Synthesis, characterization and in vitro DNA binding and cleavage studies of Cu(II)/Zn(II) dipeptide complexes

Article abstract of DOI:10.1016/j.jphotobiol.2012.12.009

Novel dipeptide complexes Cu(II)?Val-Pro (1), Zn(II)?Val-Pro (2), Cu(II)?Ala-Pro (3) and Zn(II)?Ala-Pro (4) were synthesized and thoroughly characterized using different spectroscopic techniques including elemental analyses, IR, NMR, ESI-MS and molar conductance measurements. The solution stability study carried out by UV-vis absorption titration over a broad range of pH proved the stability of the complexes in solution. In vitro DNA binding studies of complexes 1-4 carried out employing absorption, fluorescence, circular dichroism and viscometric studies revealed the binding of complexes to DNA via groove binding. UV-vis titrations of 1-4 with mononucleotides of interest viz., 5′-GMP and 5′-TMP were also carried out. The DNA cleavage activity of the complexes 1 and 2 were ascertained by gel electrophoresis assay which revealed that the complexes are good DNA cleavage agents and the cleavage mechanism involved a hydrolytic pathway. Furthermore, in vitro antitumor activity of complex 1 was screened against human cancer cell lines of different histological origin.

Full text of DOI:10.1016/j.jphotobiol.2012.12.009

Journal of Photochemistry and Photobiology B: Biology 121 (2013) 75–85
Contents lists available at SciVerse ScienceDirect
Journal of Photochemistry and Photobiology B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Synthesis, characterization and in vitro DNA binding and cleavage studies of
Cu(II)/Zn(II) dipeptide complexes
Farukh Arjmand , A. Jamsheera , D.K. Mohapatra b
a,
⇑
a
a
Department of Chemistry, Aligarh Muslim University, Aligarh 202 002, Uttar Pradesh, India
Diversity Oriented Synthesis Laboratory, Indian Institute of Chemical Technology, Hyderabad 500 607, Andhra Pradesh, India
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 August 2012
Received in revised form 29 November 2012
Accepted 19 December 2012
Available online 26 February 2013
Novel dipeptide complexes Cu(II)ꢀVal-Pro (1), Zn(II)ꢀVal-Pro (2), Cu(II)ꢀAla-Pro (3) and Zn(II)ꢀAla-Pro (4)
were synthesized and thoroughly characterized using different spectroscopic techniques including ele-
mental analyses, IR, NMR, ESI–MS and molar conductance measurements. The solution stability study
carried out by UV–vis absorption titration over a broad range of pH proved the stability of the complexes
in solution. In vitro DNA binding studies of complexes 1–4 carried out employing absorption, fluores-
cence, circular dichroism and viscometric studies revealed the binding of complexes to DNA via groove
binding. UV–vis titrations of 1–4 with mononucleotides of interest viz., 5 -GMP and 5 -TMP were also car-
ried out. The DNA cleavage activity of the complexes 1 and 2 were ascertained by gel electrophoresis
assay which revealed that the complexes are good DNA cleavage agents and the cleavage mechanism
involved a hydrolytic pathway. Furthermore, in vitro antitumor activity of complex 1 was screened
against human cancer cell lines of different histological origin.
Keywords:
Dipeptide Cu(II) and Zn(II) complexes
DNA binding profile
pBR322 DNA cleavage
In vitro anticancer activity
0
0
Ó 2013 Elsevier B.V. All rights reserved.
1
. Introduction
Small molecules binding to specific sites along DNA molecule
of peptides. Metals may be directly bound into peptides and have
been used for DNA selective recognition and/or cleavage [6,7]. It
was reported that peptide fictionalization of polypyridyl
ruthenium(II) [8,9] or rhodium(III) [10,11] metallo-intercalators
improved the selectivity of the parent intercalators, with similar
effects observed by conjugating minor groove binders with short
peptides that mimic natural protein motifs [12].
are considered as potential chemotherapeutic agents. Peptides
are attracting much attention since Novartis launched a vasopres-
sin drug analog Lypressin in 1970, presently ca. 30 peptides have
been marketed as key classes of therapeutics. Their role as media-
tors of key biological functions and their unique intrinsic proper-
ties make them particularly attractive therapeutic agents. They
can surmount the hurdles of present cancer chemotherapeutic
drugs including, little unspecific binding to molecular structures
other than the desired target, minimization of drug–drug interac-
tions and less accumulation in tissues reducing risks of complica-
tions due to intermediate metabolites. The fact that peptides
affect the tumor cells rapidly and that their secondary metabolites
are free amino acids proves that the peptides have minimized side
effects as compared to other chemotherapeutic agents that are
available currently. Recently, it has been reported that some pep-
tide derivatives show antitumor activity with little toxicity against
non-malignant cells either by triggering apoptosis [1,2] or by form-
ing ion channels/pores [3]. Furthermore, some peptides were found
to be cytotoxic against MDR cancer cells [4,5].
The rationale of our strategy is to design peptide–metal com-
plexes which prove to possess pronounced biological and better
pharmacological activity. Amongst the metal ions chosen, we have
opted for biocompatible endogenous metal ions Cu and Zn, known
for their essential role in biological living system. In particular, the
complexes based on essential metals are less toxic than those with
non-essential ones. Cu(II) complexes are known to play a significant
role either in naturally occurring biological systems or as pharmaco-
logical agents [13,14] whereas the importance of zinc for stabiliza-
tion of protein loops in enzymes, zinc fingers, etc., has generated
new interest in the field of Zn coordination chemistry [15,16]. The
discovery of the ‘zinc fingers’ has triggered intensive research on
the interaction of proteins with Zn ions [17,18]. The investigation
of Cu(II)/Zn(II)-peptide complexes is of scientific and technological
importance, since such systems may be regarded as models for both
protein–DNA and antitumor agent-DNA interactions.
Peptides are versatile and powerful ligands for a range of metal
ions since they contain a variety of potential donor centers. Metal-
lopeptide systems are unique and can affect the biological activity
Herein we report, the synthesis, spectroscopic characterization,
DNA binding and cleavage studies of dipeptide complexes of Cu(II)
and Zn(II). The synthetic strategy involves peptide moiety as a scaf-
fold for our complexes thereby, these complexes would exhibit
desirable therapeutic properties for more efficacious and safer drug
⇑
Corresponding author. Tel.: +91 571 2703893.
E-mail address: farukh_arjmand@yahoo.co.in (F. Arjmand).
1
011-1344/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotobiol.2012.12.009

7
6
F. Arjmand et al. / Journal of Photochemistry and Photobiology B: Biology 121 (2013) 75–85
administration owing to (a) better cellular uptake, (b) improved
aqueous solubility, (c) selectivity and specificity towards the target
at the molecular level and (d) reduced toxicity due to biocompati-
ble endogenously friendly ligand and metal ions. In vitro DNA bind-
ing studies revealed that these complexes are avid DNA binding
agents; thereby they can act as potent chemotherapeutics. Further-
more, DNA cleavage activity of 1 and 2 with pBR322 DNA has been
carried out, in addition in vitro cytotoxicity of complex 1 was
tested against various human cancer cell lines.
plates at an appropriate cell density to give optical density in the
linear range (from 0.5 to 1.8) and were incubated at 37 °C in CO
incubator for 24 h. Stock solutions of the complexes were prepared
as 100 mg/mL in DMSO and four dilutions i.e. 10 L, 20 L, 40 L,
80 L, in triplicates were tested, each well receiving 90 L of cell
suspension and 10 L of the drug solution. Appropriate positive
control (Adriamycin) and vehicle controls were also run. The plates
with cells were incubated in CO incubator with 5% CO for 24 h
followed by drug addition. The plates were incubated further for
8 h. Termination of experiment was done by gently layering the
cells with 50 L of chilled 30% TCA (in case of adherent cells) and
0% TCA (in case of suspension cell lines) for cell fixation and kept
at 4 °C for 1 h. Plates were stained with 50 L of 0.4% SRB for
0 min. The bound SRB was eluted by adding 100 L 10 mM Tris
pH 10.5) to each of the wells. The absorbance was read at
40 nm with 690 nm as reference wave length. All the experiments
2
l
l
l
l
l
l
2
2
4
l
2
. Experimental
5
l
2.1. Materials
2
(
5
l
Alanine, Valine, Proline, HOBT (N-Hydroxybenzotriazole) (SRL),
Cu(NO
3-dimethylaminopropyl)carbodiimide
anhydride, SOCl , TFA (trifluro acetic acid), calf thymus DNA
CT-DNA), ascorbic acid (Merck), H , 3-mercaptopropionic acid,
3
)
2
ꢀ3H
2
O, Zn(NO
3
)
2 2
ꢀ6H O
(Ranchem), EDCI (1-Ethyl-3-
were repeated three times.
(
Hydrochloride), Boc
2
(
O
2 2
2.3. Syntheses
glutathione, methyl green, DAPI, tertiary butyl alcohol, sodium
0
azide, superoxide dismutase (Sigma), guanosine-5 -monophos-
2.3.1. Synthesis of dipeptides (L , L )
1
2
0
0
phate disodium salt (5 -GMP) and thymidine-5 -monophosphate
disodium salt (5 -TMP) (Fluka) were used without further
The dipeptides used in this study were synthesized by conven-
tional solution phase methodology [23]. The Boc group was used
for N-terminal protection and the C-terminus was protected as a
methyl ester. Coupling reactions were mediated by EDCI-HOBT.
Boc-amino acid (10 mmol) was dissolved in dichloromethane
0
purification.
2.2. Methods and instrumentation
(
DCM, 20 mL). To this solution, EDCI (4.77 g, 25 mmol) was added
at 0 °C and the reaction mixture was stirred for 15 min amino acid
methylester (10 mmol) was added followed by HOBT (2.74 g,
20 mmol) to the above reaction mixture under stirring. After
Elemental analyses were performed on Elementar Vario EL III.
Fourier transform infrared spectra were recorded on Interspec
020 FTIR spectrometer in Nujol. 1H NMR spectra was obtained
2
2
4 h, the reaction mixture was filtered; the residue was washed
on a Bruker DRX-400 spectrometer. Electrospray mass spectra
were recorded on Micromass Quattro II triple quadrupol mass
spectrometer. EPR spectra of the copper complexes were recorded
on a Varian E 112 spectrometer at the X-band frequency (9.1 GHz)
at LNT. Molar conductance was measured at room temperature on
a Digisun Electronic Conductivity Bridge. Electronic spectra were
recorded on a UV-1700 PharmaSpec UV–visible Spectrophotome-
ter. Fluorescence measurements were made on Hitachi F-2500
Fluorescence Spectrophotometer. CD spectra were recorded on
Applied Photophysics Chirascan Circular Dichroism Spectrometer
with Stop Flow. Viscosity measurements were carried out from
observed flow time of CT-DNA containing solution (t > 100 s)
with DCM (30 mL) and added to the filtrate. The filtrate was
washed with 5% sodium bicarbonate and saturated sodium chlo-
ride solutions. The organic layer was dried over anhydrous Na SO ,
2 4
filtered and evaporated under reduced pressure. The crude product
was purified by column chromatography.
ꢁ
1
L
1
Semisolid mass; Yield 63.7%; IR (Nujol, cm ): 3315 (
mC@O); 1617 (masOAC@O); 1455 (m
s
m
NH);
OAC@O). H NMR
, ppm): 5.20 (1H, ANH, Boc), 4.50 (1H, m, ACH, Val), 4.25
1H, t, ACH, Pro), 3.80 (2H, t, ACH , Pro), 3.71 (3H, s, AOCH ),
.00 (2H, m, ACH , Pro), 2.25 (2H, m, ACH , Pro), 1.00 (3H, d,
ACH Val), 1.45 (9H, s, tButyl group). ESI–MS (m/z): 329
1
1
(
(
716 (
CDCl
3
2
3
2
2
2
3
,
+
[
C
16
H
28
N
2
O
5
+ H] .
0
corrected for the flow time of buffer alone (t ), using Ostwald’s Vis-
cometer at 25 ± 0.01 °C. Flow time was measured with a digital
stopwatch.
ꢁ1
L
2
Semisolid mass; Yield 67.2%; IR (Nujol, cm ): 3317 (
m
NH);
OAC@O). H NMR
, ppm): 5.35 (1H, ANH, Boc), 4.51 (1H, m, ACH, Ala), 4.45
1H, t, ACH, Pro), 3.61 (2H, t, ACH , Pro), 3.70 (3H, s, AOCH ),
.00 (2H, m, ACH , Pro), 2.25 (2H, m, ACH , Pro), 1.35 (3H, d,
ACH Ala), 1.45 (9H, s, tButyl group). ESI–MS (m/z): 301
1
1
(
(
714 (
CDCl
mC@O); 1650 (masOAC@O); 1443 (m
s
DNA binding experiments which include absorption spectral
traces, luminescence, circular dichroism and viscosity experiments
conformed to the standard methods [19,20] and practices
previously adopted by our laboratory [21]. While measuring the
absorption spectra an equal amount of DNA was added to both
the compound solution and the reference solution to eliminate
the absorbance of the CT-DNA itself, and the CD contribution by
the CT-DNA and Tris buffer was subtracted through base line
correction.
3
2
3
2
2
2
3
,
+
14 24 2 5
[C H N O + H] .
2
.3.2. Synthesis of Cu(II)ꢀVal-Pro (1)
The dipeptide, L (0.43 g, 2 mmol) was dissolved in minimum
amount (15 mL) of absolute methanol. To this a methanolic solu-
tion of Cu(NO O (0.24 g, 1 mmol) was added. The solution
1
3
2
) ꢀ3H
2
2
.2.1. In vitro antitumor studies
The cell lines used for in vitro antitumor screening activity were
was stirred for 1 h. The resultant pale blue solution was left for
slow evaporation at room temperature. The blue precipitate
obtained was filtered, washed with petroleum ether and dried in
vacuo (Scheme 1).
A498 (Renal cell), A549 (Lung), Zr-75-1 (Breast), HT29 (Colon ade-
nocarcinoma grade II cell line), A2780 (Ovary), SiHa (Uterine cer-
vix) and MCF7 (Human breast). These human malignant cell lines
were procured and grown in RPMI-1640 medium supplemented
with 10% Fetal Bovine Serum (FBS) and antibiotics to study growth
pattern of these cells. The proliferation of the cells upon treatment
with chemotherapy was determined using the Sulforhodamine B
Yield, 66.7%. IR (Nujol, cmꢁ1): 3218 (
asOAC@O); 1408 (
(m/z): 491 [C20
m
NH); 1674 (
mC@O); 1674
(m
m OAC@O); 452 (CuAN); 519 (CuAO). ESI–MS
s
+
H
34
N
4
O
6
Cu + H] . Molar Conductance,
K
M
ꢁ3
ꢁ1
2
ꢁ1
(1 ꢂ 10
UV–vis (DMSO, nm): 257, 645 nm. Anal. Calc. (%) (for C20
Cu): C 49.02; H 6.99; N 11.43. Found (%): C 49.10; H 6.94; N 11.47.
M, DMSO): 32.7
X
cm mol
(non-electrolyte).
34 4 6-
H N O
(
SRB) semi automated assay [22]. Cells were seeded in 96 well

F. Arjmand et al. / Journal of Photochemistry and Photobiology B: Biology 121 (2013) 75–85
77
Scheme 1. Schematic representation of the synthesis of complexes 1–4.
2
.3.3. Synthesis of Zn(II)ꢀVal-Pro(2)
N
12.89.
4
O
6
Zn): C 44.10; H 6.01; N 12.86. Found (%): C 44.11; H 6.03; N
This complex was synthesized by a similar procedure as
described for 1 with a methanolic solution of Zn(NO
3
)
2
ꢀ6H
2
O
(
0.29 g, 1 mmol).
3
. Results and discussion
Yield, 63.5%. IR (Nujol, cmꢁ1): 3188 (
mNH); 1665 (mC@O); 1665
1
(
(
(
m
D
asOAC@O); 1394 (
s
m OAC@O); 464 (ZnAN); 512 (ZnAO). H NMR
3.1. IR spectra
2
O, ppm): 4.52 (2H, m, ACH, Val), 4.30 (2H, t, ACH, Pro), 4.15
4H, t, ACH
2
, Pro), 3.24 (4H, NH
2
, Val), 2.35–2.00 (8H, m, ACH
2
,
+
Coordination behaviors of dipeptides to metal ions are deli-
Pro), 1.15 (6H, ACH
Molar Conductance,
3
, Val). ESI–MS (m/z): 491 [C20
H
X
34
ꢁ1
N
4
O
6
Zn-H] .
cately different among metals and peptides. The
m
(NH) band at
ꢁ
3
2
ꢁ1
K
M
(1 ꢂ 10 M, DMSO): 30.6
cm mol
ꢁ1
3
3
317 cm in the free dipeptides appeared as a broad band at
(
(
4
non-electrolyte). UV–vis (DMSO, nm): 258 nm. Anal. Calc. (%)
for C20 Zn): C 48.84; H 6.97; N 11.39. Found (%): C
8.86; H 6.94; N 11.41.
ꢁ1
117–3215 cm upon complexation. This change and relatively
(NH ) bands assigned that terminal amino group
of the peptide occupies one of the coordination sites of the metal
34 4 6
H N O
low frequency of
m
2
ꢁ1
ion [24]. The tertiary amide band at 1716 cm has been shifted
to low frequency upon complexation indicative of coordination of
amide group to the central metal ion. A distinctive difference
between the absorption of facial and meridional isomers was
observed in the AC@O stretching region; the facial isomer has only
one peak where as the meridional isomer has two split absorption
peaks [25,26]. All the complexes exhibited a strong absorption
2
.3.4. Synthesis of Cu(II)ꢀAla-Pro (3)
The dipeptide, L (0.37 g, 2 mmol) was dissolved in minimum
amount (15 mL) of absolute methanol. To this a methanolic solu-
tion of Cu(NO O (0.24 g, 1 mmol) was added. The solution
2
)
3 2
ꢀ3H
2
was stirred for 1 h. The blue precipitate appeared was filtered,
washed with petroleum ether and dried in vacuo.
ꢁ
1
ꢁ1
ꢁ1
Yield, 62.35%. IR (Nujol, cm ): 3185 (
asOAC@O); 1391 OAC@O); 480 (CuAN); 520 (CuAO).
ESI–MS (m/z): 439 [C16
m
NH); 1668 (
m
C@O); 1652
around 1660 cm
with a shoulder band at 1530 cm
in the
(m
(
m
H
s
masOAC@O region, indicating meridional geometry. msOAC@O
+
26
N
4
O
6
Cu + 5H] . Molar Conductance,
K
M
absorption band of the complexes was observed around
ꢁ3
ꢁ1
2
ꢁ1
ꢁ1
ꢁ1
(
1 ꢂ 10 M, DMSO): 33.1
X
cm mol
(non-electrolyte).
1400 cm . The large difference (>200 cm ) between the two val-
ꢁ
UV–vis (DMSO, nm): 256, 650 nm. Anal. Calc. (%) (for C16
H
26
N
4
O
6-
ues designated the coordination of COO groups to the metal ion in
Cu): C 44.28; H 6.04; N 12.91. Found (%): C 44.29; H 6.00; N 12.89.
a monodentate fashion [27]. Further evidence for complexation
was provided by the presence of medium intensity bands around
455, 515 cm corresponding to (Cu/ZnAN), (Cu/ZnAO), respec-
ꢁ1
2.3.5. Synthesis of Zn(II)ꢀAla-Pro (4)
This complex was synthesized by a similar procedure as
tively in far IR region.
described for 3 with a methanolic solution of Zn(NO
3
)
2
ꢀ6H
2
O
(
0.29 g, 1 mmol).
3.2. NMR spectra
Yield, 64%. IR (Nujol, cmꢁ1): 3215 (
m
NH); 1667 (
m
C@O); 1667 (m
as-
1
1
OAC@O); 1410 (
ppm): 4.58 (2H, m, ACH, Ala), 4.48 (2H, t, ACH, Pro), 3.24 (4H, NH
Val), 3.65 (4H, t, ACH , Pro), 2.45–2.10 (8H, m, ACH , Pro), 1.5 (6H,
ACH , Ala). ESI–MS (m/z): 478 [C16
Conductance,
m
s
OAC@O); 475 (ZnAN); 526 (ZnAO). H NMR (D
2
O,
The singlets at 1.45 and 3.70 ppm in the H NMR spectra of li-
2
,
3
gands revealed the presence of t-boc and ACO(OCH ) group respec-
2
2
tively in the ligands which disappeared upon deprotection with the
emergence of a new resonance at 12.0–12.18 ppm corresponding
to the ACOOH group. The signals at 2.25 (multiplet), 3.80 (triplet)
and 4.25 (triplet) ppm indicated the presence of proline moiety in
+
3
H
26
N
4
O
6
Zn + K + 3H] . Molar
ꢁ3
ꢁ1
2
ꢁ1
K
M
(1 ꢂ 10 M, DMSO): 31.0
X
cm mol (non-
electrolyte). UV–vis (DMSO, nm): 258 nm. Anal. Calc. (%) (for C16H
26-

7
8
F. Arjmand et al. / Journal of Photochemistry and Photobiology B: Biology 121 (2013) 75–85
the ligands. A doublet at 1.0 and a triplet at 4.51 ppm revealed the
presence of valine moiety in the ligand L whereas a doublet at
.35 and a quartet at 4.51 ppm revealed the presence of alanine
in the ligand L
Upon complexation the signals were shifted to downfield. The
absence of ACOOH resonance at 12.0–12.18 ppm in the complex
indicated the coordination of carboxylic group to Zn through
change in the position of the d–d band over a pH range 2–12 with
only minor change in intensity. The complexes also displayed sim-
ilar spectra over a period of 24 h confirming the stability of the me-
tal ion in the peptide metal complexes and also no precipitation
was observed. The non-precipitation after 24 h time period and
over a broad range of pH suggested the stability of the complexes
in solution.
1
1
2
.
2
deprotonation. The NH signal was shifted to 3.24 ppm in both
the complexes indicating the involvement of amino group in
complexation.
4
. DNA binding studies
4.1. Electronic absorption studies
3.3. Mass spectra
Electronic absorption spectroscopy is an effective method to
Electrospray mass spectra in the positive ion mode were
recorded for the free peptides and complexes 1–4. ESI–MS spectra
examine the binding modes of the metal complexes with DNA. Upon
ꢁ4
addition of incremental amounts of CT-DNA (0 ꢁ 0.2 ꢂ 10 M), the
of the di-/tetra-peptides displayed prominent peaks at m/z 329,
ꢁ
3
+
absorption bands of the complexes (0.066 ꢂ 10 M) 1–4 exhibited
hyperchromism (Fig. 1) without any shift in the intra ligand bands,
suggesting that there exists a strong interaction between the com-
plexes and DNA which is different from classical intercalation mode
of binding, since intercalation involves hypochromism. The spectral
changes of the complexes observed in presence of DNA can be ratio-
nalized in terms of groove binding [30]. The formation of DNA–pep-
tide metal complex could induce changes in the spectral
characteristics of both or either of DNA or peptide complexes [31].
Since DNA possesses several hydrogen bonding sites which are
accessible in major and minor grooves, therefore, the complexes
form hydrogen bonds via amine group of peptide moiety with N-3
of adenine or O-2 of thymine in the DNA, which may contribute to
the hyperchromism observed in the absorption spectra.
3
01 and 469 corresponding to the fragments [C16
H
28
N
2
O
5
+ H] ,
+
+
[
C
14
H
24
N
2
O
5
+ H] and [C22
36 4 7
H N O + H] , respectively. Complex 1
displayed two peaks centered at m/z 491 and 215 corresponding
+
+
to the fragments [1 + H] and [1-C10
17 2 3
H N O Cu + 2H] , respec-
tively. Two major peaks are observed for complex 2 at m/z 491
+
17 2 3-
and 215 corresponding to the fragments [2-H] and [2-C10H N O
+
Zn + 2H] , respectively. Complex 3 also displayed two prominent
+
peaks at m/z 439 and 186 which could be assigned to [3 + 5H]
+
and [3-C
H
8 13
N
2
O
3
Cu + H] , respectively. The peaks at m/z 478
and 187 in the spectrum of the complex 4 were assigned to
+
+
[
4 + K + 3H] and [4-C
.4. EPR spectra
The EPR spectra of complexes 1 and 3 acquired at LNT under the
8 13 2 3
H N O Zn + 2H] , respectively.
3
In order to quantitatively evaluate the binding magnitude of the
complexes 1–4 with DNA, the intrinsic binding constant k
of the complexes were determined from the spectral titration
curves (Table 1). From the k values, it was found that all the dipep-
b
values
magnetic field strength 3000 ± 1000G showed a spectrum, with
both parallel and perpendicular g values. The EPR spectra of the
complexes interpreted in terms of g values displayed g|| = 2.313,
b
tide complexes show more or less similar binding affinity. The
slight difference in binding behavior could be attributed to the
chain length of peptides; which enhances the hydrophobicity,
thereby resulting in higher binding affinity. The hydrophobic resi-
dues in peptides increase the stability of the peptide–DNA complex
g
g
\
= 2.131 and
gavg = 2.894 for complex 1 and g|| = 2.338,
\
= 2.145 and gavg = 2.921 for complex 3 computed from the for-
2
2
2
mula g ¼ g þ 2g =3, suggested an octahedral geometry [28].
avg
k
?
The trend g|| > g
\ e
> g (2.0023) revealed that the unpaired electron
2 2
II
was located in the d -d orbital of the Cu ions characteristic of ax-
x
y
[
32] and it is more likely that the hydrophobicity and the H-bond-
ial symmetry. The parameter G = (g|| ꢁ 2.0023)/(g ꢁ 2.0023), mea-
\
ing ability of the peptide moiety can be responsible for the higher
DNA binding affinity of complexes 1–4, since the hydrophobic res-
idues like Ala and Val can recognize the methyl on thymine base at
major groove. It is worthwhile to mention that efficiency of this
type of complexes in their biological studies has also been
observed in earlier studies [33–35].
sures the exchange interaction between the metal center in a
polycrystalline solid. G > 4 indicates negligible exchange interac-
tion in the solid complex. For complexs 1 and 3 G < 4 indicated
considerable exchange interaction in the complexes. For a Cu(II)
complex, g|| is a parameter sensitive enough to indicate covalence,
g
|| < 2.3 indicates a covalent complex whereas g|| = 2.3 or more for
an ionic environment.
0
0
4
.2. Interaction with 5 -GMP and 5 -TMP
3.5. Electronic spectra
Nucleotide–metal interactions have been reported to occur at
The electronic absorption spectra are often very helpful in the
evaluation of results furnished by other methods of structural
investigation. The UV–vis absorption spectra of the complexes 1–
in DMSO were characterized by strong intra ligand absorption
bands centered at 256–258 nm. The complexes exhibited charge
transfer bands at 275–287 nm. The complexes 1 and 3 displayed
d–d transition bands at 654 and 650 nm respectively. This spectral
feature is typical of complexes in an octahedral coordination envi-
ronment [29].
nitrogen atom of the purine and pyrimidine rings as well as oxygen
atom of the phosphate group present on the nucleotide [36]. Addi-
0
0
tion of increasing amounts of mononucleotides (5 -GMP and 5 -
TMP) to the complexes 1–4 resulted in hyperchromism (Figs. 2a
and 2b) without any substantial change in the position of the IL
band of the complexes validating interaction of the complexes to
different nucleotides. These spectral changes upon addition of
increasing amounts of mononucleotides authenticate the strong
interaction of complexes to nucleotides through electrostatic sur-
face interactions, preferably via groove binding. Metal ions pro-
mote hydrophobic interaction between nucleotides and amino
acids, and mediate interaction between proteins and nucleic acids
through formation of ternary complexes. The purine and pyrimi-
dine bases of CT-DNA become exposed due to the unwinding of
the DNA helix promoting an effective binding to these base pairs
with the transition metal complexes.
4
3
.6. Solution stability studies
The stability of the complexes 1 and 3 were determined by UV–
vis absorption titration at d–d band under identical conditions to
those used for DNA binding studies in buffered solution at various
pH values. UV–vis spectra of complexes 1 and 3 exhibited no

F. Arjmand et al. / Journal of Photochemistry and Photobiology B: Biology 121 (2013) 75–85
79
Fig. 1. UV–vis absorption spectra of complexes 1 (a), 2 (b), 3 (c) and 4 (d) in Tris HCl buffer (0.01 M, pH 7.2) upon addition of CT-DNA. Arrows indicate the change in
absorbance upon increasing DNA concentration. Inset: Plots of [DNA] vs. [DNA]/e –e for the titration of CT-DNA with complexes.
a f
4.3. Fluorescence spectral studies
Table 1
b
The binding constant (K ) values of complexes with the DNA (mean standard
deviation of ±0.12).
To further confirm the interaction between the complexes and
CT-DNA, emission experiments were carried out. At room temper-
ature, 1–4 exhibited emission bands at 390 nm when excited at
260 nm (Fig. 3). Upon addition of increasing amounts of CT-DNA
to fixed amount of complexes, the fluorescence emission intensity
was enhanced indicating the strong interaction of complexes with
DNA. Hydrophobic interactions between the complexes and poly-
electrolyte may induce changes in the excited state properties
either due to electrostatic association or intercalation [39].
Although the emission enhancement could not be regarded as a cri-
terion for binding mode, they are related to the extent to which the
complex gets into the hydrophobic environment inside the DNA
and avoid or reduce the accessibility of solvent molecules to com-
plex. The emission titration behavior suggested that the complexes
get into the hydrophobic environment inside the DNA, thereby
avoid the quenching effect of solvent water molecules and the
mobility of the complexes is restricted at the binding site ultimately
leading to decrease in vibrational mode of relaxation [40].
Complex
K
b
(M 1
ꢁ
)
Monitored at (nm)
% Hyperchromism
4
4
4
3
1
2
3
4
7.70 ꢂ 10
1.90 ꢂ 10
6.50 ꢂ 10
9.90 ꢂ 10
257
258
256
258
29
25
27
26
To compare quantitatively the affinity of the complexes to
mononucleotides, the intrinsic binding constant (k ) was also
determined (Table 2). The binding constants clearly revealed selec-
tivity for nucleobases – a higher binding propensity of Cu(II) com-
plexes for 5 -GMP while Zn(II) complexes preferentially binds to 5 -
TMP. The higher propensity of Cu(II) complexes to 5 -GMP could be
b
0
0
0
attributed to preferential binding of the complexes to the N7 nitro-
gen of 5 -GMP occurring due to the thermodynamic and kinetic
0
stability of guanine residue [37]. While in Zn(II) complexes hydro-
gen bonds could be favored between nitrogen atoms of ligand with
carbonyl oxygen of thymine. Furthermore, the presence of electron
withdrawing two oxo groups at the C2 and C3 position of the thy-
mine lowers the energy of the lone pair orbital at N3 of thymine
base which results in stronger molecular orbital interaction in
comparison to guanine resulting in preferential binding of Zn(II)
towards thymine [38].
4.4. Competitive DNA binding studies
Competitive binding studies using ethidium bromide (EB) bound
to DNA could provide affluent information regarding the DNA bind-
ing nature and relative DNA binding affinity. EB is a well known pla-

8
0
F. Arjmand et al. / Journal of Photochemistry and Photobiology B: Biology 121 (2013) 75–85
0
Fig. 2a. Absorption spectral traces of complexes in Tris HCl buffer (0.01 M, pH 7.2) upon addition of 5 -GMP, complexes 1 (a), 2 (b), 3 (c) and 4 (d) in the LF band.
0
Fig. 2b. Absorption spectral traces of complexes in Tris HCl buffer (0.01 M, pH 7.2) upon addition of 5 -TMP, complexes 1 (a), 2 (b), 3 (c) and 4 (d) in the LF band.

F. Arjmand et al. / Journal of Photochemistry and Photobiology B: Biology 121 (2013) 75–85
81
Table 2
system [42]. The quenching plots illustrated (Fig. 4) that the
quenching of EB bound to CT-DNA by the peptide complexes are
in good agreement with linear Stern–Volmer equation. From the
quenching plots Ksv values were found to be 3.26, 1.01, 1.98 and
0
0
The binding constant (K ) values of complexes 1–4 with 5 -GMP and 5 -TMP (mean
b
standard deviation of ±0.06).
Complex
0
ꢁ1
0
ꢁ1
5 -GMP (M
)
5 -TMP (M
)
ꢁ1
4
4
1
2
3
4
7.28 ꢂ 10
0.81 ꢂ 10
0.71 M for complexes 1–4, respectively.
4
4
0.99 ꢂ 10
9.10 ꢂ 10
4
4
5.60 ꢂ 10
0.62 ꢂ 10
4
4
0.59 ꢂ 10
5.72 ꢂ 10
4.5. Circular dichroic studies
Circular dichroism (CD) is a powerful method for distinguishing
nar cationic dye widely used as a sensitive fluorescence probe for
native DNA. EB emits intense fluorescence in the presence of DNA
due to its strong intercalation between the adjacent DNA base pairs.
The fluorescence intensity can be quenched by the addition of a sec-
ond molecule due to the displacement of EB from DNA [41]. The
fluorescence intensity of EB bound to DNA at 584 nm showed
remarkable decreasing trend with increasing concentration of the
complexes 1–4. The resulting decrease in fluorescence is caused
by EB changing from a hydrophobic environment to an aqueous
environment by the displacement of EB from a DNA sequence by
a quencher and the quenching is due to the reduction of the number
of binding sites on the DNA that are available to the EB. As the pep-
tide complexes were not expected to efficiently compete with the
strong intercalator EB for the intercalative binding sites, the EB dis-
placement mechanism was ruled out. So the decrease in intensity
could be due to the interaction of the complexes to DNA through
groove binding mode, releasing some EB molecules from EB-DNA
the three main DNA binding modes, namely intercalative (negative
CD), outside groove binding (positive CD), and outside stacking
(bisignate CD) [43,44]. The CD spectrum of calf thymus DNA con-
sists of a positive band at 275 nm (UV, kmax = 260 nm) caused by
base stacking and a negative band at 245 nm caused by helicity,
which are characteristic of DNA in right-handed B form. Intercala-
tors are known to enhance the intensities of both positive and neg-
ative bands whereas groove binding and electrostatic interaction of
complexes with DNA shows less or no perturbation on the base
stacking and helicity bands [45]. Incubation of B-DNA with the
peptide complexes induced marginal changes both in its positive
and negative bands retaining the basic shape (Fig. 5). The change
in the CD spectra confirms a binding event. In presence of com-
plexes, the intensities of both the positive and negative bands de-
creased. The spectral behavior suggested that the DNA binding of
the complexes induced certain conformational changes in which
the more B-like conformation is transformed into a C-like structure
Fig. 3. Emission enhancement spectra of the complexes 1 (a), 2 (b), 3 (c) and 4 (d) with increasing concentration of complexes in the absence and presence of CT-DNA in Tris
HCl buffer (0.01 M, pH 7.2). Arrows indicate change in intensity upon increasing concentration of CT-DNA.

8
2
F. Arjmand et al. / Journal of Photochemistry and Photobiology B: Biology 121 (2013) 75–85
Fig. 4. Emission spectra of EB bound to DNA in the presence of complexes 1 (a), 2 (b), 3 (c) and 4 (d) in Tris HCl buffer. Arrows indicate the intensity changes upon increasing
concentration of the complexes.
within the DNA molecule [46]. The CD spectral changes demon-
strated a groove binding mode of interaction between the com-
plexes and DNA.
observed flow time for DNA in absence and presence of the com-
plexes. The results were presented as ( ) vs. binding ratio
([complex]/[DNA]), where is the viscosity of DNA in the presence
of complex and is the viscosity of DNA alone (Fig. 6). The relative
g/g
0
g
g
0
4
.6. Viscosity studies
specific viscosity of DNA decreased with increase in the concentra-
tion of the complexes 1–4. The decrease in viscosity observed for
the complexes suggested that the hydrophobic interaction of the
peptide with DNA surface encouraged by the hydrogen bonding
interactions lead to bending (kinking) of the DNA chain.
To investigate further the binding nature of complexes to DNA,
viscosity measurements with the solutions of DNA incubated with
the complexes 1–4 have been carried out. The viscosity of DNA
solution is sensitive to the addition of metal complexes which
can bind to DNA. While classical intercalative mode causes a signif-
icant increase in viscosity of DNA solution due to separation of
base pairs at intercalation sites and increase in overall DNA length,
complexes those bind exclusively in the DNA grooves typically
cause less positive or negative or no change in DNA viscosity
4.7. Gel electrophoretic studies
To assess the DNA cleavage ability of the complexes supercoiled
pBR322 DNA (300 ng) was incubated with varying concentration of
complexes 1–4 in aqueous buffer solution (5 mM Tris HCl/50 mM
NaCl, pH 7.2) for 0.5 h without addition of a reductant (Figs. 7 and
S2). Upon gel electrophoresis of the reaction mixture, a concentra-
tion-dependent DNA cleavage was observed. With the increase in
concentration of complexes, DNA was converted from supercoiled
form (Form I) to nicked circular form (Form II) without further con-
version to linear form (Form III) suggesting single strand DNA cleav-
age. Complex 1 exhibited higher cleavage. The DNA cleavage
[
47]. The relative specific viscosity was calculated using the equa-
tion ((t ꢁ t )/t ), where t is the flow time for the buffer and t is the
0
0
0
2 2
activity of complexes 1–4 in presence activators viz., H O , 3-mer-
captopropionic acid (MPA), glutathione (GSH) and ascorbate (Asc)
was significantly enhanced (Figs. 8 and S3). The activity efficacy fol-
lowed the order H
MPA > GSH ꢃ Asc for complex 2, Asc > MPAꢃGSH > H
complex 3 and H
2
O
2
> MPA > Asc > GSH for complex 1, H
2
O
2
> -
for
2 2
O
2
O
2
> MPA > GSH ꢃ Asc for complex 4. When the
hydroxyl radical scavenger, DMSO (Figs. 8 and S3, Lane 6) and EtOH
Fig. 5. CD spectrum of CT-DNA in absence and presence of complexes 1–4.
(Lane 7) were added to the complexes 1–4, the nuclease activity was

F. Arjmand et al. / Journal of Photochemistry and Photobiology B: Biology 121 (2013) 75–85
83
1
.0
Complex 1
Complex 2
Complex 3
Complex 4
0
.5
Fig. 8. (a) Agarose gel electrophoresis pattern for the cleavage pattern of pBR322
plasmid DNA (300 ng) by complex 1 (50 M) in the presence of different activating
agents at 310 K after incubation for 30 min. Lane 1, DNA control; Lane 2,
DNA + 1 + H (0.4 M); Lane 3, DNA + 1 + MPA (0.4 M); Lane 4, DNA + 1 + GSH
0.4 M); Lane 5, DNA + 1 + Asc (0.4 M); Lane 6, DNA + 1 + DMSO (0.4 M); Lane 7,
DNA + 1 + EtOH (0.4 M); Lane 8, DNA + 1 + NaN (0.4 M); Lane 9, DNA + 1 + Super-
oxide Dismutase (15 Units); (b) Agarose gel electrophoresis pattern for the cleavage
pattern of pBR322 plasmid DNA (300 ng) by complex 2 (50 M) in the presence of
different activating agents at 310 K after incubation for 30 min. Lane 1, DNA control;
Lane 2, DNA + 2 + H O2 (0.4 M); Lane 3, DNA + 2 + MPA (0.4 M); Lane 4,
M); Lane 5, DNA + 2 + Asc (0.4 M); Lane 6, DNA + 2 + DMSO
M); Lane 7, DNA + 2 + EtOH (0.4 M); Lane 8, DNA + 2 + NaN (0.4 M); Lane 9,
DNA + 2 + Superoxide Dismutase (15 Units).
l
2
O
2
l
l
(
l
l
l
l
3
l
1
.0
1.5
2.0
2.5
1
/R = [Complex] / [DNA]
l
Fig. 6. Effect of increasing amounts of complexes 1, 2, 3 and 4 on the relative
viscosity ( ) of DNA in Tris HCl buffer (pH 7.2).
l
l
2
g
/
g
0
DNA + 2 + GSH (0.4
0.4
l
l
(
l
l
3
l
2 2
further reacts with H O via Fenton mechanism to generate a hy-
droxyl radical species that can bind to the metal ion [48]:
CuðIIÞcomplex þ DNA ! CuðIIÞ ꢁ DNA
CuðIIÞ ꢁ DNA ! CuðIÞ ꢁ DNA
Å
Å
CuðIÞ ꢁ DNA þ H
2
O
2
! CuðIIÞð OHÞ ꢁ DNA þ OH
The Cu-peptide complexes could permit molecular oxygen to
generate an -carbon stabilized radical from the C-terminal amino
a
acid present in the peptidic structure. Later, a redox reaction be-
tween this radical and the nearby Cu(II) would yield Cu(I) which
further cleaves the DNA [49].
In the complexes 1–4 the DNA cleavage is inhibited remarkably
with major groove binding agent, methyl green (Figs. 9 and S4,
Lane 2). However, no apparent inhibition of DNA damage was ob-
served in presence of DAPI, minor groove binding agent (Fig. 9,
Lane 3). This suggests major groove preference for the complexes.
This is in support of the findings from UV–vis absorption titration
that the complexes can form hydrogen bonds via amine group of
Fig. 7. (a) The cleavage patterns of the agarose gel electrophoresis for pBR322
plasmid DNA (300 ng) by 1 at 310 K after 30 min of incubation in buffer (5 mM Tris
HCl/50 mM NaCl, pH = 7.2), Lane 1, DNA control; Lane 2, DNA + 1 (10
l
M); Lane 3,
M); Lane 6,
M); (b) The cleavage patterns of the agarose gel electrophoresis for
DNA + 1 (20
DNA + 1 (50
lM); Lane 4, DNA + 1 (30 lM); Lane 5, DNA + 1 (40 l
l
pBR322 plasmid DNA (300 ng) by 2 at 310 K after 30 min of incubation in buffer
(
(
(
5 mM Tris HCl/50 mM NaCl, pH = 7.2), Lane 1, DNA control; Lane 2, DNA + 2
10
40
l
l
M); Lane 3, DNA + 2 (20
M); Lane 6, DNA + 2 (50
l
M);
M); Lane 4, DNA + 2 (30
lM); Lane 5, DNA + 2
l
ꢁ
diminished indicating the involvement of OH radicals in the cleav-
ꢁ
age process. These OH radicals participate in the oxidation of the
deoxyribose moiety, followed by hydrolytic cleavage of the sugar
phosphate backbone. Further, the addition of singlet oxygen scaven-
ger, NaN to the reaction mixture scarcely inhibited the DNA cleav-
3
age (Lane 8) suggesting non-involvement of singlet oxygen as one of
the ROS responsible for DNA breakage. In the case of SOD (superox-
ide radical scavenger) (Lane 9) the form II of the plasmid DNA was
converted to form III indicating two subsequent and proximate sin-
gle strand breaks of DNA non-randomly. The result demonstrated
that superoxide radical is not responsible for DNA scission. The pep-
tide moiety can also have an active role in the reduction of Cu(II).
Based on the above results, it could be concluded that the hydroxyl
radicals are responsible for the DNA cleavage. These OH radicals
participate in the oxidation of the deoxyribose moiety, followed
by hydrolytic cleavage of the sugar phosphate backbone.
The initial step of the possible reaction mechanisms of DNA
damage by Cu(II) complexes in the presence of hydrogen peroxide
is the interaction of Cu(II) complex with the outer sphere of DNA
strand. The second step is the reduction of Cu(II) complex to
Cu(I) complex by reducing agent, then the Cu(I)–DNA adduct
ꢁ
Fig. 9. (a) The cleavage patterns of the agarose gel electrophoresis for pBR322
plasmid DNA (300 ng) by 1 in the presence of DNA major groove binding agent
methyl green and minor groove binding agent, DAPI at 310 K after 30 min of
incubation in buffer (5 mM Tris HCl/50 mM NaCl, pH = 7.2), Lane 1, DNA control;
Lane 2, DNA + 1 + Methyl green; Lane 3, DNA + 1 + DAPI; (b) The cleavage patterns
of the agarose gel electrophoresis for pBR322 plasmid DNA (300 ng) by 2 in the
presence of DNA major groove binding agent, methyl green and minor groove
binding agent DAPI at 310 K after 30 min of incubation in buffer (5 mM Tris HCl/
50 mM NaCl, pH = 7.2), Lane 1, DNA control; Lane 2, DNA + 2 + Methyl green; Lane
3, DNA + 2 + DAPI.

8
4
F. Arjmand et al. / Journal of Photochemistry and Photobiology B: Biology 121 (2013) 75–85
Table 3
Summary of the screening data of 1 against different tumor cells.
Therefore, in vitro antitumor activity of 1 was evaluated by SRB
assay, which exhibited moderate results against A498 (Renal Cell)
and A2780 (Ovary). The studies revealed that the peptide com-
plexes exhibited apoptotic activity in addition to DNA binding
property. The design of peptide complexes could fulfill the criteria
for efficacious metal-based drugs exhibiting reduced systemic tox-
icity and base specificity towards the cancerous cells. These metal–
peptide complexes warrant their future as potential cancer chemo-
therapeutic agents.
Cell line A498 A549 Zr-75-1 HT29
A2780 SiHa MCF7
GI50
1
ADR
1
ADR
1
ADR
29.9
<10
56.5
17.4
>80
67.6
<10
122.2
58.0
176.8
116.4
>80
<10
>80
54.8
>80
>80
<10
>80
29.6
>80
31.2
<10
62.7
18.4
>80
77.9
<10
>80
49.5
>80
>80
>80
<10
>80
25.9
>80
60.1
TGI
LC50
41.4
107.3 57.2
57.1
GI50 = Growth inhibition of 50% (GI50) calculated from [(Ti–Tz)/(C–Tz)] ꢂ 100 = 50,
drug concentration result in a 50% reduction in the net protein increase.
GI50 value < 10 lg/mL is considered to demonstrate activity.
Acknowledgments
The authors are thankful to Sophisticated Test and Instrumenta-
tion Centre, Cochin University, Cochin, India for providing elemen-
tal analysis, Sophisticated Analytical Instrumentation Facility,
Panjab University, Chandigarh, India for providing NMR spectra,
Regional Sophisticated Instrumentation Center, Central Drug Re-
search Institute, Lucknow, India for providing ESI–MS, Advanced
instrumentation research facility, Jawaharlal Nehru University,
Delhi, India for providing CD facility and Advanced Centre for
Treatment, Research and Education in Cancer, Kharghar, Navi
Mumbai for carrying out antitumor activity.
peptide moiety with N-3 of adenine or O-2 of thymine in the DNA
at the major groove. Major groove binding is particularly attractive
because in biological systems the sequence specific code recogni-
tion mostly occurs by interaction between the DNA major groove
and specific protein surface motifs. The major groove offers a num-
ber of potential H-bond interaction sites to which protein motifs
can selectively bind.
5
. Antitumor activity
In vitro antitumor activity of 1 in terms of GI50 (concentration of
drug required to decrease the cell growth to 50%, compared with
that of the untreated cell number), TGI (concentration of drug
required to decrease the cell growth to 100%, compared with that
of the untreated cell number during drug incubation) and LC50
concentration of drug required to decrease the cell growth by
0% of the initial cell number prior to the drug incubation) values
were tested against A498 (Renal cell), A549 (Lung), Zr-75-1
Breast), HT29 (Colon adenocarcinoma grade II cell line), A2780
Ovary), SiHa (Uterine cervix) and MCF7 (Human breast) by SRB as-
say. Complex 1 exhibited moderate results against A498 (Renal
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jphotobiol.2012.
12.009.
(
5
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