CN109053790A - 一种溶酶体靶向的次氯酸近红外荧光探针及其制备方法和应用 - Google Patents
一种溶酶体靶向的次氯酸近红外荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种溶酶体靶向的次氯酸近红外荧光探针及其制备方法和应用,属于分析化学技术领域。本发明的技术方案要点为:一种溶酶体靶向的次氯酸近红外荧光探针,其结构式为:本发明还具体公开了该溶酶体靶向的次氯酸近红外荧光探针的制备方法及其在水环境或生物细胞体系检测中的应用。本发明的荧光探针具有近红外发射、灵敏度高、选择性好及响应迅速等特点。
Description
技术领域
本发明属于分析化学技术领域,具体涉及一种溶酶体靶向的次氯酸近红外荧光探针及其制备方法和应用。
背景技术
活性氧(ROS)与各种生理和病理进程有关,如病原体反应进程、机体衰老进程以及抵抗炎症进程。次氯酸(HClO)是活性氧中一种高效的氧化剂,通常在细胞吞噬和炎症反应过程中产生,在先天免疫***中起着重要作用。次氯酸主要分布在吞噬细胞的酸性溶酶体中,通常由在吞噬酶体中的髓过氧化物酶(MPO)催化的氯离子过氧化产生。有证据表明,生命体内错位或过量的HClO浓度表达与多种疾病有关,如神经变性、关节炎和动脉粥样硬化。例如,HClO在溶酶体中的异常积累可诱发慢性疾病,这是因为过量的次氯酸会诱导溶酶体的破裂而导致细胞死亡。此外,近来溶酶体被认为是选择性杀伤癌细胞的有力靶点,可以作为一种新型的抗癌途径(溶酶体靶向抗癌,LCD)。在诱导LCD的各种方法中,活性氧(ROS)是最常见的。然而,关于包括HClO在内的ROS诱导的癌细胞LCD的细节仍知之甚少。因此,实时监测及影像癌细胞溶酶体中HClO对于阐明LCD相关的抗癌机制和评价新的抗癌药物具有重要意义。总之,开发合适的化学工具来直接实时监测溶酶体中的次氯酸显得尤为必要。
近年来,次氯酸荧光探针的研制取得了显著进展,其中一些已成功地应用于溶酶体中次氯酸的实时检测和成像。然而,由于这些探针的短波长激发和发射(一般为λex<600nm和λem<650nm)而限制了它们在体内的实际应用,这也引发了许多问题,包括体内自发荧光的干扰、成像试剂的光漂白、对生物样品的光损伤现象。近红外荧光探针(λex>600nm和λem>650nm)能较好地解决上述问题,使生物样品的光损伤最小、组织穿透深度增加及降低背景自体荧光的干扰。然而,据我们所知,目前还没有检测溶酶体中HClO的近红外荧光探针的相关报道,导致在组织或活体中溶酶体次氯酸检测成像方面存在一定的局限性。
发明内容
针对目前还没有溶酶体靶向近红外次氯酸荧光探针报道的现状以及溶酶体内次氯酸检测所面临的问题,本发明提供了一种溶酶体靶向的次氯酸近红外荧光探针,该荧光探针具有近红外发射、灵敏度高、选择性好及响应迅速等特点。
本发明还提供了上述溶酶体靶向的近红外次氯酸荧光探针的制备方法及其在水环境或生物细胞体系检测中的应用。
本发明为解决上述技术问题采用如下技术方案,一种溶酶体靶向的次氯酸近红外荧光探针,其特征在于该荧光探针的结构式如下:
本发明所述的溶酶体靶向的次氯酸近红外荧光探针的制备方法,其特征在于具体步骤为:
步骤S1:于-78℃,在氩气保护下,将6克3-溴-N,N-二甲基苯胺与60毫升无水***加入至干燥的250毫升圆底烧瓶中,磁力搅拌5分钟使其溶解,随后将13.1毫升摩尔浓度为2.4mol/L的正丁基锂的正己烷溶液滴加至反应液中,滴加完毕后于0℃反应2小时,再将2.2毫升二氯二甲基硅烷溶于10毫升无水***中,然后滴加至上述反应液中,滴加完毕后反应至室温并搅拌过夜,加50毫升水猝灭反应,并将反应液用***萃取、用水洗涤后,用饱和NaCl水溶液洗涤,无水硫酸钠干燥,减压旋干溶剂后得到粗产品,将粗产品用硅胶柱纯化得到化合物1,其结构式如下:
步骤S2:将500mg化合物1、1260mg 2-羧基苯甲醛和37.5mg溴化铜加入到100毫升玻璃厚壁耐压管中,于140℃加热搅拌反应5小时后自然冷却至室温,然后将反应混合物溶于50毫升二氯甲烷中,用质量浓度为10%的NaOH溶液洗涤三遍,回收并旋干二氯甲烷相得到粗产品,将粗产品用硅胶柱纯化得到化合物2,其结构式如下:
步骤S3:将428.6mg化合物2、50毫升乙醇和2毫升质量浓度为85%的水合肼加入至100毫升的圆底烧瓶,于78℃反应4小时后将反应液减压蒸馏除去溶剂,将残余物用50毫升二氯甲烷溶解后,用饱和食盐水水洗二氯甲烷相,并用无水硫酸钠干燥,减压蒸馏除去二氯甲烷相得到粗产物,将粗产物用硅胶柱纯化得到化合物3,其结构式如下:
步骤S4:将221.3mg化合物3、15毫升DMF和2毫升3-(4-吗啉基)异硫氰酸丙酯加入至50毫升的圆底烧瓶,于80℃搅拌反应72小时,将反应液减压蒸馏除去溶剂后得到残余物,饱和NaCl水溶液洗涤,无水Na2SO4干燥,再将残余物用硅胶柱纯化得到目标荧光探针化合物Lyso-NIR-HClO。
本发明所述的溶酶体靶向的次氯酸近红外荧光探针在水环境或生物细胞体系选择性检测次氯酸中的应用,其中检测包括水溶液中荧光检测、细胞成像检测、组织成像检测。
本发明与现有技术相比具有以下有益效果:(1)该荧光探针的合成相对比较容易,且后处理过程相对简单;(2)该荧光探针实现了次氯酸分子高选择性高灵敏度快速检测,具有抵抗生命体内其它分子干扰的能力;(3)该荧光探针可以应用于细胞溶酶体中次氯酸的检测,通过与商业化溶酶体染料进行比较,发现此荧光探针对溶酶体具有较高的溶酶体靶向能力,两种染料的共定位系数为0.93,说明此荧光探针可以作为细胞溶酶体内次氯酸的检测探针;(4)该荧光探针具有近红外发射,能够应用于组织或者活体中次氯酸的成像检测,通过降低生命体内自发荧光背景干扰、降低对生物样品的光损伤、提高组织穿透深度等特点,以获得更加准确及稳定的光学信号及成像效果。故而,本发明中的荧光探针在次氯酸检测领域具有广阔的应用前景,对次氯酸在生物体生理和病理过程的作用机制以及溶酶体在炎症反应中的作用等研究具有重要意义。
附图说明
图1是实施例1制得的荧光探针化合物Lyso-NIR-HClO在加入不同浓度次氯酸后的荧光光谱图;
图2是实施例1制得的荧光探针化合物Lyso-NIR-HClO在加入不同浓度次氯酸后的紫外可见吸收光谱图;
图3是实施例1制得的荧光探针化合物Lyso-NIR-HClO在发射波长为680nm处的的荧光强度随次氯酸浓度(0-40μM)变化的关系曲线图,插图为荧光探针化合物Lyso-NIR-HClO在发射波长为680nm处的的荧光强度随次氯酸浓度(0-10μM)变化的关系曲线图;
图4是实施例1制得的荧光探针化合物Lyso-NIR-HClO对不同离子和分子的选择性柱状图,其中1、PBS;2、K+;3、Ca2+;4、Mg2+;5、Zn2+;6、Fe3+;7、Cu2+;8、CH3COO-;9、NO3 -;10、Cl-;11、F-;12、I-;13、Cys;14、GSH;15、Glucose;16、ATP;17、ADP;18、t-BuOO.;19、NO2 -;20、H2O2;21、ONOO-;22、O2 .-;23、.OH;24、1O2;25、NO;26、t-BuOOH;27、NaClO;
图5是pH对实施例1制得的荧光探针化合物Lyso-NIR-HClO检测次氯酸的影响图;
图6是实施例1制得的荧光探针化合物Lyso-NIR-HClO在体系中含有不同浓度的次氯酸(1μM,5μM,10μM)时,溶液在680nm处的荧光强度随时间变化关系曲线图;
图7是在HeLa细胞中实施例1制得的荧光探针化合物Lyso-NIR-HClO检测外源性次氯酸荧光成像图与商业化溶酶体定位染料LysoSensorTM Green DND-189的共定位成像图,其中a)探针浓度为5μM与LysoSensorTM Green DND-189(1μM)加入到HeLa细胞中培养30分钟后明场图,b)商业染料LysoSensorTM Green DND-189绿色通道荧光成像图,c)加入次氯酸后探针分子红色通道荧光成像图,d)绿色通道与红色通道叠加图,e)图d中粉红色椭圆内区域的绿色通道与红色通道叠加强度散点图;f)图d中粉红色直线绿色通道与红色通道叠加荧光强度比较;
图8是实施例1制得的荧光探针化合物Lyso-NIR-HClO检测HeLa中不同浓度外源性次氯酸(0μM,10μM,20μM)荧光成像图;
图9是实施例1制得的荧光探针化合物Lyso-NIR-HClO检测小鼠肝脏组织中外源性次氯酸(200μM)荧光成像图,其中a)与c)探针浓度为20μM,未加次氯酸;b)与d)探针浓度为20μM,加200μM次氯酸,成像深度均为38μm。
具体实施方式
以下通过实施例对本发明的上述内容做进一步详细说明,但不应该将此理解为本发明上述主题的范围仅限于以下的实施例,凡基于本发明上述内容实现的技术均属于本发明的范围。
实施例1
荧光探针化合物Lyso-NIR-HClO的合成
(1)化合物1的合成
于-78℃,在氩气保护下,将6克3-溴-N,N-二甲基苯胺与60毫升无水***加入至干燥的250毫升圆底烧瓶中,磁力搅拌5分钟使其溶解,随后将13.1毫升摩尔浓度为2.4mol/L的正丁基锂的正己烷溶液缓慢滴加至反应液中,滴加完毕后于0℃反应2小时,再将2.2毫升二氯二甲基硅烷溶于10毫升无水***中,然后缓慢滴加至上述反应液中,滴加完毕后反应至室温搅拌过夜,加50毫升水猝灭反应,并将反应液用***萃取,分液漏斗分取有机相,用水洗涤后(50毫升×2),饱和NaCl水溶液洗涤(50毫升×1),无水Na2SO4干燥,旋转蒸发仪蒸除溶剂得粗产品,将粗产品用硅胶柱纯化,硅胶颗粒大小为200-300目,洗脱剂体积配比为石油醚/乙酸乙酯=80:1,得到化合物1,黄色油状物,3.35g,产率75%,其合成路线如下:
(2)化合物2的合成
将500mg化合物1、1260mg 2-羧基苯甲醛和37.5mg溴化铜加入到100毫升玻璃厚壁耐压管中,封管后放置到油浴锅中于140℃加热搅拌5小时后自然冷却至室温,然后将反应混合物溶解在50毫升二氯甲烷中,用质量浓度为10%的NaOH溶液洗涤(50毫升×3),除去未反应的2-羧基苯甲醛等酸性副产物,将得到的二氯甲烷相用无水Na2SO4干燥,旋转蒸发仪蒸除溶剂得粗产品,将粗产品用硅胶柱纯化,硅胶颗粒大小为200-300目,洗脱剂体积配比为石油醚/乙酸乙酯=2:1,得到化合物2,绿色固体,0.33g,产率45%,其合成路线如下:
(3)化合物3的合成
将428.6mg化合物2、50毫升乙醇和2毫升质量浓度为85%的水合肼加入至100毫升的圆底烧瓶,于78℃反应4小时后减压除去溶剂得残余物,用50毫升CH2Cl2溶解残余物,饱和NaCl水溶液洗涤(50毫升×3),无水Na2SO4干燥,旋转蒸发仪蒸除溶剂得粗产品,将粗产品用硅胶柱纯化,硅胶颗粒大小为200-300目,洗脱剂体积配比为石油醚/乙酸乙酯=1:3,得到化合物3,象牙白固体化合物,250.2mg,产率56.5%。1H NMR(400MHz,CDCl3)δ7.93-7.89(m,1H),7.34-7.29(m,2H),6.91-6.90(m,2H),6.89-6.87(m,1H),6.76(s,1H),6.73(s,1H),6.66-6.65(d,J=3.2Hz,1H),6.64-6.63(d,J=2.8Hz,1H),3.69(s,2H),2.96(s,12H),0.61(s,3H),0.59(s,3H).HRMS(ESI):calcd for[M+H]+443.2262,found 443.2264,其合成路线如下:
(4)荧光探针化合物Lyso-NIR-HClO的合成
将化合物3(221.3mg)、15毫升DMF和2毫升3-(4-吗啉基)异硫氰酸丙酯加入至50毫升的圆底烧瓶,于80℃搅拌72小时,在减压条件下除去溶剂后用50毫升CH2Cl2溶解残余物,饱和NaCl水溶液洗涤(50毫升×3),无水Na2SO4干燥,旋转蒸发仪蒸除溶剂得粗产品,将粗产品用硅胶柱纯化,硅胶颗粒大小为200-300目,洗脱剂体积配比为二氯甲/甲醇=15:1,得到荧光探针化合物Lyso-NIR-HClO,淡黄色固体,201.0mg,产率31.9%。1H NMR(600MHz,CDCl3)δ8.00-7.99(d,J=7.2Hz,1H),7.62-7.60(m,1H),7.58-7.55(m,1H),7.15-7.14(d,J=7.2Hz,1H),6.85(d,J=1.8Hz,2H),6.60(s,1H),6.57(d,J=1.8Hz,1H),6.56(d,J=1.8Hz,1H),6.50-6.48(m,2H),5.77-5.75(m,1H),3.64(m,4H),3.22-3.18(m,2H),2.97(s,12H),2.32(s,4H),2.16-2.14(m,2H),1.31-1.26(m,2H),0.57(s,3H),0.54(s,3H).HRMS(ESI):[M+H]+calcd 629.3088,found 629.3083,其合成路线如下:
实施例2
荧光探针化合物Lyso-NIR-HClO与不同浓度次氯酸作用的荧光光谱图的测定
取实施例1制备的荧光探针化合物Lyso-NIR-HClO溶于N,N-二甲基甲酰胺(DMF)中制成10μM的储备液,从储备液中取出2毫升加入到5毫升的离心管当中,加入不同当量(0-8)的次氯酸标准溶液,用PBS缓冲溶液(10mM,pH=7.4)的溶液稀释至4毫升(DMF/PBS体积比为1:1),以620nm为激发光,狭缝宽度设置为5nm/5nm,测量其荧光光谱。荧光光谱如图1所示,随着次氯酸加入,在680nm处的荧光逐渐增强,其荧光强度与次氯酸浓度的关系如图3所示,在5.0×10-8-1.0×10-5mol/L的范围内呈线性关系,所用的荧光测定仪器为Perkin ElmerLS55荧光分光光度计。
实施例3
荧光探针化合物Lyso-NIR-HClO与不同浓度次氯酸作用的紫外可见吸收光谱图的测定
图2为实施例1制得的荧光探针化合物Lyso-NIR-HClO与不同浓度的次氯酸作用后的紫外可见吸收光谱图,次氯酸的加入量为0-40μM。从图中2可以看出,在660nm处可以观察到一个吸收峰,随次氯酸浓度的增加,其660nm处的吸光度逐渐增加。紫外可见吸收光谱测定用的仪器为TU-1900型紫外可见分光光度计(Beijing Purkinje General InstrumentCo.,Ltd.)。
实施例4
荧光探针化合物Lyso-NIR-HClO对不同分子或离子的选择性
从实施例2中的荧光探针储备液中取出2毫升加入到5毫升的离心管当中,加入待考察竞争物质的标准溶液,用PBS缓冲溶液(10mM,pH=7.4)的溶液稀释至4毫升(DMF/PBS体积比为1:1),共考察了26种竞争物质,共配置27个样品,最后一个为次氯酸。以620nm为激发光,狭缝宽度设置为5nm/5nm,测量其荧光光谱,结果如图4所示。由图4可以发现,细胞内金属离子、阴离子、中性分子、活性氮、其他活性氧对荧光探针化合物Lyso-NIR-HClO在680nm处的荧光几乎没有影响,而次氯酸溶液的加入使探针Lyso-NIR-HClO在680nm处的荧光显著增强。
实施例5
溶液pH值对荧光探针化合物Lyso-NIR-HClO测定次氯酸的荧光响应的影响
我们分别考察了pH值对空白荧光探针化合物Lyso-NIR-HClO以及Lyso-NIR-HClO+次氯酸(30μM)的两种情况下荧光强度的影响,结果如图5。在pH=3.5-8.0范围内,pH对空白荧光探针化合物Lyso-NIR-HClO的荧光强度基本无影响。而在次氯酸(30μM)存在的情况下,当pH=5.0时,荧光探针化合物Lyso-NIR-HClO的荧光强度最大,表明此时荧光探针对次氯酸的响应最好。而细胞溶酶体的pH处于4.5-5.5,从图5中可知,在pH处于4.5-5.5范围内,荧光探针化合物Lyso-NIR-HClO对次氯酸都显示出较好的响应,表明荧光探针化合物Lyso-NIR-HClO能够满足溶酶体内次氯酸检测的需求。
实施例6
荧光探针化合物Lyso-NIR-HClO与次氯酸作用的响应时间的测定
在荧光探针化合物Lyso-NIR-HClO溶液中分别加入1μM、5μM、10μM的次氯酸,配制成3个样品,在Perkin Elmer LS 55荧光分光光度计的时间模式下,分别测定含不同浓度的次氯酸样品在680nm处的荧光强度随时间的变化情况,结果如图6。
实施例7
荧光探针化合物Lyso-NIR-HClO与商业化溶酶体染料LysoSensorTM Green DND-189的溶酶体共定位实验
在HeLa细胞中,将荧光探针化合物Lyso-NIR-HClO与商业化的溶酶体染料LysoSensorTM Green DND-189进行共定位实验,说明荧光探针化合物Lyso-NIR-HClO可以定位到溶酶体中,并对溶酶体内的外源性的次氯酸进行荧光成像应用(如图7所示)。具体操作步骤如下:将荧光探针化合物Lyso-NIR-HClO的DMSO溶液和商业化溶酶体染料加入到育有HeLa细胞的培养液中,两者终浓度分别为5μM与1μM。在二氧化碳培养箱中培养30分钟后,向体系中加入一定量次氯酸水溶液,使体系次氯酸浓度为20μM,等待20分钟后用共聚焦显微镜进行成像,同时用405nm与635nm激发,在绿色通道可以观察到有绿色荧光(480-525nm)发出,此为LysoSensorTM Green DND-189发射的光,而在红色通道可以观察到有红色荧光(650-720nm)发出,此为荧光探针化合物Lyso-NIR-HClO与次氯酸响应后发出的红光,将两者用软件进行处理可以得出两种染料的共定位系数为0.93。
实施例8
荧光探针化合物Lyso-NIR-HClO对细胞外源性次氯酸的荧光成像
在HeLa细胞中,考察荧光探针化合物Lyso-NIR-HClO对外源性的次氯酸的荧光成像情况。具体操作步骤如下:将荧光探针化合物Lyso-NIR-HClO的DMSO溶液加入到育有HeLa细胞的培养液中,终浓度为5μM。在二氧化碳培养箱中培养30分钟后,向体系中加入不同量次氯酸水溶液,使体系次氯酸浓度分别为0μM、10μM、20μM,等待20分钟后用共聚焦显微镜进行成像,成像结果如图8所示。从图中可知,随着次氯酸浓度的增加,细胞红色通道的荧光强度逐渐增加,激发波长为635nm,红色通道波长收集范围为650-720nm。
实施例9
荧光探针化合物Lyso-NIR-HClO在组织中的应用
近红外的探针具有体内荧光背景低,组织穿透性强的优点。分别在两个组织样品中加入20μM的荧光探针化合物Lyso-NIR-HClO,其中第一个样品只加荧光探针化合物(图a与图c),另一个继续加入终浓度为200μM次氯酸(图b与图d),如图9所示。图a与图b是两个小鼠肝冷冻切片样品的荧光成像明场图(成像深度为38μm),图c与图d是两个小鼠肝冷冻切片样品的荧光成像荧光通道图(成像深度为38μm)。从图可以看出,荧光探针化合物Lyso-NIR-HClO具有较好的组织穿透能力,能够检测影像组织中的次氯酸。
以上实施例描述了本发明的基本原理、主要特征及优点,本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明原理的范围下,本发明还会有各种变化和改进,这些变化和改进均落入本发明保护的范围内。
Claims (3)
1.一种溶酶体靶向的次氯酸近红外荧光探针,其特征在于该荧光探针的结构式如下:
2.一种权利要求1所述的溶酶体靶向的次氯酸近红外荧光探针的制备方法,其特征在于具体步骤为:
步骤S1:于-78℃,在氩气保护下,将6克3-溴-N,N-二甲基苯胺与60毫升无水***加入至干燥的250毫升圆底烧瓶中,磁力搅拌5分钟使其溶解,随后将13.1毫升摩尔浓度为2.4mol/L的正丁基锂的正己烷溶液滴加至反应液中,滴加完毕后于0℃反应2小时,再将2.2毫升二氯二甲基硅烷溶于10毫升无水***中,然后滴加至上述反应液中,滴加完毕后反应至室温并搅拌过夜,加50毫升水猝灭反应,并将反应液用***萃取、用水洗涤后,用饱和NaCl水溶液洗涤,无水硫酸钠干燥,减压旋干溶剂后得到粗产品,将粗产品用硅胶柱纯化得到化合物1,其结构式如下:
步骤S2:将500mg化合物1、1260mg 2-羧基苯甲醛和37.5mg溴化铜加入到100毫升玻璃厚壁耐压管中,于140℃加热搅拌反应5小时后自然冷却至室温,然后将反应混合物溶于二氯甲烷中,用质量浓度为10%的NaOH溶液洗涤三遍,回收并旋干二氯甲烷相得到粗产品,将粗产品用硅胶柱纯化得到化合物2,其结构式如下:
步骤S3:将428.6mg化合物2、50毫升乙醇和2毫升质量浓度为85%的水合肼加入至100毫升的圆底烧瓶,于78℃反应4小时后将反应液减压蒸馏除去溶剂,将残余物用50毫升二氯甲烷溶解后,用饱和食盐水水洗二氯甲烷相,并用无水硫酸钠干燥,减压蒸馏除去二氯甲烷相得到粗产物,将粗产物用硅胶柱纯化得到化合物3,其结构式如下:
步骤S4:将221.3mg化合物3、15毫升DMF和2毫升3-(4-吗啉基)异硫氰酸丙酯加入至50毫升的圆底烧瓶,于80℃搅拌反应72小时,将反应液减压蒸馏除去溶剂后得到残余物,饱和NaCl水溶液洗涤,无水Na2SO4干燥,再将残余物用硅胶柱纯化得到目标荧光探针化合物Lyso-NIR-HClO。
3.权利要求1所述的溶酶体靶向的次氯酸近红外荧光探针在水环境或生物细胞体系选择性检测次氯酸中的应用,其中检测包括水溶液中荧光检测、细胞成像检测、组织成像检测。
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