Functional in vivo EPR Spectroscopy and Imaging Using Nitroxide and Trityl Radicals - Stable Radicals: Fundamentals and Applied Aspects of Odd-Electron Compounds - page 539

Half a century of continuous progress in the nitroxide chemistry has resulted in design of specific NRs for
spin labeling, 7 site-directed spin labeling, 8 EPR oximetry, 9,10 pH, 11 thiols 12,13 and NO measurements, 14-16
including in vivo functional EPR/EPRI application. 1,17 - 19 TAM radicals represent a fundamentally different
basic structure of stable organic radicals, with approximately 60 % of the unpaired electron localized
at the sterically protected central carbon atom and the remaining 40 % delocalized over the three aryl
substituents. 20 NRs have the advantages of well developed chemistry, resulting in variability of structure,
solubility, functionality, and ability to be targeted. On the other hand, TAMs have advantages over NRs in
that they offer extreme stability toward tissue redox processes, longer relaxation times, and narrower line
widths, making them particularly attractive for imaging applications. 21

16.2 Nitroxyl radicals

The first stable di- tert -alkyl nitroxide of piperidine type (TEMPO) 5 (Scheme 16.1) was synthesized fifty
years ago. The stability of the N-O group allowed chemical reactions of the nitroxides which do not involve
the radical center allowing further NR derivitization. 6,22 In the early 1960s, EPR spectral sensitivities of the
NRs to their local environment, including viscosity 23 and polarity, 24 were reported. Later on, McConnell
and colleagues established the spin labeling technique, demonstrating the EPR spectral sensitivity of bio-
logically relevant macromolecules labeled with NRs to molecular motion and microenvironment. 25,26 The
combination of EPR spin labeling with site-directed mutagenesis reactions led to the site-directed spin
labeling (SDSL) technique currently widely used in the study of protein structure and dynamics. 8,27,28
The sensitivity of NR EPR spectra to pair-wise Heisenberg spin exchange 29 or magnetic dipole - dipole
interactions, 30 provide experimental tools for the measurement of intermolecular distances and the loca-
tion of paramagnetic species, such as metal ions and oxygen. NRs were the first paramagnetic probes
used for EPR oximetry based on the spin exchange phenomenon between diradical oxygen molecule and
NRs. 10,31 - 33
Specific chemical reactions of NRs further extend their functional applications. The most well known
reaction of NRs, their one-electron reduction to EPR silent hydroxylamines, is largely responsible for the
biodegradation of the nitroxides in living tissues 34 and, in general, significantly limits many biological
applications of NRs. On the other hand, the rate of the NRs' reduction provides information on the redox
state of living tissues. 17 The other reactions of the NRs, which do not involve radical center but can be
followed by EPR, 35 include reactions of the specific NRs with protons, thiols, and nitric oxide (NO).
The functionally-enhanced NRs for EPR detection of corresponding biologically relevant molecules were
developed, namely pH-, SH-, and NO-sensitive spin probes. 35,36 The main types of the nitroxides discussed

in this chapter are shown in Scheme 16.1.

R

R

R R R

N

N

N
O

N

N
O

N
O

N

O

O

Piperidine NRs
(TEMPO: R=H)

Pyrroline
NRs

Pyrrolidine
NRs

Imidazoline
NRs

Imidazolidine
NRs

Scheme 16.1 Types of nitroxide radicals (NRs)