15N CIDNP investigations of the peroxynitric acid nitration of l-tyrosine and of related compounds

Article abstract of DOI:10.1039/b515856g

Peroxynitric acid (O₂NOOH) nitrates l-tyrosine and related compounds at pH 2-5. During reaction with O₂¹⁵NOOH in the probe of a ¹⁵N NMR spectrometer, the NMR signals of the nitration products of l-tyrosine, N-acetyl-l-tyrosine, 4-fluorophenol and 4-methoxyphenylacetic acid appear in emission indicating a nitration via free radicals. Nuclear polarizations are built up in radical pairs [ ¹⁵NO₂, PhO]^F or [¹⁵NO₂, ArH⁺]^F formed by diffusive encounters of ¹⁵NO₂ with phenoxyl-type radicals PhO or with aromatic radical cations ArH⁺. Quantitative ¹⁵N CIDNP investigations with N-acetyl-l-tyrosine and 4-fluorophenol show that the radical-dependent nitration is the only reaction pathway. During the nitration reaction, the ¹⁵N NMR signal of ¹⁵NO₃ ^- also appears in emission. This is explained by singlet-triplet transitions in radical pairs [¹⁵NO₂, ¹⁵NO ₃]^S generated by electron transfer between O ₂¹⁵NOOH and H¹⁵NO₂ formed as a reaction intermediate. During reaction of peroxynitric acid with ascorbic acid, ¹⁵N CIDNP is again observed in the ¹⁵N NMR signal of ¹⁵NO₃^- showing that ascorbic acid is oxidized by free radicals. In contrast to this, O₂¹⁵NOOH reacts with glutathione and cysteine without the appearance of ¹⁵N CIDNP, indicating a direct oxidation without participation of free radicals. The Royal Society of Chemistry.

Full text of DOI:10.1039/b515856g

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
15N CIDNP investigations of the peroxynitric acid nitration of L-tyrosine
and of related compounds
Manfred Lehnig*a and Michael Kirsch*b
Received 8th November 2005, Accepted 4th January 2006
First published as an Advance Article on the web 20th January 2006
DOI: 10.1039/b515856g
Peroxynitric acid (O2NOOH) nitrates L-tyrosine and related compounds at pH 2–5. During reaction
with O215NOOH in the probe of a 15N NMR spectrometer, the NMR signals of the nitration products
of L-tyrosine, N-acetyl-L-tyrosine, 4-fluorophenol and 4-methoxyphenylacetic acid appear in emission
•
indicating a nitration via free radicals. Nuclear polarizations are built up in radical pairs [15NO2 ,
•
•
PhO•]F or [15NO2 , ArH•+]F formed by diffusive encounters of 15NO2 with phenoxyl-type radicals PhO•
or with aromatic radical cations ArH•+. Quantitative 15N CIDNP investigations with N-acetyl-L-
tyrosine and 4-fluorophenol show that the radical-dependent nitration is the only reaction pathway.
During the nitration reaction, the 15N NMR signal of 15NO3 also appears in emission. This is
−
•
•
explained by singlet–triplet transitions in radical pairs [15NO2 , 15NO3 ]S generated by electron transfer
between O215NOOH and H15NO2 formed as a reaction intermediate. During reaction of peroxynitric
acid with ascorbic acid, 15N CIDNP is again observed in the 15N NMR signal of 15NO3 showing that
−
ascorbic acid is oxidized by free radicals. In contrast to this, O215NOOH reacts with glutathione and
cysteine without the appearance of 15N CIDNP, indicating a direct oxidation without participation of
free radicals.
RNS seem to be peroxynitrite and nitrite in the presence of perox-
Introduction
ydase or hypochlorite.12,13 Concerning peroxynitrite, an indirect
Peroxynitric acid (O2NOOH/O2NOO−; pKa 5.9) is known as an
unstable intermediate during reaction of H2O2 with either N2O5
or HNO2 since about 100 years.1 It has found growing interest
after detection in the Earth’s atmosphere as the recombination
product of free radicals HO2 and NO2 .2 Furthermore, it may be
generated under physiological conditions as the recombination
product of superoxide, O2 (HO2/O2−; pKa 4.8), and NO2 .3
In living organisms, this reaction has been suggested to be an
effective detoxification pathway for NO2 .4 During the last years,
its formation and decomposition have been studied in greater
detail, see Scheme 1.5–9
•
nitration pathway via NO2 and tyrosinyl radicals is generally
accepted.14 Peroxynitric acid might also be considered as a possible
nitration agent of tyrosine residues in biological systems.4c,15
Nitration of L-tyrosine (Tyr) with peroxynitric acid has not
been observed at pH 7.4b The purpose of the present paper is
to look for nitration reactions with peroxynitric acid at lower
pH values. 15N CIDNP investigations during the nitration of
N-acetyl-L-tyrosine (Tyrac), 4-fluorophenol (4-F–C6H4OH) and
4-methoxyphenylacetic acid (4-MeO–C6H4–CH2–COOH) will be
described for the study of the nitration mechanism of peroxynitric
acid. In preceding communications, 15N CIDNP has been applied
in proving the radical mechanism of the nitration reactions of
L-tyrosine and N-acetyl-L-tyrosine with peroxynitrite at pH 4–5 as
well as with nitrite in the presence of peroxydase or hypochlorite
at physiological pH values.16
•
•
•
•
−
•
A few oxidizing reactions with peroxynitric acid have been
reported.9a,b They were proposed to occur via different mech-
anisms, a direct one and an indirect one via free radicals
formed during the decomposition of peroxynitric acid (Scheme 1).
For demonstrating the possibility of different mechanisms, 15N
CIDNP studies during oxidation of ascorbic acid, glutathione and
cysteine will be described, too. These compounds act as scavengers
for oxidants in biological systems, especially for free radicals.17
CIDNP is used for evaluating reaction mechanisms in organic
chemistry. It leads to emission (E) and/or enhanced absorption
(A) signals in the NMR spectra of products formed during fast
radical reactions running in the probe of an NMR spectrometer
and proves the occurrence of free radicals.18–21 Especially, 15N
CIDNP has been applied to study nitration reactions of activated
aromatics by nitric acid, nitrous acid and peroxynitrous acid.16,22,23
Scheme 1 Formation and decay reactions of peroxynitric acid (see
ref. 8b, 9c, 10 and 11).
Peroxynitric acid is expected to exhibit nitrating as well as oxi-
dizing properties. Tyrosine nitration and the nitration of tyrosine
residues in proteins are used as markers for the activity of reactive
nitrogen species (RNS) in living systems.12 The most important
aOrganische Chemie, Universita¨t Dortmund, Otto-Hahn-Strasse 6, D-44221
Dortmund, Germany. E-mail: lehnig@chemie.uni-dortmund.de
bInstitut fu¨r Physiologische Chemie, Universita¨tsklinikum Essen, Hufeland-
strasse 55, D-45117 Essen, Germany. E-mail: michael.kirsch@uni-essen.de
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CIDNP is generated in radical pairs formed by the homolysis of
diamagnetic compounds from singlet states (S pairs) or by diffusive
encounters of independently generated free radicals (F pairs).
Free radicals reacting within the pairs give cage (c) products.
Free radicals which do not react within the pairs form escape
(e) products. If 15N nuclei are observed, the phase of CIDNP
effects (E, A) in the reaction products of free radicals generated by
homolysis of diamagnetic compounds 15NO2–R and by reactions
Ii is the intensity of the CIDNP signal during the ith measurement
and Dt(i, i + 1) is the time interval (2 or 3 min) between the ith
and the (i + 1)th measurement. Io is the intensity of the 15N NMR
signal of the reaction product after finishing the reaction and T1
is the longitudinal relaxation time of the nucleus investigated. For
determining E, eqn (1) is a good approximation if the reaction
time exceeds T1 by more than about one order of magnitude. This
procedure cancels differences between various runs which can
therefore be compared directly. Values determined from different
runs differ by about 15%. Chemical shifts are given in d values
relative to nitrobenzene-15N dissolved in acetonitrile as an external
reference. 15N CIDNP experiments using nitric acid or nitrous
acid as nitrating agents were performed in an analogous manner.23
•
of 15NO2 with free radicals R• is given in Scheme 2 assuming
•
g(R•) > g(NO2 ).22
Materials
O215NOOH solutions (1.57 M) were freshly prepared prior to use
as described.4a O215NOOH was also prepared in situ by addition
of H2O2 (1 M) to a solution of Na15NO2 (0.3 M or 0.15 M) in
H2O.8c,15b All the other compounds and solvents were commercial.
Nitric acid was 0.4 M in H2O and labelled with 60.3 atom% 15N
(Isotec Inc.). NaNO2 was labelled with 99.3 atom% 15N (Isotec
Inc.).
Scheme 2 15N CIDNP effects in the reaction products of free radicals
•
15NO2 and R• generated by homolysis of diamagnetic compounds
15NO2–R (S pairs) and free radical encounters (F pairs) assuming g(R•) >
•
g(NO2 ). E: emission, A: enhanced absorption.
The appearance of CIDNP proves the occurrence of radical
reactions, which does not exclude non-radical reactions leading
to the same product. To prove this, quantitative experiments have
been performed. CIDNP intensities are proportional to the prod-
uct rate formation. For quantitative investigations, the dependence
of reaction time and product concentrations is eliminated by
determining an enhancement factor E which is the ratio between
the intensity of the NMR signal immediately after formation of
the polarized product and the intensity of the NMR signal of
the product after finishing the reaction.24 The value is compared
with 15N CIDNP data obtained from various nitrating systems17,23
and calculations based on quantitative formulations of the radical
pair theory.25 This procedure will be applied during reactions
of peroxynitric acid-15N with N-acetyl-L-tyrosine and with 4-
fluorophenol, not with L-tyrosine and 4-methoxyphenylacetic acid
because of low product concentrations.
Solutions
For preparing the buffer solutions, doubly distilled water was
bubbled (2 L min−1) with synthetic air at room temperature for
20 min. Traces of transition metal ions were removed from the
buffer solutions by treatment with the heavy metal scavenger resin
Chelex 100 by gentle shaking for 18 h in the dark.26 The pH value
was adjusted with sulfuric acid and sodium hydroxide using a pH
Meter CG 825.
Capillary zone electrophoresis measurement
L-Tyrosine and 3-nitro-L-tyrosine were quantified on a Beckman
P/ACE 5000 apparatus. Separation conditions for L-tyrosine and
3-nitro-L-tyrosine were as follows: fused silica capillary (50 cm
effective length, 75 m internal diameter), hydrodynamic injection
for 5 s, temperature 30 ◦C, voltage 18 kV, normal polarity, UV
detection at 214 nm. A mixture of 50 mM sodium phosphate,
25 nM sodium borate, and 50 mM sodium dodecyl sulfate (pH 9.0)
was used as the electrolyte system. To each sample, 0.2 mM of
p-hydroxybenzoic acid was added as an internal standard.
Experimental
15N CIDNP experiments
Authentic peroxynitric acid-15N or a mixture of Na15NO2 and
H2O2 was dissolved in H2O/D2O containing phosphoric acid
(0.3 M) and NaHCO3 (0.05 M) at pH 2. After putting the reactants
into the 10 mm NMR tube, it was transferred into the probe of
the 15N NMR spectrometer (Bruker DPX 300) within 5 s and then
locked. A single pulse spectrum of the peroxynitric acid was then
taken with a pulse angle of 90◦ 2 or 3 min later. After that, the
tube was replaced, and the reactant was added to the solution.
15N NMR spectra were then taken every 2 or 3 min until the
Quantum chemical calculations
Complete basis set (CBS-Q) computations were carried out with
the Gaussian 03 (Revision A.11.3) suite of programs.27 Molecular
interactions were evaluated on the optimized gas-phase geometries
with the PCM28a procedure incorporated in Gaussian 03. Both
the PCM/(U)HF/6-31(+)G(d) and the CBS-Q methodology are
known to provide estimates within “chemical accuracy” ( 1 kcal
mol−1), as has also been demonstrated for O2NOOH-derived
reactions.28b Isotropic absolute shielding constants of the nitrogen
nucleus in a couple of compounds were calculated with the gauge-
including atomic orbital (GIAO) protocol29 at the DFT/aug-
cc-pVDZ (DFT = B1LYP and B3LYP) level of theory. The
optimization of the structure and molecular interactions with the
solvent were respected at the same level of theory.
reaction was completed. For detecting the reaction products, 15
N
NMR spectra were taken with several hundred pulses. 15N NMR
intensities I were taken directly from the spectra. During single
runs, signal intensities are proportional to concentrations within
about 5%. Signal intensities taken during different runs differ
within about 20%. An E value was determined from eqn (1).23
E = RIiDt(i, i + 1)/IoT1
(1)
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Table 1 Effect of pH on nitration of L-tyrosine (1 mM) with peroxynitric
acid (1 mM)
pH
NO2–Tyr (lM)a
7
6
5
4
3
0b
0
13 1.2
101.5 5.6
118.5 6.2
a Determined using capillary zone electrophoresis (detection limit 8 lM).
b Recovery of L-tyrosine 96.1%.
Results and discussion
Nitration of L-tyrosine with peroxynitric acid
During reaction of peroxynitric acid with L-tyrosine (Tyr) in acidic
solution, the nitration product 3-nitro-L-tyrosine (3-NO2–Tyr) is
formed [eqn (2)]. The product yield increases with decreasing pH
values from zero at pH 7 to 118.5 mM at pH 3, see Table 1.
Tyr + O2NOOH → 3-NO2–Tyr + HOOH
(2)
The unprotonated form, which is present at pH 7, is not able to
nitrate Tyr.
15N CIDNP during decomposition of peroxynitric acid-15N at pH 2
Peroxynitric acid decomposes to nitric acid in acidic solution
(Scheme 1), half-life times of 30–60 min have been found.8a,c
A
typical 15N NMR spectrum taken during the decay of O215NOOH
in H2O is given in Fig. 1a. The time dependence of 15N NMR
signal intensities I of O215NOOH (0.54 M) and 15NO3− and details
of this reaction are given in the Tables 2 and 3. The assignment of
Fig. 1 15N NMR spectra of solutions of peroxynitric acid-15N in H2O at
pH 2 and 298 K taken (a) 3 min after putting the tube in the probe (1 pulse),
(b) 3 min after adding N-acetyl-L-tyrosine (1 pulse), (c) 300 min after
adding N-acetyl-L-tyrosine (500 pulses).
Table 2 15N CIDNP during reaction of O215NOOH with organics at pH 2 and 295 K
d (ppm)a
CIDNPb
Yield (%)c
t
(min)d
1
2
Reactant
Assignment
None15b (Fig. 1a)
O215NOOH (0.54 M)
−18
9
A
N
A
E
E
E
E
A
E
E
E
E
E
A
E
—
N
A
N
A
E
E
—
100
—
2.0
—
98.0
—
—
0.2
—
—
100
—
—
100
—
100
—
100
—
20
4
15NO3
−
N-Acetyl-L-tyrosine (0.2 M) (Fig. 1b,c)
O215NOOH (0.3 M)
3-15NO2–Tyrac
(?)
−18
4
6
9
15NO3
−
1-15NO2–Tyrac
18
−18
3
13/14
−2
9
4-Fluorophenol (0.1 M) (Fig. 3)
O215NOOHe
4
2-15NO2-4-F–C6H4OH
15
=
4- NO2-4-F–C6H4
O
3-NO2-4-F–C6H4OH
15NO3
−
(?)
4
Ascorbic acid (0.15 M) (Fig. 2a)
Glutathione (0.1 M)
O215NOOHf
−18
3
15NO3
9
−
O215NOOHf
−18
9
N. o.g
0.15
8
−
15NO3
Cysteine (0.1 M)
O215NOOHf
−18
15NO3
9
−
4-Methoxyphenylacetic acid (0.02 M) (Fig. 2b)
O215NOOHf
−18
3
8
3-15NO2-4-MeO-C6H4–CH2–COOH
<0.1
100
15NO3
−
a d values against Ph15NO2, positive d values downfield. b E: emission, A: enhanced absorption, N: no CIDNP. c Product yields determined from 15N
NMR spectra taken after reaction. d Half-life time of the 15NMR signal decay of O215NOOH. e Generated in situ from Na15NO2 (0.3 M) and H2O2 (1 M).
f Generated in situ from Na15NO2 (0.15 M) and H2O2 (1 M). g Not observed.
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Table 3 15N NMR intensities Ia (a) during decay of O215NOOH (0.54 M), (b) during reaction of O215NOOH (0.3 M) with N-acetyl-L-tyrosine (0.2 M),
(c) during reaction of O215NOOHb with 4-fluorophenol (0.1 M), (d) during reaction of O215NOOHc with ascorbic acid (0.15 M) at pH 2 and 295 K
(a)
td
0
400
10
3
400
14
6
360
17
12
300
25
18
240
41
28
150
53
40
80
55
60
33
67
100
17
67
300
0
70
I(O215NOOH)
I(15NO3
)
−
(b)
td
0
3
6
9
12
100
−55
−7
15
60
18
30
−5
−2
0
21
20
2
0
0
30
3
20
0
300
0
I(O215NOOH)
750
−310
−50
500
−200
−26
330
−150
−19
125
−70
−10
−3
I(15NO3
)
−22
40
−
I(3-15NO2–Tyrac)
−4
0.8e
I(1-15NO2–Tyrac)
−17
−12
−12
−3
−2
0
0
(c)
td
0
60
10
—
—
2
200
−140
−32
−4
4
150
−150
−26
−3
6
100
−70
−12
−2
8
60
−30
−5
0
10
30
−10
−3
0
12
15
4
14
9
15
0
18
3
25
0
22
0
40
0
300
0
I(O215NOOH)
I(15NO3
)
40
−
I(2-15NO2-4-F–C6H4OH)
−2
0.08f
15
=
I(4- NO2-4-F–C6H4 O)
0
0
0
0
0
(d)
td
0
40
5
2
250
−75
4
150
−30
6
30
−4
8
7
7
10
2
12
14
—
20
22
—
20
I(O215NOOH)
I(15NO3
)
−
a I values determined from the signal-to-noise ratios using single 90◦ pulses. b O215NOOH generated in situ from Na15NO2 (0.3 M) and H2O2 (1 M).
c O215NOOH generated in situ from Na15NO2 (0.15 M) and H2O2 (1 M). d t: time after starting the reaction (in min). The spectrum at t = 0 has been taken
before adding the reactant to the solution of O215NOOH. e Determined from ◦15N NMR spectra taken after reaction (625 scans, 90◦ pulses, delay time 2
min). f Determined from 15N NMR spectra taken after reaction (400 scans, 90 pulses, delay time 3 min).
the 15NMR signals is supported by results of quantum-chemically
calculated 15N chemical shifts, see Table 4.
decreases with a half-time of about 20 min too; the magnitude
of the effect is about the same during reaction. The reaction
is finished, 95% complete, in 100 min. The 15N CIDNP effect
has also been observed at higher pH values and is built up in
The 15N NMR signal intensity of 15NO3 increases from 10 to
−
70 units during reaction showing that it is proportional to the
•
15NO3 concentration. The half-life time of 20 min taken from
radical pairs [15NO2 ,•O2H]S (Scheme 3) formed during homolysis
−
the spectra is smaller than the values given in the literature (30–
60 min). We think that this is of no importance for the nitration
experiments. It follows that 15% of O215NOOH decomposed before
taking the first spectrum, and furthermore, that the intensity of
the 15N NMR signal of O215NOOH should be about 60 units
which is much less than the 400 units observed, indicating that
it shows enhanced absorption (CIDNP of A type, Scheme 2). It
of O215NOOH (Scheme 1).15b
•
The formation of O215NOOH by recombination of 15NO2 and
•
•
HO2 via radical pairs [15NO2 ,•O2H]F leads to an E type effect16b
which is not observed under the given conditions. The 15N NMR
signal of 15NO3 appears in emission at pH 3.1.15b This has been
−
explained by electron transfer between O215NOOH and O215NOO−
(Scheme 3), which has no importance at pH 2. The reaction of
Table 4 Quantum-chemically calculated isotropic absolute shielding constants and 15N chemical shifts (d, in ppm)
Isotropic shielding constantsa
Isotropic chemical shifts
Molecule
B1LYP
B3LYP
B1LYP
B3LYP
Expb
Nitrobenzene
NO3
O2NOOH
trans-ONONO2
−125.2
−134.9
−105.6
−90.6
−121.5
−130.2
−105.8
−92.0
0.0
9.7
−19.6
184.2
−34.6
0.7
0.0
8.7
−15.7
180.0
−29.5
0.0
0
9
−18
−
−309.4
−125.9
−142.2
−121.0
−103.3
−343.6
−344.6
−291.6
−72.3
−301.5
−121.5
−141.8
−117.0
−102.8
−344.3
−339.3
−283.1
−71.4
2-NO2-4-F–C6H4OH
4-NO2-4-F–C6H4
3
=
O
17.0
20.3
13/14
−2
3-NO2-4-F–C6H4OH
4-F–C6H4–O–NO2
2-ONO-4-F–C6H4OH
3-ONO-4-F–C6H4OH
4-F–C6H4–O–ONO
N2O5
−4.2
−21.9
218.4
219.4
166.4
−52.9
−4.5
−18.7
222.8
217.8
161.6
−50.1
a Isotropic absolute shielding constants were calculated using the GIAO protocol at the DFT/aug-cc-pVDZ/DFT/aug-cc-pVDZ level of theory. During
these calculations, solvation corrections (CH3CN for nitrobenzene, H2O for all others) with the PCM solvation model were performed at the same level
of theory. b Experimental values, see Table 2 and Fig. 1–3.
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15N NMR signal of O215NOOH is enhanced, the half-time of the
reaction is shortened from 20 to 4 min and the overall reaction
time (95% yield progress) from 100 to 18 min. After reaction, the
signal due to 3-15NO2–Tyrac can only be observed by taking a
large number of scans (Fig. 1c). By taking 170 scans, a yield of
2.0% has been determined in relation to the 15NO3 yield.
−
Nitric acid and nitrous acid are effective nitration agents
of Tyrac in acidic solution.16b,22d Both are formed during the
decomposition of peroxynitric acid (Scheme 1). With the aim of
excluding them as nitrating agents, experiments were performed
with H15NO3 (0.1 M) and Na15NO2 (0.3 M) at pH 2. Tyrac is not
nitrated under these conditions, and 15N CIDNP effects are not
observed either.
Scheme 3 15N CIDNP during formation and decay of peroxynitric
acid-15N.
The occurrence of 15N CIDNP indicates that the nitration
proceeds via free radicals. The 15N CIDNP effects are identical to
those using peroxynitrous acid-15N as the nitrating agent and are
O215NOOH with H15NO2 (Schemes 1 and 3) might lead to emission
in the 15N NMR signal of 15NO3− too, which is not observed under
the applied conditions either.
•
explained as described earlier by reactions of radical pairs [15NO2 ,
•
Tyrac•]F formed by diffusive encounters of 15NO2 and phenoxyl-
Reaction of peroxynitric acid-15N with N-acetyl-L-tyrosine and
4-fluorophenol
•
type radicals Tyrac•, (Scheme 4).16 NO2 is known to generate
•
radicals GlyTyr• very efficiently from GlyTyr;30 HO2 might
For elucidating the mechanism of the nitration reaction, 15N
CIDNP studies have been performed at pH 2. During the reaction
of O215NOOH with Tyr, emission has been observed in 3-15NO2–
Tyr, but the product yield is too low for quantitative studies.
Therefore, N-acetyl-L-tyrosine (Tyrac) has been used. 15N NMR
spectra taken during the reaction of O215NOOH (0.3 M) with Tyrac
(0.2 M) at 295 K and after reaction are given in Fig. 1b,c, details of
the reaction in Table 2. The 15N NMR signal of O215NOOH shows
enhanced absorption, as described, signals at d = 4 ppm and d =
18 ppm are due to 3-nitro-N-acetyl-L-tyrosine (3-15NO2–Tyrac)
and 1-nitro-N-acetyl-L-tyrosine (1-15NO2–Tyrac) and appear in
emission. Additionally, the 15N NMR signal of 15NO3− shows E, in
contrast to the results during the decay of O215NOOH. A signal at
d = 6 ppm could not be assigned. After reaction, only the 15N NMR
signals of 3-15NO2–Tyrac and 15NO3− are observed indicating that
1-15NO2–Tyrac and the unassigned product are unstable reaction
intermediates.
readily be oxidized by phenolic compounds.31a The conclusions
are supported by calculations of Gibbs energies of the reaction
•
•
of NO2 with phenol (8.7 kcal mol−1) and of HO2 with phenol
(1.4 kcal mol−1) (Table 5, entries 1 and 2).
Scheme 4 15N CIDNP during reaction of peroxynitric acid-15N with
N-acetyl-L-tyrosine.
The enhancement factor E of the nuclear polarization has been
determined using eqn (1), see Table 6. An E value of −1100 is
derived from the 15N NMR signals of 3-15NO2–Tyrac (Table 3b).
It is comparable with that found during nitration of Tyrac with
peroxynitrous acid-15N in the presence of sodium bicarbonate (E =
−1350).16b
The time dependence of the 15N NMR signals reveals further
details of the reaction (Table 3b). After addition of Tyrac, the
Table 5 Quantum-chemically calculated Gibbs energies and aqueous solvation energies
Reactiona
DRGg
DREsolv
DRGaq
b
c
d
Entry
•
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
PhOH + NO2 → PhO• + HNO2
7.6
1.1
2.7
7.2
8.7
1.4
•
PhOH + HO2 → PhO• + H2O2
−1.3
−25.9
−8.4
127.7
−7.4
−11.8
196.6
8.1
O2NOOH + HNO2 → H2O + N2O5
−18.6
−1.1
−12.1
−3.7
−6.2
50.0
5.4
14.4
64.7
29.6
12.9
4.7
•
•
O2NOOH + HNO2 → H2O + NO2 + NO3
7.3
•
•
+
−
NO2 + NO3 → NO2 + NO3
−139.8
•
•
e
O2NOOH + N2O3 → HNO2 + NO2 + NO3
3.7
•
•
f
O2NOOH + N2O3 → HNO2 + NO2 + NO3
5.6
O2NOOH + HNO2 + H2O → O2NOOH•− + NO2 + H3O+
−146.6
−2.6
−4.1
−95.0
−109.1
38.2
•
•
•
PhOCH3 + HO2 → PhOCH2 + H2O2
•
•
PhOCH3 + NO2 → PhOCH2 + HNO2
18.5
O2NOOH + PhOCH3 → O2NOOH•− + PhOCH3
159.7
138.6
−25.2
111.0
93.6
•
+
•
•
+
−
PhOCH3 + NO2 → PhOCH3 + NO2
PhOCH3 + NO+ → PhOCH3 + NO•
•
+
•
•
+
−
−
PhOCH3 + N2O5 → PhOCH3 + NO3 + NO2
−106.3
•
•
+
PhOCH3 + NO3 → PhOCH3 + NO3
−106.4
−12.9
a Thermodynamic properties were calculated using the complete basis set (CBS-Q) methodology. b Gas phase data (kcal mol−1). c Solvation corrections
from (U)HF/6-31(+)G(d)//CBS-Q single point calculations with the PCM-UAHF solvation model for water (kcal mol−1). d DRGaq = DRGg + DRGsolv
(kcal mol−1). e trans-trans-ONONO. f cis-trans-ONONO.
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Table 6 Enhancement factors E of 15N CIDNP in nitration products of N-acetyl-L-tyrosine and 4-fluorophenol
Compound
Nitrating agent
T1 value (s)
E value
Reference
−
3-NO2–Tyrac
O15NOOCO2
24
24
96
96
96
96
96
−1350
−1100
−1090
−1202
−890
16b
O215NOOH
H15NO2
This work
23d
2-NO2-4-F–C6H4OH
H15NO3
23d
O15NOOH
16a
−
O15NOOCO2
−1110
−1250
−1222a
16a
O215NOOH
This work
23d
a Calculated following Pedersen’s treatment of the radical pair theory.25d
The nitration of 4-fluorophenol (4-F–C6H4OH) has been inves-
tigated by 15N CIDNP using different nitrating agents like nitrous
acid and nitric acid,23d peroxynitrous acid and its CO2 adduct.16a
Additionally, an E value has been calculated using quantitative
treatments of the radical pair theory.23 Furthermore, the nitration
reaction of 4-F–C6H4OH by nitrous acid has been thoroughly
investigated.31b
O215NOOH has been generated in situ using Na15NO2 (0.3 M)
and H2O2 (1 M). After that, 4-F–C6H4OH has been added to the
solution. The 15N CIDNP effects are shown in Fig. 2 and described
in the Tables 2 and 3. They are similar to those observed during the
reaction of O215NOOH with Tyrac. After adding 4-F–C6H4OH,
the intensity of the 15N NMR signal of O215NOOH is enhanced
by a factor of 3, the half-life time for the decay of peroxynitric
acid is 4 min and the reaction time 15 min (at 95% completion).
The enhancement is smaller than during reaction with authentic
peroxynitrite-15N which might be the consequence of a O215NOOH
formation yield of less than 50% under the given conditions. The
15NNMRsignalof 15NO3− alsoappearsinemission. The15NNMR
spectra of the stable product 2-nitro-4-fluorophenol (2-15NO2-4-F–
From 15N NMR spectra taken after reaction, the yield of 2-15NO2−-
4-F–C6H4OH has been determined to be 0.2% in relation to 15NO3
indicating that nitration of 4-F–C6H4OH with O2NOOH is only a
side reaction.
The 15N CIDNP effects in the nitration products are explained
in an analogous manner as described23d (Scheme 5). From the time
dependence of the 15N NMR signal of 2-15NO2-4-F–C6H4OH,
an E value of −1250 is deduced which agrees well with those
found during nitration reactions with nitrous acid, nitric acid and
peroxynitrous acid with and without bicarbonate as well as a
calculated one using Pedersen’s formulation of the radical pair
theory25d (Table 6).
Scheme 5 15N CIDNP during reaction of peroxynitric acid-15N with
4-fluorophenol.
C6H4OH) and of the intermediate 4-nitro-4-fluorocyclohexadien-
15
=
1-one (4- NO2-4-F–C6H4 O) show emission as was observed
earlier.16,23 Additionally, two small emission lines at d = −2 and
d = 4 appear. The first one is tentatively assigned to 3-15NO2-
4-F–C6H4OH. After reaction, only the 15N NMR signals due to
•
Recombination of free radicals NO2 and 4-F–C6H4O• might
lead to 4-F–C6H4–O–NO2 or nitrite-type products like 4-F–C6H4–
O–ONO, 2-ONO-4-F–C6H4OH, 3-ONO-4-F–C6H4OH and 4-
15NO3 and 2-15NO2-4-F–C6H4OH are observed. The assignment
−
of the 15N NMR signals is supported by calculating the 15N NMR
chemical shifts with the gauge-including atomic orbitals method
performed at the DFT/aug-cc-pVDZ level of theory (Table 4).
=
ONO-4-F–C6H4 O. The unassigned signal at d = 4 ppm might
be due to one of these products. Calculations of the 15N chemical
shift values show that this can be excluded, see Table 4.
The enhancement of the A type effect observed in the 15N
NMR signal of O215NOOH in the presence of Tyrac and 4-F–
•
C6H4OH is explained by scavenging of free radicals 15NO2 and
•
HO2 (Schemes 4 and 5) which cancels the E type polarization
•
built up in radical pairs [15NO2 ,.O2H]F formed by free radical
•
•
encounters of 15NO2 and HO2 (Scheme 3).
−
The E type effect in the 15N NMR signal of 15NO3 has not
been observed in the absence of phenolic compounds and must
therefore be due to a reaction of O215NOOH with Tyrac and 4-F–
C6H4OH or one of the reaction products (Schemes 4 and 5). It will
be explained as a consequence of the reaction of O215NOOH with
H15NO2 which is a reaction intermediate. This reaction is described
in the literature.8a Nuclear polarization is generated in radical pairs
•
•
[15NO2 , 15NO3 ]S formed by disproportionation between H15NO2
and O215NOOH followed by electron transfer between radicals
15NO2 and 15NO3 (Scheme 3). 15N2O5 might be an intermediate
of the reaction, since it has been reported that the dissociation
•
•
Fig. 2 15N NMR spectrum of peroxynitric acid-15N in H2O at pH 2 and
298 K taken 4 min after adding 4-fluorophenol to the solution (1 pulse).
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of N2O5 in aqueous solution to NO2 and NO3 (k > 104 s−1) is
about 5 times faster than the direct hydrolysis reaction.32 However,
no signal could be observed which might be assigned to N2O5
(Table 4). In any case, NO2+ is not stable under these conditions.33
Following this interpretation, the nuclear polarization in the 15N
+
−
•
nuclei of 15NO3− is of c type giving emission (Scheme 2, R = NO3 )
15
•
•
because of g(NO3 ) > g(NO2 ).34 An e type polarization built up
•
in radical pairs [15NO2 ,.O2H]S might be transferred to 15NO3 by
−
•
radicals 15NO2 leading to emission in the 15N NMR signal of
15NO3− too. 15N CIDNP effects of this type have been observed.16c
However, they are much weaker than c type effects.16c,24b
The explanation is supported by the results of quantum
chemical calculations (Table 5). The reactions of HNO2 with
•
•
O2NOOH leading to either N2O5 or NO2 and NO3 are predicted
•
•
to be exergonic as well as the reaction of NO2 with NO3
(entries 3–5). From an energetical point of view, the reaction
of O2NOOH with N2O3, which is always present in solutions
•
•
containing nitrous acid, might give radical pairs [NO2 , NO3 ]S
too (entries 6 and 7). A possible contribution of N2O3 to the
radical pair formation cannot be proven but it follows from the
equilibrium constant of the reaction between nitrous acid and its
corresponding anhydride (3•10−3 M−1) that the concentration of
N2O3 is some orders of magnitude lower than the concentration
of HNO2.35 The reaction of O215NOOH with H15NO2 might form
Fig. 3 15N NMR spectra of solutions of peroxynitric acid-15N in H2O
at pH 2 and 298 K taken (a) 3 min after adding ascorbic acid (1 pulse),
(b) 3 min after adding 4-methoxyphenylacetic acid (1 pulse).
•
•
radicals O215NOOH•− and 15NO2 leading to radical pairs [15NO2 ,
•
O215NOOH −]S and emission in the 15N NMR signal of 15NO3
.
−
After 6 min, the 15N NMR signal of 15NO3− changes from emission
to absorption. 9 min later, the 15N NMR signal of O215NOOH
disappears. The 15N CIDNP effects observed in the presence of
ascorbic acid are explained as described (Schemes 2 and 6).
The kinetic constants of the scavenging reactions are known
(Scheme 6). This allows us to compare the 15N CIDNP effects with
We reject this possibility because of energetic reasons (entry 8).
The 15N NMR signal of 15NO3 is generally expected to show
emission during reaction of O215NOOH with reducing agents
−
•
•
if free radicals 15NO2 and/or HO2 are involved. For proving
this, reactions of O215NOOH with the radical scavengers ascorbic
acid, glutathione and cysteine have been studied. Qualitative 15
•
N
the progress of the reaction. HO2 should be scavenged efficiently
CIDNP investigations during reactions of these compounds with
O215NOOH will be described. Nitration reactions of activated
non-phenolic aromatics might occur via free radicals too.22,23
To set about proving this, the reaction of O215NOOH with 4-
methoxyphenylacetic acid has been performed in the probe of
a 15N NMR spectrometer too.
by ascorbic acid at pH 2, and the half-life time of O2NOOH should
therefore be about 0.7 min (Schemes 1 and 6). The reaction should
be finished before taking the first spectrum after adding ascorbic
acid to the solution. The time behaviour of the 15N NMR signal
of 15NO3− is therefore caused by nuclear relaxation (T1 = 140 s 37
)
and not by the reaction. We think that this is also the case for the
decay of the signal of O215NOOH. The relaxation time of the 15
N
nucleus is unknown; it should be of the same order of magnitude
(T1 = 140 s).
Reaction of peroxynitric acid-15N with ascorbic acid, glutathione
and cysteine
The reaction of O215NOOH with glutathione is finished before it
is possible to take a 15N NMR spectrum. It is much faster than the
radical decay of O215NOOH. It follows that a direct oxidation
without participation of free radicals occurs. During reaction
of O215NOOH with cysteine, a spectrum could be taken before
peroxynitric acid-15N completely disappeared. The 15N NMR
Ascorbic acid AH2 and its anion AH− (pKa 4.25) are known to
•
•
react with HO2 as well as with NO2 to give the dehydrogenated
radical AH• (pKa −0.45) and dehydroascorbic acid A (Scheme 6).36
It should therefore be oxidized by peroxynitric acid in an indirect
manner. After adding ascorbic acid to a solution of O215NOOH in
H2O at pH 2, the 15N NMR signals of O215NOOH and of 15NO3
−
signal of 15NO3 does not show emission during this reaction.
−
show A and E as described during reaction of O215NOOH with
phenolic compounds. A 15N NMR spectrum taken 3 min after
starting the reaction of O215NOOH with ascorbic acid is shown in
Fig. 3a, the assignment of the signals and their time dependencies
in Tables 2 and 3. Additional 15N CIDNP signals are not observed.
It is concluded that there is a direct oxidation of glutathione
and cysteine by peroxynitric acid, too. More detailed conclusions
concerning the reaction mechanism cannot be drawn. Radical
reactions as described before seem not to be of any importance.
Reaction of peroxynitric acid-15N with 4-methoxyphenylacetic acid
During reaction of O215NOOH with 4-methoxyphenylacetic acid
(MeO–C6H4–CH2–COOH) the 15N NMR signals of O215NOOH
and 15NO3 appear in A and E, respectively, and the decay rate
of O215NOOH is enhanced (half-life time 8 min) as was observed
−
Scheme 6 Reaction of peroxynitric acid-15N with ascorbic acid.
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during reaction of peroxynitric acid with Tyrac, 4-F–C6H4OH
and ascorbic acid. Additionally, an emission signal is observed
at d = 3, see Fig. 3b and Table 2, which is assigned to 3-
nitro-4-methoxyphenylacetic acid (3–15NO2-4-MeO–C6H4–CH2–
COOH). After reaction, the product could not be observed by
15N NMR spectroscopy, indicating a product yield <0.1% in
relation to 15NO3−. At the beginning, the concentration of MeO–
C6H4–CH2–COOH is much smaller than that of peroxynitric acid.
systems is favoured over the electron transfer.22b,23a NO+, N2O5
and NO3 should be capable of oxidizing anisole (Table 5, entries
•
13–15) and one of them might be responsible for the formation of
ArH+. NO+ is the oxidating species under strong acidic conditions
using nitrous acid or nitric acid as nitrating agents.22a It might also
•
be formed in slow concentrations at pH 2. N2O5 and NO3 are
postulated to be present under our conditions, see Scheme 3, and
are the most probable electron acceptors.
Nevertheless, the emission in the 15N NMR signal of 15NO3 is
−
observed throughout the reaction, indicating that the nitration of
the aromatic is only a side reaction. This is confirmed by the low
yield of the nitration product.
Conclusions
Reactions of peroxynitric acid with activated aromatic compounds
and with ascorbic acid, glutathione and cysteine have been
described at pH 2 showing nitration and oxidation. They may
occur without or with participation of free radicals which has been
demonstrated by 15N CIDNP. L-Tyrosine, N-acetyl-L-tyrosine, 4-
fluorophenol and 4-methoxyphenylacetic acid are nitrated. The
quantitative analysis of the 15N CIDNP effects shows that nitration
occurs exclusively via free radicals. 15N CIDNP is observed during
reaction with ascorbic acid indicating an indirect oxidation via
free radicals. In contrast to this, the reaction with glutathione and
cysteine is faster than the decay of peroxynitric acid and does
not show 15N CIDNP indicating a direct oxidation without the
involvement of free radicals.
The generation of the nuclear polarization in the 15N NMR
signals of O215NOOH and 15NO3− is explained as above. Hydroper-
•
oxy radicals HO2 are trapped by 4-methoxyphenylacetic acid
leading to the accelerated decay of O215NOOH and the formation
of H15NO2 (Schemes 1 and 7). To our knowledge, the reactions
are not described in the literature. Calculations concerning the
•
reaction of HO2 with anisole show that the reaction might be
•
exergonic (Table 5, entry 9). The reaction of NO2 with MeO–
C6H4–CH2–COOH which might inhibit the decay of peroxynitric
acid, too, seems to be less probable (Table 5, entry 10).
At pH 7, the deprotonated peroxynitric acid does not nitrate
in situ which makes it unlikely that peroxynitric acid has any
pathophysiological importance at pH 7. However, the pH value
is significantly lower in both the stomach and the lysosomes, and
the reaction with glutathione might be fast enough to occur in
living cells. In any case, peroxynitric acid must be discussed as a
“reactive oxygen species (ROS)” as well as a “reactive nitrogen
species (RNS)”.40
Scheme 7 15N CIDNP during reaction of peroxynitric acid-15N with
4-methoxyphenylacetic acid (ArH).
The E type effect observed in the nitration product is explained
by analogy with the 15N CIDNP effects observed during nitration
reactions of activated aromatic compounds with nitric acid or
nitrous acid.22a,b,23 The nuclear polarization is built up in radical
pairs formed by diffusive encounters of 15NO2• and radical cations
of MeO–C6H4–CH2–COOH, ArH+ (Scheme 7).
Acknowledgements
The present investigation would have been impossible without the
skilled technical assistance of A. Wensing.
Radical cations ArH•+ are generated by oxidation of the
aromatic compound with reactive nitrogen species. Oxidation by
O2NOOH is excluded because of energetical reasons (Table 5,
entry 11). Additionally, the possibility of nitration reactions
with H15NO2 and H15NO3 has been investigated. At pH 1.5,
no reaction has been found using Na15NO2 (0.3 M) or H15NO3
(0.1 M). Under strong acidic conditions (∼10% sulfuric acid), 4-
methoxyphenylacetic acid is nitrated with Na15NO2 (0.3 M) as well
as with H15NO3 (0.1 M), in a similar manner as has been observed
with anisole.23b The nitration product also shows 15N CIDNP. It
follows that reactive nitrogen species other than O2NOOH and
HNO2 must be responsible for the formation of the aromatic
radical cations at pH 2.
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