Oxidative damage of aromatic dipeptides by the environmental oxidants NO2 and O3

Article abstract of DOI:10.1039/c4ob01577k

Irreversible oxidative damage at both aromatic side chains and dipeptide linkage occurs in the aromatic N- and C-protected dipeptides 7-11 upon exposure to the environmental pollutants NO₂ and O₃. The reaction proceeds through initial oxidation of the aromatic ring by in situ generated NO₃, or by NO₂, respectively, which leads to formation of nitroaromatic products. The indole ring in Phe-Trp undergoes oxidative cyclization to a pyrroloindoline. An important reaction pathway for dipeptides with less oxidisable aromatic side chains proceeds through fragmentation of the peptide bond with concomitant acyl migration. This process is likely initiated by an ionic reaction of the amide nitrogen with the NO₂ dimer, N₂O₄. This journal is

Full text of DOI:10.1039/c4ob01577k

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Oxidative damage of aromatic dipeptides by the
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environmental oxidants NO2 and O3†
Cite this: DOI: 10.1039/c4ob01577k
L. F. Gamon,a J. M. Whiteb and U. Wille*a
Irreversible oxidative damage at both aromatic side chains and dipeptide linkage occurs in the aromatic
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N- and C-protected dipeptides 7–11 upon exposure to the environmental pollutants NO2 and O3. The
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reaction proceeds through initial oxidation of the aromatic ring by in situ generated NO3 , or by NO2
,
respectively, which leads to formation of nitroaromatic products. The indole ring in Phe-Trp undergoes
oxidative cyclization to a pyrroloindoline. An important reaction pathway for dipeptides with less oxidis-
able aromatic side chains proceeds through fragmentation of the peptide bond with concomitant acyl
Received 25th July 2014,
Accepted 2nd September 2014
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DOI: 10.1039/c4ob01577k
migration. This process is likely initiated by an ionic reaction of the amide nitrogen with the NO2 dimer,
www.rsc.org/obc
N2O4.
initial damage to the acute disease state. However, a detailed
understanding of the chemical pathways by which gaseous air
Introduction
According to the World Health Organization, air pollution is pollutants damage the constituents of the RTLF is essential
responsible for the premature death of about seven million for the development of effective strategies to combat deleter-
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people each year. Nitrogen dioxide (NO2 ) and ozone (O3) are ious impacts of air pollution.
amongst the most important gaseous pollutants in the
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NO2 is moderately oxidizing [E0 (NO2 /NO2−) = 1.03 V]4a
outdoor and indoor environment, and their inhalation is and reacts only with the most reactive amino acids through
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associated with the development of respiratory tract inflam- oxidative electron transfer (ET).4b NO2 is also a potent radical
mation.1,2 The respiratory tract lining fluids (RTLF) are the trap, but reactions with closed-shell systems through addition
first biological fluids that come into contact with environ- or hydrogen abstraction are comparably slow.4c,d The biological
mental free radical and non-radical oxidants. It has been effect of O3 is attributed to its high reactivity with π systems,
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shown in in vivo studies that exposure to O3 and NO2 in iso- and O3 reacts with lipids exclusively at the CvC double bonds
lation results in significant reduction of RTLF antioxidant in unsaturated fatty acids.5a Reaction of O3 with proteins
levels.1 This weakened defence shield could provide a pathway occurs preferably at easily oxidisable side chains.5b
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for environmental oxidants to directly attack proteins and
In the polluted ambient atmosphere O3 and NO2 always
lipids present on cell surfaces or in the RTLF, which could coexist. It was found that birch pollen proteins are rapidly
lead to highly reactive protein and lipid oxidation products nitrated upon exposure to traffic pollution (which is character-
that may subsequently damage the underlying epithelial cells, ised by large concentrations of O3 and NO2 ).6a In fact, signifi-
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thereby causing inflammation.
cant synergistic effects were observed in various biological
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The effects of ground-level O3 and NO2 on lung function, molecules when these were subjected to O3/NO2 mixtures,
both in isolation and in combination, has been explored in such as increased lipid peroxidation and protein nitration.3,6
exposure studies.1–3 Unfortunately, there is only limited knowl- In particular, 3-nitrotyrosine concentrations are increased in
edge of the individual mechanistic steps that lead from the exhaled breath condensates of patients with asthma7 or cystic
fibrosis,8 and a strong immunoreactivity for nitrotyrosine in
the airway epithelium and inflammatory cells in the airways of
asthmatic patients has been found.9 The more than additive
aSchool of Chemistry and Bio21 Institute, ARC Centre of Excellence for Free Radical
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response to exposure of O3/NO2 mixtures suggests that nitrate
Chemistry and Biotechnology, The University of Melbourne, 30 Flemington Road,
Parkville, VIC 3010, Australia. E-mail: uwille@unimelb.edu.au
bSchool of Chemistry and Bio21 Institute, The University of Melbourne,
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radicals, NO3 , might be involved in these processes, which are
in situ generated via eqn (1).10
30 Flemington Road, Parkville, VIC 3010, Australia
†Electronic supplementary information (ESI) available: Synthetic procedures
and spectroscopic data for dipeptides 7–11, experimental and spectroscopic
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O3 þ NO2 ! O2 þ NO3
ð1Þ
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details for reaction of all dipeptides with NO2 and O3; crystallographic data for
It was proposed that the observed synergistic effects are due
compound 16. CCDC 1016028. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/c4ob01577k
to the toxicity of dinitrogen pentoxide, N2O5, which is formed
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through (reversible) recombination of NO2 with NO3 , and
also the hydrolysis product nitric acid (HNO3), but detailed
mechanistic studies were not performed.1,3 In fact, formation
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of NO3 through reaction of NO2 with O3 is of significant
environmental relevance, since NO3• is the most important tropo-
spheric free radical oxidant at night and responsible for
initiating chemical transformation processes in the atmos-
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phere. Although the atmospheric concentration of NO3 is less
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than that of NO2 or O3, its high reactivity towards most
organic compounds outweighs its lower abundancy.10 Thus,
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NO3 is highly electrophilic and strongly oxidising [E0 (NO3 /
NO3−) = 2.5 V],11 and also reacts through addition to π systems
and hydrogen abstraction.10 NO3• has not only a higher solubi-
Fig. 1 Aromatic dipeptides 7–11 studied in this work.
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lity in water than NO2 [KH (NO3 ) = 6.0 × 10−1 M atm−1; KH
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of the aryl radical cation 3 with NO2 and subsequent deproto-
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(NO2 ) = 1.2 × 10−2 M atm−1, at 298 K],12 but also maintains its
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nation of 4. In the absence of NO2 , benzylic deprotonation in
3 occurs first,15b‡ and the resulting radical 5 is ultimately
transformed to β-oxygenated amino acids 6.14b The high oxi-
high reactivity in water,10 with which it actually reacts only
slowly, according to: H2O + NO3 → HO• + HNO3 (k = 3 × 102
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M−1 s−1).13 On this basis, it appears highly likely that also bio-
logical molecules in the RTLF are susceptible to attack by
dation power of NO3 causes irreversible damage to all aro-
matic amino acids, even those that are inert to oxidation by
free radical and non-radical oxidants produced through
natural biochemical pathways, such as phenylalanine (1a). In
the fast reaction with tyrosine (1b) consecutive formation of
3-nitro tyrosine (2b, with x = 1) and 3,5-dinitrotyrosine (2b,
with x = 2) occurs. Tryptophan reacts through multiple path-
ways that result in both aromatic nitration and oxidative cycli-
zation through involvement of the amino acid functionality
(not shown).14a A recent multiphase kinetic study by Pöschl
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NO3 .
We have recently shown in an in vitro product study that
exposure of various N- and C-protected aromatic amino acids 1
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to O3/NO2 mixtures in either dichloromethane or acetonitrile
leads to rapid formation of nitroaromatic products
2
(Scheme 1).14a
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The reaction likely proceeds through initial NO3 induced
oxidation of the aromatic ring,15 which is followed by trapping
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et al. proposes that uptake of NO2 by proteins in the gas
phase and in the presence of O3 is a combination of a direct
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reaction of NO3 and a sequential process, where reaction of
O3 with the protein leads first to reactive oxygen intermediates,
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which react with NO2 in a second step.6c The employed
kinetic model considered tyrosine as the only target for reac-
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tion with NO2 /O3 mixtures, and did not include potential
multiple attacks at one site or at other protein residues, for
example the protein backbone. In light of our previous find-
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ings that NO3 attacks not only tyrosine, it is not unlikely that
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the direct involvement of NO3 in oxidative protein damage
may have been underestimated.
In the present study we explored the reaction of the aro-
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matic dipeptides 7–11 (Fig. 1) with NO2 and O3 in both iso-
lation and combination to simulate exposure of peptides
lining the respiratory tract surface to the atmospheric
environment.
Analysis of the reaction mixtures and identification of
major products revealed the sites in dipeptides most suscep-
tible to damage by these environmental oxidants. While
O-acetylated tyrosine (OAcTyr) is not a naturally occurring
amino acid, dipeptide 9 was included in this work to examine
how electron density at the aromatic system directs the reac-
tion outcome.
‡In the case of very electron-rich aromatic compounds, a concerted electron and
proton transfer process is likely (see ref. 15b).
Scheme 1 Reaction of NO3• with aromatic amino acids 1.
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Results and discussion
The dipeptides were protected at both the N-terminus (as acet-
amide) and C-terminus (as methyl ester; see Fig. 1). All reac-
tions were carried out at 10 °C for 20 min under exclusion of
moisture in acetonitrile by adding a measured excess amount
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of liquid NO2 (which is in equilibrium with its dimer dinitro-
gen tetroxide, N2O4) to the dipeptide 7–11, with a stream of
ozonized oxygen passing through the solution.14a§ The reac-
tions of the dipeptides with NO2• and O3 in isolation were also
explored under similar conditions. Reaction products were
isolated and purified by preparative HPLC, followed by
HR-ESI-MS and 1H NMR analysis. 13C NMR data were also
obtained for major products, where sufficient material could
be isolated. Full experimental and spectroscopic details are
given in the ESI.† Although in our experiments O3 was always
supplied as a mixture with O2, we will simply refer to these as
O3 experiments, since a direct involvement of O2 was not
observed for any reaction. It should be noted that, due to the
required repeated HPLC purifications, yields could not be
determined. Qualitative assignments for major and minor pro-
ducts were made on the basis of the UV absorptions in the
HPLC runs at different wavelengths. The aim of the present
study was the identification of ‘hot spots’ in dipeptides for
Scheme 2 Reaction of NO2• and O3 with Phe-Phe (7).
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e.g. NO3 induced oxidation of the aromatic ring, followed by
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trapping of the intermediate aryl radical cation by NO2 . The
observed para selectivity for this radical mediated nitration
(confirmed by 1H NMR) is in agreement with previous findings
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for the reaction of 1a with NO2 /O3 mixtures14a and can be
rationalized by the stability of the intermediate σ complex of
type 4a. According to the UV absorption of the reaction
mixture at λ = 220 nm, where only small signals from 12a/b
were present,¶ the major product of this reaction was the
N-acetylated phenylalanine methyl ester 1a. This assignment
was confirmed through comparison of the spectroscopic data
with those of an authentic sample. The presence of the ester
group indicates that 1a originates from the C-terminal amino
acid in 7, suggesting cleavage of the peptide bond linking the
two amino acids with concomitant migration of the N-terminal
acetyl group. The counterpart of the peptide cleavage, e.g. the
N-terminal amino acid (or possible subsequent reaction pro-
ducts), could not be obtained. Remarkably, a similar fragmen-
tation of the peptide bond also occurred in the reaction of 7
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attack by NO2 and O3 in order to assess the potential impact
for biological systems. In the biological context even seemingly
minor chemical changes often have serious pathological con-
sequences and, therefore, knowledge of explicit yields is not
required.
Reaction of NO2• and O3 with Phe-Phe (7)
Of the naturally occurring aromatic amino acids, phenyl-
alanine contains the least electron-rich aromatic ring and is
therefore comparably difficult to oxidise. The outcome of the
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with NO2 in isolation. Qualitatively, the rate of peptide frag-
mentation was slow and not affected by the presence of O3. It
is therefore reasonable to suggest that peptide cleavage in 7 is
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reaction of the Phe-Phe dipeptide 7 with O3 and NO2 in iso-
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lation and combination is shown in Scheme 2.
caused by reaction with NO2 (and not NO3 ). In contrast to
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Exposure of 7 to a mixture of NO2 and O3 for 20 min
this, a reaction between NO2 and the aromatic moieties in 7
resulted in incomplete consumption of the starting material
and formation of three products. The isomeric para-nitrated
dipeptides 12a and 12b were generated in a 1 : 1 ratio (accord-
ing to HPLC analysis at λ = 280 nm, where the UV absorption
of 7 is negligible), which indicates unselective oxidative attack
at both the N- and C-terminal amino acid. The aromatic
did not occur in the absence of O3. The possible mechanism
for the NO2 induced fragmentation of the peptide backbone
will be discussed below.
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It should be noted that dipeptide 7 did not react with O3 in
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the absence of NO2 .
nitration likely proceeds through the mechanism in Scheme 1, Reaction of NO2• and O3 with Phe-Tyr (8)
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Reaction of dipeptide 8 with a mixture of O3 and NO2
§Typical experimental procedure: To
a stirred mixture of the dipeptide
occurred highly selectively at tyrosine, resulting in complete
consumption of 8 after 20 min of reaction time and formation
of dipeptides consisting of an intact phenylalanine moiety that
is linked to either 3-nitrotyrosine (13a) or 3,5-dinitrotyrosine
(13b), respectively (Scheme 3).
(1.00 mmol) in acetonitrile (100 mL) at 10 °C was added under exclusion of
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moisture liquid NO2 (0.5 mL, 15 mmol), and a stream of ozonized O2 was
passed through the mixture at a low flow rate. Consumption of the substrates
was usually complete after 20 min of reaction time. The reaction was quenched
by addition of aq. NaHCO3 (50 mL), acetonitrile removed in vacuo and the
aqueous phase extracted with ethyl acetate. The combined organic fractions
were dried over MgSO4, concentrated and the reaction products isolated and pur-
ified by repeated preparative HPLC (see ESI† for details). It is not possible to
state the exact [NO2•] in the experiments, since an undeterminable amount of
NO2• evaporated prior to its reaction with O3. The experiments with NO2• and O3
in isolation were performed analogously.
According to UV analysis of the reaction mixture at λ = 250,
280 and 350 nm, the mono-nitrated dipeptide 13a was formed
¶It is estimated that the UV absorbance of the nitrated dipeptides 12a/b at
λ = 220 nm is approximately 1000 times higher than that of 7.
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Scheme 3 Reaction of NO2• and O3 with Phe-Tyr (8).
as major product,k which was accompanied by smaller
amounts of the di-nitrated dipeptide 13b. Formation of
dimeric products of type Phe-Tyr-Tyr-Phe, which are linked
through a covalent bond ortho to the phenolic hydroxyl group
in tyrosine (such dityrosines are commonly produced during
tyrosine oxidation in the absence of radical traps),4b was not
Scheme 4 Reaction of NO2• and O3 with Phe-OAcTyr (9).
mental conditions. On the other hand, the reaction of O3 with
dipeptide 8 led to rapid consumption of the starting material,
resulting in decomposition to many different products, which
could not be isolated and identified. This process is likely trig-
gered by initial attack of O3 at the tyrosine moiety in 8,18 since
phenylalanine does not react with O3 (see above).
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observed. The reaction of 8 with NO2 in the absence of O3 led
to formation of 13b as the major pathway, while the mono-
nitrated product 13a was not obtained. The higher degree of
aromatic nitration in the absence of O3 suggests a higher con-
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centration of dissolved NO2 under these conditions, which
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can be rationalized by the fact that the partial pressure of NO2
is higher in the absence of O2/O3.
Reaction of NO2• and O3 with Phe-OAcTyr (9)
The initial reaction of NO3• or NO2• with tyrosine likely pro-
ceeds through oxidative ET, but it is not clear whether trapping
of the resulting tyrosyl radical cation of type 3a (see Scheme 1)
Protection of the tyrosine hydroxyl group through O-acetylation
reduced significantly the susceptibility of this amino acid to
oxidative damage. Similar to the reaction of 7, Phe-OAcTyr (9)
was only incompletely consumed after 20 min exposure to a
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by NO2 occurs faster than its deprotonation to give the tyrosyl
radical (not shown), which could subsequently be quenched
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by NO2 . It can also not be excluded that, at least in the reac-
mixture of NO2 /O3, which led, according to HPLC analysis, to
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tion involving the highly reactive NO3 , direct abstraction of
a number of different products. Only the most abundant could
be isolated and identified, which are shown in Scheme 4.
UV analysis at λ = 220 nm revealed the O-acetylated tyrosine
1c, which is the C-terminal amino acid in 9, as major reaction
product, indicating that fragmentation of the peptide bond
is the dominant pathway. Minor amounts of O-acetyl-3-nitro-
tyrosine 2c and tyrosine 1b were also formed. It is reasonable
to assume that 1b is a secondary product that results from 1c,
presumably post-reaction through cleavage of the O–Ac bond
the phenolic hydrogen atom occurs. However, knowledge of
the mechanism to such detail is not essential in order to
assess the potential biological impact of the damage. Tyrosine
nitration has, in fact, serious consequences for biological
systems, since each nitro substituent lowers the pKa of tyrosine
by about 3 units.16 Under physiological conditions nitrated
tyrosine is deprotonated and generates negatively charged
hydrophilic sites in peptides, which could induce structural
changes that may lead to disruption of subunit interactions.16
Indeed, it was found that nitration of tyrosine results in inacti-
vation of a number of biologically significant proteins.17
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during aqueous work-up. If formed in the presence of NO2 /
O3, rapid oxidation to nitrotyrosine 2b would be expected. On
the other hand, 2c could be obtained via first nitration of the
O-acetylated tyrosine in 9, followed by peptide fragmentation,
or in inverse order where nitration follows fragmentation (not
shown). An additional reaction pathway for dipeptide 9 with
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It should be noted that in the reaction of 8 with NO2 , both
in the presence and absence of O3, products arising from scis-
sion of the peptide backbone could not be identified. This
shows that the fast reaction of NO2• with the tyrosine moiety in
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NO2 /O3 mixtures involves oxidative damage at the aromatic
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8 acts as efficient “sink” for NO2 with which the slower reac-
ring of the phenylalanine moiety to produce the isomeric ortho
and para nitrated dipeptides 14a/b.
tion with the peptide bond (leading to peptide fragmentation)
could not compete to a measurable extent under our experi-
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The reaction of dipeptide 9 with NO2 in the absence of O3
was slow and led predominantly to the fragmentation product
1c, but formation of very minor amounts of 1b and 2c cannot
be excluded. On the other hand, no reaction occurred between
kThe absorbance of 13a at these wavelengths is approximately 5–10 times lower
than that of 13b.
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9 and O3 in the absence of NO2 .
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Scheme 5 Reaction of NO2• and O3 with Tyr-Tyr (10).
Reaction of NO2• and O3 with Tyr-Tyr (10)
Reaction of Tyr-Tyr (10) with a mixture of NO2• and O3 resulted
in complete consumption of the dipeptide after 20 min. The
major products are the isomeric tri-nitro compounds 15a/b
and the tetra-nitrated dipeptide 15c (Scheme 5).
In addition to this, LC/MS analysis of the reaction mixture
reveals formation of a product with a high-resolution mass of
m/z 491.1409, which corresponds to a dinitrated dityrosine
dipeptide (C21H22N4O10 + H+: calcd 491.1250). Due to the
small amount, isolation and further characterization of this
product was not possible.
The site of nitration in the tri-nitro dipeptides 15a and 15b
was unequivocally confirmed by ESI mass spectrometric
sequencing using collision-induced dissociation (bx–yz
pathway).19 Thus, for 15a the fragment at m/z 251.08 corres-
ponds to the mono-nitrated N-terminal amino acid (b1:
Scheme 6 Reaction of NO2• and O3 with Phe-Trp (11).
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further confirms the high susceptibility of tyrosine to NO2
induced oxidation. Peptide cleavage to yield 2b occurred too,
but, however, only to a very minor extent under these
conditions.
Similar to the reaction of the phenylalanine-tyrosine dipep-
tide 8, reaction of 10 with O3 resulted in complete decompo-
sition, and isolation of reaction products was not possible.
+
C11H11N2O5 , calcd 251.07). The fragment at m/z 286.08 could
be assigned to the C-terminal dinitrated amino acid (y1:
+
C10H11N3O7 , calcd 286.07). Likewise, the y1 fragment of the
protonated C-terminal amino acid in 15b appears at m/z
+
241.08 (C10H13N2O5 , calcd 241.08), confirming mononitration
of the tyrosyl moiety. The corresponding b1 fragment could
not be obtained.
In addition to this, fragmentation of the peptide backbone
also occurred, but only as a very minor reaction pathway, to
Reaction of NO2• and O3 with Phe-Trp (11)
yield 3,5-dinitrotyrosine 2b, which is the former C-terminal Tryptophan is, similar to tyrosine, prone to oxidation by
amino acid moiety. From the UV absorption of the reaction radical and non-radical oxidants. The reaction of O3 with the
mixture at λ = 280 nm a product ratio for 15c/2b of ca. 5 could phenylalanine-tryptophan dipeptide 11 occurred rapidly and
be estimated. Given the high reactivity of tyrosine towards oxi- proceeded selectively at tryptophan through oxidative cleavage
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dative aromatic nitration by NO2 /O3 mixtures, it is reasonable of the indole ring. The resulting dipeptide 17 consists of an
to assume that the comparably slow fragmentation of the N′-formyl kynurenine residue and an intact phenylalanine
peptide bond occurred only after nitration of the phenolic moiety (Scheme 6).14a,20
ring.
Tryptophan was also exclusively targeted when 11 was
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Reaction of 10 with NO2 in the absence of O3 also led to exposed to a mixture of NO2 and O3. Under these conditions
the various tri- and tetra-nitrated dipeptides 15a–c, which formation of the O3 cleavage product 17 was largely sup-
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pressed, and the indole ring underwent oxidative cyclization
involving the nitrogen atom of the dipeptide linkage, to yield a
mixture of, presumably, isomeric products. Only the major
product could be isolated through crystallisation. Its structure
was confirmed by X-ray analysis as dipeptide 16, which con-
sists of an intact phenylalanine moiety and a cis-fused
N-nitroso pyrroloindoline framework possessing an angular
nitro substituent.14a While pyrroloindolines are structural
motifs in many biologically active alkaloids,21 N-nitrosamines
are known for their highly carcinogenic effects.22 The reaction
of 11 with NO2• in the absence of O3 also leads to formation of
pyrroloindoline 16, which demonstrates the ease by which
tryptophan is damaged by abundant environmental oxidants.
A mechanism for formation of 16 is proposed in the lower
part of Scheme 6,14a which consists of initial oxidation by
Scheme 7 Proposed mechanism for NO2• induced peptide cleavage.
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either NO2 or NO3 to produce indole radical cation 18.** The
distonic radical cation resonance isomer 18′ is the likely inter-
mediate involved in the subsequent steps that include radical
cent acetyl moiety and enables intramolecular nucleophilic
attack by the nitrogen atom of the dipeptide linkage.
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trapping by NO2 and nucleophilic cyclization of the amide
The resulting 5-membered ring adduct 22 can rearrange to
imide 23 through a concerted hydrogen transfer/fragmentation
sequence, followed by formation of the diazo succinimide 24.
M06-2X/cc-pVDZ calculations using N-Ac-N-NO-Gly-Gly-OMe as
simplified model for 21 (data not shown) revealed an acti-
vation barrier of some 67 kJ mol−1 for the hetero-ene type
process 22 → 23, with the sequence 22 → 24 being overall
exothermic by about 55 kJ mol−1, showing that this process is
energetically highly feasible. Subsequent hydrolysis of 24
through selective nucleophilic attack at the imide carbonyl group,
which is activated by the neighbouring diazo substituent, for
example during aqueous work-up, leads to release of the N-acetyl-
ated amino acid 1a. Although we were not able to isolate diazo
compounds of type 25 as by-products to confirm the proposed
mechanism, formation of the latter has been observed in the
cleavage of N-nitrosopeptides with amines and α-amino
esters.24 Given the high abundance of nucleophilic sites in
peptides, our findings show that activation of peptide bonds
nitrogen, which leads to the pyrroloindoline 20. It is obvious
that formation of the pyrroloindoline framework in 16 requires
some conformational flexibility of the peptide backbone. We
believe that N-nitrosation occurs only in the final step through
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reaction of 20 with N2O4 (resulting from NO2 dimerisation),
which is a known non-radical nitrosating agent.23 On the other
hand, if N-nitrosation would occur as initial step, the reduced
electron density at the indole ring would prevent such oxi-
dative cyclisation. This hypothesis was confirmed in indepen-
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dent reactions of NO2 /O3 mixtures with N- and C-protected
tryptophan, where the indole nitrogen was deactivated by
acetylation, and which did not lead to pyrroloindolines (not
shown).
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Peptide cleavage by reaction of 11 with NO2 did not occur,
•
which can be rationalized by the fact that NO2 was rapidly
consumed through the reaction with the indole ring in
tryptophan.
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through reaction with the environmental pollutant NO2 (or
NO2• induced peptide cleavage: mechanistic considerations
N2O4, respectively) may lead to significant structural changes,
which could range from rearrangement of the peptide frame-
work to backbone fragmentation.
•
To our knowledge, the NO2 induced cleavage of a peptide
bond, which was the major pathway in the reaction of dipep-
tides possessing less oxidisable aromatic side chains, has
never been reported before. We believe that this reaction,
which releases the C-terminal amino acid with concomitant
acyl transfer from the N-terminal amino acid, is not a radical
but an ionic process. Scheme 7 outlines a mechanistic ration-
ale for this fragmentation/rearrangement sequence, using the
reaction of Phe-Phe (7) as example.
N-nitrosation by N2O4 at the sterically less hindered
N-acetyl terminus in 7 occurs in the first step to give N-nitros-
amide 21. The electron-withdrawing effect caused by the
N-nitroso group14a leads to considerable activation of the adja-
Conclusion
The present model study revealed that exposure of aromatic
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N- and C-protected dipeptides to the atmospheric pollutants NO2
and O3 results in irreversible oxidative damage at both aro-
matic side chains as well as the dipeptide linkage. Whereas
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both NO2 and O3 in isolation could not induce oxidation of
the aromatic rings in Phe-Phe (7) and Phe-OAcTyr (9), these
two gases in combination led to formation of the isomeric
dipeptides 12a/b and 14a/b possessing nitrophenylalanine
residues. No preference for attack at either the N- or C-termi-
nus was found for the reaction involving dipeptide 7. The
observed strong synergistic effects caused by the combination
**It should be noted that hydrogen abstraction has recently been proposed as
initial step in the reaction of indole with NO2•; see: P. Astolfi, M. Panagiotaki,
C. Rizzoli and L. Greci, Org. Biomol. Chem., 2006, 4, 3282.
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of NO2• and O3 suggests in situ generation of NO3 . This highly that NO2 /N2O4 induced peptide fragmentation also occurs in
oxidizing radical is capable to attack even those peptide moi- tripeptides. These findings clearly indicate that the environ-
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eties that are usually inert to damage by endogenous free mental oxidants NO2 and O3, in both isolation and combi-
radical and non-radical oxidants, such as phenylalanine. The nation, can principally damage peptides to a larger extent than
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NO3 induced oxidation leads first to a phenylalanyl radical previously believed. It is therefore not unreasonable to suggest
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cation of type 3, which is subsequently trapped by NO2 . If that in biological systems oxidative damage in peptides by
•
formed in peptides, the phenylalanyl radical cation, which has NO2 and O3 may also occur even when only amino acids with
been shown in transient absorption spectroscopy studies to ‘non-vulnerable’ side chains are exposed to these environ-
• 15c
have a certain lifetime in the absence of NO2 ,
would rep- mental oxidants. We are currently exploring the reaction of the
•
resent a very strong oxidizing site. This might trigger second- NO2 /O3 system with different oligopeptides to reveal a deeper
ary inter- or intramolecular oxidation processes, for example understanding of the relative importance of peptide bond clea-
oxidative ET along the peptide chain,25 by which damage vage versus side chain oxidation in peptide damage, in particu-
could be induced at positions remote from the initial reaction lar in dependence of the amino acid sequence and
site.
In the case of dipeptides that consist of two aromatic
concentration of the oxidizing pollutants.
amino acids with different electron density, oxidative attack
occurred selectively at the more electron rich aromatic ring.
The reactions of NO2 with dipeptides containing a tyrosine
•
Acknowledgements
residue were rapid, both in the presence and absence of O3,
and led to multiple aromatic nitration. It is known from pre-
vious work that this radical mediated nitration occurs stepwise
via formation of 3-nitrotyrosine, which is subsequently trans-
formed into 3,5-dinitrotyrosine.14a This clearly shows that the
deactivating effect of one nitro group in 3-nitrotyrosine is not
Financial support by the Australian Research Council (Centre
of Excellence Scheme) and The University of Melbourne is
gratefully acknowledged.
•
sufficient to prevent further oxidation by the NO2 /O3 system.
Notes and references
The reaction involving the tryptophan containing dipeptide 11
•
with NO2 , both with O3 present or absent, was also fast and
1 Air Pollution and Health, ed. S. T. Holgate, J. M. Samet,
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•
NO2 and its dimer N2O4. While we have found previously that
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•
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•
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