A purine fluorescent probe EDPH and application thereof in detection of content of hydrogen sulfide or imaging of Hela cells

Abstract

The application discloses a purine-based fluorescent probe EDPH and application of the purine-based fluorescent probe EDPH to detection of content of hydrogen sulfide or Hela cell imaging, and a structural formula of the purine-based fluorescent probe EDPH is shown in the following formula: The purine-based fluorescent probe EDPH shows high selectivity and sensitivity to hydrogen sulfide molecules in a solution; the preparation method has simple steps, raw materials are easy to obtain, the obtained product is a solid powder, easy to store, has wide application prospect, and can be mass-produced and applied.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to a purine-based fluorescent probe EDPH and its application in detecting hydrogen sulfide content or in HeLa cell imaging. Background Technology

[0002] Hydrogen sulfide (H2S) is a toxic gas with a distinctive odor, but it is also an essential gas transfer agent in our lives. This gas molecule is produced in mammals through the endogenous metabolism of cysteine ​​(Cys) and its derivatives, catalyzed by specific enzymes. Hydrogen sulfide molecules participate in many physiological and pathological processes, playing an irreplaceable role. For example, abnormal H2S levels in human cells can lead to various diseases such as Alzheimer's disease, diabetes, Down syndrome, and cirrhosis. Furthermore, H2S is closely related to mitochondrial function. It has been reported that various reactive substances are produced in mitochondria that oxidize the body's internal environment, causing acute damage. The presence of hydrogen sulfide can prevent this damage, thus protecting cells. Therefore, visualizing the distribution and concentration of H2S in living systems is crucial for elucidating its biological role.

[0003] Organic small molecule fluorescent probes, with their unique high sensitivity, real-time imaging, and high spatiotemporal resolution, show great potential for detecting various substances. H₂S fluorescent probes are designed to utilize the reducing or nucleophilic properties of H₂S to initiate specific chemical reactions. Xu et al. synthesized a fluorescent probe containing a disulfide bond. Hong et al. developed a novel reactive near-infrared (NIR) H₂S fluorescently activated probe using 2,4-dinitrophenyl ether as the reaction site. Erman et al. pioneered a new recognition method by using electrophilic cyanate esters as H₂S recognition groups. However, most of the aforementioned probes require complex synthetic methods and long reaction times. Purines are fluorophores that can penetrate cells. Purines and their derivatives are not only valuable compounds in many biological and chemical processes but also consist of multiple large planar conjugated heterocycles with excellent fluorescence properties, making them widely suitable as good backbones for fluorescent probes. In recent years, some highly efficient fluorescent probes based on purine Schiff bases have been reported for the selective detection of various metal ions. However, highly selective fluorescent probes for detecting hydrogen sulfide from purine precursors have not yet been reported. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention aims to provide a purine-based fluorescent probe EDPH and its application in detecting hydrogen sulfide content or in HeLa cell imaging. The fluorescent probe EDPH is a Schiff base fluorescent probe based on purine and capable of rapidly detecting hydrogen sulfide molecules. It has the advantages of specific recognition of hydrogen sulfide molecules, short response time, and high sensitivity.

[0005] To address the problems in the existing technology, the technical solution adopted by this invention is as follows:

[0006] A purine-based fluorescent probe, EDPH, has the following structural formula:

[0007]

[0008] The preparation method of the above-mentioned purine-based fluorescent probe EDPH includes the following steps:

[0009] Step 1: Add 4,6-dichloro-5-aminopyrimidine and 1-naphthylamine to an organic solvent. After the solids dissolve, add concentrated hydrochloric acid, reflux and stir. After the reaction is complete, remove the organic solvent by vacuum distillation, dissolve in NaOH, extract with ethyl acetate, remove ethyl acetate by vacuum distillation, and recrystallize with methanol and water to obtain 6-chloro-N-(naphth-1-yl)pyrimidine-4,5-diamine, i.e., compound I.

[0010] Step 2: Compound I, naphthoic acid, polyphosphoric acid, and dodecyltrimethylammonium chloride were dissolved in phosphorus oxychloride and stirred under reflux. After the reaction was complete, the reaction solution was cooled to room temperature and the organic solvent was removed by vacuum distillation. An ice-water mixture was added to the reaction system under ice-water bath conditions, and the mixture was purified and eluted. The aqueous phase was removed by filtration to obtain 6-chloro-8,9-di(naphth-1-yl)-9H-purine, i.e., compound II.

[0011] Step 3: After dissolving compound II in ethanol, add hydrazine hydrate after the solid has dissolved, reflux and stir until the reaction is complete, cool to room temperature, remove the organic phase by vacuum distillation, wash the solid with organic solvent to obtain 6-hydrazine-8,9-di(naphth-1-yl)-9H-purine, i.e., compound III;

[0012] Step 4: Dissolve 3,5-dimethyl-4-hydroxybenzaldehyde and K2CO3 in DMF, stir at room temperature for 30 minutes, add 2,4-dinitrofluorobenzene, and stir the reaction at room temperature. After the reaction is complete, add 200 mL of distilled water, extract with ethyl acetate, remove the solvent under reduced pressure, and recrystallize with ethyl acetate to obtain yellow crystals, namely compound IV.

[0013] Step 5: Dissolve compounds III and IV in an organic solvent and reflux the mixture with stirring. After the reaction is complete, cool to room temperature, remove the solvent by vacuum distillation, and purify the crude product by recrystallization to obtain (E)-1-(4-(2,4-dinitrophenoxy)-3,5-dimethylbenzylmethyl)-2-(8,9-bis(naphthyl-1)-9H-purine-6-yl)hydrazine, i.e., compound EDPH.

[0014] The reaction formula for the purine-based fluorescent probe EDPH of this invention is shown below:

[0015]

[0016] As an improvement, the temperature for vacuum distillation in step 1 is 45-55℃.

[0017] As an improvement, the organic solvent used to wash the solid in step 3 is chloroform.

[0018] As an improvement, the temperature of the reflux stirring in step 4 is 75°C, and the reflux time is 2 hours.

[0019] The above-mentioned purine-based fluorescent probe EDPH is used to detect hydrogen sulfide in solution or cells.

[0020] As an improvement, the detection limit for hydrogen sulfide molecules is 41 nmol / L.

[0021] The application of the above-mentioned purine-based fluorescent probe EDPH in HeLa cell imaging.

[0022] Beneficial effects:

[0023] Compared with existing technologies, this invention provides a purine-based fluorescent probe EDPH and its application in detecting hydrogen sulfide content or in HeLa cell imaging. The purine-based fluorescent probe EDPH uses a purine ring as the fluorescent group and hydrazine hydrate and hydroxyl groups as linking groups to synthesize a fluorescent probe based on the purine parent hydrogen sulfide molecule. This preparation method uses readily available raw materials, is simple, and yields a solid powder product that is easy to store and has good stability. The use of purine derivatives as a rigid plane provides advantages such as low biotoxicity. This fluorescent probe specifically recognizes hydrogen sulfide molecules, has a short response time, and high sensitivity, exhibiting high sensitivity and selectivity for trace amounts of hydrogen sulfide molecules in solution and cells. Attached Figure Description

[0024] Figure 1 The image shows a comparison of the UV absorption spectra of the fluorescent probe for detecting hydrogen sulfide molecules prepared in Example 1 in DMSO / H2O (v / v = 9:1) solution for itself and for the fluorescent probe after the addition of hydrogen sulfide molecules.

[0025] Figure 2The fluorescent probe for detecting copper ions prepared in Example 1 is shown in the selective fluorescence spectra of different ions and organic compounds in DMSO / H2O (v / v = 9:1) solution.

[0026] Figure 3 The fluorescence spectrum response of the fluorescent probe for detecting hydrogen sulfide molecules prepared in Example 1 to different concentrations of hydrogen sulfide molecules in DMSO / H2O (v / v = 9:1) solution is shown.

[0027] Figure 4 The fluorescence response diagram of the fluorescent probe for detecting hydrogen sulfide molecules prepared in Example 1 in DMSO / H2O (v / v = 9:1) solution for selective interference detection of different ions and organic compounds is shown.

[0028] Figure 5 The response of the fluorescent probe for detecting hydrogen sulfide molecules prepared in Example 1 at different pH values ​​is shown.

[0029] Figure 6 The results of cell experiments using the fluorescent probe for detecting hydrogen sulfide molecules prepared in Example 1.

[0030] Figure 7 The mass spectrometry (MS) spectrum of the fluorescent probe EDPH prepared in Example 1 is shown below.

[0031] Figure 8 The nuclear magnetic resonance (NMR) of the EDPH fluorescent probe for detecting hydrogen sulfide prepared in Example 1. 1 H-NMR spectrum;

[0032] Figure 9 The nuclear magnetic resonance (NMR) of the EDPH fluorescent probe for detecting hydrogen sulfide prepared in Example 1. 13 C-NMR spectrum. Detailed Implementation

[0033] The present invention will now be described in detail with reference to specific embodiments.

[0034] Unless otherwise specified, the experimental methods used in this invention are conventional methods. Unless otherwise specified, all materials and reagents used in the experiments are commercially available. All reagents used in the examples are commercially available analytical grade or chemically pure.

[0035] In the examples, the various metal ion solutions are prepared by adding deionized water to chloride salt chemical reagents with a purity of over 99%, such as anhydrous cadmium chloride and anhydrous ferric chloride.

[0036] Example 1

[0037] A method for preparing a purine-based fluorescent probe EDPH includes the following steps:

[0038] (1) Preparation of compound I (6-chloro-N-(naphth-1-yl)pyrimidin-4,5-diamine)

[0039] In a 100 mL round-bottom flask, 5-amino-4,6-dichloropyrimidine (5.00 g, 30 mmol) and 1-naphthylamine (8.72 g, 61 mmol) were added and dissolved in 50 mL of methanol, followed by the addition of 5 mL of 12 mol / L HCl. The mixture was refluxed and stirred at 65 °C for 5 days. After the reaction solution cooled, the organic solvent was removed by vacuum distillation. The obtained crude product was then dissolved in 50 mL of 1 mol / L NaOH solution, and the mixture was extracted three times with ethyl acetate. Finally, ethyl acetate was removed by vacuum distillation. The organic phase was washed with 1.2 mol / L HCl, then dehydrated with saturated brine, and dried to obtain the crude product. The crude product was recrystallized from CH3OH / H2O (v / v, 1:5), and dried to obtain a light purple solid powder, compound I (6.26 g, yield 76%). 1 HNMR(400MHz,DMSO-d6)δ8.92(s,1H),7.99–7.94(m,1H),7.92–7.87(m,1H),7.85–7.81(m,1H),7.63(s,1H),7.58–7.48(m,4H),5.52(s,2H). 13 C NMR (100MHz, DMSO-d6) δ151.62,145.58,138.88,135.45,134.42,129.88,128.59,126.47,126.33,126.17,126.15,124.96,123.97,123.69.

[0040] The structural formula of the obtained compound I is:

[0041]

[0042] (2) Preparation of compound II 6-chloro-8,9-di(naphth-1-yl)-9H-purine

[0043] Compound I (1.00 g, 3.7 mmol), 1-naphthoic acid (3.2 g, 18.5 mmol), and dodecyltrimethylammonium chloride (DTAC) (0.10 g, 10 mmol) were dissolved in 25 mL of POCl3, followed by the addition of polyphosphoric acid (PPA) (5.00 g, 14.8 mmol). After the reaction was complete, the solvent was evaporated, and the residue was purified by column chromatography on silica gel using CH3OH / CH2Cl2 (v / v, 1 / 250) to give compound II (yield 47%). 1HNMR(400MHz,DMSO-d6)δ8.77(s,1H),8.30–8.24(m,1H),8.07–7.81(m,4H),7.71(dd,J=7.3 ,1.2Hz,1H),7.60–7.44(m,6H),7.40(m,J=8.3,6.8,1.3Hz,1H),7.33(dd,J=8.3,7.1Hz,1H). 13 C NMR(101MHz,DMSO-d6)δ155.8,155.1,152.5,149.5,133.9,133.2,131.5,131.3,130.6(d,J=2.9Hz) ,129.8,129.5,128.7,128.0,127.8,127.7,127.3,127.0,126.3,126.0,125.7,124.9,122.9.ESI-MS m / z:[M+H] + calcd.For C 25 H 16 ClN4407.1, found406.9.

[0044] The structural formula of the obtained compound II is:

[0045]

[0046] (3) Preparation of compound III (6-hydrazino-8,9-bis(naphthyl-1-yl)-9H-purine)

[0047] Compound II (200 mg, 0.67 mmol) was dissolved in 20 mL of ethanol, and then hydrazine hydrate was added to the flask. The mixture was stirred at 65 °C for 3 hours. After the reaction was complete (monitored by TLC), the reaction mixture was cooled to room temperature, and a large amount of yellow solid precipitated out. After filtration, the solid was washed with a large amount of cold ethanol and dried to give a white solid, compound III (183 mg, 92%). 1 H NMR (400MHz, DMSO-d6) δ9.31 (s, 1H), 8.21 (d, J = 5.6Hz, 2H), 7.94 (dd, J = 8.5, 3.3Hz, 2H), 7.86 (dd, J = 8 .9,5.3Hz,2H),7.61(d,J=7.3Hz,1H),7.53–7.43(m,5H),7.40(s,1H),7.36–7.25(m,2H),4.77(s,2H). 13C NMR (101MHz, DMSO-d6) δ153.4,149.1,133.9,133.3,131.9,131.6,130.3,130.3,129.9,129.2,128. 6,128.5,127.8,127.6,127.5,127.3,127.1,126.7,126.2,125.7,124.9,122.8,118.1,79.6.ESI-MS m / z:[M+H] + calcd.for C 25 H 19 N6403.2, found 403.2.

[0048] The structural formula of the obtained compound III is:

[0049]

[0050] (4) Preparation of compound IV (4-(2,4-dinitrophenoxy)-3,5-dimethylbenzaldehyde)

[0051] In a 100 mL round-bottom flask, 3,5-dimethyl-4-hydroxybenzaldehyde (800 mg, 5.32 mmol) and K₂CO₃ (3.742 g, 27 mmol) were added sequentially and dissolved in 30 mL of DMF. After stirring at room temperature for 30 min, 2,4-dinitrofluorobenzene was added, and the reaction was stirred at room temperature for 3 h, with the reaction monitored by TLC during the reaction. After the reaction was completed, 200 mL of distilled water was added, and the system was extracted with ethyl acetate (3 × 20 mL), followed by removal of the solvent under reduced pressure. Recrystallization from ethyl acetate gave yellow crystalline compound IV in 82% yield. 1 H NMR (400MHz, Chloroform-d) δ9.99 (s, 1H), 8.89 (d, J = 2.8Hz, 1H), 8.30 (dd, J = 9.2, 2.7Hz, 1H), 7.72 (s, 2H), 6.68 (d, J = 9.2Hz, 1H), 2.23 (s, 6H). 13 CNMR(101MHz,CHLOROFORM-D)δ191.15,154.49,153.91,141.55,138.40,134.87,132.06,131.24,129.33,129.29,122.65,115.52,16.26.

[0052] The structural formula of the obtained compound IV is:

[0053]

[0054] (5) Preparation of a fluorescent probe for detecting copper ions based on purine precursors: EDPH

[0055] Compound III (150 mg, 0.52 mmol) and compound IV (243 mg, 0.77 mmol) were dissolved in 25 mL of ethanol, and the mixture was refluxed at 75 °C for 2 hours. After the reaction solution was cooled to room temperature, a large amount of yellowish-white solid precipitated out. After filtration and collection, the solid was washed with cold ethanol to obtain the white solid probe compound EDPH (321 mg, 90%). 1 H NMR(400MHz,Chloroform-d)δ8.86(d,J=2.7Hz,1H),8.62(s,1H),8.42(s,1H),8.25(d d,J=9.2,2.7Hz,1H),7.97(s,1H),7.86(dd,J=7.7,1.5Hz,2H),7.82–7.71(m,2H),7.5 7(s,2H),7.51–7.47(m,1H),7.46(d,J=1.8Hz,1H),7.45(s,1H),7.42(dd,J=6.6,1.3H z,1H),7.37–7.32(m,2H),7.24(dd,J=7.3,1.2Hz,1H),7.15–7.08(m,1H),2.11(s,6H). 13 C NMR(101MHz,CHLOROFORM-D)δ155.28,154.18,153.29,151.43,151.14,150.52,144.10,1 41.16,138.27,134.33,133.70,132.81,131.85,131.09,130.90,130.73,130.28,130.05 ,129.30,128.80,128.71,128.64,127.76,127.33,126.98,126.63,126.52,126.19,125. 40,125.31,124.54,122.53,122.40,118.61,115.71,16.07,1.12.HR-MS(ESI)m / z:[M+H] - calcd.forC 40 H 28 N8O5700.2183, found 701.2266

[0056] The structural formula of the obtained fluorescent probe compound is:

[0057]

[0058] Mass spectra (MS) and nuclear magnetic resonance spectra of the fluorescent probes prepared in Example 1 1 H-NMR spectrum, nuclear magnetic resonance 13 The C-NMR spectra are as follows: Figure 7 , Figure 8 and Figure 9 As shown in the figure, the fluorescent probe of the present invention was successfully synthesized.

[0059] Example 2

[0060] The probe compound EDPH obtained in Example 1 was prepared into a 1 mM probe stock solution using dimethyl sulfoxide. Each ion and organic compound was prepared into a 100 mM stock solution using deionized water. 30 μL of the probe stock solution and 50 μL of hydrogen sulfide stock solution were added to 3 mL of blank solution DMSO / H2O (v / v = 9:1). The results were analyzed using a fluorescence spectrometer and a UV spectrophotometer. The maximum excitation wavelength of the fluorescent probe was found to be 500 nm, and the maximum emission wavelength was 370 nm. Specific test results are as follows:

[0061] Add 30 μL of probe stock solution and 50 μL of hydrogen sulfide molecular stock solution to 3 mL of blank stock solution (DMSO / H2O (9 / 1, v / v, pH 7.4, HEPES buffer, 0.2 mM)). Let stand for half an hour before performing UV spectroscopy. Figure 1 As shown, the probe exhibits strong absorbance at 340 nm; however, the addition of hydrogen sulfide to the probe solution causes a red shift in the wavelength corresponding to the peak (from 340 nm to a peak at 475 nm). The main reason for this red shift is that the addition of hydrogen sulfide breaks the ether bonds in the probe. Therefore, this probe can be used as a visible colorimetric probe for detecting hydrogen sulfide molecules. Figure 1 As shown, the probe has a distinct absorption peak at 340 nm.

[0062] Add 30 μL of probe stock solution and 50 μL of various ion and organic stock solutions to 3 mL of blank DMSO / H2O (v / v = 9:1). Figure 2 The graphs show the fluorescence spectra of the fluorescent probe EDPH after the addition of various ions and organic compounds. The graphs indicate that EDPH itself does not exhibit fluorescence, and the fluorescence intensity did not change significantly after the addition of ion and organic compound stock solutions. However, the addition of hydrogen sulfide to the probe solution resulted in a sudden increase in fluorescence intensity, a phenomenon known as fluorescence enhancement, and the solution changed from colorless to pale yellow. Therefore, the fluorescent probe EDPH exhibits superior selectivity for hydrogen sulfide compared to other ions.

[0063] Figure 3The image shows the fluorescence spectral response of the fluorescent probe of this invention to different concentrations of hydrogen sulfide molecules. As the concentration of hydrogen sulfide gradually increases, the fluorescence intensity of the probe at 500 nm slowly increases, eventually stabilizing at 3.4 × 10⁻⁶. 4 As can be seen from the figure, the fluorescence intensity gradually decreases with increasing equivalent amounts of hydrogen sulfide added.

[0064] To ensure that the probe is not affected by external interference in detecting hydrogen sulfide, a competitive experiment was designed. Ions other than hydrogen sulfide and sulfur-containing organic compounds were added to the probe solution, and after standing, fluorescence testing (black bar graph) was performed. The results are as follows: Figure 4 As shown in the figure, the samples exhibited no fluorescence, and the fluorescence intensity was almost identical to that of the probe itself. Next, a stock solution of hydrogen sulfide was added to the samples, and after standing again, a fluorescence test was performed (red bars). The results showed that the fluorescence of all the samples was enhanced. These experiments demonstrate that the probe has strong anti-interference capabilities and can be used to detect hydrogen sulfide molecules in complex environments.

[0065] Due to the diversity of natural environments and the stability of cell experiments, the pH range for probe testing was investigated. The pH of the blank stock solution was adjusted to 4.0, 3.0…14.0 using HCl and NaOH. Then, the fluorescence intensity of the probe response before and after at different pH values ​​at 500 nm was measured to further confirm the suitable pH range for probe EDPH testing. Results are as follows: Figure 5 As shown in the figure, the optimal detection range for the EDPH probe is pH 7.0-14.0, indicating that the EDPH probe can detect hydrogen sulfide in both neutral and weakly alkaline environments.

[0066] like Figure 6 Supported by favorable results (EDPH exhibits an "on" fluorescence response to H2S within 30 seconds, changing color from colorless to yellow, demonstrating advantages such as a large Stokes shift and fast response time to H2S), fluorescence imaging experiments were performed in HeLa cells. Laser scanning confocal microscopy revealed that neither EDPH nor HeLa cells produced fluorescence. After incubating HeLa cells with the fluorescent probe compound EDPH (10 μM) at 37°C for 30 minutes, a weak bright green fluorescence signal was observed in the cells. More importantly, when cells were incubated with hydrogen sulfide (100 μM) at 37°C for 30 minutes, intracellular fluorescence significantly increased, consistent with the results observed in solution studies. Therefore, live-cell imaging demonstrates that the probe EDPH possesses cell membrane permeability and can be used for live-cell hydrogen sulfide imaging.

[0067] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the scope of the technology disclosed in the present invention shall fall within the scope of protection of the present invention.

Claims

1. A purine-based fluorescent probe EDPH, characterized in that, The structural formula of the purine-based fluorescent probe EDPH is shown below: .

2. The method for preparing the purine-based fluorescent probe EDPH according to claim 1, characterized in that, Includes the following steps: Step 1: 4,6-Dichloro-5-aminopyrimidine and 1-naphthylamine were added to the organic solvent methanol. After the solids dissolved, concentrated hydrochloric acid was added, and the mixture was refluxed and stirred until the reaction was complete. The organic solvent was removed by vacuum distillation, and the mixture was dissolved in NaOH. Then, it was extracted with ethyl acetate, and the ethyl acetate was removed by vacuum distillation. The mixture was recrystallized from methanol and water to obtain 6-chloro-N-(naphth-1-yl)pyrimidine-4,5-diamine, i.e., compound I, with the structural formula […]. ; Step 2: Compound I, naphthoic acid, polyphosphoric acid, and dodecyltrimethylammonium chloride were dissolved in phosphorus oxychloride and stirred under reflux until the reaction was complete. The reaction solution was then completely cooled to room temperature, and the organic solvent was removed by vacuum distillation. An ice-water mixture was added to the reaction system under ice-water bath conditions for purification and elution. The aqueous phase was removed by filtration to obtain 6-chloro-8,9-di(naphth-1-yl)-9H-purine, i.e., compound II, with the structural formula […]. ; Step 3: After dissolving compound II in ethanol and allowing the solid to dissolve, hydrazine hydrate was added, and the mixture was refluxed and stirred until the reaction was complete. The mixture was then cooled to room temperature, and the organic phase was removed by vacuum distillation. The solid was washed with an organic solvent to obtain 6-hydrazine-8,9-di(naphthyl-1-yl)-9H-purine, i.e., compound III, with the structural formula [structural formula would be inserted here]. ; Step 4: 3,5-Dimethyl-4-hydroxybenzaldehyde and K₂CO₃ were dissolved in DMF and stirred at room temperature for 30 minutes. Then, 2,4-dinitrofluorobenzene was added, and the reaction was continued with stirring at room temperature. After the reaction was complete, 200 mL of distilled water was added, and the mixture was extracted with ethyl acetate. The solvent was then removed under reduced pressure, and the mixture was recrystallized from ethyl acetate to obtain yellow crystals, namely compound IV, with the structural formula [structure omitted]. ; Step 5: Dissolve compounds III and IV in an organic solvent and reflux the mixture with stirring. After the reaction is complete, cool to room temperature, remove the solvent by vacuum distillation, and purify the crude product by recrystallization to obtain (E)-1-(4-(2,4-dinitrophenoxy)-3,5-dimethylbenzylmethyl)-2-(8,9-bis(naphthyl-1)-9H-purine-6-yl)hydrazine, i.e., compound EDPH.

3. The method for preparing the purine-based fluorescent probe EDPH according to claim 1 as described in claim 2, characterized in that, The temperature for vacuum distillation in step 1 is 45-55℃.

4. The method for preparing the purine-based fluorescent probe EDPH according to claim 1 as described in claim 2, characterized in that, The organic solvent used to wash the solid in step 3 is chloroform.

5. The method for preparing the purine-based fluorescent probe EDPH according to claim 1 as described in claim 2, characterized in that, The reflux stirring temperature in step 4 is 75°C, and the reflux time is 2 hours.

6. The application of the purine-based fluorescent probe EDPH according to claim 1 in the detection of hydrogen sulfide in solution or cells.

7. The application according to claim 6, characterized in that, The detection limit for hydrogen sulfide molecules is 41 nmol / L.

8. Application of the purine-based fluorescent probe EDPH according to claim 1 in hydrogen sulfide imaging in HeLa cells.

Images