Hydrolyzable fluorescent dye compound as well as preparation method and application thereof

Abstract

The invention relates to a hydrolysable tyramine fluorescent dye compound, the compound is composed of a tyramine derivative group, an azide derivative group and a fluorescent dye, the structural formula of the hydrolysable tyramine fluorescent dye compound is as shown in the structural formula IV: # imgabs0 #, R1 and R2 are independently selected from any one or a combination of H, F, Cl, Br,-CH3 and-CH2CH3; n is equal to 1-5, m is equal to 1-6, and preferably, m is equal to 1-3; the fluorescent dye R is a fluorescein compound or a cyanine compound. According to the method, the fluorescent dye is dissociated and eluted through a mild chemical reaction after being covalently labeled with the antigen, the technical problem that traditional multi-target dyeing cannot meet the dyeing requirements of more than eight markers of customers is solved, and dyeing labeling of multiple targets on the same tissue section is conveniently achieved. Compared with the existing tyramine dye based on a tyramine signal amplification technology, the hydrolysable tyramine dye disclosed by the invention has the advantages that free radicals generated after the hydrolysable tyramine dye is oxidized are more active, the marking is faster, the sensitivity is higher, and meanwhile, the signal amplification effect is stronger.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a class of hydrolyzable fluorescent dye compounds, a preparation method thereof and an application thereof. Background Art

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

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

[0004] Tyramide Signal Amplification (TSA) is a high-density in-situ labeling technology for target proteins using horseradish peroxidase (HRP)-mediated enzymatic reactions with tyramide dyes, which can greatly improve the sensitivity and signal-to-noise ratio of detection. TSA labeling allows the dye to form a very stable covalent bond with 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, and using tyramide dyes labeled with different fluorescent dyes to achieve multi-target staining of tissue samples.

[0005] The currently commonly used multi-target staining technology uses tyramide signal amplification technology to stain the target protein sample with a covalent bond of the fluorescent dye. Due to the limitations of the detection equipment and the spectrum of fluorescent dyes, a maximum of 8 fluorescent dyes (or markers) can be labeled on the same tissue sample, which cannot meet the needs of spatial omics for multi-target labeling. In addition, although TSA technology has the effect of signal amplification, it is limited by the dye structure and cannot meet the detection needs of low-abundance markers. Summary of the invention

[0006] In order to solve the technical problem that the above-mentioned multi-target staining cannot meet the staining needs of super-multi-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 tyramine fluorescent dye compound, wherein the compound is composed of a tyramine derivative group, an azide derivative group and a fluorescent dye R.

[0008] In a preferred technical solution of the present invention, the tyramine 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, -CH2CH3.

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

[0012]

[0013] Where 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 the preferred technical scheme 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).

[0017] 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), and Sulfo-Cy7 (CAS: 477908-53-5).

[0018] In the preferred technical solution of the present invention, the tyramine derivative group and the azide derivative group undergo a condensation reaction to form an amide bond, which is then connected with 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 represented by formula IV,

[0021]

[0022] Wherein, R1 and R2 are independently selected from any one or a combination of H, F, Cl, Br, -CH3, -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 the preferred technical scheme 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).

[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), and Sulfo-Cy7 (CAS: 477908-53-5).

[0028] Another object of the present invention is to provide a compound represented by formula V,

[0029]

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

[0031] In the preferred technical scheme 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).

[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), and Sulfo-Cy7 (CAS: 477908-53-5).

[0033] Another object of the present invention is to provide a method for preparing the compound represented by formula V, which method comprises the following steps:

[0034]

[0035] R is a fluorescein or cyanine compound.

[0036] In the preferred technical scheme 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 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), and Sulfo-Cy7 (CAS: 477908-53-5).

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

[0039] In a 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 object of the present invention is to provide an intermediate represented by compound 5,

[0042]

[0043] Another object of the present invention is to provide a method for preparing compound 5, which comprises the following steps: preparing compound 5 by reacting compound 4 in the presence of a de-Boc reagent,

[0044]

[0045] In the preferred technical scheme of the present invention, the preparation method of compound 5 comprises the following steps: compound 4 is dissolved in a reaction solvent, and then a de-Boc reagent is added to react to obtain compound 5.

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

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

[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 scheme of the present invention, the preparation method of the compound 4 comprises the following steps: preparing the compound 4 by reacting the compound 3 in the presence of a desilylation ether protecting group reagent,

[0051]

[0052] In the preferred technical scheme of the present invention, the preparation method of compound 4 comprises the following steps: compound 3 is dissolved in a reaction solvent, cooled to 0°C to 4°C in an ice bath, and then a desilylation ether protecting group reagent is added to react to obtain compound 4.

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

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

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

[0056] In a preferred technical solution of the present invention, the method for preparing the compound 3 comprises the following steps: Compound 1 and compound 2 are reacted in the presence of a condensing agent to obtain compound 3.

[0057]

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

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

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

[0061] In a preferred technical scheme of the present invention, the condensing agent is selected from any one 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, or a combination thereof.

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

[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 scheme of the present invention, the base is selected from any one of an inorganic base and an organic base; preferably, the inorganic base is selected from any one of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, sodium hydroxide, lithium hydroxide, potassium hydroxide or a combination thereof; the organic base is selected from any one of triethylamine, triethanolamine, N,N-diisopropylethylamine DIEA, pyridine or a combination thereof.

[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 method for preparing compound 7, comprising the following steps:

[0069]

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

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

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

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

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

[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 scheme of the present invention, compound 5 is dissolved in a reaction solvent, cooled to -10°C to -5°C in an ice bath, compound 8 is added, and the mixture is reacted under alkaline conditions to obtain compound 9.

[0079] In a 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 scheme of the present invention, the molar ratio of the compound 5, the base and the compound 8 is 1:1-5:1-5, preferably 1:2:1.

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

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

[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 the preferred technical scheme of the present invention, compound 5 is dissolved in a reaction solvent, cooled to -10°C to -5°C in an ice bath, compound 10 is added, and the reaction is carried out under alkaline conditions to obtain compound 11.

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

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

[0091] In the 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 the preferred technical scheme of the present invention, compound 5 is dissolved in a reaction solvent, cooled to -10°C to -5°C in an ice bath, compound 12 is added, and the reaction is carried out under alkaline conditions to obtain compound 13.

[0095] In the 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 the preferred technical scheme of the present invention, the base is selected from any one of an inorganic base and an organic base; preferably, the inorganic base is selected from any one of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, sodium hydroxide, lithium hydroxide, potassium hydroxide or a combination thereof; the organic base is selected from any one of triethylamine, triethanolamine, N,N-diisopropylethylamine DIEA, pyridine or a combination thereof.

[0098] In a 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 hydrolyzing tyramine fluorescent dye, wherein:

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

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

[0103] In the preferred technical solution of the present invention, the trigger is 50mM tris(2-carbonylethyl)phosphine hydrochloride (TCEP), 20mM triphenylphosphine tris-sulfonic acid trisodium salt (TPPTS), 0.05% Tween 20, 0.05% ProClin300, and a carbonate buffer solution of pH 10.0.

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

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

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

[0107] (3) Adjust the pH value to 9-11 and make up to volume.

[0108] Another object of the present invention is to provide a method for preparing a trigger for hydrolyzing tyramine fluorescent dye, wherein the trigger contains 5-200 mM tris(2-carbonylethyl)phosphine hydrochloride (TCEP), 5-200 mM triphenylphosphine tris-sulfonate sodium (TPPTS), 0.01%-0.20% Tween 20, 0.01%-0.20% ProClin 300, and pH 8-12 25-150 mM carbonate buffer solution. The preparation method comprises the following steps:

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

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

[0111] (3) Adjust the pH value to 9-11, and make up to volume.

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

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

[0114] Another object of the present invention is to provide the use of the hydrolyzable tyramine dye compound and its trigger in fluorescent dyeing.

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

[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 liquids and solids, the percentage is volume / weight percentage; when the present invention relates to the percentage between solids and liquids, the percentage is weight / volume percentage; the rest are weight / weight percentages.

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

[0118] 1. This application realizes the dissociation and elution of fluorescent dyes through mild chemical reactions after covalent labeling of antigens with fluorescent dyes, which solves the technical problem that traditional multi-target staining cannot meet customers' needs for staining more than 8 markers, and facilitates the staining and marking of multiple targets on the same tissue section.

[0119] 2. For markers with relatively low expression abundance, the hydrolyzable tyramide dye of the present application, compared with the existing tyramide dye based on tyramide signal amplification technology, produces more active free radicals after oxidation, and has faster labeling and higher sensitivity, and also has a stronger signal amplification effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] Figure 1 Imaging diagram of the research on the detection of low-abundance targets using the hydrolyzable tyramide dye of the present invention and the traditional TSA dye;

[0121] Figure 2 The hydrolyzable tyramide dye is stained and then dissociated and eluted by a trigger to form an image;

[0122] Figure 3 Imaging of multiple rounds of staining with hydrolyzable tyramide dye. DETAILED DESCRIPTION

[0123] The following are some specific embodiments to illustrate the present invention. It is necessary to point out that the following specific embodiments are only used to further illustrate the present invention and do not limit 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] If no specific conditions are specified in the following examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The instruments, materials, reagents, etc. used in the following examples can be obtained through commercial channels unless otherwise specified.

[0125] Antibodies, enzyme-labeled antibodies, chemical reagents, etc. were all commercially available, and tissue samples were 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. Add DIEA (2.4 g, 0.019 mmol, 2.0 eq) and HBTU (3.9 g, 0.010 mmol, 1.1 eq) in sequence, and finally add compound 2 (3.4 g, 0.010, 1.1 eq) to react at room temperature. After the reaction is completed, add water, extract with EA 3 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 it to 0°C-4°C in an ice bath, and add TBAF (1N 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, and concentrate under reduced pressure to obtain 4.5 g of product compound 4 (yellow solid).

[0133] 3. Preparation of Compound 5

[0134]

[0135] Compound 4 (4.5 g, 0.007 mmol, 1.0 eq) was weighed and dissolved in anhydrous DCM (45 mL), and TFA (11.6 g, 0.102 mmol, 15.0 eq) was added and reacted at room temperature. After the reaction was completed, saturated sodium bicarbonate was used to adjust the pH to 7-8, water was added, and DCM was extracted 3 times. The organic phase was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain 2.8 g of the product compound 5 (foamy solid, 73.3%).

[0136] 1 H-NMR (500MHz, DMSO-d6): δ2.99-3.18(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),9.73(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), and 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) in sequence. After the reaction is completed, the reaction solution is directly separated and purified by reverse phase preparation to obtain 25 mg of product compound 7 (yellow solid).

[0140] 1 H-NMR (300MHz, DMSO-d6+D2O): δ3.48 (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 invention

[0142]

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

[0144] 1H-NMR (400MHz, MeOD): δ1.38(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 invention

[0146]

[0147] In a 100 mL single-mouth bottle, compound 5 (170 mg, 0.30 mmol, 1.0 eq) and DMF (5.5 mL, 50 V) were added, and the temperature was cooled to -10°C to -5°C in an ice-salt bath, and DIEA (78 mg, 0.60 mmol, 2.0 eq) and compound 10 (Sulfo-Cy5 (CAS: 146368-14-1), 113 mg, 0.15 mmol, 1.0 eq) were added. After the reaction was completed at room temperature overnight, the reaction solution was 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 hydrolyzable tyramine dye compound 13 (AF594) of the present application

[0150]

[0151] In a 100 mL single-mouth bottle, compound 5 (170 mg, 0.30 mmol, 1.0 eq) and DMF (5.5 mL, 50 V) were added, and 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) were added to the mixture under ice-salt bath cooling from -10°C to -5°C. After the reaction was completed at room temperature overnight, the reaction solution was directly separated and 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 of the hydrolyzable tyramine dye of the present application

[0154] Ratio: 50 mM pH 9.0 carbonate buffer, containing a final concentration of 10 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride), 10 mM TPPTS (sodium triphenylphosphine tris-sulfonate), 0.05% (v / v) Tween 20, 0.05% (v / v) ProClin300 (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 clean 1000 mL beaker and add 800 mL of purified water;

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

[0158] (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 fully mixed;

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

[0160] Example 6 Preparation of the trigger of the hydrolyzable tyramine dye of the present application

[0161] Ratio: 50 mM carbonate buffer with a pH of 9.0, 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), add them to 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 fully mixed;

[0166] (4) Adjust the pH to 9.0 and make up to 1000 mL.

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

[0168] Ratio: 50 mM carbonate buffer with a pH of 9.0, 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), add them to 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 fully mixed;

[0173] (4) Adjust the pH to 9.0 and make up to 1000 mL.

[0174] Example 8 Preparation of the trigger of the hydrolyzable tyramine dye of the present application

[0175] Ratio: 50 mM carbonate buffer with a pH of 11.0, containing a final concentration of 10 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).

[0176] Preparation method:

[0177] (1) Take a clean 1000 mL beaker and add 800 mL of purified water;

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

[0179] (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 fully mixed;

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

[0181] Test Example 1 Study on the detection of low-abundance targets using the hydrolyzable tyramine dye of the present invention and traditional TSA dye

[0182] Human tonsil samples were used, and the fluorescent dyes prepared by 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) were respectively used, and two different fluorescein derivatives were used for labeling (PD1 (AXB1012), PDL1 (AXB1015, commercially available, fluorescein was dissolved in DMSO, and the concentration was 1 mg / ml) and then the same image acquisition parameters were used.

[0183] The secondary antibody, DAPI, blocking solution / diluent, signal amplification solution, repair solution, traditional TSA dye, etc. in the experiment were all from AlphaTSA 7-color immunohistochemistry kit (Cat. No.: AXT37100031, commercially available), and the usage protocol was referred to the kit manual. Transparent solution Cat. No.: DZ2011.

[0184] Markers Part Number Dilution ratio PD1 AXB1012 Working fluid PDL1 AXB1015 Working fluid

[0185] Experimental steps:

[0186] 1. Take paraffin sections (3 μm thick) of human tonsils and put them in a 65-degree oven for 1 hour; dewax and hydrate the paraffin sections: soak in transparent liquid 3 times, 5 minutes each time; soak in 100% ethanol, 95% ethanol, and 80% ethanol for 3 minutes respectively; soak in distilled water 2 times, 3 minutes each time;

[0187] 2. Microwave repair, cool to room temperature: put the staining jar (containing the repair solution) into the microwave, set the microwave to high heat for 3 minutes (1 jar) until the repair solution boils; put the tissue section into the repair solution, cover it, and microwave it at low heat for 15 minutes; naturally cool 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 twice with TBST, each time for 3 minutes.

[0188] 3. Add 100μl of blocking solution, RT for 15min, shake off the blocking solution; add 100μl of primary antibody, 37 degrees for 1h; rinse 3 times with TBST, and let it stand for 3min after each wash; add 100μl of secondary antibody, 37 degrees for 10min; rinse 3 times with TBST, and let it stand for 3min after each wash; add 100μl of fluorescent dye diluted 1:100 with signal amplification solution, RT for 5min; rinse 3 times with TBST, and let it stand for 3min after each wash; repair in the microwave and cool to room temperature:

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

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

[0191] Test Example 2 Dissociation and elution studies after staining with hydrolyzable tyramide dyes and triggering

[0192] A comparative experiment was conducted using human tonsil samples for labeling, imaging after labeling with a hydrolyzable tyramide dye (Example 1), and dissociation and elution using a trigger (Example 5) after imaging. The experimental results confirmed that the dye was washed away.

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

[0194] Markers Part Number Dilution ratio CD8 AXB3051 Working fluid

[0195] Experimental steps:

[0196] 1. Take paraffin sections (3 μm thick) of human tonsils and put them in a 65-degree oven for 1 hour; dewax and hydrate the paraffin sections: soak in transparent liquid 3 times, 5 minutes each time; soak in 100% ethanol, 95% ethanol, and 80% ethanol for 3 minutes respectively; soak in distilled water 2 times, 3 minutes each time;

[0197] 2. Microwave repair, cool to room temperature: put the staining jar (containing the repair solution) into the microwave, set the microwave to high heat for 3 minutes (1 jar) until the repair solution boils; put the tissue section into the repair solution, cover it, and microwave it at low heat for 15 minutes; naturally cool 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 twice with TBST, each time for 3 minutes.

[0198] 3. Add 100μl of blocking solution, RT for 15min, shake off the blocking solution; add 100μl of primary antibody, 37 degrees for 1h; rinse 3 times with TBST, and let it stand for 3min after each wash; add 100μl of secondary antibody, 37 degrees for 10min; rinse 3 times with TBST, and let it stand for 3min after each wash; add 100μl of fluorescent dye diluted 1:100 with signal amplification solution, RT for 5min; rinse 3 times with TBST, and let it stand for 3min after each wash; repair in the microwave and cool to room temperature:

[0199] 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 / time; add 100 μl of anti-fluorescence quenching sealing agent, cover the slide with coverslip and then image.

[0200] 5. Clear the fluorescent signal of the previous round of staining: take 100uL of hydrolysis trigger of hydrolyzable tyramide fluorescent dye (Example 3), add it to the slide tissue, and treat it at 37°C for 30min; 2) rinse 3 times with TBST, and place it flat for 3min after each wash;

[0201] 6. Repeat step 4, see the result Figure 2 .

[0202] Test Example 3 Hydrolyzable tyramide dyes were used to stain 9 markers in multiple rounds of staining

[0203] The colorectal cancer sample was labeled, and the hydrolyzable tyramide dye (Example 1) was used for labeling and imaging, and a trigger (Example 5) was used for dissociation and elution after imaging. After elution, the next round of staining was performed, and this operation was repeated until 9 marker stainings were completed (plus DAPI nuclear staining, a total of 10 stainings)

[0204]

[0205]

[0206] Experimental steps:

[0207] 1. Take paraffin sections (3 μm thick) of human tonsils and put them in a 65-degree oven for 1 hour; dewax and hydrate the paraffin sections: soak in transparent liquid 3 times, 5 minutes each time; soak in 100% ethanol, 95% ethanol, and 80% ethanol for 3 minutes respectively; soak in distilled water 2 times, 3 minutes each time;

[0208] 2. Microwave repair, cool to room temperature: put the staining jar (containing the repair solution) into the microwave, set the microwave to high heat for 3 minutes (1 jar) until the repair solution boils; put the tissue section into the repair solution, cover it, and microwave it at low heat for 15 minutes; naturally cool 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 twice with TBST, each time for 3 minutes.

[0209] 3. Add 100μl of blocking solution, RT for 15min, shake off the blocking solution; add 100μl of primary antibody, 37 degrees for 1h; rinse 3 times with TBST, and place flat for 3min after each wash; add 100μl of secondary antibody, 37 degrees for 10min; rinse 3 times with TBST, and place flat for 3min after each wash; add 100μl of fluorescent dye, RT for 5min; rinse 3 times with TBST, and place flat for 3min after each wash; repair in the microwave and cool to room temperature:

[0210] 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 / time; add 100 μl of anti-fluorescence quenching sealing agent, cover the slide with coverslip and then image.

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

[0212] 6. Repeat steps 3-5 for the next 8 marker antibody stainings;

[0213] 7. Use Halo software (version V 3.6) to superimpose images to obtain multi-color images. 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 may make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

Claims

1. A hydrolyzable tyramine fluorescent dye compound, wherein: The compound is composed of a tyramine derivative group, an azide derivative group and a fluorescent dye R, The tyramine derivative group is shown in Formula I, Wherein, R1 and R2 are independently selected from any one or a combination of H, F, Cl, Br, -CH3, -CH2CH3; The azide derivative group is shown in structural formula II, Where n = 1-5, m=1-6, preferably, m=1-3; The fluorescent dye R is a fluorescein compound or a cyanine compound; Preferably, the tyramine derivative group and the azide derivative group undergo a condensation reaction to form an amide bond, which is then connected with the fluorescent dye R to form a hydrolyzable tyramine fluorescent dye compound, as shown in structural formula III:

2. The compound represented by formula IV, in, R1 and R2 are independently selected from any one of H, F, Cl, Br, -CH3, -CH2CH3 or a combination thereof; n=1-5, m=1-6, preferably, m=1-3; The fluorescent dye R is a fluorescein compound or a cyanine compound. Preferably, the fluorescein compound is selected from any one of FITC, FAM, TAMRA, Texas Red, AF594, and AF488. The cyanine compound is selected from any one of Sulfo-Cy3, Sulfo-Cy5 and Sulfo-Cy7.

3. The compound represented by formula V, R is a fluorescein compound or a cyanine compound, Preferably, the fluorescein compound is selected from any one of FITC, FAM, TAMRA, Texas Red, AF594, and AF488. The cyanine compound is selected from any one of Sulfo-Cy3, Sulfo-Cy5 and Sulfo-Cy7.

4. A method for preparing a compound represented by formula V, the method comprising the following steps: R is a fluorescein or cyanine compound, Preferably, the fluorescein compound is selected from any one of FITC, FAM, TAMRA, Texas Red, AF594, and AF488. The cyanine compound is selected from any one of Sulfo-Cy3, Sulfo-Cy5 and Sulfo-Cy7.

5. An intermediate represented by compound 5, 6. A method for preparing compound 5, comprising the following steps: preparing compound 5 by reacting compound 4 in the presence of a de-Boc reagent, 7. Any one of Compound 7, Compound 9, Compound 11, and Compound 13, 8. A method for preparing any one of compounds 7, 9, 11, and 13, comprising the following steps: preparing compound 7 from compound 5 and compound 6, Alternatively, compound 9 is prepared from compound 5 and compound 8, Alternatively, compound 11 is prepared from compound 5 and compound 10, Alternatively, compound 13 is prepared from compound 5 and compound 12, 9. A trigger for hydrolyzing a tyramine fluorescent dye, wherein: The trigger contains 5-200 mM tris(2-carbonylethyl)phosphine hydrochloride (TCEP), 5-200 mM triphenylphosphine tris-sulfonate sodium (TPPTS), 0.01%-0.20% Tween 20, 0.01-0.20% ProClin 300 and pH 8-1225-150 mM carbonate buffer solution.

10. A method for preparing a trigger for hydrolyzing tyramine fluorescent dye, the trigger comprising 5-200 mM tris(2-carbonylethyl)phosphine hydrochloride (TCEP), 5-200 mM triphenylphosphine tris-sulfonate (TPPTS), 0.01%-0.20% Tween 20, 0.01-0.20% ProClin 300, and pH 8-12 25-150 mM carbonate buffer solution, the preparation method comprising the following steps: (1) taking the required amount of sodium carbonate, TCEP (tri(2-carboxyethyl)phosphine hydrochloride), and TPPTS (sodium triphenylphosphine tris-sulfonate), add them to 70-90% of the total volume of purified water, and stir until fully dissolved to obtain a mixed solution; (2) Take the required amount of sodium bicarbonate, Tween 20, and ProClin 300, add them to the mixed solution of step (1), and stir until fully mixed; (3) Adjust the pH value to 9.0-11.0 and make up to volume.

11. Use of the hydrolyzable tyramine fluorescent dye compound according to any one of claims 1 to 3, the compound 5 according to claim 5, the compound 7 according to claim 7, the compound 9, the compound 11, the compound 13, and the trigger according to claim 9 in fluorescent staining.

12. A fluorescent staining kit comprising the hydrolyzable tyramine fluorescent dye compound as described in any one of claims 1 to 3 or any one of compound 7, compound 9, compound 11, and compound 13 as described in claim 7, and the trigger as described in claim 9.

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