Promotive effect of the platinum moiety on the DNA cleavage activity of copper-based artificial nucleases

Article abstract of DOI:10.1021/ic100001x

Copper-based artificial metallonucleases are likely to satisfy more biomedical requirements if their DNA cleavage efficiency and selectivity could be further improved. In this study, two copper(II) complexes, [CuL ¹CI₂ (1) and [CuL²CI₂] (2), and two copper(II)-platinum(II) heteronuclear complexes, [CuRL¹ (DMSO)CI ₄] (3) and [CuPtL²DMSO)CI₄] (4), were synthesized using two afunctional ligands, N-[4-(2-pyridylmethoxy)benzyl]-N,N- bis(2-pyridylmethyl)amine (L¹) and N-[3-(2pyridylmethoxy)benzyl]-N, Nbls(2-pyridylmethyl)amine (L₂). These complexes have been characterized by elemental analysis, electrospray ionization mass spectrometry, IR spectroscopy, and UV-vis spectroscopy. The DNA binding ability of these complexes follows an order of 1 < 2 < 3 < 4, as revealed by the results of spectroscopy and agarose gel electrophoresis studies. Their cleavage activity toward supercoiled pUC19 plasmid DNA is prominent at micromolar concentration levels in the presence of ascorbic acid. The introduction of a platinum(II) center to the copper(II) complexes induces a significant enhancement in cleavage activity as compared with copper(II) complexes alone. These results show that the presence of a platinum(II) center in copper(II) complexes strengthens both their DNA binding ability and DNA cleavage efficiency.

Full text of DOI:10.1021/ic100001x

Inorg. Chem. 2010, 49, 2541–2549 2541
DOI: 10.1021/ic100001x
Promotive Effect of the Platinum Moiety on the DNA Cleavage Activity of
Copper-Based Artificial Nucleases
Xindian Dong,† Xiaoyong Wang,*,‡ Miaoxin Lin,† Hui Sun,† Xiaoliang Yang,† and Zijian Guo*,†
†State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering,
‡State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University,
Nanjing 210093, People’s Republic of China
Received January 1, 2010
Copper-based artificial metallonucleases are likely to satisfy more biomedical requirements if their DNA cleavage efficiency
and selectivity could be further improved. In this study, two copper(II) complexes, [CuL1Cl2] (1) and [CuL2Cl2] (2), and two
copper(II)-platinum(II) heteronuclear complexes, [CuPtL1(DMSO)Cl4] (3) and [CuPtL2(DMSO)Cl4] (4), were synthe-
sized using two bifunctional ligands, N-[4-(2-pyridylmethoxy)benzyl]-N,N-bis(2-pyridylmethyl)amine (L1) and N-[3-(2-
pyridylmethoxy)benzyl]-N,N-bis(2-pyridylmethyl)amine (L2). These complexes have been characterized by elemental
analysis, electrospray ionization mass spectrometry, IR spectroscopy, and UV-vis spectroscopy. The DNA binding ability
of these complexes follows an order of 1 < 2 < 3 < 4, as revealed by the results of spectroscopy and agarose gel
electrophoresis studies. Their cleavage activity toward supercoiled pUC19 plasmid DNA is prominent at micromolar
concentration levels in the presence of ascorbic acid. The introduction of a platinum(II) center to the copper(II) complexes
induces a significant enhancement in cleavage activity as compared with copper(II) complexes alone. These results show
that the presence of a platinum(II) center in copper(II) complexes strengthens both their DNA binding ability and DNA
cleavage efficiency.
Introduction
generating reactive oxygen species (ROS) that oxidize DNA
and lead to strand cleavage or base modification.8,9 However,
many copper(II) complexes cleave DNA randomly and
possess only limited enzymatic activity.10,11 Therefore, the
development of artificial nucleases with improved efficiency
and selectivity is in high demand. A number of natural
nucleases require multinuclear metal centers to achieve a
synergistic effect in the process of substrate recognition and
scission.12 We and others have demonstrated the synergistic
effect between multicopper(II) centers in previous DNA
cleavage studies.13-16 Nevertheless, designing copper-based
artificial nucleases with specific and selective DNA cleavage
properties is still full of challenges.
Artificial nucleases have attracted much attention in view
of their diverse applications as potential chemotherapeutic
agents in medicine and useful cleaving reagents in molecular
biology.1-3 Transition-metal complexes constitute a rich
resource for the exploration of such nucleases because of
their structural and functional varieties.4,5 Copper complexes
are particularly attractive and most studied because of their
biologically accessible redox potential and relatively high
affinity for nucleobases.6,7 In the presence of a reductant
and dioxygen, copper complexes exert cleavage activity by
*To whom correspondence should be addressed. E-mail: boxwxy@
nju.edu.cn (X.W.), zguo@nju.edu.cn (Z.G.).
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2010 American Chemical Society
Published on Web 02/01/2010
pubs.acs.org/IC

2542 Inorganic Chemistry, Vol. 49, No. 5, 2010
Dong et al.
Figure 1. Chemical structures of complexes 1-4.
It is well-known that cisplatin-type complexes bind pre-
ferentially to the GG or AG site of DNA.17 Such a DNA
sequence preference could be used as a targeting element to
direct a nuclease moiety to specific DNA sites. In fact,
Reedjik and co-workers have reported recently that plati-
nated copper(3-Clip-Phen) complexes exhibited higher
DNA cleavage efficiency than the copper(II) complexes
alone.18-20 However, comparisons were made on complexes
with different ligands.21 We have shown that the DNA
cleavage activity of copper complexes could be largely
affected by the ligand structure;13,22 therefore the influence
of the ligand should not be excluded while the effect of the
platinum(II) center is discussed.
Elemental analysis was performed on a Perkin-Elmer 240C
analytical instrument. UV-vis spectra were determined on a
Shimadzu UV 3600 (UV-vis-near-IR) spectrophotometer.
The 1H and 13C NMR spectra were acquired on a Bruker DRX-
500 spectrometer at 298 K. The circular dichroism (CD) experi-
ments were performed on a Jasco J-810 spectropolarimeter.
Fluorescence spectra were recorded on a Perkin-Elmer LS55
luminescence spectrometer in a wavelength range of 530-
780 nm with 4 nm slits for both excitation and emission at room
temperature. Fluorescence imaging was performed with a Gel
Doc XR (BioRad), and quantification analysis was performed
with Quantity One software (version 4.6.2).
Preparation of Ligands L1 and L2. The intermediate 4-(bromo-
methyl)phenyl acetate was prepared according to the literature
method with some modifications.23 Briefly, p-cresol (30.00 g,
277.78 mmol) in aceticanhydride/pyridine (1:1, 80 mL) was stirred
at room temperature for 48 h. p-Tolyl acetate was obtained
after removal of the solvent. Bp: 120 °C. 1H NMR (CDCl3, 500
MHz, ppm): 2.31 (s, 3H, -CH3), 2.37 (s, 3H, -CH3), 6.98-7.00
(d, 2H, Ph-H), 7.19-7.21(d, 2H, Ph-H). 13C NMR (CDCl3, 500
MHz, ppm): 20.72, 20.91, 121.22, 129.86, 135.31, 148.53, 169.48.
This ester (5.70 g, 38.00 mmol) was brominated with N-bromo-
succinimide (9.60 g, 53.90 mmol) and benzoyl peroxide (0.28 g) in
CCl4 (80 mL). The exothermic reaction completed automatically
after the initial heating. The mixture was filtered, and the solvent
was removed to give a crude product, which was chromato-
graphed on silica gel (petroleum ether/ethyl acetate, 10:1, v/v),
and the intermediate was obtained. 1H NMR (CDCl3, 500 MHz,
ppm): 2.32 (s, 3H, -CH3), 4.51 (s, 2H, -CH2), 7.08-7.10 (d, 2H,
Ph-H), 7.42-7.43 (d, 2H, Ph-H). 13C NMR (CDCl3, 500 MHz,
ppm): 21.15, 32.76, 122.01, 130.28, 135.41, 150.67, 169.25.
m-Tolyl acetate and 3-(bromomethyl)phenyl acetate were pre-
pared in a similar way except that m-cresol was used in the
reaction instead of p-cresol. 1H NMR (CD3OD, 500 MHz, ppm):
2.26 (s, 3H, -CH3), 2.32 (s, 3H, -CH3), 6.91-6.93 (d, 1H,
Ph-H), 6.95 (s, 1H, Ph-H), 7.07-7.08 (d, 1H, Ph-H),
7.28-7.31 (t, 1H, Ph-H). 13C NMR (CDCl3, 500 MHz, ppm):
21.07, 21.28, 118.57, 122.22, 126.65, 129.17, 139.60, 150.75,
169.53. 1H NMR (CD3OD, 500 MHz, ppm): 2.29 (s, 3H, -CH3),
4.57 (s, 2H, -CH2), 7.05-7.06 (d, 1H, Ph-H), 7.19 (s, 1H,
Ph-H), 7.30-7.32 (d, 1H, Ph-H), 7.36-7.39 (m, 1H, Ph-H).
13C NMR (CDCl3, 500 MHz, ppm): 21.10, 32.56, 121.68, 122.28,
126.41, 129.78, 139.34, 150.87, 169.19.
In this work, we have designed two similar ligands (L1 and
L2), where rigid p-cresol or m-cresol is used to connect two
coordination centers composed of bis(2-pyridylmethyl)amine
(BPA) and 2-methylpyridine, respectively. These ligands allow
us to make direct comparisons on the DNA binding and
cleavage activity of both monocopper(II) and copper(II)-
platinum(II) complexes (Figure 1). The effect of the
platinum(II) center on the cleavage activity in 3 and 4 can
be clearly illustrated by their comparison with 1 and 2.
Experimental Section
Materials and Characterization. Reagents such as CuCl2
3
2H2O, p-cresol, m-cresol, and 2-(chloromethyl)pyridine hydro-
chloride were of analytical grade and used without further
purification. Supercoiled pUC19 plasmid DNA and 50-FAM-
end-labeled 18mer DNA were purchased from TaKaRa Bio-
technology (Dalian). Calf thymus DNA (CT-DNA), tris(hydro-
xymethyl)aminomethane (Tris), and ethidium bromide (EB)
were purchased from Sigma. The electrospray ionization mass
spectrometry (ESI-MS) spectra were recorded using an LCQ
fleet ESI-MS spectrometer (Thermo Scientific), and the isotopic
distribution patterns of the observed species were simulated
using the Isopro 3.0 program. Far-IR spectra were recorded on a
Nexus 870 FT-IR spectrometer as KBr pellets (600-100 cm-1).
(17) Jung, Y.; Lippard, S. J. Chem. Rev. 2007, 107, 1387–1407.
(18) de Hoog, P.; Boldron, C.; Gamez, P.; Sliedregt-Bol, K.; Roland, I.;
4-[[Bis(2-pyridylmethyl)amino]methyl]phenyl acetate was pre-
paredas follows. N,N-Diisopropylethylamine(DIEA; 2.35 g) and
bis(2-pyridylmethyl)amine (BPA; 3.54 g, 17.78 mmol)24 were
added to the CHCl3 solution of 4-(bromomethyl)phenyl acetate
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Pitie, M.; Kiss, R.; Meunier, B.; Reedijk, J. J. Med. Chem. 2007, 50, 3148–
3152.
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Reedijk, J. J. Biol. Inorg. Chem. 2008, 13, 575–586.
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Dewelle, J.; Mijatovic, T.; Meunier, B.; Kiss, R.; Reedijk, J. Chem.—Eur. J.
2008, 14, 3418–3426.
(23) Karlin, K. D.; Cohen, B. I.; Hayes, J. C.; Farooq, A.; Zubieta, J.
Inorg. Chem. 1987, 26, 147–153.
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P. J.; Perrin, M.; Kahn, O.; Collet, A. Inorg. Chem. 1997, 36, 2975–2981.
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(22) Chen, Z. F.; Wang, X. Y.; Li, Y. Z.; Guo, Z. J. Inorg. Chem.
Commun. 2008, 11, 1392–1396.

Article
Inorganic Chemistry, Vol. 49, No. 5, 2010 2543
(4.09 g, 17.87 mmol, 30 mL) successively. The mixture was stirred
for 2 days, and the resulting DIEA salt was eliminated by filtra-
tion. After removal of the solvent from the filtrate and purifica-
tion by silica gel chromatography (ethyl acetate/methanol,
30:1, v/v), 4-[[bis(2-pyridylmethyl)amino]methyl]phenyl acetate
was obtained. Yield: 60%. 1H NMR (DMSO-d6, 500 MHz, ppm):
2.26 (s, 3H, -CH3), 3.65 (s, 2H, -CH2), 3.73 (s, 4H, -CH2),
7.08-7.10 (d, 2H, Ph-H), 7.26-7.28 (m, 2H, Py-H), 7.44-
7.46 (d, 2H, Ph-H), 7.58-7.60 (d, 2H, Py-H), 7.79-7.82
(m, 2H, Py-H), 8.50-8.51 (d, 2H, Py-H). 13C NMR (CDCl3,
500 MHz, ppm): 21.20, 57.94, 60.11, 121.46, 122.08, 122.92,
129.80, 136.51, 136.70, 149.12, 149.83, 159.72, 169.53. ESI-MS
(m/z) found (calcd) for C21H21N3O2 (M): [M þ H]þ, 348.42
(348.17); [M þ Na]þ, 370.33 (370.15). This intermediate (2.81 g,
8.08 mmol) and 2-(chloromethyl)pyridine hydrochloride (3.00 g,
18.46 mmol) were dissolved in anhydrous acetonitrile (30 mL),
and K2CO3 (5.00 g, 36.23 mmol) was added to the solution. The
mixture was heated at 65 °C for 48 h under a nitrogen atmosphere.
After filtration and removal of the solvent, the brown-yellow
residue was purified with chromatography (ethyl acetate/ethanol,
20:1, v/v), and light-yellow N-[4-(2-pyridylmethoxy)benzyl]-N,N-
bis(2-pyridylmethyl)amine (L1) was obtained. Yield: 40%. 1H
NMR (DMSO-d6, 500 MHz, ppm): 3.57 (s, 2H, -CH2), 3.70 (s,
4H, -CH2), 5.16 (s, 2H, -CH2), 6.99-7.01 (d, 2H, Ph-H),
7.25-7.27 (m, 2H, Py-H), 7.33-7.35 (m, 2H, Ph-H; 1H,
Py-H), 7.50-7.52 (d, 1H, Py-H), 7.57-7.59 (d, 2H, Py-H),
7.78-7.81 (m, 2H, Py-H), 7.81-7.85 (m, 1H, Py-H), 8.49-8.50
(d, 2H, Py-H), 8.57-8.58 (d, 1H, Py-H). 13C NMR (DMSO-d6,
500 MHz, ppm): 57.27, 59.41, 70.85, 115.05, 122.12, 122.57,
122.99, 123.64, 130.40, 131.39, 137.03, 137.41, 149.20, 149.51,
157.25, 157.76, 159.59. ESI-MS (m/z) found (calcd) for C25H24-
N4O (M): [M þ H]þ, 397.25 (397.20); [M þ Na]þ, 419.33 (419.18);
[2M þ Na]þ, 814.92 (815.38).
Py-H). 13C NMR (DMSO-d6, 500 MHz, ppm): 57.83, 59.61,
70.78, 113.95, 115.30, 121.67, 122.04, 122.58, 122.90, 123.35,
129.79, 137.21, 137.55, 141.03, 149.27, 149.57, 157.34, 158.73,
159.65. ESI-MS (m/z) found (calcd) for C25H24N4O (M):
[M þ H]þ, 397.33 (397.20); [M þ Na]þ, 419.33 (419.18).
Synthesis of the Complexes. Complex 1 was prepared by mix-
ing methanol solutions of CuCl2 2H2O (18.80 mg, 0.11 mmol,
3
1 mL) and L1 (39.90 mg, 0.10 mmol, 3 mL) and stirring at
room temperature for 12 h. Ether was added to the mixture,
the resulting precipitate was filtered and washed with acetone
and ether, and 1 was thus obtained. Yield: 90%. Elem anal.
Found (calcd) for C25H24ON4Cl2Cu: C, 56.73 (56.55); H, 4.59
(4.56); N, 10.23 (10.55). IR (KBr pellet): νCu-Cl 267.2 cm-1
.
UV-vis [H2O; λmax, nm (ε, M-1 cm-1)]: 260 (12 500), 650 (85).
Complex 2 was prepared in the same way but using L2 as the
ligand. Yield: 94%. Elem anal. Found (calcd) for C25H24ON4-
Cl2Cu: C, 56.70 (56.55); H, 4.77 (4.56); N, 10.16 (10.55). IR (KBr
pellet): νCu-Cl 274.8 cm-1. UV-vis [H2O; λmax, nm (ε, M-1
cm-1)]: 260 (12 000), 651 (75).
Complex 3 was synthesized on the basis of complex 1. cis-
[Pt(DMSO)2Cl2]25 (28.60 mg, 0.067 mmol) was dissolved in
methanol/acetonitrile (1:6, v/v; 3 mL) and added to the metha-
nol solution of 1 (33.00 mg, 0.063 mmol, 5 mL). The mixture was
stirred at 44 °C for 48 h in the dark. Ether was added to the
mixture, and the resulting product was filtered and washed with
acetone and ether, which gave rise to 3. Yield: 93%. Elem anal.
Found (calcd) for C27H30O2N4Cl4SCuPt: C, 37.12 (37.06); H,
3.48 (3.46); N, 6.34 (6.40). IR (KBr pellet): νPt-S 438.3, νPt-Cl
334.6, νPt-Cl 311.9, νCu-Cl 270.1 cm-1. UV-vis [H2O; λmax, nm
(ε, M-1 cm-1)]: 260 (14 500), 652 (90). Complex 4 was similarly
prepared except that 2 was used as one of the reactants. Yield:
86%. Elem anal. Found (calcd) for C27H30O2N4Cl4SCuPt: C,
37.19 (37.06); H, 3.60 (3.46); N, 6.30 (6.40). IR (KBr pellet):
νPt-S 440.4, νPt-Cl 339.9, νPt-Cl 312.7, νCu-Cl 273.8 cm-1
.
UV-vis [H2O; λmax, nm (ε, M-1 cm-1)]: 259 (12 100), 650 (75).
DNA Binding Studies. The stock solution of CT-DNA was
prepared with a buffer (5 mM Tris-HCl/50 mM NaCl, pH 7.26)
and stored at 4 °C. The concentration of CT-DNA was deter-
mined by UV absorbance at 260 nm after proper dilution
with water, taking 6600 M-1 cm-1 as the molar absorption
coefficient. The ratio of the UV absorbance at 260 and 280 nm
(A260/A280) was ca 1.8, indicating that the DNA solution was
sufficiently free of protein.6
The intermediate 3-[[bis(2-pyridylmethyl)amino]methyl]phenol
was prepared by adding DIEA (1.60 g) and BPA (1.99 g, 10.00
mmol) to the CHCl3 solution of 3-(bromomethyl)phenyl acetate
(2.29 g, 10.00 mol, 30 mL) and stirring for 2 days. The precipitate
of DIEA salt was removed by filtration, and the solvent in
the filtrate was evaporated. The resulting oil was stirred with
distilled water (100 mL), methanol (100 mL), and NaOH (50 mL,
40%) for 12 h, and then the resulting mixture was neutralized
with HCl (37%). The mixture was extracted with CHCl3, and
the extraction was dried with Na2SO4. The solvent was removed,
and the crude product was purified by SiO2 column chromato-
graphy (ethyl acetate/ethanol, 20:1, v/v). 1H NMR (DMSO-d6,
500 MHz, ppm): 3.54 (s, 2H, -CH2), 3.70 (s, 4H, -CH2),
6.65-6.66 (d, 1H, Ph-H), 6.82-6.83 (d, 1H, Ph-H), 6.88
(s, 1H, Ph-H), 7.11-7.14 (m, 1H, Ph-H), 7.24-7.26, (m, 2H,
Py-H), 7.58-7.60 (d, 2H, Py-H), 7.77-7.81 (m, 2H, Py-H),
8.49-8.50 (d, 2H, Py-H), 9.38 (s, 1H, OH-H). 13C NMR
(CD3OD, 500 MHz, ppm): 58.35, 59.25, 113.89, 115.32, 119.79,
122.36, 123.29, 128.99, 137.25, 139.95, 147.96, 157.24, 159.24.
ESI-MS (m/z) found (calcd) for C19H19N3O (M): [M þ H]þ,
306.50 (306.16); [M þ Na]þ, 328.33 (328.14). The obtained inter-
mediate (1.80 g, 5.90 mmol) and 2-(chloromethyl)pyridine
hydrochloride (2.20 g, 13.41 mmol) were dissolved in anhydrous
dimethylformamide (DMF; 30 mL), and K2CO3 (5.00 g, 36.23
mmol) was added to the solution. The mixture was heated at
65 °C for 48 h under a nitrogen atmosphere. After filtration and
removal of DMF, the brown-yellow residue was purified with
chromatography (ethyl acetate/ethanol, 20:1, v/v), and light-
yellow N-[3-(2-pyridylmethoxy)benzyl]-N,N-bis(2-pyridylmethyl)-
amine (L2) was obtained. Yield: 40%. 1H NMR (DMSO-d6, 500
MHz, ppm): 3.59 (s, 2H, -CH2), 3.70 (s, 4H, -CH2), 5.19 (s, 2H,
-CH2), 6.91-6.92 (d, 1H, Ph-H), 6.98-7.00 (d, 1H, Ph-H),
7.09 (s, 1H, Ph-H), 7.24-7.27 (m, 2H, Py-H; 1H, Ph-H),
7.34-7.37, (m, 1H, Py-H), 7.50-7.51 (d, 1H, Py-H), 7.53-
7.54 (d, 2H, Py-H), 7.77-7.80 (m, 2H, Py-H), 7.81-7.84 (m,
1H, Py-H), 8.49-8.50 (d, 2H, Py-H), 8.60-8.61 (d, 1H,
CT-DNA (0.1 mM) was incubated alone or with complex 1, 2,
3, or 4 (0.1 mM) in the buffer (5 mM Tris-HCl/50 mM NaCl, pH
7.26) at 37 °C for 12 h. CD spectra were recorded at room
temperature in the wavelength range of 220-320 nm. Three
scans were performed for each spectrum.
The fluorescence of the EB-DNA system was determined in
the buffer (5 mM Tris-HCl/50 mM NaCl, pH 7.26) at room
temperature (λex=520 nm; λem=610 nm). Specifically, the CT-
DNA solution (25 μL, 0.45 mM) was added to the EB buffer
(3 mL). Aliquots of the complex solution (2.6, 2.54, 1.65, and
1.72 ꢀ 10-4 M for 1-4, respectively) were then added to the EB-
DNA solution, and the fluorescence was measured after each
addition until a 50% reduction of fluorescence appeared. The
apparent binding constant (Kapp) was calculated using the
equation
KEB½EBꢁ ¼ Kapp½complexꢁ
where KEB = 1.0 ꢀ 107 M-1, [EB] = 1.3 μM, and [complex] was
the final concentration at which a 50% reduction of the fluore-
scence had occurred.26
(25) Price, J. H.; Williamson, A. N.; Schramm, R. F.; Wayland, B. B.
Inorg. Chem. 1972, 11, 1280–1284.
(26) Lee, M.; Rhodes, A. L.; Wyatt, M. D.; Forrow, S.; Hartley, J. A.
Biochemistry 1993, 32, 4237–4245.

2544 Inorganic Chemistry, Vol. 49, No. 5, 2010
Scheme 1. Synthetic Route of the Ligands
Dong et al.
UV-vis absorption titration was carried out by adding
different concentrations of the CT-DNA solution to the com-
plex solutions with constant concentration. The changes of
the absorption intensity with increasing concentrations of
CT-DNA were recorded after equilibrium. The intrinsic binding
constant Kb of the complexes to CT-DNA was obtained by
regression analysis using the equation
Analysis of ROS. Common radical scavengers like NaN3
(1 μL, 1 M), KI (1 μL, 1 M), and dimethyl sulfoxide (DMSO;
1 μL, 100%) were added to the buffer solutions (50 mM Tris-
HCl/50 mM NaCl, pH 7.4) containing supercoiled pUC19 DNA
(200 ng) prior to the addition of each complex (1 μL, 20 μM),
respectively. The mixture was incubated at 37 °C for 10 min,
and Vc (1 μL, 10 mM) was added to initiate the reaction (Vtotal
,
10 μL). The subsequent quench, electrophoresis, and quanti-
fication procedures were performed as described above.
1=2
ðεa -εf Þ=ðεb -εf Þ ¼ ½b -ðb2 -2Kb2Ct½DNAꢁ=sÞ ꢁ=2KbCt
Results and Discussion
where b = 1 þ KbCt þ Kb[DNA]/2s, εa, εf, and εb are the extinc-
tion coefficients of the charge-transfer absorption band at a
given DNA concentration, of the free complex in solution, and
of the DNA-bound complex, respectively, Ct is the total com-
plex concentration, [DNA] is the DNA concentration in nucleo-
tides, and s is the binding site size in base pairs.27 The nonlinear
least-squares analysis was finished using Origin 8.0.
Design and Synthesis. Two multidentate ligands L1 and
L2 were designed to satisfy the coordination of copper(II)
and platinum(II) centers simultaneously (Scheme 1).
Copper(II) complexes 1 and 2 were obtained directly from
the reaction of L1 or L2 with CuCl2 2H2O in methanol at
3
room temperature, and heterodinuclear complexes 3 and 4
were prepared by the reaction of cis-[Pt(DMSO)2Cl2] with
1 or 2 in methanol, respectively. The linkers between
copper(II) and platinum(II) centers in 3 and 4 are rigid
p- and m-cresol, respectively. The platinum(II) center in
these complexes is coordinated to a 2-methylpyridine, a
DMSO, and two chloride ligands. According to the con-
figuration of [Pt(DMSO)2Cl2],25 the chlorides are most
likely in a cis conformation. The copper(II) center com-
prises a BPA and two chloride ligands, which was proven
to be an effective DNA scission motif in our previous
study.13 In this design, complexes 3 and 4 are expected to
combine the selective binding ability of the cis-platinum
moiety with the cleavage property of the Cu-BPA compo-
nent. Considering the preference of the cis-dichloroplati-
num moiety for the N7 position of neighboring adenine
and/or guanine in the major groove of DNA,29,30 the
platinum moiety in 3 and 4 may selectively direct the
copper part to the GG or AG site (vide infra).
Agarose Gel Electrophoresis. The DNA binding ability of
the complexes was tested by treating supercoiled pUC19 DNA
(200 ng) with gradient concentrations of the complex in the
buffer (50 mM Tris-HCl/50 mM NaCl, pH 7.4) until the total
volume reached 10 μL. The mixtures were incubated at 37 °C for
12 h, and the reactions were quenched by 2 μL of loading buffer
(30 mM EDTA, 36% glycerol, 0.05% xylene cyanol FF, and
0.05% bromophenol blue). The resulting solutions were loaded
onto the agarose gel (1%) and subjected to electrophoresis in a
TAE buffer (40 mM Tris acetate and 1 mM EDTA). DNA
bands were stained by EB, visualized under UV light, and
photographed.
The DNA cleavage experiments were carried out in a method
similar to that described above except that ascorbic acid (Vc;
1 μL) at a 100-fold molar concentration of each complex was
added to the reaction system as an initiator and the incubation
time was shortened to 1 h. The quantification of each DNA form
was accomplished by densitometric analysis on the agarose gel
containing EB. A correction factor of 1.47 was used for the
supercoiled DNA (form I) assessment because intercalation
of EB to form I DNA is relatively weak as compared to that
of nicked (form II) and linear DNA (form III).28 The fraction of
each DNA form was defined as the ratio of the individual band
intensity to total band intensity in a specific lane. The reported
results are the mean value of the triplicate experiments.
Characterization of the Complexes. The formation of
metal complexes and the speciation of various ionic
forms in a methanol solution were studied with ESI-MS.
(29) Cepeda, V.; Fuertes, M. A.; Castilla, J.; Alonso, C.; Quevedo, C.;
ꢀ
(27) Lahiri, D.; Bhowmick, T.; Pathak, B.; Shameema, O.; Patra, A. K.;
Ramakumar, S.; Chakravarty, A. R. Inorg. Chem. 2009, 48, 339–349.
(28) Jin, Y.; Cowan, J. A. J. Am. Chem. Soc. 2005, 127, 8408–8415.
Perez, J. M. Anti-Cancer Agents Med. Chem. 2007, 7, 3–18.
(30) Temple, M. D.; McFadyen, W. D.; Holmes, R. J.; Denny, W. A.;
Murray, V. Biochemistry 2000, 39, 5593–5599.

Article
Inorganic Chemistry, Vol. 49, No. 5, 2010 2545
Figure 2. ESI-MS spectra of complexes 1-4 in a methanol solution. Insets are the determined isotopic distribution patterns and the corresponding
simulated ones of the observed peaks.
Table 1. Assignment of the Major Observed Peaks in the ESI-MS Spectra for Complexes 1-4 (See Figure 2)
complex
species
formula
obsd m/z
calcd m/z
1
[1 - Cl]þ
C25H24ClCuN4O
C27H27CuN4O3
C50H48Cl3Cu2N8O2
C25H24ClCuN4O
C27H27CuN4O3
C50H49Cl2Cu2N8O3
C50H48Cl3Cu2N8O2
C27H30Cl3CuN4O2PtS
C29H33Cl2CuN4O4PtS
C27H30Cl3CuN4O2PtS
C29H33Cl2CuN4O4PtS
494.17-498.25
518.17-522.08
1024.83-1030.67
494.33-498.17
518.17-522.08
1006.83-1014.92
1024.67-1030.67
835.92-843.83
860.00-867.92
836.10-843.86
859.92-867.83
494.10
518.14
1025.18
494.10
518.14
1006.21
1025.18
840.04
862.07
840.04
862.07
[1 - 2Cl þ CH3COOa]þ
[1 (1 - Cl)]þ
3
2
[2 - Cl]þ
[2 - 2Cl þ CH3COO]þ
[(2 - Cl)2 þ OH]þ
[2 (2 - Cl)]þ
3
3
4
[3 - Cl]þ
[3 - 2Cl þ CH3COO]þ
[4 - Cl]þ
[4 - 2Cl þ CH3COO]þ
a CH3COO- was introduced by an eluent that contains a trace amount of CH3COOH.
As Figure 2 shows, three major peaks are observed at
m/z 494.17, 518.17, and 1024.83 for complex 1, which
could be assigned to [1 - Cl]þ, [1 - 2Cl þ CH3COO]þ,
complexes could covalently bind to DNA after losing the
labile Cl-.
The complexes were further characterized by IR spec-
tra and elemental analysis. The vibration bands for
Cu-Cl (267.2-274.8 cm-1), Pt-Cl (311.9-339.9 cm-1),
and Pt-S (438.3-440.4 cm-1) can be clearly observed in
the far-IR spectra (see Figure S1 in the Supporting
Information),31,32 and the results of elemental analysis
fit well the composition of the complexes. The involve-
ment of a cis-[Pt(DMSO)Cl2] unit in complexes 3 and 4 is
also demonstrated by the 1H NMR spectra (see Figure S2
in the Supporting Information), where the picolin pro-
tons were downfield-shifted when the copper complexes
reacted with cis-[Pt(DMSO)2Cl2].
and [1 (1 - Cl)]þ, respectively. Complex 2 also presents
3
three major peaks at m/z 494.33, 518.17, and 1024.67,
which could be attributed to [2 - Cl]þ, [2 - 2Cl þ CH3-
COO]þ, and [2 (2 - Cl)]þ, respectively. Complex 3 has
3
two major peaks at m/z 839.92 and 862.00, which corre-
spond to [3 - Cl]þ and [3 - 2Cl þ CH3COO]þ, respec-
tively. Two peaks assignable to [4 - Cl]þ and [4 - 2Cl þ
CH3COO]þ are observed at m/z 840.08 and 861.92 for
complex 4, but the latter peak is rather weak. The isotopic
distribution patterns of the above peaks match well with
the theoretical results simulated by the Isopro 3.0 pro-
gram. The detailed assignments of the peaks observed in
the spectra are listed in Table 1. ESI-MS observations
indicate that the metal-ligated skeletons of these com-
plexes are stable in solution and the chloride ligand
may dissociate from the complex. This suggests that the
(31) Whyman, R.; Hatfield, W. E. Inorg. Chem. 1967, 6, 1859–1862.
ꢀ
(32) Marques-Gallego, P.; den Dulk, H.; Brouwer, J.; Kooijman, H.;
Spek, A. L.; Roubeau, O.; Teat, S. J.; Reedijk, J. Inorg. Chem. 2008, 47,
11171–11179.

2546 Inorganic Chemistry, Vol. 49, No. 5, 2010
Dong et al.
Figure 3. UV-vis spectra of L1, L2, and complexes 1-4 (0.1 M) in an aqueous solution. Insets show closeups of the π f π* (240-280 nm) and d-d
(550-750 nm) absorption features.
Figure 4. CD spectra of CT-DNA (0.1 mM) in the presence of complexes 3 and 4, respectively, at different [complex]/[DNA] molar ratios.
Characterization of the complexes in aqueous solutions
was investigated by UV-vis spectroscopy. As shown in
Figure 3, ligand L1 shows an intense absorption band at
ca. 260 nm and two shoulder bands at around 260 nm,
which could be assigned to the n f π* and π f π*
absorption bands of pyridyls. The spectrum of copper(II)
complex 1 is similar to that of L1 except that the absorp-
tion band at 260 nm is flattened and a broad weak d-d
absorption band appears in the region of 550-750 nm
because of copper(II) coordination with pyridyls.23,33 The
formation of complex 3 from 1 barely influences the weak
d-d absorption band but further broadens the π f π*
absorption band at 260 nm because one more pyridyl is
involved in the coordination with platinum(II). The
spectral features of ligand L2 and complexes 2 and 4 are
similar to those of ligand L1 and complexes 1 and 3. These
results demonstrate that copper(II) remains bound to the
pyridyls in solution and in the presence of platinum(II).
DNA Binding Behavior of the Complexes. CD spectro-
scopy was used to probe the global changes in the DNA
conformation induced by the complexes. The CD spectra
of CT-DNA exhibit a positive band at 275 nm due to
the base stacking and a negative band at 245 nm due to the
helicity, which is characteristic of B-DNA.34 With the
addition of complex 1 or 2, the CD spectra only show
a slight change in the ellipticity for both positive and
negative bands (see Figure S3 in the Supporting In-
formation), suggesting that 1 and 2 may interact with
CT-DNA chiefly in an electrostatic mode.35 Complexes 3
and 4 perturb the base stacking and helicity bands more
considerably than 1 and 2 (Figure 4). For complex 3, both
positive and negative bands display a decrease in the
ellipticity and a slight red shift in the maximum wavelength
when the molar ratio of the complex to CT-DNA (r)
progressively increases. This is an indication of B f Z
conformational conversion, though limited, with increased
winding of the DNA helix through rotation of the bases.36
Apparently, the decrease in the ellipticity of the CD band is
mainly induced by the platinum moiety, which has been
related to the formation of short single-stranded segments
containing unpaired bases.37 For complex 4, the ellipticity
of both bands increases with respect to free DNA when
the r value is less than 0.4; however, it begins to decrease
when the ratio reaches 0.4. The change of the positive band
is similar to that induced by cisplatin,38,39 which may
reflect the contribution of the cis-platinum moiety to the
(35) Tang, S.-P.; Hou, L.; Mao, Z.-W.; Ji, L.-N. Polyhedron 2009, 28, 586–
592.
ꢀ
(36) Moradell, S.; Lorenzo, J.; Rovira, A.; Robillard, M. S.; Aviles, F. X.;
Moreno, V.; de Llorens, R.; Martinez, M. A.; Reedijk, J.; Llobet, A. J. Inorg.
Biochem. 2003, 96, 493–502.
ꢁ
ꢁ
ꢀ
ꢀ
ꢀ
ꢁ ꢀ
ꢀ
ꢀ
(37) Zaludova, R.; Zakovska, A.; Kasparkova, J.; Balcarova, Z.;
€
ꢀ
Kleinwachter, V.; Vrana, O.; Farrell, N.; Brabec, V. Eur. J. Biochem.
1997, 246, 508–517.
ꢀ
~
(33) Karlin, K. D.; Cohen, B. I. Inorg. Chim. Acta 1985, 107, L17–L20.
(34) Johnson, W. C. In Circular Dichroism: Principles and Applications;
Nakanishi, K., Berova, N., Woody, R. W., Eds.; VCH: New York, 1994;
pp 523-540.
(38) Gay, M.; Montana, A. M.; Moreno, V.; Prieto, M.-J.; Llorens, R.;
Ferrer, L. J. Inorg. Biochem. 2005, 99, 2387–2394.
(39) Gay, M.; Montana, A. M.; Moreno, V.; Prieto, M.-J.; Perez, J. M.;
Alonso, C. Bioorg. Med. Chem. 2006, 14, 1565–1572.
ꢀ
~
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Article
Inorganic Chemistry, Vol. 49, No. 5, 2010 2547
titration and the subsequent regression analysis. As
Figure 6 presents, upon the addition of an increasing
amount of CT-DNA to a solution of the complex, an
apparent hypochromism and a slight red shift (1.5-2 nm)
were observed near 260 nm, which is generally assigned to
the intercalation involving stacking interactions between
the aromatic chromophores and the base pairs of DNA.43
The Kb values of complexes 3 and 4 obtained from the
regression analysis are 4.5 ꢀ 104 and 6.9 ꢀ 104 M-1
,
respectively. These results suggest that both complexes
3 and 4 can bind to CT-DNA moderately, and 4 is
somewhat better than 3 for DNA binding, which agrees
with the above results obtained from other experiments.
The binding of complexes 3 and 4 to DNA was con-
firmed additionally with agarose gel electrophoresis. As
Figure 7 shows, the migration rate of the supercoiled
DNA (form I) is retarded and the amount of the nicked
DNA (form II) is increased after pUC19 DNA was
incubated with the complexes, which indicates that the
complexes can induce the unwinding of the DNA super-
helix. Simultaneously, the mobility of form II DNA is
slightly accelerated, which may be due to the binding of
the cis-platinum moiety to DNA shortens and condenses
the helix.44 The separation between forms I and II nar-
rows as the concentration of the complex increases. The
bands of forms I and II coalesce into one when the
concentration reaches 100 μM for complex 3 (part a, lane
5) and 60 μM for complex 4 (part b, lane 6), respectively.
This coalescence point corresponds to the amount of the
complex that is necessary for the complete removal of form
I DNA. Beyond this point, the migration rate begins to
increase again as positive supercoils are induced.45 The
results suggest that the DNA binding ability of complex 4 is
higher than that of complex 3, which is consistent with the
CD, EB, and UV-vis results discussed above. The discre-
pancy in the binding ability between 3 and 4 mainly results
from the different geometries of their ligands.
Figure 5. Fluorescence decrease (λem = 520 nm) induced by complexes
1-4 through the displacement of CT-DNA-bound ethidium bromide in
the buffer (5 mM Tris-HCl/50 mM NaCl, pH 7.26).
modification of the secondary structure of DNA. In
contrast with cisplatin, the intensity of the negative band
also increases simultaneously as compared with that of
free DNA, which may result from the influence of the
Cu-BPA component. In general, the major feature of the
spectra is the increase in the ellipticity for both bands of
CT-DNA, which is an indication of a typical intercalation
involving π-π* stacking and stabilization of the right-
handed B form of CT-DNA.40,41 The CD spectra demon-
strate that copper(II) complexes 1 and 2 exert only a small
influence on the DNA conformation, while copper-
(II)-platinum(II) complexes 3 and 4 have a more evident
impact on the conformation of DNA. The interaction
mode of these two types of complexes with DNA is
different, where the platinum(II) center plays a crucial
role. Nevertheless, the geometry of the ligands also affects
the interaction, which is evidenced by the spectral differ-
ence between 3 and 4. According to the changes in
ellipticity induced by these complexes, their perturbation
on the base-stacking and helicity bands of CT-DNA
follows an order of 1 < 2 < 3 < 4.
The binding sites of the cis-platinum moiety in different
oligodeoxyribonucleotide duplexes containing GG or AG
sequences were studied by Maxam-Gilbert footprinting
experiments.46 Preliminary results indicate that copper(II)-
platinum(II) complexes 3 and 4 indeed show some specific
binding propensity to GG and AG sequences (see the
Supporting Information). However, the binding mode
seems more complicated than that of cisplatin analogues
in the presence of an additional copper center. For this
reason, further investigation is needed to clarify the issue.
Concentration-Dependent DNA Cleavage Activity. The
DNA cleavage activity of the complexes was studied
using supercoiled pUC19 plasmid DNA by gel electro-
phoresis in the buffer (50 mM Tris-HCl/50 mM NaCl, pH
7.4) under physiologically relevant conditions. Figure 8
shows the electrophoresis results of the DNA cleavage
induced by increasing concentrations of the respective
complexes in the presence of Vc. All complexes can convert
form I DNA into form II at 1 μM (lanes 3, 7, 11, and 15),
The DNA binding behavior of the complexes was
further investigated using the EB-DNA system. EB is
an intercalator that gives a significant increase in the
fluorescence emission when bound to DNA, and its
displacement from DNA results in a decrease in the
emission intensity.42 The apparent DNA binding con-
stants (Kapp) of the four complexes were thus determined
by the EB displacement assay.26 As Figure 5 shows, the
fluorescence intensity (I) of the EB-DNA system at
610 nm decreases remarkably with the addition of the
complex, suggesting that the complex can replace the
DNA-bound EB and the binding mode involves some
intercalative interaction. However, the binding propen-
sity of these complexes is different. On the basis of the
titration profiles, Kapp of complexes 1-4 is calculated as
1.16 ꢀ 105, 1.34 ꢀ 105, 1.47 ꢀ 105, and 3.42 ꢀ 105 M-1
,
respectively. The results are in accordance with the above
CD observations.
The intrinsic binding constant (Kb) of complexes 3 and
4 to CT-DNA was determined by UV-vis absorption
(43) Baldini, M.; Belicchi-Ferrari, M.; Bisceglie, F.; Dall’Aglio, P. P.;
Pelosi, G.; Pinelli, S.; Tarasconi, P. Inorg. Chem. 2004, 43, 7170–7179.
(44) Zhao, Y. M.; He, W. J.; Shi, P. F.; Zhu, J. H.; Qiu, L.; Lin, L. P.; Guo,
Z. J. Dalton Trans. 2006, 2617–2619.
(40) Shahabadi, N.; Kashanian, S.; Purfoulad, M. Spectrochim. Acta,
Part A 2009, 72, 757–761.
(41) Efthimiadou, E. K.; Katsaros, N.; Karaliota, A.; Psomas, G. Inorg.
Chim. Acta 2007, 360, 4093–4102.
(42) Le Pecq, J. B.; Paoletti, C. J. Mol. Biol. 1967, 27, 87–106.
(45) Keck, M. V.; Lippard, S. J. J. Am. Chem. Soc. 1992, 114, 3386–3390.
(46) Comess, K. M.; Costello, C. E.; Lippard, S. J. Biochemistry 1990, 29,
2102–2110.

2548 Inorganic Chemistry, Vol. 49, No. 5, 2010
Dong et al.
Figure 6. UV-vis spectral variations of complexes 3 (45 μM) and 4 (50.7 μM) during titration with increasing concentrations of CT-DNA in the buffer
(5 mM Tris-HCl/50 mM NaCl, pH 7.26). Insets show the least-squares fit of (εa - εf)/(εb - εf) vs [DNA] for the complexes.
m-cresol-derived compounds are more active than their
p-cresol-derived counterparts. However, this influence
could be overshadowed by the impact of the metal core.
Considering all of these factors, a comprehensive order
in terms of the nuclease activity could be concluded as
1 < 2 < 3 < 4.
Time-Dependent DNA Cleavage Activity. The time
course of pUC19 DNA cleavage mediated by the com-
plexes (10 μM) was followed with agarose gel electro-
Figure 7. Agarose gel electrophoresis patterns of supercoiled pUC19
phoresis in the buffer (50 mM Tris-HCl/50 mM NaCl, pH
7.4) at 37 °C. As shown in Figure 9, for complexes 1 and 2,
the major cleavage product is form II DNA, which
accounts for ca. 80% of the total DNA during the whole
time, while form III DNA remains small (<15%) within
90 min (lanes 3-10). For complexes 3 and 4, form II DNA
is relatively low (ca. 60-70%) in the cleavage products
as compared with that for 1 and 2, while form III DNA
displays an ever-increasing trend in the testing time (lanes
11-18). At 10 min, 1 and 2 convert under 3% of the
total DNA to form III (lanes 3 and 7), while 3 and 4
convert over 10% and 20% of the total DNA to form III,
respectively (lanes 11 and 15). At 30 min, complexes 1 and
2 only transform ca. 5% of the supercoiled DNA into
form III DNA (lanes 4 and 8), but complexes 3 and 4
achieve over 20% (lanes 12 and 16). At 60 min, form I
DNA is completely converted into forms II and III DNA
by 3 and 4 (lanes 13 and 17), whereas it is still detectable
even at 90 min in the case of 1 and 2 (lanes 6 and 10).
Around 40% of the supercoiled DNA is converted into
form III DNA by complexes 3 and 4 at 90 min (lanes 14
and 18). Taken together, the cleavage efficiency is similar
between the same type of complexes, i.e., 1 vs 2 and 3 vs 4,
with that of 2 and 4 being somewhat higher than that
of 1 and 3, respectively. However, the superiority of
copper(II)-platinum(II) complexes to copper(II) com-
plexes is notable, in that the cleavage efficiency of 3 and 4
is head and shoulders above that of 1 and 2 during the
entire time course. These results lead to the same conclu-
sion as that obtained from the concentration dependent
data, that is, the cleavage efficiency of the complexes
follows an order of 1 < 2 < 3 < 4. The promotive effect
of the platinum(II) center on the DNA cleavage activity
of copper(II) complexes is thus confirmed by these ex-
periments.
plasmid DNA (200 ng) incubated with complex 3 (a) or 4 (b) in the buffer
(50 mM Tris-HCl/50 mM NaCl, pH 7.4) at 37 °C for 12 h. (a) Lane 1,
DNA control; lanes 2-7, DNA þ 3 (25, 50, 75, 100, 125, and 150 μM,
respectively). (b) Lane 1, DNA control; lanes 2-8, DNA þ 4 (20, 30, 40,
50, 60, 70, and 80 μM, respectively).
and 2 seems more active (lane 7) than the others at this
concentration. Linear DNA (form III) appears at 5 μM
for 1 and 2 (lanes 4 and 8) and at 2.5 μM for 3 and 4 (lanes
12 and 16). At 10 μM, complexes 1-3 cleave 79.5%,
94.5%, and 95.6% of form I DNA and the percentages
of form III DNA reach 2.6%, 10.3%, and 23.1% (lanes
5, 9, and 14), respectively. At the same concentration,
complex 4 completely cleaves form I DNA into forms II
and III DNA (lane 18). At 20 μM, form I DNA is totally
transformed into forms II and III DNA for complex 2
(lane 10), but it is still observable for complex 1 (lane 6).
The most impressive cleavage feature observed for these
complexes is that form III DNA appears before the
disappearance of form I DNA (lanes 4-6, 8, 9, 12-14,
16, and 17). This phenomenon indicates that the complexes
are capable of performing direct double-strand scission,17
while many copper complexes are only able to cleave single
strand successively.
The above results demonstrate that all of the complexes
designed in this study have remarkable cleavage activity,
particularly complexes 3 and 4, which show higher acti-
vity than 1 and 2 at most corresponding concentrations. A
synergistic effect between the copper(II) and platinum(II)
centers may exist in 3 and 4. Because the ligands of the
correlated complexes (1 vs 3 and 2 vs 4) and the struc-
tures of the copper(II) moieties are identical, the superior
cleavage activity of 3 and 4 manifestly indicates that
the introduction of an additional platinum(II) center to
the copper(II) complex can significantly enhance the
nuclease efficiency. The geometry of the ligands also
influences the cleavage activity. For complexes contain-
ing the same metal center or centers (1 vs 2 and 3 vs 4),
ROS Responsible for DNA Cleavage. In order to iden-
tify the ROS that are responsible for the DNA cleavage

Article
Inorganic Chemistry, Vol. 49, No. 5, 2010 2549
Figure 8. Agarose gel electrophoresis patterns of pUC19 plasmid DNA
(200 ng) after incubation with Vc (1 mM) and each complex in the buffer
(50 mM Tris-HCl/50 mM NaCl, pH 7.4) at 37 °C for 1 h. Lane 1, DNA
control; lane 2, DNA þ Vc; lanes 3-6, DNA þ Vc þ 1 (1, 5, 10, and
20 μM); lanes 7-10, DNA þ Vc þ 2 (1, 5, 10, and 20 μM); lanes 11-14,
DNA þ Vc þ 3(1, 2.5, 5,and10μM); lanes 15-18, DNA þ Vc þ 4(1, 2.5, 5,
and 10 μM).
Figure 11. Histogram showing the influence of different radical scaven-
gers on the scission of supercoiled pUC19 DNA (200 ng) by complexes
1-4 (2 μM), respectively, in the presence of Vc (1 mM) after incubation at
37 °C for 1 h.
role in the cleavage process. Hydrogen peroxide scaven-
ger KI also markedly inhibits the cleavage activity of the
complexes (lanes a6, b4, a12, and b10), suggesting that
H2O2 is involved in the cleavage reaction. Singlet oxygen
scavenger NaN3 significantly diminishes the cleavage
activity of the complexes (lanes a7, b5, a13, and b11),
1
suggesting that O2 also takes part in the cleavage me-
chanism. The involvement of 1O2 is also demonstrated by
the remarkable enhancement of the cleavage activity in
D2O (lanes a8, b6, a14, and b12), where the lifetime of 1O2
is significantly longer than that in water.27 In summary,
these complexes seem to follow some similar pathways in
the cleavage process, in which hydroxyl radicals, hydro-
gen peroxide, and singlet oxygen are crucial ROS for the
cleavage reactions.
Figure 9. Agarose gel electrophoresis patterns of pUC19 plasmid DNA
(200ng) after incubation with eachcomplex(10μM) and Vc (1 mM) in the
buffer (50 mM Tris-HCl/50 mM NaCl, pH 7.4) at 37°C for 10, 30, 60, and
90 min, respectively. Lane 1, DNA control; lane 2, DNA þ Vc; lanes 3-6,
DNAþ 1 þ Vc;lanes 7-10, DNA þ 2þ Vc;lanes11-14, DNAþ 3 þ Vc;
lanes 15-18, DNA þ 4 þ Vc.
Conclusion
Heteronuclear complexes containing copper(II) and
platinum(II) centers have displayed some intriguing nuclease
properties. The combination of a site-specific group and a
scission moiety may enhance the regional selectivity and
cleavage efficiency of an artificial nuclease. As compared
with the corresponding copper(II) complexes, the hetero-
nuclear complexes synthesized in this work exhibit an en-
hanced DNA binding ability and a superior cleavage activity
because of the preferential affinity of platinum(II) for
DNA. The geometry of the ligands also has some impact
on the cleavage property. These results strongly support the
promotive effect of the platinum(II) center in the copper-
based nuclease. This work provides a potential approach to
achieving a site-specific cleavage of DNA.
Figure 10. Agarose gel electrophoresis patterns of pUC19 plasmid
DNA (200 ng) after incubation with each complex (2 μM) and Vc
(1 mM) for 1 h at 37 °C in the absence or presence of different scavengers.
(a) Lane 1, DNA control; lane 2, DNA þ Vc; lane 3, DNA þ 1; lane 4,
DNA þ 1 þ Vc; lanes 5-8, DNA þ 1 þ Vc þ S; lane 9, DNA þ 3; lane 10,
DNA þ 3 þ Vc; lanes 11-14, DNA þ 3 þ Vc þ S. (b) Lane 1, DNA þ 2;
lane 2, DNA þ 2 þ Vc; lanes 3-6, DNA þ 2 þ Vc þ S; lane 7, DNA þ 4;
lane 8, DNA þ 4 þ Vc; lanes 9-12, DNA þ 4 þ Vc þ S. S stands for
scavenger DMSO(10%), KI(100mM), NaN3 (100mM), and D2O (70%)
sequentially in each group of lanes.
Acknowledgment. We acknowledge financial support
from the National Natural Science Foundation of China
(Grants 20631020, 90713001, 20721002, and 30870554)
and the Natural Science Foundation of Jiangsu Province
(Grant BK2008015).
reaction, experiments in the presence of different com-
mon scavengers such as NaN3, KI, and DMSO were
carried out, respectively. As Figures 10 and 11 present,
the cleavage activity of 1-4 is reduced dramatically by the
presence of hydroxyl radical scavenger DMSO (lanes a5,
b3, a11, and b9), indicating that diffusible •OH plays a key
Supporting Information Available: Far-IR, 1H and 13C NMR,
and CD spectra, details of the Maxam-Gilbert footprinting
experiements, and PAGE patterns. This material is available
free of charge via the Internet at http://pubs.acs.org.