Cysteine-activated fluorescent probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, and discloses a cysteine activated fluorescent probe as well as a preparation method and application thereof. The cysteine-activated fluorescent probe compound provided by the invention can be selectively combined with a tumor signal factor Cys, and after combination, the fluorescence intensity at the wavelength of 675nm can be enhanced by 15 times under the excitation of the wavelength of 580nm, so that a targeting effect is realized, and the cysteine-activated fluorescent probe compound has good selectivity on Cys. Traditional medicine treatment easily causes drug resistance of cancer cells, and the treatment effect is reduced. The mechanism of PDT depends on generation of active oxygen and directly induces apoptosis or necrosis of cells, and the cells are difficult to form effective resistance through gene mutation. The fluorescent probe compound provided by the invention can provide a new tool for precise treatment and improvement of treatment effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a cysteine-activated fluorescent probe and a preparation method and application thereof. Background Art

[0002] Photodynamic therapy (PDT) is a non-invasive and highly selective cancer treatment method that generates singlet oxygen ( 3 O2) or other reactive oxygen species (ROS), thereby inducing apoptosis or necrosis of tumor cells. However, the use of traditional photosensitizers still faces some challenges, including nonspecific distribution, phototoxicity, and potential damage to normal tissues. Therefore, the development of photosensitizers with targeting and bioresponsiveness has become an important research direction in the field of photodynamic therapy.

[0003] Cysteine ​​(Cys) is a key biological small molecule that plays an important role in a variety of physiological and pathological processes, such as anti-oxidative stress, metabolic regulation, and signal transduction. In some diseases (such as tumors, inflammation, and neurodegenerative diseases), the expression level of Cys is significantly abnormal. Among the various methods for detecting Cys, fluorescent probe-based detection has the inherent advantages of high sensitivity, non-invasiveness, and the ability to detect in situ, making it one of the most convenient and effective methods. It has long been considered one of the most powerful tools for detecting Cys in living systems.

[0004] At present, fluorescent probes for Cys detection can be divided into two categories: reactive fluorescent probes and coordination complex fluorescent probes, which are mainly identified through Michael addition, aldehyde cyclization, disulfide bond cleavage or other recognition mechanisms. However, most of these probes have an upper limit of detection and cannot be used in a neutral environment. More importantly, they can only be used for detection but not for tumor treatment. Summary of the invention

[0005] In order to solve the current technical problems of fluorescent probes binding to Cys, more specifically, to solve the problem of targeting Cys and activating and eliminating tumor cells, the present invention provides a cysteine-activated fluorescent probe and a preparation method and application thereof.

[0006] The specific technical scheme of the present invention is: In a first aspect, the present invention provides a cysteine-activated fluorescent probe, the structure of which is shown in formula (1):

[0007] In a second aspect, the present invention provides a method for preparing a cysteine-activated fluorescent probe, characterized in that it comprises the following steps: Step S1: dissolving compound (2) and compound (3) in a solvent and subjecting the mixture to reflux reaction to obtain compound (4); Step S2: dissolving compound (4) and acryloyl chloride in a solvent and reacting to obtain compound (1); in: The structural formula of compound (2) is: The structural formula of compound (3) is: The structural formula of compound (4) is: The structural formula of compound (1) is:

[0008] As a preferred embodiment of the above preparation method, in step S1, the solvent is ethanol.

[0009] As a preferred embodiment of the above preparation method, in step S1, the catalyst for the reflux reaction is piperidine.

[0010] As a preferred embodiment of the above preparation method, in step S1, the temperature of the reflux reaction is 75°C to 80°C.

[0011] As a preferred embodiment of the above preparation method, in step S2, the solvent is dichloromethane.

[0012] As a preferred embodiment of the above preparation method, in step S2, the catalyst for the reaction is triethylamine.

[0013] As a preferred embodiment of the above preparation method, in step S2, the reaction temperature is -5°C to 30°C.

[0014] In a third aspect, the present invention provides the use of the above fluorescent probe in imaging of cancer biomarkers.

[0015] In a fourth aspect, the present invention provides the use of the above fluorescent probe in the preparation of products based on photodynamic therapy for cancer.

[0016] Compared with the prior art, the present invention has the following technical effects: 1. The cysteine-activated fluorescent probe compound (1) provided by the present invention can selectively bind to the tumor signal factor Cys. After the binding, the fluorescence intensity at a wavelength of 675 nm can be enhanced by up to 15 times under excitation at a wavelength of 580 nm, thereby achieving a targeting effect. At the same time, the compound (1) has good selectivity for Cys.

[0017] 2. At the same time, the cysteine-activated fluorescent probe compound (1) provided by the present invention can increase the singlet oxygen yield by up to 3 times after reacting with Cys. The compound (1) has a good PDT effect. The probe compound (1) can be successfully applied to the detection of endogenous and exogenous Cys in HeLa cells, and successfully used photodynamic therapy to kill HeLa cells.

[0018] 3. Traditional drug treatments can easily induce drug resistance in cancer cells, reducing the therapeutic effect. The mechanism of PDT relies on the production of reactive oxygen species, which directly induces cell apoptosis or necrosis. It is difficult for cells to form effective resistance through gene mutation. Fluorescent probe compounds (1) can provide new tools for precise treatment and improving therapeutic efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The absorption and emission spectra of compound (1) before and after being activated by Cys, wherein -Cys indicates that it is not activated by Cys, and +Cys indicates that it is activated by Cys, (a) is the absorption spectrum, and (b) is the emission spectrum; Figure 2 The results of the study on the specificity of compound (1) to Cys; Figure 3 To study the photodynamic properties of compound (1) after being activated by Cys; Figure 4 To study the photodynamic properties of compound (1) that is not activated by Cys; Figure 5 The confocal microscopy comparison diagram of compound (1) detecting endogenous and exogenous Cys in Hela cells, wherein the upper diagram is a confocal imaging diagram, and the lower diagram is a quantitative result diagram of the fluorescence intensity in the confocal imaging diagram; Figure 6 The figure is a comparison of the photodynamic effects of compound (1) on Cys-overexpressing A549 cells before and after illumination; Figure 7 The results of the Annexin V-PI double staining method to test the photodynamic effect of compound (1) on Cys-overexpressing A549 cells before and after illumination; Figure 8 The diagram is a mechanism diagram of the fluorescence intensity enhancement of compound (1). DETAILED DESCRIPTION

[0020] In a first aspect, the present invention provides a cysteine-activated fluorescent probe, the structure of which is shown in formula (1):

[0021] The above-mentioned cysteine-activated fluorescent probe compound (1) can selectively bind to the tumor signal factor Cys to achieve a targeted effect. Compound (1) has good selectivity for Cys. Experimental verification shows that after compound (1) is activated by Cys, the fluorescence intensity at a wavelength of 675nm under excitation at a wavelength of 580nm can be enhanced by up to 15 times. Figure 8 The diagram is a mechanism diagram of the fluorescence intensity enhancement of compound (1).

[0022] In one embodiment, the present invention synthesized compound (a) and compound (b) by performing a series of screening on the fluorescent structural changes of fluorescent probe compound (1).

[0023] The structural formula of compound (a) is as follows:

[0024] The structural formula of compound (b) is as follows:

[0025] Furthermore, in one embodiment, the present invention compares the fluorescence properties of the activated fluorescent probe compound (1) with those of the compound (a) and the compound (b), and finds that the emission wavelength of the compound (1) after the reaction with cysteine ​​is 675 nm, while the emission wavelength of the compound (a) is only 590 nm and the emission wavelength of the compound (b) is 650 nm. The longer the emission wavelength, the better the penetration effect on the biological tissue, which is beneficial to the fluorescence imaging of the organism. It can be seen that the fluorescence properties of the compound after the fluorescent probe compound (1) of the present invention reacts with cysteine ​​are more superior. It is proved that the fluorescent probe compound (1) of the present invention has a more superior effect in fluorescence imaging effect and PDT performance, and this effect is unexpected.

[0026] In a second aspect, the present invention provides a method for preparing a cysteine-activated fluorescent probe, characterized in that it comprises the following steps: Step S1: dissolving compound (2) and compound (3) in a solvent and subjecting the mixture to reflux reaction to obtain compound (4); Step S2: dissolving compound (4) and acryloyl chloride in a solvent and reacting to obtain compound (1); in: The structural formula of compound (2) is: The structural formula of compound (3) is: The structural formula of compound (4) is: The structural formula of compound (1) is:

[0027] Among them, compound (2) and compound (3) are public compounds and can be obtained through existing technologies.

[0028] In one embodiment, in step S1, the solvent is ethanol.

[0029] In one embodiment, in step S1, the catalyst for the reflux reaction is piperidine.

[0030] In one embodiment, in step S1, the molar ratio of compound (2), compound (3) and piperidine is 1:1.5:0.5.

[0031] In one embodiment, in step S1, the temperature of the reflux reaction is 75°C to 80°C.

[0032] In one embodiment, in step S2, the solvent is dichloromethane.

[0033] In one embodiment, in step S2, the catalyst for the reaction is triethylamine.

[0034] In one embodiment, in step S2, the molar ratio of compound (4), triethylamine and acryloyl chloride is 1:3:2.

[0035] In one embodiment, in step S2, the reaction temperature is -5°C to 30°C.

[0036] In another embodiment, in step S2, the reaction is first carried out at a low temperature of -5°C to 5°C to avoid too intense reaction at the beginning of the reaction stage, and then the reaction is carried out at 10 to 30°C to promote the reaction and increase the reaction rate.

[0037] In a third aspect, the present invention provides the use of the above fluorescent probe in imaging of cancer biomarkers.

[0038] In a fourth aspect, the present invention provides the use of the above fluorescent probe in the preparation of products based on photodynamic therapy for cancer.

[0039] The present invention is further described below in conjunction with the embodiments and the accompanying drawings. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention.

[0040] Example 1 Preparation of Compound (4) Compound (3) (0.05 g, 0.168 mmol) and compound (2) (4-n-butyl-1,8-naphthalimide, 0.058 g, 0.244 mmol) were dissolved in ethanol, and then 2 drops of piperidine (2 μL) were added and mixed evenly to obtain a mixed solution. The mixed solution was stirred and refluxed at 78°C for 3 hours. Then, the solvent was removed by flash evaporation, and column chromatography was performed to obtain 0.053 g of compound (4) with a yield of 68%.

[0041] The obtained material was subjected to NMR analysis, and the results were as follows: The H NMR spectrum data are as follows: 1 H NMR (400MHz, Chloroform-d): δ8.63(d,J=7.9Hz,1H),8.52(d,J=3.4Hz,1H),8.36(s,1H),8. 21(d,J=8.3Hz,1H),7.51(dd,J=8.2,1.8Hz,1H),7.38(d,J=1.8Hz,1H),7.10(d,J=10.3Hz,1H ),6.91(d,J=8.2Hz,1H),6.42(d,J=8.1Hz,1H),5.83(d,J=10.3Hz,1H),3.31(ddt,J=45.6,14 .8,7.4Hz,2H),1.80(s,6H),1.52(s,2H),1.13(t,J=7.1Hz,4H),0.99(dt,J=7.1,3.5Hz,6H). The NMR carbon spectrum data are as follows: 13 C NMR (151MHz, Chloroform-d): δ196.42,165.70,163.68,163.02,134.87,134.10,131.80,130.32,1 27.03,122.89,122.87,115.18,114.93,40.31,30.20,29.70,20.37,13.84.HRMS(ESI)Calculated for C30H30IN2O3+:[M+H]+593.1296,found:593.12957. Wherein, the structural formula of compound (2) is: The structural formula of compound (3) is: The structural formula of compound (4) is:

[0042] Example 2 Preparation of Compound (1) Compound (4) (0.04 g, 0.3 mmol) and triethylamine (130 μL, 0.9 mmol) were dissolved in a dichloromethane solution (5 mL), and acryloyl chloride (50 μL, 0.6 mmol) was gradually added dropwise in an ice bath. The mixture was stirred at 0°C for 30 min, then warmed to room temperature and stirred for another 30 min to obtain a reaction mixture. The reaction mixture was extracted with DCM, and the organic phase was washed with a saturated sodium chloride solution (10 × 3 mL), dried over anhydrous Na2SO4, and purified by silica gel chromatography (E:P=1:5) to obtain compound (1) (0.020 g, yield 76%).

[0043] The obtained material was subjected to NMR analysis, and the results were as follows: The H NMR spectrum data are as follows: 1 H NMR (600MHz, Chloroform-d): δ8.61 (dd, J=7.3, 1.2Hz, 1H), 8.45 (s, 1H), 8.28 (dt, J=8.3, 1.4Hz, 1H), 7.89 (s, 1H) ,7.68–7.64(m,1H),7.54–7.50(m,1H),7.05(dd,J=17.0,10.5Hz,1H),6.48(dd,J=22.4,8.2Hz,2H),6.41(dd,J=1 7.0,1.5Hz,1H),5.93(d,J=1.4Hz,1H),4.20–4.17(m,2H),3.79(dd,J=10.2,6.8Hz,1H),3.53(dt,J=14.4,7.3Hz, 1H),3.38–3.33(m,1H),1.70(dd,J=8.1,2.3Hz,2H),1.49–1.45(m,2H),1.28–1.24(m,9H),0.99(d,J=7.2Hz,3H). The NMR carbon spectrum data are as follows: 13C NMR (151MHz, Chloroform-d): δ188.18,164.05,163.51,157.05,146.16,138.15,138.09,1 36.56,133.24,132.89,131.81,130.85,130.53,130.30,130.18,128.68,127.34,126.88, 122.95,121.53,115.18,113.17,111.77,109.06,108.84,79.61,54.87,50.84,40.21,40. 06,39.40,30.27,30.24,26.28,22.52,21.77,20.38,13.84,12.79.HRMS(ESI):Calculated for C33H32IN2O4+:[M+H]+647.1401,found:647.14014. Wherein, the structural formula of compound (4) is as follows: The structural formula of compound (1) is as follows:

[0044] Example 3 Weigh the compound (1) prepared in Example 2 and prepare it into a probe stock solution with a concentration of 1 mM using dimethyl sulfoxide. Draw 2 μL of the probe stock solution and add it to 398 μL of PBS buffer, then add Cys to it, mix well, transfer to a 96-well plate, and measure its absorbance using an ultraviolet spectrophotometer to determine the fluorescence absorption spectrum of compound (1), where the excitation wavelength is 580 nm and the emission wavelength is 675 nm. The fluorescence spectrum results are shown in Figure 1 .

[0045] The fluorescence absorption spectrum results show that after incubation with Cys, the fluorescence intensity of compound (1) at 675 nm is enhanced, indicating that the Cys response group of the probe is removed by the reaction and the fluorescence of compound (1) is released.

[0046] Example 4 2 μL of the probe mother solution prepared in Example 3 was added to 398 μL PBS buffer, and a total of 16 groups were prepared. Then 100 μM Cys, Hcy, Ala, Arg, Asp, Lys, Met, GSH, Phe, Ser, Trp, Val, Tyr, His, Gly, and blank were added respectively. Blank means adding an equal amount of PBS buffer. Then, the fluorescence intensity of each test system was detected by ultraviolet spectrophotometer, wherein the excitation wavelength was 580 nm and the emission wavelength was 675 nm. The results are as follows: Figure 2 As shown. Among them, Hcy, Ala, Arg, Asp, Lys, Met, GSH, Phe, Ser, Trp, Val, Tyr, His, Gly are used as control small molecules, and blank is used as a blank control.

[0047] From the fluorescence detection results and Figure 2 It can be seen that glutathione has a slight fluorescence response, and the fluorescence intensity of the probe does not fluctuate significantly when reacting with homocysteine. Other amino acids cannot react with compound (1). When Cys is used as a detector, the fluorescence intensity at 675nm increases significantly, and the fluorescence intensity is close to 150, which is 60 times higher than that of the blank control group, indicating that compound (1) is highly selective for cysteine.

[0048] Example 5 (1) After the reaction between compound (1) and cysteine, compound (1) is converted into compound (4). Compound (4) prepared in Example 1 is weighed and prepared into a probe stock solution of compound (4) with a concentration of 1 mM using dimethyl sulfoxide.

[0049] (2) Using a pipette, 4 μL of the mother solution of the singlet oxygen indicator ABDA (concentration of 1 mM, ABDA dissolved in dimethyl sulfoxide) was added to 395 μL of PBS (containing 1% DMSO (volume fraction), the same below) to obtain an indicator reaction solution; 1 μL of the probe mother solution of compound (4) was added to 395 μL of PBS (1% DMSO), and then mixed with the indicator reaction solution. After reacting at 37° C. for 0.5 h, the mixture was added to a 96-well plate and the ultraviolet absorption spectrum of compound (4) was measured using a fluorescent microplate reader. Then, an incandescent lamp (lamp wavelength 400 nm-780 nm, 250 V, 4 A, 20 mW / cm 2 ) for 1.5 minutes, then measure the UV absorption spectrum of compound (4), and then measure the UV absorption spectrum after 1 minute of irradiation with an incandescent lamp. After a total of 450 seconds of irradiation, the test is stopped. The results are as follows Figure 3 This step can be used to study the photodynamic properties of compound (1) after being activated.

[0050] Depend on Figure 3 It can be seen that as the illumination time increases, the absorption value gradually decreases, and the absorption value at the absorption wavelength of 378nm decreases from 0.83 to 0.33, which is a significant change, which lays a foundation for the photodynamic therapy of tumor cells. (3) The photodynamic performance of compound (1) is measured by the same method as step (2), reflecting the photodynamic performance of compound (1) when it is not activated, wherein the probe mother solution of compound (1) is prepared according to the method of Example 3. The photodynamic performance results of compound (1) are shown in Figure 4, wherein the test was stopped after the incandescent lamp was irradiated for a total of 2100 seconds. The results showed that the absorption value decreased by 0.4 after 2100 seconds of illumination, which shows that the absorption value did not change significantly with the increase of illumination time. This shows that compound (1) does not have photodynamic therapy performance, and its photodynamic therapy performance is activated only after Cys activation.

[0051] Example 6 Compound (1) was used to study the production of endogenous and exogenous cysteine ​​in HeLa cells (cervical cancer cells). The specific steps are as follows: Compound (1) was incubated with HeLa for 30 minutes, and then a confocal imaging experiment was performed. NEM is a thiol blocking agent that can remove Cys. Compound (1) and NEM were added together with HeLa and incubated for 30 minutes, and then a confocal imaging experiment was performed. Compound (1), NEM, and Cys were added together with HeLa and incubated for 30 minutes, and then a confocal imaging experiment was performed. The above confocal imaging experiment results are shown in Figure 5 .

[0052] From the results of the confocal imaging experiment, it can be seen that only compound (1) has the strongest cell fluorescence intensity. This is because Cys is overexpressed in HeLa cells, and Cys undergoes cyclization with the acryloyl ester on compound (1) to return to hydroxyl. NEM removes endogenous cysteine ​​in HeLa cells, and after adding NEM, the cell image has basically no red fluorescence. NEM removes endogenous cysteine ​​compound (1) in HeLa cells, but when Cys is added to the cell imaging image, exogenous Cys is introduced, and the fluorescence is significantly increased. Figure 7 The normalized fluorescence graph in the figure below shows the fluorescence changes intuitively, which fully demonstrates that compound (1) can detect endogenous and exogenous cysteine ​​in HeLa cells.

[0053] Example 7 (1) At 37°C, approximately 1×10 5 A549 cells were seeded in a confocal culture dish (20 mm) with 1 mL of culture medium. Before adding compound (1), the cells were allowed to adhere to the culture dish for 24 h. After incubating A549 cells with compound (1) (2.5 μM) at 37°C under light conditions for 30 min, they were washed with fresh culture medium and confocal imaging was performed; at the same time, another A549 cell was incubated with compound (1) (2.5 μM) at 37°C under light conditions for 30 min, then washed with fresh culture medium and confocal imaging was performed; at the same time, another A549 cell was incubated at 37°C under light conditions for 30 min, then washed with fresh culture medium and confocal imaging was performed. The confocal imaging results are shown in the figure. Figure 6As shown in Figures a1-a3, A549 cells were illuminated (with white light, wavelength 400nm-780nm, 16mW / cm 2 Figures b1-b3 are the imaging results after compound (1) was added to A549 cells and cultured under light-proof conditions. Figures c1-c3 are the imaging results after compound (1) was added to A549 cells and cultured under light (white light, wavelength 400nm~780nm, 16mW / cm 2 Imaging results after incubation under 37° light conditions.

[0054] like Figure 6 It can be seen that there is no fluorescence in Figures a1-a3, the living morphology of cells can be seen in Figures b1-b3 through fluorescence, and Figures c1-c3 show that the morphology of A549 cells has changed significantly, from a long spindle to a round shape, which fully proves cell apoptosis.

[0055] (2) In order to further accurately determine the effect of PDT, this step uses the Annexin V-PI double staining method (Annexin V and PI are used together to distinguish early apoptotic cells from late apoptotic cells and dead cells) to re-image the confocal imaging cells in step (1). The results are shown in Figure 7 .

[0056] Annexin V is a calcium-dependent phospholipid binding protein with a high affinity for phosphatidylserine (PS). It can specifically recognize PS on the surface of apoptotic cells, so FITC-coupled Annexin V can be used to distinguish between apoptotic cells and living cells. PI (propidium iodide) dye can bind to DNA in cells. Since the cell membranes of apoptotic cells and living cells are still intact, PI dye cannot freely pass through the cell membrane into the cell and bind to DNA. Therefore, PI dye cannot mark apoptotic cells and living cells, but only dead cells. Annexin V and PI can be used together to distinguish between early and late apoptotic cells and dead cells. Figure 7 The color change clearly shows that the cells have died, further confirming that compound (1) has a good photodynamic therapy effect.

[0057] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0058] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A cysteine-activated fluorescent probe, characterized in that: The structure is shown in formula (1):

2. A method for preparing a cysteine-activated fluorescent probe, characterized in that: The following steps are involved: Step S1: dissolving compound (2) and compound (3) in a solvent and subjecting the mixture to reflux reaction to obtain compound (4); Step S2: dissolving compound (4) and acryloyl chloride in a solvent and reacting to obtain compound (1); in: The structural formula of compound (2) is: The structural formula of compound (3) is: The structural formula of compound (4) is: The structural formula of compound (1) is:

3. The preparation method according to claim 2, characterized in that: In the step S1, the solvent is ethanol.

4. The preparation method according to claim 2, characterized in that: In the step S1, the catalyst for the reflux reaction is piperidine.

5. The preparation method according to claim 2, characterized in that: In the step S1, the temperature of the reflux reaction is 75°C to 80°C.

6. The preparation method according to claim 2, characterized in that: In the step S2, the solvent is dichloromethane.

7. The preparation method according to claim 2, characterized in that: In the step S2, the catalyst for the reaction is triethylamine.

8. The preparation method according to claim 2, characterized in that: In the step S2, the reaction temperature is -5°C to 30°C.

9. Use of the fluorescent probe according to claim 1, or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 8 in imaging of cancer biomarkers.

10. Use of the fluorescent probe according to claim 1, or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 8, in the preparation of products based on photodynamic therapy for cancer.

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