CN106220650B - 一种基于罗丹明B的Cys荧光传感器、制备方法及应用 - Google Patents
一种基于罗丹明B的Cys荧光传感器、制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种基于罗丹明B的Cys荧光传感器、制备方法及应用，以罗丹明B为前体，通过两步合成目标产物3'‑羟基‑3‑羰基‑3H‑螺[异苯并呋喃‑1,9'‑占吨]‑6'‑基(E)‑4‑((2‑(3',6'‑二(二乙胺基)‑3‑羰基螺[异二氢吲哚‑1,9'‑占吨]‑2‑基)乙基)氨基)‑4‑羰基丁‑2‑烯酸酯。本发明通过将目标产物对Cys进行检测，发现其对Cys具有很好的检测效果，同时，本发明采用的原料易得，合成步骤简单，后处理亦很方便，较易实现大规模生产。
Description
技术领域
本发明属于生物化学领域，具体涉及一种基于罗丹明B的Cys荧光传感器的制备及应用。
背景技术
半胱氨酸(Cys)即2-氨基-3-巯基丙酸，在调节细胞的各种功能方面发挥着至关重要的作用。它的主要生理作用是能够清除掉人体内的自由基，作为体内一种重要的抗氧化剂，保护许多蛋白质和酶等分子中的巯基。当Cys的含量异常时，则会带来许多健康方面的问题。例如：身体浮肿、嗜睡、肝功能损害、肌肉和脂肪的损失、增长缓慢、皮肤病灶等。另一方面，当Cys的含量高于正常值时，则会引起癌症、心血管疾病、以及老年痴呆症。鉴于Cys对人体如此至关重要，研究一种能够快速方便检测 Cys的方法具有十分重要的意义。
目前，检测Cys的方法主要有：分光光度法、荧光法、高效液相色谱(HPLC)法、高效毛细管电泳法(HPCE)、流式细胞仪法等，然而这些方法存在很多缺点，如所需仪器价格较为昂贵费时，携带不便，敏感性差，所测定的Cys浓度范围不大，对于含Cys量较小的细胞中测量则更是困难等。与此相反，由于化学发光不需要任何光源，因而在对荧光探针进行化学发光成像检测时，不存在荧光检测或者荧光成像时不可避免的光学背景的干扰，从而可以获得更低的检出限。
罗丹明类染料由于其摩尔吸光系数较大，荧光量子产率高、光谱性能优越、结构简单、易于修饰、吸收波长范围广等优势，已经被广泛应用与分子探针设计，目前，罗丹明类分子探针多用于检测Al3+，Cr3+，GSH等。
文献1(Lin S,Yoon D,Ha D,et al.Caged rhodamine-based fluorescent probefor biothiol:Selectivedetection of cysteine over homocysteine and glutathionein water[J]. Sensors and Actuators B:Chemical,2013,188:111-116.)报道了一种利用苯甲酸乙烯脂类衍生物和荧光素进行缩合，合成出一种新型的Cys传感器的方法，产率8％，但是，其产物产率、选择性并不是特别好、响应时间也过长。
文献2(Murale D P,Kim H,Choi W S,et al.Extremely selectivefluorescence detection of cysteine or superoxide with aliphatic esterhydrolysis[J].RSC Advances,2014, 4:46513-46516.)报道了一种荧光素与丙烯酸进行缩合，合成出一种新型的Cys传感器的方法，产率69％，但依然存在Hcy干扰选择性检测Cys的问题。
发明内容
本发明目的是提供一种基于罗丹明B的Cys荧光传感器、制备方法及应用。
实现本发明目的的技术解决方案是：
一种基于罗丹明B的Cys荧光传感器，该荧光传感器的结构如下：
本发明中基于罗丹明B的Cys荧光传感器的制备方法，包括以下步骤：
第一步，将马来酸酐加入到含有化合物1的三氯甲烷体系中，回流反应，反应结束后，冷却至于室温，减压除去溶剂，萃取，硅胶柱分离、真空干燥后得到化合物2；其中，化合物1的结构如下：
第二步，将化合物2加入到含有荧光素、DMAP和DCC的二氯甲烷体系中，室温反应，反应结束后冷却至室温，减压除去溶剂，萃取，硅胶柱分离最后得到目标产物即所述Cys荧光传感器。其中，化合物2的结构如下：
进一步的，第一步中，化合物1是将罗丹明B与过量的乙二胺在无水乙醇中加热回流过夜，反应完成之后，减压除去溶剂，萃取，硅胶柱分离，除去溶剂后得到。
进一步的，第一步中，化合物1与马来酸酐的摩尔比为1:1.5-2.5，反应时间为10h以上。
进一步的，第一步中，硅胶柱分离中的洗脱液为MeOH：CH2Cl2＝1:100。
进一步的，第二步中，化合物2、荧光素、DMAP和DCC的摩尔比为2：1：0.5：2 。
进一步的，第二步中，室温反应6h以上。
进一步的，第二步中，硅胶柱分离中的洗脱液为MeOH：CH2Cl2＝1:48。
本发明中所述的基于罗丹明B的荧光传感器用于检测细胞的半胱氨酸。
本发明与现有技术相比，其显著优点是：(1)本发明以罗丹明为主体合成了一种新型Cys荧光传感器，具有良好的光稳定性，长波长发射以及量子产率高等优点。(2 )本发明所选用原料成本低，合成步骤简单，后处理亦很方便，较易实现大规模生产。(3)本发明采用酸酐与伯胺缩合反应方式，合成方法简单，反应条件温和，且产率较高。(4)本发明所涉及传感器能选择性检测Cys，且敏感度较高，在检测细胞内的 Cys方面具有很大的应用前景。
附图说明
图1为本发明的化合物11H NMR。
图2为本发明的化合物113C NMR。
图3为本发明的化合物21H NMR。
图4为本发明的化合物213C NMR。
图5为本发明的目标化合物1H NMR。
图6为本发明的目标化合物13C NMR。
图7为本发明的目标化合物的紫外选择性测试图。
图8为本发明的目标化合物的荧光选择性测试图。
具体实施方式
(一)传感器化合物的合成
本发明提供了目标产物在Cys检测中的应用，发现其对Cys有很好的检测效果。本发明合成路线如下：
(二)荧光性能测试
将GSH、Cys、Hcy、Lys，Gly等不同氨基酸加入目标化合物的溶液中，进行荧光响应测试。
(三)紫外性能测试
将GSH、Cys、Hcy、Lys，Gly等不同氨基酸加入目标化合物的溶液中，吸光度光响应测试。
下面结合附图和具体实施例对本发明作进一步详细说明。
实施例1荧光化学传感器的合成
1.化合物1的合成
将罗丹明B(960mg，2mmol)与乙二胺(1.3ml，20mmol)在无水乙醇(40ml )中，控制反应温度在80℃，反应时间为12h，反应完成之后，减压除去溶剂，萃取，经硅胶柱分离得到淡黄色固体即化合物1(880mg，92％)。化合物11H NMR，13C NMR 分别如图1，图2所示。
2.化合物2的合成
将97.02mg(0.99mmol)的马来酸酐加入到3mL含有319.62mg(0.66mmol)化合物1的三氯甲烷体系中，回流反应20h，反应结束后，冷却至于室温，减压除去溶剂，萃取，经硅胶柱分离得到淡黄色固体即化合物1得到淡黄色的固体即化合物2(365mg ，95％)。化合物2 1HNMR，13C NMR分别如图3，图4所示。
3.目标化合物的合成
将1500mg化合物2(2.6mmol)再次加入到溶有332mg荧光素(1.0mmol)、490mg DMAP(0.5mmol)和440mg DCC(2.1mmol)的二氯甲烷体系中，室温反应6h，反应结束后冷却至室温，真空旋干溶剂。产物通过柱层析提纯得到1320mg(72％)淡黄色产物即目标化合物。目标化合物1H NMR，13C NMR分别如图5，图6所示。
实施例2紫外选择性能测试
目标化合物在甲醇中具有很好的溶解性，经验证，目标化合物可以溶解在MeOH:HEPES(1mM，pH＝7.4)＝1:2混合液中，配制500ml该溶液作为储备液(pH＝7.2)。
精确配置目标化合物为1×10-3mol/LMeOH-H2O混合液(1/2，V/V)，氨基酸GSH、Cys、Hcy、Lys、Gly等浓度为5×10-3mol/L水溶液,以及用MeOH：HEPES(1 mM,pH＝7.4，1/2,V/V)溶液。
紫外选择性实验如图7所示，取3ml储备液置于液体池中，加入30uL目标化合物，测其初始吸光度，然后分别加入配置好的各种氨基酸30uL，测量其稳定时的吸光度。观察图7可知，目标化合物对Cys有明显响应效果，在492nm处出现一个新峰，也即目标化合物对Cys有很好的选择性。
实施例3荧光选择性能测试
目标化合物在甲醇中具有很好的溶解性，经验证，目标化合物可以溶解在MeOH:HEPES(1mM，pH＝7.4)＝1:2混合液中，配制500ml该溶液作为储备液(pH＝7.2)。
精确配置目标化合物为1×10-3mol/LMeOH-H2O混合液(1/2，V/V)，氨基酸GSH、Cys、Hcy、Lys、Gly等浓度为5×10-3mol/L水溶液,以及用MeOH：HEPES(1 mM,pH＝7.4，1/2,V/V)溶液。
荧光选择性实验如图8所示，取3ml储备液置于液体池中，加入30uL目标化合物，测其初始荧光强度值，然后分别加入配置好的各种氨基酸30uL，测量其稳定时的荧光强度。观察图8可知，目标化合物对Cys有明显响应效果，并且在512nm处荧光强度达到最大值，也即目标化合物对Cys有很好的选择性且不受Hcy的干扰。
Claims (9)
1.一种基于罗丹明B的Cys荧光传感器，其特征在于，该荧光传感器的结构如下：
2.如权利要求1所述的基于罗丹明B的Cys荧光传感器的制备方法，其特征在于，包括以下步骤：
第一步，将马来酸酐加入到含有化合物1的三氯甲烷体系中，回流反应，反应结束后，冷却至于室温，减压除去溶剂，萃取，硅胶柱分离、真空干燥后得到化合物2；其中，化合物1的结构如下：
第二步，将化合物2加入到含有荧光素、DMAP和DCC的二氯甲烷体系中，室温反应，反应结束后冷却至室温，减压除去溶剂，萃取，硅胶柱分离最后得到目标产物即所述Cys荧光传感器，其中，化合物2的结构如下：
3.如权利要求2所述的制备方法，其特征在于，第一步中，化合物1是将罗丹明B与过量的乙二胺在无水乙醇中加热回流过夜，反应完成之后，减压除去溶剂，萃取，硅胶柱分离，除去溶剂后得到。
4.如权利要求2所述的制备方法，其特征在于，第一步中，化合物1与马来酸酐的摩尔比为1:1.5-2.5，反应时间为10h以上。
5.如权利要求2所述的制备方法，其特征在于，第一步中，硅胶柱分离中的洗脱液为MeOH：CH2Cl2＝1:100。
6.如权利要求2所述的制备方法，其特征在于，第二步中，化合物2、荧光素、DMAP和DCC的摩尔比为2：1：0.5：2。
7.如权利要求2所述的制备方法，其特征在于，第二步中，室温反应6h以上。
8.如权利要求2所述的制备方法，其特征在于，第二步中，硅胶柱分离中的洗脱液为MeOH：CH2Cl2＝1:48。
9.如权利要求1所述的荧光传感器在非诊断非治疗目的检测细胞半胱氨酸上的应用。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103524516A (zh) * | 2013-09-11 | 2014-01-22 | 江南大学 | 一种新型罗丹明类荧光探针 |
| CN103849377A (zh) * | 2014-03-24 | 2014-06-11 | 南京理工大学 | 一种基于罗丹明b的荧光传感器、制备及其应用 |
| CN104449675A (zh) * | 2014-12-04 | 2015-03-25 | 南京理工大学 | 一种基于罗丹明B的Cr3+传感器、制备及应用 |
| CN104949949A (zh) * | 2015-06-26 | 2015-09-30 | 华南师范大学 | 含有罗丹明基团及苯并呋咱基团的化合物及制备方法与应用 |
-
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103524516A (zh) * | 2013-09-11 | 2014-01-22 | 江南大学 | 一种新型罗丹明类荧光探针 |
| CN103849377A (zh) * | 2014-03-24 | 2014-06-11 | 南京理工大学 | 一种基于罗丹明b的荧光传感器、制备及其应用 |
| CN104449675A (zh) * | 2014-12-04 | 2015-03-25 | 南京理工大学 | 一种基于罗丹明B的Cr3+传感器、制备及应用 |
| CN104949949A (zh) * | 2015-06-26 | 2015-09-30 | 华南师范大学 | 含有罗丹明基团及苯并呋咱基团的化合物及制备方法与应用 |
Non-Patent Citations (5)
| Title |
|---|
| A dual emission fluorescent probe enables simultaneous detection of glutathione and cysteine/homocysteine;Xiao-Feng Yang et al.;《Chemical Science》;20140226;第5卷;第2177-2183页 * |
| A new rhodamine B-based lysosomal pH fluorescent indicator;Hong-Shui Lv et al.;《Analytica Chimica Acta》;20130628;第788卷;第177-182页 * |
| 基于罗丹明螺环衍生物的荧光探针研究进展;张晓兵等;《南京师大学报（自然科学版）》;20150630;第38卷(第2期);第1-16页 * |
| 罗丹明类阳离子荧光探针;袁跃华等;《化学进展》;20101031;第22卷(第10期);第1929-1939页 * |
| 罗丹明荧光探针在生化分析中的应用;高勇等;《化学通报》;20091231(第1期);第15-19页 * |
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