Two polypyridyl copper(ii) complexes: Synthesis, crystal structure and interaction with DNA and serum protein in vitro

Article abstract of DOI:10.1039/c3nj01107k

Two polypyridyl copper(ii) complexes, [Cu(acac)(p-CPIP)(CH ₃OH)](NO₃) (1) and [Cu(acac)(o-NPIP)(NO₃)] (2) (acac = acetylacetonate, p-CPIP = 2-(4-chlorophenyl)imidazo[4,5-f]1,10- phenanthroline, o-NPIP = 2-(2-nitrophenyl)im

Full text of DOI:10.1039/c3nj01107k

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Two polypyridyl copper(II) complexes: synthesis,
crystal structure and interaction with DNA and
serum protein in vitro†
Cite this: New J. Chem., 2014,
3
8, 955
ab
a
a
a
a
Xiao-Fei Zhao,‡ Yan Ouyang,‡ Yan-Zhao Liu, Qiao-Juan Su, He Tian,
a
a
Cheng-Zhi Xie and Jing-Yuan Xu*
3 3 3
Two polypyridyl copper(II) complexes, [Cu(acac)(p-CPIP)(CH OH)](NO ) (1) and [Cu(acac)(o-NPIP)(NO )]
(2) (acac = acetylacetonate, p-CPIP = 2-(4-chlorophenyl)imidazo[4,5-f]1,10-phenanthroline, o-NPIP =
2-(2-nitrophenyl)imidazo[4,5-f]1,10-phenanthroline), have been synthesized and characterized by single-
crystal analysis, IR and electronic spectroscopy. X-ray analyses reveal that both 1 and 2 possess a triclinic
crystal system with Pmn1 and mononuclear five-coordinated square pyramidal geometry, which were
further linked to a one-dimensional structure through p–p stacking and intermolecular hydrogen bonds.
The interaction of the Cu(II) complex with calf thymus DNA (CT-DNA) was investigated by UV-visible and
fluorescence emission spectrometry, as well as agarose gel electrophoresis. The apparent binding constant
4
À1
4
À1
(Kapp) values of 2.47 Â 10
M
for 1 and 3.04 Â 10
M
for 2 suggest moderate intercalative binding
mode between the complexes and DNA. Both complexes displayed efficient oxidative cleavage of
supercoiled DNA in the presence of external agents. In addition, fluorescence spectrometry of bovine
serum albumin (BSA) with the complexes showed that the quenching mechanism of 1 might be a dynamic
procedure, while 2 showed a combined dynamic and static quenching mechanism. For both 1 and 2, the
number of binding sites was about one for BSA. Moreover, synchronous fluorescence spectral experiments
revealed that 1 and 2 affect the microenvironment of tryptophan residues of BSA.
Received (in Victoria, Australia)
6th September 2013,
Accepted 2nd December 2013
1
DOI: 10.1039/c3nj01107k
www.rsc.org/njc
antitumor drugs, because the interaction between the small
molecule and DNA can cause DNA damage in cancer cells.
Introduction
9
,10
In recent years, metal complexes that are capable of cleaving Metal complexes containing a planar heterocyclic aromatic
DNA under physiological conditions have attracted increasing ligand, such as polypyridyl, have been at the forefront of these
attention not only because of their potential applications in investigations owing to their unusual electronic properties, diverse
artificial nucleic acid chemistry, but also because of the development chemical reactivity and peculiar structures, which resulted in their
1
–6
of metal-based anticancer agents. As we know well, cisplatin binding to DNA by many modes such as electrostatic, groove and
11
as an efficient clinical chemotherapeutic agent represents a intercalative binding. In general, polypyridyl metal complexes
major landmark in the history of metal-based anticancer drugs. displayed the intercalative binding modes to DNA for their large
12,13
However, cisplatin is associated with acquired resistance and the planar ligands and p–p overlapping interaction.
Over the past
limited spectrum of the anticancer activity, which has stimulated us few decades, it has been considered that the polypyridyl metal
to develop more effective, less toxic, target specific, and preferably complexes can probe nonradioactive nucleic acid and effectively
7
,8
14
noncovalently binding anticancer drugs. Heretofore, many studies cleave DNA. In general, the DNA cleavage mode mainly contained
15
have suggested that DNA is the primary intracellular target of oxidative cleavage, photocleavage and hydrolytic cleavage. While
most studies suggested that polypyridyl transition metal complexes
1
6
a
exhibit photocleavage ability towards DNA. However, studies
on those polypyridyl complexes hitherto have mainly focused on
fully planar ligands, and as far as we know, the complexes
containing such ligand derivatives with diverse substituents
are seldom investigated. In fact, introducing substituents
could improve the cleavage ability of polypyridyl complexes to
Tianjin Key Laboratory on Technologies Enabling Development of Clinical
Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medical University,
Tianjin 300070, P. R. China. E-mail: xujingyuan@tmu.edu.cn
b
Department of Pharmacy, The Second Affiliated Hospital of Xing Tai Medical
College in Hebei, Xingtai, 054000, P. R. China
17
†
Electronic supplementary information (ESI) available. CCDC 920155 for the
p-CPIP ligand, 801495 for 1 and 859802 for 2. For ESI and crystallographic data in
bind to DNA, such as o-NPIP based on the almost coplanar
ligand of (2-phenyl)imidazo[4,5-f]1,10-phenanthroline (PIP).
CIF or other electronic format see DOI: 10.1039/c3nj01107k
1
8,19
‡
These authors contributed equally to this work.
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The ruthenium(II) complex with o-NPIP has been synthesized Bruker AVANCE III 400 spectrometer with TMS as internal
and exhibited good DNA binding ability and photocleavage standard and (CD SO as solvent. Infrared spectroscopy on
3 2
)
2
0
ability. Inspired by these results, we synthesized a ligand KBr pellets was performed on a Bruker Vector 22 FT-IR spectro-
-(4-chlorophenyl)imidazo[4,5-f]-1,10-phenanthroline (p-CPIP) and photometer in the 4000–400 cm regions. The fluorescence
À1
2
two complexes [Cu(acac)(p-CPIP)(CH OH)](NO ) and [Cu(acac)- spectral data were obtained on a MPF-4 fluorescence spectro-
3
3
3
(o-NPIP)(NO )]. The choice of Cu was based on the fact that copper, photometer at room temperature. The Gel Imaging and documenta-
as a fundamental micronutrient for human, has bioessential tion DigiDoc-It System were assessed by Labworks Imaging and
activity and oxidative nature, which has attracted numerous Analysis Software (UVI, UK). The electronic spectra were recorded on
inorganic chemists to find the potential medical applications of a HITACHAI spectrophotometer. Emission spectra were recorded
21
copper complexes. Since Sigman found the ‘‘chemical nuclease’’ on a Shimadzu RF-5301 fluorescence spectrophotometer with
2
+
14
activity of [Cu(phen)] (phen = 1,10-phenanthroline), Cu(II) excitation at 510 nm.
complexes have been widely used as DNA footprinting agents,
Syntheses of ligands
as structural probes for DNA and as potential anticancer drugs.
Especially, Cu(II) complexes containing planar ligands have
2-(4-Chlorophenyl)imidazo[4,5-f]1,10-phenanthroline (p-CPIP).
received considerable current interest because of their strong The polypyridyl ligand of p-CPIP was obtained according to the
DNA binding ability and DNA cleavage activities via oxidation procedure in ref. 30. A mixture of 4-chlorobenzaldehyde (0.28 g,
22
and photocleavage. On the other hand, it is well known that 2 mmol), 1,10-phenanthroline-5,6-dione (0.21 g, 1 mmol) and
serum albumins, as the most abundant proteins in the circulatory glacial acetic acid (15 mL) was refluxed for about 3 h, then cooled
system, act as transporters and disposers of many endogenous and to room temperature and diluted with water (40 mL). Dropwise
23
exogenous compounds, including drugs and nutrients, mostly addition of concentrated aqueous ammonia gave yellow precipi-
through the formation of noncovalent complexes as specific binding tates, which were collected and washed with water. The crude
sites. Therefore, the absorption, distribution, metabolism and excre- products were purified by silica gel filtration (60–100 mesh,
tion properties, as well as the stability and toxicity of chemical ethanol). The principal yellow band was collected and solvent
substances, can be significantly affected by their binding activities was removed by rotary evaporation. The products were obtained,
24
to serum albumins. As a kind of serum albumin, bovine serum and then dried at 50 1C in vacuo. Then, the products were
albumin (BSA) has been extensively studied owing to its structural dissolved in ethanol and kept at room temperature for slow
25
homology with human serum albumin (HSA). Furthermore, evaporation, and gave the yellow crystals of 2-(4-chloro-
administration of platinum-based antitumor agents injected intra- phenyl)imidazo[4,5-f]1,10-phenanthroline after two days. Yield
venously prompts us to investigate the binding of this kind of 0.489 g, 74.1%. Anal. calc. for C H ClN O: C, 67.79, H, 4.40, N,
2
1
17
4
À1
complexes to BSA. As reported, 65–98% of platinum is bound by 14.49%, found: C, 67.77; H, 4.39; N, 14.51%. FT-IR (KBr, cm ):
proteins in blood plasma in one day after cisplatin treatment. Such 2964m(br), 1475(vs), 1446(vs), 1377(m), 1100(m), 1067(w), 1048(m),
interaction between platinum and proteins would lead to inactiva- 1012(w), 841(m), 801(m), 737(m), 686(m) cm . H NMR[(CD ) SO]:
3 2
26
À1 1
tion of drugs, thus affecting the therapeutic dose of drugs transferred d 13.81 (s, 1H), 9.04 (d, 2H), 8.91–8.87 (m, 2H), 8.30–8.28 (d, 2H),
2
7,28
to their target positions.
Therefore such a study is of special 7.87–7.81 (m, 2H), 7.71–7.69 (d, 2H).
2-(2-Nitrophenyl)imidazo[4,5-f]1,10-phenanthroline (o-NPIP).
interest in the evaluation of metal drugs.
Here, we report the synthesis, structural characterization This compound was synthesized in a similar way to the above
and biological properties of two polypyridyl Cu(II) complexes, one except for using 2-nitrobenzaldehyde (0.302 g, 2 mmol)
[
Cu(acac)(p-CPIP)(MeOH)](NO
3 3
) (1) and [Cu(acac)(o-NPIP)(NO )] (2). instead of 4-chlorobenzaldehyde. Yield 0.469 g, 68.1%. Anal. calc.
The results show that the two complexes can efficiently bind to DNA for C19
H
11
N
5
O
2
: C, 66.09, H, 3.19, N, 20.29%, found: C, 66.08, H,
À1
and BSA and cleave DNA in the presence of external agents.
3.17, N, 20.30%. FT-IR (KBr, cm ): 3006m(br), 1537(vs), 1364(m),
À1
1
8
(
(
08(w), 739(m), 721(w), 648(w) cm . H NMR[(CD
s, 1H), 9.05 (d, 1H), 9.01 (d, 1H), 8.95 (d, 1H), 8.86 (d, 1H), 7.94
d, 1H), 7.89–7.80 (m, 2H), 7.70 (d, 1H) and 7.60–7.53 (m, 2H).
3 2
) SO]: d 13.83
Experimental
Materials and physical measurements
Syntheses of complexes
All chemicals were purchased from commercial sources and used
3
[Cu(acac)(p-CPIP)(MeOH)](NO ) (1). The p-CPIP (0.066 g,
without further purification. Calf thymus DNA (CT-DNA), ethidium 0.2 mmol) and Cu(NO
bromide (EB), pUC19 DNA and BSA were from Sigma. Tris-HCl dissolved in MeOH and EtOH (v : v = 1 : 1) (20 mL). The solution
buffer solution was prepared using triple-distilled water. was refluxed for half an hour, and then acetylacetonate (0.09 mL)
3
)
2
Á3H
2
O (0.0429 g, 0.2 mmol) were
CT-DNA stock solution was prepared by dissolving into was added. The complex was refluxed for 3 h giving a clean blue
deionized sonicated triple-distilled water, and kept at 4 1C for solution. The reaction mixture was filtered off and kept at room
no longer than a week. The UV absorbance at 260 and 280 nm temperature for slow evaporation, and the blue crystals of
of CT-DNA solution in Tris-buffer gives a ratio of 1.8–1.9, [Cu(acac)(p-CPIP)(MeOH)](NO ) suitable for X-ray analysis were
3
2
9
indicating that the DNA was free of proteins.
collected after one day. Yield: 0.0649 g, 57% for 1. Elemental
Elemental analysis of C, H and N was carried out on a Perkin- analysis (%): calc. for C25
Elmer analyzer model 240. H NMR spectra were recorded on a 11.92%, found: C, 51.08, H, 3.73, N, 11.91%. FT-IR (KBr, cm ):
H
22ClCuN
5
O
6
: C, 51.06, H, 3.74, N,
1
À1
9
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3
1
7
078m(br), 2974m(br), 1582(v)s, 1520(vs), 1455(vs), 1378vs(br),
BSA binding studies. The protein (BSA) binding experiments
278s(br), 1101(m), 1075(m), 1014(s), 945(w), 843(m), 813(m), were performed using fluorescence quenching and UV spectro-
À1
71(w), 730(m) cm
.
photometry. The protein was prepared in the buffer solution
[
Cu(acac)(o-NPIP)(NO )] (2). The blue complex 2 was synthe- (containing 0.01 M Na HPO and NaH PO in pure aqueous
3
2
4
2
4
sized in a manner analogous to complex 1 mentioned above, medium at pH = 7.2). BSA solution concentration was controlled at
À7
with o-NPIP (0.0624 g, 0.2 mmol) in place of p-CPIP (0.066 g, 5 Â 10 M (based on the molecular weight of BSA 66 000), and the
0.2 mmol), and 20 mL of MeOH was employed as solvent. solutions were kept in the dark at 277 K for 24 h. Fluorescence
Yield: 0.061 g, 52% for 2. Elemental analysis (%): calc. for quenching measurements were done by keeping the concentration
C
48
H
36Cu
2
N
12
O
14: C, 50.88, H, 3.18, N, 14.84%; found C, 50.86, of BSA constant while adding the increasing concentration of 1 and
À1
H, 3.17, N, 14.86%. FT-IR (KBr, n/cm ): 2821(w), 1613(w), 2 at different temperatures (288, 298 and 310 K), respectively.
1
540(m), 1531(m), 1514(s), 1442(m), 1378(m), 1304(m), 1023(w), Fluorescence spectra were recorded at an excitation wavelength of
À1
816(w), 774(w), 728(w), 697(w) cm
.
290 nm and the emission spectra from 300 to 500 nm. Synchronous
fluorescence spectral experiments were carried out by the same
method as above while keeping the D values (Dl) between excitation
X-ray structure determination
Suitable single crystals of ligand p-CPIP, complexes 1 and 2 were and emission wavelengths, which are stabilized at 15 and 60 nm,
used for X-ray diffraction analyses by mounting on the tip of a respectively, at room temperature. UV spectrometry was performed
glass fiber in air. Crystal data were collected at 298(2) K for p-CPIP, using a HITACHAI spectrophotometer equipped with a 1 cm quartz
2
93(2) K for 1 and 113(2) K for 2, using a Bruker SMART CCD 1000 cell. Absorption titration experiments were done by keeping the
À5
diffractometer. Diffraction intensities for the three compounds concentration of BSA constant (3 Â 10 M) while adding the
were collected by using the o-scan technique. The structures were same concentration 1 and 2 solution, respectively. All the data
31
solved by direct methods using the program SHELXS-97 and were obtained after each successive addition of BSA solution
subsequent Fourier difference techniques, and refined anisotropi- and equilibration (ca. 10 min).
2
cally by full-matrix least-squares on F using SHELXL-97. All the
nonhydrogen atoms were refined anisotropically and all the
hydrogen atoms were located in the Fourier difference maps. Results and discussion
Measurements
We synthesized a new p-CPIP ligand, and two new complexes 1
and 2 in good yield. The blue crystals of complexes suitable for
X-ray analysis were collected after one day. The polypyridyl
metal complexes displayed the intercalative binding modes to
DNA owing to their large planar ligands to form p–p over-
DNA-binding studies. Using the UV-vis spectral method, the
relative binding of two complexes to CT-DNA was measured in
5
mM Tris-HCl/50 mM NaCl buffer (pH = 7.2). The concentration
of CT-DNA was determined from its absorption intensity at
À1 32 lapping interaction.
À1
260 nm with a molar extinction coefficient of 6600 M cm .
The absorption spectra of two complexes binding to CT-DNA were
recorded by adding CT-DNA solution at different concentrations
Crystal structure characterization
(0–35 mM) to the samples of 1 (45.5 mM) and 2 (47.6 mM) in 10% The p-CPIP ligand and two complexes have been structurally
DMF. Fluorescence quenching experiments were carried out by characterized by single crystal X-ray structural analysis (Fig. 1).
adding 1 or 2 at different concentrations to the EB-bound CT-DNA The details of crystallographic data and structure refinement
À5
À5
solution (EB = 5 Â 10 M and CT-DNA = 100 Â 10 M) in 5 mM parameters are provided in Table 1, and the selected bond
Tris-HCl/50 mM NaCl buffer (pH = 7.2). All the fluorescence lengths and angles are listed in Table 2.
intensities were obtained at room temperature with excitation
at 510 nm and emission at 592 nm.
The ligand p-CPIP crystallizes in the monoclinic crystal
system with the P2 /c space group. The basic unit contains a
1
DNA cleavage and mechanistic studies. The DNA cleavage p-CPIP and an ethanol molecule. The p-CPIP molecule contains
experiments were done by agarose gel electrophoresis, which was two planes. One is composed of N(1), N(2), N(3) and N(4), and
performed by the following literature method. pUC19 DNA in the other is composed of a benzene cycle with the substituent of
50 mM Tris-HCl/18 mM NaCl buffer (pH = 7.2) was treated with Cl(1). The basal atoms deviate from their main plane by 0.0161,
complexes 1 and 2 in the absence of additives, respectively. The 0.0133, À0.0161, and À0.0133 Å for N(1), N(2), N(3) and N(4),
samples were incubated at 37 1C for 3 h, and 4 mL of loading buffer respectively. And the Cl(1) atom deviates from the benzene ring by
was added after incubation. Then the samples were electrophoresed À0.0331 Å. The dihedral angle between the two planes is 10.21.
for 0.5 h at 100 V on 0.1% ethidium bromide (EB) gel in TAE buffer.
A perspective view of complex 1 is depicted in Fig. 1.
After electrophoresis, bands were visualized under UV light and Complex 1 crystallizes in the triclinic crystal system with the
photographed. Cleavage mechanistic investigation of pUC19 DNA Pmn1 space group. The copper atom displays a slightly distorted
was done by adding different scavengers, such as DMSO, NaN3, square pyramidal (4 + 1) geometry with t = 0.025 (t = 0 for a
superoxide dismutase enzyme (SOD), EDTA, KI and L-histine, to the perfect square pyramidal and 1 for a perfect trigonal-bipyramidal
different concentrations of complexes, and then the samples were coordination sphere according to the Addison/Reedijk geometric
3
3
incubated by the same method described for DNA cleavage, and criterion). The basal coordination positions are occupied by
further analysis was carried out by the above method. N(1) and N(2) from the p-CPIP ligand (Cu(1)–N(1) 1.980(6),
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Fig. 1 Crystal structure of [Cu(acac)(p-CPIP)(MeOH)](NO
3
) (1) and [Cu(acac)(o-NPIP)(NO
3
)] (2). Hydrogen atoms and solvent molecules are
omitted for clarity.
Cu(1)–N(2) 1.986(6) Å), and O(1) and O(2) from the acac ligand While the basal donor atoms of O(3), O(4), N(1) and N(2) deviate
(
Cu(1)–O(1) 1.884(5) and Cu(1)–O(2) 1.901(5) Å). The basal from their mean plane by À0.0298, 0.0298, 0.0319, and À0.0319 Å,
donor atoms deviate from their main plane by À0.0247, 0.0245, respectively. On the whole, complex 2 possesses a planar structure
À0.0263 and 0.0265 Å for O(1), O(2), N(1) and N(2), respectively, and with two N atoms and two O atoms situated at both sides of the
Cu(1) is displaced out of the plane towards the apex by À0.1160 Å. Cu(II) ion symmetrically at the same Cu–O bond length of
À
The apical position is occupied by the O(3) atom of MeOH (Cu(1)– 2.005(3) Å. The NO
3
anion in 1 is disordered and noncoordi-
O(3), 2.323 Å) with the distance to the plane 2.433 Å. Moreover, nated, but coordinated to the Cu atom in 2. The packing
À
there exists one noncoordinated NO
3
anion in 1, which can link diagram of 2 (Fig. S1, ESI†) shows two kinds of weak interactions
the H-bonding acceptor by weak interactions to stabilize the between two units: hydrogen bonds and p–p interactions. Hydrogen
À
structure of the complex. As can be seen from the packing diagram bonds exist between the O atoms of NO3 from one unit and
of complex 1 (Fig. S1, ESI†), the plane–plane distance of two units is N atoms of the o-NPIP ligand from the neighboring unit
3.716 Å, showing distinct p–p interactions.
(O(7)–N(3), 2.823 Å). The distances between two mononuclear
Complex 2 crystallizes with two independent molecules in units are 3.696 and 3.760 Å, respectively, showing distinct p–p
the asymmetric unit, which are almost the same except for interactions.
slight difference in bond lengths between each other. One
DNA-binding studies
[
Cu(acac)(o-NPIP)(NO
3
)] unit is depicted in Fig. 1. The coordination
environment of the Cu atom is very similar to that of 1, in which
Electronic absorption method. The electronic absorption
Cu(1) is located in a (4 + 1) environment, showing a slightly spectrometry is an effective way to investigate the interactions
distorted square-pyramidal geometry with t = 0.035. The equatorial of complexes with CT-DNA. The absorption spectra of 1 and 2 in
plane of Cu(1) is formed by two N atoms of the o-NPIP ligand and the absence and presence of CT-DNA at different concentra-
two O atoms of the acac ligand [Cu(1)–O(4) 1.904(3), Cu(1)–O(3) tions are given in Fig. 2. The results show that 1 has a very
1.914(3), Cu(1)–N(2) 2.005(3) and Cu(1)–N(1) 2.005(3) Å)]. strong absorption at ca. 283 nm, and 2 exhibits two absorptions
9
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Table 1 Crystal data and structure refinement parameters of the p-CPIP ligand, complexes 1 and 2
p-CPIP
17ClN
376.84
298(2)
1
2
Formula
Formula weight
T/K
Crystal system
Space group
C
21
H
4
O
C
25
H
22ClCuN
5
O
6
24 6 7
C H18CuN O
587.48
293(2)
Triclinic
P1%
565.99
113(2)
Triclinic
P1%
Monoclinic
P2 /n
1
a/Å
b/Å
c/Å
a/1
b/1
g/1
9.921(2)
11.098(2)
17.289(4)
90
97.50
90
1887.3(7)
4
1.326
0.221
784
8.586(17)
11.180(2)
13.844(3)
97.13(3)
104.40(3)
94.66(3)
1268.5(4)
2
8.418(3)
9.753(3)
28.363(10)
92.028(7)
90.542(6)
93.765(8)
2322.0(14)
4
3
V/Å
Z
D
À3
calc [g cm
]
1.538
1.017
602
1.619
1.001
1156
À1
m(Mo-Ka) mm
F(000)
y range for data collection
Reflections measured
Unique reflections
R(int)
3.00 to 27.48
18 344
4323
0.0646
244
3.07 to 25.05
7555
4419
0.0971
355
2.46 to 28.12
19 659
8116
0.0582
685
Parameters
GOF
1.166
0.0837, 0.1561
0.1255, 0.1722
0.972
0.0950, 0.2131
0.1818, 0.2538
1.027
0.0544, 0.1122
0.0778, 0.1225
R
R
1
, wR
, wR
2
[I > 2s(I)]
(all data)
1
2
Table 2 Selected bond lengths [Å] and bond angles [1] of complexes 1 the binding constant K
was determined using the following
b
and 2
35
eqn (1):
1
[
DNA]/(e À e ) = [DNA]/(e À e ) + 1/K (e À e )
(1)
a
f
b
f
b
b
f
Cu(1)–N(1)
Cu(1)–O(1)
Cu(1)–O(3)
O(1)–Cu(1)–O(2)
O(2)–Cu(1)–N(1)
O(2)–Cu(1)–N(2)
O(1)–Cu(1)–O(3)
N(1)–Cu(1)–O(3)
1.974(7)
1.876(6)
2.321(6)
94.6(2)
91.7(3)
169.5(3)
96.9(2)
88.7(3)
Cu(1)–N(2)
Cu(1)–O(2)
1.990(6)
1.896(6)
where [DNA] is the concentration of DNA, e , e and e corre-
a
f
b
O(1)–Cu(1)–N(1)
O(1)–Cu(1)–N(2)
N(1)–Cu(1)–N(2)
O(2)–Cu(1)–O(3)
N(2)–Cu(1)–O(3)
171.3(3)
91.4(3)
81.4(3)
92.6(2)
95.3(3)
spond to Aobsd/[complex], the extinction coefficient for the free
complex, and the extinction coefficient for the complex in the
fully bound form, respectively. The intrinsic binding constant
4
4
(K
b
) values of 2.47 Â 10 for 1 and 3.04 Â 10 for 2 were
obtained following the above equation. This suggests that the
ligands of o-NPIP and p-CPIP exhibit a similar intercalative
binding effect even with different space configurations and
2
Cu(1)–O(4)
Cu(1)–N(1)
Cu(1)–O(5)
O(4)–Cu(1)–O(3)
O(3)–Cu(1)–N(2)
O(3)–Cu(1)–N(1)
O(4)–Cu(1)–O(5)
N(2)–Cu(1)–O(5)
1.904(3)
2.004(3)
2.320(3)
94.92(12)
172.61(12)
91.41(12)
97.31(11)
86.44(11)
Cu(1)–O(3)
Cu(1)–N(2)
1.914(3)
2.005(3)
positions for the substituents of NO and Cl on the phenyl ring.
2
O(4)–Cu(1)–N(2)
O(4)–Cu(1)–N(1)
N(2)–Cu(1)–N(1)
O(3)–Cu(1)–O(5)
N(1)–Cu(1)–O(5)
91.49(13)
170.49(12) This suggests that the ligand of o-NPIP exhibits a stronger
81.78(13)
96.37(10)
89.01(12)
intercalative binding effect than p-CPIP. The interactions of
complexes (1 and 2) with DNA have shown that the complexes
are electron-acceptors and the DNA molecule is the electron-
donor. NO
group, which can accept electrons from DNA. So ligand o-NPIP
at ca. 257 nm and 290 nm, respectively. All these bands can be shows strong binding ability towards CT-DNA.
2
is a powerful electron-withdrawing group than the Cl
attributed to the intraligand p–p* transition. As the CT-DNA
Fluorescence spectroscopic methods. No luminescence is
concentration was increased, the absorption spectra of both observed for 1 and 2 at room temperature in aqueous solution,
complexes showed clearly hypochromism and red shift at the in any organic solvent examined, or in the presence of CT-DNA.
maximum peak. A 50.36% hypochromism and a 3 nm red shift To further clarify the interaction of the complexes with DNA, the
at 283 nm for 1, while a 77.7% hypochromism and a 6 nm red competitive binding experiment was carried out by adding EB,
shift at 290 nm for 2 were observed, indicating that both 1 and which is well known to be a typical indicator of the intercalation
2
bound to CT-DNA in intercalative mode. Here we suggest that probe. In the presence of CT-DNA, EB can intercalate the planar
this hypochromism and red shift should be contributed to the phenanthridinium ring between adjacent base pairs of the double
36
intercalative mode relating to a strong stacking interaction helix and emit strong fluorescence. It was reported that this
between the planar aromatic chromophore and the base pairs of enhanced fluorescence could be quenched by adding another
37
DNA. Furthermore, the extent of the hypochromism is commonly kind of molecules, so the changes in the spectra of the EB–DNA
3
4
consistent with the strength of intercalative action. In order system are often used for the interaction study between DNA
to elucidate the binding strength of the complexes with DNA, and metal complexes. The emission spectra of EB–DNA in the
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Fig. 3 Fluorescence quenching curves of EB bound to CT-DNA by 1 and
2
([complex] = 0–30.2 mM, lex = 510 nm). The arrow shows the intensity
Fig. 2 Absorption spectra of 1 and 2 (47.6 mM) in the absence and
0
changes on increasing the complex concentration. Inset: plot of I /I vs.
presence of increasing amounts of CT-DNA at room temperature in
[complex].
Tris-HCl/NaCl buffer (pH = 7.2). Inset: plot of [DNA]/(e
for absorption titration of CT-DNA with complexes.
a
À e
f
) vs. [DNA]
In this expression, [complex] is the value of a 50% reduction
7
of the fluorescence intensity of EB and KEB = 1.0 Â 10 M, [EB] =
5
0 mM. The apparent binding constants (Kapp) at room tem-
absence and presence of 1 and 2 are given in Fig. 3. The addition
of 1 and 2 to EB–DNA ([EB] = 50 mM and [CT-DNA] = 100 mM)
causes obvious reduction in the emission intensities, which
indicates that 1 and 2 can competitively bind to CT-DNA with
EB. The extent of reduction of the emission intensity gives a
measure of the binding propensity of the complex to DNA
5
À1
perature are calculated to be 4.97 Â 10 M for 1 and 1.45 Â
6
À1
1
0 M for 2, less than the binding constant of the classical
7
À1 39
intercalators and metallointercalators (10 M ). The results
show that both 1 and 2 have moderate intercalative ability to
CT-DNA. The results show that DNA EB-competitive binding
of 2 is stronger than that of 1, which is consistent with the
previous absorption spectral measurement results.
3
8
according to the classical Stern–Volmer eqn (2):
I
0
/I = 1 + Ksv[Q]
(2)
DNA cleavage studies. In order to estimate the chemical
nuclease activities of the copper complexes for DNA strand
scission, pUC19 DNA in a medium of 50 mM Tirs-HCl/NaCl
buffer (pH = 7.2) in the absence and presence of 1 and 2 under
the physiological conditions was chosen. The DNA cleavage
study was conducted by gel electrophoresis. The cleavage
activities could be assessed by the conversion of DNA from
the original supercoiled form (Form I) to the nicked form (Form
II, single-strand breaks, SSBs) and the linear form (Form III,
double-strand breaks, DSBs). When pUC19 DNA is subject to
In this expression, I and I are the fluorescence intensities in
0
the absence and presence of the quencher, respectively. Ksv is a
linear Stern–Volmer quenching constant. [Q] is the concentration of
the quencher. The quenching plot illustrates that the quenching of
EB–DNA by 1 and 2 is in agreement with the linear Stern–Volmer
equation, which also indicates that 1 and 2 effectively bind to
CT-DNA as can be seen from eqn (3):
K
EB[EB] = Kapp[complex]
(3)
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Fig. 5 (a) Gel electrophoresis diagrams showing cleavage of pUC19
DNA (0.1 mg mL ) by 1 in Tris-HCl/NaCl buffer (pH = 7.2) at different
Fig. 4 (a) Gel electrophoresis diagrams showing cleavage of pUC19 DNA
À1
À1
(0.1 mg mL ) by 1 in Tris-HCl/NaCl buffer (pH = 7.2) at different concen-
concentrations for 3 h at 37 1C. Lane 1: DNA control; lane 2: DNA + H
2
O
2
trations for 3 h at 37 1C. Lane 1: DNA control; lanes 2–8: DNA + 1 (2.08,
À5
(
1 Â 10 mM); lanes 3–8: DNA + H
2 2
O + 1 (2.08, 6.25, 14.56, 20.80, 27.04
6
.25, 14.56, 20.80, 27.04, 35.36 and 42.25 mM). (b) Gel electrophoresis
and 35.36 mM). (b) Gel electrophoresis diagrams showing cleavage
of pUC19 DNA (0.1 mg mL ) by 2 under the same conditions as above.
Lane 1: DNA control; lane 2: DNA + H
À1
diagrams showing cleavage of pUC19 DNA (0.1 mg mL ) by 2 under the
same conditions as above. Lane 1: DNA control; lanes 2–8: DNA + 2
À1
À5
2
O
2
(1 Â 10 mM); lanes 3–8: DNA +
(0.42, 1.25, 2.92, 4.17, 5.42, 7.08 and 8.28 mM).
2 2
H O + 2 (0.42, 1.25, 2.92, 4.17, 5.42 and 7.08 mM).
electrophoresis, relatively fast migration was observed for Form
I, slower migration for Form II, and Form III appeared between
Form I and Form II.
Firstly, the concentration dependent DNA cleavage by com-
plexes 1 and 2 was performed. As shown in Fig. 4, with the
increase of the complexes concentration, Form I did not
convert into Form II and Form III. So the results indicate that
complexes 1 and 2 have not exhibited effective nuclease activity
towards pUC19 DNA only by themselves. Besides a few examples
of hydrolytic cleavage, most of the DNA cleavages were mediated
40
by the copper complexes via an oxidative mechanism. Sigman
41
et al. have found that the 1,10-phenanthroline copper(II)
complex induced DNA cleavage in the presence of oxygen or
H
2
O
2
. H
2 2
O acts as an activator and can generate reactive oxygen
species (ROS) or reactive metal-oxo species (RMOS) upon reaction
with copper, which triggers the degradation of nucleic acids.
À1
Fig. 6 (a) Agarose gel showing cleavage of pUC19 DNA (0.1 mg mL
)
42
incubated by 1 (35.36 mM) in Tris-HCl/NaCl buffer (pH = 7.2) at 37 1C for
À5
Therefore, we investigated the cleavage activity of the two copper 3 h. Lane 1: DNA control; lane 2: DNA + 1 + H O2 (1 Â 10 mM); lane 3:
2
complexes in the presence of H
2
O
2
. Fig. 5 shows that the super- DNA + 1 + H
2
O
2
+ 20 mM DMSO; lane 4: DNA + 1 + H
2
O
2
+ 20 mM NaN
3
;
À1
lane 5: DNA + 1 + H
EDTA; lane 7: DNA + 1 + H
0 mM L-histidine. (b) Agarose gel showing cleavage of pUC19 DNA
2
O
2
+ 20 U mL SOD; lane 6: DNA + 1 + H
2
O
2
+ 5 mM
coiled plasmid DNA is effectively converted into Form II, which
showed that both complexes 1 and 2 exhibited higher oxidative
2
2 2 2
O + 20 mM KI; lane 8: DNA + 1 + H O +
2
DNA cleavage activity upon treatment with H
2 2
O .
À1
(
0.1 mg mL ) incubated with 2 (7.08 mM) in Tris-HCl/NaCl buffer (pH = 7.2)
À5
DNA cleavage mechanism. To check the effects of active at 37 1C for 3 h. Lane 1: DNA control; lane 2: DNA + 2 + H O (1 Â 10 mM);
2
2
oxygen species on DNA damage, we investigated the DNA cleavage lane 3: DNA + 2 + H
2
O
2
+ 20 mM DMSO; lane 4: DNA + 2 + H
2
O
2
+ 20 mM
À1
NaN
3
; lane 5: DNA + 2 + H
O
2 2
+ 20 U mL SOD; lane 6: DNA + 2 + H
2
O
O
2
+
+
in the presence of a hydroxyl radical scavenger (DMSO), singlet
oxygen quenchers (NaN and L-histidine), a superoxide scavenger
SOD), a chelating agent (EDTA) and a hydrogen peroxide scavenger
5
2
2 2
mM EDTA; lane 7: DNA + 2 + H O + 20 mM KI; lane 8: DNA + 2 + H
2
2
3
0 mM L-histidine.
(
43
(KI), respectively, under the experimental conditions. No obvious
inhibition is observed for 1 in the presence of SOD (lane 5) as are not involved in the cleavage process. When EDTA (lane 6)
shown in Fig. 6a, which suggests that the superoxide scavenger is (Fig. 6a and b) was added to the reaction mixture, the DNA
not involved in the cleavage process. And for 2, SOD and L-histidine cleavage mediated by both 1 and 2 was strongly inhibited,
have no effect on the cleavage reaction (lanes 5 and 8) (Fig. 6b), confirming that the chelating agent is the intermediate that
which indicates that the superoxide scavenger and singlet oxygen participates in the DNA scission process. The results showed
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that the EDTA, a Cu(II)-specific chelating agent that strongly binds
to Cu(II) forming a stable complex, can efficiently inhibit DNA
cleavage, indicating that Cu(II) complexes play a key role in the
cleavage process. For the copper complexes oxidative DNA cleavage,
2
1
it is proposed that Cu is reduced to Cu species by a reductant, and
1
44
Cu species form ROS subsequently by reacting with O
2 2 2
or H O .
In our work, H acts as the reductant in the absence of other
2 2
O
1
42
reductants and a coreactant with Cu . It is the origin for EDTA
quenching the Cu-induced DNA scission. KI (lane 7) (Fig. 6a and b)
significantly inhibited the DNA cleavage activity of the two
complexes, which indicated the involvement of hydrogen peroxide
in the cleavage process. This phenomenon could be explained by
the inhibitory effect of EDTA on DNA scission.
BSA studies
Fluorescence spectroscopy of BSA. Studies on the interactions
between metal complexes and proteins are of great importance
for investigating the mechanism of transport and metabolism.
Furthermore, the binding of metal complexes to proteins may
provide information on the relationship between structures and
functions of proteins.
Qualitative analysis of binding of metal compounds to BSA can
be performed by examining fluorescence spectra. The emission
spectra of BSA in the presence of increasing concentrations of 1
and 2 are recorded in the wavelength range 300–500 nm by exciting
the protein at 290 nm. Fig. 7 shows that the fluorescence intensities
of BSA obviously decrease with increasing concentrations of 1 and 2,
which indicate that 1 and 2 can effectively bind to the proteins.
Commonly, fluorescence quenching can be described by the
3
8
following Stern–Volmer eqn (4).
Fig. 7 Fluorescence spectra of BSA in the different concentrations of 1
F
0
/F = 1 + K [Q] = 1 + Ksv[Q]
q
t
0
(4)
À7
and 2 at room temperature. The concentrations of BSA are: (5 Â 10 M),
complex 1 (0–3 mM) and complex 2 (0–4 mM), respectively.
In this equation, F
sities in the absence and presence of the quencher, respectively.
Kq is the quenching rate constant of the biomolecule, Ksv is the quenching constant K for 1 and 2 is on the order of
0
and F represent the fluorescence inten-
q
1
3
À1 À1
dynamic quenching constant, t is the average life-time of the 10 L mol
s
, which is 1000-fold higher than the maximum
0
biomolecule without the quencher (t
concentration of quencher. Fig. 8 displays the Stern–Volmer 10 L mol
0
= 6.2 ns) and [Q] is the value possible for diffusion controlled quenching (i.e. 2.0 Â
10
À1 À1 46
s
). This observation suggests that the quenching
plots of the quenching of BSA emission fluorescence by 1 and 2 is not initiated by dynamic collision but by a static one. So we have
at different temperatures. Fluorescence quenching can occurby to continue exploring the quenching mechanism by UV-vis spectra.
different mechanisms, which are usually classified as dynamic
UV-vis spectroscopy. UV-vis absorption measurement is a
quenching and static quenching. The dynamic and static simple but effective method for detecting formation of complex
quenching can be distinguished by their differing dependence on formation. In a dynamic quenching mechanism, with the addition
temperature. In the case of dynamic quenching, higher tempera- of a quencher, only the excited state fluorescence molecule is
ture results in faster diffusion, and consequently, the quenching affected, which results in no change in the absorption spectra of
rate constant increases with the increasing temperature. In BSA. While, in a static quenching, a new compound is formed
contrast, in the case of static quenching, increasing tempera- between BSA and the quencher, therefore, the absorption spectra of
ture will result in decreasing complex stability and hence in BSA would be considerably affected. It appears that the UV
4
5
lowering of the value of the static quenching constant. The absorption peak goes up or down. The influences of the absorbance
corresponding Stern–Volmer quenching constant Ksv and the of BSA were observed by adding the same concentration of 1 and 2.
quenching rate constant K are given in Table 3. The results From Fig. 9, we can see that BSA absorption peaks at around
q
show that the corresponding Stern–Volmer quenching constant 280 nm due to the presence of aromatic amino acids (Trp, Tyr, and
47
K
sv increases with the rising temperature, indicating that the Phe) exist. For 1, the absorption spectra of BSA did not change,
fluorescence quenching of BSA by 1 and 2 is likely to occur by a indicating that 1 and BSA did not form a new compound. While
dynamic quenching mechanism. While the obtained bimolecular the absorption spectra increased with adding 2, indicating that
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Fig. 9 UV-vis absorption spectra of BSA in the absence and presence of 1
and 2. Black line: the absorption spectrum of BSA. Red line: the absorption
spectrum of BSA in the presence of 1 or 2 at the same concentration, c
Fig. 8 The Stern–Volmer plots of the fluorescence quenching of BSA
by 1 and 2 at different temperatures (T = 288, 298 and 310 K). Inset: plot
of F /F vs. [complex].
0
(
BSA) = c (1 or 2) = 5 Â 10À7 M.
Table 3 The quenching constants (Ksv and K
number of binding sites (n) of the BSA-1 and BSA-2 systems at different
temperatures
q
), binding constant (K
b
), the the electronic structures. So it is the possible reason why the
binding mode between complex 2 and BSA is different from
complex 1.
5
13
5
K
sv  10
K
q
 10
K
b
 10
Binding constants and the number of binding sites. It is
assumed that there are independent binding sites in the biomole-
À1
À1
À1
T (K)
(1 mol
)
(1 mol
)
(1 mol
)
n
1
2
288
1.70
2.43
2.79
2.74
3.92
4.50
1.69
1.88
2.80
b
1.07 cule. The binding constant (K ) and the number of binding sites (n)
48
2
3
98
10
1.21
1.12
can be determined by using the double logarithm eqn (5):
log((F À F)/F) = log K + n log[Q]
(5)
0
b
288
0.87
1.05
1.98
1.43
1.69
1.98
1.09
1.23
1.53
0.85
1.20
0.83
2
3
98
10
0
In this equation, F and F are the fluorescence intensities
presence and absence 1 and 2, respectively; [Q] is the total
the microenvironment of the three aromatic acid residues was concentration of 1 and 2, K is the binding constant for the
b
changed and the tertiary structure of BSA was destroyed, and this complex–protein interaction and n is the number of binding
can prove that 2 and BSA formed a novel complex. These results sites per albumin molecule. As seen from Fig. S2 (ESI†),
show that the quenching mechanism of 1 is mainly a dynamic the binding constant increases with the rising temperature,
quenching process, and that of 2 is a combined process, in which indicating that the capacity of 1 and 2 to bind to BSA is enhanced
dynamic and static quenching mechanisms are concurrent. with the increasing temperature. The values of n at the experi-
It suggested that the polypyridyl ligand, which was modified mental temperature are approximately equal to one, which
by the substituent group at different sites probably, can create indicates that there is just one independent binding site in
some interesting differences in the space configurations and BSA for 1 and 2 (Table 3).
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Synchronous fluorescence spectroscopy studies. It is reported
NJC
2 X. J. Li, K. Zheng, L. D. Wang, Y. T. Li, Z. Y. Wu and
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that synchronous fluorescence spectroscopy is frequently used to
characterize the interaction between the fluorescence probe and
49
proteins, because this technique can provide information about
the molecular environment in the vicinity of the fluorophore
50
molecules. The synchronous fluorescence spectroscopy involves
the simultaneous scanning of excitation and emission spectra on a
fluorimeter, while maintaining a fixed wavelength difference (Dl)
between them. When Dl is stabilized at 15 nm, the synchronous
fluorescence gives the characteristic information of tyrosine resi-
51
dues, whereas Dl of 60 nm indicates that of tryptophan residues.
The maximum emission wavelengths of tryptophan and tyrosine
residues are related to the polarity of their surroundings. When the
wavelengths change, it can be inferred that the protein conformation
is changed. From Fig. S4 (ESI†), it can be seen that upon addition of
8 L. F. Tan, F. Wang, H. Chao, Y. F. Zhou and C. Weng,
J. Inorg. Biochem., 2007, 101, 700–708.
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1
and 2, the maximum emission wavelength shows no shift when
Dl was equal to 15 nm. However, as seen from Fig. S5 (ESI†), the 10 A. Nori and J. Kopecek, Adv. Drug Delivery Rev., 2005, 57,
maximum emission wavelength represents obviously red shift when
609–636.
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of BSA was changed while the polarity around the tryptophan 12 M. Morshedi and H. Hadadzadeh, J. Fluoresc., 2013, 23,
52
residues increased, whereas the hydrophobicity decreased.
259–264.
1
1
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4 C. H. Ng, W. S. Wang, K. V. Chong, Y. F. Win, K. E. Neo,
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Conclusion
In this work, we have synthesized and characterized two Cu(II)
polypyridyl complexes to study the selectivity and efficiency of
DNA recognized and cleaved by binuclear Cu(II) complexes. The
interaction of complexes with calf thymus DNA (CT-DNA) was
investigated by UV-visible and fluorescence emission spectro-
metry, as well as agarose gel electrophoresis. Results suggest
moderate intercalative binding mode between the complexes
and DNA. Both 1 and 2 exhibited effective oxygen DNA cleavage
1
1
1
1
1
2
2
2
2
2
2
9 Q. L. Zhang, J. G. Liu, H. Xu, H. Li, J. Z. Liu, H. Zhou,
L. H. Qu and L. N. Ji, Polyhedron, 2001, 20, 3049–3055.
0 S. Shi, J. Liu, J. Li, K. C. Zheng, C. P. Tan, L. M. Chen and
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2 2
activity in the absence of external agents such as H O . DNA
cleavage mechanism studies show that both 1 and 2 might be
capable of promoting DNA cleavage through oxidative DNA
damage pathways. According to the results obtained from
fluorescence spectrometry of BSA, we conclude that the fluores-
cence quenching of BSA by 1 is a dynamic quenching process
because Ksv increases with increasing temperature. UV absorp-
tion spectra show that 2 involves a combined process, in which
dynamic and static quenching mechanisms are concurrent. The
details of the binding mode, specific binding sites and energy
transfer upon BSA binding with complexes are not very clear at
present and further studies are currently in progress.
3 B. X. Huang and H. Y. Kim, J. Am. Soc. Mass Spectrom., 2004,
15, 1237–1247.
4 N. Shahabadi, M. Maghsudi, Z. Kiaani and M. Pourfoulad,
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Acknowledgements
26 R. A. Alderden, M. D. Hall and T. W. Hambley, J. Chem.
Educ., 2006, 83, 728.
The work was supported by the NSFC (China) (No. 21371135)
and the Key Program of Tianjin Municipal Natural Science
Foundation (China) (No. 13JCZDJC28200).
2
2
2
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