Journal of Photochemistry and Photobiology B: Biology 141 (2014) 47–58

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Synthesis, characterization; DNA binding and antitumor activity of ruthenium(II) polypyridyl complexes A. Srishailam a, Nazar Mohammed Gabra a, Yata Praveen Kumar a, Kotha Laxma Reddy a, C. Shobha Devi a, D. Anil Kumar b, Surya S. Singh b, S. Satyanarayana a,⇑ a b

Department of Chemistry, Osmania University, Hyderabad 500007, India Department of Biochemistry, Osmania University, Hyderabad 500007, India

a r t i c l e

i n f o

Article history: Received 7 June 2014 Received in revised form 2 September 2014 Accepted 4 September 2014 Available online 16 September 2014 Keywords: Spectroscopic titrations DNA-binding Intercalative Docking Comet assay Phosphate backbone

a b s t r a c t Three new ruthenium(II) polypyridyl complexes [Ru(phen)2BrIPC]2+ (1), [Ru(bpy)2 BrIPC]2+ (2) and [Ru(dmb)2BrIPC]2+ (3) where, BrIPC = (6-bromo-3-(1H-imidazo[4,5-f] [1,10]-phenanthroline, phen = 1,10-phenanthroline, bpy = 2,20 bipyridine, dmb = 4,40 -dimethyl 2,20 bipyridine, were synthesised and characterised. DNA-binding nature was investigated by spectroscopic titrations and mode of binding was assessed by viscosity measurements. The DNA-binding constants Kb of complexes 1, 2 and 3 were determined to be in the order of 105. Experimental results showed that these complexes interact with CT-DNA by intercalative mode. Photocleavage and antimicrobial activities were complex concentration dependent, at high concentration, high activity and vice versa. MTT assay was performed on HeLa cell lines, IC50 values of complexes in the order of 3 > 2 > 1 > cisplatin. From comet assay, cellular uptake studies, we observed that complexes could enter into the cell membrane and accumulate inside the nucleus. Molecular docking studies support the DNA binding affinity with hydrogen bonding and van der Waals attractions between base pairs and phosphate backbone of DNA with metal complexes. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Medicinal inorganic chemistry and translational medicine are emerging concepts in the fields of biomedical research and health care since twenty-first century [1,2]. In the molecular biology, DNA as a carrier of genetic information is often a target for drugs. Such drugs exhibit DNA-targeted pharmacological activity because they affect DNA replication, a major step in cell growth and cell division, and correspondingly they interfere with the transcription processes and protein synthesis [3]. Based on DNA binders, investigations of bioinorganic chemistry have only existed for about the last few decades with the serendipitous discovery of the antitumor activity of cis-[Pt(NH3)2Cl2] and its derivatives [4,5]. In the search for drugs with improved clinical effectiveness, reduced toxicity and a broader spectrum of activity, other metals than platinum have been considered, such as Ru, Rh, Cu, Cd and Au etc. [6–10]. Ruthenium complexes are very promising, especially from the viewpoint of overcoming cisplatin resistance with a low general toxicity.

⇑ Corresponding author at: Department of Chemistry, Osmania University, Hyderabad, Andhra Pradesh 500007, India. Tel.: +91 40 2768233. E-mail address: [email protected] (S. Satyanarayana). http://dx.doi.org/10.1016/j.jphotobiol.2014.09.003 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.

DNA was identified as the primary molecular target of metal based anticancer drugs, the interactive mode of transition metal complexes with DNA and the potential of these complexes to act as chemotherapeutic agents have been analyzed [11]. Since the concept of intercalation into DNA was first formulated by Lerman in 1961 [12], bioinorganic research was focused on Ru(II) octahedral complexes containing at least one aromatic heterocyclic ligand for intercalation, in between base pairs. The consequences of this resulted in the study of many ruthenium complexes for biological activities, such as NAMI-A and KP109, which are entered into clinical trials [13,14]. Recently, in several laboratories, a worm research has been observed on design, synthesis and reactivity of novel ruthenium complexes, including ours [15–24]. In this report, we have synthesised three new octahedral ruthenium(II) polypyridyl complexes containing chromone based ligands (Scheme 1) and investigated their structure by Spectroscopic methods. Chromones are naturally occurring compounds which are able to cause cytotoxic effect in various types of cells. They are widely known to have anticancer, antioxidant, antiproliferative, antiHIV, anti-inflammatory and many other activities [25–27]. According to reported literature, chromone and its derivatives forming more stable and colored complex with various metals also possess comparable biological activity with cisplatin(which is an effective anticancer drug)

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[28–30]. The DNA-binding behavior was studied by absorption, emission spectral titrations and viscosity measurements. The cytotoxicity in vitro of these complexes was assessed by MTT assay (MTT = (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)). The DNA damaging studies were investigated by single cell gel electrophoresis. Cellular uptake studies were performed to analyze the entry and accumulation of complexes inside the cell in the region around the nucleus of HeLa cells.

2. Materials and methods 2.1. Materials Ruthenium(III) chloride trihydrate (RuCl33H2O), 1,10-Phenanthroline, 2,20 bipyridine, 4,40 dimethyl 2,20 bipyridine and 6bromo-4H-chromen-4-one was purchased from Sigma. All the solvents were purified before use, as per standard procedures [31]. CT (Calf Thymus) DNA was purchased from Sigma Aldrich; its solution gives a ratio of UV absorbance at 260 and 280 nm of 1.8–1.9, indicating that the DNA was sufficiently free of protein [32]. Super coiled pBR322 DNA (stored at 20 °C) was obtained from Fermentas life sciences (Genei) and MTT was purchased from HiMedia. Double distilled water was used for preparing various buffers. The human cervical carcinoma cell lines HeLa were obtained from NCCS, Pune, and maintained in RPMI 1640 medium (Sigma Aldrich Ltd.) supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 4.5 g L1 glucose, 1 non-essential amino acids and 1 antibiotics consisting of penicillin/streptomycin, gentamycin, amphotericin B, nystatin, (basal growth medium) Nexin reagent was procured from Millipore, DAPI 0.1 mg/mL was prepared by constituting a 1 mg vial and Propidium Iodide (1 mg/mL) for comet assay staining purchased from Sigma Aldrich. The cell lines were incubated in a humidified atmosphere with 5% CO2 and maintaining temperature of 37 °C. Stock solutions (50 mM) of complexes were prepared in

DMSO. The tetrazolium compound MTT (3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide) working stock at 0.4 mg/ mL was prepared by solubilising in 200 lL of DMSO and made up to the required volume with serum free medium. Ampicillin for antimicrobial studies was purchased from local pharmaceuticals. 2.2. Physicochemical measurements The UV–Visible spectrum was recorded with an Elico BL 198 spectrophotometer. Fluorescence measurements were performed on an Elico SL 174 spectrofluorimeter. IR spectra were recorded on KBr disks on a Perkin–Elmer FT-IR-1605 spectrometer. 1H NMR and 13C NMR spectra were recorded on a Bruker 400 MHz spectrometer with DMSO-d6 as solvent at RT and tetramethylsilane (TMS) as the internal standard. Microanalyses (C, H, and N) were carried out with a Perkin-Elmer 240 elemental analyzer. MTT assay in 96 well plates read with Thermo Scientific Multi Skan EX Elisa reader. Guava Easycyte 8HT Flowcytometer for flowcytometry analysis and confocal images acquired with Leica TCS-SP-5. 2.3. Synthesis and characterization Compounds 1,10-phenanthroline-5,6-dione [33], cis-[Ru(phen)2 Cl2]2H2O, cis-[Ru(bpy)2Cl2]2H2O and cis-[Ru(dmb)2Cl2]2H2O were synthesised according to the methods given in literature [34]. Synthetic scheme of Ru(II) complexes was shown in Scheme 1. 2.3.1. Preparation of BrIPC ligand A mixture of 1,10-phenanthroline-5,6-dione (0.210 g, 1.0 mM), 6-bromo-4-oxo-4H-chromone-3-carbaldehyde (0.302 g, 1.2 mM), Ammonium acetate (1.180 g, 28.8 mM) and glacial acetic acid (15 mL) were refluxed together for 4 h as per Steck and Day [35], and then cooled to room temperature and diluted with water. Drop wise addition of ammonia gave a yellow precipitate which was

Br

o

O N N

s

N

a b

Ru Ru II N

N N

g

f

c 1

5

N

h

O s

N

r

m

6

Br

h

N

f

c

e

g

N

k

i j

s

N

N

o

5

O

h

m

i

O

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e d

[Ru(bpy) 2 BrIPC] 2+

2 4

n

p

q

N

g

f

c 1

N

l

H N

r

a b

Ru Ru II

a b

O

[Ru(phen) 2 BrIPC] 2+

N

n

N

l

3

N

q

i

O

p

H N

m k j

Br o

p

q

e d 2

4

H N

r

n

3

d

BrIPC N N

6

s

N

a b

Ru II Ru N

N N

c 1

5 4

f

g

H N N

n

p

q h

m

i j

O

l

e d

2

r

Br

o

O

[Ru(dmb) 2 BrIPC] 2+

3 7

2+

Scheme 1. Structures of three Ru(II) Polypyridyl complexes; [Ru(phen)2BrIPC]

(1), [Ru(bpy)2BrIPC]2+ (2) and [Ru(dmb)2BrIPC]2+.

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collected, washed with water, dried, and purified by recrystallization from pyridine–H2O (9:1, v/v); Yield: 0.310 g (71%), Analytical data: Elemental Analysis for C22H11BrN4O2: Calc. (%): C: 59.83; H: 2.53; N: 12.61; Found: C: 60.01; H: 2.61; N: 12.73; ESI-MS (m/z): Calcd: 442, found: 443 [M+H]+. 1H NMR (DMSO-d6, 400 MHz): d: 9.00 (s, 2Hc), 7.80 (s, 2He), 7.80 (s, Hj), 7.60 (m, Hm, o), 7.40 (t, 2Hd), 6.95 (d, 1Hl´), 13C[1H] NMR (DMSO-d6, 100 MHz): 190.23 (Cq, 1C), 172.75 (Cj, 1C), 158.44 (Ca, k, 2C), 150.08 (Cc, 2C), 151.01 (Cb, m, 2C), 143.01 (Ce, 2C), 133.12 (Cb, o, 2C), 131.33 (Cs,p,f, 3C), 124.00 (Cr,g, 2C) and 118.78 (Cd, 2C), 110.37 (Ci, l, 2C), 100.46 (Cn, 1C).

was used for DNA photocleavage experiments. Buffer ‘C’ (TE – Tris-buffer and EDTA, pH = 7.6) used for pBR322 DNA dilution. The absorption titration was performed in buffer ‘A’, at a fixed complex concentration (20 lM), to which the DNA stock solution was gradually added up to the point of saturation. The mixture was allowed to equilibrate for 5 min before the spectra were recorded. The intrinsic binding constant Kb was calculated by monitoring the changes of absorption at the MLCT band with increasing concentration of DNA using the following equation [36].

2.3.2. Synthesis of [Ru(phen)2(BrIPC)](ClO4)22H2O Cis-[Ru(phen)2Cl2]2H2O (0.284 g, 0.5 mM) and BrIPC (0.221 g, 0.5 mM) was dissolved in a mixture of ethanol (25 mL) and water (15 mL), reflux it with 70 °C for 8 h under N2-atmosphere to give a clear red solution. Upon cooling, the solution was treated with saturated aq. solution of NaClO4 to give brick red ppt. Then it was washed with CH3CN–Toluene (3:1) and vacuum dried. Yield: 0.382 g (67%). Elemental Analysis for C46H31BrCl2N8O12Ru, Calc. C: 61.17; H: 3.07; N: 12.41, Found: C: 61.23; H: 3.25; N: 12.47%). ESI-MS (m/z): 452 [M – (ClO4)22H2O]. 1H NMR (DMSO-d6, 400 MHz): d 9.24–9.36 (d, 6H1, c), 8.78 (d, 6H3, e), 8.41 (s, 4H6), 8.30 (s, Hj), 8.09 (m, 2Hm, o), 8.03 (d, Hl), 7.70–7.79 (t, 6H2, d), 13 1 C[ H] NMR (DMSO-d6, 100 MHz): 173.08 (Cq, 1C), 158.62 (Cj, 1C), 157.89 (Ck, 1C), 147.21 (Cc, 1, 6C), 154.58 (Ca, 1C), 152.60 (Ch, 1C), 136.79 (C5, 4C), 145.72 (Cm, 1C), 130.42 (Ce, 3, 6C), 137.34 (Co, 1C), 128.04 (C4, b, 5C), 126.30 (C6, f, 5C), 127.02 (Cs, p, 2C), 124.91 (Cr, 1C), 121.45 (Cg, 2, 7C), 119.00 (Cl, 1C), 118.7 (Ci, 1C), 114.00 (Cn, 1C).

where [DNA] is the concentration of DNA, ea, ef and eb corresponds to the apparent absorption coefficient Aobs/[complex], the extinction coefficient for the free complex and the extinction coefficient for the complex in the fully bound form, respectively. In plots of [DNA]/(ea  ef) versus [DNA], Kb is given by the ratio of slope to the intercept. It is further extended with competitive DNA binding between EtBr and ruthenium complexes. The emission intensities were recorded with buffer ‘A’. The excitation wavelength was fixed and the emission range was adjusted before measurements. In these emission studies fixed metal complex concentration (20 lM) was taken and to this varying concentration of DNA was added. The fraction of the ligand bound was calculated from the relation Cb = Ct [(F  F0)/Fmax  F0)], where Ct is the total complex concentration, F is the observed fluorescence emission intensity at a given DNA concentration, F0 is the intensity in the absence of DNA and Fmax is when the complex is fully bound to DNA. Binding constant (Kb) was obtained from a modified Scatchard equation [37] and Scatchard plot was drawn with r/Cf vs r, where r is the Cb/[DNA] and Cf is the concentration of free complex. Viscosity experiments were carried out on Ostwald Viscometer, placed in thermostated water-bath maintained at 30 ± 0.1 °C. CT-DNA samples approximately 0.0001 M/lit were prepared by sonication in order to minimize the complexes arising from DNA flexibility [38]. Flow time was measured with a digital stopwatch, and each sample was measured at least three times, taken as average flow time into the calculation. Data was presented as (g/g0)1/3 versus binding ratio of [Ru(II)]/[DNA] [39], where g is the viscosity of DNA in the presence of the complex and g0 is the viscosity of DNA alone. Viscosity values were calculated from the observed flow time of DNA containing solutions (t > 100 s) corrected for the flow time of the buffer alone (t0) [40]. Here buffer BPE (6 mm Na2HPO4, 2 mm Na2HPO4 and 1 mm Na2EDTA, pH = 7) was used through this experiment.

2.3.3. Synthesis of [Ru(bpy)2(BrIPC)](ClO4)22H2O This complex was synthesised with similar procedure of the above complex (1), with cis-[Ru(bpy)2Cl2]2H2O (0.262 g, 0.5 mM) in place of cis-[Ru(phen)2Cl2]2H2O Yield: 0.361 g (66%). Elemental Analysis for C42H33BrCl2N8O12Ru, Calc. C: 58.71; H: 3.38; N: 13.04. Found: C: 58.79; H: 3.43; N: 13.10%.). ESI-MS (m/z): 428 [M – (ClO4)22H2O]. 1H NMR (DMSO-d6, 400 MHz): d 9.36 (s, 2H1), 8.88 (m, 6H6, c), 8.25 (d, 4H4), 8.15 (d, 2He), 8.15 (s, Hj), 8.08 (m, 3Hm, 13 C[1H] o), 7.86 (t, 4H3), 7.78 (s, Hl), 7.64 (d, 4H2), 7.37 (t, 4Hd), NMR (DMSO-d6, 100 MHz): 175.4 (Cq, 1C), 159.16 (Cj, 1C), 157.4 (C5, 4C), 163.3 (Ck, 1C), 151.87 (Cc, 6C), 146.32 (Ca, 1C), 138.5 (Ch, m, 2C), 138.03 (Ce, 2C), 138.30 (C3, 4C), 128.40 (Co, 1C), 128.24 (Cb, 1C), 127.69 (Cf, 1C), 125.49 (Cs, p, 2C), 124.92 (Cr, g, 6C), 121.98 (C2, d, 6C), 119.39 (Ci, l 2C), 115.25 (Cn, 1C). 2.3.4. Synthesis of [Ru(dmb)2(BrIPC)](ClO4)22H2O This complex was synthesised with similar procedure of the above complex (1), with cis-[Ru(dmb)2Cl2]2H2O (0.290 g, 0.5 mM) in place of cis-[Ru(phen)2Cl2]2H2O. Yield: 0.385 g (67%). Elemental Analysis for C46H41BrCl2N8O12Ru, Calc. C: 60.41; H: 4.09; N: 12.30. Found: C: 60.51; H: 4.14; N: 12.37%.). ESI-MS (m/z): 456 [M – (ClO4)22H2O]. 1H NMR (DMSO-d6, 400 MHz): 9.40 (s, 2H1), 8.71 (d, 4H6, c), 8.35 (s, 4H4), 8.10 (d, 2He), 7.97 (s, Hj), 7.85(m, Hm, o), 7.85 7.67 (t, 2Hd), 7.43 (d, 4H2), 7.17 (s, 1Hl), 2.58 (s, 12H7). 13C[1H] NMR (DMSO-d6, 100 MHz): 173.68 (Cq, 1C), 159.20 (Cj, 1C), 156.62 (C5, 4C), 150.88 (C1, 6, C, 6C), 150.03 (C3, a, 5C), 140.31 (Ch, 1C), 138.5 (Ce, m, o, 4C), 127.76 (Cb, 1C), 125.47 (C2, 4, 8C), 123.30 (Cs, f, 2C), 122.07 (Cp, g, r, 3C), 119.47 (Cl, d, 3C), 115.34 (Ci, n, l, 3C), 21.24 (C7, 4C). 2.4. DNA binding studies Buffer ‘A’ (Tris – (hydroxymethyl) amino methane hydrochloride and NaCl, pH = 7.0) was used for absorption titration, luminescence titration and viscosity measurements. Buffer ‘B’ (TAE (1x) – Tris – Buffer, glacial acetic acid and EDTA, pH = 8.0)

½DNA=ðea  ef Þ ¼ ½DNA=ðeb  ef Þ þ 1=K b ðeb  ef Þ

ð1Þ

2.5. Photo activated cleavage studies Super coiled pBR322 DNA was used for the gel electrophoresis experiments, super coiled DNA was treated with Ru(II) complexes in buffer B and the solutions were then irradiated at room temperature with a UV lamp (365 nm, 10 W). These complexes studied with concentration 20 lM to 80 lM. The samples were analyzed by electrophoresis for 1 h at 80 V on a 1.0% agarose gel in buffer B (1), pH 8.2. The gel was stained with 1 lg mL1 ethidium bromide and photographed with the GeNei gel documentation chamber. 2.6. Inhibition of bacterial growth The antimicrobial activity of the complexes was screened against viz. Escherichia coli and Staphylococcus aureus by using positive (Ampicilin) and negative (DMSO) controls respectively. A concentration of 1 mg/mL and 0.5 mg/mL of each Ru(II) complex in DMSO solution was prepared for testing against spore germination of each fungus. Filter paper discs of 5 mm size were prepared using Whatman filter paper no. 1 (sterilized in an autoclave) saturated

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with 10 lL of the Ru(II) complex dissolved in DMSO solution. The fungal culture plates were inoculated and incubated at 25 ± 0.2 °C for 24 h. The plates were then observed and the diameters of the inhibition zones (in mm) were measured and tabulated. The results were also compared with standard antimicrobial drug ampicillin at the same concentration. 2.7. Cytotoxicity assay in vitro To study the cytotoxic effect of the complexes, in vitro MTT assay was carried out [41]. HeLa cells were seeded on flat bottom 96 well plates (Orange Scientific, Belgium) at 3  103 cells/well and incubated in an incubator at 37 °C and 5% CO2. After 24 h, the cells were serum starved overnight. Complexes were then added prepared in DMSO in a concentration range of 3–100 lM, ensuring an equal volume of 200 lL across the wells of the plate. The plate was further incubated at 37 °C and 5% CO2 for 48 h. The cytotoxicity of the complexes was then tested by the addition of the yellow tetrazolium salt MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) prepared in culture medium at a working concentration of 0.4 mg/mL across the plate. The plate was further incubated for 2 h so that the MTT is reduced by the live cells, to produce a purple Formazan product. After this time, the medium was aspirated and 200 lL of DMSO (Sigma Ltd.) was added to each well. The experiment was performed in triplicates. The plate was agitated gently for 5 min before measuring the optical density at 570 nm in each well using Thermo Scientific multi-plate reader (MultiSkan EX Elisa reader). Since the absorbance correlated with the number of viable cells the percentage of viability was calculated from the absorbance. The IC50 values of the complexes were determined by plotting the percentage viability versus concentration on a logarithmic graph and the reading of the concentration at which 50% of cells are viable relative to the control. 2.8. Cellular uptake study HeLa cells (1  106 cells/mL) in growth medium were treated with 50 lM Ru(II) complexes(final DMSO concentration, 13). Electrophoresis was carried out in the alkaline solution for 30 min at 0.6 V/cm and approximately 42 mA at 4 °C. The slides were washed in chilled distilled water for 10 min to neutralize the excess alkali. The gels were air-dried for 30 min and were stained with 200 lL of PI (Propidium Iodide, 10 lg/mL) for another 20 min. The gels were de-stained with chilled distilled water, airdried and scored for comets by confocal microscopic analysis. A total of 20 comets on each gel were scored and the average tail length measured with Leica confocal microscopy software [42]. 2.10. Molecular docking studies Docking was carried out using GOLD (Genetic Optimization of Ligand Docking) software which is based on genetic algorithm (GA). This method allows as partial flexibility of protein or DNA and full flexibility of ligand. The complexes are docked to the active site of the (PDB: 2194) DNA. The interactions of these complexes with the active site are thoroughly studied using molecular mechanics calculations. During docking, the default algorithm speed was selected and the ligand binding site was defined within a 10 Å radius. After docking, the individual binding poses of each complex was observed and its interactions with the DNA was studied. 2.10.1. Gold score fitness function Gold Score performs a force field based scoring function and is made up of four components: 1. DNA-ligand hydrogen bond energy (external H-bond); 2. DNA-ligand van der Waals energy (external vdw); 3. Ligand internal van der Waals energy (internal vdw); 4. Ligand intramolecular hydrogen bond energy (internal- H-bond). The external vdw score is multiplied by a factor of 1.375 when the total fitness score is computed. This is an empirical correction to encourage DNA-ligand hydrophobic contact. The fitness function has been optimized for the prediction of ligand binding positions.

Gold Score ¼ Sðhb extÞ þ Sðvdw extÞ þ Sðhb intÞ þ Sðvdw intÞ where S (hb_ext) is the protein–ligand hydrogen bond score, S (vdw_ext) is the DNA–ligand van der Waals score, S (hb_int) is the score from an intramolecular hydrogen bond in the ligand and S (vdw_int) is the score from the intramolecular strain in the ligand. 3. Result and discussion 3.1. Synthesis and characterization Three synthesised complexes were shown in Scheme 1. 3.1.1. From absorption spectroscopy Three complexes showed MLCT peak at 460 nm, whereas ligand(BrIPC) was not with MLCT peak as shown in Fig. 1. It reveals that the formation of octahedral complexes with d6 configuration can only show a single MLCT peak from the electron transition between t2g ? A1g. 3.1.2. From IR spectra m(C@O) vibration of the ligand is at 1651; after complex formation this peak shifted to 1646 cm1. Weak bands at 623 cm1 are assigned to m(RuAN), which is not appeared in case of BrIPC.

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2

Hypochromism

(Black) Only Complex (Red) Complex + DNA

1

Absorbance

2.0

1 2 3 L

Bathochromism

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Absorption

2.5

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0.5 0 0.0 250

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Wave Length (nm)

250

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550

Wave Length (nm) Fig. 1. From above absorption spectra, three complexes 1, 2 and 3 are showing MLCT band around 455 nm, but ligand (L = ) does not show MLCT band.

Fig. 3. Absorption spectra of [Ru(phen)2(BrIPC)] complex in zoom, solid line (black) shows only with complex and dotted line (red) shows bathochromic and hypochromic shift after addition of CT-DNA. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

This indicates all six RuAN bonds with same bond length shows perfect octahedral structure. 3.1.3. 1H and 13C[H1] NMR Hc of BrIPC gives peak at d 9.00 ppm, after complex formation, same peak goes to down shift around d 8.80 ppm. 13C data has given some characteristic peaks: carbonyl carbon at d 170 ppm, adjacent to Oxygen atom appeared around d 156 ppm and at Br attached carbon appeared around d 117 ppm.

[Ru(phen)2BrIPC]2+ and [Ru(bpy)2BrIPC]2+ shown in Fig. 2, consist of three well resolved bands, the bands between 200 and 360 nm are attributed to intraligand p ? p* transitions [43]. The lowest energy band, around 460 nm is assigned to the metal–ligand charge transfer (dRu ? p*BrIPC). As the CT-DNA concentration is increased, the MLCT transition bands of complexes 1, 2 and 3 exhibit hypochromism of 11%, 9% and 7.5% and bathochromism of 23, 17 and 15 nm, respectively along with isosbestic points. As shown in Fig. 2, all three complexes showed isosbestic point. It indicates that there is equilibrium between bound DNA and the free form of the complex and further suggested only one mode of binding i.e., intercalation of compounds with DNA [44]. Hypochromism and bathochromism as shown in Fig. 3, due to binding between aromatic

3.2. DNA binding studies 3.2.1. Absorption spectroscopic studies of DNA–complex interactions Electronic absorption spectroscopy is one of the most powerful experimental techniques for probing metal complex–DNA interactions. Binding of the macromolecule leads to changes in the electronic spectrum of the metal complex. The absorption spectra of

1.5 1.0

14 -10

14

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-06

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-5

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(3)

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Isosbesticpoint 300

12

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o

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[DNA] / [Ea -Ef ] X 10

Absorbance

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16

16

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[DNA] / [Ea-E f ] X 10-10

(1) 2.5

1.0

-5

0.30 0.25 0.20 0.15 400

0.5

450

500

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0.0 250

300

350

400

450

500

Wave Length (nm) Fig. 2. The absorption spectrum of complexes [Ru(phen)2(BrIPC)]2+ (1), [Ru(bpy)2(BrIPC)]2+ (2) and [Ru(dmb)2BrIPC]2+ (3) in Tris–HCl buffer upon addition of CT-DNA. Arrow shows hypochromic and bathochromic shifts upon increase of DNA concentration. Inserted plot, [DNA]/(eb  ef) versus []DNA] for the titration of DNA with Ru(II) complex, which gives intrinsic binding constant (Kb). [DNA] = 2.5  104 M, [Complex] = 10 lM.

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Table 1 Intrinsic binding constant values (Ksv) and Ksv from absorption and fluorescence spectroscopy. Complex

Kb for Abs studies

Kb for emission studies

Ksv

[Ru(phen)2BrIPC]2+ [Ru(bpy)2 BrIPC]2+ [Ru(dmb)2 BrIPC]2+

2.26 (±0.43)  105 M1 1.87 (±0.51)  105 M1 1.19 (±0.063)  105 M1

5.10 (±0.36)  105 3.52 (±0.24)  105 2.00 (±0.57)  105

148 307 518

1.11 1.09

I0 / I

1.07 1.05 1.03

chromophore and the base pairs of DNA [45]. The intrinsic binding constants (Kb) of the complexes were in the order of 105 as shown in Table 1. Binding constant was stronger than [Ru(Phen)2(MIPC)]2+ [45], because of the presence of electron withdrawing substituent (Br in BrIPC) on the intercalative ligand which increase the DNAbinding affinity, and the electron-releasing substituent (–CH3 in MIPC) reduce the DNA affinity so electron deficient rings interact more strongly with polyanion (DNA) than electron rich rings [46]. The order of Kb values of three complexes 1 > 2 > 3. These results may be explained by the fact that, for the ancillary ligand, on going from bpy to phen, the plane area and hydrophobicity increase, leading to a greater binding affinity to DNA [47]. Moreover, complex 3 shows the less binding strength to double-helical DNA than remaining two complexes (1&2). Due to the presence of methyl groups at the 4 and 40 positions of dmb (ancillary ligand) which causes stearic hindrance and complex moiety becomes electron rich which causes decrease in the binding affinity between the CT-DNA base pairs [48]. 3.2.2. Luminescence studies of DNA-Complex interactions Emission intensity of complexes 1, 2 and 3 from their MLCT excited states upon emission around 600 nm is found to depend on the DNA concentration. These three Ru(II) complexes in the absence of DNA emit luminescence in buffer ‘A’, upon addition of

0.99

0

0.00005 0.0001 0.00015 0.0002 0.00025 0.0003 0.00035 0.0004

[Fe(CN)6] 4Fig. 5. Emission quenching of [Ru(phen)2BrIPC]2+ (1), with [Fe(CN)6]4 quencher in absence ( = absence of DNA) and presence of DNA ( = 1:30 and = 1:200).

CT-DNA (Calf thymus DNA), emission intensities of complexes [Ru(phen)2BrIPC]2+ (1), [Ru(bpy)2BrIPC]2+ (2) and [Ru(dmb)2BrIPC]2+ (3) increased as shown in Fig. 4. This implies that complexes can strongly interact with DNA and protected by DNA efficiently, from the hydrophobic environment inside the DNA helix reduces the accessibility of solvent water molecules to the duplex and the complexes mobility is restricted at the binding site, which leads to decrease in the vibrational modes of relaxation. Scatchard plots for complexes have been constructed from luminescence spectra and binding constants (Kb) were in the order of 105 as shown in Table 1. The binding constants obtained from luminescence titration with McGhee–von Hippel method is different from those obtained from absorption with the method suggested by Wolf et al. [38]. This small difference between the two sets of binding

6

+ DNA4x10 r / Cf

2200 2000

+ DNA

(2) 3500

6

3x10

6

2x10

3000

6

1x10

1800

Relative Intensity

0 0.480.510.540.570.600.630.66

1600

r

1400 1200 1000 800

2500 2000 5

1500 r / Cf

(1) 2400

Relative Intensity

1.01

1000

600 500

400 200

8x10 5 7x10 5 6x10 5 5x10 5 4x10 5 3x10 5 2x10

0 560

580

600

620

640

660

560

580

600

620

640

660

Wave length (nm)

Wave Length (nm)

1000

r / Cf

(3) 1200 Relative Intensity

0.500.520.540.560.580.600.620 r

800

2.1x10 1.8x10 1.5x10 1.2x10 9.0x10 6.0x10 3.0x10

B

6 6

+ DNA

6 6 5 5 5

0.48 0.52 0.56 0.60 0.64 r

600 400 200 0 560

580

600

620

640

Wave length (nm) Fig. 4. Luminescence of complexes [Ru(phen)2BrIPC]2+ (1), [Ru(bpy)2BrIPC]2+ (2) [Ru(dmb)2BrIPC]2+ (3) in Tris–HCl buffer upon addition of CT-DNA. Arrow shows the intensity change upon the increase of DNA concentration. Inset: Scatchard plot of above complex, which gives binding constant (Kb). [DNA] = 2.5  104 M, [Complex] = 10 lM.

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A. Srishailam et al. / Journal of Photochemistry and Photobiology B: Biology 141 (2014) 47–58

600

500

Relative Intensity

constants is due to different spectroscopy techniques and different calculation method, however they are comparable.

Complex + DNA 2+

Complex + DNA + Co + EDTA

400

2+

Complex + DNA + Co

300

Only Complex

200

100 540

560

580

600

620

640

660

Wave Length Fig. 6. Luminescence modulation routes of [Ru(phen)2BrIPC]2+ in the absence and presence of DNA by Co2+ and EDTA respectively.

1.6

EtBr Complex 1 Complex 2

1.5

Complex 3 (η /η0)1/3

1.4

1.3

1.2

1.1

1 0.015

0.02

0.025

0.03

0.035

0.04

0.045

0.05

0.055

[Complexe] / [DNA] Fig. 7. Effect of increasing amounts of EtBr( ), complexes 1( ), 2( ) and 3( ) on the relative viscosity of CT-DNA at 25 (±0.1) °C. [DNA] = 2.5  104 M.

1

2 Form II Form I

20

40

60

20

40

60 µM

3

3.2.3. Effect of anion quencher effect on luminescence studies Quencher [Fe(CN)6]4, was further supported for emission studies in the presence and absence of DNA are shown in Fig. 5, on [Ru(phen)2BrIPC]2+ complex. The complex binding to DNA can be protected from the quencher, because highly negatively charged [Fe(CN)6]4 would be repelled by the negative DNA phosphate backbone, hindering quenching of the emission of the bound complex. As illustrated in the presence of DNA complexes were efficiently quenched by [Fe (CN) 6]4resulting in linear Stern–Volmer plots. The Stern–Volmer quenching constant Ksv can be determined by using Stern–Volmer equation [40].

I0 =I ¼ 1 þ K sv ½Q 

ð2Þ

where I0 and I are the fluorescence intensities in the absence and presence of a quencher respectively, Q is the concentration of the quencher, Ksv is a linear Stern-Volmer quenching constant and values are shown in Table 1. Further interesting investigation is on photoluminescence of DNA – [Ru(phen)2BrIPC]2+ in the presence and absence of Co2+. From Fig. 6, it could be seen that complex bound to DNA (switch on), the emission intensity was quenched by Co(II) ions, it turns the light switch off [49,50]. With the addition of Co2+ to DNA – [Ru(phen)2BrIPC]2+ emission intensity decreased, due to formation of Co2+– [Ru(Phen)2BrIPC]2+ heterometallic complex [51]. However, the emission can be recovered in the presence of EDTA, thus turning the light switch on. This is because Co2+ was removed by EDTA, and Co2+ – [Ru(Phen)2BrIPC]2+ heterometallic complex cannot be formed. The present results should be of value in further developing luminescence DNA probe. 3.2.4. Determination of mode of binding Relative viscosity measurements have proved to be a reliable method for the assignment of the mode of binding of complexes to DNA. It is well-known that a classical intercalation of a ligand into DNA is known to cause a significant increase in the viscosity of a DNA solution due to an increase in the separation of the base pairs at the intercalative site and, hence, an increase in the overall DNA molecular length [52]. The change in the relative viscosity of CT-DNA in addition of complexes 1, 2 and 3 is shown in Fig. 7. With increasing the amounts of complexes 1, 2 and 3, the relative viscosity of the CT DNA solution increases steadily. Viscosity depends on its affinity to DNA, which followed the order of 1 > 2 > 3. The results suggested that these three Ru(II) complexes intercalate between the base pairs of DNA, which is consistent with our interpretation based on binding constants.

Form II

3.3. Photo activated cleavage studies

Form I 20

40

60 µM

Fig. 8. Photo activated cleavage studies of [Ru(phen)2BrIPC]2+ (1), [Ru(bpy)2BrIPC]2+ (2), [Ru(dmb)2BrIPC]2+ (3) with the concentration range of 20–60 lM.

The cleavage reactions of plasmid pBR322 DNA induced by these ruthenium (II) complexes were monitored by agarose gel electrophoresis. Gel electrophoresis separation of pBR322 DNA was studied after incubation with complexes and irradiation at 365 nm for half an hour. When electrophoresis is applied to

Table 2 Antibacterial activity of Ru(II) complexes. Complex

DMSO [Ru(phen)2BrIPC]2+ [Ru(bpy)2BrIPC]2+ [Ru(dmb)2BrIPC]2+ Ampicilin

Bacterial inhibition zone (mm) conc. (1000 lM)

Bacterial inhibition zone (mm) conc. (500 lM)

S. aureus

E. Coli

S. aureus

E. Coli

– 9.8 9.3 8.7 13.7

–

– 6.1 5.8 5.3 7.8

– 5.7 5.5 5.1 7.7

9.5 9.0 8.6 13.2

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A. Srishailam et al. / Journal of Photochemistry and Photobiology B: Biology 141 (2014) 47–58

Cytotoxicity results with HeLa Cell lines

Cell Viability %

100

Cisplan Complex 2

Complex 1 Complex 3

80 60 40 20 0

erable activity against Staphylococcus aureus and Escherichia coli at 1000 lM and 500 lM concentrations. DMSO control showed a negligible activity and ampicillin shows more activity as compared with three metal complexes. [Ru(phen)2BrIPC]2+ showed slightly higher activity than other two complexes 2 and 3. The antimicrobial activity increases as the concentration of the complexes increased. 3.5. Cytotoxicity in vitro assay

0

3.12

6.25

12.5

25

50

100

Concentraon (μM) Fig. 9. Cell viability of HeLa cell lines in vitro treatment with complexes 1, 2 and 3. Each data point is the mean ± standard error obtained from at least three independent experiments. Table 3 IC50 values of three Ru(II) complexes. Complex name

IC50 values

Cis Platin [Ru(phen)2BrIPC]2+ (1) [Ru(bpy)2BrIPC]2+ (2) [Ru(dmb)2BrIPC]2+ (3)

7.21 18.15 29.06 36.30

circular plasmid DNA, the fastest migration will be observed for DNA of closed circular conformation (Form I). If one strand is cleaved, the supercoil will relax to produce a slower moving nicked conformation (Form II). If both strands are cleaved, a linear conformation (Form III) will be generated that migrates in between, As shown in Fig. 8, no obvious cleavage was observed for the control in which metal complex was absent (DNA alone), or incubation of the plasmid with the Ru(II) complexes in the dark. With increasing concentration of complexes, the Form I decrease and Form II increase gradually. At the concentration range of 20–60 lM, three complexes can completely cleave the plasmid DNA in the sequence of concentration changes. 3.4. Antimicrobial studies The antibacterial activities of these complexes have shown the effect of concentration on S. aureus and E. coli with ampicillin as a positive control and DMSO as a negative control. From Table 2, inhibition zone data indicate that three complexes showed consid-

We have investigated the antitumor potential of the Ru(II) complexes using the 3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine the cytotoxicity of the three Ru(II) complexes against human cervical cancer cells (HeLa). Fig. 9 showed the changes in cell viability upon varying the complexes concentration from 3.125 to 100 lM after 48 h treatment. The cell viability was concentration-dependent, and increasing the concentrations of 1, 2 and 3 caused a decrease in cell viability. Comparing the IC50 values (Table 2), complexes with low cytotoxicity than cisplatin, complex 1 showed higher activity than 2 and 3 against selected tumor cell lines, this result correlated with DNA binding results. These results suggest that the complexes may have antitumor activity (see Table 3). From the viability percentage of HeLa cell lines, cytotoxicity activity was following the order of DNA binding studies. From spectroscopic and viscosity studies, we confirmed complexes bind between DNA base pairs through intercalative mode, as such, these complexes can bind to HeLa cell DNA and cause to distruction of cell. So, cytotoxicity activity order of complexes follows DNA binding activity order. 3.6. Cellular uptake studies Further investigations of the complexes were conducted based on the previously described results. Upon excitation, complexes were investigated via flow cytometry, the luminescence intensity of the cell population dramatically increased compared with the autofluorescence of untreated HeLa cells. The Median-red fluorescence intensity (MFI) values were measured in complexes 1, 2, 3 and untreated cells (control), the values are 53.0, 49.9, 23.7 and 2.39 respectively as shown in the Fig. 10. The intrinsic emission of Ru(II) complexes can be used in the design of Ru(II) complex cell imaging probes that detect the presence of DNA binding via multiple emission peaks [53,54]. Although some Ru(II) complexes can

Fig. 10. Cellular uptake analysis with red fluorescence by flow cytometry. Representative histograms: indicates cultures treated with untreated cells (control, red), 50 lM of [Ru(phen)2BrIPC]2+ (D), [Ru(bpy)2(BrIPC]2+ (E) and [Ru(dmb)2(BrIPC)]2+ (F) complexes (for 4 h). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

A. Srishailam et al. / Journal of Photochemistry and Photobiology B: Biology 141 (2014) 47–58

identify cancer cell membrane receptors and can readily accumulate in the cytoplasm of live cells, most are excluded from the nucleus and are mainly localized in the cytoplasm [55,56]. However, a certain amount of Ru(II) complexes can be efficiently transported across the plasma membrane and then accumulate in the nucleus [57,58]. Nuclear accumulation is highly desirable in anticancer agents that target genomic DNA [59]. The intracellular behavior of complexes 1, 2 and 3 were observable via confocal microscopy. The confocal microscopic images (Fig. 11) show that the complexes entered and accumulated inside the cells in the region around the nucleus, subsequently forming very sharp luminescent rings around the nucleus as indicated positions 1 (a), 2 (b) and 3 (c) shown in Fig. 11.

1

55

3.7. Comet assay The ability of Ru(II) complexes to induce apoptosis was evaluated in HeLa cell line using the comet assay. The HeLa cells were incubated with 50 lM of three Ru(II) complexes for 6 h, followed by electrophoresis and staining with PI (Propidium Iodide). The cells were scanned for the comets by Leica confocal microscopy and the average tail lengths under treatments with 1–3 complexes were recorded (Fig 12). Comet assay on HeLa cells confirms the DNA damaging effect of the complexes in the order 1 > 2 > 3 with comet tails measuring approx. 56 lm, 30 lm and 24 lm for complexes 1, 2 and 3 respectively after 6 h treatment.

a

2 b

3 c

Fig. 11. The emission imaging of the three complexes entry transportation into living HeLa cell taken by confocal microscope. Confocal images of HeLa cells stained with three complexes, [Ru(phen)2BrIPC]2+ (1), [Ru(bpy)2BrIPC]2+ (2), and [Ru(dmb)2BrIPC]2+ (3). Arrows were showing clearly that blebbing of the nuclei at 1(a), 2(b) and 3(c).

Fig. 12. Effect of 50 lM of complex [Ru(phen)2BrIPC]2+ (1), [Ru(bpy)2BrIPC]2+ (2) and [Ru(dmb)2BrIPC]2+ (3) on DNA integrity of HeLa cells treated for 6 h measured using the comet assay.

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A. Srishailam et al. / Journal of Photochemistry and Photobiology B: Biology 141 (2014) 47–58

Complex 1

Complex 3

Fig. 13. Molecular docking studies of [Ru(phen)2BrIPC]2+ (1) and [Ru(dmb)2BrIPC]2+ (3) with Gold docking software.

Table 4 The H-bond Vander Waals interactions and scores for binding of Ru(II) complexes to (PDB: 2194) DNA containing CG bases using docking calculations. Complex

H-bond donor–acceptor

Bond length (Å)

Vander Waals interactions (complex-DNA)

Bond length (Å)

Gold score fitness

Complex-1

H81-DC5:N4 O56-DA3:H62 O56-DA4:H62 N43-DA4:H62

2.561 2.620 2.659 2.548

C28-DA4:N7 H71-DA4:H8 C26-DT4:OP1

2.723 1.850 2.669

63.0201

Complex-2

H77-DC5:N4 O52-DA3:H62 O52-DA4:H62 N39-DA4:H62 N39-DC5:H41

2.399 2.321 2.189 2.340 2.219

C26-DT4:N7 C25-DT4:OP1 H77-DC5:H41 C27-DC2:OP1

2.642 2.494 1.832 2.374

58.7699

Complex-3

H77-DC5:N4 N39-DA4:H62 O52-DA3:H62 C24-DT4:OP1 C36-DT4:OP1 H69-DT3:H3 C55-DC5:OP1

2.676 2.573 2.507 2.652 2.563 1.843 2.620

H65-DC5:H41 C11-DC5:C5 H64-DC5:H5

1.776 2.886 1.048

53.5788

3.8. Molecular modelling studies

4. Conclusion

DNA–ligand interactions were identified by using a Gold docking protocol. Using GOLD, docking studies complexes were performed to investigate the poses for our scaffold in the (PDB: 2194) DNA active site [45]. To understand the nature of the interactions between the Ru(II) complexes and the (PDB: 2194) DNA, molecular docking and molecular dynamics (MD) simulation experiments were carried out. The docking result (Fig. 13) showed Ru(II) complexes shared quite similar binding sites, that they bound to DNA grooves with the O52, H77 and N39 of planar aromatic ring and spanning over adenine and cytosine. The hydrogen bonding interactions involving the energy-minimized docked poses of d(PDB: 2194) with complexes are shown in Table 4. The resulting binding energy of docked metal complexes 1, 2 and 3 were found to be 63.02, 58.76 and 53.57 kcal/mol, respectively, correlating well with the experimental DNA binding values. Thus, molecular docking study together with spectroscopic studies indicated that Ru(II) complexes 1, 2 and 3 interacts with the DNA through both noncovalent (intercalation) and H – bonding and van der Waals interactions which perhaps owe to its stronger bonding with DNA.

In this report, we synthesised three chromone based Ru(II) complexes and characterized. From spectroscopic titration, complexes binds to DNA (CT DNA) effectively in the order 1 > 2 > 3 through intercalative mode which was revealed by viscosity experiments. Molecular docking studies reveal that DNA interaction of these complexes was stabilized by hydrogen bonding between the oxygen of chromone moiety and nitrogen of DNA base pairs. Upon irradiation, these complexes can effectively cleave pBR322 DNA. These complexes showed the ability to inhibit the bacterial growth at given conditions. The hydrophobicity, cellular uptake efficiency and cytotoxic effect on cancer cells of 1–3 are correlated. The most lipophilicity and planarity of complex 1, with the ancillary ligand phen, strongly bind between DNA base pairs through p–p stacking. So complex 1, shown higher cytotoxicity and DNA binding constant (Kb) than complexes containing bpy (2) and dmb (3) and less than cisplatin, which follows DNA binding order [47,60]. Cellular uptake, accumulation into the nuclei of HeLa cell lines by Ru(II) complexes, help to promote and design novel drugs for chemotherapy.

A. Srishailam et al. / Journal of Photochemistry and Photobiology B: Biology 141 (2014) 47–58

5. Abbreviations CTCalf DAPI PI 7-AAD DMSO BrIPC dppz ESIMS IC50 MLCT MTT PBS Tris MIPC EWG ERG

DNA thymus DNA 40 ,6-diamidino-2-phenylindole dihydrochloride propidium iodide 7-aminoactinomycin D dimethyl sulfoxide 6-bromo-3-(1H-imidazo [4,5-f][1,10]phenanthroline-2-yl)-4H-chromen-4-one dipyridophenazine electrospray ionization mass spectrometry half-maximal inhibitory concentration metal-to-ligand charge transfer 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide phosphate-buffered saline tris(hydroxymethyl)aminomethane 6-methyl-3-(1H-imidazo [4,5-f] [1,10]phenanthroline-2-yl)-4H-chrome-4-one electron with drawing group electron releasing group

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