NMR and EPR studies of the reaction of nucleophilic addition of (bi) sulfite to the nitrone spin trap DMPO

Article abstract of DOI:10.1002/mrc.1226

The reaction of the nitrone spin trap 5,5-dimethylpyrroline-N-oxide (DMPO) with sodium (bi)sulfite in aqueous solutions was investigated using NMR and EPR techniques. Reversible nucleophilic addition of (bi)sulfite anions to the double bond of DMPO was observed, resulting in the formation of the hydroxylamine derivative 1-hydroxy-5,5-dimethylpyrrolidine-2-sulfonic acid, with characteristic ¹H and ¹³C NMR spectra. The reaction mechanism was suggested and corresponding equilibrium constants determined. The mild oxidation of the hydroxylamine results in the formation of an EPR-detected spectrum identical with that for the DMPO adduct with sulfur trioxide anion radical. The latter demonstrates that a non-radical addition reaction of (bi)sulfite with DMPO may contribute to the EPR detection of SO₃ ^-? radical. This possibility must be taken into account in spin trapping analysis of sulfite radical. Copyright

Full text of DOI:10.1002/mrc.1226

MAGNETIC RESONANCE IN CHEMISTRY
Magn. Reson. Chem. 2003; 41: 603–608
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mrc.1226
NMR and EPR studies of the reaction of nucleophilic
addition of (bi)sulfite to the nitrone spin trap DMPO
Dmitrii I. Potapenko,1 Elena G. Bagryanskaya,1 Vladimir V. Reznikov,2 Thomas L. Clanton3
and Valery V. Khramtsov3,4∗
1
International Tomography Center, Novosibirsk 630090, Russia
Institute of Organic Chemistry, Novosibirsk 630090, Russia
Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio 43210, USA
Institute of Chemical Kinetics and Combustion, Novosibirsk 630090, Russia
2
3
4
Received 10 March 2003; Revised 30 April 2003; Accepted 1 May 2003
The reaction of the nitrone spin trap 5,5-dimethylpyrroline-N-oxide (DMPO) with sodium (bi)sulfite in
aqueous solutions was investigated using NMR and EPR techniques. Reversible nucleophilic addition
of (bi)sulfite anions to the double bond of DMPO was observed, resulting in the formation of the
1
hydroxylamine derivative 1-hydroxy-5,5-dimethylpyrrolidine-2-sulfonic acid, with characteristic H and
13C NMR spectra. The reaction mechanism was suggested and corresponding equilibrium constants
determined. The mild oxidation of the hydroxylamine results in the formation of an EPR-detected spectrum
identical with that for the DMPO adduct with sulfur trioxide anion radical. The latter demonstrates that
−ž
a non-radical addition reaction of (bi)sulfite with DMPO may contribute to the EPR detection of SO3
radical. This possibility must be taken into account in spin trapping analysis of sulfite radical. Copyright
 2003 John Wiley & Sons, Ltd.
KEYWORDS: NMR; 1H NMR; 13C NMR; EPR; free radicals; spin trapping; nitrones; sulfite-derived radicals
(DEPMPO).17–20 However, it has been shown that some
nucleophilic agents can directly react with nitrone spin
traps, forming corresponding hydroxylamines.21–23 These
compounds, in turn, can be easily oxidized, forming radical
adducts which are identical with those formed by genuine
spin trapping, the process termed the Forrester–Hepburn
mechanism.21 Moreover, we recently observed the reaction
of nucleophilic addition of (bi)sulfite [the term (bi)sulfite
is generally used to refer to both sulfite and bisulfite
species] to the DEPMPO spin trap, with the formation
of hydroxylamine compounds, which in turn are easily
INTRODUCTION
Sulfites are widely used as preservatives in food indus-
try and play an important role in metabolic processes.1–3
The main metabolic pathway of sulfite metabolism in vivo
is based on the function of the mitochondrial enzyme sul-
fite oxidase, present largely in the liver and lung tissues
and responsible for a two-electron oxidation of sulfite to
sulfate.4 The formation of free radical intermediates, par-
ꢀž
ꢀž
ticularly sulfur-centered SO3 and oxygen-centered SO4
radicals, during sulfite metabolism via one-electron oxi-
dation has been proposed to contribute to the mecha-
nisms of its toxicity.5–7 While electron paramagnetic res-
onance (EPR) spectroscopy provides an approach for the
direct detection of SO3 paramagnetic radical anion,8 this
approach is rarely possible in biological systems owing
to the short lifetime and low stationary concentration of
the radical.9 Therefore, EPR spin trapping is a commonly
ꢀž
oxidized to DEPMPO/SO3
radical adduct.24 Therefore,
ꢀž
SO3 detection using DEPMPO may involve non-radical
addition reactions and its application requires additional
controls to prove a radical mechanism of the adduct
formation.
ꢀž
In this work, we observed a similar reaction of (bi)sulfite
nucleophilic addition to another popular nitrone spin trap,
DMPO. The mechanism is proposed and corresponding
equilibrium constants were measured.
ꢀž
used method for the detection of SO3
radical, using
mostly nitrone spin traps, such as 5,5-dimethylpyrroline-
N-oxide (DMPO)6,7,10–16 and its phosphorus-containing ana-
log, 5-(diethoxy-phosphoryl)-5-methyl-pyrrolidine-N-oxide
EXPERIMENTAL
ŁCorrespondence to: Valery V. Khramtsov, Dorothy M. Davis Heart
and Lung Research Institute, The Ohio State University, 201
DHLRI, 473 W. 12th Avenue, Columbus, Ohio 43210, USA.
E-mail: khramtsov-1@medctr.osu.edu
Contract/grant sponsor: USPHS; Contract/grant number:
GM58772.
Reagents
DMPO (5,5-dimethyl-1-pyrroline-N-oxide) was purchased
from Sigma (St. Louis, MO, USA) and diethylenetriamine-
pentaacetic acid (DTPA) from Aldrich (Milwaukee, WI,
USA).
Contract/grant sponsor: Russian Foundation for Basic Research;
Contract/grant number: 02-04-48374.
Copyright  2003 John Wiley & Sons, Ltd.

604
D. I. Potapenko et al.
Sample preparations
H2O
6
a
Freshly prepared DMPO solution in D2O, containing 0.2 mM
DTPA, was bubbled with Ar during 1 h, then sodium sulfite
was added. All solutions were titrated with HCl solution up
to required pD value. For pD measurements an OP-211/1
laboratory digital pH-meter with a WTW SenTix 61 pH
combination glass electrode was used. The pD value was
calculated according to the equation25 pD D pHobs C 0.4,
H3C
3
2
4
H
5
6
N
O
H3C
H3C
7
C5
4
CH2
H3C
1
3
where pHobs is the observed pH value. H NMR and EPR
CH2
7
2
H
C
spectra of the samples were registered. In the studies on
sulfite concentration dependence, 0.2 M KCl was added to
prevent variations in ionic strength.
8
7
6
5
4
3
2
1
0
1H- and 13C-NMR spectra
ppm
The NMR spectra were registered on Bruker Avance 200
spectrometer in 5 mm tubes.
b
ESR spectroscopy
ESR spectra were measured on a Bruker EMX spectrometer
in a 0.1 ml quartz tube. The instrument conditions were as
follows: 50 scans, modulation amplitude 0.5 G, time constant
0.64 ms, conversion time 10.24 ms, sweep width 120 G, sweep
time 10.49 s and microwave power 0.625 mW.
UV spectrophotometry
UV spectra were registered on a Shimadzu UV-1202
UV–visible spectrophotometer in quartz cuvettes of 1.0 cm
thickness. The kinetic decay of ferricyanide was determined
by measuring the absorption at 420 nm.
8
7
6
5
4
3
2
1
0
ppm
Figure 1. (a) 1H NMR spectrum of DMPO in D2O. (b) 1H NMR
spectrum of the reaction mixture containing 35 mM DMPO,
100 mM Na2SO3 and 0.2 mM DTPA. A solution of DMPO and
DTPA in D2O was bubbled with Ar for 1 h. After bubbling, solid
Na2SO3 was added. The mixture was titrated with HCl to
pHobs D 7.1 and then the NMR spectrum was registered. The
concentration of the product, P, was determined from the
integral intensity of the C6H3 and C7H3 peaks. The arrows
point to the shift of the signal of the N CH proton from
7.22 ppm in the parent nitrone to 4.05 ppm in the product, and
to appearance of two signals, at 1.09 and 1.21 ppm, from
non-equivalent C6H3 and C7H3 groups in the product.
RESULTS AND DISCUSSION
1
Figure 1 shows the H NMR spectra of DMPO (a) and the
reaction mixture of 35 mM DMPO and 100 mM Na2SO3 (b).
The main difference between the spectra is the large shift of
the signal of the N CH proton from 7.22 to 4.05 ppm,
which indicates disappearance of the double bond. The
doublet–doublet splitting of this proton signal [Fig. 1b]
can be explained by the non-equivalence of the protons
of the C3H2 group in the observed reaction product (P).
The existence of two signals from methyl groups (1.09
and 1.21 ppm) indicates non-equivalence of the C6H3 and
C7H3 groups. The 13C NMR shift of C2 (see Appendix) is
in the range of a carbon atom covalently bound with two
heteroatoms.
Figure 3 demonstrates the changes in the 1H NMR
spectrum of the reaction mixture containing 37 mM DMPO
and 100 mM sodium sulfite after addition of benzaldehyde,
which removes (bi)sulfite from reaction mixture. This leads
to the disappearance of the signals of the reaction product P
with a corresponding increase in the intensity of the DMPO
peaks. Therefore, the reaction of DMPO with sodium sulfite
is reversible, again in agreement with a similar conclusion
for the DEPMPO spin trap.24
The EPR spectrum of the reaction mixture of DMPO
and sulfite prepared as described in Fig. 1(b) did not show
any EPR signal (data not shown). The addition of 3 mM
potassium ferricyanide to the reaction mixture containing
35 mM DMPO and 100 mM Na2SO3 results in the appearance
of an EPR spectrum (Fig. 2) with hyperfine interaction
constants, aN D 14.5 G and aH D 16.06 G, which coincide
The strong dependence of the concentration of the
reaction product P on the acidity of the solution was observed
with decreases in [P] at higher pHobs (Table 1).
with the parameters of the radical adduct of DMPO with
ꢀž 6,11
While this compound can be formed by genuine
SO3
.
Based on all the data above, a mechanism of the reaction
of DMPO with (bi)sulfite is proposed similar to that for
the reaction of DEPMPO with (bi)sulfite,24 and is shown in
spin trapping of sulfite radical, the experiment shows that
oxidation of the reaction product results in the formation of
the same compound.
Copyright  2003 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2003; 41: 603–608

Nucleophilic addition of (bi)sulfite to nitrones
605
H2O
a
20 G
Figure 2. EPR spectrum of reaction mixture containing DMPO,
35 mM, sodium sulfite, 100 mM, and oxidant, K3Fe(CN)6, 3 mM
in D2O, 0.2 mM DTPA, pHobs D 7.1. Spectrometer settings
were as follows: microwave power, 0.625 mW; modulation
amplitude, 0.5 G; sweep time, 10.49 s; number of scans, 50.
Computer simulation of the spectrum gives the parameters
aN D 14.5 G and aH D 16.06 G.
9
8
7
6
5
ppm
4
3
2
1
Eqns (1)–(5).
b
K1
−
SO3
H
2−
+ SO3
H
N
O
N
P−
O−
DMPO
ꢀ1ꢁ
K2
SO3D
H
−
H
+ DSO3
9
8
7
6
5
ppm
4
3
2
1
N
O
N
O−
DMPO
P′
Figure 3. (a) 1H NMR spectrum of reaction mixture containing
37 mM DMPO and 100 mM sodium sulfite in D2O at
pHobs D 6.62 [cf. Fig. 1(b)]. (b) The same as (a) after addition of
190 mM benzaldehyde and titration of the reaction mixture to
pHobs D 6.6.
ꢀ2ꢁ
Ka
P0
P
ꢀ C DC
ꢀ3ꢁ
ꢀꢀꢀ!
ꢀꢀꢀ
−
Kb
SO3
P−
+
D+
trap, respectively; [Na2SO3]0 is the initial sodium sulfite
H
ꢀ4ꢁ
ꢀ5ꢁ
ꢀ
concentration; KaS is the acidity constant for the DSO3
N
anion; and Ka and Kb are acidity and basicity constants
for corresponding hydroxylamine compounds in D2O.
Equation (6) explains the strong dependence of the product
concentration on pHobs (Table 1). Fitting of the experimental
values to Eqn (6) shows good agreement between [P]
values obtained experimentally and those calculated using
pKaS D 7.6 and K0 D 18 l molꢀ1 (see Table 1, [DC] values
were calculated from pHobs as described in the Experimental
section). The observed difference between pKaS values of the
OD
P
KaS
ꢀ
DSO3
SO32ꢀ C DC
ꢀꢀꢀ!
ꢀꢀꢀ
The reaction scheme considers three different ioniza-
tion states of the DMPO adduct with sulfite, Pꢀ, P0
and P. The hydroxylamine derivative, P (1-hydroxy-5,5-
dimethylpyrrolidine-2-sulfonic acid anion), is the predomi-
nant form (pKb ³ 1012 –1014 l molꢀ1 26
, and degree of dissoci-
HSO3 anion in H2O (7.2)28 and DSO3 in D2O (7.6 š 0.2) is
in good agreement with the fact that for the most ionizable
groups, pK values for ionization with loss of a deuteron in
D2O are higher by ¾0.4–0.6 units than those for ionization
of the same groups with loss of proton in H2O.29
ꢀ
ꢀ
ation of sulfonic group, ˛ ³ 127).
According to Eqns (1)–(5) the product of equilibrium
constants, K0 D K1KbKaS D K2KbKa, can be calculated from
experimentally measured parameters:
The value of K0 (being a product of the equilibrium
constants) must be independent of variations in the con-
centrations of DC, DMPO and sulfite. This is in agreement
with the data shown in Table 1. The values of K0 were
calculated using pKaꢀDSO3ꢀꢁ D 7.6 and are in the range
K0 D K1KbKaS D K2KbKa
KaS C [DC]
[P]
D
ꢀ6ꢁ
[DC] ꢀ[Na2SO3]0 ꢀ [P]ꢁ[DMPO]
where [P] and [DMPO] are NMR-detected equilibrium
concentrations of the reaction product P and DMPO spin
18 š 2 l molꢀ1
.
Copyright  2003 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2003; 41: 603–608

606
D. I. Potapenko et al.
Table 1. Equilibrium concentrations of the product, P, and spin trap, DMPO, measured
by 1H NMR spectroscopy 1 h after the mixing of argon-bubbled solutions of 19 mM
°
DMPO and 63 mM sodium sulfite in D2O, 0.2 mM DTPA at 23 C and various pHobs
pHobs
[P] (mM)
[P]a
(mM)
[DMPO] (mM)
log[K0 (l molꢀ1)]a
calc
8.07 š 0.05
7.61 š 0.05
6.90 š 0.05
6.60 š 0.05
6.10 š 0.05
5.61 š 0.05
5.09 š 0.05
4.64 š 0.05
2.0 š 0.1
4.0 š 0.2
6.7 š 0.3
8.6 š 0.4
9.2 š 0.5
9.6 š 0.5
9.6 š 0.5
9.7 š 0.5
2.0
17.0 š 1.7
15.0 š 1.5
12.3 š 1.2
10.5 š 1.0
9.7 š 1.0
9.4 š 1.0
9.4 š 1.0
9.3 š 1.0
1.20 š 0.23
1.21 š 0.20
1.24 š 0.13
1.28 š 0.07
1.29 š 0.03
1.30 š 0.02
1.29 š 0.01
1.30 š 0.01
4.1
6.8
8.5
9.3
9.6
9.7
9.7
a Calculated parameters were obtained by the fitting experimental data to Eqn (6).
20.0
16.0
12.0
8.0
30
28
26
24
22
20
18
16
4.8
4.4
4.0
3.6
3.2
K
4.0
0.0
3.30 3.35 3.40 3.45 3.50 3.55 3.60 3.65 3.70
0.0
0.4
0.8
1.2
1.6
2.0
2.4
2.8
1/T ⋅103, K-1
[DMPO](C0Na SO -[P])⋅103, M2
2
3
Figure 5. Dependences of equilibrium product concentration,
Figure 4. Dependences of the equilibrium product
concentration, [P], on initial spin trap and sulfite
[P] ( , left side), and constant K0 ( , right side) on inverse of
ꢁ
°
temperature. The experimental concentrations were as follows:
concentrations. (ꢀ) Dependence of [P] on sulfite concentration;
solution of 20 mM DMPO, 0.2 mM DTPA and 0.2 M KCl in D2O
was bubbled with Ar for 1 h and solid sodium sulfite was then
added; ( ) Dependence of [P] on DMPO concentration;
DMPO, 37 mM; Na2SO3, 100 mM; DTPA, 0.2 mM; pHobs D 6.62.
The linear approximation for K0 represents fitting of the
experimental data to the equation ln K0 D A C Bꢀ1/Tꢁ, yielding
parameters A D ꢀ11.5 š 0.5 and B D ꢀ7.37 š 0.13ꢁ ð 103 K.
°
solution of DMPO, 0.2 mM DTPA in D2O was bubbled with Ar
for 1 h and solid sodium sulfite, 100 mM, was then added. The
ln K0 D A C Bꢀ1/Tꢁ, with parameters A D ꢀ11.5 š 0.5 and
B D ꢀ7.37 š 0.13ꢁ ð 103 K. Taking into account Eqn (6),
we obtain:
samples were titrated with HCl to pHobs D 7.1 and then 1
H
NMR spectra were registered. The concentration of the
product, [P], was determined from the integral intensity of the
C6H3 and C7H3 peaks.
r1H
RT
r2H
RT
r1S
R
r2S
R
ln K1 D ln K0 ꢀ ln KbKaS D ꢀ
ln K2 D ln K0 ꢀ ln KbKa D ꢀ
C
C
ꢀ7ꢁ
Equation (6) predicts that the dependences of the NMR-
detected product P on the initial concentrations of sodium
sulfite and DMPO should be linear in coordinates of
[P] versus ꢀ[DMPO]0 ꢀ [P]ꢁꢀ[Na2SO3]0 ꢀ [P]ꢁ. To confirm
this statement, the dependences of the P yield on the
initial concentrations of DMPO and sulfite were studied at
pHobs D 7.1 using 1H NMR spectroscopy and the results
are shown in Fig. 4. The observed linearization of the
experimental dependences in the performed coordinates
further supports the reaction scheme.
where r1H, r2H and r1S, r2S are the standard enthalpies
and entropies of reactions (1) and (2), respectively. These
thermodynamic characteristics can be evaluated using the
fitting parameters A and B:
r1H D r2H D ꢀ36.3 š 1.1 kJ
r1S D ꢀ95.5 š 4.7 J Kꢀ1 ꢀ R ln KbKa0
r2S D ꢀ95.5 š 4.7 J Kꢀ1 ꢀ R ln KbKaS
ꢀ8ꢁ
Figure 5 represents the temperature dependence of the
experimentally measured concentration of the product P
and constant K0 calculated using Eqn (6). The dependence
of ln K0 on 1/T is in good agreement with linear fit,
In the presence of an oxidizing agent, sulfite and
DMPO, two processes, both genuine spin trapping and
Copyright  2003 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2003; 41: 603–608

Nucleophilic addition of (bi)sulfite to nitrones
607
and hydroxylamine P as kf1 D 0.2 l molꢀ1 sꢀ1 and kf2
1.7 l molꢀ1 sꢀ1, respectively.
D
0.8
0.6
0.4
0.2
0.0
CONCLUSION
The reversible nucleophilic addition of (bi)sulfite to the
double bond of the nitrone spin trap DMPO resulting in
the formation of a hydroxylamine compound was observed.
The assignment of the product and establishment of the
mechanism of its formation were based on EPR and NMR
data, and corresponding thermodynamic parameters of the
reactions were obtained. Mild oxidation of the observed
hydroxylamine results in the formation of nitroxide radical
0
5
10
15
20
25
Time, min
which is identical with the paramagnetic adduct formed
ꢀž
by genuine EPR spin trapping of SO3
radical by the
Figure 6. Decay of K3[Fe(CN)6] in the reaction with sulfite in
the presence and absence of DMPO in argon-bubbled
phosphate buffer, pH 7.0 (0.5 mM DTPA). (ꢂ) 0.71 mM
K3[Fe(CN)6] and 14.3 mM DMPO; (ꢀ) 0.71 mM K3[Fe(CN)6] and
4.84 mM Na2SO3; ( ) 0.71 mM K3[Fe(CN)6], 4.84 mM Na2SO3
DMPO spin trap. The results presented in this paper
ꢀž
show the necessity of more careful analysis of SO3
radical spin trapping data obtained in solutions containing
sulfite, spin trap and an oxidant. Note that the redox
potentials for sulfite anions are EꢀSO3ꢀ/SO32ꢀꢁ D 0.63 V,
EꢀSO3ꢀ/HSO3ꢀꢁ D 0.84 V vs NHE.29 The redox potential
values of the various hydroxylamines are in the range
0.3–1 V.30,31 Therefore it is expected that the sulfite-oxidizing
agent may oxidize the hydroxylamine product, P, interfering
with genuine the EPR spin trapping experiment as was
shown for ferricyanide.
°
and 14.3 mM DMPO. The absorption of ferricyanide at 420 nm
was measured. Solid lines represent exponential fitting of the
data yielding the characteristic time of the accumulation of the
ferricyanide decay, ꢂ1 D 17.53 š 0.18 min in the absence (ꢀ)
and ꢂ2 D 7.37 š 0.02 min in the presence ( ) of DMPO.
°
oxidation of hydroxylamine P, may take place, resulting in
ꢀž
the formation of the same radical adduct, DMPO–SO3
.
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ꢀž
the formation of the DMPO-SO3
adduct (detected by
EPR in the presence of potassium ferricyanide, Fig. 2), we
measured the kinetics of the reduction of K3[Fe(CN)6] by
sulfite in argon-bubbled phosphate buffer, pH 7.0, in the
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of DMPO with sulfite is much faster than its oxidation,
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dCꢀOxꢁ
D ꢀkf1[Na2SO3]CꢀOxꢁ ꢀ kf2[P]CꢀOxꢁ
ꢀ9ꢁ
dt
where C(Ox) is the concentration of K3[Fe(CN)6] and kf1
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exponential form:
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ꢀ10ꢁ
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Copyright  2003 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2003; 41: 603–608

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APPENDIX
Parameters of NMR spectra of 1-hydroxy-5,5-
dimethylpyrrolidine-2-sulfonic acid (P)
C13 NMR
C2: d, 79.35 ppm, J(C,H) D 149 Hz. C3: 21.65 ppm, 134 Hz <
J(C,H) < 136 Hz. C4: t, 32.90 ppm, 132 Hz < J(C,H) <
138 Hz. C5: 73.76 ppm. C6: q, 24.63 ppm, 127 Hz < J(C,H) <
125 Hz. C7: q, 17.06 ppm, 125 Hz < J(C,H) < 127 Hz.
28. Hayon E, Treinin A, Wilf J. J. Am. Chem. Soc. 1972; 94: 47.
29. Huie RE, Neta P. J. Phys. Chem. 1984; 88: 5665.
30. Thomas G, Mohanty JG. Indian J. Chem. 1982; 21A: 451.
31. Shchukin GI, Grigor’ev IA. In Imidazoline Nitroxides, vol. 1.
Volodarsky LB (ed.). CRC Press: Boca Raton, FL, 1988; 171.
1H NMR
C2H: d-d, 4.05 ppm. C3H2, C4H2: m, 1.5–2.5 ppm; C6H3,
C7H3: 1.21 ppm, 1.09 ppm.
Copyright  2003 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2003; 41: 603–608