CN106946801B - 一种特异性识别半胱氨酸的新型荧光探针的制备与应用 - Google Patents
一种特异性识别半胱氨酸的新型荧光探针的制备与应用 Download PDFInfo
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Abstract
本发明公开了一种可以特异性识别半胱氨酸的新型荧光探针,其分子结构式如下:该荧光探针不仅能够将含巯基的半胱氨酸与不含巯基的氨基酸区分开,而且能够将半胱氨酸与结构类似的同型胱氨酸和谷光氨肽区分开。该荧光探针在检测半胱氨酸时发射红光,在生物学应用中能够降低背景干扰及光对组织细胞的伤害,此外,表现出较大的斯托克斯位移,可以减小自吸收而提高灵敏度。该荧光探针可用于环境或生物样品中的半胱氨酸的荧光传感分析,对半胱氨酸的选择性好,灵敏度高,抗干扰能力强,具有很好的应用前景。
Description
技术领域
本发明涉及的是化学分析检测技术领域,具体涉及一种可以特异性识别半胱氨酸的新型荧光探针的制备方法以及该荧光探针在体外和活细胞内检测半胱氨酸方面的应用。
背景技术
生物硫醇是许多蛋白质和小分子的重要组成部分,在细胞生命活动过程中具有重要作用。生物硫醇包括半胱氨酸(Cysteine,Cys)、同型半胱氨酸(Homocysteine,Hcy)和谷胱甘肽(Glutataione,GSH)等。半胱氨酸不仅是谷胱甘肽、乙酰辅酶和牛磺酸的前体,同时也是生物体硫铁络合物中硫配体的提供者,人体中缺少半胱氨酸会导致生长缓慢、毛发色素脱色、水肿、嗜睡、肝功能损伤、肌肉松弛、身体虚弱等症状。同型半胱氨酸和谷胱甘肽的浓度异常也可能会引起阿尔兹海默氏症、心血管疾病、癌症的发生。因此,它们在生物体内含量变化可以作为这些疾病诊断的依据。目前已经应用的技术包括高效液相色谱法、毛细管电泳法、电化学检测、光学分析和质谱鉴定,这些方法可以在体外监测半胱氨酸,不能实现在活细胞中的监测。荧光探针由于其灵敏度高、检测限低、选择性好,而且能够在活细胞中可视化检测分析物,所以研究者们开始关注将荧光探针的这项技术应用于对体外或者细胞内的半胱氨酸进行监测或者细胞荧光成像。目前已经报导了多种基于化学反应的该类探针,例如Michael加成、醛环化反应和裂解反应。其检测原理是在荧光分子内引入荧光淬灭基团,导致荧光猝灭,在半胱氨酸诱导下发生反应,荧光得以恢复,这是一种特别有效的方法。但是由于半胱氨酸和同型半胱氨酸在结构上相似,这类荧光探针很难将半胱氨酸和同型半胱氨酸特异性区分,使其在生物样品或者活体中的应用受到了限制,因此开发细胞穿透性好、斯托克斯位移大、发射波长在近红外的能够特异性识别半胱氨酸探针是很有意义的。
发明内容
本发明目的之一是提供一种合成路线简单、反应条件温和、成本较低的荧光探针合成方法;目的之二是提供一种灵敏度高、选择性好,抗干扰能力强,斯托克斯位移大,发射波长在近红外,能够对体外或者活细胞内的半胱氨酸进行监测或者细胞成像的荧光探针。
本发明解决问题采取的技术方案为,一种可以特异性识别半胱氨酸的新型荧光探针,其分子结构式如下:合成路线如下:具体合成方法如下:(1)将1,4-二乙基-1,2,3,4-四氢喹喔啉水杨醛(0.1172g,0.5mmol)溶于5mL无水二氯甲烷中,再加入三乙胺(0.14mL,1.0mmol)和丙烯酰氯(0.1358g,1.5mmol),氩气保护,室温搅拌下反应1h,用蒸馏水猝灭反应,用二氯甲烷萃取,无水硫酸钠干燥,旋蒸除去二氯甲烷,柱层析分离得到棕色油状物。产量:0.1261g。收率:87.5%。(2)将上步所得产物(0.0864g,0.3mmol)溶于7mL无水二氯甲烷中,加入三乙胺(0.042mL,0.3mmol),再加入丙二腈(0.0198g,0.3mmol),氩气保护,室温搅拌0.5h,用蒸馏水猝灭反应,用二氯甲烷萃取,无水硫酸钠干燥,旋蒸溶剂,经柱层析得到产物,真空干燥过夜,得到红色固体。产量:0.0108g。收率:10%。
本发明的荧光探针测试方法如下,将探针分子溶解在含有20%乙腈、pH为7.4的PBS缓冲溶液中,室温下进行测试。并且对低浓度的半胱氨酸可以进行定量检测,具体实施方法在实施实例中详细介绍。
本发明的荧光探针的作用机理如下,探针分子与半胱氨酸作用后,丙烯酰基部分从探针分子上离去,探针分子由无荧光变为很强的荧光,从而实现了检测半胱氨酸过程。探针分子的响应过程:
本发明的荧光探针与半胱氨酸作用后的荧光发射峰在621nm处,荧光增强了62倍。
本发明所述的探针分子合成路线简单,成本较低,对半胱氨酸的选择性好、抗干扰能力强,斯托克位移大,发射波长在近红外,该荧光探针在生物化学,环境科学等领域具有实际的应用价值。
附图说明
图1为本发明荧光探针的选择性,荧光探针(1.0×10-5mol/L)在PBS缓冲溶液(10mM,V乙腈/VPBS=2/8,pH=7.4)中,与不同种类氨基酸作用后的荧光光谱,横坐标为波长,纵坐标为荧光强度。
图2为本发明荧光探针的抗干扰能力,半胱氨酸(Cys)与其他氨基酸共存时,与荧光探针(1.0×10-5mol/L)在PBS缓冲溶液(10mM,V乙腈/VPBS=2/8,pH=7.4)中与半胱氨酸作用后的荧光强度比值(I/I0)柱状图。
图3为本发明的荧光探针(1.0×10-5mol/L)在PBS缓冲溶液(10mM,V乙腈/VPBS=2/8,pH=7.4)中,与不同浓度半胱氨酸(Cys)作用后的荧光光谱变化,横坐标为波长,纵坐标为荧光强度。
图4为本发明的荧光探针(1.0×10-5mol/L)在PBS缓冲溶液(10mM,V乙腈/VPBS=2/8,pH=7.4)中,与半胱氨酸(Cys)浓度的线性关系,横坐标为波长,纵坐标为荧光强度。
图5为本发明的荧光探针(1.0×10-5mol/L)在PBS缓冲溶液(10mM,V乙腈/VPBS=2/8,pH=7.4)中,与半胱氨酸(Cys)、同型半胱氨酸(Hcy)和谷胱甘肽(GSH)作用过程中荧光强度随时间的变化,横坐标为时间,纵坐标为荧光强度。
图6为本发明的荧光探针(1.0×10-5mol/L)在不同pH值缓冲溶液中,与半胱氨酸(Cys)作用前后的荧光强度,横坐标为pH,纵坐标为荧光强度。
图7为本发明的荧光探针(1.0×10-5mol/L)在不同条件下的细胞(Hela细胞)成像(a)探针在N-乙基顺丁烯二酰亚胺溶液处理的细胞中荧光成像(b)探针和N-乙基顺丁烯二酰亚胺溶液处理的细胞中明场成像,(c)探针在细胞中荧光成像(d)探针在细胞中明场成像,(e)探针在半胱氨酸溶液处理的细胞中荧光成像(f)探针在半胱氨酸溶液处理的细胞中明场成像。
具体实施实例
实施例1:中间产物的合成
将1,4-二乙基-1,2,3,4-四氢喹喔啉水杨醛(0.1172g,0.5mmol)溶于5mL无水二氯甲烷中,再加入三乙胺(0.14ml,1.0mmol)和丙烯酰氯(0.1358g,1.5mmol),氩气保护,室温搅拌下反应1h,用蒸馏水猝灭反应,用二氯甲烷萃取,无水硫酸钠干燥,旋蒸除去二氯甲烷,柱层析分离得到棕色油状物。产量:0.1261g。产率:87.5%。1H NMR(500MHz,CDCl3)δH 9.84(s,1H),6.97(s,1H),6.64(dd,J=17.3,1.2Hz,1H),6.37(dd,J=17.3,10.5Hz,2H),6.21(s,1H),6.08-6.01(m,1H),3.51(t,2H),3.40-3.35(m,4H),3.24(t,2H),1.22-1.17(m,6H).13C NMR(125MHz,CDCl3)δC 186.1,164.9,147.6,142.0,132.9,132.3,127.8,116.5,108.7,102.5,47.4,45.8,45.3,44.6,10.7,10.0.
实施例2:探针分子的合成
将上步所得产物(0.0864g,0.3mmol)溶于7mL无水二氯甲烷中,加入三乙胺(0.042mL,0.3mmol),再加入丙二腈(0.0198g,0.3mmol),氩气保护,室温搅拌0.5h,用蒸馏水猝灭反应,用二氯甲烷萃取,无水硫酸钠干燥,旋蒸溶剂,经柱层析得到产物,真空干燥过夜,得到红色固体。产量:0.0108g。收率:10%。探针分子的结构表征如下:1H NMR(500MHz,CDCl3)δH 7.56(d,J=2.6Hz,2H),6.68(dd,J=17.3,0.9Hz,1H),6.38(dd,J=17.3,10.5Hz,1H),6.28(s,1H),6.14(dd,J=10.5,0.9Hz,1H),3.60(t,2H),3.44(q,J=7.2,2H),3.39(q,J=7.2,2H),3.29(t,2H),1.24(t,6H).13C NMR(125MHz,CDCl3)δC 164.2,149.8,146.7,143.4,134.0,132.6,127.1,116.6,115.1,112.2,106.8,102.5,70.9,47.7,46.0,45.6,44.4,10.9,9.8.
实施例3:本发明:荧光探针的应用
将探针溶于缓冲溶液(V乙腈/VPBS=2/8,pH=7.4)中配制成1.0×10-5mol/L的溶液,向溶液中加入氨基酸(Ala,Val,Try,Phe,His,,Iso,Ser,Asp,Lys,Arg,Gly,Met,Tyr,Glu,Thr)没有引起荧光的变化,加人氨基酸(Cys,Hcy,GSH)引起了荧光变化,该荧光探针对半胱氨酸表现出高灵敏度、高选择性的识别。当半胱氨酸与干扰物质(Ala,Val,Try,Phe,His,,Iso,Ser,Asp,Lys,Arg,Gly,Met,Tyr,Glu,Thr,Hcy,GSH)共存时,探针不受干扰因素的影响,表现出来很强的抗干扰能力。该探针分子与半胱氨酸响应速度快,1分钟内即可观察到荧光的变化。探针分子在pH为7至11的范围内都可以对半胱氨酸选择性识别,表现出了生物适应的应用范围。
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1.一种特异性识别半胱氨酸的荧光探针,其结构为式Ⅰ为
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CN103351335A (zh) * | 2013-05-28 | 2013-10-16 | 中南大学 | 一种荧光增强检测含巯基氨基酸的荧光分子探针的合成及应用 |
CN104804728A (zh) * | 2015-04-27 | 2015-07-29 | 苏州罗兰生物科技有限公司 | 一种荧光增强型苯硫酚荧光探针的制备及应用 |
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