Synthesis and characterization of novel square planar copper(II)-dipeptide- 1,10-phenanthroline complexes: Investigation of their DNA binding and cleavage properties

Article abstract of DOI:10.1016/j.poly.2012.05.046

In view of the importance of aromatic moieties in DNA binding and cleavage, two new Cu(II)-dipeptide-phen (phen = 1,10-phenanthroline) complexes, viz. [Cu(II)(boc(tos)-his-trp-ome)(phen)](ClO₄)₂ (1) and [Cu(II)(boc(tos)-his-tyr-ome)(phen)](ClO₄)₂ (2) were synthesized, comprehensively characterized and their structures reported. The physico-chemical data and molecular modeling approaches support that the complexes are arranged in a distorted square planar geometry. To provide an insight on the mode and affinity of the interaction of these complexes with calf thymus (CT) DNA the following experiments were carried out: thermal denaturation (T_m), UV-Vis absorption, competitive binding, viscosity and fluorescence spectroscopy studies. These experimental results indicate that the complexes interact through stacking between the base pairs of double helix DNA. The overall binding constants (K_b) were determined as 3.33 × 10⁴ and 2.82 × 10⁴ M^-1 for 1 and 2 respectively. The complexes converted supercoiled plasmid pUC19 DNA (SC DNA) into its nicked (NC) form both in the absence (hydrolytic cleavage) and presence (oxidative cleavage) of H₂O₂. The control experiments demonstrate that no diffusible radical species were involved in the hydrolytic cleavage, but in the presence of H₂O₂, hydroxyl radical (OH) species were generated and initiated the cleavage reaction by an oxidative pathway. The rate constants for the hydrolysis of the phosphodiester bond were determined as 1.70 h^-1 and 1.82 h^-1 for 1 and 2 respectively. This amounts to a (4.7-5.0) × 10⁷-fold rate enhancement compared to non-catalyzed DNA cleavage, which is significant. These complexes exhibit better DNA binding and cleavage abilities compared to similar reported complexes.

Full text of DOI:10.1016/j.poly.2012.05.046

Polyhedron 44 (2012) 1–10
Contents lists available at SciVerse ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Synthesis and characterization of novel square planar
copper(II)–dipeptide–1,10-phenanthroline complexes: Investigation
of their DNA binding and cleavage properties
⇑
Pulimamidi Rabindra Reddy , Nomula Raju
Department of Chemistry, Osmania University, Hyderabad 500 007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In view of the importance of aromatic moieties in DNA binding and cleavage, two new Cu(II)–dipeptide–
phen (phen = 1,10-phenanthroline) complexes, viz. [Cu(II)(boc(tos)-his-trp-ome)(phen)](ClO4)2 (1) and
[Cu(II)(boc(tos)-his-tyr-ome)(phen)](ClO4)2 (2) were synthesized, comprehensively characterized and
their structures reported. The physico-chemical data and molecular modeling approaches support that
the complexes are arranged in a distorted square planar geometry. To provide an insight on the mode
and affinity of the interaction of these complexes with calf thymus (CT) DNA the following experiments
were carried out: thermal denaturation (Tm), UV–Vis absorption, competitive binding, viscosity and fluo-
rescence spectroscopy studies. These experimental results indicate that the complexes interact through
stacking between the base pairs of double helix DNA. The overall binding constants (Kb) were determined
as 3.33 ꢀ 104 and 2.82 ꢀ 104 Mꢁ1 for 1 and 2 respectively. The complexes converted supercoiled plasmid
pUC19 DNA (SC DNA) into its nicked (NC) form both in the absence (hydrolytic cleavage) and presence
(oxidative cleavage) of H2O2. The control experiments demonstrate that no diffusible radical species were
involved in the hydrolytic cleavage, but in the presence of H2O2, hydroxyl radical (OHꢀ) species were gen-
erated and initiated the cleavage reaction by an oxidative pathway. The rate constants for the hydrolysis
of the phosphodiester bond were determined as 1.70 hꢁ1 and 1.82 hꢁ1 for 1 and 2 respectively. This
amounts to a (4.7–5.0) ꢀ 107-fold rate enhancement compared to non-catalyzed DNA cleavage, which
is significant. These complexes exhibit better DNA binding and cleavage abilities compared to similar
reported complexes.
Received 16 March 2012
Accepted 22 May 2012
Available online 28 June 2012
Keywords:
Bioinorganic chemistry
Cu(II) complexes
DNA binding
Hydrolytic cleavage
Oxidative cleavage
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
to DNA, thereby cleaving the DNA and inhibiting the growth of
tumor cells, which is the basis for designing new and more efficient
The design of artificial nucleases represents an area of substan-
tial interest since nucleases have wider applications in molecular
biology in probing the structures and functions of nucleic acids
[1–4]. In contrast to natural DNA-binding enzymes, low molecular
artificial nucleases exhibit higher stability and less reactive condi-
tion dependence when they are used as an accelerator in DNA
cleavage [5,6].
antitumor drugs, and their effectiveness depends on the mode and
affinity of the binding [7–9]. Transition metal complexes that are
capable of cleaving DNA under physiological conditions are of
interest in the development of metal-based anticancer agents
[10–12]. The clinical success of cisplatin and related platinum-
based drugs, as anticancer agents that bind covalently to DNA, is
severely affected by the serious side effects, general toxicity, and
acquired drug resistance [13]. This is impetus to chemists to devel-
op innovative strategies for the preparation of more effective, less
toxic, target specific and preferably non-covalently bound antican-
cer drugs. In this regard, Cu(II) complexes are proving to be the
most promising alternatives to cisplatin as anticancer drugs.
Planar heterocyclic base complexes have been at the forefront
of these investigations because of their unusual electronic proper-
ties, diverse chemical reactivity, and peculiar structure, which
results in non-covalent interactions with DNA. Copper, being a bio-
essential transition metal ion, and its complexes, with tunable
coordination geometries in a redox active environment, could find
Many studies suggest that DNA is an important primary cellular
receptor. Many chemicals exert their antitumor effects by binding
Abbreviations: EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide); HOBt,
hydroxybenzotriazole; TFA, trifluoroacetic acid; His-trp, histidyl-tryptophan; His-
tyr, histidyl-tyrosine; phen, 1,10-phenathroline; CT-DNA, calf-thymus DNA; Tris,
Tris(hydroxymethyl)aminomethane; EB, ethidium bromide; UV–Vis, ultraviolet–
visible; DMSO, dimethyl sulfoxide; 1, [Cu(II)(boc(tos)-his-trp-ome)(phen)](ClO4)2;
2, [Cu(II)(boc(tos)-his-tyr-ome)(phen)](ClO4)2.
⇑
Corresponding author. Tel.: +91 40 27171664; fax: +91 40 27090020,
27097099.
E-mail address: profprreddy@gmail.com (P.R. Reddy).
0277-5387/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.poly.2012.05.046

2
P.R. Reddy, N. Raju / Polyhedron 44 (2012) 1–10
better applications at the cellular level. Amino acids/peptides are
the basic structural units of proteins that recognize a specific base
sequence of DNA. An amino acid with a side chain aromatic ring,
e.g. phenylalanine, tryptophan, tyrosine, etc., contributes mainly
to the stabilization of proteins through hydrophobic interactions
and the formation of hydrophilic environments [14,15].
In earlier publication [16], we isolated copper complexes of
histidine containing dipeptides with aliphatic side chains, i.e.
[CuII(HisLeu)(phen)]+ and [CuII(HisSer)(phen)]+, and studied their
DNA binding and cleavage properties. Since aromatic moieties are
known to play an important role in enhancing DNA binding and
cleavage activity, we extended our study to dipeptides with aro-
matic side chains. Here we report the synthesis, characterization,
DNA binding, hydrolytic and oxidative DNA cleavage activity of
two new complexes, [Cu(II)(boc(tos)-his-trp-ome)(phen)](ClO4)2
(1) and [Cu(II)(boc(tos)-his-tyr-ome)(phen)](ClO4)2 (2). Compared
to aliphatic dipeptide complexes, the aromatic dipeptide complexes
show better DNA binding and cleavage activity. This reveals the
importance of aromatic moieties in DNA binding and cleavage.
[boc(tos)-his-trp-ome]: 1H NMR (400 MHz, CDCl3), d: 1.40 (s,
9H), 2.34 (m, 3H), 2.93–3.90 (s, 7H), 4.40–4.95 (m, 2H), 7.10–7.85
(m, 10H), 8.03 (m, 2H), 8.05 (m, 1H), 10.10 (m, 1H); ESI-MS, m/z:
609.
[boc(tos)-his-tyr-ome]: 1H NMR (400 MHz, CDCl3), d: 1.40 (s, 9H),
2.34 (m, 3H), 2.80–3.90 (s, 7H), 5.30 (m, 1H), 4.40–4.95 (m, 2H),
6.65–7.85 (m, 9H), 8.03 (m, 2H), 8.05 (m, 1H); ESI-MS, m/z: 586.
2.2.2. [Cu(II)(boc(tos)-his-trp-ome)(phen)](ClO4)2 (1) and
[Cu(II)(boc(tos)-his-tyr-ome)(phen)] (ClO4)2 (2)
To a methanolic solution of boc-(tos)his-trp-ome (0. 609 g,
1.0 mM) for 1 or boc-(tos)his-tyr-ome (0.586 g, 1.0 mM) for 2, a
methanolic solution of Cu(OAc)2ꢂH2O (0.198 g, 1.0 mM) was added
under stirring, followed by the addition of phen (0.198 g, 1.0 mM)
in MeOH (5 mL). The reaction was continued for ca. 6 h at room
temperature and then an aq. solution of NaClO4ꢂH2O (0.36 g,
2.0 mM) was added. A yellow precipitate was isolated and it was
filtered and washed with ether, and then dried.
[1]: Anal. Calc. for C42H43Cl2CuN7O15S: C, 47.94; H, 4.12; N, 9.32;
Cu, 6.04. Found: C, 47.89; H, 4.10; N, 9.28; Cu, 6.00%. IR (KBr,
cmꢁ1): 3340br, 2926m, 1711m, 1665s, 1585s, 1518s, 1428s,
1366m, 1222m, 1166s, 1107m, 1034m, 1010s, 847s, 818m, 722s,
681m, 568m, 429s. UV–Vis [k nm; MeOH:H2O (1:10)]: 550, 395,
2. Experimental
2.1. Materials and instruments
269, 212. ESI-MS m/z: 853 [M+H]+. MP: 250 °C.
leff = 1.78 BM.
KM [Oꢁ1 cm2 Mꢁ1, 10ꢁ3 M in MeOH:H2O (1:10), 25 °C]: 120. Yield:
ꢃ85%.
EDCI, HOBt, DIPEA, TFA, LiOHꢂH2O, boc(tos)-histidine, trypto-
phan methyl ester, tyrosine methyl ester, Cu(OAc)2ꢂH2O, acetic
acid, EDTA, phen and ethidium bromide (EB) were obtained from
Sigma (99.99% purity) USA and were of analar grade. The peptides
(boc(tos)-his-trp-ome and boc(tos)-his-tyr-ome) were synthesized
by the conventional solution phase method [17]. CT-DNA was
obtained from Fluka (Switzerland). Supercoiled plasmid pUC19
DNA, agarose, Tris–HCl and Tris base were obtained from Banga-
lore Genei (India). Solvents (MeOH, CH2Cl2 and THF) were pur-
chased from Merck, India and all the chemicals were used as
supplied. The spectroscopic titrations were carried out in aerated
buffer (5 mM Tris–HCl/50 mM NaCl, pH 7.5) at room temperature
(r.t.).
[2]: Anal. Calc. for C40H42Cl2CuN6O16S: C, 46.67; H, 4.11; N, 8.16;
Cu, 6.17. Found: C, 46.63; H, 4.08; N, 8.12; Cu, 6.10%. IR (KBr,
cmꢁ1): 3349br, 2926s, 1665s, 1597m, 1502m, 1426s, 1365m,
1244m, 1176m, 1092m, 955m, 867s, 745s, 723m, 553m, 429s.
UV–Vis [k nm; MeOH:H2O (1:10)]: 540, 394, 268, 204. ESI-MS m/
z: 830 [M+H]+. MP: 265 °C;
l ,
eff = 1.76 BM. KM [Oꢁ1 cm2 Mꢁ1
10ꢁ3 M in MeOH:H2O (1:10), 25 °C]: 150. Yield: ꢃ85%.
2.3. DNA binding
2.3.1. Preparation of stock solution
Elemental analyses data were obtained from the microanalytical
Heraeus Carlo Erba 1108 elemental analyzer. The copper content
was determined on a Shimadzu AA-6300 atomic absorption spec-
trophotometer. The molar conductivity was measured on a Digisun
digital conductivity bridge (model: DI-909) with a dip type cell.
Infrared spectra were recorded on a Perkin Elmer FT-IR spectrome-
ter, in KBr pellets in the 4000–400 cmꢁ1 range. Magnetic moments
of the complexes were recorded at room temperature on a Faraday
balance (CAHN-7600) using Hg[Co(NCS)4] as the standard. Diamag-
netic corrections were made using Pascal’s constants [18]. ESI mass
spectra of the complexes were recorded using a Quattro Lc (Micro
Mass, Manchester, UK) triple quadruple mass spectrometer with
MassLynx software. UV–Vis spectra of the complexes were recorded
on a Jasco V-530 UV–Vis spectrophotometer using 1 cm quartz
micro-cuvettes. TGA analyses were obtained by a METLER TOLEDO
(TGA/SDTA 851e) thermo analyzer at 25–300 °C, with a heating rate
of 5 °C/min. The molecular modeling calculations were carried out
with a semi-empirical PM3 Hamiltonian as implemented in the
hyperchem 6.0 software program package. The DNA cleavage exper-
iments conducted using Genei (India) gel-electrophoresis equip-
ment. The gel pattern of the electrophoresis was photographed by
the Alpha-Innotec gel documentation system (USA).
Concentrated CT-DNA stock solution was prepared in 5 mM
Tris–HCl/50 mM NaCl in double distilled water at pH 7.5 and the
concentration of the DNA solution was determined by UV absor-
bance at 260 nm (e
= 6600 Mꢁ1 cmꢁ1) [19]. A solution of CT-DNA
in 5 mM Tris–HCl/50 mM NaCl (pH 7.5) gave a ratio of UV absorp-
tions at 260 and 280 nm A260/A280 of ca. 1.8–1.9, indicating that the
DNA was sufficiently free of protein [20]. All stock solutions were
stored at 4 °C and were used within 4 days. The concentration of
EB was determined spectrophotometrically at 480 nm (
e = 5680
Mꢁ1 cmꢁ1) [21].
2.3.2. Thermal denaturation (Tm) studies
Thermal denaturation studies were performed on a Shimadzu
160A spectrophotometer equipped with a thermostatic cell holder.
The Tm value of CT-DNA was determined in the absence and pres-
ence of Cu(II) complexes by keeping the concentration of DNA and
complexes 1 and 2 (30 lM) in a 1:1 ratio. The DNA samples were
continuously heated at the rate of 1 °C/min temperature increase,
while the absorption changes at 260 nm were monitored. The
melting temperature (Tm), which is defined as the temperature
where half of the total base pairs are unbound, was determined
from the midpoint of the melting curves.
DTm values were calcu-
lated by subtracting Tm of free DNA from Tm of DNA interacting
2.2. Synthesis of the peptides and Cu(II) complexes
with the complex [22].
2.2.1. boc(tos)his-trp-ome and boc(tos)his-tyr-ome
2.3.3. UV–Vis absorption spectroscopy
The dipeptides (boc(tos)-his-trp-ome and boc(tos)-his-tyr-ome)
were synthesized by adopting EDCI and HOBt as coupling agents in
the presence of DIPEA and with dry CH2Cl2 as the solvent.
Absorption spectra were recorded on Jasco V-530 UV–Vis spec-
trophotometer using 1 cm quartz micro-cuvettes. Absorption titra-
tions were performed by keeping the concentration of the

P.R. Reddy, N. Raju / Polyhedron 44 (2012) 1–10
3
complexes constant (10
CT-DNA from 0 to 10
l
M), and by varying the concentration of
the absence ([1 and 2] = 50, 100 and 150
and 2] = 5 and 10 lM) of 1 mM H2O2, dissolved in 5 mM Tris buffer
l
M) and presence ([1
l
M. In the reference cell, a DNA blank was
placed so as to cancel any absorbance due to DNA at the measured
wavelength. For complexes 1 and 2, the binding constants (Kb)
were determined from the spectroscopic titration data using the
following equation [23,24]:
at pH 7.2. The mixtures were incubated at 37 °C for a period of 3 h.
The reactions were quenched and the resulting solutions were
subjected to electrophoresis. The analysis involved loading of the
solutions onto 1% agarose gels containing 2.5 lM EB (2 lL), and
the DNA fragments separated by gel electrophoresis (60 V for 2 h
in standard Tris–acetate–EDTA (TAE) buffer, pH 8). EB-stained aga-
rose gels were imaged, and densitometric analysis of the visualized
bands was used to determine the extent of supercoiled DNA
cleavage.
Control experiments were carried out using Cu(OAc)2ꢂH2O and
the free ligands at a concentration of 1 mM. For investigation of
the mechanistic aspects, the cleavage of SC DNA was also carried
out in the presence of 1 mM DMSO, a standard hydroxyl radical
scavenger. The reaction conditions used were Cu(II) complex
½DNAꢄ=ðea
The ‘apparent’ extinction coefficient (
ꢁ
ef Þ ¼ ½DNAꢄ=ðeb
ꢁ
ef Þ þ 1=Kbðeb
ꢁ
ef
Þ
ð1Þ
e
a) was obtained by calculat-
ing Aobsd/[Cu]. The terms f and b correspond to the extinction coef-
ficients of free (unbound) and the fully bound complexes,
respectively. A plot of [DNA]/(ea f) versus [DNA] will give a slope
of 1/(eb f) and an intercept of 1/Kb(eb f). Kb is the ratio of the
e
e
ꢁ
e
ꢁ
e
ꢁ
e
slope to the intercept.
2.3.4. Competitive binding
The competitive binding experiments were performed on a
Jasco V-530 UV–Vis spectrophotometer using 1 cm quartz micro-
cuvettes. Absorption titrations were performed for complexes 1
[150
l
M for hydrolytic and 10
lM + 1 mM H2O2 for oxidative
cleavage] and 38
plasmid DNA. Each solution was incubated at 37 °C for 3 h and
lM
base-pair concentration of supercoiled
and 2 by keeping the concentration of EB (40
(40 M) constant, and by varying the complex concentration from
0 to 40 M.
lM) and DNA
analyzed according to the procedure described above. For evaluat-
ing kinetic aspects, the complex concentration was fixed at 300 lM
with different incubation times (0–120 min) under identical exper-
l
l
imental conditions.
2.3.5. Viscosity
Viscometric titrations were performed with an Ostwald
Viscometer at room temperature for EB (standard), 1 and 2. The
3. Results and discussion
concentration of DNA was 200
tions were varied from 0 to 200
l
M, the EB and complex concentra-
M and the flow times were
3.1. Synthesis and solubility
l
measured with a digital timer. Each sample was measured three
The primary dipeptide ligands were synthesized by a conven-
tional solution phase method [17] in DCM in the presence of EDCI
and HOBt as coupling agents, and their ternary Cu(II) complexes (1
and 2) were isolated on reaction with Cu(II) acetate monohydrate
in methanol, with phen as a secondary ligand (Scheme 1). The
complexes show good solubility in MeOH/EtOH:H2O (1:10)
mixture. They are non-hygroscopic and are stable in both solid
and solution phases. The analytical data for the Cu(II) complexes
were in good agreement with the molecular formula of the com-
plexes. The molar conductance values (Table 1) are in the range
for 1:2 electrolytes, no further dissociation of the complex species
([Cu(II)(boc(tos)-his-trp-ome)(phen)]2+ and [Cu(II)(boc(tos)-his-
tyr-ome)(phen)]2+ was observed. The observed magnetic moment
times for accuracy, and an average flow time was determined. Data
1/3
is presented as (
g/go)
versus [complex]/[DNA], where g is the
viscosity of DNA in the presence of complex and go that of DNA
alone. Viscosity values were calculated from the observed flow
time of DNA containing solutions (t) corrected for that buffer alone
(to),
g
= (t ꢁ to).
2.3.6. Fluorescence spectroscopy
Fluorescence spectra were recorded with a SPEX-Fluorolog
0.22 m fluorimeter equipped with a 450 W Xenon lamp. The slit
widths were 2 ꢀ 2 ꢀ 2 ꢀ 2 and the emission spectral range was
550–650 nm. A solution containing DNA and EB was titrated with
varying concentrations of 1 and 2. The concentration of DNA and
values (leff = 1.74 and 1.70 BM for 1 and 2 respectively) are in
the range for a one unpaired electron system with spin (1/2), which
represents the paramagnetic nature of the Cu(II) complexes [26].
EB was maintained at 41
was in the range 0–196
l
M and the concentration of complexes
l
M. The solutions containing DNA and EB
were mixed (equimolar ratio) and the complexes 1 and 2 were added
and allowedto equilibrate. They were excitedat 540 nm (kmax for EB)
and the fluorescence emission at 598 nm (kmax) was recorded.
Fluorescence spectra were also utilized to obtain Scatchard
plots. For this, titrations of DNA against EB in the absence and pres-
ence of the complexes were performed. The initial concentration of
3.2. Characterization of the complexes
3.2.1. IR spectra
The assignments of the infrared spectra were made on the basis
of literature and Nakamoto [27]. The IR spectra of 1 and 2 were
DNA was 20
lM and the concentrations of 1 and 2 were kept at
50 M. After each addition of EB to the solutions containing DNA
l
analyzed in comparison with their free ligand spectra. The m(N–
and the complexes, the emission spectra were recorded in the
range 550–650 nm with excitation at 540 nm at 25 °C. Corrections
were made to the data for the volume changes during the course of
the titrations. The data were analyzed by the method of Lepecq and
Paoletti [25] to obtain the bound (cb) and free (cf) concentration of
EB, and Scatchard plots were obtained by plotting rEB/cf versus rEB
(where r = cb/[DNA]).
H) vibration of the free peptides, observed at 3301 cmꢁ1 for
his-trp and 3390 cmꢁ1 for his-tyr (Fig. S1), were shifted to
3340 cmꢁ1 for 1 and 3381 cmꢁ1 for 2 upon complexation with cop-
per (Fig. S2). Similarly the imidazole m
(C@N) peak at 1674 cmꢁ1
was shifted to 1665 cmꢁ1 on interaction with Cu(II) in both
complexes, which can be attributed to the coordination of nitrogen
atoms of the amine and imino groups to copper. The peaks corre-
sponding to the free phen ring stretching frequencies m(C@C) and
2.4. DNA cleavage
m
(C@N), observed at 1502 and 1421 cmꢁ1, were shifted to 1518
and 1428 cmꢁ1 respectively, and the low energy pyridine ring
characteristic in plane and out-of-plane hydrogen bending modes
of free phen, observed at 853 and 738 cmꢁ1, were shifted to 848
and 722 cmꢁ1 respectively for 1 and 2, which is a good indication
of the coordination of the heterocyclic nitrogen to copper [27].
Electrophoresis experiments were performed using supercoiled
pUC19 plasmid DNA according to the established procedures. The
cleavage of supercoiled (SC) DNA (38
lM base pair concentration)
was accomplished by the addition of Cu(II) complexes 1 and 2 in

4
P.R. Reddy, N. Raju / Polyhedron 44 (2012) 1–10
Scheme 1. General approach for the synthesis of peptides and the Cu(II) complexes 1 and 2.
Table 1
Physico-chemical data for complexes 1 and 2.
Complex IR (cmꢁ1
)
leff ESI- UV–Vis KM
MP (°C)
(BM) MS (nm)
(Oꢁ1 cm2
m
(N–H)
m(C@N) m(M–N)
Mꢁ1
)
1
2
3340
3381
1674
1674
568
553
1.78 853 550, 395, 120
269, 212
1.76 830 540, 394, 150
268, 204
250
265
The very strong single band at 1086 cmꢁ1 for 1 and 1094 cmꢁ1 for 2
were assigned to (Cl–O) of perchlorate anions, satisfying the Wer-
m
ner’s primary valence of the complexes [28]. The new non-ligand
peaks at 568 cmꢁ1 for 1 and 553 cmꢁ1 for 2 were assigned to
m(Cu–N) vibrations [29], which also indicates that the ligands are
coordinated to copper through nitrogen atoms.
3.2.2. Electronic spectra
In the electronic spectra of the Cu(II) complexes, four absorption
bands with varied intensities were observed for 1 (Fig. 1) and 2
(Fig. S3) in MeOH:H2O solution. The intense bands at 212 and
269 nm for 1 and 206 and 268 nm for 2 were assigned to intra-li-
Fig. 1. Electronic absorption spectrum of 1. Insets: d–d transition.
gand (p–
p⁄) transitions of the phen ligand, while the less intense
band at 395 nm for 1 and 2 was assigned to LMCT transitions. Addi-
tionally, much weaker, less well defined broad bands observed in
the lower energy regions at 550 and 540 nm for 1 and 2 respectively
for 2 respectively (Fig. S5). These molecular ion species are respon-
sible for all the properties shown by the complexes.
were assigned to d–d [2B1g ? 2Eg ꢁ (
m1)(dx2ꢁy2 ? dyz)] transitions.
These transitions represent a distorted square-planar geometry.
This agrees well with the reported square-planar Cu(II) complexes
[30].
3.2.4. Thermo gravimetric analysis (TGA)
Normally, the amide group of histidine containing dipeptide
ligands is not involved in metal coordination, especially at physio-
logical pH. However, it is possible to have a five coordination
geometry around copper with one water (H2O) molecule in the
coordination sphere. To examine the presence of water molecules
in the present complexes, they were subjected to thermo gravimet-
ric analysis (TGA).
3.2.3. Mass spectra
The active chemical species of the complexes were identified by
means of electrospray ionization mass spectrometry (ESI-MS). The
mass to charge ratio peaks observed at m/z 853 and 830 were due
to the formation of the species [Cu(II)(boc(tos)-his-trp-ome)
(phen)]2+ for 1 (Fig. S4) and [Cu(II)(boc(tos)-his-tyr-ome)(phen)]2+
Water molecules present in the crystal lattices of complexes are
generally of two types – lattice water (non-coordinated to the

P.R. Reddy, N. Raju / Polyhedron 44 (2012) 1–10
5
Fig. 2. Proposed geometry and energy-minimized molecular structures, relative energies and bond lengths of 1 and 2.
metal ion) and coordinated water. The lattice water will be lost at
3.3. DNA binding
low temperatures (90–150 °C), whereas the loss of coordinated
water molecule is observed at high temperatures (150–250 °C).
The thermal decomposition stoichiometry of the analytically char-
acterized complexes was determined by thermo gravimetric anal-
ysis (TGA). The thermal behavior of the complexes was monitored
up to 500 °C in a nitrogen atmosphere. In the case of complexes 1
(Fig. S6) and 2 (Fig. S7), no weight loss was observed in the above
mentioned temperature ranges. This suggests that there is no evi-
dence for the presence of water molecules in the complexes. Based
on the literature, electronic spectra and TGA data, a distorted
square-planar geometry was confirmed for the complexes. The
complexes decomposed above 350 °C.
The Cu(II) complexes can bind to the double stranded (ds) DNA
in different modes on the basis of their structure, charge and type
of ligands. To identify the mode of binding involved between the
complexes and ds-DNA, the biophysical methods of thermal dena-
turation, UV–Vis absorption spectra, competitive binding study,
viscosity, fluorescence spectroscopy and gel-electrophoresis were
adopted.
3.3.1. Thermal denaturation studies
DNA melting studies were used in predicting the nature of bind-
ing and relative binding strength of metal complexes to the DNA
[31]. The denaturation temperatures of free and copper complex-
bound CT-DNA were examined. The melting of the helix can lead
to an increase in the absorption at 260 nm, because the extinction
coefficient of DNA bases at 260 nm in the double helical form is
much less than in the single strand form [32–37]. Thus, the helix
to coil transition temperature Tm, can be determined by monitoring
the absorbance of the DNA bases at 260 nm as a function of tem-
perature. The interaction of small molecules with double-helical
DNA may increase or decrease Tm, the temperature at which the
double helix is broken up into a single-stranded DNA. While an
increase in Tm values suggests intercalative or phosphate binding,
a decrease is typical for base binding. The thermal denaturation
profile of DNA in the absence and presence of the complexes is pro-
vided in Fig. 3. As can be seen, an increase of ꢃ6 °C for 1 and 2 was
observed in the Tm profile of the complexes as compared to free
DNA. This increase in the helix melting temperature indicates the
3.2.5. Molecular modeling studies
In the absence of crystal data, it was thought worthwhile to
obtain structural information through molecular modeling. Molec-
ular mechanics, which provides the energy minimized conforma-
tion, is
a tool of increasing importance for the structural
investigation of coordination and organometallic compounds.
These were carried out with the semi-empirical PM3 Hamiltonian
as implemented in the hyperchem 6.0 software program package.
The ball and stick representations of complexes 1 and 2 are shown
in Fig. 2. As can be seen from the figure, the complexes are
arranged in a distorted square-planar geometry at their minimum
energy state. These results also support distorted square-planar
geometries for the present complexes. The relative minimum
energies and relevant bond lengths for the complexes are also
provided.

6
P.R. Reddy, N. Raju / Polyhedron 44 (2012) 1–10
Fig. 3. Thermal denaturation profiles of free CT-DNA (N) and in the presence of 1
(d) and 2 (j). [The concentrations of 1 or 2 and DNA were 30 M.]
Fig. 4. Absorption spectra of 1 in the absence (ꢂ ꢂ ꢂ) and presence (—) of increasing
amounts of CT-DNA. Conditions: [complex] = 10 M. The arrow (;) shows the
l
l
absorbance changes upon increasing the DNA concentration. Inset: linear plot for
the calculation of the intrinsic DNA binding constant (Kb).
increased stability of the double helix when these complexes bind
to DNA. This provides a clear evidence for intercalative or phos-
phate binding of the copper complexes with DNA.
affinity of 1 than 2 for intercalative DNA binding is related to the
presence of an extended aromatic indole ring of the coordinated
his-trp, making the non-covalent interaction of the
p system of
3.3.2. UV–Vis absorption spectroscopy
the ligand with the DNA base pair more favorable and intimate
than that of the phenyl ring of his-tyr in 2. A larger aromatic moi-
ety always facilitates potential intercalative DNA binding.
To further investigate the mode of binding between the
complexes and the DNA double helix, UV-absorption studies were
carried out. Monitoring the changes in the absorption spectra of
the metal complexes upon addition of increasing amounts of
DNA is one of the most widely used methods for determining the
overall binding constants. Any interaction between the complex
and DNA is expected to perturb the ligand centered spectral tran-
sitions of the complex. The absorption intensity of the complexes
may decrease (hypochromism) or increase (hyperchromism) with
a slight increase in absorption wavelength (bathochromism) in
the presence of DNA. In general, it is assumed that hypochromism
and bathochromism are related to association (intercalation) of the
complex species with DNA [38,39]. The absorption spectra of 1 in
the absence and presence of CT-DNA are illustrated in Fig. 4. On
addition of increasing amounts of DNA to the complexes, the hypo-
chromic (23%) (19% for 2, Fig. S8) along with minor bathochromic
shifts from 269–274 nm (268–272 nm for 2) were observed. The
hypochromism and bathochromism were suggested to arise due
to the interaction between the electronic state of an intercalating
chromophore (complex) and those of the DNA bases [40,41], i.e.
3.3.3. Competitive binding
Intercalative binding was also demonstrated through competi-
tive binding experiments by UV–Vis spectroscopy using EB, a
typical indicator of intercalation [42]. The study involves the addi-
tion of the complexes to DNA pre-treated with EB and subsequent
measurement of the intensity of absorption. Free EB absorbs
around 480 nm, but on interaction with DNA the absorption inten-
sity decreases and the peak maximum shifts to a higher wave-
length [43], which is characteristic of intercalation of EB into
DNA base stacks. Fig. 5 shows that the maximal absorption of EB
at 478 nm decreased and shifted to 481 nm in the presence of
DNA, due to the intercalation of EB into the DNA base pairs. On
addition of 1 in increasing amounts, a continuous increase in
absorption was observed and the peak maximum was restored to
its original position. This suggests that the complex displaces
DNA-bound EB and occupies the intercalation sites on DNA, similar
to that of EB. Similar trends were obtained for 2 (Fig. S9).
the p⁄ orbital of the intercalated ligand can couple with the
tal of the base pairs, thus decreasing the
p⁄ transition energy
and the resulting bathochromism. Moreover the coupling orbital
p orbi-
p–
p
3.3.4. Viscosity
is partially filled by electrons, thus decreasing the transition prob-
abilities and concomitantly resulting in hypochromism. The extent
of the hypochromism is commonly consistent with the strength of
the intercalative binding. The observed spectroscopic changes are
thus consistent with intercalation of 1 and 2 into the DNA base
stacks. In order to compare the binding strengths of the complexes,
the intrinsic binding constant (Kb) for the association of the
complexes with CT-DNA (insets of the respective figures) were
determined using Eq. (1) (see Section 2) as 3.33 ꢀ 104 Mꢁ1 and
2.82 ꢀ 104 Mꢁ1 for 1 and 2 respectively. A slightly higher binding
Optical or photo-physical properties are necessary but not
sufficient to establish the mode of binding between the metal
complexes and DNA. Hence viscosity measurements were carried
out to further verify the mode of interaction of the metal
complexes towards DNA. A hydrodynamic measurement, such as
viscosity, is sensitive to length change and is regarded as the least
ambiguous and the most critical test of a binding model in solution
in the absence of crystallographic structural data [44,45]. A classi-
cal intercalation model demands that the DNA helix must lengthen
as base pairs are separated to accommodate the binding ligand/

P.R. Reddy, N. Raju / Polyhedron 44 (2012) 1–10
7
Fig. 7. Emission spectra of EB bound to CT-DNA in the absence (ꢂ ꢂ ꢂ) and presence
(—) of complex 1. [1/DNA = 0, 0.8, 1.60, 2.41, 3.19, 3.97, 4.78, kex = 540 nm, Inset:
Stern–Volmer quenching curve.]
Fig. 5. Absorption spectra of free EB (ꢂ ꢂ ꢂ) and EB bound to CT-DNA in the absence
(---) and presence (—) of increasing amount of 1. Conditions: [EB] = 40 M,
[DNA] = 40 M, [1] = (0–40
M). The arrow (") shows the absorbance changes upon
increasing the complex concentration.
3.3.5. Fluorescence spectroscopy
l
Competitive binding experiments with
a well established
l
l
fluorescence quenching experiment based on the displacement of
the intercalating drug EB from CT-DNA may give further informa-
tion about the relative binding affinity of the complexes to
CT-DNA with respect to EB. EB is a classical intercalator that gives
significant fluorescence emission intensity when it intercalates
into the base pairs of DNA. When it is replaced or excluded from
the internal hydrophobic circumstance of the DNA double helix
by other small molecules, its fluorescence emission is effectively
quenched by external polar solvent molecules such as H2O [47].
The fluorescence quenching curves of EB bound to DNA in the ab-
sence and presence of 1 are given in Fig. 7.
As shown in Fig. 7, the EB–DNA system shows a characteristic
strong emission at about 598 nm when excited at 540 nm, indicat-
ing that the intercalated EB molecules have been successfully pro-
tected by the neighboring DNA base pairs from being quenched by
H2O. A remarkable reduction in emission intensity was observed as
1 was added to the EB–DNA system, characteristic for the interca-
lative binding of 1 with DNA. Similar trends were observed for 2
(Fig. S10). The quenching efficiency for each complex was evalu-
ated by the Stern–Volmer constant Ksq, which varies with the
experimental conditions [48]:
complex, leading to an increase of DNA viscosity. In contrast, a par-
tial intercalation of the ligand/complex could bend (or kink) the
DNA helix, reduce its effective length and concomitantly its viscos-
ity. The effect of EB, 1 and 2 on the viscosity of DNA at room tem-
perature is shown in Fig. 6. The viscosity of the DNA increases
steadily with increasing concentration of the complexes. This
behavior is comparable with EB, which increases the relative spe-
cific viscosity by lengthening the DNA double helix as a result of
intercalation. The results clearly emphasize that both the com-
plexes intercalate between adjacent DNA base pairs, causing an
extension in the helix and thereby increasing the viscosity of
DNA [46].
Io=I ¼ 1 þ Ksq:r
where Io and I are the fluorescence intensities in the absence and
presence of the complex, respectively, and r is the concentration
ratio of the complex to DNA. Ksq is the linear Stern–Volmer quench-
ing constant. The quenching plots illustrate that the quenching of
emission of the EB–DNA system by the complexes is in good agree-
ment with the linear Stern–Volmer equation, which also indicates
that the complexes bind to DNA. The Ksq value is obtained as the ra-
tio of the slope to intercept. The Ksq values for complexes 1 and 2
are 0.14 and 0.13 respectively (insets of the respective figures).
Fluorescence Scatchard plots for the binding of EB to CT-DNA in
the presence of the complexes were obtained as described by
Lepecq and Paoletti [25]. From the Scatchard plots, the complexes
can be classified as exhibiting Type A or Type B behavior. Type A
behavior produces a Scatchard plot in which the slope decreases
in the presence of increasing amounts of metal complex, with no
change in the intercept on the abscissa, indicating competitive
inhibition of EB binding, viz. intercalation [49,50]. Type B behavior
Fig. 6. Effect of increasing amounts of EB (N), 1 (d) and 2 (j) on the relative
viscosities of CT-DNA at room temperature in Tris–HCl buffer. Conditions:
[DNA] = 200 lM, [EB, 1 and 2] = 0–200 lM.

8
P.R. Reddy, N. Raju / Polyhedron 44 (2012) 1–10
separately visualized by the agarose gel electrophoresis method,
whereas a simple electrostatic interaction of small molecules to
DNA does not significantly influence the SC form of DNA, thus
the mobility of the SC form of DNA does not change. These results
also confirm that the complexes bind to DNA.
The cleavage of supercoiled plasmid pUC19 DNA (2 lL) by
complexes 1 and 2 was carried out using 1% agarose gel in TAE buf-
fer (pH 8.2) and is presented in Fig. 9.
3.4.1. DNA cleavage in the absence of H2O2
Attempts were made to cleave DNA through the hydrolysis of
the phosphodiester bond. Since this process does not require any
external agents or light, it has biological significance. The DNA
cleavage experiments were performed by exposing SC DNA to
complexes 1 and 2 over a concentration range of 1–150 lM for
3 h in the absence of external agents (lanes 1–4, Fig. 9a and b for
1 and 2 respectively). The complexes show concentration depen-
dent relaxation of SC DNA to the NC form. 90% conversion of SC
DNA to NC form was achieved at 150 lM by 1 (Fig. 9a, lane 4),
while in the case of 2, 96% conversion was observed (Fig. 9b, lane
4).
Fig. 8. Fluorescence Scatchard plots for EB bound to CT-DNA in the absence (N) and
presence of 1 (d) and 2 (j). The term rEB is the concentration ratio of bound EB to
total DNA and cf is the concentration of free EB.
To ensure that the copper complexes were solely responsible for
the cleavage, control experiments were performed (Fig. 9c) under
identical experimental conditions. Free Cu(OAc)2ꢂH2O, peptides
and phen did not show any DNA cleavage activity, even after incu-
bation for 3 h at a concentration of 1 mM. These results suggest
that the complexes are responsible for the DNA cleavage and not
the individual constituents, viz. free copper or the free ligands.
illustrates both the slope and intercept changing, with complexes
exhibiting both intercalative and covalent interactions with DNA
[51]. The binding isotherms of EB and DNA in the absence and
presence of 1 and 2 were determined experimentally and are
presented in Fig. 8. As can be seen from the plots, a decrease in
slope with no change in the intercept resulted upon the addition
of 1 and 2, indicating an intercalative binding of the complexes
with DNA. Thus the results obtained from the Scatchard plots val-
idate those obtained from absorption, viscosity and emission spec-
tral studies.
3.4.2. DNA cleavage in the presence of H2O2
The cleavage was monitored by treating SC DNA with 1 and 2 in
the presence of H2O2 (lanes 5–6, Fig. 9a and b for 1 and 2 respec-
tively). A control experiment using H2O2 alone did not show any
significant DNA cleavage under similar experimental conditions
(Fig. 9c, lane 8). This proves the catalytic role of 1 and 2 in the pres-
ence of H2O2. In the presence of H2O2, complex 1 converts ꢃ89% of
3.4. DNA cleavage studies
Agarose gel electrophoresis assay, a useful method to investi-
gate various binding modes of small molecules to supercoiled
DNA, also supports the above intercalative binding mode. Interca-
lation of small molecules to plasmid DNA can loosen or cleave the
SC DNA form, which decreases its mobility rate and can be
SC DNA into the NC form at a concentration of 10 lM, while
complex 2 converts ꢃ97% at a similar concentration.
The his-tyr system (2) shows higher cleavage activity than the
his-trp system (1) due to the presence of a phenolic hydroxyl
‘OH’ functional group in the peptide side chain (his-tyr), which
Fig. 9. Agarose gel electrophoresis pattern for the cleavage of supercoiled pUC19 DNA by 1 and 2 at 37 °C in a buffer containing 5 mM Tris. HCl/5 mM aq. NaCl. (a) Lane 1, DNA
control; lane 2, DNA+ 1 (50 M); lane 3, DNA+ 1 (100 M); lane 4, DNA+ 1 (150 M); lane 5, DNA+ 1 (5 M) + H2O2 (1 mM); lane 6, DNA+ 1 (10 M) + H2O2 (1 mM); (b) lane 1,
DNA control; lane 2, DNA+ 2 (50 M); lane 3, DNA+ 2 (100 M); lane 4, DNA+ 2 (150 M); lane 5, DNA+ 2 (5 M) + H2O2 (1 mM); lane 6, DNA+ 2 (10 M) + H2O2 (1 mM); (c)
lane 1, DNA control; lane 2, DNA+ Cu(OAc)2ꢂH2O (1 mM); lane 3, DNA+ his-trp (1 mM); lane 4, DNA+ his-tyr (1 mM); lane 5, DNA+ phen (1 mM); lane 6, DNA+ 1
(150 M) + DMSO (1 mM); lane 7, DNA+ 2 (150 M) + DMSO (1 mM); lane 8, DNA+ H2O2 (1 mM); lane 9, DNA+ 1 (10 M) + H2O2 (1 mM) + DMSO (1 mM); lane 10, DNA+ 1
(10 M) + H2O2 (1 mM) + DMSO (1 mM).
l
l
l
l
l
l
l
l
l
l
l
l
l
l

P.R. Reddy, N. Raju / Polyhedron 44 (2012) 1–10
9
Scheme 2. A proposed mechanism for the oxidative cleavage of DNA by 1 and 2.
Table 2
Percentage of hydrolytic and oxidative cleavage of DNA as a function of concentration of complexes.
Entry
Complex
Hydrolytic cleavage
a[M]
Oxidative cleavage
Ref.
b%Clea
c[M]
d%Clea
1
2
3
4
5
6
7
1
2
50, 100, 150
50, 100, 150
5, 10, 25, 50, 100
10, 25, 50, 100
125, 187, 378, 437, 500
125, 187, 250, 312, 378, 437, 500
125, 250, 375, 500, 625
35, 70, 90
30, 55, 96
15, 25, 45, 100, smear
5, 8, 15, 40
45, 54, 69, 95, 97
50, 53, 55, 60, 65, 85, 90
21, 35, 42, 49, 53
5, 10
5, 10
–
–
–
76, 89
85, 97
–
–
–
–
–
This work
This work
[54]
[54]
[16]
[Cu(trp-phe)(phen)(H2O)]
[Cu(trp-phe)(phen)(H2O)]
[Cu(his-leu)(phen)]
[Cu(his-ser)(phen)]
[Cu(Hist)(trp)]
–
–
[16]
[55]
8
[Cu(Hist)(tyr)]
125, 250, 375, 500, 625
12, 21, 32, 45, 56
–
–
[55]
9
[Cu(L-Val)(bipy)(H2O)]
[Cu(L-Val)(phen)(H2O)]
[Cu(L-Val)(dpq)(H2O)]
[Cu(L-Val)(dppz)(H2O)]
–
–
–
–
–
–
–
–
50
50
50
50
34
72
86
95
[56]
[56]
[56]
[56]
10
11
12
a
b
c
Concentration of the copper(II) complexes in the absence of external agents.
% of DNA cleavage (hydrolytic).
Concentration of the copper(II) complexes in the presence of external agents (1 mM H2O2 for entries 1 and 2; 5 mM MPA for entries 9–12).
% of DNA cleavage (oxidative).
d
shows a stronger hydrogen bonding interaction with the phos-
phate oxygen of the DNA backbone than the indole ‘NH’ hydrogen
in the side chain (his-trp) of 1, thereby enhancing the cleavage
activity.
3.4.3. Mechanistic investigation of the DNA cleavage
In order to diagnose whether the cleavage mechanism followed
a hydrolytic or oxidative pathway, we monitored the quenching of
DNA cleavage in the presence of DMSO, a known hydroxyl radical
scavenger. When SC DNA was incubated with the complexes at a
concentration of 150 lM in the presence of DMSO, without adding
external agents (such as H2O2), only slight inhibition (h2%i) of DNA
cleavage was observed (Fig. 9c, lanes 6 and 7 for 1 and 2 respec-
tively). This rules out the possibility of DNA cleavage via OHꢀ-based
depurination and also a possible oxidative cleavage pathway (diox-
ygen + copper complex + traces of DNA) [52]. Hence the complexes
act as ‘‘self-activating agents’’ and cleave the ds-DNA by a hydro-
lytic mechanism.
On the other hand, in the presence of H2O2, the DNA cleavage
was almost eliminated on addition of DMSO, indicating that hydro-
xyl radicals (OHꢀ) are generated and initiated the cleavage process
(Fig. 9c, lanes 9 and 10 for 1 and 2 respectively). On the basis of this
observation, the mechanism of DNA cleavage mediated by 1 and 2
may be proposed as follows: DNA cleavage is redox-mediated – the
complexes would first interact with DNA by intercalation to form a
Cu(II)ꢂ ꢂ ꢂDNA species, followed by its reduction by an external
agent (H2O2) to a Cu(I)ꢂ ꢂ ꢂDNA species, which then generates hydro-
xyl radicals on reaction with O2. These hydroxyl radicals would
then attack DNA, causing strand scission (Scheme 2). A similar
pathway was proposed by Sigman for the oxidative cleavage reac-
tion with the bis(phen)copper complex [53].
Fig. 10. Disappearance of the supercoiled form (SC) DNA and formation of the
nicked circular (NC) form in the presence of complex 1. Conditions: [com-
plex] = 300
DNA.
lM. Data points (j) refer to SC DNA and data points (d) refer to NC
pyramidal geometry which is known to enhance the cleavage
activity around Cu(II) in the former complexes. Within the Cu–
dipeptide complexes, those containing aromatic moieties bind
and cleave DNA more effectively compared to their aliphatic
It may be seen from Table 2 [16,54–56] that the DNA binding
and cleavage affinity of Cu(II)–amino acid complexes is less
compared to Cu(II)–dipeptide complexes, in spite of the square

10
P.R. Reddy, N. Raju / Polyhedron 44 (2012) 1–10
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[Cu(II)(boc(tos)-his-tyr-ome)(phen)](ClO4)2 (2)). This clearly
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l
lM
3.4.4. Kinetics
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and increase of SC and NC DNA forms were found to fit well to a
single-exponential decay and increase, respectively. By curve fit-
ting, the following hydrolysis rates for SC DNA were obtained at
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37 °C at a fixed complex concentration of 300 l
M: 1.70 hꢁ1
(R = 0.975) for 1 and 1.82 hꢁ1 (R = 0.963) for 2. This amounts to a
(4.7–5.0) ꢀ 107-fold rate enhancement compared to non-catalyzed
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4. Conclusions
The synthesis, structural characterization, DNA binding and
cleavage properties of ternary histidine based dipeptide Cu(II)
complexes, [Cu(II)(boc(tos)-his-trp-ome)(phen)](ClO4)2 (1) and
[Cu(II)(boc(tos)-his-tyr-ome)(phen)](ClO4)2 (2), are reported in
this paper. The analytical and physicochemical data suggest that
the complexes are arranged in a square-planar geometry around
copper. The DNA binding results reveal that the complexes bind
to DNA in an intercalation mode. Their cleavage activity on
pUC19 plasmid DNA in the absence and presence of H2O2 show
that they cleave DNA efficiently. Since the mixed-ligand
complexes, containing bidentate histidine dipeptide and phen,
show a unique ability to effect DNA double strand scission in both
hydrolytic and oxidative cleavage reactions, they are considered as
better DNA binding and cleavage agents, and hence artificial
restriction enzymes in nucleic acid biochemistry.
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Acknowledgments
P.R.R. thanks the Council of Scientific and Industrial Research
(CSIR) and the University Grants Commission (UGC), New Delhi
for financial support. N.R. thanks the CSIR for a Senior Research
Fellowship.
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