CN110669005A - 一种检测半胱氨酸的荧光探针 - Google Patents

一种检测半胱氨酸的荧光探针 Download PDF

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CN110669005A
CN110669005A CN201910613664.8A CN201910613664A CN110669005A CN 110669005 A CN110669005 A CN 110669005A CN 201910613664 A CN201910613664 A CN 201910613664A CN 110669005 A CN110669005 A CN 110669005A
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cysteine
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战付旭
汲静静
张占明
郑庚修
王秋芬
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Abstract

本发明公开了一种检测半胱氨酸的荧光探针。本发明所述的荧光探针能够从多种氨基酸以及一些常见物质中识别区分半胱氨酸，该探针与Cys作用时，荧光由蓝色变成黄绿色，可实现比率型检测。该荧光探针对在环境中的半胱氨酸的检测和分析具有很好的应用。本发明的荧光探针在生物化学领域具有重要的应用价值。

Description

一种检测半胱氨酸的荧光探针
技术领域
本发明涉及分析化学领域，涉及一种检测半胱氨酸的荧光探针的制备。
背景技术
半胱氨酸(Cys)作为一种半必需的氨基酸和生物硫醇，可在人体内进行生物合成，是各种多肽或蛋白质的重要结构和功能单元。半胱氨酸(Cys)在在此各种生物学过程中发挥重要作，包括生物催化、信号转导、蛋白质周转，调节新陈代谢，维持氧化还原平衡和重金属结合。半胱氨酸不仅是谷胱甘肽、乙酰辅酶和牛磺酸的前体，而且还是生物体硫铁络合物中硫配体的提供者。人体中缺少半胱氨酸会导致生长缓慢、毛发色素脱色、水肿、嗜睡、肝功能损伤、肌肉松弛、身体虚弱等症状，同时，半胱氨酸的浓度过高也可能会引起阿尔兹海默氏症、心血管疾病、癌症等疾病。
目前，已经存在很多检测半胱氨酸的方法,例如凝胶电泳、拉曼光谱、电子顺磁共振、质谱、微电极生物传感器、光电化学传感器和细胞内核磁共振光谱,然而这些方法通常需要昂贵的仪器和复杂的制备，相反荧光成像技术具有操作简单、成本低廉、灵敏度高、选择性好等优点引起人们广泛关注。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。因此，制备能够检测半胱氨酸的比率型荧光探针是十分有意义的。
发明内容
本发明目的之一是提供一种检测半胱氨酸的荧光探针的合成方法；目的之二是提供一种检测线半胱氨酸的荧光探针。
为实现上述目的，本发明采用如下技术方案。
一种检测半胱氨酸的荧光探针，其分子结构如下：
Figure RE-GDA0002280814070000011
具体合成路线如下：
Figure RE-GDA0002280814070000021
具体合成方法如下：(1)将4-溴-1，8-萘酐和4-(2-氨基乙基)-吗啉溶于乙醇中回流。反应结束，冷却至室温，过滤收集沉积物，干燥得到化合物1。(2)将叠氮钠和化合物1加入到DMF溶液中，将反应混合物加热到100℃，反应10h。加入大量水洗，CH2Cl2萃取，干燥有机相，减压除去溶剂。得化合物2，该产品不经纯化直接用于下一步反应。(3)将化合物2和氯化铵溶于乙醇和水的混合物中。将锌粉慢慢加入混合物中加热回流1h。加入0.5 mL氨水，盐水洗涤，乙酸乙酯萃取，干燥有机相，减压除去溶剂，经柱层析分离得到化合物3。(4)将4-羟基苯甲醇和咪唑加入到DMF中，冷却到0℃，加入叔丁基二苯基氯硅烷，室温搅拌过夜。将反应液用盐水洗涤，乙酸乙酯萃取(3×20mL)，干燥有机相，减压除去溶剂，经柱层析分离得到化合物4。(5)将三甲胺和化合物4加入到CH2Cl2中，冷却到0 ℃。将丙烯酰氯加入到CH2Cl2中，冰浴下，将丙烯酰氯混合溶液滴加到反应液中，室温搅拌过夜。将反应液用5％NaHCO3溶液和盐水洗涤，干燥有机相，减压除去溶剂，经柱层析分离得到化合物5。(6)将化合物5溶于甲醇中，加入大孔树脂-15，室温搅拌6h。过滤得溶液，减压除去溶剂，经柱层析分离得到化合物6。(7)将化合物3溶于无水CH2Cl2中，降温至-15℃，加入4-二甲基氨基吡啶，搅拌10min。将甲苯光气溶液滴加到反应液中，搅拌 12h。除去多余光气，加入化合物6，0℃下反应，搅拌过夜。将反应液用水洗涤，CH2Cl2萃取(2×25mL)，干燥有机相，减压除去溶剂，经柱层析分离得到探针。
本发明的荧光探针的响应机理如下：半胱氨酸与探针分子响应后，半胱氨酸的巯基首先与丙烯酰酯的双键发生迈克尔加成反应，随后半胱氨酸的氨基进攻酯基形成一个七元环，释放出染料分子(化合物3)溶液的荧光由绿色变成蓝色。从而实现了荧光比值型检测半胱氨酸过程。探针分子的响应过程如下：
Figure RE-GDA0002280814070000031
本发明荧光探针在检测半胱氨酸的应用。
所述荧光探针在检测半胱氨酸的应用中，激发波长为410nm，检测波段为450-750nm。
本发明具有以下优点：
本发明涉及的一种检测半胱氨酸的荧光探针合成简单，选择性好，可实现比率型检测，在生物化学领域具有重要的应用价值。
附图说明
图1为探针分子结构图。
图2为探针的1H NMR谱。
图3为探针对不同浓度Cys响应的荧光光谱。
图4为探针选择性和抗干扰性图。
具体实施方式
实施例1探针的合成
Figure RE-GDA0002280814070000041
(1)将4-溴-1，8-萘酐(1g，3.61mmol)和4-(2-氨基乙基)-吗啉(1g，7.68mmol)溶于20ml乙醇中，加热回流4h。冷却至室温，过滤收集沉积物，并在室温下在真空烘箱中干燥一夜，得到化合物1(1.26g,产率：89.67％)。(2)将叠氮钠350mg(5.4mmol)溶于5mL DMF中，滴入化合物1和30ml DMF混合溶液中,加热到100℃，反应10h。加入大量水洗，CH2Cl2萃取，干燥有机相，减压除去溶剂。得化合物2，该产品不经纯化直接用于下一步反应。(3)将化合物2(0.51g,1.5mmol)和氯化铵(0.19g，3.5mmol)溶于乙醇(30ml)和水 (10ml)的混合溶液中，将锌粉(0.13g，2mmol)慢慢加入混合物中加热回流1h。加入0.5mL 氨水，盐水洗涤，乙酸乙酯萃取，干燥有机相，减压除去溶剂，经柱层析分离得到化合物3 (0.27g,产率：57.2％)。(4)将4-羟基苯甲醇(0.5g,4.03mmol)和咪唑(0.274g,4.03 mmol)加入到5mL DMF中，冷却到0℃，加入叔丁基二苯基氯硅烷(0.6g,4.03mmol)，室温搅拌过夜。将反应液用盐水洗涤，乙酸乙酯萃取(3×20mL)，干燥有机相，减压除去溶剂，经柱层析分离得到化合物4(0.78g,产率：81.2％)。(5)将三甲胺(0.29mL,2.10 mmol)和化合物4(0.5g,2.10mmol)加入到5mLCH2Cl2中，冷却到0℃。将丙烯酰氯 (0.17mL,1.9mmol)滴加到CH2Cl2中，冰浴下，将丙烯酰氯混合溶液滴加到反应液中，室温搅拌过夜。将反应液用5％NaHCO3溶液和盐水洗涤，干燥有机相，减压除去溶剂，经柱层析分离得到化合物5(0.43g,产率：70％)。(6)将化合物5(0.43g,1.47mmol)溶于10 mL甲醇中，加入大孔树脂-15(65mg)，室温搅拌6h。过滤得溶液，减压除去溶剂，经柱层析分离得到化合物6(0.22g,产率：84.6％)。(7)将化合物3(0.13g,0.41mmol)溶于无水CH2Cl2中，降温至-15℃，加入4-二甲基氨基吡啶(0.1g,0.82mmol)，搅拌10min。将甲苯光气溶液滴加到反应液中，搅拌12h。除去多余光气，加入化合物6(0.22g,1.23 mmol)，0℃下反应，搅拌过夜。将反应液用水洗涤，CH2Cl2萃取(2×25mL)，干燥有机相，减压除去溶剂，经柱层析分离得到探针(23mg,产率：10.6％)。探针1H NMR谱如图 2：1H NMR(600MHz,CDCl3)δ8.59(dd,J＝16.1,7.7Hz,2H),8.36(d,J＝8.1Hz,1H),8.19 (d,J＝8.4Hz,1H),7.77–7.72(m,1H),7.64(t,J＝9.5Hz,1H),7.47(d,J＝8.4Hz,2H),7.16 (d,J＝8.5Hz,2H),6.62(dd,J＝17.3,0.8Hz,1H),6.33(dd,J＝17.3,10.5Hz,1H),6.04(dd,J＝10.5,0.8Hz,1H),5.28(s,2H),4.32(t,J＝7.0Hz,2H),3.68(t,J＝4.2Hz,4H),2.69(t,J＝7.0Hz, 2H),2.59(s,4H).
实施例2荧光探针对Cys的响应
在DMSO中配制浓度为1mM探针储备液。配制不同浓度的Cys缓冲溶液DMSO/PBS(1/3， v/v，PBS 10mM，pH＝7.4)(浓度梯度为10eq的0-200eq)，其中探针含量为5μM，进行荧光检测(λex＝410nm)，记录475nm与540nm处的荧光强度，如图3所示。分析 I475nm/I540nm处的荧光强度与Cys浓度的线性关系，如图所示：随着Cys浓度的增加，540nm 处的吸收峰逐渐增加，475nm处的吸收峰逐渐降低，当Cys浓度达到150eq时，反应体系吸收峰值基本不再发生变化。
实施例3荧光探针的选择性
配制浓度为20mM的生物硫醇(Cys、Asp、Pro、Met、N-Ace-cys、Glu、Hcy和GSH)溶液。
将Cys和不同干扰物质的溶液与探针配制成缓冲溶液DMSO/PBS(1/3，v/v，PBS 10mM，pH＝7.4)，干扰物质终浓度为200μM，探针终浓度为5μM，摇匀后进行荧光检测 (λex＝410nm)，建立荧光强度比I475nm/I540nm与各干扰物质的柱状图，如图4所示。由图可知，荧光探针对其他生物硫醇响应值低，对Cys的I475nm/I540nm比值高，特异性好、抗干扰性能好。其他生物硫醇对探针的荧光比值几乎没有影响，能够选择性的识别Cys。

Claims (1)

1.一种检测半胱氨酸的荧光探针，其结构为：
Figure DEST_PATH_IMAGE002AAAA
。
CN201910613664.8A 2019-07-09 2019-07-09 一种检测半胱氨酸的荧光探针 Pending CN110669005A (zh)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113354584A (zh) * 2021-06-15 2021-09-07 郑州大学 一种区分Cys、Hcy和GSH的萘酰亚胺类荧光探针及其制备方法与应用
CN115521256A (zh) * 2022-10-12 2022-12-27 齐鲁工业大学 一种4-氨基-1，8-萘酰亚胺衍生物的制备方法

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* Cited by examiner, † Cited by third party
Title
BEIBEI LIANG，等: "A lysosome-targetable turn-on fluorescent probe for the detection of thiols in living cells based on a 1,8-naphthalimide derivative", 《SPECTROCHIMICA ACTA PART A: MOLECULAR AND BIOMOLECULAR SPECTROSCOPY》 *
RASIKA R. NAWIMANAGE，等: "Cascade Reaction-Based, Near-Infrared Multiphoton Fluorescent Probe for the Selective Detection of Cysteine", 《ANAL. CHEM.》 *
SOO-YEON LIM，等: "Caged rhodamine-based fluorescent probe for biothiol: Selectivedetection of cysteine over homocysteine and glutathione in water", 《SENSORS AND ACTUATORS B》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113354584A (zh) * 2021-06-15 2021-09-07 郑州大学 一种区分Cys、Hcy和GSH的萘酰亚胺类荧光探针及其制备方法与应用
CN113354584B (zh) * 2021-06-15 2023-05-16 郑州大学 一种区分Cys、Hcy和GSH的萘酰亚胺类荧光探针及其制备方法与应用
CN115521256A (zh) * 2022-10-12 2022-12-27 齐鲁工业大学 一种4-氨基-1，8-萘酰亚胺衍生物的制备方法
CN115521256B (zh) * 2022-10-12 2024-03-08 齐鲁工业大学 一种4-氨基-1，8-萘酰亚胺衍生物的制备方法

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