CN108484479B - 一种咔唑基双光子荧光探针及其制备方法和用途 - Google Patents
一种咔唑基双光子荧光探针及其制备方法和用途 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及一种咔唑基双光子荧光探针及其制备方法和用途,以实现双光子共聚焦成像可视化检测细胞溶酶体内的Cys,具有选择性高、灵敏度高、检出限低的优点。
背景技术
氨基酸是构成生物体蛋白质并与生命活动有关的最基本的物质,与生物的生命活动有着密切的关系。其中半胱氨酸(Cys),作为人体必须的氨基酸之一,是参与蛋白质合成的20种氨基酸中的一种,同时也是构建重要的谷胱甘肽的三个氨基酸之一。并且Cys在许多关键的细胞功能中起重要作用。例如,蛋白质合成,解毒和代谢等。溶酶体是真核细胞中一个酸性的细胞器,在细胞内可以起到消化、防御和调节激素分泌的作用。因此,监测细胞溶酶体中的Cys水平在生物医学研究和诊断中起着重要作用。
荧光化学探针由于具有灵敏度高、选择性好、易合成、廉价以及良好的生物应用等特点,已成为生命科学和环境科学中主要的检测工具。目前,大部分检测细胞中Cys的荧光探针均为单光子荧光探针,单光子荧光探针一般具有自荧光干扰大,激发波长小导致对细胞的光毒性大,容易发生荧光自淬灭等缺点。与单光子荧光探针相比,双光子荧光探针具有很多明显的优点,如:细胞光毒性小,不会引起荧光自淬灭,时间空间分辨率高,深的组织渗透。因而双光子荧光探针已经作为科学家们研究的一个重要课题。咔唑作为一种经典的荧光团,不仅具有大的共轭体系和共平面性能,而且具有良好的光稳定性、低毒性。以咔唑为荧光团的单光子荧光探针已经被很多文献所报道,但是作为双光子荧光探针的文献报道相对较少。
发明内容
本发明旨在提供一种咔唑基双光子荧光探针及其制备方法和用途。所要解决的技术问题是通过分子设计得到一种合适的荧光探针结构,以实现双光子共聚焦成像可视化检测细胞溶酶体内的Cys。本发明双光子荧光探针具有选择性高、灵敏度高、检出限低的优点,细胞毒性测试表明本发明荧光探针对细胞几乎没有毒性作用。
本发明咔唑基双光子荧光探针,简记为Lyso-DCHO,是以咔唑为母体,其结构式如下:
本发明咔唑基双光子荧光探针的制备方法,包括如下步骤:
步骤1:中间体1的合成
将KOH(2.0g,35.8mmol)、KI(0.4g,2.39mmol)和1,4-二溴丁烷(7.73g,35.8mmol)的混合物加热至60℃,反应1小时后缓慢加入3,6-二碘代咔唑(10g,23.9mmol),继续加热回流反应12小时;反应结束后冷却至室温,加入200mL H2O,用二氯甲烷(100mL×2)萃取混合物,将有机相蒸发,得到粗残余物,通过柱色谱法(石油/二氯甲烷=10:1作为洗脱剂)纯化,得到中间体1,6.8g,产率为51.4%。
步骤2:中间体2的合成
将中间体1(1g,1.8mmol)、4-乙炔苯甲醛(0.7g,5.4mmol)、Pd2(PPh 3)2Cl2(5.1mg,0.007mmol)、CuI(2.7mg,0.014mmol)和Et3N(5mL)在无水和无氧条件下于30℃搅拌反应12小时,反应结束后冷却至室温,将沉淀滤出并浓缩,得到粗产物,通过柱色谱(石油/二氯甲烷=4:1作为洗脱剂)纯化,得到中间体2,0.65g,产率为64.5%。
步骤3:Lyso-DCHO的合成
在氮气保护下,将中间体2(1g,1.8mmol)、KOH(0.061g,1.08mmol)、KI(0.18g,1.08mmol)和THF(10mL)的混合物加热至70℃,反应1小时后加入吗啉(6mL),继续加热回流反应36小时,反应结束后冷却至室温,过滤,蒸发滤液,得到粗产物,通过柱色谱法(石油醚/二氯甲烷/乙酸乙酯=1:1:2作为洗脱剂)纯化,得到淡黄色固体Lyso-DCHO,0.47g,产率为46.5%。
本发明Lyso-DCHO的合成过程如下:
本发明咔唑基双光子荧光探针的用途,是在定性检测细胞溶酶体内的Cys时作为检测试剂的应用。具体检测方法如下:
将本发明双光子荧光探针溶于DMSO中制得1mM的母液,取100μL的该母液于10mL容量瓶中,再用DMSO:PBS(v/v)=1:1的溶剂定容,配制成10μM的检测试剂。检测试剂分别在313nm和373nm有吸收峰;随着Cys的加入,Lyso-DCHO位于373nm的吸收峰逐渐降低,且在313nm的吸收峰逐渐升高,当Cys加入4.5倍当量时313nm的吸光度达到饱和(图2)。10μM检测试剂加入8倍当量的其他氨基酸、各种阴离子和金属阳离子作用20min后(图3的a部分),检测350-700nm范围内的荧光光谱变化,可以看出Lyso-DCHO仅对Cys有明显的荧光变化,具有专一性响应;当随着Cys(0-60μM)的不断加入,可以观察到521nm处的荧光发射峰强度逐渐减弱,且402nm处的荧光逐渐增强。在加入4.5倍当量的Cys后,402nm处的荧光强度趋于饱和(图3的b部分)。10μM检测试剂在加入Cys的浓度范围为0-5μM时,其荧光强度与Cys的浓度之间有很好的线性关系(R=3δ/k),检测浓度低至99nM(图4)。
本发明双光子荧光探针结构简单,易于合成。本发明双光子荧光探针符合ICT机理并与Cys有明确的作用位点,其中咔唑部分作为供体(D),其末端具有两个相同的接受单元(A)。在加入Cys后,会与探针结构中的两个醛基发生化学反应生成Lyso-DCHO-Cys(图1),反应前后电子云密度分布发生变化,荧光和紫外吸收性质也随之改变。在紫外灯下,肉眼就可以看出其荧光变化,荧光颜色从绿色变成蓝色光,该方法操作简单,快速灵敏。本发明双光子荧光探针对Cys具有专一性荧光响应,灵敏度高,检出限低。
附图说明
图1是本发明荧光探针分子Lyso-DCHO与Cys反应机理图。
图2是10μM探针加入Cys(0-60μM)的紫外滴定光谱图,其插图是紫外吸收峰强度比值(A313/A373)变化与Cys浓度的关系。
图3是10μM探针加入8倍当量的其他氨基酸、各种阴离子和金属阳离子作用20min后的荧光选择性谱图(图3的a部分);10μM探针加入Cys(0-60μM)的荧光滴定光谱图(图3的b部分),图3的b部分的插图是荧光发射峰强度比值(I402/I521)变化与Cys浓度的关系。
图4是10μM探针在加入Cys(0-5μM)后的荧光强度与浓度的线性关系图。
图5是探针Lyso-DCHO加入Cys前后的双光子有效吸收截面。
图6是在不同含量(0μM、10μM、20μM、30μM)的探针分子作用下的MCF-7细胞存活率图。
图7是10μM探针分子在MCF-7细胞中加入60μM的Cys后的细胞溶酶体定位成像。设置探针分子Lyso-DCHO为绿色通道(λem=500-540nm,λex=720nm);设置商品化线粒体染色剂LysoTracker Red FM为红色通道(λem=580-600nm,λex=579nm)。其中图7的a部分,图7的b部分分别为绿色通道和红色通道下的荧光共聚焦成像。图7的c部分是MCF-7细胞的明场,图7的d部分是图7的a部分、b部分、c部分的叠加,图7的e部分是图7的d部分中单个细胞的荧光强度剖面图,图7的f部分是Lyso-DCHO和LysoTracker Red FM强度的相关性分布图,重叠系数为0.90。
图8是10μM探针分子在MCF-7细胞(经1.0mM NEM预处理30min,NEM是硫醇清除剂)中加入60μM的Cys前后的双光子共聚焦细胞成像。在720nm激发下,蓝色通道中荧光发射收集范围420-460nm,绿色通道中荧光发射收集范围是500-540nm。图8的A部分是只加10μM探针Lyso-DCHO的细胞成像,图8的B部分是加探针10μM Lyso-DCHO和60μM Cys的细胞成像。
具体实施方式
下面通过实施例对本发明做进一步说明。
实施例1:荧光探针分子Lyso-DCHO的合成
1、中间体1的合成
将KOH(2.0g,35.8mmol)、KI(0.4g,2.39mmol)和1,4-二溴丁烷(7.73g,35.8mmol)的混合物加热至60℃,反应1小时后缓慢加入3,6-二碘代咔唑(10g,23.9mmol),继续加热回流反应12小时;反应结束后冷却至室温,加入200mL H2O,用二氯甲烷(100mL×2)萃取混合物,将有机相蒸发,得到粗残余物,通过柱色谱法(石油/二氯甲烷=10:1作为洗脱剂)纯化,得到中间体1,6.8g,产率为51.4%。
2、中间体2的合成
将中间体1(1g,1.8mmol)、4-乙炔苯甲醛(0.7g,5.4mmol)、Pd2(PPh 3)2Cl2(5.1mg,0.007mmol)、CuI(2.7mg,0.014mmol)和Et3N(5mL)在无水和无氧条件下于30℃搅拌反应12小时,反应结束后冷却至室温,将沉淀滤出并浓缩,得到粗产物,通过柱色谱(石油/二氯甲烷=4:1作为洗脱剂)纯化,得到中间体2,0.65g,产率为64.5%。
3、Lyso-DCHO的合成
在氮气保护下,将中间体2(1g,1.8mmol)、KOH(0.061g,1.08mmol)、KI(0.18g,1.08mmol)和THF(10mL)的混合物加热至70℃,反应1小时后加入吗啉(6mL),继续加热回流反应36小时,反应结束后冷却至室温,过滤,蒸发滤液,得到粗产物,通过柱色谱法(石油醚/二氯甲烷/乙酸乙酯=1:1:2作为洗脱剂)纯化,得到淡黄色固体Lyso-DCHO,0.47g,产率为46.5%。
1H NMR(400MHz,DMSO-d6,ppm):δ10.05(s,2H),8.57(s,2H),7.98(d,J=7.9Hz,4H),7.79(m,6H),7.74(d,J=8.5Hz,2H),4.48(t,J=6.9Hz,2H),3.52(t,J=4.2Hz,4H),2.27(t,J=6.5Hz,6H),1.85(m,2H),1.48(m,2H).13C NMR(100MHz,CDCl3,ppm):δ191.41,140.78,135.13,131.90,130.16,130.05,129.64,124.59,122.57,113.40,109.23,94.91,87.52,66.88,58.15,53.64,43.18,26.67,24.03.
实施例2:荧光探针分子的光谱测试
将本发明双光子荧光探针溶于DMSO中制得1mM的母液,取100μL的该母液于10mL容量瓶中,再用DMSO:PBS(v/v)=1:1的溶剂定容,配制成10μM的检测试剂。检测试剂分别在313nm和373nm有吸收峰;随着Cys的加入,Lyso-DCHO位于373nm的吸收峰逐渐降低,且在313nm的吸收峰逐渐升高,当Cys加入4.5倍当量时313nm的吸光度达到饱和(图2)。10μM检测试剂加入8倍当量的其他氨基酸、各种阴离子和金属阳离子作用20min后(图3的a部分),检测350-700nm范围内的荧光光谱变化,可以看出Lyso-DCHO仅对Cys有明显的荧光变化,具有专一性响应;当随着Cys(0-60μM)的不断加入,可以观察到521nm处的荧光发射峰强度逐渐减弱,且402nm处的荧光逐渐增强。在加入4.5倍当量的Cys后,402nm处的荧光强度趋于饱和(图3的b部分)。10μM检测试剂在加入Cys的浓度范围为0-5μM时,其荧光强度与Cys的浓度之间有很好的线性关系(R=3δ/k),检测浓度低至99nM(图4)。
实施例3:荧光探针分子加入Cys前后的双光子性能测试
利用双光子诱导荧光测量技术,测试荧光探针分子(Lyso-DCHO)的双光子有效吸收截面,Lyso-DCHO在760nm处具有58GM的最大双光子有效吸收截面,并且在加入Cys后在720nm处最大值变为45GM。说明探针可以应用于双光子共聚焦成像。
实施例4:细胞毒性测试
MTT(3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐)实验是根据已报道的文章操作进行细胞毒性测试。分别在同一批细胞中加入0,5,10,15μM的荧光探针分子,条件是在37℃、含5%CO2的细胞培养箱中孵育24小时,根据细胞存活度的公式:细胞存活率%=OD570(样品)/OD570(对照组)×100,可算得细胞存活率(图6)。从图6中我们可以看出,浓度为10μM时,细胞存活率有96%左右,探针浓度达到20μM时,细胞存活率还有约92%,当探针浓度达到30μM时,细胞存活率还有约89%,说明了本发明荧光探针分子对细胞无明显毒性作用,因此可以用来检测细胞中溶酶体内的Cys。
实施例5:细胞溶酶体定位测试
MCF-7细胞由DEME(invitrogen)培养液培养,成像前一天,MCF-7细胞放于激光共聚焦皿中,成像时10μM的荧光探针Lyso-DCHO的DMSO溶液加入MCF-7细胞并置于37℃、含5%CO2的细胞培养箱中孵育0.5小时,用中性的PBS缓冲溶液洗涤3次后,再往培养皿中加入0.5μM商品化溶酶体染色剂LysoTracker Red FM溶液继续孵育0.5小时,用中性的PBS缓冲溶液洗涤3次后,用双光子荧光共聚焦成像,设置探针分子Lyso-DCHO为绿色通道(λem=500-540nm,λex=720nm);设置商品化线粒体染色剂LysoTracker Red FM为红色通道(λem=580-600nm,λex=579nm)。其中图7的a部分,b部分分别为绿色通道和红色通道下的荧光共聚焦成像。图7的c部分是MCF-7细胞的明场,图7的d部分是图7的a部分、b部分、c部分的叠加,图7的e部分是图7的d部分中单个细胞的荧光强度剖面图,图7的f部分是Lyso-DCHO和LysoTracker Red FM强度的相关性分布图,重叠系数为0.90。从图7可以看出,Lyso-DCHO绝大多数定位在溶酶体内,可用于可视化检测细胞溶酶体内Cys。
实施例6:细胞成像测试
MCF-7细胞由DEME(invitrogen)培养液培养,成像前一天,MCF-7细胞放于玻底培养皿中,成像时MCF-7细胞和1.0mM NEM于37℃、含5%CO2的细胞培养箱中孵育0.5小时,之后加入10μM的荧光探针Lyso-DCHO的DMSO溶液于37℃、含5%CO2的细胞培养箱中孵育0.5小时,用中性的PBS缓冲溶液或培养液洗涤3次,用双光子荧光共聚焦成像,在720nm激发下,蓝色通道中荧光发射收集范围420-460nm,绿色通道中荧光发射收集范围是500-540nm(图8的A部分)。向上述含荧光探针的细胞培养液中加入(60μM)Cys,在37℃、含5%CO2的细胞培养箱中孵育0.5小时,用中性的PBS缓冲溶液或培养液洗涤3次,再进行双光子荧光共聚焦成像,蓝色通道内荧光明显增强,绿色通道内荧光减弱(图8的B部分)。从图8中可以看出,探针可用于细胞内Cys的双光子荧光成像。
Claims (6)
2.一种权利要求1所述的咔唑基双光子荧光探针的制备方法,其特征在于包括如下步骤:
步骤1:中间体1的合成
将KOH 35.8mmol、KI 2.39mmol和1,4-二溴丁烷35.8mmol的混合物加热至60℃,反应1小时后缓慢加入3,6-二碘代咔唑23.9mmol,继续加热回流反应12小时;反应结束后冷却至室温,加入H2O,用二氯甲烷萃取混合物,将有机相蒸发,得到粗残余物,通过柱色谱法纯化,得到中间体1;
所述中间体1的结构式如下所示:
步骤2:中间体2的合成
将中间体1 1.8mmol、4-乙炔苯甲醛5.4mmol、Pd2(PPh3)2Cl2 0.007mmol、CuI 0.014mmol和Et3N 5mL在无水和无氧条件下于30℃搅拌反应12小时,反应结束后冷却至室温,将沉淀滤出并浓缩,得到粗产物,通过柱色谱纯化,得到中间体2;
所述中间体2的结构式如下所示:
步骤3:Lyso-DCHO的合成
在氮气保护下,将中间体2 1.8mmol、KOH 1.08mmol、KI 1.08mmol和THF的混合物加热至70℃,反应1小时后加入吗啉6mL,继续加热回流反应36小时,反应结束后冷却至室温,过滤,蒸发滤液,得到粗产物,通过柱色谱法纯化,得到淡黄色固体Lyso-DCHO。
3.根据权利要求2所述的制备方法,其特征在于:
步骤1中,柱色谱法纯化时的洗脱液为石油和二氯甲烷按体积比10:1的比例混合得到。
4.根据权利要求2所述的制备方法,其特征在于:
步骤2中,柱色谱法纯化时的洗脱液为石油和二氯甲烷按体积比4:1的比例混合得到。
5.根据权利要求2所述的制备方法,其特征在于:
步骤3中,柱色谱法纯化时的洗脱液为石油醚、二氯甲烷和乙酸乙酯按体积比1:1:2的比例混合得到。
6.一种权利要求1所述的咔唑基双光子荧光探针的用途,其特征在于:所述荧光探针用于制备定性检测细胞溶酶体内的Cys的检测试剂。
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