Nucleophile addition of reduced glutathione on 2-methyl-2-nitroso compound: A combined electron paramagnetic resonance spectroscopy and electrospray tandem mass spectrometry study

Abstract

Mass spectrometry (MS) was used in conjunction with electron paramagnetic resonance (EPR) to characterize products arising from reactions between reduced glutathione (GSH) and 2-methyl 2-nitroso propane (MNP) in an oxidative medium, to evaluate the reactivity of this tripeptide as a nucleophile toward a nitroso compound. Depending on the experimental conditions, different radical species could be detected by EPR, which allowed some structural assumptions. These samples were then submitted to electrospray ionization, in both positive and negative ion modes, for structural elucidation in tandem mass spectrometry. Although the primary nitroxide products could not be detected in MS, structurally related compounds such as hydroxylamine and O-methyl hydroxylamine could be fully characterized. In the absence of light, a S-adduct was formed via a Forrester-Hepburn reaction, that is, a nucleophile addition of MNP onto the thiol function in reduced glutathione to yield a hydroxylamine intermediate, further oxidized into nitroxide. In contrast, irradiating the reaction medium with visible light could allow an inverted spin trapping reaction to take place, involving the oxidation of both MNP and GSH before the nucleophilic addition of the sulfenic acid function onto the nitrogen of MNP, yielding a so-called O-adduct. It was also found that dilution of the reaction medium with methanol for the purpose of electrospray ionization could allow nitroxides to be indirectly observed either as hydroxylamine or O-methyl hydroxylamine species. (J Am Soc

Evaluation of the Forrester–Hepburn Mechanism As an Artifact Source in ESR Spin-Trapping

“…Even though the hydroxylamine intermediate of the Forrester-Hepburn mechanism has been detected by NMR and mass spectroscopy, 14, 35 this is only a necessary but not sufficient condition for artifactual formation of radical adducts. In the past, kinetic ESR and oxygen consumption studies have been used to test for artifactual radical adduct formation.…”

Combining EPR and ESI-MS/MS to study the reactivity of alkylthiols and cysteine towards 2-methyl-2-nitrosopropane (MNP)

“…However, a by-product formed after MNP irradiation in oxidative media, namely N-tert-butoxy-N-tert-butylnitroxide (TBTBN, Scheme 1), would generate almost identical EPR signal in benzene (a N ¼ 26.6 G, g ¼ 2.0058). 7 In support to the presence of TBTBN in the medium, another well-known photo-degradation by-product of MNP, i.e. di-tert-butyl nitroxide (DTBN, Scheme 1), was also observed as a three line EPR (a N ¼ 15.4 G, g ¼ 2.0059).…”

“…1a). 7 It consisted of a large triplet due to a unique hyperfine coupling of the unpaired electron with nitrogen nucleus (a N ¼ 27.1 G, g ¼ 2.0056), the value of the hyperfine coupling constant (hfcc) a N being characteristic of an alkoxynitroxide. [13][14][15] Structural assumptions could thus be proposed for these O-adducts, as illustrated in Scheme 1a by 1a, 2a and 3a for propanethiol, octanethiol and cysteine, respectively.…”

“…[4][5][6] The addition of reduced glutathione, a tripeptide containing one cysteinyl residue, on 2-methyl-2-nitrosopropane (MNP) in oxidative media was previously studied by the combination of EPR and mass spectrometry. 7 It was then shown that different nitroxide adducts could be obtained depending on the experimental conditions. Upon white light irradiation, an alkoxynitroxide (i.e., an O-adduct) was EPR-detected, while the formation of an alkylthionitroxide (i.e., an S-adduct) was formed in the dark.…”

The Need for an Alternative to Radicals as the Cause of Fragmentation of a Thiamin‐Derived Breslow Intermediate

“…Nucleophilic additionst ob othn itroso andn itrone spin trapsmay lead to false-positive results. [18] Hydroxylamines that form from addition reactwithatmospheric oxygentogive EPR-active aminoxyl radicals.B ased on then ucleophilic properties of Breslowintermediates,suchanevent canaccount forasignal,w hich is notr elevantt ot he fragmentation process. [19] Finally, thes mall amounto fb enzaldehydet hat formswould be air-oxidized andthenp roduce radicals.…”