DNA binding and cleavage studies of copper(II) complexes with 2′-deoxyadenosine modified histidine moiety

Article abstract of DOI:10.1007/s00775-015-1282-2

This work is focused on the study of DNA binding and cleavage properties of 2′-deoxyadenosines modified with ester/amide of histidine (his⁶dA ester, his⁶dA amide) and their copper(II) complexes. To determine the coordination mode of

Full text of DOI:10.1007/s00775-015-1282-2

J Biol Inorg Chem (2015) 20:989–1004
DOI 10.1007/s00775-015-1282-2
ORIGINAL PAPER
DNA binding and cleavage studies of copper(II) complexes
with 2′‑deoxyadenosine modified histidine moiety
Justyna Borowska1 · Malgorzata Sierant2 · Elzbieta Sochacka3 · Daniele Sanna4 ·
Elzbieta Lodyga‑Chruscinska1
Received: 12 March 2015 / Accepted: 20 June 2015 / Published online: 18 July 2015
© SBIC 2015
Abstract This work is focused on the study of DNA
binding and cleavage properties of 2′-deoxyadenosines
modified with ester/amide of histidine (his6dA ester, his6dA
amide) and their copper(II) complexes. To determine the
coordination mode of the complex species potentiometric
and spectroscopic (UV–visible, CD, EPR) studies have
been performed. The analysis of electronic absorption and
fluorescence spectra has been used to find the nature of the
interactions between the compounds and calf thymus DNA
(CT-DNA). There is significant influence of the –NH2 and
–OCH3 groups on binding of the ligands or the complexes
to DNA. Only amide derivative and its complex reveal
intercalative ability. In the case of his6dA ester and Cu(II)–
his6dA ester the main interactions can be groove bind-
ing. DNA cleavage activities of the compounds have been
examined by gel electrophoresis. The copper complexes
have promoted the cleavage of plasmid DNA, but none of
the ligands exhibited any chemical nuclease activity. The
application of different scavengers of reactive oxygen spe-
cies provided a conclusion that DNA cleavage caused by
copper complexes might occur via hydrolytic pathway.
Keywords Artificial nucleases · Copper complexes ·
Histidine derivatives
Introduction
The development of synthetic nucleases capable of accel-
erating the hydrolysis of DNA has attracted considerable
scientific interest over the last 20 years. There are several
reasons for justifying the interest in such systems. First,
artificial nucleases could lead to a better understanding of
the chemistry of hydrolytic enzymes, secondly, they can be
used to detoxify pesticides and chemical weapons, which
often have phosphate ester-like structures and, thirdly, rea-
gents effectively hydrolyzing phosphodiester bonds can be
used in molecular biology as artificial restriction enzymes.
These systems are also very valuable, because they can
show different sequence selectivity to cut DNA than natural
nucleases [1].
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-015-1282-2) contains supplementary
material, which is available to authorized users.
* Justyna Borowska
juustyna.pawlak@o2.pl
* Elzbieta Lodyga-Chruscinska
elalodyg@p.lodz.pl
Transition metal complexes due to DNA binding and
cutting abilities in physiological conditions found wide
applications in chemistry of nucleic acids as sequence-
specific binding factors to DNA or as diagnostic agents in
medical applications.
Copper belongs to the so-called bioelements and plays
a critical role in biological processes. Activity of many
identified enzymes requires the presence of copper in their
active centers. Because of important function in cell metab-
olism, chemical synthesis and biological activity, copper(II)
1
Institute of General Food Chemistry, Lodz University
of Technology, ul. Stefanowskiego 4/10, 90-924 Lodz,
Poland
2
Centre of Molecular and Macromolecular Studies, Polish
Academy of Science, ul. Sienkiewicza 112, 90-363 Lodz,
Poland
3
Institute of Organic Chemistry, Lodz University
of Technology, ul. Zeromskiego 176, 90-924 Lodz, Poland
4
Istituto C.N.R. Chimica Biomolecolare, Trav. La Crucca 3,
reg. Baldinca, 07040 Sassari, Italy
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J Biol Inorg Chem (2015) 20:989–1004
complexes are the subject of research in the area of bioinor-
ganic and biological chemistry. Studies of the fragments of
nucleic acids and their complexes with copper are consid-
ered to be very important, especially in relation to biologi-
cal and clinical role of these compounds [2].
Synthesis of histidine modified 2′‑deoxyadenosines
(his6dA amide and his6dA ester)
The synthesis of 2′-deoxyadenosines modified with l-histi-
dine residue was performed according to the Scheme 1.
Nucleosides modified with amino acid residues were
used as “building blocks” of oligonucleotides with the
sequence of DNA enzymes (deoxyribozymes) [3, 4]. Deox-
yribozymes that contain nucleosides modified by the threo-
nine or histamine moiety, showed an increased potential for
hydrolysis of complementary RNA [5].
The selected modified nucleosides can serve as model
systems to clarify the structural and functional changes
occurring under the influence of transition metal ions in
DNA. In our previous work, we described the physico-
chemical and DNA cleavage properties of his6dA (histi-
dine modified 2′-deoxyriboadenosine) and its complex
with copper(II). Both the ligand and the Cu(II) complex
can effectively promote cleavage of plasmid DNA in the
absence of any reducing agents [6].
In the present work, we have firstly focused on find-
ing the stoichiometry and coordinating mode of Cu(II)
complexes with his6dA ester or his6dA amide formed in
aqueous solution. Potentiometric and spectroscopic (UV–
visible, CD, and EPR) studies have been performed. The
second step of our research was aimed to define DNA bind-
ing efficacy and DNA cleavage activity of both ligands
(his6dA ester and his6dA amide) and their complexes with
copper(II) ions. The DNA cleavage has been found only
in the case of the copper complexes. An attempt has been
made to elucidate the mechanism of DNA binding and
cleavage.
Synthesis of 3′,5′‑di‑O‑acetyl‑2′‑deoxyadenosine (b)
To the solution of 2′-deoxyadenosine a (3 g, 11.95 mmol)
in 40 mL of anhydrous pyridine 12 mL of acetic anhy-
dride (119.5 mmol) were slowly dropped and the reac-
tion mixture was stirred for 1.5 h at room temperature.
Then, after cooling in the ice bath, 10 mL of CH2Cl2
were added and the solution was washed with 10 % aq.
NaHCO3 (50 mL). The aqueous layer was extracted twice
with 30 mL of chloroform. The combined organic layers
were dried with MgSO4, the drying agent was filtered off
and chloroform evaporated under reduced pressure. The
resulting precipitate was three times evaporated with tol-
uene (3 × 15 mL) and purified by crystallization from
ethanol to give 3.18 g (9.5 mmol) of acetylated 2′-deoxy-
adenosine b (yield 79 %), TLC: Rf = 0.66, (CHCl3/
1
MeOH 80:20 v/v). H NMR (250 MHz, CDCl3) δ 2.09
(s, 3H, CH3COO), 2.14 (s, 3H, CH3COO), 2.62 (ddd,
1H, JH2′,H3 = 2.5 Hz, JH2′,H1′ = 5.9 Hz, Jgem = 14.1 Hz,
H2′), 2.96 (ddd, 1H, JH2″,H3′ = 6.3 Hz, JH2″,H1′ = 8.1 Hz,
Jgem = 14.1 Hz, H2″), 4.32–4.46 (m, 3H, H4′, H5′,
H5″), 5.43 (dt, 1H, JH3′,H2′ = JH3′,H4′ = 2.5 Hz,
JH3′,H2″ = 6.3 Hz, H3′) 5.85 (bs, 2H, NH-6), 6.46 (dd,
1H, JH1′,H2′ = 5.9 Hz, JH1′,H2″ = 8.1 Hz, H1), 7.99 (s, 1H,
H2), 8.36 (s, 1H, H8).
Synthesis of 3′,5′‑di‑O‑acetyl‑N6‑phenoxycarbonyl‑2′‑deox
yadenosine (d)
Materials and methods
Reagents
3′,5′-Di-O-acetyl-2′-deoxyadenosine b (670 mg, 2 mmol)
was dried by evaporation twice with 10 mL of anhydrous
benzene and methylene chloride (1:1 v/v). Then dissolved
in 20 mL of anhydrous methylene chloride and 1-methyl-
3-phenoxycarbonylimidazolium chloride (954 mg, 4 mmol)
was added. After the reaction mixture was stirred for 18 h
at room temperature, the solution was evaporated in vac-
uum and the residue dissolved in small amount of EtOAc.
Purification on a silica gel column using the same solvent
as the eluent gave the nucleoside d in 65 % yield (579 mg,
1.3 mmol), TLC: Rf = 0.31, (EtOAc), 1H NMR (250 MHz,
CDCl3) δ 2.07 (s, 3H, CH3COO), 2.14 (s, 3H, CH3COO),
2.68 (ddd, 1H, JH2′,H3′ = 2.5 Hz, JH2′, H1′ = 6.0 Hz,
Jgem = 14.2 Hz, H2′), 2.98 (ddd, 1H, JH2″,H3′ = 6.3 Hz,
JH2″,H1′ = 7.8 Hz, Jgem = 14.2 Hz, H2″), 4.35–4.45 (m, 3H,
H4′, H5′, H5″), 5.47 (m, 1H, H3′), 6.50 (m, 1H, H1′), 6.80–
7.45 (m, 5H, PhO), 8.22 (s, 1H, H2), 8.72 (bs, 1H, CONH),
8.81 (s, 1H, H8).
All chemicals were of reagent grade and were used without
further purification, unless otherwise noted. Plasmid pEGFP-
C1 was purchased from BD Biosciences. Agarose and eth-
idium bromide were obtained from Serva Electrophoresis,
CT-DNA, 2′-deoxyadenosine, l-histidine were purchased
from Sigma-Aldrich. Modified nucleosides: his6dA amide
and his6dA ester were prepared according to the established
procedures reported beneath. Their purity was verified by
1
HPLC, H NMR and high-resolution mass spectrometry to
be >99 %. For the experiments, Cu(II)-L systems, where
L = his6dA amide, his6dA ester, were prepared in aqueous
solutions by mixing CuCl2 and the ligands in molar ratio of
1:2, to maintain the formation of the complexes. All solu-
tions were prepared using ultra-pure water (Mili-Q Integral
Water Purification System, Merck Millipore).
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Scheme 1 Synthesis of his6dA amide and his6dA ester
Synthesis of methyl ester of N2‑{[9‑(3′,5′‑di‑O‑acetyl‑‑
d‑2′‑deoxyribofuranosyl)‑9Hpurin‑6‑yl]‑carbamoyl}‑l‑his
tidine (f)
chromatography on silica gel eluting with a gradient: 0 %
MeOH in CHCl3 to 10 % MeOH in CHCl3. The product
was obtained as white foam (173 mg, 0.32 mmol, 81 %
yield). Its NMR analysis showed the presence of trieth-
ylamine. The calculated purity of the product was 81 %
(final yield 66 %). Obtained compound f, without fur-
ther purification, was used as a substrate in subsequent
reactions, TLC: Rf = 0.07, (CHCl3/MeOH 90:10 v/v)
1H NMR (250 MHz, CDCl3) δ 2.05 (s, 3H, CH3COO),
2.15 (s, 3H, CH3COO), 2.67 (ddd, 1H, JH2′,H3′ = 2.5 Hz,
JH2′,H1′ = 5.9 Hz, Jgem = 14.1 Hz, H2′), 2.98 (m, 1H, H2″),
3.26–3.34 (m, 2H, CH2-β), 3.76 (s, 3H, OCH3), 4.13–4.42
(m, 3H, H4′, H5′, H5″), 4.94 (m, 1H, CH-α) 5.46 (m, 1H,
3′,5′-Di-O-acetyl-N6-phenoxycarbonyl-2′-deoxyadenosine
d (177 mg, 0.4 mmol) was dried by evaporation twice
with anhydrous methylene chloride and then dissolved
in the same solvent (3.6 mL). Triethylamine (124 μL,
0.88 mmol) and histidine methyl ester hydrochloride e
(106 mg, 0.4 mmol) were added. After 1 h, TLC control
(CHCl3/MeOH 90:10 v/v) indicated complete reaction
of substrates. The solvent was evaporated under reduced
pressure. Obtained solid residue was purified by column
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J Biol Inorg Chem (2015) 20:989–1004
H3′), 6.48 (m, 1H, H1′), 6.96 (bs, 1H, Im-CH-5), 7.79 (bs,
1H, Im-CH-2), 8.44 (s, 1H, H2), 8.53 (s, 1H, H8), 9.01 (bs,
1H, CONH).
conditions the same as described for the compound 1); UV
(H2O): λmax = 209.0 nm, ε = 20 941 dm3 mol−1 cm−1
,
λmax = 269.4 nm, ε = 18 274 dm3 mol−1 cm−1; (HRMS,
FAB): m/z = 432.1661, [M + H]+, calculated for
C17H22N9O5 requires 432.1666; 1H NMR (250 MHz,
D2O, DMSO) δ 2.40 (ddd, 1H, JH2′,H3′ = 3.6 Hz,
JH2′,H1′ = 6.8 Hz, Jgem = 13.3 Hz, H2′), 2.71 (m, 1H, H2″),
3.05 (m, 2H, CH2-β), 3.57 (dd, 1H, JH5′,H4′ = 4.3 Hz,
Jgem = 12.2 Hz, H5′), 3.66 (dd, 1H, JH5″,H4′ = 3.7 Hz,
Jgem = 12.2 Hz, 5″), 3.96 (m, 1H, H4′), 4.25–4.51 (signals
superimposed with the signal of water molecule: CH-α,
H3′), 6.33 (pt, 1H, JH1′,H2′ = JH1′,H2″ = 6.8 Hz, H1′), 6.99
(s, 1H, Im-CH-5), 7.61 (s, 1H, Im-CH-2), 8.50 (s, 1H, H2),
8.51 (s, 1H, H8).
Synthesis of methyl ester of N2‑{[9‑(2′‑d‑deoxyribofuranos
yl)‑9Hpurin‑6‑yl]‑carbamoyl}‑l‑histidine (his6dA ester) (1)
Methyl
ester
of
N2-{[9-(3′,5′-di-O-acetyl-d-2′-
deoxyribofuranosyl)-9Hpurin-6-yl]-carbamoyl}-l-histidine
f (140 mg, 0.26 mmol) was dissolved in 3 mL of trieth-
ylamine solution in methanol (1:9 v/v). After 3 days,
TLC control indicated complete reaction of the substrate
(CHCl3/MeOH 80:20 v/v). The solvent was evaporated
under reduced pressure and the residual solid was coevap-
orated with toluene. The obtained product was then puri-
fied by column chromatography on silica gel eluting with a
gradient: 0 % MeOH in CHCl3 to 60 % MeOH in CHCl3.
Traces of triethylamine left in the product were removed
by coevaporation several times with methanol and tolu-
ene. The desired compound 1 was obtained as white foam
(82 mg, 0.18 mmol, 68 % yield), TLC: Rf = 0.18, (CHCl3/
MeOH 80:20 v/v), HPLC: Rf = 21.95 min (XTerra®
HPLC column MS C8, 5 μm 4,6 × 150 mm, flow 1 mL/
min, 20 °C, detection at λ = 254 nm; gradient elution—
solvent A—5 mM TEAB o pH = 7.5, solvent B = AcCN;
0–25 min from 0 to 30 % of B, 25–30 min 50 % of B);
Potentiometric measurements
The deprotonation constants of the ligands (pKa) and the
stability constants of Cu(II) complexes (log β) were deter-
mined by pH-potentiometric titrations of 2.0 mL samples.
The ligand:metal molar ratio was in the range 2:1, and the
concentration of Cu(II) was 1 × 10−3 M.
Measurements was carried out at 298 K and at the con-
stant ionic strength of 0.1 M KNO3 with a MOLSPIN
pH meter (Molspin Ltd., Newcastle-upon-Tyne, UK),
equipped with a digitally operated syringe (the Molspin
DSI 0.250 mL) controlled by computer. The titrations
were performed with a carbonate-free NaOH solution of
known concentration (ca. 0.1 M) using a Russel CMAWL/
S7 semi-micro combination pH electrode, calibrated for
hydrogen ion concentration by the method of Irving et al.
[7]. The number of experimental points was 100–150 for
each titration curve. The reproducibility of the titration
points included in the evaluation was within 0.005 pH units
in the whole pH range examined (2–11.5). Protonation con-
stants of the ligand and the overall stability constants of the
complexes were calculated by SUPERQUAD program [8],
which minimizes the sum of the weighted squared residuals
between the observed and the calculated e.m.f. values. Dis-
tribution diagrams for the various systems were calculated
and plotted by the program HYSS [9].
UV (H2O): λmax = 209.4 nm, ε = 25,942 dm3 mol−1 cm−1
,
λmax = 269.2 nm, ε = 22 140 dm3 mol−1 cm−1; (HRMS,
FAB): m/z = 447.1657, [M + H]+, calculated for
C18H23N8O6 requires 447.1662; 1H NMR (250 MHz,
D2O) δ 2.43 (ddd, 1H, JH3′,H2′ = 3.4 Hz, JH2′,H1′ = 6.8 Hz,
Jgem = 14.2 Hz, H2′), 2.69 (m, 1H, H2″), 3.17 (m, 2H,
CH2-β), 3.67 (m, 5H, CH3, H5′, H5″), 4.04 (m. 1H, H4′),
4.52 (m, 1H, CH-α), 4.58–4.70 (signal superimposed
with the signal of water molecule: H3′), 6.48 (pt, 1H,
JH1′,H2′ = JH1′,H2″ = 6.8 Hz, H1′), 7.02 (s, 1H, Im-CH-5),
7.88 (s, 1H, Im-CH-2),8.27 (s, 1H, H8), 8.28 (s, 1H, H2).
Synthesis of amide of N2‑{[9‑(2′‑d‑deoxyribofuranosyl)‑9H
purin‑6‑yl]‑carbamoyl}‑l‑histidine (his6dA amide) (2)
Methyl
ester
of
N2-{[9-(3′,5′-di-O-acetyl-d-2′-
deoxyribofuranosyl)-9Hpurin-6-yl]-carbamoyl}-l-histidine
f (70 mg, 0.11 mmol) dissolved in 6 mL of methanol
saturated with gaseous ammonia. After 2 days of stirring
at room temperature TLC control (nBuOH/H2O 85:15
v/v) indicated complete disappearance of the substrate.
The solvent was evaporated under reduced pressure. The
obtained product was purified using column chromatogra-
phy on silica gel eluting with nBuOH/H2O system (85:15
v/v). Compound 2 was obtained as white foam (17 mg,
0.04 mmol, 40 % yield), TLC: Rf = 0.4, (nBuOH/H2O
85:15 v/v),); HPLC: Rf = 15.90 min (HPLC column and
Spectroscopic measurement
UV–visible (UV–Vis) spectra were recorded with a Perkin-
Elmer Lambda 11 spectrophotometer, in the same con-
centration range as used for the potentiometry. Circular
dichroism (CD) spectra were obtained with a Jobin–Yvon
CD-6 dichrograph in the ultraviolet (UV) and visible range,
using 0.05 cm and 1 cm cuvettes, respectively. The spectra
are expressed as Δε = εl − εr, where εl and εr are molar
absorption coefficients for left and right circularly polar-
ized light, respectively. Anisotropic X-band EPR spectra
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J Biol Inorg Chem (2015) 20:989–1004
993
of frozen solutions were recorded at 120 K, using a Bruker
EMX spectrometer, after the addition of ethylene glycol
to ensure good glass formation. Copper(II) stock solution
for EPR measurements were prepared from CuSO4·5H2O
enriched with 63Cu, to get better resolution of EPR spec-
tra. Metallic copper (99.3 % 63Cu and 0.7 % 65Cu) was pur-
chased from JV Isoflex, Moscow, Russia for this purpose
and converted into the sulfate. The EPR parameters were
read from the spectra (estimated uncertainties for A and g
values are 1 × 10−4 cm−1 and 0.002, respectively, in the
spectra of a single species). Measurements were performed
at the maximum concentration of each species found in
titrations.
methyl green (0.5 mM) or DAPI (4′,6-diamidino-2-phe-
nylindole) (8 μM) were applied. The general procedure of
reaction was the same as that presented above.
Ligation of nicked pEGFP‑C1
OC form of pEGFP-C1, received by the cleavage of SC
form of the plasmid by Cu(II)-L system, was isolated from
the reaction mixture using the agarose gel electrophoresis
technique and extracted from the excised gel slice using
the NucleoSpin Gel and PCR Clean-up purification sys-
tem (Macherey–Nagel), according to the manufacturer’s
protocol. The ligation reaction was performed using 25 ng
of OC form of pEGFP-C1 plasmid dissolved in water
(total volume 5 μL) and equal volume of DNA Ligation
Kit (Mighty Mix, Takara) containing T4 DNA ligase in
the ligation buffer. The ligation mixture was incubated for
30 min at room temperature to complete reaction. Imme-
diately, after that time the transformation of chemically
competent bacteria Escherichia coli, strain TOP10 (Invit-
rogen), according to routine transformation protocol with
heat shock was performed [10]. As controls for transfor-
mation procedure the (K1) untreated pEGFP-C1 plasmid;
(K2) pEGFP-C1 plasmid hydrolyzed to linear form after
treatment by nuclease BamHI without T4 DNA ligase or
(K3) with T4 DNA ligase; and (K4) pEGFP-C1 plasmid
hydrolyzed to linear form with removed phosphates at 5′
ends (alkaline phosphatase treatment, CIP—calf intes-
tinal phosphatase) with addition of T4 DNA ligase was
applied. The transformation mixtures containing compe-
tent E. coli bacteria suspension (200 μL) with ligation
mixture (10 μL) was incubated on ice for 15 min, then
tubes were put into 42 °C water bath for 30 s. and back
into ice for 2 min. 500 μL of SOC medium (2 % tryptone;
0.5 % yeast extract; 10 mM NaCl; 2.5 mM KCl; 10 mM
MgCl2; 10 mM MgSO4; 20 mM glucose) was added and
each sample was incubated at 37 °C for 1 h, with gen-
tle shaking. About 200–400 μL (7.5–15 ng of plasmid) of
resulting culture was spread on the LB-agar plates (1 %
tryptone; 0.5 % yeast extract; 1 % NaCl; 1.5 % agar) with
kanamycin (50 μg/mL), as the selection agent, and cul-
tured at 37 °C for 18 h, when resulted bacterial colonies
were counted on each LB-agar plate.
DNA studies
Cleavage of double‑stranded DNA
The cleavage reaction of pEGFP-C1 plasmid DNA (BD
Biosciences) was carried out in the mixture containing
0.25 μg of plasmid in 5 mM Tris–HCl (pH 7.5)/5 mM
NaCl buffer with various concentrations of the Cu(II) ions
(0.1; 0.2; 0.5; 1; 1.5 mM), ligands (L), i.e., his6dA amide
and his6dA ester (0.1; 0.2; 0.5; 1; 1.5 mM) or Cu(II)-L
complex systems (0.1; 0.2; 0.5; 1; 1.5 mM). For the experi-
ments, Cu(II)-L systems were prepared in aqueous solu-
tions by mixing CuCl2 and l-his6dA amide or his6dA ester
in molar ratios of 1:2 to maintain some level of unsaturated
in coordination in these labile complexes, which were then
allowed to deposit for more than 5 days. Total reaction vol-
ume was 10 μL. Samples were incubated for 16 h (over-
night) at 37 °C and the reaction was stopped by adding of
EDTA (up to 5 mM) and the 6× loading buffer (1 % SDS,
sodium dodecyl sulfate, 50 % glycerol, 0.05 % bromophe-
nol blue, 0.05 % xylenocyanol). Finally, all samples were
loaded on the 0.5 % agarose gel, containing ethidium bro-
mide (EB), 0.5 μg/mL. Electrophoresis was carried out at
130 V in TBE (Tris–borate–EDTA) buffer. Plasmid DNA
cleavage products, visible as bands in the agarose gel,
were quantified and analyzed with G-BOX system (Syn-
gene). The efficiency of the DNA cleavage was assessed by
determining the ability of the species to form open circular
(OC) or linear (L) plasmid forms from the supercoiled (SC)
form, by quantitatively estimating the intensities of appro-
priate bands using the GeneTools software (Syngene).
Additionally to standard conditions of plasmid DNA
cleavage, some reactions were performed in the presence
of several additives: standard radical scavengers such as
DMSO (0.4 M), glycerol (0.4 M), NaN3 (10 mM) or KI
(10 mM), which were included into reaction mixture before
Cu(II)-L complex addition. To characterize DNA major–
minor groove binding specificity of Cu(II)-L complex
the reaction mixtures of typical groove binders/blockers:
Absorption spectra studies
Absorption spectra titrations were carried out in phosphate
buffer (pH 7.5) at room temperature to investigate the bind-
ing affinity of the studied ligands or complexes toward
CT-DNA. The concentration of CT-DNA was determined
from the absorption intensity at 260 nm with a ε value of
6600 M−1 cm−1. Absorption titration experiments were
performed by varying the concentration of the CT-DNA
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Table 1 Protonation constants (log βHnL) and pK values of his6dA
amide and his6dA ester (standard deviations are in parentheses)
(I = 0.1 M (KNO3), T = 298 K)
Table 2 Stability constants (logβ) of complexes in Cu(II)–his6dA
amide and Cu(II)–his6dA ester systems (standard deviations are in
parentheses) (I = 0.1 M (KNO3), T = 298 K)
Ligand
log βHL
log βh2l
pK (N1)
pK (Nim)
Species
pH
Log β
His6dA amide
His6dA ester
his6dA amide
his6dA ester
6.55 ( 1)
6.52 ( 1)
9.02 ( 2)
9.02 ( 2)
2.47 ( 2)
2.5 ( 2)
6.55 ( 1)
6.52 ( 1)
CuL2
4.5
5.5
7.0
9.5
10.60 ( 2)
0.29 ( 3)
4.47 ( 2)
−3.99 ( 3)
10.54 ( 2)
0.69 ( 3)
5.81 ( 2)
−2.87 ( 3)
CuLH-1
CuL2H−1
CuL2H−2
(0–200 μM) keeping the constant concentration (10 μM)
of the ligand or complex. The absorbance (A) was recorded
after each addition of CT-DNA. While measuring the
absorption spectra an equal amount of DNA was added to
the both compound solution and the reference solution to
eliminate the absorbance of the CT-DNA itself, and phos-
phate buffer was subtracted through baseline correction.
The data were fitted to the Wolfe–Shimer equation (Eq. 1)
[11] to obtain the binding constant:
(KEB = 1.0 × 107 M−1) and CEB is the concentration of EB
(10 μM) [13].
The binding stoichiometry (n), for the ligands and the
complexes with Cu(II), were calculated using
log[(I0 − I)/I] = log Kb + n log[Q],
(4)
[DNA]
[DNA]
1
where Kb is the binding constant of ligands and their com-
plexes with DNA and n is the number of binding sites.
From the plot of log[(I0 − I/I)] versus log[Q] the number of
binding sites n have been obtained [12].
=
+
(εb − εf) Kb(εb − εf)
,
(1)
(εa − εf)
where εa, εf, and εb, are the apparent, free and bound metal
complex extinction coefficients, respectively. In particular
εf was determined by a calibration curve of the isolated
metal complex in aqueous solution, εa was determined as
the ratio between the measured absorbance and the sam-
ples concentration, A/[M]. A plot of [DNA]/(εb − εf) versus
DNA gave a slope of +1/(εb − εf) and a Y intercept equal
to 1/Kb(εb − εf) [12].
Results and discussion
Characterization of the ligands and their copper(II)
complexes
The protonation constants of the two ligands (his6dA amide
and his6dA ester), the stoichiometry and overall stability
constants of their Cu(II) complexes were determined by
potentiometric titration (Table 1, 2).
The ligands may dissociate two protons (Scheme 2).
In strong acidic region the first one can be derived from
purine nitrogen N(1) while in neutral pH the second one
from the imidazole ring Nim of histidine. The acidity of
N(1) nitrogen is lower and that of Nim donor atom is higher
as compared to the values found for his6dA, 1.97 and 7.05,
respectively [14].
Fluorescence studies
The competitive binding experiments were carried out in
Tris–HCl buffer (pH 7.2) by keeping EB-DNA solution
containing [EB] = 10 μM and [DNA] = 25 μM as con-
stant and varying the concentration of ligand or complex
(0–200 μM). Stern–Volmer quenching constant KSV of the
ligands and complexes to CT-DNA [12] were determined
from the equation
I0/I = 1 + KSV[Q],
(2)
The acidity of N(1) nitrogen donor atom depends both on
the presence of ureido group and on the nature of substitu-
ents as well [15]. It seems that the both groups –OCH3 and
–NH2 influence electron density distribution in the ligand
molecules leading to the changes of ionization constants.
In Cu(II)–his6dA amide and Cu(II–his6dA ester systems
protonated species have not been found probably because
that they are forming in a very small extent undetectable
by potentiometry. The detectable complex formation reac-
tions start in acidic solution (pH > 3). The ligand behaves
as tridentate with three protons released from purine
N(1), imidazole Nim and ureido Nam groups, respectively.
where I0 and I are the fluorescence intensities of EB-DNA
in the absence and the presence of the studied compounds,
respectively. KSV is a linear Stern–Volmer quenching con-
stant and [Q] is concentration of quencher. In the linear fit
the plot of I0/I vs [Q], KSV is given by the ratio of slope to
intercept.
The apparent binding constants were calculated using
Kapp = KEB × CEB/C50,
(3)
where C50 is the concentration of the complex that causes
50 % reduction of the initial fluorescence of EB-DNA
emission intensity, KEB is the binding constant of EB
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J Biol Inorg Chem (2015) 20:989–1004
995
Scheme 2 Protonation and deprotonation equilibria for the ligands. The charges of the ligand species have been omitted for simplicity
(Table 3) support this coordination mode which is identical
to that found in his6dA or histam6dA nucleosides [14, 16].
At neutral pH also four−nitrogen donor atoms can be
identified but one of them Nam is derived from the amide of
ureido group. In−alkaline solution the 4N donor atoms still
exist with two Nam involved in Cu(II) coordination (Fig. 2).
The charge transfer transitions visible in UV CD spec-
tra at 326 and 320 nm, respectively, and EPR parameters
(Table 3) clearly support the proposed coordination atom
sets (Figs. S2 and S3).
DNA studies
Cleavage of double‑stranded DNA
Supercoiled plasmid DNA cleavage by the ligands and their
Cu(II) complexes was studied in the absence of oxidant
like H2O2 or any reducing agents. Cu(II)–his6dA amide and
Cu(II)–his6dA ester complexes are capable to cleave double-
stranded DNA (dsDNA) at physiological pH and tempera-
ture as it was found in Cu(II)–his6dA system [14]. When
pEGFP-C1 plasmid DNA was incubated with Cu(II)–his6dA
amide or with Cu(II)–his6dA ester, the form I (SC) of the
plasmid was hydrolyzed to the form II (OC). A single cut/
nick of DNA strand relaxes the supercoiling structure of the
plasmid and leads to the form II. Figure 3 presents the aga-
rose gel electrophoresis patterns for the cleavage of pEGFP-
C1 plasmid after 16 h treatment with Cu(II)–his6dA amide
(a) or Cu(II)–his6dA ester (b) solutions. The concentration of
Cu(II)–his6dA amide and Cu(II)–his6dA ester complexes in
the reaction mixture varied from 0 to 1.5 mM. The increas-
ingly stronger conversions of form I to form II of plasmid
were observed with the increase of concentration of Cu(II)–
his6dA amide or Cu(II)–his6dA ester complexes, with the
total disappearance of the both plasmid forms at the highest
concentrations of Cu(II)–his6dA amide (1.0 and 1.5 mM) or
Cu(II)–his6dA ester (1.5 mM). It is very likely due to a strong
binding of the complexes to the plasmid DNA (Fig. 3a, b,
lines 5 and 6) and therefore the much slower migration of the
plasmid forms can be observed in the agarose gel.
Fig. 1 Comparison of copper(II) binding efficiency by his6dA
amide and his6dA ester. Metal to ligands molar ratio, Cu(II):his6dA
amide:his6dA ester/1:1:1. The concentrations of the metal and the
ligands were 1 × 10−3 M
Mono- and bis-complexes are indicated by potentiometric
data. The presence of –OCH3 or –NH2 group in histidine
moiety results in similar coordinating ability of deoxy-
adenosine ligands. The comparison of binding efficiency of
the ligands indicates that his6dA amide is more competitive
and efficient in comparison to his6dA ester over a pH range
of 4–9 (Fig. 1). Also the sequestration of copper(II) ions
between amide and ester of his6dA (52 and 47 %, respec-
tively) confirms the better chelating efficacy of the amide
especially around neutral pH.
In the whole studied pH region the stoichiometry of the
dominating copper complexes with modified deoxyaden-
osines was Cu(II):Ligand 1:2 (Fig. S1). The only difference
between them is the protonation state of the ligand. In acidic
pH a CuL2 complex was indentified with coordination
sphere of metal ion composed of {2N(1), 2Nim} two nitro-
gen atoms derived from purine base and histidine ring. The
visible spectra with the λmax band localized at 657 nm in
UV–Vis and 665 nm in CD Vis region and EPR parameters
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J Biol Inorg Chem (2015) 20:989–1004
Table 3 Spectroscopic parameters of copper complexes with his6dA ester and his6dA amide
Species
pH
Coordination mode
Spectroscopic parameters
EPR
UV–Vis
CD
G||
A|| (10−4 cm−1
)
λ (nm)
ε (dm3 cm−1 M−1
)
λ (nm)
Δε (dm3 cm−1 M−1
)
CuL2
4–5
{2N(1), 2Nim}
2.262a
2.268b
182.1a
180.0b
656a
657b
87a
58b
219a
277
330
665
209b
280
332
668
220a
252
276
327
582
733
215b
277
326
653
247a
289
323
571
733
215b
276
320
385
591
1.978a
5.631
0.146
−0.088
1.620b
0.596
0.035
−0.011
CuL2H−1
7.00
{N(1), N−am, 2Nim}
2.241a
2.248b
194.3a
192.7b
613a
629b
96a
78b
3.476a
3.263
6.469
0.320
−0.098
0.025
2.706b
1.307
0.058
−0.058
CuL2H−2
9.50
{2N−am, 2Nim}
2.241a
2.248b
194.3a
192.7b
595a
598b
120a
102b
3.248a
−0.667
0.529
−0.145
0.025
1.042b
0.954
0.244
0.085
−0.026
a
Cu–his6dA amide
his6dA ester
b
Fig. 2 Proposed structure
of CuL2H−2 complex. The
white inset shows 4N donor
atom set {N(1), N−am, 2Nim};
R = 2′-deoxyribose; L- his6dA
amide
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J Biol Inorg Chem (2015) 20:989–1004
997
Fig. 3 Agarose gel electrophoresis patterns for the cleavage of
pEGFP-C1 plasmid DNA by Cu(II)–his6dA amide complex (a),
Cu(II)–his6dA ester complex (b) or Cu(II)–his6dA complex (c) used
in various concentrations (0, 0.1, 0.2, 0.5, 1 and 1.5 mM); Line 1 con-
trol untreated plasmid DNA; line 2 plasmid DNA + 0.1 mM (a), (b)
or (c); line 3 plasmid DNA + 0.2 mM (a), (b) or (c); line 4 plasmid
DNA + 0.5 mM (a), (b) or (c); line 5 plasmid DNA + 1.0 mM (a),
(b) or (c); line 6 plasmid DNA + 1.5 mM (a), (b) or (c). The inserted
graphs present the changes of the supercoiled (SC) and open circular
(OC) form of DNA. Presented data are the average from three inde-
pendent repeats of the plasmid DNA cleavage reaction
The extent of DNA cleavage was quantified using the
GeneTools software. The results are summarized in Table
S1 (Supplementary material). With respect to them one can
conclude that up to the concentration of 0.2 mM the cleav-
age efficiency of Cu(II)–his6dA amide and Cu–his6dA com-
plexes is similar unlike that of Cu(II)–his6dA ester which is
noticeably weaker. Above this concentration the cleavage
and binding efficacy of Cu(II)–his6dA amide to DNA is the
strongest one. Free ligands his6dA amide and his6dA ester
were unable to cut plasmid DNA (Fig. S4). Free Cu(II) ions
have produced hardly any cleavage of plasmid DNA under
the concentrations studied [17–19]. The results suggest that
the Cu(II)–his6dA amide, Cu(II)–his6dA ester and Cu(II)–
his6dA complexes are the species responsible for the cleav-
age of plasmid DNA.
The mechanism of pEGFP-C1 DNA cleavage induced
by the complexes was studied using various inhibiting
agents. The reaction course was investigated in the pres-
ence of hydroxyl radical scavengers (DMSO, glycerol),
singlet oxygen quencher (NaN3) and hydrogen peroxide
scavenger (KI) under experimental conditions. Figure 4
shows no inhibition in the DNA cleavage activity of the
Cu(II)–his6dA amide, Cu(II)–his6dA ester or Cu(II)–his6dA
complexes in the presence of hydroxyl radical or hydrogen
peroxide scavengers (lines 3–5).
Slight inhibition in DNA cleavage was observed in the
presence of singlet oxygen quencher.
It suggests that the singlet oxygen species might be
involved in an oxidative cleavage pathway. However, the
involvement of singlet oxygen is rather doubtful in the
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J Biol Inorg Chem (2015) 20:989–1004
Fig. 4 Cleavage of pEGFP-C1 plasmid DNA by Cu(II)–his6dA
amide complex (a), Cu(II)–his6dA ester complex (b) or Cu(II)–
his6dA (c) (0.2 mM) in the presence of radical scavengers in 5 mM
Tris–HCl (pH 7.5)/5 mM NaCl buffer. Line 1 control untreated plas-
mid DNA; line 2 plasmid DNA + studied complexes; line 3 plasmid
DNA + DMSO (0.4 M) + studied complexes; line 4 DNA + glycerol
(0.4 M) + studied complexes; line 5 DNA + KI (10 mM) + studied
complexes; line 6 DNA + NaN3 (10 mM) + studied complexes
studied systems because this reactive form of oxygen is
usually photogenerated and in our DNA-cleavage studies
we have not used such a process. Furthermore, accord-
ing to some earlier publications, this result should not be
Ligation of nicked pEGFP‑C1
For further studies on the mechanism of DNA cleavage
the Cu(II)–his6dA amide complex was selected due to its
stronger interactions with DNA as compared to other com-
plexes. In order to confirm the hydrolytic mechanism of
plasmid DNA cleavage by the studied compounds, nicked
(OC) form II of plasmid DNA, obtained by the action of
the Cu(II)–his6dA amide complex, was ligated using T4
DNA ligase and transformed into E. coli competent cells.
The examples of successful ligation experiments, espe-
cially conversion of linear form of plasmid, result of action
of artificial chemical nucleases, into relaxed open circu-
lar form of plasmid can be already found at the literature
[22]. But in the case of dsDNA reaction with the studied
compounds, only nicked, relaxed form II, open circular OC
plasmid was observed. The linear form of plasmid has been
never achieved. Because both forms of plasmid: nicked
and relaxed, migrate in the agarose gel as one OC form
II, it was impossible to observe any results of ligation of
nicked strands of plasmid. Therefore, the E. coli transfor-
mation method and analysis of the number of obtained bac-
terial colonies were chosen for identification of hydrolytic
or oxidative mechanism of Cu(II)–his6dA amide complex
action. Bacterial transformation allows to distinguish the
both plasmid moieties: the relaxed form with repaired by
T4 DNA ligase single strand breaks—which can be ampli-
fied in the bacterial cells, and the plasmid with 5′ or 3′ ends
damaged during cleavage reaction, which are impossible to
1
interpreted as an indication that O2 is also participating
in the process; it is probably due to the affinity of sodium
azide for transition metals [20]. It was also evidenced in
many publications that Cu(II) complexes [e.g., Cu(l-His)]
[17] readily cleave plasmid DNA via the hydrolytic path-
way [21]. Taking all this into account one can suppose a
hydrolytic mechanism of DNA cleavage by the complexes
studied.
The potential binding sites of the Cu(II)–his6dA amide,
Cu(II)–his6dA ester and Cu(II)–his6dA complexes with
supercoiled plasmid pEGFP-C1 DNA were investigated using
minor and major groove binders DAPI and MG, respectively.
The supercoiled DNA was treated separately with DAPI or
MG prior to the addition of the complexes. The agarose gel
electrophoresis pattern shown in Fig. 5b clearly demonstrates
that DAPI inhibits the DNA cleavage activity of Cu(II)–
his6dA ester, suggesting that the minor groove of double
helix of DNA is the preferred site of the interaction with this
complex. In contrast, the DNA cleavage activity of Cu(II)–
his6dA amide and Cu(II)–his6dA complexes is inhibited after
the addition of MG, suggesting that these complexes have an
affinity to DNA major groove. It is very likely that the steric
clashes with DNA exterior caused by the ligands or their
complexes with Cu(II) ions dictate their DNA-binding mode
to be surface binding to minor or major groove.
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J Biol Inorg Chem (2015) 20:989–1004
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Fig. 5 Cleavage of pEGFP-C1 plasmid DNA by Cu(II)–his6dA
amide complex (a), Cu(II)–his6dA ester complex (b) or Cu(II)–
his6dA (c) (0.2 mM) in the presence of DNA groove recogni-
tion agents, reaction performed in 5 mM Tris–HCl (pH 7.5)/5 mM
NaCl buffer. Line 1 control untreated plasmid DNA; line 2 plasmid
DNA + studied complexes; line 3 plasmid DNA + DAPI (8 μM);
line 4 plasmid DNA + DAPI (8 μM) + studied complexes; line
5 plasmid DNA + MG (0.5 mM); line 6 plasmid DNA + MG
(0.5 mM) + studied complexes
repair by T4 DNA ligase or another intracellular enzymatic
activity. Because pEGFP-C1 plasmid has the kanamycin
resistance gene, transformation mixtures were applied onto
LB-agar plates containing the kanamycin as the selection
factor for bacteria cells containing plasmid. After trans-
formation, E. coli cells acquired a resistance to kanamy-
cin and they were able to grow on the medium containing
this antibiotic. After 20 h of incubation the bacterial colony
growth was observed on the plates. Four additional sam-
ples as controls of ligation and transformation procedures
were applied (Fig. 6): (K1) untreated supercoiled pEGFP-
C1 plasmid DNA, (K2) pEGFP-C1 plasmid hydrolyzed
to linear form by restriction nuclease (BamHI) without
addition of T4 DNA ligase or (K3) with T4 DNA ligase,
and (K4) pEGFP-C1 plasmid hydrolyzed to linear form
by restriction nuclease (Bam HI), after dephosphorylation
of 5′ end catalyzed by alkaline phosphatase, and after T4
DNA ligase treatment—the negative control of ligation.
Positive transformation results for K3 sample support the
correct action of T4 DNA ligase, which is able to join the
ends of linear form of plasmid after hydrolytic cleavage by
the restriction nuclease. Negative transformation results for
K4 sample indicate that T4 DNA ligase is not able to com-
bine the damaged (without 5′-phosphates) ends of plasmid.
No bacterial colonies were observed on the LB-agar plates
after transformation of samples containing the plasmid
(OC form) cleaved by the complex (Cu(II)–his6dA amide)
with or without T4 DNA ligase (K5 and K6). It suggests
that neither the T4 DNA ligase nor bacteria itself are able
to ligate the forms of pEGFP-C1 nicked during the reaction
with the complex, uptake them and amplify inside the cell.
However, after 2 weeks the bacterial colony growth was
observed on the plates K5 and K6 (K7, K8) (Fig. 6). One
of the reasons why the hydrolytic properties have not been
disclosed at the same time as that observed for K3 sam-
ple might be due to non structure-selective DNA cleavage
induced by the complex as that of the BamHI nuclease
therefore the T4 ligase could not ligate DNA in a proper
manner. It may indicate lack of cohesive termini and that is
why it makes end ligation more complex and significantly
slower.
From all the results related to DNA cleavage is diffi-
cult to unequivocally indicate that the cleavage occurs via
the hydrolytic pathway. Oxidative mechanism may not
be excluded as well although we have not determined the
involvement of hydroxyl radical or hydrogen peroxide in
DNA cleavage. The involvement of singlet oxygen is rather
impossible because this reactive form of oxygen is usually
photogenerated and in our DNA-cleavage studies we have
not used such a process.
Absorption spectral studies
Transition metal complexes can bind to DNA via both
covalent and/or non-covalent (intercalation, electrostatic
and groove binding) interactions [23]. The binding of an
intercalative molecule to DNA can be characterized by
notable intensity decrease (hypochromism) and red shift
(bathochromism) of the electronic spectral bands. On the
other hand, metal complexes, which do not intercalate or
1 3

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J Biol Inorg Chem (2015) 20:989–1004
Fig. 6 LB-agar plates of studied systems: K1 untreated pEGFP-C1;
K2 pEGFP-C1 + BamHI; K3 pEGFP-C1 + BamHI + T4 ligase;
K4 pEGFP-C1 + BamHI + alkaline phosphatase; K5 Cu(II)–his6dA
amide; K6 Cu(II)–his6dA amide + T4 ligase; K7 and K8 are K5 and
K6 after keeping at the temperature of 4 °C for several weeks
interact electrostatically with DNA may exhibit hyper-
chromism [24]. The interactions of the ligands and their
Cu(II) complexes with CT-DNA were monitored by UV–
visible spectroscopy. The absorption spectra of the stud-
ied compounds in aqueous solutions were compared in the
absence and in the presence of CT-DNA. Electronic absorp-
tion spectral data upon addition of CT-DNA and the bind-
ing constants are given in Fig. 7 and Table 4, respectively.
In the presence of increasing amounts of CT-DNA,
UV–Vis absorption spectra of all studied compounds show
slight blue shift and hyperchromism (Fig. 7). This hyper-
chromism can be attributed to external contact (surface
binding) with the duplex which could involve hydrogen
bonds of the oxygen or nitrogen atoms on the ligand with
DNA nucleobases, or electrostatic interactions between
the cationic species of the complexes and the negatively
charged phosphate groups on the DNA backbone. The
results suggest that the ligands and the complexes can bind
to CT-DNA via a groove-binding mode [25, 26]. It is also
consistent with the gel electrophoresis data (Fig. 5). In
order to quantitatively compare the binding affinity of the
compounds with CT-DNA, the intrinsic binding constants
(Kb) were determined according to the Eq. 1 by monitoring
the absorbance changes of the ligands and their complexes
with increasing concentration of CT-DNA. The Kb values
indicate the following order of binding affinity of the com-
pounds his6dA amide > Cu(II)–his6dA amide > Cu(II)–
his6dA ester > his6dA ester. The determined Kb values are
much lower than those observed for typical classical inter-
calators (ethidium bromide, EB, Kb = 1.4 × 106 M−1 in
25 mM Tris–HCl/40 mM NaCl buffer, pH 7.2) [22]. Only
in the case of his6dA amide and Cu(II)–his6dA amide one
can suppose that the ligand or the complex can be involved
in partial intercalative interactions as it was found for
many other compounds with the same order of Kb values
[27–29].
Fluorescence spectral studies
In order to get more insight into the interaction mode of
the ligand or the complexes towards DNA, the fluorescence
titration experiments have been performed. The fluores-
cence titrations, especially the EB fluorescence displace-
ment experiments, have been widely used to characterize
the interaction of compounds with DNA by following the
changes in fluorescence intensity [30]. The method is based
on a decrease of fluorescence resulting from the displace-
ment of EB from a DNA sequence by a quencher and the
quenching is due to the reduction of the number of binding
sites in the DNA available to the EB [31]. The intrinsic fluo-
rescence intensity of DNA and that of EB are low, while the
fluorescence intensity of EB will be enhanced on addition of
DNA due to its intercalation into the DNA. Therefore, EB
can be used to probe the interaction of the ligands or com-
plexes with DNA. In our experiments, as depicted in Fig. 8
for his6dA ester (a), Cu(II)–his6dA ester (b), his6dA amide
1 3

J Biol Inorg Chem (2015) 20:989–1004
1001
0.25
0.30
0.25
0.20
0.15
0.10
0.05
0.00
a
b
0.20
0.15
0.10
0.05
0.00
-0.05
240
260
280
300
240
260
280
300
λ [nm]
λ [nm]
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
d
c
230
240
250
260
270
280
290
300
230
240
250
260
270
280
290
300
λ [nm]
λ [nm]
Fig. 7 Electronic absorption spectra of his6dA ester (a), Cu(II)–
his6dA ester (b), his6dA amide (c), Cu(II)–his6dA amide (d) in the
absence and in the presence of increasing amounts of DNA concen-
tration. [Ligand] or [complex] = 10 μM, [DNA] = 0, 10, 50, 100,
150 and 200 μM. Arrow shows the absorbance changes upon increas-
ing DNA concentration
Table 4 DNA binding constant (Kb), Stern–Volmer constant (KSV), the apparent binding constant (Kapp) and number of binding sites (n) for the
ligands and their complexes with Cu(II)
Sample
KSV (M−1
)
Kb (M−1
)
Kapp (M−1
)
N
his6dA ester
1.58 ( 0.06) × 102
3.78 ( 0.04) × 102
4.93 ( 0.16) × 103
3,46 ( 0.12) × 103
4.12 ( 0.03) × 101
3.06 ( 0.05) × 102
8.11 ( 0.06) × 103
1.76 ( 0.05) × 104
4.98 ( 0.13) × 104
2.95 ( 0.11) × 104
Not determined
Nda
Nda
5.0 × 105
5.0 × 105
Nda
0.71 ( 0.11)
0.99 ( 0.09)
1.23 ( 0.06)
1.17 ( 0.05)
0.51 ( 0.06)
0.54 ( 0.08)
Cu(II)–his6dA ester
his6dA amide
Cu(II)–his6dA amide
his6dA
Cu(II)–his6dA
Not determined
Nda
The standard deviations are in parenthesis
a
50 % decrease of fluorescence intensity was not observed
(c), Cu(II)–his6dA amide (d), his6dA (e) and Cu(II)–his6dA
(f) the fluorescence intensities of EB-DNA system show a
decreasing trend with increasing concentration of studied
samples, indicating that some EB molecules are released
from EB-DNA after an exchange with the studied com-
pounds which results in the fluorescence quenching of EB.
This may be due to that the studied compounds displace
the EB from its DNA-binding sites in the competitive manner.
1 3

1002
J Biol Inorg Chem (2015) 20:989–1004
30000
25000
20000
15000
10000
5000
25000
20000
15000
10000
5000
a
b
500
550
600
650
700
750
500
550
600
650
700
750
Wavelength [nm]
Wavelength [nm]
20000
15000
10000
5000
0
c
d
15000
10000
5000
0
450
500
550
600
650
700
750
550
600
650
700
750
Wavelength [nm]
Wavelength [nm]
30000
25000
20000
15000
10000
5000
18000
16000
14000
12000
10000
8000
e
f
6000
4000
2000
500
550
600
650
700
750
800
500
550
600
650
700
750
Wavelength [nm]
Wavelength [nm]
Fig. 8 Emission spectra of EB bound to DNA in absence and pres-
ence of ligands: his6dA ester (a), his6dA amide (c) and his6dA (e)
and complexes: Cu(II)–his6dA ester (b), Cu(II)–his6dA amide (d)
and Cu(II)–his6dA (f). [EB] = 10 μM, [DNA] = 25 μM, [ligand]
or [complex] = 0, 10, 50, 100, 150, 200 μM. The arrow shows the
intensity changes on increasing the ligand and complex concentration
In order to quantitatively compare this quenching behavior,
the classical Stern–Volmer equation was employed. The plots
(Fig. 9) illustrate that the quenching of EB bound to DNA are
in good agreement with the linear Stern–Volmer equation.
The KSV and the n values of his6dA ester, his6dA amide,
his6dA and complexes: Cu(II)–his6dA ester, Cu(II)–his6dA
amide and Cu(II)–his6dA (Table 4) indicate that these com-
pounds show quenching efficiency and especially his6dA
amide and its copper complex have revealed significant
degree of binding to DNA. The binding strength of his6dA
amide and Cu(II)–his6dA amide (only those samples caused
a 50 % decrease of fluorescence intensity) with DNA was
characterized by using Eq. 3 to calculate the apparent binding
constant (Kapp). The Kapp values of his6dA amide and Cu(II)–
his6dA amide were estimated to be 5.0 × 105 M−1, support-
ing a strong interaction of these compounds with DNA. It
can be concluded that the binding of these compounds to CT-
DNA may occur by partial intercalation, since their apparent
binding constants are of the order characteristic for moderate
intercalators [32, 33]. The results are consistent with those
obtained from electronic absorption studies and point out sig-
nificant influence of the groups –NH2, –OCH3 and –COOH
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J Biol Inorg Chem (2015) 20:989–1004
1003
1.35
1.30
1.25
1.20
1.15
1.10
1.05
1.00
a
b
2.0
1.8
1.6
1.4
1.2
1.0
0.00000
0.00005
0.00010
0.00015
0.00020
0.00000
0.00005
0.00010
0.00015
0.00020
Ccomplex[M]
C[ligand][M]
1.09
1.08
1.07
1.06
1.05
1.04
1.03
1.02
1.01
1.00
c
d
1.04
1.03
1.02
1.01
1.00
0.00000
0.00005
0.00010
0.00015
0.00020
0.00000
0.00005
0.00010
0.00015
0.00020
Cligand[M]
Ccomplex[M]
1.012
1.010
1.008
1.006
1.004
1.002
e
f
1.07
1.06
1.05
1.04
1.03
1.02
1.01
0.00000
0.00005
0.00010
0.00015
0.00020
0.00000
0.00005
0.00010
0.00015
0.00020
Cligand[M]
Ccomplex[M]
Fig. 9 Stern–Volmer plot of fluorescence titrations of the ligands: his6dA ester (a), his6dA amide (c) and his6dA (e) and complexes: Cu(II)–
his6dA ester (b), Cu(II)–his6dA amide (d) and Cu(II)–his6dA (f)
on binding of the ligands/the complexes to DNA. Only the
amide derivative and its complex reveal some intercalative
ability. It may be supposed that they can acquire more heli-
coidal geometry which could facilitate intercalation.
exhibit the chelating ability towards copper(II) ions. The
complexes of 1:2 (metal:ligan−d) stoichiometry are formed
at a pH about 7. The {N(1), Nam, 2Nim} coordination mode
has been supported by spectroscopic parameters derived
from electronic absorption, CD and EPR spectra. The bind-
ing ability of these complexes towards CT-DNA has been
tested using different spectroscopic techniques. The results
suggest that the ligands and the complexes can bind to CT-
DNA via a groove-binding mode. The his6dA amide and
its complex have exhibited a greater binding propensity to
Conclusion
2′-deoxyadenosines modified in purine part of the mole-
cule with histidine moieties his6dA ester and his6dA amide
1 3

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CT-DNA. The EB competition method has revealed that
both of them are able to bind via intercalative manner as
well. The Kapp values calculated for these two systems are
similar to those found for other intercalators which can be
indicative of their tendency to act in this manner [28, 29].
The his6dA ester, his6dA and their Cu(II) complexes can
only interact via groove-binding mode to the DNA double
helix because the emission EB-DNA spectrum has been
scarcely affected by adding these compounds to the solu-
tion. The difference of binding ability of the complexes can
be derived from the differences of histidine residue incor-
porated in purine moiety of 2′-deoxyadenosine. All studied
complexes promote cleavage of pEGFP-C1. Among the
ligands only his6dA has exhibited nuclease activity [6]. The
possible cleavage mechanism can be hydrolytic pathway.
Nucleosides modified with histidine moieties can provide
“building blocks” of oligonucleotides in DNA sequence of
deoxyribozymes which in the presence of copper ions may
reveal potential for hydrolysis of complementary RNA.
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