A cell membrane-targeting H2S two-photon fluorescent probe, its preparation method and application

Abstract

This invention discloses a cell membrane-targeted H2S two-photon fluorescent probe, its preparation method, and its applications. This probe not only possesses excellent optical properties such as near-infrared fluorescence emission at 650 nm, a large Stokes shift of 140 nm, and two-photon excitation characteristics, exhibiting advantages such as low excitation-emission light scattering, low tissue absorption, and strong penetration, but also demonstrates excellent analytical and detection performance. Its pyridinium and sulfonic acid groups prevent it from entering the cell interior, allowing it to adhere only to the cell membrane surface, thus endowing the probe with excellent cell membrane localization capabilities. The probe uses a 2,4-dinitrobenzenesulfonyl group as both the H2S recognition group and a fluorescence quencher. Because the 2,4-dinitrobenzenesulfonyl group is adjacent to the pyridinium group, it is located on the outer surface of the cell membrane, enabling two-photon fluorescence imaging of H2S released transmembrane from cells.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and in particular to a cell membrane-targeting H2S two-photon fluorescent probe, its preparation method, and its application. Background Technology

[0002] H₂S is an important gaseous signaling molecule that can cross the plasma membrane and rapidly diffuse within and between cells, then transmit signals to other cells. For example, released H₂S can act on neurons and glial cells, increasing calcium levels... 2+ H2S regulates activity between neural synapses through influx. It also participates in the regulation of many signaling pathways and ion channels, playing a crucial physiological role in vasodilation, angiogenesis, neuromodulation and protection, cell proliferation, and apoptosis. The cell membrane, as the cell boundary, is an essential pathway for H2S diffusion and propagation; therefore, developing a cell membrane-targeted fluorescent probe to monitor H2S transmembrane release will be helpful in studying its related physiological processes in signal transduction.

[0003] Compared to single-photon fluorescence imaging, two-photon fluorescence imaging offers significant advantages. Two-photon imaging utilizes near-infrared wavelength excitation, which exhibits low scattering, minimal tissue absorption, and strong penetration, effectively reducing photobleaching and photodamage while avoiding interference from spontaneous background fluorescence, thereby improving the spatiotemporal resolution and accuracy of the imaging. Currently, cell membrane-targeting H2S fluorescent probes are scarce, and most are single-photon excited, resulting in short emission wavelengths, low Pearson correlation coefficients, and unknown distribution on the cell membrane, making it difficult to effectively monitor H2S released extracellularly.

[0004] Therefore, it is urgent to develop cell membrane-targeting H2S two-photon fluorescent probes with near-infrared emission wavelengths and high Pearson correlation coefficients. Summary of the Invention

[0005] The purpose of this invention is to provide a cell membrane-targeted H2S two-photon fluorescent probe, its preparation method and application. The probe of this invention can be labeled on the outer surface of the cell membrane and has both two-photon excitation characteristics and a long fluorescence emission wavelength, which can be used to directly and accurately monitor H2S released from the cell membrane to the extracellular space.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a cell membrane-targeting H2S two-photon fluorescent probe is provided, the cell membrane-targeting H2S two-photon fluorescent probe having the structure shown in formula (I):

[0008]

[0009] Wherein, R1 is a C12-C18 straight-chain alkane or olefin, and the quaternary ammonium salt is pyridinium or onium salt, including quinoline onium.

[0010] The probe is named C. 12～18 NAP-HS,

[0011] Pyridinium can also be other types of onium salts, such as quinolineium. Figure 8 C containing a quinolinetonium group 4～18 NAQ-HS and HS - Excitation and emission spectra before and after the reaction.

[0012] A second aspect of the present invention provides a method for preparing a cell membrane-targeting H2S two-photon fluorescent probe, the method comprising:

[0013] (1) 4-Bromo-1,8-naphthalenedicarboxylic anhydride and dodecyl to octadecylamine were dissolved in an organic solvent and stirred to react. The mixture was then cooled, filtered, washed, and dried to obtain compound 1 (compound 1 is N-hexadecyl-4-bromo-1,8-naphthalenedicarboxylic anhydride, structural formula see […]). Figure 1 );

[0014] (2) Compound 1 and N-hydroxyphthalimide were dissolved in an organic solvent, and K2CO3 was added and the mixture was stirred to react. After cooling to room temperature, the reaction solution was poured into ice water and acidified to pH 2.8-3.2. The solution was then filtered, washed, and vacuum dried to obtain compound 2 (compound 2 is N-hexadecyl-4-hydroxy-1,8-naphthalenediamide, structural formula see...). Figure 1 );

[0015] (3) Compound 2 and hexamethylenetetramine were dissolved in an organic solvent and stirred to react. Then, the mixture was cooled, filtered, washed, and dried to obtain compound 3 (compound 3 is N-hexadecyl-3-formyl-4-hydroxy-1,8-naphthalenediamine, structural formula see [see below]). Figure 1 );

[0016] (4) 4-Methylpyridine and 1,3-propanesulfonic acid lactone were dissolved in an organic solvent and stirred to react. The mixture was then cooled, filtered, washed, and dried to obtain compound 4 (the compound is 4-methyl-1-(3-sulfopropyl)-pyridinium, structural formula shown in [reference needed]). Figure 1 );

[0017] (5) Figure 1 Compounds 3 and 4 were dissolved in an organic solvent, tetrahydropyrrole was added dropwise, and the mixture was stirred at room temperature. After the reaction was complete, the solvent was removed by rotary evaporation, the mixture was purified, and then dried under vacuum to obtain C. 12～18 NAP;

[0018] (6) C 12～18NAP and 2,4-dinitrobenzenesulfonyl chloride were dissolved in organic solvents to give C 12～18 NAP solution and 2,4-dinitrobenzenesulfonyl chloride solution were applied to the C 12～18 Pyridine and 2,4-dinitrobenzenesulfonyl chloride solution were added sequentially to NAP solution, and then stirred at room temperature and in N2 environment. After the reaction was completed, the solution was filtered, washed and vacuum dried to obtain the cell membrane-targeting H2S two-photon fluorescent probe of claim 1.

[0019] In step (1),

[0020] The organic solvent is anhydrous ethanol. The reaction conditions include stirring at 80±5℃ for 12±2 h.

[0021] The molar ratio of 4-bromo-1,8-naphthoic anhydride to the dodecyl to octadecylamine is 1:1-3.

[0022] In step (2)

[0023] The organic solvent is dimethyl sulfoxide, the acidification solution is concentrated hydrochloric acid, and the reaction conditions include stirring at 100±5℃ for 6±2h.

[0024] The molar ratio of N-dodecyl-4-bromo-1,8-naphthalenediamide, N-hydroxyphthalimide, and K2CO3 is 1:1-2:3-6.

[0025] In step (3)

[0026] The organic solvent is trifluoroacetic acid. The reaction conditions include stirring at 75±5℃ for 12±2 h.

[0027] The molar ratio of N-dodecyl-4-hydroxy-1,8-naphthalenediamine to hexamethylenetetramine is 1:1-3.

[0028] In step (4),

[0029] The organic solvent is acetonitrile; the reaction conditions include stirring at 80±5℃ for 2.5±2h.

[0030] The molar ratio of 4-methylpyridine to 1,3-propanesulfonic acid lactone is 1:1.

[0031] In step (5),

[0032] The organic solvent is anhydrous ethanol. The reaction conditions include: stirring at room temperature for 16 ± 2 h.

[0033] The molar ratio of N-dodecyl-3-formyl-4-hydroxy-1,8-naphthalenediamide to 4-methyl-1-(3-sulfopropyl)-pyridinium is 1:1-2, and the molar ratio of N-dodecyl-3-formyl-4-hydroxy-1,8-naphthalenediamide to tetrahydropyrrole is 1 mmol / 200-800 μL.

[0034] In step (6),

[0035] The organic solvent is dichloromethane. Stirring was performed at room temperature and in an N2 environment for 12 ± 2 h.

[0036] C 12～18 The molar ratio of NAP, 2,4-dinitrobenzenesulfonyl chloride and pyridine is 1:1-3:5-20.

[0037] C 12～18 NAP-HS is characterized by a large Stokes shift of 140 nm and two-photon excitation properties.

[0038] The above-mentioned cell membrane-targeted H2S two-photon near-infrared fluorescent probes are applied in the field of H2S detection.

[0039] A third aspect of the present invention provides the application of the aforementioned cell membrane-targeting H2S two-photon fluorescent probe in the preparation of H2S detection products.

[0040] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0041] 1. The H2S two-photon fluorescent probe provided by this invention uses a structure with a pyridinium and sulfonic acid group linked at the C3 position of a naphthalimide as the two-photon fluorophore, and a 2,4-dinitrobenzenesulfonyl group as the recognition group. This probe exhibits red fluorescence at 650 nm, a large Stokes shift of 140 nm, and two-photon excitation characteristics. The fluorescence intensity of this probe is significantly enhanced after reacting with H2S, and it also shows better selectivity for H2S, enabling analysis and detection in physiological environments.

[0042] 2. Compared with existing H2S fluorescent probes, the cell membrane-targeting H2S fluorescent probe provided by this invention has a Pearson correlation coefficient as high as 0.97, which can better label the outer surface of the cell membrane.

[0043] 3. The cell membrane-targeting H2S fluorescent probe provided by this invention can realize real-time in-situ detection of H2S released into the extracellular environment, and the preparation method is relatively simple and convenient. Attached Figure Description

[0044] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0045] Figure 1 C 16 Synthetic route of NAP-HS;

[0046] Figure 2 C 16 NAP-HS and C 16 The excitation and emission spectra of NAP, where 1 is C 16 The excitation spectrum of NAP-HS, 1' is C 16 The emission spectrum of NAP-HS; 2 is C 16 The excitation spectrum of NAP, 2' is C 16 The emission spectrum of NAP;

[0047] Figure 3 C 16 Two-photon absorption cross section of NAP;

[0048] Figure 4 C 16 NAP-HS and HS at different concentrations - Fluorescence spectrum after reaction;

[0049] Figure 5 The fluorescence intensity at 650 nm and HS - Linear relationship of concentration;

[0050] Figure 6 C 16 Selectivity of NAP-HS;

[0051] Figure 7 C 16 Two-photon fluorescence imaging experiment of NAP-HS, λ ex =900nm. A is using C 16 Fluorescence imaging of cells after incubation with NAP-HS (20 μM) for 20 min; B is the cell after incubation with C. 16 Incubate with NAP-HS (20 μM) for 20 min, then use HS - Fluorescence imaging of cells after incubation at (100 μM) for 30 min; C indicates cells incubated with C20 prior to incubation. 16 Incubate with NAP-HS (20 μM) for 20 min, then use HS - Fluorescence imaging of cells after incubation at (200 μM) for 30 min;

[0052] Figure 8 C 16 NAQ-HS and C 16 The excitation and emission spectra of NAQ, where 1 is C 16 The excitation spectrum of NAQ-HS, 1' is C 16 Emission spectrum of NAQ-HS; 2 is C 16 The excitation spectrum of NAQ, 2' is C 16 The emission spectrum of NAQ; Detailed Implementation

[0053] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0054] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0055] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0056] The following will provide a detailed description of a cell membrane-targeting H2S two-photon fluorescent probe, its preparation method, and its application, in conjunction with embodiments and experimental data.

[0057] Example 1:

[0058] Intermediate product N-hexadecyl-4-bromo-1,8-naphthalenediamine ( Figure 1 Synthesis of compound 1):

[0059] 4-Bromo-1,8-naphthalenedicarboxylic anhydride (5.541 g, 20 mmol) and hexadecylamine (5.312 g, 22 mmol) were dissolved in anhydrous ethanol (150 mL), and the mixture was stirred and refluxed for 12 h. After cooling to room temperature, the reaction mixture was poured into ice water (200 mL), filtered, and the precipitate was washed with cold water and ethanol and dried under vacuum to give N-hexadecyl-4-bromo-1,8-naphthalenedicarboxylic anhydride (8.309 g, 83% yield). 1H NMR (400MHz, CDCl3) δ8.64(d,J=7.3Hz,1H),8.54(d,J=8.4Hz,1H),8.39(d,J=7.8Hz,1H),8.02(d,J=7. 8Hz,1H),7.86-7.79(m,1H),4.20-4.11(m,2H),1.72(p,J=7.5Hz,2H),1.24(s,26H),0.91-0.84(m,3H). 13 C NMR (101MHz, CDCl3) δ163.57,163.55,133.15,131.97,131.16,131.06,130.59,130.14,128.97,128.04,12 3.16,122.29,40.63,31.92,29.69,29.67,29.66,29.64,29.60,29.55,29.36,28.08,27.13,22.69,14.12.

[0060] Example 2:

[0061] Intermediate product N-hexadecyl-4-hydroxy-1,8-naphthalenediamine ( Figure 1 Synthesis of compound 2):

[0062] N-Hexadecyl-4-bromo-1,8-naphthalenedicarboximide (1.351 g, 2.7 mmol) and N-hydroxyphthalimide (489 mg, 3 mmol) were dissolved in dimethyl sulfoxide (20 mL), and then K₂CO₃ (1.244 g, 9 mmol) was added. The mixture was stirred at 100 °C for 6 h. After cooling to room temperature, the reaction solution was poured into ultrapure water (100 mL), and concentrated hydrochloric acid was added dropwise to adjust the pH of the solution to 3. The mixture was filtered, and the precipitate was washed with cold water and dried under vacuum to obtain N-hexadecyl-4-hydroxy-1,8-naphthalenedicarboximide (1.004 g, 85% yield). 1 H NMR (400MHz, DMSO-d6) δ11.84(s,1H),8.52(d,J=7.1Hz,1H),8.45(d,J=7.2Hz,1H),8.34(d,J=8.2Hz,1H),7.74(t,J= 7.8Hz,1H),7.14(d,J=8.2Hz,1H),3.99(t,J=7.5Hz,2H),1.59(p,J=7.9Hz,2H),1.18(s,26H),0.82(t,J=6.8Hz,3H). 13C NMR(101MHz,DMSO-d6)δ164.10,163.43,160.71,133.98,131.55,129.64,129.33,126.02,122.85,122.2 8,113.07,110.39,31.75,29.49,29.46,29.43,29.39,29.32,29.18,29.17,27.99,26.96,22.55,14.38.

[0063] Example 3:

[0064] Intermediate product N-hexadecyl-3-formyl-4-hydroxy-1,8-naphthalenediamine ( Figure 1 Synthesis of compound 3):

[0065] N-hexadecyl-4-hydroxy-1,8-naphthalenediamine (875 mg, 2 mmol) and hexamethylenetetramine (561 mg, 4 mmol) were dissolved in trifluoroacetic acid (20 mL), and the mixture was stirred and refluxed for 12 h. After cooling to room temperature, the reaction solution was poured into ultrapure water (40 mL), filtered, and the precipitate was washed with cold water and dried under vacuum to give N-hexadecyl-3-formyl-4-hydroxy-1,8-naphthalenediamine (698 mg, 75% yield). 1 H NMR (400MHz, CDCl3) δ13.15(s,1H),10.10(s,1H),8.73-8.61(m,3H),7.77(t,J=7.9Hz, 1H), 4.14 (t, J = 7.6Hz, 2H), 1.71 (p, J = 7.3Hz, 2H), 1.24 (s, 26H), 0.87 (t, J = 6.8Hz, 3H). 13 C NMR (101MHz, CDCl3) δ196.40,165.58,163.55,162.89,134.79,134.02,131.67,130.22,126.97,122.79,11 5.12,114.85,40.55,31.92,29.70,29.68,29.65,29.61,29.56,29.38,29.36,28.10,27.13,22.69,14.12.

[0066] Example 4:

[0067] Intermediate product 4-methyl-1-(3-sulfopropyl)-pyridinium ( Figure 1 Synthesis of compound 4):

[0068] 4-Methylpyridine (931 mg, 10 mmol) and 1,3-propanesulfonic acid lactone (1.221 g, 10 mmol) were dissolved in acetonitrile (5 mL), and then stirred and refluxed for 2.5 h. After cooling to room temperature, the precipitate was collected by filtration under reduced pressure and dried under vacuum to give 4-methyl-1-(3-sulfopropyl)pyridinium (1.838 g, yield 85%). 1 H NMR (400MHz, DMSO-d6) δ8.92(s,1H),7.98(d,J=6.4Hz,2H),4.66(t,J=6.9Hz,2H),2.51(p,J=1.9Hz,1H),2.40(t,J=7.2Hz,2H),2.20(p,J=7.1Hz,2H). 13 C NMR (101MHz, DMSO-d6) δ159.24,144.38,128.81,59.24,47.41,27.71,21.85.

[0069] Example 5:

[0070] The intermediate product yields C 16 Synthesis of NAP:

[0071] N-hexadecyl-3-formyl-4-hydroxy-1,8-naphthalenediamide (466 mg, 1 mmol) and 4-methyl-1-(3-sulfopropyl)pyridinium (216 mg, 1 mmol) were dissolved in anhydrous ethanol (40 mL), and then tetrahydropyrrole (400 μL) was added dropwise. The reaction mixture was stirred at room temperature for 16 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the residue was purified by silica gel column chromatography with CH2Cl2 / CH3OH (10:1 to 2:1, v / v) as the eluent. The solvent was removed under reduced pressure and the residue was dried under vacuum to give C 16 NAP (279 mg, yield 42%). 1 H NMR(400MHz,DMSO-d6)δ8.61(d,J=7.1Hz,2H),8.49(d,J=6.5Hz,1H),8.39(s,1H),8 .26(d,J=6.0Hz,1H),8.13(d,J=15.6Hz,1H),7.98(d,J=7.1Hz,2H),7.88(d,J=15.6 Hz,1H),7.42(t,J=7.6Hz,1H),4.50(t,J=7.0Hz,2H),4.00(t,J=7.4Hz,2H),2.47(t ,J=7.2Hz,2H),2.19(p,J=6.9Hz,2H),1.58(s,2H),1.19(s,26H),0.88-0.78(m,3H). 13CNMR(101MHz,DMSO-d6)δ177.66,164.56,163.19,155.95,143.36,143.06 ,138.70,132.46,131.99,130.84,129.78,122.96,121.89,121.53,118.8 9,116.75,101.52,57.88,56.50,47.56,45.38,31.76,29.51,29.48,29.4 5,29.40,29.29,29.18,28.24,27.64,27.08,24.09,22.56,19.01,14.40.

[0072] Example 6:

[0073] Target C 16 Synthesis of NAP-HS:

[0074] C 16 NAP (133 mg, 0.2 mmol) was dissolved in CH₂Cl₂ (10 mL), pyridine (160 μL, 2 mmol) was added, and then CH₂Cl₂ (10 mL) containing 2,4-dinitrobenzenesulfonyl chloride (80 mg, 0.3 mmol) was added dropwise. After the addition was complete, the mixture was stirred for 12 h at room temperature and under N₂ atmosphere. After the reaction was complete, the mixture was filtered, and the precipitate was washed with cold CH₂Cl₂ and dried under vacuum to obtain C. 16 NAP-HS (172 mg, yield 96%). 1 H NMR (400MHz, DMSO-d6) δ9.02(d,J=6.3Hz,2H),8.92(d,J=6.1Hz,2H),8.87(s,1H),8.61(d,J=7. 3Hz,1H),8.46(d,J=9.0Hz,1H),8.40(d,J=8.8Hz,1H),8.26(d,J=9.1Hz,1H),8.10(d,J=6.3Hz, 2H),8.02(t,J=8.4Hz,1H),7.98(d,J=6.0Hz,2H),4.66(t,J=7.1Hz,2H),4.07(t,J=7.1Hz,2H), 2.30(t,J=6.9Hz,2H), 2.20(t,J=6.8Hz,2H), 1.66(s,2H), 1.23(s,26H), 0.84(t,J=6.6Hz,3H).

[0075] Example 7:

[0076] C 16 Spectral properties of NAP-HS:

[0077] C was determined in phosphate-buffered saline (PBS, pH 7.40, containing 1 mM CTAB). 16 NAP-HS and HS - Fluorescence spectra, UV-Vis absorption spectra, and fluorescence quantum yields before and after the reaction were observed. The fluorescence quantum yield was determined using an ethanol solution of Rhodamine B (Φ...). f =0.60) was used as the standard. The measurement results are shown in Table 1.

[0078] Table 1.C 16 NAP-HS and HS - Spectroscopic properties before and after the reaction

[0079]

[0080] Experimental results show that C 16 NAP-HS itself has very weak fluorescence, and it is similar to HS. - The fluorescence intensity was significantly enhanced after the reaction. C 16 NAP-HS and C 16 The excitation and emission spectra of NAP are as follows: Figure 2 As shown, C 16 The two-photon absorption cross section of NAP is as follows: Figure 3 As shown in Figure C. 16 NAP-HS and HS at different concentrations - The fluorescence spectrum after the reaction (0.1-10 μM) is as follows: Figure 4 As shown, the fluorescence intensity at 650 nm is related to HS - The linear relationship of concentration (0.1-5 μM) is as follows: Figure 5 As shown.

[0081] Example 8:

[0082] C 16 NAP-HS selectivity for H2S:

[0083] C was examined separately 16 NAP-HS inhibits H2S and metal ions (Na+). + K + Ca 2+ Zn 2+ Mg 2+ Fe 3+ Cu 2+ Fe 3+ ), anion (Br) - I - SO4 2- SO3 2- HSO3 - CO3 2- HCO3 - NO3- NO2 - SCN - Ac - ), reactive oxygen species (H2O2, ClO) - ONOO - The response of SO3 to biothiols (Cys, Hcy, GSH) was studied, with SO3 being the most prominent. 2- HSO3 - The concentration was 50 μM, reactive oxygen species and biothiols were 200 μM, and other substances were 1 mM. Results are as follows: Figure 6 As shown, C 16 NAP-HS and HS - The fluorescence enhancement after the (10μM) reaction was much greater than that of other substances, indicating that it has better selectivity for H2S.

[0084] Example 9:

[0085] C 16 NAP-HS is used to monitor H2S released across cell membranes.

[0086] Using HeLa cells as the research subject, the labeling conditions of the probe on the cell membrane were investigated in detail, including probe toxicity, concentration, and incubation time. Using C... 16 After incubation with NAP-HS (50 μM) for 24 h, the relative viability of HeLa cells remained as high as 85%, indicating extremely low cytotoxicity. Two-photon fluorescence imaging showed that C 16 NAP-HS (20 μM) anchored to the cell membrane after 20 min of incubation. Increasing the extracellular H2S concentration significantly enhanced the red fluorescence in the cell membrane region. Figure 7 As shown. This result fully illustrates C 16 NAP-HS has cell membrane targeting and H2S detection capabilities, and can be used to monitor H2S released across the cell membrane.

[0087] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0088] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0089] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A cell membrane-targeting H2S two-photon fluorescent probe, characterized in that, The cell membrane-targeting H2S two-photon fluorescent probe has the structure shown in formula (I): （I） R1 is a C12~C18 straight-chain alkane or alkene.

2. A method for preparing the cell membrane-targeting H2S two-photon fluorescent probe according to claim 1, characterized in that, The method includes: 4-Bromo-1,8-naphthalenedicarboxylic anhydride and dodecyl-octadecylamine were dissolved in an organic solvent and stirred to react. The mixture was then cooled, filtered, washed, and dried to obtain compound 1. Compound 1 and N -Hydroxyphthalimide was dissolved in an organic solvent, and K2CO3 was added and the mixture was stirred to react. After cooling to room temperature, the reaction solution was poured into ice water and acidified to pH 2.8-3.

2. The solution was then filtered, washed, and vacuum dried to obtain compound 2. Compound 2 and hexamethylenetetramine were dissolved in an organic solvent and stirred to react. The mixture was then cooled, filtered, washed, and dried to obtain compound 3. 4-Methylpyridine and 1,3-propanesulfonic acid lactone were dissolved in an organic solvent and stirred to react. The mixture was then cooled, filtered, washed and dried to obtain compound 4. Compounds 3 and 4 were dissolved in an organic solvent, tetrahydropyrrole was added dropwise, and the mixture was stirred at room temperature. After the reaction was complete, the solvent was removed by rotary evaporation, the mixture was purified, and then dried under vacuum to obtain C. 12~18 NAP; C 12~18 NAP and 2,4-dinitrobenzenesulfonyl chloride were dissolved in organic solvents to give C 12~18 NAP solution and 2,4-dinitrobenzenesulfonyl chloride solution were applied to the C 12~18 Pyridine and 2,4-dinitrobenzenesulfonyl chloride solution were added sequentially to NAP solution, and then stirred at room temperature and in N2 environment. After the reaction was completed, the solution was filtered, washed and vacuum dried to obtain the cell membrane-targeting H2S two-photon fluorescent probe of claim 1.

3. The method according to claim 2, characterized in that: The molar ratio of the 4-bromo-1,8-naphthalenedicarboxylic anhydride to the dodecyl to octadecylamine is 1:1-3.

4. The method according to claim 2, characterized in that: The N -dodecyl-4-bromo-1,8-naphthodicarboximide, N The molar ratio of α-hydroxyphthalimide to K2CO3 is 1:1-2:3-6.

5. The method according to claim 2, characterized in that: N The molar ratio of -dodecyl-4-hydroxy-1,8-naphthalenediamide to hexamethylenetetramine is 1:1-3.

6. The method according to claim 2, characterized in that: The molar ratio of 4-methylpyridine to 1,3-propanesulfonic acid lactone is 1:

1.

7. The method according to claim 2, characterized in that: The N The molar ratio of -dodecyl-3-formyl-4-hydroxy-1,8-naphthalenediformimide and 4-methyl-1-(3-sulfopropyl)-pyridinium is 1:1-2. N The ratio of -dodecyl-3-formyl-4-hydroxy-1,8-naphthalenediformimide to tetrahydropyrrole is 1 mmol / 200-800 μL.

8. The method according to claim 2, characterized in that: C 12~18 The molar ratio of NAP, 2,4-dinitrobenzenesulfonyl chloride and pyridine is 1:1-3:5-20.

9. The application of the cell membrane-targeting H2S two-photon fluorescent probe of claim 1 in the preparation of H2S detection products, wherein the application is not for disease diagnosis or treatment purposes.

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