Chemistry Reference
In-Depth Information
15.3 Advantages of nitroxides
What are the specific advantages to nitroxides that made them so endearing to the EPR field? First of
all, they are remarkably stable in almost any solvent and over a wide pH range in aqueous solution.
The nitroxide moiety is quite tolerant to synthetic manipulations at other sites on the typically piperidine,
pyrroline or pyrollidine ring. The generalized structures, including some nitroxide rings that can be an
intergral part of a lipid or polymer chain, are depicted in Figure 15.2.
Freezing and thawing or distilling/boiling have no adverse affects on the stability of the paramagnetic
group. Since EPR does not require optical transparency and does not suffer from the magnetic susceptibility
issues frequently encountered in NMR, it is possible to work with totally opaque solutions, solids or
mixtures thereof.
EPR sensitivity is, by definition, 600 - 700 times higher per spin compared with proton NMR, with
solution detectability down to almost nanomolar levels for narrow linewidth signals. Since the EPR spectral
line shape is a major component of spin label spectral analysis, particularly with respect to nitroxides
tumbling motion, it is fairly easy to distinguish free “unattached” or unreacted labels in a sample where
incomplete dialysis or separation was impossible. Since this one-electron radical is a paramagnet, there
is a distance-dependent relaxation effect on neighboring (proton) nuclei up to distances of about 20 A
that may be quantitated in correlative NMR experiments with proteins or nucleic acids. This is based
on the same dipole - dipole interaction that is measured in nucleus - nucleus interactions and is analogous
to Forster energy transfer distance measurements in fluorescence experiments. Some applications of this
phenomenon are discussed later in this chapter.
The only real drawback of nitroxide spin labels is their susceptibility to reduction to the diamagnetic
hydroxylamine in the presence of organic or biological reducing agents. Synthetic procedures involving,
O
O
N
N
ON
O
R
1
R
2
R
R
I
II
III
(a piperidine nitroxide)
(a pyrrolidine nitroxide)
(a doxyl nitroxide)
R
1
R
2
N
R
1
R
2
N
O
O
N
O
R
1
R
2
Va
Vb
IV
(a proxyl nitroxide)
(a
cis
-azethoxyl nitroxide)
(a
trans
-azethoxyl nitroxide)
Figure 15.2
Classes of nitroxide structures. The majority of nitroxides fall within one of the three classes:
(I) 2,2,6,6-tetramethyl-piperidine-N-oxyl, (II) the 2,2,5,5-tetramethyl-pyrrolidine-N-oxyl, and (III) the 4,4-
dimethyloxazolidine-N-oxyl(doxyl)nitroxides,respectively.Twootherversatileclassesofnitroxidesaretheside
chainsubstituted2,2,5,5-tetramethyl-pyrrolidine-N-oxyl(proxyl)nitroxides(IV)andthecis-andtrans-azethoxyl
nitroxides(V).(From[4]withpermission.)
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