CN104910211A - 一种环金属铱(iii)配合物及其制备方法和在活细胞线粒体染色中的应用 - Google Patents
一种环金属铱(iii)配合物及其制备方法和在活细胞线粒体染色中的应用 Download PDFInfo
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Abstract
本发明公开了一种新型环金属铱(III)配合物及其制备方法和在活细胞线粒体染色中的应用。所述新型环金属铱(III)配合物的阳离子部分的结构式如式I所示。本发明合成得到的环金属的铱(III)配合物结构稳定,具有良好的光物理光化学性质,细胞毒性低,并表现出良好的线粒体靶向功能,是新型的活细胞线粒体双光子荧光探针。经研究表明,该配合物的光稳定性和双光子吸收截面明显优于商用线粒体染料,是潜在的优秀双光子线粒体染料。
Description
技术领域
本发明属于线粒体荧光成像技术领域。更具体地,涉及一种环金属化铱配合物及其制备方法和在活细胞线粒体染色中的应用。
背景技术
随着科学技术的发展,人类对线粒体的认识越来越深刻。线粒体的形态、尺寸、数量及分布情况具有很大的差异性,活细胞中线粒体一般呈棒状的或卵状,而在细胞分裂过程中也可变为扁盘状,枝状等。单个线粒体的长度一般为1.5~3.0μm,而人成纤维细胞线粒体可长达40μm。线粒体在细胞内的功能主要是葡萄糖的氧化磷酸化,为细胞的生命活动提供所需的能源,同时还起到调控细胞内环境的离子稳态平衡、控制细胞内源性凋亡等作用。线粒体病变或者线粒体功能发生障碍时,则会引起各种“线粒体病”,例如阿兹海默症。因此,对线粒体的研究对人类相关疾病研究和治疗具有深远意义。
通过荧光或磷光技术对线粒体成像,观察线粒体的形态变化,可作为药理及病理研究的研究依据。目前已商业化的有机小分子线粒体染料只要有以下两类:一类是线粒体膜电位依赖的如Rhodamine dyes(Rhodamine 123、TMRM 和 TMRE),Carbocyanine dyes(JC-1、JC-9 和 DiOC6及 Rosamine dyes (MitoTrackers Orange
CMTMRos 和MitoTrackers
Red CMXRos 等);但是线粒体膜电位依赖性的荧光染料会随着膜电位降低而被洗脱,不利于研究线粒体的动态变化。另一类是膜电位非依赖性的线粒体探针(MitoTracker Green FM、MitoTrackers Red FM 和MitoTrackers Deep Red),Mito Fluor Green 和 MitoID Red 等;虽然这些线粒体染料不依赖膜电位,但作为小分子有机荧光染料,
Soke 位移小,水溶性差,光漂白严重等缺点也限制着它们的应用。除此之外,上述商业化的线粒体染料均需高能量的单光子激光激发,容易产生单线态氧,不利于长时间观察线粒体的动态变化。
近年来,双光子荧光染料成为了研究热点,相比于传统的单光子染料,双光子荧光染料具有诸多优点:(1)双光子激光比单光子激光波长更长,受细胞器散射影响较小;(2)双光子可以穿透更深的样品;(3)双光子激光对细胞毒性较小。所以,双光子荧光染料比单光子荧光染料更适合观察活细胞、或用来进行定点光漂白实验。因此开发荧光量子产率高,荧光强,光稳定性好,Stoke 位移大和荧光寿命长等特点的双光子活细胞线粒体染料具有积极的意义。
发明内容
本发明要解决的技术问题是克服现有活细胞线粒体染料的缺陷和技术不足,提供一种具有双光子活细胞线粒体荧光成像功能的环金属铱(III)配合物。
本发明另一目的是提供所述环金属铱(III)配合物的制备方法。
本发明的再一目的是提供所述环金属铱(III)配合物在活细胞双光子线粒体荧光成像中的应用。
本发明上述目的通过以下技术方案实现:
一种新型环金属铱(III)配合物,其结构式如式I所示:
上述新型环金属铱(III)配合物的制备方法为:将对甲基苯甲醛和2-乙酰吡啶在乙醇中反应获得甲基苯三联吡啶(ttpy),由甲基苯三联吡啶(ttpy)和IrCl3·H2O先反应制得Ir(ttpy)Cl3前体,再由Ir(ttpy)Cl3和2-(2,4-二氟苯基)吡啶反应制得;所述的对甲基苯甲醛、2-乙酰吡啶、甲基苯三联吡啶(ttpy)、2-(2,4-二氟苯基)吡啶的结构式分别为:
。
具体地,上述新型环金属铱(III)配合物的制备方法,包括以下步骤:
S1.将对甲基苯甲醛和2-乙酰吡啶在乙醇中搅拌回流反应获得甲基苯三联吡啶(ttpy)。
S2.将S1得到的甲基苯三联吡啶(ttpy)与IrCl3·H2O在乙二醇回流中反应获得Ir(ttpy)Cl3;
S3.由Ir(ttpy)Cl3(前体)和2-(2,4-二氟苯基)吡啶(配体)在乙二醇中回流反应,反应结束后,利用饱和NH4PF6水溶液,析出橙黄色固体,即为目标化合物[Ir(ttpy)(dfppy)Cl]PF6。进一步干燥获得紫黑色粗产品,再经氧化铝柱色谱分离提纯,干燥(将溶剂用旋转蒸发仪旋干)后得到目标产物[Ir(ttpy)(dfppy)Cl]PF6。
优选地,步骤S1所述回流反应的时间为12~24小时,反应温度为室温。更优选地,步骤S1所述回流反应的时间为24小时。
优选地,步骤S2所述回流反应的时间为10~30小时,反应温度为150~190℃。更优选地,步骤S2所述回流反应的时间为15分钟,反应温度为180℃。
优选地,步骤S3所述回流反应的时间为12~24小时;反应温度为150~190 ℃。更优选地,步骤S3所述回流反应的时间为24小时,反应温度为180℃。
优选地,步骤S3所述饱和NH4PF6水溶液的质量分数为42.8%。
上述环金属铱(III)配合物在活细胞双光子线粒体荧光成像中的应用也在本发明的保护范围之内。进一步地,应用时,所述的环金属铱(III)配合物是作为活细胞双光子线粒体染料。
优选地,所述的活细胞双光子线粒体染料为Hela贴壁细胞以及HeLa 3D细胞球的双光子线粒体染料。
本发明具有以下有益效果:
本发明提供了一种新型环金属铱(III)配合物,可作为双光子线粒体靶向荧光探针。本发明合成得到的环金属的铱(III)配合物结构稳定,具有良好的光物理光化学性质,细胞毒性低,并表现出良好的线粒体靶向功能,是新型的活细胞线粒体双光子荧光探针。
本发明制备得到的环金属铱(III)配合物在双光子活细胞线粒体成像中的应用,具有以下优势:(1)具有良好的水溶性;(2)具有双光子荧光性质;(3)荧光量子产率高,荧光寿命长;(4)细胞毒性小,能对活细胞线粒体双光子荧光成像。
经研究表明,本发明的上述环金属铱(III)配合物能够对细胞(如HeLa细胞)中的线粒体双光子荧光成像,为跟踪线粒体在细胞内的变化提供有效的可视化工具,而且光稳定性和双光子吸收截面明显优于商用线粒体染料MitoTracker® Red FM,[Ir(ttpy)(dfppy)Cl]PF6是潜在的双光子线粒体染料。
附图说明
图1为本发明制备环金属铱(III)配合物所用的配体分子结构。
图2为本发明制备得到的环金属铱(III)配合物的分子结构。
图3为配体ttpy的合成途径。
图4为环金属铱(III)配合物的合成途径。
图5为环金属铱(III)配合物的紫外吸收光谱图。
图6为环金属铱(III)配合物的750nm激发下的荧光光谱图。
图7为环金属铱(III)配合物对HeLa贴壁细胞荧光成像图。
图8为环金属铱(III)配合物与商用线粒体染料MitoTracker® Red FM荧光共定位图。
图9为环金属铱(III)配合物对HeLa 3D细胞球不同深度荧光成像图。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,本发明所用试剂和材料均为市购。
以下进一步说明本发明的合成方案。为方便表述,本文对各种物质简记如下:
。
实施例
1
配体和配合物的制备方法
1、配体ttpy的合成方法:
配体的分子结构如附图1所示,配体ttpy的合成途径如附图3所示。
按照参考文献(Synlett 2005,
No. 8, 1251–1254)取2-乙酰吡啶4.84 g(40 mmol)和2. 40 g 苯甲醛(20 mmol) 溶于100 mL乙醇中。之后,再加入3.08 g(85%, 40 mmol)KOH颗粒和58 mL氨水(29.3%, 50 mmol),常温下避光搅拌25 h。过滤收集灰白色固体,并用乙醇洗涤三次,每次10 mL。再以氯仿–甲醇溶剂重结晶,得到白色固体。产量 4.2 g,产率 61 %。1H NMR (300 MHz, DMSO)
δ = 8.73 (d, J = 4.2 Hz, 2H), 8.66 (s, 2H), 8.64 (d, J = 9.0 Hz,
2H), 7.99 (t, J = 11.4 Hz, 2H), 7.80 (d, J = 6.0 Hz, 2H), 7.54 –
7.46 (m, 2H), 7.37 (d, J = 7.8 Hz, 2H), 2.39 (s, 3H)。
2、配合物[Ir(ttpy)(dfppy)Cl]PF6的合成方法:环金属铱(III)配合物的合成途径如附图4所示。
(1)制备Ir(ttpy)3
按照参考文献 (J. Am. Chem. Soc.,
1999, 121, 5009–5016) 合成方法,取配体 phtpy (91.6 mg, 0.28 mmol)和 IrCl3•H2O
(112 mg, 0.32 mmol)溶于 5 mL脱气乙二醇中加热至180℃,避光,通氩气。搅拌12分钟后,可观察到红色沉淀产生。冷却至室温,真空过滤,依次用乙醇、水和乙醚洗涤,得到橙红色固体,即为[Ir(phtpy)Cl3]。产量 180 mg,产率 90 %。
(2)制备[Ir(ttpy)(dfppy)Cl]PF6
按照参考文献 (Inorg. Chem. 2014,
53, 1487-1499) 合成方法。取[Ir(phtpy)Cl3] 93.6 mg (0.15 mmol)以及2-苯基吡啶108.5 mg(0.70 mmol)置于充满氩气的试管中,再加入5 mL乙二醇。所得混合物避光下加热至180℃,过夜。避光冷却到室温后,将反应混合物加进20 mL饱和 NH4PF6溶液,混合物在室温下搅拌1 h。真空过滤得到黄色沉淀,用水和二乙醚洗涤得到黄色固体。产量
114.5 mg,产率 53 %。ES-MS (CH3OH):
m/z 723.3 [M-PF6-Cl-]+. 1H
NMR (300 MHz, d 6-DMSO) δ = 9.87 (d, J = 5.9 Hz, 1H),
9.24 (s, 2H), 8.96 (d, J = 6.8 Hz, 2H), 8.49 (d, J = 7.3 Hz, 1H),
8.34 – 8.17 (m, 5H), 7.93 (d, J = 7.8 Hz, 1H), 7.80 (t, J = 6.7
Hz, 1H), 7.68 (d, J = 5.2 Hz, 2H), 7.54 (m, 4H), 6.91 (t, J = 7.5
Hz, 1H), 6.74 (t, J = 7.5 Hz, 1H), 6.07 (d, J = 7.6 Hz, 1H), 2.29
(s, 3H)。
3、制备得到的环金属铱(III)配合物的分子结构如附图2所示。
环金属铱(III)配合物的紫外吸收光谱图如附图5所示,在750nm激发下的荧光光谱图入附图6所示。
上述方法获得的配合物进一步进行以下实验。
实施例 2 激光共聚焦实验
1、实验方法
对数期生长的 Hela细胞在含 10%胎牛血清的DMEM 培养基中培养,细胞 接种在共聚焦显微镜专用玻底培养皿中,培养皿直径
35 mm,其中盖玻片厚度 0.085~0.13 mm,皿中心微孔直径 10 mm,5% CO2和95%空气条件下,37℃培养,贴壁生长 24小时。用5
μM配合物培育1 h,再和线粒体红色荧光染料Mitotracker-Red FM(50 nM)共染30 min,吸出培养液,然后用PBS 缓冲液洗涤3~4 次,在 Zeiss 双光子激光扫描共聚焦显微镜上成像,使用63´油镜,用
750 nm 光作为激发光源,收集 500~630
nm 范围内的荧光。
2、结果
(1)环金属铱(III)配合物对HeLa贴壁细胞荧光成像图如附图7所示。本发明的环金属铱(III)配合物在双光子光源(lex = 750 nm)激发下,能够对活细胞进行染色,而且染色过后并没有影响到细胞的生理活动,细胞没有表现出任何受损或凋亡的现象,说明该配合物是良好的活细胞双光子染料。
(2)环金属铱(III)配合物与商用线粒体染料MitoTracker® Red FM荧光共定位图如附图8所示。环金属铱(III)配合物与商用线粒体染料MitoTracker® Red FM荧光共定位系数达到R=
0.92,说明该配合物在细胞内的靶点是线粒体。
(3)环金属铱(III)配合物对HeLa 3D细胞球不同深度荧光成像图如附图9所示。HeLa 3D细胞球具有不同深度的三维结构,很方便地用于研究染料的染色深度以及进细胞能力,如图9所示,在双光子激发光源的激发下,染料的荧光能够透过将近100 mm的细胞球内部结构,表明该配合物不仅具有良好的进细胞能力,同时还能够穿透到较深的组织内部,是良好的双光子活细胞染料。
Claims (10)
1.一种新型环金属铱(III)配合物,其特征在于,其结构式如式I所示:
。
2.权利要求1所述新型环金属铱(III)配合物的制备方法,其特征在于,包括以下步骤:将对甲基苯甲醛和2-乙酰吡啶在乙醇中反应获得甲基苯三联吡啶(ttpy),由甲基苯三联吡啶(ttpy)和IrCl3·H2O先反应制得Ir(ttpy)Cl3前体,再由Ir(ttpy)Cl3和2-(2,4-二氟苯基)吡啶反应制得;
所述的对甲基苯甲醛、2-乙酰吡啶、甲基苯三联吡啶(ttpy)、2-(2,4-二氟苯基)吡啶的结构式分别为:
。
3.根据权利要求2所述制备方法,其特征在于,包括以下步骤:
S1.将对甲基苯甲醛和2-乙酰吡啶在乙醇中搅拌回流反应获得甲基苯三联吡啶(ttpy);
S2.将S1得到的甲基苯三联吡啶(ttpy)与IrCl3·H2O在乙二醇中回流反应获得前体Ir(ttpy)Cl3;
S3.由前体Ir(ttpy)Cl3和2-(2,4-二氟苯基)吡啶在乙二醇中回流反应,反应结束后,利用饱和NH4PF6水溶液,析出橙黄色固体,即为目标化合物[Ir(ttpy)(dfppy)Cl]PF6。
4.根据权利要求3所述制备方法,其特征在于,步骤S1所述回流反应的时间为12~24小时,反应温度为室温;步骤S2所述回流反应的时间为10~30小时,反应温度为150~190℃。
5.根据权利要求4所述制备方法,其特征在于,步骤S1所述回流反应的时间为24小时;步骤S2所述回流反应的时间为15分钟,反应温度为180℃。
6.根据权利要求3所述制备方法,其特征在于,步骤S3所述回流反应的时间为12~24小时;反应温度为150~190 ℃。
7.根据权利要求6所述制备方法,其特征在于,步骤S3所述回流反应的时间为24小时,反应温度为180℃。
8.根据权利要求3所述制备方法,其特征在于,步骤S3得到的橙黄色固体进一步干燥获得粗产品,再经氧化铝柱分离提纯,干燥后得到目标产物[Ir(ttpy)(dfppy)Cl]PF6。
9.根据权利要求3所述制备方法,其特征在于,步骤S3所述饱和NH4PF6水溶液的质量分数为42.8%。
10.权利要求1所述环金属铱(III)配合物在活细胞双光子线粒体荧光成像中的应用。
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