基于苯并吡喃腈的激活型半胱氨酸荧光探针

doi:10.11944/j.issn.1000-0518.2016.04.160053

摘要/Abstract

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。

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 words: cysteine, fluorescent probe, benzopyran, selectivity

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[J]. Chinese Journal of Applied Chemistry, 2016, 33(4): 397-405.

参考文献

[1]	Wood Z A,Schröder E,Harris J R,et al.Structure, Mechanism and Regulation of Peroxiredoxins[J]. Trends Biochem Sci,2003,28(1):32-40.
[2]	Refsum H,Smith A D,Ueland P M,et al.Facts and Recommendations about Total Homocysteine Determinations:An Expert Opinion[J]. Clin Chem,2004,50(1):3-32.
[3]	Gazit V,Ben-Abraham R,Vofsi O,et al.L-Cysteine Increases Glucose Uptake in Mouse Soleus Muscle and SH-SY5Y Cells[J]. Metab Brain Dis,2003,18(3):221-231.
[4]	Wang W,Rusin O,Xu X,et al.Detection of Homocysteine and Cysteine[J]. J Am Chem Soc,2005,127(45):15949-15958.
[5]	Weerapana E,Wang C,Simon G M,et al.Quantitative Reactivity Profiling Predicts Functional Cysteines in Proteomes[J]. Nature,2010,468(7325):790-795.
[6]	Seshadri S,Beiser A,Selhub J. Plasma Homocysteine as a Risk Factor for Dementia and Alzheimer's Disease[J]. New Engl J Med,2002,346(7):476-483.
[7]	Tapiero H,Townsend D,Tew K. The Antioxidant Role of Selenium and Seleno-Compounds[J]. Biomed Pharmacother,2003,57(3):134-144.
[8]	LI Yan,LIU Xiaoyan,ZHANG Shun,et al.Synthesis and Application of a Coumarin Fluorescence Labeled Peptide[J]. Chinese J Appl Chem,2014,31(12):1413-1418(in Chinese). 李燕,刘晓艳,张顺,等. 一种香豆素荧光标记多肽的合成及应用[J]. 应用化学,2014,31(12):1413-1418.
[9]	Zhao N,Wu Y H,Shi L X,et al.A Sensitive Phosphorescent Thiol Chemosensor Based on an Iridium(Ⅲ) Complex with α,β-Unsaturated Ketone Functionalized 2,2'-Bipyridyl Ligand[J]. Dalton Trans,2010,39(35):8288-8295.
[10]	Kand D,Kalle A M,Varma S J,et al.A Chromenoquinoline-Based Fluorescent Off-On Thiol Probe for Bioimaging[J]. Chem Commun,2012,48(21):2722-2724.
[11]	McMahon B K,Gunnlaugsson T. Selective Detection of the Reduced Form of Glutathione(GSH) over the Oxidized(GSSG) Form Using a Combination of Glutathione Reductase and a Tb(Ⅲ)-Cyclen Maleimide Based Lanthanide Luminescent Switch on' Assay[J]. J Am Chem Soc,2012,134(26):10725-10728.
[12]	Liu X M,Xi N,Liu S J,et al.Highly Selective Phosphorescent Nanoprobes for Sensing and Bioimaging of Homocysteine and Cysteine[J]. J Mater Chem,2012,22(16):7894-7901.
[13]	Wang P,Liu J,Lv X,et al.A Naphthalimide-Based Glyoxal Hydrazone for Selective Fluorescence Turn-On Sensing of Cys and Hcy[J]. Org Lett,2012,14(2):520-523.
[14]	Jiang W,Fu Q Q,Fan H H,et al.A Highly Selective Fluorescent Probe for Thiophenols[J]. Angew Chem,2007,119(44):8597-8600.
[15]	Zhao C C,Zhou Y,Lin Q N,et al.Development of an Indole-Based Boron-Dipyrromethene Fluorescent Probe for Benzenethiols[J]. J Phys Chem B,2010,115(4):642-647.
[16]	Jung H S,Chen X,Kim J S,et al.Recent Progress in Luminescent and Colorimetric Chemosensors for Detection of Thiols[J]. Chem Soc Rev,2013,42(14):6019-6031.
[17]	Chen X Q,Zhou Y,Peng X J,et al.Fluorescent and Colorimetric Probes for Detection of Thiols[J]. Chem Soc Rev,2010,39(6):2120-2135.
[18]	Tang B,Xing Y L,Li P,et al.A Rhodamine-Based Fluorescent Probe Containing a Se-N Bond for Detecting Thiols and Its Application in Living Cells[J]. J Am Chem Soc,2007,129(38):11666-11667.
[19]	Yang X F,Guo Y X,Strongin R M. Conjugate Addition/Cyclization Sequence Enables Selective and Simultaneous Fluorescence Detection of Cysteine and Homocysteine[J]. Angew Chem Int Ed,2011,50(45):10690-10693.
[20]	Guo Z Q,Nam S W,Park S,et al.A highly Selective Ratiometric Near-Infrared Fluorescent Cyanine Sensor for Cysteine with Remarkable Shift and Its Application in Bioimaging[J]. Chem Sci,2012,3(9):2760-2765.
[21]	Escobedo J O,Rusin O,Lim S,et al.NIR Dyes for Bioimaging Applications[J]. Curr Opin Chem Biol,2010,14(1):64-70.
[22]	TANG Kun,QIU Na,WU Pin,et al.Novel Water-soluble Asymmetric Pentamethine Cyanine Dyes:Synthesis, Fluorescent Properties and Fluorescent Labeling[J]. Chinese J Appl Chem,2014,31(11):1255-1260(in Chinese). 汤昆,邱娜,吴品,等. 新型水溶性不对称五甲川吲哚菁染料的合成,荧光性能及荧光标记[J]. 应用化学,2014,31(11):1255-1260.
[23]	Li X,Qian S J,He Q J,et al.Design and Synthesis of a Highly Selective Fluorescent Turn-On Probe for Thiol Bioimaging in Living Cells[J]. Org Biomol Chem,2010,8(16): 3627-3630.
[24]	Niu L Y,Guan Y S,Chen Y Z,et al.BODIPY-Based Ratiometric Fluorescent Sensor for Highly Selective Detection of Glutathione over Cysteine and Homocysteine[J]. J Am Chem Soc,2012,134(46):18928-18931.
[25]	Kim G J,Lee K,Kwon H,et al.Ratiometric Fluorescence Imaging of Cellular Glutathione[J]. Org Lett,2011,13(11):2799-2801.
[26]	Lin W Y,Yuan L,Cao Z M,et al.A Sensitive and Selective Fluorescent Thiol Probe in Water Based on the Conjugate 1,4-Addition of Thiols to α,β-Unsaturated Ketones[J]. Chem-Eur J,2009,15(20):5096-5103.
[27]	Shiu H Y,Chong H C,Leung Y C,et al.A Highly Selective FRET-Based Fluorescent Probe for Detection of Cysteine and Homocysteine[J]. Chem-Eur J,2010,16(11):3308-3313.
[28]	Chen X Q,Ko S K,Kim M J,et al.A Thiol-Specific Fluorescent Probe and Its Application for Bioimaging[J]. Chem Commun,2010,46(16):2751-2753.
[29]	Lin W Y,Long L L,Tan W. A Highly Sensitive Fluorescent Probe for Detection of Benzenethiols in Environmental Samples and Living Cells[J]. Chem Commun,2010,46(9):1503-1505.
[30]	Li H H,Fan J L,Wang J Y,et al.A Fluorescent Chemodosimeter Specific for Cysteine:Effective Discrimination of Cysteine from Homocysteine[J]. Chem Commun,2009,45(39):5904-5906.
[31]	Guo Z Q,Zhu W H,Zhu M M,et al.Near-Infrared Cell-Permeable Hg2+-Selective Ratiometric Fluorescent Chemodosimeters and Fast Indicator Paper for MeHg+ Based on Tricarbocyanines[J]. Chem-Eur J,2010,16(48):14424-14432.
[32]	Yin J,Kwon Y,Kim D,et al.Cyanine-Based Fluorescent Probe for Highly Selective Detection of Glutathione in Cell Cultures and Live Mouse Tissues[J]. J Am Chem Soc,2014,136(14):5351-5358.
[33]	Cao J,Zhao C C,Zhu W H. A Near-Infrared Fluorescence Chemodosimeter for Fluoride via Specific Si-O Cleavage[J]. Tetrahedron Lett,2012,53(16):2107-2110.
[34]	Kwak G,Kim H,Kang I K,et al.Charge Transfer Dye in Various Polymers with Different Polarity:Synthesis, Photophysical Properties, and Unusual Aggregation-Induced Fluorescence Changes[J]. Macromolecules,2009,42(5):1733-1738.
[35]	Li M,Wu X M,Wang Y,et al.A Near-Infrared Colorimetric Fluorescent Chemodosimeter for the Detection of Glutathione in Living Cells[J]. Chem Commun,2014,50(14):1751-1753.
[36]	Zhu W H,Huang X M,Guo Z Q,et al.A Novel NIR Fluorescent Turn-On Sensor for the Detection of Pyrophosphate Anion in Complete Water System[J]. Chem Commun,2012,48(12):1784-1786.
[37]	Li Y M,Zhang X L,Zhu B C,et al.A Highly Selective Colorimetric and “Off-On-Off” Fluorescent Probe for Fluoride Ions[J]. Anal Sci,2010,26(10):1077-1080.
[38]	XU Zhaohui. SnO Catalyzed Solvent-Free Synthesis of Dimethyl 2-[(Indol-3-yl)-methyl] malonate Derivatives[J]. Chinese J Appl Chem,2015,32(7):759-764(in Chinese). 许招会. 无溶剂氧化亚锡催化合成2-[(3-吲哚基)-甲基]丙二酸二甲酯衍生物[J]. 应用化学,2015,32(7):759-764.