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DNA-BINDING STUDY AND NUCLEASE
ACTIVITY INDUCED BY A COPPER(II), N,N’BIS[(4-METHYLPHENYL)SULFONYL]
ETHYLENEDIAMINE AND 1,10PHENANTROLINE TERNARY SYSTEM
ANDREEA BODOKI1*, ADRIANA HANGAN1, LUMINIŢA
OPREAN1, JOAQUIN BORRAS2, ALFONSO CASTINEIRAS3,
MARIUS BOJIŢĂ1
Faculty of Pharmacy, “Iuliu Haţieganu” University, Cluj-Napoca, 12 I.
Creangă street, Cluj-Napoca, Romania
2
Faculty of Pharmacy, University of Valencia, Vicent Andres Estelles s/n,
Burjassot, Valencia, Spain
3
Faculty of Pharmacy, University of Santiago de Compostela, Spain
*corresponding author: abota@umfcluj.ro
1
Abstract
The present paper reports a new method for the synthesis of a Cu(II) ternary
system using N,N’-bis[(4-methylphenyl)sulfonyl]ethylenediamine and 1,10-phenantroline
as ligands.
The binding of the complex to DNA was investigated by thermal denaturation.
Its potency as artificial nuclease has also been tested and an efficient oxidative DNA
cleavage was observed in the presence of a reducing agent (sodium ascorbate).
Rezumat
Lucrarea prezintă o nouă metodă de sinteză a unui sistem ternar al Cu(II)
utilizând ca liganzi N,N’-bis[(4-metilfenil)sulfonil]etilenediamina şi 1,10-fenantrolina.
Studiile efectuate indică o puternică interacţiune complex – ADN, interacţiune
care se traduce printr-o stabilizare a structurii ADN-ului. În prezenţa unui agent reducător
(ascorbat de sodiu), apare scindarea moleculei de ADN, activitatea nucleazică fiind similară
cu cea a complexului [Cu(1,10-fen)2]2+.



Cu(II) complex
DNA-binding
nuclease activity
INTRODUCTION
Over the past decades there has been a considerable interest in DNA
binding properties toward different types of metal complexes. Thus, many
transition metal complexes have been used as tools for understanding DNA
structure, as agents for mediation of DNA cleavage or as chemotherapeutic
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agents. Our research group has obtained and characterized several Cu(II)
complexes which are able to induce DNA cleavage, with an activity similar or
superior to that of [Cu(1,10-phen)2]2+, the first effective artificial nuclease[1-4].
Sigman [7] has shown that copper complexes of 1,10-phenantroline act
as artificial nucleases in the presence of molecular oxygen and reducing agents.
Studies have shown that the mechanism of DNA scission induced by Cu(II)
complexes involves the formation of reactive oxygen species (ROS) [6-9].
The present paper reports a new method for the synthesis of the Cu
(II) ternary system using the N,N’-bis[(4-methylphenyl)sulfonyl])
etilendiamine (H2L) and 1,10-phenantroline as ligands [5].
For the new Cu(II) ternary system, the interaction with calf-thymus
DNA was investigated by thermal denaturation. DNA cleavage induced by
the complex in the presence of reducing agent, has also been demonstrated.
MATERIALS AND METHODS
All reagents and solvents were commercially available and were
used without further purification. pUC18 was purchased from Roche
Diagnostics. Calf-thymus DNA (CT-DNA) was supplied by Sigma-Aldrich.
Synthesis
Solid copper(II) acetate monohydrate (1mmol, 199.65 mg) was
added to 20 mL of N,N’- dimethylformamide solution of H2L (0.5 mmol,
184.32 mg). A dark green solution formed immediately. Upon complete
dissolution of the copper(II) salt, 10 mL methanol solution of 1,10phenantroline monohydrate (3 mmol, 594.66 mg) was added to the system.
The dark green solution turned brown. Slow evaporation of the solvent
yielded pale green prismatic crystals, suitable for X-ray diffraction. X-ray
crystallography confirmed the structural formula [CuL(1,10-phen)2] for the
complex, structure first described by A. Sousa et al., but obtained through
an electrochemical method (figure 1) [5].
Figure 1
ORTEP drawing of [CuL(1,10-phen)2]
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Thermal denaturation
DNA melting experiments were carried out by monitoring the
absorbance (260 nm) of calf-thymus DNA (CT-DNA, 100 μM base-pairs),
at different temperatures (ranged between 25 and 90 ºC) in the absence and
presence of the complex, in a 2.5/1 DNA/complex ratio. Measurements
were performed with an Agilent 8435 spectrophotometer equipped with a
temperature-controlled sample cell. The solution containing the complex
and CT-DNA in 0.1 M borate buffer (2mM NaCl, pH 8.0) was heated with a
rate of temperature increase of 1ºC per min.
pUC18 DNA cleavage
A typical reaction with the complex was performed by mixing 7μL
of borate buffer (pH=8.0), 1μL of pUC18 (0.25μg·μL-1), 6μL of a solution
of the complex (50% DMF) at increasing concentrations between 1.8 and
6μM, and 6μL of sodium ascorbate solution (1000-fold molar excess
relative to the concentration of the complex) in borate buffer. The mixture
was incubated for 1h at 37ºC, then 3μL of a quench buffer solution
consisting of 0.25% bromophenol blue, 0.255 xylene cyanole, and 30%
glycerol was added. The solution was then subjected to electrophoresis on a
0.8% agarose gel in 0.5x TBE buffer (0.045mM TRIS, 0.045mM boric acid
and 1mM EDTA) containing 2μL per 100mL of a solution of ethidium
bromide (10 mg mL-1). Gel electrophoresis was carried out at 80V for 2 h.
The gel was photographed on a capturing gel printer plus TDI.
Mechanistic studies were performed in the presence of several
reactive oxygen species (ROS) scavengers: standard hydroxyl radical
scavengers (DMSO 0.4M, tert-butil-alcohol 0.4M, sodium formiate 0.4M
and urea 0.4M), singlet oxygen scavengers (2,2,6,6-tetramethyl-4piperidone 0.4M and DABCO 0.4M), a superoxide radical scavenger
(TIRON 100mM) and catalase 650U/ml. Methyl green, a major groove
binder (2.5μL of a 0.01 mg mL-1 solution), distamycine, a minor groove
binder (8μM), and neocuproine, a ligand that strongly bindes to Cu(I) (12,
120 and 240 µM), were also used.
RESULTS AND DISCUSSION
DNA-binding interaction – Thermal denaturation
In order to obtain information on the type of DNA – complex
interaction, the binding of the complex to CT-DNA was studied by
examining the thermal denaturation profile of DNA. The intercalation of
small molecules into the double helix has as a result an increase of melting
temperature at which the double helix denaturates into single helix DNA;
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the DNA melting temperature (Tm) is defined as the temperature at which
50% of the base pairs is unpaired [10].
The literature describes three patterns of interaction between
different structures and double stranded DNA: electrostatic interactions with
the negatively charged phosphate structures, binding interactions with the
two grooves of DNA double helix and intercalation between the staked
based pairs of native DNA. However, an increase of the Tm of less than 0.6
ºC suggests a nonspecific interaction with the phosphate groups of DNA
structure [10].
Tm of CT-DNA is 50.8 ºC in the absence of the complex and 65.9
ºC upon addition of the complex (figure 2). The significant increase of Tm
(∆Tm = 15.1 ºC) suggests that the interaction of the complex with DNA is
performed through intercalation.
0,8
1,35
DNA
0,75
1,3
0,7
1,25
0,65
1,2
0,6
1,15
DNA + complex
0,55
1,1
0,5
1,05
32
40
48
56
64
72
80
88
Temperature ( t C )
Figure 2
DNA thermal denaturation profile in the absence and presence of [CuL(1,10-phen)2]
20µM, borate buffer 1mM, NaCl 2mM (pH=8), 1%DMF ([DNA]/[C2] = 2.5/1)
Nuclease activity
The DNA cleavage activity of the complex was evaluated by the
conversion of supercoiled DNA (SC, form I) to nicked circular (NC form II)
or linearized DNA (LC form III). At concentrations of 3μM, 4.5μM and
6μM (lanes 7, 8 and 9, figure 3) the complex is able to partially convert
supercoiled DNA into nicked circular and linear DNA, activity correlated
with the increase of complex concentration.
Control experiments with CuCl2 and [Cu(1,10-phen)2]2+ were also
carried out under the same experimental conditions. At the highest
concentration assayed (6μM), no DNA strand scission was observed for
CuCl2 (lane 4, figure 3). Under the same conditions, at the highest
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concentration assayed (6μM), the complex has a similar activity to that of
[Cu(1,10-phen)2]2+ (lane 10, figure 3).
The possibility of a hydrolytic mechanism for the DNA scission
mediated by the complex was taken into consideration. The nuclease
activity of the complex was assayed in the absence of ascorbate activation
(lane 5, figure 3). No DNA cleavage was observed, indicating that a
hydrolytic mechanism is not involved.
1
2
3
4
5
6
7
8
9
10
Figure 3
1. λDNA/EcoRI + Hind III Marker; 2. supercoiled DNA control; 3. supercoiled
DNA control + ascorbate 6mM; 4. CuCl2 6 µM; 5. complex 6µM without
ascorbate; 6. complex 1.8µM; 7. complex 3µM; 8. complex 4.5µM; 9. complex
6µM; 10. [Cu(1,10-phen)2] 2+ 6µM
Nuclease activity - Mechanistic study
The mechanism of DNA cleavage mediated by the complex was
studied using ROS scavangers. The groove binding preferences were tested
in the presence of the minor groove binder distamycin and the major groove
binder methyl green. The reduction of Cu(II) to Cu(I) during the cleavage
process was evaluated with neocuproine, a ligand that strongly chelates
Cu(I) (figure 4).
1
2
3
4
5
6
7
8
9
10
11 12
13
14 15
16
Figure 4
1. λDNA/EcoRI + Hind III Marker; 2. supercoiled DNA control; 3.complex 6µM;
4.complex 6µM + DMSO 0.4M; 5.complex 6µM + tert-butyl-alcohol 0.4M;
6.complex 6µM + sodium formiate 0.4M; 7. complex 6µM + urea 0.4M;
8.complex 6µM + tetramethyl piperidone 0.4M; 9.complex 6µM + DABCO 0.4M;
10.complex 6µM + TIRON 100mM; 11.complex 6µM + neocuproine 12 µM;
12.complex 6µM + neocuproine 120 µM; 13.complex 6µM + neocuproine 240
µM; 14.complex 6µM + distamicine 8 µM; 15.complex 6µM + methyl green 1.25
µg/ml; 16.complex 6µM + catalase 650U/ml
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An inhibition of DNA cleavage is observed upon addition of tertbutyl-alcohol and sodium formiate, suggesting that hydroxyl radical is
involved in the DNA scission process (lanes 5 and 6, figure 4). A lack of
inhibition in the presence of DMSO and urea, two other standard hydroxyl
radical scavengers, can not though completely exclude the involvement of
hydroxyl radical in the DNA cleavage process (lanes 4 and 6, figure 4). The
addition of DABCO and 2,2,6,6-tethramethyl-4-piperidone (lanes 8 and 9,
figure 4) also decreases the cleavage efficiency, indicating that either singlet
oxygen (1O2) or a singlet-oxygen-like entity is one of the active oxygen
intermediates responsible for DNA scission. An inhibition of the process in
the presence of TIRON suggests that superoxide anion (·O2-) is involved in
the cleavage (lane 10, figure 4). Catalase also inhibits the nucleolytic
process (lane 16, figure 4); this confirms hydrogen peroxide (H2O2) is also
necessary in the DNA scission.
Prior treatment of pUC18 DNA with distamycine and methyl green
has no effect on the cleavage process mediated by the compound (lanes 14
and 15, figure 4). We can conclude that the studied complex does not
interact with the double strand DNA through binding to the two grooves of
DNA structure.
The presence of neocuproine at a concentration of 12μM does not
interfere with the cleavage process (lane 11, figure 4). An inhibition of the
process is though observed at higher concentrations of neocuproine (120μM
and 240μM), indicating that Cu(I) is involved in the process.
Bocarsly et al. [8, 9] have suggested a mechanism involving
hydroxyl radicals, generated by a variety of chemical and physical pathways
related to either Fenton or Haber-Weiss reaction.
Fenton mechanism:
Cu2+L + ascH2 → Cu+L + ascHCu+L + H2O2 → Cu2+L + OH- + ·OH
Haber-Weiss reaction:
Cu+L + O2 → Cu2+L + O2O2- + H2O2 → O2 + OH- + ·OH
CONCLUSIONS
A new method for the synthesis of the Cu (II) ternary system using
the N,N’-bis[(4-methylphenyl)sulfonyl])etilendiamine (H2L) and 1,10phenantroline as ligands was described.
The binding of DNA, studied by Tm measurements, indicates that
the complex interacts with the DNA base pairs through intercalation
(staking). The complex acts as an efficient chemical nuclease upon
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ascorbate activation. The participation of hydrogen peroxide, hydroxyl
radical, superoxide anion and singlet-oxygen-like entities in the DNA
scission mediated by the complex suggests a mechanism pathway involving
a Fenton or Haber-Weiss reaction.
Acknowledgement: The authors are thankful for the financial support
offered by research grants CNCSIS 1467 and PN II 61-003.
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