In mammalian tissues, ultraweak chemiluminescence arising from biomolecule oxidation has been attributed to the radiative deactivation of singlet molecular oxygen [O2 (1Δg)] and electronically excited triplet carbonyl products involving dioxetane intermediates. Herein, we describe evidence of the generation of O2 (1Δg) in aqueous solution via energy transfer from excited triplet acetone. This involves thermolysis of 3,3,4,4-tetramethyl-1,2-dioxetane, a chemical source, and horseradish peroxidase-catalyzed oxidation of 2-methylpropanal, as an enzymatic source. Both sources of excited carbonyls showed characteristic light emission at 1,270 nm, directly indicative of the monomolecular decay of O2 (1Δg). Indirect analysis of O2 (1Δg) by electron paramagnetic resonance using the chemical trap 2,2,6,6-tetramethylpiperidine showed the formation of 2,2,6,6-tetramethylpiperidine-1-oxyl. Using [18O]-labeled triplet, ground state molecular oxygen [18O2 (3Σg-)], chemical trapping of 18O2 (1Δg) with disodium salt of anthracene-9,10-diyldiethane-2,1-diyl disulfate yielding the corresponding double-[18O]-labeled 9,10-endoperoxide, was detected through mass spectrometry. This corroborates formation of O2 (1Δg). Altogether, photoemission and chemical trapping studies clearly demonstrate that chemically and enzymatically nascent excited carbonyl generates 18O2 (1Δg) by triplet-triplet energy transfer to ground state oxygen O2 (3Σg−), and supports the long formulated hypothesis of O2 (1Δg) involvement in physiological and pathophysiological events that might take place in tissues in the absence of light.

Diacetyl, methylglyoxal, and glyoxal are α-dicarbonyl catabolites prone to nucleophilic additions of amino groups of proteins and nucleobases, thereby triggering adverse biological responses. Because of their electrophilicity, in aqueous medium, they exist in a phosphate-catalyzed dynamic equilibrium with their hydrate forms. Diacetyl and methylglyoxal can be attacked by peroxynitrite (k(2) ≈ 1.0 × 10(4) M(-1) s(-1) and k(2) ≈ 1.0 × 10(5) M(-1) s(-1), respectively), a potent biological nucleophile and oxidant, yielding the acetyl radical from the homolysis of peroxynitrosocarbonyl adducts, and acetate or formate ions, respectively. We report here that glyoxal also reacts with peroxynitrite, yielding formate ion at rates at least 1 order of magnitude greater than does methylglyoxal. A triplet EPR signal (1:2:1; a(H) = 0.78 mT) attributable to hydrated formyl radical was detected by direct flow experiments. In the presence of the spin trap 2-methyl-2-nitrosopropane, the EPR spectrum displays the di-tert-butyl nitroxide signal, another signal assignable to the spin trapping adduct with hydrogen radical (a(N) = a(H) = 1.44 mT), probably formed from formyl radical decarbonylation, and a third EPR signal assignable to the formyl radical adduct of the spin trap (a(N) = 0.71 mT and a(H) = 0.14 mT). The novelty here is the detection of singlet oxygen ((1)Δ(g)) monomol light emission at 1270 nm during the reaction, probably formed by subsequent dioxygen addition to formyl radical and a Russell reaction of nascent formylperoxyl radicals. Accordingly, the near-infrared emission increases upon raising the peroxynitrite concentration in D(2)O buffer and is suppressed upon addition of O(2) ((1)Δ(g)) quenchers (NaN(3), l-His, H(2)O). Unequivocal evidence of O(2) ((1)Δ(g)) generation was also obtained by chemical trapping of (18)O(2) ((1)Δ(g)) with anthracene-9,10-divinylsulfonate, using HPLC/MS/MS for detection of the corresponding 9,10-endoperoxide derivative. Our studies add insights into the molecular events underlying nitrosative, oxidative, and carbonyl stress in inflammatory processes and aging-associated maladies.

Methylglyoxal is an α-oxoaldehyde putatively produced in excess from triose phosphates, aminoacetone, and acetone in some disorders, particularly in diabetes. Here, we investigate the nucleophilic addition of ONOO(-), known as a potent oxidant and nucleophile, to methylglyoxal, yielding an acetyl radical intermediate and ultimately formate and acetate ions. The rate of ONOO(-) decay in the presence of methylglyoxal [k(2,app) = (1.0 ± 0.1) × 10(3) M(-1) s(-1); k(2) ≈ 1.0 × 10(5) M(-1) s(-1)] at pH 7.2 and 25 °C was found to be faster than that reported with monocarbonyl substrates (k(2) < 10(3) M(-1) s(-1)), diacetyl (k(2) = 1.0 × 10(4) M(-1) s(-1)), or CO(2) (k(2) = 3-6 × 10(4) M(-1) s(-1)). The pH profile of the methylglyoxal-peroxynitrite reaction describes an ascendant curve with an inflection around pH 7.2, which roughly coincides with the pK(a) values of both ONOOH and H(2)PO(4)(-) ion. Electron paramagnetic resonance spin trapping experiments with 2-methyl-2-nitrosopropane revealed concentration-dependent formation of an adduct that can be attributed to 2-methyl-2-nitrosopropane-CH(3)CO(•) (a(N) = 0.83 mT). Spin trapping with 3,5-dibromo-4-nitrosobenzene sulfonate gave a signal that could be assigned to a methyl radical adduct [a(N) = 1.41 mT; a(H) = 1.35 mT; a(H(m)) = 0.08 mT]. The 2-methyl-2-nitrosopropane-CH(3)CO(•) adduct could also be observed by replacement of ONOO(-) with H(2)O(2), although at much lower yields. Acetyl radicals could be also trapped by added L-lysine as indicated by the presence of (ε)N-acetyl-L-lysine in the spent reaction mixture. This raises the hypothesis that ONOO(-)/H(2)O(2) in the presence of methylglyoxal is endowed with the potential to acetylate proteins in post-translational processes.

Diacetyl, like other alpha-dicarbonyl compounds, is reportedly cytotoxic and genotoxic. A food and cigarette contaminant, it is related with alcohol hepatotoxicity and lung disease. Peroxynitrite is a potent oxidant formed in vivo by the diffusion-controlled reaction of the superoxide radical anion with nitric oxide, which is able to form adducts with carbon dioxide and carbonyl compounds. Here, we investigate the nucleophilic addition of peroxynitrite to diacetyl forming acetyl radicals, whose reaction with molecular oxygen leads to acetate. Peroxynitrite is shown to react with diacetyl in phosphate buffer (bell-shaped pH profile with maximum at 7.2) at a very high rate constant ( k 2 = 1.0 x 10 (4) M (-1) s (-1)) when compared with monocarbonyl substrates ( k 2 < 10 (3) M (-1) s (-1)). Phosphate ions (100-500 mM) do not affect the rate of spontaneous peroxynitrite decay, but the H 2PO 4 (-) anion catalyzes the nucleophilic addition of the peroxynitrite anion to diacetyl. The intermediacy of acetyl radicals is suggested by a three-line spectrum ( a N = a H = 0.83 mT) obtained by EPR spin trapping of the reaction mixture with 2-methyl-2-nitrosopropane. The peroxynitrite reaction is accompanied by concentration-dependent oxygen uptake. Stoichiometric amounts of acetate from millimolar amounts of peroxynitrite and diacetyl were obtained under nonlimiting conditions of dissolved oxygen. In the presence of either l-histidine or 2'-deoxyguanosine, the peroxynitrite/diacetyl system afforded the corresponding acetylated molecules identified by HPLC-MS ( n ). These studies provide evidence that the peroxynitrite/diacetyl reaction yields acetyl radicals and raise the hypothesis that protein and DNA nonenzymatic acetylation may occur in cells and be implicated in aging and metabolic disorders in which oxygen and nitrogen reactive species are putatively involved.