An ICT-Based Hydrogen Sulfide Sensor with Good Water Solubility for Fluorescence Imaging in Living Cells

Introduction

Hydrogen sulfide (H₂S), traditionally considered to be a toxic gas with the typical smell of rotten eggs and an antioxidant or scavenger for reactive oxygen species (ROS) [1], has recently emerged as a member of the endogenous gaseous transmitter family of signaling molecules including nitric oxide (NO) and carbon monoxide (CO) [2–3]. The endogenous H₂S is produced by the enzymes (such as cystathionine β-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulphurtransferase) catalysis action of cysteine or cysteine derivatives in mammalian tissues [4–6]. H₂S plays an important physiological role in many biological processes, for instance, ischemia reperfusion injury, vasodilation, apoptosis, insulin signaling, and oxygen sensing [7–11]. However, abnormal levels of H₂S is associated with various diseases, like Alzheimer’s disease [12], Down’s syndrome [13], diabetes [10], and liver cirrhosis [14]. Therefore, it’s very significant for monitoring H₂S using sensitive, selective, and water-soluble fluorescent probe in the native biological environment.

Fluorescence imaging has been widely used as a powerful tool for monitoring biomolecules within the context of living systems with high spatial and temporal resolution [15–16]. Naphthalimide dye is the favorable building block for constructing fluorescent probes because of its excellent photophysical properties, such as high extinction coefficients, excellent quantum yields, and great photostability. In addition, azido group is well-known for sensitively and selectively responding to H₂S, the electron withdrawing azide is very easy reduced by H₂S affording the electron donating amine [17–26]. Thus, in this work, we employed the naphthalimide chromophore as the signal reporter and azido group as the responding site for H₂S to construct a H₂S probe based on internal charge transfer (ICT) process (Scheme 1). In addition, to enhance the water solubility, we introduced a hydrophilic alcoholic group to the naphthalimide core. Prior to react with H₂S, we supposed that probe Nap-N ₃ has negligible fluorescence. However, interaction of naphthalimide azide with H₂S affords 4-amino-1,8-naphthalimide, which would elicit a significant fluorescence enhancement via ICT process caused by the electron donating amine and the electron withdrawing amide. So, probe Nap-N ₃ might be employed as a turn-on fluorescent probe for detecting H₂S.

Scheme 1

A turn-on fluorescent H₂S probe based on the proposed ICT switching mechanism

Experimental

Materials and Instruments

Unless otherwise stated, all reagents were purchased from commercial suppliers and used without further purification. Solvents used were purified by standard methods prior to use. Twice-distilled water was used throughout all experiments; High resolution mass spectrometric (HRMS) analyses were measured on a Finnigan MAT 95 XP spectrometer; NMR spectra were recorded on an INOVA-400 spectrometer, using TMS as an internal standard; Electronic absorption spectra were obtained on a LabTech UV Power spectrometer; Photoluminescent spectra were recorded with a HITACHI F4600 fluorescence spectrophotometer with a 1 cm standard quartz cell; The fluorescence imaging of cells was performed with OLYMPUS FV1000 (TY1318) confocal microscopy; The pH measurements were carried out on a Mettler-Toledo Delta 320 pH meter; TLC analysis was performed on silica gel plates and column chromatography was conducted over silica gel (mesh 200–300), both of which were obtained from the Qingdao Ocean Chemicals.

Synthesis

The target compound Nap-N ₃ was readily synthesized in just two steps as shown in Scheme 2.

Scheme 2

Synthesis of H₂S sensor Nap-N₃ and the reduced product Nap-NH₂

Synthesis of Compound Nap-N₃

The mixture of compounds 4-bromo-1,8-naphthalic anhydride (82.8 mg, 0.3 mmol) and ethanolamine (90 mg, 1.5 mmol) in 5 mL of ethanol was heated at 80 °C under reflux for 4 h. After cooling to room temperature, the reaction solvent was removed under reduced pressure. The resulting residue was a light yellow solid as the crude product of intermediate compound 2, which was directly carried on the next reaction without further process. The mixture of compound 2 (16 mg, 0.05 mmol) and sodium azide (13 mg, 0.20 mmol) in 3 mL of dry N,N-dimethylformide (DMF) was heated to 90 °C for 3 h in the dark. After cooling to room temperature, the reaction mixture was poured into 100 mL of water and then extracted three times with dichloromethane. The organic phase was collected, washed with brine, and dried with anhydrous MgSO₄. The solvent was removed under reduced pressure and the solid residue was purified by flash chromatography column using methanol/dichloromethane (v/v 1:20) to afford a yellow solid as compound Nap-N ₃ (10 mg, yield 70.9 %). ¹H NMR (400 MHz, CDCl₃) δ 8.64 (d, J = 7.2 Hz, 1H), 8.58 (d, J = 8.0 Hz, 1H), 8.45 (d, J = 8.4 Hz, 1H), 7.75 (t, J = 7.8 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 4.45 (t, J = 5.2 Hz, 2H), 3.96 (t, J = 5.2 Hz, 2H).¹³C NMR (100 MHz, CDCl₃) δ 164.87, 164.50, 143.84, 132.55, 132.06, 129.13, 126.91, 124.32, 122.29, 118.52, 114.72, 61.85, 42.80. MS (EI) m/z 282.1 [M]⁺. HRMS (EI) m/z calcd for C₁₄H₁₀N₄O₃ [M]⁺: 282.0747. Found 282.0758.

Synthesis of Compound Nap-NH₂

Sodium sulfide (20 mg, 0.25 mmol) was added to the solution of compound Nap-N ₃ (14 mg, 0.05 mmol) in 3 mL DMF/H₂O (v/v 9:1) mix-solvent. The mixture was stirred for 1.5 h at room temperature in the dark. The reaction mixture was poured into 100 mL of water and then extracted two times with dichloromethane and ethyl acetate (v/v 5:1). The organic phase was collected, washed with brine, and dried with anhydrous MgSO₄. The solvent was removed under reduced pressure and the solid residue was purified by flash chromatography column using methanol/dichloromethane (v/v 1:10) to afford a yellow solid as compound Nap-NH ₂ (9.5 mg, yield 74.2 %). ¹H NMR (400 MHz, DMSO) δ 8.69 (d, J = 8.4 Hz, 1H), 8.42 (d, J = 7.2 Hz, 1H), 8.19 (d, J = 8.4 Hz, 1H), 7.65 (t, J = 8.2 Hz, 1H), 7.56 (s, 2H), 6.89 (d, J = 8.4 Hz, 1H), 4.13 (t, J = 6.6 Hz, 2H), 3.59 (m, 2H).¹³C NMR (100 MHz, DMSO) δ 164.39, 163.49, 153.23, 134.35, 131.42, 130.18, 129.92, 124.35, 122.23, 119.79, 108.57, 107.91, 58.42, 41.78.MS (EI) m/z 256.1 [M]⁺. HRMS (EI) m/z calcd for C₁₄H₁₂N₂O₃ [M]⁺: 256.0842. Found 256.0844.

Determination of the Fluorescence Quantum Yield

Fluorescence quantum yields for compounds Nap-N ₃ and Nap-NH ₂ were determined by using fluorescein (Φ _f  = 0.95 in 0.1 M NaOH aqueous solution) as fluorescence standard [27]. The quantum yields were calculated using the following equation.

$$ {\Phi}_{\mathrm{F}\left(\mathrm{X}\right)}={\Phi}_{\mathrm{F}\left(\mathrm{S}\right)}\left({A}_S{F}_X/{A}_X{F}_S\right){\left({n}_X/{n}_S\right)}^2 $$

Where Φ_F is the fluorescence quantum yield, A is the absorbance at the excitation wavelength, F is the area in the corrected emission spectrum, and n is the refractive index of the solvent used. Subscripts _S and _X refer to the standard and to the unknown, respectively.

HeLa Cells Culture

HeLa cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10 % FBS (fetal bovine serum) in an atmosphere of 5 % CO₂ and 95 % air at 37 °C.

Imaging of Exogenous H₂S in Living Cells

HeLa cells were incubated with 5.0 μM Nap-N ₃ for 20 min in an atmosphere of 5 % CO₂ and 95 % air, and then treated with 20 μM Na₂S for 10 min. Subsequently, the cells were imaged using OLYMPUS FV1000 (TY1318) confocal microscope with an excitation filter of 405 nm and emission channels of 520–570 nm (green channel).

Imaging of Endogenous H₂S in Living Cells

PC-3 cells were incubated with 200 μM cysteine for 60 min in an atmosphere of 5 % CO₂ and 95 % air, and then treated with 5.0 μM Nap-N ₃ for 20 min. Subsequently, the cells were imaged using OLYMPUS FV1000 (TY1318) confocal microscope with an excitation filter of 405 nm and emission channels of 520–560 nm (green channel).

Results and Discussion

Fluorescence Responses to H₂S

With compound Nap-N ₃ in hand, we first evaluated the capability of Nap-N ₃ to detect H₂S in aqueous buffer. The titration of H₂S to the probe Nap-N ₃ (10.0 μM) was performed in 25 mM PBS buffer (pH 7.4) with just 2 % ethanol. As designed, upon excitation at 438 nm, the free sensor displayed faint fluorescence at around 546 nm (Fig. 1a). However, addition of Na₂S (a standard source for hydrogen sulfide) elicited a significant emission enhancement, suggesting that Nap-N ₃ was reduced affording Nap-NH ₂ in the presence of H₂S and ICT process occurred. The fluorescence intensities at 546 exhibited 7.9-fold increase, it’s the ICT resonance structure of compound Nap-NH ₂ that contributes mainly to the enhanced fluorescence. The quantum yields of compounds Nap-N ₃ and Nap-NH ₂ were determined 0.0056 and 0.1175, respectively, using a reference fluorescein dye (with a quantum yield of 0.95 in 0.1 M NaOH aqueous solution). Importantly, the chemodosimeter shows an excellent linear relationship between the fluorescent emission intensities at 546 nm and the concentrations of H₂S from 0.2 to 3.0 equivalent (Fig. 1b), suggesting that the chemodosimeter is potentially useful for quantitative determination of H₂S. The ratio changes were observed in absorption spectra, as shown in Fig. 2, upon addition of H₂S, the absorption of naphthalimide azide at around 376 nm gradually faded and simultaneously a new red-shifted (62 nm) absorption band at around 438 nm (characteristic absorption of amino naphthalimide) was enhanced. There is a clear isosbestic point (at 407 nm) was appeared, suggesting that the conversion from Nap-N ₃ to Nap-NH ₂ is a concerted process.

Fig. 1

a Fluorescence spectra (λ_ex = 408 nm) of 10 μM Nap-N₃ with 0–3.5 eq. of Na₂S in 25 mM phosphate buffer (pH 7.4, containing 2 % ethanol). The inset shows the visual fluorescence color of Nap-N₃ before (left) and after (right) addition of H₂S (UV lamp, 365 nm). b The linear relationship between the fluorescence intensity at 546 nm and the concentration of H₂S

Fig. 2

Absorption spectra of probe Nap-N₃ with 0–3.5 eq. of Na₂S in 25 mM phosphate buffer (pH 7.4, containing 2 % ethanol)

Mechanism Studies

To shed light on the H₂S-triggered fluorescence turn-on response, we decided to characterize the reduced product. Incubation of Nap-N ₃ with Na₂S afforded the product, which was isolated and characterized by standard NMR and mass spectrometry (Figs. S1–3). The ¹HNMR spectra comparison of probe Nap-N ₃ and the product Nap-NH ₂ , the peak of NH₂ in Nap-NH ₂ was obviously observed, which suggested the resulting product should be reduced compound Nap-NH ₂ (Fig. 3). We further employed time-dependent density function (TD-DFT) to calculate the molecular orbital plots of Nap-N ₃ and Nap-NH ₂ by a suite of Gaussian 03 programs (6-31G(d) basis sets). As shown in Fig. 4, for the probe Nap-N ₃ , both the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) in the excited states are distributed on the naphthalimide backbone and azido group, however, the LUMO +1 is mainly located on the electron-withdrawing azido group. The theoretical data suggested the π electrons do not transfer from the azido unit to the naphthalimide moiety and ICT process could not occur in Nap-N ₃ . By contrast, in the case of Nap-NH ₂ , the LUMO +1 is primarily resided on the naphthalimide backbone, but not on the electron-donating amino group, which indicated the π electrons transfer to the naphthalimide moiety and ICT process occurs. These experimental facts and theoretical data supported our proposed ICT switching mechanism.

Fig. 3

The comparison of the partial ¹H NMR spectra of a Nap-N₃ in CD₃Cl and b Nap-NH₂ in d ₆ -DMSO

Fig. 4

Interfacial plots of the orbitals for Nap-N₃ (left) and Nap-NH₂ (right) in the excited states

Selectivity Studies

The detection limit was calculated to be 1.09 × 10⁻⁶ M (S/N = 3), which locates in the range of physiological concentration of H₂S in vivo, indicating that the probe is sensitive to H₂S and might be suitable for detecting endogenous H₂S in biological samples. To examine the selectivity, the probe Nap-N ₃ (10 μM) was treated with various biologically relevant species in the aqueous buffer, such as the representative anions, reactive oxygen species, reducing agents, small-molecule thiols, and Na₂S. As shown in Fig. 5, addition of the representative interfering species including F⁻, Cl⁻, I⁻, N₃ ⁻, CO₃ ²⁻, SO₄ ²⁻, HPO₄ ²⁻, NO₂ ⁻, NO₃ ⁻, AcO⁻, SCN⁻, citrate at 1 mM, and S₂O₃ ²⁻, SO₃ ²⁻, ClO⁻, H₂O₂, NO, ascorbic acid at 200 μM induced negligible changes. Notably, small-molecule biothiols such as glutathione (GSH) and cysteine at 1 mM triggered only a small emission intensity increase. However, just upon addition of 30 μM Na₂S could elicit obvious increase of emission intensity.

Fig. 5

Fluorescence intensity (at 546 nm) of sensor Nap-N₃ (10 μM) in the presence of various analytes in aqueous solution (pH 7.4 PBS, containing 2 % ethanol): 1, blank; 2, F⁻; 3, Cl⁻; 4, I⁻; 5, N₃ ⁻; 6, CO₃ ²⁻; 7, SO₄ ²⁻; 8, HPO₄ ²⁻; 9, NO₂ ⁻; 10, NO₃ ⁻; 11, Ac⁻; 12, SCN⁻; 13, citrate; 14, S₂SO₃ ²⁻; 15, ClO⁻; 16, H₂O₂; 17, SO₃ ²⁻; 18, NO; 19, ascorbic acid; 20, Cys; 21, GSH; 22, Na₂S

Reaction-Time

In addition, the fluorescence intensity reached its maximum at about 15 min (Fig. 6). The results suggested the probe Nap-N ₃ has a high selectivity for H₂S over other biological species.

Fig. 6

Time-dependent fluorescence intensity (at 546 nm) responses of sensor Nap-N₃ (10 μM) to 3 eq.Na₂S in PBS buffer

pH Effect

What’s more, probe Nap-N ₃ could respond well to H₂S at round physiological pH (Fig. 7). The results indicated Nap-N ₃ may be suitable for studies of H₂S in the living systems.

Fig. 7

The pH influence on the fluorescence intensity (at 546 nm) of Nap-N₃ (10 μM) in the absence (black square) or presence (red circle) of Na₂S (30 μM)

Fluorescence Imaging in Living Cells

Firstly, to examine the cell membrane permeability of naphthalimide dye, we employed compound Nap-NH ₂ for imaging living cells. The cells incubated with Nap-NH ₂ exhibited strong fluorescence, while control cells showed none fluorescence (Fig. S4), suggesting the naphthalimide dye is cell membrane permeable. Then to examine the utility of the sensor, we intended to image H₂S in living cells. For proof-of-concept, Nap-N ₃ (5 μM) was initially incubated with HeLa cells for 20 min, after rinsed three times by PBS buffer, then treated with 20 μM Na₂S for another 20 min. As shown in Fig. 8, the cells incubated with only Nap-N ₃ exhibited faint fluorescence in the green channel. However, after co-incubation with Na₂S, strong green fluorescence was observed in living cells, suggesting Nap-N ₃ was reduced by H₂S affording fluorescent compound Nap-NH ₂ . Thus, these results revealed that Nap-N ₃ is cell membrane permeable and capable of monitoring H₂S in living cells.

Fig. 8

Confocal fluorescence images of HeLa cells incubated with 5 μM Nap-N₃ for 20 min (a, b) and then 20 μM Na₂S for 20 min (c, d). Images (a, c) were acquired using 405 nm excitation and green emission channels of 520–570 nm; (b, d) bright field images. Scale bar = 40 μm

In addition to detection of extraneous H₂S, we further detected the biosynthesis H₂S inside the cells. It’s well-known that cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) could catalyze cysteine for producing H₂S in living cells [28–29]. HeLa cells were firstly incubated with 200 μM cysteine for 1 h, after rinsed three times by PBS buffer, followed addition of 5 μM Nap-N ₃ and incubated another 20 min. As exhibited in Fig. 9, in comparison with the cells loaded with only the probe, the cells co-incubated with cysteine and Nap-N ₃ elicited a marked increase of fluorescence intensity in the green channel. These results further indicated that Nap-N ₃ is capable of detecting not only external H₂S in living cells, but also endogenous H₂S biologically produced by the cells.

Fig. 9

Confocal fluorescence images of HeLa cells incubated with 5 μM Nap-N₃ only for 20 min (a, b) and 200 μM cystine for 1 h followed by 5 μM Nap-N₃ for 20 min (c, d). Images (a, c) were acquired using 405 nm excitation and green emission channels of 520–570 nm; (b, d) bright field images. Scale bar = 40 μm

Conclusions

In summary, we have introduced a high water-soluble H₂S fluorescent probe based on naphthalimide chromophore. The probe Nap-N ₃ exhibits a turn-on fluorescence signal for responding H₂S via ICT switching mechanism. It has a selectivity and sensitivity for in vitro H₂S over other various biologically relevant species, and could detect both the exogenous and endogenous H₂S in living cells.