Indole-BODIPY: a “turn-on” chemosensor for Hg²⁺ with application in live cell imaging

Abstract

A new BODIPY, 2-(bis(1-methyl-1H-indol-3-yl)methyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene bearing bis(1-methyl-1H-indol-3-yl)methyl unit at the β-pyrrolic position acting as a ‘turn-on’ chemosensor for Hg²⁺ in solution as well as in HeLa cells, is synthesized. The ‘off’ state of the probe is proposed to be a result of a photoinduced electron transfer (PET) process which gets inhibited in the presence of Hg²⁺ ions, leading to the ‘on’ state. We propose that the detection of Hg²⁺ ions proceeds via the interaction of the π-electron density of the electron rich indolic units of the BODIPY group with that of the acidic Hg²⁺ ions, restricting the PET. The sensing protocols are established by various spectroscopic/electrochemical/DFT studies.

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Introduction

The development of chemical sensors based upon emission and absorption changes has witnessed considerable advancement in recent years. However, the emission based sensors have attained an edge over the absorption based sensors owing to their ample use in biochemical labelling, electroluminescent materials, laser dyes, photonic materials etc. Among these, 4-bora-3a,4a-diaza-s-indacene (BODIPY) based probes have emerged as versatile fluorescent probes owing to their characteristic photophysical properties such as strong absorption and emission with excellent quantum yields.^1–3 Moreover, owing to the thermal stability and photochemical inertness, these have emerged as important components in a variety of applications such as photovoltaic devices, non-linear optical (NLO) materials, near infrared (NIR) absorbing dyes, etc.^4–9 Importantly, the electronic properties of BODIPY based molecular probes could be tuned by functionalisation of BODIPY core at meso-position, as well as pyrrolic at α and β positions, which has efficiently been achieved by various research groups.^4,10–15 Suitable substitution of the BODIPY probes, such as having electron donating groups on the pyrrole ring(s) show significant modulation of their electronic properties.^14–16 In continuation of our interest in the development of molecular sensing probes, we previously reported synthesis and sensing of probes based on BODIPY fluorophore appended with a dithia-dioxa-aza crown ether and ferrocene at meso-position, for Pd²⁺ and Hg²⁺/Cr³⁺, respectively.^17,18 As the functionalization of the β-pyrrolic position of the BODIPY is comparatively less studied in comparison to α-position functionalization,^4,19 herein, we report the synthesis of a BODIPY dye with bis(1-methyl-1H-indol-3-yl)methyl unit at β-pyrrolic position. The molecular electrostatic potential (MEP) calculated for bis(1-methyl-1H-indol-3-yl)methane using Gaussian 09 suite of programs²⁰ (Fig. 1), suggested electron rich indole units to interact with an acidic guest.²¹ Indeed, the dye detects Hg²⁺ ions through such interaction, which in fact constitutes one of the rare mechanisms among the reported sensing probes^22–30 for Hg²⁺ ions. Detection of mercury becomes important as all of its three forms i.e. elemental, inorganic and organic are highly toxic, and have lethal effects on the living beings caused by prenatal brain damage, cognitive and motion disorder, vision and hearing loss, central nervous and endocrine system disruptions etc.^31–37 The atmospheric contamination caused by the release of mercury through gold production, coal combustion and industrial waste, is also an important issue of concern.³⁸

Fig. 1 Molecular electrostatic potential map of bis(1-methyl-1H-indol-3-yl)methane. ED = electron density, redness indicates greatest electron density and blueness indicates the electropositive region.

Experimental section

Chemicals

Solvents (analytical grade) used for the analytical work were purchased from Thomas Baker, while the ones used for the synthetic work were of synthesis grade. The metal salts and N-methyl indole were purchased from Sigma-Aldrich and used as such. Stock solutions (0.1 M) of perchlorate salts of Li⁺, Na⁺, Mg²⁺, Ca²⁺, Pb²⁺, Ba²⁺, Mn²⁺, Ni²⁺, Cu²⁺, Hg²⁺, Cr³⁺, Fe³⁺ and nitrate salts of K⁺, Ag⁺, Zn²⁺, Cd²⁺, La³⁺, Ce³⁺, Pr³⁺, Sm³⁺, Gd³⁺, Nd³⁺, Tb³⁺ ions were prepared in double distilled water, whereas solution of 1 (5 × 10⁻⁶ M) was prepared in acetonitrile.

Instrumentation

IR spectra were recorded on a Perkin Elmer Fourier-Transform spectrophotometer in the range 400–4000 cm⁻¹ in KBr pellets. ¹H (500 MHz), ¹⁹F (282 MHz) and ¹³C NMR (125 MHz) spectra were recorded in CDCl₃ and CD₃CN on AVANCE III Bruker spectrometer. Tetramethylsilane (TMS) was used as an internal standard for ¹H and ¹³C NMR spectra, whereas, trifluoroacetic acid (TFA) was used as an external reference standard (δ: −76 ppm) for ¹⁹F NMR spectrum. Data are reported as follows: chemical shift in ppm (δ), multiplicity (s = singlet, d = doublet), integration, coupling constant J (Hz) and assignment. The NMR titrations (¹⁹F: 282 MHz ¹H: 300 MHz) were performed on JEOL-FT NMR-AL at 300 MHz. Mass spectra were recorded on a microTOF-Q II 10356 high resolution mass spectrometer (HRMS). The electrochemical studies were done on a CHI 660C electrochemical workstation with a conventional three-electrode configuration consisting of a glassy carbon and a counter electrode and a Ag/AgCl electrode as the reference. The experiments were carried out at room temperature in a 10⁻⁴ M solution of the sample in anhydrous (dried over P₂O₅) acetonitrile containing 0.01 M n-tetrabutylammonium hexafluorophosphate (TBAPF₆) as the supporting electrolyte. Deoxygenation of the solutions was achieved by purging dry nitrogen gas for 10 min and the working electrode was cleaned after each run. The voltammograms were recorded with a scan rate of 100 mV s⁻¹. Emission studies were performed on Perkin Elmer LS 55 Fluorescence Spectrometer with excitation slit width as 15 nm and emission slit width as 10 nm with off emission correction mode. UV-visible absorption spectra were recorded on a SHIMADZU 1601 PC spectrophotometer, with a quartz cuvette (path length, 1 cm). The cell holder of the spectrophotometer was maintained at 25 °C for consistency in the recordings. Melting points were determined in open capillaries and are uncorrected. The cell imaging was carried out on the NIKON A1R confocal microscope.

Quantum yield calculations

The fluorescence quantum yields were measured using 9,10-diphenylanthracene as standard having quantum yield of 0.95 in cyclohexane³⁹ using the following equation:

ϕ_u = ϕ_sF_u(1 − 10^−A_sL_s)n_u²/(1 − 10^−A_uL_u)F_sA_un_s²

where, ϕ_u and ϕ_s are the quantum yields of the test and the standard samples, respectively. A_u and A_s are the absorbance values of the test sample and the standard sample respectively, F_u and F_s are the areas of emission bands for the test sample and the reference sample, n_u and n_s are the refractive indices of test sample and standard sample solutions in their respective pure solvents.

Computational details

All theoretical calculations were carried out by using the Gaussian 09 suite of programs.²⁰ The molecular geometries of the chromophores were optimized at the DFT method employing the hybrid B3LYP functional. 6-31G* basis set was used for C, H, B, F, and N atoms whereas SDD basis set for Hg²⁺. The same model chemistry was used for the calculation of the properties of the chromophores. The first 15 excited states were calculated by using time-dependent density functional theory (TD-DFT calculations). The molecular orbital contours were plotted using Gauss view 5.0.9.

Cell culture and confocal cell imaging

Human cervix adenocarcinoma HeLa cell line was obtained from NCCS, Pune, India and maintained on Dulbecco's Modified Eagle's Medium (DMEM) supplemented with streptomycin (100 U mL⁻¹), gentamycin (100 μg mL⁻¹), 10% FBS (Sigma-Aldrich) at 37 °C and humid environment containing 5% CO₂.

For imaging, HeLa cells were cultured on 18 mm glass coverslips in 12 well plates at a concentration of 3 × 10⁴ cells per well and allowed to grow for 48 hours (till 70–80% confluence). Experiments to assess Hg²⁺ uptake were performed in the same media supplemented with different concentrations of Hg(ClO₄)₂ (20 and 30 μM) for 2 hours 30 min. Cells were washed twice with phosphate buffered saline (PBS) before incubating with 5 μM 1 in PBS for 30 min at 25 °C. The cells were again washed twice with PBS before imaging. Confocal imaging of HeLa cells was achieved using NIKON A1R confocal laser scanning microscope using diode laser with excitation at 570 nm. Imaging was carried out with Plan Apo 40× objective lens.

Procedure for synthesis and characterisation of 1

Synthesis of 5,5-difluoro-10-(p-tolyl)-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide 2. To a solution of 4-methylbenzaldehyde (0.5 g, 3.0 mmol) and pyrrole (0.68 mL, 6.0 mmol) in dry CH₂Cl₂ (150 mL), catalytic amount (one drop) of TFA was added and the mixture was stirred for 1 h under N₂ atmosphere at ambient temperature. Subsequently, the reaction mixture was treated with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (0.7 g, 3.0 mmol) and stirred for additional 30 min followed by the addition of Et₃N (15 mL). After 15 min, BF₃·Et₂O (15 mL) was added and the mixture was stirred at ambient temperature for further 3 h. After washing with saturated aqueous NaHCO₃ solution, the organic phase was separated, dried with anhydrous Na₂SO₄, filtered and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate/hexane) to afford 2 as orange coloured solid (70%); mp 160 °C. ¹H NMR (300 MHz, CDCl₃, 25 °C): δ 7.93 (s, 2H, pyrrole –CH), 7.49 (d, 2H, J = 8 Hz, ArH), 7.35 (d, 2H, J = 8 Hz, ArH), 6.97 (d, 2H, J = 4 Hz, pyrrole –CH), 6.55 (d, 2H, J = 4 Hz, pyrrole –CH), 2.48 (s, 3H, CH₃). ¹³C NMR (75 MHz, CDCl₃): δ 147.9, 143.9, 141.6, 135.1, 131.2, 130.8, 129.4, 118.58, 21.5. HRMS: m/z calculated for C₁₆H₁₃N₂BF₂, 282.0956; found: 283.3979 (M⁺ + 1).

Synthesis of 5,5-difluoro-8-formyl-10-(p-tolyl)-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide 3. A mixture of DMF (3 mL, 39 mmol) and POCl₃ (3 mL, 32 mmol) was cooled in an ice bath and stirred under N₂ for 5 min. After warming to room temperature, the reaction mixture was further stirred for 30 min. Then, BODIPY 2 (0.5 g, 0.2 mmol) in ClCH₂CH₂Cl (10 mL) was added to the reaction mixture. After raising the temperature to 80 °C, the reaction mixture was further stirred for 10 h, cooled to room temperature and slowly poured into a saturated aqueous solution of K₂CO₃ (200 mL) cooled in an ice bath. After warming to room temperature, the reaction mixture was further stirred for 1 h and extracted with CH₂Cl₂. The organic layers were combined, washed with water, dried with anhydrous NaSO₄, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography using a mixture of ethyl acetate and hexane as the eluent to give the β-formylated BODIPY 3 as reddish-brown powders (80%); mp 170 °C. ¹H NMR (500 MHz, CDCl₃): δ 9.86 (s, 1H, CHO), 8.27 (s, 1H, pyrrole –CH), 8.16 (s, 1H, pyrrole –CH), 7.50 (d, 2H, J = 8 Hz, ArH), 7.39 (d, 2H, J = 8 Hz, ArH), 7.33 (S, 1H, pyrrole –CH), 7.18 (d, 1H, J = 4 Hz, pyrrole –CH), 6.72 (d, 1H, J = 4 Hz, pyrrole –CH), 2.50 (s, 3H, CH₃). ¹³C NMR (125 MHz, CDCl₃, 25 °C): 184.8, 149.9, 142.7, 142.5, 136.8, 134.8, 134.7, 131.7, 130.7, 130.2, 129.5, 128.6, 121.2, 211.5 ppm. HRMS: m/z calculated for C₁₇H₁₃N₂OBF₂, 311.1089; found: 311.4066 (M⁺ + 1).

Synthesis of 8-(bis(1-methyl-1H-indol-3-yl)methyl)-5,5-difluoro-10-(p-tolyl)-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide 1. The compound 1 was prepared by the condensation reactions of 3 (0.1 g, 1 mmol) with N-methylindole (0.12 mL, 3 mmol), catalysed by Amberlyst 15 (20 mg) (Scheme 1). After completion (TLC), the reaction mixture was allowed to cool at room temperature and diluted with dichloromethane, filtered and concentrated under vacuum. The residue was purified by silica gel column chromatography using a mixture of ethyl acetate and hexane as the eluent to give the 1 as red solid (80%); mp 170 °C. IR (KBr): ν_max/cm⁻¹ 1608, 1473, 1572, 1540, 1280 and 739. ¹H NMR (500 MHz, CDCl₃): δ 7.82 (s, 2H, pyrrole –CH), 7.43 (d, 2H, J = 5 Hz, ArH), 7.40 (d, 2H, J = 5 Hz, ArH), 7.28 (d, 2H, J = 8 Hz, ArH), 7.24 (d, 2H, J = 8 Hz, ArH), 7.20 (dt, 2H, J = 8 Hz, J = 1 Hz, ArH), 7.02 (dt, 2H, J = 8 Hz, J = 1 Hz, ArH), 6.86 (d, 1H, J = 4 Hz, pyrrole –CH), 6.82 (s, 1H, pyrrole –CH), 6.65 (s, 2H), 6.47 (dd, 1H, J = 2 Hz, J = 4 Hz, pyrrole –CH), 5.80 (s, 1H, meso-CH), 3.69 (s, 6H, CH₃), 2.42 (s, 3H, CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 145.72, 142.35, 141.32, 137.62, 131.38, 130.48, 129.97, 129.31, 127.82, 127.24, 121.81, 119.90, 119.05, 117.26, 109.38, 32.78, 32.26, 21.47. HRMS: m/z calculated for C₃₅H₂₉N₄BF₂, 554.2453; found 554.2347 (M⁺).

Scheme 1 Synthetic route for the synthesis of compound 1: (a) (i) TFA, DCM (ii) DDQ (iii) Et₃N, BF₃OEt₂; (b) (i) POCl₃/DMF, 80 °C (ii) K₂CO₃; (c) Amberlyst 15, 80 °C. Inset: optimised structure of 1.

For the spectra see Fig. S1–S4.†

Results and discussion

Synthesis and characterisation

The new BODIPY compound 1 (Scheme 1) was synthesized by following the methods established for such derivatives.⁴⁰ The structures of 1 as well as intermediates were established using spectroscopic data.

A peak at m/z 554.2347 in the high resolution mass spectrum of 1 corresponded to the molecular formula C₃₅H₂₉N₄BF₂. In the ¹H NMR (500 MHz, CDCl₃) spectrum, two singlets (3H and 6H, respectively) at δ 2.42 and 3.69 corresponded to ArCH₃ and the two indol-1-yl methyl groups. The meso-CH appeared at δ 5.80 as a 1H singlet. While the C-1H of the BODIPY unit appeared as a 1H singlet at δ 6.82 ppm, the C-3 and C-5 H_s were observed at δ 7.82 (s, 2H). A doublet of doublet (J = 2 Hz and J = 4 Hz) at δ 6.47 was assigned to C-6H, while a 1H doublet at δ 6.86 (J = 4 Hz) corresponded to C-7H of the BODIPY unit. The protons of the C-8 aryl ring appeared as two 2H doublets (J = 5 Hz). On the other hand, the indol-2-yl H_s appeared as a 2H singlet at δ 6.65 ppm. The two sets of the C-5′ and 6′ H_s of the two indole rings appeared as two (2H) doublets of triplets (J = 8 Hz, J = 1 Hz) at δ 7.02 and δ 7.20 ppm, respectively. Similarly, the two sets of the C-4′ and 7′ H_s of the two indole rings appeared as two (2H) doublets (J = 8 Hz) at δ 7.24 and δ 7.28 ppm, respectively. The ¹³C NMR spectrum and heteronucleous single quantum coherence (HSQC) spectrum corroborated the structure of 1 and clearly established 2D heteronuclear chemical shift correlations (see ESI Fig. S5†). Before evaluating the behaviour of 1 towards various analytes, pH titrations were performed, which revealed the stability of 1over a wide pH range (see ESI Fig. S6†), suggesting the pliability of compound 1 for detection of the analyte in environmental and biological settings without resorting to buffered medium. The solution of 1 (5 × 10⁻⁶ M, in CH₃CN) was non-emissive (ϕ_f = 0.00541), which is attributed to the photoinduced electron transfer (PET) process.⁴¹ The PET mechanism is favoured when the molecular group attached to the fluorophore has the energies of its highest occupied molecular orbital, HOMO (H) and the lowest unoccupied molecular orbital, LUMO (L) levels between the H and L levels of the fluorophore. Thus, simultaneous exchange of electrons from L of the fluorophore to the L of the attached molecular group and H of the latter to H of the fluorophore can occur. Consequently, due to this PET process, the fluorophore would shift to its ground state via a non-radiative process⁴¹ thus leading to emission quenching. In order to lend support to our claim, we performed the time dependent density functional theory (TD-DFT) calculations using Gaussian 09 suite of programs.²¹ As depicted in Fig. 2, on promotion of the electron from the H−7 to L+3 in the BODIPY unit (step i), the higher energy (−5.78 eV) H−2 of bis(1-methyl-1H-indol-3-yl)methyl unit could transfer its electron density to the H−7 (−6.82 eV) of the BODIPY unit (step ii) followed by the electron transfer from L+3 (−0.12 eV) of BODIPY unit to L+1 (−0.74 eV) of bis(1-methyl-1H-indol-3-yl)methyl unit (step iii) and subsequently to H−2 (−5.91 eV) (step iv) causing quenching of the fluorescence emission. Our preliminary investigations revealed that the emission intensity of 1 was significantly enhanced upon addition of aqueous solution of Hg²⁺ ions (added as perchlorate salt) over a number of other ions including alkali and alkaline earth metal ions: Na⁺, K⁺, Ag⁺, Li⁺, Ca²⁺, Mg²⁺, Ba²⁺; transition metal ions: Cr³⁺, Mn²⁺, Fe²⁺, Fe³⁺, Ni²⁺, Cu²⁺, Zn²⁺, Pb²⁺ (added as perchlorate/nitrate salts) and lanthanides: Sm³⁺, Pr³⁺, Ce³⁺, La³⁺, Gd³⁺, Nd³⁺, Tb³⁺ (added as their nitrate salts) under similar experimental conditions (see ESI Fig. S7†). Prompted by this observation, we performed elaborate titration experiments in order to quantify the results with Hg²⁺ ions. The successive addition of an aqueous solution of Hg²⁺ ions (2.87 × 10⁻⁷ M to 3.33 × 10⁻⁵ M) to the solution of 1 (5 × 10⁻⁶ M, in CH₃CN) resulted in a steady increase in emission intensity (ϕ_f = 0.0682) as well as visually detectable change in solution fluorescence, which attained saturation when addition of 3 × 10⁻⁵ M (corresponding to 6.6 equiv.) was complete (Fig. 3). However, saturation was achieved within 2 min when an aqueous solution of Hg²⁺ corresponding to 6.6 equiv. was instantly added (see ESI Fig. S8†). We attribute this emission enhancement to the interaction of π-electron density of the electron-rich indole groups with that of the acidic Hg²⁺ ions,^42,43 restricting the PET process responsible for the radiation less decay (steps i–iv, Fig. 2). Moreover, the appropriate H orbital of the bis(1-methyl-1H-indol-3-yl)methyl unit gets lowered in energy on complexation with mercury in comparison to free 1, thus inhibiting the electron transfer (step v) to BODIPY core (Fig. 2). The detection limit calculated from the fluorescence data comes out to be 3.3 × 10⁻⁷ M which is comparable to most of the probes reported in literature (see ESI Fig. S9 and Table S1†).

Fig. 2 Comparative energies of frontier orbitals of BODIPY and bis(1-methyl-1H-indol-3-yl)methyl unit in the (free and complexed states. H = HOMO, L = LUMO).

Fig. 3 (a) Changes in the emission spectral behaviour of 1 (5 × 10⁻⁶ M, in CH₃CN) upon addition of Hg²⁺ (2.87 × 10⁻⁷ M to 3.33 × 10⁻⁵ M, in H₂O). Inset: colour change in 1 upon addition of Hg²⁺ (b) the photograph of 1 and 1 + Hg²⁺ under UV light (365 nm).

Further support of this explanation was accrued from electrochemical studies. Upon gradual addition of aqueous solution of Hg²⁺ (2 × 10⁻⁶ to 3 × 10⁻⁵ M) to the solution of 1 (1 × 10⁻⁴ M, in CH₃CN), the reversible reduction wave at −0.729 V (corresponding to the BODIPY core)⁴⁴ undergoes anodic shift to −0.643 V (Fig. 4), indicating the depleted electron density on the BODIPY core. Further the competitive experiments performed in the presence of other metal ions, did not affect the emission changes, thereby demonstrating the specificity of 1 for Hg²⁺ ions (see ESI Fig. S10†). The electronic absorption spectrum of 1 (5 × 10⁻⁶ M, in CH₃CN) is characterized by two major bands: a weak band centred at 366 nm (ε = 15 000 M⁻¹ cm⁻¹) and a strong band 520 nm (ε = 39 200 M⁻¹ cm⁻¹), and are assigned as the intramolecular charge transfer (ICT) bands on the basis of TD-DFT calculations, having main contributions from H−1 → L and H−2 → L, H−3 → L, respectively. However, upon gradual addition of aqueous solution of Hg²⁺ ions (2.87 × 10⁻⁷ M to 3.33 × 10⁻⁵ M), the absorption spectrum depicted a bathochromic shift (Fig. 5), from 520 nm to 570 nm attaining saturation at 2.87 × 10⁻⁵ M addition. The attendant hyperchromic shift of the band at 570 nm caused visual change in the color intensity of the solution. This band at 570 nm has major contributions from the low energy H−2 → L, H−3 → L transitions (ΔE = 2.61 and 2.89 eV, respectively) in comparison to the free 1 (ΔE = 3.23 and 3.76 eV, respectively) (Fig. 6). Fitting the UV-visible titration data using HypSpec, a non-linear least squares fitting programme,⁴⁵ the binding constant, log β_1 : 2 = 3.27 was obtained. The predicted 1 : 2 stoichiometry (1 : (Hg²⁺)₂) was further confirmed by the Job plot experiment (Fig. 5, inset). In support of the observed stoichiometry of the possible complex formed through the interaction of 1 and Hg²⁺, on the basis of DFT calculations, we propose that both the indole groups interact independently with the Hg²⁺ ions, as shown in the best optimised structure of the putative Hg²⁺ complex (see ESI Fig. S11†). The energy minimized structure of 1 predicts that benzene ring of one of the indole rings engages in a η⁴-coordination⁴³ to the Hg(1) with the Hg⋯C_ind distances in the range of 2.445 to 3.868 Å and are within the sum of the van der Waals radius of mercury (1.73–2.00 Å)^46–48 and that of carbon in heterocyclic aromatic compounds (1.7 Å). However, the benzene ring of the other indole ring is engaged in η²-coordination to the Hg(2) with the Hg⋯C_ind distances 3.681 and 3.778 Å. The longer Hg(2)⋯C_ind distances in comparison to Hg(1)⋯C_ind distances and the different coordination behaviours of Hg²⁺ ions can be understood as: Hg(2) is also engaged in Hg⋯F interactions (weak) with Hg(2)⋯F distances 3.403 and 3.615 Å within the sum of van der Waals radii 1.30 to 1.38 Å and 1.73 to 2.00 Å of fluorine⁴⁸ and Hg, respectively.

Fig. 4 Changes in the cyclic voltammogram of 1 (1 × 10⁻⁴ M, in CH₃CN) upon addition of Hg²⁺ solution (2 × 10⁻⁶ to 3 × 10⁻⁵ M, in H₂O).

Fig. 5 Absorption spectral changes of 1 (5 × 10⁻⁶ M, in CH₃CN) upon addition of Hg²⁺ (2.87 × 10⁻⁷ M to 3.33 × 10⁻⁵ M, in H₂O). Inset: Job's plot.

Fig. 6 Relative energies of the frontier molecular orbitals participating in the low energy electronic transitions of 1 and its complex with Hg²⁺.

In support of the predicted mercury–fluorine interactions, we recorded the changes in the ¹⁹F NMR spectrum of 1 upon adding Hg²⁺ ion solution (Fig. 7). The spectrum of 1 (δ −142.54, q, J = 30 Hz) showed a small upfield shift (Δδ = 0.35) in the fluorine signals, also accompanied with insignificant change in the coupling constant (ΔJ = 3 Hz) indicating weak interactions between the mercury and fluorine.

Fig. 7 ¹⁹F NMR spectral changes in 1 in the presence of Hg²⁺.

The interaction of Hg²⁺ with 1 was also monitored by ¹H NMR titration experiment. For that, titration of 1 was performed by adding increasing concentration of Hg(ClO₄)₂ (Fig. 8) to a solution (0.6 mL) of 1 (6.0 mM) in CD₃CN. A clear shift in the N–Me-indole signals (Δδ = 0.375 ppm) was observed, although the peak positions of p-Me-C₆H₄ (Δδ = 0.123 ppm) and/or the intensity of other protons was not significantly changed up to the addition of 0.68 equiv. (saturation point) of Hg(ClO₄)₂. However, upon adding excess amount of Hg(ClO₄)₂ the observed spectral changes (peak broadening and/or splitting) may be attributed to the heavy atom effect of Hg²⁺ ions.

Fig. 8 ¹H NMR titration of 1 (6 mM) (I) with 0.13 (II), 0.26 (III), 0.41 (IV), 0.55 (V), 0.68 (VI), 0.83 (VII), 0.96 (VIII), 1.1 (IX) and 1.25 (X), equiv. Hg(ClO₄)₂ in CD₃CN.

Since reversibility of the sensing protocol is a prerequisite for a molecular probe to be an efficient chemosensor for practical applications, we also studied the reversibility of the sensing event. Thus, the addition of an aqueous solution of cysteine to the in situ formed complex of 1 and Hg²⁺ leads to the restoration of the original non-emissive behaviour of 1 (see ESI Fig. S12†). The potential application of 1 for the detection of Hg²⁺ was further confirmed at cellular level using HeLa cells (human cervix carcinoma) as an in vitro model system. Fig. 9 shows the fluorescence images of HeLa cells treated with different concentrations of Hg²⁺ ions (0, 20 and 30 μM) and 1 (5 μM). The overlay of fluorescent and bright field images revealed the perinuclear fluorescence in cells and thereby depicting the excellent membrane permeability and subcellular localization.

Fig. 9 Confocal images of HeLa cells, supplemented with 1 (1 × 10⁻⁵ M) and Hg²⁺ (0, 20 and 30 μM).

Conclusions

In summary, we have synthesised a new BODIPY derivative which takes advantage of an electron rich bis(1-methyl-1H-indol-3-yl)methyl unit, appended at β-pyrrolic position, in detecting Hg²⁺ ions in solution as well as live cells. The detection protocol is manifested in the form of ‘turn-on’ emission for which we propose the inhibition of PET in the presence of Hg²⁺ ions prevailing in otherwise non-fluorescent BODIPY derivative.

Acknowledgements

We thank SERB-DST, New Delhi (project SB/S1/OC-45/2013) for financial assistance. NK thanks DST, New Delhi for INSPIRE fellowship and GNDU (UPE) for facilities.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: ¹H, ¹³C NMR, IR spectral data, optimised structures and their Cartesian coordinates, complete ref. 21. See DOI: 10.1039/c6ra17695j

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