The chemistry of peroxynitrite: Involvement of an ET process in the radical nitration of unsaturated and aromatic systems

Article abstract of DOI:10.1002/1099-0690(200102)2001:4<741::aid-ejoc741>3.0.co;2-q

Reactions of peroxynitrous acid, HPN, with styrene under acidic conditions lead to the oxime 1, the nitrate 2, benzaldehyde (3), and α-nitroacetophenone (4) in overall yields that depend strongly on the pH value and with a product distribution that depends on the dioxygen concentration. The results are rationalized by assuming that HPN undergoes acid-catalyzed decomposition to give nitrous anhydride, or its synthetic equivalent, which is responsible for the regioselective nitration of the styrene double bond by an ET process. The resulting β-nitrobenzyl radical 6 can, depending on the reaction conditions, undergo reversible coupling with nitric oxide to afford the nitroso derivative 7 and then the tautomeric oxime 1, or trapping by dioxygen, eventually leading to products 2, 3, and 4 through the intermediacy of the peroxynitrite derivative 8. Oxime 1 and nitrate 2 are also obtained by treating styrene with nitrous anhydride under protic conditions, the latter being produced in situ from nitric oxide/dioxygen. Similarly to styrene, 1,4-diphenylbutadiene (14) gives radicals 22 and 21 by competitive trapping at the side chain and at the aromatic ring. In turn, radicals 22 and 21 undergo β-fragmentation reactions or trapping by dioxygen with eventual formation of nitrates 16 and 17, cinnamic aldehyde (18), and the diol 15. Finally, the HPN-promoted reaction of p-cresol (27) leads to the 2-nitro derivative 28 through an initial electron-transfer process followed by in cage recombination of the resulting radical ion pair.

Full text of DOI:10.1002/1099-0690(200102)2001:4<741::aid-ejoc741>3.0.co;2-q

FULL PAPER
The Chemistry of Peroxynitrite: Involvement of an ET Process in the Radical
Nitration of Unsaturated and Aromatic Systems
Loris Grossi,*[a] Pier Carlo Montevecchi,*[a] and Samantha Strazzari[a][‡]
Keywords: Peroxynitrite / Nitration / Alkenes / Electron transfer / Nitrous anhydride
Reactions of peroxynitrous acid, HPN, with styrene under
acidic conditions lead to the oxime 1, the nitrate 2, benzal-
dehyde (3), and α-nitroacetophenone (4) in overall yields that
depend strongly on the pH value and with a product distribu-
ucts 2, 3, and 4 through the intermediacy of the peroxynitrite
derivative 8. Oxime 1 and nitrate 2 are also obtained by treat-
ing styrene with nitrous anhydride under protic conditions,
the latter being produced in situ from nitric oxide/dioxygen.
tion that depends on the dioxygen concentration. The results Similarly to styrene, 1,4-diphenylbutadiene (14) gives rad-
are rationalized by assuming that HPN undergoes acid-cata- icals 22 and 21 by competitive trapping at the side chain and
lyzed decomposition to give nitrous anhydride, or its syn-
thetic equivalent, which is responsible for the regioselective
at the aromatic ring. In turn, radicals 22 and 21 undergo β-
fragmentation reactions or trapping by dioxygen with even-
nitration of the styrene double bond by an ET process. The tual formation of nitrates 16 and 17, cinnamic aldehyde (18),
resulting β-nitrobenzyl radical 6 can, depending on the reac- and the diol 15. Finally, the HPN-promoted reaction of p-cre-
tion conditions, undergo reversible coupling with nitric oxide
sol (27) leads to the 2-nitro derivative 28 through an initial
to afford the nitroso derivative 7 and then the tautomeric ox- electron-transfer process followed by in cage recombination
ime 1, or trapping by dioxygen, eventually leading to prod-
of the resulting radical ion pair.
Introduction
either the sulfhydryl group or the alkene double bond
were required.
Peroxynitrite[2] has recently attracted the attention of
chemists and biochemists since this species appears to be
involved in the in vivo damage of several biological sub-
strates.[3] The active oxidant in these processes has been
generally believed to be the unstable peroxynitrous acid
HPN (OϭNϪOOH) (pKa ϭ 6.8; τ1/2 Ͻ 1 s),[4] which is gen-
erated from the relatively stable peroxynitrite anion PN
(OϭNϪOOϪ) at physiological pH values or lower. In vivo,
the latter is formed from the reaction of nitric oxide (NO)
with superoxide (O2·Ϫ).[3a]
To this end, we have already studied the peroxynitrite-
promoted reaction of simple thiols. In a recent paper, we
reported[9] that PN is capable of oxidizing thiolate ions to
sulfanyl radicals (and then to disulfide) under very basic
conditions, whereas at acidic pH values HPN generates a
nitrosating species, XNO, which leads to the formation of
S-nitrosothiol derivatives as the main reaction products.
Thiol nitrosation is strongly dependent on the pH value,
indicating that HPN generates the nitrosating species XNO
by an acid-catalyzed reaction.
It has been hypothesized that HPN rapidly undergoes
OϪO bond scission with formation of an ‘‘in cage’’ radical
pair, which can either act as a source of hydroxyl radicals
and NO2 (ca. 30%) or rearrange to nitric acid (70%).[5]
However, it has been reported that HPN can also oxidize
suitable substrates either by a direct mono- or di-electron
transfer mechanism or by an indirect mono-electron mech-
anism.[4,6] Among biomolecules, sulfhydryl group con-
taining proteins[7] and lipids containing polyunsaturated
fatty acids[8] are key targets, although the actual mechanism
of peroxynitrite reaction with both the sulfhydryl function
and the carbonϪcarbon double bond is far from being
fully understood.
In continuation of our studies, we report herein on the
results obtained concerning the peroxynitrite-promoted re-
action of simple alkenes carried out under conditions of
both acidic and basic pH values, where the undissociated
HPN and the dissociated PN forms, respectively, largely
predominate.
Results and Discussion
Reactions of peroxynitrite under basic conditions were
attempted by adding two molar equivalents of a 0.50 
aqueous basic solution (pH ϭ 13.5) of peroxynitrite to a
0.10  acetonitrile solution of the appropriate alkene (styr-
ene or allylbenzene). The resulting mixtures were stirred at
10 °C for 30 min, then worked up as described in the Exp.
Sect. In all cases, subsequent 1H NMR analysis of the crude
residue showed the exclusive presence of the unchanged al-
kene. No products derived from attack at the
carbonϪcarbon double bond or, in the case of allylbenzene,
at the allylic position, were detected.
In order to obtain a better understanding of these pro-
cesses, we reasoned that preliminarily studies on the peroxy-
nitrite-mediated reaction of simple molecules containing
[a]
`
Dipartimento di Chimica Organica ‘‘A. Mangini’’, Universita
di Bologna,
Viale Risorgimento 4, 40136 Bologna, Italy
E-mail: grossi@ms.fci.unibo.it
montevec@ms.fci.unibo.it
See ref.[1]
[‡]
Eur. J. Org. Chem. 2001, 741Ϫ748
WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001
1434Ϫ193X/01/0204Ϫ0741 $ 17.50ϩ.50/0
741

L. Grossi, P. C. Montevecchi, S. Strazzari
FULL PAPER
The above reactions were then repeated under acidic con- products, although in slightly lower overall yields (Table 1,
ditions under nitrogen by adding the basic solution of per- Entries 2 and 3). More significantly, we found that the relat-
oxynitrite to a solution of the alkene in a 10:3 acetonitrile/ ive yields of benzaldehyde (3), nitroacetophenone (4) and,
1.2  hydrochloric acid mixture (final pH ϭ 0). The re- particularly, nitrate 2 strongly increased at the expense of
sulting reaction mixtures were stirred for 1 h and then the oxime 1, as shown in Table 1.
worked up as described in the Exp. Sect. Even under these
The peroxynitrite-promoted reaction of styrene was re-
acidic conditions, allylbenzene was found not to react at peated under nitrogen at various pH values in the range
all. In contrast, styrene reacted to give α-nitroacetophenone between 0.0 and 5.4. As indicated by the data presented in
oxime (1), 2-nitro-1-phenylethyl nitrate (2), benzaldehyde Table 2, the relative product yields were found to be inde-
(3), and α-nitroacetophenone (4), together with an unidenti- pendent of the pH value. In contrast, the overall yield shar-
1
fied product that showed a singlet at δ ϭ 5.35 in its H ply decreased on increasing the pH value, varying from 57%
NMR spectrum. These products were formed in a ratio of at pH ϭ 0 (Table 2, Entry 1) to 13% at pH ϭ 5.4 (Table 2,
74:17:1:5:4 and in 57% overall yield based on the amount Entry 5).
of styrene used, as determined by 1H NMR analysis
(Table 1, Entry 1). Subsequent silica gel column chromato-
Table 2. Relative yields (%) of products 1, 2, 3, and 4 obtained from
reactions of styrene with peroxynitrite carried out under nitrogen at
graphy allowed the separation of compounds 1, 2, and 4.
Oxime 1 was characterized by comparison of its spectral
data with those reported in the literature,[10] whereas nitro-
acetophenone (4) was identified by comparison with an au-
thentic commercial sample. Product 2 exhibited a 1H NMR
spectrum identical to that reported in the literature[11] for
the regioisomeric 2-nitro-2-phenylethyl nitrate; the actual
structural assignment was based on its 13C NMR and mass
spectra and on the finding that hydrolysis under basic con-
ditions afforded β-nitrostyrene (5) in quantitative yield
(Scheme 1). We were unable to isolate the unknown product
by column chromatography and hence it remained unchar-
different pH values
Entry
pH
Overall yield (%)[a]
1
2
3
4
Unknown
product
1
2
3
4
5
0
57
33
29
28
13
74 17
75 12
1
5
6
7
4
7
4
4
1
2
1.4
1.7
5.4
77 15 2.5
75 11 2.5
5
5
12
78
2
2
[a]
Determined by 1H NMR with acetophenone as an internal
standard.
The main results obtained from the peroxynitrite-pro-
acterized. Benzaldehyde, formed in small amounts, was only moted reaction of styrene can be summarized as follows:
1
identified by GC/MS and H NMR analyses of the reac- Carrying out the reaction under acidic conditions gave
tion mixture.
products 1, 2, 3, and 4, whereas no products were obtained
under basic conditions. This indicates that the undissociated
form HPN, and not the peroxynitrite anion PN, is involved
as the reactive species.
Table 1. Relative yields (%) of products 1, 2, 3, and 4 obtained
from reactions of styrene with peroxynitrite carried out at pH ϭ 0
under nitrogen (A), air (B), and oxygen (C)
The relative product yields were found to be strongly de-
pendent on the oxygen concentration; under oxygen, the
nitrate 2, as well as nitroacetophenone (4) and benzal-
dehyde (3), were formed at the expense of the oxime 1,
which was the main product under nitrogen (Table 1). This
finding led us to suggest the formation of the benzyl radical
6 as a key intermediate. In turn, radical 6 can be envisaged
as formally deriving from regioselective radical nitration of
the styrene double bond, i.e. through HPN behaving as a
nitrating agent towards this double bond (Scheme 3).
However, independent experiments, in which styrene was
treated with peroxynitrite at pH ϭ 0 under nitrogen,
showed that the overall yield of reaction products 1, 2, 3,
and 4 increased with the reaction time up to 30 min. Since
HPN is very unstable (a lifetime of Ͻ 1 s has been re-
ported),[4] we can easily infer that the actual species re-
sponsible for the formation of radical 6 is derived from
HPN, but cannot be HPN itself.
Entry Conditions Overall yield (%)[a]
1 2 3 4 Unknown product
1
2
3
A
B
C
57
40
41
74 17
1
5
4
trace
Ϫ
18 62 3 17
6 63 5 27
[a]
Determined by 1H NMR with acetophenone as an internal
standard.
Moreover, results obtained by treating styrene at different
pH values (Table 2) showed that the overall yield of prod-
Scheme 1
To determine the effect of the amount of oxygen present ucts 1, 2, 3, and 4 strongly decreased on increasing the pH
in the medium, the reaction of styrene with peroxynitrite at value, notwithstanding that over the entire range examined
pH ϭ 0 was carried out under both air and oxygen. Under (0.0Ϫ5.4) HPN largely predominates over the undissociated
these conditions, compounds 1, 2, 3, and 4, as well as the form PN. On these bases, we suggest that the actual nitrat-
unknown, were again found to be the exclusive reaction ing reagent is formed from HPN by an acid-catalyzed reac-
742
Eur. J. Org. Chem. 2001, 741Ϫ748

Radical Nitration of Unsaturated and Aromatic Systems
FULL PAPER
tion, probably involving the intermediacy of its protonated bond scission with formation of a benzyloxy radical 9/nitric
form, H2PNϩ (Scheme 1).
dioxide radical pair, followed by in cage recombination.[14]
The present results parallel those we obtained regarding
the reaction of HPN with thiols,[9] which led to S-nitroso-
thiols by nitrosation of the thiol group. The S-nitrosothiol
yields, which increased with the reaction time up to 20 min,
were found to be strongly dependent on the pH value. In
this case, the involvement of the protonated form, H2PNϩ,
was also invoked in the formation of the nitrosating species.
Our overall findings lead us to infer that acid-catalyzed
decomposition of HPN leads to
a reactive species,
XϪONO, capable of behaving both as a nitrosating agent
for thiols and as a nitrating agent for the styrene double
bond. We have obtained evidence that this XϪONO species
is a synthetic equivalent of nitrous anhydride, ONϪONO,
or the anhydride itself.[12] In fact, by treating styrene with
nitrous anhydride under various conditions, the latter being
formed in situ from nitric oxide/dioxygen,[13] we obtained
results that are essentially comparable with those obtained
from the HPN-promoted reaction. When these reactions
were carried out in acetonitrile/water solutions at both
pH ϭ 7 (Table 3, Entry 4) and pH ϭ 0 (Table 3, Entries 1
and 3), we found that styrene was almost totally consumed
to give the oxime 1 and the nitrate 2 as the main products,
Scheme 3
Alternatively, the benzyloxy free radical 9 can give rise to
together with β-nitrostyrene (5) and small amounts of nitro- either benzaldehyde (3) by a β-scission process, to nitroace-
acetophenone (4) (Scheme 2).
tophenone (4) by oxidation, or to the nitrate 2 by out of
cage trapping by nitric dioxide (Scheme 3). In the reaction
Table 3. Relative yields (%) of products 1, 2, 3, 4, and 5 obtained promoted by nitric oxide/dioxygen, the latter process largely
from reactions of styrene with nitric oxide carried out under air
predominates over the formation of 3 and 4 owing to the
high nitric dioxide concentration produced under these con-
ditions (Scheme 3).
Entry
Overall yield (%)[a]
1
2
3
4
5
Unknown
product
As mentioned above, the reaction of styrene with nitric
oxide/dioxygen generated noticeable amounts of β-nitro-
styrene (5), which was most likely derived from benzyl rad-
ical 6 by an oxidation/deprotonation process (Scheme 3). At
the present time, we are unable to account for the fact that
this product was not formed in the HPN-promoted reac-
1
2
3
4
A[b]
B[b]
C[b]
D[b]
85
87
80
82
64 14 trace
45 trace
6
11
55
9
5
Ϫ
4
Ϫ
Ϫ
70 17 trace trace
37 39 trace
3
19
2
Determined by 1H NMR with acetophenone as an internal
standard. Ϫ
[a]
[b]
A: in acetonitrile acidified with 0.30 mL of 37%
hydrochloric acid; B: in neat acetonitrile; C: in acetonitrile/1  hy- tion.
drochloric acid (5:2); D: in acetonitrile/water (5:2).
It is worth noting that no oxime 1 was formed when the
reaction of styrene with nitric oxide/dioxygen was carried
out in acetonitrile in the absence of water; in this case, the
nitrate 2 and nitrostyrene (5) were the only reaction prod-
ucts (Scheme 2 and Table 3, Entry 2). According to the pro-
posed mechanism, the absence of the oxime 1 under these
latter conditions can be rationalized by assuming that
benzyl radical 6 is trapped by nitric oxide in a reversible
manner and that the acid-catalyzed tautomerization of the
resulting nitroso derivative 7 to oxime 1 can only occur in
Scheme 2
On this basis, the formation of products 1 and 2 can be a protic medium (Scheme 3). In the absence of water (or
readily explained as outlined in Scheme 3. Reaction of ni- acid), only products deriving from the irreversible trapping
trous anhydride, or its synthetic equivalent, with styrene of radical 6 by dioxygen can be formed.
could lead to the radical 6 and nitric oxide, from which the
nitroso derivative 7 and, by subsequent tautomerization, can behave as a radical nitrating agent towards the styrene
the oxime 1 could be derived. In competition with the coup- double bond. The finding that allylbenzene
At this stage a question arises, i.e. how nitrous anhydride
ling with nitric oxide, the radical 6 could be trapped by (PhϪCH2ϪCHϭCH2), in contrast to styrene, does not re-
dioxygen. Coupling of the resulting benzylperoxyl radical act with peroxynitrite under acidic conditions[15] indicates a
with nitric oxide would then give the peroxynitrite derivat- strong effect of the styrene phenyl ring in promoting the
ive 8,[14] from which the nitrate 2 could be formed by OϪO radical nitration of the alkene double bond.
Eur. J. Org. Chem. 2001, 741Ϫ748
743

L. Grossi, P. C. Montevecchi, S. Strazzari
FULL PAPER
In principle, this effect could be explained by assuming gel catalyzed decomposition. In fact, after absorption onto
that a direct radical addition of nitric dioxide (present in silica gel for 24 h, the nitrate 17 was quantitatively con-
equilibrium with nitrous anhydride) occurs, leading to the verted into nitrodiene 19, whereas the nitrate 16 was con-
resonance-stabilized benzyl radical 6. However, we have ob- verted into an 80:20 mixture of cinnamic aldehyde (18) and
tained evidence that an electron-transfer (ET) process be- nitrodiene 19. On the other hand, both 16 and 17 were
tween styrene and nitrous anhydride (or its synthetic found to be essentially stable under the reaction conditions,
equivalent) is probably involved rather than a direct radical whereas they undergo slow decomposition on standing in
nitration. From such an ET process, a radical ion pair air or rapid hydrolysis under basic conditions. Upon treat-
would initially be formed, from which radical 6 could arise ment with 10% aqueous NaOH, 17 was quantitatively con-
from in cage coupling with nitric oxide loss (Scheme 3). verted into diene 19, and 16 into cinnamic aldehyde (18)
Supporting evidence in favor of the ET mechanism was pro- (Scheme 4).
vided by the results of peroxynitrite-promoted reactions of
The hitherto unknown products 15, 16, and 17 were char-
benzalacetone (10), 2-methyl-6-[(E)-phenylmethylidene]cy- acterized on the basis of their IR, NMR, and MS data. In
1
clohexanone (12), and 1,4-diphenylbutadiene (14) carried particular, H NMR analysis showed that all three com-
out under acidic conditions.
pounds were formed as diastereomeric mixtures in the (E)
In fact, benzalacetone (10) and 2-methyl-6-[(E)-phenyl- configuration. Positive-ion electron-impact mass spectro-
methylidene]cyclohexanone (12) did not react at all with metry was not useful for these compounds, since molecular
peroxynitrite under either oxygen or nitrogen, notwith- ions were not detected. In contrast, all of them showed the
standing the presence of the styrene phenyl ring capable of molecular ion peaks in their negative-ion electron-impact
stabilizing the possible benzyl-type radical intermediates 11 mass spectra. Nitrates 16 and 17 showed, in addition to the
and 13. In terms of the ET hypothesis, the lack of reactivity negative molecular ion at m/z ϭ 314, key fragment ions at
·
of these styrene derivatives 10 and 12 can probably be at- m/z ϭ 252 [M· Ϫ NO3 ], 147 [PhϪCϭCϪNO2·Ϫ], and 46
tributed to their higher ionization potentials[16] due to the [NO2Ϫ]. The diol 15 showed the negative molecular ion at
conjugated electron-withdrawing carbonyl groups.
m/z ϭ 285 and notable fragment ions at m/z ϭ 178 [M·Ϫ
Ϫ
PhCHOH·], 123 [PhNO2·Ϫ], and 46 [NO2Ϫ]. Both 16 and
17 showed a very strong band at 1555 cmϪ1 in their IR
spectra. Elemental analysis was performed for compound
15, but not for 16 and 17 owing to the impossibility of ob-
taining pure samples. However, compound 15 has not quite
been fully characterized as it has not been possible to ascer-
1
tain the ortho or para position of the nitro group by H
NMR analysis.
In contrast, 1,4-diphenylbutadiene (14) was quantitat-
ively converted under oxygen to give the diol 15 (15%), the
1,2-adduct 16 (10%), the 1,4-adduct 17 (65%), and cinnamic
aldehyde (18) (10%) (Scheme 4). The aforementioned yields
were determined by 1H NMR analysis of the crude mixture.
Besides these products, subsequent column chromatography
also resulted in the separation of the nitrodiene 19.
According to the reaction mechanism suggested for styr-
ene, the formation of products 15؊18 can most probably be
explained by assuming that 1,4-diphenylbutadiene, owing to
its low ionization potential,[16] undergoes a facile oxidation
to the corresponding radical cation 20 (Scheme 5). This lat-
·Ϫ
ter can be trapped by the N2O3 counterion, either at the
allylic position, leading to radical 22, or at the aromatic
ring, leading to radical 21. Radical 22 can be trapped by
dioxygen at either the 2- or 4-position, ultimately leading
to peroxynitrite derivatives 25 and 26. In analogy to the
behavior exhibited by 8 (see Scheme 3), 25 and 26 can give
nitrates 16 and 17, respectively, through an OϪO bond scis-
sion/recombination process. Furthermore, peroxynitrite de-
rivative 25 can give cinnamic aldehyde (18) through an
OϪO bond scission/β-fragmentation process. In turn, rad-
ical 21 can be trapped by dioxygen to eventually afford the
peroxynitrite derivative 23. This latter might lead to oxirane
24 through attack of the electrophilic peroxidic oxygen
atom on the adjacent carbonϪcarbon double bond with
concomitant loss of NO2Ϫ. The driving force behind this
reaction can be envisaged as being the subsequent aromatiz-
ation by proton loss. From 24, the diol 15 can be smoothly
formed by hydrolysis under the reaction conditions
(Scheme 5).
Scheme 4
Independent experiments showed that 19 was formed
It is noteworthy that unchanged 1,4-diphenylbutadiene
from both nitrate derivatives 16 and 17 as a result of silica (14) was recovered in 90% yield when the reaction was car-
744
Eur. J. Org. Chem. 2001, 741Ϫ748

Radical Nitration of Unsaturated and Aromatic Systems
FULL PAPER
Scheme 6
physiological ones, allow some conclusions to be drawn
concerning the interaction mechanism of peroxynitrite with
biological targets such as the thiol function and the alkene
double bond. It appears that dissociated PN can only be-
have as a mild oxidant, capable of oxidizing the thiolate
group to sulfanyl radicals, but is incapable of oxidizing the
styrene double bond. On the other hand, under acidic con-
ditions the undissociated form, HPN, behaves as a source
of nitrous anhydride, or its synthetic equivalent, which re-
acts as either a nitrosating or nitrating agent, depending on
the nature of the substrate.
It has previously been reported that thiols are readily
converted into the corresponding S-nitrosothiols.[9] In the
present work, we show that the nitration of substrates hav-
ing suitably low ionization potentials can occur at both the
alkene double bond and the benzene ring. The reaction in-
volves initial electron transfer followed by coupling of the
resulting radical ions with loss of nitric oxide.
Reaction of styrene leads to the benzyl radical 6, which,
depending on the reaction conditions, can afford the nitroso
derivative 7 and, by reversible coupling with nitric oxide,
the tautomeric oxime 1, or undergo competitive trapping
by dioxygen, eventually leading to products 2, 3, and 4
through the intermediacy of the peroxynitrite derivative 8.
The overall yield of the reaction products decreases with
increasing pH value, whereas the relative yield ratio 1/(2 ϩ
3 ϩ 4) is strongly dependent on the dioxygen concentration.
Similarly to styrene, 1,4-diphenylbutadiene (14) gives
nitro radicals 22 and 21 by competing coupling at the side
chain and at the aromatic ring. In turn, radicals 22 and 21
undergo β-fragmentation reactions or trapping by dioxygen
with the eventual formation of nitrates 16 and 17, cinnamic
aldehyde (18), and the diol 15.
Scheme 5
ried out in the absence of oxygen (under argon or nitrogen).
Only small amounts of compounds 15Ϫ18 and some un-
identifiable products were detected by 1H NMR. This find-
ing suggests that radicals 21 and 22 undergo, in competition
with trapping by dioxygen, β-fragmentation with formation
of the starting diene 14 rather than trapping by nitric oxide,
as in the case of styrene (Scheme 5).
Trapping of radical cation 20 at the aromatic ring to give
radical 21 is consistent with our results obtained from a
brief investigation of the HPN-promoted reaction of aro-
matic substrates, i.e. acetophenone and p-cresol (27). As ex-
pected, acetophenone, due to its high ionization poten-
tial,[16] did not react at all under any conditions. In contrast,
p-cresol (27), a highly oxidizable aromatic substrate,[16] re-
acted with peroxynitrite to give 4-methyl-2-nitrophenol (28)
in a manner strongly dependent on the pH value (90% yield
at pH ϭ 0; 4% at pH ϭ 7). The formation of 28 can easily
be rationalized in terms of initial oxidation of p-cresol to
the corresponding radical cation followed by in cage re-
combination with the radical counterion, N2O3·Ϫ. The
nitrophenol 28 was also obtained in high yields (Ͼ 90%) by
treating p-cresol with nitric oxide/dioxygen in acetonitrile/
Finally, the peroxynitrite-promoted reaction of p-cresol
(27) leads to the 2-nitro derivative 28 by an initial electron-
transfer process followed by in cage recombination of the
resulting radical ion pair.
Experimental Section
General: NMR spectra were recorded with a Varian Gemini 200
(or 300) instrument using Me4Si as an internal standard. Ϫ GC-
MS analyses were performed with a Carlo Erba QMD 1000 instru-
water solution, both under acidic and neutral conditions ment. Ϫ Mass spectra were recorded with a VG 7070E instrument
operating in electron-impact ionization mode. Ϫ UV/Vis spectra
were recorded with a PerkinϪElmer Lambda 12 instrument. Ϫ IR
spectra were recorded from samples in CHCl3 solution with a
PerkinϪElmer FT-IR 1600 instrument.
(Scheme 6).
Conclusions
Our present and previous results, even though referring
Materials: Styrene, benzalacetone (10), p-cresol (27), acetophenone,
to reactions carried out under conditions very different to and allylbenzene were purchased from commercial sources. 2-
Eur. J. Org. Chem. 2001, 741Ϫ748 745

L. Grossi, P. C. Montevecchi, S. Strazzari
FULL PAPER
Methyl-6-[(E)-phenylmethylidene]cyclohexanone (12)[17] and 1,4-
nitrate 2, δ ϭ 9.98 for benzaldehyde (3), and δ ϭ 5.91 for nitroace-
tophenone (4), using acetophenone (0.30 mmol) as an internal
diphenylbutadiene (14)[18] were prepared as described in the literat-
ure. Peroxynitrite was synthesized according to the previously re- standard. The relative yields thus determined and the overall yields,
ported procedure;[9] its concentration (usually in the range
which are referred to the starting styrene, are reported in Table 1.
The reaction under nitrogen was also carried out at various pH
values (1.0, 1.4, 1.7, 5.4). The reaction mixtures obtained were ana-
lyzed as described above. The relative yields thus determined and
the overall yield, which is referred to the starting styrene, are re-
ported in Table 2.
0.45Ϫ0.50 ) was determined spectrophotometrically (ε302 ϭ 1670

Ϫ1cmϪ1).[19] Solutions kept at Ϫ18 °C showed little decomposition
over several weeks.
Reactions of Peroxynitrite under Basic Conditions. ؊ General Pro-
cedure: To a 0.10  acetonitrile solution of the appropriate alkene
(styrene or allylbenzene) (10 mL), a 0.50  aqueous solution of per-
oxynitrite (4 mL) (pH ϭ 13.5) was added with stirring at 5Ϫ10 °C.
The resulting solution was stirred for 1 h, then extracted with di-
ethyl ether. The organic layer was washed twice with water and
then the solvent was removed. In all cases, 1H NMR analysis of the
recovered residue showed the exclusive presence of the respective
unchanged alkene.
Reaction of 1,4-Diphenylbutadiene (14): The reaction was carried
out at pH ϭ 0 (3.0 mL of 1.2  hydrochloric acid added). 1H NMR
analysis of the mixture obtained from a reaction carried out under
oxygen showed the absence of starting diene and the presence of
the 1,4-adduct 17 (65%), the 1,2-adduct 16 (10%), the diol 15
(15%), and cinnamic aldehyde (18) (10%). Subsequent silica gel col-
umn chromatography gave, on gradual elution with petroleum ether
(b.p. 40Ϫ60 °C)/diethyl ether, the following products: (i) 1-Nitro-
1,4-diphenylbutadiene (19) (110 mg, 22%), identified by compar-
ison of its 1H NMR and IR data with those reported in the literat-
ure[20] {13C NMR (CDCl3, 75.5 MHz): δ ϭ 122.8 (CH), 128.4
(CH), 129.2 (CH), 129.6 (CH), 129.7 (q), 130.5 (CH), 130.7 (CH),
131.6 (CH), 136.2 (CH), 136.3 (q), 145.6 (q). Ϫ MS (EI): m/z (%) ϭ
251 [Mϩ] (25), 221 (25), 205 (85), 190 (25), 178 (35), 165 (20), 151
(15), 131 (29), 127 (50), 115 (100), 105 (85), 91 (95), 77 (90), 51
(55)}. This product was not present in the reaction mixture prior
to column chromatography. (ii) Cinnamic aldehyde (18) (30 mg,
12%). (iii) A 2:1 diastereomeric mixture of 1-nitro-2-nitrooxy-1,4-
diphenylbut-3-ene (16) (yellow oil) (35 mg, 6%) {1H NMR
(200 MHz, CDCl3): δmajor ϭ 5.28 (1 H, A part of an ABXY system,
JAB ϭ 9 Hz, JAX ϭ 6.5 Hz, JAY ϭ 1 Hz), 5.45 (1 H, B part of an
AB system, JAB ϭ 9 Hz), 5.85 (dd, 1 H, J1 ϭ 16.0 Hz, J2 ϭ 6.5 Hz),
6.65 (dd, 1 H, J1 ϭ 16.0 Hz, J2 ϭ 1 Hz), 7.20Ϫ7.55 (10 H); δminor ϭ
4.65 (t, 1 H, J ϭ 6.0 Hz), 4.95 (d, 1 H, J ϭ 6.0 Hz), 6.20 (dd, 1 H,
J1 ϭ 16.0 Hz, J2 ϭ 6.0 Hz), 6.63 (d, 1 H, J ϭ 16 Hz), 7.20Ϫ7.55
(10 H). Ϫ MS (positive-ion electron-impact method): m/z (%) ϭ
251 (5), 178 (10), 161 (10), 133 (50), 131 (35), 115 (35), 105 (50),
103 (50), 91 (100), 77 (95), 65 (30), 51 (60). Ϫ MS (negative-ion
electron-impact method): m/z (%) ϭ 314 [MϪ] (2), 252 (85), 178
(10), 147 (100), 46 (65). Ϫ IR (CHCl3): ν˜max ϭ 1650, 1555 (vs),
1535, 1490, 1450, 1365, 1310, 1280}. Elemental analysis was not
performed in view of the fact that it proved impossible to obtain a
pure sample owing to partial decomposition on the silica gel col-
Reactions of Peroxynitrite under Acidic Conditions. ؊ General Pro-
cedure: To a 0.10  acetonitrile solution (20.0 mL) of the appropri-
ate substrate {styrene, allylbenzene, benzalacetone (10), 2-methyl-
6-[(E)-phenylmethylidene]cyclohexanone (12), acetophenone, 1,4-
diphenylbutadiene (14), or p-cresol (27)} (2.0 mmol), the requisite
amount of 1.2  hydrochloric acid (3.0Ϫ6.0 mL) was added at
5Ϫ10 °C and then the appropriate gas (air, dioxygen, or nitrogen)
was bubbled through the resulting solution. After 20 min, a 0.50 
aqueous solution of peroxynitrite (8.0 mL) was added with stirring.
The reaction mixture was stirred for 1 h and then diethyl ether
(30 mL) was added. The aqueous phase was separated and the pH
value was measured (see Table 2). The organic layer was washed
twice with water and the solvent was evaporated.
Reactions of Allylbenzene, Benzalacetone (10), 2-Methyl-6-[(E)-
phenylmethylidene]cyclohexanone (12), and Acetophenone: These re-
actions were performed following the addition of 6.0 mL of 1.2 
hydrochloric acid (final pH ϭ 0). 1H NMR analyses of the resulting
reaction mixtures showed the exclusive presence of the respective
unchanged starting materials.
Reaction of Styrene: The mixture obtained from the reaction car-
ried out under nitrogen at pH ϭ 0 (6.0 mL of 1.2  hydrochloric
acid added) was chromatographed on a silica gel column. Gradual
elution with petroleum ether (b.p. 40Ϫ60 °C)/diethyl ether afforded
unchanged styrene, oxime 1[10] (125 mg, 35%), nitroacetophenone
(4) (10 mg, 3%), and the nitrate 2[11] (40 mg, 9%). Products 1 and umn and on standing in air. Compound 16 was treated with 10%
4 were identified by comparison of spectral data with those re-
aq. NaOH for 1 h; the reaction mixture was then extracted with
ported in the literature[10] and by comparison with an authentic
diethyl ether, the organic phase was separated, and the solvent was
specimen, respectively. The nitrate 2 {1H NMR (200 MHz, CDCl3): evaporated. H NMR and TLC analyses of the residue showed the
1
δ ϭ 4.58 (dd, 1 H, J1 ϭ 14.8 Hz, J2 ϭ 3.7 Hz), 4.80 (dd, 1 H, J1 ϭ
disappearance of the starting 16 and the exclusive formation of
14.8 Hz, J2 ϭ 10.2 Hz), 6.56 (dd, 1 H, J1 ϭ 10.2 Hz, J2 ϭ 3.7 Hz), cinnamic aldehyde (18). A further portion of compound 16 was
7.35Ϫ7.60 (m, 5 H). Ϫ 13C NMR (CDCl3, 50.3 MHz): δ ϭ 76.2
absorbed onto silica gel for 24 h and then extracted with diethyl
(CH2), 80.3 (CH), 127.4 (CH), 130.2 (CH), 131.1 (CH), 139.6 (C). ether. 1H NMR and TLC analyses showed the disappearance of
Ϫ MS (EI): m/z (%) ϭ 149 [Mϩ Ϫ 63] (20), 120 (40), 105 (100), 91 the starting 16 and the exclusive formation of an 80:20 mixture of
1
(40), 77 (50), 52 (60)} showed H NMR signals identical to those cinnamic aldehyde (18) and nitrodiene 19. (iv) 1-Nitro-4-nitrooxy-
reported in the literature[11] for the regioisomeric 2-nitro-2-phenyle-
thyl nitrate. Its structural assignment was based on chemical evid-
1,4-diphenylbut-2-ene (17) (yellow oil) (170 mg, 27%) {1H NMR
(200 MHz, CDCl3): δ ϭ 5.22 (0.35 H, J ϭ 5.9 Hz; lines broadened
ence: treatment with 1.0  aq. sodium hydroxide solution at room by coupling to the signal at δ ϭ 6.44; collapsing to a broad singlet
temperature for 12 h quantitatively afforded β-nitrostyrene, which
proved to be identical to an authentic specimen. GC-MS and 1H
NMR analyses of the reaction mixture showed, in addition to prod-
ucts 1, 2, and 4, the presence of a small amount of benzaldehyde
(3). The reaction at pH ϭ 0 (6.0 mL of 1.2  hydrochloric acid
upon irradiation at δ ϭ 5.9), 5.24 (0.65 H, J ϭ 5.3 Hz; lines
broadened by coupling to the signal at δ ϭ 6.44; collapsing to a
broad singlet upon irradiation at δ ϭ 5.9), 5.90 (m, 2 H), 6.44 (dd,
1 H, J1 ϭ 15.0 Hz, J2 ϭ 7.9 Hz; lines broadened by coupling to
the signal at δ ϭ 5.23; collapsing to a broad singlet upon irradiation
added) was also carried out under air, oxygen, and nitrogen. The at δ ϭ 5.9), 7.25Ϫ7.55 (10 H). Ϫ MS (positive-ion electron-impact
resulting reaction mixtures were quantitatively analyzed by 1H
NMR by considering signals at δ ϭ 5.62 for oxime 1, δ ϭ 4.58 for
method): m/z (%) ϭ 265 (10), 133 (10), 131 (20), 115 (15), 105 (90),
103 (35), 91 (30), 77 (100), 51 (50). Ϫ MS (negative-ion electron-
746
Eur. J. Org. Chem. 2001, 741Ϫ748

Radical Nitration of Unsaturated and Aromatic Systems
FULL PAPER
impact method): m/z ϭ 314 [MϪ] (1), 252 (25), 237 (15), 147 (30),
silica gel column to separate β-nitrostyrene (5) (65 mg, 45%), which
121 (10), 46 (100). Ϫ IR (CHCl3): ν˜max ϭ1550 (vs), 1535, 1490, was identified by comparison with an authentic specimen.
1450, 1360, 1280, 900 cmϪ1}. Compound 17 was treated with 10%
aq. NaOH for 1 h; the reaction mixture was then extracted with
Reaction of p-Cresol (27): The reaction mixtures obtained under
conditions A and B were analyzed by 1H NMR using aceto-
diethyl ether, the organic phase was separated, and the solvent was
phenone (0.30 mmol) as an internal standard. In both cases, 4-
methyl-2-nitrophenol (28) was found to be present in 90% yield.
1
evaporated. H NMR and TLC analyses of the residue showed the
disappearance of the starting 17 and the exclusive formation of
nitrodiene 19. A further portion of compound 17 was absorbed
onto silica gel for 24 h and then extracted with diethyl ether. 1H
NMR and TLC analyses showed the disappearance of the starting
Acknowledgments
17 and the exclusive formation of nitrodiene 19. (v) A 60:40 insep-
This work was financially supported by the Ministero dell’Univer-
arable mixture of diastereoisomeric 1,2-dihydroxy-4-(nitrophenyl)-
1-phenylbut-3-ene (15) (light-yellow oil) (75 mg, 13%) {1H NMR
(200 MHz, CDCl3): δmajor ϭ 2.50 (br. s, 2 H, OH), 4.45 (ddd, 1 H,
J1 ϭ 6.8 Hz, J2 ϭ 5.0 Hz, J3 ϭ 1.2 Hz), 4.80 (d, 1 H, J ϭ 5.0 Hz),
`
sita e della Ricerca Scientifica e Tecnologica (MURST), Rome
(funds 60% and 40%), and by the University of Bologna (funds for
selected research topics A. A. 1999Ϫ2001). We gratefully thank Mr.
Luca Zuppiroli for obtaining the mass spectra.
6.15 (dd, 1 H, J1 ϭ 16.0 Hz, J2 ϭ 6.8 Hz), 6.58 (dd, 1 H, J1
ϭ
16.0 Hz, J2 ϭ 1.2 Hz), 7.10Ϫ7.50 (m, 9 H); δminor ϭ 2.50 (br. s, 2
H, OH), 4.37 (ddd, 1 H, J1 ϭ 6.8 Hz, J2 ϭ 5.8 Hz, J3 ϭ 1.2 Hz),
4.58 (d, 1 H, J ϭ 6.8 Hz), 6.05 (dd, 1 H, J1 ϭ 16.0 Hz, J2 ϭ 5.8 Hz),
6.54 (dd, 1 H, J1 ϭ 16.0 Hz, J2 ϭ 1.2 Hz), 7.10Ϫ7.50 (m, 9 H). Ϫ
MS (positive-ion electron-impact method): m/z (%) ϭ 134 (65), 133
(95), 115 (40), 107 (75), 105 (35), 79 (75), 77 (100). MS (negative-
ion electron-impact method): m/z (%) ϭ 285 [MϪ] (10), 251 (25),
237 (25), 219 (15), 178 (95), 153 (25), 147 (45), 123 (40), 46 (100).
Ϫ IR (CHCl3): ν˜max ϭ 3560 and 3400 (br, OH), 1550, 1530, 1490,
1450, 1330 cmϪ1. Ϫ C16H15NO4 (285.3): calcd. C 67.36, H 5.30, N
4.91; found C 67.30, H 5.35, N 4.88}. 1H NMR analysis of the
mixture obtained from a reaction carried out under nitrogen
showed the presence of starting diene 14 (90% yield) together with
minor amounts of products 15Ϫ18 and other unidentified products
(10% overall yield).
[1]
In partial fulfilment of the requirements for the Ph.D. degree
in Chemical Sciences, University of Bologna.
[2]
The term ‘‘peroxynitrite’’ has been used throughout this paper
to indicate both the unprotonated and the protonated forms.
PN and HPN have been used to specifically indicate the
ONOOϪ and ONOOH forms, respectively.
[3] [3a]
J. M. Fukuto, L. J. Ignarro, Acc. Chem. Res. 1997, 30,
[3b]
149Ϫ152. Ϫ
S. Pfeiffer, B. Mayer, B. Hemmens, Angew.
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W. A. Pryor, G. L. Squadrito, Am. J. Physiol.: Lung Cell. Mol.
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J. W. Coddington, J. K. Hurst, S. V. Lymar, J. Am. Chem.
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W. A. Pryor, X. Jin, G. L. Squadrito, Proc. Natl. Acad.
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Squadrito, W. A. Pryor, G. Czapski, Free Radicals Biol. Med.
1996, 21, 965Ϫ974.
Reaction of p-Cresol (27): The reaction was performed at pH ϭ 0
(6.0 mL of 1.2  hydrochloric acid added) under nitrogen. 1H
NMR analysis of the reaction mixture using acetophenone
(0.60 mmol) as an internal standard showed the exclusive forma-
tion of 4-methyl-2-nitrophenol (28)[21] in 90% yield based on the
amount of p-cresol used. Identical results were obtained when the
same reaction was carried out under air. This reaction was also
carried out under nitrogen at pH ϭ 7. 1H NMR analysis of the
reaction mixture using acetophenone (0.6 mmol) as an internal
standard showed the exclusive formation of 4-methyl-2-nitrophenol
(28) in 4% yield based on the starting p-cresol.
[7]
[8]
R. Radi, J. S. Beckman, K. M. Bush, B. A. Freeman, J. Biol.
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H. Rubbo, R. Radi, M. Trujillo, R. Telleri, B. Kalyanaraman,
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L. Grossi, P. C. Montevecchi, S. Strazzari, Eur. J. Org. Chem.,
in press.
[9]
[10] [10a] M. L. Scheinbaum, J. Org. Chem. 1970, 35, 2790Ϫ2792. Ϫ
[10b]
M. M. Baum, E. H. Smith, J. Chem. Soc., Perkin Trans. 1
1993, 2513Ϫ2519.
[11]
H. Suzuki, T. Mori, J. Org. Chem. 1997, 62, 6498Ϫ6502.
[12]
Nitrous anhydride is a well-known nitrosating agent for thiols;
see refs.[3b,4,13a] and: G. R. Upchurch, G. N. Welch, J. Loscalzo,
Adv. Pharmacol. 1995, 34, 343؊349; R. S. Lewis, S. R. Tannen-
baum, W. M. Deen, J. Am. Chem. Soc. 1995, 117, 3933؊3939.
Reactions of Styrene and p-Cresol (27) with Nitric Oxide. ؊ General
Procedure: Styrene or p-cresol (27) (1.0 mmol) was dissolved in
either acetonitrile (14 mL) acidified with 12  hydrochloric acid
(0.30 mL) (Table 3, conditions A), in neat acetonitrile (14 mL)
(Table 3, conditions B), in a 5:2 mixture of acetonitrile/1  hydro-
chloric acid (14 mL) (Table 3, conditions C), or in a 5:2 mixture of
acetonitrile/water (14 mL) (Table 3, conditions D). The solutions
were cooled to 0 °C and nitric oxide was bubbled through them
for 60 min. After this time, TLC analysis showed almost complete
disappearance of the starting styrene or p-cresol. The reaction mix-
tures were then extracted with diethyl ether, the organic layer was
washed twice with water, and the solvent was evaporated.
[13] [13a]
J. S. Stamler, D. I. Simon, J. A. Osborne, M. E. Mullins,
D. Jaraki, T. Michel, D. J. Singel, J. Loscalzo, Proc. Natl. Acad.
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Sci. USA 1992, 89, 444Ϫ448. Ϫ
L. Grossi, S. Strazzari, J.
Org. Chem. 1999, 64, 8076Ϫ8079.
[14]
[15]
[16]
S. Padmaja, R. E. Huie, Biochem. Biophys. Res. Commun. 1993,
195, 539Ϫ544.
Neither products deriving from oxidation at the allylic position
were obtained.
The following ionization potentials (IP) have been reported in
the literature: styrene: 8.46 eV (J. M. Dyke, H. Ozeki, M. Taka-
hashi, M. C. R. Cockett, K. Kimura, J. Chem. Phys. 1992, 97,
8926Ϫ8933); benzalacetone: 8.80 eV (B. Schaldach, B. Grote-
meyer, J. Grotemeyer, H.-F. Gruetzmacher, Org. Mass Spec-
trom. 1981, 16, 410Ϫ415); 1,4-diphenylbutadiene: 8.05 eV (C.
Ruecker, D. Lang, J. Sauer, H. Friege, R. Sustmann, Chem. Ber.
1980, 113, 1663Ϫ1690); p-cresol: 8.22 eV (Y. Yagci, W. Schna-
bel, A. Wilpert, J. Bending, J. Chem. Soc., Faraday Trans. 1994,
90, 287Ϫ292); acetophenone: 9.51 eV (G. Pfister-Guillouzo, S.
Geribaldi, J.-F. Gal, Can. J. Chem. 1982, 60, 1163Ϫ1172).
R. Cornubert, P. Louis, Bull. Soc. Chim. Fr. 1938, 5, 520Ϫ534.
R. Miravalles, A. J. Guillarmod, Helv. Chim. Acta 1966, 49,
2313Ϫ2320.
Reaction of Styrene: The reaction mixtures obtained under condi-
tions A, B, C, and D were analyzed by 1H NMR using aceto-
phenone (0.30 mmol) as an internal standard to determine the
yields of the reaction products 1, 2, 4, and 5. The relative yields
thus obtained and the overall yield, which is based on the starting
styrene, are reported in Table 3. In the case of the reaction carried
out in neat acetonitrile, the mixture was chromatographed on a
[17]
[18]
Eur. J. Org. Chem. 2001, 741Ϫ748
747

L. Grossi, P. C. Montevecchi, S. Strazzari
FULL PAPER
[19]
[21]
M. N. Hughes, H. G. Nicklin, J. Chem. Soc. A 1968, 450Ϫ452.
P. Horsewood, G. W. Kirby, R. P. Sharma, J. G. Sweeny, J.
J.-M. Poirer, C. Vottero, Tetrahedron 1989, 45, 1415Ϫ1422.
[20]
Received June 19, 2000
Chem. Soc., Perkin Trans. 1 1981, 1802Ϫ1806.
[O00310]
748
Eur. J. Org. Chem. 2001, 741Ϫ748