Fluorescein derivative as well as preparation method and application thereof

Abstract

The invention provides a fluorescein derivative as well as a preparation method and application thereof. The fluorescein derivative disclosed by the invention comprises any one of 6-carboxyl fluorescein modified by an o-methylthiophene side chain, 6-carboxyl fluorescein modified by an o-benzothiophene side chain, 6-carboxyl fluorescein modified by a benzothiophene side chain and 6-carboxyl fluorescein modified by a dimethylamino o-benzothiophene side chain. Compared with traditional 6-carboxyl fluorescein, the four kinds of asymmetric fluorescein prepared by the method have obvious red shift in the aspect of emission wavelength, the Stokes shift of the four kinds of asymmetric fluorescein is large, one fluorescein derivative is higher than 100 nm, and the preparation method is simple and easy to industrialize.

Description

Technical Field

[0001] The invention relates to the technical field of organic fluorescent dye molecular synthesis, and in particular to a fluorescein derivative and a preparation method and application thereof. Background Art

[0002] In the field of biomedical testing, fluorescent labeling has the advantages of high sensitivity, high selectivity, fast response, good repeatability, simple operation and low cost compared with other detection methods. Therefore, it is widely used in molecular diagnosis, genetic testing, antibody immunoassay and other aspects.

[0003] At present, the fluorescent dyes used for labeling or derivatization mainly include rhodamine, fluorescein, cyanine, phthalaldehyde, etc. Among them, fluorescein fluorescent dyes occupy an extremely important position in the field of biological research. Fluorescein was first synthesized by Baeyer in 1871 through the Friedel-Crafts reaction of resorcinol and phthalic anhydride. It is formed by acylation and cyclodehydration condensation of electron-rich resorcinol and phthalic anhydride under acidic conditions. Fluorescein dyes are considered to be one of the most reliable probes for tracking pH changes in biological systems. The carboxyl and hydroxyl functional groups contained in its molecules can be used as pH reaction sites. The carboxyl site of fluorescein can be used as NHS activated ester for bioconjugation, drug delivery, component development of biosensors, cell biology research and fluorescent immunoassay. The hydroxyl site of fluorescein can be combined with recognition groups through esterification reaction. When specific ions are added through irreversible chemical reactions, the ester bond breaks, resulting in obvious color or fluorescence changes. The purpose of detecting different analytes can be achieved by utilizing the conversion of the two states.

[0004] The structural formula of fluorescein dye is Figure 1 As shown, the substituents R1=R2, R3=R4 are called symmetrical fluorescein; Figure 1 In the structural formula shown, the substituents R1≠R2, R3≠R4, or R1≠R2≠R3≠R4 are called asymmetric fluorescein. Figure 1 Fluorescein dyes with a medium structure can be derivatized into phosphoramidite monomers and then used directly on a DNA automatic synthesizer to modify the 5' end of a DNA probe. They can also be derivatized and loaded onto CPG or PS to directly label the 3' end of a DNA probe.

[0005] Fluorescein dyes have many advantages, such as good water solubility, relatively high fluorescence quantum efficiency, and relatively large molar extinction coefficient. Based on these advantages, even though the excitation and emission wavelengths of fluorescein are only in the visible light region, fluorescein is still widely used in biochemistry, modern biology, and medical research. Since the Stokes shift of most fluorescent dyes (such as fluorescein, rhodamine, oxazine, and anthocyanin) is very small (generally <30nm), there is a serious crosstalk between the excitation spectrum and the emission spectrum, resulting in a low signal-to-noise ratio and severe fluorescence self-quenching during imaging. Because there are few examples of asymmetric fluorescein side chain modified heterocycles, it is particularly important to design dyes with a higher Stokes shift than FAM carboxyfluorescein. Summary of the invention

[0006] In view of the above problems existing in the prior art, the object of the present invention is to provide a fluorescein derivative and a preparation method and application thereof.

[0007] The technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a fluorescein derivative, wherein the fluorescein derivative includes any one of 6-carboxyfluorescein modified with an o-methylthiophene side chain, 6-carboxyfluorescein modified with an o-phenylthiophene side chain, 6-carboxyfluorescein modified with a benzothiophene side chain, and 6-carboxyfluorescein modified with a dimethylamino o-phenylthiophene side chain;

[0009] The structural formula of the 6-carboxyfluorescein modified with the o-methylthiophene side chain is as follows:

[0010]

[0011] The structural formula of the 6-carboxyfluorescein modified with the o-phenylthiophene side chain is as follows:

[0012]

[0013] The structural formula of the benzothiophene side chain modified 6-carboxyfluorescein is as follows:

[0014]

[0015] The structural formula of the methylamino-modified 6-carboxyfluorescein with o-phenylthiophene side chain is as follows:

[0016]

[0017] In a second aspect, the present invention also provides a method for preparing the fluorescein derivative, wherein the fluorescein derivative is 6-carboxyfluorescein modified with an o-methylthiophene side chain;

[0018] The preparation method of the o-methylthiophene side chain modified 6-carboxyfluorescein comprises the following steps:

[0019] Preparation of 2-(2,4-dimethoxyphenyl)-5-methylthiophene;

[0020] 2-(2,4-dimethoxyphenyl)-5-methylthiophene is reacted with pyridine hydrochloride to obtain 4-(5-methylthiophen-2-yl)benzene-1,3-diol;

[0021] 6-carboxyfluorescein modified with o-methylthiophene side chain was obtained by reacting 4-(5-methylthiophen-2-yl)benzene-1,3-diol and 2-(2,4-dihydroxybenzoyl)terephthalic acid;

[0022] Alternatively, the fluorescein derivative is 6-carboxyfluorescein modified with an o-phenylthiophene side chain, and the preparation method of the 6-carboxyfluorescein modified with an o-phenylthiophene side chain comprises the following steps:

[0023] Preparation of 2-(2,4-dimethoxyphenyl)-5-2-phenylthiophene;

[0024] 4-(5-phenylthiophen-2-yl)benzene-1,3-diol is obtained by reacting 2-(2,4-dimethoxyphenyl)-5-phenylthiophene with pyridine hydrochloride;

[0025] 6-carboxyfluorescein modified with o-phenylthiophene side chain was obtained by reacting 4-(5-phenylthiophene-2-yl)benzene-1,3-diol with 2-(2,4-dihydroxybenzoyl)terephthalic acid.

[0026] Or, the fluorescein derivative is 6-carboxyfluorescein modified with a benzothiophene side chain;

[0027] The preparation method of the benzothiophene side chain modified 6-carboxyfluorescein comprises the following steps:

[0028] Preparation of 2-(2,4-dimethoxyphenyl)benzo[b]thiophene;

[0029] 2-(2,4-dimethoxyphenyl)benzo[b]thiophene is reacted with pyridine hydrochloride to obtain 4-(benzo[b]thiophen-2-yl)benzene-1,3-diol;

[0030] 6-carboxyfluorescein modified with benzothiophene side chain is obtained by reacting 4-(benzo[b]thiophen-2-yl)benzene-1,3-diol with 2-(2,4-dihydroxybenzoyl)terephthalic acid;

[0031] Or, the fluorescein derivative is 6-carboxyfluorescein modified with a methylamino-type o-phenylthiophene side chain;

[0032] The method for preparing the methylamino-modified 6-carboxyfluorescein with o-phenylthiophene side chain comprises the following steps:

[0033] Preparation of 4-(4-(dimethylamino)-2-hydroxybenzoyl)isophthalic acid;

[0034] 4-(5-phenylthiophen-2-yl)benzene-1,3-diol is obtained by reacting 2-(2,4-dimethoxyphenyl)-5-phenylthiophene with pyridine hydrochloride;

[0035] 6-carboxyfluorescein modified with methylamino-1-phenylthiophene side chain was obtained by reacting 4-(5-phenylthiophene-2-yl)benzene-1,3-diol and 4-(4-(dimethylamino)-2-hydroxybenzoyl)isophthalic acid.

[0036] In a third aspect, the present invention further provides an application of the fluorescein derivative or the fluorescein derivative prepared by the preparation method in immunofluorescence.

[0037] The fluorescein derivatives provided by the present invention and their preparation methods and applications have the following beneficial effects compared to the prior art:

[0038] The invention provides four asymmetric fluorescein derivatives and preparation methods thereof. Compared with traditional fluorescein, the four compounds all have obvious red shift phenomenon in fluorescence emission wavelength. Among such dyes, their Stokes shifts are all relatively large, among which the Stokes shifts of 6-carboxyfluorescein modified with o-phenylthiophene side chain and 6-carboxyfluorescein probe modified with dimethylamino o-phenylthiophene side chain are as high as about 100nm. The four asymmetric fluorescein derivatives can be derived into NHS activated esters of carboxyfluorescein, which can be applied to antibody labeling technology for immunofluorescence, and the four types of compounds can also be derived into phosphoramidite monomers directly used in DNA solid phase synthesis and used to modify the 5' end of DNA probes, and can also be loaded on CPG or PS after derivatization to directly label the 3' end of DNA probes. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0040] Figure 1 is the structural formula of conventional fluorescein dye;

[0041] Figure 2is a reaction process diagram of four asymmetric fluorescein derivatives of the present invention;

[0042] Figure 3 The UV absorption spectra of 6-carboxyfluorescein modified with o-phenylthiophene side chain (Fam-T-Ph) at different pH values;

[0043] Figure 4 The fluorescence emission spectra of 6-carboxyfluorescein modified with o-phenylthiophene side chain (Fam-T-Ph) at different pH values;

[0044] Figure 5 It is a line graph of the maximum fluorescence intensity of 6-carboxyfluorescein modified with o-phenylthiophene side chain (Fam-T-Ph) at different pH values;

[0045] Figure 6 The UV absorption spectra of 6-carboxyfluorescein modified with o-methylthiophene side chain (Fam-T-CH3) at different pH values;

[0046] Figure 7 The fluorescence emission spectra of 6-carboxyfluorescein modified with o-methylthiophene side chain (Fam-T-CH3) at different pH values;

[0047] Figure 8 It is a line graph of the maximum fluorescence intensity of 6-carboxyfluorescein modified with o-methylthiophene side chain (Fam-T-CH3) at different pH values;

[0048] Fig. 9 The UV absorption spectra of 6-carboxyfluorescein modified with benzothiophene side chain (Fam-PT) at different pH values;

[0049] Fig.10 The fluorescence emission spectra of 6-carboxyfluorescein modified with benzothiophene side chain (Fam-PT) at different pH values;

[0050] Fig.11 It is a line graph of the maximum fluorescence intensity of 6-carboxyfluorescein modified with benzothiophene side chain (Fam-PT) at different pH;

[0051] Fig.12 The UV absorption spectra of 6-carboxyfluorescein (Rh-T-Ph) modified with dimethylamino-1-phenylthiophene side chain at different pH values;

[0052] Fig.13 The fluorescence emission spectra of 6-carboxyfluorescein (Rh-T-Ph) modified with dimethylamino-1-phenylthiophene side chain at different pH values;

[0053] Fig.14It is a line graph of the maximum fluorescence intensity of 6-carboxyfluorescein (Rh-T-Ph) modified with dimethylamino-1-phenylthiophene side chain at different pH values;

[0054] Fig.15 The UV absorption spectra of four fluorescent dyes and traditional carboxyfluorescein tested under the same conditions. DETAILED DESCRIPTION

[0055] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0056] The present invention provides a fluorescein derivative, including any one of o-methylthiophene side chain modified 6-carboxyfluorescein (Fam-T-CH3), o-benzothiophene side chain modified 6-carboxyfluorescein (Fam-T-Ph), benzothiophene side chain modified 6-carboxyfluorescein (Fam-PT), and dimethylamino o-benzothiophene side chain modified 6-carboxyfluorescein (Rh-T-Ph);

[0057] Among them, 6-carboxyfluorescein modified with o-methylthiophene side chain (Fam-T-CH3) has the following structural formula:

[0058]

[0059] 6-Carboxyfluorescein modified with o-phenylthiophene side chain (Fam-T-Ph) has the following structural formula:

[0060]

[0061] 6-Carboxyfluorescein modified with benzothiophene side chain (Fam-PT) has the following structural formula:

[0062]

[0063] 6-Carboxyfluorescein (Rh-T-Ph) modified with dimethylamino-1,2-diphenylthiophene side chain has the following structural formula:

[0064]

[0065] Based on the same inventive concept, the present invention also provides a method for preparing the above-mentioned fluorescein derivatives, wherein the fluorescein derivatives are 6-carboxyfluorescein (Fam-T-CH3) modified with an o-methylthiophene side chain;

[0066] The preparation method of the above-mentioned o-methylthiophene side chain modified 6-carboxyfluorescein (Fam-T-CH3) comprises the following steps:

[0067] S11, preparing 2-(2,4-dimethoxyphenyl)-5-methylthiophene;

[0068] S12, using 2-(2,4-dimethoxyphenyl)-5-methylthiophene to react with pyridine hydrochloride to obtain 4-(5-methylthiophen-2-yl)benzene-1,3-diol;

[0069] S13. 6-carboxyfluorescein modified with o-methylthiophene side chain is obtained by reacting 4-(5-methylthiophene-2-yl)benzene-1,3-diol and 2-(2,4-dihydroxybenzoyl)terephthalic acid.

[0070] Specifically, 2-(2,4-dimethoxyphenyl)-5-methylthiophene (Compound 1) has the structural formula The specific preparation method is as follows:

[0071] Weigh 2,4-dimethoxyphenylboronic acid and tetrakistriphenylphosphine palladium into a reaction flask, replace the inert gas in the reaction system, add toluene, ethanol and alkali metal carbonate aqueous solution into the reaction flask, then add 2-bromo-5-methylthiophene into the reaction flask, reflux at 80-100°C, and monitor the reaction by TLC until the end. The reaction solution is cooled, water is added, extracted with dichloromethane, washed with saturated brine, the organic phase is dried, rotary evaporated, and separated by column chromatography to obtain a light yellow liquid product of compound 1.

[0072] In some embodiments, the molar ratio of 2-bromo-5-methylthiophene, 2,4-dimethoxyphenylboronic acid and tetrakistriphenylphosphine palladium is (0.9-1.1) mmol: (1.2-1.4) mmol: (0.03-0.04) mmol.

[0073] In some embodiments, at the step of adding the reactants, the volume ratio of toluene, ethanol and alkali metal carbonate aqueous solution is (5-7) ml: (2-3) ml: (2-3) ml;

[0074] In some embodiments, after the step of adding the above reactants and solvent is completed, the reaction temperature is raised to 80-100° C., the reaction time is 0.5-1 h, and finally compound 1 is obtained;

[0075] In some embodiments, the alkali metal carbonate used above can be any one of Cs2CO3, K2CO3, Na2CO3 and Li2CO3, and the concentration of the prepared alkali metal carbonate aqueous solution is 2.0-4.0M.

[0076] Specifically, 4-(5-methylthiophen-2-yl)benzene-1,3-diol (Compound 2) has the structural formula The preparation method of 4-(5-methylthiophene-2-yl)benzene-1,3-diol (Compound 2) is:

[0077] Weigh the prepared 2-(2,4-dimethoxyphenyl)-5-methylthiophene (Compound 1) and pyridine hydrochloride in a single-necked flask, replace the inert gas in the reaction system, and stir under reflux at 180-200°C. After the reaction is completed, cool to room temperature, add HCl aqueous solution to dissolve the solid in the reaction flask, extract with dichloromethane, wash three times with HCl aqueous solution, dry, and rotary evaporate. The crude product is separated by column chromatography to obtain Compound 2, a light pink solid product.

[0078] In some embodiments, the molar ratio of 2-(2,4-dimethoxyphenyl)-5-methylthiophene (Compound 1) to pyridine hydrochloride is (0.9-1.1) mmol: (9.0-16.5) mmol.

[0079] In some embodiments, after the step of adding the above reactants, the reaction temperature is raised to 180-200° C. and refluxed for 3-4 hours to finally obtain compound 2.

[0080] In some embodiments, the concentration of the HCl aqueous solution used in the post-reaction treatment is 1.0M to 3.0M.

[0081] Specifically, 6-carboxyfluorescein modified with an o-methylthiophene side chain (Fam-T-CH3), namely 3',6'-dihydroxy-2'-(5-methylthiophene-2-yl)-3-oxo-3H-spiro-[isobenzofuran-1,9'-xanthene]-6-carboxylic acid, is prepared by:

[0082] Weigh the 4-(5-methylthiophen-2-yl)benzene-1,3-diol (Compound 2) and 2-(2,4-dihydroxybenzoyl)terephthalic acid (Compound 7) obtained above into a flask, replace the inert gas in the reaction system, then add an organic solvent into the reaction bottle, set 60-100°C to reflux and stir, after the reaction is completed, add the reaction solution dropwise into ice water, solid precipitates, filter under reduced pressure and wash with cold water several times, dry, and separate by column chromatography to obtain a red-black solid product, which is 6-carboxyfluorescein modified with o-methylthiophene side chain (Fam-T-CH3).

[0083] In some embodiments, the molar ratio of 4-(5-methylthiophen-2-yl)benzene-1,3-diol to 2-(2,4-dihydroxybenzoyl)terephthalic acid is 1 mmol:1.0-1.1 mmol.

[0084] In some embodiments, the organic solvent added to the reaction can be methanesulfonic acid or a mixture of methanesulfonic acid and trifluoroacetic acid (volume ratio 1:1); the ratio of reactants to solvent is 1.0-1.1 mmol / 5-10 ml; that is, the volume ratio of the sum of the amounts of 4-(5-methylthiophen-2-yl)benzene-1,3-diol and 2-(2,4-dihydroxybenzoyl)terephthalic acid to the organic solvent is (1.0-1.1) mmol:(5-10) ml.

[0085] In some embodiments, 2-(2,4-dihydroxybenzoyl)terephthalic acid (Compound 7, Formula C 15 H 10 O7, CAS: 1819991-87-1) has the structural formula:

[0086] Based on the same inventive concept, the present invention also provides a method for preparing the above-mentioned fluorescein derivatives, wherein the fluorescein derivative is 6-carboxyfluorescein (Fam-T-Ph) modified with an o-phenylthiophene side chain, and the method for preparing 6-carboxyfluorescein modified with an o-phenylthiophene side chain comprises the following steps:

[0087] S21, preparation of 2-(2,4-dimethoxyphenyl)-5-2-phenylthiophene;

[0088] S22, using 2-(2,4-dimethoxyphenyl)-5-phenylthiophene and pyridine hydrochloride to react to obtain 4-(5-phenylthiophen-2-yl)benzene-1,3-diol;

[0089] S23. 6-carboxyfluorescein modified with o-phenylthiophene side chain is obtained by reacting 4-(5-phenylthiophene-2-yl)benzene-1,3-diol with 2-(2,4-dihydroxybenzoyl)terephthalic acid.

[0090] Specifically, 2-(2,4-dimethoxyphenyl)-5-2-phenylthiophene (Compound 3) has the structural formula:

[0091]

[0092] A method for preparing 2-(2,4-dimethoxyphenyl)-5-2-phenylthiophene (Compound 3), comprising:

[0093] Weigh 2,4-dimethoxyphenylboronic acid, 2-bromo-5-phenylthiophene and tetrakistriphenylphosphine palladium into a reaction flask, replace argon in the reaction system, add toluene, ethanol and an alkali metal carbonate aqueous solution into the reaction flask, reflux at 80-100° C., and monitor the completion of the reaction by TLC; cool the reaction solution, add water, extract with dichloromethane and then with saturated brine, dry the organic phase, rotary evaporate, and separate by column chromatography to obtain compound 3 as a white solid product.

[0094] In some embodiments, the molar ratio of 2-bromo-5-phenylthiophene, 2,4-dimethoxyphenylboronic acid, and tetrakistriphenylphosphine palladium is (0.9-1.1) mmol: (1.2-1.4) mmol: (0.03-0.04) mmol.

[0095] In some embodiments, at the step of adding the reactants, the volume ratio of toluene, ethanol and alkali metal carbonate aqueous solution is (5-7) ml: (2-3) ml: (2-3) ml.

[0096] In some embodiments, after the step of adding the above reactants and solvent is completed, the reaction temperature is raised to 80-100° C., the reaction time is 1-2 hours, and finally compound 3 is obtained.

[0097] In some embodiments, the alkali metal carbonate used above can be any one of Cs2CO3, K2CO3, Na2CO3 and Li2CO3, and the concentration of the prepared alkali metal carbonate aqueous solution is 2.0-4.0M.

[0098] Specifically, 4-(5-phenylthiophen-2-yl)benzene-1,3-diol (Compound 4) has the structural formula: A method for preparing 4-(5-phenylthiophene-2-yl)benzene-1,3-diol (Compound 4), comprising:

[0099] The 2-(2,4-dimethoxyphenyl)-5-phenylthiophene (Compound 3) and pyridine hydrochloride obtained above were weighed into a single-necked flask, the reaction system was replaced with an inert gas, and reflux stirring was set at 180-200°C; after the reaction, an HCl aqueous solution was added to dissolve the solid in the reaction flask, and dichloromethane was added for extraction, and then the solid was washed three times with an HCl aqueous solution, dried, and rotary evaporated. The crude product was separated by column chromatography to obtain a white solid product of Compound 4.

[0100] In some embodiments, the molar ratio of 2-(2,4-dimethoxyphenyl)-5-phenylthiophene (Compound 3) to pyridine hydrochloride is (0.9-1.1) mmol: (9.0-16.5) mmol.

[0101] In some embodiments, after the step of adding the above reactants, the reaction temperature is raised to 180-200° C. and refluxed for 3-4 hours to finally obtain compound 4.

[0102] In some embodiments, the concentration of the HCl aqueous solution used in the post-reaction treatment is 1.0M to 3.0M.

[0103] Specifically, 6-carboxyfluorescein modified with o-phenylthiophene side chain (Fam-T-Ph), namely 3',6'-dihydroxy-2'-(5-phenylthiophene-2-yl)-3-oxo-3H-spiro-[isobenzofuran-1,9'-xanthene]-6-carboxylic acid, is prepared by:

[0104] Weigh the previously obtained 4-(5-phenylthiophene-2-yl)benzene-1,3-diol (Compound 4) and 2-(2,4-dihydroxybenzoyl)terephthalic acid (Compound 7) in a flask, replace the inert gas in the reaction system, then add an organic solvent to the reaction bottle, set 60-100°C for reflux and stirring, after the reaction is completed, add the reaction solution dropwise into ice water, solid precipitates, vacuum filter and wash with cold water several times. Dry, mix and separate by column chromatography to obtain a purple-black solid product, which is 6-carboxyfluorescein modified with o-phenylthiophene side chain (Fam-T-Ph).

[0105] In some embodiments, the molar ratio of 4-(5-phenylthiophen-2-yl)benzene-1,3-diol to 2-(2,4-dihydroxybenzoyl)terephthalic acid is 1 mmol:1.0-1.1 mmol.

[0106] In some embodiments, the organic solvent added to the reaction can be methanesulfonic acid or a mixture of methanesulfonic acid and trifluoroacetic acid (volume ratio 1:1); the ratio of reactants to organic solvent is 1-1.1 mmol / 5-10 ml; that is, the volume ratio of the sum of the amounts of 4-(5-phenylthiophene-2-yl)benzene-1,3-diol and 2-(2,4-dihydroxybenzoyl)terephthalic acid to the organic solvent is (1.0-1.1) mmol:(5-10) ml.

[0107] Based on the same inventive concept, the present invention also provides a method for preparing a fluorescein derivative, wherein the fluorescein derivative is 6-carboxyfluorescein modified with a benzothiophene side chain (Fam-PT), and the method for preparing 6-carboxyfluorescein modified with a benzothiophene side chain comprises the following steps:

[0108] S31, preparing 2-(2,4-dimethoxyphenyl)benzo[b]thiophene;

[0109] S32, using 2-(2,4-dimethoxyphenyl)benzo[b]thiophene to react with pyridine hydrochloride to obtain 4-(benzo[b]thiophen-2-yl)benzene-1,3-diol;

[0110] S33. 6-carboxyfluorescein modified with benzothiophene side chain is obtained by reacting 4-(benzo[b]thiophene-2-yl)benzene-1,3-diol with 2-(2,4-dihydroxybenzoyl)terephthalic acid.

[0111] Specifically, 2-(2,4-dimethoxyphenyl)benzo[b]thiophene (Compound 5) has the structural formula:

[0112] The preparation method of 2-(2,4-dimethoxyphenyl)benzo[b]thiophene (Compound 5) comprises:

[0113] Weigh 2,4-dimethoxyphenylboronic acid, 2-bromobenzothiophene and tetrakistriphenylphosphine palladium into a reaction flask, replace argon in the reaction system, add toluene, ethanol and alkali metal carbonate aqueous solution into the reaction flask, reflux at 80-100°C, and monitor the completion of the reaction by TLC. Cool the reaction solution, add water, extract with dichloromethane and then with saturated brine, dry the organic phase, rotary evaporate, and separate by column chromatography to obtain compound 5 as a white solid product.

[0114] In some embodiments, the molar ratio of 2-bromobenzothiophene, 2,4-dimethoxyphenylboronic acid, and tetrakistriphenylphosphine palladium is (0.9-1.1) mmol: (1.2-1.4) mmol: (0.03-0.04) mmol.

[0115] In some embodiments, at the step of adding the reactants, the volume ratio of toluene, ethanol and alkali metal carbonate aqueous solution is (5-7) ml: (2-3) ml: (2-3) ml.

[0116] In some embodiments, after the step of adding the above reactants and solvent is completed, the reaction temperature is raised to 80-100° C., the reaction time is 1-2 hours, and finally compound 5 is obtained;

[0117] In some embodiments, the alkali metal carbonate used above can be any one of Cs2CO3, K2CO3, Na2CO3 and Li2CO3, and the concentration of the prepared alkali metal carbonate aqueous solution is 2.0-4.0M.

[0118] Specifically, the chemical formula of 4-(benzo[b]thiophen-2-yl)benzene-1,3-diol (Compound 6) is:

[0119] A method for preparing 4-(benzo[b]thiophen-2-yl)benzene-1,3-diol (Compound 6) comprises:

[0120] Weigh the previously obtained 2-(2,4-dimethoxyphenyl)benzo[b]thiophene (Compound 5) and pyridine hydrochloride in a single-necked flask, replace the inert gas in the reaction system, set reflux stirring at 180-200°C. After the reaction is completed, cool to room temperature, add HCl aqueous solution to dissolve the solid in the reaction flask, extract with dichloromethane, wash three times with HCl aqueous solution, dry, and rotary evaporate. The crude product is separated by column chromatography to obtain a white solid product, thereby obtaining Compound 6.

[0121] In some embodiments, the molar ratio of 2-(2,4-dimethoxyphenyl)benzo[b]thiophene (Compound 5) and pyridine hydrochloride is (0.9-1.1) mmol: (9.0-16.5) mmol;

[0122] In some embodiments, after the step of adding the above reactants, the reaction temperature is raised to 180-200° C. and refluxed for 3-4 hours to finally obtain compound 6.

[0123] In some embodiments, the concentration of the HCl aqueous solution used in the post-reaction treatment is 1.0M to 3.0M.

[0124] Specifically, the benzothiophene side chain modified 6-carboxyfluorescein (Fam-PT) is 2'-(benzo[b]thiophene-2-yl)-3',6'-dihydroxy-3-oxo-3H-spiro-[isobenzofuran-1,9'-xanthene]-6-carboxylic acid, and the preparation method thereof comprises:

[0125] Weigh the previously obtained 4-(benzo[b]thiophene-2-yl)benzene-1,3-diol (Compound 6) and 2-(2,4-dihydroxybenzoyl)terephthalic acid (Compound 7) in a flask, replace the inert gas in the reaction system, then add an organic solvent to the reaction bottle, set 60-100°C for reflux and stirring, after the reaction is completed, add the reaction solution dropwise to ice water, solid precipitates, decompress and filter, and wash with cold water several times. Dry, mix, and separate by column chromatography to obtain a purple-black solid product, which is 6-carboxyfluorescein modified with benzothiophene side chain (Fam-PT).

[0126] In some embodiments, the molar ratio of 4-(benzo[b]thiophen-2-yl)benzene-1,3-diol to 2-(2,4-dihydroxybenzoyl)terephthalic acid is 1 mmol:1.0-1.1 mmol.

[0127] In some embodiments, the organic solvent added to the reaction can be methanesulfonic acid or a mixture of methanesulfonic acid and trifluoroacetic acid (volume ratio 1:1); the ratio of reactants to solvent is 1.0-1.1 mmol / 5-10 ml; that is, the volume ratio of the sum of the amounts of 4-(benzo[b]thiophen-2-yl)benzene-1,3-diol and 2-(2,4-dihydroxybenzoyl)terephthalic acid to the organic solvent is (1.0-1.1) mmol:(5-10) ml.

[0128] Based on the same inventive concept, the present invention also provides a method for preparing a fluorescein derivative, wherein the fluorescein derivative is 6-carboxyfluorescein (Rh-T-Ph) modified with a methylamino-1,1-diphenylthiophene side chain; the method for preparing 6-carboxyfluorescein modified with a methylamino-1,1-diphenylthiophene side chain comprises the following steps:

[0129] S41, preparing 4-(4-(dimethylamino)-2-hydroxybenzoyl)isophthalic acid;

[0130] S42, using 2-(2,4-dimethoxyphenyl)-5-phenylthiophene and pyridine hydrochloride to react to obtain 4-(5-phenylthiophen-2-yl)benzene-1,3-diol;

[0131] S43. 6-carboxyfluorescein modified with methylamino-1-phenylthiophene side chain is obtained by reacting 4-(5-phenylthiophene-2-yl)benzene-1,3-diol and 4-(4-(dimethylamino)-2-hydroxybenzoyl)isophthalic acid.

[0132] Specifically, 4-(4-(dimethylamino)-2-hydroxybenzoyl)isophthalic acid (Compound 8) has the structural formula: A method for preparing 4-(4-(dimethylamino)-2-hydroxybenzoyl)isophthalic acid (Compound 8), comprising:

[0133] Add dimethylaminophenol to the reaction flask, then add toluene, and heat to 50-70°C; add trimellitic anhydride while stirring; reflux the mixture for 24 hours and cool to room temperature. Filter the residue, wash it three times with an appropriate amount of toluene, and then dissolve the solid in 60-80 ml of methanol and heat it under reflux for 10-20 minutes. Then add 20-30 ml of glacial acetic acid to remove the organic solvent under reduced pressure. Then reflux the solid in 50-70 ml of methanol for 2 hours and keep it at 4°C overnight. Filter the solid product formed, and wash the filter cake with pre-cooled methanol to obtain compound 8 as a purple solid powder.

[0134] In some embodiments, the molar ratio of dimethylaminophenol to trimellitic anhydride is 1 mmol:1.2-1.3 mmol; the dimethylaminophenol is dissolved in toluene and the heating temperature is controlled at 50-70°C.

[0135] Specifically, methylamino-modified o-phenylthiophene side chain-modified 6-carboxyfluorescein (Rh-T-Ph), namely 4-(6-(dimethylamino)-3-oxo-2-(5-phenylthiophene-2-yl)-3H-xanthene-9-yl)isophthalic acid (Rh-T-Ph), is prepared by:

[0136] Weigh the previously obtained 4-(5-phenylthiophene-2-yl)benzene-1,3-diol (Compound 4) and 4-(4-(dimethylamino)-2-hydroxybenzoyl)isophthalic acid (Compound 8) into a flask, replace the inert gas in the reaction system, then add an organic solvent into the reaction bottle, set 60-100°C for reflux and stirring, after the reaction is completed, add the reaction solution dropwise into ice water, solid precipitates, vacuum filter and wash with cold water several times. After drying, dissolve the crude product with an organic solvent, mix the sample and separate by column chromatography to obtain a purple-black solid product, which is 6-carboxyfluorescein (Rh-T-Ph) modified with methylamino-o-phenylthiophene side chain.

[0137] In some embodiments, the molar ratio of 4-(5-phenylthiophen-2-yl)benzene-1,3-diol to 4-(4-(dimethylamino)-2-hydroxybenzoyl)isophthalic acid is 1 mmol:1.0-1.1 mmol.

[0138] In some embodiments, the organic solvent added to the reaction can be methanesulfonic acid or a mixture of methanesulfonic acid and trifluoroacetic acid (volume ratio 1:1); the ratio of reactants to solvent is 1.0-1.1 mmol / 5-10 ml, that is, the volume ratio of the sum of the amounts of 4-(5-phenylthiophene-2-yl)benzene-1,3-diol and 4-(4-(dimethylamino)-2-hydroxybenzoyl)isophthalic acid to the organic solvent is (1.0-1.1) mmol:(5-10) ml.

[0139] Specifically, the synthesis routes of the o-methylthiophene side chain modified 6-carboxyfluorescein (Fam-T-CH3), o-benzothiophene side chain modified 6-carboxyfluorescein (Fam-T-Ph), benzothiophene side chain modified 6-carboxyfluorescein (Fam-PT), and dimethylamino o-benzothiophene side chain modified 6-carboxyfluorescein (Rh-T-Ph) of the present invention are as follows: Figure 2 shown.

[0140] The invention provides four asymmetric fluorescein derivatives and preparation methods thereof. Compared with traditional fluorescein, the four compounds all have obvious red shift phenomenon in fluorescence emission wavelength. Among such dyes, their Stokes shifts are all relatively large, among which the Stokes shifts of 6-carboxyfluorescein modified with o-phenylthiophene side chain and 6-carboxyfluorescein probe modified with dimethylamino o-phenylthiophene side chain are as high as about 100nm. The four asymmetric fluorescein derivatives can be derived into NHS activated esters of carboxyfluorescein, which can be applied to antibody labeling technology for immunofluorescence, and the four types of compounds can also be derived into phosphoramidite monomers directly used in DNA solid phase synthesis and used to modify the 5' end of DNA probes, and can also be loaded on CPG or PS after derivatization to directly label the 3' end of DNA probes.

[0141] The following further describes the fluorescein derivatives and preparation methods of the present application with specific examples. This section further describes the content of the present invention in conjunction with specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0142] Example 1

[0143] The present invention provides a method for preparing 6-carboxyfluorescein (Fam-T-CH3) modified with an o-methylthiophene side chain, comprising the following steps:

[0144] S11, preparation of 2-(2,4-dimethoxyphenyl)-5-methylthiophene (Compound 1):

[0145] Weigh 2,4-dimethoxyphenylboronic acid (1336.1 mg, 7.342 mmol) and add tetrakistriphenylphosphine palladium (195.79 mg, 0.169 mmol) into a reaction flask, set up a reflux condenser, replace argon in the reaction system, add 10 ml of toluene, 4 ml of ethanol and 4 ml of 2M K2CO3 aqueous solution into the reaction flask (the reaction solvent was degassed), then add 2-bromo-5-methylthiophene (177.06 mg, 5.6478 mmol) into the reaction flask by syringe, reflux at 100°C, monitor the reaction by TLC, and the reaction is completed in half an hour. The reaction solution was cooled, and an appropriate amount of water was added to dilute the reaction solution in a separatory funnel. The aqueous phase was extracted twice with dichloromethane, and the organic phase was extracted three times with saturated brine. The organic phase was dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation. The product was separated by column chromatography (PE:DCM=40:1-10:1, i.e., the eluent was PE (petroleum ether) and DCM (dichloromethane), the volume ratio of PE and DCM was 40:1-10:1, and gradient elution) to obtain a light yellow transparent liquid product, i.e., compound 1 (1020.3 mg, 77.1%). 1 H NMR (400MHz, DMSO-d6) δ7.51(d,J=8.6Hz,1H),7.20(d,J=3.6Hz,1H),6.74(dd,J=3.5,1.2Hz,1H),6 .65(d,J=2.4Hz,1H),6.57(dd,J=8.6,2.5Hz,1H),3.86(s,3H),3.79(s,3H),2.43(d,J=1.1Hz,3H). 13C NMR (101MHz, CDCl3) δ159.87,156.64,138.67,137.31,129.10,125.02,124.38,116.96,104.94,99.03,77.36,77.04,76.72,55.55,55.45,15.21;

[0146] S12, preparation of 4-(5-methylthiophene-2-yl)benzene-1,3-diol (Compound 2):

[0147] The compound 1 (500 mg, 2.136 mmol) and pyridine hydrochloride (2.999 g, 25.96 mmol) obtained above were weighed into a 10 ml single-necked flask, a reflux condenser was set, the reaction system was replaced with argon, and refluxed at 180°C for 3 hours. During the heating process, pyridine hydrochloride melted into the reaction solution to assist in stirring the reactants. After the reaction, the mixture was cooled to room temperature, and an appropriate amount of 1M HCl aqueous solution was added to dissolve the solid in the reaction flask. The residue was poured into a separatory funnel by ultrasonic treatment, and dichloromethane was added to extract the aqueous phase several times. The organic phase was washed three times with 1M HCl aqueous solution, dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation. The crude product was separated by column chromatography (DCM:CH3OH=50:1-30:1, i.e., the eluent was DCM (dichloromethane) and methanol, the volume ratio of DCM:CH3OH was =50:1-30:1, and gradient elution) to obtain a pink-purple solid product, i.e., compound 2 (380.2 mg, 86.3%). 1 HNMR(400MHz,DMSO-d6)δ9.86(s,1H),9.38(s,1H),7.29(d,J=8.5Hz,1H),7.15(d,J=3.5 Hz,1H),6.69(dd,J=3.6,1.2Hz,1H),6.52–6.27(m,1H),6.35–6.17(m,1H),2.42(s,3H). 13 CNMR(101MHz, CDCl3)δ156.63,153.76,140.56,136.10,131.06,125.95,125.55,114.17,108.06,102.89,77.35,77.03,76.71,15.32;

[0148] S13, preparation of 3',6'-dihydroxy-2'-(5-methylthiophene-2-yl)-3-oxo-3H-spiro-[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (i.e., 6-carboxyfluorescein modified with o-methylthiophene side chain (Fam-T-CH3))

[0149] Weigh the previously obtained compound 2 (100 mg, 0.3308 mmol) and 2-(2,4-dihydroxybenzoyl)terephthalic acid (compound 7) (68 mg, 0.3311 mmol) into a three-necked flask, set a reflux condenser, replace argon in the reaction system, then inject 6 ml of methanesulfonic acid into the reaction bottle, set reflux stirring at 90 °C, monitor the disappearance of the reaction raw materials by TLC, and terminate the reaction after 3 h 30 min. The reaction solution is cooled naturally, and then the reaction solution is slowly added dropwise to a beaker containing 50 ml of ice water with a dropper. Solid precipitation is observed and settles to the bottom of the beaker. Filter under reduced pressure and wash the filter cake with cold water several times. The product was dried at 50°C in vacuo overnight, and then the dried crude product was dissolved in an organic solvent. After dry mixing, the product was separated by gradient elution on a column chromatography (DCM:CH3OH=100:1-50:1, the eluent was DCM (dichloromethane) and methanol, the volume ratio of DCM:CH3OH was =50:1-30:1, gradient elution; the eluent also contained acetic acid (ACOH) with a volume concentration of 0.1%) to obtain a red-black solid product (98.5 mg, 43%), which was 6-carboxyfluorescein modified with an o-methylthiophene side chain (Fam-T-CH3). 1 H NMR (400MHz, DMSO-d6) δ13.61(s,1H),10.95(s,1H),10.19(s,1H),8.24(dd,J=8.0,1.3Hz,1H),8.13(d,J=8.0Hz,1H),7. 69(s,1H),7.07(d,J=3.6Hz,1H),6.86(s,1H),6.78(s,1H),6.75–6.64(m,2H),6.65–6.54(m,2H),2.37(d,J=1.1Hz,3H). 13 C NMR (101MHz, DMSO) δ168.34,166.52,160.15,156.04,153.11,152.22,150.80,139.24,137.97,135.89,131.48,129.84,129.65,126.69,1 25.87,125.80,125.56,124.88,119.32,113.31,110.14,109.39,103 .49,102.84,40.59,40.39,40.18,39.97,39.76,39.55,39.34,15.18.

[0150] Example 2

[0151] This embodiment provides a method for preparing 6-carboxyfluorescein (Fam-T-Ph) modified with an o-phenylthiophene side chain, comprising the following steps:

[0152] S21, preparation of 2-(2,4-dimethoxyphenyl)-5-2-phenylthiophene (Compound 3):

[0153] Weigh 2,4-dimethoxyphenylboronic acid (1978.6 mg, 10.872 mmol) and 2-bromo-5-phenylthiophene (2 g, 8.363 mmol), add tetrakistriphenylphosphine palladium (289.9 mg, 0.251 mmol) into a reaction flask, set up a reflux condenser, replace argon in the reaction system, add 20 ml of toluene, 8 ml of ethanol and 8 ml of 2M K2CO3 aqueous solution into the reaction flask (the reaction solvent was degassed), heat to 100°C, stir and reflux, monitor the reaction by TLC, and the reaction is completed in 4 hours. The reaction solution was cooled, and an appropriate amount of water was added to dilute the reaction solution in a separatory funnel. The aqueous phase was extracted twice with dichloromethane, and the organic phase was washed three times with saturated brine, and then dried over anhydrous sodium sulfate. The organic solvent was removed by rotary evaporation and separated by column chromatography (PE:DCM=10:1-5:1, i.e., the eluent was PE (petroleum ether) and DCM (dichloromethane), the volume ratio of PE and DCM was 10:1-5:1, gradient elution) to obtain white solid powder compound 3 (2.23 g, 90%). 1 H NMR (400MHz, DMSO-d6) δ7.72–7.62(m,3H),7.46(s,2H),7.41(t,J=7.7Hz,2H),7.32–7. 25(m,1H),6.70(d,J=2.4Hz,1H),6.63(dd,J=8.6,2.4Hz,1H),3.92(s,3H),3.81(s,3H). 13 C NMR (101MHz, CDCl3) δ160.26,156.82,142.68,139.24,134.78,129.06,128.83,127.0 8,125.62,125.30,123.00,116.53,105.15,99.06,77.36,77.04,76.73,55.62,55.49;

[0154] S22, preparation of 4-(5-phenylthiophene-2-yl)benzene-1,3-diol (Compound 4):

[0155] Weigh the compound 3 (1 g, 3.378 mmol) and pyridine hydrochloride (3.903 g, 33.780 mmol) obtained above into a 10 ml single-necked flask, set a reflux condenser, replace argon in the reaction system, set 180 ° C to reflux and stir for 3 hours. During the heating process, pyridine hydrochloride melts as the reaction liquid to help the reactants stir. After the reaction is completed, cool the reaction, add an appropriate amount of 1M HCl aqueous solution to dissolve the solid in the reaction bottle, ultrasonicate the residual solid, pour it into a separatory funnel, add dichloromethane to extract the aqueous phase several times, then wash the organic phase three times with 1M HCl aqueous solution, dry it with anhydrous sodium sulfate, and remove the organic solvent by rotary evaporation. The crude product is separated by column chromatography (DCM:CH3OH=70:1-40:1, the eluent is DCM (dichloromethane) and methanol, the volume ratio of DCM:CH3OH is 70:1-40:1, gradient elution) to obtain an off-white solid product compound 4 (703.4 mg, 77.6%). 1 H NMR(400MHz,DMSO-d6)δ10.12(s,1H),9.52(s,1H),7.68–7.59(m,2H),7.48–7.3 6(m,5H),7.30–7.22(m,1H),6.42(d,J=2.4Hz,1H),6.31(dd,J=8.5,2.4Hz,1H). 13 C NMR(101MHz,DMSO)δ157.30,154.00,139.58,139.36,133.62,128.44,127.67,126.36 ,124.23,123.23,122.74,111.59,106.76,102.27,39.32,39.11,38.90,38.69,38.49;

[0156] S23, preparation of 3',6'-dihydroxy-2'-(5-phenylthiophene-2-yl)-3-oxo-3H-spiro-[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (6-carboxyfluorescein modified with o-phenylthiophene side chain (Fam-T-Ph))

[0157] Weigh the previously obtained compound 4 (100 mg, 0.3308 mmol) and 2-(2,4-dihydroxybenzoyl)terephthalic acid (compound 7) (89.65 mg, 0.3341 mmol) into a three-necked flask, set a reflux condenser, replace argon in the reaction system, then inject 6 ml of methanesulfonic acid into the reaction bottle, set reflux stirring at 120 °C, monitor the disappearance of the raw materials by TLC, and terminate the reaction after 3 hours. The reaction solution is cooled naturally, and then the reaction solution is slowly added dropwise to a beaker containing 50 ml of ice water with a dropper. Solid precipitation is observed and settles to the bottom of the beaker. Filter under reduced pressure and wash the filter cake with cold water several times. The product was dried at 50°C in vacuo overnight, and then the dried crude product was dissolved in an organic solvent. After dry mixing, the product was separated by gradient elution on a column chromatography (DCM:CH3OH=200:1-50:1, the eluent was DCM (dichloromethane) and methanol, the volume ratio of DCM:CH3OH was 200:1-50:1, gradient elution; the eluent also contained acetic acid (ACOH) with a volume concentration of 0.1%) to obtain a purple-black solid product (89.65 mg, 45%), which was 6-carboxyfluorescein (Fam-T-Ph) modified with an o-phenylthiophene side chain. 1 HNMR(400MHz,DMSO-d6)δ11.20(s,1H),10.21(s,1H),8.24(dd,J=8.0,1.4Hz,1H),8.14(d,J=8.0Hz,1H),7.70(s,1H),7.6 6–7.56(m,2H),7.43–7.34(m,3H),7.33–7.24(m,2H),6.93(d,J=20.5Hz,2H),6.72(d,J=2.2Hz,1H),6.59(d,J=7.0Hz,2H). 13 C NMR (101MHz, DMSO) δ168.50,166.75,160.21,152.15,142.90,137.70,134.19,131.44,130.11,129.60,129.54,127.9 1,126.85,126.67,125.59,123.95,113.39,109.63,103.59,102.84,40.62,40.41,40.20,39.99,39.78,39.57,39.37.

[0158] Example 3

[0159] This embodiment provides a method for preparing 6-carboxyfluorescein modified with a benzothiophene side chain (Fam-PT), comprising the following steps:

[0160] S31, preparation of 2-(2,4-dimethoxyphenyl)benzo[b]thiophene (Compound 5):

[0161] Weigh 2,4-dimethoxyphenylboronic acid (827.1 mg, 4.545 mmol) and 2-bromobenzothiophene (745 mg, 3.496 mmol), add tetrakistriphenylphosphine palladium (121.2 mg, 0.104 mmol) into a reaction flask, set up a reflux condenser, replace argon in the reaction system, add 10 ml of toluene, 4 ml of ethanol and 4 ml of 2M K2CO3 aqueous solution into the reaction flask (the reaction solvent was degassed), heat to 100°C, stir and reflux, monitor the reaction by TLC, and the reaction is complete after 1 hour. The reaction solution was cooled, and an appropriate amount of water was added to dilute the reaction solution in a separatory funnel. The aqueous phase was extracted twice with two organic phases of chlorine, and then the organic phase was washed three times with saturated brine, and then dried over anhydrous sodium sulfate. The organic solvent was removed by rotary evaporation, and column chromatography was used for separation (PE:DCM=8:1, eluent was PE (petroleum ether) and DCM (dichloromethane), and the volume ratio of PE and DCM was 8:1) to obtain white solid compound 5 (827 mg, 87.5%). 1 HNMR(400MHz,DMSO-d6)δ7.91(dd,J=8.0,1.1Hz,1H),7.81(s,2H),7.69(d,J=8.6Hz,1H),7.4 1–7.21(m,2H),6.73(d,J=2.5Hz,1H),6.67(dd,J=8.6,2.5Hz,1H),3.94(s,3H),3.83(s,3H). 13 CNMR(101MHz,CDCl3)δ160.83,157.55,140.38,140.17,139.37,130.32,124.08,12 3.70,123.18,121.78,121.17,116.37,105.15,99.14,77.35,77.04,76.72,55.50;

[0162] S32, preparation of 4-(benzo[b]thiophene-2-yl)benzene-1,3-diol (Compound 6):

[0163] Weigh the previously obtained compound 5 (800 g, 2.963 mmol) and pyridine hydrochloride (4.108 g, 35.556 mmol) into a 10 ml single-necked flask, set a reflux condenser, replace argon in the reaction system, set 180°C to reflux and stir for 3 hours. During the heating process, pyridine hydrochloride melts into the reaction solution to assist in stirring the reactants. After the reaction was completed, the reaction was cooled and an appropriate amount of 1M HCl aqueous solution was added to dissolve the solid in the reaction flask. The residual solid was sonicated and poured into a separatory funnel. Dichloromethane was added to extract the aqueous phase several times, and then the organic phase was washed three times with 1M HCl aqueous solution and dried over anhydrous sodium sulfate. The organic solvent was removed by rotary evaporation and the crude product was separated by column chromatography (DCM:CH3OH=100:1-50:1, eluent was DCM (dichloromethane) and methanol, the volume ratio of DCM:CH3OH was 100:1-50:1, gradient elution) to obtain a light pink solid product, compound 6 (615.9 mg, 85.8%). 1 HNMR(400MHz,DMSO-d6)δ10.21(s,1H),9.63(s,1H),7.88(dd,J=7.8,1.1Hz,1H),7.82–7.68(m,2H),7.47(d ,J=8.5Hz,1H),7.29(dddd,J=24.4,8.3,7.2,1.3Hz,2H),6.46(d,J=2.4Hz,1H),6.35(dd,J=8.6,2.4Hz,1H). 13 C NMR(101MHz,DMSO)δ159.08,156.04,141.41,140.63,138.59,129.95,124.62,123.87,123.2 9,122.20,119.65,112.39,107.98,103.47,40.61,40.40,40.20,39.99,39.78,39.57,39.36;

[0164] S33, preparation of 2'-(benzo[b]thiophene-2-yl)-3',6'-dihydroxy-3-oxo-3H-spiro-[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (i.e., benzothiophene side chain modified 6-carboxyfluorescein (Fam-PT)):

[0165] Weigh the previously obtained compound 6 (250 mg, 0.8264 mmol) and 2-(2,4-dihydroxybenzoyl)terephthalic acid (compound 7) (200 mg, 0.8264 mmol) into a three-necked flask, set a reflux condenser, replace argon in the reaction system, then inject 10 ml of methanesulfonic acid into the reaction bottle, set reflux stirring at 100 °C, monitor the disappearance of the raw materials by TLC, and terminate the reaction after 2 hours. The reaction solution is cooled naturally, and then the reaction solution is slowly added dropwise to a beaker containing 50 ml of ice water with a dropper. Solid precipitation is observed and settled to the bottom of the beaker. Filter under reduced pressure and wash the filter cake with cold water several times. The mixture was dried at 50°C in vacuum overnight, and then the dried crude product was dissolved in an organic solvent. After dry mixing, the mixture was separated by gradient elution on a column chromatography (DCM:CH3OH=200:1-50:1, the eluent was DCM (dichloromethane) and methanol, the volume ratio of DCM:CH3OH was 200:1-50:1, and the gradient elution was performed; the eluent also contained acetic acid (ACOH) at a volume concentration of 0.1%) to obtain a dark solid product (199.37 mg, 38%), which was 6-carboxyfluorescein modified with a benzothiophene side chain (Fam-PT). 1 HNMR (400MHz, DMSO-d6) δ13.64(s,1H),11.27(s,1H),10.23(s,1H),8.31–8.23(m,1H),8.17(d,J=8.1Hz,1H),7.88(d,J=7.7Hz ,1H),7.79(s,1H),7.73(d,J=4.7Hz,2H),7.34–7.26(m,2H),6.97(d,J=12.7Hz,2H),6.74(d,J=2.3Hz,1H),6.67–6.57(m,2H). 13 C NMR(101MHz,DMSO)δ168.37,166.54,160.21,157.06,153.09,152.13,151 .78,140.21,138.96,138.79,138.13,131.59,129.67,128.22,125.90,12 4.95,124.84,124.69,123.96,122.38,122.31,118.69,113.44,110.49,1 09.42,103.78,102.90,40.60,40.39,40.18,39.98,39.77,39.56,39.35.

[0166] Example 4

[0167] This embodiment provides a method for preparing 6-carboxyfluorescein (Rh-T-Ph) modified with dimethylamino-o-phenylthiophene side chain, comprising the following steps:

[0168] S41. Preparation of 4-(4-(dimethylamino)-2-hydroxybenzoyl)isophthalic acid (Compound 8)

[0169] Dimethylaminophenol (2.20 g, 16.0 mmol) was added to the reaction flask, dissolved in 50 ml of toluene and heated to 60 ° C. Powdered trimellitic anhydride (3.84 g, 20.0 mmol) was added while stirring. The above mixture was refluxed for 24 hours and cooled to room temperature. The residue was filtered out, washed three times with an appropriate amount of toluene, and then the solid was dissolved in 80 ml of methanol and heated to reflux for 10 minutes. Then 20 ml of glacial acetic acid was added to remove the organic solvent under reduced pressure at 60 ° C. The solid was then refluxed in 50 ml of methanol for 2 hours and kept at 4 ° C overnight. The solid product formed was filtered, and the filter cake was washed with pre-cooled 50 ml of methanol to obtain compound 5 as a purple solid powder compound 8 (737.65 mg, 14%). 1 H NMR (400MHz, DMSO-d6) δ13.40(s,2H),12.36(s,1H),8.49(d,J=1.7Hz,1H),8.20(dd,J=7.9,1.7Hz,1H),7. 53(d,J=7.9Hz,1H),6.81(d,J=9.1Hz,1H),6.22(dd,J=9.2,2.5Hz,1H),6.11(d,J=2.4Hz,1H),3.01(s,6H). 13 C NMR(101MHz,DMSO)δ197.02,165.50,163.60,155.22,143.14,133.19,132.08,131.03,130.09,1 29.39,127.66,108.83,103.91,96.40,39.53,39.32,39.11,39.00,38.90,38.69,38.49,38.28;

[0170] S42, preparation of 4-(6-(dimethylamino)-3-oxo-2-(5-phenylthiophene-2-yl)-3H-xanthene-9-yl)isophthalic acid (i.e., 6-carboxyfluorescein (Rh-T-Ph) modified with dimethylamino-o-phenylthiophene side chain):

[0171] Weigh the previously obtained compound 4 (133.24 mg, 0.41 mmol) and compound 8 (100 mg, 0.372 mmol) into a three-necked flask, set a reflux condenser, replace argon in the reaction system, then inject 6 ml of methanesulfonic acid into the reaction flask, set reflux stirring at 90 °C, monitor the disappearance of the raw materials by TLC, and terminate the reaction after 3 hours. The reaction solution is cooled naturally, and then the reaction solution is slowly added dropwise to a beaker containing 50 ml of ice water with a dropper. Solid precipitation is observed and settled to the bottom of the beaker. Filter under reduced pressure and wash the filter cake with cold water several times. The mixture was dried overnight at 50 °C in vacuum, and then the dried crude product was dissolved with an organic solvent. After dry mixing, the mixture was separated by gradient elution on a column chromatography (DCM:CH3OH=100:1-40:1, the desorbent was DCM (dichloromethane) and methanol, the volume ratio of DCM:CH3OH was 100:1-40:1, and the gradient elution was performed; the eluent also contained acetic acid (ACOH) at a volume concentration of 0.1%) to obtain a dark purple solid product (91.92 mg, 44%), which was 6-carboxyfluorescein (Rh-T-Ph) modified with a dimethylamino-1,1-diphenylthiophene side chain. 1 HNMR(400MHz,DMSO-d6)δ8.48(s,1H),8.30(d,J=8.0Hz,1H),7.61(d,J=7.7Hz,2H) ,7.45–7.33(m,4H),7.29(s,2H),6.93(d,J=62.8Hz,2H),6.58(s,3H),2.99(s,6H). 13 C NMR (101MHz, DMSO-d6) δ166.67,134.28,129.55,128.99,127.89,126.50,125.58,123.78,41.77,29.46.

[0172] Performance Characterization

[0173] The above four fluorescein derivatives were subjected to UV and fluorescence analysis as follows:

[0174] Use a 1 / 10,000 electronic balance to accurately weigh appropriate amounts of the fluorescein derivatives prepared in Examples 1 to 4 above, and completely dissolve them in dimethyl sulfoxide to prepare a 1 mM stock solution for later use. Prepare 0.1 M PBS buffer solutions of different pH values ​​(range 4.0-11.0), add the above dye stock solutions to obtain a test solution with a final concentration of 10.0 μM, and perform relevant UV and fluorescence tests.

[0175] Figure 3 The UV absorption spectra of 6-carboxyfluorescein modified with o-phenylthiophene side chain (Fam-T-Ph) at different pH values; Figure 4The fluorescence emission spectra of 6-carboxyfluorescein modified with o-phenylthiophene side chain (Fam-T-Ph) at different pH values; Figure 5 This is a line graph of the maximum fluorescence intensity of 6-carboxyfluorescein modified with o-phenylthiophene side chain (Fam-T-Ph) at different pH values.

[0176] Figure 6 The UV absorption spectra of 6-carboxyfluorescein modified with o-methylthiophene side chain (Fam-T-CH3) at different pH values; Figure 7 The fluorescence emission spectra of 6-carboxyfluorescein modified with o-methylthiophene side chain (Fam-T-CH3) at different pH values;

[0177] Figure 8 This is a line graph of the maximum fluorescence intensity of 6-carboxyfluorescein modified with o-methylthiophene side chain (Fam-T-CH3) at different pH values.

[0178] Fig. 9 The UV absorption spectra of 6-carboxyfluorescein modified with benzothiophene side chain (Fam-PT) at different pH values; Fig.10 The fluorescence emission spectra of 6-carboxyfluorescein modified with benzothiophene side chain (Fam-PT) at different pH values; Fig.11 This is a line graph of the maximum fluorescence intensity of 6-carboxyfluorescein modified with benzothiophene side chain (Fam-PT) at different pH values.

[0179] Fig.12 The UV absorption spectra of 6-carboxyfluorescein (Rh-T-Ph) modified with dimethylamino-1-phenylthiophene side chain at different pH values; Fig.13 The fluorescence emission spectra of 6-carboxyfluorescein (Rh-T-Ph) modified with dimethylamino-1-phenylthiophene side chain at different pH values; Fig.14 This is a line graph of the maximum fluorescence intensity of 6-carboxyfluorescein (Rh-T-Ph) modified with dimethylamino-1-phenylthiophene side chain at different pH values.

[0180] According to the above method, four kinds of fluorescein derivative dye mother solutions were prepared respectively, and PBS buffer solution with pH=7.4 and 0.1M was added to obtain 10.0μM fluorescein derivative test solution, and its ultraviolet absorption spectrum was tested; at the same time, the traditional Fam fluorescein (i.e. 6-carboxyfluorescein, molecular formula C 21 H 12 O7) As a comparison, the same method was used to prepare the Fam fluorescein dye mother solution, and a pH = 7.4, 0.1M PBS buffer solution was added to obtain a 10.0 μM Fam fluorescein test solution, and its ultraviolet absorption spectrum was tested; the test results are as follows Fig.15 shown.

[0181] Fig.15 In the figure, FT-CH3 is Fam-T-CH3, FT-Ph is Fam-T-Ph, F-PT is Fam-PT, Rh-Ph is Rh-T-Ph, and Fam is 6-carboxyfluorescein.

[0182] from Figures 3 to 15 It can be seen that the four fluorescein derivatives of the present invention, Fam-T-Ph, Fam-T-CH3, Fam-PT, and Rh-T-Ph, all have an obvious fluorescence signal response under alkaline conditions. Compared with the traditional Fam fluorescein, they all have a good ultraviolet absorbance, and the maximum absorption is significantly red-shifted compared with the traditional Fam fluorescein.

[0183] The UV and fluorescence emission data of four fluorescein derivatives are shown in Table 1 below.

[0184] Table 1 - UV and fluorescence emission data of four fluorescein derivatives

[0185]

[0186] Table 1 max abs is the maximum absorption wavelength, λ max em represents the maximum emission wavelength, Stokes Shift represents the Stokes displacement, which can be expressed as: Stokes Shift = λ max em-λ max abs, Extinction Coefficient represents the extinction coefficient.

[0187] As can be seen from Table 1, the four asymmetric fluoresceins prepared by the present invention have an obvious red shift in emission wavelength compared with traditional 6-carboxyfluorescein, and the Stokes shift of Fam-T-Ph exceeds 100nm. The preparation method is simple and easy to commercialize. The four asymmetric fluorescein derivatives of the present invention can be used for immunofluorescence in antibody labeling technology and for modifying DNA probes in DNA solid phase synthesis.

[0188] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A fluorescein derivative, characterized in that: The fluorescein derivatives include any one of 6-carboxyfluorescein modified with o-methylthiophene side chain, 6-carboxyfluorescein modified with o-phenylthiophene side chain, 6-carboxyfluorescein modified with benzothiophene side chain, and 6-carboxyfluorescein modified with dimethylamino o-phenylthiophene side chain; The structural formula of the 6-carboxyfluorescein modified with the o-methylthiophene side chain is as follows: The structural formula of the 6-carboxyfluorescein modified with the o-phenylthiophene side chain is as follows: The structural formula of the benzothiophene side chain modified 6-carboxyfluorescein is as follows: The structural formula of the methylamino-modified 6-carboxyfluorescein with o-phenylthiophene side chain is as follows:

2. A method for preparing a fluorescein derivative as claimed in claim 1, characterized in that: The fluorescein derivative is 6-carboxyfluorescein modified with an o-methylthiophene side chain; The preparation method of the o-methylthiophene side chain modified 6-carboxyfluorescein comprises the following steps: Preparation of 2-(2,4-dimethoxyphenyl)-5-methylthiophene; 2-(2,4-dimethoxyphenyl)-5-methylthiophene is reacted with pyridine hydrochloride to obtain 4-(5-methylthiophen-2-yl)benzene-1,3-diol; 6-carboxyfluorescein modified with o-methylthiophene side chain was obtained by reacting 4-(5-methylthiophen-2-yl)benzene-1,3-diol and 2-(2,4-dihydroxybenzoyl)terephthalic acid; Alternatively, the fluorescein derivative is 6-carboxyfluorescein modified with an o-phenylthiophene side chain, and the preparation method of the 6-carboxyfluorescein modified with an o-phenylthiophene side chain comprises the following steps: Preparation of 2-(2,4-dimethoxyphenyl)-5-2-phenylthiophene; 4-(5-phenylthiophen-2-yl)benzene-1,3-diol is obtained by reacting 2-(2,4-dimethoxyphenyl)-5-phenylthiophene with pyridine hydrochloride; 6-carboxyfluorescein modified with o-phenylthiophene side chain was obtained by reacting 4-(5-phenylthiophene-2-yl)benzene-1,3-diol with 2-(2,4-dihydroxybenzoyl)terephthalic acid. Or, the fluorescein derivative is 6-carboxyfluorescein modified with a benzothiophene side chain; The preparation method of the benzothiophene side chain modified 6-carboxyfluorescein comprises the following steps: Preparation of 2-(2,4-dimethoxyphenyl)benzo[b]thiophene; 2-(2,4-dimethoxyphenyl)benzo[b]thiophene is reacted with pyridine hydrochloride to obtain 4-(benzo[b]thiophen-2-yl)benzene-1,3-diol; 6-carboxyfluorescein modified with benzothiophene side chain is obtained by reacting 4-(benzo[b]thiophen-2-yl)benzene-1,3-diol with 2-(2,4-dihydroxybenzoyl)terephthalic acid; Or, the fluorescein derivative is 6-carboxyfluorescein modified with a methylamino-type o-phenylthiophene side chain; The method for preparing the methylamino-modified 6-carboxyfluorescein with o-phenylthiophene side chain comprises the following steps: Preparation of 4-(4-(dimethylamino)-2-hydroxybenzoyl)isophthalic acid; 4-(5-phenylthiophen-2-yl)benzene-1,3-diol is obtained by reacting 2-(2,4-dimethoxyphenyl)-5-phenylthiophene with pyridine hydrochloride; 6-carboxyfluorescein modified with methylamino-1-phenylthiophene side chain was obtained by reacting 4-(5-phenylthiophene-2-yl)benzene-1,3-diol and 4-(4-(dimethylamino)-2-hydroxybenzoyl)isophthalic acid.

3. The method for preparing the fluorescein derivative according to claim 2, characterized in that: The preparation method of 2-(2,4-dimethoxyphenyl)-5-methylthiophene comprises the following steps: 2,4-dimethoxyphenylboronic acid and tetrakistriphenylphosphine palladium are placed in a reaction bottle, toluene, ethanol and an alkali metal carbonate aqueous solution are added under an inert atmosphere, and then 2-bromo-5-methylthiophene is added, and the reaction is carried out at 80-100° C. to obtain 2-(2,4-dimethoxyphenyl)-5-methylthiophene; The molar volume ratio of 2-bromo-5-methylthiophene, 2,4-dimethoxyphenylboric acid, tetrakistriphenylphosphine palladium, toluene, ethanol and alkali metal carbonate aqueous solution is (0.9-1.1) mmol: (1.2-1.4) mmol: (0.03-0.04) mmol: (5-7) ml: (2-3) ml: (2-3) ml; The alkali metal carbonate includes at least one of Cs2CO3, K2CO3, Na2CO3 and Li2CO3; The concentration of the aqueous solution of alkali metal carbonate is 2.0-4.0M.

4. The method for preparing the fluorescein derivatives according to claim 2, characterized in that: The preparation method of 4-(5-methylthiophene-2-yl)benzene-1,3-diol comprises: 2-(2,4-dimethoxyphenyl)-5-methylthiophene and pyridine hydrochloride are placed in a reaction bottle, and reacted at 180-200° C. under an inert atmosphere to obtain 4-(5-methylthiophen-2-yl)benzene-1,3-diol; The molar ratio of 2-(2,4-dimethoxyphenyl)-5-methylthiophene to pyridine hydrochloride is (0.9-1.1):(9.0-16.5); The preparation method of the o-methylthiophene side chain modified 6-carboxyfluorescein comprises: 4-(5-methylthiophene-2-yl)benzene-1,3-diol and 2-(2,4-dihydroxybenzoyl)terephthalic acid are placed in a reaction bottle, an organic solvent is added under an inert atmosphere, and the reaction is carried out at 60-100° C. to obtain 6-carboxyfluorescein modified with an o-methylthiophene side chain; The organic solvent includes at least one of methanesulfonic acid and trifluoroacetic acid; The molar ratio of 4-(5-methylthiophene-2-yl)benzene-1,3-diol to 2-(2,4-dihydroxybenzoyl)terephthalic acid is 1:(1.0-1.1); The volume ratio of the sum of the amounts of 4-(5-methylthiophene-2-yl)benzene-1,3-diol and 2-(2,4-dihydroxybenzoyl)terephthalic acid to the organic solvent is (1.0-1.1) mmol: (5-10) ml.

5. The method for preparing the fluorescein derivative according to claim 2, characterized in that: The preparation method of 2-(2,4-dimethoxyphenyl)-5-2-phenylthiophene comprises the following steps: 2,4-dimethoxyphenylboronic acid, 2-bromo-5-phenylthiophene and tetrakistriphenylphosphine palladium are placed in a reaction bottle, toluene, ethanol and an alkali metal carbonate aqueous solution are added under an inert atmosphere, and then 2-bromo-5-methylthiophene is added, and the mixture is reacted at 80-100° C. to obtain 2-(2,4-dimethoxyphenyl)-5-2-phenylthiophene; The molar volume ratio of 2-bromo-5-methylthiophene, 2,4-dimethoxyphenylboric acid, tetrakistriphenylphosphine palladium, toluene, ethanol and alkali metal carbonate aqueous solution is (0.9-1.1) mmol: (1.2-1.4) mmol: (0.03-0.04) mmol: (5-7) ml: (2-3) ml: (2-3) ml; The alkali metal carbonate includes at least one of Cs2CO3, K2CO3, Na2CO3 and Li2CO3.

6. The method for preparing the fluorescein derivative according to claim 2, characterized in that: The preparation method of the 4-(5-phenylthiophene-2-yl)benzene-1,3-diol comprises: 2-(2,4-dimethoxyphenyl)-5-phenylthiophene and pyridine hydrochloride are placed in a reaction bottle, and reacted at 180-200° C. under an inert atmosphere to obtain 4-(5-phenylthiophen-2-yl)benzene-1,3-diol; The molar ratio of 2-(2,4-dimethoxyphenyl)-5-phenylthiophene and pyridine hydrochloride is (0.9-1.1):(9.0-16.5); The preparation method of the o-methylthiophene side chain modified 6-carboxyfluorescein comprises: 4-(5-phenylthiophene-2-yl)benzene-1,3-diol and 2-(2,4-dihydroxybenzoyl)terephthalic acid are placed in a reaction bottle, an organic solvent is added under an inert atmosphere, and the reaction is carried out at 60-100° C. to obtain 6-carboxyfluorescein modified with an o-methylthiophene side chain; The organic solvent includes at least one of methanesulfonic acid and trifluoroacetic acid; The molar ratio of 4-(5-phenylthiophene-2-yl)benzene-1,3-diol to 2-(2,4-dihydroxybenzoyl)terephthalic acid is 1:(1.0-1.1); The volume ratio of the sum of the amounts of 4-(5-phenylthiophene-2-yl)benzene-1,3-diol and 2-(2,4-dihydroxybenzoyl)terephthalic acid to the organic solvent is (1.0-1.1) mmol: (5-10) ml.

7. The method for preparing a fluorescein derivative according to claim 2, characterized in that: The preparation method of 2-(2,4-dimethoxyphenyl)benzo[b]thiophene comprises the following steps: 2,4-dimethoxyphenylboronic acid, 2-bromobenzothiophene and tetrakistriphenylphosphine palladium are placed in a reaction bottle, toluene, ethanol and an alkali metal carbonate aqueous solution are added under an inert atmosphere, and then 2-bromo-5-methylthiophene is added, and the mixture is reacted at 80-100° C. to obtain 2-(2,4-dimethoxyphenyl)benzo[b]thiophene; The molar volume ratio of 2-bromobenzothiophene, 2,4-dimethoxyphenylboric acid, tetrakistriphenylphosphine palladium, toluene, ethanol and alkali metal carbonate aqueous solution is (0.9-1.1) mmol: (1.2-1.4) mmol: (0.03-0.04) mmol: (5-7) ml: (2-3) ml: (2-3) ml; The alkali metal carbonate includes at least one of Cs2CO3, K2CO3, Na2CO3 and Li2CO3.

8. The method for preparing the fluorescein derivative according to claim 2, characterized in that: The preparation method of 4-(benzo[b]thiophene-2-yl)benzene-1,3-diol comprises: 2-(2,4-dimethoxyphenyl)benzo[b]thiophene and pyridine hydrochloride are placed in a reaction bottle, and reacted at 180-200° C. under an inert atmosphere to obtain 4-(benzo[b]thiophen-2-yl)benzene-1,3-diol; The molar ratio of 2-(2,4-dimethoxyphenyl)benzo[b]thiophene to pyridine hydrochloride is (0.9-1.1):(9.0-16.5); The preparation method of the benzothiophene side chain modified 6-carboxyfluorescein comprises: 4-(Benzo[b]thiophene-2-yl)benzene-1,3-diol and 2-(2,4-dihydroxybenzoyl)terephthalic acid are placed in a reaction bottle, an organic solvent is added under an inert atmosphere, and the reaction is carried out at 60-100° C. to obtain 6-carboxyfluorescein modified with a benzothiophene side chain; The organic solvent includes at least one of methanesulfonic acid and trifluoroacetic acid; The molar ratio of 4-(benzo[b]thiophene-2-yl)benzene-1,3-diol to 2-(2,4-dihydroxybenzoyl)terephthalic acid is 1:(1.0-1.1); The volume ratio of the sum of the amounts of 4-(benzo[b]thiophene-2-yl)benzene-1,3-diol and 2-(2,4-dihydroxybenzoyl)terephthalic acid to the organic solvent is (1.0-1.1) mmol:(5-10) ml.

9. The method for preparing a fluorescein derivative according to claim 2, characterized in that: The preparation method of 4-(4-(dimethylamino)-2-hydroxybenzoyl)isophthalic acid comprises: Add dimethylaminophenol to a reaction flask, then add toluene, heat to 50-70°C, and add trimellitic anhydride to react to obtain 4-(4-(dimethylamino)-2-hydroxybenzoyl)isophthalic acid; The molar ratio of dimethylaminophenol to trimellitic anhydride is 1:(1.2-1.3); The method for preparing the methylamino-modified 6-carboxyfluorescein with o-phenylthiophene side chain comprises: 4-(5-phenylthiophene-2-yl)benzene-1,3-diol and 4-(4-(dimethylamino)-2-hydroxybenzoyl)isophthalic acid are placed in a reaction bottle, an organic solvent is added under an inert atmosphere, and the mixture is reacted at 60-100° C. to obtain 6-carboxyfluorescein modified with a benzothiophene side chain; The organic solvent includes at least one of methanesulfonic acid and trifluoroacetic acid; The molar ratio of the 4-(5-phenylthiophene-2-yl)benzene-1,3-diol to 4-(4-(dimethylamino)-2-hydroxybenzoyl)isophthalic acid is 1:(1.0-1.1); The volume ratio of the sum of the amounts of 4-(5-phenylthiophene-2-yl)benzene-1,3-diol and 4-(4-(dimethylamino)-2-hydroxybenzoyl)isophthalic acid to the organic solvent is (1.0-1.1) mmol:(5-10) ml.

10. Use of the fluorescein derivative according to claim 1 or the fluorescein derivative prepared by the preparation method according to any one of claims 2 to 9 in immunofluorescence.

Images