CN112062771B - 含杂原子多环芳烃的多类型细胞器荧光探针的合成与应用 - Google Patents
含杂原子多环芳烃的多类型细胞器荧光探针的合成与应用 Download PDFInfo
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- CN112062771B CN112062771B CN202010653277.XA CN202010653277A CN112062771B CN 112062771 B CN112062771 B CN 112062771B CN 202010653277 A CN202010653277 A CN 202010653277A CN 112062771 B CN112062771 B CN 112062771B
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- acridine
- hexafluoroantimonate
- mitochondria
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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
- C07D491/06—Peri-condensed systems
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0026—Acridine dyes
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- 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
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- G—PHYSICS
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- 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"
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- 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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- 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
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
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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
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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
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Abstract
本发明涉及一种含杂原子多环芳烃的多类型细胞器荧光探针的合成与应用。本发明所述荧光探针可对溶酶体、内质网、线粒体、线粒体‑细胞核进行特异性靶向，并应用于荧光成像、标记、示踪和生物治疗。本发明的合成方法是通过铑催化活化C＝X(X＝N,O,S,N‑R)与炔烃发生[4+2]氧化环化反应。该方法利用廉价易得的吖啶酮及其类似物，高效快速地合成了结构多样的杂原子掺杂的多环芳烃(PAHs)衍生物。与传统的反应体系相比，该方法不仅实现了高通量快速合成多种杂原子掺杂的多环芳烃(PAHs)衍生物，具有操作简便，条件温和，成本低廉等优点；同时，通过该方法能快速建立一个功能强大的荧光功能分子库，具有巨大的潜在商业价值。
Description
技术领域
本发明涉及四类具有特异性靶向多类型细胞器的杂原子掺杂多环芳烃荧光探针的合成与应用。
背景技术
随着生命科学学科的蓬勃发展，人们对亚细胞器的研究不断深入。溶酶体、内质网、线粒体和细胞核作为真核细胞的主要细胞器，承担了必要且重要的生命活动。溶酶体作为细胞器的“消化器官”，其体内含有的多种酸性水解酶使得其内环境的pH值保持在4.5-5.0范围内。作为真核细胞中的重要酸性细胞器,溶酶体具有胞内消化和调节分泌的重要功能。同时，其数量和分布异常,可能会造成肺部疾病、溶酶体贮积症以及肿瘤等各类病症.[参见：(a)Luzio,J.P.；Pryor,P.R.；Bright,N.A.Nat.Rev.Mol.Cell Biology,2007,8,622；(b)Xu,W.；Zeng,Z.；Jiang,J.-H.；Chang,Y.-T.；Yuan,L.Angew.Chem.Int.Ed.2016,55,13658；(c)Davies,B.A.；Lee,J.R.E.；Oestreich,A.J.；Katzmann,D.J.Chem.Rev.2009,109,1575；(d)Ghosh,M.；Carlsson,F.；Laskar,A.；Yuan,X.；Li,W.FEBS Lett.2011,585,623.]。内质网是连接细胞核、细胞膜和细胞质的桥梁，负责糖、脂类和蛋白质的合成与运输。生理和病理条件会干扰内质网的稳态，从而对蛋白质折叠产生负面影响，导致未折叠蛋白质的积累。功能异常通常会在内质网中积聚，进而诱发神经退行性疾病、癌症等疾病[参见：Sovolyova,N.；Healy,S.；Samali,A.；Logue,S.E.Biol.Chem.2014,395,1.]。因此，维持内质网的稳态和功能是正常细胞功能所必需的。内质网的实时跟踪对于掌握内质网复杂的生理功能过程和与内质网相关疾病的病理机制具有重要意义[参见：(a)Borgese,N.；Francolini,M.；Snapp,E.Curr.Opin.Cell Biol.2006,18,358；(b)Brodsky,J.L.；Skach,W.R.Curr.Opin.Cell Biol.2011,23,464；(c)Westrate,L.M.；Lee,J.E.Prinz,W.A.；Voeltz,G.K.Annu.Rev.Biochem.2015,84,791；(d)Zhang,H.；Hu,J.Trends CellBiol.2016,26,934.]。线粒体作为ATP的直接提供者参与了细胞的分化、调控以及凋亡过程，其数量、结构与功能的变化与阿尔兹海默症、帕金森综合征等神经退行性疾病密切相关[参见：Cui,Y.；Long,J.G.In Mitochondrial Medicine and Health,Eds.:Liu,J.K.；Wang,X.M.,Science Press,Beijing,2012,Chapter 1.]。细胞核既是细胞的中央控制枢纽，又是遗传物质染色体的主要存在部位，它在细胞的生长、代谢、分化等过程中起着不可替代的作用[参见：(a)Haraguchi,T.；Kaneda,T.；Hiraoka,Y.Genes Cells 1997,2,369；(b)Johnson,I.；Spence,M.T.Z.The Molecular Probes Handbook:A Guide toFluorescent Probes and Labeling Technologies,Life Technologies Corporation,Carlsbad,CA,11th edn,2010,ch.8.1and 12.5.]。近年来，由于其高灵敏度、高分辨率以及快速的响应时间等特性，荧光成像技术已经被广泛应用于生物学和医学等众多领域之中。细胞器荧光探针能够渗透到细胞内,选择性的与细胞器结合。利用共聚焦激光扫描显微镜(confocal laser scanning microscope,CLSM)不但可以获得溶酶体、内质网、线粒体、细胞核等细胞器的清晰荧光图像,而且可以动态观察活细胞的形态学变化[参见：(a)Liu,C.；Zhang,R.；Zhang,W.；Liu,J.；Wang,Y.-L.；Du,Z.；Song,B.；Xu,Z.P.；Yuan,J.J.Am.Chem.Soc.2019,141,8462；(b)Goujon,A.；Colom,A.；Straková,K.；Mercier,V.；Mahecic,D.；Manley,S.；Sakai,N.；Roux,A.；Stefan Matile,S.J.Am.Chem.Soc.2019,141,3380；(c)Cheng,Y.；Li,G.；Liu,Y.；Shi,Y.；Gao,G.；Wu,D.；Lan,J.；You,J.J.Am.Chem.Soc.2016,138,4730；(d)Wu,J.；Zou,Y.；Li,C.；Sicking,W.；Piantanida,I.；Yi,T.；Schmuck,C.J.Am.Chem.Soc.2012,134,1958.]。目前，可市售易得的各类型细胞器荧光探针试剂种类较少，且价格普遍昂贵。其化合物结构也相对复杂，光稳定性与毒性有待提升，对被测物的副作用影响较大。同时，大多数市售细胞器标记试剂的合成工艺难度较大和生产成本较高，不利于工业化。
稳定的多环芳香烃(Polycyclic Aromatic Hydrocarbon，简称PAH)在有机合成化学、光化学、材料化学和生物学等领域有着广泛的应用前景[参见：(a)Hicks,R.G.Org.Biomol.Chem.2007,5,1321；(b)Mishra,A.；Behera,R.K.；Behera,P.K.；Mishra,B.K.；Behera,G.B.Chem.Rev.2000,100,1973；(c)Morita,Y.；Suzuki,S.；Sato,K.；Takui,T.Nat.Chem.2011,3,197；(d)Sun,Z.；Wu,J.J.Mater.Chem.2012,22,4151；(e)Ratera,I.；Veciana,J.Chem.Soc.Rev.2012,41,303；(f)Machado,V.G.；Stock,R.I.；Reichardt,C.Chem.Rev.2014,114,10429；(g)Bosson,J.；Gouin,J.；Lacour,L.Chem.Soc.Rev.2014,43,2824；(h)Romero,N.A.；Nicewicz,D.A.Chem.Rev.2016,116,10075.]。在PAHs骨架上引入杂原子能够显著影响其光物理特性，这些杂原子掺杂的离子型骨架在荧光材料、特异性识别和生物标记等方面受到高度重视[参见：(a)Appleton,A.L.；Brombosz,S.M.；Barlow,S.；Sears,J.S.；Bredas,J.-L.；Marder,S.R.；Bunz,U.H.F.Nat.Commun.2010,1,91.(b)Liang,Z.；Tang,Q.；Xu,J.；Miao,Q.Adv.Mater.2011,23,1535.(c)Bouit,P.-A.；Escande,A.；
R.；Szieberth,D.；Lescop,C.；Nyulászi,L.；Hissler,M.；Réau,R.J.Am.Chem.Soc.2012,134,6524.(d)Bunz,U.H.F.；Engelhart,J.U.；Lindner,B.D.；Schaffroth,M.Angew.Chem.Int.Ed.2013,52,3810.(e)Mateo-Alonso,A.Chem.Soc.Rev.2014,43,6311.(f)Hirai,M.；Tanaka,N.；Sakai,M.；Yamaguchi,S.Chem.Rev.2019,119,8291.]。这些杂原子掺杂的多环骨架PAHs在荧光材料和生物标志物方面有着巨大的应用潜力。
然而，传统的杂原子掺杂的多环骨架PAHs的制备方法通常要经过繁琐的合成路线，同时在苛刻的反应条件下制备，可最终的收率不高，原子/步骤经济性较低，这使得分子骨架的多样性的愿望十分受限。本专利的目的不仅仅局限于高效快速合成某一种细胞器特异性靶向的荧光探针，而是一石多鸟，用一种方法有预见性的获得溶酶体，内质网，线粒体以及线粒体-细胞核的特异性靶向荧光探针。
发明内容
本发明的目的在于开发一个基于杂原子掺杂多环芳烃的多类型细胞器特异性靶向荧光探针试剂分子库：利用高效、简洁以及环境友好的C＝X(X＝N,O,S，N-R)与炔烃进行氧化环化，快速高效合成杂原子掺杂多环芳烃的I型、II型、III型、IV型、型V荧光分子，并将其应用于溶酶体，内质网，线粒体以及线粒体-细胞核进行特异性标记，并将其运用于活体成像、示踪和治疗等领域。
本发明利用廉价易得的吖啶酮及其类似物，通过铑催化的C＝X(X＝N,O,S,N-R)键与各类型炔烃(包括但不仅限于富电炔烃、缺电炔烃、芳基炔烃和烷基炔烃)的[4+2]氧化环化反应，高效快速地合成结构多样的杂原子掺杂的多环芳烃(PAHs)衍生物。其结构通式见附图1。
本发明解决该问题的技术方案是采用以下的原料及制备路线，如附图2：
(1)在干净、干燥的反应器中加入吖啶酮或吖啶酮类似物、炔烃、催化剂、氧化剂、添加剂和溶剂，室温下混合均匀，随后在无水无氧条件下－40～160℃反应0.1～720小时，或者在空气氛围下－40～160℃反应0.1～720小时；
(2)反应完成后将反应管冷却至室温，加入二氯甲烷将反应体系稀释，再经硅藻土过滤，并用二氯甲烷洗涤，合并滤液，减压移去溶剂，剩余物用硅胶柱层析或者中性三氧化二铝分离纯化，真空干燥。
其中，吖啶酮及其类似物的结构通式为：
其中X和Y分别为N、N-R、O、S中的一种或两种。
其中炔烃的结构通式为：
R1，R2分别为对称的或者非对称的氢、卤素、烷基、烷氧基、苄基、酯基、酰胺基、羰基、醛基、烷基氨基、羰基、硝基、氰基取代芳基或取代杂芳基等等中的一种或两种。其中烷基的碳链为碳个数为0～40的直链、支链或环链。
步骤(1)中，催化剂为二氯(五甲基环戊二烯基)合铑(III)二聚体、铑碳、三氯化铑、醋酸铑、三苯基膦羰基铑、乙酰丙酮三苯基膦羰基铑、双环辛烯氯化铑二聚体、三苯基膦氯化铑、钯碳、四(三苯基膦)钯、醋酸钯、氯化钯、二(乙腈)二氯化钯、二(苯腈)二氯化钯、1,1'-二(二苯膦基)二茂铁二氯化钯、二(三苯基膦)二氯化钯、双(二亚苄基丙酮)钯、三(二亚苄基丙酮)二钯、氯化烯丙基钯(II)二聚物、(1,5-环辛二烯)二氯化钯(II)、三氯化钌、三苯基膦氯化钌、二氯二羰基双(三苯基膦)钌、双(2-甲基烯丙基)(1,5-环辛二烯)钌(II)、对伞花烃二氯化钌二聚体中的一种或一种以上。
步骤(1)中，氧化剂为氧化银、醋酸银、碳酸银、氧气、一水合醋酸铜、醋酸铜、氯化铜、溴化铜、三氟乙酸酮、三氟甲烷磺酸铜(Ⅱ)、乙酰丙酮铜、硝酸银、六氟锑酸银、醋酸碘苯、苯醌、二氯二氰苯醌、过二硫酸钠、过二硫酸铵、过二硫酸钾、二叔丁基过氧化物中的一种或一种以上。
步骤(1)中，添加剂为六氟锑酸银、六氟锑酸钠、三氟甲磺酸银、三氟甲磺酸钠、三氟甲磺酸锌、四氟硼酸银、四氟硼酸钠、吡啶、2,2’-联吡啶、1,10-邻菲咯啉、三苯基膦、三叔丁基膦四氟硼酸盐、三环己基膦四氟硼酸盐、1,1'-联萘-2,2'-双二苯膦、2-(二叔丁基磷)-1,1'-联萘、1,2-双(二甲基瞵)乙烷、双(2-二苯基膦乙基)苯基磷、正丁基-二(1-金刚烷基)磷、1,1'-双(二苯基膦)二茂铁、1,2,3,4,5-五苯基-1’-(二叔丁基膦)二茂铁、2,2'-二(二-3,5-甲基苯基膦)-1,1'-联萘、N,N-二甲基-1-(2-联苯膦基)二茂铁乙胺、1,1'-(二苯基膦基)丙烷、二苯基(2,4,6-三甲基苯甲酰基)氧化膦、L-脯氨酸、特戊酸、乙酸、三氟乙酸、三氟甲磺酸、对甲苯磺酸、碳酸钠、碳酸氢钾、碳酸氢钠、碳酸钾、碳酸铯、特戊酸铯、磷酸钾、叔丁醇钠、叔丁醇钾、磷酸氢二钾、醋酸钠、醋酸钾、二乙胺、三乙胺、二异丙基胺、环己二胺、四丁基氯化铵、四丁基溴化铵、四丁基碘化铵、四丁基氢氧化铵、四丁基硫酸氢铵、六(亚甲基)四胺、四甲基二乙胺、二甲基二乙胺中的一种或一种以上。
步骤(1)中溶剂为二氯甲烷、二氯乙烷、三氯甲烷、甲醇、乙醇、四氢呋喃、乙醚、二甲基亚砜、苯、邻二氯苯、氯苯、甲苯、二甲苯、均三甲苯、环己烷、石油醚、叔戊醇、1,4-二氧六环、1,2-二氯乙烷、N,N-二甲基甲酰胺、N,N-二甲基乙酰胺中的一种或一种以上。
步骤(1)中，吖啶酮类似物:炔烃:催化剂:氧化剂:添加剂的摩尔比为1:(0.01～50):(0.01～10):(0.01～100):(0.01～200)。
步骤(1)反应温度为-40～160℃。
步骤(1)中反应时间为0.1～720小时。
步骤(1)中，吖啶酮类似物的反应浓度为0.0001～10mol/L。
用核磁共振氢谱(1H NMR)、碳谱(13C NMR)以及高分辨质谱证实了吖啶酮类似物与炔烃反应的最终产物的结构(如附图3)。检测所用仪器为：Bruker AV II-400MHz型核磁共振仪，其中TMS为内标，氘代CDCl3、氘代CD2Cl2、氘代乙腈和氘代丙酮为溶剂；Waters-Q-TOF-Premier(ESI)型高分辨质谱仪。
该类反应产物具有全波段荧光发射可调控的荧光特性，其荧光发射光谱可以涵盖紫外-可见光-近红外区域，波长范围为300～700nm(如附图4)。
该类反应产物的粒子粒径较小，容易穿透细胞膜进入细胞内(如附图5)。
该类反应产物可分别对溶酶体、内质网、线粒体进行特异性标记或者线粒体-细胞核的荧光标记，并实现荧光成像，(如附图6)。
反应产物的光稳定性良好，在每0.97秒扫描一次，连续扫描50次的情况下荧光发射强度仅少量减弱(如附图7)。
MTS毒性实验表明该类产物几乎没有细胞毒性(如附图8)。
光谱表征所用仪器为：HITACHI U-2910型紫外－可见分光光度计(扫描范围250～1100nm)，崛场Fluoromax-4型荧光光谱仪(扫描范围250～900nm)和莱卡TCS SP8型激光共聚焦显微镜。
与现有的市售细胞器标记试剂相比，本发明所述该类产物生物兼容性能更好，所用合成路线更加简洁、高效、环境友好，具体体现为：
1.本发明所述可对溶酶体进行特异性标记的荧光分子type I的光稳定性更好。相比于大多数市售溶酶体荧光标记试剂(通常在绿、红和深红范围：450-750nm)，本发明所述荧光分子type I在共聚焦成像时，使用激光进行多次照射后的荧光强度仅少量减弱。因此，更适合进行长时间荧光成像示踪或标记；
2.本发明所述可对溶酶体进行特异性标记的荧光分子type I属于质子型分子，对溶酶体的副作用更小。相比于大多数市售溶酶体荧光标记试剂，type I的质子化构型避免了在对溶酶体靶向后对溶酶体的不友好碱化，保证了溶酶体内环境正常的PH值；因此，更适合用于溶酶体的长期示踪观测；
3.本发明所述可对内质网进行特异性标记的荧光分子type II分子量很小、体积小，相比于大多数市售内质网光标记试剂(分子量500以上)，更容易穿透细胞膜，进入细胞；
4.本发明所述可对线粒体进行特异性标记的荧光分子type III可以十分清晰地显现线粒体的精细结构，可应用于，但不仅限于开展深入的细胞研究；
5.本发明所述可对线粒体-细胞核进行标记的荧光分子type IV可作为线粒体和细胞核的纽带，对线粒体和细胞核之间的共性进行深入研究；
6.本发明所用合成路线为C＝X(X＝N，O，S，N-R)键与炔烃直接进行的[4+2]氧化环化反应，。C–H/C–H直接交叉氧化偶联反应，与传统的制备技术相比较，缩短了较为冗长的有机合成步骤，避免了底物预活化的繁琐过程，提高了合成反应总产率，降低了总成本；
7.本发明所用的合成方法可通用于合成I型、II型、III型和IV型荧光探针分子。
8.本发明所用合成路线所用吖啶酮类似物和炔烃的化学性质稳定，易于合成，有利于降低工艺难度；
9.本发明反应温度低，反应快速，降低了能耗。
10.本发明避免产生大量废弃物，提高了原子经济性和环境友好性。
附图说明
图1为I型、II型、III型、IV型、型V分子的结构式；X、Y＝N,O,S和N-R中的一种或两种，R1、R2、R3为对称的或者非对称的氢、卤素、烷基、烷氧基、苄基、酯基、酰胺基、羰基、醛基、烷基氨基、羰基、硝基、氰基取代芳基或取代杂芳基中的一种或两种，A为六氟锑酸根、三氟甲烷磺酸根、四氟硼酸根、特戊酸根、氟离子、氯离子、溴离子、碘离子任一种阴离子；
图2为制备I型、II型、III型、IV型、型V分子的化学反应式；
图3为本发明化合物2,3-二苯基吡喃[4,3,2-kl]吖啶(II-a)的核磁氢谱图和碳谱图；
图4为本发明化合物2,3-二苯基吡喃[4,3,2-kl]吖啶(II-a)在二氯甲烷作为溶剂时的紫外-可见-近红外吸收光谱(黑色实线)和荧光发射光谱(红色实线)；
图5为本发明化合物I型、II型、III型和IV型荧光分子的动态光散射(DLS)测试数据；
图6为本发明化合物I型、II型和III型荧光分子分别与市售溶酶体深红染料LysoTrackerTM Deep Red、内质网红染料ER-TrackerTM Red(BODIPYTM TR glibenclamide)和线粒体深红染料
Deep Red FM在HepG2细胞中的共聚焦荧光成像图。以及IV型荧光分子与市售线粒体深红染料
Deep Red FM和市售细胞核染料Hoechst33342在HepG2细胞中对线粒体和细胞核共染的共聚焦荧光成像图；
图7为本发明化合物I-b和I-e的光稳定性实验图，左上角的数字代表激光扫描次数从左往右依次增加；
图8为本发明化合物I-b在HepG2细胞中的MTS细胞毒性实验结果。
具体实施方式
下面结合具体实施案例对本发明作进一步描述，将有助于对本发明的理解。但并不能以此来限制本发明的权利范围，而本发明的权利范围应以权利要求书阐述的为准。
本发明实施例中，HepG2细胞株采购于ATCC(American Type CultureCollection)公司，10％胎牛血清采购于Hyclone公司，DMEM(H)(Dulbecco's mimimumessential medium)培养基采购于美国Gibco。线粒体染料MitoTracker Green FM采购于Life Technologies公司。
实施例1：2,3-二苯基吡喃[4,3,2-kl]吖啶7-鎓六氟锑酸盐(I-a)的合成
(1)将吖啶酮(39.1mg,0.20mmol)、二苯乙炔(71.2mg,0.40mmol)、[Cp*RhCl2]2(3.1mg,5μmol,2.5mol％)，AgOAc(66.8mg,0.40mmol)，AgSbF6(13.7mg,40μmol,20mol％)，NaSbF6(51.7mg,0.20mmol)和四氢呋喃(1.0mL)加入反应管搅拌均匀后加热到80℃，反应10小时；
(2)反应完成后，将反应管冷却至室温，加入10mL二氯甲烷将反应体系稀释，再经硅藻土过滤并用10～20mL的二氯甲烷洗涤，合并滤液，减压移去溶剂，剩余物用硅胶柱层析(二氯甲烷/乙酸乙酯＝15:1,v/v)分离纯化，真空干燥后得到淡黄色固体目标产物2,3-二苯基吡喃[4,3,2-kl]吖啶7-鎓六氟锑酸盐(I-a)81.3mg，产率67％。1H NMR(400MHz,CD3CN):δ＝12.24(brs,1H),8.56-8.53(m,1H),8.18-8.13(m,2H),8.00-7.96(m,1H),7.85-7.82(m,1H),7.78-7.73(m,1H),7.57-7.51(m,5H),7.46-7.42(m,1H),7.40-7.36(m,4H),7.10-7.08(m,1H)ppm.13C NMR(100MHz,CD3CN):δ＝165.1,153.2,142.1,141.8,140.7,138.6,135.2,133.4,132.4,131.25,131.15,130.50,130.48,130.0,129.4,127.4,124.8,123.8,119.6,117.4,115.2,114.6ppm.HRMS(ESI+):计算值C27H18NO+[M]+372.1383,实测值372.1375。
实施例2：2,3-二苯基吡喃[4,3,2-kl]吖啶(II-a)的合成
(1)将吖啶酮(39.1mg,0.20mmol)、二苯乙炔(71.2mg,0.40mmol)、[Cp*RhCl2]2(3.1mg,5μmol,2.5mol％)，AgOAc(66.8mg,0.40mmol)，AgSbF6(13.7mg,40μmol,20mol％)，NaSbF6(51.7mg,0.20mmol)和四氢呋喃(1.0mL)加入反应管搅拌均匀后加热到80℃，反应10小时；
(2)反应完成后，将反应管冷却至室温，加入10mL二氯甲烷将反应体系稀释，再经硅藻土过滤并用10～20mL的二氯甲烷洗涤，合并滤液，减压移去溶剂，剩余物用中性三氧化二铝柱层析(二氯甲烷/乙酸乙酯＝10:1,v/v)分离纯化，真空干燥后得到淡黄色固体目标产物2,3-二苯基吡喃[4,3,2-kl]吖啶(II-a)55.7mg，产率75％。1H NMR(400MHz,CD2Cl2):δ＝8.28(d,J＝9.2Hz,1H),8.03(d,J＝8.8Hz,1H),7.77-7.72(m,2H),7.57-7.53(m,1H),7.47-7.41(m,6H),7.35-7.26(m,5H),6.55(d,J＝7.2Hz,1H)ppm.13C NMR(100MHz,CD2Cl2):δ＝156.5,150.8,150.3,149.8,135.0,133.8,132.6,132.5,131.3,131.0,129.6,129.5,129.3,129.3,128.5,128.3,125.0,124.3,122.0,121.5,114.6,114.4,113.8ppm.HRMS(ESI+):计算值C27H18NO[M+H]+372.1383,实测值372.1372。
实施例3：2,3-二苯基-7-甲基-7H-吡喃[4,3,2-kl]吖啶-1-鎓六氟锑酸盐(III-a)的合成
(1)将N-甲基吖啶酮(41.8mg,0.20mmol)、二苯乙炔(71.2mg,0.40mmol)、[Cp*RhCl2]2(3.1mg,5μmol,2.5mol％)，AgOAc(66.8mg,0.40mmol)，AgSbF6(13.7mg,40μmol,20mol％)，NaSbF6(51.7mg,0.20mmol)和四氢呋喃(1.0mL)加入反应管搅拌均匀后加热到80℃，反应10小时；
(2)反应完成后，将反应管冷却至室温，加入10mL二氯甲烷将反应体系稀释，再经硅藻土过滤并用10～20mL的二氯甲烷洗涤，合并滤液，减压移去溶剂，剩余物用硅胶柱层析(二氯甲烷/乙酸乙酯＝20:1,v/v)分离纯化，真空干燥后得到淡黄色固体目标产物2,3-二苯基-7-甲基-7H-吡喃[4,3,2-kl]吖啶-1-鎓六氟锑酸盐(III-a)85.7mg，产率69％。1H NMR(400MHz,acetone-d6):δ＝8.83(d,J＝8.4Hz,1H),8.56(d,J＝9.2Hz,1H),8.47-8.33(m,3H),7.94(t,J＝7.6Hz,1H),7.66(d,J＝7.6Hz,2H),7.62-7.55(m,3H),7.50-7.41(m,5H),7.27(d,J＝7.6Hz,1H),4.63(s,3H)ppm.13C NMR(100MHz,acetone-d6):δ＝164.3,153.2,143.9,143.7,141.2,139.2,136.2,133.5,132.4,131.3,130.6,130.5,130.1,129.4,127.2,125.7,124.0,118.9,117.8,115.7,115.2,115.1,37.2ppm.HRMS(ESI+):计算值C28H20NO+[M]+386.1539,实测值386.1538。
实施例4：2,3-二苯基吡喃[4,3,2-kl]吖啶-7-六氟锑酸盐(IV-a)的合成
(1)将10-甲基-N-苯基吖啶-9(10H)-亚胺(56.8mg,0.20mmol)、二苯乙炔(71.2mg,0.40mmol)、[Cp*RhCl2]2(3.1mg,5μmol,2.5mol％)，AgOAc(66.8mg,0.40mmol)，AgSbF6(13.7mg,40μmol,20mol％)，NaSbF6(51.7mg,0.20mmol)和四氢呋喃(1.0mL)加入反应管搅拌均匀后加热到80℃，反应10小时；
(2)反应完成后，将反应管冷却至室温，加入10mL二氯甲烷将反应体系稀释，再经硅藻土过滤并用10～20mL的二氯甲烷洗涤，合并滤液，减压移去溶剂，剩余物用硅胶柱层析(二氯甲烷/乙酸乙酯＝15:1,v/v)分离纯化，真空干燥后得到淡黄色固体目标产物2,3-二苯基吡喃[4,3,2-kl]吖啶-7-六氟锑酸盐(IV-a)100.2mg，产率72％。1H NMR(400MHz,CD3CN):δ＝8.07(dd,J＝8.4Hz,8.0Hz,1H),7.88(dd,J＝9.2Hz,1.2Hz,1H),7.76-7.70(m,2H),7.38-7.21(m,11H),7.09-6.99(m,5H),6.95-6.90(m,2H),4.07(s,3H)ppm.13C NMR(100MHz,CD3CN):δ＝150.5,145.8,144.6,143.9,142.4,137.8,137.6,136.1,135.6,134.2,132.2,131.9,131.0,130.63,130.55,130.4,129.7,129.4,129.2,128.9,128.2,123.2,120.6,118.1,116.2,115.3,111.8,37.4ppm.HRMS(ESI+):计算值C34H25N2 +[M]+461.2012,实测值461.2011.
实施例5：2,3-二苯基吡喃[4,3,2-kl]蒽酮-1-鎓六氟锑酸盐(V-a)的合成
(1)将氧杂蒽酮(39.2mg,0.20mmol)、二苯乙炔(71.2mg,0.40mmol)、[Cp*RhCl2]2(3.1mg,5μmol,2.5mol％)，Ag2O(46.4mg,0.20mmol,1.0equiv)，AgSbF6(13.7mg,40μmol,20mol％)，NaSbF6(51.7mg,0.20mmol)和二氯乙烷(1.0mL)加入反应管，在无水无氧条件下搅拌均匀后加热到80℃，反应10小时；
(2)反应完成后，将反应管冷却至室温，加入10mL二氯甲烷将反应体系稀释，再经硅藻土过滤并用10～20mL的二氯甲烷洗涤，合并滤液，减压移去溶剂，剩余物用硅胶柱层析(二氯甲烷/乙酸乙酯＝15:1,v/v)分离纯化，真空干燥后得到淡黄色固体目标产物2,3-二苯基吡喃[4,3,2-kl]蒽酮-1-鎓六氟锑酸盐(V-a)97.4mg，产率80％。1H NMR(400MHz,acetone-d6):δ＝8.83(d,J＝8.0Hz,1H),8.67(td,J＝8.0Hz,1.6Hz,1H),8.45-8.41(m,1H),8.24(d,J＝8.4Hz,1H),8.18(d,J＝8.8Hz,1H),7.99(t,J＝7.6Hz,1H),7.70(d,J＝8.0Hz,2H),7.62-7.60(m,4H),7.56-7.46(m,5H)ppm.13C NMR(100MHz,acetone-d6):δ＝170.0,158.7,157.2,155.8,144.9,142.0,137.8,132.6,131.9,131.6,131.1,130.8,130.6,130.5,129.6,128.8,126.9,126.6,120.7,120.2,116.9,114.9,114.0ppm.HRMS(ESI+):计算值C27H17O2 +[M]+373.1223,实测值373.1229。
实施例6：化合物2,3-二苯基吡喃[4,3,2-kl]吖啶(II-a)的紫外-可见-近红外吸收光谱图和荧光发射谱图
将化合物2,3-二苯基吡喃[4,3,2-kl]吖啶(II-a)溶于二氯甲烷中，配成1×10- 5mol/L，取2.5mL放入比色皿中,测定紫外-可见-近红外吸收以及荧光发射光谱。化合物2,3-二苯基吡喃[4,3,2-kl]吖啶(II-a)的吸收光谱最大吸收峰位于430nm；荧光发射光谱最大吸收峰位于510nm，斯托克斯位移为3648cm-1(附图4)。
实施例7：化合物2,3-二苯基吡喃[4,3,2-kl]吖啶7-鎓六氟锑酸盐(I-a)与市售溶酶体染料LysoTrackerTM Deep Red在HepG2细胞中的荧光共聚焦共成像
首先，向含有10％胎牛血清的DMEM(H)培养基中通入5％CO2，将HepG2细胞于37℃下培养24小时。将培养基去除，加入1μM化合物2,3-二苯基吡喃[4,3,2-kl]吖啶7-鎓六氟锑酸盐(I-a)的DMSO溶液于37℃下培养10-15分钟后，使用磷酸盐缓冲液清洗2～3次后；随后加入1μM市售溶酶体染料LysoTrackerTM Deep Red于37℃下培养10-15分钟。待培养结束后，再次使用磷酸盐缓冲液清洗2～3次后，将培养玻底皿经荧光共聚焦显微镜成像得到图6中的I-a。图6：I-a中，a图为化合物2,3-二苯基吡喃[4,3,2-kl]吖啶7-鎓六氟锑酸盐(I-a)的荧光成像图(激发波长：488nm,发射波长收集范围：490-570nm)。b图为市售溶酶体染料LysoTrackerTM Deep Red的荧光成像图(激发波长：633nm,发射波长收集范围：640-740nm)。c图为细胞明场图。d图为a图、b图和c图的叠加图。从d图中可以直观地看出，化合物2,3-二苯基吡喃[4,3,2-kl]吖啶7-鎓六氟锑酸盐(I-a)在细胞中的分布和市售内质网染料ER-TrackerTM Red:BODIPYTM TR glibenclamide基本一致，皮尔森系数为0.89，说明化合物2,3-二苯基吡喃[4,3,2-kl]吖啶7-鎓六氟锑酸盐(I-a)具有优异的溶酶体示踪效果，能专一性标记细胞内的溶酶体。
实施例8：化合物2,3-二苯基吡喃[4,3,2-kl]吖啶(II-a)与市售内质网染料ER-TrackerTM Red:BODIPYTM TR glibenclamide在HepG2细胞中的荧光共聚焦共成像
首先，向含有10％胎牛血清的DMEM(H)培养基中通入5％CO2，将HepG2细胞于37℃下培养24小时。将培养基去除，加入1μM化合物2,3-二苯基吡喃[4,3,2-kl]吖啶(II-a)的DMSO溶液于37℃下培养10-15分钟后，使用磷酸盐缓冲液清洗2～3次后；随后加入1μM市售内质网染料ER-TrackerTM Red:BODIPYTM TR glibenclamide于37℃下培养10-15分钟。待培养结束后，再次使用磷酸盐缓冲液清洗2～3次后，将培养玻底皿经荧光共聚焦显微镜成像得到图6中的II-a。图6：II-a中，a图为化合物2,3-二苯基吡喃[4,3,2-kl]吖啶(II-a)的荧光成像图(激发波长：405nm,发射波长收集范围：450-520nm)。b图为市售内质网染料ER-TrackerTM Red:BODIPYTM TR glibenclamide的荧光成像图(激发波长：546nm,发射波长收集范围：580-680nm)。c图为细胞明场图。d图为a图、b图和c图的叠加图。从d图中可以直观地看出，化合物2,3-二苯基吡喃[4,3,2-kl]吖啶(II-a)在细胞中的分布和市售内质网染料ER-TrackerTM Red:BODIPYTM TR glibenclamide基本一致，皮尔森系数为0.98，说明化合物2,3-二苯基吡喃[4,3,2-kl]吖啶(II-a)具有优异的内质网示踪效果，能专一性标记细胞内的内质网。
实施例9：化合物7-甲基-2,3-二丙基-7H-吡喃[4,3,2-kl]吖啶-1-六氟锑酸盐(III-e)与市售线粒体染料
Deep Red FM在HepG2细胞中的荧光共聚焦共成像
首先，向含有10％胎牛血清的DMEM(H)培养基中通入5％CO2，将HepG2细胞于37℃下培养24小时。将培养基去除，加入1μM化合物7-甲基-2,3-二丙基-7H-吡喃[4,3,2-kl]吖啶-1-六氟锑酸盐(III-e)的DMSO溶液于37℃下培养10-15分钟后，使用磷酸盐缓冲液清洗2～3次后；随后加入1μM市售线粒体染料
Deep Red FM于37℃下培养10-15分钟。待培养结束后，再次使用磷酸盐缓冲液清洗2～3次后，将培养玻底皿经荧光共聚焦显微镜成像得到图6中的III-e。图6：III-e中，a图为化合物7-甲基-2,3-二丙基-7H-吡喃[4,3,2-kl]吖啶-1-六氟锑酸盐(III-e)的荧光成像图(激发波长：488nm,发射波长收集范围：520-620nm)。b图为市售线粒体染料
Deep Red FM的荧光成像图(激发波长：633nm,发射波长收集范围：640-740nm)。c图为细胞明场图。d图为a图、b图和c图的叠加图。从d图中可以直观地看出，化合物7-甲基-2,3-二丙基-7H-吡喃[4,3,2-kl]吖啶-1-六氟锑酸盐(III-e)在细胞中的分布和市售线粒体染料
Deep Red FM基本一致，皮尔森系数为0.95，说明化合物7-甲基-2,3-二丙基-7H-吡喃[4,3,2-kl]吖啶-1-六氟锑酸盐(III-e)具有优异的线粒体示踪效果，能专一性标记细胞内的线粒体。
实施例10：化合物2,3-二苯基吡喃[4,3,2-kl]吖啶-7-六氟锑酸盐(IV-b)与市售线粒体染料
Deep Red FM和市售细胞核染料Hoechst 33342在HepG2细胞中的荧光共聚焦共成像
首先，向含有10％胎牛血清的DMEM(H)培养基中通入5％CO2，将HepG2细胞于37℃下培养24小时。将培养基去除，加入1μM化合物2,3-二苯基吡喃[4,3,2-kl]吖啶-7-六氟锑酸盐(IV-b)的DMSO溶液于37℃下培养10-15分钟后，使用磷酸盐缓冲液清洗2～3次后；随后加入1μM市售线粒体染料
Deep Red FM于37℃下培养10-15分钟后，使用磷酸盐缓冲液清洗2～3次后，随后加入1μM市售细胞核染料Hoechst 33342于37℃下培养10-15分钟。待培养结束后，再次使用磷酸盐缓冲液清洗2～3次后，将培养玻底皿经荧光共聚焦显微镜成像得到图6中的IV-b。图6：IV-b中，a图为化合物2,3-二苯基吡喃[4,3,2-kl]吖啶-7-六氟锑酸盐(IV-b)的荧光成像图(激发波长：488nm,发射波长收集范围：520-620nm)。b1图为市售线粒体染料
Deep Red FM的荧光成像图(激发波长：633nm,发射波长收集范围：640-740nm)。b2图为市售细胞核染料Hoechst 33342的荧光成像图(激发波长：405nm,发射波长收集范围：410-460nm)。c图为细胞明场图。d图为a图、b1图、b2图和c图的叠加图。从d图中可以直观地看出，化合物2,3-二苯基吡喃[4,3,2-kl]吖啶-7-六氟锑酸盐(IV-b)在细胞中的分布和市售线粒体染料
Deep Red FM与市售细胞核染料Hoechst33342基本一致，说明化合物2,3-二苯基吡喃[4,3,2-kl]吖啶-7-六氟锑酸盐(IV-b)具有优异的线粒体-细胞核示踪效果，能专一性标记细胞内的线粒体-细胞核。
实施例11：化合物化合物2,3-二苯基吡喃[4,3,2-kl]吖啶7-鎓六氟锑酸盐(I-b)的光稳定性测试
将化合物2,3-二苯基吡喃[4,3,2-kl]吖啶7-鎓六氟锑酸盐(I-b)首先，向含有10％胎牛血清的DMEM(H)培养基中通入5％CO2，将HepG2细胞于37℃下培养24小时。将培养基去除，加入1μM化合物2,3-二苯基吡喃[4,3,2-kl]吖啶7-鎓六氟锑酸盐(I-b)的DMSO溶液于37℃下培养10-15分钟后，使用磷酸盐缓冲液清洗2～3次后，将培养玻底皿在荧光共聚焦显微镜下进行荧光稳定测试(激发波长：488nm,发射波长收集范围：490-570nm)得到图7。每0.97秒扫描一次，左上角的数字为扫描次数。从图7中可以直观地看出，随着荧光共聚焦显微镜激光扫描次数的增加，化合物2,3-二苯基吡喃[4,3,2-kl]吖啶7-鎓六氟锑酸盐(I-b)在细胞中的荧光强度减弱较小。说明化合物2,3-二苯基吡喃[4,3,2-kl]吖啶7-鎓六氟锑酸盐(I-b)具有良好的荧光稳定性。
实施例12：化合物2,3-双(4-氯苯基)吡喃[4,3,2-kl]吖啶-7-六氟锑酸盐(I-b)的MTS细胞毒性实验
将处于对数生长期的HepG2细胞接种于96孔培养板中，每孔接种大约3000个细胞，在37℃下用通入5％CO2的含有10％胎牛血清的DMEM培养基中培养过夜。待细胞完全贴壁后，向其中加入不同浓度的化合物2,3-双(4-氯苯基)吡喃[4,3,2-kl]吖啶-7-六氟锑酸盐(I-b)，每组浓度另设3个复孔和空白对照孔。加样后继续培养细胞24小时，使用MTS检测法检测细胞存活率。如图8所示，在0.0～10.0μM的浓度范围内，化合物2,3-双(4-氯苯基)吡喃[4,3,2-kl]吖啶-7-六氟锑酸盐(I-b)的细胞存活率均非常高(存活率超过95％)，表明化合物2,3-双(4-氯苯基)吡喃[4,3,2-kl]吖啶-7-六氟锑酸盐(I-b)的细胞毒性非常小。
Claims (2)
1.具有特异性靶向溶酶体、内质网、线粒体、线粒体-细胞核的四类含杂原子多环芳烃荧光探针，其分子名称分别为：2,3-二苯基吡喃[4,3,2-kl]吖啶7-鎓六氟锑酸盐(I-a)、2,3-二苯基吡喃[4,3,2-kl]吖啶(II-a)、2,3-二苯基-7-甲基-7H-吡喃[4,3,2-kl]吖啶-1-鎓六氟锑酸盐(III-a)、2,3-二苯基吡喃[4,3,2-kl]蒽酮-1-鎓六氟锑酸盐(V-a)、7-甲基-2,3-二丙基-7H-吡喃[4,3,2-kl]吖啶-1-六氟锑酸盐(III-e)、2,3-双(4-氯苯基)吡喃[4,3,2-kl]吖啶-7-六氟锑酸盐(I-b)。
2.根据权利要求1所述具有特异性靶向溶酶体、内质网、线粒体、线粒体-细胞核的四类含杂原子多环芳烃荧光探针的合成方法，其特征在于(I-a)、(II-a)、(III-a)和(V-a)的合成步骤如下：
(1)分别将吖啶酮或氧杂蒽酮或N-甲基吖啶酮、二苯乙炔、催化剂，氧化剂，添加剂，和反应溶剂加入反应管，随后在无水无氧条件下－40～160℃反应0.1～720小时，或者在空气氛围下－40～160℃反应0.1～720小时，所述催化剂为[Cp*RhCl2]2，所述氧化剂为AgOAc或Ag2O，所述添加剂为NaSbF6和AgSbF6；
(2)反应完成后，将反应管冷却至室温，加入二氯甲烷将反应体系稀释，再经硅藻土过滤并用二氯甲烷洗涤，合并滤液，减压移去溶剂，剩余物用硅胶柱层析或者中性三氧化二铝柱层析分离纯化，真空干燥后得到相应的目标产物。
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