Phenylalanine Residues Are Very Rapidly Damaged by Nitrate Radicals (NO3 ⋠) in an Aqueous Environment.

Kinetic studies revealed that nitrate radicals (NO3 ⋅), which are formed through reaction of the noxious air pollutants nitrogen dioxide (NO2 ⋅) and ozone (O3 ), very rapidly oxidize phenylalanine residues in an aqueous environment, with overall rate coefficients in the 108 -109  M-1 s-1 range. With amino acids and dipeptides as model systems, the data suggest that the reaction proceeds via a π-complex between NO3 ⋅ and the aromatic ring in Phe, which subsequently decays into a charge transfer (CT) complex. The stability of the π-complex is sequence-dependent and is increased when Phe is at the N terminus of the dipeptide. Computations revealed that the considerably more rapid radical-induced oxidation of Phe residues in both neutral and acidic aqueous environments, compared to acetonitrile, can be attributed to stabilization of the CT complex by the protic solvent; this clearly highlights the health-damaging potential of exposure to combined NO2 ⋅ and O3 .

Damage of amino acids by aliphatic peroxyl radicals: a kinetic and computational study.

Absolute second-order rate coefficients for the reaction of the N- and C-protected amino acids tyrosine (Tyr), tryptophan (Trp), methionine (Met) and proline (Pro) with triethylamine-derived aliphatic peroxyl radical TEAOO˙, which was used as a model for lipid peroxyl radicals, were determined using laser flash photolysis. For Ac-Tyr-OMe a rate coefficient of 1.4 × 104 M-1 s-1 was obtained, whereas the reactions with Ac-Trp-OMe and Ac-Met-OMe were slower by a factor of 4 and 6, respectively. For the reaction with Ac-Pro-OMe only an upper value of 103 M-1 s-1 could be determined, suggesting that Pro residues are not effective traps for lipid peroxyl radicals. Density functional theory (DFT) calculations revealed that the reactions proceed via radical hydrogen atom transfer (HAT) from the Cα position, indicating that the rate is determined by the exothermicity of the reaction. In the case of Ac-Tyr-OMe, HAT from the phenolic OH group is the kinetically preferred pathway, which shuts down when hydrogen bonding with an amine occurs. In an alkaline environment, where the phenolic OH group is deprotonated, the reaction is predicted to occur preferably at Cß, likely through a proton-coupled electron transfer (PCET) mechanism.

Oxidative Damage of Aliphatic Amino Acid Residues by the Environmental Pollutant NO3·: Impact of Water on the Reactivity.

The rate of oxidative damage of aliphatic amino acids and dipeptides by the environmental pollutant nitrate radical (NO3·) in an aqueous acidic environment was studied by laser flash photolysis. The reactivity dropped by a factor of about four for amino acid residues with secondary amide bonds and by a factor of up to nearly 20 for amino acid residues with tertiary amide bonds, compared with that in acetonitrile. According to density functional theory studies, the lower reactivity is due to protonation of the amide moiety, whereas in neutral water, hydrogen bonding with the amide should have little impact on the absolute reaction rate compared with that in acetonitrile. This finding can be rationalized by the high reactivity and broad reaction pattern of NO3·. Although hydrogen bonding involving the amide group raises the energies associated with some electron transfer processes, alternative low-energy pathways remain available so that the overall reaction rate is barely affected. The undiminished high reactivity of NO3· toward aliphatic amino acid residues in a neutral aqueous environment highlights the health-damaging potential of exposure to the combined air pollutants nitrogen dioxide (NO2·) and ozone (O3).

Next-generation enhanced-efficiency fertilizers for sustained food security.

Nitrogen losses in agricultural systems can be reduced through enhanced-efficiency fertilizers (EEFs), which control the physicochemical release from fertilizers and biological nitrogen transformations in soils. The adoption of EEFs by farmers requires evidence of consistent performance across soils, crops and climates, paired with information on the economic advantages. Here we show that the benefits of EEFs due to avoided social costs of nitrogen pollution considerably outweigh their costs-and must be incorporated in fertilizer policies. We outline new approaches to the design of EEFs using enzyme inhibitors with modifiable chemical structures and engineered, biodegradable coatings that respond to plant rhizosphere signalling molecules.

Substituted 1,2,3-triazoles: a new class of nitrification inhibitors.

Nitrogen (N) fertilisers amended with nitrification inhibitors can increase nitrogen use efficiencies in agricultural systems but the effectiveness of existing commercial inhibitor products, including 3,4-dimethylpyrazole phosphate (DMPP), is strongly influenced by climatic and edaphic factors. With increasing pressure to reduce the environmental impact of large-scale agriculture it is important to develop new nitrogen-stabilising products that can give reliable and consistent results, particularly for warmer climatic conditions. We synthesised a library of 17 compounds featuring a substituted 1,2,3-triazole motif and performed laboratory incubations in two south-eastern Australian soils. In the neutral (pH 7.3) soil, the compounds N002, N013, N016 and N017, which possess short non-polar alkyl or alkynyl substituents at the triazole ring, retained NH4+-N concentrations at 35 °C soil temperature to a better extent (P < 0.001) than DMPP. In the alkaline soil (pH 8.8) N013 performed better with regards to NH4+-N retention (P = 0.004) than DMPP at 35 °C soil temperature. Overall, our data suggest that substituted 1,2,3-triazoles, which can be synthesized with good yields and excellent atom economy through 1,3-dipolar cycloaddition from readily available starting materials, are promising nitrification inhibitors performing similar to, or better than DMPP, particularly at elevated soil temperatures.

Synthesis of spirocyclic heterocycles from α,ß-unsaturated N-acyliminium ions.

The reactions of α,ß-unsaturated N-acyliminium ions, generated in situ from 4(S)-O-substitutedhydroxy-5-hydroxy-5-vinyl-N-alkylpyrrolidin-2-ones, with allylsilanes and indoles leading to the formation of spirocyclic heterocycles, are reported. Six single crystal X-ray structures and extensive 2D NMR experiments confirmed the structures and stereochemistries of these products. In addition, computational studies provided mechanistic insights and an understanding of the stereochemical outcomes of these reactions.

Ring Expansion of Thiolactams via Imide Intermediates: An Amino Acid Insertion Strategy.

The AgI -promoted reaction of thiolactams with N-Boc amino acids yields an N-(α-aminoacyl) lactam that can rearrange through an acyl transfer process. Boc-deprotection results in convergence to the ring-expanded adduct, thereby facilitating an overall insertion of an amino acid into the thioamide bond to generate medium-sized heterocycles. Application to the site-specific insertion of amino acids into cyclic peptides is demonstrated.

Oxidative damage of proline residues by nitrate radicals (NO3˙): a kinetic and product study.

Tertiary amides, such as in N-acylated proline or N-methyl glycine residues, react rapidly with nitrate radicals (NO3˙) with absolute rate coefficients in the range of 4-7 × 108 M-1 s-1 in acetonitrile. The major pathway proceeds through oxidative electron transfer (ET) at nitrogen, whereas hydrogen abstraction is only a minor contributor under these conditions. However, steric hindrance at the amide, for example by alkyl side chains at the α-carbon, lowers the rate coefficient by up to 75%, indicating that NO3˙-induced oxidation of amide bonds proceeds through initial formation of a charge transfer complex. Furthermore, the rate of oxidative damage of proline and N-methyl glycine is significantly influenced by its position in a peptide. Thus, neighbouring peptide bonds, particularly in the N-direction, reduce the electron density at the tertiary amide, which slows down the rate of ET by up to one order of magnitude. The results from these model studies suggest that the susceptibility of proline residues in peptides to radical-induced oxidative damage should be considerably reduced, compared with the single amino acid.

Reactions of a distonic peroxyl radical anion influenced by SOMO-HOMO conversion: an example of anion-directed channel switching.

In free radicals the singly occupied molecular orbital (SOMO) typically has the highest energy. Recent examples of distonic radical anions were found, however, to disobey the usual orbital configuration, with the singly occupied molecular orbital buried energetically underneath doubly occupied orbitals. This unusual ordering of electrons, which contradicts the aufbau principle, has been characterized as SOMO-HOMO orbital conversion and is expected to perturb radical anion reactivity by branching toward anion-driven over radical-driven processes. Here, we use ion trap mass spectrometry and ab initio calculations to demonstrate that SOMO-HOMO orbital conversion influences the reactivity of a distonic peroxyl radical anion. Experimentally, we generated a distonic radical anion of ß-hydroxy glutaric acid, ˙CH2CH(OH)CH2C(O)O-, and investigated its subsequent reaction with O2 in the gas phase. Theoretical calculations predict that reactions proceed through five isomeric C4H6O5˙- intermediates, two of which exhibit SOMO-HOMO conversion. The detected product ions, corresponding to loss of ˙OH + CO2, ˙OH + HCHO, HO2˙, and HO2˙ + CO2 from the peroxyl radical, can all be reconciled by the proposed reaction mechanism. Finally, we compare the oxygen recombination reaction of the distonic radical ion to the corresponding neutral radical (i.e., ˙CH2CH(OH)CH2C(O)OH). These calculations demonstrate that SOMO-HOMO conversion results in channel switching in the distonic radical anion, suppressing radical-driven mechanisms and promoting pathways that directly involve the anion site.

1,2-Addition versus homoconjugate addition reactions of indoles and electron-rich arenes to α-cyclopropyl N-acyliminium ions: synthetic and computational studies.

An investigation of the reactivity of α-cyclopropyl N-acyliminium ions towards indoles has resulted in the unprecedented synthesis of 5-cyclopropyl-5-(3-indoyl)pyrrolidin-2-ones via 1,2-addition reactions and, in the case of highly electron deficient indoles and electron rich arenes, spiroheterocycles via sequential homoconjugate and 1,2-addition reactions with often high diastereoselective control at the C-5 quaternary stereocentres. Computational studies provided support for the proposed mechanisms and stereochemical outcome of these reactions, clearly showing that the 1,2-addition pathway is kinetically controlled. In reactions where the 1,2-adduct is destabilised, for example when the arene ring is less nucleophilic, the 1,2-addition is reversible and the thermodynamically preferred homoconjugate addition and subsequent rearrangement and cyclisation reactions become the major pathway.

Oxidative Damage in Aliphatic Amino Acids and Di- and Tripeptides by the Environmental Free Radical Oxidant NO3•: The Role of the Amide Bond Revealed by Kinetic and Computational Studies.

Kinetic and computational data reveal a complex behavior of the important environmental free radical oxidant NO3• in its reactions with aliphatic amino acids and di- and tripeptides, suggesting that attack at the amide N-H bond in the peptide backbone is a highly viable pathway, which proceeds through a proton-coupled electron transfer (PCET) mechanism with a rate coefficient of about 1 × 106 M-1 s-1 in acetonitrile. Similar rate coefficients were determined for hydrogen abstraction from the α-carbon and from tertiary C-H bonds in the side chain. The obtained rate coefficients for the reaction of NO3• with aliphatic di- and tripeptides suggest that attack occurs at all of these sites in each individual amino acid residue, which makes aliphatic peptide sequences highly vulnerable to NO3•-induced oxidative damage. No evidence for amide neighboring group effects, which have previously been found to facilitate radical-induced side-chain damage in phenylalanine, was found for the reaction of NO3• with side chains in aliphatic peptides.

Reversible Photoisomerization of the Isolated Green Fluorescent Protein Chromophore.

Fluorescent proteins have revolutionized the visualization of biological processes, prompting efforts to understand and control their intrinsic photophysics. Here we investigate the photoisomerization of deprotonated p-hydroxybenzylidene-2,3-dimethylimidazolinone anion (HBDI-), the chromophore in green fluorescent protein and in Dronpa protein, where it plays a role in switching between fluorescent and nonfluorescent states. In the present work, isolated HBDI- molecules are switched between the Z and E forms in the gas phase in a tandem ion mobility mass spectrometer outfitted for selecting the initial and final isomers. Excitation of the S1 ← S0 transition provokes both Z → E and E → Z photoisomerization, with a maximum response for both processes at 480 nm. Photodetachment is a minor channel at low light intensity. At higher light intensities, absorption of several photons in the drift region drives photofragmentation, through channels involving CH3 loss and concerted CO and CH3CN loss, although isomerization remains the dominant process.

Amide Neighbouring-Group Effects in Peptides: Phenylalanine as Relay Amino Acid in Long-Distance Electron Transfer.

In nature, proteins serve as media for long-distance electron transfer (ET) to carry out redox reactions in distant compartments. This ET occurs either by a single-step superexchange or through a multi-step charge hopping process, which uses side chains of amino acids as stepping stones. In this study we demonstrate that Phe can act as a relay amino acid for long-distance electron hole transfer through peptides. The considerably increased susceptibility of the aromatic ring to oxidation is caused by the lone pairs of neighbouring amide carbonyl groups, which stabilise the Phe radical cation. This neighbouring-amide-group effect helps improve understanding of the mechanism of extracellular electron transfer through conductive protein filaments (pili) of anaerobic bacteria during mineral respiration.

Environmental Polymer Degradation: Using the Distonic Radical Ion Approach to Study the Gas-Phase Reactions of Model Polyester Radicals.

A novel precursor to the distonic O- and C-centered radical cations Oxo+O• and Oxo+C• was designed and synthesized, which represents model systems for radicals produced during polyester degradation. The precursor is equipped with a nitrate functional group, which serves as a masked site for these alkoxyl and carbon radicals that are unleashed through collision-induced dissociation (CID). Oxo+O• and Oxo+C• feature a cyclic carboxonium ion as permanent charge tag to enable monitoring their ion-molecule reactions on the millisecond to second time scale in the ion trap of the mass spectrometer. The reactions of Oxo+O• and Oxo+C• with cyclohexene, cyclohexadiene, ethyl acetate, 1,1-dimethoxyethane, and 1,2-dimethoxyethane, which exhibit structural features present in both intact and defective polyesters, were explored through product and kinetic studies to identify "hot spots" for radical-induced damage in polyesters. All reactions with Oxo+O• were extremely fast and proceeded predominantly through HAT. Oxo+C• was about two orders of magnitude less reactive and did not noticeably damage aliphatic ester moieties through hydrogen abstraction on the time scale of our experiments. Radical addition to alkene π systems was identified as an important pathway for C-radicals, which needs to be included in polymer degradation mechanisms.

Reaction of Amino Acids, Di- and Tripeptides with the Environmental Oxidant NO3. : A Laser Flash Photolysis and Computational Study.

Absolute rate coefficients for the reaction between the important environmental free radical oxidant NO3. and a series of N- and C-protected amino acids, di- and tripeptides were determined using 355 nm laser flash photolysis of cerium(IV) ammonium nitrate in the presence of the respective substrates in acetonitrile at 298±1 K. Through combination with computational studies it was revealed that the reaction with acyclic aliphatic amino acids proceeds through hydrogen abstraction from the α-carbon, which is associated with a rate coefficient of about 1.8×106 m-1 s-1 per abstractable hydrogen atom. The considerably faster reaction with phenylalanine [k=(1.1±0.1)×107 m-1 s-1 ] is indicative for a mechanism involving electron transfer. An unprecedented amplification of the rate coefficient by a factor of 7-20 was found with di- and tripeptides that contain more than one phenylalanine residue. This suggests a synergistic effect between two aromatic rings in close vicinity, which makes such peptide sequences highly vulnerable to oxidative damage by this major environmental pollutant.

Oxidative Damage of Biomolecules by the Environmental Pollutants NO2• and NO3•.

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 ß-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 well-established 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.

Synthesis of Bridged Heterocycles via Sequential 1,4- and 1,2-Addition Reactions to α,ß-Unsaturated N-Acyliminium Ions: Mechanistic and Computational Studies.

Novel tricyclic bridged heterocyclic systems can be readily prepared from sequential 1,4- and 1,2-addition reactions of allyl and 3-substituted allylsilanes to indolizidine and quinolizidine α,ß-unsaturated N-acyliminium ions. These reactions involve a novel N-assisted, transannular 1,5-hydride shift. Such a mechanism was supported by examining the reaction of a dideuterated indolizidine, α,ß-unsaturated N-acyliminium ion precursor, which provided specifically dideuterated tricyclic bridged heterocyclic products, and from computational studies. In contrast, the corresponding pyrrolo[1,2-a]azepine system did not provide the corresponding tricyclic bridged heterocyclic product and gave only a bis-allyl adduct, while more substituted versions gave novel furo[3,2-d]pyrrolo[1,2-a]azepine products. Such heterocyclic systems would be expected to be useful scaffolds for the preparation of libraries of novel compounds for new drug discovery programs.

Synthesis of Peptides by Silver-Promoted Coupling of Carboxylates and Thioamides: Mechanistic Insight from Computational Studies.

The mechanism of the recently described N→C direction peptide synthesis through silver-promoted coupling of N-protected amino acids with thioacetylated amino esters was explored by using density functional theory. Calculation of the potential energy surfaces for various pathways revealed that the reaction proceeds through silver-assisted addition of the carboxylate to the thioamide, which is followed by deprotonation and silver-mediated extrusion of sulfur as Ag2 S. The resulting isoimide is the key intermediate, which subsequently rearranges to an imide through a concerted pericyclic [1,3]-acyl shift (O-sp(2) N 1,3-acyl migration). The proposed mechanism clearly emphasises the requirement of two equivalents of Ag(I) and basic reaction conditions, which is in full agreement with the experimental findings. Alternative rearrangement pathways involving only one equivalent of Ag(I) or through O-sp(3) N 1,3-acyl migration can be excluded. The computations further revealed that peptide couplings involving thioformamides require significant conformational changes in the intermediate isoformimide, which slow down the rearrangement process.

What Are the Potential Sites of Protein Arylation by N-Acetyl-p-benzoquinone Imine (NAPQI)?

Acetaminophen (paracetamol, APAP) is a safe and widely used analgesic medication when taken at therapeutic doses. However, APAP can cause potentially fatal hepatotoxicity when taken in overdose or in patients with metabolic irregularities. The production of the electrophilic and putatively toxic compound N-acetyl-p-benzoquinone imine (NAPQI), which cannot be efficiently detoxicated at high doses, is implicated in APAP toxicity. Numerous studies have identified that excess NAPQI can form covalent linkages to the thiol side chains of cysteine residues in proteins; however, the reactivity of NAPQI toward other amino acid side chains is largely unexplored. Here, we report a survey of the reactivity of NAPQI toward 11 N-acetyl amino acid methyl esters and four peptides. (1)H NMR analysis reveals that NAPQI forms covalent bonds to the side-chain functional groups of cysteine, methionine, tyrosine, and tryptophan residues. Analogous reaction products were observed when NAPQI was reacted with synthetic model peptides GAIL-X-GAILR for X = Cys, Met, Tyr, and Trp. Tandem mass spectrometry peptide sequencing showed that the NAPQI modification sites are located on the "X" residue in each case. However, when APAP and the GAIL-X-GAILR peptide were incubated with rat liver microsomes that contain many metabolic enzymes, NAPQI formed by oxidative metabolism reacted with GAIL-C-GAILR exclusively. For the peptides where X = Met, Tyr, and Trp, competing reactions between NAPQI and alternative nucleophiles precluded arylation of the target peptide by NAPQI. Although Cys residues are favorably targeted under these conditions, these data suggest that NAPQI can, in principle, also damage proteins at Met, Tyr, and Trp residues.

Fragmentation-Rearrangement of Peptide Backbones Mediated by the Air Pollutant NO2 (.).

The fragmentation-rearrangement of peptide backbones mediated by nitrogen dioxide, NO2 (.) , was explored using di-, tri-, and tetrapeptides 8-18 as model systems. The reaction, which is initiated through nonradical N-nitrosation of the peptide bond, shortens the peptide chain by the expulsion of one amino acid moiety with simultaneous fusion of the remaining molecular termini through formation of a new peptide bond. The relative rate of the fragmentation-rearrangement depends on the nature of the amino acids and decreases with increasing steric bulk at the α carbon in the order Gly>Ala>Val. Peptides that possessed consecutive aromatic side chains only gave products that resulted from nitrosation of the sterically less congested N-terminal amide. Such backbone fragmentation-rearrangement occurs under physiologically relevant conditions and could be an important reaction pathway for peptides, in which sections without readily oxidizable side chains are exposed to the air pollutant NO2 (.) . In addition to NO2 (.) -induced radical oxidation processes, this outcome shows that ionic reaction pathways, in particular nitrosation, should be factored in when assessing NO2 (.) reactivity in biological systems.