Fluorescent Lower Rim 1,3-Dibenzooxadiazole Conjugate of Calix[4

Aug 27, 2018 - The study has been extended to the biological cells using fluorescence and confocal microscopy. While L shows strong fluorescence in He...
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Fluorescent Lower Rim 1,3-Dibenzooxadiazole Conjugate of Calix[4]arene in Selective Sensing of Fluoride in Solution and in Biological Cells Using Confocal Microscopy Bhawna Uttam, Ravinder Kandi, M. Althaf Hussain, and Chebrolu Pulla Rao* Bioinorganic Laboratory, Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India

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ABSTRACT: p-tert-Butyl-calix[4]arene was derivatized by integrating a benzooxadiazole fluorescent tag into its 1,3-arms at the lower rim to result in L and was characterized. L was titrated with 17 anions in THF and found selective for F− ions with lowest detection limit of 109 ppb. L and F− form a 1:1 complex. L self-assembles in THF to result in sheet like structures which converts into smaller spherical particles upon addition of F−. The site of interaction of F− was deduced based on 1H NMR spectroscopy and the coordination features by density functional theory (DFT) computations wherein six noncovalent interactions of the type X−H···F (where X = O, N, or C) were noticed. The sensing of F− is reversible when titrated with Ca2+, and the reversibility was demonstrated for 10 cycles without losing sensitivity. The study has been extended to the biological cells using fluorescence and confocal microscopy. While L shows strong fluorescence in HeLa cells, increasing concentrations of F− exhibited greater fluorescence quenching. Thus, L acts as a good sensor for F− in solution as well as in biological cells, a rare and unique combination for a calixarene conjugate to exhibit such sensing behavior in dual media.

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though there is no report to our knowledge where fluoride ions are being detected both in solution and in biological cells using a calixarene conjugate. This paper deals with the development of a conjugate of calix[4]arene (Figure 1) that can sense fluoride in solution as well as in the biological cells. The choice of the 7-chloro 4nitro benzofurazan (NBD) as fluorescent tag comes from its utility in cell imaging in the literature.36−39 Therefore, we report the synthesis of a fluorophore built on a calix[4]arene framework which is functionalized with NBD to result in a

INTRODUCTION In recent times, there has been an increasing demand for the design and development of robust organic molecular frameworks for ion sensing where the chemo-sensor is capable of working even in the biological cells. Among various anions that play a vital role in biological systems, fluoride is one such common anion pertinent to human health. The imbalance of the fluoride concentration in tissue can lead to health problems pertinent to dental, skeletal, kidney including urolithiasis in humans and gastric disorders, some of which are even fatal.1−4 Excessive fluoride can cause oxidative stress damage to the mitochondria and can lead to neurological disorders.5−8 Several fluoride ion sensors are known in the literature; however, only a few of these are suitable in biological imaging.9−18 Thus, molecular receptors which are versatile enough to sense fluoride ion both in solution as well as in biological cells are scarce. In this regard, the calix[4]arenes are important owing to their (i) amphiphilic nature, (ii) presence of arene core, (iii) presence of the platform suitable for organic derivatization both at lower and upper rims, and (iv) presence of flexible arms to provide suitable and selective coordination environment. Their demonstrated success in the areas of chemo-sensors, catalysis, biomimetic models, and supramolecular science provides an added advantage to calix[4]arene based molecular systems.19−23 There are only a few receptor molecules based on the calixarene platform in the literature for the detection of fluoride ions in solution,24−35 © 2018 American Chemical Society

Figure 1. Schematic representation of the design of L. Received: July 11, 2018 Published: August 27, 2018 11850

DOI: 10.1021/acs.joc.8b01761 J. Org. Chem. 2018, 83, 11850−11859

Article

The Journal of Organic Chemistry Scheme 1. Synthesis of La

a (i) 3-Bromopropyl phthalimide, K2CO3, acetonitrile, 24h at reflux. (ii) Hydrazine hydrate, C2H5OH, 12 h at reflux. (iii) 7-Chloro 4-nitro benzofurazan, pyridine, EtOH, rt for 24 h. The inset shows 1H NMR spectrum in CDCl3 corresponding to the bridge −CH2 groups of L.

lower rim 1,3-diconjugate of calix[4]arene (L) and demonstrated its potential as sensitive and selective sensor for nanomolar detection of F− in solution by UV−vis absorption and fluorescence emission spectroscopy and in biological cells by confocal and fluorescence microscopy. The binding features of F− to L were derived based on density functional theory (DFT) computational studies.

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RESULTS AND DISCUSSION Synthesis and Characterization of Lower Rim 1,3-Dibenzooxadiazole Conjugate of Calix[4]arene, L. L was synthesized in three steps starting from p-t-butyl-calix[4]arene (C4A) as given in Scheme 1 and the details given in the experimental. The precursors, viz., P1 and P2, and receptor L were well-characterized and the corresponding spectra are given in Figures SI01−SI03. The 1H NMR spectrum of L in CDCl3 shows two doublets, one at 3.398 and 3.425 and the other at 4.242 and 4.268 ppm for the bridge −CH2 groups, and the peak pattern is characteristic of the cone conformation of the calix[4]arene platform. Ion Recognition Studies of L in THF. The absorption spectra of L in THF showed two bands, one at 459 nm and the other at 334 nm. Excitation of L at 460 nm in THF resulted in strong emission with its peak maximum at 520 nm with a quantum yield of 0.69, where the quinine sulfate was used as the reference material. In order to explore the anion sensing ability of L, different ions, viz., F−, Br−, Cl−, I−, N3−, SCN−, HCO3−, H2PO4−, CO32−, NO2−, NO3−, SO42−, ADP, AMP, P2O74−, ClO4−, and HSO4− were employed in its titrations in THF, and their UV−vis absorption and fluorescence emission spectra were measured (Figure 2a). In the titration of L with F−, the 460 nm absorption band exhibited an observable redshift of 6−7 nm that is associated with the appearance of new band around 400 nm due to the complexation of F− with L as can be seen from Figure 2c. The changes observed in the absorbance of 460 nm were marginal with all the other 16 anions studied; furthermore, none of these 16 anions showed new bands in their absorption spectra, supporting the idea that the F− selectively alters the absorption spectral features of L. Since the L responds to the interaction with F− in the absorption spectra, a concentration dependent titration of L with F− resulted in a consistent increase in the absorbance of the new band that is formed, and its absorbance follows a near

Figure 2. Titration of L with 5 equiv of different anions in THF: (a) UV−vis absorption spectra and (b) fluorescence spectra. (c) Plot of emission intensity vs different anions. The inset in (c) shows the photograph of the vials containing {L + 5 equiv anion} in THF when illuminated under UV light. (d) Absorption spectral traces in the titration of L with increasing concentration of F− in THF. (e) Plot of absorbance vs [F−]/[L]. (f) Fluorescence spectral traces obtained during the titration of L with increasing concentration of F− in THF. (g) Plot of intensity vs [F−]/[L].

11851

DOI: 10.1021/acs.joc.8b01761 J. Org. Chem. 2018, 83, 11850−11859

Article

The Journal of Organic Chemistry

Figure 3. ESI-MS spectra: (a and b) L; (c and d) for the complex of [LF−].

linear trend with regard to the concentration of F− added (Figure 2d). Similar anion based titrations were carried out by measuring the fluorescence spectra of L. While all the anions studied showed only marginal change in the emission spectrum of L, the F− ion showed ∼95% quenching of the fluorescence intensity (Figure 2b), supporting the idea that even the emission spectra provides selective features for F− ion sensing among all the 17 ions studied (Figure 2g). Even in this case, a concentration-dependent titration of L with F− was carried out and showed that the data fits well to a sigmoidal plot (Figure 2e,f), in support of a complex being formed between L and F−. From the fluorescence titration data, the lowest detection limit for F− by L was obtained, 109 ppb (100 nM) (Figure SI04a), and this value is lower than that reported in the literature based on calixarene derivatives.29,32,40−42 The lowest detection limit is derived based on 3σ/m, where σ corresponds to the standard deviation of the blank measurements, and m is the slope obtained from the plot of intensity versus sample concentration. The Stern−Volmer quenching constant (KSV) for L by F− is (4.2 ± 0.23) × 103 M−1, confirming a high sensitivity of L toward F−. It is evident from the Figure SI04a that at lower F− concentrations the Stern−Volmer plot is close to linear whereas at higher concentrations it deviates from linearity and increases nearly exponentially demonstrating an amplified quenching process. The association constant and binding stoichiometry were determined by the fluorescence spectral changes occurred using Benesi−Hildebrand plot analysis which resulted in Ka of (1.6 ± 0.11) × 103 M−1 for sensing F− by L (Figure SI04b). The Ka indicates considerable interaction between F− and L. Indeed, the complexation energy estimated based on the computational calculations as given in this paper supports the interaction. The Job plot yielded 1:1 stoichiometry for the complex (Figure SI04c,d), and the same is ratified by the mass spectra where a peak corresponding to the 1:1 complex is observed at m/z = 1330.82 for the species, {[L + TBAF] − H2O − H}, while L alone showed a molecular ion peak at m/z = 1111.55. The relevant ESI-MS spectra for L and the complex are given in Figure 3a−d. Reversible Sensing of F− by L. The reversibility of sensing of F− by L has been demonstrated by using calcium perchlorate where the Ca2+ combines with F− and forms CaF2. In fact Ca2+ was used in the literature for reversibility experiments.43,44 The fluorescence intensity of L is quenched in the presence of F− ions and is enhanced back to the same level upon addition of 0.5 equiv of Ca2+ as expected (Figure 4a) owing to dicationic charge on calcium. Further addition of F− ions to this again quenches the fluorescence of L. During the 10 cycles of F− followed by Ca2+ titrations performed, L exhibited reversible behavior without much loss of sensitivity,

Figure 4. Fluorescence spectral titration showing the reversibility of L for F− sensing: (a) Titration of [L + F−] complex with Ca2+ (0 to 2.5 equiv). (b) Fluorescence intensity obtained during the titration of L (highest fluorescence intensity) with F− (quenching) and this followed by Ca2+ (enhancement). This is being carried out for consecutive 10 cycles.

where in the addition of F− results in fluorescence quenching, i.e., switch OFF, and the addition of Ca2+ enhances the fluorescence intensity, i.e., switch ON. Thereby, the L exhibits reversibility in sensing F− with the help of Ca2+ ions (Figures 4b and SI05). All these results suggest that L is a reversible sensor for F−. Supramolecular Features by Microscopy and Dynamic Light Scattering. L shows self-aggregation in THF and forms a 3D sheet kind of aggregates as revealed by SEM and TEM study (Figure 5a,c). After the addition of F− ions, these sheet type aggregates are converted into smaller spherical particles of approximately 20−30 nm size as can be noticed from Figure 5b,d. The F− induced disaggregation of L was further proven by optical microscopy images shown in Figure 5e,f. The fluorescence quenching was noticed upon addition of F− to L based on fluorescence microscopy as given in Figure 5g,h. Thus, all the microscopy results support the disaggregation of original sheets followed by reaggregation of L in the presence of F− to give spherical particles of smaller size. The disaggregation of L in the presence of F− has also been supported by the DLS measurements (Figure SI06a,b), and the average size of the hydrodynamic radius is 2818 ± 20 nm. Thus, upon addition of F−, the hydrodynamic radius was dramatically reduced by 30−40 times by exhibiting an average size of 75.5 ± 4 nm. Binding Region for F− by L Using 1H NMR Titration. 1 H NMR titrations also supported the binding of F− to L. During the titration, the concentration of L was kept constant, and the [F−]/[L] mole ratio was increased. Upon addition of tetrabutyl ammonium fluoride to L, the peak corresponding to both the NH groups at 8.50 ppm is shifted to downfield by ∼0.3 ppm indicating the interaction of F− with NH protons. The loss of 50% peak area of this N−H peak (peak a) observed during the titration of L by F− (inset plot in Figure 6) supports the removal of the proton of one of the N−H groups by F−. 11852

DOI: 10.1021/acs.joc.8b01761 J. Org. Chem. 2018, 83, 11850−11859

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Coordination Features of F− in the Complex of [LF−] by DFT Computations. The initial structure for L was built using a crystal structure given in the literature45 but by bringing some modifications as shown in Figure SI07. Furthermore, the interaction with F− was executed by placing the ion at different regions of the optimized structure of L followed by further optimization, and this resulted in the structure of the complex, [LF−] as given in Figure 7a. The

Figure 7. (a) Computationally optimized [LF−] complex at B3-LYP/ 6-31G(d,p) level. (b) Noncovalent interactions extended by F− to N− H, O−H, and C−H groups. (c) Six-coordinated geometry of [FH6] core. (d) Four-coordinated geometry of [FH4] core obtained when two of the weakest C−H interactions were removed from the coordination sphere. (Colors of the atoms: green, H; gray, C; blue, N; red, O; fluorescent green, F).

interaction energy of F− complexation with L at B3-LYP/6311++G(d,p) level of theory yielded −102 kcal/mol when BSSE and dispersion corrections are included. Indeed, the interaction energies varying from −120 to −128 kcal/mol have been reported in the literature based on the computational studies, where F− interacts with four N−H groups through hydrogen bonding.46 A keen observation of the optimized structure of the complex shows that the F− ion is not equidistant with respect to the N−H groups arising from each arm and sits in the pocket generated by the hydrogens originating from O−H of the lower rim, and N−H and C−H of both the arms of L. The experimental results obtained from

Figure 5. Left column (a, c, e, g) L and right column (b, d, f, h) L + F−. (a and b) SEM images. (c and d) TEM images. (e and f) Optical microscopy images. (g and h) Fluorescence microscopy images. Scale bar for micrographs: (a−d) 100 nm; (e, f) 100 μm; (g, h) 50 μm.

The benzofurazan ring protons observed at 8.33 (peak c) and 6.40 ppm (peak b) were upfield-shifted by 0.18 and 0.15 ppm, respectively, and this further supports that the F− binds to the core formed by the two arms of the lower rim region as schematically shown in Figure 6.

Figure 6. 1H NMR spectra for the titration of L with different equivalents of TBAF in DMSO-d6: (i) 0 (i.e., L only), (ii) 1, (iii) 2, (iv) 3, (v) 4, and (vi) 5. TBAF alone does not show any peak in 6 to 10 ppm. Inset is the plot of area under the NH peak vs [F−]/[L]. 11853

DOI: 10.1021/acs.joc.8b01761 J. Org. Chem. 2018, 83, 11850−11859

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The Journal of Organic Chemistry absorption, emission, and 1H NMR studies also support the same core for binding the F− ion as shown in Figure 7a. The optimized structure for the [LF−] complex showed six D−H··· F− (where D = O, N, or C) type of noncovalent interactions (Table 1), where three of these are relatively stronger than the Table 1. Computed Geometrical Parameters about F− in Its Complex with L Exhibiting D−H···F− (where D = O, N, or C) Type of Noncovalent Interactions D−H (Å) D

L

LF−

F−···H (Å)

D···F− (Å)

D−H···F− (deg)

N N O C C C

1.019 1.019 0.974 1.094 1.102 1.092

1.062 1.048 1.018 1.093 1.090 1.091

1.522 1.664 1.506 2.172 2.706 2.596

2.550 2.655 2.495 2.968 3.173 3.547

161.0 156.1 162.2 127.6 105.3 145.2

Figure 8. Cell cytotoxicity of TBAF (black) and L (red) on HeLa cells. The error bars are given based on repeating the same experiment for three times. The 0 μM column corresponds to only cells which is a control.

however, to a lesser extent even in nuclei. After the treatment with 50 μM of F−, the fluorescence intensity decreases up to 70%, indicating that the F− interaction with L resulted in partial quenching of the fluorescence intensity of L. The only cells without the treatment showed no fluorescence and this indeed is the control (Figure 9a−c). Mean fluorescence intensities showed that 60% fluorescence was quenched after the addition of 50 μM of F− to the L treated cells (Figure 11a). Similarly, the fluorescence microscopy studies (Figure 10) with different concentrations of F− (20, 40, 80, and 100 μM) were carried out, and we observed that the fluorescence intensity decreases with increase in the added F− concentration. At 100 μM of F−, the fluorescence intensity drops to one-third of the initial intensity (Figure 11b). Thus, the fluoride ion concentration in the cells can be easily determined both based on confocal microscopy and fluorescence microscopy.

others and results in a core of the type, [F(OH)(NH)2(CH)3], as can be noted from Figure 7b,c. The data reveals that one of the O−H of the lower rim is involved in a hydrogen bonding with F− apart from two other relatively stronger interactions of the type N−H···F−. The three other weaker interactions are of the type C−H···F−, which further augments the complexation. In the case of the three stronger interactions noted, the corresponding N−H/O−H (donor-hydrogen) bonds are significantly elongated to an extent of 0.044 Å upon interaction with F−, and the corresponding H···acceptor distances have become shorter supporting the formation of the hydrogen bonds. When all the six interactions are considered in the complex, the F− exhibits (Figure 7c) a distorted pentagonal pyramid like species, viz., F(H)6, where the atoms H-1, H-2 and H-4−H-6 form a near pentagon, while the sixth atom, H-3, occupies the pyramidal position. Out of the three C−H···F− interactions identified, only one is relatively stronger than the other two. Therefore, if only the four interactions, viz., one O− H, two N−H and one C−H, were to be considered, the F(H)4 core results in a distorted tetrahedral structure where the H··· F···H angles vary in the range of 74.5−124°, though the average angle works out to be the standard tetrahedral angle (Figure 7d). Tetrahedrally surrounded F− are not uncommon in the literature as reported both based on the computation as well as in the experimental work.47,48 Even the six-coordinated F− complex by N−H and C−H groups is reported in the literature, but their geometry and energies have not been discussed.48 Biocompatibility of L on HeLa Cells. In order to understand the biocompatibility L, MTT assay were performed on HeLa cells. The cells treated with different concentrations of L followed by incubation for 24 h showed more than 90% of the cells being viable. Even the tetrabutyl ammonium fluoride (TBAF) showed viability >85% toward HeLa cells (Figure 8). These results suggests that both L and TBAF are biocompatible based on the cell viability study carried out with the HeLa cells. Confocal and Fluorescence Imaging of HeLa Cells Using L. To understand the use of L for sensing F− in the biological systems, fluorescence and confocal microscopy studies were carried out using HeLa cells. In confocal microscopy, the cells treated with L showed high intra cellular fluorescence suggesting that L enters the cells. Green fluorescence of L was observed mostly in the cell cytoplasm,

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CONCLUSIONS AND COMPARISONS A lower rim 1,3-dibenzooxadiazole conjugate of calix[4]arene (L) has been designed to have both the binding core and the reporting moiety in its arms so that the arms can be used for selective sensing. Accordingly, L was synthesized and characterized thoroughly. Among the 17 anions studied, only the interaction of F− with L resulted in substantial changes in the absorption and fluorescence spectra. The lowest detection limit of F− is 109 ppb (100 nM), and the Job plot and the ESIMS spectra yielded a 1:1 complex. The Stern−Volmer quenching constant (KSV) and the association constant for the binding of F− to L are (4.2 ± 0.23) × 103 M−1 and (1.6 ± 0.11) × 103 M−1 confirming a high sensitivity of L toward F−. The F− sensing by L is reversible with the use of Ca2+ ions as demonstrated for 10 cycles. Both the microscopy and DLS studies support the conversion of large sheets of L into smaller spherical particles in the presence of F−. The binding core for F− and its coordination features were established using 1H NMR and DFT computational studies. The detection of F− by L in solution has been extended even to the biological cells. Since the NBD acts as both the binding core as well as the cell imaging tag, the L has been employed to detect F− inside the cells as demonstrated by fluorescence and confocal microscopy. Thus, a useful, selective, and sensitive fluorescent sensor L for the detection of F− in solution and in the biological cells has been designed, developed, and demonstrated for its utility and the highlights of this work can be gauzed from Scheme 2. 11854

DOI: 10.1021/acs.joc.8b01761 J. Org. Chem. 2018, 83, 11850−11859

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The Journal of Organic Chemistry

Figure 9. Confocal microscopy images of HeLa cells: (a) cells without treatment; (b) cells treated with L; (c) cells incubated with fluoride after treatment of L. The blue color represents nuclei stained with DAPI, green color represents L. The scale bar for all confocal microscopy images in a−c is 10 μm.

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146.9, 141.5, 133.1, 132.3, 127.9, 125.7, 125.2, 123.2, 74.4, 35.9, 34.1, 33.9, 31.9, 31.8, 31.1, 29.6. HRMS (ESI-TOF) m/z: [M + Na]+ Calcd C66H74N2NaO8 1045.5337; Found 1045.5397. Synthesis and Characterization of P2. A solution of P1 (0.50 g, 0.43 mmol) in EtOH (50 mL) was heated at reflux in the presence of hydrazine (5 mL). After 8 h of reflux, the solvent was removed under reduced pressure. The residue was dissolved in CHCl3 (50 mL) and stirred overnight to precipitate the by product and the same filtered off. The filtrate was washed twice with water (30 mL), dried using Na2SO4, and the solvent evaporated to give P2. White solid; yield (0.36 g, 90%). Melting point 236−238 °C. 1H NMR (400 MHz, CDCl3) δ 7.06 (s, 4 H, ArH), 6.80 (s, 4 H, ArH), 4.25 (d, J = 13.0 Hz, 4 H, ArCH2Ar), 4.09 (t, 4 H, OCH2), 3.34 (d, J = 13.0 Hz, 4 H, ArCH2Ar), 3.22 (t, 4H, NHCH2CH2), 2.16 (m, 4H, CH2CH2N), 1.29 (s, 18H, −C(CH3)3), 0.96 (s, 18H, −C(CH3)3). 13C NMR: 150.5, 149.6, 146.9, 141.6, 132.5, 127.7, 125.5, 125.1, 75.1, 39.8, 33.9, 33.8, 33.3, 31.7, 31.0. 13C NMR (135DEPT): 125.5 (−CH), 125.1 (−CH), 75.2 (−CH2), 39.8 (−CH2), 33.3 (−CH2), 31.7 (−CH2), 31.6 (−CH3), 31.0 (−CH3). HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C50H71N2O4 763.5408. Found 763.5440. Synthesis and Characterization of L. To a solution of 7-chloro 4-nitro benzofurazan (0.20 g, 2.2 mmol) in dry ethanol was added P2 (0.35 g, 1.0 mmol) along with a catalytic amount of dry pyridine. The reaction was stirred at room temperature for 1 day. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography using ethyl acetate/pet. ether in 20:80 as eluting medium. Orange solid. Yield (0.34 g, 70%). Melting point 224−226 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.50 (s, 2H, −NH), 8.34 (d, 2H, −ArH), 7.15 (s, 4H, -ArH), 7.13 (s, 4H, −ArH), 6.37 (d, 2H, −ArH), 4.20 (d, 4H, −CH2Ar), 4.061 (t, 4H, −OCH2), 3.83 (t, 4H, −NCH2), 3.43(d, 4H, −CH2Ar), 2.38 (m, 4H, −CH2−),

EXPERIMENTAL SECTION

Material and Methods. The tetrabutyl ammonium and sodium salts of all the anions were procured from sigma Aldrich. All the solvents used were AR-grade and were distilled and dried immediately before use. The 1H and 13C NMR spectra were measured on 400 MHz Bruker instrument. The ESI-MS spectra were recorded on a MaXis Impact-Bruker instrument; the absorption spectra were recorded on the UV−visible spectrophotometer (Cary 100 Bio). The fluorescence emission spectra were recorded on Varian-Cary eclipse fluorescence spectrophotometer. Milli Q water was used in all the measurements. The stock solutions of the receptor L and different anions were prepared in THF at 6 × 10−4 M by initially dissolving in minimum amount of water and were further diluted to give a final cuvette concentration of 20 μM in all cases. All the fluorescence titrations were carried out at λex = 460 nm. Excitation and emission slit widths used were 5 nm and a scan speed of 200 nm/min was used. Synthesis and Characterization of P1. C4A (10.0 g, 15.4 mmol) and K2CO3 (2.34 g, 17 mmol) were taken together in acetonitrile (380 mL) and refluxed for 1 h. To this was added N-(3bromopropyl) phthalimide (9.1 g, 33.9 mmol), and the mixture was continued to heat at reflux for an additional 48 h. The solvent was evaporated under vacuum, and the residue was dissolved in CHCl3. The solution was washed twice with water and brine and then dried. Evaporation of the solvent followed by precipitation from chloroform/methanol gave desired product P1. White solid; yield (10.1, 86%). Melting point 230−232 °C. 1H NMR (400 MHz, CDCl3) δ 7.74−7.76 (m, 4 H, PhtH), 7.62−7.64 (m, 4 H, PhtH), 7.35 (s, 2 H, OH), 7.04 (s, 4 H, ArH), 6.78 (s, 4 H, ArH), 4.32 (d, J = 13.0 Hz, 4 H, ArCH2Ar), 4.11 (m, 8 H, CH2N, OCH2), 3.32 (d, J = 13.0 Hz, 4 H, ArCH2Ar), 2.43 (quint, J = 7.2 Hz, 4 H, CH2CH2N), 1.28 (s, 18 H, tBu), 0.94 (s, 18 H, tBu) ppm. 13C NMR: 168.4, 150.9, 150.1, 11855

DOI: 10.1021/acs.joc.8b01761 J. Org. Chem. 2018, 83, 11850−11859

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The Journal of Organic Chemistry

Figure 10. Fluorescence images of HeLa cells treated with L (20 μM) followed by different concentration of fluoride ion. The F− concentration in μM is (a) 0; (b) 20; (c) 40; (d) 80; and (e) 100 μM. The scale for images a−e is 50 μm. 125.8, 121.6, 99.4, 74.3, 41.1, 34.5, 31.8, 31.3, 29.5, 29.7. 13C NMR (135DEPT): 137.8, 126.2, 125.8, 99.4, 74.3, 41.1, 31.8, 31.3, 29.5, 28.7. HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C62H72N8NaO10 1111.526. Found 1111.555. Characterization of [L + F−] in Situ Complex. 1H NMR (400 MHz, DMSO-d6) δ 8.71 (s, 2H, −NH), 7.92 (d, 2H, −ArH), 7.12 (s, 4H, −ArH), 7.11 (s, 4H, −ArH), 6.13 (d, 2H, −ArH), 4.20 (d, 4H, −CH2Ar), 4.061 (t, 4H, −OCH2,), 3.86 (t, 4H, −NCH2), 3.38(d, 4H, −CH2Ar,), 3.15 (t, 8H, −TBAF) 2.37 (m, 4H, −CH2−), 1.54 (m, 8H, TBAF), 1.31 (m, 8H, TBAF), 1.16 (s, 18H, −tCH3), 1.11 (s, 18H, −tCH3), 0.92 (t, 12H, TBAF). Sample Preparation for Microscopy. All the samples for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were prepared with 6 × 10−4 M L and its fluoride complex in THF, and the solutions were sonicated for 10 min before these were drop-cast on aluminum foil and copper grid respectively and allowed to dry under IR lamp.

Figure 11. (a) Histogram of fluorescence intensity obtained by confocal microscopy on HeLa cells. “None” refers to HeLa cells which were not treated. (b) Histogram of fluorescence intensity obtained by fluorescence microscopy upon treating HeLa cells with L followed by F−. “0” refers to no fluoride addition and only L. 1.18 (s, 18H, −tCH3), 1.10 (s, 18H, −tCH3,). 13C NMR: 150.5, 150.1, 147.7, 145.1, 144.4, 144.2, 142.0, 137.8, 133.5, 127.9, 126.2, 11856

DOI: 10.1021/acs.joc.8b01761 J. Org. Chem. 2018, 83, 11850−11859

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The Journal of Organic Chemistry

Scheme 2. Schematic Representation of the Highlights of F− Sensing by L in Solution, in Biological Cells, and by DFT Computations

the next day, the cells were treated with 20 μM L for 3 h along with complete media. After incubation, the media was removed and cells washed with PBS buffer three times for elimination of excess L. Ltreated cells were again incubated with 50 μM F− for 1 h, the media removed, and cells washed with PBS three times. The coverslip was fixed on a glass slide with DAPI (Invitrogen), and these were imaged using confocal laser scanning microscopy. The cells which were not treated with L were used as control. The fluorescence intensities were measured by using ZEN blue software. For fluorescence microscopy, the cells were treated with 20 μM L for 30 min in media. After incubation, the media was removed and washed with PBS buffer for three times removal of excess L. L treated cells again incubated with different concentrations of F−, i.e., 20, 40, 80, and 100 μM, for 30 min and removed the media and washed with PBS for three times. These were imaged using fluorescence microscopy.

Computational Details. Density functional theory (DFT) calculations were performed for L and its complex with F− to give [LF−] complex in order to support the conclusions derived based on the experimental data. The [LF−] complex is obtained from the geometry optimization and subsequent frequency analysis at B3-LYP/ 6-31G(d,p) level of theory. All the computations were carried out using Gaussian 09 package.49 The interaction energies were further improved by using triple-ζ quality basis set, BSSE correction along with the dispersion correction carried out using Becke−Johnson’s damping at B3-LYP-D3(BJ)/6-311++G(d,p) level of theory.50,51 Cell Culture and Cytotoxicity Studies. Human cervical cancer (HeLa) cells were obtained from NCCS Pune and were cultured in Dulbecco’s modified Eagle’s media (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin). These cultures were maintained at 37 °C, 5% CO2, humidified incubator. The media was changed every 3 days and subcultured when the cells reached confluence. The MTT assay was performed in 96-well plate with seeding density of 1.5 × 104 cells in each well in order to evaluate the cytotoxicity of L as well as tetrabutyl ammonium fluoride on HeLa cells. The cells were then treated with different concentrations of L and tetrabutylammonium fluoride (10, 20, 30, 50, 80, and 100 μM/ mL) and were incubated for 24 h at 37 °C under 5% CO2 atmosphere. After treatment, the media was removed, and 100 μL MTT reagent (5 mg/mL) was added to each well and incubated for 4 h in dark at 37 °C in the incubator. Furthermore, the medium was removed, and 200 μL of DMSO was added to each well. The absorbance was measured at 570 nm using a microplate reader (Molecular device). Confocal and Fluorescence Microscopy. HeLa cells were cultured in 6-well plate covered with glass coverslip with seed density 0.5 × 106 cells and kept for 24 h incubation. For confocal microscopy,

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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/acs.joc.8b01761.

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Spectral data for the characterization all the precursors and the final molecule L, all the titration related spectra and the corresponding data, computational related data (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. 11857

DOI: 10.1021/acs.joc.8b01761 J. Org. Chem. 2018, 83, 11850−11859

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Chebrolu Pulla Rao: 0000-0002-1004-0028 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS C.P.R. acknowledges financial support from DST/SERB {EMR/2014/000985} and the J. C. Bose National Fellowship {SB/S2/JCB-066/2015} and IIT Bombay for Institute Chair Professorship. R.K. acknowledges IIT Bombay for Institute Postdoctoral Fellowship. B.U. is thankful to CSIR for the award of Senior Research Fellowship {09/087(0835)/2015EMR-I}. We thank Dr. Sunita Joshi for and Ms. Sirilata Polepalli for some experimental help.

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