Lipid droplet hydrogen sulfide fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a lipid droplet hydrogen sulfide fluorescent probe as well as a preparation method and application thereof. The lipid droplet hydrogen sulfide fluorescent probe provided by the invention has the advantages of simplicity and convenience in operation, quick reaction, good selectivity, high sensitivity and the like in hydrogen sulfide detection. Specifically, the lipid droplet hydrogen sulfide fluorescent probe provided by the invention has a compound with a structure as shown in formula (I), or pharmaceutically acceptable salt thereof: # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of probes, and in particular to a lipid droplet hydrogen sulfide fluorescent probe and a preparation method and application thereof. Background Art

[0002] Lipid droplets are highly dynamic organelles that maintain cellular lipid and energy homeostasis and play a key role in regulating lipid and energy metabolism. Dysfunction of lipid droplets is often associated with many pathological conditions, such as metabolic diseases such as obesity, diabetes, and atherosclerosis. Hydrogen sulfide (H2S), as an important biosignaling molecule, has received extensive attention in the field of biology and related interdisciplinary research. Studies have shown that hydrogen sulfide plays a pivotal role in regulating the number and size of lipid droplets. Abnormal endogenous H2S concentration is often closely related to the occurrence of various diseases, such as cancer, Alzheimer's disease (AD), pulmonary hypertension, and diabetes. In addition, various types of cancer and hypoxic conditions often lead to dysregulation of H2S-related enzymes, which further leads to increased endogenous H2S levels and aggravates the development of cancer. Although there have been many reports on the detection of hydrogen sulfide in other organelles such as mitochondria and lysosomes, due to the highly dynamic characteristics of lipid droplets themselves, there are very limited reports on the detection of hydrogen sulfide in lipid droplets. In addition, the existing fluorescent probes have a slow detection speed, a detection limit as high as μM level, and a low sensitivity, which greatly limits their biological applications.

[0003] In summary, it is imperative to construct a fluorescent probe with a high upper detection limit for detecting lipid droplet hydrogen sulfide. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a lipid droplet hydrogen sulfide fluorescent probe and its preparation method and application, which has the advantages of high sensitivity and can detect lipid droplet hydrogen sulfide with naked eyes.

[0005] According to a lipid droplet hydrogen sulfide fluorescent probe of the first embodiment of the present invention, the lipid droplet hydrogen sulfide fluorescent probe is

[0006] A compound having a structure as shown in formula (I), or a pharmaceutically acceptable salt thereof:

[0007]

[0008] Wherein, the R1 is selected from any one of an alkyl group, a cycloalkyl group, an alkoxy group, and an aromatic substituent.

[0009] The R2 is selected from one of the following structures:

[0010]

[0011] Wherein, R3 and R4 are independently selected from C2-C20 saturated straight chain or branched alkyl groups, and R3 and R4 are the same or different.

[0012] The first embodiment of the present invention has at least the following beneficial effects:

[0013] The fluorescent probe provided by the present invention has the advantages of simple operation, rapid reaction, good selectivity and high sensitivity in hydrogen sulfide detection, and can also realize hydrogen sulfide imaging in lipid droplets. It has practical application value in the field of hydrogen sulfide detection research and can be used for biological systems and environmental monitoring, etc.

[0014] According to some embodiments of the present invention, R3 and R4 are selected from tert-butyl.

[0015] According to some embodiments of the present invention, in the R1, the alkyl group is selected from: -(CH2) n CH3, n is 0 to 18.

[0016] According to some embodiments of the present invention, in said R1, said alkoxy group is selected from: -O(CH2) n CH3, n is 0 to 18; or -(OCH2CH2) n CH3, n is 2 to 18.

[0017] According to some embodiments of the present invention, in R1, the aromatic substituent is selected from:

[0018]

[0019] n is 0 to 21.

[0020] According to a second aspect of the present invention, a method for preparing a lipid droplet hydrogen sulfide fluorescent probe comprises the following steps:

[0021] S1. Compound 2, compound 3 and a catalyst are mixed, heated for reaction and then impurities are removed to obtain compound 4, wherein the structural formula of compound 2 is shown in formula (II), and the structural formula of compound 3 is shown in formula (III):

[0022]

[0023] S2. Compound 4, 2,4-dinitrofluorobenzene and an acid-binding agent are mixed to react and remove impurities.

[0024] According to some embodiments of the present invention, in step S1, the catalyst includes piperidine.

[0025] According to some embodiments of the present invention, in step S2, the inorganic base includes at least one of potassium carbonate and cesium carbonate.

[0026] According to some embodiments of the present invention, the molar ratio of the compound 2 to the compound 3 is 1:1.

[0027] According to some embodiments of the present invention, in step S1, the mixing process further includes adding a solvent, and the solvent includes at least one of ethanol, methanol, dichloroethane and dichloromethane.

[0028] According to some embodiments of the present invention, in step S1, the impurity removal step includes: extracting, drying, evaporating, concentrating and drying to obtain a mixture, and separating by column.

[0029] According to some embodiments of the present invention, in step S2, the reaction step further comprises adding a solvent, and the solvent comprises N,N-dimethylformamide.

[0030] According to some embodiments of the present invention, the molar ratio of the compound 4, the 2,4-dinitrofluorobenzene and the inorganic base is 1:1-3:1-4.

[0031] According to some embodiments of the present invention, in step S2, the reaction temperature is 0-40°C.

[0032] According to some embodiments of the present invention, the impurity removal step includes: extraction, drying, evaporation, concentration and drying to obtain a mixture, and column separation.

[0033] According to the third aspect of the present invention, a lipid droplet hydrogen sulfide fluorescent probe is used in the preparation of an intracellular hydrogen sulfide fluorescent detection reagent.

[0034] According to the fourth aspect of the present invention, a lipid droplet hydrogen sulfide fluorescent probe is used in the preparation of an intracellular hydrogen sulfide fluorescent imaging agent.

[0035] According to some embodiments of the invention, the cells comprise HeLa cells.

[0036] According to some embodiments of the present invention, the applying step includes: mixing the lipid droplet hydrogen sulfide fluorescent probe with hydrogen sulfide in a solvent and then performing ultraviolet or fluorescence spectrum detection.

[0037] According to some embodiments of the present invention, the time for the mixed reaction of the lipid droplet hydrogen sulfide fluorescent probe and hydrogen sulfide is 20 to 45 minutes.

[0038] According to some embodiments of the present invention, the concentration of the lipid droplet hydrogen sulfide fluorescent probe is 10 -7 ~10 -2 mol / L.

[0039] According to some embodiments of the present invention, the upper detection limit of hydrogen sulfide is 51 nmol / L.

[0040] According to some embodiments of the present invention, the upper detection limit of hydrogen sulfide is 44 nmol / L.

[0041] According to some embodiments of the invention, the solvent comprises at least one of DMSO and H2O.

[0042] Under the above mixed solvent conditions, the probe has both hydrophilicity and lipophilicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0044] Figure 1 This is a high-resolution mass spectrum of the fluorescent probe LD-H2S of lipid droplet hydrogen sulfide prepared in Example 1;

[0045] Figure 2 is the structural formula of the fluorescent probe LD-H2S of lipid droplet hydrogen sulfide prepared in Example 1;

[0046] Figure 3 The synthetic route of the fluorescent probe LD-H2S of lipid droplet hydrogen sulfide prepared in Example 1;

[0047] Figure 4 This is a high-resolution mass spectrum of the lipid droplet hydrogen sulfide fluorescent probe LD-H2S-2 prepared in Example 2;

[0048] Figure 5 is the structural formula of the lipid droplet hydrogen sulfide fluorescent probe LD-H2S-2 prepared in Example 2;

[0049] Figure 6 The synthetic route of the fluorescent probe LD-H2S-2 of lipid droplet hydrogen sulfide prepared in Example 2;

[0050] Figure 7 It is the ultraviolet absorption spectrum of the solution before and after the reaction of LD-H2S and hydrogen sulfide;

[0051] Figure 8 The fluorescence spectra of the solution before and after the reaction of LD-H2S with hydrogen sulfide;

[0052] Fig. 9 This is the linear relationship between the fluorescence intensity and concentration of LD-H2S reacting with different concentrations of hydrogen sulfide.

[0053] Fig.10 This is the co-localization picture of LD-H2S in various cell organelles;

[0054] Fig.11 It is the ultraviolet spectrum of test example 5;

[0055] Fig.12 It is the ultraviolet spectrum of test example 6;

[0056] Fig.13 This is a graph showing the relationship between the HS- concentration and the probe fluorescence intensity in Test Example 7. DETAILED DESCRIPTION

[0057] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0058] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0059] In the present application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple, respectively.

[0060] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0061] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0062] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0063] Example 1

[0064] This embodiment discloses a fluorescent probe LD-H2S for lipid droplet hydrogen sulfide, and the specific steps are as follows:

[0065] S1. Compound 3 (1 mmol, 269 mg) and 4'-dimethylaminoacetophenone (1 mmol, 163 mg) were dissolved in ethanol, a catalytic amount of piperidine was added, and the mixture was heated under reflux for 24 hours to stop the reaction. The mixture was extracted with CH2Cl2 and H2O, dried over anhydrous Mg2SO4, evaporated and concentrated to obtain a mixture, and separated by column to obtain compound 4 with a yield of 63%. 1 H NMR (500MHz, Chloroform-d) δ13.03 (s, 1H), 10.50 (s, 2H), 8.03 (d, J=8.1Hz, 2H), 7.94 (d, J =8.0Hz, 2H), 7.71 (s, 2H), 7.57-7.52 (m, 2H), 7.47-7.42 (m, 2H), 2.41 (s, 6H), 1.56 (s, 3H).

[0066] S2. Compound 4 (1.0 mmol, 410 mg), 2,4-dinitrofluorobenzene (1.2 mmol, 224 mg) and potassium carbonate (1.5 mmol, 206 mg) were dissolved in DMF, and the reaction was stopped after stirring at room temperature for 8 h. The mixture was extracted with CH2C3l2 and H2O, dried over anhydrous Mg2SO4, evaporated and concentrated to obtain a mixture, and separated by column to obtain compound LD-H2S with a yield of 68%. 1 H NMR (500 MHz, Chloroform-d) δ 8.88 (s, 1H), 8.21 (s, 1H), 8.12 (d, J = 12.2 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.94 (d, J = 8.7 Hz, 2H), 7.83 (s, 1H), 7.80 (s, 1H), 7.73 (d, J = 10.3 Hz, 2H), 7.48 (t, J = 7.6 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 6.71 (d, J = 9.3 Hz, 1H), 6.67 (d, J = 8.6 Hz, 2H), 3.09 (s, 6H), 2.57 (s, 3H); its high resolution mass spectrum is as follows Figure 1 As shown, the structural formula of LD-H2S is as follows Figure 2 The synthetic route is as shown in Figure 3 As shown;

[0067] Among them, compound 1 was prepared according to the method disclosed in the literature Yang Q, Wang S, Li D, et al.Analytica Chimica Acta, 2020, 1103:202-211.

[0068] Example 2

[0069] This embodiment discloses a fluorescent probe LD-H2S-2 for lipid droplet hydrogen sulfide, and the specific steps are as follows:

[0070] S1. 1-(4-(Diphenylamino)acetophenone (42.6 mg, 0.1487 mmol) and compound 3 (40 mg, 0.1487 mmol) were dissolved in EtOH, protected by nitrogen, heated to reflux at 100°C, and the reaction was monitored by TLC. After removing the solvent, the mixture was extracted with ethyl acetate and saturated NaCl, dried with Mg2SO4, concentrated and dried, and separated by column chromatography to obtain 20 mg of a crude product of compound 6.

[0071] S2. Dissolve 2,4-dinitrofluorobenzene (17 mg, 0.0928 mmol), compound 6 (20 mg, 0.0371 mmol) and Cs2CO3 (48 mg, 0.149 mmol) in DMF, react at room temperature under nitrogen protection, and monitor the reaction by TLC. After the reaction is complete, add water to precipitate, filter and dry to obtain the probe LD-H2S-2 (11 mg, 42%). 1 H NMR (500MHz, Chloroform-d) δ8.84 (d, J=2.7Hz, 1H), 8.23–8.21 (m, 1H), 8.12 (dd, J=9 .3, 2.7Hz, 1H), 7.99–7.96 (m, 1H), 7.86–7.81 (m, 3H), 7.75 (d, J=4.5Hz, 2H), 7.73–7.7 1 (m, 1H), 7.48 (ddd, J = 8.3, 7.2, 1.2 Hz, 1H), 7.38 (ddd, J = 8.2, 7.1, 1.2 Hz, 1H), 7.36-7.31 (m, 4H), 7.19-7.14 (m, 6H), 6.99-6.95 (m, 2H), 6.70 (d, J = 9.2 Hz, 1H), 2.57 (s, 3H). Its high-resolution mass spectrum is as follows Figure 4 As shown, HRMS: m / z calcd for [C 41 H 28 N4O6S+H] +705.1802, found:705.1806, the structural formula of LD-H2S-2 is as follows Figure 5 The synthetic route is as shown in Figure 6 shown.

[0072] The preparation method of compound 3 is:

[0073] A1. 2-Hydroxy-5-methylbenzaldehyde (2000 mg, 14.7 mmol) and 2-aminothiophenol (1838 mg, 14.7 mmol) were dissolved in dry ethanol (50 mL); then, HCl (7%, 4 mL) and H2O2 (30%, 10 mL) were added and stirred at room temperature for 1 h; then, the solution was filtered, and the filter cake was washed with 50% ethanol and dried to obtain compound A (2280 mg, 64%), which was directly used in the next step; 1 H NMR (500MHz, Chloroform-d) δ12.32 (s, 1H), 7.98 (d, J=8.9Hz, 1H), 7.91 (d, J=8.8Hz, 1H), 7.51 (d, J=8. 4Hz, 1H), 7.48 (s, 1H), 7.41 (t, J=7.6Hz, 1H), 7.20 (d, J=6.4Hz, 1H), 7.01 (d, J=8.4Hz, 1H), 2.36 (s, 3H);

[0074] A1. Hexamethylenetetramine (1000 mg, 6.21 mmol) and compound A (500 mg, 6.21 mmol) were dissolved in CF3COOH and refluxed at 80°C for 6 h. The reaction was monitored by TLC. After removing the solvent, the crude product was extracted 3 times with CH2Cl2 and H2O, dried over anhydrous Mg2SO4, concentrated and dried, and the mixture was separated by a chromatographic column (PE / DCM, 1 / 1, v / v) to obtain compound 3 (491 mg, 88%). 1 H NMR (500MHz, Chloroform-d) δ13.03 (s, 1H), 10.49 (s, 1H), 8.03 (d, J=8.1Hz, 1H), 7.94 (d, J=9. 1Hz, 1H), 7.90 (s, 1H), 7.71 (s, 1H), 7.54 (t, J=7.1Hz, 1H), 7.45 (t, J=7.6Hz, 1H), 2.41 (s, 3H).

[0075] Test Example 1

[0076] Dissolve LD-H2S in THF solution to obtain probe mother solution (10 -3 mol / L), after preparing the test system of DMSO:H2O=7:3, the probe was added to obtain 10 -5mol / L LD-H2S solution, add HS - Solution (54×10 -5 mol / L), after about 30 minutes of reaction, the UV spectrum changes from Figure 7 It can be seen that a new absorption peak is generated at 500 nm, and the color of the solution changes from light yellow to orange.

[0077] Test Example 2

[0078] Dissolve LD-H2S in THF solution to obtain probe mother solution (10 -3 mol / L), after preparing the test system of DMSO:H2O=7:3, the probe was added to obtain 10 -5 mol / L LD-H2S solution, add HS - Solution (54×10 -5 mol / L), and after about 30 minutes of reaction, the change of fluorescence intensity at 655nm was tested. Figure 8 It can be seen that the fluorescence increased by about 90% after 30 minutes of reaction.

[0079] Test Example 3

[0080] Dissolve LD-H2S in THF solution to obtain probe mother solution (10 -3 mol / L), after preparing the test system of DMSO:H2O=7:3, the probe was added to obtain 10 -5 mol / L LD-H2S solution, adding different concentrations of HS - solution, and tested the change of its fluorescence intensity. Fig. 9 It can be seen that the emission peak intensity at 655nm is similar to HS - There is a good linear relationship between the concentrations, based on which the detection limit of the probe can be calculated to be 51 nM.

[0081] Test Example 4

[0082] 6×10 3 Hela cells with a concentration of 100 μg / mL were inoculated in a laser confocal dish. After culturing for 12 hours, the probe LD-H2S was co-incubated with a commercial organelle probe for 15 minutes. Then the cells were washed three times with PBS and confocal imaging was performed using a laser confocal microscope. The commercial probe for lipid droplets was BODIPY 493 / 503, the commercial probe for mitochondria was Mito-Tracker Green, the commercial probe for endoplasmic reticulum was ER-Tracker Green, the commercial probe for lysosomes was Lyso-Tracker Green, and the commercial probe for Golgi apparatus was Golgi-Tracker Green. Fig.10These are the confocal imaging data of commercial probes of the probe and other organelles, among which the colocalization coefficient of the probe LD-H2S for lipid droplets reaches 0.96, indicating that the probe has excellent targeted imaging capability for lipid droplet organelles.

[0083] Test Example 5

[0084] Dissolve LD-H2S-2 in THF solution to obtain probe mother solution (10 -3 mol / L), after preparing the test system of DMSO:H2O=9:1, the probe was added to obtain 10 -5 mol / L LD-H2S-2 solution, add HS - Solution (65×10 -5 mol / L), after about 30 minutes of reaction, the UV spectrum changes from Fig.11 It can be seen that a new absorption peak is generated at 500 nm, and the color of the solution changes from light yellow to orange.

[0085] Test Example 6

[0086] Dissolve LD-H2S-2 in THF solution to obtain probe mother solution (10 -3 mol / L), after preparing the test system of DMSO:H2O=9:1, the probe was added to obtain 10 -5 mol / L LD-H2S-2 solution, add HS - Solution (65×10 -5 mol / L), and after about 30 minutes of reaction, the change of fluorescence intensity at 650nm was tested. Fig.12 It can be seen that the fluorescence increased by about 90% after 30 minutes of reaction.

[0087] Test Example 7

[0088] Dissolve LD-H2S-2 in THF solution to obtain probe mother solution (10 -3 mol / L), after preparing the test system of DMSO:H2O=9:1, the probe was added to obtain 10 -5 mol / L LD-H2S-2 solution, adding different concentrations of HS - solution, and tested the change of its fluorescence intensity. Fig.13 It can be seen that the emission peak intensity at 650nm is similar to HS - There is a good linear relationship between the concentrations, based on which the detection limit of the probe can be calculated to be 44 nM.

[0089] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A lipid droplet hydrogen sulfide fluorescent probe, characterized in that: The lipid droplet hydrogen sulfide fluorescent probe is a compound having a structure as shown in formula (I), or a pharmaceutically acceptable salt thereof: Wherein, the R1 is selected from any one of an alkyl group, a cycloalkyl group, an alkoxy group, and an aromatic substituent; The R2 is selected from one of the following structures: Wherein, R3 and R4 are independently selected from C2-C20 saturated straight chain or branched alkyl groups, and R3 and R4 are the same or different.

2. The lipid droplet hydrogen sulfide fluorescent probe according to claim 1, characterized in that: In the R1, the alkyl group is selected from: -(CH2) n CH3, n is 0 to 18.

3. The lipid droplet hydrogen sulfide fluorescent probe according to claim 1, characterized in that: In the R1, the alkoxy group is selected from: -O(CH2) n CH3, n is 0 to 18 or -(OCH2CH2) n CH3, n is 2 to 18.

4. The lipid droplet hydrogen sulfide fluorescent probe according to claim 1, characterized in that: In the R1, the aromatic substituent is selected from: n is 0 to 21.

5. A method for preparing a lipid droplet hydrogen sulfide fluorescent probe according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: S1. Compound 2, compound 3 and a catalyst are mixed, heated for reaction and then impurities are removed to obtain compound 4, wherein the structural formula of compound 2 is shown in formula (II), and the structural formula of compound 3 is shown in formula (III): S2. Compound 4, 2,4-dinitrofluorobenzene and an acid-binding agent are mixed and reacted to remove impurities to obtain the product.

6. The preparation method according to claim 5, characterized in that: In step S1, the catalyst includes at least one of piperidine, sodium hydroxide, potassium hydroxide and calcium hydroxide.

7. The preparation method according to claim 5, characterized in that: In step S2, the acid binding agent includes at least one of potassium carbonate and cesium carbonate.

8. Use of the lipid droplet hydrogen sulfide fluorescent probe according to any one of claims 1 to 4 in the preparation of an intracellular hydrogen sulfide fluorescence detection reagent.

9. The use according to claim 8, characterized in that: The upper detection limit of the hydrogen sulfide is 100 mol / L.

10. Use of the lipid droplet hydrogen sulfide fluorescent probe according to any one of claims 1 to 4 in the preparation of an intracellular hydrogen sulfide fluorescent imaging agent.

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