Nitrone spin traps such as the most commonly used 5,5-dimethylpyrroline N
-oxide (DMPO) are important reagents for the detection of free radicals by means of ESR spin trapping [1
]. For certain mass spectrometry experiments or to investigate the fidelity of spin trapping, it is helpful to use isotopically labeled spin traps.
The di(trideuteromethyl) analog of DMPO with its mass shift of +6 compared to unlabeled DMPO can be used for dual spin-trapping mass spectrometry experiments. Here, spin traps labeled with stable isotopes (2
N or 13
C) are utilized to simplify the interpretation of mass spectrometry experiments [2
]. With equal amounts of the labeled and unlabeled spin trap present, adducts of the trapped radicals will appear as ion pairs in the mass spectrum (with the mass differences depending on the isotopes incorporated in the spin trap and on the charge state of the ion). This facilitates and clarifies the identification of radical-derived analytes.
N analog can be used to unambiguously determine the susceptibility of a particular spin-trapping experiment to the Forrester-Hepburn artifact in an ESR experiment [4
]. A Forrester-Hepburn artifact is the result of nucleophilic attack of the radical precursor on the spin trap with subsequent oxidation to the identical nitroxide radical as derived from genuine spin trapping. It is difficult to distinguish between nucleophilic attack and free-radical trapping, with normal chemical and biological control experiments being of no use. Timmins et al
. reported a method based on spin traps with different isotopes at the α- or β-positions to the nitrogen of the spin trap [4
]. The substrate is preincubated with a spin trap (first isotope), and then the spin trap labeled with the second isotope is added simultaneously with the initiation of free radical formation. Because the ESR signals are different, the origin of the signal can be determined as artifact or genuine signal. Employing that technique, we were able to identify the DMPO/•
CN radical, supposedly generated by horseradish peroxidase and hydrogen peroxide, as an artifact (unpublished data).
The classical synthesis of DMPO, as reported by Janzen et al
], is based on the synthesis of pyrrolines [6
]. Later, Le et al
] published a synthesis of 2-14
C-DMPO that avoided the direct Michael reaction of nitropropane with methyl acrylate, thereby improving the low yield and eliminating the difficult purification of this step. For the synthesis of isotopically labeled DMPO, Pou et al
] published an effective method starting from 15
N-hydroxylamine, which involved the use of hydrogen gas in an autoclave as well as a reaction with ozone derived from an ozone generator. We have developed a more facile synthetic pathway for the synthesis of DMPO based on 15
N-sodium nitrite or 2-bromopropane (D7) as the isotopically labeled starting material. The intermediate 2-nitropropane was prepared in a one-step reaction according to the reaction principle described by Kornblum et al
]. This principle has been used for nitrone spin trap synthesis [10
]. Nitropropane was then used for a DMPO synthesis similar to that of Le et al
Pathway of DMPO analog synthesis.