A viscosity and glycosidase dual-locked fluorescent probe, and a preparation method and application thereof

Abstract

The present application relates to the technical field of fluorescent probe, and discloses a fluorescent probe locked by viscosity and glycosidase, and a preparation method and application thereof.The present application provides a compound, the fluorescence emission intensity of which is enhanced with the enhancement of the solution viscosity; meanwhile, after incubation with beta-galactosidase, the fluorescence intensity of the compound at 640nm is enhanced.Therefore, in the early diagnosis imaging of ovarian cancer, the compound can be used as a fluorescent probe locked by viscosity and glycosidase, and is used for accurate imaging of ovarian cancer.The fluorescent probe of the present application greatly improves the selectivity of the probe by using a double activation mechanism, effectively avoids non-specific activation to normal tissues, and is particularly suitable for targeted imaging of specific pathological environments such as tumors.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probe technology, and in particular to a fluorescent probe with both viscosity and glycosidase dual locking, its preparation method, and its application. Background Technology

[0002] Ovarian cancer is one of the most serious malignant tumors threatening women's health. The abnormal proliferation of ovarian cancer cells leads to an increase in extracellular matrix (ECM) components, particularly the enrichment of molecules such as hyaluronic acid and collagen, resulting in increased viscosity of the tumor microenvironment. High viscosity not only affects the spread and migration of tumor cells but may also increase resistance to blood and lymphatic flow, restricting the delivery of nutrients and oxygen, creating a hypoxic environment, and thus promoting enhanced invasiveness and malignant metastasis of tumor cells. Furthermore, ovarian cancer cells are often accompanied by increased activity of various glycosidases (such as β-galactosidase and β-glucosidase). Glycosidases can break down glycosylated components in the extracellular matrix, making it easier for cancer cells to invade surrounding tissues from the primary tumor site, thereby promoting the spread and metastasis of cancer cells. The activity of glycosidases is closely related to the growth and metabolism of cancer cells.

[0003] Because ovarian cancer has an insidious onset, effective screening and early diagnosis measures are currently lacking. Monitoring ovarian cancer markers and lesion microenvironment parameters is an effective means of accurate diagnosis and treatment of cancer. Accurately distinguishing between cancerous and normal tissue is crucial for the early diagnosis and treatment of ovarian cancer.

[0004] Fluorescent probes are tools that can detect and measure chemical and biological changes in biomolecules, cells, and tissues using fluorescence signals. Molecular imaging based on fluorescence technology is a non-invasive detection method with advantages such as in-situ detection, visualization, and high spatiotemporal resolution, enabling the detection of physical parameters and biomarkers in the microenvironment of lesions.

[0005] Although numerous fluorescent probes for the targeted detection of ovarian cancer have been reported, most of the reported fluorescent probes to date only respond to one target. This mode of action easily generates false positive signals, leading to misdiagnosis. For example, patent application CN115028652A discloses a highly photosensitizing fluorescent probe for photodynamic diagnosis and treatment of ovarian cancer. This probe can bind to lysophosphatidylcholine, a biomarker of ovarian cancer, thereby achieving selective imaging of ovarian cancer cells. However, the probe provided in this patent only responds to lysophosphatidylcholine as a single target, which easily leads to misdiagnosis. Summary of the Invention

[0006] To address the imaging challenges of ovarian cancer biomarkers and further improve imaging accuracy, this invention provides a viscosity- and glycosidase-locked fluorescent probe, its preparation method, and its application.

[0007] The specific technical solution of this invention is as follows: This invention provides a fluorescent probe with both viscosity and glycosidase dual locking, the structure of which is shown in formula (1):

[0008] The compound, with the structural formula shown in formula (1), exhibits increased fluorescence emission intensity with increasing solution viscosity; simultaneously, its fluorescence intensity at 640 nm is enhanced after incubation with β-galactosidase. Therefore, in early diagnostic imaging of ovarian cancer, this compound can serve as a viscosity- and glycosidase-locked fluorescent probe for precise imaging of ovarian cancer. This dual activation mechanism significantly improves probe selectivity and effectively avoids non-specific activation of normal tissues, making it particularly suitable for targeted imaging of specific pathological environments such as tumors.

[0009] This invention also provides a method for preparing a fluorescent probe with both viscosity and glycosidase dual locking, comprising the following steps: Step S1: Dissolve compound (2) and 2,3,4,6-tetraacetoxy-α-D-pyranogalactosyl bromide in a solvent, add cesium carbonate, and react to obtain compound (3); Step S2: Dissolve compounds (3) and (4) in a solvent and react to obtain compound (5); Step S3: Dissolve compound (5) in a solvent, add saturated methanol-sodium methoxide solution, and react 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 (5) is: The structural formula of compound (1) is:

[0010] As a preferred embodiment of the above preparation method, in step S1, the solvent is acetonitrile.

[0011] As a preferred embodiment of the above preparation method, in step S1, the reaction is carried out under an inert gas atmosphere.

[0012] As a preferred embodiment of the above preparation method, in step S1, the reaction time is 6 to 10 hours.

[0013] As a preferred embodiment of the above preparation method, in step S1, the molar ratio of compound (2), 2,3,4,6-tetraacetoxy-α-D-pyranosyl bromide, and cesium carbonate is 1:1 to 3:4 to 6.

[0014] As a preferred embodiment of the above preparation method, in step S2, the reaction is a reflux reaction, and the reaction time is 6 to 10 hours.

[0015] As a preferred embodiment of the above preparation method, in step S2, the solvent is ethanol.

[0016] As a preferred embodiment of the above preparation method, in step S2, the molar ratio of compound (4) to compound (3) is 1:1 to 2.

[0017] As a preferred embodiment of the above preparation method, in step S3, the reaction temperature is 20–40°C and the reaction time is 10–30 minutes.

[0018] Further, in step S1, the post-reaction processing is as follows: the reaction solution is extracted with ethyl acetate, the organic phase is concentrated under reduced pressure, and column chromatography is performed using a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 2:1 as the eluent. The eluent containing the target product is collected and concentrated under reduced pressure to obtain compound (3).

[0019] Further, in step S2, the post-reaction processing is as follows: the reaction solution is directly concentrated under reduced pressure, and column chromatography is performed using a mixed solution of dichloromethane and methanol with a volume ratio of 20:1 as the eluent. The eluent containing the target product is collected, concentrated under reduced pressure, and compound (5) is obtained.

[0020] Further, in step S3, the post-reaction processing is as follows: the reaction solution is directly concentrated under reduced pressure, and column chromatography is performed using a mixed solution of dichloromethane and methanol with a volume ratio of 15:1 as the eluent. The eluent containing the target product is collected, concentrated under reduced pressure, and compound (1) is obtained.

[0021] Based on the above, the present invention provides an application of a fluorescent probe in ovarian cancer biomarker imaging.

[0022] Compared with the prior art, the present invention has the following technical effects: 1. This invention provides a compound with the structural formula shown in formula (1), whose fluorescence emission intensity increases with increasing solution viscosity; simultaneously, its fluorescence intensity at 640 nm is enhanced after incubation with β-galactosidase. Therefore, in early diagnostic imaging of ovarian cancer, this compound can serve as a viscosity- and glycosidase-locked fluorescent probe for precise imaging of ovarian cancer. The dual activation mechanism greatly improves the selectivity of the probe, effectively avoiding non-specific activation of normal tissues, making it particularly suitable for targeted imaging of specific pathological environments such as tumors.

[0023] 2. By marking the viscosity of the signal and glycosidase, its dual-locking significantly reduces non-specific background signals, ensuring that fluorescence is generated only under specific pathological conditions. Compared to single-activation probes, dual-locking probes are more sensitive, helping to accurately detect subtle changes in the microenvironment, which is beneficial for early diagnosis and real-time monitoring.

[0024] 3. By limiting the activation conditions of the probe to a pathological environment with high viscosity and coexistence of glycosidases, the activation of non-target tissues can be reduced, the toxic effects of the probe in non-lesion areas can be decreased, and higher safety can be ensured, making it particularly suitable for in vivo imaging and targeted therapy. Attached Figure Description

[0025] Figure 1 The above is the 1H NMR spectrum of compound (1) prepared in Example 3 of this invention.

[0026] Figure 2 This is the mass spectrum of compound (1) prepared in Example 3 of this invention.

[0027] Figure 3 The images show the absorption and emission spectra of compound (1) prepared in Example 3 of this invention before and after activation by β-galactosidase. The left image shows the absorption spectrum before and after activation, and the right image shows the emission spectrum before and after activation.

[0028] Figure 4 The images show the fluorescence absorption and emission spectra of compound (1) prepared in Example 3 of this invention at different viscosities. The left image shows the absorption spectrum at different viscosities, and the right image shows the emission spectrum at different viscosities.

[0029] Figure 5 The fluorescence emission spectra of compound (1) prepared in Example 3 of this invention in different systems including PBS, β-galactosidase, 30% Gly, and β-galactosidase + 30% Gly.

[0030] Figure 6 The images show the fluorescence absorption and emission spectra of compound (1) prepared in Example 3 of this invention in different solvent systems. The left image shows the absorption spectrum in different solvent systems, and the right image shows the emission spectrum in different solvent systems.

[0031] Figure 7 The results of the MTT assay for compound (1) prepared in Example 3 of this invention are shown.

[0032] Figure 8 Confocal fluorescence imaging of compound (1) prepared in Example 3 of this invention in HEK293 cells, HeLa cells and SKOV-3 cells. Detailed Implementation

[0033] The present invention will be further described below with reference to embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0034] In this embodiment of the invention, the structural formula of compound (1) is: 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 (5) is:

[0035] Among them, compound (4) is a publicly disclosed compound, and its preparation method can be found in the following reference: Zhang X, Huang Y, Han X, et al. Evaluating the protective effects of mitochondrial glutathione oncerebral ischemia / reperfusion injury via near-infrared fluorescence imaging[J]. Analytical chemistry, 2019, 91(22): 14728-14736.

[0036] Example 1 Preparation of compound (3) In this embodiment, compound (3) was prepared. The structural formula of compound (3) is as follows:

[0037] This embodiment is prepared according to the following steps: At room temperature, 500 mg of compound (2) (2.3 mmol) and 1894 mg of 2,3,4,6-tetraacetoxy-α-D-pyranogalactosyl bromide (4.6 mmol) were added to a round-bottom flask, along with 50 mL of acetonitrile as the reaction solvent. Then, 3752 mg of cesium carbonate (11.5 mmol) and 1635 mg of anhydrous sodium sulfate (11.5 mmol) were added. A stir bar was added to the flask, and the reaction was stirred at 500 rpm and 26 °C for 7 hours under a nitrogen atmosphere. The reaction progress was monitored by TLC. After the reaction, the product was extracted with ethyl acetate and water. The supernatant was purified by silica gel column chromatography (elution: ethyl acetate: petroleum ether = 1:2 (v / v)). The eluent containing the target product (the first compound obtained by column chromatography) was collected and concentrated under reduced pressure. The solvent was evaporated using a rotary evaporator, and the product was further dried in an oil pump to obtain a pale yellow solid (1071 mg, 1.96 mmol), which is compound (3), with a yield of 85%.

[0038] The obtained pale yellow solid was subjected to NMR analysis, and the results are as follows: 1H NMR (600MHz, Chloroform-d) δ: 10.03 (s, 1H), 7.35 (s, 1H), 5.51 (dd, J = 10.4, 8. 0Hz,1H),5.38(d,J=3.4Hz,1H),5.10–5.04(m,1H),4.81(d,J=8.0Hz,1H),4.10– 4.04(m,2H),3.80(td,J=6.9,1.3Hz,1H),3.27(q,J=5.0,4.5Hz,4H),2.81–2.67 (m,4H),2.20(s,3H),2.14(s,3H),2.01(s,3H),1.96(s,3H),1.94–1.86(m,4H).

[0039] In this embodiment, compound (2) is:

[0040] Example 2 Preparation of compound (5) In this embodiment, compound (5) was prepared. The structural formula of compound (5) is as follows:

[0041] Compound (3) (100 mg, 0.44 mmol) and compound (4) (292 mg, 0.53 mmol) were dissolved in 20 mL of ethanol and refluxed at 80 °C for 5 h. The reaction was monitored by TLC, with dichloromethane:methanol = 15:1 (v / v) as the developing solvent. Compound (5) has excellent water solubility, resulting in significant water loss during extraction. Therefore, the solvent was removed by rotary evaporation and then further dried in an oil pump to obtain a purple solid, which was compound (5).

[0042] The obtained dark blue-purple solid was subjected to NMR analysis, and the results are shown in the figure. Figure 1 The spectral data are as follows: 1H NMR (400MHz, Methanol-d4) δ: 8.86 (d, J = 15.5Hz, 1H), 8.34–8.29 (m, 1H), 8.13–8.04 (m, 2H), 7.80–7.74 (m, 2H), 7.70 (ddd, J = 8.4, 6.8, 1.3Hz, 1H), 7.57 (ddd, J = 8.1, 6.9, 1.1Hz, 1H), 7.06 (d, J = 15.6Hz, 1H), 4.78 (d ,J=7.7Hz,1H),4.05–3.98(m,4H),3.94(dd,J=3.5,1.0Hz,1H),3.73–3.64(m,2H),3.58(dd,J=11.0,6.1Hz ,1H),3.49–3.41(m,5H),3.21–3.13(m,1H),2.84–2.73(m,3H),2.06(d,J=14.1Hz,6H),1.97–1.86(m,3H).

[0043] In this embodiment, compound (3) is:

[0044] Compound (4) is:

[0045] Example 3 Preparation of compound (1) In this embodiment, compound (1) was prepared. The structural formula of compound (1) is as follows:

[0046] Compound (5) was dissolved in 20 mL of methanol, and 6 drops of saturated sodium methoxide-methanol solution were added. The reaction was carried out at room temperature for 20 min. The reaction was monitored by TLC, with the polarity being dichloromethane:methanol = 15:1. The solvent methanol was removed by rotary evaporation, and the reaction solution was purified by column chromatography (dichloromethane:methanol = 12:1 (v / v)). The eluent containing the target product was collected and concentrated under reduced pressure. The solvent was removed by rotary evaporation, and then further dried in an oil pump to obtain a deep blue-purple solid, which was compound (1), with a yield of 85%. Compound (1) was denoted as Vβ-CM.

[0047] The obtained dark blue-purple solid was subjected to NMR analysis, and the results are shown in the figure. Figure 1 The spectral data are as follows: 1H NMR (400MHz, Methanol-d4) δ: 8.86 (d, J = 15.5Hz, 1H), 8.34–8.29 (m, 1H), 8.13–8.04 (m, 2H), 7.80–7.74 (m, 2H), 7.70 (ddd, J = 8.4, 6.8, 1.3Hz, 1H), 7.57 (ddd, J = 8.1, 6.9, 1.1Hz, 1H), 7.06 (d, J = 15.6Hz, 1H), 4.78 (d ,J=7.7Hz,1H),4.05–3.98(m,4H),3.94(dd,J=3.5,1.0Hz,1H),3.73–3.64(m,2H),3.58(dd,J=11.0,6.1Hz ,1H),3.49–3.41(m,5H),3.21–3.13(m,1H),2.84–2.73(m,3H),2.06(d,J=14.1Hz,6H),1.97–1.86(m,3H).

[0048] The obtained dark blue-purple solid was analyzed by mass spectrometry, and the results are shown in the figure. Figure 2 .

[0049] In this embodiment, compound (5) is:

[0050] Example 4 A certain amount of compound (1) prepared in Example 3 was weighed and prepared into a 1 mM probe stock solution using dimethyl sulfoxide. 2 μL of the stock solution was added to 398 μL of PBS buffer. β-galactosidase (β-Gal) was added, and the mixture was incubated at 37°C for 30 min. The absorbance was measured using a UV spectrophotometer. The sample was then transferred to a 96-well plate, and the fluorescence absorption and emission spectra of compound (1) were measured. Simultaneously, the fluorescence spectrum of the unincubated compound (1) (the stock solution containing the buffer) was measured. Figure 3 . Figure 3In the diagram, Vβ-CM represents the fluorescence spectrum of compound (1) before it is excited by β-galactosidase, and Vβ-CM+β-gal represents the fluorescence spectrum of compound (1) after it is excited by β-galactosidase.

[0051] The results showed that the fluorescence intensity at 640 nm was enhanced after incubation with β-galactosidase. This indicates that the glycosidic bond of the probe was hydrolyzed by β-Gal, and the fluorescence of compound (1) was released.

[0052] Example 5: Fluorescence emission spectra of compound (1) under different viscosity systems. A certain amount of compound (1) prepared in Example 3 was weighed and prepared into a 1 mM probe stock solution using dimethyl sulfoxide. 1 μL of the probe stock solution was transferred using a pipette. Ten viscosity gradients were set in a PBS / glycerol mixture, with the proportion of glycerol ranging from 10% to 100% (volume percentage). Ten of the above 1 μL probe stock solutions were dissolved in ten PBS / glycerol mixtures, mixed thoroughly, and prepared into a fluorescent probe solution with a final concentration of 5 μM. The fluorescence spectrum was then used for spectral determination. See [image of fluorescence spectrum]. Figure 4 .

[0053] The results showed that the fluorescence emission intensity of compound (1) increased with increasing solution viscosity.

[0054] Example 6: Spectroscopic property determination of compound (1) in different systems. A certain amount of compound (1) prepared in Example 3 was weighed and prepared into a 1 mM probe stock solution using dimethyl sulfoxide. 1 μL of the fluorescent probe stock solution of compound (1) was pipetted into PBS, PBS + Gly (30%), PBS + β-Gal (8 U / mL), and PBS + Gly (30%) + β-Gal (8 U / mL), respectively, to prepare a fluorescent probe solution with a final concentration of 5 μM. The spectra were measured. Gly is glycerol, and 30% is a volume percentage. The fluorescence spectrum is shown in [reference needed]. Figure 5 .

[0055] Fluorescence assay results showed that under 590 nm excitation, the probe exhibited weak fluorescence intensity in PBS buffer (10 mM, pH = 7.4), indicating that under physiological conditions, the probe was in an unexcited state, and the fluorescent probe only showed weak fluorescence of the molecule itself. After the addition of β-Gal, the probe molecule was excited due to the reaction between the fluorescent probe and β-Gal, thus showing an increase in fluorescence intensity. Secondly, after adding 30% glycerol to increase the environmental viscosity, the free rotation within the probe was hindered, reducing the loss of non-radiative energy, which also led to an increase in fluorescence intensity. When β-Gal was present in the system and it was in a high-viscosity environment, the fluorescence effect was further enhanced due to the dual response of the probe to both viscosity and β-Gal. This indicates that compound (1) can respond to both viscosity and β-Gal simultaneously.

[0056] Example 7: Spectroscopic property determination of compound (1) in different solvents. A certain amount of compound (1) prepared in Example 3 was weighed and prepared into a 1 mM probe stock solution using dimethyl sulfoxide. 1 μL of the probe stock solution was transferred by pipette and dissolved in 199 μL of 12 different solvents: acetonitrile, methanol, dichloromethane, ethyl acetate, dimethyl sulfoxide, acetic acid, chloroform, ethanol, glycerol, dimethylformamide, and water. The solutions were thoroughly mixed to prepare a 5 μM fluorescent probe solution. The fluorescence spectrum was then measured. (See fluorescence spectrum below.) Figure 6 .

[0057] The results show that the maximum absorption wavelength of the fluorescent probe Vβ-CM hardly changes or the degree of red-shift or blue-shift is negligible under different solvent systems. Among them, the absorbance of the fluorescent probe increases significantly only in glycerol, showing a visible color enhancement effect. The above results indicate that compound (1) has good stability in different solvent systems and is only greatly affected by the viscosity of glycerol.

[0058] Example 8 MTT cell experiment of compound (1).

[0059] The MTT assay was used to detect the cytotoxicity of compound (1). The principle is that mitochondria in living cells contain succinate dehydrogenase and cytochrome C. Thiazol blue (MTT) reacts with these two enzymes to form water-insoluble blue-purple formazan crystals, which then deposit in the cells. The blue-purple formazan crystals in the cells were subsequently dissolved in DMSO, and the absorbance was measured at 550 nm using a microplate reader. Meanwhile, dead cells do not contain succinate dehydrogenase and cannot form blue-purple formazan with MTT. Therefore, cell viability is directly proportional to the deposition of blue-purple formazan; a higher OD value indicates a higher cell viability.

[0060] The detection steps are as follows: First, HELa cells were seeded at a density of approximately 2 × 10⁴ cells / well in 96-well cell culture plates and cultured for 24 h in a 37°C cell culture incubator containing 5% CO₂. Fetal bovine serum and appropriate amounts of antibodies (penicillin and streptomycin) were added to the culture medium. Then, different concentrations of compound (1) (0–50 μM) were added and incubated for 24 h. 50 μL of MTT (1 mg / mL PBS buffer) was added to each well, and incubation was continued at 37°C and 5% CO₂ for another 4 h. The culture medium was then removed, and 150 μL of DMSO was added to each well. After shaking for 5 min, the absorbance was measured at 590 nm.

[0061] The MTT test results are shown below. Figure 7 .

[0062] The results showed that compound (1) had low biotoxicity.

[0063] Confocal fluorescence imaging of compound (1) in Example 9 This embodiment performs confocal fluorescence imaging experiments on compound (1). The cells used in this embodiment are divided into normal cells, non-ovarian cancer tumor cells, and ovarian cancer cells. Among them, HEK293 cells are normal human embryonic kidney cells with low viscosity in the non-tumor cell microenvironment, HeLa cells are non-ovarian cancer human cervical cancer cells with high microenvironment viscosity but no overexpression of β-Gal, while SKOV-3, as an ovarian cancer cell, has high microenvironment viscosity and overexpression of endogenous β-Gal.

[0064] The steps in this embodiment are as follows: Under ultrasound conditions, 100 μL of DMSO solution (10 mM) of compound (1) was added dropwise to 4.9 mL of DMEM medium to prepare a sample solution for cell imaging.

[0065] Approximately 1×10⁵ HEK293, HeLa, and SKOV-3 cells were seeded into confocal culture dishes containing 1 mL of culture medium at 37°C and cultured for 24 h at 37°C in a 5% CO₂ incubator. The sample solution was then added, and the cells were incubated for 30 min at 37°C in a 5% CO₂ incubator. Finally, the cells were washed twice with PBS buffer and imaged using a laser confocal microscope (λex = 552 nm, λem = 600-650 nm). Images were processed using ImageJ with a scale bar of 25 μm, and fluorescence intensity was quantitatively analyzed.

[0066] The results showed that the probe exhibited almost no fluorescence in normal HEK293 cells. However, after stimulating cells with lipopolysaccharide (LPS) to induce an increase in intracellular viscosity, the probe showed some fluorescence in normal cells. Compared to normal HEK293 cells, the probe emitted stronger fluorescence in cancer cells (HeLa), indicating a better response to the high viscosity environment of tumor cells. In SKOV-3 cells, not only was a high-viscosity environment present, but β-Gal was also overexpressed, resulting in the probe displaying clearly visible bright red fluorescence. These experiments demonstrate that the fluorescent probe possesses good intracellular imaging capabilities and can effectively respond to both viscosity and β-Gal simultaneously.

[0067] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A fluorescent probe with both viscosity and glycosidase dual locking, characterized in that: The structural formula is shown in equation (1):

2. A method for preparing a fluorescent probe with both viscosity and glycosidase dual locking, characterized in that: Includes the following steps: Step S1: Dissolve compound (2) and 2,3,4,6-tetraacetoxy-α-D-pyranogalactosyl bromide in a solvent, add cesium carbonate, and react to obtain compound (3); Step S2: Dissolve compounds (3) and (4) in a solvent and react to obtain compound (5); Step S3: Dissolve compound (5) in a solvent, add a saturated methanol-sodium methoxide solution, and react 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 (5) is: The structural formula of compound (1) is:

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

4. The preparation method according to claim 2, characterized in that: In step S1, the reaction is carried out in an inert gas atmosphere for 6 to 10 hours.

5. The preparation method according to claim 2, characterized in that: In step S1, the molar ratio of compound (2), 2,3,4,6-tetraacetoxy-α-D-pyranosyl bromide, and cesium carbonate is 1:1 to 3:4 to 6.

6. The preparation method according to claim 2, characterized in that: In step S2, the reaction is a reflux reaction, and the reaction time is 6 to 10 hours.

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

8. The preparation method according to claim 2, characterized in that: In step S2, the molar ratio of compound (4) to compound (3) is 1:1 to 2.

9. The preparation method according to claim 2, characterized in that: In step S3, the reaction temperature is 20–40°C, and the reaction time is 10–30 minutes.

10. The application of the fluorescent probe as described in claim 1 in ovarian cancer biomarker imaging.

Images