Fe(II) phthalocyanine catalyzed oxidation of dGMP by molecular oxygen

Article abstract of DOI:10.1016/j.bmcl.2009.05.088

We show that iron(II)-phthalocyanines are able to catalyze guanosine oxidation by molecular oxygen in the presence of reducing agents such as ascorbic acid and 2-mercaptoethanol. The products of 5′-monophosphate-2′-deoxyguanosine (dGMP) oxidation were dir

Full text of DOI:10.1016/j.bmcl.2009.05.088

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4335–4338
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fe(II) phthalocyanine catalyzed oxidation of dGMP by molecular oxygen
Aleksandra A. Kuznetsova a,b, Lyudmila I. Solovyeva c, Oleg L. Kaliya c, Evgeniy A. Lukyanets c,
Dmitri G. Knorre b, Olga S. Fedorova a,b,
a Novosibirsk State University, 630090 Novosibirsk, Russia
*
b Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, 630090 Novosibirsk, Russia
c State Research Centre ‘Organic Intermediates and Dyes Institute’ (SRC ‘NIOPIK’), 103787 Moscow, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
We show that iron(II)-phthalocyanines are able to catalyze guanosine oxidation by molecular oxygen in
the presence of reducing agents such as ascorbic acid and 2-mercaptoethanol. The products of 50-mono-
phosphate-20-deoxyguanosine (dGMP) oxidation were directly analyzed using the HPLC-ESI/MS method.
The main oxidation products were 50-phospho-20-deoxy-8-oxo-7,8-dihydroguanine and the 1,N2-glyoxal
adduct of the 50-monophosphate-20-deoxyguanosine.
Received 3 April 2009
Revised 19 May 2009
Accepted 20 May 2009
Available online 27 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
DNA oxidation
Phthalocyanine
Guanine
ROS
Many efficient DNA-damaging agents have been developed over
the last several decades.1 Interest in these agents exists due to the
search for new anticancer and antiviral drugs. Among these agents,
complexes of transition metals are considered to be promising re-
agents for oxidative DNA cleavage.
The antitumor activity of transition metal complexes in the
presence of O2 and easily oxidizable substrates was first found by
Kimoto et al.,2 who used a copper glycyl-glycyl-histidine complex
with sodium ascorbate as a conjugated reducing agent. Currently,
phthalocyanine complexes of Co(II) and Fe(II) are being investi-
gated as drugs for the catalytic therapy of cancer.3
phthalocyanine in the oligonucleotide-conjugate and the positively
charged phthalocyanine in the solution. The resulting phthalocya-
nine complexes showed a significant increase in catalytic activity
compared to monomeric forms of phthalocyanines Fe(II) and
Co(II)5c and led to direct DNA strand cleavage. It was determined
that oxidation of DNA by molecular oxygen catalyzed by a complex
of Fe(II)-phthalocyanines proceeds with a higher rate than that cat-
alyzed by Co(II)-phthalocyanines; however, the latter led to a
greater extent of target DNA modification. In all cases the guanine
residues located close to the source of the oxidizing species were
the most susceptible to modification.
Among the possible oxidation sites on DNA, guanosine is the
most oxidizable nucleoside.4 There is currently no information
about guanosine-oxidation products induced by molecular oxygen
in the presence of phthalocyanines Co(II) or Fe(II).
In the present work, we investigated the oxidation of 20-deoxy-
guanosine-50-monophosphate (dGMP) as a model compound to
provide molecular insight into the structure of guanine lesions
generated by metallophthalocyanine-oligonucleotide conjugates.
We used FePcneg and its dimeric complex FePcnegꢀFePcpos immobi-
Recently, we have studied sequence-directed oxidative cleavage
of DNA with O2 and H2O2 in the presence of conjugates of cobalt(II)
and iron(II) phthalocyanines (CoPcneg and FePcneg, respectively) at-
tached to oligonucleotides.5a,b It was shown that single-stranded
DNA is efficiently damaged in complexes with these conjugates.
In addition, the site-directed modification of single-stranded DNA
by O2 and H2O2 in the presence of heterogeneous dimeric com-
plexes of negatively and positively charged Fe(II) and Co(II) phthal-
ocyanines (FePcposꢀFePcneg and CoPcposꢀCoPcneg, respectively) was
investigated.5c These complexes were formed directly on single-
stranded DNA through interaction between the negatively charged
lized on SiO2 as catalysts (Fig. 1), and reducing substrates (L-ascor-
bic acid, H2A or 2-mercaptoethanol, RSH) for dGMP oxidation by
molecular oxygen.
FePcneg–SiO2 was synthesized through the formation of an
amide bond between 3-aminopropyl modified SiO2 and an N-suc-
cinimide ester of FePcneg (see Supplementary data). The dimeric
complex FePcposꢀFePcneg–SiO2 was prepared by treatment of
6
FePcneg–SiO2 with an aqueous solution of FePcpos.
The dGMP oxidation products were directly analyzed using the
HPLC-ESI/MS method.6 The oxidation of dGMP by molecular oxygen
in the presence of FePcneg–SiO2 or FePcposꢀFePcneg–SiO2 and reduc-
ing substrate resulted in several products. The RP-HPLC traces of
these products are shown in Figure 2. Six products were identified:
* Corresponding author. Tel.: +7 383 334 6274; fax: +7 383 333 3677.
E-mail address: fedorova@niboch.nsc.ru (O.S. Fedorova).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.088

4336
A. A. Kuznetsova et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4335–4338
Free guanine (peak 1 in Figs. 2 and 3a) was formed in all cases of
dGMP oxidation (m/z = 150.1). The formation of 7,8-dihydro-8-oxo-
guanine (peak 2 in Fig. 2 and 3b) was observed only in the presence
of H2A (m/z = 166.0). Release of free bases is thought to be an indi-
cation of sugar damage.8 A 20-deoxyribose residue in the backbone
of a DNA strand has seven hydrogen atoms attached to carbon,
which in principle, are available for abstraction by an oxidizing
agent or free radicals. It is well-known that the hydroxyl radical
is able to abstract hydrogen from each 20-deoxyribose carbon of
DNA.8 Release of free base in aerobic conditions is possible when
abstraction of hydrogen atoms from C10-, C20- and C50-sites of 20-
deoxyribose occurs.8 It should be noted, that release of free bases
is also possible under DNA oxidation by high-valent metal-oxo
species such as FeV@O in activated bleomycin,9a MnV@O in manga-
nese(III) porphyrin activated by KHSO5,9b and Cu(III)@O in 1,10-
phenanthroline complex.9c Thus, the formation of free bases can
be evidenced either by OHÅ or by high-valent metal-oxo species
generation in our oxidation systems.
The formation of 1,N2-glyoxal-dGMP (peak 3 in Figs. 2 and 3c)
was observed in all cases of dGMP oxidation. However, in the
FePcposꢀFePcneg–SiO2/RSH oxidation system it was the main oxida-
tion product. The ESI-MS spectrum of 1,N2-glyoxal-dGMP showed
m/z peaks corresponding to molecular ions [MꢁH]ꢁ at 404.1 and
346.2 (loss of glyoxal moiety). The formation of the glyoxal adducts
of dG was first shown under oxidation of DNA by O2 in the pres-
ence of the Fe(II)–EDTA complex,10a and its formation could be a
consequence of sugar moiety oxidation. Besides, it was shown that
2-phosphoglycolaldehyde reacted with dG to form the 1,N2-gly-
oxal-dG adducts10b (Supplementary data, Fig. S1). The phosphogly-
colaldehyde residue can form by oxidation of the C30-atom of 20-
deoxyribose.8 Since in our conditions glyoxal-dGMP was observed
in all cases, it can be assumed that the Fe(II)–phthalocyanine com-
plex is capable of oxidizing the C30-atom of 20-deoxyribose. The
formation of the phosphoglycolaldehyde under 20-deoxyribose oxi-
dation by hydroxyl radicals should be accompanied by formation
of base propenoic acid.8 Unfortunately, we could not detect the for-
mation of base propenoic acid.
Figure 1. The structures of FePcneg and FePcpos used in this work.
guanine (Gua), 7,8-dihydro-8-oxo-guanine (8-oxoGua), 1,N2-gly-
oxal-50-monophosphate-20-deoxyguanosine (1,N2-glyoxal-dGMP),
50-monophosphate-20-deoxy-7,8-dihydro-8-oxo-guanosine (8-oxo-
dGMP), 50-aldehyde-20-deoxyguanosine (50-aldehyde-dG) and 20-
deoxy-7,8-dihydro-8-oxo-guanosine (8-oxo-dG). Identification of
the dGMP oxidation products was done using ESI-MS and by com-
parison of the retention times and UV absorbance spectra with
those of a commercial standard and described in the literature7
(Supplementary data, Table S1).
The main difference in oxidation product formation in the pres-
ence of FePcneg–SiO2 or FePcposꢀFePcneg–SiO2 was the yield. In gen-
eral, FePcposꢀFePcneg–SiO2 led to a higher yield of 8-oxo-dGMP in
the presence of H2A and 1,N2-glyoxal-dGMP in the presence of
RSH. This can be correlated with the higher catalytic activity of di-
meric complexes or with the electrostatic interaction between the
positive charged Pc and the negative charged phosphate group of
dGMP. As a result, higher degradation of dGMP occurred. Poten-
tially, both processes could contribute to oxidation.
The main difference in oxidation product formation in the pres-
ence of H2A or RSH was found in the yields of 8-oxo-dGMP and
1,N2-glyoxal-dGMP. The presence of H2A in the reaction solution
led primarily to formation of 8-oxo-dGMP. As a result, the free 8-
oxoGua base was also detected in these systems. The presence of
RSH led preferentially to the formation of 1,N2-glyoxal-dGMP. In
addition, the formation of 50-aldehyde-dGua was observed.
The compounds ultimately characterized are discussed in the
order of their retention times below. The UV and MS spectra of
the identified oxidation products of dGMP are shown in Figure 3.
Peak 4 in Figure 2 was identified as unreacted dGMP.
The formation of 8-oxo-dGMP (peak 5 in Figs. 2 and 3d) was
observed in the presence of H2A and also with low yield in the
Figure 2. HPLC profiles of reaction mixtures: oxidation of dGMP by O2 in the presence of FePcneg–SiO2 or FePcpos ꢂ FePcneg–SiO2 and H2A or RSH.

A. A. Kuznetsova et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4335–4338
4337
Figure 3. UV–vis and ESI-MS spectra of the dGMP oxidation products (dR is 20-deoxyribose and pdR is 50-monophosphate-20-deoxyribose).
FePcposꢀFePcneg–SiO2/RSH oxidation system. It should be noted that
in the FePcposꢀFePcneg–SiO2/H2A oxidation system 8-oxo-dGMP was
the main oxidation product. The formation of 8-oxo-dGMP could
also result from OHÅ generation in oxidation systems.4 A hydroxyl
radical adds to purines, giving rise to C4–OH-, C5–OH-, and C8–
OH-adduct radicals.11a C4–OH- and C5–OH-adduct radicals under-
go dehydration and form the oxidizing radicals (G–H)Å, which
The formation of 8-oxo-dG (peak 8 in Figs. 2 and 3f) was ob-
served in the presence of H2A and not observed in the presence
of RSH. It should be noted that FePcneg–SiO2 led to a higher yield
of 8-oxo-dG compared to FePcposꢀFePcneg–SiO2. The 8-oxo-dG could
be formed from dG in the same way as the 8-oxo-dGMP was
formed from dGMP.
In conclusion, the dGMP oxidation by molecular oxygen cata-
lyzed by Fe-phthalocyanine complexes was investigated. The main
oxidation products were 8-oxo-dGMP and 1,N2-glyoxal-dGMP. The
formation of 8-oxoguanine derivatives, as well as the products of
sugar oxidation (free guanine bases and 1,N2-glyoxal adduct),
could be evidence of several oxidizing species in the reaction mix-
ture: hydroxyl radicals and high valent iron-oxo species.
reconstitute guanine upon reduction. The interaction of (G–H)Å
Åꢁ
with O2
results in the formation of imidazolone and
dihydroguanidinohydantoin derivatives.11b One-electron oxidation
and one-electron reduction of C8–OH-adduct radicals lead to the
formation of 7,8-dihydro-8-oxo-guanine and 2,6-diamino-4-hy-
droxy-5-form-amidopyrimidine (FAPyG), respectively.11a We did
not detect the formation of FAPyG. This is in agreement with obser-
vations made for an isolated DNA upon exposure to HOÅ radicals in
aqueous solution. Indeed, 8-oxo-guanine was the major degrada-
Acknowledgements
tion product when DNA was exposed to
c
-rays under aerobic con-
This research was made possible in part by a Grant from RFBR
(No. 08-04-00334-a), President Grant (No. 652.2008.4), U.M.N.I.K.
No. 8775, Grant from Russian Ministry of Education and Sciences
(No. 2.1.1/1499).
ditions.11c However, a drastic increase in the yield of FAPyG at the
expense of 8-oxo-guanine was observed when O2 is absent.11d We
also did not detect the formation of imidazolone or oxazolone
derivatives. The latter is formed from imidazolone under hydro-
lytic conversion.12a It is known12b that
c-irradiation of an aerated
Supplementary data
aqueous solution of dG led predominantly to the formation of oxa-
zolone, whereas FAPyG and 8-oxo-guanine were produced with
low yield. However, when either cysteine or ascorbate was added,
even at a low concentration, a drastic decrease in the yield of oxa-
zolone was observed.12c Thus, our results are in agreement with
these observations.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.05.088.
References and notes
1. (a) Sigman, D. S. Biochemistry 1990, 29, 9097; (b) Sreedhara, A.; Cowan, J. A. J.
Biol. Inorg. Chem. 2001, 6, 337; (c) Cowan, J. A. J. Curr. Opin. Chem. Biol. 2001, 5,
634; (d) Mancin, F.; Scrimin, P.; Tecilla, P.; Tonellato, U. Chem. Commun. 2005,
2540.
2. Kimoto, E.; Tanaka, H.; Gyotoku, J.; Morishige, F.; Pauling, L. Cancer Res. 1983,
43, 824.
3. Volpin, M. E.; Vorozhtsov; G. N.; Krainova, N. J.; Levitin, I. Y.; Luzhkov, J. M.;
Lukiyanets, E. A.; Gerasimova, G. K.; Zhukova, O. S.; Kazachkina, N. I.; Kalia, O.
L.; Novodarova, G. N.; Treschalina E. M.; Syrkin, A. B.; Chissov, V. I.;
Yakubovskaya, R. I. U.S. Patent 6,004,953, 1999.
Peak 6 in Figure 2 is dG. In control experiments it was shown
that dG was present in parent dGMP.
The formation of 50-aldehyde-dGua (peak 7 in Figs. 2 and 3e) was
observed only in the FePcneg–SiO2/RSH oxidation system. The ESI-
MS spectrum of 50-aldehyde-dGua showed m/z peaks correspond-
ing to the molecular weight [MꢁH]ꢁ at 264.1 and an m/z peak
corresponding to the molecular weight [MꢁH]ꢁ of dG at 266.3.
The presence of a signal from dG could be due to the overlapping
peaks in the eluting conditions. The formation of 50-aldehyde-20-
deoxyguanosine is possible when oxidation of the C50-atom of 20-
deoxyribose occurs. The oxidizing species causing such damage
can be both hydroxyl radicals8 and high-valent metal-oxo species.9b
4. Pratviel, G.; Meunier, B. Chem. Eur. J. 2006, 12, 6018.
5. (a) Kuznetsova, A. A.; Chernonosov, A. A.; Kuznetsov, N. A.; Koval, V. V.; Knorre,
D. G.; Fedorova, O. S. Bioinorg. Chem. Appl. 2006, 2006, 1; (b) Kuznetsova, A. A.;
Solovyeva, L. I.; Fedorova, O. S. Russ. J. Bioorg. Chem. 2008, 34, 614; (c)
Kuznetsova, A. A.; Lukyanets, E. A.; Solovyeva, L. I.; Knorre, D. G.; Fedorova, O. S.
J. Biomol. Struct. Dyn. 2008, 26, 307.

4338
A. A. Kuznetsova et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4335–4338
6. All reactions were carried out according to the following standard procedure:
the reaction mixture (250 L) contained 50 mM phosphate buffer (pH 7.5),
1997, 38, 121; (c) Yu, H.; Venkatarangan, L.; Wishnoka, J. S.; Tannenbaum, S. R.
Chem. Res. Toxicol. 2005, 18, 1849.
l
1 mM dGMP, 5 mM H2A or 50 mM RSH, and 5 mg FePcneg–SiO2; the studies
were performed at 25 °C; the reducing agent was introduced in 4 additions
every 2 h and the first addition initiated the reaction. The FePcneg–SiO2 was
preliminarily treated with an aqueous solution of FePcpos (3 mg/ml) in the case
of dGMP oxidation in the presence of dimer complex. After 20 h the solution of
oxidated dGMP was separated from the Silica Gel by centrifugation and
analyzed by HPLC (chromatograph Agilent Technologies 1200 series with diode
8. (a) Pogozelski, W. K.; Tullius, T. D. Chem. Rev. 1998, 98, 1089; (b) Knorre, D. G.;
Fedorova, O. S.; Frolova, E. I. Russ. Chem. Rev. 1993, 62, 65.
9. (a) Burger, R. M. Chem. Rev. 1998, 98, 1153; (b) Mourgues, S.; Kupan, A.;
Pratviel, G.; Meunier, B. ChemBioChem 2005, 6, 2326; (c) Sigman, D. S.; Graham,
D. R.; D0Aurora, V.; Stern, A. M. J. Biol. Chem. 1979, 254, 12269.
10. (a) Murata-Kamiya, N.; Kamiya, H.; Iwamoto, N.; Kasai, H. Carcinogenesis 1995,
16, 2251; (b) Awada, M.; Dedon, P. C. Chem. Res. Toxicol. 2001, 14, 1247.
11. (a) Steenken, S. Chem. Rev. 1989, 89, 503; (b) Misiaszek, R.; Crean, C.; Joffe, A.;
Geacintov, N. E.; Shafirovich, V. J. Biol. Chem. 2004, 279, 32106; (c) Kasai, H.;
Tanooka, H.; Nishimura, S. Gann 1984, 75, 1037; (d) Douki, T.; Martini, R.;
Ravanat, J.-L.; Turesky, R.; Cadet, J. Carcinogenesis 1997, 18, 2385.
12. (a) Cadet, J.; Berger, M.; Buchko, G. W.; Joshi, P. C.; Raoul, S.; Ravanat, J.-L. J. Am.
Chem. Soc. 1994, 116, 7403; (b) Douki, T.; Cadet, J. Free Radical Res. 1996, 24,
369; (c) Douki, T.; Spinelli, S.; Ravanat, J.-L.; Cadet, J. J. Chem. Soc., Perkin Trans. 2
1999, 1875.
array detector) on an analytical reverse phase column (Nucleosil C18, 10 lm,
250 ꢂ 4.6 mm) in a linear gradient of 0–50% CH3CN in 25 mM TEA-AcOH buffer
(pH 7.5). Detection was at 250 nm. Chromatogram peaks corresponding to
different oxidation product of dGMP were collected and separately analyzed on
an ESI/MS spectrometer (Agilent 6410 Triple Quad LC/MS, Agilent
Technologies) in negative mode.
7. (a) Henle, E. S.; Luo, Y.; Gassmann, W.; Linn, S. J. Biol. Chem. 1996, 271, 21177;
(b) Murata-Kamiya, N.; Kamiya, H.; Muraoka, M.; Kaji, H.; Kasai, H. J. Radiat. Res.