A study of the glutathione metaboloma peptides by energy-resolved mass spectrometry as a tool to investigate into the interference of toxic heavy metals with their metabolic processes

To better understand the fragmentation processes of the metal-biothiol conjugates and their possible significance in biological terms, an energy-resolved mass spectrometric study of the glutathione conjugates of heavy metals, of several thiols and disulfides of the glutathione metaboloma has been carried out. The main fragmentation process of gamma-glutamyl compounds, whether in the thiol, disulfide, thioether or metal-bis-thiolate form, is the loss of the gamma-glutamyl residue, a process which ERMS data showed to be hardly influenced by the sulfur substitution. However, loss of the gamma-glutamyl residue from the mono-S-glutathionyl-mercury (II) cation is a much more energetic process, possibly pointing at a strong coordination of the carboxylic group to the metal. Moreover, loss of neutral mercury from ions containing the gamma-glutamyl residue to yield a sulfenium cation was a much more energetic process than those not containing them, suggesting that the redox potential of the thiol/disulfide system plays a role in the formal reduction of the mercury dication in the gas phase. Occurrence of complementary sulfenium and protonated thiol fragments in the spectra of protonated disulfides of the glutathione metaboloma mirrors the thiol/disulfide redox process of biological importance. The intensity ratio of the fragments is proportional to the reduction potential in solution of the corresponding redox pairs. This finding has allowed the calculation of the previously unreported reduction potentials for the disulfide/thiol pair of cysteinylglycine, thereby confirming the decomposition scheme of bis- and mono-S-glutathionyl-mercury (II) ions. Finally, on the sole basis of the mass spectrometric fragmentation of the glutathione-mercury conjugates, and supported by independent literature evidence, an unprecedented mechanism for mercury ion-induced cellular oxidative stress could be proposed, based on the depletion of the glutathione pool by a catalytic mechanism acting on the metal (II)-thiol conjugates and involving as a necessary step the enzymatic removal of the glutamic acid residue to yield a mercury (II)-cysteinyl-glycine conjugate capable of regenerating neutral mercury through the oxidation of glutathione thiols to the corresponding disulfides.     

Kinetics and Equilibria of the Thiol/Disulfide Exchange Reactions of Somatostatin with Glutathione

Rate and equilibrium constants are reported for the thiol/disulfide exchange reactions of the peptide hormone somatostatin with glutathione (GSH). GSH reacts with the disulfide bond of somatostatin to form somatostatin-glutathione mixed disulfides (Cys(3)-SH, Cys(14)-SSG and Cys(3)-SSG, Cys(14)-SH), each of which can react with another molecule of GSH to give the reduced dithiol form of somatostatin and GSSG. The mixed disulfides also can undergo intramolecular thiol/disulfide exchange reactions to re-form the disulfide bond of somatostatin or to interconvert to the other mixed disulfide. Analysis of the forward and reverse rate constants indicates that, at physiological concentrations of GSH, the intramolecular thiol/disulfide exchange reactions that re-form the disulfide bond of somatostatin are much faster than reaction of the mixed disulfides with another molecule of GSH, even though the intramolecular reaction involves closure of a 38-membered ring. Thus, even though the disulfide bond of somatostatin is readily cleaved by thiol/disulfide exchange, it is rapidly reformed by intramolecular thiol/disulfide exchange reactions of the somatostatin-glutathione mixed disulfides. By comparison with rate constants reported for analogous reactions of model peptides measured under random coil conditions, it is concluded that disulfide bond formation by intramolecular thiol/disulfide exchange in the somatostatin-glutathione mixed disulfides is not completely random, but rather it is directed to some extent by conformational properties of the mixed disulfides that place the thiol and mixed disulfide groups in close proximity. A reduction potential of -0.221 V was calculated for the disulfide bond of somatostatin from the thiol/disulfide exchange equilibrium constant.     

Studies and microdetermination of some organic sulfur functions by reduction with titanium(III) sulfate solution

Micromethods are described for the determination of organic sulfoxides and disulfides based on reduction with titanium (III) sulfate solution, the excess of which is titrated with standard ferric alum as usual. The procedures developed depended on the nature of the compound whether aromatic or aliphatic. Quantitative reduction of sulfoxides was achieved in presence of ammonium thiocyanate and sodium acetate but whereas aryl sulfoxides required heating at 70 °C for 30 minutes, the alkyl members demanded heating at 90 °C for one hour. Complete reduction of disulfides was possible in presence of acetate buffer and heating at 50 °C for 15 minutes for aromatic disulfides and at 80 °C for 30 minutes in case of the aliphatic ones. a Calculated by dividing the total titer in a ratio of 2:12. b Calculated using the total titer consumed. Sulfonyl halides, thiocyanates, and isothiocyanates do not react with the titanium(III) solution. The effect of the various sulfur functions on the determination of the nitro group with titanium(III) was also studied and various nitro-substituted sulfur compounds were successfully analyzed.     

Intermediates in the reduction of the antituberculosis drug PA-824, (6S)-2-nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine, in aqueous solution

The reduction chemistry of the new anti-tuberculosis drug PA-824, together with a more water-soluble analogue, have been investigated using pulse and steady-state radiolysis in aqueous solution. Stepwise reduction of these nitroimidazo-dihydrooxazine compounds through electron transfer from the CO(2) (-) species revealed that, unlike related nitroimidazoles, 2-electron addition resulted in the reduction of the imidazole ring in preference to the nitro group. In mildly acidic solution a nitrodihydroimidazo intermediate was formed, which was reduced further to the amine product. In both alkaline and neutral solution, an intermediate produced on 2-electron reduction was resistant to further reduction and reverted to parent compound on extraction or mass spectrometric analysis of the solution. The unusual reduction chemistry of these nitroimidazole compounds, exhibiting ring over nitro group reduction, is associated with alkoxy substitution in the 2-position of a 4-nitroimidazole. The unique properties of the intermediates formed on the reduction of PA-824 need to be considered as playing a possible role in its bactericidal action.     

A unique autocatalytic process and evidence for a concerted-stepwise mechanism transition in the dissociative electron-transfer reduction of aryl thiocyanates

Electrochemical reduction of p-methyl-, p-methoxy-, and 3,5-dinitrophenyl thiocyanates as well as p-methyl- and p-methoxyphenyl disulfides was investigated in acetonitrile at an inert electrode. This series of compounds reveals a striking change in the reductive cleavage mechanism of the S-CN bond in thiocyanates as a function of the substituent on the aryl ring of the aryl thiocyanate. With nitro substituents, a stepwise mechanism, with an anion radical as the intermediate, takes place. When electron-donating groups (methyl and methoxy) are present, voltammetric as well as convolution analyses provide clear evidence for a transition between the concerted and stepwise mechanisms based on the magnitude of the transfer coefficient alpha. Moreover, a very interesting autocatalytic process is involved during the electrochemical reduction of these compounds. This process involves a nucleophilic substitution reaction on the initial aryl thiocyanate by the electrochemically generated arenethiolate ion. As a result of this unusual process, the electrochemical characteristics (peak potential and peak width) of the investigated series are concentration dependent.     

Facile synthesis of N-10 flavin disulfides

A facile, high yield route to N-10 flavin disulfides is described. The key feature is catalytic reduction of the nitro group to an amine function in an organic sulfide using platinum sulfide on charcoal.     

Kinetic analyses of disulfide formation between thiol groups attached to linear poly(acrylamide)

Kinetic analyses were carried out for formation of disulfide crosslinkages between thiol groups on linear polymers, poly(acrylamide‐co‐N‐acrylcysteamine) (P‐SH). Disulfide crosslinkages were formed by auto‐oxidation of pendant thiol groups or through the thiol‐disulfide exchange reaction induced by addition of disulfide compounds gluthathione. In the auto‐oxidation reaction, the rate constant for disulfide formation highly depended on pH values of the reaction mixtures and the P‐SH concentrations. Gelation rate is too slow to enclose living cells into hydrogel under physiological pH 7.4. The hydrogel formation rate can be accelerated by addition of disulfides, such as oxidized glutathione. In the later case, oxygen in the reaction mixture is not consumed. The thiol‐disulfide exchange reaction is much more suitable for the cell encapsulation than the thiol auto‐oxidation reaction. These findings give a basis for enclosure of living cells in a hydrogel. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Synthesis of some polyureas based on diamino derivatives of bis-1, 3, 4-oxadiazole

Ortho-, meta, and para-nitrobenzoyl derivatives of the dihydrazides of succinic, glutaric, adipic, and azelaic acids are prepared, from which are obtained nitro and amlno derivatives of bis-1,3,4- diazole. Various conditions for synthesizing these compounds are investigated. A series of polyureas is obtained by reacting diamino derivatives of bis-1,3,4-oxadiaole with diisocyanates, and some of their properties are investigated.     

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     

Boron dipyrromethene dyes: a rational avenue for sensing and light emitting devices

Boron dipyrromethene dyes bearing nitro, amino, isocyanate and isothiocyanate functions were readily prepared under mild conditions. Various combinations allow to produce urea, diurea, thiourea, dithiourea in the 3, 4 and 5-substitution positions of the appended phenyl group. Condensation of the 3,4-substituted diamino derivative with 1,10-phenanthroline-5,6-dione and 6-formyl-2-methylpyridine allow to prepare dipyridophenazine and indole derivatives. The 3,5-dinitro-substituted indacene dye was characterized by an X-ray molecular structure showing a pronounced tilt angle of the dinitrophenyl group relative to the indacene core (approximately 84 degrees) whereas one nitro groups is basically coplanar with the phenyl ring and the second titled by approximately 21 degrees. The optical properties of these dyes reveals on/off switching of the fluorescence from the nitro to the amino compounds and further to the urea likely understood in the framework of an photoinduced electron transfer process.     

Synthesis, electrochemistry and self-assembled monolayers of novel tetrathiafulvalene (TTF) and pi-extended TTF (exTTF) disulfides

The synthesis of a new series of tetrathiafulvalene (TTF) and pi-extended TTF (exTTF) disulfides and the electrochemical properties of self-assembled monolayers derived from these compounds are described. When the intermediate bromides 3 and 7 were reacted with thiourea, followed by basic hydrolysis, the expected thiol formation was not observed and only disulfides were obtained. A mechanism is proposed to explain the self-oxidation process of these compounds. For the first time SAMs of exTTF derivatives were prepared. Electrochemical data for SAMs of 6 and 8 reveal a single two-electron chemically reversible oxidation process to form a dicationic state, typical of the exTTF system. The SAMs are stable over extended periods of time and show electrochemical stability upon repeated potential scans.     

A new method for reversible immobilization of thiol biomolecules based on solid-phase bound thiolsulfonate groups

A new method for the reversible immobilization of thiol bimolecules, e.g., thiolpeptides and thiolproteins, to beaded agarose and other solid phases is reported. The method consists of an activation and a coupling step. The activation is based on oxidation of disulfides (or thiol groups via disulfides) present in a solid phase by hydrogen peroxide at moderately acidic pH. This oxidation leads to disulfide oxides (thiolsulfinate groups of which the majority are further oxidized to thiolsulfonate). The thiolsulfonate groups react easily with thiol compounds, which become immobilized via disulfide bonds. The pH range for thiol coupling is wide (pH 5-8), but for most thiols the reaction seems to proceed faster at pH>7. The stability of the reactive group to hydrolysis, especially at neutral and weakly acidic pH, is very high. The activated gel, therefore, can be stored as a suspension at pH 5 for extended periods. The method has been used to reversibly immobilize glutathione, β-galactosidase, alcohol dehydrogenase, urease, and papain, all with exposed thiol groups as well as thiolated bovine serum albumin and sweet-potato β-amylase. Depending on the thiol content of starting thiol-agarose, thiol-sulfonate-agarose derivatives with different binding capacities can be obtained. Thus, up to 5.0 mg (16 μmol) glutathione and 15 mg thiol-protein/mL gel derivative have been immobilized.     

Distance‐dependent Enhancement in Raman Spectroscopy Probed by Conjugated Molecules with Different Molecular Lengths

In this study, the distance‐dependent enhancement effect in surface‐enhanced Raman scattering (SERS) was explored with molecules bearing different lengths of conjugated double bonds. These conjugated molecules were synthesized utilizing the diazotization‐coupling reaction allowing a thio group on one end and a nitro group on the other end. The thiol group allows the probed molecule to chemisorb on the surface of silver nanoparticles (AgNPs). The opposite end of each molecule contains a nitro group, which gives an intense SERS signal to show a fair and accurate comparison of the effect of chain length. The obtained SERS intensities were correlated with the chain lengths of these synthesized molecules, which ranged from 0.6 to 2.0 nm between the nitro and thiol groups. Based on these results, the electromagnetic field effect was mainly responsible for the signal enhancements in SERS measurements. Also, the obtained signals were exponentially decayed due to the distances of the surface of AgNPs. Based on the SERS intensities of the conjugated molecules, the contribution of CT effect to SERS for these examined molecules were limited.     

Umsetzungen von Dilithio-nitroalkanen und -allylnitroderivaten mit Carbonylverbindungen

Reactions of dilithio-nitroalkanes and dilithio-allynitroalkanes with carbonyl compounds Primary nitro compounds can by acylated via dilithium derivatives 5 with carbonic-acid derivatives to give α-nitro esters 6a – i and with carboxylic-acid esters and anhydrides to give α-nitroketones 6j – q . In the reaction of 1-nitro-1-buten with two mol-equiv. of butyllithium, the dilithium compound 10 is formed by successive Michael-addition and nitronate deprotonation. Dilithium derivatives 5 also react with ketones and benzaldehyde (→ 18a – g ); the nitro aldols 25 and 26 are likewise formed by addition of doubly deprotonated allylic nitro compounds. Some of the products have been further transformed by reduction or by Nef-reactions to the hydrochloride of the α-amino-acid 26 , to 2-amino-alcohols 28a and 28b , to α-hydroxyamino-acid esters 27a – c , to α-hydroxyimino esters 35 and 36 , to α-hydroxyimino ketones 31 and 33 , to the α-diketone 34 , and to the α-keto ester 37 .     

Hydrous pyrolysis of organic sulfur compounds: Species and distribution of secondary derivatives

The species and distributions of secondary compounds generated from eight organic sulfur compounds by way of hydrous pyrolysis were investigated. The results indicate that the formation of the secondary compounds and their structures and distribution depend on their thermal stability and the types of initial model compounds, as well as hydrous pyrolysis temperatures, while a large number and higher abundance of the secondary compounds appear to be formed mainly between 200 and 270 °C. Assignment of these secondary compounds indicates that alkyl thiols and sulfides are the most reactive compounds, producing a large number and relatively high amount of secondary organic thiols, sulfides, disulfides, sulfoxides and sulfones; while the secondary compounds generated from the thiophenic compounds are mainly low abundant methylated isomers of their own. Disulfidization, sulfidization and oxidation are the most significant mechanism(s) associated with the transformation of the initial thiols and sulfides model compounds. Alkyl thiophenes are only found to be formed from the alkyl thiol and sulfides, while it is noticed that thiophene, benzothiophene and dibenzothiophene are not genetically connected as they are not precursors of each other. Methylated thiophenic compounds are quantitatively insignificant but commonly present in the pyrolysates of thiophenes, benzothiophenes and dibenzothiophenes.     

Photooxidative coupling of thiophenol derivatives to disulfides

Disulfide bonds play an important role in determining the structure and stability of proteins and nanoparticles. Despite extensive studies on the oxidation of thiols for the synthesis of disulfides, little is known about the photooxidation of thiols, which may be a clean, safe, and economical alternative to the use of harmful and expensive metal-containing oxidants and catalysts. In this paper, we report the photooxidative coupling of thiophenol derivatives to disulfides. Para-substituted thiophenol derivatives, p-SHC(6)H(4)X (X = NO(2), COOH, Cl, and OCH(3)), are irradiated, and disulfides, X(2)(C(6)H(4))(2)S(2), are identified as the major photoproducts using Raman, UV-vis, IR, and NMR spectroscopies. For p-nitrothiophenol (pNTP), 4,4'-dinitrodiphenyldisulfide (DNDPDS) is produced in 81% yield. The product yield changes with pH, being the highest at pH ≈ 5, suggesting that both neutral thiol and anionic thiolate forms of pNTP are required for the photoreaction to occur. Excitation at 455 nm, at which the thiolate form of pNTP absorbs strongly, leads to the largest yield of DNDPDS, whereas very little DNDPDS is formed by excitation of the thiol form of pNTP at 325 nm. Our observations suggest that the photooxidation occurs via collisions of the electronically excited thiolate form of pNTP with the surrounding neutral thiol forms of pNTP. The photooxidation reaction happens regardless of the electron-withdrawing or electron-donating properties of the substituents if the pH and excitation wavelengths are properly chosen. The versatility of light and generality of the photooxidative coupling reaction of thiophenol derivatives may open new possibilities for selective and site-specific photocontrol of disulfide bond formation in biology and nanomaterial science as well as in synthetic chemistry.     

Selective reduction of peptide isothiazolidin-3-ones

Isothiazolidinones are a rare but potentially important chemical moiety in biochemistry. We report the identification of several thiol, phosphinate, and carbon nucleophiles that form covalent adducts by addition to the sulfenamide sulfur. This reduction is selective for isothiazolidinones over similar peptide disulfides. We synthesized a coumarin-based thioacid nucleophile which shows a marked fluorescence increase after addition to an isothiazolidinone sulfenamide bond. [structure: see text]     

Sulfur‐containing derivatives of 1,4‐naphthoquinone,part 1: Disulfide synthesis

Disulfides of 1,4‐naphthoquinone were synthesized, and different methods of their synthesis were investigated. High yields and purity of disulfides were obtained from the oxidation of thiol derivatives. The latter were prepared in high yields and purity from isothiuronic salts. The obtained disulfides are syntho‐ nes for compounds with a wide spectrum of biological activity. © 2005 Wiley Periodicals, Inc. Heteroatom Chem 16:205–211, 2005; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/hc.20112     

Mimetics of the Selenoenzyme Glutathione Peroxidase: Novel Structures and Unusual Catalytic Mechanisms

Glutathione peroxidase (GPx) mimetics comprise an important class of selenium-containing antioxidants that catalyze the destruction of biologically harmful peroxides in the presence of stoichiometric thiol reductants. The synthesis of two novel cyclic selenium compounds and their evaluation as GPx mimetics was achieved. The first is a cyclic seleninate ester that is formed in situ from the oxidation of allyl 3-hydroxypropyl selenide. The second is a spirodioxyselenurane that is similarly formed from di(3-hydroxypropyl) selenide. Both compounds were shown to be remarkably active catalysts in an assay based on the reduction of t-butyl hydroperoxide with benzyl thiol. The mechanisms of the catalytic cycles of the two novel selenium compounds were elucidated and were found to be distinct from each other and from that of GPx.     

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.