Bis-triazolylated-1,4-dihydropyridine - Highly selective hydrophilic fluorescent probe for detection of Fe3+

Dyes and Pigments 147 (2017) 420e428

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Bis-triazolylated-1,4-dihydropyridine - Highly selective hydrophilic fluorescent probe for detection of Fe3þ Rakesh Kumar a, *, Parveen Gahlyan a, Neha Yadav a, Mamta Bhandari b, Rita Kakkar b, Manu Dalela c, Ashok K. Prasad a a b c

Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi, 110007, India Computational Chemistry Laboratory, Department of Chemistry, University of Delhi, Delhi, 110007, India Centre for Biomedical Engineering, Indian Institute of Technology, Hauz Khas, New Delhi, 110016, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 May 2017 Received in revised form 25 August 2017 Accepted 25 August 2017 Available online 26 August 2017

A water-soluble ferric (Fe3þ) ion sensor probe has been synthesized using one-pot multicomponent Hantzsch synthesis, followed by Cu (I) catalyzed “Click reaction”. The synthesized probe C1 shows selective binding towards Fe3þ ion through a turn-off fluorescence response among various metal ions tested in aqueous medium. C1 shows an average fluorescence lifetime of 4.9  1010 s, and its selectivity towards Fe3þ ion has also been studied by Stern-Volmer plot, detection limit and binding studies. The probe C1 forms a 1:1 complex with Fe3þ ion with an association constant of (Ka ¼ 2.50  103 M1), as evident from the Benesi-Hildebrand plot. Density Functional Theory (DFT) studies of the probe reveal a square pyramidal geometry. Both Fe2þ and Fe3þ bind strongly to the probe, but the complexation energy with Fe3þ is almost double that with Fe2þ, validating the experimental results. The probe C1 has also been tested for its application in biological systems and the results of an anti-proliferation assay of the fluorescent probe C1 in the absence and presence of FeCl3 on various cell lines normal (L929) and cancerous (MCF-7, MDA-MB 231, A549 and HepG2) shows that the probe C1 would be non-cytotoxic, both in the absence and presence of Fe3þ ions in the cells. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Turn off fluorescence Stern-Volmer plot Benesi-Hildebrand plot DFT MTT assay

1. Introduction Metal ion toxicity in the environment is one of the major obstacles in the path of obtaining a green and healthy environment. Most of the attention directed to studying metal toxicity remains limited to Hg, Pb, Cr, Cd and other such heavy metals, due to which many other metals remain inadequately studied [1]. Iron (Fe), the second most abundant metal, plays a crucial role in many biological and environmental processes [2,3] and calls for the development of sensors that could recognize its presence in any system. Several oxidation states of Fe are readily stabilized by catalytic progressions [4], but ferrous (Fe2þ) and ferric (Fe3þ) ions are the two most common forms of this metal that exist in the form of oxides, hydroxides, sulfides and carbonates [5]. Even in the biological systems, Fe exists in the form of Fe2þ and Fe3þ in trace amounts for their normal metabolic process [6], including oxygen transport, DNA synthesis and electron transport [7,8], but its excess or

* Corresponding author. E-mail address: [email protected] (R. Kumar). http://dx.doi.org/10.1016/j.dyepig.2017.08.048 0143-7208/© 2017 Elsevier Ltd. All rights reserved.

deficiency leads to severe diseases such as b-thalassemia, cancer, hemochromatosis [9,10], Alzheimer's disease, liver and kidney damage [11,12]. During the process of heme synthesis and transport, Fe is required in the Fe2þ form, and the presence of Fe3þ ions disturbs the mechanism. Interference of Fe3þ in heme synthesis leads to the synthesis of hematin, hence proving lethal. Ferrous (Fe2þ) salts are quite unstable and precipitate as insoluble ferric hydroxide in drinking water. Iron also enhances unwanted bacterial growth in water, which causes deposition of sticky coating in the pipelines [13]. Hence, the determination of these two forms of iron becomes a necessity to control water contamination and prevent several health problems. There are several techniques adopted for detection of iron, like flame atomic absorption spectrometry (with detection limit 0.03 mg/L) [14], and inductively coupled plasmaatomic emission spectrometry (with detection limit 0.03 mg/L) [15]. Despite the low detection limit and high selectivity, their application is limited due to time consuming analysis and the need of advanced instrumentation [16]. This calls for the development of new techniques for their identification from the environmental as well as biological system under consideration. Fluorescent metal

R. Kumar et al. / Dyes and Pigments 147 (2017) 420e428

sensors offer a facile approach for the detection and removal of metal ions and have potential applications in the fields of biology [17], environmental science [18] and various functional materials [19,20]. Several fluorescent Fe(III) ion sensors, including derivatives of rhodamine [21], napthalamide [22,23], coumarin [24e26], fluorescein [27], BODIPY [28,29], and thiazolo-pyridine [30] have become popular because of their high sensitivity, efficiency and convenient manipulation [31]. However, their practical applications have been limited due to several shortcomings, like low water solubility, high toxicity and slow response towards Fe(III) ions [32]. The biological importance of 1,4-dihyropyridines and 1,2,3triazoles prompted us to explore a new class of fluorescent sugar based bis-triazoles linked with the 1,4-dihydropyridine ring by ether linkage for selective recognition of Fe3þ in the aqueous medium. The role of 1,2,3-triazoles formed by Cu(I) catalyzed “Click reaction’’ has already been recognized for effective binding of metal ions [33], along with their wide application in the field of medicinal chemistry [34]. In the present work, the synthesized metal sensor has been reported and investigated in detail, and shown to exhibit a nontoxic nature in various cancerous and normal cell lines. The fluorescence study reveals that the binding constant and the limit of detection of these newly synthesized compounds are comparable to those of the reported coumarin-rhodamine [35] and naphthalene-rhodamine [36] derivatives based ratiometric fluorescent iron metal sensors.

421

multiplet (m).

2.2. Synthesis

2.1. Materials and methods

2.2.1. Synthesis of diethyl 4-(2-chlorophenyl)-2,6-bis((prop-2-yn-1yloxy)methyl)-1,4-dihydropyridine-3,5-dicarboxylate (1) A mixture of 2-chlorobenzaldehyde (1.0 mmol), ethyl 3-oxo-4(prop-2-yn-1-yloxy)butanoate (2.0 mmol), ammonium acetate (1.5 mmol) and barium nitrate (0.1 mmol) was added in a round bottom flask and stirred at 90  C for about 5 h (Scheme 1). The progress of the reaction was monitored by using TLC at various time intervals. After completion of the reaction, the mixture was allowed to cool at room temperature, extracted three times with chloroform and removed any water content using anhydrous Na2SO4. The solvent was evaporated under reduced pressure and the crude product thus obtained was purified by column chromatography by using the ethyl acetate:hexane (1:19) solvent system. Compound 1 was obtained as a white solid in 70% yield having melting point: 121e123  C; IR (KBr, cm1): n 3381, 3296, 1680, 1471, 1085; 1H NMR (400 MHz, CDCl3): d 8.28 (1H, s, -NH), 7.39 (1H, dd, J ¼ 7.6, 1.3 Hz, Ar-H), 7.24e7.20 (1H, m, Ar-H), 7.13 (1H, m, Ar-H), 7.07e7.02 (1H, m, Ar-H), 5.43 (1H, s, H-4 of DHP), 4.88 (2H, d, J ¼ 16.0 Hz, -CH2O), 4.78 (2H, d, J ¼ 16.0 Hz, -CH2O), 4.28 (4H, d, J ¼ 2.3 Hz, -OCH2), 4.07 (4H, q, J ¼ 7.0 Hz, -CH2CH3), 2.49 (2H, t, J ¼ 2.3 Hz, -C≡C-H), 1.19 (6H, t, J ¼ 7.2 Hz, -CH2CH3); 13C NMR (100 MHz, CDCl3): d 167.1, 145.4, 144.6, 132.3, 131.8, 129.3, 127.6, 126.9, 101.9, 78.6, 75.6, 67.3, 59.9, 58.7, 37.1 (C-4 of DHP), 14.3; HRMS (ESI): Calculated for C25H26ClNO6 [MþH]þ 472.1522; found 472.1494.

3-(4,5-Dimethyl-2-thiozolyl)-2,5-diphenyl-tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St, Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and 1% antibiotic cocktail (contains Penicillin-Streptomycin Mixture and routinely used in tissue culture to prevent bacterial infections or contaminations in primary and secondary cells) were purchased from Lonza (Walkersville, MD, USA), while other chemicals and reagents used in the synthesis of probe C1 and metal salts were procured from Spectrochem Pvt. Ltd. and used as such without further purification. The reactions were monitored by thin-layer chromatography (TLC) on alumina coated plates (Merck silica gel 60 F 254) and the spots were visualized under UV chamber. Silica gel (100e200 mesh) was used for column chromatography to purify the compounds. One-sided open glass capillary tubes were used to record the melting points on a Buchi M-560 instrument. Infrared spectra were recorded on a SHIMADZU IR Affinity-1S FT-IR Spectrophotometer, whereas 1H and 13C NMR spectra (in CDCl3 and DMSO-d6) were recorded (in ppm) on a JNM ECX-400P (JEOL, USA) Spectrometer using tetramethylsilane (TMS) as internal reference. HRMS (High resolution mass spectrum) was carried out by using Agilent Technologies, 6530 Accurate - Mass Q-TOF LC/MS Spectrometer. Absorption spectra were recorded on Agilent Technologies, Cary Series UVeVis spectrophotometer and fluorescence emission measurements were done with the help of Varian, Cary Eclipse Fluorescence Spectrophotometer. The fluorescence lifetime experiment was recorded by HORIBA Jobin Yvon Fluorohub. Distilled water was used throughout the studies. Absorption frequencies (n) in IR are expressed in cm1, chemical shifts and coupling constants (J) in NMR in ppm (d-scale) and Hz respectively. Splitting patterns of peaks in NMR are described as singlet (s), doublet (d), triplet (t), quartet (q), doublet of doublet (dd) and

2.2.2. Synthesis of diethyl 4-(2-chlorophenyl)-2,6-bis(((1((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-1,4-dihydro pyridine-3,5-dicarboxylate(2) Compound 1 (1.0 mmol) and (2R, 3R, 4S, 5R, 6R)-2-(acetoxymethyl)-6-azidotetrahydro-2H-pyran-3,4,5-triyl triacetate (3.0 mmol) were dissolved in THF (10 mL) in a round bottom flask. A solution of CuSO4$5H2O (0.4 mmol) and sodium ascorbate (0.8 mmol) in water was added to the reaction mixture and refluxed for 1 h. The progress of the reaction was monitored by TLC. After completion of reaction, the solvent was evaporated under reduced pressure. The residue obtained was extracted three times with chloroform. The separated organic layer was dried over Na2SO4 and evaporated under reduced pressure. The crude product thus obtained was purified by column chromatography (1:25; methanol:chloroform) solvent system. Compound 2 was obtained as a white solid in 81% yield having melting point: 101e103  C; IR (KBr, cm1): n 3383, 1757, 1220, 1041; 1 H NMR (400 MHz, DMSO-d6): d 8.50 (2H, s, -N-CH), 8.44 (1H, s, -NH), 7.30e7.25 (2H, m, Ar-H), 7.21 (1H, t, J ¼ 7.40 Hz, Ar-H), 7.12 (1H, t, J ¼ 7.6 Hz, Ar-H), 6.38 (2H, dd, J ¼ 9.1, 2.9 Hz, -CH), 5.69e5.63 (2H, m, -CH), 5.56 (2H, t, J ¼ 9.5 Hz, -CH), 5.25 (1H, s, -H-4 of DHP), 5.19 (2H, t, J ¼ 9.7 Hz, -CH), 4.70e4.57 (8H, m, -CH2O, -OCH2), 4.38e4.35 (2H, m, -CH), 4.11e4.03 (4H, m, -CH2), 3.95 (4H, q, J ¼ 7.0 Hz, -CH2CH3), 2.02 (6H, s, -CH3), 1.95 (12H, d, J ¼ 6.3 Hz, -CH3), 1.75 (6H, d, J ¼ 3.7 Hz, -CH3), 1.08 (6H, t, J ¼ 7.1 Hz, -CH2CH3); 13 C NMR (100 MHz, DMSO-d6): d 170.5 (-COO), 170.1 (-COO), 169.9 (-COO), 169.0 (-COO), 166.5 (-COO), 145.7, 145.1, 144.3, 131.8, 131.6, 129.4, 128.4, 127.9, 123.8, 101.5, 84.4, 73.8, 72.6, 70.7, 68.0, 66.7, 63.9, 62.2, 59.8, 37.2 (C-4 of DHP), 31.4, 20.9, 20.9, 20.7, 20.2, 14.5; HRMS (ESI): Calculated for C53H64ClN7O24 [MþH]þ 1218.3764; found 1218.3746.

2. Experimental

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R. Kumar et al. / Dyes and Pigments 147 (2017) 420e428

Scheme 1. Schematic route for the synthesis of C1 and C1-Fe3þcomplex.

2.2.3. Synthesis of fluorescent probe diethyl 4-(2-chlorophenyl)2,6-bis(((1-((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)1,4-dihydropyridine-3,5-dicarboxylate(C1) Compound 2 (1.0 mmol) and sodium methoxide (8.0 mmol) were dissolved in methanol (5 mL) in a round bottom flask and stirred for 5 min. The reaction was monitored using TLC. After completion of the reaction, the excess base was neutralized by cationic resin Seralite SRC-120. The solvent was then evaporated under reduced pressure and washed with solvent (hexane:ethyl acetate; 1:1) to get the pure product C1. The compound was obtained as a yellowish solid in 99% yield with melting point: 110e112  C; IR (KBr, cm1):n 3375, 1687,1591; 1 H NMR (400 MHz, DMSO-d6): d 8.54 (1H, s, -NH), 8.37 (2H, s, -NCH), 7.33 (1H, d, J ¼ 6.6 Hz, Ar-H), 7.28e7.21 (2H, m, Ar-H), 7.13 (1H, t, J ¼ 7.0 Hz, Ar-H), 5.57 (2H, d, J ¼ 9.2 Hz, -CH), 5.41 (2H, d, J ¼ 6.0 Hz, -OH), 5.31 (3H, d, J ¼ 4.0 Hz, -OH, -H-5), 5.17 (2H, d, J ¼ 5.5 Hz, -OH), 4.78e4.64 (10H, m, -CH2O, -OCH2, -OH), 3.97 (4H, q, J ¼ 7.0 Hz, -CH2CH3), 3.79 (2H, dd, J ¼ 15.1, 9.0 Hz, -CH2), 3.69 (2H, dd, J ¼ 9.6, 5.5 Hz, -CH2), 3.47e3.38 (6H, m, -CH), 3.26e3.19 (2H, m, -CH), 1.09 (6H, t, J ¼ 7.0 Hz, -CH2CH3); 13C NMR (100 MHz, DMSOd6): d 166.5 (-COO), 145.7, 145.2, 143.5, 131.9, 131.6, 129.4, 128.4, 128.0, 124.0, 101.6, 88.0, 80.4, 77.4, 72.6, 70.0, 66.9, 64.0, 61.2, 59.9, 37.2 (C-4 of DHP), 14.6; HRMS (ESI): Calculated for C37H48ClN7O16 [MþH]þ 882.2912; found 882.2913. lmax (ε, M1cm1): 368 (9600). 2.2.4. Synthesis of probe C1-Fe3þcomplex The solution of the probe C1 (50 mM) was prepared in methanol (5 mL). The solution of FeCl3 (5.0 mmol) in methanol was added slowly to a solution of probe C1 (1.0 mmol) with continuous stirring for 2 h. The solid compound thus obtained (Scheme 1) after

evaporation of methanol under reduced pressure was washed with diethyl ether and dried properly under vacuum. IR (KBr, cm1): n 3379, 1624; lmax (ε, M1cm1): 359 (3480). 2.3. General method for UVevisible and fluorescence studies The interaction of the probe C1 with various metal ions was investigated by UVevisible and fluorescence studies in aqueous medium at pH ¼ 6.5. The solutions of metal ions as their chloride salts with varying concentrations (1e20 eq.; 1eq ¼ 50 mM) as guest and probe C1 (50 mM) as host were prepared separately. C1 was excited at a wavelength of 360 nm with the same excitation and emission slit, i.e. 5 cm. The aqueous solution of different metal salts was added to the solution of C1, and fluorescence and absorption changes were observed at room temperature. The average fluorescence lifetime was calculated using equation (1) [37].

¼

X

.

ai t2i ai ti

(1)

where ai ¼ normalized fluorescence; ti ¼ fluorescence lifetime and ¼ average fluorescence lifetime. 2.4. Detection limit, selectivity and quenching constant by fluorescence titration In order to analyze selectivity, the stock solutions (250 mM) of different metal salts and C1 (50 mM) were prepared in water. Test solutions were prepared by adding equal amounts of stock solution of C1 and metal salts in a quartz cuvette having 1 cm optical path length. Fluorescence changes were observed using a fluorescence

R. Kumar et al. / Dyes and Pigments 147 (2017) 420e428

spectrophotometer. For calculation of the quenching constant, the probe C1 (50 mM) was titrated against Fe3þ and Fe2þ ion with varying concentrations (1e20 eq.). The slope of the Stern-Volmer plot represents the quenching constant (Ksv) which is shown in Fig. 4(B) and the slope of the Benesi-Hildebrand plot represents the association constant (Ka), as in Fig. 6. The detection limit (D.L.) for Fe3þ and Fe2þ was calculated using the following equation: D.L. ¼ 3  s/K, where s is the standard deviation of the blank solution, and K is the slope of Fig. S5 [38]. For calculation of the standard deviation, 10 replicate measurements of the probe C1 were taken.

2.5. Computational details First-principles density functional (DF) calculations were performed using the DMol3 code [39e45]. Numerical basis sets of double zeta quality plus polarization functions (DNP), which are the numerical equivalent of the Gaussian basis, 6-31G**, but are much more accurate, were used for the computations. The exchangecorrelation contribution to the total electronic energy was treated in a spin-polarized generalized-gradient corrected (GGA) form of the local density approximation (LDA), within the framework of the Perdew-Burke-Ernzerfhof (PBE) correlation [46,47]. Gradient corrected functionals are much faster and can be efficiently used with a plane wave basis set in solid state calculations. A comparison of results with different LDA and GGA functionals for complexes had revealed that GGA/PBE is the most reliable [48]. The cores were treated using DFT semicore pseudopotentials (DSPP). The geometries of all complexes were fully optimized, without restrictions, using delocalized internal coordinates. The vibrational frequencies were calculated and it was ensured that all are real. The energy values reported here are the Gibbs energies, obtained by adding thermal corrections to the enthalpy and vibrational and electronic contributions to the entropy. The partial charges reported are the ESP charges.

2.6. In vitro cytotoxicity studies In vitro cytotoxicity potential of the fluorescent probe C1 in absence and presence of FeCl3 was evaluated using various cancer cell lines (MCF-7, MDA-MB 231, A549 and HepG2) and normal cell line (L929) using 3-(4,5-dimethyl-2-thiozolyl)-2,5diphenyltetrazolium bromide (MTT) assay. Briefly, 5  104 cells were seeded in a 96-well plate and incubated in 5% CO2 incubator at 37  C for 24 h based on a published method with slight modifications [49]. After incubation of 24 h, solutions of testing materials were added to each well and incubated for another 48 h, using serial concentrations ranging from 1 to 100 mM. After the incubation, the testing materials were removed from each well, washed with ice cold 1  PBS for 2e3 times and 50e100 mL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the insoluble formazon crystals. All the testing materials were dispersed in Dulbecco's modified Eagle's medium (DMEM) for better solubility and accuracy to check the potential. Absorbance was made using BioTek Epoch microtiter plate reader (BioTek Instruments, Inc., Winooski, VT, USA) at 450 nm. Cell viability after incubation with testing materials at different concentrations at 48 h was measured as the absorbance of treated cells normalized to control cells (without treatment). IC50 (half maximal inhibitory concentration) values were obtained by nonlinear regression using Origin 8.5 software. All experiments were separately repeated at least three times, each performed in triplicate.

423

3. Results and discussion 3.1. Synthesis In this work, a new class of probes C1 was synthesized involving the hybridization of 1,4-dihydropyridines with 1,2,3-triazoles. DHPs and triazoles are well known for their broad pharmacological spectrum [50,51]. Compound 1 was successfully synthesized by modified Hantzsch synthesis including condensation of 2chlorobenzaldehyde, ethyl 3-oxo-4-(prop-2-yn-1-yloxy)butanoate and ammonium acetate using Ba(NO3)2 as a catalyst [52], which on reaction with (2R, 3R, 4S, 5R, 6R)-2-(acetoxymethyl)-6azidotetrahydro-2H-pyran-3,4,5-triyl triacetate via click reaction [53] led to formation of compound 2. Compound C1 was then obtained by the hydrolysis of compound 2 in presence of sodium methoxide in methanol. The iron-complex of C1 was synthesized using the protocol reported in the literature (Scheme 1) [54]. 3.2. UVevisible studies C1 absorbs radiation in the UV region and shows a strong band at 368 nm. Addition of 5 equivalents of different metal ions i.e., Agþ, Ca2þ, Cr3þ, Co3þ, Cu2þ, Fe2þ, Hg2þ, Mn2þ, Ni2þ, Pb2þ, Zn2þ, Cd2þ and Naþ showed no appreciable change in the absorbance. In the case of Fe3þ ion, a large difference in the absorbance was recorded, along with a band shift from 368 nm (C1) to 359 nm (C1-Fe3þ complex). Hence it was concluded that C1 shows better complexation with Fe3þ ion among all the tested metal ions, as shown in Fig. 1. 3.3. Fluorescence studies The probe C1 absorbs radiation in the UV region and shows emission in the blue region of the visible spectra. The probe C1 was excited at 360 nm wavelength and emission was observed at 456 nm. The high Stokes shift of 96 nm may be due to extended p conjugation of the dihydropyridine ring with the two ester groups. High Stokes shift fluorophores eliminate the possibility of spectral overlap between absorption and emission, which allows less interference in detection of fluorescence [55]. For better understanding about the binding affinity of C1, fluorescence experiments were carried out with different transition, alkali and some heavy metal ions. Metal ions Agþ, Ca2þ, Cr3þ, Co3þ, Cu2þ, Hg2þ, Mn2þ, Ni2þ, Pb2þ, Zn2þ, Cd2þ and Naþ showed very little changes in the

Fig. 1. Absorption spectra of C1 (50 mM) with 5 eq. of assorted metal ions in water.

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R. Kumar et al. / Dyes and Pigments 147 (2017) 420e428 Table 1 Stern -Volmer plot (Ksv) and detection limits (D.L.).

Fig. 2. Fluorescence emission spectra of C1 (50 mM) with5 eq. of different metal ions in water.

fluorescence intensity of C1, as shown in Fig. 2, whereas Fe2þ ion and Fe3þ showed appreciable quenching. We further analyzed the fluorescence quenching of C1 by stepwise increment of 1e20 equivalents of Fe2þ and Fe3þ ions. Fe3þ showed large extent of quenching of fluorescence intensity, as shown in Fig. 3(A). We further investigated C1 for its quenching constant (Ksv) both in the case of Fe3þand Fe2þ ions using SternVolmer plot [56], as shown in Fig. 3(B) and detection limit [57] in Fig. S5 (shown in supplementary material). The higher value of Ksv indicates stronger quenching of Fe3þ ion, in spite of the presence of Fe2þ ions (Table 1). Detection limit was calculated for both Fe2þ and Fe3þ ions, which further confirmed higher sensing of Fe3þ ion. The values of the detection limit and Stern-Volmer quenching constant are presented in Table 1.

3.3.1. Selectivity & binding stoichiometry The capability of binding of Fe3þ and Fe2þ ions in the presence of other metal ions was studied in aqueous solution. Equal concentrations of either Fe2þ or Fe3þ ion and other metal ions with C1

Complex

Ksv (M1)

D.L. (mM)

C1-Fe3þ C1-Fe2þ

9.27  102 6.26  102

16.78 20.00

were taken for analyzing the selectivity. As shown in Fig. 4, no significant fluorescence quenching was observed upon addition of different metal ions, except Fe2þ and Fe3þion (black bar). In case of Fe3þ in Fig. 4(A) and Fe2þ in Fig. 4(B) in presence of other metal ions, appreciable quenching was detected (red bar). It was also observed from Fig. 4(A) that the probe C1 became selective for Fe3þ ion even in presence of Fe2þ ion. Hence, C1 acts as a selective sensor for Fe3þ ion in the presence of other metal ions. Further, the binding stoichiometry was analyzed using the Benesi-Hildebrand plot [58]. The plot suggests 1:1 binding stoichiometry of C1 with Fe3þ ion. The association constant Ka (2.49  103 M1) was calculated as the ratio of the intercept and slope of the Benesi-Hildebrand plot (Fig. 5). To understand the nature of the complex, we further recorded the FTIR and HRMS spectra. We were unable to identify the profile of the -NH peak in the FT-IR spectrum due to 8 hydroxyl groups of the sugar moiety of C1, but HRMS gave a preliminary information about the disappearance of the -NH peak displaying signals at m/z 938.5601 corresponding to [C1-Hþþ Fe3þþ Hþ].

3.3.2. Fluorescence lifetime measurements The fluorescence lifetime of C1 was measured to get more information about the fluorescent properties. C1 was excited at 360 nm and the fluorescent decay obtained was bi-exponential (shown in supplementary material Fig. S8). The average fluorescence lifetime calculated by using equation (1) was 0.49 ns (Table 2).

3.3.3. Quantum yield measurements Quantum yield measurements of C1-Fe3þ and C1-Fe2þ complexes were accomplished relative to fluorescent probe C1. The quantum yield is calculated using equation (2) [59].

Fig. 3. (A) Change in emission intensity with number of equivalents of Fe3þ and Fe2þ ions for probe C1 (50 mM) (B) Stern-Volmer plot of C1 for Fe3þ versus Fe2þ.

R. Kumar et al. / Dyes and Pigments 147 (2017) 420e428

425

Fig. 4. Selectivity of Fe3þ versus Fe2þ ions in water containing probe C1 (50 mM) þ other metal ions (black bar) and C1 þ other metal ions þ (A) Fe3þ (red bar) or (B) Fe2þ (red bar) using fluorescence studies. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

conformational scan yielded the structure shown on the left of Fig. 6. It can be seen that the structure is asymmetric. In order to explore the structures for the iron complexes, complexes of both Fe (II) and Fe (III) were constructed and the structure of the latter is reproduced on the right of Fig. 6 below. Both Fe2þ and Fe3þ complexes have similar structures, but the ligand prefers a different conformation. The complexation energies were calculated using the relation Gcomplexation ¼ Gcomplex  Gmetal-ion  Gligand

Fig. 5. Benesi-Hildebrand plot for binding of Fe3þ ion with probe C1.

Table 2 Fluorescence lifetime measurements of C1. Entry C1

a1

a2

9.71  10



FS ¼ FR $

hs hR

2

2

2.06  10

2

t1 (ns)

t2 (ns)

(ns)

0.59

1.89

0.49

Fs A $ $ R F R AS

(2)

where FR and FS are the fluorescence quantum yields of the reference (C1) and the sample, respectively; hR and hS are the refractive indexes of the reference and the sample, respectively; AR and AS are the corresponding optical densities of the reference and the sample, respectively; and FR and FS are the area under the fluorescent spectra of the reference and the sample, respectively. According to the data, the relative fluorescence quantum yield of C1-Fe3þ and C1-Fe2þ complexes was found to be 0.22 and 0.62 respectively. A significant decrease in the relative quantum yield of the C1-Fe3þ complex in comparison to the C1-Fe2þ complex also indicates selective sensing of C1 towards the Fe3þ ion. 3.4. Density functional theory (DFT) studies The ligand is conformationally very flexible. A thorough

(3)

The Fe(II) complex is found to be low spin singlet and the complexation energy is calculated as 655.2 kcal mol1, implying that the complexation is highly exergonic. As can be seen from Fig. 6, the Fe(II) ion also forms five covalent bonds, three with nitrogens and two with oxygens. The total Mayer bond order is 2.9656. The respective bond orders of N11, O23, O27, N33 and N34 (Fig. 6) with Fe are 0.7572, 0.2945, 0.3389, 0.6784 and 0.6797, respectively. Thus, Fe(II) forms stronger bonds with the nitrogens of 1,2,3 triazoles, and the one with the nitrogen of the dihydropyridine ring is strongest. This is because this nitrogen is negatively charged in the ligand (0.643), having lost a proton. On complexation, its negative charge reduces to 0.454 and the positive charge on Fe reduces to 0.357, indicating that it accepts 1.643 e from the ligand. The Fe3þ complex is also a low-spin doublet. In agreement with our experimental results, its complexation energy (1264.7 kcal mol1) is almost double that of the ferrous complex. The net partial ESP charge on Fe is 0.506, which denotes transfer of 2.496 e from the ligand, accounting for the higher stability of this complex. The bonds of Fe with N11, O23, O27, N33 and N34 are also stronger, having bond orders of 0.8292, 0.3430, 0.3699, 0.6521 and 0.6533, respectively. The Mayer total valence is 3.0532. 3.5. In vitro cytotoxicity assay The in vitro cytotoxicity assay of probe C1 in absence and presence of FeCl3 against MCF-7, MDA-MB 231, A549, HepG2 and L929 cell lines is presented in Fig. 7. The effect of C1 was measured by MTT assay in terms of IC50 values, lower the IC50 value, higher the anti-proliferation effect. As shown in Table 3, probe C1and probe C1 in presence of FeCl3 are non-cytotoxic against all the cell lines, whether normal (L929) or cancerous (MCF-7, MDA-MB 231, A549 and HepG2). These studies indicate that the metal sensing probe C1 would not cause the killing of cells while detecting the metal ion, and that the complex of C1 with Fe3þion would also be non-cytotoxic.

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R. Kumar et al. / Dyes and Pigments 147 (2017) 420e428

Fig. 6. The optimized structures of the ligand C1 and C1-Fe3þcomplex.

Fig. 7. Cytotoxicities of (A) C1 and (B) C1þFeCl3 against various cancer and normal cell lines. Cell Viability was assessed by MTT assay. Data are expressed as the percentage of viable cells exposed to testing materials, with respect to untreated controls.

4. Conclusions

Table 3 IC50 values of C1 and C1þFeCl3 in various cancer and normal cell lines. IC50 values (mM) Species

MCF-7

MDA-MB 231

A549

HepG2

L929

C1 C1þFeCl3

161.28 62.21

205.72 68.06

197.77 66.60

165.06 70.56

153.94 104.41

In conclusion, we have reported a simple and facile procedure for the synthesis of a water-soluble probe C1 for sensing Fe(II) and Fe(III) ions. It has been well-characterized by NMR, HRMS and FT-IR spectroscopy. The sensor absorbs radiation in the UV region (368 nm) and emits in the visible region (456 nm). C1 binds with both Fe2þ and Fe3þ ions in the aqueous system with detection limits 20 mM and 16.78 mM and quenching constants (Ksv) 6.26  102 M1 and 9.27  102 M1, respectively. The sensor shows 1:1

R. Kumar et al. / Dyes and Pigments 147 (2017) 420e428

stoichiometry with the Fe3þ ion, as evident from the Benesi- Hildebrand plot. From the DFT calculations, the Fe2þ complex is found to be low-spin singlet with complexation energy 655.2 kcal mol1 implying that complexation is highly exergonic, whereas the Fe3þ complex is a low-spin doublet with complexation energy 1264.7 kcal mol1, almost double that of the ferrous complex. The probe C1 may support the detection of metal ions in the biological system as well without causing cell death, as is evident from its non-cytotoxicity towards various cell lines L929 (normal), and MCF-7, MDA-MB 231, A549 and HepG2 (cancerous) in the presence or absence of Fe3þ ions. The collective studies validate C1 as a highly effective sensor for the Fe3þ ion, which is non-toxic in the biological system as well. Acknowledgements The authors gratefully acknowledge University of Delhi for financial support under R&D grant. P.G. is thankful to UGC for fellowship and USIC at this university for instrument facilities. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.dyepig.2017.08.048. References [1] Tchounwou PB, Yedjou CG, Patlolla AK, Sutton DJ. Heavy metal toxicity and the environment. NIH 2014;101:133e64. [2] Weizman H, Ardon O, Mester B, Libman J, Dwir O, Hadar Y, et al. Fluorescently- labeled ferrichrome analogs as probes for receptor-mediated, microbial iron uptake. J Am Chem Soc 1996;118:12368e75. [3] D'Autreaux B, Tucker NP, Dixon R, Spiro S. A non-haem iron centre in the transcription factor NorR senses nitric oxide. Nature 2005;437:769e72. [4] WHO. Iron in drinking-water background document for development of WHO guidelines for drinking-water quality. World Heal Organ Guidel 2003;2:1e9. [5] Elinder C-G, Friberg L, Nordberg GF, Vouk VB, editors. Handbook on the toxicology of metals, vol. 2. Elsevier; 1986. p. 276e97. [6] Carter KP, Young AM, Palmer AE. Fluorescent sensors for measuring metal ions in living systems. Chem Rev 2014;114:4564e601. [7] Chi Z, Ran X, Shi L, Lou J, Kuang Y, Guo L. Molecular characteristics of a fluorescent probe for the recognition of ferric ion based on photoresponsive azobenzene derivative. Spectrochim Acta - Part A Mol Biomol Spectrosc 2017;171:25e30. [8] Aisen P, Resnick MW, Leibold EA. Iron metabolism. Curr Opin Chem Biol 1999;3:200e6. [9] Galaris D, Skiada V, Barbout A. Redox signalling and cancer: the role of “labile” iron. Cancer Lett 2008;266:21e9. [10] Desai NK, Kolekar GB, Patil SR. Offeon fluorescent polyanthracene for recognition of ferric and fluoride ions in aqueous acidic media: application in pharmaceutical and environmental analysis. New J Chem 2014;38:4394e403. [11] Ong WY, Farooqui AA. Iron, neuroinflammation and Alzheimer's disease. Alzheimers Dis 2005;8:183e200. [12] Park GJ, You GR, Choi YW, Kim C. A naked-eye probe for simultaneous detection of iron and copper ions and its copper complex for colorimetric/ fluorescent sensing of cyanide. Sensors Actuators B Chem 2016;229:257e71. [13] Department of National Health and Welfare (Canada). Nutrition recommendations. The report of the Scientific Review Committee. Ottawa. 1990. [14] Mahmoud ME, Kenawy IMM, Hafez MMAH, Lashein RR. Removal, preconcentration and determination of trace heavy metal ions in water samples by AAS via chemically modified silica gel N-(1-carboxy-6-hydroxy) benzylidenepropylamine ion exchanger. Desalination 2010;250:61e70. [15] Alqadami AA, Abdalla MA, AlOthman ZA, Omer K. Application of solid phase extraction on multiwalled carbon nanotubes of some heavy metal ions to analysis of skin whitening cosmetics using ICP-AES. Int J Env Res Pub He 2013;10:361e74. [16] Liu SR, Wu SP. New water-soluble highly selective fluorescent probe for Fe (III) ions and its application to living cell imaging. Sensors Actuators B Chem 2012;171e172:1110e6. [17] Rajasekar M, Das TM. One-pot synthesis of fluorescein based baminoglycosylketones and their biological and material applications. RSC Adv 2014;4: 42538e45. [18] Wu K, Xiao H, Wang L, Yin G, Quan Y, Wang R. A rhodamine derivative as a highly sensitive probe for iron(III). RSC Adv 2014;4:39984e90. [19] Rajasekar M, Das TM. Synthesis, characterization and gelation studies of a novel class of rhodamine based N-glycosylamines. RSC Adv 2014;4:30976e83.

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