Bioactive M(II) Complexes of Amino Acid Based N3odonor Mixed Ligand: in Vitro Andin Silico

Bioactive M(II) complexes of amino acid based N3Odonor mixed ligand: In vitro andIn Silico DNA binding studies

Violet Dhayabaran V, Daniel Prakash T

PG and Research Department of Chemistry (DST-FIST Sponsored), Bishop Heber College (Autonomous), Tiruchirappalli – 620 017, Tamilnadu, India.

*Corresponding author: Tel.: +91-9994291637; fax: 0431-277029

E–mail address: (Violet Dhayabaran)

Supporting Material

Tables of Contents

S1. Procedure for DNA binding, cleavage, antioxidant, antimicrobial and molecular docking studies

Fig. S2. FT-IR spectrum of the ligand (L) and complexes 1 – 3

Fig. S3. UV-Visible absoprtion spectrum of the ligand (L) and complexes 1 – 3

Fig. S4.1H-NMR spectrum of the Schiff base ligand (L) and complex3

Fig. S5.13C-NMR spectrum of the Schiff base ligand (L) and complex3

Fig. S6. ESI-Mass spectrum of the ligand (L)

Fig. S7. ESI-Mass spectrum of complex 1

Fig. S8. ESI-Mass spectrum of complex 2

Fig. S9. ESI-Mass spectrum of complex 3

Fig. S10.X-band ESR spectra of complex 2at liquid nitrogen temperature

S1. Procedure for DNA binding, cleavage, antioxidant, antimicrobial and molecular docking studies

2.4.1. DNA binding experiments

The DNA binding studies were performed at room temperature by electronic absorption spectrometric experiments and were conducted with fixed concentrations of the compounds, while gradually increasing the concentration of CT-DNA.All experiments involving CT-DNA were performed in Tris–HCl buffer solution (50 mM, pH~7.2) (Tris-HCl Tris(hydroxymethyl)aminomethanehydrochloride). The CT-DNA solution gave a UV absorbance at 260-280 nm in a 1.8:1 ratio indicating that the DNA is adequately free from protein [1]. The concentration of DNA was determined by absorption spectroscopy using the ε value of 6600 M-1 cm-1 at 260 nm [2].When measuring the absorption spectra, an equal amount of DNA was added to both complex and the reference solution to eliminate the absorbance of DNA itself.All solutions were allowed to equilibrate for 5 min before measurements were made.Electrochemical titration experiments were done by keeping the concentration of the complexes constant while varying CT-DNA concentration using the solvent of Tris–HCl buffer. Solutions were deoxygenated by purging with N2 prior to measurements. The CD spectra were recorded by keeping the concentration of DNA (5 × 10-4 M) constant while varying the complex concentration from 0 to 3 × 10-5 M at room temperature with a quartz cell of 1 cm path length. Each sample solution was scanned in the range of 220–320 nm. Viscosity measurements were made using an Ubbelodhe viscometer at a constant temperature (25.0 ± 0.1 oC). CT-DNA samples of approximately 0.5 mM were prepared by sonicating in order to minimize the complexities arising from CT-DNA flexibility [3]. The compounds (1–10 µM) were titrated into the CT-DNA solution (10 µM) present in the viscometer. The flow time of each sample was measured by a digital stopwatch for three times, and an average one was calculated. Relative viscosities for CT-DNA in the presence and absence of the [CuL2Cl2]complex was calculated from the relation η = t – t0, where t is the observed flow time of DNA containing solution and t0 is that of Tris–HCl buffer alone. Data were presented as (η/η0)1/3versus binding ratio [4, 5] where where η and η0 are the viscosity of CT-DNA in the presence and absence of complex, respectively.

2.4.2. DNA cleavage studies (gel electrophoresis)

pBR322 plasmid DNA was used for all cleavage activities. In a typical experiment, the samples were subjected to electrophoresis on 1% agarose gel prepared in TBE buffer (45 mM Tris, 45 mM boric acid and 1 mM EDTA, pH~7.3) and it was incubated for 30 minutes at 37 oC. Then 20 µl from each of the incubated complex and DNA mixture was loaded on the gel with tracking dye (0.25% bromophenol blue, 40% sucrose, 0.25% xylene cyanole, and 200 mM EDTA). 5 µl H2O2 (5 mM) was added to mixture to oxidize the reactant. The gel was run at 100 V for 3 h in TBE buffer and photographed under UV light.

2.4.3. DPPH free radical scavenging assay

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the compounds was measured according to the method of Blois [47]. Various concentrations (10, 50 and 100 µg) of the test sample and the standard (ascorbic acid) were taken and the volume was adjusted to 100 mL with methanol. 5 mL of a 0.1 mM methanolic solution of DPPH was added to these tubes and shaken vigorously. DMSO was used as negative and ascorbic acid was used as positive control, respectively. The tubes were allowed to stand in the dark for 30 min at 27 °C. After incubation, the absorbance of the samples was measured at 517 nm. Percent scavenging of DPPH free radical for each concentration of each compound was calculated using the following formula [6].

% of free radical scavenging activity = {(Acontrol - Asample) /Acontrol} × 100

where Acontrol = absorbance of DPPH solution at 516 nm, Asample = absorbance of DPPH solution containing metal complex at 516 nm.

2.4.4. Antimicrobial study

The in vitro evaluation of antimicrobial activity was carried out to provide antimicrobial efficiency of the synthesised ligand and the complexes. The antimicrobial effect was tested against Gram-positive Bacillus subtilis, Staphylococcus aureus and Gram-negative Escherichia coli, Pseudomonas. aeruginosa and in vitro antifungal activity against Candida parapsilosis, Candida tropicalis, Candida kefyr and Candida albicansand the minimum inhibitory concentration (MIC) was measured.Ciprofloxacin and Nystatin were chosen as standards for antibacterial activity and antifungal activity, respectively. The nutrient agar and dextrose agar in petri plates were served as the medium for the growth of bacteria and fungi respectively. The DMSO solution of the complex was soaked in a filter paper disc of 5 mm diameter and 1 mm thickness. These discs were placed on the formerly seed plates and incubated at 37 oC. After 24 h., the inhibition zone around each disc was measured. Each test was carried out in triplicate in individual experiments and the average was reported.

2.4.5. Molecular docking study

MGL tools 1.5.4 with AutoGrid4 and AutoDock4 [7,8] were used to set up and perform blind docking calculations between the synthesised compounds and DNA sequence. The crystal structure of CT-DNA sequence d(CGCGAATTCGCG)2 dodecamer (PDB ID: 1BNA) was acquired from the Protein Data Bank. Receptor (DNA) and ligand (complex) files were prepared using AutoDock Tools. Lamarckian genetic algorithms, as implemented in AutoDock, were used to perform docking calculations. For each of the docking cases, the lowest energy docked conformation, according to the AutoDock scoring function, was selected as the binding mode. The output from AutoDock was rendered with PyMol [9].

References

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[9] DeLano WL (2004) The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA, USA. <