Tautomers of one-electron-oxidized guanosine

Article abstract of DOI:10.1002/anie.200501087

(Chemical Equation Presented) Aminic or iminic: Two tautomeric forms of oxidized guanosine have been produced by chemical radiation methods - by direct oxidation of guanosine or from the protonation of the 8-bromoguanosine electron adduct - and identified. The tautomerization from the iminic to the aminic arrangement has an activation energy of 23.0 kJ mol^-1 and occurs through a complex transition state (see scheme).

Full text of DOI:10.1002/anie.200501087

Communications
Guanosine Tautomers
DOI: 10.1002/anie.200501087
Tautomers of One-Electron-Oxidized
Guanosine**
Chryssostomos Chatgilialoglu,* Clara Caminal,
Maurizio Guerra, and Quinto G. Mulazzani
8-Bromoguanine derivatives, such as 1a and 1b, capture
electrons (eaqꢀ) with quantitative formation of the corre-
sponding debrominated nucleosides[1,2] and, therefore, they
are efficient detectors of excess electron-transfer processes.[3]
Indeed, in two recent papers 8-bromo-2’-deoxyguanosine
(1b) was incorporated in a varietyof single- and double-
Figure 1. Absorption spectra obtained from the pulse radiolysis of Ar-
purged solutions containing 1 mm 1a at pHꢁ7 with 0.25m tBuOH,
recorded 2 ms (solid line) and 45 ms (dashed line) after the pulse
(adapted from ref. [1]). The lines show the calculated vertical optical
transitions for radical 2 (dashed lines, l(f): 290 (0.128), 350 (0.054),
470 nm (0.014)) and its tautomer 3 (solid lines, l(f): 290 (0.138), 350
(0.042), 610 nm (0.051)).
bromo-9-methylguanine (1c) radical anion, protonated at N7
(as suggested previously[1]) and at anyother possible sites; no
optical transition was predicted to exist above 500 nm.
Interestingly, protonation at C8 produces loss of Brꢀ, and it
could be hypothesized that the first observable species in
pulse radiolysis had already lost Brꢀ. Consequently, we
computed the optical spectra (transition wavelengths l and
oscillator strengths f) of the deprotonated 9-methylguanine
radical cation (i.e., oxidized 9-methylguanine 2) and its
tautomers bearing an iminic substituent at C2. The computed
data for the radical 2 are reported in Figure 1 and are in good
accord with values assigned with certaintyto the oxidized
guanosine.[10] More interestingly, calculations showed that a
band is computed at wavelengths longer than 500 nm onlyfor
the tautomer 3. The computed optical transitions are also
reported in Figure 1 and are in good agreement with the
experimental spectrum recorded 2 ms after the pulse; these
data correspond to the transient of uncertain structure.
On the basis of these findings, we propose a revised
stranded oligonucleotides and G-quadruplexes, and the
ꢀ
reaction with eaq indicated that excess electron transfer is
effective.[2,4]
ꢀ
The reaction of eaq with 1a was previouslystudied by
pulse-radiolysis techniques in some detail.[1] These experi-
ments revealed the formation of two short-lived intermedi-
ates at pH ꢁ 7. Figure 1 shows the absorption spectrum of the
first observable species (solid line) obtained 2 ms after the
pulse. This species of uncertain structure decays by first-order
kinetics (k = 5.0 ” 104 sꢀ1) to produce the one-electron-oxi-
dized guanosine (dashed line).[1] In the present work we
studied the kinetics in the temperature range of 5.8–50.38C
and obtained the following Arrhenius parameters: log(A/
sꢀ1) = 8.7 ꢂ 0.4 and Ea = 23.0 ꢂ 2.5 kJmolꢀ1 (errors corre-
spond to one standard deviation).
ꢀ
mechanism for the reaction of eaq with 1 (Scheme 1). The
What is the structure of the first observable species, which
has a characteristic absorbance around 600 nm? We found
previouslythat time-dependent (TD) DFT calculations at the
B3LYP/6-31G* level[5,6] provide reliable optical transitions
for nucleoside radicals.[7,8] In neutral solution, the initial
[9]
electron adduct of 1 should be rapidlyprotonated. There-
fore, TD-B3LYP/6-31G* calculations were carried out on 8-
[*] Dr. C. Chatgilialoglu, Dr. C. Caminal, Dr. M. Guerra,
Dr. Q. G. Mulazzani
ISOF, Consiglio Nazionale delle Ricerche
Via P. Gobetti 101, 40129 Bologna (Italy)
Fax: (+39)051-639-8349
Scheme 1. Proposed mechanism for the reaction of eaqꢀ with 8-bromo-
guanosine (1a). The initial adduct 4 is rapidly protonated to give the
first observable species 5. The tautomer 6 is assigned to the second
transient species observed in the pulse-radiolysis studies (see
Figure 1).
E-mail: chrys@isof.cnr.it
[**] This research was supported in part by the European Community’s
Marie Curie Research Training Network under contract MRTN-CT-
2003-505086 (CLUSTOXDNA). We thank A. Monti and A. Martelli
for technical assistance.
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Chemie
initial electron adduct 4 is rapidlyprotonated at the C8
position to release Brꢀ and the first observable transient
species 5,[11] which should have a pKa value similar to that of
oxidized guanosine (pKa = 3.9).[10] The subsequent tautome-
rization 5!6 occurs with a log(A/sꢀ1) = 8.7, which suggests a
complex transition state, and an activation energyof
23.0 kJmolꢀ1. Another point supporting a complex transition
state is also the previouslyreported kinetic isotope effect
k(H2O)/k(D2O) = 8.0.[1]
The reaction barrier (Ea) for the direct tautomerization
3!2 is computed to be 183.7 kJmolꢀ1 at the B3LYP/6-31G*
level. Such a large value is in evident contrast with the
activation energymeasured for the
&
Figure 3. Absorption spectrum ( ) obtained from the pulse radiolysis
of Ar-purged solutions containing 0.5 mm 7 at pHꢁ7 and 0.25m
tautomerization 5!6. In analogy
with the keto–enol tautomerization
*
tBuOH, recorded 10 ms after the pulse. Absorption spectrum ( ) is
in guanine[12] and 8-oxo-7,8-dihydro-
taken from reference [10] and refers to the reaction of Br2Cꢀ with 9. The
lines show the calculated vertical optical transitions for radical 8
(R=Me) (l(f): 290 (0.063), 360 (0.036), 650 nm (0.040)).
guanine,[13] the water-assisted proton
transfer is computed to occur with a
much lower reaction barrier (the
calculated transition-state structure
Figure 2. Structure of the
transition state for the
is given in Figure 2). Indeed, the
reaction barrier is computed to be as
small as 18.8 kJmolꢀ1, in good
accord with experiment.[14]
water-assisted tautomeri-
zation 3!2 computed at
the B3LYP/6-31G* level.
Distances are in Š.
To gain insight into the different
tautomeric forms, the 8-bromo derivatives
7 and 10
(Scheme 2) were prepared, since the analogous tautomeriza-
&
Figure 4. Absorption spectrum ( ) obtained from the pulse radiolysis
of Ar-purged solutions containing 0.5 mm 10 at pHꢁ7 and 0.25m
*
)
tBuOH, recorded 2 ms after the pulse. Absorption spectrum (
obtained from the pulse radiolysis of Ar-purged solutions containing
0.1 mm 12 and 10 mm K2S2O8 at pHꢁ7 with 0.1m tBuOH, recorded
6 ms after the pulse. The lines show the calculated vertical optical tran-
sitions for radical 11 (R=Me) (l(f): 290 (0.149), 390 (0.070), 480
(0.029), 1030 nm (0.027)).
ꢀ
Scheme 2. Reactions of eaq with 8-bromoguanosine derivatives 7 and
10, of Br2Cꢀ with 9, and of SO4Cꢀ with 12. The radicals 8 and 11 are
assigned to the transient species observed in the pulse-radiolysis stud-
ies (see Figures 3 and 4, respectively).
order kinetics and their disappearance does not lead to other
transients. Furthermore, these spectra are identical to those
obtained after the oxidation of 9 byBr 2Cꢀ [10] and of 12 bySO 4Cꢀ
at pH ꢁ 7 (Figures 3 and 4).
Therefore, the spectra in Figures 3 and 4 are assigned to
radicals 8 and 11, respectively, which can be obtained either
from the reduction of 8-bromoguanine derivatives or from the
oxidation of the debrominated compounds as illustrated in
Scheme 2. TD-B3LYP/6-31G* calculations carried out on the
corresponding 9-methyl derivatives of radicals 8 and 11 fit
verywell with our assignment. Indeed, the computed optical
spectra for the radical 8 and for the radical 11[19]] are in good
agreement with the spectra shown in Figures 3 and 4,
respectively. Interestingly, both the spectrum of radical 8
and that of radical 5 have a band at 610–620 nm, which is
consistent with an iminic substitution at the C2 position.
Obviously, radical 5 can tautomerize to 6, whereas radical 8 is
blocked in one form.
tion 5!6 could not occur as a result of alkylation at NH and
NH2 moieties. g-Radiolysis of 7 and 10 in aqueous solutions at
pH ꢁ 7 was followed byproduct studies, which showed the
quantitative formation of debrominated derivatives 9 and 12,
respectively.[15] Rate constants of 1.1 ” 1010 and 8.0 ” 109 mꢀ1 sꢀ1
were determined at pH ꢁ 7 for the reactions of eaqꢀ with 7 and
10, respectively, by measuring the rate of the optical density
ꢀ
decrease of eaq as a function of the concentration of the
added nucleoside.[18] Figures 3 and 4 show the optical
absorption spectra obtained from these reactions. The e
values were calculated using G = 0.27 mmolJꢀ1, since HOC
species are scavenged bythe added tBuOH, and the HC atoms
follow another path and therefore do not contribute to the
reaction.[16] The decayof these transients follows second-
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[14] A detailed computational investigation of the water-assisted
tautomerization including the effect of the basis set and of a
polarizable continuum model for the aqueous solution will be
presented in a full account; preliminarycalculations indicate
that Ea should increase byonly3–4 kJmol ꢀ1 at the higher level of
theory.
In conclusion, our work here demonstrates the first
directlyobserved differences of the two tautomeric forms of
oxidized guanosine. The energeticallymost stable form 6 is
the one that is obtained directlybyoxidation of guanosine.
The less stable form 5 is obtained from the protonation of the
8-bromoguanosine electron adduct, and its tautomerization to
[15] Deaereated aqueous solutions containing ca. 1.5 mm 7 (or 10)
and 0.25m tBuOH at pH ꢁ 7 were irradiated under stationary-
state conditions with a total dose of 2 kGyat a dose rate of ca.
15 Gyminꢀ1 followed byHPLC analysis. [16] Compound 9 (or 12)
was the onlydetectable product, and the mass balances were
close to 100%; analysis of the data, in terms of radiation
chemical yield, gives G(ꢀ7) = 0.36, G(9) = 0.33, G(ꢀ10) = 0.33
and G(12) = 0.30 mmolJꢀ1. Taking into account that G(eaqꢀ) +
G(HC) = 0.33 mmolJꢀ1, our results lead to the conclusion that
solvated electrons and hydrogen atoms react with 7 and 10 to
yield the observed product.
6 has an activation energyof 23.0 kJmol ꢀ1
.
Experimental Section
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 HOC, HC, and eaq
were generated with concentrations of 1–20 mm. Continuous radiol-
yses were performed at room temperature using a 60Co-Gammacell,
with a dose rate of ca. 15 Gyminꢀ1. Compounds 7, 10, and 12 were
prepared following known procedures.[20]
ꢀ
[16] Radiolysis of neutral water leads to eaq (0.27), HOC (0.28), and
HC (0.062), the values in parentheses represent the radiation
chemical yields in units of mmolJꢀ1; in the presence of 0.25m
tBuOH, HOC is scavenged efficiently( k = 6.0 ” 108 mꢀ1 sꢀ1),
whereas HC reacts onlyslowly( k = 1.7 ” 105 mꢀ1 sꢀ1).[17]
[17] A. B. Ross, W. G. Mallard, W. P. Helman, G. V. Buxton, R. E.
Huie, P. Neta, NDRL-NIST Solution Kinetic Database - Ver. 3,
Notre Dame Radiation Laboratory, Notre Dame, IN and NIST
Standard Reference Data, Gaithersburg, MD, 1998, and refer-
ences therein.
Received: March 25, 2005
Revised: May23, 2005
Published online: August 17, 2005
Keywords: density functional calculations · kinetics ·
.
nucleosides · pulse radiolysis · tautomerism
[18] For eaq at 720 nm, e = 1.9 ” 104 mꢀ1 cmꢀ1, see: G. L. Hug, Natl.
ꢀ
Stand. Ref. Data Ser. U. S. Natl. Bur. Stand. 1981, No. 69.
[19] The corresponding optical transition (lone-pair NH2!p*-
(SOMO) b transition) in radical 2 is computed to occur at
830 nm with a veryweak intensity( f = 0.005). The optical-
absorption band associated with p!p*(SOMO) b spin transi-
tion is computed at 470 nm for 2 and 610 nm for 3.
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