УДК 541.49
SYNTHESES, STRUCTURES, CHARACTERIZATION, AND BIOACTIVITIES OF NEW Cd(II) COMPLEX OF 2,3-PYRAZINEDICARBOXYLATE WITH 1,10-PHENANTHROLINE
© 2015 L. Wang, Y. Y. Li, J. Ge, J. Zhang, S. K. Liang, L. F. Li, C. Ma, and E. J. Gao*
Coordination Chemistry Laboratory, Shenyang Institute of Chemical Technology, Shenyang, Liaoning, 110142 P.R. China
*E-mail: enjungao@163.com Received June 6, 2014
A novel complex [Cd(Phen)2(Pzdc)] ■ 7.5(H2O) (I), where Phen = 1,10-phenanthroline, Pzdc = 2,3-pyra-zinedicarboxylic acid, has been synthesized under room temperature conditions and characterized by IR spectroscopy, elemental analysis, and X-ray single-crystal structure analysis (CIF file CCDC no. 1023407). Its mode of interaction with fish sperm DNA (FS-DNA) have been determined by fluorescence. Gel electrophoresis assay demonstrates the ability of complex I to cleave the HL-60 DNA. Furthermore, apoptosis assay shows treatment with complex results in morphological changes of cancer cell lines. The results induce apoptosis in JEKO cells.
DOI: 10.7868/S0132344X15020103
INTRODUCTION
Cancer continue to pose a very considerable health problems threat to significant parts of the population. Chemotherapeutics has become an important method in the treatment of cancer cells. Recently, there is a great interest on the binding of transition metal complexes with DNA, owing to their possible applications as new cancer therapeuticagents and their photochemical properties that make them potential probes of DNA structure and conformation [1—3]. Some authors produced evidence to the apoptosis promoting nature of Cd including its ability to induce DNA fragmentation and chromatin condensation in kidney, blood, and liver cells [4—6].
There has been considerable interest in the design and synthesis of complexes with carboxylate ligands because carboxylates can give rise to different coordination modes with metal ions and increase recognition of the metal's role in biological systems [7, 8]. The 2,3-pyrazinedicarboxylate acid (Pzdc) with a number of potential donor atoms commonly bound to transition metals by two carboxylic groups together with two pyrazine nitrogens. 1,10-Phenanthroline (Phen) can coordinate with transition metals and disturb functions of biological systems. Phen has long been used as a component of hemoglobin models with strong coordination ability and biological activity [9—11]. A novel coordination compound has been synthesized under solvent evaporation. In this paper, complex I was characterized by IR, elemental analysis, crystal structure determination, fluorescence, gel electrophoresis and apoptotic study.
EXPERIMENTAL
Materials and measurements. All chemicals were of reagent grade quality obtained from commercial sources and used without further purification unless otherwise noted. Elemental analyses (C, H, and N) were performed on a model Finnigan EA 1112 apparatus. Infrared spectroscopy using KBr pellets were performed on a Nicolet FT-IR 470 spectrophotometer. Fluorescence measurements were performed on a PerkinElmer LS55 fluorescence spectrofluorometer. Gel electrophoresis were performed on a JS-380A gel electrophoresis spectrometer.
Synthesis of complex I. Experiments were all reagent concentration 10 mmol/L, Cd(NO3)2 • 7.5H2O aqueous solution (10 mL) and Pzdc solution (10 mL) mixing. With potassium hydroxide solution (0.1 mol/L) pH value of mixed solution will be adjusted to 4.78. After about 3 h of stirring, 10 mL of ethanol solution Phen was added. After about another 3 h of stirring, the solution was filtrated, and the filtrate was evaporated slowly in the air. Colourless crystals were obtained after 30 days.
For C30H34N6On.5Cd
anal. calcd, %: C, 46.68; H, 3.72; N, 7.26. Found, %: C, 46.70; H, 3.64; N, 7.30.
IR spectrum (v, cm-1): 3077 s, 1671 s, 3498 s, 1496 s, 1516 m, 1310 m, 1629 w, 1356 w.
X-ray structure determination. Single-crystal data of complex I were collected at 273(2) K on a Rigaku XtaLAB mini X-ray single crystal diffractometer with
Table 1. Crystal data and refinement parameters for complex I
Parameter Value
Formula weight 775.04
Crystal system Monoclinic
Space group C2/c
Unit cell dimensions:
a, A 24.436(2)
b, A 15.8797(15)
c, A 19.9150(18)
ß, deg 124.6570(10)
V, A3 6356.6(10)
Z 4
Pcalcd mg/m3 1.620
p., mm-1 0.760
/(000) 3168
e 1.63-25.06
Limiting indices -27 < h < 29, -18 < k < 14, -23 < l< 23
Reflections collected/unique 18225/5628
Rint 0.0726
Reflections with I > 2o(I) 5628
Completeness, % 99.6
Goodness of fit on F2 1.041
Number of parameters refined 469
Final R indices (I > 2a(I)) Rx = 0.0594, wR2 = 0.1276
R indices (all data) Rx = 0.0946, wR2 = 0.1432
Residual electronic density (max/min), e A-3 0.888/-0.844
MoZ« radiation (k = 0.71073 A) in the range of 1.63° < < 9 < 25.06°. The structure was solved by direct methods using SHELXS-97 [12, 13] and refined with SHELXL-97 [14]. All non-hydrogen atoms were determined with successive difference Fourier syntheses and refined by full-matrix least-squares on F 2 [15]. All hydrogens were located at the theoretical positions. Further details of the crystal data and refinement are shown in Table 1. Selected bond distances and bond angles are given in Table 2.
Supplementary material for structure I has been deposited with the Cambridge Crystallographic Data Centre (no. 1023407; deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk).
Fluorescence spectroscopic studies. The buffer solution was 50 mM Tris-HCl, pH 7.4, mixed with 10 mM NaCl. The FS-DNA was pretreated with EtBr for 2 h and then the complex and buffer were added into the DNA—EtBr system. The sample was incubated
2 h at room temperature before spectral measurements. For all fluorescence measurements, the entrance and exit slits were maintained at 10 nm. Fluorescence measurement was done using 526 nm as the excitation wavelength and the emission range was set between 540 and 750 nm.
Cleavage of HL-60 DNA. In this experiment, HL-60 DNA (extracted by ourselves) was treated with complex I (dissolved in DMF) in Tris buffer (50 mM Tris-acetate, 18 |M NaCl buffer, pH 7.2) and the contents were incubated for 1 h at room temperature. The samples were electrophoresed for 1.5 h at 120 V on 0.85% agarose gel in Tris-acetate buffer. After electrophoresis, the gel was stained with 1 mg mL-1 EtBr and photographed under UV light [16].
Apoptosis assays by flow cytometry. The ability of complex I induce apoptosis is evaluated in JEKO cells line using Annexin V conjugated with FITC and pro-pidium iodide (PI) counterstaining by flow cytometry. The JEKO cells in a suitable condition were seeded into a 6-well culture plates at 1 x 106 cells per well in a
3 mL culture medium and 12 h later the medium including the Cd(II) complex was given. After 12 h (24 h) incubation, cells were gathered, wash cells twice with cold phosphate-buffered saline and then resuspended cells in 1x Binging Buffer at a concentration of1 x 106 cells/mL. Transfer 100 |L of the solution (1 x 105 cells) to a 5 mL culture tube. Futher, add 5 |L of FITC Annexin V and 5 |L PI. Gently vortex the cells were incubated in the dark at 25°C for 15 min. Add 400 |L of 1x Binding Buffer to each tube. Analyze by flow cytometry Accuri C6, USA, within 1 h.
RESULTS AND DISCUSSION
The crystal structure of complex I was determined by X-ray crystallography (Fig. 1). The Cd2+ ion in I is coordinated by a bidentate pzdc and two bidentate
SYNTHESES, STRUCTURES, CHARACTERIZATION, AND BIOACTIVITIES OF NEW Cd(II)
Table 2. Selected bond lengths and angles for complex I
153
Bond d, Â Bond d, Â
Cd(1)-O(1) 2.267(4) Cd(1)-N(4) 2.365(5)
Cd(1)-N(3) 2.312(5) Cd(1)-N(5) 2.371(5)
Cd(1)-N(2) 2.345(5) Cd(1)-N(1) 2.360(5)
Angle ю, deg Angle ю, deg
O(1)Cd(1)N(3) 91.25(16) O(1)Cd(1)N(5) 70.69(16)
O(1)Cd(1)N(2) 86.48(16) N(3)Cd(1)N(5) 149.83(17)
N(3)Cd(1)N(2) 98.38(18) N(2)Cd(1)N(5) 104.19(17)
O(1)Cd(1)N(1) 147.98(15) N(1)Cd(1)N(5) 92.20(17)
N(3)Cd(1)N(1) 114.09(17) O(1)Cd(1)N(4) 113.84(16)
N(2)Cd(1)N(1) 71.31(17) N(3)Cd(1)N(4) 72.28(17)
N(1)Cd(1)N(4) 93.21(17) N(2)Cd(1)N(4) 157.28(17)
N(4)Cd(1)N(5) 92.63(16)
Phen. Distorted octahedral coordination geometry is comprised of N and carboxylate O from a doubly deprotonated bidentate Pzdc ligand (Cd(1)—N(5) 2.371(5), Cd(1)—N(1) 2.360(5) Â) and four N atoms from two Phen ligands (Cd(1)-N(1) 2.360(5), Cd(1)-N(2) 2.345(5), Cd(1)-N(3) 2.312(5), Cd(1)-N(4) 2.365(5) Â). The whole structure presents a three-dimensional network arrangement (Fig. 2).
Electronic absorption spectroscopy is an effective method to examine the binding mode of DNA with metal complexes [17]. The absorption spectra of complex I in the absence and presence of FS-DNA are given in Fig. 3. As the concentration is increased, changes in emission may arise from the restriction of interan-nular twisting between Phen and Pzdc. It implies that complex I interacts strongly with FS-DNA. According to the classical Stern—Volmer equation: I0/I = 1 + Ksqr [18], where I0 and I represent the fluorescence intensities in the absence and presence of the complex, respectively, and r is the concentration ratio of complex to DNA. Ksq is a linear Stern—Volmer quenching constant dependent on the ratio of the bound concentration of EtBr to the concentration of DNA. The Ksq value is obtained as the slope of I0/I versus r linear plot. The fluorescence quenching curves of DNA-bound EtBr by complex I are given in Fig. 4. The Ksq values for the complex are Ksq1 = 0.8653. Such values of quenching constant suggest that the interaction of the complex with DNA is moderate intercalation.
In past research, pBR322 plasmid DNA or pUC19 DNA were used but do not originate from cell [19, 20]. However, we analyzed DNA strand breaks in HL-60 DNA treated the complex. As shown in Fig. 5, DNA fragmentations with a characteristic laddering pattern were observed for the complex. When HL-60 DNA is conducted by electrophoresis, complex I was found t
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