Evidence of an electron-transfer mechanism in the peroxynitrite-mediated oxidation of 4-alkylphenols and tyrosine

Article abstract of DOI:10.1021/jo034089d

The mechanism of interaction of the peroxynitrite with some 4-alkylphenols and tyrosine was mainly studied by means of ESR spectroscopy and product analysis. The radical intermediates, detected as spin adducts to the 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), were identified as carbon-centered radicals to the benzene ring. The reaction seems to proceed via an electron transfer process (ET), most likely mediated by a NOx derivative, leading to the intermediacy of a phenoxyl-type radical as proved by the detection of the corresponding Pummerer-type ketone. No evidence of the formation of hydroxyl radicals, due to the homolytic cleavage of the peroxynitrite at physiological pH was obtained, even though DEPMPO hydroxyl spin adducts were detected: the latter most likely arises from the direct attack of the spin trap by the oxidant species. The possible involvement of HCO₃^-/CO₂, i.e., the formation of the nitrosoperoxycarbonate, ONOOCO₂^.-, was also investigated.

Full text of DOI:10.1021/jo034089d

Evid en ce of a n Electr on -Tr a n sfer Mech a n ism in th e
P er oxyn itr ite-Med ia ted Oxid a tion of 4-Alk ylp h en ols a n d Tyr osin e
Loris Grossi
Dipartimento di Chimica Organica “A. Mangini”, Universita` di Bologna,
Viale Risorgimento 4, I-40136 Bologna, Italy
grossi@ms.fci.unibo.it
Received J anuary 24, 2003
The mechanism of interaction of the peroxynitrite with some 4-alkylphenols and tyrosine was mainly
studied by means of ESR spectroscopy and product analysis. The radical intermediates, detected
as spin adducts to the 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), were
identified as carbon-centered radicals to the benzene ring. The reaction seems to proceed via an
electron transfer process (ET), most likely mediated by a NOx derivative, leading to the intermediacy
of a phenoxyl-type radical as proved by the detection of the corresponding Pummerer-type ketone.
No evidence of the formation of hydroxyl radicals, due to the homolytic cleavage of the peroxynitrite
at physiological pH was obtained, even though DEPMPO hydroxyl spin adducts were detected:
the latter most likely arises from the direct attack of the spin trap by the oxidant species. The
•-
possible involvement of HCO3-/CO2, i.e., the formation of the nitrosoperoxycarbonate, ONOOCO2
was also investigated.
,
•-
In tr od u ction
NO and O2 production may greatly increase the rate
of peroxynitrite formation, v ) k[NO][O2•-], reaching
potentially cytotoxic levels.
Peroxynitrite, ONOO-, is formed in vivo by the reaction
of nitric oxide, NO, with the superoxide, O2•-, eq 1:1-3
The role of the peroxynitrite as a cytotoxic species in
vivo is still debated,3 but, being in its associated form,
i.e., the peroxynitrous acid, an oxidant, it may be able to
destroy critical cellular targets. On the other hand, the
oxidizing capability of peroxynitrite toward a variety of
biomolecules has been widely investigated and demon-
strated in vitro.6 Moreover, peroxynitrite can perform
nitration of tyrosine and this modification may have
dramatic effects on proteins and enzyme functions.2
In 1952, Halpfenny and Robinson7 proposed a radical
mechanism for the reaction of peroxynitrous acid with
aromatic compounds at pH 1.4, which led to nitrated and
hydroxylated derivatives. In 1994, Van der Vliet et al.8
reported that the phenylalanine and the tyrosine are
nitrated and hydroxylated by peroxynitrite and proposed
that the hydroxylation could be caused by hydroxyl
radicals and the nitration by nitrogen dioxide. However,
an ET mechanism has been also suggested9,10 and the
intermediacy of a phenoxyl radical has been claimed
several times,9-11 even if never proved directly.
k1
NO + O2•- 98 -OONO
(1)
This reaction is diffusion controlled (k1 ) (4.3-6.7) ×
109 M-1 s-1),2 and the half-life of ONOO- under physi-
ological conditions is reported to range between 20 ms4
and 1 s,3 as the anion is, at pH ) 7.4, in equilibrium (pKa
) 6.8) with its conjugated acid, the peroxynitrous acid
(HOONO), which spontaneously decays to give mostly the
nitrate,3 eq 2:
+
+H
-OONO y-H+z HOONO f H+ + NO3
(2)
-
The biological relevance of this species was first pointed
out by Beckman et al.,5 who recognized that the peroxy-
nitrite may be formed in significant quantities under
pathological conditions, where both NO and O2 are
produced at high rates by phagocytic cells such as
macrophages, since relatively small increases in rates of
•-
* To whom corresponce should be addressed. Fax: +39-051-2093654.
(1) Moro, M. A.; Darley-Usmar, V. M.; Goodwin, D. A.; Read, N. G.;
Zamora-Pino, R.; Feelisch, M.; Radomski, M. W.; Moncada, S. Proc.
Natl. Acad. Sci. U.S.A. 1994, 91, 6702.
(2) Pfeiffer, S.; Mayer, B.; Hemmens, B. Angew. Chem., Int. Ed.
1999, 38, 1715.
(3) Fukuto, J . M.; Ignarro, L. J . Acc. Chem. Res. 1997, 30, 149.
(4) Radi, R.; Denicola, A.; Alvarez, B.; Ferre-Sueta, G.; Rubbo, H.
The Biological Chemistry of Peroxynitrite. In Nitric Oxide: Biology
and Pathobiology; Ignarro, L. J ., Ed.; Academic Press: San Diego, 2000;
p 57.
(6) (a) Radi, R.; Beckman, J . S.; Bush, K. M.; Freeman, B. A. J . Biol.
Chem. 1991, 266, 4244. (b) Radi. R.; Beckman, J . S.; Bush, K. M.;
Freeman, B. A. Arch. Biochem. Biophys. 1991, 288, 481. (c) Hogg, N.;
J oseph, J .; Kalyanaraman, B. Arch. Biochem. Biophys. 1994, 314, 153.
(d) Bartlett, D.; Church, D. F.; Bounds, P. L.; Koppenol, W. H. Free
Rad. Biol. Med. 1995, 18, 85. (e) Muijers, R. B. R.; Folkerts, G.;
Henricks, P. A. J .; Sadeghi-Hashijn, G.; Nijkamp, F. P. Life Sci. 1997,
60, 1833.
(7) Halpfenny, E.; Robinson, P. L. J . Chem. Soc. 1952, 939.
(8) Van Der Vliet, A.; O’Neill, C. A.; Halliwell, B.; Cross, C. E.; Kaur,
H. FEBS Lett. 1994, 339, 89.
(5) Beckman, J . S.; Beckman, T. W.; Chen, J .; Marshall, P. A.;
Freeman, B. A. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 1620.
(9) Pryor, W. A.; Squadrito, G. L. Am. J . Physiol. 1995, 268, L-699.
(10) Daiber, A.; Mehl, M.; Ullrich, V. Nitric Oxide 1998, 2, 259.
10.1021/jo034089d CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/15/2003
J . Org. Chem. 2003, 68, 6349-6353
6349

Grossi
analogous to that evidenced when either the DMPO13,14
(5,5-dimethyl-1-pyrroline N-oxide) or the TMIO15 (2,2,4-tri-
methyl-2H-imidazole 1-oxide) or the CDMIO15 (4-carboxy-
2,2-dimethyl-2H-imidazole 1-oxide) were used as spin
traps in the study of the peroxynitrite decomposition.
But, truly surprising was to note that conducting experi-
ments with the same amount of peroxynitrite, but dif-
ferent quantity of DEPMPO,12b the corresponding hy-
droxyl radical spin adduct was detectable at different
elapse of time: the delay was inversely proportional to
the amount of spin-trap used.16 The same test experi-
TABLE 1. ESR P a r a m eter s of DEP MP O Ad d u cts, in
Bu ffer P h osp h a te Solu tion (0.2 M, p H 7.4) a t Room
Tem p er a tu r ea
hfc (mT)
substrate
spin adduct
aP
aN
aHâ
1
2
3
4
1a
2a
3a
4a
5
4.683
4.685
4.660
4.730
4.680
1.462
1.450
1.430
1.430
1.400
2.125
2.020
2.105
2.000
1.350
1-4
a
g-Factors are lying in the range 2.0061 ( 0.0002.
ments were then curried out adding bicarbonate, i.e., in
-
the presence of the nitrosoperoxycarbonate, ONOOCO2
,
To verify the possible involvement of radical species
in the peroxynitrite-mediated oxidation of phenols, the
interaction with tyrosine 1 and 4-alkylphenols such as
4-methylphenol 2, 4-ethylphenol 3, and 4-tert-butylphenol
4, which mimic the tyrosine framework, has been studied.
Experiments, carried out in a buffer phosphate solution
at pH ) 7.4, were performed using the ESR spin trapping
technique, which is one of the most appropriate methods
available for assessing free radical formation in biological
systems. For these experiments the 5-diethoxyphospho-
ryl-5-methyl-1-pyrroline N-oxide, DEPMPO, was used; a
spin trap suitable for biological milieu because of its good
solubility in water, and due to its ability to trap carbon,
sulfur and oxygen-centered radicals, which are quite
persistent spin adducts.12
which is believed the real reacting species with biological
targets,17 but no radical species were detected by ESR.
Besides the carbon-centered radical adducts, the detec-
tion and the identification in all experiments of the
DEPMPO-hydroxyl spin adduct, independently of the
substrate investigated, could in principle support the
involvement of freely hydroxyl radicals as one of the
reacting species, Figure 1. Its formation could be ac-
counted for, as usually invoked, by the homolytic cleavage
of the -O-O- bond of the peroxynitrite, and the detec-
tion of 5 could thus be validated invoking a direct trap
of the hydroxyl radical, Scheme 1. Carbon-centered
radicals too could be accounted for by the presence of
hydroxyl radicals. For example, a benzylic-type radical
arising from the p-alkyl group of the substrates, via the
direct abstraction of hydrogen, could be involved. How-
ever, this hypothesis seems not tenable. In fact, the
radical 4a , which arises from 4, a substrate that does
not have hydrogens on the benzylic carbon of the p-alkyl
substituent, shows the same ESR spectroscopic param-
eters (hfc) as 1a -3a , Scheme 1.
Resu lts a n d Discu ssion
When substrates 1-4 were investigated by ESR spec-
troscopy, two paramagnetic species were detectable: 1a -
4a characterized by hyperfine coupling constants typical12
of a DEPMPO spin adduct deriving from the trapping of
a carbon-centered radical, the other identified as the
DEPMPO- hydroxyl radical spin adduct 5, Table 1.
Thus, for the formation of carbon-centered radicals a
different pathway has to be invoked. For instance, a
phenoxyl radical intermediate could be hypothesized, but,
of course, a completely different reaction mechanism has
to be taken into consideration (see later for discussion).
When the nitrosoperoxycarbonate was reacted,17 eq 3,
The relative amount of carbon-centered/hydroxyl ad-
ducts was depending on the nature of the substrate
investigated. However, to account for these results it was
necessary to be more acquainted with the chemical
behavior of DEPMPO. In particular, it was necessary to
verify if it could act as a trap for nitrogen-centered
radicals, such as NO or NO2, possible radical intermedi-
ates, and/or react directly with the peroxynitrite. Toward
this goal, test experiments were conducted. When
DEPMPO was reacted with pure NO or NO2, in the
absence of CO2 and at pH 7.4, no evidence of the
formation of spin adducts due to the trapping of nitrogen-
centered radicals was obtained. The DEPMPO too, as the
majority of the most common spin traps, was unable to
trap nitrogen-centered radicals. In contrast, when the
peroxynitrite was reacted with DEPMPO, a radical
species characterized by ESR spectroscopic parameters
coincident with those of the DEPMPO-hydroxyl radical
spin adduct was detected. Actually, this result was
ONOO- + CO2 f ONOOCO2- f NO2 + CO3
(3)
•-
to account for spectroscopic results, i.e., the detec-
tion of DEPMPO-carbon-centered adducts and the
DEPMPO-hydroxyl adduct, the homolysis of the -O-O-
bond could still be invoked. The formation of 5 could be
•-
justify via the direct trap of the CO3 radical, while for
(13) Lemercier, J . N.; Squadrito, G. L.; Pryor, W. A. Arch. Biochem.
1995, 321, 31.
(14) Sovitj, P.; Nguyen, S. Y.; Gladwell, T.; Rosen, G. M. Biochim.
Biphys. Acta 1995, 1244, 62.
(15) Dikalov, S.; Kirilyuk, I.; Grigor’ev, I. Biochem. Biophys. Res.
Commun.1996, 218, 616.
(16) The experiments were conducted in a buffer phosphate solution
0.2 M (pH 7.4), at room temperature, in the absence of CO2, and the
peroxynitrite concentration set at 0.84 × 10-3 mol/L. When the
DEPMPO concentration used was 5.0 × 10-2 mol/L, the spin adduct 5
was detectable almost immediately; dropping the concentration to 1.0
× 10-2 and 5.0 × 10-3 mol/L, the delay was ca. 2 and 10 min,
respectively; when the concentration was 2.0 × 10-3 mol/L no radical
species were detected within 40 min of investigation.
(17) (a) Zhang, H.; Squadrito, G. L.; Pryor, W. A. Nitric Oxide 1997,
4, 301. (b) Lemercier, J . N.; Padmaja, S.; Cueto, R.; Squadrito, G. L.;
Uppu, R. M.; Pryor, W. A. Arch. Biochem. Biophys. 1998, 345, 160. (c)
Denicola, A.; Freman, B. A.; Trujillo, M.; Radi, R. Arch. Biochem.
Biophys. 1996, 333, 49.
(11) Butler, A. R.; Rutherford, T. J .; Short, D. M.; Ridd, J . H. J .
Chem. Soc., Chem. Commun. 1997, 669.
(12) (a) Frejaville, C.; Karoui, H.; Tuccio, B.; Le Moigne, F.; Culcasi,
M.; Pietri, S.; Lauricella, R.; Tordo, P. J . Chem. Soc., Chem. Commun.
1994, 1793. (b) Karoui, H.; Hogg, N.; Frejaville, C.; Tordo, P.;
Kalyanaraman, B. J . Biol. Chem. 1996, 271, 6000. (c) Karoui, H.;
Nsanzumuhire, C.; Le Moigne, F.; Tordo, P. J . Org. Chem. 1999, 64,
1471.
6350 J . Org. Chem., Vol. 68, No. 16, 2003

Peroxynitrite-Mediated Oxidation of 4-Alkylphenols and Tyrosine
SCHEME 2
However, the homolytic cleavage of the peroxynitrite
or the nitrosoperoxycarbonate, which can be considered
the competing process to the isomerization to HNO3 and
to NO3-/CO2, respectively, takes place in a percentage
ranging between 10 and 25% maximum,19,20 and that led
us to consider the amount of free-radical species, such
as •OH or CO3•-, available for reacting, really poor.
Furthermore, the detection in blank experiments with
DEPMPO and the peroxynitrite, but not with the per-
oxycarbonate, only of the DEPMPO-hydroxyl spin adduct,
and mainly the delay of its detectability depending on
the quantity of spin trap used, leads us to hypothesize a
completely different reaction mechanism. To account for
this behavior, in fact, one needs to hypothesize that the
DEPMPO itself is oxidized by the peroxynitrite to a
diamagnetic species, which spontaneously, but slowly,
decays to a quite persistent radical species characterized
by hfc coincident with those of 5.21 This behavior is
definitely in agreement with that showed in studies on
the decomposition of peroxynitrite in the presence of
other spin traps.13-15 In particular, when the DMPO was
used, the authors13,14 concluded that although a weak
signal corresponding to the DMPO-hydroxyl radical spin
adduct is observed this does not constitute proof of the
presence of free hydroxyl radicals. As well, when TMIO
or CDMIO reacts with peroxynitrite, the authors15 state
that even though the same spin adduct as after the
reaction with OH-radicals is detected, it seems to be
formed under direct oxidation of the spin trap by peroxy-
nitrite and not under the reaction with OH-radicals,
leading to exclude the involvement of free hydroxyl
radicals deriving from the homolytic cleavage of perox-
ynitrite.
F IGURE 1. ESR spectra obtained at room temperature in a
buffer phosphate solution (pH 7.4): (a) reacting 1; radicals 1a
(*) and 5 (•) are detected; (b) reacting 2; radicals 2a (*) and 5
(•) are detected; (c) radical 5 obtained from the Fenton’s
reaction in the presence of DEPMPO.
SCHEME 1
Therefore, these results support also the hypothesis
that radical species deriving from the homolytic decay
carbon centered radicals an electron-transfer process
between CO3•- and the substrate should be hypothesized,
Scheme 2.
(19) (a) Keith, W. G.; Powell, R. E. J . Chem. Soc. A 1969, 90. (b)
Radi, R.; Cosgrove, T. P.; Beckman, J . S.; Freeman, B. A. Biochem. J .
1993, 290, (Pt.1), 51.
(20) (a) Hodges, G. R.; Ingold, K. U. J . Am. Chem. Soc. 1999, 121,
10695. (b) Coddington, J . W.; Hurst, J . K.; Lymar, S. V. J . Am. Chem.
Soc. 1999, 121, 2438.
(21) The delay in the ESR detection of 5, as a function of the
concentration of spin trap, could be explained assuming that reducing
the DEPMPO concentration the amount of the radical-precursor
decreases as well, and then a longer period of time is necessary to get
to an ESR-detectable concentration of the radical species, via ac-
cumulation.
As a matter of fact, the detection of 13CO3•-, obtained
reacting 13C-bicarbonate and peroxynitrite, has been
reported,18 but, as stated before, blank experiments
conducted between DEPMPO and the peroxynitrite in the
presence of CO2 did not allow any radical species to be
detected.
(18) Bonini, M. G.; Radi, R.; Ferrer-Sueta, G.; Da, C. Ferreirai, A.
M.; Augusto, O. J . Biol. Chem. 1999, 274, 10802.
J . Org. Chem, Vol. 68, No. 16, 2003 6351

Grossi
SCHEME 3
It is worth noting that the conversion of 2 was found
to be dependent on the presence of CO2;17a,b in particular,
at pH 7.4, the conversion was more than 80% when
HCO3-/CO2 was added. The product analysis showed the
formation of 7 and 8, yield 12.4% and 21.6%, respectively,
as the main reaction products, but no hydroxylated
derivatives. Furthermore, compounds due to the oxida-
tion of 8, 9, and 10 (<1%) and phenolic polymeric
products were also identified, Scheme 4. When the
reaction was conducted in the absence of CO2, the
conversion was much lower, ca.10%, and the detected
products were 7, yield 5%, and a trace amount of 8. The
formation of these products definitely supports the
involvement of 2b, which can be considered the precursor
of all the identified compounds and, of course, of the
radical 2a , Schemes, 2 and 3. Product analysis for the
reaction of peroxynitrite with 3 was also conducted, and
analogous results were obtained.
SCHEME 4
Con clu sion s
Although in the reaction between the tyrosine, tyro-
sine-type phenols, and the peroxynitrite or the peroxy-
carbonate the involvement of a homolytic -O-O- cleav-
•-
age, and then freely diffusing hydroxyl, CO3 and NO2
radicals, cannot be completely excluded, it seems defini-
tively most plausible that an interaction via an ET
process, leading to the formation of a phenoxyl interme-
diate, takes place, as confirmed by ESR spectroscopy and
product analysis. In particular, the detection of the
dimeric Pummerer’s ketone can be considered a definitive
prove of the ET mechanism. In this light, also the
hypothesis of the intermediacy of N2O3, arising from the
peroxynitrite, as the reacting species, seems tenable.
Finally, the DEPMPO-hydroxyl spin adduct most prob-
ably results to be formed under direct oxidation of the
spin trap, but not under the reaction with OH-radical.
The detection of 5 cannot be considered a prove of the
homolytic decay of the peroxynitrite.
Exp er im en ta l Section
Ma ter ia ls. Tyrosine, 4-methylphenol, 4-ethylphenol, and
4-tert-butylphenol were purchased from Aldrich and used as
received. The DEPMPO26 and the peroxynitrite27 were syn-
thesized according to the literature methods. For the synthesis
of the latter, a solution of H2O2 30% (10.0 mL) was diluted to
50 mL with water, chilled at 4 °C, and then added with 18
mL of NaOH (5.0 M) and 5.0 mL of 0.04 M diethylenetri-
aminepentaacetic acid (DTPA) in NaOH (0.05 M); the resulting
mixture was diluted to 100 mL, total volume, with water (the
final pH was around 13.5). Subsequently, the solution was
allowed to react with an equimolar amount of isoamyl nitrite
(12.3 mL) under vigorous stirring for 3-4 h at ca. 15 °C. The
aqueous phase was then washed with chloroform (5 × 75 mL)
to remove the contaminating isoamyl alcohol and the unre-
acted nitrite. The resulting yellow solution was treated with
an excess of MnO2 in order to destroy the unreacted H2O2 and
of the peroxynitrite cannot account for the formation of
carbon-centered radical intermediates. In contrast, a
reaction mechanism22 via an ET process seems to be more
tenable. To validate the results, the involvement of a NOx
species has been invoked too, Scheme 3.
23
In particular, the intermediacy of N2O3 has been
hypothesized to be responsible for the oxidation of
phenolic substrates. Actually, we have recently demon-
strated24 that, under the same experimental conditions,
the 4-methylphenol reacts with N2O3, via an ET process,
leading to the same products obtained when peroxynitrite
was used; in particular, the recover of the Pummerer’s
ketone 725 is considered a definitive prove of the involve-
ment of an ET process (Scheme 4).
then filtered. The yield of ONOO- was determined from its
absorption, ꢀ302 ) 1670 M-1 cm-1
.
Solutions kept at -18 °C
28
showed little decomposition over several weeks.
(22) (a) Atkinson, R.; Darnall, K. R.; Lloyd, A. C.; Winer, A. M.; Pitts,
J . N. Adv. Photochem. 1979, 11, 375. (b) Rinke, M.; Zetzsch, C. Ber.
Bunsen-Ges. Phys. Chem. 1984, 88, 55.
(23) Kissner, R.; Koppenol, W. H. J . Am. Chem. Soc. 2002, 124, 234.
(24) Grossi, L.; Montevecchi, P. C.; Strazzari, S. Eur. J . Org. Chem.
2001, 131.
(25) Barton, D. H. R.; Bruun, T. J . Chem. Soc. 1953, 603.
(26) Barbati, S. 1997. Ph.D. Thesis, University of Aix-Marseille I,
France, pp 68-82.
(27) Uppu, R. M.; Pryor, W. A. Anal. Biochem. 1996, 236, 242.
(28) Hughes, M. N.; Nicklin, H. G. J . Chem. Soc. A 1968, 450.
6352 J . Org. Chem., Vol. 68, No. 16, 2003

Peroxynitrite-Mediated Oxidation of 4-Alkylphenols and Tyrosine
An a lysis P r oced u r es. ESR analyses were carried out on
a spectrometer equipped with an X-band microwave bridge,
100 kHz modulation, and with a variable-temperature ap-
paratus. An aliquot of the reaction mixture obtained by
addition to a buffer phosphate solution (pH 7.4), the peroxy-
nitrite (final concentration 3.0 mM), the substrate (15-35
mM), and the spin trap, DEPMPO (7.5 mM), was introduced
in a quartz flat-cell placed inside the cavity of the ESR
spectrometer and immediately analyzed. GC-MS analyses
were performed using a gas chromatograph equipped with a
methyl silicone plus 5% phenyl silicone capillary column and
fid integration. All compounds were identified by comparison
of their retention times with those of authentic samples and/
or by their mass spectra. TLC chromatography was conducted
as usual; 1H NMR and mass spectra identified separated
products.
(<1%) of the 2,2′-dihydroxy-5,5′-dimethyl-3-nitrobiphenyl (9)
and the 2,2′-dihydroxy-5,5′-dimethyl-3,3′-dinitrobiphenyl (10)
were recovered; the remaining converted product was identi-
fied as a polymeric (phenolic) derivative. When the 4-ethylphe-
nol (3) was reacted, besides some unreacted substrate, the
Pummerer-like ketone and the 4-ethyl-2-nitrophenol were
identified. The yield of the latter was higher compared with
that obtained reacting (2), and that at the expenses of dimeric
products.
(b) In th e Absen ce of CO2. To keep the presence of
bicarbonate/carbon dioxide as low as possible, the buffer
phosphate solution, containing the substrate and the spin trap,
was sonicated for at least 50/60 min and purged by bubbling
of N2 for an additional 1 h just before use. The peroxynitrite,
which also was purged in advance by bubbling of N2, was then
added to the mixture and the reaction carried out under
nitrogen atmosphere. When 2 was reacted, the conversion yield
was very poor. The GC-MS analysis showed the presence of
the 4-methyl-2-nitrophenol (∼1% yield) and the 8,9b-dimethyl-
4a,9b-dihydroxy-4H-dibenzofuran-3-one (5% yield), besides, of
course, unreacted starting material. Similarly, when 3 was
investigated the 4-ethyl-2-nitrophenol and the corresponding
Pummerer’s-like ketone were detected.
P r od u ct An a lysis. Experiments were conducted on sub-
strates 2 and 3.
(a ) In th e P r esen ce of CO2. At room temperature, to 20
mL of a 0.2 M buffer phosphate solution were added 0.87 mmol
of NaHCO3 (∼25mM, i.e., close to the concentration in plasma),
1.2 mmol of substrate, and under vigorous stirring, 1.2 mmol
of peroxynitrite; the pH (7.4) was adjusted by adding HCl
(37%) dropwise. The resulting mixture was stirred for further
15 min, and the organic phase was extracted with ether (3 ×
25 mL), washed twice with water (20 mL), and then dried with
Na2SO4. In particular, when the 4-methylphenol was reacted
a high conversion yield was obtained. The GC-MS analysis
led to the identification of, besides a low quantity of unreacted
starting material, the 4-methyl-2-nitrophenol (21.6% yield) and
the 8,9b-dimethyl-4a,9b-dihydroxy-4H-dibenzofurane-3-one
(Pummerer’s ketone) (12.4% yield). However, through an
accurate quantitative TLC chromatography, minor quantities
Ack n ow led gm en t. We thank Dr. Cristina Folli for
skillful technical assistance. This work was financially
supported by Ministry of the University and Scientific
and Technological Research (MURST), Rome (Funds
60% and 40%), and by University of Bologna (Funds for
selected research topics A. A. 1999-2001).
J O034089D
J . Org. Chem, Vol. 68, No. 16, 2003 6353