CN108516984B - 一种用于检测胞嘧啶的荧光探针及其制备方法 - Google Patents
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Abstract
本发明涉及一种用于检测胞嘧啶的荧光探针及其制备方法。该荧光探针是一种含三个二甲基吡咯BODIPY基元的高荧光衍生物。所述荧光探针,是通过将三个二甲基BODIPY基元的连接于同一个烷基碳的结构,化学名为三‑([4‑(乙酰氧基苯基]‑ (4,4‑二氟‑1,3,5,7‑四甲基‑4‑硼‑3a,4a‑二吡咯))氧亚甲基丙烷。该三BODIPY在溶液状态显浅黄色,在510nm处有很强的荧光发射,该三BODIPY荧光量子产率达0.97,其与胞嘧啶络合后荧光明显增强,且荧光增强与胞嘧啶浓度呈线性关系,最低检测限达到8.2μM,其它生物分子干扰小,是一种理想的胞嘧啶快速检测传感探针。
Description
技术领域
本发明属于有机合成与分析化学技术领域,具体涉及一种用于检测胞嘧啶的荧光探针及其制备方法。
背景技术
由于荧光探针的高选择性和灵敏度,现在已被广泛应用于各类离子和小分子的检测当中。通过对探针的适当化学修饰,以便适应不同的检测底物与监测环境。众所周知,二甲基吡咯BODIPY是一个稳定的荧光染料,具有光化学稳定性强,荧光强度高,在各类环境中适应力强等特点,已成为了广受重视的荧光探针之一,目前这类荧光探针主要应用于离子检测,用于生物有机活性分子的少见报道。
胞嘧啶,学名为4-氨基-2-羰基嘧啶,是核酸(DNA和RNA)中的主要碱基组成成分之一。也可用作药物中间体。由于其对生命体独特的作用和药物功效,对其进行灵敏检测就显得非常重要。本发明就是提供一种能用于检测胞嘧啶的荧光探针及其制备方法与应用。
发明内容
本发明的一个目的之一在于提供一种能够用于胞嘧啶检测的荧光探针,通过明显的荧光增强可以灵敏地检测到胞嘧啶的存在,应用前景十分广泛。
本发明所述的一种用于检测胞嘧啶的荧光探针,是一个将三个BODIPY基元连接于同一个烷基碳的结构,其化学名为三-([4-(乙酰氧基苯基]-(4,4-二氟-1,3,5,7-四甲基-4-硼-3a,4a-二吡咯))氧亚甲基丙烷(简称三BODIPY),该荧光探针其具体结构为:
本发明另一目的在于提供一种用于检测胞嘧啶的荧光探针(三BODIPY)的制备方法。
本发明所述三BODIPY合成路线如下:
该三BODIPY的制备方法,具体包括以下步骤:
1)将三羟基丙烷与氯乙酰氯以摩尔比1:1~10比例,在二氯甲烷中加热搅拌回流,反应2~10小时,经5%氢氧化钠溶液充分洗涤去除有机层,硫酸镁干燥,过滤,减压蒸去溶剂,得到三(2-氧代-氯乙氧基甲基)丙烷,其结构式如下:
2)将三(2-氧代-氯乙氧基甲基)丙烷与羟基BODIPY以摩尔比1:3~10比例,碘化钾为催化剂,在碳酸钾-乙腈体系中加热回流12~24小时,反应结束后,产物用二氯甲烷萃取,硫酸镁干燥,过滤,浓缩滤液,经硅胶柱层析分离产物,减压旋干后得到枣红色固体,即为所述荧光探针(三BODIPY)。
本发明所制备的荧光探针(三BODIPY)在溶液状态显黄色,在510nm处有较强荧光发射,荧光量子产率0.97,能溶解于乙腈、四氢呋喃、二甲亚砜、N,N-二甲基甲酰胺等溶剂以及它们和水的混合溶剂,该荧光探针在溶液中与胞嘧啶形成1:1络合物,这种络合物的荧光比纯的三BODIPY荧光明显增强,且增强程度与胞嘧啶浓度呈线性关系,可用于环境中胞嘧啶的灵敏检测,其它无机离子和生物活性小分子如尿嘧啶、鸟嘌呤、腺嘌呤等干扰小,是一种理想的胞嘧啶快速检测传感探针。
本发明所制备的三BODIPY可对胞嘧啶进行定性、定量的检测,将浓度呈梯度变化的胞嘧啶溶液与三BODIPY溶液混合后,测定相应的荧光强度,然后以胞嘧啶的浓度为横坐标,混合体系的荧光强度为纵坐标作图,可根据荧光强度从图中读出待测溶液中的胞嘧啶浓度。
本发明的有益成果:所制备的三BODIPY在胞嘧啶存在下荧光发生显著改变,其它无机离子或生物活性小分子的存在不干扰胞嘧啶的检测,检测限为8.2μM,可用于高选择性高灵敏性地检测胞嘧啶,这对于复杂环境中胞嘧啶的检测有重要的现实应用价值。
附图说明
图1为三BODIPY具体结构式。
图2为三BODIPY与五种碱基对的紫外吸收图谱。
图3为三BODIPY与五种碱基对的荧光图谱。
图4为三BODIPY与不同浓度胞嘧啶配合的荧光图谱。
图5为三BODIPY与不同浓度胞嘧啶配合的线性拟合曲线。
图6为荧光强度随三BODIPY与胞嘧啶的摩尔分数变化图。
图7为三BODIPY与胞嘧啶在不同生物活性分子存在下的荧光变化图。
图8为三BODIPY的红外光谱图。
图9为三BODIPY的核磁共振氢谱图。
图10为三BODIPY的核磁共振碳谱图。
图11为三BODIPY的质谱图。
具体实施方式
为了进一步说明本发明,结合附图,给出以下系列具体实施例,但本发明并不受这些具体实施例的限制,任何了解该领域的技术人员对本发明的些许改动将可以达到类似的结果,这些改动也包含在本发明之中。
实施例1
1、三(2-氧代-氯乙氧基甲基)丙烷的合成步骤:
向装有25mL二氯甲烷的100mL三口烧瓶中,加入1.34g(0.01mol)三羟基丙烷和氯乙酰氯3.39g(0.03mmol),加热搅拌回流4小时,停止反应。冷却至室温,加入5%氢氧化钠溶液充分洗涤有机层3次,分出有机层,硫酸镁干燥,过滤,减压蒸去溶剂,得到三(2-氧代-氯乙氧基甲基)丙烷固体,产率85%。
2、三BODIPY的合成步骤:
向装有50mL乙腈的三口烧瓶中,加入0.363g(0.001mmol)三(2-氧代-氯乙氧基甲基)丙烷、羟基BODIPY 1.02g(0.003mmol)、碳酸钾1.0g(0.007mol)和碘化钾0.1g(0.0006mol),加热搅拌回流10小时,TLC检测原料点消失。停止反应,蒸去溶剂,加入二氯甲烷50mL和50mL的盐酸溶液(1M),萃取分层。有机层用蒸馏水30mL水洗2次,硫酸镁干燥,过滤,浓缩滤液。经硅胶柱层析分离产物(淋洗剂为二氯甲烷),减压旋干后得到枣红色固体,即为所述荧光探针(三BODIPY),产率80%。其结构式如图1所示。
本实施例制得的荧光探针三BODIPY,其分子式为C69H71B3F6N6O9。
红外光谱(KBr),v/cm-1:2958(-CH3),2923(-CH2),1550,1545(Ar),1310(Ph-O),1199(C-O-C)。
核磁氢谱(400MHz,CDCl3)δ7.19(d,J=8.0Hz,6H),7.01(d,J=8.0Hz,6H),5.96(s,6H),4.72(s,6H),4.20(s,6H),2.54(s,18H),1.39(s,18H),1.48(m,2H),0.85-0.94(m,3H)。
核磁碳谱13C NMR(100MHz,CDCl3):δppm:14.91,20.41,22.84,29.71,41.02,64.81,65.15,115.34,121.31,128.16,130.51,131.70,141.13,142.36,155.52,158.15,168.35;高分辨质谱(MS):计算值C69H71B3F6N6O9:1274.5987,测量值1256.7694(M-F-)。
本发明所制备的荧光探针三BODIPY在溶液状态显黄色,在510nm处有较强荧光发射,荧光量子产率0.97,能溶解于乙腈、四氢呋喃、二甲亚砜、N,N-二甲基甲酰胺等溶剂以及它们和水的混合溶剂,该荧光探针在溶液中与胞嘧啶形成1:1络合物,这种络合物的荧光比纯的三BODIPY荧光明显增强,且增强程度与胞嘧啶浓度呈线性关系,可用于环境中胞嘧啶的灵敏检测,其它无机离子和生物活性小分子如尿嘧啶、鸟嘌呤、腺嘌呤等干扰小,是一种理想的胞嘧啶快速检测传感探针。
本发明所制备的三BODIPY可对胞嘧啶进行定性、定量的检测,将浓度呈梯度变化的胞嘧啶溶液与三BODIPY溶液混合后,测定相应的荧光强度,然后以胞嘧啶的浓度为横坐标,混合体系的荧光强度为纵坐标作图,可根据荧光强度从图中读出待测溶液中的胞嘧啶浓度。
本实施例得到的三BODIPY进行相关测试,由核磁共振氢谱图、核磁共振碳谱图以及质谱图,确定了三BODIPY的结构为技术方案所述的的结构,如图9、图10、图11所示。
将本实施例得到的三BODIPY置于乙腈溶液中,10-4mol/L三BODIPY与10-3mol/L的五种碱基对(腺嘌呤、鸟嘌呤、胸腺嘧啶、胞嘧啶、尿嘧啶)进行紫外光谱测试,建立横坐标为波长、纵坐标为吸光度坐标系。其结果显示紫外光谱吸光度对胞嘧啶的变化最为明显,表明三BODIPY对胞嘧啶有较强的紫外光谱识别响应能力。如图2所示。
将本实施例得到的三BODIPY置于乙腈溶液中,10-4mol/L三BODIPY与10-3mol/L五种碱基对(腺嘌呤、鸟嘌呤、胸腺嘧啶、胞嘧啶、尿嘧啶)进行荧光发射测试,建立横坐标为波长、纵坐标为相对荧光强度坐标系。其结果显示荧光强度上升越大,表示三BODIPY对客体分子响应越高。在测试离子中对胞嘧啶有明显响应,说明三BODIPY对胞嘧啶能选择性识别。如图3所示。
将本实施例得到的三BODIPY置于乙腈溶液中(5×10-5mol/L),与不同当量的胞嘧啶溶液(0、0.001、0.002、0.003、0.004、0.005、0.006、0.007、0.008、0.009、0.01、0.03、0.05、0.07、0.09、0.1、0.3、0.5、0.7、0.9、1、5、10、50、100、300、500)配合,静置十分钟后测定的荧光发射状况,其结果表明在当量为500时其发射的荧光最强。如图4所示。
将本实施例得到的三BODIPY与不同浓度胞嘧啶配合的线性拟合比较,建立横坐标为胞嘧啶浓度,纵坐标为三BODIPY荧光强度变化比值的坐标系,线性拟合曲线显示加入一定浓度胞嘧啶后的三BODIPY荧光强度与未加胞嘧啶的三BODIPY荧光强度的比值,可以看出拟合曲线呈现较好的线性关系,表明通过荧光强度的变化可以定量地检测胞嘧啶浓度。如图5所示。
将本实施例得到的三BODIPY与摩尔分数变化的胞嘧啶配合荧光强度测试,建立横坐标为胞嘧啶的摩尔分数,纵坐标为三BODIPY加入胞嘧啶后的荧光强度与未加胞嘧啶的荧光强度差值坐标系。在固定的三BODIPY摩尔分数与胞嘧啶总浓度为5×10-5mol/L时,荧光强度随三BODIPY与胞嘧啶的摩尔分数变化而变化,可以看出最大差值出现在胞嘧啶摩尔分数为0.5处,说明三BODIPY与胞嘧啶的配合摩尔比为1:1。如图6所示。
将本实施例得到的三BODIPY与胞嘧啶在不同生物活性分子存在影响下的荧光变化测试。可以看出加入尿嘧啶、胸腺嘧啶、鸟嘌呤和腺嘌呤后,荧光的比值分别为0.94、0.97、0.93、0.96,均接近于1,说明这四种竞争性生物活性分子的存在,对三BODIPY和胞嘧啶的配合影响很小,也说明在多种生物活性分子共存的情况下,三BODIPY依然对胞嘧啶有好的选择性检测能力。如图7所示。
将本实施例得到的三BODIPY进行红外光谱测试,其结果是:红外光谱(KBr),v/cm-1:2958(-CH3),2923(-CH2),1550,1545(Ar),1310(Ph-O),1199(C-O-C)。如图11所示。
进行核磁氢谱测试,其结果是:(400MHz,CDCl3)δ7.19(d,J=8.0Hz,6H),7.01(d,J=8.0Hz,6H),5.96(s,6H),4.72(s,6H),4.20(s,6H),2.54(s,18H),1.39(s,18H),1.48(m,2H),0.85-0.94(m,3H)。如图9所示。
进行核磁碳谱13C测试,其结果是:NMR(100MHz,CDCl3):δppm:14.91,20.41,22.84,29.71,41.02,64.81,65.15,115.34,121.31,128.16,130.51,131.70,141.13,142.36,155.52,158.15,168.35;高分辨质谱(MS):计算值C69H71B3F6N6O9:1274.5987,测量值1256.7694(M-F-)。如10所示。
上述测试证实了三BODIPY的结构。
实施例2
1、三(2-氧代-氯乙氧基甲基)丙烷的合成步骤:
向装有30mL二氯甲烷的100mL三口烧瓶中,加入1.34g(0.01mol)三羟基丙烷和氯乙酰氯5.65g(0.05mmol),加热搅拌回流10小时,停止反应。冷却至室温,加入5%氢氧化钠溶液充分洗涤有机层3次,分出有机层,硫酸镁干燥,过滤,减压蒸去溶剂,得到三(2-氧代-氯乙氧基甲基)丙烷固体,产率88%。
2、三BODIPY的合成步骤:
向装有60mL乙腈的三口烧瓶中,加入0.363g(0.001mmol)三(2-氧代-氯乙氧基甲基)丙烷、羟基BODIPY 1.36g(0.004mmol)、碳酸钾2.0g(0.014mol)和碘化钾0.2g(0.0012mol),加热搅拌回流12小时,TLC检测原料点消失。停止反应,蒸去溶剂,加入二氯甲烷50mL和50mL的盐酸溶液(1M),萃取分层。有机层用蒸馏水30mL水洗2次,硫酸镁干燥,过滤,浓缩滤液。经硅胶柱层析分离产物(淋洗剂为二氯甲烷),减压旋干后得到枣红色固体,即为所述荧光探针,产率90%。
实施例3
1、三(2-氧代-氯乙氧基甲基)丙烷的合成步骤:
向装有40mL二氯甲烷的100mL三口烧瓶中,加入1.34g(0.01mol)三羟基丙烷和氯乙酰氯6.78g(0.06mmol),加热搅拌回流5小时,停止反应。冷却至室温,加入5%氢氧化钠溶液充分洗涤有机层3次,分出有机层,硫酸镁干燥,过滤,减压蒸去溶剂,得到三(2-氧代-氯乙氧基甲基)丙烷固体,产率82%。
2、三BODIPY的合成步骤:
向装有50mL乙腈的三口烧瓶中,加入0.363g(0.001mmol)三(2-氧代-氯乙氧基甲基)丙烷、羟基BODIPY 2.04g(0.006mmol)、碳酸钾3.0g(0.021mol)和碘化钾0.2g(0.0012mol),加热搅拌回流20小时,TLC检测原料点消失。停止反应,蒸去溶剂,加入二氯甲烷50mL和50mL的盐酸溶液(1M),萃取分层。有机层用蒸馏水30mL水洗2次,硫酸镁干燥,过滤,浓缩滤液。经硅胶柱层析分离产物(淋洗剂为二氯甲烷),减压旋干后得到枣红色固体,即为所述荧光探针,产率86%。
以上所述仅为本发明的实施例,并非因此限制本发明的保护范围,凡是利用本发明说明书及附图内容所作的等效流程变换,或直接或间接运用在其他相关的技术领域,均同理包括在本发明的专利保护范围内。
Claims (3)
1.一种用于检测胞嘧啶的荧光探针的制备方法,其特征是所述的荧光探针,是一个将三个BODIPY基元连接于同一个烷基碳的结构,其化学名为三-([4-(乙酰氧基苯基]-(4,4-二氟-1,3,5,7-四甲基-4-硼-3a,4a-二吡咯))氧亚甲基丙烷(三BODIPY),其具体结构为:
其制备方法包括以下步骤:
1)将三羟基丙烷与氯乙酰氯按摩尔比在二氯甲烷中加热搅拌回流,反应2~10小时,经5%氢氧化钠溶液充分洗涤去除有机层,硫酸镁干燥,过滤,减压蒸去溶剂,得到三(2-氧代-氯乙氧基甲基)丙烷,其结构式如下:
2)以碘化钾为催化剂,在碳酸钾-乙腈体系中,将三(2-氧代-氯乙氧基甲基)丙烷与羟基BODIPY按摩尔比加入,加热回流12~24小时,反应产物用二氯甲烷萃取,硫酸镁干燥,过滤,浓缩滤液,经硅胶柱层析分离产物,减压旋干后得到枣红色固体的荧光探针(三BODIPY)。
2.根据权利要求1所述的一种用于检测胞嘧啶的荧光探针的制备方法,其特征是三羟基丙烷与氯乙酰氯以摩尔比1:1~10。
3.根据权利要求1所述的一种用于检测胞嘧啶的荧光探针的制备方法,其特征是三(2-氧代-氯乙氧基甲基)丙烷与羟基BODIPY按摩尔比为1:3~10。
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