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Regulating glutathione-responsiveness of naphthalimide-based fluorescent probes by an oxidation strategy
Abstract
Two naphthalimide-based fluorescent probes containing a thiomorpholine (Np-NS) or a sulfoxide-morpholine (Np-NSO) component were reported. The morpholine unit of non-fluorescent Np-NS and Np-NSO can transform into the sulphone-morpholine and accompany by blue fluorescence upon oxidative stress, ascribing to the formation of sulphone-morpholine on probes. The sensing behavior displays that they can selectively respond glutathione to generate the green emission by a sulfonamide-based detection moiety both in vitro and in living cells. Interestingly, the different oxidation state of sulphur atom on thiomorpholine ring can be utilized to regulate the responsiveness of probes towards glutathione. Such oxidation strategy would provide a possibility for enhancing the response rate.
... Recently, we found that through using different oxidation states of sulfur moieties, the AIE behavior and MFC properties of TPE-based luminogens could be tuned, especially, sulfone-containing TPE derivative displayed polymorphism behavior [38]. Inspired by the work of Tang et al., and our previous reports on sulfone-based fluorescent compounds, in this work, we aimed to know how the oxidation states of sulfur moieties regulate the properties of symmetric BTPE derivatives and if the sulfone-containing BTPE possess two or more solid-states with different emission colors [39][40][41][42][43][44]. In this regard, we successfully synthesized different bis(tetraphenylethene) derivatives 1-3 which are linked by a sulfur atom in various oxidation states (Scheme 1). ...
Three novel bis(tetraphenylethene) (BTPE) derivatives 1–3 were efficiently synthesized. All the materials exhibited aggregation-induced emission characteristics, and the dynamic light scattering measurments proved that the average diameter of the oxidised analogues 2 and 3 was less than that of the thioether precursor 1 due to the oxidation of the linking S-atom. Linking two tetraphenylethylenes via a sulfur atom with different oxidation states offered compounds which showed reversible mechanofluorochromic behavior. The crystal-to-amorphous phase conversion process was responsible for the mechanofluorochromism. Most notably, sulfone-based BTPE 3 presented polymorphism due to different packing modes, and the three observable morphologies could be interconverted.
... In this regard, organic materials are not lagging behind owing to their several advantages, which includes their ease of fabrication, a wide variety of functional groups and less economical costs [4]. Among the organic class of materials, the 1,8-naphthalimides derivatives have been highlighted as fluorescent cellular imaging agents, exhibit strong fluorescence, a wide range of applications in laser dyes, organic light emitting diodes, liquid crystals, sensors, organic photovoltaics and anti-cancer compounds by targeting DNA [5][6][7][8][9][10][11][12]. The 1,8-naphthalimide derivatives possess photonic properties due to functionalization at C-4 position. ...
The present study reports the synthesis and characterization of 2,2'-(diazene-1,2-diylbis(4,1-phenylene))bis(6-(butylamino)-1H-benzo[de]isoquinoline-1,3(2 H)-dione) (4) using a dual approach consisting of experimental and computational techniques. The indigenously synthesized compound 4 is successfully characterized using UV–vis, fluorescence, FT-IR, and ¹H-MNR techniques. The experimental absorption spectra were observed in ethanol solvent at 399 nm and fluorescence emission bands were observed at 550 nm at an excitation wavelength of 400 nm. The compound 4 shows higher thermal stability. From computational point of view, the maximum absorption and emission wavelengths are observed around 377 and 510 nm, respectively, using time-dependent density functional theory (TD-DFT) methods. The average third-order NLO polarizability of compound 4 is calculated to be 407.20 × 10⁻³⁶ esu, which is about 53 times larger than the γ amplitude of para-nitroaniline pNA (a prototype NLO molecule). Additionally, we have also quantum chemically considered further derivatives of parent system 4 in the form of 4a, 4b and 4c to study the structure NLO property relationships. For derivative compounds 4a, 4b and 4c, their calculated γ amplitudes are found to be 239.97 × 10⁻³⁶, 193.66 × 10⁻³⁶, 198.59 × 10⁻³⁶ esu, respectively, which illustrate the significant effect of terminal donor/acceptor groups on the NLO response of parent compound 4. Additionally, TD-DFT calculations showed that the largest NLO response in compound 4 is due to its lower transition energy and larger oscillator strength as compared to other systems. It is believed that the present study will highlight the importance of our newly synthesized compound 4 and its derivatives owing to their good NLO response properties.
RNA cap methylations have been shown to be crucial for the life cycle, replication, and infection of ssRNA viruses, as well as for evading the host's innate immune system. Viral methyltransferases (MTases) therefore represent an attractive target for the development of compounds as tools and inhibitors. In coronaviruses, N7-methyltransferase function is localized in nsp14, which has become an increasingly important therapeutic target with the COVID-19 pandemic. In recent years, we have been developing SAH-derived bisubstrates with adenosine and an N-arylsulfonamide moiety targeting both SAM and RNA binding sites in nsp14. We report here the synthesis of 31 SAH analogues with the N-arylsulfonamide attached to the 5′-position of adenosine via different linkers such as N-ethylthioether, N-ethylsulfone, N-ethylamino or N-methyltriazole. The compounds were obtained efficiently by amine sulfonylation or click chemistry. Their ability to inhibit SARS-CoV-2 N7-MTase was evaluated and the best inhibitors showed a submicromolar inhibitory activity against N7-MTase nsp14.
Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor that plays a crucial role in cell differentiation and tumor progression, and its overexpression is closely associated with the development...
The different oxidation states of sulphur atom play a significant role on functional materials. In this work, a aryl-thioether and its sulphone substituted benzo[c][1,2,5]oxadiazole dyes were synthesized and utilized to determine thiol-containing amino acids. The result of selectivity experiments showed they detected the cysteine and homocysteine under physiological condition with negligible interference from other amino acids. In comparison to the thioether dye, the sulphone-based dye exhibited much faster response time for Cys and Hcy. However, the sulphone restricted its thiol-reactivity and bioimaging performance in living cells. By reducing the oxidation state of sulphur atom, we amazedly found that the sulfoxide-based dye still maintained high selectivity ultrafast response time for Cys/Hcy under physiological condition. It was worth mentioning that it also had high reactivity and good bioimaging performance that sulfone compounds did not have.
The first example of combining the fluorescent probe-based freeze concentration effect with N-oxide chemistry is reported for the highly sensitive and selective detection of the ferrous ion (Fe(II)). Interestingly, our...
A D-π-A-π-D type of tetraphenylene-coating benzoselenodiazole fluorescence dye with near-infrared emission has been designed and constructed. This dye shows an obvious aggregation-induced-emission behavior. In the solid state, it exhibits a reversible mechanochromism with the changes of near-infrared emission. Furthermore, this dye can be used to track the lysosomes of living cells and images in vivo.
Molecular probes suitable for different fluorescence imaging technologies can meet the requirements of different scientific researches in biological applications. In this work, a naphthalimide-cyanine based sulfonamide was used to specifically visualize the glutathione of mouse tissues with a two-photon manner for the naphthalimide moiety and a near-infrared manner for the cyanine moiety, respectively. The results showed that this probe served as a dual-model tissue-imaging agent for the visualization of glutathione with around 200 μm imaging depth in two-photon manner and 120 μm imaging depth in near-infrared manner, respectively, which provided a model for the tissue-imaging in visible and near-infrared channels.
Developing glutathione-specific fluorescent probes with novel fluorescence-responsive manners can enrich the applications of probes in living cells. In this work, we designed and synthesized a dual fluorophores (naphthalimide and dansyl) co-modified fluorescent probe, which displayed a high selectivity toward glutathione (GSH). In living cells, this probe could specifically visualize the GSH of lysosome and accompanied by two different fluorescence emissions. One is green emission that can be assigned to the nucleophilic substitution product (GSH-linked naphthalimide) while the other is orange emission that can be derived from the cleavage product (dansyl). Especially, such dual-emission bioimaging in living cells had a spatiotemporal and synchronous manner. It is expected to provide an alternative strategy for designing the functional fluorescent probes to be applied in multi-color bioimaging in future.
Different oxidation states of sulphur can be used to regulate the aggregation-induced emission (AIE) behaviours and reversible mechanofluorochromic properties of the tetraphenylethene (TPE) derivatives. With the introudction of oxygen atoms via the oxidation of thioether-containing TPE, the AIE properties and aggregation turn-on threshold of AIEgens could be regulated. The single crystal structures revealed that weak intermolecular interactions might reduce the emission intensities of 1 in the crystalline phase. In addition, one sulphone-containing TPE derivative 3 demonstrated polymorphism-dependent fluorescence of different colours due to the different packing models, resulting in a tricolored emission switching.
Photodynamic therapy (PDT) is a new treatment modality against local lesions, particularly in malignant tumors. However, higher biothiol concentrations in tumor cells will decrease the efficacy of PDT. To tackle the above challenges, a distinctive spinach‐based carbon nanomaterial called “chlorophyll‐rich biomass quantum dots” (CBQDs) with near‐infrared emission is prepared, and the CBQDs are further bound with copper ions forming CBQD‐Cu nanocomposites, which are used as a new fluorescence nanoprobe and highly efficient photosensitizer for simultaneous in vivo near‐infrared fluorescence imaging of biothiol and dual‐enhanced PDT of tumors. The mechanism of action is studied and it is shown that the binding of chlorophyll and copper ions on the surface of CBQDs reduces the energy level difference of the chlorophyll molecules, which results in an increase of reactive oxygen species (ROS) achieving enhanced PDT. On the other hand, the copper ions on the CBQD‐Cu surface can also bind with biothiol which decreases the concentration of free biothiol in tumor cells, and further enhances PDT. At the same time, the combination of copper ions and biothiol opens the fluorescence of CBQDs, which realizes near‐infrared fluorescence imaging of biothiol. Hence, the CBQD‐Cu is used as a highly effective nanomedicine for the recognition and treatment of tumors.
A tetraphenylethene (TPE) derivative substituted with a sulfonyl‐based naphthalimide unit (TPE‐Np) was designed and synthesized. Its optical properties in solution and in the solid state were investigated. Photophysical properties indicated that the target molecule, TPE‐Np, possessed aggregation‐induced emission (AIE) behavior, although the linkage between TPE and the naphthalimide unit was nonconjugated. Additionally, it exhibited an unexpected, highly reversible mechanochromism in the solid state, which was attributed to the change in manner of aggregation between crystalline and amorphous states. On the other hand, a solution of TPE‐Np in a mixture of dimethyl sulfoxide/phosphate‐buffered saline was capable of efficiently distinguishing glutathione (GSH) from cysteine and homocysteine in the presence of cetyltrimethylammonium bromide. Furthermore, the strategy of using poly(ethylene glycol)–polyethylenimine (PEG‐PEI) nanogel as a carrier to cross‐link TPE‐Np to obtain a water‐soluble PEG‐PEI/TPE‐Np nanoprobe greatly improved the biocompatibility, and this nanoprobe could be successfully applied in the visualization of GSH levels in living cells.
2 is a novel fluorescent turn-on probe for imaging biothiols based on S N Ar substitution-skeletal rearrangement strategy with dramatic fluorescence enhancement and high sensitivity.
A NIR fluorescence probe with long emission wavelength at 746 nm and high quantum yield for 0.36 to selectively detect GSH over Hcy and Cys in living systems was designed and synthesized. 2-(1,3,3-Trimethylindolin-2-ylidene) acetaldehyde was connected to rhodamine by Knoevenagel Condensation in order to elongated the emission to near infrared region. Then it was connected with ethylenediamine to get important intermediate. Finally, target molecule named RhNM was synthesized through conjugated N-[4-(carbonyl) phenyl]. RhNM shows excellent discrimination of GSH from Hcy/Cys along with obvious color changes. The fluorescence band has a rapid increase at 746 nm enhanced 106-fold after adding GSH, the quantum was calculated for 0.36. The titration experiments of GSH brought good linear relationship of intensity, then detection limit was calculate for 70 nM. The proposed mechanism of identifying GSH based on Michael addition through reactions involving the carbon-carbon double bond and intramolecular amino induced spirolactam opening. Meanwhile, the probe RhNM can work in a wide range of pH for 4-10. The probe RhNM was used for endogenous and exogenous imaging in HepG2 cells for glutathione. There was an outstanding intracellular red fluorescence occurred upon the addition of GSH incubated with HepG2 cells. The results show that it has been successfully applied for determination of GSH in diluted serum and bio-imaging of GSH in living cells with low cell toxicity. All the results indicated that the probe can be used as an ultra-sensitive, near-infrared fluorescent chemical sensor for selectively detect GSH in living cell. It has great potential in the live tissues and even in animal imaging.
We report a fluorescent probe for the selective detection of mitochondrial glutathione (GSH). The probe, containing triphenylphosphine as a mitochondrial targeting group, exhibited ratiometric and selective detection of GSH over Cys/Hcy. The probe was used for imaging mitochondrial GSH in living HeLa cells.
A novel naphthalimide-based fluorescent probe employing a sulfonamide unit as a thiol-responsive group is reported. It is capable of efficiently distinguishing GSH from cysteine and homocysteine. Bioimaging shows that it has high selectivity in living cells and can visualize the level of GSH in lysosomes. It is worth mentioning that different groups on the imide unit can affect the selectivity and reaction dynamics of the probe towards thiols.
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 that 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.
In situ
monitoring of intracellular thiol activity in cell growth and function is highly desirable. However, the discriminative detection of glutathione (GSH) from cysteine (Cys) and homocystein (Hcy) and from common amino acids still remains a challenge due to the similar reactivity of the thiol groups in these amino acids. Here we report a novel strategy for selectively sensing GSH by a dual-response mechanism. Integrating two independent reaction sites with a disulfide linker and a thioether function into a fluorescent BODIPY-based chemsensor can guarantee the synergetic dual-response in an elegant fashion to address the discrimination of GSH. In the first synergetic reaction process, the thiol group in GSH, Cys and Hcy induces disulfide cleavage and subsequent intramolecular cyclization to release the unmasked phenol-based BODIPY (discriminating thiol amino acids from other amino acids). In the second synergetic process, upon the substitution of the thioether with the nucleophilic thiolate to form a sulfenyl-BODIPY, only the amino groups of Cys and Hcy, but not that of GSH, undergo a further intramolecular displacement to yield an amino-substituted BODIPY. In this way, we make full use of the kinetically favorable cyclic transition state in the intramolecular rearrangement, and enable photophysical distinction between sulfenyl- and amino-substituted BODIPY for allowingthe discriminative detection of GSH over Cys and Hcyandthiol-lacking amino acidsunder physiological conditions. Moreover, this probe exhibits a distinguishable ratiometric fluorescence pattern generated from the orange imaging channel to the red channel, which proves the differentiation of GSH from Cys and Hcy in living cells.
A new selective fluorescent and colorimetric chemosensor for the detection of GSH was developed. The discrimination of GSH from Cys and Hcy is achieved through two emission channel detection. The detection limit of probe for GSH reached 10 nM (3 ppb). The excellent sensitivity and selectivity of probe allow the selective detection of GSH over Cys and Hcy, which can be visualized colorimetrically and/or fluorescently. The sensitive detection of GSH allowed for convenient measurement of the GSH content in human plasma. The presence of GSH in cells was demonstrated through cell imaging.
Many studies have shown that glutathione (GSH) and cysteine (Cys) / homocysteine (Hcy) levels are interrelated in biological systems. To unravel the complicated biomedical mechanisms by which GSH and Cys/Hcy are involved in various disease states, probes that display distinct signals in response to GSH and Cys/Hcy are highly desirable. In this work, we report a rhodol thioester (1) that responds to GSH and Cys/Hcy with distinct fluorescence emissions in neutral media. Probe 1 reacts with Cys/Hcy to form the corresponding deconjugated spirolactam via a tandem native chemical ligation (NCL) reaction. This intramolecular spirocyclization leads to the “quinone – phenol” transduction of rhodol dyes, and an excited-state intramolecular proton transfer (ESIPT) process between the phenolic hydroxyl proton and the aromatic nitrogen in the benzothiazole unit occurs upon photoexcitation, thus affording 2-(2’-hydroxyphenyl) benzothiazole (HBT) emission (454 nm). In the case of the tripeptide GSH, only transthioesterification takes place removing the intramolecular photo-induced electron transfer (PET) process caused by the electron deficient 4-nitrobenzene moiety giving rise to a large fluorescence enhancement at the rhodol emission band (587 nm). The simultaneous detection of GSH and Cys/Hcy is attributed to the significantly different rates of intramolecular S,N-acyl shift of their corresponding thioester adducts derived from 1. The utility of probe 1 has been demonstrated in various biological systems including serum and cells.
The first "off-on" dual-output fluorescent assay based on reaction-promoted self-assembly approach for glutathione recognition in near infrared region over other relative thiols including cysteine and homocysteine was constructed with high selectivity and large Stocks shift (about 220 nm). The fluorescence enhancement is attributed to the intermolecular interaction, which manipulates the squaraine's aggregates and results in FRET for NIR emission. The sensitivity of the sensing ensemble was further improved in buffer solution containing cationic surfactant.
Due to the biological importances of thiols, such as cysteine, homocysteine and glutathione, the development of optical probes for thiols has been an active research area in recent few years. This critical review focuses on the fluorescent or colorimetric sensors for thiols according to their unique mechanisms between sensors and thiols, including Michael addition, cyclization with aldehyde, cleavage of sulfonamide and sulfonate ester by thiols, cleavage of selenium-nitrogen bond by thiols, cleavage of disulfide by thiols, metal complexes-oxidation-reduction, metal complexes-displace coordination, nano-particles and others (110 references).
Two fluorescent, m-nitrophenol-substituted difluoroboron dipyrromethene dyes have been designed by nucleophilic substitution reaction of 3,5-dichloro-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY). Non-symmetric and symmetric probes i.e. BODIPY 1 (with one nitrophenol group at the position 3) and BODIPY 2 (with two nitrophenol groups at the positions 3 and 5) were applied to ratiometric fluorescent glutathione detection. The detection is based on the two-step nucleophilic aromatic substitution of the nitrophenol groups of the probes by glutathione in buffer solution containing CTAB. In the first stage, probe 1 showed ratiometric fluorescent color change from green (λem = 530 nm) to yellow (λem = 561 nm) due to mono-substitution with glutathione (I561 nm/I530 nm). Addition of excess glutathione caused the second stage of ratiometric fluorescent color change from yellow to reddish orange (λem = 596 nm, I596 nm/I561 nm) due to disubstitution with glutathione. Therefore, different concentration ranges of glutathione (from less to excess) could be rapidly detected by the two-stage ratiometric fluorescenct probe 1 in 5 minutes. While, probe 2 shows single-stage ratiometric fluorescent detection to GSH (from green to reddish orange, I596 nm/I535 nm). Probes 1 and 2 exhibit excellent properties with sensitive, specific colorimetric response and ratiometric fluorescent response to glutathione over other sulfur nucleophiles. Application to cellular ratiometric fluorescence imaging indicated that the probes were highly responsive to intracellular glutathione.
A naphthalimide-based ratiometric fluorescent probe for determining glutathione (GSH) was constructed by installing two oxidized morpholine (e.g. thiomorpholine-S-dioxide and morpholine-N-oxide) components on the “off-to-on” GSH probe. This probe displayed high selectivity towards GSH. As well, the bioimaging application confirmed that this probe was capable of acting as an indicator to monitor the intracellular GSH. Thus, this work provides a promising strategy to construct the ratiometric fluorescent probe.
Biothiols such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) play crucial roles in maintaining redox homeostasis in biological systems. This Mini Review summarizes the most significant current challenges in the field of thiol-reactive probes for biomedical research and diagnostics, emphasizing 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.
A novel turn-on red fluorescent BODIPY-based probe (Probe 1) for the detection of glutathione was developed. Such a probe carrys a para-dinitrophenoxy benzyl pyridinium moiety at the meso position of a BODIPY dye as self-immolative linker. Probe 1 responds selectively to glutathione with the detection limit of 109 nM over other amino acids, common metal ions, reactive oxygen species, reactive nitrogen species, and reactive sulfur species. A novel electrostatic interaction to modulate the SNAr attack of glutathione was believed to play significant role for the observed selective response to glutathione. The cleavage of dinitrophenyl ether by glutathione leads to the production of para-hydroxybenzyl moiety which is able to self-immolate through an intramolecular 1,4-elimination reaction to release the fluorescent BODIPY dye. The low toxic probe has been successfully used to detect mitochondrial glutathione in living cells.
Glutathione (GSH) ultratrace change in mitochondria of cancer cells can mildly and effectively induce cancer cells apoptosis in early stage. Thus, if GSH ultratrace change in mitochondria of cancer cells could be recognized and imaged, it will be beneficial for fundamental research of cancer therapy. There have reported a lot of fluorescent probes for GSH, but the fluorescent probe with ultrasensitivity and high selectivity for the ratio imaging of GSH ultratrace changes in mitochondria of cancer cells is scarce. Herein, based on different reaction mechanism of sulfonamide under different pH, a sulfonamide-based reactive ratiometric fluorescent probe (IQDC-M) was reported for the recognizing and imaging of GSH ultratrace change in mitochondria of cancer cells. The detection limit of IQDC-M for GSH ultratrace change is low to 2.02nM, which is far less than 1.0‰ of endogenic GSH in living cells. And during the recognition process, IQDC-M can emit different fluorescent signals at 520nm and 592nm, which results in it recognizing GSH ultratrace change on ratio mode. More importantly, IQDC-M recognizing GSH ultratrace change specifically occurs in mitochondria of cancer cells because of appropriate water/oil amphipathy (log P) of IQDC-M. So, these make IQDC-M possible to image and monitor GSH ultratrace change in mitochondria during cancer cells apoptosis for the first time.
Developing highly selective fluorescent probes for the determination of thiophenol is very significant due to its toxicity influence in biological system. In this work, we firstly found that the commercial available cyanine dye 3H-Indolium, 2-(2-(2-chloro-3-(2-(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene)-1-cyclohexen-1-yl)ethenyl)-3,3-dimethyl-1-propyl-, iodide had a remarkable capability of determining the amino-substituted thiophenols. Especially, the 2-amino thiophenol induced a fluorescence change of this cyanine dye with the largest intensity in comparison to the 3- and 4-amino thiophenols, implying Cyanine IR-780 can distinguish 2-amino thiophenols from position isomers. Despite the 2-amino thiophenol quenched the fluorescence of cyanine dye, their complex system further played a role in detecting the glutathione with high selectivity over cysteine and homocysteine. It was worth mentioning that similar selectivity can also be observed in living cells.
Amino acids containing the thiol group play significant roles in keeping biological oxidation reduction balance. Due to the important cellular function of biothiols, it is imperative to develop efficient methods to detect biothiols in complicate biological systems. In this work, we developed a cyanine-based fluorescent probe utilizing thioether as a detecting group, which showed the fluorescent "turn-on" response towards GSH and it can be used as a biomarker to determine the cellular GSH. © 2016 SIOC, CAS, Shanghai & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Glutathione (GSH), cysteine (Cys) and homocysteine (Hcy) are small biomolecular thiols that are present in all cells and extracellular fluids of healthy mammals. It is well known that each plays a separate, critically important role in human physiology and that abnormal levels of each are predictive of a variety of different disease states. Although a number of fluorescence-based methods have been developed that can detect biomolecules that contain sulfhydryl moieties, few are able to differentiate between GSH and Cys/Hcy. In this report we demonstrate a broadly applicable approach for the design of fluorescent probes that can achieve this goal. The strategy we employ is to conjugate a fluorescence-quenching 7-nitro-2,1,3-benzoxadiazole (NBD) moiety to a selected fluorophore (Dye) through a sulfhydryl-labile ether linkage to afford nonfluorescent NBD-O-Dye. In the presence of GSH or Cys/Hcy, the ether bond is cleaved with the concomitant generation of both a nonfluorescent NBD-S-R derivative and a fluorescent dye having a characteristic intense emission band (B1). In the special case of Cys/Hcy, the NBD-S-Cys/Hcy cleavage product can undergo a further, rapid, intramolecular Smiles rearrangement to form a new, highly fluorescent NBD-N-Cys/Hcy compound (band B2); because of geometrical constraints, the GSH derived NBD-S-GSH derivative cannot undergo a Smiles rearrangement. Thus, the presence of a single B1 or double B1 + B2 signature can be used to detect and differentiate GSH from Cys/Hcy, respectively. We demonstrate the broad applicability of our approach by including in our studies members of the Flavone, Bodipy and Coumarin dye families. Particularly, single excitation wavelength could be applied for the probe NBD-OF in the detection of GSH over Cys/Hcy in both aqueous solution and living cells.
Very recently, the development of fluorescent probes which are capable of specific imaging of Cys/Hcy/GSH in living cells has attracted great attention. The aim of this review is to highlight the representative examples of the specific thiol fluorescent probes reported from 2013 to 2014. However, in this review, we will restrict the discussion to specific thiol fluorescent probes developed based on organic fluorophores for bioimaging applications.
We have prepared a turn-on fluorescent probe for biothiols based on bromoketo coumarin (KC-Br). The emission intensity of coumarin chromophore is modulated by both heavy atom effect and ICT process. The probe KC-Br is intrinsically nonfluorescent, however, after reacted with thiols, bromide moiety is substituted by -SH group, which elicits a significant fluorescence increase. We surmised the free -NH2 group would further react with carbonyl in Cys/Hcy substituted intermediate product yielding to Schiff base compound KC-Cys/KC-Hcy, but not in compound KC-GSH. The internal charge transfer (ICT) effect has a stronger influence in compound KC-GSH than that in compound KC-Cys/KC-Hcy, resulting compound KC-GSH has stronger fluorescence. Thus, the probe has a good selectivity for GSH over other various biologically relevant species and even other two similar biothiols (Cys/Hcy), and could image GSH in living cells. We expect the design concept presented in this work would be widely used for design of fluorescent probes for distinguishing among of the biothiols.
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.
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
A 4-methoxythiophenol-substituted pyronin dye 1 was exploited as reaction-type fluorescent probe for biothiols Cys/Hcy and GSH. The probe itself is nonfluorescent due to the photoinduced electron transfer (PET) process. The Cys (or Hcy)-induced substitution-rearrangement cascade reaction and GSH-induced substitution reaction with the probe lead to the corresponding aminopyronin and thiopyronin dyes with distinct photophysical properties, enabling Cys/Hcy and GSH to be detected from visible and near-infrared (NIR) emission channels, respectively, in pure PB buffer with relatively fast kinetics and obvious fluorescence turn-on response. Assisted by laser scanning confocal microscope, we also demonstrated that probe 1 could simultaneously sense Cys/Hcy and GSH in B16 cells in multicolor imaging.
Although a lot of mitochondria-targeting biothiol probes have been developed and applied to cellular imaging through thiol-induced disulfide cleavage or Michael addition reactions, relatively few probes assess mitochondrial GSH with high selectivity over Cys and Hcy and with NIR fluorescence capable of noninvasive imaging in biological samples. In order to monitor mitochondrial GSH with low background autofluorescence, we designed a heptamethine-azo conjugate as an NIR fluorescent probe by introducing a tunable lipophilic cation unit as the biomarker for mitochondria and a nitroazo group as the GSH-selective reaction site as well as the fluorescence quencher. The probe exhibited a dramatic Off-On NIR fluorescence response toward GSH with high selectivity over other amino acids including Cys and Hcy. Further application to cellular imaging indicated that the probe was highly responsive to the changes of mitochondrial GSH in cells.
Glutathione (GSH), the most abundant cellular thiol, plays a crucial role in a number of different human pathologies. Near-infrared fluorescence based sensors capable of detecting intracellular GSH in vivo would be useful tools in studies aimed at gaining an understanding of the underlying mechanisms of diseases. In the study described, two near-infrared, cyanine-based fluorescence probes, 1 and 2, containing sulfonamide functional groups were prepared. Evaluation of the fluorescence changes displayed by probe 1, which contains a 2,4-dinitrobenzene sulfonamide group, shows that it can be employed to selectively detect thiols such as GSH, cysteine (Cys) and homocysteine (Hcy). In addition, the results of whole cell experiments demonstrate that 1 is cell-membrane-permeable and that it is applicable for the detection of thiols in living cells. Finally, the response of this probe to thiols can be reversed by treatment with N-methylmaleimide (NMM). Probe 2, which possesses a 5-dimethylaminonaphthyl sulfonamide group, displays high selectivity for GSH over Cys and Hcy and its response to GSH can be reversed using NMM. The potential biological utility of 2, which is cell membrane permeable, was shown by its use in fluorescence imaging of GSH in living cells. Furthermore, probe 2 can also be employed to determine changes in the intracellular levels of GSH modulated by H2O2. The properties of probe 2 enable its use in monitoring GSH in vivo in a mouse model. The results of the studies showed that intravenous injection of 2 into a mouse gives rise to a dramatic image in which strong fluorescence is emitted from various tissues of including liver, kidney, lung, and spleen. Importantly, 2 can be utilized to monitor the depletion of GSH in mouse tissue cells promoted by excessive administration of the painkiller, acetaminophen. The combined results coming from this effort suggest that the new probe will serve as efficient tools for detecting cellular GSH in animals.
A novel compound, 2-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl acrylate (probe L), was designed and synthesized as a highly sensitive and selective fluorescent probe for recognizing and detecting glutathione among cysteine, homocysteine and other amino acids. The structures of related compounds were characterized using IR, NMR and HRMS spectroscopy analysis. The probe is a non-fluorescent compound. On being mixed with glutathione in buffered EtOH:PBS=3:7 solution at pH 7.4, the probe exhibited the blue emission of the pyrazoline at 474nm and a 83-fold enhancement in fluorescence intensity. This probe is very sensitive and displayed a linear fluorescence off-on response to glutathione with fluorometric detection limit of 8.2×10(-8)M. The emission of the probe is pH independent in the physiological pH range. Live-cell imaging of HeLa cells confirmed the cell permeability of the probe and its ability to selectively discriminate GSH from Cys and Hcy in cells. The toxicity of the probe was low in cultured HeLa cells.
A chlorinated coumarin-hemicyanine dye with three potential reaction sites was exploited as fluorescent probe for biothiols. The Cys-induced substitution-rearrangement-cyclization cascade, Hcy-induced substitution-rearrangement cascade, and GSH-induced substitution-cyclizatioin cascade lead to the corresponding amino-coumarin, amino-coumarin-hemicyanine, thiol-coumarin with distinct photophysical properties, enabling Cys and GSH to be selectively detected from different emission channels at two different excitation wavelengths.
In the past few decades, the development of optical probes for thiols has attracted great attention because of the biological importance of the thiol-containing molecules such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). This tutorial review focuses on various thiol detection methods based on luminescent or colorimetric spectrophotometry published during the period 2010-2012. The discussion covers a diversity of sensing mechanisms such as Michael addition, cyclization with aldehydes, conjugate addition-cyclization, cleavage of sulfonamide and sulfonate esters, thiol-halogen nucleophilic substitution, disulfide exchange, native chemical ligation (NCL), metal complex-displace coordination, and nanomaterial-related and DNA-based chemosensors.
We report a ratiometric fluorescent sensor based on monochlorinated BODIPY for highly selective detection of glutathione (GSH) over cysteine (Cys) / homocysteine (Hcy). The chlorine of the monochlorinated BODIPY can be rapidly replaced by thiolate of biothiols through thiolhalogen nucleophilic substitution. The amino groups of Cys/Hcy, but not GSH further replace the thiolate to form amino-substituted BODIPY. The significantly different photophysical properties of sulfur and amino substituted BODIPY enable the discrimination of GSH over Cys and Hcy. The sensor was applied for detection of GSH in living cells.
Despite considerable efforts toward the development of various sophisticated spiropyrans for metal ion sensing, less attention has been paid to organic molecule sensing. One of the major difficulties for detection of organic molecules using a spiropyran is the weak and nonspecific interaction between the spiropyran and the target. Here, we report the synthesis and molecular recognition characterization of two bis-spiropyrans for dipolar molecules and their application to in vivo glutathione (GSH) fluorescent probes. Unlike the mono-spiropyrans, the newly designed bis-spiropyran molecules feature a rigidly maintained molecular cleft and two spiropyran units as binding modules. The molecular recognition is based on multipoint electrostatic interactions and structure complementarity between the opened merocyanine form of the spiropyran and the analyte. It was observed that the spiropyran 1a binds GSH in aqueous solution with high affinity (K = (7.52 +/- 1.83) x 10(4) M(-1)) and shows strong fluorescence emission upon binding. Remarkably, fluorescence output of 1a is not significantly affected by other amino acids and peptides, especially, structurally similar compounds, such as cysteine and homocysteine. Furthermore, fluorescence anisotropy and confocal fluorescent microscopy confirmed that spiropyran 1a is a comparatively good candidate for intracellular delivery and can be accumulated intensively into cells. Thus, 1a can be utilized in vivo as a GSH probe or as a marker to show the level of intracellular GSH.