Structure of radicals derived from hydroxypyrimidines in aqueous solution

Article abstract of DOI:10.1039/b204250a

EPR spectra of radicals derived from the three isomeric trihydroxypyrimidines and from 2-hydroxypyrimidine were obtained either in reduction or oxidation conditions at various pH values. Additionally, three other pyrimidine derivatives, 2-thiobarbituric a

Full text of DOI:10.1039/b204250a

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Structure of radicals derived from hydroxypyrimidines in aqueous
solution
Horácio M. Novais,a João P. Teloa and Steen Steenkenb
2
a Departamento de Engenharia Química, Instituto Superior Técnico, Av. Rovisco Pais,
P-1049-001 , Lisboa, Portugal
b Max-Planck-Institut für Strahlenchemie, D-45470, Mülheim a. d. Ruhr, Germany
Received (in Cambridge, UK) 1st May 2002, Accepted 24th May 2002
First published as an Advance Article on the web 20th June 2002
EPR spectra of radicals derived from the three isomeric trihydroxypyrimidines and from 2-hydroxypyrimidine
were obtained either in reduction or oxidation conditions at various pH values. Additionally, three other pyrimidine
derivatives, 2-thiobarbituric acid, 2-amino-4,6-dihydroxypyrimidine and 2-methyl-4,5,6-trihydroxypyrimidine, were
studied in the same conditions. Reduced radicals were produced by reaction of the pyrimidines with the hydrated
Ϫ
ؒ
Ϫ
ؒ
electron, the hydrogen atom and with the CO2 radical. In these conditions, the electron adducts as well as CO2
Ϫ
ؒ
ؒ
adducts were identified in most cases. Oxidized radicals were obtained by reaction of the substrates with OH , O
,
SO4 , and Br2 Ϫ radicals. The radicals detected and studied were either of the OH-adduct type or radicals derived
Ϫ
ؒ
ؒ
from its dehydration. The radicals were investigated by using in situ radiolysis and photolysis EPR techniques.
following a technique identical to that described for 4,6-
dihydroxy-5-methylpyrimidine.8
2-Methyl-4,5,6-trihydroxy-
Introduction
The mutagenic and lethal effects of ionizing radiation on living
systems are to a large extent due to the chemical transform-
ations induced in the DNA of the cell nucleus.1 Of the DNA
constituents, the purines and pyrimidines are more sensitive
than the deoxyribose phosphate moiety, particularly with
respect to reaction with the electrons ejected on ionization of a
molecule. Numerous studies have been performed with the aim
of understanding the sequence of events that lead from the
primary act of ionization of a purine or pyrimidine base to a
chemically recognizable species,1,2 the major tools applied being
electron paramagnetic resonance (EPR),3 pulse radiolysis,2,4
and product analysis.5 From EPR of irradiated crystals of
DNA constituents and of DNA itself,3 it has long been known
that the radicals formed by addition of one electron tend to
undergo protonation reactions, which may occur on a hetero-
atom or on a carbon atom.3 This type of reaction was later
observed to take place also in aqueous solution.2,6,7 The inde-
pendence of this reaction of environmental conditions indicates
that it has a high degree of intrinsic driving force. The question
thus arises whether this reaction is possibly a general one for
electron adducts of pyrimidines. Therefore it was considered
meaningful to extend the previous studies to the three isomeric
trihydroxypyrimidines, i.e. pyrimidine-2,4,6(1H,3H,5H )-trione
or 2,4,6-trihydroxypyrimidine (barbituric acid), dihydro-
pyrimidine-2,4,5(3H )-trione or 2,4,5-trihydroxypyrimidine
(isobarbituric acid) and dihydropyrimidine-4,5,6(1H )-trione or
4,5,6-trihydroxypyrimidine. For comparison purposes, three
other pyrimidines, 2-thiobarbituric acid, 2-amino-4,6-
dihydroxypyrimidine and 2-methyl-4,5,6-trihydroxypyrimidine,
were also studied.
pyrimidine was prepared similarly by using acetamide hydro-
chloride. The hydroxymalonate ester was obtained through
azeotropic esterification of tartronic acid (Fluka) with ethanol
and chloroform catalyzed with Amberlite resin IR-120 in the
acid form. 2-Amino-4,6-dihydroxypyrimidine was synthesized
by reaction of diethyl malonate with guanidine. All the other
substances were commercially available from Aldrich, Merck or
Fluka and were used without further purification.
The deoxygenated solutions, prepared by bubbling with
argon for about 30 minutes, typically contained either 10–100
mM formate (to scavenge H and OH radicals) or 0.1–0.5 M
tert-butyl alcohol (to scavenge OH radicals) and 1–2 mM of
the pyrimidine to react with eϪaq or H atoms. Alternatively, the
studies in oxidative media were carried out by bubbling the
solutions with N2O (to scavenge the hydrated electron). In any
case the solutions were irradiated with a beam (diameter 1 mm)
of 3 MeV electrons from a van de Graaff machine by using the
method described by Eiben and Fessenden.9
The in situ photolysis apparatus was described elsewhere.10
In this case, reductions were performed in the presence of tri-
ethylamine, and oxidations by photolysis of 25 mM potassium
persulfate or 2 mM 4-mercaptopyrimidine N-oxide.
When possible, pKa values were estimated from the depend-
ence of the relative concentrations of the acid and basic forms
of the radicals on the pH.
Results and discussion
Following our previous studies on radicals produced by
one-electron reduction7 or oxidation11 of dihydroxypyrimi-
dines like uracil, thymine, 4,6-dihydroxypyrimidine and some
of their derivatives in aqueous solution, we have extended these
investigations to the radicals derived from the isomeric tri-
hydroxypyrimidines. As before,7,11 the methods employed
involved in-situ radiolysis or photolysis of aqueous solutions of
Also relevant are one-electron oxidations, to which hydroxy-
pyrimidines are usually equally sensitive. For this reason, the
reactions with the powerful oxidants OH , Br2 and SO4
were additionally investigated.
Ϫ
Ϫ
ؒ
ؒ
ؒ
the pyrimidines and analysis by EPR of the radicals formed.
Experimental
Ϫ
ؒ
ؒ
On radiolysis of water, the radicals OH , H and e
are
aq
4,5,6-Trihydroxypyrimidine was synthesized by reaction of
formamidine hydrochloride with diethyl hydroxymalonate,
formed with yields per 100 eV of absorbed radiation as
indicated in the brackets, cf. eqn. (1),
1412
J. Chem. Soc., Perkin Trans. 2, 2002, 1412–1417
This journal is © The Royal Society of Chemistry 2002
DOI: 10.1039/b204250a

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Ϫ
ؒ
ؒ
in the equilibria. However, the possibility that some minor
tautomer may be responsible for the main radical observed
cannot be excluded.
H2O
OH (2.8), H (0.6), e aq (2.7)
(1)
The highly reactive OH radical can be scavenged by addition
of, e.g., tert-butyl alcohol to the solutions, cf. eqn. (2),
Radicals produced under reducing conditions (reactions with e؊
,
aq
؊
ؒ
ؒ
H or CO2
)
ؒ
ؒ
OH ϩ (CH3)3COH
H2O ϩ CH2(CH3)2COH (2)
2-Hydroxypyrimidine. This compound was studied as a
leaving only the reducing species, the hydrated electron and
the hydrogen atom, to react with the substrates. The resulting
radical CH2(CH3)2COH is not reactive enough to react with
model compound for trihydroxypyrimidines. On reaction with
Ϫ
eϪ at pH 4–10 in the presence of HCO2 (to scavenge OH )
ؒ
aq
ؒ
only one radical was detected, whose EPR spectrum consisted
the substrates on the ≤ ms timescale.
of a triplet (due to a pair of equivalent protons) with a coupling
constant of 12.55 G, another one of 2.20 G, a doublet (due
to one proton) of 2.55 G, a quintet (due to two nitrogens) of
1.64 G, and g = 2.0028. These parameters were interpreted in
terms of the allylic-type radical 1 (Scheme 2), formed by elec-
If it is required to remove also the H atom, propan-2-ol or
sodium formate is used instead of tert-butyl alcohol (cf. eqn.
(3)), in which case eϪaq is the main reactive radical remaining.
Ϫ
Ϫ
ؒ
ؒ
ؒ
OH (H ) ϩ HCO2
H2O (H2) ϩ CO2
(3)
The CO2 radical-anion has strongly reducing properties12
Ϫ
ؒ
and in many cases it leads to the same products as does eϪ
.
aq
However, it can also add to the pyrimidine system or to other
unsaturated molecules.
In order to perform one-electron oxidations, either the OH
radical was used, with its yield doubled by converting eϪaq into
OH radical by the use of N2O, cf. eqn. (4),
Ϫ
eϪaq ϩ H2O ϩ N2O
OH ϩ OH ϩ N2
(4)
ؒ
Scheme 2
Ϫ
Ϫ
ؒ
ؒ
or the secondary radicals Br2 or SO4 were produced via
OH (eqn. (5)) or e aq (eqn. (6)):
tron addition to the carbonyl group followed by protonation
at N1.
Ϫ
ؒ
This pattern may be compared to the behavior of the electron
adducts of uracil and its derivatives and of thymine, where pro-
tonation occurs at O4 (see Table III of ref. 7). In these cases the
protonation at O4 is favored because not only is its electron
affinity higher than that of O2, but also a delocalized, allyl-type
radical is formed (Scheme 3).
Ϫ
ϩ
ؒ
ؒ
OH ϩ Br ϩ H
H2O ϩ Br
(5)
Ϫ
Ϫ
ؒ
ؒ
Br ϩ Br
Br2
eϪaq ϩ S2O8
SO4 ϩ SO4
(6)
2Ϫ
2Ϫ
Ϫ
ؒ
ؒ
One-electron oxidations by OH usually proceed via
addition–elimination, with the addition step often unselective
and the elimination step slow (e.g. k < 103 sϪ1).13 It is for this
reason that one-electron oxidation of a system can be achieved
Ϫ
more cleanly by the use of Br2 or SO4 Ϫ, which appear to add
ؒ
ؒ
more selectively and eliminate (as 2BrϪ or as SO42Ϫ respectively)
Scheme 3
Ϫ
14
ؒ
more rapidly than does the OH /OH couple.
At pH > 10, the radical 1 formed by eϪ reaction with
The hydroxypyrimidines can in general exist in tautomeric
equilibria involving keto and enol forms.15 In aqueous solution
the keto forms are usually favored. An example of this is bar-
bituric acid where, out of the several tautomeric structures that
can be considered (cf. Scheme 1), the triketo structure largely
aq
2-hydroxypyrimidine disappeared and no new radical was seen.
The reason is probably that the aqueous electron does not react
with the enolate of 2-hydroxypyrimidine (pKa = 9.1715). Radical
1 could also be observed by photolysis of the parent compound
in the presence of triethylamine (Fig. 1a).
4,5,6-Trihydroxypyrimidine and 2-methyl-4,5,6-trihydroxy-
pyrimidine. On reduction of 4,5,6-trihydroxypyrimidine at pH =
2.5, a strongly polarized spectrum was seen which was char-
acterized by splittings from two pairs of equivalent hydrogens
(16.00 and 1.13 G), two equivalent nitrogens (2.62 G), and
g = 2.0033. This highly symmetric pattern is interpreted in terms
ؒ
of the radical 2 formed by H attack at C2 of the enolic form
and tautomerization, cf. Scheme 4:
Scheme 1
dominates with only a small contribution (≈5%) of the enol
structure.16
It is obvious that the keto forms should be easier to reduce
and the enol forms more easily oxidized. It is thus to be
expected that under reducing conditions the keto forms will
dominate the chemistry whereas under oxidizing conditions the
products will be mainly determined by the enol forms present
Scheme 4
J. Chem. Soc., Perkin Trans. 2, 2002, 1412–1417
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Table 1 Coupling constants (G) of pyrimidine–CO2 Ϫ adducts in aqueous solution
ؒ
Radical
N(1)/N(3)
H(2)
H(5)
H(6)
(N)–H
(O)–H
3
1.50d
4.98
1.55c
8.75
—
—
—
—
—
12.9
—
1.2e
—
0.18
—
0.11
—
4
0.15d
—
1.41e
2.60e
1.39
1.84
5-Me-4,6-DHPa
6
2.00d
0.41/0.29
0.68
22.75c
—
Orotic acidb
—
14.48
a 5-Methyl-4,6-dihydroxypyrimidine, adduct at C2, ref. 7. b Adduct at C6, ref. 18. c CH3. d 2N. e 2H.
Scheme 5
2,4,5-Trihydroxypyrimidine (isobarbituric acid). This com-
pound (pKa = 8.11 and 11.48)15 on reaction with eϪ at pH =
aq
7–8, yielded an EPR spectrum characterized by a triplet (due
to 2 equivalent protons) of 18.70 G, two doublets of 1.75 and
0.50 G, a quintet (due to 2 equivalent nitrogens) of 0.25 G,
and g = 2.0043. These parameters were interpreted in terms of
radical 5, formed by addition of one electron to C5 followed by
protonation at O5.
This electron addition is reasonable since the carbonyl group
at position 5 is expected to be the one with most electron-
affinity. The low nitrogen coupling is probably indicative of an
essentially exocyclic oxygen centred radical. The assignment of
the two (N)–H couplings shown in Scheme 6 can possibly be
Fig. 1 (a) EPR spectrum obtained upon photolysis of 2-hydroxy-
pyrimidine in the presence of triethylamine (radical 1); (b) EPR
spectrum obtained on electron irradiation of 4,5,6-trihydroxypyr-
imidine in the presence of sodium formate (radical 3). Q denotes the
quartz signal; (c) EPR spectrum obtained upon photolysis of 4,5,6-
trihydroxypyrimidine with potassium persulfate at pH = 8.3 (radical
14); and (d) pH = 12.1 (radical 15).
The di-keto structure for radical 2 is suggested on the basis of
the relatively large coupling constant for the (N)–H protons
and by analogy with the very similar methylene splitting of
the H-adduct of 4,6-dihydroxypyrimidine.7 The electron adduct
is assumed to be protonated because the EPR parameters are
independent of pH down to pH 2.2. Below this value the
solution turned colloidal.
Scheme 6
reversed. At pH values above 10 no other radicals formed by
electron addition to this compound were observed. The prob-
able reason is that the anion of isobarbituric acid has too low a
reactivity towards the electron.
When the reduction of the same compound was performed
in the presence of sodium formate at pH values between
5.9 and 7.2, an EPR signal, persistent even after bubbling the
solution with N2O, was observed. Radical 3 was identified as
When the reduction is performed with sodium formate
between pH 6.9 and 8.5, an EPR signal can be seen that persists
with the same or even increased intensity upon bubbling the
solution with N2O for about 30 minutes. This radical 6 is
Ϫ
Ϫ
ؒ
ؒ
the CO2 adduct at C2. Similarly, a CO2 adduct, radical 4,
was obtained with 2-methyl-4,5,6-trihydroxypyrimidine at pH
5–7 with the hyperfine coupling constants shown and g = 2.0048
(Scheme 5). It is to be noted that no other radical could be
observed from these compounds under reductive conditions.
assigned to a CO2 Ϫ adduct, as shown in Scheme 7. However, it
ؒ
must be emphasized that the assignments of both N and H
couplings shown are not unambiguous and can be reversed;
also, the small coupling of 0.11 can conversely be assigned to
the hydroxy hydrogen.
Ϫ
ؒ
Table 1 collects the known CO2 adducts of the hydroxy-
pyrimidine system.
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ؒ
Radicals formed under oxidizing conditions (reactions with OH ,
؊
؊
ؒ
ؒ
SO4 , Br2
)
2-Hydroxypyrimidine. Radicals derived from 2-hydroxypyr-
imidine by irradiation of aqueous solutions saturated with N2O
(reaction with OH radical) could only be detected at pH > 9. At
this pH a spectrum is observed that is composed of a nitrogen
quintet of 0.64 G, a hydrogen triplet of 3.8 G and g = 2.0049.
This spectrum, also obtained in the presence of 20 mM KBr,
was assigned to the secondary radical 9. This radical derives
Scheme 7
ؒ
from the OH adduct of 2-hydroxypyrimidine (not observed by
EPR) which suffers disproportionation giving, besides the
original compound, 2,5-dihydroxypyrimidine. The latter is
oxidised to give radical 9 (Scheme 10).
2,4,6-Trihydroxypyrimidine (barbituric acid). This compound
(pKa = 3.9 and 12.5)15 exists in aqueous solution mainly in the
tri-keto form in equilibrium with a small concentration
of the enolic form16 (Scheme 1). On electron irradiation of
an aqueous solution in the presence of 0.1 M tert-butyl
ؒ
alcohol (to scavenge OH ) at pH = 2.2, radical 7 was observed
(Scheme 8), whose EPR spectrum consisted of a triplet of
Scheme 10
Scheme 8
At pH ≥ 12 two new EPR spectra can be seen. One radical is
characterized by one hydrogen doublet of 17.6 G, one hydrogen
triplet of 8.65 G and one nitrogen quintet of 1.95 G (radical 10)
27.10 G, two proton doublets of 4.88 and 0.80 G, a 0.27 G
quintet and g = 2.0032. The observed radical must be derived
from the attack of the hydrogen atom on position 2 of the
enolic form.
A similarly large triplet due to two hydrogens bonded at C5 is
seen in a radical derived from uracil (aH = 32.1 G)7 and the
coupling of 4.88 G can be assigned to the hydrogen at C2,
which is of the same magnitude as, for example, the proton
coupling at the same position of radical 3.
ؒ
and can be assigned to the OH adduct to the position 5 of the
pyrimidine ring. The other EPR signal is due to radical 11, that
must be the product of OHϪ elimination from the preceding
radical and shows one hydrogen doublet of 13.45 G, one
hydrogen triplet of 8.5 G and one nitrogen quintet of 1.7 G
(Scheme 11):
2-Mercapto-4,6-dihydroxypyrimidine (2-thiobarbituric acid).
When the hydrated electron was reacted with 2-thiobarbituric
acid (pKa = 3.7 and 7.9) an EPR signal was detected between
pH 5.5 and 9.5 (radical 8). On the basis of the pH profile of
its persistence, the radical must be derived from the reaction
of eϪ with 2-thiobarbiturate mono-anion. In this case it is
aq
Scheme 11
expected that the hydrated electron adds to one of the carbonyl
groups at the 4 and 6 positions, because the oxygen has more
electron-affinity than sulfur. The symmetry pattern of the
coupling parameters indicates that the primary product of
electron addition to the mono-anion (i.e. the radical di-anion)
protonates to give radical-anion 8 (Scheme 9). It is to be noted
Noticeable is the analogy of the large doublet value of
radical 10 with those seen with OH adducts at C5 of other
hydroxypyrimidines.11,17 On the other hand, the structure of
radical 11 can be compared with that of the radical-anion
obtained from uracil by a similar mechanism.11,18
4,5,6-Trihydroxypyrimidine and 2-methyl-4,5,6-trihydroxy-
pyrimidine. On reaction of 4,5,6-trihydroxypyrimidine (4,5,6-
ؒ
THP) with OH at pH values between 3.5 and 5.5 an EPR
spectrum was detected, characterized by two equivalent
protons of 0.15 G and one proton with a 15.75 G splitting,
two equivalent nitrogens with 2.87 G and g = 2.0034. The
ؒ
radical structure is interpreted as that formed by OH addition
to C2 (radical 12). The small coupling constant of 0.15 G for
the pair of hydrogens is more consistent with the dilactim than
with the dilactam structure.
Scheme 9
At pH ≥ 5.5 lines of a new radical 14 showed up, which is
identified as the deprotonated form of the one-electron-
oxidized substrate, produced by elimination of H2O from the
OH-adduct 12. This radical has previously been described as a
secondary radical obtained from 4,6-dihydroxypyrimidine
under one-electron-oxidizing conditions.11 At pH ≈ 10 this
that there must be a rapid electron transfer between the two
carbonyl groups responsible for the symmetry of the radical-
anion.
Table 2 shows the known data for the electron and hydrogen
adducts of several hydroxypyrimidines.
J. Chem. Soc., Perkin Trans. 2, 2002, 1412–1417
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Table 2 Coupling constants (G) of pyrimidine electron or hydrogen adducts in aqueous solution
Radical
N(1)/N(3)
H(2)
H(5)
H(6)
(N)–H
2
2.62c
0.25c
0.27c
2.90c
1.45/0.2
2.45c
2.70c
2.40c
16.00d
—
4.88
—
—
—
—
1.13d
1.75/0.50
0.80
5
18.70d
—
7
8
27.10d
18.85d
32.10d
16.20d
17.95d
16.15/0.30b
—
18.8
—
—
—
1.50d
0.40
Uracila
—
4,6-DHPa
2-Me-4,6-DHPa
5-Me-4,6-DHPa
16.10
17.50b
16.15
0.55d
0.95d
0.90d
a Adduct at C5, ref. 7. b CH3. c 2N. d 2H.
radical begins to disappear to give rise to the corresponding
radical dianion 15 which was also obtained as a secondary
radical from 4,6-dihydroxypyrimidine.11
Under mildly basic conditions (pH 8.5–10) still another
radical 13 was seen with proton splittings of 9.84 and 0.50 G
and nitrogen splittings of 2.95 and 2.40 G and g = 2.0038. The
large doublet splitting of 9.84 G can be accounted for in terms
of a proton on an sp3 carbon and on this basis the radical is
identified as the ionized OH adduct to C2. Scheme 12 summar-
izes the formation of the described radicals 12 to 15.
Scheme 13
triplets (due to N) of 0.75 and 0.25 G, and g = 2.0028. These
parameters were interpreted in terms of formation of the
OH adduct to the 6-position of isobarbituric acid, radical 19
(Scheme 14). This radical was also studied by pulse radiolysis
with optical detection.19
Scheme 12
Scheme 14
The one-electron-oxidized radicals 14 and 15 could also
At low flow rates of the solution, i.e. at high conversion,
another radical, 20, with a higher g-factor of 2.0050 and having
only hyperfine constants corresponding to two equivalent
nitrogen splittings of 0.45 G, became apparent (Scheme 15).
be produced from the substrate 4,5,6-THP by one-electron
Ϫ
ؒ
oxidation, using either photochemically produced SO4 or
radiation-chemically generated Br2 Ϫ. The pKa of radical 14 was
ؒ
estimated to be 10.3.
ؒ
This radical has also been seen as a secondary product on OH
reaction with barbituric acid.18 The nature of the radical was
On oxidation of 2-methyl-4,5,6-trihydroxypyrimidine by
photolysis of aqueous solution with SO4 Ϫ and acetone at pH =
ؒ
verified by the fact that reacting dialuric acid (2,4,5,6-
3.5 an EPR spectrum could be observed that shows a hydrogen
quartet of 3.58 G, a hydrogen triplet of 0.4 G and a nitrogen
quintet of 1.53 G. This was assigned to the neutral radical
16 (Scheme 13). At pH ≥ 6.5 the monoionized radical 17 with a
hydrogen quartet of 3.53 G, a hydrogen doublet of 0.28 G and
two nitrogen triplets of 1.39 and 1.13 G can be observed. At pH
= 11 the di-ionized radical 18 is already present with a hydrogen
quartet of 4.10 G and a nitrogen quintet of 1.14 G.
Both these charged radicals 17 and 18 showed very intense
EPR signals and were also obtained as secondary radicals
from the oxidation of 2-methyl-4,6-dihydroxypyrimidine.11
pKa values of 16 and 17 were determined as 4.2 and 11.6,
respectively.
2,4,5-Trihydroxypyrimidine (isobarbituric acid). On reaction
ؒ
of this compound with OH at pH 4–7 an EPR spectrum was
obtained with hydrogen constants of 18.25, 1.02, and 0.50 G,
Scheme 15
1416
J. Chem. Soc., Perkin Trans. 2, 2002, 1412–1417

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ؒ
tetrahydroxypyrimidine) with OH at pH 4–7 produced the
same EPR spectrum. In both cases of barbituric acid and
isobarbituric acid, radical 20 is suggested to be formed by
disproportionation followed by one-electron oxidation, e.g. via
ؒ
OH addition–H2O elimination.
One-electron-oxidation of isobarbituric acid was also per-
Ϫ
formed with SO4 and with Br2 Ϫ. Under these conditions, an
EPR spectrum was observed at pH values between 2.3 and 4.0
with doublet splittings of 9.65, 3.45, and 0.20 G, nitrogen
splittings of 1.87 and 0.76 G, and g = 2.0048. These parameters
are interpreted in terms of the neutral radical 21 derived from
the enolic form of isobarbituric acid by one-electron-oxidation
followed by deprotonation. The same radical was produced by
ؒ
ؒ
Neta20 on reaction of OH with 5-halouracils at pH 2–3.
ؒ
When the pH was raised above about 5.5 the neutral radical
disappeared and lines from a new radical (22) appeared instead
(Scheme 16). The coupling constants (two hydrogen doublets of
Scheme 17
disproportionation of the OH-adduct, as found before for
isobarbituric acid (Scheme 15). This radical has some analogy
to radical-dianion 15, obtained by an analogous mechanism
from 4,6-dihydroxypyrimidine11 and by direct oxidation from
4,5,6-trihydroxypyrimidine.
Acknowledgements
HMN and JPT thank FCT for financial support through its
Centro de Processos Químicos da Universidade Técnica de
Lisboa.
Scheme 16
4.95 and 0.20 G and two nitrogen triplets of 2.20 and 1.10 G)
show that the spin density has increased on the nitrogens and
decreased on C-6, which is evidence for some electronic
rearrangement of the molecular system on further deproton-
ation. Obviously, for deprotonation to be possible one carbonyl
group has to enolize and, in the resulting delocalized radical
anion, spin density is transferred from the oxygens to the ring
nitrogens.
Above pH ≈ 11 the signal of the radical anion disappeared
giving rise to a new radical dianion 23 with the C-6 proton
splitting of 4.69 G, even smaller than that of the mono-anion,
and the difference in the coupling constants of the two nitro-
gens (2.30 and 0.87 G) is even larger, indicating a further
redistribution of spin density in favor of the heteroatoms. These
radical mono- and di-anions, 22 and 23, have previously been
References
1 For reviews see: (a) A. J. Bertinchamps, J. Hüttermann,
W. Köhnlein, R. Téoule, Eds.; Effects of Ionizing Radiation on DNA,
Springer, Berlin, 1978; (b) C. von Sonntag, The Chemical Basis of
Radiation Biology, Taylor and Francis, London, 1987.
2 S. Steenken, Chem. Rev., 1989, 89, 503.
3 For reviews see: (a) W. A. Bernhard, Adv. Radiat. Biol., 1981, 9, 199;
(b) J. Hüttermann, Ultramicroscopy, 1982, 10, 25; (c) D. M. Close,
W. H. Nelson and E. Sagstuen, in Electron Magnetic Resonance of
the Solid State, J. A. Weil, Ed.; Canadian Society for Chemistry,
Ottawa, 1987, p. 287; (d ) M. C. R. Symons, J. Chem. Soc., Faraday
Trans. 1, 1987, 83, 1.
4 For reviews see: C. von Sonntag, Int. J. Radiat. Biol., 1984, 46, 507;
C. von Sonntag and H.-P. Schuchmann, Int. J. Radiat. Biol., 1986,
49, 1.
5 For reviews see: (a) J. Cadet and M. Berger, Int. J. Radiat. Biol.,
1985, 47, 127; (b) J. Cadet, M. Berger and A. Shaw, in Mechanisms of
DNA Damage and Repair, M. G. Simic, L. Grossman, A. D. Upton,
Eds.; Plenum, New York, 1986, p. 69.
ؒ
described as secondary radicals from the reaction of OH with
uracil,18 and have also been obtained from 5-halouracils20 and
from 5-nitrouracil.21
6 D. J. Deeble, S. Das and C. von Sonntag, J. Phys. Chem., 1985, 89,
2,4,6-Trihydroxypyrimidine (barbituric acid). The reaction of
5784.
this compound with OH has been studied by Neta18 and found
ؒ
7 H. M. Novais and S. Steenken, J. Am. Chem. Soc., 1986, 108, 1.
8 R. Hull, B. J. Lovell, H. T. Openshaw and A. R. Todd, J. Chem. Soc.,
1947, 41.
to lead to formal one-electron-oxidation followed by deproton-
ation, the mechanism probably being OH addition–OHϪ
ؒ
9 K. Eiben and R. W. Fessenden, J. Phys. Chem., 1971, 75, 1186.
10 S. Steenken, W. Jaenicke-Zauner and D. Schulte-Frohlinde,
Photochem. Photobiol., 1975, 21, 21.
elimination.
2-Amino-4,6-dihydroxypyrimidine. The reaction of this com-
pound with OH produced by pulse radiolysis yielded an EPR
11 H. M. Novais and S. Steenken, J. Phys. Chem., 1987, 91, 426.
12 P. Wardman, J. Phys. Chem. Ref. Data, 1989, 18, 1637.
13 S. Steenken, J. Chem. Soc., Faraday Trans. 1, 1987, 83, 113.
14 S. Steenken, Free Radical Res. Commun., 1992, 16, 349.
15 (a) D. T. Hurst, An Introduction to the Chemistry and Biochemistry of
Pyrimidines, Purines and Pteridines, Wiley, New York, 1980; (b)
D. J. Brown, The Pyrimidines, Supplement I, 1970 and Supplement
II, 1985, Wiley, New York.
16 (a) V. I. Slesarev and B. A. Ivin, Zh. Org. Khim., 1974, 10, 113; (b)
M. V. Jovanovic and E. R. Biehl, J. Heterocycl. Chem., 1987, 24, 191.
17 H. Catterall, M. J. Davies and B. C. Gilbert, J. Chem. Soc., Perkin
Trans. 2, 1992, 1379.
ؒ
spectrum with a quintet of lines with 0.40 G, corresponding
to two equivalent nitrogens, another single nitrogen coupling
constant of 1.47 G, an hydrogen constant of 18.35 and two
equivalent hydrogen couplings with 1.56 G. The same spectrum
was obtained by photolysis in the presence of persulfate at basic
pH values (pH = 9 to 12). Since the SO4 Ϫ radical reacts mostly
ؒ
by one-electron oxidation, the radical observed in both cases
should not be the OH-adduct, but instead radical-anion 24,
resulting from OHϪ elimination (Scheme 17). It is to be noted
that there is some analogy between the observed coupling
constants of radical 24 and those of a similar radical obtained
from cytosine,22 4,6-dihydroxypyrimidine and uracil.11,18
A second EPR spectrum could also be observed super-
imposed on the previous one. This spectrum is consistent
with the secondary radical 25, which is produced from the
18 P. Neta, Radiat. Res., 1972, 49, 1.
19 M. Mori, S. Teshima, H. Yoshimoto, S. Fujita, R. Taniguchi,
H. Hatta and S. Nishimoto, J. Phys. Chem. B, 2001, 105, 2070.
20 P. Neta, J. Phys. Chem., 1972, 76, 2399.
21 P. Neta and C. L. Greenstock, Radiat. Res., 1973, 54, 35.
22 J. Geimer, K. Hildenbrand, S. Naumov and D. Beckert, Phys. Chem.
Chem. Phys., 2000, 2, 4199.
J. Chem. Soc., Perkin Trans. 2, 2002, 1412–1417
1417