Abstract
The complexes of oxovanadium(IV) with ciprofloxacin and various uni-negative bidentate ligands have been prepared and their structure investigated using spectral, physicochemical and elemental analyses. The viscosity measurement suggest that the complexes bind to DNA by intercalation. The DNA binding efficacy was determined using absorption titration to obtain the binding constant (Kb). The DNA cleavage efficacy was determined using gel electrophoresis. The DNA binding and cleavage efficacy were increased in the complexes relative to the parental ligands and metal salts. Antibacterial activity has been assayed against two Gram( − ve) i.e. Escherichia coli, Pseudomonas aeruginosa and three Gram( + ve) Staphylococcus aureus, Bacillus subtilis, Serratia marcescens microorganisms using the doubling dilution technique. The results show a significant increase in antibacterial activity in the complexes compared with parental ligands and metal salts.
Introduction
The interaction of transition metal complexes containing multidentate ligands with DNA has recently gained much attention followed by an important biological and medical roles played by potential metallointercalators. Vanadium occurs as an “essential trace” element in diverse living forms Citation1Citation2Citation3Citation4Citation5Citation6. It plays active roles in many enzymatic reactions such as halogenation of organic substrate [Citation2,Citation5,Citation6] and fixation of nitrogen through an alternative pathwayCitation2Citation7Citation8Citation9. Coordination chemistry involving vanadium is often directed towards the use of relatively hard N- and, in particular, o-based anions and to limit the growth to well-defined molecular species, chelating ligands or those containing bulky substituents are often employed. In order to gain an insight into the intricate roles of vanadium in biological system, it is advantageous to acquire information about the basic coordination chemistry of this metal employing a biologically relevant ligand donor set Citation10Citation11Citation12. Quinolone antibacterial drugs have been frequently used to treat various bacterial infections because of their broad spectrum of activity against gram-positive and gram-negative bacteria [Citation13]. The fluoroquinolones are antibacterial agents that act by forming ternary complexes with DNA gyrase and DNA topoisomerase IV on chromosomal DNA. Resistance to the compounds is generally associated with amino acid substitutions in portions of the GyrA (gyrase) and ParC (topoisomerase IV) proteins called the quinolone resistance-determining regions (QRDRs) [Citation14]. Ciprofloxacin (Cip) is one of the quinolone antibacterial agents, used in the treatment of a wide range of infections, that antagonize the A subunit of DNA gyrase [Citation14,Citation15]. In order to continue researching DNA binding model of the fluoroquinolones and their transition metal complexes [Citation16,Citation17], in this paper, we prepared the VO(IV) mixed ligand complexes of ciprofloxacin and anthranilic acid (L1), glycine (L2), β-alanine (L3), L-asparagine (L4), DL-serine (L5), o-aminophenol (L6) and DL-valine (L7). The DNA binding properties of the complexes have been investigated by ultraviolet spectroscopy, viscosity measurements and gel electrophoresis method. Experimental results indicated that complexes and ciprofloxacin can bind to DNA by intercalation modes, but the binding affinity of the complexes is much higher than that of the ligand. Antibacterial activity has been assayed against gram-positive and gram-negative bacteria using the doubling dilution technique.
Experimental
Materials and methods
All the chemicals used were of analytical grade. Vanadylsulphate, anthranilic acid (L1), glycine (L2), β-alanine (L3), L-asparagine (L4), DL-serine (L5), o-aminophenol (L6) and DL-valine (L7) were purchased from, E. Merck (India) Ltd., Mumbai. Ciprofloxacin hydrochloride was purchased from Bayer AG (Wyppertal, Germany). Luria broth, ethidium bromide, sucrose, and tris(hydroxymethyl)methylamine were purchased from Hi-media Laboratories Pvt. Ltd., India. Agarose was purchased from Sisco research lab., India. Bromophenol blue, acetic acid and EDTA were purchased from Sd fine chemicals, India. The organic solvents were purified by standard methods [Citation18].
Instrumentation physical properties
Carbon, hydrogen and nitrogen were analyzed on a model 240 Perkin Elmer elemental analyzer. Thermogravimetric analysis and differential scanning calorimetric study were performed with a model 5000/2960 SDTA, TA instrument (USA). Infrared spectra were recorded on an FT-IR Shimadzu spectrophotometer as KBr pellets in the range 4000–400 cm–1. The electronic spectra of the complexes were recorded in the range 800–200 nm on UV-160A UV–Vis. spectrophotometer, Shimadzu (Japan). The magnetic moments were measured by Gouy's method using mercury tetrathiocyanatocobaltate(II) as the calibrant (χg = 16.44 × 10− 6 cgs units at 20°C), Citizen Balance. The diamagnetic correction was made using Pascal's constant [Citation19].
UV–Vis. Spectroscopy
The electronic absorption spectroscopy is an important tool in DNA-binding studies. Concentrated solutions of metal complexes were prepared by dissolving the complexes in DMSO and diluted suitably with buffer to desire concentrations for all the experiments. The experiments were performed by maintaining a constant concentration of the complex (4 μM) while varying the nucleic acid concentration (2–20 μM). The intrinsic binding constants (Kb) for the complexes with DNA were obtained using the following equation [Citation20,Citation21].
Where [DNA] is the concentration of DNA in terms of nucleotide phosphate, [NP] the apparent absorption coefficient εf, εa and εb correspond to the extinction coefficient of the free complex, the extinction coefficient for each addition of DNA to the complex and the extinction coefficient for the complex in the fully bound form respectively.
Viscosity measurements
Viscosity measurements were carried out using an Ubbelodhe viscometer maintained at a constant temperature 27.0 ± 0.1°C in a thermostatic bath. Flow time was measured with a digital stopwatch, each sample was measured three times, and an average flow time was considered. Data are presented as (η/η0)1/3 vs. binding ratio [Citation22], where η and η0 are the viscosity of DNA in the presence and absence of complex, respectively.
Antibacterial assays- dilution method
All the bacteria were incubated and activated at 30°C for 24 h before inoculation into Luria broth for 24 h. The compounds were dissolved in DMSO and then diluted using Luria broth. Two-fold serial concentrations of the compounds were employed to determine the Minimum Inhibitory Concentration (MIC) ranging from 100 μg/mL - 0.1 μg/mL. Test cultures were incubated at 37°C (24 h). The lowest concentrations of antimicrobial agents that resulted in complete inhibition of growth were represented as MIC (μg/mL). In each case triplicate tests were performed and the average was taken as the final value [Citation23].
Gel electrophoresis
Plasmid DNA (pBR322) (100 μM) cleavage activity of mixed-ligand complexes (50 μM) was monitored using agarose gel electrophoresis. In a typical experiment supercoiled pBR322 DNA (2.5 μg/mL) in Tris-HCl (100 mM, pH 8.0) was treated with different mixed-ligand complexes. The samples were then incubated at room temperature and loaded with 0.5x loading buffer containing 40% sucrose and 0.02% bromophenol blue on 1.5% agarose gel. Electrophoresis was carried out at 100 V for 90 min. in TAE (Tris Acetate EDTA) buffer and run in duplicate. Same was stained with ethidium bromide. Same experimental conditions were maintained for control assays. The gels were viewed on UV transilluminator; images were captured with an attached camera and estimated using AlphaDigiDocTM RT. Version V.4.1.0 PC-Image software.
Synthesis of the mixed-ligand complexes
An ethanolic solution (50 mL) of VOSO4√3H2O (0.6327 g, 5 mM) was added to an ethanolic solution of (50 mL) anthranilic acid (L1) (0.3428 g, 5 mM), followed by a solution of (Cip.HCl) (0.917 g, 5 mM) in water and the reaction mixture was adjusted to pH 6.0 ∼ 7.0 with dilute NaOH solution. The resulting green solution was refluxed with stirring for 7 h, heated on a steam bath to evaporate up to half volume and kept overnight at room temperature. A fine green coloured (I) product was obtained which was washed with ether and dried in a vacuum desiccators. All the remaining compounds i.e. II-VII was prepared according to the same method but by changing ligand L1 by L2–L7 respectively. Physicochemical parameters for all the synthesised compounds are summarized in .
Table I. Chemical and physical characteristics of the complexes.
Results and discussion
Composition and properties of the mixed-ligand complexes
All the synthesized complexes are stable to air for extended period of time and soluble in DMSO, slightly soluble in ethanol and water; insoluble in benzene, acetone, acetonitrile and diethyl ether. Elemental analyses of the complexes are in good agreement with theoretical expectation. They possess high melting points indicating that the complexes are stable in air.
Spectroscopic studies of the mixed-ligand complexes
Infrared spectroscopy
The ν(C = O) stretching vibration band appears at 1708 cm− 1 in the spectra of ciprofloxacin, while in mixed-ligand complexes this band shifted towards lower energy at 1619–1680 cm− 1 (), suggests that coordination occurs through pyridone oxygen atom [Citation24]. The absorption bands observed at 1624 and 1340 cm− 1 in ciprofloxacin are assigned to be ν(COO)asy and ν(COO)sym respectively, while in mixed-ligand complexes these bands observed at 1585–1590 and 1370–1374 cm− 1. The frequency separation (Δν = ν(COO)asy – ν(COO)sym) in investigated mixed-ligand complexes is greater than 200 cm− 1, suggests that the carboxylato group posses unidentate nature [Citation25]. The absorption bands observed at 527–531 cm− 1 are attributed to ν(M-N). The absorption bands observed at 426–428 cm− 1 and 513–521 cm− 1 are attributed to ν(M-O)(Carbo) ν(M-O)(Keto) respectively, while in case of complex VI the observed band at 470 cm− 1 is due to the ν(M-O)(phenolic). The sharp band in ciprofloxacin at 3520 cm− 1 [Citation26] is due to hydrogen bonding; which is attributed to ionic resonance structure and peak observed because of stretching vibration of free hydroxyl group. This band absolutely vanished in the spectra of mixed-ligand complexes indicates deprotonation of carboxylic proton. Of particular interest are the spectroscopic features of the conformers in the 1100-850 cm− 1 region, which after screening the bands due to ligand internal stretching, reveal interesting differences in their metal-terminal oxygen (V = Ot) vibrational modes. The metal-terminal oxygen (V = Ot) band observed at ∼1000 cm− 1 region suggesting the anti conformers and vanadium has a distorted square pyramidal structure [Citation27]. The complexes have one unpaired electron, characteristic of vanadyl unit, and a fairly high V = O stretching frequency in the infra red spectrum about 1000 cm− 1, suggesting that there is no ligand (or a weakly bound solvent) in the sixth position [Citation28]. Some prominent IR band frequencies of the compounds are provided in .
Table II. Infrared spectral data of the complexes.
Electronic spectroscopy and magnetic measurements
The UV–Vis. spectra of the complexes have been recorded using UV-160A UV–Vis. spectrophotometer, Shimadzu (Japan) (). The four absorption bands are observed in mixed-ligand complexes at ∼ 810 nm for dxy → dxz(band I), ∼ 590 nm for dxy → dyz (band II), at ∼ 540 nm for 
(band III), and ∼ 405 nm for dxy → dz2 (band IV). An examination of substantiates that the parameter Δ (λ1–λ2), related to the splitting of the dyz and dxz levels, can be used for establishing the geometrical distortion. In particular, Δ (λ1–λ2), increases with increasing distortion in the following order of complexes: IV (215 nm) < II (217 nm) < VI (220 nm) = V (220 nm) < III (248 nm). The mixed-ligand complexes exhibit magnetic moment of 1.69–1.73 B.M. This value is close to the spin-only value expected for s = 1/2 system (1.73 B.M.) and may be indicative of distorted square pyramidal geometry around the VO(IV) ion.
Table III. Electronic absorption parameters of VO(IV) complexes.
Thermal analysis
The thermogravimetric analyses for the mixed-ligand complexes were carried out within a temperature range from 20–800°C in N2 atmosphere at a rate of 10°C per minute in order to establish their compositional differences as well as to ascertain the nature of associated water molecules [Citation29]. The determined temperature ranges and corresponding percent mass loss accompanying the changes in the mixed-ligand complexes on heating revealed the following things. The TG curves of mixed-ligand complexes show three-decomposition steps respectively. It has been observed that all the mixed-ligand complexes show a loss in weight corresponding to two water molecules in the range of 50–130°C which indicating that these water molecules are water of crystallization. In second step weight loss during 130–420°C is corresponding to liberation of ligands and leaving behind the oxide of metal in the temperature range 420–710°C. Suggested structure of the complexes from the above analytical facts is given in .
Figure 1. Probable structure of complex(I).
Antimicrobial activity
The complexes exhibit strong activities against two Gram( − ve) i.e. Escherichia coli, pseudomonas aeruginosa and three Gram( + ve) Staphylococcus aureus, Bacillus subtilis, Serratia marcescens microorganisms. The results concerning in vitro antimicrobial activity (MIC) of the ligands and their complexes are represented in . The ligands (L1–L7) exhibit no antimicrobial activity. The antimicrobial activity of all the complexes against the five microorganisms is much higher than metal salt, while in competition with the ciprofloxacin. It was observed that all the complexes were more potent bacteriostatic than the ligands. The inhibition activity seems to be governed in certain degree by the facility of coordination at the metal centre as well as bulkiness of the ligands. This may support the argument that some type of biomolecular binding to the metal ions or intercalation or electrostatic interactions causing the inhibition of biological synthesis and preventing the organisms from reproducing. The results of our study indicate that the compounds II, III, IV and V have good activity, by displaying high affinities towards most of the bacteria. While the compounds VI and VII have good activity against S.merscences. The strong antimicrobial activities of these compounds against tested organisms suggest further investigation on these compounds.
Table IV. In vitro antimicrobial activity of the compounds (MIC μg/mL).
The studies on the binding of the title complex to DNA
Absorption spectroscopic titrations
The application of electronic absorption spectroscopy in DNA-binding studies is one of the most useful techniques Citation30,Citation31,Citation32. Complex binding with DNA through intercalation usually results in hypsochromism and bathochromism, due to the intercalative mode involving a strong stacking interaction between an aromatic chromophore and the base pairs of DNA [Citation33]. The absorption spectra of the complex in the absence and presence of DNA are illustrated in . Change in absorbance at peak maximum shows moderate hypsochromism shift (∼ 2 nm) for each complex with increasing concentration of DNA has been monitored for an evaluation of the intrinsic binding constant, which observed in the range of 5.0 × 102 to 6.6 × 105M− 1. (Inset for the plot for the calculation of intrinsic binding constant). The binding constants are given in . The binding constant of the complexes in increasing order is III < I < VI < II ≈ V < IV < VII. These spectral characteristics are consistent with a mode of interaction that involves a stacking interaction between the complex and the base pairs of DNA, which means that the titled complexes can intercalate into the double helix structure of DNA.
Figure 2. Absorption titration spectra of complex(I).
Table V. The binding constants (Kb) of VO(IV) complexes with DNA in phosphate buffer pH 7.2.
Viscosity studies
The interaction mode between the complexes and DNA is carried out by viscosity measurements. Hydrodynamic measurements that are sensitive to length change (i.e., viscosity and sedimentation) of DNA are regarded as the least ambiguous and the most critical tests of binding mode in solution in the absence of crystallographic structural data [Citation34,Citation35]. A classical intercalation mode results in lengthening the DNA helix, as base pairs are separated to accommodate the binding ligand, leading to the increase of DNA viscosity. The effect of the complexes on the viscosity of DNA is shown in . It is found that the viscosity of DNA increases steadily with the increase of the concentration of the complex, which is similar to that of a classical intercalator EB [Citation36]. This result demonstrates that the complexes and EB bind to DNA through the same way, i.e. the classical intercalation mode. The significant increase in viscosity of the complexes is obviously due to the partial insertion of the ligand in between the DNA base pairs leading to increase in separation of base pairs at intercalation sites and hence an increase in overall DNA contour length [Citation37,Citation38].
Figure 3. The effect of the complexes on the viscosity of pBR 322 DNA.
Cleavage of plasmid pBR322 DNA
The characterization of DNA recognition by transition metal complexes has been aided by the DNA cleavage chemistry that is associated with redox-active or photoactivated metal complexes. DNA cleavage is controlled by relaxation of supercoiled circular form of pBR322 DNA into nicked circular form and linear form. When circular plasmid DNA is conducted by electrophoresis, the fastest migration will be observed for the supercoiled form (SC). If one strand is cleaved, the supercoiled will relax to produce a slower-moving open circular form (OC). If both strands are cleaved, a nicked form (NC) will be generated that migrates in between. illustrates the gel electrophoretic separations showing the cleavage of plasmid pBR322 DNA induced by the complexes under aerobic conditions. With the increase of complex concentration, the circular supercoiled DNA is converted into nicked DNA via single strand cleavage (lanes 2–10). The obtained gel was analyzed for the relative concentration of three form of the pBR 322 DNA and the relative concentrations are mentioned in . This clearly shows that the relative binding efficacy of the complexes to DNA is much higher than the binding efficacy of metal salt itself or ciprofloxacin. The different DNA-cleavage efficiency of the complexes was due to the different binding affinity of the complexes to DNA, which has been observed in other cases [Citation39,Citation40,Citation41].
Figure 4. Gel electrophoresis data with pBR322 DNA (A) Lane 1: pBR322 (Control), Lane 2: pBR322+VOSO4, Lane 3:pBR322+Cip HCl, Lane 4: pBR322+I, Lane 5: pBR322+II, Lane 6: pBR322+III, Lane 7: pBR322+IV, Lane 8:pBR322+V, Lane 9:pBR322+VI, Lane 10: pBR322+VII.
Table VI. Gel electrophoresis data of the complexes.
Acknowledgements
We wish to express our gratitude to Prof. J. S. Parmar, Head, Department of Chemistry and Head, B & R Doshi School of Biosciences, Sardar Patel University, Vallabh Vidyanagar, Gujarat, India, for providing the necessary laboratory facilities. The authors are highly thankful to the UGC for financial assistance of UGC grant 32-226/2006(SR).
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References
- ND Chasteen. Vanadium in biological systems. Ed. Dordrecht, The Netherlands: Kluwer Academic Publishers; 1990.
- H Sigel, A Vanadium Sigel, and its Role in Life. In Metal ions in biological systems. Vol. 31. New York: Marcel Dekker; 1995.
- SW Taylor, B Kammerer, E Bayer. Chem Rev 1997;97:333.
- RE Berry, EM Armstrong, RL Beddoes, D Collison, SN Ertok, M Helliwell, CD Garner. Angew Chem Int Ed 1999;38:795.
- A Messerschmidt, R Wever. Proc NatlS Acad Sci USA 1996;93:392.
- A Butler, JU Walker. Chem Rev 1993;93:937.
- RD Joerger, T Loveless, RN Pau, LA Mitchenal, BH Simon, PE Bishop. J Bacteriol 1990;172:3400.
- J Chen, J Christiansen, RC Tittsworth, BJ Hales, SJ Georg, D Coucouvanis, SP Cramer. J Am Chem Soc 1993;115:5509.
- N Pau. In: MJ Dilworth, A Glenn. Biology and biochemistry of nitrogen fixation. Elsevier: Amsterdam, The Netherlands; 1991. p 37 ff.
- A Butler, CJ Carrano. Coord Chem Rev 1991;109:61.
- A Butler, MJ Clague, GE Meiser. Chem Rev 1994;94:625.
- D Rehder. Angew Chem Int Ed Engl 1991;30:148.
- JS Wolfson, DC Hooper. Fluoroquinolone antibacterial agents. Clin Microbiol Rev 1989;2:378.
- H Yoshida, M Bogaki, M Nakamura, S Nakamura. Antimicrob Agents Chemother 1990;34:1271.
- M Trucksis, JS Wolfson, DC Hooper. J Bacteriol 1991;173 (18):5854.
- PB Pansuriya, MN Patel, P Dhandhukia, V Thakkar. J Enz Inhib Med Chem 2007;22 (4):477–487.
- PB Pansuriya, MN Patel. J Enz Inhib Med Chem 2008;23 (1):108.
- BS Furniss, AJ Hannaford, PWG Smith, AR Tatchell. Vogel's textbook of practical organic chemistry. 5th Ed. London: ELBS and Longman; 2004.
- A Weiss, H Witte. Magnetochemie. Weinheim: Verlag Chemie; 1973.
- A Wolf, GH ShimerJr, T Meehan. Biochemistry 1987;26:6392.
- Jian-Zhong Wu, Yuan Li. J Inorg Biochem 2004;98:41.
- G Cohen, H Eisenberg. Biopolymers 1969;8:45.
- RN Jones, AL Barry, TL Gaven, JA Washington, EH Lennette, A Balows, WJ Shadomy. Manual of clinical microbiology. 4th Edn. Washington, DC: American Society for Microbiology; 1984. p 972.
- I Leban, I Turel, N Bukovec. J Inorg Biochem 1997;66:241.
- D Dobrzyńska, LB Jerzykiewicz, M Duczmal. Polyhedron 2005;24:407.
- RM Silverstein, FX Webster. Spectrometric identification of organic compounds. 6th Ed. New York: John Wiley & Sons, Inc; 2004.
- S Bhattacharya, S Mukhopadhyay, S Samanta, TJR Weakley, M Chaudhury. Inorg Chem 2002;41:2433.
- P Caravan, L Gelmini, N Glover, FG Herring, H Li, JH McNeill, SJ Rettig, IA Setyawati, E Shuter, Y Sun, AS Tracey, VG Yuen, C Orvig. J Am Chem Soc 1995;117:12759.
- SH Patel, PB Pansuriya, MR Chhasatia, HM Parekh, MN Patel. J Therm Anal Cal 2008;91 (2):413.
- JK Barton, AL Raphael. J Am Chem Soc 1984;106:2172.
- TM Kelly, AB Tossi, DJ McConnell, TC Strekas. Nucleic Acids Res 1985;13:6017.
- SA Tysoe, RJ Morgan, AD Baker, TC Strekas. J Phys Chem 1993;97:1707.
- D Lawrence, VG Vaidyanathan, Unni Nair Balachandran. J Inorg Biochem 2006;100:1244.
- S Satyanaryana, JC Daborusak, JB Chaires. Biochemistry 1993;32:2573.
- S Satyanarayana, JC Dabroniak, JB Chaires. Biochemistry 1992;31:9319.
- XL Wang, H Chao, H Li, XL Hong, YJ Liu, LF Tan, LN Ji. J Inorg Biochem 2004;98:1143.
- BC Baguley, M LeBret. Biochemistry 1984;23:937.
- L Lerman. J Mol Biol 1961;3:18.
- BK Santra, PAN Reddy, G Neelakanta, S Mahadevan, M Nethaji, AR Chakravarty. J Inorg Biochem 2002;89:191.
- M Navarro, EJ Cisneros-Fajardo, A Sierralta, M Fernández-Mestre, P Silva, D Arrieche, E Marchán. J Biol Inorg Chem 2003;8:401.
- XL Wang, H Chao, H Li, XL Hong, LN Ji, XY Li. J Inorg Biochem 2004;98:423.