CN108732147B

CN108732147B - Method for detecting apoptosis process based on FRET effect

Info

Publication number: CN108732147B
Application number: CN201810366318.XA
Authority: CN
Inventors: 张寅�; 朱恒宇; 吴琦; 赵强; 刘淑娟; 黄维
Original assignee: Nanjing University of Posts and Telecommunications
Current assignee: Nanjing University of Posts and Telecommunications
Priority date: 2018-04-23
Filing date: 2018-04-23
Publication date: 2021-01-19
Anticipated expiration: 2038-04-23
Also published as: CN108732147A

Abstract

The invention provides a method for detecting an apoptosis process based on FRET effect, which mainly comprises the following steps: preparing a phosphorescent iridium complex with long service life as a donor D, preparing a short-service-life organic fluorescent small molecular compound as an acceptor A, wherein FRET effect exists between the D and the A; respectively connecting a donor D and an acceptor A to two ends of a peptide chain which can be cut by caspase only existing in the apoptosis process through a bioorthogonal reaction to form a D-A type probe, wherein the D-A type probe can be assembled outside cells and then implanted into the cells, and can also be directly assembled inside the cells; and detecting the change of the service life of the donor D or the receptor A in the D-A type probe by utilizing a confocal imaging or service life imaging technology so as to judge the apoptosis process of the cell. The invention can be applied to each stage of cell apoptosis, can distinguish each stage of cell apoptosis, and has very important application prospect in the fields of cell imaging, biomarkers, sensing and the like.

Description

Method for detecting apoptosis process based on FRET effect

Technical Field

The invention belongs to the technical field of organic photoelectric materials, and particularly relates to a D-A type probe which is based on FRET effect and can be used for detecting apoptosis and a detection application thereof in an apoptosis process.

Background

Apoptosis, also known as programmed cell death, is a natural or physiological death phenomenon of cells under the control of a series of endogenous genes, plays an important role in the aspects of guiding the formation of embryos, maintaining the balance of normal cells, regulating the growth and development of organisms, naturally aging and the like, and the specific pathways and the complex details involved in the apoptosis process are not completely understood so far. Therefore, it is very important to explore different methods for studying the apoptosis process and its specific details.

In the prior art, the detection of apoptotic cells mainly applies gel electrophoresis technology and flow cytometry, and whether the cells are apoptotic or not is judged according to the molecular change of the cells during apoptosis. Although these techniques provide useful information, they are based on the analysis of the apoptosis end point and do not allow direct and real-time observation of the apoptotic process. At present, the detection of the apoptosis process mainly utilizes the fluorescence imaging technology to detect typical characteristics in the apoptosis process, such as: changes in the cell surface Phosphatidylserine (PS) and mitochondrial membrane potential in the early stages of apoptosis, chromatin fragmentation in the late stages of apoptosis, etc. This type of technique requires determination of apoptosis by multiple results, and cannot detect each stage of apoptosis by a single probe, with the possibility of detecting false positives.

On the other hand, although the advantages of fluorescence imaging technology are outstanding, the disadvantages thereof, such as: the intensity of probe fluorescence (or phosphorescence) strongly depends on concentration, is strongly interfered by biological background fluorescence, is easily photobleached in a short time, emission bands of fluorescent probes tend to overlap, limiting the simultaneous use of multiple probes, and the like. However, fluorescence (or phosphorescence) lifetime is an inherent property of fluorescent materials, and is independent of concentration, photobleaching and excitation intensity, and compared with fluorescence imaging, fluorescence lifetime imaging technology has the advantages of wide fluorescence lifetime interval range, high resolution, small interference from background fluorescence, small overlapping range, simultaneous use and the like, and has gradually become an influential detection means in cell biology.

Disclosure of Invention

The object of the present invention is to provide a method for detecting an apoptotic process based on the FRET effect, aiming to detect an apoptotic process more efficiently and directly.

The technical solution of the invention is as follows: a method for detecting apoptotic processes based on FRET effects comprising the steps of:

s1, preparation of phosphorescent iridium complexes with long lifetimes as donors D: firstly, preparing a product which can perform bio-orthogonal reaction with azide, then connecting the product with p-nitrophenyl chloroformate, reacting the product with a substance containing amino to generate an auxiliary ligand, and then performing coordination reaction on the auxiliary ligand and an iridium dichloro bridge to prepare the phosphorescent iridium complex.

S2, preparing an organic fluorescent small molecular compound as an acceptor A, wherein the service life of the acceptor A is shorter than that of a donor D, and FRET effect exists between the acceptor A and the donor D; firstly, a product which can perform bio-orthogonal reaction with norbornene is prepared and then is connected to the organic fluorescent micromolecule compound with short service life and containing carboxyl through condensation reaction.

S3, respectively connecting the donor D and the receptor A to two ends of a peptide chain to form a D-A type probe through a bioorthogonal reaction, wherein the peptide chain is a peptide chain which can be sheared by caspase only existing in the apoptosis process, and the D-A type probe can be assembled outside cells and then implanted inside the cells or directly assembled inside the cells;

s4, detecting the life change of a donor D in the D-A type probe by using a confocal imaging or life imaging technology, and judging the apoptosis process of the cell; or detecting the life change of the receptor A in the D-A type probe to judge the apoptosis process of the cell.

Further, step S1 is carried out by reacting dibenzosuberone with trimethylsilyldiazomethane at-78 deg.C to obtain product 2; 2 is reduced by sodium borohydride to prepare a product 3; adding liquid bromine to the double bond in the product 3 through addition reaction to obtain a product 4; the product 4 reacts with LDA to prepare a product 5; the product 5 can be substituted with p-nitrophenyl chloroformate to obtain a product 6; oxidizing 4,4 '-dimethyl-2, 2' -bipyridine by selenium dioxide to obtain a product 8; the product 8 reacts with hydroxylamine hydrochloride to obtain a product 9; the product 9 is subjected to ammoniation reaction to prepare a product 10; the product 10 and the product 6 are subjected to substitution reaction to prepare an auxiliary ligand 11; the auxiliary ligand reacts with the phenylquinoline dichloro bridge to prepare the donor Ir-DIBO.

Further, step S2 is a method in which the amino group of p-cyanobenzylamine is protected with di-tert-butyl dicarbonate to obtain product 2; the product 2 reacts with acetonitrile, Zn (OTf)2 and hydrazine monohydrate to prepare a product 3; removing amino protecting groups from the product 3 under the action of concentrated hydrochloric acid to obtain amino tetrazine; the amino group on the amino tetrazine and the carboxyl group on the rhodamine B are subjected to condensation reaction to prepare the receptor tetrazine rhodamine, namely TR.

Further, the donor D has the following structural formula

Wherein the C ^ N ligand is any one of the following:

further, the receptor A has the following structural formula

Wherein A is any one of the following carboxyl-containing organic fluorescent small molecular compounds:

further, the D-A type probe has the following structural formula

Wherein XXXXXX is a connecting peptide chain selected from WEHD, LEHD, DETD, DEVD, DEHD, VEHD, LETD, LEHD.

Further, step S4 is a method, in which the peptide chain connects the long-lived donor and the short-lived acceptor, and the energy of the donor is transferred to the acceptor due to the FRET effect, so that the lifetime of the whole probe is reduced compared with that of the probe in the presence of only the donor; when the cell is in apoptosis, Caspase in the cell is activated, the Caspase cuts off a probe with a specific peptide chain so as to block FRET effect, and the phosphorescence life of the donor is recovered;

the peptide chain connects a long-life donor and a short-life acceptor, and the energy of the donor is transferred to the acceptor due to the FRET effect, so that the life of the whole probe is reduced compared with that of the probe in the presence of only the acceptor; when the cell is in apoptosis, Caspase in the cell is activated, and after the activation, the Caspase cuts off a probe with a specific peptide chain so as to block FRET effect and restore the phosphorescence lifetime of the receptor.

Therefore, the invention provides a brand-new method for detecting the apoptosis process by constructing a D-A type probe with FRET effect, wherein the donor of the probe is an iridium complex with longer service life, the acceptor is an organic fluorescent small molecular compound with shorter service life compared with the iridium complex, and the connecting peptide XXXX is a peptide chain which can be cut by caspase enzyme only existing in apoptosis. When the donor and the acceptor are linked together by a peptide chain, the energy of the donor is transferred to the acceptor due to the FRET effect, resulting in a decrease in the lifetime of the entire probe; when the cell is in the apoptosis stage, caspase is activated, the polypeptide connected with the probe is sheared, the FRET effect disappears, and the phosphorescence life of the donor is recovered, thereby achieving the purpose of detecting the apoptosis process of the cell.

The invention has very important application prospect in the fields of cell imaging, biological marking, sensing and the like, and the technical effects are shown as follows:

(1) the invention can detect the change of the service life of the probe by combining the time resolution technology, and has higher resolution than the traditional fluorescence intensity;

(2) the probe can be excited by visible light, so that the damage of an excitation light source to a biological sample is weakened;

(3) the probe can be applied to the detection of each stage of cell apoptosis, can distinguish each stage of cell apoptosis, can be simultaneously used by multiple probes, does not influence the performance of each other, has good biocompatibility, and is a good biological cell probe;

(4) the probe can be assembled in cells through bioorthogonal reaction, the assembly efficiency is high, and further purification is not needed.

Drawings

FIG. 1 is a UV-visible absorption spectrum and an emission spectrum of donor Ir-DIBO;

FIG. 2 is an image of the phosphorescent lifetime in a cell in the presence of donor Ir-DIBO alone;

FIG. 3 is a UV-VIS absorption spectrum of a rhodamine-containing acceptor;

FIG. 4 is phospholifetime imaging in cells for detection of caspase-3 enzyme probe;

FIG. 5 is life-span imaging after addition of apoptosis-inducing agents (STS);

FIG. 6 is a graph comparing the intracellular phosphorescence lifetime imaging in the presence of donor Ir-DIBO alone and probe phosphorescence lifetime distribution curves before and after apoptosis induced by STS addition.

Detailed Description

Researches show that the Caspase plays an essential role in the apoptosis process, and the Caspase exists in the whole apoptosis process all the time, and the apoptosis process is actually a cascade amplification reaction process of irreversibly hydrolyzing a substrate by the Caspase. In the process of apoptosis, the activation time periods of Caspase are different, and the existence of Caspase directly reflects the stage of apoptosis. The invention utilizes fluorescence (or phosphorescence) life imaging technology to detect and distinguish apoptosis stage by constructing a D-A type probe which is based on FRET effect and can detect whether Caspase exists; meanwhile, by changing the sequence of the connecting peptide chain of the probe, various Caspase enzymes can be detected, the detection accuracy is greatly improved, and the effect of simplifying the detection is achieved.

According to the technical scheme of the invention, the D-A type probe for detecting the apoptosis process is characterized in that a long-life donor and a short-life acceptor are connected together through a specific peptide chain, and the energy of the donor is transferred to the acceptor due to the FRET effect, so that the service life of the whole probe is reduced compared with that of the whole probe in the presence of only the donor. When the cell is apoptotic, Caspase in the cell is activated, the enzyme exists in different stages of the cell apoptosis, a plurality of Caspase enzymes detected at present exist in each stage of the cell apoptosis respectively, namely, the Caspase is activated respectively in different stages of the cell apoptosis; after activation, the enzyme cleaves the probe with the specific peptide chain to block the FRET effect and the phosphorescent lifetime of the donor is restored.

Alternatively, when a specific peptide chain links a long-lived donor and a short-lived acceptor together, the energy of the acceptor is transferred to the donor by FRET effect, thereby decreasing the lifetime of the entire probe compared to the case where only the acceptor is present. When the cell is in apoptosis, Caspase in the cell is activated, and after the activation, the enzyme cuts off a probe with a specific peptide chain so as to block FRET effect and restore the phosphorescence lifetime of the receptor.

Therefore, the cell apoptosis condition can be judged by detecting and comparing the phosphorescence lifetime of the donor or the acceptor by using the phosphorescence lifetime imaging technology.

The D-A type probe for detecting the apoptosis process has the following structural formula:

wherein, the connecting peptide chain XXXX is a peptide chain which can be cut by caspase enzyme only existing in the process of apoptosis, the left side and the right side of the peptide chain are respectively connected with a donor and an acceptor, the donor is a long-life iridium complex, and the acceptor is a short-life organic fluorescent small molecular compound. The linker peptide chain used in this example was a DEVDGK sequence directed against caspase-3 enzyme in late apoptosis, and was synthesized by Shanghai Qianzhan Biotech Co., Ltd.

Specifically, the donor preparation process is: firstly, obtaining a product 5 which can perform bio-orthogonal reaction with azide through a series of reactions, then connecting the product 5 with p-nitrophenyl chloroformate, reacting with a substance 10 containing amino to generate an auxiliary ligand 11, and performing coordination reaction on the auxiliary ligand 11 and an iridium dichloro bridge to prepare the long-life iridium complex serving as a probe donor. The synthetic route is as follows:

the preparation process of the receptor comprises the following steps: firstly, a product 4 which can perform bio-orthogonal reaction with norbornene is generated through a series of reactions, and the product 4 is connected to a short-life organic fluorescent micromolecule compound 6 containing carboxyl through condensation reaction with carboxyl, so that the preparation of the short-life organic fluorescent micromolecule serving as a probe acceptor is completed. The synthetic route is as follows:

the above donor and acceptor lifetimes are significantly different, with the donor lifetime being long and the acceptor lifetime being short. After the donor and the receptor are obtained through synthesis, the donor and the receptor can be injected into cells, and in order to avoid purification, the embodiment adopts an intracellular self-assembly mode; and (3) completing the assembly of the probe in the cell through a bioorthogonal reaction, and then detecting the change of the service life of the probe in the apoptotic cell through a confocal imaging and service life imaging technology. When the cells are assembled, the assembly is rapid and efficient, and further purification is not needed; in addition, during specific application, a proper donor and an acceptor can be selected to improve FRET efficiency, detection of caspase family enzymes can be realized by changing a connecting peptide chain, the detection sensitivity is high, mutual influence is avoided, and the detection can be used simultaneously, so that each stage of apoptosis can be distinguished.

The technical solution of the present invention will be described in detail with reference to the accompanying drawings by way of specific examples. It should be noted that the following are only exemplary embodiments to which the technical concept of the present invention is applied, and all technical solutions formed by equivalent substitutions or equivalent changes are within the scope of the present invention.

Preparation of first, auxiliary ligand

The overall preparative route for ancillary ligand 11 is shown below:

1. preparation of Compound 2

A solution of trimethylsilyldiazomethane (21.9mmol) in dichloromethane (20mL) was slowly added to a solution of dibenzosuberone (14.0mmol) and boron trifluoride etherate (21.0mmol) in dichloromethane (15mL) under nitrogen, the reaction mixture was stirred at-78 deg.C for 2 hours, then poured into ice water, the aqueous phase was extracted with dichloromethane, the organic phase was collected and washed with brine, after drying over anhydrous magnesium sulfate, the magnesium sulfate was filtered off, the organic solvent was removed by rotary evaporation, and the crude product was purified on a silica gel column to give white solid 2, yield: 78 percent. 1H NMR (400MHz, CDCl3) δ 8.26(d, J7.9 Hz,1H),7.52-7.20(m,7H),7.05(q, J16 Hz,12Hz,2H),4.06(s, 2H).

2. Preparation of Compound 3

Sodium borohydride (20mmol) was slowly added to a mixture of compound 2(10mmol) in ethanol and tetrahydrofuran (1:1, v/v, 120mL), stirred at room temperature for 7 hours, acetic acid (1mL) was slowly added to quench the reaction, the solvent was removed by rotary evaporation, the residue was dissolved in dichloromethane (100mL), the resulting solution was washed with brine (100mL) and extracted with dichloromethane, the organic phase was collected and dried over anhydrous magnesium sulfate, the magnesium sulfate was removed by filtration, and the organic solvent was removed by rotary evaporation to give 3 as a white solid (used directly in the next reaction without further purification) in 100% yield. 1H NMR (400MHz, CDCl3) δ 7.46(d, J6.7 Hz,1H), 7.25-7.07 (m,7H),6.85(q, J32 Hz,8Hz,2H),5.29(q, J16 Hz,1H),3.39(q, 2H).

3. Preparation of Compound 4

Liquid bromine (10mmol) was slowly added dropwise to a chloroform (50mL) solution of compound 3(10mmol) under a nitrogen atmosphere, and after stirring at room temperature for 0.5 hour, the organic solvent was removed by rotary evaporation, and the crude product was purified by silica gel column to give 4 as a pale yellow oil in a yield of 45%. 1H NMR (400MHz, CDCl3) δ 7.69-7.38 (m,2H), 7.27-6.88 (m,6H), 5.88-5.76 (m,1H), 5.47-5.41 (m,1H),5.31(dd, J14.4, 5.4Hz,1H), 3.77-3.56 (m,1H), 3.12-2.83 (m, 1H).

4. Preparation of Compound 5

Under nitrogen atmosphere, LDA tetrahydrofuran solution (2.0M; 8mL, 16mmol) was slowly added dropwise to compound 4(4.0mmol) tetrahydrofuran (40mL), stirred at-78 deg.C for 1 hour, transferred to room temperature and stirred for 1 hour, after the reaction was completed, water (0.5mL) was added dropwise to quench the reaction, the organic solvent was removed by rotary evaporation, and the crude product was purified by silica gel column to give 5 as a white solid in 68% yield. 1H NMR (400MHz, CDCl3) δ 7.75(d, J7.8 Hz,1H), 7.47-7.28 (m,7H),4.64(s,1H),3.11(dd, J14.7, 2.2Hz,1H), 2.99-2.83 (m, 1H).

5. Preparation of Compound 6

P-nitrophenyl chloroformate (2mmol) and pyridine (5mmol) were added to a solution of compound 5(1mmol) in dichloromethane (30mL), and after stirring at room temperature for 4 hours, the mixture was washed with brine, the collected organic phase was dried over anhydrous magnesium sulfate, the magnesium sulfate was removed by filtration, the organic solvent was removed by rotary evaporation, and the crude product was purified on a silica gel column to give 6 as a white solid in 82% yield. 1H NMR (400MHz, CDCl3) δ 8.35-8.05 (m,2H),7.62(d, J7.9 Hz,1H), 7.54-7.27 (m,9H),5.58(s,1H),3.34(dd, J15.4, 2.1Hz,1H),3.01(ddd, J16.0, 14.6,3.1Hz, 1H).

6. Preparation of Compound 8

Dissolving 4,4 '-dimethyl-2, 2' -bipyridine (10mmol) and selenium dioxide (10mmol) in 1, 4-dioxane (300mL), heating to 102 ℃, reacting for 24 hours, filtering to remove black solids when the reaction is finished, removing the solvent by rotary evaporation and dissolving in ethyl acetate, filtering to remove the solids by suction again, and removing the solvent by rotary evaporation to obtain a white solid 8 with a yield of 57%.

7. Preparation of Compound 9

Dissolving the compound 8(3mmol) in methanol (10mL), adding hydroxylamine hydrochloride (11mmol), potassium carbonate (15mmol) and water (10mL) respectively, heating to 80 ℃ for reaction for 1 hour, cooling to room temperature after the reaction is completed, pouring into cold water to precipitate a white solid, filtering and recrystallizing in methanol to obtain a white solid 9, wherein the yield is as follows: 90 percent. 1H NMR (400MHz, CDCl3) δ 8.70(d, J5.4 Hz,1H),8.61(s,1H),8.54(d, J5.1 Hz,1H),8.21(d, J12.7 Hz,2H),7.49(dd, J5.1, 1.6Hz,1H),7.18(d, J5.1 Hz,1H),5.34(t, J4.9 Hz,1H),2.46(s, 3H).

8. Preparation of Compound 10

Heating and refluxing a mixed solution of the compound 9(2mmol), ammonium acetate (5mmol), ammonia water (10mmol), ethanol (5mL) and water (5mL) for 3 hours, adding zinc powder (10mmol) in portions after the reaction starts for 30 minutes, cooling to room temperature after the reaction is completed, filtering to remove solids, removing ethanol by rotary evaporation, then adding sodium hydroxide (35mmol), generating a white precipitate, extracting an aqueous phase with dichloromethane, collecting the organic phase, drying with anhydrous magnesium sulfate, filtering to remove magnesium sulfate, and removing an organic solvent by rotary evaporation to obtain a white solid 10 with a yield of 90%. 1H NMR (400MHz, CDCl3) δ 8.62(d, J5.0 Hz,1H),8.53(d, J5.0 Hz,1H),8.33(s,1H),8.23(s,1H),7.29(d, J5.0 Hz,1H),7.14(d, J4.9 Hz,1H),3.96(d, J17.8 Hz,2H),2.44(s, 3H).

9. Preparation of ancillary ligands 11

Compound 6(2mmol) and compound 10(2mmol) were dissolved in dichloromethane (10mL) under nitrogen atmosphere and triethylamine (1mL) was added, stirred at room temperature for 12 hours, extracted with dichloromethane, the organic phase was collected, dried over anhydrous magnesium sulfate, filtered to remove magnesium sulfate, the organic solvent was removed by rotary evaporation and the crude product was purified on silica gel column to give 11 as a white solid in 78% yield. 1H NMR (400MHz, DMSO) δ 8.61-8.49 (m,2H),8.38(t, J6.2 Hz,1H), 8.36-8.19 (m,2H),7.61(d, J7.0 Hz,1H), 7.48-7.22 (m,9H),7.07(s,1H),4.31(t, J7.2 Hz,2H),3.24(d, J13.1 Hz,1H),2.79(dt, J28.3, 22.3Hz,1H),2.40(s, 3H).

Preparation of donor Ir-DIBO with main ligand of benzoquinoline

As in the above formula, 11(0.2mmol) and an iridium dichloro bridge (0.08mmol) were stirred and refluxed at 55 ℃ overnight in a mixed solvent of methylene chloride and methanol (1:3, v/v,4mL) under a nitrogen atmosphere, after which the reaction solution was cooled to room temperature, KPF6(1.4mmol) was added thereto and stirred for 4 hours, after the completion of the reaction, the organic solvent was removed by rotary evaporation, and the resulting solid was purified by column to obtain Ir-DIBO as a yellow powdery solid in a yield of 45%. 1H NMR (400MHz, CDCl3) δ 8.26-7.90 (m,11H), 7.76-7.56 (m,3H),7.33(t, J11.8 Hz,8H), 7.28-7.10 (m,6H),6.98(dq, J21.6, 7.1Hz,2H),6.81(q, J7.1 Hz,2H),6.51(ddd, J26.4, 13.3,5.7Hz,3H), 5.49-5.34 (m,1H), 4.53-4.34 (m,2H),3.23(dd, J13.3, 9.8Hz,1H),2.88(dd, J16.3, 5.0, 1H),2.46(d, J5H, 3H).

Ultraviolet-visible absorption spectrum and emission spectrum of donor Ir-DIBO

The ultraviolet-visible light spectrum and absorption spectrum testing concentrations of Ir-DIBO adopted by the invention are both 10 mu M, the testing solvent is PBS solution containing 1% DMSO with the pH value of 7.2-7.4, and the excitation wavelength is 405nm when the emission spectrum is tested.

The UV-VIS absorption and emission spectra of Ir-DIBO are shown in FIG. 1. The solid line in the figure is the ultraviolet-visible light absorption spectrum of the complex, and it can be seen that the complex shows stronger absorption in both the ultraviolet region of 250-380nm and the visible blue region of 400-500nm, and particularly the complex can be excited by visible light, so that the damage of the excitation light source to cells is greatly reduced when cell imaging experiments are performed; the dotted line is the emission spectrum of the complex, and it can be seen that the emission peak is broad, with the peak occurring at 588 nm.

Imaging of phosphorescent lifetime in cells in the presence of donor Ir-DIBO alone

The cells adopted by the invention are all human cervical carcinoma HeLa cells. The digested cells were seeded in petri dishes at 37 ℃ with 5% CO₂The culture was continued for 24 hours under the conditions of (1) to allow adhesion. Cells were washed out of necrotic tissue with PBS solution, incubated with cell culture medium of Ir-DIBO (5. mu.M, 1% DMSO) for 2 hours, washed three times with PBS solution and imaged for phosphorescence lifetime.

Images of the phosphorescent lifetime in cells in the presence of donor Ir-DIBO alone are shown in figure 2. The complex Ir-DIBO is excited by a 405nm laser, and the emission in the 550-650nm interval is collected, as shown in (a) in FIG. 2, the phosphorescence is uniformly distributed in cytoplasm, and the material is shown to enter the cells; (b) for the phosphorescence lifetime profile of the material measured by FLIM technique, the peak is found at 688ns, so the phosphorescence lifetime τ of Ir-DIBO in the cell can be considered as 688 ns.

Preparation of penta-and aminotriazines (as shown below)

1. Preparation of Compound 2

A solution of p-cyanobenzylamine (10mmol) in water (10mL) was added to a solution of NaOH (30mmol) and di-tert-butyl dicarbonate (10mmol) in water (10mL) and stirred at room temperature for 16 hours, a white precipitate formed, which was filtered and the white residue washed with water (50mL) and then dried under vacuum to give the white product 2 in 100% yield. 1H NMR (400MHz, CDCl3) δ 8.54(d, J8.4 Hz,2H),7.49(d, J8.3 Hz,2H),5.01(s,1H),4.43(d, J5.8 Hz,2H),3.09(s,3H),1.45(d, J23.4 Hz, 9H).

2. Preparation of Compound 3

Under a nitrogen atmosphere, Compound 2(2mmol), acetonitrile (39.25mmol), Zn (OTf)₂(0.96mmol) and hydrazine monohydrate (96mmol) were mixed together, heated to 70 ℃ for reaction for 72h, cooled to room temperature after completion of the reaction, sodium nitrite (39mmol) was dissolved in 5ml of water and slowly added dropwise to the above solution, followed by adjustment of pH 3 with 1M HCl, extraction with dichloromethane, collection of the organic phase, drying over anhydrous magnesium sulfate, filtration to remove magnesium sulfate, rotary evaporation to remove the organic solvent, and purification of the crude product over a silica gel column to give 3 as a reddish-purple solid in 40% yield. 1H NMR (400MHz, CDCl3) δ 8.55(d, J8.4 Hz,2H),7.50(d, J8.4 Hz,2H),5.00(s,1H),4.43(d, J5.7 Hz,2H),3.09(s,3H),1.45(d, J23.2 Hz, 9H).

3. Preparation of Compound 4

A4M solution of hydrochloric acid (8.0mmol) was added to a solution of compound 3(0.26mmol) in dichloromethane (4mL), stirred for 1 hour, and the organic solvent was removed by rotary evaporation to give a magenta solid 4, (i.e., aminotetrazine), in 100% yield. 1H NMR (400MHz, D2O) δ 8.32(t, J6.9 Hz,2H),7.57(D, J8.3 Hz,2H),4.15(D, J23.1 Hz,2H),2.85(D, J66.1 Hz, 3H).

Preparation of hexa-receptor Tetrazine Rhodamine (TR)

As shown above, compound 5(Rhodamine) (0.6mmol), 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) (0.6mmol) and 4-Dimethylaminopyridine (DMAP) (0.4mmol) were dissolved in 5mL of dichloromethane, stirred at room temperature for 30 minutes, then added to a solution of 4 (aminotetrazine, 0.4mmol) in N, N-2-methylformamide, stirred at room temperature for 24 hours, extracted with water and dichloromethane, the organic phase was collected, dried over anhydrous magnesium sulfate, filtered to remove magnesium sulfate, rotary evaporated to remove the organic solvent, and the crude product was purified by silica gel column to give the final product 6 (i.e., acceptor tetrazine Rhodamine) in 30% yield. 1H NMR (400MHz, CDCl3) δ 8.19(dd, J8.5, 1.9Hz,2H), 8.01-7.94 (m,1H), 7.52-7.41 (m,2H),7.17(dd, J8.5, 4.0Hz,2H), 7.14-7.09 (m,1H),6.26(dd, J11.0, 5.7Hz,4H),6.04(dt, J8.6, 2.7Hz,2H),4.40(s,2H),3.21(q, J7.1 Hz,8H),3.05(s,3H),1.03(t, J7.1 Hz, 12H).

Seventhly, ultraviolet-visible absorption spectrum of receptor Tetrazine Rhodamine (TR)

The Tetrazine Rhodamine (TR) spectrum test concentration adopted by the invention is 2mM, and the test solvent is PBS mixed with 1% DMSO, and the pH value of the PBS is 7.2-7.4.

The ultraviolet-visible absorption spectrum of the receptor Tetrazine Rhodamine (TR) is shown in FIG. 3. From the figure, it can be seen that the organic fluorescent small molecule shows stronger absorption at 450-600nm, and the peak appears at 540 nm. The absorption spectrum of the acceptor TR has an overlap with the emission spectrum of the donor Ir-DIBO, the condition of 'donor-acceptor pair' is satisfied, and FRET effect exists between the TR and the Ir-DIBO, so that energy can be transferred from the donor to the acceptor.

The self-assembly of the probe is accomplished by the bioorthogonal reaction of the donor and the acceptor in the cell as shown in the following figure:

eighth, phospho-luminescence lifetime imaging of probes for detecting caspase-3 enzyme in cells

Inoculating the digested HeLa cells of human cervical cancer into a culture dish, and culturing at 37 deg.C with 5% CO₂The culture was continued for 24 hours under the conditions of (1) to allow adhesion. Necrotic cells were washed with PBS solution and incubated with cell culture media with caspase-3 probe (10. mu.M, 2% DMSO)Cells were washed three times with PBS solution for 3 hours and then imaged for phosphorescence lifetime.

Phospholifetime imaging of probes used to detect caspase-3 enzyme in cells is shown in FIG. 4. The complex Ir-DIBO is excited by a 405nm laser, and the emission at 550-650nm is collected, as shown in (a) in FIG. 4, the phosphorescence is uniformly distributed in the cytoplasm, which shows that the material enters the cells; (b) for the phosphorescence lifetime profile of the material, which can be measured by FLIM technique, it can be seen that the peak is 513ns, and therefore, the phosphorescence lifetime τ of Ir-DIBO in the cell can be regarded as 513 ns.

Nine, imaging lifetime after addition of apoptosis inducer Staurosporine (STS)

Inoculating the digested HeLa cells of human cervical cancer into a culture dish, and culturing at 37 deg.C with 5% CO₂The culture was continued for 24 hours under the conditions of (1) to allow adhesion. After washing the necrotic cells with PBS solution, the cells were incubated with a cell culture solution of caspase-3 probe (10. mu.M, 2% DMSO) for 3 hours, then 2. mu.L of STS (1. mu.M) was added and incubated for 10 hours, and after washing three times with PBS solution, phosphorescence lifetime imaging was performed.

Lifetime imaging after addition of apoptosis-inducing agent (STS) is shown in FIG. 5. caspase-3 probe was excited by 405nm laser and collected at 550-650nm emission, as shown in FIG. 4 (a), the phosphorescence was uniformly distributed in the cytoplasm, indicating that the material had entered the cell; (b) in order to measure the phosphorescence lifetime distribution of the material by FLIM technology, it can be seen that the peak value is 668ns, because cell apoptosis is induced after STS is added, caspase-3 enzyme in the cell is activated, the peptide chain sequence in the probe is cut, the FRET effect existing between donor and acceptor is blocked, and the lifetime is prolonged, and it can be seen from the figure that tau is 668 ns.

The phosphorescent lifetime in cells imaged in the presence of donor Ir-DIBO alone and the probe phosphorescent lifetime distribution before and after apoptosis induced by STS addition are shown in fig. 6. As is evident from the figure, the phosphorescent lifetime in the cell is highest in the presence of donor Ir-DIBO alone, corresponding to the dashed line in the figure, τ 688 ns; when Ir-DIBO and TR are connected together by a DEVDGK peptide chain, due to the FRET effect, the energy of Ir-DIBO is transferred to TR, so that the service life of the Ir-DIBO is shortened, and the service life tau is 513ns corresponding to a solid line in the figure; when the apoptosis inducer STS is added, apoptosis is induced, caspase-3 enzyme is activated, and specific cleavage is performed on the peptide chain DEVDGK, so that FRET effect is blocked, energy transfer is eliminated, the service life is prolonged, and at the moment, tau is 668ns corresponding to a triangular line in the figure.

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited to the above embodiment, but equivalent modifications or changes made by those skilled in the art according to the present disclosure should be included in the scope of the present invention as set forth in the appended claims.

Claims

1. A method for detecting apoptotic processes based on FRET effects comprising the steps of:

s1, preparing a phosphorescent iridium complex with long service life as a donor D;

s2, preparing an organic fluorescent small molecular compound as an acceptor A, wherein the service life of the acceptor A is shorter than that of a donor D, and FRET effect exists between the acceptor A and the donor D;

s3, respectively connecting the donor D and the receptor A to two ends of a peptide chain to form a D-A type probe through a bioorthogonal reaction, wherein the peptide chain is a peptide chain which can be sheared by caspase only existing in the apoptosis process, and the D-A type probe is assembled outside cells and then implanted into the cells or directly assembled inside the cells;

2. Method for detecting apoptotic processes based on FRET effect according to claim 1, characterized in that: the method of the step S1 is that firstly, a product which can generate bio-orthogonal reaction with azide is prepared, then the product is connected with p-nitrophenyl chloroformate, and then the product reacts with a substance containing amino to generate an auxiliary ligand, and the auxiliary ligand and an iridium dichloro bridge are subjected to coordination reaction to prepare the phosphorescent iridium complex.

3. Method for detecting apoptotic processes based on FRET effect according to claim 2, characterized in that: the method of step S1 is that dibenzosuberone is used as raw material, and reacts with trimethylsilyl diazomethane at-78 ℃ to obtain product 2; 2 is reduced by sodium borohydride to prepare a product 3; adding liquid bromine to the double bond in the product 3 through addition reaction to obtain a product 4; the product 4 reacts with LDA to prepare a product 5; the product 5 can be substituted with p-nitrophenyl chloroformate to obtain a product 6; oxidizing 4,4 '-dimethyl-2, 2' -bipyridine by selenium dioxide to obtain a product 8; the product 8 reacts with hydroxylamine hydrochloride to obtain a product 9; the product 9 is subjected to ammoniation reaction to prepare a product 10; the product 10 and the product 6 are subjected to substitution reaction to prepare an auxiliary ligand 11; the auxiliary ligand reacts with the phenylquinoline dichloro bridge to prepare the donor Ir-DIBO.

4. Method for detecting apoptotic processes based on FRET effect according to claim 1, characterized in that: the method of step S2 is to prepare a product that can undergo bio-orthogonal reaction with norbornene, and then to attach it to the short-lived organic fluorescent small molecule compound having a carboxyl group through condensation reaction.

5. Method for detecting apoptotic processes based on FRET effect according to claim 4, characterized in that: step S2 is a process in which di-tert-butyl dicarbonate is used to protect the amino group on p-cyanobenzylamine to produce product 2; the product 2 reacts with acetonitrile, Zn (OTf)2 and hydrazine monohydrate to prepare a product 3; removing amino protecting groups from the product 3 under the action of concentrated hydrochloric acid to obtain amino tetrazine; the amino group on the amino tetrazine and the carboxyl group on the rhodamine B are subjected to condensation reaction to prepare the receptor tetrazine rhodamine, namely TR.

6. Method for detecting apoptotic processes based on FRET effect according to claim 1, characterized in that: the donor D has the following structural formula I or II

Wherein the C ^ N ligand is any one of the following:

7. method for detecting apoptotic processes based on FRET effect according to claim 1, characterized in that: the receptor A has the following structural formula

8. method for detecting apoptotic processes based on FRET effect according to claim 1, characterized in that: the D-A type probe has the following structural formula