Sterically shielded spin labels for in-cell EPR spectroscopy: Analysis of stability in reducing environment

Article abstract of DOI:10.3109/10715762.2014.979409

Electron paramagnetic resonance (EPR) spectroscopy is a powerful and widely used technique for studying structure and dynamics of biomolecules under bio-orthogonal conditions. In-cell EPR is an emerging area in this field; however, it is hampered by the reducing environment present in cells, which reduces most nitroxide spin labels to their corresponding diamagnetic N-hydroxyl derivatives. To determine which radicals are best suited for in-cell EPR studies, we systematically studied the effects of substitution on radical stability using five different classes of radicals, specifically piperidine-, imidazolidine-, pyrrolidine-, and isoindoline-based nitroxides as well as the Finland trityl radical. Thermodynamic parameters of nitroxide reduction were determined by cyclic voltammetry; the rate of reduction in the presence of ascorbate, cellular extracts, and after injection into oocytes was measured by continuous-wave EPR spectroscopy. Our study revealed that tetraethyl-substituted nitroxides are good candidates for in-cell EPR studies, in particular pyrrolidine derivatives, which are slightly more stable than the trityl radical.

Full text of DOI:10.3109/10715762.2014.979409

Just Accepted by Free Radical Research
Sterically shielded spin labels for in-cell EPR
spectroscopy: Analysis of stability in reducing
environment
Anil P. Jagtap, Ivan Krstic, Nitin C. Kunjir, Robert Hänsel,
Thomas F. Prisner, Snorri Th. Sigurdsson
Doi: 10.3109/10715762.2014.979409
Abstract
Electron paramagnetic resonance (EPR) spectroscopy is a powerful
and widely used technique for studying structure and dynamics of
biomolecules under bio-orthogonal conditions. In-cell EPR is an
emerging area in this field; however, it is hampered by the reducing
environment present in cells, which reduces most nitroxide spin
labels to their corresponding diamagnetic N-hydroxyl derivatives. To
determine which radicals are best suited for in-cell EPR studies, we
systematically studied the effects of substitution on radical stability
using five different classes of radicals, specifically piperidine-, imida-
zolidine-, pyrrolidine-, and isoindoline-based nitroxides as well as the
Finland trityl radical. Thermodynamic parameters of nitroxide reduc-
tion were determined by cyclic voltammetry and the rate of reduction
in the presence of ascorbate, cellular extracts and after injection into
oocytes was measured by continuous-wave (CW) EPR spectroscopy.
Our study revealed that tetraethyl-substituted nitroxides are good
candidates for in-cell EPR studies, in particular pyrrolidine deriva-
tives, which are slightly more stable than the trityl radical.
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1
Sterically shielded spin labels for in-cell EPR spectroscopy:
Analysis of stability in reducing environment
Anil P. Jagtap1, Ivan Krstic2, Nitin C. Kunjir1, Robert Hänsel3, Thomas F. Prisner2,
Snorri Th. Sigurdsson1
1University of Iceland, Department of Chemistry, Science Institute, Dunhagi 3, 107
Reykjavik, Iceland, 2Institute of Physical and Theoretical Chemistry, Goethe University
Frankfurt, Max-von-Laue-Str. 7, Frankfurt, Germany, 3Institute of Biophysical Chemistry
and Center for Biomolecular Magnetic Resonance, Goethe University, Frankfurt/Main,
Germany
Correspondence: Snorri Sigurdsson, University of Iceland, Department of
Chemistry, Reykjavík, Iceland. Tel: +354 525 4800. Fax: +354 552 8911. Email:
snorrisi@hi.is
Short title: Reductive stability of radicals
Abstract
Electron paramagnetic resonance (EPR) spectroscopy is a powerful and
widely used technique for studying structure and dynamics of biomolecules
under bio-orthogonal conditions. In-cell EPR is an emerging area in this field;
however, it is hampered by the reducing environment present in cells, which
reduces most nitroxide spin labels to their corresponding diamagnetic N-
hydroxyl derivatives. To determine which radicals are best suited for in-cell EPR
studies, we systematically studied the effects of substitution on radical stability
using five different classes of radicals, specifically piperidine-, imidazolidine-,
pyrrolidine-, and isoindoline-based nitroxides as well as the Finland trityl radical.
Thermodynamic parameters of nitroxide reduction were determined by cyclic
voltammetry and the rate of reduction in the presence of ascorbate, cellular
extracts and after injection into oocytes was measured by continuous-wave
(CW) EPR spectroscopy. Our study revealed that tetraethyl-substituted
nitroxides are good candidates for in-cell EPR studies, in particular pyrrolidine
derivatives, which are slightly more stable than the trityl radical.
Keywords: nitroxide reduction, aminoxyl radical, radical stability, spin labeling,
trityl radical

2
1. Introduction
RNA molecules have a central role in cellular processes and gene
regulation. Their three-dimensional structures and conformational dynamics
are essential for their functions as biological catalysts, structural scaffolds
and regulators of gene expression [1]. Thus, information about structure and
motion can give insights into RNA function and how it might be modulated.
Besides X-ray crystallography [2], NMR [3] and fluorescence spectroscopy
[4], pulsed electron-electron double resonance (PELDOR or DEER)
spectroscopy [5] has over the past few years demonstrated its applicability to
map the global structure of nucleic acids [6,7] and other macromolecules
[8,9] through distance measurements in the range of 1.5-8 nm, utilizing
distance-dependent magnetic dipole-dipole interaction between two
covalently attached aminoxyl (nitroxide) radicals. EPR spectroscopy also
holds promise for in-cell measurements because there is no background
interference, as can be the case with fluorescence spectroscopy. In fact,
PELDOR was recently used to study structural aspects of nucleic acids
[10,11] and proteins [12] inside intact Xenopus laevis oocytes. However, the
short lifetimes of nitroxide spin labels under cellular conditions is a severe
limitation to the general applicability of PELDOR for in-cell measurements
[13].
In addition to the use as labels for EPR spectroscopy, cyclic nitroxide
radicals are an important class of compounds for biological and medical
applications [14-16], such as contrast agents for magnetic resonance imaging
(MRI) [17], as antioxidants and as superoxide dismutase mimics [18], where
the nitroxide is involved in redox reactions by a one-electron exchange

3
between its reduced and oxidized state [19]. Additionally, nitroxide radicals
have been used to determine the partial pressure of oxygen [20] and pH [21]
values in living tissues via EPR spectroscopy. The aforementioned
applications of nitroxides in biological fluids are adversely affected when the
paramagnetic center is readily reduced to the EPR-silent hydroxylamine [22].
Since the application of nitroxides inside living cells is of growing interest,
extensive efforts have been taken in the design and synthesis of nitroxide
radicals that are more resistant towards reduction [23,24]. Several factors
affect the stability of nitroxides, such as ring size (piperidine, pyrrolidine,
isoindoline), the presence of heteroatoms within the ring (imidazolidine),
substituents (neutral or charged) and the identity of the alkyl substituents in
the positions adjacent to the nitroxide functional group. Although the
stability of a variety of nitroxides in the presence of ascorbate has been
reported [22,25-36], the biostability of radicals has invariably been
investigated under different conditions [26-30,37-41]. Furthermore, each of
these biostability studies has focused on a small number of radicals. The
study of Kinoshita et al. is similar to the work described here in terms of the
techniques and conditions used for evaluation of radical stability. They also
compared tetramethyl and tetraethyl piperidine nitroxides; however, our
work additionally includes imidazolidine and isoindoline nitroxides as well
as a triphenylmethyl (trityl) radical [37].
In this paper, we compare redox properties of a series of nitroxides varying
with ring type, substituents and charge, using several approaches with the
aim to identify radicals that are suitable for in-cell studies. A trityl radical,
which has been shown to be relatively stable under reductive conditions [42],

4
was chosen for comparison. We also determined the hyperfine coupling
constants (Aiso) of all the nitroxides, that are sensitive to the polarity around
the nitroxide moiety, by continuous wave (CW) EPR. With regards to the
nitroxides, we focused on tetraethyl-substituted radicals with pyrrolidine-,
piperidine-, isoindoline- and imidazolidine-based structures and compared
them with the more reactive tetramethyl-substituted analogues. The effect of
electronegative and charged substituents on the nitroxide rings was also
evaluated. In addition to determination of the kinetic and thermodynamic
stability of the radicals in the presence of ascorbic acid, their stability was
tested in a cytosolic extract from Xenopus leavis oocytes and inside living
oocyte cells.
2. Material and methods
2.1. Preparation of radicals
Chemicals were purchased primarily from Sigma-Aldrich Chemical
Company and Acros- Belgium, and were used without further purification.
Thin layer chromatography (TLC) was performed on glass backed TLC
plates with extra hard layer (Kieselgel 60 F254, 250 µm, Silicycle) and
compounds were visualized by UV light at 254 nm. Silica gel (230-400
mesh, 60 Å) was purchased from Silicycle, and used for flash
chromatography. Molecular mass of organic compounds were determined by
HR-ESI-MS (Bruker, MicroTof-Q).
Tetramethyl-derived spin labels 1, 2, 3 and pyrrolidine spin label 12 were
purchased, while tetraethyl piperidine derivative 4 was prepared according to
the reported procedure [37] and used for the synthesis of compounds 5 and 6

5
[37,43]. Compounds 11 and 13-15 were synthesized as previously described
[44-47].
Compounds 8 and 10 were prepared using a modification of previously
reported method [48]. In the reported procedure for the synthesis of 8,
compound 16a was oxidized and then nitrated to obtain 18a. However,
nitration gave multiple spots in our hands and, therefore, compound 16a was
first nitrated to obtain compound 17a (Scheme 1) [49]. Subsequent oxidation
with m-CPBA yielded compound 18a in moderate yields, which was
converted to 8 using the previously published protocol [48]. This modified
procedure was also used for the synthesis of the corresponding tetraethyl
derivative 10. Preparation of 8 using this strategy has recently been reported
by Mileo et al. [50].
1,1,3,3-Tetramethyl-5-nitroisoindoline 18a
To a solution of compound 17a (0.9 g, 4 mmol) in CH2Cl2 (5 mL) at 0 °C
was added a solution of m-CPBA (1.4 g, 8.17 mmol) in CH2Cl2 (6 mL). The
resulting solution was stirred at 0 °C for 1 h and then at 24 °C for 2h. The
reaction mixture was diluted with CH2Cl2 (10 mL), the organic layer washed
successively with an aqueous NaOH solution (2.5 N, 2 x 10 mL) and brine
(10 mL). The organic layer was dried over anhydrous Na2SO4, filtered and
then concentrated in vacuo. The crude reaction mixture was purified by
column chromatography (5% MeOH in CH2Cl2) to yield 18a as a yellow
solid (0.9 g, 94% yield). HR-ESI-MS: 258.0975 (M+Na), calcd. 235.0953 for
C12H15N2O3.
Tetraethyl-5-nitroisoindoline 18b

6
To a solution of compound 17b (0.120 g, 0.43 mmol) in CH2Cl2 (2 mL) at 0
°C was added a solution of m-CPBA (0.089 g, 0.5 mmol) in CH2Cl2 (1 mL).
The resulting solution was stirred at 0 °C for 1 h and then at 24 °C for 3h.
The reaction mixture was diluted with CH2Cl2 (5 mL), the organic layer
washed successively with an aqueous NaOH solution (2.5 N, 2 x 10 mL) and
brine (10 mL). The organic layer was dried over anhydrous Na2SO4, filtered
and then concentrated in vacuo. The crude reaction mixture was purified by
column chromatography (30 % EtOAc in petroleum ether) to yield 18b as a
yellow solid (0.1 g, 79% yield). HR-ESI-MS: 314.1602 (M+Na), calcd.
291.1601 for C16H23N2O3
2.2. Cyclic voltammetry
Cyclic voltammetry experiments were performed in a situ 4 mM phosphate
buffer (PBS) solution (pH 7.2) containing 1 mM radical concentration. A
three-electrode cell arrangement was used encompassing glassy carbon
working electrode, which was polished before each measurements, a
reference electrode Ag/AgCl/KCl(3M), and a double-wire platinum counter-
electrode. The following parameters were used: 0.1 V/s scan rate, 0.0 V
starting potential, 1.0 V upper vertex potential, -1.5 lower vertex potential, -
0.00244 V cathodic step potential, 0.00244 V anodic step potential. In case of
6, the starting potential was -0.2 V, because of similar anodic and cathodic
peak potentials.
2.3. Monitoring reduction of radicals in ascorbic acid and in cell extract
using CW-EPR
Preparation of the crude cytoplasmic extracts from Xenopus laevis oocytes
was as previously described [10]. 19 µL of 5 mM ascorbic acid solution in 4

7
mM PBS (pH = 7.4), or 19 µL of cell extract were mixed with 1 µL of a 4
mM radical solution (final radical concentration 200 µM). The solution was
transferred into an EPR tube (1 mm inner diameter, Wilmad, USA) and the
EPR signal intensity was measured as a function of time using a Bruker E500
CW-EPR spectrometer at X-band (9.43 GHz), using the following settings:
9.43 GHz modulation frequency, 40.96 ms time constant, 1.0 mW
microwave power, 40.96 ms conversion time, 1.0 G modulation amplitude,
1024 number of points, 70 G field sweep and 160-170 sweeps.
2.4. Monitoring reduction of radicals inside cells by CW-EPR
Oocytes from Xenopus laevis trapped in stage VI, characterized with ca.1
mm diameter and ca.1 µL volume were used [10]. Samples were prepared by
microinjection of ca. 40 nL of 4 mM spin label stock solution into 50 oocytes
and subsequent incubation at room temperature for a variable time (0, 15, 30,
60, 120 min) before freezing the cells in liquid nitrogen, until the radical
concentration was determined by CW-EPR: microwave power 0.2 mW,
modulation amplitude 2.0 G, number of scans 10, temperature 125 K.
3. Results and discussion
3.1. Selection of radicals
Five classes of spin labels were investigated, each of which was based on a
specific cyclic nitroxide, i.e. piperidine- (Figure 1A), isoindoline- (Figure
1B), imidazolidine- (Figure 1C), pyrrolidine-derivatives (Figure 1C) or a
trityl radical (Figure 1C). The piperidine series served to investigate the
effects of the substituents on the ring, all of which were electron
withdrawing, except for the alkyl groups flanking the nitroxide. The
isoindolines (Figure 1B) had ionizable groups to facilitate solubility in

8
aqueous solutions, a sulfonate in compounds 7 [51,52] and 9 [51,52], and a
tetraalkylammonium ion in compounds 8 and 10. Each type of nitroxide was
prepared as tetramethyl- and tetraethyl-derivatives. The trityl radical 15 was
the only carbon-based radical in this study.
3.2. Thermodynamic parameters for reduction in the presence of ascorbic
acid
Ascorbic acid, a reducing agent present to some extent in biological
systems [53-55], is commonly used for evaluation of radical stability [29]
and was used to investigate the thermodynamic parameters of the radicals. To
determine the Gibbs free energy (∆G) and the reaction equilibrium constant
(K) for the reduction, the redox potential of each radical was measured by
cyclic voltammetry. The half-wave potentials correspond to standard redox
potentials as long as the diffusion coefficients of the reduced and oxidized
state are equal, which is generally the case.
A typical voltammogram of a nitroxide, recorded by sweeping the potential
of a glassy carbon electrode between -1.5 and 1 V (vs Ag/AgCl/KCl) in
both the anodic and the cathodic direction, respectively, contains two peak
couples (Figure S4A). The first peak couple appears at the anodic potential of
the voltammogram and originates from the reversible oxidation of the
nitroxide to an oxoammonium cation (Figure S4B). The peak separation is
close to the theoretical value of 59 mV for a one electron transfer. This
reaction is not important in a biological context, where nitroxide radicals are
reduced. The second peak-couple represents the reduction of the nitroxide
(Figure S4C) and the corresponding oxidation of hydroxylamine. In this case,
the electron transfer to the nitroxide is coupled to a chemical reaction,

9
making the peak separation larger. Since the redox behavior of trityl radicals
differs significantly from nitroxides, 15 was not studied by cyclic
voltammetry.
The half-wave potentials (E1/2) were determined from the cyclic
voltammograms as the midpoint between the oxidation and the reduction
peak potentials for the second peak-couple: E1/2 = (Epox+Epred)/2 (Table 1).
The tetramethyl-substituted piperidines had the highest E1/2 value (ca. -0.07
V), while the tetraethyl-substituted isoindoline- and pyrrolidine-derived
nitroxides displayed the lowest redox potential (-0.32 V), making them the
most resistant to reduction. The difference between the standard redox
potential of ascorbic acid (E0 = -0.15 V vs Ag/AgCl/KCl) and the measured
E1/2 of each nitroxide, were used to determine ∆G and K for the reduction of
the nitroxide with ascorbate [56] (Table 1). The ΔG values for the reduction
were negative for the tetramethyl-derived piperidines, thus making the
nitroxide reduction energetically favorable. In contrast, the tetraethyl-
substituted nitroxides yielded ΔG values of about 6-16 kJ/mol and small
equilibrium constants, showing that their equilibrium lies towards the educts.
Using the equilibrium constants determined from the cyclic voltammetry
measurements and known starting concentrations of educts, the equilibrium
concentrations of the nitroxide radicals were calculated, normalized to the
starting concentration ([N]eq/[N]0) and expressed in Table 1 as percentages.
The equilibrium concentrations of the nitroxides in reaction with ascorbic
acid were also determined experimentally by following the intensity of the
low-field nitroxide line of the EPR spectra as a function of time (Figure 2A).
The decaying EPR signal was fitted with the pseudo-first order reaction

10
kinetic implicitly including the equilibrium signal as a fit parameter (see 3.3).
This signal originates from radicals present at equilibrium and was compared
with the equilibrium concentration of nitroxides that were calculated from the
redox potentials. Comparison of the last two columns in Table 1 shows an
excellent agreement between the equilibrium concentrations of the nitroxide
radicals as measured by EPR spectroscopy and those calculated from the
thermodynamic parameters. Thus, knowledge of the redox properties of
radicals can be used to predict with high precision the equilibrium
concentration for their reduction with ascorbate anion.
3.3. Kinetics of the reduction of radicals in ascorbic acid solution
The large excess of ascorbate that was used for reduction of radicals
ensured (pseudo) first-order kinetics and allowed fitting of the decay curve
with a first order exponential function:
, where
[N] is the concentration of the radical at time t, [N]0 is the initial
concentration and k′ is the pseudo first order rate constant. [N]eq is a constant
offset given by the thermodynamic equilibrium of nitroxide radicals under
steady state conditions. Dividing k′ by the concentration of ascorbic acid
yielded the bimolecular rate constants (Table 2).
The calculated bimolecular rate constants confirmed that the rate of reduction
depends on several factors: the size of the nitroxide ring system, the nature of
the substituents and shielding of the nitroxide moiety (Figure 2A, Figure S1).
Replacing the methyl groups adjacent to the nitroxides with ethyl groups had
the largest impact on radical stability, presumably by sterically restricting
access of reductants to the nitroxide [22,25]. The hyperfine coupling
constants (Aiso) of the radicals were determined from their CW-EPR spectra

11
(Table S1) and confirmed that the ethyl-derivatized nitroxides had a lower
Aiso value than the corresponding methyl derivatives, as expected for a more
hydrophobic environment. The steric effect was most pronounced in the case
of the piperidine derivatives, where the tetraethyl-substituted nitroxides 4, 5
and 6 were found to be ca. 100 times more stable against reduction than the
tetramethyl-derivatives 1, 2 and 3. This data is in agreement with the recently
published study of Kajer et al [57].It has been shown that cyclohexyl or
pyran groups do not further increase radical stability [22,58], making
tetraethyl-derived nitroxide radicals good candidates for in vivo EPR.
Other substituents also affected stability of the radicals, although to a lesser
extent than the alkyl groups adjacent to the nitroxides. Positively charged
substituents decreased the stability of the radicals, presumably by attracting
the ascorbate anion. For example, 3, where the amino group is protonated at
pH 7, is three-fold more reactive than 2. The imidazolidine 13 was
considerably less stable than the other tetraethyl derivatives, presumably due
to protonation [44]. For the isoindoline derivatives, the effect of charge was
not as pronounced, presumably because the charge is farther from the
nitroxide functional group; the positively charged 8 is less than two-fold
more reactive than the negatively charged 7, while no difference was
observed for the tetraethyl-substituted compounds 9 and 10.
The largest effect on the nitroxide stability of the tetramethyl-substituted
nitroxides was caused by the structure of the nitroxide-bearing ring. The five-
membered pyrrolidine, imidazolidine and isoindoline-derivatives were three-
to ten-fold more stable towards reduction than the six-membered piperidine
derivatives [32]. Similar rates of reduction for 7 and 12 indicate that the

12
pyrrolidine- and isoindoline-rings have similar stability. The slowest
reduction rate was found to be that for the negatively charged tetraethyl
pyrrolidine derivative 14.
3.4. Radical stability in cell extract
The rate of radical reduction was also investigated in cytoplasmic extracts of
oocytes and as in the case of ascorbate reduction, all tetramethyl substituted
radicals were rapidly reduced in the cellular extract (Figure 2B, Figure S2).
The EPR time traces (Figure 2B) did not follow a single exponential decay,
presumably due to the presence of various reducing agents (ascorbate,
glutathione, cysteine, NADH, NADPH) and a significant amount of
dissolved oxygen in the cell extract. Therefore, signal intensities after 2 h of
reaction time were used for comparing the stability of the radicals in the
cytosolic fluid. As seen from Figure 2B and Table 2, the most resistant
radical is the pyrrolidine-derivative 14, which was 95% intact after 2 h,
followed by the tetraethyl-piperidines 4 and 5 (75%), tetraethyl-
imidazolidine and tetraethyl-piperidine 6 (65%), trityl (60%) and tetraethyl-
isoindoline (50%). The different relative stability of isoindoline vs. piperidine
nitroxides in the cell extract, compared to ascorbate reduction, is presumably
due the presence of different reducing agents in the cell. In addition, different
rates of re-oxidation of the corresponding hydroxylamines to nitroxides by
oxidants in the cell extract, including molecular oxygen, could also be a
contributing factor [59].
3.5. Radical stability inside cells
As stated above, the primary aim of this study was to compare, under
identical experimental conditions, the stability of potential spin labels for

13
investigation of biomolecules in living cells by pulsed EPR spectroscopy.
Therefore, the ultimate test was their persistence in oocyte cells. Charged
radicals 6, 9, 10, 14 and 15 gave reduction profiles that are very similar to
those recorded in cell extracts (Table 2), presumably because they were
distributed in the cytosol which should yield similar reduction kinetics to cell
extracts (see Figure S2 and S3 for decay curves for all radicals in cytosolic
extract and inside cells). In contrast, the reduction kinetics for non-charged
radicals 4, 5 and 13 inside cells were much faster than in the cell extracts,
e.g. only about 15% of 5 remained after 2 h. Radical 6, containing the same
ring size but carrying a protonated amino group, retained 65% of the signal.
A possible explanation for this discrepancy is that the non-charged radicals
could have entered intracellular membranes and been reduced rapidly by the
electron transport chain components in the mitochondrial membrane [60].
Pyrrolidine 14 was the most persistent radical, retaining more than 85% of
the signal, with the trityl radical 15 following closely behind (70%).
4. Conclusions
In conclusion, introduction of bulky ethyl groups next to a nitroxide group
leads to significant stabilization against reduction by both ascorbic acid and
the reductants present in living cells, yielding radicals that are more stable in
cells than trityl radical 15. The tetraethyl-substituted pyrrolidine-based
nitroxide carrying a carboxylic group (14) demonstrated superior stability
against reduction due to combination of sterical shielding, ring size and
charge, all of which are factors that should be taken into account for the
design of spin labels for in vivo studies. Charged and neutral radicals showed
different relative stabilities in cell extracts than in cells, indicating that

14
conjugation to biomolecules could have a large effect on the stability of these
radicals. The trityl radical exhibited considerable stability towards reduction,
especially in cells.
Acknowledgements
This work was supported by the Icelandic Research Fund (120001021), the
Deutsche Forschungsgemeinschaft (SFB 902, Molecular principles of RNA-
based regulation) and by a doctoral fellowship to A. P. Jagtap from the
University of Iceland Research Fund. We thank Dr. S. Jonsdottir for
assistance in collecting analytical data for structural characterization of
compounds.
Declaration of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of the paper.

15
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20
Table 1. Half-wave potentials (E1/2) of
nitroxide radicals, Gibbs free energy (ΔG)
and equilibrium constants (K) for reduction of
nitroxides with ascorbic acid, and equilibrium
concentrations of the nitroxides (calculated
and measured).
E§
ΔG†
[N]eq/[N]0 (%)
1/2
Rad.*
K = e-(ΔG/RT)
[V]
[kJ/mol]
calculated
measured
1.2 ± 0.1
53 ± 3
-0.08
-0.27
-0.08
-0.27
-0.07
-0.23
-0.16
-0.32
-0.16
-0.32
-0.16
-0.21
-0.18
-0.32
-7 ± 2
12 ± 1
-7 ± 2
12 ± 1
-7 ± 2
8 ± 1
16 ± 5
0.009 ± 0.003
16 ± 5
1.4 ± 0.5
59 ± 6
1
4
1.4 ± 0.5
59 ± 6
1.4 ± 0.1
55 ± 2
2
0.009 ± 0.003
16 ± 5
5
0.9 ± 0.5
33 ± 6
1.5 ± 0.3
31 ± 2
3
0.04 ± 0.02
0.7 ± 0.3
6
7
1 ± 1
4.5 ± 2.0
82 ± 4
5.0 ± 0.5
84 ± 2
16 ± 1
0.5 ± 1
16 ± 1
1 ± 1
0.0012 ± 0.0005
0.8 ± 0.3
9
3.8 ± 1.2
82 ± 4
4.8 ± 0.5
76 ± 2
8
0.0012 ± 0.0005
0.7 ± 0.3
10
11
13
12
14
4.5 ± 2.0
22 ± 5
5 ± 2
6 ± 1
0.09 ± 0.04
0.3 ± 0.1
16 ± 1
3 ± 1
9 ± 3
11 ± 1
16 ± 1
0.0012 ± 0.0005
86 ± 4
91± 7
*Radicals; §For the half-wave potential (E1/2) the error
is ±0.01;
†
.

21
Table 2. Half-life of the radicals and their
corresponding second order reaction rate
constant for reduction with ascorbic acid,
EPR signal intensities after incubation (2 h)
in ascorbic acid, in the cell extracts and inside
oocyte cells.
2nd Order
rate const.
[(M·s)-1]
Extent of reduction†
Rad.*
Lifetime§ [s]
Asc.¶
Cell
extract
0
Oocyte
cells
-
158 ± 1
2600 ± 50
165 ± 2
5.75 ± 0.04
0.066 ± 0.001
5.51 ± 0.005
0.058 ± 0.002
16.8 ± 0.7
1
57
1
1
4
75
0
34
-
2
2640 ± 60
54 ± 2
58
1
78
0
15
-
5
3
1480 ± 40
365 ± 5
0.115 ± 0.003
0.44 ± 0.01
31
5
65
0
66
-
6
7
1980 ± 50
220 ± 3
0.079 ± 0.003
0.74 ± 0.01
84
5
45
0
51
-
9
8
2350 ± 60
100 ± 2
0.081 ± 0.001
1.62 ± 0.04
78
5
52
0
55
-
10
11
13
12
14
15
1940 ± 30
2160 ± 20
85000 ± 30000
10500 ± 1000
0.084 ± 0.002
0.079 ± 0.001
0.002 ± 0.001
0.016 ± 0.002
19
14
94
74
66
38
94
60
25
-
88
70
*Radicals. The starting concentration of radicals was 200
mM and concentration of ascorbic acid 5 mM, except
for spin label 1-3 where it was 1 mM; §calculated as t1 =
1/k′; †listed as a percentage, after 2 h of reaction time;
¶ascorbic acid.

22
Figure legends
Figure 1. Radicals for evaluation of reductive stability. (A) Piperidine-based
nitroxides. (B) Isoindoline-based nitroxides. (C) Imidazolidine- and
pyrrolidine-based nitroxides and a trityl radical.
O
OH
NH2
A
N
O
N
O
N
O
1
2
3
O
OH
NH2
N
O
N
O
N
O
6
4
5
B
I
N O
N O
N
NaO3S
8
7
I
N O
N O
NaO3S
N
9
10
COOH
C
N
N
O
N
O
3
12
11
S
S
S
S
COOH
N
15
COONa
N
O
N
O
13
14

23
Figure 2. (A) Reduction of selected radicals with ascorbic acid (200 µM
conc. of radical and 5 mM ascorbic acid in PBS buffer, pH 7.2, except for
compounds 1-3, when ascorbate conc. was 1 mM). The EPR signal intensity
is plotted as a function of time. (B) Decay curves for the radicals in a
cytosolic extract.
A
B

24
Scheme 1. Syntheses of isoindoline-derived radicals 18a and b, used for
synthesis of 8 and 10; R is either methyl (a) or ethyl (b).
R
R
R
R
R
R
HNO3
m-CPBA
CH2Cl2
NH
R
NH
R
N O
R
H2SO4
O2N
O2N
R
R
R
16a,b
17a,b
18a,b