Oxidative Damage of Biomolecules by the Environmental Pollutants

(5) NO3•, which is rapidly photolyzed during daytime, is a highly reactive electrophilic radical and the most important nighttime tropospheric oxida...
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Oxidative Damage of Biomolecules by the Environmental Pollutants NO2• and NO3• Luke F. Gamon and Uta Wille* School of Chemistry and Bio21 Institute, The University of Melbourne, Parkville, VIC 3010, Australia S Supporting Information *

CONSPECTUS: Air pollution is responsible for the premature death of about 7 million people every year. Ozone (O3) and nitrogen dioxide (NO2•) are the key gaseous pollutants in the troposphere, which predominantly result from combustion processes. Their inhalation leads to reactions with constituents in the airway surface fluids (ASF) of the respiratory tract and/or lungs. ASF contain small molecular-weight antioxidants, which protect the underlying epithelial cells against oxidative damage. When this defense system is overwhelmed, proteins and lipids present on cell surfaces or within the ASF become vulnerable to attack. The resulting highly reactive protein and lipid oxidation products could subsequently damage the epithelial cells through secondary reactions, thereby causing inflammation. While reactions of NO2• with biological molecules are considered to proceed through radical pathways, the biological effect of O3 is attributed to its high reactivity with π systems. Because O3 and NO2• always coexist in the polluted ambient atmosphere, synergistic effects resulting from in situ formed strongly oxidizing nitrate radicals (NO3•) may also require consideration. For example, in vitro product studies revealed that phenylalanine, which is inert not only to oxidants produced through biochemical processes, but also to NO2• or O3 in isolation, is damaged by NO3•. The reaction is initiated by oxidation of the aromatic ring and, depending on the availability of NO2•, leads to formation of nitrophenylalanine or β-nitrooxyphenylalanine, which could serve as marker for NO3•-induced oxidative damage in peptides. More easily oxidizable aromatic amino acids are directly attacked by NO2• and are converted to the same products independent of whether O3 is also present. Remarkably, NO2•-induced oxidative damage in peptides occurs not only through the wellestablished radical oxidation of peptide side chains, but also through an unprecedented fragmentation/rearrangement of the peptide backbone. This process is initiated by a nonradical N-nitrosation of a peptide bond involving the dimer of NO2•, i.e., N2O4, and contracts the peptide chain in the N → C direction by expelling one amino acid residue with simultaneous fusion of the remaining molecular termini, thereby forming a new peptide bond. This peptide cleavage could potentially be highly relevant for peptide segments with “nonvulnerable” side chains closer to the terminus that are not tied up in complex secondary and tertiary structures and therefore accessible for environmental oxidants. Likewise, NO2• reacts with cholesterol at the CC moiety through an ionic mechanism, which leads to formation of 6-nitrocholesterol in the presence of moisture. Contrary to common belief, this clearly shows that ionic chemistry, in particular nitrosation reactions by intermediately formed NO+, requires consideration when assessing NO2• toxicity. This conclusion is supported by recent work by Colussi et al. (Enami, S.; Hoffmann, M. R.; Colussi, A. J. Absorption of inhaled NO2. J. Phys. Chem. B. 2009, 113, 7977−7981), who showed that anions in the airway surfaces fluids mediate NO2• absorption by catalyzing its hydrolytic disproportionation into NO2−/HNO2 and NO3−. These findings could be the key to our understanding why NO2•, despite its low water solubility, has such pronounced biological effects in vivo.

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INTRODUCTION

According to the World Health Organization (WHO), air pollution is responsible for the premature death of about 7 million people every year.1 The key gaseous pollutants in the © 2016 American Chemical Society

Received: May 8, 2016 Published: September 26, 2016 2136

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resulting oxidation products could damage epithelial cells through secondary reactions, thereby causing inflammation. It has been suggested that O3 and NO2• may modulate airway diseases by increasing the release of pro-inflammatory mediators from airway epithelial cells.12 Reactions of NO2• with biological molecules are believed to proceed through radical pathways.13,14 NO2• is a moderately strong one-electron oxidant [E0 (NO2•/NO2−) = 1.03 V vs NHE]6 and radical trap, but reactions with closed-shell compounds through addition to π systems or hydrogen atom transfer (HAT) are usually slow,15 except for lipids, where abstraction of allylic hydrogens could lead to lipid peroxidation.14 NO2• exists in a temperature-dependent equilibrium with its dimer dinitrogen tetroxide, N2O4, which is a known nonradical nitrosating agent through its isomeric ionic form [NO+NO3−].16 In light of this and the recent finding that anions in the ASF mediate NO2• absorption by catalyzing a similar disproportionation into NO3− and NO2−/HNO2 (which is a precursor of NO+ in the acidic environment resulting from hydrolysis),17 it surprises that ionic pathways have not been considered in reactions of NO2• with biological molecules. The biological effect of O3 is attributed to its high reactivity with π systems, and O3-induced damage in lipids occurs exclusively at alkene moieties in unsaturated fatty acids.18 With peptides, O3 usually reacts with readily oxidizable side chains, such as those present in tyrosine, tryptophan, or methionine residues.19 In the polluted ambient atmosphere, O3 and NO2• always coexist, and significant synergistic effects were found in exposure studies with NO2•/O3 mixtures compared to the single gases.20−23 A mass spectrometric investigation on lipid oxidation suggested that the observed enhanced O3 toxicity in the presence of NO2• could be due to in situ formed NO3•.24 Further, the more than additive airway response to the combination of O3 and NO2• could result from the toxicity of dinitrogen pentoxide, N2O5, which is produced through (reversible) recombination of NO3• with NO2• (reaction 5), in addition to nitric acid (HNO3) formed through hydrolysis of N2O5.20,21 However, NO3•, which is considerably more soluble in water than NO2• [KH (NO3•) = 0.60 mol kg−1 bar−1; at 298 K],10 reacts also directly with organic molecules through oxidative electron transfer (ET), radical addition and HAT.5 The mechanism through which air pollution exposure leads to respiratory tract diseases is still only poorly understood. This lack of knowledge can, at least in part, be attributed to the design of many existing studies, which were primarily aimed at exploring the response of biological systems to pollutant exposure without identifying reaction products. This Account summarizes the findings from our in vitro model studies on oxidative damage in biological molecules caused by exposure to NO3• and to NO2• in the absence and presence of O3 to gain understanding of the chemical conversions that could principally occur and to reveal possible synergisms arising from combination of these pollutants. The work focused on amino acids and short oligopeptides possessing mainly aromatic side chains, which were used as model systems for peptides in the ASF, but also includes the reaction of cholesterol as an example for lipids.

troposphere, the lowest layer of the atmosphere, are ozone (O3), nitrogen dioxide (NO2•), volatile organic compounds (VOCs) and various other open- and closed shell nitrogen species, such as the nitrate radical (NO3•), nitric acid, alkyl nitrites, and nitrates. Increasing air pollution has significant implications including health problems, loss of crops, regional weather alterations, and even global climate change.1,2 The free radical NO2•, a toxic brown gas and contributor to photochemical smog, is formed through oxidation of nitrous oxide (NO•), for example by peroxyl radicals, ROO•, (reaction 1) or O3 (reaction 2) (Chart 1). Combustion processes, in Chart 1. Cycling of NO2• in the Troposphere (Selected Reactions)

particular traffic exhaust, are a major source for tropospheric NO•, which is continuously regenerated during daytime through photolysis of NO2•, thereby leading to formation of O3 (reaction 3). Average 24 h outdoor concentrations of up to 0.21 ppm for NO2• have been reported.3 Typical [O3] in the northern hemisphere are in the range of 35−40 ppb, with considerably higher concentrations occurring episodically throughout the year depending on the weather.4 At night, the fast cycling between NO2• and NO• ceases, and NO2• reacts with O3 through O atom transfer to form NO3• (reaction 4) with a rate coefficient of k = 3.2 × 10−17 cm3 molecule−1 s−1 at 298 K in the gas phase.5 NO3•, which is rapidly photolyzed during daytime, is a highly reactive electrophilic radical and the most important nighttime tropospheric oxidant [E0 (NO3•/NO3−) = 2.3−2.5 V vs NHE].6 In polluted urban areas, [NO3•] of up to 400 ppt have been measured.7 NO3• is also produced indoors, mainly through inefficient burning of solid fuels and biomass, and through oxidation of NO2• by Criegee intermediates that result from reaction of VOCs with O3 (mainly from outdoor-indoor transport).8 The complexity of reactions occurring between the many components in the atmosphere presents a significant challenge in determining the adverse health effects of the single pollutants. Any biochemical effect caused by inhalation of air pollutants, the primary route of exposure, will take place in the airway surface fluids (ASF) of the respiratory tract and/or lungs.9 Because both NO2• and O3 are only moderately soluble in water [KH (NO2•) = 0.04 mol kg−1 bar−1, KH (O3) = 0.013 mol kg−1 bar−1; at 298 K],10 the majority of these pollutants will reach the lower respiratory tract, where they eventually dissolve in the ASF throughout the conducting airways of the lung. The ASF contain small molecular-weight antioxidants, most importantly glutathione, ascorbic and uric acid, which act as first line of defense to protect the underlying epithelial cells against oxidative damage, for example by environmental pollutants. However, when oxidative damage exceeds the capacity of the antioxidant defense system, proteins and lipids present on cell surfaces or in the ASF are attacked.11 The 2137

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Scheme 2. Proposed Mechanism of the NO3•-Induced Oxidation of Phenylalanine

AMINO ACIDS AND PEPTIDES

Reaction of NO3• with N- and C-Protected Amino Acids in the Absence of NO2•, O3, and O2

NO3• was generated in situ in acetonitrile from cerium(IV) ammonium nitrate (CAN) through photoinduced ET in the presence of the respective substrate (Chart 2).25 Chart 2. Generation of NO3• in Solution

These conditions enabled to study the reactions of NO3• “in isolation” without interference by other radical or nonradical oxidants typically present in the environment. The reactions were quenched through the addition of water, followed by extraction and purification by chromatography. All experiments were performed with N- and C-protected amino acids to mimic an extended peptide structure. Phenylalanine (1a), which is usually inert to radical and nonradical oxidants produced through natural metabolic pathways, was completely consumed upon reaction with an excess of NO3• (3 equiv of CAN was typically used). The nitrate ester 2a was obtained as the major product, in addition to alcohol 3a, the β-keto amino acid 4a and alkene 5a (Scheme 1).25

NO3• (or CAN, respectively), they were still obtained when [CAN] = [1a]. Under these conditions, 1a was only incompletely consumed, which suggests that phenylalanine, once damaged, is more susceptible to further oxidation than the intact amino acid. Contrary to chemical synthesis, which aims at high yields, in biological systems even small modifications can have major impacts, for example if the damaged amino acid is critical for signaling processes or protein−protein interactions.28 NO3•-induced oxidation of methionine 11 led to nondiastereoselective formation of sulfoxide 12 (Scheme 3).29 The

Scheme 1. Reaction of Phenylalanine and Substituted Derivatives with NO3•

Scheme 3. Reaction of Methionine with NO3•

Competition experiments involving phenylalanines 1a−c with different electron density at the aromatic ring, as well as laser flash photolysis and computational studies26 provided support for a mechanism initiated by oxidative ET, in agreement with rate data in the literature.27 The resulting aryl radical cation 6 subsequently undergoes deprotonation to yield the benzyl radical 7 (Scheme 2). A second ET leads to the benzyl cation 8, which is followed by recombination with nitrate ions from the sterically less hindered face to give the β-nitrate threo-2a with high diastereoselectivity. A minor pathway involves proton loss in 8 to give alkene 5a. Further oxidation of 2a leads to the αnitrooxy-substituted benzyl cation 9 after deprotonation, which recombines with nitrate to form the dinitrate 10. The latter hydrolyses to the β-keto phenylalanine 4a, as indicated by the observation of a partially hydrolyzed product possessing a benzylic hydroxyl and nitrate substituent (not shown). Alcohol 3a likely results from hydrolysis of nitrate 2a. Although formation of all products requires more than one equivalent of

reaction might proceed through a two-electron oxidation at sulfur by NO3•, followed by trapping of the cationic intermediate 13 with water during workup (pathway a). However, since quenching the reaction with H218O did not give 18O containing sulfoxide 12, this mechanism can be excluded.29 We therefore suggest an addition/elimination process, which involves a short-lived methionine-NO3• adduct 14 with a two-center-three-electron O∴S bond (pathway b), similar to the adduct resulting from addition of hydroxyl radicals to methionine.30 Subsequent homolytic β-fragmentation leads to 12 with release of NO2•. 2138

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[NO+NO3−].16 Apart from the carcinogenicity of N-nitrosamides,33 this clearly demonstrates that ionic chemistry, specifically nitrosations by NO+, contributes to the reactivity of NO2• toward biological molecules. Interestingly, neither NO2• nor O3 in isolation reacted with the aromatic ring in phenylalanine. Nitrophenylalanine 16 could therefore be regarded as a marker for combined NO2•/O3 damage. Reaction of tyrosine 15a with NO2• in both absence and presence of O3 gave nitrotyrosines 17a and 18a, along with the respective N-nitroso products 17b and 18b.31 Tyrosine is a common target for oxidation processes in biological systems, and formation of nitrotyrosine occurs in a wide range of inflammatory−immune responses.28 For example, patients with asthma show increased 3-nitrotyrosine concentrations in the exhaled breath condensate34 and a strong immune reactivity for nitrotyrosine in the airway epithelium and inflammatory cells in the airways.35 This is significant in light of our finding that reaction of tyrosine with NO2• could provide a direct pollutionderived pathway to nitrotyrosines in the airways. Protection of the hydroxyl group in tyrosine dramatically lowered its susceptibility toward oxidative damage. Thus, while O-acetyl tyrosine 15b did not react with either NO2• or O3 in isolation, exposure to both oxidants led to formation of 3nitrotyrosine derivative 19a through reaction with in situ generated NO3•. In addition, NO2•-mediated N-nitrosation also occurred to give 19b. Similar to tyrosine, tryptophan 21 is also susceptible to oxidation. The reaction with NO2•, both in the presence or absence of O3, gave cis-fused pyrroloindolines 22 possessing a bridgehead nitro substituent as major products (Scheme 5).31,36

Reaction of NO3• with N- and C-Protected Amino Acids in the Presence of NO2•, O3, and O2

In the environment NO3• is always accompanied by other oxidants, in particular NO2•, O3 and O2. Knowledge of the contribution of the various species to pollution derived oxidative damage is therefore crucial. In situ generation of NO3• through reaction of excess NO2• ([NO2•] ca. 0.16 M) in a stream of ozonized O2 (reaction 4) in either dichloromethane or acetonitrile, followed by neutralization with aqueous bicarbonate enabled exploration of the combined effects of these pollutants. Under these conditions, phenylalanine 1a was oxidized to nitrophenylalanine 16a (Scheme 4).31 Scheme 4. Reaction of Phenylalanine and Tyrosines with NO2• and O3

Scheme 5. Reaction of Tryptophan with NO2• and O3

Products arising from benzylic oxidation, which were formed in the reaction with NO3• in the absence of NO2•, O3 and O2, were not obtained. This outcome suggests trapping of the initially formed radical cation 6 by recombination with NO2•,32 which obviously outcompetes loss of the benzylic proton that occurs in the absence of NO2• (see Scheme 2). Deprotonation in the resulting σ-complex 20 leads to 16a. The mechanism is supported by the ortho- and para-selectivity of this reaction, which is similar to electrophilic aromatic nitrations suggesting a common intermediate.32 Control experiments revealed that 1a did not react under our experimental conditions with a mixture of concentrated nitric and sulfuric acids (Scheme 4), which clearly shows that formation of 16a does not involve NO2+. The concurrently occurring N-nitrosation of the amide moiety to give 16b is caused by N2O4 through the ionic form

Ring nitration through ET and formation of 22a occurred when the reaction was performed in a nonpolar solvent (dichloromethane), whereas NO2•-mediated N-nitrosation to give 22b was observed in acetonitrile, which, due to its higher polarity, might provide additional stabilization of the ionic structure of N2O4. Diastereomeric pyrroloindolines with 2139

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Accounts of Chemical Research opposite configuration at the fused cyclopentyl rings and a bridgehead hydroxyl group were byproducts in this reaction (not shown).31,36 Formation of the pyrroloindoline framework is likely initiated by oxidative ET at the more electron-rich heteroaromatic ring to give the resonance stabilized radical cation 23 (Scheme 5). The observed bridgehead nitro substituent suggests trapping of 23 by NO2•, which is followed by a cisselective nucleophilic cyclization involving the amide group. Although tryptophan is also readily attacked by O3 (not shown),19 in the presence of NO2• this reaction was largely suppressed, indicating that aromatic oxidation by NO2• and/or in situ generated NO3• occurs faster than ozonolysis. Similar to the reaction involving NO3• (see Scheme 3), treatment of methionine 11 with NO2•/O3 yielded a mixture of sulfoxide 12 and sulfone 26 (Scheme 6). The former was the

Scheme 7. Reaction of Phenylalanine-Containing Dipeptides with NO2• and O3

Scheme 6. Reaction of Methionine with NO2• and O3

with NO2• exhaustive nitration of both aromatic rings was found in both absence or presence of O3, giving the isomeric tri- and tetranitro dipeptides 30c−e (Scheme 8).36 Scheme 8. Reaction of Tyrosine-Containing Dipeptides with NO2• and O3 only isolable product in the reaction of 11 with NO2•, while the reaction of 11 with O3 led to a 1:1 mixture of 12 and 26.29 Thus, formation of methionine sulfone could be considered as a marker for O3 pollution, whereas methionine sulfoxide is a nonspecific oxidation product resulting from O3, NO2• or NO3• pollution, respectively. Reaction of NO3• with Dipeptides in the Presence of NO2•, O3, and O2

Dipeptides provide an excellent model for assessing relative reactivities of different side chains toward oxidative damage by NO3• or NO2• (in either the absence or presence of O3), as well as the role of the peptide bond in these processes. Interestingly, in the reaction of diphenylalanine 27a with NO2•/O3 mixtures, nitration of one aromatic ring to give 28a/b was only a minor pathway with no preference for the N- or the C-terminal residue (Scheme 7).36 The major product, phenylalanine 1a, was formed through an unexpected cleavage of the peptide bond. Closer inspection revealed that this fragmentation was caused by NO2• and did not require O3. Likewise, in the reaction of NO2•/O3 mixtures with the dipeptide 27b fragmentation of the peptide backbone also occurred as major pathway. The N-acetylated amino group in the resulting tyrosine 15b suggests that this fragmentation occurs with concomitant migration of the acetyl moiety from the N-terminus of the dipeptide to the C-terminal amino acid. On the other hand, aromatic nitration by in situ generated NO3•, which took place exclusively at the phenylalanine residue to give 28c/d, was only a minor reaction. The NO2•-mediated peptide fragmentation was largely suppressed in dipeptides consisting of aromatic amino acids with readily oxidizable side chains. Exposure of phenylalaninetyrosine 29a to NO2• in the absence and presence of O3 led to mono- and dinitration of tyrosine to give 30a and 30b as the major products, respectively, leaving the less oxidizable phenylalanine residue intact. In the reaction of dityrosine 29b

Similarly, selective oxidation of the tryptophan residue occurred when phenylalanine-tryptophan 31 was exposed to NO2• or NO2•/O3 mixtures (Scheme 9). The major product, pyrroloindoline 32 was formed through oxidative cyclization involving an internal peptide bond, which requires considerable conformational flexibility. The reactions of tripeptides 33, which have a central tryptophan residue, became very unselective. The HRMS data of the complex reaction mixture indicate formation of small amounts of cyclized products 34. In the case of 33a, peptide fragmentation to give the nitrotryptophan-containing dipeptide 35 also occurred as a minor pathway.37 This suggests that NO2•-induced formation of pyrroloindolines could occur with sterically unhindered tryptophan residues located at the peptide terminus, where both flexibility and accessibility of the peptide chain is comparatively high. Dipeptide 36 reacted with NO2•/O3 mixtures through selective oxidation of the methionine residue to give the sulfoxide 37 and the sulfone 38 (Scheme 10).29 Similar to the reaction of the single amino acid 11, 37 is the sole product in the reaction of NO2•, whereas both 37 and 38 are formed in the reaction with O3. NO2•-induced peptide cleavage did not occur. 2140

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mechanistic insight was obtained by studying the reaction of NO2• with a series of di-, tri-, and tetrapeptides (Table 1).38 These data show that (i) the process requires a secondary amide (entries 4, 7 vs 5, 8) and leads to expulsion of sterically unhindered amino acid residues, preferably glycine; (ii) steric hindrance at the peptide bond (caused by large α-substituents) lowers the reaction efficiency (entries 3 and 11); (iii) tri- and tetrapeptides containing several glycine residues are shortened through successive glycine expulsion (entries 4, 9, and 10); and (iv) peptides with sequential aromatic amino acids eliminate the least sterically encumbered N-terminal residue (entry 7). Overall, the peptide chain is contracted in the N → C direction by expelling one amino acid residue at a time with simultaneous fusion of the remaining molecular termini. This continues until “accessible” peptide bonds are no longer available and/or NO2• is depleted. 1 H NMR mechanistic studies for the reaction of diphenylalanine 27a with NO2• revealed that successive amide N-nitrosation to give 39 occurs, starting at the less sterically hindered N-terminus, which suggests involvement of dimeric NO2• as nitrosating agent (Scheme 11).16 The actual

Scheme 9. Reaction of Tryptophan-Containing Di- and Tripeptides with NO2• and O3

Scheme 11. Mechanistic Studies of the NO2•-Mediated Peptide Fragmentation Scheme 10. Reaction of a Methionine-Containing Dipeptide with NO2• and O3

Reaction of NO2• with Short Oligopeptides

The NO2•-mediated backbone fragmentation in dipeptides was a very minor pathway (if at all), when side chain oxidation provided an efficient “sink” for NO2•. On the other hand, because of the potential biological impact of such backbone scissions for peptide sequences with inert side chains,

fragmentation requires an aqueous environment at a physiologically relevant near neutral pH and leads to the shortened peptide with release of the C-terminal amino acid as αhydroxylated derivative 40.38

Table 1. NO2•/N2O4-Mediated Peptide Backbone Fragmentation entry

peptidec

products

1 2 3 4 5 6 7 8 9 10 11

AcNH-Gly-Phe-CO2Me AcNH-Ala-Phe-CO2Me AcNH-Val-Phe-CO2Med AcNH-Gly-Gly-Phe-CO2Me PhthN-Gly-Gly-Phe-CO2Me AcNH-Gly-Ala-Phe-CO2Me AcNH-(4-OAc)Phe-Phe-(4-OAc)Phe-CO2Me PhthN-(4-OAc)Phe-Phe-(4-OAc)Phe-CO2Me AcNH-Gly-Gly-Gly-Phe-CO2Me PhthN-Gly-Gly-Gly-Phe-CO2Me AcNH-Val-Val-Gly-Phe-CO2Med

AcNH-Phe-CO2Me AcNH-Phe-CO2Me AcNH-Phe-CO2Mee AcNH-Phe-CO2Me PhthN-Gly-Phe-CO2Me AcNH-Ala-Phe-CO2Me (40%)f + AcNH-Phe-CO2Me (60%) AcNH-Phe-(4-OAc)Phe-CO2Mee no reaction AcNH-Gly-Gly-Phe-CO2Me (20%)f + AcNH-Gly-Phe-CO2Me (30%) + AcNH-Phe-CO2Me (50%) PhthN-Gly-Gly-Phe-CO2Me (10%)f + PhthN-Gly-Phe-CO2Me (90%) AcNH-Val-Val-Phe-CO2Mee

a Product purity at least 80% (determined by 1H NMR) unless stated otherwise. bConditions: NO2•/N2O4, acetonitrile, 10 °C, 10 min, then aqueous NaHCO3. cAc = acetyl, Phth = phthaloyl, Gly = glycine, Ala = alanine, Phe = phenylalanine, Val = valine, (4-OAc)Phe = O-acetyltyrosine. dReaction time 4.5 h. eConsiderable formation of unidentified decomposition products. fProduct ratio in % determined by 1H NMR.

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Accounts of Chemical Research These findings are consistent with the mechanism in Scheme 12, where N-nitrosation of dipeptide 41 activates the acetyl

diastereoselective formation of nitroimine nitrates 50b,c occurred exclusively (Scheme 13).46

Scheme 12. Proposed Mechanism of the NO2•-Mediated Peptide Fragmentation

Scheme 13. Reaction of Cholesterols with NO2•

Even in the case of unprotected cholesterol 49a a reaction with the π system was observed, clearly demonstrating the susceptibility of the alkene moiety to oxidative damage. In the presence of moisture the reaction with NO2• led to 6nitrocholesterol, as shown exemplary for the reaction of the Oacetylated derivative 49c. Intuitively, formation of 51c could be rationalized through a radical pathway, consisting of NO2• addition to the less hindered site of the alkene, followed by hydrogen abstraction, for example by NO2•,14 which restores the π system. However, the finding that 51c is also rapidly formed in the reaction of NO2• with moistened 50c strongly suggests an ionic process involving [NO+NO3−]. The mechanism in Scheme 14 proposes initial electrophilic addition of NO+ to the CC bond, which is followed by recombination of 52 with NO3− from the sterically unhindered face to give the nitroso nitrate ester 53.

group in 42 for attack by the nearby amide nitrogen. The resulting pyrrolidinone 43 rearranges to the diazotic acid 44 through concerted hydrogen migration−fragmentation, which is associated with a moderate activation barrier (Ea) and transfers the acyl residue onto the adjacent peptide bond.36 Deprotonation of 44 upon neutralization triggers a cyclization/fragmentation process in 45 to release the shortened peptide, e.g., 47, and oxadiazole 46, which is ultimately converted into the hydroxy acid 48.38 It is worth noting that multiple backbone scission in polylysine and polyarginine by NO2• in water at pH 7.4 has been reported previously, but detailed mechanistic studies were not performed.39 In summary, although oxidation of reactive side chains by NO2• is considerably faster than the NO2•-mediated peptide cleavage,36 the latter could potentially be highly relevant in segments with “nonvulnerable” side chains closer to the peptide terminus, which are not tied up in complex secondary and tertiary structures that limit access for environmental oxidants.

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Scheme 14. Proposed Mechanism for the Reaction of Cholesterol with NO2•

LIPIDS

Reaction of NO2• with Cholesterol

Cholesterol (49a) is the most abundant neutral lipid in the epithelial lining fluid in the lower airways,40 where it is directly exposed to oxidizing pollutants.41 It has been shown that cholesterol reacts with O3 at the alkene moiety leading to formation of bioactive oxysterols,42 which, if not eliminated, induce apoptosis and cytotoxicity.43 NO2•, on the other hand, is believed to damage cholesterol largely by reaction with the hydroxyl group to give cholesteryl nitrite, in addition to radical processes occurring at the alkene.44,45 However, a detailed product analysis under various experimental conditions revealed a distinctly different outcome for the reaction with NO2•. When O-protected cholesterols 49b,c were exposed in the absence of moisture to either gaseous NO2•, or to NO2• dissolved in aprotic organic solvents, 2142

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Accounts of Chemical Research Tautomerization yields oxime 54, which is subsequently converted to the nitroimine 50 through reaction with NO+.47 In the presence of water elimination of nitric acid occurs through nucleophilic allylic substitution at the imine nitrogen in 50, which is followed by protonation of the nitro group in 55. Subsequent heterolytic N−N bond cleavage in 56 yields 6nitroso cholesterol 57 and nitrous acid. M06-2X/6-311++G** calculations for a simplified model system predict a modest Ea of 51 kJ mol−1 and considerable exothermicity (ΔE = −109 kJ mol−1) for the latter process.46 In the final step, 57 is oxidized to 51 by NO2•,48 which is, in fact, the only reaction in the entire sequence where NO2• acts as a radical species. It can be assumed that 6-nitrocholesterol 51 would be ultimately obtained in the aqueous environment of the airways, where it could serve as marker for NO2• pollution.

precursor of NO+ in the acidic environment created by this process),17 our findings could be the key to our understanding why NO2•, despite its low water solubility, has such pronounced biological effects in vivo. These results provide the basis for future work in aqueous environments to gain fundamental understanding of the chemistry at the air/ASF interface.

CONCLUSIONS This work provided new insight into oxidative damage in selected amino acids and small peptides caused by the environmental pollutants NO3• and NO2• in the absence and presence of O3. In vitro product and mechanistic model studies enabled identification of important reaction pathways, which provide useful guidelines for future epidemiological investigations. A clear synergism resulting from combined exposure to NO2• and O3 was found only in the reaction with phenylalanine, which indicates that in situ formed strongly oxidizing NO3• is responsible for these transformations. More easily oxidizable amino acids are directly attacked by NO2• and converted to the same products practically independent of the presence of O3. These findings suggest that the observed synergism in epidemiological exposure studies to NO2•/O3 mixtures might possibly result from a faster destruction of the ASF antioxidant defense shield under these conditions. Kinetic data for the reaction of important ASF constituents with NO2• and NO3• are therefore urgently required to assess the relevance of individual reactions in the biological context. Our studies revealed a novel fragmentation/rearrangement in peptides, which involves NO2•/N2O4 and proceeds through an ionic mechanism that is initiated by N-nitrosation of the peptide bond. This enables contraction of the peptide chain in the N → C direction by expelling one amino acid residue with simultaneous fusion of the remaining molecular termini to form a new peptide bond. Thus, NO2• damages peptides through both radical oxidation of reactive side chains as well as through nonradical peptide backbone fragmentation/rearrangement. Apart from the potential biological impact of the latter process, this finding is particularly significant, since it contradicts the generally accepted view that oxidative stress from environmental radicals, in particular damage resulting in peptide backbone cleavage, proceeds through radical pathways.49 Likewise, the reaction of NO2• with the π system in cholesterol occurs without involvement of radical steps. The important role of moisture on the reaction outcome shows that careful analysis of the products under different experimental conditions is crucial for obtaining detailed knowledge of the reaction mechanism. Although it is currently unknown what factors determine whether NO2• reacts through radical or through ionic pathways involving NO+, our model studies, although largely performed in aprotic organic solvents, clearly reveal the importance to include ionic chemistry when assessing NO2• toxicity. In light of the recent observations that anions mediate NO2• absorption in the ASF through hydrolytic disproportionation into nitrate and nitrite (which is the

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.accounts.6b00219. Experimental details for Schemes 3, 4, 6, 9, and 10 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +61 (0)3 8344 2425. Fax: +61 (0)3 9347 8189. Email: [email protected]. Author Contributions

The manuscript was written through equal contribution of both authors, who have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. Biographies Luke F. Gamon received his B.Sc. (2009) and M.Sc. (2011) at The University of Melbourne, Australia. He has recently obtained his Ph.D. degree in the area of oxidative damage of biological molecules by radical air pollutants under the supervision of Assoc. Prof. Uta Wille. He received an Endeavour Postdoctoral Research Fellowship (2016) to support the study of protein oxidation in Prof. Mike Davies' laboratory at the University of Copenhagen, Denmark. Uta Wille received her Diploma and Ph.D. degrees from the University of Kiel, Germany (1988 and 1993) in atmospheric chemistry under the supervision of Prof. Ralph N. Schindler. She completed her Habilitation in Organic Chemistry at the same institution in 1999, where she merged atmospheric radicals with organic synthesis. During her Habilitation she undertook a postdoctoral stay with Prof. Bernd Giese at the University of Basel, Switzerland. She had a position as Privatdozent at the University of Kiel (1999−2002) and was appointed as Lecturer in the School of Chemistry of The University of Melbourne in 2003, followed by promotion to Senior Lecturer (2006) and Associate Professor (2011). She has been a Chief Investigator in the Centre of Excellence for Free Radical Chemistry and Biotechnology of the Australian Research Council (2005−2013).

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ACKNOWLEDGMENTS This work was supported by the Australian Research Council under the Centre of Excellence Scheme, the University of Melbourne and the National Computing Infrastructure facility (NCI).

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