Rhodium-catalyzed disulfide exchange reaction

A system of RhH(PPh3)4, trifluoromethanesulfonic acid, and (p-tol)3P catalyzes the disulfide exchange reaction. Treatment of two symmetrical dialkyl disulfides with the catalyst provides an equilibrium mixture of three disulfides within 15 min in refluxing acetone. The catalyst is active after reaching the equilibrium, and addition of a disulfide to the mixture changes the ratio of the products. The use of 4 mol equiv excess of one of the disulfides provides the unsymmetrical disulfide in a yield exceeding 80%. Disulfide-containing peptides also undergo an exchange reaction. The reactions of diaryl disulfides and dialkyl disulfides are even faster, and reach equilibrium within 5 min at room temperature in the presence of the rhodium complex and 1,2-bis(diphenylphosphino)ethane (dppe). This exchange reaction is considerably affected by the substituents on the disulfides. Treatment of diphenyl disulfide, di(p-tolyl) disulfide, and bis(sec-butyl) disulfide yields phenyl p-tolyl disulfide at room temperature with unchanged bis(sec-butyl) disulfide; random disproportionation occurs at reflux. The rhodium catalysis can be used for the exchange reaction of disulfides and diselenides giving selenosulfides as well as disulfides and ditellurides giving tellurinosulfides.     

Disulfide bond cleavage induced by a platinum(II) methionine complex

The cleavage of a disulfide bond and the redox equilibrium of thiol/disulfide are strongly related to the levels of glutathione (GSH)/oxidized glutathione (GSSG) or mixed disulfides in vivo. In this work, the cleavage of a disulfide bond in GSSG induced by a platinum(II) complex [Pt(Met)Cl2] (where Met = methionine) was studied and the cleavage fragments or their platinated adducts were identified by means of electrospray mass spectrometry, high-performance liquid chromatography, and ultraviolet techniques. The second-order rate constant for the reaction between [Pt(Met)Cl2] and GSSG was determined to be 0.4 M(-1) s(-1) at 310 K and pH 7.4, which is 100- and 12-fold faster than those of cisplatin and its monoaqua species, respectively. Different complexes were formed in the reaction of [Pt(Met)Cl2] with GSSG, mainly mono- and dinuclear platinum complexes with the cleavage fragments of GSSG. This study demonstrated that [Pt(Met)Cl2] can promote the cleavage of disulfide bonds. The mechanistic insight obtained from this study may provide a deeper understanding on the potential involvement of platinum complexes in the intracellular GSH/GSSG systems.     

A TICS-fluorophore based probe for dual-color GSH imaging

Cascading reactions in fluorophores accompanied by the replacement of different fluorescence wavelengths can be used to develop luminescent materials and reactive fluorescent probes. Based on multiple signal channels, the selectivity of probes can be improved and the range of response to guest molecule recognition can be expanded. By regulating the position, number, and activity of active sites in fluorophores, fluorescent probes that successively react with thiol and amino groups in cysteine (Cys), homocysteine (Hcy) have been developed, which can only react with the thiol group of GSH. In this paper, we report the first probe capable of cascading nucleophilic substitution reaction with the thiol group and amino group of GSH at a single reaction site, and showed the dual-color recognition of GSH, which improved the selectivity of GSH also was an extension of GSH probes. The probe Rho-DEA was based on a TICS fluorophore, and the intramolecular cascade nucleophilic substitution reaction occurs with Cys/Hcy. The thiol substitution of the first step reaction with Cys/Hcy was quenched due to intersystem crossing to triplet state, so GSH can be selectively recognized from the fluorescence signal. Rho-DEA has the ability of mitochondrial localization, and finally realized in situ dual-color fluorescence recognition of GSH in mitochondria.     

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.     

Characterization of alkali induced formation of lanthionine, trisulfides, and tetrasulfides from peptide disulfides using negative ion mass spectrometry

Peptide disulfides are unstable under alkaline conditions, resulting in the formation of products containing lanthionine and polysulfide linkages. Electrospray ionization mass spectrometry has been used to characterize major species obtained when cyclic and acyclic peptide disulfides are exposed to alkaline media. Studies on a model cyclic peptide disulfide () and an acyclic peptide, oxidized glutathione, bis (^γGlu - Cys - Gly - COOH), are described. Disulfide cleavage reactions are initiated by the abstraction of C^αH or C^βH protons of Cys residues, with subsequent elimination of H₂S or H₂S₂. The buildup of reactive thiol species which act on intermediates containing dehydroalanine residues, rationalizes the formation of lanthionine and polysulfide products. In the case of the cyclic peptide disulfide, the formation of cyclic products is facilitated by the intramolecular nature of the Michael addition reaction of thiols to the dehydroalanine residue. Mass spectral evidence for the intermediate species is presented by using alkylation of thiol groups as a trapping method. Mass spectral fragmentation in the negative ion mode of the peptides derived from trisulfides and tetrasulfides results in elimination of S₂.     

Determination of penicillamine, penicillamine disulfide and penicillamine-glutathione mixed disulfide by high-performance liquid chromatography with electrochemical detection

Methodology is described for the simultaneous determination of D-penicillamine, penicillamine disulfide and the penicillamine-glutathione mixed disulfide, as well as glutathione and glutathione disulfide, in human plasma, erythrocytes and urine. The various thiols and disulfides are separated by reversed-phase ion-pairing liquid chromatography with detection by an electrochemical detector with dual gold/mercury amalgam electrodes in series. The thiols are detected at the downstream electrode; the disulfides are reduced at the upstream electrode and then detected as the thiols at the downstream electrode. Detection limits (at a signal-to-noise ratio of 2.0) are in the picomole range for 20 microliters of injected solution for all compounds except penicillamine disulfide, which has a detection limit of 600 pmol in 20 microliters. A convenient method is described for preparation of the penicillamine-glutathione mixed disulfide by thiol/disulfide exchange with standardization of the solution by 1H NMR spectroscopy.     

Electro-generated reactive oxygen species at Au surface as an indicator to explore glutathione redox chemistry and quantification

The thiol to disulfide inter-conversion plays vital role in the glutathione (GSH) metabolism in human body. Mimicking the GSH conversion to disulfide by electro-generated reactive oxygen species at gold electrode interface and the use of electro-reduction of the produced disulfides for the electro-analysis of low levels of GSH is reported.     

The gas-phase H/D exchange mechanism of protonated amino acids

A mass spectrometry and Density Functional Theory study of gas-phase H/D exchange in protonated Ala, Cys, Ile, Leu, Met, and Val is reported. Site-specific rate constants were determined and results identify the alpha-amino group as the protonation site. Lack of exchange on the Cys thiol group is explained by the absence of strong intramolecular hydrogen bonding within the reaction complex. In aliphatic amino acids the presence of a methyl group at the beta-C atom was found to lower the site-specific H/D exchange rate for amino hydrogens. Study of the exchange mechanism showed that isotopic exchange occurs in two independent reactions: in one, only the carboxylic hydrogen is exchanged and in the other, both carboxylic and amino group hydrogens exchange. The proposed reaction mechanisms, calculated structures of various species, and a number of structural findings are consistent with experimental data.     

Drug interactions with potential rubber closure extractables: Identification of thiol–disulfide exchange reaction products of captopril and thiurams

Mixtures of thiuram disulfides are frequently used as accelerators in rubber stoppers for injectables and sterilized powders for injection. Rapid reactions of thiuram disulfides between themselves and with thiols yield mixed disulfides due to thiol–disulfide exchange. The possibility of exchange reactions of thiuram disulfides extracted from rubber stoppers and drug products containing pendant thiol groups have not been reported in the analysis of potential stopper extractables. In this paper we report the formation and identification of mixed thiuram disulfides of N,N,N′,N′-dimethylthiuram disulfide (TMTD), N,N,N′,N′-dibutylthiuram disulfide (TBTD), and captopril (a thiol-containing drug). A reversed-phase HPLC method was developed for the determination of TMTD, TBTD, captopril and their disulfides in aqueous vehicles, using a YMC ODS AQ column at 35 °C and mobile phases A and B consisting of acetonitrile:water:trifluoroacetic acid (TFA) (20:80:0.1) and acetonitrile:TFA (100:0.1), respectively. The captopril–TBTD and captopril–TMTD disulfides were identified by MS, with molecular ions at m/z 420.9 and m/z of 337.1, respectively. Possible structures for the fragment ions in the spectra are provided. Mixed captopril–thiuram formation was studied as a function of pH. Captopril–TMTD formation was enhanced at pH 6.0, reaching a maximum of 31.3% in 4.1 h. At pH 4.0 and 2.2, the mixed captopril adduct product was still detected in solution after 20 h. The impact of the formation of mixed disulfide products of thiol-containing drugs with thiurams in the HPLC profile of extractables and leachables studies is discussed.     

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.     

Dissociation of disulfide‐intact somatostatin ions: the roles of ion type and dissociation method

The dissociation chemistry of somatostatin‐14 was examined using various tandem mass spectrometry techniques including low‐energy beam‐type and ion trap collision‐induced dissociation (CID) of protonated and deprotonated forms of the peptide, CID of peptide‐gold complexes, and electron transfer dissociation (ETD) of cations. Most of the sequence of somatostatin‐14 is present within a loop defined by the disulfide linkage between Cys‐3 and Cys‐14. The generation of readily interpretable sequence‐related ions from within the loop requires the cleavage of at least one of the bonds of the disulfide linkage and the cleavage of one polypeptide backbone bond. CID of the protonated forms of somatostatin did not appear to give rise to an appreciable degree of dissociation of the disulfide linkage. Sequential fragmentation via multiple alternative pathways tended to generate very complex spectra. CID of the anions proceeded through CH₂? S cleavages extensively but relatively few structurally diagnostic ions were generated. The incorporation of Au(I) into the molecule via ion/ion reactions followed by CID gave rise to many structurally relevant dissociation products, particularly for the [M+Au+H]²⁺ species. The products were generated by a combination of S? S bond cleavage and amide bond cleavage. ETD of the [M+3H]³⁺ ion generated rich sequence information, as did CID of the electron transfer products that did not fragment directly upon electron transfer. The electron transfer results suggest that both the S? S bond and an N? C_α bond can be cleaved following a single electron transfer reaction. Copyright © 2009 John Wiley & Sons, Ltd.     

Important role of the 3-mercaptopropionamide moiety in glutathione: promoting effect on decomposition of the adduct of glutathione with the oxoammonium ion of TEMPO

Cyclic voltammetry of TEMPO in aqueous 0.1 M NaOH in the presence of glutathione (GSH) or cysteine (Cys) indicated the following points: (i) Both of the thiols rapidly formed adducts 3 with oxoammonium ion 1 anodically generated from TEMPO. (ii) 3 generated from GSH entered a succeeding reaction that generated N-oxide anion 2- (the reduced TEMPO). (iii) 3 produced from Cys remained intact over the time scale of voltammetry. A structural feature of GSH was considered to contribute to the observed behavior of this tripeptide. Possible structural features were evaluated by screening various thiols on the basis of whether they provided GSH-like voltammetric results. The 3-mercaptopropionamide group with an amide hydrogen in GSH was determined to be responsible for the observed difference between GSH and Cys. The likely function is to transform 3 from GSH into a 5-imino-1,2-oxathiolane intermediate, thereby releasing 2-. Product analysis for reactions of model thiols representing GSH and Cys with 1 provided support for this argument and suggested that the reaction of GSH or Cys with 1 would produce the corresponding disulfides, regardless of whether a five-membered ring intermediate was formed. The proposed function of the 3-mercaptopropionamide moiety of GSH may provide useful insight for the molecular design of exogenous thiol compounds as novel drugs for the treatment of GSH-depletion-related disorders.     

Intramolecular disulfide bond between catalytic cysteines in an intein precursor

Protein splicing is a self-catalyzed and spontaneous post-translational process in which inteins excise themselves out of precursor proteins while the exteins are ligated together. We report the first discovery of an intramolecular disulfide bond between the two active-site cysteines, Cys1 and Cys+1, in an intein precursor composed of the hyperthermophilic Pyrococcus abyssi PolII intein and extein. The existence of this intramolecular disulfide bond is demonstrated by the effect of reducing agents on the precursor, mutagenesis, and liquid chromatography-mass spectrometry (LC-MS) with tandem MS (MS/MS) of the tryptic peptide containing the intramolecular disulfide bond. The disulfide bond inhibits protein splicing, and splicing can be induced by reducing agents such as tris(2-carboxyethyl)phosphine (TCEP). The stability of the intramolecular disulfide bond is enhanced by electrostatic interactions between the N- and C-exteins but is reduced by elevated temperature. The presence of this intramolecular disulfide bond may contribute to the redox control of splicing activity in hypoxia and at low temperature and point to the intriguing possibility that inteins may act as switches to control extein function.     

Switching the Switch: Ligand Induced Disulfide Formation in HDAC8

Human histone deacetylase 8 is a well-recognized target for T-cell lymphoma and particularly childhood neuroblastoma. PD-404,182 was shown to be a selective covalent inhibitor of HDAC8 that forms mixed disulfides with several cysteine residues and is also able to transform thiol groups to thiocyanates. Moreover, HDAC8 was shown to be regulated by a redox switch based on the reversible formation of a disulfide bond between cysteines Cys₁₀₂ and Cys₁₅₃. This study on the distinct effects of PD-404,182 on HDAC8 reveals that this compound induces the dose-dependent formation of intramolecular disulfide bridges. Therefore, the inhibition mechanism of HDAC8 by PD-404,182 involves both, covalent modification of thiols as well as ligand mediated disulfide formation. Moreover, this study provides a deep molecular insight into the regulation mechanism of HDAC8 involving several cysteines with graduated capability to form reversible disulfide bridges.     

Characterization of disulfide linkages and disulfide bond scrambling in recombinant human macrophage colony stimulating factor by fast-atom bombardment mass spectrometry of enzymatic digests

Fast-atom bombardment mass spectrometry was used to study disulfide bonding patterns in heat-denatured human recombinant macrophage colony stimulating factor (rhM-CSF). The heat-denaturated protein was studied by analysis of the pattern of peptides in the proteolytic digests. Native rhM-CSF is a homodimer with intramolecular disulfide linkages between Cys7–Cys90, Cys48–Cys139, and Cys102–Cys146 and intermolecular linkages between Cys31-Cys31, and the pairs Cys157 and Cys159. Brief heating for 1 min leads to partial disulfide bond scrambling. In addition to the native disulfide bonds between Cys7–Cys90, Cys48–Cys139, and Cys31-Cys31, nonnative disulfide bonds were detected between Cys48–Cys90 and Cys48–Cys102. When heated for 5 min the disulfide bonds of rhM-CSF are completely scrambled and lead to nonnative intramolecular disulfide bonds between Cys48–Cys102 and Cys90–Cys102 and one intermolecular disulfide bond between Cys102–Cys102.     

Highly selective fluorescence turn-on probe for glutathione

An activated coumarin-3-phenyl enone (1) by an intramolecular hydrogen bond has shown a selective and ratiometric response toward GSH and Cys over other natural amino acids through the Michael addition reaction of a thiol group to 1. When GSH was added to 1, a prominent color change together with a fluorescence turn-on property was observed so that submillimolar GSH was detectable.     

Fluorescent coumarin thiols measure biological redox couples

In this report we present a new chemical probe, 3-HTC, that can reversibly and ratiometrically measure the thiol-disulfide equilibrium of biological systems. 3-HTC is composed of a coumarin that has a thiolate directly conjugated to its extended aromatic π system while formation of a disulfide attenuates this conjugation. The fluorescence and absorption properties of 3-HTC are therefore very sensitive to the redox state of its thiol. 3-HTC reacts reversibly with thiols and disulfides enabling its use to measure dynamic GSH/GSSH ratios in vitro as well as to monitor the reversible redox status of whole cell lysates.     

Substituent effects on the chain‐transfer behavior of 7‐methylene‐2‐methyl‐1,5‐dithiacyclooctane in the presence of disulfides and thiols

The chain‐transfer behavior of 7‐methylene‐2‐methyl‐1,5‐dithiacyclooctane was investigated in the presence of four chain‐transfer agents: thiophenol (PhSH), thiobenzoic acid (BzSH), diphenyl disulfide (PhSSPh), and dibenzoyl disulfide (BzSSBz). The chain‐transfer constants for these compounds at 60 °C were 0.38 (PhSH), 0.76 (BzSH), 0.24 (PhSSPh), and 0.05 (BzSSBz). The variations in the thiol chain‐transfer constants could be explained in terms of the stability of the resulting radicals. The chain transfer to the disulfides, however, appeared to be determined by the electronic character of the disulfide bond, and this suggests that the transfer took place via an addition–fragmentation mechanism. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 4421–4425, 2002     

Nanoparticles comprising a mixed monolayer for specific bindings with biomolecules

This work presents a strategy of using mixed monolayer protected nanoparticles for specific interactions with target biological molecules. The mixed monolayer is composed of a shielding component and a capture component. The shielding component utilizes ethylene glycol oligomers to prevent nonspecific binding with biomolecules. The capture component is chosen to specifically interact with the target of interest, such as a protein molecule. Such a concept was demonstrated by two synthetic systems. The first one is gold nanoparticles protected by a mixed monolayer of tri(ethylene glycol) thiol (EG(3)-SH) and tiopronin (Tp), which was prepared by a one-step synthesis. Surface chemical composition studies using (1)H NMR spectroscopy revealed that the reactivity of EG(3)-SH is 3 times as high as that of Tp in the nanoparticle formation. Gel electrophoresis analysis identified a critical ratio of (EG(3)-S-)/Tp on the nanoparticle surface above which no nonspecific binding occurred. By further derivatizing Tp into a biotin group, we synthesized Au(-S-EG(3))(n)/Tp-biotin particles that bind specifically to streptavidin with negligible nonspecific binding. The second system is gold nanoparticles protected by a mixed monolayer of EG(3)-SH and glutathione (GSH). By controlling the feeding ratio of EG(3)-SH and GSH, we made Au(-S-EG(3))(n)/GSH particles that bind specifically to gultathione-S-transferase (GST) with negligible nonspecific binding.     

Direct identification of a novel disulfide bond linkage system of new isolated isomer (isomer V) in recombinantly produced h-IGF-I

Insulin-like growth factor I (IGF-I or somatomedin C) is a serum polypeptide with three intramolecular disulfide bonds. In the course of synthesis by the recombinant DNA method, three disulfide bond isomers, all of which have Cys18-Cys61 with three combinations of two disulfide bonds formed by Cys6, Cys47, Cys48 and Cys52, were identified. Natural type, isomer II, was proved to have a Cys6-Cys48, Cys18-Cys61, Cys47-Cys52 disulfide bond system. Now, the fourth isomer, isomer V which doesn't have Cys18-Cys61 disulfide, has been isolated, and its novel disulfide bond linkage system was identified by a chemical synthetic method. The supposed conformation constrained in 3D structure for isomer V would be discussed for its biological activity.