Spectroscopic Investigations , Anti-bacterial Activities and DNA-interactions of Metal Complexes ( Cr ( III ) , Zn ( II ) , Ni ( II ) ) Containing Phendione Ligand

The transition metal complexes [Cr(acac)2(phendione)]Cl, [Zn(acac)2(phendione)], [Ni(acac)2(phendione)], [Zn(phendione)3](ClO4)2 and [Ni(phendione)2](ClO4)2 were synthesized from 1, 10-phenanthroline-5, 6-dione (phendione). The phendione ligands and its metal complexes were characterized by FTIR, UV-Visible spectroscopy, PL study and in some cases by GC-MS. The red emission band of complex [Zn(acac)2(phendione)] was observed by excitation at 250 nm. DNA binding activities of the complexes were studied by DNA Gel Electrophoresis. DNA binding studies of the synthesized compounds showed that the metal complexes are strong DNA binding and cleaving agents. The [Ni(phendione)2] (ClO4)2 complex was the most effective DNA binding and cleaving agent and [Cr(acac)2(phendione)]Cl complex shows relatively lower affinity to the DNA. The metal complexes easily cause DNA degradation or cleavage (after 2.5 h) with genomic DNA rather than plasmid DNA. Antimicrobial activity studies revealed that all metal complexes of phendione showed efficient resistance to bacteria. The [Cr(acac)2(phendione)]Cl complex showed most potential antimicrobial activities against the bacteria Escherichia coli with an annular radius of the inhibitory zone, 4.33.


Introduction
The development of new materials that exhibit extensive optical and biological activities such as DNA binding, DNA cleaving, antimicrobial sensibility have become potential for the last few decades in the medicinal and pharmaceutical fields.The metal complexes with the ligand containing heterocyclic ring systems are very promising candidates for such applications [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17].The ongoing interests in the pharmacological field for the development of better therapeutic agents which can bind and damage DNA is rationalized by the fact that many diseases, including cancer occur because of aberrant gene expression.Transition metal complexes are the potential candidates in this field due to their tunable coordination environments and versatile physiochemical properties for designing highly sensitive diagnostic agents for medicinal applications as exemplified by the chemotherapeutic agents like the cis-platin or bleomycins.In our present work we intended to synthesize some transition metal complexes containing phendione.We have chosen 3d transition metals namely Cr, Zn, and Ni because these metals are bio-essential and ubiquitous in biological systems.The reason for choosing the phendione ligand is that metal complexes of this ligand have been reportedly show huge biological activities such as DNA binding and damaging activities and also their metal complexes have been reported to show NLO (Nonlinear Optical) properties for device applications.In addition phendione is an amazing chelating ligand that can form monomeric as well as polymeric coordination complexes.The term "phenanthroline" is used to denote any one of the three heterocyclic ring systems.Our recent study is concerning phendione ligands, which is originated from phenanthroline [2,3].Phendione based metal complex chemistry has been growing rapidly due to promising electrical, optical and biological applications [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23].In our present study we reported the synthesis of several metal complexes containing phendione ligand with their characterization, optical as well as biological properties.

Synthesis of [Cr(acac) 2 (phendione)]Cl complex
[Cr(acac) 2 Cl]•2H 2 O was synthesized using a similar method [25].In an ethanolic solution of [Cr(acac) 2 Cl]•2H 2 O (0.020 g, 0.07 mmol) taken in a round bottomed flask a chloroform solution of phendione (0.0148 g, 0.07 mmol) was added drop wise.This solution was stirred at room temperature for 6 h.After completion of the reaction the solution was kept for the evaporation of the solvent or solvent was reduced by a rotary evaporator and allowed to stand for several days.This was collected by filtration and washed with dichloromethane and recrystallized from methanol.GC-MS peak was observed at m/z 435.

Synthesis of [Zn(acac) 2 (phendione)] complex
Zn(acac) 2 •H 2 O was synthesized according to the literature [26].In an ethanolic solution of Zn(acac) 2 •H 2 O (0.01248 g, 0.07 mmol) taken in a round bottomed flask a chloroform solution of phendione (0.0148 g, 0.07 mmol) was added drop wise.This solution was stirred at room temperature for 6 h.After completion of the reaction the solution was kept for the evaporation of the solvent or solvent was reduced by a rotary evaporator and allowed to stand for several days.This was collected by filtration and washed with chloroform.GC-MS peak was observed at m/z 447.

Synthesis of [Ni(acac) 2 (phendione)] complex
Ni(acac) 2 •2H 2 O was synthesized according to the literature method [27].In an ethanolic solution of Ni(acac) 2 •2H 2 O (0.01362 g, 0.07 mmol) in a round bottomed flask a chloroform solution of phendione (0.0148 g, 0.07 mmol) was added drop wise.This solution was stirred at room temperature for 6 h.After completion of the reaction the solution was kept for the evaporation of the solvent or solvent was reduced by a rotary evaporator and allowed to stand for several days.This was collected by filtration and washed with chloroform.GC-MS was observed at m/z 441.

Synthesis of [Zn(phendione) 3 ](ClO 4 ) 2 complex
In an aqueous solution of ZnCl 2 (137 mg, 1 mmol) taken in a round bottomed flask 20 mL ethanolic solution of phendione (157.5 mg, 3 mmol) was slowly added.The resulting solution was stirred at room temperature for 24 h.The complex was precipitated as a pale yellow solid by addition of sodium perchlorate (150 mg, 3 mmol).The product [Zn(phendione) 3 ](ClO 4 ) 2 was collected by suction filtration, washed with cold water and diethylether and then air dried [28].

Antimicrobial activities study
Antimicrobial activities of the synthesized metal complexes were performed against both Gram-negative (Eschericha coli, Proteus mirabilis, klebsiella pneumonia and pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria.The antibacterial activity was studied by modified Kirby-Bauer disk diffusion method.In brief, the pure cultures of organisms were sub cultured in nutrient broth at 37°C on a rotary shaker at 120 rpm.For bacterial growth, a lawn of culture was prepared by spreading the 20 µL fresh culture having 10 6 colony-forming units (CFU)/mL of each test organism on nutrient agar plates with the help of a sterile glass-rod spreader.Plates were left standing for 10 min to let the culture get absorbed.Then 6 mm wells were punched into the nutrient agar plates for testing antimicrobial activity.Wells were sealed with one drop of molten agar (0.8% agar) to prevent leakage of compounds from the bottom of the wells.Using a micropipette, 50 µL (500 µg) of the sample compounds suspension was poured onto each of five wells on all plates.After overnight incubation at 37°C the different levels of zones of inhibitions were measured [29].

Results and Discussions
Phendione was synthesized by the oxidation of 1, 10-phenanthrolines using KBr and mixture of conc.sulfuric and nitric acids as oxidant.Characterization of phendione was accomplished by melting point, UV-visible and IR spectroscopic methods.Our results matched well with those of literature [8].Melting point of phendione was found to be 258-260°C which is consistence with the literature.The electronic spectrum of phendione recorded in acetone solution exhibited two absorption bands in the UV region.The absorption band at 359 nm is assigned to n → π* of C=N bonds.The strong absorption band centered at 451 nm is assigned to n → π* of the C=O group of phendione ligand.IR spectrum of phendione clearly exhibited characteristic peaks at 1688 cm -1 and at 1564 cm - 1 which are ascribable to a stretching frequency of the C=O and C=N bands respectively in the ligand [9].
In chromium complex [Cr(acac) 2 Cl]•2H 2 O characteristic absorption peaks appeared at around 535 nm and 388 nm can be assigned to E→T 2g and n transitions respectively.For the [Cr(acac) 2 (phendione)]Cl complex the characteristic absorption peaks at around 541 nm and 451 nm can be assigned to the E→T 2g and n π * transitions respectively (Fig. 1).This confirms the presence of chromium in the complex.The FTIR spectrum of phendione clearly exhibited stretching frequency of the C=O band at 1689 cm -1 .This band is shifted in the [Cr(acac) 2 (phendione)]Cl complex and found at 1703 cm -1 which is reasonable since the C=O moieties are far removed from the site of coordination of this ligand with the metal ion [9,10].In IR spectrum of the starting chromium complex [Cr(acac) 2 Cl]•2H 2 O this new band is absent, which confirms the presence of phendione in the [Cr(acac) 2 (phendione)]Cl complex.
The electronic spectrum of [Zn(acac) 2 (phendione)] complex recorded in acetone solution exhibited two absorption bands in the UV-Visible region.The absorption band at 325 nm (a) is assigned to n → π* of C=N bonds.The strong absorption band centered at 451 nm (b) is assigned to n→π* of the C=O group of phendione ligand (Fig. 2).Fig. 2. Absorption spectra for the [Zn(acac) 2 (phendione)] complex.
The C=O band is also found to be shifted in the [Zn(acac) 2 (phendione)] complex compared to that of phendione and found at 1716 cm -1 .In IR spectrum of the starting complex Zn(acac) 2 •2H 2 O this band is not observed.It may prove the presence of phendione in coordination to Zn(II) in the [Zn(acac) 2 (phendione)] complex (Fig. 3).[Zn(phendione) 3 ](ClO 4 ) 2 complex was synthesized by the reaction of ZnCl 2 •6H 2 0 and phendione maintaining a molar ratio of 1:3.The electronic spectrum of the complex that was recorded in acetonitrile solution (Fig. 4) exhibited one band in the visible region and three absorption bands in the UV region.The strong absorption band centered at 487 nm (a) is assigned to n π* of the C=O group of phendione ligand.The three bands centered at 253, 293 and 314 nm (b) are assigned to ligand centered (π π*) transitions [6,[20][21][22].The solvent dependency of n π* energy on the solvent polarity, a well known phenomenon, is the reason behind such an assignment [17,22].Compared with that of Zn(acac) 2 •2H 2 O complex the FTIR spectrum of the [Zn(phendione) 3 ](ClO 4 ) 2 complex confirms the existence of the phendione ligand.The IR band is observed to be shifted in the [Zn(phendione) 3 ](ClO 4 ) 2 complex and found at 1696 cm -1 compared to the C=O band of phendione at 1689 cm -1 .This band is absent in starting Zn(acac) 2 •2H 2 O complex, which proves the presence of phendione in [Zn(phendione) 3 ](ClO 4 ) 2 .Besides, the very strong absorption at around 640 cm -1 , the bands at 940 cm -1 and 1090 cm -1 are assigned to ν as (ClO 4 -), ν s (ClO 4 -) and δ(ClO 4 -) of ionic perchlorate in [Zn(phendione) 3 ](ClO 4 ) 2 .

Wavelength (nm)
A bs orbance complex.Thus, IR as well as UV-Visible spectra studies confirmed the formation of metal complexes with phendione and acac ligands.
The strong blue emission band at 427 nm is observed.PLE spectrum with several bands at 350, 375, 395, 428 and 461 nm are observed when monitored at 510 nm (Fig. 10).

PL spectra of [Ni(phendione) 2 ](ClO 4 ) 2 complex
PL spectra of [Ni(phendione) 2 ](ClO 4 ) 2 complex was also measured in acetone (Fig. 11).Emission bands are observed at 403, 430 and 454 nm when excited at 355 nm [21].The blue emission bands are observed at 430 and 454 nm.The complex also showed emission bands at 374 nm in UV region and at 403 nm in visible regions (violet emission).Emission bands are observed at 406, 429 and 460 nm when excited at 379 nm [21].The blue emission bands are observed at 406, 429 and 460 nm.PLE bands are observed at 332, 379 and 392 nm when monitored at 429 nm [21].This complex showed PL spectra mostly in visible region.

PL spectra of [Ni(acac) 2 (phendione)] complex
PL spectra of [Ni(acac) 2 (phendione)] was measured in acetonitrile (Fig. 12).Emission bands are observed at around 335, 395 and 435 nm when excited at 298 nm [21].The strongest blue emission band is observed at around 435 nm.The emission bands of metal complex are observed at 360, 370, 410, 428 and 455 nm when excited at 340 nm.Excitation at 410 nm provided emissions at 440, 453 and 470 nm.Therefore, the metal complexes with phendione and acac ligands showed varieties of characteristic PL peaks in UV, Visible and NIR regions in solution.The compounds that showed PL peaks in NIR regions may be assigned to octahedral geometry.

Antibacterial activity assay
The antibacterial activities of synthesized compounds were tested against five bacterium, namely Proteus mirabilis(a), Escherichia coli(b), Pseudomonas aeruginosa(c), Klebsiellapneumoniae(d) and Staphylococcus Aurius(e).The annular radius of the inhibitory zones created by the antibacterial agents (tested compounds) was measured [29].The results of the antibiogram sensing ability of the synthesized compounds are given below in the following Table 2 and Fig. 13 for [Cr(acac) 2 (phendione)]Cl complex.
The [Cr(acac) 2 (phendione)]Cl complex showed highest antibacterial activities against Escherichia coli (Table 2 and Fig. 13).The antibacterial activity results revealed that the [Zn(acac) 2 (phendione)] complex showed highest antibacterial activities against Klebsiellapneumoniae.The highest antibacterial activity against Staphylococcus aureus is observed for [Zn(phendione) 3 ](ClO 4 ) 2 complex.The [Ni(phendione) 2 ](ClO 4 ) 2 complex showed highest antibacterial activities against Staphylococcus aureus.It is obvious from the antibacterial activity testing result that phendione related complexes showed enormous antibacterial activities.The huge antibacterial activities of the phendione related compounds can be attributed to the fact that phendione has free quinoid functional group with redox properties which can cause the bacterial cell breaking and binding to the protein synthesizing enzyme thus inhibiting the protein synthesis for bacterial growth resulting in the death of microbes.The [Cr(acac) 2 (phendione)]Cl complex showed the highest antibacterial activities among the metal complexes under investigation.The [Cr(acac) 2 (phendione)]Cl complex showed most potential antimicrobial activities against the bacteria Escherichia coli with an annular radius of the inhibitory zone, 4.33 and then against Pseudomonas aeruginosa (4.17) and Staphylococcus aureus(4.17),respectively.

DNA binding studies
DNA binding activities of the synthesized complexes were studied by agarose gel electrophoresis method [17].The DNA binding was studied by varying time for the metal complexes of the phendione ligand.DNA was extracted from bacteria by extraction kit and the binding activity of the synthesized products were studied for both genomic and plasmid DNA.The result is shown in Fig. 14.It is apparent that all the metal complexes under investigation bind to DNA effectively.As seen in Lane 5, [Cr(acac) 2 (phendione)]Cl complex intercalate into DNA less strongly then other complexes.It is also evident that Zn and Ni complexes not only intercalate into DNA strongly but also cause DNA degradation or cleavage after 2.5 h.
The DNA binding activities of the metal complexes were also studied after 2.5 h of incubation with plasmid DNA.From the result it is obvious that the metal complexes bind to DNA such weakly that after 2.5 h cannot intercalate into DNA much except [Ni(phendione) 2 ](ClO 4 ) 2 .It is observed that [Ni(phendione) 2 ](ClO 4 ) 2 complex not only intercalate into DNA but also causes DNA degradation or cleavage after 2.5 h.This implies that [Ni(phendione) 2 ](ClO 4 ) 2 complex is a better intercalated into DNA more rapidly than does the other complexes in case of plasmid DNA.The [Ni(phendione) 2 ](ClO 4 ) 2 complex bind to plasmid DNA mostly through electrostatic interaction between the phosphate groups of DNA and the N-H group of the ligand.From the above results, we can come to the conclusion that most of the metal complexes under study easily cause DNA degradation or cleavage after 2.5 h with genomic DNA rather than plasmid DNA.The DNA binding activities of the metal complexes were also studied after 16 h of incubation with plasmid DNA.This result is shown in the Fig 16 .We can see that all complexes not only intercalate into DNA but also cause DNA degradation or cleavage after 16 h.In summary, all the metal complexes can easily cause DNA degradation or cleavage after 16 h with plasmid DNA.
These apparent results can be rationalized in terms of both intercalation and degradation of DNA by the metal complexes under investigation.From the DNA binding study we observed that, the most effective intercalator is [Ni(phendione) 2 ](ClO 4 ) 2 complex, whose intercalation to DNA causes the inhibition of DNA intercalation of ethidium bromide.Thus the DNA band appears to be almost absent.All the metal complexes also effectively degrade or cleave DNA strands which are apparent from increased mobility of the DNA bands.Intercalation of metal complexes to plasmid DNA is observed after 16 h of incubation.However, in case of genomic DNA most metal complexes except [Cr(acac) 2 (phendione)]Cl intercalated rapidly only after 2.5 h of incubation.

Conclusion
The transition metal complexes [Cr(acac) 2 (phendione)]Cl, [Zn(acac) 2 (phendione)], [Ni(acac) 2 (phendione)], [Zn(phendione) 3 ](ClO 4 ) 2 and [Ni(phendione) 2 ](ClO 4 ) 2 were synthesized from phendione.The phendione ligands and its metal complexes were characterized by FTIR, UV-Visible spectroscopy and PL study and in some cases by Mass.The red emission band of complex [Zn(acac) 2 (phendione)] was observed by excitation at 250 nm.DNA binding activities of the complexes were studied by DNA Gel Electrophoresis.DNA binding studies of the synthesized compounds showed that the metal complexes are strong DNA binding and cleaving agents.The [Ni(phendione) 2 ] (ClO 4 ) 2 complex was the most effective DNA binding and cleaving agent and [Cr(acac) 2 (phendione)]Cl complex showed relatively lower affinity for DNA binding and cleaving.The metal complexes easily caused DNA degradation or cleavage (after 2.5 h) with genomic DNA rather than with plasmid DNA.Intercalation of most of the metal complexes to plasmid DNA was observed after 16 h of incubation.Antimicrobial activity studies revealed that, all metal complexes of phendione showed efficient resistance to bacteria.The [Cr(acac) 2 (phendione)]Cl complex showed most potential antimicrobial activities against the bacteria Escherichia coli with an annular radius of the inhibitory zone, 4.33 and then against Pseudomonas aeruginosa (4.17) and Staphylococcus aureus(4.17),respectively.The transition metal complexes can cause the bacterial cell breaking and binding to the protein synthesizing enzyme, thus inhibiting the protein synthesis for bacterial growth resulting in the death of microorganisms.

Fig. 15
Fig.15shows that the metal complexes also bind to genomic DNA effectively after 16 h of incubation and only Lane 5, [Cr(acac) 2 (phendione)]Cl complex intercalate less into DNA.The Zn and Ni complexes not only intercalate into DNA but also cause DNA degradation or cleavage after 16 h.The DNA binding activities of the metal complexes were also studied after 16 h of incubation with plasmid DNA.This result is shown in the Fig16.We can see that all complexes not only intercalate into DNA but also cause DNA degradation or cleavage after 16 h.In summary, all the metal complexes can easily cause DNA degradation or cleavage after 16 h with plasmid DNA.These apparent results can be rationalized in terms of both intercalation and degradation of DNA by the metal complexes under investigation.From the DNA binding study we observed that, the most effective intercalator is [Ni(phendione) 2 ](ClO 4 ) 2 complex, whose intercalation to DNA causes the inhibition of DNA intercalation of ethidium bromide.Thus the DNA band appears to be almost absent.All the metal complexes also effectively degrade or cleave DNA strands which are apparent from increased mobility of the DNA bands.Intercalation of metal complexes to plasmid DNA