A class of hydrolyzable fluorescent dye compounds, their preparation methods and their applications

Abstract

The present invention relates to a hydrolyzable tyramine fluorescent dye compound. Among them, the compound is composed of a tyramine derivative group, an azide derivative group and a fluorescent dye. The structural formula of the hydrolyzable tyramine fluorescent dye compound is as shown in Structural Formula IV: Among them, R1 and R2 are independently selected from any one or a combination of H, F, Cl, Br, -CH3, -CH2CH3; n = 1-5, m = 1-6, preferably, m = 1-3; the fluorescent dye R is a fluorescein compound or a cyanine compound. The present invention realizes the dissociation and elution of the fluorescent dye through a mild chemical reaction after covalently labeling the antigen with the fluorescent dye, solves the technical problem that traditional multi-target staining cannot meet the staining requirements of customers for more than 8 markers, and facilitates the staining and labeling of multiple targets on the same tissue section. For markers with relatively low expression abundance, compared with the existing tyramine dyes based on tyramine signal amplification technology, the free radicals generated after oxidation of the hydrolyzable tyramine dye of the present invention are more active, the labeling is faster and the sensitivity is higher, and at the same time, the signal amplification effect is also stronger.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to a class of hydrolyzable fluorescent dye compounds, their preparation methods and their applications. Background Art

[0002] Spatial omics is another research hotspot following single-cell sequencing technology. It mainly studies the relative positional relationships of cells in tissue samples to reveal the impact of cell spatial distribution relationships on diseases, and can make up for the defect that single-cell sequencing technology cannot obtain cell spatial distribution information.

[0003] With the rise of spatial omics, the assessment of the tumor microenvironment / tumor immune microenvironment can be used to guide the medication, companion diagnosis or prognosis of tumor patients. Multiplex staining based on tumor tissues has become an important means to achieve the clinical transformation of the tumor microenvironment and spatial omics research.

[0004] Tyramide Signal Amplification (TSA) is an in-situ labeling technology for high-density tyramide dyes on target proteins using an enzyme-catalyzed reaction mediated by horseradish peroxidase (HRP), which can greatly improve the detection sensitivity and signal-to-noise ratio. TSA labeling forms a very stable covalent bond between the dye and the tissue target protein. During microwave repair, the antibody complex is removed while the binding of the tyramide dye to the tissue target protein is not affected, thus overcoming the limitation of antibody species on staining. Multiplex staining of tissue samples is achieved using tyramide dyes labeled with different fluorescent dyes.

[0005] Currently, the commonly used multiplex staining technology stains by covalently binding fluorescent dyes to tissue target protein samples through tyramide signal amplification technology. Due to the limitations of detection equipment and the fluorescence dye spectrum, at most 8 fluorescent dyes (or markers) can be labeled on the same tissue sample, which cannot meet the needs of spatial omics for ultra-high target labeling. In addition, although the TSA technology has a signal amplification effect, it is limited by the dye structure and cannot meet the detection requirements of low-abundance markers. Summary of the Invention

[0006] In order to solve the above technical problem that multiplex staining cannot meet the staining requirements of ultra-high target markers, the present application provides a class of hydrolyzable tyramide fluorescent dyes and reagents that trigger their hydrolysis and structural breakage to remove fluorescent groups.

[0007] The object of the present invention is to provide a hydrolyzable tyramide fluorescent dye compound, wherein the compound consists of a tyramide derivative group, an azide derivative group, and a fluorescent dye R.

[0008] In a preferred technical solution of the present invention, the tyramide derivative group is as shown in Formula I.

[0009]

[0010] Wherein, R1 and R2 are independently selected from any one or a combination of H, F, Cl, Br, -CH3, and -CH2CH3.

[0011] In a preferred technical solution of the present invention, the azide derivative group is as shown in Structural Formula II,

[0012]

[0013] wherein, n = 1 - 5,

[0014] m = 1 - 6, preferably, m = 1 - 3.

[0015] In a preferred technical solution of the present invention, the fluorescent dye R is a fluorescein compound or a cyanine compound.

[0016] In a preferred technical solution of the present invention, the fluorescein compounds are selected from any one of FITC (CAS: 1173 - 43 - 9), FAM (CAS: 76823 - 03 - 5), TAMRA (CAS: 98181 - 63 - 6), Texas Red (CAS: 216972 - 99 - 5), AF594 (CAS: 1638544 - 48 - 5), and AF488 (CAS: 1374019 - 99 - 4).

[0017] In a preferred technical solution of the present invention, the cyanine compounds are selected from any one of Sulfo - Cy3 (CAS: 1424150 - 38 - 8), Sulfo - Cy5 (CAS: 146368 - 14 - 1), and Sulfo - Cy7 (CAS: 477908 - 53 - 5).

[0018] In a preferred technical solution of the present invention, the tyramine derivative group and the azide derivative group form an amide bond through a condensation reaction, and then are connected to the fluorescent dye R to form a hydrolyzable tyramine fluorescent dye compound, as shown in Structural Formula III:

[0019]

[0020] Another object of the present invention is to provide a compound shown in Formula IV,

[0021]

[0022] wherein, R1 and R2 are independently selected from any one or a combination of H, F, Cl, Br, -CH3, and -CH2CH3;

[0023] n = 1 - 5,

[0024] m = 1 - 6, preferably, m = 1 - 3;

[0025] R is a fluorescein compound or a cyanine compound.

[0026] In a preferred technical solution of the present invention, the fluorescein compound is selected from any one of FITC (CAS: 1173 - 43 - 9), FAM (CAS: 76823 - 03 - 5), TAMRA (CAS: 98181 - 63 - 6), Texas Red (CAS: 216972 - 99 - 5), AF594 (CAS: 1638544 - 48 - 5), AF488 (CAS: 1374019 - 99 - 4).

[0027] In a preferred technical solution of the present invention, the cyanine compound is selected from any one of Sulfo - Cy3 (CAS: 1424150 - 38 - 8), Sulfo - Cy5 (CAS: 146368 - 14 - 1), Sulfo - Cy7 (CAS: 477908 - 53 - 5).

[0028] Another object of the present invention is to provide a compound shown in Formula V,

[0029]

[0030] R is a fluorescein compound or a cyanine compound.

[0031] In a preferred technical solution of the present invention, the fluorescein compound is selected from any one of FITC (CAS: 1173 - 43 - 9), FAM (CAS: 76823 - 03 - 5), TAMRA (CAS: 98181 - 63 - 6), Texas Red (CAS: 216972 - 99 - 5), AF594 (CAS: 1638544 - 48 - 5), AF488 (CAS: 1374019 - 99 - 4).

[0032] In a preferred technical solution of the present invention, the cyanine compound is selected from any one of Sulfo - Cy3 (CAS: 1424150 - 38 - 8), Sulfo - Cy5 (CAS: 146368 - 14 - 1), Sulfo - Cy7 (CAS: 477908 - 53 - 5).

[0033] Another object of the present invention is to provide a preparation method of a compound shown in Formula V, and the method comprises the following steps:

[0034]

[0035] R is a fluorescein or cyanine compound.

[0036] In the preferred technical solution of the present invention, the fluorescein compound is selected from any one of FITC (CAS: 1173-43-9), FAM (CAS: 76823-03-5), TAMRA (CAS: 98181-63-6), Texas Red (CAS: 216972-99-5), AF594 (CAS: 1638544-48-5), and AF488 (CAS: 1374019-99-4).

[0037] In the preferred technical solution of the present invention, the cyanine compound is selected from any one of Sulfo-Cy3 (CAS: 1424150-38-8), Sulfo-Cy5 (CAS: 146368-14-1), and Sulfo-Cy7 (CAS: 477908-53-5).

[0038] In the preferred technical solution of the present invention, the base is selected from any one of inorganic bases and organic bases; preferably, the inorganic base is selected from any one or a combination of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, sodium hydroxide, lithium hydroxide, and potassium hydroxide; the organic base is selected from any one or a combination of triethylamine, triethanolamine, DIEA, and pyridine.

[0039] In the preferred technical solution of the present invention, the molar ratio of the compound 5 to the base is 1:1 - 5.

[0040] In the preferred technical solution of the present invention, the reaction temperature is 10 - 30 °C, preferably 20 - 25 °C.

[0041] The purpose of the present invention is to provide an intermediate shown by the compound 5.

[0042]

[0043] Another purpose of the present invention is to provide a preparation method of the compound 5, and this method includes the following steps: the compound 5 is prepared from the compound 4 in the presence of a de-Boc reagent.

[0044]

[0045] In the preferred technical solution of the present invention, the preparation method of the compound 5 includes the following steps: the compound 4 is dissolved in a reaction solvent, and then a de-Boc reagent is added, and the compound 5 is prepared by reaction.

[0046] In the preferred technical solution of the present invention, the de-Boc reagent is selected from any one or a combination of trifluoroacetic acid, hydrochloric acid, phosphoric acid, TBAF, TMSI, TMSOTf, and ZnBr2.

[0047] In the preferred technical solution of the present invention, the molar ratio of the compound 4 to the de-Boc reagent is 1:10 - 25, preferably 1:15 - 20.

[0048] In the preferred technical solution of the present invention, the reaction solvent is selected from any one or a combination of THF, DMF, and DCM.

[0049] In the preferred technical solution of the present invention, the reaction temperature is 10 - 30 °C, preferably 20 - 25 °C.

[0050] In the preferred technical solution of the present invention, the preparation method of the compound 4 includes the following steps: the compound 4 is prepared from the compound 3 in the presence of a desilyl ether protecting group reagent.

[0051]

[0052] In the preferred technical solution of the present invention, the preparation method of the compound 4 includes the following steps: the compound 3 is dissolved in a reaction solvent, cooled to 0 °C - 4 °C in an ice bath, and then a desilyl ether protecting group reagent is added, and the compound 4 is prepared by reaction.

[0053] In the preferred technical solution of the present invention, the mass-to-volume ratio of the compound 3 to the desilyl ether protecting group reagent is 1:1 - 10, preferably 1:5 - 6.

[0054] In the preferred technical solution of the present invention, the desilyl ether protecting group reagent is selected from Bu4N + F - 、any one or a combination of AcOH and TBAF.

[0055] In the preferred technical solution of the present invention, the reaction solvent is selected from any one or a combination of THF, DMF, and DCM.

[0056] In the preferred technical solution of the present invention, the preparation method of the compound 3 includes the following steps: the compound 3 is prepared from the compound 1 and the compound 2 in the presence of a condensing agent.

[0057]

[0058] In the preferred technical solution of the present invention, the preparation method of the compound 3 includes the following steps: the compound 1 is dissolved in a reaction solvent, and then a condensing agent and the compound 2 are added in sequence, and the compound 3 is prepared under alkaline conditions.

[0059] In the preferred technical solution of the present invention, the molar ratio of the compound 1, the condensing agent, and the compound 2 is 1:1 - 2:1 - 2, preferably 1:1.1:1.1.

[0060] In the preferred technical solution of the present invention, the molar ratio of Compound 1, base, condensing agent, and Compound 2 is 1:1-2:1-2:1-2, preferably 1:2:1.1:1.1.

[0061] In the preferred technical solution of the present invention, the condensing agent is selected from any one or a combination of DCC, DIC, EDCI, ethyl chloroformate, sulfonyl chloride, Boc anhydride, HATU, HBTU, HCTU, TBTU, TSTU, TNTU, PyBOP, DPP-Cl, DECP, DPPA, MPTA, BOP-Cl, thionyl chloride, and oxalyl chloride.

[0062] In the preferred technical solution of the present invention, the reaction solvent is selected from any one or a combination of THF, DMF, and DCM.

[0063] In the preferred technical solution of the present invention, the reaction temperature is 10-30 °C, preferably 20-25 °C.

[0064] In the preferred technical solution of the present invention, the base is selected from any one of inorganic bases and organic bases; preferably, the inorganic base is selected from any one or a combination of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, sodium hydroxide, lithium hydroxide, and potassium hydroxide; the organic base is selected from any one or a combination of triethylamine, triethanolamine, N,N-diisopropylethylamine DIEA, and pyridine.

[0065] Another object of the present invention is to provide any one of the following compounds,

[0066]

[0067]

[0068] Another object of the present invention is to provide a preparation method of Compound 7, comprising the following steps:

[0069]

[0070] In the preferred technical solution of the present invention, the preparation method of Compound 7 comprises the following steps: Compound 5 is dissolved in a reaction solvent, and then Compound 6 is added, and Compound 7 is prepared under alkaline conditions.

[0071] In the preferred technical solution of the present invention, the molar ratio of Compound 5 and Compound 6 is 1:1-5, preferably 1:1.1.

[0072] In the preferred technical solution of the present invention, the molar ratio of Compound 5, base, and Compound 6 is 1:1-5:1-5, preferably 1:3:1.1.

[0073] In the preferred technical solution of the present invention, the base is selected from any one of inorganic bases and organic bases; preferably, the inorganic base is selected from any one or a combination of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, sodium hydroxide, lithium hydroxide, potassium hydroxide; the organic base is selected from any one or a combination of triethylamine, triethanolamine, N,N-diisopropylethylamine (DIEA), pyridine.

[0074] In the preferred technical solution of the present invention, the reaction solvent is selected from any one or a combination of THF, DMF, DCM.

[0075] In the preferred technical solution of the present invention, the reaction temperature is 10 - 30 °C, preferably 20 - 25 °C.

[0076] Another object of the present invention is to provide a method for preparing compound 9, comprising the following steps:

[0077]

[0078] In the preferred technical solution of the present invention, compound 5 is dissolved in the reaction solvent, cooled to -10 °C to -5 °C in an ice bath, and compound 8 is added, and the reaction is carried out under alkaline conditions to obtain compound 9.

[0079] In the preferred technical solution of the present invention, the molar ratio of compound 5 to compound 8 is 1:1 - 5, preferably 1:1.

[0080] In the preferred technical solution of the present invention, the molar ratio of compound 5, base, and compound 8 is 1:1 - 5:1 - 5, preferably 1:2:1.

[0081] In the preferred technical solution of the present invention, the base is selected from any one of inorganic bases and organic bases; preferably, the inorganic base is selected from any one or a combination of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, sodium hydroxide, lithium hydroxide, potassium hydroxide; the organic base is selected from any one or a combination of triethylamine, triethanolamine, N,N-diisopropylethylamine (DIEA), pyridine.

[0082] In the preferred technical solution of the present invention, the reaction solvent is selected from any one or a combination of THF, DMF, DCM.

[0083] In the preferred technical solution of the present invention, the reaction temperature is 10 - 30 °C, preferably 20 - 25 °C.

[0084] Another object of the present invention is to provide a method for preparing compound 11, comprising the following steps:

[0085]

[0086] In a preferred technical solution of the present invention, compound 5 is dissolved in a reaction solvent, and compound 10 is added while cooling to -10°C to -5°C in an ice bath, and compound 11 is prepared by reacting under basic conditions.

[0087] In a preferred technical solution of the present invention, the molar ratio of compound 5 to compound 10 is 1:1 - 5, preferably 1:1.

[0088] In a preferred technical solution of the present invention, the molar ratio of compound 5, base, and compound 10 is 1:1 - 5:1 - 5, preferably 1:2:1.

[0089] In a preferred technical solution of the present invention, the base is selected from any one of inorganic bases and organic bases; preferably, the inorganic base is selected from any one or a combination of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, sodium hydroxide, lithium hydroxide, potassium hydroxide; the organic base is selected from any one or a combination of triethylamine (TEA), triethanolamine, N,N - diisopropylethylamine DIEA, and pyridine.

[0090] In a preferred technical solution of the present invention, the reaction solvent is selected from any one or a combination of THF, DMF, and DCM.

[0091] In a preferred technical solution of the present invention, the reaction temperature is 10 - 30°C, preferably 20 - 25°C.

[0092] Another object of the present invention is to provide a method for preparing compound 13, comprising the following steps:

[0093]

[0094] In a preferred technical solution of the present invention, compound 5 is dissolved in a reaction solvent, and compound 12 is added while cooling to -10°C to -5°C in an ice bath, and compound 13 is prepared by reacting under basic conditions.

[0095] In a preferred technical solution of the present invention, the molar ratio of compound 5 to compound 12 is 1:1 - 5, preferably 1:1.

[0096] In a preferred technical solution of the present invention, the molar ratio of compound 5, base, and compound 10 is 1:1 - 5:1 - 5, preferably 1:2:1.

[0097] In a preferred technical solution of the present invention, the base is selected from any one of inorganic bases and organic bases; preferably, the inorganic base is selected from any one or a combination of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, sodium hydroxide, lithium hydroxide, potassium hydroxide; the organic base is selected from any one or a combination of triethylamine, triethanolamine, N,N - diisopropylethylamine DIEA, and pyridine.

[0098] In the preferred technical solution of the present invention, the reaction solvent is selected from any one of THF, DMF, DCM or a combination thereof.

[0099] In the preferred technical solution of the present invention, the reaction temperature is 10 - 30 °C, preferably 20 - 25 °C.

[0100] Another object of the present invention is to provide a trigger for hydrolyzed tyramine fluorescent dye, wherein,

[0101] The trigger contains 5 - 200 mM tris(2 - carboxyethyl)phosphine hydrochloride (TCEP), 5 - 200 mM sodium triphenylphosphine trisulfonate (TPPTS), 0.01% - 0.20% Tween 20, 0.01% - 0.20% ProClin 300, and a carbonate buffer solution with pH 8 - 12 and 25 - 150 mM.

[0102] In the preferred technical solution of the present invention, the trigger contains 10 - 100 mM tris(2 - carboxyethyl)phosphine hydrochloride (TCEP), 10 - 100 mM sodium triphenylphosphine trisulfonate (TPPTS), 0.05% - 0.10% Tween 20, 0.05% - 0.10% ProClin 300, and a carbonate buffer solution with pH 9 - 11 and 50 - 100 mM.

[0103] In the preferred technical solution of the present invention, the trigger is a carbonate buffer solution containing 50 mM tris(2 - carboxyethyl)phosphine hydrochloride (TCEP), 20 mM trisodium triphenylphosphine trisulfonate (TPPTS), 0.05% Tween 20, 0.05% ProClin 300, and pH 10.0.

[0104] In the preferred technical solution of the present invention, the preparation method of the trigger is as follows:

[0105] (1) Take the required amounts of sodium carbonate, TCEP (tris(2 - carboxyethyl)phosphine hydrochloride), and TPPTS (sodium triphenylphosphine trisulfonate), add them to purified water accounting for 70 - 90% of the total volume, and stir until fully dissolved to obtain a mixed solution;

[0106] (2) Take the required amounts of sodium bicarbonate, Tween 20, and ProClin 300, add them to the mixed solution in step (1), and stir until fully mixed;

[0107] (3) Adjust the pH value to 9 - 11, make up the volume, and that's it.

[0108] Another object of the present invention is to provide a method for preparing a trigger for a hydrolyzable tyramine fluorescent dye, the trigger containing 5 - 200 mM tris(2 - carboxyethyl)phosphine hydrochloride (TCEP), 5 - 200 mM trisodium triphenylphosphine trisulfonate (TPPTS), 0.01% - 0.20% Tween 20, 0.01% - 0.20% ProClin 300, and a carbonate buffer solution with a pH of 8 - 12 and a concentration of 25 - 150 mM. The preparation method includes the following steps:

[0109] (1) Take the required amounts of sodium carbonate, TCEP (tris(2 - carboxyethyl)phosphine hydrochloride), and TPPTS (trisodium triphenylphosphine trisulfonate), add them to purified water accounting for 70 - 90% of the total volume, and stir until fully dissolved to obtain a mixed solution;

[0110] (2) Take the required amounts of sodium bicarbonate, Tween 20, and ProClin 300, add them to the mixed solution in step (1), and stir until fully mixed;

[0111] (3) Adjust the pH value to 9 - 11, make up the volume to obtain the product;

[0112] In a preferred technical solution of the present invention, the trigger contains 10 - 100 mM tris(2 - carboxyethyl)phosphine hydrochloride (TCEP), 10 - 100 mM trisodium triphenylphosphine trisulfonate (TPPTS), 0.05% - 0.10% Tween 20, 0.05% - 0.10% ProClin 300, and a carbonate buffer solution with a pH of 9 - 11 and a concentration of 50 - 100 mM.

[0113] In a preferred technical solution of the present invention, the trigger is a carbonate buffer solution containing 50 mM tris(2 - carboxyethyl)phosphine hydrochloride (TCEP), 20 mM trisodium triphenylphosphine trisulfonate (TPPTS), 0.05% Tween 20, 0.05% ProClin 300, and a pH of 10.0.

[0114] Another object of the present invention is to provide the application of the hydrolyzable tyramine dye compound of the present invention and its trigger in fluorescence staining.

[0115] Another object of the present invention is to provide a fluorescence staining kit, which includes the hydrolyzable tyramine dye compound and the trigger of the present invention.

[0116] Unless otherwise specified, when the present application relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between a liquid and a solid, the percentage is volume / weight percentage; when the present invention relates to the percentage between a solid and a liquid, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0117] Compared with the prior art, the present application has the following beneficial technical effects:

[0118] 1. In the present application, after the antigen is covalently labeled with a fluorescent dye, the fluorescent dye can be dissociated and eluted through a mild chemical reaction, solving the technical problem that traditional multi-target staining cannot meet the customer's staining requirements for more than 8 markers, and facilitating the staining and labeling of multiple targets on the same tissue section.

[0119] 2. For markers with relatively low expression abundance, compared with the existing tyramide dyes based on tyramide signal amplification technology, the free radicals generated after the oxidation of the hydrolyzable tyramide dye in the present application are more active, the labeling is faster and more sensitive, and at the same time, the signal amplification effect is stronger. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] Figure 1 Imaging diagrams of the detection and research of low-abundance targets by the hydrolyzable tyramide dye of the present invention and traditional TSA dyes;

[0121] Figure 2 Imaging diagram of dissociation and elution after staining with the hydrolyzable tyramide dye using a trigger;

[0122] Figure 3 Imaging diagrams of multi-round staining with the hydrolyzable tyramide dye. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0123] The following lists a part of specific embodiments to illustrate the present invention. It is necessary to point out here that the following specific embodiments are only used to further illustrate the present invention and do not represent a limitation on the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the present invention still fall within the protection scope of the present invention.

[0124] For those not specifying specific conditions in the following embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For the instruments, materials, reagents, etc. used in the following embodiments, unless otherwise specified, they can all be obtained through commercial channels.

[0125] Antibodies, enzyme-labeled antibodies, chemical reagents, etc. are all commercially available, and tissue samples are all from hospitals.

[0126] Example 1 Preparation of the hydrolyzable fluorescein isothiocyanate derivative compound 7 (5-FITC) of the present invention

[0127] 1. Preparation of compound 3

[0128]

[0129] Weigh compound 1 (4.4 g, 0.009 mmol, 1.0 eq) and dissolve it in DMF. Then add DIEA (2.4 g, 0.019 mmol, 2.0 eq) and HBTU (3.9 g, 0.010 mmol, 1.1 eq) successively. Finally, add compound 2 (3.4 g, 0.010, 1.1 eq), and react at room temperature. After the reaction is completed, add water, extract with EA three times, collect the organic phase, wash with saturated sodium chloride, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and purify by silica gel column chromatography to obtain 5.3 g of product compound 3 (colorless oil).

[0130] 2. Preparation of Compound 4

[0131]

[0132] Weigh compound 3 (5.3 g, 0.007 mmol, 1.0 eq) and dissolve it in THF (106 mL). Cool the solution to 0 °C - 4 °C in an ice bath, and add TBAF (1 N in THF, 26.5 mL). After the reaction is completed, add water, extract with EA three times, collect the organic phase, wash with saturated sodium chloride, dry with anhydrous sodium sulfate, concentrate under reduced pressure to obtain 4.5 g of product compound 4 (yellow solid).

[0133] 3. Preparation of Compound 5

[0134]

[0135] Weigh compound 4 (4.5 g, 0.007 mmol, 1.0 eq) and dissolve it in anhydrous DCM (45 mL). Add TFA (11.6 g, 0.102 mmol, 15.0 eq), and react at room temperature. After the reaction is completed, adjust the pH to 7 - 8 with saturated sodium bicarbonate, add water, extract with DCM three times, collect the organic phase, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and purify by silica gel column chromatography to obtain 2.8 g of product compound 5 (foamy solid, 73.3%).

[0136] 1 1H-NMR (500 MHz, DMSO-d6): δ 2.99 - 3.18 (2H, m), 3.50 - 3.51 (2H, m), 3.76 (2H, m), 3.90 - 4.18 (2H, m), 4.18 (2H, s), 4.18 - 4.26 (2H, m), 5.19 (1H, m), 6.76 (2H, d), 6.97 - 7.03 (2H, dd), 7.2 (1H, m), 7.40 - 7.50 (7H, m), 7.64 (2H, d), 7.91 (3H, t), 8.7 (1H, t), 9.7 (1H, s).

[0137] 4. Preparation of Compound 7

[0138]

[0139] Weigh compound 5 (100 mg, 0.178 mmol, 1.0 eq) and dissolve it in anhydrous DMF (2 mL). Then add TEA (54 mg, 0.534 mmol, 3.0 eq) and compound 6 (FITC (CAS: 1173 - 43 - 9), 76 mg, 0.196 mmol, 1.1 eq) successively. After the reaction is completed, the reaction solution is directly purified by reverse-phase preparative separation to obtain 25 mg of product compound 7 (yellow solid).

[0140] 1 1H-NMR (300 MHz, DMSO-d6 + D2O): δ 3.48 (2H, m), 3.72 (6H, m), 4.08 (2H, s), 4.14 - 4.18 (2H, m), 5.05 (1H, m), 6.16 - 6.22 (4H, m), 6.74 - 6.80 (4H, m), 6.88 - 6.98 (3H, m), 7.06 - 7.09 (1H, m), 7.37 - 7.43 (7H, m), 7.52 - 7.55 (3H, m), 7.75 (1H, s), 8.16 (1H, br).

[0141] Example 2 Preparation of the Hydrolyzable Tyramine Dye Compound 9 (Sulfo-Cy3) of the Present Application

[0142]

[0143] Add compound 5 (170 mg, 0.30 mmol, 1.0 eq), DMF (5.5 mL), and add DIEA (78 mg, 0.60 mmol, 2.0 eq) at -10 °C to -5 °C under an ice-salt bath. Then add compound 8 (Sulfo-Cy3 (CAS: 1424150 - 38 - 8), 110 mg, 0.15 mmol, 1.0 eq). After the reaction is completed at room temperature overnight, the reaction solution is directly purified by reverse-phase preparative separation to obtain 57 mg of pink flocculent solid compound 9.

[0144] 1H-NMR (400 MHz, MeOD): δ 1.38 (5H, t), 1.64 (2H, t), 1.73 (14H, d), 2.17 (2H, t), 3.36 (2H, t), 3.46 (2H, t), 3.80 - 3.82 (2H, m), 3.93 - 4.00 (3H, m), 4.05 - 4.09 (1H, m), 4.13 (2H, s), 4.15 - 4.18 (4H, m), 5.01 (1H, t), 6.40 - 6.48 (2H, m), 6.74 (2H, d), 6.89 - 6.94 (2H, m), 7.05 - 7.08 (1H, m), 7.27 (1H, d), 7.31 - 7.35 (6H, m), 7.41 - 7.43 (2H, m), 7.50 (2H, d), 7.88 - 7.94 (4H, m), 8.50 (1H, t).

[0145] Example 3 Preparation of the Hydrolyzable Tyramine Dye Compound 11 (Sulfo-Cy5) of the Present Application

[0146]

[0147] In a 100 mL single-necked flask, add Compound 5 (170 mg, 0.30 mmol, 1.0 eq), DMF (5.5 mL, 50V). While cooling to -10 °C to -5 °C in an ice-salt bath, add DIEA (78 mg, 0.60 mmol, 2.0 eq), Compound 10 (Sulfo-Cy5 (CAS: 146368 - 14 - 1), 113 mg, 0.15 mmol, 1.0 eq). After the reaction is completed overnight at room temperature, the reaction solution is directly separated and purified by reverse-phase preparation to obtain 54 mg of Compound 11.

[0148] [M + H] + m / z 1199.43.

[0149] Example 4 Preparation of the Hydrolyzable Tyramine Dye Compound 13 (AF594) of the Present Application

[0150]

[0151] In a 100 mL single-necked flask, add compound 5 (170 mg, 0.30 mmol, 1.0 eq), DMF (5.5 mL, 50 V), and cool it in an ice-salt bath to -10 °C to -5 °C. Then add DIEA (78 mg, 0.60 mmol, 2.0 eq), and compound 12 (AF594 (CAS: 1638544-48-5), 153 mg, 0.15 mmol, 1.0 eq). After the reaction is completed overnight at room temperature, the reaction solution is directly purified by reverse-phase preparation to obtain 58 mg of compound 13.

[0152] [M+H] + m / z 1265.38.

[0153] Example 5 Preparation of the Trigger for the Hydrolyzable Tyramine Dye of the Present Application

[0154] Ratio: 50 mM carbonate buffer with a pH of 9.0, which contains a final concentration of 10 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride), 10 mM TPPTS (triphenylphosphine trisulfonate), 0.05% (v / v) Tween 20, 0.05% (v / v) ProClin 300 (commercially available from sigma, product number 48914-U, containing 3% isothiazolinone) (the above v / v is mL / mL).

[0155] Preparation method:

[0156] (1) Take a 1000 mL beaker that has been cleaned and add 800 mL of purified water;

[0157] (2) Weigh 1.59 grams of anhydrous sodium carbonate, 2.866 grams of TCEP (tris(2-carboxyethyl)phosphine hydrochloride), and 5.684 grams of TPPTS (triphenylphosphine trisulfonate), add them to the above beaker, and stir on a magnetic stirrer until fully dissolved;

[0158] (3) Weigh 2.94 grams of sodium bicarbonate, measure 0.5 mL of Tween 20, and 0.5 mL of ProClin 300, add them to the above beaker, and stir on a magnetic stirrer until fully mixed;

[0159] (4) Adjust the pH value to 9.0 and make up the volume to 1000 mL.

[0160] Example 6 Preparation of the Trigger for the Hydrolyzable Tyramine Dye of the Present Application

[0161] Ratio: 50 mM pH 9.0 carbonate buffer containing a final concentration of 100 mM TCEP, 10 mM TPPTS, 0.05% (v / v) Tween 20, and 0.05% (v / v) ProClin 300 (the above v / v is mL / mL).

[0162] Preparation method:

[0163] (1) Take a clean 1000 mL beaker and add 800 mL of purified water;

[0164] (2) Weigh 1.59 g of anhydrous sodium carbonate, 28.66 g of TCEP (tris(2-carboxyethyl)phosphine hydrochloride), and 5.684 g of TPPTS (sodium triphenylphosphine tris-metasulfonate) into the above beaker and stir on a magnetic stirrer until fully dissolved.

[0165] (3) Weigh 2.94 g of sodium bicarbonate, 0.5 mL of Tween 20, and 0.5 mL of ProClin 300, add them to the above beaker, and stir on a magnetic stirrer until thoroughly mixed;

[0166] (4) Adjust the pH to 9.0 and adjust the volume to 1000 mL.

[0167] Example 7 Preparation of the trigger of the hydrolyzable tyramine dye of the present application

[0168] Ratio: 50 mM pH 9.0 carbonate buffer containing a final concentration of 10 mM TCEP, 100 mM TPPTS, 0.05% (v / v) Tween 20, and 0.05% (v / v) ProClin 300 (the above v / v is mL / mL).

[0169] Preparation method:

[0170] (1) Take a clean 1000 mL beaker and add 800 mL of purified water;

[0171] (2) Weigh 1.59 g of anhydrous sodium carbonate, 2.866 g of TCEP (tris(2-carboxyethyl)phosphine hydrochloride), and 56.84 g of TPPTS (sodium triphenylphosphine tris-metasulfonate) into the above beaker and stir on a magnetic stirrer until fully dissolved.

[0172] (3) Weigh 2.94 g of sodium bicarbonate, 0.5 mL of Tween 20, and 0.5 mL of ProClin 300, add them to the above beaker, and stir on a magnetic stirrer until thoroughly mixed;

[0173] (4) Adjust the pH to 9.0 and adjust the volume to 1000 mL.

[0174] Example 8 Preparation of Trigger for Hydrolyzable Tyramine Dye of the Present Application

[0175] Ratio: 50 mM carbonate buffer with pH 11.0, containing 10 mM TCEP, 10 mM TPPTS, 0.05% (v / v) Tween 20, and 0.05% (v / v) ProClin 300 (v / v herein refers to mL / mL).

[0176] Preparation Method:

[0177] (1) Take a 1000 mL beaker that has been cleaned and add 800 mL of purified water;

[0178] (2) Weigh 1.59 grams of anhydrous sodium carbonate, 2.866 grams of TCEP (tris(2-carboxyethyl)phosphine hydrochloride), and 5.684 grams of TPPTS (trisodium triphenylphosphine trisulfonate), add them to the above beaker, and stir on a magnetic stirrer until completely dissolved;

[0179] (3) Weigh 2.94 grams of sodium bicarbonate, measure 0.5 mL of Tween 20, and 0.5 mL of ProClin 300, add them to the above beaker, and stir on a magnetic stirrer until completely mixed;

[0180] (4) Adjust the pH value to 11.0 and make up the volume to 1000 mL.

[0181] Test Example 1 Research on Detection of Low-Abundance Targets by Hydrolyzable Tyramine Dye and Traditional TSA Dye of the Present Invention

[0182] Using human tonsil samples, respectively using the fluorescent dyes prepared in the present invention (Example 1, Example 2) and the existing fluorescent dyes based on tyramide signal amplification technology (TSA) (XTSA 520, XTSA 570, commercially available), after labeling with two different fluorescein derivatives (PD1 (AXB1012), PDL (AXB1015), commercially available, fluorescein was dissolved with DMSO at a concentration of 1 mg / ml), the same image acquisition parameters were used.

[0183] In the experiment, the secondary antibody, DAPI, blocking solution / diluent, signal amplification solution, repair solution, traditional TSA dye, etc. were all from the AlphaTSA 7-color immunohistochemistry kit (product number: AXT37100031, commercially available), and the usage protocol referred to the kit instruction manual. The product number of the clearing solution: DZ2011.

[0184] Marker Article No. Dilution Ratio PD1 AXB1012 Working Solution PDL1 AXB1015 Working Solution

[0185] Experimental Steps:

[0186] 1. Take human tonsil paraffin sections (3 μm thick) and place them in an oven at 65 °C for 1 h; dewax and hydrate the paraffin sections: soak in the clearing solution 3 times, 5 min each time; soak in 100% ethanol, 95% ethanol, and 80% ethanol for 3 min respectively; soak in distilled water 2 times, 3 min each time.

[0187] 2. Repair with a microwave oven and cool to room temperature: Place the staining cylinder (containing the repair solution) in the microwave oven, set the microwave oven to high heat for 3 min (1 cylinder) until the repair solution boils; place the tissue section in the repair solution, cover it, and microwave at low heat for 15 min; cool naturally to room temperature (about half an hour). Wipe off the residual liquid near the sample, draw a circle with a hydrophobic pen, rinse once with ultrapure water, and rinse 2 times with TBST, 3 min each time.

[0188] 3. Add 100 μl of blocking solution, incubate at RT for 15 min, and discard the blocking solution; add 100 μl of the primary antibody and incubate at 37 °C for 1 h; rinse 3 times with TBST, place it flat for 3 min after each wash; add 100 μl of the secondary antibody and incubate at 37 °C for 10 min; rinse 3 times with TBST, place it flat for 3 min after each wash; add 100 μl of the fluorescent dye diluted 1:100 with the signal amplification solution and react at RT for 5 min; rinse 3 times with TBST, place it flat for 3 min after each wash; repair with a microwave oven and cool to room temperature:

[0189] 4. Add 100 μl of DAPI working solution for staining at room temperature for 5 min; wash: wash 3 times with distilled water, 5 min each time; add 100 μl of anti-fluorescence quenching mounting medium, cover with a coverslip and image, and the results are shown in Figure 1 。

[0190] The results confirmed that the fluorescent dye synthesized in the present invention has a stronger signal under the same conditions.

[0191] Test Example 2 Study on dissociation and elution of the hydrolyzable tyramine dye after staining with a trigger

[0192] Label human tonsil samples, image after labeling with a hydrolyzable tyramine dye (Example 1), and perform a comparative experiment on dissociation and elution with a trigger (Example 5) after imaging. The experimental results confirmed that the dye was washed off.

[0193] The antibodies used in the experiment are as follows:

[0194] Marker Article No. Dilution Ratio CD8 AXB3051 Working Solution

[0195] Experimental procedure:

[0196] 1. Take human tonsil paraffin sections (3 μm thick) and place them in an oven at 65 °C for 1 h; dewax and hydrate the paraffin sections: soak in the clearing solution 3 times, 5 min each time; soak in 100% ethanol, 95% ethanol, and 80% ethanol for 3 min each; soak in distilled water 2 times, 3 min each time.

[0197] 2. Repair with a microwave oven and cool to room temperature: Place the staining cylinder (containing the repair solution) in the microwave oven, set the microwave oven to high heat for 3 min (1 cylinder) until the repair solution boils; place the tissue section in the repair solution, cover, and microwave at low heat for 15 min; cool naturally to room temperature (about half an hour). Wipe off the residual liquid near the sample, draw a circle with a hydrophobic pen, rinse once with ultrapure water, and rinse 2 times with TBST, 3 min each time.

[0198] 3. Drop 100 μl of the blocking solution, incubate at RT for 15 min, and discard the blocking solution; drop 100 μl of the primary antibody, incubate at 37 °C for 1 h; rinse 3 times with TBST, lay flat for 3 min after each wash; drop 100 μl of the secondary antibody, incubate at 37 °C for 10 min; rinse 3 times with TBST, lay flat for 3 min after each wash; add 100 μl of the fluorescent dye diluted 1:100 with the signal amplification solution, react at RT for 5 min; rinse 3 times with TBST, lay flat for 3 min after each wash; repair with a microwave oven and cool to room temperature:

[0199] 4. Drop 100 μl of the DAPI working solution for staining, incubate at room temperature for 5 min; wash: wash 3 times with distilled water, 5 min each time; drop 100 μl of the anti-fluorescence quenching mounting medium, cover with a coverslip and image.

[0200] 5. Remove the fluorescence signal from the previous round of staining: Take 100 uL of the hydrolysis trigger of the hydrolyzable tyramine fluorescent dye (Example 3), add it to the tissue on the slide, and treat at 37 °C for 30 min; 2) Rinse 3 times with TBST, lay flat for 3 min after each wash.

[0201] 6. Repeat step 4, and the results are shown in Figure 2 。

[0202] Test Example 3 The hydrolyzable tyramine dye is used for staining research of 9 markers through multiple rounds of staining

[0203] Label with colorectal cancer samples, image after labeling with the hydrolyzable tyramine dye (Example 1), and use the trigger (Example 5) for dissociation and elution after imaging. After elution, perform the next round of staining, and repeat this operation until staining of 9 markers is completed (a total of 10 stainings including DAPI nuclear staining).

[0204]

[0205]

[0206] Experimental procedures:

[0207] 1. Take human tonsil paraffin sections (3 μm thick) and place them in an oven at 65 °C for 1 h; dewax and hydrate the paraffin sections: soak in the clearing solution 3 times, 5 min each time; soak in 100% ethanol, 95% ethanol, and 80% ethanol for 3 min respectively; soak in distilled water 2 times, 3 min each time;

[0208] 2. Repair by microwave oven and cool to room temperature: Place the staining cylinder (containing the repair solution) in the microwave oven, set the microwave oven to high heat for 3 min (for 1 cylinder) until the repair solution boils; put the tissue section into the repair solution, cover it, and microwave at low heat for 15 min; cool naturally to room temperature (about half an hour). Wipe off the residual liquid near the sample, draw a circle with a hydrophobic pen, rinse once with ultrapure water, and rinse 2 times with TBST, 3 min each time.

[0209] 3. Drop 100 μl of the blocking solution, incubate at RT for 15 min, and discard the blocking solution; drop 100 μl of the primary antibody, incubate at 37 °C for 1 h; rinse 3 times with TBST, and place it flat for 3 min after each wash; drop 100 μl of the secondary antibody, incubate at 37 °C for 10 min; rinse 3 times with TBST, and place it flat for 3 min after each wash; add 100 μl of the fluorescent dye, react at RT for 5 min; rinse 3 times with TBST, and place it flat for 3 min after each wash; repair by microwave oven and cool to room temperature:

[0210] 4. Drop 100 μl of the DAPI working solution for staining, incubate at room temperature for 5 min; wash: wash 3 times with distilled water, 5 min each time; drop 100 μl of the anti-fluorescence quenching mounting medium, cover with a coverslip and image.

[0211] 5. Remove the fluorescence signal of the previous round of staining: Take 100 uL of the hydrolysis trigger of the hydrolyzable tyramide fluorescent dye (Example 3), add it to the tissue on the slide, and treat at 37 °C for 30 min; 2) Rinse 3 times with TBST, and place it flat for 3 min after each wash;

[0212] 6. Repeat steps 3 - 5 for the subsequent staining of 8 marker antibodies;

[0213] 7. Use Halo software (version V 3.6) to perform image overlay to obtain a multi-color image, and the results are shown in Figure 3 .

[0214] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or deformations according to the present invention. As long as they do not depart from the spirit of the present invention, they shall fall within the scope of protection of the claims of the present invention.

Claims

1. The compound shown in Formula IV, ； Ⅳ Among them, R1 and R2 are independently selected from any one of H, F, Cl, Br, -CH3, and -CH2CH3; R is a fluorescein compound or a cyanine compound; The fluorescein compounds are selected from any one of FITC (CAS: 1173-43-9), FAM (CAS: 76823-03-5), TAMRA (CAS: 98181-63-6), Texas Red (CAS: 216972-99-5), AF594 (CAS: 1638544-48-5), and AF488 (CAS: 1374019-99-4); The cyanine compounds are selected from any one of Sulfo-Cy3 (CAS: 1424150-38-8), Sulfo-Cy5 (CAS: 146368-14-1), and Sulfo-Cy7 (CAS: 477908-53-5).

2. The compound shown in Formula V, ； Ⅴ R is a fluorescein compound or a cyanine compound; The fluorescein compounds are selected from any one of FITC (CAS: 1173-43-9), FAM (CAS: 76823-03-5), TAMRA (CAS: 98181-63-6), Texas Red (CAS: 216972-99-5), AF594 (CAS: 1638544-48-5), and AF488 (CAS: 1374019-99-4); The cyanine compounds are selected from any one of Sulfo-Cy3 (CAS: 1424150-38-8), Sulfo-Cy5 (CAS: 146368-14-1), and Sulfo-Cy7 (CAS: 477908-53-5).

3. A method for preparing the compound shown in Formula V as claimed in claim 2, the method comprising the following steps: ； R is a fluorescein or cyanine compound; The fluorescein compounds are selected from any one of FITC (CAS: 1173-43-9), FAM (CAS: 76823-03-5), TAMRA (CAS: 98181-63-6), Texas Red (CAS: 216972-99-5), AF594 (CAS: 1638544-48-5), and AF488 (CAS: 1374019-99-4); The cyanine compounds are selected from any one of Sulfo-Cy3 (CAS: 1424150-38-8), Sulfo-Cy5 (CAS: 146368-14-1), and Sulfo-Cy7 (CAS: 477908-53-5).

4. The preparation method according to claim 3, wherein the base is selected from any one of inorganic bases and organic bases; wherein, The inorganic base is selected from any one or a combination of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, sodium hydroxide, lithium hydroxide, and potassium hydroxide; the organic base is selected from any one or a combination of triethylamine, triethanolamine, DIEA, and pyridine.

5. The preparation method according to claim 3, wherein the molar ratio of the compound 5 to the base is 1:1 - 5.

6. The preparation method according to any one of claims 3 - 5, wherein the reaction temperature is 10 - 30 °C.

7. The preparation method according to claim 6, wherein the reaction temperature is 20 - 25 °C.

8. An intermediate represented by the compound 5 5。 9. A preparation method of the compound 5 according to claim 8, the method comprising the following steps: the compound 4 is prepared to obtain the compound 5 in the presence of a de - Boc reagent 。 10. The preparation method according to claim 9, wherein the preparation method of the compound 5 comprises the following steps: the compound 4 is dissolved in a reaction solvent, and then a de - Boc reagent is added, and the compound 5 is prepared by reaction.

11. The preparation method according to claim 10, wherein the de - Boc reagent is selected from any one or a combination of trifluoroacetic acid, hydrochloric acid, phosphoric acid, TBAF, TMSI, TMSOTf, ZnBr2.

12. The preparation method according to claim 10, wherein the molar ratio of the compound 4 to the de - Boc reagent is 1:10 - 25.

13. The preparation method according to claim 12, wherein the molar ratio of the compound 4 to the de - Boc reagent is 1:15 - 20.

14. The preparation method according to claim 10, wherein the reaction solvent is selected from any one or a combination of THF, DMF, DCM.

15. The preparation method according to claim 10, wherein the reaction temperature is 10 - 30 °C.

16. The preparation method according to claim 15, wherein the reaction temperature is 20 - 25 °C.

17. The preparation method according to claim 9, wherein the preparation method of the compound 4 comprises the following steps: the compound 3 is prepared to obtain the compound 4 in the presence of a de - silyl ether protecting group reagent 。 18. The preparation method according to claim 17, wherein the preparation method of the compound 4 comprises the following steps: the compound 3 is dissolved in a reaction solvent, cooled to 0 °C - 4 °C in an ice bath, and then a de - silyl ether protecting group reagent is added, and the compound 4 is prepared by reaction.

19. The preparation method according to claim 17, wherein the mass - to - volume ratio of the compound 3 to the de - silyl ether protecting group reagent is 1:1 - 10.

20. The preparation method according to claim 19, wherein the mass - to - volume ratio of the compound 3 to the de - silyl ether protecting group reagent is 1:5 - 6.

21. The preparation method according to claim 18, wherein the de - silyl ether protecting group reagent is selected from any one or a combination of AcOH, TBAF.

22. The preparation method according to claim 18, wherein the reaction solvent is selected from any one or a combination of THF, DMF, DCM.

23. The preparation method according to claim 17, wherein the preparation method of the compound 3 comprises the following steps: the compound 1 and the compound 2 are prepared to obtain the compound 3 in the presence of a condensing agent 。 24. The preparation method according to claim 23, wherein the preparation method of the compound 3 comprises the following steps: the compound 1 is dissolved in a reaction solvent, and then a condensing agent and the compound 2 are added in sequence, and the compound 3 is prepared under basic conditions.

25. The preparation method according to claim 23, wherein the molar ratio of compound 1, the condensing agent, and compound 2 is 1:1-2:1-2.

26. The preparation method according to claim 25, wherein the molar ratio of compound 1, the condensing agent, and compound 2 is 1:1.1:1.

1.

27. The preparation method according to claim 23, wherein the molar ratio of compound 1, the base, the condensing agent, and compound 2 is 1:1-2:1-2:1-2.

28. The preparation method according to claim 27, wherein the molar ratio of compound 1, the base, the condensing agent, and compound 2 is 1:2:1.1:1.

1.

29. The preparation method according to claim 23, wherein the condensing agent is selected from any one or a combination of DCC, DIC, EDCI, ethyl chloroformate, sulfonyl chloride, Boc anhydride, HATU, HBTU, HCTU, TBTU, TSTU, TNTU, PyBOP, DPP-Cl, DECP, DPPA, MPTA, BOP-Cl, thionyl chloride, and oxalyl chloride.

30. The preparation method according to claim 24, wherein the reaction solvent is selected from any one or a combination of THF, DMF, and DCM.

31. The preparation method according to claim 24, wherein the reaction temperature is 10-30 °C.

32. The preparation method according to claim 31, wherein the reaction temperature is 20-25 °C.

33. The preparation method according to claim 24, wherein the base is selected from any one of inorganic bases and organic bases.

34. The preparation method according to claim 33, wherein the inorganic base is selected from any one or a combination of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, sodium hydroxide, lithium hydroxide, and potassium hydroxide.

35. The preparation method according to claim 33, wherein the organic base is selected from any one or a combination of triethylamine, triethanolamine, N,N-diisopropylethylamine (DIEA), and pyridine.

36. Any one of compound 7, compound 9, compound 11, and compound 13 。 37. A preparation method of compound 7, comprising the following steps: 。 38. The preparation method according to claim 37, wherein the preparation method of compound 7 comprises the following steps: compound 5 is dissolved in a reaction solvent, and then compound 6 is added, and compound 7 is prepared under alkaline conditions.

39. The preparation method according to claim 38, wherein the molar ratio of compound 5 and compound 6 is 1:1-5.

40. The preparation method according to claim 39, wherein the molar ratio of compound 5 and compound 6 is 1:1.

1.

41. The preparation method according to claim 38, wherein the molar ratio of compound 5, the base, and compound 6 is 1:1-5:1-5.

42. The preparation method according to claim 41, wherein the molar ratio of compound 5, the base, and compound 6 is 1:3:1.

1.

43. The preparation method according to claim 38, wherein the base is selected from any one of inorganic bases and organic bases.

44. The preparation method according to claim 43, wherein the inorganic base is selected from any one or a combination of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, sodium hydroxide, lithium hydroxide, and potassium hydroxide.

45. The preparation method according to claim 43, wherein the organic base is selected from any one or a combination of triethylamine, triethanolamine, N,N - diisopropylethylamine (DIEA), and pyridine.

46. The preparation method according to claim 38, wherein the reaction solvent is selected from any one or a combination of THF, DMF, and DCM.

47. The preparation method according to claim 38, wherein the reaction temperature is 10 - 30 °C.

48. The preparation method according to claim 47, wherein the reaction temperature is 20 - 25 °C.

49. A preparation method of compound 9, comprising the following steps: 。 50. The preparation method according to claim 49, wherein compound 5 is dissolved in a reaction solvent, cooled to -10 °C to -5 °C in an ice bath, and compound 8 is added, and the reaction is carried out under basic conditions to obtain compound 9.

51. The preparation method according to claim 50, wherein the molar ratio of compound 5 to compound 8 is 1:1 - 5.

52. The preparation method according to claim 51, wherein the molar ratio of compound 5 to compound 8 is 1:

1.

53. The preparation method according to claim 50, wherein the molar ratio of compound 5, base, and compound 8 is 1:1 - 5:1 - 5.

54. The preparation method according to claim 53, wherein the molar ratio of compound 5, base, and compound 8 is 1:2:

1.

55. The preparation method according to claim 50, wherein the base is selected from any one of inorganic bases and organic bases.

56. The preparation method according to claim 55, wherein the inorganic base is selected from any one or a combination of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, sodium hydroxide, lithium hydroxide, and potassium hydroxide.

57. The preparation method according to claim 55, wherein the organic base is selected from any one or a combination of triethylamine, triethanolamine, N,N - diisopropylethylamine (DIEA), and pyridine.

58. The preparation method according to claim 50, wherein the reaction solvent is selected from any one or a combination of THF, DMF, and DCM.

59. The preparation method according to claim 50, wherein the reaction temperature is 10 - 30 °C.

60. The preparation method according to claim 59, wherein the reaction temperature is 20 - 25 °C.

61. A preparation method of compound 11, comprising the following steps: 。 62. The preparation method according to claim 61, wherein compound 5 is dissolved in a reaction solvent, cooled to -10 °C to -5 °C in an ice bath, and compound 10 is added, and the reaction is carried out under basic conditions to obtain compound 11.

63. The preparation method according to claim 62, wherein the molar ratio of compound 5 to compound 10 is 1:1 - 5.

64. The preparation method according to claim 63, wherein the molar ratio of compound 5 to compound 10 is 1:

1.

65. The preparation method according to claim 62, wherein the molar ratio of compound 5, base, and compound 10 is 1:1 - 5:1 - 5.

66. The preparation method according to claim 65, wherein the molar ratio of the compound 5, the base, and the compound 10 is 1:2:

1.

67. The preparation method according to claim 62, wherein the base is selected from any one of inorganic bases and organic bases.

68. The preparation method according to claim 67, wherein the inorganic base is selected from any one or a combination of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, sodium hydroxide, lithium hydroxide, and potassium hydroxide.

69. The preparation method according to claim 67, wherein the organic base is selected from any one or a combination of triethylamine (TEA), triethanolamine, N,N - diisopropylethylamine (DIEA), and pyridine.

70. The preparation method according to claim 62, wherein the reaction solvent is selected from any one or a combination of THF, DMF, and DCM.

71. The preparation method according to claim 62, wherein the reaction temperature is 10 - 30 °C.

72. The preparation method according to claim 71, wherein the reaction temperature is 20 - 25 °C.

73. A preparation method of a compound 13, comprising the following steps: 。 74. The preparation method according to claim 73, wherein the compound 5 is dissolved in a reaction solvent, cooled to -10 °C to -5 °C in an ice bath, and the compound 12 is added, and the reaction is carried out under basic conditions to obtain the compound 13.

75. The preparation method according to claim 74, wherein the molar ratio of the compound 5 and the compound 12 is 1:1 - 5.

76. The preparation method according to claim 75, wherein the molar ratio of the compound 5 and the compound 12 is 1:

1.

77. The preparation method according to claim 74, wherein the molar ratio of the compound 5, the base, and the compound 10 is 1:1 - 5:1 - 5.

78. The preparation method according to claim 77, wherein the molar ratio of the compound 5, the base, and the compound 10 is 1:2:

1.

79. The preparation method according to claim 74, wherein the base is selected from any one of inorganic bases and organic bases.

80. The preparation method according to claim 79, wherein the inorganic base is selected from any one or a combination of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, sodium hydroxide, lithium hydroxide, and potassium hydroxide.

81. The preparation method according to claim 79, wherein the organic base is selected from any one or a combination of triethylamine, triethanolamine, N,N - diisopropylethylamine (DIEA), and pyridine.

82. The preparation method according to claim 74, wherein the reaction solvent is selected from any one or a combination of THF, DMF, and DCM.

83. The preparation method according to claim 74, wherein the reaction temperature is 10 - 30 °C.

84. The preparation method according to claim 83, wherein the reaction temperature is 20 - 25 °C.

85. The application of any one of the compounds according to any one of claims 1 - 2, the compound 7 according to claim 36, the compound 9, the compound 11, and the compound 13 in fluorescence staining.

86. A fluorescence staining kit, comprising a compound as described in any one of claims 1-2 or any one of compound 7, compound 9, compound 11, and compound 13 as described in claim 36, and a trigger. The trigger contains 5-200 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 5-200 mM triphenylphosphine tris(m-sulfonate) sodium salt (TPPTS), 0.01%-0.20% Tween 20, 0.01%-0.20% ProClin 300, and a carbonate buffer solution with a pH of 8-12 and a concentration of 25-150 mM.

87. The fluorescence staining kit as described in claim 86, wherein the trigger contains 10-100 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 10-100 mM triphenylphosphine tris(m-sulfonate) sodium salt (TPPTS), 0.05%-0.10% Tween 20, 0.05%-0.10% ProClin 300, and a carbonate buffer solution with a pH of 9-11 and a concentration of 50-100 mM.

88. The fluorescence staining kit as described in claim 87, wherein the trigger is a carbonate buffer solution containing 50 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 20 mM triphenylphosphine tris(m-sulfonate) trisodium salt (TPPTS), 0.05% Tween 20, 0.05% ProClin 300, and a pH of 10.

0.

89. The fluorescence staining kit as described in any one of claims 86-88, wherein the preparation method of the trigger is as follows: (1) Take the required amounts of sodium carbonate, TCEP (tris(2-carboxyethyl)phosphine hydrochloride), and TPPTS (triphenylphosphine tris(m-sulfonate) sodium salt), add them to purified water accounting for 70-90% of the total volume, and stir until fully dissolved to obtain a mixed solution; (2) Take the required amounts of sodium bicarbonate, Tween 20, and ProClin 300, add them to the mixed solution in step (1), and stir until fully mixed; (3) Adjust the pH value to 9-11, make up the volume to the mark, and obtain the trigger.

Images