CN110041296B - 一种特异性双光子成像荧光探针及制备方法与应用 - Google Patents
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Abstract
Description
技术领域
本公开属于生物分析检测技术领域,涉及半胱氨酸的检测,具体涉及一种特异性双光子成像荧光探针及制备方法与应用。
背景技术
这里的陈述仅提供与本公开有关的背景信息,而不必然构成现有技术。
抑郁症是一类全球性的精神疾病,具有较高的患病率、复发率、死亡率、致残率,严重危害人类的健康。不幸的是,目前对于抑郁症病理生理学的了解还是非常不完善。现有研究发现抑郁症与大脑中的氧化应激—氧化还原失衡密切相关。半胱氨酸(Cys)是由甲硫氨酸的去甲基化以及胱氨酸的还原产生的氨基酸。正常水平的Cys(30-200μM)能够保持各种蛋白质构象,参与细胞内重要的抗氧剂谷胱甘肽(GSH)的合成。同时半胱氨酸常作为一种解毒“药物”,直接参与细胞内的还原过程和磷脂代谢,具有保护细胞免受氧化损伤的功能。因此,Cys在细胞内氧化还原平衡中起到重要的作用,Cys的水平异常必然会导致大脑中氧化还原稳态的变化。
荧光成像技术因其实时、可视、对生物体损伤小等优点,成为从分子水平上研究细胞及活体内生物学事件与疾病的发生发展的重要成像方法。据本公开发明人所知,近年来已经发展了许多检测Cys的荧光探针,基于硫醇的亲核性与还原性。但是,经过发明人研究发现,这些探针也可以与Hcy、GSH反应,导致现有方法的选择性差,不能准确地对活体脑部Cys的浓度变化进行实时、原位成像检测。因此,亟需发展用于特异性、高灵敏度识别Cys的工具,指示抑郁小鼠活体大脑中Cys的变化。
发明内容
为了解决现有技术的不足,本公开的目的是提供一种特异性双光子成像荧光探针及制备方法与应用,该荧光探针不仅能够对Cys进行高特异性、高灵敏度的检测,而且具有性质稳定、细胞毒性低、易通过血脑屏障等优点。
为了实现上述目的,本公开的技术方案为:
一方面,一种式Ⅰ化合物,其结构式如下所示:
另一方面,一种上述式Ⅰ化合物的制备方法,包括以香豆素120及化合物1为原料通过以下反应路线制备:
其中,R为卤素或羟基。
第三方面,一种上述式Ⅰ化合物在检测半胱氨酸中的应用。
第四方面,一种特异性双光子成像荧光探针,包括上述式Ⅰ化合物。
第五方面,一种上述式Ⅰ化合物或荧光探针在检测细胞和/或脑部中半胱氨酸中的应用。
第六方面,一种半胱氨酸的检测方法,将上述式Ⅰ化合物或荧光探针放入待测溶液中进行反应,然后进行荧光检测。
本公开中的荧光探针由Cys的特异性识别基团硫代甲酸苯酯和荧光团香豆素120发生酰胺化反应生成。该荧光探针中存在碳硫双键,碳硫双键具有强吸电子效应,降低了氨基的给电子能力,导致香豆素环的供吸电子效应减弱,荧光降低。检测半胱氨酸时,半胱氨酸的巯基进攻碳硫双键发生亲核加成反应,随后苯氧基离去碳硫双键恢复,氨基继续进攻碳硫双键发生亲核加成反应。通过连续的两次亲核加成反应,探针与Cys之间形成稳定的五员环,破坏了碳硫双键,恢复了香豆素环的供吸电子效应,使荧光增强,从而实现对Cys的特异性检测。
本公开的有益效果为:
1、本公开提供的荧光探针对Cys特异性识别,灵敏度高,细胞毒性低,光稳定性好,具有良好的双光子性质。
2、本公开提供的荧光探针可以穿过血脑屏障进入大脑。
3、利用本公开提供的荧光探针在细胞和活体成像中,采用754nm双光子激发波长,对细胞损伤小,穿透深度大,可以实时精确追踪细胞及活体脑部Cys的真实水平变化。
4、本公开的荧光探针合成简单、产率高、容易提纯。
附图说明
构成本公开的一部分的说明书附图用来提供对本公开的进一步理解,本公开的示意性实施例及其说明用于解释本公开,并不构成对本公开的不当限定。
图1为本公开实施例例制备的TCS的核磁共振氢谱图;
图2为本公开实施例例制备的TCS的核磁共振碳谱图;
图3为本公开实施例制备的TCS与Cys反应的原理图;
图4为本公开实施例制备的TCS与Cys反应前后的荧光表征图,A为单光子荧光图,B为双光子荧光图;
图5为本公开实施例制备的TCS对Cys的选择性实验表征图,A为检测活性氧和金属离子的选择性柱状图,1.blank;2.CA;3.VC;4.NaS2O3;5.SO3 2-;6.NaNO2;7.Glucose;8.Ca2+;9.Mg2+;10.Zn2+;11.Al3+;12.Mn2+;13.Na+;14.K+;15.Cu2+;16.Fe2+;17.Cu+;18.Fe3+;19.ONOO-;20.O2·-;21.ROO·;22.ClO-;23.·OH;24.H2O2;25.1O2;26.NO;27.TBHP;28.DTT;29.Cys,B为检测氨基酸的选择性柱状图,1.blank,2.Phe;3.Ile,4.Asp,5.His,6.Thr,7.Tyr,8.Glu,9.Met,10.Gly,11.Arg,12.Lys,13.Ser,14.Val,15.Pro,16.Cystine,17.SeCys2,18.SeC,19.DTT,20.Peptide:RGDPGC,21.Peptide:CRGDPG,22.Peptide:CRGDPC,23.Cys.;
图6为本公开实施例制备的TCS,TCS对PC12细胞的双光子荧光成像图,A为只用TCS孵育的细胞成像图,B为加入Cys的细胞成像图,C为加入Hcy的细胞成像图,D为加入GSH的细胞成像图,E为加入Cys和H2O2的细胞成像图,F为加入Cys和NEM的细胞成像图,G为加入DTT的细胞成像图,H为加入DTT和NEM的细胞成像图,I、J分别为A-D和E-H的数据输出图;
图7为本公开实施例制备的TCS对抑郁症模型中小鼠脑部的表征图,A为双光子荧光成像图,B为A荧光强度数据输出图。
具体实施方式
应该指出,以下详细说明都是示例性的,旨在对本公开提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本公开所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本公开的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
现有荧光探针检测Cys选择性差、无法准确对活体脑部Cys的浓度变化进行检测等不足,为了解决如上的技术问题,本公开提出了一种特异性双光子成像荧光探针及制备方法与应用。
本公开的一种典型实施方式,提供了一种式Ⅰ化合物,其结构式如下所示:
本公开的另一种实施方式,提供了一种上述式Ⅰ化合物的制备方法,包括以香豆素120及化合物1为原料通过以下反应路线制备:
其中,R为卤素或羟基。
该实施方式的一种或多种实施例中,香豆素120与化合物1进行酰胺化反应后获得。
为了提高式Ⅰ化合物的收率,该实施方式的一种或多种实施例中,R为卤素。与R为羟基相比,R为卤素时,获得的式Ⅰ化合物的收率更高。为了降低成本,该系列实施例中,R为Cl。
该实施方式的一种或多种实施例中,反应的温度为25~30℃。当温度为27.5~28.5℃时,反应效果更好。
该实施方式的一种或多种实施例中,反应体系在惰性气氛下进行。所述惰性气氛采用的气体为氮气、氩气等提供。为了降低生产成本,反应体系在氮气气氛下进行。
该实施方式的一种或多种实施例中,香豆素120及化合物1的摩尔比为1:0.9~1.1。
该实施方式的一种或多种实施例中,反应体系采用的溶剂为四氢呋喃。采用四氢呋喃能够更好的将香豆素120及化合物1溶解,从而增加反应效率。
本公开的第三种实施方式,提供了一种上述式Ⅰ化合物在检测半胱氨酸中的应用。
本公开的第四种实施方式,提供了一种特异性双光子成像荧光探针,包括上述式Ⅰ化合物。
本公开的第五种实施方式,提供了一种上述式Ⅰ化合物或荧光探针在检测细胞和/或脑部中半胱氨酸中的应用。所述应用以非疾病的诊断或治疗为目的。
本公开的第六种实施方式,提供了一种半胱氨酸的检测方法,将上述式Ⅰ化合物或荧光探针放入待测溶液中进行反应,然后进行荧光检测。
该实施方式的一种或多种实施例中,采用单光子荧光成像,激发波长为340nm,发射波段为360~550nm。
该实施方式的一种或多种实施例中,采用双光子荧光成像,激发波长为754nm,发射波段为390~550nm。
为了使得本领域技术人员能够更加清楚地了解本公开的技术方案,以下将结合具体的实施例详细说明本公开的技术方案。
实施例
荧光探针(TCS)的合成
首先在氮气保护下将香豆素120(0.176g,1.0mmol)溶解在四氢呋喃(10mL)中,然后加入硫代氯甲酸苯酯(135μL,1.0mmol)。接下来将混合物在28℃和氮气下搅拌12小时。反应完成后,将混合物真空浓缩。通过薄板层析法提纯,洗脱剂为乙酸乙酯:环己烷=2:1,得到淡黄色固体,即荧光探针,记为TCS。最后,将TCS溶解Tris缓冲液(1%DMSO,pH=7.4)中进行进一步表征和性能评价。
TCS的质谱及核磁表征
HRMS(ESI)m/z calcd.for C17H13NO3S[M+H]+calculated 312.0688,found312.0670.NMRdata:1HNMR(400MHz,d6-DMSO),图1所示:δ8.12(s,1H),7.79(d,2H),7.49(t,2H),7.31(t,1H),7.23(d,2H),6.35(s,1H),5.77(s,1H),2.42(s,3H).13C NMR(101MHz,d6-DMSO),图2所示:δ160.27,157.81,153.40,129.84,127.33,126.67,122.88,122.43,119.28,118.14,116.98,115.67,114.19,113.71,18.50.
荧光检测原理如图3所示,检测半胱氨酸时,半胱氨酸的巯基进攻碳硫双键发生亲核加成反应,随后苯氧基离去碳硫双键恢复,氨基继续进攻碳硫双键发生亲核加成反应。通过连续的两次亲核加成反应,探针与Cys之间形成稳定的五员环,破坏了碳硫双键,恢复了香豆素环的供吸电子效应。
荧光检测具体步骤
对照组:取10μL 50mM的TCS(溶于DMSO),然后用990μL的Tris(0.01M)定容至1mL;实验组:取10μL 50mM的TCS,取10μLCys(0-10mM)、980μL的Tris(0.01M)定容至1mL。分别孵育15min,用F-4600日立荧光光谱仪扫描得到荧光光谱,其中激发波长为340nm,发射波长范围为360~550nm。
荧光检测表征结果如下:
TCS对Cys的荧光响应。单光子荧光成像的激发波长为340nm,发射波段为360~550nm。图4A描述的是TCS(50μM)对Cys(0、2、4、5、7、10、17、20、24、30、35、40、50、100μM)的荧光响应情况。可以看出,在443nm处荧光强度随着Cys浓度增大,荧光逐渐增强。做443nm处荧光强度随浓度变化的线性图,如图4B所示,得到线性范围为0~35μM,斜率(k)为17.25,检测限为3σ/k=0.16μM(σ为10组50μMTCS在443nm处荧光强度数值的标准偏差)。双光子荧光成像的激发波长为754nm,发射波段为390~550nm。图4C描述的是TCS(50μM)与Cys(100μM)反应前后荧光强度的变化情况。可以看出,探针与反应后导致443nm处荧光增强。可以说明发明TCS可以用于Cys的检测。
TCS对Cys的选择性。将50μM的TCS溶解在Tris缓冲液(含1%DMSO,pH=7.4)中。取46份相同溶液,分别向溶液中加入活性氧,金属离子和氨基酸,然后进行荧光扫描(Ex=340nm)。图5A(1.blank;2.CA(柠檬酸);3.VC(抗坏血酸);4.NaS2O3;5.SO3 2-;6.NaNO2;7.Glucose(葡萄糖);8.Ca2+;9.Mg2+;10.Zn2+;11.Al3+;12.Mn2+;13.Na+;14.K+;15.Cu2+;16.Fe2+;17.Cu+;18.Fe3+;19.ONOO-;20.O2·-;21.ROO·10;22.ClO-;23.·OH;24.H2O2;25.1O2;26.NO;27.TBHP(过氧化叔丁醇);28.DTT(二硫苏糖醇);29.Cys)其他浓度均为100μM)、5B(1.blank,2.Phe(苯丙氨酸);3.Ile(异亮氨酸),4.Asp(天冬氨酸),5.His(组氨酸),6.Thr(甘氨酸),7.Tyr(苏氨酸),8.Glu(谷氨酸),9.Met(甲硫氨酸),10.Gly(甘氨酸),11.Arg(精氨酸),12.Lys(赖氨酸),13.Ser(丝氨酸),14.Val(缬氨酸),15.Pro(脯氨酸),16.Cystine(胱氨酸),17.SeCys2(硒代胱氨酸),18.SeC(硒代半胱氨酸),19.DTT(二硫苏糖醇),20.Peptide:RGDPGC(Arg-Gly-Asp-Pro-Gly-Cys),21.Peptide:CRGDPG(Cys-Arg-Gly-Asp-Pro-Gly),22.Peptide:CRGDPC,(Cys-Gly-Asp-Pro-Gly-Cys),23.Cys.)为443nm处荧光数据输出图(ONOO-浓度为10-7M,O2·-,ROO·,1O2,NO,TBHP浓度均为10μM其他物质均为100μM)。可以看出,TCS可以特异性的检测Cys。
TCS对PC12细胞中的Cys的双光子共聚显微成像检测。PC12细胞中加入50μM的TCS,37℃孵育15min后,进行双光子共聚焦显微成像。图6A为只用TCS孵育的细胞成像图,图6B为加入Cys的细胞成像图,图6C为加入Hcy的细胞成像图,图6D为加入GSH的细胞成像图,图6E为加入Cys和H2O2的细胞成像图,图6F为加入Cys和NEM的细胞成像图,图6G为加入DTT的细胞成像图,图6H为加入DTT和NEM的细胞成像图,图6I,6J分别为6A-D和6E-H的数据输出图,可以说明本发明TCS可以用于细胞中Cys的成像检测。
TCS对抑郁症模型中小鼠脑部Cys的双光子成像检测。抑郁模型(CUMS)参照文献(X.Wang,P.Li,Q.Ding,C.Wu,W.Zhang and B.Tang,AngewandteChemie(Internationaled.in English),2019,131,4722-4726.)建立。CUMS模型小鼠又可分为易感组(Susceptible)和高抗组(Resilient),其中易感组表现出明显的抑郁行为。成像前,先将小鼠麻醉,随后腹腔注射0.3mg/kg的TCS。孵育10分钟后,进行脑部的双光子成像。图7-Control组为正常组的成像图,图7-Susceptible为易感组成像图,图7-Resilient组为高抗组的成像图,(图7中第一排图为2D成像图,第二排为3D成像图,图7B为Control,Susceptible,Resilient组的荧光强度数据输出图)可以说明本发明TCS可以用于活体大脑中Cys的成像检测。
以上所述仅为本公开的优选实施例而已,并不用于限制本公开,对于本领域的技术人员来说,本公开可以有各种更改和变化。凡在本公开的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本公开的保护范围之内。
Claims (10)
3.如权利要求2所述的式Ⅰ化合物的制备方法,其特征是,R为卤素。
4.如权利要求2所述的式Ⅰ化合物的制备方法,其特征是,R为Cl。
5.如权利要求2所述的式Ⅰ化合物的制备方法,其特征是,反应的温度为25~30℃。
6.如权利要求2所述的式Ⅰ化合物的制备方法,其特征是,反应的温度为27.5~28.5℃。
7.如权利要求2所述的式Ⅰ化合物的制备方法,其特征是,反应体系在惰性气氛下进行。
8.如权利要求2所述的式Ⅰ化合物的制备方法,其特征是,反应体系采用的溶剂为四氢呋喃。
9.如权利要求2所述的式Ⅰ化合物的制备方法,其特征是,香豆素120及化合物1的摩尔比为1:0.9~1.1。
10.如权利要求1所述的式Ⅰ化合物在制备检测半胱氨酸的特异性双光子成像荧光探针中的应用。
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