Synthesis and spin-trapping properties of a new spirolactonyl nitrone

Article abstract of DOI:10.1021/jo702434u

(Chemical Equation Presented) Spin trapping using electron paramagnetic resonance (EPR) spectroscopy is commonly employed for the identification of transient radicals in chemical and biological systems. A spirolactonyl-nitrone with rigid H-bond acceptor, 7-oxa-1-azaspiro[4.4]non-1-en-6-one 1-oxide, CPCOMPO, has been synthesized and characterized, and its spin-trapping properties were investigated. The rate of formation of CPCOMPO-O₂H was determined using competition kinetics between the superoxide/hydroperoxyl radical (O₂^?-/HO₂^?) trapping by CPCOMPO and the spontaneous dismutation of this radical in aqueous media. The rate constant of 60 M^-1·s^-1 is the highest rate constant thus far observed at neutral pH for any nitrones using the kinetic method employed. Decay kinetics were also experimentally investigated for CPCOMPO-O₂H. The effect of rigid H-bond acceptor on the stability of the CPCOMPO-O₂H were computationally rationalized and compared to that of EMPO-O₂H, which has flexible H-bond acceptor, and results show the need of a "loose" H-bond acceptor for improved adduct stability.

Full text of DOI:10.1021/jo702434u

Synthesis and Spin-Trapping Properties of a New Spirolactonyl
Nitrone
Yongbin Han,†,‡ Beatrice Tuccio,§ Robert Lauricella,§ Antal Rockenbauer,|
Jay L. Zweier,‡ and Frederick A. Villamena*,†,‡
Department of Pharmacology and Center for Biomedical EPR Spectroscopy and Imaging, The DaVis Heart
and Lung Research Institute, and the DiVision of CardioVascular Medicine, Department of Internal
Medicine, College of Medicine, The Ohio State UniVersity, Columbus, Ohio 43210, Laboratory Chimie
ProVence-UMR 6264, UniVersity of ProVence-CNRS, Faculty of Sciences, Saint Jerome 13397 Marseille
Cedex 20, France, and Chemical Research Center, Institute of Structural Chemistry,
H-1025 Budapest, Pusztaszeri 59, Hungary
frederick.Villamena@osumc.edu
ReceiVed NoVember 13, 2007
Spin trapping using electron paramagnetic resonance (EPR) spectroscopy is commonly employed for the
identification of transient radicals in chemical and biological systems. A spirolactonyl-nitrone with rigid
H-bond acceptor, 7-oxa-1-azaspiro[4.4]non-1-en-6-one 1-oxide, CPCOMPO, has been synthesized and
characterized, and its spin-trapping properties were investigated. The rate of formation of CPCOMPO-
•-
O2H was determined using competition kinetics between the superoxide/hydroperoxyl radical (O2
/
•
HO2 ) trapping by CPCOMPO and the spontaneous dismutation of this radical in aqueous media. The
rate constant of 60 M-1‚s-1 is the highest rate constant thus far observed at neutral pH for any nitrones
using the kinetic method employed. Decay kinetics were also experimentally investigated for CPCOMPO-
O2H. The effect of rigid H-bond acceptor on the stability of the CPCOMPO-O2H were computationally
rationalized and compared to that of EMPO-O2H, which has flexible H-bond acceptor, and results show
the need of a “loose” H-bond acceptor for improved adduct stability.
Introduction
of O2 to superoxide radical anion (O2•-) can lead to the
production of some of the most highly oxidizing species known
to be generated in biological system such as hydroxyl (HO•),
peroxynitrite (ONOO-), and oxidized gluthatione radical anion
(GSSG•-).1
Spin trapping is described by the addition of short-lived free
radicals to a nitrone spin trap which then gives a paramagnetic
spin adduct with longer half-life that is detectable by electron
paramagnetic resonance (EPR) spectroscopy (Scheme 1).6 The
pyrroline-based nitrones have been the reagent of choice for
spin-trapping applications due to their ability to give finger-
printable EPR spectrum upon addition to most radicals. Among
the commonly used cyclic spin traps are 5,5-dimethyl-1-
pyrroline N-oxide (DMPO), 5-diethoxyphosphoryl-5-methyl-1-
Free radicals play an important role in the initiation of
oxidative damage to key biomolecular components of the cell.1
Free radicals in unregulated concentrations have been central
mediators in the pathogenesis of various diseases, such as
ischemia-reperfusion injury,2 inflammation,3 cancer,4 and car-
diovascular diseases5 to name a few. The one electron reduction
* To whom correspondence should be addressed. Tel: 614-292-8215. Fax:
614-688-0999.
† Department of Pharmacology, The Ohio State University.
‡ Center for Biomedical EPR Spectroscopy and Imaging, The Ohio State
University.
§ Laboratory Chimie Provence-UMR 6264, University of Provence-CNRS.
| Chemical Research Center, Institute of Structural Chemistry.
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10.1021/jo702434u CCC: $40.75 © 2008 American Chemical Society
Published on Web 03/11/2008
J. Org. Chem. 2008, 73, 2533-2541
2533

Han et al.
SCHEME 1. Spin-trapping by Cyclic Nitrones
FIGURE 1. Possible stabilization of superoxide adduct of DEPMPO
and EMPO using “loose” H-bond acceptors.
pyrroline N-oxide (DEPMPO), and 5-ethoxycarbonyl-5-methyl-
1-pyrroline N-oxide (EMPO), which have shown numerous
applications in the fields of fuel cell technology,7,8 nanomate-
rials,9,10 photodynamic therapy,11 and environmental remedia-
tion12 and in the investigation of simple organic transforma-
tions.13 Superoxide radical anion has also been detected using
spin trapping from melanosomes,14 mitochondria,15-18 photo-
synthetic systems,19-21 nitric oxide synthase (NOS),22-24 en-
dothelial cells,25,26 human neutrophils,27,28 and reperfused heart
tissue.29-32 Nitrones have been employed as therapeutic agents
in the treatment of acute stroke and ischemia-reperfusion
injuries.33,34 In in vivo systems, PBN and DMPO exhibit
therapeutic properties in stroke models,35 improvement in
cerebral blood flow,36 and NO-releasing properties.37
FIGURE 2. Possible stabilization of O2 and HO• adducts of
•-
CPCOMPO with a rigid hydrogen bond acceptor.
•-
Although DMPO is the most popular spin trap for O2
detection, it is still confronted by certain limitations such as
•-
poor O2 trapping ability and the relatively short half-life of
•-
the O2 adduct formed, thus making its formation impossible
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to detect in in vivo systems.38 There is, therefore, a need to
•-
undertstand factors that governed spin trap reactivity to O2
and the stability of its O2•- adduct. Theoretical and experimental
studies showed that the presence of an electron-withdrawing
substituent (such as the phosphoryl and alkoxycarbonyl moieties)
in the C-5 position of the pyrroline ring increases the reactivity
of nitrones to various radical species compared to the unsub-
stituted nitrone, DMPO.39,40 Moreover, the presence of intramo-
lecular H-bonding in O2 (Figure 1) or HO• adducts of
•-
phosphoryl and alkoxycarbonyl substituted nitrones has a
profound effect on the stability of the spin adduct.41,42
Among the newly conceptualized nitrone that we have
previously theoretically investigated is 6,7-oxa-1-azaspiro[4.4]-
non-1-en-6-one 1-oxide (CPCOMPO), which may show a
promising spin-trapping property due to its predicted high rate
constant for O2•- trapping (Figure 2).39,40 We have previously39
predicted that CPCOMPO exhibited high positive charge density
on the nitronyl-C, and the CPCOMPO-OH shows intramo-
lecular -CdO---H-O- H-bond interaction with an electronic
property similar to that of DEPMPO-OH. The CPCOMPO-
O2H also shows strong intramolecular H-bond interaction
between CdO or N-O moieties and the hydroperoxyl-H with
predicted H-bond distances of 1.9-2.0 Å indicating a possible
stabilization of the O2•- adduct.41 This work will investigate if
cyclic nitrone with rigid spirolactonyl hydrogen-bond acceptor
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2534 J. Org. Chem., Vol. 73, No. 7, 2008

Synthesis of a New Spirolactonyl Nitrone
SCHEME 2. Synthesis of CPCOMPO
•-
at C-5 position could considerably enhance (or worsen) the O2
and HO• adducts’ stability compared to those with “loose”
H-bond acceptors found in EMPO and DEPMPO.
Results and Discussion
Synthesis and Characterization. The preparation of CP-
COMPO is shown in Scheme 2 demonstrating a new methodol-
ogy for the synthesis of cyclic nitrones without employing the
usual reductive cyclization of nitro aldehydes. Boc-deprotection
of 3 was achieved in CH2Cl2 using TFA to afford compound 4.
The oxidation of secondary amine to nitrone using the conven-
tional reagents such as Na2WO4-H2O2,21 m-CPBA,22 and
23
HOAc-H2O2 were inefficient. The use of methyltrioxorhe-
nium(VII) (MTO)-H2O2,43-45 however, oxidized 4 to nitrones
and gave a relatively high yield (see the Experimental Section).
Spectroscopic data are consistent with the presence of nitrone
1
moiety with H NMR peaks at 7.0 ppm and IR peaks at 1583
cm-1 and 1226 cm-1 for CdN and N-O, respectively.46 The
structure of CPCOMPO was also confirmed by X-ray crystal-
lography with a racemic space group of P21/c (Figure 3).
FIGURE 4. X-band EPR spectra of CPCOMPO-O2H generated from
(a) light- riboflavin (36%): aN ) 12.6 G, aâ-H )10.3 G; HO• adduct
(38%); alkyl adduct (9%): aN ) 13.8 G, aâ-H ) 22.1 G; unknown
triplet product (17%): aN ) 14.2 G; (b) KO2 in DMSO, I (81%): aN
) 12.9 G, aâ-H )10.3 G, aγ-H ) 1.4 G; II (11%): aN ) 13.2 G, aâ-H
) 11.1 G, aγ-H ) 1.0 G; HO• adduct (7%); (c) xanthine-xanthine
oxidase, I (62%): aN ) 13.0 G, aâ-H )10.0 G, aγ-H ) 1.5 G; II
(17%): aN ) 13.1 G, aâ-H ) 11.5 G, aγ-H ) 1.1 G; HO• adduct (20%);
FIGURE 3. ORTEP figure for CPCOMPO.
Spin Trapping Studies. (a) Superoxide Radical Anion. In
this study, superoxide, O2•-, and its protonated form, HO2 , were
•
not distinguished since both radicals lead to the same EPR-
detectable spin adduct CPCOMPO-O2H in aqueous media.
With the aim of simplifying the notation, the term “superoxide”
will be used to designate both superoxide and hydroperoxyl
radicals as a whole. Similarly, the symbol “O2•-” will represent
either protonated or nonprotonated forms of the radical. Figure
(d) H2O2 in pyridine, I (43%), aN ) 13.2 G, aâ-H ) 11.2 G, aγ-H
)
0.9 G; II (57%), aN ) 12.3 G, aâ-H ) 9.6 G, aγ-H ) 1.6 G. Simulated
spectra are shown as trace plots.
•-
spectra reveal the presence of O2 adduct and significant
contamination from the HO• adduct (the HO• adduct can be
4 shows the EPR spectra of CPCOMPO-O2H, the O2•-/HO2
adduct, generated from various radical sources and gave a
common spectral profile. For simplicity, computer simulation
of the spectra only assumed the presence of two species. The
•
•-
formed from the decomposition of the O2 adduct). Compu-
tational studies show that the formation of cis-CPCOMPO-
-
O2 is more energetically preferred by 3.1 kcal/mol over the
trans adduct at the PCM/B3LYP/6-31+G**//B3LYP/6-31G*
level (Table 1). Our previous studies47-49 showed that at neutral
(43) Saladino, R.; Neri, V.; Cardona, F.; Goti, A. AdV. Synth. Catal. 2004,
346, 639.
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Nitroxides; John Wiley & Sons: New York, 1989.
•-
pH the initial addition of O2 to nitrones occurs followed by
(47) Villamena, F. A.; Hadad, C. M.; Zweier, J. L. J. Phys. Chem. A
2005, 109, 1662.
(48) Villamena, F. A.; Merle, J. K.; Hadad, C. M.; Zweier, J. L. J. Phys.
Chem. A 2005, 109, 6083.
J. Org. Chem, Vol. 73, No. 7, 2008 2535

Han et al.
TABLE 1. Free Energies of Reaction (in kcal/mol) of CPCOMPO
with Various Radicals at the PCM/B3LYP/6-31+G**//B3LYP/
6-31G* Level at 298 K
adducts
∆Grxn,aq
adducts
∆Grxn,aq
•
•-
HO2
O2
cis
trans
-5.2
-4.9
cis
trans
9.2
12.3
HO•
SO3•- (S-centered)
effect of water at the PCM/B3LYP/6-31+G**//B3LYP/6-31G*.
However, prediction of the aâ-H was overestimated by about
1.9 G. In order to improve the predicted aΝ and aâ-H values
for CPCOMPO-O2H values, a model that includes explicit
water molecule interaction was employed at the same level of
theory. The Boltzamman-averaged aΝ and aâ-H values for the
two conformations of cis CPCOMPO-O2H‚(H2O)2 were 7.5
and 3.4 G, respectively, while aΝ ) 12.0 G and aâ-H ) 9.3 G
were predicted for the trans adducts. Although the trends in the
relative aΝ and aâ-H values for the cis and trans adducts for
CPCOMPO-O2H versus CPCOMPO-O2H‚(H2O)2 are similar,
the latter gave less accurate values. The use of three explicit
molecules followed similar trends (except for aN) in the relative
values for cis and trans isomers but accuracy is greatly
compromised. On the basis of four preferred conformations for
each of the cis and trans CPCOMPO-O2H‚(H2O)3 adducts, the
calculated Boltzamman-averaged values for cis adducts were
cis
trans
-35.4
-38.6
cis
trans
1.8
3.0
•
CH3 CHOH
SO3•- (O-centered)
cis
trans
-16.2
-19.1
cis
trans
5.9
3.8
t-BuO•
MeS•
cis
trans
-12.8
-17.9
cis
trans
-6.7
-6.4
•-
CO2
cis
trans
-12.9
-13.8
SCHEME 3. Mechanism of the Formation of
CPCOMPO-O2H in Neutral pH
aΝ ) 11.0 G and aâ-H ) 2.6 G and aΝ ) 10.2 G and aâ-H
)
8.3 G for the trans adducts. It should be noted that the qualitative
trends in the hfsc values are more relevant in this study since
â-hydrogen hfsc seems to be more problematic to predict
accurately. It has been noted in previous works51,53 that factors
such as dynamic effects due to the variation in the pyramidal-
ization of the nitroxides moiety as well as sensitivity of the
aâ-H to the fluxional conformation of the hydroperoxyl moiety
should be taken into account for the accurate prediction of hfsc’s.
Figure 4 shows the experimental spectra of the CPCOMPO-
O2H from various O2•- generating systems. The spectrum arising
from the use of light-riboflavin O2•- generating system (Figure
4a) gave a significant contamination amount of an unknown
decomposition product with a triplet feature and a C-centered
radical adduct. Superoxide adduct concentrations generated from
KO2, xanthine-xanthine oxidase, and H2O2-pyridine (Figure
4b-d) are significantly high (>80%) compared to the HO•
adduct with negligible presence of artifactual adducts as
observed for the light-riboflavin system. Due to the presence
of multiple species in Figure 4a, the decomposition of CP-
COMPO-O2H to its diastereomeric components by simulation
was not successful. However, Figure 4b,c gave reasonably clean
spectra that allow the simulation of the two diastereomeric forms
of CPCOMPO-O2H. In both cases, the major adduct formed
gave lower hfsc values for nitrogen and â-H compared to the
minor adduct formed. These results may suggest that the major
adduct formed is the cis-CPCOMPO-O2H consistent with the
proton abstraction from water by the hydroperoxide to form
the final adduct nitrone-O2H as evidenced by the endoergic
free energies and low bimolecular rate constant of this reaction
at pH 7.0.50 It can, therefore, be assumed that the major
diastereoisomer of CPCOMPO-O2H formed in solution is the
cis isomer as determined by the preference of cis-CPCOMPO-
-
O2 formation over the trans isomer prior to protonation (see
Scheme 3).
The calculated hyperfine splitting constants (hfsc) (ax) at the
PCM/B3LYP/6-31+G**//B3LYP/6-31G* for the respective cis-
and trans-CPCOMPO-O2H, and by only considering the bulk
dielectric effect of water solvent, gave lower hfsc’s for the cis
isomer, i.e., aΝ ) 10.5 G and aâ-H ) 6.8 G, compared to the
trans adduct with aΝ ) 11.7 G and aâ-H ) 8.2 G. An extensive
computational study on the prediction of hfsc’s of DMPO-
OOH and DMPO-OH adducts was previously reported51 and
showed that the overall effect of the level of theory on the
Boltzmann-weighted hfsc’s for DMPO-OOH conformations is
negligible whether at the B3LYP/6-31+G**//B3LYP/6-31G*
or CBS-QB3. Furthermore, it has been demonstrated by Saracino
et al.52 that the presence of two explicit water molecules
interaction with nitroxides bearing no â-hydrogen (CP and CT),
and with consideration of the bulk dielectric effect of water,
gave significant improvement in the calculated aΝ value
compared to in the absence of water interaction.
•-
Similarly, we showed51 improvement in the Boltzamman-
average values of the calculated aΝ of the various conformers
of DMPO-OOH relative to the experimental aΝ using model
that includes two explicit water molecules and bulk dielectric
thermodynamic preference of O2 addition to CPCOMPO at
the cis position and the qualitative predicted trends in the hfsc’s
for the cis and trans CPCOMPO-O2H. However, simulation
of the EPR spectrum in pyridine (Figure 4d) yielded two
diastereoisomeric adducts in almost equal yields in which the
species with a lower aâ-H of 9.6 G is still slightly more
predominant (57%) than the species with higher aâ-H of 11.2
G at 43%. It should be noted that by using H2O2-pyridine, the
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Hadad, C. M.; Zweier, J. L. J. Am. Chem. Soc. 2007, 129 8177.
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Chem. A 2005, 109, 6089.
•-
O2 adduct is formed not through spin trapping but via
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L.; Ruzzi, M.; Corvaia, C.; Barone, V. J. Phys. Chem. A 2002, 106, 10700.
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2536 J. Org. Chem., Vol. 73, No. 7, 2008

Synthesis of a New Spirolactonyl Nitrone
FIGURE 5. X-band EPR spectra of various radical adducts of CPCOMPO: (a) hydroxyl radical: g ) 2.00550, aN ) 13.9 G, aâ-H ) 13.4 G; (b)
R-hydroxyethyl radical (marked by *) (43%): g ) 2.00528, aN ) 14.7 G, aâ-H ) 21.0 G; OR radical (marked by o) (42%): g ) 2.00539, aN
)
14.0 G, aâ-H ) 13.3 G; (c) tert-butoxy radical: g ) 2.00541, aN ) 14.0 G, aâ-H ) 13.2 G; (d) sulfite radical, I (79%): g ) 2.00530, aN ) 13.7
G, aâ-H ) 15.4 G; II (21%): g ) 2.00532, aN ) 13.9 G, aâ-H ) 12.1 G); (e) glutathionyl radical: g ) 2.00561, aN ) 14.6 G, aâ-H ) 13.4 G; (f)
carbon dioxide radical anion: g ) 2.00523, aN ) 14.5 G, aâ-H ) 17.3 G. Simulated spectra are shown as trace plots.
nucleophilic addition of the HOO- and subsequent oxidation
of the resulting hydroxylamine to nitroxide as previously
proposed.54,55
The SO3•- adduct gave a high proportion of the cis and trans
forms as shown in Figure 5d with identical g factors and almost
similar aN values. The observed hfsc values for the CP-
COMPO-SO3 was close to those observed in the SO3•- adducts
of EMPO57 and DMPO.58 Computational studies show that
although the formation of CPCOMPO-SO3 is endoergic, the
free energy difference for the formation of S- and O-centered
adducts is only about 0.8 kcal/mol according to Table 1, with
the cis and trans isomers being favored for the S- and O-centered
adducts, respectively. Due to the similarities in the energetics
of S- and O-centered CPCOMPO-SO3 formation, it can be
assumed that both products are formed in solution as shown in
Figure 6. Natural population analysis (NPA) of the charge
densities for the trigonal pyramidal, SO3•-, gave a -0.98 e
charge on each of the 3 O atoms and a +1.94 e positive charge
on the S atom. Spin density distribution is 19% and 43% on
each of the O and S atoms, respectively. The high spin density
(b) Hydroxyl and Other Radicals. The EPR spectrum of
CPCOMPO-OH generated from the Fenton reaction is shown
in Figure 5a. Unlike the HO• adduct of EMPO and BMPO (or
BocMPO),56 the hfsc’s for the cis and trans products for
CPCOMPO are difficult to discern. Figure 5b-f shows the EPR
•
spectra for the R-hydroxyethyl (CH2 CHOH), tert-butoxy (t-
BuO•), sulfite (SO3•-), glutathione (GS•), and carbon dioxide
anion (CO2•-) radical adducts of CPCOMPO, each with
distinctive hfsc parameters.
The calculated free energies of reaction at the PCM/B3LYP/
6-31+G**// B3LYP/6-31G* level for the formation of the
various adducts are shown in Table 1. Unlike in the formation
of HO2• and O2•- adduct in which the cis isomers are possibly
preferred, the formation of trans isomers are more favored for
•-
the HO•, CH3 CHOH, t-BuO•, and CO2 adducts (relative
Boltzmann ratios of the cis and trans adduct are shown in the
Supporting Information). The thermodynamics of GS• addition
can be represented by the addition of MeS• for simplicity and
gave almost the same exoergic free energies of ∼-6.5 kcal/
mol for the cis and trans adduct formation.
•
•-
distribution on the O and S atoms of SO3 could explain the
favorability of formation of both O- and S-centered CP-
COMPO-SO3. The dependence of the mode of radical addition
to nitrones on the spin density distribution of the radical had
been discussed in our previous works.59
Kinetics and Computational Studies. (a) Rate of Forma-
tion of CPCOMPO-O2H Adduct. The kinetic method used
in the present work, based on a competition between the
superoxide trapping and its spontaneous dismutation at pH 7,
has been extensively described elsewhere and was employed
(54) Barbati, S.; Clement, J. L.; Olive, G.; Roubaud, V.; Tuccio, B.;
Tordo, P. 13P labeled cyclic nitrones: A new class of spin traps for free
radicals in biological milieu. In Free Radicals in Biology, EnVironment;
Minisci, F., Ed.; Kluwer Academic Publishers: Dordrecht, The Netherlands,
1997; p 39.
(55) Tuccio, B.; Zeghdaoui, A.; Finet, J.-P.; Cerri, V.; Tordo, P. Res.
Chem. Intermed. 1996, 22, 393.
(56) Villamena, F. A.; Hadad, C. M.; Zweier, J. L. J. Phys. Chem. A
2003, 107, 4407.
(57) Olive, G.; Mercier, A.; Le Moigne, F.; Rockenbauer, A.; Tordo, P.
Free Radical Biol. Med. 2000, 28, 403.
(58) Potapenko, D. I.; Bagryanskaya, E. G.; Reznikov, V. V.; Clanton,
T. L.; Khramtsov, V. V. Magn. Reson. Chem. 2003, 41, 603.
J. Org. Chem, Vol. 73, No. 7, 2008 2537

Han et al.
FIGURE 7. Experimental (black) and calculated (red) kinetic curves
indicating the time-dependent changes in the CPCOMPO-O2H con-
centration as a function of CPCOMPO concentration at pH 7.0.
FIGURE 6. Sulfite radical anion adducts of CPCOMPO. See Table 1
for the respective free energies of reaction.
TABLE 2. Rate Constants for the Spin Trapping of Superoxide by
Nitrones (kt) and for the Decay of Nitrone/Superoxide Spin Adducts
(kd) at pH 7.0
SCHEME 4. Reactions 1-4 Considered in the Kinetic
Model
curve
[CPCOMPO]/mM
kd/10-3 s-1
kt/M-1 s-1
a
b
c
10
50
120
6.1 ( 0.2
4.8 ( 0.2
9.6 ( 0.2
60 ( 4
order decay of CPCOMPO-O2H, and for the spin trapping of
superoxide, respectively, and Y represents the EPR-silent
products. The apparent rate constant for the trapping reaction
by the nitrones, kt, has been demonstrated50 to be pH dependent
•
•-
and includes the contribution of both HO2 and O2 trapping.
SCHEME 5. Rate Equations 5-8 Considered in the Kinetic
Model
At pH 7, kdis is equal to 635 × 103 M-1‚s-1 50
, and the rate eqs
5-8 given in Scheme 5 can be written on the basis of these
reactions.
Figure 7 shows the formation and decay of CPCOMPO-
O2H as a function of time after deconvolution of the experi-
mental plots. As shown in Table 2, curve fitting of the three
experimental plots yielded kinetic rate constants, kt and kd. The
calculated rate of formation was kt ) 60 ( 4 M-1‚s-1, which
is higher compared to that observed at pH 7.2 for EMPO60-62
and AMPO,40 of 10.9 and 25.2 M-1‚s-1, respectively, using the
same kinetic method as previously reported. The theoretically
determined rate constant of 3.1 M-1‚s-1 (at the PCM/mPW1K/
for several linear and cyclic nitrones.40,60-62 The method does
not require calibration of the initial superoxide concentration.
EPR spectra of CPCOMPO-O2H were recorded as a function
of time at various CPCOMPO concentrations, and kinetic curves
were obtained after treatment of these spectra using both singular
value decomposition (SVD) and pseudo-inverse deconvolution
methods. Modeling these curves allowed the determination of
the rate constants kt and kd for the superoxide trapping and the
adduct decay reactions, respectively.
•-
6-31+G** level) for the trapping of O2 by CPCOMPO is
intermediate in reactivity compared to other nitrones considered
in our previous study.40 Using the same experimental conditions
and method of kinetic analysis, the second-order rate constant
reported for other nitrones are as follows (in M-1‚s-1): DEPO
(31.1);60 AMPO (25.2);40 EMPO (10.9);62 DEPMPO (3.95);62
BocMPO (3.45);63 DMPO (2.0).62 Therefore, the experimental
rate constant of 60 M-1‚s-1 for CPCOMPO is by far the fastest
experimentally observed for any nitrones.
The kinetic model used can be described by reactions 1-4
(Scheme 4), in which kdis, kd, and kt are the rate constants for
the superoxide spontaneous dismutation, for the pseudo-first-
•-
The reactivity of the carbonyl-C to O2 was also explored
since, like nitrones, the carbonyl group maybe susceptible to
nucleophilic attack from O2•-. Figure 8 shows the addition
reaction of O2•- to the carbonyl-C moiety of CPCOMPO. The
(59) Villamena, F. A.; Locigno, E. J.; Rockenbauer, A.; Hadad, C. M.;
Zweier, J. L. J. Phys. Chem. A 2006, 110, 13253.
(60) Allouch, A.; Roubaud, V.; Lauricella, R.; Bouteiller, J.-C.; Tuccio,
B. Org. Biomol. Chem 2005, 3, 2458.
(61) Lauricella, R.; Allouch, A.; Roubaud, V.; Bouteiller, J.-C.; Tuccio,
B. Org. Biomol. Chem. 2004, 2, 1304.
•-
free energy of O2 addition to the carbonyl-C trans to the
nitronyl moiety is 11.7 kcal/mol endoergic and more preferred
(62) Lauricella, R. P.; Bouteiller, J.-C. H.; Tuccio, B. N. Phys. Chem.
Chem. Phys. 2005, 7, 399.
(63) Allouch, A.; Roubaud, V.; Lauricella, R.; Bouteiller, J.-C.; Tuccio,
B. Org. Biomol. Chem. 2003, 1, 593.
2538 J. Org. Chem., Vol. 73, No. 7, 2008

Synthesis of a New Spirolactonyl Nitrone
hydroperoxyl-H and, therefore, may be responsible for their
longer half-lives. The effect of trans adduct, however, on the
•-
overall half-lives of the O2 adduct should cancel out since
the N-O---H-O2 is the common H-bond interaction among
the trans adducts of CPCOMPO, EMPO, and BMPO as we
previously reported47,49,65 with the N-O moiety in all of the
adducts having similar conformation. The inductive effect of
substituents at the C-5 position on the C-N bond breaking
process, however, may also play an important role in the
unimolecular decomposition of the adducts regardless of the
presence of intramolecular H-bonding and is now currently being
investigated in our laboratory.
FIGURE 8. Superoxide radical cis (left) and trans (right) addition to
the carbonyl-C of CPCOMPO.
The kinetics of decay of CPCOMPO-OH was also investi-
gated using H2O2 photolysis. An analysis of the decay plots for
CPCOMPO-OH and EMPO-OH fit pure first order for the
first 17 min of decay. The calculated first-order rate constants
for CPCOMPO-OH and EMPO-OH decomposition were 1.13
( 0.03 × 10-3 s-1 and 5.66 ( 0.29 × 10-4 s-1, respectively.
The corresponding half-lives of ∼ 10.2 min for CPCOMPO-
OH and 20.4 min for EMPO-OH are significantly different
from each other. The most preferred diastereomeric product for
CPCOMPO-OH is the trans form (99% Boltzmann weighed)
while equal preference for cis and trans adduct formation has
been predicted for EMPO-OH,66,67 suggesting that intramo-
lecular CdO----HO H-bonding is not present in CPCOMPO-
OH adduct and possibly plays a role in the stabilization of
EMPO-OH adduct.
Conclusion
CPCOMPO was synthesized and characterized, and its spin-
trapping properties were investigated and found to exhibit
discernible EPR spectral profiles for various spin adducts.
•-
Computational studies using DFT show O2 addition to
lactonyl-O is less favored compared to addition to nitronyl-C.
Using a kinetic approach for the evaluation of rate constant,
CPCOMPO gave second-order rate constant for HO2 /O2
FIGURE 9. Hydroxyl and hydroperoxyl adducts of CPCOMPO
showing intramolecular H-bonding interactions.
•
•-
addition of kt ) 60 M-1‚s-1, which is the highest rate constant
observed thus far for a nitrone using the kinetic model developed
by Tuccio et al. The half-life of CPCOMPO-O2H, however,
seems to be compromised by the presence of a rigid H-bond
acceptor. Therefore, strong intramolecular H-bonding in the
presence of flexible H-bond acceptor should be taken into
account in the design of new spin traps for longer HO2-adduct
stability. Moreover, since spirolactone moiety is found in natural
products68 and have exhibited biological activity such as
antagonist of the aldosterone receptors of the epithelial cells,69
than the corresponding cis addition reaction. Considering that
the calculated free energy of O2•- addition to the nitronyl-C is
9.2 kcal/mol (Table 1), which is about 2.5 kcal/mol more
preferred than addition to carbonyl-C, it can be inferred that
the most preferred reaction of O2•- to CPCOMPO is via addition
to the nitronyl-C.
(b) Decay Rates of CPCOMPO-O2H and CPCOMPO-
OH Adducts. The half-life of CPCOMPO-O2H was found to
be only t1/2 ∼ 2.4 min when the concentration of CPCOMPO
was 50 mM (see Table 2). Although the decay rate of nitrone
O2H depends on many factors, such as the nitrone concentration
•-
the presence of spirolactonyl moiety along with the high O2
scavenging ability of the nitronyl group in CPCOMPO can have
interesting pharmacological applications.
•-
or the O2 generating system used, this value is significantly
shorter compared to that of BMPO (or BocMPO)64 and EMPO
with t1/2 ∼ 9 and 14 min, respectively. This kinetic result
suggests that rigid H-bond acceptor has a significant effect on
the HO2-adduct stability. This instability of CPCOMPO-O2H
is probably due to the formation of a rigid 8- or 7-membered
ring system (assuming that cis adduct is more favored than the
trans adduct based on Table 1) that is formed upon intramo-
lecular H-bonding of hydroperoxyl-H with carbonyl-O and
nitronyl-O, respectively (see Figure 9). Unlike the EMPO and
BMPO analogues, the ester moiety is “loose” and can have
flexibility in maintaining the H-bonding interaction with the
Experimental Section
Pyrrolidine-1,2-dicarboxylic Acid 1-tert-Butyl Ester 2-Methyl
Ester (2). Compound 2 was prepared according to the procedure
described elsewhere70 but using proline 1 (26.2 g, 228 mmol) as a
(65) Villamena, F. A.; Merle, J. K.; Hadad, C. M.; Zweier, J. L. J. Phys.
Chem. A. 2007, 111, 9995.
(66) Villamena, F. A.; Hadad, C. M.; Zweier, J. L. J. Phys. Chem. A
2003, 107, 4407.
(67) Villamena, F. A.; Hadad, C. M.; Zweier, J. L. J. Am. Chem. Soc.
2004, 126, 1816.
(68) Ramsay, L. E.; Shelton, J. R.; Wilkinson, D.; Tidd, M. J. Br. J.
Clin. Pharm. 1976, 3, 607.
(64) Villamena, F. A.; Zweier, J. L. J. Chem. Soc., Perkin Trans. 2 2002,
1340.
(69) Doggrell, S. A.; Brown, L. Expert Opin. InVest. Drugs 2001, 10,
943.
J. Org. Chem, Vol. 73, No. 7, 2008 2539

Han et al.
starting material to yield 37.0 g (98%) of crude proline methyl ester
hydrochloride. The crude salt (20 g, 120 mmol) was used without
further purification to give compound 2 (23.8 g, 86%). H NMR
(400 MHz, CDCl3, ppm): δ 1.39 and 1.43 (s, Boc rotamers 9H),
1.80-2.00 (m, 3H), 2.12-2.28 (m, 1H), 3.31-3.56 (m, 2H), 3.73
(s, 3H), 4.19-4.36 (m, 1 H). IR (neat, cm-1) νmax 2976, 2881, 1747,
1695, 1392, 1365, 1199, 1157, 1119, 1088, 1033, 999, 974, 922,
888, 858, 772. Identical NMR and IR spectra were observed
previously by Kurokawa et al.71
high sensitivity resonator at room temperature. Unless otherwise
indicated, the instrument settings used for general spectral acquisi-
tion are as follows: microwave power, 20 mW; modulation
amplitude, 1.2 G; receiver gain, 3.56 × 105; scan time 21 s; time
constant, 41 s; sweep width, 80 G. All the spin trapping studies
were carried out in a phosphate buffer (PBS) (10 mM) at pH 7.0
containing 100 µM diethylene triamine pentaacetic acid (DTPA).
Sample cells used were 50 µL quartz or glass capillary tubes for
UV or non-UV irradiation experiments, respectively. The spectrum
simulation was carried out using an automatic fitting program.72
1
6-Oxo-7-oxa-1-azaspiro[4.4]nonane-1-carboxylic Acid tert-
Butyl Ester (3). To the compound, pyrrolidine-1,2-dicarboxylic
acid 1-tert-butyl ester 2-methyl ester 2 (4 g, 17.39 mmol), in dry
THF (100 mL) was slowly added lithium diisopropylamide (1.8
M, 18 mL) at -20 °C and the solution stirred for 30 min. The
solution was allowed to warm to room temperature and stirred for
1 h. Ethylene oxide gas was slowly bubbled through the solution
for 30 min at -20 °C, and the resulting mixture was stirred at room
temperature for 12 h. The progress of reaction was monitored by
TLC using hexane/EtOAc (7:3). The reaction was quenched by the
addition of water and extracted with EtOAc (3 × 30 mL), and the
collected organic layer was dried over anhydrous Na2SO4 and
filtered. The solvent was removed in vacuo, and the residue was
purified by passing through the silica gel column using hexane-
EtOAc (7:3) as eluent to give the crystalline compound, 6-oxo-7-
oxa-1-azaspiro[4.4]nonane-1-carboxylic acid tert-butyl ester 3 (3
g, 33.4%): 1H NMR (400 MHz, CDCl3, ppm) δ 1.32 (s, 9H), 1.64-
2.14 (m, 5H), 2.53-2.88 (m, 1H), 3.40 (m, 2H), 4.07 (m, 1H),
4.24-4.43 (m, 1H); IR (neat, cm-1) νmax 2975, 2878, 1777, 1690,
1388, 1367, 1205, 1157, 1132, 1107, 1026, 982, 961, 931, 854,
770, 730, 693; EI-MS calcd for C12H19NO4 m/z 241.13, found
241.07; HRMS calcd for C12H19NNaO4+ (M + Na+) m/z 264.1212,
found 264.1201.
Spin Trapping. (a) Superoxide Radical Anion. (i) Light
Riboflavin. A 50 µL oxygenated PBS solution containing 0.1 mM
riboflavin and 50 mM CPCOMPO was irradiated with 150 W light
source positioned at 12 cm away from the sample cavity. (ii)
Xanthine-Xanthine Oxidase (X-XO). A 50 µL PBS solution
containing 100 µM DTPA, 0.4 mM xanthine, 0.5 unit/mL xanthine
oxidase, and 50 mM CPCOMPO was used. (iii) KO2-Generating
System. Superoxide adduct was generated by mixing 25 µL of 100
mM CPCOMPO in PBS and 50 µL of 100 mM KO2 in DMSO.
(iv) H2O2/Pyridine System. Pseudo-superoxide adduct was gener-
ated by mixing 50 mM CPCOMPO with 160 mM H2O2 in pyridine.
(b) Hydroxyl Radical. PBS solution containing 0.2% H2O2 and
50 mM CPCOMPO was irradiated for 5 min using a low-pressure
mercury vapor lamp at 254 nm wavelength. (c) Miscellaneous
•
Radicals. Spin trapping of SO3•-, CO2•-, CH3 CHOH, GS•, and
tBuO• was carried out in 50 µL PBS solution containing 50 mM
CPCOMPO, 0.2% H2O2, and 100 mM of the respective radical
source NaHCO2, Na2SO3, ethanol, GSSG, and (tBuO)2. Each of
the mixtures was irradiated with UV for a period of 5 min.
Kinetic Studies (a) Rate Constant of CPCOMPO-O2H
Formation and Decay. Detailed descriptions of the kinetic
procedure are given elsewhere,60,61 and the method used will be
only briefly reiterated here. Measurements were made at pH 7 in
a 10 mM PBS using a X-XO superoxide generating system. In a
typical experiment, a solution contains CPCOMPO (10, 50 or 120
mM), 0.4 mM xanthine, 0.5 unit/mL xanthine oxidase, and
3-carboxy-2,2,5,5-tetramethylpyrrolidin-1-oxyl (3-CP, 1.0 µM) as
internal reference. An EPR spectrum of the solution was recorded
every 21 s (first recorded at the 42nd s) for ca. 1 h. Noise reduction
was accomplished using the singular value decomposition (SVD)
procedure. The kinetic curves of the adduct concentration [CP-
COMPO-O2H] as a function of time were obtained after decon-
volution of the signal using the pseudo-inverse method.61 This
approach leads to the curves representing only the time evolution
of the sole adduct CPCOMPO-O2H without any contribution from
the HO• adduct from a series of EPR spectra composed of mixtures
of paramagnetic species with different kinetic formation. These
calculations were achieved using a homemade computer program
written in FORTRAN, using subroutines given in Numerical
Recipes.73
Computer modeling of the kinetic curve obtained was achieved
using the homemade program Kalidaphnis (formerly Daphnis). The
use of this program written in FORTRAN has been described in
several papers,74,75 and it can be obtained upon request from the
authors. It allows fitting of experimental curves as signal amplitude
or concentration by numerical integration of appropriate rate
equations. Application of the standard least-square method yields
pertinent kinetic parameters and their respective error values. Using
this approach, the curves obtained at the three different initial
concentrations of CPCMPO (10, 50, and 120 mM) were considered
7-Oxa-1-azaspiro[4.4]nonan-6-one (4). To compound 3 (1.20
g, 5.0 mmol) in CH2Cl2 (10 mL) was slowly added trifluoroacetic
acid (TFA) (1 mL) at 0 °C. The mixture was allowed to warm to
room temperature and stirred for overnight. The solvent was
removed and basified with potassium carbonate and then extracted
with EtOAc (3 × 50 mL). The organic layer was collected and
dried over anhydrous sodium sulfate and then filtered. The solvent
was evaported in vacuo to afford the amine 4 (0.52 g, 76%) and
was used without further purification. 1H NMR (400 MHz, CDCl3,
ppm) δ 1.88-2.10 (m, 4H), 2.23 (m, 1H), 2.33(m, 1H), 3.05 (m,
1H), 3.28 (m, 1H), 4.21 (q, 1H), 4.39 (m, 1H); IR (neat, cm-1
max 3630, 3534, 2983, 1775, 1422, 1379, 1282, 1220, 1191, 1158,
)
ν
1113, 1023, 959, 793, 693; EI-MS calcd for C7H11NO2 m/z 141.1,
+
found 141.0; HRMS calcd for C7H11NNaO2 (M + Na+) m/z
164.0687, found 164.0697.
7-Oxa-1-azaspiro[4.4]non-1-en-6-one, 1-Oxide (CPCOMPO).
Methyltrioxorhenium(VII) (17.5 mg, 0.07 mmol), EtOH (5 mL),
pyridine (28 µL, 0.35 mmol), and 30% H2O2 (511 µL, 4.5 mmol)
were added sequentially to a 25-mL round bottom flask. A solution
of the amino compound 4 (0.5 g, 3.55 mmol) in EtOH (5 mL) was
added dropwise at 0 °C. After the mixture was stirred at room
temperature for 1 h, 10 mL of water was added. The mixture was
extracted with DCM (3 × 30 mL), and the organic phase was dried
over MgSO4 concentrated, and purified on silica column using 5%
MeOH in DCM (Rf ) 0.15) to give white solid product (0.314 g,
57%): 1H NMR (250 MHz, CDCl3, ppm) δ 2.20-2.35 (m, 2H),
2.78 (m, 2H), 3.02 (m, 1H), 3.23 (m, 1H), 4.42 (m, 1H), 4.71 (q,
1H), 6.98 (s, 1H); 13C NMR (250 MHz, CDCl3, ppm) δ 26.2, 30.0,
32.3, 66.6, 78.3, 134.8, 173.8; IR (neat, cm-1) ν 3405, 1769, 1582,
1382, 1226, 1167, 1018; EI-MS calcd for C7+H9NO3 m/z 155.06,
found 155.0; HRMS calcd for C7H9NNaO3 (M + Na+) m/z
178.0480, found 178.0475.
(72) Rockenbauer, A.; Korecz, L. Appl. Magn. Reson. 1996, 10, 29.
(73) Press, W. H.; Teukolsky, S. A.; Vetterling, W. T.; Flannery, B. P.
Numerical Recipes in FORTRAN. The Art of Scientific Computing, 2nd ed.;
Cambridge University Press: Cambridge, 1992.
EPR Measurements. General Methods. EPR measurements
were carried out on a Bruker EMX spectrometer equipped with
(74) Mathieu, C.; Tuccio, B.; Lauricella, R.; Mercier, A.; Tordo, P. J.
Chem. Soc., Perkin Trans. 2 1997, 2501.
(75) Tuccio, B.; Lauricella, R.; Frejaville, C.; Bouteiller, J.-C.; Tordo,
P. J. Chem. Soc., Perkin Trans. 2 1995, 295.
(70) Dondoni, A.; Perrone, D. Org. Synth. 2000, 77, 64.
(71) Kurokawa, M.; Shindo, T.; Suzuki, M.; Nakajima, N.; Ishihara, K.;
Sugai, T. Tetrahedron: Asymmetry 2003, 14, 1323.
2540 J. Org. Chem., Vol. 73, No. 7, 2008

Synthesis of a New Spirolactonyl Nitrone
together and modeled using the rate equations shown in Scheme
5, which then led to the kt and kd values.
structures was negligible, i.e., S2 ) 0.75. Conformational search
of the superoxide adduct complexes with explicit water molecules
were carried out using SPARTAN at the MMFF level.
(b) Decay Kinetics of Hydroxyl Radical Adducts. In a typical
kinetic study, 50 µL of solution containing 50 mM of CPCOMPO
or EMPO and 0.33 mM H2O2 was UV irradiated for 3 min in the
cavity. The lowest field peak decay was monitored as a function
of time over a period of 2680 s after the light source was turned
off.
Acknowledgment. This work is supported by NIH Grant
No. HL081248. A.R. acknowledges support from the Hungarian
Scientific Research Fund, OTKA T-469153. The Ohio Super-
computer Center (OSC) is acknowledged for generous compu-
tational support of this research. We thank Prof. David Hart of
the OSU Chemistry Department for helpful suggestions and Dr.
Judith Galluci for the X-ray measurements. J.L.Z. acknowledges
support from the NIH grants HL38324, HL63744, and HL65608.
Computational Method. Density functional theory (DFT)76,77
was applied in this study to determine the optimized geometry,
vibrational frequencies, and single-point energy of all stationary
points.78-81 The effect of aqueous solvation was also investigated
using the polarizable continuum model (PCM).82-86 All calculations
were performed using Gaussian 0387 at the Ohio Supercomputer
Center. Single-point energies were obtained at the B3LYP/6-
31+G** level based on the optimized B3LYP/6-31G* geometries,
and the B3LYP/6-31+G**//B3LYP/6-31G* wave functions were
used for natural population analyses (NPA).88 These calculations
used six Cartesian d functions. Stationary points for CPCOMPO
and the CPCOMPO spin adducts have zero imaginary vibrational
frequency as derived from a vibrational frequency analysis (B3LYP/
6-31G*). A scaling factor of 0.9806 was used for the zero-point
vibrational energy (ZPE) corrections for the B3LYP/6-31G* level.89
Spin contamination for all of the stationary point of the radical
Supporting Information Available: Spectral characterization
data; computional data; crystallographic information files (CIF).
This material is available free of charge via the Internet at
http://pubs.acs.org.
JO702434U
(87) Frisch, M. J. T. G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M.
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