CN108148056A - 比率型近红外半胱氨酸荧光探针 - Google Patents
比率型近红外半胱氨酸荧光探针 Download PDFInfo
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- C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
- C07D417/10—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing aromatic rings
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Abstract
本发明涉及比率型近红外半胱氨酸荧光探针。本发明的探针可以与半胱氨酸作用,且具有高灵敏性和选择性的特点,可在半胱氨酸高灵敏性和高选择性的识别中应用,或者其可测定样品中半胱氨酸的浓度。
Description
技术领域
本发明涉及检测半胱氨酸比率型近红外荧光探针。本发明的探针可以与半胱氨酸作用,且具有高灵敏性和选择性的特点,可在半胱氨酸高灵敏性和高选择性的识别中应用,或者其可测定样品中半胱氨酸的浓度。
背景技术
硫醇的种类有很多种,但小分子生物硫醇有三种,分别为同型半胱氨酸、谷胱甘肽和半胱氨酸。而其中半胱氨酸存在于生物组织细胞的很多小分子和蛋白质中,是组成它们的重要物质,并且半胱氨酸在哺乳动物调节系统调节多种生理和病理过程中起着相当重要的作用,主要是通过半胱氨酸的还原性特质能够被氧化为氧化性的物质维持细胞环境中的氧化还原平衡。同型半胱氨酸在生物体中起着重要的作用,它是甲硫氨酸在一些反应中的中间产物,一旦形成后有两种去路,一是重新转化为甲硫氨酸,二是在一些物质的催化下形成半胱氨酸。谷胱甘肽,是人体中最丰富的非蛋白巯基,在细胞内有许多的功能,包括解毒自由基和过氧化物,调节细胞生长和蛋白质的功能,和维护免疫功能,所以,谷胱甘肽的浓度可以作为评估氧化还原状态和在生物解毒状态在有机体中地位的指标。但半胱氨酸在人体中是起着至关重要的氨基酸之一,它可以改变蛋白质分子之间的二硫键来减弱蛋白质空间结构,从而使蛋白质能够伸展开来。另外半胱氨酸还具有解毒、抗衰老、改变炎症以及预防治疗放射性疾病。大量调查表明,超出正常水平的生物硫醇已被证明与人类疾病的产生有关,如缓慢生长、肝受损和皮肤受损。半胱氨酸不足会导致许多疾病,包括生长阻滞、肝损坏、嗜睡、肌肉软弱和脂肪损失。人类血液中高浓度的同型半胱氨酸是造成神经管效果、骨质疏松症和炎症性肠病等疾病的危险因素。
目前,用来定量检测半胱氨酸的方法多种多样,主要方法包括:滴定方法、紫外吸收法、分子荧光分析法、发射光谱分析法、质谱分析法等。几种方法相比之下,利用分子荧光分析的荧光探针有着明显的优势,包括高选择性、具有高灵敏度、能够快速反应、及时检测、细胞成像等。目前用来检测半胱氨酸的探针有很多,但是比率型近红外的荧光探针且能够进行生物成像的很少。
另一方面,比率型荧光探针能够利用相邻的两个峰之间的增减值来减轻周围环境对半胱氨酸检测的影响,所以具有更大的实用价值。
综上,建立一种能够检测半胱氨酸的比率型近红外荧光探针是十分有意义的。
发明内容
本领域急需一种高灵敏性和高选择性比率型测定半胱氨酸的探针,从而能够有效检测特别是能够在样本中检测半胱氨酸。为此,本发明提出了一种新颖的检测半胱氨酸的探针,该探针可直接使用,且能够高灵敏和高选择性比率型识别半胱氨酸。本发明涉及的探针可对半胱氨酸的进行高灵敏性和高选择性测定。
具体而言,本发明提供了一种识别半胱氨酸的探针,其结构如下:
其中:R1,R2,R3,R4,R5,R6,R7,R8,R9,R10,R11,R12和R13为独立地选自由氢原子、直链或支链烷基、直链或支链烷氧基、磺酸基、酯基和羟基组成的组;且其中的R1,R2,R3,R4,R5,R6,R7,R8,R9,R10,R11,R12和R13可以相同或不同。
优选地,本发明中所述直链或支链烷基、直链或支链烷氧基是C1-10的烷基或烷氧基。
优选地,本发明中所述直链或支链烷基、直链或支链烷氧基是C1-5的烷基或烷氧基。
优选地,本发明中所述直链或支链烷基、直链或支链烷氧基是C1-3的烷基或烷氧基。
优选地,本发明中所述直链或支链烷基为甲基、乙基、丙基、正戊基、2-甲基丁基、异丁基、或4-甲基庚基。
优选地,本发明中所述直链或支链烷氧基为甲氧基、乙氧基或丙氧基。
优选地,本发明中R1,R2,R3,R4,R5,R6,R7,R8,R9,R10,R11,R12和R3为独立地选自氢原子、甲基、乙基、丙基、甲氧基、乙氧基或丙氧基。
优选的,本发明的比率型探针是:
本发明的比率型荧光探针可与半胱氨酸进行作用,导致荧光光谱的变化,从而实现对半胱氨酸的比率型定量检测。
具体而言,本发明的半胱氨酸比率型荧光探针分别与Cys、Gly、Pro、Leu、Val、Thr、和Ala进行作用均不能导致荧光光谱的明显改变,从而实现对半胱氨酸的选择性识别,进而可任选地用于排除这些氨基酸的存在对半胱氨酸的定量测定的干扰。
本发明的半胱氨酸比色探针的稳定性好,能够长期保存使用。
进一步的,本发明的半胱氨酸探针能够高灵敏和高选择性比率型测定半胱氨酸,且不需要做进一步的有机合成修饰,有利于商业化的推广应用。
附图说明
图1:探针对于不同浓度的半胱氨酸有很好的响应值。探针(5μM)与半胱氨酸(0,0.1,0.2,0.30.4,0.5,0.6,0.7,0.8,0.9,1,1.5,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7,7.5,8μM)反应10min后放入荧光分光光度计进行检测。
图2:在浓度为0到8μM时有很好的线性,检出限为1μM。探针(5μM)与半胱氨酸(0,0.1,0.2,0.30.4,0.5,0.6,0.7,0.8,0.9,1,1.5,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7,7.5,8μM)反应10min后放入荧光分光光度计进行检测。
图3:在其它氨基酸(Cys、Gly、Pro、Leu、Val、Thr、Ala)浓度为100μM时,探针(5μM)对它们的荧光响应值,以及再在里面加入半胱氨酸后探针的响应值。检测溶液体系均为乙醇∶水=5∶5(v/v)含PBS=5mM,pH=7.4,测试温度为25℃。
具体实施方式
本发明提供了一种高灵敏和高选择性比率型测定半胱氨酸的探针及其光谱性能。
本发明的半胱氨酸比率型探针具有以下结构通式:
其中:R1,R2,R3,R4,R5,R6,R7,R8,R9,R10,R11,R12和R13为独立地选自由氢原子、直链或支链烷基、直链或支链烷氧基、磺酸基、酯基和羟基组成的组;且其中的R1,R2,R3,R4,R5,R6,R7,R8,R9,R10,R11,R12和R13可以相同或不同。
优选地,本发明中所述直链或支链烷基、直链或支链烷氧基是C1-10的烷基或烷氧基。
优选地,本发明中所述直链或支链烷基、直链或支链烷氧基是C1-5的烷基或烷氧基。
优选地,本发明中所述直链或支链烷基、直链或支链烷氧基是C1-3的烷基或烷氧基。
优选地,本发明中所述直链或支链烷基为甲基、乙基、丙基、正戊基、2-甲基丁基、异丁基、或4-甲基庚基。
优选地,本发明中所述直链或支链烷氧基为甲氧基、乙氧基或丙氧基。
优选地,本发明中R1,R2,R3,R4,R5,R6,R7,R8,R9,R10,R11,R12和R13为独立地选自氢原子、甲基、乙基、丙基、甲氧基、乙氧基或丙氧基。
较优选地,本发明中R1,R2,R3,R4,R5,R6,R7,R8,R9,R10,R11,R12和R13选自氢、甲基、乙基或丙基;最优选地,R1,R2,R3,R4,R5,R6,R7,R8,R9,R10,R11,R12和R13均为H。
本发明的能够高灵敏和高选择性比率型测定半胱氨酸的探针的显著特征是能够高灵敏和高选择性地识别半胱氨酸,和/或在氨基酸的存在下能够准确对半胱氨酸进行定量分析。
本发明的探针具有如下优点:
1、对半胱氨酸有着十分好的选择性,而对于别的氨基酸则无明显的响应;
2、探针响应时间为20min,因此可以作为快速检测半胱氨酸的手段;和/或
3、该探针的检出限为1μM,可以定量的检测1到8μM的半胱氨酸,灵敏度比较高。
该探针与半胱氨酸反应后有一个170nm的蓝移,这在检测半胱氨酸的比率型近红外类的探针中是比较少的。本发明也为合成一种比率型探针提出了一个新的方法。
下面将通过借助以下实施例来更详细地说明本发明。以下实施例仅是说明性的,应该明白,本发明并不受下述实施例的限制。
实施例1探针制备
2,6-二甲酰基苯酚合成与提纯
用天平称取苯酚940.5mg和六次甲基四胺2.80g放入100mL的玻璃反应瓶中,加入30mL的三氟乙酸,放入一个洗干净的小磁子,之后利用84-1磁力搅拌控温电热套加热回流,记录反应时间,反应过程中利用薄层层析硅胶板跟踪进程,约需反应6h,反应完全后,关掉反应。合成反应见式I:
式I:2,6-二甲酰基苯酚的合成
准备一个100mL的烧杯,在其中倒进50mL左右的蒸馏水。在反应停止,冷却后,将反应溶液滴加至事先准备好的烧杯中,
用滴管搅拌均匀,将烧杯中的液体倒入到分液漏斗中,再在其中添加20mL左右的二氯甲烷,扣紧塞子使劲摇晃几下,之后将分液漏斗放置在铁架上静置几分钟,使液体分层。由于二氯甲烷的密度比水大,所以二氯甲烷溶液在下面,我们的产物是有机物,一般不溶于水,从而萃取的过程中完全溶于二氯甲烷。打开分液漏斗的活塞,使二氯甲烷从下部流出。然后再在分液漏斗中剩余的液体中加入20mL左右的二氯甲烷萃取,如此反复重复3到5次,使产品完全进入二氯甲烷。
在萃取完之后,秤重一个25mL的玻璃反应瓶,用这个烧瓶将我们萃取所得到的二氯甲烷液体用旋转蒸发仪旋干。然后取少许旋干的物质在4mL的离心管中,再加入少量的二氯甲烷使其溶解,用玻璃毛细管移取少许溶液,在薄层层析硅胶板上点板,之后将薄层层析硅胶板放入含有约1mL二氯甲烷的玻璃广口瓶中,约静待3min,等到二氯甲烷液体快将薄层层析硅胶板全部浸湿时用镊子将薄层层析硅胶板取出,用手提式紫外分析仪中的365nm的荧光灯激发,可以观察到一个荧光点,在254nm的荧光灯激发时,没有其它点,说明萃取旋干后获得的固体比较纯,可以直接投下一步反应。等烧瓶冷却后再次称重,得到产品的重量为976.5mg,产率为65.1%。
2-甲酰基-6-苯并噻唑苯酚的合成与提纯
将上述得到的产品2,6-二甲酰基苯酚称取450.0mg和邻氨基苯硫酚称取375.6mg放入100mL的玻璃反应瓶里,再加入25mL无水乙醇溶液,使两种药品完全溶解,放入洗干净的小磁子,之后将玻璃反应瓶放在84-B强磁力搅拌器上室温搅拌。在搅拌的过程中加入9mmol的37%的盐酸溶液,最后加入18mmol的30%的过氧化氢溶液。继续在室温下搅拌30min左右。合成反应见式II。
式II:2-甲酰基-6-苯并噻唑苯酚的合成
反应完成,利用滴管将固体物质吸出,然后抽滤,用无水乙醇冲洗。
移取少许上层产品在离心管里,溶在少量的二氯甲烷里,用玻璃毛细管移取少许溶液,在薄层层析硅胶板上点板,之后将点好的硅胶板放进含有约1mL二氯甲烷的玻璃广口瓶里,约静待3min,等到二氯甲烷液体快将薄层层析硅胶板全部浸湿时用镊子将薄层层析硅胶板取出,用手提式紫外分析仪中的365nm的荧光灯激发,可以观察到一个荧光点,在254nm的荧光灯激发时,有一个很小的黑点,说明抽滤得到的固体比较纯,由于这不是最终产品,纯度达到80%以上就可以用于下一步反应,所以我们没有再进一步提纯,直接用于下一步反应。
抽滤一段时间,直至固体完全干透后,利用药匙将固体从漏斗中移取至25mL的小玻璃反应瓶中,事先对小玻璃反应瓶进行称重,放入产品后再次称重,从而得到产品的重量为521.6mg,产率为68.2%。
N-甲基-2-甲基喹啉鎓盐的制备及提纯
用天平称取300mg 2-甲基喹啉放入洗干净的50mL的玻璃反应瓶中,倒进20mL的碘甲烷,放入磁子,用84-1磁力搅拌控温电热套加热回流4个小时左右。合成反应见式III。
式III:N-甲基-2-甲基喹啉鎓盐的合成
反应完之后将烧瓶取下,待冷却后,有固体析出,用抽滤装置进行抽滤,得到固体,称重后约285mg,产率为98%。
2-(N-甲基-2-乙烯基)喹啉-6-苯并噻唑苯酚的合成及提纯
称取产物2-甲酰基-6-苯并噻唑苯酚255mg和产物N-甲基-2-甲基喹啉鎓盐285mg放入洗干净的50mL的玻璃反应瓶里,倒进20mL左右的无水乙醇,尽量使2种药品完全溶解,之后放进小磁子,用84-1磁力搅拌控温电热套加热回流,反应12个小时。
式IV:2-(N-甲基-2-乙烯基)喹啉-6-苯并噻唑苯酚的合成
反应结束后,通过旋蒸去除反应溶剂无水乙醇,并加入15mL的二氯甲烷使其溶解。然后将溶液滴加至色谱柱中,得到纯品的重量为108.9mg,产率为20.8%。
1H-NMR结果的分析
氢谱数据如下:1H-NMR(400MHz,DMSO-d6)δ(*10-6):4.23(s,3H),7.16(t,J=7.6Hz,1H),7.25-7.27(m,4H),7.52(t,J=7.0Hz,1H),7.59(t,J=7.0Hz,1H),7.64(d,J=16.9Hz,1H),7.90(d,J=8.0Hz,1H),7.99(d,J=8.0Hz,1H),8.10(d,J=8.0Hz,1H),8.15(d,J=16.9Hz,1H),8.20(d,J=8.0Hz,1H),8.22(d,J=7.6Hz,1H),8.83(d,J=7.6Hz,1H),13.32(s,1H)。以上数据符合2-(N-甲基-2-乙烯基)喹啉-6-苯并噻唑苯酚的结构式特征。
13C-NMR结果的分析
碳谱数据如下:13C-NMR(100MHz,DMSO-d6)δ(*10-6):46.28,114.26,116.27,117.19,118.64,119.16,121.32,121.76,123.18,123.53,123.97,124.38,125.47,126.29,127.62,130.36,131.28,131.38,134.25,144.47,150.18,152.47,155.29,167.36。以上数据符合2-(N-甲基-2-乙烯基)喹啉-6-苯并噻唑苯酚的结构式特征。
实施例2溶液配制
称取5.22mg的最终产品,配制成1mM的探针母液。称取12.1mg的半胱氨酸配制成10mM的半胱氨酸(Cys)母液。
从探针母液中移取50μL液体稀释至10mL,配成5μM的探针检测液。将10mL探针检测液一分为二,在其中的一份中加入25μL的半胱氨酸母液,浓度为50μM。
上述用于配制溶液的体系均为乙醇:水=5:5(v/v)含PBS=5mM,pH=7.4。
实施例3响应时间对探针识别半胱氨酸的影响
和实施例2所述一样配成5μM的探针检测液,之后快速加入半胱氨酸母液使其浓度为10μM,摇晃几下,使溶液中的物质分配均匀后将其快速放进荧光分光光度计中,设置荧光分光光度计在每30秒扫描一次,由此得到不同时间的响应光谱,记录光谱图并保存,检测时室温为25℃。检测溶液体系仍然为乙醇∶水=5∶5(v/v)含PBS=5mM,pH=7.4。结果如图1所示。从荧光光谱图上看出纯探针溶液在700nm处峰值比较高,在530nm处基本上没有峰,加入半胱氨酸后,光谱图为在700nm处的峰值慢慢减小,在530nm处的峰值慢慢增高。
实施例4探针对半胱氨酸的定量影响
同样配制5μM的探针检测液,并将探针检测液加入比色管中定容至10mL。移取半胱氨酸溶液依次加入比色管中,使半胱氨酸的浓度最终为:0,0.1,0.2,0.30.4,0.5,0.6,0.7,0.8,0.9,1,1.5,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7,7.5,8μM并使其分布均匀,检测溶液体系均为乙醇:水=5:5(v/v)含PBS=5mM,pH=7.4,放置10min后用荧光分光光度计在激发波长为430nm下进行检测并保存好光谱图。检测时室温为25℃。结果如图2所示。探针对于不同浓度的半胱氨酸有很好的响应值。在浓度为0到8μM时有很好的线性,检出限为1μM。由此可见,本发明所合成的探针可以通过比较相邻两个峰的荧光强度的方法检测半胱氨酸并有良好的灵敏度。
实施例5探针识别半胱氨酸时抗干扰性
准备7个比色管加入探针溶液定容到10mL,第一个中只加入半胱氨酸10μM,剩下的依次加入Cys、Gly、Pro、Leu、Val、Thr、Ala,浓度为100μM,然后再加入半胱氨酸10μM。同样静置10min后,从左侧第一个比色管开始,依次放入荧光分光光度计中检测,并保存好数据。检测溶液体系为乙醇:水=5:5(v/v)含PBS=5mM,pH=7.4,检测时室温为25℃。结果如图3所示。
从图中可以看出,在将其它硫醇溶液加入到探针溶液中反应一段时间后,溶液中探针的荧光强度没有太大的变化,说明没有响应,但是当再在里面加入半胱氨酸后,探针就会出现比较高的响应值。因此,探针对半胱氨酸有良好的选择性、抗干扰性。
由此可见,本发明的探针是一个对半胱氨酸有高选择性和很好的抗干扰性的探针。
虽然用上述实施方式描述了本发明,应当理解的是,在不背离本发明的精神的前提下,本发明可进行进一步的修饰和变动,且这些修饰和变动均属于本发明的保护范围之内。
Claims (8)
1.化合物,其具有以下结构
其中:R1,R2,R3,R4,R5,R6,R7,R8,R9,R10,R11,R12和R3为独立地选自由氢原子、直链或支链烷基、直链或支链烷氧基、磺酸基、酯基和羟基组成的组;且其中的R1,R2,R3,R4,R5,R6,R7,R8,R9,R10,R11,R12和R13可以相同或不同。
2.根据权利要求1所述的化合物,其中所述直链或支链烷基、直链或支链烷氧基是C1-10的烷基或烷氧基。
3.根据权利要求1所述的化合物,其中所述直链或支链烷基、直链或支链烷氧基是C1-5的烷基或烷氧基。
4.根据权利要求1所述的化合物,其中所述直链或支链烷基、直链或支链烷氧基是C1-3的烷基或烷氧基。
5.根据权利要求1所述的化合物,其中所述直链或支链烷基为甲基、乙基、丙基、正戊基、2-甲基丁基、异丁基、或4-甲基庚基。
6.根据权利要求1所述的化合物,其中所述直链或支链烷氧基为甲氧基、乙氧基或丙氧基。
7.根据权利要求1所述的化合物,其中:R1,R2,R3,R4,R5,R6,R7,R8,R9,R10,R11,R12和R13为独立地选自氢原子、甲基、乙基、丙基、甲氧基、乙氧基或丙氧基。
8.根据权利要求1所述的化合物,其为如下结构的化合物:
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