CN115594713A - Small molecule probe for extracting and purifying mitochondria and preparation method and application thereof - Google Patents
Small molecule probe for extracting and purifying mitochondria and preparation method and application thereof Download PDFInfo
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- CN115594713A CN115594713A CN202211260542.3A CN202211260542A CN115594713A CN 115594713 A CN115594713 A CN 115594713A CN 202211260542 A CN202211260542 A CN 202211260542A CN 115594713 A CN115594713 A CN 115594713A
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- triphenylphosphine
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Abstract
The invention provides a small molecular probe for extracting and purifying mitochondria and a preparation method and application thereof, solves the problems that mitochondria cannot be effectively separated from magnetic beads and the biocompatibility is low when the mitochondria are extracted by using magnetic beads at present, and provides an effective way for mitochondrial implantation and future industrial development. The small molecule probe designed by the invention comprises a mitochondrion targeting group, a polyethylene glycol connecting group, a light controllable cutting group and an azide group which are sequentially bonded; the material is connected with DBCO-magnetic beads through non-copper catalytic click chemistry, and under the irradiation of ultraviolet light with the wavelength of 365-400nm, a light-controllable cutting group is broken, so that mitochondria and the magnetic beads are separated.
Description
Technical Field
The invention belongs to biological and new medical technology, and particularly relates to a small molecular probe for extracting and purifying mitochondria and a preparation method and application thereof.
Background
Aging is a major high risk factor for various chronic diseases, and in the face of the increasingly severe aging society of China, various chronic diseases will bring heavy burden to families and society. Only one neurodegenerative disease, the probability of suffering from neurodegenerative diseases in the aged 65 years and older is statistically about 10%, and the proportion thereof will gradually increase with age. Mitochondria are one of the most important organelles in a cell, and play a crucial role in the basic function and survival of the cell. On the one hand, as a "power plant" for cells, mitochondria produce Adenosine Triphosphate (ATP), the most important energy storage molecule required to maintain cell survival, mainly through oxidative phosphorylation. On the other hand, mitochondria release Reactive Oxygen Species (ROS) when damaged, and signals that cytochrome C promotes apoptosis. Thus, once mitochondrial homeostasis is disrupted, it can lead to cellular ATP deficiency, mitochondrial damage, can result in excess ROS or insufficient cellular energy supply, and ultimately cell death. Many studies have shown that mitochondrial damage plays a critical role in the development of many chronic diseases including neurodegenerative diseases, cancer and cardiovascular diseases. However, currently, for the treatment of mitochondrial damage, the standard protocols are mainly aimed at the disease symptoms, such as antioxidant drugs, etc., but most protocols are palliative and not permanent, and there is no clear clinical data to support the effectiveness of these protocols.
Cell therapy has great application prospect as an emerging therapeutic technology. Cell therapy the regimen for mitochondrial damage is to inject healthy mitochondria directly into the mitochondria-damaged tissue. Animal experiments have demonstrated that freshly extracted mitochondria, after injection into mouse heart tissue, can be harvested by cardiomyocytes and significantly enhance cardiac activity. Therefore, cell therapy based on mitochondria has very wide application prospect, and the obtainment of mitochondria with high activity and high purity is a crucial part of the mitochondrial cell therapy.
At present, various methods for obtaining high-purity mitochondria are reported, and the most widely used method is component separation centrifugation, which utilizes an ultra-high speed centrifuge to separate and purify different organelles and components in cell lysate. However, the differential centrifugation (GC method) has high requirements for instruments and equipment and operation of laboratory personnel, and extracted mitochondria generally contain pollutants such as lysosomes, endoplasmic reticulum and peroxisomes at the same time.
Several groups of recent subjects utilize antibodies to the mitochondrial membrane protein TOM22 to specifically non-covalently link mitochondria in cell lysates. The methods greatly improve the extraction purity of mitochondria, have simple operation process, avoid using an ultra-high speed centrifuge and a complicated and unstable experimental operation process, and effectively purify the captured mitochondria by using magnetic beads and magnetic field force; however, this method also has certain disadvantages, which limit further mitochondrial implantation and future industrialization development. Firstly, mitochondrial targeting groups based on antibodies and polypeptides are expensive to prepare and transport; secondly, after the mitochondria are purified, firm irreversible connection is formed between the mitochondria and the magnetic beads; as a non-biodegradable magnetic bead, the degree of influence of the mitochondria-antibody-magnetic bead combination on the activity of acquiring mitochondria is unknown. After the implantation of the mitochondrial cells, the magnetic beads with large particle sizes and non-biodegradation also have potential safety hazards on the target cells and tissues after the implantation.
Disclosure of Invention
The invention aims to solve the problems that mitochondria cannot be effectively separated from magnetic beads and the biocompatibility is low when the mitochondria are extracted by using the magnetic beads at present, and limits the implantation of the mitochondria and the future industrial development, and provides a small molecular probe for extracting and purifying the mitochondria and a preparation method and application thereof.
The conception of the invention is as follows:
for the current situation that mitochondria can not be effectively separated from magnetic beads when being extracted by magnetic beads, the research team of the invention considers that the irreversible connection between the magnetic beads and the mitochondria is changed, the magnetic beads and the mitochondria can be reversibly connected, and a multifunctional mitochondrial targeting small molecular probe is designed by combining a photocleavable group and a mitochondrial targeting group, thereby solving the problem that the magnetic beads can not be separated from the mitochondria in the current mitochondrial magnetic bead extraction mode.
In order to achieve the purpose, the technical solution provided by the invention is as follows:
a multifunctional mitochondrion targeting small molecule probe is characterized in that:
the mitochondrion targeting group, the polyethylene glycol connecting group, the optical controllable cutting group and the azide group are sequentially bonded;
wherein the mitochondrial targeting group is for specific binding to the outer mitochondrial membrane;
the polyethylene glycol connecting group is used for connecting the mitochondrion targeting group with the light controllable cutting group;
the optical controllable cutting group can be broken under the irradiation of light with the wavelength of 365-400 nm;
the azide groups are useful for non-copper catalyzed click chemistry.
Further, the probe is (1- (4- (2-azidoethoxy) -5-methoxy-2-nitrophenyl) -3, 14-dioxo-2, 7, 10-trioxa-4, 13-diazacyclohex-16-yl) triphenylphosphine; the molecular structure is as follows:
the preparation method of the multifunctional mitochondrial targeting small molecule probe is characterized by comprising the following steps:
1) Synthesis of (15, 15-dimethyl-3, 14-dioxo-7, 10-dioxo-4, 13-diazadecyl) triphenylphosphine and 4- (2-azidoethoxy) -5-methoxy-2-nitrobenzyl (2, 5-dioxopyrrolidin-1-yl) carbonate, respectively
Wherein the synthesis steps of (15, 15-dimethyl-3, 14-dioxo-7, 10-dioxo-4, 13-diazadecyl) triphenylphosphine are as follows:
synthesis of (2-carboxyethyl) triphenylphosphine
Adding triphenylphosphine into acetonitrile solution containing 3-bromopropionic acid, stirring at 60-80 deg.C (preferably 80 deg.C), detecting by thin layer chromatography until the reaction is completed, vacuum concentrating the reaction solution, and extracting the residue with organic solvent (such as chloroform, dichloromethane or ethyl acetate, preferably chloroform);
adding ether into the organic phase to precipitate a product, collecting the product, washing for multiple times, and spin-drying to obtain (2-carboxyethyl) triphenylphosphine;
synthesis of (15, 15-dimethyl-3, 14-dioxo-7, 10-dioxo-4, 13-diazadecyl) triphenylphosphine
In an inert atmosphere (N) 2 ) Dissolving the (2-carboxyethyl) triphenylphosphine obtained in the step I in ice-bath anhydrous dichloromethane, and adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 1-hydroxybenzotriazole into the system; adding N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) pivalic amide and N-methylmorpholine after 15-30min (preferably 15 min), reacting at normal temperature, detecting by thin-layer chromatography until the reaction is completed, adding water to quench the reaction, extracting with dichloromethane, washing and drying the organic phase (the concrete process is that the organic phase is washed by saturated sodium bicarbonate solution and saturated saline water in sequence, and the organic layer is dried by anhydrous magnesium sulfate), filtering and concentrating to obtain (15, 15-dimethyl-3, 14-dioxo-7, 10-dioxo-4, 13-diazadecyl) triphenylphosphine;
the synthesis of 4- (2-azidoethoxy) -5-methoxy-2-nitrobenzyl (2, 5-dioxopyrrolidin-1-yl) carbonate was as follows:
(1) synthesis of 4- (2-bromoethoxy) -3-methoxybenzaldehyde
Dissolving vanillin in acetonitrile, adding 1, 2-dibromoethane and potassium carbonate, reacting at normal temperature, and detecting by thin-layer chromatography until the reaction is finished; filtering the salt formed in the reaction system, concentrating the filtrate to obtain a yellow oily product, standing and converting the yellow oily product into a white precipitate; further purifying the crude product on a silica gel column, eluting to give 4- (2-bromoethoxy) -3-methoxybenzaldehyde, using as eluent PE: EA = 2;
(2) synthesis of 4- (2-bromoethoxy) -5-methoxy-2-nitrobenzaldehyde
Adding excessive cooling nitric acid into the 4- (2-bromoethoxy) -3-methoxybenzaldehyde obtained in the step (1) at 0-4 ℃ (preferably 0 ℃), stirring for 15-30min (preferably 25 min), heating to room temperature for reaction, detecting by thin layer chromatography until the reaction is completed, adding water for quenching reaction, filtering and collecting precipitate, and washing with ice water for multiple times to obtain 4- (2-bromoethoxy) -5-methoxy-2-nitrobenzaldehyde;
(3) synthesis of (4- (2-bromoethoxy) -5-methoxy-2-nitrophenyl) methanol
Dissolving the 4- (2-bromoethoxy) -5-methoxy-2-nitrobenzaldehyde obtained in the step (2) in ethyl acetate, keeping the whole process away from light (for example, wrapping a reaction container by using an aluminum foil), then adding a sodium hydroxide solution dissolved with sodium borohydride into the mixture, stirring the mixture at room temperature for reaction, neutralizing the reaction solution by using hydrochloric acid, extracting the reaction solution by using ethyl acetate for multiple times, combining organic layers, drying the organic layers (drying by using anhydrous magnesium sulfate), filtering the mixture, and concentrating the organic layers under reduced pressure to obtain a light yellow solid crude mixture;
the crude mixture was purified by flash column chromatography (EA: PE =1 4) to give (4- (2-bromoethoxy) -5-methoxy-2-nitrophenyl) methanol;
(4) synthesis of (4- (2-azidoethoxy) -5-methoxy-2-nitrophenyl) methanol
In an inert gas (N) 2 ) Under protection, dissolving the (4- (2-bromoethoxy) -5-methoxy-2-nitrophenyl) methanol obtained in the step (3) in N, N-dimethylformamide, adding sodium azide, stirring and reacting under the condition of keeping away from light at 60-80 ℃ (preferably 60 ℃), detecting by thin layer chromatography until the reaction is completed, diluting the reaction solution with ethyl acetate, washing and drying an organic phase, filtering, and concentrating to obtain the (4- (2-azidoethoxy) -5-methoxy-2-nitrophenyl) methanol (sequentially using water and saturated NaHCO) 3 And saturated brine, and finally dried over anhydrous magnesium sulfate, filtered and evaporated to obtain (4- (2-azidoethoxy) -5-methoxy-2-nitrophenyl) methanol);
(5) synthesis of 4- (2-azidoethoxy) -5-methoxy-2-nitrobenzyl (2, 5-dioxopyrrolidin-1-yl) carbonate
Adding the (4- (2-azidoethoxy) -5-methoxy-2-nitrophenyl) methanol obtained in step (4) to acetonitrile in which triethylamine and N, N-disuccinimidyl carbonate are dissolved, at room temperature under an inert gas (N) 2 ) Stirring under the protection condition for reaction, and detecting by thin-layer chromatography until the reaction is finished; the reaction solution was concentrated under reduced pressure, the solvent was removed, and the residue was purified by flash column chromatography (EA: PE = 1;
2) Synthesis of probe (1- (4- (2-azidoethoxy) -5-methoxy-2-nitrophenyl) -3, 14-dioxo-2, 7, 10-trioxa-4, 13-diazacyclohexan-16-yl) triphenylphosphine
2.1 Dissolving (15, 15-dimethyl-3, 14-dioxo-7, 10-dioxo-4, 13-diazadecyl) triphenylphosphine obtained in step 1) in dichloromethane, and slowly adding trifluoroacetic acid dropwise in ice bath; then moving to normal temperature and stirring for reaction, and detecting by thin-layer chromatography until the reaction is finished; then, performing vacuum rotary evaporation, and pumping for multiple times by using a dichloromethane belt until trifluoroacetic acid is removed to obtain a product A;
2.2 Dissolving the product A obtained in the step 2.1) in anhydrous acetonitrile, and sequentially adding triethylamine and the anhydrous acetonitrile solution of the 4- (2-azidoethoxy) -5-methoxy-2-nitrobenzyl (2, 5-dioxopyrrolidin-1-yl) carbonate obtained in the step 1); the reaction was stirred at room temperature away from light and was detected by thin layer chromatography until the reaction was complete and concentrated in vacuo to give the crude product which was purified by flash column chromatography (methanol: dichloromethane =1 10) to give (1- (4- (2-azidoethoxy) -5-methoxy-2-nitrophenyl) -3, 14-dioxo-2, 7, 10-trioxa-4, 13-diazacyclohex-16-yl) triphenylphosphine.
Further, in step i, the equivalent ratio of triphenylphosphine to 3-bromopropionic acid is 1; stirring for 24h;
in step II, the equivalent ratio of (2-carboxyethyl) triphenylphosphine, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, 1-hydroxybenzotriazole, N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) pivaloamide and N-methylmorpholine is 1; the reaction time is 4-24h;
in the step (1), the equivalent ratio of vanillin to 1, 2-dibromoethane to potassium carbonate is 1; the reaction time is 24-48h, and the standing time is 24-48h;
in the step (2), the equivalent ratio of the 4- (2-bromoethoxy) -3-methoxybenzaldehyde to the nitric acid is 1; heating to room temperature and reacting for 1-2h;
in the step (3), the equivalent ratio of 4- (2-bromoethoxy) -5-methoxy-2-nitrobenzaldehyde to sodium borohydride is 1;
in the step (4), the equivalent ratio of (4- (2-bromoethoxy) -5-methoxy-2-nitrophenyl) methanol to sodium azide is 1; stirring for 12-48h;
in the step (5), the equivalent ratio of (4- (2-azidoethoxy) -5-methoxy-2-nitrophenyl) methanol to triethylamine to N, N-disuccinimidyl carbonate is 1; the stirring time is 1-3h;
step 2), the equivalent ratio of (15, 15-dimethyl-3, 14-dioxo-7, 10-dioxo-4, 13-diazadecyl) triphenylphosphine, triethylamine and 4- (2-azidoethoxy) -5-methoxy-2-nitrobenzyl (2, 5-dioxopyrrolidin-1-yl) carbonate is 1; and in the step 2.1), stirring and reacting for 1-5h.
Meanwhile, the invention provides the application of the multifunctional mitochondrial targeting small molecular probe in extraction and purification of mitochondria.
TPP-magnetic beads with mitochondria targeting function, which is characterized in that: the multifunctional mitochondrial targeting small molecular probe is obtained by connecting a non-copper click chemistry and DBCO-magnetic beads;
wherein, the DBCO-magnetic bead is obtained by connecting diphenyl cyclooctyne-active ester (DBCO-NHS) and magnetic beads modified by amino;
the surface layer of the amino modified magnetic bead is polystyrene, the magnetic core is superparamagnetic ferroferric oxide, the particle size is 50-1000nm (preferably 200nm, under the particle size, the extraction amount is the most), the sedimentation coefficient is 3-6s, and the relevant R value of an immunochemiluminescence detection curve is more than 0.99000.
The preparation method of the TPP-magnetic bead with the mitochondrial targeting function is characterized by comprising the following steps of:
s1, washing amino magnetic beads with DMSO for 2-4 times (preferably 2 times) under the action of a strong magnetic field, and then washing with anhydrous DMF for 1-2 times (preferably 1 time); then dispersing the amino magnetic beads in anhydrous DMF, adding TEA and DBCO-NHS solution, and carrying out ultrasonic reaction to obtain DBCO-magnetic beads;
s2, under the action of a strong magnetic field, cleaning DBCO-magnetic beads with DMF for 2-4 times, dispersing the DBCO-magnetic beads into the DMF, adding the multifunctional mitochondrial targeting small molecular probe of claim 1, and connecting the DBCO-magnetic beads through non-copper click chemistry to obtain the TPP-magnetic beads.
The TPP-magnetic bead with the mitochondria targeting function is applied to extraction and purification of mitochondria.
The method for extracting the high-purity mitochondria is characterized by comprising the following steps:
A1. co-culturing the multifunctional mitochondrial targeting small molecule probe of claim 1 and viable cells for 6-18h, and then lysing to obtain a cell lysate;
A2. adding the cleaned DBCO-magnetic beads into the A1 cell lysate, connecting the cleaned DBCO-magnetic beads with a multifunctional mitochondrial targeting small molecular probe through non-copper click chemistry, and grabbing mitochondria;
the DBCO-magnetic bead is obtained by connecting diphenyl cyclooctyne-active ester (DBCO-NHS) and an amino modified magnetic bead;
A3. attracting magnetic beads in the A2 cell lysate by using a strong magnetic field, separating mitochondria captured by the magnetic beads from components of other cell lysates, and collecting the cell lysate containing the mitochondria;
A4. irradiating the cell lysate obtained by A3 by ultraviolet light with the wavelength of 365-400nm (at least 100J), so that nitro electrons on a small molecular benzene ring are transferred to an ortho position and are broken to form a carbonyl group, thereby separating magnetic beads; and then separating the magnetic beads from the mitochondria connected with the small molecules by using a strong magnetic field, and taking supernatant fluid to obtain the high-purity mitochondria.
Alternatively, the first and second electrodes may be,
B1. cleaning the TPP-magnetic beads with the mitochondria targeting function of claim 6, adding the cleaned TPP-magnetic beads into cell lysate, incubating for 6-18h, and grabbing mitochondria;
B2. separating the magnetic beads grasping the mitochondria from other components of the cell lysate by using a strong magnetic field, and collecting the cell lysate containing the mitochondria;
B3. irradiating B2 with 365-400nm ultraviolet (at least 100J) to obtain cell lysate, transferring nitro electrons on small molecule benzene ring to ortho position, and breaking to form carbonyl group to separate magnetic bead; and then separating the magnetic beads from the mitochondria connected with the small molecules by using a strong magnetic field, and taking supernatant fluid to obtain the high-purity mitochondria.
The mechanism of the invention is as follows:
after the small molecular probe is contacted with mitochondria, mitochondria can be efficiently grabbed through a bioorthogonal reaction between the small molecular probe and magnetic beads (78% of mitochondria grabbing is realized by every 250mg of magnetic beads), and the mitochondria and the magnetic beads are dissociated in a response manner under a light induction condition, so that the extraction and purification of the mitochondria are realized, and a key basis is finally provided for the implantation treatment of the mitochondria, namely, the small molecular probe for targeting is designed, and the extracted mitochondria and the magnetic beads are separated through light control (ultraviolet with the wavelength of 365-400 nm), so that the interference of the magnetic beads on subsequent experiments and the influence of the activity of the mitochondria are reduced; compared with the commercial kit which is commonly used at present, the extraction purity is higher, the quantity is more, and the activity of mitochondria is not influenced.
The invention has the advantages that:
1. the raw materials for synthesizing the small molecular probe are easy to obtain, the cost is low, the synthesis steps are simple, and the small molecular probe has good solubility (10 mg/mL in DMSO and 1mg/mL in water) due to the polyethylene glycol connecting group, can be directly used for extraction and purification of mitochondria in a biological sample, and can be commercialized.
2. The multifunctional mitochondrial targeting small molecular probe can realize mitochondrial targeting and photoinduced release at the same time, solves the problem that the existing magnetic bead cannot be separated from mitochondria, and is the design with the smallest influence on mitochondria at present.
3. The multifunctional mitochondrial targeting micromolecule probe is used for extracting and purifying mitochondria, the purity of the mitochondria is 2 times that of a commercial kit, the extraction efficiency of the mitochondria is 6 times that of the commercial kit, and the mitochondria has excellent performance and application prospect.
Drawings
FIG. 1 is a diagram of a mitochondrial extraction and purification process using the probe of the present invention, wherein A is a diagram of mitochondrial extraction and purification based on a multifunctional targeted small molecule mitochondrial probe and magnetic beads; b is a chemical schematic diagram of the surface of the magnetic beads in the process;
FIG. 2 is a synthetic route of the small molecule probe of the present invention;
FIG. 3 illustrates the principle of light cutting according to the present invention;
FIG. 4 is a graph of three methods of mitochondrial grasping;
FIG. 5 is a schematic representation of the ATP kit used in the detection of mitochondrial activity according to the present invention;
FIG. 6 shows the principle of ATP detection by the ATP kit;
FIG. 7 is a graph of data on the number of mitochondria extracted by two methods measured by flow cytometry;
FIG. 8 is a graph showing analysis of data on the purity of extracted mitochondria by flow cytometry;
FIG. 9 is a graph of an analysis of the number of active mitochondria extracted per tray of T25 cells measured by flow cytometry (set as 100% in the present method);
FIG. 10 is a diagram showing data analysis of mitochondrial ATP activity.
Detailed Description
The invention is described in further detail below with reference to the following figures and specific examples:
the mechanism of the invention for extracting and purifying mitochondria is shown in figure 1; the method specifically comprises the following steps:
1. synthesis of multifunctional mitochondrion targeting small molecule probe HW1
The designed molecular structure and the synthesis process of the small molecular probe are shown in figure 2, and the small molecular probe mainly comprises 1) a mitochondrion targeting group, 2) a polyethylene glycol connecting group, 3) a light controllable cutting group, 4) and an azide group for non-copper catalytic click chemistry which are sequentially bonded. The synthesis part is simple and clear, and the target probe can be obtained quickly. Wherein the thread grainThe body targeting group is Triphenylphosphine (TPP) aiming at mitochondria, and has the advantages of simple and stable synthesis, transportation and preparation, low price and the like of micromolecule TPP compared with an antibody by carrying out high-selectivity targeting on mitochondrial membrane potential. The other important part is a light controllable cutting group, under the irradiation of ultraviolet light with UV =365-400nm, the nitro group on the benzene ring generates electron transfer to generate CO 2 And an amino group, which is cleaved to separate mitochondria from magnetic beads (fig. 3). Although the mitochondrial targeting group and the optical controllable cutting group can be directly connected, after the magnetic bead is connected, the magnetic bead can influence the targeting of the mitochondrial targeting group and the extraction of mitochondria due to the fact that the distance between the mitochondrial targeting group and the magnetic bead is too close, so that the polyethylene glycol connecting group for connecting the mitochondrial targeting group and the optical controllable cutting group is introduced.
The method solves the problems that mitochondria extracted by using magnetic beads can not be effectively separated from the magnetic beads and the biocompatibility is low.
2. Chemical modification of nano magnetic bead surface
The aminated modified magnetic bead is a super paramagnetic functionalized magnetic microsphere. Compared with the traditional magnetic beads, the magnetic beads have the characteristics of faster magnetic responsiveness, good dispersibility, extremely low non-specific adsorption, richer binding sites and the like, can be conveniently and efficiently combined with various ligands in a high-loading manner under the action of special chemical reagents, can be used as a good base material for subsequent treatment such as coating, adsorption, chemical modification and the like, and are important carrier tools in medical and molecular biology research.
Therefore, the invention selects the amino modified magnetic beads produced by Baimeige biology company as a carrier tool. The surface layer of the magnetic beads is polystyrene, the magnetic cores are superparamagnetic ferroferric oxide, the particle size selected in the embodiment is 200nm, the sedimentation coefficient is 3-6s, and the R value related to an immunochemiluminescence detection curve is greater than 0..99000.
Firstly, connecting DBCO-NHS with amino modified magnetic beads to obtain DBCO-magnetic beads; and then connecting the synthesized small molecular probe HW1 with the DBCO-magnetic bead by using non-copper click chemistry to finally obtain the TPP-magnetic bead with the mitochondria targeting function.
3. Mitochondria grasping based on multifunctional mitochondria targeting small molecule probe
Currently, the commercial method for extracting mitochondria is to separate the organelles of cell lysate by multi-component centrifugation to obtain mitochondria. As shown in fig. 4, the present invention employs two other methods, which are different from the commercial method of extracting mitochondria.
The first method is that the connected DBCO-magnetic beads are firstly connected with a small molecular probe HW1 outside cells by non-copper click chemistry, and then are incubated with cell lysate for a period of time to achieve the purpose of capturing mitochondria;
the second method is that the small molecular probe HW1 and living cells are co-cultured for a period of time, then the cells are cracked, and DBCO-magnetic beads are added to be connected through non-copper click chemistry, so that the purpose of grabbing mitochondria is achieved.
4. Mitochondrial purification and light controlled release
The mitochondria captured by the magnetic beads can be separated from other components of cell lysate by attracting the magnetic beads through the two methods by using a strong magnetic field; then, ultraviolet irradiation with UV =365-400nm is carried out, nitro electrons on a small molecule benzene ring are transferred to an ortho-position to be broken to form carbonyl, and thus magnetic beads are separated; finally, separating the magnetic beads from the mitochondria connected with the small molecules by using a strong magnetic field, and taking supernatant fluid to obtain the mitochondria with high purity, wherein the principle of photocleavage is shown in figure 3.
5. Functional characterization of mitochondria after purification
The activity of the purified mitochondria is detected by using a purchased ATP kit, and the activity of the purified mitochondria is qualitatively analyzed mainly according to the ATP amount generated by the purified mitochondria with high purity. Then, the purified high-purity mitochondria are transplanted into cells with mitochondria defects, and the survival of the cells and the ATP production in the cells and the change of other markers which are closely related to the mitochondrial activity are observed to distinguish the activity of the mitochondria.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
1) Synthesis of (2-carboxyethyl) triphenylphosphine TPP-COOH
Triphenylphosphine (1.31g, 5.0 mmol) was added to a solution of 3-bromopropionic acid (0.78g, 5.5 mmol) dissolved in 50mL acetonitrile. The resulting mixture was stirred at 80 ℃ for 24h. Thin layer chromatography detects the reaction is complete, concentrate in vacuo and extract the residue with a minimum amount of chloroform. The product was precipitated by addition of ether, the supernatant was removed two to three times to wash off excess 3-bromopropionic acid, and the resulting precipitate was spin dried to give (2-carboxyethyl) triphenylphosphine TPP-COOH as a yellow oily liquid (2.01g, 97%). (2-carboxyethyl) triphenylphosphine TPP-COOH: 1 H NMR(500MHz,CDCl 3 )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.
2) Synthesis of (15, 15-dimethyl-3, 14-dioxo-7, 10-dioxo-4, 13-diazadecyl) triphenylphosphine TPP-PEG-Boc
In N 2 (2-carboxyethyl) triphenylphosphine TPP-COOH (134mg, 0.4 mmol) was dissolved in anhydrous DCM at 0 ℃ in ice bath under an atmosphere, and EDC (95.8mg, 0.5 mmol) and HOBt (67.5mg, 0.5 mmol) were added to the above system; adding N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) pivaloyl amide NH after 15min 2 Boc (112mg, 0.45mmol) was reacted with N-methylmorpholine (60. Mu.L, 0.5 mmol) at ambient temperature for 24h. Detecting by thin layer chromatography until the reaction is completed, adding water to quench the reaction, extracting with DCM, and then using saturated NaHCO 3 The solution was washed with brine, and the organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated to give the product (15, 15-dimethyl-3, 14-dioxo-7, 10-dioxo-4, 13-diazadecyl) triphenylphosphine TPP-PEG-Boc (215mg, 95%) as an oil. (15, 15-dimethyl-3, 14-dioxo-7, 10-dioxo-4, 13-diazadecyl) triphenylphosphine TPP-PEG-Boc: 1 H NMR(500MHz,CDCl 3 )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.
3) Synthesis of 4- (2-bromoethoxy) -3-methoxybenzaldehyde Br-vanillin
Dissolving vanillin (1g, 6.6 mmol) in 60mL acetonitrile, adding 1, 2-dibromoethane (6.2g, 32.8mmol) and potassium carbonate (4.54g, 32.8mmol), reacting at normal temperature for 48h, and detecting by thin layer chromatography until the reaction is completed. The salt formed in the reaction system was filtered, precipitated thoroughly with acetonitrile, and the filtrate was concentrated by vacuum evaporation to give a yellow oily product which was converted to a white precipitate after standing for 48 h. The crude product was further purified on a silica gel column using PE: EA =2 as eluent for the pure product to give 4- (2-bromoethoxy) -3-methoxybenzaldehyde Br-vanillin (1.04g, 61%) as a white solid product. 4- (2-bromoethoxy) -3-methoxybenzaldehyde Br-vanillin: 1 H NMR(500MHz,CDCl 3 )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/z calcd:259.10,found:257.99.
4) 4- (2-Bromoethoxy) -5-methoxy-2-nitrobenzaldehyde Br-vanillin-NO 2 Synthesis of (2)
4- (2-bromoethoxy) -3-methoxybenzaldehyde Br-vanillin (1.5g, 8.00mmol) was placed in an oven-dried flask, cooled nitric acid (50 mL, excess) was added at 0 ℃, the reaction mixture was stirred at 0 ℃ for 25min, then warmed to room temperature for 1h. The reaction was terminated with 100mL of cold water by TLC until completion, and the precipitate was collected by filtration and washed with ice water (3X 30 mL). The obtained product 4- (2-bromoethoxy) -5-methoxy-2-nitrobenzaldehyde Br-vanillin-NO 2 (1.53g, 82%) was used without further purification. 4- (2-Bromoethoxy) -5-methoxy-2-nitrobenzaldehyde Br-vanillin-NO 2 : 1 HNMR(500MHz,CDCl 3 )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,CDCl 3 )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.
5) (4- (2-Bromoethoxy)-5-methoxy-2-nitrophenyl) methanol Br-vanillyl phenol NO 2 Synthesis of (2)
4- (2-bromoethoxy) -5-methoxy-2-nitrobenzaldehyde Br-vanillin-NO 2 (0.5g, 1.64mmol) was dissolved in 50mL of ethyl acetate and wrapped with aluminum foil. Sodium borohydride (186mg, 4.92mmol) in sodium hydroxide (50mL, 1mol/L) was then added to the mixture solution and stirred at room temperature for 2h. The reaction was neutralized with 1mol/L hydrochloric acid and extracted with ethyl acetate (3X 30 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give a pale yellow solid. The crude mixture was purified by flash column chromatography (EA: PE =1 4) to give the desired product Br-vanillyl phenol-NO 2 (437.8mg, 87%). (4- (2-Bromoethoxy) -5-methoxy-2-nitrophenyl) methanol Br-Vanillyl-NO 2 : 1 H NMR(500MHz,CDCl 3 )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,CDCl 3 )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.
6) (4- (2-Azidoethoxy) -5-methoxy-2-nitrophenyl) methanol NVOC-N 3 Synthesis of (2)
In N 2 Under the protection, adding (4- (2-bromoethoxy) -5-methoxy-2-nitrophenyl) methanol Br-vanillyl phenol-NO 2 (300mg, 0.98mmol) in DMF, adding sodium azide (195mg, 3 mmol), stirring at 60 deg.C away from light for about 48h, detecting by thin layer chromatography until the reaction is completed, diluting with ethyl acetate, washing with water once, and adding saturated NaHCO 3 Washing once, washing once with saturated brine, finally drying with anhydrous magnesium sulfate, filtering, evaporating to obtain the required product (4- (2-azidoethoxy) -5-methoxy-2-nitrophenyl) methanol NVOC-N 3 (181.5mg, 69%). (4- (2-Azidoethoxy) -5-methoxy-2-nitrophenyl) methanol NVOC-N 3 : 1 H NMR(500MHz,CDCl 3 )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,CDCl 3 )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:268.23,found:268.08.
7) Synthesis of 4- (2-azidoethoxy) -5-methoxy-2-nitrobenzyl (2, 5-dioxopyrrolidin-1-yl) carbonate NVOC-NHS
The (4- (2-azidoethoxy) -5-methoxy-2-nitrophenyl) methanol NVOC-N 3 (200mg, 0.74mmol) was added to Et dissolved 3 N (151mg, 1.5 mmol) and N, N-disuccinimidyl carbonate (220mg, 0.95mmol) in 3mL of MeCN. At room temperature and N 2 Stirring for 1.5h under the protection condition, and detecting by thin layer chromatography until the reaction is finished. Concentrated under reduced pressure, the solvent was removed and the residue was purified by flash column chromatography (EA: PE = 1. 4- (2-azidoethoxy) -5-methoxy-2-nitrobenzyl (2, 5-dioxopyrrolidin-1-yl) carbonate NVOC-NHS: 1 H NMR(500MHz,CDCl 3 )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 C NMR(125MHz,CDCl 3 )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.
8) Synthesis of (1- (4- (2-azidoethoxy) -5-methoxy-2-nitrophenyl) -3, 14-dioxo-2, 7, 10-trioxa-4, 13-diazacyclohex-16-yl) triphenylphosphine HW1
(15, 15-dimethyl-3, 14-dioxo-7, 10-dioxo-4, 13-diazadecyl) triphenylphosphine TPP-PEG-Boc (22.6 mg, 0.05mmol) was dissolved in DCM and 0.3mL of trifluoroacetic acid was slowly added dropwise thereto while cooling on ice. Then the mixture is moved to the normal temperature and stirred for 1h, and the thin layer chromatography detects that the reaction is finished. It was then rotary evaporated in vacuo and stripped with DCM 5-6 times until the trifluoroacetic acid was removed and the product used without further purification.
The resulting product was dissolved in 5mL of anhydrous acetonitrile, triethylamine (20. Mu.L, 0.15 mmol) was added, and 4- (2-azidoethoxy) -5-methoxy-2 dissolved in anhydrous acetonitrile was added-nitrobenzyl (2, 5-dioxopyrrolidin-1-yl) carbonate (25mg, 0.06mmol). The reaction was stirred at room temperature away from light, detected by thin layer chromatography until the reaction was complete, and concentrated in vacuo to give the crude product which was purified by flash column chromatography (MeOH: DCM =1 10) to give the desired product (27mg, 71%). (1- (4- (2-azidoethoxy) -5-methoxy-2-nitrophenyl) -3, 14-dioxo-2, 7, 10-trioxa-4, 13-diazacyclohex-16-yl) triphenylphosphine: 1 HNMR(500MHz,MeOD)δppm:7.98(dd,J＝7.5,1.8Hz,2H),7.99-7.91(m,2H),7.91(d,J＝1.5Hz,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＝6.2,3.0Hz,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,CDCl 3 )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.
9) Synthesis of DBCO-magnetic beads and TPP-magnetic beads
Taking 50 mu L of amino magnetic beads from 50mg/mL, washing twice with 500 mu L of DMSO under the action of a strong magnetic field, washing once with 500 mu L of anhydrous DMF, dispersing the amino magnetic beads in 174.2 mu L of anhydrous DMF, adding 3.3 mu L of TEA and 320 mu L of 25mmol/L DBCO-NHS solution, and reacting for about 5 hours under 100Hz ultrasound to obtain the DBCO-magnetic beads.
50 mu L of DBCO-magnetic beads are taken, washed three times by 200 mu L of DMF under the action of a strong magnetic field, then redispersed in 50 mu L of DMF, then 72 mu L of 17mg/mL HW1 is added, and connection is carried out by copper-free click chemistry to obtain TPP-magnetic beads. TPP-magnetic beads were washed twice with 200. Mu.L DMSO and once with 200. Mu.L PBS before use.
10 ) cell culture
Mitochondrion-specific Green Fluorescent Protein (GFP) -transfected human hepatoma cell HepG-2 was cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 1% streptomycin and penicillin, and incubated at 37 deg.C with 5% CO 2 The incubator of (1). The cell line continuously expresses GFP with mitochondrial targeting,therefore, mitochondria in cells are all provided with green fluorescence (FITC channel), and the extracted mitochondria can be conveniently quantified.
11 Mitochondrial extraction experiments
Three groups, including control group (commercial extraction tool), experimental group 1 and experimental group 2, were designed for each experiment, and the conditions were kept consistent during the extraction process, except for the different methods used.
Dividing HepG-2 cells in the same batch of culture medium into three parts, wherein one part of the HepG-2 cells is co-cultured with the small molecular probe HW1 for 12h and then simultaneously cracking the three parts of the HepG-2 cells to obtain corresponding cell lysate.
Control group: firstly, centrifuging the obtained cell lysate for the first time, separating cell debris from organelles, and taking supernatant fluid; then carrying out second centrifugation to separate mitochondria from other organelles and taking the precipitate; the resulting pellet was divided into two portions, one 70 μ L for testing mitochondrial activity and the other 200 μ L for testing mitochondrial number.
Experimental group 1: washing TPP-magnetic beads synthesized outside cells twice with DMSO, washing with PBS once, adding into cell lysate, and incubating for 60min under 4 deg.C constant temperature oscillator; then separating the magnetic beads grabbed with the mitochondria from other components of the cell lysate by utilizing a strong magnetic field; at the moment, the obtained magnetic beads grasping the mitochondria are divided into 70 mu L for testing the activity of the mitochondria and 200 mu L for testing the number of the mitochondria; then dispersing two parts of magnetic beads with mitochondria in a mitochondrial buffer solution again, and then carrying out ultraviolet shearing irradiation for 10min under a low-temperature environment; then separating the supernatant under the action of a strong magnetic field to obtain two parts of liquid to be detected.
Experimental group 2: the cells were co-cultured with HW1 for 12h before cell lysis, then lysed together with the above two groups, and DBCO-magnetic beads were added to the cell lysate, and incubated for 60min at 4 ℃ with a constant temperature shaker, and the subsequent operations were identical to those of experiment group 1.
12 Mitochondrial Activity test and its principles
As shown in FIGS. 5 and 6, the mitochondrial Activity assay SystemThe characteristics of a proprietary thermostable luciferase are utilized to allow reaction conditions to generate stable "luminescent signals while inhibiting endogenous enzymes (e.g., ATPase) released during cell lysis. The release of ATPase interferes with accurate measurement of ATP.
The nature of the reagent overcomes problems caused by factors such as ATPase that interfere with ATP.
In order to examine the extraction method of the present invention and the extraction method of the existing kit, the number and purity of mitochondria extracted were also analyzed, and the results are as follows:
as shown in FIG. 7, A-B represent the mitochondrial purity of the extract of Experimental group 1 as measured by flow cytometry; C-D represents the mitochondrial purity as measured by flow cytometry extracted with commercial kits (control). The pictures are from left to right: the size (y-axis) GPF signal (x-axis) distribution of all particles is detected by flow cytometry, wherein P2 gate represents the detection of mitochondria and P1 gate represents the detection of cell debris in all particles; distribution of GFP (i.e., FITC) signal intensity of particles within the P1 gate; a profile of GFP signal intensity of particles in P2 gate; distribution of GFP signal intensity of integrated P1 and P2 gated particles. Further extracted and analyzed by P2 gate fraction (i.e. mitochondrial purity) and mitochondrial number (i.e. total particle number P2 gate fraction) in the figures are presented in fig. 8 and fig. 9, respectively. As can be seen from the figure, the mitochondria extracted by the invention are far superior to the mitochondria extraction kit in the current market in terms of the purity (> 2 times) and efficiency (> 10 times) of mitochondria extraction.
FIG. 8 is a graph showing the composition ratio of mitochondria in the extracted solution system according to flow cytometry analysis, wherein higher mitochondrial ratio indicates higher purity of extracted mitochondria; data are shown as mean ± standard deviation (N = 4). Student's t-test, P × <0.01 this data shows that the mitochondrial purity extracted by the present invention is significantly higher than the mitochondrial concentration extracted by the commercial kit.
FIG. 9 shows the ratio of the number of mitochondria extracted from the same number of cells according to flow cytometry analysis. The higher the number of mitochondria, the higher the extraction efficiency. Data are shown as mean ± standard deviation (N = 2). Student's t-test, P × <0.1. This set of data shows that the extraction efficiency of the present invention is significantly higher than that of the commercial kits.
FIG. 10 is a schematic view of a process for utilizing
Mitochondrial activity profiles obtained with the reagents. Data are shown as mean ± standard deviation (N = 2). Student's t-test, ns: not significant fluorescent. At the same mitochondrial concentration, mitochondrial ATP synthesis capacity extracted by the two methods was similar.
In addition to the above embodiments, the present invention also provides other embodiments within the aforementioned process range, and the synthesized small molecule probe can solve the problem that the magnetic beads cannot be separated from mitochondria at present, and has a promising development prospect.
While the invention has been described with reference to specific embodiments, the invention is not limited thereto, and various equivalent modifications or substitutions can be easily made by those skilled in the art within the technical scope of the present disclosure.
Claims (9)
1. A multifunctional mitochondrial targeting small molecule probe, characterized in that:
the mitochondrion targeting group, the polyethylene glycol connecting group, the optical controllable cutting group and the azide group are sequentially bonded;
wherein the mitochondrial targeting group is for specific binding to the outer mitochondrial membrane;
the polyethylene glycol connecting group is used for connecting the mitochondrion targeting group with the light controllable cutting group;
the optical controllable cutting group can be broken under the irradiation of light with the wavelength of 365-400 nm;
the azide groups are useful for non-copper catalyzed click chemistry.
2. The multifunctional mitochondrial-targeted small molecule probe of claim 1, wherein: the probe is (1- (4- (2-azidoethoxy) -5-methoxy-2-nitrophenyl) -3, 14-dioxo-2, 7, 10-trioxa-4, 13-diazacyclohexane-16-yl) triphenylphosphine; the molecular structure is as follows:
3. the method for preparing the multifunctional mitochondrial targeting small molecule probe of claim 1, comprising the steps of:
1) Synthesis of (15, 15-dimethyl-3, 14-dioxo-7, 10-dioxo-4, 13-diazadecyl) triphenylphosphine and 4- (2-azidoethoxy) -5-methoxy-2-nitrobenzyl (2, 5-dioxopyrrolidin-1-yl) carbonate, respectively
Wherein the synthesis steps of (15, 15-dimethyl-3, 14-dioxo-7, 10-dioxo-4, 13-diazadecyl) triphenylphosphine are as follows:
synthesis of (2-carboxyethyl) triphenylphosphine
Adding triphenylphosphine into acetonitrile solution containing 3-bromopropionic acid, stirring at 60-80 deg.C (preferably 80 deg.C), detecting by thin layer chromatography until the reaction is completed, vacuum concentrating the reaction solution, and extracting the residue with organic solvent;
adding ether into the organic phase to precipitate a product, collecting the product, washing for multiple times, and spin-drying to obtain (2-carboxyethyl) triphenylphosphine;
synthesis of (15, 15-dimethyl-3, 14-dioxo-7, 10-dioxo-4, 13-diazadecyl) triphenylphosphine
Under inert atmosphere, dissolving the (2-carboxyethyl) triphenylphosphine obtained in the step I in ice-bath anhydrous dichloromethane, and then adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 1-hydroxybenzotriazole into the system; adding N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) pivaloyl amide and N-methylmorpholine after 15-30min, reacting at normal temperature, detecting by thin-layer chromatography until the reaction is finished, adding water to quench the reaction, extracting by using dichloromethane, washing and drying an organic phase, filtering and concentrating to obtain (15, 15-dimethyl-3, 14-dioxo-7, 10-dioxo-4, 13-diazadecyl) triphenylphosphine;
the synthesis of 4- (2-azidoethoxy) -5-methoxy-2-nitrobenzyl (2, 5-dioxopyrrolidin-1-yl) carbonate was as follows:
(1) synthesis of 4- (2-bromoethoxy) -3-methoxybenzaldehyde
Dissolving vanillin in acetonitrile, adding 1, 2-dibromoethane and potassium carbonate, reacting at normal temperature, and detecting by thin-layer chromatography until the reaction is finished; filtering the salt formed in the reaction system, concentrating the filtrate to obtain a yellow oily product, standing and converting the yellow oily product into a white precipitate; further purifying the crude product on a silica gel column, eluting to obtain 4- (2-bromoethoxy) -3-methoxybenzaldehyde;
(2) synthesis of 4- (2-bromoethoxy) -5-methoxy-2-nitrobenzaldehyde
Adding excessive cooling nitric acid into the 4- (2-bromoethoxy) -3-methoxybenzaldehyde obtained in the step (1) at 0-4 ℃, stirring for 15-30min, then heating to room temperature for reaction, detecting by thin-layer chromatography until the reaction is finished, adding water to quench the reaction, filtering and collecting precipitate, and washing for multiple times to obtain 4- (2-bromoethoxy) -5-methoxy-2-nitrobenzaldehyde;
(3) synthesis of (4- (2-bromoethoxy) -5-methoxy-2-nitrophenyl) methanol
Dissolving the 4- (2-bromoethoxy) -5-methoxy-2-nitrobenzaldehyde obtained in the step (2) in ethyl acetate, keeping the whole process away from light, then adding a sodium hydroxide solution dissolved with sodium borohydride into the mixture, stirring the mixture at room temperature for reaction, neutralizing the reaction solution with hydrochloric acid, extracting the reaction solution with ethyl acetate for multiple times, combining organic layers, drying, filtering and concentrating the organic layers under reduced pressure to obtain a light yellow solid crude mixture;
the crude mixture was purified by flash column chromatography to give (4- (2-bromoethoxy) -5-methoxy-2-nitrophenyl) methanol;
(4) synthesis of (4- (2-azidoethoxy) -5-methoxy-2-nitrophenyl) methanol
Under the protection of inert gas, dissolving the (4- (2-bromoethoxy) -5-methoxy-2-nitrophenyl) methanol obtained in the step (3) in N, N-dimethylformamide, adding sodium azide, stirring for reaction at 60-80 ℃ away from light, detecting by thin-layer chromatography until the reaction is completed, diluting a reaction solution by using ethyl acetate, washing and drying an organic phase, filtering, and concentrating to obtain (4- (2-azidoethoxy) -5-methoxy-2-nitrophenyl) methanol;
(5) synthesis of 4- (2-azidoethoxy) -5-methoxy-2-nitrobenzyl (2, 5-dioxopyrrolidin-1-yl) carbonate
Adding the (4- (2-azidoethoxy) -5-methoxy-2-nitrophenyl) methanol obtained in the step (4) into acetonitrile dissolved with triethylamine and N, N-disuccinimidyl carbonate, stirring at room temperature under the protection of inert gas for reaction, and detecting by thin-layer chromatography until the reaction is finished; concentrating the reaction solution under reduced pressure, removing the solvent, and purifying the residue by flash column chromatography to obtain 4- (2-azidoethoxy) -5-methoxy-2-nitrobenzyl (2, 5-dioxopyrrolidin-1-yl) carbonate;
2) Synthesis of probe (1- (4- (2-azidoethoxy) -5-methoxy-2-nitrophenyl) -3, 14-dioxo-2, 7, 10-trioxa-4, 13-diazacyclohex-16-yl) triphenylphosphine
2.1 Dissolving (15, 15-dimethyl-3, 14-dioxo-7, 10-dioxo-4, 13-diazadecyl) triphenylphosphine obtained in step 1) in dichloromethane, and slowly adding trifluoroacetic acid dropwise in ice bath; then moving to normal temperature and stirring for reaction, and detecting by thin-layer chromatography until the reaction is finished; then, performing vacuum rotary evaporation, and pumping for many times by using a dichloromethane belt until trifluoroacetic acid is removed to obtain a product A;
2.2 Dissolving the product A obtained in the step 2.1) in anhydrous acetonitrile, sequentially adding triethylamine and the anhydrous acetonitrile solution of 4- (2-azidoethoxy) -5-methoxy-2-nitrobenzyl (2, 5-dioxopyrrolidin-1-yl) carbonate obtained in the step 1), stirring at room temperature in the dark for reaction, detecting by thin layer chromatography until the reaction is completed, performing vacuum concentration to obtain a crude product, and purifying by flash column chromatography to obtain (1- (4- (2-azidoethoxy) -5-methoxy-2-nitrophenyl) -3, 14-dioxo-2, 7, 10-trioxa-4, 13-diazacyclohexan-16-yl) triphenylphosphine.
4. The method for preparing the multifunctional mitochondrial targeting small molecule probe according to claim 3, wherein the method comprises the following steps:
in the step I, the equivalent ratio of triphenylphosphine to 3-bromopropionic acid is 1-5; stirring for 24h;
in step ii, the equivalent ratio of (2-carboxyethyl) triphenylphosphine, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, 1-hydroxybenzotriazole, N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) pivaloamide, N-methylmorpholine is 1; the reaction time is 4-24h;
in the step (1), the equivalent ratio of vanillin, 1, 2-dibromoethane and potassium carbonate is 1; the reaction time is 24-48h, and the standing time is 24-48h;
in the step (2), the equivalent ratio of the 4- (2-bromoethoxy) -3-methoxybenzaldehyde to the nitric acid is 1; heating to room temperature and reacting for 1-2h;
in the step (3), the equivalent ratio of 4- (2-bromoethoxy) -5-methoxy-2-nitrobenzaldehyde to sodium borohydride is 1;
in the step (4), the equivalent ratio of (4- (2-bromoethoxy) -5-methoxy-2-nitrophenyl) methanol to sodium azide is 1; stirring for 12-48h;
in the step (5), the equivalent ratio of (4- (2-azidoethoxy) -5-methoxy-2-nitrophenyl) methanol to triethylamine to N, N-disuccinimidyl carbonate is 1; stirring for 1-3h;
step 2), the equivalent ratio of (15, 15-dimethyl-3, 14-dioxo-7, 10-dioxo-4, 13-diazadecyl) triphenylphosphine, triethylamine and 4- (2-azidoethoxy) -5-methoxy-2-nitrobenzyl (2, 5-dioxopyrrolidin-1-yl) carbonate is 1; and in the step 2.1), stirring and reacting for 1-5h.
5. The use of the multifunctional mitochondrial targeting small molecule probe of claim 1 for extracting and purifying mitochondria.
6. TPP-magnetic beads with a mitochondrion targeting function, characterized in that:
the multifunctional mitochondrial targeting small molecule probe of claim 1, which is obtained by connecting DBCO-magnetic beads through non-copper click chemistry;
wherein the DBCO-magnetic bead is obtained by connecting diphenyl cyclooctyne-active ester (DBCO-NHS) and magnetic beads modified by amino;
the surface layer of the magnetic bead modified by the amino is polystyrene, the magnetic core is superparamagnetic ferroferric oxide, the particle size is 50-1000nm, the sedimentation coefficient is 3-6s, and the R value related to an immunochemiluminescence detection curve is greater than 0.99000.
7. The method for preparing TPP-magnetic beads with mitochondrial targeting function according to claim 6, comprising the following steps:
s1, washing amino magnetic beads with DMSO (dimethyl sulfoxide) for 2-4 times under the action of a strong magnetic field, and then washing with anhydrous DMF (dimethyl formamide) for 1-2 times; then dispersing the amino magnetic beads in anhydrous DMF, adding TEA and DBCO-NHS solution, and carrying out ultrasonic reaction to obtain DBCO-magnetic beads;
s2, under the action of a strong magnetic field, cleaning DBCO-magnetic beads with DMF for 2-4 times, dispersing the DBCO-magnetic beads into the DMF, adding the multifunctional mitochondrial targeting small molecular probe of claim 1, and connecting the DBCO-magnetic beads through non-copper click chemistry to obtain the TPP-magnetic beads.
8. Use of the TPP-magnetic beads with mitochondrial targeting function according to claim 6 for the extraction and purification of mitochondria.
9. A method for extracting high-purity mitochondria, which is characterized by comprising the following steps:
A1. co-culturing the multifunctional mitochondrial targeting small molecule probe of claim 1 and viable cells for 6-18h, and then lysing to obtain a cell lysate;
A2. adding the cleaned DBCO-magnetic beads into the A1 cell lysate, connecting the cleaned DBCO-magnetic beads with a multifunctional mitochondrial targeting small molecular probe through non-copper click chemistry, and grabbing mitochondria;
the DBCO-magnetic bead is obtained by connecting diphenyl cyclooctyne-active ester (DBCO-NHS) and an amino modified magnetic bead;
A3. attracting magnetic beads in the A2 cell lysate by using a strong magnetic field, separating mitochondria captured by the magnetic beads from components of other cell lysates, and collecting the cell lysate containing the mitochondria;
A4. irradiating the cell lysate A3 by ultraviolet with the wavelength of 365-400nm to transfer nitro electrons on a small molecular benzene ring to an ortho-position to be broken to form carbonyl so as to separate magnetic beads; then separating the magnetic beads from the mitochondria connected with the small molecules by utilizing a strong magnetic field, and taking supernatant fluid to obtain the mitochondria with high purity.
Alternatively, the first and second liquid crystal display panels may be,
B1. cleaning the TPP-magnetic beads with the mitochondria targeting function of claim 6, adding the cleaned TPP-magnetic beads into cell lysate, incubating for 6-18h, and grabbing mitochondria;
B2. separating the magnetic beads grasping the mitochondria from other components of the cell lysate by using a strong magnetic field, and collecting the cell lysate containing the mitochondria;
B3. irradiating B2 with 365-400nm ultraviolet to obtain cell lysate, transferring nitro electrons on the small molecule benzene ring to the ortho position to break to form carbonyl, and separating magnetic beads; and then separating the magnetic beads from the mitochondria connected with the small molecules by using a strong magnetic field, and taking supernatant fluid to obtain the high-purity mitochondria.
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