Suppression of thiol exchange reaction in the determination of reduced-form thiols by high-performance liquid chromatography with fluorescence detection after derivatization with fluorogenic benzofurazan reagent, 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate and 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole

The derivatization of the reduced-form thiols with SBD-F (7-fluoro-2,1,3-benzoxadiazole-4-sulfonate) and ABD-F (4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole) was studied. The yields of the derivatives of the reduced-form thiols (cysteine, homocysteine, reduced-form glutathione) with SBD-F at 60 degrees C for 45 min in the borate buffer (pH 9.3) were significantly decreased in the presence of the oxidized-form thiols (cystine, homocystine, oxidized-form glutathione) because of the thiol exchange reaction between the reduced-form and the oxidized-form thiols. The use of ABD-F at low temperature enabled the suppression of these thiol exchange reactions, and the recommended conditions were below 5 degrees C for 90 min in borate buffer (pH 9.3). These results suggest that ABD-F is a preferred derivatization reagent for the accurate determination of the reduced-form thiols in samples containing the oxidized-form thiols. In addition, it was also suggested that the derivatization of the reduced-form thiols should also be performed at low temperature when derivatization reagents such as o-phthalaldehyde (OPA) and monobromobimane (BrB) are used.     

Self-assembled monothiol-terminated hyperbranched polyglycerols on a gold surface: a comparative study on the structure, morphology, and protein adsorption characteristics with linear poly(ethylene glycol)s

Monothiol-terminated hyperbranched polyglycerols (HPGs) were synthesized by ring-opening polymerization of glycidol from partially deprotonated 2,2'-dihydroxyethane disulfide as the initiator and subsequent reduction of the disulfide group. Two molecular weights of HPG thiols were synthesized. The molecular weights of the polymers were determined by MALDI-TOF analysis, and the presence of thiol was verified by Ellman's assay. The self-assembly of HPG thiols on gold was studied and compared with that of linear poly(ethylene glycol) (PEG) thiols utilizing various surface analysis techniques. Monothiol-functionalized HPGs readily adsorbed to a gold surface and formed highly uniform thin films on the surface. The graft density of the HPG layer decreased with an increase in the molecular weight of the polymer. The amount of polymer on the surface increased with increasing incubation concentration and saturated above 6 g/L polymer concentration. Generally, HPG thiols gave lower graft density compared to linear PEG thiols of similar molecular weight. AFM morphological studies showed that HPG thiols form more uniform and smooth surface films compared to PEG thiols. Incubation of a polymer-coated surface (HPG thiols and PEG thiols) with bovine serum albumin and immunoglobulin showed that the high molecular weight hyperbranched polyglycerol was more resistant to protein adsorption than linear PEG of similar molecular weight or lower molecular weight HPG. The protein adsorption decreased with increasing graft density of the HPG chains on the surface. Our results show that HPG could be a good alternative to PEG in the development of nonfouling functional surfaces.     

Detecting thiols in a microchip device using micromolded carbon ink electrodes modified with cobalt phthalocyanine

This paper describes the fabrication and evaluation of a chemically modified carbon ink microelectrode to detect thiols of biological interest. The detection of thiols, such as homocysteine and cysteine, is necessary to monitor various disease states. The biological implications of these thiols generate the need for miniaturized detection systems that enable portable monitoring as well as quantitative results. In this work, we utilize a microchip device that incorporates a micromolded carbon ink electrode modified with cobalt phthalocyanine to detect thiols. Cobalt phthalocyanine (CoPC) is an electrocatalyst that lowers the potential needed for the oxidation of thiols. The CoPC/carbon ink composition was optimized for the micromolding method and the resulting microelectrode was characterized with microchip-based flow injection analysis. It was found that CoPC lowers the overpotential for thiols but, as compared to direct amperometric detection, a pulsed detection scheme was needed to constantly regenerate the electrocatalyst surface, leading to improved peak reproducibility and limits of detection. Using the pulsed method, cysteine exhibited a linear response between 10-250 microM (r(2) = 0.9991) with a limit of detection (S/N = 3) of 7.5 microM, while homocysteine exhibited a linear response between 10-500 microM (r(2) = 0.9967) with a limit of detection of 6.9 microM. Finally, to demonstrate the ability to measure thiols in a biological sample using a microchip device, the CoPC-modified microelectrode was utilized for the detection of cysteine in the presence of rabbit erythrocytes.     

Synthesis of alkenyl sulfides through the iron-catalyzed cross-coupling reaction of vinyl halides with thiols

We report here the iron-catalyzed cross-coupling reaction of alkyl vinyl halides with thiols. While many works are devoted to the coupling of thiols with alkyl vinyl iodides, interestingly, the known S-vinylation of vinyl bromides and chlorides is limited to 1-(2-bromovinyl)benzene and 1-(2-chlorovinyl)benzene. Investigation on the coupling reaction of challenging alkyl vinyl bromides and chlorides with thiols is rare. Since the coupling of 1-(2-bromovinyl)benzene and 1-(2-chlorovinyl)benzene with thiols can be performed in the absence of any catalyst, here we focus on the coupling of thiols with alkyl vinyl halides. This system is generally reactive for alkyl vinyl iodides and bromides to provide the products in good yields. 1-(Chloromethylidene)-4-tert-butyl-cyclohexane was also coupled with thiols, giving the targets in moderate yields.     

Colorimetric and ratiometric fluorescent chemosensor based on diketopyrrolopyrrole for selective detection of thiols: an experimental and theoretical study

A colorimetric and ratiometric fluorescent thiol probe was devised with diketopyrrolopyrrole (DPP) fluorophore. The probe gives absorption and emission at 523 and 666 nm, respectively. In the presence of thiols, such as cysteine, the absorption and emission band shifted to 479 and 540 nm, respectively. Correspondingly, the color of the probe solution changed from purple to yellow, and the fluorescence changed from red to yellow. The emission intensity at 540 nm was enhanced by 140-fold. The Stokes shift of probe 1 (107 nm) is much larger than the unsubstituted DPP fluorophore (56 nm). Mass spectral analysis demonstrated that besides the expected Michael addition of thiols to the C═C bonds, the CN groups of the malonitrile moieties also react with thiols to form 4,5-dihydrothiazole structure. Probe 1 was used for fluorescence imaging of intracellular thiols. In the presence of thiols, both the green and red channel of the microscopy are active. With removal of the intracellular thiols, signal can only be detected through the red channel; thus, ratiometric bioimaging of intracellular thiols was achieved. The ratiometric response of probe 1 was rationalized by DFT calculations. Our complementary experimental and theoretical studies will be useful for design of ratiometric/colorimetric molecular probes.     

Titrimetric determination of thiols: Tetrathionate, iron(III), cystine and hexacyanoferrate(III) as thiol reagents

Four analytical reagents, tetrathionate, iron(III), cystine and hexacyanoferrate(III) have been tested with respect to their specificity for oxidation of thiols to disulphides. Of a number of thiols studied, most have a strong tendency to oxidize beyond the disulphide stage with several of the commonly employed reagents. Tetrathionate, cystine and hexacyanoferrate(III) function in phosphate buffer of pH 7, but iron(III) does not require rigid control of pH, although the solution should be only feebly acidic. The reagents were used in excess and the thiosulphate or cysteine formed in the reaction of thiols with tetrathionate or cystine respectively was determined. The residual iron(III) was measured by adding ascorbic acid or mercaptoacetic acid and titrating with 2,6-dichlorophenolindophenol or iodine monochloride respectively; surplus hexacyanoferrate(III) was back-titrated with ascorbic acid. All four reagents react selectively with thiols even in the presence of several possible interfering substances and afford results that are accurate and precise.     

RAFT polymerization of vinyl methacrylate and subsequent conjugation via enzymatic thiol‐ene chemistry

The polymerization of vinyl methacrylate (VMA) allows the synthesis of polymers with pendant double bonds. When this polymerization was undertaken in the presence of 2‐cyanopropyl dithiobenzoate as reversible addition–fragmentation chain transfer agent, it led almost exclusively to vinylester functional sidegroups, which were available for further reactions. The vinylester functionality could not be functionalized using common thiol‐ene catalysts, but could be activated using Candida antarctica lipase B (CAL‐B) (Novozyme 435). The reaction between PVMA and various thiols in N, N‐dimethyl formamide in the presence of CAL‐B led exclusively to the formation of the anti‐Markovnikov product. The rate of reaction between PVMA and 1‐butanethiol was monitored using ¹H NMR. The reaction was complete within 72 h. Similar results were obtained with other small‐sized thiols such as 2‐mercaptoethanol, 3‐mercaptopropionic acid, and 2‐(trimethylsilyl)ethanethiol, while more bulky thiols, such as secondary thiols, thiols with long alkyl chains, and sterically demanding thiols, such as mono(6‐deoxy‐6‐mercapto)‐β‐cyclodextrin, only led to lower conversions. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Speciation and determination of thiols in biological samples using high performance liquid chromatography-inductively coupled plasma-mass spectrometry and high performance liquid chromatography-Orbitrap MS

An analytical method was developed for the determination of thiols in biological samples. Reverse phase chromatography coupled to ICP quadrupole MS or Orbitrap MS was employed for the separation and detection of thiols. For the determination of total thiols, oxidized thiols were reduced using dithiothreitol (DTT). Reduction efficiencies for species of interest were found to be close to 100%. Reduced thiols were derivatized by p-hydroxymercuribenzoate (PHMB) and then separated on a C8 column. Optimization of the extraction, separation and detection steps of the HPLC-ICP-MS and HPLC-Orbitrap MS methods was carried out. Detection limits for cysteine, homocysteine, selenocysteine, glutathione, selenomethionine and cysteinyl-glycine were found to be 18, 34, 39, 12, 128 and 103 fmol, respectively, using HPLC-Orbitrap MS and 730, 1110, 440, 1110 and 580 fmol for cysteine, homocysteine, selenocysteine, glutathione, and cysteinyl-glycine using HPLC-ICP-MS. Contrary to expectation, the LODs and RSDs are higher for the HPLC-ICP-MS instrument, therefore HPLC-Orbitrap MS was used for the determination of thiols in yeast samples. Three different brands of baker's yeast and a selenized yeast were analyzed. The GSH and cysteine levels found in these samples ranged from 4.45 to 17.87 μmol g(-1) and 0.61 to 1.32 μmol g(-1), respectively.     

Spectrophotometric determination of biologically active thiols with eosin, silver(I) and adenine.

A simple and highly sensitive spectrophotometric method for the determination of biologically active thiols based on the fading of eosin-silver(I)-adenine ternary complex was established. In the determination of 6-mercaptopurine (MP), Beer's law was obeyed in the range 0.02-0.30 microg ml(-1), with an effective molar absorptivity at 562 nm and the relative standard deviation being 3.5 x 10(5) dm3 mol(-1) cm(-1) and 0.72% (n = 5). Analytical data for various biologically active thiols were determined with the proposed method. This method is about 5-10 times more sensitive than the conventional spectrophotometric methods. A compound having a disulfide bond (-S-S-), such as cystine, could also be determined by the conversion of disulfides to free thiols with the sulfite ion. The procedure was successfully applied to assays of various biologically active thiols in actual medicines.     

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.     

L-Cysteine Determination Based on Tyrosinase Amperometric Biosensors without Interferences from Thiolic Compounds

This paper presents an enzymatic analysis method for selective detection of L-cysteine (L-Cys) without interferences from other thiols like cysteamine (CA) and mercaptoacetic acid (MAC). The amperometric biosensors are based on tyrosinase (Tyr) that converts catechol to o-quinone. The L-Cys detection is based on the fact that all thiols (including L-Cys) react with o-quinone producing electroinactive compounds and only interfering thiols (CA and MAC) inhibit Tyr. One sample was analyzed twice: with Tyr immobilized on WE surface to quantify enzyme inhibition by thiolic interferents and with Tyr free in solution to investigate the reactions between quinone and all thiolic compounds.     

Thiol metabolomics of endothelial cells using capillary liquid chromatography mass spectrometry with isotope coded affinity tags

Thiol and disulfide levels are critical to maintaining the redox potential of a cell. Perturbations of these levels are important in disease pathogenesis. To improve endogenous mammalian metabolome quantitation, thiol specific tagging, extraction and relative quantitation were undertaken. Reduced and oxidized thiol (disulfide) metabolites from endothelial cells were tagged and extracted using cleavable isotope coded affinity tags (cICAT). Extracted cICAT labeled thiols were analyzed using capillary reverse phase liquid chromatography coupled to mass spectrometry (capLC-MS) with positive mode electrospray ionization. Reactions between thiol metabolite standards and the reactive group of cICAT indicate completion by 8h at pH 9 with no apparent disulfide formation. cICAT labeled reduced thiols from endothelial cells showed 1-5% RSD using ratiometric quantitation of isotopes and 6-17% RSD based on signal intensity alone. Sample injection was optimized to 16 pmol. Using high mass accuracy MS, 75 putative thiol metabolites were detected in all experimental samples. Treatment of endothelial cells with 2,3-dimethoxy-5-methyl-1,4-benzoquinone (BQ) shows decreased levels in 28 putative reduced thiols and increased levels of 27 putative disulfides. Treatment of endothelial cells with 30 mM glucose resulted in 22 putative reduced thiols with decreased levels and 7 putative disulfides with increased concentration. Thiols were identified based on accurate mass within 3 ppm and analysis of fragmentation patterns. Using higher collision induced dissociation (HCD), shared product ions between different thiols led to the analysis of thiols from the cysteine-glutathione (Cys-GSH) pathway. Specific reduced thiols and disulfides in this pathway revealed changes different from the overall trends of thiols/disulfides. This suggests varying regulation of the Cys-GSH pathway distinct from other thiol-containing pathways and dependence on the type of environmental stimulus. These results indicate the utility of analyzing reduced thiols and disulfides in eukaryotic samples.     

Distribution of low-density lipoprotein-bound low-molecular-weight thiols: a new analytical approach

We have recently demonstrated that low-density lipoprotein (LDL) apoprotein is able to bind the most concentrated plasma thiols such as cysteine, cysteinylglycine, and homocysteine by disulfide linkage. However, the LIF CE assay employed to measure linked thiols was not sensitive enough to verify whether low concentrated plasma thiols as glutathione and glutamylcysteine are also linked to apoprotein. By modifying sample treatment and electrophoretic parameters we set up a new method with an LOQ of about 1.5 nmol/L, by which we demonstrate that LDL apoprotein binds all physiological plasma thiols. The increased sensitivity was obtained by drying released apoB thiols after reduction treatment, dissolving them directly in a low volume of derivatization buffer and decreasing the dilution factor of derivatized sample before CE injection. Moreover, by increasing the concentration of the electrolyte buffer, we improved the selectivity of peaks, in particular between glutathione (GSH) and the impurity peak derived from unreacted 5-iodoacetamidofluorescein, which in the previous electrophoretic conditions were overlapped. The method optimization, reached by searching the best combination between sample matrix and CE run buffer, is fully described. Given the potential pathologic significance of protein thiolation, the proposed method may be useful to understand the mechanisms and the balances that regulate the interaction between thiols and -SH free groups of proteins.     

Existence of low-molecular-weight thiols in Caenorhabditis elegans demonstrated by HPLC-fluorescene detection utilizing 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide

A highly sensitive and simple method using HPLC-fluorescence detection with 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide (DAABD-Cl) as a fluorogenic reagent demonstrated the existence of the low-molecular-weight thiols in the extract of Caenorhabditis elegans (C. elegans). The method includes derivatization of the thiols with DAABD-Cl at 40 degrees C for 10 min in borate buffer (pH 9.0) containing TCEP, CHAPS and EDTA, separation of the derivatives on an ODS column and fluorometric determination of the derivatives at 510 +/- 15 nm with excitation at 400 +/- 15 nm. The identification of the thiols was made by HPLC-electrospray ionization mass spectrometry (LC-MS) following isolation of the derivatives using HPLC-fluorescence detection. Low-molecular-weight thiols were found to exist in the extract of C. elegans, such as cysteine, cysteinylglycine, gamma-glutamylcysteine, reduced glutathione and two other unidentified thiol compounds, confirming the existence of the 'glutathione cycle' in C. elegans similar to the mammalian body.     

Bifunctional Fluorescent Probe for Sequential Sensing of Thiols and Primary Aliphatic Amines in Distinct Fluorescence Channels

Thiols and primary aliphatic amines (PAA) are ubiquitous and extremely important species in biological systems. They perform significant interplaying roles in complex biological events. A single fluorescent probe differentiating both thiols and PAA can contribute to understanding the intrinsic inter‐relationship of thiols and PAA in biological processes. Herein, we rationally constructed the first fluorescent probe that can respond to thiols and PAA in different fluorescence channels. The probe exhibited a high selectivity and sensitivity to thiols and PAA. In addition, it displayed sequential sensing ability when the thiols and PAA coexisted. The application experiments indicated that the probe can be used for sensing thiols and PAA in human blood serum. Moreover, the fluorescence imaging of endogenous thiols and PAA as well as antihypertensive drugs captopril and amlodipine in living cells were successfully conducted.     

Regioselective copper(I)-catalyzed C–H hydroxylation/C–S coupling: expedient construction of 2-(styrylthio)phenols

Regioselective copper(I)-catalyzed C–H hydroxylation/C–S coupling of aryl thiols with vinyl halides was developed. Starting from substituted aryl thiols and vinyl halides, various 2-(styrylthio)phenol derivatives were efficiently prepared. The application of the synthetic methodology to generate the bioactive organic intermediate was also exemplified.     

Intercalation of gaseous thiols and sulfides into Ag+ ion-exchanged aluminum dihydrogen triphosphate

Ag(+) ion-exchanged layered aluminum dihydrogen triphosphate (AlP) with the interlayer distance of 0.85 nm was synthesized by the ion-exchange of proton in triphosphate with Ag(+) ion. The amount of exchanged Ag(+) ion depended on the concentration of AgNO(3) aqueous solution. Ag(+) ion-exchanged AlP adsorbed gaseous thiols and sulfides into the interlayer region. The adsorption amounts of thiols were more than those of sulfides, thiols with one mercapto group > thiol with two mercapto groups > sulfides, and depended on the amount of exchanged Ag(+) ion in the interlayer region. The thiols with one mercapto group were intercalated to expand the interlayer distance of Ag(+) ion-exchanged AlP, whereas there was no expansion in the adsorption of sulfide. In the case of thiol with two mercapto groups, there was observed contraction of the interlayer distance through the bridging with Ag(+) ions of the upper and lower sides of the interlayer region.     

Electroanalytical Exploitation of Nitroso Phenyl Modified Carbon‐Thiol Interactions: Application to the Low Voltage Determination of Thiols

The electrochemical generation of nitrosophenyl groups covalently attached to graphite powder (nitrosophenylcarbon) from carbon powder chemically modified with nitrophenyl groups and their subsequent reaction with thiols (glutathione, cysteine and homocysteine) has been investigated as a method by which the later can be quantified. The modified carbon powder was immobilized onto a basal plane pyrolytic graphite electrode and characterized by cyclic voltammetry by scanning between 1.0 V and ?1.0 V vs. SCE in phosphate buffer (pH 7). Square wave voltammetry (SWV) was used for the determination of thiols and the SWV parameters were optimized. The nitrosophenylcarbon is electrogenerated from nitrophenylcarbon and can chemically oxidize thiols to disulfides. Subsequent reduction of nitrosophenylcarbon to phenylhydroxylaminecarbon during the square wave voltammetric process leads to a decrease in the reductive current. This can be correlated to the concentration of thiol present within the medium. The cyclic voltammetric responses of basal plane pyrolytic graphite electrode, edge plane pyrolytic graphite electrode, glassy carbon electrode and boron‐doped diamond electrode in the direct oxidation of thiols were also investigated and all were found to have a significantly higher overpotential compared to the described method using nitrosophenylcarbon.     

Kinetic Resolution of sec‐Thiols by Enantioselective Oxidation with Rationally Engineered 5‐(Hydroxymethyl)furfural Oxidase

Various flavoprotein oxidases were recently shown to oxidize primary thiols. Herein, this reactivity is extended to sec‐thiols by using structure‐guided engineering of 5‐(hydroxymethyl)furfural oxidase (HMFO). The variants obtained were employed for the oxidative kinetic resolution of racemic sec‐thiols, thus yielding the corresponding thioketones and nonreacted R‐configured thiols with excellent enantioselectivities (E≥200). The engineering strategy applied went beyond the classic approach of replacing bulky amino acid residues with smaller ones, as the active site was additionally enlarged by a newly introduced Thr residue. This residue established a hydrogen‐bonding interaction with the substrates, as verified in the crystal structure of the variant. These strategies unlocked HMFO variants for the enantioselective oxidation of a range of sec‐thiols.     

The oxidation of aqueous thiols on a graphite intercalation compound adsorbent

Aqueous phase adsorption of thiols onto a graphite intercalation compound (GIC) adsorbent was shown to be accompanied by surface assisted oxidation of the thiols to their disulfide dimer. This effect was studied for three thiols; ethanethiol, propanethiol and sec-butanethiol. Unlike the case of thiol oxidation on activated carbons in aqueous conditions, the generation of dimer did not affect the adsorbent’s ability to oxidise the thiols in solution in the concentration range studied (0–100 ppm). This was attributed to the non-porous, crystalline nature of the adsorbent. This meant that once formed the dimers migrated from the active sites for thiol oxidation, believed to be mainly at the edges of the graphite planes, onto the hydrophobic basal plane where they could be adsorbed. Oxidation of the GIC adsorbent in the anodic compartment of an electrochemical cell was shown to attenuate the adsorbent’s ability to oxidise thiols. It was inferred that this was a result of a decrease in the amount of CO groups at the plane edges caused by this oxidation process.