Effects of the Ligand Structure of Cu(II) Complexes on Oxidative DNA Cleavage

Article abstract of DOI:10.1002/bkcs.12370

Cu complexes were synthesized by substituting the hydrogen of the amine group of basic ligand 2,2′-dipicoylamine (dpca) (complex 2) with CH₃CO (complex 1), phenyl (complex 3), and methyl (complex 4), respectively, and their DNA cleavage activity was investigated using linear dichroism (LD) and electrophoresis. The DNA cleavage efficiencies of Cu complexes 3 and 4 with phenyl and methyl, which are electron-donating functional groups, turned out to be the highest, and LD magnitudes rapidly decreased at 260 nm. In particular, Cu complex 3 showed a rapid LD magnitude reduction to 63% of the total for 90 min, and to 50% of the total at 12 min. DNA cleavage efficiencies were high in the order of phenyl > methyl > HCH₃CO, and the highest DNA cleavage efficiency was observed in the presence of electron-donating groups. The electrophoresis results are also consistent with the changes in LD spectra over time. The Cu complexes (1–4) were found to cleave DNA through oxidative pathways, and the major reaction oxygen species involved in DNA cleavage were the superoxide radical (·O₂^?), singlet oxygen (¹O₂), and hydroxyl radical (·OH).

Full text of DOI:10.1002/bkcs.12370

Article
DOI: 10.1002/bkcs.12370
BULLETIN OF THE
J. H. Han et al.
KOREAN CHEMICAL SOCIETY
Effects of the Ligand Structure of Cu(II) Complexes on Oxidative DNA
Cleavage
§,
Ji Hoon Han,†,¶ Ji Hoon Kim,† Maeng-Joon Jung,‡ Seog K. Kim,†, and Yoon Jung Jang
*
*
†Department of Chemistry, Yeungnam University, Gyeongsan City 38541, Republic of Korea.
*E-mail: seogkim@yu.ac.kr
‡Department of Chemistry, Kyungpook National University, Daegu 41566, Republic of Korea
§College of Basic Education, Yeungnam University, Gyeongsan City 38541, Republic of Korea.
*E-mail: jyj5014@ynu.ac.kr
¶Present address: Department of Applied Chemistry, Andong National University, Andong-si 1375,
Republic of Korea
Received June 8, 2021, Accepted July 15, 2021
Cu complexes were synthesized by substituting the hydrogen of the amine group of basic ligand 2,20-
dipicoylamine (dpca) (complex 2) with CH3CO (complex 1), phenyl (complex 3), and methyl (complex 4),
respectively, and their DNA cleavage activity was investigated using linear dichroism (LD) and electro-
phoresis. The DNA cleavage efficiencies of Cu complexes 3 and 4 with phenyl and methyl, which are
electron-donating functional groups, turned out to be the highest, and LD magnitudes rapidly decreased at
260 nm. In particular, Cu complex 3 showed a rapid LD magnitude reduction to 63% of the total for
90 min, and to 50% of the total at 12 min. DNA cleavage efficiencies were high in the order of
phenyl > methyl > HCH3CO, and the highest DNA cleavage efficiency was observed in the presence of
electron-donating groups. The electrophoresis results are also consistent with the changes in LD spectra
over time. The Cu complexes (1–4) were found to cleave DNA through oxidative pathways, and the major
reaction oxygen species involved in DNA cleavage were the superoxide radical (ꢀO2ꢁ), singlet oxygen
(1O2), and hydroxyl radical (ꢀOH).
Keywords: Metal complex, DNA cleavage, Oxidative cleavage, supercoil DNA, Scavenger
Micro Linear Dichroism
Introduction
ester bonds. It is catalyzed by enzymes, chemically or by
radiation. Cu complexes are known to cleave DNA through
different mechanisms including hydrolysis and oxidative
pathways.15,16 Oxidative pathways generally produce free
radicals or singlet oxygen (1O2) or ꢀOH radical species in
solutions containing light or oxidation-reduction active
metal complexes. These reactive oxygen species damage
sugars and bases to form non-natural fragment DNA.17,18
In the previous study, the lab investigated the DNA
cleavage effects of [M(2,20-bipyridine)2(NO)3](NO)3 (M =
Cu(II), Zn(II), and Cd(II)) complexes and found that Cu
complexes, of transition metals, cleave supercoiled DNA
and double stranded DNA the most effectively.
This result emphasizes that the role of the center metal in
DNA cleavage is very important.19 DNA cleavage by Cu
complexes also depends on the structure of the ligands
bound to the center metal and the nature of the ligands.17–19
The laboratory has already reported that the DNA cleavage
efficiency of [Cu(2,20-Dipicoylamine(dpca))-(NO3)2] complex
in which N atom in dpca is directly coordinated to Cu is low.
In the present study, new Cu(II) complexes, including [Cu
(dpca)(NO3)2](dpca = 2,20-dipycolylamine, complex 1) [Cu
(dpcaa)(NO3)2](dpcaa = 2,20-dipycolylacetamide, complex
Deoxyribonucleic acid (DNA) is biologically very impor-
tant.1 DNA contains information in the form of genes,
which is crucial for a variety of functions. DNA is the
major target molecule for most anticancer and antiviral
therapies based on cell biology.2–7 In particular, studies on
DNA interactions with transitional metal complexes con-
taining multidentate aromatic ligands have interesting prop-
erties including a role as a probe in the research on DNA
structure and forms as well as potential contribution to the
development of new therapeutic agents, generating consid-
erable interest in many researchers.8,9 Among the metals
that compose these transition metal complexes, Cu is an
important trace element in plants and animals and forms
complexes in various biological processes.10–12
Cu(II) complexes have been studied as medicinal treat-
ments that have potential for the treatment of many dis-
eases, including cancer.13,14 Thus, research on Cu
complexes have been actively carried out in the fields of
biological and inorganic chemistry. DNA cleavage is the
cleavage of basic constituents, such as bases and sugars,
through oxidative pathways or hydrolysis of phosphate
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BULLETIN OF THE
ISSN (Print) 0253-2964 | (Online) 1229-5949
KOREAN CHEMICAL SOCIETY
2), [Cu(dpcb)(NO3)2](dpcb = 2,20-dipycolylaminobenzene,
complex 3), and [Cu(medpca)(NO3)2](medpca = N-methyl-
2,20-dipycolylamine, complex 4), were synthesized by
substituting the hydrogen attached to N of the basic ligand
structure 2, 20-dipicoylamine (dpca) with electron-donating
and electron-withdrawing groups and the DNA cleavage effi-
ciencies of the Cu (II) were compared (Scheme 1). It is
expected that this study will provide much information for
understanding the mechanism of damage to DNA.
below).
2,2-dipycolylacetaamide
2,20-Dipicoylamine
(dpca),20
and
2,20-
(dpcaa),21
dipycolylaminobenzene (dpcab)22 and N-methyl-2,20-
dipicolylamine (medpca)23 were synthesized as reported
previously. Complexes 1–4 [Cu(dpca)(NO3)2], [Cu(dpcaa)
(NO3)2], [Cu(dpcab)(NO3)2], and [Cu(medpca)(NO3)2]
were synthesized and characterized using a method reported
elsewhere.18 All reactions were run in
atmosphere.
a
nitrogen
Synthesis of Ligands and Complexes
Synthesis
of
2,20-Dipicoylamine
(dpca),
2,2-
Experimental
Dipycolylacetaamide (dpcaa), 2,20-Dipycolylaminobenzene
(dpcab), and N-methyl-2,20-dipicolylamine (medpca)
2,20-Dipicoylamine (dpca)
Chemicals. All chemicals including solvents were pur-
chased from (Sigma-Aldrich, St. Louis, MO) and used in
the synthesis were of reagent grade and without further
purification. NMR spectra were recorded on a Varian
600 spectrometer. Chemical shifts (δ) are reported in ppm,
relative to tetramethylsilane Si(CH3)4. Elemental analysis
for carbon and hydrogen was carried out by using Scientific
Flash EA 1112 HT elemental analyzer (Thermo Fisher Sci-
entific, MA). The pBR322 plasmid DNA (referred to as
scDNA) stock solution (1 mg/mL) was purchased from
New England Biolabs (Ipswich, MA). Double-stranded
highly polymerized calf thymus DNA (dsDNA) was
obtained from Worthington Biochemicals (Lakewood, NJ)
and dissolved in 5 mM cacodylate buffer pH 7.0. The con-
centration of dsDNA was determined using a molar extinc-
tion coefficient of ε258nm = 6600 Mꢁ1 cmꢁ1. The
concentrations of the Cu(II) complexes were measured
spectrophotometrically using the extinction coefficientsꢁo1f
Dissolve 2-aminomethylpyridine (0.541 g, 5 mmol) in
10 mL of MeOH. Add 2-pyridinecarboxaldehyde (0.535 g,
5 mmol) to this solution. The color of the solution turns
dark brown immediately. Slowly add sodium borohydride
(0.378 g, 10 mmol) during stirring at room temperature for
10 h. As the reaction progresses, the color of the solution
changes to yellowish. Continue stirring at room temperature
for more than 2 h. Remove the solvent from the solution
through evaporation, and put 10 mL of distilled water and
slowly add concentrated hydrochloric acid to neutralize the
solution. The neutralized solution is extracted with 10 mL
CH2Cl2. Repeat this process five times. Add an appropriate
amount of MgSO4 to the separated organic layer and shake
sufficiently to remove water from the organic layer, and fil-
ter out MgSO4. Evaporate the organic layer with water
removed once more and obtain orangish-yellow liquid. This
is purified through silica gel column chromatography. Ethyl
acetate: EtOH: n-Hexane (7:2:1) is used as developer. This
process is repeated several times to obtain the final dark
yellow liquid (0.697 g).
ε260nm = 8000 Mꢁ1 cmꢁ1, ε252nm = 8400 Mꢁ1 cm
,
ε256nm = 10 750 Mꢁ1 cmꢁ1, and ε254nm = 10 600 cmꢁ1
Mꢁ1 for Cu(II) complex 1, 2, 3, and 4, respectively. All
experiments were carried out in a 5 mM cacodylate buffer,
pH = 7.0, except for the electrophoresis measurements (see
Yield: 697 mg, 70%.
1H NMR(300 MHz, MeOH) 3.93 (s, 5H) 7.27–7.31 (m,
2H) 7.47–7.5 (d, 2H) 7.34–7.28 (m, 4H) 7.76–7.81 (t,2H)
8.48–8.49 (d, 2H).
13C NMR(300 MHz, MeOH): 54.7), 122.84, 123.84,
138.93, 149.55, 150.141, 160.16.
2,2-Dipycolylacetaamide (dpcaa)
Dissolve dipicolyamine (dpca) (0.9 g, 5 mmol) in 30 mL of
CH2Cl2 and lower the temperature of the solution using an
ice bath and add triethylamine (1 mL, 7 mmol) to slow
down the reaction rate. Slowly add acetyl chloride (0.6 mL,
8 mmol) to this solution. After stirring for approximately
3 h, the ice bath is removed and the solution is further
stirred at room temperature for an hour. This solution is
extracted with water and CH2Cl2 approximately five times,
and an appropriate amount of MgSO4 is added to the sepa-
rated organic layer. After sufficiently shaking it to remove
water from the organic layer, MgSO4 is filtered out. Evapo-
rate the organic layer with water removed once more to
obtain dark yellow liquid. This is purified through silica gel
Scheme 1. Chemical structures of the [Cu(dpcaa)(NO3)2] (com-
plex1), [Cu(dpca)(NO3)2] (complex 2), [Cu(dpcb)(NO3)2] (com-
plex 3), and [Cu(medpca)(NO3)2] (complex 4) used in the this
study.
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Oxidative DNA Cleavage
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column chromatography. Ethyl acetate: EtOH (5:1) is used
as developer. This process is repeated several times to
obtain the final dark yellow liquid (0.724 g).
Yield: 724 mg, 60%.
approximately five times. The extracted organic layers are
combined and dried with MgSO4, which is filtered out
through a vacuum filter, and the remaining filtrate is rotary
evaporated to remove the solvent. The obtained mixture is
liquid mixed with sticky solids. To this liquid, add an
appropriate amount of diethyl ether and store it in the
refrigerator for a day. Collect the solution dissolved in
diethyl ether and evaporate an appropriate amount of
diethyl ether to obtain golden color liquid. The obtained
liquid is purified through silica gel column chromatogra-
phy. Ethyl acetate: EtOH (4:1) is used as a developing sol-
vent. This process is repeated several times to obtain the
final golden color liquid.
1H NMR (300 MHz, MeOH) 2.25 (s, 3H), 4.72 (s, 2H),
4.76 (s, 2H), 7.28–7.34 (m, 4H), 7.74–7.78 (m, 2H), 8.42–
8.52 (m, 2H).
13C NMR (300 MHz, MeOH) 21.5, 52, 55.4, 123, 124,
138.5, 139, 149.5, 150.3, 157.6, 158.2, 174.4.
2,20-Dipycolylaminobenzene (dpcab)
The solution obtained by dissolving [pretreated
*(Chloromethyl) pyridine hydrochloride] (Chloromethyl)
pyridine hydrochloride (9.5 g, 58 mmol) in 20% aqueous
sodium hydroxide (NaOH) is extracted with CH2Cl2 three
to five times and the organic layer is dried with MgSO4,
which is removed through filtering. Evaporate the solution
and remove the solvent. By using 2-chloromethylpyridine
with solvent removed, the ligand (dpcab) is synthesized.
Dissolve aniline (1.8 g, 19 mmol) in 50 mL of benzene.
During stirring of the solution, slowly add trimethylamine
Yield: 576 mg, 60%.
1H NMR: 2.25 (s, 3H), 3.74 (s, 4H), 7.26–7.31 (m, 2H)
7.59–7.62 (d, 2H), 7.77–7.83 (m, 2H), 8.44–8.46 (d, 2H).
13C NMR: 42.62, 63.97, 123.98, 125.21, 139.01, 149.2,
159.86.
Synthesis of Complexes [Cu(dpcaa)(NO3)2] (Complex 1)
[Cu(dpca)(NO3)2] (Complex 2), [Cu(dpcab)(NO3)2]
(Complex 3), and [Cu(medpca)(NO3)2] (Complex 4). To
the solution of Cu(NO3)2ꢀ3H2O(0.28 g, 1.2 mmol) in
30 mL of MeOH, slowly add the appropriate ligand
(1 mmol) dissolved in 5 mL of MeOH. Let it react at room
temperature for a day. After the reaction has been com-
pleted, the solution is concentrated. Then, carefully add
diethyl ether to obtain crystals. The crystals obtained are
dissolved again in MeOH and recrystallized with diethyl
ether. Wash them using diethyl ether as solvent to obtain
the final blue crystals.
ꢂ
(5.8 g, 57 mmol) and raise the temperature up to 80 C. At
ꢂ
80 C, dissolve the pretreated 2-(chloromethyl) pyridine in
a little amount of benzene and slowly add it to the solution
over five times. Maintain the temperature for 2 h. Slowly
cool it to room temperature to obtain a solution. This solu-
tion is concentrated and extracted first with diethyl ether
and NaHCO3 aqueous solution. The obtained organic layer
is extracted once again with brine (NaCl aqueous solution)
approximately five times. At this time, the NaHCO3 aque-
ous solution removes the inorganic by-products. The
extracted organic layers are combined and dried with
MgSO4. The solids are filtered through a vacuum filter, and
the remaining filtrate is rotary evaporated to remove the
solvent. At this time, a mixture of solids and liquid is
obtained. The mixture is purified through silica gel column
chromatography. Ethyl acetate is used as developer. This
process is repeated several times to obtain the final brown
crystals (2.62 g).
[Cu(dpcaa)(NO3)2] (complex 1) yield: 59% (257 mg).
Elemental
analysis
Calcd
(%)
for
C14H15N5O7Cuꢀ0.5H2O: C, 38.40; H, 3.65; N, 15.99.
Found: C, 38.62; H, 3.49; N,15.72.
[Cu(dpca)(NO3)2] (complex 2) yield: 75% (290 mg).
Elemental analysis Calcd (%) for C12H13N5O6Cu: C,
37.26; H, 3.39; N, 18.11. Found: C, 36.84; H, 3.37;
N, 17.42.
[Cu(dpcab)(NO3)2] (complex 3) yield: 64%, (302 mg).
Elemental analysis Calcd (%) for C18H17N5O6Cuꢀ$
0.5H2O: C, 45.77; H, 3.81; N, 14.83. Found: C, 46.08; H,
3.68; N, 14.43.
[Cu(medpca)(NO3)2] (complex 4) yield: 60%, (240 mg).
Elemental analysis Calcd (%) for C13H15N5O6Cu: C,
38.95; H, 3.77; N, 17.47. Found: C, 38.92; H,
3.77; N, 16.92.
Cleavage of pBR322 Plasmid DNA. The appropriate
amount of ascorbic acid and the metal complexes was
added to a scDNA solution in a 5 mM cacodylate buffer
(pH 7.0) for the conventional cleavage experiment. The
mixture was incubated for 1 h at room temperature.
The reaction was quenched by the addition of a stopping
buffer containing 7 mM EDTA, 0.15% bromophenol blue,
0.15% xylene cyanol, and 75% glycerol. The mixtures were
placed on a 1% agarose gel for electrophoresis at 25 V,
400 mA for 400 min. The gel was stained with Tris–
Yield: 2620 mg, 50%.
1H NMR: 4.82 (s, 4H), 6.64–6.67 (m, 3H), 7.05–7.11 (t,
2H) 7.27–7.35 (m, 4H), 7.71–7.73 (t, 2H), 8.49–
8.51 (d, 2H).
13C NMR: 57.95, 113.73, 118.34, 122.79, 123.6, 130.37,
139.14, 149.81, 150.4, 160.27.
N-Methyl-2,20-Dipicolylamine (medpca)
Dissolve bpma (1 g, 5 mmol) in 10 mL of acetonitrile, and
2 mL of dimethylformamide is added after diluting in
10 mL of acetonitrile. Slowly add methyliodide (0.637 g,
4.5 mmol) to this solution. Let it react at room temperature
while stirring for approximately 2 h. As methyliodide is
very toxic, this process is done in the hood. This solution
is concentrated and extracted first with CH2Cl2 and
NaHCO3 aqueous solution. The obtained organic layer is
extracted once again with brine (NaCl aqueous solution)
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Oxidative DNA Cleavage
KOREAN CHEMICAL SOCIETY
acetate–EDTA (TAE) buffer containing 0.5 μg/mL
ethidium bromide, 20 mM Tris acetate and 1 mM EDTA,
and visualized by UV transillumination and photographed
using an Olympus C-5060 camera. The integrated densities
of the bands were quantified using Total Lab TL100 image
analysis software (Nonlinear Dynamic Ltd., USA). The
amounts of scDNA were corrected by a factor of 1.4 based
on the weaker binding of ethidium to this structure.
three shapes of plasmid DNA can be easily distinguished
through agarose gel electrophoresis. As shown in Figure 1,
when there is no Cu metal complex, scDNA shows two
clear bands: a supercoiled band (Form I) and a relaxed cir-
cular form (Form II). When metal complexes are present,
cleavage activity is evident depending on concentration
changes. At the lowest complex concentration of 10 μM,
no noticeable cleavage efficiency change was observed, but
Form II showed a slight increase. When concentrations
increased to 30 and 50 μM, the cleavage effects of Cu com-
plexes 3 and 4 are clearly observed.
In particular, when the concentration of Cu complex
3 increased to 30 μM, the strand was completely cleaved
that Form I disappeared and Form II and Form III became
clear. In the case of Cu complex 4, the same pattern was
observed at 50 μM. Therefore, at the same concentration,
the scDNA cleavage efficiency of Cu complex 3 was the
highest, followed by Cu complex 4. In addition, it was
observed that these Cu complexes showed increase in
cleavage efficiency depending on incubation time
(Supporting Information Figure S1).
When incubation time was set at 1 h, clear cleavage
activity on scDNA was not observed in Cu complexes
1 and 2, but when incubation time increased to 3 h, there
were remarkable changes: Form III was observed at most
concentrations except at 10 μM. However, despite the
increase in Cu incubation time, the order of scDNA cleav-
age efficiency of metal complexes did not change. Figure 2
shows the results of an electrophoresis experiment to com-
pare the cleavage activities of all complexes at a Cu
complex concentration of 30 μM for an hour of incubation
time. The relative amounts of Form I, Form II, and Form
III related to Figure 2 are summarized in Table 1.
When there is no Cu complex, scDNA showed 89.88%
of Form I and 10.12% of Form II. Also, in the presence of
ascorbate, which is a reducing agent, similar rates (89.05%,
10.95%) were observed, which means that the effect of
ascorbate on the cleavage of scDNA on electrophoresis is
negligible. In the presence of Cu complexes 1 and 2, cleav-
age effect on scDNA was not noticeable, but the relative
rates of Form II increased from 10.12 (only scDNA) to
16.44 (complex 1) and 15.82 (complex 2), respectively. In
the case of Cu complex 3, Form I completely disappeared
and the rate of Form II increased to 94.86%, although it
was not completely distinguished, and the rate of Form II
was 5.14%. Compared with Cu complex 3, Cu complex
4 had relatively lower cleavage efficiency with relative rates
of 51.07% (Form I) and 48.93% (Form II). Form III was
not observed in any complex except Cu complex 3.
Linear Dichroism Spectroscopic Measurements. Linear
dichroism (LD) is defined by the difference in the absorp-
tion of polarized parallel and perpendicular radiation rela-
tive to the laboratory reference axis of the oriented
sample.24–26 The use of LD as a tool for detecting dsDNA
cleavage in real time is described elsewhere.17–19,24
As it was described, decrease in LD intensity reflects
increase in flexibility of dsDNA as a result of the cleavage
of one strand and/or decrease in the DNA length by cut of
both DNA strands. The time-dependent decrease in LD at
260 nm and the LD spectrum were recorded on a J-810
spectropolarimeter (Jasco, Tokyo, Japan) using a micro-
volume, thermostatically controlled inner rotating Wada-
type cuvette cell purchased from Kromak Ltd. (Dunmow,
GB). The results were fitted to two exponential decay cur-
ves using the OriginPro8.0 program (OriginLab Co., North-
ampton, MA, USA). The goodness of fit was evaluated
using the residuals.
Results and Discussion
Cleavage of pBR322 scDNA Probed by Electrophoresis.
Cleavage activity of Cu complexes 1, 2, 3, and 4 was mea-
sured through electrophoresis at biophysical condition
(pH = 7) using supercoiled pBR322 plasmid DNA
(Figure 1). If a strand of scDNA (Form I) is cleaved, it
appears in a nick circular form (Form II) and when cleaved
once again, it appears in a linear form (Form III). These
Figure 1. Cleavage of supercoiled pBR322 DNA by the 10, 30,
and 50 μM of Cu complexes (1–4) detected by 1% agarose gel
electrophoresis. Lane 1: pBR322 DNA; lanes 2–4: in the presence
the complex 1; lanes 5–7: in the presence of the complex 2; lanes
8–10: complex 3; and lanes 11–12: in the presence of the complex
4. The sample mixtures were incubated for 1 h. The gel was run
Effect of Reactive Oxygen Scavengers. The four kinds of
Cu complexes used in this scDNA cleavage study were
synthesized by substituting the hydrogen of the amine
group of basic ligand 2,20-dipicoylamine (dpca) (complex
2) with CH3CO (complex 1), phenyl (complex 3), and
methyl (complex 4), respectively. The cleavage of scDNA
by Cu (II) complexes were not shown without the addition
for 400 min under 25 V. [scDNA]
acid] =100 μM.
= 100 μM, [ascorbic
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KOREAN CHEMICAL SOCIETY
Figure 2. Cleavage of supercoiled pBR322 DNA by the 30 μM of
Cu complexes (1–4) detected by 1% agarose gel electrophoresis.
Lane 1: pBR322 DNA; lanes 2: in the presence of ascorbate; lane
3: in the presence the complex 1; lane 4: in the presence of the
complex 2; lane 5: complex 3; and lane 6: in the presence of the
complex 4. The sample mixtures were incubated for 1 h. The gel
was run for 400 min under 25 V. [scDNA] = 100 μM, [ascorbic
acid] = 100 μM.
Figure 3. Cleavage of supercoiled pBR322 DNA by complexes
1–4 and the effect of various reactive oxygen species scavengers
detected by 1% agarose gel electrophoresis. The numbers in each
panel represent the same experimental conditions. Panel (a), (b),
(c), and (d) for complex 1, complex 2, complex 3, and complex
4, respectively: Lane 1: scDNA; lane 2: scDNA + ascorbate; lanes
3–6: in the presence of 1 mM tiron, 5 mM sodium azide, 30 mM
DMSO, and 0.125 unit of catalase, respectively. [scDNA] =
100 μM, [Cu(II) complex] = 30 μM, and [ascorbate] = 100 μM.
The mixtures were incubated for 1 h and the gel was run for
400 min under 25 V.
of any external reagents. This indicates that scDNA cleav-
age by Cu (II) complexes occur due to other factors that
can be generated by them. Cleavage by Cu (II) complexes
can be achieved through oxidative pathways as well as
hydrolysis pathways.17,18,27–30 Reaction oxygen species
(ROS) generated from the interaction of Cu(II) complexes
with O2 are considered to be important factors in scDNA
cleavage.17,18 To investigate how singlet oxygen reaction
was carried out in the presence of various ROS scavengers.
Figure 3 and Table 2 show the results of electrophoresis
experiments to examine the effects of ROS scavengers on
scDNA cleavage by Cu complexes (1–4) and the relative
rates of Form I, Form II, and Form III. In the case of Cu
complex 1, in the presence of 1 mM tiron (lane 3), which is
a superoxide radical (ꢀO2ꢁ) scavenger (except catalase);
5 mM sodium azide (lane 4), which is a singlet oxygen
(1O2) scavenger; and 50 mM dimethylsulfoxide (DMSO)
(lane 5), which is a Hydroxyl radical (ꢀOH) scavenger,
cleavage efficiency was slightly inhibited. With no scaven-
ger, that is, when there is only Cu complex 1, the relative
rates of Form I and Form II were 75.20% and 24.08%,
respectively. However, the rates were 97.71% and 2.29% ꢁin
complex 2. Slight changes were identified in the presence
of tiron, which is a superoxide radical (ꢀO2ꢁ) scavenger;
sodium azide, a singlet oxygen (1O2) scavenger; and
DMSO, a hydroxyl radical (ꢀOH) scavenger.
Cu complex 3 displayed the most apparent effect of ROS
scavengers on the cleavage of scDNA. Form I completely
disappeared and Form II and Form III were clearly visible
when no scavenger is present. However, in the presence of
5 mM sodium azide, a singlet oxygen (1O2) scavenger; and
50 mM DMSO, a hydroxyl radical (ꢀOH) scavenger, the
relative rates of Form I and Form II were 83.81% and
16.19%, and 93.08% and 6.92%, respectively. These rates
are similar to those observed when only scDNA is present
(Form I: 95.62%, Form II: 4.38%) (Table 2). The results
demonstrate that the major ROS in the cleavage of scDNA
by Cu complex 3 are the singlet oxygen (1O2) and hydroxyl
radical (ꢀOH). In addition, they show that at least scDNA
cleavage by Cu complex 3 does not involve the superoxide
radical (ꢀO2ꢁ) among ROS. It was observed that scDNA
the presence of tiron, which is a superoxide radical (ꢀO2
)
scavenger; 98.67% and 1.33% in the presence of sodium
azide, a singlet oxygen (1O2) scavenger; and 95.82% and
4.18% in the presence of DMSO, a hydroxyl radical (ꢀOH)
scavenger. These changes in rates indicate the decrease in
cleavage efficiency. Similar results were observed in Cu
Table 1. Relative amount (%) of the Form (I), Form (II), and Form (III) in the presence of various Cu(II) complexes.
Lane
1 (scDNA)
2 (scDNA + ascorbate)
3 (complex 1)
4 (complex 2)
5 (complex 3)
6 (complex 4)
Form 1 (%)
Form 2 (%)
Form 3 (%)
89.88
10.12
89.05
10.95
83.56
16.44
84.18
15.82
51.07
48.93
94.86
5.14
The data were read from the electrophoresis data shown in Figure 2. [scDNA] = 100 μM, [ascorbate] = 100 μM, and [Cu(II) complex] = 30 μM.
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Oxidative DNA Cleavage
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Table 2. Effect of the various ROS scavengers on scDNA
cleavage.
singlet oxygen in scDNA cleavage and the effect of super-
oxide radical (ꢀO2ꢁ) is negligible.
These ROS are generated by the interaction of Cu
(II) complexes and O2. First, Cu (II) complexes binds to
ascorbate, which is a reducing agent, to form Cu (I),
which binds to dissolved oxygen to form the superoxide
radical (ꢀO2ꢁ). The latter again receives electrons and
forms the hydrogen peroxide (H2O2), which can also
receive electrons and generated the hydroxyl radical
(ꢀOH). The mechanism can be summarized in the follow-
ing reaction series.
(a)
Form I
95.62
Form II
Form III
Only DNA
DNA + complex 3
Trion
Sodium azide
DMSO
4.38
98.01
97.17
16.19
6.92
1.99
2.83
83.81
93.08
12.59
Catalase
77.64
9.76
(b)
CuðIIÞ complexþascorbate ! CuðIÞþ ꢀascorbateꢁ þHþ
Form I
Form II
Form III
Â
Ã
CuðIÞ complexþO2 ! CuðIÞꢁO2 ! CuðIIÞꢁ ꢀO2ꢁ
Only DNA
DNA + complex 4
Trion
Sodium azide
DMSO
98.62
29.72
78.64
69.46
40.19
50.97
1.38
! CuðIIÞ complexþ ꢀO2ꢁ
70.28
21.36
30.54
59.81
49.03
2eꢁþ2Hþ
2ꢀOꢁ2
!
2H2O2
e
ꢁþHþ
Catalase
H2O2
2ꢀOH
ƒƒ!
The data were obtained from Figure 3 for Cu(II) complex 3 (a), 4 (b).
[scDNA] = 100 μM, [ascorbate] = 100 μM, and [Cu(II)
complex] = 30 μM.
1
O2 ! O2
The mechanism begins with the reduction of Cu
(I).[17–19 The reductivity of Cu complexes can vary
greatly depending on the structure and nature of the
bound ligand.17–19 In particular, it is established that Cu
bound to a ligand having electron-donating groups can
be further reduced.18 This is because a ligand with
electron-donating groups can, when bound to Cu,
increase its electron density, which improves its red-
uctivity. Based on the ligand (dpca) attached to Cu com-
plex 2 used in this study, that of Cu complex 1 belongs
to the electron-withdrawing group, while those of Cu
complexes 3 (dpcab) and 4 (medpca) belong to the
electron-donating group. As reported in the previous
study, it was observed that Cu complexes 3 and 4 with
electron-donating groups had higher scDNA cleavage effi-
ciency. However, although the ligand of Cu complex
3 had a lower electron-donating ability than the ligand of
Cu complex 4, the scDNA cleavage efficiency of Cu
complex 3 was remarkably high. In general, the phenyl
group has a lower degree of electron-withdrawing activity
than the methyl group. But in the ligand of Cu complex
3, the H of the 2,20-dipicoylamine (dpca) group was
substituted by the phenyl that was stabilized through the
conjugation of the π electrons of pyridine on both sides
of the amine and those of the phenyl, and this led to the
increase in the electron density of the ligand. Therefore,
it is considered that the phenyl group became more
active in electron-donating activity than that of Cu com-
plex 4, affected by the surrounding functional groups,
and this explains the high scDNA cleavage efficiency of
Cu complex 3.
cleavage is slightly inhibited even in the presence of cata-
lase, and the amount related to Form II tends to be slightly
lower than in the absence of scavengers. The cleavage of
scDNA by Cu complex 3 appears to involve all the
hydroxyl radical, singlet oxygen, and H2O2. The most
important role is probably performed by the hydroxyl radi-
cal (ꢀOH) generated by Cu complex 3 in the solution. The
effect of ROS scavengers on scDNA cleavage by Cu com-
plex 4 can be summarized as follows. While tiron, a super-
oxide radical (ꢀO2ꢁ) scavenger, had almost no effect on
scDNA cleavage by Cu complex 3, it caused the most
apparent changes in scDNA cleavage by Cu complex
4. The relative rates of Form I and Form II were 78.64%
and 21.36%, respectively, which are almost opposite to the
rates of 29.72% (Form I) and 70.28% (Form II) that scDNA
cleaved by Cu complex 4 showed in the absence of scaven-
gers. This suggests that the major ROS of scDNA by Cu
complex 4 is the superoxide radical (ꢀO2ꢁ). The results of
electrophoretic analysis conducted in the presence of vari-
ous ROS scavengers revealed that all Cu complexes 1–4
cleave scDNA through oxidative pathways, although the
scDNA cleavage efficiencies of Cu complexes 1 and 2 were
low and thus the cleavage factors were not clear. Except for
the case of Cu complex 3, the common major ROS engaꢁg-
ing in scDNA cleavage was the superoxide radical (ꢀO2
)
and its effect was the most apparent in Cu complex 4. How-
ever, the results suggest that the hydroxyl radical and sin-
glet oxygen as well as the superoxide radical (ꢀO2ꢁ) are
involved in scDNA cleavage to some extent. Meanwhile,
Cu complex 3 involves the hydroxyl radical (ꢀOH) and
Another important ROS in scDNA cleavage by Cu com-
plex 3 is the singlet oxygen (1O2). It can arise from oxygen
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BULLETIN OF THE
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Oxidative DNA Cleavage
KOREAN CHEMICAL SOCIETY
by light without electron transport and can also be gener-
ated in the complex process of producing ROS. However,
no definitive mechanism for the formation of singlet oxy-
gen (1O2) was identified in this study.
results of the electrophoresis, meaning that it has the
highest cleavage efficiency. To understand the highest
dsDNA cleavage efficiency of Cu complex 3, two factors
should be taken into consideration. They are electron-
donating ability and the magnitude of interaction with
DNA. As reported in the previous study, high electron-
donating ability and binding affinity to DNA lead to great
effect on DNA cleavage.19,31,32
As the absorption regions of Cu complexes 1–4 and
DNA coincide with each other, the DNA affinity of the Cu
complexes cannot be measured. Therefore, the DNA cleav-
age effect related to the latter remains uncertain. Thus, the
results of this experiment suggest that the electron-donating
ability of Cu complex 3 is the most important factor in the
difference in oxidative dsDNA cleavage efficiency between
Cu complex 3 and the others.
Changes in the LD Spectrum with Time in the Presence
of Cu Complexes. LD is the most powerful tool for mea-
suring the length changes in dsDNA and scDNA. The mag-
nitude of the LD signal of DNA depends on various factors
including flexibility and DNA length. More flexibility and
shorter DNA result in a decrease in orientation, which con-
sequently leads to a decrease in the magnitude of the LD
signal.17–19
Figure 4 shows the LD spectra measured 90 min after
adding ascorbate to DNA and Cu complexes 1–4,
respectively.
DNA exhibits a negative LD spectrum in the DNA
absorption region.
The maximum LD intensity of DNA was observed at
ꢃ258 nm when no Cu complex was present (Figure 4,
curve a). The addition of Cu complexes and the reducing
reagent ascorbate to DNA did not cause any chemical shift.
As a result of measuring the LD spectra after adding Cu
complexes to DNA over time, no change was observed
(data not shown). However, it was observed that the LD
signal magnitude of DNA-ascorbate was slightly reduced
than when only DNA is present (Figure 4, curve b).
Although it did not occur during the electrophoretic experi-
ment, it was found that the DNA was slightly cleaved by
ascorbate after 90 min. In the presence of Cu complexes,
the LD spectra were further reduced in magnitude and the
LD signal magnitudes of complex 1, 2, 3, and 4 decreased
for 36%, 56%, 77%, and 74%, respectively, compared with
that of DNA-ascorbate.
Cleavage of dsDNA Probed by LD. This real-time LD
technique is based on the fact that the magnitude of LD in
the DNA absorption region solely reflects the flexibility
and length of DNA if the other factors, including the opti-
cal density, viscosity, temperature, and flow rate (in the
flow orientation case), are kept constant. Figure 5 shows
the changes in the magnitudes of LD signal at 260 nm by
adding Cu complexes 1–4 to dsDNA.
Changes in the magnitudes of LD signal over time were
very large and fast in Cu complex 3 in a short time. At
6 min, the LD magnitude decreased by 40% in Cu complex
3, while reduction by only 14% in Cu complex 4 and less
than 0.1% in Cu complexes 1 and 2 were observed. The
initial changes in the LD magnitudes occurred very fast:
within 6 min in Cu complex 3 and 20 min in Cu complex
4, after which the change rates slowed down. In contrast to
this, the Cu complexes 1 and 2 showed no changes in the
magnitudes of LD signal for almost 10 min. But
the changes became more apparent over time. This suggests
This change in the magnitude of LD signal was most
apparent in Cu complex 3, which is consistent with the
Figure 4. LD spectrum of the dsDNA-Cu complex (1–4) adducts
at 90 min after mixing [dsDNA]
=
100 μM, [Cu
Figure 5. Decrease in the LD magnitude at 260 nm of the dsDNA
with respect to time in the presence of complexes 1, 2, 3, and
4. The best-fit exponential decay curves, LD = a1 exp (ꢁt/τ1)
+ a2 exp (ꢁt/τ2) for Cu complex 3 and LD = a exp (ꢁt/τ) for
4 are denoted by the black solid curve. [DNA] = 100 μM,
[ascorbate] = 100 μM, and [Cu(II) complex] = 30 μM.
complex] = 30 μM, and [ascorbate] = 100 μM. Curves a and b
denote the LD spectrum of only DNA and DNA-ascorbate,
respectively. Curves c, d, e, and f are the LD spectrum of the
dsDNA-complex 1, 2, 3, and 4 + ascorbate at 90 min after
mixing, respectively.
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BULLETIN OF THE
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Oxidative DNA Cleavage
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Table 3. Rate constants and the relative amplitude obtained from
proper fitting equation for Cu complex 3 and 4.
composed of two exponential components and the fast one
represents the cleavage of one of the two strands of DNA
and the slow one the cleavage of another strand around the
cleaved site. This leads to a change in the magnitude of
the LD signal due to changes in the fluidity and length of
DNA. The change of the magnitude of LD signal in Cu
complex 4 occurred as the primary reaction. This implicates
the cleavage of one of the two strands in DNA, and this
increased the fluidity of DNA and resulted in the decrease
in the LD signal.
a1
k1 (minꢁ1
)
a2
k2 (minꢁ1
)
Cu complex 3
Cu complex 4
0.41
1
0.037
0.045
0.59
0.45
that Cu complexes 3 and 4 effectively increase the flexibil-
ity of dsDNA or shorten the length of dsDNA in a short
time, whereas such effects of Cu complexes 1 and 2 are
negligible. However, dsDNA cleavage efficiency also
increased in Cu complexes 1 and 2 over time. After 90 min
of the reaction, the cleavage efficiencies of Cu complexes
1–4 were 24%, 57%, 63%, and 69%, respectively. The
scDNA cleavage efficiency is the highest in Cu complex
Conclusion
New Cu(II) complexes were synthesized by substituting the
H
of the amine group in the basic ligand 2,20-
Dipicoylamine (dpca), with the phenyl and methyl groups
that are electron-donating functional groups, and the acetyl
group that is an electron-withdrawing group, to investigate
their DNA cleavage activity. All Cu complexes cleaved
dsDNA and scDNA through oxidative pathways, and Cu
complexes with the electron-donating groups had higher
cleavage activity than those with the electron-withdrawing
group. DNA cleavage efficiency was higher in the order of
phenyl > methyl > HCH3CO and Cu complex 3 showed the
highest DNA cleavage activity. The ROS engaging in DNA
cleavage were the superoxide radical (ꢀO2ꢁ), singlet oxygen
(1O2), and hydroxyl radical (ꢀOH).
4
after 90 min of the reaction; however, as Cu
complex 3 cleaved scDNA very rapidly at the initial stage
of the reaction, there can be an error in the initial value
when t = 0 in the y-axis (LD/LDt=0) of the spectrum in
Figure 5, which is the reason why it is difficult to make
comparison. The magnitude of LD signal reached zero
within 90 min in no complex. Table 3 shows the results of
a decay curve analysis on Cu complexes 3 and 4 with the
best DNA cleavage efficiency. As a result of analyzing
using residuals, the decrease in the LD magnitude of Cu
complex 3 was not fitted to the simple primary or second-
ary reaction rates, but the sum of two component exponen-
tial decays [Eq. (1)], which is the sum of two first order
kinetics; while that of Cu complex 4 was fitted to the first
order exponential decay function [Eq. (2)].
Acknowledgment. This work was supported by the
National Research Foundation of Korea (Grant No. NRF-
2017R1D1A3B03031788) and by the 2016 Yeungnam
University Research Grant.
LDðtÞ ¼ a1expðꢁt=τ1Þþa2expðꢁt=τ2Þ
LDðtÞ ¼ a1expðꢁt=τ1Þ
ð1Þ
ð2Þ
Conflict of Interest. The authors declare no conflict of
interest.
Cu complex 3 consists of two exponential components
Supporting Information. Additional supporting informa-
tion may be found online in the Supporting Information
section at the end of the article.
and the reaction times (τ) are 26.80 min (slow component,
k1 = 3.7 ꢄ 10ꢁ2) and 2.2 min (fast component, k2
4.5 ꢄ 10ꢁ1), respectively.
=
Their relative amplitudes are 0.41 and 0.59,
respectively. The reaction time (τ) in Cu complex 4 is
22.05 min and the primary reaction rate constant is
4.5 ꢄ 10ꢁ2. In the LD experiment, the decrease in the LD
signal over time is generally influenced by DNA orientation
and optical factors. However, in this experiment, the optical
factor was constant since the surrounding environment such
as the concentrations and temperatures of the complexes
was maintained the same during the measurement of the
changes in the LD signal over time.
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