Rhodamine derived colorimetric and fluorescence mercury(II) chemodosimeter for human breast cancer cell (MCF7) imaging

Abstract

Condensation of rhodaminehydrazone with naphthalene-1-carboxaldehyde generates a colourless probe, RDHDNAP that can selectively detect Hg²⁺ through generation of a pink color along with significant fluorescence enhancement. The binding constant and lowest detection limit for Hg²⁺ are 2.0 × 10⁵ M⁻¹ and 3 × 10⁻⁷ M respectively. Hg²⁺ imaging in human breast cancer cells (MCF7) under a fluorescence microscope is achieved.

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Mercury, one of the heavy metals, is widely used in different industries¹ and has several other sources like volcanic emissions, gold mining, and solid waste incineration.² It can easily permeate biological membranes such as the skin and gastrointestinal tissues. Being thiophilic in nature, it has high affinity for proteins and enzymes³ and causes neurological problems,⁴ myocardial infarction,⁵ DNA damage, Minamata disease,⁶ and other chronic diseases.⁷ The US Environmental Protection Agency (USEPA) has recommended 2 ppb inorganic Hg²⁺ as maximum tolerance level in drinking water.⁸ Hence, ultra-trace level selective determination of Hg²⁺ at physiological condition is very demanding.⁹ Conventional available techniques include atomic absorption/emission spectroscopy, neutron activation analysis, inductively coupled plasma mass spectrometry, cold vapour atomic fluorescence spectrometry, electrochemical sensing and X-ray microanalysis etc. However, methods based on absorption/emission spectroscopy have several advantages, viz. fast, non-destructive, economic, user friendly involving facile sample preparation process. Several known photo sensing processes include photo induced electron transfer (PET),¹⁰ photo-induced charge transfer (PCT),¹¹ fluorescence resonance energy transfer (FRET),¹² intermolecular charge transfer (ICT),¹³ chelation enhanced fluorescence (CHEF),¹⁴ perturbation of optical transitions and polarisabilities and intra-molecular charge transfer (ICT).¹⁵ Due to spin orbit coupling, Hg²⁺ generally quenches fluorescence upon binding to a probe. Amongst several Hg²⁺ selective fluorescence sensors,¹⁶ rhodamine based probes have several advantages like large absorption coefficient, high fluorescence quantum yield, absorption and emission in the visible region.^17,18 The spirolactam form of rhodamine-B is colourless with an absorption band ∼250 nm that undergoes significant fluorescence enhancement along with colour generation in presence of specific analyte(s), attributed to the spirolactam ring opening.¹⁹ Choice of solvent plays a crucial role for biological studies and green analysis. Most of the rhodamine-based Hg²⁺ sensors function in CH₃CN–H₂O solution,²⁰ toxic to the living system, while several others used in EtOH–H₂O (2 : 1 or 1 : 1, v/v).²¹ The present probe functions in much greener media, EtOH–H₂O (3 : 7, v/v). The facile synthesis, structural confirmation by single crystal X-ray crystallography and functioning in a greener solvent place the present probe (RDHDNAP) in an advantageous position for colorimetric and fluorescence recognition of intra-cellular Hg²⁺ in human breast cancer cell (MCF7).

Results and discussion

Single crystals of RDHDNAP (1) and its model compound (2) containing pyrene are grown from their ethanol solution and characterized by X-ray crystallography. The most significant structural data are listed in Table 1. The molecular diagram of 1, presented in Fig. 1 (top) shows the formation of spirolactam moiety having hydrazone functional group. The bond lengths and angles subtended at the spiro atom are consistent with the existence of quaternary carbon centre. In agreement, the spirolactam and xanthene rings adopt almost an orthogonal arrangement with an Ω angle between their least square planes being 85.15(2)°. The formation of the N–N=C functional group is corroborated by N(32)–N(34) and N(34)–C(35) bond lengths with values of 1.376(2) and 1.281(2) Å, respectively.

Table 1 Selected bond lengths (Å) and angles (°) of (1) and (2)

Compound	1	2
C(7)–C(8)	1.519(2)	1.509(3)
C(8)–C(9)	1.517(2)	1.524(3)
C(8)–C(25)	1.527(2)	1.522(4)
C(8)–N(32)	1.494(2)	1.506(3)
N(32)–N(34)	1.376(2)	1.389(3)
N(34)–C(35)	1.281(2)	1.272(4)
C(31)–N(32)	1.386(2)	1.391(3)
C(7)–C(8)–C(25)	110.64(12)	111.6(2)
C(9)–C(8)–C(7)	109.82(12)	110.91(19)
C(9)–C(8)–C(25)	111.43(14)	111.5(2)
N(32)–C(8)–C(7)	112.44(13)	111.37(19)
N(32)–C(8)–C(9)	112.65(12)	111.38(19)
N(32)–C(8)–C(25)	99.52(12)	99.61(19)
C(31)–N(32)–C(8)	114.07(13)	114.0(2)
N(34)–N(32)–C(8)	127.55(12)	127.9(2)
N(34)–N(32)–C(31)	117.25(14)	117.9(2)
C(35)–N(34)–N(32)	118.14(14)	119.7(2)

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Fig. 1 X-ray crystal structure of RDHDNAP in two different views: molecular diagram of the overall structure with relevant atom labelling scheme (top); self-assembly of two molecules of 1 via π⋯π and C–H⋯π interactions (bottom). The dashed lines indicate the distances between the naphthalene's C–H and the closest aromatic ring.

In the crystal structure, pairs of RDHDNAP molecules are self-assembled in a centro-symmetric arrangement, stabilized by π⋯π stacking interactions with an inter-planar and shift distances between the naphthalene rings of 3.714(1) and 1.336(2) Å, respectively. In addition, two C–H⋯π intermolecular edge-to-face interactions with distances of 2.49 Å have been observed between both partners, along with two intra-molecular ones with distances of and 2.77 Å, as illustrated in Fig. 1 (bottom).

Effect of pH on the emission characteristics of RDHDNAP and its Hg²⁺ adduct has been examined. Fig. 2 reveals higher emission intensity of free RDHDNAP at lower pH, attributed to spirolactam ring opening. In presence of Hg²⁺, RDHDNAP shows higher emission intensity at pH 6 to 10, enabling its detection and estimation at physiological pH. Addition of Hg²⁺ to colourless RDHDNAP causes instant pink colouration, along with appearance of 558 nm peak due to Hg²⁺ induced ring-opening of the probe (Fig. 3). This new peak gradually increases upon incremental addition of Hg²⁺.

Fig. 2 Effect of pH on emission intensity of RDHDNAP (20 μM) (black) and RDHDNAP–Hg²⁺ system (red) in HEPES buffered (0.1 M) solution (ethanol–water = 3/7, v/v, pH 7.4); λ_ex = 550 nm, λ_em = 591 nm.

Fig. 3 Changes in the absorption spectra of RDHDNAP (20 μM) upon gradual addition of Hg²⁺ (1.0, 2.5, 7.5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 400, 600 and 800 μM); inset: naked eye colour of free RDHDNAP and [RDHDNAP–Hg²⁺] system. Same solvent media as noted in Fig. 2 caption.

Free RDHDNAP exhibits a very weak emission at 591 nm (λ_ex = 550 nm) characteristics of its close spirolactam ring structure which gradually increases upon gradual addition of Hg²⁺ (Fig. 4). This is attributed to Hg²⁺ assisted hydrolysis of RDHDNAP (Fig. 5), corroborated from the mass spectrum of the end product (Fig. S1, ESI†). Initially, Hg²⁺ assisted hydrolysis of RDHDNAP leads to ring opening, followed by rapture of imine bond. Further, hydrazine unit is dislodged yielding free open ring rhodamine-B unit.

Fig. 4 Changes in the emission spectra of RDHDNAP (20 μM) upon gradual addition of Hg²⁺ (0.1, 0.2, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 25.0, 50.0, 100.0, 200.0, 400.0, 600.0 and 800.0 μM, λ_ex = 550 nm). Inset: corresponding UV light exposed colour change. Same solvent media as noted in Fig. 2 caption.

Fig. 5 Proposed Hg²⁺ sensing mechanism by RDHDNAP.

In presence of Hg²⁺, fluorescence quantum yield of RDHDNAP increases 55.6 fold (from 0.0079 to 0.44) (ESI). Plot of emission intensities vs. added Hg²⁺ concentration is sigmoidal (Fig. S2, ESI†). Other bio-relevant and common cations viz. Mn²⁺, Cd²⁺, Ni²⁺, Co²⁺, Mg²⁺, Fe³⁺, Hg²⁺, Cu²⁺, Ca²⁺, Pb²⁺, Zn²⁺, Fe²⁺ and Pb²⁺ fail to cause any significant colour change of RDHDNAP, either naked eye or under UV light (Fig. 6).

Fig. 6 Changes in the emission spectra of RDHDNAP (20 μM) in presence of different cations (1000 μM). Inset: naked eye (top) and UV light exposed (bottom) colours of RDHDNAP in presence of said cations, viz. Mn²⁺ (1), Cd²⁺ (2), Ni²⁺ (3), Co²⁺ (4), Mg²⁺ (5) Fe³⁺ (6), Hg²⁺(7), Cu²⁺ (8), Ca²⁺ (9), Pb²⁺ (10), Zn²⁺ (11), Fe²⁺ (12) and Pb²⁺ (13).

Probably, due to strong paramagnetic effect, none other than Cu²⁺ interfere a little (Fig. 7). Job's plot (Fig. S3, ESI†) shows 1 : 1 (mole ratio) binding interaction between RDHDNAP and Hg²⁺. The association constant obtained following Benesi–Hildebrand method²² is 2.0 × 10⁵ M⁻¹ (Fig. S4, ESI†). RDHDNAP can detect as low as 3 × 10⁻⁷ M Hg²⁺, very much competitive to the reported values for other rhodamine based Hg²⁺ sensors useful for cell imaging studies, and listed in tabular form in ESI.†

Fig. 7 Relative emission intensities of RDHDNAP + Hg²⁺ system in presence of different cations: [RDHDNAP + Hg²⁺] (black); RDHDNAP–Hg²⁺ + cations (100 eq.) (red); λ_ex = 550 nm, λ_em = 591 nm. Same solvent media as noted in Fig. 2 caption.

Although, Yao et al. have reported its analogues pyrene derivative (2)²³ as Hg²⁺ sensor, however, the performance of the present probe, RDHDNAP is better in terms of LOD (100 times lower). Additionally, Hg²⁺ imaging in human breast cancer cell (MCF7) is possible with RDHDNAP. Fig. 8 shows the single crystal X-ray structure of 2. The bond lengths and bond angles around the spiro carbon centre as well as for the hydrazine functional group are similar to those found for 1. The most relevant structural feature in the crystal packing of the pyrene analogue is the formation of dimeric self-assembled molecules assisted by C–H⋯π stacking interactions between extended pyrene rings at an inter-planar distance of 3.998(2) Å, and shitted by 1.981(3) Å. Furthermore, two molecular entities adopt a ladder type spatial disposition (Fig. 8, bottom).

Fig. 8 X-ray crystal structure of 2 in two different views: molecular diagram of the overall structure with relevant atom labelling scheme (top); space filling representation showing the self-assembly of two pyrene derivatives in a ladder type arrangement (bottom).

Due to poor water solubility, intra-cellular Hg²⁺ imaging using the pyrene probe (1) remains unsuccessful.

Human breast cancer cell imaging

Human breast cancer cells (MCF7) treated only with RDHDNAP, (without Hg²⁺) are used as control. RDHDNAP can detect intracellular Hg²⁺ very efficiently (Fig. 9).

Fig. 9 Fluorescence microscope image of MCF7 cells: (A) and (B) are photographs after incubating cells for 2 h with Hg²⁺ (50 μM) and RDHDNAP (20 μM) respectively while (C) is the photograph of cells after incubating with 50 μM Hg²⁺ for 2 h, followed by addition of 20 μM RDHDNAP.

The application of this new method is substantiated by analyzing Hg²⁺ content in several industrial waste water samples and comparing the results obtained from a reference method²⁴ (Table 2). The close proximity of the results of a certified reference material by the present method clearly indicates its acceptability for Hg²⁺ determination in real samples.

Table 2 Analysis of certified reference materials and industrial waste water samples

Sample	Hg^2+ found^a (μg g^−1, present method)	Hg^2+ found (μg g^−1, reference method)
a Average value, N = 3.b NIES certified value: 1.2 ± 0.2 μg g^−1.
Industrial waste water (station 1)	141.2 ± 0.2	140.7 ± 0.4
Industrial waste water (station 2)	179.2 ± 0.4	179.8 ± 0.2
Industrial waste water (station 3)	165.2 ± 0.4	165.6 ± 0.3
Industrial waste water (station 4)	225.7 ± 0.6	226.4 ± 0.6
NIES certified sample^b	1.15 ± 0.2	1.14 ± 0.2

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Conclusion

Single crystal X-ray structurally characterised rhodaminehydrazone derivative, RDHDNAP is an excellent colorimetric and fluorescence probe for Hg²⁺ at physiological pH. Its Hg²⁺ detection limit is 3 × 10⁻⁷ M. RDHDNAP senses Hg²⁺ via hydrolysis mechanism, useful to monitor intracellular Hg²⁺ accumulation in human breast cancer cell.

Experimental

General procedure

Rhodamine B, naphthalene-1-carboxaldehyde and pyrene-1-carboxaldehyde have been purchased from Sigma Aldrich (India). Spectroscopic grade solvents have been used. Other chemicals are of analytical reagent grade and used without further purification. Milli-Q 18.2 MΩ cm⁻¹ water has been used as and when required. Shimadzu UV-Vis-2450 spectrophotometer has been used for measuring the absorption spectra. FTIR spectra are recorded on a Shimadzu FTIR spectrometer. Mass spectra are obtained using QTOF Micro YA 263 mass spectrometer in ES positive mode. ¹H NMR spectra have been recorded using Bruker Advance 300 (300 MHz) instrument in CDCl₃. Steady-state fluorescence experiments have been performed using Hitachi F-4500 spectrofluorimeter. The pH of the solutions has been measured using Systronics digital pH meter (model 335, India). The industrial waste water samples have been collected from Damodar river (Durgapur-Raniganj Industrial belt, West Bengal, India), filtered through 0.45 μm Millipore membrane filter and analyzed using RDHDNAP.

Synthesis of rhodaminehydrazone unit

N-(Rhodamine-B) lactam-hydrazine is prepared by refluxing Rhodamine B and hydrazine hydrate for 3 h following literature method.²⁵

Synthesis of RDHDNAP. Both N-(rhodamine-B) lactam-hydrazine (0.050 g, 0.1016 mmol) and naphthalene-1-carboxaldehyde (0.015 g, 0.1016 mmol) are dissolved in 10 mL ethanol and mixed drop wise under stirring condition. Then the reaction mixture is refluxed for 6 h (Scheme 1). After filtration, the filtrate is kept undisturbed. About 3 days later, light pink crystals, suitable for single crystal X-ray diffraction have been obtained. The ESI-MS spectrum of RDHDNAP generates a peak at m/z, 595 (calcd, 594) corresponding to [RDHDNAP + H]⁺ (Fig. S5, ESI†). FTIR spectrum shows a peak at 1728 cm⁻¹, attributed to the imine bond (Fig. S6, ESI†). ¹H NMR (CDCl₃, 300 MHz, Fig. S7, ESI†): δ, ppm: 1.12 (12H, t, 6.873 Hz), 3.30941 (8H, d, 6.87 Hz), 6.2656 (2H, d, 8.11 Hz), 6.49759 (2H, s), 6.60144 (2H, d, 8.562), 7.16257 (1H, d, 6.867), 7.38252 (3H, t, 7.257), 7.5112 (2H, s), 7.75220 (2H, d, 7.581), 7.87405 (1H, d, 6.189), 8.03211 (2H, d, 6.984), 9.25623 (1H, s). ¹³C NMR (CDCl₃, 300 MHz, Fig. S8, ESI†): δ, ppm: 165.00, 153.06, 151.94, 148.93, 146.36, 133.54, 133.35, 131.05, 130.90, 129.97, 129.30, 128.30, 128.11, 126.84, 126.44, 125.62, 125.26, 124.21, 123.83, 123.37, 108.16, 105.94, 97.95, 65.91, 44.31, 12.57.

Scheme 1 Synthesis of RDHDNAP.

Synthesis of RDHDTCA–Hg²⁺ adduct. To ethanol solution of RDHDNAP (0.10 g), 2 mL Hg(NO₃)₂·H₂O (0.057 g, 0.1680 mmol) solution in ethanol is added drop wise and stirred for 5 min. The solvent is evaporated using rotary evaporator while deep pink residue is obtained. The ESI-MS spectrum of [RDHDNAP–Hg²⁺] system generates peaks at m/z 457.0 and 443.2, FTIR/cm⁻¹, ν(C=O) 1585 (Fig. S9, ESI†).

X-ray crystallography

The single crystal X-ray data of RDHDNAP (1) and analogous pyrene derivative (2) are collected with monochromated Mo-Kα radiation (λ = 0.71073 Å) on a Bruker SMART Apex II diffractometer equipped with a CCD area detector at 150(2) K and at room temperature, respectively. The crystal is positioned at 35 mm from the CCD and the spots are measured using counting time of 80 and 100 s for 1 and 2, respectively. Data reduction is carried out using the SAINT-NT software package.²⁶ A multi-scan absorption correction is applied to all intensity data of each compound using the SADABS program.²⁷ Both the structures are solved by combination of direct methods with subsequent difference Fourier syntheses and refined by full matrix least squares on F² using the SHELX-2013 suite.²⁸ Hydrogen atoms bound to carbon are inserted at ideal geometric positions. Molecular and the crystal packing diagrams are drawn with the PyMOL software package.²⁹ The crystal data together with refinement details are given in Table 3.

Table 3 Crystal data and refinement parameters of 1 and 2

Complex	1	2
Empirical formula	C39H38N4O2	C45H40N4O2
Mw	594.73	668.81
Crystal system	Monoclinic	Monoclinic
Space group	P21/n	P21/c
a [Å]	11.5411(14)	9.273(4)
b [Å]	11.5412(10)	16.808(7)
c [Å]	23.608(2)	23.315(9)
β [°]	92.592(4)	100.688(15)
V [Å^3]	3141.4(6)	3571(2)
Z	4	4
Dc [Mg m^−3]	1.258	1.244
μ [mm^−1]	0.078	0.077
Reflections collected	20 130	20 045
Unique reflections, [Rint]	6437 [0.0366]	7267 [0.0788]
Final R indices
R1, wR2 [I > 2σI]	0.0456, 0.1027 [4432]	0.0673, 0.1798 [3736]
R1, wR2 (all data)	0.0760, 0.1163	0.1297, 0.2194

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Cytotoxicity assay

In vitro cytotoxicity is measured using the colorimetric methyl thiazolyl tetrazolium (MTT) assay against human breast cancer cells (MCF7). Cells are seeded into 24-well tissue culture plate in presence of 500 μL Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C and 5% CO₂ atmosphere for overnight and then incubated for 12 h in presence of RDHDNAP at different concentrations (10–100 μM). Then cells are washed with PBS buffer and 500 μL supplemented DMEM medium is added. Subsequently, 50 μL MTT (5 mg mL⁻¹) is added to each well and incubated for 4 h. Next, violet formazan is dissolved in 500 μL sodium dodecyl sulfate solution in water–DMF mixture. The absorbance of solution is measured at 558 nm using microplate reader. The cell viability is determined by assuming 100% cell viability for cells without RDHDNAP (Fig. 10).

Fig. 10 Cell viability of RDHDNAP towards MCF7 cells after 12 h incubation.

In vitro cell imaging

Human breast cancer cell line, MCF7 is grown in DMEM (Sigma, St. Louis, USA) supplemented with 10% fetal bovine serum (Sigma, St. Louis, USA), 2 mM glutamine, 100 U mL⁻¹ penicillin–streptomycin solution (Gibco, Invitrogen, USA) in the presence of 5% CO₂ at 37 °C. For in vitro imaging study, the cells are seeded in 6 well culture plate with a seeding density of 10⁵ cells per well. After reaching 60–70% confluence, the previous complete media is replaced with serum free media, supplemented with Hg²⁺ and RDHDNAP at a concentration of 50 and 20 μM followed by incubation for 2 h to facilitate uptake of cation or RDHDNAP by cells. The cells are then observed under an inverted microscope (Dewinter, Italy) at different magnifications to examine any adverse effect on cellular morphology. RDHDNAP treated cells are then incubated with Hg²⁺ for 15–30 min and observed under an inverted fluorescence microscope (Dewinter, Italy) at different magnifications using blue filter. Images are taken through an attached CCD camera with the help of BioWizard 4.2 software. Control experiment is performed using the medium devoid of metal salt.

Acknowledgements

Authors sincerely thank UGC-DAE-CRS-Kolkata for financial support. B. Kumari sincerely thanks BU for fellowship. S. Lohar is thankful to CSIR for fellowship. Authors sincerely acknowledge CAS (B. U.) for assistance. Suggestions from reviewers to improve the quality of the MS are gratefully acknowledged.

Notes and references

1. (a) Q. Wang, D. Kim, D. D. Dionysiou, G. A. Sorial and D. Timberlake, Environ. Pollut., 2004, 131, 323; (b) E. M. Nolan and S. J. Lippard, Chem. Rev., 2008, 108, 3443; (c) C. M. L. Carvalho, E.-H. Chew, S. I. Hashemy, J. Lu and A. Holmgren, J. Biol. Chem., 2008, 283, 11913; (d) G. Guzzi and C. A. M. La Porta, Toxicology, 2008, 244, 1.
2. (a) R. Von Burg and M. R. Greenwood, Metals, VCH, Weinheim, 1991, p. 1045; (b) A. Renzoni, F. Zino and E. Franchi, Environ. Res., 1998, 77, 68; (c) K. Rurack, M. Kollmannsberger, U. Resch-Genger and J. Dauhb, J. Am. Chem. Soc., 2000, 122, 968.
3. (a) B. R. Von, J. Appl. Toxicol., 1995, 15, 483; (b) H. H. Harris, I. J. Pickering and G. N. George, The Chemical Form of Mercury in Fish, Science, 2003, 301, 1203.
4. J. Gutknecht, J. Membr. Biol., 1981, 61, 61.
5. G. N. George, R. C. Prince, J. Gailer, G. A. Buttigieg, M. B. Denton, H. H. Harris and I. J. Pickering, Chem. Res. Toxicol., 2004, 17, 999.
6. M. Harada, Crit. Rev. Toxicol., 1995, 25, 1.
7. G. D. Huy, M. Zhang, P. Zuo and B.-C. Ye, Analyst, 2011, 136, 3289.
8. Mercury Update: Impact on Fish Advisories. EPA Fact Sheet EPA-823-F-01-011, EPA, Office of Water, Washington, DC, 2001.
9. (a) Z. Xu, S. J. Han, C. Lee, J. Yoon and D. R. Spring, Chem. Commun., 2010, 46, 1679; (b) M. Suresh, S. Mishra, S. K. Mishra, E. Suresh, A. K. Mandal, A. Shrivastav and A. Das, Org. Lett., 2009, 11, 2740.
10. J. S. Kim and D. T. Quang, Chem. Rev., 2007, 107, 3780.
11. (a) S. K. Kim, J. H. Bok, R. A. Bartsch, J. Y. Lee and J. S. Kim, Org. Lett., 2005, 7, 4839; (b) H. J. Kim, S. K. Kim, J. Y. Lee and J. S. Kim, J. Org. Chem., 2006, 71, 6611.
12. (a) B. W. van der Meer, G. Coker III and S. Y. Simon, Resonance Energy Transfer, Theory and Data, VCH, Weinheim, 1994; (b) I. Leray, F. O'Reilly, J. L. Habib Jiwan, J. P. Soumillion and B. Valeur, Chem. Commun., 1999, 795; (c) B. Liu, F. Zeng, G. Wu and S. Wu, Chem. Commun., 2011, 47, 8913; (d) R. K. Castellano, S. L. Craig, C. Nuckolls and J. Rebek, J. Am. Chem. Soc., 2000, 122, 7876.
13. (a) Z. Xu, Y. Xiao, X. Qian, J. Cui and D. Cui, Org. Lett., 2005, 7, 889; (b) J. B. Wang, X. F. Qian and J. N. Chi, J. Org. Chem., 2006, 71, 4308.
14. (a) N. C. Lim, J. V. Schuster, M. C. Porto, M. A. Tanudra, L. Yao, H. C. Freake and C. Bruckner, Inorg. Chem., 2005, 44, 2018; (b) S. Guha, S. Lohar, A. Banerjee, A. Sahana, A. Chaterjee, S. K. Mukherjee, J. S. Matalobos and D. Das, Talanta, 2012, 91, 18; (c) S. Das, A. Sahana, A. Banerjee, S. Lohar, S. Guha, J. S. Matalobos and D. Das, Anal. Methods, 2012, 4, 2254.
15. S. A. de Silva, A. Zavaleta, D. E. Baron, O. Allam, E. V. Isidor, N. Kashimura and J. M. Percapio, Tetrahedron Lett., 1997, 38, 2237.
16. (a) A. Kaur, K. Sharma, S. Kaur, N. Singh and N. Kaur, RSC Adv., 2013, 3, 6160; (b) B. N. Ahmed and P. Ghosh, Dalton Trans., 2011, 40, 12540.
17. (a) S. Adhikari, A. Ghosh, S. Mandal, A. Sengupta, A. Chattopadhyay, J. S. Matalobos, S. Lohar and D. Das, Dalton Trans., 2014, 43, 7747; (b) S. Mandal, A. Banerjee, S. Lohar, A. Chattopadhyay, B. Sarkar, S. K. Mukhopadhyay, A. Sahana and D. Das, J. Hazard. Mater., 2013, 261, 198; (c) M. J. Culzoni, A. Muňoz de la Peňa, A. Machuca, H. C. Goicoechea and R. Babiano, Anal. Methods, 2013, 5, 30.
18. (a) A. Banerjee, A. Sahana, S. Lohar, I. Halui, S. K. Mukhopadhyay, D. A. Safin, M. G. Babashkina, M. Bolte, Y. Garcia and D. Das, Chem. Commun., 2013, 49, 2527; (b) B. Bag and A. Pal, Org. Biomol. Chem., 2011, 9, 4467; (c) X. Guan, W. Lin and W. Huang, Org. Biomol. Chem., 2014, 12, 3944.
19. (a) H. Wang, Y. Li, S. Xu, Y. Li, C. Zhou, X. Fei, L. Sun, C. Zhang, Y. Li, Q. Yang and X. Xu, Org. Biomol. Chem., 2011, 9, 2850; (b) X. Zhang, X. J. Huang and Z. J. Zhu, RSC Adv., 2013, 3, 24891.
20. (a) J. H. Soh, K. M. K. Swamy, S. K. Kim, S. Kim, S. H. Lee and J. Yoon, Tetrahedron Lett., 2007, 48, 5966; (b) Y. Zhou, K. Chu, H. Zhen, Y. Fang and C. Yao, Spectrochim. Acta, Part A, 2013, 106, 197; (c) H. N. Kim, M. H. Lee, H. J. Kim, J. S. Kim and J. Yoon, Chem. Soc. Rev., 2008, 37, 1465.
21. (a) Y. Zhou, X. Y. You, Y. Fang, J. Y. Li, K. Liu and C. Yao, Org. Biomol. Chem., 2010, 8, 4819; (b) Z. Zhou, M. Yu, H. Yang, K. Huang, F. Li, T. Yi and C. Huang, Chem. Commun., 2008, 3387; (c) H. Yang, Z. Zhou, K. Huang, M. Yu, F. Li, T. Yi and C. Huang, Org. Lett., 2007, 9, 4729.
22. H. A. Benesi and J. H. Hildebrand, J. Am. Chem. Soc., 1949, 71, 2703.
23. K. H. Chu, Y. Zhou, Y. Fang, L. H. Wang, J. Y. Li and C. Yao, Dyes Pigm., 2013, 98, 339.
24. E. M. Soliman, M. B. Saleh and S. A. Ahmed, Anal. Chim. Acta, 2004, 523, 133.
25. X. F. Yang, X. Q. Guo and Y. B. Zhao, Talanta, 2002, 57, 883.
26. Bruker SAINT-plus, Bruker AXS Inc., Madison, Wisconsin, USA, 2007.
27. G. M. Sheldrick, SADABS, University of Göttingen, Germany, 1996.
28. G. M. Sheldrick, Acta Crystallogr., Sect. A: Found. Crystallogr., 2008, 64, 112–122.
29. The PyMOL Molecular Graphics System, Version 1.5.0.4, Schrödinger, LLC.

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Footnote

† Electronic supplementary information (ESI) available. CCDC 961580 and 900558. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4ra14624g

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