Compound, fluorescent cluster, immunofluorescent cluster, and preparation method and application of compound, fluorescent cluster and immunofluorescent cluster

Abstract

The invention relates to the field of fluorescent probes, in particular to a compound, a fluorescent cluster, an immunofluorescent cluster and a preparation method and application of the compound, the compound is shown in the formula (I), the compound has complete water solubility and has the high signal-to-noise ratio of fluorescence imaging, the fluorescent cluster prepared from the compound also has complete water solubility, active functional groups are clear and controllable, and the immunofluorescent cluster has the high fluorescence imaging signal-to-noise ratio and the high fluorescence imaging signal-to-noise ratio. The fluorescent cluster is high in fluorescence brightness and high in photobleaching resistance, the fluorescent cluster can be coupled with a nano antibody under the condition of pure water to obtain an immunofluorescent cluster, the antibody activity is not affected, and the fluorescent cluster can be used for a cell immunofluorescent staining probe. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of fluorescent probes, and in particular to a compound, a fluorescent cluster, an immunofluorescent cluster, and a preparation method and application thereof. Background Art

[0002] Immunofluorescence staining technology refers to the technology of quantitatively analyzing or localizing characteristic antigens of biological samples such as cells by labeling antibodies with fluorescent molecules and other luminescent agents, and using instruments such as fluorescence microscopes. It is of great significance in the fields of microbiology, immunology, pathology and immunohistochemistry. Nanobody (Nb) is a new type of antibody derived from camelids and is currently the smallest molecular fragment with complete antigen recognition function. Its relative molecular mass is 12-15kd, which is only one-tenth of traditional antibodies. It can specifically bind to hidden antigens on the cell surface that are difficult for traditional antibodies to recognize, which helps to achieve highly sensitive immunofluorescence staining.

[0003] The signal intensity of fluorescent molecules labeled on the surface of nanoantibodies is an important factor in determining the quality of their fluorescence imaging and analysis results. Traditional fluorescent molecules rely on specific fluorescent groups to emit fluorescence. When the number of excitations is too many or the excitation time is too long, the structure of the fluorophore will undergo irreversible changes and will no longer emit fluorescence, resulting in photobleaching, which seriously affects the analysis of the sample's fluorescence signal. Cyanine-type and pyrrole boron-type fluorescent molecules have a certain resistance to photobleaching, but they are highly hydrophobic and are prone to denaturation and inactivation of antibodies when coupled to the surface of small-sized nanoantibodies. Fluorescent materials such as carbon quantum dots, upconversion nanomaterials, and rare earth complexes have good resistance to photobleaching, but they also have various shortcomings, such as unstable fluorescence signals, low quantum yields, high detection costs, and complex preparation processes.

[0004] Aggregation-induced emission (AIE) material is a new type of fluorescent material, which is characterized by limiting the heat energy generated by the internal movement of molecules by molecular aggregation, thereby promoting the output ratio of light energy, thereby generating a fluorescent signal. The fluorescent signal generated by the aggregation state of AIE materials has the advantages of good stability and strong resistance to photobleaching, and is widely expected in the field of biological imaging. Tetraphenylethylene is a cheap and readily available AIE molecular matrix that can emit fluorescent signals in an aggregated state. However, tetraphenylethylene is extremely hydrophobic and only dissolves in organic solvents. In other words, only in a reaction environment containing organic solvents such as ethanol and DMSO can the AIE molecular matrix be coupled with nano antibodies. However, organic solvents can destroy the structure of nano antibodies, thereby affecting the activity of nano antibodies, and then affecting the effect of immunofluorescence staining. Therefore, although the AIE molecular matrix has the above-mentioned advantages, its non-water solubility hinders its application in immunofluorescence staining.

[0005] The inventors found that only when the AIE molecules can be coupled with nanoantibodies in a non-organic solvent-dependent reaction environment can the application of AIE molecules in nanoantibody immunofluorescence staining be truly realized. To achieve this goal, the inventors tried to explore how to solve the problem that the AIE molecules with hydrophobic properties must rely on an organic solvent environment when coupled with nanoantibodies. For example, coupling is achieved in an aqueous environment, such as pure water, phosphate buffer (PBS), 2-(N-morpholino)ethanesulfonic acid buffer (MES), and tris(hydroxymethyl)aminomethane hydrochloride buffer (TRIS), which can avoid organic reagents from damaging the nanoantibody structure and ensure the activity of the nanoantibody, thereby achieving highly sensitive immunofluorescence staining.

[0006] In order to achieve the above-mentioned coupling with nanoantibodies in an aqueous environment, the inventors found that the above purpose can be achieved by modifying the AIE molecules to be completely water-soluble. The literature (ACS Appl. Mater. Interfaces 2021, 13, 17, 19625–19632) increased the water solubility of tetraphenylethylene by introducing a sulfonic acid group (-SO3). Although the hydrophilicity of the molecule was increased, it was also impossible to achieve its dissolution in a 100% pure water solvent. It is not a completely water-soluble AIE molecule. Therefore, coupling it or its clusters to nanoantibodies must still be carried out in a reaction environment containing an organic solvent, and the application of AIE molecules in nanoantibody immunofluorescence staining will not be achieved. Summary of the invention

[0007] The present invention aims to solve the problem of coupling AIE molecules or their clusters with nanoantibodies without using organic solvents as the reaction environment, so as to achieve the coupling in a water environment, thereby avoiding the phenomenon that the organic solvent destroys the structure of the nanoantibody during the coupling, and avoiding the coupling affecting the activity of the nanoantibody, resulting in the problem of difficulty in staining.

[0008] In order to achieve the above object, the first aspect of the present invention provides a compound (UTPS2), wherein the compound is as shown in formula (I):

[0009]

[0010] The second aspect of the present invention provides a method for preparing a compound, which comprises the following steps:

[0011] S110. The mixed solution of sodium ethoxide and ethanol is added to the first mixed solution to obtain a second mixed solution;

[0012] adding a mixed solution of 1,4-butanesultone and ethanol to the second mixed solution to perform a first reaction;

[0013] Then, a first purification is performed to obtain a first intermediate product, and the first intermediate product is shown in formula (ⅠⅠ):

[0014]

[0015] Wherein, the first mixed solution is a mixed solution of [1,1-bis(4-bromophenyl)-2-bis(4-hydroxyphenyl)]ethylene and ethanol under an inert atmosphere;

[0016] S120. adding 4-pyridylboric acid, potassium hydroxide, and tetrakis(triphenylphosphine)palladium to a mixed solution of the first intermediate product and acetonitrile to carry out a second reaction;

[0017] A second purification is then performed to obtain a second intermediate product, and the second intermediate product is shown in formula (III):

[0018]

[0019] S130. dissolving the second intermediate product and 5-chloromethyluracil in N,N-dimethylformamide under an inert atmosphere to carry out a third reaction;

[0020] After a third purification, the compound described in the first aspect is obtained.

[0021] The third aspect of the present invention provides a method for preparing a fluorescent cluster, which comprises mixing the compound described in the first aspect, single-stranded DNA, and a solvent to obtain the fluorescent cluster.

[0022] The fourth aspect of the present invention provides a fluorescent cluster (UTPS2 / ssDNA) prepared by the preparation method described in the third aspect.

[0023] The fifth aspect of the present invention provides a method for preparing an immunofluorescent cluster, wherein the fluorescent cluster described in the fourth aspect is coupled to a nanobody in the water environment.

[0024] The sixth aspect of the present invention provides an immunofluorescent cluster (EGFR Nb-UTPS2 / ssDNA) prepared by the preparation method described in the fifth aspect.

[0025] The seventh aspect of the present invention provides use of the compound described in the first aspect, the fluorescent cluster described in the fourth aspect, or the immunofluorescent cluster described in the sixth aspect in the preparation of an immunofluorescent staining reagent.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] On the first aspect, the compound UTPS2 prepared by the present invention is an AIE molecule with complete water solubility. The fluorescent clusters prepared therefrom can be coupled with nanoantibodies under pure water conditions. Compared with the AIE molecules in the prior art that must use organic solvents as the coupling environment to couple with nanoantibodies, the phenomenon of organic reagents destroying the structure of nanoantibodies is avoided, thereby avoiding the problem of reducing the activity of nanoantibodies, and can truly realize the use of AIE molecules for immunofluorescence staining.

[0028] In the second aspect, there is a large Stokes shift between the fluorescence emission peak and the absorption peak of the compound prepared by the present invention, which is beneficial to improving the signal-to-noise ratio of fluorescence imaging.

[0029] On the third aspect, the present invention uses the precise recognition of AU base complementary pairing to prepare fluorescent clusters. The terminally modified UTPS2 / ssDNA fluorescent clusters provided are not only completely water-soluble, but also have clear and controllable active functional groups, and can be arbitrarily coupled to the surface of the target antibody without affecting its activity, and are universal. At the same time, it also has the advantages of high fluorescence brightness and strong resistance to photobleaching.

[0030] In the fourth aspect, the Nb-UTPS2 / ssDNA immunofluorescence cluster provided by the present invention has the advantages of good water solubility, resistance to photobleaching, stable fluorescence signal, and ease of availability in the application of immunofluorescence staining, thereby achieving sensitive and stable immunofluorescence staining and analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the high resolution mass spectrum of the intermediate product 1 prepared in Example A1;

[0032] Figure 2 is the high resolution mass spectrum of the intermediate product 2 prepared in Example A1;

[0033] Figure 3 is the high resolution mass spectrum of Compound A1 prepared in Example A1;

[0034] Figure 4 are the absorption and emission spectra of compound A1 prepared in Example A1 and comparative example DA4; wherein (a) the absorption spectrum of compound A1, (b) the emission spectrum of compound A1, and (c) the absorption and emission spectrum of DA4;

[0035] Figure 5 is a graph showing the variation of the fluorescence signal intensity of the fluorescent cluster B1 prepared in Example B1 and comparative examples DB1 and DB2 with the number of irradiation times;

[0036] Figure 6 is a graph showing the fluorescence intensity of the fluorescent cluster B2 prepared in Example B2 and comparative examples DB1 and DB2 as a function of the solar light irradiation time;

[0037] Figure 7 It is a fluorescence performance diagram of the immunofluorescence cluster C1 prepared in Example C1 and comparative examples DC1 and DC2. DETAILED DESCRIPTION

[0038] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0039] The fluorescence signal generated by the aggregation state of AIE materials has the advantages of good stability and strong resistance to photobleaching, and is widely expected in the field of biological imaging. The parent AIE molecule is extremely hydrophobic. The existing modified AIE molecules have improved hydrophilicity, but still have hydrophobicity. Some modified AIE molecules also take advantage of the hydrophilic and hydrophobic properties to achieve aggregation and form fluorescent clusters by hydrophobic force.

[0040] However, the inventors found that if the AIE molecule is hydrophobic, it is only soluble in organic solvents, that is, the AIE molecule must be coupled to the nanobody using an organic solvent as the reaction environment, and an organic solvent such as ethanol or DMSO must be used as the reaction environment. Although the AIE molecule matrix can be coupled to the nanobody, the organic solvent destroys the structure of the nanobody, thereby affecting the activity of the nanobody and the immunofluorescence staining effect. Therefore, although the AIE molecule matrix has the above advantages, the coupling condition that an organic solvent must be used as the reaction environment hinders its application in nanobody immunofluorescence staining.

[0041] The inventors found that only when the AIE molecules can be coupled with nano antibodies without the need to use organic solvents as a reaction environment can the application of AIE molecules in immunofluorescence staining be truly realized. To achieve this goal, the inventors tried to explore how to achieve AIE molecules with hydrophobic properties to overcome the reliance on organic solvents when coupled with nano antibodies. For example, coupling in an aqueous environment can avoid organic reagents from damaging the structure of nano antibodies, ensure the activity of nano antibodies, and thus achieve nano antibody immunofluorescence staining. Further, in order to achieve the above-mentioned coupling with nano antibodies in an aqueous environment, the inventors unexpectedly discovered that the above purpose can be achieved by modifying the AIE molecules to be completely water-soluble.

[0042] To this end, the first aspect of the present invention provides a compound (UTPS2), wherein the compound is as shown in formula (I),

[0043]

[0044] The compounds of the present invention are completely water-soluble, but their AIE parent molecules and intermediate products (Formula (ⅠⅠ), Formula (ⅠⅠⅠ)) are not completely soluble in water.

[0045] The compound of the invention has high fluorescence brightness, stable fluorescence signal and strong anti-photobleaching ability.

[0046] In the present invention, the pyridine group enables a large Stokes shift between the fluorescence emission peak and the absorption peak, and has a high signal-to-noise ratio of fluorescence imaging.

[0047] The second aspect of the present invention provides a method for preparing a compound, which comprises the following steps:

[0048] S110. The mixed solution of sodium ethoxide and ethanol is added to the first mixed solution to obtain a second mixed solution;

[0049] adding a mixed solution of 1,4-butanesultone and ethanol to the second mixed solution to perform a first reaction;

[0050] Then, a first purification is performed to obtain a first intermediate product, and the first intermediate product is shown in formula (ⅠⅠ):

[0051]

[0052] Wherein, the first mixed solution is a mixed solution of [1,1-bis(4-bromophenyl)-2-bis(4-hydroxyphenyl)]ethylene and ethanol under an inert atmosphere;

[0053] S120. adding 4-pyridylboric acid, potassium hydroxide, and tetrakis(triphenylphosphine)palladium to a mixed solution of the first intermediate product and acetonitrile to carry out a second reaction;

[0054] A second purification is then performed to obtain a second intermediate product, and the second intermediate product is shown in formula (III):

[0055]

[0056] S130. dissolving the second intermediate product and 5-chloromethyluracil in N,N-dimethylformamide under an inert atmosphere to carry out a third reaction;

[0057] After a third purification, the compound described in the first aspect is obtained.

[0058] According to the present invention, in step S110, the molar ratio of [1,1-bis(4-bromophenyl)-2-bis(4-hydroxyphenyl)]ethylene, sodium ethoxide and 1,4-butane sultone is 1:2-3:5-8.

[0059] According to the present invention, in step S120, the molar ratio of the first intermediate product, 4-pyridylboric acid, potassium hydroxide and tetrakis(triphenylphosphine)palladium is 1:2-3:8-12:2-4.

[0060] According to the present invention, in step S120, the first purification includes column chromatography.

[0061] According to the present invention, in step S120, the second purification includes column chromatography.

[0062] According to the present invention, in step S130, the molar ratio of the second intermediate product to 5-chloromethyluracil is 1:2-5.

[0063] According to the present invention, in step S130, the third purification includes semi-preparative liquid chromatography separation and purification.

[0064] According to the present invention, in step S110, in the mixed solution of [1,1-bis(4-bromophenyl)-2-bis(4-hydroxyphenyl)]ethylene and ethanol, the concentration of [1,1-bis(4-bromophenyl)-2-bis(4-hydroxyphenyl)]ethylene is 0.8-1.2 mol / L.

[0065] According to the present invention, in step S110, in the mixed solution of ethanol and sodium ethoxide, the concentration of the sodium ethoxide is 3-5 mol / L.

[0066] According to the present invention, in step S110, a mixed solution of ethanol and sodium ethoxide is added dropwise to the first mixed solution and stirred for 0.5-2h.

[0067] According to the present invention, in step S110, in the mixed solution of 1,4-butane sultone and ethanol, the concentration of 1,4-butane sultone is 1-2 mol / L.

[0068] According to the present invention, in step S110, the eluents in the first purification are dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 1:0.4-0.6.

[0069] According to the present invention, in step S120, in the mixed solution of the first intermediate product and acetonitrile, the concentration of the first intermediate product is 10-20 mol / L.

[0070] According to the present invention, in step S120, the second purification further includes rotary evaporation, wherein the product obtained from the second reaction is subjected to the rotary evaporation to obtain a solid product, and after the solid product is dissolved in methanol, the supernatant is collected and subjected to the column chromatography of the second purification.

[0071] According to the present invention, in step S120, the eluent of the column chromatography of the second purification is dichloromethane, methanol and triethylamine, and the volume ratio of triethylamine, dichloromethane and triethylamine is 1:600-800:300-400.

[0072] According to the present invention, in step S130, the concentration of the second intermediate product is 8-15 mol / L.

[0073] According to the present invention, in step S110, the conditions of the first reaction include: the first reaction temperature is 20-30° C., and the first reaction time is 20-28 h.

[0074] According to the present invention, in step S120, the conditions of the second reaction include: under vacuum conditions, the second reaction temperature is 70-90° C., and the second reaction time is 8-16 hours.

[0075] According to the present invention, in step S130, the conditions of the third reaction include: the third reaction temperature is 30-60° C., and the third reaction time is 45-50 h.

[0076] The third aspect of the present invention provides a method for preparing a fluorescent cluster, which comprises mixing the compound described in the first aspect, single-stranded DNA, and a solvent to obtain the fluorescent cluster.

[0077] In the present invention, the fluorescent cluster is completely water-soluble and can be arbitrarily coupled to the surface of the target nano-antibody under pure water conditions without affecting its activity. It also has the advantages of high fluorescence brightness and strong resistance to photobleaching.

[0078] According to the present invention, the mixing conditions include: mixing time of 5-30 min; pH of 5-12.

[0079] According to a specific embodiment of the present invention, the mixing conditions include: mixing time of 10-15 min; pH of 6-8.

[0080] According to the present invention, the single-stranded DNA is adenine single-stranded DNA.

[0081] According to the present invention, the single-stranded length of the single-stranded DNA is 5-30A.

[0082] According to a specific embodiment of the present invention, the single-stranded length of the single-stranded DNA is 20-25A.

[0083] Specifically, when other conditions remain unchanged and the single-strand length of the single-stranded DNA is 20A, the fluorescence intensity of the fluorescent cluster reaches a saturation value.

[0084] According to the present invention, the molar ratio of the compound to the single-stranded DNA is 3-10:1.

[0085] According to a specific embodiment of the present invention, the molar ratio of the compound to the single-stranded DNA is 3.5-5:1.

[0086] Specifically, when other conditions remain unchanged, when the molar ratio of the compound to the single-stranded DNA is 5:1, the fluorescence intensity of the fluorescent cluster reaches a saturation value.

[0087] According to the present invention, the solvent is at least one of water, phosphate buffered saline (PBS), 2-(N-morpholino)ethanesulfonic acid buffer (MES), and tris(hydroxymethyl)aminomethane hydrochloride buffer (TRIS).

[0088] According to a specific embodiment of the present invention, the solvent is water and / or phosphate buffer.

[0089] According to the present invention, the single-stranded DNA terminal is modified with at least one of an amino group, a carboxyl group and a thiol group.

[0090] The fourth aspect of the present invention provides a fluorescent cluster (UTPS2 / ssDNA) prepared by the preparation method described in the third aspect.

[0091] The fifth aspect of the present invention provides a method for preparing an immunofluorescent cluster, wherein the fluorescent cluster described in the fourth aspect is coupled to a nanobody under pure water conditions.

[0092] The sixth aspect of the present invention provides an immunofluorescent cluster (EGFR Nb-UTPS2 / ssDNA) prepared by the preparation method described in the fifth aspect.

[0093] In the present invention, the immunofluorescence cluster has the advantages of good water solubility, resistance to photobleaching, stable fluorescence signal, and easy availability in the application of immunofluorescence staining, thereby realizing sensitive and stable immunofluorescence staining analysis.

[0094] The seventh aspect of the present invention provides use of the compound described in the first aspect, the fluorescent cluster described in the fourth aspect, or the immunofluorescent cluster described in the sixth aspect in the preparation of an immunofluorescent staining reagent.

[0095] The present invention will be described in detail below through examples.

[0096] Test description:

[0097] Method for determining the induced luminescence properties of compounds: water is selected as a good solvent, anhydrous ethanol is selected as a poor solvent, and different proportions of water / ethanol mixed solvents (V / V) are prepared, and the water contents are 10%, 30%, 50%, 70%, 90%, and 100%, respectively. 1 μM of the compound is dissolved in mixed solvents with different water contents, and the emission spectra of the compound in solvents with different water / ethanol ratios are measured using an ELISA instrument.

[0098] Fluorescence signal intensity test method of fluorescent clusters: 1 μM fluorescent clusters were added to a 96-well plate, and the plate was excited once every 30 seconds using an ELISA reader for a total of 30 times, and the fluorescence emission intensity was recorded every 5 times.

[0099] The anti-photobleaching performance testing method of the fluorescent clusters is as follows: 1 μM fluorescent cluster solution is irradiated under sunlight for 0, 1, 2, 3, 4, 5, 10, 20, and 30 minutes respectively, and then the fluorescence signal intensity is tested using an enzyme-labeled instrument.

[0100] Affinity K of immunofluorescence clusters D Test method: SPR technology was used for determination. The instrument used was Biacore. The antigen was coupled to the detection channel. The coupling method was recommended by the equipment manufacturer. After data fitting, the affinity K was analyzed. D

[0101] Test method for the anti-photobleaching performance of immunofluorescence clusters: A431 cells (EGFR positive) were taken and diluted to 1×10 5 Cell suspension to be used was prepared at a concentration of 10 cells / mL. After that, 200 μL of the cell suspension to be used was aspirated and added to a 96-well plate and cultured in a cell culture incubator for 24 hours. 1 μM EGFR nanoantibody immunofluorescence cluster probe was added to the well plate of A431 cells and incubated in a cell culture incubator for 1 hour. Wash three times with PBS under light-proof conditions, continuously irradiate under a fluorescence microscope (intensity 65 μW), and take fluorescent photos every 30 seconds. Image J software was used to quantitatively analyze the cell fluorescence intensity. Test method for the anti-photobleaching performance of immunofluorescence clusters for cell immunostaining: A431 cells (EGFR positive) were aspirated and diluted to 1×10 5 Cells / mL of standby cell suspension were added. Afterwards, 200 μL of the standby cell suspension was drawn and added to a 96-well plate and cultured in a cell culture incubator for 24 hours. 1 μM of EGFR nanobody immunofluorescence clusters and EGFR nanobody-FITC were added to the well plates of A431 cells, respectively, and incubated in a cell culture incubator for 1 hour. PBS was added to wash three times under light-proof conditions, continuously irradiated under a fluorescence microscope (intensity 65 μW), and fluorescent photos were taken every 30 seconds. The cell fluorescence intensity was quantitatively analyzed using Image J software. The commercial fluorescent molecule FITC was coupled to the EGFR nanobody to obtain an EGFR nanobody-FITC fluorescent probe as a control group. It is understandable that FITC can be replaced by other commonly used fluorescent molecules in the art, such as FAM.

[0102] In the following examples and comparative examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if no manufacturer is specified, are all conventional products that can be obtained through commercial channels.

[0103] Synthesis of compounds

[0104] Example A1: The preparation method of compound A1 (UTPS2) is as follows:

[0105] (1) 1 mM [1,1-bis(4-bromophenyl)-2-bis(4-hydroxyphenyl)]ethylene was added to 10 mL of anhydrous ethanol under argon protection. Then, a mixture of 2 mM sodium ethoxide and 10 mL of anhydrous ethanol was added dropwise and stirred for 1 hour. Then, a mixture of 5 mM 1,4-butanesulfonic acid lactone and 5 mL of anhydrous ethanol was added and stirred vigorously at room temperature for 24 hours, and a large amount of white solid appeared. The product was purified by silica gel column chromatography with the eluent being dichloromethane / methanol in a volume ratio of 7:3. Intermediate product 1 was obtained with a yield of 65%. High-resolution mass spectrometry analysis of intermediate product 1 was performed, and the results were as follows: Figure 1 .

[0106] (2) Dissolve 1 mM intermediate product 1 in 15 mL acetonitrile, add 2 mM 4-pyridylboronic acid, 10 mM potassium hydroxide, and 2 mM tetrakis(triphenylphosphine)palladium, put all in, evacuate, heat under reflux at 80°C with stirring. After reacting for 12 hours, a solid product is obtained by rotary evaporation. Dissolve the solid product in methanol and recover the supernatant. Purify the supernatant by silica gel column with the eluent being dichloromethane / methanol / triethylamine in a volume ratio of 700:300:1. Obtain intermediate product 2 with a yield of 43%. High-resolution mass spectrometry analysis of the second intermediate product 2 is performed, and the results are as follows: Figure 2 .

[0107] (3) 1 mM intermediate 2 and 2 mM 5-chloromethyluracil were dissolved in 10 mL N,N-dimethylformamide solvent under nitrogen protection and reacted at 50°C for 48 hours. The product was separated and purified by semi-preparative liquid chromatography to obtain compound A1 (Formula (I)) with a yield of 45%. Compound A1 was subjected to high-resolution mass spectrometry analysis, and the results were as follows: Figure 3 .

[0108] Comparative Example DA1: According to step (1) of Example A1, intermediate product 1 (formula (II)) was obtained as compound DA1.

[0109] Comparative Example DA2: According to steps (1) and (2) of Example A1, intermediate product 2 (formula (III)) was obtained as compound DA2.

[0110] Comparative Example DA3: According to the prior art, an N-hydroxysuccinimide-modified AIE molecule NHS-CSPP was synthesized as compound DA3.

[0111] Comparative Example DA4: Commercial DAPI stain.

[0112] The commercial DAPI stain was mixed with the cell DNA for 10 minutes, and 1 μM DAPI-DNA mixed solution was taken and the absorption and emission spectra were measured using an ELISA reader. The results are shown in the attached figure. Figure 4 (c) as shown.

[0113] Compound A1 was tested for its induced luminescence performance, and the results are shown in the attached Figure 4 As shown in (a) and (b).

[0114] The water solubility of compounds A1, DA1 and DA2 was tested.

[0115] Compounds A1, DA1, and DA2 were dissolved in PBS buffer and the emission spectra were measured using an ELISA reader. The fluorescence intensity is shown in Table 1 below:

[0116] Table 1

[0117] Compound Fluorescence signal intensity A1 2516±1332 DA1 5145±876 DA2 6137±986

[0118] As shown in Table 1, A1 has good dispersibility in 100% pure water system and no fluorescence signal. Compared with A1, the fluorescence signals of DA1 and DA2 are enhanced by 2 times and 2.5 times, respectively, proving that they aggregate to varying degrees in pure water solution and cannot be completely dissolved.

[0119] Compounds A1, DA1, and DA2 were subjected to LC / MS high-resolution mass spectrometry and 1 H NMR test.

[0120] LC / MS high-resolution mass spectrometry was performed using Agilent Q-TOF 6550. ESI-mode, voltage 3200 V; flow rate: 0.3 ml / min; injection volume 1 ul; Gas Temp 150°C; drying gas 15 L / min; sheath gas temp 350°C; sheath gas flow 12 L / min, mass spectrometry scanning range: 100-1000 m / z.

[0121] 1H NMR measurements were performed using a Bruker AVANCE NEO 400 MHz and reported in ppm. When reporting peak multiplicities, the following abbreviations may be used: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br-s (broad singlet), dd (doublet of doublets), dt (doublet of triplets). When reported, coupling constants (J) are reported in Hertz (Hz).

[0122] Spectral data of compound A1

[0123] 1 H NMR (400MHz, DMSO) δ9.14(d,J=6.8Hz,2H),8.41(d,J=6.8Hz,2H),7.76(d,J=7.8 Hz,1H),7.44(d,J=8.5Hz,2H),7.36–7.29(m,6H),7.27–7.19(m,1H),7.15(d,J=8 .5Hz,4H),6.76(d,J=8.5Hz,4H),5.51(d,J=1.0Hz,2H),4.01(t,J=6.0Hz,4H),2. 62(t,J=5.9Hz,4H),1.83(dddd,J=12.7,6.9,6.1,1.0Hz,4H),1.78–1.71(m,4H).

[0124] C 44 H 42 N3O 10 S2 2- MS (ESI) theoretical value: 836.95; m / z found value: [M-2Na] 836.87.

[0125] Spectral data of compound DA1 (intermediate product 1)

[0126] 1 H NMR (400MHz, DMSO) δ7.66(d,J=8.4Hz,2H),7.39(d,J=8.4Hz,2H),7.34–7.26(m,4H),7.19–7.14(m,1H),7. 07(d,J=8.5Hz,4H), 6.76(d,J=8.5Hz,4H), 3.93(t,J=6.1Hz,4H), 2.96(t,J=6.1Hz,4H), 1.86–1.73(m,8H).

[0127] C 34 H 33 BrO8S2 2- MS (ESI) theoretical value: 713.66; m / z measured value: 1 / 2[M-2Na] 357.04.

[0128] Spectral data of compound DA2 (intermediate product 2)

[0129] 1 H NMR (400MHz, DMSO) δ8.68(d,J=5.9Hz,2H),7.71(d,J=8.5Hz,2H),7.65(d,J=6.1Hz,2H),7.52(d,J=8.5Hz,2H),7.37–7.29(m, 4H),7.25–7.20(m,1H),7.17(d,J=8.5Hz,4H),6.75(d,J=8.4Hz,4H),4.09–3.96(m,4H),2.69–2.56(m,4H),1.88–1.70(m,8H).

[0130] C 39 H 37 NO8S2 2- MS (ESI) theoretical value: 711.85; m / z measured value: 1 / 2[M-2Na] 355.60.

[0131] Synthesis of fluorescent clusters

[0132] Fluorescence intensity conditions of fluorescent clusters: Single-stranded DNA (ssDNA-20A) with a chain length of 20A at different concentrations (0, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 3.0 μM) was mixed with 10 μM compound A1 in a TRIS buffer at pH 6.5 for 12 minutes, and the emission spectrum was measured using an ELISA reader. The fluorescence intensity is shown in Table 2 below:

[0133] Table 2

[0134] ssDNA-20A concentration (μM) Fluorescence signal intensity 0 2571±1836 0.4 6684±2142 0.8 13103±1741 1.2 16891±1539 1.6 24676±3282 2.0 32647±1331 2.4 32902±2547 3.0 32354±1263

[0135] As shown in Table 2, as the concentration of SSDNA-20A increases, the fluorescence intensity reaches a saturation value when the ssDNA concentration increases to 2.0 μM.

[0136] 10 μM of compound A1 and 2.0 μM of ssDNA with chain lengths of 0, 5, 10, 15, 20, and 25 A (ssDNA-5A-ssDNA-30A) were mixed in water for 10 minutes, and the emission spectrum was measured using an ELISA instrument. The fluorescence intensity is shown in Table 3 below:

[0137] Table 3

[0138] ssDNA chain length (A) Fluorescence signal intensity ssDNA-0A 2646±1332 ssDNA-5A 8324±2145 ssDNA-10A 17103±2328 ssDNA-15A 27892±3330 ssDNA-20A 31263±1521 ssDNA-25A 32645±1775

[0139] As shown in Table 3, when no ssDNA is added, compound A1 is in a dispersed state and does not fluoresce. As the length of the added ssDNA increases, the fluorescence intensity increases. When the length of the ssDNA is 20A, the fluorescence intensity reaches a saturation value.

[0140] Example B1: 8 μM of compound A1 and 2.0 μM of amino-modified ssDNA-22A were mixed in a phosphate buffer at pH 7.4 for 15 minutes to obtain fluorescent cluster B1.

[0141] Example B2: 7 μM of compound A1 and 2.0 μM of carboxyl-modified ssDNA-20A were mixed in a phosphate buffer at pH 8.0 for 14 minutes to obtain fluorescent cluster B2.

[0142] Example B3: 10 μM of compound A1 and 2.3 μM of carboxyl-modified ssDNA-23A were mixed in a phosphate buffer at pH = 6.5 for 20 minutes to obtain fluorescent cluster B3.

[0143] Comparative Example DB1: Commercial FITC fluorescent molecule.

[0144] Comparative Example DB2: Commercial Cy2 fluorescent molecule.

[0145] The fluorescence signal intensity of the fluorescent clusters B1, DB1, and DB2 was tested. The results are shown in the attached figure. Figure 5 shown.

[0146] The anti-photobleaching performance of the fluorescent clusters B2, DB1, and DB2 was tested. The results are shown in the attached figure. Figure 6 shown.

[0147] Synthesis of immunofluorescent clusters

[0148] Example C1: EDC and NHS were used to activate the carboxyl-modified fluorescent cluster B3 in MES buffer, and the activated fluorescent cluster B3 was coupled with the anti-epidermal growth factor receptor nanobody to obtain the targeted immunofluorescent cluster C1 (EGFRNb-UTPS2 / ssDNA).

[0149] Comparative Example DC1: The commercial carboxylated Cy2 fluorescent molecule was dissolved in the organic solvent DMSO to prepare a solution with a concentration of 10 mM. The carboxyl group of the Cy2 fluorescent molecule was activated by EDC / NHS reaction and then coupled to the anti-epidermal growth factor receptor nanobody to obtain the Cy2-labeled nanobody DC1 (EGFRNb-Cy2).

[0150] Comparative Example DC2: Commercial FITC fluorescent molecules were dissolved in DMSO to prepare a solution with a concentration of 10 mM. 100 μL of the solution was added to the phosphate buffer of the EGFR nanobody (200 μM, 900 μL) and coupled in the nanobody to obtain FITC-labeled nanobody DC2 (EGFR Nb-FITC).

[0151] Comparative Example DC3: Unconjugated anti-epidermal growth factor receptor nanobody DC3.

[0152] Comparative Example DC4: Compound DA3 was dissolved in an organic solvent DMSO to prepare a solution with a concentration of 10 mM. 5 μL of the solution was taken and added to an aqueous Na2HCO3 solution (6.8 nmol, 495 μL) of EGFR nanobody for coupling reaction to obtain CSPP-labeled nanobody DC4 (EGFR Nb-CSPP).

[0153] Affinity K was measured for immunofluorescence clusters C1, DC1, DC3, and DC4 D Test, Affinity K D As shown in Table 4. The anti-photobleaching performance of cell immunostaining was tested for the immunofluorescence clusters C1, DC1, and DC2. The results are shown in the attached table. Figure 7 shown.

[0154] Table 4

[0155] C1 DC1 DC3 DC4 <![CDATA[Affinity K D > <![CDATA[3.7×10 -8 ]]> <![CDATA[2.6×10 -7 ]]> <![CDATA[4.6×10 -8 ]]> <![CDATA[8.2×10 -7 ]]>

[0156] From the results in Table 4, we can see that compared with the unmodified original EGFR nanobody DC3, the affinity of nanobody C1 after modification with the fluorescent cluster probe has no significant change, proving that the coupling modification of the water-soluble fluorescent cluster has no significant effect on the structure and performance of the nanobody. The commercial Cy2 has strong lipid solubility and is also a hydrophobic molecule. It also needs to be coupled with the nanobody in an organic solvent environment. The above coupling of hydrophobic molecules and nanoantibodies has structural damage to the nanoantibody, resulting in the affinity of the nanoantibody DC1 after it is modified on the surface of the nanoantibody reduced to 2.6×10 -7 , which is one order of magnitude lower than the original nanobody, indicating that it has an impact on the structure and performance of the nanobody. Compared with C1 and DC4, when 1% DMSO organic solvent is added during the nanobody coupling process, the affinity of DC4 nanobody decreases by one order of magnitude, proving that the reaction in a non-pure aqueous solution environment will lead to a decrease in the activity of the nanobody.

[0157] Depend on Figure 4 It can be seen that compound A1 has good solubility in 100% pure water solvent, and the benzene ring can rotate freely, so no fluorescence signal is generated. As the water content gradually decreases, compound A1 aggregates in the solvent, and a maximum absorption peak of 329nm appears ( Figure 4 (a)), and the concurrent maximum emission wavelength is 512nm ( Figure 4 (b)) Fluorescence signal. After commercial DAPI binds to DNA, the absorption peak is 360nm and the emission peak is 460nm ( Figure 4 (c)). The above results show that the AIE fluorescent molecule UTPS2 is completely water-soluble and can form aggregates and emit fluorescence in poor solvents. The Stokes shift is larger than that of the commercial DAPI stain, which is beneficial to reduce the image signal-to-noise ratio.

[0158] Depend on Figure 5 It can be seen that the fluorescence intensity of the fluorescent cluster B1 is significantly higher than that of the commercial Cy2 fluorescent dye and FITC fluorescent dye. The fluorescence signal of the fluorescent cluster B1 has no obvious attenuation after 30 excitations, while the fluorescence of the commercial Cy2 and FITC fluorescent molecules has obvious attenuation after 5 excitations, and the fluorescence intensity is further weakened or even disappears after 30 irradiations, indicating that compared with commercial fluorescent molecules, the fluorescent cluster provided by the present invention has a stronger fluorescent signal and better anti-photobleaching performance.

[0159] Depend on Figure 6 It can be seen that the fluorescence intensity of commercial Cy2 and FITC fluorescent molecules gradually decreases with the extension of sunlight irradiation time. The fluorescence signal of FITC fluorescent molecules almost disappears after 5 minutes of illumination, while the fluorescence brightness of fluorescent cluster B2 does not decay significantly after illumination, indicating that it has good anti-photobleaching performance.

[0160] Depend on Figure 7 It can be seen that after 60 minutes of continuous irradiation, the fluorescence performance of C1-stained cells did not change significantly. However, the fluorescence intensity of cells stained by DC2 in the control group decreased by about 85% after 10 minutes of continuous irradiation, and the fluorescence intensity of cells stained by DC1 decreased by about 50% after 60 minutes of continuous irradiation, indicating that the immunofluorescence clusters provided by the present invention have good anti-photobleaching performance in immune cell staining experiments.

Claims

1. A compound, characterized in that The compound is shown in formula (I):

2. A method for preparing a compound, characterized in that: The following steps are involved: S110. The mixed solution of sodium ethoxide and ethanol is added to the first mixed solution to obtain a second mixed solution; adding a mixed solution of 1,4-butanesultone and ethanol to the second mixed solution to perform a first reaction; Then, a first purification is performed to obtain a first intermediate product, and the first intermediate product is shown in formula (ⅠⅠ): Wherein, the first mixed solution is a mixed solution of [1,1-bis(4-bromophenyl)-2-bis(4-hydroxyphenyl)]ethylene and ethanol under an inert atmosphere; S120. adding 4-pyridylboric acid, potassium hydroxide, and tetrakis(triphenylphosphine)palladium to a mixed solution of the first intermediate product and acetonitrile to carry out a second reaction; A second purification is then performed to obtain a second intermediate product, and the second intermediate product is shown in formula (III): S130. dissolving the second intermediate product and 5-chloromethyluracil in N,N-dimethylformamide under an inert atmosphere to carry out a third reaction; After a third purification, the compound according to claim 1 is obtained.

3. The preparation method according to claim 2, characterized in that: in, In step S110, the molar ratio of [1,1-bis(4-bromophenyl)-2-bis(4-hydroxyphenyl)]ethylene, sodium ethoxide and 1,4-butane sultone is 1:2-3:5-8; Wherein, in step S120, the molar ratio of the first intermediate product, 4-pyridylboric acid, potassium hydroxide and tetrakis(triphenylphosphine)palladium is 1:2-3:8-12:2-4; Wherein, in step S120, the first purification includes column chromatography; Wherein, in step S120, the second purification includes column chromatography; Wherein, in step S130, the molar ratio of the second intermediate product to 5-chloromethyluracil is 1:2-5; Wherein, in step S130, the third purification includes semi-preparative liquid chromatography separation and purification.

4. The preparation method according to claim 2 or 3, It is characterized in that in, In step S110, in the mixed solution of [1,1-bis(4-bromophenyl)-2-bis(4-hydroxyphenyl)]ethylene and ethanol, the concentration of [1,1-bis(4-bromophenyl)-2-bis(4-hydroxyphenyl)]ethylene is 0.8-1.2 mol / L; Wherein, in step S110, in the mixed solution of ethanol and sodium ethoxide, the concentration of the sodium ethoxide is 3-5 mol / L; Wherein, in step S110, a mixed solution of ethanol and sodium ethoxide is added dropwise to the first mixed solution and stirred for 0.5-2h; Wherein, in step S110, in the mixed solution of 1,4-butane sultone and ethanol, the concentration of 1,4-butane sultone is 1-2 mol / L; Wherein, in step S110, the eluents in the first purification are dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 1:0.4-0.6; Wherein, in step S120, in the mixed solution of the first intermediate product and acetonitrile, the concentration of the first intermediate product is 10-20 mol / L; Wherein, in step S120, the second purification further comprises rotary evaporation, wherein the product obtained by the second reaction is subjected to the rotary evaporation to obtain a solid product, the solid product is dissolved in methanol, and the supernatant is collected and subjected to the column chromatography of the second purification; Wherein, in step S120, the eluent of the column chromatography of the second purification is dichloromethane, methanol and triethylamine, and the volume ratio of triethylamine, dichloromethane and triethylamine is 1:600-800:300-400; Wherein, in step S130, the concentration of the second intermediate product is 8-15 mol / L.

5. The preparation method according to any one of claims 2 to 4, characterized in that: in, In step S110, the conditions of the first reaction include: the first reaction temperature is 20-30°C, and the first reaction time is 20-28h; Wherein, in step S120, the conditions of the second reaction include: under vacuum conditions, the second reaction temperature is 70-90° C., and the second reaction time is 8-16 h; Wherein, in step S130, the conditions of the third reaction include: the third reaction temperature is 30-60° C., and the third reaction time is 45-50 h.

6. A method for preparing a fluorescent cluster, characterized in that: The method comprises mixing the compound according to claim 1, single-stranded DNA, and a solvent to obtain the fluorescent cluster; Preferably, the compound is linked to the single-stranded DNA through complementary base pairing during the mixing. Wherein, the mixing conditions include: Preferably, the mixing time is 5-30 min, more preferably 10-15 min; the pH is 5-12, more preferably 6-8; Preferably, the single-stranded DNA is adenine single-stranded DNA; Preferably, the single-stranded length of the single-stranded DNA is 5-30A, preferably 20-25A; Preferably, the molar ratio of the compound to the single-stranded DNA is 3-10:1, preferably 3.5-5:1; Preferably, the solvent is at least one of water, phosphate buffer, 2-(N-morpholine)ethanesulfonic acid buffer, and tris(hydroxymethylaminomethane) hydrochloride buffer, preferably water and / or phosphate buffer; Preferably, the single-stranded DNA end is modified with at least one of an amino group, a carboxyl group and a thiol group.

7. The fluorescent cluster obtained by the preparation method according to claim 6.

8. A method for preparing an immunofluorescent cluster, characterized in that: The fluorescent cluster according to claim 7 is coupled to the nanobody in the water environment.

9. The immunofluorescence clusters obtained by the preparation method according to claim 8.

10. Use of the compound according to claim 1, the fluorescent cluster according to claim 7 or the immunofluorescent cluster according to claim 9 in preparing an immunofluorescent staining reagent.

Images