Inclusion complexes of EMPO derivatives with 2,6-di-O-methyl-β- cyclodextrin: Synthesis, NMR and EPR investigations for enhanced superoxide detection

Article abstract of DOI:10.1039/b606062e

The free radical trapping properties of eight 5-alkoxycarbonyl-5-methyl-1- pyrroline N-oxide (EMPO) type nitrones and those of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) were evaluated for trapping of superoxide anion radicals in the presence of 2,6-di-O-methyl-β-cyclodextrin (DM-β-CD). ¹H-NMR titrations were performed to determine both stoichiometries and binding constants for the diamagnetic nitrone-DM-β-CD equilibria. EPR titrations were then performed and analyzed using a two-dimensional EPR simulation program affording 1: 1 and 1: 2 stoichiometries for the nitroxide spin adducts with DM-β-CD and the associated binding constants after spin trapping. The nitroxide spin adducts associate more strongly with DM-β-CD than the nitrones. The ability of the nitrones to trap superoxide, the enhancement of the EPR signal intensity and the supramolecular protection by DM-β-CD against sodium l-ascorbate reduction were evaluated. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b606062e

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Inclusion complexes of EMPO derivatives with 2,6-di-O-methyl-b-
cyclodextrin: synthesis, NMR and EPR investigations for enhanced
superoxide detection†
David Bardelang,a Antal Rockenbauer,b Hakim Karoui,a Jean-Pierre Finet,*a Inga Biskupska,a
Karol Banaszaka and Paul Tordoa
Received 28th April 2006, Accepted 8th June 2006
First published as an Advance Article on the web 30th June 2006
DOI: 10.1039/b606062e
The free radical trapping properties of eight 5-alkoxycarbonyl-5-methyl-1-pyrroline N-oxide (EMPO)
type nitrones and those of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) were evaluated for trapping of
superoxide anion radicals in the presence of 2,6-di-O-methyl-b-cyclodextrin (DM-b-CD). 1H-NMR
titrations were performed to determine both stoichiometries and binding constants for the diamagnetic
nitrone–DM-b-CD equilibria. EPR titrations were then performed and analyzed using a
two-dimensional EPR simulation program affording 1 : 1 and 1 : 2 stoichiometries for the nitroxide spin
adducts with DM-b-CD and the associated binding constants after spin trapping. The nitroxide spin
adducts associate more strongly with DM-b-CD than the nitrones. The ability of the nitrones to trap
superoxide, the enhancement of the EPR signal intensity and the supramolecular protection by
DM-b-CD against sodium L-ascorbate reduction were evaluated.
Introduction
Reactive oxygen species (ROS) have benefited from intense atten-
tion because of their implication in oxidative stress and a range
of pathological processes.1 Since the discovery of DMPO or PBN
as spin traps, significant advances to find more efficient molecular
systems trapping the ROS with enhanced lifetime of the resulting
spin adducts have been realized with the synthesis of better nitrone
spin traps.2,3 Among recently reported nitrones, EMPO analogues
(Fig. 1) are efficient spin traps for the detection of carbon and
oxygen centered free radicals by electron paramagnetic resonance
(EPR) spectroscopy.3 Unfortunately, use of these traps for the
detection of ROS in biological media is limited by the presence
of species that reduce instantaneously the nitroxide spin adducts
into EPR silent hydroxylamines.4 To overcome these difficulties,
Fig. 1 Chemical structures of DM-b-CD and EMPO.
cyclodextrins (CDs) are effective in (i) increasing the EPR signal
of transient free radicals by the spin trapping-EPR technique is
intensity, (ii) lengthening the life time of superoxide spin adducts,
quite recent. Chen and co-workers11 have studied the trapping
and (iii) protecting the nitroxide spin adducts resulting from spin
of carbon-centered radicals with nitroso compounds stabilized
trapping against enzymatic or chemically reductive species.5
by cyclodextrins. However, cyclic nitrones are a better choice to
For paramagnetic guests, EPR was successfully used to study
trap superoxide as illustrated by 5-diethoxyphosphoryl-5-methyl-
inclusion complexes of stable free radicals in cyclodextrins6 for
1-pyrroline N-oxide (DEPMPO) in the absence of cyclodextrin.2a
molecular6c,7 and chiral8 recognition and in terms of multimodal
Inclusion of PBN nitrones in b-cyclodextrin12 and especially their
inclusion complexes.6b,c,9 Bioreduction experiments were also per-
methylated derivatives led to improved detection of superoxide
formed with persistent nitroxides to test their ability to be pro-
radical in vitro and its observation under reductive conditions.5c
tected against reduction by ascorbic acid10 through inclusion in a
Moreover, the binding of PBN–superoxide spin adducts resulted
cyclodextrin. However, the use of macrocycles during the detection
in multiple equilibria due to the heteroditopic character of
the PBN skeleton. This situation was analyzed in terms of
microscopic binding constants and site interaction parameter
(cooperativity).5c,13 However, in spite of the recognized trapping
properties of PBN, the oxygen free radicals derived spin adducts
are less stable than those resulting from the trapping by EMPO
analogues. In addition, with EMPO analogues, only 1 : 1 inclusion
complexes are expected between the CDs and both the nitrones
and the spin adducts. Therefore, we decided to investigate the
aLaboratoire SREP, UMR 6517 CNRS et Universite´s d’Aix-Marseille 1,
2 et 3, Avenue Escadrille Normandie Niemen, Marseille, 13397, France.
E-mail: jean-pierre.finet@up.univ-mrs.fr; Fax: + 33 491 288 758; Tel: + 33
491 288 927
bChemical Research Center, Institute for Structural Chemistry, H-1525,
Budapest, PO Box 17, Hungary
† Electronic supplementary information (ESI) available: Synthesis yields,
NMR spectra and additional figures. See DOI: 10.1039/b606062e
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influence of DM-b-CD on the spin trapping of superoxide anion
radical by a series of EMPO analogues.
and CMMPO 9d for which 2D-NOESY spectra were necessary
for a non ambiguous assignment. The methylene protons of the
pyrroline ring exhibited three multiplet signals and were also
assigned using 2D-NOESY in D2O (ESI†).
Results and discussion
Synthesis
Continuous variation method
The continuous variation method (Job’s plot)15 was used to
determine the stoichiometry of the inclusion complexes. NMR
samples were prepared by mixing equimolar amounts of freshly
made stock solutions of b-CD and nitrone (10 mM) in different
volumes leading to the desired values of the ratio SH = [H]0/([H]0 +
A range of EMPO ester derivatives was selected to cover a large
scale of binding constants with DM-b-CD, considering that the
more the nitrones are associated with the cyclodextrin, the less they
can trap free radicals.5c Nitrones 2d and 4d to 9d were prepared
by an adaptation of the four step procedure of Zhao et al.3c
(Scheme 1 and Electronic Supplementary Information, ESI†),
whereas EMPO 3d was prepared by the procedure of Olive et al.3a
[G]0), with the total products concentration kept constant (Vtot
=
500 lL) while the molar fractions of both components varied
from 0 to 1 by steps of 0.1. The averaged values were monitored
as a function of the cyclodextrin and nitrone concentrations. In
preliminary experiments with D2O solutions of the nitrones in
the presence of DM-b-CD, significant 1H-NMR complexation
induced shifts (CIS) showed the presence of inclusion complexes.
The mean values of the affected protons between the free and
included guests suggested a fast exchange process between both
species on the NMR timescale. However, the signal of the methyl
group that substitutes the pyrroline ring was split into two signals
during the titration whatever the nitrone. In spite of limited
changes detectable in the 1H-NMR spectra in the case of DMPO
1d, MeMPO 2d and EMPO 3d in the presence of DM-b-CD, a
1 : 1 stoichiometry was derived from the Job’s analysis of the CIS
values for all the considered nitrones (see Fig. 3 for an example
with CMMPO as a guest).
Scheme 1 General synthetic route to ester functionalized EMPO type
nitrones (x refers to the alkoxy group, Fig. 2).
Fig. 3 Job’s diagram for CMMPO 9d–DM-b-CD equilibrium. (HCD3
HCD5 = protons inside the DM-b-CD cavity, see Fig. 1).
,
Fig. 2 Chemical structure of DMPO and of the synthesized EMPO and
analogues.
NMR study
Structure–complexation induced shift correlations
The characteristic features of the inclusion complexes between
EMPO-type nitrones and 2,6-di-O-methyl-b-cyclodextrin were
determined by 1H-NMR studies. To study the effect of the host–
guest association, the use of NMR spectroscopy was selected, as
For each nitrone, the maximum CIS values were derived from the
two extreme spectra (free of CD and maximum concentration of
CD), affording information on the inclusion mode of the guests
inside the CD cavity (Table 1). Nitrones 1d, 2d and 3d, bearing
poorly hydrophobic groups, showed only moderate CIS. Because
of the good water solubility of these nitrones, they did not exhibit
great affinity for the hydrophobic inner cavity of DM-b-CD. On
the other hand, substitution of the ester function by bulkier alkyl
groups resulted in a greater affinity for the CD cavity. Indeed,
whereas the CIS of the nitronyl proton H7 decreased from nitrone
1
it provides specifically H and 13C complexation induced shifts
(CIS) upon inclusion and represents a powerful tool to investigate
1
molecular edifices in solution.14 The H-NMR signals of all the
nitrones were first carefully assigned for D2O solution of the
nitrones alone (ESI†). All the signals related to the ester part
of the nitrones were easily attributed except for sBuMPO 5d
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Table 1 1H NMR CIS values of EMPO analogues in the presence of
DM-b-CD
to a difference in the nitrone conformations implying that, during
the titration, the C–H8 bond interacts with the asymmetric cone
16
=
of the adjacent C O bond.
Nitrone Structure
H7
H8
H9
H10
However, this hypothesis does not explain the CIS observed in
the case of benzyl ester nitrone 6d, since H8 is the only proton
to give detectable and high CIS, while the rest of the molecule is
not affected. X-Ray crystallographic analyses of the benzyl ester
nitrone 6d and of the corresponding saturated cyclohexylmethyl
ester nitrone 9d helped us to understand this behaviour (ESI†).
BnMPO 6d adopts a folded down (cis) conformation (ESI†)
with the phenyl group slightly facing the nitrogen atom of the
nitrone function. On the other hand, CMMPO 9d exhibits an
unfolded geometry (trans) with the cyclohexyl group opposite to
a
1d
−0.03 +0.03
+0.04
a
2d
3d
−0.05 −0.03 +0.04
−0.05 −0.07 +0.04 +0.01
−0.04 −0.14 +0.03 +0.04
˚
the pyrroline ring (ESI†). The 5.6 A distance between the positively
4d
charged nitrogen atom and the p cloud for BnMPO 6d is too large
to allow a stabilizing effect (angle between the 6-membered and
the 5-membered ring planes of around 51◦, ESI†). Nevertheless,
the folded face-to-face geometry could be explained by a p–p and a
5d
7d
8d
−0.03 −0.16 +0.08 +0.08
−0.05 +0.05 +0.01 +0.08
−0.02 −0.15 +0.05 +0.10
˚
pyrrolinyl CH–p interaction (around 2.8 and 2.7 A respectively for
each CH–p distance, see Fig. 4a and ESI†) that probably stabilizes
each phenyl group.17
a
9d
6d
−0.10 +0.06 +0.10
a
a
−0.32 +0.04
a |Dd| < 0.01 ppm.
2d to 9d (except in the case of 7d), the CIS observed on the alkyl
ester region (H10) increased. Thus, the part of the nitrone that
is included in the CD cavity seems to change, depending on the
nature of the ester group. Replacement of the small methyl ester
group by the bulkier tert-butyl or neo-pentyl group shifts the site,
that is preferentially recognized by the CD, from the pyrroline
ring to the ester moiety. In parallel, the general strength of the
association is directly correlated with the nature of the ester group
(see binding constants in Table 2). Although the fit between alkyl
groups and the inside cavity of DM-b-CD should be better for the
cyclohexyl group (and optimal for the adamantyl group, although
not studied in the present series), the highest CIS were not observed
in the case of nitrone 9d. Moreover, the usually good affinity of the
phenyl group for the inner b-CD cavity was not observed in the
case of BnMPO 6d. Instead of recording a significant CIS value for
the aromatic protons (H10), a relatively big CIS was observed for
the pyrroline proton H8. Besides, the absolute value for H8 CIS
increased from +0.03 to +0.32 ppm on going from nitrone 1d to
nitrone 6d (in the order displayed in Table 1 except for nitrones 7d
and 9d). This trend is unusual since, in most cases, the H8 proton
exhibits the highest CIS value compared to the other protons with
the exclusion of other pyrroline ring protons. This might be related
Fig. 4 Weak stabilizing interactions in the crystal structure of BnMPO
6d and yin-yang-like disposition of two nitrone molecules.
The weak interactions (Fig. 4a) may be related to the observed
relative disposition of two molecules of 6d that is reminiscent of
the yin-and-yang symbol (Fig. 4b). The folded conformation of
BnMPO 6d can prevent the phenyl group from being included
in the CD cavity due to steric hindrance (this would explain the
absence of CIS for aromatic protons H10). In contrast to the
BnMPO nitrone 6d, the CMMPO nitrone 9d adopts an unfolded
geometry allowing the cyclohexyl moiety to be complexed by the
DM-b-CD. One hydrogen bond per molecule can be observed
between the nitronyl proton of one molecule of 9d and the carbonyl
˚
oxygen of a second one (≈2.4 A distance, Fig. 5a). An almost
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toward experimental points afforded the equilibrium constants
(Fig. 7).
Fig. 5 Opposite disposition of the ester moieties in the crystal structure
of CMMPO 9d leading to a sheet-like structure in the solid state.
Fig. 7 Calculation of binding constants by non linear curve fitting of the
chemical shift of the suitable EMPO 3d protons investigated as a function
of DM-b-CD concentration.
face-to-face disposition of the pyrroline rings is observed (Fig. 5b
and ESI†) with the cyclohexyl moieties far apart. This alternation
of hydrophobic cyclohexyl and hydrophilic nitrone packing zones
leads to an overall sheet-like structure (see ESI†).
All protons affording convergent fit were considered. The weak
intensity of the H8 signal made its detection rather tedious up to
35 mM of CD. Thus the CD concentration domain was limited
in this case (Table 2). The useful range of CD concentrations
has also been restricted for nPtMPO 8d and CMMPO 9d since a
poor fit was obtained, if all points were considered.8d Correlation
coefficients exceeded 0.99 except for a few cases. Calculations
afforded estimations of maximum CIS and the value of binding
constants related to the considered proton, with the errors
estimated as previously described.12 Finally, when more than one
binding constant was estimated for a nitrone because of the
different influence of the solvent on the nitrone protons in the
complex, an averaged binding constant value was considered to
give a more significant equilibrium constant with DM-b-CD.19 The
first aim was to detect a clear trend in the nitrone complexation
by DM-b-CD. The results are reported in Table 2. The values
of the binding constants were also used for the EPR titrations
to estimate the binding constants between the spin adducts and
DM-b-CD after superoxide trapping. In agreement with the CIS
studies, the highly water soluble nitrones presented moderate
binding constants (4 to 43 M−1 from MeMPO 2d to sBuMPO 5d),
with only slight differences upon the considered protons. In the
case of bulky ester groups (like tert-butyl), the binding constants
exceeded 200 M−1. The independent values are homogeneous for
CMMPO 9d, close to 960 M−1 but more dispersed for tBuMPO
7d and nPtMPO 8d. Nevertheless, a clear trend can be observed
in the series with binding constant values increasing regularly
with the size of the ester group from 4 M−1 for the smallest
(MeMPO 2d) to 962 M−1 for the bulkiest (CMMPO 9d). These
calculated maximum CIS and binding constant values agree well
with the previous analysis of experimental CIS and with the
continuous variation method. On the other hand, it must be
remembered that the values estimated from equilibria occurring in
deuterated water are probably slightly overestimated compared to
the experiments in water.20 So, nitrones 1d to 7d showed moderate
binding constants toward DM-b-CD in line with good trapping
properties (sufficient availability of the trap toward free radicals).
On the contrary, nPtMPO 8d and CMMPO 9d were expected to
Thus, the solid state information may explain the absence of
CIS for the aromatic protons of molecule 6d due to the folded
geometry of the nitrone, contrary to the unfolded one for nitrone
9d. However, the effect of the solvent can play an important role on
the nitrone geometry and, in spite of this structural information,
the observed CIS for H8 of nitrone 6d in solution with the exclusion
of the other protons cannot be fully rationalized.
Calculation of association constants16
The 1H-NMR titration technique was used to evaluate the binding
constants. The whole sets of nitrone protons chemical shifts were
monitored with the guest concentration kept constant, while the
DM-b-CD concentration was increased (Fig. 6).
Fig. 6 Chemical shift evolution for selected guest protons during 1H
NMR titration of tBuMPO 7d in the presence of DM-b-CD (R =
[CD]/[nitrone]).
Formation of 1 : 1 complexes was assumed from the results
of the continuous variation method. The Macomber model was
then chosen to calculate the binding constants from the titration
curves.18 The non linear curve fitting procedure of these curves
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Table 2 Binding constants and maximum 1H-NMR CIS for the different nitrones based on the selected protons, toward DM-b-CD
Entry
Nitrone
Proton
H7
K
Hi/M−1
24 ( 4)
|Dd| (ppm)
r2
[CD]/mM
0.5–141
K
NMR/M−1
DMPO
0.035 ( 0.003)
>0.994
24
MeMPO
EMPO
H7
H8
4 ( 1)
3 ( 1)
0.128 ( 0.019)
0.102 ( 0.019)
>0.988
>0.972
0.5–141
0.5–141
4
12
27
43
H7
H8
12 ( 3)
12 ( 2)
0.078 ( 0.009)
0.113 ( 0.012)
>0.996
>0.997
0.5–141
0.5–141
iPrMPO
sBuMPO
H7
H8
32 ( 6)
22 ( 2)
0.052 ( 0.004)
0.218 ( 0.011)
>0.991
>0.998
0.5–141
0.5–95
H8
H9
H10
78 ( 7)
22 ( 4)
28 ( 5)
0.213 ( 0.008)
0.104 ( 0.006)
0.103 ( 0.008)
>0.991
>0.992
>0.992
0.5–35
0.5–141
0.5–141
BnMPO
H8
207 ( 13)
0.343 ( 0.007)
>0.998
0.5–95
207
tBuMPO
nPtMPO
H7
H10
379 ( 51)
80 ( 8)
0.051 ( 0.002)
0.085 ( 0.002)
>0.987
>0.982
0.5–141
0.5–141
230
429
H8
H9
H10
678 ( 156)
294 ( 50)
314 ( 29)
0.157 ( 0.008)
0.033 ( 0.002)
0.074 ( 0.002)
>0.992
>0.982
>0.995
0.5–35
0.5–35
0.5–35
CMMPO
H9
H10
H11
843 ( 130)
1052 ( 104)
990 ( 103)
0.039 ( 0.001)
0.092 ( 0.002)
0.093 ( 0.002)
>0.988
>0.997
>0.995
0.5–35
0.5–35
0.5–35
962
trap less efficiently the superoxide in the presence of DM-b-CD
Calculations of association constants
due to their higher affinity toward the CD cavity.
In the EPR titration experiments, the global amount of cyclodex-
trin is expected to be shared between all products susceptible to
be complexed: the nitrones and their superoxide adducts (Fig. 8).
The nitrone concentration being much larger than that of the
nitroxides, the proportion of CD occupied for complexation of
the nitrones is crucial for estimation of the free CD, which can be
EPR study
Preliminary EPR spectra of the spin adducts of superoxide trapped
by nitrones 1d–9d in the presence of DM-b-CD were recorded
under identical experimental conditions. In a previous study,5c we
found that decreasing intensity of the EPR spectrum is associated
with increasing binding constants of the corresponding nitrone–
CD couples. In the present series of nitrones, EMPO 3d, sBuMPO
5d and BnMPO 6d showed the highest EPR spectrum intensity.
However, in the case of the two bulkiest nitrones (nPtMPO 8d
and CMMPO 9d) the spin trapping reaction is impeded (nitrones
exhibiting the larger binding constants with cyclodextrins) com-
pared to the smaller nitrones 1d–7d. The high signal intensity in the
case of nitrones 3d, 5d and 6d can be assigned to the known good
stability of the superoxide spin adducts, allowing a more efficient
accumulation of the paramagnetic species compared to the case
of other nitrones. Thus, the affinity of the trap for the cyclodextrin
is important to predict the trapping efficiency; but the intrinsic
stability of the superoxide spin adduct plays also an important
role for free radical detection. The EPR signal enhancement can
be attributed to inclusion of the nitroxide spin adduct in the CD
unless the parent nitrone is too strongly complexed by the CD to
allow good trapping.
estimated by KNMR
.
Fig. 8 Schematic representation of equilibria occurring before and after
spin trapping of superoxide by EMPO analogues in the presence of
DM-b-CD.
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Table 3 Calculated values of binding constants and EPR parameters for
spin trapping of superoxide in the presence of DM-b-CD
As previously reported in the case of PBN derivatives used as
spin traps,5c the EPR spectra intensities are also correlated to
the added CD concentration (Fig. 9). The pattern of the EPR
spectra shows continuous variation in the titrations presenting
the superimposition of free and included species. The association
properties of pyrroline type nitrone spin adducts differ from
those of the open chain nitrones: for the ring compound, only
two types of association can be observed contrary to the PBN
derivatives, where two different single and one double association
can be formed. In the EMPO type series, efforts to distinguish
the free and complexed species of the cis and trans superoxide
spin adducts by 2D-EPR simulations21 were not successful due
to the poor resolution. Although the fit become better, the large
number of adjusted parameters made the confidence of the spectral
decomposition rather poor. A further difficulty is the presence
of a chemical exchange phenomenon3a that is also a source of
error, which can prevent the most sophisticated analysis. For these
reasons, we considered only a simpler rigid superimposed model
concerning the 1 : 1 association.
e
Nitrone
DMPO
Param.a
1 : 0b
1 : 1c
1 : 2d
KNMR
af
aN/mT
aH/mT
1.413
1.137
0.118
1.343
1.104
0.121
343
1.250
1.102
0.106
113
1.247
1.103
0.107
207
1.249
1.090
0.114
266
1.249
1.078
0.118
367
1.250
1.063
0.119
645
1.267
1.088
0.113
658
1.265
1.039
0.124
892
1.334
1.048
0.177
603
1.248
1.003
0.124
129
1.248
0.999
0.132
307
1.253
1.082
0.102
2 985
1.259
1.072
0.100
3 510
1.251
1.082
0.075
7 340
1.271
1.080
0.110
18 500
1.268
1.090
0.061
3 950
1.243
1.094
0.105
133700
24
14.3
ꢀ
aH /mT
K/M−1
aN/mT
aH/mT
MeMPO
EMPO
1.306
1.084
0.101
4
28.3
17.3
9.9
8.5
3.1
2.9
2.1
2.2
ꢀ
aH /mT
K/M−1
aN/mT
aH/mT
1.317
1.086
0.103
12
ꢀ
aH /mT
K/M−1
aN/mT
aH/mT
iPrMPO
sBuMPO
BnMPO
tBuMPO
nPtMPO
CMMPO
1.320
1.096
0.102
27
ꢀ
aH /mT
K/M−1
aN/mT
aH/mT
1.321
1.085
0.104
43
ꢀ
aH /mT
K/M−1
aN/mT
aH/mT
1.317
1.077
0.101
207
230
429
962
ꢀ
aH /mT
K/M−1
aN/mT
aH/mT
1.333
1.097
0.106
ꢀ
aH /mT
K/M−1
aN/mT
aH/mT
1.317
1.074
0.105
ꢀ
aH /mT
K/M−1
aN/mT
aH/mT
1.326
1.086
0.106
1.278
1.048
0.118
2 144
ꢀ
aH /mT
K/M−1
a EPR spectra parameters. b Parameters for the free nitroxides. c Parameters
for the 1 : 1 complexes. d Parameters for the 1 : 2 complexes. e In M−1. f a =
K
1 : 1/KNMR
CD. Further evidence for the association is given by the high field
lines of the spectrum that have smaller amplitude than the other
lines in the cases of 1 : 1 and 1 : 2 species, a clear indication of the
restriction of the molecular rotation upon inclusion (see Fig. 9).
The possibility for the 2D EPR simulation program to take
into account the previously calculated KNMR greatly helped for a
reliable determination of nitroxide–CD binding constants. As for
PBN, the 1 : 1 nitroxide binding constants are found always larger
for the included spin adducts than for the parent nitrones toward
DM-b-CD complexation (Fig. 10).5c
Fig. 9 Selected EPR spectra of EMPO 3d superoxide spin adduct with
increasing concentration of DM-b-CD: superoxide spin adduct (a) without
CD, (b) with CD 3 mM, (c) 6 mM, (d) 9 mM, (e) 12 mM, (f ) 25 mM, (g)
100 mM.
The trend in the values of K1 : 1 for the nitroxide–CD pair is the
same as for KNMR considering the changes in the ester function.
Indeed, a clear increase of K1 : 1 with bulkier nitroxide ester parts
can be seen. On the other hand, the a ratio (K1 : 1/KNMR) also
named selectivity is decreasing with the general size of the spin
adducts depending on the nitrone considered.
In spite of these limitations, 2D-EPR simulations implying
free (1 : 0), single associated (1 : 1) and double associated
(1 : 2) nitroxides gave the best results. Thus, the possibility of
encapsulation followed by dimerization of DM-b-CD at high CD
concentrations was once again observed.
The results of 2D spectral decompositions of the EPR titrations
are reported in Table 3.
A convincing proof of spin adduct encapsulation in the CD
cavity is given by the decrease of the nitrogen splitting constant aN
that reflects the lower polarity of the environment in the vicinity
of the nitroxide function, which is the case of inner cavity of the
This trend can be explained by the differences in size and
polarity for each nitrone and corresponding nitroxide considered.
Indeed, the effect on complexation when the superoxide spin
adduct is formed is more intense for the smaller nitrones than for
the bigger nitrones. On the other hand, the larger calculated K1 : 2
binding constants compared to K1 : 1 support the occurrence of
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Fig. 10 Comparison of (spin adduct) nitroxide–CD binding constants
(dark grey) and nitrone–CD binding constants (light grey) for all the
considered nitrones.
Fig. 11 Protection of the EMPO-OOH nitroxide spin adduct by
DM-b-CD (50 mM) against sodium L-ascorbate reduction. (a) EMPO-
OOH EPR signal in the absence of cyclodextrin. (b) Signal obtained after
addition of SOD and sodium L-ascorbate (c) EMPO-OOH EPR signal
in the presence of DM-b-CD. (d) 1 min 15 s after addition of SOD and
sodium L-ascorbate (0.1 mM). (e) After 2 min. (f ) After 2 min 45 s. (g)
After 3 min 30 s.
DM-b-CD dimerization with the superoxide adducts as included
guest at high CD concentration. Nevertheless, calculation of the
stepwise binding constants according to Connors13 shows a non
cooperative complexation process.
This is probably due to the difference in polar surfaces of
the superoxide spin adducts (ester hydrophobic part versus more
hydrophilic hydroperoxy-nitroxide group). Complexation of the
ester function can be expected easier than complexation of the
hydroperoxy-nitroxide counterpart.
Table 4 Maximum time of EPR observation of the superoxide spin
adducts under reductive conditions (0.1 mM sodium L-ascorbate)
Entry
Nitrone
tobs
Bioreducing experiments
1d
2d
3d
4d
5d
6d
7d
8d
9d
DMPO
MeMPO
EMPO
iPrMPO
sBuMPO
BnMPO
tBuMPO
nPtMPO
CMMPO
3 min
2 min 50 s
3 min 30 s
2 min 20 s
4 min 10 s
1 min 40 s
1 min
Despite the efficient spin trapping properties of EMPO, its
derivatives were only seldom applied as traps in biological media
since the resulting spin adducts are instantaneously reduced. It
is promising, however, that the cyclodextrins proved effective
in protecting the superoxide spin adducts against enzymatic
and molecular reduction in the case of DEPMPO5a and PBN
derivatives as spin traps.5b,5c In the case of the superoxide adducts
of EMPO analogues 1d–9d under reductive conditions, the EPR
spectrum of EMPO superoxide spin adduct can be recorded up to
3 minutes in the presence of DM-b-CD (Fig. 11).
1 min 40 s
2 min 20 s
Conclusion
The other EMPO analogue nitrones as spin traps did not exhibit
a higher observed time except in the case of sBuMPO 5d (up to 4
minutes: see Table 4).
EMPO type nitrones are significantly complexed by 2,6-di-O-
methyl-b-cyclodextrin (DM-b-CD) and the complexation strongly
depends on the nature of the ester function. Trapping of superoxide
radical by these nitrones does not hinder the complexation process
by the DM-b-CD. The strength of the spin adduct recognition
even increases by formation of the less polar nitroxide group
that replaces the strongly polar nitrone structure after spin trap-
ping. The stabilization induced by complexation affords a better
detection due to the enhanced EPR spectrum intensities, unless
the too strongly complexed nitrones block the trapping process.
Contrary to PBN, the difference in polarity for the heteroditopic
EMPO superoxide spin adduct (ester side and hydroperoxy-
nitroxide group) induces a non-cooperative situation. Indeed, at
high DM-b-CD concentration a second complexation effectively
occurs, but less efficient than the first one. Nevertheless, DM-b-
CD dimerizes to encapsulate the unstable superoxide spin adducts.
Thus, cyclodextrins should promote the use of nitrone type spin
traps for the direct observation and the characterization of free
radicals, in particular, the ROS species in ex vivo or in vivo EPR
experiments.
Moreover significant differences in the decrease of the EPR
signal intensity (linked to the nitroxide concentration) were
monitored after the first minute of reduction (data not shown).
However, this trend cannot be correlated to the maximum time
of observation of the nitroxides since it is affected both by the
ability to trap the superoxide and by the stability of the resulting
spin adducts. The nitroxide spin adducts derived from EMPO
3d and sBuMPO 5d exhibited the longest observation time of
the signal, and these nitrones produced also the greatest EPR
spectrum intensity all things being otherwise equal. That would
explain that, in spite of a rapid loss of signal for EMPO-OOH
EPR spectrum, the signal observation time is still rather long.
Finally, we did not observe any direct correlation between the
estimated nitroxide binding constants toward DM-b-CD and the
resistance abilities of these complexed spin adducts toward L-
ascorbate. However, the presence of DM-b-CD undoubtedly slows
down the reductive process.
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Cyclohexylmethyl 2-bromopropanoate 9a. 66%, dH 0.92–1.05
(m, 2H), 1.14–1.33 (m, 3H), 1.67–1.77 (m, 6H), 1.83 (d, J 7.0,
3H), 3.92–4.04 (m, 2H), 4.37 (q, J 6.9, 1H).
Experimental
General
Cyclohexylmethyl 2-nitropropanoate 9b. Flash chromatogra-
phy (pentane–CH3OH, 18 : 2), 60%, dH 0.9–1.02 (m, 2H), 1.13–
1.32 (m, 3H), 1.64–1.77 (m, 6H), 1.80 (d, J 7.0, 3H), 3.90–4.04 (m,
2H), 5.22 (q, J 7.1, 1H).
All commercially available chemicals and organic solvents were
used as received without further purification. 2,6-Di-O-methyl-
b-cyclodextrin was purchased from Acros Organics. Xanthine
oxidase (XOD) and diethylenetriaminepentaacetic acid (DTPA)
were from Sigma Chemical Co. Crude materials were purified by
flash chromatography on Merck silica gel 60 (0.040–0.063 mm).
1H-NMR and 13C-NMR spectra were recorded with a Bruker DPX
300 spectrometer at 300.13 MHz and 75.54 MHz respectively.
Chemical shifts (d) are reported in ppm for a solution of the
nitrone in CDCl3 with internal reference Me4Si (Euriso-Top, CEA
Saclay, 99.80%). J values are reported in Hz. The assignments of
NMR signals were facilitated by the use of DEPT135 and NOESY
sequence in D2O if necessary. Melting points were measured on a
Bu¨chi B-540 apparatus and are uncorrected.
Cyclohexylmethyl 1-oxo-4-nitro-4-methylpentanoate 9c. 97%,
dH 0.89–1.01 (m, 2H), 1.13–1.31 (m, 3H), 1.61–1.75 (m, 6H), 1.80
(s, 3H), 2.42–2.65 (m, 4H), 4.02 (d, J 6.0, 2H), 9.77 (s, 1H).
5-Cyclohexylmethoxycarbonyl-5-methyl-1-pyrroline N-oxide 9d.
Flash chromatography (pentane–AcOEt–CH3OH, 12 : 5 : 3), 9%,
colourless crystals, mp 52 ◦C; dH 0.91–1.03 (m, 2H), 1.13–1.31 (m,
3H), 1.62–1.73 (m, 6H), 1.73 (s, 3H), 2.12–2.22 (m, 1H), 2.55–2.85
(m, 3H), 4.01 (m, 2H), 6.97 (t, J 2.6, 1H); dC 20.97, 25.55, 25.85,
26.24, 29.50, 32.55, 36.95, 71.17, 79.05, 134.59, 169.93. Calcd for
C13H21NO3, 0.2 H2O (239.31 g mol−1) C, 64.28; H, 8.88; N, 5.77.
Found C, 64.28; H, 8.81; N, 5.70%.
The nitrones were prepared by the procedure described by
Zhao et al.3c and only the characteristic physical data of the new
compounds are reported.
NMR measurements
5-Methoxycarbonyl-5-methyl-1-pyrroline N-oxide 2d,3g 5-
ethoxycarbonyl-5-methyl-1-pyrroline N-oxide 3d,3a 5-(1-methyl-
ethoxy)carbonyl-5-methyl-1-pyrroline N-oxide 4d,3f 5-(2-butoxy-
carbonyl)-5-methyl-1-pyrroline N-oxide 5d3f and 5-(1,1-dimethyl-
ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide 7d3c were prepared
by literature procedures.
All spectra were recorded at 300 K in D2O (Euriso-Top, CEA
Saclay, 99.90%), the residual HOD signal (d 4.79 ppm) being used
as the internal reference.14
Continuous variation method (Job’s plot)
This technique was used as described previously for similar
nitrone–cyclodextrin equilibria (10 mM final concentration).5c
Benzyl 1-oxo-4-nitro-4-methylpentanoate 6c. 95%, dH 1.79 (s,
3H), 2.46–2.59 (m, 4H), 5.23 (s, 2H), 7.31–7.40 (m, 5H), 9.70 (s,
1H).
Binding constant calculations
The results obtained in a precedent work for PBN analogue–
cyclodextrin titrations helped in choosing the final nitrone con-
centration (3 mM) as well as the work range of DM-b-CD
concentration (from 0.5 to 141 mM). The NMR experiments were
performed as described previously.5c CIS values were determined
by comparison of 1H-NMR spectra of the nitrone alone and
the mixture containing the nitrone and the maximum DM-b-CD
concentration.
5-Benzyloxycarbonyl-5-methyl-1-pyrroline N-oxide 6d. Flash
chromatography (CH2Cl2–EtOH 95, 18.5 : 1.5), 10%, colourless
crystals, mp 62 ◦C; dH 1.75 (s, 3H), 2.10–2.20 (m, 1H), 2.52–2.80
(m, 3H), 5.18 (d, J 12.4, 1H), 5.28 (d, J 12.3, 1H), 6.96 (t, J 2.6, 1H),
7.32–7.40 (m, 5H); dC 20.94, 25.84, 32.34, 67.69, 79.08, 127.98,
128.42, 128.61, 134.82, 135.12, 169.69. Calcd for C13H15NO3, 0.1
CH2Cl2 (233.27 g mol−1) C, 65.08; H, 6.34; N, 5.79. Found C,
65.27; H, 6.58; N, 5.69%.
EPR measurements
2,2-Dimethylpropyl 2-bromopropanoate 8a. 97%, dH 0.97 (s,
9H), 1.84 (d, J 6.9, 3H), 3.82 (d, J 10.5, 1H), 3.92 (d, J 10.6,
1H), 4.41 (q, J 7.0, 1H).
EPR spectra were recorded at room temperature using a Bruker
ESP 300 EPR spectrometer at 9.5 GHz (X-band) employing 100
kHz field modulation. All the solutions were prepared in a chelex-
treated phosphate buffer (0.1 M, pH 7.4).
2,2-Dimethylpropyl 2-nitropropanoate 8b. Flash chromatogra-
phy (pentane–CH3OH, 19.5 : 0.5), 38%, dH 0.94 (s, 9H), 1.82 (d, J
7.2, 3H), 3.89 (d, J 10.4, 1H), 3.95 (d, J 10.4, 1H), 5.25 (q, J 7.2,
1H).
Superoxide trapping
The EMPO analogue (25 mM), diethylenetriaminepentaacetic
acid (DTPA 0.5 mM) and hypoxanthine (HX 0.2 mM) were mixed
in the presence of desired concentrations of DM-b-CD. To this
oxygenated solution (bubbled during 1.5 min), xanthine oxidase
(XOD 0.05 U mL−1) was added to generate superoxide.
2,2-Dimethylpropyl
92%, dH 0.94 (s, 9H), 1.81 (s, 3H), 2.44–2.66 (m, 4H), 3.90 (s, 2H),
9.78 (s, 1H).
1-oxo-4-nitro-4-methylpentanoate
8c.
5-(2,2-Dimethylpropoxycarbonyl)-5-methyl-1-pyrroline N-oxide
8d. Flash chromatography (CH2Cl2–CH3OH, 18.5 : 1.5), 42%,
white solid, mp 50–51 ◦C; dH 0.96 (s, 9H), 1.74 (s, 3H), 2.13–2.23
(m, 1H), 2.57–2.86 (m, 3H), 3.90 (d, J 10.5, 2H), 6.97 (t, J 2.6, 1H);
dC 20.97, 25.83, 26.32, 31.54, 32.51, 75.13, 79.05, 134.36, 169.83.
Calcd for C11H19NO3, 0.1 H2O (213.28 g mol−1) C, 61.43; H, 9.00;
N, 6.51. Found C, 61.53; H, 8.96; N, 6.48%.
Binding constant calculations
EPR spectra were recorded at a fixed time after generation of
superoxide (within five minutes) and a constant nitrone con-
centration (25 mM) was used. The amount of cyclodextrin was
progressively increased (from 3 to 170 mM) to detect changes in
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the spectral pattern of the EPR signal characteristic of superim-
position between the free and included superoxide spin adducts.
Deconvolution of EPR spectra from EPR titrations using the two-
dimensional simulation software21 afforded stoichiometries and
binding constants of the nitroxide spin adducts toward DM-b-CD.
J. Vasquez-Vivar, H. Karoui, C. Nsanzumuhire, P. Marta´sek, P. Tordo
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1818–1823; (b) M. Lucarini, B. Luppi, G. F. Pedulli and B. P. Roberts,
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references therein.
Sodium L-ascorbate reduction
Reaction mixture of EMPO analogues (25 mM) with DTPA
(0.5 mM), HX (0.2 mM) and DM-b-CD (50 mM) were prepared
followed by addition of XOD (0.05 U mL−1) to start the trapping
reaction. When the maximum of EPR signal intensity was reached
(around 16 minutes), superoxide dismutase (SOD 50 U mL−1) was
added to the solution to stop the trapping of superoxide followed
by subsequent addition of sodium L-ascorbate solution (0.1 mM
final concentration).
X-Ray crystal structure determinations
Colourless prism type single crystals of compounds 6d and 9d
suitable for X-Ray diffraction were obtained by slow evaporation
of their solution in a pentane–Et2O mixture within two days.
Crystal data for BnMPO 6d. C13H15NO3, M = 233.26, mon-
˚
oclinic, a = 10.2450(2), b = 5.5640(10), c = 21.3930(6) A, b =
◦
3
˚
102.24(8) , U = 1191.75(5) A , T = 293(2) K, space group P21/c,
Z = 4, l(Mo-Ka) = 0.093 mm−1, 3901 reflections measured, 3514
unique, Rint = 0.036, final R = 0.058.‡
7 Y. Kotake and E. G. Janzen, Free Radical Res. Commun., 1990, 10,
103–108.
Crystal data for CMMPO 9d. C13H21NO3, M = 239.31,
orthorhombic, a = 9.5780(10), b = 7.9730(10), c = 35.1840(6)
8 (a) J. Michon and A. Rassat, J. Am. Chem. Soc., 1979, 101, 995–996;
(b) J. Michon and A. Rassat, J. Am. Chem. Soc., 1979, 101, 4337–4339;
(c) E. G. Janzen and Y. Kotake, J. Am. Chem. Soc., 1988, 110, 7912–
7913; (d) P. Franchi, M. Lucarini, E. Mezzina and G. F. Pedulli, J. Am.
Chem. Soc., 2004, 126, 4343–4354.
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10 (a) C. Ebel, K. U. Ingold, J. Michon and A. Rassat, Nouv. J. Chim.,
1985, 9, 479–485; (b) C. Ebel, K. U. Ingold, J. Michon and A. Rassat,
Tetrahedron Lett., 1985, 26, 741–744; (c) M. Okazaki and K. Kuwata,
J. Phys. Chem., 1985, 89, 4437–4440.
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Soc., Perkin Trans. 2, 1998, 1709–1714; (b) Y. Chen, H.-L. Chen, Q.-C.
Yang, X.-Y. Song, C.-Y. Duan and T. C. W. Mak, J. Chem. Soc., Dalton
Trans., 1999, 629–634; (c) D.-Y. Han, Z.-P. Bai and H.-L. Chen, Wuji
Huaxue Xuebao, 1999, 15, 507–508; (d) X. Song, Y. Chen and H. Chen,
New J. Chem., 2001, 25, 985–988.
◦
3
˚
˚
A, b = 90.00 , U = 2686.84(6) A , T = 293(2) K, space group
Pbca, Z = 8, l(Mo-Ka) = 0.083 mm−1, 3360 reflections measured,
2912 unique, Rint = 0.061, final R = 0.0541.‡
Acknowledgements
The financial support of the Hungarian Scientific Research Fund
through grants OTKA T-046953 and NKFP 1/A/005/2004
“MediChem2” is gratefully acknowledged. The authors thank the
Conseil Re´gional Provence Alpes Coˆte d’Azur and TROPHOS
Company for financial support. The Socrates program is also
acknowledged for partial financial support of K. Banaszak
and I. Biskupska. Sandrine Lambert, Dr Michel Giorgi, Dr
Sylvain Marque and Dr Robert Faure are thanked for various
contributions. DMPO was generously provided by Radical Vision
(Marseille).
12 D. Bardelang, J.-L. Clement, J.-P. Finet, H. Karoui and P. Tordo,
J. Phys. Chem. B, 2004, 108, 8054–8061.
13 (a) K. A. Connors, Binding Constants: the measurement of molecular
complex stability, John Wiley and Sons, New York, 1987; (b) K. A.
Connors, Chem. Rev., 1997, 97, 1325–1357.
14 H. J. Schneider, F. Hacket, V. Ru¨diger and H. Ikeda, Chem. Rev., 1998,
98, 1755–1785.
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Lin, B. Perly and D. Wouessidjewe, J. Pharm. Sci., 1990, 79, 643–646.
16 L. Fielding, Tetrahedron, 2000, 56, 6151–6170.
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