Host-guest supramolecular probe for accelerating nitroreductase detection and its application

Abstract

Host-guest supramolecular probes and applications for accelerating nitroreductase detection belong to the fields of organometallic supramolecular, bioinorganic and biofluorescent probes. The supramolecular host-guest probe platform, which is assembled based on the metal organic cage main structure constructed based on the cofactor structure and the nitroreductase small molecule fluorescent probe, can be used for the rapid quantitative detection of nitroreductase in tumors, simplifying the enzyme-catalyzed double substrate process to a single substrate process. The traditional fluorescent sensor that depends on the cofactor concentration is no longer interfered by the cofactor, and has a rapid fluorescent signal response to the detection of the target enzyme. There is a linear relationship between the fluorescence emission change of the host-guest probe and the nitroreductase concentration, and compared with the simple small molecule probe, the host-guest probe shows better sensitivity to the target enzyme. This host-guest supramolecular fusion strategy provides an effective tool for the new rapid quantitative detection of nitroreductase and provides a new method for biological tracing.

Description

Technical Field

[0001] The invention belongs to the field of metal organic supramolecular, bioinorganic and biofluorescent probes, and specifically relates to a preparation method of a host-guest supramolecular probe for rapid quantitative detection of nitroreductase and its application in solution, cell and living body detection. Background Art

[0002] Tissue hypoxia is an important physiological indicator of many tumors and is closely related to various physiological activities such as tumor growth, infection, and spread and metastasis. Tumor tissue hypoxia causes a slightly acidic growth environment (pH 6.5-7.0) for cancer cells, and hypoxia can lead to abnormal expression of various hypoxia enzymes related to it. In hypoxic tumor tissues, nitroreductase is at an overexpression level, and its content and activity are higher than those of normal cells. At present, many fluorescent probes targeting nitroreductase have been developed for tumor cell imaging and cancer diagnosis. These fluorescent probes are reduced and activated by nitroreductase overexpressed in hypoxic tumor cells, producing obvious changes in fluorescence signals that are different from normal cells, thereby achieving early diagnosis of cancer. Nitroreductase consumes the cofactor nicotinamide adenine dinucleotide (NADH) through a "ping-pong" mechanism to reduce a variety of nitro compounds, which is the basic principle for designing these nitroreductase fluorescent probes. Although great progress has been made in the research on fluorescent probes for evaluating nitroreductase content, they usually require tens of minutes of reaction to reach fluorescence emission equilibrium even when there is a large amount of excess NADH (tens to hundreds of times the molar amount) in the solution. At this point, it will obviously affect the detection efficiency and accuracy of nitroreductase. The concentration and expression level of nitroreductase usually vary greatly in different cells and tumors, and even in the same cells. When a certain amount of traditional nitro fluorescent probe is used for detection, the unknown concentration of cofactor NADH and nitroreductase, two variables, will cause the probe emission to change significantly over time. It is difficult for traditional probes to exclude the interference of NADH concentration on the detection results, and the enzyme content and fluorescence response do not show a stable linear relationship. Therefore, it is still a challenging subject for traditional probe methods to detect nitroreductase content and accurately distinguish the changes in enzyme activity in normal and disease states. Summary of the invention

[0003] The purpose of the present invention is to propose a host-guest supramolecular probe for rapid quantitative detection of nitroreductase in hypoxic tumors, including its preparation method and biological test application, a metal organic cage complex as a NADH mimetic fused with a guest molecule to form a quasi-molecule to react with nitroreductase, and a strategy to change the original ping-pong mechanism double substrate reaction into a single substrate reaction. By reducing the types of substrates and shortening the distance between each substrate, the reaction kinetics are changed.

[0004] The technical solution of the present invention is as follows:

[0005] A nitroreductase fluorescent substrate, the structural formula of the fluorescent substrate is:

[0006]

[0007] in, A method for preparing a nitroreductase fluorescent substrate comprises the following steps:

[0008]

[0009] (1) Using acenaphthenequinone and 4-nitro-o-phenylenediamine as raw materials, preparing the compound shown in Intermediate 1; the molar ratio of acenaphthenequinone:4-nitro-o-phenylenediamine is 1:1-1.1;

[0010] (2) Using intermediate 1, stannous chloride and 37% hydrochloric acid as raw materials and concentrated hydrochloric acid as solvent, heating under reflux to prepare synthetic intermediate 2, wherein the molar ratio of intermediate 1 to stannous chloride is 1:4-6;

[0011] (3) Using intermediate 2 and nitro-substituted product as raw materials and carbonate as base, the nitroreductase fluorescent substrate was prepared in DMF at room temperature;

[0012] The nitro substituent is p-nitrobenzyl bromide, 2-(chloromethyl)-5-nitrothiophene, 2-(chloromethyl)-5-nitrofuran or 1-(2-chloroethyl)-2-nitro-1H-imidazole;

[0013] The molar ratio of the intermediate 2: nitro substituent: carbonate is 1:1.1-1.3:2-2.2;

[0014] The host-guest supramolecular probe comprises a host part and a guest part, wherein the guest part is the fluorescent substrate according to claim 1. The host part is a metal organic cage complex, wherein the metal ion in the metal organic cage complex is a zinc ion;

[0015] The structure of the ligand precursor compound DPM of the metal organic cage complex is as follows:

[0016]

[0017] The preparation method of the ligand precursor compound DPM is as follows:

[0018]

[0019] (1) Using 4-bromobenzoic acid and 2-morpholinoethanolamine as raw materials, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine as catalysts, compound intermediate 3 was prepared;

[0020] The molar ratio of 4-bromobenzoic acid: 2-morpholinoethanolamine: N, N'-dicyclohexylcarbodiimide: 4-dimethylaminopyridine is 1.1-1.3: 1: 1.5-1.7: 1-1.2;

[0021] (2) using methyl propiolate, benzaldehyde and ammonium acetate as raw materials to prepare compound intermediate 4; the molar ratio of ammonium acetate:methyl propiolate:benzaldehyde is 4-4.4:2-2.2:1;

[0022] (3) Using intermediate 3, intermediate 4 and a base as raw materials, a compound represented by intermediate 5 is prepared in an organic solvent;

[0023] The base is sodium carbonate, potassium carbonate, cesium carbonate or an organic base;

[0024] The organic solvent is selected from acetone, N,N-dimethylformamide or 1,4-dioxane;

[0025] The molar ratio of intermediate 3:intermediate 4 is 1.1-1.3:1;

[0026] (4) reacting the intermediate 5 with 80% hydrazine hydrate to obtain a compound represented by a ligand precursor compound DPM; the molar ratio of the intermediate 5 to 80% hydrazine hydrate is 1:5 to 1:10;

[0027] The metal organic cage complex is of Zn3DPM3 type.

[0028] The preparation method of the metal organic cage complex is:

[0029] A self-assembly reaction was carried out using a ligand precursor compound DPM, 2-formylpyridine and zinc perchlorate hydrate, wherein the molar ratio of DPM:2-formylpyridine:zinc perchlorate hydrate was 1:2:1.

[0030] The molar ratio of the host part to the guest part was 1:1.

[0031] The host-guest supramolecular probe is applied to the quantitative detection of nitroreductase.

[0032] The host-guest supramolecular probe is used to prepare a reagent for detecting nitroreductase

[0033] Based on the host-guest supramolecular probe structure, basic ligand building blocks and enzyme catalytic reactions, e.g. Figure 1 shown.

[0034] Among them, host-guest probe The invention comprises a metal organic cage complex main molecule part (Zn-MPPB) and a nitroreductase fluorescent substrate guest molecule part (NAQA). The main body is composed of a ligand precursor (DPM), 2-formylpyridine and a metal ion (M n+). The guest part is connected by a fluorophore and a p-nitrobenzyl group (Formula I). ​​The reaction principle of the probe: Under the action of nitroreductase, the nitrobenzene part of the host-guest probe is reduced to hydroxylamine benzene, and the structure of the fluorescent substrate changes significantly, so the corresponding fluorescence response is turned on. The metal organic cage complex, as a NADH mimetic, fuses with the substrate to form a quasi-molecular structure, catalyzes the reaction with the enzyme, and changes the double substrate reaction of the ping-pong mechanism into a single substrate reaction. By reducing the types of substrates and shortening the distance between the substrates, the reaction kinetics are changed.

[0035] The dihydropyridine part of the ligand precursor DPM has a similar structure to NADH and serves as the active part of the NADH mimetic for proton and electron transfer;

[0036] The method for preparing the metal organic cage complex comprises the following steps:

[0037]

[0038] (1) Using 4-bromobenzoic acid and 2-morpholinoethanolamine as raw materials, N,N'-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) are used to catalyze esterification in a dichloromethane solution to prepare the compound represented by intermediate 3; the molar ratio of 4-bromobenzoic acid: 2-morpholinoethanolamine: DCC: DMAP is 1.1:1:1.5:1;

[0039] (2) Using methyl propiolate, benzaldehyde, ammonium acetate and glacial acetic acid as raw materials to prepare the compound represented by intermediate 4; the molar ratio of ammonium acetate: methyl propiolate: benzaldehyde is 4:2:1;

[0040] (3) Using intermediate 3, intermediate 4, a base (excess, ≥ 2 eq) and an organic solvent as raw materials to prepare the compound shown in intermediate 5; the base is sodium carbonate, potassium carbonate, cesium carbonate or an organic base; the organic solvent is selected from acetone, N,N-dimethylformamide or 1,4-dioxane: the molar ratio of intermediate 3:intermediate 4 is 1.1:1;

[0041] (4) reacting the intermediate 5 with 80% hydrazine hydrate to obtain the compound shown in DPM; the molar ratio of the intermediate 5 to 80% hydrazine hydrate is 1:5 to 1:10;

[0042] (5) A self-assembly reaction is carried out with DPM, 2-formylpyridine and zinc perchlorate hydrate to obtain a final metal organic cage complex Zn-MPPB; the molar ratio of DPM:2-formylpyridine:zinc perchlorate hydrate is 1:2:1.

[0043] In the guest structure, the fluorophore part of the fluorescent substrate of the enzyme-catalyzed reaction can be, but is not limited to, acenaphthene[1,2-b]quinoxaline group, etc.;

[0044] The method for preparing the fluorescent substrate comprises the following steps:

[0045]

[0046] (1) Using acenaphthenequinone and 4-nitro-o-phenylenediamine as raw materials, preparing the compound shown in Intermediate 1; the molar ratio of acenaphthenequinone:4-nitro-o-phenylenediamine is 1:1;

[0047] (2) The intermediate 1, stannous chloride and 37% hydrochloric acid were used as raw materials and concentrated hydrochloric acid was used as solvent. The structure of the synthetic intermediate 2 was prepared by heating under reflux as a nitroreductase fluorescent substrate. The molar ratio of the intermediate 1 to the stannous chloride was 1:4.

[0048] (3) Using the intermediate 2 shown, p-nitrobenzyl bromide (or 2-(chloromethyl)-5-nitrothiophene, 2-(chloromethyl)-5-nitrofuran, 1-(2-chloroethyl)-2-nitro-1H-imidazole and other groups that can be activated by nitroreductase reduction) as raw materials, potassium carbonate or cesium carbonate as a base, react in DMF at room temperature to prepare a structure of formula I or formula II as a nitroreductase fluorescent substrate; the molar ratio of the intermediate 2: p-nitrobenzyl bromide: potassium carbonate is 1:1.1:2.

[0049] The host-guest supramolecular probe preparation method comprises the following steps:

[0050] 1) Dissolving a dihydropyridine ligand precursor DMP, 2-formylpyridine and a desired coordinated zinc perchlorate hydrate in an acetonitrile solvent respectively and mixing them thoroughly, precipitating a metal organic complex by solvent evaporation crystallization, solvent diffusion crystallization or adding a small polar miscible solvent, and confirming the structure M3N3 by nuclear magnetic resonance, mass spectrometry or single crystal structure, wherein M is a metal ion and N is a dihydropyridine ligand;

[0051] 2) Mixing the above metal organic complex and nitroreductase fluorescent substrate in a solution to obtain a host-guest probe, and performing one-dimensional nuclear magnetic resonance (NMR) 1 H- 1 H NOESY spectrum, 2D 1 H- 1 H NOESY, 1 H- 1 The host-guest formation was confirmed by H DOSY and other nuclear magnetic resonance methods. Meanwhile, high-resolution mass spectrometry, host-guest UV titration, job's plot and other methods were used to verify the stable formation of the host-guest probe.

[0052] The host-guest probe is used to detect nitroreductase in a solvent.

[0053] A host-guest supramolecular probe for rapid quantitative detection of nitroreductase in solvents. Test method: Add host-guest probe (0-5μM) to the newly configured nitroreductase HEPES buffer (10.0mM, pH 6.8, 25℃) to conduct fluorescence titration experiments, UV titration and fluorescence kinetic experiments. The supramolecular probe formed by the host metal organic cage with cofactor NADH function and the substrate molecule with fluorescence indication function fuses the substrate and coenzyme mimetic into a quasi-molecular structure, so that the original double substrate catalytic process of nitroreductase on coenzyme NADH and nitro substrate is optimized to a single substrate process. The detection of nitroreductase achieves rapid equilibrium and is no longer affected by NADH concentration. Therefore, the change in the detected fluorescence intensity is only related to the change in the nitroreductase content, realizing rapid quantitative detection of nitroreductase.

[0054] The host-guest probe is used to detect nitroreductase in cells.

[0055] A host-guest supramolecular probe for rapid quantitative detection of nitroreductase in cells, the test method: DMEM culture medium (added with 10% FBS, 1% double antibody) is used to culture MCF-7 and 231 cells, and 1640 culture medium (added with 10% FBS, 1% double antibody) is used to culture A549 cells. After culturing cells for 6-12 hours under normoxic conditions, Anaero Pack and Anaero Pouch-Micro Aero (Mitsubishi Gas Chemical Company, Inc.) with different oxygen content environments (20%, 8%, 0.1%), the host-guest supramolecular probe is used for incubation for 5 minutes, and laser confocal fluorescence imaging experiments of cells under different oxygen contents are performed. Cells are cultured for 6-12 hours under the same oxygen content environment (for example, all at 0.1% O2), and then the host-guest supramolecular probe is used for different incubation times (such as 0-20 minutes) to carry out cell fluorescence imaging over time experiments.

[0056] The host-guest probe is used to detect nitroreductase in living animals.

[0057] A type of host-guest supramolecular probe for rapid quantitative detection of nitroreductase in living mice. The test method is: use the required cancer cells (A549, MCF-7) to model tumors in nude mice, and inject the host-guest supramolecular probe described in the above into the mice during the tumor formation period (the general injection concentration and range are 10-200μM, 50-200μL), and conduct an imaging experiment of the mouse tumor fluorescence changes over time.

[0058] Beneficial effects of the present invention:

[0059] By introducing NADH, the most common cofactor in anaerobic enzymes, into the framework of the metal organic cage Zn-MPPB, the present invention proposes a supramolecular probe based on Used for bio-tracing imaging of the catalytic activity of nitroreductases in aqueous solutions and biological systems. As a quasi-molecular structure, it can directly react with nitroreductase, optimizing the dual-substrate enzyme catalysis process of natural nitroreductase, nitro substrate and NADH into a simpler single-substrate catalysis process. This method eliminates the interference of different concentrations on the detection results in the detection of hypoxic enzymes, and the linear relationship between fluorescence intensity and enzyme content is expected to reach a rapid equilibrium. At the same time, including the substrate in the main cage containing NADH can reduce the transport time of external NADH and shorten the distance between the substrate and the cofactor active site, thereby enabling a rapid signal response to the content level of hypoxic enzymes.

[0060] The host-guest supramolecular strategy can enhance the response speed of the probe and have stronger detection capabilities. Through rational design, multifunctional metal-organic cages with hydrophobic cavities of a certain size and unique chemical conversion capabilities, together with guest probe molecules, interact with nitroreductase, which will eliminate the excessive demand for NADH, promote the delivery of the probe, and accelerate the response to the enzyme.

[0061] NADH mimics are integrated into the framework of metal-organic cages to give the metal-organic cages the ability of NADH. Specific nitroreductase fluorescent probes are screened as guest molecules, and NADH mimic metal-organic cages are used as host molecules. The two are assembled to form host-guest probes, which can effectively promote the electron transfer process between the host cage and the guest molecules. Through hydrophilic and hydrophobic interactions and intermolecular interactions, hydrophobic guest molecules are stably encapsulated in the hydrophobic cavity of the metal-organic cage, forming a quasi-molecular structure together. As a whole, the quasi-molecular structure reacts with nitroreductase through a single substrate process, which increases the reaction rate. Traditional small molecule probes and NADH react with nitroreductase through a "ping-pong" mechanism double substrate reaction process, which takes more time. The cage framework is fine-tuned by introducing amide and morpholine groups to increase hydrogen binding sites, improve water solubility and biocompatibility, and thus better interact with the probe and nitroreductase.

[0062] To some extent, the metal-organic cage can be used as an efficient carrier to transfer the probe to the nitroreductase, and can also be used as a cofactor mimic of the nitroreductase to reduce the probe, demonstrating the versatility and key connection of the metal-organic cage to the guest probe molecule and the nitroreductase. These advantages weaken the interference of the cofactor NADH on the nitroreductase assay results, speed up the detection efficiency, and enable the host-guest probe to quickly and accurately quantify the nitroreductase, which can be used to distinguish and quantify the different enzyme activities in cells under normal and disease conditions.

[0063] In the present application, the metal-organic cage and the guest molecule are significantly optimized and adjusted compared with the previous structure. Each ligand of the metal-organic cage introduces a p-formamide phenyl part, which increases the tail chain of the ligand and expands the cavity size of the metal-organic cage. At the same time, the introduced amide group increases the hydrogen bonding site. The above optimizes the cavity size and increases the hydrogen bonding site to better accommodate the guest molecule. The guest substrate molecule is connected to the nitroreductase trigger group by introducing a larger, more rigid, and more sensitive fluorescent signal response large conjugated planar fluorophore structure. Before and after the enzyme reaction, the fluorescence signal growth multiple is greatly enhanced compared with before, so that the detection effect and sensitivity are better. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Schematic diagram of the basic building block of the host-guest probe and its rapid response to nitroreductase in vivo.

[0065] Figure 2 yes One Dimension 1 H- 1 H NOESY and 2D 1 H- 1 H NOESY spectrum.

[0066] Figure 3 yes of 1 H- 1 H DOSY spectra, Zn-MPPB and High-resolution mass spectrum.

[0067] Figure 4 These are the UV titration diagrams of Zn-MPPB and NAQA and the UV titration diagrams of Zn-MPPB and enzyme molecules.

[0068] Figure 5 Zn-MPPB and Fluorescence stability graph.

[0069] Figure 6 yes Titration experiment and kinetic experiment diagram of the reaction of NAQA-NADH with nitroreductase respectively.

[0070] Figure 7 yes The double reciprocal Michaelis-Menten equation diagrams of the reactions of NAQA-NADH and nitroreductase respectively.

[0071] Figure 8 Various interferences Selectivity and competition plots for the reaction with nitroreductase.

[0072] Fig. 9 The figure is the MTT experiment analysis of the toxicity of Zn-MPPB and NAQA to A549 and MCF-7 cells.

[0073] Fig.10 yes Fluorescence imaging of MCF-7 cells treated with the same incubation time but different hypoxia treatments and the same hypoxia but different incubation time in the control group.

[0074] Fig.11 yes Flow cytometric analysis of A549 and MCF-7 cells in the control group.

[0075] Fig.12 It is an injection And the NAQA fluorescence imaging of MCF-7 tumor-bearing mice over time. DETAILED DESCRIPTION

[0076] The present invention proposes a structure-based host-guest supramolecular probe for a strategy for rapid quantitative detection of nitroreductase in hypoxic tumors. The host-guest probe exhibits rapid quantitative detection characteristics in solvent, cell and in vivo experiments. The present invention is further described below in conjunction with examples.

[0077] A dihydropyridine ligand hydrazide precursor DPM for assembling a NADH mimetic metal organic cage complex, wherein the dihydropyridine active group has the function of NADH transmitting electrons, and the hydrazide group is easy to coordinate and assemble with metal ions and 2-aldehyde pyridine. The ligand precursor is the compound shown in the following figure DPM:

[0078]

[0079] A method for preparing a metal organic cage complex for NADH mimics comprises the following steps:

[0080]

[0081] (1) Using 4-bromobenzoic acid and 2-morpholinoethanolamine as raw materials, N,N'-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) are used to catalyze esterification in a dichloromethane solution to prepare the compound represented by intermediate 3; the molar ratio of 4-bromobenzoic acid: 2-morpholinoethanolamine: DCC: DMAP is 1.1:1:1.5:1;

[0082] (2) Using methyl propiolate, benzaldehyde, ammonium acetate and glacial acetic acid as raw materials to prepare the compound represented by intermediate 4; the molar ratio of ammonium acetate: methyl propiolate: benzaldehyde is 4:2:1;

[0083] (3) Using intermediate 3, intermediate 4, a base (excess, >2 eq) and an organic solvent as raw materials to prepare the compound shown in intermediate 5; the base is sodium carbonate, potassium carbonate, cesium carbonate or an organic base; the organic solvent is selected from acetone, N,N-dimethylformamide or 1,4-dioxane: the molar ratio of intermediate 3:intermediate 4 is 1.1:1;

[0084] (4) reacting the intermediate 5 with 80% hydrazine hydrate to obtain the compound shown in DPM; the molar ratio of the intermediate 5 to 80% hydrazine hydrate is 1:5 to 1:10;

[0085] (5) A self-assembly reaction is carried out with DPM, 2-formylpyridine and zinc perchlorate hydrate to obtain a final metal organic cage complex Zn-MPPB; the molar ratio of DPM:2-formylpyridine:zinc perchlorate hydrate is 1:2:1.

[0086] : An M3N3 type metal-organic cage complex assembled from a dihydropyridine ligand and a metal ion, wherein M is a metal ion and N is a dihydropyridine ligand.

[0087] The host-guest probe uses an NADH analog metal organic cage complex as a main part and a nitro fluorescent substrate of nitroreductase as a guest part; the host-guest probe is assembled from the NADH analog metal organic cage complex and the nitro fluorescent substrate, and the molar ratio of the NADH analog metal organic cage complex to the nitroreductase fluorescent substrate is 1:1.

[0088] The nitroreductase fluorescent substrate has a structure of Formula I or Formula II, and the preparation method of the nitroreductase fluorescent substrate is:

[0089]

[0090] (1) Using acenaphthenequinone and 4-nitro-o-phenylenediamine as raw materials, preparing the compound shown in Intermediate 1; the molar ratio of acenaphthenequinone:4-nitro-o-phenylenediamine is 1:1;

[0091] (2) Using intermediate 1, stannous chloride and 37% hydrochloric acid as raw materials and concentrated hydrochloric acid as solvent, heating under reflux to prepare the structure of synthetic intermediate 5 as a nitroreductase fluorescent substrate; the molar ratio of intermediate 4 to stannous chloride is 1:4.

[0092] (3) Using the intermediate 2 shown, p-nitrobenzyl bromide (or 2-(chloromethyl)-5-nitrothiophene, 2-(chloromethyl)-5-nitrofuran, 1-(2-chloroethyl)-2-nitro-1H-imidazole and other groups that can be activated by nitroreductase reduction) as raw materials, potassium carbonate or cesium carbonate as a base, a structure of formula I or formula II is prepared in DMF at room temperature as a nitroreductase fluorescent substrate; the molar ratio of the intermediate 2: p-nitrobenzyl bromide: potassium carbonate is 1:1.1:2.

[0093] Preparation method of host-guest probe assembled from metal-organic cage complex and nitroreductase fluorescent substrate:

[0094] (1) dissolving a dihydropyridine ligand precursor, 2-formylpyridine and a desired coordinated metal salt in an organic solvent, mixing and stirring them thoroughly, precipitating a metal organic complex by solvent evaporation crystallization, solvent diffusion crystallization or adding a small polar miscible solvent, and confirming the structure M3N3 by nuclear magnetic resonance, mass spectrometry or single crystal structure, wherein M is a metal ion and N is a dihydropyridine ligand;

[0095] (2) The above metal organic complex and the nitroreductase fluorescent substrate are mixed in a solution to obtain a host-guest probe, and then the host-guest probe is detected by one-dimensional nuclear magnetic resonance. 1 H- 1 H NOESY spectrum, 2D 1 H- 1 H NOESY, 1 H- 1 The host-guest formation was confirmed by H DOSY and other nuclear magnetic resonance methods. Meanwhile, high-resolution mass spectrometry, host-guest UV titration, job's plot and other methods were used to verify the stable formation of the host-guest probe.

[0096] Metal-organic cage complexes were fused with nitroreductase fluorescent substrates to form host-guest supramolecular probes for the quantitative detection of nitroreductase.

[0097] The host-guest probe is applied to the quantitative detection of nitroreductase in a solvent, and 0-5 μM of the host-guest probe is added to the nitroreductase HEPES buffer to carry out ultraviolet titration experiment and fluorescence kinetic experiment.

[0098] Example 1: Synthesis and preparation of host-guest supramolecular probe

[0099] The The synthesis preparation method includes the synthesis of metal organic cage complex, the synthesis of nitroreductase fluorescent substrate and Preparation.

[0100] Example 1 Synthesis of Nitroreductase Fluorescent Substrate:

[0101]

[0102] (1) Compound 1 was synthesized using acenaphthenequinone and 4-nitro-o-phenylenediamine as raw materials

[0103] Under argon protection at room temperature, acenaphthenequinone (1.82 g, 10 mmol) and 4-nitro-o-phenylenediamine (1.53 g, 10 mmol) were stirred in 20 ml of glacial acetic acid. The temperature was raised to 80 ° C and refluxed for 12 h. The reaction progress was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature, filtered, washed with water, vacuum dried, and purified by column chromatography to obtain a light yellow powder compound 1 with a yield of 2.80 g and a yield of 93.6%. 1 H NMR (500MHz, CF3COOD) δ8.92(s,1H),8.44–8.31(m,3H),8.19(d,J=9.0Hz,1H),8.12(d,J=8.0Hz,1H),8.04(d,J=8.0Hz,1H),7.66(dd,J=15.8,7.7Hz,2H). 13 C NMR(101MHz,CF3COOD)δ150.04,146.72,144.13,134.81,131.86,131.69,130.07, 129.91,125.67,125.40,124.13,122.98,121.58,121.07,120.38,118.60,118.48.

[0104] (2) Compound 2 was prepared using compound 1, concentrated hydrochloric acid and stannous chloride as raw materials

[0105] Compound 1 (1.50 g, 5.0 mmol) and stannous chloride (3.80 mg, 20 mmol) were stirred and refluxed in concentrated hydrochloric acid (40 mL) at 80 ° C. The reaction progress was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature. The precipitate was filtered and washed with 1M HCl, and the pH was adjusted to 5-9. Vacuum drying and column chromatography purification gave brown powder compound 2, with a yield of 1.10 g and a yield of 81.8%. 1 H NMR (500MHz, DMSO-d6) δ8.28(d,J=6.9Hz,1H),8.18(d,J=6.9Hz,1H),8.14(d,J=8.2Hz,1H),8.09(d,J=8. 2Hz,1H),7.86(d,J=8.9Hz,1H),7.84–7.78(m,2H),7.25(dd,J=8.9,1.9Hz,1H),7.14(s,1H),6.13(s,2H). 13C NMR(126MHz,DMSO-d6)δ153.20,150.50,148.19,143.20,134.36,134.32,132.18,131.89, 130.00,129.51,129.29,128.78,128.65,128.20,121.35,120.96,120.25,106.88.ESI-MS m / z:[M+H]calculated for[C 18 H 12 N3] + 270.1026, found 270.1032.

[0106] (3) Synthesis of NAQA using compound 2 and p-nitrobenzyl bromide as raw materials

[0107] At room temperature, compound 2 (0.27 g, 1.0 mmol) and potassium carbonate (0.69 mg, 5.0 mmol) were dissolved in DMF (40 mL). After mixing for 10 min, p-nitrobenzyl bromide (0.26 g, 1.2 mmol) was added in batches and the reaction was continued under stirring for 12 h. TLC monitored the reaction progress. After the reaction was completed, the reaction solution was cooled to room temperature. It was added to 100 ml of saturated brine, solids were precipitated, filtered, washed with water, and dried in vacuo. Column chromatography purification gave a yellow powder compound NAQA with a yield of 263 mg and a yield of 65.0%. 1 H NMR (400MHz, DMSO-d6) δ8.25–8.18(m,4H),8.17(d,J=8.2Hz,1H),8.12(d,J=8.2Hz,1H),7.90(d,J=9.0Hz,1H),7.82(ddd,J=8.2,7.1 ,3.5Hz,2H),7.71(d,J=8.8Hz,2H),7.45(t,J=6.1Hz,1H),7.36(dd,J=9.1,2.6Hz,1H),6.87(d,J=2.5Hz,1H),4.66(d,J=6.0Hz,2H). 13 CNMR(126MHz,DMSO-d6)δ153.19,149.31,148.54,147.93,146.56,143.18,134.74,134.35,132.04,131.75,12 9.94,129.56,129.51,128.91,128.78,128.48,128.10,123.66,121.50,121.04,120.56,104.27,45.69.ESI-MS m / z:[M+H]calculated for[C 25 H17 N4O2] + 405.1346, found 405.1336.

[0108] Example 2 Synthesis of Nitroreductase Fluorescent Substrate

[0109]

[0110] (1) Under argon protection at room temperature, acenaphthenequinone (1.82 g, 10 mmol) and 4-nitro-o-phenylenediamine (1.53 g, 10 mmol) were stirred in 20 ml of glacial acetic acid. The temperature was raised to 80°C and refluxed for 12 h. The reaction progress was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature, filtered, washed with water, vacuum dried, and purified by column chromatography to obtain compound 1.

[0111] (2) Compound 1 (1.50 g, 5.0 mmol) and stannous chloride (3.80 mg, 20 mmol) were stirred and refluxed in concentrated hydrochloric acid (40 mL) at 80°C. The reaction progress was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature. The precipitate was filtered and washed with 1 M HCl, and the pH was adjusted to 5-9. It was dried in vacuo to obtain compound 2.

[0112] (3) Compound 2 (0.27 g, 1.0 mmol) and potassium carbonate (0.69 mg, 5.0 mmol) were dissolved in DMF (40 mL) at room temperature. After mixing for 10 min, 2-(chloromethyl)-5-nitrothiophene (0.21 g, 1.2 mmol) was added in batches and the reaction was continued under stirring for 12 h. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was cooled to room temperature. The solid was added to 100 ml of saturated brine, filtered, washed with water, dried in vacuo, and the target compound was obtained by column chromatography. ESI-MS m / z: 400.0837.

[0113] Example 3 Synthesis of Nitroreductase Fluorescent Substrate

[0114]

[0115] (1) Under argon protection at room temperature, acenaphthenequinone (1.82 g, 10 mmol) and 4-nitro-o-phenylenediamine (1.53 g, 10 mmol) were stirred in 20 ml of glacial acetic acid. The temperature was raised to 80°C and refluxed for 12 h. The reaction progress was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature, filtered, washed with water, vacuum dried, and purified by column chromatography to obtain compound 1.

[0116] (2) Compound 1 (1.50 g, 5.0 mmol) and stannous chloride (3.80 mg, 20 mmol) were stirred and refluxed in concentrated hydrochloric acid (40 mL) at 80°C. The reaction progress was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature. The precipitate was filtered and washed with 1 M HCl, and the pH was adjusted to 5-9. It was dried in vacuo to obtain compound 2.

[0117] (3) Compound 2 (0.27 g, 1.0 mmol) and potassium carbonate (0.69 mg, 5.0 mmol) were dissolved in DMF (40 mL) at room temperature. After mixing for 10 min, 1-(2-chloroethyl)-2-nitro-1H-imidazole (0.21 g, 1.2 mmol) was added in batches and the reaction was continued with stirring for 12 h. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was cooled to room temperature. The solid was added to 100 ml of saturated brine, filtered, washed with water, dried in vacuo, and the target compound was obtained by column chromatography. ESI-MS m / z: 394.1044.

[0118] Example 4 Synthesis of Metal-Organic Cage Complex Zn-MPPB

[0119] The synthetic route is as follows:

[0120]

[0121] (1) Compound 3 was prepared from 4-bromobenzoic acid and 2-morpholinoethanolamine

[0122] 4-Bromobenzoic acid (2.10 g, 10 mmol), DCC (3.09 g, 15 mmol) and DMAP (1.22 g, 10 mmol) were stirred in an ice bath in 40 ml THF. Then 2-morpholineethanol (1.30 g, 10 mmol) was slowly added in batches, and the reaction was continued in an ice bath under stirring until room temperature for 16 h. TLC monitored the reaction progress. After the reaction was completed, the reaction solution was poured into 100 ml saturated brine. Dichloromethane extraction, saturated sodium carbonate washing, water washing, and vacuum drying were performed. Column chromatography purification gave white powder compound 3, with an output of 3.01 g and a yield of 96%. 1 H NMR (400MHz, DMSO-d6) δ8.50(t,J=5.5Hz,1H),7.78(d,J=8.5Hz,2H),7.68(d,J=8. 5Hz,2H),3.59–3.54(m,4H),3.41–3.33(m,4H),2.46(t,J=7.0Hz,2H),2.41(s,2H). 13C NMR(126MHz,DMSO-d6)δ165.71,134.16,131.74,129.73,125.25,66.68,57.74,53.76,37.11.ESI-MS m / z:[M+H]calculated for[C 13 H 18 BrN2O2] + 313.0546, found 313.0551.

[0123] (2) Compound 4 was prepared from ammonium acetate, benzaldehyde and methyl propiolate

[0124] Methyl propionate (1.68 g, 20 mmol), benzaldehyde (1.06 g, 10 mmol) and ammonium acetate (2.31 g, 30 mmol) were refluxed in glacial acetic acid (4.0 mL) at 80°C for 12 hours. After the reaction was completed and cooled, the solid product was filtered and washed with Et2O (10 mL×3) to obtain a yellow powder crude product compound 4, which was recrystallized from ethanol with a yield of 1.28 g and a yield of 46%. 1 H NMR (400MHz, DMSO) δ7.39(s,2H),7.26–7.18(m,4H),7.15–7.08(m,1H),4.75(s,1H),3.95(br,1H),3.54(s,6H). 13 C NMR(126MHz,DMSO)δ166.75,147.11,135.08,127.95,127.50,126.15,105.57,50.82,36.85.NMR(126MHz,DMSO).ESI-MS m / z:[M+H]calculated for C 15 H 16 NO4 + 274.1074, found 274.1074.

[0125] (3) Compound 5 (DMCPPDD) was prepared from Compound 3 and Compound 4

[0126] Under argon protection, compound 4 (3.00 g, 11 mmol) and cesium carbonate (10.65 g, 25 mmol) were stirred in anhydrous DMF (40 ml) at room temperature for 1 h. Compound 3 (3.13 g, 10 mmol), CuI (190 mg, 1.0 mmol) and pyridine-2-carboxylic acid (246 mg, 2 mmol) were added to the reaction solution respectively, and refluxed at 130 ° C for 36 h. TCL was used to monitor the reaction progress. After the reaction was completed, the mixture was cooled to room temperature, poured into 300 ml of saturated brine, and filtered to obtain a solid. The mixture was washed with water, dried in vacuo, and purified by column chromatography to obtain a white powder compound 5 with a yield of 3.40 g and a yield of 67%. 1 H NMR (400MHz, DMSO-d6) δ8.50(t,J=5.4Hz,1H),7.98(d,J=8.5Hz,2H),7.77(s,2H),7.60(d,J=8.6Hz,2H),7.32–7.25(m,4H),7 .21–7.14(m,1H),4.83(s,1H),3.61(s,6H),3.59–3.56(m,4H),3.42(dd,J=12.7,6.4Hz,2H),2.51–2.46(m,2H),2.42(s,4H). 13 C NMR (126MHz, DMSO-d6) δ166.14,165.02,145.29,144.16,134.99,131.73,128.90,128. 19,127.79,126.63,119.71,110.17,66.19,57.33,53.29,51.35,37.02,36.60.ESI-MS m / z:[M+H]calculated for[C 28 H 33 N3O6] + 506.2286, fond 506.2291.

[0127] (4) Preparation of ligand precursor compound DPM using compound 5 and hydrazine hydrate as raw materials

[0128] A mixed solution of 80% hydrazine hydrate (10 ml) and compound 5 (505 mg, 1.0 mmol) was stirred and refluxed at 120°C for 12 hours. The reaction progress was monitored by TCL. After the reaction was completed, the mixture was cooled to room temperature, collected and vacuum dried. The crude product was purified by flash column chromatography to obtain a light yellow solid compound DPM ligand precursor with a yield of 81 mg and a yield of 16%. 1H NMR (400MHz, DMSO-d6) δ9.22(s,2H),8.47(t,J=5.6Hz,1H),7.95(d,J=8.8Hz,2H),7.63(s,2H),7.60(d,J=8.8Hz,2H),7.27–7.19(m,4 H),7.12(t,J=6.8Hz,1H),5.17(s,1H),4.27(s,4H),3.62–3.53(m,4H),3.41(dd,J=12.9,6.5Hz,2H),2.50–2.45(m,2H),2.42(s,4H). 13 C NMR(126MHz,DMSO-d6)δ166.39,165.18,145.25,144.49,129.88,128.68,128.04,1 28.00,127.73,126.21,117.86,117.74,112.93,66.19,57.40,53.30,36.57.ESI-MS m / z:[M+H]calculated for[C 26 H 33 N7O4] + 506.2510, fond 506.2502.

[0129] (5) Synthesis of metal organic cage Zn-MPPB using compound DPM, 2-formylpyridine and zinc perchlorate hydrate as raw materials

[0130] Under argon protection, compound DPM (101 mg, 0.2 mmol), 2-formylpyridine (43 mg, 0.4 mmol) and zinc perchlorate hexahydrate (138 mg, 0.2 mmol) were reacted in 10 ml of anhydrous acetonitrile at 70 ° C for 17 hours. After the reaction was completed, it was cooled to room temperature, and ether was added to the reaction solution while stirring vigorously to precipitate a yellow precipitate. A yellow solid was obtained by high-speed centrifugation and vacuum drying to obtain a yellow powder product Zn-MPPB with a yield of 139 mg and a yield of 93%. 1 H NMR(400MHz,DMSO-d6)δ12.15(br,3H),9.59(br,2H),8.85–8.31(m,12H),8.27–8.11(m,6H),8.08–7.84(m,18H ),7.77–7.64(m,6H),7.59–7.19(m,18H),7.14(s,3H),5.42(s,3H),4.11–3.60(m,18H),3.54–3.05(m,18H).ESI MS m / z:[H6Zn3(MPPB)3] 6+=374.6128,[H5Zn3(MPPB)3] 5+ =449.3338,[H6Zn3(MPPB)3(ClO4 - )] 5+ =469.1253,[H4Zn3(MPPB)3] 4+ =561.4143,[H5Zn3(MPPB)3(ClO4 - )] 4+ =586.4042,[H6Zn3(MPPB)3(2ClO4 - )] 4+ =611.3928,[H3Zn3(MPPB)3] 3+ =748.2159,[H4Zn3(MPPB)3(ClO4 - )] 3+ =781.5339,[H5Zn3(MPPB)3(2ClO4 - )] 3+ =814.8527,[H6Zn3(MPPB)3(3ClO4 - )] 3+ =848.1718.

[0131] Example 5 Preparation

[0132] The obtained Zn-MPPB compound and NAQA were dissolved in deuterated DMSO, respectively, and solutions of equal concentration were prepared and mixed in a 1:1 ratio. 1 H- 1 H NOESY spectrum ( Figure 2 A) Two-dimensional 1 H- 1 H NOESY Figure 2 B) 1 H- 1 H DOSY( Figure 3 A) NMR spectrum. 1 H- 1 H NOESY and 2D 1 H- 1 The H NOESY spectrum results show that the NH protons of NAQA interact with multiple protons in the Zn-MPPB compound. 1 H- 1 The H DOSY spectrum results show that after the formation of host-guest, the metal organic cage and the guest molecule have similar diffusion coefficients. Figure 3 B and 3C) Results ESI MS m / z: [H3Zn3(MPPB)3(NAQA)] 2+=882.9246 proves the existence of host-guest. All the above experiments prove that Zn-MPPB and NAQA interact with each other and can form a host-guest structure.

[0133] Example 6-9: Solvent property test based on host-guest supramolecular probe

[0134] Host-Guest Supramolecular Probes The solvent test was carried out according to the literature method, and NAQA was set as the control group.

[0135] Example 6 stability

[0136] The stability of host-guest supramolecular probes determines their application capabilities and scope. The HH interaction of a mixture of Zn-MPPB and NAQA (1:1) was tested in deuterated DMSO. Figure 2 ), it was found that the NH of NAQA had obvious interactions with other H of Zn-MPPB. 1 H- 1 H DOSY spectrum ( Figure 3 A) The results show that after the host-guest formation, the metal organic cage and the guest molecule have similar diffusion coefficients. The NMR results show that Subject-object structure. Stability in aqueous environments plays a decisive role in its application in biological testing. Then, microcalorimetric titration and UV-visible absorption titration experiments were performed in DMSO-HEPES buffer (v / v, DMSO 90%, 25°C). The binding free energy was calculated to be -8.5 kcal / mol and the binding constant was 1.7×10 6 M -1 The interaction between Zn-MPPB and NAQA reduces the energy of the system and can proceed spontaneously. Figure 4 A) The calculated binding constant is 1.1×10 6 M -1 The free binding energy calculated by molecular docking experiment is -9.62kcal / mol. The binding constants calculated by various experiments have good matching. In addition, the free binding energy of Zn-MPPB and Fluorescence intensity stability experiment was carried out under solvent conditions ( Figure 5 ), UV absorption stability test, and pH UV stability test. The results showed that Zn-MPPB and The UV absorption stability is almost unchanged within 30 minutes, and the fluorescence intensity stability has a slight change within 20 minutes, but it remains stable overall. According to the subsequent host-guest probe and enzyme reaction kinetics experiments, cell imaging tests, flow cytometry tests, and mouse imaging tests, the test time did not exceed 30 minutes, indicating that the host-guest probe can exist stably under the test conditions. These experimental results prove The host-guest probe can be used for biological testing. This part innovatively uses a variety of testing methods and means to jointly prove the existence and stability of the host-guest probe, which has indicative and normative standards for the application of host-guest chemistry in the biological field.

[0137] Example 7 Interaction between Zn-MPPB and nitroreductase

[0138] Only by interacting with nitroreductase can the host-guest probe strategy be used for nitroreductase activity detection. First, nitroreductase and Zn-MPPB were titrated by UV in HEPES ( Figure 4 B), the calculated binding constant is 6.9×10 6 M -1 . The circular dichroism experiment of nitroreductase and Zn-MPPB proved that the secondary and tertiary structures of nitroreductase maintained their configurations before and after the addition of Zn-MPPB, and the enzyme activity was not affected. Particle size analysis showed that the average particle size of nitroreductase was 6.3nm. After the addition of Zn-MPPB, the average particle size increased to 7.7nm, indicating that Zn-MPPB interacted with nitroreductase and could attach to nitroreductase. Through molecular docking experiments, the binding free energy of NAQA binding to the nitroreductase binding pocket was calculated to be -8.68kcal / mol, and the binding free energy of Zn-MPPB binding to the nitroreductase binding pocket was -11.12kcal / mol. Calculations show that after Zn-MPPB and NAQA bind to nitroreductase, the system energy decreases and can proceed spontaneously. In order to verify the binding of Zn-MPPB to nitroreductase, MALDI-TOF MS test was performed before and after Zn-MPPB was added to nitroreductase. The experiment found that before adding Zn-MPPB, the characteristic peak of nitroreductase mass spectrum [MH + ]=49698, indicating that it is a dimer form; after adding Zn-MPPB, the characteristic peak of the mass spectrum of nitroreductase-Zn-MPPB [MH + ]=50154; Compared with the mass spectrometry data of nitroreductase, the formation of nitroreductase-Zn-MPPB complex causes nitroreductase to lose one molecule of NAD + (C 21 H 28 N7O 14P2, Mr = 664.4), 2 molecules of FMN (C 17 H 21 N4NaO9P, Mr = 478.3) and 9 molecules of H2O. The mass spectrometry data verified that the enzyme can bind to Zn-MPPB to form a complex. The binding constants calculated from these experiments have a good match, indicating that under the test conditions, Zn-MPPB and nitroreductase can interact, so the host-guest supramolecular probe may be used to detect nitroreductase activity in biological environments.

[0139] Example 8 Solvent fluorescence test with NAQA and nitroreductase

[0140] The nitroreductase reaction was carried out in HEPES buffer (10.0 mM, pH 6.8, 25°C). Or NAQA fluorescence response test to characterize its activity, excitation light 440nm, emission range 500 to 700nm, fluorescence titration and fluorescence kinetics ( Figure 6 A, 6B, 6D and 6E) tests. In the experimental group test (host-guest probe 5μM, nitroreductase 0-5μg / ml), it was found that the host-guest probe can quickly respond to nitroreductase and complete the fluorescence enhancement response within 30 seconds. The comparison group (NAQA 5μM, NADH 15μM, nitroreductase 0-5μg / ml) takes more than fifteen minutes for the fluorescence intensity to slowly reach equilibrium, and the final intensity is significantly lower than the host-guest probe experimental group. The change of emission peak intensity at 540nm before and after the reaction of small molecule probe NAQA with enzyme. After the reaction with 5μg / ml nitroreductase, the fluorescence intensity of the host-guest probe increased by more than 36 times, while that of the small molecule probe was about 29 times, indicating that the host-guest probe has a certain enhancement effect on the enzyme response. Taking the fluorescence emission at 540nm as the reference, the relationship between the relative fluorescence intensity and the reaction time and the amount of nitroreductase was plotted ( Figure 6 C and 6F), it can be seen that because The fluorescence signal of the experimental group and the nitroreductase reaction was rapidly balanced, and the fluorescence intensity hardly changed with time. The fluorescence intensity was only linearly correlated with the enzyme content. However, the fluorescence intensity of the NAQA control group was related to both the time change and the nitroreductase content, and the relationship between the fluorescence intensity and the nitroreductase content could not be accurately quantified. It can be used for ultra-fast and accurate quantitative detection of nitroreductase in solution. Using the Michaelis equation, further fluorescence kinetic experiments and calculations ( Figure 7 ), compared with the NAQA control group and the nitroreductase reaction, The maximum reaction rate and turnover number TON of the experimental group reacting with nitroreductase increased by at least 10 times, indicating that compared with the traditional small molecule probe method, this host-guest probe strategy can more quickly and accurately detect nitroreductase quantitatively.

[0141] Example 9 Other biomolecule interfering substances Response to nitroreductase fluorescence test

[0142] The internal environment of life is very complex. For subsequent cell and biological tests, it is necessary to exclude interference from other ions or molecules in a complex physiological environment and still have an accurate response to nitroreductase. Common interfering species in the cell environment were selected, including glucose (10mM), glutathione (GSH, 1mM), ATP (1mM), vitamin C (1mM), various amino acids (1mM L-Ala, L-Cys, L-Hcy, L-His, L-Phe, L-Pro, etc.), glucose oxidase (Glu ox , 10 μg ml -1 ), cytochrome C (Cyt C, 10 μg ml -1 ) and bovine serum albumin (BSA, 10 μg ml -1 ) respectively with (5μM) or NAQA (5μM) for interference Response to nitroreductase fluorescence test (eg Figure 8 Compared with nitroreductase (5 μg / ml), these interfering species did not cause The obvious fluorescence changes of NAQA and NAQA indicate that they can recognize and detect nitroreductase with high selectivity. Competition assays with these interfering analytes. After (5μM) was mixed evenly with the above-mentioned interferents, and then nitroreductase was added, the fluorescence response was still turned on super quickly, and its response time and intensity did not change significantly, showing good consistency. It has good selectivity and competitive ability among these interferents and is able to maintain structural stability.

[0143] Example 10: Cellular Experimental Test Based on Host-Guest Supramolecular Probe

[0144] (1) Cytotoxicity experiments of Zn-MPPB and NAQA

[0145] The cells were inoculated in 96-well plates, and 0, 1, 2, 5, 10, 20 μM Zn-MPPB or NAQA were added to the cells for 24 h. The MTT assay was performed (eg Fig. 9), and the toxicity of the test compounds to MCF-7 and A549 cells.

[0146] (2) Cell imaging test at different hypoxia levels

[0147] Cancer cells (including MCF-7 and A549 cells) treated with different hypoxia levels (20%, 8%, 0.1% O2) for 6 h were used After incubation for 5 min, laser confocal imaging was performed with an excitation wavelength of 458 nm and a fluorescence signal collection range of 500-600 nm. Fig.10 A. Use (5μM) was used as the experimental group, and NAQA (5μM) was used as the control group. After incubation with MCF-7 cells treated with different hypoxia for 5 minutes, cell imaging tests were performed. The experimental results showed that as the degree of cell hypoxia increased, the fluorescence intensity of all tested cells gradually increased. This phenomenon was more obvious in the experimental group, and its fluorescence intensity was significantly higher than that in the control group. The fluorescence of the treated group was much higher than that of the normoxia and 8% O2 control groups.

[0148] (3) Fluorescence intensity imaging test of cells treated with 0.1% O2 hypoxia over incubation time

[0149] After 6 h of 0.1% O2 hypoxia treatment, various cancer cells (including MCF-7 and A549 cells) were treated with After incubation for different time (0, 1, 3, 5, 10, 20 min), laser confocal imaging was performed with an excitation wavelength of 458 nm and a fluorescence signal collection range of 500-600 nm. Fig.10 B. Use (5μM) was used as the experimental group, and NAQA (5μM) was used as the control group. The MCF-7 cells treated with 0.1% O2 hypoxia were incubated for different time periods and then subjected to cell fluorescence imaging tests. The experimental results showed that as the drug incubation time increased, The cell fluorescence intensity of the experimental group quickly reached the maximum within five minutes, and the subsequent increase in incubation time did not significantly change the cell fluorescence intensity. This shows that the cell fluorescence intensity can reach equilibrium after incubation for 5 minutes, and the subsequent increase in incubation time to 20 minutes did not significantly change the cell fluorescence intensity; while in the NAQA control group, the cell fluorescence intensity gradually increased slowly with the increase in incubation time, but was significantly weaker than Experimental group. The results of cell imaging fluorescence kinetic test showed that the host-guest probe can quickly trace the degree of cell hypoxia and its corresponding nitroreductase by bioluminescence.

[0150] Embodiment 11 Flow cytometric analysis of cells treated with 0.1% O2 hypoxia

[0151] Well-growing MCF-7 and A549 cells were inoculated into culture flasks and cultured for 24 hours until the cells almost covered the bottom of the flask. Then, the culture medium was replaced and 0.1% O2 hypoxia treatment was performed for 6 hours. (5 μM) was used as the experimental group, NAQA (5 μM) was used as the control group for incubation for 5 min, and cells without drug addition were used as the blank control group. After treatment, the cells were dissociated and washed twice with PBS. The collected cells were dispersed in 1 ml of PBS and analyzed by flow cytometry (λ ex =488nm, FITC 533 / 30nm), such as Fig.11 The results showed that NAQA or Compared with the blank control group, the relative fluorescence intensity of the cells in the incubated cell group was significantly stronger than that in the blank control group. The relative fluorescence intensity of the cultured cells was also stronger than that of the NAQA control group, which verified the ability of the host-guest probe to rapidly image the nitroreductase of cancer cells after a short incubation period. These results are consistent with the results of cell confocal imaging, confirming that The excellent imaging performance of the host-guest probe demonstrates the ability of the host-guest probe to be applied in flow cytometry to rapidly detect NTR in hypoxic tumor cells.

[0152] Example 12 In vivo experimental test of host-guest supramolecular probe in mice

[0153] The nude mice were injected with tumors in the armpits to establish MCF-7 tumor-bearing mouse models. After the tumors had grown for 1-2 weeks, the mice were injected (100 μL, 100 μM) was used to perform imaging tests of tumor fluorescence changes over time (0, 2, 5, 10 min) in living mice (λ ex / λ em =450 / 570nm)(e.g. Fig.12 ). NAQA (100 μL, 100 μM) was set as the control group. The test results showed that The fluorescence intensity of the tumor of the mice in the experimental group quickly produced a strong fluorescence signal within 20 minutes, while the fluorescence of the tumor of the mice in the control group increased slowly over time and was still very weak at 20 minutes. Compared with traditional biological probes, it has the ability to image tumors more quickly.

Claims

1. A nitroreductase fluorescent substrate, characterized in that: The general structural formula of the fluorescent substrate is: ； Among them, R is .

2. The method for preparing a nitroreductase fluorescent substrate according to claim 1, characterized in that: The following steps are involved: ； (1) Using acenaphthenequinone and 4-nitro-o-phenylenediamine as raw materials, preparing the compound represented by intermediate 1; the molar ratio of acenaphthenequinone:4-nitro-o-phenylenediamine is 1:1-1.1; (2) Using intermediate 1, stannous chloride and 37% hydrochloric acid as raw materials and concentrated hydrochloric acid as solvent, heating under reflux to prepare synthetic intermediate 2, wherein the molar ratio of intermediate 1 to stannous chloride is 1:4-6; (3) Using intermediate 2 and p-nitrobenzyl bromide as raw materials and carbonate as base, the nitroreductase fluorescent substrate was prepared in DMF at room temperature; The molar ratio of the intermediate 2: p-nitrobenzyl bromide: carbonate is 1:1.1-1.3:2-2.

2.

3. A host-guest supramolecular probe for accelerating nitroreductase detection, characterized in that: The host-guest supramolecular probe comprises a main part and a guest part, the guest part is the fluorescent substrate according to claim 1, the main part is a metal organic cage complex Zn-MPPB, the metal ion in the metal organic cage complex is a zinc ion; the molar ratio of the main part to the guest part is 1:1; the metal organic cage complex is of Zn3DPM3 type; The preparation method of the metal organic cage complex Zn-MPPB is: A ligand precursor compound DPM, 2-formylpyridine and zinc perchlorate hydrate are used for self-assembly reaction to obtain Zn-MPPB, wherein the molar ratio of DPM:2-formylpyridine:zinc perchlorate hydrate is 1:2:1; The structure of the ligand precursor compound DPM of the metal organic cage complex is as follows: 。 4. The host-guest supramolecular probe for accelerating nitroreductase detection according to claim 3, characterized in that: The preparation method of the ligand precursor compound DPM is as follows: ； (1) Using 4-bromobenzoic acid and 2-morpholinoethylamine as raw materials, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine as catalysts, compound intermediate 3 was prepared; The molar ratio of 4-bromobenzoic acid: 2-morpholineethylamine: N,N'-dicyclohexylcarbodiimide: 4-dimethylaminopyridine is 1.1-1.3:1:1.5-1.7:1-1.2; (2) Using methyl propiolate, benzaldehyde and ammonium acetate as raw materials to prepare compound intermediate 4; the molar ratio of ammonium acetate:methyl propiolate:benzaldehyde is 4-4.4:2-2.2:1; (3) Using intermediate 3, intermediate 4 and a base as raw materials, a compound represented by intermediate 5 is prepared in an organic solvent; The base is sodium carbonate, potassium carbonate, cesium carbonate or an organic base; The organic solvent is selected from acetone, N,N-dimethylformamide or 1,4-dioxane; The molar ratio of intermediate 3:intermediate 4 is 1.1-1.3:1; (4) The intermediate 5 is reacted with 80% hydrazine hydrate to obtain the compound represented by the ligand precursor compound DPM; the molar ratio of the intermediate 5 to 80% hydrazine hydrate is 1:5 to 1:

10.

5. Use of the host-guest supramolecular probe for accelerating nitroreductase detection according to any one of claims 3-4, characterized in that: The host-guest supramolecular probe is applied to the quantitative detection of nitroreductase for non-disease diagnosis and treatment purposes.

6. Use of the host-guest supramolecular probe for accelerating nitroreductase detection according to any one of claims 3 to 4, characterized in that: The host-guest supramolecular probe is used for preparing a reagent for detecting nitroreductase.

Images