A method for analyzing 8:2 fluoromodulin-target protein interactions based on confocal fluorescence imaging
By generating an 8:2FTOH-Glu(OtBu)-FITC complex and utilizing confocal fluorescence imaging, the problems of expensive equipment and complexity in analyzing the interaction between 8:2FTOH and target proteins in existing technologies were solved, and economical and efficient interaction analysis was achieved. It was revealed that 8:2FTOH is a direct ligand of AHR and is applicable to the analysis of multi-organ inflammation and injury.
Patent Information
- Application Number
- CN202510129310.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-05
- Publication Date
- 2025-09-26
- Estimated Expiration
- 2045-02-05
AI Technical Summary
Existing technologies make it difficult to efficiently and economically analyze the interaction between 8:2 fluorotelomer alcohol (8:2FTOH) and target proteins, especially the aryl hydrocarbon receptor (AHR). In addition, existing methods require expensive equipment, complex operations, and have prominent non-specific binding problems.
Fmoc-Glu(OtBu)OH was used as a linker reagent to react with 8:2FTOH to generate an 8:2FTOH-Glu(OtBu)-FITC complex, and its interaction with the target protein was observed and analyzed by confocal fluorescence imaging.
It has achieved a simple, economical and intuitive observation and analysis of the interaction between 8:2FTOH and target proteins. It was discovered for the first time that 8:2FTOH is a direct ligand of AHR and can be used to analyze its mediated multi-organ inflammation and damage.
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Figure CN119959197B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of molecular biology, and in particular to an 8:2 fluoromodulin-target protein interaction analysis method based on confocal fluorescence imaging. Background Art
[0002] The linear alkane contaminant 8:2-fluorotelomer alcohol (8:2FTOH) is a novel persistent organic pollutant that accumulates in the food chain and is widely used in commercial and industrial products such as paints, polishes, coatings, adhesives, and electronics. The widespread use of 8:2FTOH makes it ubiquitous in the environment. 8:2FTOH can enter animals and humans through multiple pathways, including air, soil, and food, and poses risks of hepatotoxicity, nephrotoxicity, developmental toxicity, immunotoxicity, and endocrine disruption. Although the levels of 8:2FTOH in the environment and the health risks it poses are concerning, little research has yet to investigate the mechanisms of 8:2FTOH-induced tissue damage and inflammation.
[0003] The aryl hydrocarbon receptor (AHR) is a highly conserved ligand-activated transcription factor. It is a central sensor involved in multiple environmental responses and a key player in the toxicity induced by environmental pollutants. In recent years, studies have reported that PFAS can activate the AHR and mediate oxidative stress and inflammation through this protein. However, these studies only demonstrated, through virtual docking technology, that PFAS can bind to the AHR and cause increased expression of the AHR pathway-related gene CYP1A1. The reported AHR ligands are all aromatic planar hydrocarbons, and no studies have yet confirmed that 8:2FTOH is a direct ligand of the AHR.
[0004] Currently, the analysis of interactions between small molecules and proteins relies primarily on four techniques: fluorescence polarization immunoassay (FPIA), isothermal calorimetry (ITC), and surface plasmon resonance (SPR). However, these methods suffer from disadvantages such as expensive equipment and reagents, complex procedures, difficulty in obtaining and immobilizing proteins, and nonspecific binding. FITC fluorescence imaging observation offers the advantages of simple experimental conditions, no need for additional equipment, and ease of operation. However, there are currently no reports on the analysis of the interaction between 8:2FTOH and AHR using FITC fluorescence imaging. Summary of the Invention
[0005] The purpose of the present invention is to provide an 8:2 fluoromodulin-target protein interaction analysis method based on confocal fluorescence imaging to solve the problems existing in the above-mentioned prior art. The present invention uses Fmoc-Glu (OtBu) OH as a linker reagent and a linear alkane pollutant 8:2FTOH to produce an 8:2FTOH-Glu (OtBu) -FITC complex with strong fluorescence, and then observes and analyzes the strength of the interaction between 8:2FTOH and the target protein through a confocal microscope. The present invention is simple and intuitive, highly economical and easy to operate; compared with previous molecule-protein interaction technologies, the experimental conditions are simple and no additional instruments and equipment are required.
[0006] To achieve the above object, the present invention provides the following solutions:
[0007] The present invention provides a method for analyzing the interaction between 8:2 fluoromodulin and target protein based on confocal fluorescence imaging, comprising the following steps:
[0008] After 8:2FTOH, Fmoc-Glu(OtBu)OH and the catalyst are mixed and reacted, washing, drying over anhydrous magnesium sulfate and first vacuum drying are performed in sequence to obtain 8:2FTOH-Fmoc-Glu(OtBu);
[0009] The Fmoc group of the 8:2FTOH-Fmoc-Glu(OtBu) was removed, and the mixture was vacuum dried for a second time. Fluorescein was added to carry out a fluorescence reaction, and the mixture was purified to obtain 8:2FTOH-Glu(OtBu)-FITC;
[0010] The 8:2FTOH-Glu(OtBu)-FITC was co-incubated with cells, and the interaction between the compound and the target protein was determined by fluorescence localization analysis.
[0011] Preferably, the target protein is aryl hydrocarbon receptor.
[0012] Preferably, the molar concentration ratio of the 8:2FTOH, Fmoc-Glu(OtBu)OH and the catalyst is 1:1.2:1.
[0013] Preferably, the mixing reaction time is 22 hours and the temperature is 20°C.
[0014] Preferably, the preparation used for removing the Fmoc group is 50% polyvinyl pyrrolidone.
[0015] Preferably, the catalyst is 4-dimethylaminopyridine.
[0016] Preferably, the fluorescein is isothiocyanate.
[0017] Preferably, the purification column used in the purification is a G25 rapid desalting column.
[0018] Preferably, the temperature of the first vacuum drying is 60° C. and the time is 0.5 h;
[0019] And / or, the temperature of the second vacuum drying is 60° C. and the time is 2 hours.
[0020] Preferably, the temperature of the fluorescence reaction is 4° C. and the time is 4 hours.
[0021] The present invention discloses the following technical effects:
[0022] The present invention establishes a method for observing and analyzing the interaction between 8:2FTOH and the aryl hydrocarbon receptor in cells based on confocal fluorescence imaging. The method is simple, intuitive, and highly economical. Specifically, the present invention uses Fmoc-Glu(OtBu)OH as a linker reagent with the linear alkane pollutant 8:2FTOH to produce an 8:2FTOH-Glu(OtBu)-FITC complex with strong green fluorescence. The interaction strength between 8:2FTOH and the target protein (AHR) can then be observed and analyzed using a confocal microscope. The present invention is simple, intuitive, economical, and easy to operate. Compared with previous molecule-protein interaction technologies, the experimental conditions are simple and no additional instruments or equipment are required.
[0023] At the same time, the present invention discovered and reported for the first time that the straight-chain alkane pollutant 8:2FTOH is a direct ligand of AHR. Therefore, the interaction analysis method provided by the present invention can be used to analyze the multi-organ inflammation and damage mediated by 8:2FTOH through activation of AHR. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0025] Figure 1 This is the synthesis process of 8:2FTOH-Glu(OtBu)-FITC;
[0026] Figure 2 The mass spectrum of 8:2FTOH-Glu(OtBu);
[0027] Figure 3 This is the mass spectrum of 8:2FTOH-Glu(OtBu)-FITC;
[0028] Figure 4These are confocal fluorescence images of 8:2FTOH-Glu(OtBu)-FITC and AHR at different incubation times; 8:2FTOH+FITC refers to the treatment with 8:2FTOH-Glu(OtBu)-FITC; and FITC refers to the treatment with FITC only. DETAILED DESCRIPTION
[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0030] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.
[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.
[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.
[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0034] Example 18: Establishment of Synthesis Conditions for 2FTOH-Fmoc-Glu(OtBu)
[0035] (1) Optimization of the synthesis ratio of 8:2FTOH-Fmoc-Glu(OtBu)
[0036] The synthesis ratio was optimized according to Table 1, with the yield of 8:2FTOH-Fmoc-Glu(OtBu) as the selection criterion.
[0037] The specific steps are as follows:
[0038] According to Ratio 1 and Ratio 2 in Table 1, accurately weigh the corresponding amounts of 8:2FTOH, Fmoc-Glu(OtBu)OH, and catalyst DMAP (4-dimethylaminopyridine) into a 50 mL round-bottom flask containing 15 mL of dichloromethane (DCM). After complete dissolution, stir at room temperature (25°C) for 24 hours. After the reaction is complete, add an equal volume of DCM and wash twice with an equal volume of distilled water. After washing, dry the mixture over anhydrous magnesium sulfate and vacuum dry it at 60°C for 0.5 hours to obtain 8:2FTOH-Fmoc-Glu(OtBu). Calculate the yield by weight.
[0039] Table 1 Compound ratio
[0040]
[0041] The results showed that when the molar concentration ratio of 8:2FTOH, Fmoc-Glu(OtBu)OH and DMAP was 1:1.2:1, the yield of 8:2FTOH-Fmoc-Glu(OtBu) was the highest (Table 1).
[0042] (2) Optimization of synthesis temperature and time of 8:2FTOH-Fmoc-Glu(OtBu)
[0043] Further optimization was performed based on the temperature and synthesis time of the previous step. The ratio of 8:2FTOH, Fmoc-Glu(OtBu)OH, and DMAP was 1:1.2:1. The temperature and synthesis time were optimized according to Table 2, with the yield of 8:2FTOH-Fmoc-Glu(OtBu) as the selection criterion.
[0044] The specific steps are as follows:
[0045] Accurately weigh the corresponding masses of 8:2FTOH, Fmoc-Glu(OtBu)OH, and catalyst DMAP at a molar ratio of 1:1.2:1 into a 50 mL round-bottom flask containing 15 mL of dichloromethane (DCM). Once completely dissolved, stir at 20°C, 25°C, and 30°C for 22, 24, and 26 hours, respectively. After the reaction, add an equal volume of DCM and wash twice with an equal volume of distilled water. After washing, dry the product over anhydrous magnesium sulfate and vacuum dry it at 60°C for 0.5 hour to obtain 8:2FTOH-Fmoc-Glu(OtBu). The yield was calculated by weight.
[0046] Table 2 Yield (%) of 8:2FTOH-Fmoc-Glu(OtBu) at different temperatures and synthesis times
[0047] Temperature (℃) / synthesis time (h) 22 24 26 20 94 88 84 25 89 77 76 30 82 75 77
[0048] The results showed that the yield of 8:2FTOH-Fmoc-Glu(OtBu) was the highest when stirred at 20°C for 22 h (Table 2).
[0049] Example 28: Establishment of Synthesis Conditions for 2FTOH-Glu(OtBu)-FITC
[0050] Screening of de-Fmoc reagents: The specific types of de-Fmoc reagents are shown in Table 3, and the yield of 8:2FTOH-Glu(OtBu)-FITC is used as the selection criterion.
[0051] The synthesis process of 8:2FTOH-Glu(OtBu)-FITC is as follows Figure 1 The specific steps are as follows:
[0052] 8:2FTOH, Fmoc-Glu(OtBu)OH, and catalyst DMAP were accurately weighed at a molar ratio of 1:1.2:1 into a 50 mL round-bottom flask containing 15 mL of dichloromethane (DCM). After complete dissolution, the mixture was stirred at 20°C for 22 hours. After completion of the reaction, an equal volume of DCM was added and the product was washed twice with an equal volume of distilled water. After washing, the product was dried over anhydrous magnesium sulfate and vacuum-dried at 60°C for 0.5 hours to obtain 8:2FTOH-Fmoc-Glu(OtBu). The Fmoc group of 8:2FTOH-Fmoc-Glu(OtBu) was removed by using 50% polyvinylpyrrolidone solution, acetone, and diethylamine, respectively, with stirring at room temperature for 30 minutes. The product was then vacuum-dried at 60°C for 2 hours to obtain 8:2FTOH-Glu(OtBu). The product was dissolved in dimethylformamide (DMF), 0.1 mM fluorescein isothiocyanate (FITC) was added, and the reaction was incubated at 4°C for 4 hours. The reaction product was purified by G25 rapid desalting column to obtain 8:2FTOH-Glu(OtBu)-FITC, and then dried under vacuum at 60°C and weighed to calculate the yield. 8:2FTOH-Glu(OtBu) and 8:2FTOH-Glu(OtBu)-FITC were detected by mass spectrometry. The mass spectrum of 8:2FTOH-Glu(OtBu) is shown in Figure 2 As shown, the mass spectrum of 8:2FTOH-Glu(OtBu)-FITC is as shown Figure 3 shown.
[0053] Table 3 Yield (%) of 8:2FTOH-Glu(OtBu)-FITC under different Fmoc removal reagents
[0054] Fmoc removal reagent Yield (%) 50% polyvinylpyrrolidone 82 acetone 50 Diethylamine 66
[0055] The results showed that the yield was the highest when 50% polyvinylpyrrolidone was used as the Fmoc removal reagent (Table 3).
[0056] Example 38: Establishment of 2FTOH-Glu-FITC and Cell Incubation Time
[0057] 8:2FTOH, Fmoc-Glu(OtBu)OH, and catalyst DMAP were accurately weighed at a molar ratio of 1:1.2:1 into a 50 mL round-bottom flask containing 15 mL of dichloromethane (DCM). After complete dissolution, the mixture was stirred at 20°C for 22 hours. After completion of the reaction, an equal volume of DCM was added and the product was washed twice with an equal volume of distilled water. After washing, the product was dried over anhydrous magnesium sulfate and vacuum-dried at 60°C for 0.5 hours to obtain 8:2FTOH-Fmoc-Glu(OtBu). The Fmoc group of 8:2FTOH-Fmoc-Glu(OtBu) was removed by stirring with 50% polyvinylpyrrolidone solution at room temperature for 30 minutes and then vacuum-dried at 60°C for 2 hours to obtain 8:2FTOH-Glu(OtBu). The product was dissolved in dimethylformamide (DMF), 0.1 mM FITC was added, and the reaction was incubated at 4°C for 4 hours. The reaction product was purified by G25 rapid desalting column to obtain 8:2FTOH-Glu(OtBu)-FITC. After incubating 8:2FTOH-Glu(OtBu)-FITC with cells for 1 and 2 hours, the culture medium containing the complex was removed and the immunofluorescence assay of AHR was performed. The colocalization of 8:2FTOH-Glu(OtBu)-FITC (green fluorescence) and AHR (red fluorescence) was observed under a confocal microscope. The results are shown in Figure 2. Figure 4 The results showed that the imaging effect of 8:2FTOH-Glu(OtBu)-FITC and AHR was the best when incubated for 2 hours, and co-localization between 8:2FTOH and AHR could be observed.
[0058] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.
Claims
1. A method for analyzing the interaction between 8:2 fluoromodulin and target protein based on confocal fluorescence imaging, characterized in that: The following steps are involved: 8:2 fluorotelomer alcohol (8:2 FTOH), Fmoc-Glu(OtBu) OH and the catalyst were mixed and reacted in a flask containing dichloromethane. After adding dichloromethane, the mixture was washed, dried over anhydrous magnesium sulfate and dried under vacuum to obtain 8:2 FTOH-Fmoc-Glu(OtBu). The Fmoc group of the 8:2 FTOH-Fmoc-Glu (OtBu) is removed, vacuum drying is performed for a second time, fluorescein is added to perform a fluorescence reaction, and purification is performed to obtain 8:2 FTOH-Glu (OtBu)-FITC; the fluorescein is isothiocyanate; The 8:2 FTOH-Glu(OtBu)-FITC was co-incubated with cells, and the interaction between the compound and the target protein was determined by fluorescence colocalization analysis.
2. The interaction analysis method according to claim 1, characterized in that The target protein is the aryl hydrocarbon receptor.
3. The interaction analysis method according to claim 1, characterized in that The molar concentration ratio of the 8:2 FTOH, Fmoc-Glu(OtBu)OH and the catalyst is 1:1.2:
1.
4. The interaction analysis method according to claim 1, characterized in that The mixing reaction was carried out for 22 hours at a temperature of 20°C.
5. The interaction analysis method according to claim 1, characterized in that The preparation used for removing the Fmoc group is 50% polyvinyl pyrrolidone.
6. The interaction analysis method according to claim 1 or 3, characterized in that The catalyst is 4-dimethylaminopyridine.
7. The interaction analysis method according to claim 1, characterized in that The purification column used in the purification is a G25 fast desalting column.
8. The interaction analysis method according to claim 1, characterized in that The first vacuum drying temperature is 60°C and the time is 0.5h; And / or, the temperature of the second vacuum drying is 60° C. and the time is 2 hours.
9. The interaction analysis method according to claim 1, characterized in that The temperature of the fluorescence reaction is 4° C. and the time is 4 hours.