A ratiometric fluorescent probe for detection of exogenous mitochondrial SO₂ based on a FRET mechanism

Abstract

A novel imidazo[1,5-a]pyridine-hemicyanine based ratiometric fluorescent probe for detection of mitochondrial SO₂ was designed and synthesized. The probe is based on a fluorescence resonance energy transfer (FRET) mechanism. It exhibits high selectivity and sensitivity towards SO₃²⁻ with a fast response time (3 min) and detection limit of 0.13 μM. Further, it showed low cytotoxicity and was successfully applied to image exogenous mitochondrial SO₂ in cells.

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Introduction

Sulfur dioxide (SO₂), which used to be considered as a toxic environmental pollutant, is now considered to be a new possible signal molecule following nitric oxide, carbon monoxide and hydrogen sulfide.^1–4 Cancers, neurological disorders and cardiovascular diseases could be caused by high exogenous SO₂ levels. As exogenous SO₂ is produced via oxidation of some sulphur-containing amino acids and hydrogen sulfide in mitochondria,^5,6 it is extremely important to develop selective, sensitive and rapid methods for SO₂ detection in mitochondria.

When hydrated in aqueous media, SO₂ can be transformed into its derivatives bisulfite (HSO₃⁻) and sulfite (SO₃²⁻). Therefore, methods for the detection of HSO₃⁻/SO₃²⁻ such as electrochemistry, chromatography, titration and capillary electrophoresis have been developed.^7–10 However, those methods can not realize imaging in cells.

Since the first sulfite fluorescent probe was reported by Chang group in 2010,¹¹ numerous probes based on nucleophilic reactions with aldehydes, Michael additions, dequenching of levulinate and coordinative interactions have been developed in recent years.^12–29 Despite the remarkable progress achieved, limitations such as long detection times and poor water solubility still remain. More importantly, those intensity-based probes are susceptible to factors like external environment, substrate concentration and instrument sensitivity.

Ratiometric fluorescent probes are more advantageous than intensity-based ones. Förster resonance energy transfer (FRET) mechanism is most widely used to construct well-performing ratiometric probes.^30–32 To date, some well-behaved ratiometric fluorescent probes for SO₂ derivatives have been developed.^33–36 However, there is great room for improvement since these probes are still subject to some drawbacks such as unsatisfactory detection limits, long response time, and poor selectivity over H₂S. Recently, we successfully synthesized the imidazole[1,5-a]pyridine via a tandem reaction.^37–39 Some fluorescent probes based on this new fluorophore for Cu²⁺, and Hg²⁺ have been constructed subsequently.^40,41 Continuing our efforts to search for new fluorophore and extend their applications,^42–53 herein, we report a new FRET platform for the rapid detection of SO₂. Imidazole[1,5-a]pyridine was selected as donor, hemicyanine dyad as receptor, and piperazine as connection unit. The probe IPIN-SO₂ can detect SO₃²⁻ rapidly (3 min) and sensitively in a wide pH range of 5–10. More importantly, IPIN-SO₂ can be used for imaging exogenous mitochondrial SO₂ in cells.

Experimental

Materials and apparatus

UV-vis spectra were recorded on a U-2600 UV-vis spectrometer (Hitachi) and fluorescence spectra were recorded on a RF-5301PC luminescence spectrophotometer (Shimadzu) at room temperature. ¹H NMR and ¹³C NMR spectra were measured on a Bruker Avance 400 (400 MHz) spectrometer (CDCl₃ as solvent and tetramethylsilane (TMS) as an internal standard). HRMS spectra were recorded on a Q-TOF6510 spectrograph (Agilent). Nikon fluorescence inverted microscope (Ti 2-U) was used to record cell imaging. All reagents and solvents were purchased from commercial sources and used without further purification. Metal ion solution was prepared by dissolving the deionized water with metal chloride as raw material. The anionic solution was prepared by dissolving sodium containing compounds into deionized water. Deionized water was used in the whole absorption and fluorescence detection process.

Cell culture and imaging

Brain glioma cells were cultured in RPMI-1640 containing 10% bovine serum in a 5% CO₂/95% air incubator at 37 °C. For cell imaging experiments, the growth medium was removed and replaced by RPMI-1640 without CS. The cells were incubated in a 1 μM IPIN-SO₂ incubator at 37 °C and 5% CO₂/95% air for 30 minutes. The cells were washed with PBS three times and cell images were obtained via an inverted fluorescence microscope from Ti 2-U (Nikon, ECLIPSE, equipped with FRET system, Mercury lamp light source). For the probe, the excitation light source is 395/25 nm and the emission collected through filter is 605/55 nm and 460/50 nm. The colocalization experiments have been carried out through Laser Scanning Confocal Microscope (FV1000, Olympus). For the probe, the excitation light source is 405 nm and the emission collected is 560–620 nm. For the MitoTracker@ Deep Red, the excitation light source is 633 nm and the emission collected through filter is 650–700 nm.

Synthesis

Synthesis of compound 3. Compound 1 and 2 were synthesized according to the literature.⁵

Compound 1 (315 mg, 1 mmol) was dissolved in ethanol (15 mL), then compound 2 (190 mg, 1 mmol) was added, and 3 drops of piperidine were added. After heating and refluxing for 12 hours, the solvent was removed under reduced pressure. A deep red solid was obtained which was used for the next step without further purification.

Synthesis of the probe IPIN-SO₂. Compound 4 were synthesized according to the literature.³³

Compound 4 (253 mg, 1 mmol) was added to 30 mL dichloromethane and then DMAP (183 mg, 1.5 mmol) and EDC (288 mg, 1.5 mmol) were added. After stirring at room temperature for half an hour, compound 3 (488 mg, 1 mmol) was added and stirred for 12 hours at room temperature. Then the solvent was removed under reduced pressure to afford crude compound IPIN-SO₂, which was purified on a silica gel column (C₂H₅OH : CH₂Cl₂ = 1 : 100; yields: 68.9%). ¹H NMR (400 MHz, CDCl₃) δ 8.20 (d, J = 8.0 Hz, 2H), 8.09 (d, J = 12.0 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.58–7.50 (m, 7H), 7.02 (d, J = 8.0 Hz, 2H), 6.72 (d, J = 8.0 Hz, 1H), 4.86 (q, J = 8.0 Hz, 2H), 3.86 (s, 4H), 3.66 (s, 4H), 2.97 (t, J = 8.0 Hz, 2H), 1.81 (m, 6H), 1.60 (m, 5H), 1.44 (m, 2H), 0.97 (t, J = 8.0 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 179.3, 167.6, 154.9, 154.6, 142.6, 140.6, 138.7, 135.2, 129.5, 128.5, 124.1, 122.6, 120.9, 117.9, 114.2, 113.4, 112.3, 106.8, 51.4, 46.7, 44.5, 42.9, 29.0, 27.6, 26.3, 22.5, 22.0, 14.0, 13.8. HRMS: ([M]⁺); calcd for C₃₆H₄₁ClN₅O: 594.2994; found: 594.3003.

Results and discussion

Synthesis of the probe IPIN-SO₂

The synthetic route of probe IPIN-SO₂ is shown in Scheme 1. Probe IPIN-SO₂ was obtained as deep purple solid powder in 89.5% yield through classical condensation of compound 3 and compound 4. The probe IPIN-SO₂ was characterized by ¹H NMR, ¹³C NMR and HRMS.

Scheme 1 Synthesis of probe IPIN-SO₂.

UV-vis and fluorescence spectra response of IPIN-SO₂

As shown in Fig. 1, IPIN-SO₂ is more selective to SO₃²⁻ compared with other competitive ions, which do not cause any significant absorption changes in the visible region, which can be used as a “naked eye” chemical colorimeter. When added 10 equiv. SO₃²⁻, the solution was obviously lighter from pink (Fig. 1, inset).

Fig. 1 Ultraviolet absorption spectra of IPIN-SO₂ (10 μM) in 0.1 M PBS (pH = 7.4) buffer solution with addition of 100 μM of various species (SO₃²⁻, AcO⁻, Br⁻, H₂PO₄⁻, Cl⁻, CO₃²⁻, HCO₃⁻, F⁻, HPO₄²⁻, I⁻, NO₂⁻, NO₃⁻, S₂O₃²⁻, SO₄²⁻, HS⁻, Fe³⁺, Ca²⁺, Cu²⁺, K⁺, Na⁺, Zn²⁺), GSH (glutathione), Hcy (homocysteine), Cys (cysteine).

The interaction between IPIN-SO₂ and SO₃²⁻ was further studied by UV-vis spectroscopic titration in 0.1 M PBS (pH = 7.4) buffer solution. IPIN-SO₂ showed characteristic absorption at 500 nm. However, when the sulfite was added to the IPIN-SO₂ solution, the absorption peak at 500 nm decreased rapidly with the increase of sulfite concentration as shown in Fig. S1.† When 10 equiv. amount of SO₃²⁻ was added to IPIN-SO₂ solution, the fluorescence intensity immediately increased at 475 nm and the fluorescence intensity at 580 nm decreased significantly. By contrast, the other competitive cations did not cause any significant fluorescence changes, indicating that IPIN-SO₂ has a better selectivity for SO₃²⁻ in the fluorescence spectrum as shown in Fig. 2.

Fig. 2 Fluorescence spectra of IPIN-SO₂ (10 μM) in 0.1 M PBS (pH = 7.4) buffer solution with addition of 100 μM of various species (λ_ex = 380 nm).

As shown in Fig. 3, the fluorescence titration process of IPIN-SO₂ was recorded. In IPIN-SO₂ aqueous solution, with the increase of SO₃²⁻ concentration, the fluorescence intensity increases at 475 nm and decreases at 580 nm, and the two emission peaks can be well separated (105 nm). The results show that the developed FRET system can effectively avoid the overlap of emission spectra and ensure the high resolution and accuracy of the determination. Moreover, when SO₃²⁻ concentration increased from 0 μM to 30 μM, the fluorescence intensity ratio increased from 0.29 to 12.78, about 44 times. When SO₃²⁻ concentration was in the range of 1.5–4.0 μM, there was a good linear relationship as shown in Fig. S2.†

Fig. 3 Fluorescence spectra of IPIN-SO₂ (10 μM) with the addition of SO₃²⁻ (0–30 equiv.) in 0.1 M PBS (pH = 7.4) buffer solution (λ_ex = 380 nm).

According to LOD = 3σ/k (σ is the standard deviation of ten blank solutions and k is the slope of the linear calibration plot between the fluorescence intensity and the concentration of SO₃²⁻), the detection limit was as low as 0.13 μM.

In addition, the interference experiments were carried out under the coexistence of various species (Fig. 4). Background ions did not interfere with fluorescence intensity. Sulfite-induced fluorescence enhancement (I₄₇₅/I₅₈₀) remained unaffected by the coexistence of other species.

Fig. 4 Ratiometric fluorescence responses I₄₇₅/I₅₈₀ of IPIN-SO₂ (10 μM) upon the addition of 10 equiv. SO₃²⁻ in the presence of 100 μM background ions in PBS (pH = 7.4) buffer solution.

Kinetic study

In Fig. 5, the time course of fluorescence response of IPIN-SO₂ aqueous solution with SO₃²⁻ is shown. The fluorescence intensity ratio (I₄₇₅/I₅₈₀) reached the maximum value in 3 min when 10 equiv. SO₃²⁻ was added and the fluorescence ratio of IPIN-SO₂ almost remained unchanged with time, which indicates that IPIN-SO₂ can be used as a fast response SO₃²⁻ probe.

Fig. 5 Time dependent increase of IPIN-SO₂ (10 μM) fluorescence intensities after addition of 10 equiv. SO₃²⁻ in PBS (pH = 7.4) solution (λ_ex = 380 nm).

Effect of pH

As shown in Fig. 6, in order to detect SO₃²⁻ efficiently and selectively, the effect of different acid concentrations on IPIN-SO₂ was studied to find the suitable pH range. In PBS buffer solution, the fluorescence titration curves of IPIN-SO₂ and IPIN-SO₂ have no obvious change between pH 5.0 and 10.0 of fluorescence intensity ratio (I₄₇₅/I₅₈₀), indicating that the sensor IPIN-SO₂ and the sensor IPIN-SO₂ existing in SO₃²⁻ are stable within this pH range.

Fig. 6 Ratio of fluorescent intensities at 475 nm and 580 nm for IPIN-SO₂ (10 μM) in the presence of SO₃²⁻ (10 equiv.) at varied pH values (λ_ex = 380 nm).

Mechanism

As shown in Scheme 2, once the IPIN-SO₂ energy donor is excited, FRET will enter the hemicyanine group from the imidazole[1,5-a]pyridine fluorescent group, which may weaken or even quench the fluorescence of the imidazole[1,5-a]pyridine fluorescent group. Interruption of the p–π conjugation in the hemicyanine fluorophore results in increasing the energy of its first singlet level above that of the donor group. In addition, both the probe IPIN-SO₂ and the donor can be activated at 380 nm, but the receptor cannot, which further confirms the FRET process in probe IPIN-SO₂.

Scheme 2 Proposed sensing mechanism of IPIN-SO₂ with SO₃²⁻.

To further clarify the proposed mechanism, HRMS of the reaction product was conducted. A clear mass (m/z 676.2735) of adduct appeared after the probe reacted with SO₃²⁻ (Fig. S8†).

Cell imaging

As probe IPIN-SO₂ shows excellent optical response to SO₃²⁻ in vitro, cell imaging of IPIN-SO₂ has been further studied in glioma cells. IPIN-SO₂ fluorescence is stable in living cell (Fig. S3†) and cytotoxicity is negligible at 1–16 μM concentration (Fig. S4†). Because cationic cyanine dyes may accumulate in mitochondria,^54,55 colocalization assays were performed with MitoTracker@ Deep Red FM and IPIN-SO₂ (Fig. 7). The fluorescence of IPIN-SO₂ and MitoTracker@ Deep Red FM has a significant overlap, and the overlap coefficient is 0.948 (Fig. 7d), indicating that IPIN-SO₂ was well distributed in mitochondria.

Fig. 7 Glioma cells were incubated with IPIN-SO₂ (0.1 μM) for 1 h, followed by MitoTracker@ Deep Red (0.01 μM) for 0.5 h. (a) The fluorescence image of MitoTracker@ Deep Red FM. λ_ex = 633 nm, λ_em = 650–700 nm. (b) The fluorescence image of probe IPIN-SO₂. λ_ex = 405 nm, λ_em = 560–620 nm. The red fluorescence was colored as green for discrimination. (c) Merged images of (a) and (b). (d) Colocalization coefficient (Pearson's coefficient) of IPIN-SO₂ and MitoTracker@ Deep Red was 0.948.

Then the probe IPIN-SO₂ was applied to SO₃²⁻ imaging in living glioma cells. When the glioma cells were incubated with probe IPIN-SO₂ for 1 hour, the red channel showed strong fluorescence and blue channel weak fluorescence (Fig. S5†). When incubated for 0.5 hour with different concentrations of Na₂SO₃, the cells showed enhanced fluorescence of red channel and decreased fluorescence of blue channel.

Furthermore, we studied whether the probes could be used to detect exogenous bisulfite in cells. Glioma cells were incubated with probe IPIN-SO₂ for 1 hour, washed with PBS solution three times, incubated with 0.5 mM GSH (glutathione) and 0.25 mM Na₂S₂O₃ for 0.5 hour, and then photographed with fluorescence inverted microscope to observe obvious fluorescence changes in glioma cells (Fig. 8). On the contrary, no significant fluorescence changes were observed when the glioma cells incubated with IPIN-SO₂ were incubated only with GSH or Na₂S₂O₃. These results indicate that the probe can detect exogenous bisulfite in glioma cells.

Fig. 8 The first line (vertically): glioma cells were incubated with IPIN-SO₂ (0.1 μM) for 1 h; the second line: glioma cells were incubated with IPIN-SO₂ (0.1 μM) for 1 h, and then with 0.5 mM GSH and 0.25 mM Na₂S₂O₃ for another 0.5 h; the third line: glioma cells were incubated with probe (0.1 μM) for 1 h, then with 0.5 mM GSH for 0.5 h.

Conclusions

In summary, a novel FRET-based ratio fluorescence probe of imidazole[1,5-a]pyridine substituted hemicyanines has been developed. IPIN-SO₂ has unique selectivity and high sensitivity (detection limit 0.13 μM) for SO₃²⁻, which can detect SO₃²⁻ rapidly (3 min) over a wide pH range of 5 to 10. It is noteworthy that the new ratio fluorescence probe avoids automatic fluorescence, severe self-quenching and fluorescence detection errors. More importantly, the probe has been successfully applied to the identification of exogenous SO₂ in mitochondria in cells.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the Science Fund of Shandong Province for Excellent Young Scholars (ZR2017JL015), the Natural Science Foundation of China (21602153) and the Natural Science Foundation of Shandong Province (ZR2018LB014).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: ¹H NMR, ¹³C NMR and MS spectra of probe, and additional cell images. See DOI: 10.1039/c8ra10328c

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