CN111285835A - 一种含有磺酰胺基结构的罗丹明pH值荧光探针及其应用 - Google Patents
一种含有磺酰胺基结构的罗丹明pH值荧光探针及其应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及一种含有磺酰胺基结构的罗丹明pH值荧光探针,特别涉及一种含有磺酰胺基结构的罗丹明pH值荧光探针的制备和应用。
背景技术
pH 值是生理学、药物学、病理学等研究中的重要参数。人体细胞内的pH在许多生理、病理过程中起着重要的作用(Nature Rev. Mol. Cell. Biol., 2010, 11(1), 50-61)。酸性或碱性过强会引起细胞功能紊乱,导致心、肺病变或神经类疾病,严重时甚至会有生命危险。因此监测细胞内pH值的变化可以为研究生理和病理过程提供重要信息。测定pH值一般用玻璃电极,但由于存在电化学干扰、可能的机械损伤等缺陷而不适于活体细胞的pH 监测(Cell Mol. Biol., 2000, 46(8), 1361-1374)。荧光探针检测细胞pH值属于非侵入性方法,不会破坏样品,同时具有灵敏度高、选择性好,仪器设计灵活,试样量少,操作简单,适用于高通量筛选等特点,是检测细胞pH值的理想方法(Trends in AnalyticalChemistry, 2010, 29(9), 1004-1013)。
一般情况下正常的细胞内存在两个主要的pH值范围:细胞质主要处于 6.8~7.4之间,酸性细胞器的pH值为4.5~6.0。针对这两个范围的pH值探针已经有大量文献报道,并已分别用于细胞质pH值的定性研究和酸性细胞器的检测(高等学校化学学报, 2010, 13(6), 1148-1151)。但是对于癌症细胞而言,由于通过糖酵解途径的厌氧代谢异常导致其细胞的pH值比正常细胞平均低0.55个单位,其pH值大概处于5.8-7.7的范围(Bioorganic &Medicinal Chemistry Letters 22 (2012) 2440-2443)。以此异常pH值为靶标的核磁、电化学癌症诊断方法已有文献报道。由于目前在此区间有线性响应的pH值荧光探针较少,所以基于pH值荧光探针的癌症诊断方法鲜见文献报道。因此开发一种在弱酸性范围(pH=5.0-7.0)有线性响应的pH值荧光探针对于癌症研究和癌症诊断具有重要的意义。此外,目前已开发出的pH值荧光探针大多数存在灵敏度较差,线性范围较窄等缺点。因此开发一种在弱酸性范围(pH=5.0-7.0)有线性响应的pH值荧光探针仍然是该领域的研究热点。
罗丹明是含有氧杂蒽结构的染料,具有很高的吸光系数、较长的激发和发射波长、较高的荧光量子产率和良好的光稳定性等优点,作为荧光探针的母体已大量的用于构建pH值荧光探针(Chem. Rev., 2012, 112, 1910-1956)。当罗丹明基pH值探针的内酰胺结构处于螺环状态时几乎没有荧光,当罗丹明内酰胺的羰基质子化时,可导致探针的五元螺环中的碳氮键断裂,形成开环结构,荧光强度显著增强,从而实现对pH值的选择性响应(Org.Biomol. Chem. , 2014, 12, 526-533)。所以罗丹明基pH值荧光探针大多是在pH值由大变小时呈现荧光增强,鲜见pH值由小变大时荧光增强的罗丹明基pH值探针。
磺酰胺基的磺酰部分是强吸电子基团,可以使旁边N的电子云密度下降导致N上的氢具有酸性。一些磺酰胺基化合物在弱酸性条件下就可以电离出质子,成为磺酰胺基负离子(Org. Lett. 2019, 21, 1641-1644)。本发明根据这一原理将磺酰基引入罗丹明顶环的氨基,构建了一种含有磺酰胺基结构的罗丹明pH值荧光探针。在酸性条件下,磺酰胺基的吸电子作用使罗丹明荧光淬灭,当溶液的酸性减弱至pH值大于探针磺酰胺基的pKa时,磺酰胺基上的氢电离,该基团转变为带负电荷的给电子基团,此时罗丹明的荧光就恢复了。因此本探针可以实现pH值由小变大过程的荧光增强响应。
发明内容
本发明的目的是提供一种含有磺酰胺基结构的罗丹明pH值荧光探针及其应用,该罗丹明pH值荧光探针可以实现pH值由小变大过程的荧光增强响应,同时具有选择性好、水溶性好的优点,具有在生物细胞中检测pH值的应用前景。
本发明采用的技术方案是:
一种含有磺酰胺基结构的罗丹明pH值荧光探针的结构通式如(I)所示:
(I)
其中:
R2、R3可以同时为氢,或者R2、R3选自氢、2-6个碳原子烷基,或R2和R3组成构成一个4-8个碳原子的环烷基。
所述的一种含有磺酰胺基结构的罗丹明pH值荧光探针的制备方法如下:
将罗丹明类化合物加入有机溶剂中,冷却至0℃后在搅拌下缓慢加入磺酰氯类化合物和缚酸剂,充分搅拌5 min。升温到一定温度后反应一定时间,减压蒸除溶剂,将反应混合物加入水中,用乙酸乙酯萃取三次,合并有机相,用MgSO4 充分干燥。减压蒸除溶剂,硅胶层析分离纯化得目标产物。
上述方法中所述的有机溶剂指DMF、DMSO、吡啶其中的一种或者其混合物。
上述方法中所述的缚酸剂指吡啶、碳酸钠、碳酸钾中的一种。
上述方法中所述的有机溶剂为吡啶,则可以不用缚酸剂。
上述方法中所述的反应温度为室温~60℃。
上述方法中所述的反应时间是2~5小时。
本发明所述的一种含有磺酰胺基结构的罗丹明pH值荧光探针用于对细胞pH值的检测。
本发明的有益效果是:本发明所述的一种含有磺酰胺基结构的罗丹明pH值荧光探针,能选择性识别氢离子,并且抗干扰性强,灵敏度高,水溶性好可以在水溶液中使用。由于其特殊的响应原理,这种探针对pH值由小变大过程具有荧光增强响应。细胞影像实验表明探针可以对细胞的pH值产生荧光响应。另外,探针的pKa值可以通过改变R1基团在5.38~6.31范围内进行调控,相应的pH值线性响应区间的调控范围在4.60~7.20之间,完全可以满足对不同生物细胞的pH值检测,尤其有望应用于癌症的诊断和相关研究。
附图说明
图1是实施例6制备的罗丹明pH值荧光探针3在不同pH值的荧光光谱。
图2是实施例6制备的罗丹明pH值荧光探针3的pH值线性响应区间图。
图3是实施例6制备的罗丹明pH值荧光探针3对常见物质的抗干扰图(其中编号1-25是pH = 4.20条件下探针3(10 μM)与常见物质(50 μM)共存时的荧光强度,这些常见物质包含金属离子(K+、Ca2+、Na+、Mg2+、Al3+、 Zn2+、 Fe3+、 Cu2+、 Mn2+、 Co2+)、氨基酸(Cys、Glu、Hls、Trp)、多肽(GSH)、活性氧物种(H2O2、HClO)和常见阴离子(F-、CH3COO-、HCO3 -、HSO3 -、SO4 2-、S2-、NO2-、S2O3 2-);编号26是pH = 7.40条件下探针3(10 μM)的荧光强度。激发波长为400 nm)。
图4是实施例6制备的罗丹明pH值荧光探针3的可逆性测试图。
图5是实施例6制备的罗丹明pH值荧光探针3的细胞影像图。
具体实施方式
实施例1 罗丹明化合物1的合成
罗丹明化合物1
称取3-二乙基氨基酚(1.65 g,10.00 mmol)于100 mL 烧瓶中,加入20 mL经分子筛干燥过的甲苯,在60℃下缓慢搅拌至完全溶解。加入邻苯二甲酸酐(1.50 g,约10.13 mmol),在N2保护下,混合物回流反应12h。用稀盐酸溶液(ω= 20%)调节至pH≤4,得到***沉淀。将产物抽滤,用蒸馏水充分洗涤,干燥得4-二乙氨基酮酸粗品0.70 g,收率42.50%。
称取4-二乙氨基酮酸粗品1 g(3.2 mmol)于100 mL烧瓶中,加入10 mL甲烷磺酸,在50℃下搅拌至充分溶解,加入间氨基苯酚0.4 g(3.80 mmol),在N2保护下加热至120℃持续反应10 h。反应完毕后冷却至室温,用0.1 M Na2CO3溶液调节至pH≤5,再用乙酸乙酯(3×20 mL)充分萃取,合并有机相,用MgSO4干燥抽滤,减压蒸除溶剂,硅胶层析过柱(V二氯甲烷:V甲醇=1:1),得红色固体0.40 g,收率40.35%。1H NMR (400 MHz, CDCl3) δ 8.03 (d, J = 7.1Hz, 1H), 7.57 (dtd, J = 19.6, 7.3, 1.1 Hz, 2H), 7.14 (d, J = 7.2 Hz, 1H),6.66 (d, J = 9.0 Hz, 1H), 6.57 (d, J = 8.6 Hz, 1H), 6.50 (d, J = 2.2 Hz, 1H),6.45 (d, J = 2.5 Hz, 1H), 6.39 (dd, J = 9.1, 2.5 Hz, 1H), 6.33 (dd, J = 8.6,2.2 Hz, 1H), 3.36 (q, J = 7.1 Hz, 4H), 1.16 (t, J = 7.1 Hz, 6H). 13C NMR (101MHz, CDCl3) δ 169.64, 153.87, 150.95, 150.48, 133.51, 129.58, 129.38, 129.21,125.99, 125.10, 112.47, 110.18, 109.05, 107.12, 100.53, 97.15, 44.60, 12.44.
实施例2 罗丹明化合物2的合成
罗丹明化合物2
以3-二甲基氨基酚为原料,采用实施例1的方法制备罗丹明化合物2。红色固体0.39 g,收率40.52%。1H NMR (400 MHz, CDCl3) δ 8.03 (d, J = 7.1 Hz, 1H), 7.60 (dtd, J =19.6, 7.3, 1.1 Hz, 2H), 7.12 (d, J = 7.2 Hz, 1H), 6.66 (d, J = 9.0 Hz, 1H),6.56 (d, J = 8.6 Hz, 1H), 6.40 (d, J = 2.2 Hz, 1H), 6.45 (d, J = 2.5 Hz, 1H),6.39 (dd, J = 9.1, 2.5 Hz, 1H), 6.33 (dd, J = 8.6, 2.2 Hz, 1H), 3.34(q, J =7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 169.64, 153.87, 150.95, 150.48,134.54, 129.58, 129.38, 129.21, 125.99, 124.95, 112.50, 110.18, 109.11,107.12, 100.53, 97.15, 44.12.
实施例3 罗丹明化合物3的合成
罗丹明化合物3
以3-环戊氨基酚为原料,采用实施例1的方法制备罗丹明化合物3。 红色固体0.43 g,收率42.65%。1H NMR (400 MHz, CDCl3) δ 8.02 (d, J = 7.1 Hz, 1H), 7.56 (dtd, J =19.6, 7.3, 1.1 Hz, 2H), 7.14 (d, J = 7.2 Hz, 1H), 6.66 (d, J = 9.0 Hz, 1H),6.57 (d, J = 8.6 Hz, 1H), 6.52 (d, J = 2.2 Hz, 1H), 6.50 (d, J = 2.5 Hz, 1H),6.42 (dd, J = 9.1, 2.5 Hz, 1H), 6.34 (dd, J = 8.6, 2.2 Hz, 1H), 3.35 (q, J =7.1 Hz, 4H), 1.17 (t, J = 7.1 Hz, 4H). 13C NMR (101 MHz, CDCl3) δ 168.94,153.77, 151.05, 150.51, 133.51, 129.58, 129.38, 129.21, 125.98, 125.10,112.47, 110.18, 109.05, 107.12, 100.53, 97.15, 44.56, 42.19, 15.21, 12.34.
实施例4 探针1的合成
将罗丹明化合物1 0.20 g(0.52 mmol)加入10 mL吡啶中,反应液在冰水浴中冷却后,在搅拌下缓慢加入4-硝基苯磺酰氯0.11 g(0.58 mmol),搅拌5 min。升温至20℃反应2 h,减压蒸除吡啶。将反应混合物加入50 mL水中,用乙酸乙酯(3×20 mL)充分萃取,合并有机相,用MgSO4 充分干燥,减压蒸除溶剂,硅胶层析过柱(V石油醚:V乙酸乙酯=3:1),得红色固体0.1691 g,收率57.01%。1H NMR (400 MHz, CDCl3) δ 8.16 (d, J = 8.9 Hz, 2H), 8.05– 7.96 (m, 3H), 7.63 (dtd, J = 20.9, 7.4, 1.0 Hz, 2H), 7.16 – 7.05 (m, 2H),6.61 (dd, J = 8.6, 2.1 Hz, 1H), 6.53 (t, J = 8.3 Hz, 2H), 6.41 (d, J = 2.5Hz, 1H), 6.35 (dd, J = 8.9, 2.6 Hz, 1H), 3.35 (q, J = 7.1 Hz, 4H), 1.16 (t, J= 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 170.24, 152.74, 152.30, 151.97,149.97, 149.87, 144.82, 138.54, 134.96, 129.69, 129.00, 128.79, 128.43,127.02, 125.12, 124.13, 115.70, 115.57, 108.72, 107.86, 104.46, 97.34, 44.41,12.33.
实施例5 探针2的合成
按照实施例4的方法合成得红色固体0.1820 g,收率58.96%。 1H NMR (400 MHz,CDCl3) δ 8.00 (dd, J = 19.4, 7.6 Hz, 3H), 7.67 – 7.57 (m, 4H), 7.17 – 7.10(m, 2H), 6.68 (dd, J = 8.6, 2.2 Hz, 1H), 6.60 – 6.52 (m, 2H), 6.43 (d, J =2.5 Hz, 1H), 6.36 (dd, J = 9.0, 2.6 Hz, 1H), 3.34 (q, J = 7.0 Hz, 4H), 1.15(t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 170.26, 152.72, 152.26,149.76, 142.60, 138.57, 134.92, 134.52, 134.20, 129.63, 128.96, 128.76,127.64, 126.97, 126.11, 126.08, 125.03, 124.34, 124.01, 121.63, 115.47,115.32, 108.60, 107.66, 104.46, 97.34, 86.13, 44.35, 12.29.
实施例6 探针3的合成
按照实施例4的方法合成得红色固体0.1534 g,收率47.71%。 1H NMR (400 MHz,CDCl3) δ 8.50 (d, J = 8.5 Hz, 1H), 8.39 (d, J = 8.7 Hz, 1H), 8.31 (dd, J =7.4, 1.3 Hz, 1H), 8.00 – 7.96 (m, 1H), 7.61 – 7.43 (m, 4H), 7.13 – 7.02 (m,3H), 6.60 (dd, J = 8.6, 2.2 Hz, 1H), 6.49 (dd, J = 9.8, 8.7 Hz, 2H), 6.37 (d,J = 2.5 Hz, 1H), 6.30 (dd, J = 8.9, 2.6 Hz, 1H), 3.31 (q, J = 7.1 Hz, 4H),2.83 (s, 6H), 1.13 (t, J = 7.0 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 169.68,152.66, 152.55, 152.06, 151.82, 149.42, 138.69, 134.71, 133.86, 130.96,130.13, 129.68, 129.41, 129.37, 128.78, 128.60, 126.84, 124.74, 123.84,122.99, 118.30, 115.17, 114.96, 114.65, 108.28, 107.11, 104.53, 97.36, 84.12,45.23, 44.30, 12.32.
实施例7 探针4的合成
按照实施例4的方法合成得红色固体0.1712 g,收率60.60%。1H NMR (400 MHz,CDCl3) δ 8.01 (d, J = 7.3 Hz, 1H), 7.90 – 7.81 (m, 2H), 7.67 – 7.56 (m, 2H),7.16 – 7.09 (m, 2H), 7.04 (t, J = 8.6 Hz, 2H), 6.68 (dd, J = 8.6, 2.2 Hz,1H), 6.54 (dd, J = 8.7, 4.0 Hz, 2H), 6.42 (d, J = 2.4 Hz, 1H), 6.34 (dd, J =9.0, 2.5 Hz, 1H), 3.34 (q, J = 7.1 Hz, 4H), 1.15 (t, J = 7.1 Hz, 6H). 13C NMR(101 MHz, CDCl3) δ 169.99, 166.38, 163.84, 152.67, 152.42, 152.22, 149.62,138.64, 134.88, 129.94, 129.84, 129.58, 128.95, 128.73, 126.93, 124.94,123.95, 116.39, 116.17, 115.45, 115.23, 108.52, 107.67, 104.56, 97.42, 85.15,44.38, 12.32.
实施例8 探针5的合成
以罗丹明化合物3为原料,按照实施例4的方法合成得红色固体0.1578 g,收率54.25%。 1H NMR (400 MHz, CDCl3) δ 8.03 – 7.99 (m, 1H), 7.79 – 7.74 (m, 2H),7.61 (dtd, J = 20.3, 7.4, 1.1 Hz, 2H), 7.34 – 7.29 (m, 2H), 7.14 – 7.10 (m,2H), 6.67 (dd, J = 8.6, 2.2 Hz, 1H), 6.54 (dd, J = 8.8, 2.6 Hz, 2H), 6.42 (d,J = 2.5 Hz, 1H), 6.34 (dd, J = 9.0, 2.6 Hz, 1H), 3.34 (q, J = 7.0 Hz, 4H),1.15 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 170.09, 152.64, 152.45,152.20, 149.62, 139.43, 138.57, 137.37, 134.89, 129.57, 129.27, 128.93,128.70, 128.53, 126.89, 124.91, 123.93, 115.39, 115.22, 108.48, 107.65,104.43, 97.37, 85.15, 44.34, 12.33.
实施例9 探针6的合成
以罗丹明化合物2为原料,按照实施例4的方法合成得红色固体0.1246 g,收率44.42%。 1H NMR (400 MHz, CDCl3) δ 8.02 – 7.98 (m, 1H), 7.74 (d, J = 8.4 Hz,2H), 7.65 – 7.55 (m, 2H), 7.19 (d, J = 8.1 Hz, 2H), 7.11 (dd, J = 7.8, 1.4Hz, 2H), 6.69 (dd, J = 8.6, 2.3 Hz, 1H), 6.57 – 6.51 (m, 2H), 6.41 (d, J =2.5 Hz, 1H), 6.33 (dd, J = 9.0, 2.6 Hz, 1H), 3.33 (q, J = 7.0 Hz, 4H), 2.33(s, 3H), 1.15 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 169.79, 152.62,152.18, 149.47, 143.98, 138.80, 135.74, 134.78, 129.68, 129.47, 128.90,128.66, 127.09, 126.88, 124.79, 123.86, 115.15, 114.86, 108.34, 107.35,104.57, 97.40, 84.25, 44.32, 21.38, 12.32.
实施例10 探针7的合成
按照实施例4的方法合成得红色固体0.1659 g,收率57.44%。 1H NMR (400 MHz,CDCl3) δ 7.99 (dt, J = 7.3, 0.9 Hz, 1H), 7.80 – 7.75 (m, 2H), 7.60 (dtd, J =19.6, 7.3, 1.2 Hz, 2H), 7.13 – 7.08 (m, 2H), 6.88 – 6.81 (m, 2H), 6.69 (dd, J= 8.6, 2.2 Hz, 1H), 6.54 (dd, J = 12.3, 8.7 Hz, 2H), 6.41 (d, J = 2.6 Hz,1H), 6.32 (dd, J = 9.0, 2.6 Hz, 1H), 3.77 (s, 3H), 3.33 (q, J = 7.1 Hz, 4H),1.14 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 169.79, 163.07, 152.66,152.62, 152.18, 149.49, 138.92, 134.79, 130.20, 129.47, 129.26, 128.89,128.66, 126.88, 124.78, 123.88, 115.10, 114.88, 114.22, 108.33, 107.32,104.55, 97.39, 84.24, 55.41, 44.30, 12.32.
实施例11 探针8的合成
按照实施例4的方法合成得红色固体0.0852 g,收率33.35%。 1H NMR (400 MHz,CDCl3) δ 8.02 (d, J = 7.4 Hz, 1H), 7.64 (dtd, J = 22.5, 7.4, 1.0 Hz, 2H),7.21 – 7.17 (m, 2H), 6.77 (dd, J = 8.6, 2.3 Hz, 1H), 6.68 (d, J = 8.6 Hz,1H), 6.56 (d, J = 8.9 Hz, 1H), 6.44 (d, J = 2.5 Hz, 1H), 6.36 (dd, J = 8.9,2.6 Hz, 1H), 3.36 (q, J = 7.1 Hz, 4H), 3.16 – 3.10 (m, 2H), 1.84 (dq, J =10.2, 7.5 Hz, 2H), 1.17 (t, J = 7.1 Hz, 6H), 1.01 (t, J = 7.5 Hz, 3H). 13C NMR(101 MHz, CDCl3) δ 169.64, 152.69, 152.56, 149.62, 138.96, 134.84, 129.57,129.39, 128.77, 127.06, 124.91, 123.97, 115.49, 114.29, 108.48, 106.73,104.66, 97.48, 83.99, 53.52, 44.41, 17.14, 12.74, 12.39.
实施例12 探针9的合成
将罗丹明化合物1 0.20 g(0.52 mmol)加入10 mL DMF中,反应液在冰水浴中冷却后,在搅拌下缓慢加入二甲胺基磺酰氯0.083 g(0.58 mmol)和无水Na2CO3 1.09 g(1 mmol),搅拌5 min。升温至60℃反应5 h。减压蒸除DMF,将反应混合物加入50 mL水中,用乙酸乙酯(3×20 mL)充分萃取,合并有机相,用MgSO4充分干燥,减压蒸除溶剂,硅胶层析过柱(V二氯甲烷:V甲醇=20:1),得紫色固体0.0730 g,收率28.50%。 1H NMR (400 MHz, CDCl3) δ 7.99 (d, J= 7.5 Hz, 1H), 7.66 – 7.54 (m, 3H), 7.19 (dt, J = 7.5, 1.0 Hz, 1H), 6.80 (t,J = 1.3 Hz, 1H), 6.61 (d, J = 1.2 Hz, 2H), 6.56 (d, J = 8.9 Hz, 1H), 6.44 (d,J = 2.6 Hz, 1H), 6.33 (dd, J = 8.9, 2.6 Hz, 1H), 3.35 (q, J = 7.1 Hz, 4H),3.01 (s, 6H), 1.16 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 169.69,154.09, 153.65, 153.19, 153.02, 152.38, 149.39, 134.51, 129.17, 128.74,128.40, 127.27, 124.61, 124.00, 117.28, 113.07, 108.02, 105.31, 97.55, 84.87,44.33, 12.43.
实施例13 探针性能测试
选取实施例6制备的罗丹明pH值荧光探针3为例进行性能测试。
1)荧光探针3在不同pH值条件下的荧光光谱测定。将一定量的探针3储备液(1 mM)分别加入不同pH值的缓冲液中,使探针3的最终浓度为10 μM。对所得的样品进行荧光光谱测定,结果如图1所示(激发光波长400nm)。因为探针3具有两个荧光发射峰,所以采用472nm和576 nm处的荧光强度比例与pH值构建函数关系。结果显示pH值范围为5.6到7.0之间为该探针的线性响应区间(R2=0.9907)。结果如图2所示。对于其他探针,则采用576 nm处的荧光强度与pH值构建函数关系。
2)荧光探针3对常见物质的抗干扰实验。取一定量的探针3储备液(1 mM)加入pH =4.20的缓冲液中,再分别加入一定量的常见物质,使所得样品中探针3的浓度为10 μM,常见物质的浓度为50 μM。这里所指的常见物质包含金属离子(K+、Ca2+、Na+、Mg2+、Al3+、 Zn2+、 Fe3 +、 Cu2+、 Mn2+、 Co2+)、氨基酸(Cys、Glu、Hls、Trp)、多肽(GSH)、活性氧物种(H2O2、HClO)和常见阴离子(F-、CH3COO-、HCO3 -、HSO3 -、SO4 2-、S2-、NO2-、S2O3 2-)。另外取一定量的探针3储备液(1mM)加入pH = 7.40的缓冲液中,得到探针3的浓度为10 μM的样品。将以上样品进行荧光光谱测定,检测波长为576 nm,激发波长为400 nm,所得数据见图3。可以看出常见物质不影响探针3对pH值的响应,说明探针3具有良好的抗干扰性。
3)荧光探针3的可逆性测试。在探针3(50 μM)的水溶液中加入一定量的NaOH(0.1M)溶液将样品的pH值调至7.4,测定样品在576 nm处的荧光强度;然后加入一定量的HCl(0.1 M)溶液将样品的pH值调至4.2,测定样品在576 nm处的荧光强度(激发波长为 400nm)。如此重复操作五次。所得数据见图4。由图4可知,该探针的荧光强度随着pH值的升降而强弱变化,呈现良好的可逆性。
4)荧光探针3的细胞影像。在盛有SKBR3细胞的培养皿中加入80μL的探针储备液(1mM),保持在37oC及5% CO2条件下孵育30分钟。用PBS溶液(0.01M)冲洗培养皿 3 次。在40倍共聚焦荧光显微镜下观察,细胞区域有明显的荧光出现(如图5)。可以看出在共聚焦成像实验中细胞形态健康,说明该探针对细胞没有明显的毒性。这一结果表明探针3有可能用于检测细胞的pH值。
Claims (8)
2.如权利要求1所述的一种含有磺酰胺基结构的罗丹明pH值荧光探针的制备方法,其特征在于,包括如下步骤:
将罗丹明类化合物加入吡啶中,冷却至0℃后在搅拌下缓慢加入磺酰氯类化合物,充分搅拌5 min,升温到一定温度后反应一定时间,减压蒸除溶剂,将反应混合物加入水中,用乙酸乙酯萃取三次,合并有机相,用MgSO4 充分干燥,减压蒸除溶剂,硅胶层析分离纯化得目标产物。
3.如权利要求1所述的一种含有磺酰胺基结构的罗丹明pH值荧光探针的制备方法,其特征在于,包括如下步骤:
将罗丹明类化合物加入有机溶剂中,冷却至0℃后在搅拌下缓慢加入磺酰氯类化合物和缚酸剂,充分搅拌5 min,升温到一定温度后反应一定时间,减压蒸除溶剂,将反应混合物加入水中,用乙酸乙酯萃取三次,合并有机相,用MgSO4 充分干燥,减压蒸除溶剂,硅胶层析分离纯化得目标产物。
4.根据权利要求3所述的一种含有磺酰胺基结构的罗丹明pH值荧光探针的制备方法,其特征在于,所述的有机溶剂选自DMF、DMSO、吡啶中的一种或者两种以上的混合物。
5.根据权利要求3所述的一种含有磺酰胺基结构的罗丹明pH值荧光探针的制备方法,其特征在于,所述的缚酸剂选自吡啶、碳酸钠、碳酸钾中的一种。
6.根据权利要求2或3所述的一种含有磺酰胺基结构的罗丹明pH值荧光探针的制备方法,其特征在于,所述的反应温度为20~60℃。
7.根据权利要求2或3所述的一种含有磺酰胺基结构的罗丹明pH值荧光探针的制备方法,其特征在于,所述的反应时间是2~5小时。
8.如权利要求1所述的一种含有磺酰胺基结构的罗丹明pH值荧光探针用于对细胞pH值的检测。
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