CN112940011B - 一种溶酶体靶向的比率型次氯酸荧光探针 - Google Patents
一种溶酶体靶向的比率型次氯酸荧光探针 Download PDFInfo
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Abstract
本发明公布了一种溶酶体靶向的比率型次氯酸荧光探针，属于化学分析检测技术领域，其分子结构式如下：
在检测过程中，该探针具有选择性好、灵敏度高、抗干扰能力强等优点。同时，该探针具有靶向溶酶体的能力。这些优良的性能表明该荧光探针在环境及生物等领域具有重要的应用价值。
Description
技术领域
本发明属于化学分析检测技术领域，具体涉及一种溶酶体靶向的比率型次氯酸荧光探针，以及该探针在检测次氯酸中的应用。
背景技术
活性氧物种包括单线态氧、过氧化氢、超氧自由基以及次氯酸等，在哺乳动物生命体系的生理和病理过程中起着重要的作用。其中，次氯酸是最重要的一种活性氧物种，在髓过氧化物酶(MPO)的帮助下，过氧化氢和次氯化物可以在免疫细胞中合成。次氯酸不仅对细胞中的病原体具有致死作用，而且在微生物的免疫防御中也起着非常重要的作用。但是，次氯酸的过量生产会引起许多疾病，例如癌症，肺损伤，关节炎，动脉血管硬化等。因此，开发具有高选择、高灵敏度检测次氯酸的方法是很有意义。
荧光探针因其操作方便、无损检测、时空分辨率高等优点，已经成为次氯酸检测的常用方法。近来年，有一些关于次氯酸荧光探针的文献报道，但大部分该类荧光探针属于开关型，这类探针受环境的影响较大。然而，比率型探针能克服这一缺点，受环境影响较小。由于具有靶向能力的探针，可用于阐明次氯酸的亚细胞分布并解释次氯酸的生物学功能，因此具有靶向能力及快速响应的次氯酸的比率型荧光探针仍是挑战。
发明内容
为克服现有技术的不足，本发明旨在提供一种溶酶体靶向的比率型次氯酸荧光探针。
本发明中的荧光探针，其分子结构如下：
本发明中的荧光探针反应条件温和、成本较低，通过以下合成路线制备：
本发明中的荧光探针检测机制如下：
荧光探针本身发射红色荧光，与次氯酸反应后，酚噻嗪上的硫原子被氧化为亚砜，探针的荧光从红色变为绿色，因此探针可以实现对次氯酸的检测。
本发明的荧光探针灵敏度高。荧光探针与次氯酸的荧光响应测试在PBS缓冲液(pH＝7.4，10mM，1％CTAB)中进行。探针本身在610nm处发射红色荧光，当次氯酸加入后，535nm处有绿色荧光产成(激发波长为440nm)，且随次氯酸浓度的增加，535nm处的荧光强度与610nm处的荧光强度的比值明显增大，当次氯酸的浓度达到800μM时，I535/I610达到最大，与空白探针溶液相比增强了73倍。且I535/I610的值与浓度在0-700μM范围内的次氯酸有很好的线性关系，线性相关系数是0.9912。根据信噪比S/N＝3，计算出探针的检测限为0.58μM。
本发明的荧光探针选择性好。在PBS缓冲液(pH＝7.4，10mM，1％CTAB)中，探针分子本身发射红色荧光，在加入100倍当量次氯酸之后，I535/I610增大了73倍。而加入阴离子(HS-，SO3 2-，S2O3 2-)，阳离子(Na+，K+，Mg2+，Ca2+)，生物硫醇(GSH，Hcy，Cys)以及活性氧(H2O2，1O2，·OH，·OtBu，ROO·，ONOO-，NO)后，I535/I610基本上保持不变。
本发明的荧光探针抗干扰能力强。在其他检测物(Na+，K+，Mg2+，Ca2+，HS-，SO3 2-，S2O3 2-，GSH，Hcy，Cys，H2O2，1O2，·OH，·OtBu，ROO·，ONOO-，NO)存在的情况下不影响检测次氯酸的效果。
本发明的荧光探针响应速度快。在与次氯酸作用后，荧光强度立即发生改变，I535/I610在1min内即可达到平衡。
本发明的荧光探针具有较低的细胞毒性。RAW264.7细胞与探针(0μM，5μM，10μM，15μM，20μM)在37℃培育24h后，存活率均在95％以上。
本发明的荧光探针能靶向溶酶体。通过探针(0.2mM)与溶酶体标记探针Lyso-Tracker Red(0.2mM)共染色RAW264.7细胞的荧光成像，分析得到绿色通道(探针的荧光)和蓝色通道(Lyso-Tracker Red的荧光)的皮尔逊相关系数为0.77。
附图说明
图1为荧光探针的合成路线。
图2为荧光探针(10.0μM)响应不同浓度次氯酸的紫外-可见吸收光谱。横坐标为波长，纵坐标为吸光度。
图3(a)为本发明的荧光探针(10.0μM)在PBS缓冲溶液(10mM，pH＝7.4，1.0mMCTAB)中与不同浓度次氯酸作用后荧光光谱变化，横坐标为波长，纵坐标为荧光强度。(b)荧光探针(10.0μM)与不同浓度次氯酸作用后，I535/I610值的变化，横坐标为浓度，纵坐标为荧光强度比值I535/I610。
图4为本发明的荧光探针(10.0μM)在PBS缓冲溶液(10mM，pH＝7.4，1.0mM CTAB)中与次氯酸作用过程中I535/I610随浓度变化的线性关系，横坐标为浓度，纵坐标为荧光强度比值I535/I610。
图5为本发明的荧光探针(10.0μM)在不同pH的PBS缓冲溶液(10mM,1.0mM CTAB)中，探针与次氯酸作用前后的荧光强度比值的变化，横坐标为波长，纵坐标为荧光强度比值I535/I610。
图6为本发明的荧光探针(10.0μM)在PBS缓冲溶液(10mM，pH＝7.4，1.0mM CTAB)中分别与100倍当量的次氯酸以及其他离子((0)ClO-，(1)Na+，(2)K+，(3)Mg2+，(4)Ca2+，(5)HS-，(6)SO3 2-，(7)S2O3 2-，(8)GSH，(9)Hcy，(10)Cys，(11)H2O2，(12)1O2，(13)·OH，(14)·OtBu，(15)ROO·，(16)ONOO-，(17)NO)作用后荧光强度比值的柱状图，横坐标为不同检测物，纵坐标为荧光强度比值I535/I610。
图7为本发明的荧光探针(10.0μM)在其他干扰物((0)ClO-，(1)Na+，(2)K+，(3)Mg2+，(4)Ca2+，(5)HS-，(6)SO3 2-，(7)S2O3 2-，(8)GSH，(9)Hcy，(10)Cys，(11)H2O2，(12)1O2，(13)·OH，(14)·OtBu，(15)ROO·，(16)ONOO-，(17)NO)存在下与100倍当量次氯酸作用后荧光强度比值柱状图。测试体系为PBS缓冲溶液(10mM，pH＝7.4，1.0mM CTAB)，横坐标为不同检测物，纵坐标为荧光强度比值I535/I610。
图8为本发明的荧光探针(10.0μM)在PBS缓冲溶液(10mM，pH＝7.4，1.0mM CTAB)中分别与不同浓度次氯酸(0μM,200μM,400μM,600μM,1000μM)作用过程中，I535/I610随时间的变化，横坐标为时间，纵坐标为荧光强度比值I535/I610。
图9为本发明的荧光探针检测RAW264.7细胞内次氯酸的共聚焦细胞成像。A1-A4为在37℃时用探针(10.0μM)培育细胞20min的成像效果。B1-B4为在37℃时，先用次氯酸(1.0mM)培育细胞20min，随后用探针(10.0μM)培育细胞30min的成像效果。C1-C4在37℃时将细胞先用LPS(2mg/mL)和PMA(2mg/mL)培育2h,再用探针(10.0μM)培育30min的成像效果。
图10为本发明的荧光探针对细胞的毒性试验。横坐标为探针浓度即5.0×10-6mol/L、1.0×10-5mol/L、1.5×10-5mol/L、2.0×10-5mol/L，纵坐标为细胞的成活率。
图11为本发明的荧光探针(2.0μM)与溶酶体标记探针Lyso-Tracker Red(0.2μM)共染色RAW264.7细胞的溶酶体靶向荧光成像。(a)探针的绿色通道。(b)Lyso-Tracker Red染料的红色通道。(c)叠加了绿色和红色通道。(d)明场图像。(e)绿色荧光通道与红色荧光通道的相关性。
具体实施实例
实施例1：化合物2的合成
化合物1(2.306g，10.0mmol)和正丁基溴(2.765g，20mmol)溶解于25mL无水二甲基亚砜中，再加入氢氧化钠(0.800g，20.0mmol)和催化量的碘化钾，100℃，回流6h，待反应完成后，冷却到室温，用二氯甲烷萃取三次，减压旋转蒸干溶剂，得到粗产品，用柱层析分离纯化(石油醚/二氯甲烷＝2/1，v/v)得到无色油状液体产品2。产量：2.5935g；产率：95.0％。
实施例2：化合物3的合成
在氩气保护下，将N,N-二甲基甲酰胺(0.51mL，7.2mmol)缓慢加入三氯氧磷(0.6mL，7.2mmol)中，冰水浴搅拌15min，将中间体2(1.6767g，6mmol)溶于无水N,N-二甲基甲酰胺中，再加入到上述溶液中，60℃回流4h，反应完毕后，将混合物倒入50mL冰水中，然后用10％氢氧化钠溶液中和，用二氯甲烷萃取三次，有机相用无水硫酸钠干燥，减压旋转蒸干溶剂，得到粗产品，用柱层析分离纯化得到黄色固体产品3。产量：1.3146g；产率：70.1％。
实施例3：化合物4的合成
将铝粉(0.2025g，3.6mmol)加入到无水乙腈(15mL)中，并在室温搅拌5min。向该混合液中分批添加碘(0.0306g，2.55mmol)，并在氮气保护下搅拌。将化合物3(0.9530g，3.0mmol)溶于无水乙腈(15mL)，滴加至该混合物中。将得到的悬浮液回流6h。冷却至室温后，将混合物用100mL乙酸乙酯萃取3次。有机相用无水硫酸钠干燥，减压旋转蒸干溶剂，得到粗产品，用柱层析分离纯化(石油醚/二氯甲烷＝1/1，v/v)得到黄色油状液体产品4。产量：0.7891g；产率：87.3％。
实施例4：化合物5的合成
化合物4(0.6130g，2.1mmol)，丙二酸二乙酯(0.8011g，5.0mmol)溶于15mL无水乙醇中，再加入哌啶(400μL，4.1mmol)和乙酸(200μL，3.5mmol)，室温下搅拌7h，将反应液减压旋转蒸干溶剂，得到粗产品，用柱层析分离纯化得到黄色固体产品5。产量：0.4859g；产率：60.5％。
实施例5：化合物6的合成
称取化合物5(0.2504g，0.6mmol)，氢氧化钠(0.0760g，2.0mmol)于15mL无水甲醇中，回流0.5h，将反应液减压旋转蒸干溶剂，溶解在50mL二氯甲烷中，用10％HCl溶液中和，用水洗涤后，有机层经无水硫酸钠干燥，减压旋转蒸干溶剂并进一步的真空干燥，得到牛血红色固体产品6。产量：0.2663g；产率：90.1％。
实施例6：探针的合成
化合物6(0.0993g，0.3mmol)，2-吗啉乙醇(0.07861g，0.6mmol)溶于10mL二氯甲烷中，再加入1-乙基-(3-二甲氧基)碳二亚胺盐酸盐(0.0635g，0.28mmol)和4-二甲氧基吡啶(0.0640g，0.28mmol)，室温下反5h，将反应液减压旋转蒸干溶剂，得到粗产品，用柱层析分离纯化(乙醇/二氯甲烷＝1:10，v/v)得到牛血红色固体产品，即探针分子(0.0745g，57％)。HRMS(ESI)m/z:calcd for C26H28N2O5S[M+H]+,481.1719；found,481.1782.1H NMR(400MHz,CDCl3)δ8.31(s,1H),7.13(d,J＝15.8Hz,2H),7.05(d,J＝6.3Hz,1H),6.95(t,J＝7.2Hz,1H),6.87(d,J＝8.1Hz,1H),6.59(s,1H),4.42(t,J＝5.9Hz,2H),3.80(t,2H),3.68(t,4H),2.73(t,J＝5.9Hz,2H),2.54(t,4H),1.75(m,J＝7.5Hz,2H),1.42(m,J＝7.3Hz,2H),0.92(t,J＝7.4Hz,3H).13C NMR(100MHz,CDCl3)δ163.4,157.1,156.6,151.0,148.0,142.2,127.8,127.4,126.1,124.0,122.9,121.0,116.2,113.1,112.7,101.9,66.9,62.8,58.2,57.0,53.9,48.3,28.4,20.0,18.4,13.7.
实施例7：探针分子在细胞中对外源性次氯酸的检测
RAW264.7细胞首先用含次氯酸(1.0mM)的PBS缓冲液(10.0mM，pH＝7.4)孵育20min，用PBS缓冲液漂洗3次；再与含探针(10.0μM)的PBS缓冲液孵育30min，用PBS缓冲液漂洗3次后，用激光共聚焦荧光显微镜进行细胞荧光成像，可以看到强烈的绿色荧光信号。在对照实验中，RAW264.7细胞仅用含探针(10.0μM)的PBS缓冲液(10.0mM，pH＝7.4)孵育20min，用PBS缓冲液漂洗后，用激光共聚焦荧光显微镜进行细胞荧光成像，可以观察到极微弱的绿色荧光信号和较强的红色荧光信号。
实施例8：探针分子在细胞中对内源性次氯酸的检测
先将RAW264.7细胞分别用LPS(2mg/mL)和PMA(2mg/mL)培育2h，用PBS缓冲液漂洗3次；再与含探针(10.0μM)的PBS缓冲液孵育30min，用PBS缓冲液漂洗3次后，用激光共聚焦荧光显微镜进行细胞荧光成像，可以观察到强烈的绿色荧光信号和微弱的红色荧光信号。
实施例9：探针分子对溶酶体的靶向能力
RAW264.7细胞首先用含Lyso-Tracker Red(0.2mM)的PBS缓冲(10.0mM，pH＝7.4)孵育20min，用PBS缓冲液漂洗3次；再与含探针(10.0μM)的PBS缓冲液孵育30min，用PBS缓冲液漂洗3次后，用激光共聚焦荧光显微镜进行细胞荧光成像，可以观察到的绿色荧光信号和红色荧光信号有较好的重叠。
Claims (1)
1.一种溶酶体靶向的比率型次氯酸荧光探针，其特征在于，其结构式为：
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