Method for designing fluorescent probes for protein detection

Abstract

The present application relates to the technical field of fluorescent chemical sensor, and particularly relates to a design method of a fluorescent probe for protein detection. The method comprises the following steps: (a) obtaining a PDB file of a target protein; calculating the cavity size of the target protein; (b) determining an AIE group, and modifying a hydrophilic group and / or a hydrophobic group on the AIE group to obtain a series of AIE molecules; (c) calculating the configuration with the lowest structure energy and the corresponding size of each AIE molecule; screening the AIE molecules meeting the requirements of entering the cavity of the target protein; (d) performing molecular docking of the screened AIE molecules and the target protein, adjusting the structure of the AIE molecules through the molecular docking result, and designing the fluorescent probe for protein detection. The present application can realize the customization of the fluorescent probe responding to a specific protein according to the specific protein.

Description

Technical Field

[0001] This invention relates to the field of fluorescent chemical sensor technology, and in particular to a method for designing a fluorescent probe for protein detection. Background Technology

[0002] Proteins are the material basis of all life and the main carriers of life activities. Changes in protein content in the body are closely related to various physiological activities. For example, caspase is highly active in apoptotic cells, the content of calreticulin on the surface of tumor cells and apoptotic cells is much higher than that in normal cells, and cancer cells can cause abnormal levels of certain marker proteins in the blood. Therefore, protein detection is an important method for assessing the health status of an organism.

[0003] Currently, there are various methods for protein detection, such as immunoassay, instrumental detection, bioprobes, surface plasmon resonance (SPR), and electrophoresis. Most of these methods suffer from drawbacks such as high equipment costs and complex operation. Bioprobes, however, offer the most convenient and rapid method. In particular, the discovery of aggregation-induced emission (AIE) materials has solved the problem of fluorescence quenching in the aggregated state of traditional bioprobes, leading to widespread attention for AIE bioprobes in protein detection. Proteins contain numerous irregularly shaped cavities. When AIE probes with specific structures enter these cavities, they are subjected to various forces from the protein's amino acid residues, thus restricting their movement and exhibiting a point-lit fluorescence response.

[0004] While AIE probes are one of the most convenient methods for protein detection, current research primarily relies on experimental work. This involves first synthesizing fluorescent probes with AIE properties, and then experimentally verifying whether the probes specifically respond to certain proteins. It relies heavily on trial and error, lacking theoretical guidance. Therefore, there is an urgent need for theoretical guidance to predict which structurally specific molecules can respond to a particular protein, enabling the targeted synthesis of probe molecules for the target protein.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] One object of the present invention is to provide a method for designing fluorescent probes for protein detection, so as to effectively design fluorescent probes that can bind to proteins for protein detection.

[0007] Another object of the present invention is to provide a method for screening proteins that can specifically respond to AIE molecules.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0009] The design method for fluorescent probes used in protein detection includes the following steps:

[0010] (a) Obtain the PDB file of the target protein; calculate the cavity size of the target protein;

[0011] (b) Determine the AIE motif and modify the AIE motif with hydrophilic and / or hydrophobic groups to regulate the hydrophilicity and hydrophobicity of the molecule, thereby obtaining a series of AIE molecules;

[0012] (c) Calculate the lowest energy configuration of each AIE molecule using Chem3D software, and calculate the size of each lowest energy configuration; screen out AIE molecules that meet the requirements for entering the cavity of the target protein;

[0013] (d) The selected AIE molecules are molecularly docked with the target protein. The structure of the AIE molecules is adjusted based on the molecular docking results to design a fluorescent probe for protein detection.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] (1) The method of the present invention optimizes the design steps of fluorescent probes by using molecular docking technology to design probes that can bind to proteins based on the structure of the target protein. It can realize the "customization" of fluorescent probes that respond to specific proteins, and is simple to operate and has low design cost.

[0016] (2) The method of the present invention expands the application scope of AIE molecules by using molecular docking technology to screen out proteins that can specifically respond to AIE molecules based on existing methods. It can screen out probes that may specifically respond to certain proteins from previously reported AIE molecules, simplifying the design process of fluorescent probes. Attached Figure Description

[0017] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0018] Figure 1 The dimensions of the two main active cavities of human serum albumin in Example 1 of this invention;

[0019] Figure 2 The sizes of the four polyarylpyrrole molecules in Example 1 of this invention;

[0020] Figure 3 This is the result of the docking of TPPP-2Na with human serum albumin molecules in Example 1 of the present invention;

[0021] Figure 4 This is the result of the docking of TPPP-2Na-C with human serum albumin molecules in Example 1 of the present invention;

[0022] Figure 5 This is the result of the docking of TPPP-2Na-3C with human serum albumin molecules in Example 1 of the present invention;

[0023] Figure 6 This is the result of the docking of TPPP-4Na with human serum albumin molecules in Example 1 of the present invention;

[0024] Figure 7 The image shows the fluorescence response of TPPP-4Na to human serum albumin molecules in Example 1 of this invention. The excitation wavelength was 380 nm, and the concentration of TPPP-4Na was 1 × 10⁻⁶. -5 mol / L, human serum albumin concentration: 0.5 mg / mL;

[0025] Figure 8 The particle size distributions of the TPPP-4Na solution, the mixed solution of TPPP-4Na and human serum albumin, and the human serum albumin solution in Example 1 of this invention are shown below.

[0026] Figure 9 This is the result of the docking of TPPP-2Na with bovine serum albumin molecules in Example 2 of the present invention;

[0027] Figure 10 This is the result of the docking of TPPP-2Na with acetylcholinesterase molecules in Example 2 of the present invention;

[0028] Figure 11 This is the result of the docking of TPPP-2Na with butyrylcholinesterase molecules in Example 2 of the present invention;

[0029] Figure 12 This is the result of the docking of TPPP-2Na with lipase molecules in Example 2 of the present invention;

[0030] Figure 13 This is the result of the docking of TPPP-2Na with lysozyme molecules in Example 2 of the present invention;

[0031] Figure 14 This is the result of the docking of TPPP-2Na with pepsin molecules in Example 2 of the present invention;

[0032] Figure 15 This is the result of the docking of TPPP-2Na with trypsin molecules in Example 2 of the present invention;

[0033] Figure 16 This is the result of the docking of TPPP-2Na with γ-globulin molecules in Example 2 of the present invention;

[0034] Figure 17 The image shows the fluorescence response of TPPP-2Na to eight proteins—bovine serum albumin, butyrylcholinesterase, acetylcholinesterase, trypsin, pepsin, lipase, lysozyme, and γ-globulin—in Example 2 of this invention. The excitation wavelength was 320 nm, and the concentration of TPPP-2Na was 1 × 10⁻⁶. -5 mol / L, protein concentration: 0.5 mg / mL;

[0035] Figure 18 The particle size distributions of the TPPP-2Na solution, the TPPP-2Na and bovine serum albumin mixed solution, and the bovine serum albumin solution in Example 2 of this invention are shown below.

[0036] Figure 19 This is the result of the docking of TPPP-2Na-C with bovine serum albumin molecules in Example 3 of the present invention;

[0037] Figure 20 This is the result of the docking of TPPP-2Na-C with acetylcholinesterase molecules in Example 3 of the present invention;

[0038] Figure 21 This is the result of the docking of TPPP-2Na-C with butyrylcholinesterase molecules in Example 3 of the present invention;

[0039] Figure 22 This is the result of the docking of TPPP-2Na-C with lipase molecules in Example 3 of the present invention;

[0040] Figure 23 This is the result of the docking of TPPP-2Na-C with lysozyme molecules in Example 3 of the present invention;

[0041] Figure 24 This is the result of the docking of TPPP-2Na-C with pepsin molecules in Example 3 of the present invention;

[0042] Figure 25 This is the result of the docking of TPPP-2Na-C with trypsin molecules in Example 3 of the present invention;

[0043] Figure 26 This is the result of the docking of TPPP-2Na-C with γ-globulin molecules in Example 3 of the present invention;

[0044] Figure 27The image shows the fluorescence response of TPPP-2Na-C to eight proteins—bovine serum albumin, butyrylcholinesterase, acetylcholinesterase, trypsin, pepsin, lipase, lysozyme, and γ-globulin—in Example 3 of this invention. The excitation wavelength was 310 nm, and the concentration of TPPP-2Na-C was 1 × 10⁻⁶. -5 mol / L, protein concentration: 0.5 mg / mL;

[0045] Figure 28 The particle size distributions are shown for the TPPP-2Na-C solution, the TPPP-2Na-C and bovine serum albumin mixed solution, and the bovine serum albumin solution in Example 3.

[0046] Figure 29 This is the result of the docking of TPPP-2Na-3C with bovine serum albumin molecules in Example 4 of the present invention;

[0047] Figure 30 This is the result of the docking of TPPP-2Na-3C with acetylcholinesterase molecules in Example 4 of the present invention;

[0048] Figure 31 This is the result of the docking of TPPP-2Na-3C with butyrylcholinesterase molecules in Example 4 of the present invention;

[0049] Figure 32 This is the result of the docking of TPPP-2Na-C with lipase molecules in Example 4 of the present invention;

[0050] Figure 33 This is the result of the docking of TPPP-2Na-3C with lysozyme molecules in Example 4 of the present invention;

[0051] Figure 34 This is the result of the docking of TPPP-2Na-3C with pepsin molecules in Example 4 of the present invention;

[0052] Figure 35 This is the result of the docking of TPPP-2Na-3C with trypsin molecules in Example 4 of the present invention;

[0053] Figure 36 This is the result of the docking of TPPP-2Na-3C with γ-globulin molecules in Example 4 of the present invention;

[0054] Figure 37 The image shows the fluorescence response of TPPP-2Na-3C to eight proteins—bovine serum albumin, butyrylcholinesterase, acetylcholinesterase, trypsin, pepsin, lipase, lysozyme, and γ-globulin—in Example 4 of this invention. The excitation wavelength was 310 nm, and the concentration of TPPP-2Na-3C was 1 × 10⁻⁶. -5 mol / L, protein concentration: 0.5 mg / mL;

[0055] Figure 38 The particle size distributions are shown for the TPPP-2Na-3C solution, the TPPP-2Na-3C and bovine serum albumin mixed solution, and the bovine serum albumin solution in Example 4. Detailed Implementation

[0056] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0057] The design method for fluorescent probes used in protein detection includes the following steps:

[0058] (a) Obtain the PDB file of the target protein; calculate the cavity size of the target protein;

[0059] (b) Determine the AIE motif and modify the AIE motif with hydrophilic and / or hydrophobic groups to regulate the hydrophilicity and hydrophobicity of the molecule, thereby obtaining a series of AIE molecules;

[0060] (c) Calculate the lowest energy configuration of each AIE molecule using Chem3D software, and calculate the size of each lowest energy configuration; screen out AIE molecules that meet the requirements for entering the cavity of the target protein;

[0061] (d) The selected AIE molecules are molecularly docked with the target protein. The structure of the AIE molecules is adjusted based on the molecular docking results to design a fluorescent probe for protein detection.

[0062] Molecular docking studies the electrostatic, hydrogen bond, hydrophobic, and van der Waals interactions between active sites of small drug molecules and biomolecules to achieve spatial matching. It continuously optimizes their spatial positioning, relative positions, and dihedral angles of rotatable bonds, ultimately selecting the appropriate site based on a scoring function. Molecular docking can calculate the interactions between ligands and receptors, and can be used to explain the interaction mechanisms between AIE probes and proteins.

[0063] In a specific embodiment of the present invention, the PDB file of the target protein is downloaded and obtained from a PDB database.

[0064] In a specific embodiment of the present invention, the cavity size of the target protein is calculated using Pymol software.

[0065] In a specific embodiment of the present invention, the AIE unit includes at least one of the following: tetraphenylethylene system, polyaryl-1,3-butadiene system, polyarylpyrrole system, and polyaryl-benzopyrrole system. Examples include tetraphenylethylene, tetraphenyl-1,3-butadiene, 1,2,5-triphenylpyrrole, and 1,2,4,5-tetraphenylbenzopyrrole.

[0066] In a specific embodiment of the present invention, the method for regulating the hydrophilicity and hydrophobicity of molecules includes: modifying the AIE motif with hydrophilic and / or hydrophobic groups to restrict the movement of AIE molecules in the cavity of the target protein.

[0067] In a specific embodiment of the present invention, the restricted movement means that the rotation of more than half of the benzene rings in the AIE molecule is restricted within the cavity of the target protein. The software can visually display whether the molecule is subject to forces from amino acid residues in the protein. When no force is applied, the benzene rings in the AIE molecule can rotate around the single bonds; when subjected to forces from amino acid residues, the rotation of the benzene rings is restricted. When the movement of more than half of the benzene rings in the AIE molecule is restricted, the movement of the AIE molecule within the cavity of the target protein is considered restricted.

[0068] In practice, this also includes: when the protein is water-soluble, modifying the AIE motif with hydrophilic groups; and simultaneously introducing hydrophobic groups to regulate the compatibility of the AIE molecule within the protein cavity.

[0069] In a specific embodiment of the present invention, the hydrophilic group includes a carboxyl salt group and / or a sulfonate group; the hydrophobic group includes an alkyl group and / or a phenyl group.

[0070] In a specific embodiment of the present invention, the AIE molecule that meets the requirements for entering the cavity of the target protein satisfies the following condition: the size of the lowest energy configuration of the AIE molecule is smaller than the cavity size of the target protein.

[0071] In a specific embodiment of the present invention, the dimensions of the lowest-energy configurations of the AIE molecule are calculated using Pymol software.

[0072] In a specific embodiment of the present invention, the molecular docking is performed using AutoDock Vina software. Further, in the molecular docking, each AIE molecule docks with the target protein 10 times, resulting in 20 configurations per docking. The configuration with the lowest binding energy in each docking is selected as the optimal configuration for that docking. The interaction between the AIE molecule and the protein in each optimal configuration is analyzed using Pymol software, and the optimal configuration with the highest probability of occurrence is selected. The interaction between the AIE molecule and the target protein is then analyzed using Pymol software. Based on the analysis results, the structure of the AIE molecule is adjusted.

[0073] In a specific embodiment of the present invention, prior to the molecular docking, the following is further included:

[0074] The AutoDock Tools software was used to sequentially remove water molecules from the protein, add hydrogen, and balance its charge, and then output a receptor file in PDBQT format.

[0075] The AutoDock Tools software was used to perform hydrogenation on AIE molecules and to detect torsion centers and torsion bonds, and then output ligand files in PDBQT format.

[0076] Open the ligand and receptor files in AutoDock Tools software, set the docking box, and ensure that the size of the docking box includes the entire region of the receptor to guarantee that it covers all areas where the ligand may bind.

[0077] The degree of interaction between the AIE molecule and the target protein determines the strength of their binding. According to the mechanism of restricted luminescence of AIE molecules, the force that restricts the "rotor" part of the ligand molecule contributes to the luminescence of the molecule. The more force that restricts the "rotor" part, the stronger the luminescence of the molecule. This allows for the adjustment of the molecular structure to be restricted as much as possible.

[0078] In a specific embodiment of the present invention, the adjustment method includes:

[0079] If the analysis results show that the AIE molecule is not subjected to electrostatic and hydrogen bonding in the cavity of the target protein, then functional groups containing O and / or N are added to the AIE molecule.

[0080] If the analysis results show that more than half of the benzene rings in the AIE molecule are not restricted by forces within the cavity of the target protein, then alkyl groups are added to the benzene rings of the AIE molecule and / or the number of benzene rings is increased in the AIE molecule.

[0081] If the analysis results show that the forces acting on the AIE molecule in the cavity of the target protein all come from one side of the molecule, then a benzene ring or heterocycle is introduced onto the benzene ring to increase the size of the molecule.

[0082] In a specific embodiment of the present invention, the method further includes: (e) synthesizing AIE molecules according to the design results and verifying the binding of the AIE molecules to the target protein. Further, the verification includes: adding AIE molecules to a PBS solution of the target protein and detecting changes in the fluorescence intensity and particle size of the solution.

[0083] The concentration of the target protein in the PBS solution can be 0.5 mg / mL. AIE molecules are added to the solution to achieve a concentration of 1 × 10⁻⁶. -5 mol / L. Furthermore, if the solution shows significant fluorescence enhancement upon the addition of AIE molecules, and the protein does not aggregate, indicating a decrease in the amount of AIE molecule aggregates, it suggests that AIE molecules can bind to proteins. The binding mechanism involves AIE molecules entering the protein cavity, and the AIE molecules are constrained by sufficient intermolecular forces within the protein cavity, resulting in significant fluorescence enhancement.

[0084] In a specific embodiment of the present invention, before calculating the cavity size of the target protein, the method further includes: calculating the active sites on human serum albumin using Protein Plus software, excluding active sites on the protein surface and smaller active sites. Further, the smaller active sites refer to active sites with a depth < the molecular length of the AIE motif. For example, a depth < The active site.

[0085] This invention also provides a method for screening proteins that specifically respond to AIE molecules, comprising the following steps:

[0086] (A) Obtain the PDB file of the AIE molecule and the PDB file of the protein to be screened;

[0087] (B) Perform molecular docking between the AIE molecule and the protein to be screened, and analyze the molecular docking results to determine the protein that the AIE molecule can specifically respond to.

[0088] In a specific embodiment of the present invention, the method for obtaining the PDB file of the AIE molecule includes: drawing the AIE molecule structure using Chemdraw, calculating the configuration with the lowest energy of the AIE molecule structure using Chem3D software, and saving it as a PDB file.

[0089] In a specific embodiment of the present invention, the protein to be screened includes at least one of serum albumin, acetylcholinesterase, butyrylcholinesterase, lipase, lysozyme, pepsin, trypsin, and γ-globulin.

[0090] In a specific embodiment of the present invention, the PDB file of the protein is downloaded from a PDB database.

[0091] In a specific embodiment of the present invention, the molecular docking is performed using AutoDock Vina software. Further, in the molecular docking, the AIE molecule docks with each protein to be screened 10 times, resulting in 20 configurations per docking. The configuration with the lowest binding energy in each docking is selected as the optimal configuration for that docking. Pymol software is used to analyze the interaction between the AIE molecule and the protein in each optimal configuration, and the optimal configuration with the highest probability of occurrence is selected for further analysis.

[0092] In practice, the docking results are visualized using Pymol software to show the interaction between AIE molecules and proteins.

[0093] In a specific embodiment of the present invention, the analysis includes: determining whether the AIE molecule has entered the cavity of the protein, and the magnitude of the force exerted on the AIE molecule by the protein cavity.

[0094] In a specific embodiment of the present invention, the method further includes: (C) verifying the binding of the AIE molecule to the screened protein based on the analysis results. Further, the verification includes: adding the AIE molecule to a PBS solution of the screened protein and detecting changes in the fluorescence intensity and particle size of the solution.

[0095] The protein concentration in the PBS solution can be 0.5 mg / mL. Adding AIE molecules to the solution brings the AIE molecule concentration to 1 × 10⁻⁶. -5 mol / L. Furthermore, if the solution shows significant fluorescence enhancement upon the addition of AIE molecules, and the protein does not aggregate, the amount of AIE molecules in aggregates decreases. This indicates that AIE molecules exhibit a spot-lighting fluorescence response to the protein, and the response mechanism involves AIE molecules entering the protein cavity, with molecular motion restricted by amino acid residues, thus resulting in enhanced fluorescence.

[0096] In practice, molecular docking is performed using the AutoDock Vina software. Specific methods can be found in: J. Eberhardt, D. Santos-Martins, AFTillack, and S. Forli. (2021). AutoDock Vina 1.2.0: New Docking Methods, Expanded Force Field, and Python Bindings. Journal of Chemical Information and Modeling. and O. Trott, A. Jolson, AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. Journal of Computational Chemistry 31 (2010) 455-461.

[0097] Example 1

[0098] This embodiment provides a method for designing a fluorescent probe for the detection of human serum albumin, including the following steps:

[0099] (1) Download the PDB file of human serum albumin from the PDB database, and use Pymol or Discovery Studio software to delete the redundant ligands in the protein PDB file, keeping only the protein structure.

[0100] The active sites on human serum albumin were calculated using Protein Plus software, excluding protein surface and smaller active sites.

[0101] The size of the major active cavity on human serum albumin was calculated using Pymol software, such as... Figure 1 As shown.

[0102] (2) Molecular design

[0103] The polyaryl pyrrole moiety was selected. Considering the water solubility of human serum albumin, a hydrophilic group was added to the polyaryl pyrrole to increase its hydrophilicity. Common hydrophilic groups include carboxylates and sulfonates. Considering that sulfonates may disrupt the secondary structure of proteins, a carboxylate was introduced onto the polyaryl pyrrole to increase its hydrophilicity.

[0104] Protein cavities are hydrophobic regions, while carboxylates are strong hydrophilic groups. Therefore, we consider introducing hydrophobic groups to regulate the hydrophilicity and hydrophobicity of the molecule.

[0105] Based on the above considerations, four polyarylpyrrole carboxylates with different hydrophilicity and hydrophobicity were designed, and their structures are as follows:

[0106]

[0107] (3) Calculation of AIE molecule size

[0108] The molecular structures of the four polyarylpyrroles were drawn using ChemDraw software. The lowest-energy configurations for each structure were calculated using Chem3D software, and the dimensions of the lowest-energy configurations were measured using Pymol software. (Specific details are as follows...) Figure 2 As shown, the sizes of the four AIE molecules all meet the size requirements for entering the cavity of human serum albumin.

[0109] (4) Molecular docking

[0110] The molecular structures of TPPP-2Na, TPPP-2Na-C, TPPP-2Na-3C and TPPP-4Na were drawn using Chemdraw software. Then, the configuration with the lowest molecular energy was calculated using Chem3D and saved as a PDB file.

[0111] The AutoDock Tools software was used to remove water molecules, add hydrogen, and balance the charge of human serum albumin, and then the receptor file was output in PDBQT format.

[0112] The AutoDock Tools software was used to perform hydrogenation on AIE molecules and to detect torsion centers and torsion bonds, and then the ligand file was output as a PDBQT file.

[0113] Open the ligand and receptor files in AutoDock Tools software, set the docking box, and ensure that the size of the docking box covers the entire region of the receptor to ensure that it includes all regions where the ligand may bind.

[0114] Set the docking parameters, generate 20 configurations each time, and export the parameter file;

[0115] Using Autodock Vina, four AIE small molecules were sequentially docked with human serum albumin, with each small molecule docked 10 times. The configuration with the lowest binding energy in each docking was the optimal configuration for that docking. The optimal configuration with the highest probability among the 10 dockings was selected, and the interaction between the AIE small molecules and the protein was analyzed using Pymol software.

[0116] The docking results of the four AIE small molecules with human serum albumin are as follows: Figures 3-6 As shown in the diagram, the docking results clearly show that TPPP-4Na experiences the strongest interactions in human serum albumin, with all four benzene rings interacting with amino acid residues. Therefore, the TPPP-4Na molecule was ultimately chosen.

[0117] (5) The preparation methods of TPPP-4Na include:

[0118] To a 100 mL round-bottom flask equipped with a magnetic stirrer, methyl 4-aminobenzoate (0.02 mol), methyl 4-aldehyde benzoate (0.02 mol), p-toluenesulfonic acid (0.002 mol), and glacial acetic acid (50 mL) were added sequentially. The reaction mixture was heated to 90 °C and stirred for 30 min. Then, 2,3-butanedione (0.01 mol) was slowly added to the reaction mixture using a syringe. After the addition was complete, stirring was continued for 3 h, during which a yellow powder precipitate was observed to form in the reaction solution. The crude product was obtained by vacuum filtration. The collected filter cake was washed with acetic acid to obtain a pale yellow powder. The crude product was purified by recrystallization from a mixed solvent of chloroform and ethyl acetate to obtain an intermediate product.

[0119] Add 0.001 mol of intermediate product and 50 mL of tetrahydrofuran to a 100 mL round-bottom flask equipped with a magnetic stirrer, and heat under reflux to dissolve the intermediate product. Weigh 2.00 g of NaOH and dissolve it in 8 mL of methanol. Gradually add the methanol-NaOH solution to the reaction solution. At this point, the reaction solution becomes turbid, and an insoluble precipitate forms. After heating under reflux for about 8 hours, the reaction solution gradually becomes clear. Continue heating and stirring for 12 hours. Remove the reaction solvent by rotary evaporation, add 15 mL of H2O and 2.00 g of NaOH, and continue heating under reflux for 24 hours. After the reaction is complete, slowly add the reaction solution to tetrahydrofuran to precipitate the product TPPP-4Na. Filter under reduced pressure and collect the filter cake. Dissolve the obtained crude product in 20 mL of H2O, then slowly add it to tetrahydrofuran. Filter under reflux and collect the filter cake. Test the pH of the filtrate with precision pH paper. Repeat the process to remove residual NaOH from the product until the pH of the filtrate is close to neutral. Finally, the collected product was dried to obtain TPPP-4Na. The prepared TPPP-4Na was characterized, and the data are as follows:

[0120] 1 H NMR (400MHz, D2O, ppm): δ7.88-7.77(m,4H),7.75-7.64(m,4H),7.19(dd,J=8.6,1.9Hz,4H),7.17-7.12(m,4H),6.37(s,2H).

[0121] 13C NMR (101MHz, D2O, ppm): δ175.13,174.65,163.48,141.67,135.37,133.63,133.42,131.88,129.97,128.95,127.46,124.29,96.23.

[0122] MALDI-TOF-MS (m / z): C 34 H 18 N2O8 4- The calculated molecular weight was 582.11, and the molecular weight characterized by mass spectrometry was 585.13.

[0123] The above structural characterization results indicate that the synthesized compound is TPPP-4Na.

[0124] The aggregation-induced emission (AIE) properties of TPPP-4Na in water and tetrahydrofuran systems were verified, confirming that TPPP-4Na has significant AIE properties.

[0125] (6) Verify the binding of TPPP-4Na to human serum albumin.

[0126] Weigh 6.74 mg of compound TPPP-4Na and dissolve it in 10 mL of PBS to prepare a concentration of 1 × 10⁻⁶. -3 Prepare an adult serum albumin PBS solution by dissolving 0.5 mg of human serum albumin in 0.99 mL of PBS buffer.

[0127] Take 3 sample vials and label them 1 to 3. Add 0.99 mL of PBS buffer to sample vial 1, and add 0.99 mL of human serum albumin PBS solution to sample vials 2 and 3. Add 10 μL of TPPP-4Na PBS solution to sample vials 1 and 2, and add 10 μL of PBS solution to sample vial 3.

[0128] Sample vial #1 contains TPPP-4Na solution with a concentration of 1×10⁻⁶. -5 mol / L; Sample vial #2 contains a mixed solution of TPPP-4Na and human serum albumin, with TPPP-4Na concentration of 1×10 mol / L. -5 The concentration of human serum albumin is 0.5 mg / mL; sample vial #3 contains human serum albumin solution with a concentration of 0.5 mg / mL.

[0129] Fluorescence emission spectroscopy was performed on the solutions in sample vials 1 and 2, with an excitation wavelength of 380 nm, to obtain the following results: Figure 7The fluorescence response spectrum of TPPP-4Na to human serum albumin is shown in the figure. As shown, the fluorescence intensity of the TPPP-4Na and human serum albumin mixture is significantly enhanced compared to sample vial #1.

[0130] Particle size analysis was performed on sample bottles 1-3, such as... Figure 8 As shown, after adding TPPP-4Na to the human serum albumin solution, the particle size of human serum albumin did not change significantly, but the particle size intensity of TPPP-4Na in the range of 100–300 nm decreased significantly, indicating that TPPP-4Na molecules entered the cavity of human serum albumin.

[0131] The changes in fluorescence intensity and particle size indicate that TPPP-4Na molecules can bind to human serum albumin. The molecules can enter the cavity of human serum albumin and are subjected to forces sufficient to restrict molecular rotation in the cavity, resulting in a significant enhancement of fluorescence in the cavity of human serum albumin.

[0132] Example 2

[0133] This embodiment provides a method for screening proteins that can specifically respond to existing AIE molecules, including the following steps:

[0134] (1) The preparation methods of TPPP-2Na include:

[0135] The preparation method of the intermediate product refers to the preparation method of DPP-C1 in patent CN109180685A. Methyl 4-aminobenzoate (0.02 mol), benzaldehyde (0.02 mol), p-toluenesulfonic acid (0.002 mol), and glacial acetic acid (50 mL) were added sequentially to a 100 mL round-bottom flask equipped with a magnetic stirrer. The reaction mixture was heated to 90 °C and stirred for 30 min. Then, 2,3-butanedione (0.01 mol) was slowly added to the reaction solution using a syringe. After the addition was complete, stirring was continued for 3 h, and a yellow powder precipitate was observed to form in the reaction solution. The crude product was obtained by vacuum filtration. The collected filter cake was washed with acetic acid to obtain a pale yellow powder. The crude product was purified by recrystallization using a mixed solvent of chloroform and ethyl acetate to obtain the intermediate product.

[0136] Add 0.001 mol of intermediate product and 50 mL of tetrahydrofuran to a 100 mL round-bottom flask equipped with a magnetic stirrer, and heat under reflux to dissolve the intermediate product. Weigh 2.00 g of NaOH and dissolve it in 8 mL of methanol. Gradually add the methanol-NaOH solution to the reaction solution. At this point, the reaction solution becomes turbid, and an insoluble precipitate forms. After heating under reflux for about 8 hours, the reaction solution gradually becomes clear. Continue heating and stirring for 12 hours. Remove the reaction solvent by rotary evaporation, add 15 mL of H2O and 2.00 g of NaOH, and continue heating under reflux for 24 hours. After the reaction is complete, slowly add the reaction solution to tetrahydrofuran to precipitate the product TPPP-2Na. Filter under reduced pressure and collect the filter cake. Dissolve the obtained crude product in 20 mL of H2O, then slowly add it to tetrahydrofuran. Filter under reflux and collect the filter cake. Test the pH of the filtrate with precision pH paper. Repeat the process to remove residual NaOH from the product until the pH of the filtrate is close to neutral. Finally, the collected product was dried to obtain the product TPPP-2Na.

[0137] The prepared TPPP-2Na was characterized, and the data are as follows:

[0138] 1 H NMR (400MHz, D2O, ppm): δ7.79 (d, J = 8.2Hz, 4H), 7.24 (d, J = 3.7Hz, 4H), 7.23-7.09 (m, 10H), 6.43-6.25 (m, 2H).

[0139] 13 C NMR (101MHz, 90% D2O+10% THF-d8, ppm): δ172.56, 141.57, 135.56, 134.50, 133.22, 131.64, 130.41, 128.30, 127.61, 126.48, 123.72, 95.41.

[0140] MALDI-TOF-MS (m / z): C 32 H 20 N2O4 2- The calculated molecular weight was 496.14, and the mass spectrometry characterization showed a molecular weight of 497.15.

[0141] The above structural characterization results indicate that the synthesized compound is TPPP-2Na.

[0142] The aggregation-induced emission properties of TPPP-2Na in water and tetrahydrofuran systems were verified, confirming that TPPP-2Na has obvious AIE properties.

[0143] (2) Molecular docking

[0144] The molecular structure of TPPP-2Na was drawn using Chemdraw software, and then the configuration with the lowest molecular energy was calculated using Chem3D and saved as a PDB file.

[0145] Download PDB files for eight proteins from the PDB database: bovine serum albumin, butyrylcholinesterase, acetylcholinesterase, trypsin, pepsin, lipase, lysozyme, and gamma globulin.

[0146] Use Pymol or Discovery Studio software to remove redundant ligands from protein PDB files, retaining only the protein structure.

[0147] The AutoDock Tools software was used to sequentially remove water molecules from the protein, add hydrogen, and balance its charge, and then output a receptor file in PDBQT format.

[0148] The AutoDock Tools software was used to perform hydrogenation on AIE molecules and to detect torsion centers and torsion bonds, and then output ligand files in PDBQT format.

[0149] Open the ligand and receptor files in AutoDock Tools software, set the docking box, and ensure that the size of the docking box covers the entire region of the receptor to ensure that it includes all regions where the ligand may bind.

[0150] Set the docking parameters, generate 20 configurations each time, and export the parameter file;

[0151] Using Autodock Vina, AIE molecules were sequentially docked to each protein, with each protein docked 10 times. The configuration with the lowest binding energy in each docking was the optimal configuration for that docking. The optimal configuration with the highest probability among the 10 dockings was selected, and the interaction between AIE molecules and proteins was analyzed using Pymol software.

[0152] The docking results of TPPP-2Na with eight proteins—bovine serum albumin, butyrylcholinesterase, acetylcholinesterase, trypsin, pepsin, lipase, lysozyme, and γ-globulin—are as follows: Figures 9-16 As shown in the diagram, the docking results indicate that TPPP-2Na can bind to the cavity of bovine serum albumin (BSA), and all four benzene rings of the molecule are restricted by intermolecular forces. In contrast, most other proteins bind to the protein surface, and even when bound to a protein cavity, the intermolecular forces restrict the molecule significantly. Therefore, based on the docking results, BSA is the most likely candidate to bind to TPPP-2Na, suggesting that TPPP-2Na may be useful for the detection of BSA.

[0153] (3) Fluorescence response test of TPPP-2Na molecules to proteins

[0154] Weigh 5.42 mg of compound TPPP-2Na and dissolve it in 10 mL of PBS to prepare a concentration of 1 × 10⁻⁶. -3 mol / L -1 TPPP-2Na PBS solution;

[0155] Weigh 0.5 mg of each protein and dissolve it in 0.99 mL of PBS buffer to prepare protein PBS solutions;

[0156] Take 9 sample vials and label them 1 to 9. Add 0.99 mL of PBS buffer to sample vial 1, and add 0.99 mL of PBS solution of 8 proteins to sample vials 2 to 9 respectively.

[0157] Add 10 μL of TPPP-2Na PBS solution to each of the nine sample vials sequentially, so that the concentration of TPPP-2Na in each sample vial is 1 × 10⁻⁶. -5 The concentration of protein in the sample vials was 0.5 mg / mL, except for sample vial #1 which contained no protein.

[0158] Fluorescence emission spectroscopy was performed on the solutions in sample vials 1-9 with an excitation wavelength of 320 nm to obtain the following results: Figure 17 The fluorescence response spectra of TPPP-2Na to different proteins are shown. The fluorescence intensity of sample vial 1 is taken as I0, and the fluorescence intensity of the other sample vials is taken as I. The I / I0 ratio reflects the fluorescence response of TPPP-2Na molecules to different proteins. As shown in the figure, the fluorescence intensity of the TPPP-2Na mixed solution with bovine serum albumin is significantly enhanced compared to sample vial 1, while the fluorescence intensity of the TPPP-2Na mixed solution with other proteins does not show a significant enhancement compared to sample vial 1.

[0159] (4) Binding of TPPP-2Na with bovine serum albumin

[0160] Weigh 5.42 mg of compound TPPP-2Na and dissolve it in 10 mL of PBS to prepare a concentration of 1 × 10⁻⁶. -3 Prepare a bovine serum albumin PBS solution by dissolving 0.5 mg bovine serum albumin in 0.99 mL of PBS buffer.

[0161] Take 3 sample vials and label them 1 to 3. Add 0.99 mL of PBS buffer to sample vial 1, and add 0.99 mL of bovine serum albumin PBS solution to sample vials 2 and 3.

[0162] Add 10 μL of TPPP-2Na PBS solution to sample vials 1 and 2, and add 10 μL of PBS solution to sample vial 3.

[0163] Sample vial #1 contains TPPP-2Na solution with a concentration of 1×10⁻⁶. -5 mol / L; Sample vial #2 contains a mixed solution of TPPP-2Na and bovine serum albumin, with TPPP-2Na concentration of 1×10⁻⁶ mol / L. -5 The concentration of bovine serum albumin (BSA) is 0.5 mg / mL; sample vial #3 contains BSA solution with a concentration of 0.5 mg / mL.

[0164] Particle size analysis was performed on sample bottles 1-3, such as... Figure 18 As shown, after adding TPPP-2Na to the bovine serum albumin solution, the particle size of bovine serum albumin did not change significantly, but the particle size intensity of TPPP-2Na in the range of 100–300 nm decreased significantly, indicating that TPPP-2Na molecules entered the cavity of bovine serum albumin.

[0165] The changes in fluorescence intensity and particle size indicate that TPPP-2Na molecules can bind to bovine serum albumin (BSAL), and the molecules can enter the cavity of BSAL. In the cavity, they are subjected to forces sufficient to restrict molecular rotation, resulting in a significant enhancement of fluorescence in the BSAL cavity of TPPP-2Na.

[0166] Example 3

[0167] This embodiment provides a method for screening proteins that can specifically respond to existing AIE molecules, including the following steps:

[0168] (1) The preparation methods of TPPP-2Na-C include:

[0169] 0.02 mol of 4-aminobenzoate, 0.02 mol of 4-methylbenzaldehyde, 0.002 mol of p-toluenesulfonic acid, and 50 mL of glacial acetic acid were added sequentially to a 100 mL round-bottom flask equipped with a magnetic stirrer. The reaction mixture was heated to 90 °C and stirred for 30 min. Then, 0.01 mol of 2,3-butanedione was slowly added to the reaction mixture using a syringe. After the addition was complete, stirring was continued for 3 h, during which a yellow powder precipitate was observed to form in the reaction solution. The crude product was obtained by vacuum filtration. The collected filter cake was washed with acetic acid to obtain a pale yellow powder. The crude product was purified by recrystallization from a mixed solvent of chloroform and ethyl acetate to obtain an intermediate product.

[0170] Add 0.001 mol of the intermediate product and 50 mL of tetrahydrofuran to a 100 mL round-bottom flask equipped with a magnetic stirrer, and heat under reflux to dissolve the intermediate product. Weigh 2.00 g of NaOH and dissolve it in 8 mL of methanol. Gradually add the methanol-NaOH solution to the reaction solution. At this point, the reaction solution becomes turbid, and an insoluble precipitate forms. After heating under reflux for about 8 hours, the reaction solution gradually becomes clear. Continue heating and stirring for 12 hours. Remove the reaction solvent by rotary evaporation, add 15 mL of H2O and 2.00 g of NaOH, and continue heating under reflux for 24 hours. After the reaction is complete, slowly add the reaction solution to tetrahydrofuran to precipitate the product TPPP-2Na-C. Filter under reduced pressure and collect the filter cake. Dissolve the obtained crude product in 20 mL of H2O, then slowly add it to tetrahydrofuran. Filter under reflux and collect the filter cake. Test the pH value of the filtrate using precision pH paper. Repeat the process to remove residual NaOH from the product until the pH of the filtrate is close to neutral. Finally, dry the collected product to obtain the product TPPP-2Na-C.

[0171] The prepared TPPP-2Na-C was characterized, and the data are as follows:

[0172] 1 H NMR (400MHz, D2O, ppm): δ8.00-7.76 (m, 4H), 7.28 (d, J = 8.4Hz, 4H), 7.15 (d, J = 1.5Hz, 8H), 6.50 (s, 2H), 2.31 (s, 6H).

[0173] 13 C NMR(101MHz,90% D2O+10% THF-d8, ppm): δ172.53,141.74,136.19,135.38,134.26,131.37,130.41,130.33,128.84,127.54,123.70,95.01,20.26.

[0174] MALDI-TOF-MS (m / z):C 34 H 24 N2O4 2- The calculated molecular weight was 524.17, and the mass spectrometry characterization showed a molecular weight of 525.18.

[0175] The above structural characterization results indicate that the synthesized compound is TPPP-2Na-C.

[0176] The aggregation-induced emission (AIE) properties of TPPP-2Na-C in water and tetrahydrofuran systems were verified, confirming that TPPP-2Na-C has significant AIE properties.

[0177] (2) Molecular docking

[0178] The molecular structure of TPPP-2Na-C was drawn using Chemdraw software, and then the configuration with the lowest molecular energy was calculated using Chem3D and saved as a PDB file.

[0179] Download PDB files for eight proteins from the PDB database: bovine serum albumin, butyrylcholinesterase, acetylcholinesterase, trypsin, pepsin, lipase, lysozyme, and gamma globulin.

[0180] Use Pymol or Discovery Studio software to remove redundant ligands from protein PDB files, retaining only the protein structure.

[0181] The AutoDock Tools software was used to sequentially remove water molecules from the protein, add hydrogen, and balance its charge, and then output a receptor file in PDBQT format.

[0182] The AutoDock Tools software was used to perform hydrogenation on AIE molecules and to detect torsion centers and torsion bonds, and then output ligand files in PDBQT format.

[0183] Open the ligand and receptor files in AutoDock Tools software, set the docking box, and ensure that the size of the docking box covers the entire region of the receptor to ensure that it includes all regions where the ligand may bind.

[0184] Set the docking parameters, generate 20 configurations each time, and export the parameter file;

[0185] Using Autodock Vina, AIE molecules were sequentially docked to each protein, with each protein docked 10 times. The configuration with the lowest binding energy in each docking was the optimal configuration for that docking. The optimal configuration with the highest probability among the 10 dockings was selected, and the interaction between AIE molecules and proteins was analyzed using Pymol software.

[0186] The docking results of TPPP-2Na-C with eight proteins—bovine serum albumin, butyrylcholinesterase, acetylcholinesterase, trypsin, pepsin, lipase, lysozyme, and γ-globulin—are as follows: Figures 19-26As shown in the figure, the docking results indicate that TPPP-2Na-C can bind to the cavity of bovine serum albumin (BSA), and three of the four benzene rings of the molecule are restricted by intermolecular forces. In contrast, most other proteins bind to the protein surface, and even when bound to a protein cavity, the intermolecular forces restrict the molecule significantly. Therefore, based on the docking results, BSA is the most likely candidate to bind to TPPP-2Na-C, suggesting that TPPP-2Na-C may be useful for the detection of BSA.

[0187] (3) Fluorescence response test of TPPP-2Na-C molecule to protein

[0188] Weigh 5.70 mg of compound TPPP-2Na-C and dissolve it in 10 mL of PBS to prepare a concentration of 1×10⁻⁶. -3 mol / L -1 TPPP-2Na-C PBS solution;

[0189] Weigh 0.5 mg of each protein and dissolve it in 0.99 mL of PBS buffer to prepare protein PBS solutions;

[0190] Take 9 sample vials and label them 1 to 9. Add 0.99 mL of PBS buffer to sample vial 1, and add 0.99 mL of PBS solution of 8 proteins to sample vials 2 to 9 respectively.

[0191] Add 10 μL of TPPP-2Na-C PBS solution to each of the nine sample vials sequentially, so that the concentration of TPPP-2Na-C in each vial is 1 × 10⁻⁶. -5 The concentration of protein in the sample vials was 0.5 mg / mL, except for sample vial #1 which contained no protein.

[0192] Fluorescence emission spectroscopy was performed on the solutions in sample vials 1-9 with an excitation wavelength of 310 nm to obtain the following results: Figure 27 The figure shows the fluorescence response spectra of TPPP-2Na-C to different proteins. The fluorescence intensity of sample vial 1 is taken as I0, and the fluorescence intensity of the other sample vials is taken as I. The I / I0 ratio reflects the fluorescence response of TPPP-2Na-C to different proteins. As shown in the figure, the fluorescence intensity of the TPPP-2Na-C mixed solution with bovine serum albumin is significantly enhanced compared to sample vial 1, while the fluorescence intensity of the TPPP-2Na-C mixed solution with other proteins does not show a significant enhancement compared to sample vial 1.

[0193] (4) Binding of TPPP-2Na-C with bovine serum albumin

[0194] Weigh 5.70 mg of compound TPPP-2Na-C and dissolve it in 10 mL of PBS to prepare a concentration of 1×10⁻⁶. -3 Prepare a bovine serum albumin PBS solution by dissolving 0.5 mg bovine serum albumin in 0.99 mL of PBS buffer.

[0195] Take 3 sample vials and label them 1 to 3. Add 0.99 mL of PBS buffer to sample vial 1, and add 0.99 mL of bovine serum albumin PBS solution to sample vials 2 and 3.

[0196] Add 10 μL of TPPP-2Na-C PBS solution to sample vials 1 and 2, and add 10 μL of PBS solution to sample vial 3.

[0197] Sample vial #1 contains TPPP-2Na-C solution with a concentration of 1×10⁻⁶. -5 mol / L; Sample vial #2 contains a mixed solution of TPPP-2Na-C and bovine serum albumin, with TPPP-2Na-C concentration of 1×10⁻⁶ mol / L. -5 The concentration of bovine serum albumin (BSA) is 0.5 mg / mL; sample vial #3 contains BSA solution with a concentration of 0.5 mg / mL.

[0198] Particle size analysis was performed on sample bottles 1-3, such as... Figure 28 As shown, after adding TPPP-2Na-C to the bovine serum albumin solution, the particle size of bovine serum albumin did not change significantly, but the particle size intensity of TPPP-2Na-C in the range of 100–300 nm showed a significant decrease, indicating that TPPP-2Na-C molecules entered the cavity of bovine serum albumin.

[0199] The changes in fluorescence intensity and particle size indicate that TPPP-2Na-C molecules can bind to bovine serum albumin (BSAL), and the molecules can enter the cavity of BSAL. In the cavity, they are subjected to forces sufficient to restrict molecular rotation, resulting in a significant fluorescence enhancement of TPPP-2Na-C in the BSAL cavity.

[0200] Example 4

[0201] This embodiment provides a method for screening proteins that can specifically respond to existing AIE molecules, including the following steps:

[0202] (1) The preparation methods of TPPP-2Na-3C include:

[0203] 0.02 mol of 4-aminobenzoate, 0.02 mol of 4-isopropylbenzaldehyde, 0.002 mol of p-toluenesulfonic acid, and 50 mL of glacial acetic acid were added sequentially to a 100 mL round-bottom flask equipped with a magnetic stirrer. The reaction mixture was heated to 90 °C and stirred for 30 min. Then, 0.01 mol of 2,3-butanedione was slowly added to the reaction mixture using a syringe. After the addition was complete, stirring was continued for 3 h, during which a yellow powder precipitate was observed to form in the reaction solution. The crude product was obtained by vacuum filtration. The collected filter cake was washed with acetic acid to obtain a pale yellow powder. The crude product was purified by recrystallization from a mixed solvent of chloroform and ethyl acetate to obtain an intermediate product.

[0204] Add 0.001 mol of the intermediate product and 50 mL of tetrahydrofuran to a 100 mL round-bottom flask equipped with a magnetic stirrer, and heat under reflux to dissolve the intermediate product. Weigh 2.00 g of NaOH and dissolve it in 8 mL of methanol. Gradually add the methanol-NaOH solution to the reaction solution. At this point, the reaction solution becomes turbid, and an insoluble precipitate forms. After heating under reflux for about 8 hours, the reaction solution gradually becomes clear. Continue heating and stirring for 12 hours. Remove the reaction solvent by rotary evaporation, add 15 mL of H2O and 2.00 g of NaOH, and continue heating under reflux for 24 hours. After the reaction is complete, slowly add the reaction solution to tetrahydrofuran to precipitate the product TPPP-2Na-3C. Filter under reduced pressure and collect the filter cake. Dissolve the obtained crude product in 20 mL of H2O, then slowly add it to tetrahydrofuran. Filter under reflux and collect the filter cake. Test the pH value of the filtrate using precision pH paper. Repeat the process to remove residual NaOH from the product until the pH of the filtrate is close to neutral. Finally, dry the collected product to obtain the product TPPP-2Na-3C.

[0205] The prepared TPPP-2Na-3C was characterized, and the data are as follows:

[0206] 1 H NMR (400MHz, D2O, ppm): δ7.80 (d, J = 8.3Hz, 4H), 7.22 (d, J = 8.0Hz, 4H), 7.11 (s, 8H), 6.43 (s, 2H), 2.79 (p, J = 6.9Hz, 2H), 1.11 (d, J = 6.9Hz, 12H).

[0207] 13C NMR(101MHz,90% D2O+10%THF-d8,ppm): δ172.32,147.08,141.66,135.41,134.66,131.53,130.92,130.43,127.51,126.17,123.69,95.01,33.44,23.28.

[0208] MALDI-TOF-MS (m / z): C 38 H 32 N2O4 2- The calculated molecular weight was 580.24, and the molecular weight was determined to be 581.25 by mass spectrometry.

[0209] The above structural characterization results indicate that the synthesized compound is TPPP-2Na-3C.

[0210] The aggregation-induced emission (AIE) properties of TPPP-2Na-3C in water and tetrahydrofuran systems were verified, confirming that TPPP-2Na-3C has significant AIE properties.

[0211] (2) Molecular docking

[0212] The molecular structure of TPPP-2Na-3C was drawn using Chemdraw software, and then the configuration with the lowest molecular energy was calculated using Chem3D and saved as a PDB file.

[0213] Download PDB files for eight proteins from the PDB database: bovine serum albumin, butyrylcholinesterase, acetylcholinesterase, trypsin, pepsin, lipase, lysozyme, and gamma globulin.

[0214] Use Pymol or Discovery Studio software to remove redundant ligands from protein PDB files, retaining only the protein structure.

[0215] The AutoDock Tools software was used to sequentially remove water molecules from the protein, add hydrogen, and balance its charge, and then output a receptor file in PDBQT format.

[0216] The AutoDock Tools software was used to perform hydrogenation on AIE molecules and to detect torsion centers and torsion bonds, and then output ligand files in PDBQT format.

[0217] Open the ligand and receptor files in AutoDock Tools software, set the docking box, and ensure that the size of the docking box covers the entire region of the receptor to ensure that it includes all regions where the ligand may bind.

[0218] Set the docking parameters, generate 20 configurations each time, and export the parameter file;

[0219] Using Autodock Vina, AIE molecules were sequentially docked to each protein, with each protein docked 10 times. The configuration with the lowest binding energy in each docking was the optimal configuration for that docking. The optimal configuration with the highest probability among the 10 dockings was selected, and the interaction between AIE molecules and proteins was analyzed using Pymol software.

[0220] The docking results of TPPP-2Na-3C with eight proteins—bovine serum albumin, butyrylcholinesterase, acetylcholinesterase, trypsin, pepsin, lipase, lysozyme, and γ-globulin—are as follows: Figures 29-36 As shown in the figure, the docking results indicate that TPPP-2Na-3C can bind to the cavity of bovine serum albumin (BSA), and three of the four benzene rings of the molecule are restricted by intermolecular forces. In contrast, most other proteins bind to the protein surface, and even when bound to a protein cavity, the intermolecular forces restrict the molecule significantly. Therefore, based on the docking results, BSA is the most likely candidate to bind to TPPP-2Na-3C, suggesting that TPPP-2Na-3C may be useful for the detection of BSA.

[0221] (3) Fluorescence response test of TPPP-2Na-3C molecule to protein

[0222] Weigh 6.26 mg of compound TPPP-2Na-3C and dissolve it in 10 mL of PBS to prepare a concentration of 1×10⁻⁶. -3 mol / L -1 TPPP-2Na-3CPBS solution;

[0223] Weigh 0.5 mg of each protein and dissolve it in 0.99 mL of PBS buffer to prepare protein PBS solutions;

[0224] Take 9 sample vials and label them 1 to 9. Add 0.99 mL of PBS buffer to sample vial 1, and add 0.99 mL of PBS solution of 8 proteins to sample vials 2 to 9 respectively.

[0225] Add 10 μL of TPPP-2Na-3C PBS solution to each of the nine sample vials sequentially, so that the concentration of TPPP-2Na-3C in each sample vial is 1 × 10⁻⁶. -5 The concentration of protein in the sample vials was 0.5 mg / mL, except for sample vial #1 which contained no protein.

[0226] Fluorescence emission spectroscopy was performed on the solutions in sample vials 1-9 with an excitation wavelength of 310 nm to obtain the following results: Figure 37The fluorescence response spectra of TPPP-2Na-3C to different proteins are shown. The fluorescence intensity of sample vial 1 is taken as I0, and the fluorescence intensity of the other sample vials is taken as I. The I / I0 ratio reflects the fluorescence response of TPPP-2Na-3C to different proteins. As shown in the figure, the fluorescence intensity of the TPPP-2Na-3C mixed solution with bovine serum albumin is significantly enhanced compared to sample vial 1, while the fluorescence intensity of the TPPP-2Na-3C mixed solution with other proteins does not show a significant enhancement compared to sample vial 1.

[0227] (4) Binding of TPPP-2Na-3C with bovine serum albumin

[0228] Weigh 6.26 mg of compound TPPP-2Na-3C and dissolve it in 10 mL of PBS to prepare a concentration of 1×10⁻⁶. -3 Prepare a bovine serum albumin PBS solution by dissolving 0.5 mg bovine serum albumin in 0.99 mL of PBS buffer.

[0229] Take 3 sample vials and label them 1 to 3. Add 0.99 mL of PBS buffer to sample vial 1, and add 0.99 mL of bovine serum albumin PBS solution to sample vials 2 and 3.

[0230] Add 10 μL of TPPP-2Na-3C PBS solution to sample vials 1 and 2, and add 10 μL of PBS solution to sample vial 3.

[0231] Sample vial #1 contains TPPP-2Na-3C solution with a concentration of 1×10⁻⁶. -5 mol / L; Sample vial #2 contains a mixed solution of TPPP-2Na-3C and bovine serum albumin, with TPPP-2Na-3C concentration of 1×10⁻⁶ mol / L. -5 The concentration of bovine serum albumin (BSA) is 0.5 mg / mL; sample vial #3 contains BSA solution with a concentration of 0.5 mg / mL.

[0232] Particle size analysis was performed on sample bottles 1-3, such as... Figure 38 As shown, after adding TPPP-2Na-3C to the bovine serum albumin solution, the particle size of bovine serum albumin did not change significantly, but the particle size intensity of TPPP-2Na-3C in the range of 100–300 nm showed a significant decrease, indicating that TPPP-2Na-3C molecules entered the cavity of bovine serum albumin.

[0233] The changes in fluorescence intensity and particle size indicate that TPPP-2Na-3C molecules can bind to bovine serum albumin (BSAL), and the molecules can enter the cavity of BSAL. In the cavity, they are subjected to forces sufficient to restrict molecular rotation, resulting in a significant enhancement of fluorescence in the BSAL cavity of TPPP-2Na-3C.

[0234] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for designing a fluorescent probe for the detection of human serum albumin, characterized in that, Includes the following steps: (a) Obtain the PDB file of the target protein; calculate the cavity size of the target protein; (b) Determine the AIE motif and modify the AIE motif with hydrophilic and / or hydrophobic groups to regulate the hydrophilicity and hydrophobicity of the molecule, thereby obtaining a series of AIE molecules; (c) Calculate the lowest energy configuration of each AIE molecule using Chem3D software, and calculate the size of each lowest energy configuration; screen out AIE molecules that meet the requirements for entering the cavity of the target protein; (d) Molecular docking is performed between the screened AIE molecules and the target protein. The structure of the AIE molecules is adjusted based on the molecular docking results to design a fluorescent probe for protein detection. (e) Synthesize AIE molecules based on the design results and verify the binding of the AIE molecules to the target protein; the verification includes: adding AIE molecules to a PBS solution of the target protein and detecting changes in the fluorescence intensity and particle size of the solution; The AIE molecule in step (e) is a compound with the following structure: 。 2. The method according to claim 1, characterized in that, The PDB file of the target protein was downloaded from the PDB database; the cavity size of the target protein was calculated using Pymol software.

3. The method according to claim 1, characterized in that, The AIE molecule that meets the requirements for entering the cavity of the target protein satisfies the following condition: the size of the lowest energy configuration of the AIE molecule is smaller than the cavity size of the target protein; The sizes of the lowest-energy configurations of the AIE molecule were calculated using Pymol software.

4. The method according to claim 1, characterized in that, The method for regulating the hydrophilicity and hydrophobicity of molecules includes: modifying the AIE motif with hydrophilic and / or hydrophobic groups to restrict the movement of AIE molecules in the cavity of the target protein; The hydrophilic groups include carboxyl groups and / or sulfonate groups; the hydrophobic groups include alkyl groups and / or phenyl groups.

5. The method according to claim 1, characterized in that, The molecular docking was performed using AutoDock Vina software; In the molecular docking process, each AIE molecule docks with the target protein 10 times, resulting in 20 configurations per docking. The configuration with the lowest binding energy in each docking is selected as the optimal configuration for that docking. The interaction between the AIE molecule and the protein in each optimal configuration is analyzed using Pymol software, and the optimal configuration with the highest probability of occurrence is selected. The interaction between the AIE molecule and the target protein is then analyzed using Pymol software. Based on the analysis results, the structure of the AIE molecule is adjusted.

6. The method according to claim 1, characterized in that, In step (d), the adjustment method includes: If the analysis results show that the AIE molecule is not subjected to electrostatic and hydrogen bonding in the cavity of the target protein, then functional groups containing O and / or N are added to the AIE molecule. If the analysis results show that more than half of the benzene rings in the AIE molecule are not restricted by forces within the cavity of the target protein, then alkyl groups are added to the benzene rings of the AIE molecule and / or the number of benzene rings is increased in the AIE molecule. If the analysis results show that the forces acting on the AIE molecule in the cavity of the target protein all come from one side of the molecule, then a benzene ring or heterocycle is introduced onto the benzene ring to increase the size of the molecule.

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