A multimodal probe with dual functions of trace positioning and target protein enrichment capture and preparation method and application thereof

Abstract

The application discloses a kind of multi-modal probes with tracer positioning and target protein enrichment capture bifunction, and preparation method and application thereof, and relates to the field of biochemistry.The structure formula of the multi-modal probe is:the application is based on the principle of chemistry biology, and develops a kind of multi-modal probe with diketopyrrolo structure and azide substitution group simultaneously.The multi-modal probe can react with oxygen-rich olefin-substituted drug molecules to produce specific fluorescence under LED lamp irradiation.Based on azide substitution group, click reaction can occur with alkyne capture group, so the multi-modal probe simultaneously has the functions of in vivo tracer positioning and target protein enrichment capture, and can realize fluorescence labeling and drug target monitoring on protein level, living cells, tissues and in vivo small animals.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochemistry, in particular to a multi-modal probe with dual functions of tracer positioning and target protein enrichment capture and a preparation method and application thereof. BACKGROUND

[0002] Chemical biology, as an interdisciplinary subject, is committed to studying the structure and function of molecules in living organisms by integrating principles and technologies of chemistry and biology. In recent years, with the continuous development of fluorescent probe and biomarker technologies, multi-modal molecular probes based on fluorescent labeling and biological specificity have gradually attracted widespread attention. Multi-modal molecular probes are composed of targeting elements and elements that can generate optical, electrical, magnetic, and other signals. The targeting elements can include small drug molecules, polypeptides, proteins, and antibodies, while the signal-generating elements usually use radionuclides, fluorescent dyes, metals for nuclear magnetic resonance detection, and radioactively labeled isotopes. Multi-modal probes can achieve molecular imaging and positioning through multiple ways under different observation scales and conditions, providing more possibilities for biomedical research and clinical applications.

[0003] In order to overcome the limitations of light stability and fluorescence quantum yield of traditional fluorescent labels, researchers have developed various new types of multi-modal molecular probes. Among them, nanocarriers play a key role as an important carrier form. Nanocarriers have a large surface area, providing abundant sites for the connection of various multi-modal labels. However, the particle size and distribution of nanoparticles directly affect their safety, kinetic behavior in the body. In addition, factors such as the composition, surface charge properties, and surface modification of nanoparticles also affect their behavior in the body.

[0004] Multi-modal probes can be used for multi-modal imaging at the cellular level and in live animal models, and have potential biomedical research and clinical diagnostic application value, which is of great significance for biomedical research and clinical applications. For example, Chinese patent (CN112521373A) discloses the preparation and application of a multi-modal probe. This patent rapidly constructs a multi-modal probe for molecular imaging based on a biocompatible reaction "one-pot three-step" cascade reaction. The probe uses an IR780 derivative with near-infrared fluorescence as the skeleton, and can realize multifunctional near-infrared fluorescence detection through successive substitution reactions and click reactions. Chinese patent (CN113105360A) discloses a multi-modal probe FN and its preparation method and application. The chemical formula of the multi-modal probe FN involved in this patent is C 18 H 13 FN2O2, which has strong specificity, good selectivity, and high sensitivity, can detect Cu 2+ , S 2- in vitro and in cells. 2+, S 2- In addition, the Gd-Chelate Fluorophore Conjugate (Gd-FPC) probe is based on the combination of gadolinium metal ions (Gd 3+ ) and fluorescent dyes, and by synthesizing fluorescent dyes with gadolinium ion chelate ligands, a multimodal detection with both magnetic resonance imaging and fluorescence imaging can be achieved, which can be used for biological labeling and tissue imaging (Ravoori, M K, et al. Scientific reports, 2022; 6: 38991). The Cy5.5-Bisphosphonate Conjugate (Cy5.5-BPC) probe is based on Cy5.5 fluorescence and bisphosphonate, and has the functions of fluorescence imaging and near-infrared spectral imaging, which can be used for early diagnosis and observation of skeletal diseases (Damasco JA, et al. Talanta., 2023; 256: 124308). The NIRF-CMR Probe (NIRF-CMP) probe combines near-infrared fluorescent dyes and nuclear magnetic resonance (NMR) technology, and can simultaneously realize near-infrared fluorescence imaging and nuclear magnetic resonance imaging (Wang N, et al. Photodiagnosis Photodyn Ther. 2023; 42: 103325).

[0005] In summary, these multimodal molecules can realize the monitoring of small molecule drugs at the levels of living organisms, living cells, tissues and proteins. By utilizing click chemistry reactions, biological orthogonal reactions and nanoparticles as carriers, these probes have high selectivity and sensitivity, and can realize the enrichment, localization and imaging of target proteins, providing new methods and means for the study of small molecule drug target organs and target proteins. However, there are few reports of multimodal probe molecules that integrate in vivo tracking and localization, target protein enrichment and capture. SUMMARY

[0006] The purpose of the present application is to provide a multimodal probe with the functions of tracking and localization and target protein enrichment and capture, and a preparation method and application thereof, to solve the problems existing in the prior art. The multimodal probe has diketopyrrolo structure and azide substitution group at the same time, and can react with oxygen-rich olefin-substituted drug molecules under LED lamp irradiation to produce specific fluorescence. Based on the azide substitution group, a click reaction with alkyne capture group can occur, so the multimodal probe has the functions of in vivo tracking and localization and target protein enrichment and capture.

[0007] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0008] The present application provides a multimodal probe with the functions of tracking and localization and target protein enrichment and capture, and the structural formula is as follows:

[0009]

[0010] The application further provides a preparation method of the multimodal probe, comprising the step of reacting 1-(azidomethyl)pyrene and sodium periodate under the action of a catalyst to obtain the multimodal probe.

[0011] Further, the catalyst is ruthenium trichloride.

[0012] Further, the preparation method of the 1-(azidomethyl)pyrene comprises:

[0013] After the reaction of 1-pyrenemethanol and azidophosphoric acid diphenyl ester, 1,8-diazabicycloundec-7-ene is added to continue the reaction to obtain the 1-(azidomethyl)pyrene.

[0014] Further, after the reaction of the 1-pyrenemethanol and the azidophosphoric acid diphenyl ester for 5 minutes at 0 DEG C, the 1,8-diazabicycloundec-7-ene is added to continue the reaction.

[0015] Further, after the addition of the 1,8-diazabicycloundec-7-ene, the temperature for the continued reaction is 25 DEG C, and the reaction time is 2 hours.

[0016] Further, the ratio of the 1-pyrenemethanol, the azidophosphoric acid diphenyl ester and the 1,8-diazabicycloundec-7-ene is 10 mmol:12 mmol:1630 muL.

[0017] The application further provides the use of the multimodal probe in the preparation of a product for tracing and locating an oxygen-rich olefin-labeled drug molecule and / or enriching and capturing a target protein of the drug molecule.

[0018] The application further provides a product for tracing and locating an oxygen-rich olefin-labeled drug molecule and / or enriching and capturing a target protein of the drug molecule, and the active ingredient comprises the multimodal probe.

[0019] The application further provides the use of the multimodal probe or the product in the tracing and locating of an oxygen-rich olefin-labeled drug molecule and / or the enrichment and capture of a target protein of the drug molecule.

[0020] The application discloses the following technical effects:

[0021] In order to better realize the functions of in-vivo tracing and locating of an integrated target molecule and enrichment and capture of a target protein, the application develops a multimodal probe AMPD which simultaneously has a diketopyrene structure and an azide substituent based on the principle of chemical biology, and the multimodal probe can simultaneously undergo biological orthogonal reactions with an oxygen-rich olefin-labeled small-molecule drug and an alkyne-modified magnetic capture microsphere, and can realize fluorescence labeling and drug target monitoring on the protein level, living cells, tissues and living small animals.

[0022] The multimodal probe AMPD of the present application has the following technical effects:

[0023] 1. The optimal excitation wavelength is 395 nm, and the optimal emission wavelength is 500 nm, with a large gap between excitation and emission, avoiding mutual interference and improving the signal resolution of the probe.

[0024] 2. The photo-induced biological orthogonality has good concentration responsiveness. By measuring the fluorescence intensity, the concentration change of the target substance can be accurately evaluated, which can be used for quantitative analysis and detection.

[0025] 3. The multimodal probe molecule AMPD can specifically recognize oxygen-rich olefin-labeled drug molecules, and by selecting a detection wavelength above 500 nm, the interference of the body's own fluorescence is effectively avoided, improving the accuracy of target localization labeling.

[0026] 4. The multimodal probe molecule AMPD can induce specific biological orthogonality under ice blue LED light, and can be used for labeling oxygen-rich olefin-labeled drug small animals and tissue cell imaging analysis and cell sorting, with high biological safety.

[0027] In summary, the multimodal probe has high selectivity and sensitivity, high biological safety, and can realize specific enrichment and imaging analysis of target proteins at the in vivo level through various biological orthogonality reactions, and has wide application potential in the fields of fluorescence tracing, drug targeted therapy and mechanism research. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0029] Figure 1 is the synthesis route of the multimodal probe AMPD of the present application; wherein DPPA represents azidophosphoric acid diphenyl ester; THF represents tetrahydrofuran; DCM represents dichloromethane;

[0030] Figure 2 is the nuclear magnetic resonance detection result of the multimodal probe AMPD;

[0031] Figure 3 is the high-resolution mass spectrometry detection result of the multimodal probe AMPD;

[0032] Figure 4 is the nuclear magnetic resonance hydrogen spectrum of oxygen-rich olefin-berberine;

[0033] Figure 5 The nuclear magnetic resonance carbon spectrum of the oxygen-enriched olefin- bergenin;

[0034] Figure 6 The light-induced reaction conditions between the multimodal probe AMPD and the oxygen-enriched olefin-labeled molecule bergenin and the fluorescence spectrum of the orthogonal reaction product; wherein, A is the ultraviolet or visible light-induced condition; B is the fluorescence spectrum of the cross-linking product of the oxygen-enriched olefin- bergenin and the AMPD probe at different concentrations;

[0035] Figure 7 The in vivo imaging analysis results of the multimodal probe AMPD and the oxygen-enriched olefin-labeled molecule bergenin on the glass catfish; wherein, A is the fluorescence imaging diagram; B is the statistical result;

[0036] Figure 8 The gel electrophoresis (A) and fluorescence imaging (B) analysis results of the multimodal probe AMPD and the oxygen-enriched olefin-labeled molecule bergenin on the ex vivo tissue lysate protein of the administered mouse; in A and B, 0 represents the control, and 1 represents the experimental group;

[0037] Figure 9 The flow cytometry sorting diagram of the oxygen-enriched olefin-labeled molecule bergenin based on the multimodal probe AMPD on the target cells of the administered mouse lung tissue;

[0038] Figure 10 The silver staining analysis results of the gel electrophoresis of the target protein enrichment capture condition of the oxygen-enriched olefin-labeled molecule bergenin based on the multimodal probe AMPD in the target cells of the administered mouse lung. DETAILED DESCRIPTION

[0039] The various illustrative embodiments of the present application will now be described in detail below, which should not be considered limiting on the present application, but rather as a description of certain aspects, features, and embodiments of the present application.

[0040] It should be understood that the terms used in the present application are merely used to describe particular embodiments and are not intended to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value within the stated range, and any other stated value or intermediate value within the stated range, is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0041] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not construed as an admission that it is prior art with respect to the present application.

[0042] Many modifications and variations of this application of the application described herein will be apparent to those of ordinary skill in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0043] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed materials and methods.

[0044] Example 1 Synthesis of the multi-modal probe AMPD

[0045] The synthetic route of the multi-modal probe AMPD is shown in Figure 1 The specific synthesis steps are as follows:

[0046] 1) Put 2320 mg (10 mmol, 1.0 eq) of 1-pyrenemethanol and 3310 mg (12 mmol, 1.2 eq) of diphenyl phosphazide into a 100 mL round-bottom flask, add 50 mL of anhydrous tetrahydrofuran solution under nitrogen protection, and react at 0°C for 5 minutes; then slowly drop 1630 μL of 1,8-diazabicycloundec-7-ene, and react at 25°C for 2 hours. After TLC detection shows that the raw material is completely reacted, quench the reaction with 1 mol / L hydrochloric acid. After extraction with ethyl acetate and sufficient washing with saturated brine, dry the organic phase with anhydrous sodium sulfate, and then concentrate under vacuum, and purify with a silica gel column to obtain 2342 mg of 1-(azidomethyl)pyrene with a yield of 91.1%.

[0047] 2) 771 mg (3 mmol, 1.0 eq) 1-(azidomethyl)pyrene was placed in a 50 mL round bottom flask, and a mixture of 5 mL dichloromethane and 5 mL acetonitrile was added under nitrogen protection, and a solution of 5136 mg (24 mmol, 8.0 eq) sodium periodate and 621 mg (3 mmol, 1.0 eq) ruthenium trichloride (metal salt) mixed with water (5 mL) was slowly added under ice bath, and the reaction was carried out at room temperature (25°C) for 12 hours. After TLC detection showed that the raw material disappeared, saturated sodium thiosulfate was added to quench the reaction, and the organic phase was extracted with ethyl acetate and washed with saturated brine. After drying over anhydrous sodium sulfate, the organic phase was concentrated under vacuum, and purified by silica gel column to obtain 292.2 mg of 1-(azidomethyl)pyrene-4,5-dione, i.e. the multi-modal probe AMPD, with a yield of about 34%.

[0048] The multi-modal probe AMPD prepared in this example is a light yellow light powder with a melting point of 152°C, a molecular formula of C 17 H9N3O2, a molecular weight of 287.0695, and is easily soluble in dichloromethane, ethanol, acetone, chloroform and the like, and slightly soluble in water, and has the following structural formula:

[0049]

[0050] As shown in Figure 2 , the nuclear magnetic resonance detection (NMR) detection result of AMPD is as follows: 1 H NMR (400 MHz, CDCl3) δ 8.51 (dd, J = 13.6, 7.5 Hz, 2H), 8.21 (d, J = 7.9 Hz, 1H), 8.03 (d, J = 9.1 Hz, 1H), 7.95 (d, J = 9.1 Hz, 1H), 7.84-7.74 (m, 2H), 7.53 (d, J = 8.6 Hz, 1H), 4.91 (s, 2H).

[0051] As shown in Figure 3 , the high resolution mass spectrometry (HRMS) detection of AMPD shows that the high resolution mass spectrum HRMS of [M+Na] + is 310.0587, and the measured value is 310.0590, which is basically consistent with the theoretical value.

[0052] Example 2 Synthesis of oxygen-rich olefin-bailcain

[0053] In this application, the active natural product bailcain is taken as an example, and an oxygen-rich olefin-modified drug molecule oxygen-rich olefin-bailcain is prepared, which is used to investigate the tracking and capture efficiency of the multi-modal probe AMPD prepared in Example 1 on the oxygen-rich olefin drug molecule and the target protein.

[0054] The preparation method of oxygen-rich olefin-bailcain is as follows:

[0055] 1) 100 mg (1.14 mmol, 1 eq) of chloro-oxoalkene, 4-dimethylaminopyridine (0.228 mmol, 0.2 eq) and 259 mg (1.36 mmol, 1.2 eq) of 4-toluenesulfonyl chloride were added to a 25 mL round bottom flask containing 5 mL of anhydrous dichloromethane, followed by the addition of 230 mg of triethylamine (2.28 mmol, 2 eq) to the system, stirring at 25 °C for 12 hours. The progress of the reaction was monitored by TLC. After the reaction was completed, the mixture was extracted with ethyl acetate (3 x 10 mL). The organic phase was repeatedly washed with saturated brine, and the combined organic phase was dried over anhydrous sodium sulfate and then dried under reduced pressure to obtain the crude product. After purification by silica gel column (petroleum ether: ethyl acetate = 4: 1), about 207 mg of yellow liquid intermediate OTs-oxoalkene was obtained, with a yield of 75%.

[0056] 2) 200 mg (0.61 mmol, 1 eq) of Bergenin and 100 mg (1.2 mmol, 2 eq) of K2CO3 were put into a 50 mL round bottom flask, and 10 mL of anhydrous DMF was added under nitrogen protection, followed by the slow addition of 177 mg (0.73 mmol, 1.2 eq) of the above activated OTs-oxoalkene, stirring for 12 hours. After the complete reaction of the raw material was detected by TLC, the reaction was quenched with 1 mol / L dilute hydrochloric acid, and extracted with ethyl acetate and saturated NaCl, and the combined organic phase was dried over anhydrous Na2SO4 and then concentrated under vacuum to obtain the crude product. After purification by column chromatography (dichloromethane:methanol = 20:1), 109 mg of oxoalkene-Bergenin (white powder) was obtained, with a yield of 45%.

[0057] 3) The product oxoalkene-Bergenin was detected by nuclear magnetic resonance (NMR), and the results are shown in Figures 4-5 1 HNMR (400 MHz, DMSO-d6) δ 8.56 (s, 1H), 7.13 (s, 1H), 6.58 (d, J = 7.6 Hz, 1H), 5.68 (d, J = 5.4 Hz, 1H), 5.46 (d, J = 5.8 Hz, 1H), 5.04 (d, J = 10.5 Hz, 1H), 4.92 (s, 1H), 4.32-4.22 (m, 3H), 4.11-3.97 (m, 4H), 3.88-3.83 (m, 1H), 3.79 (s, 3H), 3.67 (td, J = 8.9, 5.4 Hz, 1H), 3.63-3.55 (m, 1H), 3.46 (dd, J = 12.3, 6.3 Hz, 1H), 3.24-3.17 (m, 1H). 13 ​CNMR (100MHz, DMSO-d6) δ 163.3, 152.1, 151.8, 148.0, 141.7, 118.5, 118.1, 106.5, 87.4, 81.8, 79.7, 73.7, 72.0, 70.7, 67.5, 66.5, 61.1, 60.1. This compound was confirmed as the target product.

[0058] Example 3 Characterization of the properties of the multimodal probe AMPD

[0059] The multimodal probe AMPD(10) prepared in Example 1 was used respectively. -5 (mol / L) and the oxygen-rich olefin-bergenin (10 mol / L) prepared in Example 2 -5 The solution (mol / L) was dissolved in a mixture of PBS and acetonitrile. Using a multi-functional microplate reader (Spark, Austria), UV-Vis light of different wavelengths from 330 nm to 520 nm was applied for excitation to investigate the efficiency of photo-initiated bioorthogonal reactions. The results are as follows: Figure 6 As shown in Figure A, the best initiation efficiency is achieved under 365nm ultraviolet light, but the bioorthogonal reaction between oxygen-rich olefins and pyrene-dione can also be initiated under ice-blue visible light at 435nm to 460nm.

[0060] Similarly, a mixed solution of the two probes at the same dosage was taken and irradiated with an ice-blue LED lamp (SMD 5050LED) for 20 minutes. The fluorescence spectrum of the multimodal AMPD-oxygen-enriched olefin-bergenin fluorescent product was examined using a multifunctional microplate reader (Spark, Austria). The results are as follows: Figure 6 As shown in Figure B, the optimal excitation wavelength was determined to be 395 nm, and the optimal emission wavelength was determined to be 500 nm. With increasing concentration of oxygen-rich olefin-bergenin (10... -7 -10 -4 mol / L), which is related to the multimodal probe AMPD (10 -4 The fluorescence intensity of photoinitiated crosslinking at mmol / L was also enhanced, showing good concentration dependence, at 10 mmol / L. -7 -10 -4 It exhibits good dose-response responsiveness within the mol / L concentration range.

[0061] Example 4: Application of Multimodal Probe AMPD in In vivo Imaging of Model Animals

[0062] The present embodiment selects mode animal glass cat catfish as the research object, through the administration of oxygen-enriched olefin drugs, to investigate the feasibility of the multi-modal probe AMPD in vivo fluorescence imaging. 20 glass cat catfish (Shandong Yixie Biological Technology Co., Ltd.) were randomly divided into four groups: control group (scutellarein 6.75 μmol / L), low concentration group (0.75 μmol / L), medium concentration group (2.25 μmol / L) and high concentration group (6.75 μmol / L) of oxygen-enriched olefin-scutellarein (prepared in Example 2). First, the glass cat catfish was given to be placed in the oxygen-enriched olefin-scutellarein aqueous solution of different groups overnight, and then the water was replaced for 3 times to ensure that there was no residual drug in the solution. Subsequently, the glass cat catfish of all groups was placed in 10 μmol / L of multi-modal probe AMPD (prepared in Example 1) solution, and the ice blue LED light source of the above-mentioned Example 3 was used to irradiate the glass cat catfish sample for 20 minutes to induce the photocatalytic bio-orthogonal reaction of the multi-modal probe AMPD and the oxygen-enriched olefin-scutellarein. Subsequently, the fluorescence imaging analyzer (Huayue Enterprise Group Co., Ltd.) was used to perform fluorescence in vivo detection at 395 nm excitation wavelength and 550 nm emission wavelength. The experimental results are shown in Figure 7 The control group of scutellarein prototype drug did not produce specific fluorescence. The multi-modal probe AMPD and the oxygen-enriched olefin-scutellarein produced specific fluorescence signals on the in vivo glass cat catfish, and were positively correlated with the concentration of the oxygen-enriched olefin-scutellarein. This indicates that the multi-modal probe AMPD probe has good safety under in vivo drug administration, and can induce crosslinking reaction under ice blue LED visible light source, which can be used for in vivo imaging research in biomedical research and clinical practice.

[0063] Example 5 Application of multi-modal probe AMPD in drug target organ distribution investigation

[0064] In order to investigate the distribution of scutellarein in the target organs of mice, the experiment was divided into control group and experimental group. The control group was given 50 mg / kg / day of scutellarein prototype drug by gavage, and the experimental group was given 50 mg / kg / day of oxygen-enriched olefin-scutellarein (prepared in Example 2) by gavage, which lasted for one week. After the experiment, the heart, liver, spleen, lung and kidney tissues of the mice were taken, ground and centrifuged to obtain tissue protein lysate. The above tissue protein lysate was mixed with 10 -4 mol / L of multi-modal probe AMPD (prepared in Example 1) to irradiate for 20 minutes using the ice blue LED light source of the above-mentioned Example 3. The above protein solution was quantified using BCA kit (Beijing Solabio Technology Co., Ltd.), and the same amount of protein was subjected to SDS-PAGE gel electrophoresis separation. And referring to the above-mentioned Example 4, the fluorescence imaging analyzer was used to perform gel fluorescence imaging detection at 395 nm excitation wavelength and 550 nm emission wavelength. The experimental results are shown inFigure 8 As shown in Figure 6B, compared with the gel analysis of CBB (Coomassie Brilliant Blue) staining, Figure 8 Figure 6C (middle A), the experimental group detected fluorescence signals in the protein samples of liver and lung tissues, while no specific fluorescence was detected in other organs (middle B). The experimental results show that the multi-modal probe AMPD has the advantage of being used for drug target organ positioning and distribution investigation. Figure 8

[0065] Example 6 Application of multi-modal probe AMPD in drug target cell sorting

[0066] Referring to the experimental method of Example 5, after 50 mg / kg / day of oxygen-enriched olefine-ankangmycin was administered for a week, the lung tissue was cut and subjected to collagenase digestion to prepare a single cell suspension. Density gradient centrifugation was used to separate the cells, and the obtained living cells were suspended in 1640 cell culture medium containing 10 -5 mol / L multi-modal probe AMPD. The ice blue LED light source of Example 3 was used for irradiation for 20 minutes, the supernatant was removed by centrifugation, and then the labeled cells were resuspended in 1640 cell storage solution. The fluorescence-labeled cells were sorted using the cell sorter of FACSAria Fusion (USA). As shown in Figure 6D, about 57.63% of the lung cells were labeled after continuous administration for one week. The multi-modal probe AMPD can well identify oxygen-enriched olefine-labeled drug molecules on living cells, opening up a new way for non-invasive labeling and sorting of target cells. Figure 9

[0067] Example 7 Application of multi-modal probe AMPD in drug target protein capture

[0068] Referring to Example 6, the AMPD probe-labeled cells after the above oxygen-enriched olefine-ankangmycin administration were collected, and the following target protein capture enrichment operation was performed. The specific cell lysis process includes: treating the cells with RIPA lysis solution to release the intracellular proteins. After centrifugation, 1 mL of supernatant was taken, and 1 μL of biotinylated alkyne-modified streptavidin magnetic microspheres was added according to the literature method (Zhang M et al., FEBS Journal, 2020: 287: 1816). 10 -4 mol / L ascorbic acid sodium and 10 -4 mol / L copper sulfate were used as catalysts for click reaction at 4°C overnight. The magnetic microspheres were collected, 20 μL of SDS-PAGE loading buffer was added, and the sample was heated at 100°C for 5 minutes. Subsequently, 12.5% SDS-PAGE gel electrophoresis was used for separation, and alkaline silver staining reagent (Tianjin Dingguo Biotechnology Co., Ltd.) was used for staining. As shown in Figure 6E, compared with the control group, the experimental group can detect specific target proteins enriched. Figure 10 As shown in Figure 6E, compared with the control group, the experimental group can detect specific target proteins enriched.​​

[0069] The above-described embodiments are merely intended to describe the preferred modes of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements to the present application made by those skilled in the art, without departing from the design spirit of the present application, should fall within the scope of the present application as defined by the claims.

Claims

1. A multimodal probe with dual functionalities of oxygen-rich olefin tracer localization and target protein-enriched capture, characterized in that, The structural formula is shown as follows: 。 2. A method of preparing a multi-modal probe as claimed in claim 1, characterized in that, The step of reacting 1-(azidomethyl)pyrene and sodium periodate under the action of a catalyst to obtain the multi-modal probe; The catalyst is ruthenium trichloride.

3. The production method according to claim 2, characterized by, The preparation method of the 1-(azidomethyl)pyrene comprises: After the reaction of 1-pyrenemethanol and diphenyl phosphorazide, 1,8-diazabicycloundec-7-ene is added for further reaction to obtain the 1-(azidomethyl)pyrene.

4. The production method according to claim 3, characterized by, The 1-pyrenemethanol and the diphenyl phosphorazide are reacted at 0°C for 5 minutes, and then the 1,8-diazabicycloundec-7-ene is added for further reaction.

5. The preparation method according to claim 4, characterized in that, After the 1,8-diazabicycloundec-7-ene is added, the temperature for further reaction is 25°C, and the reaction time is 2 hours.

6. The production method according to claim 5, wherein The ratio of the 1-pyrenemethanol, the diphenyl phosphorazide and the 1,8-diazabicycloundec-7-ene is 10 mmol:12 mmol:1630 μL.

7. Use of the multi-modal probe of claim 1 in the preparation of a product for tracing and positioning an oxygen-rich olefin-labeled drug molecule and / or enriching and capturing a target protein of the drug molecule.

8. A product for tracing the localization of an oxygen-enriched olefin-labeled drug molecule and / or enrichment of a target protein that captures the drug molecule, characterized in that, The active ingredient comprises the multi-modal probe of claim 1.

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