Damage of aromatic amino acids by the atmospheric free radical oxidant NO3 in the presence of NO2, N2O4, O3 and O2

Article abstract of DOI:10.1039/c0ob01186j

Analysis of the products formed in the reaction of NO₃ with the N- and C-protected aromatic amino acids 1-5, which was performed under conditions that simulate exposure of biosurfaces to environmental pollutants, revealed insight how this impor

Full text of DOI:10.1039/c0ob01186j

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PAPER
Damage of aromatic amino acids by the atmospheric free radical oxidant
∑
∑
NO3 in the presence of NO2 , N2O4, O3 and O2†
Catrin Goeschen,a Natalia Wibowo,a Jonathan M. Whiteb and Uta Wille*a
Received 16th December 2010, Accepted 7th February 2011
DOI: 10.1039/c0ob01186j
∑
Analysis of the products formed in the reaction of NO3 with the N- and C-protected aromatic amino
acids 1–5, which was performed under conditions that simulate exposure of biosurfaces to
environmental pollutants, revealed insight how this important atmospheric free-radical oxidant can
∑
cause irreversible damage. In general, NO3 induced electron transfer at the aromatic ring is the
exclusive initial pathway in a multi-step sequence, which ultimately leads to nitroaromatic compounds.
∑
In the reaction of NO3 with tryptophan 5 tricyclic products 12 and 13 are formed through an
intramolecular, oxidative cyclization involving the amide moiety. In addition to this, strong indication
for formation of N-nitrosamides was obtained, which likely result from reaction with N2O4 through an
independent non-radical pathway.
∑
The adverse health effects of ambient air pollutants have been
addressed in numerous epidemiological studies.1 In particular, a
link has been proposed between increases in respiratory morbidity
and hospital admissions for asthma and increased concentrations
of atmospheric ozone, O3, and nitrogen oxides, NOx (NOx is
a generic term for the free radical species nitrogen oxide and
NO3 is the most important oxidant in the atmosphere at night
(where it is also formed through the reaction in eqn (1)), and its
reactivity towards most organic compounds is many orders of
∑
magnitude higher than that of either NO2 or O3, respectively.5
∑
Since NO3 can react via oxidative electron transfer (ET; E0
∑
-
(NO3 /NO3 ) = 2.3 V vs. NHE),6 hydrogen abstraction (HAT),
or addition to p-systems, every organic molecule is principally
susceptible to attack by this free radical species.5 In the absence of
organic reaction partners, NO3∑ can recombine with NO2∑ to form
dinitrogen pentoxide, (N2O5) in an equilibrium reaction, which
∑
nitrogen dioxide, NO∑ and NO2 , respectively), but the underlying
∑
mechanism is not clear.2 It has been suggested that O3 and/or NO2
may modulate airway diseases, such as asthma, by increasing the
release of inflammatory mediators from bronchial epithelial cells
and that the cells of asthmatic patients may be more susceptible
to the adverse effects of these pollutants.3 Interestingly, an
investigation of the pulmonary effects of a combined exposure to
O3 and NO2∑ in mice revealed that changes in the lung metabolism
∑
serves as a reservoir for NO3 .
Although the chemistry of NO3 in the atmosphere has been
∑
extensively studied during the past decades,5 only very few
links between this free radical oxidant and oxidative damage in
biomolecules that are exposed to the atmospheric environment
have been made. The most important constituents of the res-
piratory tract surface lining fluid, which covers the respiratory
tract epithelial cells, are, besides antioxidants such as glutathione,
vitamin C and uric acids, metal binding proteins.7 A recent
investigation showed that birch pollen proteins are efficiently
nitrated in polluted air.8 This suggests that protein nitration may
play a central role in the promotion of allergies by air pollutants
∑
were synergistic.4 This might suggest that not NO2 and O3 as
individual species are the actual culprit behind these adverse health
∑
effects, but instead highly reactive nitrate radicals, NO3 , which are
∑
formed through the reaction of NO2 with O3 (eqn (1)):
∑
∑
(1)
NO2 + O3 → NO3 + O2
aARC Centre of Excellence for Free Radical Chemistry and Biotechnology,
School of Chemistry and BIO21 Molecular Science and Biotechnology
Institute, The University of Melbourne, 30 Flemington Road, Parkville, VIC
3010, Australia. E-mail: uwille@unimelb.edu.au; Fax: +61 3 9347 8189;
Tel: +61 3 8344 2425
∑
and a direct involvement of NO3 in this process, especially since
its reaction with water, which is the major constitutent of the
respiratory tract lining fluid, is slow.5 In a recent product study,
we have shown for the first time that aromatic amino acids are
bSchool of Chemistry and BIO21 Molecular Science and Biotechnology
Institute, The University of Melbourne, 30 Flemington Road, Parkville, VIC
3010, Australia
† Electronic supplementary information (ESI) available: Experimental
conditions and spectroscopic data for all reaction products, X-ray data
for 12a. CCDC reference number 810818. For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/c0ob01186j
∑
irreversibly oxidatively damaged by reaction with NO3 , which was
generated in situ by irradiation of ceric(IV) ammonium nitrate
(CAN) at l = 350 nm.9 Although this is in principle a very
∑
convenient methodology to produce NO3 on preparative scale in
solution, because of the considerable oxidation power of CAN
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itself,‡ its thermal reaction with some more easily oxidizable
amino acids can complicate analysis of the products formed in the
yields could not be determined for any of the reactions under
investigation. However, since this study is aimed at obtaining
insight into the nature of the products in order to assess the
potential impact of such chemical modifications for biological
systems, exact yields are only of minor importance.
∑
reaction with NO3 . In addition, under environmental conditions
∑
NO3 is not an isolated reactant, but is always accompanied by
other radicals and non-radical oxidants, such as NO2∑ (which is in
equilibrium with its dimer dinitrogen tetroxide, N2O4), O3, and O2,
respectively, whichprincipallycould alltrap reactive intermediates.
Thus, in order to explore the role of such additional reactants on
∑
(a) The reaction of NO2 /N2O4 with amino acids
∑
Analysis of the reactions of all amino acids with NO2 /N2O4 in
∑
the products formed during the NO3 induced oxidative damage of
both presence and absence of O3 revealed formation of small
to considerable amounts of new products, which underwent
fragmentation upon ionization (ESI-MS) to reveal a molecular
mass that was apparently identical with the respective parent
amino acid, but which showed longer retention times on the HPLC
column. Assignment of these compounds will be exemplified for
aromatic amino acids, we have performed detailed product studies
on the reaction of N- and C-protected§ L-phenylalanine (1a), L-
∑
tyrosines (2a–4a) and L-tryptophan (5) with NO3 (Fig. 1), which
was in situ generated through the reaction shown in eqn (1). These
experiments could be considered as model to simulate exposure
of proteins lining the respiratory tract surface to the atmospheric
environment.
∑
the reaction of phenylalanine 1a with NO2 , where isolation by
HPLC gave a yellowish material, which we propose is N-nitroso
phenylalanine 1b (Scheme 1). 1H NMR analysis of this compound
revealed characteristic differences compared to the spectrum of
1a, such as the absence of the amide proton signal, and significant
downfield shifts of the proton at the a-carbon from d = 4.88 ppm
(in 1a) to d = 5.55 ppm in 1b, of one of the benzylic protons
from d = 3.14 ppm (in 1a) to d = 3.42 ppm in 1b, and of the N-
acetyl protons from d = 1.98 ppm (in 1a) to d = 2.60 ppm in 1b.
The magnitude of the observed downfield shifts are in excellent
1
agreement with H NMR data of N-nitrosated peptide bonds in
literature.11 In addition to this, the IR spectrum of 1b showed
the absence of the N–H stretching vibration and a significant
shift of the carbonyl stretching vibration to higher wavenumbers,
which is in accordance with an electron-withdrawing substituent
at the amide nitrogen (see ESI†). We believe that 1b is formed by
reaction of 1a with N2O4, which has been shown to act as effective
non-radical nitrosating agent for amides.ꢀ The mechanism of this
reaction has been explored in detail and will not be discussed
here.12 N-Nitrosamide 1b is expected to undergo cleavage of
the N–N bond in the mass spectrometer, and the lability of
1b was demonstrated by the observation that decomposition to
the parent amino acid 1a occurred upon standing in solution at
room temperature. The finding that the atmospheric pollutant
Fig. 1
Results and Discussion
The reactions were performed at +10 ◦C under exclusion of
moisture in dichloromethane by adding a measured excess amount
of liquid NO2∑ to the amino acid, with a stream of O3 in O2 passing
through the solution.10 Consumption of the amino acids was
usually complete after 20 min of reaction time.¶ After work-up the
reaction products were isolated and purified by preparative HPLC,
followed by NMR and ESI-MS analysis. Unless stated otherwise,
product ratios were obtained from the 1H NMR spectrum of the
reaction mixture through integration of the acetyl proton signals,
which showed sufficiently different chemical shifts for the starting
amino acids and the reaction products. Detailed spectroscopical
data are given in the ESI.† In addition, control reactions were also
performed where the amino acids were separately treated with
∑
system NO2 /O3 leads to rapid formation of N-nitrosoamides is
significant, since these species are the most broadly acting and
among the most potent carcinerogens.13 It should be noted that,
∑
either NO2 /N2O4 or an O2/O3 mixture, respectively. It should be
noted that, due to the required repeated purifications by HPLC,
‡ E0 (Ce4+/Ce3+) = 1.61 V vs. NHE [V. Nair and A. Deepthi, Chem. Rev.,
2007, 107, 1862].
§ Protection of the N- and C-terminus in the amino acids was performed
to simulate the peptide bond in the protein.
Scheme 1 Suggested N-nitrosation of phenylalanine 1a.
¶ Typical experimental procedure: To a stirred mixture of the amino acid
(1.00 mol) in anhydrous dichloromethane (15 mL) at 10 ◦C was added
liquid NO2 (0.5 mL, 15 mmol), and ozonized O2 was bubbled through
∑
ꢀ It cannot be excluded that this new product is derived from N-
∑
the mixture at a low flow rate. After 20 min, the reaction was quenched
by addition of aq. NaHCO3 (10 mL), the phases were separated and
the aqueous phase was extracted with dichloromethane. The combined
organic fractions were dried over MgSO4, concentrated and the reaction
products isolated and purified by repeated preparative HPLC (see also
nitration through reaction of the amide moiety with NO2 /N2O4 [T. Kaiya,
K. Nakamura, M. Tanaka, N. Miyaka and K. Kohda, Chem. Pharm. Bull.,
2004, 52, 570; B. C. Challis, D. E. C. Shuker, D. H. Fine, E. U. Goff and
G. A. Hoffman, Acta Cient. Compostel. 1982, 19, 153]. Compound 10b
was the only case where we were able to obtain high resolution ESI-
MS data, which support formation of an N-nitroso amide. Unfortunately,
all N-nitroso amides obtained in this work were too unstable to allow
identification through chemical derivatization.
∑
ESI†). It is not possible to state the exact [NO2 ] in our experiments, since
∑
an undeterminable amount of NO2 evaporated prior to its reaction with
O3.
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∑
in contrast to other studies,14 a reaction of NO2 and/or N2O4
with the aromatic residue in the amino acids 1–5 was not observed
under our experimental conditions.
∑
(b) The reaction of NO3 with phenylalanine 1a
∑
The reaction of NO3 with phenylalanine 1a led to formation of
the isomeric ring-nitrated products 6a (Scheme 2). NMR and GC
analysis revealed that the para and ortho substituted isomers were
the major products, but very minor amounts of the meta nitrated
product were also obtained. In addition to this, N-nitrosation was
also found to occur, leading to both ortho- and para-6b, whereas
meta-6b, if formed, was below the detection limit of the HPLC. The
1
∑
N-nitrosamides 6b were assigned on the basis of their H NMR
Scheme 3 Proposed mechanism for the reaction of NO3 with phenylala-
data, which showed similar characteristic downfield shifts for the
protons adjacent to the amide moiety as observed for 1b. No
reaction occurred between 1a and an O2/O3 mixture in the control
experiment.
nine 1a.
radical cation 7 is stabilized through loss of a benzylic proton,
thus leading to the benzyl radical intermediate 9 with a restored
aromatic ring, which undergoes further reactions (not shown).††
From these findings, we conclude that the radical cation 7 must
have a certain lifetime that enables trapping by suitable reagents,
∑
for example NO2 , prior to benzylic deprotonation. However, a
reaction of 7 (or any other radical intermediates) with either O2 or
the solvent, which were present in excess in our experiments, was
not observed.
Interestingly, under biochemical conditions the aromatic ring in
phenylalanine is practically inert to oxidation. Our finding that this
amino acid is readily damaged by the environmental free radical
∑
Scheme 2 Products of the reaction of NO3 with phenylalanine 1a.
∑
oxidant NO3 has a potentially considerable biological impact for
This reaction outcome strongly contrasts the findings from our
previous experiments, where NO3 was generated in the absence
of NO2 by photolysing CAN.9 Under the latter conditions, the
reaction involving 1a only resulted in products with an oxidized
benzylic position, whereas the aromatic ring remained apparently
intact. We will restrict the mechanistic discussions to reactions
∑
proteins. Thus, since the radical cation intermediate 7 is in turn
also a strong oxidant, it might trigger an electron transfer cascade
within the protein that could potentially lead to oxidative damage
at remote positions.15
∑
∑
∑
(c) The reaction of NO3 with tyrosines 2a–4a
involving NO3 , since the observed N-nitrosation likely results
from an independent non-radical side reaction of the amide moiety
with N2O4, which could principally occur before and/or after the
Tyrosine 2a, which is very easily oxidized under biological
conditions, rapidly reacts with NO3 to give a mixture of 3-
∑
∑
NO3 reaction with the amino acid.
The different reaction pathways in dependence of the NO3
nitrotyrosine 4a, 3,5-dinitrotyrosine 10a and their respective N-
nitroso compounds 4b and 10b with a ratio of 4a : 4b : 10a : 10b =
16 : 1.4 : 1 : 1.8 (Scheme 4). Through an independent experiment,
∑
source can be rationalized by the mechanism proposed in
∑
Scheme 3, where NO3 induced oxidation (oxidative ET) of the
∑
∑
where 4a was reacted with NO3 in the presence of NO2 and O3/O2,
it was established that the bis-nitrated tyrosine 10a results from
aromatic ring leads to the radical cation 7,9 which is trapped by
∑
NO2 to give the s-complex 8. Rearomatization through deproto-
∑
NO3 induced oxidation of 3-nitrotyrosine 4a. From a synthetic
nation yields nitrophenylalanine 6a.** A mechanism proceeding
through an intermediate of type 8 is supported by the observed
strong preference for ortho- and para-substituted products, which
is in line with the selectivity of electrophilic aromatic substitutions
involving alkylaromatic compounds and which proceed via the
point of view, this is an interesting finding, since nitration of such
strongly deactivated aromatic rings requires usually much harsher
conditions.16
Formation of the nitroaromatic ring in both 4 and 10 is
likely to proceed through an oxidative ET/radical recombina-
tion/deprotonation mechanism in analogy to that shown in
Scheme 3. Trapping of any radical intermediates by O2 was not
observed. Similarly, reaction of the O-acetylated tyrosine 3a with
∑
same intermediate.10 In contrast to this, in the absence of NO2
∑
(for example, when NO3 is generated by CAN photolysis) the
∑
∑
** It should be noted that the NO2 /N2O4 equilibrium constant favours the
NO3 leads to the 3-nitrotyrosine derivative 11a, in addition to
dimer in solution [T. F. Redmond and B. B. Wayland, J. Phys. Chem., 1968,
72, 1626], and N2O4 can be oxidized with O3 to give N2O5. Kinetic studies
in the gas-phase revealed that the latter reacts with unsaturated compounds
by several orders of magnitude slower than NO3∑ [C. Pfrang, R. S. Martin,
C. E. Canosa-Mas and R. P. Wayne, Phys. Chem. Chem. Phys., 2006,
8, 354], and N2O5 does not nitrate deactivated aromatic compounds in
solution (see ref. 13). The inert and nonpolar solvent dichloromethane
was used in these experiments to avoid ionic dissociation of any N2O5
present in the system.
the corresponding N-nitroso product 11b (11a : 11b = 1 : 1.35).
In contrast to the reaction of tyrosine 2a, formation of an O-
acetylated 3,5-dinitrotyrosine derivative was not observed in our
experiments. It is further important to note that none of the
†† We have shown that formation of 9 does not occur in a direct step
∑
through benzylic hydrogen abstraction in 1a by NO3 (see ref. 9).
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∑
Scheme 4 Products of the reaction of NO3 with tyrosines 2a–4a.
tyrosines 2a–4a underwent a reaction with the O3/O2 mixture
∑
in the absence of NO2 .
∑
Scheme 5 Products of the reaction of tryptophan 5 with NO3 and with
O3.
Nitration of the aromatic ring caused by environmental free
radicals might have serious consequences, in particular, if it
occurs on tyrosine residues in proteins. Since the pKa, which is
10 in tyrosine, decreases to about 7.5 in 3-nitrotyrosine, under
physiological conditions 3-nitrotyrosine exists in its anionic form.
Formation of a negatively charged hydrophilic site in proteins has
severe structural implications, since subunit interactions can be
disrupted.
but with an OH substituent at the bridgehead carbon so that the
structure can be tentativly assigned as 13a/b.
Formation of compounds 12/13 obviously proceeds under
involvement of the amide functionality in 5. Scheme 6 shows the
∑
(d) The reaction of NO3 with tryptophan 5
∑
In the reaction of tryptophan 5 with NO3 four products were
formed. According to the respective protonated molecular ions
[M + H+] in the LC/ESI-MS of the crude reaction mixture,
the two major products were isomers possessing formally two
additional NO2 moieties, compared to the starting amino acid 5.
The two minor compounds were also isomeric species containing
one additional NO2 and OH group (Scheme 5). After isolation
and purification by preparative HPLC, we were delighted that
one of the major products readily crystallized and could be
analysed by X-ray. This revealed the tricyclic compound 12a,
which contained a nitrated aromatic ring and two cis-fused
cyclopentyl rings featuring a nitro substituent at the bridgehead
carbon of the bicyclo[3.3.0] system, which is cis to the ester
group at C9. On the basis of the available spectroscopic data
and mechanistic considerations (see below), we believe that the
other isomer possessing two NO2 substituents (e.g. 12b) must be a
diasteroisomer of 12a, but it was not possible from the 1H NMR
data to deduce the stereochemistry at C7 and C8.‡‡ In the case of
the two minor products, unfortunately only one isomer could be
isolated in an amount sufficient for NMR analysis. Based on the
general similarity of its NMR spectra with those for 12a/b (see
ESI†), however, we assume that both minor isomers should have
a similar tricyclic framework possessing also a nitroaromatic ring,
‡‡ A change of the stereochemistry at the a-carbon of the amino acid as
Scheme 6 Proposed mechanism for the reaction of NO3∑ with tryptophan
5 in the presence of NO2 .
∑
a result of the reaction with NO3 can in principle not be excluded, but
∑
seems to be not very likely.
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proposed reaction mechanism, where nitration of the aromatic
ring occurs para to the dominating activating amino substituent
through a pathway similar to that outlined in Scheme 3. The
Conclusions
∑
This first detailed product analysis of the reaction of NO3 with
aromatic amino acids, which was performed under conditions
that simulate exposure of biosurfaces to atmospheric pollutants,
revealed clear pathways how this important atmospheric free-
radical oxidant can cause irreversible damage to biolocially
important molecules. All amino acids under investigation reacted
∑
resulting nitrotryptophan 15 can be further oxidized by NO3
at the indole-nitrogen to give radical cation 16, which is in
resonance with the distonic isomer 16¢. Recombination with
∑
∑
X∑ (X∑ = NO2 , NO3 or O2, see below) from either above or
below the molecular plane of 16¢ yields cation 17, which acts as
acceptor in the subsequent nucleophilic cyclization of the amide-
nitrogen by which the tricyclic framework is established. The
∑
∑
rapidly with NO3 , which was in situ generated by treating NO2
with an O3/O2 mixture, leading ultimately to aromatic ring
nitration. There are no indications that the initial reaction of
∑
major pathway involves trapping of 16¢ by NO2 , which gives the
∑
NO3 occurs at any other site of the amino acid except the
observed dinitro compounds 12a/b. The minor products 13 could
aromatic ring. No influence of residual O2 on product formation
in the reactions involving phenylalanine 1 and tyrosines 2a–4a
was found. In the reaction with tryptophan 5 trapping of radical
intermediates by O2 cannot be excluded, but this would represent
only a very minor pathway. Also, in none of the reactions under
investigation products arising from a reaction involving the solvent
were observed.
be formed through two different pathways. Trapping of 16¢ by
NO3 gives nitrate ester 18, which may undergo hydrolysis to the
∑
corresponding alcohol 13 during work-up and/or purification of
the reaction products. If trapping of 16¢ occurs through reaction
with O2, subsequent decomposition of the resulting peroxyl radical
19 could also lead to 13. The latter pathway would represent
∑
the only case in the NO3 induced oxidative damage of aromatic
∑
In the reaction of NO3 with tryptophan 5 the damage was not
amino acids so far, where residual oxygen would be involved in
product formation. It should be noted that the reactions outlined
in Scheme 6, e.g. those leading to nitration of the aromatic ring and
those resulting in oxidative cyclization involving the amide moiety
could also proceed in reverse order. Interestingly, the finding that
limited to the aromatic ring system. Oxidation occurred also at
the nitrogen atom of the pyrrol ring leading to formation of an
intermediate carbocation 17, which is quenched by intramolecular
nucleophilic cyclization involving the amino acid functionality.
Because of the reduced flexibility of the backbone, a similar
cyclization is not very likely to occur in proteins. On the other
hand, a cationic intermediate of type 17 in a protein could be
trapped by any nucleophile available in the reaction system, for
example water or hydroxyl- and thiyl groups of cysteine, threonine
or serine residues, respectively, which could lead to significant
structural changes in the protein.
∑
in the reaction of tryptophan 5 with NO2 /O3 N-nitroso amides
were not formed as by-products, can be taken as indication that
the radical-induced oxidative cyclization of the amide moiety must
occur much faster than N-nitrosation.
The stereochemistry in 12a, which features a cis arrangement
between the ester group at C9 and the NO2 substituent, can
be rationalized through attack of the amide nitrogen N1 at C8
from the less sterically hindered site anti to the NO2 substituent
at C7 (which in turn results from bottom face recombination
Another important outcome of this study is the finding that
∑
NO3 can even damage phenylalanine, which is usually inert
∑
to oxidation by free radical and non-radical oxidants produced
through natural biochemical pathways. Thus, since the initially
formed aryl radical cation 7 has a certain lifetime, it would
represent a very strong oxizing site in a protein, which might
trigger an oxidative ET along the peptide chain that could lead
to damage at positions remote from the initial reaction site at the
interface between biological surface and environment. Our future
work is aimed at exploring this hypothesis in detail, using specially
designed peptides as model substrates.
of NO2 with 16¢). It is likely that the isomeric product 12b
could possess inverted configurations at C7 and C8, which
∑
result from recombination of NO2 with 16¢ from the top
face.§§
∑
The observed participation of the amide group in the NO3
induced oxidation of tryptophan 5 could be unique to our
experimental system, where the reaction with the isolated amino
acid was studied. In a protein the less flexible backbone may
prevent the conformational change required for such a cyclization.
On the other hand, a positive charge similar to that of intermediate
17 in proteins is a potential target for attack by any nucleophile
present in the system through both intra- and intermolecular
pathways, which could lead to significant structural modificatios
in the protein.
Whereas NO2∑ does not undergo a noticeable chemical reaction
with the aromatic amino acids investigated in this study, we have
strong experimental indications that N-nitrosamides are formed
through an independent, non-radical pathway by reaction with
∑
N2O4, which is in equilibrium with NO2 . Although we have
not further explored this reaction, our results reveal a “dual
Tryptophan 5 was the only amino acid in this study, which
reacted rapidly with a mixture of O3/O2. The product of this
reaction was identified as dicarbonyl compound 14 (Scheme 5),17
which results from ozonolysis of the more electron-rich N-
heterocycle. This product was not observed when the reaction was
∑
mode of action” of the atmospheric pollutant system NO2 /O3
∑
in its reactions with aromatic amino acids, e.g. (i) NO3 initiated
nitration of the aromatic ring, and (ii) N-nitrosation of the peptide
bond by N2O4, thus generating highly labile and carcinogenic
species.
It should be noted that formation of nitrated aromatic amino
acids is a general feature observed in a wide range of inflammatory-
immune responses, which are conditions associated with in-
creased levels of oxidative stress.18 In vivo studies revealed that
3-nitrotyrosine concentrations are increased in exhaled breath
condensate of patients with asthma19 or cystic fibrosis,20 and a
∑
performed in the presence of NO2 , which indicates that formation
∑
of NO3 and its reaction with 5 occurred significantly faster than
reaction of 5 with O3.
§§ It cannot be excluded, however, that the NO2 substituent in 13b occupies
a different position on the aromatic ring.
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strong immunoreactivity for nitrotyrosine in the airway epithelium
and inflammatory cells in the airways of asthmatic patients has
been found.21 Since nitration of proteins has been observed to
6 P. Neta and R. E. Huie, J. Phys. Chem., 1986, 90, 4644; L. Eberson,
Adv. Phys. Org. Chem., 1981, 18, 79; E. Baciocchi, T. Del Giacco, S. M.
Murgia and G. V. Sebastiani, J. Chem. Soc., Chem. Commun., 1987,
1246 and cited literature.
7 E. R. Pacht and W. B. Davis, J. Appl. Phys., 1988, 64, 2092; C. E. Cross,
A. von der Vlieth, C. A. O’Neill, S. Louie and B. Halliwell, Environ.
Health Perspect., 1994, 102, 185.
∑
occur in polluted air, our results suggest that NO3 formed through
∑
reaction of NO2 and O3 in close vicinity to biological surfaces,
such as the respiratory tract lining fluid, could potentially be the
real causer of certain pollution-derived diseases.
8 T. Franze, M. G. Weller, R. Niessner and U. Po¨schl, Environ. Sci.
Technol., 2005, 39, 1673.
9 D. C. E. Sigmund and U. Wille, Chem. Commun., 2008, 2121.
10 H. Suzuki and T. Mori, J. Chem. Soc., Perkin Trans. 2, 1997, 1265 and
cited literature.
Acknowledgements
11 J. Garcia, J. Goncalez, R. Segura and J. Vilarrasa, Tetrahedron, 1984,
This work was supported by the Australian Research Council
under the Centres of Excellence Program. Technical support by
Mr John Karas from the BIO21 Institute is gratefully acknowl-
edged. We thank Associate Professor Craig Hutton for helpful
discussions.
40, 3121.
12 R. W. Darbeau, R. S. Pease and E. V. Perez, J. Org. Chem., 2002, 67,
2942 and cited literature.
13 See for example: W. Lijinsky, N-Nitrosamines, in: ACS Symposium
Series, 1979, 101, 165.
14 K. Kikugawa, T. Kato and Y. Okamoto, Free Radical Biol. Med., 1994,
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