Synthesis of a mitochondria-targeted spin trap using a novel Parham-type cyclization

Article abstract of DOI:10.1016/j.tet.2009.07.081

A new cyclic nitrone spin trap, [4-(3′,3′-dibutyl-2′-oxy-3′H-isoindol-5′-yloxy)butyl]triphenylphosphonium bromide (MitoSpin), bearing a lipophilic cation has been prepared by a route that involves a novel Parham-type lithiation-cyclization of an isocyanat

Full text of DOI:10.1016/j.tet.2009.07.081

Tetrahedron 65 (2009) 8154–8160
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of a mitochondria-targeted spin trap using a novel
Parham-type cyclization
,
Caroline Quin a, Jan Trnka b, Alison Haya, Michael P. Murphy b, Richard C. Hartley a
*
a Centre for the Chemical Research of Ageing, WestCHEM Department of Chemistry, University of Glasgow, Glasgow, G12 8QQ, UK
b MRC Mitochondrial Biology Unit, Hills Road, Cambridge, CB2 0XY, UK
a r t i c l e i n f o
a b s t r a c t
A new cyclic nitrone spin trap, [4-(30,30-dibutyl-20-oxy-30H-isoindol-50-yloxy)butyl]triphenylphospho-
nium bromide (MitoSpin), bearing a lipophilic cation has been prepared by a route that involves a novel
Parham-type lithiation–cyclization of an isocyanate to give the isoindolinone core. MitoSpin accumulates
in a membrane potential dependent way in energized mitochondria and its oxidation could potentially
be used in the study of oxidative stress resulting from reactive oxygen species generated in mitochondria.
Ó 2009 Elsevier Ltd. All rights reserved.
Article history:
Received 1 May 2009
Received in revised form 9 July 2009
Accepted 28 July 2009
Available online 6 August 2009
1. Introduction
cation.9,10 These lipophilic cations easily permeate biological
membranes due to their hydrophobicity and large ionic radius, and
Mitochondria play a central role in energy metabolism and cell
death, and consequently mitochondrial dysfunction contributes to
many pathologies.1 It has been suggested that reduction of oxygen
to superoxide by the mitochondrial respiratory chain ultimately
leads to the production of highly reactive oxygen-centered radicals
and carbon-centered radicals, which are then responsible for many
of these pathologies and for aging on a cellular and whole organism
level.2,3 Nitrones 1 react with these highly reactive oxygen-cen-
tered and carbon-centered radicals (Yꢀ) to give nitroxides 2 that are
more stable and longer lived (Scheme 1).4 The nitroxides 2 can be
detected by EPR spectroscopy and the hyperfine splittings observed
can often be used to identify the radical that led to their formation.
Thus, nitrones can be used as so-called spin traps for the study of
radical processes in biological samples and there is interest in
making spin traps that would accumulate within mitochondria and
report on mitochondrial radical production.5–8
the large mitochondrial membrane potential (150–170 mV, nega-
tive inside) causes the several-hundred fold accumulation of these
lipophilic cations into the mitochondrial matrix in accordance with
the Nernst equation.9–11 This approach has been used to target
antioxidants to mitochondria in cells, in vivo and in patients as
a therapeutic approach10,12,13 and to drive the accumulation of
probe molecules within mitochondria.14–16 Recently, three different
spin traps have been targeted to mitochondria by conjugating them
to TPP cations: a
-phenyl-N-tert-butylnitrone (PBN),5 5-tert-butoxy-
carbonyl-5-methyl-1-pyrroline N-oxide (BMPO),8 and 5-(diethoxy-
phosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO)6,7 (Fig. 1).
The PBN derivative 3 produces long-lived nitroxide radicals with
carbon-centered radicals, but not with oxygen-centered radicals,
while the targeted BMPO and DEPMPO spin traps 4 and 5 also react
O
O
N
Ph3P
Br
Small molecules can be targeted to mitochondria by conjugation
to lipophilic cations such as the alkyltriphenylphosphonium (TPP)
t
Bu
MitoPBN 3
O
Y
PPh3
Br
O
N
O
Y
Y
R2
R2
R2
MitoBMPO 4
+
R1
N
O
R1
N
O
R1
N
O
2
1
O
PPh3
Br
Scheme 1. Spin-trapping with nitrones.
O
N
H
N
O
PO(OEt)2
MitoDEPMPO 5
* Corresponding author.
E-mail address: rich@chem.gla.ac.uk (R.C. Hartley).
Figure 1. Mitochondria-targeted spin traps.
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.07.081

C. Quin et al. / Tetrahedron 65 (2009) 8154–8160
8155
with oxygen-centered radicals to generate relatively stable spin
adducts, which can be identified by their hyperfine couplings.
One problem with nitroxides is that they are rapidly converted
into EPR-silent products in vivo through their reduction by ascor-
bate, glutathione, and ubiquinol,17 and there is interest in
developing spin traps that generate adducts that are resistant to
such reduction. 1,1,3,3-Tetraalkylisoindolin-2-yloxyl radicals18,19
are reduced more slowly than many,18 and we designed MitoSpin 6
as a spin trap that would be targeted to mitochondria by a TPP
cation and was expected to generate sterically protected (albeit less
so than the tetralkyl derivatives) isoindolin-2-yloxyl radicals 7 by
reaction with highly reactive oxygen-centered and carbon-cen-
tered radicals (Yꢀ) (Scheme 2). The nitrone moiety would be con-
jugated with an electron-donating alkoxy group to enhance
reactivity with electron-deficient oxygen-centered radicals, and
since the benzylic C–N bond of spin adducts 7 would be held in
To assess whether MitoSpin 6 accumulated within mitochondria
driven by the membrane potential we used an ion-selective elec-
trode that responds to the triphenylphosphonium (TPP) moiety
(Fig. 2). The electrode was inserted into a stirred, thermostated
incubation chamber and showed the expected logarithmic re-
sponse to five sequential additions each increasing the concentra-
tion of MitoSpin 6 by 1 mM to give a final concentration of 5 mM
(Fig. 2, arrowheads show additions). Isolated mitochondria were
then added in the presence of the respiratory inhibitor rotenone to
prevent generation of a membrane potential from endogenous
substrates. This led to a slight decrease in the concentration of
MitoSpin due to the expected adsorption of the compound to the
mitochondrial surface.26,27 Addition of the respiratory substrate
succinate to generate a mitochondrial membrane potential led to
a rapid decrease in the concentration of MitoSpin 6, consistent with
its uptake into the mitochondrial matrix on induction of a mem-
brane potential. To confirm that the uptake was due to the mito-
chondrial membrane potential, we next added the uncoupler
carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP),
which abolished the mitochondrial membrane potential and led to
a rapid efflux of MitoSpin 6 from the mitochondria. The electrode
response to hydrophobic TPP compound is semi-quantitative due
to electrode drift.26 Even so, we can estimate that under these
conditions the steady state concentration of MitoSpin 6 outside
a ring perpendicular to the
p system, this conjugation should not
encourage the fragmentation of the spin adducts. The alkyl groups
would serve the dual role of encouraging interaction with the
mitochondrial inner membrane (the site of the main source of free
radicals within mitochondria) and sterically protecting the radical
center in the adduct, while the presence of two tails rather than one
should also discourage micelle formation.20
energized mitochondria is approximately 3 mM and that the re-
O
mainder of this is accumulated inside the mitochondria. From the
incubation chamber volume and the intramitochondrial volume
Ph3P
N
O
Br
(w0.6 m
L/mg protein28) this corresponds to approximately 3 mM
MitoSpin 6
concentration of MitoSpin 6 inside mitochondria, consistent with
the w1000-fold accumulation by the mitochondria expected from
the Nernst equation for mitochondria with the expected mito-
chondrial membrane potential values of 150–170 mV.9 Therefore,
these data are consistent with uptake of MitoSpin 6 into mito-
chondria driven by the mitochondrial membrane potential,
indicating that MitoSpin 6 behaves in a similar way to other hy-
drophobic TPP-conjugated compounds and that on addition to
mitochondria it is rapidly taken up into the matrix.29
Y
O
Ph3P
Br
N
Y
O
7
Scheme 2. Intended spin-trapping of radicals (Yꢀ) by MitoSpin 6.
2. Results and discussion
Next MitoSpin 6 was assessed as a spin trap. The Fenton reaction
between iron(II) ions and hydrogen peroxide was used to generate
hydroxyl radicals (Scheme 4) in the presence of MitoSpin 6, and the
EPR spectrum obtained at a range of concentrations of each re-
actant was a narrow triplet (Fig. 3). The same spectrum was
obtained when MitoSpin and hydrogen peroxide were irradiated
with UV light. The signal was very stable (72 h, without significant
change), but could be abolished by treatment of the solution with
glutathione or ascorbic acid. This narrow triplet is not consistent
with the hydroxy adduct 21 and is assigned as MitoSpinox 23
Fevig and co-workers had prepared a nitrone related to Mito-
Spin 6, via Lewis-acid mediated cyclization of an isocyanate to give
an isoindolinone core.21 However, in preliminary studies, we found
a similar cyclization unsatisfactory, so MitoSpin 6 was prepared by
first brominating 3-methoxyphenylacetic acid 8 to give acid 9. This
was converted into ester 10 and two consecutive enolate alkyl-
ations produced ester 12 via ester 11. Conversion to the free acid 13
could be achieved in base or by SN2 reaction with lithium iodide,
and reaction with diphenylphosphoryl azide (DPPA) followed im-
mediately by Curtius rearrangement of the acyl azide gave iso-
cyanate 14. Rather than hydrolyzing the isocyanate 14 to give an
amine for transition metal-mediated carbonylation with carbon
monoxide,22 we decided to investigate whether a Parham-type
procedure23 involving lithium–bromine exchange followed by
intramolecular reaction between the resulting aryllithium and the
isocyanate would give amide 15 directly. Couture and co-workers
had shown that isoindolinones could be made from carbamates in
this way,24 and intermolecular reaction between phenyllithium and
an isocyanate to produce an acyclic amide was known,25 but the
intramolecular version of this reaction was new. Therefore, we
were delighted to find that the isocyanate 14 gave amide 15 in high
yield when treated with tert-butyllithium. Reduction to give amine
16 proceeded smoothly. Oxidation of amine 16 to nitrone 17 fol-
lowed by deprotection to give phenol 19 was successful, but
reversing the steps to give the phenol 18 first was more efficient.
Finally, coupling with commercially available phosphonium salt 20
gave MitoSpin 6, albeit in modest yield (Scheme 3).
(Scheme 5), which has no b-hydrogen atom. A related nitroxide,
5,5-dimethyl-2-pyrrolidone-1-oxyl (DMPOX), has a similar small
hyperfine splitting of AN¼7.1 G for coupling with the nitrogen nu-
cleus.30 Under the Fenton conditions it seems likely that the orig-
inally generated hydroxy adduct 21 is oxidized to the EPR-silent
hydroxamic acid 22 (represented as the more thermodynamically
stable tautomer31) by reaction with iron(III) ions. A similar reaction
has been reported for DMPO.32 Hydroxamic acid 22 could also be
formed by disproportionation of nitroxide 21 or by abstraction of
a hydrogen atom from the nitroxide 21 by a second hydroxyl rad-
ical. Reaction between hydroxamic acid 22 and a hydroxyl radical
would give MitoSpinox 23. Reduction of MitoSpinox 23 with glu-
tathione or ascorbic acid is not surprising as the electron-with-
drawing carbonyl group means that the O–H bond dissociation
energy of the hydroxamic acid 22 will be at least 20 kJ molꢁ1 higher
than those of simple hydroxylamines.33
The high propensity of MitoSpin 6 to be oxidized means that it
will not be useful for distinguishing between different radicals in
mitochondria. However, it is taken up by mitochondria and reacts

8156
C. Quin et al. / Tetrahedron 65 (2009) 8154–8160
(a) LDA, THF
MeO
MeO
Br2, CHCl3
–78 °C
(b) BuI
CO2H
CO2R
MeO
CO2Me
Br
8
9 R = H, 100%
Br
MeOH
cat. H2SO4
11 91%
10 R = Me, 98%
(a) LDA, THF
–78 °C
(b) BuI
DPPA, Et3N
PhMe, 0 °C
then reflux
t
2 eq. BuLi
MeO
THF, –78 °C
MeO
MeO
NCO
CO2R
NH
Br
Br
O
15 93%
4 eq. BH3•THF, THF
5 eq. H2O2
14 97%
12 R = Me, 93%
5 M KOH(aq)
DMSO, heat
13 R = H, 84%
50 mol% NaWO4
H2O-MeOH
MeO
MeO
NH
N O
16 99%
17 53%
3.5 eq. BBr3
CH2Cl2, reflux
5 eq. H2O2
50 mol% NaWO4
H2O-MeOH
HO
HO
NH
N
O
18 93%
19 61%
Br
2 eq.
Ph3P
Br
20
CsCO3, MeOH, reflux
O
Ph3P
N
O
Br
MItoSpin 6 24%
Scheme 3. Synthesis of MitoSpin 6.
Figure 2. Uptake of MitoSpin by energized mitochondria. An electrode sensitive to the
TPP moiety of MitoSpin was inserted into 3 mL KCl buffer supplemented with 4 g/mL
rotenone with stirring at 37 ꢂC and five consecutive 1
M additions of MitoSpin were
m
m
used to calibrate the response of the ion-selective electrode (arrowheads). The addi-
tion of rat liver mitochondria (mitos; 1 mg protein/mL), succinate (10 mM), and FCCP
(1 mM) are indicated. This result is typical of three independent measurements.
Figure 3. EPR spectrum of MitoSpinox 23, g¼2.0071, AN¼7.6 G, the bar indicates the
scale for the magnetic field strength in Gauss.
Fe2 + H2O2
Fe3 + HO + HO
Scheme 4.
believed to be responsible for much of the endogenous oxidative
stress in cells, gives it potential as a therapeutic antioxidant.34
In conclusion, we have demonstrated a novel Parham-type cy-
clization of an isocyanate to access an isoindolone skeleton, and
used this as the key step in a concise synthesis of a new spin trap,
MitoSpin 6. MitoSpin 6 is a potentially useful mitochondria-tar-
geted spin trap and could also be used therapeutically or as a probe
to investigate mitochondrial oxidative stress in various models.
with oxidizing agents to give a strong, simple EPR spectrum, so it
may be useful for the detection of oxidative stress in mitochondria,
when natural antioxidants are depleted. Indeed, its oxidation
products could also be quantified by other techniques such as
HPLC–MS as markers of oxidative stress. Furthermore, MitoSpin’s
ability to reduce oxidizing species, and its accumulation at the sites

C. Quin et al. / Tetrahedron 65 (2009) 8154–8160
8157
0.61–0.48 (2H, m). dC (CDCl3, 100 MHz): 159.30 (C), 144.48 (C),
135.10 (CH), 133.82 (d, J¼9.9 Hz, CH), 133.58 (CH), 130.55 (d,
J¼12.6 Hz, CH), 126.82 (C), 120.89 (CH), 118.84 (d, J¼85.8 Hz, C)
114.24 (CH), 108.41 (CH), 84.18 (C), 67.19 (CH2), 37.21 (CH2), 29.62
(d, J¼16.6 Hz, CH2), 24.83 (CH2), 22.51 (CH2), 22.05 (CH2), 19.52
(CH2), 13.90 (CH3). IR (KBr, cmꢁ1): 3051, 2929–2858, 1683, 1611,
1586, 1526. LRMS (FAB/NOBA): 578 [Mþ (phosphonium cation),
100]. HRMS: 578.3201. C38H45NO2P requires 578.3188.
O
Ph3P
Ph3P
N
O
O
Br
MitoSpin 6
HO
O
N
3.1.3. (20-Bromo-50-methoxyphenyl)acetic acid 9. Bromine (8.3 mL,
162 mmol) was added dropwise to a solution of 3-methoxy-
phenylacetic acid 8 (25.7 g, 154 mmol) in CHCl3 (100 mL). The
resulting mixture was allowed to stir overnight at rt and then
poured into 20% aqueous sodium thiosulfate solution (300 mL).
The layers were separated and the aqueous layer was extracted
with CHCl3 (200 mL). The combined organics were washed with
20% aqueous sodium thiosulfate solution, dried over MgSO4 and
concentrated in vacuo to give the aryl bromide 7 as an off-white
solid (37.9 g, 100%); a small portion was recrystallized from EtOAc/
hexane to give plates. Mp 108–109 ꢂC. dH (CDCl3, 400 MHz): 7.46
(1H, d, J¼8.8 Hz), 6.85 (1H, d, J¼3.0 Hz), 6.74 (1H, dd, J¼8.8,
3.0 Hz), 3.80 (2H, s), 3.78 (3H, s). dC (CDCl3, 100 MHz): 176.50 (C),
158.95 (C), 134.27 (C), 133.40 (CH), 117.29 (CH), 115.46 (C), 114.84
(CH), 55.51 (CH3), 41.49 (CH2). IR (KBr cmꢁ1): 2949–2856, 1692,
Br
21
OH
Fe3
Fe2 + H
O
Ph3P
Br
N
OH
22
O
HO
H2O
O
Ph3P
Br
N
O
O
MitoSpinox 23
ꢀ
ꢀ
1594, 1571. LRMS (EIþ), m/z: 246 [Mþ
(
81Br), 28%)], 244 [Mþ
Scheme 5.
(
79Br), 28%], 201 (20), 199 (20), 165 (100). HMRS: 243.9734.
ꢀ
C9H9BrO3 requires (Mþ ) 243.9735. 1H and 13C NMR matches
literature.35
3. Experimental
3.1. General
3.1.4. Methyl 2-(20-bromo-50-methoxyphenyl)acetate 10. Concentrated
sulfuric acid (6 drops) was added to a solution of the carboxylic acid 9
(37.9 g, 155 mmol) in methanol (150 mL). The resulting mixture was
stirred at rt overnight. The reaction was concentrated in vacuo to ap-
proximately 20 mL and the mixture taken up in EtOAc and washed
with 1 M NaOH(aq), then with brine. The organics were dried over
MgSO4 and concentrated in vacuo to give ester 10 (37.6 g, 98%) as
a yellow oil, which crystallized on standing to give prisms. Mp:
38–40 ꢂC (lit.36 oil). dH (CDCl3, 400 MHz): 7.43 (1H, d, J¼8.8 Hz), 6.84
(1H, d, J¼3.0 Hz), 6.70 (1H, dd, J¼8.8, 3.0 Hz), 3.77 (3H, s), 3.74 (2H, s),
3.71 (3H, s). dC (CDCl3, 100 MHz): 170.98 (C), 158.99 (C), 135.04 (C),
133.38 (CH), 117.22 (CH), 115.44 (C), 114.64 (CH), 55.53 (CH3), 52.29
(CH3), 41.73 (CH2). IR (thin layer, cmꢁ1) 3007, 2952, 1743. LRMS (EIþ),
3.1.1. Synthesis. Reactions under an inert atmosphere were carried
out using oven-dried or flame-dried glassware. Solutions were
added via syringe. THF was freshly distilled from sodium benzo-
phenone. Dichloromethane was distilled from CaH2 prior to use.
Reagents were obtained from commercial suppliers and used
without further purification unless otherwise stated. 1H and
13C NMR spectra were obtained on a Bruker DPX/400 spectrometer
operating at 400 and 100 MHz, respectively. 1H NMR spectra in
CDCl3 are referenced to residual chloroform at 7.26 ppm and in
CD3OD are referenced to residual methanol at 3.31 ppm. 13C NMR
spectra in CDCl3 are referenced to CDCl3 at 77.16 ppm and in CD3OD
are referenced to CD3OD at 49.00 ppm. DEPT was used to assign the
signals in the 13C NMR spectra as C, CH, CH2, or CH3. All coupling
constants are measured in Hz. Mass spectra (MS) were recorded on
a Jeol JMS700 (MStation) spectrometer. Some IR spectra were
obtained employing a Golden GateÔ attachment that uses a type
IIa diamond as a single reflection element so that the IR spectrum of
the compound (solid or liquid) could be directly detected (thin
layer) without any sample preparation.
ꢀ
ꢀ
m/z: 260 [Mþ
( (
81Br), 16%], 258 [Mþ 79Br), 16], 179 (100). HMRS:
ꢀ
257.9892 and 259.9871. C10H1179BrO3 requires (Mþ ) 257.9892, and
ꢀ
81
C10
H
BrO3 requires (Mþ ) 259.9872. IR matches literature and
11
1H NMR spectrum is similar, but not the same as that recorded in
a different solvent (CCl4).36
3.1.5. Methyl 2-(20-bromo-50-methoxyphenyl)hexanoate 11. n-BuLi
(43 mL, 2.5 M in hexane, 109 mmol) was added dropwise to a stir-
red solution of diisopropylamine (15 mL, 109 mmol) in dry THF
(150 mL) at ꢁ78 ꢂC under argon ensuring that the internal tem-
perature remained below ꢁ60 ꢂC. The resulting solution was
allowed to stir for 15 min and a solution of methyl ester 10 (25 g,
99 mmol) in dry THF (200 mL) was added dropwise ensuring that
the internal temperature remained below ꢁ60 ꢂC. The reaction
mixture was allowed to stir for 15 min and then butyl iodide
(12.0 mL, 109 mmol) was added dropwise. The mixture was
allowed to warm to rt and stirred overnight. The reaction was
quenched by the addition of ice cold H2O and extracted with Et2O.
The organic layer was washed with H2O, dried over MgSO4, and
concentrated in vacuo to give a brown oil. The oil was purified using
flash chromatography on silica gel eluting with hexane followed by
hexane/Et2O (3:2) to afford the ester 11 as a yellow oil (20.7 g, 91%).
Rf [SiO2, hexane/Et2O (3:2)]: 0.63. dH (CDCl3, 400 MHz): 7.43 (1H, d,
J¼8.8 Hz), 6.93 (1H, d, J¼3.1 Hz), 6.67 (1H, dd, J¼8.8, 3.1 Hz), 4.09
3.1.2. [4-(30,30-Dibutyl-20-oxy-30H-isoindol-50-yloxy)butyl]triphenyl-
phosphonium bromide (MitoSpin) 6. A stirred solution of nitrone 19
(500 mg, 1.9 mmol), cesium carbonate (686 mg, 2.10 mmol), and
4-bromobutyltriphenylphosphonium bromide (1.83 g, 3.83 mmol)
in dry MeOH (15 mL) were heated to reflux for 48 h. The mixture
was then partitioned between H2O and EtOAc, and the aqueous
phase was extracted with EtOAc (2ꢃ). The combined organic
extracts were dried over MgSO4 and concentrated in vacuo to give
a yellow solid foam. Recrystallization from EtOAc/hexane gave
MitoSpin 6 as an amorphous solid (298 mg, 24%). Mp: decomposes
at 84 ꢂC. dH (CDCl3, 400 MHz): 7.92–7.87 (6H, m), 7.80–7.77 (3H,
m), 7.70–7.66 (7H, m), 7.22 (1H, d, J¼8.4 Hz), 6.85 (1H, dd, J¼8.4,
2.0 Hz), 6.74 (1H, d, J¼2.0 Hz), 4.18 (2H, t, J¼5.8 Hz), 4.06–4.03
(2H, m), 2.35–2.25 (2H, m), 2.13–2.05 (2H, m), 1.96–1.63 (4H, m),
1.24–1.13 (4H, m), 0.99–0.82 (2H, m), 0.75 (6H, t, J¼6.4 Hz),

8158
C. Quin et al. / Tetrahedron 65 (2009) 8154–8160
(1H, t, J¼7.5 Hz), 3.77 (3H, s), 3.65 (3H, s), 2.07–1.98 (1H, m), 1.78–
1.70 (1H, m), 1.36–1.21 (4H, m), 0.82 (3H, t, J¼6.9 Hz). dC (CDCl3,
100 MHz): 174.04 (C), 159.21 (C), 139.87 (C), 133.46 (CH), 115.28
(CH), 114.34 (C), 114.34 (CH), 55.50 (CH3), 52.13 (CH3), 49.99 (CH),
33.16 (CH2), 29.62 (CH2), 22.58 (CH2), 13.98 (CH3). IR (thin layer,
then quenched with saturated ammonium chloride solution and
the mixture was extracted with Et2O (3ꢃ). The combined organics
were washed with brine, dried over MgSO4, and concentrated in
vacuo to giva a brown oil. Flash chromatography on silica gel eluting
with hexane/EtOAc (4:1) gave the isocyanate 14 as an oil (20.8 g,
97%). Rf [SiO2, hexane/Et2O (4:1)]: 0.78 dH (CDCl3, 400 MHz): 7.47
(1H, d, J¼8.8 Hz), 7.25 (1H, d, J¼2.8 Hz), 6.67 (1H, dd, J¼8.8, 2.8 Hz),
3.82 (3H, s), 2.74–2.66 (2H, m), 1.90–1.83 (2H, m), 1.43–1.22 (6H, m),
1.00–0.84 (2H, m), 0.70 (6H, t, J¼7.6 Hz). dC (CDCl3, 100 MHz):
158.78 (C), 141.40 (C), 136.22 (CH), 121.87 (C), 117.41 (CH), 113.62
(CH), 109.49 (C), 70.77 (C), 55.55 (CH3), 40.84 (CH2), 26.59 (CH2),
22.73 (CH2), 14.07 (CH3). IR (Thin layer, cmꢁ1) 3010–2850, 2261,
ꢀ
cmꢁ1) 3010–2800, 1737, 1595, 1572. LRMS (EIþ), m/z: 316 [Mþ
ꢀ
(
81Br), 49%], 314 [Mþ
(
79Br), 49%], 257 (12), 314 (12), 235 (100).
ꢀ
HMRS: 314.0518 and 316.047. C14H1979BrO3 requires (Mþ ) 314.0518,
ꢀ
81
and C14
H
BrO3 requires (Mþ ) 316.0499.
19
3.1.6. Methyl 2-(20-bromo-50-methoxyphenyl)-2-butylhexanoate
12. n-BuLi (39 mL, 2.5 M in hexane, 99 mmol) was added drop-
wise to a stirred solution of diisopropylamine (14 mL, 99 mmol)
in dry THF (150 mL) at ꢁ78 ꢂC under argon ensuring that the
internal temperature remained below ꢁ60 ꢂC. The resulting so-
lution was allowed to stir for 15 min and a solution of ester 11
(28.4 g, 90.1 mmol) in dry THF (200 mL) was added dropwise
ensuring that the internal temperature remained below ꢁ60 ꢂC.
The reaction mixture was allowed to stir for 15 min and then
butyl iodide (11 mL, 99 mmol) was added dropwise. The mixture
was allowed to warm to rt and stirred overnight. The reaction was
quenched by the addition of ice cold H2O and extracted with
Et2O. The organic layer was washed with H2O, dried over MgSO4,
and concentrated in vacuo to give a brown oil. Flash chroma-
tography on silica gel eluting with hexane followed by hexane/
Et2O (3:2) afforded the ester 12 as a yellow oil, which crystallized
on standing and was washed with hexane to give the ester as a an
amorphous solid (22.9 g, 93%). Mp: 53–54 ꢂC. Rf [SiO2, hexane/
Et2O (3:2)]: 0.65. dH (CDCl3, 400 MHz): 7.45 (1H, d, J¼8.7 Hz), 6.91
(1H, d, J¼3.0 Hz), 6.65 (1H, dd, J¼8.7, 3.0 Hz), 3.81 (3H, s), 3.67
(3H, s), 2.20–2.12 (2H, m), 1.99–1.92 (2H, m), 1.32–1.24 (4H, m),
1.11–1.05 (2H, m), 0.96–0.90 (8H, m). dC (CDCl3, 100 MHz): 175.94
(C), 158.36 (C), 142.97 (C), 134.90 (CH), 116.87 (CH), 114.33 (C),
111.94 (CH), 55.41 (CH3), 54.48 (C), 52.15 (CH3), 33.26 (CH2), 26.15
(CH2), 23.11 (CH2), 14.01 (CH3). IR (thin layer, cmꢁ1) 3010–2800,
ꢀ
ꢀ
1592, 1569. MS, (EIþ), m/z: 355 [Mþ 81Br), 36%], 353 [Mþ 79Br),
( (
36], 298 (100), 296 (100), 242 (60), 240 (61), 201 (39), 199 (36), 174
ꢀ
(81). HMRS: 353.0991 and 355.0968. C17H2479BrNO2 requires (Mþ
)
ꢀ
81
353.0990 and C17
H
24
BrNO2 requires (Mþ ) 355.0970.
3.1.9. 3,3-Dibutyl-2,3-dihydro-5-methoxyisoindol-1-one 15. tBuLi
(24 mL, 1 M in cyclohexane, titrated using 1,3-diphenyl acetone-p-
tosylhydrazone, 24 mmol) was added dropwise to a stirred solution
of the isocyanate 14 (4.00 g, 11.8 mmol) in dry THF (100 mL) at
ꢁ78 ꢂC under argon. The resulting mixture was allowed to warm to
rt and stirred overnight before ice cold water was added. The
mixture was extracted with EtOAc and the organic layer was
washed with brine, dried over MgSO4, and concentrated in vacuo to
afford amide 15 as an amorphous solid (3.02 g, 93%). Mp: 96–97 ꢂC.
dH, (CDCl3, 400 MHz): 7.71 (1H, d, J¼8.4 Hz), 6.93 (1H, dd, J¼8.4,
2.2 Hz), 6.87 (1H, s), 6.75 (1H, d, J¼2.2 Hz), 3.87 (3H, s), 1.88–1.72
(4H, m), 1.24–1.14 (6H, m), 0.84–0.76 (8H, m). dC (CDCl3, 100 MHz):
170.56 (C), 163.16 (C), 153.23 (C), 125.17 (CH), 125.06 (C), 113.85
(CH), 106.50 (CH), 64.80 (C), 55.75 (CH3), 39.24 (CH2), 25.62 (CH2),
22.90 (CH2), 14.01 (CH3). IR (KBr, cmꢁ1) 3286, 2955–2850, 1693,
ꢀ
1661, 1606, 1488. LRMS (EIþ), m/z: 275 (Mþ , 1.5%), 218 (100). HRMS:
ꢀ
275.1884. C17H25O2N requires (Mþ ) 275.1885.
ꢀ
ꢀ
1732, 1596. LRMS (EIþ), m/z: 372 [Mþ
(
81Br), 3%], 370 [Mþ
(
79Br),
3.1.10. 1,1-Dibutyl-2,3-dihydro-6-methoxy-1H-isoindole 16. Borane/
THF complex (51 mL of a 1 M solution in THF, 51 mmol) was added
over 20 min to a stirred solution of amide 15 (3.50 g, 12.7 mmol) in
dry THF (50 mL) at 0 ꢂC under argon and the mixture allowed to stir
for 1 h. After this time the mixture was heated to reflux for 48 h. The
reaction was then quenched by the slow addition of ice cold H2O
(100 mL). 1 M NaOH(aq) was added and the mixture extracted with
Et2O (2ꢃ). The combined organic extracts were washed with brine
(100 mL), dried over MgSO4, and then concentrated in vacuo to give
an oil 16 (3.30 g, 99%). dH (CDCl3, 400 MHz): 7.16 (1H, d, J¼8.4 Hz),
6.77 (1H, dd, J¼8.4, 2.4 Hz), 6.61 (1H, d, J¼2.4 Hz), 4.14 (2H, s), 3.82
(3H, s), 2.05–1.65 (4H, m), 1.26–1.23 (6H, m), 1.10–1.04 (2H, m), 0.89
(6H, t, J¼7.2 Hz). The amine was dissolved in Et2O (20 mL) and
ethereal HCl was added affording the salt as a white precipitate.
Mp: decomposed at 210 ꢂC. dH (CDCl3, 100 MHz): 10.19 (2H, s), 7.17
(1H, d, J¼8.4 Hz), 6.88 (1H, dd, J¼8.4, 2.2 Hz), 6.59 (1H, d, J¼2.2 Hz),
4.60 (2H, s), 3.82 (3H, s), 2.13–1.96 (4H, m), 1.57–1.48 (2H, m), 1.42–
1.23 (6H, m), 0.91 (6H, t, J¼7.2 Hz). dC (CDCl3, 100 MHz): 160.37 (C),
143.48 (C), 125.85 (C), 124.00 (CH), 114.45 (CH), 108.28 (CH), 75.76
(C), 55.76 (CH3), 48.62 (CH2), 37.40 (CH2), 25.73 (CH2), 22.82 (CH2),
14.05 (CH3). IR (KBr, cmꢁ1): 3010–2800, 2717, 1621, 1588. MS, (CIþ),
m/z: 262 [MþHþ (amine), 82%], 218 (24), 79 (100). HRMS: 262.2172.
C17H28ON requires [MþHþ (amine)] 262.2171.
3], 291 (100). HRMS: 372.1125 and 370.1143. C18H2781BrO3 re-
ꢀ
ꢀ
quires (Mþ ) 372.1125 and C18H2779BrO3 requires (Mþ ) 370.1144.
3.1.7. 2-(20-Bromo-50-methoxyphenyl)-2-butylhexanoic acid 13. 5 M
KOH(aq) (25 mL) was added to a stirred solution of ester 12 (10.0 g,
27.0 mmol) in DMSO (150 mL) and the resulting mixture was
heated to reflux overnight. The reaction was allowed to cool, was
acidified with 5 M HCl(aq), and extracted with EtOAc (3ꢃ). The
combined organic extracts were washed with brine, dried over
MgSO4, and concentrated in vacuo to afford the carboxylic acid 13
as an off-white solid (8.08 g, 84%). A small portion was recrystal-
lized from DCM to give needles. Mp: 163–165 ꢂC. dH (CDCl3,
400 MHz): 7.46 (1H, d, J¼8.6 Hz, H-30), 6.91 (1H, d, J¼3.0 Hz, H-60),
6.66 (1H, dd, J¼8.6 Hz, 3.0 Hz, H-40), 3.80 (3H, s, CH3O), 2.24–2.16
(2H, m, CH2), 1.99–1.92 (2H, m, CH2), 1.33–1.11 (6H, m, 3ꢃCH2),
0.99–0.92 (2H, m, CH2), 0.88 (6H, t, J¼7.2 Hz, 2ꢃCH3). dC (CDCl3,
100 MHz): 181.71 (C), 158.39 (C), 142.34 (C), 135.02 (CH), 116.99
(CH), 114.48 (C), 112.18 (CH), 55.46 (CH3), 54.54 (C), 32.94 (CH2),
26.09 (CH2), 23.12 (CH2), 14.03 (CH3). IR (KBr, cmꢁ1): 3010–2955,
ꢀ
2610, 1703, 1597, 1570. LRMS (EIþ), m/z: 358 [Mþ
(
81Br), 2%], 370
ꢀ
ꢀ
[Mþ
(
79Br), 2], 291 (Mþ –ꢀBr, 100), 85 (35). HRMS: 356.0986 and
ꢀ
358.0977. C17H2579BrO3 requires (Mþ ) 356.0987 and C17H2581BrO3
ꢀ
requires (Mþ ) 358.0969.
3.1.11. 1,1-Dibutyl-6-methoxy-1H-isoindole-2-oxide 17. The hydro-
chloride salt of amine 16 (500 mg, 1.68 mmol) was dissolved in
MeOH (5 mL) then NaOH (70.5 mg, 1.68 mmol), Na2WO4 (58.0 mg,
0.17 mmol), and 30% H2O2 in H2O (0.57 mL, 5.04 mmol) were
added. The resulting mixture was then allowed to stir at rt under
argon for 3 days. The reaction was then quenched with 20% Na2S2O3
in brine and extracted into EtOAc (3ꢃ). The organic extracts were
3.1.8. 1-Bromo-2-(50-isocyanatonon-50-yl)-4-methoxybenzene
14. Triethylamine (9.2 mL, 66 mmol) was added to a stirred solu-
tion of carboxylic acid 13 (21.5 g, 60.2 mmol) in dry toluene
(250 mL) at 0 ꢂC under argon. Diphenylphosphoryl azide (14.3 mL,
66 mmol) was then added and the resulting mixture was allowed to
stir for 15 min before heating to reflux for 18 h. The reaction was

C. Quin et al. / Tetrahedron 65 (2009) 8154–8160
8159
combined, dried over MgSO4, and concentrated to give an orange
oil. Column chromatography, on silica gel, eluting with EtOAc/
hexane (3:7) gave the nitrone 17 as a pale orange solid (157 mg,
34%). dH (CDCl3, 400 MHz): 7.67 (1H, s), 7.25 (1H, d, J¼8.4 Hz), 6.88
(1H, dd, J¼8.4, 1.6 Hz), 6.75 (1H, d, J¼1.6 Hz), 3.87 (3H, s), 2.14–2.07
(2H, m), 1.87–1.79 (2H, m), 1.30–1.11 (4H, m), 1.02–0.91 (2H, m,
CH2), 0.77 (6H, t, J¼7.2 Hz), 0.64–0.58 (2H, m). dC (CDCl3, 100 MHz):
160.09 (C), 144.51 (C), 133.69 (C), 126.98 (C), 120.95 (CH), 113.01
(CH), 108.41 (CH), 84.23 (C), 55.75 (CH3), 37.33 (CH2), 24.90 (CH2),
22.61 (CH2), 13.95 (CH3). IR (KBr, cmꢁ1): 3100–2800, 1588, 1525.
a 10 mM solution). The solution was then immediately transferred
to a quartz flat cell and placed in the EPR spectrometer for analysis.
3.1.16. Reaction between MitoSpin 4 and hydroxyl radicals generated
by UV irradiation. Aqueous hydrogen peroxide (150
mL of a 100 mM
solution) was added to a solution of MitoSpin 4 in DMF (150
mL of
a 30 mM solution) in a quartz cuvette and the solutionwas irradiated
with UV light (254 nm, 12 W lamp) for 20 min before transferring to
a quartz flat cell and placed in the EPR spectrometer for analysis.
ꢀ
LRMS (EIþ), m/z: 275 (Mþ , 18%), 258 (82), 219 (100), 202 (41), 176
3.1.17. Mitochondrial incubations. Rat liver mitochondria were
prepared by homogenization followed by differential centrifuga-
tion in 250 mM sucrose, 5 mM Tris–HCl, 1 mM EGTA, pH 7.4.37
Protein concentration was determined by the biuret assay using
BSA as a standard.38 All incubations were at 30 ꢂC in KCl buffer
(120 mM KCl, 1 mM EGTA, 10 mM HEPES, pH 7.2) supplemented
ꢀ
(96), 160 (32). HRMS: 275.1887. C17H25O2N requires (Mþ ) 275.1885.
3.1.12. 3,3-Dibutyl-2,3-dihydro-1H-isoindol-5-ol 18. BBr3 (54 mL of
a 1 M solution in DCM, 54 mmol) was added dropwise to a solution
of amine 16 (4.00 g, 15.3 mmol) in dry DCM (100 mL) at 0 ꢂC under
argon. The reaction mixture was then allowed to warm to rt for
72 h. After this time the reaction was quenched with ice cold water.
The mixture was basified with 1 M NaOH(aq) and extracted with
DCM (3ꢃ). The combined organics were washed with brine, dried
over MgSO4, and concentrated in vacuo to give phenol 18 as an
amorphous solid (3.52 g, 93%). Mp: 110–111 ꢂC. dH (CD3OD,
400 MHz): 7.21 (1H, d, J¼8.3 Hz), 6.84 (1H, dd, J¼8.3, 2.2 Hz), 6.65
(1H, d, J¼2.2 Hz), 4.46 (2H, s), 2.09–2.02 (2H, m), 1.93–1.85 (2H, m),
1.45–1.32 (6H, m), 1.26–1.16 (2H, m), 0.88 (6H, t, J¼7.1 Hz). dC
(CD3OD, 100 MHz): 159.74 (C), 143.85 (C), 125.14 (C), 125.04 (CH),
117.61 (CH), 110.34 (CH), 76.59 (C), 49.65 (CH2), 37.42 (CH2), 26.53
(CH2), 23.79 (CH2), 14.17 (CH3). IR (KBr, cmꢁ1): 3433, 3310, 2955–
2931, 2858, 1612, 1481, 1465. LRMS (CIþ), m/z: 248 [MþHþ (amine),
100%], 190 (25). HRMS: 248.2010. C16H26NO requires [MþHþ
(amine)] 248.2015.
with 10 mM succinate and 4 mg/mL rotenone. A TPP-selective
electrode was constructed and used as previously described26 to
measure MitoSpin accumulation into rat liver mitochondria.
Acknowledgements
We thank SPARC and the BBSRC for the purchase of the bench-
top EPR spectrometer used. The University of Glasgow for Caroline
Quin0s studentship. The Wellcome Trust for funding Alison Hay.
Gates Cambridge Trust for funding Jan Trnka.
References and notes
1. (a) Wallace, D. C. Science 1999, 283, 1482–1488; (b) Lin, M. T.; Beal, M. F. Nature
2006, 443, 787–795.
2. Finkel, T. Nat. Rev. Mol. Cell Biol. 2005, 6, 971–976.
3. Murphy, M. P. Biochem. J. 2009, 417, 1–13.
4. Rosen, G. M.; Britigan, B. E.; Halpern, H. J.; Pou, S. Free Radicals: Biology and
Detection by Spin Trapping; OUP: Oxford, 1999.
5. Murphy, M. P.; Echtay, K. S.; Blaikie, F. H.; Asin-Cayuela, J.; Cocheme, H. M.;
Green, K.; Buckingham, J. A.; Taylor, E. R.; Hurrell, F.; Hughes, G.; Miwa, S.;
Cooper, C. E.; Svistunenko, D. A.; Smith, R. A.; Brand, M. D. J. Biol. Chem. 2003,
278, 48534–48545.
6. Hardy, M.; Chalier, F.; Ouari, O.; Finet, J. P.; Rockenbauer, A.; Kalyanaraman, B.;
Tordo, P. Chem. Commun. 2007, 1083–1085.
7. Hardy, M.; Rockenbauer, A.; Vasquez-Vivar, J.; Felix, C.; Lopez, M.; Srinivasan, S.;
Avadhani, N.; Tordo, P.; Kalyanaraman, B. Chem. Res. Toxicol. 2007, 20,1053–1060.
8. Xu, Y.; Kalyanaraman, B. Free Radical Res. 2007, 41, 1–7.
9. Ross, M. F.; Kelso, G. F.; Blaikie, F. H.; James, A. M.; Cocheme, H. M.; Filipovska,
A.; Da Ros, T.; Hurd, T. R.; Smith, R. A.; Murphy, M. P. Biochemistry (Mosc) 2005,
70, 222–230.
10. Murphy, M. P.; Smith, R. A. Annu. Rev. Pharmacol. Toxicol. 2007, 47, 629–656.
11. Murphy, M. P.; Smith, R. A. J. Adv. Drug Delivery Rev. 2000, 41, 235–250.
12. Smith, R. A. J.; Porteous, C. M.; Coulter, C. V.; Murphy, M. P. Eur. J. Biochem. 1999,
263, 709–716.
13. Smith, R. A. J.; Porteous, C. M.; Gane, A. M.; Murphy, M. P. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 5407–5412.
3.1.13. 3,3-Dibutyl-2-oxy-3H-isoindol-5-ol 19. Sodium tungstate
(1.00 g, 3.0 mmol) and hydrogen peroxide (2.1 mL, 90 mmol) were
added to a stirred solution of amine 18 (1.50 g, 6.1 mmol) in dry
methanol (20 mL) and the resulting mixture was stirred at rt for
72 h. The reaction mixture was then quenched with 20% aqueous
sodium sulfite solution and extracted with DCM (3ꢃ). The com-
bined organic extracts were washed with brine, dried over MgSO4,
and concentrated in vacuo to give an orange foam. The foam was
triturated with EtOAc to give phenol 19 as an off-white solid
(980 mg, 61%). A small portion was recrystallized from hexane/
EtOAc to give prisms. Mp: 193–195 ꢂC. dH (CD3OD, 400 MHz): 8.12
(1H, s), 7.32 (1H, d, J¼8.4 Hz), 6.81 (1H, dd, J¼8.4, 2.0 Hz), 6.74 (1H,
d, J¼2.0 Hz), 2.14–2.01 (2H, m),1.94–1.78 (2H, m),1.23–1.06 (4H, m),
0.95–0.85 (2H, m), 0.80 (6H, t, J¼6.4 Hz), 0.74–0.70 (2H, m, CH2).
dC (CD3OD, 100 MHz): 160.82 (C), 146.81 (C), 139.29 (CH), 125.71 (C),
123.72 (CH), 116.73 (CH), 110.07 (CH), 85.11 (C), 37.69 (CH2), 25.88
(CH2), 23.38 (CH2), 14.14 (CH3). IR (KBr, cmꢁ1): 3439 (OH), 2954–
14. Burns, R. J.; Smith, R. A. J.; Murphy, M. P. Arch. Biochem. Biophys.1995, 322, 60–68.
15. Lin, T. K.; Hughes, G.; Muratovska, A.; Blaikie, F. H.; Brookes, P. S.; Darley-Usmar,
V.; Smith, R. A. J.; Murphy, M. P. J. Biol. Chem. 2002, 277, 17048–17056.
16. Robinson, K. M.; Janes, M. S.; Pehar, M.; Monette, J. S.; Ross, M. F.; Hagen, T. M.;
Murphy, M. P.; Beckman, J. S. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15038–15043.
17. Trnka, J.; Blaikie, F. H.; Smith, R. A. J.; Murphy, M. P. Free Radical Biol. Med. 2008,
44, 1406–1419.
ꢀ
ꢀ
ꢀ
2857, 1587, 1537. LRMS (EIþ), m/z: 261 (Mþ , 10%), 244 (Mþ ꢁ OH,
ꢀ
ꢀ
23), 216 (Mþ ꢁH2O and HCN, 58), 205 (Mþ ꢁC4H8, 69%), 188
(Mþ ꢁ OH and C4H8, 37), 162 (Mþ ꢁC4H8 and ꢀPr, 100), 146 (Mþ ꢁ Bu
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
and BuH, 32). HRMS: 261.1728. C16H23NO2 requires (Mþ ) 261.1729.
18. (a) Marx, L.; Chiarelli, R.; Guiberteau, T.; Rassat, A. J. Chem. Soc., Perkin Trans. 1
2000, 1181–1182; (b) Marx, L.; Rassat, A. Angew. Chem., Int. Ed. 2000, 39, 4494–
4496; (c) Reid, D. A.; Bottle, S. E.; Micallef, A. S. Chem. Commun. 1998, 1907–1908.
19. The oxidation and reduction of this type of radical has been studied: Blinco, J.
P.; Hodgson, J. L.; Morrow, B. J.; Walker, J. R.; Will, G. D.; Coote, M. L.; Bottle, S. E.
J. Org. Chem. 2008, 73, 6763–6771.
20. (a) Cooper, A. Biophysical Chemistry; Royal Society of Chemistry: Cambridge,
2004; Chapter 1; (b) Shearman, G. C.; Ces, O.; Templer, R. H.; Seddon, J. M.
J. Phys. Condens. Matter 2006, 18, S1105–S1124.
21. Fevig, T. L.; Bowmen, S. M.; Janowick, D. A.; Jones, B. K.; Munson, H. R.;
Ohlweiler, D. F.; Thomas, C. E. J. Med. Chem. 1996, 39, 4988–4996.
22. Orito, K.; Horibata, A.; Nakamura, T.; Ushito, H.; Nagasaki, H.; Yaguchi, M.;
Yamashita, S.; Tokuda, M. J. Am. Chem. Soc. 2004, 126, 14342–14343.
23. (a) Parham, W. E.; Bradsher, C. K. Acc. Chem. Res. 1982, 15, 300–305; (b) Clayden, J.
Organolithiums: Selectivity for Synthesis; Pergamon: London, 2002; Chapter 3.
24. (a) Couture, A.; Deniau, E.; Lamblin, M.; Lorion, M.; Grandclaudon, P. Synthesis
2007, 1434–1437; (b) Lamblin, M.; Couture, A.; Deniau, E.; Grandclaudon, P. Org.
3.1.14. EPR spectroscopy. EPR spectra were acquired on a bench-top
EPR machine with a permanent magnet and a magnetic sweep
circuit (center of field¼0.345 T, sweep width 25 mT) operating at
a frequency of 9.8 GHz (X-band). Acquisition parameters: RG
3.99ꢃ103, 2.76 mW, MA 0.5 G. Hyperfine couplings were derived
from simulations and the g value calculated using strong pitch as
the reference.
3.1.15. Reaction between MitoSpin 4 and hydroxy radicals in the
presence of iron ions. Aqueous iron(II) sulfate (100
solution) and aqueous hydrogen peroxide (100 L of a 1 mM solu-
tion) were added to a solution of the MitoSpin 4 in DMF (100 L of
mL of a 1 mM
m
m

8160
C. Quin et al. / Tetrahedron 65 (2009) 8154–8160
Biomol. Chem. 2007, 5, 1466–1471; (c) Lamblin, M.; Couture, A,; Deniau, E.;
30. Rosen, G. M.; Rauckman, E. J. Mol. Pharmacol. 1980, 17, 233–238; Løvstad, R. A.
BioMetals 2003, 16, 435–439.
31. Saldyka, M.; Mielke, Z. Vib. Spectrosc. 2007, 45, 46–54.
32. Makino, K.; Hagi, A.; Ide, H.; Murakami, A. Can. J. Chem. 1992, 70, 2818–2827.
33. Luo, Y.-R. Handbook of Bond Dissociation Energies in Organic Compounds; CRC:
Boca Raton, 2003.
34. Review of nitrones as therapeutics: Floyd, R. A.; Kopke, R. D.; Choi, C.-H.; Foster,
S. B.; Doblas, S.; Towner, R. A. Free Radical Biol. Med. 2008, 45, 1361–1374.
35. Jeong, J. H.; Weinreb, S. M. Org. Lett. 2006, 8, 2309–2312.
36. Ambros, R.; von Angerer, S.; Wiegrebe, W. Arch. Pharm. (Weinheim) 1988, 321,
481–486.
37. Chappell, J. B.; Hansford, R. G. In Subcellular Components: Preparation and
Fractionation; Birnie, G. D., Ed.; Butterworths: London, 1972; pp 77–91.
38. Gornall, A. G.; Bardawill, C. J.; David, M. M. J. Biol. Chem. 1949, 177, 751–766.
Grandclaudon, P. Synthesis 2006,1333–1338; (d) Moreau, A.; Lorion, M.; Couture,
A.; Deniau, E.; Grandclaudon, P. J. Org. Chem. 2006, 71, 3303–3305; (e) Moreau, A.;
Couture, A.; Deniau, E.; Grandclaudon, P. Tetrahedron 2006, 62, 5787–5790.
25. Kuramochi, K.; Watanabe, H.; Kitahara, T. Synlett 2000, 397–399.
26. Asin-Cayuela, J.; Manas, A. R.; James, A. M.; Smith, R. A.; Murphy, M. P. FEBS Lett.
2004, 571, 9–16.
27. Smith, R. A.; Kelso, G. F.; James, A. M.; Murphy, M. P. Methods Enzymol. 2004,
382, 45–67.
28. Ross, M. F.; Da Ros, T.; Blaikie, F. H.; Prime, T. A.; Porteous, C. M.; Severina, I. I.;
Skulachev, V. P.; Kjaergaard, H. G.; Smith, R. A.; Murphy, M. P. Biochem. J. 2006,
400, 199–208.
29. Ross, M. F.; Prime, T. A.; Abakumova, I.; James, A. M.; Porteous, C. M.; Smith, R.
A. J.; Murphy, M. P. Biochem. J. 2008, 411, 633–645.