WO2021120653A1 - Cofactor-substrate probe platform for fast quantitative detection of tumor hypoxia related enzyme - Google Patents

Cofactor-substrate probe platform for fast quantitative detection of tumor hypoxia related enzyme Download PDF

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WO2021120653A1
WO2021120653A1 PCT/CN2020/109921 CN2020109921W WO2021120653A1 WO 2021120653 A1 WO2021120653 A1 WO 2021120653A1 CN 2020109921 W CN2020109921 W CN 2020109921W WO 2021120653 A1 WO2021120653 A1 WO 2021120653A1
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substrate
cofactor
metal
nadh
organic
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焦扬
段春迎
张磊
高旭
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大连理工大学
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/18—Metal complexes
    • C09K2211/188—Metal complexes of other metals not provided for in one of the previous groups

Definitions

  • the invention belongs to the field of metal-organic supramolecular, bio-inorganic and biological fluorescent probes, and specifically relates to a method for preparing a host-guest supramolecular probe for rapid quantitative detection of hypoxic enzymes and its application in solution, cell, and living body detection .
  • Hypoxia is an important indicator of many cancers and is closely related to the various physiological activities of tumors.
  • many substrate-based enzymatic fluorescent probes use targeted hypoxia activation methods to identify the location and expression level of hypoxic enzymes in cancer cells, and are used to detect abnormal levels of hypoxic enzymes in solutions, cancer cells and small animals. Detection and imaging are essential for the early diagnosis and monitoring and treatment of cancer.
  • the concentration and expression level of hypoxic enzymes are usually very different in different cells and tumors, and even the same cells have different concentrations under different conditions.
  • the two variables of unknown concentration of cofactor and enzyme will cause the probe emission to change significantly over time.
  • the detection of hypoxic enzyme content in the body is highly dependent on the content of cofactors.
  • researchers add a large excess (tens of times to Several hundred times the molar amount) of the cofactor to eliminate the interference of the cofactor concentration in the enzyme-catalyzed reaction, so that the cofactor concentration phase control no longer restricts the maximum reaction rate of the hypoxic enzyme catalyzed substrate, and the response signal is only related to the enzyme content.
  • This method eliminates the limitation of the concentration of cofactors on the fluorescence opening rate of the enzyme-catalyzed probe, so as to achieve accurate and rapid detection.
  • the cofactor-substrate-based probes are combined into a supramolecular system in which there is a clear stoichiometric ratio between the cofactor and the substrate, and the host and guest molecules close to each other will promote electron transfer Efficiency, this will make the reaction rate of the probe and the enzyme not be disturbed by the concentration of endogenous cofactors, which is very helpful for rapid quantitative detection.
  • the purpose of the present invention is to propose a cofactor-substrate probe platform for rapid quantitative detection of tumor hypoxia-related enzymes, including its preparation method and biological test application, and metal-organic cage complexes as NADH mimics and The fusion of the substrate into an excimer reacts with the enzyme to catalyze the reaction, which turns the multi-substrate reaction into a single-substrate reaction strategy.
  • the host-guest probe Zn-MPB ⁇ L-NO 2 includes the metal-organic cage complex body part (Zn-MPB) and the nitroreductase fluorescent substrate guest part (L-NO 2 ).
  • the main part is an M 3 N 3 type metal-organic cage complex composed of ligand (formula I) and metal ion (M n+ ).
  • the guest part is connected by a fluorophore and a p-nitrobenzyl group (Formula II, Formula III).
  • the reaction principle of the probe Under the action of NTR, the nitrophenyl group of the host-guest probe is reduced to aminobenzene and further intramolecular cleavage.
  • the structure of the fluorescent substrate changes obviously, so the corresponding fluorescent response is turned on.
  • Metal-organic cage complexes are used as NADH mimics and substrates to fuse into excimers and enzyme-catalyzed reactions, turning multi-substrate reactions into single-substrate reactions, reducing the types of substrates, shortening the distance between substrates, and promoting The reaction kinetics changes.
  • the structure of the dihydropyridine part of the ligand in formula I is similar to NADH, as the active part of the NADH mimic, used for proton and electron transfer;
  • the method for preparing the ligand includes the following steps:
  • R 1 is a group that provides coordination atoms
  • R 1 is 2-pyridyl, 2-mercaptophenyl, 2-thiophene, 2-pyrrole, quinoline or isoquinoline
  • R 2 is phenyl, hydrogen, Halogen, cyano, benzyloxy.
  • the preparation method of the fluorescent substrate includes the following steps:
  • R 3 is the fluorophore part of the enzyme-catalyzed fluorescent substrate, which can be, but is not limited to, 2-phenyl-3a,11b-dihydro-1H-phenanthrene[9,10-d]imidazole group, naphthalene Group, quinoxaline group, porphyrin or porphin group, etc.;
  • R 3 is 2-phenyl-3a,11b-dihydro-1H-phenanthrene[9,10-d]imidazolyl
  • the preparation method of the cofactor-substrate host-guest probe platform includes the following steps:
  • the dual-substrate catalytic process with substrate is optimized as a single-substrate process. Make the detection of nitroreductase quickly reach equilibrium, no longer affected by NADH content, so the detected fluorescence intensity change is only related to the change of nitroreductase content, realizing rapid quantitative detection of nitroreductase.
  • a type of cofactor-substrate host-guest probe for the quantitative and rapid detection of nitroreductase in cells test method: configure DMEM medium (add FBS 10%, double antibody 1%) for culturing MCF-7 and 231 Cells, configure 1640 medium (add FBS 10%, double antibody 1%) for culturing A549 cells. After setting different oxygen content environment (20%, 8%, 0.1%) to culture cells for 6-12h, incubate with cofactor-substrate supramolecular probe for 1 min to perform laser confocal fluorescence imaging of cells under different oxygen content experiment.
  • a type of cofactor-substrate host-guest probe for the quantitative and rapid detection of nitroreductase in mice Inject the cofactor-substrate supramolecular probe described in the injection (generally, the injection concentration and range are 10-200 ⁇ M, 50-200 ⁇ L), and perform the imaging experiment of mouse tumor fluorescence over time.
  • Zn-MPB ⁇ L- NO 2 cofactor-substrate-based supramolecular probe
  • NTR nitroreductase
  • 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 achieve a rapid balance.
  • the inclusion of the substrate in the host containing NADH can reduce the transit time of external NADH and shorten the distance between the substrate and the active site of the cofactor, thereby enabling rapid signal response to the level of hypoxic enzyme.
  • the main application objects of the drug mainly applicable to the fields of biomimetic catalysis, tumor imaging diagnosis and treatment.
  • the invention is mainly aimed at the accurate detection of the activity of a broad-spectrum cancer marker hypoxic enzyme to diagnose cancer.
  • the present invention can also be used as an imaging agent for guiding tumor resection surgery. It can be said that it has broad application prospects in the medical and health field.
  • Figure 1 is a diagram based on the structure of the cofactor-substrate probe platform, the basic ligand components and the enzyme-catalyzed reaction.
  • Figure 2 is the NOESY NMR spectrum of Zn-MPB ⁇ L-NO 2 and the experimental graph of the fluorescence selectivity of the interferent.
  • Figure 3 is the fluorescence kinetic test diagram of the reaction between L-NO 2 or Zn-MPB ⁇ L-NO 2 and NTR.
  • Figure 4 is a CCK8 experiment to analyze the toxicity of Zn-MPB and L-NO 2 to MCF-7 cells.
  • Figure 5 is the fluorescence imaging of L-NO 2 and Zn-MPB ⁇ L-NO 2 on MCF-7 cells treated with different hypoxia.
  • Figure 6 is the fluorescence imaging images of L-NO 2 and Zn-MPB ⁇ L-NO 2 on MCF-7 cells treated with 0.1% hypoxia at different incubation times.
  • Fig. 7 is a graph of fluorescence imaging of MCF-7 tumor-forming mice injected with L-NO 2 and Zn-MPB ⁇ L-NO 2 over time.
  • the present invention proposes a strategy based on a cofactor-substrate probe platform and used for ultra-fast quantitative detection of tumor hypoxic enzymes.
  • the host-guest probe exhibits the characteristics of ultra-fast quantitative detection in solvent, cell and in vivo experiments.
  • the present invention will be further introduced in conjunction with the embodiments.
  • Example 1 Synthesis and preparation of supramolecular host-guest probes with cofactor-substrate structure
  • the synthetic preparation method of Zn-MPB ⁇ L-NO 2 includes the synthesis of host metal-organic cage complex, the synthesis of NTR fluorescent substrate and the preparation of Zn-MPB ⁇ L-NO 2.
  • NTR fluorescent substrate The synthesis of NTR fluorescent substrate includes the following steps:
  • the synthesis of the host metal-organic cage complex Zn-MPB includes the following steps:
  • Methyl propionate (1.68 g, 20 mmol), benzaldehyde (1.06 g, 10 mmol) and ammonium acetate (2.31 g, 30 mmol) were refluxed at 80°C for 12 hours in glacial acetic acid (4.0 mL). After the reaction was completed and cooled, the solid product was suction filtered and washed with Et 2 O (10 mL ⁇ 3) to obtain the crude yellow powder compound 3, which was recrystallized from ethanol. The yield was 1.32 g and the yield was 48%.
  • Reflux compound 2 (1.79 g, 12 mmol), compound 3 (2.73 g, 10 mmol) and potassium carbonate (0.35 g, 25 mmol) in acetone for more than 12 hours.
  • TCL detects the progress of the reaction.
  • the reaction is completed, it is cooled and filtered.
  • the organic phase was collected, and the solid was washed thoroughly with dichloromethane, and the organic phase was collected.
  • the solvent was removed, and the crude product was purified by column chromatography (ethyl acetate/petroleum ether, 1:10, v/v) to obtain a pale yellow solid DMPDD with a yield of 2.04 g and a yield of 53%.
  • the preparation of Zn-MPB ⁇ L-NO 2 includes the following steps:
  • the obtained Zn-MPB compound and L-NO 2 were separately dissolved in deuterated DMSO, and a solution of equal concentration was prepared and mixed 1:1.
  • the NOESY NMR spectrum is shown in Figure 2A.
  • the NH protons of L-NO 2 and multiple protons in the Zn-MPB compound have mutual leases, indicating that a host-guest structure is formed.
  • ESI MS m/z: H 2 Zn 3 (MPB) 3 (L-NO 2 )) 2+ 1166.17.
  • Example 2 Solvent property test based on the cofactor-substrate probe platform
  • the stability of the cofactor-substrate supramolecular probe platform determines its application capability and scope.
  • the HH interaction of Zn-MPB and L-NO 2 mixture (1:1) was tested in deuterated DMSO, and it was found that the NH of L-NO 2 had obvious interaction with other H of Zn-MPB, indicating that Zn- was formed.
  • the stability of Zn-MPB ⁇ L-NO 2 in an aqueous environment plays a decisive role in its application in biological testing.
  • DMSO Tris-HCl buffer v/v, DMSO 90%, 25°C
  • microcalorimetric titration and ultraviolet-visible light absorption titration experiments were carried out.
  • the free energy of binding calculated by microcalorimetric titration is -9.43 kcal/mol, and the binding constant is 8.33 ⁇ 10 6 M ⁇ 1 , indicating the interaction between Zn-MPB and L-NO 2.
  • the binding constant was calculated by ultraviolet-visible light absorption titration to be 1.70 ⁇ 10 6 M ⁇ 1 .
  • the binding constants calculated by the two experiments have a good match, indicating that Zn-MPB ⁇ L-NO 2 exists stably under the test conditions.
  • the interaction of Zn-MPB ⁇ L-NO 2 with NTR indicates that the host-guest probe strategy may be used for the detection of NTR activity.
  • the calculation shows that the energy of the system decreases after the combination of Zn-MPB and L-NO 2 with NTR, and it can proceed spontaneously.
  • MALDI-TOF MS test was performed before and after adding Zn-MPB to NTR.
  • Microcalorimetric titration and ultraviolet-visible light absorption titration experiments were further carried out in Tris-HCl buffer (v/v, DMSO 1%, 25°C) to verify their interaction.
  • the binding constant was calculated by microcalorimetric titration to be 1.92 ⁇ 10 6 M ⁇ 1 .
  • the stated interaction between Zn-MPB and L-NO 2 reduces the energy of the system and can proceed spontaneously.
  • the binding constant was calculated by ultraviolet-visible light absorption titration to be 1.30x10 6 M ⁇ 1 .
  • the binding constants calculated by the two experiments have a good match, indicating that Zn-MPB and NTR can interact under the test conditions, and supramolecular probes may be used to detect NTR activity in a biological environment.
  • the fluorescence intensity of the L-NO 2 control group is related to time changes and NTR content, and the relationship between fluorescence intensity and NTR content cannot be accurately quantified. It shows that Zn-MPB ⁇ L-NO 2 can be used for ultra-fast and accurate quantitative detection of NTR in solution.
  • the maximum reaction rate and the number of conversions TON of the Zn-MPB ⁇ L-NO 2 experimental group reacting with NTR increased by at least 100 times. It shows that compared with the traditional probe method, the coenzyme-fluorescent substrate host-guest probe strategy can quantitatively detect NTR more quickly and accurately.
  • MPB ⁇ L-NO 2 responds to the nitroreductase fluorescence test (Figure 2B). Compared with NTR (5 ⁇ g/ml), these interfering substances did not cause obvious fluorescence changes of Zn-MPB ⁇ L-NO 2 and L-NO 2 , indicating that they can identify and detect NTR with high selectivity.
  • Example 3 Cell experiment test based on the cofactor-substrate probe platform
  • Inoculate cultured cells on a 96-well plate and add 0, 1, 2, 5, 10, 20 ⁇ M Zn-MPB or L-NO 2 to the cells, respectively, and incubate for 24 h, and then pass the CCK8 analysis experiment ( Figure 4) ), to test the toxicity of the compound to MCF-7 cells.
  • Carcinoma cells including MCF-7, 231 and A549 cells treated with different levels of hypoxia (20%, 8%, 0.1% O 2 ) for 6 hours were incubated with Zn-MPB ⁇ L-NO 2 (1 ⁇ M) for 1 min , Perform laser confocal imaging, excitation wavelength 488 nm, fluorescence signal collection range 508-608 nm.
  • Zn-MPB ⁇ L-NO 2 (1 ⁇ M) as the experimental group
  • L-NO 2 (1 ⁇ M) as the control group
  • they were incubated with different hypoxia-treated MCF-7 cells for 1 min. Imaging test.
  • the experimental results found that as the degree of cell hypoxia increased, the fluorescence intensity of all the tested cells gradually increased.
  • the phenomenon of the Zn-MPB ⁇ L-NO 2 experimental group was more obvious, and its fluorescence intensity was significantly higher than that of the control group. .
  • the fluorescence of the Zn-MPB ⁇ L-NO 2 treatment group was much higher than that of the control group.
  • Example 4 Mice in vivo experimental test based on the cofactor-substrate probe platform
  • This embodiment is the same as the best embodiment.
  • Metal-organic polyhedral cages have the ability of enzymes to efficiently catalyze the conversion of substrates.
  • researchers use metal-organic cages with specific hydrophilic-hydrophobic cavities as a unique host to catalyze chemical transformations.
  • the cofactor mimics are directly introduced into the framework of the metal-organic cage complex, so that the catalytic active site is close to the substrate, and the effective electron transfer process between the reactants is promoted.
  • Encapsulation of luminescent probes based on enzyme substrates in "molecular containers" assembled by cofactors provides an important tool that can be used to distinguish and quantitatively detect different enzyme activities in normal and disease states.
  • This novel cofactor-substrate structure host-guest supramolecular probe fusion strategy enables the redox reaction of the luminescent substrate, cofactor and enzyme to occur effectively, showing excellent kinetic characteristics.

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Abstract

Provided is a cofactor-substrate probe platform, which is used for the fast quantitative detection of a tumor hypoxia related enzyme. The probe platform uses an NADH mimetic metal organic cage-like complex assembled by a dihydropyridine ligand as a subject part, uses a fluorogenic substrate as a guest part, and obtained by assembly. The present invention provides a fusion strategy of a cofactor mimetic and a fluorogenic substrate, so that an enzyme catalysis double-substrate process is simplified to be a single-substrate process, a fluorescence sensor is no longer interfered by the cofactor and has ultrafast fluorescence signal response to the detection of a target enzyme, and the fluorescence intensity of a cofactor-substrate probe is linearly related to the content of the target enzyme. The fusion strategy provides an effective tool for the fast quantitative detection of a hypoxia enzyme, and can be used for biological tracing, disease diagnosis, and the like.

Description

辅因子—底物探针平台用于肿瘤缺氧相关酶的快速定量检测Cofactor-substrate probe platform for rapid quantitative detection of tumor hypoxia-related enzymes 技术领域Technical field
本发明属于金属-有机超分子、生物无机和生物荧光探针领域，具体涉及用于快速定量检测缺氧酶的主-客体超分子探针的制备方法及在溶液、细胞、活体检测方面的应用。The invention belongs to the field of metal-organic supramolecular, bio-inorganic and biological fluorescent probes, and specifically relates to a method for preparing a host-guest supramolecular probe for rapid quantitative detection of hypoxic enzymes and its application in solution, cell, and living body detection .
背景技术Background technique
缺氧是许多癌症的一项重要指标，与肿瘤的各种生理活动密切相关。目前，许多基于底物的酶促荧光探针通过使用靶向低氧激活的方法鉴定癌细胞中低氧酶的位置和表达水平，用于溶液、癌细胞和小动物体内低氧酶异常水平的检测和成像，对癌症的早期诊断和监测治疗至关重要。然而，缺氧酶的浓度和表达水平通常在不同的细胞和肿瘤中差别很大，甚至相同的细胞处于不同条件下其浓度也有差异。当一定量的传统探针用于检测时，未知浓度的辅因子和酶两个变量都会导致探针发射随时间发生显着变化，传统探针很难排除辅因子浓度对检测结果的干扰，酶含量与荧光响应没有显示出稳定的线性关系。因此，传统的探针方法来定量检测酶含量，准确区分正常和疾病状态的酶活性变化仍然是一项具有挑战性的课题。Hypoxia is an important indicator of many cancers and is closely related to the various physiological activities of tumors. At present, many substrate-based enzymatic fluorescent probes use targeted hypoxia activation methods to identify the location and expression level of hypoxic enzymes in cancer cells, and are used to detect abnormal levels of hypoxic enzymes in solutions, cancer cells and small animals. Detection and imaging are essential for the early diagnosis and monitoring and treatment of cancer. However, the concentration and expression level of hypoxic enzymes are usually very different in different cells and tumors, and even the same cells have different concentrations under different conditions. When a certain amount of traditional probes are used for detection, the two variables of unknown concentration of cofactor and enzyme will cause the probe emission to change significantly over time. It is difficult for traditional probes to exclude the interference of cofactor concentration on the detection result. The content and fluorescence response did not show a stable linear relationship. Therefore, the traditional probe method to quantitatively detect enzyme content and accurately distinguish the changes in enzyme activity between normal and disease states is still a challenging subject.
技术问题technical problem
对于传统的探针来说其对体内缺氧酶含量的检测高度依赖于辅因子的含量，通常在溶剂检测辅因子依赖性的缺氧酶实验中，研究人员通过加入大量过量（几十倍到几百倍摩尔量）的辅因子，从而消除酶催化反应中辅因子浓度干扰，使得辅因子浓度相控制不再制约缺氧酶催化底物的最大反应速率，响应信号只与酶含量相关。这种方法消除了辅因子的浓度对酶催化探针荧光开启速率的限制，以实现精确、快速的检测。但是细胞中检测时，不同种类的细胞，甚至在具有不同缺氧条件的相同细胞中，缺氧酶和相应辅因子的表达水平是不固定的，而且也无法实现溶剂实验中辅因子大大过量的条件。因此，将基于辅因子-底物的探针组合到一个超分子系统中，该体系中辅因子和底物两者之间具有明确的化学计量比，并且相互接近的主客体分子将促进电子转移效率，这将使探针与酶的反应速率不被内源辅因子浓度干扰，对于快速定量检测很有帮助。For traditional probes, the detection of hypoxic enzyme content in the body is highly dependent on the content of cofactors. Usually in the solvent detection of cofactor-dependent hypoxic enzyme experiments, researchers add a large excess (tens of times to Several hundred times the molar amount) of the cofactor to eliminate the interference of the cofactor concentration in the enzyme-catalyzed reaction, so that the cofactor concentration phase control no longer restricts the maximum reaction rate of the hypoxic enzyme catalyzed substrate, and the response signal is only related to the enzyme content. This method eliminates the limitation of the concentration of cofactors on the fluorescence opening rate of the enzyme-catalyzed probe, so as to achieve accurate and rapid detection. However, when testing in cells, different types of cells, even in the same cells with different hypoxic conditions, the expression levels of hypoxic enzymes and corresponding cofactors are not fixed, and it is impossible to achieve a large excess of cofactors in solvent experiments. condition. Therefore, the cofactor-substrate-based probes are combined into a supramolecular system in which there is a clear stoichiometric ratio between the cofactor and the substrate, and the host and guest molecules close to each other will promote electron transfer Efficiency, this will make the reaction rate of the probe and the enzyme not be disturbed by the concentration of endogenous cofactors, which is very helpful for rapid quantitative detection.
技术解决方案Technical solutions
本发明的目的是提出一种基于辅因子-底物探针平台用于肿瘤缺氧相关酶的快速定量检测，包括其制备方法和生物测试应用，金属-有机笼状配合物作为NADH模拟物与底物融合成准分子与酶催化反应，将多底物反应变为单底物反应的策略。通过减少底物种类，缩短各底物之间距离，促进反应动力学改变。The purpose of the present invention is to propose a cofactor-substrate probe platform for rapid quantitative detection of tumor hypoxia-related enzymes, including its preparation method and biological test application, and metal-organic cage complexes as NADH mimics and The fusion of the substrate into an excimer reacts with the enzyme to catalyze the reaction, which turns the multi-substrate reaction into a single-substrate reaction strategy. By reducing the types of substrates, shortening the distance between the substrates, and promoting the change of reaction kinetics.
本发明的技术方案如下：The technical scheme of the present invention is as follows:
基于辅因子-底物探针平台结构、基本配体组成单元及酶催化反应，如图1所示。Based on the cofactor-substrate probe platform structure, basic ligand components and enzyme-catalyzed reactions, as shown in Figure 1.
其中，主-客体探针Zn-MPB⊃L-NO 2包括金属-有机笼状配合物主体部分（Zn-MPB）和硝基还原酶荧光底物客体部分（L-NO 2）。主体部分由配体（式I）和金属离子（M n+）组成的M 3N 3型金属-有机笼状配合物。客体部分由荧光团和对硝基苄基连接（式II、式III）。探针的反应原理：在NTR作用下，主-客体探针的硝基苯基团被还原成氨基苯，并进一步分子内裂解，荧光底物结构明显发生变化，因此相应的荧光响应随之开启。金属-有机笼状配合物作为NADH模拟物与底物融合成准分子与酶催化反应，将多底物反应变为单底物反应，通过减少底物种类，缩短各底物之间距离，促进反应动力学改变。 Among them, the host-guest probe Zn-MPB⊃L-NO 2 includes the metal-organic cage complex body part (Zn-MPB) and the nitroreductase fluorescent substrate guest part (L-NO 2 ). The main part is an M 3 N 3 type metal-organic cage complex composed of ligand (formula I) and metal ion (M n+ ). The guest part is connected by a fluorophore and a p-nitrobenzyl group (Formula II, Formula III). The reaction principle of the probe: Under the action of NTR, the nitrophenyl group of the host-guest probe is reduced to aminobenzene and further intramolecular cleavage. The structure of the fluorescent substrate changes obviously, so the corresponding fluorescent response is turned on. . Metal-organic cage complexes are used as NADH mimics and substrates to fuse into excimers and enzyme-catalyzed reactions, turning multi-substrate reactions into single-substrate reactions, reducing the types of substrates, shortening the distance between substrates, and promoting The reaction kinetics changes.
式I中配体的二氢吡啶部分结构与NADH类似，作为NADH模拟物活性部分，用于质子和电子传递；The structure of the dihydropyridine part of the ligand in formula I is similar to NADH, as the active part of the NADH mimic, used for proton and electron transfer;
所述的配体制备方法，包括如下步骤：The method for preparing the ligand includes the following steps:
Figure 144192dest_path_image001
Figure 144192dest_path_image001
其中：R 1为提供配位原子的基团，R 1采用2-吡啶基、2-巯基苯基、2-噻吩、2-吡咯、喹啉或异喹啉；R 2为苯基、氢、卤素、氰基、苄氧基。 Among them: R 1 is a group that provides coordination atoms, R 1 is 2-pyridyl, 2-mercaptophenyl, 2-thiophene, 2-pyrrole, quinoline or isoquinoline; R 2 is phenyl, hydrogen, Halogen, cyano, benzyloxy.
（1）以丙炔酸甲酯、R 2-CHO、醋酸铵和冰醋酸为原料制备中间体1所示化合物；所述醋酸铵：丙炔酸甲酯：R 2-CHO的摩尔比为4:2:1； (1) Prepare the compound shown in Intermediate 1 with methyl propiolate, R 2 -CHO, ammonium acetate and glacial acetic acid as raw materials; the molar ratio of ammonium acetate: methyl propiolate: R 2 -CHO is 4 :2:1;
（2）以所示中间体1、4-（2-氯乙基）吗啉、碱、有机溶剂为原料制备中间体2所示化合物；所述碱为碳酸钠、碳酸钾、碳酸铯或有机碱；所述有机溶剂选自丙酮、N,N-二甲基甲酰胺或1,4-二氧六环；所述中间体1：4-（2-氯乙基）吗啉的摩尔比为1:1.1；(2) Prepare the compound shown in Intermediate 2 with the indicated intermediate 1, 4-(2-chloroethyl)morpholine, alkali, and organic solvent as raw materials; the alkali is sodium carbonate, potassium carbonate, cesium carbonate or organic Alkali; the organic solvent is selected from acetone, N,N-dimethylformamide or 1,4-dioxane; the molar ratio of the intermediate 1:4-(2-chloroethyl)morpholine is 1:1.1;
（3）以中间体2和80%水合肼反应，得到中间体3所示化合物；中间体2与80%水合肼的摩尔比1:5到1:10；(3) Intermediate 2 is reacted with 80% hydrazine hydrate to obtain the compound shown in Intermediate 3. The molar ratio of intermediate 2 to 80% hydrazine hydrate is 1:5 to 1:10;
（4）以中间体3和R 1-CHO在醋酸催化下反应，得到最终配体化合物式I结构；所述中间体3：R 1-CHO的摩尔比1:2。 (4) Intermediate 3 and R 1 -CHO are reacted under the catalysis of acetic acid to obtain the final ligand compound formula I structure; the molar ratio of intermediate 3: R 1 -CHO is 1:2.
其中：当R 1为2-吡啶基，R 2为苯基时，其制备方法如下： Wherein: when R 1 is 2-pyridyl and R 2 is phenyl, the preparation method is as follows:
Figure 530174dest_path_image002
Figure 530174dest_path_image002
（1）以丙炔酸甲酯、苯甲醛、醋酸铵和冰醋酸为原料制备中间体1所示化合物；所述醋酸铵：丙炔酸甲酯：苯甲醛的摩尔比为4:2:1； (1) Prepare the compound shown in Intermediate 1 with methyl propiolate, benzaldehyde, ammonium acetate and glacial acetic acid as raw materials; the molar ratio of ammonium acetate: methyl propiolate: benzaldehyde is 4:2:1 ；
（2）以所示中间体1、卤代化合物4-（2-氯乙基）吗啉、碱（过量，>2eq）、有机溶剂为原料制备中间体2所示化合物；所述碱为碳酸钠、碳酸钾、碳酸铯或有机碱；所述有机溶剂选自丙酮、N,N-二甲基甲酰胺或1,4-二氧六环；所述中间体1：4-（2-氯乙基）吗啉的摩尔比为1:1.1；(2) Prepare the compound shown in Intermediate 2 with the shown intermediate 1, halogenated compound 4-(2-chloroethyl)morpholine, base (excess, >2eq), and organic solvent as raw materials; the base is carbonic acid Sodium, potassium carbonate, cesium carbonate or organic base; the organic solvent is selected from acetone, N,N-dimethylformamide or 1,4-dioxane; the intermediate 1: 4-(2-chloro The molar ratio of ethyl)morpholine is 1:1.1;
（3）以中间体2和80%水合肼反应，得到中间体3所示化合物；中间体2与80%水合肼的摩尔比1:5到1:10；(3) Intermediate 2 is reacted with 80% hydrazine hydrate to obtain the compound shown in Intermediate 3. The molar ratio of intermediate 2 to 80% hydrazine hydrate is 1:5 to 1:10;
（4）以中间体3和2-醛基吡啶在醋酸催化下反应，得到最终配体化合物式I结构；所述中间体3：2-醛基吡啶的摩尔比1:2。(4) Intermediate 3 and 2-aldehyde pyridine are reacted under the catalysis of acetic acid to obtain the final ligand compound formula I structure; the molar ratio of the intermediate 3: 2-aldehyde pyridine is 1:2.
所述的荧光底物制备方法，包括如下步骤：The preparation method of the fluorescent substrate includes the following steps:
Figure 44332dest_path_image003
Figure 44332dest_path_image003
客体结构中，R 3为酶催化的荧光底物的荧光团部分，可以是且不仅限于2-苯基-3a，11b-二氢-1H-菲[9,10-d]咪唑基团、萘基团、喹喔啉基团、卟啉或卟吩基团等； In the guest structure, R 3 is the fluorophore part of the enzyme-catalyzed fluorescent substrate, which can be, but is not limited to, 2-phenyl-3a,11b-dihydro-1H-phenanthrene[9,10-d]imidazole group, naphthalene Group, quinoxaline group, porphyrin or porphin group, etc.;
（1）选择上述羟基、酚羟基或氨基荧光团R 3-OH、R 3-NH 2； (1) Select the above-mentioned hydroxyl, phenolic hydroxyl or amino fluorophore R 3 -OH, R 3 -NH 2 ;
（2）以上述荧光团和对硝基苄溴为原料，合成式II或式III结构，作为硝基还原酶荧光底物。(2) Using the above-mentioned fluorophore and p-nitrobenzyl bromide as raw materials, the structure of formula II or formula III is synthesized as the fluorescent substrate of nitroreductase.
当R 3为2-苯基-3a，11b-二氢-1H-菲[9,10-d]咪唑基时，可采用以下方法进行式Ⅱ或式Ⅲ的合成： When R 3 is 2-phenyl-3a,11b-dihydro-1H-phenanthrene[9,10-d]imidazolyl, the following methods can be used to synthesize Formula II or Formula III:
Figure 33016dest_path_image004
Figure 33016dest_path_image004
（1）以4-羟基苯甲醛（或4-氨基苯甲醛）、对硝基苄溴为原料，制备中间体4（或中间体5）所示化合物；所述4-羟基苯甲醛（或4-氨基苯甲醛）：对硝基苄溴的摩尔比为1:1；(1) Using 4-hydroxybenzaldehyde (or 4-aminobenzaldehyde) and p-nitrobenzyl bromide as raw materials to prepare the compound shown in Intermediate 4 (or Intermediate 5); the 4-hydroxybenzaldehyde (or 4 -Aminobenzaldehyde): the molar ratio of p-nitrobenzyl bromide is 1:1;
（2）以所示中间体4（或中间体5）、9, 10-菲咯醌、醋酸铵（>10 eq）为原料，醋酸作溶剂，加热回流制备合成式Ⅱ或式Ⅲ的结构，作为硝基还原酶荧光底物；所述中间体4（或中间体5）：9, 10-菲咯醌的摩尔比为3:1。(2) Using the indicated intermediate 4 (or intermediate 5), 9, 10-phenanthroquinone, and ammonium acetate (>10 eq) as raw materials, acetic acid as the solvent, heating and refluxing to prepare the structure of synthetic formula II or formula III, As a fluorescent substrate for nitroreductase; the molar ratio of intermediate 4 (or intermediate 5): 9, 10-phenanthroquinone is 3:1.
所述的辅因子-底物主客体探针平台制备方法，包括如下步骤：The preparation method of the cofactor-substrate host-guest probe platform includes the following steps:
1）选择上述式I中配体与所需配位金属盐分别溶于有机溶剂（乙腈、乙醇等），选取一定比例的这两种溶液充分混合搅拌8h，通过溶剂挥发结晶、溶剂扩散结晶或加入小极性互溶溶剂沉淀出金属-有机配合物，通过核磁、质谱或单晶结构等数据确认结构M 3N 3； 1) Select the ligand and the required coordination metal salt in the above formula I to dissolve in an organic solvent (acetonitrile, ethanol, etc.), select a certain proportion of these two solutions, mix and stir them for 8 hours, and crystallize through solvent volatilization, solvent diffusion or The metal-organic complex is precipitated by adding a small polar miscible solvent, and the structure M 3 N 3 is confirmed by data such as nuclear magnetic, mass spectrometry or single crystal structure;
2）以上述配体与金属盐得到的金属-有机笼状配合物和硝基还原酶荧光底物混合，通过质谱、核磁或单晶确认辅因子-底物主客体探针的结构，分析金属-有机笼状配合物与荧光底物的化学计量比。2) The metal-organic clathrate complex obtained from the above ligand and metal salt is mixed with the fluorescent substrate of nitroreductase, and the structure of the cofactor-substrate host-guest probe is confirmed by mass spectrometry, nuclear magnetism or single crystal, and the metal is analyzed -The stoichiometric ratio of organic cage complex to fluorescent substrate.
所述的辅因子-底物主客体探针在溶剂中检测硝基还原酶的应用。The application of the cofactor-substrate host-guest probe for detecting nitroreductase in a solvent.
一类用于溶剂中硝基还原酶定量快速检测的辅因子-底物主客体探针，测试方法：向新配置的硝基还原酶Tris-HCl缓冲液（10.0 mM, pH 7.4, 25℃）中加入辅因子-底物主客体探针（0-5 μM）进行荧光滴定实验和荧光动力学实验。具有辅因子NADH功能的主体金属-有机笼与具有荧光指示功能的底物分子形成的超分子探针，将底物和辅酶模拟物融合成准分子结构，使硝基还原酶原有的辅酶NADH和底物双底物催化过程优化为单底物过程。使硝基还原酶的检测快速达到平衡，不再受NADH含量的影响，因此检测的荧光强度变化只与硝基还原酶含量变化有关，实现快速对硝基还原酶的定量检测。A type of cofactor-substrate host-guest probe for the quantitative and rapid detection of nitroreductase in solvents, test method: to newly configured nitroreductase Tris-HCl buffer (10.0 mM, pH 7.4, 25℃) Add cofactor-substrate host-guest probe (0-5 μM) to perform fluorescence titration experiment and fluorescence kinetics experiment. A supramolecular probe formed by the main metal-organic cage with the function of cofactor NADH and the substrate molecule with the function of fluorescent indicator, fusion of the substrate and the coenzyme mimic into an excimer structure, making the original coenzyme NADH of nitroreductase The dual-substrate catalytic process with substrate is optimized as a single-substrate process. Make the detection of nitroreductase quickly reach equilibrium, no longer affected by NADH content, so the detected fluorescence intensity change is only related to the change of nitroreductase content, realizing rapid quantitative detection of nitroreductase.
所述的辅因子-底物主客体探针在细胞中检测硝基还原酶的应用。The application of the cofactor-substrate host-guest probe for detecting nitroreductase in cells.
一类用于细胞中硝基还原酶定量快速检测的辅因子-底物主客体探针，测试方法：配置DMEM培养基（加入FBS 10%， 双抗1%）用于培养MCF-7和231细胞，配置1640培养基（加入FBS 10%， 双抗1%）用于培养A549细胞。设置不同氧含量环境（20%，8%，0.1%）培养细胞6-12h后，再用辅因子-底物超分子探针进行孵育1 min，进行不同氧含量下细胞的激光共聚焦荧光成像实验。相同氧含量环境（例如都在0.1% O 2）下培养细胞6-12 h，然后再辅因子-底物超分子探针对细胞进行不同的孵育时间（如0-10 min），开展细胞荧光成像随时间变化实验。 A type of cofactor-substrate host-guest probe for the quantitative and rapid detection of nitroreductase in cells, test method: configure DMEM medium (add FBS 10%, double antibody 1%) for culturing MCF-7 and 231 Cells, configure 1640 medium (add FBS 10%, double antibody 1%) for culturing A549 cells. After setting different oxygen content environment (20%, 8%, 0.1%) to culture cells for 6-12h, incubate with cofactor-substrate supramolecular probe for 1 min to perform laser confocal fluorescence imaging of cells under different oxygen content experiment. Culture the cells for 6-12 h under the same oxygen content environment (for example, all in 0.1% O 2 ), and then use the cofactor-substrate supramolecular probe to incubate the cells for different incubation times (for example, 0-10 min) to develop cell fluorescence Imaging changes over time experiment.
所述的辅因子-底物主客体探针在活体动物中检测硝基还原酶的应用。The application of the cofactor-substrate host-guest probe for detecting nitroreductase in living animals.
一类用于小鼠活体内硝基还原酶定量快速检测的辅因子-底物主客体探针，测试方法：利用所需的癌细胞对裸鼠进行种瘤，在成瘤期对小鼠进行注射中所述的辅因子-底物超分子探针（一般注射浓度和范围为10-200 μM、50-200 μL），进行小鼠瘤体荧光随时间变化成像实验。A type of cofactor-substrate host-guest probe for the quantitative and rapid detection of nitroreductase in mice Inject the cofactor-substrate supramolecular probe described in the injection (generally, the injection concentration and range are 10-200 μM, 50-200 μL), and perform the imaging experiment of mouse tumor fluorescence over time.
本发明的有益效果：The beneficial effects of the present invention:
通过将缺氧酶中最常见的辅因子NADH模拟物引入金属有机胶囊Zn-MPB的骨架中，我们在此提出了一种基于辅因子-底物的超分子探针（Zn-MPB⊃L-NO 2），用于水溶液和生物系统中对缺氧酶硝基还原酶（NTR）的催化活性进行生物示踪成像。Zn-MPB⊃L-NO 2作为准分子结构可以直接一起与NTR反应，将天然NTR与硝基底物以及NADH的双底物酶催化过程优化成更简单的单底物催化过程。该方法在缺氧酶检测中消除了不同浓度对检测结果的干扰，荧光强度和酶含量之间的线性关系有望达到快速平衡。同时，将底物包含在含有NADH的宿主中可以减少外部NADH的转运时间，并缩短底物与辅因子活性位点之间的距离，从而能够对缺氧酶的含量水平进行快速的信号响应。 By introducing NADH mimics, the most common cofactor in hypoxic enzymes, into the framework of metal organic capsule Zn-MPB, we here propose a cofactor-substrate-based supramolecular probe (Zn-MPB⊃L- NO 2 ), used for biological tracer imaging of the catalytic activity of hypoxic enzyme nitroreductase (NTR) in aqueous solutions and biological systems. Zn-MPB⊃L-NO 2 as a quasi-molecular structure can directly react with NTR together, optimizing the double-substrate enzymatic catalysis process of natural NTR, nitrate 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 achieve a rapid balance. At the same time, the inclusion of the substrate in the host containing NADH can reduce the transit time of external NADH and shorten the distance between the substrate and the active site of the cofactor, thereby enabling rapid signal response to the level of hypoxic enzyme.
1、该药品主要应用对象：主要适用于仿生催化、肿瘤成像诊断治疗等领域。1. The main application objects of the drug: mainly applicable to the fields of biomimetic catalysis, tumor imaging diagnosis and treatment.
2、市场及价格定位：由于生活水平的提高，人类寿命得到大幅提高，而老年人群里更易患癌症；另一方面，现代人们的不健康生活方式，吸烟、感染、职业暴露、环境污染、不合理膳食、遗传因素等都会导致癌症发病率增加。2018年CA: A Cancer Journal for Clinicians（影响因子223）期刊上一篇文章总结了当年全球肿瘤统计情况，结果显示全球185个国家地区估计有1819万癌症新增病例以及960万癌症死亡病例。而我国每天约有1万人确诊癌症，相当于平均每分钟就有7个人确诊癌症，我国癌症患病率处于国际中等偏上水平。癌症的早期诊断对于患者的治愈率和存活时间有着巨大的提升，该发明主要针对广谱的癌症标志物缺氧酶的活性的准确检测，来诊断是否患癌。另一方面，本发明还可以作为指导肿瘤切除手术显像剂。可以说在医疗健康领域有着广阔的应用前景。 2. Market and price positioning: Due to the improvement of living standards, human life span has been greatly improved, and the elderly are more susceptible to cancer; on the other hand, modern people's unhealthy lifestyles, smoking, infection, occupational exposure, environmental pollution, and unreasonable Diet, genetic factors, etc. will cause the incidence of cancer to increase. In 2018, an article in CA: A Cancer Journal for Clinicians (Impact Factor 223) summarized the global tumor statistics that year. The results showed that there were an estimated 18.19 million new cancer cases and 9.6 million cancer deaths in 185 countries around the world. In my country, about 10,000 people are diagnosed with cancer every day, which is equivalent to an average of 7 people diagnosed with cancer every minute. The prevalence of cancer in my country is at an internationally higher level. Early diagnosis of cancer has greatly improved the cure rate and survival time of patients. The invention is mainly aimed at the accurate detection of the activity of a broad-spectrum cancer marker hypoxic enzyme to diagnose cancer. On the other hand, the present invention can also be used as an imaging agent for guiding tumor resection surgery. It can be said that it has broad application prospects in the medical and health field.
3、应用效益：癌症早期症状不明显，目前对早期癌症的筛查比较复杂昂贵，因此很多患者往往错过最佳治疗时间。将本发明应用到癌症早期诊断可快速对病症进行筛查，而且廉价易行，使得普通民众都能用得起。本专利所建立的方法将有助于解决这一问题，因此具有巨大的潜在经济价值。3. Application benefits: Early cancer symptoms are not obvious, and the current screening for early cancer is more complicated and expensive, so many patients often miss the best treatment time. The application of the present invention to the early diagnosis of cancer can quickly screen for diseases, and is cheap and easy to implement, making it affordable for ordinary people. The method established by this patent will help to solve this problem, so it has huge potential economic value.
4、推广价值：据统计数据显示，全球癌症药物市场规模从2013年的729亿美元扩大至2017年的1106亿美元，复合年增长率达12.8%。随着癌症药物市场的进一步扩大，预计2030年以前，全球肿瘤市场销售额将超4000亿美元。本专利涉及的化合物目前还未形成产品，其相关产品一旦市场化，其市场价值将达到百亿元级别。4. Promotional value: According to statistics, the global cancer drug market has expanded from US$72.9 billion in 2013 to US$110.6 billion in 2017, with a compound annual growth rate of 12.8%. With the further expansion of the cancer drug market, it is estimated that by 2030, the global oncology market sales will exceed US$400 billion. The compound involved in this patent has not yet formed a product, and once its related products are marketed, their market value will reach the level of tens of billions of yuan.
附图说明Description of the drawings
图1是基于辅因子-底物探针平台结构、基本配体组成单元及酶催化反应图。Figure 1 is a diagram based on the structure of the cofactor-substrate probe platform, the basic ligand components and the enzyme-catalyzed reaction.
图2是Zn-MPB⊃L-NO 2的NOESY核磁谱图以及干扰物对其荧光选择性实验图。 Figure 2 is the NOESY NMR spectrum of Zn-MPB⊃L-NO 2 and the experimental graph of the fluorescence selectivity of the interferent.
图3是L-NO 2或Zn-MPB⊃L-NO 2与NTR反应的荧光动力学测试图。 Figure 3 is the fluorescence kinetic test diagram of the reaction between L-NO 2 or Zn-MPB⊃L-NO 2 and NTR.
图4是CCK8实验分析Zn-MPB和L-NO 2分别对MCF-7细胞的毒性实验图。 Figure 4 is a CCK8 experiment to analyze the toxicity of Zn-MPB and L-NO 2 to MCF-7 cells.
图5是L-NO 2和Zn-MPB⊃L-NO 2对不同缺氧处理MCF-7细胞的荧光成像图。 Figure 5 is the fluorescence imaging of L-NO 2 and Zn-MPB⊃L-NO 2 on MCF-7 cells treated with different hypoxia.
图6是L-NO 2和Zn-MPB⊃L-NO 2对0.1%缺氧处理的MCF-7细胞不同孵育时间的荧光成像图。 Figure 6 is the fluorescence imaging images of L-NO 2 and Zn-MPB⊃L-NO 2 on MCF-7 cells treated with 0.1% hypoxia at different incubation times.
图7是注射L-NO 2以及Zn-MPB⊃L-NO 2对MCF-7成瘤小鼠荧光随时间成像图。 Fig. 7 is a graph of fluorescence imaging of MCF-7 tumor-forming mice injected with L-NO 2 and Zn-MPB⊃L-NO 2 over time.
具体实施方式Detailed ways
本发明提出了一种基于辅因子-底物探针平台并用于肿瘤缺氧酶的超快定量检测的策略，该主客体探针在溶剂、细胞以及活体实验中表现出超快定量检测的特征，下面结合实施例对本发明做进一步介绍。The present invention proposes a strategy based on a cofactor-substrate probe platform and used for ultra-fast quantitative detection of tumor hypoxic enzymes. The host-guest probe exhibits the characteristics of ultra-fast quantitative detection in solvent, cell and in vivo experiments. In the following, the present invention will be further introduced in conjunction with the embodiments.
实施例1：辅因子-底物结构的超分子主客体探针的合成制备Example 1: Synthesis and preparation of supramolecular host-guest probes with cofactor-substrate structure
所述的Zn-MPB⊃L-NO 2合成制备方法包括主体金属有机笼状配合物合成、NTR荧光底物合成以及Zn-MPB⊃L-NO 2的制备。 The synthetic preparation method of Zn-MPB⊃L-NO 2 includes the synthesis of host metal-organic cage complex, the synthesis of NTR fluorescent substrate and the preparation of Zn-MPB⊃L-NO 2.
1.      NTR荧光底物合成，包括如下步骤：1. The synthesis of NTR fluorescent substrate includes the following steps:
Figure 316230dest_path_image005
Figure 316230dest_path_image005
（1）以对羟基苯甲醛和对硝基苄溴为原料合成化合物1(1) Synthesis of compound 1 using p-hydroxybenzaldehyde and p-nitrobenzyl bromide as raw materials
室温下，将4-羟基苯甲醛（1 g，8.20 mmol）、对硝基苄基溴（1.7 g，8.20 mmol）、碳酸钾（1.7 g，12.30 mmol）和N，N-二甲基甲酰胺（10 ml）的混合物在60℃下加热2小时。TCL监测反应进程，反应完成后将混合物冷却至室温，并倒入冷水（50 ml）中。抽滤得到灰白色固体，用水（10 ml×3）洗涤，真空浓缩并通过柱层析纯化，得到4-（4-硝基-苄氧基）-苯甲醛，产量1.56 g，产率74％。 1H NMR (400 MHz, CDCl 3) δ 9.91 (s, 1H), 8.27 (d, J = 8.3 Hz, 2H), 7.87 (d, J = 8.4 Hz, 2H), 7.62 (d, J = 8.3 Hz, 2H), 7.09 (d, J = 8.4 Hz, 2H), 5.27 (s, 2H). 13C NMR (101 MHz, CDCl 3) δ 189.61, 161.91, 146.79, 142.26, 131.05, 129.64, 126.64, 122.95, 114.07, 67.83. ESI-MS m/z: [M+H] calculated for C 14H 12NO 4 + 258.0761, found 258.0767。 At room temperature, combine 4-hydroxybenzaldehyde (1 g, 8.20 mmol), p-nitrobenzyl bromide (1.7 g, 8.20 mmol), potassium carbonate (1.7 g, 12.30 mmol) and N,N-dimethylformamide (10 ml) of the mixture was heated at 60°C for 2 hours. TCL monitored the progress of the reaction. After the reaction was completed, the mixture was cooled to room temperature and poured into cold water (50 ml). The off-white solid was obtained by suction filtration, washed with water (10 ml×3), concentrated in vacuo and purified by column chromatography to obtain 4-(4-nitro-benzyloxy)-benzaldehyde with a yield of 1.56 g and a yield of 74%. 1 H NMR (400 MHz, CDCl 3 ) δ 9.91 (s, 1H), 8.27 (d, J = 8.3 Hz, 2H), 7.87 (d, J = 8.4 Hz, 2H), 7.62 (d, J = 8.3 Hz , 2H), 7.09 (d, J = 8.4 Hz, 2H), 5.27 (s, 2H). 13 C NMR (101 MHz, CDCl 3 ) δ 189.61, 161.91, 146.79, 142.26, 131.05, 129.64, 126.64, 122.95, 114.07, 67.83. ESI-MS m/z: [M+H] calculated for C 14 H 12 NO 4 + 258.0761, found 258.0767.
（2）以化合物1和9, 10-菲咯醌为原料合成NTR荧光底物L-NO 2 (2) Synthesis of NTR fluorescent substrate L-NO 2 using compounds 1 and 9, 10-phenanthroquinone as raw materials
将9, 10-菲咯醌（166 mg，0.8 mmol），4-（4-硝基苄氧基）-苯甲醛（617 mg，2.4 mmol）和乙酸铵（123 mg，16 mmol）的混合物在冰醋酸（4 mL）中加热搅拌回流。反应完成后，将冷却至室温，抽滤收集所得浅黄色固体，并用过量的水和甲醇洗涤除去起始原料，DMSO中重结晶，产量295 mg，收率83％。 1H NMR (500 MHz, DMSO) δ13.32 (s, 1H), 8.85 (dd, J = 16.6, 8.3 Hz, 2H), 8.59 (d, J = 7.8 Hz, 1H), 8.54 (d, J = 7.9 Hz, 1H), 8.28 (dd, J = 8.7, 1.9 Hz, 4H), 7.78 (d, J = 8.5 Hz, 2H), 7.76-7.70 (m, 2H), 7.66-7.61 (m, 2H), 7.28 (d, J = 8.8 Hz, 2H), 5.39 (s, 2H). 13C NMR (126 MHz, DMSO) δ 158.81, 149.08, 147.06, 144.76, 136.88, 128.30, 127.75, 127.48, 127.43, 127.40, 127.03, 126.96, 125.10, 124.97, 124.03, 123.67, 123.63, 123.60, 122.39, 121.82, 115.25, 68.20. ESI-MS m/z: [M-H] calculated for C 28H 18N 3O 3 - 446.1499, found 446.1490。 Combine 9, 10-phenanthroquinone (166 mg, 0.8 mmol), 4-(4-nitrobenzyloxy)-benzaldehyde (617 mg, 2.4 mmol) and ammonium acetate (123 mg, 16 mmol) in a mixture Heat and stir to reflux in glacial acetic acid (4 mL). After the reaction was completed, the mixture was cooled to room temperature, and the obtained light yellow solid was collected by suction filtration, washed with excess water and methanol to remove the starting materials, and recrystallized from DMSO. The yield was 295 mg, and the yield was 83%. 1 H NMR (500 MHz, DMSO) δ13.32 (s, 1H), 8.85 (dd, J = 16.6, 8.3 Hz, 2H), 8.59 (d, J = 7.8 Hz, 1H), 8.54 (d, J = 7.9 Hz, 1H), 8.28 (dd, J = 8.7, 1.9 Hz, 4H), 7.78 (d, J = 8.5 Hz, 2H), 7.76-7.70 (m, 2H), 7.66-7.61 (m, 2H), 7.28 (d, J = 8.8 Hz, 2H), 5.39 (s, 2H). 13 C NMR (126 MHz, DMSO) δ 158.81, 149.08, 147.06, 144.76, 136.88, 128.30, 127.75, 127.48, 127.43, 127.40, 127.03 ., 126.96, 125.10, 124.97, 124.03, 123.67, 123.63, 123.60, 122.39, 121.82, 115.25, 68.20 ESI-MS m / z: [MH] calculated for C 28 H 18 N 3 O 3 - 446.1499, found 446.1490.
2.      主体金属有机笼状配合物Zn-MPB合成，包括如下步骤：2. The synthesis of the host metal-organic cage complex Zn-MPB includes the following steps:
Figure 732168dest_path_image006
Figure 732168dest_path_image006
（1）以2-吗啉乙醇、二氯亚砜为原料制备如图化合物2(1) Use 2-morpholine ethanol and thionyl chloride as raw materials to prepare compound 2 as shown in the figure
将二氯亚砜（4.76 g，40 mmol）和10 ml甲苯加入到圆底烧瓶中，并在冰浴中搅拌15分钟。 然后，将2-吗啉-4-基-乙醇（1.31 g，10 mmol）溶解在甲苯（5 ml）中，并用滴液漏斗缓慢滴加到冰浴的圆底烧瓶中。0℃下反应2小时后，将逐渐升温到90℃下再回流8小时。反应完成后，将混合物在冷盐水（50 ml）中淬灭。然后将溶液的pH调节至10，用乙酸乙酯萃取，柱层析纯化，得到浅褐色油状化合物2（4-（2-氯乙基）-吗啉），产量1.33 g，收率89％。 1H NMR (400 MHz, CDCl 3) δ 3.75 – 3.69 (m, 4H), 3.59 (t, J = 6.9 Hz, 2H), 2.72 (t, J = 6.9 Hz, 2H), 2.55 – 2.48 (m, 4H). 13C NMR (101 MHz, CDCl 3) δ 66.77, 60.13, 53.52, 40.63. ESI-MS m/z: [M+H] calculated for C 6H 13ClNO + 150.0680, found 150.0677。 Thionyl chloride (4.76 g, 40 mmol) and 10 ml of toluene were added to the round bottom flask and stirred in an ice bath for 15 minutes. Then, 2-morpholin-4-yl-ethanol (1.31 g, 10 mmol) was dissolved in toluene (5 ml) and slowly dropped into an ice-bathed round bottom flask using a dropping funnel. After reacting at 0°C for 2 hours, the temperature is gradually increased to 90°C and refluxed for 8 hours. After the reaction was completed, the mixture was quenched in cold brine (50 ml). Then the pH of the solution was adjusted to 10, extracted with ethyl acetate, and purified by column chromatography to obtain the light brown oily compound 2 (4-(2-chloroethyl)-morpholine), the yield was 1.33 g, and the yield was 89%. 1 H NMR (400 MHz, CDCl 3 ) δ 3.75 – 3.69 (m, 4H), 3.59 (t, J = 6.9 Hz, 2H), 2.72 (t, J = 6.9 Hz, 2H), 2.55 – 2.48 (m, 4H). 13 C NMR (101 MHz, CDCl 3 ) δ 66.77, 60.13, 53.52, 40.63. ESI-MS m/z: [M+H] calculated for C 6 H 13 ClNO + 150.0680, found 150.0677.
（2）以醋酸铵、苯甲醛和丙炔酸甲酯为原料制备化合物3(2) Preparation of compound 3 with ammonium acetate, benzaldehyde and methyl propiolate as raw materials
将丙酸甲酯（1.68 g，20 mmol），苯甲醛（1.06 g，10 mmol）和乙酸铵（2.31 g，30 mmol）在冰醋酸（4.0 mL）中80℃回流12小时。 待反应完成冷却后，将固体产物抽滤并用Et 2O（10 mL×3）洗涤，得到黄色粉末粗产物化合物3，将其用乙醇重结晶，产量1.32 g，产率48％。 1H NMR (400 MHz, DMSO) δ 7.39 (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). 13C NMR (126 MHz, DMSO) δ 166.75, 147.11, 135.08, 127.95, 127.50, 126.15, 105.57, 50.82, 36.85. NMR (126 MHz, DMSO). ESI-MS m/z: [M+H] calculated for C 15H 16NO 4 + 274.1074, found 274.1074。 Methyl propionate (1.68 g, 20 mmol), benzaldehyde (1.06 g, 10 mmol) and ammonium acetate (2.31 g, 30 mmol) were refluxed at 80°C for 12 hours in glacial acetic acid (4.0 mL). After the reaction was completed and cooled, the solid product was suction filtered and washed with Et 2 O (10 mL×3) to obtain the crude yellow powder compound 3, which was recrystallized from ethanol. The yield was 1.32 g and the yield was 48%. 1 H NMR (400 MHz, DMSO) δ 7.39 (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 (126 MHz, DMSO) δ 166.75, 147.11, 135.08, 127.95, 127.50, 126.15, 105.57, 50.82, 36.85. NMR (126 MHz, DMSO). ESI-MS m/z: [ M+H] calculated for C 15 H 16 NO 4 + 274.1074, found 274.1074.
（3）以化合物2和化合物3为原料制备化合物4（DMPDD）(3) Preparation of compound 4 (DMPDD) using compound 2 and compound 3 as raw materials
将化合物2（1.79 g，12 mmol），化合物3（2.73 g，10 mmol）和碳酸钾（0.35 g，25 mmol）在丙酮中回流12小时以上，TCL检测反应进程，反应完成后冷却并过滤，收集有机相，并将固体用二氯甲烷充分洗涤， 收集有机相。完成后，除去溶剂，并将粗产物通过柱色谱法纯化（乙酸乙酯/石油醚，1：10，v/v），得到浅黄色固体DMPDD，产量2.04 g，收率53％。 1H NMR (400 MHz, CDCl 3) δ 7.36 – 7.31 (m, 2H), 7.25 – 7.20 (m, 4H), 7.17 – 7.12 (m, 1H), 4.87 (s, 1H), 3.75 – 3.71 (m, 4H), 3.63 (s, 6H), 3.50 (t, J = 6.1 Hz, 2H), 2.63 (t, J = 6.1 Hz, 2H), 2.56 – 2.52 (m, 4H). 13C NMR (101 MHz, CDCl 3) δ 167.36, 146.69, 137.92, 128.04, 128.00, 126.45, 108.19, 66.85, 58.28, 53.62, 51.63, 51.29, 37.05. ESI-MS m/z: [M+H] calculated for C 21H 27N 2O 5 + 387.1914, found 387.1911. Reflux compound 2 (1.79 g, 12 mmol), compound 3 (2.73 g, 10 mmol) and potassium carbonate (0.35 g, 25 mmol) in acetone for more than 12 hours. TCL detects the progress of the reaction. After the reaction is completed, it is cooled and filtered. The organic phase was collected, and the solid was washed thoroughly with dichloromethane, and the organic phase was collected. After completion, the solvent was removed, and the crude product was purified by column chromatography (ethyl acetate/petroleum ether, 1:10, v/v) to obtain a pale yellow solid DMPDD with a yield of 2.04 g and a yield of 53%. 1 H NMR (400 MHz, CDCl 3 ) δ 7.36 – 7.31 (m, 2H), 7.25 – 7.20 (m, 4H), 7.17 – 7.12 (m, 1H), 4.87 (s, 1H), 3.75 – 3.71 (m , 4H), 3.63 (s, 6H), 3.50 (t, J = 6.1 Hz, 2H), 2.63 (t, J = 6.1 Hz, 2H), 2.56 – 2.52 (m, 4H). 13 C NMR (101 MHz , CDCl 3 ) δ 167.36, 146.69, 137.92, 128.04, 128.00, 126.45, 108.19, 66.85, 58.28, 53.62, 51.63, 51.29, 37.05. ESI-MS m/z: [M+H] calculated for C 21 H 27 N 2 O 5 + 387.1914, found 387.1911.
（4）以化合物4和水合肼为原料，合成酰肼化合物5(4) Using compound 4 and hydrazine hydrate as raw materials, synthesize hydrazide compound 5
将80％水合肼（20 ml）和化合物4（3.86 g，10 mmol）的混合溶液在120℃下搅拌回流12小时以上。 反应完成后，将其收集并真空干燥。将粗产物通过快速柱色谱法纯化（CH 2Cl 2/CH 3OH，40∶1，v/v），得到浅黄色固体化合物5。产量3.46 g，收率90％。 1H NMR (400 MHz, DMSO) δ 8.63 (br, 2H), 7.31 (d, J = 7.1 Hz, 2H), 7.17 (t, J = 7.4 Hz, 2H), 7.12 (s, 2H), 7.08 (t, J = 7.3 Hz, 1H), 4.98 (s, 1H), 4.41 (br, 4H), 3.61 – 3.55 (m, 4H), 3.49 (t, J = 5.9 Hz, 2H), 2.54 – 2.48 (m, 2H), 2.44 (s, 4H). 13C NMR (101 MHz, CDCl 3) δ 172.08, 139.20, 132.99, 132.93, 131.18, 112.88, 71.53, 58.40, 55.57, 40.60. ESI-MS m/z: [M+H] calculated for C 19H 27N 6O 3 + 387.2139, found 387.2134。 A mixed solution of 80% hydrazine hydrate (20 ml) and compound 4 (3.86 g, 10 mmol) was stirred and refluxed at 120° C. for more than 12 hours. After the reaction was completed, it was collected and dried under vacuum. The crude product was purified by flash column chromatography (CH 2 Cl 2 /CH 3 OH, 40:1, v/v) to obtain compound 5 as a pale yellow solid. The yield is 3.46 g, and the yield is 90%. 1 H NMR (400 MHz, DMSO) δ 8.63 (br, 2H), 7.31 (d, J = 7.1 Hz, 2H), 7.17 (t, J = 7.4 Hz, 2H), 7.12 (s, 2H), 7.08 ( t, J = 7.3 Hz, 1H), 4.98 (s, 1H), 4.41 (br, 4H), 3.61 – 3.55 (m, 4H), 3.49 (t, J = 5.9 Hz, 2H), 2.54 – 2.48 (m , 2H), 2.44 (s, 4H). 13 C NMR (101 MHz, CDCl 3 ) δ 172.08, 139.20, 132.99, 132.93, 131.18, 112.88, 71.53, 58.40, 55.57, 40.60. ESI-MS m/z: [ M+H] calculated for C 19 H 27 N 6 O 3 + 387.2139, found 387.2134.
（5）以化合物为原料合成配体H 2MPB (5) Synthesis of ligand H 2 MPB using compounds as raw materials
将化合物5（3.86 g，10 mmol）加入到含有2-吡啶醛（2.68 g，25 mmol）的乙醇溶液（50 mL）中。 加入几滴乙酸后，将混合物在80℃下加热搅拌12小时以上。反应完成后冷却过滤收集浅黄色固体产物，用乙醇洗涤，真空干燥并从甲醇中重结晶，得到纯的化合物H 2MPB。产量4.95 g，收率88％。 1H NMR (500 MHz, DMSO) δ11.36 (s, 2H), 8.57 (d, J = 4.8 Hz, 2H), 8.29 (s, 2H), 7.86 (d, J = 7.8 Hz, 2H), 7.83 (td, J = 7.6, 1.7 Hz, 2H), 7.47 (s, 2H), 7.38 – 7.36 (m, 2H), 7.35 (s, 2H), 7.22 (t, J = 7.6 Hz, 2H), 7.12 (t, J = 7.3 Hz, 1H), 5.31 (s, 1H), 3.64 (s, 2H), 3.60 (s, 4H), 2.64 (s, 2H), 2.50 (s, 4H). 13C NMR (126 MHz, DMSO) δ 163.82, 153.52, 149.37, 146.96, 145.03, 136.65, 135.86, 127.92, 127.65, 126.10, 123.91, 119.51, 108.26, 66.28, 57.75, 53.20, 50.81, 35.80. ESI-MS m/z: [M+H] calculated for C 31H 33N 8O 3 + 565.2670, found 565.2670。 Compound 5 (3.86 g, 10 mmol) was added to an ethanol solution (50 mL) containing 2-pyridine aldehyde (2.68 g, 25 mmol). After adding a few drops of acetic acid, the mixture was heated and stirred at 80°C for more than 12 hours. After the completion of the reaction, the light yellow solid product was collected by cooling and filtration, washed with ethanol, dried in vacuum and recrystallized from methanol to obtain the pure compound H 2 MPB. The yield was 4.95 g, and the yield was 88%. 1 H NMR (500 MHz, DMSO) δ11.36 (s, 2H), 8.57 (d, J = 4.8 Hz, 2H), 8.29 (s, 2H), 7.86 (d, J = 7.8 Hz, 2H), 7.83 (td, J = 7.6, 1.7 Hz, 2H), 7.47 (s, 2H), 7.38 – 7.36 (m, 2H), 7.35 (s, 2H), 7.22 (t, J = 7.6 Hz, 2H), 7.12 ( t, J = 7.3 Hz, 1H), 5.31 (s, 1H), 3.64 (s, 2H), 3.60 (s, 4H), 2.64 (s, 2H), 2.50 (s, 4H). 13 C NMR (126 MHz, DMSO) δ 163.82, 153.52, 149.37, 146.96, 145.03, 136.65, 135.86, 127.92, 127.65, 126.10, 123.91, 119.51, 108.26, 66.28, 57.75, 53.20, 50.81, 35.80. ESI-MS m/z: [M +H] calculated for C 31 H 33 N 8 O 3 + 565.2670, found 565.2670.
（6）Zn-MPB的组装合成(6) Assembly and synthesis of Zn-MPB
将六水合高氯酸锌（18.6 mg，0.05 mmol）和H 2MPB（28.5 mg，0.05 mmol）溶于CH 3CN（10 ml）中，得到黄色澄清溶液。搅拌4小时后，将溶液倒入乙醚中，得到黄色沉淀，离心分离并真空干燥，得到橘黄色粉末，产量35 mg，产率75％。 1H NMR (400 MHz, DMSO) δ 11.45 (br, 6H), 8.44 (m, 12H), 7.93 (m, 12H), 7.47 (m, 12H), 7.35 (d, J = 5.8 Hz, 6H), 7.22 (s, 6H), 7.14 (t, J = 7.2 Hz, 3H), 5.32 (s, 3H), 3.77 (s, 6H), 3.62 (s, 12H), 2.77 – 2.50 (m, 18H).ESI MS m/z: [H 2Zn 3(MPB) 3] 2+ = 942.54。 Dissolve zinc perchlorate hexahydrate (18.6 mg, 0.05 mmol) and H 2 MPB (28.5 mg, 0.05 mmol) in CH 3 CN (10 ml) to obtain a clear yellow solution. After stirring for 4 hours, the solution was poured into ether to obtain a yellow precipitate, which was centrifuged and dried in vacuum to obtain an orange powder with a yield of 35 mg and a yield of 75%. 1 H NMR (400 MHz, DMSO) δ 11.45 (br, 6H), 8.44 (m, 12H), 7.93 (m, 12H), 7.47 (m, 12H), 7.35 (d, J = 5.8 Hz, 6H), 7.22 (s, 6H), 7.14 (t, J = 7.2 Hz, 3H), 5.32 (s, 3H), 3.77 (s, 6H), 3.62 (s, 12H), 2.77 – 2.50 (m, 18H).ESI MS m/z: [H 2 Zn 3 (MPB) 3 ] 2+ = 942.54.
3. Zn-MPB⊃L-NO 2的制备，包括如下步骤： 3. The preparation of Zn-MPB⊃L-NO 2 includes the following steps:
将得到的Zn-MPB化合物与L-NO 2分别溶解在氘代DMSO中，配置等浓度的溶液，并1:1混合。其NOESY核磁谱如图2A所示。L-NO 2的N-H质子与Zn-MPB化合物中多个质子间具有相互租用，说明形成了主客体结构。ESI MS m/z: H 2Zn 3(MPB) 3(L-NO 2)] 2+ = 1166.17. The obtained Zn-MPB compound and L-NO 2 were separately dissolved in deuterated DMSO, and a solution of equal concentration was prepared and mixed 1:1. The NOESY NMR spectrum is shown in Figure 2A. The NH protons of L-NO 2 and multiple protons in the Zn-MPB compound have mutual leases, indicating that a host-guest structure is formed. ESI MS m/z: H 2 Zn 3 (MPB) 3 (L-NO 2 )) 2+ = 1166.17.
实施例2：基于辅因子-底物探针平台的溶剂性质测试Example 2: Solvent property test based on the cofactor-substrate probe platform
辅因子-底物探针平台（Zn-MPB⊃L-NO 2）的溶剂测试参照文献方法进行，并设置L-NO 2为对照组，具体步骤为： The solvent test of the cofactor-substrate probe platform (Zn-MPB⊃L-NO 2 ) is carried out according to the literature method, and L-NO 2 is set as the control group. The specific steps are as follows:
（1）Zn-MPB⊃L-NO 2稳定性 (1) Zn-MPB⊃L-NO 2 stability
辅因子-底物超分子探针平台的稳定性决定了其应用能力和范围。在氘代DMSO中测试Zn-MPB和L-NO 2混合物（1:1）的H-H相互作用，发现L-NO 2的N-H与Zn-MPB的其他H有明显的相互作用，说明形成了Zn-MPB⊃L-NO 2主客体结构。Zn-MPB⊃L-NO 2在水性环境中的稳定性对于其在生物测试应用中起着决定性作用。在DMSO Tris-HCl 缓冲液 (v/v, DMSO 90%, 25℃)中进行微量量热滴定和紫外-可见光吸收滴定实验。通过微量量热滴定计算出结合自由能为-9.43 kcal/mol，结合常数为8.33×10 6 M − 1，说明的Zn-MPB和L-NO 2相互作用。通过紫外-可见光吸收滴定计算出结合常数为1.70×10 6 M − 1。两种实验计算出的结合常数具有良好的匹配性，说明在测试条件下，Zn-MPB⊃L-NO 2稳定存在。 The stability of the cofactor-substrate supramolecular probe platform determines its application capability and scope. The HH interaction of Zn-MPB and L-NO 2 mixture (1:1) was tested in deuterated DMSO, and it was found that the NH of L-NO 2 had obvious interaction with other H of Zn-MPB, indicating that Zn- was formed. MPB⊃L-NO 2 host-object structure. The stability of Zn-MPB⊃L-NO 2 in an aqueous environment plays a decisive role in its application in biological testing. In DMSO Tris-HCl buffer (v/v, DMSO 90%, 25℃), microcalorimetric titration and ultraviolet-visible light absorption titration experiments were carried out. The free energy of binding calculated by microcalorimetric titration is -9.43 kcal/mol, and the binding constant is 8.33×10 6 M − 1 , indicating the interaction between Zn-MPB and L-NO 2. The binding constant was calculated by ultraviolet-visible light absorption titration to be 1.70×10 6 M − 1 . The binding constants calculated by the two experiments have a good match, indicating that Zn-MPB⊃L-NO 2 exists stably under the test conditions.
（2）超分子探针与NTR相互作用(2) Interaction between supramolecular probe and NTR
Zn-MPB⊃L-NO 2与NTR相互作用才能说明主客体探针策略可能用于NTR活性检测。首先通过分子对接计算，算出了L-NO 2与NTR结合口袋绑定的结合自由能为-8.78 kcal/mol，Zn-MPB⊃L-NO 2与NTR结合口袋绑定的结合自由能为-11.03kcal/mol。计算说明Zn-MPB和L-NO 2与NTR结合后体系能量降低，可自发进行。为了进一步通过实验验证Zn-MPB与NTR结合，将Zn-MPB加入NTR前后进行MALDI-TOF MS测试，实验发现加入Zn-MPB前，NTR质谱的特征峰[M-H +]=24715；加入Zn-MPB后，NTR-Zn-MPB质谱的特征峰[M-H +]=25159，相比于NTR质谱数据，NTR-Zn-MPB复合物的形成使得NTR失去了一分子FMN (C 17H 20N 4NaO 9P,M = 478.3)、一分子NADH (C 21H 27N 7Na 2O 14P 2,M = 709.4)和14分子 H 2O。质谱数据验证了酶可以稳定的和Zn-MPB结合形成复合物。在Tris-HCl 缓冲液 (v/v, DMSO 1%,25℃)中进一步进行了微量量热滴定和紫外-可见光吸收滴定实验来验证它们的相互作用。通过微量量热滴定计算出结合常数为1.92×10 6 M − 1。说明的Zn-MPB和L-NO 2相互作用使得系统能量降低，可自发进行的。通过紫外-可见光吸收滴定计算出结合常数为1.30x10 6 M − 1。两种实验计算出的结合常数具有良好的匹配性，说明在测试条件下，Zn-MPB和NTR能相互作用，超分子探针可能用于生物环境检测NTR活性。 The interaction of Zn-MPB⊃L-NO 2 with NTR indicates that the host-guest probe strategy may be used for the detection of NTR activity. First, through molecular docking calculations, it is calculated that the binding free energy of L-NO 2 and the NTR binding pocket is -8.78 kcal/mol, and the binding free energy of Zn-MPB⊃L-NO 2 and the NTR binding pocket is -11.03 kcal/mol. The calculation shows that the energy of the system decreases after the combination of Zn-MPB and L-NO 2 with NTR, and it can proceed spontaneously. In order to further verify the binding of Zn-MPB to NTR through experiments, MALDI-TOF MS test was performed before and after adding Zn-MPB to NTR. The experiment found that before adding Zn-MPB, the characteristic peak of NTR mass spectrum [MH + ]=24715; adding Zn-MPB Later, the characteristic peak of the NTR-Zn-MPB mass spectrum [MH + ]=25159. Compared with the NTR mass spectrum data, the formation of the NTR-Zn-MPB complex caused NTR to lose a molecule of FMN (C 17 H 20 N 4 NaO 9 P,M = 478.3), one molecule of NADH (C 21 H 27 N 7 Na 2 O 14 P 2 , M = 709.4) and 14 molecules of H 2 O. Mass spectrometry data verified that the enzyme can stably combine with Zn-MPB to form a complex. Microcalorimetric titration and ultraviolet-visible light absorption titration experiments were further carried out in Tris-HCl buffer (v/v, DMSO 1%, 25℃) to verify their interaction. The binding constant was calculated by microcalorimetric titration to be 1.92×10 6 M − 1 . The stated interaction between Zn-MPB and L-NO 2 reduces the energy of the system and can proceed spontaneously. The binding constant was calculated by ultraviolet-visible light absorption titration to be 1.30x10 6 M − 1 . The binding constants calculated by the two experiments have a good match, indicating that Zn-MPB and NTR can interact under the test conditions, and supramolecular probes may be used to detect NTR activity in a biological environment.
（3）Zn-MPB⊃L-NO 2与L-NO 2溶剂对比荧光测试 (3) Zn-MPB⊃L-NO 2 and L-NO 2 solvent contrast fluorescence test
在Tris-HCl 缓冲液 (10.0 mM, pH 7.4, 25℃)中进行NTR对Zn-MPB⊃L-NO 2或L-NO 2的荧光响应测试，表征其活性，激发光468 nm，发射范围490到670 nm，进行荧光滴定和荧光动力学（图3A，3B）测试。在Zn-MPB⊃L-NO 2实验组测试（主客体探针5 μM，NTR 0-5 μg/ml）中，发现其快速响应NTR，五秒内即可完成荧光增强响应。而对比组（L-NO 2 5 μM，NADH 15 μM，NTR 0-5 μg/ml）则需要十分钟以上，荧光强度才会缓慢达到平衡状态，且最终强度明显小于主客体探针实验组。取530 nm处荧光发射为基准，绘制荧光相对强度与反应时间和NTR量关系图（图3C，3D），可以看出因为Zn-MPB⊃L-NO 2实验组与NTR超快平衡，荧光强度几乎不随时间变化，荧光强度只与NTR含量线性相关。而L-NO 2对比组荧光强度与时间变化以及NTR含量都有关，无法很好地准确定量荧光强度与NTR含量的关系。说明Zn-MPB⊃L-NO 2可以用于溶液中NTR的超快速准确定量检测。通过进一步荧光动力学实验和计算，相比于L-NO 2对照组与NTR反应，Zn-MPB⊃L-NO 2实验组与NTR反应的最大反应速率和转换次数TON提高了至少100倍以上。说明相比于传统的探针方法，辅酶-荧光底物主客体探针策略能够更快速、更准确的定量检测NTR。 The fluorescence response of NTR to Zn-MPB⊃L-NO 2 or L-NO 2 was tested in Tris-HCl buffer (10.0 mM, pH 7.4, 25℃) to characterize its activity. Excitation light 468 nm, emission range 490 To 670 nm, perform fluorescence titration and fluorescence kinetics (Figure 3A, 3B) test. In the test of the Zn-MPB⊃L-NO 2 experimental group (host-guest probe 5 μM, NTR 0-5 μg/ml), it is found that it responds quickly to NTR, and the fluorescence enhancement response can be completed within five seconds. For the comparison group (L-NO 2 5 μM, NADH 15 μM, NTR 0-5 μg/ml), it takes more than ten minutes for the fluorescence intensity to slowly reach equilibrium, and the final intensity is significantly lower than the host-guest probe experimental group. Taking the fluorescence emission at 530 nm as the benchmark, plot the relationship between the relative fluorescence intensity and the reaction time and the amount of NTR (Figure 3C, 3D). It can be seen that because the Zn-MPB⊃L-NO 2 experimental group and the NTR ultra-fast equilibrium, the fluorescence intensity It hardly changes with time, and the fluorescence intensity is only linearly related to the NTR content. The fluorescence intensity of the L-NO 2 control group is related to time changes and NTR content, and the relationship between fluorescence intensity and NTR content cannot be accurately quantified. It shows that Zn-MPB⊃L-NO 2 can be used for ultra-fast and accurate quantitative detection of NTR in solution. Through further fluorescence kinetic experiments and calculations, compared with the L-NO 2 control group reacting with NTR, the maximum reaction rate and the number of conversions TON of the Zn-MPB⊃L-NO 2 experimental group reacting with NTR increased by at least 100 times. It shows that compared with the traditional probe method, the coenzyme-fluorescent substrate host-guest probe strategy can quantitatively detect NTR more quickly and accurately.
（4）其他生物分子干扰物对Zn-MPB⊃L-NO 2响应硝基还原酶荧光测试 (4) Fluorescence test of nitroreductase response of other biomolecular interference substances to Zn-MPB⊃L-NO 2
细胞内环境十分复杂，考虑到要将Zn-MPB⊃L-NO 2应用于后续的细胞及生物测试，必须在复杂的生理环境中排除其他离子或分子的干扰，依然对NTR具有准确的响应。选取细胞环境中常见干扰物种葡萄糖（50 mM）、二硫苏糖醇（DTT 10 mM）、各种氨基酸（ 1 mM 的D-Glu，L-Tyr，L-Pro，L-Arg，L-Asp和H-Cys-OH•HCl等）和血清白蛋白（BSA 1 μg/ mL）分别与Zn-MPB⊃L-NO 2 (5 μM) 或L-NO 2 (5 μM)进行干扰物对Zn-MPB⊃L-NO 2响应硝基还原酶荧光测试（如图2B）。相比于NTR （5 μg/ml），这些干扰物质没有引起Zn-MPB⊃L-NO 2和L-NO 2 的明显荧光变化，说明它们能够高选择性的对NTR进行识别检测。进行了Zn-MPB⊃L-NO 2和Zn-MPB在血浆及培养基中主体金属-有机笼状配合物的荧光测试（激发光375 nm）。发现在十分钟内，Zn-MPB⊃L-NO 2和Zn-MPB在血浆和配置好的DMEM细胞培养基中其主体笼Zn-MPB的荧光强度无明显变化，表明主体笼能够稳定存在于血浆和细胞培养基中，为后续细胞和生物提供了前提保证。 The intracellular environment is very complicated. Considering that Zn-MPB⊃L-NO 2 is to be used in subsequent cell and biological tests, the interference of other ions or molecules must be excluded in the complex physiological environment, and it still has an accurate response to NTR. Select common interference species in the cell environment: glucose (50 mM), dithiothreitol (DTT 10 mM), various amino acids (1 mM D-Glu, L-Tyr, L-Pro, L-Arg, L-Asp) And H-Cys-OH•HCl, etc.) and serum albumin (BSA 1 μg/mL), respectively, interfere with Zn-MPB⊃L-NO 2 (5 μM) or L-NO 2 (5 μM). MPB⊃L-NO 2 responds to the nitroreductase fluorescence test (Figure 2B). Compared with NTR (5 μg/ml), these interfering substances did not cause obvious fluorescence changes of Zn-MPB⊃L-NO 2 and L-NO 2 , indicating that they can identify and detect NTR with high selectivity. The fluorescence test of the host metal-organic cage complex of Zn-MPB⊃L-NO 2 and Zn-MPB in plasma and culture medium (excitation light 375 nm) was carried out. It was found that within ten minutes, Zn-MPB⊃L-NO 2 and Zn-MPB had no significant changes in the fluorescence intensity of the main cage Zn-MPB in the plasma and the configured DMEM cell culture medium, indicating that the main cage can stably exist in the plasma In the cell culture medium, it provides a prerequisite guarantee for subsequent cells and organisms.
实施例3：基于辅因子-底物探针平台的细胞实验测试Example 3: Cell experiment test based on the cofactor-substrate probe platform
（1）Zn-MPB和L-NO 2对细胞毒性实验 (1) Cytotoxicity test of Zn-MPB and L-NO 2
在96孔板上接种培养细胞，并向细胞中分别加入0，1，2，5，10，20 μM的Zn-MPB或L-NO 2进行孵育24 h后，通过CCK8分析实验（如图4），测试化合物对MCF-7细胞的毒性。 Inoculate cultured cells on a 96-well plate, and add 0, 1, 2, 5, 10, 20 μM Zn-MPB or L-NO 2 to the cells, respectively, and incubate for 24 h, and then pass the CCK8 analysis experiment (Figure 4) ), to test the toxicity of the compound to MCF-7 cells.
（2）Zn-MPB⊃L-NO 2不同缺氧程度或CoCl 2处理细胞成像测试 (2) Zn-MPB⊃L-NO 2 different degree of hypoxia or CoCl 2 treatment cell imaging test
对不同缺氧程度（20%，8%，0.1% O 2）处理6h的癌细胞（包括MCF-7、231和A549细胞）使用Zn-MPB⊃L-NO 2（1 μM）孵育1 min后，进行激光共聚焦成像，激发波长488 nm，荧光信号收集范围508-608 nm。如图5，使用Zn-MPB⊃L-NO 2（1 μM）作为实验组，L-NO 2（1 μM）作为对照组，分别与不同缺氧处理的MCF-7细胞孵育1 min后进行细胞成像测试。实验结果发现，随着细胞缺氧程度增加，所有测试的各种细胞的荧光强度逐渐增加，其中Zn-MPB⊃L-NO 2实验组这一现象更为明显，其荧光强度明显高于对照组。0.1%缺氧程度的细胞，Zn-MPB⊃L-NO 2处理组的荧光远高于对比组。 Carcinoma cells (including MCF-7, 231 and A549 cells) treated with different levels of hypoxia (20%, 8%, 0.1% O 2 ) for 6 hours were incubated with Zn-MPB⊃L-NO 2 (1 μM) for 1 min , Perform laser confocal imaging, excitation wavelength 488 nm, fluorescence signal collection range 508-608 nm. As shown in Figure 5, using Zn-MPB⊃L-NO 2 (1 μM) as the experimental group and L-NO 2 (1 μM) as the control group, they were incubated with different hypoxia-treated MCF-7 cells for 1 min. Imaging test. The experimental results found that as the degree of cell hypoxia increased, the fluorescence intensity of all the tested cells gradually increased. Among them, the phenomenon of the Zn-MPB⊃L-NO 2 experimental group was more obvious, and its fluorescence intensity was significantly higher than that of the control group. . In 0.1% hypoxic cells, the fluorescence of the Zn-MPB⊃L-NO 2 treatment group was much higher than that of the control group.
（3）利用Zn-MPB⊃L-NO 2对0.1%O 2缺氧处理的细胞进行荧光强度随孵育时间成像测试 (3) Use Zn-MPB⊃L-NO 2 to perform imaging test of fluorescence intensity with incubation time on cells treated with 0.1% O 2 hypoxia
对0.1% O 2缺氧处理的各种癌细胞（包括MCF-7、231和A549细胞）使用Zn-MPB⊃L-NO 2（1 μM）孵育不同时间（0，1，3，5，10 min）后，进行激光共聚焦成像，激发波长488 nm，荧光信号收集范围508-608 nm。如图6，使用Zn-MPB⊃L-NO 2（1 μM）作为实验组，L-NO 2（1 μM）作为对照组，分别与0.1%缺氧处理的MCF-7细胞孵育不同时间后进行细胞荧光成像测试。实验结果发现，随着药物孵育时间增加，Zn-MPB⊃L-NO 2实验组的细胞荧光强度没有明显改变，孵育1 min即可使细胞的荧光强度达到平衡，后续增加孵育时间到10 min对细胞荧光强度没有明显变化；而L-NO 2对照组中，随孵育时间增加细胞荧光强度逐渐缓慢增加，但整体上明显弱于Zn-MPB⊃L-NO 2实验组。细胞成像荧光动力学测试结果表明，能够快速对细胞缺氧程度及其相应的NTR进行生物荧光示踪。 Incubate various cancer cells (including MCF-7, 231 and A549 cells) treated with 0.1% O 2 hypoxia with Zn-MPB⊃L-NO 2 (1 μM) for different times (0, 1, 3, 5, 10) min), laser confocal imaging was performed, excitation wavelength was 488 nm, and fluorescence signal collection range was 508-608 nm. As shown in Figure 6, using Zn-MPB⊃L-NO 2 (1 μM) as the experimental group and L-NO 2 (1 μM) as the control group, they were incubated with 0.1% hypoxia-treated MCF-7 cells for different periods of time. Cell fluorescence imaging test. The results of the experiment found that with the increase of drug incubation time, the fluorescence intensity of the cells in the Zn-MPB⊃L-NO 2 experimental group did not change significantly. The fluorescence intensity of the cells can be balanced after 1 min incubation, and the subsequent increase in the incubation time to 10 min The cell fluorescence intensity did not change significantly; while in the L-NO 2 control group, the cell fluorescence intensity gradually increased with the increase of incubation time, but the overall fluorescence intensity was significantly weaker than that of the Zn-MPB⊃L-NO 2 experimental group. Cell imaging fluorescence dynamics test results show that it can quickly trace the degree of cell hypoxia and its corresponding NTR with biofluorescence.
实施例4：基于辅因子-底物探针平台的小鼠活体实验测试Example 4: Mice in vivo experimental test based on the cofactor-substrate probe platform
将裸鼠腋下注射种瘤，分别建立MCF-7荷瘤小鼠模型，待成瘤后，注射Zn-MPB⊃L-NO 2（100 μL，100 μM）进行活体小鼠肿瘤荧光随时间变化（0，2，5，10 min）成像测试（λ ex/λ em = 470/530 nm）（如图7）。设置L-NO 2（100 μL，100 μM）作为对照组。测试结果表明，Zn-MPB⊃L-NO 2实验组小鼠瘤体荧光强度在10 min内即可达到峰值，而对照组小鼠瘤体荧光随时间增加缓慢，在10 min依然没达到平衡。Zn-MPB⊃L-NO 2相比于传统生物探针，具有更快速对瘤体成像的能力。 Nude mice were injected with seed tumors under the armpits to establish MCF-7 tumor-bearing mouse models. After tumor formation, Zn-MPB⊃L-NO 2 (100 μL, 100 μM) was injected to change the fluorescence of tumors in vivo with time (0, 2, 5, 10 min) imaging test (λ ex /λ em = 470/530 nm) (Figure 7). Set L-NO 2 (100 μL, 100 μM) as a control group. The test results show that the tumor fluorescence intensity of the mice in the Zn-MPB⊃L-NO 2 experimental group can reach the peak within 10 minutes, while the tumor fluorescence of the mice in the control group increases slowly with time and still does not reach equilibrium in 10 minutes. Compared with traditional biological probes, Zn-MPB⊃L-NO 2 has the ability to image tumors more quickly.
本发明的实施方式Embodiments of the present invention
本实施方式同最佳实施方式。This embodiment is the same as the best embodiment.
工业实用性Industrial applicability
金属-有机多面体笼具有酶高效催化底物转化的能力，研究人员使用具有特定亲-疏水腔的金属-有机笼作为独特的主体来催化化学转化。将辅因子模拟物直接引入到金属-有机笼状配合物的骨架中，使催化活性位点靠近底物，并促进反应物之间有效的电子转移过程。将基于酶底物的发光探针封装在辅因子组装的“分子容器”中，得到一种可用于区分和定量检测正常状态和疾病状态下不同酶活性的重要工具。这种新型辅因子-底物结构的主-客体超分子探针融合策略使发光底物、辅因子和酶的氧化还原反应能够有效发生，表现出出色的动力学特性。Metal-organic polyhedral cages have the ability of enzymes to efficiently catalyze the conversion of substrates. Researchers use metal-organic cages with specific hydrophilic-hydrophobic cavities as a unique host to catalyze chemical transformations. The cofactor mimics are directly introduced into the framework of the metal-organic cage complex, so that the catalytic active site is close to the substrate, and the effective electron transfer process between the reactants is promoted. Encapsulation of luminescent probes based on enzyme substrates in "molecular containers" assembled by cofactors provides an important tool that can be used to distinguish and quantitatively detect different enzyme activities in normal and disease states. This novel cofactor-substrate structure host-guest supramolecular probe fusion strategy enables the redox reaction of the luminescent substrate, cofactor and enzyme to occur effectively, showing excellent kinetic characteristics.
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Claims (8)

  1. 一种用于组装NADH模拟物金属有机笼状配合物的二氢吡啶配体H 2MPB，其特征在于：二氢吡啶配体具备NADH传输电子的功能，该配体为式Ⅰ 所示化合物： A dihydropyridine ligand H 2 MPB for assembling NADH mimics metal-organic cage complexes, characterized in that: the dihydropyridine ligand has the function of NADH electron transport, and the ligand is a compound represented by formula I:
    Figure 301586dest_path_image001
    Figure 301586dest_path_image001
    其中：among them:
    R 1为提供配位原子的基团，R 1采用2-吡啶基、2-巯基苯基、2-噻吩、2-吡咯、喹啉或异喹啉； R 1 is a group that provides a coordination atom, and R 1 is 2-pyridyl, 2-mercaptophenyl, 2-thiophene, 2-pyrrole, quinoline or isoquinoline;
    R 2为苯基、氢、卤素、氰基、苄氧基。 R 2 is phenyl, hydrogen, halogen, cyano, or benzyloxy.
  2. 根据权利要求1所述的一种用于NADH模拟物金属有机笼状配合物的二氢吡啶配体的制备方法，其特征在于，包括如下步骤：The method for preparing dihydropyridine ligands for NADH mimics metal-organic clathrate complexes according to claim 1, characterized in that it comprises the following steps:
    Figure 712976dest_path_image002
    Figure 712976dest_path_image002
    （1）以丙炔酸甲酯、R 2-CHO、醋酸铵和冰醋酸为原料制备中间体1所示化合物；所述醋酸铵：丙炔酸甲酯：R 2-CHO的摩尔比为4:2:1； (1) Prepare the compound shown in Intermediate 1 with methyl propiolate, R 2 -CHO, ammonium acetate and glacial acetic acid as raw materials; the molar ratio of ammonium acetate: methyl propiolate: R 2 -CHO is 4 :2:1;
    （2）以所示中间体1、卤代化合物4-（2-氯乙基）吗啉、碱、有机溶剂为原料制备中间体2所示化合物；所述碱为碳酸钠、碳酸钾、碳酸铯或有机碱；所述有机溶剂选自丙酮、N,N-二甲基甲酰胺或1,4-二氧六环；所述中间体1：4-（2-氯乙基）吗啉的摩尔比为1:1.1；所述中间体1：碱的摩尔比为1:2-4；(2) Prepare the compound shown in Intermediate 2 with the shown intermediate 1, the halogenated compound 4-(2-chloroethyl)morpholine, a base, and an organic solvent as raw materials; the base is sodium carbonate, potassium carbonate, and carbonic acid Cesium or organic base; the organic solvent is selected from acetone, N,N-dimethylformamide or 1,4-dioxane; the intermediate 1: 4-(2-chloroethyl)morpholine The molar ratio is 1:1.1; the molar ratio of the intermediate 1: base is 1:2-4;
    （3）以中间体2和80%水合肼反应，得到中间体3所示化合物；中间体2与80%水合肼的摩尔比1:5-10；(3) Intermediate 2 is reacted with 80% hydrazine hydrate to obtain the compound shown in Intermediate 3. The molar ratio of intermediate 2 to 80% hydrazine hydrate is 1:5-10;
    （4）以中间体3和R 1-CHO在醋酸催化下反应，得到最终配体化合物式I结构；所述中间体3：R 1-CHO的摩尔比1:2。 (4) Intermediate 3 and R 1 -CHO are reacted under the catalysis of acetic acid to obtain the final ligand compound formula I structure; the molar ratio of intermediate 3: R 1 -CHO is 1:2.
  3. 根据权利要求1所述的二氢吡啶配体组装得到的NADH模拟物金属有机笼状配合物，其特征在于：所述二氢吡啶配体与金属离子组装得到的M 3N 3型金属有机笼状配合物，M为金属离子，N为二氢吡啶配体。 The NADH mimic metal-organic cage complex obtained by assembling a dihydropyridine ligand according to claim 1, characterized in that: an M 3 N 3 type metal-organic cage obtained by assembling the dihydropyridine ligand and a metal ion Like complex, M is a metal ion and N is a dihydropyridine ligand.
  4. 根据权利要求3所述的NADH模拟物金属-有机笼状配合物装配的辅因子-底物探针平台，其特征在于：所述探针平台采用NADH模拟物金属-有机笼状配合物作为主体部分，硝基还原酶荧光底物作为客体部分，辅因子-底物探针平台由NADH模拟物金属-有机笼状配合物和硝基荧光底物组装得到，NADH模拟物金属-有机笼状配合物与硝基还原酶荧光底物的摩尔比为1:1。The cofactor-substrate probe platform for assembly of NADH mimics metal-organic cage complexes according to claim 3, wherein the probe platform uses NADH mimics metal-organic cage complexes as the main body Part, nitroreductase fluorescent substrate as the guest part, the cofactor-substrate probe platform is assembled from NADH mimic metal-organic cage complex and nitro fluorescent substrate, NADH mimic metal-organic cage complex The molar ratio of substance to nitroreductase fluorescent substrate is 1:1.
  5. 根据权利要求4所述的NADH模拟物金属-有机笼状配合物装配的辅因子-底物探针平台，其特征在于：所述硝基还原酶荧光底物为式Ⅱ或式Ⅲ的结构，硝基还原酶荧光底物的制备方法为：The cofactor-substrate probe platform assembled by NADH mimic metal-organic cage complexes according to claim 4, characterized in that: the nitroreductase fluorescent substrate is a structure of formula II or formula III, The preparation method of nitroreductase fluorescent substrate is:
    Figure 885331dest_path_image003
    Figure 885331dest_path_image003
    其中：R 3为酶催化的荧光底物的荧光团部分，R 3采用2-苯基-3a，11b-二氢-1H-菲[9,10-d]咪唑基、萘基、喹喔啉基、卟啉基或卟吩基； Among them: R 3 is the fluorophore part of the enzyme-catalyzed fluorescent substrate, and R 3 is 2-phenyl-3a,11b-dihydro-1H-phenanthrene[9,10-d]imidazolyl, naphthyl, quinoxaline Group, porphyrin group or porphyrin group;
    采用R 3-OH或R 3-NH 2与硝基苄溴为原料，合成式Ⅱ或式Ⅲ的结构，作为硝基还原酶荧光底物。 Using R 3 -OH or R 3 -NH 2 and nitrobenzyl bromide as raw materials, the structure of formula II or formula III is synthesized as the fluorescent substrate of nitroreductase.
  6. 根据权利要求4所述的NADH模拟物金属-有机笼状配合物装配的辅因子-底物探针平台的制备方法，其特征在于：The preparation method of the cofactor-substrate probe platform assembled by NADH mimic metal-organic cage complexes according to claim 4, characterized in that:
    （1）将二氢吡啶配体与所需配位金属盐分别溶于有机溶剂中，将两种溶液充分混合搅拌，通过溶剂挥发结晶、溶剂扩散结晶或加入小极性互溶溶剂沉淀出金属-有机配合物，通过核磁、质谱或单晶结构确认结构M 3N 3，M为金属离子，N为二氢吡啶配体； (1) Dissolve the dihydropyridine ligand and the required coordination metal salt in an organic solvent separately, mix the two solutions thoroughly and stir, and precipitate the metal through solvent volatilization crystallization, solvent diffusion crystallization or adding a small polar miscible solvent- For organic complexes, the structure M 3 N 3 is confirmed by nuclear magnetism, mass spectrometry or single crystal structure, where M is a metal ion and N is a dihydropyridine ligand;
    （2）将上述金属-有机配合物和硝基还原酶荧光底物混合，得到辅因子-底物主客体探针。(2) Mix the above-mentioned metal-organic complex and the fluorescent substrate of nitroreductase to obtain a cofactor-substrate host-guest probe.
  7. 根据权利要求4所述的NADH模拟物金属-有机笼状配合物装配的辅因子-底物探针平台的应用，其特征在于：NADH模拟物金属-有机笼状配合物与硝基还原酶荧光底物融合成辅因子-底物超分子探针平台应用于硝基还原酶的定量检测。The application of the cofactor-substrate probe platform assembled by NADH mimics metal-organic cage complexes according to claim 4, characterized in that: NADH mimics metal-organic cage complexes and nitroreductase fluorescence The substrate fusion becomes a cofactor-substrate supramolecular probe platform for the quantitative detection of nitroreductase.
  8. 根据权利要求7所述的NADH模拟物金属-有机笼状配合物装配的辅因子-底物探针平台的应用，其特征在于：所述辅因子-底物探针平台应用于溶剂中的硝基还原酶的定量检测，向辅因子-底物主客体探针Tris-HCl缓冲液中加入硝基还原酶0-5 μg/mL进行荧光滴定实验和荧光动力学实验。The application of a cofactor-substrate probe platform assembled by NADH mimics metal-organic cage complexes according to claim 7, characterized in that: the cofactor-substrate probe platform is applied to nitrate in a solvent For the quantitative detection of base reductase, add nitroreductase 0-5 μg/mL to the cofactor-substrate host-guest probe Tris-HCl buffer for fluorescence titration experiments and fluorescence kinetics experiments.
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