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S-Oxygenation of Thiocarbamides V: Oxidation of Tetramethylthiourea by Chlorite in Slightly Acidic Media Tabitha Chigwada, Wilbes Mbiya, Kudzanai Chipiso, and Reuben Hazvienzani Simoyi J. Phys. Chem. A, Just Accepted Manuscript • Publication Date (Web): 12 Jun 2014 Downloaded from http://pubs.acs.org on June 28, 2014

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The Journal of Physical Chemistry

S-Oxygenation of Thiocarbamides V: Oxidation of Tetramethylthiourea by Chlorite in Slightly Acidic Media

by

Tabitha Chigwada† Wilbes Mbiya, Kudzanai Chipiso and Reuben H. Simoyi*ǂ Department of Chemistry, Portland State University, Portland, OR 97207-0751, USA. ǂ School of Chemistry and Physics, University of KwaZulu-Natal, Westville Campus, Durban 4014. South Africa. † Permanent address: Eugene C. Bennett Department of Chemistry, West Virginia University, Morgantown, WV 26506-6045, USA.

ACS Paragon Plus Environment

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Abstract: 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(Me2N)C=S + H2O → Me2N(Me2N)C=O + SO42- + 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 with other substituted thioureas, can only stabilize intermediate oxoacids, before formation of sulfate, in the form of zwitterions. In 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 re-form ClO2-. Oxidation of TTTU by chlorite has a complex dependence on acid due to chlorous acid dissociation and protonation of the thiol group on TTTU in high acid conditions which renders the thiol center a less effective nucleophile. Keywords: Kinetics S-oxides Metabolic activation Zwitterions Genotoxcity


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Introduction Thiocarbamides represent a vast group of biologically active compounds.1 The simplest and most active member of this group is thiourea.2,3,4 Substituted thioureas present physiological activities that are heavily dependent on the degree and type of substituents attached to the parent thiourea.2 Thiourea is known to be very efficient as a protectant against paraquat5 because of its ability to deactivate superoxide ion.5,6 Dimethylthiourea, on the other hand, is an efficient hydroxyl radical scavenger,7,8 capable of reversing cardiac left ventricular remodeling and failure that occurs after myocardial infarction as well as also being an effective protection against photic injury.9,10,11,12,13 Despite this, DMTU, however, failed to ameliorate reperfusioninduced skeletal muscle injury from leukocyte-derived reactive oxygen metabolites14. It is also a known edematogenic agent15 and yet inhibits cigarette smoke-induced broncho-constriction by scavenging hydroxyl radicals derived from smoke.16,17 The ambiguity in its physiological effects can only be rationalized by an evaluation of the mechanistic basis of its bioactivation. Our initial motivation in studying oxidation mechanisms of organosulfur compounds such as DMTU and thiocarbamides in general, were research publications that reported that a good indication of the scavenging abilities of DMTU could be correlated with an increase in concentrations of dimethylthiourea dioxide (sulfinic acid of DMTU) rather than the disulfide.18 Tetramethylthiourea is well established as a teratogen.19 It is a fetotoxin that produced malformations in the tail, palate and extremities of surviving rat fetuses. None of the other substituted thioureas are as potent. Thioureas, in general, differentially induce physiological effects based on their structures.20 This would suggest that their bioactivation mechanisms might differ; especially their oxidation mechanisms.20 This manuscript represents Part V in a series of work devoted to elucidating the Soxygenation mechanisms of a series of substituted thioureas.21,22,23,24 We report, here, on the


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oxidation mechanism of tetramethylthiourea by mildly acidic chlorite solutions. Tetramethylthiourea differs from other substituted thioureas in that it does not possess a detachable proton on one of the nitrogen groups, and thus is unable to form stable oxo-acids such as sulfinic and sulfonic acids without resorting to formation of a zwitterion. These zwitterions are extremely unstable especially in the presence of nucleophiles in which a nucleophilic attack on the carbon center of the CN2 unit is extremely facile. Our preliminary investigations have shown that the reaction dynamics presented by the oxidation of TTTU by similar oxidants are very different from those observed in the oxidations of thiourea, DMTU, and trimethylthiourea.24

Experimental Section Materials. The following analytical grade chemicals were used without purification: perchloric acid (72%), 1,1,3,3, tetramethyl-2-thiourea (TTTU) (Acros), barium chloride, sodium chloride, sodium chlorite (Aldrich), sodium perchlorate and soluble starch (Fisher). Commercially available sodium chlorite varied in purity (78-88%) with the main impurities being chloride and carbonate. The sodium chlorite was recrystallized once from a water/ethanol mixture to bring the assay value to 95%. These were stored in a desiccator in small batches of 5 g or less to avoid possible explosions.25 The recrystallized chlorite was standardized iodometrically by adding excess acidified potassium iodide and titrating liberated iodine against sodium thiosulfate with freshly prepared starch as an indicator.26 Chlorine dioxide was prepared by the standard method of oxidizing sodium chlorate in a sulfuric acid/oxalic acid mixture.27 The stream was passed through a sodium carbonate solution before being collected in ice-cold water at 4 oC at a pH of ~ 3.5. Standardization of ClO2 was also accomplished by iodometric techniques through addition of


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excess acidified potassium iodide and back-titrating the liberated iodine against standard sodium thiosulfate. The chlorine dioxide was stored in an acidic medium in a volumetric flask wrapped in aluminium foil at 4°C. Chlorine dioxide was also standardized jointly by its molar absorptivity coefficient of 1,265 M-1cm-1 at 360 nm on a Perkin-Elmer Lambda 25S UV/Vis spectrophotometer.

1.4 1.2 c 1.0

Absorbance

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b

0.8 0.6

a

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Wavelength Figure 1: UV absorption spectrum of (a) 5.000 x 10-5 M tetramethylurea (product), (b) 1.5 x 10-5 M tetramethylthiourea (c) 9.08 x 10-4 M ClO2 showing peaks for TTTU at 248 nm (,max = 16,608 M-1 cm-1) and chlorine dioxide at 360 nm (,max = 1,265 M-1 cm-1) respectively. There is no interference from the substrate or from the product at 360 nm. The absorptivity coefficient of the product, at less than 200 M-1 cm-1, is negligible at 248 nm.

Methods. All experiments were carried out at 25°C and an ionic strength of 1.0 M (sodium perchlorate). The ClO2-/ClO2/TTTU reactions were monitored spectrophotometrically at λ = 360


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nm as reactions were performed in excess chlorite so as to utilize the formation of chlorine dioxide as an indicator of reaction progress. TTTU has an absorption peak at 248 nm (,max = 16,608 M-1 cm-1) which was utilized to follow its rate of depletion in the initial stages (only). Figure 1 shows spectra of TTTU (reactant), tetramethylurea, TTU (product) and chlorine dioxide. TTTU’s absorbance does not contribute to the absorption at 360 nm but chlorine dioxide contributes significantly to the absorbance at 248 nm. The product of oxidation, tetramethylurea, contributes very insignificantly at 248 nm with an absorptivity coefficient of less than 200 M-1 cm-1. Despite chlorine dioxide’s contribution to the absorbance observed at 248 nm, its formation is delayed (see Figures 2, 3a and 4b) and thus in the beginning of the reaction, before formation of chlorine dioxide, observed activity in the peak at 248 nm could be attributed solely to concentration changes in TTTU. Kinetics measurements were performed on a Hi-Tech Scientific double mixing SF-61DX2 stopped-flow spectrophotometer. The data from the spectrophotometer were amplified and digitized via an Omega Engineering DAS-50/1 16-bit A/D board interfaced to a computer for storage and data analysis. Stoichiometric determinations were carried out by mixing various ratios of chlorite and TTTU (in acidic medium to avoid alkaline disproportionation of ClO2)28 in stoppered volumetric flasks and scanning them spectrophotometrically for formation of ClO2 after an incubation of up to two days. Qualitative and quantitative analysis of sulfate produced was performed through its precipitation as BaSO4. For reactions run in excess chlorite conditions, the excess oxidizing power was evaluated by addition of excess acidified iodide, which was titrated against standard thiosulfate with freshly prepared starch as an indicator. Tests for adventitious metal ion catalysis.29 Water used for preparing reagent solutions was obtained from a Barnstead Sybron Corporation water purification unit capable of producing both distilled and de-ionized water (Nanopure). Not much difference was observed in the


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general reaction kinetics observed with pure distilled de-ionized water and those run in the presence metal ion chelators such as EDTA. Inductively-Coupled Plasma Mass Spectrometry (ICPMS) was utilized to quantitatively evaluate the concentrations of a number of metal ions in the water used for our reaction medium. ICPMS analysis showed negligible concentrations of iron, copper and silver and approximately 1.5 ppb of cadmium and 0.43 ppb in lead.

2.0 1.8 1.6 1.4 Absorbance

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Wavelength/nm

Figure 2:


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Figure 2: Absorbance scans of TTTU during oxidation by chlorite in aqueous acidic medium. Traces were collected every 60 second reaction time intervals. [TTTU]0 = 5 x 10-4 M, [ClO2-]0 = 4 x 10-3 M, and [HClO4]0 = 0.1 M. Note that the peak of chlorine dioxide starts to form before the peak at 248 nm has vanished. This is in agreement with data presented in Figure 6b.

RESULTS Reaction dynamics. Figure 2 shows the observed activity of the reaction in the UV region. Initially the peak at 248 goes down with no activity being observed at 360 nm. After a short induction time, while the peak at 248 nm is still decreasing, the 360 nm peak starts to rise. The final absorption reading at 360 nm is attained after very long incubation periods, some in the regions of days. Most of the rapid increase in the peak at 360 nm is accomplished within the first 5 – 10 s. Further increases after this period are very slow. Chlorine dioxide formed after 10 s is due to the disproportionation of chlorite solutions in acidic media:30

5ClO2- + 4H+ → 4ClO2(aq) + 2H2O + Cl-

(R1)

Reaction stoichiometry. Through a combination of spectrophotometric, gravimetric and titrimetric techniques, the stoichiometry of the reaction was deduced to be:

2ClO2- + Me2N(Me2N)C=S + H2O → Me2N(Me2N)C=O + SO42- + 2Cl- + 2H+ (R2)

Stoichiometry R2 was the highest ratio, R, of chlorite to TTTU that could be used without forming chlorine dioxide as a final product (excluding spurious and/or transient ClO2 formations). ESI-MS spectral data showed that production of Me2N(Me2N)C=O , TTU, was not quantitative. TTU broke down into many hydrolysis fragments that included dimethylurea, but


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none of these fragments altered the 2:1 ratio of oxidant to reductant that was needed to completely consume TTTU and give quantitative formation of sulfate. Figure 3a shows effect of altering chlorite concentrations (effectively altering R) while keeping all other species constant. In this series of experiments, a chlorite concentration that does not satisfy stoichiometry R3 will not produce chlorine dioxide and hence will not show an induction period (see Figure 3b).The induction period will be infinite, and the inverse of the induction period will be zero.


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1.4

f e

1.2

d c

Absorbance at 360 nm

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1.0 b 0.8 a 0.6

0.4

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0.0 0

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2

3

4

5

Time/seconds

Figure 3a: Figure 3a: The effect of chlorite on the absorbance traces obtained at 360 nm. Chlorite concentrations are directly proportional to the amount of ClO2 formed and inversely proportional to the induction period at fixed TTTU concentrations. [TTTU]0 = 1.25 x 10-3 M, [HClO4]0 = 0.125 M, I(NaClO4) = 1.0 M, [ClO2-]0 = (a) 5.00 x 10-3 M, (b) 6.25 x 10-3 M, (c) 7.50 x 10-3 M, (d) 8.75 x 10-3 M, (e) = 10.00 x 10-3 M, (f) 11.25 x 10-3 M.


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4.0

3.5

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2.5 I/Tind

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[NaClO2] x 103

Figure 3b: Figure 3b: Plot of 1/Tind vs [ClO2-] using stochiometric excess of chlorite, showing that an inverse relationship exists for a long range of chlorite concentrations. [TTTU]0 = 1.25 x 10-3 M, [HClO4]0 = 0.125 M, I(NaClO4) = 1 M The intercept value (extrapolated to 1/T = 0) gives a chlorite concentration of 2.5 x 10-3 M, which is the exact stoichiometry predicted in stoichiometric reaction R1.


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1.2

b

1.0 a Absorbance at 360nm

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Figure 4a:


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Figure 4a: Absorbance traces at 360 nm showing effect of low acid concentrations on the oxidation of TTTU by chlorite. In this case there is no well-defined induction period. [TTTU]0 = 1.25 x 10-3 M, [ClO2-]0 = 5.00 x 10-3M, I(NaClO4) = 1 M, [HClO4]0 = (a) 0.005 M, (b) = 0.010 M. Acid catalyzes formation of chlorine dioxide until the pH of the reaction medium exceeds pKa of chlorous acid.

a 0.8 b Absorbance at 360nm

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Figure 4b: Figure 4b: Absorbance traces at 360 nm showing effect of high acid concentrations on the oxidation of TTTU by chlorite. In this case there is a well-defined induction period which in not affected by the amount of acid. Concentration of acid is inversely proportional to the amount of chlorine dioxide formed. The reaction now proceeds predominantly through chlorous acid.


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[TTTU]0 = 1.25 x 10-3 M, [ClO2-]0 = 5.00 x 10-3 M, I(NaClO4) = 1 M, [HClO4]0 = (a) 0.1 M, (b) 0.2 M, (c) 0.3 M, (d) 0.4M, (e) 0.5 M.

An extrapolation of the linear plot of inverse of induction period vs. chlorite concentrations gives the exact amount of chlorite needed for stoichiometry R3. For a fixed amount of TTTU of 0.00125 M, the intercept value of chlorite deduced by this plot is 0.0026 M; which is very close to stoichiometry R3.

Reaction Kinetics. Figures 3a and b show that there is an inverse dependence (to the first power) of the induction time with chlorite concentrations. Such a correlation would indicate that the reactions that consume TTTU and those that form chlorine dioxide, though they both involve chlorite, react at different time scales. The ratio of oxidant to reductant of at least 4 ensures that all TTTU is oxidized to TTU. If reaction between product chlorine dioxide and TTTU was slow, then one would expect oligooscillatory behavior in chlorine dioxide production. The effect of acid depends on its strength. Figure 4a shows the effect of acid at low concentrations (0.005 – 0.025 M). There is no real discernible induction period (cf. Figure 3a with [H+]0 = 0.125 M). However, in general there is an increase in the rate of formation of chlorine dioxide in this acid range. High acid concentrations (Figure 4b) reverse this trend. The now well-defined induction period is independent of acid concentrations and there is a decrease in rate and amount of chlorine dioxide formed as acid is increased. This behavior could arise from the fact that acid retards reactions that form chlorine dioxide or catalyzes reactions that consume it. The effect of TTTU shown in Figure 5 is expected for conditions in which chlorite is in overwhelming excess over TTTU. There is very little variation in the induction period, but high TTTU concentrations will show higher and more rapid production of chlorine dioxide after the induction period.


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f e

0.8 d c Absorbance at 360nm

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0.6 b

0.4

a

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5

10

15

20

Time/seconds Figure 5:

Figure 5: “Peacock-tail type” traces derived from variation of [TTTU]0 in large excess of [ClO2-]0. The induction period stays the same although the rate of formation of chlorine dioxide increases. The amount of chlorine dioxide formed is directly proportional to the amount [TTTU]0. [ClO2-]0 = 5.00 x 10-3 M, [HClO4]0 = 0.125 M, I(NaClO4) = 1 M, [TTTU]0 = (a) 2.5 x 10-4 M, (b) 5.0 x 10-4 M, (c) 7.5 x 10- 4M, (d) 10.0 x 10-4 M, (e) 12.5 x 10-4 M, (f) 15.0 x 10-4 M.


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Data collected at 248 nm. Figure 6a shows absorbance traces at 248 nm, one of the absorption peaks of TTTU. It shows a 2-phase reaction progress in which initially there is rapid decrease in absorbance followed by region where there is no change in absorbance. The last stage involves a rapid autocatalytic drop in absorbance to the final absorbance that arises from mainly from

1.4

1.2

Absorbance at 248nm

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Time/seconds Figure 6a: Figure 6a: Effect of chlorite on the depletion of TTTU in its oxidation by chlorite monitored at 248 nm. The data displays two-stage kinetics. The products of the TTTU oxidation and chlorine


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dioxide give the residual absorbance that can be seen in the absorbance-time data at the end of the reaction. Acid concentrations are essentially buffered in this series of experiments. [TTTU]0 = 7.5 x 10-5 M, [HClO4]0 = 0.125 M, I(NaClO4) = 1 M, [ClO2-]0 = (a) 5.0 x 10-4 M, (b) 6.0 x 10-4 M, (c) 7.0 x 10-4 M, (d) 8.0 x 10-4 M, (e) 9.0 x 10-4 M, (f) 10.0 x 10-4 M.

1.6

1.4 b a

1.2 Absorbance

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Time/seconds Figure 6b Figure 6b: Two traces superimposed at (a) 248 nm and (b) 360 nm. Formation of chlorine dioxide commences before all TTTU and its intermediates have been fully oxidized. [ClO2-]0 = 5.00 x 10-4 M, [TTTU]0 = 5.0 x 10-5 M, [HClO4]0 = 0.125 M. The traces at 360 nm have been magnified 10X.


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chlorine dioxide and to a lesser extent from the TTU product. The initial phase of this reaction has first order dependence on chlorite concentrations. The rate of the second phase of the reaction is also catalyzed by chlorite. What might appear like a shutting down of the reaction about 5 s into the reaction is due to the formation of some TTTU intermediate which later either decomposes or is further oxidized to obtain final products.

1.4

1.2

1.0 Absorbance at 248nm

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0.8

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10 11 12 13

Time/seconds

Figure 6c: Effect of acid on the depletion of TTTU in its oxidation by chlorite monitored at 248 nm. The data also displays two-stage kinetics. Note the change in absorbance traces from sigmoidal autocatalysis at low acid to basic zero order as acid is increased past the pKa of chlorous acid. [TTTU]0 = 7.5 x 10-5 M, [ClO2-]0 = 5.00 x 10-4M, I(NaClO4) = 1 M, [HClO4]0 = (a) 0.005 M, (b) 0.010 M, (c) 0.015 M, (d) 0.020 M, (e) 0.025 M

This can be rationalized from the following analysis of our data: final product, TTU, does not absorb substantially at 248 nm. The observed absorbance at the end of the reaction is from


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chlorine dioxide. The observed lack of change in absorbance at 248 nm is partly derived from the fact that contribution from chlorine dioxide and depletion of TTTU effectively nullify each other.

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1.0


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Time/s Figure 6d: A continuation of the acid increase shown in Figure 6c which now displays a saturation in rate of reaction for the first phase of the reaction with acid followed by a retardation of the second stage of the reaction. No autocatalytic behavior is observed in the first stage of the reaction. [TTTU]0 = 1.0 x 10-4 M.

Figure 6b superimposes a typical absorbance trace taken at 360 nm with one taken at 248 nm. It shows that chlorine dioxide formation commences before the end of the first stage of the reaction and continues at a slower rate after the end of this stage. Of note is the fact that chlorine dioxide continues during the period when there is no activity in the absorbance at 248


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nm. Acid has opposite effects on the two stages of the reaction. Figure 6c shows a series of traces taken at low acid concentrations. Low acid delivers sigmoidal decay kinetics in the first stage and as acid is slowly increased, this sigmoidal trace gives way to normal decay trace with a concave shape (see also Figure 6d). The second stage is very rapid at low acid and in higher acid environments, the intermediate lingers a while longer. In conditions of high acid, coupled with high excess of oxidant; R š10; pseudo-first order kinetics are never attained (Figure 6d), and the rate of decay of TTTU in the first stage becomes insensitive to variations in acid, but the second phase is still inhibited by acid. TTTU gives a first order dependence to its rate of depletion (Figures 7) at all acid concentrations.

2.0

1.6 Absorbance at 248nm

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Figure 7: Depletion of TTTU in its oxidation by chlorite. The data also displays two-stage kinetics. Chlorite is in excess over TTTU. Baseline absorbances obtained at the end of the reaction are


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from chlorine dioxide. [ClO2-]0 = 5.00 x 10-4M, [HClO4]0 = 0.125 M, (NaClO4) = 1 M, [TTTU]0 = (a) 2.5 x 10-5 M, (b) 5.0 x 10-5 M, (c) 7.5 x 10-5 M, (d) 10.0 x 10-5 M, (e) 12.5 x 10-5 M.

1.4

1.2

Absorbance at 248nm

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1.0

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10

Time/seconds Figure 8

Figure 8: Effect of low acid concentrations on the oxidation of TTTU by chlorine dioxide. Rate of depletion of TTTU increases with increase in acid concentrations. This, however, quickly saturates as in Figures 6c and d. [TTTU]0 = 7.5 x 10-5 M, [ClO2]0 = 5.00 x 10-4M, I(NaClO4) = 1 M, [HClO4]0 = (a) 0.005 M, (b) 0.010 M, (c) 0.015 M, (d) 0.020 M, (e) 0.025 M.


- 21 -


Chlorine dioxide oxidations. Many chlorine dioxide oxidations are retarded by acid, but Figure 8 shows that acid catalyzes the oxidation of TTTU at low acid concentrations. This is in agreement with experimental data in Figure 4a taken at the same low acid concentrations in which the rate of formation on chlorine dioxide increases with acid and yet at higher acid concentrations the effect is inhibitory. However, some mild form of autocatalysis is also observed.

0.8

0.7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 48

0.6

0.5

0.4

0.3 e

f

d

c

b

a

0.2

0.1 0

5

10

15

20

Time/s Figure 9: Effect of chlorine dioxide in the depletion of TTTU. Rate of depletion of TTTU increases with increase in chlorine dioxide concentrations. [TTTU]0 = 5.0 x 10-5 M, [HClO4]0 = 0.125 M. [ClO2]0 = (a) 1.35 x 10-4 M, (b) 1.8 x 10-4 M, (c) 2.25 x 10-4 M, (d) 2.7 x 10-4 M, (e) 3.6 x 10-4 M, (f) 4.5 x 10-4 M.

Figure 9a shows that the rate of depletion of TTTU in high acid and excess chlorine dioxide shows nearly zero order kinetics in TTTU. This is possible if there is a rapid and irreversible first


- 22 -

Page 23 of 48

step of the reaction that involves TTTU with the subsequent rate-determining steps not involving TTTU.

0.7

0.6


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.5 g

0.4

f 0.3

e

0.2

d c

0.1

a

b

0.0 0

1

2

3

4

5

Time/s Figure 10a: Effect of chlorine dioxide in the oxidation of TTTU. The adduct observed in Figures 9 a and b does not absorb at 360 nm. The initial absorbances observed are the ones expected from contribution from chlorine dioxide on its own before its depletion commences. [TTTU]0 = 1.0 x 10-2 M, [HClO4]0 = 0.125 M. [ClO2]0 = (a) 4.5 x 10-5 M, (b) 9.0 x 10-5 M, (c) 1.35 x 10-4 M, (d) 1.8 x 10-4 M, (e) = 2.7 x 10-4M, (f) 3.6 x 10-4 M, (g) 4.5 x 10-4 M.

In high acid, chlorine dioxide depletion follows nearly first order dependence (Figure 10a). Data utilized for Figure 10 were derived from experiments performed in high excess of TTTU such that all chlorine dioxide is consumed at the end of the reaction. The use of high acid simplifies the kinetics by using HClO2 as the oxidant with little to no contribution from ClO2-. Figure 10b


- 23 -


shows a variation of TTTU while keeping ClO2 constant. Nearly pseudo-first order kinetics are observed, but the rate of consumption of ClO2 does not seem to be affected by TTTU.

0.35

0.30


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 48

0.25

0.20

0.15

0.10

0.05

0.00 0.0

0.5

1.0

1.5

2.0

Time/s

Figure 10b: Variation of TTTU in its oxidation by chlorine dioxide in close to pseudo-first order kinetics environment. [ClO2]0 = 1.8 x 10-4 M, [HClO4]0 = 0.1 M. [TTTU]0 = (a) 2.50 x 10-3 M, (b) 3.75 x 10-3 M, (c) 5.00 x 10-3 M, (d) 6.25 x 10-3 M, (e) 7.50 x 10-3 M. Rate increases (a) to (e). Trace (e) is the fastest and has the lowest residual absorbance.

The observed differences in base-lines are due to the varied input concentrations of TTTU and not to any changes in the kinetics. The surprising experimental finding was that chloride appears to have no effect on the reaction rate (Figure 11). It appeared to be more relevant in affecting the disproportionation of chlorite instead (reaction R2). All traces shown in Figure 11 gave the same initial rates. The differences observed in initial absorbances is due to the catalytic effect of


- 24 -

Page 25 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


chloride on the disproportionation of chlorite, which is the initial intermediate formed as the reduction of chlorine dioxide commences.

MECHANISM Nature of intermediate species. Figures 6a, b, d and 7 show the formation of a long-lived intermediate species about 10 s into the reaction which later decays after a short incubation period. This species appears to be more stable in acid and decomposes much more reluctantly in high acid environments. This species is the S-oxide of TTTU, (Me2N)2CS→O. This is a totally unexpected result, totally unprecedented in all our studies of organosulfur chemistry. Never had a sulfenic acid been stabilized without extensive steric hindrance around the S-O bond.31,32 Any intermediate species between TTTU and its urea analogue, TTU, has to exist in a zwitterionic form without any resonance form available to stabilize the C – S bond. Example below is a putative sulfonic acid zwitterion of TTTU:

Me2N

O C

Me2N

S

O O

In this structure, the electronegative oxygen atoms render the sulfur atom partially positive. Xray analysis of the sulfonic acids of thiourea and dimethylthiourea has consistently shown a 4electron 3-center unit with a positive charge delocalized over the 3 atoms. With both the C- and S-atoms positively charged, the subsequent C-S bond will be exceedingly weak. The molecule with such a framework would be very weak and would decompose easily through cleavage of the C-S bond. In extreme cases, such a molecule would not form. Consistently, research in our lab and other labs has proved that thiourea oxoacids are extremely unstable and decompose


- 25 -


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 48

readily in basic environments.33 Substituted thioureas with an available proton on one of the nitrogen atoms: methyl, dimethyl and trimethylthiourea can form non-polar resonance forms:

The sulfenic acid is the suggested intermediate based on the following experiment: TTTU was mixed, in a 50 % acetonitrile solution with exactly 2 equivalents of hydrogen peroxide. This solution was stirred, for over 2 hours at -10 0C. The resulting solution was allowed to thaw and stand overnight at room temperature. After 12 hours, long, colorless flat triclinic-like crystals were formed. The supernatant solution also gave needle-like crystals after adding 50 % pet ether. Both sets of crystals, including the supernatant liquid gave a spectrum with a peak at 224 nm. None of the crystals diffracted for X-ray analysis. They appeared to be composed of several species. The product of this synthesis was characterized as the (zwitterionic) S-oxide (Me2N)2CS→O by ESI-MS spectra (vide infra). The absence of a proton on the nitrogen atoms precludes the formation of a true sulfenic acid. The intermediate species formed in this reaction did not give the same spectrum as TTTU (and thus assumed to be the S-oxide). Another synthesis in which only one mole of H2O2 was used and another in which three moles were used gave the same result. Our initial use of two moles was to ensure formation of the sulfinic acid


- 26 -

Page 27 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


which was expected to be the most stable oxo-acid.34,35 Since use of 1, 2 and 3-mole equivalents gave essentially the same result, it was plausible to assume that the S-oxide is the most stable metabolite. The basic environment allows the solvent, water, to act as a nucleophile which can attack the positively charged 4-electron 3-center carbon atom, eliminating the sulfur group, and forming urea. High acid environments stabilize this zwitterion, and hence Figures 6c and d show a slower decomposition of this intermediate species with acid.

ESI – MS data were used to determine both products and nature of the intermediate species observed in Figures 6 a – d. Initially, a stoichiometric ratio of chlorite to TTTU was mixed in slightly acidic solutions. An ESI spectrum was taken about 60 s into the reaction, way before the reaction had reached completion. It was assumed that both products and intermediates would be detected in this time range. Figure 11 shows the ESI-MS spectrum produced in from this experiment. This spectrum shows the expected peak for the substrate TTTU at m/z = 133.07 as well as a peak for the product, tetramethylurea at 117.10. The surprising feature of this spectrum is the observation of a strong peak for the tetramethylthiourea S-oxide at m/z = 149.07. This is the only plausible structure after addition of an atom of oxygen. This is shown in structure 1. The S-oxide is formed if one of the pi-electrons on the S center is donated fully to the more electronegative oxygen atom.


- 27 -


Structure I

TTTUCLO2 #12-19 RT: 0.16-0.22 AV: 8 NL: 2.02E7 T: FTMS + c ESI Full ms [70.00-300.00] 133.07982

100 95 90 117.10243 85 80 88.02158 75 70 65 R e la tiv e A b u n d a n c e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 48

60 55 50 45 40 35 30

149.07476

25 20

131.08183

135.06871

15 10

101.10745

5

90.01736 98.54174 93.09108

0 85

90

95

100

139.08455 120.04797 125.06879

106.53025 115.07576 105

110

115

163.09049 155.06180 157.05765 164.92098

144.92789

120

125

130

135

140

145

150

155

160

165

170

181.06480 174.70825 175

180

185

m/z

Figure 11: Mixture of the stoichiometric amounts of ClO2- : TTTU. Spectrum was taken 60 s into the reaction. Both the substrate, TTTU and product, TTU can be observed at m/z 117.10 and 117.10. A strong peak for the S-oxide can be observed at 149.07.

To conclusively determine the nature and type of metabolites formed in the oxidation of TTTU, a clean 2-electon oxidant was used to successively oxidize TTTU by 2-electron equivalents: aqueous bromine: Br2(aq) + 2e- → 2Br- ; E0 = 1.08 V The reduction product is only bromide with no other possibilities. Thus, addition of one equivalent of bromine should oxidize the sulfur center in TTTU to the sulfenic acid, 2 equivalents


- 28 -

189.51733 190

199.16979 195

200

Page 29 of 48

to the sulfinic acid and 3 equivalents to the sulfonic acid. A full oxidation of TTTU to its urea analogue needs 4 equivalents of bromine. These oxoacids will only be formed if they are stable. In all oxidations, TTTU will form its most stable metabolite from the equivalents of oxidant used.

TTTUBR1 #4-11 RT: 0.12-0.21 AV: 8 NL: 2.55E6 T: FTMS + c ESI Full ms [70.00-300.00] 88.02142 100 95 90 85 80 75 70 65 R e la tive A b u n d a n ce

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


60 55 50 45 40 35 133.07953

30 25

149.07458

20 15 10 90.01722

5

91.79922

0 85

90

95

101.10726 106.53007 100

105

110

116.08556 115

120

127.04345

135.07529

125

135

130

142.77563

140 m/z

145

152.05308 150

155

163.09026 160

165

172.56651 179.01808 170

175

180

189.41528 185

190

Figure 12a: Spectrum obtained after 5 min of addition of 1:1 equivalents of bromine to TTTU. Only the S-oxide is observed, as well as a hydrolysis product, dimethylurea.

In Figure 12a, one equivalent of aqueous bromine was added to TTTU. The spectrum shows the expected m/z peak for TTTU at 133.08 and the S-oxide at 149.07. A strong peak for dimethylurea is also observed at 88.02 which should be a hydrolysis product which is not part of the oxidation of TTTU.


- 29 -

199.16969 195

200


TTTUBR2 #6 RT: 0.12 AV: 1 NL: 1.14E7 T: FTMS + c ESI Full ms [70.00-300.00] 88.02145 100 95 90 85 80 149.07451

75

133.07953

70 65 R e lative A b u nd a n ce

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 48

60 55 50 45 40 35 30 25 20 151.07033

15 10 5 0 85

90

116.08552

101.10726

90.01723

97.76492

106.53007

95

105

100

110

135.07530

120.73164 127.04335 115

120

125

130

155.06149 146.98845

135

140

145

150

155

163.09021 160

165

171.05656 170

189.44363 181.01619

175

180

185

m/z

Figure 12b: Spectrum obtained after addition of 2 equivalents of bromine. The strongest peak is for the S-oxide at 149.07 with negligibly-small peaks for the sulfinic acid and sulfonic acid at 155.06 and 171.06, respectively. Dimeric TTTU shows up at 132.07.

No other higher oxidation state oxoacids are observed. Figure 12b shows the addition of 2 equivalents of bromine. The S-oxide peak now increases at the expense of the substrate peak. This S-oxide peak is so dominant that the peaks observed for the sulfinic acid and for the sulfonic acid at 155.08 and 171.06, respectively; can be considered negligible. A peak for the dimeric TTTU, (Me2N)2CS-SC(NMe2)2, at 132.07 begins to grow at this oxidant to reductant ratio.


- 30 -

196.89413 190

195

200

Page 31 of 48

TTTUBR3 #4 RT: 0.10 AV: 1 NL: 1.29E8 T: FTMS + c ESI Full ms [70.00-300.00] 132.07199

100 95 90 85 80 75 70 65 R e la tive A b u n d a n c e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


60 55 50 45

149.07484

40 35 30 134.06766 25 20

88.02164

15 10 5

90.01745

0 85

90

117.10255 101.10754 106.53028 115.53570 122.00691 127.04372 97.99147 95

100

105

110

115

120

125

130

179.01848

163.09050 151.07050 139.08440 135

140

161.05852 145

150

155

160

189.45851

172.56664 165

170

175

180

185

190

198.10837 195

m/z

Figure 12c: addition of three equivalents of bromine to one equivalent of TTTU. The largest peak is for the dimeric TTTU species at 132.07. The product, dimethylurea, at this ratio, is negligible at 117.10.

Addition of three equivalents, as shown in Figure 12c; gives predominantly the dimeric species and the S-oxide. These are the most stable metabolites. The dimer is very stable, and when


- 31 -

200


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 48

formed, will accumulate and be oxidized much more slowly to dimethylurea. In excess of oxidant, TTTU is successively oxidized to the product without the accumulation of any metabolites. Formation of dimer, in the absence of excess oxidizing power is facile, after protonation of the S-oxide. It will then combine with and unreacted TTTU molecule to produce the dimer:

Structure II. Dimeric TTTU species. Molar mass is 264.14. With the double positive charge, m/z is the observed 132.07 in Figure 12c.

Initial stages of the reaction. The search for a plausible mechanism for this reaction should start at the data in Figure 6b. The short induction period with respect to absorptivity measurements at 360 nm contains a very rapidly decreasing absorbance at 248 nm. Next, the data in Figure 6c in which the consumption profiles of TTTU change with acid concentrations have to be rationalized. Our mechanism should also be able to explain the apparent insensitivity of the reaction’s induction period to acid concentrations at low acid (Figure 4a) and (at high acid concentrations), the depressed chlorine dioxide formation with acid observed in Figure 4b. Figure 6c data suggests that there exists more than one oxidizing species in the reaction mixture and abundance of each species is dependent on the pH of the reaction medium. The pKa of chlorous acid is approximately 1.72 and so in the pH range of 1 to 3 one expects very active changes in the relative concentrations of chlorous acid and of chlorite anion. Thus, the change


- 32 -

Page 33 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


in observed kinetics with acid in this range can be attributed to the fact that both HClO2 and ClO2- are active oxidants. We can set up a mass balance equation on the chlorine (III) species:

[Cl(III)]T = [HClO2] + [ClO2]

(1)

Equation (1) links the two reactive species such that we can evaluate the following concentrations of HClO2 and ClO2- with respect to acid concentrations:

[Cl ( III )]T [ClO2 ] = ; 1 + K a [H + ] −

−1

[ HClO2 ] =

K a [ H + ][Cl ( III )]T

(2)

−1

1 + K a [H + ]

Ka is the acid dissociation constant for chlorous acid. HClO2 concentrations dominate below pH 2 and above this pH up to pH 4 there will be a mixture of both protonated and unprotonated forms of chlorine (III) species. Since reaction is first order in chlorite and in TTTU (in the initial stages), we can write the rate of reaction as:

Rate = -d[TTTU]/dt = [TTTU]{k1[ClO2-] + k2[HClO2]}

(3)

Which simplifies to:

− d [TTTU ]] [TTTU ][Cl ( III )]T = {k1 + k 2 K a −1[ H + ]} −1 + dt 1 + K a [H ]

(4)

Equation (4) can be handled within certain concentration limits in which we can eliminate one of the terms. For example, in high acid conditions, [ClO2-] .0 and [HClO2] . [Cl(III)]T; this simplifies equation (5) to:


- 33 -


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 48

rate = k2[Cl(III)]0[TTTU]

(5)

We can calculate a limiting k2 value in high acid concentrations (e.g. Figure 6a) and this delivered a value of 4.55±1.3x102 M-1 s-1. By assuming a pKa = 1.72 and using known values for initial reagent concentrations, initial rate equation (4) can be calculated at each data point and a value for k1 is evaluated. Our experimental data deduced a value of k1 = 0.98±0.15x102 M-1 s-1. Foe the direct reaction of chlorine dioxide with TTTU, a series of experiments were next run at very high oxidant to reductant ratios and high acid concentrations such that [H+]0, [ClO2]0 >> [TTTU] in search of pseudo-first order kinetics. This treatment is shown in Figure 10b. Our rate of reaction became:

-d[TTTU]/dt = kobs[ClO2]; kobs = k3[TTTU] = constant

(5)

This treatment showed that the order of the reaction with respect to [ClO2] is unity and k3 was evaluated as 1.55±0.04x102 M-1 s-1.

Chlorine dioxide formation. The initial stages of the reaction involve the formation of the reactive oxychlorine species, HOCl. HOCl can then either further oxidize the substrate or react with chlorite to form chlorine dioxide. The first metabolite to be formed is the surprisingly stable sulfenic acid zwitterionic species which exists as the S-oxide:

ClO2- + (Me2N)2C=S + H+ → (Me2N)2CS→O + HOCl

(R4)

ClO2- + HOCl + H+ ⇄ Cl2O2 + H2O

(R5)

ClO2- + Cl2O2 → 2ClO2(aq) + Cl-

(R6)


- 34 -

Page 35 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Since reaction R5 is considered fast, the rate of formation of chlorine dioxide becomes dependent upon rate of formation of HOCl from the oxidation reactions of TTTU. This sequence, R4 – R6 is sufficient to explain data in Figure 5 in which higher concentrations of TTTU gave higher rates of formation of chlorine dioxide. The observed complex acid dependence can be explained through reaction rate equation (4). Low acid concentrations approximate the denominator to unity, and one would observe a mild acid catalysis based on the second numerator term in acid. As acid is increased, equation (4) predicts saturation where acid will no longer have an effect. Further increase in acid, past 0.1 M will result in a retardation of the reaction. This further retardation with acid is derived from the protonation of the TTTU which occurs at a much higher acid concentrations than those required for protonation of chlorous acid which has a pKa of 1.72 and should saturate undissociated chlorous acid at ambient acid concentrations of 0.01 M.

(Me2N)2C=S + H+ º [(Me2N)2C=S-H]+ ; Kb

(R7)

If the protonated species is inert to attack by HClO2, then reaction rate equation (4) can be rewritten as equation (6):

[TTTU ][Cl ( III )]T − d [TTTU ]] = {k1 + k 2 K a −1[ H + ]} −1 + + dt (1 + K a [ H ])(1 + K b [ H ])

(6)

to account for the retardation due to the formation of protonated TTTU. Equation (6) can then account for the mild catalysis at low acid, insensitivity at intermediate acid concentrations, and the retardation observed at high acid concentrations. The formation of chlorine dioxide will mimic the rate of formation HOCl which is controlled by equation (6).


- 35 -


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 48

Consumption of TTTU. Figures 6a-d, 7a, and 8 show that consumption of TTTU occurs in two distinct stages. The first stage gives way to a slow stage followed by (what appears to be) a rapid decay to form products. The expected decay route for TTTU should involve the following pathway shown in Scheme I:

Scheme I: Expected generic pathway for the oxidation of thiocarbamides.

Figure 12b shows all three metabolites; 149.07 for the sulfenic acid, 155.06 for the sulfinic and 171.06 for the sulfonic acid. Abundances of the sulfinic and sulfonic acids, however, are so insignificant such that Scheme I is not the dominant pathway. Figure 11, which involves oxidation by chlorite, does not even show evidence for the sulfonic acid; despite a small peak for the sulfinic acid. Dimer formation was extensive in Figures 12b and c; environments in which oxidant was in stoichiometric minority. Figure 11a shows the minor peak for the dimer at m/z = 132.07. This, however, is not the dominant route of oxidation of TTTU because a dimer


- 36 -

Page 37 of 48

represents, effectively, a terminal product due to its slow further reaction. All dimeric species are known for being more stable and less soluble in polar solvents. The presence of unreacted TTTU, in the presence of the S-oxide, encourages facile dimer formation. In stoichiometric excess of oxidant the S-oxide proceeds directly urea and oxidatively-unsaturated sulfur adducts. These adducts could be directly responsible for the genotoxicity and teratogenicity observed with TTTU. This further oxidation of the S-oxide could be by either ClO2- or HOCl: (Me2N)2CS→O + HOCl + H2O → (Me2N)2C=O + HSO2- + Cl- + 2H+

(R8)

Reaction R8 is an irreversible entropy-driven process in which the sulfenic acid breaks up. The sulfoxylate anion is easily oxidized to sulfate

Consumption of chlorine dioxide. The main route for chlorine dioxide consumption is its direct reaction with TTTU. Chlorine dioxide should react very slowly or should be inert to the zwitterion sulfur oxo-acids. Further oxidation of these oxo-acids will be accomplished through chlorite and HOCl. The first step of the reaction of chlorine dioxide with TTTU involves the rapid formation of an intimate adduct of the two species. This is free energy driven: the sulfur center is nucleophilic with two lone pair of electrons while the chlorine center has an unpaired electron. The formation of the adduct, [(Me2N)2CSClO2], should be rapid:

O

O C

: :

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


S

+

. Cl :

C

O

S

Cl O

(R9)


- 37 -


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 48

Our experimental data in Figure 9b show that this adduct is formed within milliseconds of mixing of the two reagents. This adduct formation has been suggested in other chlorine dioxide oxidations. 36 Further reaction of this adduct will involve a hydrolysis

[(Me2N)2CSClO2] + H2O → (Me2N)2CS→O + HClO2- + H+

(R10)

The Cl(II) species further reacts with ClO2 to form ClO2-: HClO2- + ClO2(aq) → 2ClO2- + H+

(R11)

The product of reaction R11 could either be chlorite or chlorous acid depending on the pH of reaction solution. Chlorite is a more labile and active oxidizing agent that chlorine dioxide. It can oxidize in the 2-electron steps that are facile for sulfur oxidations. Reactions R10 + R11 show that one TTTU molecule produces 2 molecules of ClO2-. If chlorite oxidations are faster than chlorine dioxide’s, we expect constant reaction rate enhancement. This is indeed the case as the data in Figures 6c and 8 show. High acid concentrations should destroy this ‘autocatalysis’ as the reaction will predominantly proceed through HClO2. Data produced with high acid concentrations as in Figures 6a, 6b, 10a and 10b indeed does not show any hint of sigmoidal decay kinetics which could be indicative of autocatalysis. Involvement of free radicals. The possible involvement of free radicals was investigated in this manuscript. Chlorine dioxide is a radical and can produce an EPR spectrum even in the absence of a trap. There are two potential problems associated with detecting chlorine dioxide – mediated radicals. The first involves possible oxidation of the trap by chlorine dioxide.37 The second involves the generation of a second trapping agent, different from DMPO, that is generated by the oxidation of DMPO. Figure 13a shows a series of EPR spectra derived from ClO2 : TTTU mixtures. The spectrum is complex and appears to involve more than one radical


- 38 -

Page 39 of 48

species. The chlorine dioxide DMPO adduct radical is well known from its hyperfine coupling constants and its 1:2:2:1 signature spectrum. It appears to be combined with another radical species from the complex spectrum observed in Figure 13a. Addition of TTTU effectively quenches the EPR signal. This suggests that the overall mechanism of chlorine dioxide oxidation of TTTU occurs predominantly by a non-radical pathway. Further confirmation was derived from series of experiments of chlorite with TTTU (see Figure 13b). In this set of experiments TTTU was varied at constant chlorite and acid conditions.

Effect of TTTU Variation in Chlorine Dioxide on EPR Signal Intensity 3x106

2x106

EPR Signal Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(a) 1x106 (e) 0

-1x106

-2x106

-3x106

0.mM

0.4 mM

0.2 mM

3460

0.8 mM

3470

3480

1.2 mM 3490

Magnetic Field (Gauss) Fixed: [DMPO]0 = 0.1 M, [ClO2]0 = 2.0 x 10-3 M and varied [TTTU] 0 = (a) 0 mM, (b) 0.2 mM, (c) 0.4 mM, (d) 0.8 mM and (e) 1.2 mM.

The observed complex EPR spectrum showed an increase in radical species with increase in TTTU. This EPR spectrum decayed at the end of the reaction. This conforms to our initial


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observations that showed an increase in chlorine dioxide production with increase in TTTU since reaction R4 controls formation of chlorine dioxide and is dependent on concentrations of TTTU (see Figure 5).

Effect of TTTU variation on EPR Signal

(e)

4e+5

EPR Signal Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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2e+5 (a) 0

-2e+5

-4e+5

(b)

(a)

-6e+5 3450

3460

3470

(c) 3480

(d)

(e) 3490

3500

Gauss

+

-

Fixed: [DMPO]0 = 0.1 M, [H ]0 = 0.1 M, [ClO2 ]0 = 0.125 M and varied [TTTU]0 = (a) 0 M, (b) 10 mM, (c) 20 mM, (d) 40 mM and (e) 80 mM.

Figure 13b: EPR spectra generated from the chlorite – TTTU reaction. Spectra were taken after 60 s of incubation. Spectral intensity increases with TTTU concentrations. Spectra is closer to the expected 1:2:2:1 intensity for the chlorine dioxide adduct with DMPO. The dissymmetry is due to the dynamic nature of the signal.

Overall reaction mechanism and scheme: The reaction mixture effectively contains three separate reactions; all operating at approximately the same rates, hence the observed complex behavior. The first reaction is the oxidation of TTTU by chlorite to yield chloride and TTU. The


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second is the reaction of excess chlorite with HOCl which is an intermediate of the first reaction, to produce chlorine dioxide. The last reaction involves the reaction of chlorine dioxide with TTTU. None of these reactions can be separated. A full modeling of the mechanism was not possible because activity of chlorine dioxide in acidic conditions could not be determined. Using the kinetics constants determined for k1, k2 and k3; initial rates for chlorite – TTTU and chlorine dioxide – TTTU could be modeled. However, there was substantial overlap in absorbances of TTTU and ClO2(aq) such that none of the absorbance measurements could be isolated. What might appear like an autocatalytic decay in Figures 6a and d is effectively a combination of the consumption of TTTU combined with that of ClO2(aq). The lack of absorbance activity prior to this rapid decay is derived from a combination of the increase in ClO2(aq) absorbance coupled with the decrease in TTTU absorbance; resulting in no change in observed absorbances.

CONCLUSIONS. This particular study in the 5-part series of oxidations of substituted thioureas is the most unique out of all of them. TTTU is rapidly and the most easily oxidized of the series of substituted thioureas studied in our lab. Most of the thiourea compounds used as therapeutic drugs are in the ring-cyclized form such as propylthiouracil and methimazole.20,38,39,40 Their bioactivation mechanisms pass through stable metabolites. TTTU cannot form a stable metabolite until formation of TTU. The change in oxidation state of the sulfur center involves 8 electrons. Since these 8 electrons cannot be delivered in one step; this results in the formation of very highly reactive metabolic intermediates which cannot be stabilized in the physiological pH of 7.4. The S-oxide produced in this oxidation, stabilized below pH 2, rapidly decomposes in the physiological environment. Since ESI-MS data could not detect any substantial metabolites after the S-oxide; it is reasonable to assume that the sulfur center is cleaved off from the CN2


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moiety at the +2 oxidation state. This is a highly reactive state.41,42 It can combine with DNA and biological proteins, thereby exerting its genotoxicity and teratogenicity.43 All adverse effects of thiourea-based drugs are related to their oxidations.44

AKNOWLEDGMENTS: This work was supported by Grant Number CHE 1056366 from the National Science Foundation and partial funding from the University of KwaZulu-Natal, South Africa.


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Graphical abstract:


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Graphical abstract:


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