Acceleration of hydrolytic DNA cleavage by dicopper(II) complexes with p-cresol-derived dinucleating ligands at slightly acidic pH and mechanistic insights

Article abstract of DOI:10.1246/bcsj.20180353

Four dicopper(II) complexes, [Cu₂(ˉ-X)(bcmp)](ClO₄)₂ [X = OH (1a) and X = Cl (1b)], [Cu₂(ˉ-OH)(Me₄bcmp)]-(ClO₄)₂ (2), and [Cu₂(bcc)](ClO₄)₃ (3),

Full text of DOI:10.1246/bcsj.20180353

Document type: Article
Selected Paper
Acceleration of Hydrolytic DNA Cleavage by Dicopper(II) Complexes
with p-Cresol-Derived Dinucleating Ligands at Slightly Acidic pH
and Mechanistic Insights
Masahito Kodera,* Yuki Kadoya, Kenta Aso, Katsuki Fukui, Akiko Nomura,
Yutaka Hitomi, and Hiroaki Kitagishi
Department of Molecular Chemistry and Biochemistry, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan
E-mail: mkodera@mail.doshisha.ac.jp
Received: November 17, 2018; Accepted: December 26, 2018; Web Released: January 22, 2019
Masahito Kodera
Masahito Kodera is a Professor at the Department of Molecular Chemistry and Biochemistry, Graduate
School of Science and Engineering, Doshisha University. He received his Ph.D. at the Engineering
Graduate School, Kyoto University in 1987. He started his career as an Assistant Professor at the Institute
for Molecular Science in 1987, and moved to Kyushu University. He was an Assistant Professor at
Kyushu University from 1988 to 1993, and moved to Doshisha University. He was Lecturer at Doshisha
University from 1993 to 1994, Associate Professor from 1994 to 2001, and has been Professor from 2001
until now.
Abstract
1. Introduction
Four dicopper(II) complexes, [Cu2(¯-X)(bcmp)](ClO4)2
[X = OH (1a) and X = Cl (1b)], [Cu2(¯-OH)(Me4bcmp)]-
(ClO4)2 (2), and [Cu2(bcc)](ClO4)3 (3), were synthesized with
three p-cresol-derived ligands, 2,6-bis(1,4,7-triazacyclononyl-
methyl)-4-meth-ylphenol (Hbcmp), 2,6-bis(1,4,7-triaza-4,7-
Recently, there have been considerable interests in the devel-
opment of metal complexes for medical purposes by block-
ing DNA replication through binding and/or cutting DNA.1
Various platinum(II) complexes, cisplatin2 and its derivatives,
such as oxaliplatin,3 lobaplatin,4 and nedaplatin,5 are used as
anticancer drugs. Since these anticancer drugs are accompanied
with heavy side-effects and resistance problems, metal com-
plexes that bind and/or cut DNA of cancer cells selectively are
required to solve these problems.
It is well known that the microenvironment in cancer cells
is significantly different from that in normal cells.6 As one of
the key features of cancer cells, tumor extracellular pH is often
acidic.7 This is mainly caused by aerobic glycolysis. On the
other hand, intracellular pH of tumors is neutral to alkaline.7
Various dinuclear metal complexes capable of promoting
hydrolytic DNA cleavage have been synthesized for the
purpose of developing a new type of anticancer drugs.1a-1c,8
Montagner reported that a dicopper(II) complex with Hbcmp
[Cu2(¯-OH)(bcmp)](NO3)2 (1c) hydrolytically cleaved super-
coiled plasmid pUC19 DNA (SC-DNA) at pH 8.2, and showed
high cytotoxic activity to cancer cells, causing apoptosis.9 The
pH-dependent hydrolytic DNA cleavage by 1c, however, has
not been reported. The selective apoptosis of cancer cells by
using pH-dependent DNA cleavage of metal complexes is
desirable for the development of anticancer drugs that are less
toxic, target specific, and with reduced side effects. Even if
hydrolytic cleavage is reversible as repair enzymes repair DNA
damage in cancer cells, pH-dependent hydrolytic DNA cleav-
dimethylcyclonon-ylmethyl)-4-methylphenol
(HMe4bcmp),
and 2,6-bis(1,4,7,10-tetrazacyclododecylmethyl)-4-methylphe-
nol (Hbcc) to study hydrolytic DNA cleavage. Crystal struc-
tures of 1a, 1b, 2, and 3 were determined by X-ray analysis.
The pH titrations and spectroscopic studies in the complex-
ations of the ligands with copper(II) perchlorate revealed that
the dicopper core structures of 1a, 2, and 3 in the solid state are
kept at pH 5-9 in an aqueous solution. DNA binding abilities
of 1a, 2, and 3 were examined by isothermal titration calo-
rimetry (ITC). DNA cleavage studies were carried out by using
supercoiled plasmid pUC19 DNA. 1a largely accelerated
hydrolytic DNA cleavage at pH 5-6 but not at pH 7-8. This is
the first example of pH-dependent DNA cleavage by a dicopper
complex. Inhibition studies with specific DNA binders, 4¤,6-
diamidino-2-phenylindole and methyl green, suggested that 1a
accelerates the DNA cleavage via GC-specific binding. The
mechanistic insights into the pH-dependent DNA cleavage are
proposed on the basis of the crystal structures, structures in
aqueous solutions, DNA binding modes, and DNA cleavage
activities of 1a, 1b, 2, and 3.
Keywords: Dicopper(II) complex
j
DNA cleavage
j
Mechanism
Bull. Chem. Soc. Jpn. 2019, 92, 739–747 | doi:10.1246/bcsj.20180353
© 2019 The Chemical Society of Japan | 739

age may provide a new concept for a DNA cleavage study as
well as the development of anti-cancer drugs.
bined and dried over Na2SO4. After filtration and concentra-
tion, the product was obtained as yellow solid. Yield: 2.83 g
(98%). H NMR (500 MHz, CDCl3); ¤/ppm: 7.67 (d, J = 8.0
Hz, 2H, Ph), 7.30 (d, J = 8.0 Hz, 2H, Ph), 3.21-3.29 (m, 4H,
CH2), 2.87-2.94 (m, 4H, CH2), 2.69 (s, 4H, CH2), 2.42 (s, 3H,
CH3), 2.40 (s, 6H, CH3).
1
In this study, we synthesized four dicopper(II) complexes
[Cu2(¯-OH)(bcmp)](ClO4)2 (1a), [Cu2(¯-Cl)(bcmp)](ClO4)2
(1b), [Cu2(¯-OH)(Me4bcmp)](ClO4)2 (2), and [Cu2(bcc)]-
(ClO4)2 (3) to study the pH-dependence of hydrolytic DNA
cleavage by the dicopper complexes, and found that 1a largely
accelerated the hydrolytic cleavage of supercoiled plasmid
pUC19 DNA at pH 5.0-6.0, but did not at pH 7.0-8.0. Since
usually hydrolytic cleavage of DNA is accelerated under basic
conditions, the large acceleration at pH 5-6 is quite an unusual
example. For example, purple acid phosphatases specifically
accelerate the hydrolysis of phosphate ester at acidic pH. Here,
we propose mechanistic insights into the pH-dependent activity
control in the hydrolytic DNA cleavage of 1a.
1,4-Dimethyl-1,4,7-triazacyclononane.
1,4-Dimethyl-7-
tosyl-1,4,7-triazacyclononane (3.24 g, 10.4 mmol) was added to
conc. H2SO4 (15 mL) and heated at 120 °C for 36 h under N2.
The mixture was cooled to room temperature, and 12 M aque-
ous NaOH was slowly added until pH became 10. Generated
Na2SO4 was filtered off, and extracted with CHCl3 (5 © 40 mL).
The product was obtained from the extracts as yellow oil. Yield:
0.933 g (57%). 1H NMR (500 MHz, CDCl3); ¤/ppm: 2.65-2.71
(m, 4H, CH2), 2.50-2.57 (m, 8H, CH2), 2.41 (s, 6H, CH3).
2,6-Bis(N,N¤-4,7-dimethyl-1,4,7-triazacyclonon-1-ylmeth-
yl)-4-methylphenol Pentahydrochloride (HMe4bcmp¢5HCl).
1,4-Dimethyl-1,4,7-triazacyclononane (0.365 g, 2.32 mmol)
and 2,6-bis(chloromethyl)-4-methylphenol (0.215 g, 1.05
mmol) were dissolved in MeCN (40 mL) and Et3N (0.34 mL,
2.44 mmol) was added. The solution was refluxed for 12 h
under N2. After concentration, the remainder was purified by
column chromatography (alumina, CHCl3/MeOH) to give 2,6-
bis(N,N¤-4,7-dimethyl-1,4,7-triazacyclonon-1-ylmethyl)-4-
2. Experimental
Materials. All ordinary reagents were purchased and used
as received unless otherwise noted. A supercoiled plasmid
pUC19 DNA was purchased from Nippon Gene CO., LTD. A
33 mer oligo DNA 5¤-d(GAC TCC ACA GTG CAT ACG TGG
GCT CCA ACA GGT)-3¤ and the complementary strand were
purchased from Thermo Fisher Scientific, from which a 33
mer double-strand DNA was prepared. Two p-cresol-derived
ligands, 2,6-bis(1,4,7-triazacyclononylmethyl)-4-methylphenol
(Hbcmp) and 2,6-bis(1,4,7,10-tetrazacyclododecylmethyl)-4-
methylphenol (Hbcc) were prepared according to literature.10
Measurements. Elemental analyses (C, H, and N) were
carried out on a Perkin-Elmer Elemental Analyzer 2400 II. UV-
vis absorption spectra were recorded on an Agilent 8454 UV
spectroscopy system. The pH measurement was carried out on
a Horiba Laqua electrode. Electrospray ionization mass spectra
(ESI MS) were recorded on a JEOL JMS-T100CS spectrom-
eter. Infrared (IR) spectra were recorded on a Shimadzu Single
1
methylphenol as yellow oil. Yield: 0.330 g (70%). H NMR
(500 MHz, CDCl3); ¤/ppm: 6.87 (s, 2H, Ph), 3.72 (s, 4H, CH2),
2.84-2.89 (m, 8H, CH2), 2.63-2.69 (m, 16H, CH2), 2.35 (s,
12H, CH3), 2.23 (s, 3H, CH3). The yellow oil product (1.19 g,
2.66 mmol) was dissolved in EtOH (27 mL) and 12 M HCl
(9 mL) was added. The mixture was concentrated to dryness.
After filtration and washing with EtOAc, the product was
obtained as white solid. Yield: 1.41 g (2.24 mmol, 84%). Anal.
calcd for HMe4bcmp¢5HCl¢5H2O: C, 41.76; H, 8.55; N, 11.69.
Found: C, 41.69; H, 8.83; N, 12.22. 1H NMR (500 MHz, D2O);
¤/ppm: 7.39 (s, 2H, Ph), 4.21 (s, 4H, CH2), 3.10-3.79 (m, 24H,
CH2), 2.94 (s, 12H, CH3), 2.34 (s, 3H, CH3).
Dicopper Complexes 1a, 1b, 2, and 3. To a solution of
Hbcmp (0.149 mmol) in H2O (10 mL) was added a solution
of Cu(ClO4)2¢6H2O (126.6 mg, 0.342 mmol) in H2O (4 mL).
The solution was neutralized with 1.0 M aqueous NaOH under
N2. After concentration, [Cu2(¯-OH)(bcmp)](ClO4)2 (1a) was
obtained as green solid and recrystallized from Et2O/MeCN
to give green crystals suitable for the X-ray analysis. Yield:
34.8 mg (44.5 ¯mol, 30%). Anal. calcd for [Cu2(¯-OH)bcmp]
(ClO4)2¢MeCN¢0.5H2O: C, 35.30; H, 5.41; N, 12.53. Found:
1
Reflection HATR IR Affinity-1 MIRacle 10. H NMR spectra
were recorded on a JEOL ECA-500RX spectrometer using
Me4Si or TSP as an internal standard.
1-Tosyl-1,4,7-triazacyclononane. 1,4,7-Tritosyl-1,4,7-tri-
azacyclononane (7.84 g, 13.2 mmol) and phenol (9.43 g, 100
mmol) were dissolved in 30% HBr solution in AcOH (110 mL).
The solution was stirred for several hours at 30 °C until evolu-
tion of HBr ceased and then heated at 90 °C for 2 days. The
HBr salt of the product generated was collected by filtration
and washed with Et2O. The solid was dissolved in 1.5 M aque-
ous NaOH (100 mL) and extracted with CHCl3 (4 © 15 mL).
The combined organic layer was dried over Na2SO4. After
filtration and concentration, the product was obtained as
C, 35.48; H, 5.50; N, 12.21. ESI MS (H2O/MeCN m/z);
+
[1a ¹ ClO4] : 632.93. IR (KBr); v/cm¹1: 3586-2864, 1613,
~
1
white solid. Yield: 2.70 g (72%). H NMR (500 MHz, CDCl3);
1479, 1458, 1375, 1082. [Cu2(¯-Cl)(bcmp)](ClO4)2 (1b) was
obtained from Hbcmp¢6HCl. Yield: 11.9 mg (31%). Anal.
calcd for [Cu2(¯-Cl)bcmp](ClO4)2¢MeCN: C, 34.88; H, 5.09;
N, 12.38. Found: C, 35.00; H, 5.14; N, 12.20. ESI MS (H2O/
MeCN m/z); [1b ¹ ClO4]+: 650.89. v~/cm¹1: 3620-2872,
1614, 1476, 1446, 1354, 1103. [Cu2(¯-OH)(Me4bcmp)](ClO4)2
(2) was obtained from HMe4bcmp. Yield: 94.3 mg (52%).
Anal. calcd for [Cu2(¯-OH)Me4bcmp](ClO4)2¢MeCN¢2H2O:
C, 37.46; H, 6.17; N, 11.33. Found: C, 37.72; H, 6.26; N,
11.08. ESI-MS (H2O/MeCN m/z); [2 ¹ ClO4]+: 689.04.
¤/ppm: 7.69 (d, J = 8.0 Hz, 2H, Ph), 7.31 (d, J = 8.0 Hz, 2H,
Ph), 3.16-3.21 (m, 4H, CH2), 3.05-3.11 (m, 4H, CH2), 2.89
(s, 4H, CH2), 2.43 (s, 3H, CH3).
1,4-Dimethyl-7-tosyl-1,4,7-triazacyclononane.
Formic
acid (10 mL) and formaldehyde (10 mL) were added dropwise
to a solution of 1-tosyl-1,4,7-triazacyclononane (2.62 g, 9.25
mmol) in H2O (3 mL) at 0 °C. The mixture was stirred for 30
min, and refluxed at 110 °C for 15 h. To the resultant mixture
was added 12 M HCl (6 mL), and concentrated to dryness. To
the residue was added 1.5 M aqueous NaOH (20 mL) and
extracted with CHCl3 (4 © 20 mL). The extracts were com-
v/cm¹1: 3613-2868, 1614, 1481, 1472, 1431, 1366, 1082.
~
[Cu2(bcc)](ClO4)3 (3) was obtained from Hbcc and recrystal-
740 | Bull. Chem. Soc. Jpn. 2019, 92, 739–747 | doi:10.1246/bcsj.20180353
© 2019 The Chemical Society of Japan

lized from Et2O/MeCN/MeOH. Yield: 0.119 g (50%). Anal.
calcd for [Cu2bcc](ClO4)3¢MeOH¢1.5H2O: C, 32.52; H, 5.67;
N, 11.67. Found: C, 32.46; H, 5.41, N; 11.81. ESI MS (H2O/
MeCN m/z); [3 ¹ ClO4]+: 801.12, [3 ¹ 2ClO4]2+: 351.08.
v~/cm¹1: 3620-293, 1616, 1470, 1441, 1381-1306, 1099.
Structure Determination of Single Crystals. The crystal
structures of 1a, 1b, 2 and 3 were determined on a Rigaku R-
AXIS RAPID diffractometer using multi-layer mirror mono-
chromated Cu-Kα radiation. The data were collected at a
temperature of ¹160 « 1 °C or ¹180 « 1 °C to a maximum
pH 5.0 and 5.5 (MES)) containing NaCl (10 mM) in the
presence of DAPI or methyl green (5, 10, 50 ¯M), and 1a
(10 ¯M) was added and incubated at 37 °C for 1 h in the dark.
Progress of the reaction was evaluated as shown in the DNA
cleavage study.
3. Results and Discussion
Syntheses and Structures of Dicopper Complexes. The
chemical structures of 2,6-bis(1,4,7-triazacyclononylmethyl)-4-
methylphenol (Hbcmp), 2,6-bis(1,4,7-triaza-4,7-dimethylcyclo-
nonylmethyl)-4-methylphenol (HMe4bcmp), and 2,6-bis-
(1,4,7,10-tetrazacyclododecylmethyl)-4-methylphenol (Hbcc)
are shown in Scheme 1. Hbcmp and Hbcc have triazacyclo-
nonane (tacn) and tetraazacyclododecane (cyclen) as pendant
groups at 2,6-positions of the p-cresol moiety, respectively, and
prepared according to the literature.10 HMe4bcmp is a new
ligand where four macrocyclic NH groups of Hbcmp are fully
methylated. The synthetic route of HMe4bcmp is shown in
Scheme 2. These ligands form dicopper(II) complexes [Cu2-
(¯-X)(bcmp)](ClO4)2 [X = OH (1a) and X = Cl (1b)], [Cu2-
(¯-OH)(Me4bcmp)](ClO4)2 (2), and [Cu2(bcc)](ClO4)3 (3). The
complexes were structurally characterized by X-ray analysis,
and characterized by elemental analysis and various spectro-
scopic measurements. As described below, the ligands stabilize
the dinuclear structures at pH 5-9 in an aqueous solution.
ORTEP views of 1a, 1b, 2, and 3 are shown in Figure 1. The
crystallographic data are shown in Table S1, and the selected
bond distances and angles are shown in Table S2. In 1a, two
Cu(II) ions are incorporated into a bcmp ligand with the ¯-
OPh-¯-OH double bridge, where each Cu ion takes distorted
square pyramidal geometry with three N-atoms of a pendant
tacn and two O-atoms of the ¯-OPh-¯-OH bridge. 1b is struc-
turally almost the same as 1a but ¯-OH of 1a is replaced by
¯-Cl. In 2, two Cu(II) ions are incorporated into a Me4bcmp
ligand with the ¯-PhO-¯-OH double bridge. The overall
structures of 1a and 2 are almost the same except for NCH3
2θ value of 136.5°. The linear absorption coefficient, ¯, for
¹1
Cu-Kα radiation is 31.825, 38.394, 39.633, or 44.927 cm
.
An empirical absorption correction was applied. The data were
corrected for Lorentz and polarization effects. Crystallographic
data of the complexes are shown in Table S1. Crystallographic
data reported in this manuscript have been deposited with
Cambridge Crystallographic Data Centre as supplementary
publication no. CCDC-000000. Copies of the data can be
obtained free of charge via CCDC Website.
pH Change and Spectral Studies in Complexation of
Hbcmp, HMe4bcmp, and Hbcc. A solution of each ligand
(0.5 mM) and Cu(ClO4)2¢6H2O (1.0 mM) in H2O (2.0 mL) was
placed in a cuvette, and titrated with an aqueous NaOH
(0.05 M) at 25 °C. The pH change was monitored on a pH
meter. The electronic absorption and ESI MS spectra were
measured using the same solution after measuring pH.
Isothermal Titration Calorimetry (ITC).
ITC mea-
surements were performed at 37 °C on a Malvern MacroCal
VP-ITC. The solution of the 33 mer oligo ds-DNA (25 ¯M) in
a buffered aqueous solution ((MES, 10 mM) containing NaCl
(10 mM)) was prepared in the cell. The solution of 1a, 2, or 3
(1.0 mM) in a buffered aqueous solution (MES, 10 mM) con-
taining NaCl (10 mM) was prepared in the syringe. The 25
aliquots (10 ¯L each) of the solution of 1a, 2, or 3 were added
into the DNA solution to measure the heat of complexation.
The raw data was corrected by subtracting the heat of dilution,
and the titration curve thus obtained was analyzed using
ORIGIN software. Each ITC data was collected by two inde-
pendent measurers and the reproducible data was employed.
DNA Cleavage Study. DNA cleavage activity of 1a, 1b, 2,
and 3 were evaluated by using pUC19 DNA and followed
by agarose gel electrophoresis. pUC19 DNA (0.50 ¯g/¯L) was
incubated with the dicopper complex (10 ¯M) in an aqueous
buffer solution (10 mM, pH 5.0, 5.5, 5.9, 6.0 (MES), 7.4 (Tris-
HCl), and 8.2 (TAPS)) containing NaCl (10 mM) at 37 °C in
the dark. An aliquot was taken from the solution at 0, 1, 2, 5,
and 10 h, and the reaction was quenched upon addition of load-
ing buffer (0.025% bromophenol blue, 0.025% xylene cyanol
FF, 1.0 mM EDTA and 30% glycerol). Each sample was loaded
onto a 1% agarose gel in TAE (Tris/acetate/EDTA) buffer. The
gels were subjected to electrophoresis for 1 h at 100 V, followed
by staining with ethidium bromide (0.5 ¯g/mL) for 1 h. Gel
bands were visualized using UV transilluminator and photo-
graphed using a Vilber Lourmat ECX-20-M, and were quan-
tified using a correction factor of 1.06 for the reduced stain
uptake of Form I. All data for the conversion of Form I to
Form II are shown in Figure S5-S13 and Table S4-S12.
Inhibition of the DNA Cleavage of 1a. pUC19 DNA (0.50
¯g/¯L) was dissolved in an aqueous buffer solution (10 mM,
Scheme 1. Chemical structures of supporting ligands.
Scheme 2. Synthetic scheme of H2Mebcmp.
Bull. Chem. Soc. Jpn. 2019, 92, 739–747 | doi:10.1246/bcsj.20180353
© 2019 The Chemical Society of Japan | 741

(A)
(B)
(A)
(B)
(C)
(D)
(C)
(D)
Figure 2. pH change of an aqueous solution containing
each ligand (0.5 mM), (A) Hbcmp, (C) HMe4bcmp, or (D)
Hbcc, and Cu(ClO4)2¢6H2O (1.0 mM) upon titration with
an aqueous NaOH solution (0.05 M) at 25 °C. In the case of
(B), 1b was used instead of the ligand, and the pH change
was monitored in the same way.
Figure 1. ORTEP diagrams of the cationic parts of (A) 1a,
(B) 1b, (C) 2, and (D) 3 (ORTEP plot; unlabeled open
ellipsoids represent carbon atoms).
groups in 2. In 3, two Cu(II) ions are incorporated into a bcc
ligand with the ¯-PhO bridge, and each Cu takes square
pyramidal geometry with four N-atoms of a pendant cyclen and
a O-atom of the ¯-OPh bridge. The Cu£Cu distances of 1a and
2 are 2.9985(7) and 3.033(1) ¡, close to each other, but shorter
than 3.878(1) ¡ of 3. This may reflect differences of the bridg-
ing structures where the ¯-PhO-¯-OH doubly bridged struc-
tures of 1a and 2 shorten the Cu£Cu distances as compared
with the ¯-PhO singly bridged one of 3. The coordination
geometry is examined by the τ-values that vary from 0, for an
idealized square pyramidal, to 1, for an idealized trigonal
bipyramidal.11 The τ-values, τCu(1) = 0.243 and τCu(2) = 0.228
Dicopper Core Structures at pH 5-9 in an Aqueous
Solution. To examine dicopper core structures at pH 5-9 in an
aqueous solution, complexations of the ligands were monitored
by pH and spectroscopic measurements. To an aqueous solu-
tion of a 1:2 mixture of each ligand and Cu(ClO4)2¢6H2O was
added standardized aqueous NaOH, and the pH change was
monitored on a pH meter and the electronic absorption and
electrospray ionization mass (ESI MS) spectra were measured.
The plots of the pH change vs molar equivalent of NaOH added
are shown in Figure 2. The electronic absorption and ESI MS
spectra at various pH are shown in Figures S2 and S3,
respectively.
for 1a, τCu(1) = 0.217 and τCu(2) = 0.206 for 2, and τCu(1)
=
0.038 and τCu(2) = 0.066 for 3, show that 1a and 2 are distorted
square pyramidal similar to each other and 3 is regular square
pyramidal.
In the complexations of Hbcmp and HMe4bcmp, 2 eq of
¹
OH was consumed by pH 5.0 as shown in Figure 2(A) and
In spite of the similar bridging structures of 1a and 2, the
accessibility to the ¯-OH group is significantly different. The
O-atom of ¯-OH in 1a protrudes by 0.842 ¡ from the mean
plane defined by four N-atoms of macrocyclic NH groups
(Figure S1), clearly showing that the ¯-OH group is easy to
access from outside. On the other hand, the O-atom of ¯-OH in
2 is surrounded by the NCH3 groups that sterically hinder the
access to the ¯-OH group, where the deviation of the O-atom of
¯-OH from the mean plane defined by four C-atoms of NCH3
groups is 0.001 ¡ (Figure S1). Moreover, the Cu-O¯-OH bond
distances 1.971(2) and 1.980(3) ¡ of 1a are significantly longer
than 1.930(3) and 1.925(4) ¡ of 2. These data indicate that the
¯-OH bridge of 1a weakly bonds to two Cu(II) ions. These
structural features suggest that the ¯-OH bridge of 1a is more
reactive in the nucleophilic attack to an external substrate such
as a phosphate moiety of DNA than that of 2.
(C). This can be assigned to the release of two protons
belonging to the phenol of each ligand and to a H2O bonded to
dicopper(II) core to form ¯-OPh-¯-OH bridge. This is con-
sistent with pKa value of the H2O bonded to the dicopper(II)
core, 4.69, reported for 1c.9 The electronic absorption spectra in
complexation of Hbcmp and HMe4bcmp at pH 5-9 are almost
the same as those of 1a and 2 in an aqueous solution, respec-
tively (Figure S2). The spectra of 1a and 2 exhibit two bands at
around 340-380 nm assignable to phenoxo-Cu(II) and hydroxo-
Cu(II) LMCT (ligand-to-metal charge transfer) bands, taking
account of the reported data that the LMCT bands appear at
350-400 nm in related dicopper(II) complexes.12 Moreover, the
bridging structures of 1a and 2 are confirmed by the ESI MS
spectra shown in Figure S3. The ESI MS spectra of 1a and 2 at
pH 5-9 in aqueous solutions show the major peaks at m/z 632
and 689 corresponding to [bcmp + 2Cu(II) + OH + ClO4]+
742 | Bull. Chem. Soc. Jpn. 2019, 92, 739–747 | doi:10.1246/bcsj.20180353
© 2019 The Chemical Society of Japan

and [Me4bcmp + 2Cu(II) + OH + ClO4]+, respectively. These
results show that the ¯-OPh-¯-OH bridges of 1a and 2 are kept
at pH 5-9. Upon addition of aqueous NaOH to 1b in an aqueous
(A)
(B)
¹
solution, 1 eq of OH was consumed by pH 5.0, (Figure 2(B)),
showing that ¯-Cl of 1b was replaced by ¯-OH, and thus, 1b is
converted to 1a in an aqueous solution.
¹
In the complexation of Hbcc, 1 eq of OH was consumed by
pH 5.0 (Figure 2(D)). The electronic absorption spectra in the
complexation of Hbcc at pH 5-9 are the same as that of 3 in an
aqueous solution (Figure S2), which exhibits a weak band at
410 nm assignable to the phenoxo-Cu(II) LMCT.13 The ESI MS
spectra of 3 at pH 5-9 in an aqueous solution show the major
peak at m/z 801 corresponding to [bcc + 2Cu(II) + 2ClO4]+
(Figure S3). These results show that the ¯-OPh bridged struc-
ture of 3 is kept at pH 5-9 in an aqueous solution.
(C)
(D)
Thus, the ¯-OPh-¯-OH bridges of 1a and 2 and the ¯-OPh
bridge of 3 are kept intact at pH 5-9 in H2O. It was report-
ed that the ¯-OPh-¯-OH bridge is kept intact at pH 5-12
in dicopper(II) complex with 2,6-bis(dipyridylmethylamino-
methyl)-4-methyl-phenol, a p-cresol-derived heptadentate di-
nucleating ligand.14a Thus, the ¯-OPh-¯-OH bridging struc-
tures are common in the dicopper(II) complexes with the p-
cresol-derived heptadentate ligands in the wide range of pH.
On the other hand, the ¯-OPh singly bridged structure of 3 is
kept with the bcc ligand under the same conditions.
(E)
DNA Binding of Dicopper(II) Complexes. The pH and
structural effect on the DNA binding ability of 1a, 2, and 3
were explored by isothermal titration calorimetry (ITC). By
the ITC measurement, the binding enthalpy (¦H°) is directly
obtained, and the entropy (¦S°) and the binding constant (K)
are derived from curve fitting and free energy relationship. A
linear 33 mer oligo double-stranded DNA was used for the
measurement of the DNA binding constant where the hydro-
lytic DNA cleavage by the dicopper complexes can be elimi-
nated because of the low reactivity of linear DNA. The isother-
mal calorimetric titration curves were recorded for the titration
of 1a into the 33 mer oligo double-stranded DNA solution in
10 mM of MES buffer at pH 5.0, 5.5, and 6.0 in the presence
of NaCl (10 mM) to keep a constant ionic strength. Moreover,
the titration curves for DNA binding of 2 and 3 were obtained
at pH 6.0 under the same conditions. The thermodynamic
parameters, ¦H°, ¦S°, and K, are shown in Table S3. Negative
¦H° and positive ¦S° were obtained in all measurements,
showing that DNA binding of 1a, 2, and 3 are enthalpically and
entropically favorable. The ITC profiles and the corresponding
molar ratio plots are shown in Figure 3.
The DNA binding constants of 1a determined at pH 5.0, 5.5,
and 6.0 are 1.81 « 0.84 © 105, 1.23 « 0.43 © 105, and 1.05 «
0.20 © 105 M¹1, respectively (Table S3), and slightly increas-
ing with lowering pH, indicating that a binding complex of 1a
with DNA is stabilized by the protonation. Since the structure
of 1a is kept intact at pH 5.0-9.0 in an aqueous solution, 1a is
not protonated in the pH domain. Thus, the protonation occurs
in the binding complex of 1a with DNA. The ¦H° and ¦S°
values obtained at pH 5.0, 5.5, and 6.0 are ¹0.57 « 0.08,
¹1.08 « 0.24, and ¹1.39 « 0.19 (kcal¢mol¹1) and 22.2, 19.8,
and 18.5 (cal¢mol¹1¢K¹1), respectively (Table S3). Both ¦H°
and ¦S° values increased with lowering pH, suggesting that the
protonation may occur at the phosphate moiety of the DNA
Figure 3. Isothermal calorimetric titration curves: 1a at
pH 5.0 (A), 5.5 (B), 6.0 (C), 2 at pH 6.0 (D), and 3 at
pH 6.0 (E). Experimental conditions: A solution of 1a
(1 ¯M) in a syringe was added, in an equal interval 25
times, to a solution of the linear 33 mer ds-DNA (25 ¯M)
in the cell in the presence of NaCl (10 mM) in an aqueous
buffer solution at pH 5.0, 5.5, and 6.0 (MES, 10 mM) at
37 °C. In the case of 2 and 3, titrations were conducted in
the same manner at pH 6.0 (MES, 10 mM).
main chain in the binding complex to induce dehydration.
Thus, the increase in the binding constant of 1a with DNA at
low pH is entropy-driven.
Moreover, the DNA binding abilities of 2 and 3 may be
explained from both hydrophobic and electrostatic interactions
with DNA. The DNA binding constants of 2 and 3 determined
at pH 6.0 are 2.64 « 0.97 © 105, and 2.63 « 0.53 © 105 M
¹1
,
respectively (Table S3). These values are almost the same.
Interestingly, however, the ¦H° and ¦S° values, ¹0.48 « 0.06
(kcal¢mol¹1) and 23.2 (cal¢mol¹1¢K¹1) for 2, and ¹2.26 «
0.11 (kcal¢mol¹1) and 17.5 (cal¢mol¹1¢K¹1) for 3, are largely
different between 2 and 3. The increase of entropy shows the
increase of degree of freedom in the system. Therefore, the
largest ¦S° value 23.2 indicates that hydrophobic interaction of
Bull. Chem. Soc. Jpn. 2019, 92, 739–747 | doi:10.1246/bcsj.20180353
© 2019 The Chemical Society of Japan | 743

(A)
(B)
2 with DNA plays a key contribution in the complexation
where dehydration from 2 and DNA causes the ¦S° value. The
hydrophobic interaction is important because 2 is the most
hydrophobic in 1a, 1b, 2, and 3. The negative enthalpy shows
the formation of some new bonds including H-bond and
electrostatic interaction. In this case, the least exothermic ¦H°
value ¹0.48 in the complexation of 2 with DNA indicates that
the bonding interactions are not so important. This is reason-
able because 2 does not have NH groups capable of forming H-
bonds. On the basis of the same consideration, ¦H° and ¦S°
values ¹2.26 and 17.5 in complexation of 3 with DNA clearly
show that H-bond and electrostatic interaction largely contrib-
ute, and the hydrophobic interaction is not so important. This is
reasonable because 3 has six NH groups with net charge 3+,
and thus, the most hydrophilic. The ¦H° values of 1a, 2, and
3 at pH 6 are ¹1.39, ¹0.48, and ¹2.26, respectively. 1a and
3 are more exothermic than 2. This shows that the H-bonding
interactions of the NH groups play a key role in the DNA
binding. Thus, the chemical understandings of the DNA bind-
ing abilities are deduced from thermodynamic parameters ¦H°
and ¦S°. Moreover, these results show that the interaction of
1a, 2, and 3 with DNA is controlled with chemical modifica-
tions of the pendant macrocyclic groups.
(C)
(D)
(E)
Acceleration of Hydrolytic Cleavage of Supercoiled
Plasmid pUC19 DNA with Dicopper(II) Complexes in
Slightly Acidic pH.
Montagner et al. reported that 1c
Figure 4. pH-dependent profile for DNA cleavage pro-
moted by (A) Blank, (B) 1a, (C) 1b, (D) 2, and (E) 3,
respectively. Experimental conditions: [NaCl] = 10 mM,
[buffer] = 10 mM (pH 5.0, 5.5, 5.9, 6.0 (MES), 7.4 (Tris-
HCl), and 8.2 (TAPS)), [pUC19 DNA] = 50 ¯Mbp,
[complex] = 10 ¯M at 37 °C for 0, 1, 2, 5, and 10 h.
Experiments were carried out at least three times.
promoted hydrolytic cleavage of supercoiled plasmid pUC19
DNA (SC-DNA, Form I) in an aqueous buffer solution at
pH 8.2 at 37 °C,9 and that the reaction was dependent on con-
centration of 1c where at 50 ¯M of 1c, almost complete nick-
ing of Form I to Form II was observed in 3 h, but at 5 ¯M of
1c, Form II was not observed. In our study, the reaction of
SC-DNA with 1a (10 ¯M) was examined at 37 °C in aqueous
solutions at pH 5.0, 5.5, 5.9, 6.0, 7.4, and 8.2. In the absence of
the dicopper(II) complex, the DNA cleavage was not observed
at all as shown in Figure 4(A) of the blank experiment. Time
courses of the decrease of Form I in the reaction of 1a at
pH 5.0-8.2 are shown in Figure 4(B). The DNA cleavage was
largely accelerated at pH 5.0-6.0. By using such a small
amount, 10 ¯M, of 1a, 80% conversion of Form I to Form II
was attained at pH 5.0 for 5 h. Interestingly, however, no DNA
cleavage activity was observed at pH 7.4 and 8.2. Tris buffer
used at pH 7.4 is known to coordinate Cu(II) ion,14b but in our
case, gave no influence because the electronic spectra of 1a in
aqueous solutions using MES, Tris-HCl, and TAPS as buffer
were completely the same each other. As shown in Figure 4(C),
1b shows exactly the same activity as 1a. This is because 1b is
rapidly converted to 1a at pH 5-9 in an aqueous solution.
To examine the possibility of oxidative DNA cleavage by
1a, the reaction was carried out in the presence of H2O2 (100-
500 ¯M), but no acceleration occurred (Figure S4). Moreover,
it was examined in the presence of NaN3 and dimethyl sulf-
oxide that are known to inhibit oxidative DNA cleavage by
singlet oxygen and hydroxyl radical, respectively, but the DNA
cleavage of 1a was not inhibited (Figure S4). Thus, it is
demonstrated that the reaction of 1a is not the oxidative DNA
cleavage.
one of the distinctive natures of cancer cells, it is known that
pH7a-7c and concentrations of reactive oxygen species (ROS)15
in cancer cells are significantly different from those in normal
cells. If we could control DNA cleavage activity of metal
complexes depending on pH and ROS concentrations, it
may serve to develop new types of anti-cancer drugs that selec-
tively kill cancer cells but not normal cells. Therefore, pH-
dependent acceleration of the hydrolytic DNA cleavage by 1a
may provide some insight into the development of a new type
of anti-cancer drugs to reduce side-effects. It is necessary to
investigate the cytotoxicity of 1a to cancer cells in our future
study.
Inhibition of DNA Cleavage with Specific DNA Binders.
As estimated from the DNA binding constant of 1a, ca 80-90%
of the total amount of 1a used bonds to DNA under the reaction
conditions. Thus, it may be reasonable that the large accel-
eration of the hydrolytic DNA cleavage by 1a is attained via
the specific binding to DNA.
There are mainly four modes in non-covalent binding of
metal complexes to ds-DNA, an intercalation, an electrostatic
interaction with the DNA main chain, and a minor or a major
groove binding. It is known that metal complexes having about
a thousand molecular weight are suitable for the minor groove
binding because of groove size.16 Generally, the minor groove
binders have crescent-shape structures with cationic centers at
This is the first example of pH-dependent acceleration of
hydrolytic DNA cleavage by synthetic metal complex. As
744 | Bull. Chem. Soc. Jpn. 2019, 92, 739–747 | doi:10.1246/bcsj.20180353
© 2019 The Chemical Society of Japan

(A)
Figure 5. Chemical structures of 4¤,6-diamidino-2-phenyl-
indole (DAPI) and methyl green.
the both ends of compounds. These structural features match
the overall structure of 1a. The major groove binding may be
possible but not so important because the molecular size of 1a
is much smaller than the space of the major groove. To clarify a
DNA binding mode of 1a, we carried out inhibition experi-
ments using 4¤,6-diamidino-2-phenylindole (DAPI),17 a minor
groove binder, and methyl green,18 a major groove binder. The
chemical structures of DAPI and methyl green are shown in
Figure 5.
(B)
Time courses in the DNA cleavage of 1a (10 ¯M) in the
presence of DAPI (5.0-50 ¯M) at pH 5.0 are shown in
Figure 6(A). The inhibition rates at 10 h are estimated from
these data as 19, 50, and 87% in the presence of 10, 20, and
50 ¯M of DAPI, respectively, and increases with increase of
the concentration of DAPI. Thus, the DNA cleavage of 1a is
inhibited by DAPI. The inhibition rates, however, are too small,
Figure 6. DNA cleavage by 1a in the presence of (A) DAPI
and (B) methyl green at pH 5.0. Experimental conditions:
[NaCl] = 10 mM, [buffer] = 10 mM (MES), [pUC19
DNA] = 50 ¯M bp, [1a] = 10 ¯M, and [DAPI] = [methyl
green] = 5, 10, 20, and 50 ¯M at 37 °C for 0, 1, 2, 5, and
10 h. Experiments were carried out at least three times.
¹1
considering from DNA binding constants, 4.7 « 0.3 © 106 M
reported for DAPI with ct-DNA in pH 5.0 acetate buffer at
25 °C19 and 1.81 « 0.84 © 105 M¹1 determined in this study for
1a with a linear 33 mer oligo ds-DNA in pH 5.0 MES buffer at
37 °C. The binding constant of DAPI is nearly 25-fold larger
than that of 1a. This means that the DNA cleavage of 1a may
be almost completely inhibited in the presence of the same
concentration of DAPI. The inhibition rate, however, was only
19% at the same concentration of DAPI. It is known that DAPI
strongly bonds to DNA at the AT-rich site as a minor groove
binder but weakly at the GC-rich site as an intercalator.20 The
binding constant of DAPI with GC-DNA was reported to be
shown that the NH groups of 1a form H-bonds with a phos-
phate moiety of the DNA main chain. Thus, the DNA binding
ability of 1a is enhanced by not only the intercalation of a
cresol moiety but also the H-bonds of the NH groups.
Mechanistic Insights into the pH-Dependent Activity
Control in Hydrolytic DNA Cleavage of 1a. Purple acid
phosphatases are widely distributed enzymes in biological
systems, and catalyze hydrolytic cleavage of phosphate ester at
slightly acidic pH.21 This has a ¯-OH bridged dimetal active
center where the ¯-OH bridge directly attacks phosphate ester
as a nucleophile to accelerate hydrolysis.22 Thus, it is likely
that the ¯-OH bridge is a key structure responsible for the large
acceleration of hydrolytic DNA cleavage of 1a. So, we exam-
ined the DNA cleavage of 2, having a ¯-OH bridge similar to
1a, under the same reaction conditions to gain some insight
into the mechanism of the acceleration in the hydrolytic DNA
cleavage. As shown by time courses of decrease of Form I
(Figure 4(D)), however, 2 showed no activity in the DNA
cleavage at pH 5-8. The hydrolytic DNA cleavage activities of
1a and 2 are totally different from each other in spite of similar
bridging structures. This drastic difference may be explained by
nucleophilicity of the ¯-OH bridge and neighboring-group
participation of the NH groups in 1a. As shown by the crystal
structures of 1a and 2, the ¯-OH bridge of 1a protrudes from
the mean plane defined by four N-atoms of the NH groups by
0.842 ¡, and thus, can easily access the phosphate ester in
DNA. On the other hand, the ¯-OH bridge of 2 is difficult to
¹1 20
¹1
1.2 © 105 M
,
slightly smaller than 1.81 © 105 M of 1a.
The low inhibition rate of DAPI can be explained from these
binding constants, and thus, DAPI blocks the DNA binding of
1a at GC-site. Therefore, acceleration of the DNA cleavage by
1a may be attained through intercalation to the GC-site.
The results of inhibition experiments using a major groove
binder, methyl green, are shown in Figure 6(B). The inhibition
rates at 10 h are 12, 26, and 64% in the presence of 10, 20, and
50 ¯M of methyl green, and slightly lower than 19, 50, and
87% by DAPI. Thus, DAPI is a stronger inhibitor than methyl
green. Methyl green is a major groove binder,17b and reported
to retard DNA cleavage of deoxyribonuclease I, a minor groove
binder.17b Thus, methyl green inhibits DNA binding of 1a at
the major groove, and also retards DNA cleavage of 1a at the
minor groove. This may be the reason for the relatively weak
inhibition of methyl green compared with DAPI.
It may be reasonable that a cresol moiety of 1a acts as an
intercalator. The cresol ring of 1a, however, is smaller than the
aromatic ring of DAPI. From the DNA binding studies, it was
Bull. Chem. Soc. Jpn. 2019, 92, 739–747 | doi:10.1246/bcsj.20180353
© 2019 The Chemical Society of Japan | 745

access due to the steric hinderance of the NCH3 groups. The
nucleophilicity of the ¯-OH bridge may be responsible for the
difference of the DNA cleavage activity between 1a and 2.
Thus, it is proposed that the DNA cleavage of 1a proceeds
through the nucleophilic attack of the ¯-OH bridge.
Neighboring-group participation by the macrocyclic NH
groups of 1a may play a key role in the DNA cleavage. This is
reasonable because they enforce the DNA binding of 1a
through the H-bonds as described in the DNA biding studies.
Moreover, it is well-known that NH groups located near the
metal center can accelerate the metal complex-based hydrolytic
cleavage of phosphate esters.23 In this situation, in order to
clarify the role of the macrocyclic NH groups, 3 that has six
NH groups was used for the DNA cleavage. Time courses in
the reactions of 3 at pH 5.0-8.2 are shown in Figure 4(E). In
the comparison of the time courses shown in Figure 4(B) and
(E), 1a is more reactive than 3. The relatively low reactivity of
3 may be due to the absence of the ¯-OH bridge. This further
supports that the ¯-OH bridge is responsible for the DNA
cleavage. Interestingly, however, 3 accelerated the DNA cleav-
age without a ¯-OH bridge, where the NH groups can activate
a phosphate ester of the DNA main chain by the H-bonds and
hydrolytic DNA cleavage may be attained by nucleophilic
attack of H2O molecule. On the other hand, in 2, the NCH3
groups do not form the H-bond to the phosphate ester, resulting
in almost no activity of 2 in the DNA cleavage. In the com-
parison of DNA cleavage activity among 1a, 2, and 3, it is
proposed that both the ¯-OH bridge and the NH groups are the
key structures for the hydrolytic DNA cleavage.
One of the mechanistic insights deduced from the DNA
binding studies is that the dehydration occurs by the pro-
tonation to the phosphate moiety in the DNA binding complex
of 1a. Another mechanistic insight is the H-bonding interaction
of the NH groups in 1a to the phosphate diester in the DNA
main chain to potentially activate it for the DNA cleavage. The
mechanism proposed on the basis of these features is shown in
Figure 7. In the first step of the hydrolytic DNA cleavage, 1a
binds to the GC-site as an intercalator to form the DNA binding
complex. The DNA binding complex is stabilized by H-bonds
of the NH groups with a phosphate moiety in the DNA main
chain. In this stage, the binding complex is weakly protonated
and entropically stabilized by dehydration, and the phosphate
diester is activated by the protonation for hydrolytic cleavage.
The protonation may decrease the transition energy in the
nucleophilic attack of the ¯-OH bridge to the phosphate ester.
Here, the large acceleration of the hydrolytic DNA cleavage of
1a at pH 5-6 is also explained from the pKa values of phos-
phate monoester. The pKa1 and pKa2 values of deoxyribose-
phosphate monoester are reported to be 1-2 and 5-6, respec-
tively.24 The pKa2 value, 5-6, is exactly the same as the pH
value at which the hydrolytic DNA cleavage of 1a is accel-
erated. Thus, stabilization of the product monoester is the driv-
ing force of the large acceleration of the DNA cleavage by 1a
at pH 5-6. Finally, the dicopper complex is released from the
product monoester. Thus, it is proposed that hydrolytic DNA
cleavage of 1a proceeds through the three steps as follows, (1)
the DNA binding, (2) the stabilization of the transition state
by protonation to give the phosphate monoester, and (3) the
release of 1a.
Figure 7. Proposed mechanism of the hydrolytic DNA
cleavage by 1a.
4. Conclusion
In this study, we synthesized dicopper complexes, 1a, 1b, 2,
and 3 with three p-cresol-derived ligands. The crystal struc-
tures revealed that overall structures of 1a and 2 are similar but
the accessibility to the ¯-OH bridge is different. The dicopper
core structures in the crystals are kept at pH 5-9 in an aqueous
solution as shown by pH titrations and spectroscopic measure-
ments in the complexations of the ligands with Cu(II) ions
upon addition of a standardized aqueous NaOH. DNA binding
mode of 1a was examined by ITC measurements and inhibition
experiments with specific DNA binders DAPI and methyl
green, showing that 1a bonds to DNA at the GC-site as an
intercalator with assistance of the H-bond of the NH groups to
a phosphate moiety in the DNA main chain. Moreover, the
thermodynamic parameters, ¦H°, ¦S°, and K, indicated that
the DNA binding is entropically and enthalpically favorable
and the binding ability of 1a is slightly enhanced by the
protonation at pH 5-6. 1a accelerated cleavage of supercoiled
plasmid pUC19 DNA from Form I to Form II at pH 5-6. This
is the first example of large acceleration of hydrolytic DNA
cleavage by synthetic metal complexes at pH 5-6, though
purple acid phosphatases accelerate hydrolysis of phosphate
ester at acidic conditions. The comparison of the DNA cleav-
age activity among 1a, 2, and 3 revealed that the ¯-OH bridge
accelerates the DNA cleavage as a nucleophile and the NH
746 | Bull. Chem. Soc. Jpn. 2019, 92, 739–747 | doi:10.1246/bcsj.20180353
© 2019 The Chemical Society of Japan

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transition state in the DNA cleavage by 1a is stabilized by
DNA binding and protonation to the DNA binding complex.
Mechanistic insights are proposed on the basis of these studies.
The pH dependent activity control in the DNA cleavage of 1a
may provide some insights into the development of new cancer
drugs to reduce the side effects.
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This work was supported by MEXT-Supported Program of
the Strategic Research Foundation at Private University, 2015-
2019.
Supporting Information
X-ray structural data for the dicopper complexes, distances
of O-atom of ¯-OH bridge from the mean planes of 1a and 2,
electronic absorption and ESI MS spectra in the complexation
of ligands, DNA cleavage by 1a in the presence of H2O2, NaN3,
or dmso, agarose gel electrophoresis profiles for all DNA
cleavage studies, bond distances and angles for the dicopper
complexes, data for the fractions of Form I, Form II, and
Form III formed. This material is available on https://doi.org/
10.1246/bcsj.20180353.
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