CN109232499A - 水溶性香豆素基荧光探针的合成及其生化应用 - Google Patents

水溶性香豆素基荧光探针的合成及其生化应用 Download PDF

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CN109232499A
CN109232499A CN201811316361.1A CN201811316361A CN109232499A CN 109232499 A CN109232499 A CN 109232499A CN 201811316361 A CN201811316361 A CN 201811316361A CN 109232499 A CN109232499 A CN 109232499A
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water
fluorescent probe
base fluorescent
synthesis
coumarin base
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李和平
赵超
尚金艳
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Changsha University of Science and Technology
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Abstract

本发明涉及化学结构式A所示的水溶性香豆素基荧光探针的制备方法及其在检测L－半胱氨酸领域的应用。A式中R1、R2、R3、R4选自：氢、氘、氟、氯、溴、氨基、C1~C2烷基；R5选自：氢、C1~C2烷基。

Description

水溶性香豆素基荧光探针的合成及其生化应用
技术领域
本发明涉及化合物的制备方法，具体是水溶性香豆素基荧光探针的制备方法及其在检测L－半胱氨酸（Cys）方面的应用。
背景技术
L-半胱氨酸(Cys)是一种含巯基脂肪族天然氨基酸，在中性环境下易被空气氧化成胱氨酸，它直接参与蛋白质的合成编码，同时它又是生物体内硫铁络合物中硫配体的提供者，人体中的Cys水平异常会引起毛发色素脱色、生长缓慢、肝功能损伤、肌肉松弛、肥胖、阿尔茨海默氏病和心血管病等临床症状, 因此, 在生物条件下对Cys进行有效检测意义重大。
在现有的检测方法中, 荧光分析法具有操作简单、检测限低、灵敏度高和高选择性等优点, 受到广泛关注，因此，以具有良好生物相容性和荧光性能的香豆素为母体，引入亲水性基团，合成基于香豆素衍生物的水溶性荧光探针，在Cys的检测方面具有良好应用前景。
发明内容
本发明的目的在于提供化学结构式A所示的水溶性香豆素基荧光探针的制备方法及其在检测Cys方面的应用。
式中R1、R2、R3、R4选自：氢、氘、氟、氯、溴、氨基、C1 ~C2烷基；R5选自：氢、C1~C2烷基。
本发明的目的在于提供水溶性香豆素基荧光探针的制备方法，其特征在于它的制备操作是：以二氯甲烷为溶剂，三乙胺为催化剂，以化学结构式B所示的3-氰基-7-羟基香豆素与R5-CH=CHCOCl在冰-水浴条件下反应0.5~3.0 h，制得A所示的水溶性香豆素基荧光探针，制备反应如下：
式中R1、R2、R3、R4选自：氢、氘、氟、氯、溴、氨基、C1 ~C2烷基；R5选自：氢、C1~C2烷基。
本发明与现有技术相比具有如下优点：
首次合成如A式所示的水溶性香豆素基荧光探针，并首次发现其具有检测Cys的性能；合成在二氯甲烷溶剂中,三乙胺为催化剂，冰-水浴条件下即可进行。
具体实施方式
实施例1
优选化合物3-氰基-7-丙烯酰氧基香豆素的合成。
将1.0 mmol的3-氰基-7-羟基香豆素溶于20.0 mL二氯甲烷中,加入1.0 mL三乙胺,冰-水浴条件下加入1.0 mmol丙烯酰氯，搅拌30 min至反应完全, 将混合物溶于10.0mL乙酸乙酯中，用去离子水洗涤2-3次，所得有机相用无水硫酸钠干燥，旋蒸除去溶剂，乙酸乙酯重结晶，抽滤，真空干燥，得化合物3-氰基-7-丙烯酰氧基香豆素,产率为84.1%，1HNMR（300MHz，CDCl3），δ5.71-6.26（m,3H,-CH=CH2）, δ6.83-7.24(m, 3H, Ar-H), δ8.12(s, 1H,C4-H)ppm; MS(m/z)，242（M++1）。
实施例2
水溶性香豆素基荧光探针（A）的合成。
式中R1、R2、R3、R4选自：氢、氘、氟、氯、溴、氨基、C1 ~C2烷基；R5选自：氢、C1~C2烷基。
按实施例1的方法制备化学结构式A所示的水溶性香豆素基荧光探针。
实施例3
优选化合物3-氰基-7-丙烯酰氧基香豆素随不同Cys加入量的荧光谱图的变化。
取3-氰基-7-丙烯酰氧基香豆素溶于pH=7.4的PBS缓冲溶液配成浓度为2.0 mM的探针母液，将Cys溶于pH=7.4的PBS缓冲溶液配制成100.0 mM的Cys母液，25℃下用荧光光谱仪测量当探针3-氰基-7-丙烯酰氧基香豆素浓度为1mM时，Cys浓度分别为0, 0.1, 0.2,0.4, 0.8, 1.2, 1.8 mM时的荧光发射光谱（激发波长=410nm），发现荧光发射光谱的荧光强度随着Cys浓度的升高而荧光强度不断增大，发射波长为460nm处的荧光强度由0逐步增大到1350。

Claims (2)

1.水溶性香豆素基荧光探针，其结构特征如化学结构式A所示，式中R1、R2、R3、R4选自：氢、氘、氟、氯、溴、氨基、C1 ~C2烷基；R5选自：氢、C1~C2烷基。
2.如权利要求1所述的水溶性香豆素基荧光探针在检测L－半胱氨酸中的应用。
CN201811316361.1A 2018-11-07 2018-11-07 水溶性香豆素基荧光探针的合成及其生化应用 Pending CN109232499A (zh)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103570701A (zh) * 2013-11-01 2014-02-12 山西大学 一种香豆素衍生物及其制备方法和应用
CN103755672A (zh) * 2014-01-26 2014-04-30 大连理工常熟研究院有限公司 一种用于识别半胱氨酸的特异性荧光探针及其应用
CN103773361A (zh) * 2014-02-25 2014-05-07 山东大学 一种以香豆素为母核的半胱氨酸荧光探针及其应用
US20180093963A1 (en) * 2015-04-16 2018-04-05 Council Of Scientific & Industrial Research Novel coumarin derivative for detection of cysteine and process for the synthesis thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103570701A (zh) * 2013-11-01 2014-02-12 山西大学 一种香豆素衍生物及其制备方法和应用
CN103755672A (zh) * 2014-01-26 2014-04-30 大连理工常熟研究院有限公司 一种用于识别半胱氨酸的特异性荧光探针及其应用
CN103773361A (zh) * 2014-02-25 2014-05-07 山东大学 一种以香豆素为母核的半胱氨酸荧光探针及其应用
US20180093963A1 (en) * 2015-04-16 2018-04-05 Council Of Scientific & Industrial Research Novel coumarin derivative for detection of cysteine and process for the synthesis thereof

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