CN109232626B - 一种基于二氟硼氧基香豆素的so2比率型荧光探针 - Google Patents
一种基于二氟硼氧基香豆素的so2比率型荧光探针 Download PDFInfo
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Abstract
Description
技术领域
本发明属于荧光探针领域,具体涉及一种检测二氧化硫/亚硫酸(氢)盐的荧光探针。
背景技术
二氧化硫(SO2)是大气中主要污染物之一,进入呼吸道后,因其易溶于水,故大部分被阻滞在上呼吸道,在湿润的粘膜上生成具有腐蚀性的亚硫酸、硫酸和硫酸盐,使刺激作用增强。又由于亚硫酸钠和亚硫酸氢钠的防腐、抗氧化作用而被广泛用作食品添加剂,所以SO2与人类健康的关系非常密切。再者,SO2被认为是“第四种气体信号分子”,其在生物体的浓度与其功能密切相关,因此,设计合成近红外双光子的SO2荧光探针对于研究其在生物体内的功能研究具有重要的意义。目前,针对检测二氧化硫的分子荧光探针已有文献报道,但这些常见的荧光探针响应时间较长、散射干扰等缺陷,这样有可能影响在复杂生理环境中对二氧化硫检测的应用。
发明内容
针对现有技术中的探针在检测二氧化硫/亚硫酸(氢)盐存在响应时间较长、散射干扰等问题,本发明提供一种以二氟硼氧基香豆素为荧光团的新型SO2探针,检测时间短。
为实现上述目的,本发明采用如下技术方案。
一种基于二氟硼氧基香豆素检测二氧化硫/亚硫酸(氢)盐的荧光探针,其结构式如式(I)所示:
式(I)。
上述荧光探针的制备方法,包括以下步骤:
(1)苯酚与丙二酸在三氯氧磷中加热回流反应,得到化合物1:
(2)化合物1与4-二乙胺基苯酚在甲苯中加热回流反应,分离、纯化得化合物2:
(3)化合物2和乙酸酐在吡啶中加热回流反应,分离、纯化得化合物3:
(4)化合物3和三氟化硼乙醚在甲苯中回流加热反应,分离、纯化得化合物4:
(5)化合物4和对苯二甲醛在浓硫酸存在下于冰乙酸中回流加热反应,分离、纯化得荧光探针:
步骤(1)中苯酚与丙二酸的摩尔比为1:1。
步骤(2)中化合物1与4-二乙胺基苯酚的摩尔比为1:1。
步骤(3)中化合物2和乙酸酐的摩尔比为1:1.2。
步骤(4)中化合物3与三氟化硼乙醚的摩尔比为1:2。
步骤(5)中化合物4与对苯二甲醛的摩尔比为1:1.2。
步骤(1)-(5)中,所述加热温度为110-120℃。
步骤(2)中所述分离纯化步骤为将反应体系冷却至室温,减压过滤,滤饼用乙醇洗涤2-3次,真空干燥,所得粗产物以乙醇重结晶,得到提纯品。
步骤(3)中所述分离纯化步骤为将反应体系冷却至室温,减压过滤,滤饼用乙醇洗涤2-3次,真空干燥,然后粗产物均进行柱层析纯化,层析淋洗液为二氯甲烷:石油醚=1:1(v/v)。
步骤(4)中所述分离纯化步骤为将反应体系冷却至室温,然后加入石油醚中,减压过滤,滤饼用乙醇洗涤2-3次,真空干燥即得。
步骤(5)中所述分离纯化步骤为将反应体系冷却至室温,真空旋干溶剂,然后粗产物均进行柱层析纯化,层析淋洗液为二氯甲烷:乙醇=50:1(v/v)。
一种上述荧光探针在检测溶液和细胞内亚硫酸(氢)盐的应用。
本荧光探针的检测机理如下:
本发明的荧光探针以二氟硼氧基香豆素为荧光团,含有碳碳双键。探针本身具有近红外的荧光,在亚硫酸氢盐或亚硫酸盐存在下,可采用亲核加成方式进攻不饱和C=C键,发出二氟硼氧基香豆素的荧光,表现为在710 nm处所发射的荧光由强变弱,在480 nm处所发射的荧光由弱变强。可根据两个波长下的荧光变化检测亚硫酸氢盐或亚硫酸盐的存在。
本发明具有以下优点:
本发明的荧光探针结构新颖、检测速度快,能够采用化学方法大量合成。能够成功应用于细胞成像,所述探针对于监测活细胞中SO2衍生物有潜在应用价值,在生物体分析检测中显示出很大的优越性。
附图说明
图1为探针的1H NMR谱;
图2为探针检测不同浓度的亚硫酸氢钠的荧光强度;
图3为探针在PBS缓冲液中的光稳定性;
图4为探针的离子选择性测试;
图5为探针的细胞成像应用。
具体实施方式
下面结合实施例和附图对本发明做进一步说明,但本发明不受下述实施例的限制。
实施例1 探针的合成
(1)化合物1的合成
在50 mL的圆底烧瓶中,加入苯酚(1.88 g,20 mmol)与丙二酸(2.08 g,20 mmol)混合,向其中加入20 mL三氯氧磷并加热至110 ℃,回流搅拌3 h,然后将溶剂旋干,冷却至室温,将反应体中的上层透明黄棕色油状液体缓慢倒出,得到化合物1,产率:65%,粗产物无需纯化可直接参与下一步反应;
(2)化合物2的合成
在50 mL的圆底烧瓶中,加入化合物1(1.02 g,4 mmol)与4-二乙胺基苯酚(660mg,4 mmol)混合,向其中加入20 mL甲苯,并加热至115 ℃,回流搅拌7 h,冷却至室温,出现大量浅黄色沉淀,减压过滤,滤饼用乙醇洗涤2-3次,真空干燥,所得粗产物以乙醇重结晶可得到提纯品化合物2,产率:81%;
(3)化合物3的合成
在50 mL的圆底烧瓶中,向其中加入吡啶20 mL,加入化合物2(699 mg,3 mmol),乙酰乙酸乙酯(306 mg,3 mmol),加热到120 ℃后,回流6 h,冷却至室温,出现浅黄色沉淀,减压过滤,滤饼用乙醇洗涤3次,真空干燥,得粗产物,然后进行柱层析纯化,层析淋洗液为二氯甲烷:石油醚=1:1(v/v),除去溶剂得淡黄色固体,即为化合物2,产率:68%;
(4)化合物4的合成
在50 mL的圆底烧瓶中,加入20 mL甲苯,加入化合物3(550 mg,2 mmol),加热温度至115 ℃,在氮气保护下向其中缓慢滴加三氟化硼乙醚500 μL,回流搅拌半小时,然后冷却至室温后加石油醚后,析出浅黄色沉淀,减压过滤,滤饼用乙醇洗涤2-3次,真空干燥,得淡黄色固体,即为化合物4,产率:98%;
(5)荧光探针的合成
在30 mL的圆底烧瓶中,向其中加入10 mL冰乙酸,然后加入化合物4(323 mg,1mmol)和对苯二甲醛(160 mg,1.2 mmol)后加热搅拌,当温度达到60 ℃后,滴加500 μL的浓硫酸,加热至120 ℃回流8 h后减压蒸馏去除溶剂,所得粗产物进行柱层析纯化,层析淋洗液为二氯甲烷:甲醇=50:1,得荧光探针,产率:60%。探针的1H NMR谱如图1。1H NMR (400MHz, DMSO) δ 10.07 (s, 1H), 8.45 (d, J = 15.8 Hz, 1H), 8.25 (d, J = 15.8 Hz,1H), 8.04 (q, J = 8.4 Hz, 4H), 7.85 (d, J = 9.4 Hz, 1H), 6.93 (dd, J = 9.4,2.2 Hz, 1H), 6.67 (d, J = 2.2 Hz, 1H), 3.60 (q, J = 7.0 Hz, 4H), 1.26–1.12(m, 6H)。
实施例2 不同浓度的亚硫酸氢钠中探针的荧光强度变化
配制浓度为1 mM的实施例1所得荧光探针的二甲基亚砜测试母液溶液待用。
配制探针终浓度为10 μM,含20 %乙腈溶液的PBS溶液(pH 7.4),分别与不同浓度的亚硫酸氢钠(0μM、10μM、20μM、40μM、60μM、90μM、120μM、150μM、180μM、210μM、250μM、280μM、300μM)充分作用,进行荧光检测(λex=410 nm,λem=480 nm;λex=520 nm,λem=710 nm)。得各体系中荧光强度,建立荧光强度与亚硫酸氢钠浓度标准曲线,结果如图2所示。由图2可知,随着亚硫酸氢钠浓度的增加,480 nm处的荧光强度逐渐增加,710 nm处的荧光强度逐渐降低;当亚硫酸氢钠浓度达到300 μM时,反应体系荧光强度达到饱和状态。
实施例3 探针在PBS缓冲液中的稳定性
配制浓度为1 mM的实施例1所得荧光探针的二甲基亚砜测试母液溶液待用。
配制探针终浓度为10 μM,含20%乙腈溶液的PBS溶液(pH 7.4),与亚硫酸氢钠(300μM)充分作用,每隔5分钟进行荧光检测(λex=410 nm,λem=480 nm;λex=520 nm,λem=710 nm)。得不同时间点体系中480nm与720nm处荧光强度比值,建立I480/I720与时间的标准曲线。如图3所示,所述探针在90min内的稳定性良好。
实施例4 探针的选择性
配制浓度为1 mM的实施例1所得荧光探针的二甲基亚砜测试母液溶液待用。配制浓度为100 mM的各种不同离子,氨基酸和活性氧/活性氮,不同活性硫溶液作为备用。
配制探针终浓度为10 μM,含20%乙腈溶液的PBS溶液(pH 7.4),加入30当量的各离子溶液或30当量的各氨基酸溶液,用PBS缓冲液定容,体系pH为7.4,摇匀后进行荧光检测(λex=410 nm,λem=480 nm;λex=520 nm,λem=710 nm),建立荧光强度与各离子的柱状图,如图4所示,其中,1-25号加入的分别是:探针,氯化钙,氯化钴,硫酸铜,硫酸铁,硫酸亚铁,碘化钾,硫酸钠,氯化镁,亚硫酸钠,硝酸钾,氟化钠,亚硝酸钠,硫酸镍,氯化亚锡,硫酸锌,硫酸银过氧化叔丁醇,过氧化叔丁基,过氧化氢,次氯酸钠,同型半胱氨酸,半胱氨酸,谷胱甘肽,硫氢化纳,亚硫酸氢钠。测试离子的浓度为300 μM,氨基酸的浓度为300 μM,活性氧活性氮浓度为300 μM。测试溶液的荧光发射光谱,由图4可以发现,其他离子(或氨基酸)对探针的荧光几乎没有影响。
实施例5 探针细胞成像
将适当密度的HeLa细胞接种到两个灭菌的35 mm成像培养皿中,在CO2培养箱(温度为37℃,5% CO2)中培养,待细胞贴壁后,向培养皿中加入实施例1所得荧光探针,使其终浓度均为5 μM。继续培养0.5 h,其中一个加入适量亚硫酸氢钠水溶液,使终浓度为150 μM,孵育0.5 h后,随后进行细胞成像(激发波长405 nm,检测波段为425-475 nm;激发波长561nm,检测波长为570-620 nm),结果如图5所示,其中a1-d1为探针孵育细胞成像,a2-d2为探针与SO2衍生物孵育的细胞成像;而a1、a2为明场成像获得的图像,b1、b2为绿通道成像获得的图像,c1、c2为红通道成像获得的图像,d1,d2为三图像叠加后的图像。由图5可知,只加入探针时,细胞具有红色荧光,加入亚硫酸氢钠后,细胞红色荧光消失,而产生强烈的绿色荧光信号。
Claims (6)
3.根据权利要求2所述的制备方法,其特征在于,步骤(1)中,苯酚与丙二酸的摩尔比为1:1;步骤(2)中,化合物1与4-二乙胺基苯酚的摩尔比为1:1;步骤(3)中,化合物2和乙酸酐的摩尔比为1:1.2;步骤(4)中,化合物3与三氟化硼乙醚的摩尔比为1:2;步骤(5)中,化合物4与对苯二甲醛的摩尔比为1:1.2。
4.根据权利要求2所述的制备方法,其特征在于,步骤(1)-(5)中,所述加热温度为110-120℃。
5.根据权利要求2所述的制备方法,其特征在于,步骤(2)中所述分离纯化步骤为将反应体系冷却至室温,减压过滤,滤饼用乙醇洗涤2-3次,真空干燥,所得粗产物以乙醇重结晶,得到提纯品;
步骤(3)中所述分离纯化步骤为将反应体系冷却至室温,减压过滤,滤饼用乙醇洗涤2-3次,真空干燥,然后粗产物均进行柱层析纯化,层析淋洗液为体积比为1:1的二氯甲烷:石油醚;
步骤(4)中所述分离纯化步骤为将反应体系冷却至室温,然后加入石油醚中,减压过滤,滤饼用乙醇洗涤2-3次,真空干燥即得;
步骤(5)中所述分离纯化步骤为将反应体系冷却至室温,真空旋干溶剂,然后粗产物均进行柱层析纯化,层析淋洗液为体积比为50:1的二氯甲烷:甲醇。
6.一种如权利要求1所述的荧光探针在制备定性与定量检测溶液和细胞内亚硫酸氢盐试剂中的应用。
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