Synthesis, structural characterization and DNA interaction studies on a mononuclear copper complex: Nuclease activity via self-activation

Article abstract of DOI:10.1016/j.inoche.2011.01.008

Mononuclear copper(II) complex [Cu(Pyimpy)(Cl)(ClO₄)] has been synthesized from a tridentate ligand Pyimpy (Pyimpy = 1-phenyl-1-(pyridin-2-yl)- 2-(pyridin-2-ylmethylene)hydrazine) and characterized by elemental analysis, electronic absorption and IR spectroscopy. Molecular structure of 1 was determined by single crystal X-ray diffraction. Structural index parameter (τ) calculation supported distorted square pyramidal geometry around the metal centre. The stabilization of copper(II) centre was examined by electrochemical studies. DNA interaction studies were investigated by absorption spectral studies and EthBr displacement assay. Nuclease activity studies afforded cleavage of pBR322 plasmid DNA via self-activating mechanism. Investigation of mechanism indicated the possible role of reactive oxygen species in DNA cleavage.

Full text of DOI:10.1016/j.inoche.2011.01.008

Inorganic Chemistry Communications 14 (2011) 489–492
Contents lists available at ScienceDirect
Inorganic Chemistry Communications
journal homepage: www.elsevier.com/locate/inoche
Synthesis, structural characterization and DNA interaction studies on a mononuclear
copper complex: Nuclease activity via self-activation
⁎
Kaushik Ghosh , Pramod Kumar, Nidhi Tyagi, Udai P. Singh, Nidhi Goel
Department of Chemistry, Indian Institute of Technology, Roorkee, Roorkee-247667, Uttarakhand, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Mononuclear copper(II) complex [Cu(Pyimpy)(Cl)(ClO4)] has been synthesized from a tridentate ligand
Pyimpy (Pyimpy = 1-phenyl-1-(pyridin-2-yl)-2-(pyridin-2-ylmethylene)hydrazine) and characterized by
elemental analysis, electronic absorption and IR spectroscopy. Molecular structure of 1 was determined by
single crystal X-ray diffraction. Structural index parameter (τ) calculation supported distorted square
pyramidal geometry around the metal centre. The stabilization of copper(II) centre was examined by
electrochemical studies. DNA interaction studies were investigated by absorption spectral studies and EthBr
displacement assay. Nuclease activity studies afforded cleavage of pBR322 plasmid DNA via self-activating
mechanism. Investigation of mechanism indicated the possible role of reactive oxygen species in DNA
cleavage.
Received 20 October 2010
Accepted 6 January 2011
Available online 14 January 2011
Keywords:
Copper complex
Crystal structure
Electrochemistry
DNA binding
Nuclease activity
© 2011 Elsevier B.V. All rights reserved.
There has been considerable interest in the recent years for design
and synthesis of DNA cleaving reagents for their applications in
biotechnology and medicine [1–3]. Transition metal ions show diverse
structural features, variable oxidation and spin states and redox
properties in different complexes. These properties could be exploited
to discover novel artificial nucleases. Among the first row transition
elements, copper has got a special interest in this regard since the
discovery of first chemical nuclease by Sigman et al. [4]. Biologically
relevant copper has high affinity for the nucleobases and copper
complexes possess biologically accessible redox properties [5]. There
are several reports of copper complexes and their nuclease activity
studies, however few reports [6,7] are there in the literature where
copper complexes are acting as artificial nucleases without the
addition of any oxidizing or reducing agent. This type of nuclease
activity could be possible via a self-activating mechanism [6] and/or
through a hydrolytic pathway [7]. Our quest was to synthesize such
type of molecule and herein we report the synthesis and character-
ization of a novel copper complex [Cu(Pyimpy)(Cl)(ClO4)] (1) derived
from tridentate ligand Pyimpy. Complex 1 was successful in cleaving
DNA in absence of any oxidizing or reducing agents. Molecular
structure of 1 was determined by single crystal X-ray diffraction.
Redox property of the metal centre was examined by electrochemical
studies. DNA interaction with calf thymus DNA (CT DNA) and
nuclease activity with pBR322 plasmid were investigated and results
of our mechanistic studies will be scrutinized in this communication.
Schiff-base ligand Pyimpy (Scheme 1) was synthesized by the
condensation of pyridine 2-aldehyde and 2-(1-phenylhydrazinyl)
pyridine [8]. Pyimpy was reacted with Cu(ClO4)2·6H2O in methanol
and a green solution was obtained. Addition of one equivalent of
Et4NCl to the above solution gave rise to [Cu(Pyimpy)(Cl)(ClO4)] (1)
(shown in Scheme 1). In IR spectra of 1, splitting of peaks near
1090 cm−1 indicated the presence of coordinated perchlorate ion.
However, solution conductivity measurement gave rise to 1:1
electrolyte [8]. Hence we speculate the dissociation of perchlorate
ion in solution. Complex 1 afforded strong charge transfer band at
384 nm in UV–vis spectra. Oxidation state 2+ for copper in complex 1
was confirmed by magnetic susceptibility measurements at room
temperature.
Molecular structure of complex [Cu(Pyimpy)(Cl)(ClO4)] (1) is
depicted in Fig. 1 and the matrix parameters are described in Table S1.
In crystal structure, two pyridine nitrogen (NPy) and one azomethine
nitrogen (NIm) bind to the metal centre in mer fashion. The ligand has
three six-membered rings, among them, two pyridine rings are in the
same plane whereas the other phenyl ring is roughly perpendicular
(82.92°) to the ligand binding plane. The structural index parameter
[9] (τ) was determined to describe the geometry around penta
coordinated metal centre. In complex 1 the τ value was calculated to
be 0.174 and the geometry of the complex around the metal centre
was described as distorted square pyramid (Scheme S1, details are
given in the supporting information). In the complex the square plane
consisted of two NPy and one NIm donors along with a Cl− ligand
whereas ClO−4 ligand occupied in axial position. The copper centre
was 0.13 Å above the plane generated by N1, N2, N4 and Cl1. Cu–NPy
distances (~2.00 Å) and Cu–Nim distance ~1.97 Å are consistent with
the reported data [10]. The equatorial Cu–Cl bond distance (2.21 Å) is
⁎
Corresponding author. Tel.: +91 1332 285547; fax: +91 1332 2735601.
E-mail address: ghoshfcy@iitr.ernet.in (K. Ghosh).
1387-7003/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.inoche.2011.01.008

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K. Ghosh et al. / Inorganic Chemistry Communications 14 (2011) 489–492
Scheme 1. Schematic conversion of Pyimpy into [Cu(Pyimpy)(ClO4)(Cl)] (1).
smaller than the values (2.34 Å–2.43 Å) reported in the literature
[5,11] and even shorter than the values (2.26 Å and 2.28 Å) reported
[12] for the equatorial Cu–Cl bond. The axial Cu–OClO4 bond distance
was close to the value reported by Palaniandavar et al. [7a].
Fig. 2. Absorption spectra of complex [1]=50 μM, in 0.1 M phosphate buffer (pH 7.2)
containing 5% DMF in the presence of increasing amounts of [DNA]=0–90 μM. Arrows
show the absorbance changes upon increasing DNA concentration.
The redox behavior of Cu(II) in complex 1 was investigated by
cyclic voltammetry at a glassy carbon electrode using Ag/AgCl
reference electrode. Cyclic voltammogram of 1 in DMF solution at
298 K is shown in Fig. S2. The complex showed irreversible cyclic
voltammogram with a cathodic peak near −0.060 V possibly due to
CuII/CuI couple. The anodic peak near + 0.488 V vs. Ag/AgCl probably
satisfy the condition for self activation predicted by Reedijk et al. [6b].
We have done all the DNA interaction studies in phosphate buffer
(0.1 M, pH 7.2) and there was negligible change in absorbtion spectra
of 1 after one month. Electronic absorption spectroscopic techniques
were used to investigate the binding of DNA with metal complexes. To
achieve this, the absorption spectra of 1 in the absence and presence
of CT-DNA at different concentrations were measured (Fig. 2). The
binding constant Kb for 1 has been found to be 5.2×103 M−1. The
observed binding constant was similar to be values obtained for other
copper complexes [13] but much smaller than the classical inter-
calators and metallointercalators where binding constants were
reported to be in the order of 107 M−1 [14]. During the titration
with DNA we found some interesting spectral changes. The spectral
changes showed a blue shift (37 nm) for the charge transfer band and
concomitant formation of an isosbestic point at 358 nm. This type of
spectral changes were possibly due to new species generation in
presence of DNA [15]. Fate of 1 with the variation of pH was
investigated and we found no change in UV–visible spectra in the pH
range 3–10 [15]. We have invesigated ESI-MS spectral studies of
complex 1 in water. The data clearly expressed the presence of copper
complex in aqueous solution (detailed are shown in supporting
information). We performed the titration experiment in H2O (MilliQ)
instead of that in buffer. The change in absorption spectra of com-
plex 1 in H2O (MilliQ) afforded spectral changes similar to the
data depicted in Fig. 2. These data indicated that phosphate group
was not involved for such changes. It may be possible that copper in
presence of DNA got attached to the nucleic acid base(s) because of
its high affinity [3,16] and a new species was generated during
DNA interaction. More insight into this DNA binding event is under
progress.
We have examined the competitive binding of ethidium bromide
(EthBr) vs complex 1 with CT DNA using fluorescence spectral studies
to get better insight into DNA binding events. Fluorescence quenching
spectra of 1 were recorded (Fig. 3) and Stern–Volmer constant Ksv was
found to be 2.45×104 M−1. Kb and Ksv values for complex 1 indicated
moderate interaction with DNA [13,17].
We extended our DNA interaction studies by examining the
nuclease activity of the complex 1. The cleavage of supercoiled
pBR322 DNA by 1 and formation of nicked (NC) and linear (LC) DNA
were studied by several gel electrophoresis experiments in Tris–boric
acid–EDTA buffer (TBE). Following important observations were
obtained from Fig. 3. First, nuclease activity of 1 was observed
(Fig. 4, lane 3–9) in absence of any oxidizing or reducing agents and
Fig. 1. ORTEP plot of [Cu(Pyimpy)(Cl)(ClO4)] showing the atom numbering scheme.
Thermal ellipsoids are drawn at 30% probability. Selected bond lengths (Å) and angles
(deg): Cu(1)–N(1) 2.004(4), Cu(1)–N(2) 1.973(3), Cu(1)–N(4) 1.996(4), Cu(1)–Cl(1)
2.2133(15), Cu(1)–O(3) 2.524, N(1)–Cu(1)–N(2) 79.88(15), N(1)–Cu(1)–N(4) 159.59
(15), N(1)–Cu(1)–Cl(1) 99.03(12), N(2)–Cu(1)–N(4) 79.95(14), N(2)–Cu(1)–Cl(1)
170.05(10), N(4)–Cu(1)–Cl(1) 100.30(11).
Fig. 3. Fluorescence emission spectra of the EB–DNA in 0.1 M phosphate buffer (pH 7.2)
containing 5% DMF in the absence (dashed line) and presence (solid line) of complex 1.
[EB]=5 μM, [DNA]=25 μM, [Cu(Pyimpy)(Cl)(ClO4)]=0–14.38 μM, λex =250 nm.

K. Ghosh et al. / Inorganic Chemistry Communications 14 (2011) 489–492
491
Fig. 4. Gel electrophoresis separations showing the cleavage of supercoiled pBR322
DNA (100 ng) by complex 1. Incubated at 37 °C for 1.5 h. lane 1, DNA control; lane 2,
DNA+10% DMF; lanes 3–9, DNA+1=10, 25 40, 50, 60, 80, 100 μM respectively; lanes
10–14, DNA+1 (100 μM)+incubation time 5, 15, 30, 60 and 90 min respectively.
Fig. 6. Gel electrophoresis separations showing the cleavage of supercoiled pBR322
DNA (100 ng) by complex 1 (50 μM) in presence of radical scavengers (20 mM). lane 1,
DNA; lane 2, DNA+Cu(ClO4)2∙6H2O (100 μM); lane 3, DNA+1 (50 μM); lane 4–10,
DNA+1 (50 μM)+DMSO, ethanol, urea, catalase (1 U), D2O, L-histidine, NaN3,
respectively.
the amount of NC DNA was found to be increased with the increase in
concentration of 1 (10–100 μM) without the formation of LC form of
DNA. Second, enhancement of DNA cleavage activity was observed
due to variation of incubation time (Fig. 4, lane 10–14).
that complex 1 generated reactive oxygen species (ROS) which were
responsible for nuclease activity. Hence we found that 1 is a novel
example of a copper complex by which nuclease activity happened via
self-activation and to the best of our knowledge the single example of
such type of DNA cleaving agent available in the literature was by
Reedijk et al. [6]. However, our results derived from machenistic
investigation was not similar to the data reported by Reedijk and
coworkers.
In conclusion, mononuclear copper(II) complex [Cu(Pyimpy)(Cl)
(ClO4)] (1) derived from tridentate ligand Pyimpy has been
synthesized and characterized. Molecular structure of complex 1
was determined by X-ray crystallography. DNA interaction by
absorption spectral studies showed formation of a new species during
interaction. This molecule exhibited nuclease activity in absence of
any oxidizing or reducing agents. The cleavage efficiency was
dependent both on the complex concentration and on the incubation
period. Inhibition of nuclease activity was observed in the presence of
radical scavengers and possible role of reactive oxygen species was
speculated. Details of mechanistic investigation on this interesting
activity of this complex, bilogical activity studies and reactivity studies
on other related complexes are currently in progress.
In this next experiment depicted in Fig. 5, we examined the DNA
cleavage activity in presence of oxidizing or reducing agent. Nuclease
activity of 1 in presence of H2O2 or 2-mercaptoethanol (BME)
increased the cleavage activity as compared to the previous
experiment done by complex itself. Moreover, it has been found out
that in the presence of H2O2 or 2-mercaptoethanol 50 μM concentra-
tion of 1 showed the complete conversion of SC form to NC and LC
form of DNA (Fig. 5, lane 7, 8). The results of our control experiment
were described in Fig. S4. These data clealy indicated that Pyimpy
itself was not responsible for DNA cleavage activity.
Investigation of the mechanism becomes very much important
when copper complexes exhibit nuclease activity in absence of any
external reagents. There are several reports in the literature where
such type of activity was described, however, following reports will be
of our interest at this point. In certain reports authors explained such
type of DNA clevage activity via hydrolytic pathway because nuclease
activity was not inhibited by radical scavengers [7]. Later on religation
experiment was examined to prove hydrolytic cleavage. If nuclease
activity was inhibited by the presence of radical scavengers we could
speculate the possible role of reactive oxygen species (ROS) in
nuclease activity. Hence we decided to perform first the investigation
of nuclease actvity in presence of radical scavengers (Fig. 6).
Involvement of reactive oxygen species (hydroxyl radical, super-
oxide ion, singlet oxygen and hydrogen peroxide) in nuclease activity
could be diagnosed by monitoring the quenching of DNA cleavage in
the presence of radical scavengers in solution [5]. The hydroxyl
radical scavengers like DMSO, ethanol and urea showed the complete
inhibition of nuclease activity (Fig. 6, lane 4–6). These results
suggested that hydroxyl radicals may be involved in the cleavage
process. Addition of singlet oxygen scavengers like L-histidine and
NaN3 (Fig. 6, lane 9,10) showed complete inhibition of nuclease. So
these results suggested that 1O2 or any other singlet oxygen-like
entity may participate in the DNA strand scission. However, we did
not observed enhancement of nuclease activity in presence of D2O
[18] (Fig. 6, lane 8). Probable participation of hydrogen peroxide was
excluded due to enhancement of nuclease activity upon addition of
catalase (Fig. 6, lane 7). On the basis of above observations we predict
Acknowledgments
KG is thankful to DST, New Delhi, India through the SERC FAST
Track project (SR/FTP/CS-44/2006) for financial support. PK and NT
are thankful to CSIR, India for financial assistance. We are thankful to
IITR for instrumental facilities. UPS is thankful to IIT Roorkee for single
crystal X-ray facility. We would like to thank Mr. Shibdas Banerjee for
his help.
Appendix A. Supplementary material
The experimental and crystal structure details are in supporting
information. The CCDC No. for [Cu(Pyimpy)(Cl)(ClO4)] is 773744. The
data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/
retrieving.html. Supplementary data to this article can be found
online at doi:10.1016/j.inoche.2011.01.008.
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