Fe2+

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Article Cite This: ACS Omega 2018, 3, 18646−18655

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Pyrrolo[3,4‑c]pyridine-Based Fluorescent Chemosensor for Fe3+/Fe2+ Sensitivity and Their Application in Living HepG2 Cells Pampa Maity,† Barnali Naskar,† Sanchita Goswami,† Chandraday Prodhan,‡ Tandrima Chaudhuri,§ Keya Chaudhuri,‡ and Chhanda Mukhopadhyay*,† †

Department of Chemistry, University of Calcutta, 92 APC Road, Kolkata 700009, India Molecular & Human Genetics Division, CSIR−Indian Institute of Chemical Biology, 4 Raja S. C. Mallick Road, Kolkata 700032, India § Department of Chemistry, Dr. B. N. Dutta Smriti Mahavidyalaya, Burdwan 713407, India Downloaded via 79.110.25.158 on December 28, 2018 at 17:00:43 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: New 2H-pyrrolo[3,4-c]pyridine-1,3,6(5H)-trione-based fluorophores have been synthesized by a single-step procedure in an ambient reaction condition. By this protocol, a total of 44 new compounds were generated and the photophysical property of 23 compounds was studied in detail. These fluorophores show good fluorescence activity and illustrate high selectivity for Fe3+/Fe2+ cation in the 10−7 (M) range. Notably, this 2H-pyrrolo[3,4-c]pyridine-1,3,6(5H)-trione moiety is first used as a turn-off chemosensor for the Fe3+/Fe2+ cation. In addition, this probe was further applied for imaging Fe3+ in living HepG2 cells.

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INTRODUCTION Small-molecule organic fluorophores are an important candidate in the multidisciplinary field of chemosensing, photovoltaic, confocal microscopy, organic light-emitting diode technology, and polymer and textile industry because of their enormous potential.1 Hence, they have attracted considerable attention to a broader scientific community including biology, chemistry, and material science.2 Toward that end, development of fluorogenic probes for selective sensing of metal ions of biological interests has certainly become relevant. Of the different metal ions such as iron, aluminum, copper, and zinc, iron is the most abundant essential trace element present in the human body.3 Because of the high affinity toward oxygen, iron is critically engaged in oxygen transport in all the tissues.4 However, it is really important to have a certain balance of the iron content in the human body. Whereas the deficiency could lead to anemia, hemochromatosis, diabetes, liver damage, and cancer, the excess of iron could cause severe damage to nucleic acids, lipids, and proteins, which further accelerates the possibility of serious diseases like Alzheimer’s, Parkinson’s, and so forth.5 Therefore, the pursuit of detection of iron with a high level of selectivity and sensitivity is a decisive aspect. In this regard, © 2018 American Chemical Society

various analytical methods and electrochemical methods have come up for critical detection of metal ions.6 However, timeconsuming sample pretreatments, along with strenuous procedures and expensive equipment limit their extensive use.7 Thus, an alternative approach considering the low cost, high sensitivity, easy monitoring of target ions, and nondestructive in nature could prove effective. Herein, the fluorescent sensors could be marked as appropriate means because of their aforementioned advantages.8 Although, fluorescence-based sensing has got a substantial deal of recognition, Fe3+ selective fluorescent probes are sporadic because of their paramagnetic nature, resulting in fluorescence quenching.9 Although there are several instances where turnon fluorescence responses by Fe3+ have been observed, the selectivity was significantly disrupted by the presence of other common metal ions such as Cu2+ and Cr3+.10 Hence, there is high demand to design an easy but selective and sensitive fluorescence-based Fe3+ sensing active core with cost-effective synthetic strategy. Received: August 20, 2018 Accepted: December 14, 2018 Published: December 28, 2018 18646

DOI: 10.1021/acsomega.8b02110 ACS Omega 2018, 3, 18646−18655

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Table 1. Substrate Scope of Selective Dialkylated Productsa

In a recent advancement, we have developed the 2Hpyrrolo[3,4-c]pyridine-1,3,6(5H)-trione core by multicomponent reaction in aqueous medium and its detailed photophysical property was further investigated.11 It is due to its comparatively lower quantum yield that further substitution in the active core is rather very necessary. Keeping this in perspective, herein, we report a novel, easy-to-make, Nsubstituted pyrrolo[3,4-c]pyridine derivative which can detect Fe3+/Fe2+ fluorimetrically. Although there are several turn-off and turn-on probes reported in the literature for determination of Fe3+/Fe2+ selectively, these probes have some drawbacks such as synthetic difficulties, ligand cytotoxicity, low fluorescence quantum yield, and cross-sensitivity with other common metal cations. Besides this, we have prepared this pyrrolo[3,4-c]pyridine derivative via an easy and simple procedure and quantum yield of this probe is quite high. This is so far the first report in which the N-substituted 2Hpyrrolo[3,4-c]pyridine-1,3,6(5H)-trione has been used as an Fe3+/Fe2+ sensor.

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RESULTS AND DISCUSSION Synthesis. Herein, we report the synthesis of highly substituted 2H-pyrrolo[3,4-c]pyridine-1,3,6(5H)-trione fluorophore and its fluorometric attempt toward effective detection of Fe3+/Fe2+ ions (Table 1). The selective sensing has been extended in HepG2 cells. They have been very easily synthesized using unsubstituted pyrrolo[3,4-c]pyridine triones and alkyl halides (benzyl, allyl, ethyl, n-propyl, n-butyl) in the presence of anhydrous K2CO3 and dimethylformamide (DMF) at room temperature (25−30 °C) in a single-step procedure. This reaction is highly efficient and offers the opportunity toward a broad substrate scope (Tables 2 and 3). To determine the best optimal condition, the reaction has been screened first in different solvent mediums. Because of the low soluble nature of compound 1x in ethanol, methanol, and water, it produces very trace amounts of product in these solvents at room temperature. Although there is a certain improvement in the percentage of product formation by following the refluxing condition, it does not exceed the 60% yield. On the other hand, in DMF medium at room temperature, the product yield reaches up to 88% (Scheme 1). In a reaction between 1x and 2y, there is a possibility of formation of two different products, that is, 3xy and 4xy. The compound 1x contains two labile H atoms which can be easily substituted by alkyl groups. When 1 equivalent of compound 1x and 2 equivalents of compound 2y are present, compound 3xy is formed where both the −NH and −NH2 groups get substituted by alkyl groups. On the other hand, in the presence of 1 equivalent of compound 1x and 1 equivalent of compound 2y, compound 4xy has formed as the −NH group is more labile than −NH2. In the presence of 2 equivalents of benzyl bromide, allyl bromide, ethyl iodide, dialkylated product, that is 3xy is formed. However, in the case of n-propyl bromide and n-butyl bromide, compound 4xy is formed as a major product even if 2 equivalents of n-propyl bromide and n-butyl bromide are used. This can be attributed to the steric crowding of the long chain of this group that leads to 4xy as the major product and compound 3xy as a minor product. An assembly of 44 compounds were synthesized following this strategy and compounds were characterized by 1H, 13C NMR, HRMS, IR, elementary analysis, and X-ray single-crystal analysis (Figure 1).

a

Reaction conditions: substrates 1x (1 mmol) and 2y (2 mmol) were stirred with anhydrous K2CO3 at 25−30 °C for 12 h in 7 mL DMF. b Isolated yields.

Table 2. Synthesized Monoalkylated Products

a

Reaction conditions: substrates 1x (1 mmol) and 2y (1 mmol) were stirred with anhydrous K2CO3 at 25−30 °C for 12 h in 7 mL of DMF. b Isolated yields.

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Table 3. Substrate of Di and Monoalkylated Products

a Reaction conditions: substrates 1x (1 mmol) and 2y (2 mmol) were stirred with anhydrous K2CO3 (2 mmol) at 25−30 °C for 12 h in 7 mL of DMF. bIsolated yields.

Scheme 1. General Scheme of Fluorophore Synthesis

Figure 1. Oak Ridge Thermal Ellipsoid Plot (ORTEP) diagram of the compound 3ea (CCDC no. 1862004).

Photophysical Property. Photophysical properties of chemosensors in the presence of Fe3+: To investigate the UV−vis spectral changes of chemosensors (3aa, 3ca, 3da, 3fa, 3ab, 3cb, 3db, 3fb, 3ac, 3cc, 3dc, 3fc, 3ad, 3cd, 3dd, 3fd, 3ae, 3ce, 3de, 3fe, 4ac, 4cd, 4ce), the

measurements were conducted in dimethyl sulfoxide (DMSO)/H2O (1:9, v/v) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH = 7.4) solution. 18648


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Adding different concentrations of Fe3+ to the solution of chemosensors, notable changes in the spectral output occur (Figures S90−112, Supporting Information). The fluorescence spectral properties of chemosensors (3aa, 3ca, 3da, 3fa, 3ab, 3cb, 3db, 3fb, 3ac, 3cc, 3dc, 3fc, 3ad, 3cd, 3dd, 3fd, 3ae, 3ce, 3de, 3fe, 4ac, 4cd, 4ce) were investigated in DMSO/H2O (1:9, v/v) HEPES buffer (pH = 7.4) solution. The free chemosensors exhibited significant fluorescence intensity. On stepwise addition of different concentrations of Fe3+ to the solution of chemosensors, a gradual decrease in the fluorescence intensity occurred (Figure 2). In order to evaluate

ranging from polar protic to nonpolar solvent which has been depicted in Table S1, Supporting Information. To further assess the coordination of chemosensors with Fe3+, electrospray ionization (ESI) mass spectra were recorded. The ESI-mass spectrometry spectra of compound 3ab (Figure 4) showed an m/z peak at 336.1344 whereas in the case of the

Figure 2. Emission spectra of 3ab (5 × 10−6 M) in the presence of increasing amounts of [Fe3+] (0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, and 10 (×10−6) M in DMSO/H2O (1:9, v/v) HEPES buffer (pH = 7.4) solution (λex = 365.98 nm, λem = 486.09 nm). Inset: Fluorescence emission intensity of 3ab at 486.09 nm as a function of [Fe3+].

the selectivity and suitability of chemosensors (3aa, 3ab, 3ac, 3ad) for practical applications as chemosensors for Fe3+, competitive experiments were carried out in the presence of various metal ions such as Li+, Na+, K+, Ca2+, Mg2+, Mn2+, Ba2+, Cu2+, Ni2+, Co2+, Zn2+, Cd2+, Hg2+, Pb2+, Sr2+, Cr3+, and Al3+-in DMSO/H2O (1:9, v/v) HEPES buffer (pH = 7.4) solution. It was observed that other metal ions did not provide any interference toward selective detection of Fe3+ except Fe2+, indicating excellent selectivity for Fe3+ and Fe2+ over these competing cations (Figure 3). We have also investigated the effect of photophysical properties of chemosensors (3aa, 3ca, 3da, 3fa, 3ab, 3cb, 3db, 3fb, 3ac, 3cc, 3dc, 3fc, 3ad, 3cd, 3dd, 3fd, 3ae, 3ce, 3de, 3fe, 4ac, 4cd, 4ce) in different solvents

Figure 4. (a) HRMS of compound 3ab. (b) Mass peak for the compound 3ab and Fe3+ and (c) Job’s plot for determination of stoichiometry of the Fe3+/3ab complex in solution.

compound-Fe3+ complex the m/z peak is observed at 195.0275. This suggested the 1:1 binding mode of Fe3+ and chemosensors. The chemosensor/Fe3+ stoichiometry was determined for 3aa, 3ab, 3ac, 3ad from Job’s plot analysis in DMSO/H2O (1:9, v/v) HEPES buffer (pH = 7.4) solution and it was found to be 1:1 (Figures S136−138, Supporting Information). The binding constant of chemosensors (3aa, 3ab, 3ac, 3ad) with Fe3+ was calculated by the Benesi−Hildebrand equation (Figures 5 and S139−141, Supporting Information). The binding constant (K) and the detection limit (LOD) of the chemosensors (3aa, 3ab, 3ac, 3ad) (Figures 5 and S143−145, Supporting Information) were measured using fluorescence titration data (Table 4). The binding constant of chemosensor 3ab with Fe2+ was also calculated and it has been shown that this value is comparatively lower than that of chemosensor 3ab with the Fe3+ complex. The fluorescence titration data, Benesi−Hildebrand equation, and the LOD of the chemosensor 3ab with Fe2+ are shown in Figures S135, 142, and 146 respectively (Supporting Information). The binding interactions of chemosensors (3aa, 3ab, 3ac, 3ad) with Fe3+ were investigated. As Na2 ethylenediaminetetraacetic acid (EDTA) offers a very good chelating environment for Fe3+, an increase in the fluorescence intensity takes place, which revealed regeneration of the free chemosensor (3aa, 3ab, 3ac, 3ad). Further addition of Fe3+ to the solution mixture, immediately quenches the emission intensity (Figures

Figure 3. Fluorescence quenching efficiency of 3ab (5 × 10−6 M) in the presence of 4 equiv of different cations except 2 equiv of Fe3+ in solution [the magenta bar portion]. Fluorescence intensity of a mixture of 3ab (5 × 10−6 M) with other metal ions (20 × 10−6 M) followed by addition of Fe3+ (10 × 10−6 M) to the HEPES buffer (pH = 7.4) solution [the cyan bar portion] (λex = 365.98 nm, λem = 486.09 nm). 18649


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Table 4. Binding Constant (K) and Limit of Detection Were Studied with Complexation Properties of the Compounds (3aa, 3ca, 3da, 3fa, 3ab, 3cb, 3db, 3fb, 3ac, 3cc, 3dc, 3fc, 3ad, 3cd, 3dd, 3fd, 3ae, 3ce, 3de, 3fe, 4ac, 4cd, 4ce) compound 3aa 3ca 3da 3fa 3ab

3cb 3db 3fb 3ac 3cc

Figure 5. (a) Benesi−Hildebrand plot 1/(F0 − F) vs 1/[Fe3+] for complexation between 3ab and Fe3+ derived from the emission titration curve and (b) LOD of 3ab for Fe3+ as a function of [Fe3+].

3dc 3fc 3ad

S150−152). These results clearly indicated reversibility and reusability of the receptors (3aa, 3ab, 3ac, 3ad) (Figure 6). To elucidate the practical application of chemosensors (3aa, 3ab, 3ac, 3ad), pH titration experiments were carried out to investigate the effects of pH on the fluorescent chemosensors in DMSO/H2O (1:9, v/v) solution and pH was adjusted with the help of HCl/NaOH. As shown in Figure S153 (Supporting Information), in the pH range of 6.0−9.0, a distinct fluorescence was observed in the absence of Fe3+. Upon the addition of Fe3+, fluorescence quenching is observed in the pH 6.0−9.0 region, suggesting suitability of the chemosensors (3aa, 3ab, 3ac, 3ad) under physiological conditions at pH = 7.4 which is required for biological and environmental applications. Theoretical Calculation. Density functional theory (DFT) and time-dependent DFT (TDDFT) calculation of ligand 3ab is carried out to examine the electronic structure of the ligand and its metal complexes. Compound 3ab does not show much solvatochromism. As 3ab contains N and O donor in close proximity its anionic form 3ab−1 is very much resonance stabilized via these two tautomeric forms (shown in Figure 7); thus, −NH proton of the ligand is very much labile. Thus, photoinduced electron transfer (PET) is very much possible in 3ab. Frontier molecular orbital picture of 3ab and its anionic form are almost similar (Figure 8) just with slight minimization of band gap (from 4.04 eV of 3ab to 3.42 eV of its anion). TDDFT results also show mainly highest occupied molecular orbital (HOMO) to lowest unoccupied molecular orbital (LUMO) transition predominates on excitation. Anion (3ab−1) has higher oscillators strength, that is, higher

3cd 3dd 3fd 3ae 3ce 3de 3fe 4ac 4cd 4ce

quantum yielda (Φf) 0.62 0.06 0.62 0.05 0.62 0.04 0.62 0.04 0.63 0.03 0.03 0.63 0.02 0.64 0.02 0.63 0.02 0.65 0.03 0.66 0.03 0.67 0.03 0.67 0.03 0.64 0.04 0.64 0.04 0.67 0.04 0.68 0.04 0.62 0.03 0.64 0.04 0.65 0.04 0.63 0.03 0.64 0.03 0.65 0.03 0.65 0.03

(free 3aa) (3aa + Fe3+) (free 3ca) (3ca + Fe3+) (free 3da) (3da + Fe3+) (free 3fa) (3fa + Fe3+) (free 3ab) (3ab + Fe3+) (3ab + Fe2+) (free 3cb) (3cb + Fe3+) (free 3db) (3db + Fe3+) (free 3fb) (3fb + Fe3+) (free 3ac) (3ac + Fe3+) (free 3cc) (3cc + Fe3+) (free 3dc) (3dc + Fe3+) (free 3fc) (3fc + Fe3+) (free 3ad) (3ad + Fe3+) (free 3cd) (3cd + Fe3+) (free 3dd) (3dd + Fe3+) (free 3fd) (3fd + Fe3+) (free 3ae) (3ae + Fe3+) (free 3ce) (3ce + Fe3+) (free 3de) (3de + Fe3+) (free 3fe) (3fe + Fe3+) (free 4ac) (4ac + Fe3+) (free 4cd) (4cd + Fe3+) (free 4ce) (4ce + Fe3+)

binding constant (K) (M−1)

LODb (M)

3.25 ± 0.04 × 105

4.03 × 10−7

3.47 ± 0.06 × 105

4.14 × 10−7

3.56 ± 0.03 × 105

4.06 × 10−7

3.34 ± 0.04 × 105

4.19 × 10−7

3.84 ± 0.05 × 105 3.24 ± 0.04 × 105

3.87 × 10−7 4.19 × 10−7

3.16 ± 0.08 × 105

4.28 × 10−7

3.64 ± 0.06 × 105

4.21 × 10−7

3.34 ± 0.05 × 105

4.04 × 10−7

3.59 ± 0.07 × 105

4.12 × 10−7

3.15 ± 0.03 × 105

4.15 × 10−7

3.34 ± 0.08 × 105

4.23 × 10−7

3.64 ± 0.06 × 105

4.11 × 10−7

3.53 ± 0.04 × 105

4.37 × 10−7

3.34 ± 0.09 × 105

4.34 × 10−7

3.62 ± 0.03 × 105

4.35 × 10−7

3.53 ± 0.06 × 105

4.29 × 10−7

3.63 ± 0.07 × 105

4.17 × 10−7

3.45 ± 0.05 × 105

4.09 × 10−7

3.25 ± 0.02 × 105

4.07 × 10−7

3.32 ± 0.03 × 105

4.11 × 10−7

3.42 ± 0.05 × 105

4.05 × 10−7

3.64 ± 0.07 × 105

4.12 × 10−7

3.53 ± 0.04 × 105

4.33 × 10−7

Error in measurement ±3%. b = ±4%, where b is the standard deviation.

a

absorptivity which was also found experimentally during titration absorbance of 3ab increases (Figure S94). The ground-state geometry of the metal complexed 3ab has been optimized with MMFF calculation. From the experiment, it is seen that Fe3+ forms complexes with 3ab in a 1:1 stoichiometry. Quantum chemical structure optimizations were 18650


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Figure 6. Fluorescence emission spectra of chemosensor (3ab) in the presence of Fe3+ ion followed by addition of EDTA. Figure 9. (a) Frontier molecular orbital (FMO) picture of groundstate optimized (3ab−Fe)2+ shows ligand-to-metal charge transfer (LMCT). (b) Optimized geometry of (3ab−Fe)2+ adduct.

solvation. The energies of both HOMO and LUMO of 3ab were stabilized upon complexing with metal ion. The frontier molecular orbital picture of Figure 8a indicates that in 3ab, HOMO is primarily centered over the pyridine ring, whereas LUMO is extended toward the fused fivemembered ring structure, thus simply indicating intramolecular charge transfer from the pyridinium ring to the fused ring. For the ferric ion complex the metal ion centre was calculated to be closer to the oxygen donor might inhibit the photo induced electron transfer (PET). This can be predicted from Figure 9 because of a decrease in HOMO density on the donor centers compared to that in uncomplexed 3ab in Figure 8a. Thus, it might be stated that through inhibition of PET, 3ab can sense the iron (3+) ions and the intensity of fluorescence of 3ab needs to enhance in the presence of such metal ions. However, Fe3+ being paramagnetic in nature causes massive quenching of fluorescence (Figure 2). We have proposed a plausible mechanistic pathway to generate the chemosensor/Fe3+ ensemble in Scheme 2. The

Figure 7. Tautomeric form of compound 3ab.

Scheme 2. Plausible Mechanism for the Response toward Fe3+

Figure 8. FMO picture of ground state optimized 3ab and its anionic form.

performed accordingly (Figure 9). Figure 9b shows that Fe3+ was precisely centered over the oxygen with a metal−ligand distance of 2.95 Å. In order to obtain information about the role of frontier molecular orbitals in the extent of interaction, a single-point density-based calculation using the MPW1PW91/ 6-31G method has been employed for the metal ion interactions and the ground-state energy parameters have been presented in Table S2 (Supporting Information). The band gap of 3ab (4.04 eV) was shifted to low energy, that is, to longer wavelength on interaction with metal ion, which effect was not observed experimentally. Again, in the case of the 3ab−1 ion, it has a similar band gap (3.41 eV), whereas in the case of Fe3+, the band gap further decreases (0.52 eV). The difference between theoretically calculated band gap and experimental excitation parameters might arise due to

change in fluorescence signature of the chemosensors as a result of binding to Fe3+ might be attributed to deprotonation of −NH allowing charge transfer from chemosensor to Fe3+. Biological Application. The fluorescence emission property of 3ab was further evaluated in vitro because of the quenching of the emission after selective binding of 3ab with Fe3+ ions. The effective dose of ligand 3ab was determined by evaluating the cell viability of HepG2 cells in the presence of different concentrations of 3ab with the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. No significant decrease in cell viability was found up to 40 μM concentration of 3ab, suggesting that concentrations lower than 40 μM will be appropriate to study the in vitro 18651


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fluorescence properties of 3ab. Higher than 90% of HepG2 cells are viable at 10 μM of 3ab, which was used as the treatment dose for further experiments. No fluorescence emission was found when HepG2 cells were treated with 10 μM of Fe3+ ions. However, 10 μM of ligand 3ab exhibited prominent blue intracellular fluorescence in HepG2 cells, mostly localized throughout the cytoplasm of the cells, but hardly in the nucleus. Meanwhile, the intracellular fluorescence emission of 10 μM of ligand 3ab got significantly quenched in the presence of 10 μM of Fe3+ ions because of their binding with the ligand. Increasing the Fe3+ ions concentration from 10 to 20 μM resulted in concentrationdependent quenching of the intracellular fluorescence. Thus, the ligand 3ab can function as a potential biodetector of Fe3+ ions with reduced cytotoxic effect (Figures 10 and 11).

biocompatible and low cytotoxic nature it can be easily used for Fe3+ ion detection in biological samples.

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EXPERIMENTAL SECTION General Information. A Bruker 300 MHz instrument was used for 1H and 13C NMR spectra at 300 and 75 MHz, respectively. DEPTQ-135 experiments were performed on Bruker 300 MHz instruments at 75 MHz. Chemical shifts are reported in parts per million downfield from an internal tetramethylsilane reference. Coupling constants (J) are reported in hertz, and spin multiplicities are represented by the symbols s (singlet), brs (broad singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). Using a PerkinElmer Spectrophotometer, RX/FT-IR system IR spectra were recorded. Band positions are reported in reciprocal centimeters (cm−1). The CHN analyses were performed on a 2400 Series II CHNS Analyzer, PerkinElmer USA. Melting points were determined on an electrical melting point apparatus with an open capillary. The progress of the reaction was checked by thin-layer chromatography (TLC) using a 300−400 mesh silica gel. All the available reagents were purchased from commercial sources and used without purification. All the solvents used during reactions were distilled for purity. The ESI-mass spectroscopy was performed on a Qtof Micro YA263 mass spectrometer. The absorption spectra were obtained using a Hitachi UV−vis U-3501 spectrophotometer and emission spectra were recorded on a PerkinsElmer LS55 fluorimeter.12 General Procedure for the Synthesis of Compounds. Pyrrolo[3,4-c]pyridine-1,3,6(5H)-trione (1 mmol) was mixed with anhydrous K2CO3 (2 or 1 mmol) and alkyl halide (2 or 1 mmol) in a 25 mL round-bottomed flask containing 7 mL of dry DMF. Then, it was stirred at room temperature (25−30 °C) for 12 h and the progress of the reaction was monitored by TLC. The spot for pyrrolo[3,4-c]pyridine-1,3,6(5H)-trione disappeared after 12 h. After that, the reaction mixture was extracted with ethyl acetate and it was subjected to column chromatography using 60−120 mesh silica gel and petroleum ether−ethyl acetate mixture as the eluent and the pure products 3xy were obtained at 10−12% ethyl acetate in petroleum ether and 4xy at 20−25% ethyl acetate in petroleum ether, respectively.13 Evaluation of Fluorescence Quantum Yield in Solution. Fluorescence quantum yield was determined using quinine sulfate (ΦR = 0.546 in 0.1 M H2SO4) as standard in

Figure 10. Percentage of HepG2 cells viable after treatment with different concentrations (1−100 μM) of 3ab (measured by MTT assay).

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CONCLUSIONS In this work, we have developed a new 2H-pyrrolo[3,4c]pyridine-1,3,6(5H)-trione-based fluorescent chemosensor. This probe exhibits a very good fluorescence sensing ability to Fe3+/Fe2+ cation over other common cations. This probe formed a 1:1 complex with the metal ion, which can be confirmed by Job’s plot, ESI-mass spectroscop,y and Benesi Hildebrand equation. Here, EDTA has been used as a coordinating agent that regenerates the free ligand from its complex form and this free ligand further forms a 1:1 complex with metal ion. Moreover, an in vitro fluorescent cell imaging study of this probe demonstrates that because of its

Figure 11. Representative image of the fluorescence emission of 10 μM of 3ab in HepG2 cells captured at 40× magnification. Absence of fluorescence signal from HepG2 cells was observed in the presence of 10 μM of Fe3+ ions, whereas a prominent fluorescence signal was found in the presence of 10 μM of 3ab ligand. However, the fluorescence emission gradually quenched with increasing concentration of Fe3+ ions. 18652


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concentration of 105 cells/well were treated with different concentrations of 3ab (1−100 μM) and incubated for 24 h at 37 °C. Cell-viability was determined by adding MTT solution (500 μg/mL) following incubation at 37 °C for 4 h. The intracellular formazan formed by reduction of MTT was solubilized in 0.04 N acidic isopropyl alcohol, and absorbance at 590 nm was determined with a 96-well plate reader (PHERAstar FSX Multimode Microplate Reader, BMG LABTECH, Germany). Cell viability was expressed as a percentage of the absorbance optical density (OD) corresponding to cells without treatment. Characterization of in Vitro Fluorescence. To characterize the fluorescence emission property of 3ab, HepG2 cells were seeded over a coverslip at a concentration of 3 × 105 cells, and then incubated with 10 μM of 3ab at 37 °C for 60 min. Evaluation of quenching of fluorescence emission of 3ab after binding with intracellular Fe3+ ions was done by treating HepG2 cells with 10 and 20 μM of Fe3+ ions at 37 °C for 60 min, followed by subsequent incubation with 10 μM of 3ab at 37 °C for 60 min. Fluorescence microscopy was done with Leica DM3000 (Germany) at 40× magnification.

methanol solution. The quantum yield was calculated using the following equation. ϕs = ϕR

η2 As Abs R × × S2 AR AbsS ηR

where the A terms denote the integrated area under the fluorescence curve, Abs denotes absorbance, η is the refractive index of the medium, and Φ is the fluorescence quantum yield. Subscripts S and R denote the respective parameters for the studied sample and reference, respectively. Sample Preparation for UV−Vis and Fluorescence Spectral Studies. A stock solution of chemosensors (3aa, 3ca, 3da, 3fa, 3ab, 3cb, 3db, 3fb, 3ac, 3cc, 3dc, 3fc, 3ad, 3cd, 3dd, 3fd, 3ae, 3ce, 3de, 3fe, 4ac, 4cd, 4ce) for UV−visible and emission titration studies were prepared in DMSO/H2O (1:9, v/v) HEPES buffer (pH = 7.4) solution and for various metal ions were prepared in DMSO/H2O (2:8, v/v) medium. In UV−visible titration experiment, a stock solution of chemosensors (3aa, 3ca, 3da, 3fa, 3ab, 3cb, 3db, 3fb, 3ac, 3cc, 3dc, 3fc, 3ad, 3cd, 3dd, 3fd, 3ae, 3ce, 3de, 3fe, 4ac, 4cd, 4ce) 10−3 (M) were filled separately in a quartz optical cell of 1.0 cm optical path length to achieve a final concentration of solution of compounds 5 × 10−6 (M) in 2000 μL and metal ions were added using a micropipette to a solution of these chemosensors. For emission titration experiment, 10 μL of 10−3 (M) solution of each compounds (3aa, 3ca, 3da, 3fa, 3ab, 3cb, 3db, 3fb, 3ac, 3cc, 3dc, 3fc, 3ad, 3cd, 3dd, 3fd, 3ae, 3ce, 3de, 3fe, 4ac, 4cd, 4ce) were taken in a quartz optical cell of 1.0 cm optical path length in 2000 μL and then stock solutions of metal ions were added gradually to it by using a micropipette. Spectral data were recorded at 1 min after the addition of the metal ions for both titrations. For the titration experiment, we used the cations, viz. [Li+, Na+, K+, Ca2+, Mg2+, Mn2+, Ba2+, Cu2+, Ni2+, Co2+,Fe3+, Fe2+, Zn2+, Cd2+, Hg2+, Pb2+, Sr2+, Al3+ and Cr3+] as their chloride salts. Determination of Fe3+ in DMSO/H2O (1:9, v/v) HEPES Buffer (pH = 7.4) Solution. Using the fluorescence titration data, the LOD was determined on the basis of 3σ/K.14 In Figures S143−S145 reveals a good linear correlation between the value of relative fluorescence (ΔF = F0 − F) and the concentration of Fe3+ ions with a correlation coefficient (R2) of 0.9988. A linear regression curve was then fitted to these normalized ratio data, and the point at which this line crossed the coordinate axis was considered as the LOD. The LOD was calculated using the following equation. σ DL = K × S

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b02110. Characterization, 1H, 13C NMR spectra, and full spectroscopic data of all compounds (PDF) Crystallographic data (CIF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Sanchita Goswami: 0000-0002-1045-2508 Chhanda Mukhopadhyay: 0000-0003-2065-3378 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS One of the authors (P.M.) would like to thank the University Grants Commission (UGC), New Delhi, for her fellowship (SRF). We would also like to thank CAS-V, Department of Chemistry, University of Calcutta, for funding as a departmental project.

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where K = 2 or 3 (we take 3 in this case), σ is the standard deviation of the blank solution, and S is the slope of the calibration curve. Cell Culture for in Vitro Studies. In vitro studies were done to identify the biological application of ligand 3ab using human liver carcinoma cell line HepG2 obtained from NCCS, Pune, India; the cells were maintained in complete growth medium containing Dulbecco’s modified Eagle’s medium, 10% fetal calf serum, 1% penicillin−streptomycin (10 mg/mL stock) in CO2-Incubator at 5% CO2 concentration, and 37 °C temperature. Determination of Cytotoxicity by MTT Assay. In order to determine the cytotoxic effect of ligand 3ab on living mammalian cells, HepG2 cells seeded in a 96-well plate at a

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