Photogeneration of 2-deoxyribonolactone in benzophenone-purine dyads. Formation of ketyl-C1′ biradicals

Article abstract of DOI:10.1021/ol801514v

(Chemical Equation Presented) Photolysis of the title dyads under aerobic conditions leads to a 2-deoxyribonolactone derivative. Laser flash photolysis reveals that the process occurs from the short-lived benzophenone-like triplet excited state. A mechanism involving intramolecular electron transfer with the purine bases (adenine, guanine, or 8-oxoadenine) as donors is proposed.

Full text of DOI:10.1021/ol801514v

ORGANIC
LETTERS
2008
Vol. 10, No. 20
4409-4412
Photogeneration of
2-Deoxyribonolactone in
Benzophenone-Purine Dyads.
Formation of Ketyl-C1′ Biradicals
Cecilia Paris, Susana Encinas, Nourreddine Belmadoui, Mar´ıa J. Climent, and
Miguel Angel Miranda*
Instituto de Tecnolog´ıa Qu´ımica UPV-CSIC/Departamento de Qu´ımica, UniVersidad
Polite´cnica de Valencia, AVenida de los Naranjos S/N, 46022 Valencia, Spain
mmiranda@qim.upV.es
Received July 3, 2008
ABSTRACT
Photolysis of the title dyads under aerobic conditions leads to a 2-deoxyribonolactone derivative. Laser flash photolysis reveals that the
process occurs from the short-lived benzophenone-like triplet excited state. A mechanism involving intramolecular electron transfer with the
purine bases (adenine, guanine, or 8-oxoadenine) as donors is proposed.
Chemical modification of DNA produces dramatic conse-
quences in the normal function of living systems, due to
alterations of the genetic material during cell replication.1
Oxidatively generated damage to DNA may be induced by
a number of agents, most frequently free radicals, reactive
oxygen species (ROS), UV light, or ionizing radiations.2 The
main target units are the nucleobases and the sugar moiety.
Loss of a base is one of the most frequent lesions produced
in DNA and has been shown to be highly mutagenic.3
Formation of abasic sites modifies the spatial conformation
on the double helix and interferes with the correct function
(2) (a) Chatgilialoglu, C.; O′Neill, P. Exp. Gerontol. 2001, 36, 1459–
1471. (b) Cadet, J.; Berger, M.; Douki, T.; Morin, B.; Raoul, S.; Ravanat,
J. L.; Spinelli, S. Biol. Chem. 1997, 387, 1275–1286. (c) Burrows, C.;
Muller, J. G. Chem. ReV. 1998, 98, 1109–1151. (d) Armitage, B. Chem.
ReV. 1998, 98, 1171–1200.
(3) (a) Sato, K.; Greenberg, M. G. J. Am. Chem. Soc. 2005, 127, 2806–
2807. (b) Dedon, P. Chem. Res. Toxicol. 2008, 21, 206–219. (c) Xue, L.;
Greenberg, M. M. J. Am. Chem. Soc. 2007, 129, 7010–7011.
(1) Wang, Y. Chem. Res. Toxicol. 2008, 21, 276–281.
10.1021/ol801514v CCC: $40.75
Published on Web 09/23/2008
2008 American Chemical Society

of the repair enzyme machinery.4 These abasic sites may
arise from generation of a C1′ radical and subsequent
formation of a 2-deoxyribonolactone (dL), and abstraction
of the H1′ hydrogen at the anomeric carbon by an appropriate
agent is the common initiation step. Sugiyama reported that
irradiation of 5-halouracil inserted in DNA generates dL in
the adjacent adenine, by a mechanism involving initial
formation of a C1′ sugar radical.5 On the other hand,
Chatgilialoglu, Newcomb, and co-workers have found that
reaction of the 2′-deoxyuridin-1′-yl radical with molecular
oxygen leads to dL as the final product.6
which has been widely employed for mechanistic studies on
nucleosides.9 The structural variations were designed to
evaluate the influence of the cisoid vs transoid spatial
arrangement (1a vs 1b), the base reactivity (series 1a, 2, 3),
and the length of the spacer (1a vs 4).10
The dyads were synthesized by condensation of KP or its
glycine derivative (KPGly) with 2′-deoxyadenosine (dA), 2′-
deoxyguanosine (dG), or 8-oxo-7,8-dihydro-2′-deoxyadenos-
ine (8-oxodA), using a carbodiimide as an activating agent.
Further details can be found in the Supporting Information
section (pages S2-S12).
Even though it is generally agreed that DNA oxidation
primarily occurs at guanine sites, Schuster, Majima, and
others have found that the adenine radical cation may also
be involved in charge-migration processes.7 In spite of the
importance of this nucleobase, comparatively little informa-
tion is available on its oxidation chemistry and specifically
on the generation of dL by photosensitization.
With this background, the aim of the present work is the
use of several benzophenone (BP)-purine dyads (Scheme
1) to perform a mechanistic study on the photosensitized
Upon UVA irradiation (350 nm) of 5′-KP-dA, 5′-KP-dG,
and 5′-KP-8-oxodA under oxygen in acetonitrile-water (4:1
v/v), the same 2-deoxyribonolactone (5) was isolated in all
cases (see spectra in pages S13-S16).
After confirming photochemical formation of the dL lesion
in the dyads, time-resolved studies were performed. Selective
excitation of the BP chromophore was achieved at 308 and
355 nm. As expected, the typical triplet-triplet BP absorption
maximum at λ ) 530 nm was detected in acetonitrile
solutions (inset of Figure 1). The triplet lifetimes (τT) were
Scheme 1. Structures of the Investigated Compounds
Figure 1. Triplet decays of S-ketoprofen (sbs), 3′-KP-dA (s9s),
and 5′-KP-dA (s2s) monitored at 530 nm following laser
excitation (308 nm), in acetonitrile solutions (1 × 10 -4 M). Inset:
Triplet-triplet transient absorption spectrum for 5′-KP-dA recorded
following laser excitation (308 nm) in deareated acetonitrile, 30 ns
after the laser pulse.
oxidation of adenine, with special emphasis on the possible
formation of dL as a lesion resulting from this type of
process.8 In these dyads, the BP unit is provided by
S-ketoprofen (KP), a well-known photosensitizing drug,
strongly dependent on the position in which the KP unit was
linked to the sugar moiety: for 3′-KP-dA, the decay was
similar to that of isolated ketoprofen, whereas for 5′-KP-
dA, the decay was much faster and occurred in the submi-
crosecond time scale (Figure 1). This can be safely attributed
to a more effective interaction between the chromophores
(4) Mishina, Y.; Duguid, E. M.; He, C. Chem. ReV. 2006, 106, 215–
232.
(5) (a) Xu, Y.; Sugiyama, H. J. Am. Chem. Soc. 2004, 126, 6274–6279.
(b) Xu, Y.; Sugiyama, H. Angew. Chem., Int. Ed. 2006, 45, 1354–1362.
(6) Chatgilialoglu, C.; Ferreri, C.; Bazzanini, R.; Guerra, M.; Choi, S.-
Y.; Emanuel, C. J.; Horner, J. H.; Newcomb, M. J. Am. Chem. Soc. 2000,
122, 9525–9533.
(9) (a) Lhiaubet-Vallet, V.; Encinas, S.; Miranda, M. A. J. Am. Chem.
Soc. 2005, 127, 12774–12775. (b) Encinas, S.; Belmadoui, N.; Climent,
M. J.; Gil, S.; Miranda, M. A. Chem. Res. Toxicol. 2004, 17, 857–862. (c)
Bosca´, F.; Mar´ın, M. L.; Miranda, M. A. Photochem. Photobiol. 2001, 74,
637–655.
(7) (a) Kawai, K.; Takada, T.; Nagai, T.; Cai, X.; Sugimoto, A.;
Fujitsuka, M.; Majima, T. J. Am. Chem. Soc. 2003, 125, 16198–16199. (b)
Schlientz, N.; Schuster, G. B. J. Am. Chem. Soc. 2003, 125, 15732–15733.
(8) Bosca´, F.; Miranda, M. J. Photochem. Photobiol. B: Biol. 1998, 43,
1–26.
(10) (a) Belmadoui, N.; Encinas, S.; Climent, M. J.; Miranda, M. A.
Chem.-Eur. J. 2006, 12, 553–561. (b) Lhiaubet-Vallet, V.; Belmadoui,
B.; Climent, M. J.; Miranda, M. A. J. Phys. Chem. B 2007, 111, 8277–
8282.
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Org. Lett., Vol. 10, No. 20, 2008

absorbance at 425 nm (maximum naphthalene triplet forma-
tion), and R ) constant.
in the cisoid dyad, as previously observed for thymine
analogues.10 The same trend was observed for 5′-KP-dG and
5′-KP-8-oxodA, whose decays also occurred in the submi-
crosecond range.
Operating in this way, the values determined for the KP
triplet lifetimes in the dyads were 30 ns for 5′-KP-dA, 9 ns
for 5′-KP-dG, and 12 ns for 5′-KP-8-oxodA. On the basis
of these results, estimation of the involved rate constants
was made using eq 2
Thus, in the 5′-dyads, the intramolecular interaction
between KP and the purine base resulted in a significant
quenching of the excited BP-like triplet state. This could in
principle be associated with an electron transfer process;
however, the charge separated species were not detected
under the employed experimental conditions, probably due
to a very fast back electron transfer. To stabilize such species
and to enhance the prospects of detecting the purported
intermediates, water was added to the system. As a matter
of fact, laser flash photolysis (LFP) of acetonitrile-water
solutions (4:1 v/v) of 5′-KP-dA, 5′-KP-dG, and 5′-KP-8-
oxodA yielded new transients with maxima at 550 nm
(Figure 2), assigned to the characteristic BP-ketyl radical
kintra ) (1 ⁄ τT - 1 ⁄ τ0)
(2)
where kintra ) intramolecular quenching rate constant, τ0 )
triplet lifetime of free KP, and τT ) triplet lifetime in the
dyads. The values obtained (3.3 × 107 s-1, 1.1 × 108 s-1,
and 8.3 × 107 s-1 for 5′-KP-dA, 5′-KP-dG, and 5′-KP-8-
oxodA) are in agreement with the expectations for an electron
transfer mechanism. Thus, to demonstrate the viability of
the process, the free energy changes (∆G, kJ/mol) were
calculated using the Rehm-Weller eq 3
∆G ) 96.5[E0(D+• ⁄ D) - E0(A ⁄ A-•)] - ∆Eoo
(3)
where E0(D+•/D) is the one-electron oxidation potential of
the nucleobases; E0(A/A-•) is the one-electron reduction
potential of the photosensitizer, and ∆Eoo is the triplet
excited-state energy (KP or BP), determined by spectroscopic
measurements. In this context, negative ∆G increments for
the intermolecular process in related D/A systems have been
reported by Lhiaubet et al.13 for dA (∆G ) -59 kJ/mol, E0
) 1.18 V vs SCE) and dG (∆G ) -71 kJ/mol, E0 ) 1.05
V vs SCE) nucleosides, when S-KP was used as an acceptor
(E0 ) -1.24 V vs SCE, ∆Eoo ) 292.6 kJ/mol) in aqueous
media. These findings are consistent with our results for
dyads 1a, 2, and 3 by laser flash photolysis. Even though no
E0 values were available for 3, it can be assumed that the
relative free energy changes would be dG > 8-oxodA >
dA, in accordance with the nucleoside reactivities.
For the same donor-acceptor pair, the electron transfer
process should be sensitive to the length of the spacer. To
evaluate the influence of this factor, 5′-KPGly-dA was
studied by time-resolved experiments under the same condi-
tions. The transient absorption spectrum displayed the two
typical T-T bands with maxima at 330 and 530 nm (see
pages S18-S19 in Supporting Information). Its lifetime (0.4
µs) was much longer than that of 5′-KP-dA (0.03 µs), and
the rate constant determined by application of eq 2 was
accordingly lower (2.2 × 106 s-1). This constitutes a further
support for the proposed electron transfer mechanism.
Scheme 2 summarizes the sequential steps leading from the
dyads to the final 2-deoxyribonolactone product. Here,
deprotonation of the purine radical cation at C1′ could be
achieved by the BP-like radical anion. Assistance of the
aqueous medium seems to be esential, as otherwise the ketyl
radical could not reach C1′. The (KP)•- absorption (630 nm)
was not observed in the nanosecond time scale. However,
the corresponding (KPH)• band (550 nm) was clearly detected
in the LFP experiments. Previously, Cadet and co-workers
have isolated dL after UVA irradiation of 2′-deoxyguanosine
(dG) in the presence of BP, and its formation has been
attributed to photochemical electron transfer, followed by
deprotonation of the radical cation at the C1′ position. The
Figure 2. Transient absorption spectra of 5′-KP-dA (sOs), 5′-
KP-dG (sbs), and 5′-KP-8-oxodA (s9s) in acetonitrile-water
solutions, 30 ns after the 308 nm laser pulse.
moieties.11 At very early stages (<30 ns), the KP triplet
excited states were also detected, although the features of
the BP-ketyl radicals were already present. Expectedly, the
use of mixed organic-aqueous media also increased the
electron transfer efficiency, thus shortening the triplet
lifetimes.
Hence, to obtain an accurate measurement of the τT values,
the naphthalene (NP) quenching method developed by
Scaiano was employed.12 When increasing amounts of NP
were added to the dyads, deactivation of the KP triplet
excited state (selectively excited at 355 nm) was ac-
companied by generation of the NP triplet at 425 nm. A
double reciprocal plot of the absorbance increment (∆A425
)
against NP concentration (Supporting Information, see pages
S17-S18) provided the τT values upon application of eq 1
1 ⁄ ∆A425 ) R + (R ⁄ kqτT)[NP]-1
(1)
where kq ) intermolecular rate constant for quenching of
KP by NP (4 × 109 M-1 s-1), ∆A425 ) end of pulse
(11) Bensasson, R.; Gramain, J. C. J. Chem. Soc., Faraday Trans. I
1980, 76, 1801–1810.
(12) Scaiano, J. C.; McGimpsey, W. G.; Leigh, W. J.; Jakobs, S. J. Org.
Chem. 1987, 52, 4540–4544.
(13) Lhiaubet, V.; Paillous, N.; Chouini-Lalanne, N. Photochem. Pho-
tobiol. 2001, 74, 670–678.
Org. Lett., Vol. 10, No. 20, 2008
4411

be 0.17 µs for 5′-KP-dA, 0.21 µs for 5′-KP-dG, and 0.30 µs
for 5′-8-oxo-dA: they were remarkably shorter than those
previously reported for the KP-derived ketyl radical (>20
µs) and for the nucleoside C1′ radicals (typically 10-20
µs).16,6 Biradicals are known to live in the submicrosecond
range, and their deactivation proceeds through a complex
combination of different processes, including intersystem
crossing to the singlet biradicals, back hydrogen transfer,
radical coupling, etc.17
Scheme 2
.
Summary of the Sequential Steps Leading from the
Dyads to 5′-KP-dL
Although direct observation of purine-derived C1′ radicals
as transient species has not been reported yet, Chatgilialoglu,
Newcomb, and co-workers have performed time-resolved
studies on pyrimidine analogues.6 Thus, generation of
ketyl-C1′ biradicals is potentially interesting as a model for
kinetic measurements on C1′ radical reactivity with oxygen
or reducing agents, which is relevant in connection with the
oxidation and repair of this lesion. In the present work, these
hardly detectable sugar radicals are made visible through the
KP-reduced moiety, which absorbs in a longer wavelength
region and is therefore more convenient for experimental
purposes.
In summary, a 2-deoxyribonolactone is formed by pho-
tolysis of KP-tethered purine nucleosides, through the
generation of C1′-ketyl biradicals. Transient absorption
spectroscopy supports an intramolecular electron-transfer
mechanism. These results are potentially applicable for direct
kinetic measurements on C1′ sugar radical reactivity.
BP-sensitized irradiation of a dinucleotide (dTdG) leads to
formation of dL at the dG site. In the absence of BP or in
the presence of a phthalocyanine derivative as a type II
photosensitizer, no significant dL formation is observed.14
In addition, 2-deoxyribonolactone has been identified among
the 7-methylpyrido[3,4-c]psoralen photosensitized degrada-
tion products of 2′-deoxyadenosine in the solid state.15
Estimation of the ketyl-C1′ biradical quantum yields in
the dyads was done by determining the maximum transient
absorbance of optically matched acetonitrile-water solutions
(A ) 0.25) at 570 nm, after 308 nm LFP excitation. Using
KP as a standard for comparison (Φ ) 1), the obtained
results (Φ ) 0.82 for 5′-KP-dA, Φ ) 0.95 for 5′-KP-dG,
and Φ ) 0.88 for 5′-KP-8-oxodA) were in agreement with
the relative oxidizability of the bases. The biradical lifetimes
were determined from the first-order decays and found to
Acknowledgment. We thank the financial support given
by the EU to M. A. M., grant “Selective Formation and
Biochemistry of Oxidative Clustered DNA Damage” (CLUS-
TOXDNA, MRTN-CT-2003-505086). We also thank Dr. V.
Lhiaubet-Vallet for her assistance in the experimental part.
Supporting Information Available: Experimental pro-
cedures of synthesis, photolysis, characters of compounds,
and LFP data. This material is available free of charge via
the Internet at http://pubs.acs.org.
OL801514V
(16) Monti, S.; Sortino, S.; De Guidi, G.; Marconi, G. J. Chem. Soc.,
Faraday Trans. 1997, 93, 2269–2275
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(14) Buchko, G. W.; Cadet, J. Can. J. Chem. 1992, 70, 1827–1832.
(15) Voituriez, L.; Cadet, J. Photochem. Photobiol. 1999, 70, 152–158.
(17) Andreu, I.; Bosca´, F.; Sanchez, L.; Morera, I. M.; Camps, P.;
Miranda, M. A. Org. Lett. 2006, 8, 4597–4600.
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