CN110143954A - 一种香豆素衍生物及其合成方法和应用 - Google Patents
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Abstract
本发明提供了一种香豆素衍生物及其合成方法和在半胱氨酸检测中的应用，所述香豆素衍生物的中文名称为(E)‑7‑(二乙基氨基)‑3‑(3‑(4‑((7‑硝基苯并[c][1,2,5]恶二唑‑4‑基)氧基)苯基)丙烯酰基)‑2H‑吡喃‑2‑酮，英文名称为(E)‑7‑(diethylamino)‑3‑(3‑(4‑((7‑nitrobenzo[c][1,2,5]oxadiazol‑4‑yl)oxy)phenyl)acryloyl)‑2H‑chromen‑2‑one，命名为CNS。本发明同时提供了一种超灵敏检测半胱氨酸的方法，该方法基于所述香豆素衍生物，在PBS/DMSO(v/v,9/1,pH＝7.4)溶液中通过荧光分光光度计检测半胱氨酸的含量。该方法实现了超灵敏简便检测半胱氨酸。
Description
技术领域
本发明涉及香豆素衍生物，具体属于一种香豆素衍生物及其合成方法，以及该衍生物在超灵敏检测半胱氨酸中的应用。
背景技术
研究表明，半胱氨酸的来源途径之一是胱硫醚β-合酶(CBS)和胱硫醚γ-裂解酶(CSE)对同型半胱氨酸的硫转移；同时，半胱氨酸也可以通过γ-谷氨酰半胱氨酸合成酶(GCS)和谷氨酰胺合成酶(GS)的连续作用转化为谷胱甘肽。因此，作为细胞内活性硫物质的转运站，半胱氨酸在生物体中起着重要作用。半胱氨酸作为一种还原性物质，与维持氧化还原的稳态，蛋白质翻译和折叠，以及信号转导等生理过程方面有着紧密的联系。正常血浆中Cys的总浓度为250μM，细胞中半胱氨酸的总浓度为30～200μM。同时，研究表明，半胱氨酸浓度的异常往往与某些疾病有关，如心脑血管疾病，阿尔茨海默病，抑郁症，肠道损伤甚至癌症。
近年来，荧光探针因其设计选择性好、具有低细胞毒性、可原位检测等优异的性能而引起了许多人的关注，已成为生物医学发展中具有广阔前景的课题之一。
在本发明中，合成了一种基于香豆素的化合物，通过半胱氨酸和化合物在反应前后荧光信号的变化，实现半胱氨酸的超灵敏、特异性检测。
发明内容
本发明的目的是提供一种香豆素衍生物及其合成方法，以及该衍生物在检测半胱氨酸中的应用，该衍生物用于检测半胱氨酸时检出限低，选择性好。
本发明提供的一种香豆素衍生物，中文名称为((E)-7-(二乙基氨基)-3-(3-(4-((7-硝基苯并[c][1,2,5]恶二唑-4-基)氧基)苯基)丙烯酰基)-2H-吡喃-2-酮，英文名称为(E)-7-(diethylamino)-3-(3-(4-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)oxy)phenyl)acryloyl)-2H-chro men-2-one，命名为CNS。其结构式为：
CNS的合成方法，步骤为：
1)按摩尔比1:1～1.5将3-乙酰基-7-二乙基氨基-香豆素与对羟基苯甲醛在无水乙醇中搅拌溶解；加入催化量的哌啶，逐渐加热至回流，回流后搅拌该混合溶液12～24小时，待反应完全后，减压除去溶剂，经硅胶柱层析纯化，得到橙色粉末即化合物1；
2)将化合物1和NBD-Cl按摩尔比1:1～1.5一同溶解于无水乙醇中，随后加入催化量的TFA,常温下搅拌12～24小时，然后将反应溶液倒入冰水中，搅拌2～2.5小时，用适量的二氯甲烷萃取，收集有机相，干燥，减压除去溶剂后经硅胶柱层析纯化，得到橙色固体CNS。
作为优选：步骤1)中3-乙酰基-7-二乙基氨基-香豆素和对羟基苯甲醛的摩尔比为1:1.2。步骤2)中化合物1和NBD-Cl的摩尔比为1:1.1。
上述合成的香豆素衍生物CNS用于超灵敏检测半胱氨酸。
一种检测半胱氨酸的方法，包括如下步骤：
(1)、配制pH＝7.4、浓度为10mM的PBS缓冲溶液，配制2mM的半胱氨酸水溶液，配置2mM的CNS的DMSO溶液；
(2)、取1800μLPBS缓冲溶液、200μL DMSO、0.5μL CNS的DMSO溶液于荧光比色皿中，在荧光分光光度仪上检测，随着半胱氨酸的加入，555nm处的荧光强度的逐渐增强；
(3)、在10个比色皿中，各加入1800μL PBS缓冲溶液、200μL DMSO、0.5μM CNS的DMSO溶液，分别加入半胱氨酸溶液的量为0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1μM，5min后在荧光光谱仪上测定555nm处荧光强度为181、228.7、272.1、316.2、359.3、396.4、469.7、513.2、576.3、670.6，以半胱氨酸浓度为横坐标，以荧光强度为纵坐标绘制图，得到半胱氨酸浓度的工作曲线；线性回归方程为：F-F0＝506.91818c+121.16818,R2＝0.9862，c的单位为10-6mol/L；
(4)、测定样品溶液时，将测得的荧光强度代入线性回归方程，即可求得半胱氨酸的浓度。
与现有技术相比，本发明的有益效果：
1、本发明香豆素衍生物CNS的合成只需两步，合成方法简单，操作方便；
2、本发明将香豆素衍生物CNS用于半胱氨酸检测，将NBD设计为识别位点，由于NBD氨基衍生物优异的光学性质，检出限远远小于其他检测半胱氨酸荧光探针，为0.02μM；
3、检测手段简单，只需要借助荧光光谱仪即可实现；
4、检测信号明显，为增强型荧光；
5、通过生物学应用，探针可成为研究半胱氨酸在活体中功能的良好传感器，也为疾病的诊断和治疗提供了新的途径。
附图说明
图1实施例1制备的CNS的核磁氢谱图
图2实施例1制备的CNS的核磁碳谱图
图3实施例1制备的CNS的质谱图
图4 CNS与半胱氨酸作用的荧光发射图
图5 CNS测定半胱氨酸的工作曲线
图6 CNS与各种分析物的荧光柱状图
图7 CNS测定半胱氨酸的时间曲线
图8 CNS测定内、外源性半胱氨酸的细胞成像图
图9 CNS测定不同浓度外源性半胱氨酸的细胞成像图
图10 CNS测定内、外源性半胱氨酸的活体(斑马鱼)成像图
具体实施方式
下面结合实施例和附图对本发明做进一步说明，但本发明保护范围不受下述实施例的限制。
实施例1
CNS的制备和表征
1)按摩尔比1:1.2将3-乙酰基-7-二乙基氨基-香豆素与对羟基苯甲醛在无水乙醇中搅拌溶解；加入催化量的哌啶，逐渐加热至回流，回流后搅拌该混合溶液12～24小时，待反应完全后，减压除去溶剂，经硅胶柱层析纯化，得到橙色粉末即化合物1；
2)将化合物1和NBD-Cl按摩尔比1:1.1一同溶解于无水乙醇中，随后加入催化量的TFA,常温下搅拌12～24小时，然后将反应溶液倒入冰水中，搅拌2～2.5小时，用适量的二氯甲烷萃取，收集有机相，干燥，减压除去溶剂后经硅胶柱层析纯化，得到橙色固体CNS(0.18g,37％yield)。1H NMR(600MHz,DMSO-d6)8.67–8.60(m,2H),8.00(d,J＝16.5Hz,1H),7.95(d,J＝6.7Hz,2H),7.76(d,J＝16.4Hz,1H),7.71(d,J＝8.3Hz,1H),7.51(d,J＝7.3Hz,2H),6.84(t,J＝10.8Hz,2H),6.63(s,1H),3.32(s,4H),1.16(s,6H),(见图1)。13C NMR(151MHz,DMSO-d6)δ186.0,160.4 158.8,154.8,153.6,153.1,149.0,145.9,144.9,140.8,135.9,134.1,132.9,131.3,131.1,130.1,126.4,121.9,115.9,110.8,110.7,108.4,96.4,45.0,12.9.(见图2)。ESI-MS m/z:[M+Na]+calcd for 549.1380；Found 549.1377(见图3)。
实施例2
在荧光比色皿中，加入1800μL PBS缓冲溶液、200μL DMSO、0.5μM CNS的DMSO溶液，逐渐加入半胱氨酸水溶液(0-1μM)，每加入一次，5min后在荧光光谱仪上测定555nm处荧光强度，荧光强度逐渐增强(见图4)。
实施例3
配制pH＝7.4、浓度为10mM的PBS缓冲溶液，并用DMSO配制2mM CNS的溶液，配制2mM半胱氨酸水溶液；在10个比色皿中，取1800μL PBS缓冲溶液、200μL DMSO、0.5μM CNS的DMSO溶液，分别加入半胱氨酸溶液的量为0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1μM，5min后在荧光光谱仪上测定555nm处荧光强度为181、228.7、272.1、316.2、359.3、396.4、469.7、513.2、576.3、670.6，以半胱氨酸浓度为横坐标，以荧光强度为纵坐标绘制图，得到半胱氨酸浓度的工作曲线；线性回归方程为：F-F0＝506.91818c+121.16818,R2＝0.9862，c的单位为10-6mol/L(见图5)。
实施例4
配制pH＝7.4、浓度为10mM的PBS缓冲溶液，配制2mM CNS的DMSO溶液，配制2mM的半胱氨酸水溶液；在荧光比色皿中，加1800μL PBS缓冲溶液、200μL DMSO和0.5μM CNS的DMSO溶液，再分别加入10倍当量的其它分析物和半胱氨酸：1.Arg,2.Asn,3.Asp,4.Br-,5.Cl-,6.CN-,7.CO3 2-,8.F-,9.Trp,10.Glu,11.Gly,12.GSH,13.H2S,14.Hcy,15.I-,16.Leu,17.Lys,18.NO2 -,19.Phe,20.Thr,21.Val,22.Cys,23.Hyp,24.Try,25.GAGB,26.Tyr,27.SO4 2-)的水溶液，在荧光分光光度仪上检测，绘制不同分析物对应的555nm处荧光强度柱状图(见图6)。半胱氨酸使得检测体系在555nm处荧光强度明显升高，其它的分析物基本没有引起检测体系荧光强度的变化。
实施例5
配制pH＝7.4、浓度为10mM的PBS缓冲溶液，配制2mM CNS的DMSO溶液，配制2mM的半胱氨酸水溶液；在荧光比色皿中，加1800μL PBS缓冲溶液、200μL DMSO和0.5μM CNS的DMSO溶液，再加入1μL半胱氨酸水溶液，在555nm处测得其反应时间为300秒.(见图7)。
实施例6
配制pH＝7.4、浓度为10mM的PBS缓冲溶液，并用DMSO配制2mM CNS的溶液，配制2mM半胱氨酸水溶液；把5μL CNS的DMSO溶液加入到2mL的PBS中；将探针溶液加入Hela细胞培养液中，使得其浓度为5μM，与Hela细胞在37℃下，孵育15分钟后用PBS溶液冲洗三次，进行激光共聚焦成像，此时细胞内有微弱荧光；把20mΜNEM(N-乙基马来酰亚胺：细胞内硫醇清除剂)溶液加入到2mL的PBS中，将溶液加入Hela细胞培养液中，在37℃下孵育20分钟后用PBS溶液冲洗三次，进行激光共聚焦成像，细胞内无荧光；向孵育过NEM的细胞中再加入5μM的探针溶液，在37℃下孵育15分钟后用PBS溶液冲洗三次，进行激光共聚焦成像，此时细胞在荧光成像仪下显示出明显黄色荧光(见图8)；将探针溶液加入Hela细胞培养液中，使得其浓度为5μM，与Hela细胞在37℃下，孵育15分钟后用PBS溶液冲洗三次，再加入外源的半胱氨酸，使其浓度分别为30μM、50μM、100μM、200μM，在37℃下，孵育15分钟，随后均用PBS缓冲溶液冲洗三次，进行激光共聚焦成像，此时细胞在荧光成像仪下显示出不同程度的黄色荧光，如图9。
实施例7
配制2mM CNS的DMSO溶液，配制2mM半胱氨酸水溶液；当探针CNS(10μM，15分钟)与未处理的斑马鱼直接孵育时，在黄色通道中发出强荧光信号。然而，探针在NEM预处理的斑马鱼中孵育，荧光信号强度显着降低。NEM和探针预处理后Cys再灌注时，黄色通道的荧光强度再次增强(见图10)。实验结果表明，探针CNS可以对斑马鱼内源性和外源性Cys进行特异性成像。
上述实验结果说明CNS是检测活体内半胱氨酸良好的候选者。
Claims (8)
1.一种香豆素衍生物CNS，其特征在于，结构式为：
2.如权利要求1所述的一种香豆素衍生物CNS的合成方法，其特征在于，包括如下步骤：
1)按摩尔比1:1～1.5将3-乙酰基-7-二乙基氨基-香豆素与对羟基苯甲醛在无水乙醇中搅拌溶解；加入催化量的哌啶，逐渐加热至回流，回流后搅拌该混合溶液12～24小时，待反应完全后，减压除去溶剂，经硅胶柱层析纯化，得到橙色粉末即化合物1；
2)将化合物1和NBD-Cl按摩尔比1:1～1.5一同溶解于无水乙醇中，随后加入催化量的TFA,常温下搅拌12～24小时，然后将反应溶液倒入冰水中，搅拌2～2.5小时，用适量的二氯甲烷萃取，收集有机相，干燥，减压除去溶剂后经硅胶柱层析纯化，得到橙色固体CNS。
3.如权利要求2所述的一种香豆素衍生物CNS的合成方法，其特征在于，所述步骤1)中3-乙酰基-7-二乙基氨基-香豆素和对羟基苯甲醛的摩尔比为1:1.2。
4.如权利要求2所述的一种香豆素衍生物CNS的合成方法，其特征在于，所述步骤2)中化合物1和NBD-Cl的摩尔比为1:1.1。
5.如权利要求1所述的香豆素衍生物CNS在半胱氨酸检测中的应用。
6.一种特异性检测半胱氨酸的方法，其特征在于，包括如下步骤：
(1)、配制pH＝7.4、浓度为10mM的PBS缓冲溶液，配制2mM的半胱氨酸水溶液，配置2mM的权利要求1所述CNS的DMSO溶液；
(2)、取1800μL PBS缓冲溶液、200μL DMSO、0.5μL CNS的DMSO溶液于荧光比色皿中，在荧光分光光度仪上检测，随着半胱氨酸的加入，555nm处的荧光强度的逐渐增强；
(3)、在10个比色皿中，各加入1800μL PBS缓冲溶液、200μL DMSO、0.5μM CNS的DMSO溶液，分别加入半胱氨酸溶液的量为0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1μM，5min后在荧光光谱仪上测定555nm处荧光强度为181、228.7、272.1、316.2、359.3、396.4、469.7、513.2、576.3、670.6，以半胱氨酸浓度为横坐标，以荧光强度为纵坐标绘制图，得到半胱氨酸浓度的工作曲线；线性回归方程为：F-F0＝506.91818c+121.16818,R2＝0.98623)，c的单位为10-6mol/L；
(4)、测定样品溶液时，将测得的荧光强度代入线性回归方程，即可求得半胱氨酸的浓度。
7.如权利要求1所述的香豆素衍生物CNS在制备细胞成像试剂中的应用。
8.如权利要求1所述的香豆素衍生物CNS在制备活体成像试剂中的应用。
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110526908A (zh) * | 2019-09-06 | 2019-12-03 | 渤海大学 | 基于2-苯乙烯基吲哚盐类衍生物长波发射可区分检测Cys/Hcy荧光探针及其应用 |
| CN111233804A (zh) * | 2020-03-03 | 2020-06-05 | 山西大学 | 苯并吡喃氧鎓离子-香豆素衍生物及其合成方法和应用 |
| CN111303072A (zh) * | 2020-02-27 | 2020-06-19 | 山西大学 | 一种区分检测半胱氨酸的试剂及其合成方法和应用 |
| CN112939918A (zh) * | 2021-02-05 | 2021-06-11 | 山西大学 | 一种香豆素衍生物ctt及其合成方法和应用 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103788076A (zh) * | 2014-01-22 | 2014-05-14 | 山西大学 | 一种检测半胱氨酸的试剂和方法 |
| CN106432164A (zh) * | 2016-09-26 | 2017-02-22 | 山西大学 | 一种香豆素衍生物docopa及其制备方法和应用 |
-
2019
- 2019-06-03 CN CN201910474790.XA patent/CN110143954A/zh active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103788076A (zh) * | 2014-01-22 | 2014-05-14 | 山西大学 | 一种检测半胱氨酸的试剂和方法 |
| CN106432164A (zh) * | 2016-09-26 | 2017-02-22 | 山西大学 | 一种香豆素衍生物docopa及其制备方法和应用 |
Non-Patent Citations (1)
| Title |
|---|
| PENG WANG，ET AL.: "A novel DCM-NBD conjugate fluorescent probe for discrimination of Cys/Hcy from GSH and its bioimaging applications in living cells and animals", 《SENSORS AND ACTUATORS B》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110526908A (zh) * | 2019-09-06 | 2019-12-03 | 渤海大学 | 基于2-苯乙烯基吲哚盐类衍生物长波发射可区分检测Cys/Hcy荧光探针及其应用 |
| CN110526908B (zh) * | 2019-09-06 | 2022-10-25 | 渤海大学 | 基于2-苯乙烯基吲哚盐类衍生物长波发射可区分检测Cys/Hcy荧光探针及其应用 |
| CN111303072A (zh) * | 2020-02-27 | 2020-06-19 | 山西大学 | 一种区分检测半胱氨酸的试剂及其合成方法和应用 |
| CN111233804A (zh) * | 2020-03-03 | 2020-06-05 | 山西大学 | 苯并吡喃氧鎓离子-香豆素衍生物及其合成方法和应用 |
| CN112939918A (zh) * | 2021-02-05 | 2021-06-11 | 山西大学 | 一种香豆素衍生物ctt及其合成方法和应用 |
| CN112939918B (zh) * | 2021-02-05 | 2022-07-19 | 山西大学 | 一种香豆素衍生物ctt及其合成方法和应用 |
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