Pharmacology & Pharmacy, 2011, 2, 122-126
doi:10.4236/pp.2011.23016 Published Online July 2011 (
Copyright © 2011 SciRes. PP
Unsaturated Keto and Exomethylene
Pyranonucleoside Analogues of Thymine and
Uracil Exhibit Potent Antioxidant Properties
Chrysoula Spanou1, Niki Tzioumaki2, Stella Manta2, Panagiotis Margaris1,2, Dimitrios Kouretas1,
Dimitri Komiotis2, Kalliopi Liadaki1*
1Department of Biochemistry and Biotechnology, Laboratory of Animal Physiology, University of Thessaly, Larissa, Greece;
2Department of Biochemistry and Biotechnology, Laboratory of Organic Chemistry, University of Thessaly, Larissa, Greece.
Received March 29th, 2011; revised April 30th, 2011; accepted June 22nd, 2011.
Nucleoside analogues play an important role in the development of antitumor and antiviral agents. Specific sugar
modified pyranonucleosides, like the keto and exocyclic methylene nucleosides, have been studied for their biological
properties, but there is little information regarding their antioxidant activity. The present study reports the antioxidant
activity of a series of α,β-unsaturated 2’- or 4’- keto and exomethylene 5’-hydroxymethyl-lacking pyranonucleosides.
The antioxidant activity was evaluated using an in vitro assay which is based on the capacity to protect DNA strand
scission induced by peroxyl radicals (ROO). The majority of the tested nucleoside analogues exhibit potent antioxidant
properties against ROO radicals. We conclude that the presence of a carbon-carbon double bond at α,β-disposition to
exomethylene group at position 2 of the sugar moiety and the substitution of thymine with uracil improves the antioxi-
dant capacity of these analogues.
Keywords: D-Lyxopyranonucleoside Derivatives, D-Arabinonucleoside Derivatives, DNA Damage, Peroxyl Radicals.
1. Introduction
Nucleosides are structural modules of nucleic acids with
fundamental importance in all living systems [1]. They
constitute the basis for development of antitumor and
antiviral agents because they act as selective inhibitors of
key enzymes involved in cancer or viral replication [2],
or as nucleic acid chain terminators which interrupt cel-
lular replication [3-6]. Sugar modified pyranonucleosides
are recognized as an important class of biologically ac-
tive molecules [7-12]. Among them, unsaturated keto
[13-15], as well as exomethylene pyranonucleoside ana-
logues [16-19], exhibit interesting antitumor and antiviral
properties, while early studies demonstrated that the
presence of a primary hydroxyl and hydroxymethyl group
in the sugar moiety does not seem to be critical for bio-
logical activity [18,20-21].
In order to investigate the antioxidant properties of
nucleoside analogues we have previously synthesized a
new class of unsaturated 3’-fluoro-4’-ketonucleosides,
that of N4-benzoyl cytosine and N6-benzoyl adenine, re-
spectively [19]. Most of the aforementioned com-
pounds showed significant ability to protect DNA from
the strand breaking activity of ROO radicals. Further-
more, those nucleoside analogues containing an α,β-un-
saturated keto system were the most potent against the
activity of ROO radicals.
In extending these studies the antioxidant activity of a
series of α,β-unsaturated 2’- or 4’- keto and exomethyl-
ene 5’-hydroxymethyl-lacking pyranonucleoside analo-
gues was investigated. Specifically, the present study is
the first attempt to correlate structural modifications of
the aforementioned nucleoside analogues with the ability
to inhibit ROO radicals-induced DNA damage.
2. Results
Five out of the eight tested nucleoside analogues inhib-
ited the DNA damage induced by ROO radicals (Table
1). Compound 4 was the most potent as it exhibited 22%
inhibition of radical-induced DNA damage at the con-
centration of 20 μΜ. It should be noted that all com-
pounds had no effect on plasmid conformation when
they were tested alone at the highest concentration.
Unsaturated Keto and Exomethylene Pyranonucleoside Analogues of Thymine and Uracil Exhibit Potent Antioxidant
Figure 1. Effect of nucleoside analogue 4 on peroxyl radi-
cal-induced plasmid DNA strand scission. Bluescript-SK +
plasmid DNA (1 μg/10 μL) was incubated in the presence of
2.5 mM AAPH for 45 min in the dark and the reaction
products were analyzed in 0.8% agarose gel. Lane 1: nega-
tive control. Lane 2: 2.5 mM AAPH. Lanes 3-7: AAPH plus
5, 10, 20, 50, 100 μM of the nucleoside analogue respectively.
Lane 8: plasmid DNA plus 100 μM of the nucleoside ana-
logue. OC: open circular; SC: supercoiled.
The presence of an exomethylene group at 2’ position
compared to the 4’ position of the sugar moiety seems to
be important for the antioxidant properties of the nucleo-
side analogues. Specifically, compounds 1 and 3 which
contain the exomethylene group at 4’ position of the
sugar moiety had no activity, while their corresponding
compounds 2 and 4 which contain the exomethylene
group at 2’ position exhibited potent antioxidant activity
(Table 1). Compound 2 inhibited ROO radicals to 16%
and 36% at 50 and 100 μM respectively and compound 4
was a potent inhibitor even at 20 μΜ (Table 1 and Fig-
ure 1). The differences in the potency observed between
compounds 2 and 4 can be attributed to the different nu-
cleobase (thymine and uracil respectively).
Similar to the exomethylene group the effect of the
position of an unsaturated keto system in these nucleo-
side analogues was examined. Compound 5 had no in-
hibitory activity at any concentration, while compound 6
had antioxidant activity only at the highest tested con-
centration (100 μΜ). Both compounds have thymine as
nucleobase but differ at the position of the unsaturated
keto system. It is possible that the translocation of the
keto group at position 2’ favors the antioxidant activity
of the compounds. However, this is not the case when
thymine is replaced by uracil, as exhibited by the similar
antioxidant capacities of compounds 7 and 8 (22% and
19% inhibition at 100 μΜ respectively).
3. Discussions
The present study reports the antioxidant properties of
α,β-unsaturated keto and exomethylene D-arabino- and
D-lyxo-pyranonucleoside analogues with thymine and
uracil as heterocyclic base. Specifically, these nucleoside
analogues were evaluated for the ability to inhibit ROO
radicals-induced DNA damage.
Our results demonstrate that the α,β-unsaturated 2’-
exomethylene nucleosides exhibit potent antioxidant
activities. This property is further reinforced when the
nucleobase thymine is replaced by uracil. The potent
antioxidant properties of these compounds can be ex-
plained by a radical stabilization resonance effect, which
can be attributed to their structural properties. It should
be mentioned that the α,β-unsaturated 4’-exomethylene
nucleosides had no antioxidant properties.
In contrast to the exomethylene group the influence of
the keto group in the antioxidant properties of these
compounds is less efficient. The α,β-unsaturated 2’-keto
nucleosides exhibit antioxidant properties only at con-
centrations of 100 μM. The 2’-keto and the 4’-keto uracil
nucleoside analogues showed similar antioxidant proper-
ties which does not apply to the 2’-keto and the 4’-keto
thymine analogues. These results point to a nucleobase
preference since the substitution of thymine with the
smaller uracil leads to compounds with increased anti-
oxidant abilities. It seems that uracil might be beneficial
for the interaction of these compounds with the specific
4. Conclusions
The results of this study demonstrate that the presence of
a carbon-carbon double bond at α,β-disposition to exo-
methylene group at 2’-position of the sugar moiety and
uracil as nucleobase improves the antioxidant capacity of
the nucleoside analogues. These might be necessary
structural modifications that favor the interaction of these
nucleosides with the radicals. ROO radicals are involved
as a major initiating factor in lipid peroxidation chain
reactions [22]. Thus, the ability of the tested compounds
to protect DNA strand breakage by scavenging peroxyl
radicals could suggest that these compounds may also
prevent lipid peroxidation. Based on the above findings it
would be interesting to further investigate the potential
effectiveness of these nucleoside analogues in the pre-
vention and probably the treatment of diseases caused by
overproduction of free radicals. Further in vitro studies
are required to elucidate the exact mechanisms involved
in the antioxidant activity of these compounds.
5. Experimental
5.1. General
2.2’-azo-bis-2-amidinopropane dihydrochloride (AAPH)
was purchased from Sigma-Aldrich (St Louis MO, USA).
Bluescript-SK + plasmid DNA was isolated from a large
scale bacterial culture. All chemicals and solvents used
were of the highest quality commercially available.
5.2. Nucleoside analogues
Nucleoside analogues 1, 3, 5 and 7 were previously syn-
Copyright © 2011 SciRes. PP
Unsaturated Keto and Exomethylene Pyranonucleoside Analogues of Thymine and Uracil Exhibit Potent Antioxidant
Copyright © 2011 SciRes. PP
Table 1. Antioxidant properties of nucleosides analogues against ROΟ radical induced DNA damage.
% Inhibition
Compounds 5 μΜ 10 μΜ 20 μΜ 50 μΜ 100 μΜ
NI NI NI 16 ± 1†* 36 ± 3*
NI NI 22 ± 1* 21 ± 3* 25 ± 2*
NI NI NI NI 9 ± 1*
NI NI NI NI 22 ± 3*
NI NI NI NI 19 ± 2*
NI: no significant inhibition. Values are the means ± SΕ of the percent inhibition from three independent experiments. *p < 0.05 when compared with control
(plasmid DNA plus AAPH). Thy: Thymine, U: Uracil.
Unsaturated Keto and Exomethylene Pyranonucleoside Analogues of Thymine and Uracil Exhibit Potent Antioxidant
thesized [18] and analogues 2, 4, 6 and 8 were also pre-
viously synthesized [23]. All analogues were freshly
prepared in DMSO.
5.3. Peroxyl Radical-Induced DNA Strand
Scission Assay
The assay was performed using the method described by
Chang et al. [24]. Peroxyl radicals were generated from
thermal decomposition of AAPH. The reaction mixture
(10 μL) containing 1 μg Bluescript-SK + plasmid DNA,
2.5 mM AAPH in phosphate-buffered saline (PBS: 137
mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM
KH2PO4) and the tested product at different concentra-
tions (5, 10, 20, 50, 100 μM) was incubated in darkness
for 45 min at 37˚C. AAPH was added last right before
incubation. The reaction was terminated by the addition
of 3 μL loading buffer (0.25% bromophenol blue and
30% glycerol) and analyzed in 0.8% agarose gel elec-
trophoresis at 70 V for 1 h. The gels were stained with
ethidium bromide (0.5 μg/mL), destained with water,
photographed by UV translumination using the Vilber
Lourmat photodocumentation system (DP-001.FDC)
(Torcy, France) and analyzed with Gel-Pro Analyzer
version 3.0 (MediaCybernetics, Silver Spring, USA).
Each experiment was carried out in triplicate. The use of
DMSO at the tested concentrations did not affect the
results of the assay.
5.4. Inhibition of Free Radical-Induced DNA
The induction of DNA strand breaks by peroxyl (ROO)
was measured by the conversion of supercoiled Blue-
script-SK + plasmid double stranded DNA to the open
circular conformation analyzed in agarose gel electro-
phoresis. Preventive activity of the tested samples was
assessed by the inhibition of conversion of supercoiled
(unnicked) conformation to open circular (nicked). The
percentage inhibition of radical-induced DNA strand
cleavage by the tested compounds was calculated using
the following equation:
inhibition SS
where So is the percentage of supercoiled conforma- tion
in the negative control sample (plasmid DNA alone), Sp
is the percentage of supercoiled conformation in the
positive control sample (plasmid DNA with the radical
initiating factor) and S is the percentage of supercoiled
conformation in the sample containing plasmid DNA, the
tested compound and the radical initiating factor. It
should be noted that prior to treatment Bluescript-SK +
plasmid DNA contained approximately 10% - 20% open
circular DNA.
5.5. Statistical Analysis
All results are expressed as mean ±SD (n = 3). Statisti-
cal computations were carried out using the SPSS 13.0
software. For statistical analysis, one-way ANOVA was
applied followed by Dunnett’s test for multiple pair-wise
comparisons. Dose response relationships were exam-
ined by Spearman’s correlation analysis. Differences
were considered significant at p < 0.05.
6. Acknowledgements
This work has been funded by the Postgraduate Pro-
grams of “Biotechnology-Quality Assessment in Nutri-
tion and the Environment” and “Molecular Biology and
Genetics Applications-Diagnostic Markers” of the De-
partment of Biochemistry and Biotechnology of the
University of Thessaly.
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