WO2021103700A1 - 一种可响应硝基还原酶的乏氧探针化合物及其制备与应用 - Google Patents

一种可响应硝基还原酶的乏氧探针化合物及其制备与应用 Download PDF

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WO2021103700A1
WO2021103700A1 PCT/CN2020/111752 CN2020111752W WO2021103700A1 WO 2021103700 A1 WO2021103700 A1 WO 2021103700A1 CN 2020111752 W CN2020111752 W CN 2020111752W WO 2021103700 A1 WO2021103700 A1 WO 2021103700A1
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hypoxia
nitroreductase
probe
formula
suction filtration
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刘小龙
刘景丰
郑爱仙
孙海燕
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福建医科大学孟超肝胆医院
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    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
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    • C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
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    • 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"
    • 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
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    • G01N21/64—Fluorescence; Phosphorescence
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  • the tumor microenvironment plays an important role in the occurrence, development and metastasis of tumors.
  • the tumor microenvironment is relatively stable, not easily affected by environmental factors, and resistant to drug resistance, which has important research significance.
  • the tumor microenvironment mainly has the characteristics of hypoxia, slightly acidic environment, reducing environment, and up-regulation of the expression of specific biological enzymes.
  • the tumor's slightly acidic environment is caused by the high metabolism of glucose by cancer cells and the production of acidic lactic acid products.
  • hypoxic area An important feature of hypoxic cells in the tumor area is the high expression of reductase, which mainly includes nitroreductase, quinone reductase, azo reductase and so on. If these tumor microenvironmental factors such as acidity, reducibility, and hypoxia are not considered, only in vitro cell-level studies are used to construct cancer diagnosis and treatment methods. When they are applied to animals and humans, good reproducible effects are often not obtained. Therefore, imaging research on the tumor microenvironment has important theoretical significance and application prospects.
  • Fluorescence imaging technology is inseparable from the support of fluorescent probes.
  • Fluorescence imaging technology based on fluorescent probes has the advantages of visualization, in-situ, and non-destructive, so it is widely used in biological imaging analysis at the cell and living level.
  • small-molecule fluorescent probes have the advantages of easy synthesis and modification, easy adjustment of the spectrum, high fluorescence quantum yield, and good biocompatibility. They can be induced by intramolecular charge transfer (ICT) and light induction.
  • Electron transfer (PET), fluorescence resonance energy transfer (FRET) and other different principles construct fluorescent probes with high sensitivity, good selectivity, and fast responsiveness, so as to realize the research of responsiveness to specific target substances.
  • rhodamine dyes have the advantages of high fluorescence quantum yield, large molar absorption coefficient, good light stability, wide pH application range, and good biocompatibility.
  • the "off-on" response mechanism of rhodamine can be used to construct various enhanced fluorescent probes, which can well avoid the interference of background fluorescence, and thus is widely used in the analysis and detection of various biomolecules.
  • Intramolecular charge transfer refers to the process in which molecules with push-pull electronic structures undergo intramolecular electron transfer in an excited state to form intramolecular positive and negative charge separation. Its macroscopic performance is the red shift or blue shift of the spectrum.
  • the basic principle is that the two ends of the fluorophore in the fluorescent probe are connected to the electron-donating group and the electron-withdrawing group in the form of conjugate bonds to form a conjugated system with a "push-pull" effect.
  • the recognition unit in the electronic group interacts with the target molecule, it affects the performance of the fluorophore and causes spectral changes.
  • the fluorescent dyes can be constructed to specifically respond through the intramolecular charge transfer mechanism (ICT)
  • ICT intramolecular charge transfer mechanism
  • the purpose of the present invention is to provide a method for preparing a fluorescent probe compound that can respond to nitroreductase, and its application in hypoxia analysis and imaging.
  • the present invention also relates to a method for preparing the fluorescent probe compound, and the method includes:
  • the ratio of the amount of the hydroxyl-containing coumarin derivative to the 4-diethylamino keto acid substance in step (1) is 1:1.
  • the ratio of the amount of the intermediate, potassium carbonate and nitrobenzyl bromide substance in step (2) is 1:1.2:1.2.
  • step (1) the separation and purification method described in step (1) is as follows: the solid obtained by suction filtration is separated and purified by column chromatography with dichloromethane and methanol in a volume ratio of 100:1 to 3:1 as eluents. After removing the solvent by rotary evaporation, the sample was slurried with ethyl acetate and dichloromethane, and the solid obtained by suction filtration was dried under vacuum to obtain the intermediate represented by formula (VI).
  • the separation and purification method described in step (2) is as follows: the solid obtained by suction filtration is separated and purified by column chromatography with petroleum ether and ethyl acetate in a volume ratio of 10:1 to 0:1 as eluents. The collected samples are rotary evaporated to remove the solvent, and then be slurried with ethyl acetate and methanol respectively, and the solid obtained by suction filtration is dried in a vacuum to obtain the fluorescent probe compound represented by formula (I).
  • Compound (I) has a positive charge and usually participates in the reaction in the form of a salt (such as hydrochloride).
  • the nitrobenzyl group in this compound is an electron-withdrawing group, resulting in weak intramolecular charge transfer (ICT).
  • ICT intramolecular charge transfer
  • the compound responds to hypoxia-related nitroreductase, the absorption and fluorescence signals change significantly.
  • the compound has high sensitivity and strong specificity to hypoxia-related nitroreductase, and can be used for analysis, detection and imaging research.
  • the invention also relates to the application of the fluorescent probe compound in hypoxia analysis and imaging.
  • the probe is used for the analysis and detection of hypoxia-related nitroreductase.
  • the probe is used for fluorescence imaging of hypoxic cells.
  • the fluorescent probe compound of the present invention can realize a highly sensitive and highly specific response to the hypoxia-related nitroreductase of the tumor microenvironment; secondly, the compound is prepared through a condensation reaction, and the molecular The degree of conjugation is significantly improved, so that the absorption and fluorescence emission wavelengths are significantly red-shifted compared with dyes such as coumarin and rhodamine, so the hypoxia probe has less interference when it is used in analysis and imaging applications; the probe uses intramolecular charge transfer
  • the mechanism (ICT) is constructed as a signal-enhancing probe with a small background signal.
  • Figure 1 shows the synthesis route and response principle of the hypoxia probe of the present invention
  • Figure 2 is a hydrogen NMR spectrum characterization diagram of the intermediate of the present invention.
  • Figure 3 is a hydrogen NMR spectrum characterization diagram of the hypoxia probe of the present invention.
  • Figure 4 is the absorption (A) and fluorescence (B) spectral characterization diagrams of the intermediates and hypoxia probes of the present invention
  • Fig. 5 shows the investigation of the response of the probe to hypoxia-related nitroreductase through absorption (A) and fluorescence (B) spectrograms of the present invention
  • Figure 6 is a graph showing the changes in the fluorescence spectra of the hypoxia probe of the present invention after adding different concentrations of nitroreductase (A) and the changes in the fluorescence spectra of the probe after adding ions, redox molecules, amino acids, proteins and other substances (B);
  • Figure 7 shows the cell survival of normal liver cells 7701 and liver cancer cells 7721 after incubating with different concentrations of hypoxia probes for 24 hours;
  • Figure 8 shows the application of the hypoxia probe of the present invention for fluorescence imaging of liver cancer cells in hypoxic and non-hypoxic states
  • Fig. 9 is the fluorescence imaging of the tumor site of the tumor-bearing nude mice with the hypoxia probe of the present invention.
  • hypoxia probe ie the fluorescent probe compound (I)
  • the structure was characterized by hydrogen NMR spectroscopy, and the results are shown in Figure 3.
  • the absorption and fluorescence spectra are shown in Figure 4. Compared with the intermediate, the absorption of the hypoxic probe is significantly blue-shifted, and the fluorescence emission at about 660 nm disappears.
  • Figure 5 shows the absorption and fluorescence changes of the hypoxia probe when reduced nicotinamide adenine dinucleotide (NADH) and nitroreductase (NTR) are added separately and at the same time. It can be found that when NADH and nitroreductase are added separately, the absorption of the solution at about 600nm and the fluorescence emission at 660nm are not significantly changed. Since NADH itself absorbs at 350nm, the absorption of the solution at this wavelength after adding NADH Significantly enhanced.
  • NADH nicotinamide adenine dinucleotide
  • NMR nitroreductase
  • FIG. 6A shows the change of the fluorescence signal of the probe after adding the nitroreductase in the concentration range of 0.025-5 ⁇ g/mL. It can be found that the fluorescence signal of the solution will increase with the increase of the concentration of nitroreductase.
  • GSH, DTT, 5mM amino acids
  • Arg, Try, Cys, His, Leu, Glu, 5mM amino acids
  • BSA HSA, 5mg/mL; SOD, GOx, 5 ⁇ g/mL
  • 50 ⁇ M NADH at 37°C Half an hour
  • the present invention also investigates the cytotoxicity of the hypoxia probe.
  • the CCK-8 cell viability assay was used to analyze the cytotoxicity of the cells after 24 hours of incubation with different concentrations of fluorescent probes. The results are shown in Figure 7. It can be seen that the probe has no obvious toxicity to cancer cells and normal cells within a certain concentration range. Therefore, the probe is not only suitable for analysis and detection research, but also for fluorescence imaging research at the cellular level.
  • the present invention further investigates the application of hypoxia probe for cell fluorescence imaging. After inoculating cancer cells in a 35mm petri dish, incubate in hypoxia (1% oxygen) and normoxia for 24 hours, then react with 5 ⁇ g/mL probe for a period of time and stain the nucleus with DAPI, and finally image with a confocal fluorescence microscope In the study, the cells that were not treated with the probe were used as controls, and the excitation wavelength was 633nm. As shown in Figure 8, no matter whether the cells are under normoxia or hypoxia, when there is no reaction with the probe, red fluorescence cannot be observed after 633nm laser irradiation.
  • the present invention further investigates the use of hypoxia probes for fluorescence imaging studies at the animal level.
  • Tumor-bearing nude mice were obtained by subcutaneously injecting HeLa cells into the back of BALB/c nude mice. When the tumor grows to an appropriate size, two nude mice with different tumor sizes are used for in vivo fluorescence imaging studies.
  • Figure 9 shows the fluorescence imaging image of tumor-bearing nude mice injected with 100 ⁇ L 30 ⁇ g/mL hypoxia probe into the tumor as a function of injection time, and the fluorescence imaging image of the tumor isolated 180 minutes after the probe injection. The image of nude mice before injection was used as a control.
  • the excitation wavelength is at 640nm
  • the background fluorescence of the hypoxia probe itself is also quite low, so when the probe was just injected, no fluorescence was observed at the tumor site of the mouse (0 min).
  • hypoxia is easily produced at the tumor site, which leads to overexpression of nitroreductase. Since the hypoxia probe is activated by the over-expressed nitroreductase, it can be observed that the fluorescence intensity of the tumor site will gradually increase with the passage of time when the probe is injected.
  • the in vitro fluorescence imaging of the tumor further confirms that the fluorescent signal is indeed at the tumor site, which is consistent with the fact that nitroreductase is expressed in hypoxic tumors. These results can confirm that the prepared hypoxia probe can be used for animal-level hypoxia imaging.
  • the imaging research of tumor microenvironment has important theoretical significance and application prospects.
  • the present invention mainly provides a hypoxia probe capable of responding to nitroreductase and a preparation method thereof.
  • the probe can achieve a highly sensitive and specific response to hypoxia-related nitroreductase in the tumor microenvironment, and has the following advantages: 1)
  • the probe is prepared by a condensation reaction, and the degree of molecular conjugation is significantly improved, thereby The absorption and fluorescence wavelengths are significantly red-shifted compared with dyes such as coumarin and rhodamine, and the interference is less when performing analysis and imaging applications; 2)
  • the probe is constructed by the intramolecular charge transfer mechanism (ICT), which is a signal-enhanced probe. Needle, the background signal is small.
  • ICT intramolecular charge transfer mechanism

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Abstract

本发明涉及一种可响应硝基还原酶的荧光探针化合物(I)制备方法，以及其在乏氧分析与成像中的应用。本发明荧光探针可实现对肿瘤微环境乏氧相关硝基还原酶的高灵敏、高特异性的响应；其次，该化合物通过缩合反应制得，分子的共轭程度明显提高，从而吸收和荧光发射波长较香豆素和罗丹明等染料发生明显红移，所以乏氧探针进行分析与成像应用时，干扰更小；该探针通过分子内电荷转移机制(ICT)构建，为信号增强型探针，背景信号较小。

Description

一种可响应硝基还原酶的乏氧探针化合物及其制备与应用 (一)技术领域
本发明涉及一种可响应硝基还原酶的荧光探针化合物制备方法，以及其在乏氧分析与成像中的应用。
(二)背景技术
近年来，人们对癌症的研究越来越深入，发现肿瘤微环境对肿瘤的发生、发展和转移起着重要作用。肿瘤微环境与具有明显个体差异的肿瘤细胞本身相比，相对比较稳定，不易受环境因素影响，不易产生抗药性，具有重要的研究意义。肿瘤微环境主要具有乏氧、微酸性环境、还原性环境、特定生物酶表达上调等特点。肿瘤微酸环境是由癌细胞对葡萄糖的高代谢并生成酸性的乳酸产物造成的。此外，肿瘤细胞的生长速度远快于其内部血管的产生速度，而且快速生长的肿瘤细胞需要从周围血管中吸收更多的氧气和营养物质来满足其生长需要，从而很容易在实体肿瘤中产生乏氧区域。肿瘤区域乏氧细胞的一个重要特征是还原酶高表达，主要包括硝基还原酶、醌还原酶、偶氮还原酶等。若不考虑这些微酸性、还原性、乏氧等肿瘤微环境因素，只基于体外细胞水平的研究构建癌症诊断与治疗方法，将其应用于动物和人体时，往往不能获得良好的重现效果。因此，对肿瘤微环境进行成像研究具有重要理论意义与应用前景。
荧光成像技术的发展离不开荧光探针的支持。基于荧光探针的荧光成像技术由于具有可视化、原位、无损等优点，广泛应用于细胞及活体水平的生物成像分析。常用的荧光探针中，小分子荧光探针因其具有容易合成与修饰、光谱易调节、荧光量子产率高、生物相容性良好等优点，可通过 分子内电荷转移(ICT)、光诱导电子转移(PET)、荧光共振能量转移(FRET)等各种不同的原理构建灵敏度高、选择性好、响应性快的荧光探针，从而实现对特定目标物质的响应性研究。例如，罗丹明类染料具有荧光量子产率高、摩尔吸收系数大、光稳定性好、pH适用范围广、生物相容性好等优点。此外，罗丹明的“关-开”响应机理能够用于构建各种增强型荧光探针，能够很好的避免背景荧光的干扰，从而被广泛应用于各种生物分子的分析与检测。
分子内电荷转移(ICT)是指具有推拉电子结构的分子在激发态时发生分子内电子转移，形成分子内正负电荷分离的过程，其宏观表现是光谱的红移或者蓝移。其基本原理是荧光探针中的荧光团两端分别以共轭键形式连接供电子基团和吸电子基团而形成具有“推拉”作用的共轭体系。当电子基团中的识别单元与目标分子相互作用时，对荧光团性能产生影响从而造成光谱变化。利用含羟基的香豆素衍生物与4-二乙氨基酮酸一起制备能够用于构建响应型荧光探针的荧光染料,该荧光染料可通过分子内电荷转移机制(ICT)构建能够特异性响应乏氧相关硝基还原酶的增强型荧光探针，从而进行生物分析与荧光成像。
(三)发明内容
本发明目的是提供一种可响应硝基还原酶的荧光探针化合物制备方法，以及其在乏氧分析与成像中的应用。
本发明采用的技术方案是：
一种可响应硝基还原酶的荧光探针化合物，其结构如式(I)所示：
Figure PCTCN2020111752-appb-000001
本发明还涉及制备所述的荧光探针化合物的方法，所述方法包括：
(1)式(II)所示含羟基的香豆素衍生物与式(III)所示4-二乙氨基酮酸溶于磺酸并在90～100℃搅拌反应10～20小时，冷却到室温后加入水，抽滤所得固体分离纯化，得到式(VI)所示中间体；
Figure PCTCN2020111752-appb-000002
(2)式(VI)所示中间体溶于N，N-二甲基甲酰胺，然后加入碳酸钾和硝基苄溴，并在50～60℃搅拌反应12～18小时，反应完全后加入水，抽滤所得固体分离纯化，得到式(I)所示荧光探针化 合物。
优选的，步骤(1)中所述含羟基的香豆素衍生物与4-二乙氨基酮酸物质的量之比为1:1。
优选的，步骤(2)中所述中间体、碳酸钾和硝基苄溴物质的量之比为1:1.2:1.2。
具体的，步骤(1)中所述分离纯化方法如下：抽滤所得固体以体积比为100:1～3:1的二氯甲烷和甲醇作为洗脱剂，用柱层析分离纯化，将收集的样品旋蒸除去溶剂后再用乙酸乙酯和二氯甲烷打浆处理，抽滤所得固体真空烘干，得到式(VI)所示中间体。
具体的，步骤(2)中所述分离纯化方法如下：抽滤所得固体以体积比为10:1～0:1的石油醚和乙酸乙酯作为洗脱剂，用柱层析分离纯化，将收集的样品旋蒸除去溶剂后再分别用乙酸乙酯和甲醇打浆处理，抽滤所得固体真空烘干，得到式(I)所示荧光探针化合物。
化合物(I)带有正电荷，通常以盐(比如盐酸盐)的形式参与反应。该化合物中的硝基苄基为吸电子基团，导致分子内电荷转移(ICT)较弱。随着与硝基还原酶反应，化合物中的硝基被还原，然后通过重排-消除反应释放出荧光分子，从而使得化合物的分子内电荷转移(ICT)增强。该化合物在响应乏氧相关的硝基还原酶时，吸收和荧光信号发生显著变化。该化合物对乏氧相关硝基还原酶的响应灵敏度高、特异性强，可用于分析检测与成像研究。
本发明还涉及所述的荧光探针化合物在乏氧分析与成像中的应用。
具体的，所述探针用于乏氧相关硝基还原酶的分析检测。
或者，所述探针用于乏氧细胞荧光成像。
本发明的有益效果主要体现在：本发明荧光探针化合物可实现对肿瘤微环境乏氧相关硝基还原酶的高灵敏、高特异性的响应；其次，该化合物通过缩合反应制得，分子的共轭程度明显提高，从而吸收和荧光发射波长较香豆素和罗丹明等染料发生明显红移，所以乏氧探针进行分析与成像应用时，干扰更小；该探针通过分子内电荷转移机制(ICT)构建，为信号增强型探针，背景信号较小。
(四)附图说明
图1为本发明乏氧探针的合成路线以及响应原理；
图2为本发明中间体的核磁氢谱表征图；
图3为本发明乏氧探针的核磁氢谱表征图；
图4为本发明中间体和乏氧探针的吸收(A)和荧光(B)光谱表征图；
图5为本发明通过吸收(A)和荧光(B)光谱图考察探针对乏氧相关硝基还原酶的响应情况；
图6为本发明乏氧探针加入不同浓度的硝基还原酶后的荧光光谱变化图(A)以及探针加入离子、氧化还原分子、氨基酸、蛋白质等各种其他物质后的荧光光谱变化图(B)；
图7为正常肝细胞7701和肝癌细胞7721分别孵育不同浓度的乏氧探针24小时后的细胞生存情况；
图8为本发明乏氧探针用于肝癌细胞在乏氧和非乏氧状态下的荧光成像；
图9为本发明乏氧探针用于荷瘤裸鼠肿瘤部位的荧光成像。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
实施例1：乏氧探针的合成方法及应用
1、中间体的合成(合成路线参见图1)：
10g含羟基的香豆素衍生物与等摩尔量的15.3g 4-二乙氨基酮酸溶于100mL磺酸,并在95℃搅拌反应16小时，冷却到室温后加入200mL水，抽滤减压得到的固体混合物用二氯甲烷和甲醇(100/1～3/1)作为洗脱剂，用柱层析分离纯化，将收集的样品旋蒸除去溶剂后再用150mL乙酸乙酯和二氯甲烷(1:1)打浆处理，抽滤所得固体真空烘干，即为中间体(VI)。利用核磁氢谱表征其结构，其结果见图2。其吸收和荧光谱图见图4。
2、乏氧探针的合成：
5g中间体溶于35mL的N，N-二甲基甲酰胺(DMF)中，然后加入1.2倍摩尔量的1.44g碳酸钾和2.24g硝基苄溴，并在50℃搅拌反应16小时。反应完后，加入250mL水到上述溶液，抽滤所得固体混合物用石油醚和乙酸乙酯(10/1～0/1)作为洗脱剂，用柱层析分离纯化，将收集的样品旋蒸除去溶剂后再用25mL乙酸乙酯和甲醇分别打浆处理，抽滤所得固体真空烘干，即为乏氧探针(即荧光探针化合物(I))。利用核磁氢谱表征其结构，其结果见图3。其吸收和荧光谱图见图4，与中间体相比，乏氧探针的吸收发生明显蓝移，并且660nm左右的荧光发射消失。
乏氧探针对硝基还原酶的响应能力：
5μg/mL的荧光探针在10mM PBS(pH＝7.4)溶液中与还原态烟酰胺腺嘌呤二核苷酸(NADH)(50μM)以及不同浓度的硝基还原酶(NTR)(0.025～5μg/mL)在37℃反应半小时，然后检测其吸收和荧光变化情况。
图5为乏氧探针在单独和同时加入还原态烟酰胺腺嘌呤二核苷酸(NADH)和硝基还原酶(NTR)情况下的吸收和荧光变化情况。可以发 现，单独加入NADH和硝基还原酶时，溶液在600nm左右的吸收和在660nm处的荧光发射都没有明显变化，由于NADH本身在350nm处有吸收，所以加入NADH后溶液在该波段的吸收有明显增强。当同时加入NADH和硝基还原酶时，溶液的吸收和荧光都明显增强，说明以NADH作为电子供体时，该探针能够响应乏氧相关的硝基还原酶，使得溶液的吸收和荧光发生变化。并且加入的硝基还原酶浓度越大时，信号变化越明显。图6A显示的是探针加入0.025～5μg/mL浓度范围的硝基还原酶后的荧光信号变化情况，可以发现溶液的荧光信号会随着硝基还原酶浓度的升高而升高。
特异性实验：
5μg/mL的荧光探针在10mM PBS(pH＝7.4)溶液中分别与金属离子(Na +、K +、Ca 2+、Mg 2+，5mM)、氧化还原分子(AA、H 2O 2、GSH、DTT，5mM)、氨基酸(Arg、Try、Cys、His、Leu、Glu，5mM)和蛋白质(BSA、HSA，5mg/mL；SOD、GOx，5μg/mL)以及50μM的NADH在37℃反应半小时，然后检测其荧光变化情况。从图6B可以发现，分析这些物质时，溶液的荧光信号都没有发生明显变化。这个结果可以看出，该探针对乏氧相关硝基还原酶具有特异响应能力。
细胞毒性实验：
本发明另外还考察了乏氧探针的细胞毒性。96孔板接种正常肝细胞7701和肝癌细胞7721后，利用CCK-8细胞活力检测实验分析细胞孵育不同浓度的荧光探针24小时后的细胞毒性，其结果如图7所示。可以看出，该探针在一定浓度范围内对癌细胞和正常细胞都没有明显毒性。因此，该探针不仅适用于分析检测研究，也适用于细胞等水平的荧光成像研究。
荧光成像实验：
本发明进一步考察了乏氧探针用于细胞荧光成像的研究。35mm的培养皿接种癌细胞后，分别在乏氧(1％氧气)和常氧状态孵育24小时，然后与5μg/mL探针反应一段时间并用DAPI进行细胞核染色，最后用共聚焦荧光显微镜进行成像研究，以未用探针处理的细胞作为对照，激发波长为633nm。如图8所示，不管常氧还是乏氧状态的细胞，当没有与探针反应时，633nm激光照射后不能观测到红色荧光。常氧状态下的细胞与乏氧探针反应后再用激光进行照射时，也只能观察到微弱的红色荧光。而当乏氧状态下的细胞与乏氧探针反应后再用激光进行照射时，则能观察到很明显的红色荧光。这个结果说明，本发明构建的探针确实能够用于乏氧成像。
动物水平荧光成像实验：
本发明进一步考察了乏氧探针用于动物水平的荧光成像研究。荷瘤裸鼠通过皮下注射HeLa细胞到BALB/c裸鼠背部获得。当肿瘤生长到适当大小，使用两只肿瘤大小不同的裸鼠进行体内荧光成像研究。图9显示的是肿瘤内注射了100μL 30μg/mL乏氧探针的荷瘤裸鼠随注射时间变化的荧光成像图，以及探针注射180min后分离出来的肿瘤的荧光成像图。注射之前的裸鼠成像图则作为对照。如图所示，当激发波长在640nm处时，裸鼠的自发荧光是非常低的。此外，乏氧探针本身的背景荧光也相当低，因此当探针刚刚注射时，小鼠的肿瘤部位没有观察到荧光(0min)。据报道，乏氧容易在肿瘤部位产生，从而导致硝基还原酶过表达。由于乏氧探针被过表达的硝基还原酶所活化，因此可以观察到肿瘤部位的荧光强度会随着注射探针时间的推移而逐渐增强。肿瘤的离体荧光成像图进一步证实，荧光信号确实在肿瘤部位，这与硝基还原酶在乏氧肿瘤中表达这一事实是相一致的。这些结果可以证实，所制备的乏氧探针可用于动物水平 的乏氧成像。
结论：
对肿瘤微环境进行成像研究具有重要理论意义与应用前景。本发明主要提供一种可响应硝基还原酶的乏氧探针及其制备方法。该探针可实现对肿瘤微环境乏氧相关硝基还原酶的高灵敏、高特异性的响应，具有如下优势：1)该探针通过缩合反应制得，分子的共轭程度明显提高，从而吸收和荧光波长较香豆素和罗丹明等染料发生明显红移，进行分析与成像应用时，干扰更小；2)该探针通过分子内电荷转移机制(ICT)构建，为信号增强型探针，背景信号较小。

Claims (9)

  1. 一种可响应硝基还原酶的荧光探针化合物，其结构如式(I)所示：
    Figure PCTCN2020111752-appb-100001
  2. 制备权利要求1所述的荧光探针化合物的方法，所述方法包括：
    (1)式(II)所示含羟基的香豆素衍生物与式(III)所示4-二乙氨基酮酸溶于磺酸并在90～100℃搅拌反应10～20小时，冷却到室温后加入水，抽滤所得固体分离纯化，得到式(VI)所示中间体；
    Figure PCTCN2020111752-appb-100002
    (2)式(VI)所示中间体溶于N，N-二甲基甲酰胺，然后加入碳酸钾和硝基苄溴，并在50～60℃搅拌反应12～18小时，反应完全后加入水，抽滤所得固体分离纯化，得到式(I)所示荧光探针化合物。
  3. 如权利要求2所述的方法，其特征在于步骤(1)中所述含羟基的香豆素衍生物与4-二乙氨基酮酸物质的量之比为1:1。
  4. 如权利要求2所述的方法，其特征在于步骤(2)中所述中间体、碳酸钾和硝基苄溴物质的量之比为1:1.2:1.2。
  5. 如权利要求2所所述的方法，其特征在于步骤(1)中所述分离纯化方法如下：抽滤所得固体以二氯甲烷和甲醇作为洗脱剂，用柱层析分离纯化，将收集的样品旋蒸除去溶剂后再用乙酸乙酯和二氯甲烷打浆处理，抽滤所得固体真空烘干，得到式(VI)所示中间体。
  6. 如权利要求2所述的方法，其特征在于步骤(2)中所述分离纯化方法如下：抽滤所得固体以石油醚和乙酸乙酯作为洗脱剂，用柱层析分离纯化，将收集的样品旋蒸除去溶剂后再分别用乙酸乙酯和甲醇打浆处理，抽滤所得固体真空烘干，得到式(I)所示荧光探针化合物。
  7. 权利要求1所述的荧光探针化合物在乏氧分析与成像中的应用。
  8. 如权利要求7所述的应用，其特征在于所述乏氧探针用于乏氧相关硝基还原酶的分析检测。
  9. 如权利要求7所述的应用，其特征在于所述乏氧探针用于乏氧细胞荧光成像。
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