Unsaturated Keto and Exomethylene Pyranonucleoside Analogues of Thymine and Uracil Exhibit Potent Antioxidant Properties

doi:10.4236/pp.2011.23016

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Pharmacology & Pharmacy, 2011, 2, 122-126

doi:10.4236/pp.2011.23016 Published Online July 2011 (http://www.scirp.org/journal/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.

Email: [email protected]

Received March 29th, 2011; revised April 30th, 2011; accepted June 22nd, 2011.

ABSTRACT

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

Properties

123

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

radicals.

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-

Unsaturated Keto and Exomethylene Pyranonucleoside Analogues of Thymine and Uracil Exhibit Potent Antioxidant

Properties

124

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

ΝΙ‡ ΝΙ ΝΙ ΝΙ ΝΙ

NI NI NI 16 ± 1†* 36 ± 3*

ΝΙ ΝΙ ΝΙ ΝΙ ΝΙ

NI NI 22 ± 1* 21 ± 3* 25 ± 2*

ΝΙ ΝΙ ΝΙ ΝΙ ΝΙ

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

Properties

125

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

Damage

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





00

(1)

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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