Oxyhalogen-Sulfur Chemistry: Kinetics and Mechanism of Oxidation

Article pubs.acs.org/JPCA

Oxyhalogen-Sulfur Chemistry: Kinetics and Mechanism of Oxidation of N‑Acetyl Homocysteine Thiolactone by Acidified Bromate and Aqueous Bromine Wilbes Mbiya,† Boyoung Choi,† Bice S. Martincigh,‡ Moshood K. Morakinyo,† and Reuben H. Simoyi*,†,‡ †

Department of Chemistry, Portland State University, Portland, Oregon 97207-0751, United States School of Chemistry and Physics, University of KwaZulu-Natal, Westville Campus, Private Bag X54001, Durban 4000, Republic of South Africa

‡

ABSTRACT: 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 Nacetyl 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 × 102 M−1 s−1.

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INTRODUCTION Of late, our laboratory has been interested in the chemistry of homocysteine1 as part of a broader study on biological thiols. Not only in the physiological environment, sulfur chemistry is also relevant in environmental chemistry2 and in agriculture as the major ingredient of most pesticides.3−8 There have been several studies that have shown that there is a link between elevated levels of homocysteine and cardiovascular disease9−14 as well as other diseases such as osteoporosis,15−19 Alzheimer’s disease, renal failure, and birth defects.15,18−22 In 2006, the United States Centers for Disease Control and Prevention (CDC) provided strong and encouraging evidence that, as a result of folic acid fortification (homocysteine lowering) in the North American food supply, there have been significantly fewer deaths due to stroke.23 There is, however, a vast number of other studies which show no link between homocysteine and cardiovascular disease.24 Thus homocysteine is biologically an enigma. The mechanism of homocysteine in aiding the onset of cardiovascular disease is not yet known, and it has not yet been confirmed in randomized studies that homocysteine lowering using vitamin therapy reduces cardiovascular disease risk. To develop more appropriate targeted therapy, an enhanced understanding of the © 2013 American Chemical Society

molecular mechanisms of the role of homocysteine in disease is needed. Our laboratory has proceeded to study a number of biologically active thiols by subjecting them to powerful oxidants, most in much higher concentrations than those obtained in the physiological environment.25−31 The benefits of such a format are to obtain oxidation metabolites at rates faster than those obtained from the use of microsomal fractions.32−36 The generated oxidation metabolites can then be evaluated for biological activity. With the nucleophilicity of the sulfur center, most of the bioactivations of the sulfur center are oxidative.37 Thus the oxidation of biologically active thiols and thiocarbamides represents the fate of most of these compounds in the physiological environment. Sulfur compounds are very fluxional in their effect in the physiological environment. A small difference in their structure could result in great differences in their physiological functions and effects. For example, while phenylthiourea is highly toxic to rats, diphenylthiourea is innocuous.38,39 Cysteine, an essential Received: August 19, 2013 Revised: October 24, 2013 Published: October 24, 2013 13059

dx.doi.org/10.1021/jp408304e | J. Phys. Chem. A 2013, 117, 13059−13069

The Journal of Physical Chemistry A

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bromate and aqueous bromine are also precursors to the biologically significant oxidant, hypobromous acid, HOBr.61

amino acid, and, together with its derivative, cystine, play an important structural role in proteins.40 It is also acknowledged as an effective antioxidant.41−44 The addition of one extra CH2 in the chain gives homocysteine, a toxic thiol.45,46 One reason given for the toxicity of homocysteine is its ability to form a five-membered thiolactone which can modify proteins by forming an amide bond with lysine residues of biological proteins and peptides (Scheme 1).47

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EXPERIMENTAL SECTION Materials. The following reagents were used without further purification: N-acetyl homocysteine thiolactone (NAHT), sodium bromate, sodium bromide, bromine, sodium chloride, and sodium thiosulfate (Fisher). All of the major reactants were assumed to be of high purity so there was no need for standardization. Bromine solutions, being volatile, were kept capped and were standardized before each set of experiments. Methods. Experiments were carried out at 25 ± 0.1 °C. Ionic strength was maintained at 1.0 M (NaClO4) in all experiments. Most of the reaction’s kinetics determinations were performed on a Hi-Tech Scientific SF-61DX2 doublemixing stopped-flow spectrophotometer. Reaction progress was followed by monitoring the production of Br2 at 390 nm. The direct reactions between bromine and NAHT were monitored by following the consumption of bromine at 390 nm on the stopped-flow spectrophotometer. Figure 1 shows the super-

Scheme 1

Surprisingly, a substituted homocysteine thiolactone, Nacetyl homocysteine thiolactone, (2-acetamido-4-mercaptobutyric acid γ-thiolactone, NAHT) has been found to have many beneficial physiological roles.48−50 NAHT is an important free radical scavenger and is able to increase the superoxide dismutase (SOD) levels in the body, while the parent compound, homocysteine thiolactone, has never been observed to have any reactive oxygen species (ROS) deactivating effects. NAHT is a very efficient agonist for restoring alkaline phosphatase levels following methyl mercury chloride poisoning.51−53 It is also widely used in preparing β-hCG COOH peptide carrier conjugates of predictable composition.54 The two thiolactones, except for the acetyl group, are structurally similar (see Structures I and II, below).

Figure 1. UV−vis spectral scans of N-acetyl homocysteine thiolactone, bromine, and the product of N-acetyl homocysteine thiolactone and acidified bromate. Bromine has two wavelengths, 265 and 390 nm. NAcetyl thiolactone absorbs at 235 nm, and there was no interference on the bromine peak at 390 nm which was used to quantify bromine concentrations.

NAHT (medically known as citiolone) contains a blocked αamino group. NAHT has several interesting pharmaceutical applications. For example NAHT has been used as a mucolytic and muco-regulating drug.55 When NAHT is combined with antibiotic ampicillin, it reduces the severity of the clinical pattern, with rapid improvement and a reduction of the main symptoms of febrile infection of the respiratory system.56,57 It is also frequently used for the treatment of chronic hepatitis.55,58 Clinically controlled studies have shown that NAHT has been shown to be more effective in the treatment of acute bronchitis and bronchopneumopathy than a sister mucolytic drug Nacetylcysteine.57,59 It is a well-tolerated drug and an effective fluidifier. Even though, mucolytic drugs have rarely been implicated in the fixed drug eruption etiology, several episodes of fixed exanthema related to NAHT intake have been reported.55 Although several studies have been performed on the reactivity of NAHT, it has not, however, been adequately characterized.60 We report, in this manuscript, on a kinetics and mechanistic study of its reaction with strong oxidants: aqueous bromine and acidic bromate so as to evaluate the metabolic pathway of this important organosulfur compound. Acidic

imposition of the three spectra for NAHT, product solution of NAHT and acidic bromate, and aqueous bromine. The bromine peak is isolated from the organic species, and thus formation of bromine could be followed without any interference. All solutions were prepared using doubly distilled deionized water from a Barnstead Sybron Corporation 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 negative mode. All EPR spectra were recorded on a Bruker Biospin e-scan spectrometer designed to perform EPR measurements in the X-band range at room temperature. Stoichiometric Determinations. Stoichiometric determinations were performed by an iodometric titration method in which varying amounts of bromate were reacted with fixed concentrations of NAHT in highly acidic conditions. The 13060



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excess bromate was evaluated by addition of acidified excess iodide with the liberated iodine titrated against standard sodium thiosulfate with freshly prepared starch as indicator. Bromine−NAHT stoichiometric determinations were performed in excess bromine with excess bromine determined spectrophotometrically as well as iodometrically. In both cases, rather than a single static value determination of stoichiometry, a graphical analysis was utilized. Graphical methods were not dependent on the determination of the exact concentrations of any reagent, as long as the same reagent solutions were utilized throughout.

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RESULTS AND DISCUSSION Stoichiometry. A series of experiments were performed with a constant NAHT concentration of 2 mM while varying excess acidified bromate. The same standard thiosulfate solution was used in a titrimetric analysis of excess oxidizing power left after complete consumption of the substrate, NAHT. Figure 2a shows the graphical plot of the volume of thiosulfate needed for the fixed concentration of bromate used. This plot is linear with an intercept of 1.9 mM bromate for null volume of thiosulfate needed. This is the concentration of bromate needed to just oxidize NAHT with no bromate left to react with iodide for the iodometric titration. This suggests a 1:1 stoichiometry. The addition of BaCl2 did not afford the white BaSO4 precipitate, indicating that the C−S bond in NAHT was not cleaved and no sulfate was formed. Bromine center goes from +5 in bromate, to −1 in bromide; which is a 6-electron transfer. The sulfur center in NAHT is at oxidation state of −2; and to balance out the electrons, if oxidation is occurring only on the sulfur center, the oxidation state of sulfur in the product is +4, which is a sulfonic acid. Thus the five-membered thiolactone ring opens up to afford the sulfonic acid. Further experiments using ESI-MS in excess oxidant showed only one product, at m/z = 224.0233, with no other products appearing on the spectrum (see Figure 2b). The opening of the ring gives a carboxylic acid and a sulfonic acid, N-acetyl homocysteine sulfonic acid. Here, let us assume NAH−SO3H represents Nacetylhomocysteine sulfonic acid (see Structure III, below), so the stoichiometry is then: NAHT + BrO3− → NAH−SO3H + Br−

(R1)

Figure 2. (a) Stoichiometric plot generated from iodometric titration of sodium thiosulfate against liberated I2 following complete oxidation of N-acetyl homocysteine thiolactone in acidic iodate. For a fixed Nacetyl homocysteine thiolactone concentration of 0.002 M, the intercept on the [BrO3−] axis is 0.0019 M, suggesting a stoichiometric ratio for [BrO3−/NAHT] at 1:1. (b) Product ESI-MS spectrum taken in the negative mode for stoichiometric concentrations of the bromate to NAHT (1:1 ratio) in 0.20 M percloric acid. No other products are observed except to the sulfonated thiolactone after ring opening. (c) Stoichiometric determination of bromine-N-acetyl homocysteine thiolactone reaction by bromine titration. Fixed [NAHT]0 = 1 × 10−4 M and varied [Br2] and the absorbance of the remaining bromine was plotted against initial bromine concentration. The intercept on the [Br2] axis is 0.0003 M giving a 1:3 ratio [NAHT:Br2].

A stoichiometric determination was also made for the direct oxidation of NAHT with aqueous bromine. This was performed both iodometrically and spectrophotometrically. For the spectrophotometric determination, excess bromine was used, and final bromine absorbance plotted against initial bromine concentration. The intercept of the expected linear plot represents the amount of bromine needed to just oxidize NAHT. Figure 2c shows such a plot from our experimental data. NAHT was held constant at 0.10 mM. The intercept of this plot shows that 0.3 mM aqueous bromine is needed to just oxidize NAHT completely, with no aqueous bromine left; 13061



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suggesting a 1:3 stoichiometry and oxidation to the same substituted homocysteine sulfonic acid, the one observed in stoichiometry R1: NAHT + 3Br2 + 3H 2O → NAH−SO3H + 6Br − + 6H+ (R2)

Kinetics. Since the reaction was characterized by the formation of bromine, thus reaction R1 had to proceed to afford the bromide needed for formation of aqueous bromine from the bromate−bromide reaction: BrO3− + 6H+ + 5Br− → 3H 2O + 3Br2(aq)

(R3)

The observed reaction is thus a combination of R1 and R3 that consumes the bromide formed in R1 and converts to aqueous bromine (5R1 + R3): 5NAHT + 6BrO3− + 6H+ → 5NAH−SO3H + 3Br2(aq) + 3H 2O

Figure 4. Absorbance traces showing the effect of varying NAHT concentrations on the amount of bromine formed. [H+]0 = 0.2 M; [BrO3−]0 = 0.06 M; [NAHT]0 = (a) 0.006 M, (b) 0.007 M, (c) 0.008 M, (d) 0.009 M, (e) 0.010 M, and (f) 0.011 M. The traces show an increase in iodine formed as NAHT concentration increases from a to f.

(R4)

Production of bromine occurs after the rate of reaction that produces aqueous bromine R3 exceeds 1 that consumes aqueous bromine R2. Figure 3 shows a series of spectral

is based on the rates of reactions R3 and R2. When the rate of R2 is greater than R3, no bromine production will be observed. The rate of reaction R2 is dependent on NAHT concentrations, and thus higher initial NAHT concentrations enhance reaction R2 and delay the onset of bromine production. If reactions were run in extremely high [BrO3−]0/[NAHT]0 ratios, ca. 15 and above, no variation in induction period is observed (data not shown). Only differences observed would be the rate of formation of bromine after this invariant induction period and the final bromine concentrations which are 60% of the initial NAHT concentrations. This has been observed in similar kinetics systems that proceed through an induction period.62 No simple linear relationship was obtained between the induction period and the initial concentrations of NAHT. Figure 5a shows a series of kinetics traces taken in excess bromate, while keeping [NAHT]0 constant. These traces show oligooscillatory behavior with respect to bromine concentrations. Two peaks in bromine concentrations are observed. The first peak is just after the end of the induction period and is sharp and distinct. The second peak corresponds to the expected bromine concentrations based on stoichiometry R4. All of the traces shown in Figure 5a, after the reaction had gone to completion, gave the same concentration of bromine, since this was determined by the concentration of NAHT (vide supra). A plot of inverse of initial bromate concentrations vs the induction period is linear (see Figure 5b). Such a plot is always useful in double-checking our derivation of the reaction stoichiometry R1. In Figure 5b, the intercept on the induction period axis should give the expected stoichiometry R1. The intercept value would correspond to the bromate concentration that gives an induction period of infinity, which is the highest concentration of bromate needed just before formation of bromine as a final product. This would be stoichiometry R1 if the rate of reaction that consumes bromine, R2, is much faster than reactions that form bromine. Hence the formation of bromine would indicate complete consumption of NAHT. If statement above is correct, in Figure 5b, one expects an intercept value of 5.00 mM of bromate if R1 is the prevailing stoichiometry. Figure 5b shows an intercept closer to 1.00 mM

Figure 3. Absorbance scans of NAHT during oxidation by acidifed bromate. The traces were collected every 5 s reaction time intervals. [NAHT]0 = 0.002 M, [BrO3−]0 = 0.006 M, and [H+]0 = 0.20 M.

scans taken every 5 s between 250 and 700 nm. The only activity observed is at the bromine absorption peak. The emergence of the peak at 390 nm is sudden and rapid. Figure 4 shows a series of kinetics experiments carried out in excess acidified bromate over NAHT. The final expected product will be aqueous bromine with no bromide, according to stoichiometry R4. In this format, final bromine concentrations obtained were determined by initial concentrations of NAHT with expected bromine concentrations at 0.6[NAHT]0 The reaction seems to have three phases: An initial quiescent phase in which no activity is observed with respect to bromine production (induction period), followed by a rapid increase in bromine at the end of the induction period, and the final phase is a slow bromine production rate which terminates after stoichiometry R4 is attained. All traces shown in Figure 4 were truncated before the full amount of bromine as per stoichiometry R4 was attained. At these fixed bromate concentrations, higher NAHT concentrations gave longer induction periods. This is expected since bromine production 13062



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Figure 5. (a) Absorbance traces showing the effect of varying bromate concentrations on the rate of reaction. [NAHT]0 = 0.005 M; [H+]0 = 0.2 M; [BrO3−]0 = (a) 0.004 M, (b) 0.005 M, (c) 0.006 M, and (d) 0.007 M. (b) Linear relationship between inverse of induction period and initial bromate concentrations. This suggests that the rate of reaction that is a prerequisite for the formation of bromine, which is dependent on bromate concentrations to the first power.

Figure 6. (a) Absorbance traces showing the effect of varying acid concentrations. [NAHT]0 = 0.005 M; [BrO3−]0 = 0.06 M; [H+]0 = (a) 0.18 M, (b) 0.2 M, (c) 0.22 M, and (d) 0.24 M. The induction time decreased with increasing [H+]0 indicating acid catalysis of the prerequisite reaction for formation of bromine. (b) In this range of acid concentrations, induction time is dependent on the square of the initial acid concentrations.

bromate needed for formation of bromine. This suggests that oxidation of NAHT to the sulfonic acid is not a prerequisite for the production of bromine. Production of bromine can commence after formation of a less oxidatively saturated metabolite such as the sulfenic acid or the sulfone. This metabolite should be relatively stable and can accumulate during the reaction’s progress in the presence of the highly oxidizing environment containing aqueous bromine. Figure 6a shows the effect of acid in excess bromate conditions. These traces also exhibited oligooscillatory dynamics. The transient peaks decreased with increase in initial acid concentrations, indicating that the reaction that consumes the transient intermediate is inhibited by acid. Figure 6b shows that, in this range of acid concentrations, there is an inverse square acid dependence on the induction period (see Figure 6b). At higher acid concentrations, this linearity was lost. Bromide is a product of the reaction (stoichiometry R1), and it also catalyzes the reaction (see Figure 7). Bromide shortens the induction period and also increases the final bromine concentrations obtained due to the expected enhancement of reaction R3 after complete consumption of the substrate. The transient peaks are not as sharp as in reactions without added bromide. The direct reaction of bromine and NAHT was extremely rapid (see Figures 8 and 9), and it was over within

Figure 7. Absorbance traces showing the effect of varying bromide concentration. [NAHT]0 = 0.005 M, [H+]0 = 0.2 M, [BrO3−]0 = 0.06 M, [Br−]0 = (a) 0 M, (b) 0.0001 M, (c) 0.0002 M, (d) 0.0003 M, (e) 0.0004 M, (f) 0.0005 M, and (g) 0.0006 M. Addition of bromide (from trace b to g) reduced the induction time and increased the amount of bromine formed. 13063



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experimentally. Thus there has to be another source of bromide ions in experiments run without added bromide. We assume this is the reaction between the acidified bromate with NAHT in a two-electron oxidation. H+ + BrO3− ⇌ HBrO3

(R6)

HBrO3 + NAHT → HBrO2 + NAHTSO (Structure IV)

(R7)

The product of reaction R7 could result in ring-opening to afford a sulfenic acid or retention of the ring structure to give a sulfoxide (see Structure IV).

Figure 8. Absorbance traces showing the effect of varying N-acetyl homocysteine thiolactone concentration on bromine depletion. [Br2]0 = 0.015 M and varied [NAHT]0 = (a) 0.002 M, (b) 0.003 M, (c) 0.004 M, (d) 0.005 M, and (e) 0.006 M.

Sulfenic acids are known to be unstable and can only be stabilized in sterically hindered versions.65,66 ESI-MS data in Figure 10a shows that a 1:1 mixture of NAHT and aqueous bromine gives predominantly the sulfoxide (Structure IV). No other product is observed at this two-electron oxidation of the sulfur center. HBrO2 can further oxidize NAHT until it is reduced to bromide: HBrO2 + NAHT → HOBr + NAHTSO

(R8)

HOBr + NAHT → H+ + Br − + NAHTSO

(R9)

Combining reactions R6−R9 gives the following transient stoichiometry, S4: BrO3− + 3NAHT + Br − → 2Br − + 3NAHSO

Figure 9. Absorbance traces showing the effect of varying bromine concentration. Fixed [NAHT]0 = (a) 0.001 M and varied [Br2]0 = 0.005 M, (b) 0.0075 M, (c) 0.01 M, (d) 0.01 M, and (e) 0.015 M.

This stoichiometry gives an ever-increasing concentration of bromide with each step. The establishment of bromide concentrations shifts the rate-determining step to reaction R5 as expected from acidic bromate conditions. Kinetics data in Figure 7 show catalysis by bromide, but not in the mode of autocatalysis which would have predicted an exponential increase in rate with the addition of the autocatalyst. This is expected, since the generation of bromide in stoichiometry S4 is through at least three two-electron reduction steps of acidic bromate, and not through a single reaction step. Oxidation of NAHT. ESI-MS data suggest that oxidation of the thiolactone proceeds through successive addition of oxygen on the sulfur center from the sulfoxide to the sulfone and finally to the sulfonic acid. The predominance of the sulfoxide after a one-equivalent oxidation suggests that there is a special stability associated with this metabolite over the sulfone (see Figure 10a). Figure 10b shows that, with two equivalents of oxidant, the expected sulfone disproportionates into the product sulfonic acid of the substrate thiolactone. There is no evidence of ring-opening before formation of the sulfonic acid. Overall Reaction Scheme. Aqueous bromine is formed from a single reaction, the reverse of the hydrolysis reaction of bromine:

100 ms. Types of absorbance traces obtained indicated bimolecular kinetics. Both series of experiments delivered first order kinetics in NAHT and aqueous bromine.

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MECHANISM The observed kinetics implicate the standard oxybromine kinetics that involves the following composite rate-determining step:63,64 BrO3− + Br − + 2H+ ⇌ HBrO2 + HOBr

(S4)

(R5)

This reaction generates the active oxidizing species since bromate, itself, is inert as an oxidant. Those reactions run with bromide initially added in the reaction mixture as in Figure 7 utilize reaction R5 as the rate-determining step. Those without added bromide have to generate bromide first to enable R5 to resume. Standard bromate solutions contain trace amounts of bromide at approximately 10−6 M. These trace bromide concentrations can initiate formation of the reactive species as in reaction R5. However, using these concentrations of bromide in a rough kinetics model of the reaction derives induction periods which are much longer than those obtained

HOBr + Br − + H+ ⇌ Br2(aq) + H 2O 13064

(R11)



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Figure 10. (a) Negative ESI-MS spectrum of a [NAHT−Br2] solution of ratio at a 1:1 stoichiometric ratio using 10:90 methanol−water as solvent. Spectrum was taken after two minutes of mixing the reagents together. Only one product is obtained, the sulfoxide. (b) This spectrum was obtained two minutes into the reaction of a 1:2 ratio of [NAHT]0:[Br2]0. This spectrum shows a sulfone and the product sulfonic acid, but no sulfoxide at m/z = 174.

reductant will be the difference between reaction R11 and reactions of bromine with NAHT and its oxidation intermediates before formation of the sulfonic acid. Figures 8

This is a very rapid reaction, with a forward rate constant of 8.9 × 109 M−2 s−1 and a reverse rate constant of 110 s−1.64 The rate of formation of bromine before complete consumption of 13065



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and 9 show that reaction of bromine with NAHT is extremely rapid and that formation of bromine indicates that all NAHT would have been depleted. The special stability of the sulfoxide suggests that its further oxidation to the sulfone is relatively slower. The transient peak in the absorbance at 390 nm can thus be explained by a contribution from two effects. The first involves the accumulation of the sulfoxide due to its stability. Thus formation of bromine can commence before the full oxidation of the thiolactone to the sulfonic acid. Br2 + NAHT + H 2O → NAHTSO + 2Br − + 2H+; kR12

(R12)

Br2 + NAHTSO + H 2O → NAHT−SO2 + 2Br − + 2H+; kR13

(R13)

In this case, kR12 > kR13; and hence bromine would accumulate, even in the presence of the sulfoxide. In this case one would expect coexistence of bromine and the sulfoxide. Since reaction is run in excess oxidant, the sulfoxide will be depleted and formation of bromine will then proceed according to the standard oxybromine kinetics of reaction R3. The other contribution to the transient peak arises from the presence of both aqueous bromine and bromide. This will result in the formation of tribromide, Br3−: Br2 + Br − ⇌ Br3−;

Keq = 17

(R14)

While aqueous bromine has an absorptivity coefficient of 142 M−1 cm−1 at 390 nm, Br3−’s absorptivity coefficient at the same wavelength is 1006 M−1 cm−1. Figure 11a shows the UV−vis spectra generated from aqueous bromine and mixtures of bromine with varying sodium bromide concentrations. Spectra show an isosbestic point for aqueous bromine and tribromide of 440 nm and an elevated absorbance for the tribromide and bromine mixtures at 390 nm. The absorptivity at 440 nm is 91 M−1 cm−1. Thus the linear relationship between bromine concentrations and absorbance is lost. The formation of bromine in the presence of bromide will deliver a higher rate of increase in absorbance at 390 nm than would have been expected from an increase in bromine concentrations in the absence of bromide. The observed initial peak would thus arise partly from the equilibrium of eq R14 shifting to the right but without a concomitant increase in bromine concentrations. The depletion of bromine, through reaction R11, would give a decrease in absorbance that is not a linear function of the concentration of bromine according to the Lambert−Beer law. Reactions monitored at 440 nm also displayed the transient peak, indicating that bromine concentrations do display oligooscillatory behavior. Figure 11b shows absorbance traces taken at 440 nm. Since the absorptivity coefficient is lower at 440 nm, overall absorbances recorded are lower. Thus there exists an intermediate in the oxidation of the thiolactone that is stable enough to coexist with aqueous bromine. An examination of Figure 8 clearly shows that this intermediate is the sulfoxide which is the metabolite obtained after addition of one oxygen atom to the thiolactone, or its two-electron oxidation. All traces in Figure 8 have the same constant initial bromine concentrations of 0.015 M, and yet the starting absorbances are not constant for all of those traces. The absorbance traces show that there is an initial step in the oxidation of NAHT by bromine that is so rapid that it cannot

Figure 11. (a) UV−vis spectra of aqueous bromine with successive addition of crystals of sodium bromide. The increase in relative tribromide concentrations increases the measured absorbance at 390 nm without an increase in aqueous bromine concentrations. The isosbestic point was deduced to be 440 nm. (b) Bromate−thiolactone reactions monitored at 440 nm, the bromine−tribromide isosbestic point. The same oligooscillations observed at 390 nm are also observed at this wavelength, indicating that these oligooscillations are not derived solely from the absorptivity differences between bromine and tribromide at 390 nm.

be captured by our stopped-flow instrument which has a mixing time of 1 ms. This step consumes 1 mole of NAHT for every mole of bromine. In trace a, with 0.015 M aqueous bromine, the expected initial absorbance of bromine should be 2.31. Instead, an initial absorbance of 1.81 is observed, which is equivalent to the absorbance of 0.015 M (bromine) − 0.002 M (NAHT) = 0.013 M (bromine). 0.013 M aqueous bromine will give the observed absorbance of 1.81. Trace e has an initial concentration of 0.006 M NAHT, and the initial absorbance of bromine observed is that corresponding to 0.009 M bromine (1.28). This establishes that reaction R11 is extremely rapid to the point of being diffusion-controlled. This is not unusual with electrophilic reactions of bromine on a soft nucleophilic sulfur center.67,68 Bromide needed to activate reaction R5 is derived from the sequence of reactions R6−R9, culminating in everincreasing bromide concentrations according to stoichiometry S4. This transfers rate-determining step to reaction R5. Even though bromide is a product, it does not act as an autocatalyst; its effect on the reaction is linear, as expected from standard mass-action kinetics due to a time-lag through sequence of 13066



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Table 1. Full Reaction Scheme for the Bromate−Thiolactone Reactiona reaction

reaction

kf; kr

M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15

BrO3− + NAHT + H+ → HBrO2 + NAHSO HBrO2 + NAHT → HOBr + NAHSO HOBr + NAHT → Br− + H+ + NAHSO BrO3− + 2H+ + Br− ⇄ HBrO2 + HOBr HBrO2 + Br− + H+ ⇄ 2HOBr HOBr + Br− + H+ ⇄ Br2 + H2O HBrO2 + NAHSO → HOBr + NAHSO2 HBrO2 + NAHSO2 → HOBr + NAHSO3 HOBr + NAHSO → NAHSO2 + Br− + H+ HOBr + NAHSO2 → NAHSO3H + Br− + H+ Br2(aq) + NAHT + H2O → NAHSO + 2Br− + 2H+ Br2(aq) + NAHSO + H2O → NAHSO2 + 2Br− + 2H+ Br2(aq) + NAHSO2 + H2O → NAHSO3H + 2Br− + 2H+ NAHSO2 + NAHSO2 ⇄ NAHSO + NAHSO3H NAHSO + NAHSO ⇄ NAHT + NAHSO2H

3.0 × 10−1; 0 1.0 × 103; 0 5.0 × 103; 0 2.1; 1.0 × 10−4 2.0 × 106; 2.0 × 10−5 8.9 × 109; 1.1 × 102 3.0 × 101; approximately 0 5.0 × 103 1.5 × 102 1.0 × 104 2.94 × 102 1.0 × 102 2.5 × 103 1.0 × 10−3 1.0 × 10−4

a Table Legend: forward and reverse rate constants, separated by a semicolon. Except for reactions involving water, the units of kinetics rate constants derived from the reactions’ molecularity.

sulfonic acid and the thiolactone, with only 2 equiv of the sulfonic acid formed as expected from the overall stoichiometric eq R2. The full mechanism of the reaction is shown in Table 1. There are three major oxidizing species in the reaction mixture: HBrO2, HOBr, and Br2(aq). There are also three reducing species in solution: the thiolactone, the sulfoxide, and the cyclic sulfone. Bromous acid was used in the initiation sequence of reactions (reaction M2) and in the oxidation of the sulfoxide and sulfone (reactions M7 and M8). We could also equally ignore all contributions from bromous acid by taking advantage of its rapid disproportionation to hypobromous acid (reaction M5). There was no loss in accuracy of the model by assuming that HBrO2 only participated as in reaction M5, apart from the initiation sequence M1−M3. Reactions M4−M6 are the wellknown oxybromine reactions whose kinetics constants were sourced from literature values.63,64,72 Reactions M7−M13 involved the reduction of a bromine center counpled to the oxidation of a sulfur center, and all of these reactions were assumed to be irreversible. The rate constant for reaction M11 was derived from this study. Subsequent oxidation of the sulfoxide, M12, was made slower than reaction M11. The last two reactions were disproportionation reactions for stoichiometric consistency. The reaction was followed through formation of aqueous bromine, whose concentration was determined by its absorbance at 390 nm (Figure 1). Modeling the reaction using bromine production was not possible due to the presence of three unknown concentrations: Br2, Br−, and Br3−, while only having two equations in the reaction network. One is reaction R14, and the other involves the total absorbance observed at 390 nm:

reactions R6−R9 before it can be utilized. Bromide is known to be the control species in the generation of oscillatory behavior in the Belousov−Zhabotinskyi reaction: its critical concentration determines the dominant reaction.69 Bromous acid, HBrO2, is the autocatalytic species in acidic bromate oxidations.70 Generation of bromous acid is derived from BrO2, a one-electron oxidant.63 This would result in a oneelectron oxidation of the sulfur center, resulting in the formation of radical species. BrO3− + HBrO2 + H+ → 2BrO2 · + H 2O

(R15)

BrO2 · + H+ + e− → HBrO2

(R16)

The use of standard DMPO traps did not show any formation of radicals, in contrast with other oxidations of organosulfur compounds by acidic bromate.71 Thus, pathway R15−R16 is insignificant in the reaction system being reported in this manuscript. Figure 10b, with 2 equiv of bromine for 1 equiv of NAHT, shows that there are extensive disproportionation reactions involving the sulfur center. Two equivalents of aqueous bromine involve a four-electron oxidation of the sulfur center in the thiolactone to give either a sulfinic acid or the cyclic sulfone. ESI data in Figure 10b indicate that the ring remains closed as a sulfone. Furthermore, the sulfone is readily oxidized to the stable sulfonic acid with the concomitant opening of the ring in an irreversible process. Entropy terms for a reclosing of the ring are prohibitive. 2NAH−SO2 → NAH−SO3H + NAHSO

(R17)

While the sulfonic acid formed in reaction R17 is terminal and cannot be oxidized further, the sulfoxide can further oxidize (reaction R13) or disproportionate R18: 2NAHSO → NAHT + NAHSO2

A TOT = A Br2 + A Br3−

(R18)

(R19)

Since both Br2 and Br3− concentrations vary with time, the necessary third equation involving mass balance could not be derived. Modeling the reaction utilizing the mechanism in Table 1 gave the expected induction period and final bromine concentrations obtained but could not successfully simulate the rate of formation of bromine at the end of the induction period. The model always derived a rate lower than the rate experimentally observed. This was expected due to the much higher absorptivity of tribromide over bromine at 390 nm.

The ESI spectrum in Figure 10b surprisingly shows a low abundance of the stable sulfoxide, in favor of the cyclic sulfone and the sulfonic acid product. Several experiments were performed at this oxidant-to-reductant ratio of 2:1. They all showed negligible traces of the sulfoxide due to reaction R18. Reaction R18 is a slow rearrangement process that is essential for stoichiometric consistency in conditions of excess reductant. On prolonged standing the favored products will be the 13067



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AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by Research Grant no. CHE 1056311 from the National Science Foundation. REFERENCES

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