Journal of Inorganic Biochemistry xxx (2015) xxx–xxx

JIB-09768; No of Pages 7

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Journal of Inorganic Biochemistry

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Oxidative DNA cleavage by Cu(II) complexes: Effect of periphery substituent groups

Wei Wang a, Young Ae Lee a, Gyeongwon Kim a, Seog K. Kim a,⁎, Ga Ye Lee b, Jinheung Kim b,Youngmee Kim b, Gyeong Jin Park c,d, Cheal Kim c,d,⁎a Department of Chemistry, Yeungnam University, Gyeongsan City, Gyeong-buk 712-749, Republic of Koreab Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, Republic of Koreac Department of Fine Chemistry, Seoul National University of Science and Technology, Seoul 139-743, Republic of Koread Department of Interdisciplinary Bio IT Materials, Seoul National University of Science and Technology, Seoul 139-743, Republic of Korea

⁎ Corresponding authors.E-mail addresses: seogkim@yu.ac.kr (S.K. Kim), chealk

http://dx.doi.org/10.1016/j.jinorgbio.2015.07.0150162-0134/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: W. Wang, et al., Ox(2015), http://dx.doi.org/10.1016/j.jinorgbio

a b s t r a c t

a r t i c l e i n f o

Article history:Received 11 March 2015Received in revised form 5 June 2015Accepted 15 July 2015Available online xxxx

Keywords:Cu complexesDNA cleavageDipicolylamineLinear dichroism

A series of structurally-related [Cu(R-benzyl-dipicolylamine)(NO3)2] complexes, where R = methoxy- (1),methyl- (2), H- (3), fluoro- (4), and nitro-group (5), were synthesized, and their activity on DNA cleavage wasinvestigated by linear dichroism (LD) and electrophoresis. The addition of a benzyl group to the dipicolylamineligand of the [Cu(dipicolylamine)(NO3)2] complex (A), i.e., the [Cu(benzyl-dipicolylamine)(NO3)2] complex(3), caused significant enhancement in the efficiency of oxidative cleavage of both super-coiled (sc) and doublestranded (ds) DNA, as evidenced by the electrophoresis pattern and faster decrease in the LD intensity at 260 nm.The efficiency inDNA cleavagewas also alteredwith furthermodifications of the benzyl group by the introduction ofvarious substituents at the para-position. The cleavage efficiency appeared to be the largest when themethyl groupwas attached. The order of efficiency in DNA cleavage wasmethyl Nmethoxy≈H N fluoro≈ nitro group.When anelectron-withdrawing group was introduced, the cleavage efficiency decreased remarkably. The reactive oxygenspecies involved in the cleavage process were the superoxide radical and singlet oxygen. A possible mechanismfor this variation in the DNA cleavage efficiency was proposed.

© 2015 Elsevier Inc. All rights reserved.

1. Introduction

Biomedical inorganic chemistry has been a fascinating area ofresearch because of its potential applications in biology and medicinalchemistry [1–4]. Transition metal complexes have huge potential inmany applications because of their cationic character, modularity, reac-tivity, redox chemistry, photoreactions and well-defined three dimen-sional structures. Such properties of metal complexes have led to theiruse as anticancer reagents [3], artificial nuclease [4–6], acceptors ordonors in the DNA mediated electron/hole/energy transfer [7,8], andprobes for nucleic acids structures [9–12]. Few synthetic nucleases cancreate both single- and double-strand breaks in duplex DNA [13–16].Single-strand DNA (ssDNA) cleavage and double-strand DNA (dsDNA)cleavage represent different pathways. Double-strand breaks arebelieved to be biologically more important as sources of cell lethalitythan a single strand break because they are repaired less readily byDNA repair mechanisms [17,18].

Recently, the catalytic effect of [M(2,2′-bipyridine)2(NO)3](NO)3(M = Cu(II), Zn(II) and Cd(II)) was examined and the Cu(II) complexeffectively cleaved both super-coiled DNA and double-stranded DNA[19], highlighting the importance of the central metal ion. Cu(II) com-plexes of phenanthrolines and other heteroaromatic ligands have been

im@seoultech.ac.kr (C. Kim).

idative DNA cleavage by Cu(II.2015.07.015

studied widely as a transition metal based complexes because of theircatalytic effect on nucleic acid cleavage [19–30,33–35]. As an example,bis(1,10-phnanthroline)Cu(II) complexes, which can recognize a specif-ic sequence with high affinity, showed high DNA cleavage efficiencythrough direct abstraction of the H1′ hydrogen of the deoxyribose moi-ety. This complex has been usedwidely as a footprint reagent for nucleicacids and proteins [20]. The cleavage efficiency of synthetic nucleasecan be tuned by the appropriate selection of the ligand. The[Cu(II)(pyrimol)Cl] complex was reported to exhibit efficient self-activated DNA cleavage and cytotoxicity against the L1210murine leuke-mia and A2780 human ovarian carcinoma cell lines [21]. Some multinu-clear copper(II) complexes were reported to promote DNA cleavageefficiently by oxidizing deoxyribose or nucleobase moieties selectively[22–24]. Recently, the cleavage efficiency of the Cu(II) complexes with astructurally-related ligand, i.e., [Cu(2,2′-bipyridine)2(NO3)]NO3, [Cu(2,2′-dipyridylamine)2(NO3)2], and [Cu(dipicolylamine)(NO3)2] complexes,was investigated [33–35]. The [Cu(2,2′-bipyridine)2(NO3)]NO3 complexand the [Cu(dipicolylamine)(NO3)2] complex exhibited the highest andlowest efficiency in DNA cleavage. Although these results suggest thatthe efficiency in DNA cleavage depends on the ligand structure and prop-erty, only two reports to date have been demonstrated on the electroniceffects at 4-position of the benzene ring on ligand [33–35].

In this study, a new series of the Cu(II) complexes were synthesizedand characterized: benzyl-2,2′-dipicolyamine-Cu(II) complexes towhich a methoxy-, methyl-, H-, fluoro- and nitro-group is attached

) complexes: Effect of periphery substituent groups, J. Inorg. Biochem.

2 W. Wang et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx

at the 4-position of the benzyl group (Fig. 1). By comparing the DNAcleavage efficiency of these Cu(II) complexes, the effect of electronwithdrawing and donating substituents at the benzyl group on DNAcleavage was investigated.

2. Experimental

2.1. Materials

All chemicals were purchased from Sigma-Aldrich and used as re-ceived. NMR spectrawere recorded on a Varian 400 spectrometer. Chem-ical shifts (δ) are reported in ppm, relative to tetramethylsilane Si(CH3)4.Absorption spectra were recorded at room temperature using a PerkinElmer model Lambda 2S UV/Vis spectrometer. Electrospray ionizationmass spectra (ESI-mass) were collected on a Thermo Finnigan (San Jose,CA, USA) LCQTM Advantage MAX quadrupole ion trap instrument.Elemental analysis for carbon, nitrogen, and hydrogen was carried outby using a Flash EA 1112 elemental analyzer (thermo) in the OrganicChemistry Research Center of Sogang University, Korea. The pBR322plasmid DNA (referred to as scDNA) stock solution (1 mg/mL) waspurchased fromNew England Biolabs (MA, USA). Double stranded highlypolymerized calf thymus DNA (dsDNA) was obtained fromWorthingtonBiochemicals (NJ, USA) and dissolved in 5 mM cacodylate buffer pH 7.0.

Fig. 1. Summary of the synthetic route and chemical structure of the Cu(II) complexes investigarespectively. [Cu(dipicolylamine)(NO3)2], denoted by complex A in the text, is also shown.

Please cite this article as: W. Wang, et al., Oxidative DNA cleavage by Cu(II(2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.07.015

The concentration of dsDNA was determined using a molar extinctioncoefficient of ε260 nm = 6700 M−1 cm−1.

Themolar extinction coefficient of the Cu(II) complexes 1, 2, 3 and 4at 254 nmwas determined to be 12,500M−1 cm−1, 16,700 M−1 cm−1,9800 M−1 cm−1 and 9500 M−1 cm−1, respectively. The molar extinc-tion coefficient of complex 5 was 15,100 M−1 cm−1 at 259 nm. Allexperiments were carried out in a 5 mM cacodylate buffer, pH = 7.0,except for the electrophoresis measurements (see below). N-(4-Methylbenzyl)-1-(pyridin-2-yl)-N-(pyridin-2-ylmethyl)methanamine(L2), bis(2-pyridylmethyl)benzylamine (L3), and N-(4-nitrobenzyl)-1-(pyridin-2-yl)-N-(pyridin-2-ylmethyl)methanamine (L5) weresynthesized as reported previously [36–38]. Complex A [Cu(2,2′-dipicolylamine)2(NO3)2] was synthesized and characterized using amethod reported elsewhere [19].

2.2. Synthesis of N-(4-methoxybenzyl)-1-(pyridin-2-yl)-N-(pyridin-2-ylmethyl)methanamine (L1) and N-(4-fluorobenzyl)-1-(pyridin-2-yl)-N-(pyridin-2-ylmethyl)methanamine (L4)

L1was synthesized by the coupling of 2-picolyl chloride hydrochloride(1.84 g, 11 mmol) and (4-methoxyphenyl)methanamine (0.665 mL,5mmol) in 10mL ofwater and acetonitrile (1/1) at 70 °C. To this solution,5 mL of aqueous NaOH (0.898 g, 22 mmol) was added dropwise over aperiod of 30 min and the resulting mixture was stirred for an additional

ted in this study. The substituent R is CH3O\\, CH3\\, H\\, F\\and NO2\\for complex 1 to 5,

) complexes: Effect of periphery substituent groups, J. Inorg. Biochem.

3W. Wang et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx

2 h. The cooled solution was extracted three times with 20 mL of methy-lene chloride, and the combined organic layers were dried with Na2SO4,and filtered. The solvent was removed in vacuo and the product wasobtained as a deep brown oil. Yield: 1.48 g (92.8%). IR (KBr):ν(cm−1) = 1589(m), 1510(s), 1432(m). 1H NMR (CDCl3, 400 MHz): δ8.51 (d, 2H), 7.66 (t, 2H), 7.6 (d, 2H), 7.32 (d, 2H), 7.14 (t, 2H), 6.85 (d,2H), 3.79 (s, 4H), 3.78 (s, 3H), 3.62 (s, 2H). 13C NMR (DMSO-d6,400 MHz): δ 159.01, 149.46, 137.23, 131.05, 130.53, 123.11, 122.77,115.20, 114.33, 59.56, 57.40, 55.63, 55.66. HRMS (ESI): m/z calcd. forC20H21N3O + H+ ([M + H+]), 320.17; found, 320.27. Anal. calcd forC20H21N3O: C, 75.21; H, 6.63; N, 13.16. Found: C, 75.57; H, 6.20; N, 13.14%.

L4 was synthesized by the same method as L1. Yield: 1.42 g (92.6%).IR (KBr): ν(cm−1) = 1589(m), 1507(s), 1436(m). 1H NMR (CDCl3,400 MHz): δ 8.52 (d, 2H), 7.66 (t, 2H), 7.56 (d, 2H), 7.36 (t, 2H), 7.15(t, 2H), 6.99 (t, 2H), 3.78 (s, 4H), 3.64 (s, 2H). HRMS (ESI): m/z calcdfor C19H18FN3 + H+ ([M + H+]), 308.15; found, 308.17. Anal. calcdfor C19H18FN3: C, 74.25; H, 5.90; N, 13.67. Found: C, 74.43; H, 6.13; N,13.85%.

2.3. Synthesis of [Cu(L1)(NO3)2](1), [Cu(L2)(NO3)2](2), [Cu(L3)(NO3)2](3),and [Cu(L4)(NO3)2](4)

Cu(NO3)2·2.5H2O (0.28 g, 1.2 mmol) was added to a stirred solutionof L1 (0.32 g, 1 mmol) inmethanol (30mL). The solution was stirred for1 day at room temperature until a precipitate formed. The resultingprecipitate was filtered and washed 5 times with diethylether. The yieldwas 0.43 g (84.6%). IR (KBr): ν(cm−1) = 1609(m), 1511(m), 1413(s).HRMS (ESI): m/z calcd. for C20H21CuN4O4

+: 444.09 [L1 + Cu + NO3]+:found, 444.07 (Fig. S1). UV–vis λmax (nm) (ε, M−1 cm−1) (H2O): 254(12,500) (Fig. S6). Anal. calcd for C20H21CuN5O7 (506.96): C, 47.38; H,4.18; N, 13.81. Found: C, 47.22; H, 4.43; N, 13.50%.

[Cu(L2)(NO3)2](2) was synthesized by the same method as 1. Theyield was 0.41 g (84.0%). IR (KBr): ν(cm−1) = 1610(w), 1418(s).HRMS (ESI): m/z calcd. for C20H21CuN4O3

+: 428.09 [L2 + Cu + NO3]+:found, 428.07 (Fig. S2). UV–vis λmax (nm) (ε, M−1 cm−1) (H2O): 254(16,700) (Fig. S6). Anal. Calcd for C20H21CuN5O6 (490.96): C, 48.93; H,4.31; N, 14.26. Found: C, 49.21; H, 4.12; N, 13.92%.

[Cu(L3)(NO3)2](3) was synthesized by the same method as 1. Theyield was 0.42 g (88.9%). IR (KBr): ν(cm−1) = 1611(m), 1419(s).HRMS (ESI): m/z calcd. for C19H19CuN4O3

+: 414.07 [L3 + Cu + NO3]+:found, 414.08 (Fig. S3). UV–vis λmax (nm) (ε, M−1 cm−1) (H2O): 254(9800) (Fig. S6). Anal. Calcd for C19H19CuN5O6 (476.93): C, 47.85; H,4.02; N, 14.68. Found: C, 47.43; H, 3.77; N, 15.03%.

[Cu(L4)(NO3)2](4) was synthesized by the same method as 1. Theyield of complex 4was 44.1 mg (89.1%). IR (KBr): ν(cm−1) = 1610(w),1509(w), 1418(s). HRMS (ESI): m/z calcd. for C19H18CuFN4O3

+: 432.07[L4 + Cu + NO3]+: found, 432.07 (Fig. S4). UV–vis λmax (nm) (ε,M−1 cm−1) (H2O): 254 (9500) (Fig. S6). Anal. calcd for C19H18CuFN5O6

(494.92): C, 46.11; H, 3.67; N, 14.15. Found C, 46.37; H, 3.97; N, 13.85%.[Cu(L5)(NO3)2](5) was synthesized by the same method as 1. The

yield was 0.46 g (87.9%). IR (KBr): ν(cm−1) = 1610(m), 1522(m),1421(m), 1343(m), 1288(s). HRMS (ESI): m/z calcd. for C19H18CuN5O5

+:459.06 [L5 + Cu + NO3]+: found, 459.07 (Fig. S5). UV–vis λmax (nm) (ε,M−1 cm−1) (H2O): 259 (15,100) (Fig. S6). Anal. calcd for C19H18CuN6O8

(521.93): C, 43.72; H, 3.48; N, 16.10. Found C, 43.54; H, 3.69; N, 16.15%.

2.4. X-ray data collection and structure determination

A blue rod-type crystal (4), approximately 0.10 mm × 0.03 mm ×0.03mm in size, was used for X-ray crystallographic analysis. The diffrac-tion data for the compound was collected on a Bruker SMART APEXdiffractometer equipped with a monochromator using a Mo Kα (k =0.71073 Å) incident beam. The crystal was mounted on a glass fiber.The CCD data was integrated and scaled using the BRUKER-SAINT soft-ware package, and the structure was solved and refined using SHEXTLV6.12. All hydrogen atoms were located in the calculated positions.

Please cite this article as: W. Wang, et al., Oxidative DNA cleavage by Cu(II(2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.07.015

Table S1 lists the crystallographic data including the bond lengths andangles (Supporting Information). Structural information was depositedat the Cambridge Crystallographic Data Centre (CCDC 978432).

2.5. Cleavage of pBR322 plasmid DNA

The appropriate amount of ascorbic acid and the metal complexeswas added to a scDNA solution in a 5 mM cacodylate buffer (pH 7.0)for the conventional cleavage experiment. The mixture was incubatedfor 30 min at room temperature. The reaction was quenched by theaddition of a stopping buffer containing 7mM EDTA, 0.15% bromophenolblue, 0.15% xylene cyanol and 75% glycerol. The mixtures were placed ona 1% agarose gel for electrophoresis at 25 V, 400 mA for 400 min. The gelwas stained with Tris–acetate–EDTA (TAE) buffer containing 0.5 μg/mLethidium bromide, 20 mM Tris acetate and 1 mM EDTA, and visualizedby UV trans-illumination and photographed using an Olympus C-5060camera. The integrated densities of the bands were quantified usingTotal Lab TL100 image analysis software (Nonlinear Dynamic Ltd., USA).The amounts of scDNA were corrected by a factor of 1.4 based on theweaker binding of ethidium to this structure.

2.6. LD and other spectroscopic measurements

Linear dichroism (LD) is defined by the difference in the absorptionof polarized parallel and perpendicular radiation relative to the labora-tory reference axis of the oriented sample [39–41]. The use of LD as atool for detecting dsDNA cleavage in real-time is described elsewhere[19,33–35,39]. As it was described, decrease in LD intensity reflectsincrease in flexibility of dsDNA as a result of the cleavage of one strandand/or decrease in the DNA length by cut of both DNA strands. Recordedtime-dependent LD intensity was fitted by single or double exponentialdecay curves from which the rate constant can be calculated. An Origin8.0 program was used for curve fitting. The absorption spectra wererecorded on a Shimadzu 1601PC. The time-dependent decrease in LDat 260 nm and the LD spectrum were recorded on a J-810 spectropolar-imeter (Jasco, Tokyo, Japan) using a micro-volume, thermostatically-controlled inner rotating Wada-type cuvette cell purchased fromKromak Ltd. (Dunmow, GB). The results were fitted to one or two expo-nential decay curves using the OriginPro8.0 program (OriginLab Co.,Northampton, MA, USA). The goodness of fit was evaluated using theresiduals. The electrochemical experiments using a three-electrode one-compartment cell were conducted using a potentiostat (CH Instruments,Model 630C). The electrochemical measurements were conducted usingan Ag/AgCl reference electrode, coiled platinum counter electrode andglassy carbon electrode (Bioanalytical Systems Inc., A = 0.071 cm2).

3. Results and discussion

3.1. Synthesis and characterization of the Cu complexes

Ligands, L2, L3, and L5, were synthesized as previously reported[36–38], whereas L1 and L4 were obtained by coupling of 2-picolylchloride hydrochloride and two equimolar amounts of (4-methoxyphenyl)methanamine or 4-fluoro-benzylamine, respec-tively, in a mixture of water and acetonitrile (1/1) (Fig. 1). Theproducts were obtained as dark brownish oils.

The copper complexes (1, 2, 3, 4 and 5)were synthesized by reactingequimolar amounts of Cu(NO3)2·2.5H2O and ligands L1, L2, L3, L4 and L5in CH3OH (Fig. 1). The complexes were characterized by IR spectrosco-py, ESI-mass spectrometry (Figs. S1–S5, Supporting information), UV–vis spectrum (Fig. S6, Supporting information), elemental analysis. TheIR spectra of the complexes (1–5) have some common features. Theydisplayed a strong absorption band at approximately 1288 cm−1 thatwas attributable to the ν(N–O) band of nitrate and a series of bandsover 1410–1610 cm−1 region, which were characteristic of pyridylgroups [42].

) complexes: Effect of periphery substituent groups, J. Inorg. Biochem.

Table 1Crystal data and structure refinement for complex 4.

Empirical formula C19H18CuFN5O6

Formula weight 494.92Temperature 170(2) KWavelength 0.71073 ÅCrystal system MonoclinicSpace group P2(1)/cUnit cell dimensions a = 15.735(3) Å α = 90.00°

b = 8.2390(16) Å β = 107.12(3)°c = 16.465(3) Å γ = 90.00°

Volume 2040.0(7) Å3

Z 4Density (calculated) 1.611 Mg/m3

Absorption coefficient 1.128 mm−1

Crystal size 0.10 × 0.03 × 0.03 mm3

Reflections collected 10,712Independent reflections 4003 [R(int) = 0.1415]Data / restraints / parameters 4003/0/289Goodness-of-fit on F2 0.783Final R indices [I N 2σ(I)] R1 = 0.0636, wR2 = 0.1321R indices (all data) R1 = 0.1644, wR2 = 0.1549Extinction coefficient 0.0146 (11)Largest diff. peak and hole 0.585 and −0.688 e·Å−3

4 W. Wang et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx

The positive-ion mass spectra for the complexes 1–5 indicated thatthe peaks (at m/z = 444.07, 428.07, 414.07, 432.07 and 459.06) areassignable to the [Cu(L)(NO3)]+ complex species [calcd, m/z = 444.07,428.09, 414.08, 432.07 and 459.07] (Figs. S1–S5). The observed isotopicpatterns were well consistent with the calculated ones. UV–vis absorp-tion spectra of complexes 1, 2, 3 and 4 showed a sharp band (ε1 =12,500 M−1 cm−1, ε2 = 16,700 M−1 cm−1, ε3 = 9800 M−1 cm−1 andε4 = 9500 M−1 cm−1 ) at 254 nm in H2O (Fig. S6), whereas onlythat of complex 5 showed a red-shifted sharp band (ε5 =15,100 M−1 cm−1) at 259 nm (Fig. S6). In the visible region, all theCu complexes displayed very weak bands around 650 nm (inset ofFig. S6). In order to gain a more detailed insight into the nature of thecopper complexes (1–5), UV–vis spectroscopic studies for 1 and 2were carried out at different pH values (Figs. S7 and S8), because Luis'group, recently, well established the relationship between the coordi-nation environments and pH values [31,32]. In both cases, only a singleband at pH range of 5–10was observed, indicating the presence of onlya complex species.

Complex 4was also characterized by an analysis of the single crystalX-ray structure, which is shown in Fig. 2. Table 1 lists the crystal dataand structure refinement for the complex 4. L4 coordinates the Cu2+

ion in a planar conformation, and two nitrate ions complete a distortedbipyramidal geometry around the Cu2+ ion. Two pyridine nitrogenatoms of the tridenate L4 ligand occupy the axial positions, and theoxygen atoms of the nitrate ligands coordinate Cu2+ in a plane[Cu(1)–O(41), 2.158(5); Cu(1)–O(51), 2.133(5) Å] (Table S1). The Cu–Npy distances [1.938(5) and 1.950(5) Å] and Cu–Namine distance[2.093(5) Å] (Table S1) are similar to the general observation that theaxial Cu–Npy distances are shorter than the Cu–Namine distance [31,32].

3.2. Effect of benzyl group on sc and dsDNA cleavage

The LD signal in the DNA absorption region is expected to be negativewith a shape symmetrical to a normal absorption spectrum, as expected

Fig. 2. Structure of complex 4. The displacement ellipsoids are shown at the 30%probability level.

Please cite this article as: W. Wang, et al., Oxidative DNA cleavage by Cu(II(2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.07.015

from the set-up adopted in this study [40,41]. Themagnitude of LD in theDNA absorption region depends solely on the flexibility and length ofDNA when the other conditions, including the temperature, concentra-tion, shear gradient, and viscosity are held constant. The flexibility ofdsDNA increases when one of the strands of dsDNA is cleaved. Anincrease in dsDNA flexibility results in a decrease in the ability of orien-tation, resulting in a decreased LD magnitude. The cleavage of bothstrands at a similar place shortens the DNA molecule, which also resultsin a lower LD magnitude in the DNA absorption region. Using this con-cept, LD is an excellent tool for probing the cleavage of sc and dsDNAin real-time [19,33–35,39,43–46].

Fig. 3(A) shows the decrease in the LD magnitude at 260 nm as afunction of time for dsDNA in the presence of complex A and complex3. The LD spectra of complex 3 at the time of mixing and 30 min aftermixing are inserted. With time, the magnitude of LD in the presenceof complex 3 decreased significantly but the extent of the decrease inthe presence of complex A was much less, suggesting that complex 3cleaves dsDNA more effectively than complex A. Fig. 3(B) shows thegel electrophoresis separations of complex 3- and complex A-inducedcleavage of pBR322 scDNA. In lane 1, pBR322 DNA exhibited twobands in the absence of the Cu(II) complexes, as expected. The relativeamount of the intense band corresponding to Form-I (super-coiledform) was 81.4%, and the other less intense band reflecting form-II(circular DNA) was 18.6%. In the presence of complex A (lanes 2–4),the band corresponding to Form II, relaxed circular scDNA, was moreintense. Increasing the concentration of complex A from 10 to 50 μMdid not affect: the relative amount of scDNA was constantly ~30%. Incontrast, complex 3 produced a significant amount of Form II scDNA,which was accompanied by a decrease in the amount of Form IscDNA, even at the lowest concentration of 10 μM (lane 5). The relativeamounts of Form I and Form II DNAwere 50.2% and 49.8%, respectively.In the presence of 30 and 50 μM complex 3, a new band correspondingto Form III, which is a linear form of scDNA, appeared and the bandcorresponding to scDNA disappeared (lanes 6 and 7, respectively). Inthe presence of 30 μM complex 3, 91.1% of Form II and 8.9% of Form IIIwere apparent. A similar value was obtained in the presence of 50 μMcomplex3. This suggests that complex 3was significantlymore effectivein the cleaving scDNA than complex A as it was noted previously forthe dsDNA case. Therefore, the addition of a benzyl group to thedipicolylamine ligand of complex A caused significant enhancement inthe cleavage efficiency of both sc and dsDNA.

) complexes: Effect of periphery substituent groups, J. Inorg. Biochem.

Fig. 3. (A) Change in the LD magnitude at 260 nm of the dsDNA in the presence of theCu(II) complexes. Curves 1 and 2 denote the LD change in the presence of complex 3and complex A, respectively. [DNA] = 200 μM and [Cu(II) complex] = 50 μM.(B) Cleavage of super-coiled pBR322DNAby complex3 and complexA at various complexconcentrations. Lane 1: scDNA; lane 2–4: in the presence of 10 μM, 30 μMand 50 μMof thecomplex A and lanes 5–7 in the presence of the complex 3 at the same concentrations.[DNA] = 50 μM and [ascorbate] = 200 mM. The mixtures were incubated for 30 minand the gel was run for 400 min under 25 V. The length of column was 7.5 cm.

Fig. 4. Decrease in the LD magnitude at 260 nm of the dsDNA with respect to time in thepresence of complexes 2, 3, and 5. The decrease for complex 1was similar to that of com-plex3 and the result obtained from complex 4was similar to that of complex 5. The best fitexponential decay curves, LD = a exp(−t/τ), are denoted by the solid curve. [DNA] =200 μM, [ascorbate] = 200 μM and [Cu(II) complex] = 50 μM.

5W. Wang et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx

3.3. Effect of the electron withdrawing and donating substituents at thebenzyl group on DNA cleavage

The effect of the benzyl-dipicolylamine ligand was tested further byintroducing a range of substituents, i.e., methoxy-, methyl-, fluoro- andnitro-groups, to the 4 position of the benzene ring (Fig. 1). Similar to com-plex 3, all complexes exhibited cleavage activity for dsDNA, as evidencedby the decrease in the LDmagnitude at 260 nm. A decrease in the DNA LDintensity in the presence of other oxidative metal complexes was report-ed. For example [19,31,32], the [Cu(2,2′-bipyridine)(NO3)](NO3) and[Cu(2,2′-bipyridylamine)2(NO3)](NO3) complexes caused a reducedDNA LD signal: The DNA LD signal almost disappeared within 20 minafter mixing both complexes due to the cleavage of dsDNA. The patternof the time-dependent decrease in the LD signal was described by thesum of two exponential curves. The rapidly decreasing component wasassigned to the cleavage of the first DNA strand causing an increase inthe DNA flexibility, whereas the slow step was attributed to the cleavageof the second strand that occurred near the first cleavage site. The latterstep induced shortening of the DNA length. One of the reactive oxygenspecies, the superoxide radical (O2

−•) was found to be responsible forthe cleavage ofDNA inboth steps by the [Cu(2,2′-bipyridine)(NO3)](NO3)and [Cu(2,2′-bipyridylamine)2(NO3)](NO3) complexes (see followingsection).

Complexes 1–5 also caused a decrease in the DNA LD magnitude at260 nm (Fig. 4). The pattern of the time-dependent decrease in DNA

Please cite this article as: W. Wang, et al., Oxidative DNA cleavage by Cu(II(2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.07.015

LD for complex 1 resembled that of complex 3. The pattern of complex4 was similar to that of complex 5. In contrast to the reports of otherCu(II) complexes [19,30–32], the decrease was found to be single expo-nential decay (LD = a exp(− t/τ)). This is in contrast to the resultsobtained from other metal complexes including Cu(II) complexes,which produced two component exponential curves. Therefore, thepossibility of cleavage of the complementary strand near the firstcleaved site resulting in a shortening of DNA is unlikely to have occurredin the present case. The reaction time τ obtained from the best fit curvewas 14.5, 15.7, 17.6, and 16.6 min for complexes 2, 1 ≈ 3, 4, and 5,respectively. These reaction times correspond to the rate constant ofthe first order reactions of 1.15 × 10−3 s−1, 1.06 × 10−3 s−1,0.95 × 10−3 s−1, and 1.00 × 10−3 s−1, respectively. Although com-plexes 1–3, particularly complex 2, showed the largest rate constant,these values were in a similar range. Therefore, it is difficult to drawany significance. On the other hand, the remaining intensity of theDNA LD signal showed remarkable differences. The DNA LD signal40 min after mixing was the lowest for complex 2, being 37% of theinitial DNA LD signal. Those were 48% for complexes 1 and 3. The DNALD signals of complexes 4 and 5were the lowest at 54% and 57%, respec-tively. The magnitude of the DNA LD signal is affected by a range of fac-tors. On the other hand, the experimental conditions in this study werekept constant, and the decay of DNA LD appeared to be single exponen-tials. Therefore, the decrease in DNA LD intensity mainly reflects theincrease in flexibility. Although the difference is not large, introducingelectron donating groups to the para-position of the benzyl ring resultedin an enhancement of the cleavage activity of the Cu(II) complexes,whereas substituting the same position with an electron withdrawinggroup reduced the activity.

Fig. 5 shows the gel electrophoresis separations of the Cu(II)complex-induced cleavage of pBR322 scDNA. Table 2 lists the relativeamounts of cleaved DNA in the presence of various Cu(II) complexes,which are shown in lane 2 s in panels 1–5. In lane 1 of each panel inthe absence of Cu(II) complexes, scDNA exhibited a clear bandcorresponding to Form-I (super-coiled form) and a less intense bandrepresenting Form-II (circular form). The DNA cleavage activity wasclearly observed in the presence of complexes 1–5 (lane 2 in eachpanel). In the case of complexes 1–3, in which the electron donatinggroup was attached to the para-position of the benzyl ring, the bandcorresponding to the super-coiled form disappeared and that of Form-II became increasingly intense (Table 2). A band that represents Form-III (nicked linear form) was also observed for all three complexes. Forexample, the presence of complex 1 caused the appearance of 88.7% cir-cular DNA and 11.3% linear DNA. This ensures that complexes 1–3 effi-ciently cleave the scDNA. In contrast, when the electron withdrawinggroup was introduced (complexes 4 and 5), the efficiency of scDNA

) complexes: Effect of periphery substituent groups, J. Inorg. Biochem.

1 2 3 4 5 6

1

2

3

1 2 3 4 5 6

4

5

Fig. 5. Cleavage of super-coiled pBR322 DNA by complexes 1 to 5 and the effect of variousreactive oxygen species scavengers detected by 1% agarose gel electrophoresis. Thenumbers on the panel denote the complex number. Lane 1: scDNA; lane 2: scDNA +Cu(II) complex; lanes 3–6: in the presence of 1 mM tiron, 5 mM sodium azide, 0.125unit of catalase and 30 mM DMSO, respectively. [DNA] = 50 μM, [Cu(II) complex] =50 μM and [ascorbate] = 200 mM. The mixtures were incubated for 30 min and the gelwas run for 400 min under 25 V.

Table 2Relative amount (%) of cleaved DNA in the presence of the Cu(II) complexes. The datawere read from the electrophoresis data shown in Fig. 5.

Form I Form II Form III

scDNA 81.4 18.6Complex 1 88.7 11.3Complex 2 75.9 24.1Complex 3 88.9 11.1Complex 4 53.1 46.9Complex 5 51.2 48.4

Table 3Effect of various ROS scavengers on scDNA cleavage. The data were obtained fromFig. 5(3) for complex 3 as an example.

Form I Form II Form III

scDNA 81.4 18.6scDNA + complex 3 88.9 11.1Tiron 77.3 22.7Sodium azide 79.4 20.6Catalase 66.5 33.5DMSO 53.1 46.9

6 W. Wang et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx

cleavage decreased significantly (panels 4 and 5, lane 2). Although someincrease in the density of the band corresponding to Form-II was noticed,the extent of the increase was far less effective (Fig. 5). In contrast to thecase of complexes 1–3, the intense Form-I band remained. Furthermore,

Please cite this article as: W. Wang, et al., Oxidative DNA cleavage by Cu(II(2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.07.015

the band representing Form-III did not appear. In addition to the time-dependent DNA LD measurement, the replacement of H at the para-position of the benzyl ring by the electron withdrawing group resultedin a decrease in the DNA cleavage efficiency.

3.4. Effect of reactive oxygen scavengers

Various reactive oxygen species (ROS) can participate in the DNAcleaving process [47–50]. Lanes 3–6 in Fig. 5 show the effect of ROSscavengers on scDNA cleavage. Table 3 lists the relative amount ofscDNA and cleaved DNA. In the case of complexes 1–3, the presence of1 mM tiron, which is a superoxide radical (O2

−•) scavenger, had a largeinhibitory effect on the cleavage of scDNA (lanes 3). 5 mM sodiumazide, a singlet oxygen (1O2) scavenger also inhibited the cleavage aseffectively as tiron (lane 4). In the complex 3 case, for an example,relative amount of Forms I and II was 79.4% and 20.6% in the presenceof sodium azide and was 77.3% and 22.7% in the presence of tiron,respectively. This result is in contrast to the production of relativeamount of 88.9% Form II and 11.1% Form III obtained with the complex3 in the absence of ROS scavengers (Table 3). For complexes 1–3 (panels1–3, Fig. 5), the band representing Form-I remained visible in the pres-ence of either tiron or sodiumazide, whereas that for Form-IIwas signif-icantly less intense compared to the result obtained in their absence.The inhibitory effects of tiron and sodium azide appear to be larger forcomplex 3 than complexes 1 and 2. The effect of the 0.125 unit ofcatalase and 20 mM DMSO, which are hydroxyl radical (•OH) andH2O2 scavengers, is also shown in lanes 5 and 6, respectively. In thecase of complexes 1 and 2 (panels 1 and 2, Fig. 5), the DMSO andcatalase scavengers had a very small inhibitory effect for the cleavageprocess. The remaining Form-I band shown in lanes 5 and 6 (panel 3,Fig. 5) suggests that catalase and DMSO also had an inhibitory effect inthe complex 3 case, albeit to a lesser extent than those of sodiumazide and tiron. The scDNA cleavage process was also inhibited by thepresence of sodium azide and tiron in complexes 4 and 5. The densityof the Form-II band in the presence of sodium azide and tiron (lanes 3and 4, panels 4 and 5, Fig. 5) was lower than that in the absence ofROS scavengers (lane 2), whereas the effect of catalase and DMSO wasnegligible. Overall, the main ROS involved in the cleavage process ofDNA induced by a series of Cu(II) complexes investigated in this studyappear to be the singlet oxygen and superoxide radical.

An electrophoresis study in the presence of various ROS scavengersrevealed the superoxide radical (O2

−·) to be one of the main species

) complexes: Effect of periphery substituent groups, J. Inorg. Biochem.

7W. Wang et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx

involved in the scDNA cleavage reaction induced by complexes 1–5. Theoxygen radical might be produced by the coordination of O2 to theCu(I) complex through the following reaction:

Cu IIð Þcomplexþ ascorbate→Cu Ið Þ þ ascorbate− � þHþ

Cu Ið Þcomplexþ O2 ⇌ Cu Ið Þ−O2 ⇌ Cu IIð Þ−O2−�½ �⇌ Cu IIð Þcomplexþ O2

−� :

For this reaction, the Cu(I) complex, which is essential, must bereduced from the Cu(II) complexes. Although there is nodirect evidencefor the reduction of Cu(II) complexes 1–5 to Cu(I), the reduction of theCu(II) complexes that bind to DNA [51,52] or to amine groups [53–55]has been reported. Faster DNA cleavage by Cu complexes 1 withelectron-donating groups suggests that the second step is the rate-determining step. It is conceivable that introducing electron donatinggroup to thebenzylmoiety enriches the negative charges at coordinatednitrogen atom resulting in strengthen the Cu–N bond. Consequently,the O2 bound at the opposite side will be removed easily causingenhanced cleavage rate.

Another important ROS in the DNA cleavage process induced bycomplexes 1–5 is singlet oxygen. Although the transition from theground state oxygen molecule to a singlet state is forbidden despitethe low energy difference between these two states, the involvementof singlet oxygen was confirmed by the electrophoresis experiment.The origin of singlet oxygen in the system adopted in this study isunclear.

4. Conclusion

The replacement of a H atom of the dipicoylamine ligand with abenzyl group of the [Cu(dipicolylamine)(NO3)2] complex enhancedthe efficiency of oxidative cleavage of both sc and dsDNA. Introducingan electron donating group at the para-position resulted in a furtherincrease in efficiency, whereas the presence of an electronwithdrawinggroup reduced the efficiency. The ROS involved in the cleavage processwere the superoxide radical and singlet oxygen.

Acknowledgments

This study was supported by an internal research grant ofYeungnam University and the Korea Research Foundation (NRF-2014R1A2A1A11051794).

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jinorgbio.2015.07.015.

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