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应用化学
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应用化学  2016, Vol. 33 Issue (4): 397-405    DOI: 10.11944/j.issn.1000-0518.2016.04.160053
  研究论文 本期目录 | 过刊浏览 | 高级检索 |
基于苯并吡喃腈的激活型半胱氨酸荧光探针
杨婷婷,郭志前,邵安东,赵平(),朱为宏
华东理工大学精细化工研究所 上海 200237
A Turn-On Fluorescent Probe for Cysteine Based on Benzopyran
YANG Tingting,GUO Zhiqian,SHAO Andong,ZHAO Ping(),ZHU Weihong
Institute of Fine Chemicals,East China University of Science & Technology,Shanghai 200237,China
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摘要 

设计并合成了基于苯并吡喃腈为母体单元的近红外激活型荧光探针(E)-2-(苯并吡喃腈基)乙烯基-5-(二乙氨基)丙烯酸苯酯(DCM-AC),其结构中的丙烯酰酯键作为氨基酸激活反应的响应基团。 研究结果表明,探针分子DCM-AC对半胱氨酸具有高灵敏、选择性光谱响应,不仅能观察到明显的颜色变化,而且探针在710 nm处的荧光发射强度显著增强,相应的荧光增强比值与半胱氨酸的浓度(1.0~8.0 μmol/L)呈现良好的线性关系。 探针DCM-AC对半胱氨酸的检出限为2.8×10-7 mol/L,能选择性检测半胱氨酸区别于结构类似的高半胱氨酸和谷胱甘肽,且不受其它氨基酸物质干扰。 通过质谱、核磁和紫外吸收光谱研究了DCM-AC检测半胱氨酸的反应激活机理:半胱氨酸先通过巯基与DCM-AC上的丙烯酰酯双键发生亲核加成,然后环化脱除内酰胺环状化合物。

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杨婷婷
郭志前
邵安东
赵平
朱为宏
关键词 半胱氨酸荧光探针苯并吡喃腈选择性    
Abstract

A benzopyran-based turn-on near-infrared probe (E)-2-(benzopyran)ethenyl-5-(diethylamino)phenyl acrylate(DCM-AC) has been successfully synthesized. Acrylate bond is specifically designed for the response units of amino acid. The results show that the spectral response of DCM-AC towards cysteine is highly sensitivity and selectivity. With increasing the concentration(1.0~8.0 μmol/L) of cysteine, a significant color change can be observed and the fluorescent intensity of DCM-AC at 710 nm increases gradually, showing a good linear relationship with concentration. The limit of detection of DCM-AC for cysteine is determined as 2.8×10-7 mol/L. DCM-AC exhibits a highly selective probe for cysteine over homocysteine and glutathione, with no interference of other substances. The recognition mechanism of the DCM-AC to cysteine is verified by mass spectrum,1H NMR titration and absorption spectra, indicating that cysteine thiol groups attack at acrylate double bond of DCM-AC by addition reaction, and finally removed as the cyclic lactam compound.

Key wordscysteine    fluorescent probe    benzopyran    selectivity
收稿日期: 2016-02-01           接受日期: 2016-03-02
基金资助:国家自然科学基金项目(61306022)资助
通讯作者: 赵平     E-mail: pzhao@ecust.edu.cn
引用本文:   
杨婷婷, 郭志前, 邵安东, 赵平, 朱为宏. 基于苯并吡喃腈的激活型半胱氨酸荧光探针[J]. 应用化学, 2016, 33(4): 397-405.
YANG Tingting, GUO Zhiqian, SHAO Andong, ZHAO Ping, ZHU Weihong. A Turn-On Fluorescent Probe for Cysteine Based on Benzopyran. Chinese Journal of Applied Chemistry, 2016, 33(4): 397-405.
链接本文:  
http://yyhx.ciac.jl.cn/CN/10.11944/j.issn.1000-0518.2016.04.160053      或      http://yyhx.ciac.jl.cn/CN/Y2016/V33/I4/397
Scheme 1Synthesis of fluorescence probe DCM-AC
图1不同pH值下DCM-OH(10 μmol/L)的紫外吸收光谱(A)和DCM-AC(10 μmol/L)与DCM-OH(10 μmol/L)在710nm处的荧光强度(B),溶液体系为V(DMSO):V(PBS)=50:50(37 ℃)
Fig.1Absorption spectra of DCM-OH(A) and fluorescence intensity of DCM-AC(10 μmol/L) and DCM-OH(10 μmol/L)(B) at 710 nm and different pH in V(DMSO):V(PBS)=50:50(37 ℃) solution
图2DCM-AC(10 μmol/L)滴定不同浓度(0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 2.0, 3.0, 4.0化学计量)的半胱氨酸10 min后的紫外吸收光谱(A)和荧光光谱(B),溶液体系为V(DMSO):V(PBS)=50:50(37 ℃, pH=7.4)
Fig.2Absorption(A) and fluorescence(B) spectra of DCM-AC(10 μmol/L) with the titration of Cys(0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 2.0, 3.0, 4.0 chemometric numbers) at 10 min in V(DMSO):V(PBS)=50:50(37 ℃, pH=7.4) solution
图3DCM-AC(10 μmol/L)与5倍化学计量(50 μmol/L)的半胱氨酸、高半胱氨酸、谷胱甘肽滴定后及空白DCM-AC的荧光强度比值I/I0随时间变化的曲线图(A)和荧光强度随时间变化的线性拟合直线(B),溶液体系为V(DMSO):V(PBS)=50:50(37 ℃, pH=7.4)
Fig.3Time-dependent spectra of fluorescence intensity ratio I/I0(A) and liner fit of fluorescence intensity(B) of DCM-AC(10 μmol/L) with the addition of Cys, Hcy, GSH(50 μmol/L) and blank in V(DMSO):V(PBS)=50:50(37 ℃, pH=7.4) solution
图4DCM-AC(10 μmol/L)与50倍化学计量(500 μmol/L)的干扰物滴定前后的荧光强度比值图(从左到右依次是:空白,硫化氢(H2S)、谷氨酸(Glu)、精氨酸(Arg)、丙氨酸(Ala)、亮氨酸(Leu)、异亮氨酸(Ile)、脯氨酸(Pro)、苯丙氨酸(Phe)、丝氨酸(Ser)、苏氨酸(Thr)、酪氨酸(Tyr)、天冬氨酸(Asp)、天冬酰胺(Asn);M&Cys表示干扰物与半胱氨酸共存),溶液体系为V(DMSO):V(PBS)=50:50(37 ℃, pH=7.4)
Fig.4Relative fluorescence intensity of DCM-AC(10 μmol/L) with analytes(500 μmol/L). From left to right in the order: H2S, glutamic acid, arginine, alanine, leucine, isoleucine, proline, phenylalanine, serine, threonine, tyrosine, aspartic acid, asparagine; M:a mixture of disruptor and cysteine) in V(DMSO):V(PBS)=50:50(37 ℃, pH=7.4) solution
图5DCM-OH和DCM-AC(10 μmol/L)与5.0倍化学计量半胱氨酸反应前后的紫外吸收光谱,溶液体系为V(DMSO):V(PBS)=50:50(37 ℃, pH=7.4)
Fig.5Absorption spectra of DCM-OH and DCM-AC(10 μmol/L) before and after addition Cys(5.0 chemometric) in V(DMSO):V(PBS)=50:50(37 ℃, pH=7.4) solution
图6DCM-AC滴加半胱氨酸前(A)、后(B)的质谱图
Fig.6Mass spectra of probe DCM-AC before(A) and after(B) addition of Cys
图7DCM-AC滴加半胱氨酸后在DMSO-d6中的1H NMR谱图
Fig.7Partial 1H NMR spectra of probe DCM-AC upon addition of Cys in DMSO-d6

a.the isolated aggregates after DCM-AC reacted with Cys; b.DCM-OH; c.DCM-AC

Scheme 2Fluorescence response mechanism between DCM-AC and cysteine
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