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6Фталоцианины Phthalocyanines
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http://macroheterocycles.isuct.ru
Сообщение Communication
Dimeric Fe-Co Phthalocyanine Complex as a Reagent for the Selective Damage of Nucleic Acids
Alexander A. Chernonosov,ab@ Ludmila I. Solov'eva,c Evgenii A. Luk'yanets,c Dmitrii G. Knorre,a and Ol'ga S. Fedorovaab
aInstitute of Chemical Biology and Fundamental Medicine, 630090 Novosibirsk, Russia bNovosibirsk State University, 630090 Novosibirsk, Russia cInstitute of Organic Intermediates and Dyes, 103787 Moscow, Russia @Corresponding author E-mail: sandy@niboch.nsc.ru
The complexes of Fe11 and Co11 with phthalocyanines are extremely good catalysts for oxidation of organic compounds with molecular oxygen and hydrogen peroxide. Their solubility and reactivity are increased by conjugation with oligonucleotides and by formation of dimeric complexes between negatively and positively charged phthalocyanines. In our work such complexes were formed directly on single-stranded DNA through interaction between negatively charged Co11 phthalocyanine in conjugate and positively charged Fe" phthalocyanine in solution. The resulting oppositely charged phthalocyanine complexes showed significant increase of catalytic activity compared with monomeric forms of phthalocyanines Fe11. The site-directed modification of single- and double-stranded DNA by H2O2 in the presence of dimeric complexes of negatively charged Co11 and positively charged Fe11 phthalocyanines was detected. These complexes catalyzed the DNA oxidation with high efficacy and led to direct DNA strand cleavage.
Keywords: Fe11 and Co11 phthalocyanines, nucleic acid, catalysis.
Introduction
Many natural and synthetic compounds that could selectively modified various cell constituents including DNA are studied as potential anti-cancer drugs during past decades. Complexes of phthalocyanines (Pcs) with the ions of transition metals (Fe, Co, etc) represent an interesting type of reactive group due to their ability to catalyze the formation of reactive oxygen species. Currently, phthalocyanine complexes of Co11 and Fe11 are being investigated as drugs for the catalytic therapy of cancer.[1]
The main problem of phthalocyanine usage in cancer therapy is their low solubility in water solutions. The solubility could be increased by Pcs conjugation with 6 or 8 charged substituents. But this method did not increase the specificity of resulting reagents.
On the other hand, using any affinity groups as substituents it is possible to increase the selectivity of action of the resulting conjugates. For example, the conjugation of Pcs with oligonucleotides^ which are of interest as potential anti-cancer drugs themselves,[3] could has a positive synergetic effect in therapy of cancer.
In recent investigations, the sequence-directed oxidative cleavage of DNA with O2 and H2O2 in the presence of conjugates of Co11 and Fe11 phthalocyanines attached to oligonucleotides were studied.[4,5] It was shown that single-stranded DNA is efficiently damaged in complexes with these conjugates. Moreover, the activity of Pcs could be increased by forming dimeric complexes between negatively and positively charged phthalocyanines.
The site-directed modification of single-stranded DNA by O2 and H2O2 in the presence of heterogeneous dimeric
complexes of negatively and positively charged Fe11 and Co11 phthalocyanines (FeIIPc FeIIPc and CoIIPc CoIIPc ,
r j v pos neg pos neg'
respectively) was investigated in our previous work.[6] These complexes were formed directly on single-stranded DNA through interaction between the negatively charged phthalocyanine in the oligonucleotide-conjugate and the positively charged phthalocyanine in the solution. The resulting phthalocyanine complexes showed a significant increase in catalytic activity compared to monomeric forms of phthalocyanines Fen and CoII[6] and led to direct DNA strand cleavage. It was determined that oxidation of DNA by molecular oxygen catalyzed by a complex of FeII-phthalocyanines proceeds with a higher rate than that catalyzed by CoII-phthalocyanines; however, the latter led to a higher yield of target DNA modification.
In current work we decided to combine two phthalocyanines to form heterogeneous metal dimeric complex of negatively charged CoII and positively charged Fen phthalocyanines (FeIIPc CoIIPc ). We study the
pos neg
modification of single-and double-stranded DNA by such complex.
Experimental
Chemicals and reagents. Acrylamide, N,N'-methylene-bisacrylamide, urea, acetonitrile, DMF (Fluka, Switzerland), Tris and piperidine, were used. All solutions were prepared with double-distilled water using ultrapure reagents. Hydrogen peroxide (stabilized, more than 30%) was purchased from Fluka. T4 polynucleotide kinase was purchased from Sibenzyme (Russia). [y-32P]ATP (> 3000Ci/mmol) was purchased from Biosan (Russia). All experiments were carried out at 25 °C in a buffer containing 0.16 M NaCl, 0.02 M sodium phosphate (pH 7.4), and 1 mM EDTA.
Макрогетероциклы /Macroheterocycles 2011 4(2) 135-137 © ISUCT Publishing
135
Dimeric Fe-Co Phthalocyanine Complex
Phthalocyanines. Cobalt(II) tetra-4-carboxyphtha-locyanine CoPc was prepared and characterized as described previously.171 Octakis-4,5-(iV-P-aminoethyl-P'-iV,iV-diethylammonioethoxycarbonyl) phthalocyanine CoPcpos and FeP-cpos complexes were prepared with high yields by quaternization of the respective octakis-4,5-(P-chloroethoxycarbonyl)phthalocyanine complexes171 with VV-diethylethylenediamine by heating them in V-methylpyrrolidone in the presence of catalytic amounts of anhydrous sodium iodide.[6]
Oligonucleotides and conjugates. The oligonucleotides 5'd(AATGGGAATAAAAAAAAAAA)3' and 5'd(TATTCCCATT)3' (ODN) were synthesized and purified in the same way as reported previously.[6] Concentrations of the oligonucleotides were determined from their absorbance at 260 nm.[8] The 5'-termini of the oligonucleotides were 32P-labeled using a standard procedure with T4-polynucleotide kinase and [y-32P]ATP (> 3000 Ci/mmol).[9] The concentration of the labeled oligonucleotide solution did not exceed 2.5 •10-7 M. The oligonucleotide derivative CoPc-NH-(CH2)6-0-5'pd(TATTCCCATT)3' CoIIPc-ODN was synthesized using a previously reported solid phase method[10] with a 30- 40% yield.
Oxidation of the target oligonucleotides. Oxidation of DNA target by hydrogen peroxide was carried out in DNA duplex (Figure 1). The concentrations of the target DNA and phthalocya-nine-oligonucleotide derivatives were equimolar (1.010-6 M), the concentration of the 32P-labeled DNA-target was 1.010-8 M. The concentrations of the hydrogen peroxide was 1.010-3 M. The reaction was initiated by adding H2O2 or the reducing agents. Aliquots (10 ^l) were taken from the reaction mixture at different times and immediately transferred into polypropylene tubes containing 200 ^l of 2% LiClO4 in acetone. The precipitate was pelleted by centrifugation, washed twice with 80% ethanol and once with acetone, and dried under vacuum. The samples were then either treated or not with 1 M piperidine.[11] The products of the modification were separated by 20% PAGE in the presence of 7 M urea. After electrophoresis, the gel was exposed to CP-BU X-ray film (Agfa-Gevaert, Belgium) for 10-20 h at -10 °C. Autoradiograms were quantitated using Gel-Pro Analyzer 3.0 software (Media Cybernetics, MD). The yield of modification was calculated as the ratio of the areas of the product peaks to the sum of the areas of the product and the initial oligonucleotide peaks. The error did not exceed 20%.
Fe"Pc interacts with DNA and is located at the 5'-end of
pos
DNA target. The total modification yield was about 25% in the presence of H2O2.
5'-[32P]ÀATGGGAATAAAAAAAAAaX-3' DNA target
TTACCCTTAT-ConPcneg ConPcneg-ODN
„Ilr
Fe Pc,
•pos
Rz
R2 FenPcpos
RI = R2 = R = COOC2H4NtC2H5)2C2H4NH2 Me = Fe(II)
ConPcneg-ODN
Rl = COOH, R2 = H, Me - Co(D) R = pO(CH2)6NH-ODN
Figure 1: Structures of the DNA duplex and nucleotide sequences of the DNA target and CoIIPc -ODN; and the structures of the Pcs
° neg '
used in this work.
20
18
16
~ 14 c 0)
■g 12 S
a> 10 o>
a % 8
<u
Ô 6 <
Z 4 a
] PcFe(ll)
A19A18A17A16A15A14A13A12A11A10 T9 AB A7 G6 G5 G4 Nucleic acid residues
Results and Discussion
Oxidation of DNA target by O2 and H2O2 in the presence of complexes of oppositely charged phthalocyanines of ConPc and FenPc was studied within the DNA duplex
neg pos A
containing two parts (Figure 1). The first part is a double-stranded fragment (A1-A10) formed between the oligonucleotide part of the phthalocyanine-oligonucleotide derivative and the complementary sequence of the single-stranded target. The other part is a single-stranded fragment (A11-A20) of the target, not involved in duplex formation. This region of the target DNA is located in close distance to the phthalocyanine moiety in ConPcneg-ODN and, therefore can be highly modified. Free FenPcpos can interact in solution with both double- and single-stranded parts of complex and negatively charged Pc residues.
In case of free Fe"Pc the nucleotide oxidation de-
pos
pended on the region of interaction between target and FeIIPc . As can be seen from Figure 2A the nucleotides G4-
pos
A7 are the mostly modified region with 5-8% modification yield. As far as we did not observe any preferable oxidation of other nucleotides, we could make a conclusion that free
20-
18-
sS 16-
+J* c 14-
S
g 12-
a)
S) IS 10-
>
ra a> 8-
o 6-
<
z
D 4-
2-
0-
I complex PcCo(ll) -PcFe(ll)
B
........
A19A18A17A16A15A14A13A12A11A10 T9 AS A7 G6 G5 G4 Nucleic acid residues
Figure 2. Distribution of base modifications in the target DNA by free FeIIPc (A) and by dimeric complex FeIIPc CoIIPc -ODN
pos v ' J r pos neg
(B). The modifications were revealed by treatment with 1 M pip-eridine.
The distribution of nucleotide modifications was
more selective in the case of FenPc ConPc -ODN compos neg
136
Макрогетероциmbl /Macroheterocycles 2011 4(2) 135-137
A. A. Chernonosov et al.
plex. As for FenPcpos the G4-A7 region was oxidized with 3-4% modification yield, but A12 and A13 nucleotides were mainly modified with 19% and 8% yield, respectively (Figure 2B). Such selective oxidation corresponds to expected location of the reactive complex (FenPc ConPc
A A v pos neg
) near A10-A15 region. The total modification yield was about 50% that in 2-fold higher than in the case of free FeIIPc . The oxidation in the G4-A7 region in both cases
pos
seems to be due to reactivity and localization of the free FenPc within DNA duplex.
pos
In comparison with the catalytic activities of the
FenPc FenPc and ConPc ConPc complexes reportpos neg pos neg
ed previously[6] the reactivity of the heterogeneous complex studied in this work was similar to the last one. Thus, the change of Co" into Fe" in Pcpos did not significantly influence the modification yield and DNA oxidation rate within first 24 hours, although the selectivity of action was increased.
Conclusions
Overall, this study indicates that heterogeneous dimeric complex Fe"Pc ConPc -ODN possesses the increased
pos neg
catalytic activity in the oxidative degradation of DNA with H2O2 in comparison with monomeric phthalocyanines.[412]
Acknowledgements. This research was made possible in part by a Grant from the Russian Foundation for Basic Research (No. 11-04-01377), President Grant (No. 3185.2010.4) and Grant from Russian Ministry of Education and Sciences (No. 2.1.1/10697).
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Received 29.04.2011 Accepted 20.06.2011
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