One-electron reduction of N-chlorinated and N-brominated species is a source of radicals and bromine atom formation

Article abstract of DOI:10.1021/tx100325z

Hypochlorous (HOCl) and hypobromous (HOBr) acids are strong bactericidal oxidants that are generated by the human immune system but are implicated in the development of many human inflammatory diseases (e.g., atherosclerosis, asthma). These oxidants react readily with sulfur- and nitrogen-containing nucleophiles, with the latter generating N-halogenated species (e.g., chloramines/bromamines (RR′NX; X = Cl, Br)) as initial products. Redox-active metal ions and superoxide radicals (O₂ ^?-) can reduce N-halogenated species to nitrogen- and carbon-centered radicals. N-Halogenated species and O₂ ^?- are generated simultaneously at sites of inflammation, but the significance of their interactions remains unclear. In the present study, rate constants for the reduction of N-halogenated amines, amides, and imides to model potential biological substrates have been determined. Hydrated electrons reduce these species with k₂ > 10⁹ M^-1 s^-1, whereas O₂^?- reduced only N-halogenated imides with complex kinetics indicative of chain reactions. For N-bromoimides, heterolytic cleavage of the N-Br bond yielded bromine atoms (Br^?), whereas for other substrates, N-centered radicals and Cl^-/Br^- were produced. High-level quantum chemical procedures have been used to calculate gas-phase electron affinities and aqueous solution reduction potentials. The effects of substituents on the electron affinities of aminyl, amidyl, and imidyl radicals are rationalized on the basis of differential effects on the stabilities of the radicals and anions. The calculated reduction potentials are consistent with the experimental observations, with Br^? production predicted for N-bromosuccinimide, while halide ion formation is predicted in all other cases. These data suggest that interaction of N-halogenated species with O ₂^?- may produce deleterious N-centered radicals and Br^?.

Full text of DOI:10.1021/tx100325z

ARTICLE
pubs.acs.org/crt
One-Electron Reduction of N-Chlorinated and N-Brominated Species
Is a Source of Radicals and Bromine Atom Formation
,
†,||
‡
†
‡
§
David I. Pattison,* Robert J. O’Reilly, Ojia Skaff, Leo Radom, Robert F. Anderson, and
†,||
Michael J. Davies
†
The Heart Research Institute, 7 Eliza Street, Newtown, NSW 2042, Australia
‡
School of Chemistry and ARC Centre of Excellence for Free Radical Chemistry and Biotechnology, University of Sydney, Sydney, NSW
2
006, Australia
§
Department of Chemistry, University of Auckland, Auckland, New Zealand
Faculty of Medicine, University of Sydney, Sydney, NSW 2006, Australia
S Supporting Information
b
ABSTRACT: Hypochlorous (HOCl) and hypobromous
HOBr) acids are strong bactericidal oxidants that are generated
(
by the human immune system but are implicated in the
development of many human inflammatory diseases (e.g.,
atherosclerosis, asthma). These oxidants react readily with
sulfur- and nitrogen-containing nucleophiles, with the latter
generating N-halogenated species (e.g., chloramines/broma-
0
mines (RR NX; X = Cl, Br)) as initial products. Redox-active
•
-
metal ions and superoxide radicals (O2 ) can reduce N-
•-
halogenated species to nitrogen- and carbon-centered radicals. N-Halogenated species and O2 are generated simultaneously at
sites of inflammation, but the significance of their interactions remains unclear. In the present study, rate constants for the reduction
of N-halogenated amines, amides, and imides to model potential biological substrates have been determined. Hydrated electrons
9
-1 -1
•-
reduce these species with k > 10 M
s
, whereas O2 reduced only N-halogenated imides with complex kinetics indicative of
2
•
chain reactions. For N-bromoimides, heterolytic cleavage of the N-Br bond yielded bromine atoms (Br ), whereas for other
-
-
substrates, N-centered radicals and Cl /Br were produced. High-level quantum chemical procedures have been used to calculate
gas-phase electron affinities and aqueous solution reduction potentials. The effects of substituents on the electron affinities of
aminyl, amidyl, and imidyl radicals are rationalized on the basis of differential effects on the stabilities of the radicals and anions. The
•
calculated reduction potentials are consistent with the experimental observations, with Br production predicted for N-
bromosuccinimide, while halide ion formation is predicted in all other cases. These data suggest that interaction of N-halogenated
•
-
•
species with O2 may produce deleterious N-centered radicals and Br .
1
. INTRODUCTION
thiocyanate ions to hypochlorous (HOCl), hypobromous
HOBr), and hypothiocyanous (HOSCN) acids.2
,3
(
A major feature of the human inflammatory response is the
HOCl and HOBr are strong oxidants and potent antibacterial
recruitment of phagocytic white blood cells (neutrophils,
eosinophils, monocytes, or macrophages) to sites of inflam-
mation and their subsequent activation. The latter results in a
respiratory burst that consumes oxygen and generates a high
agents that react rapidly with nucleophilic targets (reviewed in
ref 4); in biological systems, this is comprised primarily of sulfur-
and nitrogen-containing moieties. Thus, in proteins the primary
sites of attack by HOCl and HOBr are free thiols (Cys side-
chain), thioethers (Met side-chain), disulfide bonds (cystine),
•
-
flux of superoxide radicals (O2 ) via an NADPH oxidase
NOX2) complex (reviewed in ref 1). The O2• subsequently
-
(
amines (Lys side-chain), and other nitrogenous side-chains (e.g.,
rapidly dismutates to generate hydrogen peroxide (H O )
and O2.
2
2
4-7
His).
dues.
In contrast, HOSCN specifically targets thiol resi-
HOCl and HOBr also react with nitrogen-containing
8-10
Simultaneous to the respiratory burst, activated leukocytes
moieties in DNA/RNA bases, phospholipids and some
release a range of inflammatory mediators, including protease
polysaccharides.4
,11-15
These reactions result in halogenation
2
,3
and peroxidase enzymes, from intracellular granules. Myelo-
peroxidase (MPO) is a major protein component of the phago-
cytic granules of neutrophils, monocytes, and some tissue
macrophages, while eosinophil peroxidase (EPO) is released
from eosinophils. These enzymes utilize the H O generated
during the respiratory burst to oxidize chloride, bromide, and
of the nucleophilic nitrogen center to form N-halogenated
0
0
species (RR N-Cl or RR N-Br), which retain the oxidizing
2
Received: September 21, 2010
Published: February 23, 2011
2
2
r 2011 American Chemical Society
371
dx.doi.org/10.1021/tx100325z
|Chem. Res. Toxicol. 2011, 24, 371–382

Chemical Research in Toxicology
ARTICLE
capacity of HOCl and HOBr but have relatively long half-
Scheme 1. Structures of the N-Halogenated (X = Cl or Br)
Species Investigated in This Study
1
6-19
a
lives.
potentially important intermediates in the biological effects of
The properties of N-halogenated species render them
2
0-23
HOCl and HOBr.
N-Halogenated species react via multiple pathways (reviewed
in refs 2 and 24), including: (i) one-electron reduction to yield
radicals, (ii) halogen transfer to regenerate the parent com-
pound, or (iii) hydrolysis (via an imine) to yield carbonyl
derivatives. Of particular interest in the current studies is the
one-electron reduction of N-halogenated species; these pro-
2
þ
þ
cesses are stimulated by transition metal ions (Fe , Cu , or
3
þ 12,16,24,25
•- 26-28
Ti In the case of chloramines and
most N-brominated species, nitrogen- and carbon-centered
)
and O2
.
radicals and halide ions are generated,1
2,16,25,29
but for some N-
0,31
brominated species (e.g., N-bromosuccinimide3 ) heterolytic
cleavage of the N-Br bond may result in the formation of imidyl
•
anions and bromine atoms (Br ).
These reactions are of potential importance as both O2•- and
HOCl/HOBr (and hence N-halogenated species) are generated
concurrently by activated leukocytes, thereby raising the possi-
•
-
bility that O2 and N-halogenated species may interact at
inflammatory loci. A key determinant of the importance of these
reactions in vivo are the relative rate constants for reaction of the
a
(
a) N-Chloro- or N-bromo-succinimide (NCS or NBS) and (b) N-
bromoglutarimide (NBG) are models of biological substrates that
contain the imide moiety (R-C(O)-NH-C(O)-R ) including some
nucleobases and antioxidants. (c) N-Bromo-4-hydroxy-2-pyrrolidinone
4,11,17,20,32,33
0
N-halogenated species with biological targets,
and
•
-
the rate constants for O2 dismutation (reviewed in ref 34).
Many of these values have been determined, but the rates of
(NBr-HOP) and (d) N-bromo or N-chloro glycine anhydride ((Gly)
or (Gly) Cl) were used as models of amide groups, as found primarily in
protein backbones. (e) N-Chloro- and N-bromo-6-amino-caproic acid
CANCl or CANBr) were used to model the chloramines/bromamines
2
Br
•
-
reduction of N-halogenated species by O are unknown. In the
2
2
•
-
current study, the reactions of O2 with N-halogenated species
have been investigated by pulse radiolysis techniques; the N-
halogenated compounds investigated (Scheme 1) were chosen
to model nitrogenous motifs found in biological molecules that
are known to react with HOCl/HOBr. The products of one-
electron reduction of N-brominated species have been probed as
these reactions are potentially a novel source of highly damaging
bromine atoms in vivo. High-level quantum-chemistry calcula-
tions have been utilized to provide further insight into the
mechanisms of these reactions; these data suggest that the
observed processes are general phenomena for N-halogenated
species.
(
formed on the Lys side-chain of proteins, as well as other primary
amines.
before use and kept on ice for <1 h; the stock solutions were diluted
into the reaction mixture immediately prior to acquisition of the kinetic
data. For stability studies and determination of extinction coefficients,
the stock solutions were diluted to the desired concentration with
phosphate buffer.
2
.4. Assay of Concentrations of N-Halogenated Species.
25
N-Halogenated species were quantified as reported previously using
-thio-(2-nitrobenzoic acid) (TNB; ca. 40 μM). The extinction coeffi-
5
-
1
-1 36
cient (ε) for TNB at 412 nm was taken as 14150 M cm
.
2.5. Kinetic Studies. The concentrations of the N-halogenated
2
. EXPERIMENTAL PROCEDURES
species were determined by measuring their absorbance spectra (Ocean
Optics HR 4000 Composite-grating spectrometer) before and after
3
7
2
.1. Materials. Sodium formate and phosphate buffer reagents for
experiments, utilizing published extinction coefficients and those in
Table 1. Concentrations of N-chlorosuccinimide (NCS) and N-bromo-
succinimide (NBS) were calculated on a mass basis with subsequent
dilution. Reaction mixtures contained 10-500 μM of each substrate in 5
mM phosphate buffer (pH 7.2) solutions.
pulse radiolysis were obtained from Merck; potassium thiocyanate was
from Riedel-de Haen. All other chemicals were obtained from Sigma/
Aldrich/Fluka (Castle Hill, NSW, Australia) and used without further
purification. HOCl solutions were standardized by measuring the
absorbance at 292 nm at pH 12 (ε292( OCl) = 350 M cm ). All
solutions were prepared using water purified by a Millipore “Milli-Q”
filtration system, and phosphate buffers utilized for stability studies and
extinction coefficient determination were also treated with Chelex resin
-
-1
-1 35
Time-resolved optical absorption and kinetic studies were carried out
at room temperature (22 ( 1 °C) using the University of Auckland’s
Pulse Radiolysis Facility, which utilizes a 4 MeV linear accelerator to
deliver 200 ns electron pulses of 2.5-6 Gy dose to a 2 cm path length
cell. The optical detection system and dosimetry method have been
(BioRad, Hercules, CA, USA) to remove contaminating metal ions.
3
8
2
.2. Preparation of Hypobromous Acid. HOBr was prepared
reported. The radiolysis of water produces three well-characterized
6,14
as described previously by mixing HOCl (40 mM) with a 1.125-fold
excess of NaBr (45 mM). After 5 min, the resulting HOBr solution was
diluted with phosphate buffer to the required concentration.
radicals as well as molecular products (eq 1; μM per absorbed dose of 1
-1
Gy (J kg ) given in parentheses).
2
.3. Preparation of N-Halogenated Species. Concentrated
stocks of the N-halogenated species were prepared by adding HOCl or
HOBr to a solution containing a 2- to 100-fold excess of parent
compound in phosphate buffer and were vortexed during and after
the addition of HOCl/HOBr to ensure efficient mixing. For kinetic
experiments, these concentrated stocks were prepared immediately
For experiments designed to study the reactions of CO2•-, solutions
containing sodium formate (0.2 M) were presaturated with N O gas for
2
15 min before pulse radiolysis. The addition of formate ions (to scavenge
3
72
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Chemical Research in Toxicology
ARTICLE
Table 1. Extinction Coefficients (ε) Determined for the N-
Brominated Species Utilized in These Studies
level in conjunction with the conductor-like polarizable continuum
a
48,49
model (CPCM) with Bondi radii.
This approach provides solvation
energies (ΔGsolv) with a mean absolute deviation from experimental
ε (M- cm-1
1
)
substrate
NBG
λ (nm)
-1
results of 15.2 kJ mol for a test set of 70 species including neutrals,
5
0
cations, and anions. As the electron-addition reaction does not involve
changes in the number of molecules, no change-of-state correction is
required to account for the change from the gas phase (1 atm) to
solution (1 M). Combination of ΔG(gas) and ΔGsolv yields ΔG(aq)
260
220 ( 10
82 ( 9
2
80
260
75
(
Gly)
2
Br
270 ( 10
162 ( 7
404 ( 7
240 ( 20
110 ( 10
2
þ
values that correspond to 298 K and 1 M H . Absolute reduction
CANBr
290
250
potentials have then been calculated from ΔG(aq), and finally these are
NBr-HOP
o
converted to reduction potentials (E ) relative to the normal hydrogen
2
80
electrode (NHE). This involves subtracting the estimated absolute
a
51
All values were calculated from absorbance data at five or six different
reduction potential of 4.43 V for hydrogen from the absolute reduction
concentrations, determined in replicate, and repeated on two separate
occasions, resulting in n > 10 for all values. The errors quoted for each
species are presented as 95% confidence intervals. However, for NBr-
HOP (250 and 280 nm) and NBG (260 nm), the nonbrominated parent
also exhibits a small absorbance at the stated wavelengths resulting in a
small systematic error in the reported extinction coefficient.
potentials of the radicals.
2.7. Statistical Analysis. All error bars shown correspond to
mean ( 1 SD (n > 3 determinations). The errors reported on extinction
coefficients and rate constants are 95% confidence limits. However, due
to the moderate instability of some of these N-halogenated species, these
errors indicate the reproducibility of the assays and kinetic determina-
tions, and do not include any systematic errors due to the decomposition
of the N-halogenated species. All statistical analyses were carried out
using Origin Pro 7 (OriginLab Corp.) software.
•
•
the OH radicals and H atoms) and saturation with N
2
O gas (to convert
-
•
the hydrated electrons (e ) into OH radicals) results in the exclusive
aq
•
-
-1 39
•-
generation of CO
2
in a yield of 0.68 μM Gy . Reactions of O
2
with the substrates were studied in the same formate-containing
-
solutions which were saturated with O
2
gas, where the e aq are
3. RESULTS
•
-
scavenged by O , and the CO2 radical anions undergo fast electron
(in equilibrium with its protonated
form) in the same yield (eqs 2-6). Reactions of the eaq species were
studied in N -saturated solutions containing BuOH (0.5 M) to sca-
venge the OH radicals and H-atoms (eq 7) to form an inert, nonredu-
2
•
-
transfer to exclusively form O
2
3.1. Stability Studies and Determination of Extinction
40
-
Coefficients for Relevant N-Halogenated Species. The N-
halogenated species investigated (Scheme 1) were prepared by
the addition of HOCl or HOBr (100 μM or 2 mM) to a 2- to 100-
fold molar excess of the nonhalogenated parent compounds. N-
Chlorosuccinimide (NCS) and N-bromosuccinimide (NBS)
were commercial products. The stabilities of the N-brominated
species were assessed at 22 °C by directly monitoring their UV/
visible spectra (200-320 nm) and/or assaying the concentration
t
2
•
•
-
cing radical on the alcohol. The reactions of O
2
were monitored at 260
2
rapidly dismutates, its
or 280 nm and those of e-aq at 620 nm. As O
•-
stability was also monitored in the absence of any substrates. Bromine
•
•-
atom (Br ) formation was monitored by quantifying Br
2
at 360 nm,
through its fast reaction with added bromide ions (1 to 10 mM; eq 8).
HO =H þ HCO2 f CO2•- þ H2O=H2
•
•
-
ð2Þ
ð3Þ
0
25
of active halogens (HOX or RR NX species) by the TNB assay,
over periods of up to 3 h. Data for related N-chlorinated species
have been reported.37
-
aq
•
-
e
þ N2O þ H2O f HO þ HO þ N2
CO2•- þ O2 f O2•- þ CO2
For N-bromo-4-hydroxy-2-pyrrolidinone (NBr-HOP), N-
bromoglutarimide (NBG), and N-bromo glycine anhydride
ð4Þ
ð5Þ
(
(Gly) Br), the UV/visible spectra and stabilities were indepen-
2
dent of the molar excess of the nonhalogenated parent. Thus,
kinetic studies (see later) were carried out with low molar
excesses of substrate over oxidant to minimize interference from
the nonhalogenated parents. The yield (relative to the initial
HOBr concentration) in each case was ca. 85-90%. NBr-HOP
and NBG did not show any decay at room temp over 90 min,
-
aq
•-
e
þ O2 f O2
HO2• h H þ O2
þ
•-
ð6Þ
•
•
•
HO =H þ ðCH3Þ COH f CH2 ðCH3Þ COH þ H2O=H2 ð7Þ
3
2
whereas (Gly) Br decayed to 60- 70% of the initial concentra-
2
Br þ Br u Br2•-
•
-
ð8Þ
tion over this time. As this decay would induce systematic errors
into the kinetic data, the stability of (Gly) Br was also monitored
2
at 0 °C; under these conditions, minimal decomposition was
2
.6. Theoretical Procedures. Standard ab initio and density
41-43
observed over 3 h.
functional theory calculations
radicals and anions relevant to the one-electron reduction of nitrogen
halides using the Gaussian 03 (G03 ) and Gaussian 09 (G09 )
programs. Gas-phase structures and harmonic vibrational frequencies
were carried out for a selection of
In contrast, the presence of excess 6-amino-caproic acid, used
in the preparation of N-bromo-6-amino-caproic acid (CANBr),
resulted in significant changes in the UV/visible spectra. This is
consistent with equilibration between bromamines and dibro-
44
45
were obtained at the B3-LYP/6-31G(2df,p) level. Gas-phase enthalpies
52
and free energies were obtained at the G4 level,46 including corrections
mamines, and therefore, CANBr stock solutions were prepared
with either a 50- or 100-fold excess of the parent compound over
HOBr to minimize dibromamine formation; under these condi-
tions, CANBr accounted for ca. 85% of the HOBr added.
The stability of CANBr was not investigated further as the
closely related species, N-ε-bromo-N-R-acetyl-lysine, undergoes
4
7
(
calculated using appropriately scaled vibrational frequencies ) for
zero-point vibrational energies, thermal energies, and entropies. These
energies allow gas-phase electron affinities and gas-phase free energies
for electron addition to be evaluated.
The effect of bulk water was estimated using G03 through the
calculation of free energies of solvation (in water) at the HF/6-31þG(d)
28
minimal decay over 1 h at 37 °C; thus, CANBr is unlikely to
3
73
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Chemical Research in Toxicology
ARTICLE
result, a definitive second-order rate constant for the reaction of
•-
O2 with NBG could not be determined despite clear evidence
for a rapid reaction. If it is assumed that the decay at 260 nm
monitors only O2• and the fast component of the decay is due to
-
•
-
6
the initial reaction of O with NBG, a lower limit of ca. 3 ꢀ 10
2
-
1 -1
M
s
can be determined. However, it should be noted that
it is not possible mathematically to distinguish which of the
decay components corresponds to the O2• reaction with
NBG. Furthermore, the final absorbance (after 10 ms) of the
signal monitored at 260 nm showed extensive bleaching (up to
-
∼
0.01 absorbance units; Figure 1a), indicating that detection at
60 nm not only probes the O2• concentration but also is
-
2
influenced by the concentration of NBG (consistent with the
extinction coefficient reported in Table 1). The large bleach in
signal at 260 nm upon completion of reaction suggests that
ca. 23 μM NBG was consumed (using the extinction coefficient
•
-
in Table 1), which is much greater than the 2-4 μM O
2
generated during the electron pulse. Further evidence for
enhanced NBG loss was obtained when samples were subjected
to a second electron pulse without flushing of the sample cell. In
this case, no decay of the O2• signal at 260 nm was detected,
consistent with complete consumption of the NBG following
the first electron pulse.
-
In order to verify that the reduction of NBG was mediated by
O2• and not its precursor CO2 (eqs 1-5), these experiments
-
•-
were repeated under an N O atmosphere. Under these condi-
2
tions, the transient spectrum obtained (with increasing absor-
bance from 320 nm into the UV region, with a maximum at ca.
•
-
Figure 1. Kinetic traces obtained to follow the reaction of O
2
•- 53,54
(
generated in an O -saturated solution containing 0.2 M sodium formate
250 nm) is consistent with the presence of CO
2
.
The rate
2
-
and 5 mM phosphate buffer (pH 7.2)) with (a) N-bromoglutarimide (0
μM (O) and 62 μM (b)) at 260 nm and (b) N-bromosuccinimide (0
μM (O), 100 μM (9), and 250 μM (2)) at 280 nm. Although these
wavelengths were chosen to follow O2 consumption, both NBG and
NBS also exhibit absorption at these wavelengths, resulting in a
significant bleaching of the signals obtained as NBG and NBS are
consumed.
of decay of CO2• (at 260 nm) was unchanged by the absence or
presence of 50 μM NBG, suggesting that NBG does not react
with CO2• over these time scales.
-
•
-
3.2.2. N-Bromosuccinimide. The reaction of NBS (100 or
50 μM) with O2 was investigated using the standard condi-
•-
2
tions, but with the signal monitored at 280 nm. At both NBS
concentrations, the absorption at 280 nm decayed over a few
milliseconds, while in the absence of NBS, no decay was observed
over 5 ms (Figure 1b). The decay curves obtained with 100 μM
NBS were fitted with a single exponential decay yielding a kobs ca.
decay significantly over 90 min on ice, as confirmed during the
kinetic experiments.
The extinction coefficients (ε) for the N-brominated species
-
1
1200 s , while the data at 250 μM NBS required a double
(
Table 1) were determined by using multiple dilutions of stock
exponential decay to fit accurately, with the observed rate con-
-
1
solutions and plotting the observed absorbance (at the wave-
length of interest) versus the concentration (as determined
by the TNB assay). The derived ε values are consistent with
those for other N-brominated species52 and were subsequently
used to quantify the stock solutions used in the kinetic studies. ε
values for the N-chlorinated species have been reported
stant for the fast component ranging from 4600 to 8300 s
-
1
and the slower component ca. 500 s . These data did not allow
to be accurately
determined due to the complexity of the decays, but a lower limit
was determined, on the basis of the fast
component of the kinetic decay at 280 nm and the assumption
•-
a second-order rate constant for NBS þ O
2
7
-1 -1
of 1 ꢀ 10 M
s
3
7
•-
previously.
that this monitored only O
2
consumption. However, as with
3
.2. Rate Constants for the Reduction of N-Halogenated
NBG, a considerable bleaching of the signal was observed at 280
nm consistent with extensive consumption of NBS (indicating
chain reactions are occurring), and exposure of the sample to a
second electron pulse did not result in decay of the 280 nm signal
over 5 ms.
Species by Superoxide. Initial experiments established condi-
tions (0.2 M sodium formate, buffered to pH 7.0-7.2 with 5 mM
sodium phosphate, gassed with O for 10-15 min) under which
2
•
-
reproducible O
production could be monitored by pulse
2
radiolysis. Under these standard conditions, no decay of the
3.2.3. N-Chlorosuccinimide. The reaction of NCS with O2•-
was initially investigated in the standard formate/phosphate
solutions. Addition of NCS (100-270 μM) to these solutions
resulted in a concentration-dependent increase in the rate of
•-
O2 signal was observed (at 260 or 280 nm) over time scales up
to 5 ms (Figure 1).
3
.2.1. N-Bromoglutarimide. Addition of NBG (40-90 μM)
•-
•-
to the above system resulted in an enhanced rate of decay of O2
decay of the O2 signal at 280 nm. The resulting data were fitted
(
measured at 260 nm; Figure 1a). Biphasic kinetic parameters
were required to fit the decays. The observed rate constants
with a single exponential decay, and the first-order rate constants
were plotted against the NCS concentration, yielding a second-
-
1
5
-1 -1
derived from the fast component varied from 230 to 5600 s
but did not correlate with the NBG concentration present. As a
,
order rate constant of (8 ( 2) ꢀ 10 M
s
for the reaction of
NCS with O2• . With higher NCS concentrations (540 μM),
-
3
74
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Chemical Research in Toxicology
ARTICLE
(
eq 9, where a reflects the excess of parent used in preparing the
N-brominated species).
Gradient ¼ k ðN - BrÞ þ a ꢀ k ðparentÞ
ð9Þ
The rate constant for this second process was determined by
2
2
-
examining the decay of e
in the presence of glutarimide
aq
(
200 μM) under identical conditions; this yielded an approximate
9
-1 -1
rate constant of 7 ꢀ 10 M s . These data suggest that the
-
second-order rate constant for the reaction of NBG with e
is
aq
10
-1 -1
>
10
M
s
(estimated using eq 9 (a = 10)). Unfortunately,
accurate determination of this rate constant is not feasible as the rate
-
of decay of e under these conditions is very fast (t1/2 < 1 μs), and
aq
the reaction with glutarimide is a major contributor to the observed
decay, with the pseudo-first-order rate constant for the consumption
-
of e by glutarimide double that of NBG under these conditions.
aq
-
A transient spectrum (250-400 nm) obtained 25 μs after e
aq
reduction of NBG (66 μM) displayed a relatively weak absor-
bance with a maximum at 360 nm. This spectrum did not show
any change in features over time, decayed completely by 500 μs,
•
-
and is consistent with the production of low levels of Br2 via
bromine atom formation.
3
.3.2. (Gly) Br. The reduction of (Gly) Br (11-33 μM) by
2
2
e- was monitored under conditions identical to those for NBG.
aq
-
As with NBG, the decay of e
is a composite rate of the
aq
reactions with (Gly) Br and the excess (Gly) , but the rate of
2
2
-
e
decay measured at 620 nm is clearly faster in (Gly) Br-
aq
2
Figure 2. (a) Second-order plots for the quenching of hydrated electrons
containing solutions when compared with control solutions
containing comparable (Gly) , but no (Gly) Br (Figure 2b). A
(monitoredat620 nm) byN-bromoglutarimide (9), N-bromo-4-hydroxy-2-
2
2
pyrrolidinone (2), and N-bromo glycine anhydride ((). The gradients of
linear plot of kobs v1ersus [(Gly) Br] (Figure 2a) yielded a
2
these linear plots correspond to composite second-order rate constants for the
0
-1 -1
-
gradient of 3.0 ꢀ 10
M
s . This value corresponds to the
reactions of e aq with both the N-brominated species and their nonbromi-
sum of the second-order rate constants for (Gly) Br and the 10-
2
nated parents (present in excess; see text). (b) Kinetic traces for the decay
-
fold excess of parent (Gly) . The second-order rate constant for
2
(Gly) reacting with e
of e (monitored at 620 nm) in the presence of (Gly) (190 μM) alone
aq
2
-
(
O) or (Gly) Br (22 μM) in the presence of a 10-fold excess of (Gly) (b).
2
aq
has been established previously as ca.
suggesting k > 1 ꢀ 10 M
2
2
9
-1 -1 37,59
10 -1 -1
Single exponential fits to the data are overlaid for both kinetic traces.
1.5 ꢀ 10 M
s
,
2
s
(by the
2
-
use of eq 9 with a = 10) for the reaction of e with (Gly) Br. As
aq
interfering reactions of NCS with the primary radiolytically
for NBG, more accurate determination of this rate constant from
these data is not appropriate as parent (Gly) consumes approxi-
generated radicals resulted in complex kinetics.
3
2
.2.4. Other N-Halogenated Species. The reactions of O2•
-
-
mately 50% of the e aq under the experimental conditions.
-
with the other N-halogenated species shown in Scheme 1 were
However, these data (Figure 2a) suggest that reaction of e aq
also investigated using the standard formate/phosphate solu-
tions. The rate of decay of the O2 signal (monitored at 280
nm) was unchanged on addition of CANBr (90 μM), CANCl
with (Gly)
In addition to the kinetics of reaction of (Gly)
the transient spectra (250-400 nm) were monitored over 1.5 ms
with 66 μM (Gly) Br. At short time scales (ca. 1 μs), a spectrum
displaying absorbance maxima at ca. 260 and 360 nm was
observed (Figure 3, solid lines). The intensity of this signal
reached a maximum after 25 μs, before decaying back to baseline
within 1 ms of the initial pulse. The relative intensities of the
absorption peaks at 260 and 360 nm remained the same over all
time scales, indicating that the spectrum is due to a single species.
2
Br is slower than that for NBG.
•
-
Br with e-aq
,
2
(
265 μM), (Gly) Cl (170 μM), or NBr-HOP (60 μM), indicat-
2
2
•
-
ing that O2 does not react with these N-halogenated species
over these time scales.
3
.3. Rate Constants for the Reduction of N-Halogenated
Species by Hydrated Electrons. In the light of the above
observations, approximate rate constants for the one-electron
-
reduction of the N-brominated species by e were determined;
aq
corresponding data for N-chlorinated species have been
When the experiments were repeated with parent (Gly) , this
transient spectrum was no longer present, indicating that this
2
3
0,37,55-58
published.
.3.1. N-Bromoglutarimide. Reduction of NBG (7-18 μM)
3
intermediate species is formed via the reduction of (Gly) Br.
2
-
by e was investigated in N -saturated solutions containing 0.5
3.3.3. NBr-HOP. For the reaction of NBr-HOP (11-42 μM)
aq
2
t
-
M BuOH with 5 mM sodium phosphate buffer (pH 7.2). The
loss of e
exponential decays were fitted to yield observed rate constants
with e (Figure 2a), the excess parent HOP was found to react
aq
-
-
was monitored at 620 nm over 10-20 μs, and the
slowly with e aq. Thus, the gradient of the linear plot of k
aq
obs
-
versus [NBr-HOP] yields the second-order rate constant for e
aq
5
-1
6
-1
9
-1 -1
ranging from 7.2 ꢀ 10 s to 2.1 ꢀ 10 s (Figure 2a). The
with NBr-HOP of k = (6.4 ( 0.9) ꢀ 10 M
s
directly. No
2
resulting second-order rate constant was calculated as k = 1.1 ꢀ
further intermediates were detected between 250 and 450 nm
over the time scales investigated (up to 9 ms).
2
1
1
-1 -1
1
0 M
s
when only the NBG concentration was considered.
-
3.3.4. CANBr. For the reaction of CANBr with e-aq, single
exponentialfittingof the decaytracesmonitored at 620 nmyielded
However, it is not possible for e to react with such a high rate
aq
constant, suggesting that this rate constant is a composite value
-
6
-1
5 -1
for the reaction of e
with NBG as well as excess glutarimide
reproducible kobs values of 1.1 ꢀ 10 s and 5.7 ꢀ 10 s at
aq
3
75
dx.doi.org/10.1021/tx100325z |Chem. Res. Toxicol. 2011, 24, 371–382

Chemical Research in Toxicology
ARTICLE
Figure 3. Transient spectra obtained following the reduction of N-
bromo glycine anhydride by hydrated electrons in the presence (dashed
lines, open symbols) or absence (solid lines, solid symbols) of added
NaBr (1 mM). The spectra represent those measured immediately after
the electron pulse (9,0), after 25 μs (2,Δ), and 1.5 ms (b,O).
8
7 μM and 45 μM, respectively. These values yield a second-order
-
10
rate constant for the reaction of CANBr with e of ca. 1.2 ꢀ 10
aq
-1 -1
M
s .
.4. Formation of Bromine Atoms on the Reduction of N-
3
Brominated Species. It has been shown experimentally (at pH
3
0
31
9
.3) and predicted theoretically that reduction of N-bromo-
-
•-
succinimide by e or O2 results in heterolytic cleavage of the
aq
•
N-Br bond to give imidyl anions and free bromine atoms (Br ).
Spectra consistent with the formation of low levels of the Br2•
-
Figure 4. Kinetic traces (360 nm) obtained following the reaction of N-
•
-
bromosuccinimide (100 μM) with O
2
2
(generated in an O -saturated
radical species were also detected during the reduction of NBG
-
solution containing 0.2 M sodium formate and 5 mM phosphate buffer
(pH 7.2)) in the absence (9) and presence (b) of 1 mM NaBr. Panel a
shows the growth of the signal attributed to Br
showing the subsequent decay of this signal over 5 ms.
by e in the current studies. In order to confirm the formation
aq
•
of Br upon one-electron reduction of the N-brominated species
•
-
2
over 200 μs, with b
in Scheme 1, selected reactions were repeated in the presence of
-
•-
excess Br with the potential generation of Br monitored at
2
3
0
•
3
60 nm.
3
3.4.2. N-Bromoglutarimide. The formation of Br following
one-electron reduction of NBG (66 μM) by e aq was probed (at
360 nm) in the presence or absence of added 1 mM NaBr. In the
presence of added NaBr, the signal was approximately 3-fold
30
-
.4.1. N-Bromosuccinimide. The previous studies on the
•
-
•
reduction of NBS by O2 to form Br were repeated at pH 7.2
to mimic biological pH values, using the standard formate/
phosphate solutions supplemented with 1 mM sodium bromide
4
-1
greater in intensity, with a rate of growth (kobs ∼ 6 ꢀ 10 s
)
(
NaBr). NBS (100 μM) was added to the above solution
very similar to that in the absence of NaBr, providing good
•-
•
immediately prior to use to minimize thermal reactions between
NBS and Br , and the formation of Br was monitored at 360
nm. In the absence of added Br , a small exponential growth was
evidence for the formation of Br
2
and, by inference, Br . This
-
•-
signal subsequently decayed over a period of ca. 3 ms with
unresolved mixed-order kinetics.
2
-
4
-1
observed over 200 μs (k, 1.3 ꢀ 10 s ), probably due to the
3.4.3. Other N-Brominated Species. The reactions of
-
Br (48 μM) and CANBr (87 μM) with e-aq were
presence of low concentrations of Br in NBS. Addition of 1 mM
(Gly)
2
NaBr enhanced the signal intensity ca. 10-fold, with the growth
examined in a similar manner; in both cases, there was no
significant signal enhancement at 360 nm. In agreement with
this observation, the transient spectra (250-400 nm) obtained
4
-1
phase having k, 2 ꢀ 10 s (Figure 4a). These signals decayed to
baseline over 5 ms (Figure 4b) with an observed first-order rate
3
-1
constant of ca. 10 s ; these decay rates are of the same order as
following the reduction of (Gly) Br in the presence of 1 mM
2
•
-
those obtained for Br2 produced in an N O-saturated solution
(Figure 3, solid lines and symbols) or 10 mM NaBr demon-
strated only a minor increase in absorbance at 360 nm compared
2
containing 5 mM phosphate buffer (pH 7.2) and 1 mM NaBr
•
(
data not shown).
The above experiments were repeated in BuOH/phosphate
solutions to investigate the reactions of NBS (100 μM) with
with the absence of NaBr. These data suggest that Br is not
t
generated on reduction of (Gly) Br or CANBr.
2
3.5. Theoretical Investigations. High-level quantum chem-
istry computations have been undertaken to help rationalize the
experimental observations. Initially, the gas-phase electron affi-
nities (EA) were calculated for a selection of species relevant to
the one-electron reduction of N-halogenated species. The EA is
defined as the energy liberated when an electron is added to a
neutral species (i.e., the negative of the energy change for the
reaction shown in eq 10). A positive EA thus corresponds to a
-
e
aq. The traces obtained at 360 nm in the presence of 1 mM
NaBr displayed an enhancement in signal intensity (as seen with
•
-
-
O2 ), confirming that e -mediated reduction of NBS also
aq
•
results in a signal consistent with the formation of Br . The first-
order rate constant for the growth of Br2 under these condi-
•
-
•-
tions was considerably faster than that with the O
system
(7 ( 1) ꢀ 10 s ), while the subsequent decay could not be
fitted accurately with a single exponential function.
2
4
-1
(
•
situation in which the electron is bound to the neutral species X .
3
76
dx.doi.org/10.1021/tx100325z |Chem. Res. Toxicol. 2011, 24, 371–382

Chemical Research in Toxicology
ARTICLE
the EA of CH NH actually being lower (by 30.7 kJ mol-1) than
•
Table 2. Calculated Gas-Phase Electron Affinities (EA),
3
•
Radical Stabilization Energies (RSE), and Anion Stabilization
that of the parent compound NH . However, substitution of one
2
-
1
of the hydrogen atoms of NH2• with the electron-withdrawing
Energies (ASE) (G4, 298 K, kJ mol ), Aqueous Free
solv
Energies of Solvation (ΔΔG , HF/6-31þG(d), kJ mol-1),
CF substituent leads to an increase in the EA of the resulting
3
o
a
•
-1
•
and Reduction Potentials (E , V)
radical (CF NH ) by 189.1 kJ mol compared with NH . This
3
2
significant increase in EA can be partially attributed to a relative
h
Eo
species
NH •
CH
CF
imidazolyl•
HCONH•
EA
RSE
ASE ΔΔGsolv
-1
destabilizing effect in the radical (RSE = -5.4 kJ mol
,
b
c
consistent with previous investigations e.g., ref 66 and references
2
74.3 (74.4)
43.6 (41.7)
263.4
0.0
0.0
1.5
-360.5
-326.1
0.06
NH•
NH•
therein), but is predominantly due to the large stabilizing effect in
3
32.2
-5.4
47.0
-0.63
-
1
the anion (ASE = 183.7 kJ mol ).
3
183.7 -260.0
225.6 -238.4
0.97
d
Analogously, substitution with a single (electron-with-
252.9 (252.1)
279.8
0.64
•
drawing) formyl substituent, as in the case of HCONH , leads
-26.9 178.6 -278.0
-43.9 265.2 -225.2
1.24
-
1
to a much higher EA (279.8 kJ mol ). This increase can likewise
(
CHO)
2
N•
383.4
1.90
be attributed to the existence of a small destabilizing effect
succinimidyl• 384.8 e
-56.9 253.6 -241.4
e
1.97 (2.22)
1.82 (1.92)
2.34 (2.41)
i
i
i
-1
of -26.9 kJ mol in the radical (consistent with previous
Br•
Cl•
327.5 (324.5)
f
-276.9
66
-1
data ) but a substantial stabilizing effect (of 178.6 kJ mol
)
g
345.7 (348.7)
-309.3
in the anion. A second formyl substituent, as exists in the
a
b
•
Experimental values are in parentheses. Experimental value from ref
1. Experimental value from ref 60. Experimental value from ref 63.
The gas-phase electron affinity and radical stabilization energy of the
succinimidyl radical are based on the cyclic ( A ) structure rather than
(CHO) N radical, leads to further destabilization of the radical
2
c
d
-
1
6
(
(
RSE = -43.9 kJ mol ) and further stabilization of the anion
e
-1
ASE = 265.2 kJ mol ), although the effect of the second
2
0
substituent is smaller than that of the first. The combined effect
the lower energy f acyclic carbon-centered β-(isothiocyanatocar-
g
of destabilization of the radical and substantial stabilization of the
bonyl)ethyl radical. Experimental value from ref 64. Experimental
•
h
-
anion leads to the high EA of the (CHO) N radical (383.4
2
value from ref 65. Defined as the difference between ΔGsolv (X ) and
-
1
•
i
kJ mol ). Similar contributions lead to the high EA of the
ΔGsolv (X ). Experimental values from ref 55.
-
1
succinimidyl radical (384.8 kJ mol ).
The gas-phase EAs (Table 2) of both Cl (345.7 kJ mol-1) and
•
The systems investigated provide representative examples of the
species investigated experimentally. Thus, the EAs have been
calculated (Table 2) for N-centered radicals derived from the
halogenated derivatives of two amines (CH NH and CF NH ),
an amide (HCONH ), two imides (succinimidyl radical and
CHO) N ), and chlorine and bromine atoms. Good agreement
was observed between the calculated (G4) and available
gas-phase EAs (Table 2), with the differences
between theory and experiment all less than 5 kJ mol
•
-1
•
Br (327.5 kJ mol ) are lower than those of the (CHO) N
2
-
1
radical (383.4 kJ mol ) and the succinimidyl radicals (384.8 kJ
•
•
-
1
3
3
mol ). This indicates that in the gas-phase, one-electron
•
reduction of both the N-chloro and N-bromo derivatives of these
•
•
•
(
2
imides should give rise to Cl /Br and the imidyl anions rather
-
-
than Cl /Br and the imidyl radicals.
6
0-65
experimental
In order to allow a more meaningful comparison of the
calculated results with experiments in aqueous solution, the
influence of aqueous solvation on the thermodynamics of one-
electron reduction was investigated. The calculations for
-
1
.
X þ e f X-
•
-
ð10Þ
-
•
ΔΔGsolv = ΔGsolv(X ) - ΔG (X ) (Table 2) yield large
solv
In order to facilitate analysis of the calculated EAs for the
various N-centered radicals (Table 2), it is convenient to define
the radical stabilization energy (RSE) of these species as the
energy change for the formal reaction presented in eq 11. The
RSE measures the effect of the substituent X on the stability of
the radical XNH relative to its effect in the closed-shell molecule
XNH . A positive value indicates relative stabilization of the
negative values, reflecting the considerably greater solvation of
anions compared with that of neutral radicals. The calculated
o
reduction potentials (E ) are given in Table 2.
The solvation energies are very sensitive to the model emo-
ployed for the small N-centered anions, and the calculated E
values consequently have considerably greater associated uncer-
•
tainty than the underlying gas-phase EAs. As a result, the
2
agreement between calculated and experimental5
5,67
reduction
radical. Similarly, the anion stabilization energy (ASE) is defined
as the energy change for the formal reaction shown in eq 12. The
ASE measures the effect of the substituent X on the stability of
potentials is typically poorer. The solvation energies vary quite
widely for the different species, dependent in part on mole--
cular size. For example, the solvation energy for the smaller Cl
the anion XNH- relative to its effect in the neutral XNH . A
2
-
1
positive value indicates relative stabilization of the anion. Finally•,
(-309.3 kJ mol ) is considerably larger than that for the bulkier
-1
the effect of the substituent X on the EA of the radical XNH
succinimidyl anion (-241.4 kJ mol ).
•
•
•
[
ΔEA(XNH ) = EA(XNH ) - EA(NH )] is related simply to
the RSE((XNH ) and ASE(XNH ) values (eq 13).
As a result of the differential solvation effects, in water the
2
•
-
succinimidyl radical no longer gives rise to the most favorable
o
one-electron reduction. The calculated E (1.97 V) is less than
•
•
XNH þ NH3 f XNH2 þ NH2
ð11Þ
ð12Þ
ð13Þ
•
o
that for Cl (E = 2.34 V), which therefore undergoes preferential
-
one-electron reduction in solution. Br also has a greater
-
-
XNH þ NH3 f XNH2 þ NH2
solvation energy than the succinimidyl anion, but in this case,
it is not sufficient to override the gas-phase ranking of EAs, with
o
•
-
•
the one-electron reduction (E = 1.82 V) slightly less favorable
ΔEAðXNH Þ ¼ ASEðXNH Þ - RSEðXNH Þ
o
than that for the succinimidyl radical (E = 1.97 V). The
o
•
These calculations show that a methyl substituent has a
calculated E for the acyclic (CHO) N radical is also greater
2
•
•
considerably greater stabilizing effect in CH NH (RSE = 32.2
than that of Br (1.90 V vs 1.82 V) indicating that the imidyl
radical, and not Br , would undergo preferential reduction.
3
-
1
-
-1
•
kJ mol ) than in CH NH (ASE = 1.5 kJ mol ). This leads to
3
3
77
dx.doi.org/10.1021/tx100325z |Chem. Res. Toxicol. 2011, 24, 371–382

Chemical Research in Toxicology
ARTICLE
Once a theoretical basis for the experimental observations was
established, it was investigated whether a correlation exists
between the rates of one-electron reduction of N-halogenated
species and the thermodynamics of their reduction. Using the
reduction of MeNHCl and MeNHBr as probes (eq 14), (gas-
phase) enthalpies were calculated for these processes. These
calculations suggest that the reduction of MeNHCl is associated
consistent with the current results where no intermediates (other
than Br2• ) were observed for NBS or NBG. •
-
In the current studies, the formation of Br on reduction of
NBS or NBG by O2• or e
-
-
was demonstrated by the
aq
enhancement of a transient signal at 360 nm in the presence of
-
•-
1 mM Br , consistent with the formation of Br . The detection
2
of low yields of Br2• in the absence of added Br is believed to
be due to low levels of Br /Br in the commercial NBS, and the
-
-
-
1
-
with a reduced exothermicity (99.5 kJ mol ) compared with
2
-
1
-
that of MeNHBr (122.1 kJ mol ). This ordering parallels the
faster observed reduction rates for the N-bromo species and
suggests that the relative strengths of N-X bonds is an important
factor governing the reduction rates.
preparation of NBG from Br -containing HOBr. For both NBS
and NBG, the Br2• signal displayed a small rapid rise, followed
-
4
5 -1
by a growth phase with first-order rate constants of 10 -10 s
Lind et al. utilized the observed rate of formation of Br
derive the second-order rate constant for the reaction of O
.
•
-
to
2
•
-
-
•
-
2
MeNHX þ e f MeNH þ X
ð14Þ
8
-1 -1
with NBS (k , 5 ꢀ 10 M
s ), with the assumption that the
2
rate-determining step in this reaction was the reduction of NBS
-
30
at Br concentrations >1 mM. With the current data, this
4. DISCUSSION
approach yields an approximate second-order rate constant of 2
8
-1 -1
•-
.1. Absolute Rate Constants for e-aq-Mediated Reduc-
ꢀ
10 M
s
for the reaction of NBS with O consistent with
2
4
30
tion of N-Halogenated Species. It is well established that one-
electron reduction of N-halogenated species results in the
heterolytic cleavage of the N-X bond to yield an anion and a
radical species. Rate constants for the reduction of N-chlorinated
previous reports. This value is considerably larger than the
lower limit derived above from the consumption of O
•
-
at 260
2
nm, and provides a more robust estimate of this rate constant
given that it is mathematically impossible to distinguish, from a
single data set, which of two consecutive reactions are faster. A
-
9
-1
species by e
are close to diffusion-controlled (k g 10 M
aq
37,55-58,68
2
-
1
-
s
).
The fastest reactions are for the N-chloroimides,
similar approach can be employed to analyze the e aq-mediated
with rate constants (k ) for N-chloroglutarimide and N-chlor-
reduction of NBS and NBG, yielding second-order rate constants
2
1
0
-1 -1
10
-1 -1
8
8
-1 -1
osuccinmide of 1.9 ꢀ 10
M
s
and 1.6 ꢀ 10
M
M
s
,
of 6.7 ꢀ 10 and 9 ꢀ 10 M
s
respectively, which are
5
5,68
10
-1
-1)
10 -1 -1
respectively.
Similar high values (ca. 1.5 ꢀ 10
s
markedly lower than those obtained (>10
and this work) from direct quenching of e aq (>600 nm). It is
possible that this discrepancy arises from NBS and NBG
M
s
from ref 30
-
have been reported for the N-chloroamides of the cyclic dipep-
3
7
•-
•-
tides of (Gly) and (Ala) . Reduction of monochloramine
2
2
-
(
1
e
NH Cl) by e
occurs with a similar rate constant (k , 2.2 ꢀ
having lifetimes that are measurable on the time scale of pulse
radiolysis, similar to that reported by Lind et al. for the reduction
2
aq
2
1
0
-1
s-1
56,57
0
M
), while the rate constants for the
-
•-
-mediated reduction of N-chlorinated primary amines
of N-bromophthalimide (PBr) to form PBr (λmax, 325 nm)
aq
9
-1 -1
5
-1
• 30
(
RNHCl) vary from 6.1-9.3 ꢀ 10 M
s
(e.g., (k (CA-
which decomposes relatively slowly (k < 10 s ) to form Br .
2
-
9
-1 -1 37
•-
NCl þ e ), 9.3 ꢀ 10 M
s
). The N-chlorinated species
However, the reported lifetime of NBS is too short to measure
on the pulse radiolysis time scale. Thus, it is more likely that the
aq
-
30
with the lowest measured rate constant with e
is (N-chloro,
aq
-1 -1 58
9
•-
N-phenyl)glycine with k = 2.9 ꢀ 10 M
s
;
this lower
relatively slow rates of growth of Br
2
arise from chain reactions
2
•
reactivity is presumably due to perturbation of the electron
density at the nitrogen center by the phenyl ring.
that release Br as one of the radical chain carriers.
4
.3. Initiation of Chain Reactions Following the Reduction
of N-Brominated Imides. Complex chain reactions have been
identified in synthetic bromination reactions mediated by NBS,
though the identities of the chain carriers has been a source of
In contrast to these N-chlorinated species, few data are
-
available for N-brominated compounds with e aq, with the only
reported rate constant that we are aware of being for N-
6
9,70
1
0
-1 -1 30
debate.
Pulse radiolysis studies on one-electron reduction of
have demonstrated that multiple radicals can be
bromosuccinimide with k = 2.9 ꢀ 10
M
s
.
This rate
2
NBS3
0,55,68,71
constant is 1.8 times faster than the corresponding reaction for
NCS. Thus, the approximate rate constants (>1010
M
-1
s )
-1
involved, including competing chains involving succinimidyl
•
• 30,71
•-
radicals (S ) and Br .
The current data for O2 -mediated
determined here for N-bromoglutarimide, (Gly) Br, and CANBr
2
reduction of NBS and NBG are consistent with such processes, as
the extent of NBS/NBG loss (estimated from the bleaching of
the signal at 260 or 280 nm) is ca. 10 to 20 times the amount of
compare favorably with those expected on the basis of the
corresponding N-chloro data. Typically, a small increase in rate
constant is observed for the N-bromo compared with the N-
chloro species; this may, in part, be attributed to the relative
weakness of N-Br compared with N-Cl bonds (see above).
•-
O2 generated by the electron pulse. The occurrence of chain
-
reactions in the e aq-mediated reduction of NBS and NBG is not
apparent in the current studies, as the reaction was not mon-
4
.2. Species Generated by Reduction of N-Halogenated
Imides. In all cases, reduction of the N-chlorinated species has
been attributed to the cleavage of the N-Cl bond to yield the
itored below 300 nm where NBS/NBG absorb. However, the
•-
slow production of the Br
signal (360 nm; reaction 8)
2
-
-
corresponding N-centered radical and Cl . This process is
compared with the consumption of e aq (620 nm) is consistent
•
believed to occur via the formation of a radical anion intermedi-
with secondary Br formation (eqs 15 and 16) during chain
•- 55
71
ate. This species has been observed in the case of NCS , but
reactions, as described previously.
3
7,56-58
not with other N-chlorinated species.
The rate constant
•
-
•
•
for the decomposition of NCS to the succinimidyl radical and
CH ðCH Þ COH þ Br f BrCH ðCH Þ COH þ Br
2
3 2
2
2
3 2
5
-1 55
chloride has previously been determined as 8 ꢀ 10 s , similar
5
-1
•-
ð15Þ
to that found here (∼ 7 ꢀ 10 s ). In contrast to NCS , which
55
•-
has a moderate lifetime at room temperature, NBS has only
•
-
-
•
30
S þ Br f S þ Br
ð16Þ
been detected at 77 K by EPR spectroscopy. This observation is
3
78
dx.doi.org/10.1021/tx100325z |Chem. Res. Toxicol. 2011, 24, 371–382

Chemical Research in Toxicology
ARTICLE
•
Scheme 2. Proposed Reaction Mechanism for the Reduction
the production of Br , which can initiate radical chain reactions of
of N-Bromo Glycine Anhydride ((Gly) Br) by Hydrated
moderate length (ca. 10-20). These observations are consistent
2
Electrons (e-aq), with Initial Formation of an Amidyl Radical
and a Subsequent Rapid 1,2-Shift to Yield an R-Carbon-
Centered Radical
with E values obtained from high-level quantum chemical
o
computations. Furthermore, preliminary calculations (O’Reilly
et al., unpublished data) on more biologically relevant imide-
containing substrates indicate, for example, that one-electron
•
reduction of N-bromouracil would be expected to generate Br
and the nitrogen-centered uracil anion, whereas the reverse is
true for N-chlorouracil.
These reactions of N-halogenated imides may be of biological
importance as it is well established, for example, that the levels of
5
-bromo- and 5-chloro-uracil are markedly elevated in inflam-
7
3,74
matory tissue and atherosclerotic lesions in humans.
In
4
.4. Radicals Generated by the Reduction of the N-Bro-
addition, levels of parent uracil are massively elevated in human
mamide, (Gly) Br. In contrast to the complex behavior ob-
inflammatory tissue (up to 600 μM, ca. 1000-fold higher than
plasma concentrations ). The mechanism of formation of the
2
-
73
served with the N-bromo imides, e aq reduction of (Gly) Br
2
resulted in a transient with absorption maxima at 260 and 360 nm
modified bases is unclear, but may well involve N-chlorinated or
N-brominated intermediates (reviewed in ref 4). In addition to
uracil, these imide moieties are present in thymine and purine
metabolites such as xanthine and urate (plasma concentrations
(
Figure 3). A similar transient has been reported for the reaction
•
59
of HO with (Gly) , but control experiments with (Gly) alone
2
2
confirmed that this reaction is not the source of this intermediate.
75
The observed absorption is different from that arising from the
are ∼5 μM and 200-300 μM, respectively ), all of which react
-
11
reaction of (Gly) with e aq, with this yielding a spectrum with a
readily with HOCl, and should react readily with HOBr
2
single absorption maximum that rises into the UV region
(reviewed in ref 4). These observations suggest that the in vivo
formation of N-brominated and N-chlorinated imide species is
likely to occur at sites of inflammation, particularly within the
phagosomes of activated white cells that phagocytose foreign
5
9
(
λmax < 240 nm). Thus, it is proposed that the reaction of
-
(
Gly) Br with e
cleaves the N-Br bond to form an N-
2
aq
- •
centered radical and Br (rather than Br ). The resulting N-
centered radical undergoes a rapid 1,2-shift to yield the same
carbon-centered radical generated on reaction of HO with
organisms and apoptotic cells.
•
•-
It is known that O
and H O are also generated in
2
2 2
(
Gly) (Scheme 2). This mechanism is well established for N-
phagosomes by NOX enzymes as part of the inflammatory
2
16,24
1
•-
chlorinated N-acetyl amino acids,
and the corresponding
response. While concentrations of O
2
are typically considered
radical has been detected by EPR spin-trapping studies of
to be quite low (submicromolar), modeling studies suggest that
2
9
•-
(
Gly) Br.
in the small volume of the phagosome, O
2
is generated at a flux
2
-
1
4
.5. Theoretical Investigations. The calculated reduction
of 5.2 mM s and reaches a steady state concentration of 25
μM.76 Thus, while the current kinetic studies cannot provide
potentials in aqueous solution (Table 2) are consistent with the
experimental findings. In the first place, the present calculations
confirm that one-electron reduction of N-chloro- or N-bromo-
amides or -amines should give rise to N-centered radicals plus
•-
definitive rate constants for the interaction of O2 with N-
halogenated imides due to the initiation of chain reactions, these
reactions are clearly favorable with relatively fast rate constants
-
-
•
•
6
-1 -1
Cl /Br rather than N-centered anions plus Cl /Br because the
halides are better able than the amido or amino moieties to
accommodate the negative charge. In contrast, one-electron
(>10 M
s ). These data suggest that the production of
activated N-halogenated species in the presence of elevated
•-
phagosomal O
concentrations might induce tissue damage
2
•
reduction of N-bromosuccinimide should yield Br plus succini-
in vivo through the formation of destructive organic radicals and
•
midyl anion, but this preference is small. Finally, the one-
Br , especially as these species can elicit efficient chain reactions.
electron reduction of the N-Cl bond of N-chlorosuccinimide
-
is predicted to yield the succinimidyl radical and Cl preferen-
tially because of the larger E for Cl than for the succinimidyl
radical.
o
•
’
ASSOCIATED CONTENT
S
Supporting Information. Gaussian archive entries for
b
Comparison of the calculated reduction potentials for the
B3-LYP/6-31G(2df,p) optimized geometries for all structures
investigated (Table S1) and G4 electronic energies, vibrational
corrections, free energies of solvation, and G4 free energies
(Table S2). This material is available free of charge via the
Internet at http://pubs.acs.org.
radicals of interest with the experimentally derived value for the
•
-
72
O2(aq)/O2
couple (reevaluated recently as -0.18 V )
•-
(aq)
established that reduction by O2 is thermodynamically favor-
able for all of the radicals investigated, except MeNH and
possibly NH2 . This is an important conclusion as it resolves the
-
-
apparent paradox that only N-halogenated imides are reduced by
•-
’ AUTHOR INFORMATION
O2 on the pulse radiolysis time scale, whereas steady state
•
-
studies show that O
species.
does reduce the other N-halogenated
2
Corresponding Author
*
2
6-28
Tel:þ61-2-8208-8900. Fax: þ61-2-9565-5584. E-mail: pattisond@
4
.6. Conclusions. The current kinetic studies have shown
hri.org.au.
that N-halogenated species readily undergo one-electron reduc-
tion. In the case of the N-chlorinated species, and many of the N-
Funding Sources
-
•-
brominated species, one-electron reduction by e
or O2
This work was supported by grants from AINSE Ltd (Award No.
AINGRA07035), the Australian Research Council (ARC Cen-
tres of Excellence grant CE0561607, and Discovery program
aq
6,24,29,68
results in the production of N-centered radicals.1
How-
ever, in N-bromoimides the favored reduction pathway leads to
3
79
dx.doi.org/10.1021/tx100325z |Chem. Res. Toxicol. 2011, 24, 371–382

Chemical Research in Toxicology
ARTICLE
grants DP0988311 and DP1095821), and the National Heart
Foundation (Grants in Aid: G09S4313 and G08S3769).
absolute rate constants and assessment of biological relevance. Biochem.
J. 422, 111–117.
(11) Prutz, W. A. (1998) Interactions of hypochlorous acid with
pyrimidine nucleotides, and secondary reactions of chlorinated pyrim-
’
ACKNOWLEDGMENT
idines with GSH, NADH, and other substrates. Arch. Biochem. Biophys.
3
49, 183–191.
We thank Drs. Sujata Shinde and Andrej Maros, and Mr.
(12) Rees, M. D., Hawkins, C. L., and Davies, M. J. (2003)
Grant Woodroffe for technical expertise at the Auckland Pulse
Radiolysis Facility, and Drs. Clare Hawkins and Martin Rees for
invaluable discussions. The generous allocations of computing
time from the National Computational Infrastructure (NCI)
National Facility and from Intersect Australia Ltd. are also
gratefully acknowledged.
Hypochlorite-mediated fragmentation of hyaluronan, chondritin sul-
fates, and related N-acetyl glycosamines: Evidence for chloramide
formation, free radical transfer reactions and site-specific fragmentation.
J. Am. Chem. Soc. 125, 13719–13733.
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Hypochlorous acid mediated oxidation of lipid components present in
low-density lipoproteins: absolute rate constants, product analysis and
computational modeling. Chem. Res. Toxicol. 16, 439–449.
’
ABBREVIATIONS
(14) Skaff, O., Pattison, D. I., and Davies, M. J. (2007) Kinetics of
•
t
ASE, anion stabilization energy; Br , bromine atoms; BuOH,
hypobromous acid-mediated oxidation of lipid components and anti-
oxidants. Chem. Res. Toxicol. 20, 1980–1988.
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emerging role in pathology. Pharmacol. Ther. 115, 400–409.
tert-butanol; CANBr, N-bromo-6-amino-caproic acid; CANCl,
•
N-chloro-6-amino-caproic acid; Cl , chlorine atoms; CPCM,
0
conductor-like polarizable continuum model; DTNB, 5,5 -dithi-
-
o
(16) Hawkins, C. L., and Davies, M. J. (1998) Reaction of HOCl
obis-(2-nitrobenzoic acid); e , hydrated electron; E , reduc-
aq
with amino acids and peptides: EPR evidence for rapid rearrangement
and fragmentation reactions of nitrogen-centered radicals. J. Chem. Soc.,
Perkin Trans. 2, 1937–1945.
tion potential; EA, electron affinity; EPO, eosinophil peroxidase;
(
Gly) , cyclic Gly dipeptide (also known as glycine anhydride);
2
G03, Gaussian 03 program; G09, Gaussian 09 program; G4,
(17) Pattison, D. I., Hawkins, C. L., and Davies, M. J. (2007)
Gaussian 4 theory; (Gly) Br, N-bromo glycine anhydride;
2
Hypochlorous acid-mediated protein oxidation: How important are
chloramine transfer reactions and protein tertiary structure?. Biochem-
istry 46, 9853–9864.
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Heinecke, J. W. (1998) Human neutrophils employ the myeloperox-
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family of reactive aldehydes. J. Biol. Chem. 273, 4997–5005.
(
Gly) Cl, N-chloro glycine anhydride; HOBr, the equilibrium
2
-
mixture of hypobromous acid and its anion, OBr , at
physiological pH 7.4; HOCl, the equilibrium mixture of hypo-
-
chlorous acid and its anion, OCl , at physiological pH 7.4; MPO,
myeloperoxidase; NBG, N-bromoglutarimide; NBr-HOP, N-
bromo-4-hydroxy-2-pyrrolidinone; NBS, N-bromosuccinimide;
NCS, N-chlorosuccinimide; NHE, normal hydrogen elect-
rode; RSE, radical stabilization energy; TNB, 5-thio-2-nitroben-
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