CN114195749B - 一种荧光染料fp-1及其制备方法和用途 - Google Patents
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Abstract
本发明公开了一种荧光染料FP‑1及其制备方法,涉及荧光染料技术领域,制备方法包括以下步骤:将4‑甲基‑7‑羟基‑8‑甲酰基香豆素和苯甲酰乙酸乙酯溶解于有机溶剂中,向其中滴加哌啶,得到反应液,将反应液在20~30℃下搅拌反应3~6小时,反应完成后进行过滤,对所得滤饼进行重结晶处理,得到荧光染料FP‑1;本发明的荧光染料FP‑1,可以在DMF/水(v/v=1:1)时,通过荧光增强检测半胱氨酸,最低检测限为43μM,并且在半胱氨酸浓度为25~750μM时,可利用荧光染料FP‑1定量检测半胱氨酸。
Description
技术领域
本发明涉及荧光染料技术领域,具体说是一种荧光染料FP-1及其制备方法和用途。
背景技术
半胱氨酸(Cys)、同型半胱氨酸(Hcy)和谷胱甘肽(GSH)是生物体内最常见也最重要的生物硫醇,这些生物硫醇具有独特的亲核性与氧化还原性质,因此对于维持人体健康方面发挥着不可替代的作用。半胱氨酸的缺少可能导致儿童成长缓慢以及肌肉萎缩等严重健康问题,而细胞中同型半胱氨酸浓度的异常通常与心血管疾病与阿尔兹海默症密不可分,谷胱甘肽在蛋白质的合成中起到极为关键的调节作用,其浓度的异常会引起新陈代谢的紊乱,因此准确的测定半胱氨酸、同型半胱氨酸和谷胱甘肽等生物硫醇的含量具有重要的意义。
目前对于生物硫醇的检测方法有液相色谱法和毛细管电泳法,但是现有检测方法需要使用昂贵的仪器、对操作人员的专业技能要求高、以及需要对待测样品进行预处理,相比之下,荧光检测法具有操作简便和灵敏度高等优点,因此越来越受到人们的关注。由于半胱氨酸在生物体内可转化成谷胱甘肽,因此两者在检测时对荧光探针的灵敏度要求更高,因此在半胱氨酸和谷胱甘肽共存时专属检测半胱氨酸的荧光探针材料亟待开发。
发明内容
为解决上述问题,本发明的目的是提供一种荧光染料FP-1及其制备方法和用途。
本发明为实现上述目的,通过以下技术方案实现:
一种荧光染料FP-1,其结构式如下:
本发明还包括荧光染料FP-1的制备方法,包括以下步骤:
将4-甲基-7-羟基-8-甲酰基香豆素和苯甲酰乙酸乙酯溶解于有机溶剂中,向其中滴加哌啶,得到反应液,将反应液在20~30℃下搅拌反应3~6小时,反应完成后进行过滤,对所得滤饼进行重结晶处理,得到荧光染料FP-1;
其中4-甲基-7-羟基-8-甲酰基香豆素、苯甲酰乙酸乙酯、哌啶和有机溶剂的摩尔体积比为1mol:0.9~1.5mol:3~4L;
4-甲基-7-羟基-8-甲酰基香豆素和苯甲酰乙酸乙酯的质量和与哌啶的质量比为1:0.05~0.1。
优选的制备方法,所述有机溶剂为乙腈或/和乙酸乙酯。
优选的制备方法,所述有机溶剂为乙腈。
优选的制备方法,重结晶采用的溶剂由乙腈和乙醇混合得到。
优选的制备方法,重结晶采用的溶剂由乙腈和乙醇按照体积比4~9:1混合得到。
本发明还包括荧光染料FP-1的用途,用于选择性检测半胱氨酸。
本发明相比现有技术具有以下优点:
本发明的荧光染料FP-1,可以在DMF/水(v/v=1:1)时,通过荧光增强检测半胱氨酸,最低检测限为43μM,并且在半胱氨酸浓度为25~750μM时,可利用荧光染料FP-1定量检测半胱氨酸;
本发明的荧光染料FP-1的制备方法,反应步骤少,原料易得,方便易行,成本低,对环境友好,所合成的新的荧光染料FP-1可方便地用于定性和定量检测半胱氨酸,产生良好的经济效益和社会效益。
附图说明
图1为荧光染料FP-1的红外光谱图;
图2为荧光染料FP-1的核磁共振氢谱;
图3为荧光染料FP-1的核磁共振碳谱;
图4为荧光染料FP-1溶液(Blank)及加入不同氨基酸后的荧光发射光谱图;
图5为荧光染料FP-1和Cys溶液中加入其它氨基酸后在445 nm处荧光强度变化图;
图6为荧光染料FP-1溶液(20 μM)中加入不同浓度Cys后的荧光发射光谱图;
图7为荧光染料FP-1溶液(20 μM)中加入不同浓度Cys后在445 nm处荧光强度变化图。
具体实施方式
本发明的目的是提供一种荧光染料FP-1及其制备方法和用途,通过以下技术方案实现:
一种荧光染料FP-1,其结构式如下:
本发明还包括荧光染料FP-1的制备方法,合成路线为
;
包括以下步骤:
将4-甲基-7-羟基-8-甲酰基香豆素和苯甲酰乙酸乙酯溶解于有机溶剂中,向其中滴加哌啶,得到反应液,将反应液在20~30℃下搅拌反应3~6小时,反应完成后进行过滤,对所得滤饼进行重结晶处理,得到荧光染料FP-1;
其中4-甲基-7-羟基-8-甲酰基香豆素、苯甲酰乙酸乙酯、哌啶和有机溶剂的摩尔体积比为1mol:0.9~1.5mol:3~4L;
4-甲基-7-羟基-8-甲酰基香豆素和苯甲酰乙酸乙酯的质量和与哌啶的质量比为1:0.05~0.1。
优选的制备方法,所述有机溶剂为乙腈或/和乙酸乙酯。
优选的制备方法,所述有机溶剂为乙腈。
优选的制备方法,重结晶采用的溶剂由乙腈和乙醇混合得到。
优选的制备方法,重结晶采用的溶剂由乙腈和乙醇按照体积比4~9:1混合得到。
本发明还包括荧光染料FP-1的用途,用于选择性检测半胱氨酸,本发明的荧光染料FP-1可以在以下常见氨基酸中特异性检测出半胱氨酸,常见氨基酸包括缬氨酸、酪氨酸、亮氨酸、天冬酰胺、色氨酸、组氨酸、精氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、谷氨酸、甘氨酸、谷胱甘肽和同型半胱氨酸,均不会对半胱氨酸的检测产生干扰。
本发明的原料4-甲基-7-羟基-8-甲酰基香豆素为黄色固体,可外购或参照文献报道的方法(Jin L, Tan X, Dai L, Zhao C, Wang W, Wang Q. A novel coumarin-basedfluorescent probe with fine selectivity and sensitivity for hypochlorite andits application in cell imaging. Talanta, 2019, 202: 190-197)合成。
以下结合具体实施例来对本发明作进一步的描述。
实施例1
一种荧光染料FP-1,其结构式如下:
。
实施例2
实施例1所述一种荧光染料FP-1的制备方法,包括以下步骤:
将204g 4-甲基-7-羟基-8-甲酰基香豆素和172.8g苯甲酰乙酸乙酯溶解于3L乙腈中,向其中滴加18.84g哌啶,得到反应液,将反应液在20℃下搅拌反应6小时,反应完成后进行过滤,对所得滤饼进行重结晶处理,得到荧光染料FP-1。
实施例3
实施例1所述一种荧光染料FP-1的制备方法,包括以下步骤:
将204g 4-甲基-7-羟基-8-甲酰基香豆素和288g苯甲酰乙酸乙酯溶解于4L乙酸乙酯中,向其中滴加49.2g哌啶,得到反应液,将反应液在30℃下搅拌反应3小时,反应完成后进行过滤,对所得滤饼进行重结晶处理,得到荧光染料FP-1。
实施例4
实施例1所述一种荧光染料FP-1的制备方法,包括以下步骤:
将204g 4-甲基-7-羟基-8-甲酰基香豆素和192g苯甲酰乙酸乙酯溶解于有机溶剂中,向其中滴加23.5g哌啶,得到反应液,将反应液在25℃下搅拌反应4小时,反应完成后进行过滤,对所得滤饼进行重结晶处理,得到荧光染料FP-1;
所述有机溶剂由乙腈和乙酸乙酯按照体积比1:1混合得到。
实施例5
实施例1所述一种荧光染料FP-1的制备方法,包括以下步骤:
将204g 4-甲基-7-羟基-8-甲酰基香豆素和230g苯甲酰乙酸乙酯溶解于有机溶剂中,向其中滴加25g哌啶,得到反应液,将反应液在25℃下搅拌反应4小时,反应完成后进行过滤,对所得滤饼进行重结晶处理,得到荧光染料FP-1;
所述有机溶剂由乙腈。
对实施例5中的滤饼进行重结晶处理时,采用不同的重结晶试剂对产物FP-1的产率和纯度进行检测,其中纯度按照高效液相色谱法进行检测,结果如表1所示。
表1 重结晶试剂的选择对产品收率和纯度的影响
由表1的结果可以看出,重结晶的溶剂选为乙腈和乙醇时,产品的纯度在98%以上,并且重结晶采用的溶剂由乙腈和乙醇按照体积比4~9:1混合时,产品的收率能达到85%以上,并且纯度也能在99.0%以上。
对上述实施例所得的荧光染料FP-1进行红外光谱、核磁共振氢谱和核磁共振碳谱对荧光染料FP-1的结构进行了表征,结果如下:
氢谱数据:如图1所示,数据整理如下:1H NMR (400 MHz, DMSO-d6): δ (ppm)8.03 (s, 1H), 7.73 (d, J = 8 Hz, 1H), 7.58-7.54 (m, 2H), 7.40-7.37 (m, 3H),6.92-6.89 (m, 1H), 6.30 (s, 1H), 4.02 (q, J = 8 Hz, 2H), 2.41 (t, J = 8 Hz,3H), 0.99 (s, 3H).
碳谱数据:如图2所示,数据整理如下: 13C NMR (101 MHz, DMSO-d6): δ (ppm)191.0, 159.32, 158.60, 157.20, 153.45, 150.07, 138.04, 135.96, 133.99,129.93, 129.61, 128.70, 126.48, 115.67, 113.32, 112.52, 56.00, 18.53, 18.33,18.26, 13.70.
红外数据:如图3所示,数据整理如下: IR (KBr pellet): ν (cm‒1) 3297.3 (酚羟基O‒H的伸缩振动), 3078.4 (=C‒H伸缩振动), 2992.6 (C‒H伸缩振动), 1702.4 (C=O伸缩振动), 1620.0 (C=C伸缩振动), 1588.6 (苯环上C=C伸缩振动), 1477.8 (CH2中C‒H变形振动), 1387.6 (CH3中C‒H变形振动), 1363.5 (CH3中C‒H变形振动), 1265.6 (C‒O伸缩振动), 1177.9 (C‒O‒C伸缩振动), 1058.8 (苯酚C‒O伸缩振动), 972.5 (=C‒H变形振动), 819.6 (苯环上=C‒‒H变形振动), 699.6 (苯环上=C‒H变形振动)。
测得荧光染料FP-1的比移值Rf = 0.52 (石油醚/乙酸乙酯 = 2:1, v/v)。以上结果证明荧光染料FP-1的结构正确。
对上述实施例所得的荧光染料FP-1在混合溶剂DMF/水(1:1, v/v)中的溶液及加入不同氨基酸后的荧光发射光谱,如图4所示,激发波长为335 nm。荧光染料FP-1溶液(20 μM)在445 nm处显示出中等强度的荧光发射峰,加入Cys后荧光强度显著增强,而加入其它氨基酸包括缬氨酸(Val)、酪氨酸(Tyr)、亮氨酸(Leu)、天冬酰胺(Asn)、色氨酸(Trp)、组氨酸(His)、精氨酸(Arg)、赖氨酸(Lys)、甲硫氨酸(Met)、苯丙氨酸(Phe)、谷氨酸(Glu)、甘氨酸(Gly)、GSH和Hcy后荧光强度未发生明显变化,表明染料FP-1可作为荧光增强型探针,用于Cys的荧光检测。
为了考察荧光染料FP-1在荧光检测Cys时的抗干扰性,测定了荧光染料FP-1的DMF/水(1:1, v/v)溶液(20 μM)中加入Cys (200 μM)以及再加入其它氨基酸(200 μM)后的荧光发射光谱,激发波长设定为335 nm。将光谱图中445 nm处荧光强度变化表示在图5中,从图中可以看出,利用荧光染料FP-1对Cys进行荧光检测时,只有共存氨基酸为Hcy时对检测产生一定的影响,其它氨基酸的共存不会对检测产生干扰,表明染料FP-1作为荧光增强型探针检测Cys具有良好的抗干扰性能。
设定激发波长为335 nm,测定了荧光染料FP-1的DMF/水(1:1, v/v)溶液(20 μM)中加入不同浓度Cys (10, 25, 50, 150, 250, 500, 750 μM)后的荧光发射光谱图,如图6所示,浓度从大到小对应曲线由高到低,随着加入Cys浓度的增加,体系在445 nm处的荧光发射峰强度逐渐增大。将光谱图中445 nm处荧光强度随加入Cys浓度的变化作图,得到图7。在加入Cys浓度为25~750 μM范围内,445 nm处的荧光发射峰强度与加入Cys浓度呈现良好的线性相关,通过线性拟合得到线性方程式Y = 748.54X + 204284,线性相关系数R2 =0.9952,计算得到荧光染料FP-1检测Cys的最低检测限为43 μM。在Cys浓度为25~750 μM时,可利用荧光染料FP-1定量检测Cys。
Claims (2)
1.一种荧光染料FP-1,其特征在于:所述荧光染料FP-1的结构式如下:
。
2.权利要求1所述荧光染料FP-1在制备选择性检测半胱氨酸的检测试剂中的应用。
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