Human cystathionine gamma-lyase-responsive fluorescent dye and preparation method and application thereof

Abstract

The present application relates to a kind of human cysteine gamma-cleaving enzyme (hCSE) response fluorescent dye and its preparation method and application.The excitation wavelength of the fluorescent dye is 500-630nm, and the emission wavelength is 650-850nm.The dye can be selectively imaged hCSE enzyme on living cells rapidly, with high signal-to-noise ratio, compared with prior art, cell fluorescence signal enhancement can reach 10 times.In addition, the dye is the first fluorescent probe that can be used for in vivo identification and imaging of hCSE enzyme, by intratumoral injection, with small dose, tumor fluorescence signal can be significantly enhanced in a short time.The application direction includes hCSE enzyme fluorescence detection and imaging, imaging preparation, hCSE enzyme targeted tumor light-driven treatment related preparation, hCSE enzyme targeted tumor chemotherapy prodrug preparation, hCSE enzyme targeted radioimaging preparation, and as a tool for biomarker research and drug development.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, and in particular relates to a human cystathionine γ-lyase responsive fluorescent dye, its preparation method and application. Background Technology

[0002] Human cystathionine γ-lyase (hCSE) is a key enzyme in the transsulfurization pathway. Using pyridoxal phosphate as a coenzyme, it catalyzes the cleavage of the C-γ-S bond of L-cystathionine to produce L-cysteine, α-butanone, and ammonia. This is the only pathway for L-cysteine ​​synthesis in the human body. hCSE can also catalyze the formation of hydrogen sulfide (H2S), a gaseous signaling molecule, from L-cysteine ​​or L-homocysteine. Recent studies have shown that hCSE is a potential tumor marker. For example, the metabolic level of hCSE is significantly elevated in breast cancer cells and tissues, and even higher in triple-negative breast cancer cells and tissues. Overexpressed hCSE and the resulting increase in endogenous H2S can promote breast cancer cell proliferation, migration, and invasion through signal transduction and the activator of transcription 3 (ACT3) and vascular endothelial growth factor (VEGF) signaling pathways. Therefore, the identification and detection of hCSE is of significant medical and clinical importance for a deeper understanding of its biological and pathological functions, as well as for tumor diagnosis, treatment, and prognosis.

[0003] Current methods for detecting hCSE enzyme mainly include Western blotting, immunohistochemistry, quantitative reverse transcription polymerase chain reaction (RT-PCR), methylene blue assay for H2S, and 5,5-dithiobis(2-nitrobenzoic acid) colorimetric assay for L-cysteine. None of these methods are suitable for rapid detection of hCSE enzyme at the live cell level, hindering in-situ studies of hCSE enzyme. Activated fluorescent probes, due to their ease of operation, high sensitivity, and high biocompatibility, have been widely used for in-situ detection of cancer biomarkers at the cellular and in vivo levels. Han et al. reported the only activated hCSE enzyme fluorescent probe to date, CSEP, using L-Hcy as the recognition group and flexible 2-mercaptoethylaminocarbamate as a severable linker, successfully detecting hCSE enzyme in frozen sections of human hepatocellular carcinoma cells LM3, zebrafish, and septic rat liver tissue. However, the reaction between the CSEP probe and hCSE enzyme is slow, and the fluorescence imaging signal-to-noise ratio is low, limiting its further application in cells and in vivo. Therefore, there is an urgent need to develop a recognition structure for rapid identification of hCSE enzymes, thereby improving the recognition rate and imaging signal-to-noise ratio of hCSE enzymes. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a class of human cystathionine γ-lyase (hCSE) responsive fluorescent dyes, which can be used for rapid, high signal-to-noise ratio recognition of hCSE enzymes at the cellular and in vivo levels. This fluorescent dye can serve as a substrate for hCSE enzymes; the products catalyzed by hCSE enzyme cleavage carry thiol functional groups with strong nucleophilic properties, allowing for further intramolecular cyclization reactions that release fluorescent signals.

[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] Wherein, R1 and R2 are each independently selected from one of hydrogen atom, methyl, ethyl, propyl, butyl, and pentyl;

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

[0009] In a further preferred embodiment of the above-described technical solution, the fluorescent dye is selected from one of the following: Cy3, Cy5, Cy7 cyanine dyes, FITC, Alexa Fluor 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, a further preferred option is:

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

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

[0013] The near-infrared dyes mentioned above, such as IRDye 800CW and IRDye 700DX;

[0014] The photosensitizing drug is selected from compounds such as porphyrins, phthalocyanines, and niloblues.

[0015] In a further preferred embodiment of the technical solution described above, the molecular structure prepared according to the present invention is as follows:

[0016]

[0017] In a further preferred embodiment of the above-described technical solution, the excitation wavelength of the hCSE enzyme-responsive fluorescent dye is 500-630 nm, and the emission wavelength is 650-850 nm. Even more preferably, 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 described in this invention are as follows:

[0019] First, the hCSE enzyme-responsive fluorescent dye described in this invention can rapidly, with a high signal-to-noise ratio, and selectively image hCSE enzymes on live cells. Previously reported activating hCSE enzyme fluorescent probes, such as CSEP, showed only about a 1.5-fold increase in fluorescence signal after 0.5 hours of incubation with live cells (Analytical Chemistry, 2022, 94, 1203). In contrast, the fluorescent dye described in this invention showed an approximately 10-fold increase in fluorescence signal after 0.5 hours of incubation with live cells (experimental group). Furthermore, in the control group, live cells pretreated with the hCSE enzyme inhibitor DL-propargylglycine (PAG) and then incubated with the fluorescent dye described in this invention showed a fluorescence signal intensity approximately 33% lower than the experimental group, indicating that the fluorescent dye described in this invention "lights up" the live cells by specifically recognizing the hCSE enzyme.

[0020] Second, the fluorescent dye described in this invention is the first fluorescent probe that can be used for in vivo imaging of hCSE enzymes. Existing detection methods, including the recently reported fluorescent probe CSEP, have failed to image hCSE enzymes at the in vivo level. The hCSE enzyme-responsive fluorescent dye described in this invention, administered via intratumoral injection at a small dose (5 nmol), can "illuminate" xenograft breast cancer within a short time (approximately 60 minutes), with the average fluorescence signal of the tumor increasing by approximately 6 times (experimental group). In the control group, after administering the hCSE enzyme inhibitor DL-propyneglycine (PAG) (100 nmol) via peritumoral injection, followed by intratumoral injection of the fluorescent dye described in this invention (5 nmol), the average fluorescence signal intensity at the tumor site was approximately 50% lower than that in the experimental group, indicating that the fluorescent dye described in this invention "illuminates" xenograft breast cancer by specifically recognizing and responding to hCSE enzymes.

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

[0022] (1) Take intermediate 2 with general formula 2, add anhydrous dichloromethane and piperidine mixed solution to react fully, dilute with dichloromethane, wash with sodium citrate buffer and saturated saline, dry, filter, concentrate to obtain a light yellow oily substance.

[0023] (2) Add anhydrous dichloromethane to the oily substance prepared in step (1), and under alkaline conditions at -5 to 5°C, add p-nitrophenyl chloroformate and stir thoroughly. Then, restore the reaction to room temperature and continue the reaction. Add a photosensitive dye or photosensitive drug to the reaction solution and stir thoroughly. Concentrate and purify to obtain a pure product.

[0024] (3) Add a mixed solution of dichloromethane and trifluoroacetic acid to the pure product prepared in step (1), and then react, concentrate, crystallize and purify to obtain an hCSE enzyme-responsive fluorescent dye with general formula I.

[0025]

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

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

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

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

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

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

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

[0033] A third aspect of this application is to protect the use of the fluorescent dye for non-disease diagnostic and therapeutic purposes, and in biological and medical diagnostic preparations related to hCSE enzymes.

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

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

[0036] The hCSE enzyme-responsive fluorescent dye of this invention can rapidly, with a high signal-to-noise ratio, and selectively image hCSE enzymes on living cells. The hCSE enzyme-responsive fluorescent dye of this invention is the first fluorescent probe that can be used for in vivo identification and imaging of hCSE enzymes, which has important biological and medical significance for studying hCSE enzymes 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 yet been fully explored, mainly due to the lack of effective research tools. The hCSE enzyme-responsive fluorescent dyes described in this invention can provide a class of efficient tools for the study of hCSE enzyme-related signaling pathways and the diagnosis and treatment of related diseases. Attached Figure Description

[0038] Figure 1 The NMR of the fluorescent probe DCM-hCSE described in this invention 1 H-chart;

[0039] Figure 2 The NMR of the fluorescent probe DCM-hCSE described in this invention 13 C spectrum;

[0040] Figure 3 This is the high-resolution mass spectrum of the fluorescent probe DCM-hCSE described in this invention;

[0041] Figure 4 This invention evaluates the cytotoxicity of the fluorescent probe DCM-hCSE.

[0042] Figure 5 The results are time-fluorescence imaging of the fluorescence probe DCM-hCSE recognizing the endogenous hCSE enzyme in MDA-MB-231 cells as described in this invention.

[0043] Figure 6 These are fluorescence imaging images of the fluorescent probe DCM-hCSE described in this invention recognizing the endogenous hCSE enzyme in cancer cells MCF-7 and MDA-MB-231;

[0044] Figure 7 The fluorescent probe DCM-hCSE described in this invention is used for fluorescence imaging of xenograft triple-negative breast cancer. Detailed Implementation

[0045] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

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

[0047] Example 1

[0048] The synthetic route for the fluorescent probe DCM-hCSE is shown below:

[0049]

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

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

[0052] L-homocysteine ​​(5.3670 g, 20 mmol) was dissolved in 160 mL of 1,4-dioxane in a 500 mL three-necked flask. 180 mL of 10% NaHCO3 solution was added to the reaction flask, and the flask was placed in an ice-water bath. Di-tert-butyl dicarbonate (9.6030 g, 44 mmol) was added at approximately 0 °C. The mixture was allowed to return to room temperature and stirred overnight. For post-treatment, the pH of the reaction solution was adjusted to 4 with NaHSO4 solution, and the solution was extracted with ethyl acetate (2 × 50 mL). The organic layers were combined, washed with saturated brine (2 × 50 mL), dried over anhydrous Na2SO4, filtered, and concentrated to obtain a white solid product.

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

[0054] Compound 1-1 (4.6858 g, 10 mmol) was dissolved in 35 mL of DMF in a 100 mL flask. Cesium carbonate (3.2582 g, 10 mmol) was added, and the mixture was cooled to 0 °C in an ice-water bath. MeI (3.6000 g, 25 mmol) was added dropwise to the flask, and the reaction was allowed to proceed overnight. The solvent was removed under vacuum, and the mixture was diluted with ethyl acetate and washed with water and saturated brine, respectively. The mixture was dried over anhydrous Na2SO4 and filtered. The organic layer was concentrated and purified by column chromatography to give 4.21 g of a 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 compounds 1-3

[0058] Compounds 1-2 (4.2214 g, 8.5 mmol) were dissolved in 30 mL of tetrahydrofuran in a 100 mL reaction flask. Under nitrogen protection, tributylphosphine (2.5779 g, 12.75 mmol) was added, and the mixture was stirred for 15 min. Then, 30 mL of water was added, and the reaction was allowed to proceed overnight. The tetrahydrofuran was removed by rotary evaporation, and the concentrated solution was diluted with ethyl acetate. The solution was washed with NaHSO4 and saturated brine, dried over anhydrous Na2SO4, filtered, and the organic layer was concentrated. The solution was purified by column chromatography to give 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 dissolved in 60 mL of dichloromethane in a 250 mL reaction flask. Triphenylphosphine (2.8850 g, 11 mmol) and hexachloroethane (2.7600 g, 11 mmol) were added under nitrogen protection. The reaction was stopped after 4 h. Saturated sodium bicarbonate solution was added, and the mixture was extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give 2.55 g of 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] Compounds 1-3 (1.8 g, 7.22 mmol) were placed in a 50 mL reaction flask. Under nitrogen protection, 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 sequentially. The mixture was reacted overnight at room temperature. After finishing, the product was diluted with ethyl acetate (50 mL) and washed with water and saturated brine, respectively. The product was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (petroleum ether: ethyl acetate = 4:1) to give 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 carried out for 1 hour, the reaction solution was concentrated, and diluted with dichloromethane. The solution was washed successively with sodium citrate buffer (pH = 5) and saturated saline, 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). The mixture was in an ice-water bath for 10 minutes at approximately 0°C. Anhydrous N,N-diisopropylethylamine (5 equivalents) was added dropwise. After stirring in the ice-water bath for 1 hour, the mixture was allowed to return to room temperature and reacted for another hour to obtain compound 3-3. Fluorescent dye DCM (133.92 mg, 349.24 μmol) was then added, and the reaction proceeded overnight. The mixture was concentrated, slurryed, and purified 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 prepared pure Boc-DCM-hCSE. The mixture was then reacted thoroughly, 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 N5O7S + ,660.2486).

[0073] Example 2

[0074] Cytotoxicity evaluation of DCM-hCSE.

[0075] After incubating MDA-MB-231 cells with 0-20 μM dye DCM-hCSE for 24 hours, the cytotoxicity of DCM-hCSE was determined using the thiazolyl 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 even at high concentrations, the dye DCM-hCSE has no obvious cytotoxicity and high biocompatibility, and can be safely used in cells and in vivo to detect and identify hCSE enzymes.

[0077] Example 3

[0078] Time-fluorescence intensity statistics of DCM-hCSE recognition of endogenous hCSE enzyme in MDA-MB-231 cells. MDA-MB-231 breast cancer cell culture medium was prepared, and MDA-MB-231 cells were cultured in laser confocal microscopy dishes. After the cell abundance reached 60%, DCM-hCSE (5 μM) was added, and the cells were cultured further at 37℃ under a 5% CO2 atmosphere. Cell fluorescence images were acquired at different time points (0, 15, 30, 45, 60, 75, 90, 105, 120, 135, and 150 min) using a STELLARIS 5 laser confocal scanning fluorescence microscope.

[0079] The results are as follows Figure 5 As shown in Figure A, significant fluorescence enhancement occurred in MDA-MB-231 breast cancer cells within a short period (approximately 60 minutes), demonstrating that the dye DCM-hCSE can rapidly recognize and respond to hCSE enzymes in living cells. Figure B shows that the fluorescence of MDA-MB-231 cells increased more than tenfold after incubation with the dye DCM-hCSE for 2 hours, proving that DCM-hCSE can be effectively used for hCSE enzyme recognition imaging in living cells.

[0080] Example 4

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

[0082] Medium was prepared for breast cancer cells MCF-7 and MDA-MB-231, and the cells were cultured in laser confocal imaging dishes. Once the cell abundance reached 60%, the experimental group cells were added with DCM-hCSE (5 μM) and incubated at 37°C and 5% CO2 for 2 h. The control group cells were first incubated with the hCSE enzyme inhibitor DL-propargylglycine (PAG, 1 mM) for 30 min, then added with DCM-hCSE (5 μM) and incubated at 37°C and 5% CO2 for 2 h. Cells were washed twice with phosphate-buffered saline (PBS), and fluorescence images were acquired using a STELLARIS 5 laser confocal scanning fluorescence microscope.

[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, by about 33%, proving that the dye DCM-hCSE can be used for the specific recognition of hCSE enzyme in breast cancer cells.

[0084] Example 5

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

[0086] 6-week-old BABL / c nude mice were subcutaneously injected with MDA-MB-231 cells (5*10). 6 (in PBS), the tumor grew to 200 mm. 3The treatment was performed on both sides. In the experimental group, mice received intratumoral injection of DCM-hCSE (100 μM, dispersed in 50 μL PBS). In the control group, mice were first injected adjacent to the tumor with the hCSE enzyme inhibitor DL-propargylglycine (PAG, 2 mM, dispersed in 50 μL PBS), and two hours later, DCM-hCSE (100 μM, dispersed in 50 μL PBS) was injected intratumorally. Near-infrared fluorescence imaging of the tumor and the entire mouse body at different time points (0, 15, 30, 45, 60, 75, 90, 105, 120, 135, and 150 min) was acquired using a PerkinElmer IVIS spectrometer.

[0087] The results are as follows Figure 7 As shown in Figure A, the xenograft breast cancer in the experimental group exhibited significant fluorescence enhancement within a short period (approximately 60 minutes). In the control group, the average fluorescence signal intensity at the tumor site was significantly lower than that in the experimental group, demonstrating that the dye DCM-hCSE can rapidly and specifically recognize and respond to hCSE enzymes in vivo. Figure B shows that compared to the experimental group, the average fluorescence signal intensity at the tumor site in the control group was approximately 50% lower, demonstrating that the dye DCM-hCSE can be effectively used for in vivo recognition imaging of hCSE enzymes.

[0088] The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A class of human cystathionine γ-lyase-responsive fluorescent dyes, characterized in that: The dye has the following DCM-hCSCE structure: The fluorescent dye has an excitation wavelength of 500-630 nm and an emission wavelength of 650-850 nm.

2. The method for preparing the fluorescent dye as described in claim 1, characterized in that: Includes the following steps: (1) Take intermediate 3-1, add anhydrous dichloromethane and piperidine mixed solution to react fully, dilute with dichloromethane, wash with sodium citrate buffer and saturated saline, dry, filter, concentrate to obtain a light yellow oily substance; (2) Add anhydrous dichloromethane to the oily substance prepared in step (1), and under alkaline conditions at -5 to 5°C, add p-nitrophenyl chloroformate and stir thoroughly. Then, restore the reaction to room temperature and continue the reaction. Add fluorescent dye to the reaction solution and stir thoroughly. Concentrate and purify to obtain the pure product. (3) Add a mixed solution of dichloromethane and trifluoroacetic acid to the pure product prepared in step (2), and then react, concentrate, crystallize and purify to obtain the fluorescent dye as described in claim 1; 。 3. The method for preparing the fluorescent dye according to claim 2, characterized in that: In step (1), the volume ratio of anhydrous dichloromethane to piperidine in the mixed solution 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 intermediate 3-1 to alkali is 1:(4-10); the molar ratio of intermediate 3-1 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 fluorescent dye to intermediate 3-1 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 mixed solution of dichloromethane and trifluoroacetic acid is (1-4):

1.

9. The application of the fluorescent dye as described in claim 1 for non-disease diagnosis and treatment purposes; the application is the fluorescence detection and fluorescence imaging of hCSE enzyme, and the development of fluorescence imaging agents.

Images