| Peroxynitrite (PN; O=NOO-) and its acid, peroxynitrous acid (O=NOOH; pKa ≈ 6.2) are two non-radical oxidants formed primarily in nitric oxide (*NO) producing biological systems. It is known that spontaneous decomposition of O=NOOH can be a significant source of hydroxyl radicals (*OH; up to 40 mol %) while the reaction of PN with endogenous carbon dioxide (CO 2) can result in the formation of carbonate radical (CO3*-; up to 40 mol %). In both reactions, *NO2 is the second free radical species formed in yields up to 40 mol% (based on the combined concentration of O=NOO- and O=NOOH). Methionine (Met), an essential amino acid (AA), has been shown to be highly reactive towards *OH, *NO2, CO3*- and the like. The present work focuses on synthesis and characterization of a set of tripeptides containing reactive (Met) and non-reactive amino acids (Alanine (Ala) and Glycine (Gly)), and examines their reactivity towards free radical oxidants formed during the H+- and CO2-catalyzed decomposition of O=NOO-. For the sake of comparison, the oxidation of Met or Met-containing dipeptide (Met-Gly) was also studied in this system. Several tripeptides including Ac-Ala-Met-Gly, Ac-Ala-Met-Gly-NH2 and Ala-Met-Gly were synthesized using the protocol of solid phase peptide synthesis (SPPS). Followed by Fmoc deprotection and subsequent purification by reversed phase (RP) HPLC, the peptides were characterized by 1H-NMR and high resolution mass spectrometry. The stoichiometry of oxidation of Met, Met-Gly, Ala-Met-Gly, and Ac-Ala-Met-Gly in PN-mediated reactions was determined based on the initial and final concentrations measured by RP-HPLC. It was found that in reactions carried out in the presence or absence of added bicarbonate, the yields of oxidation of Met and Met-containing peptides were always below 40 mol% (based on the initial concentration of O=NOO- plus O=NOOH), meaning that the reaction in both cases is mediated predominantly through free radical oxidants and not by O=NOO- or O=NOOH. In contrary to our expectations, the yields of oxidation were somewhat lower when substrates were oxidized with PN in the presence of added carbonated species. At pH 7.0, the reduction potential of CO3*-/HCO 3- (1.78 V) is lower than that of HO*/HO- (2.3 V). The lower reduction potential of CO3*-/HCO 3- couple makes CO3*- a less reactive but more selective oxidant than that of *OH towards Met and other biological targets. Ideally, this should have resulted in higher yields of overall oxidation of Met and Met-containing peptides by PN carried out in the presence of added carbonated species. The discrepancy between the observed and expected yields of Met oxidation reactions performed in the absence of added carbonated species suggests that there exists a mechanism that compensates for higher yields. It is possible that this compensatory mechanism is the direct oxidation of Met and Met-containing peptides by O=NOOH. This explanation is consistent with the facts that the bimolecular reaction between O=NOOH and Met is about 181+/-8 M-1s-1 at pH 7.4 and 25°C. |
