CN109293528B - 一种异氟尓酮硝基的衍生物及其合成方法和应用 - Google Patents
一种异氟尓酮硝基的衍生物及其合成方法和应用 Download PDFInfo
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- C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
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- C07C255/35—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by halogen atoms, or by nitro or nitroso groups
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Abstract
本发明提供了一种异氟尓酮硝基衍生物及其合成方法和应用,所述的衍生物是(E)‑2‑(5,5‑二甲基‑3‑(4‑硝基苯乙烯基)环己‑2‑烯‑1‑亚基)丙二腈。衍生物的合成步骤:氮气保护下,将异氟尓酮和丙二腈加入无水乙醇中,哌啶催化下搅拌回流10‑15小时,加入蒸馏水析出灰黑色固体,正庚烷重结晶,得到黄色晶体化合物1;氮气保护下,将化合物1和对硝基苯甲醛,在哌啶催化下,乙醇中回流6‑10小时,旋蒸除去乙醇,将得到的混合物通过柱色谱分离得到橙黄色固体异氟尓酮硝基衍生物。所述的异氟尓酮硝基衍生物可用于溶液中硫化氢的快速检测。
Description
技术领域
本发明涉及有机小分子荧光探针及H2S的检测,具体涉及一种异氟尓酮硝基的衍生物(IS-NO2)及其合成方法和H2S检测中的应用。
背景技术
硫化氢(H2S)是一种气体信号小分子。人的血清中H2S的含量为30-100μmol/L,人脑中的含量则高达160μmol/L。当人体内H2S含量较低时,会导致糖尿病患者的伤口难以愈合,然而当H2S含量较高时,会对生物体造成严重损害,如心脏、脑功能的障碍,呼吸系统损伤,而且使得阿尔茨海默病的发病几率大大增加。因此,发展有效且方便快捷的检测硫化氢的方法迫在眉睫。与传统的检测硫化氢的手段(电化学法,比色法,气相色谱法,金属硫化物沉淀法等)相比,有机小分子荧光探针检测,方便快捷且成本较低,因此开发一个有机小分子荧光探针具有很重要的意义。
为了更加深入的了解硫化氢的相关生物学生理学方面的功能和作用机理,建立快速便捷、灵敏度高、选择性好、能满足环境样品检测和生物体系检测需要的次氯酸分析方法,具有重要的研究意义和应用价值。荧光成像分析法具有灵敏度高,选择性好,操作简单,能够对分析物进行实时定量检测,异佛尔酮染料具有近红外的发光,高的荧光量子产率和良好的的化学热稳定性,够在生物环境下高效的检测生物分子并且具有较高的时空分辨率等优点,因此设计合成以异氟尓酮为荧光团的硫化氢探针具有重要意义。
发明内容
本发明的目的在于提供一种异氟尓酮硝基的衍生物及其合成方法,以及该衍生物可以用于快速检测硫化氢。
本发明提供的一种异氟尓酮硝基的衍生物,中文名:((E)-2-(5,5-二甲基-3-(4-硝基苯乙烯基)环己-2-烯-1-亚基)丙二腈;英文名:(E)-2-(5,5-dimethyl-3-(4-nitrostyryl)cyclohex-2-en-1-ylidene)malononitrile;结构式为:
本发明提供的一种异氟尓酮硝基的衍生物IS-NO2的合成方法,包括如下步骤:
1)在氮气保护下,在乙醇中按摩尔比1:1-1.5加入异氟尓酮和丙二腈,以及催化量的哌啶,在75℃下搅拌回流10-15小时;然后将反应液倒入1-2倍的蒸馏水中放置过夜,将放置过夜的溶液减压过滤得到黑色固体,用正庚烷重结晶,得到黄色晶体化合物1;
2)在氮气保护下,按摩尔比1:1-1.5将化合物1和对硝基苯甲醛,以及催化量的哌啶,在乙醇中搅拌回流6-10小时,冷却至室温后旋转蒸发除去乙醇,得到的混合物通过柱色谱分离得到橙黄色固体IS-NO2。
3)步骤1)中异氟尓酮和丙二腈的摩尔比优选为1:1.5。
4)步骤1)中搅拌回流时间优选为12小时。
5)步骤2)中化合物1和对硝基苯甲醛的摩尔比优选为1:1.2。
6)步骤2)中搅拌回流时间优选为8小时。
所述的异氟尓酮硝基衍生物可用于溶液中硫化氢的检测。
本发明提供的一种检测H2S的方法,包括如下步骤:
(1)、配制pH=7.4、浓度为10mM的PBS缓冲溶液,配制2mM的如权利要求1所述的IS-NO2的DMSO溶液;
(2)、将2000μL pH 7.4的PBS缓冲溶液、50μL如权利要求1所述的IS-NO2的DMSO溶液加到荧光比色皿中,在荧光分光光度仪上检测,随着待测样的加入,565nm的荧光强度逐渐减弱,650nm的荧光逐渐增强;
(3)、配制pH=7.4、浓度为10mM的PBS缓冲溶液,并用DMSO配制2mM IS-NO2的溶液,配制2mM硫化氢水溶液;在6个比色皿中,取2000μL PBS缓冲溶液50μM IS-NO2的DMSO溶液,分别加入硫化氢溶液的量为5、10、15、20、30、35,5min后在荧光光谱仪上测定650nm和565nm处荧光强度,两者的比值为0.39,0.49,0.54,0.62,0.76,0.95,以硫化氢浓度为横坐标,以荧光强度比值为纵坐标绘制图,得到硫化氢浓度的工作曲线;线性回归方程为Y=0.254X+0.2006,X单位为10-6mol/L。
(4)、测定样品溶液时,将测得的荧光强度比值代入线性回归方程,即可求得硫化氢的浓度
与现有技术相比,本发明具有如下优点和效果:
1)本发明提供了一种异氟尓酮硝基衍生物(IS-NO2),可用于溶液中H2S的快速检测。
2)本发明提供了一种检测H2S的方法,是在PBS(pH 7.4)缓冲溶液中,通过荧光分光光度计检测H2S。该方法对H2S实现了比率型检测,并显示了高的选择性,且是一种发射波长在近红外区的检测试剂,对生物的光毒害性小。该方法检测过程简便、检测结果准确。
附图说明:
图1实施例1制备的IS-NO2的核磁氢谱图
图2实施例1制备的IS-NO2的核磁碳谱图
图3实施例1制备的IS-NO2的质谱图
图4IS-NO2与H2S作用的荧光发射图
图5IS-NO2与H2S作用的紫外吸收图
图6IS-NO2测定硫化氢的工作曲线
图7IS-NO2与各种还原性物质的的荧光发射图
图8IS-NO2与H2S作用的时间关系图
图9IS-NO2与H2S作用的细胞成像图
具体实施方式:
实施例1(E)-2-(5,5-二甲基-3-(4-硝基苯乙烯基)环己-2-烯-1-亚基)丙二腈的合成及表征。
1)在氮气保护下,在无水乙醇中加入异氟尓酮(10mmol,1.38g),丙二腈(15mmol,0.99g),搅拌条件下滴入100μL的哌啶,在75℃的温度下搅拌回流12小时;然后将反应产物倒入250mL的烧杯中,再倒入50mL的冰蒸馏水,放置过夜;将放置过夜的溶液减压过滤,得到灰黑色固体,固体用正庚烷重结晶,得到黄色晶体化合物1;0.92g,产率:49.46%。
2)在氮气保护下,将上述黄色晶体化合物1(10mmol,1.86g)、对硝基苯甲醛(12mmol,1.81g),在100μL哌啶的催化下,无水乙醇中75℃搅拌回流8小时,冷却至室温后旋转蒸发除去乙醇,将反应得到的混合物通过柱色谱分离(洗脱剂为二氯甲烷:乙酸乙酯=6:1)得到橙黄色固体,((E)-2-(5,5-二甲基-3-(4-硝基苯乙烯基)环己-2-烯-1-亚基))丙二腈异氟尓酮硝基衍生物IS-NO2;1.52g,产率:56%。
表征结果见图1、2、3。
实施例2
配制pH=7.4、浓度为10mM的PBS缓冲溶液,并用DMSO配制2mM的IS-NO2的溶液;把将2000μL pH 7.4的PBS缓冲溶液及50μL IS-NO2的DMSO溶液加到荧光比色皿中,取H2S(2mM)的溶液,逐渐用微量进样器加到此比色皿中,加样的同时在荧光分光光度仪上检测,随着H2S的加入,565nm的荧光强度逐渐减弱,660nm的荧光逐渐减弱。荧光发射图见图4。(图4中的插图,在黑暗条件下用365nm的紫外灯照射,拍摄的照片)。
实施例3
配制pH=7.4、浓度为10mM的PBS缓冲溶液,并用DMSO配制2mM的IS-NO2的溶液;把将2000μL pH 7.4的PBS缓冲溶液及50μL IS-NO2的DMSO溶液加到比色皿中,取H2S(2mM)的溶液配制pH=7.4、浓度为10mM的PBS缓冲溶液,并用DMSO配制2mM的IS-NO2溶液,逐渐用微量进样器加到此比色皿中,加样的同时在紫外分光光度仪上检测,随着H2S的加入,325nm的紫外吸收逐渐增强,425nm的紫外吸收逐渐减弱,500nm的紫外吸收逐渐增强。紫外吸收图见图5。(图5插图:将上述溶液在日光环境下拍摄的照片)。
实施例4
配制pH=7.4、浓度为10mM的PBS缓冲溶液,并用DMSO配制2mM IS-NO2的溶液,配制2mM硫化氢水溶液;在6个比色皿中,取2000μL PBS缓冲溶液50μM IS-NO2的DMSO溶液,分别加入硫化氢溶液的量为5、10、15、20、30、35,5min后在荧光光谱仪上测定650nm和565nm处荧光强度,两者的比值为0.39,0.49,0.54,0.62,0.76,0.95,以硫化氢浓度为横坐标,以荧光强度比值为纵坐标绘制图,得到硫化氢浓度的工作曲线;线性回归方程为Y=0.254X+0.2006,X单位为10-6mol/L,工作曲线见图6。
实施例5
配制pH=7.4、浓度为10mM的PBS缓冲溶液,并用DMSO配制2mM的IS-NO2溶液;在9个荧光比色皿中,各加入将2000μL pH 7.4的PBS缓冲溶液和50μL IS-NO2的DMSO溶液,再将第二个比色皿中加入700μL H2S,第三个到第九个比色皿中加入700μL(2×10-2M)的各种分析物SO3 2-,I-,Br-,Cl-,OH-,F-,Sn2+在荧光分光光度仪上检测,绘制不同分析物对应的650/565相对荧光强度比值R的柱状图(见图7)。(图7插图:将上述溶液在日光环境下拍摄,得到插图上层的照片,在黑暗条件下,用365nm的紫外灯照射条件下拍摄,得到插图下层的照片)。
经实验证明,其它分析物不干扰上述体系对H2S的测定。
实施例6
配制pH=7.4、浓度为10mM的PBS缓冲溶液,并用DMSO配制2mM的IS-NO2溶液;在荧光比色皿中,各加入将2000μL pH 7.4的PBS缓冲溶液和50μL IS-NO2的DMSO溶液,取H2S(2mM)的溶液700μL加入到体系中,在荧光分光光度仪上检测,每隔30秒进行一次纪录,见图8。(图8插图:在650nm处,纪录最大荧光发射,以时间为横坐标,在650nm处荧光发射为纵坐标,绘制的时间和荧光强度的关系图)。
实施例7
配制pH=7.4、浓度为10mM的PBS缓冲溶液,并用DMSO配制2mM的IS-NO2溶液。首先将50μL IS-NO2的DMSO溶液与2000μL pH 7.4的PBS缓冲溶液共同孵育在HepG-2培养皿上30分钟。30分钟后,用pH 7.4的PBS缓冲溶液冲洗三次。冲洗完毕后,将H2S(2mM)的溶液500μL与2000μL pH 7.4的PBS缓冲溶液共同孵育在HepG-2培养皿上30分钟,然后再用pH 7.4的PBS缓冲溶液冲洗三次,之后用共聚焦显微镜进行拍摄,得到图9。
Claims (2)
2.异氟尓酮硝基衍生物IS-NO2在制备溶液中H2S检测的试剂中的应用,其特征在于,包括如下步骤:
(1)、配制pH=7.4、浓度为10 mM的PBS缓冲溶液,配制2 mM的如权利要求1所述的IS-NO2的DMSO溶液;所述IS-NO2的结构式为:
(2)、将2000 μL pH 7.4的PBS缓冲溶液、50 μL IS-NO2的DMSO溶液加到荧光比色皿中,在荧光分光光度仪上检测,随着待测样的加入,565 nm的荧光强度逐渐减弱,650 nm的荧光逐渐增强;
(3)、配制pH=7.4、浓度为10 mM的PBS缓冲溶液,并用DMSO配制2 mM IS-NO2的溶液,配制2 mM硫化氢水溶液;在6个比色皿中,取2000 µL PBS缓冲溶液50 µM IS-NO2的DMSO溶液,分别加入硫化氢溶液的量为5、10、15、20、30、35,5min后在荧光光谱仪上测定650 nm 和565nm 处荧光强度,两者的比值为0.39,0.49,0.54,0.62,0.76,0.95,以硫化氢浓度为横坐标,以荧光强度比值为纵坐标绘制图,得到硫化氢浓度的工作曲线;线性回归方程为Y=0.254X+0.2006,X单位为10-6 mol/L;
(4)、测定样品溶液时,将测得的荧光强度比值代入线性回归方程,即可求得硫化氢的浓度。
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