A 1:2 copper(II)-tripeptide complex for DNA binding and cleavage agent under physiological conditions

Article abstract of DOI:10.1002/cbdv.200800079

A 1:2 copper-tripeptide complex, [Cu^II(Boc-His-Gly-His-OMe) ₂]²⁺, was synthesized and structurally characterized. The absorption band at 577 nm suggests a square-planar geometry around Cu ^II. The DNA-binding and

Full text of DOI:10.1002/cbdv.200800079

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
A 1:2 Copper(II)–Tripeptide Complex for DNA Binding and Cleavage
Agent under Physiological Conditions
by Pulimamidi R. Reddy*a), Pallerla Manjulaa), Tushar K. Chakrabortyb), and Rajarshi Samantab)
a) Department of Chemistry, Osmania University, Hyderabad – 500007, India
(phone: þ91-40-27171664; fax: þ91-40-27090020; e-mail: rabi_pr@rediffmail.com)
b) Indian Institute of Chemical Technology, Hyderabad – 500007, India
A 1:2 copper–tripeptide complex, [CuII(Boc-His-Gly-His-OMe)2]2þ, was synthesized and structur-
ally characterized. The absorption band at 577 nm suggests a square-planar geometry around CuII. The
DNA-binding and DNA-cleavage properties of the CuII complex were investigated. The complex binds
to calf thymus DNA (CT DNA) in an intercalative fashion and cleaves plasmid pUC-19 DNA
hydrolytically at micromolar concentrations under physiological conditions. The intrinsic binding
constant (Kb ¼1.2 ꢀ102 mꢁ1) for the binding of Cu–tripeptide complex with DNA suggests that this
complex is suitable for rapid diffusion on the pharmacological time scale.
Introduction. – The field of nucleic acid-targeted drug design has been the subject of
several investigations in recent years [1–10]. This is because the DNA-binding proteins
such as restriction endonucleases and transcriptional factors recognize specific DNA
sequences to carry out the precise genetic manipulations ongoing in a living system.
Low-molecular-weight bioactive ligands, which also recognize and interact with DNA,
are of chemical, biological, and medical significance as potential artificial gene
regulators or cancer chemotherapeutic agents. The remarkable biological potencies of
these bioactive ligands are thought to be a consequence of their ability to bind to
cellular DNA, and produce single or double stranded breaks with great efficiency.
Metallopeptides are unique among metal-based nucleic acid-binding agents in their
ability to incorporate and position along the periphery of a well-defined metal-complex
framework the same chemical functional groups used by proteins and peptide-based
natural product antitumor agents for the molecular recognition of DNA and RNA [11].
Metallopeptides of the general form CuII- or NiII(Gly-Gly-His) have contributed to our
understanding of fundamental nucleic acid recognition and reactivity phenomena in
the context of both proteins and low-molecular-weight agents [12]. Our laboratory has
been involved in the development of nucleic acid-targeted drug design under
physiological conditions [13–16]. Here, we report the synthesis and characterization
of the complex [CuII(Boc-His-Gly-His-OMe)2]2þ (Scheme) along with its DNA-
binding and DNA-cleavage properties. The Cu complex binds to DNA in an
intercalation mode (Kb ¼1.2 ꢀ102 mꢁ1) and converts supercoiled plasmid pUC-19
DNA into a nicked circular form at micromolar concentration.
Results and Discussion. – Characterization of the Complex. The IR spectra of the
Cu complex indicating the coordination of the 1H-imidazole N-atom with the metal,
ꢀ 2009 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
765
Scheme
and the appearance of a new band at 568 cmꢁ1 confirms the N-atom ligation around Cu.
ESI Mass spectra were utilized to identify the composition of the Cu complex (Fig. 1).
The molecular-ion peak at m/z 512 ([MþNH4]þ ) confirms the 2 :1 peptide–Cu
complex. Accordingly, N(4) ligation around Cu has been proposed (Fig. 2).
The UV profile of CT DNA at a fixed concentration and increasing concentrations
of the Cu complex was monitored at 260 nm in Tris·HCl buffer (pH 7.5). In the
absence of DNA, no significant absorbance was observed in the range of 240–280 nm,
neither for the free peptide nor for the complex [CuII(tripeptide)2]. However, an
increase in UV absorbance was observed on the addition of the Cu complex to CT
DNA (Fig. 3,a). The absorption maximum (lmax) of CT DNAwas also shifted from 257
to 253 nm. This indicates the denaturation or a conformational change of the DNA
duplex.
The thermal denaturation profile of DNA in the absence and presence of the Cu
complex is provided in Fig. 3,b. A slight increase of 3–48 was observed in the Tm profile
of the complex as compared to free DNA. As it is well-known, the increase of Tm means
an intercalative and/or phosphate binding, whereas a decrease is an indicator of binding
to the bases. These results provide an evidence for intercalative and/or phosphate
binding of the CuII complex with DNA. It seems that the first interaction of DNA with
the complex is due to an electrostatic interaction between the positively charged

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Fig. 1. ESI Mass spectrum of the Cu complex
Fig. 2. Energy-minimized molecular structure of [CuII(Boc-His-Gly-His-OMe)2]2þ. Optimized calcu-
lated bond lengths (in ꢂ) and bond angles (in degrees) of Cu and the interacting N-atoms: CuꢁN(1)
1.922, CuꢁN(2) 1.947, CuꢁN(3) 1.945, CuꢁN(4) 1.936, CuꢁN(1)ꢁN(2) 100.9, CuꢁN(1)ꢁN(3) 104.5,
CuꢁN(1)ꢁN(4) 115.4.
complex and the negatively charged DNA phosphate backbone. This is followed by an
intercalative mode of binding.
Ethidium bromide (EB) emits intense fluorescence light in the presence of DNA
due to its strong intercalation between the adjacent DNA base pairs. It was reported
that the enhanced fluorescence can be quenched by the addition of a second molecule

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
767
Fig. 3. a) UV/VIS Absorption spectra of [CuII(Boc-His-Gly-His-OMe)2]2þin the absence (bottom) and
presence (top) of increasing amounts of the complex. b) Thermal denaturation profile and differential
melting curves (inset) of calf thymus DNA (75 mm) before (A) and after (B) addition of Cu complex
(75 mm). D is the difference in absorbance.
[17][18]. The quenching extent of EB fluorescence bound to DNA was used to
determine the extent of binding between the second molecule and DNA. The emission

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
spectra of EB bound to CT DNA in the absence and the presence of the CuII complex is
given in Fig. 4,a. The addition of the CuII complex to DNA pretreated with EB caused
appreciable reduction in emission intensity, indicating that the complex competes with
EB in binding to DNA.
According to the Stern–Volmer equation [18]:
I0/I¼1þKsq ·r
where I0 and I are the fluorescence intensities in the absence and the presence of
complex, respectively, Ksq is a linear Stern–Volmer quenching constant dependent on
the ratio of the concentrations of bound EB to unbound EB, and r is the concentration
ratio of the complex to DNA. The quenching plot (Fig. 4,a) illustrates that the
quenching of EB bound to DNA by the CuII complex is in good agreement with the
linear Stern–Volmer equation, which indicates that the complex binds to DNA. In the
plot of I0/I vs. [complex]/[DNA], Ksq is given by the ratio of slope to intercept, and the
Ksq value for the CuII complex is 0.14.
Fluorescence Scatchard plots for the binding of EB to CT DNA in the presence of
the Cu complex were obtained as described by Lepecq and Paoletti [19]. The binding
isotherms of EB and DNA in the absence and presence of the complex were
determined experimentally and presented in Fig. 4,b. The Scatchard plots in the
presence of the complex result in the decrease of slope as compared to the complex free
plot, with no change in the intercept (Fig. 4,b). These results confirm an intercalative
binding of the CuII complex with DNA [19].
The cleavage reaction on supercoiled pUC-19 DNA was monitored by agarose-gel
electrophoresis. When supercoiled DNA was subjected to electrophoresis, a relatively
fast migration was observed for the intact supercoiled DNA (Form I). If scission occurs
on one strand (nicking), the supercoiled DNA will relax to generate a slower-moving
open circular form (Form II). If both strands are cleaved, a linear form (Form III) that
migrates between Form I and Form II will be generated. When supercoiled pUC-19
DNA was incubated at 378 for 4 h in 5 mm Tris·HCl/5 mm NaCl at pH 7.5 in the
presence of Cu complex, it was converted from supercoiled (Form I) to nicked circular
form (Form II; Fig. 5,a). Although this complex did not require the addition of an
external agent, we were keen to discount the possibility that the DNA cleavage
occurred via an OH radical-based depurination pathway. When supercoiled pUC-19
DNA was incubated with higher amounts of the Cu complex in the presence of DMSO
or glycerol, no significant change in the cleavage was observed. This clearly indicates
that this complex-mediated cleavage in the absence of exogenous coreactants does not
proceed via either diffusible OH radicals or free superoxide [20].
The cleavage of pUC-19 DNA by the Cu complex has been kinetically
characterized by quantification of supercoiled and nicked DNA. The observed
distribution of supercoiled and nicked DNA in an agarose gel provides a measure of
the extent of hydrolysis of the phosphodiester bonds in each plasmid DNA, and the
data were used to perform a simple kinetic analysis. Fig. 5,b, shows the time-course plot
for the decrease of Form I and formation of Form II during the reaction under mild
conditions by the Cu complex. The decrease of Form I fits well to a single exponential
decay curve, and the increase of Form II also fits well to a single exponential curve.

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
769
Fig. 4. a) Fluorescence emission spectra of ethidium bromide (EB) bound to CT DNA in the absence
(top) and presence (bottom) of 1:2 CuII –tripeptide complex. [Complex]/[DNA]¼0, 0.24, 0.48, 0.72, 0.94,
1.18; lex ¼540 nm. Inset: Stern–Volmer quenching plot. b) Fluorescence Scatchard plots for the binding
II
of EB to CT DNA in the absence ( ) and presence ( ) of [Cu (Boc-His-Gly-His-OMe)2]2þ. The term rEB
~
&
is the concentration ratio of bound EB to total DNA, and cf is the concentration of free EB.

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Fig. 5. a) Agarose-gel electrophoresis patterns for the cleavage of pUC-19 DNA by the Cu complex. Lane
1: DNA control; Lanes 2–5: [Cu complex]¼125, 250, 350, 500 mm, resp. NC is the nicked circular and SC
*
the supercoiled form of pUC-19 DNA, resp. b) Disappearance of supercoiled ( , Form I) and formation
~
of nicked circular ( , Form II) forms of plasmid DNA in the presence of Cu complex (250 mm) as a
function of the incubation time (pH 7.5, temp. 378).
From these curve fits, the hydrolysis rate constants at 378 and at a complex
concentration of 250 mm were determined to be 2.82ꢀ10ꢁ5 sꢁ1 (R¼0.979) for the
Cu complex.
Recently, Long and co-workers [21] have shown by NMR and molecular-simulation
model studies that NiII(Gly-Gly-His) (1:1 complex) binds to DNA through minor-
groove contacts, and established that it acts as a mini model for minor-groove binding.
The [CuII(Boc-His-Gly-His-OMe)2]2þ complex with a square-planar geometry per-
fectly fits into the model suggested by them. In their complex, the DNA contacts are
through His NH, terminal amine NꢁH H-atoms, and H-bond formation through the
AT region, whereas in our complex the contacts are most likely through imidazole NH
H-atoms.

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
771
It was observed recently [2] that the DNA binding constant below 104 mꢁ1 was
assumed to allow a sufficiently rapid diffusion on the pharmacological time scale, and
even moderate values of DNA binding (105 –106 mꢁ1) can limit diffusion. With a Kb
value of 1.2ꢀ102 mꢁ1 along with its cleavage ability, the complex [CuII(Boc-His-Gly-
His-OMe)2]2þ has a potential to be developed as a nucleic acid-targeted drug.
The authors thank the University Grants Commission (New Delhi) and the Department of Science
and Technology (Government of India) for financial support.
Experimental Part
General. Boc-NH-His(Tos)-Gly-OH, NH2-His(Tos)-OMe, and ethidium bromide (EB; 99.99%
purity) were obtained from Sigma (Germany). Cu(OAc)2 ·H2O (AnalaR grade) was purchased from
Merck. All other chemicals and solvents (spectroscopic grade) were purchased from commercial sources
and used without further purification. Doubly dist. H2O was used to prepare the buffer solns. Calf thymus
(CT) DNA was obtained from Fluka (Switzerland). Plasmid pUC-19 DNA, agarose gel, and Tris·HCl
(Tris¼Tris(hydroxymethyl)aminomethane) buffer were obtained from Bangalore Genei (India). The
spectroscopic titrations were carried out in aerated buffer (5 mm Tris·HCl, 50 mm NaCl, pH 7.5) at r.t.
Molar conductivity: Digisun DI-909 digital conductivity bridge with a dip-type cell, using a 10ꢁ3 m soln. of
the complex in MeOH. Magnetic susceptibility: Faraday balance (CAHN-7600) at r.t. using
Hg[Co(CNS)4] as standard. Diamagnetic corrections were made by using Pascalꢃs constants [22]. EPR
Spectra: Jeol (JES-FA200) X-band spectrometer. UV/VIS Spectra: Shimadzu 160A spectrophotometer
(800–200 nm); lmax in nm. IR Spectra: Perkin-Elmer FT IR spectrometer; KBr pellets, ˜n in cmꢁ1
.
1H-NMR Spectra: Varian Gemini FT-NMR spectrometer at 200 MHz in CDCl3. ESI-MS: Micro Mass
quattro Lc triple-quadrupole mass spectrometer with MassLynx software (Manchester, UK); in m/z.
Elemental analyses: Heraeus Carlo Erba 1108 elemental analyzer. Cu Content: Perkin-Elmer 2380
atomic absorption spectrometer.
Peptide Synthesis. For the synthesis of the tripeptide Boc-NH-His(Tos)-Gly-His(Tos)-OMe
(C34H42N7O10S2), the procedure of Varenkamp and co-workers [23] was applied. To a soln. of Boc-
NH-His(Tos)-Gly-OH in anh. CH2Cl2 at 08 under N2, 1-hydroxybenzotriazole (HOBt) was added under
stirring, followed by 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDCl). After
15 min at 08, NH2-His(Tos)-OMe (dissolved in CH2Cl2 and neutralized with EtN(i-Pr)2) was added to the
mixture, which was allowed to reach r.t., and stirring was continued for 6 h. The reaction was quenched
with a sat. aq. NH4Cl soln. and extracted with AcOEt. The combined org. layers were washed with a sat.
aq. NH4Cl soln., sat. aq. NaHCO3 soln., and brine, dried (Na2SO4), and evaporated in vacuo. The residue
was purified by CC (SiO2 (60–120 mm); CHCl3/MeOH). IR (KBr): 3407m (NꢁH), 1670s (COꢁN),
1750w (COOR), 1080 (imidazole N). 1H-NMR (200 MHz, CDCl3): 8.10–7.04 (m, 14 H); 5.68 (d, J ¼ 7.5,
1 H); 4.92–4.69 (m, 1 H); 4.53–4.34 (m, 1 H); 3.97–3.78 (m, 2 H); 3.66 (s, 3 H); 3.19–2.89 (m, 4 H); 2.43
(s, 6 H); 1.42 (s, 9 H). ESI-MS: 773 ([MþH]þ ). Anal. calc. for C34H42N7O10S2: C 56.50, H 5.81, N 13.57;
found: C 56.49, H 5.45, N 13.58.
Synthesis of the Metal Complex. A soln. of Boc-NH-His(Tos)-Gly-His(Tos)-OMe (140 mg,
0.181 mmol) in anh. MeOH (10 ml) was stirred for 1 h. Then, the pH of the soln. was adjusted to ca.
7.0 with Et3N. To this soln., Cu(OAc)2 ·H2O (19 mg, 0.0906 mmol) in MeOH was added, and stirring was
continued for 6 h. The resulting green colored precipitate was filtered off, washed with hot MeOH, and
dried to afford the Cu complex (Scheme). Yield: 50%. Lm (Molar conductance) 1.20ꢀ10ꢁ3 Wꢁ1 cm2
molꢁ1. M.p. 2408. UV/VIS (MeOH): 577. IR (KBr): 3446m (NꢁH), 1684s (COꢁN), 1756w (COOR),
1122 (imidazole N), 568 (CuꢁN). ESI-MS: 512 ([2 MþNH4]þ ). Anal. calc. for C40H58N14O12Cu: C
48.53, H 5.86, N 19.81, Cu 6.42; found: C 48.50, H 5.83, N 19.82, Cu 6.45.
DNA Binding. Preparation of a CT DNA Soln. A conc. CT DNA stock soln. was prepared in 5 mm
Tris·HCl/50 mm NaCl in H2O at pH 7.5, and the concentration of the DNA soln. was determined by UV
absorbance at 260 nm. The molar absorption coefficient was taken as 6600 mꢁ1 cmꢁ1 [24]. The soln. of CT
DNA in 5 mm Tris·HCl/50 mm NaCl (pH 7.5) gave a ratio of UVabsorption at 260 and 280 nm, A260/A280
,

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of ca. 1.8–1.9, indicating that the DNAwas sufficiently free of protein [25]. All stock solns. were stored at
48 and used within one week. The concentration of EB was determined spectrophotometrically at 480 nm
(e¼5680 mꢁ1cmꢁ1) [26].
Absorption-Spectroscopic Studies. Absorption spectra were recorded on a Jasco V-530 UV/VIS
spectrophotometer using 1-cm quartz microcuvettes. Increasing known amounts of the Cu complex in
MeOH was added to DNA. The experiments were carried out at r.t. in 5 mm Tris·HCl/50 mm NaCl
(pH 7.5). After each addition, the mixture was shaken and kept for ca. 5 min, and the absorbance was
recorded.
Thermal-Denaturation Studies. Thermal-denaturation studies were recorded on a Shimadzu 160A
spectrophotometer equipped with a thermostated cell holder. DNA (75 mm) was treated with the
complex (75 mm) in buffer (5 mm Tris·HCl/50 mm NaCl) at pH 7.5. The samples were continuously
heated at a rate of 18/min, while the absorption changes at 260 nm were monitored. Values for the melting
temperature (Tm) and for the melting interval (DT) were determined according to the procedures
reported in [27]. Differential melting curves were obtained by numerical differentiation of the
experimental melting curves.
Intercalator Displacement by Fluorescence Spectroscopy. Fluorescence spectra were recorded with a
SPEX- Fluorolog 0.22 m fluorimeter equipped with a 450-W Xe lamp. The slit widths were 2ꢀ2ꢀ2ꢀ2,
and the emission spectral range was 550–650 nm. All fluorescence titrations were carried out in 5 mm
Tris·HCl/50 mm NaCl (pH 7.5) at r.t. The soln. containing DNA and EB was titrated with varying
concentrations of the CuII complex. The solns. were excited at 540 nm, and the fluorescence emission,
which corresponds to 565 nm, was recorded. The samples were shaken and kept for 2–3 min for
equilibrium, and then the spectra were recorded. The DNA concentration was always 54 mm. The
concentrations of the complex was in the range of 0–77 mm, and the EB concentration was 43 mm.
Fluorescence spectra were also used to obtain Scatchard plots. For these, titrations of DNA against
EB in the absence and presence of the CuII complex was performed. Initial concentration of DNA in
5 mm Tris·HCl/50 mm NaCl was 34 mm. After each addition of EB to the solns. containing DNA and the
metal complex, the emission spectra were recorded from 550 to 650 nm with 540-nm excitation at r.t.
Corrections were made for the volume changes during the course of titrations. The data were analyzed by
the method of Lepecq and Paoletti [19] to obtain bound (cb) and free (cf) concentrations of EB. Scatchard
plots were obtained by plotting rEB/cf vs. rEB (where rEB ¼cb/[DNA]).
Electrophoresis Experiments. Agar-gel electrophoresis was performed with pUC-19 DNA in Tris·
HCl buffered solns. at pH 7.5. DNA-Cleavage studies were performed by adding varying concentrations
of the CuII complex (0–500 mm) to 2 ml of pUC-19 DNA (0.5 mg/ml; 10 mm Tris·HCl, 1 mm EDTA,
pH 8.0) to 5 mm Tris·HCl/50 mm NaCl (total volume 16 ml). After mixing, the DNA solns. were
incubated at 378 for 4 h. Kinetic analysis was carried out at fixed concentrations of DNA and CuII
complex with different incubation times under identical experimental conditions. The reactions were
quenched by the addition of bromophenol blue and the mixtures were analyzed. Experiments were
carried out with 1% agar gels run at 50 V for 4 h in TAE (Tris/AcOH/EDTA) buffer. The gels were
stained with EB (0.5 mg/ml) and photographed under UV light.
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Received February 24, 2008