Fluorescent Thiol Research Articles

Thiol-sensitive probe enables dynamic electrochemical assembly of serum protein for detecting SARS-Cov-2 marker protease in clinical samples

The poor situational awareness about the spreading of the virus especially in the underdeveloped regions calls for novel virus assays of low cost and simple operation. Currently, such assays are exclusively restricted to nucleic acid detection. In this investigation, a virus protein serum assay has been proposed in a one-step and reagent-less route. Specifically, in this assay, the main protease of the virus is targeted by a short probe mimicking its substrate. While the probe-protein interaction brings them together, a fluorescent thiol targeting molecule reacts with the free thiol groups on the target protein near the probe, generating a fluorescence signal proportional to the concentration of the target. This induces an electroactive 2D peptide nano-network on the sensing surface only in the presence of the target protein. The sensitivity of the method is enhanced through potential electrochemical scanning during incubation with serum samples. The successful detection of the virus marker protein in the serum of the infected patients encourages further development of incorporation of this method into clinical practice.

Design, Synthesis, and Characterization of Bis(7-(N-(2-morpholinoethyl)sulfamoyl)benzo[c][1,2,5]oxadiazol-5-yl)sulfane for Nonprotein Thiol Imaging in Lysosomes in Live Cells.

Thiols are critical to cellular structures and functions. Disturbance of cellular thiols has been found to affect cell functions and cause various diseases. Intracellularly, thiols were found unevenly distributed in subcellular organelles. Probes capable of detecting subcellular thiol density in live cells are valuable tools in determining thiols' roles at the subcellular level. The subcellular organelle lysosome is the place where unwanted macromolecules are removed through degradation by hydrolytic enzymes. The degradation also serves as a regulation of a variety of cellular functions such as autophagy, endocytosis, and phagocytosis to maintain cellular homeostasis. Thiols are found to be involved in the lysosomal degradation process. A probe that can detect lysosomal thiols in live cells will be a valuable tool in unveiling the roles of thiols in lysosomes. We would like to report bis(7-(N-(2-morpholinoethyl)sulfamoyl)benzo[c][1,2,5]-oxadiazol-5-yl)sulfane (BISMORX) as a thiol specific fluorogenic agent for live cell nonprotein thiol (NPSH) imaging in lysosomes through fluorescence microscopy. BISMORX itself shows no fluorescence and reacts readily with a NPSH to form a fluorescent thiol adduct with excitation and emission wavelengths of 380 and 540 nm, respectively. BISMORX does not react with compounds containing nucleophilic functional groups other than thiols such as -OH, -NH2, and -COOH. No reaction was observed either when BISMORX was mixed with protein thiols. BISMORX was able to image, quantify, and detect the change of NPSH in lysosomes in live cells. A colocalization experiment with LysoTracker Red DND-99 confirmed that the thiols imaged by BISMORX were indeed lysosomal thiols.

Unbiased Thiol-Labeling and Top-Down Proteomic Analyses Implicate Multiple Proteins in the Late Steps of Regulated Secretion.

Regulated exocytosis enables temporal and spatial control over the secretion of biologically active compounds; however, the mechanism by which Ca2+ modulates different stages of exocytosis is still poorly understood. For an unbiased, top-down proteomic approach, select thiol- reactive reagents were used to investigate this process in release-ready native secretory vesicles. We previously characterized a biphasic effect of these reagents on Ca2+-triggered exocytosis: low doses potentiated Ca2+ sensitivity, whereas high doses inhibited Ca2+ sensitivity and extent of vesicle fusion. Capitalizing on this novel potentiating effect, we have now identified fluorescent thiol- reactive reagents producing the same effects: Lucifer yellow iodoacetamide, monobromobimane, and dibromobimane. Top-down proteomic analyses of fluorescently labeled proteins from total and cholesterol-enriched vesicle membrane fractions using two-dimensional gel electrophoresis coupled with mass spectrometry identified several candidate targets, some of which have been previously linked to the late steps of regulated exocytosis and some of which are novel. Initial validation studies indicate that Rab proteins are involved in the modulation of Ca2+ sensitivity, and thus the efficiency of membrane fusion, which may, in part, be linked to their previously identified upstream roles in vesicle docking.

Thiol-specific fluorogenic agent for live cell non-protein thiol imaging in lysosomes.

Thiol molecules play a significant role in cellular structures and functions. These molecules are distributed in cells unevenly at the subcellular level. Disturbance of cellular thiols has been associated with various diseases and disorders. Probes that are able to detect subcellular thiol density in live cells are valuable tools in determining thiols' roles at the subcellular level. Lysosomes are a subcellular organelle involved in the degradation of macromolecules through the action of proteolytic enzymes. The degradation not only serves as a way to dispose of unwanted macromolecules but also a way to regulate a variety of cellular functions such as autophagy, endocytosis, and phagocytosis to maintain cell homeostasis. A probe that can detect lysosomal thiols in live cells will be useful in unveiling the roles of thiols in lysosomes. Currently, limited probes are available to detect lysosomal thiols in live cells. We would like to report 4,4'-{[7,7'-thiobis(benzo[c][1,2,5]oxadiazole-4,4'-sulfonyl)]bis(oxy))bis(naphthalene-2,7-disulfonicacid) (TBONES) as a thiol-specific fluorogenic agent for lysosomal thiol imaging in live cells through fluorescence microscopy. TBONES exhibits no fluorescence and readily reacts with non-protein thiols to form fluorescent thiol adducts with λex = 400nm and λem = 540nm. No reaction was observed when TBONES was mixed with compounds containing nucleophilic functional groups other than thiols such as -OH, -NH2, and -COOH. No reaction was observed either when TBONES was mixed with protein thiols. When incubated with cells, TBONES selectively and effectively imaged lysosomal thiols in live cells. Imaging of lysosomal thiols was confirmed by a co-localization experiment with LysoTracker™ Blue DND-22.

Investigating ROS, RNS, and H2S-Sensitive Signaling Proteins.

The modification of proteins is a key way to alter their activity and function. Often thiols, cysteine residues, on proteins are attractive targets for such modification. Assuming that the thiol group is accessible then reactions may take place with a range of chemicals found in cells. These may include reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), reactive nitrogen species such as nitric oxide (NO), hydrogen sulfide (H2S), or glutathione. Such modifications often are instrumental to important cellular signaling processes, which ultimately result in modification of physiology of the organism. Therefore, there is a need to be able to identify such modifications. There are a variety of techniques to find proteins which may be altered in this way but here the focus is on two approaches: firstly, the use of fluorescent thiol derivatives and the subsequent use of mass spectrometry to identify the thiols involved; secondly the confirmation of such changes using biochemical assays and genetic mutants. The discussion will be based on the use of two model organisms: firstly the plant Arabidopsis thaliana (both as cell cultures and whole plants) and secondly the nematode worm Caenorhabditis elegans. However, these tools, as described, may be used in a much wider range of biological systems, including human and human tissue cultures.

Thiol Specific and Mitochondria Selective Fluorogenic Benzofurazan Sulfide for Live Cell Nonprotein Thiol Imaging and Quantification in Mitochondria.

Cellular thiols are divided into two major categories: nonprotein thiols (NPSH) and protein thiols (PSH). Thiols are unevenly distributed inside the cell and compartmentalized in subcellular structures. Most of our knowledge on functions/dysfunctions of cellular/subcellular thiols is based on the quantification of cellular/subcellular thiols through homogenization of cellular/subcellular structures followed by a thiol quantification method. We would like to report a thiol-specific mitochondria-selective fluorogenic benzofurazan sulfide {7,7'-thiobis( N-rhodamine-benzo[c][1,2,5]oxadiazole-4-sulfonamide) (TBROS)} that can effectively image and quantify live cell NPSH in mitochondria through fluorescence intensity. Limited methods are available for imaging thiols in mitochondria in live cells especially in a quantitative manner. The thiol specificity of TBROS was demonstrated by its ability to react with thiols and inability to react with biologically relevant nucleophilic functional groups other than thiols. TBROS, with minimal fluorescence, formed strong fluorescent thiol adducts (λex = 550 nm, λem = 580 nm) when reacting with NPSH confirming its fluorogenicity. TBROS failed to react with PSH from bovine serum albumin and cell homogenate proteins. The high mitochondrial thiol selectivity of TBROS was achieved by its mitochondria targeting structure and its higher reaction rate with NPSH at mitochondrial pH. Imaging of mitochondrial NPSH in live cells was confirmed by two colocalization methods and use of a thiol-depleting reagent. TBROS effectively imaged NPSH changes in a quantitative manner in mitochondria in live cells. The reagent will be a useful tool in exploring physiological and pathological roles of mitochondrial thiols.

Preparation of Fluorescent Thiol Group‐Functionalized Silica Microspheres for the Detection and Removal of Silver Ions in Aqueous Solutions

We demonstrate that silica microspheres can act as a sensitive fluorescent sensor and adsorbent of Ag+ in aqueous media. These thiol‐functionalized silica microspheres are doped with quantum dots (QDs) using organosilane chemistry in a one‐step preparation. Ligand exchange takes place between the thiolated organosilane and acid‐capped QDs, making the doping easy. Ag+ adsorption by the silica microspheres causes the decrease of fluorescence intensity of the QDs. The detection limit for Ag+ is found to be 10 μmol/L. The abundance of thiol groups on the surface of the microspheres could effectively remove Ag+ through strong interaction. When microspheres with a diameter of 1.1 μm are used as the adsorbents, the adsorption capacity for Ag+ reached 102 mg/g. This excellent adsorption ability is due to the abundance of thiol groups that act as the active sites, facilitating the adsorption of the massive metal ions on the surface of the microspheres. Furthermore, the adsorption isotherm data follows the Freundlich model. The structure and content of the silica microspheres were investigated by scanning and high‐resolution transmission electron microscopy, energy dispersive X‐ray spectroscopy, and Raman analysis, and the fluorescence properties were characterized by fluorescence microscopy.

Non-protein thiol imaging and quantification in live cells with a novel benzofurazan sulfide triphenylphosphonium fluorogenic compound.

Thiols (-SH) play various roles in biological systems. They are divided into protein thiols (PSH) and non-protein thiols (NPSH). Due to the significant roles thiols play in various physiological/pathological functions, numerous analytical methods have been developed for thiol assays. Most of these methods are developed for glutathione, the major form of NPSH. Majority of these methods require tissue/cell homogenization before analysis. Due to a lack of effective thiol-specific fluorescent/fluorogenic reagents, methods for imaging and quantifying thiols in live cells are limited. Determination of an analyte in live cells can reveal information that cannot be revealed by analysis of cell homogenates. Previously, we reported a thiol-specific thiol-sulfide exchange reaction. Based on this reaction, a benzofurazan sulfide thiol-specific fluorogenic reagent was developed. The reagent was able to effectively image and quantify total thiols (PSH+NPSH) in live cells through fluorescence microscopy. The reagent was later named as GUALY's reagent. Here we would like to report an extension of the work by synthesizing a novel benzofurazan sulfide triphenylphosphonium derivative [(((7,7'-thiobis(benzo[c][1,2,5]oxadiazole-4,4'-sulfonyl))bis(methylazanediyl))bis(butane-4,1-diyl))bis(triphenylphosphonium) (TBOP)]. Like GUALY's reagent, TBOP is a thiol-specific fluorogenic agent that is non-fluorescent but forms fluorescent thiol adducts in a thiol-specific fashion. Different than GUALY's reagent, TBOP reacts only with NPSH but not with PSH. TBOP was effectively used to image and quantify NPSH in live cells using fluorescence microscopy. TBOP is a complementary reagent to GUALY's reagent in determining the roles of PSH, NPSH, and total thiols in thiol-related physiological/pathological functions in live cells through fluorescence microscopy. Graphical Abstract Live cell imaging and quantification of non-protein thiols by TBOP.

Discovery of selective cystathionine β-synthase inhibitors by high-throughput screening with a fluorescent thiol probe.

A high-throughput assay was developed to identify inhibitors of cystathionine β-synthase (CBS), which is one of three key enzymes involved in H2S biosynthesis. Screening of 6491 natural compounds identified several selective CBS inhibitors, which suppressed the proliferation of HT29 cancer cells, with IC50 values of less than 10 μM.

Rapid labeling of amino acid neurotransmitters with a fluorescent thiol in the presence of o-phthalaldehyde.

LIF detection often requires labeling of analytes with fluorophores; and fast fluorescent derivatization is valuable for high-throughput analysis with flow-gated CE. Here, we report a fast fluorescein-labeling scheme for amino acid neurotransmitters, which were then rapidly separated and detected in flow-gated CE. This scheme was based on the reaction between primary amines and o-phthalaldehyde in the presence of a fluorescent thiol, 2-((5-fluoresceinyl)aminocarbonyl)ethyl mercaptan (FACE-SH). The short reaction time (<30 s) was suited for on-line mixing and derivatization that was directly coupled with flow-gated CE for rapid electrophoretic separation and sensitive LIF detection. To maintain the effective concentration of reactive FACE-SH, Tris(2-carboxyethyl)phosphine was added to the derivatization reagents to prevent thiol loss due to oxidation. This labeling scheme was applied to the detection of neurotransmitters by coupling in vitro microdialysis with online derivatization and flow-gated CE. It is also anticipated that this fluorophore tagging scheme would be valuable for on-chip labeling of proteins retained on support in SPE.

A fluorescence high throughput screening method for the detection of reactive electrophiles as potential skin sensitizers

Skin sensitization is an important toxicological end-point in the risk assessment of chemical allergens. Because of the complexity of the biological mechanisms associated with skin sensitization, integrated approaches combining different chemical, biological and in silico methods are recommended to replace conventional animal tests. Chemical methods are intended to characterize the potential of a sensitizer to induce earlier molecular initiating events. The presence of an electrophilic mechanistic domain is considered one of the essential chemical features to covalently bind to the biological target and induce further haptenation processes. Current in chemico assays rely on the quantification of unreacted model nucleophiles after incubation with the candidate sensitizer. In the current study, a new fluorescence-based method, ‘HTS-DCYA assay’, is proposed. The assay aims at the identification of reactive electrophiles based on their chemical reactivity toward a model fluorescent thiol. The reaction workflow enabled the development of a High Throughput Screening (HTS) method to directly quantify the reaction adducts. The reaction conditions have been optimized to minimize solubility issues, oxidative side reactions and increase the throughput of the assay while minimizing the reaction time, which are common issues with existing methods. Thirty-six chemicals previously classified with LLNA, DPRA or KeratinoSens™ were tested as a proof of concept. Preliminary results gave an estimated 82% accuracy, 78% sensitivity, 90% specificity, comparable to other in chemico methods such as Cys-DPRA. In addition to validated chemicals, six natural products were analyzed and a prediction of their sensitization potential is presented for the first time.

A highly selective and sensitive fluorescent thiol probe through dual-reactive and dual-quenching groups.

A new fluorescent probe installed with dual-reactive and dual-quenching groups was rationally designed for highly selective and sensitive sensing of biothiols. The sensitivity of the probe toward thiols was significantly improved by dual-quenching effects. Furthermore the selectivity of the probe was also greatly enhanced by installation of dual-reactive groups.

Rapid and thiol-specific high-throughput assay for simultaneous relative quantification of total thiols, protein thiols, and nonprotein thiols in cells.

Thiol groups in biological molecules play a significant role in various physiological functions and pathological conditions. Thiols are divided into two major groups: protein thiols and nonprotein thiols. Numerous methods have been reported for thiol assays. Most of these methods have been developed for glutathione, the principal nonprotein thiol, despite the fact that cellular protein thiols are more abundant than glutathione. Further, these methods usually involve a process of biological sample preparation followed by a separation method, and they are time-consuming. We reported previously a series of thiol-specific fluorogenic benzofurazan sulfides. These nonfluorescent benzofurazan sulfides react rapidly and specifically with a thiol to form a strong fluorescent thiol adduct. The rapid reaction, thiol-specific and fluorogenic nature of the sulfides successfully yielded an application of one of the sulfides for relative quantitation of total thiols in live cells through fluorescence microscopy. In this work, we employed the same compound to develop the first high-throughput method for simultaneous monitoring of protein thiols, nonprotein thiols, and total thiols in cells in a 96-well plate on a fluorescence microplate reader at λex = 430 nm and λem = 520 nm, respectively. The method is rapid and sensitive, and has been validated by an HPLC thiol assay method. The method can detect thiols with cell concentrations as low as 500 cells/well. We also demonstrated that the method can readily monitor changes in cellular thiol levels. Although the method cannot provide an absolute quantification for thiols because fluorescence intensity of different thiol adducts varies, it provides an accurate measurement of relative quantification, relative to the control. The method will be a valuable tool in thiol-related biomedical/pharmaceutical research.

A turn-on green fluorescent thiol probe based on the 1,2-addition reaction and its application for bioimaging

The nucleophilic nature of thiols is used to develop a novel, sensitive, and highly effective sensor. In this work, we developed a new fluorescent probe for thiols based on fluorescein and maleic anhydride. Among the tested amino acid, only GSH, Cys, Hcy, could turn on the fluorescence emission suggesting that the system was a highly selective sensor for thiols. As a typical biological thiol, GSH was used for further examining the fluorescence response of probe. It is found that probe can detect GSH quantitatively with a detection limit as low as 0.026μM in aqueous/DMSO solution. Moreover its potential for biological applications was confirmed by employing it for fluorescence imaging of thiol in living cells.

Hydrogen sulfide deactivates common nitrobenzofurazan-based fluorescent thiol labeling reagents.

Sulfhydryl-containing compounds, including thiols and hydrogen sulfide (H2S), play important but differential roles in biological structure and function. One major challenge in separating the biological roles of thiols and H2S is developing tools to effectively separate the reactivity of these sulfhydryl-containing compounds. To address this challenge, we report the differential responses of common electrophilic fluorescent thiol labeling reagents, including nitrobenzofurazan-based scaffolds, maleimides, alkylating agents, and electrophilic aldehydes, toward cysteine and H2S. Although H2S reacted with all of the investigated scaffolds, the photophysical response to each scaffold was significantly different. Maleimide-based, alkylating, and aldehydic thiol labeling reagents provided a diminished fluorescence response when treated with H2S. By contrast, nitrobenzofurazan-based labeling reagents were deactivated by H2S addition. Furthermore, the addition of H2S to thiol-activated nitrobenzofurazan-based reagents reduced the fluorescence signal, thus establishing the incompatibility of nitrobenzofurazan-based thiol labeling reagents in the presence of H2S. Taken together, these studies highlight the differential reactivity of thiols and H2S toward common thiol-labeling reagents and suggest that sufficient care must be taken when labeling or measuring thiols in cellular environments that produce H2S due to the potential for both false-positive and eroded responses.

A Unique Family of Rigid Analogues of the GFP Chromophore with Tunable Two‐Photon Action Cross‐Sections for Biological Imaging

Conformationally restricted analogues of the GFP chromophore have been synthesized. The spectroscopic properties of GCTPOC, a unique family with tunable two-photon action cross-sections, were investigated. GCTPOC could be used as a robust two-photon platform for development of a two-photon fluorescent thiol sensor, which could stain endogenous thiols both in vitro and in vivo.

A fluorescence-based assay for the measurement of S-adenosylhomocysteine hydrolase activity in biological samples

The methylation of DNA, RNA, and proteins plays crucial roles in numerous biological processes, including epigenetic control, virus replication, and cell differentiation. In mammals, the rate-limiting step of the S-adenosylmethionine-dependent methylation process is exclusively controlled by S-adenosylhomocysteine (S-AdoHcy) hydrolase (SAHH). SAHH hydrolyzes S-AdoHcy to adenosine and homocysteine (Hcy) and is therefore a potential therapeutic target for various diseases, including cancer, malaria, and viral diseases. However, a simple and highly sensitive assay for the evaluation of SAHH activity, particularly for drug discovery, had not yet been developed. Here we present the development of a fluorescence-based assay for the measurement of SAHH activity in biological samples. We combined the advantages of the detection of fluorescent thiol groups in Hcy by ThioGlo1 with the S-AdoHcy-driven enzyme-coupled reaction. Our results confirmed the reliability of the proposed assay for the measurement of the SAHH activity of purified SAHH and showed the potential of this assay for the measurement of the SAHH activity of biological samples. Therefore, the proposed SAHH activity assay may be utilized in clinical laboratories and in high-throughput screenings for the identification of new SAHH inhibitors with potentially beneficial effects on numerous pathologies.

Benzofurazan Sulfides for Thiol Imaging and Quantification in Live Cells through Fluorescence Microscopy

Thiol groups play a significant role in various cellular functions. Cellular thiol concentrations can be affected by various physiological or pathological factors. A fluorescence imaging agent that can effectively and specifically image thiols in live cells through fluorescence microscopy is desirable for live cell thiol monitoring. Benzofurazan sulfides 1a-1e were synthesized and found to be thiol specific fluorogenic agents except 1d. They are not fluorescent but form strong fluorescent thiol adducts after reacting with thiols through a sulfide-thiol exchange reaction. On the other hand, they exhibit no reaction with other biologically relevant nucleophilic functional groups such as -NH(2), -OH, or -COOH revealing the specificity for the detection of thiols. Sulfide 1a was selected to confirm its ability to image cellular thiols through fluorescence microscopy. The compound was demonstrated to effectively image and quantify thiol changes in live cells through fluorescence microscopy using 430 and 520 nm as the excitation and emission wavelengths, respectively. The quantification results of total thiol in live cells obtained from fluorescence microscopy were validated by an high-pressure liquid chromatography/ultraviolet (HPLC/UV) total thiol assay method. The reagents and method will be of a great value to thiol redox-related research.

Design of OFF/ON fluorescent thiol probes based on coumarin fluorophore

This paper presents a series of first- and second-generation click-modified coumarin-based fluorescent probes for thiols. These molecules demonstrate high turn-on fluorescent response and good selectivity towards aromatic thiols over other relevant reactive sulfur species, reactive oxygen species and common nucleophiles. Moreover, probe 1a can detect thiols in the reduced rabbit plasma sample. Therefore, this approach provides a particularly impressive tool for detecting thiol in biological systems.

Analysis of alpha‐1‐acid glycoprotein isoforms using CE‐LIF with fluorescent thiol derivatization

The analysis of glycoprotein isoforms is of high interest in the biomedical field and clinical chemistry. Many studies have demonstrated that some glycoprotein isoforms could serve as biomarkers for several major diseases, such as cancers and vascular diseases, among others. Capillary zone electrophoresis (CZE) is a well-established technique to separate glycoprotein isoforms, however, it suffers from limited sensitivity when UV-Vis detection is used. On the other hand, with laser-induced fluorescence (LIF) detection, derivatization reaction to render the proteins fluorescent can destroy the resolution of the isoforms. In this work, a derivatization procedure through the thiol groups of glycoproteins using either 5-(iodoacetamide) fluorescein (5-IAF) or BODIPY iodoacetamide is presented with the model protein of alpha-1-acid glycoprotein (AGP). The derivatization process presented enabled high-resolution analysis of AGP isoforms by CZE-LIF. The derivatization procedure was successfully applied to label AGP from samples of serum and secretome of artery tissue, enabling the separation of the AGP isoforms by CE-LIF in natural samples at different concentration levels.