Nuclease activity via self-activation and anticancer activity of a mononuclear copper(II) complex: Novel role of the tertiary butyl group in the ligand frame

Article abstract of DOI:10.1021/ic2016676

The copper complex [Cu(^tBuPhimp)(Cl)] (1) derived from tridentate ligand ^tBuPhimpH having N₂O donors was synthesized, and its molecular structure was determined. A phenoxyl radical complex was generated in solution at room temperature using Ce(IV). The nuclease and anticancer activities of 1 were investigated. The roles of the tert-butyl group and singlet oxygen in the DNA cleavage activity were also discussed.

Full text of DOI:10.1021/ic2016676

Communication
pubs.acs.org/IC
Nuclease Activity via Self-Activation and Anticancer Activity of a
Mononuclear Copper(II) Complex: Novel Role of the Tertiary Butyl
Group in the Ligand Frame
Kaushik Ghosh,*,† Pramod Kumar,† Varun Mohan,† Udai P. Singh,† Sahba Kasiri,‡
and Subhrangsu S. Mandal‡
†Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247667, India
‡Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, Texas 76012, United States
S
* Supporting Information
phenoxyl radical complexes and this radical-mediated cleavage
ABSTRACT: The copper complex [Cu(tBuPhimp)(Cl)]
(1) derived from tridentate ligand BuPhimpH having
of DNA.13 In our recent report, we found that generated
phenoxyl radicals in copper complexes were not stable at room
temperature; however, stabilization of phenoxyl radical
complexes was found in zinc complexes. This was due to the
formation of phenoxo-bridged dinuclear zinc complexes.12,13
Investigation of the literature revealed that phenoxyl radical
complexes were stabilized by a −SR group at the ortho position
to the phenolato function and/or bulky alkyl group in the
phenyl ring bearing a phenolato donor.14,15
t
N2O donors was synthesized, and its molecular structure
was determined. A phenoxyl radical complex was
generated in solution at room temperature using Ce(IV).
The nuclease and anticancer activities of 1 were
investigated. The roles of the tert-butyl group and singlet
oxygen in the DNA cleavage activity were also discussed.
In this endeavor, we tried to examine the increased stability
of the phenoxyl radical at room temperature and study the
nuclease activity. No complex among the family of complexes
derived from ligand PhimpH ([Cu(Phimp)(X)] where X =
nteraction of metal complexes with DNA and transition-
metal-complex-mediated DNA damage are important areas
I
of chemical research because of their applications in nucleic
acid chemistry and cancer research.1 Moreover, DNA/RNA
cleavage is a fundamental reaction in gene regulation and gene
therapy and is important for programmed cell death.2 Among
complexes of first-row transition elements, copper complexes
received special attention because copper is one of the essential
elements in biology and several metalloproteins need copper
for their activity.3 Biologically relevant copper has a high affinity
for the nucleobases and copper complexes possess biologically
accessible redox properties.3,4 Investigation of the literature5,6
revealed that oxidative DNA cleavage reactions by copper
complexes are mediated by reactive oxygen species (ROS)
produced via the oxidation of CuII to CuIII by an oxidizing agent
and the reduction of CuII to CuI by a reducing agent.
Alternatively, transient-metal-bound species formulated as
[CuOH]2+, [CuOOH]+, and [CuO]+ are also reported to be
responsible for DNA strand scission.6 It has also been reported
that the copper complexes, derived from proper ligand(s),
could cleave DNA without any external agent in the presence of
light.7
However, the addition of such an oxidizing or reducing agent
or illumination of light is not conducive for in vivo applications
of such complexes;8 hence, it would be of interest to find a self-
activated system that would not require any type of activation
to generate reactive species for DNA cleavage activity. Only a
few reports on the nuclease activity via self-activation are
available in the literature.8−10 Recently, we have reported a
mononuclear copper(II) complex that afforded cleavage of
pBR322 plasmid DNA via self-activation.11
H2O, Phimp−, CH3COO−, SCN−, and NO2 ) exhibited DNA
−
cleavage activity without the addition of an oxidizing or
reducing agent.12 Herein we report the synthesis and
characterization of a copper complex derived from a tert-
butyl-group-substituted PhimpH {tBuPhimpH, (E)-2,4-di-tert-
butyl-6-[[phenyl(pyridin-2-yl)hydrazono]methyl]phenol; Fig-
ure 1} and its nuclease activity studies. To the best of our
knowledge, there is no report where the role of the radical
stabilizing group in the nuclease activity via self-activation as
well as the anticancer activity was described.
Reaction of the deprotonated tBuPhimpH with CuCl2·2H2O
afforded [Cu(tBuPhimp)(Cl)] (1). Details of the synthetic
procedures and spectroscopic characterization are described in
the Supporting Information. The molecular structure of
[Cu(tBuPhimp)(Cl)]·CH3OH (1·CH3OH) afforded a dis-
torted square-planar geometry (Figure 1) around the metal
center having meridional spanning of ligand. The distances
around the metal center were consistent with the reported
values.12 Details of the structural features and magnetic
properties are described in the Supporting Information.
The cyclic voltammogram of complex 1 exhibited a cathodic
peak near −0.820 V due to a CuII/CuI couple (Figure 2).
Phenolato oxygen stabilized higher oxidation states, and this
negative potential was consistent with the reported data.12
Interestingly, we found a quasi-reversible redox couple near
+1.0 V, which clearly indicated ligand-centered oxidation, i.e.,
the generation of a phenoxyl radical (1•+) complex (E1/2
=
As a part of our ongoing research on the interaction of
Received: August 2, 2011
Published: February 28, 2012
copper complexes with DNA,11,12 we were exploring the
© 2012 American Chemical Society
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Communication
The nuclease activity of 1 has been studied using supercoiled
(SC) pBR322 DNA, and the extent of DNA cleavage was
measured by gel electrophoresis. Contrary to our previous
results,12 we found a different type of nuclease activity where
the disappearance of both bands (SC and NC DNA) was
observed. An increase in the concentration of 1 (∼50 μM)
afforded the disappearance of DNA bands. Hence, 1 exhibited
excellent DNA cleavage activity in the absence of an external
agent. Variation of the incubation time for the DNA cleavage
activity (Figure 4b, lanes 10−15) clearly expressed the activity
Figure 1. Schematic representation of tBuPhimpH and ORTEP
drawing (50% probability level) of 1·CH3OH with an atom numbering
scheme. Solvent molecules and hydrogen atoms are omitted for clarity.
Selected bond length (Å) and bond angles (deg): Cu1−O1 1.8660(9),
Cu1−N1 1.9546(9), Cu1−N2 1.9812(10), Cu1−Cl1 2.2479(3); O1−
Cu1−N1 91.21(4), O1−Cu1−N2 162.46(4), O1−Cu1−Cl1 92.24(3),
N1−Cu1−N2 81.01(4), N1−Cu1−Cl1 172.99(3), N2−Cu1−Cl1
97.27(3).
Figure 4. Gel electrophoresis separations showing the cleavage of SC
pBR322 DNA (40 ng) by 1 in a buffer containing 10% acetonitrile and
incubated at 37 °C for 2 h. (a) Lane 1: DNA control. Lane 2: DNA +
10% acetonitrile. Lane 3: DNA + tBuPhimpH (100 μM). Lanes 4−11:
DNA + 1 = 5, 10, 20, 30, 40, 50, 75, and 100 μM, respectively. (b)
Lane 1: DNA control. Lane 2: DNA + 1 (50 μM). Lanes 3−9: DNA +
1 (50 μM) + DMSO, urea, ethanol, NaN3, L-histidine, D2O, and
catalase, respectively. Lane 10: DNA control. Lanes 11−15: DNA + 1
(50 μM) + 10, 30, 60, 90, and 120 min of incubation, respectively.
within 30 min. These observations indicated extensive DNA
degradation and DNA cleavage at multiple positions.17
In certain reports,5b,11 authors explained self-activated DNA
cleavage via a hydrolytic pathway because the nuclease activity
happened in the absence of any external agent and was not
inhibited by radical scavengers. If the nuclease activity was
inhibited by the presence of radical scavengers, one could
speculate the possible role of ROS in the nuclease
activity;5,6,8−12 hence, we investigated the nuclease activity in
the presence of radical scavengers (Figure 4b, lanes 1−9).
In the search for a mechanism, the inhibition of the nuclease
activity was studied in presence of dimethyl sulfoxide (DMSO),
ethanol, urea, NaN3, L-histidine, D2O, and catalase.5,6 The
addition of singlet-oxygen scavengers NaN3 and L-histidine
(Figure 4b, lanes 6 and 7) caused complete inhibition of the
nuclease activity. These results suggested that 1O2 or any other
singlet-oxygen-like entity may participate in DNA strand
scission.5−9 Moreover, enhancement of the nuclease activity
in the presence of D2O also supports the above observation
(Figure 4b, lane 8).7b,9 A comparison of the nuclease activity in
the presence and absence of oxygen clearly indicated the role of
oxygen (Figure S7 in the Supporting Information). On the
basis of above observations, we speculate that 1 generated
singlet oxygen and/or singlet-oxygen-like ROS, which were
responsible for the nuclease activity. Hence, we found that 1 is
a novel example of a copper complex by which nuclease activity
happened via self-activation.
Figure 2. Cyclic voltammogram of a 10−3 M solution of 1 in CH2Cl2
in the presence of 0.1 M TBAP as the supporting electrolyte and Ag/
AgCl as the reference electrode; scan rate 0.1 V s−1. Inset: Cyclic
voltammograms at 50, 100, 200, 300, 400, and 500 mV s−1 scan rates.
+1.014 V and ΔEp = 0.158 V).16 Repeated scans as well as
different scan rates clearly expressed the generation of a
phenoxyl radical complex and regeneration of 1. This was also
supported by UV−vis spectral data (Figure S5).
The generation of a phenoxyl radical complex was performed
by oxidation of 1 with (NH4)2[CeIV(NO3)6] (CAN) in an
acetonitrile solution at room temperature. The green color of
complex 1 was converted into a beige-colored solution by CAN
during ligand-centered oxidation, and the formation of
phenoxyl radical complex [1]•+ was supported by the
characteristic peaks in UV−vis spectra (Figure S5(a)). Upon
the addition of CAN, the charge-transfer band of the copper
complex 1 at 422 nm (8075 M−1 cm−1) decreased and the
appearance of a new band at 416 nm (6240 M−1 cm−1) and a
broad band at 565 nm (1990 M−1 cm−1) with an isosbestic
point at 437 nm was observed because of the formation of a
phenoxyl radical complex. The intensities of the characteristic
bands near 416 and 565 nm were dependent on the
concentration of CAN. A total of 2 equiv of CAN was required
for the complete conversion of 1 into radical complex [1]•+,
and there was no change in the UV−vis spectra upon the
further addition of CAN (Figure S5(a)). We also examined the
decomposition of the radical complex and regeneration of 1
(Figure S5(b)). The decomposition of radical complex [1]•+
was observed within ∼40 min (Figure S5(b), inset).
These data prompted us to study the anticancer activity of 1
on MCF-7 cells. Preliminary data afforded an IC50 value of 4.76
0.14 μM in cell viability assay, which was found to be better
than the IC50 value obtained for cisplatin (17.98 1.18) in the
same experiment.18 Differential interference contrast (DIC)
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Inorganic Chemistry
Communication
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Figure 5. DIC images of the MCF-7 cells that were treated with 10
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In summary, novel complex 1 exhibited quasi-reversible
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ASSOCIATED CONTENT
* Supporting Information
Details of the synthesis, characterization, X-ray analysis, and a
CIF file. This material is available free of charge via the Internet
at http://pubs.acs.org.
■
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AUTHOR INFORMATION
Corresponding Author
*E-mail: ghoshfcy@iitr.ernet.in.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
K.G. is thankful to DST, New Delhi, India, for SERC FAST
Track project (SR/FTP/CS-44/2006) support. P.K. and V.M.
are thankful to CSIR, MHRD and UGC India, respectively, for
financial assistance. U.P.S. is thankful to IIT Roorkee for its
single-crystal X-ray facility. S.S.M. is thankful for NIH Grant
1R15 ES019129-01.
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