CN114014892A - 一种特异性识别超氧阴离子的bodipy类荧光探针及其制备方法和应用 - Google Patents
一种特异性识别超氧阴离子的bodipy类荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种特异性识别超氧阴离子的BODIPY类荧光探针及其制备方法和应用,基于BODIPY结构的、含有二苯基磷酸酯识别基团的荧光增强探针BOIPDY‑T,对超氧阴离子具有高度的选择性。此外,该探针可在极宽的pH范围内对超氧阴离子实现检测,而且探针自身的荧光不受环境pH的影响,这对它的实际应用是非常有利的;探针BOIPDY‑T具光学性质优良,低pH敏感性,高灵敏度,高选择性,可以很容易在化学分析检测领域具有广阔的应用前景。
Description
技术领域
本发明涉及荧光探针技术领域,尤其是涉及一种特异性识别超氧阴离子的BODIPY类荧光探针及其制备方法和应用。
背景技术
在生物体内的各种活性氧(ROS)中,通过线粒体呼吸链的氧(O2)产生的超氧阴离子是生物体内极为重要的杀菌氧化剂,对人体健康起着保护作用。然而,超氧阴离子的过度产生与一系列病理疾病有关,如阿尔茨海默病、类风湿性关节炎、心脏病、糖尿病,甚至癌症。此外,生物体内产生的超氧阴离子寿命很短,通常在产生地点的几十微米范围内转化为其他ROS。尽管超氧阴离子浓度的变化在体内起着非常重要的作用,但其低浓度和短寿命使得检测超氧阴离子非常困难。因此,在复杂的生物系统中对超氧阴离子进行特异性测量和跟踪,对阐明相关疾病的确切发病机制很有意义。目前为止,已经报道了许多关于超氧阴离子的化学小分子荧光探针。例如公开号为CN107337681A、CNCN111100184A等。
但是,这些现有技术并不是基于BODIPY的探针。BODIPY家族成员作为一大荧光染料,由于其优异的光谱和光物理性能,包括优异的环境稳定性,以及大的摩尔消光系数、高的量子产率等,已被广泛应用于功能性荧光生物探针母核。截止目前,该荧光生物探针母核并未实现将BODIPY优异的性能应用于超氧阴离子特异性识别上,因此有待改进。
发明内容
有鉴于现有技术的上述问题,本发明的目的是克服现有技术中的不足,提出一种特异性识别超氧阴离子的BODIPY类荧光探针及其制备方法和应用。
本申请提供的一种特异性识别超氧阴离子的BODIPY类荧光探针,所述荧光探针分子式为C33H30BF2N2O4P,其结构式为:
一种特异性识别超氧阴离子的BODIPY类荧光探针的制备方法,包括如下步骤:
将T-Br、BODIPY-COOH和DBU混合物溶于无水四氢呋喃中制得混合溶液A,在氩气保护下室温搅拌,待反应完毕后将溶剂减压旋干;柱层析进一步纯化,用洗脱剂洗脱后得到探针BODIPY-T;
其中T-Br的结构式为
其中BODIPY-COOH的结构式为
其中DBU的结构式为
进一步的,所述T-Br的制备方法包括如下步骤:
S1、原料混合、反应:将T-OH、NBS和DMTU混合物溶于二氯甲烷中,制得体积摩尔浓度为0.1-1.0mol/L的混合溶液B,二氯甲烷亦在其中起到稀释反应物的作用,在室温条件下搅拌,得反应液;
S2、制备中间体T-Br:将反应液用水洗涤3次,接着将二氯甲烷用无水硫酸钠干燥,最后将溶剂减压旋干后得到中间体T-Br;
其中T-OH的结构式为
其中NBS的结构式为
其中DMTU的结构式为
进一步的,所述洗脱剂为石油醚和乙酸乙酯混合液,两者体积比为2:1。
进一步的,所述T-Br,BODIPY-COOH和DBU的摩尔比为9:5:10。
进一步的,所述T-OH,NBS和DMTU的摩尔比为1:1.5:0.45。
进一步的,所述室温为15-30℃,优选为25℃。
进一步的,所述混合溶液A的体积摩尔浓度为0.5-1.0mol/L。
特异性识别超氧阴离子的BODIPY类荧光探针在检测、识别环境中或生物样品中超氧阴离子的检测试剂、水中超氧阴离子浓度测定或者标记物中的用途。
进一步的,用于制备正常细胞和活体体内的超氧阴离子的检测试剂或者标记物中的应用。
进一步的,超氧阴离子的BODIPY类荧光探针测定超氧阴离子的检测方法如下:
通过荧光分光光度法,以480nm为激发波长,在530nm的波长处测定超氧阴离子的荧光强度;样品的检测限为0.062微摩尔。
通过采用上述检测方法:本发明的荧光探针其本身荧光微弱,在与超氧阴离子作用后迅速有很强的荧光发射,最大发射波长为530nm,本发明的荧光探针的斯托克斯位移约60nm,其最大吸收波长为470nm附近,与超氧阴离子反应后最大发射波长为530nm。
综上所述,本申请包括以下有益技术效果:
1、BODIPY-COOH的酯化是设计荧光探针的独特技术,因为酯化的衍生物是吸光度红移且荧光量子产率急剧下降,而羧酸化的衍生物是吸收蓝移且有较高荧光的。在本发明中,利用BODIPY-COOH作为母核,利用二苯基磷酸酯为超氧阴离子识别基团设计并合成了一种新的超氧阴离子荧光探针(BODIPY-T),用于超灵敏和选择性地检测活细胞中的内源性超氧阴离子而不受细胞内氧化还原剂和pH值的干扰;
2、本申请的荧光探针还可用于水中超氧阴离子浓度测定和细胞中超氧阴离子成像的研究;
3、本发明的荧光探针对超氧阴离子有很好的选择性,在pH等于7.4的PBS缓冲溶液中,探针溶液荧光强度微弱,加入20μM超氧化钾,荧光强度逐渐增强,5分钟后增大到最初的12.9倍左右。而同样条件下分别加入其他可能干扰的氨基酸和离子,探针的荧光强度并没有明显变化,对超氧阴离子具有高度的选择性;
4、本发明的荧光探针对超氧阴离子的检测表现出很高的灵敏度。探针溶液的荧光强度,随着增加超氧阴离子浓度而递增,大约在加入4倍量超氧阴离子时达到峰值。在0到2.4倍量超氧阴离子的区间内,探针溶液荧光强度与超氧阴离子浓度有很好的线性关系;
5、探针在非常宽的pH范围内能对超氧阴离子进行准确检测,且自身荧光不受pH影响;
6、本发明的BODIPY衍生物作为探针的荧光母核,BODIPY衍生物的结构决定其荧光性质十分优异。通过修饰形成的探针本身荧光可以几乎完全掩蔽,与超氧阴离子作用数分钟后,荧光强度迅速增强(荧光增强倍数大于12倍),而且具有很高的量子产率。检测限低至0.062微摩尔,灵敏度高,选择性和抗干扰性能好;
综上所述,本发明的荧光探针是一种便捷,灵敏,适用于体外和活细胞内部检测超氧阴离子的工具,在化学分析检测领域具有广阔的应用前景。
附图说明
图1为本发明荧光探针制备过程示意图;
图2本发明中间体T-Br的质谱谱图;
图3本发明荧光探针BODIPY-T的核磁氢谱谱图;
图4本发明荧光探针BODIPY-T的核磁碳谱谱图;
图5本发明荧光探针BODIPY-T的质谱谱图;
图6为本发明的荧光探针(5μM)在PBS:DMSO 4:1缓冲溶液(pH=7.4)中,加入递增浓度超氧阴离子(0-20μM),反应5分钟后的吸收光谱图和荧光光谱图;
图7为本发明的荧光探针与超氧阴离子反应机理图;
图8为本发明的荧光探针(5μM)在PBS:DMSO 4:1缓冲溶液(pH=7.4)中,加入递增浓度超氧阴离子(0-20μM),反应5分钟后的荧光强度变化曲线。其插图为本发明的荧光探针(10μM)在PBS:DMSO 4:1缓冲溶液(pH=7.4)中,加入递增浓度超氧阴离子(0-12μM)的线性关系图;
图9为本发明荧光探针的各种不同离子和分子选择性的柱状图。其中,彩色柱分别代表以下物质(20μM)与探针(5μM)作用后的荧光强度:氯化锰,硫酸钠,硝酸银,硫酸亚铁,硫酸铁,半胱氨酸,谷胱甘肽,硫化钠,碳酸氢钠,硫氢化钠,硫酸钠,水合肼,硝酸,次氯酸,过氧化氢,超氧化钾,水;
图10为本发明荧光探针在不同pH环境对超氧阴离子响应情况及荧光变化图;
图11为本发明的荧光探针的MTT毒性实验与探针浓度梯度(最终浓度)变化图;
图12为本发明的荧光探针(5μM)在不同条件下RAW264.7细胞中成像结果;
图13为图6-12的彩色原图的二维码。
具体实施方式
以下结合附图1-13对本申请作进一步详细说明。
本申请实施例公开一种特异性识别超氧阴离子的BODIPY类荧光探针的制备方法。
该荧光探针的合成路线如图1所示。
将T-OH(325mg,1.0mmol,1.0eq.)和NBS(265mg,1.5mmol,1.5eq.)混合物溶于5mL二氯甲烷中,加入DMTU(47mg,0.45mmol,0.45eq.),此时混合溶液的体积摩尔浓度为0.59mol/L,室温搅拌2小时。反应完毕混合溶液用二氯甲烷稀释,然后用水洗涤三次,接着将二氯甲烷用无水硫酸钠干燥,最后将溶剂减压旋干后得到中间体T-Br 227mg,产率70%。其质谱图,如图2所示。
HRMS(ESI)calculated for C19H17BrO2P+,[M+H]+,387.0144,found,387.0142.
将中间体T-Br(193mg,0.45mmol),BODIPY-COOH(75.0mg,0.25mmol)溶于5mL无水四氢呋喃中,然后加入DBU(76.0μL,0.5mmol),此时混合溶液的体积摩尔浓度为0.24mol/L,在氩气保护下室温搅拌过夜小时。反应结束后减压旋干去除四氢呋喃,将残余物溶于二氯甲烷,用1N盐酸水溶液洗涤两次,在硫酸钠上干燥,并蒸发至干。柱层析进一步纯化,残余物在硅胶上以石油醚/乙酸乙酯(2/1v/v)为洗脱剂进行纯化,得到探针BODIPY-T 82mg,产率55%。其核磁图谱和质谱图,如图3-5所示。
1HNMR(400MHz,CDCl3)δ=7.92-7.89(m,2H),7.89-7.86(m,2H),7.58-7.53(m,2H),7.50-7.45(m,4H),7.33(d,J=8.4Hz,2H),7.25(d,J=8.4Hz,2H),6.00(s,2H),5.29(s,2H),2.51(s,6H),1.90(s,6H).13CNMR(101MHz,CDCl3)δ=164.9,157.7,151.5,141.2,132.7,131.9,131.8,131.0,130.0,128.8,128.7,121.2,121.13,121.08,67.9,14.8,12.6.HRMS(ESI)calculated for C33H31BF2N2O4P+,[M+H]+,599.2077,found,599.2076.
本发明的荧光探针对超氧阴离子的检测机理详情如下所述:BODIPY的羧基被酯化后,酯基的强吸电子效应导致荧光被掩蔽掉。BODIPY-T的磷酸酯被超氧阴离子氧化后,导致分子内发生消除反应,引发酯键断裂导致羧基暴露,发射很强的荧光。响应过程中探针的吸收和荧光变化如图6所示,荧光探针与超氧阴离子反应机理如图7所示。
实验分析:
一、荧光强度与超氧阴离子浓度的关系
取5μM荧光探针的PBS:DMSO 4:1缓冲溶液(pH=7.4),加入递增浓度超氧阴离子(0-20μM),分别有0μM、2μM、4μM、6μM、8μM、10μM、12μM、14μM、16μM、18μM、20μM,反应5分钟后,其荧光光谱图如图8a所示,其荧光强度与超氧阴离子的浓度的线性关系图如图8b所示,计算得到该分子的检测限可以达到0.062微摩,适合微量检测。可见本发明的荧光探针具有良好的应用前景。
二、荧光探针抗干扰能力检测
取探针分子溶液(5μM)于在PBS:DMSO 4:1缓冲溶液(pH=7.4)中配成待测溶液,再分别加入各种干扰物分子,氯化锰,硫酸钠,硝酸银,硫酸亚铁,硫酸铁,半胱氨酸,谷胱甘肽,硫化钠,碳酸氢钠,硫氢化钠,硫酸钠,水合肼,硝酸,次氯酸,过氧化氢,超氧化钾,水,反应5分钟后测量,其结果如图9所示,溶液的荧光几乎没有明显变化,而只加入超氧阴离子的探针溶液荧光大幅度增强(约12倍),可见该荧光探针能够对超氧阴离子实现专属识别。本发明的荧光探针对超氧阴离子的检测具有很强的抗干扰能力。
三、荧光探针在不同pH环境对超氧阴离子响应情况及荧光变化
取探针分子溶液(5μM)于不同pH(pH=2、3、4、5、6、7、8、9、10、11、12)的磷酸盐缓冲液中配成待测溶液,分别测量其荧光强度,然后分别加入20μM超氧化钾,反应5分钟后测量,其结果如图10所示。探针分子在不同pH的缓冲溶液中,荧光强度保持稳定,而在不同pH缓冲也中与超氧化钾后荧光增强约12倍,且荧光强度不受pH影响。可见本发明的荧光探针具有良好的抗环境pH干扰能力。
四、荧光探针的MTT毒性实验
RAW264.7细胞接种于4孔培养皿(1.3×104个/孔),放置于细胞培养箱中,使之完全贴壁。之后更换新鲜培养液,加入200μL不同浓度100μM、80μM、60μM、40μM,20μM、10μM、5μM、0μM的荧光探针分散液,培养24h后,在酶标仪上将波长设定为570nm,测定96孔板每孔溶液的吸光度(OD值),按下列公式计算细胞生存率:细胞存活率=(OD待测组-OD空白组)/(OD细胞组-OD空白组)×100%。从图11中看出,细胞存活率大于80%,基本无毒性。
五、荧光探针(5μM)在不同条件下RAW264.7细胞中成像结果
为了探究探针的生物应用性,我们利用探针BODIPY-T检测细胞水平的超氧阴离子含量。将RAW264.7细胞在Dulbecco’s modified Eagle’s medium(DMEM)中培养。荧光成像前,将RAW264.7细胞接种于4孔培养皿(1.3×104个/孔),培养1天。
第一组RAW264.7细胞只与BODIPY-T孵育。
第二组RAW264.7细胞先用PMA处理30分钟,再用含BODIPY-T(5μM)的培养基中处理30分钟。
第三组RAW264.7细胞先用LPS处理30分钟,再用含BODIPY-T(5μM)的培养基中处理30分钟。
第4组RAW264.7细胞先用LPS处理30分钟,再用含Tiron(活性氧清除剂)处理30分钟,最后用含BODIPY-T(5μM)的培养基中处理30分钟。共聚焦成像前,将培养皿中用过的培养基废弃,用磷酸盐缓冲液清洗3次。
最后所有组细胞用激光显微镜成像,其中激发波长为488nm,收集波段为515-580nm。第一组对照细胞荧光微弱(图12a),第二组而用PMA预先处理的细胞,在绿色通道中观察到强烈的荧光(图12b)。第三组加入了LPS超氧阴离子诱发剂预处理的细胞,也能观察到强烈的绿色荧光后(图12c)。第四组先用LPS处理,再用Tiron处理的细胞,则荧光强度明显弱于第二组和第三组(图12d),这一结果表明BODIPY-T可以检测到活细胞中超氧阴离子含量的差异。以上结果验证了BODIPY-T可以作为一种良好的荧光探针选择性检测活细胞中的超氧阴离子。
其中:
图12中的a1、b1、c1和d1均代表细胞中明场成像图;
图12中的a2、b2、c2和d2均代表细胞中荧光成像图;
图12中的a3、b3、c3和d3均代表细胞中Merge图。
为便于对图6-12更直观的观察,特附上图6-12的彩色原图的二维码。具体的,通过手持终端对图3的二维码进行扫描。
以上均为本申请的较佳实施例,并非依此限制本申请的保护范围,故:凡依本申请的结构、形状、原理所做的等效变化,均应涵盖于本申请的保护范围之内。
Claims (10)
4.根据权利要求3所述的一种特异性识别超氧阴离子的BODIPY类荧光探针的制备方法,其特征在于:所述洗脱剂为石油醚和乙酸乙酯混合液,两者体积比为2:1。
5.根据权利要求4所述的一种特异性识别超氧阴离子的BODIPY类荧光探针的制备方法,其特征在于:所述T-Br,BODIPY-COOH和DBU的摩尔比为9:5:10。
6.根据权利要求5所述的一种特异性识别超氧阴离子的BODIPY类荧光探针的制备方法,其特征在于:所述T-OH,NBS和DMTU的摩尔比为1:1.5:0.45。
7.根据权利要求6所述的一种特异性识别超氧阴离子的BODIPY类荧光探针的制备方法,其特征在于:所述混合溶液A的体积摩尔浓度为0.5-1.0mol/L。
8.权利要求1所述的特异性识别超氧阴离子的BODIPY类荧光探针在检测、识别环境中或生物样品中超氧阴离子的检测试剂、水中超氧阴离子浓度测定或者标记物中的用途。
9.根据权利要求8所述的一种特异性识别超氧阴离子的BODIPY类荧光探针的应用,其特征在于:用于制备正常细胞和活体体内的超氧阴离子的检测试剂或者标记物中的应用。
10.根据权利要求8所述的一种特异性识别超氧阴离子的BODIPY类荧光探针的应用,其特征在于:超氧阴离子的BODIPY类荧光探针测定超氧阴离子的检测方法如下:
通过荧光分光光度法,以480nm为激发波长,在530nm的波长处测定超氧阴离子的荧光强度;样品的检测限为0.062微摩尔。
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