Oxidative DNA Cleavage, Formation of μ-1,1-Hydroperoxo Species, and Cytotoxicity of Dicopper(II) Complex Supported by a p-Cresol-Derived Amide-Tether Ligand

Article abstract of DOI:10.1021/acs.inorgchem.9b02093

Metal complexes to promote oxidative DNA cleavage by H₂O₂ are desirable as anticancer drugs. A dicopper(II) complex of known p-cresol-derived methylene-tether ligand Hbcc [Cu₂(bcc)]³⁺ did not promote DNA cleavag

Full text of DOI:10.1021/acs.inorgchem.9b02093

Communication
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Oxidative DNA Cleavage, Formation of μ‑1,1-Hydroperoxo Species,
and Cytotoxicity of Dicopper(II) Complex Supported by a p‑Cresol-
Derived Amide-Tether Ligand
Yuki Kadoya,† Katsuki Fukui,† Machi Hata,† Risa Miyano,† Yutaka Hitomi,† Sachiko Yanagisawa,‡
Minoru Kubo,‡ and Masahito Kodera*,†
†Department of Molecular Chemistry and Biochemistry, Doshisha University, Kyotanabe Kyoto 610-0321, Japan
‡Department of Life Science, University of Hyogo, Kouto 2-1, Ako Kamigori Hyogo 678-1297, Japan
S
* Supporting Information
cells can be used to reduce the side-effect of anticancer drugs if
ABSTRACT: Metal complexes to promote oxidative
DNA cleavage by H2O2 are desirable as anticancer
drugs. A dicopper(II) complex of known p-cresol-derived
methylene-tether ligand Hbcc [Cu2(bcc)]3+ did not
promote DNA cleavage by H2O2. Here, we synthesized
a new p-cresol-derived amide-tether one, 2,6-bis(1,4,7,10-
tetrazacyclododecyl-1-carboxyamide)-p-cresol (Hbca-
mide). A dicopper(II) complex of the new ligand
[Cu2(μ-OH)(bcamide)]2+ was structurally characterized.
This complex promoted the oxidative cleavage of
supercoiled plasmid pUC19 DNA (Form I) with H2O2
at pH 6.0−8.2 to give Forms II and III. The reaction was
largely accelerated in a high pH region. A μ-1,1-
hydroperoxo species was formed as the active species
and spectroscopically identified. The amide-tether com-
plex is more effective in cytotoxicity against HeLa cells
than the methylene-tether one.
we develop metal complexes capable of specifically activating
H2O2 in cancer cells. So far, many complexes of copper,1a,14
iron,15 and various metals1d,16 have been reported to accelerate
the oxidative DNA cleavage, but in most of them, large excess
amounts of reducing reagents, such as 3-mercaptopropionic
acid,14d glutathione,14d,e dithiothreitol,15b,d and ascorbic
acid,14a−c,15a are essential. Amounts of reducing agents,
however, are lower in cancer cells than in normal cells.
Therefore, metal complexes capable of accelerating the
oxidative DNA cleavage by H2O2 without reductants are
desirable as anticancer drugs.
Previously, we reported that a dicopper(II) complex with p-
cresol-derived methylene-tether ligand [Cu2(bcc)](ClO4)3 (1)
and related complexes accelerate hydrolytic DNA cleavage at
pH 5−6,17 but 1 did not accelerate the DNA cleavage with
H2O2. In this study, we synthesized a new p-cresol-derived
amide-tether ligand 2,6-bis(1,4,7,10-tetraazacyclododecyl-1-
carboxamide)-p-cresol (Hbcamide) and found that its
dicopper(II) complex [Cu2(μ-OH)(bcamide)](ClO4)2 (2)
largely accelerates the oxidative DNA cleavage by H2O2
without reductants.
arious metal complexes have been developed as
Vanticancer drugs that block DNA replication through
binding and/or cutting DNA.1 In particular, platinum(II)
complexes, for example, cisplatin2 and its derivatives, such as
oxaliplatin,3 lobaplatin,4 and nedaplatin,5 are capable of
strongly binding DNA to block the replication and are
clinically used as anticancer drugs.6 However, dosing of this
type of anticancer drugs is accompanied by heavy side-effects
and platinum-resistance problems.7 As one of the other types
of anticancer drugs clinically used, bleomycin is known to
cleave double-strand DNA (ds-DNA).8 It has a metal binding
site as a pentadentate donor including an amide donor. The
iron complex of bleomycin reacts with dioxygen molecules
and/or hydrogen peroxide to form the activated bleomycin,
proposed as a hydroperoxoiron(III) complex, to oxidatively
cleave ds-DNA.9 This, however, causes the problem of side-
effects in the lungs with the long-time use.10
The methylene- and amide-tether ligands, Hbcc and
Hbcamide, used in this study have two tetraazacyclododecane
(cyclen) pendant groups tethered at 2,6-positions of p-cresol
by −CH2− and −CO− groups, respectively (Scheme 1). Hbcc
and [Cu2(bcc)](ClO4)2 (1) were prepared as previously
reported.17 Hbcamide and [Cu2(μ-OH)(bcamide)](ClO4)2
(2) are new compounds, and the synthetic details are shown
Scheme 1. Chemical Structures of Hbcc and Hbcamide
It is well-known that the microenvironment in the cancer
cells is significantly different from that in normal cells.11 As one
of the key features of cancer cells, the concentration of reactive
oxygen species (ROS) including H2O2 is relatively higher in
cancer cells than in normal cells.12 This is mainly caused by
mitochondrial malfunction and increased metabolic activity of
cancer cells.13 Thus, the overproduction of H2O2 in cancer
Received: July 13, 2019
© XXXX American Chemical Society
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DOI: 10.1021/acs.inorgchem.9b02093
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Communication
in the Supporting Information. Caution! Perchlorate salts are
potentially explosive and should be handled with great caution.
The amide-tether complex 2 was structurally characterized
by X-ray analysis (see Table S1 for the crystallographic data
and Table S2 for the selected bond distances and angles). The
crystal structure of 1 was previously reported.17 ORTEP views
of 1 and 2 are shown in Figure 1A and B, respectively. In 2,
Figure 2. Time courses for the decrease of percent of Form I upon
reaction of pUC19 DNA (50 μM bp) with 1 (red) or 2 (purple; 50
μM) in the presence of H2O2 (0.5 mM) at pH 6.0 (10 mM MES, 10
mM NaCl) at 37 °C.
the reaction of 1 is caused by hydrolytic DNA cleavage as
previously reported.17 On the other hand, in the presence of
H2O2, the amide-tether complex 2 significantly accelerated the
decrease of percent of Form I as shown in Figure 2. The
hydrolytic DNA cleavage activity of 2 was almost the same as
that of 1 (see Figure S8). The oxidative DNA cleavage is
confirmed by the fact that Form III produced in the reaction is
not converted to form II by treatment with T4 DNA ligase
(Figure S20). Thus, 2 specifically promoted oxidative DNA
cleavage with H2O2, where 2 activates H2O2 for the oxidative
DNA cleavage because H2O2 did not cleave DNA by itself in
the absence of 2. Moreover, the oxidative DNA cleavage rate
increased with an increase of concentrations of both 2 and
H2O2 as shown in Figures S9 and S10 and Tables S6. These
results suggest that the reaction proceeds via formation of the
active species from 2 and H2O2. The active species was
spectroscopically identified as described below.
The pH dependence of the oxidative DNA cleavage was
further examined by using the amide-tether complex 2 in
detail. A decrease of percent of Form I and increase of percent
of Form III in the reactions at pH 6.0, 7.4, and 8.2 in the
presence of H2O2 (0.3 mM) are shown in Figure S4. As shown
in Figure S4A, the decrease of percent of Form I is accelerated
at higher pH. Moreover, it was found that the formation rate of
Form III is drastically enhanced at higher pH as shown in
Figure S4B. At pH 8.2, 50% of form I was converted to Form
III in 5 h, but an almost negligible amount of Form III was
detected at pH 6.0. This suggested that the formation of the
active species is accelerated at high pH. Form III is a linear
DNA formed by the cleavage of two phosphate-ester main
chains at the near positions of ds-DNA. Actually, 2 showed
relatively high oxidative DNA cleavage activity in the presence
of H2O2 as compared with a tricopper complex reported
before.14 Thus, the active species has a strong oxidation ability
capable of cleaving ds-DNA, and this is similar to the activated
bleomycin.
Figure 1. ORTEP diagrams of each cationic part of 1 (A) and 2 (B).
Hydrogen atoms are omitted for clarity.
two Cu(II) ions are incorporated into a bcamide ligand and
bridged by the endogenous μ-PhO and the exogenous μ-OH
groups, where each Cu ion takes square pyramidal geometry
with three N atoms of a pendant cyclen and two O atoms of
the μ-OPh-μ-OH bridge. As previously reported, however,
there is no μ-OH bridge in 1 where a Cu ion is coordinated by
four N-atoms of pendant cyclen and the O-atom of the μ-PhO
bridge. The Cu···Cu distance 3.047(11) Å of 2 is shorter than
the 3.878(1) Å of 1, due to the μ-PhO-μ-OH double bridge in
2. The tether groups make these distinct differences in the Cu
coordination structures between 1 and 2.
The structure of 2 in an aqueous solution was examined by
the spectroscopic studies. The ESI-MS spectrum of 2 in an
aqueous solution is shown in Figure S1. The major peak
appeared at m/z 745 corresponding to [bcamide + 2Cu(II) +
OH + ClO4]+. This shows that the μ-OPh-μ-OH bridge of 2 is
kept in an aqueous solution. In the electronic absorption
spectrum of 2 (Figure S2), two distinct bands appear at 326
and 390 nm assignable to HO− and PhO− to Cu(II) LMCT
bands. These are similar to the LMCT bands at 340−400 nm
reported for the related μ-OPh-μ-OH double bridged
dicopper(II) complexes.17,18
The reactivities of 1 and 2 in the DNA cleavage were
examined using supercoiled plasmid pUC19 DNA (form I) as
a substrate in the absence and presence of H2O2 under
physiological pH 6.0−8.2 at 37 °C. Forms I, II, and III of DNA
denote supercoiled close-circular, nicked circular, and linear
double-strand DNA, respectively. Form I is converted to
Forms II and III in the DNA cleavage. The amounts of Forms
I, II, and III were analyzed by agarose gel electrophoresis. The
gel patterns and time courses are shown in Figures S3−S12
and Tables S3−S7. The decreases of percent of Form I versus
time in the reactions using 1 and 2 at pH 6.0 in the presence of
H2O2 (0.5 mM) are shown in Figure 2. The decreased rate of
Form I in the reaction of the methylene-tether complex 1 in
the presence of H2O2 is exactly the same as that in the absence
of H2O2. Thus, 1 did not accelerate the oxidative DNA
cleavage with H2O2 at all. The decrease of percent of Form I in
The oxidative DNA cleavage studies in the presence of
various inhibitors, NaN3 (singlet oxygen scavenger), DMSO
(hydroxy radical scavenger), and KI (superoxide scavenger),
were also carried out to identify the active species that may be
formed in the reaction of 2 with H2O2. The results are shown
in Figures S11 and S12 and Tables S7. The addition of NaN3
(0.5 mM) and DMSO (0.5 mM) to the reaction of 2 slightly
enhanced the DNA cleavage but did not inhibit at all,
suggesting that both a singlet oxygen and a diffusible hydroxy
radical are not the active species in the DNA cleavage. The
B
DOI: 10.1021/acs.inorgchem.9b02093
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Communication
reaction was only slightly inhibited when KI (0.5 mM) was
added to the reaction of 2. In this case, KI acts as a reductant,
and thus, H2O2 by itself and a Cu-bonded peroxide complex
are possible candidates as the active species in the DNA
cleavage. The former is not the active species because the DNA
cleavage did not occur when only H2O2 was used. Both 2 and
H2O2 are essential for DNA cleavage.
To detect the active species, the reactions of complexes, 1
and 2, with H2O2 were monitored on the electronic absorption
spectra. Upon the addition of H2O2 to a solution of the
methylene-tether complex 1, any spectral change did not occur
(Figure S13), showing that 1 does not bind H2O2. This is
consistent with no reactivity of 1 in the oxidative DNA
cleavage with H2O2. On the other hand, the amide-tether
complex 2 reacted with H2O2 to give clear bands at 340 (ε =
5600 M−1 cm−1) and 398 (ε = 4800 M−1 cm−1) nm (Figure
S13). The band at 398 nm is similar to HO2− to Cu(II) LMCT
bands around 390−400 nm reported for various μ-1,1-
hydroperoxodicopper(II) complexes,19 indicating that a μ-
1,1-hydroperoxodicopper(II) species [Cu2(O2H)(bcamide)]2+
(3) may be formed in the reaction of 2 with H2O2. Formation
Figure 4. A proposed chemical structure of the μ-1,1-
hydroperoxodicopper(II) complex 3.
tetraguaiacol as an oxidized product, and the typical absorption
bands appeared at 412 and 470 nm.21 The concentration of
tetraguaiacol obtained was estimated as 0.05 mM from
absorbance at 470 nm (ε = 26.6 mM−1 cm−1;21a,22 Figure
S17B) where 0.2 mM of 3 was used to form tetraguaiacol
because 4 equiv of 3 is consumed to convert guaiacol to
tetraguaiacol. Thus, the yield of the tetraguaiacol was ca. 80%
on the basis of 3 used. These results clearly show 3 is the active
species in the reaction. The increase of tetraguaiacol was
monitored at 500 nm, and the reaction rate was estimated as
2.93 × 10−2 min−1 (Figure S17C). Guaiacol was not oxidized
only by H2O2. These results indicate that 3 is the direct
oxidant in the oxidative DNA cleavage.
Finally, we examined cytotoxicity of the methylene-tether
complex 1 and the amide tether complex 2 against HeLa cells.
HeLa is a human cell line derived from cervical cancer cells
taken from Henrietta Lacks, a patient who died of cancer. The
cytotoxicity was determined by means of MTT assay.23 The
details are described in the Supporting Information. The IC50
values (Table S8 and Figure S20) clearly show that 2 is more
effective in the cytotoxicity than 1. This is proportional to the
high DNA cleavage ability of 2.
−
of 3 was not affected by Et3N, small phosphate anions, and N3
ion as shown in Figure S14B−E. The formation of 3 is shown
by CSI MS spectra, having two major peaks at m/z 331 and
761 corresponding to [bcamide +2Cu(II) + O2H]2+ and
[bcamide +2Cu(II) + O2H + ClO4]+, respectively (Figure
S15). When 18O-labeled H218O2 was used instead of H216O2,
these shifted to m/z 333 and 765, demonstrating that two O
atoms of 3 come from H2O2 (Figure S16). The CSI MS
spectrum measured upon the reaction of 2 with H2O2 in H2O
gave two major peaks at m/z 331 and 761 (Figure S21),
showing that 3 is formed in the oxidative DNA cleavage in
H2O. Moreover, the formation of 3 in H2O was also observed
on the electronic absorption spectrum (Figure S22).
The resonance Raman spectra of 3 are shown in Figure 3. A
clear band appeared at 897 cm−1 upon reaction of 2 with
In this study, we synthesized a new amide-tether ligand and
its dicopper(II) complex [Cu2(μ-OH)(bcamide)](ClO4)2 (2)
and found that the amide-tether complex 2 largely accelerated
the oxidative DNA cleavage with H2O2. Moreover, 2 reacts
with H2O2 to form the active species, which was spectroscopi-
cally identified as a μ-1,1-hydroperoxodicopper(II) complex 3.
These results may shed light on the development of new
anticancer drugs to reduce the heavy side-effect.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.9b02093.
■
S
Figure 3. (A) Resonance Raman spectra of 3 prepared from 2 and
excess amounts of H216O2 (red) or H218O2 (blue) in MeOH at −30
°C with excitation at 405 nm. (B) A difference spectrum of A. S and *
mean solvent and H2O2.
Detailed experimental procedures, syntheses of ligands
and complexes, Figures S1−S22, Tables S1−S8, and
Schemes S1 and S2 (PDF)
H216O2. By using H218O2 instead of H216O2, this band shifted
to 842 cm−1 due to the isotope shift (55 cm−1). These data are
similar to the νO−O band at 892 cm−1 and an isotope shift of 52
cm−1 of μ-1,1-hydroperoxodicopper(II) complex with the p-
cresol derived ligand.20 Thus, 3 is proposed as a μ-1,1-
hydroperoxodicopper(II) complex shown in Figure 4.
The reaction of 3 (0.25 mM) with guaiacol (7.5 mM) was
monitored by the electronic absorption spectral change (Figure
S17), where 3 readily oxidized guaiacol to give corresponding
Accession Codes
CCDC 1937747 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk, or by contacting The Cam-
bridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
C
DOI: 10.1021/acs.inorgchem.9b02093
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Communication
(10) Adamson, I. Y. R. Pulmonary toxicity of bleomycin. Environ.
Health Perspect. 1976, 16, 119−126.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: mkodera@mail.doshisha.ac.jp.
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Normal and Tumor Stem Cells. Adv. Cancer Res. 2014, 122, 1−67.
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Oxygen Species Homeostasis in Cancer Cells and Implications for
Cancer Therapy. Clin. Cancer Res. 2013, 19, 4309−4314. (b) Sza-
trowski, T. P.; Nathan, C. F. Production of Large Amounts of
hydrogen peroxide by Human Tumor Cells. Cancer Res. 1991, 51,
794−798.
ORCID
Yutaka Hitomi: 0000-0002-2448-296X
Masahito Kodera: 0000-0001-9979-1743
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was supported by MEXT-Supported Program of the
Strategic Research Foundation at Private University, 2015−
2019.
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DOI: 10.1021/acs.inorgchem.9b02093
Inorg. Chem. XXXX, XXX, XXX−XXX