Thiol Amino Acids Research Articles

Theoretical study of the binding mechanism between anticancerous drug mercaptopurine and gold nanoparticles using a cluster model.

Mercaptopurine is an effective anticancer medicine yet known with serious adverse reactions, thus requiring further attempts to enhance its biological targeting. Small gold clusters Aun (n = 2-10) were used as model reactants to simulate the surface of gold nanoparticles. The computed results show that the drug molecules tend to anchor on the gold clusters at the S atom with the associated binding energies varying from -50 to -34 kcal mol-1 (in vacuum) and from -42 to -28 kcal mol-1 (in aqueous solution). Furthermore, the adsorption of the drug onto the gold surface is considered as a reversible process, and the mechanism of drug releasing was found to be triggerable by internal factors, such as a pH change or the concentrated presence of thiol amino acids in cancerous protein structures. Calculations based on density functional theory (DFT) were performed to probe the nature of interactions between the drug and gold nanoparticles. Structural features, thermodynamic parameters, bonding characteristics, and electronic properties of the resulting complexes were investigated at the PBE//cc-pVTZ/cc-pVDZ-PP level.

A highly selective ratio fluorescence probe for detecting cysteine and its bioimaging application

Cysteine is an important thiol amino acid that is abundantly present in cells and has an important function in various life processes of the human body. Therefore, it is of great significance to develop an accurate method for monitoring cysteine concentration in cells. We designed and synthesized a novel FRET-ICT-based ratiometric fluorescence and colorimetric probe (BD-C) for detecting cysteine. As cysteine was gradually added, the fluorescence intensity of probe BD-C in buffer solution decreased at 614 nm and increased at 428 nm. The logarithm of the fluorescence intensity ratio to cysteine exhibited favorable linearity in the concentration range of 0.5–14.0 μM, with a detection limit of 395 nM. The energy transfer efficiency of this FRET system is up to 98.8%. BD-C demonstrated preeminent selectivity for cysteine and could specifically react with cysteine, though other sulfhydryl amino acids, such as GSH and Hcy, were present. Furthermore, BD-C exhibited outstanding targeting ability to the endoplasmic reticulum (ER) and was successfully applied in cellular imaging.

Solvato-Controlled Assembly and Structural Transformation of Emissive Poly-NHC-Based Organometallic Cages and Their Applications in Amino Acid Sensing and Fluorescence Imaging.

Stimuli-induced structural transformation of supramolecular cages has drawn increasing attention because of their sensitive feature to external variations as model systems to simulate biological processes. However, combining structural transformation and useful functions has remained a difficult task. This study reports the solvato-controlled self-assembly of two unique topologies with different emission characteristics, a water-soluble Ag8 L4 cage (A) and an Ag4 L2 cage (B), produced from the same sulfonate-pendant tetraphenylethene (TPE) bridged tetrakis-(1,2,4-triazolium) ligand. Both cages show interesting solvent-responsive reversible structural transformation, and the change of fluorescence signals can efficiently track the process. Additionally, water-soluble cage A exhibits unique properties in thermochromism, thiol amino acid sensing, and subcellular imaging in aqueous media.

Chiral nanocrystals grown from MoS2 nanosheets enable photothermally modulated enantioselective release of antimicrobial drugs

The transfer of the concept of chirality from molecules to synthesized nanomaterials has attracted attention amongst multidisciplinary teams. Here we demonstrate heterogeneous nucleation and anisotropic accumulation of Au nanoparticles on multilayer MoS2 planes to form chiroptically functional nanomaterials. Thiol amino acids with chiral conformations modulate asymmetric growth of gold nanoarchitectures on seeds of highly faceted Au/MoS2 heterostructures. Consequently, dendritic plasmonic nanocrystals with partial chiral morphologies are synthesized. The chirality of dendritic nanocrystals inherited from cysteine molecules refers to the structural characteristics and includes specific recognition of enantiomeric molecules. With integration of the intrinsic photothermal properties and inherited enantioselective characteristics, dendritic Au/MoS2 heterostructures exhibit chirality-dependent release of antimicrobial drugs from hydrogel substrates when activated by exogenous infrared irradiation. A three-in-one strategy involving synthesis of chiral dendritic heterostructures, enantioselective recognition, and controlled drug release system is presented, which improves nanomaterial synthetic technology and enhances our understanding of crucial chirality information.

Cysteine mitigates the effect of NaCl salt toxicity in flax (Linum usitatissimum L) plants by modulating antioxidant systems

Agriculture, the main water-consuming factor, faces a global water scarcity crisis. Saline water is an alternative water source, while excess NaCl decreases plant growth and productivity of crops. L-cysteine (Cys) is a promising thiol amino acid in plant growth and development. Flax; Linum usitatissimum L. is an economical plant with low salt tolerance. NaCl salt stress at 50 and 100 mM inhibited the growth parameters, the photosynthetic pigments, total soluble sugars, total phenols, and amino nitrogen in flax plants. Salt stress led to a marked rise in proline and lipid peroxidation and altered the protein profile. Foliar application of cysteine at 0.8 and 1.6 mM mitigates the unfriendly effects of NaCl stress on flax plants. Cysteine enhanced the growth traits, photosynthetic pigments, amino nitrogen, total phenols, and new polypeptides in NaCl-stressed plants. However, cysteine declined the total sugars, proline, the activity of peroxidase, and ascorbate peroxidase. The results confirmed that cysteine had reductant properties. Furthermore, it decreased the NaCl oxidative stress and maintained the stability of membranes by lowering lipid peroxidation. Overall, the redox capacity of L-cysteine is the cause behind its potential counteracting of the adverse effects of NaCl toxicity on the growth of flax plants.

Synthesis and characterization of carnitine-based ionic liquids and their evaluation as additives in cyclodextrin-electrokinetic chromatography for the chiral separation of thiol amino acids

In this study, new chiral ionic liquids based on the non-protein amino acid L-carnitine as cationic chiral counterpart and several anions (bis(trifluoromethane)sulfonimide (NTf2−), L-lactate− or Cl−) as counterions were synthesized. Moreover, three different salts based on L-carnitine were also synthesized and the other three were commercially acquired and used for comparison. The synthesized ionic liquids and salts were characterized by nuclear magnetic resonance, fourier transform infrared spectroscopy, high-performance liquid chromatography-mass spectrometry, and elemental analysis. Subsequently, they were used as additives to establish a γ-CD-based dual system for the enantiomeric separation of cysteine and homocysteine (previously derivatized with fluorenylmethoxycarbonyl chloride) by capillary electrokinetic chromatography. The effect of the nature of the anionic counterions and the presence of different substituents on the L-carnitine molecule on the chiral separation of both amino acids was investigated. The enantioseparations obtained with each dual system studied were compared in terms of the enantiomer effective mobilities (μeff) and the effective electrophoretic selectivity (αeff). Practically, all the dual systems evaluated exhibited substantially improved enantioseparations for the two amino acids compared with the single γ-CD system.

High-throughput and high-sensitivity capillary electrophoresis\u2013mass spectrometry method for sulfur-containing amino acids

Biological thiol amino acids have been suggested as biomarkers for pathological changes because they are reactive chemicals that participate in various physiological processes. In this study, multisegmented injection capillary electrophoresis–mass spectrometry with online sample preconcentration was used for analysis of thiol amino acids and intermediates of sulfur metabolism in human glioma cell line U-251 with high accuracy, throughput, and sensitivity. This was achieved using multiple, large-volume injections for online sample preconcentration. The 16 intermediates of sulfur metabolism had a good linear correlation coefficient range of 0.984–1 and the limit of detection range was 1.4–203.9 ng/mL. The recovery ranges of most amino acids were 88.1–114.5%, 89.0–104.3%, and 76.9–104.5% at 0.3, 0.75, and 1.5 μg/mL, respectively. The relative standard deviation ranges for the inter- and intra-day precision were 1.8–10.7% and 4.3–18.8%, respectively. Compared with the traditional injection method, the analytical time for compounds in sulfur metabolism was reduced to 4 min/sample, the method throughput was enhanced five times, and the sensitivity was increased 14.4–33.1 times. Customized injection sequences were applied in experimental optimization. The developed method simplified the experimental optimization to one injection and is suitable for the analysis of sulfur metabolites in biological samples and has high sensitivity, throughput, speed, and accuracy.

Novel styrylbenzimidazolium-based fluorescent probe for reactive sulfur species: Selectively distinguishing between bisulfite and thiol amino acids

In this study, a new fluorescent probe containing dicyanovinyl moiety has been designed and synthesized. Fluorescent probe based on styrylbenzimidazolium derivative was reported for the effective detection of bisulfite and distinguish it from biothiols by exploiting dicyanovinyl group as the recognition site. The photophysical properties of the novel styrylbenzimidazolium derivative were assessed by determination of absorption and fluorescence spectra, fluorescence quantum yield, and fluorescence lifetime. Its spectroscopic behavior towards various analytes has been evaluated in aqueous media at a pH of 7.4. The highest increase in fluorescence intensity of compound 5 in the presence of different analytes was observed for sodium bisulfite and the limit of detection was estimated to be 0.25 μM. The styrylbenzimidazolium dye was applied to detect bisulfite in various wine sample using fluorimetry. Finally, the ability of this novel probe to detect HSO3− in red wine samples was evaluated.

Updates on Versatile Role of Putative Gasotransmitter Nitric Oxide: Culprit in Neurodegenerative Disease Pathology

Nitric oxide (NO) is a versatile gasotransmitter that contributes in a range of physiological and pathological mechanims depending on its cellular levels. An appropriate concentration of NO is essentially required for cellular physiology; however, its increased level triggers pathological mechanisms like altered cellular redox regulation, functional impairment of mitochondrion, and modifications in cellular proteins and DNA. Its increased levels also exhibit post-translational modifications in protein through S-nitrosylation of their thiol amino acids, which critically affect the cellular physiology. Along with such modifications, NO could also nitrosylate the endoplasmic reticulum (ER)-membrane located sensors of ER stress, which subsequently affect the cellular protein degradation capacity and lead to aggregation of misfolded/unfolded proteins. Since protein aggregation is one of the pathological hallmarks of neurodegenerative disease, NO should be taken into account during development of disease therapies. In this Review, we shed light on the diverse role of NO in both cellular physiology and pathology and discussed its involvement in various pathological events in the context of neurodegenerative diseases.

Hydrolysis of homocysteine thiolactone results in the formation of Protein-Cys-S-S-homocysteinylation.

An increased level of homocysteine, a reactive thiol amino acid, is associated with several complex disorders and is an independent risk factor for cardiovascular disease. A majority (>80%) of circulating homocysteine is protein bound. Homocysteine exclusively binds to protein cysteine residues via thiol disulfide exchange reaction, the mechanism of which has been reported. In contrast, homocysteine thiolactone, the cyclic thioester of homocysteine, is believed to exclusively bind to the primary amine group of lysine residue leading to N-homocysteinylation of proteins and hence studies on binding of homocysteine thiolactone to proteins thus far have only focused on N-homocysteinylation. Although it is known that homocysteine thiolactone can hydrolyze to homocysteine at physiological pH, surprisingly the extent of S-homocysteinylation during the exposure of homocysteine thiolactone with proteins has never been looked into. In this study, we clearly show that the hydrolysis of homocysteine thiolactone is pH dependent, and at physiological pH, 1 mM homocysteine thiolactone is hydrolysed to ~0.71 mM homocysteine within 24 h. Using albumin, we also show that incubation of HTL with albumin leads to a greater proportion of S-homocysteinylation (0.41 mol/mol of albumin) than N-homocysteinylation (0.14 mol/mol of albumin). S-homocysteinylation at Cys34 of HSA on treatment with homocysteine thiolactone was confirmed using LC-MS. Further, contrary to earlier reports, our results indicate that there is no cross talk between the cysteine attached to Cys34 of albumin and homocysteine attached to lysine residues.

Cellular glutathione levels in HL-60 cells during respiratory burst are not correlated with ultra-weak photon emission

Recently, ultra-weak photon emission (UPE) was developed as a novel tool for measuring oxidative metabolic processes, as its generation is related to reactive oxygen species (ROS). Both an imbalance in ROS or the uncontrolled production of ROS can lead to oxidative stress, which is commonly associated with many diseases. In addition to playing several biological functions, the thiol amino acid glutathione has an important antioxidant function in the body's defense against ROS. Specifically, glutathione is an important endogenous antioxidant that helps maintain oxidant levels. At the cellular level, glutathione is present in its reduced form (GSH) at relatively high concentrations (in the millimolar range) and in its oxidized form (GSSG) at low concentrations (in the micromolar range). Thus, the GSH/GSSG ratio is often used as an indicator of cellular redox state. Here, we used the HL-60 cell line as a model system in order to determine whether UPE is correlated with intracellular GSH and GSSG levels. HL-60 cells were differentiated into neutrophil-like cells and then stimulated to undergo respiratory burst. We then recorded UPE in real time for 9000 seconds and used capillary electrophoresis coupled to mass spectrometry to measure GSH and GSSG levels in cell extracts. We found that although respiratory burst significantly decreased the GSH/GSSG ratio, this change was not significantly correlated with the UPE profile.

Cysteine-Cystine Photoregeneration for Oxygenic Photosynthesis of Acetic Acid from CO2 by a Tandem Inorganic-Biological Hybrid System.

Tandem "Z-scheme" approaches to solar-to-chemical production afford the ability to independently develop and optimize reductive photocatalysts for CO2 reduction to multicarbon compounds and oxidative photocatalysts for O2 evolution. To connect the two redox processes, molecular redox shuttles, reminiscent of biological electron transfer, offer an additional level of facile chemical tunability that eliminates the need for solid-state semiconductor junction engineering. In this work, we report a tandem inorganic-biological hybrid system capable of oxygenic photosynthesis of acetic acid from CO2. The photoreductive catalyst consists of the bacterium Moorella thermoacetica self-photosensitized with CdS nanoparticles at the expense of the thiol amino acid cysteine (Cys) oxidation to the disulfide form cystine (CySS). To regenerate the CySS/Cys redox shuttle, the photooxidative catalyst, TiO2 loaded with cocatalyst Mn(II) phthalocyanine (MnPc), couples water oxidation to CySS reduction. The combined system M. thermoacetica-CdS + TiO2-MnPc demonstrates a potential biomimetic approach to complete oxygenic solar-to-chemical production.

A Safety Evaluation of a Nature-Identical l-Ergothioneine in Sprague Dawley Rats.

l-(+) Ergothioneine is a naturally occurring thiol amino acid with antioxidant properties and potential benefits as a dietary supplement. Despite its century-old identification and wide distribution in human food, little is known of its mechanism of action and safety. The nature-identical biomimetic of l-(+) ergothioneine, produced by Mironova Labs and supplied as Mironova (EGT+), has been investigated in the present studies for its mutagenic and toxicologic potential. In a plate incorporation and preincubation assay with Salmonella typhimurium strains TA98, 100, 1,535, and 1,537 and Escherichia coli WP2uvrA strain, at dose concentrations of 1.58, 5, 15.8, 50, 158, 500, 1,580, and 5,000 μg/plate with and without metabolic activation, no cytotoxicity or mutagenicity was observed. Following a preliminary 28-day study, a repeated dose 90-day gavage study at dose levels of 0, 400, 800, and 1,600 mg/kg body weight (bw)/d in Sprague Dawley rats, in which dose-proportional systemic absorption was confirmed by plasma analysis, no adverse clinical, body weight/gain, food consumption and efficiency, clinical pathology, or histopathological changes associated with the administration of the nature-identical ergothioneine were observed. In conclusion, EGT+ administered over 90 days was well tolerated with a no adverse effect level at 1,600 mg/kg bw/d, the highest dose tested for male and female rats. In addition, the nature-identical test substance, EGT+ was not mutagenic in a bacterial reverse mutation assay at plate concentrations of up to 5,000 μg/mL in the presence or absence of metabolic activation.

Evaluation of transnitrosating ability of N-nitrosoguanidines to alkyl thiols and thiol amino acids

The transfer of the nitroso group from 1-nitroso-1-methyl-3-tolylsulfonylguanidine (NOTSG) and 1-nitroso-1-methyl-3-benzoylguanidine (NOBMG) to some thiols, including the amino acid cysteine, was studied in a pH range between 7 and 12.The measured apparent bimolecular rate constant of transnitrosation (ktrapp) revealed a bell-shaped pH dependence that clearly indicates that both nitrosoguanidines react through the corresponding neutral form, and the nucleophiles in the thiolate anion form to give the corresponding S-nitrosothiol. Regarding cysteine, the existence of three macroscopic acidity constants influenced the kinetic behavior of the transnitrosation reaction. Transnitrosation rates (ktr) of the two possible nucleophilic species were obtained and it was found that NOBMG has lower thiol transnitrosation capacity due to the lower electron-withdrawing effect of benzoyl group and to the possible stabilization of the anionic structure as a consequence of the establishment of the intramolecular hydrogen bond. The ktr values of the studied nucleophiles were calculated and a Brønsted-type plot was established giving unexpected negatives βnuc(βnuc(NOBMG)=−0,17 and βnuc(NOTSG)=−0,11). The atypical βnuc values were attributed to the need for previous desolvation of the nucleophile.

Synthesis, spectroscopic characterisation, and potential application of dyes containing a carbostyril skeleton as sensors for thiols

Several dyes based on a carbostyril skeleton were synthesised and characterised using proton nuclear magnetic resonance spectroscopy and electron ionisation mass spectrometry. Their basic spectroscopic properties, such as absorption and emission spectra, were also measured. The behaviour of the obtained dyes in the absence and presence of thiol amino acids and other compounds under various conditions were studied. These dyes contain various moieties in the structure, acting as Michael acceptors. Results show that the studied compounds have the potential to act as colorimetric sensors for thiols, but the benzothioazolium dyes are less effective than dicyanoethylene derivatives.

Dyes derived from 3‐formyl‐2(1H)‐quinolone – synthesis, spectroscopic characterisation, and their behaviour in the presence of sulfhydryl and non‐sulfhydryl amino acids

Novel dyes based on a 3‐formyl‐2(1H)‐quinolone skeleton were synthesised and characterised using 1H nuclear magnetic resonance spectroscopy and mass spectrometry. The spectroscopic properties of these dyes, such as their absorption spectra, emission spectra, and quantum fluorescence yields, were also examined. The behaviour of the obtained compounds at a pH of 7.4 in the absence and in the presence of thiol amino acids, such as l‐cysteine, l‐glutathione, and N‐acetyl‐l‐cysteine, were studied. The spectroscopic responses of the tested dyes towards other amino acids were also investigated. A reference compound was synthesised to understand the reaction mechanism between the thiols and the obtained dyes. The experimental results show that the synthesised dyes have the potential to act as sensors for thiols.

A dual-response BODIPY-based fluorescent probe for the discrimination of glutathione from cystein and homocystein.

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 allowing the discriminative detection of GSH over Cys and Hcy and thiol-lacking amino acids under 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.

L-Cysteine and L-AP4 microinjections in the rat caudal ventrolateral medulla decrease arterial blood pressure.

The thiol amino acid L-cysteine increases arterial blood pressure (ABP) when injected into the cerebrospinal fluid space in conscious rats, indicating a pressor response to centrally acting L-cysteine. A prior synaptic membrane binding assay suggests that L-cysteine has a strong affinity for the L-2-amino-4-phosphonobutyric acid (L-AP4) binding site. The central action of L-cysteine may be vial-AP4 sensitive receptors. The present study investigated cardiovascular responses to L-cysteine and L-ap4 microinjected into the autonomic area of the caudal ventrolateral medulla (CVLM) where inhibitory neurons regulate ABP via pre-sympathetic vasomotor neurons. Both the injection of L-cysteine and L-AP4 in the CVLM sites identified with L-glutamate produced the same depressor and bradycardic responses in urethane-anesthetized rats. Neither a prior antagonist microinjection of MK801 for the N-methyl-D-aspartate (NMDA) receptor nor CNQX for the non-NMDA receptor attenuated the responses to L-cysteine, but the combination of the two receptor blocking with an additional prior injection abolished the response. In contrast, either receptor blockade alone abolished the response to L-AP4, indicating distinct mechanisms between responses to L-cysteine and L-AP4 in the CVLM. The results indicate that the CVLM is a central active site for L-cysteine's cardiovascular response. Central L-cysteine's action could be independent of the L-AP4 sensitive receptors. Cardiovascular regulation may involve endogenous L-cysteine in the CVLM. Further multidisciplinary examinations are required to elaborate on L-cysteine's functional roles in the CVLM.

Genome wide DNA methylation profiling for epigenetic alteration in coronary artery disease patients

The alteration in the epigenome forms an interface between the genotype and the environment. Epigenetic alteration is expected to make a significant contribution to the development of cardiovascular disease where environmental interactions play a key role in disease progression. We had previously shown that global DNA hypermethylation per se is associated with coronary artery disease (CAD) and is further accentuated by high levels of homocysteine, a thiol amino acid which is an independent risk factor for cardiovascular disease and is also a key modulator of macromolecular methylation. We have identified 72 differentially methylated regions (DMRs) that were hypermethylated in CAD patients in the background of varying homocysteine levels. Following deep bisulfite sequencing of a few of the selected DMRs, we found significantly higher methylation in CAD cases. We get six CpG sites in three DMRs that included the intronic region of C1QL4 gene and upstream region of CCDC47 and TGFBR3 genes. To the best of our knowledge, this is the first study to identify hypermethylated regions across the genome in patients with coronary artery disease. Further validation in different populations is necessary for this information to be used for disease risk assessment and management.

S-carbamoylation impairs the oxidant scavenging activity of cysteine: Its possible impact on increased LDL modification in uraemia

Carbamoylation is the non-enzymatic reaction of cyanate with amino-, hydroxy- or thiol groups. In vivo, amino group modification (N-carbamoylation) resulting in altered function of proteins/amino acids has been observed in patients suffering from uraemia due to urea-derived cyanate. Uraemia has been linked to impaired antioxidant defense. As thiol-compounds like cysteine, N-acetyl cysteine and GSH have oxidant scavenging properties one may speculate that thiol-group carbamoylation (S-carbamoylation) may impair their protective activity. Here we report on the effect of S-carbamoylation on the ABTS free radical and HOCl scavenging property of cysteine as well on its ability to protect LDL from atherogenic modification induced by AAPH generated peroxylradicals or HOCl. The results show that S-carbamoylation impaired the ABTS free radical and HOCl scavenging property of the thiol-compounds tested. The ability of the thiols to protect LDL from lipid oxidation and apolipoprotein modification was strongly diminished by S-carbamoylation. The data indicate that S-carbamoylation could impair the free radical and HOCl scavenging of thiol-amino acids reducing their protective property against LDL atherogenic modification by these oxidant species. As S-carbamoylation is most effective at pH 7 to 5 in vivo thiol-carbamoylation may especially occur at sites of acidic extracellular pH as in hypoxic/inflammatory macrophage rich areas like the atherosclerotic plaque where increased LDL oxidation has been found and may contribute to the higher oxidative stress in uraemia.