Active peptide fluorescent probe as well as preparation method and application thereof
By developing active peptide fluorescence probes, real -time monitoring the transfer and metabolic process of Hempote Mushroom Active Peptide in the body, solving the problem of difficulty monitoring existing technology, and realizing the basic support for researching its immune regulation mechanism.
Patent Information
- Application Number
- CN202411481843.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-23
- Publication Date
- 2025-01-24
AI Technical Summary
The prior art is difficult to monitor the transport, absorption and metabolic processes of active peptides of ceruleus in real time in vivo, resulting in increased difficulty in studying its immune regulation mechanism.
Develop an active peptide fluorescent probe to verify its biological safety and accuracy through small animal live imaging and in vitro cell experiments, and monitor the transfer and metabolic processes of the active peptides in the body in real time.
A real -time and accurate monitoring of the transfer and metabolic process of the activated peptide of Hemplars of Hemper, reducing the cost and complexity of research, and providing the foundation for studying its immune regulation mechanism.
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Figure CN119350442A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and specifically relates to an active peptide fluorescent probe and a preparation method and application thereof. Background Art
[0002] Hericium erinaceus is a traditional edible mushroom that can be used as both medicine and food. Current research on Hericium erinaceus mainly focuses on compounds such as Hericium erinaceus polysaccharides and Hericium erinaceus proteins, but research on Hericium erinaceus peptides is still incomplete.
[0003] In the early stage, researchers (Yu Yihan, Preparation of Hericium erinaceus active peptides and their immunomodulatory effects [D], 2021) obtained a highly immune Hericium erinaceus active octapeptide LPGKVIAS through separation and purification, structural identification and in vitro immune activity screening. The amino acid sequence is Leu-Pro-Gly-Lys-Val-Ile-Ala-Ser and the molecular weight is 784.5Da. However, the mechanism of its transport, digestion and absorption in the body is still unclear.
[0004] At present, the methods for studying the transport, absorption and metabolism of active substances mainly include in vitro intestinal cell models, drug metabolism kinetics research and isotope tracing technology.
[0005] In vitro intestinal cell models: mainly include two-dimensional and three-dimensional cell culture models. Two-dimensional culture is to culture single-cell or multi-cell cell models in a plane or Transwell chamber, while three-dimensional culture is to culture in an extracellular matrix to provide a microenvironment that is closer to physiological and dynamic characteristics. This method only simulates the absorption and metabolic process of the intestine in vitro and cannot completely replace the in vivo environment. The results are inaccurate and the cost is high.
[0006] Pharmacokinetics: Study the absorption, distribution, metabolism and excretion of drugs in the body. This method can provide detailed dynamic information of drugs in the body, including the half-life and clearance rate of drugs. However, this method cannot simulate complex physiological and pathological conditions, resulting in certain deviations in the results, and requires a large number of animal experiments and clinical data, which is cumbersome and costly.
[0007] Isotope tracing technology: It is a microanalytical method that uses isotopes as tracers to mark the research object, that is, introducing isotope atoms into the research object, and using mass spectrometers, nuclear magnetic resonance instruments and other analytical instruments to measure the changes in their position, quantity and other changes after the reaction, thus becoming a means of studying the transport, absorption and metabolic processes of active substances. However, this method has relatively high requirements for instruments and equipment, especially when labeling multiple elements at the same time, it requires ultra-high-resolution mass spectrometry for measurement, and derivatization is required when necessary. In addition, due to the small number of stable isotopes that can be used as tracers and their high prices, its scope of application is subject to certain restrictions.
[0008] Therefore, it is of great significance to construct a fluorescent probe that can monitor the transport, absorption and metabolic process of Hericium erinaceus active peptides in real time in vivo. Summary of the invention
[0009] Purpose of the invention: The purpose of the present invention is to provide an active peptide fluorescent probe and its preparation method and application in view of the deficiencies of the prior art. The present invention constructs an active peptide fluorescent probe that can monitor the transport, absorption and metabolism of Hericium erinaceus active peptides in real time in vivo. Through in vitro cell experiments, it is verified that the active peptide probe has no cytotoxicity, has biosafety, and can be applied to biological research; through small animal in vivo imaging, it is verified that the active peptide probe can more conveniently, quickly and accurately monitor the transport, absorption and metabolism of the Hericium erinaceus active peptides in real time in the living body, thereby providing a basis for studying its immune regulation mechanism.
[0010] Technical solution: The purpose of the present invention is achieved through the following technical solution:
[0011] The present invention provides an active peptide fluorescent probe having the following structural formula:
[0012]
[0013] The present invention also provides a method for preparing the above-mentioned active peptide fluorescent probe, comprising the following steps:
[0014] (1) N,N-dimethylformamide, dichloromethane and phosphorus oxychloride are mixed, added to cyclohexanone dissolved with dichloromethane, and heated and stirred until the reaction is completed; the reaction solution is cooled to room temperature, and post-treated to obtain compound 1;
[0015] (2) Mix 2,3,3-trimethyl-3H-indole and 1,3-propane sultone in acetonitrile, heat and stir until the reaction is complete; cool the reaction solution to room temperature, add it to an ice water-ether mixture solution, collect the red precipitate, wash, separate and purify it to obtain compound 2;
[0016] (3) dissolving compound 1, compound 2 and sodium acetate in a mixed solution of n-butanol-benzene, heating under reflux for reaction, and after the reaction is complete, removing the solvent to obtain a crude product, which is purified to obtain compound 3;
[0017] (4) dissolving compound 3, 3-mercaptopropionic acid and triethylamine in DMF and stirring at room temperature. After the reaction is complete, removing the solvent to obtain a crude product; purifying to obtain compound 4;
[0018] (5) Compound 4 was dissolved in DMF, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate HATU and N,N-diisopropylethylamine DIPEA were added, and p-fmoc was added after stirring. After reacting at room temperature for 2 hours under nitrogen protection, piperidine was added to the reaction solution and stirring was continued. After the reaction was completed, the reaction product was treated before purification using semi-preparative reverse-phase high performance liquid chromatography; the product was separated and purified using a preparative high performance liquid chromatography column, and the obtained solution was freeze-dried to obtain the active peptide fluorescent probe;
[0019] The reaction route is as follows:
[0020]
[0021] In the present invention, there are two amino groups on the active octapeptide of Hericium erinaceus, and one of the amino groups is protected by fmoc to obtain compound p-fmoc, which is synthesized by Nanjing GenScript Biotech Co., Ltd. Compound 4 binds to the unprotected amino site in p-fmoc, and fmoc is detached to obtain probe Cy-p.
[0022] In step (1), the heating temperature is 80° C. and the heating time is 3 h.
[0023] In step (2), the heating temperature is 80° C. and the heating time is 12-24 hours.
[0024] In step (3), the volume ratio of n-butanol to benzene is 1:1.5.
[0025] The present invention also provides application of the active peptide fluorescent probe in monitoring the active peptide of Hericium erinaceus.
[0026] The amino acid sequence of the Hericium erinaceus active peptide is Leu-Pro-Gly-Lys-Val-Ile-Ala-Ser, that is, the Hericium erinaceus active octapeptide LPGKVIAS.
[0027] The present invention adopts the above-mentioned active peptide fluorescent probe to monitor the in vivo transport and absorption process of Hericium erinaceus active peptides through small animal in vivo imaging.
[0028] In order to understand the mechanism of action of Hericium erinaceus active peptide in vivo, the present invention synthesized the above-mentioned active peptide fluorescent probe; the distribution position of Hericium erinaceus active peptide at different time points in vivo was understood through small animal living imaging; the distribution and final metabolism of Hericium erinaceus active peptide in various organs of the animal body at different time points were understood through in vitro organ imaging.
[0029] The present invention also provides the use of the active peptide fluorescent probe in the preparation of a preparation for monitoring the in vivo transport and absorption process of the Hericium erinaceus active peptide.
[0030] Beneficial effects:
[0031] (1) The present invention constructs an active peptide fluorescent probe that can monitor the active peptide of Hericium erinaceus in real time in vivo. Through in vitro cell experiments, it is verified that the active peptide probe has no cytotoxicity, has biosafety, and can be applied to biological research; through small animal in vivo imaging, it is verified that the active peptide probe can more conveniently, quickly, and accurately monitor the process of transport, absorption and metabolism of the active peptide of Hericium erinaceus in real time in the living body, thereby providing a basis for studying its immune regulation mechanism.
[0032] (2) Compared with the traditional in vitro intestinal cell model and drug metabolism kinetics research, the active peptide fluorescent probe of the present invention can monitor the exact location of active substances in living organisms in real time, has high sensitivity in signal detection and accurate results, and the probe synthesis process is simple and convenient with low cost.
[0033] (3) Compared with isotope tracing technology, the active peptide fluorescent probe of the present invention has lower requirements for detection equipment and is more widely used. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 The mass spectrum of the fluorescent probe Cy-p prepared in Example 1 of the present invention;
[0035] Figure 2 This is a cell viability graph of the fluorescent probe Cy-p prepared in Example 1 of the present invention incubated with RAW264.7 cells for 24 hours;
[0036] Figure 3 The biodistribution diagram of Cy-p in healthy mice after oral administration is shown in Figure 2. Figure 3 A is a schematic diagram of Cy-p visualization;
[0037] Figure 3 B is the fluorescence intensity image of the whole body of mice from 0 to 48 hours; Figure 3 C is the in vitro imaging of the main organs of mice (heart, liver, spleen, lung, kidney, intestine, and stomach) from 0 to 24 hours. DETAILED DESCRIPTION
[0038] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.
[0039] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.
[0040] Example 1 Preparation of fluorescent probe Cy-p
[0041] The synthetic route is as follows:
[0042]
[0043] 1. Preparation of Compound 1
[0044] First, N,N-dimethylformamide (DMF) (7.33 g, 7.7 mL, 100 mmol), dichloromethane (DCM) (20 mL) and phosphorus oxychloride (15.62 g, 101.89 mmol) were mixed in an ice-water bath (0°C), and then added to cyclohexanone (3.16 mL, 30.57 mmol) dissolved in dichloromethane (37 mL), and then stirred at 80°C for 3 h until the reaction was completed. The reaction solution was cooled to room temperature, poured into ice water and kept overnight to obtain compound 1.
[0045] 2. Preparation of Compound 2
[0046] 2,3,3-Trimethyl-3H-indole (2 g, 12.48 mmol) and 1,3-propane sultone (4.57 g, 37.44 mmol) were mixed in 15 mL of acetonitrile and heated and stirred at 80 ° C overnight until the reaction was completed. After the reaction solution was cooled to room temperature, it was added dropwise to a mixed solution of ice water-ether (1:1, v / v). The red precipitate was collected, washed with petroleum ether (30 mL), and gradient eluted using methanol (MeOH) and DCM (1:30-200) as eluents to obtain compound 2.
[0047] 3. Preparation of Compound 3
[0048] Compound 1 (0.50 g, 2.90 mmol), compound 2 (1.64 g, 5.81 mmol) and sodium acetate (0.48 g, 5.81 mmol) were dissolved in a mixed solution of 50 mL of n-butanol-benzene (1:1.5, v / v), and refluxed at 110°C for 12 h using a Dean-Stark apparatus. After the reaction was completed, the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography using MeOH / DCM (1:25-200) as an eluent to obtain compound 3.
[0049] 4. Preparation of Compound 4
[0050] Compound 3 (0.2 g, 0.28 mmol), 3-mercaptopropionic acid (0.06 g, 0.57 mmol) and triethylamine (0.06 g, 0.57 mmol) were dissolved in 5 mL DMF, and stirred overnight at room temperature using a Dean-Stark apparatus. After the reaction was complete, the solvent was removed under reduced pressure to obtain a crude product. Using MeOH / DCM (1:10-200) as an eluent, the crude product was purified by silica gel column chromatography to obtain compound 4.
[0051] 5. Preparation of fluorescent probe Cy-p
[0052] (1) Synthesis reaction preparation: 100 mg of compound 4 was dissolved in a reaction bottle containing 5 mL of N,N-dimethylformamide (DMF).
[0053] (2) Addition of activator and base: 60 mg of activator HATU and 30 μL of organic base DIPEA were added to the solution and stirred for 15 minutes.
[0054] (3) Addition of reactants: 130 mg of compound p-fmoc (purity ≥ 95%, purchased from Nanjing GenScript Biotech Co., Ltd.) was added to the stirred solution, and the mixture was reacted at room temperature for 2 hours under nitrogen protection.
[0055] (4) Termination of reaction: 1 mL of piperidine was added to the reaction mixture and stirred at room temperature for 30 minutes to terminate the reaction.
[0056] (5) Preparation before purification: The reaction product was treated before purification using semi-preparative reverse phase high performance liquid chromatography (RP-HPLC). The sample was dissolved to prepare a solution with a concentration of 10 mg / mL and filtered through a 0.22 μm filter membrane.
[0057] (6) Purification: Purification was carried out using a Zorbax 300SB-C18 column in a preparative HPLC system. The mobile phases used were 0.1% trifluoroacetic acid in ultrapure water (phase A) and 0.1% trifluoroacetic acid in acetonitrile (phase B). Elution was performed in a gradient elution mode, and the detection wavelength was set to 220 nm.
[0058] (7) Collection and drying: The corresponding components are automatically collected according to the appearance of the chromatographic peaks. After the same components collected multiple times are mixed, the solvent is removed by vacuum concentration and freeze drying technology to finally obtain the purified product in the form of green powder, i.e., the fluorescent probe Cy-p. Its mass spectrum is shown in Figure 1 .
[0059] ESI-MS: m / z, calculated value C 75 H 109 N 11 O 17 S 32 - ,765.86,measured value: 765.76.
[0060] Example 2 Cytotoxicity Test
[0061] The MTT method was used to determine the toxicity of the fluorescent probe Cy-p. When the adhesion rate of RAW264.7 cells (Shanghai Fudan IBS Cell Bank) in the culture dish reached 90%, they could be plated. The original culture medium in the culture dish was removed, and the cells were collected into a centrifuge tube. The cells were diluted with the prepared complete culture medium (DMEM culture medium, 90% + high-quality fetal bovine serum, 10%) and blown into a single cell suspension. The cell count was counted on a cell counting plate and the cell density was adjusted to 5×10 4 / mL, and then the cells were inoculated into 96-well plates. After overnight attachment, RAW264.7 cells were stimulated with fluorescent active peptide Cy-p at concentrations of 0, 1, 2, 5, 10, and 20 μmol / L (the group with a concentration of 0 μmol / L was the CT group), and 5 parallels were made for each gradient. After 24 hours of co-incubation at room temperature, 20 μL of MTT solution was added to each well. After incubation in the incubator for another 4 hours, the culture medium was discarded, 200 μL of DMSO was added to each well, and the plates were shaken on a low-speed shaker for 10 minutes to mix well. The absorbance was detected at a wavelength of 490 nm.
[0062] Figure 2 This is a cell viability graph of RAW264.7 cells incubated with Cy-p for 24 hours. From the graph, it can be found that after the Cy-p compound was co-cultured with the cells for 24 hours, it had no significant effect on the proliferation of RAW264.7 cells compared with the CT group in the concentration range of 0-20 μmol / L, indicating that the Cy-p compound has no cytotoxicity at 0-20 μmol / L, has the advantage of high biosafety, and can be used for in vivo imaging.
[0063] Example 3 Transport and metabolism of Hericium erinaceus active peptides in mice
[0064] Male ICR and C57 BL / 6 mice (4 weeks old, 18 ± 2.00 g) were purchased from Henan Scobes Biotechnology Co., Ltd. (Anyang, China, Certificate No.: SCXK (Yu) 2020-0005). Before the experiment, mice were free to eat and drink water, and the experiment was conducted after three days of acclimatization. Before administration, the mice were fasted for 12 h and free to drink water. 21 mice were randomly divided into 7 groups (n = 3 per group).
[0065] like Figure 3As shown in A, the Cy-p prepared in Example 1 was prepared into a 200 μmol / L stock solution with DMSO, and then diluted to 20 μmol / L with physiological saline solution. 200 μL of Cy-p probe sample (concentration of 20 μmol / L) was administered to each mouse by gavage. In vivo imaging of mice was performed at 0h, 1h, 2h, 6h, 8h, 12h, 24h and 48h after administration.
[0066] Next, in order to identify its fluorescence signals in specific tissues and organs, we conducted an in vitro optical imaging study. The Cy-p prepared in Example 1 was prepared into a 200 μmol / L stock solution with DMSO, and then diluted to 20 μmol / L with physiological saline solution. 200 μL of the probe sample (at a concentration of 20 μmol / L) was administered to each mouse by gavage. At 1h, 6h, 12h, and 24h after administration, the mice were euthanized, the mice were quickly dissected, and then the isolated organs were imaged and analyzed for fluorescence.
[0067] Figure 3 B is the tissue distribution image of Cy-p after oral administration in mice. It was found that obvious fluorescence was observed in the abdomen of mice 1 hour after oral administration. 2 hours after oral administration, the fluorescence intensity of the abdomen of mice decreased slightly compared with the previous time point. At the next time point 6 hours, the fluorescence intensity began to weaken. Weak fluorescence was observed after 24 hours, and the fluorescence completely disappeared after 48 hours.
[0068] Figure 3 C is the fluorescence imaging of Cy-p mouse ex vivo organs. It was found that the fluorescence intensity gradually decreased over time, which may be caused by absorption and excretion. Cy-p reached the stomach and small intestine of mice 1 hour after gavage; at 6 hours, Cy-p mainly completed the process of transport from the stomach to the intestine of mice, and at this time, the active peptide of Hericium erinaceus entered the large intestine of mice. After 12 hours and 24 hours, the fluorescence disappeared. This shows that Cy-p stayed in the stomach and intestine for 6 hours. In addition, obvious Cy-p fluorescence signals were detected in the lungs, indicating that it was absorbed into the systemic circulation. This finding supports the ability of Cy-p to cross the gastrointestinal barrier and enter the systemic circulation.
[0069] Based on in vitro cytotoxicity experiments, it was verified that the active peptide probe had no cytotoxicity, was biosafe, and could be used in biological research; based on the changes in fluorescence intensity of in vivo imaging of small animals and in vitro imaging of organs, it was verified that the active peptide probe Cy-p could more conveniently, quickly, and accurately monitor the transport, absorption and metabolism of the Hericium erinaceus active peptide in real time in living bodies, thereby providing a basis for studying its immune regulation mechanism.
[0070] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.
Claims
1. An active peptide fluorescent probe, characterized in that: It has the following structural formula:
2. A method for preparing the active peptide fluorescent probe according to claim 1, characterized in that: The following steps are involved: (1) N,N-dimethylformamide, dichloromethane and phosphorus oxychloride are mixed, added to cyclohexanone dissolved with dichloromethane, and heated and stirred until the reaction is completed; the reaction solution is cooled to room temperature, and post-treated to obtain compound 1; (2) Mix 2,3,3-trimethyl-3H-indole and 1,3-propane sultone in acetonitrile, heat and stir until the reaction is complete; cool the reaction solution to room temperature, add it to an ice water-ether mixture solution, collect the red precipitate, wash, separate and purify it to obtain compound 2; (3) dissolving compound 1, compound 2 and sodium acetate in a mixed solution of n-butanol-benzene, heating under reflux for reaction, and after the reaction is complete, removing the solvent to obtain a crude product, which is purified to obtain compound 3; (4) dissolving compound 3, 3-mercaptopropionic acid and triethylamine in DMF and stirring at room temperature. After the reaction is complete, removing the solvent to obtain a crude product; purifying to obtain compound 4; (5) Compound 4 was dissolved in DMF, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate HATU and N,N-diisopropylethylamine DIPEA were added, and p-fmoc was added after stirring. After reacting at room temperature for 2 hours under nitrogen protection, piperidine was added to the reaction solution and continued to stir. After the reaction was completed, the reaction product was purified by semi-preparative reverse phase high performance liquid chromatography. The product was separated and purified by a preparative high performance liquid chromatography column, and the obtained solution was freeze-dried to obtain the active peptide fluorescent probe. The reaction route is as follows:
3. The preparation method according to claim 2, characterized in that: In step (1), the heating temperature is 80° C. and the heating time is 3 h.
4. The preparation method according to claim 2, characterized in that: In step (2), the heating temperature is 80° C. and the heating time is 12-24 hours.
5. The preparation method according to claim 2, characterized in that: In step (3), the volume ratio of n-butanol to benzene is 1:1.
5.
6. Use of the active peptide fluorescent probe according to claim 1 in monitoring active peptides of Hericium erinaceus.
7. The use according to claim 6, characterized in that: The amino acid sequence of the Hericium erinaceus active peptide is Leu-Pro-Gly-Lys-Val-Ile-Ala-Ser.
8. The use according to claim 6, characterized in that: The active peptide fluorescent probe according to claim 1 is used to monitor the transport and absorption process of Hericium erinaceus active peptide in vivo through small animal in vivo imaging.
9. Use of the active peptide fluorescent probe according to claim 1 in the preparation of a preparation for monitoring the transport and absorption process of Hericium erinaceus active peptides in vivo.