CN113072559A - 一类亲脂性含有癸酸酯结构的罗丹明pH荧光探针及其制备方法和应用 - Google Patents
一类亲脂性含有癸酸酯结构的罗丹明pH荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开一类亲脂性含有癸酸酯结构的罗丹明pH荧光探针及其制备方法和应用。所述荧光探针的结构通式如(Ⅰ)所示。用罗丹明B与三氯氧磷反应制备罗丹明B酰氯，再与丝氨醇制备罗丹明B丝氨醇，随后将其溶解于干燥的二氯甲烷中，加入N,N‑二异丙基乙胺、4‑二甲氨基吡啶和癸酰氯，室温搅拌36‑48小时，经硅胶柱层析提纯，分别制得RK‑1和RK‑2荧光探针。该探针均具有较好的溶解性和pH可逆性(3.0‑9.0)，吸光度和荧光强度均不受金属离子的干扰。长脂肪连有助于其进入亲脂性的环境中，能够定位于细胞的细胞膜上。此外，大型蚤成像实验表明这类探针具有较好的微生物染色性，能够对大型蚤及其酸性消化道进行荧光成像。
Description
技术领域
本发明涉及罗丹明pH荧光探针，特别涉及亲脂性含有癸酸酯结构的罗丹明pH荧光探针及其应用。
背景技术
物质的跨膜运输是维持细胞正常生命活动的基础之一，包括透过脂膜的简单扩散和通过载体及通道来转运等，脂膜一般指具有磷脂双分子层结构或其他脂溶性物质的膜结构，包括细胞膜，角质层等。细胞膜由磷脂双分子层及镶嵌在其上的蛋白质载体构成。维持细胞之间的跨膜运输的膜转运蛋白主要分为载体蛋白和通道蛋白。这一类蛋白是由富含固醇脂、甘油三酯的疏水性内核和由蛋白质、磷脂、胆固醇等组成的外壳构成的球状微粒。其中通道蛋白是跨膜运输的亲水通道，允许适当大小的离子通过。在脂膜的离子通道中，质子通道维持着细胞内环境的稳定，同时保持着胞液中各类细胞器pH的稳定。为了便于研究膜上pH通道在质子传递过程中所发挥的重要作用，急需研发质子通道可视化的荧光成像试剂。荧光探针是能够满足上述要求的最优选择，其具有操作简单、可视化强、灵敏度高、可以实现微环境检测、不会破坏样品等优点。此外，因其对细胞没有损害性，而且具有高选择性、便捷的可视化成像的特性，与其他技术相比具有着显著的优势。为了研究细胞膜上的H+通道和水蛋白在细胞内pH调控过程中的作用，研发具有亲脂性膜定位的高灵敏度、强抗干扰及具有较窄的pH响应窗口的荧光探针，并将其用于细胞膜和大型蚤的荧光成像，具有重要意义。
发明内容
本发明的目的是提供一类亲脂性含有癸酸酯结构的罗丹明pH荧光探针，该罗丹明pH荧光探针可以定位于细胞膜并快速响应、高灵敏度检测大型蚤体内pH的变化。
本发明采用的技术方案是：一类亲脂性含有癸酸酯结构的罗丹明pH荧光探针，具有通式(Ⅰ)的结构，
其中，
一类亲脂性含有癸酸酯结构的罗丹明pH荧光探针的制备方法，包括如下步骤：
1)取罗丹明B与三氯氧磷，以1,2-二氯乙烷为溶剂，90℃下加热回流反应3-5小时，蒸掉溶剂1,2-二氯乙烷，制备罗丹明B酰氯；
2)于步骤1)所得罗丹明B酰氯中，加入丝氨醇、三乙胺、乙醇和乙腈的混合溶液，室温下搅拌24小时，经硅胶柱层析提纯，制备罗丹明B丝氨醇；
3)将罗丹明B丝氨醇溶解于干燥的二氯甲烷中，依次加入N,N-二异丙基乙胺、4-二甲氨基吡啶和癸酰氯，室温搅拌36-48小时，所得反应物用二氯甲烷萃取，取下层有机相，干燥有机相，所得产物经硅胶柱层析提纯。
优选地，上述的制备方法，所得产物经硅胶柱层析提纯，具体为，将产物注入硅胶柱中，以体积比为10:1的二氯甲烷和乙酸乙酯混合物为洗脱剂进行洗脱获得产物RK-1；所述产物RK-1的结构式如(Ⅱ)所示，
优选地，上述的制备方法，所得产物经硅胶柱层析提纯，具体为，将产物注入硅胶柱中，以体积比为5:1的二氯甲烷和乙酸乙酯混合物为洗脱剂进行洗脱获得产物RK-2；所述产物RK-2的结构式如(Ⅲ)所示，
优选地，上述的制备方法，按摩尔比，罗丹明B丝氨醇:N,N-二异丙基乙胺:4-二甲氨基吡啶:癸酰氯＝1:10:0.3:4。
本发明提供的一类亲脂性含有癸酸酯结构的罗丹明pH荧光探针作为荧光试剂在细胞膜定位荧光成像中的应用。
优选地，所述细胞为MCF-7细胞。
本发明提供的一类亲脂性含有癸酸酯结构的罗丹明pH荧光探针作为荧光试剂在浮游生物体内pH荧光成像中的应用。
优选地，所述浮游生物体为大型蚤。
本发明的有益效果是：
1、本发明，利用癸酰氯和羟基的酯化反应，将亲脂性的长碳链引入罗丹明螺酰亚胺结构中，合成制备了RK-1和RK-2。本发明制备的RK-1和RK-2均对H+响应迅速，在pH3.0-7.0的范围内光谱响应灵敏，并且具有良好的酸碱光谱响应可逆性。RK-1荧光探针在pH3.0-6.0范围内呈现出较敏感的光谱响应，其pKa为3.51(±0.02)(Intensity)和3.19(±0.07)(Abs)；RK-2借助于分子内氢键的辅助作用，在pH 3.5-7.0范围内呈现出较敏感的光谱响应，其pKa为4.68(±0.05)(Intensity)和4.26(±0.001)(Abs)。
2、本发明制备的探针均具有较好的溶解性和pH可逆性(3.0-9.0)，吸光度和荧光强度均不受金属离子的干扰。此外，该探针还具有较好的亲脂性和生物兼容性。RK-1能够快速定位聚集于MCF-7细胞膜以及聚集于大型蚤的身体各处；RK-2能够快速聚集于大型蚤的消化道内，对膜结构和大型蚤进行荧光成像。
3、本发明制备的探针，长脂肪连有助于其进入亲脂性的环境中，能够定位于细胞的细胞膜上。此外，大型蚤成像实验表明这类探针具有较好的微生物染色性，能够对大型蚤及其酸性消化道进行荧光成像。
附图说明
图1是实施例1制备的RK-1pH变化的荧光的光谱响应。
图2是实施例1制备的RK-2pH变化的荧光的光谱响应。
图3是实施例1制备的RK-1在pH 7.2时对金属干扰性的吸收响应。
图4是实施例1制备的RK-2在pH 7.2时对金属干扰性的吸收响应。
图5是实施例1制备的RK-1在pH 7.2时对金属干扰性的荧光响应。
图6是实施例1制备的RK-2在pH 7.2时对金属干扰性的荧光响应。
图7是实施例1制备的RK-1的可逆性测试。
图8是实施例1制备的RK-2的可逆性测试。
图9是实施例1制备的RK-1在细胞膜上定位成像。
图10是实施例1制备的RK-1对浮游动物大型蚤的荧光显微成像。
图11是实施例1制备的RK-2对浮游动物大型蚤的荧光显微成像。
具体实施方式
实施例1一类亲脂性含有癸酸酯结构的罗丹明pH荧光探针
反应式如下：
制备方法：
1)将1摩尔的罗丹明B与3摩尔的POCl3加入到干燥的1,2-二氯乙烷中，90℃下加热回流反应3-5小时，冷却至室温后，蒸掉溶剂1,2-二氯乙烷，制得罗丹明B酰氯。
2)将1摩尔丝氨醇和3摩尔三乙胺溶于1mL乙醇和30mL乙腈溶液中，然后将混合溶液加入到步骤1)所得罗丹明B酰氯中，室温下搅拌24小时，经硅胶柱层析提纯，得到罗丹明B丝氨醇。
3)将1mol罗丹明B丝氨醇溶于200ml干燥的二氯甲烷中，加入10mol N,N-二异丙基乙胺、0.3mol 4-二甲氨基吡啶和4mol癸酰氯，室温下搅拌36-48小时，所得反应物用二氯甲烷萃取，取下层有机相，用无水硫酸镁干燥有机相，所得产物经硅胶柱层析提纯。
3.1)产物注入硅胶柱中，以体积比为10:1的二氯甲烷和乙酸乙酯混合物为洗脱剂进行洗脱，获得淡粉色油状产物RK-1；产物RK-1的核磁结构表征数据：
RK-1:1H NMR(400MHz,CDCl3)δ7.90(dd,J＝5.8,2.8Hz,1H),7.53–7.43(m,2H),7.13–7.04(m,1H),6.48(d,J＝8.9Hz,2H),6.39(d,J＝2.5Hz,2H),6.30(dd,J＝8.9,2.5Hz,2H),4.30(dd,J＝11.3,6.7Hz,2H),4.13(dd,J＝11.2,6.7Hz,2H),3.59(dd,J＝13.3,6.6Hz,1H),3.45–3.24(m,9H),2.06(td,J＝7.4,1.8Hz,5H),1.48(dd,J＝14.3,7.2Hz,5H),1.18(t,J＝7.0Hz,14H),0.89(t,J＝6.9Hz,7H).
3.2)产物注入硅胶柱中，以体积比为5:1的二氯甲烷和乙酸乙酯混合物为洗脱剂进行洗脱，获得淡粉色油状产物RK-2；产物RK-2的核磁结构表征数据：
RK-2：1H NMR(400MHz,CDCl3)δ7.91(dd,J＝5.8,2.8Hz,1H),7.57–7.42(m,2H),7.10(dd,J＝5.7,2.7Hz,1H),6.48(dd,J＝8.8,6.2Hz,2H),6.39(t,J＝2.4Hz,2H),6.35–6.22(m,2H),4.94–4.76(m,1H),4.55(dd,J＝10.9,8.4Hz,1H),3.87(dd,J＝11.0,5.9Hz,1H),3.70(dd,J＝12.0,4.4Hz,1H),3.58–3.49(m,1H),3.43–3.26(m,8H),2.36(t,J＝7.5Hz,1H),2.11–2.04(m,2H),1.54–1.42(m,2H),1.40–1.22(m,20H),1.19(t,J＝7.0Hz,13H),0.95–0.82(m,5H).
实施例2应用试验
本应用试验采用实施例1制备的RK-1及RK-2荧光探针进行。
一、荧光光谱的测定
分别将RK-1及RK-2荧光探针溶解于乙醇水溶液(1:1,v:v)中，配制浓度为2×10- 5mol/L的RK-1及RK-2荧光探针溶液。
将RK-1及RK-2荧光探针溶液分别用氢氧化钠和盐酸调节pH，分别制备pH范围在2-12不同pH值的溶液，测试荧光光谱，结果如图1和图2所示，图1及图2中每条线分别代表不同pH值的测试结果，最下端的线代表最高pH值为12，往上pH值依次降低最上端为pH值最低为2。结果表明，随着pH值的降低，荧光强度不断增强。RK-1探针在pH处于3.0～6.0范围内较为敏感，其荧光强度增强15倍多；RK-2探针在pH处于3.5～7.0范围内较为敏感，其荧光强度增强了200多倍。
二、金属离子干扰性测定
分别将RK-1及RK-2荧光探针溶解于乙醇/Tris-HCl缓冲体系水溶液(1:1,v:v)中，配制浓度为1×10-5mol/L的RK-1及RK-2荧光探针溶液，调节pH为7.2。分别取浓度为1×10- 5mol/L，pH为7.2的RK-1及RK-2荧光探针溶液，各加入探针摩尔量的10倍的金属盐水溶液，进行荧光光谱和吸收光谱的测定，取最大吸收峰和最大荧光峰处的强度依据所加入的金属离子种类作图，结果如图3-6所示。
图3和图4是制备的RK-1及RK-2在pH 7.2时对金属干扰性的吸收光谱响应。图5和图6是制备的RK-1及RK-2在pH 7.2时对金属干扰性的荧光光谱响应。图3、图4、图5、图6中，1-空白，2-K+，3-Al3+，4-Ca2+，5-Cr3+，6-Mn2+，7-Mg2+，8-Pb2+，9-Fe3+，10-Cd2+，11-Hg2+，12-Fe2 +，13-Na+，14-Cu2+，15-Zn2+。结果显示，金属离子对RK-1及RK-2的吸收与荧光光谱均没有影响，即本发明的RK-1及RK-2荧光探针不受金属离子的干扰。
三、可逆性测试
光谱测定RK-1及RK-2荧光探针在pH为3.0左右和9.0左右荧光变化的可逆性。用NaOH，HCl调节pH在3.0和9.0左右，进行荧光光谱测定。
由图7和图8可知，RK-1及RK-2荧光探针响应迅速，荧光强度变化稳定且可逆。
四、荧光显微成像
首先，向培养有MCF-7的细胞中加入RK-1，培养5min后，用PBS溶液清洗，然后将pH4.0的PBS缓冲液加入培养皿中，将该培养皿置于共聚焦显微镜下进行观察。
如图9所示，在pH4.0的PBS溶液中，探针RK-1呈现出红色荧光，这表明RK-1能够定位聚集于MCF-7细胞的细胞膜上，并指示体系的pH。
其次，向培养的大型蚤中，分别加入RK-1及RK-2荧光探针的二甲基亚砜溶液，混合均匀后，染色5min后，将该培养皿置于共聚焦显微镜下进行观察。
如图10所示，亮场下的大型蚤生长状态良好；经过RK-1荧光探针染色后，如图10所示，可以观测到大型蚤的体内各处呈现强烈的红色荧光，实验结果表明RK-1荧光探针能够聚集于大型蚤的体内各处，并显示其体内呈现酸性。
如图11所示，亮场下的大型蚤生长状态良好；经过RK-2荧光探针染色后，如图11所示，可以观测到大型蚤的消化道内呈现强烈的红色荧光，实验结果表明RK-2荧光探针能够聚集于大型蚤的肠道内，并显示肠道呈现酸性。
Claims (9)
1.一类亲脂性含有癸酸酯结构的罗丹明pH荧光探针，其特征在于，具有通式(Ⅰ)的结构，
其中，
或，R1＝H，
2.权利要求1所述的一类亲脂性含有癸酸酯结构的罗丹明pH荧光探针的制备方法，其特征在于，包括如下步骤：
1)取罗丹明B与三氯氧磷，以1,2-二氯乙烷为溶剂，90℃下加热回流反应3-5小时，蒸掉溶剂1,2-二氯乙烷，制备罗丹明B酰氯；
2)于步骤1)所得罗丹明B酰氯中，加入丝氨醇、三乙胺、乙醇和乙腈的混合溶液，室温下搅拌24小时，经硅胶柱层析提纯，制备罗丹明B丝氨醇；
3)将罗丹明B丝氨醇溶解于干燥的二氯甲烷中，依次加入N,N-二异丙基乙胺、4-二甲氨基吡啶和癸酰氯，室温搅拌36-48小时，所得反应物用二氯甲烷萃取，取下层有机相，干燥有机相，所得产物经硅胶柱层析提纯。
3.根据权利要求2所述的制备方法，其特征在于，所得产物经硅胶柱层析提纯，具体为，将产物注入硅胶柱中，以体积比为10:1的二氯甲烷和乙酸乙酯混合物为洗脱剂进行洗脱获得产物RK-1；所述产物RK-1的结构式如(Ⅱ)所示，
4.根据权利要求2所述的制备方法，其特征在于，所得产物经硅胶柱层析提纯，具体为，将反应物注入硅胶柱中，以体积比为5:1的二氯甲烷和乙酸乙酯混合物为洗脱剂进行洗脱获得产物RK-2；所述产物RK-2的结构式如(Ⅲ)所示，
5.根据权利要求2所述的制备方法，其特征在于，按摩尔比，罗丹明B丝氨醇:N,N-二异丙基乙胺:4-二甲氨基吡啶:癸酰氯＝1:10:0.3:4。
6.权利要求1所述的一类亲脂性含有癸酸酯结构的罗丹明pH荧光探针作为荧光成像试剂在细胞膜定位荧光成像中的应用。
7.根据权利要求6所述的应用，其特征在于，所述细胞为MCF-7细胞。
8.权利要求1所述的一类亲脂性含有癸酸酯结构的罗丹明pH荧光探针作为荧光成像试剂在浮游生物体内pH荧光成像中的应用。
9.根据权利要求8所述的应用，其特征在于，所述浮游生物体为大型蚤。
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