CN111362958A - 一种特异性识别还原型gsh的罗丹明类荧光探针及其制备方法与应用 - Google Patents
一种特异性识别还原型gsh的罗丹明类荧光探针及其制备方法与应用 Download PDFInfo
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- CN111362958A CN111362958A CN202010241269.4A CN202010241269A CN111362958A CN 111362958 A CN111362958 A CN 111362958A CN 202010241269 A CN202010241269 A CN 202010241269A CN 111362958 A CN111362958 A CN 111362958A
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- fluorescent probe
- rhodamine
- probe
- gsh
- reduced gsh
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Images
Classifications
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- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
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Abstract
本发明公开一种特异性识别还原型GSH的罗丹明类荧光探针及其制备方法与应用,基于罗丹明B结构的、含有联烯酰胺识别基团的比色型荧光增强探针RBA,对GSH具有高度的选择性。此外,该探针可以通过两个简单的酰化反应合成,这对它的实际应用是非常有利的。联烯酰胺容易合成,可以连接到相对复杂的分子上,在室温下长期稳定,易于和硫醇反应,而不与生物体系中常见的其它亲核试剂(胍、咪唑、羟基、胺、羧基等)反应,在化学分析检测领域具有广阔的应用前景。
Description
技术领域
本发明属于分析化学技术领域,涉及一种特异性识别还原型GSH的罗丹明类荧光探针及其制备方法与应用。
背景技术
谷胱甘肽(GSH)是细胞内含量最丰富的非蛋白硫醇,具有多种细胞功能,包括维持细胞内氧化还原活性、异种代谢、细胞内信号转导和基因调控。更具体地说,GSH可以使蛋白质中的半胱氨酸硫醇基处于还原状态,并通过捕获破坏核糖核酸和缺氧核糖核酸的自由基来保护细胞免受氧化应激。一般而言,细胞内GSH水平的变化与许多疾病有关,如白细胞减少症、牛皮癣、肝损伤、阿尔茨海默病、帕金森氏病、冠心病、癌症和获得性免疫缺陷综合征(AIDS)。由于GSH的重要性,迫切需要灵敏的方法对其进行选择性检测。
最近的研究导致了各种测定GSH水平的常规技术的发展,如高效液相色谱(HPLC)、毛细管电泳(CE)、伏安法、电化学分析、液相色谱-质谱(LCMS)、傅立叶变换红外光谱(FTIR)和荧光光谱。在已报道的检测技术中,荧光检测已被证明是最方便的。荧光检测是一种重要的检测方法简单易行,无创,检测下限低。荧光探针的最大优点是它可以用于细胞内检测。
然而,由于GSH、半胱氨酸(Cys)和高半胱氨酸(Hcy)具有相似的结构和性质,因此从Cys和Hcy中鉴别CSH是一项非常具有挑战性的任务。虽然到目前为止已经报道了许多区分GSH和Cys/Hcy的化学探针,但很多探针都存在合成困难等缺陷。在这些探针中,罗丹明类探针表现出优异的性能,引起了人们对设计多功能GSH探针的兴趣。最具代表性的罗丹明基还原型GSH探针,但这些探针以马来酰亚胺、丙烯酰胺和α-卤酰基为识别基团,通过亲电反应与GSH的巯基共价连接,在羧基的作用下形成罗丹明荧光团的开环结构。然而,这些探针的识别基团仍有一个或多个缺点,一些基团与巯基以外的亲核基团如氨基、羧基、胍基和咪唑基有潜在的反应,这将影响最终检测结果的准确性。此外,马来酰亚胺和硫醇形成可逆的共价键,可以被一些含硫醇的分子和其他反应物逆转。为了克服上述缺点,需要寻找一种选择性高、不可逆的能与硫醇反应的官能团。
发明内容
针对目前还原型GSH检测的现状,本发明的目的是提供一种特异性识别还原型GSH的罗丹明类荧光探针的合成方法,并将该探针应用于水中还原型GSH含量和活细胞中还原型GSH成像的研究。
本发明解决问题采取的技术方案如下,
一种能特异性识别还原型GSH的罗丹明类荧光探针,分子探针分子式为C35H40N4O3,其化学结构如下:
本发明还提供一种能特异性识别还原型GSH的罗丹明类荧光探针的制备方法,将摩尔比为1∶1.2-3的3-丁炔酸和2-氯-1-甲基吡啶碘化物混合物溶于无水二氯甲烷中制备的混合溶液的体积摩尔浓度为0.44-1.5mol/L,氩气保护下室温搅拌,得到羧酸活化中间体,随后将羧酸活化中间体慢慢滴入结构式RBN的反应液中,再慢慢滴入与2-氯-1-甲基吡啶碘化物等量的无水三乙胺,反应完毕将溶剂减压,减压旋干;柱层析进一步纯化,用洗脱剂洗脱后得到结荧光探针产物,
其中结构式RBN的结构式为
作为进一步改进,所述3-丁炔酸和2-氯-1-甲基吡啶碘化物的摩尔比为1∶1.5。
作为进一步改进,所述柱层析采用200-300目硅胶进行层析。
作为进一步改进,所述洗脱剂为石油醚和乙酸乙酯混合液,两者体积比为3∶1。
本发明制备的一种能特异性识别还原型GSH的罗丹明类荧光探针的应用范围可以为在制备检测、识别环境中或生物样品中谷胱甘肽的检测试剂或者标记物中的应用,但是不限于此范围。
进一步的的应用范围为,还原型GSH的罗丹明类荧光探针在制备检测、识别环境中或生物样品中谷胱甘肽的检测试剂或者标记物中的应用,其特征在于,所述还原型GSH的罗丹明类荧光探针用于制备正常细胞和活体体内的谷胱甘肽的检测试剂或者标记物中的应用。
进一步的,所述样品的检测限为1微摩尔。
本发明还提供还原型GSH的罗丹明类荧光探针测定谷胱甘肽的检测方法,其特征在于,通过荧光分光光度法,以560nm为激发波长,在585nm的波长处测定谷胱甘肽的荧光强度。
本发明的荧光探针其本身几乎无荧光,在与还原型GSH作用后迅速有很强的荧光发射,最大发射波长为585nm,本发明的荧光探针的斯托克斯位移约23nm,其最大吸收波长为562nm,与还原型GSH反应后最大发射波长为585nm。
本发明的荧光探针对还原型GSH有很好的选择性,在pH等于7.4的PBS缓冲溶液中,探针溶液荧光强度微弱,加入10倍当量的还原型GSH,荧光强度逐渐增强,10分钟后增大到30分钟时的45倍左右。而同样条件下分别加入其他可能干扰的氨基酸和离子,探针的荧光强度并没有明显变化。
本发明的荧光探针识别基团联烯酰胺具有很强的抗干扰能力,能特异性的与巯基发生反应而不和氨基,羧基,咪唑,胍基等常见生物亲核试剂反应。
本发明的荧光探针对还原型GSH的检测表现出很高的灵敏度。探针溶液的荧光强度,随着增加还原型GSH浓度而递增,大约在加入20倍量还原型GSH时达到峰值。在0到10倍量还原型GSH的区间内,探针溶液荧光强度与还原型GSH浓度有很好的线性关系。
本发明的优点:在本发明中,我们开发了一种基于罗丹明B结构的、含有联烯酰胺识别基团的比色型荧光增强探针RBA,对GSH具有高度的选择性。此外,该探针可以通过两个简单的酰化反应合成,这对它的实际应用是非常有利的。联烯酰胺容易合成,可以连接到相对复杂的分子上,在室温下长期稳定,易于和硫醇反应,而不与生物体系中常见的其它亲核试剂(胍、咪唑、羟基、胺、羧基等)反应。在细胞中的共聚焦成像实验也证明了探针对GSH的选择性和灵敏度,以及GSH在细胞内的识别能力。最后,基于荧光测量和液质联用分析,提出了选择性检测GSH的潜在机制。
本发明的罗丹明衍生物作为探针的荧光母核,合成步骤仅两步,原料廉价易得。应用于荧光探针领域,母核的性质优异,量子产率较高,可以利用其合成多种新型探针。该罗丹明衍生物的结构决定其荧光性质十分优异。通过修饰形成的探针本身荧光可以几乎完全掩蔽,与还原型GSH作用数分钟后,荧光强度迅速增强(荧光增强倍数大于45倍),而且具有很高的量子产率。检测限低至1微摩尔,灵敏度高,选择性和抗干扰性能好。
综上所述,本发明的荧光探针是一种便捷,灵敏,适用于体外和活细胞内部检测还原型GSH的工具,在化学分析检测领域具有广阔的应用前景。
附图说明
图1为本发明荧光探针制备过程示意图;
图2本发明荧光探针RBA的核磁氢谱谱图;
图3本发明荧光探针RBA的核磁碳谱谱图;
图4本发明荧光探针RBA的质谱谱图;
图5为本发明荧光探针荧光响应示意图;
图6为本发明荧光探针的各种不同离子和分子选择性和抗干扰能力的柱状图。其中,从序号1到25黑色柱分别代表以下物质(100μM)与探针(10μM)作用后的荧光强度:丙氨酸,天冬氨酸,精氨酸,Cys,甘氨酸,谷氨酸,组氨酸,高胱氨酸,脯氨酸,色氨酸,酪氨酸,水,NaCl,KCl,MgSO4,CaCl2,ZnCl2,CuSO4,NaF,MgCl2,NaSO4,Na2CO3,NaHS,NaOCl,水;灰色柱代表随后加入还原型GSH的响应情况;
图7为本发明的荧光探针(10μM)在磷酸盐缓冲溶液(pH=7.4)中,加入递增浓度还原型GSH(0-1000μM),反应60分钟后的荧光光谱图;
图8为本发明的荧光探针(10μM)在PBS缓冲溶液(pH=7.4)中,加入递增浓度还原型GSH(0-1000μM),反应60分钟后的荧光强度变化曲线。其插图为本发明的荧光探针(10μM)在PBS缓冲溶液(pH=7.4)中,加入递增浓度还原型GSH(0-100μM)的线性关系图;
图9为本发明的荧光探针专一性验证的高效液相色谱图。(A)荧光探针分子(10μM)与10倍量组氨酸作用后的液相色谱图。(其中18.19分钟为探针RBA的色谱峰)(B)荧光探针分子(10μM)与10倍量精氨酸作用后的液相色谱。(C)荧光探针分子(10μM)与10倍量谷氨酸作用后的液相色谱图。(D)荧光探针分子(10μM)与10倍量还原型Cys作用后的液相色谱图(其中17.13分钟为探针RBA与Cys偶联物的色谱峰)。(E)荧光探针分子(10μM)与10倍量组氨酸作用后的液相色谱图。(其中16.59分钟为探针RBN与还原型GSH偶联物的色谱峰);
图10探针RBA和Cys偶联物的质谱谱图;
图11探针RBA和GSH偶联物的质谱谱图;
图12为本发明的荧光探针的MTT毒性实验,探针浓度梯度(最终浓度):20μM、10μM、5μM,2.5μM、1.25μM、0.625μM、0μM(对照),细胞存活率大于80%,基本无毒性;
图13为本发明的荧光探针(10μM)在不同条件下MCF-7细胞中成像结果。(A)未加探针处理的细胞中荧光成像、明场成像以及Merge图。(B)加入RBA溶液处理的细胞中荧光成像、明场成像以及Merge图;(C)RBA在加入N-乙基马来酰胺溶液处理的细胞中荧光成像、明场成像以及Merge图;(D)RBA在加入N-乙基马来酰胺溶液处理后又补加还原型GSH处理细胞中荧光成像、明场成像以及Merge图。使用543nm激发,比例尺:100微米。
具体实施实例
下面结合实施例对本发明作进一步说明,但本发明并不限于以下实施例。
实施例1:
该荧光探针的合成路线如图1所示
将3-丁炔酸(84mg,1.0mmol)和2-氯-1-甲基吡啶碘化物(1.5eq,1.5mmol)溶于3mL二氯甲烷中,氮气保护下室温搅拌一个小时.随后将该反应液慢慢滴入484mg,1.1mmol罗丹明酰胺的反应液中,再慢慢滴入三乙胺(0.2mL,1.5mmol),反应完毕将溶剂减压旋干;200-300目硅胶进行层析进一步纯化,体积比为3∶1的石油醚和乙酸乙酯混合液洗脱,得到白色固体荧光探针320mg,产率38%,其核磁图谱和质谱图,如图2-4所示。
1H NMR(300MHz,CDCl3)δ1.09(t,J=6.88Hz,12H)2.30(br.s.,0.75H)3.02(br.0.75H)3.23(br.s.,3.5H)3.24-3.33(m,8H)5.17(d,J=6.42Hz,0.5H)5.46(t,J=6.42Hz,0.25H)6.20(m,J=8.62Hz,2H)6.30(s,2H)6.36(m,J=8.80Hz,2H)7.01(br.s.,1H)7.37(br.s.,2.75H)7.83(br.s.,1H);
13C NMR(75MHz,CDCl3)12.62,27.34,39.59,40.41,44.37,65.48,73.68,77.26,97.80,104.85,108.25,122.86,123.91,128.14,128.46,130.56,132.74,148.93,153.29,153.81,166.55,169.58.
ESI-HRMS calculated for C34H39N4O3 +[M+H]+,551.3017;found,551.3014.
实施例2:
本发明的荧光探针对还原型GSH的检测机理详情如下所述,罗丹明B的羧基与乙二胺作用后,所得的探针分子荧光由于分子内关环的原因导致荧光被掩蔽掉。联烯酰氨与还原型GSH的巯基发生共价偶联,由于GSH的微酸性环境,荧光母核开环,发射很强的荧光。响应过程如图5所示。
实施例3
取探针分子溶液(10μM)于磷酸盐缓冲溶液(pH=7.4)中配成待测溶液,再分别加入各种离子和分子,丙氨酸,天冬氨酸,精氨酸,Cys,甘氨酸,谷氨酸,组氨酸,高胱氨酸,脯氨酸,色氨酸,酪氨酸,,NaCl,KCl,MgSO4,CaCl2,ZnCl2,CuSO4,NaF,MgCl2,NaSO4,Na2CO3,NaHS,NaOCl,水,反应60分钟后测量,其结果如图6所示,溶液的荧光几乎没有明显变化,而只加入还原型GSH的探针溶液荧光大幅度增强(约45倍),可见该荧光探针能够对还原型GSH实现专属识别。当加入过其他离子与分子的溶液中随后补加入还原型GSH,反应60分钟后测量,探针溶液荧光依然显著增强,本发明的荧光探针对还原型GSH的检测具有很强的抗干扰能力。加入10μM荧光探针在磷酸盐缓冲溶液(pH=7.4)中,加入递增浓度还原型GSH(0-1000μM),分别有0.1eq、0.2eq、0.5eq、0.8eq、1eq、3eq、5eq、8eq、10eq、20eq、50eq、80eq、1000eq,反应60分钟后,其荧光光谱图如图7所示,该探针分子检测限低,可以达到1微摩,适合微量检测。其荧光强度与还原型GSH的浓度的线性关系图如图8所示,可见本发明的荧光探针具有良好的生物适应能力及应用前景。
实施例4
为了确定探针RBA的联烯酰胺基团对硫醇基团的特异性,选择液质联用对偶联产物的进行研究,以确定联烯酰胺基团的选择。当探针RBA与组氨酸反应时(图9A)、精氨酸(图9B)和谷氨酸(图9C)。这些色谱图显示了同样的结果。在18.19min只出现一个保留峰,质谱进一步证实保留峰的成分是探针RBA(图4)。当探针RBA与Cys反应时(图4D),液相色谱图在17.13min出现一个新的峰,质谱进一步证实该峰的成分是产物RBA-Cys(图10)。同样,当用探针RBA和GSH(图9E)反应时,在16.59min的液相色谱图上出现了一个新的峰,质谱进一步证实该峰的成分是产物RBA-GSH(图11)。以上实验结果表明,只有含硫醇的分子能和探针反应,从而形成了预期的结构RBA-Cys或RBA-GSH,这表明联烯酰胺基团对硫醇的选择性优于一些典型的生理亲核试剂。Cys和GSH和探针虽然都能结合,但是GSH比Cys多一个羧基,呈酸性,RBA在酸性条件下才能产生荧光。所以RBA-Cys的产物不产生荧光,RBA-GSH的产物有荧光。RBA对GSH的检测具有特异性。
实施例5
MCF-7细胞接种于4孔培养皿(1.3×104个/孔),放置于细胞培养箱中,使之完全贴壁。之后更换新鲜培养液,加入20μL不同浓度20μM、10μM、5μM,2.5μM、1.25μM、0.625μM、0μM的荧光探针分散液,培养12h后,在酶标仪上将波长设定为570nm,测定96孔板每孔溶液的吸光度(OD值),按下列公式计算细胞生存率:细胞存活率=(OD待测组-OD空白组)/(OD细胞组-OD空白组)×100%。从图12中看出,细胞存活率大于80%,基本无毒性。
实施例6
为了探究探针的生物应用性,我们利用探针RBA检测细胞内GSH含量。将MCF-7细胞在Dulbecco’s modified Eagle’s medium(DMEM)中培养。荧光成像前,将细胞接种于4孔培养皿(1.3×104个/孔),培养1天。第一组MCF-7细胞不与RBA或其他试剂孵育。第二组细胞用含RBA(10μM,含1‰四氢呋喃)的培养基中处理5h,第三组细胞先用N-乙基马来酰亚胺(0.5mM)处理60分钟,然后用含RBA(10μM)的培养基处理5h,第4组先用N-乙基马来酰亚胺(0.5mM)预处理60分钟,然后用磷酸盐缓冲液洗涤3次,再用GSH培养液(0.5mM)处理30分钟。最后用10μM的RBA液更换用过的培养基,继续培养5小时,共聚焦成像前,将培养皿中用过的培养基废弃,用磷酸盐缓冲液清洗3次。最后所有组细胞用激光显微镜成像,其中激发波长为543nm,收集波段为552-617nm。第一组对照细胞没有显示任何荧光(图13A),而当与RBA孵育5小时后,在红色通道中观察到强烈的荧光(图13B)。第三组加入了GSH清除剂后,细胞的荧光强度明显减弱(图13C)。随后向细胞中加入0.5mM的GSH(NEM预处理)导致红色荧光的发射(图13D),这一结果表明RBA可以检测到活细胞中GSH浓度的差异。以上结果验证了RBA可以作为一种良好的荧光探针选择性检测活细胞中的GSH。
Claims (9)
3.权利要求2所述的一种特异性识别还原型GSH的罗丹明类荧光探针的制备方法,其特征在于,所述3-丁炔酸和2-氯-1-甲基吡啶碘化物的摩尔比为1∶1.5。
4.权利要求2所述的一种特异性识别还原型GSH的罗丹明类荧光探针的制备方法,其特征在于,所述柱层析采用200-300目硅胶进行层析。
5.权利要求2所述的一种特异性识别还原型GSH的罗丹明类荧光探针的制备方法,其特征在于,所述洗脱剂为石油醚和乙酸乙酯混合液,两者体积比为3∶1。
6.根据权利要求1所述的还原型GSH的罗丹明类荧光探针在制备检测、识别环境中或生物样品中谷胱甘肽的检测试剂或者标记物中的应用。
7.根据权利要求6所述的还原型GSH的罗丹明类荧光探针在制备检测、识别环境中或生物样品中谷胱甘肽的检测试剂或者标记物中的应用,其特征在于,所述还原型GSH的罗丹明类荧光探针用于制备正常细胞谷胱甘肽的检测试剂或者标记物中的应用。
8.根据权利要求6所述的还原型GSH的罗丹明类荧光探针在制备检测、识别环境中或生物样品中谷胱甘肽的检测试剂或者标记物中的应用,其特征在于,所述样品的检测限为1微摩尔。
9.根据权利要求1所述的还原型GSH的罗丹明类荧光探针测定谷胱甘肽的检测方法,其特征在于,通过荧光分光光度法,以560nm为激发波长,在585nm的波长处测定谷胱甘肽的荧光强度。
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