Mito-DEPMPO synthesized from a novel NH2-reactive DEPMPO spin trap: A new and improved trap for the detection of superoxide

Article abstract of DOI:10.1039/b616076j

Mito-DEPMPO, a new DEPMPO analogue bearing a triphenylphosphonium group, was synthesized via a novel NH₂-reactive DEPMPO. The half-life of the Mito-DEPMPO superoxide adduct was estimated to be ca. 40 min. Using Mito-DEPMPO, reactive oxygen spec

Full text of DOI:10.1039/b616076j

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Mito-DEPMPO synthesized from a novel NH -reactive DEPMPO spin
2
trap: a new and improved trap for the detection of superoxide{
a
a
a
Micael Hardy, Florence Chalier, Olivier Ouari, Jean-Pierre Finet, Antal Rockenbauer,
a
b
c
Balaraman Kalyanaraman and Paul Tordo*
a
Received (in Cambridge, UK) 3rd November 2006, Accepted 22nd November 2006
First published as an Advance Article on the web 2nd January 2007
DOI: 10.1039/b616076j
Mito-DEPMPO, a new DEPMPO analogue bearing a
triphenylphosphonium group, was synthesized via a novel
NH -reactive DEPMPO. The half-life of the Mito-DEPMPO
2
superoxide adduct was estimated to be ca. 40 min. Using Mito-
DEPMPO, reactive oxygen species generated in intact mito-
chondria were detected and characterized by EPR.
Scheme 1
Reactive oxygen-centered radicals (ROR) play a key role in
numerous physiological and pathological processes including
aging, cancer, atherosclerosis, neurodegenerative diseases and
targeting of spin traps to increase trapping efficiency and enhance
biocompatibility in biological systems. From more selective spin-
trapping experiments, structural and kinetic information about the
production and decay of free radicals should be obtained, as well
as information on the biological significance of the trapped
radicals. More precise studies will permit better knowledge of the
etiology and pathogenesis of numerous diseases. Recently, few
works have detailed the preparation of a-phenyl-N-tert-butyl
1
diabetes. A considerable amount of progress has been made over
the past decade concerning their roles as second messengers in
1,2
various physiological and pathological processes.
However,
selective detection of ROR still poses major limitations, as most
ROR are highly reactive species with very short lifetimes in
biological systems. In the past 15 years, the EPR spin-trapping
technique has developed into one of the most powerful methods
9
10
nitrone (PBN)-based spin traps exhibiting lectins, mitochondria,
11
12
cell membranes, and sulfhydryl-containing peptide targeting
properties. However, the PBN conjugated nitrones exhibited
limited spin-trapping properties toward oxygen-centered radicals.
With the aim of developing an efficient spin trap that can be used
as a starting block for further conjugation to relevant moieties such
as targeting groups, labels or drugs, the synthesis of a DEPMPO-
based spin trap bearing a reactive group for amino- and thiol-
coupling in water media was performed.
3
for detecting ROR.
Spin-trapping is a technique in which short-lived free radicals
react with a nitrone spin trap to form a more persistent nitroxide
spin adduct that can be conveniently detected by direct EPR.
Typically, the spin adducts exhibit a characteristic hyperfine
splitting pattern, enabling the structural characterization of the
4
short-lived radical species. However, there are still numerous
limitations with respect to cellular applications of spin-trapping
due to the instability of spin adducts, bioreduction to EPR-silent
products, and compartmentalization of radical generation in
We report herein the synthesis of NHS-DEPMPO, 5, a
DEPMPO analogue bearing at C4 an activated carbonate function
cis to the phosphoryl group (Scheme 2). The facile coupling of 5
to arms involving either a biotin or a triphenylphosphonium
group led to nitrones 6 and 7 (Scheme 2). Preliminary results on
superoxide spin-trapping with 5 and 7 are also reported.
4
cellular systems. In the past decade, a significant improvement
has been made with regard to enhancing the chemical stability of
ROR-trapped spin adducts with the development of new spin
5
6
7
traps such as DEPMPO, EMPO and BMPO (Scheme 1). More
recently, using b-cyclodextrin to encapsulate the DEPMPO-OOH
adduct, Karoui et al. reported a 7-fold enhancement in in vitro
NHS-DEPMPO was prepared according to a four-step synthe-
tic sequence as described in Scheme 3 with a 25% overall yield.{
Biotin was selected because of its high binding affinity to avidin
8
adduct stability (t1/2 = 96 min).
15
21
(K
assoc. = 1.7 6 10
M ) and its successful application in many
Despite these improvements in spin-trap design and spin-adduct
sequestration, significant challenges remain with respect to selective
1
3
biomedical assays. Because of the increasing importance of
the chemical biology of reactive oxygen species (ROS) in
a
SREP, UMR 6517 CNRS et Universit e´ s Aix-Marseille 1, 2 et 3, Centre
de Saint J e´ r oˆ me, 13013 Marseille, France.
E-mail: tordo@up.univ-mrs.fr; Fax: +33 491 288 758;
Tel: +33 491 106 009
b
Chemical Research Center, Institute of Structural Chemistry, H-1525
Budapest, PO Box 17, Hungary
c
Dept of Biophysics and Free Radical Research Center, Medical College
of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226,
USA. E-mail: balarama@mcw.edu; Tel: +1 414 456-4000
{
Electronic supplementary information (ESI) available: Preparation of
mitochondria, synthesis of 5, 6 and 7, and spectrometer settings. See DOI:
0.1039/b616076j
1
Scheme 2
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Scheme 3 Reagents and conditions: i, PBu
DIBAL-H, CH Cl , 278 uC; iii, Zn/NH Cl, H
DSC, Et N, CH CN, rt; v, biotinylamidopropylammonium trifluoro-
acetate, Et N, DMSO, rt; vi, (2-aminoethyl)triphenylphosphonium
bromide, Et N, CH Cl , rt.
3
, C
6
H
12–CH
2 2
Cl (9 : 1), rt; ii,
Fig. 1 Spin-trapping of superoxide radical by NHS-DEPMPO 5 and
2
2
4
2
O/THF (10 : 1), rt; iv,
Mito-DEPMPO 7: (a) EPR spectrum obtained after 10 min incubation of
3
3
a mixture containing hypoxanthine (HX) (0.4 mM), xanthine oxidase
3
2
1
(
XO) (0.04 U mL ), diethylenetriaminepentaacetic acid (DTPA) (1 mM)
3
2
2
and NHS-DEPMPO (20 mM) in phosphate buffer (0.1 M, pH 7.3); (b) as
in (a) but with Mito-DEPMPO. The gray lines represent the computer
simulations of the spectra.
1
4
mitochondrial diseases, it appeared that accumulating a spin trap
within mitochondria will increase the mechanistic understanding of
1
0
the role of ROS. Murphy et al. developed a novel approach by
one observed with DEPMPO/OOH. With NHS-DEPMPO it is
reasonable to assume that trapping of superoxide is stereospecific,
and occurs on the less hindered face of the pyrroline N-oxide ring,
yielding only the trans form of the NHS-DEPMPO/OOH adduct.
The spectrum of this adduct was easily calculated with the ROKI
which PBN, conjugated to the triphenylphosphonium cation
(Mito-PBN), was selectively targeted to mitochondria. Mito-PBN
was shown to be more potent than ‘non-targeted’ PBN in
inhibiting mitochondrial oxidation. Although the conjugation to a
triphenylphosphonium cation helps ‘target’ PBN to mitochondria,
the Mito-PBN oxy-radical adducts are still as unstable as the PBN
oxy-radical adducts. The EPR detection and characterization of
Mito-PBN adducts are still ambiguous.
16
program, assuming a chemical exchange between the two
conformers in a 5.6 : 4.4 ratio (Table 1). These results are
in agreement with those obtained with cis 4-PhDEPMPO, a
15
DEPMPO analogue bearing a phenyl group in the 4-position.
Spin-trapping experiments coupled with EPR detection were
performed in a phosphate buffer solution (pH 7.3) using NHS-
DEPMPO 5 as the spin trap. Trapping superoxide using two
different generating systems (Hypoxanthine/Xanthine Oxidase and
The superoxide spin-trapping experiments were also conducted
with Mito-DEPMPO and very similar spectra were obtained as
shown in Fig. 1(b). No superoxide adduct was detected when SOD
was added to the trapping reaction mixture. In the presence of
15
21
2
KO /18-C-6 systems) led to the detection of a persistent signal
glutathione peroxidase (10 U mL ) and reduced glutathione
1.2 mM) only the signal due to the hydroxyl radical spin adduct,
shown in Fig. 1(a). Using the HX/XO system, the signal was
(
2
1
cancelled when SOD (600 U mL ) was previously added to the
trapping reaction mixture. Moreover, when glutathione peroxidase
Mito-DEPMPO/OH, was observed. The hyperfine splitting
constant’s (hfsc’s) parameters obtained by simulation exhibited
very close values to those of NHS-DEPMPO/OOH (Table 1). The
half-life times of Mito-DEPMPO/OOH and DEPMPO/OOH
were determined following the same experimental procedure, i.e.
2
1
(
10 U mL ) and reduced glutathione (1.2 mM) were added, only
the signal of the hydroxyl radical spin adduct, NHS-DEPMPO/
OH, was observed. Based on these additional experiments, the
signal shown in Fig. 1(a) can be unambiguously assigned to
the NHS-DEPMPO/OOH spin adduct (Table 1). The eight-line
spectrum of NHS-DEPMPO/OOH is clearly less complex than the
1
5
the monitoring of their EPR signal decay. Surprisingly, the half-
life of the Mito-DEPMPO/OOH adduct was estimated to be ca.
40 min which is 2.5 and 45 times more than those reported for the
Table 1 Simulated EPR hyperfine splitting constants for radical adducts of Mito-DEPMPO and NHS-DEPMPO
2
1 a
b
Adducts Generating system
Diastereoisomer Conformer k/s
P
a /mT
a
N
/mT
a
H
/mT
aHc/mT
7
7
5
7
a
-OOH
HX/XO, phosphate buffer trans (100%)
HX/XO, phosphate buffer trans (100%)
T
T
T
T
1
2
1
2
(56%)
(44%)
(69.7%) 1.16 6 10
(30.3%)
1.38 6 10
5.224
5.347
5.327
5.201
1.327
1.252
1.279
1.297
1.066
1.285
1.237
1.013
0.039, 0.019,
0.074, 0.019, 0.048 (2), 0.083
0.05 (3), 0.048 (2), 0.044, 0.016
-OOH
21
b
Exchange rate constants in s
.
Number of equivalent protons are given in parentheses.
1
084 | Chem. Commun., 2007, 1083–1085
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quantifying superoxide anions generated in biological systems. To
our knowledge, this is the first experiment where ROR were
detected from intact mitochondria by using the EPR spin-trapping
technique.
The authors would like to thank Professor N. G. Avadhani
and Dr S. Srinivasan for supplying mitochondria. This work was
supported by National Institutes of Health grant HL07305-1 and
Hungarian National Research Fund OTKA T-046953.
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Fig. 2 Spin-trapping from intact mitochondria: (a) EPR spectrum
obtained in the presence of isolated mitochondria (200 mg of proteins)
with Mito-DEPMPO 7 (50 mM) in phosphate buffer, pH 7.3 after 20 min
of incubation and then adding succinate (100 mM) at 37 uC; (b) as in (a)
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DEPMPO/OOH and DMPO/OOH adducts respectively.
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8
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1
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for both spectra was observed suggesting that the trapping of
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mitochondria [Fig. 2(c) and 2(d)]. These results indicate that
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1
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1
In summary, NHS-DEPMPO is a versatile precursor for the
preparation and targeting of a site-directed DEPMPO-based spin
trap. Owing to its increased affinity for superoxide anions, Mito-
DEPMPO may become the trap of choice for detecting and
1
1
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Chem. Commun., 2007, 1083–1085 | 1085