DNA Binding Properties of Two Ruthenium ( III ) Complexes Containing Schiff Bases Derived from Salicylaldehyde : Spectroscopic and Electrochemical Evidence of CT DNA Intercalation

The interaction of CT DNA by two anionic Ru(III) complexes with N-substituted salicylidenimine ligands was investigated by spectroscopic titration and cyclic voltammetry. The result gives a surprising evidence for intercalation of DNA by the negatively charged complex species containing non typical intercalating ligands with Kb of order 10 M. Na[RuCl2(N-R-5-X-salim)2], where R represents butyl or phenyl and X = H, Cl, were characterized on the basis of elemental analysis, MALDI-TOF mass spectrometry, infrared, UV / visible spectroscopic measurements and cyclic voltammetry. (doi: 10.5562/cca2216)


INTRODUCTION
Ruthenium compounds have been the subject of great interest and impressive development in last decades for many reasons, especially due to their catalytic 1−4 and anticancer activities. 5−11 Ruthenium generally demonstrates affinity toward N-donor molecules such as proteins and DNA. The mechanism of the action of antitumor-active ruthenium compounds is not entirely known. Closely, it is thought that the complexes containing chlorides, or other easily leaving groups, can hydrolyze in vivo, allowing the covalent binding of the nucleobases from DNA to ruthenium. 12 Many Ru(II) and Ru(III) compounds with numerous different ligands are reasonably synthesized and investigated for the purpose of possible application in medicine and catalysis. There are no many complexes containing Schiff bases derived from salicyladehyde described in literature, although such ligands generally show considerable stereochemical flexibility and capability to tune the reduction potential of metal center, acting as N-or N,O ligands. 13,14 Some of them are described as very good catalysts in organic synthesis because the presence of salicylaldimine in the molecule increases the stability and catalytic activity. 15 Such ligands are derived from 2,4,6-tris substituted phenylamines and salicylaldehyde containing attached quaternary ammonium group to increase hydrophilic character. 16−18 Furthermore, some ruthenium complexes with salicyladi-mines showed significant biological activity against some bacteria, in contrast to the less active free ligands. 19 In addition, two dichloro-bis[N-phenyl-5substituted-salicylideniminato-N,O]ruthenate(III) are described as electrochemical mediators for the low potential amperometric determination of ascorbic acid. 20,21 We aimed to investigate interaction of Ru(III) complexes containing simple Schiff bases derived from salicylaldehyde with DNA and we report spectroscopic and electrochemical evidence of CT DNA intercalation by two dichloro-bis[N-substituted-5-X-salicylideniminato-N,O]ruthenate(III) compounds, hereinafter Na[RuCl 2 (N-R-5-X-salim) 2 ] where R represents butyl or phenyl, while X = H, Cl.

Physical Measurements
Elemental analyses were performed on a Perkin Elmer 2400 Series CHNS/O Analyzer. Mass spectra were obtained on a matrix-assisted laser desorption / ionization-time-of-flight MALDI-TOF/TOF mass spectrometer (4800 Plus MALDI TOF/TOF analyzer, Applied Biosystems Inc., Foster City, CA, USA) equipped with Nd:YAG laser operating at 355 nm with firing rate 200 Hz in the negative ion reflector mode. 1600 shots per spectrum was taken with mass range 10−1500 Da, focus mass 500 Da and delay time 100 ns. Small amount of sample (on pipette tip) was resuspended in 10 µl of DHB MALDI matrix (5 mg/mL; dissolved in 50/50 acetonitrile/water, v/v) and 1 µl was spotted on MALDI plate. The spectrum was internally calibrated providing measured mass accuracy within 5 ppm of theoretical mass riboflavin and 3-aminosalicylic acid were used as internal calibrants in negative ion mode.
The infrared spectra were recorded as KBr pellets on a Perkin Elmer spectrum BX FTIR System in the region 4000−400 cm −1 . UV/visible spectra, hydrolyses and CT DNA binding were measured on a Perkin Elmer lambda 35 spectrophotometer. Hydrolysis experiments were performed by adding the concentrated solution of complexes in DMSO to water buffered solutions (phosphate buffer, pH 7.5). The stock solution of CT DNA was prepared in Tris-HCl buffer at pH 7.4 and stored at 4 °C maximally 1−4 days. The concentration of DNA was calculated on the basis of extinction coefficient 6600 M −1 cm −1 at 260 nm. 23 The purification of DNA by phenol extraction methods improved the ratio of UV absorbance at 260 and 280 nm A 260 /A 280 ca. 1.8, indicating that DNA was satisfactory free from proteins. The absorption of DNA itself was removed by adding the same amount of DNA in reference solutions as in ruthenium complex-DNA solutions. Concentrated stock solutions of complexes were prepared by initial dissolving compounds in small amount of DMSO and diluting to the required concentrations.
Cyclic voltammograms of Na[RuCl 2 (N-R-5-Xsalim) 2 ] were recorded on an electrochemical workstation Autolab potentiostat/galvanostat (PGSTAT 12) in DMF (N,N-dimethylformamide) solution with sodium perchlorate as supporting electrolyte using glassy carbon working electrode and Ag/AgCl reference electrode in the range of potential of −1.5 to +0.1 V, with scan rate rate 0.8 V s −1 . Working electrode was polished prior to measurements with 1 μm diamond paste. Electrochemical titrations of ruthenium compounds with CT DNA were recorded by cyclic voltammetry in Tris-HCl buffered water solution (pH 7.4), at ambient temperature in 5 mL-volume conical compartment self-made cell. The volume of the cell is adapted to the experiments meaning to the required volumes of Na[RuCl 2 (N-R-5-X-salim) 2 ] solutions (2 mL) and added μL-amounts (0−60) of CT DNA.

Synthesis and Spectroscopic Studies
Synthesis Na[RuCl 2 (N-R-5-X-salim) 2 ] compounds were synthesized from RuCl 3 and freshly prepared ligands, butylsalicylidenime (R = C 4 H 9 , X = H) and N-phenyl-5-chlorosalicylidiemine (R= C 6 H 5 , H = Cl), in absolute ethanol solutions in molar ratio 1 : 2. The water suspended in liquid Schiff base was not removed before the use because the synthesis of final product was not affected by the presence of water; moreover it is known that, in some cases, the traces of water in butylsalicylidenimine assists replacement of leaving group by Schiff base in starting compound. 24

UV/Visible Spectroscopy
Electronic spectra of free Schiff bases and corresponding complexes were recorded in CH 2 Cl 2 solutions. Na[RuCl 2 (N-Bu-5-H-salim) 2 ] exhibits one broad absorption band centered about 415 nm arising from intraligand transition of whole molecule of Schiff base. Compared to free ligand , when Ru−N bond is formed, the lone pair on nitrogen atom becomes stabilized and absorption assigned to n → π* showed hypsohromic shift (blue shift) for 7 nm. In Na[RuCl 2 (N-Ph-5-Clsalim) 2 ] this absorption appears around 495 nm. Weakly defined broad absorption centered around 350 nm, assigned to the transition of -HC=N-group, is superimposed by Cl → Ru(III) LMCT transition in Na[RuCl 2 (N-Bu-5-H-salim) 2 ] while in phenyl derivative a well defined band appears around 338 nm. Weak broad absorption for both compounds, centered on 600 nm in the region of d-d spin allowed transition of low spin t 2g 5 Ru(III), can be assigned to ( 2 T 2g → 2 A 2g ). 25

Behavior in Solutions
Understanding of behavior of compounds in solution, e.g. hydrolysis and electron transfer processes are thought to be very important for potential biological activity and possible antitumor purpose of complex compounds. The hydrolyses of Na[RuCl 2 (N-R-5-Xsalim) 2 ] compounds under physiological condition in phosphate buffer (pH 7.5; 0.1 M NaCl) designates reasonable stabilization of ruthenium(III) after chelation and blue shift of the LMCT bands over the time.

Cyclic Voltammetry Measurements
Ru(III)/Ru(II) reduction potentials change with ligand environment and is thought to be very important for possible antitumor properties of a compound. The electronic effect of nitrogen from azomethine group and phenolate oxygen on reduction potential is different. More electronegative and smaller oxygen atom, hard in character stabilizes Ru(III), while nitrogen, as softer, prefers lower oxidation state. Kinetically, Ru(II) compounds are more labile than Ru(III) which are believed to can be activated by reduction in vivo. Few compounds like gluthation in cells and ascorbic acid in blood are thought to contribute to the reduction in vivo.  2 ], in addition to peak-topeak separation values and apparent reduction waves as result of stabilization of Ru(III) after coordination trough phenolic oxygen from salycilidenimine, 27,28 suggest the quasi-reversible one-electron transfer processes ( Figure 4, Table 1).

Spectroscopic Study
Interaction of metal complex compounds with DNA is taken as important initial signal about possible evaluation of biological properties of a compound. Activity of ruthenium compounds toward DNA, as a key target for anticancer drugs, may originate from either their covalent interaction with DNA nucleobases or non-covalent binding such as electrostatic interaction of positively charged species with phosphate backbone and intercalation as well. Electronic absorption is very useful method to determine the binding properties of metal complexes   Weak bathochromic shifts, hypochromism and constant binding values of order 10 4 M −1 indicate that both compounds, containing Schiff bases derived from salicylaldehyde, act as moderate DNA-intercalators. 30 Electrochemical Study Electrochemical method is useful complement method for UV / visible spectroscopic investigation of metal complex intercalative mode binding to DNA. Here we describe electrochemical measurements of redox couples Ru III complex / Ru II complex in the presence of increasing amounts of CT DNA (Figures 7 and 8, Tables 2  and 3).
Shifts of E 1/2 for both compounds toward more positive values, with increasing concentration of added DNA, suggest different ability of Ru(II) and Ru(III) compounds to bind DNA as a result of intercalation mode of binding. Changes in peak-to-peak separation, after addition of DNA, indicate an increase in reversibility of one-electron redox processes.

CONCLUSION
Some ruthenium complexes with planar and fused aromatic π-electronic acceptor system or similar aromatic heterocyclic rings are described as DNA intercalators which are able to keep base pair separated distorting double helix structure of DNA. 31,32 Less extended planar ligands may cause partial intercalation due to omitting of the ancillary ligands and phosphate backbone e.g. in the cases of some 1,10-phenanthroline compounds. 33,34 Although two easily leaving chlorides in the structures of Na[RuCl 2 (N-R-5-X-salim) 2 ] do not exclude a priori covalent binding or even electrostatic interaction of neutral and positively charged complex species produced by loss of chlorides, our spectroscopic and electrochemical investigations suggest an intercalative mode of binding with constant of order 10 4 M −1 . Possibly, this is the first report on anionic Ru(III) compounds with Schiff bases derived from salicyladehyde and simple amines which act as DNA intercalators.