Chemical radiation studies of 8-bromoguanosine in aqueous solutions

Article abstract of DOI:10.1021/ja991785f

Chemical radiolytic methods were used to investigate the reactions of hydrated electrons (e_aq^-) with 8-bromoguanosine (8-Br-Guo) as a function of pH. γ-Radiolysis of 8-Br-Guo in aqueous solutions followed by product studies showed the formation of guanosine (Guo) as a single product at various pH. In D₂O solutions the quantitative incorporation of deuterium at the 8-position was also observed. Pulse radiolysis revealed the instantaneous formation of a guanosine radical cation (Guo^.+ or its deprotonated forms) in acid or basic solutions. The same transient species results from the reaction of H^. with 8-Br-Guo at pH 3, as well as from the reaction of (CH₃)₂CO^.- with 8-Br-Guo at pH 13. In neutral solution, the initial electron adduct was rapidly protonated to give the first observable transient species that decays by first-order kinetics (k = 5 × 10⁴ s^-1) to produce the Guo(-H⁺)^. radical once again. Tailored experiments allowed the reaction mechanism to be defined in some detail.

Full text of DOI:10.1021/ja991785f

1900
J. Am. Chem. Soc. 2000, 122, 1900-1907
Chemical Radiation Studies of 8-Bromoguanosine in Aqueous
Solutions
Marcella Ioele,† Rita Bazzanini,† Chryssostomos Chatgilialoglu,*,† and
Quinto G. Mulazzani*,‡
Contribution from the I.Co.C.E.A. and F.R.A.E., Consiglio Nazionale delle Ricerche, Via P. Gobetti 101,
40129 Bologna, Italy
ReceiVed May 28, 1999. ReVised Manuscript ReceiVed NoVember 15, 1999
-
Abstract: Chemical radiolytic methods were used to investigate the reactions of hydrated electrons (eaq
)
with 8-bromoguanosine (8-Br-Guo) as a function of pH. γ-Radiolysis of 8-Br-Guo in aqueous solutions followed
by product studies showed the formation of guanosine (Guo) as a single product at various pH. In D2O solutions
the quantitative incorporation of deuterium at the 8-position was also observed. Pulse radiolysis revealed the
“instantaneous” formation of a guanosine radical cation (Guo•+ or its deprotonated forms) in acid or basic
solutions. The same transient species results from the reaction of H• with 8-Br-Guo at pH 3, as well as from
the reaction of (CH3)2CO•- with 8-Br-Guo at pH 13. In neutral solution, the initial electron adduct was rapidly
protonated to give the first observable transient species that decays by first-order kinetics (k ) 5 × 104 s-1
)
to produce the Guo(-H+)• radical once again. Tailored experiments allowed the reaction mechanism to be
defined in some detail.
Introduction
malian and bacterial cells, makes them more sensitive to the
lethal effects of ionizing and ultraviolet radiations.9 This property
of 5-Br-uracil derivatives has been exploited in clinical studies10
and has been used for mapping protein-nucleic acid interac-
tions.11 The addition of an electron to 5-Br-uracil derivatives
affords the adduct 1 which fast releases a bromide ion with the
formation of the vinyl radical 2 (eq 1).5 Species 1 has also been
During the last two decades a number of publications have
appeared referring to the biological and immunological activities
of 8-bromoguanosine (8-Br-Guo) and its 3′,5′-cyclic mono-
phosphate. For example, it has been reported that the metabolic
processing of 8-Br-Guo by guanosine specific enzymes is
minimal as a consequence of the molecular conformational
changes.1 The presence of a bulky substituent such as the
bromine atom at the C8 position of guanosine destabilizes the
normal anti orientation of the base, sterically constraining the
molecule to the syn conformation.2 This arrangement, together
with the change in the sugar conformation, is reflected in a
modification of the 13C and 1H NMR spectra, giving a
characteristic upfield shift of the C2′ signal and a downfield
shift of the H2′ signal. More recently, 8-bromoguanosine-3′,5′-
cyclic monophosphate has been found to be capable of decreas-
ing basal and growth factor-stimulated DNA synthesis in
primary aortic smooth muscle cells from newborn rats, display-
ing an antimitogenic effect similar to some NO-releasing agents.3
The interaction of ionizing and ultraviolet radiations with
5-bromo-substituted pyrimidine nucleosides has been intensively
investigated.4-8 It has been shown, for example, that 5-bromo-
2′-deoxyuridine, when incorporated into DNA of both mam-
reported by Saito and co-workers in the photoreaction of duplex
(4) Danziger, R. M.; Hayon, E.; Langmuir, M. E. J. Phys. Chem. 1968,
72, 3842. Langmuir, M. E.; Hayon, E. J. Chem. Phys. 1969, 51, 4893.
Zimbrick, J. D.; Ward, J. F.; Myers, L. S., Jr. Int. J. Radiat. Biol. 1969, 16,
505.
(5) Adams, G. E.; Willson, R. L. Int. J. Radiat. Biol. 1972, 22, 589.
Patterson, L. K.; Bansal, K. M. J. Phys. Chem. 1972, 76, 2392. Bansal, K.
M.; Patterson, L. K.; Schuler, R. H. J. Phys. Chem. 1972, 76, 2386. Bhatia,
K.; Schuler, R. H. J. Phys. Chem. 1973, 77, 1888. Wagner, B. O.; Klever,
H.; Schulte-Frohlinde, D. Z. Naturforsch. 1974, 28b, 86. Rivera, E.; Schuler,
R. H. J. Phys. Chem. 1983, 87, 3966.
(6) Nese, C.; Yuan, Z.; Schuchmann, M. N.; von Sonntag, C. Int. J.
Radiat. Biol. 1992, 62, 527.
(7) Fuciarelli, A. F.; Sisk, E. C.; Zimbrick, J. D. Int. J. Radiat. Biol.
1994, 65, 409. Fuciarelli, A. F.; Sisk, E. C.; Miller, J. H.; Zimbrick, J. D.
Int. J. Radiat. Biol. 1994, 66, 505. Beach, C.; Fuciarelli, A. F.; Zimbrick,
J. D. Radiat. Res. 1994, 137, 385.
(8) Ito, S.; Saito, I.; Matsuura, J. J. Am. Chem. Soc. 1980, 102, 7535.
Swanson, B. J.; Kutzer, J. C.; Koch, T. H. J. Am. Chem. Soc. 1981, 103,
1274.
(9) Ogata, R.; Gilbert, W. Proc. Natl. Acad. Sci. 1977, 74, 4973. Cadet,
J.; Vigny, P. In Biorganic Photochemistry; Morrison, H., Ed.; Wiley: New
York, 1990; Vol. 1, pp 1-272.
(10) Mitchell, J. B.; Russo, A.; Kinsella, T. J.; Glatstein, E. Int. J. Radiat.
Oncol. Biol. Phys. 1986, 12, 1513.
† I.Co.C.E.A.
‡ F.R.A.E.
(1) Goodman, M. G.; Weigle, W. O. Proc. Natl. Acad. Sci. U.S.A. 1981,
78, 7604. Goodman, M. G.; Weigle, W. O. J. Immunol. 1982, 129, 2715.
Goodman, M. G.; Weigle, W. O. J. Immunol. 1983, 130, 2580. Goodman,
M. G.; Weigle, W. O. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 862. Koo, G.
C.; Jewell, M. E.; Manyak, C. L.; Sigal, N. H.; Wicker, L. S. J. Immunol.
1988, 140, 3249.
(2) Uesugi, S.; Ikehara, M. J. Am. Chem. Soc. 1977, 99, 3250. Jordan,
F.; Niv, H. Biochim. Biophys. Acta 1977, 476, 265. Uesugi, S.; Ohkubo,
M.; Urata, H.; Ikehara, M.; Kobayashi, Y.; Kyogoku, Y. J. Am. Chem. Soc.
1984, 106, 3675. Polissou, M.; Theophanides, T. Inorg. Chim. Acta 1987,
137, 195.
(11) Blatter, E. E.; Ebright, Y. W.; Ebright, R. H. Nature 1992, 359,
650. Willis, M. C.; Hicke, B. J.; Uhlenbeck, O. C.; Cech, T. R.; Koch, T.
H. Science 1993, 262, 1255.
(3) Dhaunsi, G. S. M.; Kaur, C.; Hassid, A. Am. J. Pharm. 1997, 272,
1342.
10.1021/ja991785f CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/23/2000

8-Bromoguanosine in Aqueous Solutions
J. Am. Chem. Soc., Vol. 122, No. 9, 2000 1901
oligonucleotides contaning a 5′-ABrU-3′ sequence.12 These
authors proposed single electron transfer from the adjacent
adenine moiety of the 5′-side to the photoexcited 5-Br-uracil
residue. In turn, vinyl radical 2 either abstracts a hydrogen (from
a hydrogen donor5,6 and/or from the closest sugar unit of the
backbone12) or induces cross-linking to amino acid deriva-
tives.11,13 To our knowledge the analogous electron adducts of
8-bromo purine derivatives have not been reported to date.14
aromatic compounds.18 Herein we report detailed chemical
radiation studies of 8-Br-Guo in aqueous solution where this
mechanistic scenario is partially true. In particular, we have
investigated the reaction of 8-Br-Guo with the reducing spe-
cies: eaq-, H•, (CH3)2CO•-.
Results and Discussion
Generation of Radicals.19 Radiolysis of neutral water leads
to the species eaq-, HO•, and H• as shown in eq 2 where the
values in parentheses represent the yields expressed in terms
of G-values (molecules/100 eV of absorbed radiation). The
reactions of eaq- with the substrates were studied by irradiating
deoxygenated solutions containing 0.25 M t-BuOH or 0.13 M
i-PrOH. In the presence of t-BuOH, only HO• radicals are
scavenged (eq 3, k3 ) 6.0 × 108 M-1 s-1), whereas both HO•
and H• species react with i-PrOH (eqs 4 and 5, k4 ) 1.9 × 109
M-1 s-1, k5 ) 7.4 × 107 M-1 s-1). In alkaline solution, in the
The effect of bromination was also considered for macro-
molecules such as RNA and DNA, where early studies showed
the occurrence of mutations and changes in the amino acid
composition of an isolated protein.15 Bromination produces
significant biochemical modifications of certain nucleic acids,
the most important being the formation of 5-bromopyrimidine
and 8-bromopurine nucleotides.16 In their recent report Sevilla
and co-workers studied with ESR spectroscopy the electron
transfer occurring within bromine-doped DNA, which contained
5-bromo-6-hydroxy-5,6-dihydrothymine, 5-bromocytosine, and
8-bromoguanine moieties in a 0.2:1:0.23 ratio.16 The observed
paramagnetic species that result from the electron scavenging
are assigned to pyrimidine-type adducts. Based on comparison
with some available data, they suggested that 8-Br-dGuo is not
expected to compete effectively for the electron with the other
brominated moieties, although they pointed out the experimental
difficulties associated with the determination of 8-Br-dGuo as
an electron trap in the system.
-
presence of t-BuOH, additional eaq is produced from the
reaction of H• with HO- (eq 6, k6 ) 2.2 × 107 M-1 s-1),
whereas at acidic pH (e3) the reaction of eaq- with H+ to give
H• (eq 7, k7 ) 2.3 × 1010 M-1 s-1) becomes relevant.
Interestingly, von Sonntag and co-workers have shown that
the electron adducts (and/or their heteroatom-protonated forms)
of thymine, adenine, and cytosine transfer an electron to 5-Br-
uridine, whereas the electron adduct of guanine does not behave
accordingly.6 They concluded that guanine can serve as the
ultimate sink for the electron which is in antithesis with the
general thought of the electron transfer within the DNA
molecule, i.e., the electron loss resides at the guanine moieties
and the electron gain is at the pyrimidine moieties.17
The (CH3)2CO•- species was generated by irradiating N2O-
saturated solutions containing 0.13 M i-PrOH at pH 13. The
presence of N2O transforms efficiently eaq- into the O•- radical
(eq 8, k8 ) 9.1 × 109 M-1 s-1). The HO• radical [pKa(HO•) )
11.9] is in equilibrium with its conjugated base O•- (eq 9/-9,
k9 ) 1.2 × 1010 M-1 s-1, k-9 ) 1 × 108 s-1). Hydrogen
abstraction from i-PrOH by HO•, H•, and O•- produces (CH3)2C-
(•)OH which deprotonates to give (CH3)2CO•- (eq 10, pKa )
12.03).20 The radical anion is a better reductant than the neutral
radical, (E°[(CH3)2CO,H+/(CH3)2C(•)OH] ) -1.39 V, E°[(CH3)2-
CO/(CH3)2CO•-)] ) -2.1 V).21,22
On the basis of these observations, we thought that 8-Br-
Guo could be prompted to capture electrons and perhaps it loses
the bromine ion rapidly like the 5-Br-uracil derivatives, to give
the corresponding radical in the 8-position. This expectation is
further strengthened by a very recent report which has shown
that debromination of 8-Br-dGuo can be achieved in a quantita-
tive yield by methylene blue and visible light similar to bromo-
(12) Sugiyama, H.; Fujimoto, K.; Saito, I. J. Am. Chem. Soc. 1995, 117,
2945. Sugiyama, H.; Fujimoto, K.; Saito, I.; Kawashima, H.; Sekine, T.;
Ishido, Y. Tetrahedron Lett. 1996, 37, 1805. Cook, G. P.; Greenberg, M.
M. J. Am. Chem. Soc. 1996, 118, 10025. Sugiyama, H.; Fujimoto, K.; Saito,
I. Tetrahedron Lett. 1997, 38, 8057. Fujimoto, K.; Sugiyama, H.; Saito, I.
Tetrahedron Lett. 1998, 39, 2137.
(13) Norris, C. L.; Meisenheimer, P. L.; Koch, T. H. J. Am. Chem. Soc.
1996, 118, 5796 and references therein.
(14) Photolysis (at 254 nm) of 8-bromo-2′-deoxyadenosine in deareated
aqueous solution gave 5′,8-cyclo-2′-deoxyadenosine in a low yield. See:
Romieu, A.; Gasparutto, D.; Molko, D.; Cadet, J. J. Org. Chem. 1998, 63,
5245.
The radicals CO2•- [E°(CO2/CO2•-) ) -1.91 V] and CH2O•-
•
[the conjugated base of CH2OH: pKa(•CH2OH) ) 10.71;
E°(CH2O/CH2O•-) ) -1.81 V]21 were generated by irradiating
N2O-saturated solutions at pH 13 containing 0.1 M sodium
formate or methanol, respectively. The rate constants for the
scavenging of H• and HO• radicals by the formate ion are 2.1
(15) Jones, A. S.; Woodhouse, D. L. Nature 1959, 183, 1603. Brammer,
K. W. Biochim. Biophys. Acta 1963, 72, 217.
(16) Razskazovskii, Y.; Swarts, S. G.; Falcone, J. M.; Taylor, C.; Sevilla,
M. D. J. Phys. Chem. B 1997, 101, 1460 and references therein.
(17) For example, see: Steenken, S. Chem. ReV. 1989, 89, 503. Bernhard,
W. A. J. Phys. Chem. 1989, 93, 2187. Sevilla, M. D.; Becker, C.; Yan, M.;
Summerfield, S. R. J. Phys. Chem. 1991, 95, 3409. Cullis, P. M.;
McClymont, J. D.; Malone, M. E.; Mather, A. N.; Podmore, I. D.; Sweeney,
M. C.; Symons, M. C. R. J. Chem. Soc., Perkin Trans. 2 1992, 1409.
Steenken, S.; Jovanovic, S. V. J. Am. Chem. Soc. 1997, 119, 617. Holmlin,
R. E.; Dandliker, P. J.; Barton, J. K. Angew. Chem., Int. Ed. Engl. 1997,
36, 2714. Kelley, S. O.; Jackson, N. M.; Hill, M. G.; Barton, J. K. Angew.
Chem., Int. Ed. Engl. 1999, 38, 941. Giese, B.; Wessely, S.; Spormann,
M.; Lindemann, U.; Meggers, E.; Michel-Beyerle, M. E. Angew. Chem.,
Int. Ed. Engl. 1999, 38, 996.
(18) Venkatarangan, L.; Yang, D.-H.; Epling, G. A.; Basu, A. K.
Tetrahedron Lett. 1999, 40, 1441.
(19) (a) Buxton, G. V.; Greenstock, C. L.; Helman, W. P.; Ross, A. B.
J. Phys. Chem. Ref. Data, 1988, 17, 513 and references therein. (b) Ross,
A. B.; Mallard, W. G.; Helman, W. P.; Buxton, G. V.; Huie, R. E.; Neta,
P. NDRL-NIST Solution Kinetic Database, Ver. 3; Notre Dame Radition
Laboratory: Notre Dame, IN and NIST Standard Reference Data: Gaith-
ersburg, MD, 1998.
(20) Laroff, G. P.; Fesseden, R. W. J. Phys. Chem. 1973, 77, 1283.
(21) Schwarz, H. A.; Dodson, R. W. J. Phys. Chem. 1989, 93, 409.
(22) All the redox potentials given in this work are vs NHE.

1902 J. Am. Chem. Soc., Vol. 122, No. 9, 2000
Ioele et al.
Table 1. Rate Constants (M-1 s-1)a for the Reaction of eaq-, H•,
•-
and Br2 with 8-Bromoguanosine (8-Br-Guo) and Guanosine (Guo),
Together with pKa Values of the Two Nucleosides
8-Br-Guo (pH)b
Guo (pH)
pKac
1.4, 8.4, 12.4
1.1 × 1010 (7)
5.0 × 109 (13)
4.5 × 108 (3)
4.5 × 107 (7)
3.0 × 108 (13)
1.9, 9.25, 12.33d
6 × 109 (7)e,f
k(eaq- + S)
k(eaq- + S-)
k(H• + S)
k(Br2•- + S)
k(Br2•- + S-)
9 × 107 (12)e,f
5 × 108 (6.5)e
3.9 × 107 (7)g
2.5 × 108 (13)d
a At 22 ( 2 °C; estimated to be accurate to (10%. b This work.
c pKa = 12.4 refers to the deprotonation of the sugar moiety. d From
ref 23. e From ref 26. f From ref 6. g From ref 27.
Figure 1. Effect of irradiation dose on the consumption of 8-Br-Guo
and the formation of Guo from the continuous irradiation of vacuum
degassed solutions containing ca. 1.5 mM 8-Br-Guo and 0.25 M
t-BuOH at pH 3 (O, b), ∼7 (4, 2), and 13 (0, 9).
× 108 and 3.2 × 109 M-1 s-1, respectively; the rate constants
for the scavenging of these radicals by methanol are 2.6 × 106
and 9.7 × 108 M-1 s-1, respectively.
The dibromine radical anion, Br2•-, was generated by
irradiating N2O-saturated solutions containing 0.1 M KBr. Under
these conditions, at pH 7, the HO• radical is converted into the
dibromine radical anion through the process shown in eq 11
(k11 ) 1.1 × 1010 M-1 s-1). At pH 13, the O•- species also
generates Br2 radicals through reaction 12 (k12 ) 2.2 × 108
•-
M-1 s-1).
Acid-Base Properties of 8-Br-Guo. At pH ∼7, the spectrum
of 8-Br-Guo shows two bands at 210 and 265 nm with ꢀ ) 2.1
× 104 and 1.5 × 104 M-1 cm-1, respectively. Since the spectrum
is pH dependent, the acid-base properties of the species could
be determined by following the changes in absorbance at
selected wavelengths. From the analysis of the optical density
vs pH curves, the pKa values reported in Table 1 were obtained.
For comparison purposes, in Table 1 the pKa values for Guo
are also reported.23 For 8-Br-Guo the pKa value of 1.4,
representing the protonation of one of the nitrogen sites, is
slightly lower than the corresponding value of 1.9 for Guo. The
value of pKa ) 8.4 obtained for 8-Br-Guo represents the
deprotonation of the NH group and is almost one pKa unit lower
than the corresponding value reported for Guo, i.e., 9.25. The
difference probably arises from the presence of the electron-
withdrawing bromine atom in the 8-position.24 The value of pKa
∼ 12.4 observed for 8-Br-Guo and Guo represents a deproton-
ation from the sugar moiety, presumably from the 2′ position,
where the alkoxy anion formed is stabilized by an intramolecular
hydrogen bond with the neighbor OH group.25
γ-Radiolysis of 8-Br-Guo. Aqueous solutions of 8-Br-Guo
(ca. 1.5 mM) and t-BuOH (0.25 M) at pH 3, ∼7, and 13 were
prepared. Control experiments showed that 8-Br-Guo is stable
at the various pH values used for at least 3 days. The samples
containing 10 mL of deoxygenated solution were irradiated
under stationary-state conditions with a dose rate of ca. 27 Gy/
min followed by HPLC analysis. In all experiments, the
formation of Guo was identified by comparison of the retention
time with an authentic sample. At pH ∼7 and 13 mass balances
obtained by HPLC using 8-Br-Guo and Guo as external
standards were close to 100%. At pH ∼7 bromide ion was
Figure 2. G-values as a function of irradiation dose for the consump-
tion of 8-Br-Guo (3, 0) and the formation of Guo (1, 9) from the
continuous irradiation of vacuum degassed solutions containing ca. 1.5
mM 8-Br-Guo and 0.13 M i-PrOH or 0.25 M t-BuOH at pH 13. The
lines are the linear fit to the average data of G[-(8-Br-Guo)] and
G(Guo).
detected by HPLC and its yield was found to be identical with
that of Guo. At pH 3 Guo was the predominant product,
although small amounts of a second product which has not yet
been identified were also formed. Figure 1 shows the consump-
tion of 8-Br-Guo and the formation of Guo. The concentrations
of 8-Br-Guo and Guo decreased and increased, respectively, as
the dose increased in the range of 0-1.5 kGy. Furthermore,
the efficiency of debromination decreased with decreasing the
pH of the medium.
Analysis of the data in terms of G-values showed that (i) at
pH 13 G[-(8-Br-Guo)] and G(Guo) are close to 3.2 and
independent of the dose (Figure 2), (ii) at pH 7 G[-(8-Br-Guo)]
and G(Guo) are close to 3.2 when the line is extrapolated at
zero dose, and (iii) at pH 3 the G[-(8-Br-Guo)] and G(Guo)
are close to 2 when the lines are extrapolated at zero dose (see
Supporting Information). Taking into account that G(eaq-) +
G(H•) ) 3.2 (eq 2) and that hydrogen atoms are converted into
solvated electrons at pH 13 (eq 6) whereas the solvated electrons
are partially converted into hydrogen atoms at pH 3 (eq 7), our
results lead to the conclusion that all solvated electrons, and to
some minor extent the hydrogen atoms, react quantitatively with
8-Br-Guo to yield Guo. In this respect, the reaction could also
be a useful synthetic tool.18 In fact, a sample irradiated for 3 h
at pH 13 afforded Guo in 84% yield as determined by HPLC
using an external standard (see Supporting Information).
When i-PrOH replaced t-BuOH at pH 13, the efficiency of
the 8-Br-Guo conversion to Guo increased appreciably. Figure
2 shows that the G-values in i-PrOH are more than twice those
(23) Jovanovic, S. V.; Simic, M. G. J. Phys. Chem. 1986, 90, 974 and
references therein.
(24) A more pronounced decrease in the pKa (from 9.5 to 8.1) is observed
when comparison is made between uracil6 and bromouracil.23
(25) Saenger, W. Principles of Nucleic Acid Structure; Springer-
Verlag: New York, 1984.

8-Bromoguanosine in Aqueous Solutions
J. Am. Chem. Soc., Vol. 122, No. 9, 2000 1903
Figure 4. Absorption spectra obtained from the pulse radiolysis at
pH 13 of (4) 1 mM 8-Br-Guo, 0.25 M t-BuOH, Ar-purged, 2 µs after
the pulse; (O) 1 mM Guo, 0.1 M KBr, N2O-saturated, 30 µs after the
pulse; and (b) 1 mM 8-Br-Guo, 0.1 M KBr, N2O-saturated, 30 µs after
the pulse. Inset: pH dependence of absorption at 720 nm obtained from
the pulse radiolysis of Ar-purged solutions containing 1 mM 8-Br-
Guo and 0.25 M t-BuOH; dose ) 22 Gy, optical path ) 2.0 cm. The
solid line represents the pKa equation fit to the data; the inflection point
was obtained at pH 10.4.
Figure 3. 1H NMR spectra in the range of 5.8 to 8.4 ppm obtained
after irradiation of 8-Br-Guo in H2O (upper spectrum) or in D2O (lower
spectrum) at pH 13.
Scheme 1
Figure 5. Plot of kobs vs [8-Br-Guo] for the decay of eaq- (λmon ) 720
nm) and for the buildup of product (λmon) 310 nm) from the pulse
radiolysis of Ar-purged solutions containing 0.25 M BuOH abd 0.1 M
NaOH. Insets: Decay observed at 720 nm (upper left) and buildup
observed at 310 nm (lower right) in the presence of 0.5 mM 8-Br-
Guo; optical path ) 2.0 cm, dose per pulse ) 22 Gy. The solid lines
represent the first-order kinetic fits to the data.
is a facile process.28 Arrhenius expressions are available for these
reactions which are governed by pseudo-first-order rate con-
stants. These kinetic data suggest that under our conditions the
hydrogen-deuterium exchange is unimportant. In fact, control
experiments performed on a solution of Guo under the same
experimental conditions showed no such exchange. Therefore,
the mechanism reported in Scheme 1 cannot be correct since
the hydrogen in the 8-position originated from water.
in t-BuOH. At pH 13 the (CH3)2C(•)OH radical is mainly in
the form of its conjugated base (see eq 10/-10) which is a good
-
reductant. Therefore, we suggest that at pH 13 both eaq and
(CH3)2CO•- are able to reduce 8-Br-Guo.
From a mechanistic point of view, these results suggest that
-
the reaction of eaq with 8-Br-Guo produces Guo in a manner
-
similar to the reaction of eaq with 5-bromouridine which
Pulse Radiolysis Studies in Basic Solutions. The optical
produces uridine. Thus the addition of an electron to 8-Br-Guo
results in the release of a bromide ion and the corresponding
radical at the 8-position that reacts with t-BuOH or i-PrOH to
afford Guo (Scheme 1).
-
absorption spectrum obtained after the reaction of eaq with
8-Br-Guo at pH 13, taken 2 µs after the pulse, is shown in Figure
4 (open triangles). The spectrum contains three bands centered
at 310, 480, and 720 nm, respectively. The ꢀ-values were
To investigate the origin of the hydrogen donor the reactions
were also performed in D2O. The samples containing 8-Br-Guo
(ca. 1.5 mM) and t-BuOH (0.25 M) in D2O at different pH were
irradiated with a dose of 1.5 kGy followed by 1H NMR analysis.
To our surprise, in all cases a deuterium atom was incorporated
in the 8-position, i.e., 8-D-Guo. Figure 3 shows for comparison
-
calculated using G ) 3.2, which represents the sum of G(eaq
)
and G(H•) since the HO• radicals are scavenged by the t-BuOH
(cf. eqs 2 and 3). The time profile of the formation of this
-
transient (Figure 5, right inset) or the disappearance of eaq
(26) Candeias, L. P.; Wolf, P.; O’Neill, P.; Steenken, S. J. Phys. Chem.
1992, 96, 10302.
(27) Candeias, L. P.; Steenken, S. J. Am. Chem. Soc. 1989, 111, 1094.
(28) Lane, M. J.; Thomas, C. J., Jr. Biochemistry 1979, 18, 3839.
Livramento, J.; Thomas, C. J., Jr. J. Am. Chem. Soc. 1974, 96, 6529.
Tomasz, M.; Olson, J.; Mercado, C. M. Biochemistry 1972, 11, 1235.
1
the H NMR spectra in the range of 5.5 to 8.5 ppm of the
reactions carried out at pH 13 in H2O and D2O, respectively. It
is well-known from the literature that hydrogen-deuterium
exchange at the C8 position of guanosine and related nucleotides

1904 J. Am. Chem. Soc., Vol. 122, No. 9, 2000
Ioele et al.
Scheme 2
(Figure 5, left inset)29 leads to a pseudo-first-order rate constant,
k
obs, and was measured at a number of different 8-Br-Guo
concentrations. From the slope of the linear plot, the bimolecular
rate constant was found to be 5.0 × 109 M-1 s-1 (Figure 5). In
comparison with the analogous reactions of Guo (see Table 1),
the presence of bromine increases the rate constant of the
reduction by a factor of 55. The transient decays by second-
order kinetics with 2k ) 6.8 × 108 M-1 s-1, and its decay does
not give rise to absorbing species at λ > 320 nm.
The above-described spectrum resembled in shape and
ꢀ-values those obtained by the reaction of Guo with oxidants
•-
•- 27
such as SO4 or Br2
.
To closely compare the spectra of
•-
oxidized Guo, we also examined the reactions of Br2 with
Guo and 8-Br-Guo.30 The reactions were carried out in N2O-
saturated solutions cantaining 0.1 M KBr (vide infra). The
optical absorption spectra obtained at pH 13 after the completion
Figure 6. Absorption spectra obtained from the pulse radiolysis of
Ar-purged solutions containing 1 mM 8-Br-Guo: (b) at pH ∼7 in the
presence of 0.13 M i-PrOH, taken 2 µs after the pulse, and (4) at pH
3 in the presence of 0.25 M t-BuOH, taken 8 µs after the pulse.
•-
of the reactions of Guo and 8-Br-Guo with Br2 (30 µs after
× 108 M-1 s-1. On the other hand, no reaction was observed
the pulse) are also shown in Figure 4. The spectra obtained from
the oxidation of Guo (open circles) and from the reduction of
8-Br-Guo (open triangles) are identical. Furthermore, the OD
change at 720 nm for reduced 8-Br-Guo follows a pKa curve
profile with an inflection point at pH 10.4 (Figure 4, inset and
Scheme 2) which is in good agreement with the inflection point
at pH 10.7 reported for the oxidized Guo.27 On the other hand,
the spectrum obtained from the oxidation of 8-Br-Guo (solid
circles) showed a similar shape although the ꢀ-values were
higher than those obtained for the oxidized Guo. A similar pKa
value was also measured (Scheme 2). The rate constants for
•-
between the species CO2 and CH2O•- with 8-Br-Guo.
At pH 13, 8-Br-Guo and Guo are present in their deprotonated
forms at the base moiety, i.e., 8-Br-Guo(-H+) and Guo(-H+),
respectively (Table 1 and Scheme 2). There is no doubt that
the species obtained from the reaction of 8-Br-Guo(-H+) with
eaq or (CH3)2CO•- is identical with that obtained from the
-
reaction of Guo(-H+) with Br2•-. The electron-adduct of 8-Br-
Guo(-H+), upon elimination of Br-, would give a species
equivalent to Guo(-2H+)•- with a pKa ) 10.4 (Scheme 2).
Although the reaction may seem stoichiometrically simple, the
γ-radiolysis results show that the mechanism is not. Attempts
to detect any intermediate have been unsuccessful. For example,
the kinetics at 720 (representing mainly the decay of eaq-) and
310 nm (representing mainly the formation of Guo(-2H+)•-)
were the same even at the highest concentration of the substrate
(0.5 mM) used for these experiments. In other words, the
formation of Guo(-2H+)•- is “instantaneous” with a rate constant
higher than 107 s-1, i.e., the process should be complete in less
than 300 ns.
•-
the reactions of Br2 with 8-Br-Guo were determined (dose
per pulse ) 9-23 Gy) by measuring the rate of optical density
•-
decrease of Br2 as a function of the concentration of 8-Br-
Guo.29 The bimolecular rate constant was found to be 3.0 ×
108 M-1 s-1, which is similar to that of the analogous reaction
of Guo (Table 1). Oxidized 8-Br-Guo and oxidized Guo decay
by second-order kinetics with 2k ) 1.5 × 108 and 2 × 108
M-1 s-1, respectively.
The reactivity of the (CH3)2CO•- radical anion toward 8-Br-
Guo at pH 13 was also examined. It was found that (CH3)2CO•-
Pulse Radiolysis Studies in Neutral Solutions. A rate
constant of 1.1 × 1010 M-1 s-1 was determined for the reactions
reacts with 8-Br-Guo with k ) 8.3 × 107 M-1 s-1 giving a
-
of 8-Br-Guo with eaq at pH ∼7 by measuring the rate of the
-
optical density decrease of eaq- as a function of the concentration
of the added nucleoside.29 This value is slightly higher than the
analogous reaction of Guo at the same pH (Table 1).6,26 Figure
6 shows the optical absorption spectrum obtained from the
reaction of 8-Br-Guo with eaq- (solid circles). The ꢀ-values were
calculated using G ) 2.6, since the H• and HO• species are
scavenged by the i-PrOH and therefore do not contribute to the
reaction.31 The same spectrum that contains a sharp band with
transient identical with that obtained after reduction by eaq
.
This transient decays by second-order kinetics with 2k ) 1.9
(29) For eaq- at 720 nm, ꢀ ) 1.9 × 104 M-1cm-1; for Br2•- at 360 nm,
ꢀ ) 1 × 104 M-1cm-1: Hug, G. L. Natl. Stand. Ref. Data Ser., Natl. Bur.
Stand. 1981, No. 69.
(30) The reaction of Br2•- with Guo has previously been studied by pulse
radiolysis at pH 12-13.23, 27 However, we were obliged to repeat some of
these experiments since some information of interest to us was not given
in these papers.

8-Bromoguanosine in Aqueous Solutions
J. Am. Chem. Soc., Vol. 122, No. 9, 2000 1905
) 1.4 × 108 M-1 s-1. In addition, the spectra, recorded after
completion of the decay of the first transient obtained in the
absence or in the presence of phosphates were found to be
identical with each other. Furthermore, the rate of decay at 620
nm increases with increasing [H+], so that at pH 4, 2 µs after
the pulse, the band at 620 nm has already disappeared (Figure
7, open triangles). From the kinetic analysis of a fast component
observed at pH 4 and from computer modeling of the system,37
the rate constant for the reaction with H+ was estimated to be
+
kH = 1 × 1010 M-1 s-1
.
To obtain information about the reactivity of the transient
species originating from the reactions of 8-Br-Guo with eaq- at
pH ∼7, its redox properties were investigated toward methyl
viologen (MV2+) and N,N,N′,N′-tetramethyl-p-phenylenediamine
(TMPD).38,39 The experiments were performed in Ar-purged
solutions containing, in addition to 1 mM 8-Br-Guo and 0.25
M t-BuOH, 20-100 µM MV2+ or TMPD. The results showed
that this radical is unable to reduce MV2+ whereas it is able to
effect the oxidation of TMPD.42 Inasmuch as the radical derived
from t-BuOH is unreactive toward TMPD, we concluded that
the TMPD•+ radical cation formed in the system originates
solely from the species obtained from the reactions of eaq- and
H• with 8-Br-Guo. The yield of TMPD•+ increased with
increasing 8-Br-Guo concentration, approaching a plateau that
corresponded to 80% (lower limit) of the sum of the yields of
Figure 7. Absorption spectra obtained from the pulse radiolysis (dose
) 24 Gy) of Ar-purged solutions containing 1 mM 8-Br-Guo at pH
∼7 with 0.25 M t-BuOH, taken 2 (9) and 45 µs (4) after the pulse; at
pH 7.7 (buffered with phosphates), with 0.25 M t-BuOH, taken 2 µs
after the pulse (b); and at pH 4 with 0.25 M t-BuOH, taken 2 µs after
the pulse (3). Insets: Time dependence of absorption at 620 nm from
the pulse radiolysis of Ar-purged solutions containing 1 mM 8-Br-
Guo and 0.13 M i-PrOH at pH ∼7 in H2O (a) and in D2O (b); dose per
pulse ) 9 Gy; optical path ) 2.0 cm. The solid lines represent the
first-order kinetic fits to the data from which k(H2O) ) 5.0 × 104 s-1
and k(D2O) ) 6.2 × 103 s-1 were obtained.
λmax ) 310 nm and a broad band in the 500-650 nm region
was obtained by replacing i-PrOH with t-BuOH (Figure 7, solid
squares). The decay of this transient (monitored at 620 nm)
follows first-order kinetics.33 The measured rates were found
to increase slightly with increasing doses, which indicates the
presence of second-order contributions owing to radical-radical
reactions. To minimize this effect, the rate was measured at a
low dose (9 Gy), where k ) 5.0 × 104 s-1 was obtained (Figure
7, inset a).34 When H2O was replaced by D2O, the decay at
620 nm became slower, with k ) 6.2 × 103 s-1 (Figure 7, inset
b), i.e., the solvent kinetic isotope effect (kH/kD) is 8.0.
Furthermore, the disappearance of this species gives rise to a
new transient the spectrum of which, taken 45 µs after the pulse,
is also shown in Figure 7 (open triangles). The last transient is
-
eaq and H•. A rate constant of k ) 1.7 × 109 M-1 s-1 was
measured.44,45
The mechanism we conceived for the reactions discussed
above is illustrated in Scheme 3. The observed initial transient
at neutral pH is assigned to radical 4. That is, the reaction of
-
eaq with 8-Br-Guo should afford the radical anion 3 which is
expected to be rapidly protonated to yield the neutral radical
4.46 This intermediate, which is unreactive toward MV2+ and
molecular oxygen, efficiently oxidizes TMPD to give the
corresponding cation. The decay of 4 follows a first-order
kinetics to give Guo(-H+)• with a rate constant of 5.0 × 104
s-1. By replacing H2O with D2O as the reaction medium, a
kinetic isotopic effect kH/kD ) 8.0 was determined which
•-
very similar to that obtained after the oxidation of Guo by Br2
(36) Rafi, A.; Sutton, H. C. Trans. Faraday Soc. 1965, 61, 877. Treinin,
A.; Hayon, E. J. Am. Chem. Soc. 1975, 97, 1716. Wong, D.; Di Bartolo, B.
J. Photochem. 1975, 4, 249-268.
at pH ∼7, taken 100 µs after the pulse to allow the completion
of the Br2 decay.35
•-
(37) Curtis, A. R.; Sweetenham, W. P. FACSIMILE/CHEKMAT Users’
Manual; AERE 12805, UKAEA, 1988.
It was also found that the rate of decay at 620 nm is
influenced by phosphates. The absorption spectra obtained 2
µs after the pulse at pH ∼7 in the presence and in the absence
of phosphates are almost superimposable (Figure 7). However,
the presence of phosphates accelerated the decay at 620 nm,
with kobs increasing linearly with [H2PO4-], resulting in kphosp
(38) MV2+, E°(MV2+/MV•+) ) -0.45 V and TMPD, E°(TMPD•+
/
TMPD) ) 0.27 V.39 The rate constants for the reduction of MV2+ and/or
oxidation of TMPD, and the associated yields of MV•+ and/or TMPD•+
,
were determined at 602 and 565 nm, respectively, which represent the
40
absorption maxima of MV•+ (ꢀ ) 1.37 × 104 M-1 cm-1
)
and TMPD•+
(ꢀ ) 1.25 × 104 M-1 cm-1),41 respectively.
(39) Wardman, P. J. Phys. Chem. Ref. Data 1989, 18, 1637 and references
(31) (a) By examining a N2O-saturated solution containing 8-Br-Guo
and i-PrOH we established that the (CH3)2C(•)OH radical does not react
with 8-Br-Guo at pH 7. (b) On the other hand, some reactivity of the
(CH3)2C(•)OH radical toward 8-Br-Guo was instead observed at pH 3.32
(32) This behavior is similar to that previously observed in the case of
adenosine which does not react with the 2-propanol radical at pH 7 whereas
at pH 2 it does, see: Moorthy, P. N.; Hayon, E. J. Am. Chem. Soc. 1975,
97, 3345.
therein.
(40) Watanabe, T.; Honda, K. J. Phys. Chem. 1982, 86, 2617.
(41) Fujita, S.; Steenken, S. J. Am. Chem. Soc. 1981, 103, 2540.
(42) It must be noted that, under the conditions employed, i.e., [8-Br-
Guo] g 10 [TMPD], the competition between 8-Br-Guo and TMPD for
eaq- is well in favor of 8-Br-Guo since k(eaq- + TMPD) ) 9.1 × 107 M-1
s-1 43
.
(43) Rao, P. S.; Hayon, E. J. Phys. Chem. 1975, 79, 1063.
(33) The addition of molecular oxygen has no effect on the decay of
this transient species.
(44) The plot kobs vs [TMPD] shows an intercept corresponding to k )
2 × 104 s-1. This suggests a parallel path (possibly related to the second-
order contributions observed for the decay at 620 nm) for the decay of the
precursor of TMPD•+ that would account for about 10% diminution of the
yield of TMPD•+. Therefore, the reaction of 8-Br-Guo and eaq-/H• generates
species possessing oxidazing properties in nearly quantitative yiels.
(45) The TMPD•+ radical cation is also formed in the presence of 0.01
M of phosphates. Since the formation, yield, and decay of TMPD•+ are
similar in the absence and presence of phosphates, this suggests that both
radical 4 and Guo(-H+)• (Scheme 3) oxidize TMPD with comparable rate
constants. However, at higher concentrations of TMPD, the major path is
the reaction of TMPD with radical 4.
(34) This rate constant was found to be independent of the nature and
concentration of the HO• scavengers t-BuOH or i-PrOH (0.1-1.0 M). This
observation rules out the possibility that the transient decays by reacting
with the alcohol, for example by abstracting from it a hydrogen atom.
(35) The reaction of Br2•- with Guo is relatively slow (Table 1) so that
the disproportionation reaction of Br2•-, 2k ) 3.8 × 109 M-1s-1 at the
ionic strength ) 0.1, had to be taken into account when the ꢀ-values of
•-
oxidized Guo at pH 7 were calculated. The disproportionation of Br2
-
generates Br- and Br3 which absorbs strongly at 266-270 nm (ꢀ ∼ 4 ×
104 M-1cm-1).36 The difference between the spectra of oxidized Guo and
the spectrum of reduced 8-Br-Guo in the 300 nm region could be due to
the effect of the tail of the spectrum of Br3- (see Supporting Information).
(46) Conductometric detection showed to be the case for the reaction of
-
eaq with Guo.27

1906 J. Am. Chem. Soc., Vol. 122, No. 9, 2000
Ioele et al.
Scheme 3
Figure 8. Absorption spectra obtained from the pulse radiolysis of an
Ar-purged solution containing 1 mM 8-Br-Guo and 0.25 M t-BuOH at
pH 3, taken 1 (b) and 8 µs (3) after the pulse; dose ) 24 Gy; optical
path ) 2.0 cm. Insets: Time profile of the absorptions at 310 (a) and
400 nm (b). The solid lines represent the first-order kinetic fits to the
data.
Scheme 4
suggests the involvement of proton transfer or a complex
transition state.47 A transition state such as 5 is expected to be
involved since this reaction is accelerated by phosphate and H+
as found to be the case. It is also worth emphasizing that this
mechanistic proposal is consistent with the γ-radiolysis studies
which showed the incorporation of H or D atoms at C8 derived
from H2O and D2O, respectively.48,50
Pulse Radiolysis Studies in Acidic Solutions. Figure 6 (open
triangles) shows the optical absorption spectrum obtained from
the reaction of 8-Br-Guo with eaq- and H• at pH 3. The ꢀ-values
were calculated using G ) 3.2, which represents the sum of
G(eaq-) and G(H•).
The spectrum at pH 3 was generated in two steps: the first
was complete in less than 1 µs and corresponds to the reaction
of 8-Br-Guo with eaq- (vide infra), the second was complete in
about 8 µs and assigned to the reaction of 8-Br-Guo with H•.
Figure 8 shows that the two processes generated the same
spectrum which contains one band centered at 310 nm and two
broad and less intense bands around 400 and 500 nm. This
transient spectrum resembles in shape and ꢀ-values those
(47) Williams, J. M.; Kreevoy, M. M. In AdVances in Physical Organic
Chemistry; Gold, V., Ed.; Academic Press: London, 1968; Vol. 6, p 63.
Saunders, W. H., Jr. In Techniques of Chemistry; Lewis, E. S., Ed.; Wiley:
New York, 1974; Vol. 6, p 211.
•-
obtained by the reactions of Guo with SO4 which produced
the guanosine radical cation (Guo•+).27 Furthermore, a pKa =
3.8 is observed for this transient which matched well with the
reported value of 3.9 for Guo•+.27
(48) One referee suggested that an alternative mechanism may be present
which is the loss of a free bromine atom (Br•) from the radical ion 3, giving
the 8-guanosinyl anion which is directly protonated to the product; the
oxidation of TMPD (Scheme 3) could then be attributed to the Br•. Although
this proposal could explain some of the observed results,49 it is easy to
verify since it is well-known that in the presence of Br- the Br• is converted
into the detectable species Br2•-, and therefore easily monitored.19b Pulse
irradiation of Ar-purged solutions at pH ∼7 and 13 containing 2 M t-BuOH
and 1 mM 8-Br-Guo produced the same results both in the presence and in
the absence of 5 mM Br-. In other words, the presence of 5 mM Br- did
The rate constant for the reaction of H• with 8-Br-Guo was
obtained at pH 3 (in the presence of 0.25 M t-BuOH)31b from
the linear dependence of the rate of the second buildup at 310
and 400 nm at different 8-Br-Guo concentrations (0.5 and 1.0
mM). Examples of this analysis are shown in Figure 8, insets
a and b. A value of 4.5 × 108 M-1 s-1 was obtained that is
similar to that for the reaction of H• with Guo (Table 1).26
The reaction mechanism could involve the addition of H• to
the double bond located between N7 and C8 to give the amino
radical 6, followed by a very fast heterolytic cleavage of the
â-CBr bond (Scheme 4).51 In the transition state, the C-Br bond
•-
not show detectable amounts of Br2 nor did it affect the kinetics for the
formation of the spectrum of the oxidized species.
(49) This mechanism has been shown to operate in other systems, e.g.,
Br• released from the reaction of eaq- with N-bromosuccinimide, see: Lind,
J.; Shen, X.; Eriksen, T. E.; Merenyi, G.; Eberson, L. J. Am. Chem. Soc.
1991, 113, 4629.
(50) Recent ab initio calculations on the 8-Br-Guo•- (intermediate 3 in
Scheme 3) indicate that the unpaired electron resides at the six-membered
ring mainly at the C atom bonded to the three nitrogens. These calculations
suggest that the intermediate 3 can release neither a bromide ion nor a
bromine atom. (We are grateful to Dr. M. Guerra for sharing these
unpublished results with us).
(51) At pH 3 the protonated form of 8-Br-Guo is ca. 5% (Scheme 4). It
-
is expected that eaq reacts rapidly also with this form to give Guo•+ via
intermediate 4 (see Scheme 3).

8-Bromoguanosine in Aqueous Solutions
J. Am. Chem. Soc., Vol. 122, No. 9, 2000 1907
buffered with phosphates (Merck). For the determination of the solvent
kinetic isotope effect, D2O (Aldrich, 99.8 atom % D) was used.
is expected to be strongly polarized, with the positive charge
being delocalized on the neighboring nitrogens, and the interac-
tion of H+ with Br should facilitate the ejection of the bromide
ion. However, a transition state such as 5 could also account
for the reaction of the H• atom with 8-Br-Guo to give Guo+•
and ultimately Guo.
Pulse Radiolysis. Pulse radiolysis with optical absorption detection
was performed by using a 12 MeV linear accelerator, which delivered
20-200 ns electron pulses with doses between 5 and 50 Gy, by which
OH•, H•, and eaq are generated with 1-20 µM concentrations.55 The
-
pulse irradiations were performed at room temperature (22 ( 2 °C) on
samples contained in Spectrosil quartz cells of 2 cm optical path length.
Solutions were protected from the analyzing light by means of a shutter
and appropriate cutoff filters. The bandwidth used throughout the pulse
radiolysis experiments was 5 nm. The radiation dose per pulse was
monitored by means of a charge collector placed behind the irradiation
cell and calibrated with an N2O-saturated solution containing 0.1 M
Conclusions
The affinity of the solvated electron with 8-Br-Guo is very
-
high. The rate constants of eaq with 8-Br-Guo and its
deprotonated form is much faster than the corresponding
reactions of Guo or the analogous reaction of other nucleosides19
and comparable to 5-bromouridine.4,5 The results described
HCO2 and 0.5 mM methyl viologen, using Gꢀ ) 9.32 × 104 at 602
-
nm.56 G(X) represents the number of molecules of species formed per
-
demonstrate that the reaction of 8-Br-Guo with eaq in neutral
100 eV of energy absorbed by the system.
and acidic solutions leads quantitatively to Guo. The reaction
mechanism is described in Scheme 3 and shows that radical 4
is involved as an intermediate followed by the ejection of a
bromide ion to give the deprotonated form of the guanosine
radical cation via the proposed transition state 5. In basic
solution the reaction is still more efficient, although the
mechanism is not straightforward.52 Therefore, the mechanistic
Continuous Radiolyses. Continuous radiolyses were performed at
room temperature (22 ( 2 °C) on 10 mL samples using a 60Co-
Gammacell with a dose rate of ∼27 Gy min-1. The exact absorbed
radiation dose was determined with the Fricke chemical dosimeter by
taking G(Fe3+) ) 15.5.57 The reactions of 8-Br-Guo with eaq and H•
-
were investigated using vacuum degassed solutions containing 1.5 mM
substrate and 0.25 M t-BuOH that were irradiated with appropriate
doses. The solutions irradiated at pH 3 and 13 were neutralized with
the required amount of NaOH, 0.1 and HCl 0.1 M, respectively,
followed by HPLC or NMR analysis. Qualitative and quantitative HPLC
studies were performed on a Waters Associates Unit equipped with a
model 600 multisolvent delivery system and controller, a 486 tunable
absorbance detector, and a Millenium 2010 data workstation. The
aqueous solutions of the samples were loaded after suitable neutraliza-
tion onto a reverse-phase analytical column (Waters Spherisorb,
S5ODS2, C18, 150 × 4.6 mm, 3 mm), eluted in water with a 0-90%
methanol gradient over 30 min at a flow rate of 1 mL/min, and detected
at 254 nm. Absolute concentrations of 8-Br-Guo and Guo were
interpolated from external standard calibration curves. The absolute
concentration of the bromide ion released was detected at 210 nm using
KBr as external standard. 1H NMR spectra were performed on a Varian
Mercury 400 MHz.
-
scenario of the debromination of 8-Br-Guo by the action of eaq
is different from those determined for the bromoaryl and
bromovinyl derivatives.18 Furthermore, it should be noted that
under continuous radiolysis in the whole range of pH we studied,
how the Guo+• or its deprotonated forms afford Guo is still
unclear.53,54 Further work on this intriguing mechanism as well
as on the analogous reactions of 8-bromopurine derivatives is
in progress.
Experimental Section
Materials. 8-Bromoguanosine (Aldrich) and guanosine (Fluka) were
used as received. N,N,N′,N′-Tetramethyl-p-phenylenediamine was puri-
fied by sublimation. Sodium formate (Merck), i-PrOH (C. Erba for
fluorimetry), t-BuOH (Fluka, puris.), KBr (Merck, Suprapur), and 1,1′-
dimethyl-4,4′-bipyridinium dichloride (methyl viologen, MV2+) (Aldrich
or Sigma) were employed. Solutions were freshly prepared using water
purified with a Millipore (Milli-Q) system; their pH was the “natural”
pH (∼7) or was adjusted with HClO4 or NaOH (Merck, Suprapur) or
Acknowledgment. We thank A. Monti and Dr. A. Martelli
for assistance with the pulse radiolysis experiments. We also
thank Ciba Specialty Chemicals for a grant to M.I. and R.B.
(52) We believe a mechanistic scheme similar to that at neutral solution
is the most appropriate one. We suggest that at pH 13 the deprotonated
form (at the N1 position) of intermediate 4 could lose a bromide ion rapidly
with the concomitant 1,2H shift. The driving force should be the delocal-
ization of the negative charge which favors a C-Br polarized bond.
(53) It has been suggested that the hydration of the guanine radical cation
in double-stranded DNA may be responsible for the formation of 8-oxo-
dGuo moiety.54 For example, see: Kasai, H.; Yamaizumi, Z.; Berger, M.;
Cadet, J. J. Am. Chem. Soc. 1992, 114, 9692. Cullis, P. M.; Malone, M. E.;
Merson-Davies, L. A. J. Am. Chem. Soc. 1996, 118, 2775. Meggers, E.;
Michel-Beyerle, M. E.; Giese, B. J. Am. Chem. Soc. 1998, 120, 12950.
(54) However, it is well-known that 8-oxo-Guo is not derived from the
Guo•+ (Candeias, L. P.; Steenken, S. J. Am. Chem. Soc. 1992, 114, 699)
which is in agreement with our results.
Supporting Information Available: G-values dependence,
kinetic data, and absorption spectra (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
JA991785F
(55) Hutton, A.; Roffi, G.; Martelli, A. Quad. Area Ric. Emilia Romagna
1974, 5, 67.
(56) Mulazzani, Q. G.; D’Angelantonio, M.; Venturi, M.; Hoffman, M.
Z.; Rodgers, M. A. J. J. Phys. Chem. 1986, 90, 5347.
(57) Spinks, J. W. T.; Woods, R. J. An Introduction to Radiation
Chemistry, 3rd ed.; Wiley: New York, 1990; p 100.