439 nm owing to an intramolecular charge-transfer mechanism. Thus, dual-emission ratiometric measurement with a large redshift (Dl = 93 nm) and significant changes in the ratio (F500/F439) of the emission intensity (R/R0 up to 27) is estab-
lished. Moreover, the sensor H2L displays excellent selectivity response, high sensitive fluorescence enhancement, and strong binding ability to Cd2 + . Coordination properties of H2L towards Cd2 + were fully investigated by absorption/fluorescence spectroscopy, which indicated the formation of a 2:1 H2L/Cd2 + complex. All complexes were characterized by X-ray crystallography, and TD-DFT calculations were performed to understand the origin of optical selectivity shown by H2L. Two-photon fluorescence microscopy experiments have demonstrated that H2L could be used in live cells for the detection of Cd2 + .
Introduction
Therefore, it is of great significance to develop excellent methods for the quantification of Cd2 + in environment or living cells. As one of powerful means and techniques for detecting metal ions, fluorescent sensors have attracted increasing attention in recent years because of simplicity, high sensitivity and selectivity, instantaneous response, and in vivo applications, which convert interaction of the target species with the receptor unit into an optical signal changing.[4] The main challenge of fluorescent sensors for Cd2 + is to discriminate it from Zn2 + . As a result of being in the same group of the periodic table and closed-shell d10 electronic configuration, they usually possess similar chemical behaviors, including similar spectral changes after interactions with fluorescent sensors.[5] So far, a few fluorescent Cd2 + probes have been reported, and most of them are intensity-based. As is well-known, the emission intensity is dependent on many other factors, such as emission collection efficiency, sample environment, sensor concentration, bleaching, optical path length, and illumination intensity.[5, 6] In contrast, a ratiometric sensor exhibits spectral shift in absorption and emission spectra upon binding to the analyte of interest, and the ratio of emission intensities at two different wavelengths can be used to evaluate the analyte concentration, thus reducing the interference induced by the unknown local sensor concentration and the deviations in detecting conditions.[7] Moreover, two-photon (TP) probes that can selectively detect Cd2 + are very rare, which utilize two photons of lower energy for the excitation and have evolved into a widely used method in biomedical research.[8] To date, sever-
Cadmium, an important heavy and transition metal, is widely used in many industrial and agricultural processes, thus resulting in serious pollution to air, water, and soil. It is one of the highly toxic metals and listed seventh on the Top 20 Hazardous Substances Priority List by the Agency for Toxic Substances and Disease Registry and US Environmental Protection Agency (EPA).[1] Cd2 + can accumulate in ecosystems and humans owing to its long biological half-life (15–20 years for humans) through the soil-plant-animal-human food chain and inhaling of cigarette smoke.[2] Either short-term or long-term exposure to Cd2 + can lead to mutations and cancer, such as renal dysfunction, calcium metabolism disorders, and pulmonary, prostatic, and renal cancer.[3] Nevertheless, cellular Cd2 + uptake and carcinogenic mechanisms are still poorly understood. [a] Dr. Z. Shi, Q. Han, Dr. L. Yang, H. Yang, Dr. X. Tang, Dr. W. Dou, Z. Li, Y. Zhang, Y. Shao, Prof. Dr. W. Liu Key Laboratory of Nonferrous Metals Chemistry and Resources Utilization of Gansu Province and State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering Lanzhou University, Lanzhou 730000 (P. R. China) Fax: (+ 86) 931-8912582 E-mail: [email protected] [b] L. Guan School of Life Sciences Lanzhou University, Lanzhou 730000 (P. R. China) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201404224. Chem. Eur. J. 2015, 21, 290 – 297
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Full Paper al ratiometric Cd2 + sensors have been developed and successfully used in biological applications recently.[9] Chemists still need to design novel methods that can quantify Cd2 + and overcome limitations, including poor selectivity and water solubility, small spectral shift, or operation at non-physiological pH. In our previous research, we have successfully designed and synthesized HL, a combination of 8-HQ or 8-AQ, which showed a bright “switch-on” optical response based on the photoinduced electron-transfer (PET) mechanism upon binding Cd2 + .[6d] However, recognition of Cd2 + using HL must be performed in absolute ethanol and easily suffers from interference from anions. These disadvantages encouraged us to improve sensing properties by incorporating with auxiliary receptors. According to the ionic radius and the limitation of coordination numbers, we hope that introducing a hydroxymethyl at the 2-position of 8-HQ would generate a novel sensor with high selectivity and sensitivity for Cd2 + . Herein, we present our design and synthesis of sensor H2L, featuring a modified 8-HQ. As expected, metal-ion complexation and accompanying selectivity of H2L is enhanced. H2L showed redshifts in both emission and absorption wavelength upon binding Cd2 + that is based on the ICT mechanism in aqueous media. Furthermore, we carefully studied its complexation with Cd2 + by fluorescence/absorption titration, X-ray crystallography, and TD-DFT calculations. Above all, the complex of H2L with Cd2 + exhibits a large two-photon absorption cross section at 700 nm and TP fluorescence imaging have demonstrated that H2L is membrane-permeable and can detect intracellular Cd2 + .
X-ray crystallography was obtained by slow evaporation from a CH3CN/CH3OH solution. All of the complexes were fully characterized by 1H NMR, 13C NMR, ESI-MS, FTIR, and elemental analysis or X-ray diffraction measurement. Spectroscopic measurements of H2L were performed under physiological pH conditions (Tris-HCl, 25 mm, pH 7.40, H2O/ EtOH 8:2, v/v). The UV/Vis spectrum of H2L exhibits an absorption maximum at 302 nm at room temperature, which can be assigned to the p–p* transitions of the quinoline groups (Figure 1).[6d] When titrated by Cd(ClO4)2 (0–2.0 equiv), the absorb-
Figure 1. UV/Vis spectral changes of 0.10 mm H2L upon addition of Cd2 + (0– 0.3 mm) in Tris-HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v) at room temperature. Inset: Absorbance changes at * 302 and * 342 nm as a function of the Cd2 + concentration, indicating the 2:1 stoichiometry for H2L-Cd2 + .
Results and Discussion ance of H2L at 302 nm gradually decreased with an increasing concentration of Cd2 + (Figure 1). Moreover, a new absorption band appeared at 342 nm with a distinct isosbestic point at 325 nm, suggesting chelation-elicited effective ICT. The absorbance at 342 and 302 nm changed linearly with the concentration of Cd2 + up to a molar ratio (H2L/Cd2 + ) of 2:1, and there was saturation at even higher [Cd2 + ] (Figure 1, inset). Upon addition of Zn2 + , similar changes in the absorption spectrum were observed (Supporting Information, Figure S1), indicating the similar coordination pattern between H2L-Cd2 + and H2LZn2 + . These indicate the 2:1 binding stoichiometry between H2L and Cd2 + , Zn2 + , which was further confirmed by Job’s plots (Supporting Information, Figures S2 and S3), fluorescence titrations, and crystal structures. The optical properties of H2L are mainly dominated by the quinoline platforms. We investigated its fluorescence properties in the presence of common metal ions such as Li + , Na + , K + , Ca2 + , Mg2 + , Al3 + , Cr3 + , Mn2 + , Fe3 + , Co2 + , Ni2 + , Cu2 + , Ag + , Zn2 + , Cd2 + , and Hg2 + in Tris—HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v). As shown in Figure 2, the free sensor H2L displayed an emission maximum at around 407 nm upon excitation at 325 nm. Physiologically important metal ions and most heavy and transition-metal ions, including Li + , Na + , K + , Ca2 + , Mg2 + , Cr3 + , Ni2 + , Mn2 + , Hg2 + , Ag + , and Al3 + , induced negligible changes in the emission profiles. Fe3 + , Co2 + , and Cu2 +
The synthesis of H2L is shown in Scheme 1. Compound 2 was synthesized by the reaction of 8-hydroxyquinaldine with 1 using anhydrous potassium carbonate in refluxing CH3CN. Oxidation of active methyl group of 2 to formyl with SeO2 followed by reduction using NaBH4 afforded H2L. Significantly, the metal complex of [Cd(H2L)2(ClO4)2]H2O was obtained through the reaction of H2L with Cd(ClO4)2. A suitable single crystal for
Scheme 1. Synthesis of H2L. Chem. Eur. J. 2015, 21, 290 – 297
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Full Paper arly with the total concentration of Cd2 + up to a mole ratio (H2L/Cd2 + ) of 2:1 (Figure 3, inset). Job’s plot and ESI-MS (m/z [Cd(H2L)2(ClO4)] + 930.9) also confirmed the binding stoichiometry of 2:1 between H2L and Cd2 + . Moreover, introduction of Cd2 + turned the visual emission of the sensor from blue to green (Supporting Information, Figure S4), which further supports the ratiometric fluorescence response. Based on the fluorescence titration studies, the association constant (log K) of H2L for Cd2 + was calculated to be 10.86 using Thordarson’s fitting program,[10] suggesting the strong binding ability of H2L with Cd2 + . The method detection limits (MDL) was measured to be 23.63 nm,[11] which is lower than the maximum limit (45 nm) for drinking water emphasized by The US Environmental Protection Agency (EPA).[12] Furthermore, the direct proof experiment for the trace Cd2 + determination was performed (Supporting Information, Figure S5). To establish the practical application of H2L, we carried out an experiment to monitor residual Cd2 + in a reactor using H2L. A CH3CN (10 mL) solution of Cd(ClO4)2·6H2O (4.2 mg) was stirred in three flasks for two days at room temperature. After the solution was poured out, the three flasks were treated with different washing procedures respectively (washing with H2O (1 10 mL); washing with H2O (3 10 mL); and washing with H2O (3 10 mL) and CH3OH (3 10 mL)). Subsequently, H2L solution (Tris-HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v)) was added into the three flasks and R was determined to be 3.23, 1.68, and 1.06 respectively. The results demonstrate that our detection system could function well in Cd2 + analysis in environmental samples. The sensing process of Cd2 + by H2L was found to be reversible. This was demonstrated by the addition of an ethylenediaminetetraacetic acid (EDTA) aqueous solution to the glowing H2L-Cd2 + solution, which can almost recover the original emission of H2L (Supporting Information, Figure S6). These indicate that H2L is an excellent ratiometric fluorescent sensor with high sensitivity and reversibility. In the selectivity experiment, Zn2 + also showed some ratiometric changes as similar as that of Cd2 + (Figure 2). To understand the distinction between the interaction of H2L with Cd2 + and Zn2 + , we also investigated the fluorescence titration spectra of Zn2 + (0–1.2 equiv) to H2L (Figure 4). Similarly, the emission spectrum showed a redshift to 495 nm with an isoemissive point at 465 nm. Therefore, it may be difficult to avoid the interference of Zn2 + for detecting trace Cd2 + as there are some obvious overlap in the fluorescent spectra for the Cd2 + and Zn2 + determinations as shown in Figure 3 and Figure 4. Efforts are under way to design and synthesize analogues, which can be better used for detecting trace Cd2 + in the water media. Job’s plot (Supporting Information, Figure S3) and crystal structure (see below) confirmed the binding stoichiometry of 2:1 between H2L and Zn2 + . The binding constant (log K) of H2L for Zn2 + was determined to be 6.98 using a fitting program, which was much less than that for Cd2 + (10.86).[10] These results imply that highly selective fluorescent response may arise from selective combination. The structures of the Cd2 + and Zn2 + complexes with H2L are shown in Figure 5 and the Supporting Information, Figure S8, respectively. Selected bond lengths and bond angles are given
Figure 2. Fluorescence emission spectra of 50 mm H2L in the presence of different metal ions (1 mm for s-block and 0.1 mm for d-block metal ions) in Tris-HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v). lex = 325 nm. Inset: Visual fluorescent photographs of H2L-Zn2 + , H2L, and H2L-Cd2 + in Tris-HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v). The photographs were taken under a handheld UV/Vis (365 nm) lamp.
quenched the fluorescence in varied degrees, which always meet in the other metal ion sensors. Interestingly, on addition of Cd2 + , the emission band at 407 nm had disappeared with the appearance of a new striking increased band at 500 nm. Although Zn2 + could induce similar redshifts of fluorescence spectra, the enhancements in fluorescence intensity at 495 nm are negligible compared with that induced by Cd2 + . These results indicate that H2L shows excellent selectivity for Cd2 + in aqueous media. The fluorescence titration spectra of Cd2 + to H2L (50 mm) are displayed in Figure 3. Addition of gradually increasing concentrations of Cd2 + caused a significant effect on the emission profile: the emission intensity at 407 nm gradually decreased, and simultaneously, a new peak appeared at 500 nm with a large redshift of 93 nm and dramatically enhanced intensity, which can be attributed to the ICT structure in the excited state of the H2L-Cd2 + complex.[9a] A well-defined isoemissive point at 439 nm was noted. The emission ratio at 500 and 439 nm (F500/F439) increased by about 27-fold and changed line-
Figure 3. Fluorescence emission spectra of 50 mm H2L upon addition of Cd2 + (0–0.2 mm) in Tris-HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v) at room temperature. lex = 325 nm. Inset: Ratio (F500/F439) changes as a function of the Cd2 + concentration, indicating the 2:1 stoichiometry for H2L-Cd2 + . Chem. Eur. J. 2015, 21, 290 – 297
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Full Paper square-bipyramidal coordination geometry. These results imply that H2L would grasp Cd2 + better than Zn2 + . The larger Cd2 + (ionic radii r + = 0.96 for Cd2 + and r + = 0.74 for Zn2 + ) may be more suitable for the binding pocket of H2L, resulting in a high affinity for Cd2 + .[9b, 15] It has been noted that five-membered chelate rings favor the complexation of larger metal ions, whereas six-membered chelate rings favor the complexation of smaller metal ions.[15] The new emission band (500 nm) of H2L-Cd2 + may arise from two effects. First, after binding Cd2 + , the intramolecular hydrogen bond (N···H···O) is broken, and the radiationless relaxation is forbidden.[16] Second, the coordination of Cd2 + to 8-HQ induces an intramolecular electron transfer process (ICT) from the nitrogen atom of the heterocycle to the metal ion. Consequently, an obvious redshift in both emission and absorption wavelength could be observed. Time-dependent density functional theory (TD-DFT) calculations were performed to clarify the Zn2 + /Cd2 + optical discrimination by H2L. As illustrated in Figure 6 a, the absorption band at about 302 nm of H2L was calculated at 324 nm, which was assigned to the S0!S2 monoelectronic vertical transition. This transition corresponds to HOMO!LUMO + 1 (f = 0.1202).
Figure 4. Fluorescence emission spectra of 50 mm H2L upon addition of Zn2 + in Tris-HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v) at room temperature. lex = 325 nm. Inset: Ratio (F500/F439) changes as a function of the Zn2 + concentration.
Figure 5. ORTEP (ellipsoids set at 30 % probability) of [Cd(H2L)2(ClO4)2]H2O. Hydrogen atoms, disordered perchlorate, and solvent molecules are omitted for clarity.
in the Supporting Information, Table S2 and S3. They crystallize in the monoclinic system from methanol/acetonitrile solvent, with space group P2/n and P2/c, respectively. We can see that H2L chelates Cd2 + with 2:1 stoichiometry, and the asymmetric unit consists of H2L and half of Cd2 + . Cd2 + is eight-coordinate and surrounded by two carbonyl oxygen atoms, two 8-HQ oxygen atoms, two 8-HQ nitrogen atoms, and two hydroxymethyl oxygen atoms from two H2L ligands, which forms three five-membered chelate rings with each one H2L ligand. The bond length of Cd1N3 is 2.322 (4) , and the Cd1O distance ranges from 2.367 (3) to 2.498 (3) (Supporting Information, Table S2), which are within the reported extent (2.26–2.52 for CdN and 2.20–2.59 for CdO).[13] In Zn(H2L)2(ClO4)2 as demonstrated in the Supporting Information, Figure S8, although Zn2 + adopts coordination conformation similar to Cd2 + in Cd(H2L)2(ClO4)2, Zn1O2 distance is 2.464(3) and it is beyond the reported bond length range (1.95–2.37 for ZnO).[14] Consequently, Zn2 + is six-coordinate and forms a distorted Chem. Eur. J. 2015, 21, 290 – 297
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Figure 6. Interface plots of the molecular orbitals calculated for respective compounds in the gas phase involved in the principal singlet vertical electronic transitions evaluated by TD-DFT: a) H2L, b) H2L-Cd2 + complex, c) H2LZn2 + complex. Black and gray parts on molecular orbitals refer to the different phases of the molecular wave functions, where the isovalue is 0.02 au.
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Full Paper For [Cd(H2L)2]2 + (Figure 6 b), the two experimental absorption bands at about 342 nm and 302 nm were calculated at 334 nm (HOMO1!LUMO, f = 0.0772, p–p*) and 309 nm (HOMO!LUMO + 1, f = 0.3106), respectively; these transitions involve ICT processes within the two ligands. While in the case of [Zn(H2L)2]2 + (Figure 6 c), the two experimental absorption bands were calculated at 319 nm (HOMO1!LUMO + 1, f = 0.0943) and 308 nm (HOMO!LUMO + 1, f = 0.3189, p–p*), respectively. Some of differences between experimental and theoretical absorption bands are perhaps caused by the solvent effects. These results strongly imply that the optical selectivity shown by H2L arises from different types of transition process among the respective compounds of H2L with metal ions.[17] To further understand the nature of interactions between sensor H2L and Cd2 + , 1H NMR titration was carried out (Figure 7). It can be seen that the NMR signals of methylene (CH2(a) and CH2(b)) and hydroxy protons of H2L are significant-
Figure 8. Selectivity of H2L for Cd2 + in the presence of other metal ions in Tris-HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v). lex = 325 nm. Black bars represent the addition of an excess of the appropriate metal ion (1 mm for Li + , Na + , K + , Ca2 + , Mg2 + ; 0.1 mm for all other metal ions) to a 50 mm solution of H2L. White bars represent the subsequent addition of 0.1 mm Cd(ClO4)2(H2O)6 to the solution.
ure S11, the fluorescence intensity of H2L-Cd2 + is hardly interfered with by these anions. Along with excellent selectivity for practical applications, it is essential that the probe can be operated in the physiological pH range. Therefore, we investigated the pH effect on the fluorescence response of H2L in the absence and presence of Cd2 + . The emission peak of H2L is observed at approximately 406 nm at neutral pH, and no dramatic change is expected within a broad pH range of 4.0–12.0.[18] At pH 13, owing to the deprotonation of the NH moiety of the aminoquinoline, a new redshifted emission band around 486 nm with slight enhancements could be observed (Supporting Information, Figure S12). The complexation of Cd2 + induces a fluorescence-observable increase of H2L at 500 nm and decrease at 407 nm within a pH range of 5–13. As can be seen from the Supporting Information, Figure S13, the fluorescence intensity ratio (F500/F439) of Cd2 + -H2L continues increasing in the pH 4.0–6.0 range, which may be due to the competition of H + . Moreover, H2L and the Cd2 + -H2L complex showed a stable F500/ F439 ratio under neutral and alkaline conditions. These results demonstrated that H2L can be used in complex system (environment samples or living systems) as a ratiometric fluorescent probe for Cd2 + without interference from other cations, anions, and pH effects. Having established the utility of H2L by one-photon spectroscopy, we have investigated the two-photon properties of H2L and its complexes using the TP induced fluorescence measurement technique. As shown in Figure 9, H2L shows no obvious two-photon action cross-section, and Zn2 + can induce weak enhancement in TP action cross-section around 700 nm. However, its Cd2 + complex has satisfactory efficiency in TP sensitization at 700 nm. This indicates the capability of H2L for detecting Cd2 + by the TP fluorescence. Figure 10 shows that changes in the TP excited fluorescence spectra (lex = 700 nm) of H2L titration with Cd2 + are qualitatively similar to those of corresponding one-photon excited fluorescence spectra. The fluorescence intensity at 515 nm dramatically enhanced with increasing concentrations of Cd2 + and increases linearly with
Figure 7. 1H NMR spectra (400 MHz) of H2L and H2L-Cd2 + in CD3CN.
ly shifted to the down-field direction upon titration with Cd2 + ion, indicating that 8-HQ is metal ion receptor to effectively interact with Cd2 + . However, addition of Cd2 + induces a marked up-field shift of the NH signal from 11.14 to 10.68, suggesting the increase of electron density of 8-AQ in the H2L-Cd2 + system. This is due to the effective ICT process upon binding Cd2 + . Owing to similar coordination patterns of H2L-Zn2 + and H2L-Cd2 + , 1H NMR signal changes of H2L upon addition of Zn2 + ion are similar to that of Cd2 + (Supporting Information, Figure S9). The emission response of H2L with different metal ions revealed a remarkable selectivity for Cd2 + (Figure 2). However, the most important criterion for a selective sensor is the ability to detect a specific species in a complex system. Therefore, we investigated the stability of the H2L-Cd2 + complex in the presence of other metal ions in Tris—HCl (25 mm, pH 7.20), H2O/ EtOH (8:2, v/v; Figure 8; Supporting Information; Figure S10). Most representative metal ions have little effect on the fluorescence of H2L for Cd2 + detection. Owing to competition coordination, Fe3 + slightly quenched the fluorescence of H2L-Cd2 + but clearly detectable (Supporting Information, Figure S10). The anion responses to the detection systems were further investigated. As shown in the Supporting Information, FigChem. Eur. J. 2015, 21, 290 – 297
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Figure 9. Two-photon excitation spectra of H2L with and without Cd2 + /Zn2 + in Tris-HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v).
Figure 11. TP image of SMMC-7721 cells. lex = 900 nm. a) Bright-field transmission image of cells incubated with 100 mm H2L for 2 h at 37 8C; b) TP image of the cells shown in (a); c) an overlay image of (a) and (b); d) brightfield transmission image of cells supplemented with 100 mm H2L for 2 h and then further incubated with 100 mm Cd(NO3)2 for 1 h at 37 8C; e) TP image of the cells shown in (d); f) an overlay image of (d) and (e); g) bright-field transmission image of cells that after treatment with H2L and Cd(NO3)2 and subsequent treatment with 200 mm EDTA for 30 min at 37 8C. h) TP image of the cells shown in (g); i) an overlay image of (g) and (h). Scale bars: 10 mm.
Cd2 + were further treated with EDTA (200 mm for 30 min) that decreases the level of Cd2 + , it showed a weaker fluorescent signal (Figure 11 g–i). These cell experiments show that H2L is cell-membrane-permeable and can be used to monitor Cd2 + reversibly in vivo.
Figure 10. Two-photon emission spectra of 50 mm H2L upon addition of Cd2 + (0–0.2 mm) in Tris-HCl (25 mm, pH 7.40), H2O/EtOH (8:2, v/v) at room temperature. lex = 700 nm. Inset: 1) Enlarged figure from 385 nm to 415 nm of two-photon emission spectra; 2) emission intensity (515 nm) changes as a function of the Cd2 + concentration.
Conclusion
the total concentration of Cd2 + up to a mol ratio (H2L/Cd2 + ) of 2:1 (Figure 10, inset), indicating the capability of H2L for quantitatively detecting Cd2 + by the TP fluorescence. Therefore, on the basis of 2:1 stoichiometry and TP fluorescence titration data, the association constant (log K) of with Cd2 + was calculated to be 10.69, which is comparable to that based on onephoton excited fluorescence titration spectra. Because of extremely weak two-photon action cross section, H2L shows no obvious TP fluorescence, so it showed two-photon off–on changes for Cd2 + . In vitro studies demonstrated the ability of H2L to detect Cd2 + with excellent properties. We next sought to investigate the utility of H2L for monitoring the intracellular (living SMMC7721 cells) exogenous Cd2 + under two-photon excitation. SMMC-7721 cells treated with 100 mm H2L alone in the growth medium for 2 h at 37 8C gave a very weak fluorescence (Figure 11 a–c). However, the cells were subsequently incubated with Cd2 + (100 mm) for another 1 h and it displayed green fluorescence (Figure 11 d–f). When the cells exposed to H2L and Chem. Eur. J. 2015, 21, 290 – 297
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We have successfully designed and synthesized a ratiometric fluorescent sensor (H2L) for Cd2 + , and evaluated the Cd2 + fluorescence sensing and binding properties of H2L in aqueous media. It shows high selectivity and sensitivity for Cd2 + , reversible fluorescent response, as well as two-photon absorption upon binding with Cd2 + . The sensor provides dual-emission ratiometric detection of Cd2 + with a significant red-shift in emission and remarkable changes in the ratio (F500/F439) of the emission intensity. Two-photon imaging demonstrates that H2L is membrane-permeable and can monitor the Cd2 + in living cells. The Cd2 + selective fluorescence response is attributed to the enhanced selective binding ability of H2L by auxiliary receptor. Introducing of auxiliary binding site can prevent anion from coordinating and enhance the binding ability and selectivity of ligands toward analytes. The special size selectivity may give some insight into how to construct fluorescent sensors with special properties, which is very useful for probe design in the future. 295
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(w), 1539 (vs), 1485 (s), 1428 (m), 1321 (w), 1254 (m), 1114 (m), 1064 (m); ESI-MS: m/z [(M + 1) + ]: 360.3.
Synthesis of H2L and complexes
Synthesis of [Cd(H2L)2(ClO4)2]H2O: A methanol solution (8 mL) of Cd(ClO4)2·6H2O (0.0210 g, 0.05 mmol) was added to a magnetically stirred acetonitrile solution (5 mL) of H2L (0.0179 g, 0.05 mmol). The mixture was stirred in air for 30 min. It was filtered and kept in air. The transparent colorless single crystals suitable for X-ray crystallography were obtained on slow evaporation of the filtrate within 2 days. Anal. calcd for [Cd(H2L)2(ClO4)2]H2O: C 48.13, H 3.46, N 8.02; found: C 48,03, H 3.27, N 7.97; FTIR (KBr pellet, cm1): 3375 (br), 1663 (s), 1550 (vs), 1484 (m), 1434 (m), 1325 (m), 1265 (m), 1126 (s), 1095 (vs). ESI-MS m/z [Cd(H2L)2(ClO4)] + : 930.9. Synthesis of [Zn(H2L)2(ClO4)2]H2O: A methanol solution (8 mL) of Zn(ClO4)2·6H2O (0.0286 g, 0.05 mmol) was added to a magnetically stirred acetonitrile solution (3 mL) of H2L (0.0179 g, 0.05 mmol). The mixture was stirred in air for 30 min. It was filtered and kept in air. The transparent colorless single crystals suitable for X-ray crystallography were obtained on slow evaporation of the filtrate within 2 days. Anal. calcd for [Zn(H2L)2(ClO4)2]H2O: C 50.39, H 3.62, N 8.40; found: C 50.27, H 3.43, N 8.29; FTIR (KBr pellet, cm1): 3421 (br), 1655 (s), 1554 (s), 1513 (m), 1488 (m), 1325 (m), 1268 (m), 1121 (s), 1096 (vs). ESI-MS m/z [Zn(H2L)(ClO4)] + : 522.0.
Preparation of 2-((2-methylquinolin-8-yl)oxy)-N-(quinolin-8-yl)acetamide (2): Compound 1 (1.10 g, 5.00 mmol) and K2CO3 (1.38 g, 10 mmol) were added to a solution of 8-hydroxyquinadine (0.80 g, 5.00 mmol) in degassed CH3CN (100 mL). The reaction mixture was refluxed overnight. After removal of the insoluble materials by filtration, the filtrate was evaporated. The residue was dissolved in EtOAc (100 mL), and the organic layer was washed with water (2 100 mL) and brine (100 mL), dried over MgSO4, and concentrated. The crude residue was purified by chromatography (SiO2, EtOAc/ petroleum ether = 1:2) to give 2 as a white solid (1.55 g, 90 %). Mp: 160.1 161.5 8C. Anal. calcd for C20H17N3O2 : C 73.45, H 4.99, N 12.24; found: C 73.03, H 4.81, N 11.96; 1H NMR (400 MHz, CDCl3) d = 2.86 (s, 3 H), 5.03(s, 2 H), 7.19(d, J=7.6 Hz, 1 H), 7.32–7.43 (m, 4 H), 7.49–7.55 (m, 2 H), 8.02(d, J=8.0 Hz, 1 H), 8.10 (d, J = 8.0 Hz, 1 H), 8.78(d, J=2.4 Hz, 1 H), 8.87 (d, J = 7.2 Hz, 1 H), 11.25(s, 1 H); 13 C NMR (100 MHz, CDCl3) d = 25.67, 69.44, 111.20, 116.96, 121.29, 121.44, 122.10, 122.56, 125.41, 127.02, 127.72, 127.87, 133.92, 135.93, 138.85, 139.93, 148.35, 152.90, 158.21, 167.18; FT-IR (KBr pellet, cm1): 3315 (br), 1686 (s), 1534 (vs), 1505 (m), 1488 (m), 1426 (m),1384 (w), 1326 (m), 1236 (m), 1116 (m); ESI-MS m/z [(M + 1) + ]: 344.3.
Crystallographic studies
Preparation of 2-((2-formylquinolin-8-yl)oxy)-N-(quinolin-8-yl)acetamide (3): A solution of 2 (0.65 g, 1.88 mmol) in 1,4-dioxane (70 mL) was added in several portions to a vigorously stirred hot suspension of SeO2 (0.24 g, 2.1 mmol) in 1,4-dioxane (50 mL). The mixture was stirred for 10 h at approximately 80 8C, after which deposited selenium was filtered off and the solution was evaporated under reduced pressure. The crude residue was purified by chromatography (SiO2, EtOAc/petroleum ether = 1:1) to give 3 as a white solid (0.60 g, 90 %). Mp: 211.2–211.5 8C. Anal. calcd for C21H15N3O3 : C 70.58, H 4.23, N 11.76; found: C 70.72, H 4.09, N 11.54. 1H NMR (400 MHz, CDCl3) d = 5.07 (s, 2 H), 7.30 (dd, J = 0.8, 7.2 Hz, 1 H), 7.46 (dd, J = 4.4, 8.4 Hz, 1 H), 7.57–7.60 (m, 3 H), 7.64 (t, J = 8.4 Hz, 1 H), 8.13 (d, J = 8.4 Hz, 1 H), 8.17 (dd, J = 1.6, 8.4 Hz, 1 H), 8.34 (d, J = 8.4 Hz, 1 H), 8.75 (dd, J = 1.6, 4.0 Hz, 1 H), 8.89 (dd, J = 3.6, 5.6 Hz, 1 H), 10.41 (d, J = 0.8 Hz, 1 H), 11.29 (s, 1 H); 13C NMR (100 MHz, CDCl3) d = 69.09, 111.45, 117.22, 118.01, 121.32, 121.74, 122.44, 127.19, 128.05, 129.60, 131.39, 133.87, 136.23, 137.29, 138.91, 140.18, 148.48, 151.76, 154.02, 166.42, 193.95; FT-IR (KBr pellet, cm1): 3321 (m), 1707 (s), 1679 (s), 1615 (w), 1545 (vs), 1491 (m), 1426 (m), 1382 (m), 1322 (s), 1239 (m), 1112 (m); ESI-MS m/z [(M + 1) + ]: 358.3.
Single-crystal X-ray diffraction measurements were collected with an Agilent Technologies SuperNova diffractometer operating at 50 kV and 30 mA using Mo Ka radiation (l = 0.71073 ). Data collection and reduction were performed using CrysAlis PRO, Agilent Technologies, version 1.171.36.28. Empirical absorption correction using spherical harmonics, implemented in SCALE3 ABSPACK scaling algorithm. The structure was solved by direct methods and refined by full-matrix least-squares on F 2 using the SHELXL97 program package. Crystal data and details of the structure determination for H2L, [Cd(H2L)2(ClO4)2]H2O and [Zn(H2L)2(ClO4)2]H2O are summarized in the Supporting Information, Table S1. CCDC 998925, 956497, and 973060 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data request/cif.
Computational methods Theoretical calculations were carried out by using Gaussian 09 (Rev. A.02)[19] programs at Density Functional Theory (DFT) and TD-DFT levels. A double-& quality basis set consisting of Hay and Wadt’s effective core potentials (LANL2DZ)[20] was employed for the Zn and Cd atom[21] and a 6–31G* basis set[22] for the H, C, N, and O atoms.[23] TD-DFT calculation for the S0 !Sn transitions using the same functional and basis set were then performed based on the single-crystal structures at ground states. Typically, the lowest 100 singlet roots of the nonhermitian eigenvalue equations were obtained to determine the vertical excitation energies. Oscillator strengths were deduced from the dipole transition matrix elements (for singlet states only).
Preparation of 2-((2-(hydroxymethyl)quinolin-8-yl)oxy)-N-(quinolin-8-yl)acetamide (H2L): Excess solid NaBH4 (0.076 g, 2.0 mmol) was added to the methanol solution of 3 (0.60 g, 1.69 mmol) in portions and the mixture was stirred for 30 min at room temperature. After removal of the solvent, the residue was partitioned between CH2Cl2 and 0.1 m HCl. The organic layer was dried (Na2SO4) and concentrated. The crude product was purified by chromatography (SiO2, petroleum ether/EtOAc 1:5, v/v) to afford H2L as a white solid (0.43 g, 71 %). Mp: 167.3–167.9 8C. Anal. calcd. for C21H17N3O3 : C 70.18, H 4.77, N 11.69; found: C 69.12, H 4.69, N 11.36; 1H NMR (400 MHz, CDCl3) d = 4.89 (s, 2 H), 5.02 (s, 2 H), 7.12 (dd, J = 2.0, 6.4 Hz, 1 H), 7.31 (d, J = 8.4 Hz, 1 H), 7.42–7.49 (m, 3 H), 7.54 (d, J = 4.4 Hz, 2 H), 8.10 (d, J = 8.4 Hz, 1 H), 8.13 (dd, J = 1.6, 8.0 Hz, 1 H), 8.91 (t, J = 4.4 Hz, 1 H), 9.15 (dd, J = 1.6, 4.4 Hz, 1 H), 11.22 (s, 1 H);13C NMR (100 MHz, CDCl3) d = 64.25, 68.05, 110.16, 117.11, 119.03, 120.98, 121.68, 122.45, 126.19, 126.82, 127.94, 128.77, 133.63, 136.06, 136.63, 138.38, 138.79, 149.76, 152.33, 158.33, 166.36; FT-IR (KBr pellet, cm1): 3397 (br), 1672 (s), 1609 Chem. Eur. J. 2015, 21, 290 – 297
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Cell culture and two-photon fluorescence microscopy imaging SMMC-7721 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum in an atmosphere of 5 % CO2 and 95 % air at 37 8C humidified air for
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Full Paper 24 h. One day before imaging, the cells were passaged and plated in phenol red-free medium on glass coverslips in 24-well plates. H2L in the culture media (a solution of H2L in DMSO (0.01 m) was diluted into DMEM at 100 mm) was added to the cells, and the cells were incubated for 2 h at 37 8C. After washing twice with PBS buffer, the cells were further incubated with 100 mm Cd2 + for 1 h. For EDTA experiment, the cells incubated with H2L and Cd2 + were further treated with 200 mm EDTA for 30 min. The treated cells were imaged by a two-photon fluorescence microscopy (Olympus FV 1000 MPE) with a mode-locked laser source set at wavelength 900 nm.
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