CN103044947B - 一类尼罗蓝荧光染料,其制备方法及应用 - Google Patents
一类尼罗蓝荧光染料,其制备方法及应用 Download PDFInfo
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Abstract
一类尼罗蓝荧光染料,其制备方法及应用。所述的尼罗蓝荧光染料具有如下结构通式I。本发明所述的尼罗蓝类荧光染料具有优异的光化学物理性质,例如高的摩尔消光系数、良好的光稳定性、高的荧光量子产率等优点。对细胞的内环境不敏感,具有良好的化学稳定性和光稳定性,而且可以解决通常溶酶体探针对细胞高毒性和光漂白严重等的弊端。可应用于溶酶体荧光特异性标记。
Description
技术领域
本发明涉及一类荧光探针化合物及其制备方法和用途,该化合物适用于精细化工领域中细胞溶酶体的标记。
背景技术
溶酶体是细胞内的一种非常重要的亚细胞器,其内包含能够分解废弃材料和细胞碎片的酸性水解酶,他们能够消化过量的或磨损的细胞器、食物粒子,并且能够吞没病毒或细菌。
溶酶体的主要作用是消化作用,是细胞内的消化器官,细胞自溶,防御以及对某些物质的利用均与溶酶体的消化作用有关。同时,越来越多的事实证明,溶酶体在许多生理、药理及病理过程中均起重要作用。随着溶酶体的研究日益受到基础医学研究工作者及临床学家的重视,因此,开发出摩尔吸光系数大,荧光量子产率高;荧光发射波长处于长波且有较大的斯托克斯位移的荧光探针,在对溶酶体进行标记、监测的应用过程中有着重要意义。
荧光探针具有非侵入性、易操作、能够实现可视化检测等特点,受到普遍的重视。目前许多溶酶体的定位染料都包含一条弱碱性链,可以使探针向酸性的溶酶体细胞器聚集。近年来,研究者开发出了多种溶酶体的标记性染料,一部分已经商业化。可是这些染料共有的一个缺点是激发、发射波长都较短(~350-550nm),在进行活细胞共聚焦成像时,对活细胞造成很大的损伤,同时,此类荧光染料容易发生光漂白,并易受到自发荧光的干扰,这一系列的不足都严重的影响了染料的应用性能。因此开发一种激发、发射波长长,光稳定性好,对细胞具有低毒性的溶酶体探针具有重要意义。
尼罗蓝类荧光染料具有优异的光化学物理性质,例如高的摩尔消光系数、良好的光稳定性、高的荧光量子产率等优点。对细胞的内环境不敏感,具有良好的化学稳定性和光稳定性,而且可以解决通常溶酶体探针对细胞高毒性和光漂白严重等的弊端。
目前公开报道的几类尼罗蓝染料包括1SBPO、2SBPO、Bona11、Bona12等(USPatent Number:6,140,500,2000年10月31日;Development of water-soluble far-redfluorogenic dyes for enzyme sensing,Tetrahedron62(2006)578-585),但这些已公开的化合物不易透过细胞膜进入活细胞内部,并且无法实现溶酶体定位染色。
发明内容
本发明的目的在于提供一类具有优良性能的溶酶体标记性探针的合成及应用。
本发明首先公开一类尼罗蓝荧光染料,具有如下结构通式I:
通式I中:
R1和R2独立地选自H、C1-9烷基、由C1-9烷基任意取代的苯基、由C1-9烷基任意取代的萘基、卤素、OR7、N(R7)2、氰基、(CH2CH2O)nH、(CH2)mCOOM和(CH2)mSO3M;
R3和R4各自独立地选自H、(CH2)2NH2、2-吗啉乙基、C1-6烷基、C1-6烷基任意取代的苯基、C1-6烷基任意取代的萘基、卤素、羟基、巯基、氰基、硝基、卤代烷基、烷基氨基、酰氨基;
R5和R6各自独立地选自H、C1-6烷基、由C1-9烷基任意取代的苯基、C1-9烷基任意取代的萘基、卤素、氰基、(CH2CH2O)nH、(CH2)mCOOM或(CH2)mSO3M;
n、m各自为0-9的整数;
M为H、K、Na、Li、NH4、NH3R7、NH2(R7)2、NH(R7)3或N(R7)4;
R7为H、C1-9烷基或CH2CH2OH。
上述本发明所提供的的结构通式I的尼罗蓝荧光探针的最大发射峰在687nm处,在pH为4.5-6.5(溶酶体pH范围)时,荧光基本保持不变,因此可用于活细胞溶酶体的定位与监测。用1μM浓度的I在37℃孵化12小时后细胞存活率可达98%,且光稳定性好。结构I与LysoSensorTM Green DND-189(以下均简称为LysoSensor Green)在活细胞内溶酶体的染色位置基本相同,Pearson系数在0.95以上。此外,结构I的尼罗蓝类荧光染料还具有高的摩尔消光系数、良好的光稳定性和高的荧光量子产率等优点。
因此,本发明的具有通式I结构的尼罗蓝染料在准确标记溶酶体的基础上解决了通常溶酶体探针对细胞具有高毒性和光漂白严重等的弊端。相对于激发、发射波长都较短(~350-550nm)的商业化溶酶体探针来说,I在进行活细胞共聚焦成像时,能很大程度的降低对活细胞造成的损伤,从而在标记以及监测细胞的整个凋亡过程中有很好的效果。
鉴于此,本发明进一步的目的是提供所述的尼罗蓝荧光染料在制备溶酶体荧光特异性标记试剂中的应用。尤其是在制备用于监测细胞凋亡的试剂中的应用。
附图说明
本发明附图6幅:
图1是对本发明的荧光探针化合物NBB和NBC在不同溶剂中的吸收、发射光谱的测定。探针化合物NBB和NBC的浓度为5μM,测试体系为水、乙醇、甲醇、丙酮、DMSO等。图1a是NBB在不同测试体系中的吸收光谱;图1b是NBB在不同测试体系中的发射光谱;图1c是NBC在不同测试体系中的吸收光谱;图1d是NBC在不同测试体系中的发射光谱;
图2是对本发明的荧光探针化合物NBB和NBC进行pH荧光滴定测试。探针化合物NBB和NBC的浓度为5μM,测试体系为HEPES缓冲溶液。图2a和2b是NBB对pH的荧光滴定光谱和滴定曲线,激发波长为649nm;图2c和2d是NBC对pH的滴定曲线,激发波长为649nm;
图3是本发明的探针化合物NBB和NBC与市售染料LysoSensor Green的光稳定性对比测试。
图4是本发明的探针化合物NBB和NBC和LysoSensor Green在人乳腺癌细胞(MCF-7)中的荧光成像。荧光探针化合物NBB和NBC的浓度是50nM,LysoSensorGreen的浓度是1μM。
图5是本发明的探针化合物NBB和NBC和LysoSensor Green在人乳腺癌细胞(MCF-7)中的复染荧光成像。荧光探针化合物NBB和NBC的浓度是2μM,LysoSensor Green的浓度是1μM。
图6是本发明的探针化合物NBB和NBC的MTT细胞毒性测试。
具体实施方式
本发明所述的尼罗蓝荧光染料,具有如下结构通式I:
具体实施方式之一,所述的R3(或R4)为2-氨基乙基或2-吗啉乙基。优选2-氨基乙基。
具体实施方式之二,所述的R1、R2各自独立地选自氢或甲基;优选甲基。
进一步优选的具体实施方案,R4(或R3)、R5和R6各自独立地选自氢或甲基;最优选H。
最为优选地,本发明所述的罗丹明荧光染料包括化合物NBB和NBC:
本发明中使用的各种原料均可市售获得,或者可通过本领域技术人员公知的方法或现有技术中公开的方法由本领域公知的原料简单地制备得到。
应认识到,本发明化合物中的各种环取代基有一些可在上述步骤进行之前或刚完成后,通过标准的芳族取代反应来引入或通过常规的官能团修饰来产生,这包括在本发明的方法步骤方面。这种反应和修饰包括例如取代基通过芳族取代反应的引入、取代基的还原、取代基的烷基化和取代基的氧化。用于这些过程的试剂和反应条件是化学领域公知的。芳族取代反应的具体实例包括用浓硝酸引入硝基,用例如酰卤和路易斯酸(如三氯化铝)在Friedel Crafts条件下引入酰基,用烷基卤和路易斯酸(如三氯化铝)在Friedel Crafts条件下引入烷基,和引入卤素基团。修饰的具体实例包括通过例如用镍催化剂进行催化氢化或者用铁在盐酸存在下进行加热处理,将硝基还原成氨基;将烷硫基氧化成烷基亚磺酰基或烷基磺酰基。
除另有说明外,本文中使用的术语具有以下含义。
本文中使用的术语“烷基”包括直链烷基和支链烷基。如提及单个烷基如“丙基”,则只特指直链烷基,如提及单个支链烷基如“异丙基”,则只特指支链烷基。例如,“C1-6烷基”包括C1-4烷基、C1-3烷基、甲基、乙基、正丙基、异丙基和叔丁基。类似的规则也适用于本说明书中使用的其它基团。
本文中使用的术语“卤素”包括氟、氯、溴和碘。
下述非限制性实施例可以使本领域的普通技术人员更全面地理解本发明,但不以任何方式限制本发明。
实施例1.荧光探针化合物NBB的合成:
荧光探针化合物NBB的合成:
将化合物2(0.45g,2.4mmol)加入10mL乙醇的100mL单口烧瓶中,冰浴下搅拌,量取5-(二甲氨基)-2-亚硝基苯酚(0.29g,2.0mmol),将固体加入到单口烧瓶中,滴加1毫升浓盐酸,冰浴搅拌4小时,减压蒸除溶剂。重结晶分离得到带有绿色金属光泽的固体NBB(92%)。1H NMR(400MHz,MeOD),δ:1.29(s,6H),3.38(q,J=8Hz,2H),3.98(t,J=8Hz,2H),6.89(d,1H),7.01(s,1H),7.26(q,1H),7.82(t,J=8Hz,1H),7.89(q,J=8Hz,2H),8.48(d,J=8Hz,1H),8.91(d,J=8Hz,1H),13C NMR(100MHz,MeOD),δ:39.68,48.44,48.30,47.08,95.79,96.38,114.88,124.38,124.39,129.64,132.00,132.32,132.45,133.80,148.55,152.92,156.84,157.62ppm;TOF MS:m/z calcd forC20H21N4O+[M]+:333.17,found:333.1705
实施例2.荧光探针化合物NBC的合成:
荧光探针化合物NBC的合成:
将化合物3(0.61g,2.4mmol)加入10mL乙醇的100mL单口烧瓶中,冰浴下搅拌,量取5-(二甲氨基)-2-亚硝基苯酚(0.29g,2.0mmol),将固体加入到单口烧瓶中,滴加1毫升浓盐酸,冰浴搅拌4小时,减压蒸除溶剂。重结晶分离得到带有绿色金属光泽的固体NBC(53%)。1H NMR(400MHz,MeOD),δ:3.33(m,J=4Hz,8H),3.63(s,3H),4.01(s,5H),4.23(d,J=4Hz,2H),7.00(d,J=4Hz,1H),7.18(s,1H),7.41(d,J=8Hz,1H),7.87(m,1H),7.96(t,J=8Hz,2H),8.54(d,J=8Hz,1H),8.97(d,J=8Hz,1H);13C NMR(100MHz,MeOD),δ:38.11,39.98,48.08,48.30,48.44,52.27,54.59,63.65,93.25,95.75,116.66,123.02,123.30,124.15,129.46,131.29,131.64,132.00,132.91,133.13,148.55,151.92,156.80,157.58ppm;TOF MS:m/z calcd for C24H27N4O2 +[M]+:403.21,found:403.2143
实施例3.荧光探针化合物NBB和NBC在不同溶剂中的吸收发射光谱
将荧光探针化合物NBB和NBC分别选择不同溶剂(如水、乙醇、甲醇、丙酮、DMSO等)配成浓度为5μM的溶液,并分别对其吸收、发射光谱进行测定。图1a是NBA在不同测试体系中的吸收光谱;图1a是NBB在不同测试体系中的吸收光谱;图1b是NBB在不同测试体系中的发射光谱;图1c是NBC在不同测试体系中的吸收光谱;图1d是NBC在不同测试体系中的发射光谱;从图中我们可以看出,在中性和偏酸性溶剂中,化合物NBB和NBC的吸收和发射光谱均在近红外范围内,当溶剂呈现弱碱性时,由于染料分子共轭结构的改变,吸收和发射光谱会出现一定程度的蓝移。
实施例4.荧光探针化合物NBB和NBC对pH荧光滴定实验
将5μM的探针化合物NBB加入到HEPES缓冲液(pH7.4)中,通过加入盐酸缓慢调节pH至3,记录响应的荧光强度,激发波长为649nm测试结果显示于图2a和2b图中。从图中可以看出,随着pH的下降,探针化合物最大发射峰665nm处的荧光强度逐渐增强,pH为3.5时基本饱和。同样条件下,激发波长调整为649nm,通过Sigmoidal得到NBB和NBC缓冲溶液的相对荧光强度的变化分别如图2a和2c,pKa值分别为9.89(图2b)和9.97(图2d)。在pH为4.5-6.5(溶酶体pH范围)时,NBB和NBC的荧光强度没有明显变化,表明NBB和NBC均可用于溶酶体的标记与监测。实施例5.荧光探针化合物NBB和NBC与LysoSensor Green的光稳定性对比测试
将化合物NBB和NBC分别配成5uM的HEPES缓冲溶液装入可以密封的比色皿中,将商业化溶酶体定位染料LysoSensor Green配成50uM的PBS缓冲溶液装入可以密封的比色皿中。使用50g/L的亚硝酸钠溶液盛于一个长方体玻璃缸中做截止滤光器,滤去波长小于400nm的紫外光。另外,亚硝酸钠溶液也能起到冷阱的作用,使样品的温度保持常温。测定样品的初始吸光度值后,选用500W碘钨灯作为光源,距离样品30cm处,通电光照,计时。隔10分钟,30分钟,1小时后,测量样品光照后的荧光强度。如图3所示,经过4小时光照后,化合物NBB、NBC和LysoSensor Green在HEPES缓冲液和PBS缓冲液中分别褪色2%、3%和5%。结果显示,本发明设计的探针化合物NBB和NBC具有较强的光稳定性。
实施例6.荧光显微镜下观察探针化合物NBB和NBC对MCF-7活细胞内染色
MCF-7细胞在DEME(invitrogen)中用10%的FCS(invitrogen)培养。共聚焦荧光成像实验前一天,细胞种在专用的细胞共聚焦培养皿中。第二天,向其中加入50nM的探针化合物NBB/NBC,保持在37℃及5%CO2条件下,孵育30分钟,然后用磷酸缓冲溶液冲洗3遍后,进行共聚焦成像。
细胞的培养密度为2×105cells/mL。成像所用仪器为Olympus FV1000-IX81倒置显微镜,100倍油镜。激发光为639nm激发,收集660-690nm波段。
图4a/d是白光图,图4b/e是荧光成像图,图4c/f分别是图4a/d和图4b/e的叠加图。溶酶体在细胞内并不是均一分布的,而是在细胞质中散落分布,溶酶体的pH在4.5-5.5的范围内。NBB和NBC的pKa均大于9.5,因此三者在pH为4.5-5.5的环境中能发出较稳定的荧光。从图4中细胞的发光部分可以看出,探针化合物NBB和NBC进入到了溶酶体细胞器中。
实施例7.荧光显微镜下观察探针化合物NBB、NBC与LysoSensor Green对MCF-7活细胞的复染
LysoSensor Green是一种商品化线粒体绿色荧光探针,可以用于活细胞内溶酶体特异性荧光染色。将探针化合物NBB和NBC与LysoSensor Green对MCF-7进行活细胞染色,比较它们对活细胞的染色位置,能够进一步确定NBB和NBC对溶酶体的特异性荧光标记。
MCF-7细胞在DEME(invitrogen)中用10%的FCS(invitrogen)培养。共聚焦荧光成像实验前一天,细胞种在专用的细胞共聚焦培养皿中。第二天,向其中分别加入2μM的探针化合物NBB和NBC,保持在37℃及5%CO2条件下,孵育30分钟,然后用磷酸缓冲溶液冲洗3遍。再加入1μM的市售染料LysoSensor Green,保持在37℃及5%CO2条件下,孵育15分钟,然后用磷酸缓冲溶液冲洗3遍后,进行共聚焦成像。
细胞的培养密度为2×105cells/mL。成像所用仪器为Olympus FV1000-IX81倒置显微镜,100倍油镜。NBB和NBC的激发光为639nm激发,收集660-690nm波段;LysoSensor Green的激发波长为488nm,收集495-515nm波段。
从图5中可以看出,在MCF-7细胞中,探针化合物NBB、NBC和LysoSensor Green的染色位置基本吻合,共区域化系数达到了0.95以上。表明NBB和NBC很好地定位于溶酶体细胞器中。
实施例8.探针化合物NBB和NBC的细胞毒性测试
将要检测的HeLa和Raw264.7细胞用0.25%胰蛋白酶消化,用含10%胎牛血清的RPMI-1640培养液配成单个细胞悬液,以每孔103~104个细胞接种于96孔培养板中,每孔体积200μL;将培养板移入孵箱中,37℃,5%CO2及饱和湿度下培育24小时后,分别加入染料浓度为0.5μM和1.0μM,继续培养12小时;每孔加入MTT溶液(5mg/mL)20μL,孵育4小时,终止培养,小心吸掉孔内培养上清液。然后,每孔加入150μL的DMSO,振荡10分钟,使结晶物充分溶解;在酶标仪上测定各孔550nm处的吸光度,计算细胞存活率:试验组光吸光度/对照组吸光度值×100%。
从图6中可以看出,探针化合物NBB和NBC对Hela和Raw264.7细胞无明显细胞毒性,孵育12小时后,细胞存活率可以达到96%以上。
Claims (6)
1.一类尼罗蓝荧光染料,具有如下结构通式I:
通式I中:
R1和R2独立地选自H、C1-9烷基、由C1-9烷基任意取代的苯基、由C1-9烷基任意取代的萘基、卤素、OR7、N(R7)2、氰基、(CH2CH2O)nH、(CH2)mCOOM和(CH2)mSO3M;
R3和R4中一个是H,另一个为(CH2)2NH2或2-吗啉乙基;
R5和R6各自独立地选自H、C1-6烷基、由C1-9烷基任意取代的苯基、C1-9烷基任意取代的萘基、卤素、氰基、(CH2CH2O)nH、(CH2)mCOOM或(CH2)mSO3M;
n、m各自为0-9的整数;
M为H、K、Na、Li、NH4、NH3R7、NH2(R7)2、NH(R7)3或N(R7)4;
R7为H、C1-9烷基或CH2CH2OH。
2.权利要求1所述的荧光染料,其特征在于,所述的R1和R2各自独立地选自H或C1-9烷基。
3.权利要求2所述的荧光染料,其特征在于,所述的R1和R2各自独立地选自H或甲基。
4.权利要求3所述的荧光染料,其特征在于,所述的R1和R2均为甲基。
6.权利要求1所述的尼罗蓝荧光染料在制备溶酶体荧光特异性标记试剂中的应用。
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