Biological Thiols Research Articles

TDDFT study on a fluorescent probe for distinguishing analogous thiols based on smiles rearrangement

The complete excited-state sensing mechanism of a fluorescent probe capable of distinguishing cysteine/homocysteine and glutathione from analogous biological thiols has been investigated. Using a TDDFT method, the nature of the fluorescence differences in the detection of thiols by the probe has been explained at the molecular level. Calculation results imply that the probe undergoes photoinduced electron transfer (PET) from the fluorophore to the nitrobenzooxadiazole (NBD)-based acceptor in the excited state. In the presence of a thiol, the NBD moiety is cleaved and the red fluorescence emission of the fluorophore is enhanced through inhibition of the PET process. The sulfur-substituted NBD-thiol product is predisposed to undergo excited-state torsion, leading to fluorescence quenching. However, for cysteine and homocysteine, their appropriate distances lead to Smiles rearrangements with relatively low activation energies (26.60 kJ/mol and 42.94 kJ/mol, respectively) and the emission of a distinct green fluorescence at ambient temperature. It has been theoretically confirmed that the distance between two reactive sites, such as sulfhydryl and amino moieties, can be used to distinguish different thiols, thus providing rational support for the control of fluorescence activity and the design of probe molecules.

Discrimination and Quantification of Glutathione by Cu+-Based Nanozymes.

Glutathione (GSH) is the most abundant low-molecular-weight biological thiol in vivo and has been linked to several diseases. The accurate quantification of GSH is therefore crucial for disease diagnosis and monitoring. In this study, we prepared self-assembled Cu(I)-Cys (cysteine) nanozymes through a two-step procedure. The Cu(I)-Cys nanoparticles exhibited peroxidase-mimicking activity. Upon the addition of H2O2, they were able to oxidize 3,3,5,5-tetramethylbenzidine (TMB) into oxTMB, resulting in a measurable increase in UV-Vis absorption at 655 nm. However, in the presence of GSH, oxTMB was reduced back to TMB, leading to a decrease in UV-Vis absorption at 655 nm. By utilizing these changes in the absorption intensity, we achieved the sensitive detection of GSH with a detection limit of 2.13 μM. Moreover, taking advantage of the different peroxidase-mimicking activities of Cu(I)-Cys nanoparticles at various pH values, a sensor array with Cu(I)-Cys nanoparticles at pH 4 and pH 5 was constructed. The discrimination of GSH among Cys and ascorbic acid was achieved and the practicability of the sensor array in human serum was validated. This novel approach holds significant promise for the precise discrimination and quantification of GSH and its potential applications in disease diagnosis and therapeutics.

Tumor-Targeting Probe for Dual-Modal Imaging of Cysteine In Vivo.

Cysteine (Cys) is a crucial biological thiol that has a vital function in preserving redox homeostasis in organisms. Studies have shown that Cys is closely related to the development of cancer. Thus, it is necessary to design an efficient method to detect Cys for an effective cancer diagnosis. In this work, a novel tumor-targeting probe (Bio-Cy-S) for dual-modal (NIR fluorescence and photoacoustic) Cys detection is designed. The probe exhibits high selectivity and sensitivity toward Cys. After reaction with Cys, both NIR fluorescence and photoacoustic signals are activated. Bio-Cy-S has been applied for the dual-modal detection of Cys levels in living cells, and it can be used to distinguish normal cells from cancer cells by different Cys levels. In addition, the probe is capable of facilitating dual-modal imaging for monitoring changes in Cys levels in tumor-bearing mice. More importantly, the excellent tumor-targeting ability of the probe greatly improves the signal-to-noise ratio of imaging. To the best of our knowledge, this is the first Cys probe to combine targeting and dual-modal imaging performance for cancer diagnosis.

Fluorescent Coumarin-based Probe for Detection of Biological Thiols

Abstract: Nature-derived thiols such as cysteine, homocysteine, and reduced glutathione play diverse and important roles in both plant and animal cells. In plants, they maintain redox homeostasis and affect photosynthesis and signal transduction processes. In animals, changes in the levels of biothiols have been linked to cardiovascular disease, Alzheimer's disease, neuropsychiatric disorders, or stroke. The detection of biothiols in biological samples is, therefore, very important. For this purpose, rapid, sensitive, and non-invasive methods for their sensing in living cells are highly desirable. : This perspective reviews the past five years of advances in coumarin fluorescent probes for biothiol detection. The design of the probes, the mechanisms of thiol detection, and the applications in live cell imaging are presented. The classification of probes based on the coumarin backbone was made on the basis of the mode of their interactions with the analyte.

A ratiometric fluorescent probe for the detection of biological thiols based on a new supramolecular design

A new ratiometric fluorescent probe is designed and prepared based on the concept of supramolecular encapsulation and dye competition. This supramolecular probe is based on two commercially-available dyes, one common guest and a simple-to-synthesize host. Fluorescence spectroscopy confirms that the supramolecular probe is capable of detecting thiols quantitatively with a broad linear region in phosphate buffered saline or fetal bovine serum. Mechanistic study shows a reaction between thiol specie and the guest to alter the distribution of encapsulated dyes. The supramolecular probes are demonstrated to quantitatively detect extracellular biological thiols by plate reader, which shows it keeps its effectiveness in complex buffered systems.

A naphthalimide-based fluorescent probe with mitochondria targeting for GSH sensing and cancer cell recognition

GSH is an important biological thiol which involves many biological processes, and also an biomarker for cancer. So it's urgent to develop fluorescent probes with excellent sensitivity and high selectivity for GSH detection. Herein, we develop a new GSH-responsive fluorescent probe TPP-Naph-SS-Fc based on PET (photoinduced electron transfer) mechanism. TPP-Naph-SS-Fc contains naphthalimide fluorephore, the ferrocene quencher, the disulfide cleavage bond and the triphenylphosphine mitochondrial localizer. It shows a fluorescence turn-on response toward GSH for cleavage the disulfide bond, which result in the blocking of PET process. Especially, this probe can localize in mitochondria and make real-time imaging of GSH, also demonstrate the ability to distinguish between cancerous and normal cells.

Bioimaging of glutathione variation for early diagnosis of hepatocellular carcinoma using a liver-targeting ratiometric near-infrared fluorescent probe.

Reliable biomarkers are crucial for early diagnosis of diseases and precise therapy. Biological thiols (represented by glutathione, GSH) play vital roles in the antioxidant defense system for maintaining intracellular redox homeostasis in organisms. However, the aberrant variation in the cellular concentration of GSH correlates with diverse diseases including cancer. Here, a ratiometric near-infrared fluorescent probe CyO-Disu is constructed for the specific sensing of GSH variation in live cells and mice models of hepatic carcinoma (HCC). CyO-Disu features three key elements, a response moiety of bis(2-hydroxyethyl) disulfide, a near-infrared fluorescence signal transducer of heptamethine ketone cyanine, and a targeting moiety of D-galactose. By virtue of its liver-targeting capability, CyO-Disu was utilized for evaluating GSH fluctuations in primary and metastatic hepatoma living cells. To evaluate the efficacy of CyO-Disuin vivo, orthotopic HCC and pulmonary metastatic hepatoma mice models were employed for GSH imaging using two-dimensional and three-dimensional fluorescence molecular tomographic imaging systems. The bioimaging results offered direct evidence that GSH displayed varied concentrations during the progression of HCC. Therefore, the as-synthesized probe CyO-Disu could serve as a potential powerful tool for the early diagnosis and precise treatment of HCC using GSH as a reliable biomarker.

A dual-response NIR fluorescent probe for separately and continuously recognizing H2S and Cys with different fluorescence signals and its applications.

Given the significant interactions between hydrogen sulfide (H2S) and cysteine (Cys) in organisms, a dual-site multi-purpose fluorescent probe (Cy-NP) for H2S and Cys was synthesized. Cy-NP is composed of two fluorophores: naphthalimide that emits in the visible region of 500-600 nm, and cyanine dye that emits in the NIR region of 700-800 nm. Cy-NP showed admirable sensitivity and selectivity for identifying H2S and Cys by fluorescent signals with limits of detection as low as 0.15 μM and 1.4 μM, respectively. Furthermore, other biological thiols (especially GSH and Hcy) showed no positive response to Cy-NP compared with H2S and Cys. The chemical mechanism of Cy-NP with H2S and Cys in DMF/PBS (1/1, v/v, pH = 7.4) solution was verified by HRMS and DFT calculations. Further, Cy-NP was successfully applied to monitor H2S released in raw meat and adapted to detect H2S and Cys in MCF-7 cells independently and continuously.

Photoswitchable spiropyran molecules for specific sensing of thiols and fluorescent inks

This work presents promising photoswitching-based spiropyran molecules as fluorescent probes, which can be used not only for sensitive and selective detection of biological thiols but also as fluorescent inks for information encryption.

Chemoselective bicyclobutane-based mass spectrometric detection of biological thiols uncovers human and bacterial metabolites.

Sulfur is an essential element of life. Thiol-containing metabolites in all organisms are involved in the regulation of diverse biological processes. Especially, the microbiome produces bioactive metabolites or biological intermediates of this compound class. The analysis of thiol-containing metabolites is challenging due to the lack of specific tools, making these compounds difficult to investigate selectively. We have now developed a new methodology comprising bicyclobutane for chemoselective and irreversible capturing of this metabolite class. We utilized this new chemical biology tool immobilized onto magnetic beads for the investigation of human plasma, fecal samples, and bacterial cultures. Our mass spectrometric investigation detected a broad range of human, dietary and bacterial thiol-containing metabolites and we even captured the reactive sulfur species cysteine persulfide in both fecal and bacterial samples. The described comprehensive methodology represents a new mass spectrometric strategy for the discovery of bioactive thiol-containing metabolites in humans and the microbiome.

Electrochemical reactions of highly active nitroxyl radicals with thiol compounds.

Nitroxyl radicals are known to electrochemically oxidize thiols as well as alcohols and amines. In this study, a preliminary investigation of the electrochemical reaction of thiols with 9-azabicyclo[3.3.1]nonane N-oxyl (ABNO), 2-azaadamantane N-oxyl (AZADO), and nortropine N-oxyl (NNO), which are highly active due to their bicyclo structures, for use in electrochemical analysis was performed and the results were compared with those for a typical nitroxyl radical compound, 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO). Mercaptopropane sulfonic acid (MPS) was used as a model compound to investigate the electrochemical response in aqueous solution. In addition, electrochemical detection of glutathione, a biological thiol molecule, was performed.

Biochemical control of the mitochondrial protein MitoNEET by biological thiols and lipid-derived electrophiles

MitoNEET is a mitochondrial [2Fe-2S]-containing protein known for its involvement in cellular metabolism, iron regulation, and oxidative stress. The protein has been associated with diseases ranging from diabetes to Parkinson's disease, prompting the development of compounds designed to target mitoNEET selectively. Unfortunately, drug development is limited due to a lack of mechanistic understanding of how mitoNEET integrates into pathophysiological processes, and biological compounds that govern mitoNEET function are still ill-defined. We demonstrate an oxygen-dependent reaction with biological thiols catalyzed by mitoNEET. Specifically, we observed that mitoNEET converts L-cysteine to cystine. Finally, we showed that reduced glutathione (L-GSH) regulates the reactivity of two lipid-derived biomarkers of oxidative stress, 4-HNE and 4-ONE, towards mitoNEET. We found that exposure to L-GSH before treatment with either of the electrophilic aldehydes prevents the formation of a covalently linked mitoNEET dimer. Meanwhile, adding L-GSH after electrophile treatment recovers mitoNEET from the 4-HNE induced modification but not from the modification induced by 4-ONE. These results indicate a possible role for mitoNEET in thiol-mediated oxidative stress and may facilitate the development of drugs designed to modulate mitoNEET activity to improve pathophysiological states.

Fluorescent Sensing of Glutathione and Related Bio-Applications.

Glutathione (GSH), as the most abundant low-molecular-weight biological thiol, plays significant roles in vivo. Abnormal GSH levels have been demonstrated to be related to the dysfunction of specific physiological activities and certain kinds of diseases. Therefore, the sensing of GSH is emerging as a critical issue. Cancer, with typical high morbidity and mortality, remains one of the most serious diseases to threaten public health. As it is clear that much more concentrated GSH is present at tumor sites than at normal sites, the in vivo sensing of GSH offers an option for the early diagnosis of cancer. Moreover, by monitoring the amounts of GSH in specific microenvironments, effective diagnosis of ROS levels, neurological diseases, or even stroke has been developed as well. In this review, we focus on the fluorescent methodologies for GSH detection, since they can be conveniently applied in living systems. First, the fluorescent sensing methods are introduced. Then, the principles for fluorescent sensing of GSH are discussed. In addition, the GSH-sensing-related biological applications are reviewed. Finally, the future opportunities in in the areas of fluorescent GSH sensing-in particular, fluorescent GSH-sensing-prompted disease diagnosis-are addressed.

Spherical Nucleic Acid Probe Based on 2′-Fluorinated DNA Functionalization for High-Fidelity Intracellular Sensing

Traditional spherical nucleic acids (SNAs) based on gold nanoparticles (AuNPs) assembled through Au-S covalent bonds are widely used in DNA-programmable assembly, biosensing, imaging, and therapeutics. However, biological thiols and other chemical substances can break the Au-S bonds and cause response distortion during the application process, specifically in cell environments. Herein, we report a new type of SNAs based on 2'-fluorinated DNA-functionalized AuNPs with excellent colloidal stability under high salt conditions (up to 1 M NaCl) and over a broad pH range (1-14), as well as resistance to biothiols. The fluorinated spherical nucleic acid probe (Au/FDNA probe) could detect targeted cancer cells with high fidelity. Compared to the traditional thiolated DNA-functionalized AuNP probe (Au-SDNA probe), the Au/FDNA probe exhibited a higher sensitivity to the target and a lower signal-to-background ratio. Furthermore, the Au/FDNA probe could discriminate target cancer cells in a mixed culture system. Using the proposed FDNA functionalization method, previously developed SNAs based on AuNPs could be directly adapted, which might open a new avenue for the design and application of SNAs.

Coumarin-Ensembled Vanadium(V) Compounds and Their Affinity Studies Toward Biological Thiols Probed by Fluorescence Spectroscopy.

Fluorescence spectroscopic studies of a pair of new oxido-vanadium(V) compounds with biological thiols, such as homocysteine (Hcy), cysteine (Cys), and glutathione (GSH), have been investigated in this article. Despite notable progress in vanadium-thiol chemistry, no attention has been paid to exploring vanadium-based optical probes to study their interaction with biothiols. For this purpose, two oxido-vanadium(V) compounds, 1 and 2, have been prepared involving a tridentate ONO donor-based luminescent coumarin-derived ligand. Single crystal X-ray diffraction analysis, NMR (1 H, 13 C, and 51 V) spectroscopy, XPS, and DFT calculations have been used to establish their identities. The vanadium center in these compounds has a distorted octahedral environment. In compound 2, a methanol molecule is coordinated to the vanadium(V) center in the trans position of the terminal oxido moiety. The latter exerts a strong trans-labilizing influence on the coordinating methanol. Both 1 and 2 are weakly fluorescent. Photophysical investigations of the vanadium complexes in aqueous media at physiological pH (7.4) in the presence of various biothiols and amino acids showed significant fluorescence enhancement (83-fold) of the vanadium complexes, specifically with Hcy. The specific affinity of the complexes for Hcy remained unchanged even in the presence of other biothiols and amino acids. Kinetic investigation reveals pseudo-first order behavior of the compound with Hcy. Mechanistic studies have manifested that Hcy-induced reduction triggers the decomplexation of the vanadium compound, followed by hydrolysis and subsequent cyclization. Time-correlated single photon counting suggested that the radiative rate constant (kr ) of 1 and 2 in the presence of Hcy serves as the prime factor for the fluorescence enhancement of the medium. Compound 1 has been tested efficiently for Hcy measurement in blood plasma rendering it suitable for practical applications.

A benzotriazole-coumarin derivative as a turn-on fluorescent probe for highly efficient and selective detection of homocysteine and its bioimaging application

Biological thiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) play essential roles in physiological and pathological processes, abnormal concentration of biothiols could lead to various diseases. Due to the structure similarity, it’s difficult to design fluorescence probes to selectively detect the common biothiols. Herein, a series of fluorescent molecules based on benzotriazole and coumarin moieties were designed and synthesized. Among these molecules, (E)-3-(2-(1H-benzo[d][1–3]triazol-1-yl)vinyl)-4-chloro-7-(diethylamino)–2H-chromen-2-one (BTAC (e)) exhibits high selectivity towards Hcy. When different biothiols were introduced, the probe BTAC(e) could react with Hcy by a thiol-halogen SNAr nucleophilic substitution-rearrangement reaction and exhibited enhancement in fluorescence. With the introduction of benzotriazole, BTAC(e) could respond to Hcy more quickly. At the same time, this probe exhibited excellent selectivity towards Hcy, it turns out that Cys and GSH have little influence on the fluorescence of BTAC(e). Under the optimized conditions, the fluorescence intensity has a good linear relationship with the concentration of Hcy in the range of 2.0 × 10-7 to 7.0 × 10-6 mol L-1 and the limit of detection for Hcy is 1.55 × 10-8 mol L-1. Moreover, probe BTAC (e) has been applied for imaging Hcy in living cells and the experimental result is satisfactory.

HPLC Study of Product Formed in the Reaction of NBD-Derived Fluorescent Probe with Hydrogen Sulfide, Cysteine, N-acetylcysteine, and Glutathione

Hydrogen sulfide (H2S) and its bioderivatives analogs, such as L-cysteine (L-Cys) and glutathione (GSH), are ubiquitous biological thiols in the physiological and pathological processes of living systems. Their aberrant concentration levels are associated with many diseases. Although several NBD-based fluorescence probes have been developed to detect biological thiols, the HPLC-detection of H2S, GSH, L-Cys, and N-acetylcysteine-specific products has not been described. Herein, a novel NBD-derived pro-coumarin probe has been synthesized and used to develop a new strategy for the triple mode detection of H2S and such thiols as GSH, L-Cys, and NAC. Hydrogen sulfide and those biothiols at physiological pH release fluorescent coumarin from the probe and cause a significant fluorescence enhancement at 473 nm. The appropriate NBD-derived product for H2S, L-Cys, GSH, and NAC has a different color and retention time that allows distinguishing these biological thiols meaning the probe has a great possibility in the biological application. Fluorescent imaging combined with colorimetric and HPLC detection of H2S/biothiol-specific product(s) brings a potential tool for confirming the presence of biological thiols and determining concentrations in various aqueous biological samples.

DNA Nanostructure-Guided Plasmon Coupling Architectures

DNA Nanostructure-Guided Plasmon Coupling Architectures

A novel near infrared probe with large Stokes shift for detection of H2S in living cells

Hydrogen sulfide (H2S) is an important gas signal molecule, which participates in a variety of pathological and physiological processes. In this paper, a novel near-infrared fluorescence probe (NIR-H2S) with azido as the reaction site was designed and synthesized. NIR-H2S utilizes the reducibility of H2S to detect H2S, which effectively avoids the interference of biological thiols. NIR-H2S is a D-π-A dye, and thiophene is the π-bridge, which can not only enhance the π conjugation of the molecules but also increase the vibrational flexibility of the molecules. Therefore, NIR-H2S shows a significant near-infrared (NIR) turn-on signal at 693 nm, with a large Stokes shift (258 nm). Cell imaging experiments show that NIR-H2S has high contrast imaging ability and can be used to detect H2S in cells. The excellent performance of NIR-H2S indicates that NIR-H2S is one of the valuable candidate probes for the detection of H2S in living cells.

Steric Control in Activator-Induced Nucleophilic Quencher Detachment-Based Probes: High-Contrast Imaging of Aldehyde Dehydrogenase 1A1 in Cancer Stem Cells.

Turn-on fluorescence probes can visualize the enzyme activity with high contrast. We have established a new turn-on mechanism, activator-induced nucleophilic quencher detachment (AiQd), and developed AiQd-based turn-on fluorescence probes for the detection of enzymes. Herein, we demonstrate that the precise steric control efficiently quenches the fluorescence of AiQd-based turn-on probes before the enzymatic transformation. Theoretical calculation appropriately predicted the ratio of the fluorescence-quenched closed-ring form of probes. βC5S-A, which has a sterically demanding methyl group at the β-position of a fluorescence-quenching nucleophilic mercapto group, showed a low background signal. βC5S-A responded to aldehyde dehydrogenase 1A1 (ALDH1A1) with high selectivity, thereby enabling high-contrast live imaging of cancer stem cells (signal-to-noise ratio >10). The ALDH1A1-responsiveness of βC5S-A was not significantly affected by amino acids and biological thiols, such as cysteine and glutathione.