Nitroxide Radicals: Properties, Synthesis and Applications - Stable Radicals: Fundamentals and Applied Aspects of Odd-Electron Compounds - page 219

In vivo , the nitroxide spin adducts can be rapidly reduced to the corresponding EPR silent hydroxy-
lamines, due to the presence of relatively high concentrations of reducing agents like reduced glutathione
(GSH), L-ascorbate anion, and so on. The protection of spin adducts toward bioreduction is one of the
most important current challenges for the EPR-ST study of free radicals released in biological systems and
also to other important biological applications of nitroxides.
As already mentioned in Section 5.7, inclusion of nitroxides in the cavity of various hosts appears as a
promising approach to achieve their protection in biological milieu. When cyclodextrin is used in buffer
solutions to encapsulate superoxide adducts of various pyrroline- N -oxides, a sevenfold enhancement in sta-
bility and a partial protection against glutathione peroxidase- and L-ascorbate anion-induced reduction were
reported. 331 The same enhancement was observed during the trapping of glutathiyl radical with PBN. 332
Bardelang reported the first synthesis of a nitrone grafted to a permethylated -cyclodextrin. 333 β Villamena

-CD-AMPO, a cyclic nitrone grafted on a cyclodextrin (Scheme 5.68). 334

published the synthesis of β

-CD-DEPMPO 119 , 333 Scheme 5.68) was synthesized using
NHS-DEPMPO ( 116 ) as versatile building block. Very promising preliminary in vitro results have been
obtained with 119 : its superoxide adduct has a half-life of about 36 minutes and showed a greatly improved
resistance to bioreduction processes compared to what was observed with DEPMPO in the presence of
cyclodextrins. 333

A DEPMPO-appended β -cyclodextrin ( β

5.11.1 Immuno spin trapping
Mason and coworkers have developed a new highly specific and sensitive technique capable of detecting
protein 335 and DNA radicals. 336 This technique was named “immuno spin trapping” and associates EPR
spin trapping with immuno assays. DMPO ( 110 ) is used as the spin trap (Scheme 5.69). After a protein
radical has been trapped by DMPO, the nitroxide spin adduct goes to one-electron oxidation, converting
it to an EPR silent DMPO-protein adduct.
Then, this DMPO-protein adduct can be detected by polyclonal antibodies (anti-DMPO). This immuno
technique was initially reported for the study and the identification of protein-derived free radical
species generated during the reaction of myo- 338a and hemoglobin 338b,337 with hydrogen peroxide.
Since the first paper by Mason et al . in 2002 reporting the advantages of this technique, more than
20 papers have been published investigating free radical intermediate production during cell protein
damaging processes. 338,339

5.11.2 Conclusion
The goal of getting spin traps that afford nitroxide spin adducts exhibiting half-lives from 15 to 40 minutes
has actually been achieved. However, combining a higher persistency for the nitroxide spin adducts and a
faster addition of the free radical species on the nitrone should lead to higher steady state concentrations in
solution, facilitating the free radical detection. Associated with an improved protection against bioreduction
and the high performance EPR equipments available today, EPR-spin trapping should represent a unique
tool to investigate the fate of free radicals in biological systems.

H 3 C
H 3 C

H 3 C
H 3 C

Protein H 3 C

H 3 C

[Ox]

+ Protein

H Protein

N
O

N
O

H N

O

Scheme 5.69 Trapping of protein radical by DMPO