Convective instabilities derived from dissipation of chemical energy.

Oxidation reactions of a series of organosulfur compounds by chlorite are excitable, autocatalytic, and exothermic and generate a lateral instability upon being triggered by the autocatalyst. This article reports on the convective instabilities derived from the reaction of chlorite and thiourea in a Hele-Shaw cell. Reagent concentrations used for the development of convective instabilities delivered a temperature jump at the wave front of 2.1 K. The reaction zone was 2 mm and due to normal cooling after the wave front, this induced a spike rather than the standard well-studied front propagation. Localized spatiotemporal patterns develop around the wave front. This exothermic autocatalytic reaction has solutal and thermal contributions to density changes that act in opposite directions due to the existence of a positive isothermal density change in the reaction. The competition between these effects generates thermal plumes. The fascinating feature of this system is the coexistence of plumes and fingering in the same solution as the front propagates through the Hele-Shaw cell. Wave velocities of descending and ascending fronts are oscillatory. Fingers and plumes are generated in alternating frequency as the front propagates. This generates hot and cold spots within the Hele-Shaw cell, and subsequently spatiotemporal inhomogeneities. The small ΔT at the wave front generated thermocapillary convection which competed effectively with thermogravitational forces at low Eötvös numbers. A simplified reaction-diffusion-convection model was derived for the system. Plume formation is heavily dependent on boundary effects from the cell dimensions.

Electrochemical versus Enzymatic in Vitro Oxidations of 6-propyl-2-thiouracil: Identification, Detection, and Characterization of Metabolites.

6-Propylthiouracil, PTU, is a well-known antithyroid drug that has been the mainstay of treatment of Graves' disease. It is, however, also associated with liver toxicity and idiosyncratic toxicity. These toxicities are generally associated with metabolites derived from its bioactivation. In this manuscript, bioactivation of PTU was studied via two separate techniques: electrochemical oxidation and through the use of human liver microsomes. The aim of this work was to compare the bioactivation products of these two techniques. The electrochemical technique was studied online with a mass spectrometer, EC/ESI/MS. The microsomal oxidations were studied in tandem with liquid chromatography. The EC/ESI/MS technique was devoid of the normal reducing biological matrix prevalent in microsomal incubations. The predominant product at 400 mV was the dimeric PTU species with negligible formation of other metabolites. At higher potentials, complete desulfurization of PTU was observed with formation of sulfate. No sulfonic acid was observed, suggesting that the cleavage of the C-S bond was effected at the sulfinic acid stage, releasing a highly reducing sulfur species which is known to give rise to genotoxicity. The microsomal oxidations, surprisingly, showed formation of the unstable sulfenic acid, the S-oxide. Further incubation showed both the sulfinic and sulfonic acids. None of the systems showed any adducts with nucleophiles such as glutathione, showing that none of the reactive metabolites were stable enough to be adducted to nucleophiles in both the biological matrix and the electrochemical oxidizing environment.

Oxyhalogen-Sulfur Chemistry: Kinetics and Mechanism of Oxidation of 1,3-Dimethylthiourea by Acidic Bromate.

The mechanism of oxidation of the well-known radical scavenger dimethylthiourea, DMTU, by acidic bromate was studied. The stoichiometry of the reaction is 4:3: 4BrO3- + 3CS(NHMe)2 + 3H2O → 3SO42- + 3CO(NHMe)2 + 6H+ + 4Br-. In excess acidic bromate, the reaction stoichiometry is 8:5: 8BrO3- + 5CS(NHMe)2 + H2O → 5SO42- + 5CO(NHMe)2 + 4Br2 + 2H+. In excess bromate, the reaction displays well-defined clock reaction characteristics in which initially there is a quiescent period before formation of bromine. The direct reaction of aqueous bromine with DMTU, with a bimolecular rate constant of k = (1.95 ± 0.15) × 105 M-1 s-1, is much faster than reactions that form bromine such that formation of bromine indicates complete consumption of DMTU. ESI spectrometry showed evidence for an oxidation pathway that passes through the sulfenic, sulfinic, and sulfonic acids before formation of sulfate. In contrast to the oxidation of tetramethylthiourea, these oxoacid intermediates are not as abundant or as stable. The final product of oxidation was dimethylurea, the desulfurized DMTU. EPR spectroscopy implicates more than one set of radical species. The absence of the dimeric DMTU species, even in excess reductant indicates negligible formation of thiyl radicals. This also precludes substantial formation of the sulfenic acid intermediate which would form the dimer from a condensation-type reaction with unreacted DMTU. A 20-step reaction mechanism network was modeled which gave a reasonable fit with experimental data.

Kinetics and Mechanism of Bioactivation via S-Oxygenation of Anti-Tubercular Agent Ethionamide by Peracetic Acid.

The kinetics and mechanism of the oxidation of the important antitubercular agent, ethionamide, ETA (2-ethylthioisonicotinamide), by peracetic acid (PAA) have been studied. It is effectively a biphasic reaction with an initial rapid first phase of the reaction which is over in about 5 s and a second slower phase of the reaction which can run up to an hour. The first phase involves the addition of a single oxygen atom to ethionamide to form the S-oxide. The second phase involves further oxidation of the S-oxide to desulfurization of ETA to give 2-ethylisonicotinamide. In contrast to the stability of most organosulfur compounds, the S-oxide of ETA is relatively stable and can be isolated. In conditions of excess ETA, the stoichiometry of the reaction was strictly 1:1: CH3CO3H + Et(C5H4)C(═S)NH2 → CH3CO2H + Et(C5H4)C(═NH)SOH. In this oxidation, it was apparent that only the sulfur center was the reactive site. Though ETA was ultimately desulfurized, only the S-oxide was stable. Electrospray ionization (ESI) spectral analysis did not detect any substantial formation of the sulfinic and sulfonic acids. This suggests that cleavage of the carbon-sulfur bond occurs at the sulfenic acid stage, resulting in the formation of an unstable sulfur species that can react further to form more stable sulfur species. In this oxidation, no sulfate formation was observed. ESI spectral analysis data showed a final sulfur species in the form of a dimeric sulfur monoxide species, H3S2O2. We derived a bimolecular rate constant for the formation of the S-oxide of (3.08 ± 0.72) × 102 M-1 s-1. Oxidation of the S-oxide further to give 2-ethylisonicotinamide gave zero order kinetics.

Kinetics and Mechanism of Oxidation of Methimazole by Chlorite in Slightly Acidic Media.

The kinetics and mechanism of the oxidation of methimazole (1-methyl-3H-imidazole), MMI, by chlorite in mildly acidic environments were studied. It is a complex reaction that gives oligo-oscillations in chlorine dioxide concentrations in excess chlorite conditions. The stoichiometry is strictly 2:1, with the sulfur center being oxidized to sulfate and the organic moiety being hydrolyzed to several indeterminate species. In excess MMI conditions over chlorite, the sulfinic acid and sulfonic acid were observed as major intermediates. The sulfenic acid, which was observed in the electrochemical oxidation of MMI, was not observed with chlorite oxidations. Initial reduction of chlorite produced HOCl, an autocatalytic species in chlorite oxidations. HOCl rapidly reacts with chlorite to produce chlorine dioxide, which, in turn, reacts rapidly with MMI to produce more chlorite. The reaction of chlorine dioxide with MMI is competitive, in rate, with the chlorite-MMI and HOCl-ClO2(-) reactions. This explains the oligo-oscillations in ClO2 concentrations.

Characterization and comparative analysis of 2,4-toluene diisocyanate and 1,6-hexamethylene diisocyanate haptenated human serum albumin and hemoglobin.

Diisocyanates (dNCOs) are low molecular weight chemical sensitizers that react with autologous proteins to produce neoantigens. dNCO-haptenated proteins have been used as immunogens for generation of dNCO-specific antibodies and as antigens to screen for dNCO-specific antibodies in exposed individuals. Detection of dNCO-specific antibodies in exposed individuals for diagnosis of dNCO asthma has been hampered by poor sensitivities of the assay methods in that specific IgE can only be detected in approximately 25% of the dNCO asthmatics. Apart from characterization of the conjugates used for these immunoassays, the choice of the carrier protein and the dNCO used are important parameters that can influence the detection of dNCO-specific antibodies. Human serum albumin (HSA) is the most common carrier protein used for detection of dNCO specific-IgE and -IgG but the immunogenicity and/or antigenicity of other proteins that may be modified by dNCO in vivo is not well documented. In the current study, 2,4-toluene diisocyanate (TDI) and 1,6-hexamethylene diisocyanate (HDI) were reacted with HSA and human hemoglobin (Hb) and the resultant adducts were characterized by (i) HPLC quantification of the diamine produced from acid hydrolysis of the adducts, (ii) 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay to assess extent of cross-linking, (iii) electrophoretic migration in polyacrylamide gels to analyze intra- and inter-molecular cross-linking, and (iv) evaluation of antigenicity using a monoclonal antibody developed previously to TDI conjugated to Keyhole limpet hemocyanin (KLH). Concentration-dependent increases in the amount of dNCO bound to HDI and TDI, cross-linking, migration in gels, and antibody-binding were observed. TDI reactivity with both HSA and Hb was significantly higher than HDI. Hb-TDI antigenicity was approximately 30% that of HSA-TDI. In conclusion, this data suggests that both, the extent of haptenation as well as the degree of cross-linking differs between the two diisocyanate species studied, which may influence their relative immunogenicity and/or antigenicity.

Reactivity measurement in estimation of benzoquinone and benzoquinone derivatives' allergenicity.

Benzoquinone (BQ) and benzoquinone derivatives (BQD) are used in the production of dyes and cosmetics. While BQ, an extreme skin sensitizer, is an electrophile known to covalently modify proteins via Michael Addition (MA) reaction whilst halogen substituted BQD undergo nucleophilic vinylic substitution (SNV) mechanism onto amine and thiol moieties on proteins, the allergenic effects of adding substituents on BQ have not been reported. The effects of inserting substituents on the BQ ring has not been studied in animal assays. However, mandated reduction/elimination of animals used in cosmetics testing in Europe has led to an increased need for alternatives for the prediction of skin sensitization potential. Electron withdrawing and electron donating substituents on BQ were assessed for effects on BQ reactivity toward nitrobenzene thiol (NBT). The NBT binding studies demonstrated that addition of EWG to BQ as exemplified by the chlorine substituted BQDs increased reactivity while addition of EDG as in the methyl substituted BQDs reduced reactivity. BQ and BQD skin allerginicity was evaluated in the murine local lymph node assay (LLNA). BQD with electron withdrawing groups had the highest chemical potency followed by unsubstituted BQ and the least potent were the BQD with electron donating groups. The BQD results demonstrate the impact of inductive effects on both BQ reactivity and allergenicity, and suggest the potential utility of chemical reactivity data for electrophilic allergen identification and potency ranking.

Detailed mechanistic studies into the reactivities of thiourea and substituted thiourea oxoacids: decompositions and hydrolyses of dioxides in basic media.

Dioxides of methylthiourea (methylaminoiminomethanesulfinic acid, MAIMSA) and dimethylthiourea (dimethylaminoiminomethanesulfinic acid, DMAIMSA) were synthesized and, together with thiourea dioxide (aminoiminomethanesulfinic acid, AIMSA), were studied with respect to their decompositions and hydrolyses in basic aqueous media. All three were stable in acidic media and existed as zwitterions with the positive charge spread out on the 4-electron 3-center N-C-N skeleton and the negative charge delocalized over the two oxygen atoms. All three are characterized by long and weak C-S bonds that are easily cleaved in polar solvents through a nucleophilic attack on the positively disposed carbon center, followed by cleavage of the C-S bond. The sulfur moiety leaving groups are highly unstable, reducing, and rapidly oxidized to S(IV) as hydrogen sulfite in the presence of oxidant. In aerobic conditions, molecular oxygen is a sufficient and efficient oxidant that can oxidize, at diffusion-controlled limits, the highly reducing sulfur species in one-electron steps, thus opening up a cascade of possibly genotoxic reactive oxygen species, commencing with the superoxide anion radical. Radical formation in these decompositions was confirmed by electron paramagnetic resonance techniques. In strongly basic media, decomposition of the dioxides to yield sulfoxylate (SO2(2-), HSO2(-)) is irreversible and, in anaerobic environments, will disproportionate to yield more stable sulfur species from HS(-) to SO4(2-). Decomposition products were dependent on concentrations of molecular oxygen. Solutions open to the atmosphere, with availability to excess oxygen, gave the urea analogue of the thiourea and sulfate, while in limited oxygen conditions hydrogen sulfite and other mixed oxidation states sulfur oxoanions are obtained. DMAIMSA has the longest C-S bond at 0.188 nm and was the most reactive. MAIMSA, with the shortest at 0.186 nm, was the least reactive. Electrospray ionization-mass spectrometry data managed to detect all of the formerly postulated intermediates.

Kinetics and mechanistic investigation into the possible activation of imidazolium trans-[tetrachloridodimethylsulfoxideimidazoleruthenate(III)], NAMI-A, by 2-mercaptoethane sulfonate.

Imidazolium trans-[tetrachloridodimethylsulfoxideimidazoleruthenate(III)], NAMI-A, is a promising antimetastatic prodrug with high specificity for metastatic cancer cells. Limited activity of NAMI-A against primary tumor suggests that its use in combination with other anticancer drug(s) might present a more desirable therapeutic outcome. The mechanism of activation and action of this prodrug is still largely unknown. The biological targets, as well, have not yet been delineated. The kinetics and mechanism of interaction of NAMI-A with 2-mercaptoethane sulfonate, MESNA, a chemoprotectant, have been studied spectrophotometrically under pseudo-first order conditions of excess MESNA. The reaction is characterized by initial reduction of NAMI-A and formation of dimeric MESNA as evidenced by electospray ionization mass spectrometry. A first order dependence on both NAMI-A and MESNA was obtained and a bimolecular rate constant of 0.71 ± 0.06 M(-1) s(-1) was deduced. Activation parameters determined (ΔS(≠) = -178.12 ± 0.28 J K(-1) mol(-1), ΔH(≠) = 20.64 ± 0.082 kJ mol(-1) and ΔG(≠) = 75.89 ± 1.76 kJ mol(-1) at 37 ± 0.1 °C and pH 7.4) are indicative of formation of an associative intermediate prior to product formation and subsequent hydrolysis of the reduced complex. Our results suggest that MESNA might be able to activate the prodrug while still protecting against toxicity when given in a regimen involving NAMI-A and chemotherapy drug(s) that induce bladder and kidney toxicities.

S-oxygenation of thiocarbamides V: oxidation of tetramethylthiourea by chlorite in slightly acidic media.

The reaction between tetramethylthiourea (TTTU) and slightly acidic chlorite has been studied. The reaction is much faster than comparable oxidations of the parent thiourea compound as well as other substituted thioureas. The stoichiometry of the reaction in excess oxidant showed a complete desulfurization of the thiocarbamide to yield the corresponding urea and sulfate: 2ClO2(-) + (Me2N)2C ═ S + H2O → (Me2N)2C ═ O + SO4(2-) + 2Cl(-) + 2H(+). The reaction mechanism is unique in that the most stable metabolite before formation of the corresponding urea is the S-oxide. This is one of the rare occasions in which a low-molecular-weight S-oxide has been stabilized without the aid of large steric groups. ESI-MS data show almost quantitative formation of the S-oxide and negligible formation of the sulfinic and sulfonic acids. TTTU, in contrast to other substituted thioureas, can only stabilize intermediate oxoacids, before formation of sulfate, in the form of zwitterions. With a stoichiometric excess of TTTU over oxidant, the TTTU dimer is the predominant product. Chlorine dioxide, which is formed from the reaction of excess chlorite and HOCl, is a very important reactant in the overall mechanism. It reacts rapidly with TTTU to reform ClO2(-). Oxidation of TTTU by chlorite has a complex dependence on acid as a result of chlorous acid dissociation and protonation of the thiol group on TTTU in high-acid conditions, which renders the thiol center a less effective nucleophile.

Oxyhalogen-sulfur chemistry: kinetics and mechanism of oxidation of chemoprotectant, sodium 2-mercaptoethanesulfonate, MESNA, by acidic bromate and aqueous bromine.

The oxidation of a well-known chemoprotectant in anticancer therapies, sodium 2-mercaptoethanesulfonate, MESNA, by acidic bromate and aqueous bromine was studied in acidic medium. Stoichiometry of the reaction is: BrO3(-) + HSCH2CH2SO3H → Br(-) + HO3SCH2CH2SO3H. In excess bromate conditions the stoichiometry was deduced to be: 6BrO3(-) + 5HSCH2CH2SO3H + 6H(+) → 3Br2 + 5HO3SCH2CH2SO3H + 3H2O. The direct reaction of bromine and MESNA gave a stoichiometric ratio of 3:1: 3Br2 + HSCH2CH2SO3H + 3H2O → HO3SCH2CH2SO3H + 6Br(-) + 6H(+). This direct reaction is very fast; within limits of the mixing time of the stopped-flow spectrophotometer and with a bimolecular rate constant of 1.95 ± 0.05 × 10(4) M(-1) s(-1). Despite the strong oxidizing agents utilized, there is no cleavage of the C-S bond and no sulfate production was detected. The ESI-MS data show that the reaction proceeds via a predominantly nonradical pathway of three consecutive 2-electron transfers on the sulfur center to obtain the product 1,2-ethanedisulfonic acid, a well-known medium for the delivery of psychotic drugs. Thiyl radicals were detected but the absence of autocatalytic kinetics indicated that the radical pathway was a minor oxidation route. ESI-MS data showed that the S-oxide, contrary to known behavior of organosulfur compounds, is much more stable than the sulfinic acid. In conditions where the oxidizing equivalents are limited to a 4-electron transfer to only the sulfinic acid, the products obtained are a mixture of the S-oxide and the sulfonic acid with negligible amounts of the sulfinic acid. It appears the S-oxide is the preferred conformation over the sulfenic acid since no sulfenic acids have ever been stabilized without bulky substituent groups. The overall reaction scheme could be described and modeled by a minimal network of 18 reactions in which the major oxidants are HOBr and Br2(aq).

Pyridoxylamine reactivity kinetics as an amine based nucleophile for screening electrophilic dermal sensitizers.

Chemical allergens bind directly, or after metabolic or abiotic activation, to endogenous proteins to become allergenic. Assessment of this initial binding has been suggested as a target for development of assays to screen chemicals for their allergenic potential. Recently we reported a nitrobenzenethiol (NBT) based method for screening thiol reactive skin sensitizers, however, amine selective sensitizers are not detected by this assay. In the present study we describe an amine (pyridoxylamine (PDA)) based kinetic assay to complement the NBT assay for identification of amine-selective and non-selective skin sensitizers. UV-Vis spectrophotometry and fluorescence were used to measure PDA reactivity for 57 chemicals including anhydrides, aldehydes, and quinones where reaction rates ranged from 116 to 6.2 × 10(-6) M(-1) s(-1) for extreme to weak sensitizers, respectively. No reactivity towards PDA was observed with the thiol-selective sensitizers, non-sensitizers and prohaptens. The PDA rate constants correlated significantly with their respective murine local lymph node assay (LLNA) threshold EC3 values (R(2) = 0.76). The use of PDA serves as a simple, inexpensive amine based method that shows promise as a preliminary screening tool for electrophilic, amine-selective skin sensitizers.

Oxyhalogen-sulfur chemistry: kinetics and mechanism of oxidation of N-acetyl homocysteine thiolactone by acidified bromate and aqueous bromine.

N-acetyl homocysteine thiolactone (NAHT), medically known as citiolone, can be used as a mucolytic agent and for the treatment of certain hepatic disorders. We have studied the kinetics and mechanisms of its oxidation by acidic bromate and aqueous bromine. In acidic bromate conditions the reaction is characterized by a very short induction period followed by a sudden and rapid formation of bromine and N-acetyl homocysteine sulfonic acid. The stoichiometry of the bromate-NAHT reaction was deduced to be: BrO3(-) + H2O + CH3CONHCHCH2CH2SCO → CH3CONHCHCH2CH2(SO3H)COOH + Br(-) (S1) while in excess bromate it was deduced to be: 6BrO3(-) + 5CH3CONHCHCH2CH2SCO + 6H(+) → 3Br2 + 5CH3CONHCHCH2CH2(SO3H)COOH + 2H2O (S2). For the reaction of NAHT with bromine, a 3:1 stoichiometric ratio of bromine to NAHT was obtained: 3Br2 + CH3CONHCHCH2CH2SCO + 4H2O → 6Br(-) + CH3CONHCHCH2CH2(SO3H)COOH + 6H(+) (S3). Oxidation occurred only on the sulfur center where it was oxidized to the sulfonic acid. No sulfate formation was observed. The mechanism involved an initial oxidation to a relatively stable sulfoxide without ring-opening. Further oxidation of the sulfoxide involved two pathways: one which involved intermediate formation of an unstable sulfone and the other involves ring-opening coupled with oxidation through to the sulfonic acid. There was oligooscillatory production of aqueous bromine. Bromide produced in S1 reacts with excess bromate to produce aqueous bromine. The special stability associated with the sulfoxide allowed it to coexist with aqueous bromine since its further oxidation to the sulfone was not as facile. The direct reaction of aqueous bromine with NAHT was fast with an estimated lower limit bimolecular rate constant of 2.94 ± 0.03 × 10(2) M(-1) s(-1).

Kinetics and mechanism of the oxidation of N-acetyl homocysteine thiolactone with aqueous iodine and iodate.

The kinetics of N-acetyl homocysteine thiolactone (NAHT) oxidation by aqueous iodine and iodate were studied by spectrophotometric techniques. The iodate-NAHT reaction is slow and results in the formation of N-acetyl homocysteine thiolactone sulfoxide as the sole product (NAHTSO). The stoichiometry of the reaction was deduced as: IO3(-) + 3NAHT → I(-) + 3NAHTSO (S1). In excess iodate conditions, the iodide produced in S1 is oxidized to give iodine: IO3(-) + 5I(-) + 6H(+) → 3I2 + 3H2O (S2). Thus in excess iodate conditions the overall stoichiometry of the reaction is a linear combination of S1 and S2 that eliminates iodide, 5S1 + S2: 2IO3(-)+ 5NAHT+ 2H(+) → I2 + 5NAHTSO + H2O. There was a 1:1 stoichiometry for the NAHT - I2 reaction: NAHT+ I2 + H2O → NAHTSO +2I(-) + 2H(+) (S3). All reactions, S1, S2 and S3 occur simultaneously and since they are all comparable in rate; complex dynamics were observed. Iodide catalyzes S1 and S2 but inhibits S3. Iodide is a product of both S1 and S3. It has the most profound effect on the overall global dynamics observed. The overall reaction scheme which involved S1, S2 and S3 was modeled by a simple 12-reaction mechanistic scheme which gave a very good fit to experimental data.

Characterization of methylene diphenyl diisocyanate-haptenated human serum albumin and hemoglobin.

Protein haptenation by polyurethane industrial intermediate 4,4'-methylene diphenyl diisocyanate (MDI) is thought to be an important step in the development of diisocyanate (dNCO)-specific allergic sensitization; however, MDI-haptenated albumins used to screen specific antibody are often poorly characterized. Recently, the need to develop standardized immunoassays using a consistent, well-characterized dNCO-haptenated protein to screen for the presence of MDI-specific IgE and IgG from workers' sera has been emphasized and recognized. This has been challenging to achieve due to the bivalent electrophilic nature of dNCOs, leading to the capability to produce multiple cross-linked protein species and polymeric additions to proteins. In the current study, MDI was reacted with human serum albumin (HSA) and hemoglobin (Hb) at molar ratios ranging from 1:1 to 40:1 MDI/protein. Adducts were characterized by (i) loss of available 2,4,6-trinitrobenzene sulfonic acid (TNBS) binding to primary amines, (ii) electrophoretic migration in polyacrylamide gels, (iii) quantification of methylene diphenyl diamine following acid hydrolysis, and (iv) immunoassay. Concentration-dependent changes in all of the above noted parameters were observed, demonstrating increases in both number and complexity of conjugates formed with increasing MDI concentrations. In conclusion, a series of bioanalytical assays should be performed to standardize MDI-antigen preparations across lots and laboratories for measurement of specific antibody in exposed workers that in total indicate degree of intra- and intermolecular cross-linking, number of dNCOs bound, number of different specific binding sites on the protein, and degree of immunoreactivity.

Oxyhalogen-sulfur chemistry: kinetics and mechanism of oxidation of captopril by acidified bromate and aqueous bromine.

By nature of their nucleophilicity, all thiol-based drugs are oxidatively metabolized in the physiological environment. The key to understanding the physiological role of a hypertension drug, (2S)-1-[(2S)-2-methyl-3-sulfanylpropanoyl]pyrrolidine-2-carboxylic acid, medically known as captopril is through studying its oxidation pathway: its reactive intermediates and oxidation products. The oxidation of captopril by aqueous bromine and acidified bromate has been studied by spectrophotometric and electrospray ionization techniques. The stoichiometry for the reaction of acidic bromate with captopril is 1:1, BrO3(-) + (C4H6N)(COOH)(COCHCH3CH2)-SH → (C4H6N)(COOH)(COCHCH3CH2)-SO3H + Br(-), with reaction occurring only at the thiol center. For the direct reaction of bromine with captopril, the ratio is 3:1; 3Br2 + (C4H6N)(COOH)(COCHCH3CH2)-SH + 3H2O → (C4H6N)(COOH)(COCHCH3CH2)-SO3H + 6HBr. In excess acidic bromate conditions the reaction displays an initial induction period followed by a sharp rise in absorbance at 390 nm due to rapid formation of bromine. The direct reaction of aqueous bromine with captopril was much faster than oxidation of the thiol by acidified bromate, with a bimolecular rate constant of (1.046 (±0.08) × 10(5) M(-1) s(-1). The detection of thiyl radicals confirms the involvement of radicals as intermediates in the oxidation of Captopril by acidified BrO3(-). The involvement of thiyl radicals in oxidation of captopril competes with a nonradical pathway involving 2-electron oxidations of the sulfur center. The oxidation product of captopril under these strong oxidizing conditions is a sulfonic acid as confirmed by electrospray ionization mass spectrometry (ESI-MS), iodometric titrations, and proton nuclear magnetic resonance ((1)H NMR) results. There was no evidence from ESI-MS for the formation of the sulfenic and sulfinic acids in the oxidation pathway as the thiol group is rapidly oxidized to the sulfonic acid. A computer simulation analysis of this mechanism gave a reasonably good fit to the experimental data.

Substituent effects on the reactivity of benzoquinone derivatives with thiols.

Benzoquinone (BQ) is an extremely potent electrophilic contact allergen that haptenates endogenous proteins through Michael addition (MA). It is also hypothesized that BQ may haptenate proteins via free radical formation. The objective of this study was to assess the inductive effects (activating and deactivating) of substituents on BQ reactivity and the mechanistic pathway of covalent binding to a nucleophilic thiol. The BQ binding of Cys34 on human serum albumin was studied, and for reactivity studies, nitrobenzenethiol (NBT) was used as a surrogate for protein binding of the BQ and benzoquinone derivatives (BQD). Stopped flow techniques were used to determine pseudofirst order rate constants (k) of methyl-, t-butyl-, and chlorine-substituted BQD reactions with NBT, whereas electron pair resonance (EPR) studies were performed to investigate the presence of the free radical mediated binding mechanism of BQD. Characterization of adducts was performed using mass spectrometry and nuclear magnetic resonance spectroscopy (NMR). The rate constant values demonstrated the chlorine-substituted (activated) BQD to be more reactive toward NBT than the methyl and t-butyl-substituted (deactivated) BQD, and this correlated with the respective EPR intensities. The EPR signal, however, was quenched in the presence of NBT suggesting MA as the dominant reaction pathway. MS and NMR results confirmed adduct formation to be a result of MA onto the BQ ring with vinylic substitution also occurring for chlorine-substituted derivatives. The binding positions on BQ and NBT/BQ(D) stoichiometric ratios were affected by whether the inductive effects of the substituents on the ring were positive or negative. The reactivity of BQ and BQD is discussed in terms of the potential relationship to potential allergenic potency.

Detailed mechanistic investigation into the S-nitrosation of cysteamine.

The nitrosation of cysteamine (H2NCH2CH2SH) to produce cysteamine-S-nitrosothiol (CANO) was studied in slightly acidic medium by using nitrous acid prepared in situ. The stoichiometry of the reaction was H2NCH2CH2SH + HNO2 → H2NCH2CH2SNO + H2O. On prolonged standing, the nitrosothiol decomposed quantitatively to yield the disulfide, cystamine: 2H2NCH2CH2SNO → H2NCH2CH2S-SCH2CH2NH2 + 2NO. NO2 and N2O3 are not the primary nitrosating agents, since their precursor (NO) was not detected during the nitrosation process. The reaction is first order in nitrous acid, thus implicating it as the major nitrosating agent in mildly acidic pH conditions. Acid catalyzes nitrosation after nitrous acid has saturated, implicating the protonated nitrous acid species, the nitrosonium cation (NO+) as a contributing nitrosating species in highly acidic environments. The acid catalysis at constant nitrous acid concentrations suggests that the nitrosonium cation nitrosates at a much higher rate than nitrous acid. Bimolecular rate constants for the nitrosation of cysteamine by nitrous acid and by the nitrosonium cation were deduced to be 17.9 ± 1.5 (mol/L)-1 s-1 and 6.7 × 104 (mol/L)-1 s-1, respectively. Both Cu(I) and Cu(II) ions were effective catalysts for the formation and decomposition of the cysteamine nitrosothiol. Cu(II) ions could catalyze the nitrosation of cysteamine in neutral conditions, whereas Cu(I) could only catalyze in acidic conditions. Transnitrosation kinetics of CANO with glutathione showed the formation of cystamine and the mixed disulfide with no formation of oxidized glutathione (GSSG). The nitrosation reaction was satisfactorily simulated by a simple reaction scheme involving eight reactions.

Production, characterization and utility of a panel of monoclonal antibodies for the detection of toluene diisocyanate haptenated proteins.

Diisocyanates (dNCOs) are highly reactive low molecular weight chemicals used in the manufacture of polyurethane products and are the most commonly reported cause of occupational asthma. Mechanistic disease studies and development of biomonitoring and research tools, such as monoclonal antibodies (mAbs) have been hampered by dNCOs' ability to self-polymerize and to cross-link biomolecules. Toluene diisocyanate (TDI)-specific monoclonal antibodies (mAbs), with potential use in immunoassays for exposure and biomarker assessments, were produced and reactivities characterized against mono- and diisocyanate and dithioisocyanate protein conjugates. In general, TDI reactive mAbs displayed stronger recognition of isocyanate haptenated proteins when the NCO was in the ortho position relative to the tolyl group, and were capable of discriminating between isocyanate and isothiocyanate conjugates and between aromatic and aliphatic dNCOs. Preliminary studies using TDI vapor exposed cells suggest potential utility of these mAbs for both research and biomonitoring.