Heat shock protein 90 fluorescent probe based on CY5 carboxylic acid as well as preparation method and application of heat shock protein 90 fluorescent probe

Abstract

The invention relates to the technical field of fluorescent probes, in particular to a CY5 carboxylic acid-based heat shock protein 90 fluorescent probe as well as a preparation method and application thereof, the chemical structural formula of the fluorescent probe is as follows: # imgabs0 #, and the probe shows excellent specificity and can accurately recognize and combine HSP90 protein in cells. Secondly, the introduction of CY5 fluorophore endows the probe with excellent optical properties, including longer excitation and emission wavelength, high quantum yield and good light stability, which effectively reduce background interference and improve signal-to-noise ratio, so that long-time observation and dynamic tracking research become possible.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a heat shock protein 90 fluorescent probe based on CY5 carboxylic acid, a preparation method and an application thereof. Background Art

[0002] Heat shock protein 90 (HSP90) is a protein that plays an important role in cells and is a cytoplasmic protein. It mainly exists in the form of homodimers, its activity is controlled by ATP, and is widely present inside and outside the cells of various species. The activity of HSP90 is regulated by post-translational modifications, such as phosphorylation, acetylation, and SUMOylation, which make it a target of cancer signal transduction mechanisms. Research reports have found that HSP90 is associated with a variety of diseases, including breast cancer, melanoma, lung cancer, liver cancer, bladder cancer, and other cancers, as well as some autoimmune diseases and neurodegenerative diseases.

[0003] HSP90 is mainly divided into the following four subtypes:

[0004] HSP90α (inducible): mainly present in the cytoplasm;

[0005] HSP90β (structural type): also mainly exists in the cytoplasm;

[0006] GRP94: mainly found in the endoplasmic reticulum;

[0007] TRAP-1: Mainly exists in the mitochondrial matrix.

[0008] Given the important role of HSP90 in the occurrence and development of diseases, the development of HSP90 detection tools with strong specificity and high sensitivity has become a research hotspot in recent years. Fluorescence labeling technology, as a non-invasive and highly sensitive detection method, has been widely used in HSP90 research.

[0009] In order to meet different research needs, some companies have developed highly specific, multi-application, and multi-genus HSP90 antibodies. And provide a variety of conjugated direct-labeled antibodies, including fluorescent antibodies. For example, the HSP90 antibody developed by stressmarq (item number: smc-107) can be used in WB, IHC, ICC / IF, IP, ELISA and other reactions, and can react with a variety of species including humans, mice, rats, rabbits, dogs, chickens, fish, etc., and the antibody can provide nearly 17 conjugated direct-labeled antibodies, such as HRP, AP, biotin, streptavidin, FITC, and ATTO series full-spectrum fluorescent dyes. In related studies, fluorescent HSP90 antibodies can be used for specific experiments and detections. For example, through immunofluorescence technology, the distribution and expression of HSP90 in cells can be observed; in some single-molecule studies, fluorescently labeled HSP90 is used to track its dynamic changes and interactions with other molecules.

[0010] However, existing HSP90 fluorescent probes still have some limitations. For example, the HSP90 antibody (Cat. No.: SMC-107) developed by Stressmarq can be applied to a variety of experimental techniques, but its fluorescent labeling process is complicated and the labeling efficiency is unstable, resulting in poor reproducibility of experimental results. In addition, most of the existing HSP90 fluorescent probes on the market are based on traditional fluorophores, such as FITC, which have poor photostability and are prone to photobleaching, which is not conducive to long-term observation and dynamic tracking research.

[0011] On the other hand, some research teams have tried to develop small molecule-based HSP90 fluorescent probes, such as the HSP90 inhibitors described in patent CN103582642A. Although such probes have good cell permeability, they often lack sufficient specificity and are prone to produce false positive signals, affecting the accuracy of experimental results.

[0012] In addition, the performance of existing HSP90 fluorescent probes varies greatly in different cell types, especially in distinguishing the expression levels of HSP90 in normal cells and cancer cells. This problem limits the application of HSP90 probes in cancer diagnosis and research.

[0013] CY5 carboxylic acid belongs to the near-infrared fluorophore family and has the following notable characteristics:

[0014] High photostability: CY5 carboxylic acid can maintain stable fluorescence intensity under long-term irradiation, which is suitable for long-term imaging and detection.

[0015] Low background interference: Its fluorescence emission wavelength is in the near-infrared region, which can effectively reduce the interference of spontaneous luminescence and scattered light of biological samples and improve the detection signal-to-noise ratio.

[0016] Strong penetrating power: Near-infrared light has strong tissue penetrating power, facilitating imaging and detection of deep tissues.

[0017] Structural compatibility: The carboxylic acid group in the CY5 carboxylic acid structure is easy to form stable covalent bonds with biological molecules such as proteins and peptides through chemical modification.

[0018] Therefore, chemical modification can achieve specific binding of CY5 carboxylic acid to HSP90, reduce nonspecific signals, and improve detection accuracy. The high fluorescence intensity and low background interference of CY5 carboxylic acid are helpful for detecting low expression levels of HSP90. High photostability facilitates dynamic research and long-term observation of changes in HSP90.

[0019] The structure of CY5 carboxylic acid is easy to bind to HSP90 through chemical modification to form a stable complex, and the existing chemical modification and fluorescent labeling technologies provide mature technical support for its binding.

[0020] However, there is no report on fluorescent probes that specifically bind to HSP90 using CY5 carboxylic acid. Therefore, developing a fluorescent probe for HSP90 based on CY5 carboxylic acid is not only technologically innovative, but also of great significance for promoting the application of HSP90 in disease diagnosis and research. Summary of the invention

[0021] The present invention aims to solve the technical problems in the prior art, and provides a heat shock protein 90 fluorescent probe based on CY5 carboxylic acid, a preparation method and its application, and achieves the specific binding of CY5 carboxylic acid with HSP90 through chemical modification, thereby improving the sensitivity and specificity of HSP90 detection, reducing false positive signals, and accurately distinguishing the expression level of HSP90 in different cell types. The fluorescent probe of the present invention has a wide range of application prospects, and will provide a more efficient, more sensitive and more specific detection tool for the study of HSP90, and further promote the application of HSP90 in disease diagnosis and treatment.

[0022] The heat shock protein 90 fluorescent probe based on CY5 carboxylic acid is characterized in that the chemical structural formula of the fluorescent probe is as follows:

[0023]

[0024] The preparation method of the heat shock protein 90 fluorescent probe based on CY5 carboxylic acid, the specific reaction formula is as follows:

[0025]

[0026] The following steps are involved:

[0027] (1) SM1 and SM2 are mixed in a ratio of 0.8 to 1.5 molar equivalents;

[0028] (2) adding DMF solvent to the mixture to dissolve it;

[0029] (3) adding an organic base DIPEA, a condensing agent HBTU and HOBt to the solution in sequence;

[0030] (4) stirring the reaction at room temperature for 1 to 2 hours;

[0031] (5) adding water to the reaction solution and extracting with an organic solvent;

[0032] (6) drying the obtained organic phase;

[0033] (7) Purify the target product CY5-HSP90001 by column chromatography or reverse phase C18 chromatography.

[0034] Preferably, in step (1), the molar ratio of SM1 to SM2 is 0.9 to 1.2:1.

[0035] Preferably, the amount of DMF used in step (2) is 5-10 mL / g.

[0036] Preferably, in step (3):

[0037] The amount of DIPEA used is 2 to 5 molar equivalents;

[0038] The amount of HBTU used is 1.5 to 2.5 molar equivalents;

[0039] The amount of HOBt used is 1.8 to 3 molar equivalents.

[0040] Preferably, the organic solvent used in step (5) is ethyl acetate or dichloromethane.

[0041] A heat shock protein 90 fluorescent probe based on CY5 carboxylic acid is prepared by the method described.

[0042] Preferably, the molecular weight of the probe is 904.34, and the probe is stored at 2-8°C.

[0043] The application of the fluorescent probe in detecting heat shock protein 90 in human hepatic stellate cells LX-2, human liver cancer cells HCCLM3 and PLC / PRF / 5 comprises the following steps:

[0044] (1) LX-2, HCCLM3 and PLC / PRF / 5 cells were inoculated into laser confocal microscopy dishes and cultured until the cell density reached 80%;

[0045] (2) Aspirate the culture medium, add 1 mL of 4% paraformaldehyde to each well, and fix at 37°C for 5 to 10 minutes;

[0046] (3) Aspirate the fixative and add 1 mL of 4% paraformaldehyde again, and fix at 37°C for 15 minutes;

[0047] (4) Wash cells three times with PBS, 5 minutes each time;

[0048] (5) Add 1 mL of PBS containing 0.1% TritonX-100 to each well and let stand at room temperature for 15 to 20 minutes to permeabilize the membrane;

[0049] (6) Wash cells with PBS three times, 5 minutes each time;

[0050] (7) Add 1 mL of PBS containing 1% BSA to each well and incubate at room temperature for 30 minutes for blocking;

[0051] (8) Aspirate the blocking solution, add the fluorescent probe diluted with PBS containing 0.01% TritonX-100, and incubate at 4°C overnight;

[0052] (9) Wash cells with PBS three times, 5 minutes each time;

[0053] (10) Add 0.5 mL of 1:1000 diluted DAPI dye to each well and incubate at room temperature for 10 minutes;

[0054] (11) Remove the DAPI dye and wash the cells three times with PBS, 5 minutes each time;

[0055] (12) Add 200 μL of anti-fluorescence quencher to the center of the laser confocal dish;

[0056] (13) The cells were observed and photographed using an Olympus laser confocal microscope.

[0057] Preferably, the working concentration of the fluorescent probe is 2.5 μg / mL, and the dilution ratio is 1:800.

[0058] The present invention creatively designs a multi-component synergistic heat shock protein 90 fluorescent probe based on CY5 carboxylic acid by deeply studying the regulatory mechanism of tumor metabolic network. Among them, the bromine atom in the molecular structure of 3-bromopyruvic acid has a unique electronic effect, which can enhance its binding ability with metabolic enzymes. The pyruvic acid skeleton can effectively intervene in the terminal reaction of the glycolysis pathway. As a histamine receptor antagonist, lonidamine can not only regulate the pH value of the tumor microenvironment, but also regulate energy metabolism by affecting the histamine signaling pathway. Metformin forms a multi-level regulation of tumor energy metabolism by inhibiting mitochondrial complex I and activating the AMPK signaling pathway.

[0059] When these three components are combined in a specific mass ratio (0.1-0.8:0.1-0.8:0.1-0.8), they exhibit significant synergistic effects:

[0060] First, at the molecular level, the action sites of the three components are distributed in key metabolic nodes such as glycolysis, mitochondrial oxidative phosphorylation and AMPK signaling pathway, forming a comprehensive metabolic network regulation.

[0061] Secondly, at the cellular level, the combination simultaneously reduced ATP production, inhibited glucose uptake and lactate production, and significantly weakened the metabolic plasticity of tumor cells.

[0062] Again, at the pharmacodynamic level, the combination showed a strong reversal effect on multiple drug-resistant tumor cell lines, with the reversal multiple reaching up to 9.2 times, which is an effect that cannot be achieved by a single component.

[0063] The outstanding advantages and unexpected technical effects of the present invention include:

[0064] The probe should have the following characteristics: high specificity, high sensitivity, good photostability, excellent cell permeability, and stable performance in different cell types. At the same time, the synthesis method of the probe should be simple and reliable, and easy to mass produce and apply.

[0065] The present invention aims to solve the above technical problems and provides a method for preparing a heat shock protein 90 fluorescent probe based on CY5 carboxylic acid and its application. The method couples the CY5 fluorophore with the HSP90 specific recognition fragment through an optimized synthesis route, and successfully prepares the HSP90 fluorescent probe CY5-HSP90001 with both high specificity and excellent optical properties.

[0066] The CY5-HSP90001 probe of the present invention exhibits significant advantages in multiple aspects. First, the probe exhibits excellent specificity and can accurately identify and bind to HSP90 protein in cells. Second, the introduction of the CY5 fluorophore gives the probe excellent optical properties, including longer excitation and emission wavelengths, high quantum yield and good photostability, which effectively reduce background interference, improve the signal-to-noise ratio, and make long-term observation and dynamic tracking research possible.

[0067] More importantly, the probe of the present invention exhibits stable performance in a variety of cell types. In particular, in the comparative experiments of human hepatic stellate cells LX-2 and human liver cancer cells HCCLM3 and PLC / PRF / 5, the CY5-HSP90001 probe clearly demonstrated its ability to distinguish the expression levels of HSP90 in normal cells and cancer cells. This feature not only provides a powerful tool for the study of the role of HSP90 in the occurrence and development of cancer, but also lays the foundation for the development of cancer diagnosis methods based on HSP90 expression levels.

[0068] In addition, the synthesis method provided by the present invention is simple and reliable, and has good repeatability and scalability. By optimizing the reaction conditions, such as solvent selection, condensation agent dosage and reaction time, high yield and high purity product preparation is achieved. This makes it possible to mass produce and widely apply the CY5-HSP90001 probe.

[0069] In summary, the CY5-HSP90001 probe of the present invention has made significant progress in specificity, sensitivity, optical properties and cell applicability, and effectively solved many technical problems existing in existing HSP90 fluorescent probes. The successful development of this probe provides a powerful new tool for HSP90-related research, disease diagnosis and drug development, and is expected to promote the further development of the HSP90 research field and play an important role in future clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 This is the immunofluorescence of the CY5-HSP90001 probe (blue: nucleus, DAPI; green: cytoplasm, CY5-HSP90001 probe, scale bar 10μm). In LX-2, HCCLM3 and PLC / PRF / 5 cells, the fluorescence signal of the CY5-HSP90001 probe can be well detected. The fluorescence is distributed in the cytoplasm, and its fluorescence intensity in human liver cancer cells HCCLM3 and PLC / PRF / 5 is higher than that in human hepatic stellate cells LX-2.

[0071] Figure 2 is the H NMR spectrum of the fluorescent probe of the present invention. DETAILED DESCRIPTION

[0072] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0073] Example 1

[0074] A heat shock protein 90 fluorescent probe based on CY5 carboxylic acid, the chemical structure of the fluorescent probe is as follows:

[0075]

[0076] Example 2

[0077] The preparation method of the heat shock protein 90 fluorescent probe based on CY5 carboxylic acid, the specific reaction formula is as follows:

[0078]

[0079] The method comprises the following steps:

[0080] First, SM1 and SM2 are mixed at a ratio of 0.8 molar equivalents. The selection of this ratio can ensure the complete reaction while minimizing the generation of by-products. Secondly, DMF solvent is added to the mixture in an amount of 5 mL / g. DMF, as a polar aprotic solvent, can dissolve the reactants well and promote the reaction.

[0081] Then, organic base DIPEA (2 molar equivalents), condensing agent HBTU (1.5 molar equivalents) and HOBt (1.8 molar equivalents) are added to the solution in sequence. The addition of DIPEA can neutralize the acid generated during the reaction and maintain the pH value of the reaction system; the synergistic effect of HBTU and HOBt can effectively activate the carboxyl group and promote the formation of amide bonds.

[0082] Next, the reaction was stirred at room temperature for 1 hour. Carrying out the reaction at room temperature can reduce energy consumption and is also beneficial to the stability of the product. After the reaction is completed, water is added to the reaction solution and extracted with ethyl acetate. The addition of water can terminate the reaction, and ethyl acetate as an organic extractant can effectively separate the target product.

[0083] Finally, the obtained organic phase is dried and purified by column chromatography to obtain the target product CY5-HSP90001. Column chromatography can effectively remove by-products and unreacted raw materials generated in the reaction and improve the purity of the product.

[0084] Preferably, in an embodiment of the present invention, the molecular weight of the obtained product CY5-HSP90001 is 904.34, HNMR (500MHz, CDCl3) δ7.64 (d, J=7.4Hz, 1H), 7.50-7.32 (m, 1H), 7.13 (dd, J=15.6, 7.9Hz, 1H), 6.60 (dd, J=32.0, 13.7Hz, 1H), 5.35 (s, 1H), 4.12-4.03 (m, 1H), 3.80-3.67 (m, 1H), 3.63 (s ,1H),3.49(s,1H),3.16(s,1H),3.00(s,1H),2.80(s,1H),2.43(t,J=7.1Hz,1H),2.27–2.19(m,1H),2.01(d,J=6.3Hz,1H),1.85(s,1H),1.73(s,1H),1.55(s,7H),1.44(d,J=6.5Hz,1H),0.88(t,J=6.8Hz,1H), this precise molecular weight is conducive to subsequent quantitative analysis and application. In addition, storing the product at 2-8℃ can ensure its long-term stability and extend its service life.

[0085] Example 3

[0086] This embodiment provides another method for preparing a heat shock protein 90 fluorescent probe based on CY5 carboxylic acid. The method comprises the following steps:

[0087] First, SM1 and SM2 were mixed in a ratio of 1.2 molar equivalents. The selection of this ratio can ensure the complete conversion of SM2 and improve the yield of the target product. Secondly, DMF solvent was added to the mixture in an amount of 8 mL / g. A larger amount of DMF can increase the solubility of the reactants and is conducive to the uniform reaction.

[0088] Then, organic base DIPEA (3.5 molar equivalents), condensing agent HBTU (2 molar equivalents) and HOBt (2.4 molar equivalents) were added to the solution in sequence. Higher concentrations of reagents can accelerate the reaction rate and shorten the reaction time.

[0089] Next, the reaction was stirred at room temperature for 1.5 hours. Extending the reaction time can ensure that the reaction is fully carried out and improve the product yield. After the reaction is completed, water is added to the reaction solution and extracted with dichloromethane. Dichloromethane as an extraction agent can improve the extraction efficiency, especially for some by-products that are difficult to dissolve in ethyl acetate.

[0090] Finally, the obtained organic phase is dried and purified by reverse phase C18 chromatography to obtain the target product CY5-HSP90001. Reverse phase C18 chromatography has a good separation effect for compounds with strong polarity, and can further improve the purity of the product.

[0091] Example 4

[0092] This embodiment provides a method for preparing a heat shock protein 90 fluorescent probe based on CY5 carboxylic acid, the method comprising the following steps:

[0093] First, SM1 and SM2 were mixed at a ratio of 1.5 molar equivalents. This ratio represents the upper limit of the ratio of SM1 to SM2 in the method of the present invention, which can ensure the complete reaction of SM2 and evaluate the effect of excess SM1 on the reaction.

[0094] Next, DMF solvent was added to the mixture in an amount of 10 mL / g. This amount represents the upper limit of the amount of DMF used in the present invention, which can fully dissolve the reactants and evaluate the effect of the amount of solvent on the reaction.

[0095] Then, organic base DIPEA (5 molar equivalents), condensing agent HBTU (2.5 molar equivalents) and HOBt (3 molar equivalents) were added to the solution in sequence. These amounts represent the upper limits of the amounts of each reagent in the present invention, and the effects of high concentration reagents on the reaction can be evaluated.

[0096] Next, the reaction was stirred at room temperature for 2 hours. This time represents the upper limit of the reaction time in the present invention, which can ensure the complete progress of the reaction and evaluate the effect of long-term reaction on the product.

[0097] Finally, water was added to the reaction solution, and the mixture was extracted with dichloromethane. The obtained organic phase was dried and purified by reverse phase C18 chromatography to obtain the target product CY5-HSP90001.

[0098] Preferably, in the embodiments of the present invention, the purity of the obtained product can reach more than 98%, and the yield is about 85%. Such high-purity and high-yield products can be directly used in subsequent cell experiments without further purification.

[0099] Example 5

[0100] This embodiment provides another method for preparing a heat shock protein 90 fluorescent probe based on CY5 carboxylic acid, the method comprising the following steps:

[0101] First, SM1 and SM2 are mixed at a ratio of 1.1 molar equivalents. This ratio represents the middle value of the ratio of SM1 to SM2 in the method of the present invention, which can balance the reaction efficiency and the utilization rate of raw materials.

[0102] Next, DMF solvent was added to the mixture in an amount of 7.5 mL / g, which represents the middle value of the amount of DMF used in the present invention and can provide a suitable reaction environment.

[0103] Then, organic base DIPEA (3 molar equivalents), condensing agent HBTU (1.8 molar equivalents) and HOBt (2.2 molar equivalents) were added to the solution in sequence. These amounts represent the intermediate values ​​of the amounts of each reagent in the present invention, which can balance the reaction efficiency and reagent cost.

[0104] Next, the reaction was stirred at room temperature for 1.5 hours, which represents the middle value of the reaction time in the present invention, and can improve production efficiency while ensuring complete reaction.

[0105] Finally, water was added to the reaction solution, and the mixture was extracted with a mixed solvent of ethyl acetate and dichloromethane (volume ratio 1:1). This mixed solvent can combine the advantages of the two solvents and improve the extraction efficiency. The obtained organic phase was dried, and the target product CY5-HSP90001 was obtained by a combined purification method of column chromatography and reverse phase C18 chromatography.

[0106] Preferably, in the embodiments of the present invention, the purity of the obtained product can reach more than 99%, and the yield is about 90%. Such high-purity and high-yield products are not only suitable for cell experiments, but also for applications with higher requirements such as in vivo imaging of animals.

[0107] Example 6

[0108] This example provides a method for using the prepared CY5-HSP90001 fluorescent probe in detecting heat shock protein 90 in human hepatic stellate cells LX-2, human liver cancer cells HCCLM3 and PLC / PRF / 5. The method comprises the following steps:

[0109] First, LX-2, HCCLM3 and PLC / PRF / 5 cells were inoculated in laser confocal dishes and cultured until the cell density reached 80%, which ensured that the cells were in a good growth state and was conducive to subsequent experiments.

[0110] Secondly, fix the cells with 4% paraformaldehyde twice, each for 15 minutes. Paraformaldehyde can effectively maintain the morphological structure of cells and prevent the cells from being deformed or damaged in subsequent operations.

[0111] Then, the membrane was permeabilized with PBS solution containing 0.1% TritonX-100 for 15 minutes. As a non-ionic surfactant, TritonX-100 can gently increase the permeability of the cell membrane, which is conducive to the entry of the fluorescent probe into the cell.

[0112] Next, the cells were blocked with PBS solution containing 1% BSA for 30 minutes. BSA can block nonspecific binding sites, reduce background fluorescence, and improve signal specificity.

[0113] Again, add CY5-HSP90001 fluorescent probe at a working concentration of 2.5 μg / mL (dilution ratio 1:800) and incubate overnight at 4°C. Low temperature incubation can reduce nonspecific binding and improve the binding efficiency of the probe to the target protein.

[0114] Finally, the nuclei were stained with DAPI dye for 10 minutes and observed and photographed under a laser confocal microscope. DAPI can specifically stain the cell nucleus, which helps to determine the location and morphology of the cells.

[0115] Preferably, in the embodiment of the present invention, the CY5-HSP90001 fluorescent probe can detect obvious fluorescence signals in LX-2, HCCLM3 and PLC / PRF / 5 cells, and the signals are mainly distributed in the cytoplasm. This distribution pattern is consistent with the subcellular localization of HSP90, proving the high specificity of the probe. In addition, the fluorescence intensity observed in human liver cancer cells HCCLM3 and PLC / PRF / 5 is significantly higher than that in human hepatic stellate cells LX-2, which is consistent with the high expression of HSP90 in cancer cells, demonstrating the potential application value of the probe in cancer research and diagnosis.

[0116] Through the above examples, it can be seen that the heat shock protein 90 fluorescent probe based on CY5 carboxylic acid provided by the present invention not only has good cell permeability and specificity, but also shows excellent detection effects in various cell types. The successful development of this fluorescent probe provides a new tool for HSP90-related research and disease diagnosis, and is expected to promote the development of related fields.

[0117] Example 7: Pharmacological evaluation of heat shock protein 90 fluorescent probe based on CY5 carboxylic acid

[0118] This example aims to evaluate the pharmacological properties of the heat shock protein 90 fluorescent probe CY5-HSP90001 based on CY5 carboxylic acid. The evaluation includes the following steps:

[0119] First, the basic information of the CY5-HSP90001 probe was determined. The results showed that the molecular weight of the probe was 904.34, which was consistent with the theoretical calculation value, confirming the correctness of the synthetic product. In addition, it was found that the probe could be stably stored at 2-8°C, which provided convenience for subsequent experiments and applications.

[0120] Secondly, the preparation method of the probe was optimized. Through experiments, the best preparation scheme was determined: weigh 1 mg of CY5-HSP90001 powder and dissolve it in 0.5 mL of DMSO to make a mother solution. When used, dilute the mother solution with PBS containing 0.01% TritonX-100 to a working concentration of 2.5 μg / mL (dilution ratio 1:800). This preparation method not only ensures the full dissolution of the probe, but also improves the cell permeability of the probe by adding a small amount of TritonX-100.

[0121] Then, cell immunofluorescence experiments were performed to evaluate the performance of the probe. Human hepatic stellate cells LX-2, human hepatoma cells HCCLM3 and PLC / PRF / 5 were selected as experimental subjects. These cell lines were selected based on the important role of HSP90 in the occurrence and development of liver cancer, while also taking into account the necessity of normal hepatic stellate cells as controls.

[0122] The experimental steps are as follows: First, the cells were inoculated in a laser confocal dish and cultured to 80% density. Then, the cells were fixed with 4% paraformaldehyde, which can well maintain the morphological structure of the cells. Subsequently, the cells were permeabilized with a PBS solution containing 0.1% TritonX-100, which can increase the permeability of the cell membrane and facilitate the probe to enter the cell interior.

[0123] Furthermore, the cells were blocked with PBS solution containing 1% BSA to reduce nonspecific binding. Then, the prepared CY5-HSP90001 probe was added and incubated overnight at 4°C. Finally, the cell nuclei were stained with DAPI and observed and photographed under an Olympus laser confocal microscope.

[0124] The experimental results are exciting. Obvious fluorescence signals were observed in all three cell types, and the signals were mainly distributed in the cytoplasm. This distribution pattern is highly consistent with the known subcellular localization of HSP90, confirming the excellent specificity of the probe. It is particularly noteworthy that the fluorescence intensity observed in human liver cancer cells HCCLM3 and PLC / PRF / 5 was significantly higher than that in human hepatic stellate cells LX-2. This result is consistent with the high expression of HSP90 in cancer cells, demonstrating the great potential of this probe in cancer research and diagnosis.

[0125] Finally, the experimental data were quantitatively analyzed. The results showed that the CY5-HSP90001 probe can produce clear fluorescence signals at a low concentration of 2.5 μg / mL, and its sensitivity is comparable to that of commercial fluorescent probes on the market. This not only proves the high efficiency of the probe, but also implies its potential commercial value.

[0126] In summary, this example fully demonstrates the excellent performance of the CY5-HSP90001 probe in detecting HSP90 expression in cells. The probe not only has high specificity and sensitivity, but also can effectively distinguish the expression levels of HSP90 in normal cells and cancer cells. These characteristics make the CY5-HSP90001 probe a powerful tool for studying the role of HSP90 in the occurrence and development of liver cancer, and also provide new ideas for the development of liver cancer diagnosis methods based on HSP90.

[0127] In the future, the team plans to further expand the range of cell types and explore the potential application of the probe in other cancer types. In addition, the team will study the performance of the probe in living animal models to lay the foundation for its eventual application in clinical diagnosis.

[0128] The CY5-HSP90001 probe of the present invention has the following important applications:

[0129] 1. Cell biology research: CY5-HSP90001 probe can be used to study the localization, distribution and dynamic changes of HSP90 in cells. Through immunofluorescence technology, researchers can observe the expression pattern of HSP90 in different cell types and different physiological states. This is of great significance for a deep understanding of the role of HSP90 in cell physiology and pathology.

[0130] 2. Disease diagnosis: This probe shows a stronger fluorescence signal in human liver cancer cells, which makes it a promising new tool for liver cancer diagnosis. In addition, since HSP90 is associated with many types of cancer, this probe may also have application potential in the diagnosis of other cancers such as breast cancer, melanoma, lung cancer, and bladder cancer.

[0131] 3. Drug development: The CY5-HSP90001 probe can be used to screen HSP90 inhibitors and study the interaction between drugs and HSP90. In the process of new drug development, researchers can use this probe to quickly evaluate the effects of potential drugs on HSP90, thereby accelerating the drug screening process.

[0132] 4. Biological process research: This probe helps to deeply explore the mechanism of action of HSP90 in various physiological and pathological processes. For example, it can be used to study the dynamic changes of HSP90 in protein folding, cell stress response, signal transduction and other processes.

[0133] 5. Single-molecule studies: Using the fluorescent properties of the CY5-HSP90001 probe, researchers can conduct dynamic studies of HSP90 at the single-molecule level, tracking its movement within cells and its interactions with other molecules.

[0134] 6. Flow cytometry application: This probe can be used in flow cytometry to achieve rapid quantitative analysis of HSP90 expression levels in a large number of cells. This has important applications in studying tumor heterogeneity and screening specific cell populations.

[0135] 7. High-throughput screening: In drug development and basic research, the CY5-HSP90001 probe can be used to establish a high-throughput screening platform to rapidly evaluate the effects of a large number of compounds or genes on HSP90 expression and function.

[0136] 8. Personalized medical research: By detecting the expression level of HSP90 in patient samples, this probe may provide an important reference for personalized cancer treatment and help doctors choose the most suitable treatment plan.

[0137] 9. Autoimmune disease research: Given the important role of HSP90 in the immune system, this probe can also be used to study the expression changes of HSP90 in autoimmune diseases, providing new ideas for the diagnosis and treatment of related diseases.

[0138] 10. Neurodegenerative disease research: HSP90 is associated with certain neurodegenerative diseases. The CY5-HSP90001 probe may play an important role in the research of diseases such as Alzheimer's disease and Parkinson's disease.

[0139] In conclusion, the CY5-HSP90001 probe has broad application prospects in many fields such as basic research, clinical diagnosis and drug development, and provides a powerful new tool for HSP90-related research and disease diagnosis and treatment.

[0140] The present invention provides a novel heat shock protein 90 (HSP90) fluorescent probe CY5-HSP90001, which exhibits excellent performance and broad application prospects in many aspects. The species reactivity, application range, usage method and storage conditions of the probe will be described in detail below.

[0141] First, regarding species reactivity, the CY5-HSP90001 probe is designed primarily for human HSP90 protein. However, based on the high conservation of the HSP90 protein sequence among different species, it is reasonable to expect that the probe can also be used for studies of mouse and rat samples. This cross-species application potential facilitates comparative biological studies and animal model experiments. Nevertheless, it is recommended that researchers should conduct necessary validation experiments when applying the probe to samples other than human to ensure the specificity and effectiveness of the probe.

[0142] Secondly, in terms of application scope, the CY5-HSP90001 probe is mainly suitable for cell immunofluorescence (ICC / IF) and flow cytometry (FlowCyt). In the cell immunofluorescence experiment, clear positive signals were successfully detected in human hepatic stellate cells LX-2, human liver cancer cells HCCLM3 and PLC / PRF / 5. This not only confirms the effectiveness of the probe, but also reveals its potential in distinguishing the expression levels of HSP90 in normal cells and cancer cells. For flow cytometry applications, although large-scale verification has not yet been carried out, based on the fluorescence properties of the probe, there is reason to believe that it is also applicable to this technology platform.

[0143] Further, regarding the method of use, the recommended starting dilution is 1:800. Specifically, the probe stock solution can be prepared first (for example, 1 mg dissolved in 0.5 mL DMSO), and then diluted to the working concentration with PBS containing 0.01% TritonX-100 when used. It is worth noting that the dilution of 1:800 is only the recommended starting concentration. Considering that different experimental conditions and cell types may affect the sensitivity of the probe, it is strongly recommended that users determine the optimal dilution or concentration through preliminary experiments. This optimization process can ensure the best signal-to-noise ratio under specific experimental conditions.

[0144] Finally, regarding storage conditions, the CY5-HSP90001 probe should be stored in liquid form in DMSO. In order to ensure the long-term stability of the probe, it is recommended to store it in an environment of -20°C. This low-temperature storage can effectively reduce the degradation rate of the probe molecule and extend its service life. In addition, it is recommended to store the probe in small portions to reduce the effect of repeated freezing and thawing on the stability of the probe. When using, the required amount should be slowly thawed at room temperature to avoid vigorous shaking to maintain the activity of the probe.

[0145] In summary, the CY5-HSP90001 probe exhibits good properties in terms of species reactivity, application range, usage methods, and storage conditions. These properties make it a powerful tool for studying HSP90 in the fields of cell biology, disease diagnosis, and drug development. It is expected that this new probe will bring new breakthroughs to HSP90-related research and play an important role in future clinical applications.

[0146] The above description is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A heat shock protein 90 fluorescent probe based on CY5 carboxylic acid, characterized in that: The chemical structural formula of the fluorescent probe is as follows:

2. A method for preparing a heat shock protein 90 fluorescent probe based on CY5 carboxylic acid as claimed in claim 1, characterized in that: The specific reaction formula is as follows: The following steps are involved: (1) SM1 and SM2 are mixed in a ratio of 0.8 to 1.5 molar equivalents; (2) adding DMF solvent to the mixture to dissolve it; (3) adding an organic base DIPEA, a condensing agent HBTU and HOBt to the solution in sequence; (4) stirring the reaction at room temperature for 1 to 2 hours; (5) adding water to the reaction solution and extracting with an organic solvent; (6) drying the obtained organic phase; (7) Purify the target product CY5-HSP90001 by column chromatography or reverse phase C18 chromatography.

3. The preparation method according to claim 1, characterized in that: In the step (1), the molar ratio of SM1 to SM2 is 0.9 to 1.2:

1.

4. The preparation method according to claim 2, characterized in that: The amount of DMF used in the step (2) is 5-10 mL / g.

5. The preparation method according to claim 2, characterized in that: In the step (3): The amount of DIPEA used is 2 to 5 molar equivalents; The amount of HBTU used is 1.5 to 2.5 molar equivalents; The amount of HOBt used is 1.8 to 3 molar equivalents.

6. The preparation method according to claim 1, characterized in that: The organic solvent used in step (5) is ethyl acetate or dichloromethane.

7. A heat shock protein 90 fluorescent probe based on CY5 carboxylic acid, characterized in that: The probe is prepared by the method according to any one of claims 2 to 6.

8. The fluorescent probe according to claim 7, characterized in that The molecular weight of the probe is 904.34 and the probe is stored at 2-8°C.

9. The use of the fluorescent probe according to claim 7 in detecting heat shock protein 90 in human hepatic stellate cells LX-2, human hepatoma cells HCCLM3 and PLC / PRF / 5, characterized in that , including the following steps: (1) LX-2, HCCLM3 and PLC / PRF / 5 cells were inoculated into laser confocal microscopy dishes and cultured until the cell density reached 80%; (2) Aspirate the culture medium, add 1 mL of 4% paraformaldehyde to each well, and fix at 37°C for 5 to 10 minutes; (3) Aspirate the fixative and add 1 mL of 4% paraformaldehyde again, and fix at 37°C for 15 minutes; (4) Wash cells three times with PBS, 5 minutes each time; (5) Add 1 mL of PBS containing 0.1% TritonX-100 to each well and let stand at room temperature for 15 to 20 minutes to permeabilize the membrane; (6) Wash cells with PBS three times, 5 minutes each time; (7) Add 1 mL of PBS containing 1% BSA to each well and incubate at room temperature for 30 minutes for blocking; (8) Aspirate the blocking solution, add the fluorescent probe diluted with PBS containing 0.01% TritonX-100, and incubate at 4°C overnight; (9) Wash cells with PBS three times, 5 minutes each time; (10) Add 0.5 mL of 1:1000 diluted DAPI dye to each well and incubate at room temperature for 10 minutes; (11) Remove the DAPI dye and wash the cells three times with PBS, 5 minutes each time; (12) Add 200 μL of anti-fluorescence quencher to the center of the laser confocal dish; (13) The cells were observed and photographed using an Olympus laser confocal microscope.

10. The use according to claim 9, characterized in that ,The working concentration of the fluorescent probe is 2.5 μg / mL, and the dilution ratio is 1:800.

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