A Highly Selective Fluorescent Probe for Imaging Hydrogen Sulfide in Living HeLa Cells

As an important endogenous gasotransmitter, hydrogen sulfide (H2S) has been implicated with a variety of biological processes and has attracted more and more attention for its key role in a wide range of pathological processes. However, lacking tools for H2S-specific in situ detection, the changes of endogenous H2S levels in the pathological progression of diseases are still unclear. In this work, a turn-on fluorescent probe (BF2-DBS) has been designed and synthesized by two-step reactions using 4-diethylaminosalicylaldehyde and 1,4-dimethylpyridinium iodide as raw materials. Probe BF2-DBS displays high selectivity and sensitivity to H2S with a large Stokes shift and good anti-interference ability. The practical application of probe BF2-DBS to detect endogenous H2S was evaluated in living HeLa cells.


Introduction
Hydrogen sulfide (H 2 S) is widely known as a colorless, toxic gas with rotten egg smell in the past decades. H 2 S also plays a key role in biological systems, and has recently been recognized as the third endogenous gaseous signaling molecule [1,2] besides nitric oxide (NO) and carbon monoxide (CO). In mammalian systems, it is produced endogenously from L-cysteine and catalyzed by two pyridoxal-5'-phosphate-dependent enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) [3]. Endogenous hydrogen sulfide has a variety of biological effects in the cardiovascular system, such as vasorelaxation, inhibiting vascular smooth muscle cell proliferation, and showing myocardial negative inotropic effects. It also plays a pathophysiological role in many diseases including Alzheimer's disease [4], diabetes [5], liver cirrhosis [6], renal/cardiovascular diseases [7], and cancers [8]. Therefore, there is an intense need to develop methods for detection of H 2 S in order to exhibited excellent sensing performance toward H 2 S with 40-fold fluorescence enhancement, a rapid response time, excellent sensitivity and good selectivity. In light of these unique properties, BF 2 -DBS has been successfully applied for the fluorescence imaging of H 2 S in living cells, demonstrating that it would be an effective tool for H 2 S detection in living systems. We believe that the development of this probe is a significant breakthrough for visualizing endogenous H 2 S within living biological samples and opens up further opportunities to study its cellular biochemistry.

Reagents and Instrumentation
All reagents and instrumentation were shown in Supporting Information.

Synthesis of Probe BF 2 -DBS
Synthesis of compound 1. 4-diethyl aminosalicylaldehyde (1mmol, 200 mg) and p-aminophenol (1mmol, 110 mg) were dissolved in appropriate amount of ethanol, then p-toluene sulfonic acid (p-TsOH) (0.5mmol, 95 mg) was dissolved in 2mL H 2 O and added to the mixed special system. Stir the mixture at 80℃ for 3 h. The reaction process was monitored by TLC. After the reaction was over, the mixture coolant was brought to room temperature and concentrated by vacuum, and yellow solid crude product was obtained by filtration. The crude product was obtained by column chromatography, about 227 mg, and the yield was about 80%. 1  Synthesis of compound 2. The obtained compound 1 (1.5mmol, 425 mg) was dissolved in dichloromethane solution. At 0℃, 3mL triethylamine and 3mL boron trifluoride ether were added for about 30 min at room temperature. Then 5% sodium bicarbonate solution was added to quench the reaction, extract the organic layer, and then dry with anhydrous Na2SO4 and concentrate in vacuum. The crude product was purified by silica gel column to obtain about 100 mg orange solid with yield of about 20%. 1  Synthesis of probe BF 2 -DBS. Compound 2 (137 mg, 0.41mmol) was dissolved in an appropriate amount of dichloromethane and anhydrous triethylamine (about 54µL) was added. Then 2, 4-dinitrobenzene sulfonyl chloride was dissolved in dichloromethane. The mixture was dropped into the mixing system at 0℃ and stirred overnight at room temperature. After the reaction was completed, the crude product was concentrated in vacuum and purified by silica gel column. 78 mg of orange solid was obtained, with a yield of about 35%. 1

Synthesis and Sensing Mechanism
As shown in scheme 1, we used 4-(diethylamino)-salicylaldehyde react with 4-aminophenol to get the fluorophore 2. Since 2,4-dinitro-1-fluorobenzene is both a good electronwithdrawing group and a selective reporting group for H 2 S, we used it to react with fluorophore 2 and obtained the probe BF 2 -DBS ( 1 H NMR, 13 C NMR and HRMS in Supplementary data). The asymmetric electronic structure in this fluorophore will contribute to the large stokes shift. The strong electron pull-push along the conjugated structure is beneficial to the strong fluorescence intensity. Thus, the desired optical properties including large stokes shift, bright emission as well as sensitive response, will be integrated into this probe. To validate H 2 S-induced thiolysis of the BF 2 -DBS probe, high performance liquid chromatography (HPLC) analysis was conducted ( Figure S1, Supporting Information). The data showed that BF 2 -DBS (HPLC retention time, TR = 5.9 min) was converted to compound 2 (TR = 3.0 min).

Spectroscopic Properties
The capability of BF 2 -DBS for recognizing H 2 S was investigated by monitoring fluorescent intensities after adding various concentrations of NaHS. When different concentrations of NaHS were introduced into the BF 2 -DBS system, the fluorescence emission at 500 nm significantly enhances with the increasing concentrations of NaHS (Fig. 1). A linear range from 0 to 40 µM was obtained with a limit of detection of 76 nM (3σ/k), indicating that BF 2 -DBS has high sensitivity toward H 2 S and has the potential to track the changes of H 2 S levels in the biosystems.
At present, tens of minutes or even an hour is needed to achieve complete reaction for most H 2 S probes, which greatly hinders the application of probes in real-time detection. Therefore, we explored the time-dependent experiment of BF 2 -DBS to H 2 S (Fig. 2a, original fluorescence spectrum changes were shown in figure S2 in supporting information). The fluorescence intensity of BF 2 -DBS increased rapidly after adding H 2 S, and reached stability within 20 min. These data illustrated the good reactivity of BF 2 -DBS, which was beneficial to the rapid detection of H 2 S. The selectivity of BF 2 -DBS toward H 2 S, was investigated against various biological relevant species, such as amino acids, cations, active oxygen species, anions and thiols. Under identical conditions, addition of 20 equiv of various analytes produced negligible fluorescence as compared with that of H 2 S (Fig. 2b, original fluorescence and absorption spectrum

Visualizing H 2 S in Living Cells
Inspired by above promising results, we further evaluated whether probe could be used to monitor endogenous H 2 S in living cells. We performed the imaging analysis on the HeLa cells. Firstly, we had the HeLa cells incubated only with the BF 2 -DBS (10 µM) for 30 min. As shown in Fig. 4, a strong green fluorescence signal was observed (Fig. 4, A1), indicating probe BF 2 -DBS could detect the endogenous changes were shown in figure S3 and figure S4 in supporting information). These results demonstrated that BF 2 -DBS could be employed for specific recognition of H 2 S.
A suitable range of pH response is one of the key factors for the successful application of the probe in biological systems (pH = 7.4). Therefore, the applicable pH range of BF 2 -DBS (10 µM) was explored. As shown in Fig. 3a, negligible fluorescence of BF 2 -DBS changes was observed at the pH range of 3-9 (original fluorescence spectrum changes were shown in figure S5 in supporting information). The cytotoxicity and biocompatibility of BF 2 -DBS were also evaluated on HeLa cells by using standard methyl thiazolyl tetrazolium (MTT) assay (Fig. 3b). The cell viability remained at a high level after 24 h incubation with  Funding Not applicable.

Data Availability Not applicable.
Code Availability Not applicable.

Declarations
Ethics Approval For this type of study, the ethical approval was not required, because this study does not involve animal manipulation. H 2 S in living HeLa cells. While, pretreating the cells with 1 mM NEM (a thiol scavenger) and propargylglycine (PPG, known as a commercial inhibitor for CBS and CSE) for 1 h, cells in group B1 exhibited the negligible fluorescence in the collection channel of 450-550 nm under the excitation of 405 nm, because endogenous hydrogen sulfide is removed by NEM and its production is also inhibited by PGG. However, as shown in panel C1, after addition of NaHS (40 µM), the fluorescence intensity in panel C1 increased obviously. These results indicates that the probe BF 2 -DBS penetrated into the cells and released the fluorescence by the trigger of endogenous or exogenous H 2 S.

Conclusion
In summary, a novel fluorescence probe for H 2 S was designed and synthesized. On the basis of the interaction of 2, 4-dinitrophenyl ester group with H 2 S, probe BF 2 -DBS exhibited a robust "turn-on" response to H 2 S with high sensitivity and selectivity over other thiols. Cell imaging experiments have been carried out and the results indicate that probe BF 2 -DBS can be used to image endogenous H 2 S in living HeLa cells. We expect that probe BF 2 -DBS Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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