A small molecule probe for extracting and purifying mitochondria and its preparation method and application

Abstract

The present invention provides a small molecule probe for extracting and purifying mitochondria, as well as its preparation method and application. This approach addresses the current problem of using magnetic beads to extract mitochondria, which often results in ineffective separation of mitochondria from the beads and low biocompatibility. It provides an effective approach for mitochondrial implantation and future industrial development. The small molecule probe designed in the present invention includes a mitochondrial targeting group, a polyethylene glycol linker, a photo-controlled cleavage group, and an azide group, all bonded in sequence. The probe is connected to DBCO-magnetic beads via non-copper-catalyzed click chemistry. Upon irradiation with ultraviolet light at a wavelength of 365-400 nm, the photo-controlled cleavage group cleaves, thereby separating the mitochondria from the beads.

Description

Technical Field

[0001] The present invention belongs to biological and new medical technology, and specifically relates to a small molecule probe for extracting and purifying mitochondria, and a preparation method and application thereof. Background Art

[0002] Aging is a major risk factor for many chronic diseases. Faced with my country's increasingly aging society, various chronic diseases will place a heavy burden on families and society. For neurodegenerative diseases alone, the prevalence of neurodegenerative diseases among those aged 65 and older is estimated to be approximately 10%, and this rate increases with age. Mitochondria, as one of the most important organelles within cells, play a crucial role in basic cellular function and survival. As the cell's "powerhouse," mitochondria primarily produce adenosine triphosphate (ATP), the essential energy storage molecule required for cell survival, through oxidative phosphorylation. Furthermore, when damaged, mitochondria release reactive oxygen species (ROS) and cytochrome C, among other signals that promote apoptosis. Therefore, disruption of mitochondrial homeostasis can lead to cellular ATP deficiency and mitochondrial damage, which can result in excessive ROS or insufficient cellular energy supply, ultimately leading to cell death. Numerous studies have demonstrated that mitochondrial damage plays a key role in the development and progression of many chronic diseases, including neurodegenerative diseases, cancer, and cardiovascular disease. However, the current standard treatment for mitochondrial damage mainly focuses on addressing disease symptoms, such as antioxidants, but most of these treatments only treat the symptoms and not the root cause, and there is a lack of clear clinical data to support the effectiveness of these treatments.

[0003] As an emerging therapeutic technology, cell therapy has great application prospects. The cell therapy solution for mitochondrial damage is to inject healthy mitochondria directly into the mitochondrially damaged tissue. Animal experiments have shown that freshly extracted mitochondria can be taken up by cardiomyocytes after being injected into mouse heart tissue, and significantly enhance cardiac activity. Therefore, mitochondrial-based cell therapy has very broad application prospects, and obtaining highly active and high-purity mitochondria is a crucial part of mitochondrial cell therapy.

[0004] Currently, various methods for obtaining high-purity mitochondria have been reported. The most widely used method is fractionation, which uses an ultrahigh-speed centrifuge to separate and purify different organelles and components in a cell lysate. However, differential centrifugation (GC) requires high instrumentation and experimental personnel, and the extracted mitochondria often contain contaminants such as lysosomes, endoplasmic reticulum, and peroxisomes.

[0005] Recently, several research groups have used antibodies targeting the mitochondrial membrane protein TOM22 to specifically and non-covalently bind mitochondria from cell lysates. These methods significantly improve the purity of mitochondrial extraction and simplify the process, avoiding the use of ultracentrifuges and the complex and unstable experimental procedures. They also utilize magnetic beads and magnetic fields to effectively purify captured mitochondria. However, these methods also have drawbacks that limit their further application in mitochondrial implantation and future industrialization. First, the preparation and transportation costs of antibody- and peptide-based mitochondrial targeting moieties are high. Second, after mitochondrial purification, a strong, irreversible bond is formed between the mitochondria and the magnetic beads. Furthermore, as the magnetic beads are non-biodegradable, the extent to which the mitochondria-antibody-magnetic bead conjugate affects the activity of the retrieved mitochondria remains unknown. Furthermore, the large size and non-biodegradable nature of the magnetic beads during subsequent mitochondrial implantation also pose safety risks to the target cells and tissues. Summary of the Invention

[0006] The purpose of the present invention is to solve the problem that mitochondria cannot be effectively separated from the magnetic beads and have low biocompatibility when currently using magnetic beads to extract mitochondria, which limits mitochondrial implantation and future industrial development, and to provide a small molecule probe for extracting and purifying mitochondria, as well as its preparation method and application.

[0007] The concept of the present invention:

[0008] Regarding the current problem of using magnetic beads to extract mitochondria, the mitochondria cannot be effectively separated from the magnetic beads. The research team of this invention considered starting from changing the irreversible connection between the magnetic beads and the mitochondria, converting the connection between the magnetic beads and the mitochondria into a reversible connection. By combining the photo-cleavable group with the mitochondrial targeting group, a multifunctional mitochondrial-targeting small molecule probe was designed, thereby solving the problem of the magnetic beads being unable to be separated from the mitochondria in the current mitochondrial magnetic bead extraction method.

[0009] To achieve the above objectives, the technical solutions provided by the present invention are:

[0010] A multifunctional mitochondrial-targeting small molecule probe with the following unique features:

[0011] It includes a mitochondrial targeting group, a polyethylene glycol linking group, a light-controllable severing group, and an azide group bonded in sequence;

[0012] Wherein, the mitochondrial targeting group is used to specifically bind to the mitochondrial outer membrane;

[0013] The polyethylene glycol linking group is used to connect the mitochondrial targeting group to the light-controllable cleavage group;

[0014] The photo-controllable cleavage group can be broken under the irradiation of light with a wavelength of 365-400nm;

[0015] The azide group is used for non-copper catalyzed click chemistry.

[0016] Furthermore, the probe is (1-(4-(2-azidoethoxy)-5-methoxy-2-nitrophenyl)-3,14-dioxo-2,7,10-trioxa-4,13-diazacyclohexane-16-yl)triphenylphosphine; its molecular structure is:

[0017]

[0018] The method for preparing the multifunctional mitochondrial-targeting small molecule probe is special in that it comprises the following steps:

[0019] 1) Synthesis of Compound 3 and Compound 8

[0020] Among them, the synthesis process of compound 3 is:

[0021]

[0022] The specific steps are as follows:

[0023] I. Synthesis of Compound 1

[0024] Add triphenylphosphine to an acetonitrile solution containing 3-bromopropionic acid, stir and react at 60-80° C. (preferably 80° C.), and detect by thin layer chromatography until the reaction is complete. Concentrate the reaction solution in vacuo and extract the residue with an organic solvent (such as chloroform, dichloromethane or ethyl acetate, preferably chloroform);

[0025] Ether was added to the organic phase to precipitate the product, which was collected and washed several times, and then spin-dried to obtain compound 1;

[0026] II. Synthesis of Compound 3

[0027] Under an inert atmosphere (N2), compound 1 obtained in step I is dissolved in anhydrous dichloromethane in an ice bath, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole are added to the above system; after 15-30 minutes (preferably 15 minutes), compound 2 and N-methylmorpholine are added, and the reaction is carried out at room temperature. Thin layer chromatography is performed until the reaction is complete, and water is added to quench the reaction. The reaction is extracted with dichloromethane, and the organic phase is washed and dried (specifically, the organic phase is washed with saturated sodium bicarbonate solution and saturated brine, and the organic layer is dried over anhydrous magnesium sulfate), filtered, and concentrated to obtain compound 3;

[0028] The synthetic process of compound 8 is as follows:

[0029]

[0030] The specific steps are as follows:

[0031] ①. Synthesis of compound 4

[0032] Vanillin was dissolved in acetonitrile, and 1,2-dibromoethane and potassium carbonate were added. The reaction was carried out at room temperature and detected by thin layer chromatography until the reaction was complete. The salt formed in the reaction system was filtered, and the filtrate was concentrated to obtain a yellow oily product, which converted to a white precipitate upon standing. The crude product was further purified on a silica gel column to obtain compound 4 by elution. PE:EA = 2:1 was used as the eluent for elution.

[0033] ②. Synthesis of compound 5

[0034] At 0-4°C (preferably 0°C), an excess of cooled nitric acid was added to the compound 4 obtained in step ①, and the mixture was stirred for 15-30 min (preferably 25 min). The mixture was then warmed to room temperature for reaction. Thin layer chromatography was performed to detect when the reaction was complete. Water was added to quench the reaction, and the precipitate was collected by filtration and washed with ice water several times to obtain compound 5.

[0035] ③. Synthesis of compound 6

[0036] Compound 5 obtained in step ② was dissolved in ethyl acetate, and the mixture was kept away from light throughout the process (for example, by wrapping the reaction vessel with aluminum foil). Then, a sodium hydroxide solution containing sodium borohydride was added thereto, and the reaction was stirred at room temperature. The reaction solution was neutralized with hydrochloric acid and extracted with ethyl acetate several times. The organic layers were combined, dried (over anhydrous magnesium sulfate), filtered, and concentrated under reduced pressure to obtain a crude mixture of a light yellow solid;

[0037] The crude mixture was purified by flash column chromatography (EA:PE=1:4) to give compound 6;

[0038] ④. Synthesis of Compound 7

[0039] Under the protection of inert gas (N2), compound 6 obtained in step ③ was dissolved in N,N-dimethylformamide, sodium azide was added, and the mixture was stirred at 60-80°C (preferably 60°C) in the dark to react. Thin layer chromatography was performed until the reaction was complete. The reaction solution was diluted with ethyl acetate, and the organic phase was washed and dried, filtered, and concentrated to obtain (4-(2-azidoethoxy)-5-methoxy-2-nitrophenyl)methanol (washed with water, saturated NaHCO3, and saturated brine, and finally dried over anhydrous magnesium sulfate, filtered, and evaporated to obtain compound 7;

[0040] ⑤. Synthesis of Compound 8

[0041] Compound 7 obtained in step ④ was added to acetonitrile in which triethylamine and N,N-disuccinimidyl carbonate were dissolved. The mixture was stirred at room temperature under inert gas (N2) protection to react. Thin layer chromatography was performed until the reaction was complete. The reaction solution was concentrated under reduced pressure to remove the solvent. The residue was purified by flash column chromatography (EA:PE=1:2) to obtain compound 8.

[0042] 2) Synthesis of the probe (1-(4-(2-azidoethoxy)-5-methoxy-2-nitrophenyl)-3,14-dioxo-2,7,10-trioxa-4,13-diazacyclohexan-16-yl)triphenylphosphine

[0043]

[0044] 2.1) Compound 3 obtained in step 1) was dissolved in dichloromethane, and trifluoroacetic acid was slowly added dropwise under an ice bath. The mixture was then stirred at room temperature for reaction, and the reaction was monitored by thin-layer chromatography until completion. The mixture was then vacuum evaporated and evaporated with dichloromethane several times to remove the trifluoroacetic acid, yielding compound 9.

[0045] 2.2) Compound 9 obtained in step 2.1) was dissolved in anhydrous acetonitrile, and triethylamine and the anhydrous acetonitrile solution of compound 8 obtained in step 1) were added sequentially; the reaction was stirred at room temperature in the dark, and the reaction was monitored by thin-layer chromatography until completion. The crude product was concentrated in vacuo to obtain the crude product, which was purified by flash column chromatography (methanol:dichloromethane = 1:10) to obtain the target product (1-(4-(2-azidoethoxy)-5-methoxy-2-nitrophenyl)-3,14-dioxo-2,7,10-trioxa-4,13-diazacyclohexan-16-yl)triphenylphosphine.

[0046] Furthermore, in step I, the equivalent ratio of triphenylphosphine to 3-bromopropionic acid is 1:1-5 (preferably 1:1.1); the stirring time is 24 hours;

[0047] In step II, the equivalent ratio of (2-carboxyethyl)triphenylphosphine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, compound 2, and N-methylmorpholine is 1:1-5:1.1-5:1.1-5:1.1-5; the reaction time is 4-24 hours;

[0048] In step ①, the equivalent ratio of vanillin, 1,2-dibromoethane, and potassium carbonate is 1:2-5:2-5; the reaction time is 24-48 hours, and the standing time is 24-48 hours;

[0049] In step ②, the equivalent ratio of compound 4 to nitric acid is 1:50-200; the temperature is raised to room temperature and the reaction is carried out for 1-2 hours;

[0050] In step ③, the equivalent ratio of compound 5 and sodium borohydride is 1:2-5, and the reaction is stirred for 2-4 hours, preferably 2 hours;

[0051] In step ④, the equivalent ratio of compound 6 to sodium azide is 1:3-10; the stirring time is 12-48h;

[0052] In step ⑤, the equivalent ratio of compound 7, triethylamine and N,N-disuccinimidyl carbonate is 1:2-5:1.1-3; the stirring time is 1-3 hours;

[0053] In step 2), the equivalent ratio of compound 3, triethylamine and compound 8 is 1:2-5:1.1-3; and in step 2.1), the reaction is stirred for 1-5 hours.

[0054] At the same time, the present invention provides the use of the multifunctional mitochondria-targeting small molecule probe in extracting and purifying mitochondria.

[0055] The TPP-magnetic beads with mitochondrial targeting function are special in that they are obtained by connecting the multifunctional mitochondrial targeting small molecule probe to DBCO-magnetic beads through non-copper click chemistry;

[0056] Among them, DBCO-magnetic beads are obtained by linking diphenylcyclooctyne-active ester (DBCO-NHS) with amino-modified magnetic beads;

[0057] The surface layer of the amino-modified magnetic beads is polystyrene, the magnetic core is superparamagnetic ferroferric oxide, the particle size is 50-1000 nm (preferably 200 nm, at this particle size, the extraction amount is the largest), the sedimentation coefficient is 3-6s, and the immunochemiluminescence detection curve correlation R value is >0.99000.

[0058] The method for preparing the TPP-magnetic beads with mitochondrial targeting function is special in that it comprises the following steps:

[0059] S1. Wash the amino magnetic beads 2-4 times (preferably 2 times) with DMSO under a strong magnetic field, and then wash them 1-2 times (preferably 1 time) with anhydrous DMF; then disperse the amino magnetic beads in anhydrous DMF, add TEA and DBCO-NHS solution, and sonicate to obtain DBCO-magnetic beads;

[0060] S2. The DBCO-magnetic beads are first washed with DMF 2-4 times under a strong magnetic field, and then dispersed in DMF. The multifunctional mitochondrial-targeting small molecule probe according to claim 1 is then added and connected by non-copper click chemistry to obtain TPP-magnetic beads.

[0061] The application of the above-mentioned TPP-magnetic beads with mitochondrial targeting function in extracting and purifying mitochondria.

[0062] A method for extracting high-purity mitochondria, which is special in that it includes the following steps:

[0063] A1. Co-culturing the multifunctional mitochondrial-targeting small molecule probe according to claim 1 with viable cells for 6-18 hours, and then lysing them to obtain a cell lysate;

[0064] A2. Add washed DBCO-magnetic beads to the A1 cell lysate and connect them to a multifunctional mitochondria-targeting small molecule probe via non-copper click chemistry to capture mitochondria.

[0065] DBCO-magnetic beads are obtained by linking diphenylcyclooctyne-active ester (DBCO-NHS) with amino-modified magnetic beads;

[0066] A3. Use a strong magnetic field to attract the magnetic beads in the A2 cell lysate, separate the mitochondria captured by the magnetic beads from other components of the cell lysate, and collect the cell lysate containing the mitochondria.

[0067] A4. Irradiate the cell lysate obtained in A3 with ultraviolet light at a wavelength of 365-400 nm (at least 100 J), causing the nitro group on the benzene ring of the small molecule to transfer electrons to the adjacent position, breaking it to form a carbonyl group, thereby detaching the magnetic beads. A strong magnetic field is then used to separate the magnetic beads from the mitochondria attached to the small molecule, and the supernatant is collected to obtain high-purity mitochondria.

[0068] or,

[0069] B1. After washing the TPP-magnetic beads having mitochondrial targeting function according to claim 6, adding them to the cell lysate and incubating for 6-18 hours to capture mitochondria;

[0070] B2. Use a strong magnetic field to separate the magnetic beads containing the mitochondria from the other components of the cell lysate, and collect the cell lysate containing the mitochondria;

[0071] B3. Irradiate the cell lysate obtained in B2 with ultraviolet light at a wavelength of 365-400 nm (at least 100 J of irradiation) to transfer the electrons of the nitro group on the benzene ring of the small molecule to the adjacent position, breaking it to form a carbonyl group, thereby detaching the magnetic beads. A strong magnetic field is then used to separate the magnetic beads from the mitochondria attached to the small molecule, and the supernatant is collected to obtain high-purity mitochondria.

[0072] Mechanism of the present invention:

[0073] After the small molecule probe of the present invention comes into contact with mitochondria, it can efficiently capture mitochondria (78% mitochondrial capture is achieved for every 250 mg of magnetic beads) through a bioorthogonal reaction with the magnetic beads, and responsively dissociate the mitochondria from the magnetic beads under light-induced conditions, thereby achieving mitochondrial extraction and purification, and ultimately providing a key basis for mitochondrial implantation therapy. That is, the present invention designs a targeted small molecule probe that separates the extracted mitochondria and magnetic beads through light control (ultraviolet with a wavelength of 365-400nm), thereby reducing the interference of magnetic beads on subsequent experiments and the impact of mitochondrial activity; compared with the currently commonly used commercial kits, the extraction purity is higher, the quantity is larger, and the activity of the mitochondria is not affected.

[0074] The advantages of the present invention are:

[0075] 1. The raw materials for synthesizing the small molecule probe of the present invention are readily available, low in cost, and the synthesis steps are simple. In addition, due to the polyethylene glycol linker, the small molecule probe has good solubility (10 mg / mL in DMSO and 1 mg / mL in water), and can be directly used for the extraction and purification of mitochondria in biological samples, making its commercialization possible.

[0076] 2. The multifunctional mitochondrial-targeting small molecule probe of the present invention can simultaneously achieve mitochondrial targeting and light-induced release, solving the current problem that magnetic beads cannot be separated from mitochondria. It is the design that has the least impact on mitochondria.

[0077] 3. The multifunctional mitochondrial-targeted small molecule probe of the present invention is used to extract and purify mitochondria, with a purity twice that of commercial kits and an extraction efficiency six times that of commercial kits, showing superior performance and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 The following are steps for extracting and purifying mitochondria using the probe of the present invention, wherein A is a schematic diagram of mitochondrial extraction and purification based on the multifunctional mitochondrial targeting small molecule probe and magnetic beads; B is a schematic diagram of the surface chemistry of the magnetic beads during the process;

[0079] Figure 2 This is the synthetic route of the small molecule probe of the present invention;

[0080] Figure 3 This is the light-section principle of the present invention;

[0081] Figure 4 Three methods for grabbing mitochondria;

[0082] Figure 5 This is a diagram illustrating the detection process of the ATP kit used to detect mitochondrial activity in the present invention;

[0083] Figure 6 The principle of ATP test kit for ATP detection;

[0084] Figure 7 The data graphs show the number of mitochondria extracted by two methods measured by flow cytometry;

[0085] Figure 8 This is a data analysis diagram of the purity of extracted mitochondria measured by flow cytometry;

[0086] Figure 9 The figure shows the analysis of the number of active mitochondria extracted from each plate of T25 cells measured by flow cytometry (set as 100% in this method);

[0087] Figure 10 This is a data analysis diagram of mitochondrial ATP activity. DETAILED DESCRIPTION

[0088] The present invention will be described in further detail below with reference to the accompanying drawings and specific embodiments:

[0089] The mechanism of extracting and purifying mitochondria of the present invention is as follows: Figure 1 As shown; specifically including:

[0090] 1. Synthesis of the multifunctional mitochondrial-targeting small molecule probe HW1

[0091] The present invention intends to design and synthesize a small molecule probe, the design molecular structure and synthesis process of the probe are as follows Figure 2 As shown, the main components include 1) mitochondrial targeting group, 2) polyethylene glycol linking group, 3) light-controllable cleavage group, 4) and azide group for non-copper catalyzed click chemistry, which are bonded in sequence. The synthesis part is relatively simple and clear, and the target probe can be obtained relatively quickly. Among them, the mitochondrial targeting group is triphenylphosphine (TPP) for mitochondria, which targets the mitochondrial membrane potential with high selectivity, and compared with antibodies, small molecule TPP has the advantages of simplicity, stability and low price in synthesis, transportation and preparation. Another important part is the light-controllable cleavage group. Under the irradiation of UV = 365-400nm ultraviolet light, the nitro group on the benzene ring undergoes electron transfer to produce CO2 and amino group, which breaks it and separates the mitochondria from the magnetic beads ( Figure 3 Although the mitochondrial targeting group and the photo-controlled cleavage group can be directly linked, the proximity of the mitochondrial targeting group to the magnetic beads after connection can affect the targeting of the mitochondrial targeting group and thus the extraction of mitochondria. Therefore, a polyethylene glycol linker was introduced to connect the mitochondrial targeting group and the photo-controlled cleavage group. This design not only ensures the success rate of mitochondrial extraction, but also improves the solubility of the small molecule probe.

[0092] This method will solve the current problems of mitochondria extracted using magnetic beads that cannot be effectively separated from the magnetic beads and have low biocompatibility.

[0093] 2. Surface chemical modification of nanomagnetic beads

[0094] Amino-modified magnetic beads are superparamagnetic functionalized magnetic microspheres. Compared to traditional magnetic beads, they exhibit faster magnetic responsiveness while maintaining excellent dispersibility, extremely low nonspecific adsorption, and a richer array of binding sites. They can efficiently and conveniently bind to a variety of ligands at high loadings using specialized chemical reagents. They serve as an excellent base material for subsequent processing such as coating, adsorption, and chemical modification, making them an important carrier tool in medical and molecular biology research.

[0095] Therefore, the present invention uses amino-modified magnetic beads produced by Biomag Biotech as a carrier tool. The beads have a polystyrene surface and a superparamagnetic ferroferric oxide core. In this example, the particle size selected is 200 nm, the sedimentation coefficient is 3-6 seconds, and the immunochemiluminescence detection curve has an R value > 0.99000.

[0096] First, DBCO-NHS was connected to amino-modified magnetic beads to obtain DBCO-magnetic beads; then, the synthesized small molecule probe HW1 was connected to the DBCO-magnetic beads using non-copper click chemistry to finally obtain TPP-magnetic beads with mitochondrial targeting function.

[0097] 3. Mitochondrial Capture Based on Multifunctional Mitochondrial-Targeted Small Molecule Probes

[0098] The current commercial method for extracting mitochondria is to first separate the organelle layers of the cell lysate through multiple fraction centrifugation to obtain mitochondria. Figure 4 As shown, different from the commercial method of extracting mitochondria, the present invention adopts two other methods.

[0099] The first method is to first connect the connected DBCO-magnetic beads to the small molecule probe HW1 outside the cell using non-copper click chemistry, and then incubate with cell lysate for a period of time to achieve the purpose of capturing mitochondria;

[0100] The second method is to first co-culture the small molecule probe HW1 with living cells for a period of time, then lyse the cells, and then add DBCO-magnetic beads to connect them through non-copper click chemistry to achieve the purpose of capturing mitochondria.

[0101] IV. Mitochondrial Purification and Photocontrolled Release

[0102] The two methods mentioned above use a strong magnetic field to attract magnetic beads and separate the mitochondria captured by the magnetic beads from other components of the cell lysate; then, by ultraviolet irradiation at UV = 365-400nm, the nitro electrons on the benzene ring of the small molecule are transferred to the adjacent position to form a carbonyl group, thereby detaching the magnetic beads; finally, a strong magnetic field is used to separate the magnetic beads from the mitochondria connected to the small molecule, and the supernatant is taken to obtain high-purity mitochondria. The principle of optical sectioning is as follows Figure 3 shown.

[0103] V. Functional Characterization of Purified Mitochondria

[0104] Purified mitochondrial activity is tested using a purchased ATP kit. This qualitative analysis is based on the amount of ATP produced by the purified, high-purity mitochondria. Mitochondrial activity is then assessed by transplanting the purified, high-purity mitochondria into cells with mitochondrial defects. Cell survival, intracellular ATP production, and changes in several other markers closely related to mitochondrial activity are then observed to determine mitochondrial activity. Specific embodiments

[0106] 1) Synthesis of compound 1TPP-COOH

[0107] Triphenylphosphine (1.31 g, 5.0 mmol) was added to a 50 mL acetonitrile solution containing 3-bromopropionic acid (0.78 g, 5.5 mmol). The resulting mixture was stirred at 80°C for 24 h. Thin-layer chromatography was performed until the reaction was complete, and the residue was concentrated under vacuum and extracted with a minimum amount of chloroform. Ether was added to precipitate the product, and the supernatant was removed and washed two or three times to remove excess 3-bromopropionic acid. The resulting precipitate was then dried to obtain the yellow oily liquid compound 1TPP-COOH (2.01 g, 97%). (2-Carboxyethyl)triphenylphosphine TPP-COOH: 1 H NMR (500MHz, CDCl3) δppm: 7.59-7.96 (m, 15H), 2.96-3.03 (m, 2H), 3.72-3.79 (m, 2H). MS: m / z calcd: 335.36, found: 335.12.

[0108] 2) Synthesis of compound 3TPP-PEG-Boc

[0109] Under a nitrogen atmosphere, compound 1TPP-COOH (134 mg, 0.4 mmol) was dissolved in anhydrous DCM at 0°C in an ice bath. EDC (95.8 mg, 0.5 mmol) and HOBt (67.5 mg, 0.5 mmol) were then added to the system. After 15 minutes, compound 2NH2-Boc (112 mg, 0.45 mmol) and N-methylmorpholine (60 μL, 0.5 mmol) were added and the mixture was allowed to react at room temperature for 24 hours. Thin-layer chromatography confirmed that the reaction was complete, and water was added to quench the reaction. The mixture was extracted with DCM, washed with saturated NaHCO3 solution, and then with saturated brine. Finally, the organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain the oily product, compound 3TPP-PEG-Boc (215 mg, 95%). Compound 3TPP-PEG-Boc: 1 H NMR (500MHz, CDCl3) δppm: 9.12 (s, 1H), 7.86-7.66 (m, 15H), 5.45 (s, 1H), 3.80 (q, J=12.2, 9.7Hz, 2H), 3.66-3.57 (m, 6H), 3.53 (t, J=5.2Hz, 2H), 3.38 (q,J=5.9Hz,2H),3.30(q,J=5.4Hz,2H),2.93(ddd,J=11.6,7.9,5.3Hz,2H ),1.42(s,9H),1.30-1.23(m,2H),0.87(tdq,J=10.3,7.0,4.0,3.5Hz,4H). 13 C NMR (125MHz, CDCl3) δppm: 173.3, 155.9, 135.0, 130.4, 117.9, 79.5, 70.1, 41.3, 28.4, 19.0. MS: m / z calcd: 565.67, found: 565.28.

[0110] 3) Synthesis of compound 4Br-vanillin

[0111] Vanillin (1g, 6.6mmol) was dissolved in 60mL of acetonitrile, and then 1,2-dibromoethane (6.2g, 32.8mmol) and potassium carbonate (4.54g, 32.8mmol) were added. The reaction was allowed to proceed at room temperature for 48h, and the reaction was detected by thin layer chromatography until completion. The salt formed in the reaction system was filtered and thoroughly precipitated with acetonitrile. The filtrate was then concentrated by vacuum evaporation to obtain a yellow oily product, which turned into a white precipitate after standing for 48h. The crude product was further purified on a silica gel column using PE:EA=2:1 as the eluent for the pure product to obtain a white solid product compound 4Br-vanillin (1.04g, 61%). 4-(2-Bromoethoxy)-3-methoxybenzaldehydeBr-vanillin:1 HNMR (500MHz, CDCl3) δppm: 9.87 (s, 1H), 7.48-7.41 (m, 2H), 6.99 (d, J = 8.0Hz, 1H), 4.42 (t ,J=6.6Hz,2H),3.94(s,3H),3.71(t,J=6.6Hz,2H).MS:m / zcalcd:259.10,found:257.99.

[0112] 4) Synthesis of compound 5Br-vanillin-NO2

[0113] Compound 4 (1.5 g, 8.00 mmol) was placed in an oven-dried flask. Cold nitric acid (50 mL, excess) was added at 0°C. The reaction mixture was stirred at 0°C for 25 minutes and then allowed to warm to room temperature for 1 hour. The reaction was complete as determined by thin-layer chromatography. The reaction was then terminated with 100 mL of cold water. The precipitate was collected by filtration and washed with ice water (3 × 30 mL). The resulting product, 5Br-vanillin-NO2 (1.53 g, 82%), was used without further purification. Compound 5Br-vanillin-NO2: 1 HNMR (500MHz, CDCl3) δppm: 10.46 (s, 1H), 7.64 (s, 1H), 7.44 (s, 1H), 4.49 (t, J = 6.3Hz, 2H), 4.04 (s, 3H), 3.75 (t, J = 6.3Hz, 2H). 13 C NMR (125MHz, CDCl3) δppm: 190.84, 152.91, 150.04, 130.86, 126.38, 112.45, 109.88, 68.77, 64.62, 56.12, 28.20. MS: m / z calcd: 304.10, found: 304.97.

[0114] 5) Synthesis of compound 6Br-vanillin-NO2

[0115] Compound 6Br-vanillin-NO2 (0.5 g, 1.64 mmol) was dissolved in 50 mL of ethyl acetate and wrapped with aluminum foil. Sodium borohydride (186 mg, 4.92 mmol) in sodium hydroxide (50 mL, 1 mol / L) was then added to the mixture solution and stirred at room temperature for 2 h. The reaction was neutralized with 1 mol / L hydrochloric acid and extracted with ethyl acetate (3×30 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated under reduced pressure to obtain a light yellow solid. The crude mixture was purified by flash column chromatography (EA:PE=1:4) to obtain the desired product Br-vanillin-NO2 (437.8 mg, 87%). Compound 6Br-vanillin-NO2:1 H NMR (500MHz, CDCl3) δppm: 7.76 (s, 1H), 7.24 (s, 1H), 5.02-4.98 (m, 2H), 4.42 (t, J = 6.4Hz, 2H), 4.02 (s, 3H), 3.72 (t, J = 6.3Hz, 2H), 2.63 (s, 1H). 13 C NMR (125MHz, CDCl3) δppm: 154.57, 146.42, 139.56, 133.31, 111.59, 110.78, 69.38, 62.80, 56.55, 28.26. MS: m / z calcd: 306.11, found: 306.99.

[0116] 6) Synthesis of compound 7NVOC-N3

[0117] Under N2 protection, compound 6Br-vanillin-NO2 (300 mg, 0.98 mmol) was dissolved in DMF, sodium azide (195 mg, 3 mmol) was added, and the mixture was stirred at 60°C in the dark for about 48 hours. Thin-layer chromatography was performed until the reaction was complete. The mixture was diluted with ethyl acetate, washed once with water, then once with saturated NaHCO3, and once with saturated brine, and finally dried over anhydrous magnesium sulfate, filtered, and evaporated to obtain the desired product, compound 7NVOC-N3 (181.5 mg, 69%). Compound 7NVOC-N3: 1 H NMR (500MHz, CDCl3) δppm: 7.75 (s, 1H), 7.24 (s, 1H), 5.00 (s, 2H), 4.27 (t, J = 5.0Hz, 2H ),4.02(d,J=1.5Hz,3H),3.71(t,J=5.0Hz,2H),2.98(s,1H),2.90(s,1H),2.66(s,1H). 13 C NMR (125MHz, CDCl3) δppm:177.49,172.04,167.79,167.13,139.19,133.80,129.89,129.72,129.37,129 .23,128.78,126.05,122.51,115.77,56.49,52.60,29.22,25.33.MS:m / zcalcd:268.23,found:268.08.

[0118] 7) Synthesis of Compound 8NVOC-NHS

[0119] Compound 7NVOC-N3 (200 mg, 0.74 mmol) was added to 3 mL of MeCN dissolved with Et3N (151 mg, 1.5 mmol) and N,N-disuccinimidyl carbonate (220 mg, 0.95 mmol). The mixture was stirred at room temperature under N2 protection for 1.5 h, and the reaction was detected by thin-layer chromatography until completion. The mixture was concentrated under reduced pressure to remove the solvent, and the residue was purified by flash column chromatography (EA:PE=1:2) to obtain the desired product, compound 8NVOC-NHS (250 mg, 83%), as a light yellow solid. Compound 8NVOC-NHS: 1 H NMR (500MHz, CDCl3) δppm: 7.79 (s, 1H), 7.07 (s, 1H), 5.79 (s, 2H), 4.26 (t, J = 5.0Hz, 2H), 4.06 (s, 3H), 3.70 (t, J = 5.0Hz, 2H), 2.86 (s, 4H). 13 CNMR (125MHz, CDCl3) δppm:177.49,172.04,167.79,167.13,139.19,133.80,129.89,129 .72,129.37,129.23,128.78,126.05,122.51,115.77,56.49,52.60,29.22,25.33.MS:m / z calcd:409.31,found:409.09.

[0120] 8) Synthesis of (1-(4-(2-azidoethoxy)-5-methoxy-2-nitrophenyl)-3,14-dioxo-2,7,10-trioxa-4,13-diazacyclohexan-16-yl)triphenylphosphine HW1

[0121] Compound 3TPP-PEG-Boc (22.6 mg, 0.05 mmol) was dissolved in DCM and 0.3 mL of trifluoroacetic acid was slowly added dropwise under an ice bath. The mixture was then stirred at room temperature for 1 hour and monitored by thin-layer chromatography until the reaction was complete. The mixture was then vacuum evaporated and pumped 5-6 times with DCM to remove the trifluoroacetic acid. The resulting product, compound 9, was used without further purification.

[0122] The resulting product was dissolved in 5 mL of anhydrous acetonitrile, triethylamine (20 μL, 0.15 mmol) was added, and then compound 8 (25 mg, 0.06 mmol) dissolved in anhydrous acetonitrile was added. The reaction system was stirred at room temperature in the dark and monitored by thin-layer chromatography until the reaction was complete. The crude product was concentrated in vacuo and purified by flash column chromatography (MeOH:DCM = 1:10) to obtain the desired product (27 mg, 71%). (1-(4-(2-azidoethoxy)-5-methoxy-2-nitrophenyl)-3,14-dioxo-2,7,10-trioxa-4,13-diazacyclohexan-16-yl)triphenylphosphine: 1 H NMR(500MHz,MeOD)δppm:7.98(dd,J=7.5,1.8Hz,2H),7.99-7.91(m,2H),7.91(d,J=1.5 Hz,2H),7.91-7.84(m,6H),7.83(dd,J=8.1,3.6Hz,4H),7.29(s,1H),5.48(s,2H),4.36- 4.30(m,3H),4.05(s,3H),3.83-3.73(m,2H),3.73(t,J=4.8Hz,2H),3.69(dd,J=6.2,3.0 Hz,2H),3.67-3.60(m,4H),3.54(t,J=5.4Hz,2H),3.39-3.41(m,4H),2.81-2.77(m,2H). 13 C NMR (125MHz, CDCl3) δppm: 162.59, 162.36, 158.67, 156.33, 147.64, 136.70, 134.13, 130.82, 119. 18,118.49,115.50,70.96,70.23,69.58,68.77,56.33,49.90,40.56,39.88,18.50,18.06.MS:m / z calcd:759.78,found:759.29.

[0123] 9) Synthesis of DBCO-magnetic beads and TPP-magnetic beads

[0124] Take 50 μL from 50 mg / mL amino magnetic beads, wash twice with 500 μL DMSO under a strong magnetic field, and then wash once with 500 μL anhydrous DMF. Then disperse the amino magnetic beads in 174.2 μL anhydrous DMF, add 3.3 μL TEA and 320 μL 25 mmol / L DBCO-NHS solution, and react under 100 Hz ultrasound for about 5 hours to obtain DBCO-magnetic beads.

[0125] 50 μL of DBCO magnetic beads were washed three times with 200 μL of DMF under a strong magnetic field. The beads were then dispersed in 50 μL of DMF. 72 μL of 17 mg / mL HW1 was added and linked via copper-free click chemistry to produce TPP magnetic beads. Before use, the TPP magnetic beads were washed twice with 200 μL of DMSO and once with 200 μL of PBS.

[0126] 10) Cell culture

[0127] Human HepG-2 hepatocellular carcinoma cells transfected with mitochondria-specific green fluorescent protein (GFP) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% streptomycin, and penicillin in a 37°C incubator containing 5% CO₂. This cell line constitutively expresses mitochondria-targeted GFP, resulting in green fluorescence (FITC channel) in the mitochondria within the cells, facilitating quantification of extracted mitochondria.

[0128] 11) Mitochondrial extraction experiment

[0129] Three groups were designed for each experiment, including a control group (commercial extraction tool), experimental group 1 and experimental group 2. Except for the different methods used in the extraction process, other conditions remained the same.

[0130] HepG-2 cells in the same batch of culture medium were divided into three parts, one of which was co-cultured with the small molecule probe HW1 for 12 hours, and then the three parts of cells were lysed at the same time to obtain the corresponding cell lysates.

[0131] Control group: The obtained cell lysate was centrifuged for the first time to separate the cell debris from the organelles, and the supernatant was taken; then the mitochondria were separated from other organelles by centrifugation for the second time, and the precipitate was taken; the precipitate was divided into two parts, one part of 70 μL was used to test the mitochondrial activity, and the other part of 200 μL was used to detect the number of mitochondria.

[0132] Experimental Group 1: The TPP-magnetic beads synthesized outside the cells were washed twice with DMSO and once with PBS, and then added to the cell lysate and incubated in a constant temperature oscillator at 4°C for 60 minutes; the magnetic beads with mitochondria captured were then separated from other components of the cell lysate using a strong magnetic field; the magnetic beads with mitochondria captured were then divided into 70 μL for testing mitochondrial activity and 200 μL for detecting the number of mitochondria; the two parts of magnetic beads with mitochondria were then redispersed in the mitochondrial buffer and subjected to ultraviolet shear irradiation for 10 minutes at a low temperature; the supernatant was then separated under the action of a strong magnetic field to obtain the two parts of liquid to be tested.

[0133] Experimental Group 2: This portion of cells was co-cultured with HW1 for 12 h before cell lysis, and then lysed at the same time as the above two groups. DBCO-magnetic beads were then added to the cell lysate, and the cells were also incubated in a constant temperature oscillator at 4°C for 60 min. Subsequent operations were the same as those in Experimental Group 1.

[0134] 12) Mitochondrial activity test and its principle

[0135] like Figure 5 and Figure 6 As shown, the mitochondrial activity assay system utilizes the properties of a proprietary thermostable luciferase to enable reaction conditions that produce a stable "luminescent" luminescent signal while inhibiting endogenous enzymes (such as ATPase) released during cell lysis. The release of ATPase would interfere with accurate ATP measurement. The properties of the reagent overcome the problems caused by factors such as ATPase that interfere with ATP.

[0136] In order to verify the extraction method of the present invention and the extraction method of the existing kit, the number and purity of the extracted mitochondria were also analyzed, and the results are as follows:

[0137] like Figure 7 As shown, AB represents the purity of mitochondria extracted from experimental group 1 measured by flow cytometry; CD represents the purity of mitochondria extracted by a commercial kit (control group) measured by flow cytometry. From left to right, the pictures are: the size (y-axis) and GPF signal (x-axis) distribution of all particles detected by flow cytometry, where the P2 gate represents the detection of mitochondria in all particles, and the P1 gate represents the detection of cell fragments in all particles; the distribution of GFP (i.e. FITC) signal intensity of particles in the P1 gate; the distribution of GFP signal intensity of particles in the P2 gate; the distribution of GFP signal intensity of particles in the integrated P1 and P2 gates. The proportion of P2 gate (i.e. mitochondrial purity) and the number of mitochondria (i.e. total number of particles * P2 gate proportion) in the figure were further extracted and analyzed, and displayed respectively. Figure 8 and Figure 9 As can be seen from the figure, the mitochondria extracted by the present invention are far superior to the mitochondrial extraction kits currently on the market in terms of purity (>2 times) and efficiency (>10 times).

[0138] Figure 8 Figure 3 is a graph showing the proportion of mitochondrial components in the extracted solution system based on flow cytometric analysis. A higher mitochondrial proportion indicates a higher purity of the extracted mitochondria. Data are presented as mean ± standard deviation (N=4). Student's t-test, P**<0.01. This data shows that the purity of mitochondria extracted by the present invention is significantly higher than the concentration of mitochondria extracted using a commercial kit.

[0139] Figure 9 This is the ratio of mitochondrial numbers extracted from the same number of cells, as analyzed by flow cytometry. A higher mitochondrial count indicates a higher extraction efficiency. Data are presented as mean ± standard deviation (N = 2). Student's t-test, P** < 0.1. This data demonstrates that the extraction efficiency of the present invention is significantly higher than that of commercial kits.

[0140] Figure 10 To utilize Figure 1. Mitochondrial activity obtained using the two reagents. Data are presented as mean ± SD (N = 2). Student's t-test, ns: not significant. At the same mitochondrial concentration, the mitochondrial ATP synthesis capacity extracted by the two methods was similar.

[0141] In addition to the above embodiments, the present invention also carries out other embodiments within the aforementioned process range. The synthesized small molecule probes can solve the problem that the current magnetic beads cannot be separated from mitochondria, and have excellent development prospects.

[0142] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with this technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed in the present invention, and these modifications or replacements should all be included in the scope of protection of the present invention.

Claims

1. A multifunctional mitochondrial-targeting small molecule probe, characterized by: It includes a mitochondrial targeting group, a polyethylene glycol linking group, a light-controllable severing group, and an azide group bonded in sequence; Wherein, the mitochondrial targeting group is used to specifically bind to the mitochondrial outer membrane; The polyethylene glycol linking group is used to connect the mitochondrial targeting group to the light-controllable cleavage group; The photo-controllable cleavage group can be broken under the irradiation of light with a wavelength of 365-400nm; The azide group is used for non-copper catalyzed click chemistry; The probe is (1-(4-(2-azidoethoxy)-5-methoxy-2-nitrophenyl)-3,14-dioxo-2,7,10-trioxa-4,13-diazacyclohexane-16-yl)triphenylphosphine; its molecular structure is:

2. The method for preparing the multifunctional mitochondrial-targeting small molecule probe according to claim 1, comprising the following steps: 1) Synthesis of Compound 3 and Compound 8 Among them, the synthesis process of compound 3 is: The specific steps are as follows: I. Synthesis of Compound 1 Triphenylphosphine was added to an acetonitrile solution containing 3-bromopropionic acid, and the mixture was stirred at 60-80°C and tested by thin layer chromatography until the reaction was complete. The reaction solution was concentrated in vacuo and the residue was extracted with an organic solvent. Ether was added to the organic phase to precipitate the product, which was collected and washed several times, and then spin-dried to obtain compound 1; II. Synthesis of Compound 3 Under an inert atmosphere, compound 1 obtained in step I was dissolved in anhydrous dichloromethane in an ice bath, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole were added to the above system; after 15-30 minutes, compound 2 and N-methylmorpholine were added, and the reaction was carried out at room temperature. TLC was used to detect the completion of the reaction, and water was added to quench the reaction. The reaction was extracted with dichloromethane, and the organic phase was washed and dried, filtered, and concentrated to obtain compound 3; The synthetic process of compound 8 is as follows: The specific steps are as follows: ①. Synthesis of compound 4 Vanillin was dissolved in acetonitrile, and 1,2-dibromoethane and potassium carbonate were added. The reaction was carried out at room temperature and detected by thin layer chromatography until the reaction was complete. The salt formed in the reaction system was filtered and the filtrate was concentrated to obtain a yellow oily product, which turned into a white precipitate after standing. The crude product was further purified on a silica gel column to obtain compound 4. ②. Synthesis of compound 5 At 0-4°C, add excess cooled nitric acid to compound 4 obtained in step ①, stir for 15-30 minutes, then warm to room temperature for reaction. Thin layer chromatography is used to detect when the reaction is complete. Water is added to quench the reaction, and the precipitate is collected by filtration and washed several times to obtain compound 5; ③. Synthesis of compound 6 Compound 5 obtained in step ② was dissolved in ethyl acetate, and the mixture was kept away from light throughout the process. A sodium hydroxide solution containing sodium borohydride was then added thereto, and the reaction was stirred at room temperature. The reaction solution was neutralized with hydrochloric acid and extracted with ethyl acetate several times. The organic layers were combined, dried, filtered, and concentrated under reduced pressure to obtain a pale yellow solid crude mixture; The crude mixture was purified by flash column chromatography to give compound 6; ④. Synthesis of Compound 7 Under inert gas protection, compound 6 obtained in step ③ was dissolved in N,N-dimethylformamide, sodium azide was added, and the mixture was stirred at 60-80°C in the dark for reaction. Thin layer chromatography was performed until the reaction was complete. The reaction solution was diluted with ethyl acetate, and the organic phase was washed and dried, filtered, and concentrated to obtain compound 7; ⑤. Synthesis of Compound 8 Compound 7 obtained in step ④ was added to acetonitrile in which triethylamine and N,N-disuccinimidyl carbonate were dissolved. The reaction was stirred at room temperature under inert gas protection and detected by thin layer chromatography until the reaction was complete. The reaction solution was concentrated under reduced pressure to remove the solvent, and the residue was purified by flash column chromatography to obtain compound 8. 2) Synthesis of the probe (1-(4-(2-azidoethoxy)-5-methoxy-2-nitrophenyl)-3,14-dioxo-2,7,10-trioxa-4,13-diazacyclohexan-16-yl)triphenylphosphine 2.1) Compound 3 obtained in step 1) was dissolved in dichloromethane, and trifluoroacetic acid was slowly added dropwise under an ice bath. The mixture was then stirred at room temperature for reaction, and the reaction was monitored by thin-layer chromatography until completion. The mixture was then vacuum evaporated and evaporated with dichloromethane several times to remove the trifluoroacetic acid, yielding compound 9. 2.2) Compound 9 obtained in step 2.1) was dissolved in anhydrous acetonitrile, and triethylamine and the anhydrous acetonitrile solution of compound 8 obtained in step 1) were added sequentially. The reaction was stirred at room temperature in the dark and monitored by thin-layer chromatography until the reaction was complete. The crude product was concentrated in vacuo to obtain the crude product, which was purified by flash column chromatography to obtain the target product (1-(4-(2-azidoethoxy)-5-methoxy-2-nitrophenyl)-3,14-dioxo-2,7,10-trioxa-4,13-diazacyclohexan-16-yl)triphenylphosphine.

3. The method for preparing the multifunctional mitochondrial-targeting small molecule probe according to claim 2, characterized in that: In step I, the equivalent ratio of triphenylphosphine to 3-bromopropionic acid is 1:1-5; the stirring time is 24 hours; In step II, the equivalent ratio of (2-carboxyethyl)triphenylphosphine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, compound 2, and N-methylmorpholine is 1:1-5:1.1-5:1.1-5:1.1-5; the reaction time is 4-24 hours; In step ①, the equivalent ratio of vanillin, 1,2-dibromoethane, and potassium carbonate is 1:2-5:2-5; the reaction time is 24-48 hours, and the standing time is 24-48 hours; In step ②, the equivalent ratio of compound 4 to nitric acid is 1:50-200; the temperature is raised to room temperature and the reaction is carried out for 1-2 hours; In step ③, the equivalent ratio of compound 5 and sodium borohydride is 1:2-5, and the reaction is stirred for 2-4 hours; In step ④, the equivalent ratio of compound 6 to sodium azide is 1:3-10; the stirring time is 12-48h; In step ⑤, the equivalent ratio of compound 7, triethylamine and N,N-disuccinimidyl carbonate is 1:2-5:1.1-3; the stirring time is 1-3 hours; In step 2), the equivalent ratio of compound 3, triethylamine and compound 8 is 1:2-5:1.1-3; and in step 2.1), the reaction is stirred for 1-5 hours.

4. Use of the multifunctional mitochondrial-targeting small molecule probe according to claim 1 in extracting and purifying mitochondria.

5. A TPP-magnetic bead with mitochondrial targeting function, characterized in that: The multifunctional mitochondrial-targeting small molecule probe according to claim 1 is connected to DBCO-magnetic beads through non-copper click chemistry; Among them, DBCO-magnetic beads are obtained by linking diphenylcyclooctyne-active ester (DBCO-NHS) with amino-modified magnetic beads; The surface layer of the amino-modified magnetic beads is polystyrene, the magnetic core is superparamagnetic ferrosoferric oxide, the particle size is 50-1000 nm, the sedimentation coefficient is 3-6 s, and the R value associated with the immunochemiluminescence detection curve is greater than 0.99000.

6. The method for preparing the TPP-magnetic beads with mitochondrial targeting function according to claim 5, characterized in that: The following steps are involved: S1. Wash the amino magnetic beads 2-4 times with DMSO and 1-2 times with anhydrous DMF under a strong magnetic field. Then, disperse the amino magnetic beads in anhydrous DMF and add TEA and DBCO-NHS solution. Ultrasonication is performed to obtain DBCO-magnetic beads. S2. The DBCO-magnetic beads are first washed with DMF 2-4 times under a strong magnetic field, and then dispersed in DMF. The multifunctional mitochondrial-targeting small molecule probe according to claim 1 is then added and connected by non-copper click chemistry to obtain TPP-magnetic beads.

7. Use of the TPP-magnetic beads with mitochondrial targeting function according to claim 5 in extracting and purifying mitochondria.

8. A method for extracting high-purity mitochondria, characterized in that: The following steps are involved: A1. Co-culturing the multifunctional mitochondrial-targeting small molecule probe according to claim 1 with viable cells for 6-18 hours, and then lysing them to obtain a cell lysate; A2. Add washed DBCO-magnetic beads to the A1 cell lysate and connect them to a multifunctional mitochondria-targeting small molecule probe via non-copper click chemistry to capture mitochondria. DBCO-magnetic beads are obtained by linking diphenylcyclooctyne-active ester (DBCO-NHS) with amino-modified magnetic beads; A3. Use a strong magnetic field to attract the magnetic beads in the A2 cell lysate, separate the mitochondria captured by the magnetic beads from other components of the cell lysate, and collect the cell lysate containing the mitochondria. A4. Irradiate the cell lysate obtained in A3 with ultraviolet light at a wavelength of 365-400 nm, causing the nitro group on the benzene ring of the small molecule to transfer electrons to the adjacent position, forming a carbonyl group, thereby detaching the magnetic beads. A strong magnetic field is then used to separate the magnetic beads from the mitochondria attached to the small molecule, and the supernatant is collected to obtain high-purity mitochondria. or, B1. After washing the TPP-magnetic beads with mitochondrial targeting function according to claim 5, add them to the cell lysate and incubate for 6-18 hours to capture mitochondria; B2. Use a strong magnetic field to separate the magnetic beads containing the mitochondria from the other components of the cell lysate, and collect the cell lysate containing the mitochondria; B3. Irradiate the cell lysate obtained in B2 with ultraviolet light at a wavelength of 365-400nm, causing the nitro group on the benzene ring of the small molecule to transfer electrons to the adjacent position, breaking to form a carbonyl group, thereby detaching the magnetic beads. A strong magnetic field is then used to separate the magnetic beads from the mitochondria attached to the small molecule, and the supernatant is collected to obtain high-purity mitochondria.

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