HPLC simultaneous analysis of thiols and disulfides: on-line reduction and indirect fluorescence detection without derivatization

A liquid chromatography method with indirect fluorescence detection has been developed for simultaneous detection of cysteine, cystine, homocysteine, homocystine, glutathione and glutathione disulfide. After separation in their native forms, a post-column solution of tris(2-carboxyethyl)phosphine (TCEP) at 32 mM, pH 8 is added to reduce the disulfides on-line to the corresponding thiol. The effluent is then merged with a second post-column solution of the highly fluorescent complex Cd(HQS)(2)(2-). The cadmium is complexed by the eluting thiols, effectively quenching the fluorescence. Optimization of the separation, the on-line reduction and the indirect fluorescence detection are discussed. Detection limits from 0.3-4.3 microM (0.04 to 2.6 ppm) are achieved for the six analytes in a 20 min separation.     

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.     

Synthesis of fluorescent long-chain thiols/disulfides as building-blocks for self-assembled monolayers preparation

New symmetrical disulfides together with the corresponding thiols bearing fluorescent end-groups have been synthesized as building-blocks for self-assembled monolayers (SAMs). The synthesis has been accomplished starting from aromatic nitrogen heterocycles in three steps. The conversion of the tosylated intermediate into the final disulfide is accomplished by use of sodium hydrogen sulfide (NaSH). Both products (thiols and disulfides) were isolated and characterized.      

Determination of sulbutiamine and its disulfide derivatives in human plasma by HPLC using on-line post-column reactors and fluorimetric detection

Summary A new direct HPLC procedure for the simultaneous determination of sulbutiamine (Arcalion) and other thiamine disulfides in human plasma has been developed. The method involves an automated solidphase extraction on octadecylsilyl (C18) cartridges and chromatographic separation of the compounds on an RP Select B column with gradient elution using methanol and phosphate buffer. Detection was by fluorescence of the resulting thiochromes obtained from two on-line post-column reactors. Optimization of post-column reaction parameters has been achieved. This method has been proved to be highly selective for the determination of the thiamine disulfide derivatives and quantitation limits of 5 ng·ml^–1 were obtained for each compound in human plasma. Linearity was in the range 5–200 ng·ml^–1. Precision and accuracy were also demonstrated by within-day and between-day assays, and showed the good reliability of the method.     

Photochemical desulfurization of thiols and disulfides

A visible light-induced desulfurization process for thiols and disulfides using triethylphosphite and triethylborane is reported. The reaction can be effected on a range of organic molecules having either primary, secondary or tertiary thiol groups and disulfides without the need of protecting groups. Thus, after treating l-cystine 7, l-cystine dimethylester 8, thioctic amide 9 and glutathione disulfide 10, first with tributylphosphine, later with triethylborane/triethylphosphite under irradiation in a one-pot reaction, the corresponding desulfurized compounds l-Ala, l-Ala, 1-octanamide and γ-l-Glu-l-Ala-Gly, respectively, are prepared in high yields with retention of configuration.     

Zn(II) complexes of glutathione disulfide: structural basis of elevated stabilities

Glutathione disulfide (GSSG), a long disregarded redox partner of glutathione (GSH), is thought to participate in intracellular zinc homeostasis. We performed a concerted potentiometric and NMR spectroscopic study of protonation and Zn(II) binding properties of GSSG ((γECG)(2)) and a series of its nine analogs with C-terminal modifications, tripeptide disulfides: (γECS)(2), (γECE)(2), (γECG-NH(2))(2), (γECG-OEt)(2), and (γEcG)(2); dipeptide disulfides, (γEC)(2) and (γEC-OEt)(2); and mixed disulfides, γECG-γEC and γECG-γEC-OEt. The acid-base and Zn(II) complexation properties in this group of compounds are strictly correlated to average C-terminal electrostatic charges. In particular, it was demonstrated that GSSG assumes a bent (head-to-tail) conformation in solution at neutral pH, which is controlled by electrostatic attraction between the protonated γ-amino groups of the Glu residue and the deprotonated C-terminal Gly carboxylates. This interaction modulates the ability of GSSG to coordinate Zn(II), both indirectly, by affecting the basicities of the amino groups, and directly, through the participation of the Gly carboxylates in the outer coordination sphere of the Zn(II) ion. A specific coiled structure of the major [Zn-GSSG](2-) complex is additionally stabilized by the formation of hydrogen bonds between glycinyl carboxylates and two Zn(II)-coordinated water molecules. The elevated stability of Zn(II)-GSSG complexes was demonstrated by competition with FluoZin-3, a fluorescent sensor with high Zn(II) affinity, commonly used in in vitro and in vivo studies. The potential biological functions and reactivity of GSSG complexes of Zn(II) ions are discussed.     

Effects of reducing agents on the determination of thiolic compounds in the presence of their disulfides using bimane pre-column derivatization

Summary The influence of reducing agents (sodium borohydride, tributylphosphine, dithioerythritol and dithiothreitol) on the conversion of disulfides into their parent thiols, with specific application to cysteine was investigated. Dithioerythritol and dithiothreitol were found to be most suitable for this reaction. A contact time of one hour at room temperature provided quantitative reduction as tested using cystine as a pure disulfide standard. A modified fluorescence labelling procedure with monobromobimane followed by reversed-phase HPLC allows quantitation of the parent thiol and the disulfide content when completing the labelling reaction with and without preliminary treatment with reducing agent. The effects of various bimane reducing agent ratios on the yield of the reaction are discussed. Precautions should be taken when dealing with complex matrices with respect to reagent concentrations and ratios.     

"Forbidden" disulfides: their role as redox switches

Seminal studies by Richardson and Thornton defined the constraints imposed by protein structure on disulfide formation and flagged forbidden regions of primary or secondary structure seemingly incapable of forming disulfide bonds between resident cysteine pairs. With respect to secondary structure, disulfide bonds were not found between cysteine pairs: A. on adjacent beta-stands; B. in a single helix or strand; C. on non-adjacent strands of the same beta-sheet. In primary structure, disulfide bonds were not found between cysteine pairs: D. adjacent in the sequence. In the intervening years it has become apparent that all these forbidden regions are indeed occupied by disulfide-bonded cysteines, albeit rather strained ones. It has been observed that sources of strain in a protein structure, such as residues in forbidden regions of the Ramachandran plot and cis-peptide bonds, are found in functionally important regions of the protein and warrant further investigation. Like the Ramachandran plot, the earlier studies by Richardson and Thornton have identified a fundamental truth in protein stereochemistry: "forbidden" disulfides adopt strained conformations, but there is likely a functional reason for this. Emerging evidence supports a role for forbidden disulfides in redox-regulation of proteins.     

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.     

Characterization of the disulfides of bio-thiols by electrospray ionization and triple-quadrupole tandem mass spectrometry

Glutathione and other intracellular low molecular mass thiols act both as the major endogenous antioxidant and redox buffer system and, as recently highlighted, as an important regulator of cellular homeostasis. Such cellular functions are mediated by protein thiolation, a newly recognized post-translational modification which involves the formation of mixed disulfides between GSH and key disulfide-linked Cys residues in the native protein structure. It is also well known that thiol-seeking heavy metals, such as mercury, cadmium and lead, may interfere in this regulatory system, thus disrupting the cellular functioning. To identify such mixed disulfides in order to investigate their biological role, 15 homo- and heterodimeric disulfides were prepared by air oxidation of binary mixtures containing cysteine, homocysteine, penicillamine, N-acetylcysteine, N-acetylpenicillamine and glutathione and their protonated molecules were characterized by mass spectrometry. Collisionally activated unimolecular decomposition of protonated homo- and heterodimeric disulfides generated by electrospray ionization gives rise to fission of the disulfide system both between the two sulfur atoms and across the C--S bonds, to yield structurally specific fragments which allow one to define the structure of the compounds and to discriminate between isomeric compounds. Fission between the sulfur atoms yields a pair of R--S(+) ions and, in some cases, also the complementary fragments corresponding to the protonated amino acids. Fission across the C--S bonds mainly occurs in the disulfides of N-acetylcysteine and N-acetylpenicillamine and gives rise to non-S-containing fragments formally similar to those obtained from some mercapturic acids. The complementary fragments, formally connected as R--S--S(+) ions are also observed. Fragmentation of glutathione disulfides mainly shows the characteristic loss of the terminal gamma-linked glutamic acid and little, if any, fragmentation of the disulfide system.     

Mass spectral fragmentation of bis(1-arylimino-1-alkyl/arylthiomethyl) disulfides under positive electrospray ionization conditions

The mass spectral fragmentation of bis(1-arylimino-1-alkyl/arylthiomethyl) disulfides under positive electrospray ionization conditions has been investigated. The protonated molecular ions predominantly produce trisulfide thiocarbonimidate ions by elimination of a molecule of arylisonitriles. The trisulfide thiocarbonimidate ions further lose a sulfur atom to yield disulfide thiocarbonimidate ions and then eliminate a molecule of thiols or disulfanes to give rise to the corresponding dithioisonitriliums or thioisonitriliums, respectively. Both alkyl dithio and thioisonitriliums can undergo a McLafferty-type rearrangement to yield the corresponding protonated arylisothiocyanoates. The major characteristic of bis(1-arylimino-1-alkyl/arylthiomethyl) disulfides is loss of diverse neutral molecules, such as isonitriles, thiols, thioaldehydes, and disulfanes under positive electrospray ionization conditions.     

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.     

Synthesis of glycosyl disulfides containing an α-glycosidic linkage

A wide range of symmetrical and unsymmetrical glycosyl disulfides is synthesized with focus on the use of α-glycosyl thiols. Oxidation of α-glycosyl thiols with iodine leads to symmetrical α,α-glycosyl disulfides, while unsymmetrical disulfides are readily synthesized from α- and β-glycosyl thiols under the action of DDQ. Thus, glycosyl disulfides containing at least one α-glycosidic linkage are made available.     

Urinary mesna and total mesna measurement by high performance liquid chromatography with ultraviolet detection

We describe in this report a method for determination of mesna and total mesna in urine by high performance liquid chromatography with ultraviolet detection. The method involves a treatment of the urine sample with tri-n-butylphosphine in order to convert mesna disulfides to its reduced counterpart mesna, ultraviolet labelling with 2-chloro-1-methylquinoluinium tetrafluoroborate, reversed-phase HPLC separation, and detection and quantitation at 350 nm. The result corresponds to total mesna that is sum of mesna, dimesna and its mixed disulfides with endogenous thiols. For determination of mesna the reduction step is omitted. Content of disulfide forms of mesna can be calculated by subtracting the concentration of mesna from the total mesna concentration. The separation of 2-S-quinolinium derivatives of mesna from those of endogenous urinary thiols and internal standard was achieved on an analytical Waters Nova-Pak C18 (150 mm × 3.9 mm, 5 μm) column. A mixture of an aqueous solution of pH 2.3, 0.05 M trichloroacetic acid and acetonitrile (88:12, v/v) was used as a mobile phase at flow rate of 1.2 ml/min and ambient temperature.The assay for mesna and total mesna in urine was proved to be linear over the studied ranges of 0.2-30 and 0.2-800 nmol/ml urine, respectively. The mean recoveries over the calibration ranges were 95.4% for mesna and 99.7% for total mesna. The lower limits of detection and quantitation were 0.1 and 0.2 nmol/ml for both the procedures, respectively. The imprecision did not exceed 8.5%. No interference from endogenous substances was observed.     

Oxidation/reduction interconversion of thiols and disulfides using hydrogen and oxygen catalyzed by a rhodium complex

RhH(PPh₃)₄ catalyzes reduction of disulfides to thiols by hydrogen and RhH(PPh₃)₄/1,4-bis(diphenylphosphino)butane (dppb) catalyzes oxidation of thiols to disulfides by oxygen.     

Simultaneous determination of thiols and disulfides by high-performance liquid chromatography with fluorescence detection

The proposed simultaneous determination of thiols and disulfides requires 4- (aminosulfonyl)- 7-fluor-2,1,3,-benzoxadiazole (ABD-F) as the pre-column derivatization reagent for thiols and ammonium 7-fluoro-2,1,3,-benzoxadiazole-4-sulfonate (SBD-F) for disulfides followed by chromatography. The thiols and disulfides in solution are treated with ABD-F at 60°C for 5 min in pH 9.3 borate buffer containing 1 nM disodium-EDTA. After removal of excess of ABD-F with ethyl acetate, the remaining disulfides in the aqueous phase are treated with SBD-F at 60°C for 20 min in the presence of tri-n-butylphosphine, a reducing agent. The ABD-thiols and SBD-thiols thus produced are separated by reversed-phase chromatography and detected by fluorimetry (380- nm excitation, 510 - nm emission). SBD-cystein, SBD-homocystein, ABD-homocysteine, ABD-cysteine, SBD-glu- tathione, ABD-homocystein, SBD-N-acetylcystein, ABD-glutatione and ABD-N-acetylcysteine are well separated by linear gradient elution from 0.15 M H₃PO₄/CH₃CN (96:4) to 0.15 M H₃PO₄/CH₃CN (85:15) over 30 min followed by isocratic elution with 0.15 M H₃PO₄/CH₃CN (85:15) fro 10 min. The detection limits for the derivatives are in the range 0.09–0.9 pmol. When the method was applied to the determination of thiols and disulfides in rat tissues, cystein (0.75 μmol g^-1) and cystine (0.62 μmol g^-1) were obtained in kidney and reduced glutathione (1.4–3.4 μmol g^-1) was observed in liver, spleen, heart and testicle.     

Characterization of organic disulfides by paper electrophoresis after reduction to 2-alkyl(aryl)thio-1-methylpyrinium lodides

Summary A procedure is outlined that permits characterization of alkyl and aryl disulfides. The procedure utilizes the reduction of disulfides with lithium aluminium hydride or sodium bis(2-methoxyethoxy)aluminium hydride to the corresponding thiols followed by reaction with 2-chloro-1-methylpyridinium iodide. 2-Alkyl(aryl)thio-1-methyl-pyridinium iodides so formed are separated by high-voltage paper electrophoresis in boric acid — diethanolamine — EDTA buffer at pH 7.6; the relative mobilities measured after visualization with iodine vapor. Several disulfides, including primary, secondary, tertiary and aryl compounds, have been characterized in this way.Presented in part at the Ninth International Symposium on Organic Sulfur Chemistry, June 9–14, 1980, Riga, USSR.     

On-line redox derivatization liquid chromatography using double separation columns and one derivatization unit

A new on-line redox derivatization technique using double separation columns and one redox derivatization unit was presented for enhancement of separation selectivity of HPLC. This on-line redox derivatization HPLC system consisted of two separation columns and one redox derivatization unit placed between them. The redox reaction proceeds in the derivatization unit so that an analyte compound migrates as its original form in the first column, while as its oxidized or reduced form in the second column. The retention of the analytes is controlled by the lengths of the two separation columns in this system. We adopted a small column packed with porous graphitic carbon (PGC) as a redox derivatization unit and two C18 silica columns treated with hexadecyltrimethylammonium chloride as separation columns. The redox activity of PGC and the efficiency of the on-line redox derivatization HPLC system for enhancement of separation selectivity were investigated using EDTA complexes of some metal ions. Original untreated PGC and PGC treated with hydrogen peroxide completely oxidized Co(II)-EDTA and converted it to Co(III)-EDTA, while the other metal complexes eluted as their original oxidation states throughout the system. Selective separation and determination of cobalt in a reference copper alloy by the developed method were demonstrated.     

High-performance liquid chromatography of sulfur-containing amino acids and related compounds with amperometric detection at a modified electrode

Organic disulfides generally are not oxidized at bare electrodes under conditions that are suited to routine amperometric detection, and thiols are typically oxidized in a manner that leads to partial blockage of the surface. Modification of a carbon electrode with a film of Ru(III,IV) oxide stabilized with cyanocross-links permits the amperometric detection of cystine, cysteine, glutathione, methionine, and glutathione disulfide under conditions compatible with their chromatographic separation on a strong cation-exchange column. Detection limits of 0.2-0.6 microM and linear dynamic ranges of at least 1-50 microM were obtained. The electrode was stable for at least 11 days with a pH 1 citrate, phosphate mobile phase.     

部分还原和分步序列测定法定位虎纹捕鸟蛛毒素-Ⅳ二硫键

结合部分还原和分步序列测定法确定了虎纹捕鸟蛛毒素-的二硫键配对方式.在pH=3和40℃的条件下与还原剂三羧甲基磷酸(TCEP)反应10min,利用RP-HPLC分离并分别收集含有一对和两对二硫键被还原的中间体,分别与0.5mol/L碘乙酰胺溶液(pH=8.3)反应1min,使游离巯基烷基化后,测定各中间体的氨基酸序列,从而确定虎纹捕鸟蛛毒素-的3对二硫键分别为Cys2-Cys17,Cys9-Cys24和Cys-Cys31(1-4,2-5和3-6).