Oxyhalogen-sulfur chemistry - Kinetics and mechanism of the bromate oxidation of cysteamine

Article abstract of DOI:10.1139/V08-031

The kinetics and mechanism of the oxidation of the biologically important molecule, cysteamine. by acidic bromate and molecular bromine have been studied. In excess acidic bromate conditions, cysteamine is oxidized to N-brominated derivatives, and in excess cysteamine the oxidation product is taurine according to the following stoichiometry: BrO₃^- + H ₂NCH₂CH₂SH → H₂NCH ₂CH₂SO₃H + Br^-. There is quantitative formation of taurine before N-bromination commences. Excess aqueous bromine oxidizes cysteamine to give dibromotaurine: 5Br₂ + H ₂NCH₂CH₂SH + 3H₂O → Br ₂NCH₂CH₂SO₃H + 8Br^- + 8H⁺, while excess cysteamine conditions gave monobromotaurine. The oxidation of cysteamine by aqueous bromine is effectively diffusion-controlled all the way to the formation of monobromotaurine. Further formation of dibromotaurine is dependent on acid concentrations, with highly acidic conditions inhibiting further reaction towards formation of dibromotaurine. The formation of the N-brominated derivatives of taurine is reversible, with taurine regenerated in the presence of a reducing agent such as iodide. This feature makes it possible for taurine to moderate hypobromous acid toxicity in the physiological environment.

Full text of DOI:10.1139/V08-031

416
Oxyhalogen–sulfur chemistry — Kinetics and
mechanism of the bromate oxidation of
cysteamine
Moshood K. Morakinyo, Edward Chikwana, and Reuben H. Simoyi
Abstract: The kinetics and mechanism of the oxidation of the biologically important molecule, cysteamine, by acidic
bromate and molecular bromine have been studied. In excess acidic bromate conditions, cysteamine is oxidized to N-
brominated derivatives, and in excess cysteamine the oxidation product is taurine according to the following stoichiometry:
BrO3 + H2NCH2CH2SH ÷ H2NCH2CH2SO3H + Br–. There is quantitative formation of taurine before N-bromination
–
commences. Excess aqueous bromine oxidizes cysteamine to give dibromotaurine: 5Br2 + H2NCH2CH2SH + 3H2O ÷
Br2NCH2CH2SO3H + 8Br– + 8H+, while excess cysteamine conditions gave monobromotaurine. The oxidation of
cysteamine by aqueous bromine is effectively diffusion-controlled all the way to the formation of monobromotaurine. Fur-
ther formation of dibromotaurine is dependent on acid concentrations, with highly acidic conditions inhibiting further re-
action towards formation of dibromotaurine. The formation of the N-brominated derivatives of taurine is reversible, with
taurine regenerated in the presence of a reducing agent such as iodide. This feature makes it possible for taurine to mod-
erate hypobromous acid toxicity in the physiological environment.
Key words: cysteamine, hypobromous acid, toxicities, antioxidant.
Résumé : On a étudié la cinétique et le mécanisme de l’oxydation de la molécule biologiquement importante, cystéa-
mine, par le bromate acide et le brome moléculaire. Dans des conditions de bromate acide en excès, la cystéamine est
oxydée en dérivés bromés et, dans un excès de cystéamine, le produit d’oxydation est la taurine qui est formée en
d’après la stoechiométrie: BrO3 + H2NCH2CH2SH ÷ H2NCH2CH2SO3H + Br–. Il y a formation quantitative de taurine
–
avant que la N-bromation commence. Un excès de brome en milieu aqueux provoque une oxydation de la cystéamine
avec formation de dibromotaurine: 5Br2 + H2NCH2CH2SH + 3H2O ÷ Br2NCH2CH2SO3H + 8Br– + 8H+, alors que les
conditions dans lesquelles la cystéamine est en excès conduit à la formation de la monobromotaurine. L’oxydation de
la cystéamine par le brome aqueux est effectivement une réaction sous contrôle de la diffusion jusqu’à la formation de
la monobromotaurine. La formation subséquente de dibromotaurine dépend des concentrations d’acide alors que les
conditions fortement acides inhibent la réaction subséquente pouvant conduire à la formation de la dibromotaurine. La
formation des dérivés N-bromés de la taurine est réversible, la taurine pouvant être régénérée en présence d’un agent
réducteur, tel un ion iodure. Cette caractéristique fait que la taurine peut réduire le caractère toxique de l’acide hypo-
bromeux dans un environnement physiologique.
Mots-clés : cystéamine, acide hypobromeux, toxicités, antioxydant.
[Traduit par la Rédaction] Morakinyo et al. 425
Introduction
the kidney and liver significantly higher (3). In general,
thiols are readily oxidized to disulfides in the presence of
mild oxidants, and in the presence of more powerful oxi-
dants S-oxygenation can occur, which yields, successively,
sulfenic, sulfinic, and sulfonic acids. In some cases, the C–S
bond cleaves to give sulfate as the final oxidation product
(2, 4). Those thiols without an amino group adjacent to the
sulfhydryl group are easily oxidized to sulfate and a mixture
of alcohols, aldehydes, and carboxylic acids (depending on
the amount and strength of oxidant). α-Aminothiols are very
reactive, but will not easily cleave the C–S bond, and can
thus be regenerated for further use after oxidation to the S-
oxide or the disulfide (5). Sulfinic and sulfonic acids formed
from thiols with an amino group on the α or β position have
been found to be quite stable (6, 7).
Sulfur metabolism in biological systems is believed to be
the main source of thiols (1, 2). The most important thiols in
biological chemistry are cysteine, homocysteine, and
glutathione. Glutathione is the most abundant non-protein
thiol in mammals, an adult human having about 30 g
glutathione widely distributed in most tissues, with levels in
Received 30 August 2007. Accepted 6 February 2008.
Published on the NRC Research Press Web site at
canjchem.nrc.ca on 5 April 2008.
M.K. Morakinyo, E. Chikwana,1 and R.H. Simoyi.2
Department of Chemistry, Portland State University, Portland,
OR 97207-0751, USA.
1Present address: Department of Chemistry & Biochemistry,
California State University-Chico, CA 95929-0210, USA.
2Corresponding Author (e-mail: rsimoyi@pdx.edu).
In living systems, S-oxygenation of the thiol group in the
physiological environment can be initiated by two
microsomal systems: one involving cytochrome P-450 sys-
Can. J. Chem. 86: 416–425 (2008)
doi:10.1139/V08-031
© 2008 NRC Canada

Morakinyo et al.
417
Scheme I.
tem of enzymes and the other based on the flavin-containing
monooxygenase (8–10). In the presence of these enzyme
systems and cofactor-like compounds such as sulfide and
hydroxylamine, cysteamine is oxidized to the sulfinic acid
derivative, hypotaurine. Most metabolic pathways, however,
give hypotaurine as a precursor to taurine (the sulfonic acid).
Scheme I encompasses nearly all possible reactions of
cysteamine in the physiological environment.
Cysteamine and its major metabolite, hypotaurine, are
known to be excellent scavengers of HOCl and other reac-
tive oxygen species (ROS) and are more likely to act as anti-
oxidants in vivo than taurine (3, 11, 12). These ROS can
cause damage to DNA, proteins, and lipids in addition to
causing radical damage and oxidative stress in animal cells
(11, 13, 14). In addition to protection against radical damage
in DNA, cysteamine can also act as a repair agent for DNA
through the formation of the protective RSSR•–, which then
reacts with the DNA•+ radical ion to regenerate DNA and
form cystamine, the cysteamine disulfide (2, 3). The ability
of cystamine to reversibly form disulfide links with the
sulfhydryl groups at or near the active sites of enzymes is
also important in regulation of several essential metabolic
pathways (15, 16).
[R1] H2O2 + Br– + Myeloperoxidase/H+ ÷
HOBr + H2O
Taurine and its precursors, e.g., hypotaurine and
cysteamine have been suggested as possible moderators of
HOBr toxicity by forming longer-lived and less oxidizing
products in their reactions with HOBr (24).
Experimental
Our research group embarked on a series of studies to elu-
cidate the kinetics and mechanisms of the oxidation of
cysteamine and its S-oxides. Previous studies done in our
laboratory on the cysteamine sulfinic acid, hypotaurine, have
shown that it reacts quite rapidly with chlorite to give a mix-
ture of taurine, monocloro- and dichloro-taurine (17). Oxida-
tion is believed to occur simultaneously at the sulfur center
(to give the sulfonic acid) and at the nitrogen center (to give
the chloramines). The oxidation of taurine by chlorite (18)
and bromate (19) showed that oxidation occurs only on the
nitrogen centre to give the corresponding N-derivatives. The
reaction was exceedingly slow, even with the most powerful
oxidizing agents such as acidic bromate. The formation of
N-bromo- and N-chloro-taurines is suggested as a possible
mechanism by which taurine can moderate the oxidative tox-
icity of halogens and hypobromous and hypochlorous acid in
the slightly basic physiological environments (20).
A recent study from our laboratory on the oxidation of
cysteamine by iodine and acidic iodate showed that these
mild oxidants only oxidized cysteamine to its dimer,
cystamine (21). In this study, stronger oxidants bromine and
acidic bromate have been used. The aim of the study re-
ported in this manuscript was to investigate whether these
oxidants also produced cystamine as a viable product or
whether the oxidation proceeded to hypotaurine or all the
way to taurine as well as the N-bromotaurines. Bromate is a
precursor to HOBr, a particularly damaging oxidizing spe-
cies in the physiological environment. Stimulated granulo-
cytes produce oxidizing agents (especially H2O2) and secrete
granular proteins into the extracellular medium, which con-
tributes to their antimicrobial, cytotoxic, and cytolytic activi-
ties (22). Each group of cells contains a specific peroxidase,
which catalyzes the reactions of hydrogen peroxide with
halogens. The enzyme, myeloperoxidase, which is abundant
in neutrophils, catalyzes the oxidation of Br– ions by H2O2
to yield HOBr as a reactive and oxidatively damaging prod-
uct (23).
Materials
Cysteamine hydrochloride (CA, 2-aminoethanethiol hydro-
chloride) 98%, cystamine, bromine, potassium iodide, potas-
sium bromide (Sigma-Aldrich), sodium perchlorate (98%)
(Acros), sodium bromate, perchloric acid (72%), soluble
starch, sodium thiosulfate, and hydrochloric acid (Fisher)
were used without further purification. The concentration of
bromine was determined by standardization against
thiosulfate solution after addition of excess acidic iodide.
Spectrophotometry was used as a complementary technique
by measuring bromine absorbance at 390 nm where the ex-
tinction coefficient had been deduced to be 142 mol–1 L cm–1.
This standardization was carried out before each series of ki-
netic experiments owing to the volatile nature of bromine. CA
solutions were prepared just before use and not kept for more
than 24 h. All solutions were prepared using distilled and
deionized water from a Barnstead Sybron Corporation water-
purification unit. Inductively coupled plasma mass spectrome-
try (ICPMS) analysis showed negligible concentrations of
iron, copper, and silver and approximately 1.5 ppb of cad-
mium and 0.43 ppb in lead as the most abundant metal ions,
which was not enough to affect the overall reaction kinetics
and mechanism (25).
Methods
All experiments were carried out at 25.0 0.1 °C and a con-
stant ionic strength of 1.0 mol/L (NaClO4). CA, sodium per-
chlorate, and perchloric acid solutions were mixed in one
vessel and bromate (or bromine) solutions in another. A
PerkinElmer Lambda 25 UV–vis spectrophotometer interfaced
to a Pentium IV computer was used for most of the kinetics
–
and stoichiometric determinations. The slower BrO3 – CA re-
actions were monitored on the UV–vis spectrophotometer by
monitoring bromine absorbance at 390 nm. Faster reactions
were followed on a Hi-Tech Scientific SF61 – DX2 double
mixing stopped-flow spectrophotometer.
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Can. J. Chem. Vol. 86, 2008
Fig. 1. (a) Iodometry titration for stoichiometry analysis of prod-
Results
uct solutions for a constant [CA]o and [H+]o of 0.002 mol/L and
–
0.2 mol/L, respectively. [BrO3 ] = (a) 0.004 mol/L, (b)
Stoichiometry
–
0.005 mol/L, (c) 0.006 mol/L, (d) 0.007 mol/L, (e) 0.008 mol/L,
(f) 0.009 mol/L, (g) 0.010 mol/L. This intercept represents the
maximum amount of bromate tolerated by the reaction system
before permanent bromine production is observed. (b) Plot of
absorbance at infinity (A∞) vs. [Br2]o from the bromine oxidation
of cysteamine. The intercept, as expected, is consistent with the
stoichiometry equation proposed for the direct Br2 – CA reac-
tion. [CA] = 0.0003 mol/L, [Br2]intercept = 0.0015 mol/L.
The stoichiometry of the BrO3 – cysteamine reaction in
acidic medium is very complex. The reaction showed simul-
taneous oxidations at both the nitrogen and sulfur centers to
give N-derivatives of the cysteamine sulfonic acid. Unlike
what had been observed with other small organic sulfur mol-
ecules subjected to strong oxidizing agents, there is no
cleavage of the C–S bond as evidenced by the lack of sulfate
production. The reaction gives a mixture of products:
H2NCH2CH2SO3H,
Br(H)NCH2CH2SO3H,
and
1
Br2NCH2CH2SO3H, all of which give nearly identical H
NMR spectra in the strongly acidic medium necessary for
bromate oxidations.
Iodometric titrations in excess bromate conditions gave a
reproducible stoichiometry consistent with a 6e– oxidation of
the sulfur center to the sulfonic acid, taurine, according to
the following stoichiometric ratio:
–
[R2] BrO3 + H2NCH2CH2SH ÷
H2NCH2CH2SO3H + Br–
In this titrimetric method, excess acidic bromate was re-
acted with a fixed amount of cysteamine and the excess oxi-
dizing power left after complete consumption of cysteamine
determined by addition of excess acidified iodide followed
by a thiosulfate titration. Figure 1a shows a plot of
thiosulfate titer vs. initial bromate concentrations. The plot
is linear, as expected, with the intercept on the bromate con-
centration axis (zero thiosulfate titer) representing the exact
amount of bromate needed to oxidize cysteamine. Data
shown in Fig. 1a were taken with cysteamine fixed at
0.0020 mol/L. The intercept concentration on the bromate
axis is 0.0021 mol/L, which is very close, within statistical
error, to the 1:1 stoichiometric ratio of reaction [R2]. The
UV spectrum of the product solution, on the other hand,
shows a strong absorption peak at 244 nm, which is consis-
tent with the formation of dibromotaurine. This is even more
evident when Br– is deliberately added to the reaction mix-
ture to produce predominantly the dibromotaurine after pro-
longed standing.
–
[R3] 5BrO3 + 3H2NCH2CH2SH + Br– + 6H+ ÷
3Br2NCH2CH2SO3H + 6H2O
and so all contribution to the absorbance at this wavelength
was attributed solely to bromine. Figure 1b shows the data
derived from such a series of experiments for a fixed con-
centration of 3.0 × 10–4 mol/L cysteamine. The intercept
concentration of the [Br2]0 axis is 1.5 × 10–3 mol/L, which
represents the exact amount of bromine needed to consume
3 × 10–4 mol/L cysteamine, which confirms the 1:5
stoichiometry.
Monobromotaurine and dibromotaurine can be quantified
in the reaction product by their rapid reaction with excess io-
dide to produce iodine, which could be determined spectro-
photometrically and (or) titrimetrically:
Reaction [R3] is preceded by the formation of
monobromotaurine:
–
[R4] 4BrO3 + 3H2NCH2CH2SH + 3H+ ÷
3(Br)HNCH2CH2SO3H + Br– + 3H2O
Direct reaction of cysteamine with aqueous bromine
showed a clean 1:5 stoichiometric ratio:
[R5] 5Br2 + H2NCH2CH2SH + 3H2O ÷
Br2NCH2CH2SO3H + 8Br– + 8H+
Stoichiometry [R5] was deduced by mixing excess bro-
mine solutions with a fixed amount of cysteamine and mea-
suring the residual absorbance of bromine at 390 nm. None
of the mono- or di-bromotaurine products absorb at 390 nm,
[R6] (Br)HNCH2CH2SO3H + 2I– + H+ ÷
H2NCH2CH2SO3H + I2 (aq) + Br–
© 2008 NRC Canada

Morakinyo et al.
419
1
Fig. 2. H NMR spectrum showing (A) cysteamine,
Reaction [R6] is the reason why iodometric titrations in
excess bromate only showed the formation of the sulfonic
acid taurine (stoichiometry [R2]), and not the N-bromo de-
rivatives. Their formation was masked, since they rapidly
oxidized the iodide to iodine.
(B) cysteamine + BrO3 + H+, (C) hypotaurine, (D) hypotaurine
–
+ BrO3 + H+, (E) taurine, and (F) taurine + BrO3 + H+.
–
–
Product identification
Dibromotaurine was identified by its distinct UV spec-
trum featuring a sharp strong peak at 244 nm and a weaker
peak at 336 nm (26). In addition to a peak also at 336 nm,
monobromotaurine has an absorption peak at 288 nm (27)
with a weak absorptivity coefficient of only 425 mol–1 L cm–1.
Bromine oxidations of cysteamine always produced mixtures
of the mono- and di-bromotaurines, with the
monobromotaurine’s peak at 336 nm completely obscured
by the contribution from dibromotaurine. Highly acidic envi-
ronments favored monobromotaurine over dibromotaurine,
since the ratio of the absorbance at 288:244 increased with a
decrease in pH. This ratio also increased if taurine was
added to the stoichiometric solution [R5]. Quantitative for-
mation of monobromotaurine could be achieved by mixing
an exact 1:4 ratio of cysteamine to bromine:
Fig. 3. (a) Effect of varying bromate concentration on the oxida-
tion of CA by bromate. There is an increase in the rate of bro-
mine formation as the bromate concentration is increased. [CA]o
[R7] 4Br2 + H2NCH2CH2SH + 3H2O ÷
= 0.005 mol/L, [H+]o = 0.2 mol/L, [BrO3 ] = (a) 0.05 mol/L, (b)
–
BrHNCH2CH2SO3H + 7Br– + 7H+
0.06 mol/L, (c) 0.07 mol/L, (d) 0.08 mol/L, (e) 0.09 mol/L, (f)
1H NMR spectra were also used to prove that the
cysteamine carbon skeleton of two sets of methylene protons
is not disturbed during its oxidation. Figure 2 shows the se-
ries of spectra representing the reactants and products. Spec-
trum A is of pure cysteamine in neutral pH showing the
methylene proton triplets centered at 2.78 and 3.15 ppm.
Spectrum B is of the product of cysteamine–bromate reac-
tion in acidic medium. The two methylene triplets appear at
3.4 and 4.08 ppm with a larger coupling constant than ob-
served with pure cysteamine. Spectrum C is of pure
hypotaurine, while D is of the oxidation product of
hypotaurine with acidic bromate. Spectrum E is the standard
spectrum of taurine in D2O at nearly neutral pH. The appear-
ance of the methylene peaks upfield compared with the other
spectra is due to the comparatively higher pH conditions uti-
lized in the acquisition of this spectrum. Spectrum F is the
product of the taurine–bromate reaction. Spectra B, D, and F
are identical, showing that cysteamine, hypotaurine, and
taurine are all oxidized by acidic bromate to the same final
product. The two smaller triplets appearing on the bromate–
taurine spectrum are due to the fact that this reaction pro-
duces a mixture of products, which also include the oxime,
H(OH)NCH2CH2SO3H (28).
0.10 mol/L, (g) 0.11 mol/L. (b) Linear plot of reciprocal induc-
tion time vs. [BrO3 ] for data shown in Fig. 3a.
–
Reaction kinetics
As with all bromate oxidations, this reaction is only viable
in highly acidic environments. The reaction could be moni-
tored by following the appearance of bromine in excess
bromate conditions. In this configuration, the reaction ini-
tially showed a quiescent period with no formation of bro-
mine. We shall call this period the “induction period”. The
end of the induction period is signaled by the formation of
bromine. Figure 3a shows a series of typical bromine
absorbance traces derived from varying the initial bromate
concentrations. At the end of the induction period, there is a
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420
Can. J. Chem. Vol. 86, 2008
Fig. 4. (a) Effect of progressively increasing [H+] at constant
rapid formation of bromine, which eventually gives way to a
slower rate of formation until the excess bromate or bromide
formed from the reaction mixture has been exhausted, thus
–
–
[CA] and [BrO3 ]. [CA]o = 0.005 mol/L, [BrO3 ]o = 0.05 mol/L,
[H+] = (a) 0.20 mol/L, (b) 0.25 mol/L, (c) 0.30 mol/L, (d)
0.35 mol/L, (e) 0.40 mol/L, (f) 0.45 mol/L, (g) 0.50 mol/L, (h)
0.60 mol/L. (b) Plot showing the linear dependence of induction
–
shutting down the BrO3 /Br– reaction:
–
[R8] BrO3 + 5Br– + 6H+ ÷ 3Br2 + 3H2O
time on the reciprocal of [H+]o for data shown in Fig 4a.
2
Figure 3b shows that the length of this induction period
has an inverse linear dependence upon initial bromate con-
centrations. This would suggest that the precursor reaction to
the formation of bromine depends on bromate concentra-
tions to the first power. Figure 4a shows the reaction’s de-
pendence on acid concentrations, with Fig. 4b showing that
the induction period follows an inverse squared dependence
on acid that tends towards saturation as acid concentrations
are further increased. The biphasic bromine formation in
Fig. 4a is worth noting. Though higher acid concentrations
reduce the induction period, they also reduce the amount of
bromine formed in the rapid first step. The rate of formation
of bromine in the slower phase appeared insensitive to varia-
tions in acid concentrations, and they also appear to be zero-
–
order. Figure 5 shows that for as long as [BrO3 ]0 » [CA]0,
then the induction period will be invariant, but the rate and
amount of bromine formed after the induction period varies
with initial cysteamine concentrations. This trend would be
expected, since reaction [R8] is catalyzed by bromide, and
higher CA concentrations would produce higher bromide
concentrations.
The direct reaction of bromine with CA was so rapid that
its rate approached the detection limit of our stopped-flow
apparatus. The only way to slow the reaction down so that it
could be observed on our stopped-flow ensemble was to
flood the reaction with bromide so that the reaction being
observed was between CA and tribromide (see Fig. 6) (29,
30).
–
[R9] Br2 + Br– º Br3 ; Keq = 17
–
[R10] 3Br3 + H2NCH2CH2SH + 3H2O ÷
H2NCH2CH2SO3H + 6H+ + 9Br–
The reaction involves a very fast initial step that is com-
plete in less that 50 ms followed by a slower step that takes
about 1.0 s. The initial step involves the consumption of ap-
proximately 3 equiv. of bromine to 1 equiv. of CA. This is
equivalent to oxidizing CA to taurine:
for the Br2 – CA reaction, but it appeared to stunt the second
slower part of the reaction in which bromamines are formed
(see Fig. 8).
Mechanism
The rapid Br2 – CA reaction implies that in both Figs. 1a
and 3a, formation of bromine indicates complete consump-
tion of CA. Any bromine formed in the presence of CA will
be rapidly consumed and will not accumulate. The bromine–
hypotaurine reaction was also found to be as rapid as the Br2
– CA reaction; which implies that reaction [R2] has to run
its course before bromine production commences.
The inverse bromate dependence and inverse square acid
dependence of the induction period implies that the rate-
determining step to reaction [R11] is the well-known stan-
dard bromate initiation reaction:
[R11] 3Br2 + H2NCH2CH2SH + 3H2O ÷
H2NCH2CH2SO3H + 6Br– + 6H+
The formation of bromamines after formation of taurine
appears to constitute the slower stage of the reaction. Fig-
ure 7 shows a series of experiments in which bromine is in
excess of the stoichiometric amounts needed to satisfy reac-
tions [R5] and [R7]. Trace a is a control experiment, which
was not flooded with bromide. It shows that the reaction is
complete within the mixing time of the stopped-flow instru-
ment. The rest of the other traces show one single rapid step
without a slower second step. No acid was added to this se-
ries of kinetics runs, and this appears to enhance the nor-
mally slower second step. The full 5:1 stoichiometry [R5] is
attained within 0.5 s. Acid, however, was not inhibitory
enough to allow for the determination of kinetics constants
–
[R12] BrO3 + 2H+ + Br– º HBrO2 + HOBr
We base our assertion on the following logic. The direct
reaction of bromine and cysteamine (Figs. 6–8) is so rapid
(close to diffusion-controlled) and much faster than the reac-
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Morakinyo et al.
421
Fig. 7. Effect of varying [Br2]o on the direct reaction of Br2 vs.
CA in excess bromide monitored at 390 nm. [CA] =
0.0003 mol/L, [Br–] = 1.0 mol/L, [Br2] = (a) 0.001 68 mol/L (no
bromide), (b) 0.001 68 mol/L, (c) 0.002 10 mol/L, (d)
0.002 50 mol/L, (e) 0.002 93 mol/L, (f) 0.003 35 mol/L.
Fig. 5. “Peacock-tail type” traces obtained from variation of
[CA]o in excess [BrO3 ]o conditions. The final Br2(aq) increases
–
–
with [CA]o, but the induction period does not change. [BrO3 ] =
0.1 mol/L, [H+] = 0.2 mol/L, [CA] = (a) 0.005 mol/L, (b)
0.006 mol/L, (c) 0.007 mol/L, (d) 0.008 mol/L, (e) 0.009 mol/L,
(f) 0.010 mol/L, (g) 0.011 mol/L.
Fig. 8. Effect of acid on the bromine–CA reaction. Acid is
mildly inhibitory on the first initial rapid phase of the reaction
but shuts down the second slower section of the reaction, which
involves formation of bromamines. [Br2]o = 0.002 45 mol/L,
[CA]o = 0.0004 mol/L, [H+] = (a) no acid, (b) 0.006, (c) 0.008,
(d) 0.010, (e) 0.012, (f) 0.014 mol/L.
Fig. 6. Effect of varying CA on the direct reaction of Br2 vs.
CA in excess bromide monitored at 390 nm. [Br2] =
0.00215 mol/L, [Br–] = 1.0 mol/L, [CA] = (a) 0.0001 mol/L (b)
0.0002 mol/L (c) 0.0003 mol/L (d) 0.0004 mol/L (e)
0.0005 mol/L (f) 0.0007 mol/L.
bromide (reaction [R14]) will make its effect as an oxidant
negligible.
[R14] HBrO2 + Br– + H+ º 2HOBr
tion of bromate and cysteamine such that formation of bro-
mine should indicate complete consumption of cysteamine.
The time taken to achieve complete consumption of
cysteamine (the induction period monitored in Figs. 3b and
4b), is inversely proportional to the rate of reaction (i.e.,
consumption of cysteamine).
If one re-writes reaction [R13] and substitutes CA with
any 2-electron reductant (i.e., the 2 electrons can be supplied
by any oxidizable species in the reaction mixture), then a
linear combination of R12 + 3R13 + R14 gives an overall re-
action stoichiometry that is autocatalytic in Br–:
Further reaction can occur between the reactive species
HBrO2 and HOBr with CA and its oxidative by-products.
–
[R15] BrO3 + 6H+ + Br– + 6e– ÷ 2Br– + 3H2O
[R13] HOBr + H2NCH2CH2SH ÷
H2NCH2CH2SOH + Br– + H+
As long as reaction [R12] is the rate-determining step, Br–
will be an autocatalyst. All bromate solutions contain trace
amounts of bromide in the reaction mixture, which have
While HBrO2 can also act as oxidizing agent as in reac-
tion [R13], its rapid disproportionation in the presence of
© 2008 NRC Canada

422
Can. J. Chem. Vol. 86, 2008
Table 1.
No.
Reaction
kf, kr
–
M1
M2
M3
M4
M5
M6
M7
M8
BrO3 + 2H+ + Br– º HBrO2 + HOBr
2.1, 1.00 × 10–4
HBrO2 + Br– + H+ º 2HOBr
HOBr + Br– + H+ º Br2 + H2O
2.00 × 106, 2.00 × 10–5
8.9 × 109, 1.10 × 102
4.00 × 107, 2.00 × 10–10
1.50 × 109, 8.8 × 107
1.00 × 107, 1.00 × 109
1.00 × 106, 5.0 × 108
6.45
2HBrO2 º BrO3 + HOBr + H+
–
Br2 + Br– º Br3
–
BrO3 + H+ º HBrO3
–
H+ + HBrO3 º H2BrO3
+
H2BrO3 + H2NRSH ÷ HBrO2 + H2NRSOH + H+
+
M9
HBrO2 + H2NRSH ÷ HOBr + H2NRSOH
5.00 × 103
M10
M11
M12
M13
M14
M15
M16
M17
M18
HOBr + H2NRSH ÷ H+ + Br- + H2NRSOH
HOBr + H2NRSOH ÷ H+ + Br– + H2NRSO2H
HOBr + H2NRSO2H ÷ H+ + Br– + H2NRSO3H
Br2 + H2NRSH + H2O ÷ 2H+ + 2Br– + H2NRSOH
Br2 + H2NRSOH + H2O ÷ 2H+ + 2Br– + H2NRSO2H
5.00 × 106
5.00 × 105
1.00 × 103
5.00 × 108
2.50 × 108
Br2 + H2NRSO2H + H2O ÷ 2H+ + 2Br– + H2NRSO3H 1.70 × 103
Br2 + H2NRSO3H ÷ BrHNRSO3H + H+ + Br–
Br2 + BrHNRSO3H º Br2HNRSO3H + H+ + Br
2BrHNRSO3H + H+ º Br2HNRSO3H + H2NRSO3H
3.30
7.00 × 10–2, 1.05×10–3
5.00, 1.00 × 10–2
Note: The units for the kinetics constants in the third column are derived from the reaction’s
molecularity except where water is involved, in which case water was ignored.
been measured at approximately 5.0 × 10–6 mol/L. Quantita-
tively, are these sufficient to initiate the reaction? A com-
puter simulation of the reaction while utilizing these trace
bromide concentrations gave much slower reaction kinetics
and longer induction periods when compared with the exper-
imental traces. This suggests that there should be another
pathway that generates bromide ions, which will initiate re-
action [R12]. Another pathway that exists for initial bromide
formation involves oxidation by protonated bromic acid
(31–33):
[R12] is rate-determining up to the stage where cysteamine
is completely converted to taurine.
All kinetics data collected imply that the oxidation of CA
to taurine is much faster than the subsequent N-bromination
reactions. Since bromate oxidations are only viable at low
pH conditions, in all bromate oxidations of CA studied, the
CA should be fully protonated on the amino group:
+
[R21] H2NCH2CH2SH + H+ º HSCH2CH2NH3 ; Kb
The protonation of the amino group should retard the
electrophilic bromination of this group:
–
[R16] H+ + BrO3 º HBrO3
+
[R17] H+ + HBrO3 º H2BrO3
+
[R22] HSCH2CH2NH3 + Br2 º
HSCH2CH2NHBr + Br– + 2H+
+
[R18] H2BrO3 + H2NCH2CH2SH ÷
H2NCH2CH2SOH + HBrO2 + H+
An intermediate such as HSCH2CH2NHBr should be very
unstable and should rapidly hydrolyze to the sulfenic acid,
since the thiol group is much more easily oxidizable than the
N-containing group:
[R19] HBrO2 + H2NCH2CH2SH ÷
H2NCH2CH2SOH + HOBr
Bromide ions will thus be generated by the successive re-
duction of bromate to bromide. Adding the sequence R16 +
R17 + R18 + R19 + R13 and assuming each reductant can
be represented by 2 e’s; the overall reaction representing ini-
tial bromide formation will be similar to [R15] without the
autocatalysis:
[R23] HSCH2CH2NHBr + H2O ÷
H2NCH2CH2SOH + Br– + H+
N-bromination of the sulfenic acid and hypotaurine will
also be insignificant owing to kinetics factors in which the
oxidation of the sulfur group is much more rapid than N-
bromination (see Table 1).
–
[R20] BrO3 + 6H+ + 6e– ÷ Br– + 3H2O
The autocatalytic route will take over and become domi-
nant as the reaction proceeds and rate of bromide production
shifts from the kinetics derived from [R20] to those derived
from [R15]. As bromide ions accumulate, their rate of pro-
duction ceases to be the rate-determining step, and reaction
[R12] takes over as the overall rate-determining step. Data
shown in Figs. 3b and 4b strongly suggests that reaction
N-bromination kinetics
In neutral to slightly basic environments, reaction [R24]
should be very rapid and almost diffusion-controlled.
[R24] HO3SCH2CH2NH2 + Br2 ÷
HO3SCH2CH2NHBr + Br– + H+
© 2008 NRC Canada

Morakinyo et al.
423
Scheme II.
Our stopped-flow ensemble, with a mixing time of 3 ms,
could not catch this step, nor the next step that involves for-
mation of the dibromo derivative:
[R25] HO3SCH2CH2NHBr + Br2 º
HO3SCH2CH2NBr2 + Br– + H+
This reaction is slower in highly acidic medium because
reactions of type [R22] are slower than those of type [R24].
Electrophilic attack on the N-center by bromine is retarded
by the protonation of the nitrogen center. Kinetics data in
Figs. 6 and 7 show first-order kinetics in both bromine and
cysteamine. If one was to assume, in a limiting case, that the
route of bromination through [R22] is negligible, then the
initial rate of N-bromination would be given by the equation,
Bromine formation
There is only one reaction in the reaction mixture respon-
sible for bromine formation:
[R28] HOBr + Br– + H+ º Br2(aq) + H2O
d[Br2] k24[HO3SCH2CH2NH2]T[Br2]0
[1]
Rate = −
=
dt
1 + Kb[H+ ]
[HO3SCH2CH2NH2]T is the total cysteamine sulfonic acid
concentration before it is partitioned into the protonated and
unprotonated forms. Retardation by bromide ions can be
mathematically handled in the same manner, in which rate of
reaction becomes
Since both HOBr and Br2 react rapidly with cysteamine
(and its metabolite, hypotaurine), then formation of bromine
is an indicator that cysteamine and its metabolites have all
been oxidized to at least the taurine stage. The formation of
bromine at the end of the induction period should depend in
some manner on the rate of formation of bromide (assuming
excess of acidic bromate). The full reaction network respon-
sible for bromine formation is complex, and can be depicted
by the network shown in Scheme II. To simplify this
scheme, only the relevant reactions after cysteamine has
been oxidized to taurine (presented as RNH2 in the scheme)
are included. It is a bromide-controlled network due to bro-
mide’s role as an autocatalyst in the reaction network. It pro-
vides a positive feedback loop that feeds into reaction A and
accelerates the reaction.
This network scheme can explain the rate of bromine for-
mation after the induction period as well as the biphasic na-
ture of bromine production. Without excess bromate the
feedback loop cannot exist, since reaction [R12] is a precur-
sor to all autocatalysis. The reaction network relies on process
B being fast (reaction [R28]). Our simulations also showed that
HOBr does not accumulate to concentrations higher than
10–6 mol/L for the duration of the reaction. The fact that pro-
cess C is faster than E delivers the biphasic bromine forma-
tion. Since bromine formation depends on bromide formation,
the faster N-bromination of taurine ensures a fast rate of for-
mation of bromide and subsequently a high rate of initial for-
mation of bromine. Higher acid concentrations retard the
formation of dibromotaurine. Figure 4a illustrates this effect:
high acid concentrations catalyze the formation of HOBr,
leading to higher bromine formation (processes A + B). This
in turn leads to higher bromide formation (process C), which
feeds back into process A. Acid catalysis of A is stronger
than acid retardation of C. However, acid retardation of E is
significant and when most of the monobromotaurine has been
formed, the subsequent bromination to dibromotaurine is
slower (in high acid), and the equilibrium of reaction [R25] is
shifted to the left. With process E slower, bromide formation
is slower, and subsequently bromine formation is slow, as ob-
served in the slower bromine formation in the second phase.
The zero-order kinetics observed in bromine formation is due
to the fact that this section is no longer solely dependent on
d[Br2] k24[HO3SCH2CH2NH2]T[Br2]0
[2]
−
=
dt
{1 + Kb[H+ ]}{1 + Keq[Br− ]}
In none of our experiments were the retardations by Br–
and H+ effective enough for the use of eqs. [1] and [2] in de-
termining bimolecular rate constant k24. We could only use
initial rate data and assuming bimolecular kinetics in Fig. 8
to estimate that k24 should be larger than 105 mol–1 L s–1.
Reliable values for k24 can be derived from relaxation tech-
niques (34). In the physiological environment and in basic
conditions, in general, the major route of N-bromination (re-
action [R26]) is through hypobromous acid (35), since the
bromine hydrolysis reaction favors hypobromous acid at
high pH and aqueous bromine in acidic conditions (36).
[R26] HO3SCH2CH2NH2 + HOBr ÷
HO3SCH2CH2NHBr + H2O
N-bromination in bromate oxidations is predominantly
through aqueous bromine, since bromate oxidations only
proceed in acidic environments (37).
The acid retardation observed for the reaction shown in
Fig. 8 may suggest that reaction [R26] is slower than [R24].
Previous studies have confirmed that the monobromo-and
dibromo- derivatives are affected by acid, with highly acidic
conditions favoring monobromotaurine (37). This means that
while [R24] can proceed quantitatively to the product
monobromotaurine, [R25] is an equilibrium condition in
which high acid concentrations in the products can swing
the equilibrium towards monobromotaurine formation. The
addition of taurine to dibromotaurine solutions saw the de-
crease in the peak at 244 nm (dibromotaurine) and the emer-
gence of a peak at 288 nm (monobromotaurine):
[R27] HO3SCH2CH2NH2 + HO3SCH2CH2NBr2 º
2HO3SCH2CH2NHBr
© 2008 NRC Canada

424
Can. J. Chem. Vol. 86, 2008
Fig. 9. Simulated results (symbols) of traces a and c (solid lines)
from Fig. 3a showing the effect of bromate variation on CA oxi-
dation.
spective of the other reaction rates. The initiation reactions
were important in establishing the time taken before bro-
mine formation commenced, but a few seconds into the re-
action reactions M6–M10 ceased to be effective. Since we
could not harness the bromination reactions due to their
speed, the lower-limit-estimated rate constants were effec-
tive, since ultimately, formation of bromine was controlled
by M1. Figure 9 shows a reasonably good fit to the data on
bromate-dependence data shown in Fig. 3a. A good fit was
also obtained with acid-dependence traces shown in Fig. 4a.
The model was able to satisfactorily predict both bromate-
and acid-dependence effects with respect to induction period
and rate of formation of bromine at the end of this induction
period.
Conclusion
The reversible nature of N-bromination of the taurine
formed after a full oxidation of cysteamine suggests that
cysteamine and its oxidation metabolites are capable of mod-
erating hypobromous acid and hypochlorous acid toxicities by
forming these long-lived brominated derivatives. The ease of
regeneration of taurine in reducing conditions means that this
moderation can proceed indefinitely in a cyclic manner.
process A, but a much more complex network. Primarily,
equilibrium [R25] acts as a valve in the release of bromide
and maintains a constant release of bromide ions, as in drug-
suspension reactions.
Overall reaction mechanism and simulations
Acknowledgement
The overall mechanism that can explain the observed
global dynamics of the system on the basis of Scheme II is
shown in Table 1. The first group of reactions M1–M4 and
M5 are the well-known oxybromine reactions whose kinetics
constants are well known (31, 33, 39–41). These were not
varied during the simulations and were accepted as “fixed”.
The next set of reactions M6–M10 constitute the initiation
reactions whose sole purpose is to produce bromide ions
(autocatalytically). Protolytic reactions such as M6 and M7
are normally diffusion-controlled. The use of diffusion-
controlled rate constants for these reactions rendered the
simulations too stiff, necessitating hours of computer time
instead of mere seconds with the use of the kinetics con-
stants used in Table 1. The adopted kinetics constants did
not deliver any different simulation results when compared
to the use of the diffusion-controlled kinetics constants. The
third set of reactions M11–M15 constitute the oxidation of
cysteamine to taurine. The major oxidants are HOBr and
Br2. Even though this set of reactions is fast, they are all
controlled (after the initiation reactions), by reaction [R12]
(M1 in the table). Rapid reaction [R28] (M3 in table) is also
controlled by [R12], since production of Br– is a prerequisite
reagent for formation of HOBr. The N-bromination reactions
are listed in the last section, M16–M18. We have assumed in
all these simulations that we are dealing with a protonated
taurine molecule on the amino group, since the pH condi-
tions necessary for the activity of bromate as an oxidant are
low enough to protonate that group. Since we did not and
could not quantify the degree of acid retardation by the reac-
tion, we felt that this was a reasonable assumption. Reaction
M18 was a necessary disproportionation reaction in high-
acid conditions. Even in low-acid conditions, reaction M18
ensured that stoichiometry [R7] was attained first before
stoichiometry [R5]. The simplicity of the simulations was
derived from the bottle-neck offered by reaction M1, irre-
This work was supported by research grant no. CHE
0614924 from the National Science Foundation.
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