CN107082751B - 一种具有Cu2+识别功能的双光子荧光探针及其制备方法和用途 - Google Patents
一种具有Cu2+识别功能的双光子荧光探针及其制备方法和用途 Download PDFInfo
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Abstract
本发明公开了一种具有Cu2+识别功能的双光子荧光探针及其制备方法和用途，其中具有Cu2+识别功能的双光子荧光探针的结构式为：本发明双光子荧光探针在紫外‑可见光区可以选择性地识别Cu2+，不受其它金属离子的干扰，实验证明探针分子与铜离子形成的配合物具有良好的水溶性和生物相容性。生物学实验表明，本发明双光子荧光探针可以透过细胞膜，靶向在细胞线粒体和斑马鱼的肝部及胃部，显示黄绿色荧光，加入外源性铜离子后，探针在生物体内的荧光几乎完全淬灭，随着与Cu2+配位能力更强的组氨酸加入，探针HL被释放，荧光基本恢复。这一研究结果的发现，本发明双光子荧光探针作为Cu2+识别荧光探针具有重要的实用价值。
Description
技术领域
本发明涉及一种双光子荧光探针及其制备方法和用途，具体地说是一种具有Cu2+识别功能的双光子荧光探针及其制备方法。
背景技术
铜离子作为人体必要的微量元素之一，与许多生理过程有着紧密联系。它在体内的平衡对有机体的新陈代谢具有至关重要的作用，但机体对于细胞内铜离子的含量有着十分严格的调控机制，非正常生理浓度的铜离子会导致一系列疾病，例如朊病毒疾病、老年痴呆症等一些神经退行性疾病。另外，铜离子被视为环境污染的金属离子之一，过量的铜离子也会对动植物造成严重危害。因此，制备检测生命体系和环境中铜离子的探针对于生命科学、环境科学都有着重要的意义。目前，大多数铜离子探针，仅适用于短波激发的单光子荧光显微技术，由于受背景干扰较大，组织渗透能力弱，限制了该类探针在生物体内的应用。近红外发光(长波激发)的双光子吸收材料，相对于单光子吸收材料而言，它适用于双光子荧光显微技术，具有组织自发荧光弱、自吸收少、渗透能力强等优点，为有效地检测生物体内铜离子提供了有利条件。近期，虽报道了一些铜离子识别的双光子荧光探针，但是，具有良好水溶性和生物相容性并且能够检测生物体内铜离子的双光子荧光探针还鲜为报道，目前仍急需探究。
申请人对本申请的主题进行了如下的文献检索：
1、xueshu.baidu.com网检索结果：(2017/3/23)
2、中国期刊网检索结果：
检索方式一：
篇名-Cu2+识别的双光子荧光探针：无相关文献。
篇名-一种Cu2+识别的双光子荧光探针--席夫碱化合物及其制备方法：无相关文献。
检索方式二：
全文-Cu2+识别的双光子荧光探针：无相关文献。
全文-一种Cu2+识别的双光子荧光探针--席夫碱化合物及其制备方法：无相关文献。
发明内容
本发明旨在提供一种具有Cu2+识别功能的双光子荧光探针及其制备方法和用途。本发明考虑到探针分子的性质与识别金属离子特性之间的密切关系，我们用具有强给电子能力及较高反应活性的N,N-二乙基水杨醛，与含有季铵盐基团的苯胺衍生物反应，制备出具有特定配位点、良好水溶性和生物相容性的D–π–A共轭结构的席夫碱化合物(HL)，通过X-ray单晶衍射确定了其结构，并在HEPES-buffer溶液中进行了识别金属离子活性筛选，结果表明HL 分子在紫外-可见光区可以选择性地识别Cu2+，不受其它金属离子的干扰，实验证明探针分子与铜离子形成的配合物具有良好的水溶性和生物相容性。生物学实验表明，探针HL可以透过细胞膜，靶向在细胞线粒体和斑马鱼的肝部及胃部，显示黄绿色荧光，加入外源性铜离子后，探针在生物体内的荧光几乎完全淬灭，随着与Cu2+配位能力更强的组氨酸加入，探针HL被释放，荧光基本恢复。这一研究结果的发现，HL作为Cu2+识别荧光探针具有重要的实用价值。
本发明具有Cu2+识别功能的双光子荧光探针，为席夫碱化合物(HL)，其结构式为：
本发明具有Cu2+识别功能的双光子荧光探针的制备方法，包括如下步骤：
1、化合物Ⅰ的合成
将50.00g(47.6mmol)二乙醇胺溶解在80mL DMSO中，加入20mL(18.8mmol)对氟硝基苯，140℃反应3.5h；待反应液冷却至室温后，倒入1000mL水中(有大量黄色固体粉末析出)，常温下搅拌10min后抽滤，水洗两遍，真空干燥8-12h，得黄色粉末Ⅰ40.00g，即为化合物Ⅰ，产率94％。
2、化合物Ⅱ的合成
将5.00g(22mmol)化合物Ⅰ溶解在30mL乙腈中，在冰盐浴条件下逐滴滴加2mL(11mmol)PBr3，然后加热回流6h；待反应液冷却至室温后倒入冰水中，用饱和Na2CO3溶液缓慢调至中性，此时有大量黄色固体粉末析出，抽滤，用乙醇重结晶，真空干燥8-12h，得淡黄色棒状晶体Ⅱ5.78g，为化合物Ⅱ，产率75％。
3、化合物Ⅲ的合成
将4.00g(11.4mmol)化合物Ⅱ溶解在20mL THF中，加入40mL 40％的三甲胺水溶液，用100mL高压反应釜在90℃反应12h；减压除去有机溶剂，冷却至室温后，缓慢滴加含有8.00g(45.6mmol)六氟磷酸铵的水溶液，此时有橘黄色固体析出，抽滤，真空干燥24 h，得化合物Ⅲ6.2g，产率89％。
4、化合物Ⅳ的合成
将2.00g(3.3mmol)化合物Ⅲ用20mL甲醇溶解在50mL的史莱克烧瓶中，依次加入0.10g 5％Pd/C和1mL水合肼，在氮气保护下回流反应12h；趁热滤除Pd/C，减压除去溶剂，将粗产物用无水乙醇加热洗涤，趁热抽滤得白色固体1.43g，即为化合物Ⅳ，产率75％。
5、目标产物HL的合成
将0.50g(2.61mmol)4-N N’-二羟乙基水杨醛加热溶解在15mL乙醇中，滴加2滴冰醋酸，搅拌5min后加入1.00g(1.75mmol)化合物Ⅳ，回流反应12h后有黄色固体析出，趁热抽滤，用乙腈和乙醇(v/v＝1:1)重结晶得黄褐色针状晶体1.02g，即为目标产物HL，产率73％。
本发明合成路线如下：
本发明双光子荧光探针在检测生物体内铜离子时可以作为Cu2+识别荧光探针应用。
本发明双光子荧光探针能够可逆性地识别细胞内外源性铜离子，在识别细胞内铜离子后荧光淬灭，并且在组氨酸的作用下实现双光子荧光探针的荧光恢复。
本发明的有益效果体现在：
1、HL的合成原料易得，成本低，反应条件温和，处理简单。
2、目标分子中引入季铵盐基团，既可以调节分子内推拉电子能力，从而产生非线性光学效应，又能很大程度上增加探针的水溶性和生物相容性，便于实现在生物体内和环境中微量元素检测方面的应用。
3、HL作为Cu2+探针选择性好、检测限低、灵敏度高，且可以通过组氨酸实现可逆性。在 HEPES-buffer溶液中分别加入不同金属离子后，仅当铜离子存在时，HL的紫外可见吸收强度迅速减弱，同时在短波位置上出现一个新的吸收峰(图2)，单光子和双光子荧光发射强度明显减弱(图2，图3)，随着组氨酸的加入，HL的光谱又基本恢复。
4、HL具有很好的细胞膜通透性，以720nm为激发波长时，观察到其双光子荧光显影结果(图4，图5)，这种长波激发短波发射的光具有对细胞和组织损伤小、渗透能力强的特点， HL可以靶向细胞线粒体，可逆性地识别细胞内外源性铜离子。目前，不存在类似的荧光探针，具有较强的商业价值。
附图说明
图1是HL的晶体结构图(为了便于观察，省略氢原子和两个六氟磷酸根)，CCDC号为1537634。说明本发明合成的HL是一个结构明确的新型席夫碱化合物。
图2是HL在HEPES-buffer溶液中与金属离子作用的吸收光谱图(a)和发射光谱图(b)。该图说明了无论是紫外-可见吸收光谱还是发射光谱，HL都能实现对铜离子的选择性识别。
图3是探针HL识别Cu2+可逆性的研究。(a)单光子荧光光谱图；(b)HL荧光“on-off”循环次数；(c)双光子荧光光谱图；(d)HL加入Cu2+和组氨酸前后的有效双光子吸收截面变化图。该图说明HL加Cu2+荧光淬灭，随着组氨酸的加入荧光基本恢复，HL作为铜离子探针具有良好的可逆性。
图4HL加入Cu2+和组氨酸前后肝癌细胞的双光子荧光成像研究结果。(a)双光子作用图； (b)细胞内荧光强度分析图；(c)流式细胞仪分析图。该图说明，探针HL可以靶向细胞线粒体，可逆性地识别细胞内的外源性铜离子。
图5HL加入Cu2+和组氨酸前后斑马鱼的双光子荧光成像研究结果。(a)HL双光子显影图；(b)HL加Cu2+双光子显影图；(c)HL加Cu2+再加组氨酸的双光子显影图。该图说明，探针HL可以进入斑马鱼肝部和胃部，可逆性地识别活体内的外源性铜离子。
具体实施方式
1、化合物Ⅰ的合成
将50.00g(47.6mmol)二乙醇胺溶解在80mL DMSO中，加入20mL(18.8mmol)对氟硝基苯，140℃反应3.5h；待反应液冷却至室温后，倒入1000mL水中(有大量黄色固体粉末析出)，常温下搅拌10min后抽滤，水洗两遍，真空干燥8-12h，得黄色粉末Ⅰ40.00g，即为化合物Ⅰ，产率94％。
1H-NMR(400MHz,d6-DMSO,ppm)δ:8.01(d,J＝9.5Hz,2H),6.82(d,J＝9.5Hz,2H),4.86(t, J＝5.1Hz,2H),3.65-3.54(m,8H)。
2、化合物Ⅱ的合成
将5.00g(22mmol)化合物Ⅰ溶解在30mL乙腈中，在冰盐浴条件下逐滴滴加2mL(11mmol)PBr3，然后80℃加热回流6h；待反应液冷却至室温后倒入冰水中，用饱和Na2CO3溶液缓慢调至中性，此时有大量黄色固体粉末析出，抽滤，用乙醇重结晶，真空干燥8-12h，得淡黄色棒状晶体Ⅱ5.78g，为化合物Ⅱ，产率75％。
1H-NMR(400MHz,d6-DMSO,ppm),δ:7.80(d,J＝7.9Hz,2H),7.50(dd,J＝18.9,8.0Hz,2H,), 4.13(s,2H,),3.59(s,2H),3.44(t,J＝10.8Hz,2H),3.37(t,J＝8.0Hz,2H),3.24(d,J＝14.2Hz, 2H),2.42(s,2H).13C-NMR(100MHz,d6-DMSO,ppm)δ28.81,61.65,112.32,124.23, 137.42,155.72.ESI-MS:m/z,calcd:349.93,found:351.92([M]+H+)。
3、化合物Ⅲ的合成
将4.00g(11.4mmol)化合物Ⅱ溶解在20mL THF中，加入40mL 40％的三甲胺水溶液，用100mL高压反应釜在90℃反应12h；减压除去有机溶剂，冷却至室温后，缓慢滴加含有8.00g(45.6mmol)六氟磷酸铵的水溶液，此时有橘黄色固体析出，抽滤，真空干燥24 h，得化合物Ⅲ6.2g，产率89％。
1H-NMR(D2O,400MHz,ppm)δ:8.15(d,J＝9.2,2H),6.83(d,J＝9.6,2H),4.00(t,J＝8.0,4H), 3.49(t,J＝7.6,4H),3.18(s,18H)。
4、化合物Ⅳ的合成
将2.00g(3.3mmol)化合物Ⅲ用20mL甲醇溶解在50mL的史莱克烧瓶中，依次加入0.10g 5％Pd/C和1mL水合肼，在氮气保护下70℃回流反应12h；趁热滤除Pd/C，减压除去溶剂，将粗产物用无水乙醇加热洗涤，趁热抽滤得白色固体1.43g，即为化合物Ⅳ，产率75％。
5、目标产物HL的合成
将0.50g(2.61mmol)4-N N’-二羟乙基水杨醛加热溶解在15mL乙醇中，滴加2滴冰醋酸，搅拌5min后加入1.00g(1.75mmol)化合物Ⅳ，回流反应12h后有黄色固体析出，趁热抽滤，用乙腈和乙醇(v/v＝1:1)重结晶得黄褐色针状晶体1.02g，即为目标产物HL，产率73％。
IR(KBr,cm-1):3443,2982,1627,1519,837,560.1H-NMR(d3-MeCN,400MHz,ppm)δ:8.57(s, 1H),7.31(d,J＝8.3,3H),6.98(d,J＝8.0,2H),6.32(t,J＝18.1,1H),6.16(s,1H),3.72(t,7.2,4H), 3.45(t,J＝7.3,8H),3.15(s,18H),1.18(t,J＝7.0,6H).13C-NMR(100MHz,d3-MeCN)δ:159, 155,148,143,141,134,122,117,116,104,61,56,45,43,12.ESI-MS:m/z,calcd:745.29,found: 227.68([M-2PF6 –]2+/2)；Anal.Calc.forC27H45ON5F12P2:C,43.49；H,6.08；N,9.39.Found: C,43.48；H,6.06；N,9.41。
【目标分子HL的生物学研究】
1、活细胞内Cu2+的检测
人体肝癌细胞(HepG2)与低浓度(10μmol)的HL在含有5％CO2气体和95％空气的培养箱中37℃下培养0.5h，然后在其中加入铜离子培养0.5h，最后加入His(组氨酸)，再培养0.5h。双光子激光共聚焦显影结果表明，目标分子不仅可以透过细胞膜靶向线粒体部位，而且识别线粒体中的铜离子后荧光淬灭，在组氨酸的作用在实现了探针的荧光恢复。
2、斑马鱼体内Cu2+的检测
将孵化五天的斑马鱼放在HEPES-buffer溶液中，加入HL(5μmol)，在常温下培养2h，然后在其中加入铜离子培养0.5h，最后加入His(组氨酸)，再培养0.5h。双光子激光共聚焦显影结果表明，目标分子不仅可以进入斑马鱼的胃部和肝部，而且识别斑马鱼体中的铜离子后荧光淬灭，在组氨酸的作用下实现了探针的荧光恢复。
Claims (4)
1.一种具有Cu2+识别功能的双光子荧光探针，其特征在于其结构式为：
2.一种权利要求1所述的具有Cu2+识别功能的双光子荧光探针的制备方法，其特征在于包括如下步骤：
(1)化合物Ⅰ的合成
将50.00g二乙醇胺溶解在80mL DMSO中，加入20mL对氟硝基苯，140℃反应3.5h；待反应液冷却至室温后，倒入水中，有黄色固体粉末析出，常温下搅拌后抽滤，水洗，真空干燥8-12h，得黄色粉末Ⅰ，即为化合物Ⅰ；
(2)化合物Ⅱ的合成
将5.00g化合物Ⅰ溶解在30mL乙腈中，在冰盐浴条件下逐滴滴加2mL PBr3，然后加热回流6h；待反应液冷却至室温后倒入冰水中，用饱和Na2CO3溶液调至中性，有黄色固体粉末析出，抽滤，用乙醇重结晶，真空干燥8-12h，得淡黄色棒状晶体，即为化合物Ⅱ；
(3)化合物Ⅲ的合成
将4.00g化合物Ⅱ溶解在20mL THF中，加入40mL 40％的三甲胺水溶液，用100mL高压反应釜在90℃反应12h；减压除去有机溶剂，冷却至室温后，缓慢滴加含有8.00g六氟磷酸铵的水溶液，有橘黄色固体析出，抽滤，真空干燥24h，得化合物Ⅲ；
(4)化合物Ⅳ的合成
将2.00g化合物Ⅲ用20mL甲醇溶解在50mL的史莱克烧瓶中，依次加入0.10g 5％Pd/C和1mL水合肼，在氮气保护下回流反应12h；趁热滤除Pd/C，减压除去溶剂，将粗产物用无水乙醇加热洗涤，趁热抽滤得白色固体，即为化合物Ⅳ；
(5)目标产物HL的合成
将0.50g 4-N N’-二羟乙基水杨醛加热溶解在15mL乙醇中，滴加2滴冰醋酸，搅拌5min后加入1.00g化合物Ⅳ，回流反应12h后有黄色固体析出，趁热抽滤，用乙腈和乙醇重结晶得黄褐色针状晶体1.02g，即得目标产物。
3.一种权利要求1所述的具有Cu2+识别功能的双光子荧光探针的用途，其特征在于：所述双光子荧光探针在制备生物体内Cu2+识别荧光探针中的应用。
4.一种权利要求1所述的具有Cu2+识别功能的双光子荧光探针的应用，其特征在于：所述双光子荧光探针能够可逆性地识别细胞内外源性铜离子，在识别细胞内铜离子后荧光淬灭，并且在组氨酸的作用下实现双光子荧光探针的荧光恢复；所述应用为非治疗和/或诊断目的。
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