CN110590726A - 一种同时区分Cys/Hcy和GSH的开关型荧光探针 - Google Patents

一种同时区分Cys/Hcy和GSH的开关型荧光探针 Download PDF

Info

Publication number
CN110590726A
CN110590726A CN201910826098.9A CN201910826098A CN110590726A CN 110590726 A CN110590726 A CN 110590726A CN 201910826098 A CN201910826098 A CN 201910826098A CN 110590726 A CN110590726 A CN 110590726A
Authority
CN
China
Prior art keywords
hcy
cys
gsh
fluorescent probe
probe
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201910826098.9A
Other languages
English (en)
Other versions
CN110590726B (zh
Inventor
宋相志
钟浩
张会
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Central South University
Original Assignee
Central South University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Central South University filed Critical Central South University
Priority to CN201910826098.9A priority Critical patent/CN110590726B/zh
Publication of CN110590726A publication Critical patent/CN110590726A/zh
Application granted granted Critical
Publication of CN110590726B publication Critical patent/CN110590726B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/42—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms in positions 2 and 4
    • C07D311/56—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms in positions 2 and 4 without hydrogen atoms in position 3
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Optics & Photonics (AREA)
  • Pathology (AREA)
  • Engineering & Computer Science (AREA)
  • Materials Engineering (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明公开一种具有内质网靶向检测Cys/Hcy和GSH的开关型荧光探针的制备方法以及该荧光探针在体外和活细胞内质网内检测Cys/Hcy和GSH方面的应用，其结构式为：我们将识别基团和定位基团通过染料的桥连构建到一个分子上，通过三种生物硫醇亲核性的差异达到区分生物硫醇的目的。此外该系列化合物有望在生物医药，光电以及环境保护领域有着较好的应用前景。

Description

一种同时区分Cys/Hcy和GSH的开关型荧光探针
技术领域
本发明属于化学分析检测技术领域，具体涉及一种内质网靶向的同时区分检测Cys/Hcy和GSH开关型荧光探针的制备方法及该荧光探针在体外和生物体内检测Cys/Hcy和GSH的应用。
背景技术
半胱氨酸(Cys)、同型半胱氨酸(Hcy)和谷胱甘肽(GSH)是三种重要的生物硫醇，在人体生理过程中起着至关重要的作用，它们的含量与人体各种疾病的发生，监测和治疗密切相关。半胱氨酸是合成蛋白质过程中的一种必需氨基酸，它含量的异常会导致皮肤损伤、毛发脱皮、水肿、肌肉、脂肪流失和肝脏损伤等多种疾病。同型半胱氨酸有维持细胞稳态的重要作用，同型半胱氨酸含量的异常也与心血管疾病、骨质疏松症等各种疾病有关。谷胱甘肽是目前已知最丰富的生物硫醇，也是一种重要的抗氧化剂，在细胞对抗氧化应激方面发挥着重要作用。然而，细胞中谷胱甘肽的表达异常与艾滋病感染、癌症等疾病密切相关。并且，有研究表明这三种生物硫醇之间有着非常密切的关系。在胱硫醚-β-合成酶的作用下，同型半胱氨酸可与丝氨酸作用形成胱硫醚，胱硫醚在胱硫醚-γ-裂解酶作用下形成半胱氨酸。在γ-谷氨酰半胱氨酸合成酶和谷胱甘肽合成酶作用下，半胱氨酸与谷氨酸和甘氨酸形成谷胱甘肽。
内质网是细胞中最大的细胞器，它与许多细胞生理过程密切相关。最近的研究表明，内质网能影响各种生物硫醇的溶度。但遗憾的是，目前尚无内质网靶向的同时检测Cys/Hcy和GSH的荧光探针，无法对内质网中Cys/Hcy和GSH 进行实时监测，以监测内质网与Cys/Hcy和GSH之间的相互作用。因此，开发一种内质网靶向的同时区分检测Cys/Hcy和GSH的荧光探针非常重要。
发明内容
本发明目的之一是提供一种合成路线简单、反应条件温和、成本较低的内质网靶向荧光探针；目的之二是提供一种灵敏度高、选择性好，抗干扰能力强，开光型，能够对体外或者活细胞内的Cys/Hcy和GSH进行快速监测或者细胞成像的荧光探针，其结构如下：
其合成路线为：
具体合成方法：将化合物1(200mg，4mmol),化合物2(64mg，4mmol) 和无水三乙胺(485.38mg，4.8mmol)溶于8mL无水乙腈中，在氮气保护下室温搅拌1.5h。反应液减压旋蒸除去溶剂，柱层析色谱得黄色固体ER-CP(160mg， 65％)。
本发明的探针的机理如下：
本发明的荧光探针的作用机理如下，在探针分子中，对硝基苯硫醚键作为生物硫醇的识别基团，当对硝基苯硫醚键与荧光团通过化学键相连时，由与该识别基团的光诱导电子转移效应(PET)，导致该分子没有荧光。当生物硫醇与该探针反应时，探针的苯硫醚键被切断，使光诱导电子转移效应消失，荧光团荧光释放出来。其中，探针与Cys和Hcy反应时，分别生成ER-CP-S-Cys和 ER-CP-S-Hcy。ER-CP-S-Cys经过快速的分子内重排反应生成蓝色荧光的 ER-CP-N-Cys,ER-CP-S-Hcy则经过较慢的分子内重排反应生成蓝色荧光的 ER-CP-S–Hcy。探针与GSH反应时，生成不能分子内重排的绿色荧光的 ER-CP-S-GSH。因此，通过荧光信号和响应时间可以区分检测Cys(蓝色荧光，蓝色荧光出现较快)/Hcy(蓝色荧光，蓝色荧光出现较慢)和GSH(绿色荧光)。同时我们将荧光团和内质网靶向基团对甲基苯磺酰胺通过化学键连接，使得该荧光探针能在内质网中富集。
本发明的荧光探针具有首次实现细胞内内质网中区分检测Cys/Hcy和 GSH的特征。同时还有合成路线简单，灵敏度高等优点。
附图说明
图1为本发明荧光探针(10.0uM)在PBS缓冲液中(10mM，pH＝7.4，含 20％DMSO)与20倍当量Cys(A)，Hcy(B)或GSH(C)反应随时间变化的吸收光谱。
图2本发明荧光探针(10.0uM)在含20％二甲基亚砜(DMSO)的PBS缓冲液 (10mM，pH＝7.4)中，第一列为分别与Cys、Hcy和GSH反应的荧光光谱(A1-C1)。第二列探针响应后荧光强度分别与Cys、Hcy和GSH的浓度呈线性关系(A2-C2)。 A,B在370nm处激发，C在427nm处激发。
图3为本发明荧光探针(10.0uM)的在PBS缓冲液(10mM，pH＝7.4，含 20％DMSO)中,室温分别与对照，Hcy,Cys,GSH,Tyr,Ser,Ile,Arg,Met,His,Val, Thr,Phe,Lys,Ala,Gly,NaNO3,NaNO2,Na2SO4,KO2,KCl,KI,Na2SO3等反应记录的荧光光谱。A，B为473nm处激发，C为541nm处激发。A,C响应38min， B响应210min。
图4为本发明荧光探针(10.0uM)在存在和不存在Cys(15.0倍当量，A)时， Hcy(20.0倍当量，B)和GSH(8.0倍当量，C)。发射的荧光随pH变化的效应.A， B为473nm处激发，C为541nm处激发。A,C响应38min，B响应210min。
图5A为本发明探针(5.0uM)与人类宫颈癌细胞孵育30min后的共聚焦显微镜图。其中B人类宫颈癌细胞与NEM(1.0mM)处理30min后，再与Cys 和荧光探针孵育30min的人类宫颈癌细胞共聚焦显微镜图。C为人类宫颈癌细胞与NEM(1.0mM)处理30min后，再与探针和Hcy孵育90min的人类宫颈癌细胞共聚焦显微镜图,D为人类宫颈癌细胞与NEM(1.0mM)处理30min后，再与探针和GSH孵育30min的人类宫颈癌细胞共聚焦显微镜图，E为探针单独与NEM(1.0mM)处理后的人类宫颈癌细胞，再与探针孵育30min的人类宫颈癌细胞共聚焦显微镜图。激发波长为405nm；蓝色通道为450nm-480nm，绿色通道为520nm-550nm。图片标尺：20um。
图6人类宫颈癌细胞经1.0mM NEM预处理30min，再用0.3mM Cys处理30min，再与本发明探针(5.0uM)和ER-trackerred(1.0uM)共同孵育30min。(A) 为明场图像；(B)为蓝色通道(激发波长405nm，发射从450nm到480nm)；(C) 为红色通道(激发波长535nm，发射从600nm到630nm)；(D)为图(B)和图(C) 的合并图像。图片标尺：20um。
图7为本发明的荧光探针与Cys响应的高分辨质谱。
图8为本发明的荧光探针与Hcy响应的高分辨质谱。
图9本发明的荧光探针与GSH响应的高分辨质谱。
图10为本发明的荧光探针(10.0uM)与20倍当量的Cys，Hcy和GSH的时间响应曲线。A1-C1为370nm处激发，A2-C2为427nm处激发。狭缝：5/5nm，电压700V。
图11为本发明的荧光探针(10.0uM)在PBS缓冲液中(10mM，pH＝7.4，含20％DMSO)并且存在/不存在20倍当量Cys时荧光强度随时间的变化。激发波长370nm。狭缝：5/5nm，电压700V。
图12为本发明的荧光探针(10.0uM)在PBS缓冲液中(10mM，pH＝7.4，含20％DMSO)并且存在/不存在20倍当量Hcy时荧光强度随时间的变化。激发波长370nm。狭缝：5/5nm，电压700V。
图13为本发明的荧光探针(10.0uM)在PBS缓冲液中(10mM，pH＝7.4，含20％DMSO)并且存在/不存在20倍当量GSH时荧光强度随时间的变化。激发波长370nm。狭缝：5/5nm，电压700V。
图14为本发明的荧光探针(10.0uM)与人类宫颈癌细胞孵育24h后的细胞可见百分比图。
图15为本发明荧光探针和ER-Tracker Red的强度相关图。
具体实施实例
实施例1：探针分子的合成
探针ER-CP的合成
将化合物1(200mg，4mmol),化合物2(64mg，4mmol)和无水三乙胺(485.38mg，4.8mmol)溶于8mL无水乙腈中，在氮气保护下室温搅拌1.5h。反应液减压旋蒸除去溶剂，柱层析色谱得黄色固体ER-CP(160mg，65％)。
1H NMR(300MHz，DMSO-d6)δ8.46(t，J＝5.2Hz，1H)，8.11(d，J＝8.6 Hz，2H)，7.65(d，J＝7.9Hz，2H)，7.59–7.44(m，3H)，7.45–7.27(m，3H)， 6.62(d，J＝11.5Hz，2H)，3.19(d，J＝6.5Hz，4H)，2.74(d，J＝6.3Hz，2H)， 2.38(s，3H)，1.09(t，J＝6.5Hz，6H)。
13C NMR(75MHz,DMSO-d6)δ146.2，144.1，143.3，143.2，137.7，130.1， 128.9，128.5，127.0，125.1，124.7，110.2，106.7，97.2，44.5，42.1，29.5，21.4， 12.7。
HRMS(ESI)m/z:计算值C29H30N4O7S2[M+Na]+:633.1448；找到 633.1454。
实施例2：本发明的荧光探针的应用
本发明荧光探针(10.0uM)在在PBS缓冲液(10mM，pH＝7.4，含 20％DMSO)中分别与对照，Tyr,Ser,Ile,Arg,Met,His,Val,Thr,Phe,Lys,Ala,Gly, NaNO3,NaNO2,Na2SO4,KO2,KCl,KI,Na2SO3等反应引起探针本身发射峰的明显变化，而当加入Cys后，较快时间内能观察到蓝色荧光，当加入Hcy后，较慢时间内能观察到蓝色荧光，当加入GSH后，能观察到绿色荧光，说明该探针具有优良的选择性，且能区分检测Hcy/Cys和GSH。而与ER-Tracker Red共染实验，皮尔逊(氏)同域化(0.90)，表面本发明荧光探针能靶向内质网。而细胞毒性实验，细胞成像实验说明探针具有好的生物适用前景。

Claims (1)

1.一种区分检测Cys/Hcy和GSH的开关型荧光探针，其结构式如下:
CN201910826098.9A 2019-09-04 2019-09-04 一种同时区分Cys/Hcy和GSH的开关型荧光探针 Active CN110590726B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910826098.9A CN110590726B (zh) 2019-09-04 2019-09-04 一种同时区分Cys/Hcy和GSH的开关型荧光探针

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910826098.9A CN110590726B (zh) 2019-09-04 2019-09-04 一种同时区分Cys/Hcy和GSH的开关型荧光探针

Publications (2)

Publication Number Publication Date
CN110590726A true CN110590726A (zh) 2019-12-20
CN110590726B CN110590726B (zh) 2022-10-04

Family

ID=68857252

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910826098.9A Active CN110590726B (zh) 2019-09-04 2019-09-04 一种同时区分Cys/Hcy和GSH的开关型荧光探针

Country Status (1)

Country Link
CN (1) CN110590726B (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN116804630A (zh) * 2023-08-03 2023-09-26 中拓生物有限公司 一种血清同型半胱氨酸测定试剂盒

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107298654A (zh) * 2017-05-19 2017-10-27 济南大学 一种检测内质网中硫化氢的荧光探针
CN108727326A (zh) * 2018-07-06 2018-11-02 广西师范学院 识别半胱氨酸和谷胱甘肽的荧光探针及制备方法与应用
CN110078665A (zh) * 2019-05-16 2019-08-02 济南大学 一种内质网靶向的检测次氯酸的荧光探针和应用
CN110156688A (zh) * 2019-05-22 2019-08-23 济南大学 一种靶向内质网检测极性的荧光探针及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107298654A (zh) * 2017-05-19 2017-10-27 济南大学 一种检测内质网中硫化氢的荧光探针
CN108727326A (zh) * 2018-07-06 2018-11-02 广西师范学院 识别半胱氨酸和谷胱甘肽的荧光探针及制备方法与应用
CN110078665A (zh) * 2019-05-16 2019-08-02 济南大学 一种内质网靶向的检测次氯酸的荧光探针和应用
CN110156688A (zh) * 2019-05-22 2019-08-23 济南大学 一种靶向内质网检测极性的荧光探针及其应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HUI ZHANG ET AL.: "A lysosome-targetable fluorescent probe for the", 《RSC ADV.》 *
HUI ZHANG ET AL.: "An endoplasmic reticulum-targetable fluorescent", 《ORG. BIOMOL. CHEM.》 *
JING LIU ET AL.: "Simultaneous Fluorescence Sensing of Cys and GSH from Different", 《J. AM. CHEM. SOC.》 *
WENQIANG CHEN ET AL.: "Unexpected reaction patterns enable", 《CHEM.COMMUN.》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN116804630A (zh) * 2023-08-03 2023-09-26 中拓生物有限公司 一种血清同型半胱氨酸测定试剂盒
CN116804630B (zh) * 2023-08-03 2024-03-05 中拓生物有限公司 一种血清同型半胱氨酸测定试剂盒

Also Published As

Publication number Publication date
CN110590726B (zh) 2022-10-04

Similar Documents

Publication Publication Date Title
Zhang et al. Pyridinium substituted BODIPY as NIR fluorescent probe for simultaneous sensing of hydrogen sulphide/glutathione and cysteine/homocysteine
Qiu et al. A selective and sensitive fluorescent probe for homocysteine and its application in living cells
Ding et al. Smart probe for rapid and simultaneous detection and discrimination of hydrogen sulfide, cysteine/homocysteine, and glutathione
Zhang et al. Meso-heteroaryl BODIPY dyes as dual-responsive fluorescent probes for discrimination of Cys from Hcy and GSH
Wang et al. Thiazolyl substituted NBD as fluorescent probe for the detection of homocysteine and cysteine
Zhang et al. A simple and readily available fluorescent turn-on probe for cysteine detection and bioimaging in living cells
WO2012111818A1 (ja) 蛍光プローブ
CN110590726B (zh) 一种同时区分Cys/Hcy和GSH的开关型荧光探针
Zhai et al. A dual emission fluorescent probe enables simultaneous detection and discrimination of Cys/Hcy and GSH and its application in cell imaging
Zhu et al. A novel colorimetric and ratiometric fluorescent probe for cysteine based on conjugate addition-cyclization-elimination strategy with a large Stokes shift and bioimaging in living cells
CN106046012B (zh) 一种香豆素类生物硫醇荧光探针及其制备方法
CN110563689A (zh) 一种特异性检测活细胞中的半胱氨酸的长波长发射荧光探针及其制备方法和应用
Song et al. A lysosome-targetable fluorescent probe for simultaneous detection and discrimination of Cys/Hcy and GSH by dual channels
Yang et al. A fluorescent dyad with large emission shift for discrimination of cysteine/homocysteine from glutathione and hydrogen sulfide and the application of bioimaging
Zhu et al. A simple highly specific fluorescent probe for simultaneous discrimination of cysteine/homocysteine and glutathione/hydrogen sulfide in living cells and zebrafish using two separated fluorescence channels under single wavelength excitation
Wang et al. A novel NBD-based fluorescent turn-on probe for the detection of cysteine and homocysteine in living cells
Roubinet et al. A FRET-based probe for fluorescence sensing of sulfide/sulfite analytes, using a novel long-wavelength water-soluble 7-hydroxycoumarin as reporter fluorophore
CN110498786B (zh) 一种检测半胱氨酸/同型半胱氨酸和谷胱甘肽的新型比值型荧光探针
Anila et al. A Cysteine-Specific Fluorescent Switch for Monitoring Oxidative Stress and Quantification of Aminoacylase-1 in Blood Serum
Song et al. Anaphthalimide-based fluorescent probe for mercapto-containing compounds
CN105601658B (zh) 一种能够区分生物硫醇的荧光探针的制备与应用
Tan et al. A simple lysosome-targeted fluorescent probe based on flavonoid for detection of cysteine in living cells
Cai et al. A rational design of fluorescent probes for specific detection and imaging of endogenous formaldehyde in living cells
CN109928940B (zh) 基于碱性蓝-3的检测次氯酸的近红外荧光探针分子的制备
KR20140135483A (ko) 넓은 범위의 pH 측정이 가능한 비율계량적 pH 프로브

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant