CN110407835A - 咪唑并[1,2-a]吡啶近红外比率型pH荧光探针及其制备和应用 - Google Patents
咪唑并[1,2-a]吡啶近红外比率型pH荧光探针及其制备和应用 Download PDFInfo
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Abstract
本发明公开了基于咪唑并[1,2‑a]吡啶近红外比率型pH荧光探针及其制备方法，以及该探针用于活细胞和大肠杆菌内极度酸性pH变化的测定。在惰性气体保护下，将4‑二甲基氨基肉桂醛或其衍生物，4‑氯苯胺和碳酸氢钠溶于甲醇，加热回流反应后；和2‑(4‑甲氧基苯基)‑7‑甲基咪唑并[1,2‑a]吡啶溶于N,N‑二甲基甲酰胺，以叔丁醇钾为碱性试剂，加热回流反应；浓缩后，经硅胶柱分离。该探针在极度酸性条件下其发射波长大于600nm处于近红外区并且呈现比率发射荧光特性，同时兼具对H+高度灵敏性、良好的选择性和大的Stokes位移等优点。
Description
技术领域
本发明涉及荧光探针，具体属于咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针及其制备方法和应用。
背景技术
氢离子是重要的细胞内物质之一，在调控细胞、酶和组织功能中发挥着重要的作用。不同的原核生物以及真核细胞的不同亚细胞器内的pH值从碱性到强酸性不等。许多疾病与细胞内异常的氢离子浓度有关，严重时会引发诸如炎症、癌症和阿尔茨海默症等。因此，对细胞内pH进行灵敏、准确的实时监测，可以为分子水平上研究细胞的生理和毒理过程提供重要的信息。
在检测细胞内pH的众多方法中，荧光分析法具有非破坏性、高灵敏性、响应速度快、高信噪比以及能够连续检测pH变化的快速动力学过程等特性。此外，结合激光共聚焦显微镜，荧光成像技术成为分子水平上进行实时原位监测细胞内pH的重要手段。其中，近红外荧光探针的发射波长为650-900nm，在这一范围内细胞的自发荧光较弱，可以很好的规避背景干扰而获得更高的准确性，而且使用发射波长大于600nm的光，不仅能减少光毒性，还能渗透组织达数厘米，甚至可以对活体组织进行造影，这大大增加了对疾病监测和诊断的准确性，同时兼顾的比率荧光发射特性，可以通过同时记录两个不同发射波长的荧光强度，并计算它们的比值可以有效避免例如：溶剂极性、探针浓度在细胞内负染不均、温度、仪器等其他环境因素的干扰，从而达到对分析物精确的定量分析。迄今为止，文献报道了许多性能优异的比率型pH荧光探针，但该类探针大多数适用于弱酸性亚细胞器(如：溶酶体和内涵体，pH 4.0-5.5)和近中性细胞质(pH 6.8-7.4)内pH的检测。遗憾的是，对于极度酸性(pH<4)环境下，基于近红外比率型pH荧光探针的研发缺乏关注，导致这方面的pH荧光探针种类十分有限。虽然对于大多数生物来说，极度酸性是致命的，但是大量的微生物包括“嗜酸菌”和幽门螺杆菌已经进化到可以在这种极度酸性条件下存活。还有肠道致病菌，它能够经过哺乳动物极度酸性的胃液(pH 0.9-1.5)到达小肠，引起致命的感染。这就迫切需要开发有效的比率型极度酸性pH荧光探针，并且兼具大的Stokes位移，高的灵敏性、良好的选择性、光稳定性以及低毒性等特性，应用于细胞及大肠杆菌内极度酸性的检测。
发明内容
本发明的目的之一是提供咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针；目的之二是提供该探针的制备方法，该方法工艺简单、成本低廉；目的之三是提供该探针的用途，即在检测细胞内酸性pH变化中的应用，以及在检测大肠杆菌内极度酸性pH变化中的应用。该探针对H+有高的灵敏度和选择性，且对pH变化的检测表现为成比率发射，并具有大的Stokes位移，能够有效减小激发光和细胞或生物样品自身荧光的干扰。
本发明提供的一种咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针，结构式如下：
本发明提供的咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针的制备方法，合成路线如下：
本发明提供的咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针的制备方法，包括如下步骤：
(1)在惰性气体保护下，将4-二甲基氨基肉桂醛，4-氯苯胺溶于无水乙醇，加热回流4小时后，将反应液冷却至室温，倒入大量的蒸馏水中，用二氯甲烷溶剂萃取，减压蒸馏；
(2)将步骤(1)所得产物和和2-(4-甲氧基苯基)-7-甲基咪唑并[1,2-a]吡啶溶于N,N-二甲基甲酰胺，以叔丁醇钾为碱性试剂，在90℃下加热回流14-15小时，将反应液冷却至室温，倒入大量的蒸馏水中，用二氯甲烷溶剂萃取，减压蒸馏；
(3)将步骤(2)所得产物，经硅胶柱分离纯化，洗脱剂为体积比1：1的乙酸乙酯和正己烷溶液，即得所需产物。
本发明提供的咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针在检测细胞内pH变化中的应用。
本发明提供的咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针在检测细胞内pH变化中的应用，包含如下步骤：将细胞与咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针在37℃、5％CO2的孵育箱中共同孵育15分钟，用磷酸盐缓冲液洗涤，加入尼日利亚菌素孵育细胞10分钟，用激光共聚焦显微镜检测。
本发明提供的咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针在检测大肠杆菌内极度酸性pH变化中的应用。
本发明提供的咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针在检测大肠杆菌内极度酸性pH变化中的应用，包含如下步骤：大肠杆菌与咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针于摇床中共同孵育2小时，用激光共聚焦显微镜检测。
本发明提供的咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针在检测大肠杆菌内极度酸性pH变化中的应用，共同孵育时的pH<4。
与现有极度酸性pH荧光探针相比，本发明合成的咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针具有以下优点：(1)本探针在酸性条件下其发射荧光>600nm属于近红外区，可以很好的规避背景干扰而获得更高的准确性，减少光毒性，渗透组织达数厘米，对活体组织进行造影。(2)本探针属于比率发射型pH荧光探针，能够有效消除因探针负染不匀、生物样品厚度不均、溶剂、温度以及设备等因素引起的误差，从而得到比非比率型荧光探针更准确的测定结果。(3)咪唑并[1,2-a]吡啶环是药物中间体，具有很好的生物相适性。(4)该探针通过可见光激发/发射，可有效的克服光漂白以及细胞自发荧光的干扰；(5)超大的Stokes位移(90nm)，有利于减小成像过程中激发光的干扰；(6)对H+响应具有很高的灵敏度和选择性，不受常见金属离子和生物体内中一些常见氨基酸等物质的干扰；(7)本探针是基于分子内电荷转移(ICT)原理设计的，N,N-二甲基苯甲醛或其衍生物和咪唑并[1,2-a]吡啶环分别为给电子基团和吸电子基团，在强酸性条件下，咪唑并[1,2-a]吡啶基团上N原子发生质子化导致其吸电子能力增强，从而加强了整个分子体系的ICT效应，使得该探针的紫外吸收和荧光发射光谱发生红移，而且在自然光和紫外灯下溶液颜色明显改变，可通过裸眼观察。(8)该探针具有很好的细胞通透性，采用激光共聚焦显微成像技术可进行及活细胞和大肠杆菌内极度酸性pH变化的检测；(9)本探针的合成步骤简单，产率高，具有商品化应用价值。
附图说明
图1.本发明实施例2中探针随pH值变化的紫外吸收光谱图。
图2.本发明实施例3中探针随pH值变化的荧光发射光谱图。
图3.本发明实施例4中探针的F650nm/F560nm随pH值变化的Boltzmann函数关系图。
图4.本发明实施例5中探针在常见阴阳离子和生物体内一些常见氨基酸、活性氧、活性氮等物质存在下，对H+的选择性。
图5.本发明实施例6中探针分别在pH 3.00，pH 4.00，pH 7.40时，与HeLa细胞共同孵育30min的激光共聚焦成像图。
图6.本发明实施例7中探针与大肠杆菌(E.coli)在pH 1.0～7.4的条件下共同孵育2h的激光共聚焦成像图。
具体实施方法：
实施例1：
1.探针4-((1E,3E)-4-(2-(4-甲氧基苯基)咪唑并[1,2-a]吡啶-7-基)丁-1,3-二烯-1-基)-N,N-二甲基苯胺的制备：
(1)在惰性气体保护下，将4-二甲基氨基肉桂醛(10mmol，1.75g)，4-氯苯胺(10mmol，1.29g)，溶于15mL甲醇溶液中，加热回流，反应4h。将反应液冷却至室温，倒入大量的蒸馏水中，用二氯甲烷溶剂萃取，减压蒸馏除去有机溶剂，制得产物粗品；
(2)将(1)中所得的粗产物(10mmol，2.84g)，2-(4-甲氧基苯基)-7-甲基咪唑并[1,2-a]吡啶(10mmol，2.38g)和叔丁醇钾(25mmol，2.81g)溶于20mL DMF中，在90度下加热反应15h。将得到的固体溶于水后用CH2Cl2萃取，减压蒸馏除去溶剂制得产物粗品；
(3)将产物粗品经硅胶柱分离纯化，V乙酸乙酯：V正己烷＝1：1为洗脱剂，得到淡黄色固体(2.48g，63％)。1H NMR(400MHz,CDCl3)δ(ppm):2.99(s,6H),3.86(s,3H),6.56-6.80(m,5H),6.96-7.00(m,4H),7.34(d,J＝8.0Hz,2H),7.46(s,1H),7.72(s,1H),7.87(d,J＝8.0Hz,2H),7.99(d,J＝4.0Hz,1H).13C NMR(100MHz,CDCl3)δ(ppm):13.0,21.5,28.6,30.5,39.3,54.2,111.2,113.1,123.8,126.2,126.7,129.2.
实施例2
将实施例1制得的探针用DMSO(二甲亚砜)配制浓度为1mM的储备液保存。实验中用H2O/DMSO(V/V＝4/1)体系将探针稀释为终浓度10μM，用1mol/L的HCl调节该体系的pH值，并记录其紫外吸收光谱(图1)。随着pH值的降低，短波长处的吸收峰逐渐下降，长波长处的吸收峰显著增强，并且在431nm附近存在一个等吸收点。
实施例3
用H2O/DMSO(V/V＝4/1)体系将实施例1制得的探针稀释为终浓度10μM，用1mol/L的HCl调节该体系的pH值，固定激发波长为431nm，记录其荧光发射光谱(图2)。随着pH值的降低，560nm处的荧光强度逐渐降低，而650nm处出现一个新的发射峰并显著增强，同时在611nm附近出现等发射点。
实施例4
将实施例1制得的探针浓度保持在10μM，在DMSO/水(体积比为2:1)体系中用用高浓度小体积的HCl和NaOH溶液调节pH值，以349nm为激发波长，记录其荧光发射光谱(图3)。随着pH值的降低，526nm处的荧光峰逐渐减弱，而456nm处的荧光峰逐渐增强。通过Boltzmann函数拟合F650nm/F560nm值与pH变化曲线，计算pKa值为3.10(图3)。
实施例5
将实施例1制得的探针浓度保持在10μM，分别考察该探针在金属离子以及生物体中常见氨基酸等物质存在下，对H+的选择性。如图4所示，分别在不同pH(pH 7.00,pH 3.20和pH 2.40)条件下，探针对上述物质几乎没有响应，证明该探针对H+具有很高的选择性。图4中物质的顺序和浓度依次为：1.空白；2.Mg+(0.2mM)；3.Pb+(0.2mM)；4.Cd2+(0.2mM)；5.Ca2+(0.2mM)；6.Hg2+(0.2mM)；7.Co2+(0.2mM)；8.K+(0.2mM)；9.Cu2+(0.2mM)；10.Ba2+(0.2mM)；11.Ag+(0.2mM)；12.Mn2+(0.2mM)；13.Zn2+(0.2mM)；14.天冬氨酸(0.2mM)；15.半胱氨酸(0.2mM)；16.苯基丙氨酸(0.2mM)；17.精氨酸(0.2mM)；18.赖氨酸(0.2mM)。
实施例6
将贴壁的HeLa细胞与实施例1制得的探针在pH 7.4的条件下，于37℃、5％CO2的孵育箱中共同孵育15min，然后分别用磷酸盐缓冲液(pH 7.4和pH 3.0)轻轻洗涤3次，除去多余的探针，再加入nigericin(5mg mL-1)继续孵育细胞10min，在激光共聚焦显微镜下观察。固定激发波长为405nm和488nm，收集发射波段分别为绿色通道(500-570nm)和红色通道(610-660nm)。当pH降低时，细胞的绿色荧光明显减弱，红色荧光增强(图5)。
实施例7
将接种好的大肠杆菌(E.coli)与实施例1制得的探针分别在pH 1.0,2.0,3.0,4.0,5.0,6.0和7.4的条件下于摇床中共同孵育2h，在激光共聚焦显微镜下观察。分别固定激发波长为405nm和488nm，收集发射波段分别为绿色通道(500-570nm)和红色通道(610-660nm)。当pH值降至极度酸性1.0时，大肠杆菌的绿色荧光几乎猝灭，而红色荧光显著增强(图6)。
Claims (8)
1.一种咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针，其特征在于结构式如下：
2.根据权利要求1所述咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针的制备方法，其特征在于，合成路线如下：
3.根据权利要求2所述咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针的制备方法，其特征在于，包括如下步骤：
(1)在惰性气体保护下，将4-二甲基氨基肉桂醛，4-氯苯胺溶于无水乙醇，加热回流4小时后，将反应液冷却至室温，倒入大量的蒸馏水中，用二氯甲烷溶剂萃取，减压蒸馏；
(2)将步骤(1)所得产物和和2-(4-甲氧基苯基)-7-甲基咪唑并[1,2-a]吡啶溶于N,N-二甲基甲酰胺，以叔丁醇钾为碱性试剂，在90℃下加热回流14-15小时，将反应液冷却至室温，倒入大量的蒸馏水中，用二氯甲烷溶剂萃取，减压蒸馏；
(3)将步骤(2)所得产物，经硅胶柱分离纯化，洗脱剂为体积比1：1的乙酸乙酯和正己烷溶液，即得所需产物。
4.根据权利要求1所述咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针在检测细胞内pH变化中的应用。
5.根据权利要求4所述咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针在检测细胞内pH变化中的应用，其特征在于，包含如下步骤：将细胞与咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针在37℃、5％CO2的孵育箱中共同孵育15分钟，用磷酸盐缓冲液洗涤，加入尼日利亚菌素孵育细胞10分钟，用激光共聚焦显微镜检测。
6.根据权利要求1所述咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针在检测大肠杆菌内极度酸性pH变化中的应用。
7.根据权利要求6所述咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针在检测大肠杆菌内极度酸性pH变化中的应用，其特征在于，包含如下步骤：大肠杆菌与咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针于摇床中共同孵育2小时，用激光共聚焦显微镜检测。
8.根据权利要求7所述咪唑并[1,2-a]吡啶类近红外比率型pH荧光探针在检测大肠杆菌内极度酸性pH变化中的应用，其特征在于，共同孵育时的pH<4。
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