A Multi‐signal Fluorescent Probe with Multiple Binding Sites for Simultaneous Sensing of Cysteine,Homocysteine, and Glutathione

A novel fluorescent probe was developed by integrating chlorinated coumarin and benzothiazolylacetonitrile and exploited for simultaneous detection of cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). Featuring four binding sites and different reaction mechanisms for different biothiols, this probe exhibited rapid fluorescence turn‐on for distinguishing Cys, Hcy, and GSH with 108‐, 128‐, 30‐fold fluorescence increases at 457, 559, 529 nm, respectively, across different excitation wavelengths. Furthermore, the probe was successfully applied to the fluorescence imaging of endogenous Cys and GSH and exogenous Cys, Hcy, and GSH in living cells.     

A Multi‐signal Fluorescent Probe with Multiple Binding Sites for Simultaneous Sensing of Cysteine,Homocysteine, and Glutathione

A novel fluorescent probe was developed by integrating chlorinated coumarin and benzothiazolylacetonitrile and exploited for simultaneous detection of cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). Featuring four binding sites and different reaction mechanisms for different biothiols, this probe exhibited rapid fluorescence turn‐on for distinguishing Cys, Hcy, and GSH with 108‐, 128‐, 30‐fold fluorescence increases at 457, 559, 529 nm, respectively, across different excitation wavelengths. Furthermore, the probe was successfully applied to the fluorescence imaging of endogenous Cys and GSH and exogenous Cys, Hcy, and GSH in living cells.     

Construction of a Selective Fluorescent Probe for GSH Based on a Chloro‐Functionalized Coumarin‐enone Dye Platform

Glutathione (GSH), the most abundant intracellular biothiol, protects cellular components from damage caused by free radicals and reactive oxygen species (ROS), and plays a crucial role in human pathologies. A fluorescent probe that can selectively sense intracellular GSH would be very valuable for understanding of its biological functions and mechanisms of diseases. In this work, a 3,4‐dimethoxythiophenol‐substituted coumarin‐enone was exploited as a reaction‐type fluorescent probe for GSH based on a chloro‐functionalized coumarin‐enone platform. In the probe, the 3,4‐dimethoxythiophenol group functions not only as a fluorescence quencher through photoinduced electron transfer (PET) to ensure a low background fluorescence, but also as a reactive site for biothiols. The probe displays a dramatic fluorescence turn‐on response toward GSH with the long‐wavelength emission (600 nm) and significant Stokes shift (100 nm). The selectivity of the probe toward GSH over cysteine (Cys), homocysteine (Hcy), and other amino acids was demonstrated. Assisted by laser‐scanning confocal microscopy, we have demonstrated that the probe could specifically sense GSH over Cys/Hcy in human renal cell carcinoma SiHa cells.     

Design of fluorescent probes with optimized responsiveness and selectivity to GSH

We designed and synthesized a series of BODIPY based probes with fast and distinct ratiometric responsiveness for discriminative detection of GSH from Cys and Hcy. The discriminative detection is based on the different products obtained by the S_NAr between probes and thiol-containing amino acids. The amino group of the obtained thioether from the reaction with Cys or Hcy but not GSH would trigger an intramolecular nucleophilic substitution through five- or six-membered cyclic transition state, finally yielding an amino substituted derivative. To achieve highly discriminative detection and fast response, a series of structure modifications and improvements have been made by elongating the π-conjugation and introducing electron withdrawing groups, finally affording probe BOD-DBNPF with optimized responsiveness and selectivity. Importantly, BOD-DBNPF was successfully used for the selective detection of GSH from Cys with distinct fluorescent ratiometric responses in living HeLa cells.     

Rational Design of an Ultrasensitive and Highly Selective Chemodosimeter by a Dual Quenching Mechanism for Cysteine Based on a Facile Michael‐Transcyclization Cascade Reaction

Differentiation of biologically important thiols, such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) is still a challenging task. Herein, we present a novel fluorescent chemodosimeter capable of selectively detecting Cys over other biothiols including Hcy and GSH and other amino acids by a facile thiol‐Michael addition/transcyclization rearrangement cascade click process. The unique transcyclization step is critical for the selectivity as a result of the kinetically favorable formation of a six‐membered ring with the Cys Michael adduct. Moreover, the probe adopts a distinctive dual quenching mechanism—photoinduced electron transfer (PET) and photoinduced intramolecular charge transfer (ICT) to deliver a drastic turn‐on fluorescence response only at the Cys‐selective transcylization step. The judicious selection of strong electron‐withdrawing naphthalimide fluorophore with maleimide group enhances the electrophilicity and thus reactivity for the cascade process leading to fast detection and ultrasensitivity with a detection limit of 2.0 nm (S/N=3). The probe has demonstrated its practical utility potential in Cys imaging in live cells.     

Distinguishable multi-substance detection based on three-channel NIR fluorescent probe in physiology and pathology of living cells and zebrafish

Mitochondria is the main organelle for the production of reactive sulfur species (RSS), such as homocysteine (Hcy), cysteine (Cys), glutathione (GSH) and sulfur dioxide (SO₂). These compounds participate in a large number of physiological processes and play an extremely important role in maintaining the balance of life systems. Abnormal concentration and metabolism are closely related to many diseases. Due to their similarities in chemical properties, it is challenging to develop a single fluorescent probe to distinguish them simultaneously. Here, we synthesized the probe PI-CONBD with three fluorophores, NBD-Cl and benzopyranate as the reaction sites of GSH/Cys/Hcy and SO₂, respectively. Three biothiols all could cleavage ether bond to release benzopyrylium and coumarin moiety, which emitted red and blue fluorescence, but Cys/Hcy also could do intramolecular rearrangement after nucleophilic substitution, resulting in yellow fluorescence. Thus the probe can distinguish Cys/Hcy and GSH. Subsequently, only SO₂ could quench red fluorescence by adding CC of benzopyrylium. The probe also could localize well in mitochondria by oxonium ion for all kinds of cells. The probe not only could detect above sulfur-containing active substances of intracellular and extracellular but also monitor the level of them under oxidative stress and apoptosis process in living cells and zebrafish.     

A colorimetric,ratiometric and water-soluble fluorescent probe for simultaneously sensing glutathione and cysteine/homocysteine

A chlorinated coumarin-aldehyde was developed as a colorimetric and ratiometric fluorescent probe for distinguishing glutathione (GSH), cystenine (Cys) and homocysteine (Hcy). The GSH-induced substitution-cyclization and Cys/Hcy-induced substitution-rearrangement cascades lead to the corresponding thiol-coumarin-iminium cation and amino-coumarin-aldehyde with distinct photophysical properties. The probe can be used to simultaneously detect GSH and Cys/Hcy by visual determination based on distinct different colors – red and pale-yellow in PBS buffer solution by two reaction sites. From the linear relationship of fluorescence intensity and biothiols concentrations, it was determined that the limits of detection for GSH, Hcy and Cys are 0.08, 0.09 and 0.18 μM, respectively. Furthermore, the probe was successfully used in living cell imaging with low cell toxicity.     

选择性检测谷胱甘肽的荧光探针

本文设计合成了一种基于BODIPY衍生物选择性检测谷胱甘肽的比率式荧光探针1。荧光探针1中BODIPY的3位连有苯乙炔基团,5位连有咪唑盐离去基团,利用其与谷胱甘肽和半胱氨酸反应机理的不同实现了对谷胱甘肽的选择性检测。紫外可见吸收光谱和荧光光谱实验结果表明探针分子1与谷胱甘肽反应后的光谱发生明显红移,可以实现对谷胱甘肽的比率式检测。探针分子1对谷胱甘肽有极高的选择性,不受其它氨基酸尤其是半胱氨酸的干扰。荧光滴定实验表明探针分子1可实现对谷胱甘肽的定量检测,检测限为3.3×10-8 mol/L。探针分子成功地应用于活体细胞中检测谷胱甘肽。     

Rational design of a bifunctional fluorescent probe for distinguishing Hcy/Cys from GSH with ideal properties

By pairing two fluoropho res according to their optical prope rties such as absorption spectral overlap and absorptivity,fluorescent quantum yield and emission spectral separation,a bifunctional fluorescent probe,TQBF-NBD,was rationally designed and synthesized to discriminatively sense Hcy/Cys and GSH with good selectivity and sensitivity.It is noted that this probe could work under a single-wave length excitation and displayed a mega-large Stokes shift.TQBF-NBD reacted with Hcy/Cys to give a mixed green-red fluorescence and displayed a red fluorescence upon the treatment with GSH.Distinguishable imaging of intracellular Hcy/Cys from GSH with the help of TQBF-NBD was realized in living cells and zebrafish.     

A fluorescence turn-on probe for cysteine and homocysteine based on thiol-triggered benzothiazolidine ring formation

We synthesized a new coumarin-based probe TP, containing a disulfide moiety, to detect biothiols in cells. A fluorescence turn-on response is induced by the thiol–disulfide exchange of the probe, with subsequent intramolecular benzothiazolidine ring formation giving rise to a fluorescent product. The probe exhibits an excellent selectivity for cysteine (Cys) and homocysteine (Hcy) over glutathione (GSH) and other amino acids. The fluorescent probe also exhibits a highly sensitive fluorescence turn-on response to Cys and Hcy with detection limits of 0.8 μM for Cys and 0.5 μM for Hcy. In addition, confocal fluorescence microscopy imaging using RAW264.7 macrophages demonstrates that the probe TP could be an efficient fluorescent detector for thiols in living cells.     

A multisite-binding fluorescent probe for simultaneous monitoring of mitochondrial homocysteine,cysteine and glutathione in live cells and zebrafish

Homocysteine(Hcy), cysteine(Cys) and glutathione(GSH) play crucial roles in redox homeostasis during mitochondria functions. Simultaneous differentiation and visualization of mitochondrial biothiols dynamics are significant for understanding cell metabolism and their related diseases. Herein, a multisitebinding fluorescent probe(MCP) was developed for simultaneous sensing of mitochondrial Cys, GSH and Hcy from three fluorescence channels for the first time. This novel probe exhibited rapid fluor...     

A TICS-fluorophore based probe for dual-color GSH imaging

Cascading reactions in fluorophores accompanied by the replacement of different fluorescence wavelengths can be used to develop luminescent materials and reactive fluorescent probes. Based on multiple signal channels, the selectivity of probes can be improved and the range of response to guest molecule recognition can be expanded. By regulating the position, number, and activity of active sites in fluorophores, fluorescent probes that successively react with thiol and amino groups in cysteine (Cys), homocysteine (Hcy) have been developed, which can only react with the thiol group of GSH. In this paper, we report the first probe capable of cascading nucleophilic substitution reaction with the thiol group and amino group of GSH at a single reaction site, and showed the dual-color recognition of GSH, which improved the selectivity of GSH also was an extension of GSH probes. The probe Rho-DEA was based on a TICS fluorophore, and the intramolecular cascade nucleophilic substitution reaction occurs with Cys/Hcy. The thiol substitution of the first step reaction with Cys/Hcy was quenched due to intersystem crossing to triplet state, so GSH can be selectively recognized from the fluorescence signal. Rho-DEA has the ability of mitochondrial localization, and finally realized in situ dual-color fluorescence recognition of GSH in mitochondria.     

Exceptional time response,stability and selectivity in doubly-activated phenyl selenium-based glutathione-selective platform

A phenyl-selenium-substituted coumarin probe was synthesized for the purpose of achieving highly selective and extremely rapid detection of glutathione (GSH) over cysteine (Cys)/homocysteine (Hcy) without background fluorescence. The fluorescence intensity of the probe with GSH shows a ∼100-fold fluorescent enhancement compared with the signal generated for other closely related amino acids, including Cys and Hcy. Importantly, the substitution reaction with the sulfhydryl group of GSH at the 4-position of the probe, which is doubly-activated by two carbonyl groups, occurs extremely fast, showing subsecond maximum fluorescence intensity attainment; equilibrium was reached within 100 ms (UV-vis). The probe selectivity for GSH was confirmed in Hep3B cells by confocal microscopy imaging.     

Dual‐Emission Channels for Simultaneous Sensing of Cysteine and Homocysteine in Living Cells

The development of a fluorescent probe to distinguish between cysteine (Cys) and homocysteine (Hcy) is always a challenge owing to their structural similarity, and the simultaneous detection of Cys and Hcy by utilizing different emission channels is especially difficult. In this work, we designed and synthesized a new fluorescent probe to differentiate between Cys and Hcy on the basis of a coumarin derivative with a chlorine atom and an α,β‐unsaturated aldehyde. Cys and Hcy induced different cascade reactions with the probe, which led to different products with distinct photophysical properties. The nonfluorescent probe responded to Cys and emitted strong blue fluorescence, whereas it reacted with Hcy and generated yellow fluorescence without interference from glutathione. In addition, the probe was successfully applied to distinguish between Cys and Hcy in living cells.     

特异性识别半胱氨酸荧光探针的合成及细胞成像研究

基于光诱导电子转移(PET)机制,利用Cys亲核性较强,能够与探针分子发生亲核取代反应,使丙烯酰基离去,使探针分子体系内PET过程失效,合成了一种特异性识别半胱氨酸的荧光探针。当向探针溶液分别加入多种测试物时,除与Cys结构类似的Hcy和GSH会引起探针溶液微弱的荧光变化外,其他氨基酸均不会引起探针溶液荧光强度的变化,该探针对Cys具有良好的选择性和灵敏度,可在生理条件下检测Cys,并且区分Hcy和GSH。同时,该探针成功实现了细胞内Cys的荧光成像,为在生物学及医学中的实际应用建立了一种特异性识别Cys的分析方法。     

Fluorescent Probes with Multiple Binding Sites for the Discrimination of Cys,Hcy, and GSH

Biothiols such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) play crucial roles in maintaining redox homeostasis in biological systems. This Minireview summarizes the most significant current challenges in the field of thiol‐reactive probes for biomedical research and diagnostics, emphasizing the needs and opportunities that have been under‐investigated by chemists in the selective probe and sensor field. Progress on multiple binding site probes to distinguish Cys, Hcy, and GSH is highlighted as a creative new direction in the field that can enable simultaneous, accurate ratiometric monitoring. New probe design strategies and researcher priorities can better help address current challenges, including the monitoring of disease states such as autism and chronic diseases involving oxidative stress that are characterized by divergent levels of GSH, Cys, and Hcy.     

基于新型不对称BODIPY的GSH荧光探针

本文通过在BODIPY母体中引入丙二腈,设计合成了一个新型不对称BODIPY荧光染料CN-B-Cl。由于丙二腈强吸电作用,荧光染料CN-B-Cl具有优异的化学活性,能够与含巯基的化合物在buffer体系中迅速发生芳香亲核取代反应;与GSH反应生成硫取代BODIPY,而与Cys/Hcy反应生成氮取代BODIPY。根据不同取代基BODIPY化合物发光性能的不同,该荧光探针可选择性区分GSH与Cys/Hcy。     

Target‐Triggered NIR Emission with a Large Stokes Shift for the Detection and Imaging of Cysteine in Living Cells

Background autofluorescence from biological systems generally reduces the sensitivity of a fluorescent probe for imaging biological targets. Addressing this challenge requires the development of fluorescent probes that produce emission in the near‐infrared region. Herein, we report the design and synthesis of a fluorescent probe that generates an NIR emission with a large Stokes shift upon the selective response to Cys over Hcy and GSH. The probe is designed to consist of two Cys‐sensing sites, an acrylate ester and an aldehyde installed ortho to each other. The reaction of the probe with Cys triggers an excited state intramolecular proton transfer process upon photo‐excitation, thereby producing an NIR emission with a large Stokes shift. Accordingly, this probe hold great promise for the selective detection of Cys in biological systems. We further demonstrate the capacity of this probe for Cys imaging in living cells.     

Simultaneous Visualization of Endogenous Homocysteine,Cysteine, Glutathione,and their Transformation through Different Fluorescence Channels

Studying numerous biologically important species simultaneously is crucial to understanding cellular functions and the root causes of related diseases. Direct visualization of endogenous biothiols in biological systems is of great value to understanding their biological roles. Herein, a novel multi‐signal fluorescent probe was rationally designed and exploited for the simultaneous sensing of homocysteine (Hcy), cysteine (Cys), and glutathione (GSH) using different emission channels. This probe was successfully applied to the simultaneous discrimination between and visualization of endogenous Hcy, Cys, GSH, and their transformation in living cells.     

A Novel Fluorescent Probe Based on Perylene Derivative for Hg2+ Ions and Biological Thiols and its Application in Live Cell Imaging and Theoretical Calculations

The selective and sensitive detection of biothiols; cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) in aqueous solutions is of considerable importance because of their pivotal roles in maintaining the reducing environment in the cells. This study describes a strategy for the determination of biothiols based on the PDI/Met‐Hg²⁺complex platform. We designed and fabricated methionine modified perylene diimide molecule as a selective sensing probe for Hg²⁺ ions in aqueous solutions ( PDI/Met‐Hg ²⁺). The complex between perylene bisimide derivative ( PDI/Met) and Hg²⁺ was investigated and it demonstrated turn‐on fluorescence response for the detection of the biological thiols. Besides, PDI/Met displayed fluorescence quenching response in the presence of mercury ions and the emission intensity of PDI/Met‐Hg²⁺ was recovered after transferring biothiols (Cys, Hcy, and GSH). Thus, PDI/Met could be utilized as a fluorescent chemosensor for the sequential recognition of mercury ions and biological thiols.