CN117586247B - 一种具有粘度和半胱氨酸特异性响应的线粒体靶向近红外荧光探针及其制备方法和应用 - Google Patents
一种具有粘度和半胱氨酸特异性响应的线粒体靶向近红外荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开一种具有粘度和半胱氨酸特异性响应的线粒体靶向近红外荧光探针及其制备方法和应用,属于荧光探针技术领域。探针的分子式为C37H38N5O4S+,结构式如下:。在本发明中,基于扭曲分子内电荷转移TICT和替代重排传感机制的选择性,设计了一种线粒体靶向的近红外探针NVCP,并表现出较大的斯托克斯位移、化学稳定性、优良的生物相容性和精确的线粒体定位。基于上述优点,NVCP可以敏感地研究活细胞中线粒体粘度和Cys的动态变化。
Description
技术领域
本发明属于荧光探针技术领域,具体涉及一种具有粘度和半胱氨酸特异性响应的线粒体靶向近红外荧光探针及其制备方法和应用。
背景技术
缺血性中风(IS)以脑局部缺血缺氧为特征,导致神经功能缺损和残疾,随着全球人口老龄化,已成为一个重要的公共卫生问题。早期识别IS是改善及时干预和预后的关键。线粒体功能障碍是缺血后对葡萄糖和缺氧最直接的反应,与IS后的早期事件密切相关,包括活性氧(ROS)介导的氧化应激、N-甲基-D-天冬氨酸(NMDA)和谷氨酸诱导的兴奋性毒性。大量研究证明,维持线粒体功能对神经元的活动和生存至关重要。
粘度是微环境中反映蛋白质、脂质、多糖等物质的流动状态的一个重要参数,对细胞内生物分子之间的信号传递和相互作用具有重要意义。线粒体作为细胞器的能量供应细胞的有特定粘度(63cp),而异常线粒体粘度可能影响执行功能的线粒体粘度的增加减少电子传递链的活动,紧随其后的是凋亡和自噬。粘度异常粘度与疾病密切相关,如阿尔茨海默病(AD)、糖尿病、癌症和IS。因此,原位监测线粒体粘度的变化对于了解细胞功能的表现和阐明IS的发展机制具有重要意义。
半胱氨酸(Cys)是一种含硫氨基酸,占蛋白质组成的2%。线粒体中的Cys还通过脂肪酸氧化、氨基酸分解、有氧代谢和磷酸化等化学反应参与能量的产生和传递。此外,研究人员发现Cys衍生物N-乙酰-L-半胱氨酸(NAC)具有作为溶栓治疗IS的溶栓药物,这是一种临床常用的抗氧化药物。因此,监测线粒体Cys动态变化的反应性成像工具的发展有助于IS的研究。
荧光成像技术具有操作简单、选择性好、灵敏度高、实时原位分析等优点,已成为生物医学研究中不可缺少的检测方法。在荧光成像技术的基础上,开发了一系列用于成像的荧光探针。然而,目前还没有能同时检测粘度和生物硫醇的近红外探针,这些荧光探针的响应指标主要为pH、硫醇还原酶、金属蛋白酶、一氧化氮、过氧亚硝酸盐离子、Fe2+、H2S和粘度。虽然这些探针为IS荧光成像提供了可视化工具,但大多数都存在长荧光发射波、小斯托克位移和单指标检测等问题。
针对目前的不足,本发明旨在设计和合成一种具有粘度和Cys(NVCP)特异性响应的线粒体靶向近红外荧光探针,用于原位追踪IS过程中粘度和Cys的时空分布。
发明内容
本发明通过分子设计,合成出了一种具有粘度和Cys特异性响应的线粒体靶向近红外荧光探针NVCP,并进一步提供了探针NVCP的制备方法和应用。本发明目标探针,无特别说明均用NVCP替代。
为实现上述技术目的,本发明所采用的技术方案为:
一种具有粘度和半胱氨酸特异性响应的线粒体靶向近红外荧光探针,探针的分子式为C37H38N5O4S+,结构式如下:
一种粘度和半胱氨酸特异性响应的线粒体靶向近红外荧光探针的制备方法,包括以下制备步骤:
(1)将500mg4-甲基喹啉、521mg对氨基苯甲醛和960mgZn(OAc)2混合在5mL乙酸酐中,在氮保护下在150℃下回流5h;冷却至室温后,用饱和碳酸氢钠水溶液和二氯甲烷萃取去除乙酸酐,收集有机相,通过真空蒸馏得到粗产物,以体积比为1:1的CH2Cl2/
石油醚为洗脱液,经色谱层析纯化,得到浅黄色粉末化合物3,化合物3的结构式为:
(2)500mg将化合物3、434mg4-羟基苯乙基溴溶解在10mL乙腈中,在80℃下搅拌过夜;溶剂在减压下浓缩以得到粗产品,粗产物以体积比=5:1的CH2Cl2/CH3OH为洗脱液进行色谱层析纯化,得到紫色粉末NVCD,NVCD的结构式为:
(3)将396mg化合物NVCD、276mg碳酸钾溶解于10mL无水N,N-二甲基甲酰胺中,在室温氮保护下激活30min,然后加入579mg化合物5,加热至55℃过夜反应;所得到的混合物依次用乙酸乙酯和饱和盐水洗涤;用体积比为5:1的CH2Cl2/CH3OH作为洗脱液进行柱层析纯化,得到紫色粉末终产物荧光探针NVCP。
进一步的,步骤(3)化合物5的结构式为:
本发明探针NVCP的合成路线为:
一种粘度和半胱氨酸特异性响应的线粒体靶向近红外荧光探针的应用,利用荧光光谱仪检测水环境中的粘度和Cys;所述探针利用共聚焦荧光显微镜检测离体活细胞环境中的粘度和Cys,以获取中间信息。
本发明荧光探针NVCP,可特异性定位于细胞线粒体。向细胞内加入一定量的莫能菌素,随后加入探针和碧云天商用线粒体定位剂(绿色)共同培养,能检测到探针的荧光信号和线粒体定位剂(绿色)的荧光信号重叠。
本发明荧光探针本身无荧光,与Cys反应后在592nm荧光增强,线性范围为0–400μM,检测限为37nM;与Gly反应在695nm处荧光强度逐渐增强,粘度从1.4cp变化到834cp,表现出22倍的荧光增强,且Log I695 nm与Logη荧光在0.5~2.0之间的曲线图呈直接线性关系,该探针表现出很高的灵敏度;该荧光探针在pH 4.1-6时显示出微弱的荧光发射,而在pH7.0-9.0时表现出显著的荧光增强,这与生理pH范围相一致;该荧光探针对粘度和Cys表现出很好的选择性,不受其他干扰物的影响。
与空白组对比,经N-乙基马来酰亚胺(NEM,硫醇阻断剂)预处理后的细胞荧光信号明显降低,而添加了Cys的NEM细胞的荧光强度明显强于NEM组的细胞,说明NVCP对细胞内Cys肯定敏感。经莫能菌素(Moe)或制霉菌素(Nys)预处理后的细胞比仅加载NVCP的对照组细胞表现出更强的红色荧光信号,这些结果表明了NVCP在活细胞中检测外源性/内源性Cys的敏感性。
有益效果
本发明的一种具有粘度和Cys特异性响应的线粒体靶向近红外荧光探针NVCP在粘度和Cys存在下荧光发生显著变化,可以用于高灵敏度的检测粘度和Cys,同时,并表现出较大的斯托克斯位移、化学稳定性、优良的生物相容性和精确的线粒体定位。更为重要的是,探针NVCP可以敏感地研究离体活细胞中线粒体粘度和Cys的动态变化。
附图说明
图1为本发明实施例1中化合物3的1HNMR图谱;
图2为本发明实施例1中化合物3的13CNMR图谱;
图3为本发明实施例1中化合物3的HRMS图谱;
图4为本发明实施例1中化合物NVCD的1HNMR图谱;
图5为本发明实施例1中化合物NVCD的HRMS图谱;
图6为本发明实施例1中探针NVCP的1HNMR图谱;
图7为本发明实施例1中探针NVCP的13CNMR图谱;
图8为本发明实施例1中探针NVCP的HRMS图谱;
图9为本发明实施例1中探针NVCP与Cys作用前后的紫外可见吸收光谱图,其中,横坐标为波长,纵坐标为紫外吸收强度,黑线为纯探针溶液,红线为加入Cys响应后的溶液;
图10本发明实施例2中探针NVCP随不同浓度Cys的加入荧光谱图的变化情况图,其中横坐标为波长,纵坐标为荧光强度。从下至上,Cys浓度依次为0、10、20、30、50、100、150、200、250、300、400、500μmol/L;
图11为本发明实施例2中探针NVCP与Cys在430nm激发,592nm发射处荧光强度的线性方程图;
图12为本发明实施例2中探针NVCP与Cys在0~100min时间范围内的荧光强度曲线图;
图13为本发明实施例2中探针NVCP不同干扰分析物的选择性和干扰分析物对待测物的竞争性柱状荧光数据图;其中:黑色表示探针对干扰分析物的选择性;红色代表干扰分析物对Cys的竞争性;
图14为本发明实施例2中探针NVCP对Cys及其他分析物的线性荧光响应图;
图15为本发明实施例2中探针NVCP在pH 3~10范围内与Cys(500μM)在592nm处的荧光强度曲线图;黑线为纯探针溶液,红线为加入Cys响应后的溶液;
图16为本发明实施例2中探针NVCP在缓冲液和甘油(Gly)中吸收光谱图;其中,横坐标为波长,纵坐标为紫外吸收强度。黑线为纯探针溶液,红线为加入甘油(Gly)响应后的溶液;
图17为本发明实施例2中探针NVCP在不同粘度的水-甘油混合物中的荧光光谱;其中,横坐标为波长,纵坐标为荧光强度,荧光探针的浓度为10μM,Gly浓度依次为:2.7、3、3.3、3.7、4.5、4.75、5、7.9、10.8、24.3、30、36、45、95、193、396、834cp;
图18为本发明实施例2中探针NVCP与甘油在550nm激发,695nm发射处log I 695与logη之间的线性关系;
图19为本发明实施例2中探针NVCP不同干扰分析物的选择性和干扰分析物对待测物的竞争性柱状荧光数据图;其中:黑色表示探针对干扰分析物的选择性;红色代表干扰分析物对甘油(Gly)的竞争性;
图20为本发明实施例2中探针NVCP在pH为3-10的范围内,与0%、20%、80%Gly响应荧光强度曲线图;
图21为本发明实施例2中探针NVCP在不同溶剂中的吸收光谱图;
图22为本发明实施例2中探针NVCP在不同溶剂中的荧光响应图;
图23为本发明实施例6中探针NVCP与PC12细胞存活率实验结果图;
图24为本发明实施例6中探针NVCP和Mito-tracker green处理的PC12细胞的共聚焦激光荧光显微镜图像;
图25为本发明实施例6中探针NVCP在PC12细胞中与内、外源性Cys的荧光成像图;
图26为本发明实施例6中探针NVCP与莫能菌素和制霉菌素预处理的PC12细胞的共聚焦激光荧光成像。
具体实施方式
下面结合具体实施例对本发明的技术方案做进一步说明,但不限于此。
实施例1
化合物3的合成:
将4-甲基喹啉(500mg,3.5mmol)、对氨基苯甲醛(521mg,3.5mmol)和Zn(OAc)2(960mg,5.3mmol)混合在5mL乙酸酐中,在氮保护下在150℃下回流5h。采用薄层色谱法检测反应结果。冷却至室温后,用饱和碳酸氢钠水溶液和二氯甲烷萃取去除乙酸酐。收集有机相,通过真空蒸馏得到粗产物。以CH2Cl2/石油醚(v/v,1:1)为洗脱液,经色谱层析纯化,得到浅黄色粉末化合物3(650.5mg,产率64%)。1HNMR(500MHz,CDCl3):δ8.84-8.84(d,J=4.6Hz,1H),8.23-8.21(d,J=8.4Hz,1H),8.14-8.13(d,J=8.5Hz,1H),7.71-7.68(t,J=7.6Hz,1H),7.59-7.53(td,J=13.5,7.9Hz,3H),7.51-7.49(d,J=8.5Hz,2H),7.31-7.29(d,J=16.0Hz,1H),6.72-6.70(d,J=8.5Hz,2H),2.99(s,6H).13C NMR(126MHz,CDCl3):δ129.2,128.5,128.2,127.5,125.4,125.2,124.1,122.5,119.9,116.4,115.0,111.2,110.6,76.3,76.1,75.8,39.2.HRMS[M+H]+found,275.1548,calculated for C19H19N2 +,275.1453。合成路线如下:
化合物NVCD的合成:
将化合物3(500mg,1.8mmol)、4-羟基苯乙基溴(434mg,2.1mmol)溶解在10mL乙腈中,在80℃下搅拌过夜。溶剂在减压下浓缩以得到粗产品。粗产物以CH2Cl2/CH3OH(v/v,5:1)为洗脱液进行色谱层析纯化,得到紫色粉末NVCD(241mg,产率26%)。1H NMR(500MHz,DMSO):δ9.57(s,1H),9.07-9.05(d,J=8.5Hz,1H),8.89-8.87(d,J=6.6Hz,1H),8.52-8.50(d,J=9.0Hz,1H),8.27-8.25(d,J=6.7Hz,1H),8.22-8.19(m,2H),8.03-8.00(d,J=15.6Hz,1H),7.98-7.95(t,J=7.6Hz,1H),7.90-7.88(d,J=8.5Hz,2H),6.95-6.93(d,J=8.0Hz,2H),6.83-6.82(d,J=8.6Hz,2H),6.69-6.67(d,J=8.0Hz,2H),5.10-5.08(t,J=7.0Hz,2H),3.38-3.13(dd,J=13.2,5.8Hz,2H),3.07(s,6H).HRMS[M]+found,395.2128,calculated for C27H27N2O+,395.2123.合成路线如下:
探针NVCP的合成:
将化合物NVCD(396mg,1.0mmol)、碳酸钾(276mg,2.0mmol)溶解于10mL无水N,N-二甲基甲酰胺中,在室温氮保护下激活30min,然后加入化合物5(579mg,2.0mmol),加热至55℃过夜反应。所得到的混合物依次用乙酸乙酯和饱和盐水洗涤。用CH2Cl2/CH3OH(v/v,5:1)作为洗脱液进行柱层析纯化,得到紫色粉末NVCP(200mg,收率为21%)。1H NMR(500MHz,CH3OH):δ8.88-8.86(d,J=8.6Hz,1H),8.56-8.55(d,J=6.4Hz,1H),8.46-8.44(d,J=8.8Hz,1H),8.24-8.21(t,J=7.7Hz,1H),8.11-8.10(d,J=6.3Hz,1H),8.00-7.99(d,J=7.7Hz,1H),7.91-7.88(d,J=15.5Hz,1H),7.80-7.78(d,J=8.4Hz,2H),7.23-7.21(d,J=7.9Hz,2H),7.18-7.17(d,J=7.9Hz,2H),6.83-6.82(d,J=8.4Hz,2H),6.57-6.56(d,J=7.6Hz,1H),6.06-6.02(d,J=17.2Hz,1H),5.51-5.50(d,J=9.3Hz,1H),5.20-5.18(t,J=5.6Hz,2H),3.71-3.68(s,2H),3.41-3.37(dd,J=14.0,6.9Hz,4H),3.12(s,6H),1.15-1.12(t,J=7.0Hz,6H).13C NMR(126MHz,CH3OH):δ175.1,155.5,154.1,153.9,151.8,147.6,146.8,146.5,146.0,139.2,137.4,136.4,136.1,135.7,132.5,132.0,129.7,127.6,127.4,125.0,124.2,123.0,122.1,119.3,114.4,113.4,113.0,110.7,79.2,78.9,78.6,58.3,49.5,49.3,49.2,49.0,48.8,48.7,48.5,43.6,40.3,35.6,32.8,31.9,23.5,15.1.HRMS[M]+found,648.2641,calculated for C37H38N5O4S+,648.2645.合成路线如下:
实施例2
荧光探针NVCP与Cys作用的溶液配制
取实施例1制备的探针NVCP溶于DMF中,制成浓度为10mM探针母液;将半胱氨酸配制成浓度为100mM的母液,最终配制成2mL探针浓度为10μM的PBS测试液(30%DMF,25mM,pH7.4)。
实施例3
荧光探针NVCP与Cys作用的紫外可见吸收光谱性质的测定
在2mL探针浓度为10μM的PBS测试液(30% DMF,25mM,pH7.4)中加入500μM的Cys,测定荧光探针与Cys作用的紫外光谱图,如图9所示。紫外可见吸收光谱测定用的仪器为天美/UV2600型紫外可见分光光度计。图9为本发明实施例1中探针NVCP与Cys作用前后的紫外可见吸收光谱图;横坐标为波长,纵坐标为紫外吸收强度。黑线为纯探针溶液,红线为加入Cys响应后的溶液。
实施例4
荧光探针NVCP与不同浓度的Cys反应的荧光光谱变化
在2mL探针浓度为10μM的PBS测试液(30% DMF,25mM,pH7.4)中分别加入不同浓度(0-500μM)的Cys。用荧光光谱仪测试探针分别与不同浓度Cys反应液的荧光光谱变化(激发波长为430nm,收集420-900nm处的荧光发射峰),荧光光谱变化情况如图10所示,随着不同浓度Cys浓度的加入,在592nm处的荧光强度值逐渐增强。图10中,横坐标为波长,纵坐标为荧光强度。从下至上,Cys浓度依次为0、10、20、30、50、100、150、200、250、300、400、500μmol/L。
如图11所示,探针荧光强度和Cys浓度在0-400μmol/L具有良好的线性关系,当荧光强度达到最大值时,比探针空白液的荧光强度增强63倍。所用的荧光测定仪器为Hitachi/F-7100荧光分光光度计。
实施例5
荧光探针NVCP与Cys随时间变化的荧光强度
在2mL探针浓度为10μM的PBS测试液(30% DMF,25mM,pH7.4)中加入500μM的Cys,测试探针与Cys随时间变化的荧光强度,由图12可见,随着时间增加,592nm处的荧光强度逐渐增强,70min后荧光强度达到峰值。
实施例6
荧光探针NVCP对不同干扰分析物的选择性和竞争性研究
将2mL探针浓度为10μM的PBS测试液(30% DMF,25mM,pH7.4)分别配成含100μM分析物和含100μM分析物、500μMCys的测试液:H2S、GSH、Hcy、K+、Ca2+、Mg2+、DL-lysine、DL-proline、DL-serine、D-phenylalanine、L-alanine、L-glutamine、L-arginine、L-leucine、L-threonine、L-valine、L-histidine、Glycine。反应70min后检测测试液的荧光光谱变化。由图13、14可以发现,相对于空白测试液,加入不同干扰分析物的测试液荧光强度没有明显变化,加入Cys测试液的荧光强度发生了显著增强。而待测物的荧光强度不受其他干扰分析物的影响,实验结果说明探针NVCP对Cys具有良好的选择性和竞争性。
实施例7
荧光探针NVCP在不同pH溶液中对Cys的光谱测定
取2μL实施例2荧光探针母液,5μL 100mM Cys母液配置成2mL不同pH的测试液(30%DMF,25mM),pH分别为3、4、5、6、7、7.4、8、9、10,检测其随pH变化的荧光光谱。由图15可见,在pH为7.4-10时有良好的响应,表明探针具有较好的生物相容性。
实施例8
荧光探针NVCP与Gly作用的紫外可见吸收光谱性质的测定
将200μL DMF,1800μL Gly配置成2mL探针浓度为10μM的测试液,测定荧光探针与Gly作用的紫外光谱图。吸收光谱如图16所示。
实施例9
荧光探针NVCP与不同浓度的Gly反应的荧光光谱变化
在2mL探针浓度为10μM的测试液中加入不同体积的Gly,检测荧光探针与不同体积Gly作用的荧光光谱(激发波长为550nm,收集540-900nm处的荧光发射峰),荧光光谱变化情况如图17所示,随着不同体积Gly的加入,在695nm处的荧光强度值逐渐增强,也就是说,反应体系荧光强度随粘度增加而逐渐增强。如图18所示,与Gly反应在695nm处粘度从1.4cp变化到834cp的荧光强度逐渐增强,LogI695nm与Logη荧光在0.5~2.0之间的曲线图呈直接线性关系(图18)。
实施例10
荧光探针NVCP对不同干扰分析物的选择性和竞争性研究
取2μL实施例2荧光探针母液,分别配成2mL含300μM分析物和含300μM分析物、Gly(1% DMF)的测试液:Ag+、K+、Na+、Ca2+、Cu2+、Fe2+、Zn2+、Mg2+、Cl-、ClO-、ONOO-、O2 .-、Br-、SO4 2-、Fe3+、Hcy、GSH、Cys、H2O2、H2S。由图19可以发现,相对于空白测试液,加入不同干扰分析物的测试液荧光强度没有明显变化,加入Gly测试液的荧光强度发生了显著增强。而待测物的荧光强度不受其他干扰分析物的影响,实验结果说明探针NVCP对Gly具有良好的选择性和竞争性。
实施例11
荧光探针NVCP在不同pH溶液中对Gly的光谱测定
取2μL实施例2荧光探针母液,分别加入0%、20%、80%Gly配置成2mL不同pH的测试液,pH分别为3、4、5、6、7、7.4、8、9、10,检测其随pH变化的荧光光谱。由图20可以发现,在pH4.0-6.0时显示出微弱的荧光发射,而在pH7.0-9.0时表现出显著的荧光增强,这与生理pH范围相一致。
实施例12
探针NVCP在不同溶剂中的荧光响应
取2μL实施例2荧光探针母液,分别加入不同溶剂配置成2mL测试液,检测探针对不同溶剂的荧光响应。由图21可以发现,除甘油外,探针NVCP的荧光强度几乎不受溶剂的影响。
实施例13
探针NVCP在不同溶剂中的吸收光谱
取2μL实施例2荧光探针母液,分别加入不同溶剂配置成2mL测试液,检测探针在不同溶剂中的吸收光谱。由图22可以发现,除甘油外,探针NVCP在不同极性溶剂中的吸收峰几乎没有变化。
实施例14
探针NVCP在PC12中细胞存活率实验
取对数生长期的细胞以5-10×104/mL的密度接种于96孔板,每孔100μL细胞悬液,在37℃,5%CO2条件的恒温培养箱孵育24h。吸弃培养基,将探针的DMSO溶液与培养基一起配制成8个浓度梯度(0μM、5μM、10μM、15μM、20μM)的溶液,每孔加入100μL溶液,孵育24h。孵育结束后,用1mL注射器吸取96孔板内的培养基,各孔避光加入20μL含MTT的基础培养基(MTT终浓度为0.5mg/ml),培养箱避光孵育4h,孵育结束后,用1mL移液枪吸取培养基,每孔加100μLDMSO溶液,置37℃摇床上低速震摇10min使甲瓒充分溶解。酶标仪测定490nm波长处的吸光度(OD)值,用空白孔(培养基+MTT)校准吸光度值,按以下公式计算各组细胞存活率:
细胞相对存活率(%)=(实验组OD值-空白组OD值)/(对照组OD值-空白组OD值)×100%。实验重复6次,数据表示为mean±SD。
如图23所示,当探针NVCP的浓度达到20μM时,细胞的存活率可达90%,由此表明探针NVCP具有较小的细胞毒性。
实施例15
探针NVCP和Mito-tracker green处理的PC12细胞的共聚焦激光荧光显微镜图像
将PC12细胞提前用10μM制酶菌素处理30min,再与Mito-Tracker green(1μM)和探针NVCP(10μM)同时孵育30min,然后用PBS洗涤细胞,成像。显微成像采用共聚焦激光扫描显微镜(CLSM,Zeiss LMS880)。Mito-Tracker green激发波长为488nm,收集发射波长范围为500-520nm;探针的激发波长为594nm,收集发射波长范围为640-710nm。如图24所示,NVCP的红色通道荧光图像和Mito-Tracker gree的绿色通道荧光图像具有与良好的皮尔逊系数(0.94)。用NVCP和Mito-Tracker gree染色的感兴趣的线性区域(ROI)在强度曲线上也显示出密切的同步性。比例尺=10μM。
实施例16
探针NVCP与PC12细胞中内、外源性Cys的荧光成像
对于内源性Cys成像,细胞与探针NVCP孵育,在抑制实验中,细胞用NEM(200μM,30min)处理,然后与NVCP孵育。对于外源性Cys成像,细胞用NEM预处理,并与外源性Cys(300μM,30min)孵育。从CLSM软件中获得荧光图像,绿色通道在540-600nm处采集,激发时间为488nm。结果如图25所示,与空白组对比,经N-乙基马来酰亚胺(NEM,硫醇阻断剂)预处理后的细胞荧光信号明显降低,说明NVCP对细胞内Cys肯定敏感。此外,添加到Cys的NEM细胞的荧光强度明显强于NEM组的细胞。这些结果表明了NVCP在活细胞中检测外源性/内源性Cys的敏感性,表明NVCP在Cys的生物成像方面具有巨大的应用潜力。比例尺=20μM。
实施例17
探针NVCP与PC12细胞中粘度的荧光成像
细胞与或不与10μM莫能菌素(Moe)和制霉菌素(Nys)一起孵育30min,所有细胞都与NVCP(10μM)再孵育30min。在成像前,用PBS洗涤三次,去除残留的探针。从CLSM获得荧光图像,红色通道在640-710nm采集,激发长为594nm。如图26所示,莫能菌素(Moe)或制霉菌素(Nys)预处理后的细胞比仅加载NVCP的对照组细胞表现出更强的红色荧光信号,表明Moe和Nys刺激后线粒体粘度增加。以上结果表明,NVCP可用于监测生命***中线粒体粘度和Cys的变化。比例尺=20μM。
需要说明的是,上述实施例仅仅是实现本发明的优选方式的部分实施例,而非全部实施例。显然,基于本发明的上述实施例,本领域普通技术人员在没有做出创造性劳动的前提下所获得的其他所有实施例,都应当属于本发明保护的范围。
Claims (4)
1.一种具有粘度和半胱氨酸特异性响应的线粒体靶向近红外荧光探针,其特征在于,探针的分子式为C37H38N5O4S+,结构式如下:
。
2.一种权利要求1所述粘度和半胱氨酸特异性响应的线粒体靶向近红外荧光探针的制备方法,其特征在于,包括以下制备步骤:
(1)将500 mg4-甲基喹啉、521 mg对氨基苯甲醛和960 mgZn(OAc)2混合在5mL乙酸酐中,在氮保护下在150℃下回流5h;冷却至室温后,用饱和碳酸氢钠水溶液和二氯甲烷萃取去除乙酸酐,收集有机相,通过真空蒸馏得到粗产物,以体积比为1:1的CH2Cl2/石油醚为洗脱液,经色谱层析纯化,得到浅黄色粉末化合物3,化合物3的结构式为:;
(2)将500mg化合物3、434mg4-羟基苯乙基溴溶解在10mL乙腈中,在80℃下搅拌过夜;溶剂在减压下浓缩以得到粗产品,粗产物以体积比=5:1的CH2Cl2/CH3OH为洗脱液进行色谱层析纯化,得到紫色粉末NVCD,NVCD的结构式为:;
(3)将396mg化合物NVCD、276mg碳酸钾溶解于10mL无水N,N-二甲基甲酰胺中,在室温氮保护下激活30min,然后加入579 mg化合物5,加热至55℃过夜反应;所得到的混合物依次用乙酸乙酯和饱和盐水洗涤;用体积比为5:1的CH2Cl2/CH3OH作为洗脱液进行柱层析纯化,得到紫色粉末终产物荧光探针NVCP;
步骤(3)化合物5的结构式为:。
3.一种权利要求1所述粘度和半胱氨酸特异性响应的线粒体靶向近红外荧光探针的应用,其特征在于,用于检测离体活细胞环境中的粘度和半胱氨酸,以获取中间信息。
4.一种权利要求1所述粘度和半胱氨酸特异性响应的线粒体靶向近红外荧光探针的应用,其特征在于,用于检测水环境中的粘度和半胱氨酸。
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106588912A (zh) * | 2016-11-30 | 2017-04-26 | 济南大学 | 一种可区分检测半胱氨酸/高半胱氨酸、谷胱甘肽和硫化氢的荧光探针及其制备方法、应用 |
CN108623510A (zh) * | 2018-04-26 | 2018-10-09 | 济南大学 | 一种靶向线粒体近红外发射的粘度荧光探针及其合成方法 |
CN111518071A (zh) * | 2020-05-20 | 2020-08-11 | 湘潭大学 | 一种半胱氨酸近红外荧光探针的制备和应用 |
CN114605376A (zh) * | 2022-04-21 | 2022-06-10 | 济南大学 | 一种检测半胱氨酸和粘度的双功能荧光探针及其制备 |
CN114773288A (zh) * | 2022-04-18 | 2022-07-22 | 南华大学附属南华医院 | 一种区别检测半胱氨酸和同型半胱氨酸/谷胱甘肽的荧光探针及其制备方法和应用 |
CN115650897A (zh) * | 2022-10-25 | 2023-01-31 | 广西师范大学 | 一种同时检测Cys和线粒体粘度的荧光探针及其制备方法和应用 |
CN116178349A (zh) * | 2023-02-22 | 2023-05-30 | 许昌学院 | 一种检测半胱氨酸的高尔基体靶向近红外荧光探针、其制备方法和应用 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9618453B2 (en) * | 2011-10-11 | 2017-04-11 | The Hong Kong University Of Science And Technology | Aggregation induced emission of fluorescent bioprobes and methods of using the same |
-
2023
- 2023-11-16 CN CN202311532752.8A patent/CN117586247B/zh active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106588912A (zh) * | 2016-11-30 | 2017-04-26 | 济南大学 | 一种可区分检测半胱氨酸/高半胱氨酸、谷胱甘肽和硫化氢的荧光探针及其制备方法、应用 |
CN108623510A (zh) * | 2018-04-26 | 2018-10-09 | 济南大学 | 一种靶向线粒体近红外发射的粘度荧光探针及其合成方法 |
CN111518071A (zh) * | 2020-05-20 | 2020-08-11 | 湘潭大学 | 一种半胱氨酸近红外荧光探针的制备和应用 |
CN114773288A (zh) * | 2022-04-18 | 2022-07-22 | 南华大学附属南华医院 | 一种区别检测半胱氨酸和同型半胱氨酸/谷胱甘肽的荧光探针及其制备方法和应用 |
CN114605376A (zh) * | 2022-04-21 | 2022-06-10 | 济南大学 | 一种检测半胱氨酸和粘度的双功能荧光探针及其制备 |
CN115650897A (zh) * | 2022-10-25 | 2023-01-31 | 广西师范大学 | 一种同时检测Cys和线粒体粘度的荧光探针及其制备方法和应用 |
CN116178349A (zh) * | 2023-02-22 | 2023-05-30 | 许昌学院 | 一种检测半胱氨酸的高尔基体靶向近红外荧光探针、其制备方法和应用 |
Non-Patent Citations (2)
Title |
---|
有机双光子线粒体内活性小分子荧光探针研究进展;杨志广;李运林;陈亚红;郭雷;张建夫;彭鹏;;分析测试学报;20160525(05);138-145 * |
杨志广 ; 李运林 ; 刘增臣 ; 田丰收 ; 彭鹏 ; .有机巯基小分子生物荧光探针研究进展.分析试验室.2015,(11),124-130. * |
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