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.     

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.     

Development of a Small Molecule Probe Capable of Discriminating Cysteine,Homocysteine, and Glutathione with Three Distinct Turn‐On Fluorescent Outputs

The simultaneous discrimination of Cys, Hcy, and GSH by a single probe is still an unmet challenge. The design and synthesis of a small molecule probe MeO‐BODIPY‐Cl (BODIPY=boron dipyrromethene) is presented, which can allow Cys, Hcy, and GSH to be simultaneously discriminated on the basis of three distinct fluorescence turn‐on responses. The probe reacts with these thiols to form sulfenyl‐substituted BODIPY, which is followed by intramolecular displacement to yield amino‐substituted BODIPY. The kinetic rate of the intramolecular displacement reaction determines the observed different sensing behavior. Therefore, the probe responds to Cys, Hcy, and GSH with fluorescence turn‐on colors of yellow, yellow and red, and red, respectively. With this promising feature in hand, the probe was successfully used in imaging of Cys, Hcy and GSH 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...     

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。     

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.     

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.     

Lighting‐Up of the Dye Malachite Green with Mercury(II)–DNA and Its Application for Fluorescence Turn‐Off Detection of Cysteine and Glutathione

This work describes a novel strategy for the highly sensitive and selective detection of cysteine (Cys) and glutathione (GSH) based on the Hg²⁺–AGRO100–malachite green (MG) complex system. The dye MG, which has a very low quantum yield in aqueous solution by itself, can bind with the thymine‐rich DNA AGRO100 in the presence of Hg²⁺ ions to generate a striking fluorescence intensity enhancement of 1000‐fold. As sulfur‐containing amino acids, Cys and GSH effectively sequester Hg²⁺ ions from the Hg²⁺–AGRO100–MG complex structure to switch the ‘lit‐up’ chemosensor to the ‘off’ state (about a 50‐fold fluorescence intensity decrease), thus providing a facile, but effective, method to probe for Cys/GSH. The fluorescence titration, UV absorption, CD, and Raman spectra provide some insight into the structural and chemical basis for the enhancement effect. The formation of the Hg²⁺–AGRO100–MG complex significantly affects the electronic structure and conformation of the MG molecule by leading to an extended π system, which is the likely origin of the observed striking fluorescence intensity enhancement. Notably, the proposed sensing platform exhibits exquisite selectivity and sensitivity toward Cys/GSH with limits of detection of 5 nM for Cys and 10 nM for GSH, respectively. Furthermore, the straightforward assay design avoids labeling of the probe, uses only commercially available materials, and still displays comparable sensitivity and excellent selectivity.     

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.     

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 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.     

基于萘酰亚胺的生物硫醇探针的合成及对含硫醇氨基酸的检测

合成了以4-羟基萘酰亚胺为荧光团,2,4-二硝基苯磺酰氧基为特异性识别基团的生物硫醇探针4-(2,4-二硝基苯磺酰氧基)-正丁基-1,8-萘酰亚胺(DNSBN).吸收光谱和荧光光谱结果表明, DNSBN对半胱氨酸(Cys)、同型半胱氨酸(Hcy)和谷胱甘肽(GSH)3种生物硫醇分子具有高效的检测识别能力,不受其它17种天然氨基酸的干扰.同时,通过荧光滴定实验证实了此探针是一种比率型探针,555 nm处的荧光强度与溶液中的生物硫醇分子浓度在0 ~ 20 μmol/L范围内呈良好的线性关系,对Cys、Hcy和GSH的检出限(3σ)分别为25.9、92.0和77.9 nmol/L.而吸收光谱、荧光光谱和质谱表征数据显示,生物硫醇与2,4-二硝基苯磺酸酯发生亲核取代反应并导致磺酸酯的分解.随着识别基团的解离,探针分子的d-PeT (donor-excited photoinduced electron transfer) 效应被解除,并出现非常明显的比色与荧光变化.HeLa细胞成像实验表明,探针DNSBN具有良好的生物相容性,能够对细胞外源性生物硫醇分子进行检测.     

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.     

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.     

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.     

Design of activatable red-emissive assay for cysteine detection in aqueous medium with aggregation induced emission characteristics

A novel water-soluble red-emissive AIE fluorescence probe for cysteine (Cys) in situ was prepared and the performance of selectivity and sensitivity has been carefully investigated in this study. The probe was established on the electrostatic interaction of sulfonate functionalized tetraphenylethene (TPE) and polycation generated by the reaction between a polymer bearing dinitrobenzenesulfonate groups and Cys. From the experimental results, it was easy to distinguish Cys from glutathione (GSH) and homocysteine (Hcy) with a detection limit of 73 nmol/L. The assay system also possessed strong anti-interference ability against multitudinous amino acids. The Stokes shift was 142 nm and the emission ranged from 550 nm to 850 nm. In addition, double responses in fluorescence and ultraviolet-visible spectra also make the red-emissive assay ideal for sensitive detection and quantification of Cys for most purposes, especially in-situ monitoring of Cys in aqueous medium.     

Rearrangement regulated cysteine fluorescent probe for cellular oxidative stress evaluation induced by copper(II)

Cysteine (Cys) plays an important role in regulating cellular redox balance. But due to the constant changes in the concentration of Cys in organisms, fast response sensors are urgent required for practical application. In this work, a fluorescent probe with a fast response was developed by linking coumarin derivatives containing α,β-unsaturated ketones to NBD. The PET effect made the system non-fluorescent. When the probe reacted with Cys, the bond between the coumarin derivative and the NBD was cut off, meanwhile a rapid rearrangement and reactive site passivation occurred. Then two fluorophores with the same emission peak are released, among them, strong fluorescence signal of NBD dominated. Thus, although the similar reaction occurred for Hcy, the rate of NBD derivative rearrangement was slow, in a short time, fluorescence signal was still weak. As for GSH, cleavage could occur, but no rearrange within the NBD molecule due to GSH with large volume. Because of strong fluorescent emission, this probe was successfully used in biological imaging about cell and zebrafish. More importantly, the probe was successfully used to evaluate the oxidative stress caused by copper(II) in living cells. This fluorescence strategy and application will provide a new way of studying intracellular oxidative stress processes and damage.     

Benzothiazole‐Pyimidine‐Based BF2 Complex for Selective Detection of Cysteine

Due to the similar structure and reactivity of cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), the simultaneous discrimination of Cys over Hcy and GSH by a single fluorescent sensor is still a great challenge. In this work, a benzothiazole‐pyimidine‐based boron difluoride complex ( BPB ) was developed as a new fluorescent sensor for Cys. The sensor exhibits a highly selective “turn‐on” response to cysteine over Hcy, GSH and other amino acids in aqueous solution at physiological pH. The observed pseudo‐first‐order rate constant for the reaction of BPB with Cys was calculated to be about 0.062 min⁻¹. The detection limit of this sensor for Cys was determined to be 332 nm, and bioimaging of exogenous Cys by this sensor was successfully applied in living cells, thus indicating that this sensor holds great potential for biological applications.     

Installation of efficient quenching groups of a fluorescent probe for the specific detection of cysteine and homocysteine over glutathione in solution and imaging of living cells

Herein, we report the synthesis and characterisation of a new fluorescent probe 4-(7-nitro-benzo[1,2,5]oxadiazol-4-yl)-benzaldehyde (NBOB) installed with quenching groups for highly selective and sensitive sensing of biothiols. The probe itself is non-fluorescent due to the presence of quenching groups and photoinduced electron transfer (PET) process. Thus, sensitivity of the probe towards thiols was significantly improved by quenching effects. NBOB has been shown to exhibit selective reactivity towards cysteine (Cys) and homocysteine (Hcy) over glutathione (GSH) under stoichiometric conditions. The response mechanism was proved by ¹H NMR, LCMS and theoretical calculation. The probe NBOB has been shown to react with Cys present in Vero cells by fluorescence microscopy.