WO2023092814A1 - 一种氨基取代色烯并喹啉型荧光标记物及其制备和应用 - Google Patents

一种氨基取代色烯并喹啉型荧光标记物及其制备和应用 Download PDF

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WO2023092814A1
WO2023092814A1 PCT/CN2021/143133 CN2021143133W WO2023092814A1 WO 2023092814 A1 WO2023092814 A1 WO 2023092814A1 CN 2021143133 W CN2021143133 W CN 2021143133W WO 2023092814 A1 WO2023092814 A1 WO 2023092814A1
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chromenoquinoline
amino
fluorescent marker
substituted
fluorescent
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葛健锋
朱明森
喻情
孙如
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苏州大学
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C07D491/044—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
    • C07D491/052—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring the oxygen-containing ring being six-membered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
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    • 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"
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C09K2211/1033—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with oxygen
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
    • C09K2211/1048—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms with oxygen

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  • the invention belongs to fluorescent labeling technology, in particular to an amino-substituted chromenoquinoline fluorescent label and its preparation and application.
  • lipid droplet As a dynamic independent organelle, lipid droplet is an important place for neutral lipid storage in eukaryotic cells. In the process of lipid coordination, it can promote the absorption, metabolism, transportation and signal transduction of lipids in cells, and then maintain The smooth progress of various physiological activities (reference: J.-W.Shi, Y.Tian, X.-L.Zhang, et al., An AIEgen-Based Fluorescent Probe for Highly Selective and Specific Imaging of Lipid Droplets in L02 and HepG2 Cells, Sensors and Actuators B: Chemical, 2019 (284) 545-552); Mitochondria are composed of a double-membrane system and are organelles involved in energy metabolism and cell homeostasis in eukaryotic cells, so they are involved in the activities of the entire living body Acting as a power source for energy exchange, a metabolic factory and an information transduction center, it is the center of cell metabolism (reference: X.-Y.Li,
  • Nile Red and BODIPY are commonly used commercial lipid droplet markers, and NileRed derivatives lack specificity for lipid droplets during the labeling process, and in some cases will stain other organelles; BODIPY derivatives are due to the The relatively small Stokes displacement also limits its use.
  • most of the commonly used commercial markers are some salt structures, such as triphenylphosphine salt, pyridinium salt and indole salt, etc. They can be located in mitochondria, but there are also some problems.
  • the present invention utilizes different aromatic amines and coumarin derivatives to obtain a series of heterocyclic compounds with chromenoquinolines through one-step reaction on the basis of controlling the feed ratio, so that it can be used in biological applications , capable of imaging lipid droplets or mitochondria.
  • the invention provides an amino-substituted chromenoquinoline-type fluorescent marker, which has one or more chromenoquinoline rings in the structural center of the amino-substituted chromenoquinoline-type fluorescent marker, and the chromenoquinoline-type fluorescent marker There are one or more dialkylamino groups around the ring ring.
  • the amino-substituted chromenoquinoline fluorescent marker is synthesized from an aromatic amine and a coumarin fluorescent precursor through a one-step reaction.
  • aromatic amine is 1-naphthylamine (5a), N,N-diethyl-p-phenylenediamine (5b) or p-phenylenediamine (5c).
  • the fluorescent precursor of coumarin is 4-chloro-3-formylcoumarin (5d) or 7-(N,N-diethylamino)-4-chlorocoumarin-3-formaldehyde (5e);
  • the molar ratio of the aromatic amine to the coumarin-based fluorescent precursor is 1:1-2.
  • the aromatic amine and the coumarin-based fluorescent precursor react to synthesize an amino-substituted chromenoquinoline fluorescent marker under the action of a catalyst; the catalyst is a Lewis acid.
  • the Lewis acid is aluminum trichloride, boron trifluoride, sulfur trioxide or iron bromide.
  • reaction solvent is tetrahydrofuran; the molar ratio of the aromatic amine to the catalyst is 1:1-2.
  • the invention also provides the application of an amino-substituted chromenoquinoline fluorescent marker as an organelle fluorescent marker.
  • the 1a, 1b and 1c amino-substituted chromenoquinoline fluorescent markers act on lipid droplet imaging; the 1d amino-substituted chromenoquinoline fluorescent markers act on mitochondria.
  • the present invention discloses a series of novel heterocyclic fluorescent markers for the first time.
  • the introduction of nitrogen heterocycle improves the biocompatibility of the compound, which is more conducive to the application in the biological field, and is more effective in aromatic amines and coumarin derivatives.
  • the reaction process is convenient and quick, and the target product can be obtained with one-step operation and can be located in lipid droplets or mitochondria, which has biological application value.
  • the nitrogen-containing heterocycle as the center of this type of structure improves biocompatibility, and N,N-diethylamino, as a commonly used strong electron-donating group, is located at both ends of the structure, which improves the solubility of the compound in organic solvents and also affect its optical properties.
  • Fluorescent markers that can be localized to lipid droplets or mitochondria were synthesized by ring-forming reaction between inexpensive and readily available aromatic amines and classic coumarin derivatives. In addition to maintaining the excellent fluorescence properties of coumarin, this kind of fluorescent markers can finally turn the coumarin derivatives without targeting ability into fluorescent probes that can target lipid droplets or mitochondria by modifying the structure. Needle. More importantly, the whole process of reaction based on two cheap and easy-to-obtain raw materials is simple, convenient and fast.
  • the invention utilizes cheap and easy-to-obtain aromatic amine compounds and tetrahydrofuran aprotic solvent as the organic solvent for the reaction, and anhydrous aluminum trichloride participates in it, so as to ensure the smooth progress of the reaction and has practical application value.
  • the fluorescence imaging of lipid droplets or mitochondria can be realized.
  • fluorescent markers have low cytotoxicity and little damage to biological samples in the process of cell imaging, and have large Stokes shift background interference, which has great scientific significance and commercial value.
  • Fig. 1 is the synthesis route of fluorescent marker 1a-1d involved in the present invention
  • FIG. 1 is the photostability of fluorescent markers 1a-1d
  • Fig. 3 is the ultraviolet-visible absorption spectrum and fluorescence spectrum of fluorescent marker 1a-1d in chloroform
  • Fig. 4 is the cytotoxicity test diagram of fluorescent marker 1a-1d
  • Fig. 5 is a cell imaging diagram of fluorescent markers 1a-1d in HeLa cells.
  • laser confocal microscopy was used for cell imaging; the green light channel was excited at 405 nm, and fluorescent signals in the range of 468-550 nm were collected; the red light channel was excited at 561 nm, and fluorescent signals in the range of 570-750 nm were collected.
  • the synthetic route of the embodiment of the present invention is shown in Figure 1, wherein 5a-5e represent the numbers of different raw materials.
  • the ratio of raw materials and the purification method adopt conventional ratios or conventional purification methods.
  • the examples are schematic representations, and the present invention verifies the correctness of the product structure by proton nuclear magnetic spectrum, carbon nuclear magnetic spectrum and high-resolution mass spectrometry.
  • the compound p-phenylenediamine 5c was further used to synthesize the dye 1d.
  • the photostability is calculated according to the change of absorption intensity at different times before and after irradiation to calculate the remaining absorption rate.
  • the photostability of the fluorescent markers is 1a: 46%, 1b: 43%, 1c: 76%, 1d: 71%, Cy7: 5.3%. It can be seen that the dyes 1a-1d and cyanine dyes It has higher photostability.
  • fluorescent marker 1a concentration is 10 ⁇ M in dichloromethane, as shown in Figure 3, fluorescent marker 1a has maximum absorption at 452nm ( Figure 3 (a)); Fluorescent marker 1a There is the highest fluorescence intensity at 532nm ( Figure 3(e)), the excitation wavelength at this time is 428nm, and the slit width is 3nm/3nm.
  • fluorescent marker 1b The ultraviolet absorption and fluorescence emission spectra of fluorescent marker 1b (concentration is 10 ⁇ M) in dichloromethane, as shown in Figure 3, fluorescent marker 1b has maximum absorption (Figure 3 (b)) at 473nm place; Fluorescent marker 1b There is the highest fluorescence intensity at 562nm (Fig. 3(f)), the excitation wavelength at this time is 407nm, and the slit width is 3nm/3nm.
  • fluorescent marker 1c concentration is 10 ⁇ M in dichloromethane, as shown in Figure 3, fluorescent marker 1c has maximum absorption at 375nm ( Figure 3 (c)); Fluorescent marker 1c There is the highest fluorescence intensity at 546nm ( Figure 3(g)), the excitation wavelength at this time is 461nm, and the slit width is 3nm/3nm.
  • the ultraviolet absorption and fluorescence emission spectra of the fluorescent marker 1d (with a concentration of 10 ⁇ M) in dichloromethane, as shown in Figure 3, the fluorescent marker 1d has a maximum absorption at 429nm (Figure 3 (d)); the fluorescent marker 1d There is the highest fluorescence intensity at 495nm ( Figure 3(h)), the excitation wavelength at this time is 430nm, and the slit width is 5nm/3nm.
  • the present invention also tested the cytotoxicity of fluorescent markers 1a-1d.
  • the viability of HeLa cells in the presence of these dyes was measured by using the CCK-8 method. HeLa cells were incubated with different concentrations of dyes (2, 4, 6, 8 and 10 ⁇ M) for 6 h. As shown in Figure 4, the cytotoxicity test results of the fluorescent markers 1a-1d showed that they had good cell viability and were suitable for live cell imaging.
  • cell survival rate (%) (A sample -A b )/(A c -A b ), wherein A c : negative control (including culture medium and cells, no dye to be tested added), A b : Blank (including the dye to be tested and culture medium, without adding cells), A sample : test group (including culture medium, cells and dye to be tested).
  • fluorescent marker 1a was prepared as a stock solution using dimethyl sulfoxide, and then added to regular cell culture medium, so that fluorescent marker 1a was in the cell culture
  • concentration in the medium was 6 ⁇ M, and then Nile Red (100 nM), a lipid droplet red marker, was added respectively, and HeLa cells were co-cultured with HeLa cells in a saturated humidity, 37 ° C, 5% CO 2 incubator for 5 min (the same as the following experiments); then buffered with PBS After three washes, the cells were imaged using a confocal laser microscope.
  • the green light channel is excited at 405nm to collect fluorescent signals in the range of 468-550nm
  • the red light channel is excited at 561nm to collect fluorescent signals in the range of 570-750nm.
  • the results are shown in Figure 5, where (a) is the bright field, (b) is the cell imaging image of the fluorescent marker 1a, (c) is the cell imaging image of the lipid droplet red marker, (d) is the green light channel and Overlay of the red channel, (e) is the fluorescence intensity of the ROI line in the overlay, (f) is the colocalization experiment, and their colocalization coefficient is 0.95.
  • the experimental method used is the same as that of fluorescent marker 1a above, as shown in Figure 5, (g) is the bright field, (h) is the fluorescent marker The cell imaging image of 1b, (i) is the cell imaging image of lipid droplet red marker, (j) is the overlay image of green light channel and red light channel, (k) is the fluorescence intensity of ROI line in the overlay image, (l ) is a colocalization experiment, and their colocalization coefficient is 0.97. The results showed that fluorescent marker 1b, like probe 1a, had good targeting ability to lipid droplets in HeLa cells.
  • the experimental method of the fluorescent marker 1c is the same as that of the fluorescent marker 1a above, as shown in Figure 5, (m) is the bright field, (n) is the cell imaging image of the fluorescent marker 1b, (o) is the lipid droplet The cell imaging image of the red marker, (p) is the overlay image of the green light channel and the red channel, (q) is the fluorescence intensity of the ROI line in the overlay image, (r) is the colocalization experiment, and their colocalization coefficient is 0.85.
  • lipid droplets in HeLa cells were imaged fluorescently.
  • dimethyl sulfoxide to prepare the fluorescent marker 1d into a mother solution, and then add it to the regular cell culture medium, so that the concentration of the fluorescent marker 1d in the cell culture medium is 8 ⁇ M, and HeLa cells are in saturated humidity , 37°C, and 5% CO 2 incubator for 5 minutes; then, after washing three times with PBS buffer, the cells were imaged using a laser confocal microscope. The green light channel was excited at 405nm, and the fluorescent signal in the range of 468-550nm was collected.
  • the present invention discloses for the first time a series of amino-substituted chromenoquinoline fluorescent markers based on the ring-forming reaction of aromatic amines and coumarin derivatives, which can realize the detection of lipid droplets and neutral structures. Fluorescence imaging of mitochondria.
  • the present invention makes the compound have extremely strong fluorescence intensity in dichloromethane organic solvent after forming a ring through one-step reaction, and has high optical stability compared with cyanine dyes, and when it is further applied to biological cell imaging, the cell Low toxicity, less damage to biological samples, high selectivity and ability to quickly stain and image cells. Imaging results indicated that the compounds were specifically targeted to lipid droplets or mitochondrial organelles.

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Abstract

氨基取代色烯并喹啉型荧光标记物及其制备方法和应用。所述荧光标记物利用芳香胺类化合物的氨基与两种邻位含氯的醛基香豆素进行一步成环反应制备得到，其具有优异荧光发射性能的同时，在不同的有机溶剂中均具有良好的光稳定性，有效解决了菁类染料的光稳定性差的问题，同时可以在细胞水平上对不同细胞器进行标记。氮杂环的引入使得化合物的生物相容性得到了提高，为生物应用奠定了良好的基础。反应的原料廉价易得且合成标记物的步骤简单、容易操作，在不降低标记物性能的同时可以有效地降低生产成本，具有一定的商业价值。

Description

一种氨基取代色烯并喹啉型荧光标记物及其制备和应用 技术领域
本发明属于荧光标记技术，具体涉及一种氨基取代色烯并喹啉型荧光标记物及其制备和应用。
背景技术
脂滴作为一个动态的独立的细胞器，它是真核细胞中性脂质储存的重要场所，在脂质协调过程中，可以促进细胞中脂质的吸收，代谢，运输和信号传导，进而来维持各项生理活动的顺利进行(参考：J.-W.Shi,Y.Tian,X.-L.Zhang,et al.,An AIEgen-Based Fluorescent Probe for Highly Selective and Specific Imaging of Lipid Droplets in L02 and HepG2 Cells,Sensors and Actuators B:Chemical,2019(284)545-552)；线粒体由双膜系统组成，是真核细胞内参与能量代谢和细胞内稳态的细胞器，因此在整个生命体的活动中充当能量交换的动力源，代谢工厂和信息传导中心，是细胞新陈代谢的中枢(参考：X.-Y.Li,Y.-M.Hu,H.-M.Ma,et al.,Mitochondria-Immobilized Near-Infrared Ratiometric Fluorescent pH Probe to Evaluate Cellular Mitophagy,Anal Chem,2019(91)11409-11416)。在复杂的细胞环境中，对脂滴和线粒体的状态进行观察和检测显得极其重要(参考：D.I.Danylchuk,P.H.Jouard,A.S.Klymchenko,Targeted Solvatochromic Fluorescent Probes for Imaging Lipid Order in Organelles under Oxidative and Mechanical Stress,J.Am.Chem.Soc,2021(143)912-924)。就目前的检测手段来讲，荧光标记由于选择性好，灵敏度高，操作方便，一直活跃在研究者的视线中，近年来，标记脂滴和线粒体的染料类荧光探针也越来越多(参考：(1)J.-Y.Ni,R.Sun,J.-F.Ge,et al.,Convenient Construction of Fluorescent Markers for Lipid Droplets with 1,8-Naphthalimide Unit,Dyes and Pigments,2021(186)109003.(2)W.Ma,B.Xu,Y.-J.Xu,et al.,The Application of Amide Units in the Construction of Neutral Functional Dyes for Mitochondrial Staining,J.Mater.Chem.B,2021(9)2524)。
目前常用的细胞器商用标记物在经过长期反复使用的过程中，一些缺点就随之慢慢的凸显出来。Nile Red和BODIPY的衍生物是常用的脂滴商用标记物，而NileRed类衍生物在标记的过程中对脂滴缺乏特异性，在一些情况下会对其它细胞器进行染色；BODIPY类衍生物由于斯托克斯位移比较小也限制了其使用。在标记线粒体的过程中，常用的商用标记物大多是一些盐类结构，例如三苯基膦盐、吡啶盐和吲哚盐等，它们能够定位于线粒体的同时也存 在一些问题，这些阳离子进入细胞后会改变线粒体的膜电位，进而使细胞的微环境发生改变，影响生命体的生理活动(参考：Y.Wang,B.Xu,R.Sun,J.-F.Ge,et al.,The Application of Nitrogen Heterocycles in Mitochondrial-Targeting Fluorescent Markers with Neutral Skeletons,J.Mater.Chem.B,2020(8)7466)。
发明内容
为了解决上述问题，本发明利用不同的芳香胺与香豆素类衍生物在控制投料比的基础上，通过一步反应得到一系列具有色烯并喹啉的杂环化合物，使其在生物应用中，能够对脂滴或线粒体进行成像。
本发明提供一种氨基取代色烯并喹啉型荧光标记物，所述氨基取代色烯并喹啉型荧光标记物结构中心有一个或多个色烯并喹啉环，所述色烯并喹啉环的周边有一个或多个二烷基氨基。
优选的，其结构式如1a-1d中任一所示：
Figure PCTCN2021143133-appb-000001
优选的，所述氨基取代色烯并喹啉型荧光标记物中的色烯并喹啉环旁有1-2个苯环。
优选的，所述氨基取代色烯并喹啉型荧光标记物由芳香胺和香豆素类荧光前体通过一步反应合成。
进一步地，所述芳香胺为1-萘胺(5a)、N,N-二乙基对苯二胺(5b)或对苯二胺(5c)。
进一步地，所述香豆素荧光前体为4-氯-3-甲酰基香豆素(5d)或7-(N,N-二乙基氨基)-4-氯香豆素-3-甲醛(5e)；
Figure PCTCN2021143133-appb-000002
进一步地，所述芳香胺和香豆素类荧光前体的摩尔比为1:1-2。
进一步地，所述芳香胺和香豆素类荧光前体在催化剂作用下反应合成氨基取代色烯并喹啉型荧光标记物；所述催化剂为路易斯酸。
进一步地，所述路易斯酸为三氯化铝、三氟化硼、三氧化硫或溴化铁。
进一步地，反应溶剂为四氢呋喃；所述芳香胺和催化剂的摩尔比为1：1-2。
本发明还提供一种氨基取代色烯并喹啉型荧光标记物作为细胞器荧光标记的应用。
进一步地，所述1a、1b和1c氨基取代色烯并喹啉型荧光标记物作用于脂滴成像；所述1d氨基取代色烯并喹啉型荧光标记物作用于线粒体。
本发明的技术方案相比现有技术具有以下优点：
本发明首次公开了一系列新型的杂环荧光标记物，氮杂环的引入提高了化合物的生物相容性，更有助于在生物领域的应用，而且在芳香胺与香豆素类衍生物反应的过程中方便快捷只需一步操作，就可得到目标产物且可以定位于脂滴或线粒体，具有生物应用价值。
含氮杂环作为此类结构的中心，提高了生物相容性，N,N-二乙氨基作为常用的强给电子基位于结构的两端，在提高化合物在有机溶剂中溶解性的同时也影响其光学性质。
将廉价易得的芳香胺与经典的香豆素类衍生物通过成环反应合成了可以定位于脂滴或线粒体的荧光标记物。此类荧光标记物在保持香豆素优良的荧光性能以外，通过对结构进行修饰，把没有靶向能力的香豆素类衍生物最终变成了可以对脂滴或线粒体进行靶向的荧光探针。更为重要的是基于两种廉价易得的原料进行反应的整个过程操作简单且方便快捷。
本发明利用廉价易得的芳香胺类化合物并且使用四氢呋喃非质子性溶剂作为反应的有机溶剂，无水三氯化铝参与其中，保证反应顺利进行具有实际应用价值。
本发明的杂环类荧光探针分别与HeLa细胞共培育之后，可以实现对脂滴或线粒体的荧光成像。这类荧光标记物在细胞成像过程中细胞毒性低对生物样品损伤小，具有大的斯托克斯位移背景干扰小，具有极大的科学意义和商业价值。
附图说明
图1为本发明涉及的荧光标记物1a-1d的合成路线；
图2为荧光标记物1a-1d的光稳定性；
图3为荧光标记物1a-1d在氯仿中的紫外-可见吸收光谱和荧光光谱；
图4为荧光标记物1a-1d的细胞毒性测试图；
图5为荧光标记物1a-1d在HeLa细胞中的细胞成像图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地 理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
以下实施例中，利用激光共聚焦显微镜进行细胞成像；绿光通道使用405nm激发，收集468-550nm范围内的荧光信号；红光通道使用561nm激发，收集570-750nm范围内的荧光信号。
本发明实施例的合成路线参见附图1，其中5a-5e代表不同原料的编号。本发明的化合物合成中，原料比例以及纯化方法采用常规比例或者常规纯化方法，实施例为示意性表述，且本发明通过核磁氢谱、核磁碳谱以及高分辨质谱验证产物结构的正确性。
实施例1
染料1a合成的步骤如下：
取化合物N,N-二乙基对苯二胺5b(1.0毫摩尔，393.0毫克)，化合物5d(1.0毫摩尔,500.0毫克)和三氯化铝(1.0毫摩尔，319.0毫克)溶解于20.0毫升的四氢呋喃中，然后回流3到4个小时，TLC检测反应进度；降至室温后，直接通过旋转蒸发仪除去溶剂，经柱层析(洗脱剂：纯二氯甲烷)分离得到纯净染料1a，橙色固体，175.3毫克，产率为44.5％。表征为：核磁氢谱 1H NMR(300MHz,DMSO-d 6)δ(ppm)8.97(s,1H,Ar-H),8.57(s,1H,Ar-H),8.58(d,J＝5.7Hz,1H,Ar-H),8.02(d,J＝9.6Hz,Ar-H),7.68(d,J＝8.1Hz,1H,Ar-H),7.56(t,J＝14.4Hz,1H,Ar-H),7.42(d,J＝8.7Hz,2H,Ar-H),δ7.20(s,1H,Ar-H),3.53(q,J＝6.0Hz,4H,2×CH 2),2.50(s,6H,2×CH 3).核磁碳谱 13C NMR(151MHz,CDCl 3)δ(ppm)161.9,151.9,144.9,137.4,130.8,130.2,123.1,117.1,115.8,103.8,44.7,12.4.高分辨质谱HRMS(ESI +)m/z理论值C 20H 18N 2O 2 +,[M+Na] +:341.1260,实测值:341.1223。
实施例2
染料1b合成的步骤如下：
取化合物N,N-二乙基对苯二胺5b(1.0毫摩尔，176.16毫克)，化合物5e(1.0毫摩尔，300.0毫克)和三氯化铝(1.0毫摩尔，142.9毫克)溶解于20.0毫升的四氢呋喃中，然后回流3到4个小时，TLC检测反应进度；降至室温后，直接通过旋转蒸发仪除去溶剂，经柱层析(洗脱剂：纯二氯甲烷)分离得到纯净染料1b，橙红色固体，66.8毫克，产率为23％。其表征为：核磁氢谱 1HNMR(300MHz,DMSO-d 6)δ(ppm)8.87(s,1H,Ar-H),8.29(d,J＝6.6Hz,1H,Ar-H),7.91(d,J＝7.2Hz,1H,Ar-H),7.60(d,J＝6.6Hz,Ar-H),7.13(s,1H,Ar-H),6.77(t,J＝6.6Hz,1H,Ar-H),6.56(s,1H,Ar-H),3.46(q,J＝18Hz,8H,4×CH 2),1.17(q,J＝6.0Hz,12H,4×CH 3).核磁碳谱 13C NMR(151MHz,CDCl 3)δ(ppm)162.7,153.9,145.5,137.9,129.5,128.3,125.4,123.0,114.8,108.9,104.6,98.1,44.7,12.6.高分辨质谱HRMS(ESI +)m/z理论值：C 24H 27N 3O 2 +,[M+H] +:390.2176,实测值:390.2217。
实施例3
染料1c合成的步骤如下：
取化合物1-萘胺5a(1.0毫摩尔，153.6毫克)，化合物5e(1.0毫摩尔，300毫克)和三氯化铝(1.0毫摩尔，142.9毫克)溶解于20.0毫升的四氢呋喃中，然后回流3到4个小时，TLC检测反应进度；降至室温后，直接通过旋转蒸发仪除去溶剂，经柱层析(洗脱剂：纯二氯甲烷)分离得到纯净染料1c，亮黄色固体，123.7毫克，产率41.2％。其表征为：核磁氢谱 1H NMR(300MHz,DMSO-d 6)δ(ppm)9.33(d,J＝5.4Hz,1H,Ar-H),9.01(s,1H,Ar-H),8.53(d,J＝6.9Hz,1H,Ar-H),8.04(d,J＝5.4Hz,Ar-H),7.98(d,J＝6.6Hz,1H,Ar-H),7.84(q,J＝9.0Hz,3H,Ar-H),6.83(d,J＝6.3Hz,1H,Ar-H),6.56(s,1H,Ar-H),3.46(q,J＝5.1Hz,4H,2×CH 2),1.16(q,J＝9.9Hz,6H,2×CH 3).核磁碳谱 13C NMR(151MHz,CDCl 3)δ(ppm)162.3,154.7,150.5,149.8,138.9,135.1,130.8,129.7,127.8,127.0,126.1,125.6,124.4,109.1,98.1,45.1,12.5.高分辨质谱HRMS(ESI +)m/z理论值：C 24H 20N 2O 2 +，[M+Na] +:391.1417,实测值:391.1433。
实施例4
为了验证苯环的两边对称位置都能进行成环反应，进一步利用化合物对苯二胺5c合成了染料1d。
染料1d合成的一般步骤如下：
取化合物对苯二胺5c(1.0毫摩尔，55.09毫克)，化合物5e(2.0毫摩尔，285.0毫克)和三氯化铝(2.0毫摩尔，135.8毫克)溶解于25.0毫升的四氢呋喃中，然后回流6到7个小时，TLC检测反应进度；降至室温后，直接通过旋转蒸发仪除去溶剂，经柱层析(洗脱剂：二氯甲烷/甲醇(40/1，v/v))分离得到纯净染料1d，橙黄色固体，108.73毫克，产率21.8％。其表征为：核磁氢谱 1H NMR(300MHz,CDCl 3-d 6)δ(ppm)7.94(d,J＝9.0Hz,1H,Ar-H),7.86(d,J＝9.0,1H,Ar-H),6.88(s,2H,Ar-H),6.75(d,J＝7.8Hz,2H,Ar-H),6.59(d,J＝8.7,2H,Ar-H),6.37(s,2H,Ar-H),3.44(m,8H,4×CH 2),1.20(t,J＝14.1Hz,12H,4×CH 3).高分辨质谱HRMS(ESI +)m/z理论值：C 24H 20N 2O 2 +,[M+H] +:559.2340,实测值:559.4016。
效果对比1
对上述制备的染料1a-1d(浓度为10μM)进行光稳定性测试，首先称取相应质量的染料1a-1d和参照物Cy7，将其分别溶解在乙腈中(浓度为10μM)，用飞利浦碘钨灯(500W)照射所有样品，灯与样品间的距离设为25cm。在灯和样品之间放置一个8cm厚的亚硝酸钠(60克每升)冷阱，以消除热量和短波长光。连续照射6h，其中每半小时进行一次紫外、荧光测试，六小时后，光稳定性根据照射前后不同时间吸收强度的变化来计算剩余吸收率。如图2所示，荧光标记物光稳定性分别是1a：46％，1b：43％，1c：76％，1d：71％，Cy7：5.3％， 可以看出染料1a-1d与菁类染料相比有着较高的光稳定性。
效果对比2
对上述制备的染料1a-1d(浓度为10μM)在二氯甲烷中的紫外吸收和荧光发射性质进行了测试，横坐标为波长，纵坐标分别为吸光度或荧光强度，结果如图3所示。
荧光标记物1a(浓度为10μM)在二氯甲烷中的紫外吸收和荧光发射光谱，如图3所示，荧光标记物1a在452nm处有最大吸收(图3(a))；荧光标记物1a在532nm处有最高的荧光强度(图3(e))，此时的激发波长为428nm，狭缝宽度为3nm/3nm。
荧光标记物1b(浓度为10μM)在二氯甲烷中的紫外吸收和荧光发射光谱，如图3所示，荧光标记物1b在473nm处有最大吸收(图3(b))；荧光标记物1b在562nm处有最高的荧光强度(图3(f))，此时的激发波长为407nm，狭缝宽度为3nm/3nm。
荧光标记物1c(浓度为10μM)在二氯甲烷中的紫外吸收和荧光发射光谱，如图3所示，荧光标记物1c在375nm处有最大吸收(图3(c))；荧光标记物1c在546nm处有最高的荧光强度(图3(g))，此时的激发波长为461nm，狭缝宽度为3nm/3nm。
荧光标记物1d(浓度为10μM)在二氯甲烷中的紫外吸收和荧光发射光谱，如图3所示，荧光标记物1d在429nm处有最大吸收(图3(d))；荧光标记物1d在495nm处有最高的荧光强度(图3(h))，此时的激发波长为430nm，狭缝宽度为5nm/3nm。
效果对比3
此外，本发明还测试了荧光标记物1a-1d的细胞毒性。通过使用CCK-8方法测量在这些染料的存在下HeLa细胞的活力。将HeLa细胞分别与不同浓度的染料(2、4、6、8和10μM)孵育6h。如图4所示，荧光标记物1a-1d的细胞毒性测试结果表明：它们具有良好的细胞生存力并且适合用于活细胞成像。
图4中，细胞存活率(％)＝(A sample–A b)/(A c–A b)，其中A c：阴性对照(包括培养基和细胞，无待测染料添加)，A b：空白(包括待测染料和培养基，无细胞添加)，A sample：测试组(包括培养基、细胞和待测染料)。
效果对比4
为了测试荧光标记物1a与商用脂滴标记物相比的荧光标记能力，使用二甲基亚砜将荧光标记物1a配制成母液，随后加入常规细胞培养基中，使得荧光标记物1a在细胞培养基中的浓度为6μM，随后分别加入脂滴红色标记物Nile Red(100nM)，与HeLa细胞在饱和湿度、37℃、5％CO 2培养箱共同培养5min(以下实验相同)；然后经PBS缓冲液洗三次后，利用激光共聚焦显微镜进行细胞成像。绿光通道选用405nm激发，收集468-550nm范围内的荧 光信号，红光通道使用561nm激发，收集570-750nm范围内的荧光信号。结果如图5所示，其中(a)为明场，(b)为荧光标记物1a的细胞成像图，(c)为脂滴红色标记物的细胞成像图，(d)为绿光通道和红光通道的叠加图，(e)为叠加图中ROI线的荧光强度，(f)为共定位实验，它们的共定位系数为0.95。结果表明，荧光标记物1a的荧光图像与商用线粒体红色标记物Nile Red的荧光图像在细胞中的分布情况一致，且强度相近，表明荧光标记物1a在HeLa细胞中具有脂滴标记能力，可作为脂滴绿色标记物。
效果对比5
为了验证荧光标记物1b(6μM)对HeLa细胞脂滴的标记效果，采用的实验方法与上述荧光标记物1a相同，如图5所示，(g)为明场，(h)为荧光标记物1b的细胞成像图，(i)为脂滴红色标记物的细胞成像图，(j)为绿光通道和红光通道的叠加图，(k)为叠加图中ROI线的荧光强度，(l)为共定位实验，它们的共定位系数为0.97。结果表明荧光标记物1b同探针1a一样，在HeLa细胞中都对脂滴具有很好的靶向能力。
效果对比6
荧光标记物1c(5μM)的实验方法与上述荧光标记物1a一样，如图5所示，(m)为明场，(n)为荧光标记物1b的细胞成像图，(o)为脂滴红色标记物的细胞成像图，(p)为绿光通道和红光通道的叠加图，(q)为叠加图中ROI线的荧光强度，(r)为共定位实验，它们的共定位系数为0.85。以上的结果表明荧光标记物1c同探针1a和1b一样，在HeLa细胞中都对脂滴具有很好的靶向能力。
效果对比7
为了验证化合物1d是否与探针1a-1c一样，都可以对HeLa细胞中的脂滴进行荧光成像。对其进行细胞实验，使用二甲基亚砜将荧光标记物1d配制成母液，随后加入常规细胞培养基中，使得荧光标记物1d在细胞培养基中的浓度为8μM，与HeLa细胞在饱和湿度、37℃、5％CO 2培养箱共同培养5min；然后经PBS缓冲液洗三次后，利用激光共聚焦显微镜进行细胞成像。绿光通道选用405nm激发，收集468-550nm范围内的荧光信号，结果发现1d并没有对细胞中的脂滴进行染色，其染色部位类似于线粒体。为了验证这个现象再次进行共定位细胞成像，在原来的细胞培养基中加入线粒体商用红色标记物CMARos(100nM)，结果如图5所示，(s)为明场，(t)为荧光标记物1d的细胞成像图，(u)为线粒体红色标记物的细胞成像图，(v)为绿光通道和红光通道的叠加图，(w)为叠加图中ROI线的荧光强度，(x)为共定位实验，它们的共定位系数为0.90。这一现象表明染料1d具有线粒体的标记能力，可以作为线粒体的绿色标记物。
可以看出，本发明首次公开了基于芳香胺类与香豆素类衍生物的成环反应，合成了一系列氨基取代色烯并喹啉型荧光标记物，可以实现对脂滴以及中性结构对线粒体的荧光成像。本发明在通过一步反应成环之后使化合物在二氯甲烷有机溶剂中具有极强的荧光强度，并且与菁类染料相比具有高的光学稳定性，同时在进一步应用于生物细胞成像时，细胞毒性低，对生物样品损伤小，选择性高且能够快速地对细胞染色且成像。成像结果表明了此类化合物对脂滴或线粒体细胞器具有特异的靶向性。
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (9)

  1. 一种氨基取代色烯并喹啉型荧光标记物，其特征在于：所述氨基取代色烯并喹啉型荧光标记物的结构中心有一个或多个色烯并喹啉环，所述色烯并喹啉环的周边有一个或多个二烷基氨基。
  2. 根据权利要求1所述的氨基取代色烯并喹啉型荧光标记物，其特征在于，其结构式如1a-1d中任一所示：
    Figure PCTCN2021143133-appb-100001
  3. 根据权利要求1或2所述的氨基取代色烯并喹啉型荧光标记物，其特征在于：所述氨基取代色烯并喹啉型荧光标记物由芳香胺和香豆素类荧光前体反应合成。
  4. 根据权利要求3所述的氨基取代色烯并喹啉型荧光标记物，其特征在于：所述芳香胺为1-萘胺、N,N-二乙基对苯二胺或对苯二胺。
  5. 根据权利要求3所述的氨基取代色烯并喹啉型荧光标记物，其特征在于：所述香豆素荧光前体为4-氯-3-甲酰基香豆素或7-(N,N-二乙基氨基)-4-氯香豆素-3-甲醛。
  6. 根据权利要求3所述的氨基取代色烯并喹啉型荧光标记物，其特征在于：所述芳香胺和香豆素类荧光前体的摩尔比为1:1-2。
  7. 根据权利要求3所述的氨基取代色烯并喹啉型荧光标记物，其特征在于：所述芳香胺和香豆素类荧光前体是在催化剂作用下反应合成氨基取代色烯并喹啉型荧光标记物；所述催化剂为路易斯酸。
  8. 根据权利要求7所述的氨基取代色烯并喹啉型荧光标记物，其特征在于：所述芳香胺和催化剂的摩尔比为1：1-2。
  9. 如权利要求1-8中任一项所述的氨基取代色烯并喹啉型荧光标记物作为细胞器荧光标记的应用。
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