Novel mononuclear zinc complexes with 2,2′-dimethyl-4,4′-bithiazole: Synthesis, crystal structure and DNA-binding studies

Polyhedron 42 (2012) 153–160

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Novel mononuclear zinc complexes with 2,20 -dimethyl-4,40 -bithiazole: Synthesis, crystal structure and DNA-binding studies Zohreh Mehri Lighvan a, Anita Abedi b,⇑, Maryam Bordar c a

Young Researcher Club, North Tehran Branch, Islamic Azad University, Tehran, Iran Department of Chemistry, North Tehran Branch, Islamic Azad University, P.O. Box 19585-936, Tehran, Iran c Department of Chemistry, Faculty of Science, The University of Qom, P.O. Box 37185-359, Qom, Iran b

a r t i c l e

i n f o

Article history: Received 17 January 2012 Accepted 8 May 2012 Available online 18 May 2012 Keywords: Zinc complexes Crystal structure DNA interaction Fluorimetry CD spectroscopy

a b s t r a c t Two neutral mononuclear zinc complexes with 2,20 -dimethyl-4,40 -bithiazole (dm4bt), [Zn(dm4bt)Br2] (1) and [Zn(dm4bt)I2] (2) were synthesized and characterized by elemental analysis, IR, UV–Vis and NMR spectroscopy and their structures were studied by single-crystal diffraction. These complexes have a bidentate nitrogenous ligand with two halide anions attached to a zinc metal in a distorted tetrahedral geometry. The interaction ability of the two complexes with native calf thymus DNA (CT-DNA) has been monitored as a function of the metal complex–DNA molar ratio by UV–Vis absorption spectrophotometry, fluorescence spectroscopy, circular dichroism (CD) and thermal denaturation studies. The intrinsic binding constants Kb of complexes 1 and 2, with CT-DNA obtained from UV–Vis absorption studies were 3.47 ± 0.02  104 M1 and 3.19 ± 0.02  104 M1, respectively. Both complexes exhibit luminescent properties in the absence and presence of CT-DNA and the fluorescence study ascertain the interaction of 1 and 2 with CT-DNA. Moreover the addition of the complexes to CT-DNA (1:2) led to an increase of the melting temperature of DNA up to around 2.7 °C. Further fluorimetric studies were performed using methylene blue (MB) as a fluorescence probe, indicating low intercalative interaction of the complexes with CT-DNA. The CD study also points to groove binding mode in the complexes rather than intercalation mode. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction There has been substantial interest in metal-based small molecules because of their relevance in the development of new reagents for biotechnology and medicine [1–3]. The metal complexes containing multidentate aromatic ligands are very important due to their capacity for binding and cleaving DNA under physiological conditions [4,5]. These efforts stem from the development of novel chemotherapeutics and highly sensitive diagnostic agents [6]. Metal complexes are known to bind to DNA in either a non-covalent or a covalent fashion. In covalent binding, the labile ligands of the complexes are replaced by a nitrogen base of DNA such as guanine N7 [7]. Non-covalent DNA interactions include three binding modes: intercalation, groove (surface) binding and external static electronic effects, along the outside of the DNA helix [8]. Zinc is one of the most abundant trace elements present in biological systems as well as biological processes and several metallo-proteins contain this element [9–11]. Zn(II) plays a central key in zinc fingers, it presents unique abilities to facilitate rewinding of melted DNA, it induces hydrolysis of DNA and RNA and it can ⇑ Corresponding author. Tel.: +98 21 22262563; fax: +98 21 22222512. E-mail address: [email protected] (A. Abedi). 0277-5387/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.poly.2012.05.006

promote B–Z transitions of DNA, suggest binding ability to DNA from the nucleo-bases and the phosphate groups [12] where the theoretical studies demanded the stabilization energy of zinc with base-N to be further than other bioactive metals such as copper and nickel [13]. Therefore the biological activity, especially DNA interaction ability of zinc complexes have been the subject of a large number of studies [14–16]. On the other hand, bithiazole is a moiety of bleomycins (BLMs), a natural antibiotic, used clinically in combination chemotherapy in the treatment of germ cell tumors, lymphomas, Kaposi’s sarcoma, cervical cancers, and squamous cell carcinomas of the head and neck [17–19] where the bithiazole tail of bleomycin plays an important role in the interaction of bleomycin with DNA [20,21]. The bithiazole domain is responsible for multiple modes of DNA binding including partial intercalation and binding within the minor groove [22]. As part of our continuing research on metal complexes with bithiazole [23–25], in the present work, our experimental approach to the design of the proposed anticancer zinc complexes with bithiazole derivatives, 2,20 -dimethyl-4,40 -bithiazole, involves three stages: (i) synthesis and identifying of the complexes by means of elemental analysis, IR, UV–Vis, NMR and X-ray diffraction methods, (ii) improving their ability to interact with DNA using UV–Vis and

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fluorescence spectroscopy, and (iii) the investigation of their interaction mechanism with DNA by thermal denaturation, fluorescence study of MB–DNA and circular dichroism (CD) spectra. Reports on zinc complexes with bithiazole are limited [23,26–29] and to our knowledge, there is no report on the biological investigation of zinc-bithiazole complexes. 2. Experimental 2.1. Materials The reagents and chemicals were purchased from commercial sources and used as received without further purification. The ligand 2,20 -dimethyl-4,40 -bithiazole (dm4bt) was prepared according to our previous report [23]. Calf thymus DNA (CT-DNA) was obtained from Sigma. The stock solution of CT-DNA gave a ratio 1.8–1.9 in UV absorbance at 260 and 280 nm (A260/A280) to check DNA purity, indicating that the DNA was sufficiently free of protein contamination [30]. The DNA concentration per nucleotide was determined by absorption spectroscopy using the molar absorption coefficient (e = 6600 M1 cm1 at 260 nm) [31,32]. The stock solutions were stored at 5 °C and were consumed within 4 days. All the experiments involving interactions of the compounds with DNA were carried out in doubly distilled water buffer containing 5 mM Tris–HCl [Tris(hydroxymethyl)-aminomethane] and 50 mM NaCl, and adjusted to pH 7.4 with hydrochloric acid. 2.2. Physical measurements The UV–Vis spectra were recorded on a Varian Cary 100 UV–Vis spectrophotometer using a 1 cm path length cell. The fluorescence spectra were recorded on a Varian Cary eclipse spectrofluorometer. Infrared spectra (4000–250 cm1) of solid samples were taken as 1% dispersions in KBr pellets using a Shimadzu-470 spectrometer. 1 H NMR spectra were acquired on a Bruker AC-300 MHz spectrometer at ambient temperature in DMSO-d6. The melting points are uncorrected and were obtained by a Kofler Heizbank Rechart type 7841 melting point apparatus. Elemental analyses were performed using a Heraeus CHN–O Rapid analyzer. The Tm spectra were recorded on a Varian BioCary-100 UV–Vis spectrophotometer using a 1 cm path length cell. Circular dichroism measurements were carried out on a Jasco-810 spectropolarimeter at room temperature with a rectangular quartz cell of 1 cm path.

in acetonitrile (30 ml). The resulting solution was stirred at 50 °C for 2 h, and was then filtered and left at room temperature. After a week, it began to produce colorless prismatic crystals of 2 (yield 0.094 g, 72%). 1H NMR dH (DMSO-d6): 2.68 (s, 3H, Me) and 7.75 (s, 1H, Ar). 13C NMR dC (DMSO-d6): 19.3 (Me), 115.6, 149.8 and 166.8 (Ar). IR (KBr, cm1): 3080 (mC–H, Ar), 2907 (mC–H, Me), (mC@C), 2825, 1563 (mC@C), 1528 (mC@N), 1431 (mC–C), 1374, 1287, 1210 (mC–N), 1155, 977, 770 (mS–C), 637, 586 (mZn–N) and 259 (mZn–I). UV–Vis (DMF) kmax: 232 nm. Anal. Calc.: C, 18.64; H, 1.55; N, 5.43. Found: C, 18.51; H, 1.53; N, 5.39%. 2.5. Crystal structure determination and refinement The X-ray diffraction measurements were made on a Bruker APEX II CCD area detector diffractometer (Mo Ka radiation, graphite monochromator, k = 0.71073 Å). For [Zn(dm4bt)Br2] (1) and [Zn(dm4bt)I2] (2), colorless prismatic crystals with dimensions of 0.35  0.35  0.30 mm3, and 0.21  0.14  0.08 mm3, were respectively chosen and mounted on a glass fiber and used for data collection. Cell constants and an orientation matrix for data collection were obtained by least square refinements of diffraction data from 3425 unique reflections for 1 and 3144 for 2. Data were collected at a temperature of 173(2) °C [100(2) K] to a maximum 2h value of 57.98° for 1 and 54.00° for 2, in a series of x scans in 1° oscillations. Semi-empirical absorption corrections were carried out using the program SADABS [33]. The structures were solved by direct methods using the program SHELXS-97 [34]. The refinement and all further calculations were carried out using SHELXL-97 [34]. The C-bound H-atoms were included in calculated positions and treated as riding atoms using SHELXL-97 default parameters. The non-H atoms were refined anisotropically, using weighted fullmatrix least-squares on F2. Software packages APEX2 (data collection), SAINT (cell refinement and data reduction) and SHELXTL (molecular graphics and publication material) were also used [34,35]. A summary of the crystal data, experimental details and refinement results has been given in Table 1.

Table 1 Crystallographic and structure refinement data for compounds 1 and 2.

Formula Formula weight T (K) k (Å) Crystal system Space group Crystal size (mm) a (Å) b (Å) c (Å) b (°) V (Å3) Z Dcalc (g cm3) h range for data collection F(0 0 0) Absorption coefficient (mm) Index ranges

2.3. Synthesis of [Zn(dm4bt)Br2] (1) ZnBr2 (0.080 g, 0.355 mmol) was dissolved in water (10 ml), mixed with methanol (30 ml) and reacted with 2,20 -dimethyl4,40 -bithiazole (0.070 g, 0.356 mmol) [23] dissolved in acetonitrile (30 ml). The resulting mixture was stirred at 50 °C for 2 h and then filtered and left at room temperature. After a week, it began to produce pale yellow prismatic crystals of 1 (yield 0.102 g, 68%). 1H NMR dH (DMSO-d6): 2.69 (s, 3H, Me) and 7.75 (s, 1H, Ar). 13C NMR dC (DMSO-d6): 19.3 (Me), 115.6, 149.8 and 166.7 (Ar). IR (KBr, cm1): 3149, 3090 (mC–H, Ar), 2983 (mC–H, Me), 2914, 1559 (mC@C), 1527(mC@N), 1436 (mC–C), 1212 (mC–N), 1148, 977, 795 (mS–C), 639, 575 (mZn–N) and 251 (mZn–Br). UV–Vis (DMF) kmax: 252 nm. Anal. Calc.: C, 22.80; H, 1.90; N, 6.64. Found: C, 22.63; H, 1.88; N, 6.59%.

Data collected Unique data (Rint) Parameters, restraints Final R1, wR2a (observed data) Final R1, wR2a (all data) Goodness-of-fit on F2 (S) Largest difference in peak and hole (e Å3)

2.4. Synthesis of [Zn(dm4bt)I2] (2) Complex 2 was prepared according to the procedure described for complex 1. ZnI2 (0.080 g, 0.250 mmol) was dissolved in water (10 ml), mixed with methanol (30 ml) and reacted with 2,20 -dimethyl-4,40 -bithiazole (0.050 g, 0.255 mmol) [23] dissolved

a

1

2

C8H8Br2N2S2Zn 421.47 100(2) 0.71073 monoclinic P21/n 0.35  0.35  0.30 8.8711(8) 12.4900(12) 12.1499(12) 106.236(2) 1292.5(2) 4 2.166 2.39–28.99 808 8.374 12 6 h 6 12 17 6 k 6 17 16 6 l 6 16 15 089 3425 (0.0378) 138, 0 0.0213, 0.0481 0.0288, 0.0494 1.006 0.500, 0.695

C8H8I2N2S2Zn 515.45 100(2) 0.71073 monoclinic P21/n 0.21  0.14  0.08 9.0969(11) 12.7098(13) 12.9660(12) 105.388(7) 1445.4(3) 4 2.369 2.29–27.00 952 6.232 11 6 h 6 11 11 6 k 6 16 16 6 l 6 14 9628 3144 (0.0185) 138, 0 0.0241, 0.0671 0.0253, 0.0678 1.005 0.537, 2.333

R1 = R||Fo|  |Fc||/R|Fo|, wR2 = [R(w(Fo2  Fc2)2)/Rw(Fo2)2]1/2.

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3. Results and discussion 3.1. Synthesis and spectroscopic characterization of 1 and 2 We previously reported the synthesis and crystal structure of dm4bt and its zinc chloride complex [23]. The synthesis of 1 and 2 were achieved by the addition of dm4bt to a solution of zinc halides in a 1:1 M ratio, according to Eq. (1) where the reaction yields were relatively high. Complexes of 1 and 2 are stable in air and can be used directly for routine analyses.

ZnX2 þ dm4bt ! ½Znðdm4btÞX2 

ð1Þ

X ¼ Br; complex 1; X ¼ I; complex 2 NMR investigation can help to investigate the interaction between bithiazole moiety of BLM with DNA, especially in the absence of structural data [36,37]. The NMR spectra of both complexes in DMSO-d6 are very close and similar to that of free dm4bt [23]. 1H NMR shows a singlet for the methyl group at 2.7 ppm and another singlet in the aromatic region at 7.8 ppm for aromatic C–H, in both complexes. Moreover 13C NMR exhibits one aliphatic carbon of methyl at 19.3 ppm and three aromatic carbons at 115.6, 149.8 and 166.8 ppm as expected. NMR spectra for both compounds establish the presence of one type of material in the DMSO solution. This means that the halogen ligands are not exchanged with DMSO molecules. Infrared spectra for the complexes present several bands in the range 3100–700 cm1 corresponding to vibrations of the heterocycle ligand. The vibration bands present at around 3010 and 2900 cm1 in 1 and 2 are assigned as m(C–HAr) and m(C–HMe), respectively. Bands in the range of 1200–1600 cm1 are assigned to vibration modes of C–N, C–C, C@N and C@C bonds. Strong bands at 795 and 770 cm1 are assigned to S–C vibrations in the IR spectra of 1 and 2, respectively. Far infrared spectra of the complexes exhibit metal moiety vibrations. Zn–N and Zn–halogen stretching vibrations are observed at 575 and 251 cm1 for complex 1, and at 586 and 259 cm1 for complex 2. The UV–Vis spectra of the DMF solution of the complexes show signals in the near-UV region (252 nm for 1 and 232 nm for 2), which can be assigned to the ligand-centered p ? p⁄ transitions of bithiazole. 3.2. Crystal structure of complexes 1 and 2 Crystallographic data for 1 and 2 are given in Table 1, and selected bond lengths and angels are presented in Table 2. Fig. 1 shows ORTEP diagrams of complexes 1 and 2. As shown in the figure, both complexes are monomer and their asymmetric unit contains one independent molecule [Zn(dm4bt)X2] (X = Br in 1 and X = I in 2). The Zn atom is four-coordinated in a distorted tetrahedral configuration by two halogen atoms and two N atoms from the chelating 2,20 -dimethyl-4,40 -bithiazole ligand which is coordinated to the Zn in a bidentate fashion. Table 2 Selected bond lengths (Å) and angles (°) of complexes 1 and 2.

Zn1–N1 Zn1–N2 Zn1–X1 Zn1–X2 N1–Zn1–N2 N1–Zn1–X1 N1–Zn1–X2 N2–Zn1–X1 N2–Zn1–X2 X1–Zn1–X2

1, X = Cl

2, X = I

2.0746(18) 2.0454(18) 2.3491(4) 2.3292(4) 81.05(7) 105.39(5) 123.30(5) 110.98(5) 110.39(5) 119.300(13)

2.053(3) 2.083(3) 2.5389(5) 2.5435(4) 80.46(10) 108.13(7) 113.34(7) 121.66(7) 108.22(7) 118.757(15)

Fig. 1. ORTEP view of the complexes 1 (a) and 2 (b) with the atom numbering scheme. Displacement ellipsoids for non-H atoms are drawn at the 50% probability level.

The Zn–N bond lengths are in the range of 2.0454(18) and 2.083(3) Å, and in close agreement with Zn–N bond length in tetrahedral zinc complex with bipyridine [26]. It can be concluded that the coordination ability of nitrogen from bithiazole is similar to bipyridine. The bite angle of N1–Zn–N2 is 81.05(7)° and 80.46(10)° in 1 and 2, which significantly deviate from the ideal 109.5° and is comparable with the value of 74.9° in [Zn(bpy)Cl2]. These small bite angles, accompanied with the different bond distances of Zn–N and Zn–halogen are the main factors accounting for the distortion in tetrahedral geometry around zinc. In 1, the Zn–Br bond lengths are 2.0454(18) and 2.083(3) Å, whereas Zn–I distances are 2.3491(4) and 2.5389(5) Å in 2, which are comparable with these bond distances in similar compounds [38]. The two thiazole rings are relatively planar with N1–C1–C5–N2 dihedral angle of 3.0(3)° in 1 and 1.5(4)° in 2. The intermolecular p. . .p contacts between the adjacent bithiazole rings contribute to packing stabilization, performing dimer fragment, as shown in Fig. 2, where the distance between two adjacent thiazole ring planes is 3.5738(13) Å for complex 1 and 3.6987(18) Å for complex 2, showing slightly weaker p–p interaction in 2, due to the steric influence of iodine ions. 3.3. DNA-binding mode and affinity 3.3.1. Electronic absorption titration Electronic absorption spectroscopy is universally employed to examine the binding characteristics of metal complexes with DNA [39,40]. The UV–Vis absorption spectra of Zn complexes show an intense absorption band, assigned to the p–p⁄ transition of the aromatic ligand. As shown in Fig. 3, the addition of increasing amounts of DNA perturbs the spectrum pattern of the two

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DNA are similar and in good agreement with the Kb value for other reported zinc complexes with DNA [43]. The values of Kb have been described in the literature for classical intercalators (ethidiumbromide–DNA) whose binding constant have been found to be in the order of 106–107 M1 [44,45]. This result suggests that partial intercalations between the base pairs is the main mode of interaction of the Zn(II) complexes with DNA and it is also likely that the compound binds to the helix via groove mode [46]. 3.3.2. Fluorescence spectra It is known that CT-DNA does not give emission, but the titled complexes display luminescent properties. Hence, the binding ability of the two complexes with DNA was also investigated by fluorescence spectroscopy. A proper fluorescence spectrum was obtained with irradiation at k = 386 nm for 1. When the DNA solution was added gradually to compound 1, the fluorescence intensity was enhanced that illustrates the complex interact with DNA and the hydrophobic environment inside the DNA helix reduces the accessibility of solvent DMF to the complex and it restricts the complex mobility at the binding site which results in a decrease of the vibrational modes of relaxation and thus higher emission intensity [43]. In spite of complex 1, complex 2 emits properly with irradiation at k = 683 nm where with increasing amounts of DNA, quenched fluorescence is observed (Fig. 4). It is possible that the slower rate diffusion near the DNA, more vibrational modes and transition state level due to existence of ancillary iodine ligands as heavy atoms can cause decrease in fluorescence intensity in complex 2. Whether this distinction in fluorescence behavior implies on different interaction mode of two complexes with DNA cannot be simply justified, with consideration of high similarity of their properties in other experiments. However both enhancement and quenching in this experience confirm the efficient interaction of two compounds with CT-DNA. Fig. 2. Arrangement of p–p stacked ring systems of bithiazole in crystal packing diagram of 1 (a) and 2 (b). Hydrogen atoms are omitted for clarity.

complexes significantly, where they hypochromisms as well as bathochromism. The hypochromism reached 18% and 21%, with bathochromism at about 0.8 and 0.5 nm, for complexes 1 and 2, respectively. These spectral characteristics obviously suggest that the titled complexes most likely interact with DNA through a mode of stacking interaction between the aromatic bithiazole ligand of the complexes and the base pairs of DNA. Crystal packing studies confirmed that bithiazole ligands in two Zn complexes are able to be involved in p. . .p stacking interactions (see Fig. 2). For the quantitative investigation of the binding strength of the complexes to CT-DNA, UV spectrophotometric titration was performed [8,41]. The intrinsic binding constants Kb of the complexes with CTDNA were calculated using the following function equation [42]:

½DNA=ðea  ef Þ ¼ ½DNAðeb  ef Þ þ 1=K b ðeb  ef Þ where [DNA] is the concentration of DNA in base pairs and the apparent absorption coefficients ea, ef and eb correspond to Aobsd/ Zn complex concentration, the extinction coefficient of the free zinc complex and the extinction coefficient of the compound in the fully DNA-bound form, respectively. In plots [DNA]/(ea  ef) versus [DNA] (Fig. 3, inset), the Kb value, obtained by the linear fit of data was 3.47 ± 0.02  104 M1 for 1 and 3.19 ± 0.02  104 M1 for 2, given by the ratio of the slope to the intercept. This means that the binding strengths of 1 and 2 with

3.3.3. DNA–MB displacements To monitor the interaction mode of the complexes with CT-DNA, a fluorescence assay of the probe organic molecules was applied. Methylene blue (MB), a photosensitizer drug, showing promising applications in biological straining and diagnosis of diseases including carcinoma [47–49] is a planar dye molecule, where most studies indicated that (at low ionic strength buffer and low concentration of DNA) the major binding mode of MB with DNA was through intercalation [50]. Firstly, a basic understanding of the spectral behavior of MB seems essential, then CT-DNA pretreated with MB as a fluorescence probe (in a DNA to MB concentration ratio of 10), as the emission intensity of MB is quenched on adding CT-DNA. This emission quenching phenomenon displays the strong stacking interaction of MB with the adjacent DNA base pairs, as expected. The variation of emission spectra of the MB–DNA solutions with increasing amounts of complexes 1 and 2 is shown in Fig. 5. In two cases, with the increase of the concentration of the complexes, the emission band of the MB–DNA solutions increased in intensity but only slightly. It is known that the increase of the fluorescence intensity is due to releasing free MB molecules from DNA–MB complex, demanding the formation of metal complex–DNA instead of intercalator MB. When the probe fluorescence intensity is sufficiently close to the corresponding pure MB fluorescence intensity, it means a complete recovery of MB [51]; whereas this was not observed in our experience (Fig. 5). Our studies demand a partial replacing of MB by the complexes from the MB–DNA system, which is a partial intercalative mode of binding between the titled compounds and CT-DNA.

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a

1.2

1

Absorbance

0.8

0.6

0.4

0.2

0 210

230

250

270

290

310

Wavelength (nm)

b

1.8 1.6 1.4

Absorbance

1.2 1 0.8 0.6 0.4 0.2 0 210

220

230

240

250

260

270

280

290

300

310

Wavelength (nm) Fig. 3. (a) UV–Vis absorption spectra of complex 1 (70 lM) in Tris–HCl buffer in the presence of increasing amounts of CT-DNA (0, 49.5, 98.0, 145.6, 192.3, 238.1, 283.0 and 327.0 lM DNA) and (b) UV–Vis absorption spectra of complex 2 (50 lM) in the presence of increasing amounts of CT-DNA (0, 24.9, 49.5, 73.9, 98.3, 145.6, 192.3, 238.0 and 327.0 lM DNA). The arrow indicates the absorbance change upon increasing DNA concentration. The inset is plot of DNA concentration/|(eb  ef)| vs. DNA concentration for the titration of DNA to complexes.

3.3.4. Thermal denaturation studies The melting of DNA is an important tool for studying the interaction of transition metal ion with nucleic acid. Thermal denaturation studies of CT-DNA with the complexes provide evidence for the ability of the complexes to stabilize the double-stranded DNA [52]. It is well known that when the temperature in the solution increases, the double-stranded DNA gradually dissociates to single strands. The extinction coefficient of the DNA base at 260 nm in the double-helical form is much less than that in the single strand form hence, melting of the helix leads to an increase in the absorption at this wavelength. Thus, the helix to coil transition temperature can be determined by monitoring the absorbance of the DNA bases at k = 260 nm as a function of temperature [53]. This transition of double stranded DNA to single stranded DNA is termed as the melting temperature of DNA (Tm) [54]. The thermal behavior

of DNA in the presence of complexes can offer some information on the interaction of the complexes with DNA. Fig. 6 shows that in the absence of complexes 1 and 2, the Tm of CT-DNA was 81.62 ± 0.2 °C under our experimental conditions. The melting point increased by 2.72 ± 0.02 and 2.57 ± 0.02 °C for complexes 1 and 2 in a Zn complex to DNA concentration ratio of 1:2. Common DNA metallo-intercalators cause DTm values of 10–14 °C [55,56]. The moderately increased DTm with the titled zinc complexes suggests a partial intercalative and the groove bindings of these complexes to DNA. This experiment confirms our previous evidence [57,58]. 3.3.5. CD spectroscopy CT-DNA is the B-form DNA, a right-handed double helix with the base pairs stacked in the center and the base plane perpendic-

158

a

360 310

Fluorescence intensity (a.u.)

Fluorescence intensity (a.u.)

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260 210 160 110 60 10 347

397

447

497

165 145

Free MB

125 105 85 65 45 25 5 654

547

674

694

Wavelength (nm) 250

200

150

100

50

0 676

686

696

706

716

726

736

746

b

165

Fluorescence intensity (a.u.)

Fluorescence intensity (a.u.)

b

714

734

754

774

794

754

774

794

Wavelength (nm)

145

Free MB

125 105 85 65 45 25 5 654

756

674

694

Wavelength (nm)

ular to the helical axis causing two well-known grooves; the major and minor grooves. CT-DNA exhibits two conservative CD bands, a positive band at 277 nm due to base stacking and a negative band at 245 nm due to right handed helicity which is characteristic of DNA in the right-handed B form [55]. These bands are sensitive towards binding of any small molecule or drug and hence CD spectroscopy is useful in monitoring the conformational variations of DNA in the presence of the studied complexes in solution [59]. CD spectral variations of CT-DNA in the UV region were recorded by the respective addition of complexes 1 and 2, as shown in Fig. 7. Intercalative interaction of classical intercalators such as methyleneblue with DNA leads to enhancements in the intensity of the 275 nm band and a decrease in the intensity of the 245 nm band [60]. Whereas incubation of the titled zinc complexes with CT-DNA in a molar ratio of 0.5 causes decrease in the intensity of the 275 nm band with no considerable changes in the intensity of the 245 nm band. These changes are indicative of a groove binding nature in the binding of these complexes [61–63]. However positive band (275 nm) exhibits redshift to 283 nm upon addition of the complexes that suggests a partial interaction between bithiazole rings of the two complexes and base pairs of DNA may exist [64].

734

Wavelength (nm) Fig. 5. Fluorescence emission spectra of MB and DNA–MB in the absence and presence of complexes 1 (a) and 2 (b); (kex = 630 nm; kem = 688–804 nm; 5 lM MB; 50 lM DNA; 0, 14.7, 29.1, 43.06, 56.6, 95.0, 107.1, 130.4, 166.6, 230.7, 285.7 and 333.3 lM complex).

0.85 DNA

0.8

1 2

0.75

Absorbance

Fig. 4. (a) Fluorescence emission spectra of complex 1 (413 lM) in the presence of increasing amounts of CT-DNA, in Tris–HCl buffer (kex = 300 nm; kem = 350– 550 nm; 0, 40.0, 79.3, 136.8, 192.6, 264.6, 367.7, 527.6 and 674.2 lM DNA) and (b) fluorescence emission spectra of complex 2 (325 lM) in the presence of increasing amounts of CT-DNA in Tris–HCl buffer (kex = 665 nm; kem = 670–770 nm; 0, 98.6, 192.6, 367.7, 449.4, 527.6, 674.1 and 933.4 lM DNA). The arrow indicates the emission intensity changes upon increasing DNA concentration.

714

0.7 0.65 0.6 0.55 50

60

70

80

90

100

Temperature °C Fig. 6. Plots of the changes of absorbance at 260 nm of CT-DNA (75 lM) on heating in the absence and the presence of the complexes (37.5 lM) in 5 mM Tris–HCl with 50 mM NaCl.

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References

5 DNA

4

1

CD (medg)

3 2

2

1 0 -1 -2 -3 220

159

240

260

280

300

320

Wavelength (nm) Fig. 7. CD spectra of CT-DNA (100 lM) in the absence and presence of the complexes (50 lM), in 5 mM Tris–HCl with 50 Mm NaCl.

4. Conclusions Bioactive zinc complexes in DNA cleavage may be considered important in the context of non-toxic metal centers, in comparison with heavy-metal contained pt-based anticancer drugs, such as cisplatin and carboplatin. In a summary of our study, firstly two new zinc complexes with bioactive bithiazole ligand [Zn(dm4bt)X2] (X = Br and I) were synthesized and fully characterized. Their Xray single crystal analysis revealed that the thiazole rings of ligand can participate in p–p stacked interaction. The bioactivity investigation by UV–Vis and fluorescence spectroscopy demands that the titled complexes strongly interact with native CT-DNA. Further investigation about their binding modes suggests groove binding mode of the complexes rather than intercalative mode, resulting from low melting point variation of DNA, low alteration of DNA-bound MB absorption spectrum and the CD study of CT-DNA in the presence of the zinc complexes. This may be explained based on probable hydrogen bonds between nitrogen and sulfur atoms of bithiazole and DNA base pairs promoting groove binding and steric influence of methyl groups inhibiting intercalation of bithiazole. Furthermore our experience showed that there is no considerable difference between two complexes 1 (contained Br) and 2 (contained I) as they exhibit close interaction properties. The studies on in vitro properties of titled compounds as well as biological investigation of un-substituted relevant zinc-bithiazole compounds (for comparison) are currently ongoing in our laboratory.

Acknowledgments We wish to acknowledge the financial support from the Young Researcher Club, North Tehran Branch, Islamic Azad University.

Appendix A. Supplementary data CCDC 851521 and 851522 contain the supplementary crystallographic data for 1 and 2, respectively. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail: [email protected].

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