CN105837609B - 一种快速检测半胱氨酸的荧光探针的制备及其应用 - Google Patents
一种快速检测半胱氨酸的荧光探针的制备及其应用 Download PDFInfo
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Abstract
本发明涉及一种快速检测半胱氨酸的荧光探针的制备其及应用，探针化合物的结构如I所示。其具体制备方法是：在4‑氰基联苯酚的3号位上以多聚甲醛引入醛基，得到的化合物II与吡咯发生亲核加成生成化合物III；化合物III先后与DDQ和三氟化硼‑乙醚反应得到化合物Ⅳ，最后与氯乙酰氯发生取代反应生成探针化合物Ⅰ。该探针化合物可用于半胱氨酸的快速分析检测，具有灵敏度高、检测限低等特点。
Description
技术领域
本发明涉及一种快速检测半胱氨酸的荧光探针化合物的制备及其应用，属于荧光探针技术领域。
技术背景
生物体内有着各种不同的活性物种，它们有着特殊的生理功能，并对生命过程起着至关重要的影响。在众多组成生命的物质中，巯基化合物起着不可代替的重要作用。半胱氨酸作为一种重要的巯基氨基酸，参与蛋白质的合成和细胞新陈代谢。人体中半胱氨酸的缺乏会导致儿童生长缓慢，皮肤损伤，身体虚弱等健康问题，而过量的半胱氨酸则会诱发神经性中毒。同型半胱氨酸通常源于蛋氨酸在体内代谢。临床研究显示，它能够刺激血管平滑肌增生，造成血管内皮损伤，与心血管疾病、糖尿病、癌症等慢性病有着密切的关系。谷胱甘肽是细胞内含量最高的非蛋白巯基化合物，起着维持机体氧化还原动态平衡的作用以防止细胞内过量氧化态物质的堆积。因此，详尽的监测细胞中此类巯基化合物分布、水平波动对了解生命过程、研究生物体病理有着重要的科学意义。
基于光学检测的荧光探针分析方法，具有高灵敏度、高选择性、可原位检测、对细胞损伤小等优势，目前很多研究人员正在致力于这项研究。在现有技术中，CN104119263 A提供了一种基于花菁素的半胱氨酸荧光探针化合物，CN104447421 A提供了一种基于2-羟基-6-乙酰基萘的半胱氨酸探针化合物，但是此两种化合物都存在制备方法复杂，不能达到更低浓度的检测限以及实现半胱氨酸的快速检测。
发明内容
针对现有技术的不足，本发明提供一种半胱氨酸荧光探针化合物，可以灵敏快速地检测半胱氨酸。
本发明还提供所述半胱氨酸荧光探针化合物的制备方法与应用。
本发明的技术方案如下：
一种半胱氨酸荧光探针化合物，具有式I所示的结构：
本发明所述的半胱氨酸荧光探针化合物的制备方法，包括如下步骤：
（1）在氮气保护下，将4-氰基联苯酚溶于乙腈中，分别依次加入适量无水氯化镁、三乙胺和多聚甲醛，反应在70℃下进行5小时，得到化合物II；
（2）在氮气保护下，将化合物II溶于干燥的二氯甲烷中，加入过量吡咯和催化量的三氟乙酸，该反应在常温下进行1小时，得到化合物Ⅲ；
（3）在氮气保护下，将化合物III溶于干燥的二氯甲烷中，加入适量DDQ并反应1.5小时，反应结束后，取过量三乙胺和三氟化硼-乙醚溶液依次加入，反应在常温下进行22小时，得到化合物Ⅳ；
（4）在氮气保护下，将化合物Ⅳ，溶于干燥的二氯甲烷中，加入过量的三乙胺和氯乙酰氯，反应在冰盐浴下进行4小时，得到探针化合物I。
根据本发明，优选的，步骤（1）所述4-氰基联苯酚、无水氯化镁、三乙胺和多聚甲醛的摩尔比为1:1.5:4:15；
根据本发明，优选的，步骤（2）所述化合物Ⅱ、三氟乙酸和吡咯的摩尔比为1:0.1:20；
根据本发明，优选的，步骤（3）所述化合物Ⅲ、DDQ、三乙胺和三氟化硼-乙醚的摩尔比为1:1.2:10:15；
根据本发明，优选的，步骤（4）所述化合物Ⅳ、三乙胺和丙烯酰氯的摩尔比为1:1.2:1.2；
根据本发明，优选的，步骤（1）-（4）全程在氮气保护下进行；
更为详细的，本发明的半胱氨酸荧光探针化合物的制备方法，步骤如下：
(a) 在氮气保护下，取3.1 g（16 mmol）的4-氰基联苯酚溶于50 ml乙腈中，向其中加入2.8 g（24 mmol）的无水MgCl2和8.2 ml（60 mmol）的无水三乙胺，最后加入干燥的多聚甲醛6.1 g（220 mmol），在70℃下反应8小时。反应完成后，溶液用大量的稀盐酸酸化，二氯甲烷萃取，硅胶柱分离，得到白的固体产物Ⅱ1.8 g，产率52%。
(b) 在氮气保护下，取1.0 g（4.48 mmol）的化合物Ⅱ，置于100 ml三口烧瓶中，向其中加入30 ml干燥的二氯甲烷，搅拌至完全溶解。再加入7.5 ml（0.11 mol）吡咯和催化量的三氟乙酸，反应在是常温进行一小时。待反应结束后，饱和碳酸钠溶液洗涤，无水硫酸钠干燥，旋蒸除去溶剂，硅胶柱分离，得灰白色化合物Ⅲ 0.7 g，产率43%。
(c) 在氮气保护下，取0.65g（1.91 mmol）化合物Ⅲ溶于50 ml干燥的二氯甲烷中，向溶液中缓慢加入0.5 g（2.2 mmol）DDQ，反应在常温下进行1.5小时。反应结束后，再加入3.0 ml（22 mmol）三乙胺，反应十分钟后，缓慢加入4.0 ml（32 mmol）三氟化硼乙醚，反应在常温下进行22小时。粗产物水洗，二氯甲烷萃取，硅胶柱纯化，得到红色固体化合物Ⅳ0.3g，产率41%。
(d) 在氮气保护下，取0.07 g（0.26 mmol）化合物Ⅳ加入100 ml的三口烧瓶中，加入10ml干燥的二氯甲烷溶解，冰浴降温至0℃，再加入0.04 ml（0.31 mmol）干燥的三乙胺和0.026 ml（约0.31 mmol）的氯乙酰氯。反应在冰盐浴下进行4小时停后，加入少量的水淬灭反应。粗产物经过水洗、萃取、干燥、硅胶柱纯化，得到化合物Ⅰ0.038g，产率约为41%。
本发明所述的半胱氨酸荧光探针化合物，可应用水体环境中的半胱氨酸含量的快速检测。
进一步优选的，所述荧光探针用于在pH 7.4的乙腈/水（1:1，v/v）溶液中半胱氨酸的快速检测。最低可探测的半胱氨酸浓度为19.1nM。
本发明通过实验验证，所述荧光探针在pH 7.4的乙腈/水（1:1，v/v）溶液中，以360nm波长的光源作为激发光，其溶液在530 nm处有很强的绿色荧光，随着半胱氨酸的加入，溶液荧光在10分钟内迅速淬灭；而加入同型半胱氨酸和谷胱甘肽的时候，荧光也会发生淬灭，但是响应时间较长；加入其他氨基酸后溶液的荧光没有明显变化。本发明的荧光探针化合物对半胱氨酸具有很高的选择性。
本发明的荧光探针化合物在pH 7.4的乙腈/水（1:1，v/v）溶液中，随着半胱氨酸浓度的增加，其荧光逐渐发生淬灭。且荧光强度与半胱氨酸浓度成反比例线性关系，据此线性关系，可以定量测试溶液中半胱氨酸的含量。
与现有的检测技术相比，本发明荧光探针具有以下优点：
1、本发明的荧光探针化合物对半胱氨酸选择性好，灵敏度高，检测限极低；
2、本发明的荧光探针化合物所用原料都是常规试剂，合成简单，产率高；
3、本发明的荧光探针化合物对半胱氨酸的响应速度快，实现了快速分析；
4、本发明的荧光探针化合物对同型半胱氨酸和谷胱甘肽也有响应，通过响应时间分析，可以也区别此两种物质，实现了一种探针分子的多功能检测。
附图说明
图1为本发明的荧光探针化合物的核磁氢谱图。
图2为本发明的荧光探针在pH 7.4的乙腈/水（1:1，v/v）溶液中，与不同浓度的半胱氨酸响应后的荧光强度工作曲线图。
图3为本发明的荧光探针化合物在pH 7.4的乙腈/水（1:1，v/v）溶液中，与半胱氨酸、同型半胱氨酸以及谷胱甘肽响应后，荧光强度随时间的变化。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明，但不限于此。实施例中的各种原料均来自于市场购买。
实施例1、荧光探针分子的合成
(a)在氮气保护下，取3.1 g（16 mmol）的4-氰基联苯酚溶于50 ml乙腈中，向其中加入2.8 g（24 mmol）的无水MgCl2和8.2 ml（60 mmol）的无水三乙胺，最后加入干燥的多聚甲醛6.1 g（220 mmol），在70℃下反应8小时。反应完成后，溶液用大量的稀盐酸酸化，二氯甲烷萃取，硅胶柱分离，得到白的固体产物Ⅱ1.8 g，产率52%。
(b)在氮气保护下，取1.0 g（4.48 mmol）的化合物Ⅱ，置于100 ml三口烧瓶中，向其中加入30 ml干燥的二氯甲烷，搅拌至完全溶解。再加入7.5 ml（0.11 mol）吡咯和催化量的三氟乙酸，反应在常温进行一小时。待反应结束后，饱和碳酸钠溶液洗涤，无水硫酸钠干燥，旋蒸除去溶剂，硅胶柱分离，得灰白色化合物Ⅲ 0.7 g，产率43%。
(c)在氮气保护下，取0.65g（1.91 mmol）化合物Ⅲ溶于50 ml干燥的二氯甲烷中，向溶液中缓慢加入0.5 g（2.2 mmol）DDQ，反应在常温下进行1.5小时。反应结束后，再加入3.0 ml（22 mmol）三乙胺，反应十分钟后，缓慢加入4.0 ml（32 mmol）三氟化硼乙醚，反应在常温下进行22小时。粗产物水洗，二氯甲烷萃取，硅胶柱纯化，得到橘红色固体化合物Ⅳ0.3g，产率约为41%。
(d)在氮气保护下，取0.07 g（0.26 mmol）化合物Ⅳ加入100 ml的三口烧瓶中，加入10ml干燥的二氯甲烷溶解，冰浴降温至0℃，再加入0.04 ml（0.31 mmol）干燥的三乙胺和0.026 ml（0.31 mmol）的氯乙酰氯。反应在冰盐浴下进行4小时停后，加入少量的水淬灭反应。粗产物经过水洗、萃取、干燥、硅胶柱纯化，得到化合物Ⅰ0.038g，产率约为41%。核磁氢谱如图1。
实施例2、探针分子对不同巯基氨基酸的荧光响应实验
取本发明的荧光探针化合物，溶解到乙腈中，配制成50 μM 浓度的探针分子母液；取一定量的半胱氨酸、同型半胱氨酸和谷胱甘肽，分别溶于100ml 超纯水中，配制成10-2 M浓度的不同氨基酸母液；取2.38g 4-羟乙基哌嗪乙磺酸，溶于100 ml 超纯水中，用氢氧化钠调价pH到7.4，配制成缓冲浓度为0.1 M的缓冲溶液。
移取1ml 探针分子母液，向其中加入4 ml 乙腈，1 ml HEPES缓冲溶液，3.7 ml 超纯水，分别向其中加入0.3 ml Cys、Hcy和GSH母液。立即开始扫描其荧光强度随时间的变化。如图2，结果表明可以根据反应时间以及进行的程度来快速区分不同的巯基氨基酸。
实施例3、探针分子对不同浓度的半胱氨酸的荧光响应实验
移取13组1 ml 探针分子母液，向其中加入4 ml乙腈，1 ml HEPES缓冲溶液，分别加入3.7 ml、3.725 ml、3.75 ml、3.775ml、3.80ml、3.825ml、3.85ml、3.875ml、3.90ml、3.925ml、3.950ml、3.975ml、4.0 ml超纯水，和0.3 ml、0.275 ml、0.25 ml、0.225ml、0.20ml、0.175ml、0.150ml、0.125ml、0.100ml、0.075ml、0.050ml、0.025ml、0.0 ml Cys母液。室温下培养十分钟，以360 nm波长的光源激发，测试其荧光发射光谱。如图3所示，结果表明探针分子能够准确表征不同浓度的半胱氨酸。
Claims (1)
1.一种半胱氨酸荧光探针化合物，具有式I所示的结构。
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