Electrocatalytic Oxidation of 2‐Thiouracil and 2‐Thiobarbituric Acid at a Carbon‐Paste Electrode Modified with Cobalt Phthalocyanine

Voltammetric behavior of two mercaptopyrimidine derivatives (2‐thiouracil and 2‐thiobarbituric acid) has been studied by cyclic voltammetry at a cobalt phthalocyanine (CoPc)‐modified carbon‐paste electrode. The results of voltammetric determinations showed that the CoPc in the matrix of modified electrode acts as catalyst for electrooxidation of these thiols (RSH), lowering the overpotential of the reaction and significantly increasing the sensitivity for detection of thiols in neutral conditions. The results of voltammetric and polarization measurements in solutions with various pHs were used for prediction of the mechanism of electrocatalytic oxidation at the surface of modified electrode. These results showed that at the modified electrode, electrochemical oxidation of thiolate anion (RS^?) is the rate‐determining step. It was found that the modified electrode exhibits good selectivity for catalytic oxidation of mercaptopyrimidines over other biologically important mercaptans such as cysteine, glutathione and thioglycolic acid. The results demonstrate that the peak current for thiol oxidation has a linear variation with the concentration in the range of 1×10^?2–1×10^?5 M. This system can be used for sensitive and selective voltammetric detection of mercaptopyrimidine derivatives.     

Electrochemical Reduction of Ellman's Reagent: A Novel Selective Detection Protocol for Thiol Compounds

Herein we detail a novel approach to the detection of total sulfhydryl thiol species based on an electrochemical adaptation of the classical Ellman's reaction. The ability to electrochemically reduce the nitro moieties of both the parent 5,5‐dithiobis(2‐nitrobenzoic acid) (DTNB) and the resulting 5‐thio‐2‐nitrobenzoic acid (TNBA) species provides a selective means of detecting thiol compounds. The use of the reductive sweep means that electrode passivation as a result of oxidative electrochemical procedures can be avoided and the selectivity improved through positioning the analytical signal within a region where few interferences reside. The electrochemical protocol enables the detection of the thiols in both acidic and basic media, which the colorimetric method is unable to do.     

Electrochemical detection of thiols in biological media

The utilisation of catechol as an electrochemical indicator for the presence of sulphydryl thiols (RSH) has been investigated. The electrochemical oxidation of the catechol within tissue culture media was examined with the influence exerted on the redox chemistry by cysteine evaluated in terms of the development of an analytical protocol. The electro-generation of o-quinone was found to be followed by a 1,4-addition reaction with available cysteine such that an increase in the current, attributed to the re-oxidation of the thiol-catechol adduct, could be exploited as means of quantifying the concentration of the thiol. The selectivity of the reaction has been assessed with no interference from lysine, tyrosine, methionine or cystine. Other amino acids possessing sulphydryl thiol functionalities (homocysteine and glutathione) were, however, found to react through a similar route to that observed with cysteine.     

Living polymerization of styrene initiated by mercaptan/ε‐caprolactam

The bulk polymerization of styrene initiated by ?‐caprolactam (CL) and n‐dodecyl mercaptan (RSH) has been explored. This novel polymerization system shows living characteristics. For example, the molecular weight of the resulting polymers increases with conversion, and the system has the ability to form diblock copolymers and so forth. The polymer chain end contains thiol and lactam structures, which we have investigated with Fourier transform infrared, ¹H NMR, and ¹³C NMR techniques. Electron spin resonance spectra and theoretical calculations by the Hartree–Fock methods have been used to examine the mechanism. The results reveal that the initial polymerization starts from thiol via a chain‐transfer reaction, and the propagation proceeds by the insertion of a monomer between the terminal group and the intermediate structure of lactam. Finally, the polymerization kinetics have been examined. The polymerization rate varies linearly with the concentration of CL and RSH, and this confirms the mechanism. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 4976–4993, 2004     

Photoassisted Synthesis of Luminescent Mannose–Au Nanodots for the Detection of Thyroglobulin in Serum

We have employed mannose‐modified gold nanodots (Man–Au NDs) as a luminescence sensor for the detection of the thyroid‐cancer marker thyroglobulin (Tg) in homogeneous solutions. The luminescent Man–Au NDs are prepared through the reaction of 2.9 nm‐diameter gold nanoparticles (Au NPs) with 11‐mercapto‐3,6,9‐trioxaundecyl‐α‐D ‐mannopyranoside (Man‐RSH) under the irradiation of a light‐emitting diode (LED). We have found that the irradiation enhances the quantum yield (～11 %), alters the emission wavelength and lifetimes, and shortens the preparation time. A luminescence assay has been developed for Tg based on the competition between Tg and Man–Au NDs for the interaction with the concanavalin A (Con A). Because luminescence quenching of the Man–Au NDs by Con A is inhibited by Tg selectivity, we have obtained a highly sensitive and selective assay for Tg.     

Thiol‐disulfide redox equilibria of glutathione metaboloma compounds investigated by tandem mass spectrometry

The thiol group of cysteine plays a pivotal role in structural and functional biology. We use mass spectrometry to study glutathione‐related homo‐ and heterodimeric disulfides, aiming at understanding the factors affecting the redox potentials of different disulfide/thiol pairs. Several electrospray ionization (ESI)‐protonated disulfides of cysteamine, cysteine, penicillamine, N‐acetylcysteine, N‐acetylpenicillamine, γGluCySH, HSCyGly, and glutathione were analyzed on a triple quadrupole instrument to measure their energy‐resolved tandem mass spectra. Fission of the disulfide bond yields RSH*H⁺ and RS⁺ ions. The logarithm of the intensity ratio of the RS⁺/RSH*H⁺ fragments in homodimeric disulfides is proportional to the normal reduction potential of their RSSR/RSH pairs determined by nuclear magnetic resonance (NMR) in solution, the more reducing ones yielding the higher ratios. Also in some R₁S‐SR₂ disulfides, the ratio of the intensities of the RSH + H⁺ and RS⁺ ions of each participating thiol shows a linear relationship with the Nernst equation potential difference of the corresponding redox pairs. This behavior allows us to measure the redox potentials of some disulfide/thiol pairs by using different thiol‐reducing probes of known oxidoreductive potential as reference. To assist understanding of the fission mechanism of the disulfide bond, the fragments tentatively identified as ‘sulfenium’ were themselves fragmented; accurate mass measurement of the resulting second‐generation fragments demonstrated a loss of thioformaldehyde, thus supporting the assigned structure of this elusive intermediate of the oxidative stress pathway. Understanding this fragmentation process allows us to employ this technique with larger molecules to measure by mass spectrometry the micro‐redox properties of different disulfide bonds in peptides with catalytic and signaling biological activity. Copyright © 2008 John Wiley & Sons, Ltd.     

Kinetics and mechanism for reduction of the anticancer prodrug trans,trans,trans-[PtCl2(OH)2(c-C6H11NH2)(NH3)] (JM335) by thiols

The reduction of the platinum(IV) prodrug trans,trans,trans-[PtCl2(OH)2(c-C6H11NH2)(NH3)] (JM335) by L-cysteine, DL-penicillamine, DL-homocysteine, N-acetyl-L-cysteine, 2-mercaptopropanoic acid, 2-mercaptosuccinic acid, and glutathione has been investigated at 25 degrees C in a 1.0 M aqueous perchlorate medium with 6.8 < or = pH < or = 11.2 using stopped-flow spectrophotometry. The stoichiometry of Pt(IV):thiol is 1:2, and the redox reactions follow the second-order rate law -d[Pt(IV)]/dt = k[Pt(IV)][RSH]tot, where k denotes the pH-dependent second-order rate constant and [RSH]tot the total concentration of thiol. The pH dependence of k is ascribed to parallel reductions of JM335 by the various protolytic species of the thiols, the relative contributions of which change with pH. Electron transfer from thiol (RSH) or thiolate (RS-) to JM335 is suggested to take place as a reductive elimination process through an attack by sulfur at one of the mutually trans chloride ligands, yielding trans-[Pt(OH)2(c-C6H11NH2)(NH3)] and RSSR as the reaction products, as confirmed by 1H NMR. Second-order rate constants for the reduction of JM335 by the various protolytic species of the thiols span more than 3 orders of magnitude. Reduction with RS- is approximately 30-2000 times faster than with RSH. The linear correlation log(kRS) = (0.52 +/- 0.06)-pKRSH--(2.8 +/- 0.5) is observed, where kRS denotes the second-order rate constant for reduction of JM335 by a particular thiolate RS- and KRSH is the acid dissociation constant for the corresponding thiol RSH. The slope of the linear correlation indicates that the reactivity of the various thiolate species is governed by their proton basicity, and no significant steric effects are observed. The half-life for reduction of JM335 by 6 mM glutathione (40-fold excess) at physiologically relevant conditions of 37 degrees C and pH 7.30 is 23 s. This implies that JM335, in clinical use, is likely to undergo in vivo reduction by intracellular reducing agents such as glutathione prior to binding to DNA. Reduction results in the immediate formation of a highly reactive platinum(II) species, i.e., the bishydroxo complex in rapid protolytic equilibrium with its aqua form.     

A comparative kinetic study for the oxidation of 2‐mercaptoethanol by di‐μ‐oxo‐bis(1,4,7,10‐tetraazacyclododecane)‐dimanganese(III,IV) and di‐μ‐oxo‐bis(1,4,8,11‐tetraazacyclotetradecane)‐dimanganese(III,IV) complexes: Influence of copper(II)

A comparative kinetic study of the reactions of two mixed valence manganese(III,IV) complexes with macrocyclic ligands, [L¹Mn^IV(O)₂Mn^IIIL¹], 1 (L¹ = 1,4,7,10‐tetraazacyclododecane) and [L²Mn^IV(O)₂Mn^IIIL²], 2 (L² = 1,4,8,11‐tetraazacyclotetradecane) with 2‐mercaptoethanol (RSH) has been carried out by spectrophotometry in aqueous buffer at (30 ± 0.1)°C. Rate of the reactions between the oxidants and the reductant was found to be negligibly slow with no systematic dependence on either redox partners. Externally added copper(II) (usually 5 × 10^?7 mol dm^?3), however, increases the rate of the reduction of 1 and 2 significantly. In the presence of catalytic amount of copper(II), the rate of the reaction is nearly proportional to [RSH] at lower concentration of the reductant but follows a saturation kinetics at higher concentration of the latter for the reaction between 1 and the thiol. Reaction rate was found to be strongly influenced by the variation of acidity of the medium and the observed kinetics suggests that the two reductant species ([Cu(RSH)]²⁺ and [Cu(RS)]⁺) are significant for the reaction between 1 and the thiol. The dependence of the rate on [RSH] for the reduction of 2 by the thiol was complex and rationalized considering two equilibria involving the catalyst (Cu²⁺) and the reductant. The pH rate profile suggests that both the μ‐O protonated [Mn^III(O)(OH)Mn^IV] and the deprotonated [Mn^III(O)₂Mn^IV] forms of the oxidant 2 become important. The kinetic results presented in this study indicate the domination of outer‐sphere path. © 2003 Wiley Periodicals, Inc. Int J Chem Kinet 36: 129–137, 2004     

Electrochemically Initiated Catalytic Oxidation of 5‐Thio‐2‐Nitrobenzoic Acid (TNBA) in the Presence of Thiols at a Boron Doped Diamond Electrode: Implications for Total Thiol Detection

The electrochemical response of 5,5‐dithiobis(2‐nitrobenzoic acid) (DTNB) to increasing additions of thiol species has been examined at a boron doped diamond electrode. A reaction has been shown to occur with a range of biologically relevant thiols and proceeds via a CECC' process. A total thiol detection methodology has been developed showing that the sensitivities of the standard addition plots are independent of the individual thiol species added to the solution. The analytical utility of the reaction process has been assessed using chronoamperometry with the corresponding data producing detection limits of 5.7 μM, 4.4 μM and 5.8 μM for the detection of cysteine, homocysteine and glutathione respectively.     

Preliminary Study on Reverse Pulse Amperometric Detection in Capillary Electrophoresis for Simultaneous Determination of Cysteine and Cystine

Simultaneous determination of cysteine (RSH) and cystine (RSSR), two important sulfur-containing amino acids, in capillary electrophoresis (CE) has been an analytical task. Dual-microelectrode amperometric detection seems to be a good scheme, but significant difficulty in electrode construction and poor detection limit for RSSR determination remain major short-comings. In reverse pulse amperometric (RPA) detection, the applied potentials are repeatedly pulsed back and forth between the reducing initial potential (e.g., E_i = ?1.4 V) and the oxidizing final potential (e.g., E_f = 0.0 V) at a single, gold-mercury amalgam (Au/Hg) microelectrode. At E_i, RSSR is reduced to RSH which causes catalytical oxidation of the Au/Hg amalgam microelectrode when the potential is pulsed to E_f. The resulting anodic current is then recorded. Therefore, by using RPA detection after CE separation, RSH and RSSR can be simultaneously determined.     

HPLC determination of thiol drugs in pharmaceutical formulations using ethacrynic acid as a precolumn ultraviolet derivatization reagent

Summary A high performance liquid chromatographic method has been developed for the determination of aliphatic thiol drugs, such as N-acetyl-L-cysteine, captopril and mercaptopropionylglycine in pharmaceutical formulations. The procedure involves a precolumn derivatization of the thiol drug with ethacrynic acid followed by reversedphase HPLC separation and UV detection. The conditions for a rapid and selective reaction of the thiols with ethacrynic acid have been investigated. The method proved to be suitable for a reliable and selective quality control of commercial dosage forms of the examined thiol drugs.     

Ratiometric detection of mitochondrial thiols with a two-photon fluorescent probe

We report a ratiometric two-photon probe (SSH-Mito) for mitochondrial thiols. This probe shows a marked blue-to-yellow emission color change in response to RSH, a significant two-photon cross section, good mitochondrial thiol selectivity, low cytotoxicity, and insensitivity to pH over the biologically relevant pH range, allowing the direct visualization of RSH levels in live cells as well as in living tissues at 90-190 μm depth without interference from other biologically relevant species through the use of two-photon microscopy.     

Voltammetric Determination of Surface‐Confined Biomolecules with N‐(2‐Ethyl‐ferrocene)maleimide

A thiol‐specific electroactive cross‐linker, N‐(2‐ethyl‐ferrocene)maleimide (Fc‐Mi), has been used to tag surface‐confined peptides containing cysteine residues or oligodeoxynucleotides (ODNs) whose 3′ ends have been modified with thiol groups. The peptides studied herein include both the oxidized and reduced forms of glutathione and a hexapeptide. Cyclic voltammograms (CVs) of the Fc‐Mi groups attached to the surfaces were used to quantify the total number of cysteine residues that are tagged and/or can undergo facile electron transfer reactions with the underlying electrodes. A quartz crystal microbalance was used in conjunction with CV to estimate the total number of cysteine groups labeled by Fc‐Mi per peptide molecule. By comparing to mass spectrometric studies, it is confirmed that not all of the Fc‐Mi linked to the cysteine groups can participate in the electron transfer reactions. The methodology is further extended to the determination of ODN samples in a sandwich assay wherein the thiol linker on the 3′ end can be tagged with Fc‐Mi. The analytical performance was evaluated through determinations of a complementary ODN target and targets with varying numbers of mismatching bases. ODN samples as low as 10 fmol can be detected. Such a low detection level is remarkable considering that no signal amplification scheme is involved in the current method. The approach is shown to be sequence‐ and/or structure‐specific and does not require sophisticated instrumentation and complex experimental procedure.     

Voltammetric Detection of Captopril on Graphite Screen Printed Electrodes

We report the electrochemical detection of captopril on commercially available screen printed electrodes (GSPE); it exploits the silver residue left behind on a GSPE during its manufacturing process and involves the catalytic formation of the silver thiol complex (Ag⁺+RSH→AgSR+H⁺) at potentials corresponding to the oxidation of silver. The oxidation of the silver thiol complex was found to vary linearly with the captopril concentration up to 0.8 mM. A sensitivity of 13.34±0.58 μA mM^?1 is reported with a limit of detection of 4.27±0.18 μM.     

Access to Versatile β‐Cyclodextrin Scaffolds through Guest‐Mediated Monoacylation

Herein, we report the selective mono‐derivatization of heptakis[6‐deoxy‐6‐(2‐aminoethylsulfanyl)]‐β‐CD ( 1 ) through a guest‐mediated covalent capture strategy. The use of guests functionalized with cleavable linkers enables the installation of an amine‐orthogonal thiol group on the primary rim of 1 as a handle for further transformations to the β‐CD scaffold. Applying this methodology, two novel monoderivatized β‐CDs were obtained in good yield and high purity. Both of these monoacylated CDs were amenable to facile linker cleavage and further modification at the resulting thiol group. This methodology can be applied towards the synthesis heterofunctionalized β‐CD constructs for analyte sensing, drug delivery, and other applications.     

Effects of aromatic thiols on thiol-disulfide interchange reactions that occur during protein folding

The folding of disulfide containing proteins from denatured protein to native protein involves numerous thiol-disulfide interchange reactions. Many of these reactions include a redox buffer, which is a mixture of a thiol (RSH) and the corresponding disulfide (RSSR). The relationship between the structure of RSH and its efficacy in folding proteins in vitro has been investigated only to a limited extent. Reported herein are the effects of aliphatic and especially aromatic thiols on reactions that occur during protein folding. Aromatic thiols may be particularly efficacious as their thiol pK(a) values and reactivities match those of the in vivo catalyst, protein disulfide isomerase (PDI). This investigation correlates the thiol pK(a) values of aromatic thiols with their reactivities toward small molecule disulfides and the protein insulin. The thiol pK(a) values of nine para-substituted aromatic thiols were measured; a Hammett plot constructed using sigma(p-) values yielded rho = -1.6 +/- 0.1. The reactivities of aromatic and aliphatic thiols with 2-pyridyldithioethanol (2-PDE), a small molecule disulfide, were determined. A plot of reactivity versus pK(a) of the aromatic thiols had a slope (beta) of 0.9. The ability of these thiols to reduce (unfold) the protein insulin correlates strongly with their ability to reduce 2-PDE. Since the reduction of protein disulfides occurs during protein folding to remove mismatched disulfides, aromatic thiols with high pK(a) values are expected to increase the rate not only of protein unfolding but protein folding as well.     

A sensitive and selective detection method for thiol compounds using novel fluorescence probe

In this work, a sensitive and selective detection method based on fluorescence resonance energy transfer (FRET) was developed for analyzing thiol compounds by using a novel fluorescent probe. The new fluorescent probe contains a disulfide bond which selectively reacts with nucleophilic thiolate through the thiol-disulfide exchange reaction. An obvious fluorescence recovery can be observed upon addition of the thiol compound in the fluorescent probe solution due to the thiol-disulfide exchange reaction and the destruction of FRET. This novel probe was successfully used to determine dithiothreitol (DTT), glutathione (GSH) and cysteine (Cys). The limits of detection (LOD) were 2.0 μM for DTT, 0.6 μM for GSH, and 0.8 μM for Cys. This new detection method was further investigated in the analysis of compound amino acid injection.     

Covalent Adaptable Networks with Tunable Exchange Rates Based on Reversible Thiol–yne Cross‐Linking

The design of covalent adaptable networks (CANs) relies on the ability to trigger the rearrangement of bonds within a polymer network. Simple activated alkynes are now used as versatile reversible cross‐linkers for thiols. The click‐like thiol–yne cross‐linking reaction readily enables network synthesis from polythiols through a double Michael addition with a reversible and tunable second addition step. The resulting thioacetal cross‐linking moieties are robust but dynamic linkages. A series of different activated alkynes have been synthesized and systematically probed for their ability to produce dynamic thioacetal linkages, both in kinetic studies of small molecule models, as well as in stress relaxation and creep measurements on thiol–yne‐based CANs. The results are further rationalized by DFT calculations, showing that the bond exchange rates can be significantly influenced by the choice of the activated alkyne cross‐linker.     

Avoiding Thiol Compound Interference: A Nanoplatform Based on High‐Fidelity Au–Se Bonds for Biological Applications

Gold nanoparticles (Au NPs) assembled through Au?S covalent bonds have been widely used in biomolecule‐sensing technologies. However, during the process, detection distortions caused by high levels of thiol compounds can still significantly influence the result and this problem has not really been solved. Based on the higher stability of Au?Se bonds compared to Au?S bonds, we prepared selenol‐modified Au NPs as an Au‐Se nanoplatform (NPF). Compared with the Au‐S NPF, the Au‐Se NPF exhibits excellent anti‐interference properties in the presence of millimolar levels of glutathione (GSH). Such an Au‐Se NPF that can effectively avoid detection distortions caused by high levels of thiols thus offers a new perspective in future nanomaterial design, as well as a novel platform with higher stability and selectivity for the in vivo application of chemical sensing and clinical therapies.     

Kinetic Potentiometric Determination of some Thiols with Iodide Ion‐Sensitive Electrode

Abstract

This paper describes a kinetic potentiometric method for the determination of thiols (RSH): l‐cysteine (cys), N‐acetyl‐l‐cysteine (NAC), l‐glutathione (glu), and d‐penicillamine (pen). The proposed method is based on the reaction of formation the sparingly soluble salts, RSAg, between RSH and Ag⁺ ions. During this reaction potential‐time curves were recorded by using an electrochemical cell with commercial iodide selective electrode. The rectilinear calibration graphs are obtained in the RSH concentration range from 1.0×10^?5 to 1.0×10^?3 M. The applicability of the proposed method was demonstrated by determination of chosen compounds in pharmaceutical dosage forms.