Copper(II) complexes of acylhydrazones: Synthesis, characterization and DNA interaction

Article abstract of DOI:10.1002/aoc.2955

Two new acylhydrazone copper(II) complexes of 4-hydroxy-N′-[(1E)-1- (4-methylphenyl)ethylidene]benzohydrazide (HL¹) and 4 ethyl [4-({(2E)-2-[1-(4-methylphenyl)ethylidene]hydrazinyl}carbonyl)phenoxy]acetate (HL²) have been synthesized and characterized. The structures of both acylhydrazone and copper(II) complexes were identified by elemental analysis, infrared spectra, UV-visible electronic absorption spectra, magnetic susceptibility measurements, TGA and powder X-ray diffraction. DNA binding and DNA cleavage activities of the synthesized copper complexes were examined by using UV-visible titration and agarose gel electrophoresis, respectively. The effect of complex concentration on the DNA cleavage reactions in the absence and presence of H₂O₂ was also investigated. The results indicate that all the complexes bind slightly to calf thymus DNA and cleavage pBR322 DNA. The mechanistic studies demonstrate that a hydrogen peroxide-derived species and singlet oxygen (¹O₂) are the active oxidative species for DNA cleavage. Copyright 2013 John Wiley & Sons, Ltd. Two new acylhydrazone copper(II) complexes of 4-hydroxy-N'-[(1E)-1-(4-methylphenyl) ethyl-idene]benzohydrazide and 4 ethyl [4-({(2E)-2-[1-(4-methylphenyl)- ethylidene]hydrazinyl}carbonyl)-phenoxy]acetate have been synthesized and characterized. DNA binding and DNA cleavage activities of the synthesized copper(II) complexes were examined. The mechanistic studies show that a species derived from hydrogen peroxide and singlet oxygen are the active oxidative species for DNA cleavage.

Full text of DOI:10.1002/aoc.2955

Full Paper
Received: 23 May 2012
Revised: 11 October 2012
Accepted: 21 November 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/aoc.2955
Copper(II) complexes of acylhydrazones:
synthesis, characterization and DNA interaction
Cansu Gökçe and Ramazan Gup*
Two new acylhydrazone copper(II) complexes of 4-hydroxy-N0-[(1E)-1-(4-methylphenyl)ethylidene]benzohydrazide (HL1) and 4
ethyl [4-({(2E)-2-[1-(4-methylphenyl)ethylidene]hydrazinyl}carbonyl)phenoxy]acetate (HL2) have been synthesized and
characterized. The structures of both acylhydrazone and copper(II) complexes were identified by elemental analysis, infrared
spectra, UV–visible electronic absorption spectra, magnetic susceptibility measurements, TGA and powder X-ray diffraction.
DNA binding and DNA cleavage activities of the synthesized copper complexes were examined by using UV-visible titration
and agarose gel electrophoresis, respectively. The effect of complex concentration on the DNA cleavage reactions in the
absence and presence of H2O2 was also investigated. The results indicate that all the complexes bind slightly to calf thymus
DNA and cleavage pBR322 DNA. The mechanistic studies demonstrate that a hydrogen peroxide-derived species and singlet
oxygen (1O2) are the active oxidative species for DNA cleavage. Copyright © 2013 John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
Keywords: acylhydrazone; copper(II) complexes; DNA binding and cleavage
purchased from Fluka and Sigma-Aldrich and used without
further purification. Calf thymus DNA (CT-DNA) was purchased
Introduction
The chemistry of transition metal complexes has been exten-
sively investigated in recent years to assess their potential appli-
cations in bioinorganic chemistry.[1–8] In biological systems metal
ions are always coordinated to ligands rather than existing as free
ions, and many transition metal complexes have been identified
as anticancer, antitubercular, antibiotic, antimicrobial, antifungal
and herbidical agents.[9–13] Copper complexes have a long his-
tory of medicinal use and their antitumor properties have
attracted attention recently as they are less toxic than complexes
of non-essential metals such as platinum.[14,15] It is well recog-
nized that copper complexes have the ability to induce the hy-
drolysis or oxidative cleavage of DNA, and Cu(II) complexes are
among the most extensively studied chemical nucleases.[16–22]
Usually, the DNA cleavage activity of Cu(II) complexes is observed
in the presence of oxidizing or reducing co-reactants. Despite
many studies of the structure and DNA interactions of transition
metal complexes with potential as anticancer agents, because
of serious side effects of many of the drugs development of
new transition metal complexes is still required.
from Sigma- Aldrich. pBR322 DNA was purchased from Ferman-
tas. 1H and 13C NMR spectra were recorded on a Bruker
400 MHz spectrometer in DMSO-d6 with tetramethylsilane as
the internal standard. IR spectra were recorded on a PerkinElmer
1605 FT-IR spectrometer as KBr pellets. The electronic spectra of
the ligands and complexes were recorded on a UV-1601
Shimadzu spectrophotometer. Carbon, hydrogen and nitrogen
analyses were carried out on a LECO 932 CHNS analyzer and
metal contents were determined by inductively coupled
plasma–atomic absorption spectroscopy (ICP-AES) using a DV
2000 PerkinElmer instrument. Room temperature magnetic
susceptibility measurements were carried out on powdered
samples using a Sherwood Scientific MK1 Model Gouy magnetic
susceptibility balance. Melting points were determined on an
Electrothermal IA 9100 digital melting point apparatus. Thermo-
gravimetric analysis was carried out in a dynamic nitrogen
atmosphere (20 ml minꢀ1) with a heating rate of 20 ꢁC minꢀ1
using a PerkinElmer Pyris 1 TGA thermal analyzer in the Central
Laboratory at Middle East Technical University. The X-ray diffrac-
tion (XRD) analyses of the powder samples were recorded with a
Rigaku Corporation X-ray diffractometer (SmartLab model)
analyzer in the Central Laboratory at Mugla Sıtkı Koçman Univer-
sity. All the diffraction patterns were obtained by using CuKa1-
radiation, with a graphite monochromator at 5ꢁ minꢀ1 scanning
rate. 4-Hydroxy-N0-[(1E)-1-(4-methylphenyl)ethylidene]benzohy-
drazide (HL1) was synthesized as described previously.[23]
Acylhydrazones have a number of metal binding sites and can act
as neutral or monoanionic bidentate or tridentate ligands, depend-
ing on the substituents and the reaction conditions. Furthermore,
they can coordinate to metals as either the keto or the enol tauto-
meric form, making them attractive as ligands. Herein, we described
the synthesis and characterization of new Cu(II) complexes of acylhy-
drazones and their DNA binding and cleavage activities.
Experimental
Chemistry
*
Correspondence to: Ramazan Gup, Department of Chemistry, Mugla Sıtkı
Koçman University, 48000 Kotekli-Mugla, Turkey. E-mail: rgup@mu.edu.tr
All chemicals used were analytical reagent grade. Copper(II)
acetate, ethyl 4-hydroxybenzoate, 4-methylacetophenone, ethyl
bromoacetate, K2CO3, acetone and hydrazine monohydrate were
Department of Chemistry, Mugla Sıtkı Koçman University, 48000Kotekli-Mugla,
Turkey
Appl. Organometal. Chem. 2013, 27, 263–268
© 2013 John Wiley & Sons, Ltd.

C. Gökçe and R. Gup
Synthesis of 4 ethyl [4-({(2E)-2-[1-(4-methylphenyl)ethylidene]hydrazinyl}
H2O2 (5 mM) was added to the mixture to oxidize the reactant.
Finally, the reaction mixture was diluted with the Tris buffer
(100mM Tris, pH 8) to a total volume of 30 ml. After that reaction
mixtures were incubated at 37 ꢁC for 2 h. Samples (20 ml) were then
incubated at 37 ꢁC and loaded with 4 ml loading dye (0.25% bromo-
phenol blue, 0.25% xylene cyanol, 30% glycerol, 10 mmol etyhyle-
nediaminetetraacetic acid (EDTA)) on a 1% agarose gel containing
1 mg mlꢀ1 of EtBr. The gel was run at 100 V for 3 h in Tris–borate–
EDTA (TBE) buffer and photographed under UV light. To test for
the presence of reactive oxygen species generated during strand
scission, reactive oxygen intermediate scavengers were added
alternately to the reaction mixture. These scavengers were DMSO
(100mM), KI (100 mM), NaN3 (100 mM) and EDTA (100 mM). Samples
were treated as described above.
carbonyl)-phenoxy]acetate (HL2)
A mixture of HL1 (10 mmol, 2.683 g), ethyl bromoacetate
(10 mmol, 1.670 g) and dry K2CO3 (10 mmol, 1.380 g) in 40 ml
acetone was refluxed with stirring for 18 h and poured into
200 ml cold water. The white precipitate formed was filtered and
washed with water and finally recrystallized from ethanol–water.
Yield 62 %; m.p. 135 ꢁC; UV (EtOH, nm) 298, 222; IR (KBr)
(n, cmꢀ1) 3229 (NH), 1764 (C=O)ester, 1648 (C=O)amide, 1607
(C=N), 1385 (C-N), 1263 and 1084 (C-O-C); 1H NMR (CDCl3,
a
ppm) d 1.25 (t, J = 7.1, CH3), 2.25 (s, 3H, H3Cc-Ar), 2.31 (s, 3H,
N=C-bCH3), 4.23 (q, J = 7.1, OCH2CH3), 4.57 (s, Ar-OCH2), 6.85
(d, 2H, J = 8.3, ArHd), 7.78 (d, 2H, J = 8.3, ArHe), 7.11 (d, 2H,
J = 8.2, ArHf), 7.88 (d, 2H, J = 8.2, ArHg), 9.37 (s, 1H, NH); 13C
NMR (CDCl3, ppm) 168.7 (C14), 160.5 (C8), 154.0 (C6), 139.8
(C12), 135.3 (C5), 132.5 (C2), 129.3 (C9), 128, 6 (C4), 126.8 (C10),
114.5 (C3), 113.9 (C11), 65.3 (C13), 61.7 (C15), 21.5 (C7), 19.4
(C1), 14.4 (C16). Anal. (% calc./found) for C20H22N2O4 C: 67.78/
67.40, H: 6.26/6.36, N: 7.90/7.69.
Results and Discussion
HL1 was synthesized by refluxing 4-hydroxybenzoydrazide with
p-methyl acetophenone in the presence of two drops of glacial
acetic acid in absolute ethanol.[17] The reaction of this compound
with ethyl bromoacetate in the presence of dry K2CO3 in dry
acetone gave HL2. The reactions proceeded smoothly, producing
the corresponding acylhydrazones in good yields (Scheme 1).
The acylhydrazone ligand is soluble in common organic solvents
but insoluble in water. The structure of the ligand was elucidated
Synthesis of Cu(II) complexes
A solution of 1 mmol copper(II) acetate dihydrate in EtOH
(10 ml) was added to a hot solution containing triethylamine
(2 mmol, 0.202 g) and 2 mmol HL1 and HL2 in absolute ethanol
(25 ml) with stirring. The reaction mixture was refluxed for 3 h.
On standing over night, the complexes were separated,
collected by filtration and finally washed with water.
1
by elemental analyses, FT-IR, electronic absorption, and H and
13C NMR (Scheme 1). The complexes were synthesized by react-
ing the acylhydrazones with copper(II) acetate in a molar ratio
of 2:1. Attempts to isolate crystals suitable for single X-ray diffrac-
tion were unsuccessful. Therefore, the overall geometry of the
complexes was inferred from the X-ray powder diffraction data,
thermal and elemental analysis, IR and electronic spectra.
For [Cu(L1)2]: green complex; yield 81%; m.p. 234ꢁC. meff = 1.69
BM; UV (DMF, nm) 384, 300; FT-IR (KBr, cmꢀ1): 3309 b (O–H),
1610 s (C=N–N=C), 1364 (C-N), 1237 s and 1166m (C–O–C). Anal.
(% calc./found) for C32H30CuN4O4 C: 64.26/63.94, H: 5.06/5.14,
N: 9.37/9.09, Cu: 10.62/10.31.
For [Cu(L2)2]: dark-grey complex; yield 79%; m.p. 139 ꢁC. meff =1.64
BM; UV (DMF, nm) 381.5 298.5; FT-IR (KBr, cmꢀ1): 1762 s (C= =O)ester
,
1H and 13C NMR spectroscopy
1606 m (C=N–N=C), 1206 s and 1169 m (C–O–C). Analysis (% calc./
found) for C40H44CuN4O9 C: 60.94/60.71, H: 5.63/5.49, N: 7.12/7.09,
Cu: 8.06/8.13.
The main 1H NMR signals for each of the acylhydrazone com-
pounds are given in the Experimental section. Formation of the
HL2 was confirmed by the absence of the OH proton signal at
10.18 ppm assigned to the starting material HL1. The D2O
exchangeable singlet resonance at 9.37 ppm for HL2 ligand is
assigned to NH protons. The singlet and triplet signals observed
at 4.57 ppm and at 4.23 ppm are attributed to methylene protons
DNA Binding
Electronic absorption titrations
All experiments involving the interaction of the complexes with
CT-DNA were carried out in water buffer containing 5 mM Tris
[tris(hydroxymethyl)aminomethane] and 50 mM NaCl, and ad-
justed to pH 7.3 with HCl. The solution of CT-DNA in the buffer
gave a ratio of UV absorbance of 1.8–1.9:1 at 260 and 280 nm, in-
dicating that the CT-DNA was sufficiently free of protein.[24] The
CT-DNA concentration per nucleotide was determined spectro-
photometrically by employing an extinction coefficient of
6600 Mꢀ1 cmꢀ1 at 260 nm.[25] An appropriate amount of metal
complex was dissolved in a solvent mixture of 1% DMF and
99% Tris–HCl buffer. Absorption titration experiments were per-
formed by maintaining the metal complex concentration as con-
stant while gradually increasing the concentration of CT-DNA
within 0–100 mM.
1
of –CH2OPh and –OCH2CH3 groups, respectively, in the H NMR
1
spectra of HL2. The other obtained values for H NMR chemical
shifts of HL2 are given in the Experimental section. These
data are in agreement with those previously reported for similar
compounds.[23,26–33]
1
The H NMR spectral assignments of the new acylhydrazone
compound are also supported by the 13C NMR spectrum. The
characteristic chemical shifts of the carbonyl (C8) and azo-
methine (C6) groups of the HL2 are observed at 160.5 and
154.0 ppm. The signal at 168.7 ppm is assignable to the ester
carbonyl group (C14) of ligand HL2. The chemical shifts for the
carbon atoms of the aromatic rings were recorded between
113.9 and 139.8 ppm. The signals observed between 14.4 and
65.3 ppm are attributed to the aliphatic carbon atoms. These
assignments are in good agreement with those previously
reported for similar compounds.[23,26–33] These results strongly
suggest that the proposed acylhydrazone compound has been
DNA cleavage
pBR322 plasmid DNA was used for all cleavage activities. In a typical
experiment, 7ml plasmid DNA (50 ng mlꢀ1) was mixed with different
concentrations of complexes (10, 25, 50,100 and 150mM dissolved
in DMF) to determine optimum activation concentration. 5ml
1
formed. In the H and 13C NMR spectra of the HL1 and HL2 only
one signal was observed for all hydrogens and carbons,
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Appl. Organometal. Chem. 2013, 27, 263–268

Copper(II) complexes of acylhydrazones
Scheme 1. Schematic diagram showing the synthesis of acylhydrazones and copper(II) complex. (i) NH2NH2.H2O, EtOH, reflux 4 h; (ii) 4-methylaceto-
phenone, EtOH, AcOH, reflux 4 h; (iii) ethyl bromoacetate, K2CO3, acetone, reflux 15 h; (iv) Cu(OAc).2H2O, EtOH, reflux 2 h.
indicating that both acylhydrazones are in the E configura-
tion.[34,35] Owing to the paramagnetic nature of the copper(II)
complexes, their 1H and 13C NMR spectra could not be obtained.
evidence concerning the structure of the metal complexes,
powder XRD recorded at 2θ = 10–70ꢁ was performed (supporting
information Fig. S1). Both copper complexes show sharp peaks,
indicating that the above complexes are crystalline in nature.
The XRD patterns for complexes are quite similar and suggest
that the complexes have similar structure.
IR spectroscopy
The IR spectrum of HL1 shows a broad O–H stretching vibration
at 3296 cmꢀ1. However, in the IR spectrum of HL2 this band
disappears, indicating that complete condensation takes place.
The characteristic amide I band of new hydrazone is observed at
1648 cmꢀ1. On the other hand, the band observed at 1764 cmꢀ1
is assigned to the ester carbonyl stretching vibration, which is
also characteristic for carboxylic esters. The amide NH stretching
band of HL1 was not observed in the IR spectra, probably due to
overlapping with the broad OH stretching frequency. However,
these bands are observed clearly at 3229 cmꢀ1 in IR spectra of
the HL2 ligand, which is hydroxyl group free. The other character-
istic IR peaks of acylhydrazone ligand synthesized in this work are
given in the Experimental section. These values are in accord with
those previously reported for such compounds.[23,26–28,31–33,36–38]
To determine the modes of the acylhydrazone ligands coordi-
nation with Cu(II) ion, we compared the IR spectra of the metal
free ligands with those of the complexes. The IR spectra of
the copper(II) complexes lack absorptions due to amide I
[n(C= =O)], n(C= =N)imine and n(N–H), but show a new band
between 1610 and 1606 cmꢀ1 probably due to n(C= =N–N= =C)
stretching, suggesting that the NH proton is likely lost via depro-
tonation induced by the metal and the resulting enolic oxygen
and the azomethine nitrogen coordinate to the copper.[29–33]
Therefore, from the IR spectra, it is concluded that acylhydra-
zones act as mono anionic bidentate ligands coordinating
through the azomethine nitrogen and the enolic oxygen. In
the IR spectra of the [Cu(L4)2] complex, the (C= =O)ester stretch-
ing vibration is observed almost at the same position as in the
metal-free ligand, indicating non-participation of the ester
carbonyl in coordination.
Thermal Analyses
According to TGA curves, thermal degradation of [Cu(L1)2] complex
occurred in two steps (supporting information Fig. S2). The first step
within the temperature range 240–460 ꢁC corresponds to the
removal of the organic part of the acylhydrazone ligand. The last step
is the decomposition of copper(II) complexes, leaving metallic ash as
a residue. On the other hand, the thermal degradation of [Cu(L2)2]
occurred in three steps. This complex showed the loss of one mole
water molecule within the temperature range 170–190 ꢁC. The other
steps of decomposition of this complex involve the removal of the
organic part of ligand, leaving metallic ash as a residue.
Electronic Absorption Spectra
The electronic spectra of acylhydrazones exhibited maxima at
299 nm and 298 nm for HL1 and HL2, respectively, corresponding
to the n ! p* electronic transitions.[26,27,31] Electronic absorption
spectra of the complexes were recorded in DMF solution. By com-
paring the electronic absorption spectra of the free hydrazone
ligands and their copper complexes, it was observed that the
maxima bands of the free ligands showed a slight red shift. Fur-
thermore, in the electronic spectra of the copper complexes there
is a band in the visible region, 384 nm for [Cu(L1)2] and 382 nm for
[Cu(L2)2], attributed to LMCT transition. Unfortunately the
expected weak d–d transition in the visible region for the one-
electron paramagnetic (meff = 1.69 and 1.64 BM) complexes cannot
be detected even with concentrated solutions. It may be lost in
the low energy tail of the charge transfer transition.[32,33,39,40]
DNA Binding Studies
XRD Analysis
Electronic absorption titrations
Single-crystal XRD could not be employed to confirm the struc-
tures of the complexes since attempts to isolate crystals suitable
for single X-ray diffraction were unsuccessful. To obtain further
Absorption titration is an effective method to examine the bind-
ing mode of DNA with metal complexes.[41–45] Therefore, the
Appl. Organometal. Chem. 2013, 27, 263–268
© 2013 John Wiley & Sons, Ltd.
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C. Gökçe and R. Gup
binding interaction of the complexes bearing acylhydrazone
ligands with CT-DNA was monitored by UV-visible spectroscopy.
The absorption spectra of the above four complexes recorded
in the absence and presence of increasing amounts of CT-DNA
are given in Fig. 1. The absorption spectra of the complexes are
affected by increasing concentration of CT-DNA. The spectra
show clearly that the addition of DNA to the solutions of the
copper(II) complexes causes hypochromism with a red shift.
Upon the addition of DNA, the band at 294 nm for the complex
[Cu(L1)2] exhibited hypochromism of about 1.79% together with
a 1 nm red shift (Table 1). Similarly, the band at 290 nm for
complex [Cu(L2)2] showed hypochromism (1.38%), accompanied
by a red shift of 1 nm. The bands at 222 nm for both copper(II)
complexes did not show any shift, nor any change in absorption
of intensity. A distinct isosbestic point observed at 306 nm for
complex Cu(L1)2, and at 305 nm for complex Cu(L1)2, indicates
the existence of a single mode of binding. The observed
hypochromism could be attributed to a slight stacking interac-
tion between the aromatic chromophores of the complexes and
DNA base pairs consistent with the intercalative binding mode,
while the red shift is evidence of the stabilization of the CT-
DNA duplex. The binding constant, Kb, was determined by using
the following equation:
Table 1. Effect of CT-DNA on the absorbance bands and binding
constant of the complexes and adducts
ꢀ1
Complex
lmax (nm)
Δl (nm)
H (%)
Kb (M
)
Free
Bound
Cu(L1)2
Cu(L2)2
294
290
295
291
1.0
1.0
1.79
1.38
7.1 ꢃ 103
4.0 ꢃ 103
active in the absence of H2O2 as their electrophoresis patterns
are similar to that of control DNA (lane 11). In contrast, in the
presence of hydrogen peroxide both complexes show nucle-
ase activity. As shown in Fig. 2, an increase in complex
concentration gives rise to an increase in the DNA cleavage.
In lanes 1–3 for the complex [Cu(L1)2], the circular supercoiled
DNA band is completely lost and forms I and III are present.
With concentration of 75 mM (lane 4), only the linear DNA band
is observed. On the other hand, in lane 5 no bands are observed.
When the concentration of the [Cu(L1)2] is increased to 100 mM, the
circular supercoiled DNA degrades completely into small pieces.
Interestingly, this complex shows slight nuclease activity at high
concentrations of the complex in the absence of hydrogen perox-
ide. At 75 and 100 mM a small amount of the supercoiled DNA was
converted to the nicked DNA as seen in lanes 9 and 10 in Fig. 2. It
is reasonably expected in the absence of an oxidant agent the
DNA cleavage is hydrolytic. On the other hand, it is also
reported that the DNA cleavage mediated by the some cop-
per and zinc complexes occurs via an oxidative mechanism
without any reducing agent.[21,48–50] The oxidative DNA clea-
vages in these works are suggested to be ligand-based. For
this reason, and keeping this in mind, it may be concluded
that the remarkable amount of organic radical in the complex
[Cu(L1)2] can be generated, probably from phenol moiety of
the ligand, at high concentrations. This non-diffusible radical
may cause an oxidative DNA cleavage in the absence of
hydrogen peroxide. Further studies are underway to elucidate
the cleavage mechanism.
½DNAꢂ ðea ꢀ ef Þ ¼ ½DNAꢂ ðeo ꢀ ef Þ þ 1 Kbðeo ꢀ ef Þ
where [DNA] is the concentration of DNA in base pairs, ea, ef and
eo correspond to Aobsd / [M], the extinction coefficient of the
complexes and the extinction coefficient of the complex in the
fully bound form, respectively, and Kb is the intrinsic binding
constant. The ratio of the slope to intercept in the plot of [DNA]
(ea – ef) versus [DNA] gives the value of Kb for the complex. The
obtained binding constants of these complexes are much lower
than those observed for typical classical intercalators, ethidium
DNA (Kb = 1.4 ꢃ 106
M
ꢀ1). On the other hand, the binding
constant of [Cu(L1)2] complex is higher than that of [Cu(L2)2].
DNA Cleavage Studies
The ability of the copper complexes to cleave DNA in the presence
and absence of H2O2 was studied by gel electrophoresis using the
supercoiled form of pBR322 at 37 ꢁC in TBE buffer.[5–8,46,47] We
prepared five different concentrations of the complexes with a
range from 10 to 100 mM. The progress of the cleavage reaction with
increasing concentrations of each copper complex in the presence
and absence of H2O2 is given in Fig. 2. From the results shown in
lanes 6–10 for [Cu(L1)2] and lanes 18–22 for [Cu(L1)2], it can
be deduced that both copper complexes are not nuclease
[Cu(L2)2] is found to cleave the supercoiled DNA to nicked and
linear DNA forms at the same time at low concentrations in the
presence of hydrogen peroxide. With 10 and 25 mM concentra-
tions (lanes 13 and 14), the intensity of form I is found to
decrease, while the intensities of forms II and III increase. It is also
seen that the intensity of nicked DNA is much higher than that of
form III. When the complex concentration is at 50 mM, the circular
supercoiled form disappeared completely and the intensity of
form III increased compared with intensity of form III in lane 13
Figure 1. Electronic spectra of Cu(II) complexes in the presence of increasing amounts of CT-DNA. [CT-DNA] = 0–100 mM. (a) [Cu(L1)2]; (b) [Cu(L2)2]. (––)
presence of T-DNA; (- - -) absence of CT-DNA.
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Copper(II) complexes of acylhydrazones
Figure 2. Agarose gel electrophoresis of pBR322 plasmid DNA treated with different concentrations of copper(II) complexes. Lane 1, Cu(L1)2 (10 mM) +
DNA + H2O2; lane 2, Cu(L1)2 (25 mM) + DNA + H2O2; lane 3, Cu(L1)2 (50 mM) + DNA + H2O2; lane 4, Cu(L1)2 (75 mM) + DNA + H2O2; lane 5, Cu(L1)2 (100 mM) +
DNA + H2O2; lane 6, Cu(L1)2 (10 mM) + DNA; lane 7, Cu(L1)2 (25 mM) + DNA; lane 8, Cu(L1)2 (50 mM) + DNA; lane 9, Cu(L1)2 (75 mM) + DNA; lane 10, Cu(L1)2
(100 mM) + DNA; lane 11, DNA control; lane 12, 1 kb marker; lane 13, Cu(L2)2 (10 mM) + DNA + H2O2; lane 14, Cu(L2)2 (25 mM) + DNA + H2O2; lane 15, Cu
(L2)2 (50 mM) + DNA + H2O2; lane 16, Cu(L2)2 (75 mM) + DNA + H2O2; lane 17, Cu(L2)2 (100 mM) + DNA + H2O2; lane 18, Cu(L2)2 (10 mM) + DNA; lane 19, Cu
(L2)2 (25 mM) + DNA; lane 20, Cu(L2)2 (50 mM) + DNA; lane 21, Cu(L2)2 (75 mM) + DNA; lane 22, Cu(L2)2 (100 mM) + DNA.
and 14. With a complex concentration of 75 and 100 mM, no band
is observed in lanes 16 and 17, probably due to complete degra-
dation of DNA into small pieces. This complex does not cause any
DNA damage in the absence of hydrogen peroxide regardless of
the concentration of the copper complex (lanes 18–22 in Fig. 2).
peroxide.[56] Similarly, the significant reduction in the ability of
the complexes to cleavage DNA in the presence of KI suggests
that a hydrogen peroxide-derived species is the reactive species
that actually cleaves DNA. Neither complex exhibits DNA damage
activity in the presence of EDTA (lanes 2 and 6). EDTA, a metal
chelating agent that strongly binds to metal ions forming a stable
complex, can efficiently inhibit DNA cleavage, indicating that
copper ions play a key role in the cleavage.
Mechanism of DNA cleavage
The cleavage mechanisms of pBR322 DNA induced by [Cu(L1)2]
and [Cu(L2)2] complexes were investigated and clarified in the
presence of different scavenging agents, including a hydroxyl
radical scavenger (DMSO),[51,52] a hydrogen peroxide scavenger
(KI),[53,54] a singlet oxygen scavenger (NaN3)[51,54] and a chelating
agent (EDTA).[51,55] Figure 3 clearly shows the near disappearance
of form II in the presence of the hydroxyl scavenger, DMSO
(Lanes 4 and 8). This demonstrates that hydroxyl radicals are
the active oxidative species that promote the DNA cleavage by
the complexes. While azide is ineffective for the [Cu(L2)2] complex
(lane 5), addition to DNA + [Cu(L1)2] significantly inhibited the
nuclease activity of the complex (lane 1), indicating that singlet
oxygen radical is probably involved in the DNA cleavage reaction
of the [Cu(L1)2]. The reason of this difference may be explained by
the fact that L1 has a hydroxyl group which is effective in the
formation of singlet oxygen in the presence of hydrogen
Conclusions
Two new copper(II) complexes of two acylhydrazones have been
synthesized and characterized using different spectroscopic
techniques. Studies on the binding of both copper complexes
with DNA show that they can bind to CT-DNA due to a chelating
effect. The types of binding mode of the titled complexes are
almost the same. The DNA cleavage capabilities of the copper
(II) complexes in the presence of H2O2 revealed that they cleave
the circular supercoiled DNA to nicked DNA and linear DNA
under aerobic conditions. The DNA cleavage is clearly concentra-
tion dependent. [Cu(L1)2] exhibited better nuclease activity than
[Cu(L2)2] since it can cause DNA damage even in the absence of
hydrogen peroxide.
Acknowledgements
We thank the Scientific Research Projects Foundation of Mugla
Sıtkı Koçman University for financial support of this work.
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