Kinetics of Oxidation of Tetramethylthiourea by Aqueous Bromine and

ARTICLE pubs.acs.org/JPCA

S-Oxygenation of Thiocarbamides IV: Kinetics of Oxidation of Tetramethylthiourea by Aqueous Bromine and Acidic Bromate Risikat O. Ajibola and Reuben H. Simoyi* Department of Chemistry, Portland State University, Portland, Oregon 97207-0751, United States ABSTRACT: The kinetics and mechanism of oxidation of tetramethylthiourea (TTTU) by bromine and acidic bromate has been studied in aqueous media. The kinetics of reaction of bromate with TTTU was characterized by an induction period followed by formation of bromine. The reaction stoichiometry was determined to be 4BrO3- þ 3(R)2CdS þ 3H2O f 4Br- þ 3(R)2CdO þ 3SO42- þ 6Hþ. For the reaction of TTTU with bromine, a 4:1 stoichiometric ratio of bromine to TTTU was obtained with 4Br2 þ (R)2CdS þ 5H2O f 8Br- þ SO42- þ (R)2CdO þ 10Hþ. The oxidation pathway went through the formation of tetramethythiourea sulfenic acid as evidenced by the electrospray ionization mass spectrum of the dynamic reaction solution. This S-oxide was then oxidized to produce tetramethylurea and sulfate as final products of reaction. There was no evidence for the formation of the sulfinic and sulfonic acids in the oxidation pathway. This implicates the sulfoxylate anion as a precursor to formation of sulfate. In aerobic conditions, this anion can unleash a series of genotoxic reactive oxygen species which can explain TTTU’s observed toxicity. A bimolecular rate constant of 5.33 ( 0.32 M-1 s-1 for the direct reaction of TTTU with bromine was obtained.

’ INTRODUCTION There are a number of pharmacophores that are based on simple sulfur chemistry. The simplest one is the sulfonamide moiety. Several drugs that cover a wide range of diseases are based on this simple chemistry. They include the following: diuretics such as furosemide,1-3 clopamide, and thiazide; anticonvulsants such as sultiame;4 COX-2 inhibitors celecoxib5,6 and acetazolamide;7 and well-known antibiotics sulfamethoxazole (Bactrim) and sulfadimethoxine.8 Another well-known sulfurbased pharmacophore is the thioureido group (simplest member thiourea) where the four possible substitutions on the two nitrogens are populated by different substituents. The two nitrogens can also be part of a cycle as is the case with ethylenethiourea,9 methimazole10 (1-methyl-3H-imidazole-2-thione), and 6-propylthiouracil.

Methimazole is used to treat hyperthyrodism, a condition that usually occurs when the thyroid gland is producing too much thyroid hormone.11 Compounds with this thiouredo functionality constitute a phenomenally diverse group of biologically active compounds. No other pharmacophore possesses such a wide range of biological activity. The ease of synthesis of substituted thioureas12-14 means that there are now hundreds of these analogues available which have not yet been characterized.15,16 r 2011 American Chemical Society

The general approach involves varying substituents R1 and R3 while leaving R2 and R4 as protons so as not to impose steric hindrance around the thiocarbonyl bond. This thiourea class of compounds is presently being utilized as rodenticides,17,18 bactericides, goitrogenics,19-24 antitubercular,15,25,26 antimalarials,27 antithrombotics, and barbiturates28 among others. Further, ethionamide, an amide prodrug, is one of only a few drugs that (in combination with gatifloxacin29) treats CNS tuberculosis which has lodged in the meninges.29-31 New research into this pharmacophore is now directed toward anticancer and anti-HIV derivatives.13,32,33 Recent studies have reported on the effects of thioureas on human health without any mechanistic rationalization of these effects. For example, thiourea reacts with superoxide to yield a sulfhydryl compound.34 This has been promulgated as a possible explanation for its protective effect against paraquat. Mixtures of thiourea and superoxide dismutase were effective antidotes to paraquat toxicity in an HL60 cell culture system.34 Thioureas also inhibited the reduction of cytochrome c by the xanthine/xanthine oxidase superoxide generating system and the release of iron from ferritin by superoxide radicals. Dimethylthiourea, DMTU (R1 = R3 = Me), is a small, highly diffusible molecule that efficiently scavenges toxic oxygen metabolites in vitro and reduces oxidative injury in many biological systems.35 DMTU is one of the most efficient scavengers known for hydrogen peroxide, hydroxyl radical, and hypochlorous acid.36 Although in most Received: December 31, 2010 Revised: February 3, 2011 Published: March 14, 2011 2735

dx.doi.org/10.1021/jp1124052 | J. Phys. Chem. A 2011, 115, 2735–2744

The Journal of Physical Chemistry A

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Figure 1. Spectral scans of TTTU, TTU, bromine, and tribromide. TTTU absorbs in the UV region at 248 nm; TTU has no significant absorbance in either the near-UV or the vis region. Bromine solutions absorb at two wavelengths, 266 and 390 nm. The 390 nm peak is isolated and can be used to quantify bromine concentrations.

situations it is effective in decreasing oxidant-mediated injury, it has, however, inexplicably, on occasions, failed to reduce injury in some biological systems where oxygen metabolites were ostensibly causing damage.37 Despite aforementioned clinical applications, substituted thioureas are generally known to be toxic.38 Although the mechanisms of toxicity are not entirely understood, studies have shown the metabolic transformation of the thiourea moiety to highly reactive S-oxygenated metabolites accounts for the major physiological effects.39-42 The inherent toxicities of thiourea compounds have been attributed to the formation of sulfur oxo-acids.42 Slight differences in structures of these compounds may result in expression of significantly different physiological roles. For instance, the difference in the number of substituents in the case of phenylthiourea and diphenylthiourea accounts for the toxicity and innocuity of the two compounds, respectively.18,43 Our group recently embarked on a series of studies to elucidate the oxidation mechanisms of structurally related thiocarbamides since small changes in substituents afford varied metabolic effects.44-46 S-Oxygenation kinetics of the series thiourea, dimethylthiourea, and trimethylthiourea have been studied.47,48 Tetramethylthiourea (TTTU) presents structural uniqueness from the other three. With all four positions on the nitrogens occupied by methyl groups, none of its oxo-acids are able to exist in non-zwitterionic forms. Stable non-zwitterionic structures are possible only with TTTU and with the final oxidation product, tetramethylurea, TTU. Animal studies have shown TTTU to be one of the most potent known teratogens.49,50 It induces hepatotoxicity43 and has been shown to induce thyroid hyperplasia and tumor formation following prolonged exposure.51 In this manuscript, we report on the oxidation of TTTU by aqueous bromine and bromate under acidic conditions.

’ EXPERIMENTAL SECTION Materials. The following reagents were used without further purification: sodium bromate, perchloric acid (70-72%), sodium bromide, bromine, sodium chloride, sodium thiosulfate (Fisher), and barium chloride (Sigma-Aldrich). Tetramethylthiourea (Acros)

Figure 2. (a) Stoichiometric determination of bromate-TTTU reaction by iodometric titration. A fixed TTTU concentration of 3.00 10-3 M was reacted with varying amounts of bromate. Excess oxidizing power was quantified as iodine by titration with standard thiosulfate solution in the presence of excess acidified iodide. The plot of the volume of thiosulfate against bromate concentrations gave the concentration of bromate needed for complete consumption of TTTU. (b) Spectral changes observed every 30 s of the stoichiometric 3:4 solution, [TTTU]o = 1.0 10-4 M, [BrO3-]o = 1.33 10-4 M, and [Hþ]o = 0.1 M. The TTTU peak at 248 nm decreases with time but did not disappear completely as the product TTU also absorbs around this wavelength. As expected, there was no formation of bromine at 390 nm.

was recrystallized from 80% acetonitrile and characterized by X-ray crystallography. TTTU solutions were standardized spectrophotometrically using a molar extinction coefficient of 17 868 M-1cm-1 at 248 nm as determined in this study (see Figure 1). Bromine solutions, being volatile, were kept capped and standardized spectrophotometrically before each set of experiments. Methods. The kinetics of reaction were carried out at a constant temperature of 25 ( 0.5 C and ionic strength of 1.0 M using sodium perchlorate. The direct reactions between bromine and TTTU were monitored by following consumption of bromine at 390 nm (ε = 142 M-1 cm-1), and those involving observations of the consumption of the TTTU peak at 248 nm were monitored on a Hi-Tech Scientific SF-61DX2 doublemixing stopped flow spectrophotometer. Slower reactions were studied on a Perkin-Elmer Lambda 25 UV/vis spectrophotometer. Figure 1 shows the different UV-vis spectra for the reactant 2736

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Table 1. Gravimetric Determination of Sulfate Formation through Precipitation as Barium Sulfate [TTTU]

[BrO3- ]

BaSO4

[BaSO4]

(mol)

(mol)

produced (g)

(mol)

-3

Yield (%)

-3

3 10 3 10-3

4.0 10 4.0 10-3

0.6839 0.6929

2.9302 10 2.9687 10-3

97.67 98.96

3 10-3

4.0 10-3

0.6920

2.9649 10-3

98.83

-3

-3

0.6874

2.9452 10-3

98.17

0.6858

2.9383 10-3

97.94

3 10

4.0 10

3 10-3

4.0 10-3

-3

(TTTU), product (TTU), bromine, and tribromide. TTU has no discernible absorption peak in the visible region. Bromine has an isolated peak at 390 nm. Bromine and tribromide have an isosbestic point at 464 nm which was not utilized because of its low absorptivity coefficient (ε = 91 M-1 cm-1). Initial rates of consumption of TTTU could be monitored at 248 nm before interference from tribromide commences. Water used for preparing reagent solutions was obtained from a Barnstead Sybron Corp. water purification unit capable of producing both distilled and deionized water (Nanopure). Mass spectra of product solutions were taken on a Thermo Scientific LTQ-Orbitrap Discovery mass spectrometer (San Jose, CA) equipped with an electrospray ionization source operated in the positive mode. Stoichiometric Determinations. Stoichiometric determinations were performed by an iodometric titration method in which varying amounts of bromate were reacted with fixed concentrations of TTTU in highly acidic conditions. The excess bromate was evaluated by addition of acidified excess iodide with the liberated iodine next titrated against standard sodium thiosulfate. The stoichiometry was also confirmed by gravimetric analysis via BaSO4 precipitation.

’ RESULTS Stoichiometry. In the reaction of TTTU with bromate, variable stoichiometries were obtained depending on the ratio of oxidant to reductant. Varying amounts of bromate were reacted with a fixed concentration of TTTU under acidic conditions. Excess bromate from the reaction solutions after a 24-h incubation period was determined by addition of excess acidified iodide and titration of the librated iodine against standardized sodium thiosulfate. The extrapolation to zero of the plot of volume of thiosulfate against bromate concentrations (Figure 2a) corresponds to the exact amount of bromate needed to completely consume the initial TTTU concentration, [TTTU]o, without any excess bromate left to form bromine. Figure 2a shows that 0.0030 M solution of TTTU needs a 0.003987 M solution of bromate for its complete consumption. This results in a 4:3 ratio of bromate to TTTU according to stoichiometry R1

4BrO3 - þ 3ðRÞ2 CdS þ 3H2 O f 4Br- þ 3ðRÞ2 CdO þ 3SO4 2- þ 6Hþ

ðR1Þ

where R = NMe2. Figure 2b shows rapid spectral scans taken at 30 s intervals of a stoichiometric solution of TTTU and bromate in aqueous acidic medium. This is a 4:3 ratio solution in which aqueous bromine is not an expected product. This figure shows a monotonic decrease in the 248 nm peak which corresponds to depletion of TTTU. The stoichiometry R1 was confirmed by gravimetric quantification of sulfate as barium sulfate (Table 1) showing that,

Figure 3. Stoichiometric determination of bromine-TTTU reaction by iodometric titration. Fixed 9.80 10-4 M TTTU was reacted with varying amounts of bromine. The plot of the volume of thiosulfate used against the varying bromine concentrations gave 4.09 10-3 M as the bromine concentration needed for complete consumption of TTTU.

quantitatively, all the sulfur present ended up as sulfate at completion of reaction. Bromine formation, however, was observed at an oxidant to reductant ratio R ([BrO3-]o/[TTTU]o) greater than 4/3. In high excess of bromate, the limiting stoichiometry with respect to bromine formation was 8BrO3 - þ 5ðRÞ2 CdS þ H2 O f 4Br2 þ 5ðRÞ2 CdO þ 5SO4 2- þ 2Hþ

ðR2Þ

Stoichiometry R2 is a linear combination of R1 and the BrO3-/ Br- reaction that consumes Br- formed in R1 (reaction R4, vide infra). The stoichiometry for the direct reaction of bromine with TTTU was evaluated using a combination of iodometric titration (Figure 3) and spectrophotometric techniques. In the spectrophotometric technique, varying amounts of excess aqueous bromine concentrations were reacted with a fixed amount of TTTU and the final absorbance at 390 nm noted. A plot was then made of final absorbance vs initial bromine concentrations. Extrapolating gives the amount of bromine needed to completely oxidize the fixed amount of TTTU. Both methods gave a 4:1 ratio of bromine to TTTU (reaction R3) 4Br2 þ ðRÞ2 CdS þ 5H2 O f 8Br- þ SO4 2þ ðRÞ2 CdO þ 10Hþ

ðR3Þ

Product Analysis. The positive mode electrospray ionization mass spectrum of solution containing a stoichiometric ratio of TTTU and bromate in 50:50 methanol/water was taken after 2 min (Figure 4a). This dynamic solution showed interesting peaks that indicate a specific pathway for this oxidation reaction that proceeds through the sulfenic acid. Formation of sulfenic acid, an intermediate in TTTU oxidation, was confirmed by the 149 m/z peak (Figure 4a). Peaks at 117 and 133 m/z represent TTU, product of reaction, and substrate TTTU, respectively. The spectrum shown in Figure 4b represents the same stoichiometric solution taken after 24 h to ensure completion of reaction. It shows the disappearance of the sulfenic acid peak and a strong peak for the expected product, TTU. Of note is the fact that the 2737



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Figure 4. (a) Positive ESI-MS spectrum of a stoichiometric TTTU-BrO3- solution using 50:50 methanol:water as solvent. This spectrum was taken 2 min into the reaction. (b) Positive ESI-MS spectrum of a stoichiometric TTTU-BrO3- solution taken after 24 h of the start of reaction.

only visible intermediate formed is the one represented by the m/z = 149 peak. This peak represents the S-oxide of TTTU, (R)2CSfO, which is the only structure possible in the absence of ionizable protons on the N atoms to facilitate formation of a CdN double bond. The positive charge is delocalized over the sp2-hybridized carbon and the two nitrogens, and the negative

charge is on the oxygen. The dioxide and trioxides, contrary to other thiourea analogues, were not viable.52,53 Reaction Kinetics. Reactions of TTTU with bromate monitored at the absorption wavelength of bromine, λmax = 390 nm were characterized by an initial period in which no absorbance activity was observed. We labeled this period as the 2738



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Figure 6. (a) Effect of bromate. [TTTU]o = 4.0 10-3 M; [Hþ]o = 0.30 M; [BrO3-]o = (a) 0.016, (b) 0.018, (c) 0.020, (d) 0.022, (e) 0.024, (f) 0.026, and (g) 0.028 M. (b) Plot of the inverse of the induction time against bromate concentrations of the data shown in Figure 6a. The [BrO3-]-axis intercept confirms the stoichiometry R1.

Figure 5. (a) Effect of low acid. [BrO3-]o = 0.25 M; [TTTU]o = 2.5 10-3 M; [Hþ]o = (a) 0.025, (b) 0.027, (c) 0.030, (d) 0.035, and (e) 0.037 M. Final bromine concentration is independent of [Hþ]o as the same amount was formed at completion of reaction. (b) Effect of high acid. [TTTU]o = 2.5 10-3 M; [BrO3-]o = 0.20 M; [Hþ]o = (a) 0.10, (b) 0.12, (c) 0.14, (d) 0.16, (e) 0.20, and (f) 0.22 M. Induction time decreases with increasing acid concentrations. (c) Plot of induction time against square of the inverse initial acid concentrations of the data shown in Figure 5b.

‘induction time’. The length of induction time is determined by initial reagent concentrations. Other TTTU-bromate reactions were monitored by the consumption of TTTU at 248 nm. Reactions of bromine with TTTU were followed by monitoring the decomposition of bromine at 390 nm.

Effect of Acid. An increase in acid concentration decreased the induction period (Figure 5a and 5b). The final amount of bromine is independent of the initial acid concentration. The reactions shown in Figure 5a and 5b have a large excess of BrO3-, and the final concentrations of bromine are determined by [TTTU]0. Acid acted as a typical catalyst in that it did not alter this ratio, only increased the rate at which this ratio was attained. In conditions of low acid (Figure 5a), formation of bromine was biphasic: with an initial slow rate of formation followed by a more rapid formation after the induction period. At highly acidic conditions, there was an immediate and rapid formation of bromine (Figure 5b). This formation was so rapid that it initially ‘overshot’ the expected 5:4 TTTU to Br2 ratio. In all traces shown in Figure 5, the final observed bromine absorbance was 0.28, which is equivalent to 0.002 M Br2, the expected stoichiometric equivalent that can be derived from 0.0025 M TTTU. In both cases, there exists a square inverse acid dependence on the induction period (see plot in Figure 5c). This suggests that processes leading to commencement of bromine formation are dependent on acid to the second power in their rate-determining step. Deviation from the square inverse acid dependence occurs at high acid concentrations. Effect of Bromate. An increase in bromate concentrations at fixed concentrations of both TTTU and acid is accompanied by a decrease in induction period (Figure 6a). The induction period 2739



Figure 7. Effect of TTTU variation in overwhelming excess of bromate. “Peacock-tail-type” traces showing that the induction time is independent of [TTTU]o in conditions of large excess of oxidant. Final amount of bromine is dependent on [TTTU]o. [BrO3-] = 0.25 M; [Hþ] = 0.25 M; [TTTU]o = (a) 0.50 10-3, (b) 1.0 10-3, (c) 1.5 10-3, (d) 2.0 10-3, (e) 2.5 10-3, (f) 3.0 10-3, and (g) 3.5 10-3 M.

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Figure 9. Superimposition of the trace of bromine formation at 390 nm on TTTU consumption at 248 nm.

Figure 8. Effect of bromide. Bromide ions have a catalytic effect on the reaction by reducing the observed induction time. [TTTU]o = 4.0 10-3 M; [BrO3-]o = 0.10 M; [Hþ]o = 0.15 M; [Br-]o = (a) 0.0, (b) 1.0 10-3, (c) 2.0 10-3, (d) 3.0 10-3, and (e) 4.0 10-3 M.

depended inversely on bromate concentrations as shown in Figure 6b. The plot of inverse induction time against bromate concentration (Figure 6b) was extrapolated to infinite induction time to give the bromate concentration that will completely react with [TTTU]o with no formation of bromine. The intercept on the [BrO3-] axis corresponds to a stoichiometric amount of bromate as shown by R1, i.e., with a fixed concentration of 0.0045 M TTTU, the BrO3- concentration as Tind f ¥ is 0.006 M, which is the expected 4:3 bromate to TTTU ratio. Effect of TTTU. The effect of TTTU observed in overwhelming excess of bromate is shown in Figure 7. The final amount of bromine formed depended on [TTTU]o. Thus, the final amount of bromine produced increased with an increase in [TTTU]o. This would be expected from stoichiometry R2. The induction time, however, is independent of [TTTU]o since the experimental conditions have [BrO3-]o . [TTTU]o. Figure 7 shows the

Figure 10. (a) Effect of acid variation on the consumption of TTTU at 248 nm. [TTTU]o = 1.0 10-4 M; [BrO3-]o = 3.0 10-4 M; [Hþ]o = (a) 0.100, (b) 0.125, (c) 0.150, (d) 0.175, (e) 0.200, and (f) 0.225 M. (b) Effect of TTTU variation at 248 nm. [BrO3-]o = 3.0 10-4 M; [Hþ]o = 0.10 M; [TTTU]o = (a) 0.50 10-4, (b) 0.60 10-4, (c) 0.70 10-4, (d) 0.80 10-4, and (e) 0.90 10-4 M.

peacock-tail-type kinetic traces that have been observed in previous studies.54,55 2740



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Scheme 1

Figure 11. Effect of TTTU variation on bromine oxidation of TTTU at 390 nm.[Br2]0 = 9.50 10-3 M; [TTTU]o = (a) 1.125 10-3, (b) 1.25 10-3, (c) 1.375 10-3, (d) 1.500 10-3, and (e) 1.625 10-3 M.

Effect of Bromide. An increase in bromide concentrations increased the rate of bromine formation and decreased the observed induction period (Figure 8). This also led to an increase in the amount of final bromine formed. Increased bromine formation with an increase in bromide concentrations can be explained by an extraneous reaction R4 where bromide fuels this reaction BrO3 - þ 5Br- þ 6Hþ f 3Br2 þ 3H2 O

’ REACTION MECHANISM The reaction kinetics and dynamics seem to conform to the well-known bromate-bromide reaction kinetics in acidic medium.54,56 There is implied acid dependence in rate of second order from the dependence of the induction period. Bromate-bromide kinetics involve consecutive protonation of bromate anion to give protonated bromic acid which appears to be the reactive oxidizing species ðR5Þ

k2 , k-2 ; K a2 -1

ðR6Þ

HBrO3 þ Hþ T H2 BrO3 þ ;

The rate-determining step now becomes the two-electron oxidation of TTTU by protonated bromic acid, reaction R7

ðR4Þ

Curiously, bromide is a product of the reaction while it is also a reactant, and yet the reaction dynamics do not display the autocatalysis characteristics such as sigmoidal decay kinetics. Observed Kinetics at 248 nm. Figure 9 shows a synchronized comparison of absorbance traces taken at 248 and 390 nm for the same solution run at excess bromate. Bromine formation appears to only commence after TTTU is completely consumed. This is, however, not strictly correct. At the point of formation of bromine, the absorbance observed at 248 nm is a contribution from depletion of TTTU and formation of TTU and tribromide, which both have significant absorptivity coefficients at this wavelength. Thus, a lack of absorbance activity at 248 nm as observed in Figure 9 does not imply that depletion of TTTU has ceased. At this absorption wavelength of TTTU, the effects of acid and TTTU were observed on the consumption of TTTU by acidified bromate (see Figure 10a and 10b). An increase in acid concentration increased the rate of TTTU consumption (Figure 10a). The rate of TTTU consumption also increased with increasing TTTU concentrations (Figure 10b). This plot shows significant absorption at the wavelength of observation after completion of the reaction. This absorption is expected and due to the accumulation of reaction product, TTU (see Figure 1). Initial rate measurements were thus confined to the first 2% of the reaction before commencement of absorbance contribution from products and intermediates. Bromine-TTTU Reaction. Acid has an inhibitory effect on reaction of TTTU with bromine. The effect of TTTU variation is seen in Figure 11. An increase in TTTU concentration increased the rate of bromine consumption. Initial rate plots showed that the reaction has a linear dependence on both bromine and TTTU. This resulted in a bimolecular rate constant of 5.33 ( 0.32 M-1 s-1 for the direct reaction of TTTU with bromine.

BrO3 - þHþ T HBrO3 ; k1 , k-1 ; K a1 -1

H2 BrO3 þ þ ðRÞ2CdS f HBrO2 þ ðRÞ2 CSO þ Hþ ; k3 ðR7Þ Here, (R)2CSO is the zwitterionic form of the sulfenic acid The acid-base equilibria can be quantified in the following equations ½HBrO3 ¼ K a1 -1 ½BrO3 - ½Hþ ; ½H2 BrO3 þ ¼ K a2 -1 ½HBrO3 ½Hþ

ð1Þ

The overall rate of reaction is given by v¼ -

d½ðRÞ2 CdS ¼ k3 ½H2 BrO3 þ ½ðRÞ2 CdS dt

ð2Þ

From the sequence R5-R7, the initial rate of disappearance of TTTU is then derived to be vo ¼

d½ðRÞ2 CdS a þ 2 ¼ k3 Kb K-1 ½BrOð3Þ 3 o ½ðRÞ2 CdSo ½H o dt

From previously established mechanisms,46 the sulfenic acid formed in R7 is supposed to be unstable and should be rapidly oxidized to sulfinic acid or form the disulfide with unreacted TTTU. ESI -MS spectral data show, however, a very strong signal for the sulfenic acid (see Figure 4a). Of all the possible sulfur oxoacid metabolites before formation of sulfate and TTU, the sulfenic acid shows the strongest signal and abundance. The lack of ionizable protons on the two nitrogen atoms of TTTU precludes the formation of resonance-stabilized non-zwitterionic forms of any oxo-acids of TTTU. Thus, one expects the sulfenic, sulfinic, and sulfonic acids to be zwitterionic, with a positive charge spread out on a 3-center four-electron system around an sp2-hybridized carbon center.53 The negative charge is then delocalized over the oxygen atoms attached to the sulfur center. The sulfenic acid of TTTU is thus particularly viable because it can 2741



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Scheme 2

form a stable S-oxide as in the case of thionicotinamide.57 The first step in the oxidation of TTTU can be depicted as in Scheme 1. A comparable, well-established structure of thionicotinamide S-oxide has the structure

Subsequent oxidation of the sulfenic acid to sulfate does not proceed through the standard sulfinic to sulfonic acid sequence. Those zwitterion oxo-acids will be unstable due to steric issues that would be associated with the methyl groups on the two nitrogen atoms and the tetrahedral sulfur center bearing the oxygen atoms.53 Our attempts to synthesize these oxo-acids failed, and they could not be isolated. ESI-MS spectra do not show even their transient formation (cf. the sulfenic acid which was observed in abundance in the Figure 4a spectrum). The sulfenic acid should be very reactive in the polar aqueous environment used for these sets of studies. In water, the sulfenic acid will form an equilibrium mixture with its hydrated form. The cleaving of the C-S bond occurs at this point, leaving a very highly reducing sulfur leaving group which is rapidly oxidized to sulfate. The sequence is depicted in Scheme 2. Elimination of the highly reducing sulfur species, HSOH, and its oxidation to bisulfite should occur rapidly since it would be extremely unstable. In aerobic conditions, the sulfoxylate anion, formed after first oxidation of HSOH, should react, at diffusion controlled rates, with oxygen to produce a series of genotoxic reactive oxygen species SO2 2- þ O2 f SO2 - þ O2 -

ðR8Þ

SO2 - þ O2 f SO2 þ O2 -

ðR9Þ

Direct experimental evidence is available for the formation of the sulfoxyl anion radical in aerobic decompositions of thiourea dioxides in basic and slightly basic media.58,59 Dismutation of superoxide radical can yield peroxide, and reaction of the sulfoxylate anion with peroxide can yield the highly damaging hydroxyl radical. The positively disposed carbon atom on the zwitterion should be vulnerable to attack by a nucleophilic solvent such as water. Table 2 shows all the possible reactions that can occur in acidic TTTU-bromate solutions. Apart from reaction initiation through series of reactions R5-R7; initiation can also occur through trace amounts of bromide ions which exist in equilibrium with bromate ions (ca. 10-5 M).56 These bromide ions can initiate the cascade of reactions M1-M4, resulting in formation of the reactive species HBrO2, HOBr, and Br2. These (reactive species) will carry the bulk of the oxidations since the bromate ion itself is considered inert as an oxidant. Only trace amounts of bromide ions are needed to initiate such a cascade of reactions since bromide is regenerated. In fact, addition of M1 þ M2 þ 3M8 gives the following overall stoichiometric equation which shows cubic autocatalysis in bromide ions BrO3 þ 2Br þ 3ðRÞ2 CdS f 3ðRÞ2 CSO þ 3Br

ðR10Þ

The expected autocatalytic increase in bromide with time should fuel reaction M1, which produces the reactive and oxidizing species HBrO2 and HOBr. With the rate-determining step being M1, one would still expect second-order kinetics in acid as observed in Figure 5c. However, utilizing 5 10-5 M bromide as the initial concentration (the accepted concentration of bromide in equilibrium with 1.0 M bromate solutions), a rough simulation gives induction periods in the regions of hours, much slower than observed kinetics in Figures 5a and 5b. Utilizing the other initiation pathway involves reaction M6, the direct reaction of the protonated bromic acid with TTTU. The overall initiation reaction sequence will be M4 þ M5 þ M6 þ M7 þ M8 BrO3 þ 3ðRÞ2 CdS f 3ðRÞ2 CSO þ Br

2742

ðR11Þ



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Table 2. Putative Reactions Possible in Acidic TTTU-Bromate Mixtures no. M1

reaction BrO3

-

þ Br

-

þ 2H þ T HBrO2 þ HOBr

M2

HBrO2 þ Br - þ 2H þ T 2HOBr

M3

HOBr þ H þ þ Br - T Br2 þ H2 O

M4

BrO3 - þ H þ T HBrO3

M5

HBrO3 þ H þ T H2 BrO3þ

M6

H2 BrO3 þ þ ðRÞ2 CdS f HBrO2 þ ðRÞ2 CSO þ H þ

M7

HBrO2 þ ðRÞ2 CdS f HOBr þ ðRÞ2 CSO

M8

HOBr þ ðRÞ2 CdS f ðRÞ2 CSO þ H þ þ Br -

M9

HOBr þ ðRÞ2 CSO f ðRÞ2 CSO2 þ H þ þ Br -

M10

HOBr þ ðRÞ2 CSO2 f HSO3 þ ðRÞ2 CdO þ H þ

M11

Br2 þ ðRÞ2 CdS þ H2 O f ðRÞ2 CSO þ 2H þ þ 2Br -

M12

Br2 þ ðRÞ2 CSO þ H2 O f ðRÞ2 CSO2 þ 2H þ þ 2Br -

M13

Br2 þ ðRÞ2 CSO2 þ H2 O f ðRÞ2 CSO3 þ 2H þ þ 2Br -

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

M14 Br2 þ ðRÞ2 CSO3 þ 2H2 O f ðRÞ2 CdO þ SO4 2 - þ 4H þ þ 2Br M15

ðRÞ2 CSO3 þ H2 O f ðRÞ2 CdO þ HSO3 - þ H þ

M16

HOBr þ HSO3 - f SO4 2 - þ 2H þ þ Br -

’ CONCLUSION The oxidation mechanism of TTTU by oxybromine species is remarkably different from that subscribed to by thiourea and other substituted thioureas that have the ability to form stabilized sulfur oxo-acids. While most other thioureas exhibit their toxicities in aerobic basic environments (pH > 8) due to the formation of the sulfoxyl anion radical,41 TTTU can form the same sulfoxyl anion radical and a cascade of reactive oxygen species in aerobic conditions and at a wider range of pH values because any oxidation step after the sulfenic acid (irrespective of pH) will produce genotoxic oxygen-based and sulfur-based radicals. While all these substituted thioureas can act as goitrogenics, TTTU gives thyroid tumors51 at much lower dosages, has the most powerful teratogenicity of all N-alkylated thioureas,60 and is the most effective in inhibitting thyroid hormone synthesis by being most efficient in abstracting the necessary Iþ atom.61

’ ACKNOWLEDGMENT This work was supported by Research Grant Number CHE 0614924 from the National Science Foundation. ’ REFERENCES

The overall activity of this initiation sequence is production of Br-, which is then used in reaction M1 to fuel the reaction (second-order kinetics in acid). In both initiation scenarios, bromide is expected to catalyze the reaction. This is shown in Figure 8. If the reaction initiation proceeds solely through trace amounts of bromide that exist in bromate solutions (M1 þ M2 þ 3M8), then the cubic autocatalysis would dominate and the reaction would be sensitive to minute changes in bromide ion. Figure 8 shows that bromide catalyzes the reaction but not in the form of an autocatalyst in which we expect sigmoidal kinetics. Figure 8 data show that bromide catalyzes the reaction in a linear fashion, in the form of reaction M1 in Table 2. Thus, the reaction proceeds predominantly through M6 (or R7). Table 2 is a comprehensive reaction scheme, applicable to all oxidations of thioureas by oxybromine species. From our proposed mechanism, most of the reactions in Table 2 are not needed for TTTU oxidations due to TTTU’s inability to form nonzwitterionic oxo-acid intermediates before formation of sulfate and the urea analogue. The TTTU oxidation mechanism involves reactions M1-M8 and then a composite reaction that involves Scheme 2. ðRÞ2 CSO þ 3H2 O f ðRÞ2 CSO þ HSO3 - þ 5Hþ þ 4eðR12Þ Composite reaction R12 is next followed by reaction M16. While one might use all the oxidants in the reaction medium (HBrO2, HOBr, and Br2(aq)), use of only one, HOBr, as in reactions M8 and M16 is still justified because reactions M2 and M3 rapidly establish their equilibria.

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Kinetics of Oxidation of Tetramethylthiourea by Aqueous Bromine and

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