Ratiometric sensing of fluoride and acetate anions based on a BODIPY-azaindole platform and its application to living cell imaging

Abstract

A new BODIPY-azaindole based fluorescent sensor 1 was designed and synthesized as a new colorimetric and ratiometric fluorescent chemosensor for fluoride. The binding and sensing abilities of sensor 1 towards various anions were studied by absorption, emission and ¹H NMR titration spectroscopies. The spectral responses of 1 to fluoride in acetonitrile–water were studied: an approximately 69 nm red shift in absorption and ratiometric fluorescent response was observed. The striking light yellow to deep brown color change in ambient light and green to blue emission color change are thought to be due to the deprotonation of the indole moiety of the azaindole fluorophore. From the changes in the absorption, fluorescence, and ¹H NMR titration spectra, proton-transfer mechanisms were deduced. Density function theory and time-dependent density function theory calculations were conducted to rationalize the optical response of the sensor. Results were supported by confocal fluorescence imaging and MTT assay of live cells.

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Introduction

The design and development of artificial molecular systems for the sensing of biologically important anions has been the focus of a recent flurry of work.¹ Among the anions, the fluoride ion (F⁻) is of particular interest owing to its established role in dental care and clinical treatment for osteoporosis.² An acute intake of a large dose or chronic ingestion of lower doses of F⁻ can result in gastric and kidney disorders, dental and skeletal fluorosis, and urolithiasis in humans, and even death.³ For these reasons, an improved method for the detection and sensing of F⁻ with high selectivity is of current interest in the chemosensor research field. A variety of fluorogenic chemosensors showing absorbance or fluorescence changes upon binding with F⁻ have been reported.⁴ However, most of them show a turn-off response in emission spectra and modest selectivity. To increase the selectivity and sensitivity, ratiometric measurements are utilized, which involve the observation of changes in the ratio of the intensities of the absorption or the emission at two wavelengths.⁵ Ratiometric fluorescent probes have an important feature: they permit signal ratio and thus increase the dynamic range and provide built-in correction for environmental effects. The perceived color change is useful not only for the ratiometric method of detection but also for rapid visual sensing. Up to now, many investigations have been conducted to create ratiometric fluorescent probes for cations.⁶ In contrast, only a few ratiometric fluorescent sensors for F⁻ and acetate (AcO⁻) have been reported in the literature.⁷ Thus, realization of ratiometric measurements for F⁻ and AcO⁻ are still a challenge.

Boron dipyrromethene (BODIPY)⁸ dyes have been chosen as fluorophore signaling units because of their widely accepted superiority e.g., high fluorescence quantum yields, high molar absorptivity, high photostability, visible wavelength absorption and modular nature enabling facile functionalization, etc., compared with other fluorophore signaling units. These favourable features have made it possible for BODIPY dyes to be widely used as fluorophore cores for the construction of fluorescent sensors/labels,⁹ light harvesting systems,¹⁰ photodynamic therapy agents¹¹ and molecular logic gate systems.¹²

An azaindole unit placed at the meso-position of the BODIPY core plays a crucial role in photoinduced-electron-transfer (PET)-based modulation of emission. Typically, for efficient ICT (internal charge transfer), the donor and acceptor are located in the 7- and 3-positions, respectively.¹³ On the other hand, it is known that BODIPY dyes undergo a PET process with a functional group at the meso-position.¹⁴ Previous work by others¹⁵ has demonstrated that a phenoxy substituent at the meso (8) position of the BODIPY core is a very strong PET donor. The spectral signature of PET is quenching of fluorescence without any significant changes in the emission wavelength.¹⁶ Again, PET is an effective signalling mechanism employed in the design of ratiometric fluorescent sensors.¹⁷ Thus, we envisioned that ratiometric fluorescent sensors for fluoride anions and acetate could be constructed by exploiting the PET properties of both azaindole and BODIPY dyes.

Current studies are aimed at designing and synthesizing ratiometric molecular probes with colorimetric and fluorimetric assays to selectively detect the presence of a target anion over a wide range of other interfering anions. However, to the best of our knowledge, there are no reports on ratiometric fluorescent sensors based on a BODIPY-azaindole platform using the PET mechanism. Here, we have developed a new BODIPY-azaindole based probe 1, which enables naked eye and dual channel (absorption and fluorescence) detection of F⁻ and AcO⁻ ions, respectively.

Results and discussion

Scheme 1 outlines the synthesis of the BODIPY-azaindole based probe 1. Probe 1 was prepared through the condensation reaction of azaindole aldehyde with 2,4-dimethylpyrrole according to a published procedure.¹⁸ The structures of the sensors were characterized on the basis of ¹H and ¹³C NMR and HRMS (Fig. S1–S3, ESI†).

Scheme 1 Synthesis of probe 1.

The interaction of probe 1 with anions was investigated through spectrophotometric titrations by adding a standard solution of the tetrabutylammonium salt of anions to a 7 : 3 CH₃CN : H₂O solution (0.02 M HEPES buffer, pH 7.2) of the probe. The free probe 1 displayed an intense absorption band at 501 nm and a weak absorption at 350 nm. The main band at 501 nm is the typical absorption band of BODIPY dyes. Upon addition of 0.5 molar equiv. of fluoride ions, little change was observed at 501 nm. However, the addition of a further amount of fluoride ions, induced new absorption bands at 419 and 254 nm. The peak at 350 nm, the π–π* transition of the chromophore, disappeared gradually, and there was simultaneous growth of a new strong absorption band centred at 419 nm, which is the charge transfer (CT) band. The absorption band at 350 nm decreased and a new red shift band at 419 nm appeared and developed due to the hydrogen binding interaction between indolic NH of probe 1 and fluoride anions. The light yellow color of the probe solution turned reddish-brown at the same time. Four well-defined isosbestic points at 266, 290, 369 and 460 nm were observed (Fig. 1a), indicating the formation of a new species upon treatment of probe 1 with F⁻. The stoichiometry of the 1-fluoride interaction was confirmed to be 1 : 1 from the Job’s plot (Fig. S4, ESI†). Analogous investigations were carried out on a variety of anions such as Cl⁻, Br⁻, I⁻, NO₃⁻, SO₄²⁻, AcO⁻, HSO₄⁻, and H₂PO₄⁻. Specifically, the receptor 1 in aqueous organic solvent changed color in the presence of F⁻ and AcO⁻ anions (Fig. S5, ESI†). The most pronounced effect was the F⁻-induced color change from yellow to deep reddish-brown. Other anions such as Cl⁻, Br⁻, I⁻, NO₃⁻, SO₄²⁻, HSO₄⁻, and H₂PO₄⁻ did not induce any spectral response (Fig. 1b). It is worth noting that the absorption band was not altered by less basic anions such as Cl⁻, Br⁻, I⁻, NO₃⁻, SO₄²⁻, HSO₄⁻, and H₂PO₄⁻. These results provide strong evidence that the red shifted peak at 419 nm is probably due to the deprotonation of the indole N–H of probe 1.¹⁹ The binding constant for F⁻ was determined to be 3.43 × 10⁴ M⁻¹ (Fig. S6, ESI†). The detection limit of fluoride was about 1.21 μM (S6, ESI†).

Fig. 1 Changes in the UV/vis absorption spectra of sensor 1 (4.5 μM) in 7 : 3 CH₃CN : H₂O solution (0.02 M HEPES buffer, pH 7.2) (a) in the presence of F⁻ anions only (0–20 equiv.) and (b) in the presence of various anions (0–300 equiv. except F⁻ and AcO⁻:0–20 equiv.).

Upon addition of more water or methanol to the solution of probe 1 in the presence of F⁻ and AcO⁻ anions, respectively, the absorption of the two systems were recovered. This may be attributed to the formation of hydrogen bonds between the anions and protic solvent, which compete with the binding to probe 1. The fluoride- and acetate-induced deprotonation process is fully reversible, the addition of polar protic solvents (H₂O and CH₃OH) results in a reverse color change from reddish-brown to yellow. This is presumably because protic solvents compete with the NH moiety for F⁻ or AcO⁻, moreover, the presence of a relatively high amount of protic solvent does not favor the formation of deprotonated receptor 1.

The anion-binding properties of probe 1 were then studied in 7 : 3 CH₃CN : H₂O solution (0.02 M HEPES buffer, pH 7.2) by emission spectroscopy. The results obtained were in good agreement with those of UV/vis absorption spectroscopy, the sensor exhibited a ratiometric fluorescent response to F⁻ anions. Upon excitation at 350 nm, the free sensor displayed an intense emission band at 512 nm due to an efficient PET process from the lone pair of electrons on the azaindole moiety, which has inherent electron donating property, to the excited BODIPY fluorophore.²⁰ When the concentration of F⁻ increased, the new emission band at 425 nm was gradually enhanced (Fig. 2a), this is attributed to local emission (LE) due to the presence of the azaindole moiety, while the intensity of emission at 512 nm decreased correspondingly.

Fig. 2 (a) Fluorescence spectra (excitation at 350 nm) of sensor 1 (4.5 μM) in 7 : 3 CH₃CN : H₂O solution (0.02 M HEPES buffer, pH 7.2) in the presence of (a) 0–200 equiv. of F⁻ and (b) the fluorescence intensity ratio (I₄₂₅/I₅₁₂) as a function of equiv. of fluoride anions.

A clear iso-emission point was observed at 492 nm. With the addition of 200 equiv. of F⁻, the emission band at 512 nm was quenched efficiently, this could be attributed to the enhanced PET process upon binding of F⁻. Thus, the sensor provided a significant ratiometric fluorescent response (I₄₂₅/I₅₁₂) to F⁻ anions as seen in Fig. 2b. However, upon excitation at 419 nm, treatment of the sensor with F⁻ anions afforded no noticeable emission. This is the typical behavior of BODIPY-based CT sensors: the CT emission band is almost nonfluorescent when excited at the CT absorption band.²¹ Among the anions investigated, only AcO⁻ induced similar spectral changes (Fig. S7, ESI†). The spectral behavior revealed that deprotonation of the NH fragment by F⁻ and not hydrogen bonding to it is responsible for the drastic color change, as a result of altering the optical properties of the chromogenic and fluorogenic azaindole skeleton.

Interestingly, when TBAF (tetra-n-butylammoniumfluoride) was added to the optimized 7 : 3 CH₃CN : H₂O solution (0.02 M HEPES buffer, pH 7.2) of 1, an apparent color change from light yellow to deep brown in ambient light, as shown in the inset of Fig. 1a, could be observed by the naked eye. Upon progressive addition of TBAF, the intensity at 350 nm gradually decreased, and a large bathochromic shift (69 nm) of the maximum could be observed at 419 nm (Fig. 1a). This was not observed with other anions except acetate ion. It has been noted that the deprotonation of receptor 1 is also induced by the basic anion AcO⁻, and appearance of the band at 419 nm and development of the light brown color were observed after large excess addition (Fig. 3). The results support our expectation that 1 could serve as a sensitive naked-eye probe for F⁻. The fluoride sensing process was also clearly seen not only by color change but also by bright fluorescence under a UV lamp. During the fluorometric titration of 1 with F⁻ ions the green color solution of the receptor became deep blue. Comparative fluorescence changes upon addition of various anions to compound 1 are shown in Fig. 3. The dramatic combination of anion-specific response/nonresponse makes receptor 1 an especially effective colorimetric anion sensor under the solution-phase conditions. The above results indicate that receptor 1 exhibits excellent selective sensing for F⁻ (F⁻ > AcO⁻ ≫ other anions) in aqueous organic solvent.

Fig. 3 Relative fluorescence changes of 1 (4.5 μM) after treatment with various relevant analytes (200 equiv. for F⁻ and AcO⁻, 300 equiv. for other anions) in 7 : 3 CH₃CN : H₂O solution (0.02 M HEPES buffer, pH 7.2). The inset shows a photograph of visible color (top) under ambient light and visual fluorescence color (bottom) under a hand-held UV lamp (366 nm).

When the deprotonation reaction of 1 was carried out in a mixture of anions containing Cl⁻, Br⁻, I⁻, NO₃⁻, SO₄²⁻, HSO₄⁻, and H₂PO₄⁻, no fluorescence appeared, but a strong fluorescence emission appeared only when fluoride anion was added to this mixture (Fig. 3). These observations suggest that compound 1 is highly selective toward F⁻ even in the presence of a complex mixture of anions. The experimental results suggest that compound 1 shows high selectivity in colorimetric and fluorescent sensors for the fluoride anion.

¹H NMR titration experiments in CDCl₃ were conducted to look into the nature of the new peak formed in UV-vis fluoride titration spectra. To confirm this fact, ¹H NMR interactions of receptor 1 with F⁻ and AcO⁻ (Fig. S8, ESI†) were carried out in CDCl₃. It was found that the indolic NH proton signal (δ = 13.03 ppm) in the drastic downfield shift (Δδ = 0.70 ppm) of the spectrum broadened and then finally disappeared with an increase in the concentration of F⁻.

In Fig. 4, the majority of signals on the aryl rings shift upfield distinctly after the addition of 1 equiv. of fluoride ions owing to the through-bond effects, and complete disappearance of the signals for the NH protons (13.03 ppm) was also observed at the same time. These observations clearly support the theory that the proton transfer interaction between compound 1 and F⁻ involves the indolic NH proton. Fluoride has a high affinity towards hydrogen and could easily induce N–H bond cleavage. Interestingly, after the addition of 2.0 equiv. of fluoride ions, a new 1 : 2 : 1 triplet signal at 15.8 ppm appeared, which is ascribed to the FHF⁻dimer.²² The existence of this new species indicates the deprotonation of the NH group. The deprotonation of the receptor was confirmed by the Bronsted acid–base reaction between 1 and [Bu₄N]⁺OH⁻. This deprotonation process was also confirmed by the identical UV-vis spectral changes observed in the titration experiment with tetrabutylammonium hydroxide (TBAOH) to that observed for fluoride ions (Fig. S9, ESI†). A stepwise increase in the concentration of TBAOH produced results analogous to those found in the case of F⁻ ion and other ions less basic than OH⁻ (pK_a = 30 in CH₃CN). To elucidate the mechanism of interaction, the pH titration was carried out in the presence of fluoride ion in aqueous acetonitrile solution. Similar results were obtained at a high pH range (Fig. S10, ESI†). With the deprotonation of 1, the charge delocalizes on the entire conjugated π-system, which shifts the aryl rings further upfield. From the ¹H NMR titration results, it was found that 1 and fluoride form a hydrogen-bond complex upon addition of 1 equiv. of fluoride ions, while almost no changes were observed for the UV-vis spectra; with an increase of fluoride concentration, a new deprotonated species appeared, and the CT band of UV-vis spectra formed quickly. These results suggest that the sensor–fluoride interaction is indeed a two-step process at low fluoride concentration as shown in Scheme 2, the sensor–fluoride interaction is authentic hydrogen binding, and with an increase of the fluoride ions, excess fluoride interacts with the sensor–fluoride complex and induces deprotonation of the sensor.

Fig. 4 Change in partial ¹H NMR spectra of 1 upon addition of F⁻ in CDCl₃ solvent: (1) 0, (2) 0.5, (3) 1.0, and (4) 2.0 equiv. F⁻.

Scheme 2 Proposed binding mode of sensor 1 with F⁻ anions.

To gain insight into the optical response of sensor 1 to F⁻ anions, sensor 1 and the corresponding deprotonated species were examined by density function theory (DFT) and time-dependent density function theory (TDDFT) calculations at the B3LYP/6-31 G(d) level of the Guassian 03 program²³ (Fig. S11, ESI†). The geometry optimizations for 1 and deprotonated species were done in a cascade fashion starting from semiempirical PM2 followed by ab initio HF to DFT B3LYP by using various basis sets, viz., PM2 → HF/STO-3G → HF/3-21G → HF/6-31G → B3LYP/6-31G(d,p). As shown in Fig. 5, for sensor 1, both the HOMO and LUMO are distributed at the BODIPY part. In addition,the planar BODIPY ring fragment is almost perpendicular to the adjacent azaindole part with a dihedral angle of 89.5°. Thus, the ICT character of the sensor is only modest due to the presence of azaindole part into the meso position. While for the deprotonated species, the HOMO is distributed in both the ring system of azaindole and BODIPY, and the LUMO is mostly located between the BODIPY unit and the meso position of the carbon. This indicates that the deprotonated product bears ICT character among the fluorophore moieties, which is consistent with the design strategy. Furthermore, the energy gap between the HOMO and LUMO of the deprotonated product is smaller than that of sensor 1, which is in good agreement with the red shift in the absorption observed upon treatment of sensor 1 with F⁻ anions.

Fig. 5 Calculated (DFT B3LYP/6-31 G(d) level) geometry and HOMO−LUMO energy levels and the interfacial plots of the orbitals for sensor 1 and the deprotonated species.

To explain the formation of the LE band at 425 nm with excitation at 350 nm upon addition of sensor 1 to F⁻ anions, sensor 1 and the deprotonated product were optimized by DFT calculations. The formation of the LE emission band at 425 nm in the deprotonated product is likely due to the nonplanar and nonconjugated character of the meso position. In addition, we also performed time-dependent density function theory (TD-DFT) calculations for both the deprotonated product and the sensor 1. In the case of the deprotonated product, TD-DFT calculations provide a calculated absorption band at 406 nm belonging to the S₀ → S₃ energy state (Table S1) (S12, ESI†). The main contributing transition (∼67.40%) for S₀ → S₃ energy state arises from HOMO-1 → LUMO. This is consistent with the absorbance band at 419 nm obtained experimentally. The LE band mostly originates from azaindole moiety (Fig. S13, ESI†), further confirming that the LE band originates from the azaindole moiety in the deprotonated product. By contrast, as shown in Fig. S14 ESI,† no allowed singlet state transitions are located on the azaindole moiety in sensor 1. Thus, the LE emission from the azaindole moiety is not present in the sensor.

The probe has thermodynamically favourable binding properties to F⁻ and forms a 1 F⁻ complex which gives emission spectra in the visible range. To further demonstrate the practical biological application of the probe, fluorescence imaging experiments were carried out in living cells, particularly for the sensitive detection of intracellular F⁻. The well-established MTT assay, which is based on mitochondrial dehydrogenase activity of viable cells, was adopted to study cytotoxicity of the above mentioned compounds at varying concentrations. Fig. 6 shows that the probe compound did not exert any adverse effect on cell viability, the same is the case when cells were treated with varying concentrations of F⁻. However, exposure of HCT cells to 1 F⁻ complex resulted in a decline in cell viability above a concentration of 25 μM. The effect was more pronounced at higher concentrations and showed an adverse cytotoxic effect in a dose-dependent manner which is in agreement with previous literature reports suggesting cytotoxic and anti-proliferative effects of 1 F⁻ complex on cancer cells.²⁴ The viability of HCT cells was not influenced by the solvent (DMSO) as evidenced in Fig. 6, leading to the conclusion that the observed cytotoxic effect could be attributed to the 1 F⁻ complex.

Fig. 6 MTT assay to determine the cytotoxic effect of probe 1 and 1 F⁻ complex on HCT cells.

The results obtained in the in vitro cytotoxic assay suggested that, in order to pursue fluorescence imaging studies of 1 F⁻ complex in live cells, it would be prudent to choose a working concentration of 20 μM for the probe compound. Hence, to assess the effectiveness of compound 1 as a probe for intracellular detection of F⁻ by confocal microscopy, RAW cells were treated with 20 μM F⁻ for 1 h followed by 10 μM probe solution to promote formation of 1 F⁻ complex. Fluorescence microscopic studies revealed a lack of fluorescence for RAW cells when treated with either probe compound or F⁻ alone (Fig. 7). Upon incubation with F⁻ followed by probe compound a striking switch- ON fluorescence was observed inside RAW cells, which indicated the formation of 1 F⁻ complex, as observed earlier in solution studies. Further, an intense green fluorescence was conspicuous in the cytoplasmic region of RAW cells which indicates that the probe could readily cross the membrane barrier, permeate into RAW cells, and rapidly sense intracellular F⁻. It is significant to mention here that bright field images of treated cells did not reveal any gross morphological changes, which suggested that RAW cells were viable. These findings open up the avenue for future in vivo biomedical applications of the sensor.

Fig. 7 Confocal microscopic images of RAW 264.7 control cells (a–d) and F⁻ treated (e–g) cells pretreated with Probe 1. All the images were acquired with a 40× objective lens. F⁻ treated cells do not emit fluorescence as control. (a) Bright field image of the cells (b) nuclei counter stained with DAPI fluorescent stain (1 μg mL⁻¹) (c) only probe 1 at c = 1.0 × 10⁻⁵ M concentration (excitation wave length 405 nm and emission range 540–550 nm) (d) overlay image of (b) and (c) in dark field (e) bright field image of RAW cells treated with probe 1 and F⁻ (f) cells treated with Probe 1 (c = 1.0 × 10⁻⁵ M) and F⁻ (c = 2.0 × 10⁻⁵ M) (g) nuclei counterstained with DAPI fluorescent stain (1 μg mL⁻¹) excited at 405 nm (h) Overlapping image of (f) and (g).

Conclusion

In conclusion, we have developed a new simple system (1) BODIPY-azaindole based ratiometric fluorescent sensor for fluoride anions in organo-aqueous media. The system provides chromogenic and fluorogenic dual signals by displaying (i) a deep brown color and (ii) a strong blue fluorescence from an initially light yellow and green fluorescent solution, upon exposure to fluoride. The response time was only a few seconds in acetonitrile, and we were able to easily follow the response by either absorption or emission changes. The drastic color change and strong fluorescence change of 1 are simply driven by hydrogen bonding interaction between the indolic NH proton and F⁻. DFT and TD-DFT calculations were conducted to rationalize the optical response of the sensor. The sensitivity of compound 1 to F⁻ was demonstrated in living cells, and cell toxicity assay reveals that probe 1 can be used for selective imaging of F⁻ in living cells. Moreover, the simple probe design presented here may contribute to the development of more efficient and more useful dual-mode anion probes.

Experimental

General information and materials

All the solvents were of analytical grade. All anionic compound such as tetrabutylammonium salts of F⁻, Cl⁻, Br⁻, I⁻, NO₃⁻, SO₄²⁻, AcO⁻, HSO₄⁻, and H₂PO₄⁻ were purchased from Sigma–Aldrich Chemical Co., stored in desiccators under vacuum containing self-indicating silica, and used without any further purification. Solvents were dried according to standard procedures. Unless stated otherwise, commercial grade chemicals were used without further purification. Elix Millipore water was used throughout all the experiments. All the reactions were magnetically stirred and monitored by thin-layer chromatography (TLC) using Spectrochem GF254 silica gel coated plates. Column chromatography was performed with silica gel. The ¹H NMR spectra were recorded on a Bruker–300 MHz spectrometer. Mass spectra were carried out using a Water's QTOF Micro YA 263 mass spectrometer. The ¹H NMR chemical shift values are expressed in ppm (δ) relative to CHCl₃ (δ = 7.19 ppm). UV-visible and fluorescence spectra measurements were performed on a JASCO V530 and a Photon Technology International (PTI-LPS-220B) spectrofluorimeter respectively. The following abbreviations are used to describe spin multiplicities in ¹H NMR spectra: s = singlet; d = doublet; t = triplet; m = multiplet. Elemental analyses were performed with a Perkin-Elmer 2400 elemental analyzer.

General method of UV-vis and fluorescence titration

By UV-vis method. UV-vis spectra were recorded on a JASCO V-530 spectrophotometer using a dissolution cell of 10 mm path and the fluorescence spectra were recorded on a Perkin Elmer Model LS 55 spectrophotometer using a fluorescence cell (10 mm). For UV-vis and fluorescence titrations, stock solution of receptor was prepared (c = 1 × 10⁻⁵ mL⁻¹) in 7 : 3 CH₃CN:H₂O solution (0.02 M HEPES buffer, pH 7.2). The solution of the guest anions using their TBAX salts in the order of 1 × 10⁻⁴ mL⁻¹ was also prepared in 7 : 3 CH₃CN : H₂O solution (0.02 M HEPES buffer, pH 7.2). The test solution of sensor 1 was prepared by the proper dilution method. The spectra of these solutions were recorded by means of UV-vis and fluorescence methods. The substrate binding interaction was calculated according to the Benesi–Hildebrand equation.

Here A₀ is the absorbance of receptor in the absence of guest, A is the absorbance recorded in the presence of added guest, ε₀ and ε are the corresponding molar absorption co-efficient and K_B represents the substrate binding interaction with guest.

Synthesis

Synthesis of the compound 1. 7-Azaindole-3-carboxaldehyde (3) (0.15 g, 1.03 mmol) and 2,4-dimethylpyrrole (0.23 g, 2.39 mmol) were dissolved in dry CH₂Cl₂ (60 mL) under a nitrogen atmosphere. Trifluoroacetic acid (TFA) (7.9 μL, 0.103 mmol) was added, and the mixture was stirred at room temperature overnight. After TLC monitoring showed complete disappearance of the aldehyde, a solution of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (0.54 g, 2.395 mmol) in anhydrous CH₂Cl₂ (30 mL) was added. This mixture was further stirred for 5 h, washed with water three times, dried over anhydrous Na₂SO₄, filtered, and concentrated to dryness. The resulting compound was roughly purified by using aluminum oxide column chromatography to give a brown powder as the crude compound 2 (about 0.147 g, 0.464 mmol, yield of the first step is 45%). This product 2 was used without further purification in the next step. The brown powder (0.147 g, 0.464 mmol) and NEt₃ (3 mL) were dissolved in anhydrous CH₂Cl₂ (70 mL) under a nitrogen atmosphere. The solution was stirred at room temperature for 45 min, and BF₃–OEt₂ (3 mL) was subsequently added. This mixture was stirred for 6 h where upon the complexation was found to be completed by TLC monitoring. The mixture was washed thoroughly with water and brine, dried over anhydrous Na₂SO₄, filtered, and evaporated under vacuum. The crude compound was purified by silica gel column chromatography (eluent: 10% EtOAc in hexane) to give a reddish brown solid as the pure compound 1 (0.086 g, 0.237 mmol, yield of the second step is 51%, yield overall 23%): mp > 250 °C.

¹H NMR (CDCl₃, 300 MHz) δ(ppm): 13.029 (s, 1H, NH), 8.395 (d, 1H, J = 5.7 Hz), 8.336 (d, 1H, J = 8.4 Hz), 7.547 (s, 1H), 7.454 (t, 1H, J = 7.5 Hz), 5.945 (s, 2H), 2.507 (s, 6H), 1.257 (s, 6H).

¹³C NMR (CDCl₃, 75 MHz) δ(ppm): 154.46, 150.99, 148.51, 143.71, 142.59, 129.32, 126.90, 125.55, 121.29, 118.63, 116.75, 113.88, 14.36, 13.89.

TOF MS ES+, m/z = 365.1748 [M+1]⁺, calc. for C₂₀H₁₉BF₂N₄ = 364.1996. Anal. calcd for C₂₀H₁₉BF₂N₄: C, 65.96; H, 5.26; N, 15.38 Found: C, 65.84; H, 5.29; N, 15.32%.

Studies on live cells. Frozen human colorectal carcinoma cell line HCT 116 (ATCC:CCL-247) were obtained from the American Type Culture Collection (Rockville, MD, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM, Sigma Chemical Co., St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Invitrogen), penicillin (100 μg mL⁻¹), and streptomycin (100 μg mL⁻¹). The RAW 264.7 macrophages were obtained from NCCS, Pune, India and maintained in DMEM containing 10% (v/v) fetal calf serum and antibiotics in a CO₂ incubator. Cells were initially propagated in 25 cm² tissue culture flask in an atmosphere of 5% CO₂ and 95% air at 37 °C humidified air till 70–80% confluency.

Fluorescent imaging studies. For fluorescent imaging studies, RAW cells, 1 × 10⁻⁵ cells in 150 μL media were seeded on a sterile 12 mm diameter poly-L-lysine coated coverslip and kept in a sterile 35 mm covered Petri dish and incubated at 37 °C in a CO₂ incubator for 24–30 h. The next day the cells were washed three times with phosphate buffered saline (pH 7.4) and fixed using 4% paraformaldehyde in PBS (pH 7.4) for 10 minutes at room temperature washed with PBS followed by permeabilization using 0.1% saponin for 10 minutes. Then the cells were incubated with c = 2.0 × 10⁻⁵ M F⁻ dissolved in 100 μL DMEM at 37 °C for 1 h in a CO₂ incubator and observed under an Andor spinning disk confocal microscope. The cells were again washed thrice with PBS (pH 7.4) to remove any free metal and incubated in DMEM containing probe to a final concentration of 1.0 × 10⁻⁵ M followed by washing with PBS (pH 7.4) three times to remove excess probe outside the cells and images were captured as before. Coverslips were mounted on slides using a permanent mounting medium supplemented by DAPI (1 μg mL⁻¹) and stored in the dark before microscopic images were acquired.

Cytotoxicity assay of live cells. The cytotoxic effects of probe, F⁻ and probe-F⁻ complex were determined by MTT assay following the manufacturer's instruction (MTT 2003, Sigma-Aldrich, MO). HCT cells were cultured into 96-well plates (approximately 10⁴ cells per well) for 24 h. The next day the media was removed and various concentrations of probe, F⁻ and probe-F⁻ complex (0, 15, 25, 50, 75, and 100 μM) made in DMEM were added to the cells and incubated for 24 h. Solvent control samples (cells treated with DMSO in DMEM), no cells and cells in DMEM without any treatment were also included in the study. Following incubation, the growth media was removed, and fresh DMEM containing MTT solution was added. The plate was incubated for 3–4 h at 37 °C. Subsequently, the supernatant was removed, the insoluble colored formazan product was solubilized in DMSO, and its absorbance was measured in a microtiter plate reader (Perkin-Elmer) at 570 nm. The assay was performed in triplicate for each concentration of probe, F⁻ and 1-F⁻ complex. The OD value of the wells containing only DMEM medium was subtracted from all the readings to get rid of the background influence. Data analysis and calculation of standard deviation was performed with Microsoft Excel 2007 (Microsoft Corporation).

Acknowledgements

We thank the DST-West Bengal [Project no. 124(Sanc.)/ST/P/S&T/9G-17/2012] for financial support. RM thanks the DST, New Delhi for a fellowship. We are also thankful to the Annesur Rahaman Centre for High Performance Computing, IACS, Kolkata for providing us with computational time.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3an01663c

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