Mitochondria-targeted spin traps: Synthesis, superoxide spin trapping, and mitochondrial uptake

Article abstract of DOI:10.1021/tx500032e

Development of reliable methods and site-specific detection of free radicals is an active area of research. Here, we describe the synthesis and radical-trapping properties of new derivatives of DEPMPO and DIPPMPO, bearing a mitochondria-targeting tripheny

Full text of DOI:10.1021/tx500032e

Article
pubs.acs.org/crt
Mitochondria-Targeted Spin Traps: Synthesis, Superoxide Spin
Trapping, and Mitochondrial Uptake
Micael Hardy,† Florent Poulhes,† Egon Rizzato,† Antal Rockenbauer,∥ Karol Banaszak,† Hakim Karoui,†
́
Marcos Lopez,⊥,# Jacek Zielonka,‡,§ Jeannette Vasquez-Vivar,‡,§ Savitha Sethumadhavan,‡,§
Balaraman Kalyanaraman,‡,§ Paul Tordo,*,† and Olivier Ouari*,†
†Aix Marseille Universite,
́
CNRS, ICR UMR 7273, 13397 Marseille, France
‡Department of Biophysics and §Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United
States
∥Department of Physics, Budapest University of Technology and Economics and MTA-BME Condensed Matter Research Group,
Budafoki ut 8, 1111 Budapest, Hungary
⊥Biomedical Translational Research Group, Biotechnology, Fundacion Cardiovascular de Colombia, Floridablanca, Santander,
Colombia
#Graduate Program of Biomedical Sciences, Faculty of Health, Universidad del Valle, Cali, Colombia
S
* Supporting Information
ABSTRACT: Development of reliable methods and site-specific detection of free radicals is an active area of research. Here, we
describe the synthesis and radical-trapping properties of new derivatives of DEPMPO and DIPPMPO, bearing a mitochondria-
targeting triphenylphosphonium cationic moiety or guanidinium cationic group. All of the spin traps prepared have been
observed to efficiently trap superoxide radical anions in a cell-free system. The superoxide spin adducts exhibited similar spectral
properties, indicating no significant differences in the geometry of the cyclic nitroxide moieties of the spin adducts. The
superoxide adduct stability was measured and observed to be highest (t1/2 = 73 min) for DIPPMPO nitrone linked to
triphenylphosphonium moiety via a short carbon chain (Mito-DIPPMPO). The experimental results and DFT quantum
chemical calculations indicate that the cationic property of the triphenylphosphonium group may be responsible for increased
superoxide trapping efficiency and adduct stability of Mito-DIPPMPO, as compared to the DIPPMPO spin trap. The studies of
uptake of the synthesized traps into isolated mitochondria indicated the importance of both cationic and lipophilic properties,
with the DEPMPO nitrone linked to the triphenylphosphonium moiety via a long carbon chain (Mito10-DEPMPO) exhibiting
the highest mitochondrial uptake. We conclude that, of the synthesized traps, Mito-DIPPMPO and Mito10-DEPMPO are the
best candidates for potential mitochondria-specific spin traps for use in biologically relevant systems.
•−
needed. Mechanistic studies on the role of O2 in oxidative
stress processes is made particularly difficult by its low steady-
state concentration and the lack of highly specific probes that
allow its unequivocal identification and quantification. Electron
paramagnetic resonance (EPR) in combination with the spin-
trapping technique is the method of choice to detect and
INTRODUCTION
■
There is increased evidence for the involvement of superoxide
radical anions (O2•−) in cell damage and disease via direct
effects or through formation of secondary reactive oxygen and
nitrogen species (ROS, RNS).1−3 The role of these species in
the pathogenesis and progression of diseases such as athero-
sclerosis,4 diabetes,5 cancer,6 and neurodegenerative diseases7,8
is, however, supported by indirect evidence due to the
difficulties of direct detection of ROS and RNS in vivo. To
gain information on the mechanisms controlling ROS
production at the molecular level, the development of new
reliable and efficient techniques for their detection9,10 is
11−15
•−
characterize radicals such as O2
.
In the spin-trapping
technique, a spin trap, typically a nitrone or nitroso probe, is
introduced into the system under investigation to scavenge
Received: January 30, 2014
Published: June 2, 2014
© 2014 American Chemical Society
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Scheme 1. Chemical Structure of Spin Traps 6−12
radicals which are too short-lived to be directly detected by
EPR. The EPR spectra of the resulting persistent nitroxide spin
adducts can usually be recorded, and the spectral analysis can
provide valuable information on the chemical identity and
dynamics of the radical species produced in the investigated
system. However, different drawbacks still limit the use and
reliability of the spin trapping for in cell and in vivo studies, and
its application to the characterization of O2•−and other oxygen-
centered radicals still needs to be improved. Important progress
has been made over the last several years, notably with the
development of new spin traps that form superoxide spin
adducts exhibiting half-lifetimes significantly longer (17−45
min)16−20 than that observed for the most widely used nitrone
spin trap, DMPO (∼1 min).21−23 Recent research in the
oxidative stress field has focused on the development of
targeted probes for detecting reactive species in cells.24,25
Oxygen can be partially reduced by mitochondrial electron
transfer protein complexes I and III to O2•−, leading to cell
dysfunction.26,27 Therefore, targeting spin traps to mitochon-
dria might help to characterize the contribution of mitochon-
drial O2•−. Lipophilic cations such as triphenylphosphonium
(TPP+) and N-alkylpyridinium ions have been shown to be
effective mitochondria-targeting agents. Their uptake across the
mitochondrial inner membrane is enhanced by the mitochon-
drial membrane potential (ΔΨ) according to the Nernst
equation, with a predicted ∼10-fold accumulation of the cation
within mitochondria for every ∼60 mV increase in ΔΨ.26,28,29
In recent years, TPP+-conjugated probes have been used in
numerous studies focused on mitochondria-associated re-
sponses. For instance, Murphy et al.30 have determined that
MitoQ, a TPP+-conjugated ubiquinone antioxidant, accumu-
lates up to several-hundred-fold in mitochondria matrix and
selectively protected mitochondria, both in vitro and in vivo
from the oxidative damage.31 Mitochondria-targeted cyclic and
linear nitrones have been studied, but usually these reagents
have limited spin-trapping properties.29,32,33 Recently, we
reported the synthesis and the spin-trapping properties of the
TPP+-conjugated DEPMPO spin trap (Mito-DEPMPO).16,34
Using this new reagent, we demonstrated that the detection of
the superoxide radical anion generated from intact isolated
mitochondria is feasible. This result can be explained by the
accumulation of Mito-DEPMPO in mitochondria, but we have
also shown that, compared with DEPMPO, the rate of trapping
of superoxide with Mito-DEPMPO is about two times higher,
•−
and that the half-life of the O2 adduct is about 2.5 times
longer. However, the reasons for these differences between
DEPMPO and Mito-DEPMPO are not clear, and we
speculated that electrostatic interactions between the TPP+
cation and the O2•− or/and the presence of stabilizing H-bonds
•−
in the O2
adduct might contribute to the observed
improvement in superoxide-trapping properties of Mito-
DEPMPO.
To better understand the spin-trapping properties of
mitochondria-targeted nitrones and to optimize the influence
of the TPP+ cation on the spin trapping of O2•−, we have
synthesized a series of Mito-DEPMPO analogues 6−12
(Scheme 1). In the series Mito-DIPPMPO 6, Mito5-DIPPMPO
7, and Mito10-DEPMPO 9, the length of the linker (C4-
CH2OC(O)NH(CH2)n-TPP+) between the C4 and the TPP+
moiety increases (n = 2, 5, and 10, respectively). Two TPP+
moieties are present in Mito-bis-DIPPMPO 8 (n = 2), and a
neutral −N(H)CPh3 group mimicking the lipophilicity of TPP+
was introduced in TritA-DEPMPO 10 (n = 2); finally, the
TPP+ cation was replaced by a guanidium group in Gua-
DIPPMPO 11 and Agm-DIPPMPO 12 (n = 2 and 4,
respectively).
NHS-DIPPMPO and NHS-DEPMPO were used as
precursors for the synthesis of the series of derivatives 6−12,
illustrating the versatility of the postfunctionalization step
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(Scheme 2). For synthetic convenience, NHS-DIPPMPO was
mostly used.
a
Scheme 2
Figure 1. Geometry of compound 4 from X-ray diffraction analysis
(Pov-Ray view).
hypoxanthine/xanthine oxidase (HX/XO) and KO2/18-crown-
6-ether/DMSO (KO2/CE/DMSO) in phosphate buffer. Figure
2 shows the EPR spectra obtained after 10 min incubation of a
mixture containing HX (0.4 mM), XO (0.04 U mL−1), DTPA
(1 mM), and the spin trap (20 mM of Mito-DIPPMPO; 8
Mito-bis-DIPPMPO; 7 Mito5-DIPPMPO, and 9 Mito10-
DEPMPO) in oxygen-bubbled phosphate buffer (0.1 M, pH
7.3). The computer-calculated EPR spectra, obtained using the
parameters reported in Table 1, are shown as gray lines (Figure
2).
a
Reagents and conditions: (i) PBu3, C6H12/CH2Cl2, rt; (ii) DIBAL-H,
CH2Cl2, −78°C; (iii) Zn/NH4Cl, H2O/THF, rt; (iv) DSC, Et3N,
CH3CN, rt.
Hereafter, we describe the synthesis of compounds 6−12,
their spin-trapping properties, and the binding/uptake proper-
ties of compounds 6−10 to energized mitochondria.
In the presence of superoxide dismutase (SOD), no EPR
signal was detected (Figure 2b,d,f,h). Furthermore, for
compounds 6−9, identical EPR signals were observed using
either the HX/XO or the (KO2/CE/DMSO) system, the
signals being particularly long-lasting with the former. After 30
min, a weak additional signal (<10%) is observable and was
assigned to the HO adduct. These results establish that the
EPR signals shown in Figure 2 can be unambiguously assigned
RESULTS AND DISCUSSION
■
Synthesis. NHS-DIPPMPO 5 was prepared (32% overall
yield) in a four-step synthetic sequence (Scheme 2) following
the procedure described by Hardy et al.16 (Supporting
Information).
•−
to the corresponding O2 spin adducts.
Nitrofuranone 2 was obtained in 74% yield by reacting
nitrophosphonate 1 with 2(5H)-furanone in the presence of
tributylphosphine as catalyst. Reduction of 2 by DIBAL-H at
−78 °C led to hemiacetal 3 in good yield (75%). Reductive
cyclization of compound 3 in the presence of zinc and
ammonium chloride afforded nitrones 4 and 4′ as a mixture of
cis/trans diastereoisomers, which were separated on silica gel
column chromatography (60% yield for 4). Compound 4 was
recrystallized in Et2O/pentane (8:2), and the geometry
obtained by X-ray diffraction confirmed the cis position of the
diisopropylphosphonyl group relative to the hydroxymethylene
moiety (Figure 1).
All spectra appear as doublets (31P coupling) of significantly
distorted quartets resulting from close couplings with the 14N
1
and Hβ; they were calculated (Table 1) using the EPR/ROKI
program.35 For all of the series, the best fit was obtained
assuming that the spin-trapping reaction yields only the trans-
diastereoisomer (−OOH and −P(O)(OR)2 groups in a trans
geometry) and the existence of a chemical exchange between
two conformational sites T1 and T2 composed of rapidly
exchanging conformers. In the case of the DEPMPO-OOH and
DIPPMPO-OOH spin adducts, the same assumptions were
made to account for the dramatic alternate line width observed
on the spectra of the trans-diastereoisomers.34−38
Reaction of diastereoisomer 4 with N,N′-disuccinimidyl-
carbonate (DSC) in the presence of triethylamine afforded
NHS-DIPPMPO 5 in 95% yield.
Compounds 6−12 were obtained by reacting NHS-
DIPPMPO or NHS-DEPMPO with the amino function of
the appropriate side chain, with yields ranging from 50 to 91%
(Scheme 3 and Supporting Information).
Except TritA-DIPPMPO 10 and Mito10-DEPMPO 9, all
compounds were soluble up to 50 mM concentration in water
and in phosphate buffer solutions.
EPR/Spin Trapping. EPR/Spin Trapping of Superoxide.
The O2•− trapping properties (Scheme 4) of compounds 6−12
were evaluated using two different O2•−-generating systems:
No EPR signals were observed when mixing Agm-DIPPMPO
11 and Gua-DIPPMPO 12 with the HX/XO system. This
result is consistent with the inhibitory effect of the guanidine
salts on the XO activity.39 Incubations with KO2/CE/DMSO
led to detection of very similar EPR signals to Mito-DIPPMPO-
OOH (Figure 3), confirming the ability of the probes to trap
O2•−, consistent with the inhibitory effects of spin traps 11 and
12 on the XO activity.
Within the series of superoxide adduct of compounds 6−12,
the values of the determined EPR parameters are similar and
close to the values obtained previously for Mito-DEPMPO-
OOH (Table 1), suggesting that the modification of the C4
side chain does not significantly change the geometry of the
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Scheme 3. Synthesis of Compounds 6−12
Scheme 4. Spin-Trapping Experiments
Figure 2. Spin trapping of superoxide radical using Mito-DIPPMPO (6), Mito-bis-DIPPMPO (7), Mito5-DIPPMPO (8), and Mito10-DEPMPO (9).
(a) EPR spectrum obtained after 10 min incubation of a mixture containing hypoxanthine (HX) (0.4 mM), xanthine oxidase (XO) (0.04 U mL−1),
DTPA (1 mM), and 6 (20 mM) in oxygen-bubbled phosphate buffer (0.1 M, pH 7.3). (b) Same as in (a) but in the presence of SOD (600 U mL−1).
(c) Same as in (a) but containing 7 (20 mM). (d) Same as in (c) but in the presence of SOD (600 U mL−1). (e) Same as in (a) but containing 8 (20
mM). (f) Same as in (e) but in the presence of SOD (600 U mL−1). (g) Same as in (a) but containing 9 (20 mM) and 20% DMSO. (h) Same as in
(g) but in the presence of SOD (600 U mL−1). Gray lines: calculated spectra (Table 1). Spectrometer settings: microwave power, 10 mW (a−h);
modulation amplitude, 0.7 G (a−h); smooth point, 1 (a−h); gain 75 dB (a−h); sweep time, 41.94 s (a−h); conversion time, 40.96 ms (a−h).
preferred conformers of these spin adducts. However, as it is
shown below, the C4 side chain has a strong influence on their
stability.
stopped by the addition of a large amount of SOD and the
decay kinetics of the O2 adduct was monitored by following
the changes in EPR signal intensity. The kinetic studies were
performed at 23 °C in 50 μL capillaries, setting the microwave
power of the EPR spectrometer at 20 mW. The signal decay
was monitored during 70 min, recording successive spectra
•−
Decay Kinetics of the Superoxide Adduct. Once the
•−
concentration of the O2 spin adduct has reached a plateau
•−
(after ∼9 min), further formation of the O2 spin adduct was
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Table 1. Calculated EPR Parameters for the Superoxide Spin Adduct of Mito-DEPMPO and Newly Synthesized Nitrones 6−12
a
spin adduct
diastereoisomer
site
k (s−1
)
⟨aP⟩ (G)
⟨aN⟩ (G)
⟨aHβ⟩ (G)
Mito-DEPMPO-OOH
trans (100%)
T1 (70%)
T2 (30%)
T1 (88%)
T2 (12%)
T1 (54%)
T2 (46%)
T1 (60%)
T2 (40%)
T1 (46%)
T2 (54%)
T1 (79%)
T2 (21%)
T1 (74%)
T2 (26%)
0.1 × 108
4.2 × 108
2.5 × 108
5.1 × 108
0.67 × 108
0.8 × 107
0.35 × 107
53.27
52.01
53.41
48.05
53.07
45.01
54.03
51.86
54.54
51.48
53.7
12.79
12.97
12.86
12.76
12.83
12.69
12.80
12.82
12.72
12.80
12.8
12.37
10.13
12.13
7.38
Mito-DIPPMPO-OOH (6-OOH)
Mito-bis-DIPPMPO-OOH (8-OOH)
Mito5-DIPPMPO-OOH (7-OOH)
Mito10-DEPMPO-OOH (9-OOH)
Agm-DIPPMPO-OOH (11-OOH)
Gua-DIPPMPO-OOH (12-OOH)
trans (100%)
trans (100%)
trans (100%)
trans (100%)
trans (100%)
trans (100%)
12.17
6.06
12.78
10.57
14.66
9.00
11.9
51.8
13.3
9.5
53.2
12.8
12.4
51.5
13.1
9.4
a
Exchange rate constants in s−1. For DMPO-OOH spin adduct, a theoretical study in an explicit water solution based on a combined QM/MM/MD
protocol showed that the EPR spectrum can be explained by two sites in chemical exchange. Moreover, it was demonstrated that each site consists of
an equilibrium between the two main five-membered ring conformations of DMPO (3T4 and 4T3). For all spin adducts, the g values were very close
and measured to be 2.006(4).
Figure 3. Spin trapping of superoxide radical using Gua-DIPPMPO 11 and Agm-DIPPMPO 12. (i) EPR spectrum obtained after 2 min incubation
of a mixture containing the KO2/CE/DMSO (5 mM/5 mM/5%) system and 12 (25 mM) in a phosphate buffer (0.1 M, pH 7.3). (ii) Same as in (i)
but with 11 (50 mM). Gray lines: calculated spectra (Table 1). Spectrometer settings: microwave power, 10 mW; modulation amplitude, 0.7 G; time
constant, 1.28 ms; gain 105; sweep time, 21 s; conversion time, 20.48 ms.
Figure 4. Kinetics of decay of (a) Mito-bis-DIPPMPO-OOH (8-OOH) and (b) Mito-DIPPMPO-OOH (6-OOH).
every 42 s (Figure 4). All of the recorded spectra were
simulated using the Rocky program.35 The decay curves for the
superoxide spin adducts of 6−9 are shown in Supporting
Information, and the apparent half-lifetimes are listed in Table
2. It is worth noting that the half-lifetime values depend
strongly on the experimental conditions and various parameters
such as temperature, microwave power, EPR cell (capillaries,
AquaX cell) must be carefully controlled in order to get
reproducible results.
The values of apparent half-lifetimes (t1/2) for the superoxide
adducts are reported in Table 2. As one might expect, given the
results obtained with Mito-DEPMPO,16 a ratio of 2.6 was
found between the apparent half-lifetime of Mito-DIPPMPO-
OOH (n = 2) and that observed with the parent nitrone
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Table 2. Apparent Half-Lifetime Values for Superoxide
Adducts of DIPPMPO and 6−9 and the Ratio Values of t1/2
X-OOH/t1/2 DIPPMPO-OOH
spin adducts
DIPPMPO-OOH
t1/2 (min)
ratio
28
73
1
Mito-DIPPMPO-OOH (6-OOH)
Mito5-DIPPMPO-OOH (7-OOH)
Mito-bis-DIPPMPO-OOH (8-OOH)
2.61
1.31
1.05
0.91
0.78
36.6
29.3
25.5
22
a
DIPPMPO-OOH
a
Mito10-DEPMPO-OOH (9-OOH)
a
In 0.1 M phosphate buffer/DMSO mixture (80:20).
DIPPMPO-OOH (this ratio amounts to 2.4−2.5, in the case of
Mito-DEPMPO-OOH and DEPMPO-OOH). However, when
the spacer arm linking the TPP+ group and the pyrroline ring
was made longer (7, Mito5-DIPPMPO, and 9, Mito10-
DEPMPO) and when two triphenylphosphonium groups are
appended (8, Mito-bis-DIPPMPO), the stability of the
corresponding superoxide adducts is very close to that observed
for the parent nitrone spin adducts. For Agm-DIPPMPO and
Gua-DIPPMPO, attempts to perform a kinetic study using the
KO2/CE/DMSO system were frustrated due to the poor
reproducibility of the procedure, in addition to decreased
Figure 5. EPR spectra of radical adducts of Mito-bis-DIPPMPO (8)
and Mito10-DEPMPO (9). (a) EPR spectrum obtained 10 min after
reduction of the 8-OOH (a) and 9-OOH (b) adducts (Figure 2) by
adding GPx (10 U mL−1) and GSH (1.2 mM) to the incubation
mixture and bubbling argon gas for 2 min. Gray lines: calculated
spectra (Table 3). Spectrometer settings: microwave power, 30 mW;
modulation amplitude, 0.7; time constant, 1.28 ms; gain 105; sweep
time, 20.4 ms; conversion time, 41.9.
•−
persistency of the O2 adducts. Measurement of TritA-
DEPMPO-OOH (10) half-lifetime was not possible because of
the low solubility of TritA-DEPMPO.
EPR Characterization of Hydroxyl and 1-Hydroxyethyl
Radicals. Hydroxyl Radical. When a Fenton system was used
in phosphate buffer to generate HO• in the presence of
compounds 6−9, complex signals of low intensity were
observed (data not shown). These signals likely correspond
to the superimposition of the spectra of hydroxyl spin adducts
and the spin adducts of radicals resulting from hydrogen
abstraction of HO• on compounds 6−9. In the presence of an
excess of EtOH, hydrogen abstraction generates mainly the 2-
hydroxylethyl radical whose spin adducts are clearly identified
(Figure 6). These signals are diminished in intensity in the
presence of catalase, indicating that these signals depend on
hydrogen peroxide breakdown. 12 line EPR spectra corre-
sponding to the HO• adducts of 6, 8, and 9 (Figures 5 and 6)
were obtained by reduction of the corresponding superoxide
adducts with glutathione peroxidase/glutathione (GPx/GSH),
and the hyperfine coupling constants are listed in Table 3.
Quantum-Mechanical Calculations. Under the exper-
imental conditions we used for our spin-trapping experiments,
the apparent half-lifetime of Mito-DIPPMPO-OOH is the
longest ever observed for the superoxide spin adduct of a spin
trap belonging to the pyrroline N-oxide series. In an attempt to
rationalize this result, we undertook a density functional theory
(DFT) approach to the structure of Mito-DIPPMPO (6) and
Mito-DIPPMPO-OOH (6-OOH).
The structures of four conformers (6A to 6D; see Supporting
Information) have been obtained for 6, and one of lower
energy (6A) is shown in Figure 7. For all the calculated
conformers, the geometry of the pyrroline ring and its two C5
substituents are almost the same and very close to that
determined by X-ray diffraction for compound 4 (Figure 1).
This geometry is characterized by an envelope at C4 and a
pseudoaxial (iPrO)2P(O)- substituent with its PO bond
directed toward the pyrroline ring. The five calculated
conformers differ by the geometry of the C4 substituent. For
conformer 6A, the geometry of the (iPrO)2P(O)- and C4
Figure 6. Spin trapping of carbon-centered radical by Mito-bis-
DIPPMPO (8) and Mito10-DEPMPO (9). (a) Signal obtained after 15
min incubation of a mixture containing Mito-bis-DIPPMPO (20 mM),
H2O2 (2 mM), FeSO4 (2 mM), EtOH (15%), and DTPA (1 mM) in
phosphate buffer (0.1 M, pH 7.3). (b) Signal obtained after 1 min
incubation of a mixture containing Mito10-DEPMPO (20 mM), H2O2
(2 mM), FeSO4 (2 mM), EtOH (15%), and DTPA (1 mM) in
phosphate buffer (0.1 M, pH 7.3). Gray lines: calculated spectra
(Table 3). Spectrometer settings: microwave power, 30 mW (a,b);
modulation amplitude, 0.7 (a,b); time constant, 1.28 ms (a,b); gain,
105 (a,b); sweep time, 20.4 ms (a,b); conversion time, 41.9 (a,b).
substituents is in agreement with the trans addition of
superoxide leading to the observed trans-diastereoisomer spin
adduct. Furthermore, the kinetics of addition should benefit
•−
from the electrostatic interaction of O2 with the positive
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Table 3. EPR Parameters of Hydroxyl and Carbon-Centered Spin Adducts
spin adduct
generating system
aP (G)
aN (G)
aHβ (G)
Mito-bis-DIPPMPO-OH
HX/XO and then GPx/GSH
Fe2+, H2O2, EtOH (15%)
HX/XO and then GPx/GSH
Fe2+, H2O2, EtOH (15%)
HX/XO and then GPx/GSH
Fe2+, H2O2, EtOH (5%)
52.4
56.8
53.1
57.5
52.9
57.6
13.6
14.3
13.6
14.3
13.4
14.2
10.5
19.8
10.5
19.7
10.2
19.4
Mito-bis-DIPPMPO-CH(OH)CH3
Mito-DIPPMPO-OH
Mito-DIPPMPO-CH(OH)CH3
Mito10-DEPMPO-OH
Mito10-DEPMPO-CH(OH)CH3
Figure 7. Calculated DFT [B3LYP-6-31G(d,p), PCM (water)] structures of the lowest energy conformers of Mito-DIPPMPO (6A) and Mito-
DIPPMPO-OOH (6-OOHA).
charges brought by the TPP+ moiety, and we indeed observed
Table 4. Percentage of Mitochondria Uptake Determined by
that it increases by a factor 2 compared with DIPPMPO.
The structures of four conformers 6-OOHA to 6-OOHD
(Figure 7 and Supporting Information) have been obtained for
the superoxide adduct of DIPPMPO. The conformers 6-
OOHA,B,C correspond to the trans addition of superoxide to 6,
and their energies are very close (ΔEmax = 2.93 kJ·mol−1). For
these three conformers, the positively charged phosphorus
atom P8 is close to the hydroperoxyl group (P8−O10 = 4.94 Å
for 6-OOHA, Figure 7), and stabilizing interactions can be
established between the positive hydrogen atoms (d = +0.31,
Mulliken charge) of one phenyl group and the oxygen lone
pairs (H9−O10 = 2.51 Å for 6-OOHA, Figure 7). These
interactions of the TPP+ moiety with the hydroperoxyl group
likely contribute to the stabilization of the species, resulting in
the especially long half-lifetime observed. For conformer 6-
OOHD, the distance P8−O10 is much higher (10.53 Å) and its
energy is 6.10 to 9.02 kJ·mol−1 higher compared to that of
conformers 6-OOHA,B,C. For these latter conformers, the
agreement between the calculated and the experimental
hyperfine coupling constants is satisfying [for 6-OOHA, the
lowest energy conformer, AN = 1.00 mT (1.28)exp; AP = 5.00
mT (5.27)exp; AHβ = 1.13 mT (1.17)exp].
HPLC/UV−Vis Detection
spin traps
mitochondrial uptake (%)
DIPPMPO
2.4
29
Mito-DIPPMPO (6)
Mito5-DIPPMPO (7)
Mito10-DEPMPO (9)
Mito-bis-DIPPMPO (8)
TritA-DEPMPO (10)
21.1
58
25.6
0
decrease in compounds’ concentration during incubation with
energized (with succinate) mitochondria, as compared to
mitochondria without succinate. The effects of triphenyl-
phosphonium (TPP+) in improving their affinity for mitochon-
dria is clear from the investigated series, where little effects are
seen for DIPPMPO and no membrane potential-dependent
uptake is observed for TritA-DEPMPO 10. We conclude that
highly delocalized cationic charge on the lipophilic phenyl rings
is necessary for the accumulation in mitochondria in membrane
potential-dependent manner. Surprisingly, the presence of two
TPP+ in Mito-bis-DIPPMPO 8 did not increase the uptake,
which may be due to the close vicinity of the two moieties.40
The best uptake was obtained with Mito10-DEPMPO 9,
probably due its higher hydrophobicity and amphiphilic
properties. The mitochondrial uptake of Mito10-DEPMPO 9
and Mito-bis-DIPPMPO 8 has been independently confirmed
by LC-MS/MS analysis of the extracts of mitochondrial pellets,
indicating significantly higher accumulation of compounds 8
and 9, as compared to the nontargeted spin trap, DIPPMPO
(data not shown).
Mitochondrial Uptake Studies. The mitochondrial bind-
ing/uptake of compounds 6−10 was evaluated from the
decrease of the compounds’ concentration after incubation
during 30 min with respiring mitochondria. No kinetic study
was performed, and the method was based on the quantification
of the compounds remaining in the supernatant (see
Experimental Section). The results of mitochondrial uptake
experiments reported in Table 4 represent the percentage of
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1.4), 23.4 (s), 23.3 (d, J = 1.4), 16.2 (d, J = 1.4), 16.1 (d, J = 1.4);
HMRS calcd for C12H22NO7P, [C12H22NO7P + NH4]+, 341.1472;
found 341.1474.
CONCLUSIONS
■
•−
With the aim to develop tools for the study of O2 formation
in mitochondria, we have prepared a series of DIPPMPO and
DEPMPO derivatives, with a substituent in the 4-position of
the pyrroline ring, having different lengths and bearing a TPP+
cation or a guanidinium group as the chain end. We have
studied the spin-trapping properties of these new spin traps and
determined their mitochondria uptake. All of the compounds
bearing a TPP+ cation efficiently react with O2•−, forming stable
adducts. The results of quantum-mechanical calculations
4-(1-Diisopropyloxyphosphoryl-1-nitroethyl)-2-hydroxytetra-
hydrofuran 3. Product 3 was obtained as a yellow oil (3 g, 75%)
corresponding to a mixture of four diastereoisomers: 31P NMR
1
(121.49 MHz) δ 14.58 (44%), 14.75 (22%), 14.95 (34%); H NMR
(300.13 MHz) δ 1.40−1.25 (12H, m), 1.66 and 1.78 and 1.83 (3H, 3d,
J = 14.4, 14.5, 14,0), 1.98−1.87 (1H, m), 3.57−3.32 (1H, m), 3.94−
3.60 (2H, m), 4.21−4.01 (1H, m), 4.85−4.62 (2H, m), 5.51 (1H, t, J =
3.2); 13C NMR (75.47 MHz) δ 97.8 (s), 97.7 (s), 91.4 (CIV, d, J =
148.4), 73.8 (d, J = 6.7), 73.7 (d, J = 6.7), 73.5 (d, J = 7.3), 67.1 (d, J =
0.9), 66.9 (d, J = 0.9), 43.0 (s), 42.3 (s), 35.2, (s), 34.9 (s), 34.8 (s),
24.2 (d, J = 7.8), 24.5 (d, J = 5.0), 24.4 (d, J = 5.9), 15.2 (s), 14.9 (s);
HMRS calcd for C12H24NO7P, [C12H22NO7P + H]+, 326.1363; found
326.1363.
5-Diisopropyloxyphosphoryl-5-methyl-4-hydroxymethyl-1-pyrro-
line N-Oxide 4 and 4′. Nitrones 4 and 4′ were obtained in 60% yield
(7 g) corresponding to a mixture of two diastereoisomers. (4R*,5R*)-
4-HMDIPPMPO 4: 31P NMR (121.49 MHz) δ 21.19; 1H NMR
(300.13 MHz) δ 1.31 (3H, d, J = 6.2), 1.32 (3H, d, J = 6.0), 1.37 (3H,
d, J = 6.2), 1.41 (3H, d, J = 6.2), 1.70 (3H, d, J = 14.5), 2.75−2.37
(3H, m), 3.92−3.82 (2H, m), 4.18 (1H, m), 4.95−4.71 (2H, m), 6.88
(1H, dt, J = 2.4, 2.4); 13C NMR (75.47 MHz) δ 134.1 (1C, d, J = 7.7),
77.0 (1C, d, J = 152.0), 73.3 (1C, d, J = 7.1), 72.8 (1C, d, J = 7.7), 62.4
(1C, d, J = 5.5), 49.3 (1C, d, J = 5.7), 29.2 (1C, s), 24.2 (1C, d, J =
2.7), 24.0 (1C, d, J = 2.7), 24.8 (1C, d, J = 6.0), 23.6 (1C, d, J = 5.5),
21.4 (1C, d, J = 1.6); ESI-MS m/z 293 [M + H]+; HMRS calcd for
C12H24NO5P, [C12H25NO5P + H]+, 294.1465; found 294.1465.
(4S*,5R*)-4-HMDIPPMPO 4′: 31P NMR (121.49 MHz) δ 21.97;
1H NMR (300.13 MHz) δ 1.31 and 1.32 (12H, 2d, J = 6.2, 6.2), 1.57
•−
suggest that the addition of the negatively charged O2 to
Mito-DIPPMPO could be facilitated by the attraction exerted
by the TPP+ group. Moreover, in the most stable conformers of
Mito-DIPPMPO-OOH, the interactions of the TPP+ moiety
with the hydroperoxyl group likely contribute to the
stabilization of the species, resulting in the especially long
half-lifetime observed.
Results from the mitochondria uptake studies showed the
key role of the TPP+ moiety in the targeting properties. It
should be noted, however, that other factors such as the
accessibility of the TPP+ group or the amphiphilic properties of
the molecule can modulate the mitochondria uptake, which
cannot readily be predicted, and experimental determination is
required. Spin traps that can be compartmentalized or localized
at particular biological sites are part of the toolbox required for
maximizing the amount of useful information, as it is now clear
that data obtained from multiple techniques are often required
to obtain a definitive picture on the formation and role of free
radicals in biological systems. The apparent half-lifetime of the
O2•− adduct and the mitochondrial uptake are 73 min and 30%
for Mito-DIPPMPO (6) and 22 min and 60% for Mito10-
DEPMPO (9), making these new spin traps suitable candidates
for mitochondrial superoxide trapping.
(3H, d, J = 16.0), 2.60−2.34 (2H, m), 3.14−2.72 (2H, m), 3.79−3.69
(1H, m), 4.2−3.90 (1H, m), 4.90−4.67 (2H, m), 6.82 (1H, dt, J = 2.7,
2.6); 13C NMR (75.47 MHz) δ 134.2 (1C, d, J = 8.8), 76.7 (1C, d, J =
159.7), 73.0 (1C, d, J = 7.1), 72.1 (1C, d, J = 7.7), 61.4 (1C, d, J = 6.0),
40.9 (1C, s), 30.0 (1C, d, J = 4.9), 24.2 (1C, d, J = 2.7), 23.9 (1C, d, J
= 4.4), 23.8 (1C, d, J = 4.9), 23.5 (1C, d, J = 6.6), 14.2 (1C, s).
(4R*,5R*)-5-Diisopropyloxyphosphoryl-5-methyl-4-(succinimidyl-
oxycarbonyloxymethyl)-1-pyrroline N-Oxide 5. Nitrone NHS-
DIPPMPO 5 was obtained as a white crystal (1.4 g, 100%): 31P
EXPERIMENTAL SECTION
■
Materials. CH2Cl2 was distilled under dry argon atmosphere in the
presence of P2O5. All reagents were used as received without further
purification. The reactions were monitored by TLC on silica gel and
by 31P NMR. Crude materials were purified by flash chromatography
1
NMR (81.01 MHz) δ 18.0; H NMR (300.13 MHz) δ 1.41−1.26
(12H, m), 1.66 (3H, d, J = 14.0), 2.90−2.63 (3H, m), 2.8 (4H, s),
4.63−4.48 (1H, m), 4.86−4.67 (3H, m), 6.92 (1H, m); 13C NMR
(50.32 MHz) δ 168.5 (2C, s), 151.3 (1C, s), 133.5 (1C, d, J = 7.3),
75.9 (1C, d, J = 149.8), 73.9 (1C, d, J = 6.4), 72.1 (1C, d, J = 7.8), 70.5
(1C, d, J = 3.2), 45.6 (1C, d, J = 2.3), 30.0 (1C, d, J = 0.9), 25.4 (2C,
s), 24.5 (1C, d, J = 1.4), 23.8 (1C, d, J = 1.4), 23.7 (1C, d, J = 1.8),
23.4 (1C, d, J = 7.3), 20.3 (1C, s); HMRS calcd for C17H27N2O9P,
[C17H27N2O9P + H]+, 435.1527; found 435.1527.
on silica gel 60 (0.040−0.063 mm). 31P NMR, H NMR, and 13C
1
NMR spectra were recorded with 300 or 400 spectrometers at 121.49,
300.13, and 75.54 MHz, respectively. 31P NMR data were taken in
1
CDCl3 using 85% H3PO4 as an external standard with broad-band H
1
decoupling. H NMR and 13C NMR data were taken in CDCl3 using
TMS or CDCl3 as internal reference, respectively. Chemical shifts (δ)
are reported in parts per million and coupling constant J values in
Hertz. The assignments of NMR signals were facilitated by use of the
DEPT 135 sequence for all of the nitrones. High-resolution MS
(HRMS) experiments were performed with a mass spectrometer
equipped with an electrospray ionization source operated in the
positive ion mode. In this hybrid instrument, ions were measured
using an orthogonal acceleration time-of-flight (oa-TOF) mass
analyzer.
Synthesis of the Nitrone Mito-DIPPMPO 6. Mito-DIPPMPO 6.
To a mixture of NHS-DIPPMPO (0.2 g, 0.46 mmol) and (2-
aminoethyl)triphenylphosphonium bromide (0.18 g, 0.46 mmol) in
CH2Cl2 (15 mL) was added at room temperature under inert
atmosphere triethylamine (141 μL, 1.06 mmol). The reaction mixture
was stirred for 3 h. The solution was washed with 8 mL of distilled
water and extracted three times with CHCl3. The organic layers were
dried over Na2SO4, and the solvent distilled under reduced pressure.
Purification of the crude product by flash chromatography on silica gel
(CH2Cl2/EtOH 80:20) afforded a white powder (0.28 g, 86%),
corresponding to Mito-DIPPMPO 6: 31P NMR (121.49 MHz) δ 17.8,
20.9; 1H NMR (300.13 MHz) δ 1.38−1.27 (12H, m), 1.62 (3H, d, J =
14.1), 2.68−2.52 (3H, broad band), 3.57−3.68 (2H, m), 3.95−3.33
(2H, m), 4.17−4.07 (1H, m), 4.36−4.29 (1H, m), 4.79−4.67 (2H, m),
6.91 (1H, m), 7.52 (1H, t, J = 6.0), 7.82−7.63 (15H, broad band); 13C
NMR (75.47 MHz) δ 156.5 (1C, s), 135.2 (3C, d, J = 2.9), 134.6 (1C,
d, J = 7.3), 133.6 (6C, d, J = 10.3), 130.5 (6C, d, J = 12.4), 117.5 (3C,
d, J = 86.5), 75.1 (1C, d, J = 149.6), 73.4 (1C, d, J = 6.6), 71.8 (1C, d, J
= 8.0), 64.5 (1C, s), 46.4 (1C, d, J = 2.2), 35.2 (1C, s), 30.7 (1C, s),
24.5 (1C, d, J = 1.5), 24.0 (1C, d, J = 3.7), 25.8 (1C, d, J = 5.1), 23.6
Synthesis of Nitrone NHS-DIPPMPO 5. The synthesis was
performed by adapting the procedure described by Hardy et al.41 The
nitrophosphonate 1 has already been described.42
4-(1-Diisopropyloxyphosphoryl-1-nitroethyl)tetrahydrofuran-2-
one 2. Product 2 was obtained as a yellow oil (25 g, 74%)
corresponding to a mixture of two diastereoisomers: 31P NMR (121.49
MHz) δ 12.84 (60%), 12.95 (40%); 1H NMR (300.13 MHz) δ 1.40−
1.30 (12H, m), 1.75 (3H, d, J = 14.4), 2.74−2.36 (2H, m), 3.82−3.63
(1H, m), 4.54−4.07 (2H, m), 4.86−4.66 (2H, m); 13C NMR (75.47
MHz) δ 174.7 (CIV, s), 174.6 (CIV, s), 90.5 (CIV, d, J = 148.0), 90.2 (d,
J = 147.1), 74.4 (s), 74.3 (s), 74.2 (d, J = 1.8), 74.0 (d, J = 1.4), 68.4
(d, J = 2.8), 68.2 (s), 40.2 (s), 39.7 (s), 30.3 (d, J = 2.8), 29.9 (d, J =
8.3), 24.1 (d, J = 3.2), 23.9 (d, J = 1.8), 23.8 (d, J = 1.8), 23.5 (d, J =
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(1C, d, J = 7.3), 23.3 (1C d, J = 48.4), 20.3 (1C, s); HMRS calcd for
[C33H43N2O6P2]+, Br−, [C33H43N2O6P2]+, 625.2591; found 625.2587.
Synthesis of Nitrone Mito-bis-DIPPMPO 8. Bis-[2-(triphenyl-
phosphonium bromide)ethyl]amine A. A mixture containing bis(2-
bromoethyl)amine43 (7 g, 0.03 mol) and triphenylphosphane (16 g,
0.061 mol) in acetonitrile (50 mL) was refluxed for 48 h. The solvent
was distilled under reduced pressure. Purification of the crude product
by flash chromatography on a silica gel (CH2Cl2/EtOH 80:20)
afforded a brown solid A (12 g, 52%): 31P NMR (121.49 MHz) δ
23.56; 1H NMR (300.13 MHz) δ 3.06−3.12 (4H, m), 3.72−3.80 (4H,
m), 7.60−7.82 (30H, m); 13C NMR (75.47 MHz) δ 134.6 (6C, d, J =
2.9), 133.8 (12C, d, J = 10.3), 130.4 (12C, d, J = 12.5), 118.5 (6C, d, J
= 86.6), 41.7 (2C, s), 23.7 (2C, d, J = 50.6); ESI-MS m/z 297.6 [M +
H]++.
(10-Aminodecyl)triphenylphosphonium Bromide C. To a solution
of B (7 g, 0.011 mol) in EtOH (70 mL) was added hydrazine (0.54
mL, 0.011 mol). The mixture was refluxed for 15 h. The solvent is
distilled, and the impurity was recrystallized using a mixture Et2O/
EtOH (2/1). The product purified by flash chromatography on silica
gel (CH2Cl2/EtOH 80:20) afforded a yellow solid C (4 g, 73%): 31P
1
NMR (121.49 MHz) δ 24.61; H NMR (300.13 MHz) δ 7.95−7.73
(15H, m), 3.70−3.55 (2H, m), 2.80−2.70 (2H, m), 1.60−1.40 (6H,
m), 1.35−1.10 (10H, m). MS calcd for [C28H37NP]+, Br−,
[C28H37NP]+, 418.2; found 418.2.
Mito10-DEPMPO 9. To a mixture of NHS-DEPMPO (0.25 g, 0.61
mmol) and (10-aminodecyl)triphenylphosphonium bromide C (0.32
g, 0.62 mmol) in CH2Cl2 (20 mL) was added at room temperature
under inert atmosphere triethylamine (0.23 mL, 1.61 mmol). The
reaction mixture was stirred for 3 h and then washed with water (15
mL). The organic layer was dried over Na2SO4, and the solvent was
distilled under reduce pressure. Purification of the crude product by
flash chromatography on silica gel (CH2Cl2/EtOH 70:30) afforded a
white powder (0.31 g, 64%), corresponding to Mito10-DEPMPO 9:
Mito-bis-DIPPMPO 8. To a mixture of NHS-DIPPMPO (0.2 g, 0.46
mmol) and bis[2-(triphenylphosphonium bromide)ethyl]amine A
(0.37 g, 0.46 mmol) in CH2Cl2 (5 mL) was added at room
temperature under inert atmosphere triethylamine (141 μL, 1.06
mmol). The reaction mixture was stirred for 3 h. The solution was
washed with 8 mL of distilled water and extracted three times with
CHCl3. The organic layers were combined and dried over Na2SO4,
and the solvent was removed under reduce pressure. Purification of the
crude product by flash chromatography on silica gel (CH2Cl2/EtOH
80:20) afforded a white powder (0.25 g, 50%), corresponding to Mito-
1
31P NMR (121.49 MHz) δ 20.58, 25.48; H NMR (300.13 MHz) δ
1.39−1.16 (17H, m), 1.50−1.40 (2H, m), 1.65−1.57 (3H, m), 1.68
(3H, d, J = 14.0), 2.80−2.55 (3H, m), 3.19−3.06 (2H, m), 3.90−3.60
(3H, m), 4.30−4.10 (5H, m), 4.56−4.45 (1H, m), 6.97 (1H, dt, J =
2.4, 2.4), 7.92−7.62 (15H, m); 13C NMR (75.47 MHz) δ 156.1 (1CIV,
s), 134.8 (1C, d, J = 8.2), 134.9 (3C, d, J = 3), 133.7 (6C, d, J = 9.7),
130.4 (6C, d, J = 12.6), 118.5 (3CIV, d, J = 85.5), 76.2 (1C, d, J =
155.7), 64.3 (1C, d, J = 6.3), 63.9 (1C, s), 62.5 (1C, d, J = 8.0), 46.7
(1C, d, J = 2.3), 41.0 (1C, s), 30.4 (1C, s), 30.2 (1C, s), 29.7 (1C, s),
29.0 (1C, s), 28.9 (2C, s), 26.52 (1C, s), 23.0 (1C, d, J = 49.3), 22.6
(1C, d, J = 4.0), 22.7 (1C, d, J = 49.3), 20.3 (1C, s), 16.4 (1C, d, J =
5.7), 16.3 (1C, d, J = 5.7); HRMS calcd for [C39H55N2O6P2]+, Br−,
[C39H55N2O6P2]+, 709.3530; found 709.3529.
1
bis-DIPPMPO 8: 31P NMR (121.49 MHz) δ 17.61, 21.89; H NMR
(300.13 MHz) δ 1.38−1.26 (12H, m), 1.53 (3H, d, J = 13.8), 2.82−
2.62 (3H, m), 4.10−3.90 (4H, m), 4.35−4.12 (4H, m), 4.60−4.39
(2H, m), 4.79−4.62 (2H, m), 6.83 (1H, m), 7.60−7.98 (30H, m); 13C
NMR (75.47 MHz) δ 154.9 (1C, s), 134.8 (3C, d, J = 2.9), 134.7 (3C,
d, J = 2.9), 134.6 (1C, d, J = 8.8), 134.2 (6C, d, J = 7.3), 134.1 (6C, d, J
= 7.3), 130.4 (6C, d, J = 12.5), 130.3 (6C, d, J = 12.5), 117.9 (3C, d, J
= 86.6), 117.8 (3C, d, J = 86.8), 75.8 (1C, d, J = 149.6), 73.5 (1C, d, J
= 6.6), 71.7 (1C, d, J = 7.3), 64.3 (1C, d, J = 2.2), 45.8 (1C, d, J = 2.2),
42.9 (1C, s), 42.6 (1C, s), 29.2 (1C, s), 24.5 (1C, s), 24.0 (1C, d, J =
4.4), 23.9 (1C, d, J = 4.4), 23.4 (1C, d, J = 7.3), 23.1 (1C, d, J = 46.2),
22.1 (1C, d, J = 47.7), 20.3 (1C, s); HMRS calcd for
[C53H61N2O6P3]2+, 2Br−, [C53H61N2O6P3]2+, 457.1866; found
457.1868.
Synthesis of Nitrone TritA-DEPMPO 10. TritA-DEPMPO 10. To
a mixture of NHS-DEPMPO (0.15 g, 3.6 mmol) and N-tritylethylene-
diamine hydrobromide (0.14 g, 3.6 mmol) in CH2Cl2 (10 mL) was
added triethylamine (103 μL, 1.61 mmol) at room temperature under
argon. The reaction mixture was stirred for 5 h and then washed with
water (15 mL). The organic layer was dried over Na2SO4, and the
solvent was distilled under reduced pressure. Purification of the crude
product by flash chromatography on a silica gel (CH2Cl2/EtOH
97:03) afforded a white powder (0.2 g, 91%), corresponding to TritA-
Synthesis of Nitrone Mito5-DIPPMPO 7. Mito5-DIPPMPO 7. To
a mixture of NHS-DIPPMPO (0.200 g, 0.46 mmol) and (2-
aminopentyl)triphenylphosphonium bromide44 (0.190 g, 0.46 mmol)
in CH2Cl2 (20 mL) was added at room temperature under inert
atmosphere triethylamine (147 μL, 6 mmol). The reaction mixture
was stirred for 3 h. The solution was washed with 8 mL of distilled
water and extracted three times with CHCl3. The organic layers were
combined and dried over Na2SO4, and the solvent was removed under
reduce pressure. Purification of the crude product by flash
chromatography on silica gel (CH2Cl2/EtOH 80:20) afforded a
white powder (0.171 g, 50%), corresponding to Mito5-DIPPMPO 7:
1
DEPMPO 10: 31P NMR (121.49 MHz) δ 19.75; H NMR (300.13
MHz) δ 7.49−7.43 (5H, m), 7.33−7.28 (4H, m), 7.27−7.20 (6H, m),
7.18 (1H, t, J = 1.2), 7.01 (1H, m), 5.08 (1H, t, J = 4.2), 4.62−4.53
(1H, m), 4.38−4.12 (5H, m), 3.37−3.21 (2H, m), 2.84−2.57 (3H, m),
2.30 (2H, t, J = 6.0), 1.73 (3H, d, J = 14.0), 1.36 (6H, dt, J = 7.0); 13C
NMR (75.47 MHz) δ 156.1 (1CIV, s), 145.7 (3CIV, s), 134.5 (1C, d, J
= 8.0), 128.5 (6C, s), 127.9 (6C, s), 126.4 (3C, s), 76.1 (1C, d, J =
160.6), 70.6 (1CIV, s), 64.3 (1C, d, J = 6.3), 64.1 (1C, s), 62.4 (1C, d, J
= 7.4) 46.7 (1C, s), 43.6 (1C, s), 41.7 (1C, s), 30.3 (1C, s), 20.3 (1C,
s), 16.3 (1C, d, J = 5.7), 16.2 (1C, d, J = 5.7); HRMS calcd for
[C32H40N3O6P], [C32H40N2O6P]+, 594.2728; found 594.2735.
Synthesis of Nitrone Gua-DIPPMPO 11. Gua-DIPPMPO 11. To
a mixture of (2-aminoethyl)guanidine (0.13 g, 1,1 mmol) in
acetonitrile (5 mL) was added NHS-DIPPMPO 5 (0.4 g, 0.92
mmol) in 10 mL of anhydrous acetonitrile following by the addition of
N-ethyldiisopropylamine (0,24 mL, 1,38 mmol). The reaction mixture
was stirred overnight. The solvent was removed under reduce pressure.
Purification of the crude product by flash chromatography on basic
alumina (CH2Cl2/EtOH 85:15) afforded a pale yellow powder (0.23 g,
60%): 31P NMR (121.49 MHz, D2O) δ 17.81; 1H NMR (300.13
MHz) δ 6.91 (1H, d, J = 3.02), 4.78−4.67 (2H, m), 4.59−4.53 (1H,
m), 4.41−4.35 (1H, m), 3.87−3.69 (2H, m), 3.65−3.57 (2H, m),
2.78−2.52 (3H, m), 1.64 (3H, d, J = 14), 1.35−1.27 (12H, m); 13C
NMR (75.47 MHz) δ 161.5 (1CIV, s), 158.5 (1C, s), 144.7 (1C, d, J =
8.0), 76.6 (1CIV, d, J = 153.74 Hz), 75.7 (1C, d, J = 8.0), 75.58 (1C, d,
J = 8.0), 61.8 (1C, d, J = 3.5), 49.1 (1C, d, J = 2.3), 43.4 (2C, s), 31.7
(1C, s), 24.1 (1C, d, J = 3.4), 23.9 (1C, d, J = 3.4), 23.7 (1C, d, J =
5.2), 23.5 (1C, d, J = 5.2), 19.89 (1C, d, J = 1.7); ESI-MS/MS [M +
H+] 422.22.
1
31P NMR (121.49 MHz) δ 18.0, 24.3; H NMR (300.13 MHz) δ
1.38−1.26 (12H, m), 1.62 (3H, d, J = 14.0), 1.71−1.57 (6H, m),
2.72−2.61 (3H, broad band), 3.13−3.09 (2H, m), 3.73−3.66 (2H, m),
4.21−4.16 (1H, m), 4.45−4.40 (1H, m), 4.77−4.69 (2H, m), 6.29
(1H, t, J = 5.5), 6.91 (1H, m), 7.82−7.63 (15 H, broad band); 13C
NMR (75.47 MHz) δ 156.6 (1C, s), 135.0 (3C, d, J = 2.9), 135.5 (6C,
d, J = 10.3), 130.4 (6C, d, J = 12.5), 118.2 (3C, d, J = 85.8), 75.9 (1C,
d, J = 150.4), 73.2 (1C, d, J = 6.6), 71.6 (1C, d, J = 7.3), 64.0 (1C, s),
46.5 (1C, d, J = 2.2), 39.9 (1C, s), 30.7 (1C, s), 29.5 (1C, s), 28.3 (1C,
s), 26.9 (1C, d, J = 16.7), 24.5 (1C, d, J = 1.5), 23.9 (1C, d, J = 3.7),
23.7 (1C, d, J = 5.4), 23.4 (1C, d, J = 6.6), 22.5 (1C, d, J = 49.9), 21.8
(1C, d, J = 4.4), 20.3 (1C, s); HMRS calcd for [C36H49N2O6P2]+, Br−,
[C36H49N2O6P2]+, 667.3060; found 667.3060.
Synthesis of Nitrone Mito10-DEPMPO 9. (10-Phthalimido-
decyl)triphenylphosphonium Bromide B. A mixture containing
bromophthalimide (7 g, 0.019 mol) and triphenylphosphane (5 g,
0.019 mol) in acetonitrile (60 mL) was refluxed for 15 h. The solvent
was distilled under reduced pressure. Purification of the crude product
by flash chromatography on a silica gel (CH2Cl2/EtOH 80:20)
afforded a white solid B (9 g, 73%): MS calcd for [C36H39NO2P]+,
Br−, [C36H39NO2P]+, 548.3; found 548.3.
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Synthesis of Nitrone Agm-DIPPMPO 12. Agm-DIPPMPO 12.
To a mixture of agmantine (0,21 g, 0,84 mmol) in acetonitrile (5 mL)
was added NHS-DIPPMPO (0.28 g, 0.64 mmol) in 8 mL of
anhydrous acetonitrile followed by the addition of N-ethyldiisopropyl-
amine (0.33 mL, 1.94 mmol). The reaction mixture was stirred
overnight. The solvent was removed under reduce pressure.
Purification of the crude product by flash chromatography on basic
alumina (CH2Cl2/EtOH 75:25) afforded a pale yellow powder (0.277
ABBREVIATIONS
■
DEPMPO, 5-(diethoxyphosphoryl)-5-methylpyrroline N-oxide;
DIPPMPO, 5-(diisopropyloxyphosphoryl)-5-methylpyrroline
N-oxide; DMPO, 5,5-dimethylpyrroline N-oxide; NHS-
DIPPMPO, (N-hydroxysuccinimidyl-DIPPMPO); EMPO, 5-
ethoxycarbonyl-5-methylpyrroline N-oxide; HX, hypoxanthine;
Mito-DIPPMPO, (4R*,5R*)-5-(diisopropyloxyphosphoryl)-5-
methyl-4-[({[2-(triphenylphosphonio)ethyl]carbamoyl}oxy)-
methyl]pyrroline N-oxide bromide; Mito10-DEPMPO,
(4R*,5R*)-5-(diethoxyphosphoryl)-5-methyl-4-[({[2-
(triphenylphosphonio)decyl]carbamoyl}oxy)methyl]pyrroline
N-oxide bromide; Mito5-DIPPMPO, (4R*,5R*)-5-(diiso-
propyloxyphosphoryl)-5-methyl-4-[({[2-(triphenyl-
phosphonio)pentyl]carbamoyl}oxy)methyl]pyrroline N-oxide
bromide; Mito-bis-DIPPMPO, (4R*,5R*)-5-(diisopropyloxy-
phosphoryl)-5-methyl-4-[({bis[2-(triphenylphosphonio)ethyl]-
carbamoyl}oxy)methyl]pyrroline N-oxide bromide; TrA-
DEPMPO, 5-(diethoxyphosphoryl)-5-methyl-4-[({trityl-2-aza-
ethyl}carbamoyl)oxymethyl]pyrroline N-oxide; Gua-DIPPM-
PO, 5-(diisopropyloxyphosphoryl)-5-methyl-4-[({[2-
(guanidino)ethyl]carbamoyl}oxy)methyl]-5-methyl-1-pyrroline
N-oxide; Agm-DIPPMPO, 5-(diisopropyloxyphosphoryl)-5-
methyl-4-[({[2-(guanidino)buthyl]carbamoyl}oxy)methyl]-5-
methyl-1-pyrroline N-oxide; SOD, superoxide dismutase; XO,
xanthine oxidase; ROS, reactive oxygen; RNS, reactive nitrogen
species
1
g, 95%): 31P NMR (121.49 MHz, D2O) δ 18.88; H NMR (300.13
MHz) δ 7,36 (1H, d, J = 2.8), 4.85−4.75 (2H, m), 4.36−4.29 (1H, m),
4.20−4.14 (1H, m), 3.14−3.05 (4H, m), 2.98−2.60 (3H, m), 1.67
(3H, d, J = 14.9), 1.60−1.50 (4H, m), 1.31−1.26 (12H, m); 13C NMR
(75.47 MHz) δ 162.9 (1CIV, s), 158.9 (1C, s), 144.3 (1C,d, J = 7.5),
77.3 (1CIV, d, J = 153.7), 75.6 (1C, d, J = 7.5), 75.39 (1C, d, J = 8.0),
64.6 (1C, d, J = 2.8), 46.2 (1C, d, J = 2.3), 41.51 (1C, s), 40.6 (1C, s),
30.7 (1C, s), 26.9 (1C, s), 26.0 (1C, s), 24.1 (1C, d, J = 2.9), 24.0 (1C,
d, J = 4.0), 23.7 (1C, d, J = 6.9), 23.6 (1C, d, J = 4.6), 19.99 (1C, s);
ESI-MS/MS [M + H+] 450.25.
Mitochondrial Uptake Studies. Mitochondria were isolated from
rat heart as described by Sethumadhavan et al.45 Briefly, freshly
isolated heart tissue was homogenized in modified Chappell Perry
medium: 10 mM HEPES, 100 mM KCl, 1 mM EGTA, 5 mM MgSO4,
1 mM ATP, and 0.2% BSA, pH 7.4. Homogenates were centrifuged at
700g for 15 min at 4 °C. The supernatant was transferred to a cold
clean tube and subjected to high-speed centrifugation (10 000g, 15
min, 4 °C). The final supernatant was discarded, and the
mitochondrial pellet was washed twice. Finally, the pellets were
resuspended in storage buffer: 10 mM HEPES, 100 mM KCl, and 1
mM EGTA, pH 7.4. Mitochondrial protein was quantified by
bicinchoninic acid method and used immediately for uptake assays.
Incubations were performed in 10 mM HEPES buffer (pH = 7.2)
containing KCl (120 mM), EGTA (1 mM), succinate (5 mM) with 10
μM of the spin trap solution. Aliquots of the supernatant were
collected at 0 and 30 min of incubation for HPLC analysis. Aliquots of
the initial solutions and samples at time 0 and 30 min were analyzed,
and the initial reagent solution after 30 min was also reinjected to
confirm the stability of the compounds tested over the course of
experiment. After incubation, the mixtures were centrifugated (10 min
× 1000g, 4 °C), and supernatant was analyzed by HPLC with UV−vis
absorption detection.
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ASSOCIATED CONTENT
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S
* Supporting Information
1H, 31P, 13C NMR, and EPR spectra, X-ray data and quantum-
mechanical calculations. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: olivier.ouari@univ-amu.fr.
*E-mail: paul.tordo@univ-amu.fr.
Funding
This work was supported by Aix-Marseille University, CNRS,
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A.R. thanks the Hungarian Science Fund for partial funding of
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NOTE ADDED AFTER ASAP PUBLICATION
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This article was published ASAP on June 13, 2014. Author
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