Three mononuclear oxovanadium complexes [VO(Hbid)(CF 3PIP)] (1) (Hbid=(E)-2-(2-hydroxybenzylideneamino) isoindoline-1,3-dione, CF 3PIP=2-(2-trifluoromethyl phenyl)imidazole[4,5-f][1,10] phenanthroline), [VO(Hbid)(m-CF 3PIP)]; (2) (m-CF 3PIP=2-(3-trifluoromethyl phenyl)imidazole [4, 5-f][1,10]phenanthroline) and [VO(Hbid)(p-CF 3PIP)]; (3) (p-CF 3PIP=2-(4-trifluoromethyl phenyl) imidazole[4,5-f][1,10]phenanthroline) have been synthesized and characterized by elemental analysis, IR, molar conductance, ES-MS and 1H NMR. The DNA-binding properties of these complexes were studied by using UV-Vis absorption titration, fluorescence spectra, viscosity measurements and thermal denaturation studies. The results show that 1, 2 and 3 interact with calf thymus DNA (CT-DNA) by intercalation modes and the magnitude of their intrinsic binding constants ( Kb values) follows the order: 2 < 1 < 3. Furthermore, their photocleavage properties with pBR322 plasmid DNA were investigated by agarose gel electrophoresis experiments. The DNA cleavage capacity of complex 3 is also stronger than that of 1 and 2.
The development of transition metal complexes which are actually to be utilized in a wide range of biochemistry and medicine as well as therapeutic medication against tumor cells has achieved fantastic step forward in past decades [
Complexes with Schiff base Hbid, where Hbid=(E)-2-(2-hydroxybenzylideneamino) isoindoline-1,3-dione, displayed chemical nuclease activity by partial intercalation thus ability to inhibit the growth of both Gram-pos. and Gram-neg. bacteria [
In the present article, three mononuclear oxovanadium complexes [VO(Hbid)(CF3PIP)] (1), [VO(Hbid)(m- CF3PIP)] (2), [VO(Hbid)(p-CF3PIP)] (3), (Hbid=(E)-2-(2-hydroxybenzylideneamino)isoindoline-1,3-dione, CF3PIP=-(2-trifluoromethyl phenyl)imidazole [4,5-f][1,10]phenanthroline, m-CF3PIP=2-(3-trifluoromethyl phenyl)imidazole[4,5-f][1,10]phenanthroline, p-CF3PIP=2-(4-trifluoromethyl phenyl)imidazole[4,5-f][1,10] phenanthroline) have been synthesized and characterized by elemental analysis, IR, molar conductance, ES-MS and 1H NMR. The DNA-binding properties of these three complexes were well studied by UV-Vis titration, fluorescence spectra, viscosity measurements and thermal denaturation studies. Photocleavage reactions with pBR322 supercolied plasmid DNA were investigated by agarose gel electrophoresis experiments. The compounds employed in this work are shown in Scheme 1.
Scheme 1. Structure of VO(Hbid)(CF3PIP) (1), R1=CF3, R2=H, R3=H; VO(Hbid) (m-CF3- PIP) (2), R1=H, R2=CF3, R3=H; VO(Hbid) (p-CF3PIP) (3), R1=H, R2=H, R3=CF3.
All chemicals used in the synthesis and physical measurements were analytical reagent grade or biochemical quality and without further purification unless otherwise specified. 1,10-Phenanthroline was obtained from Guangzhou Chemical Reagent Factory. VOSO4∙xH2O were purchased from Alfa Aesar, N-Aminophthalimide were purchased from TCI, CT-DNA and pBR322 supercoiled plasmid DNA were obtained from Sigma. Tris buffer 1 (Tris=tris(hydroxyl-methyl)aminomethane) containing 5 m∙mol∙L−1 Tris-HCl and 50 m∙mol∙L −1 NaCl (pH = 7.2) was used for absorption titration, fluorescence emission and viscosity measurements. Tris buffer 2 containing 50 m∙mol∙L−1 Tris-HCl and 18 m∙mol∙L−1 NaCl (pH = 7.2) was used for the gel electrophoresis experiments. A phosphoric acid buffer containing 1.5 m∙mol∙L−1 Na2HPO4, 0.5 m∙mol∙L−1 NaH2PO4 and 0.25 m∙ mol∙L−1 Na2H2EDTA (H4EDTA=N,N'-ethane-1,2-diylbis[N-(carboxymethyl) glycine]) (pH = 7.0) was used for thermal denaturation. A solution of CT-DNA in buffer 1 gave a ratio of UV absorbance at 260 and 280 nm of 1.8 - 1.9:1, indicating that the DNA was sufficiently free of protein [
Microanalysis (C, H, and N) was carried out with a PerkinElmer 240Q elemental analyzer. Electrospray mass spectra (ES-MS) were recorded on an LCQ system (Finnigan MAT, USA) using methanol as mobile phase. 1H NMR spectra were recorded on a Varian-500 spectrometer. All chemical shifts are given relative to tetramethylsilane (TMS). Infrared spectra were recorded on a Bomem FTIR model MB102 instrument using KBr pellets method. UV-Vis spectra were recorded on a Shimadzu UV-3101 PC spectrophotometer at room temperature. Emission spectra were recorded on a Perkin-Elmer Lambda 55 spectrofluorophotometer. Molar conductivities in DMF (1 m∙mol∙L−1) solution at room temperature were measured using a DDS-307 digital direct reading conductivity meter.
CF3PIP was synthesized through a modification of a previously reported procedure [
CF3PIP: Yield: 61%. Anal. Found: C, 65.48; H, 3.14; N, 15.35; Calcd for C20H11F3N4: C, 65.93; H, 3.04; N, 15.28. 1H NMR (DMSO-d6, 500 MHz) δ: 9.03 (s, 2H, J = 8.7 Hz, ArH), 9.02 (s, 2H, J = 8.6 Hz, ArH), 8.47 (m, 1H, J = 8.3 Hz, ArH), 8.45 (m, 1H, J = 8.2 Hz, ArH), 7.96 (m, 1H, J = 8.1 Hz, ArH), 7.82 (br, 3H, J = 7.6 Hz, ArH), 3.32 (s, 1H, -NH). 13C NMR (DMSO-d6, 500 MHz) δ: 148.9 C(15), 148.0 C(1,12), 143.7 C(5,14), 133.8 C(3ꞌ,5ꞌ), 129.7 C(1ꞌ), 129.5 C(3), 129.1 C(7), 126.6 C(2ꞌ,6ꞌ), 125.9 C(4ꞌ), 125.8 C(-CF3), 124.8 C(8), 124.7 C(4), 123.4 C(9), 123.3 C(10), 122.6 C(6,13). ES-MS: (CH3OH): m/z 365.0 ([M+H]+), 387.0 ([M+Na]+). IR (KBr disk): ν (cm−1) = 3406.8 (s, N-H), 3078.2 (m, C-H), 1606.7 (s), 1551.8 (m), 1483.0 (s, C=C), 1315.3 (vs, C-F). Conductance (Ω−1∙cm2∙mol−1): 9.2.
m-CF3PIP: Yield: 58%. Anal. Found: C, 65.67; H, 3.13; N, 15.42; Calcd for C20H11F3N4: C, 65.93; H, 3.04; N, 15.38. 1H NMR (DMSO-d6, 500 MHz) δ: 8.99 (m, 2H, J = 8.8 Hz, ArH), 8.83 (d, 2H, J = 8.7 Hz, ArH), 8.56 (m, 2H, J = 8.2 Hz, ArH), 7.78 (m, 1H, J = 8.0 Hz, ArH), 7.75 (m, 3H, J = 7.5 Hz, ArH). 3.38 (s, 1H, -NH). 13C NMR (DMSO-d6, 500 MHz) δ: 148.2 C(15), 148.0 C(1,12), 143.6 C(5,14), 135.3 C(3ꞌ,5ꞌ), 132.5 C(1ꞌ), 130.3 C(3), 129.8 C(7), 128.2 C(2ꞌ,6ꞌ), 127.9 C(4ꞌ), 127.0 C(-CF3), 126.8 C(8), 126.7 C(4), 126.0 C(9), 124.8 C(10), 123.8 C(6,13). ES-MS: (CH3OH): m/z 365.0 ([M+H]+), 387.0 ([M+Na]+). IR (KBr disk): ν (cm−1) = 3241.6 (s, N-H), 3065.8, (m, C-H), 1607.2 (m), 1566.7 (s), 1548.5 (m, C=C), 1312.2 (vs, C-F). Conductance (Ω−1∙ cm2∙mol−1): 9.6.
p-CF3PIP: Yield: 66%. Anal. Found: C, 65.78; H, 3.24; N, 15.32; Calcd for C20H11F3N4: C, 65.93; H, 3.04; N, 15.38. 1H NMR (DMSO-d6, 500 MHz) δ: 9.07 (d, 2H, J = 8.6 Hz, ArH), 8.84 (d, 2H, J = 8.4 Hz, ArH), 8.04 (d, 1H, J = 8.1 Hz, ArH), 7.97 (d, 1H, J = 7.8 Hz, ArH), 7.91 (d, 1H, J = 7.9 Hz, ArH), 7.83 (t, 3H, J = 7.7 Hz, ArH). 3.40 (s, 1H, -NH). 13C NMR (DMSO-d6, 500 MHz) δ: 149.3 C(15), 147.7 C(1,12), 143.6 C(5,14), 131.6 C(3ꞌ,5ꞌ), 131.4 C(1ꞌ), 130.1 C(3), 129.9 C(7), 129.6 C(2ꞌ,6ꞌ), 128.4 C(4ꞌ), 125.5 C(-CF3), 125.2 C(8), 123.2 C(4ꞌ), 123.1 C(4), 122.4 C(9), 122.3 C(10), 121.6 C(6,13). ES-MS: (CH3OH): m/z 365.0 ([M+H]+), 387.0 ([M+Na]+). IR (KBr disk): ν (cm−1) = 3371.2 (s, N-H), 3065.8 (w, C-H), 1608.2 (s), 1565.5 (m, C=C), 1326.5 (vs, C-F). Conductance (Ω−1∙cm2∙mol−1): 8.6.
[VO (Hbid) (CF3PIP)] was prepared by the synthetic steps similar to a previously reported method [
This compound was synthesized by a similar procedure as for the complex 1, with m-CF3PIP (0.364 g, 1 m∙mol) in place of CF3PIP. Yield: 43%. Anal. Found: C, 60.36; H, 2.91; N, 12.06; Calcd for C35H20F3N6O4V: C, 60.35; H, 2.89; N, 12.07. 1H NMR (DMSO-d6, 500 MHz) δ: 8.87 (s, 1H, CH=N), 8.59 (dd, 2H, ArH, J = 8.5 Hz), 8.44 (d, 2H, ArH, J = 8.5 Hz), 8.36 (br, 6H, ArH, J = 8.3 Hz), 8.27 (d, 1H, J = 8.2 Hz), 8.08 (d, 1H, ArH, J = 7.7 Hz), 7.72 (dd, 2H, ArH, J = 7.8 Hz), 7.34 (d, 2H, ArH, J = 6.6 Hz), 6.48 (dd, 2H, ArH, J = 7.6 Hz), 5.20 (s, 1H, -NH). ES-MS (CH3OH): m/z: 695.1 ([M-H]−), 727.1 ([M + CH3OH − H]−). IR (KBr disk): ν (cm−1)=3428.3 (N-H), 3027.0 (m, C=C-H), 1721.9 (s, HC=N), 1607.5 (vs, C=O), 1312.2 (s, -CF3), 968.8 (s, V=O), 713.7 (m, V-N). Conductance (Ω−1∙cm2∙mol−1): 9.7.
This complex was prepared using a similar procedure described for complex 1. Yield: 36%. Anal. Found: C, 60.28; H, 2.93; N, 12.06; Calcd for C35H20F3N6O4V: C, 60.35; H, 2.89; N, 12.07. 1H-NMR (DMSO-d6, 500MHz) δ: 9.12 (s, 1H, CH=N), 8.86 (d, 2H, ArH, J = 8.5 Hz), 8.63 (d, 2H, ArH, J = 8.4 Hz), 8.44 (br, 6H, ArH, J = 8.2 Hz), 8.26 (d, 1H, J = 8.4 Hz), 8.12 (d, 1H, ArH, J = 7.8 Hz), 7.80 (d, 2H, ArH, J = 7.2 Hz), 7.34 (d, 2H, ArH, J = 6.8 Hz), 6.62 (t, 2H, ArH, J = 7.8 Hz), 5.12 (s, 1H, -NH). ES-MS (CH3OH): m/z: 695.1 ([M-H]−), 727.1 ([M + CH3OH-H]−). IR (KBr disk): ν (cm−1) = 3401.6 (N-H), 2982.9, 3077.2 (m, C=C-H), 1725.7 (vs, HC=N), 1609.4 (vs, C=O), 1314.5 (s, -CF3), 956.6 (s, V=O), 730.9 (m, V-N). Conductance (Ω−1∙cm2∙mol−1): 11.8.
Absorption titration of the oxovanadium complexes in buffer 1 were performed using a fixed concentration of the oxovanadium complexes (20 μ∙mol∙L−1) to which the DNA stock solutions were added. The oxovanadium-DNA solutions were incubated at room temperature for 3 mins before the absorption spectra were recorded. In order to further elucidate the binding strength of the complexes, the intrinsic binding constant Kb with CT-DNA was obtained by monitoring the change in the absorbance of the ligand transfer band with increasing amounts of DNA. Kb was then calculated using the following equation [
where [DNA] is the concentration of DNA in the base pairs, and εa – εf and εb refer to the corresponding apparent absorption coefficient Aobsd/[Vanadium], the extinction coefficient for the free oxovanadium complexes and the extinction coefficient for the oxovanadium complexes in the fully bound form, respectively. In plots of [DNA]/(εa – εf) versus [DNA], Kb is obtained by the ratio of the slope to the intercept.
Viscosity measurements were carried out with an Ubbelohde viscometer maintained at a constant temperature of (28.0 ± 0.1) C in a thermostatic bath. Flow time was measured with a digital stopwatch, and each sample was measured five times to obtain the average flow time. Date are presented as (h/h0)1/3 versus binding ratio [
Thermal denaturation studies were carried out with Shimadzu UV-3101 PC spectrophotometer equipped with a Peltier temperature-controlling programmer (±0.1˚C). The melting curves were obtained by measuring the absorbance at 260 nm for solutions of CT-DNA (80 μ∙mol∙L−1) in the absence and presence of oxovanadium complex [20 μ∙mol∙L−1] as a function of the temperature was scanned from 50˚C to 90˚C at a speed of 5˚C min−1. The melting temperature (Tm) was taken as the mid-point of the hyperchromic transition.
The photoinduced cleavage activity of supercoiled pBR322 DNA by the oxovanadium complexes was studied by performing agarose gel electrophoresis experiment, pBR322 DNA (0.1 μg) was treated with the complexes in buffer 2 in different concentrations, and the solutions were incubated at 37˚C in the incubator for 1 h. The samples were analyzed by electrophoresis for 2 h at 90 V in tris-boric buffer containing 0.8% agarose gel. The gel was stained with 0.1 μL/ml DuRed nucleic acid gel stain and photographed under UV light on an Alpha Innotech IS-5500 fluorescence chemiluminescence and visible imaging system [
The Schiff base ligand was prepared by a reaction of salicylaldehyde with N-Aminophthalimide in the appropriate mole ratios using absolute ethanol as solvent [
According to the IR spectra of phenanthroline-based ligands show absorption at ca. 3400 cm−1 (imidazole N-H), 3065 - 3078 cm−1 (C=C-H), 1483-1608 cm−1 (C=C), 1312-1326 cm−1 (C-F), respectively. In parallel, IR spectra absorption of complexes 1-3 observed at 3401-3425 cm−1 for (imidazole N-H), 2982 - 3077 cm−1 for (C=C-H), 1607 - 1725 cm−1 for (C=O) and (-CH=N), 1312 - 1314 cm−1 for (-CF3), 713 - 730 cm−1 for (V-N), respectively. The strong (VO) band at 956 - 974 cm−1 could be cleared identified for the formulation of the complex.
Electronic spectra of complexes 1-3 show an intense band at ca. 272 nm assignable to π―π* transition of aromatic rings of phenanthroline-based ligands [
In the 1H NMR spectra of phenanthroline-based ligands showed peaks of aromatic hydrogen (ArH), imidazole secondary amine (NH) proton. However, in the 1H NMR spectra of complexes, the peaks of imine (CH=N) proton were observed, which affirmed that the Schiff base ligand was coordinated to vanadium.
In the ES-MS spectra of CF3PIP, m-CF3PIP and p-CF3PIP showed peaks at m/z 365 ([M+H]+) and 387 ([M + Na]+). The molecular ion peaks of three complexes at m/z 695 ([M-H]−) and 727 ([M+CH3OH-H]−). Elemental analysis, ES-MS, IR and 1H NMR data of all the compounds are in good agreement with the expected structures.
The molar conductance values of phenanthroline-based ligands and complexes 1-3 in DMF (1 m∙mol∙L−1) are shown in
Compounds | Ω−1∙cm2∙mol−1 |
---|---|
CF3PIP | 9.2 |
m-CF3PIP | 9.6 |
p-CF3PIP | 8.6 |
VO(Hbid)(CF3PIP) | 10.7 |
VO(Hbid)(m-CF3PIP) | 9.7 |
VO(Hbid)(p-CF3PIP) | 11.8 |
For the purpose of evaluating the capacity of oxovanadium complexes interacting with CT-DNA, the electronic spectra of complexes 1, 2 and 3 in the absence and presence of increasing amounts of CT-DNA are shown in
In order to elucidate the binding strengths of these complexes, the intrinsic binding constant Kb were calculated by monitoring the changes of absorbance in the ligand transfer bands with increasing amounts of CT-DNA. The Kb values of complexes 1-3 were calculated as 7.40 × 104 (mol∙L−1)−1, 5.42 × 104 (mol∙L−1)−1 and 1.25 × 105 (mol∙L−1)−1, respectively, indicating that they strongly interact with DNA by intercalation. And the DNA-bind- ing activities of these three complexes are higher than that of reported mononuclear oxovanadium complexes VO(SAA)(phen) (4.50 × 104 M−1) and VO(MOSAA) (phen) (2.95 × 104 M−1) [
The interaction of the complexes (20 μ∙mol∙L−1) with CT-DNA was investigated using fluorescence emission titration experiment in the Tris buffer 1 at room temperature. The emission spectra of complexes 1, 2 and 3 in the absence and presence of CT-DNA exhibit luminescence in Tris buffer, with a maxima appearing at 442 nm, 443 nm and 440 nm respectively, which are shown in
To further clarify the nature of the binding interaction between both oxovanadium complexes and DNA in the absence of X-ray structural data or NMR spectra, viscosity measurements that are regarded as the least controversial and the most rigorous means of testing the binding model of DNA in solution [
The effects of complexes 1, 2 and 3 on the viscosity of CT-DNA are shown in
DNA melting experiments are generally applied to investigate the extent of intercalation, which were carried out by monitoring the intensity of DNA bases at 260 nm at different temperatures, both in the absence and presence of oxovanadium complexes. Thermal behaviors of DNA in the presence of compounds can give insight into their conformational changes when the temperature is raised and offer information about the interaction strength of complexes with DNA. As a rule, with the temperature in the solution rising, the double-stranded DNA will gradually dissociate to single strands and generate a hyperchromic effect on absorption spectra of DNA bases. Thus, the melting temperature Tm, which is defined as the temperature where half of the total base pairs are un- bounded, is usually introduced. Generally, Tm will increase considerable when intercalative binding occurs, since intercalation of the complexes into DNA base pairs causes stabilization of base stacking and hence raises the melting temperature of the double-stranded DNA [
The melting curves of CT-DNA in the absence and presence of complexes 1-3 are shown in
The experimental data reveals that complexes in collaboration with phenanthroline-substituted auxiliary li-
gands exhibit appreciable DNA intercalative activities, in addition, complex 3 showed a larger ΔTm, indicating that 3 exhibits stronger DNA-binding affinity when interacting with DNA, in conformity with the electronic absorption titration.
The interaction mode between these oxovanadium complexes and plasmid DNA were further investigated by agarose gel electrophoresis experiments. When circular plasmid DNA is subject to electrophoresis, relatively fast migration will be observed for the intact supercoiled form (form Ι). If scission occurs on one strand (nicking), the supercoil will relax to generate a slower moving open circular form (form II). If both strands are cleaved, a linear form (form III) migrating between form I and II will be generated [
As is shown both in
In this paper, three novel Schiff base oxovanadium complexes, [VO(Hbid)(CF3PIP)] (1), [VO(Hbid)(m-CF3PIP)] (2), [VO(Hbid)(p-CF3PIP)] (3) have been synthesized and characterized by EA, ES-MS, IR, 1H NMR and molar conductance. Their DNA-binding activities with CT-DNA were evaluated using UV-Vis titration, viscosity measurements and thermal denaturation. These three oxovanadium complexes interact with CT-DNA by intercalation modes. Their photocleavage activities of supercoiled plasmid DNA were investigated in the presence of H2O2 probably via generating hydroxyl radical, the results also show efficiently oxidative cleavage activities, in
conformity with DNA-binding behaviors of the complexes. In addition, complex 3 exhibits a higher DNA- binding affinity in contrast to complexes 1 and 2, indicating that the electrochemical characteristics of phenanroline plane and the existence of substituted strong electronic-withdrawing group (-CF3) at the different locations introduced on the aromatic ring of phenanroline-based ligands plays an important role in the DNA-binding affinity.
We gratefully acknowledge financial support for this work by the Science and technology Research Project of Guangdong Province (No.2012B031800431), P. R. China and Cultivation of Natural Science Joint Fund of the First Affiliated Hospital and the Scientific & Technical Department of Guangdong Pharmaceutical University (No. 2014-36).