CN110511203B - 芥子气荧光探针及其制备、应用 - Google Patents
芥子气荧光探针及其制备、应用 Download PDFInfo
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Abstract
本发明公开了一类芥子气荧光探针及其制备、应用；该类探针以氧杂蒽/硅杂蒽/磷杂蒽为荧光母体，以硫酮为识别基团，其结构通式如式(I)所示。该探针合成方法简单，反应时间短，灵敏度高，其检测限为0.8μM。该探针首次实现了生物样本中的芥子气的检测；在环境科学和医学研究中具有广阔的应用前景。
Description
技术领域
本发明属于化学分析检测技术领域，具体涉及一种芥子气荧光探针及其制备、应用。
背景技术
芥子气(sulfur mustard,SM)是难防难治化学战剂的典型代表，被称为“毒剂之王”。芥子气是广泛的烷基化毒剂，可引起体内多靶点多脏器的中毒损伤，其毒理作用机制十分复杂，迄今为止尚未阐明。芥子气中毒后极难治愈，且预后较差，临床多以改善症状的方式进行治疗。
目前传统的分析方法在芥子气的检测上存在一定的局限性。气相色谱分析芥子气时，存在热不稳定，分析时间较长，不能实现体内或细胞水平的动态检测；而同位素示踪法虽然具有灵敏度高，能动态分析的优点，但也存在无法区分芥子气原型与芥子气代谢产物，实验危险性较高；液相色谱样本前处理流程复杂，可导致长时间处理过程中芥子气降解，且需要用到同位素来定位；基于胶体金的原子吸收法存在内源性巯基化合物干扰的问题。因此发展灵敏度高、选择性好，能“实时”动态跟踪芥子气的分析与检测技术是具有重大意义。近年来，小分子荧光探针技术逐渐成为生命科学、环境科学领域不可或缺的研究手段。
申请号为201910054522.2的中国发明专利申请公开了一种基于硫-π相互作用检测芥子气模拟物的荧光材料及其制备方法和应用。其实现对芥子气模拟物-2-氯乙基乙基硫醚具有超高灵敏性(10ppb)和选择性荧光响应的微米球聚集体，其荧光量子产率为20～60％，具有较高的灵敏度。该检测方法属于高分子荧光材料，与小分子荧光探针存在区别。更重要的是芥子气与芥子气模拟物存在一定差别，该荧光材料只测试了其对芥子气模拟物的检测效果，其对芥子气的检测效果尚不明确。此外，上述高分子荧光材料，无法实现生物样本和活细胞水平的芥子气或者芥子气模拟物浓度检测。
发明内容
本发明的目的在于针对上述技术存在的不足提供一种芥子气荧光探针；该探针是一类新型的特异性检测芥子气的荧光探针，以氧杂蒽/硅杂蒽/磷杂蒽为荧光母体，以硫酮为识别基团，具有式I所示结构：
本发明的再一个目的是提供式I所示荧光探针在检测芥子气中的应用。本发明以活细胞中的应用为例子，可通过以下步骤实现：将式I所示探针溶液加入到细胞培养液中孵育，孵育5分钟后，然后用磷酸盐缓冲液(pH＝7.4)洗涤去除多余的探针，观察记录细胞荧光强度，本发明提供的荧光探针的特征在于它本身几乎无荧光信号，但可与芥子气发生迅速的反应，生成强荧光产物，实现大幅的荧光增强，从而实现芥子气荧光强度的定量分析。
本发明的目的是通过以下技术方案来实现的：
第一方面，本发明涉及一类荧光探针，所述荧光探针的化学结构通式如式(I)所示：
第二方面，本发明涉及一种所述的荧光探针在检测芥子气中的用途。
作为本发明的一个实施方案，利用所述荧光探针对粉末、有机溶剂、土壤或生物体系中芥子气进行定性或定量测定。
作为本发明的一个实施方案，所述生物体系包括细胞样本、血浆、组织匀浆、活细胞或动物组织。
第三方面，本发明涉及一种利用所述的荧光探针检测细胞样本中芥子气的方法，所述方法包括如下步骤：
S1、正常处理细胞，处理后将荧光探针溶液加入到细胞培养液中孵育6～10分钟，用磷酸盐缓冲液洗涤去除多余的探针；
S2、观察记录细胞荧光强度，通过细胞的荧光强度来反应芥子气浓度的高低。
第四方面，本发明涉及一种所述的荧光探针的制备方法，所述方法包括如下步骤：
A1、化合物A
与水混合，100～220℃反应5～12小时，冷却过滤得化合物B
R1为氧或氮；
A2、所述化合物B溶于二氯甲烷和吡啶，在0～40℃下滴入三氟甲磺酸酐的二氯甲烷溶液0～40℃下反应2～5个小时，得化合物C
A3、以DMSO为溶剂，所述化合物C与化合物D
在90～120℃下反应3～ 18小时，得化合物E
A4、所述化合物E溶于甲苯，在常温下加入劳森试剂在90～120℃下反应20～60 分钟，得所述荧光探针。
所述化合物B与三氟甲磺酸酐的用量比为0.01mol:6.5mL～0.01mol:10mL；所述化合物C与化合物D的摩尔比为1:10～1：30；所述化合物E和劳森试剂的摩尔比为 1:1～1:3。
第五方面，本发明涉及一种所述的荧光探针的制备方法，所述方法包括如下步骤：
B1、化合物a
碳酸钾、R2-I在乙腈中80～150℃反应过夜，得化合物 b
B2、化合物b以及40％的甲醛水溶液在R2-COOH中80℃反应过夜，反应液加入到冰水中，加入氢氧化钠水溶液将pH调到6～8，到化合物c
B3、化合物c溶于四氢呋喃，-10～-60℃氮气保护下滴加仲丁基锂，-10～-60℃反应2～6小时，加入二甲基二氯化硅，得化合物d
B4、化合物d溶于丙酮，少量多次加入高锰酸钾，0～-20℃反应1～8小时，得化合物e
B5、化合物e、劳氏试剂溶于甲苯80～150℃氮气保护下反应0.5～2小时，得荧光探针
所述化合物a、R2-I的摩尔比为1：4～1：10；所述化合物b、R2-COOH的摩尔比为1：1～1：6。
所述化合物c、二甲基二氯化硅的摩尔比为1：1～1：8；所述化合物e和劳森试剂的质量比为1:2～1:5。
本发明的荧光探针可与样本中的芥子气发生特异性的反应，从而识别样本中是否存在芥子气。该类探针本身几乎无荧光，与芥子气反应后可生成具有强荧光的产物，因而可用于样本中芥子气的灵敏检测。荧光探针法不仅可用于检测细胞样本、血浆、组织匀浆中的芥子气，还可用于活细胞和动物组织中芥子气的动态检测。具体而言，本发明具有以下有益效果：
(1)探针反应速度较快，10分钟内可完成检测；
(2)灵敏度高，检测限低达0.8μM；
(3)具有良好的透膜性，可用于水样和细胞水平的芥子气含量检测；
(4)选择性好，不与常见的生物体内物质发生反应。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1是荧光探针I-1(A)和I-2(B)与不同浓度的芥子气反应前后的荧光变化；
图2是荧光探针I-1(A)和I-2(B)对芥子气的选择性；
图3是荧光探针I-1和I-2检测活细胞中芥子气；其中，A为I-1与芥子气孵育0min，B为I-1与芥子气孵育5min，C为I-1与芥子气孵育10min，D为I-1与芥子气孵育15min， E为I-2与芥子气孵育0min，F为I-2与芥子气孵育5min，G为I-2与芥子气孵育10min， H为I-2与芥子气孵育15min；
图4是荧光探针I-1的核磁共振氢谱；
图5是荧光探针I-1的核磁共振碳谱；
图6是荧光探针I-1的高分辨质谱；
图7是荧光探针I-2的核磁共振氢谱；
图8是荧光探针I-2的核磁共振碳谱；
图9是荧光探针I-2的高分辨质谱。
具体实施方式
下面结合实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干调整和改进。这些都属于本发明的保护范围。
实施例1、芥子气荧光探针的制备
化合物I-1的合成
参照上述反应路线，将原料1(5.0g,0.02mol)与水(65mL)混合于闷罐，220 度反应6小时，冷却后过滤得到固体，然后将固体在真空下旋干得到淡黄色固体化合物2。将化合物2(3.0g,0.01mol)溶于二氯甲烷(300mL)和吡啶(12mL)，在零度下滴入三氟甲磺酸酐(6.5mL)的二氯甲烷(20mL)溶液，在常温下反应两个小时，然后用水淬灭，二氯甲烷层再用1mol/L的稀盐酸和饱和食盐水洗涤，硫酸钠干燥，浓缩后得到淡黄色固体化合物3。化合物3(3.0g,6.09mmol)溶于DMSO(17mL)，向其中加入二乙胺(4.5g,60.97mmol)，在90度下反应18个小时，然后溶于乙酸乙酯，再缓慢加入到水中，有机层分液后再用饱和食盐水洗涤后，用无水硫酸钠干燥，旋干得到粗品。粗品用硅胶过柱纯化(石油醚：乙酸乙酯＝10:1～4:1)得到黄色固体化合物4。化合物4 (770mg,2.27mmol)溶于甲苯(20mL)，在常温下加入劳森试剂(1.1g,2.73mmol)，在90度下反应20分钟。反应液用乙酸乙酯稀释，然后用水和饱和食盐水洗涤，无水硫酸钠干燥，旋干得到粗品。粗品用硅胶过柱纯化(二氯甲烷为洗脱剂)得到红色固体化合物I-1(核磁共振氢谱如图4，核磁共振碳谱如图5，高分辨质谱如图6)。1H NMR(400MHz,CDCl3)δ8.66(d,J＝9.3Hz,2H),6.70(dd,J＝9.3,2.6Hz,2H),6.40(d,J＝2.5Hz, 2H),3.47(q,J＝7.1Hz,8H),1.25(t,J＝7.1Hz,12H).13C NMR(101MHz,CDCl3)δ 195.37,153.42,152.53,132.01,119.67,110.25,95.37,44.91,12.64.HRMS(ESI,m/z):Calcd. for[C21H26N2OS+H]+,355.1844；Found,355.2010.
实施例2、芥子气荧光探针的制备
化合物I-2的合成
参照上述反应路线，将化合物5，碳酸钾，碘乙烷在100mL的乙腈中80度反应过夜，过滤，滤液旋干过柱得到油状物化合物6。原料6以及40％的甲醛水溶液在20mL 的乙酸中80度反应过夜，反应液加入到冰水中，加入氢氧化钠水溶液将pH调到8，乙酸乙酯萃取并旋干过柱得到化合物7。化合物7溶于20mL四氢呋喃，-60度氮气保护下滴加仲丁基锂，-60度反应2小时，加入二甲基二氯化硅，恢复室温，反应过夜，直接旋干投入下一步。化合物8溶于20mL丙酮，少量多次加入高锰酸钾，零度反应2小时，过滤旋干过柱纯化得到70mg油状物化合物9。化合物9 50mg，劳氏试剂106mg溶于5mL甲苯90度氮气保护下反应1小时，直接过柱纯化得到20mg绿色金属状固体I-2 (核磁共振氢谱如图7，核磁共振碳谱如图7，高分辨质谱如图9)。1H NMR(400MHz, CDCl3)δ8.78(d,J＝9.1Hz,2H),6.79–6.60(m,4H),3.46(q,J＝7.1Hz,8H),1.23(t,J＝ 7.1Hz,12H),0.43(s,6H).13C NMR(101MHz,CDCl3)δ190.79,162.36,148.89,136.05, 135.79,112.86,44.72,12.78.HRMS(ESI,m/z):Calcd.for[C23H32N2SSi+H]+,397.2134； Found,397.2317.
实施例3、荧光探针I-1和I-2与不同浓度的芥子气的反应前后的荧光变化
将荧光探针少量溶解于乙腈，分别加入不同浓度的芥子气，探针的终浓度为20μM。使用550nm/650nm激发，记录溶液在最大发射波长下的荧光强度，反应15分钟后，I-1 和I-2溶液中的荧光强度达到最大，可知I-1和I-2的反应时间均在15min以内。将I-1 和I-2与不同浓度的芥子气反应，观察反应后荧光强度，结果如图1所示；由图1可知，探针的荧光强度可以随着芥子气浓度的升高而升高。
实施例4、荧光探针I-1和I-2对芥子气的选择性
将DMSO溶解后的荧光探针，将探针分别加入含有或不含有芥子气的不同溶液中，从1到21分别为空白对照、芥子气、DCP、Na2S、谷胱甘肽、半胱氨酸、三氯化磷、盐酸、碘乙烷、氰化钾，分别记录其溶液的荧光强度，如图2所示；由图2可知，I-1 和I-2均可选择性的与芥子气反应，不与其他化合物反应。
实施例5、荧光探针I-1和I-2检测活细胞中的芥子气
在37℃、5％CO2的条件下，将HaCaT细胞接种于细胞培养液中培养。细胞生长密度为80％，胰酶消化，接种于共聚焦拍照用的腔室盖玻片上。向细胞先后加入I-1(10 μM)或I-2(10μM)。处理后将芥子气加入到细胞培养液中孵育，然后用磷酸盐缓冲液(pH ＝7.4)洗涤去除多余的探针。用激光共聚焦显微镜进行观察、拍照，其激发波长为514 nm/633nm，从图3中可以观察到细胞中的荧光强度逐渐增强，I-1到10min后荧光强度进入平台期，I-2到15min达到平台期，表明探针可以在细胞水平上检测芥子气，且检测时间分别为10min和15min，结果如图3所示。
以上对本发明的具体实施例进行了描述。需要理解的是，本发明并不局限于上述特定实施方式，本领域技术人员可以在权利要求的范围内做出各种变形或修改，这并不影响本发明的实质内容。
Claims (3)
1.一种荧光探针在制备检测生物体系中芥子气的探针中的用途，所述荧光探针的结构式为：
所述荧光探针可以对生物体系中芥子气进行定性或定量测定。
2.根据权利要求1所述的用途，其特征在于，所述生物体系包括细胞样本、血浆、组织匀浆、活细胞或动物组织。
3.一种非诊断、非治疗性的利用荧光探针检测细胞样本中芥子气的方法，其特征在于，所述荧光探针的结构式为：
所述方法包括如下步骤：
S1、正常处理细胞，处理后将荧光探针溶液加入到细胞培养液中孵育6～10分钟，用磷酸盐缓冲液洗涤去除多余的探针；
S2、观察记录细胞荧光强度，通过细胞的荧光强度来反映 芥子气浓度的高低。
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