Human cystathionine gamma-lyase response fluorescent dye as well as preparation method and application thereof

Abstract

The invention relates to a human cystathionine gamma-lyase (hCSE) response fluorescent dye as well as a preparation method and application thereof. The excitation wavelength of the fluorescent dye is 500-630 nm, and the emission wavelength of the fluorescent dye is 650-850 nm. The dye can rapidly and selectively image hCSE enzyme on living cells at a high signal-to-noise ratio, and compared with the prior art, the dye has the advantage that the cell fluorescence signal can be enhanced by 10 times. Besides, the dye is a first fluorescent probe capable of being used for in-vivo recognition imaging of hCSE enzyme, and tumor fluorescence signals can be remarkably enhanced within a short time by applying a small dosage in an intratumor injection mode. The application directions comprise hCSE enzyme fluorescence detection and imaging, imaging preparations, hCSE enzyme targeted tumor light-driven treatment related preparations, hCSE enzyme targeted tumor chemotherapy prodrug preparations, hCSE enzyme targeted radiation imaging preparations and tools for biomarker research and drug development.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical engineering, and in particular relates to a human cystathionine gamma-lyase responsive fluorescent dye and a preparation method and application thereof. Background Art

[0002] Human cystathionine γ-lyase (hCSE) is one of the key enzymes in the transsulfurization pathway. It uses pyridoxal phosphate as a coenzyme and can catalyze the cleavage of the C-γ-S bond of L-cystathionine to generate L-cysteine, α-ketobutyric acid and ammonia. This is the only way for the human body to synthesize L-cysteine. hCSE can also catalyze L-cysteine ​​or L-homocysteine ​​to generate the gas signaling molecule hydrogen sulfide (H 2 S). Recent studies have shown that human cystathionine γ-lyase is a potential tumor marker. For example, the metabolic level of hCSE enzyme in breast cancer cells and tissues is significantly increased, and it has a higher metabolic level in triple-negative breast cancer cells and tissues. Overexpression of hCSE enzyme is associated with increased endogenous H 2 S can promote breast cancer cell proliferation, migration and invasion through the signal transducer and activator of transcription 3 signaling pathway and the vascular endothelial growth factor signaling pathway. Therefore, the identification and detection of hCSE enzyme has important medical and clinical significance for in-depth analysis of the biological and pathological functions of the enzyme, as well as tumor diagnosis, treatment and prognosis.

[0003] The existing detection methods of hCSE enzyme mainly include protein blotting, immunohistochemistry, quantitative reverse transcription polymerase chain reaction, methylene blue assay, 2 The above methods are not suitable for rapid detection of hCSE enzyme at the level of living cells, which hinders the in situ study of hSCE enzyme. Activated fluorescent probes have been widely used for in situ detection of cancer markers at the cell and living body levels due to their simple operation, sensitive detection and high biocompatibility. Han et al. used L-Hcy as the recognition group and flexible 2-mercaptoethylcarbamate as the cleavable linker to report the only activated hCSE enzyme fluorescent probe CSEP so far, which was successfully used to detect hCSE enzyme in human liver cancer cell LM3, zebrafish and frozen sections of liver tissue of septic rats. However, the reaction of probe CSEP with hCSE enzyme is slow, and the fluorescence imaging signal-to-noise ratio is low, which limits its further promotion and application in cells and living bodies. Therefore, it is urgent to develop a recognition structure for rapid recognition of hCSE enzyme to improve the recognition rate and imaging signal-to-noise ratio of hCSE enzyme. Summary of the invention

[0004] In view of the above problems, the present invention provides a class of human cystathionine γ-lyase (hCSE enzyme) responsive fluorescent dyes, which can be used to quickly identify hCSE enzymes at the cellular and living body levels with a high signal-to-noise ratio. The fluorescent dye can be used as a substrate for the hCSE enzyme, and the product after catalytic cleavage by the hCSE enzyme carries a thiol functional group with a strong nucleophilic property, which can continue to undergo an intramolecular cyclization reaction to release a fluorescent signal.

[0005] In view of this, the first aspect of the present invention provides a class of hCSE enzyme-responsive fluorescent dyes having the following general structural formula I:

[0006]

[0007] Among them, the R 1 , R 2 Each independently selected from a hydrogen atom, a methyl group, an ethyl group, a propyl group, a butyl group, and a pentyl group;

[0008] The F is a fluorescent dye or a photosensitizing drug.

[0009] In the technical solution described above, it is further preferred that the fluorescent dye is selected from one of Cy3, Cy5, Cy7 cyanine dyes, FITC, AlexaFluor series dyes, Dylight series dyes, fluorescein and its derivatives, rhodamine and its derivatives, BODIPY series dyes, and near-infrared dyes.

[0010] For the technical solution described above, it is further preferred that:

[0011] The Alexa Fluor series dyes, such as Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647, etc.;

[0012] The Dylight series dyes, such as Dylight 488, Dylight 550, Dylight 650, etc.;

[0013] The near-infrared dyes described, such as IRDye 800CW, IRDye 700DX, etc.;

[0014] The photosensitizing drug is selected from porphyrin compounds, phthalocyanine compounds, Nile blue compounds, etc.

[0015] In the technical solution described above, it is further preferred that the molecular structure prepared in the embodiment of the present invention is as follows:

[0016]

[0017] In the technical solution described above, it is further preferred that the excitation wavelength of the hCSE enzyme-responsive fluorescent dye is 500-630 nm, and the emission wavelength is 650-850 nm. It is further preferred that the excitation wavelength of the hCSE enzyme-responsive fluorescent dye is 550-630 nm, and the emission wavelength is 680-750 nm.

[0018] The specific advantages of the hCSE enzyme-responsive fluorescent dye of the present invention are as follows:

[0019] First, the hCSE enzyme-responsive fluorescent dye described in the present invention can rapidly, with high signal-to-noise ratio, and selectively image the hCSE enzyme on living cells. After the activated hCSE enzyme fluorescent probe CSEP reported to be incubated with living cells for 0.5 hours, the cell fluorescence signal only showed about 1.5 times the fluorescence enhancement (Analytical Chemistry, 2022, 94, 1203). In comparison, after the fluorescent dye described in the present invention was incubated with living cells for 0.5 hours, the cell fluorescence signal enhancement can reach about 10 times (experimental group). In addition, in the control group, the living cells were pretreated with the hCSE enzyme inhibitor DL-propargylglycine (PAG) and then incubated with the fluorescent dye described in the present invention. The fluorescence signal intensity was about 33% lower than that of the experimental group, indicating that the fluorescent dye described in the present invention "lights up" the above-mentioned living cells by specifically recognizing the hCSE enzyme.

[0020] Second, the fluorescent dye of the present invention is the first fluorescent probe that can be used for in vivo identification and imaging of hCSE enzyme. Existing detection methods, including the recently reported fluorescent probe CSEP, have not been used to image hCSE enzyme at the in vivo level. The hCSE enzyme-responsive fluorescent dye of the present invention can be administered in a small dose (5nmol) by intratumoral injection, and can "light up" xenografted breast cancer in a short time (about 60 minutes), and the average fluorescence signal of the tumor is enhanced by about 6 times (experimental group). In the control group, after the hCSE enzyme inhibitor DL-propargylglycine (PAG) (100nmol) was administered by paratumoral injection, the fluorescent dye of the present invention (5nmol) was injected intratumorally, and the average fluorescence signal intensity at the tumor was about 50% lower than that of the tumor in the experimental group, indicating that the fluorescent dye of the present invention "lights up" xenografted breast cancer by specifically recognizing and responding to the hCSE enzyme.

[0021] The second aspect of the present invention provides a method for preparing the above fluorescent dye, comprising the following steps:

[0022] (1) taking the intermediate 2 having the structure of the general formula 2, adding anhydrous dichloromethane and a mixed solution of piperidine to fully react, diluting with dichloromethane, washing with sodium citrate buffer and saturated brine, drying, filtering, and concentrating to obtain a light yellow oily substance;

[0023] (2) adding anhydrous dichloromethane to the oily substance prepared in step (1), adding p-nitrophenyl chloroformate at -5-5°C under alkaline conditions and stirring thoroughly, returning to room temperature to continue the reaction, adding a photosensitizing dye or a photosensitizing drug to the reaction solution and stirring thoroughly, concentrating and purifying to obtain a pure product;

[0024] (3) Adding a mixed solution of dichloromethane and trifluoroacetic acid to the pure product prepared in step (1), and then fully reacting, concentrating, crystallizing, and purifying to obtain an hCSE enzyme-responsive fluorescent dye having the general formula I.

[0025]

[0026] More preferably, the volume ratio of anhydrous dichloromethane to piperidine in the mixed solution of anhydrous dichloromethane and piperidine in step (1) is 4-8:1;

[0027] More preferably, the alkaline agent providing alkaline conditions in the above step (2) is selected from one of N,N-diisopropylethylamine, triethylamine, 4-dimethylaminopyridine, sodium carbonate, potassium carbonate, and cesium carbonate, and the molar ratio of the intermediate 2 to the alkaline agent is 1:(4-10);

[0028] Wherein, the molar ratio of intermediate 2 to p-nitrophenyl chloroformate is 1:(1-2.5);

[0029] More preferably, the molar ratio of the intermediate 2 to the alkali agent is 1:(4-5), and the molar ratio of the intermediate 2 to p-nitrophenyl chloroformate is 1:(1-1.2);

[0030] More preferably, in the above step (2), the molar ratio of the photosensitizing dye or photosensitizing drug added to the reaction solution to the intermediate 2 is (1.0-1.5):1;

[0031] More preferably, the volume ratio of the oily substance prepared in the above step (2) to anhydrous dichloromethane is 1:(10-30);

[0032] More preferably, the volume ratio of dichloromethane to trifluoroacetic acid in the mixed solution of dichloromethane and trifluoroacetic acid in step (3) is (1-4):1;

[0033] The third aspect of the present application is to protect the use of the fluorescent dye in biological and medical diagnostic preparations related to hCSE enzyme for non-disease diagnosis and treatment purposes.

[0034] Furthermore, the above applications include but are not limited to: hCSE enzyme fluorescence detection and fluorescence imaging, imaging preparations, hCSE enzyme-targeted tumor light-driven therapy-related preparations, hCSE enzyme-targeted tumor chemotherapy prodrug preparations, hCSE enzyme-targeted radioimaging preparations, and tools for biomarker research or drug development, etc.

[0035] The beneficial effects of the present invention compared with the prior art are as follows:

[0036] The hCSE enzyme-responsive fluorescent dye of the present invention can rapidly, with a high signal-to-noise ratio, and selectively image the hCSE enzyme on living cells; the hCSE enzyme-responsive fluorescent dye of the present invention is the first fluorescent probe that can be used for in vivo identification and imaging of the hCSE enzyme, which has important biological and medical significance for studying the hCSE enzyme at the in situ living cell level, especially at the living animal level.

[0037] In summary, the biological functions and medical application potential of hCSE enzymes have not been fully explored. One of the main reasons is the lack of effective research tools. The hCSE enzyme-responsive fluorescent dye described in the present invention can provide a type of efficient tool for the study of hCSE enzyme-related signal pathways and the diagnosis and treatment of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The fluorescent probe DCM-hCSE of the present invention is 1 H-Figure;

[0039] Figure 2 The fluorescent probe DCM-hCSE of the present invention is 13 C spectrum;

[0040] Figure 3 It is a high-resolution mass spectrum of the fluorescent probe DCM-hCSE of the present invention;

[0041] Figure 4 It is the cytotoxicity evaluation of the fluorescent probe DCM-hCSE of the present invention;

[0042] Figure 5 It is the time-fluorescence imaging intensity result of the fluorescent probe DCM-hCSE of the present invention recognizing the endogenous hCSE enzyme in MDA-MB-231 cells;

[0043] Figure 6 It is a fluorescence imaging picture of the fluorescent probe DCM-hCSE of the present invention recognizing the endogenous hCSE enzyme of cancer cell MCF-7 and MDA-MB-231;

[0044] Figure 7 The fluorescent probe DCM-hCSE of the present invention is used for fluorescent imaging of xenograft triple-negative breast cancer. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present application to clearly describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments in the present application belong to the scope of protection of this application.

[0046] In the description of this application, it should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to this application. The following specific examples illustrate the hCSE enzyme recognition structure and its preparation method and application of the present invention:

[0047] Example 1

[0048] The synthesis route of the fluorescent probe DCM-hCSE is as follows:

[0049]

[0050] The synthesis process of the fluorescent probe DCM-hCSE is as follows:

[0051] (1) Synthesis of Compound 1-1

[0052] Take L-homocystine (5.3670g, 20mmol) in a 500mL three-necked flask, dissolve it in 160mL of 1,4-dioxane, add 180mL of 10% NaHCO3 solution to the reaction bottle, and place the three-necked flask in an ice-water bath, add di-tert-butyl dicarbonate (9.6030g, 44mmol) at about 0°C, return the mixture to room temperature, and stir overnight. Post-treatment: adjust the pH of the reaction solution to 4 with NaHSO4 solution, extract the solution with ethyl acetate (2×50mL), combine the organic layers, wash the organic layers with saturated brine (2×50mL), dry and filter with anhydrous Na2SO4, and concentrate the organic layer to obtain a white solid product.

[0053] (2) Synthesis of Compound 1-2

[0054] Take compound 1-1 (4.6858 g, 10 mmol) in a 100 mL flask, dissolve it in 35 mL of DMF, add cesium carbonate (3.2582 g, 10 mmol), cool to 0°C in an ice-water bath, add MeI (3.6000 g, 25 mmol) dropwise into the flask, react overnight, remove the solvent in vacuo, dilute with ethyl acetate, wash with water and saturated brine respectively, dry over anhydrous Na2SO4 and filter, concentrate the organic layer, and purify by column chromatography to obtain 4.21 g of white solid product with a yield of 90.34%.

[0055] 1 H NMR (400MHz, DMSO) δ7.33(d,J=7.9Hz,2H),4.16–4.03(m,2H),3.63(s,6H),2.79–2.66(m,4H),2.11–1.98(m,2H),1.97–1.84(m,2H),1.38(s,18H).

[0056] 13 CNMR(101MHz,DMSO)δ172.65,155.52,78.35,52.20,51.88,33.86,30.16,28.14.

[0057] (3) Synthesis of Compound 1-3

[0058] Compound 1-2 (4.2214 g, 8.5 mmol) was placed in a 100 mL reaction bottle and dissolved in 30 mL of tetrahydrofuran. Under nitrogen protection, tributylphosphine (2.5779 g, 12.75 mmol) was added. After stirring for 15 min, 30 mL of water was added. The reaction was allowed to proceed overnight. The tetrahydrofuran was removed by rotary evaporation to obtain a concentrated solution, which was diluted with ethyl acetate, washed with NaHSO4 and saturated brine respectively, dried over anhydrous Na2SO4 and filtered. The organic layer was concentrated and purified by column chromatography to obtain 3.8615 g of a yellow oily product with a yield of 91.19%.

[0059] 1 H NMR (400MHz, DMSO-d6) δ7.28(d,J=8.0Hz,1H),4.13(td,J=7.9,6.3Hz,1H),3.62(d,J=4.5Hz,3H) ,2.48-2.42(m,1H),2.37-2.31(m,1H),1.86(p,J=7.2Hz,2H),1.38(s,9H),1.18(t,J=7.1Hz,1H).

[0060] (4) Synthesis of Compound 2-1

[0061] Fmoc-serine methyl ester (3.4113 g, 10 mmol) was placed in a 250 mL reaction bottle and dissolved in 60 mL of dichloromethane. Triphenylphosphine (2.8850 g, 11 mmol) and hexachloroethane (2.7600 g, 11 mmol) were added under nitrogen protection. The reaction was terminated after 4 h. Saturated sodium bicarbonate solution was added, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain 2.55 g of a white solid product with a yield of 70.83%.

[0062] 1 HNMR (400MHz, DMSO) δ8.06(d,J=8.2Hz,1H),7.90(d,J=7.5Hz,2H),7.73(d,J=7.5Hz,2H),7.42(t,J=7.4Hz,2H),7.33(t,J=7.4Hz,2H),4 .50–4.43(m,1H),4.33(d,J=7.0Hz,2H),4.24(t,J=6.8Hz,1H),3.91(dd,J=11.3,4.4Hz,1H),3.82(dd,J=11.2,7.8Hz,1H),3.67(s,3H).

[0063] 13 C NMR (101MHz, DMSO) δ169.54,155.92,143.70,127.66,127.07,125.25,120.13,65.89,55.34,52.42,46.57,43.76.

[0064] (5) Synthesis of Compound 3-1

[0065] Compound 1-3 (1.8 g, 7.22 mmol) was placed in a 50 mL reaction bottle, and anhydrous DMF (15 mL), compound 2-1 (2.47 g, 6.88 mmol), cesium carbonate (2.24 g, 6.88 mmol) and potassium iodide (1.14 g, 6.88 mmol) were added in sequence under nitrogen protection. The mixture was reacted at room temperature overnight, and the mixture was diluted with ethyl acetate (50 mL) for post-treatment, and washed with water and saturated brine, respectively, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (petroleum ether: ethyl acetate = 4:1) to obtain 1.1 g of a yellow oily product with a yield of 28%.

[0066] 1 H NMR (400MHz, DMSO) δ7.92–7.87(m,3H),7.72(d,J=7.4Hz,2H),7.42(t,J=7.4Hz,2H),7.36–7.29(m,3H),4.31(d,J=7.1Hz,2H),4.26–4.17(m,2H),4. 12–4.06(m,1H),3.64(s,3H),3.61(s,3H),2.89(ddd,J=14.1,9.3,5.1Hz, 1H),2.81–2.71(m,1H),2.61–2.52(m,2H),1.90–1.79(m,2H),1.37(s,9H).

[0067] 13C NMR (101MHz, DMSO) δ172.76,171.35,155.93,155.56,143.74,140.72,127.64,127.06,125 .23,120.12,78.32,65.77,59.75,52.50,52.11,51.83,46.59,32.18,30.79,28.14,27.98.

[0068] (6) Synthesis of DCM-hCSE

[0069] Compound 3-1 (200 mg, 349.24 μmol) was placed in a reaction flask, and a mixed solvent of anhydrous dichloromethane and piperidine (5 mL) was added (anhydrous dichloromethane: piperidine = 4:1, volume ratio). The reaction was allowed to react for 1 hour. The reaction solution was concentrated and diluted with dichloromethane, washed with sodium citrate buffer (pH = 5) and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 3-2, which was directly used to synthesize compound 3-3. p-Nitrophenyl chloroformate (77.43 mg, 384.16 μmol) and compound 3-2 were dissolved in anhydrous dichloromethane (3 mL), ice-water bath for 10 minutes at about 0°C, anhydrous N, N-diisopropylethylamine (5 equivalents) was added dropwise, and stirred in an ice-water bath for 1 hour, and then the reaction was continued at room temperature for 1 hour to obtain compound 3-3, and fluorescent dye DCM (133.92 mg, 349.24 μmol) was added to react overnight, concentrated, and slurried to purify to obtain compound Boc-DCM-hCSE. A mixed solution of dichloromethane and trifluoroacetic acid (2 mL) (anhydrous dichloromethane: trifluoroacetic acid = 1:1, volume ratio) was added to the pure Boc-DCM-hCSE prepared above, and then fully reacted, concentrated, crystallized, and purified to obtain 42 mg of a purple-red solid product with a yield of 18.23%.

[0070] 1 H NMR(400MHz,MeOD)δ8.81(d,J=8.3Hz,1H),7.81(t,J=7.6Hz,1H),7.77–7.68(m,2H),7.57(d, J=8.6Hz,1H),7.47(t,J=7.6Hz,1H),6.82(t,J=7.9Hz,2H),6.62(d,J=8.7Hz,1H),6.43(s,1H ),4.59–4.51(m,1H),4.20–4.14(m,1H),3.80(s,6H),3.44(dd,J=13.9,6.9Hz,4H),3.24–3.1 5(m,1H),3.02–2.93(m,1H),2.75(t,J=7.3Hz,2H),2.24–2.04(m,2H),1.20(t,J=7.0Hz,6H).

[0071] 13 C NMR(101MHz,MeOD)δ172.53,170.53,161.14,156.84,154.31,153.88,153.16,151.99,135.81,135.54,131.32,126.76,126.26,120.11, 118.92,118.65,117.42,116.29,115.30,110.92,106.75,106.41,59 .87,55.14,53.83,53.26,52.71,45.67,34.50,31.20,28.10,12.93.

[0072] HRMS(ESI):m / z 660.2482[M+H](calcd for C 34 H 38 N 5 O 7 S + ,660.2486).

[0073] Example 2

[0074] Cytotoxicity evaluation of DCM-hCSE.

[0075] After MDA-MB-231 cells were incubated with 0-20 μM DCM-hCSE for 24 h, the cytotoxicity of DCM-hCSE was determined using the thiazole blue colorimetric method (MTTassay).

[0076] The results are as follows Figure 4 As shown, after incubating MDA-MB-231 cells with 15 μM dye DCM-hCSE for 24 hours, the survival rate was higher than 90%, proving that the dye DCM-hCSE has no obvious cytotoxicity even at higher concentrations, has high biocompatibility, and can be safely used to detect and identify hCSE enzymes in cells and living organisms.

[0077] Example 3

[0078] Time-fluorescence intensity statistics of DCM-hCSE recognition of endogenous hCSE enzyme in MDA-MB-231 cells. Prepare breast cancer cell MDA-MB-231 culture medium, culture MDA-MB-231 cells in laser confocal culture dish, add DCM-hCSE (5 μM) after cell abundance reaches 60%, and incubate at 37°C and 5% CO 2The cells were cultured under the gas environment. Laser confocal scanning fluorescence microscope STELLARIS 5 was used to collect cell fluorescence images at different time points (0, 15, 30, 45, 60, 75, 90, 105, 120, 135 and 150 min).

[0079] The results are as follows Figure 5 As shown in Figure A, breast cancer cells MDA-MB-231 can undergo significant fluorescence enhancement in a short period of time (about 60 minutes), proving that the dye DCM-hCSE can quickly identify and respond to hCSE enzymes in living cells. Figure B shows that the fluorescence of the dye DCM-hCSE is enhanced more than ten times when incubated with MDA-MB-231 cells for 2 hours, proving that DCM-hCSE can be well used for hCSE enzyme recognition imaging in living cells.

[0080] Example 4

[0081] DCM-hCSE recognizes endogenous hCSE enzyme in cancer cells MCF-7 and MDA-MB-231.

[0082] Prepare breast cancer cell culture medium for MCF-7 and MDA-MB-231 cells, and culture MCF-7 and MDA-MB-231 cells in laser confocal imaging culture dishes. When the cell abundance reaches 60%, DCM-hCSE (5 μM) is added to the cells in the experimental group and incubated at 37°C and 5% CO 2 The cells were incubated at 37°C and 5% CO for 2 h. The cells in the control group were first incubated with the hCSE enzyme inhibitor DL-propargylglycine (PAG, 1 mM) for 30 min, and then DCM-hCSE (5 μM) was added. 2 The cells were incubated for 2 h. The cells were washed twice with phosphate buffered saline (PBS), and the fluorescence images of the cells were collected using a laser confocal scanning fluorescence microscope STELLARIS 5.

[0083] The results are as follows Figure 6 As shown, compared with the experimental group, the fluorescence signal intensity of the control group was significantly reduced and was about 33% lower, proving that the dye DCM-hCSE can be used for the specific identification of hCSE enzyme in breast cancer cells.

[0084] Example 5

[0085] DCM-hCSE fluorescence imaging of breast cancer xenografts.

[0086] 6-week-old BABL / c nude mice were subcutaneously injected with MDA-MB-231 cells (5*10 6 / in PBS), tumors grew to 200mm 3The experimental group of mice was injected with DCM-hCSE (100μM, dispersed in 50μL PBS) intratumorally, and the control group of mice was first injected with hCSE enzyme inhibitor DL-propargylglycine (PAG, 2mM, dispersed in 50μL PBS) adjacent to the tumor for 2 hours, and then injected with DCM-hCSE (100μM, dispersed in 50μL PBS) intratumorally. PerkinElmer IVIS spectrometer was used to collect near-infrared fluorescence images of tumors and mice at different time points (0, 15, 30, 45, 60, 75, 90, 105, 120, 135 and 150min).

[0087] The results are as follows Figure 7 As shown in Figure A, in the experimental group, in a short period of time (about 60 minutes), xenograft breast cancer can undergo obvious fluorescence enhancement. In the control group, the average fluorescence signal intensity at the tumor is significantly lower than that of the experimental group, proving that the dye DCM-hCSE can quickly and specifically identify and respond to the hCSE enzyme in vivo. Figure B shows that compared with the experimental group, the average fluorescence signal intensity at the tumor in the control group is about 50% lower, proving that the dye DCM-hCSE can be well used for in vivo recognition imaging of the hCSE enzyme.

[0088] The embodiments of the present application are described above in conjunction with the accompanying drawings. In the absence of conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present application, ordinary technicians in this field can also make many forms without departing from the purpose of the present application and the scope of protection of the claims, all of which are within the protection of the present application.

Claims

1. A human cystathionine γ-lyase responsive fluorescent dye, characterized in that: The dye has the structure of the following general formula I: Wherein, R1 and R2 are each independently selected from one of hydrogen atom, methyl, ethyl, propyl, butyl and pentyl; The F is a fluorescent dye or a photosensitizing drug; The excitation wavelength of the fluorescent dye is 500-630nm, and the emission wavelength is 650-850nm.

2. The method for preparing a fluorescent dye according to claim 1, wherein: The steps include: (1) taking the intermediate 2 having the structure of the general formula 2, adding anhydrous dichloromethane and a mixed solution of piperidine to fully react, diluting with dichloromethane, washing with sodium citrate buffer and saturated brine, drying, filtering, and concentrating to obtain a light yellow oily substance; (2) adding anhydrous dichloromethane to the oily substance prepared in step (1), adding p-nitrophenyl chloroformate at -5-5°C under alkaline conditions and stirring thoroughly, returning to room temperature to continue the reaction, adding a photosensitizing dye or a photosensitizing drug to the reaction solution and stirring thoroughly, concentrating and purifying to obtain a pure product; (3) adding a mixed solution of dichloromethane and trifluoroacetic acid to the pure product prepared in step (2), reacting sufficiently, concentrating, crystallizing, and purifying to obtain an hCSE enzyme-responsive fluorescent dye having the general formula I; 3. The method for preparing the fluorescent dye according to claim 2, characterized in that: The volume ratio of anhydrous dichloromethane to piperidine in the anhydrous dichloromethane and piperidine mixed solution in step (1) is 4-8:

1.

4. The method for preparing the fluorescent dye according to claim 2, characterized in that: The alkaline agent providing alkaline conditions in step (2) is selected from one of N,N-diisopropylethylamine, triethylamine, 4-dimethylaminopyridine, sodium carbonate, potassium carbonate, and cesium carbonate.

5. The method for preparing the fluorescent dye according to claim 2, characterized in that: The molar ratio of the intermediate 2 to the alkali agent is 1:(4-10); the molar ratio of the intermediate 2 to p-nitrophenyl chloroformate is 1:(1-2.5).

6. The method for preparing the fluorescent dye according to claim 2, characterized in that: The molar ratio of the photosensitizing dye or photosensitizing drug to the intermediate 2 is (1.0-1.5):

1.

7. The method for preparing the fluorescent dye according to claim 2, characterized in that: The volume ratio of the oily substance to anhydrous dichloromethane is 1:(10-30).

8. The method for preparing the fluorescent dye according to claim 2, characterized in that: The volume ratio of dichloromethane to trifluoroacetic acid in the dichloromethane and trifluoroacetic acid mixed solution is (1-4):

1.

9. Use of the dye according to claim 1 in biological and medical research preparations related to hCSE enzyme for purposes other than disease diagnosis and treatment.

10. The use according to claim 9, characterized in that: The applications include: fluorescence detection and fluorescence imaging of hCSE enzyme; research and development of imaging preparations; research on hCSE enzyme-targeted light-driven therapy-related preparations; research on hCSE enzyme-targeted chemotherapy prodrug preparations; research and development of hCSE enzyme-targeted radioimaging preparations; as a tool for biomarker research; and as a tool for drug development research.

Images