Intercalating Compound And Determination Of Dna Binding Biology Essay

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Pharmachemistry Report for 2010. In this research we have made a new plantinum based drug, developed at the University of Western Sydney and is called as phenSS[1,10-Phenanthroline](1S,2S-diaminocyclohexane ) plantinum(II) Cl2]. The drug shows high degree of potential as an anticancer agent. The drug is also analyzed for its purity and identification by UV and NMR spectoscopy. The partition coefficient of the drug is also determined to analyze critically how easily it can cross cell membranes after dissolution into hydrophobic fluids like fats or lipids. We also analyzed the effect of the substituent on the hydrophobicity factor. The Kirby -Bauer disc test was used to determine the antibacterial activity of the drug. The drug is completely investigated by testing under this method by taking many possible combination of a series of bacteria and agar medium. Deoxyribonucleic acid, DNA, is a molecule of great biological significance. The site at which drug targets the DNA can activate or inhibit DNA Molecule protein synthesis. We studied the DNA binding constant to analyze how the drug targets in inhibitory mode, to destroy cells for antitumor and antibiotic action.

It has been proved that a number of factors had a substantial influence on drug activity. For example a substituent that had a particular effect on one of these factors with one class of drug, might have the same effect on the other drug. Known covalent intercalation between the base pairs of DNA and positively charged metallointercalators has been an area of wide study. A series of square planar platinum(II) compounds incorporating methylated derivatives of phen, 4-methyl-1,10-phenanthroline (4-Mephen), 5-methyl-1,10-phenanthroline (5-Mephen), 4,7-dimethyl-1,10-phenanthroline (4,7-Me2phen), 5,6-dimethyl-1,10-phenanthroline (5,6-Me2phen) and 3,4,7,8-tetramethyl-1,10-phenanthroline (3,4,7,8-Me4phen) were synthesised and the relationship between their structure and biological activity investigated. The biological activity of these compounds was quantified using the in vitro cytotoxicity assay against the Murine leukaemia cell line. Large variation in cytotoxicities with different methylation was observed. The 5- and 5,6-methylated derivatives of phen displayed a greater biological activity. Binding constants were determined using circular dichroism spectroscopy (CD) and induced circular dichroism (ICD). ICD was used to highlight any differences in the spectra.

The metal complexes studied: (a) [Pt(en)(phen)]2 , (b) [Pt(en)-(4-Mephen)]2 , (c) [Pt(en)(5-Mephen)]2 , (d) [Pt(en)(4,7-Me2phen)]2 ,(e) [Pt(en)(5,6-Me2phen)]2 and (f ) [Pt(en)(3,4,7,8-Me4phen)]2 .

It is proved that DNA properties can be influenced by intercalation and are reported as important steps for studying the methods used to change the genetic information of an organism. However, this is also true that not all compounds that intercalate with DNA show anti tumor activity as many clinically tested drugs are capable of intercalative binding. In this experiment, the effect of substituent is also tested hypothetically that how the position of the substituent methyl groups or the number of methyl groups may determine the difference in biological activity of the drug.

Drastic effects are observed on the hydrophobic nature of the drug and on its activity due to different substituents. Hydrophobic nature of a drug is important as it controls the dissolution of the drug into fats or lipids. In this experiment, we determine the hydrophobicity of the drug by examining its partition coefficients in water and octanol.

Partition Coefficient

P= [Molar Concentration in octanol] / [Molar concentration in water]

The antibacterial activities of the drug is determined by the Kirby-Bauer Disc Bioassay.

In this experiment, the rates of diffusion of different sample drugs through the agar media is studied to determine the intercalation between the types of tested samples and bacteria / agar environment. It also determines the effect of bacterial environment on the tested sample on the basis of diameter of inhibition zones.

Alison Rodger, Bengt Nordén,(1997) Circular Dichroism and Linear Dichroism, Oxford University Press.Victor A. Bloomfield, Donald M. Crothers, Ignacio Tinoco,(2000), University Science Books.



The melting points were determined (uncorrected) on a Gallenkamp Melting Point apparatus. UV spectra were recorded on a Varian Cary 1E Spectrophotometer and CD spectra were obtained with a Jasco J-810 Spectropolarimeter both at room temperature.


Potassium tetrachloroplatinate(II) (K2[PtCl4]) was purchased from Precious Metals Online. ct-DNA, Trizma (tris(hydroxymethyl)aminomethane hydrochloride) and all deuterated solvents were purchased from Aldrich Chemical Company. CM-25 Sephadex and Waters C-18 Sep-pak columns were obtained from Pharmacia. Aqueous solutions were prepared with Milli-Q water (Millipore, MA). Oligonucleotides were purchased from Geneworks Inc. All other reagents and solvents were of analytical grade.


Synthesis of Plantinum Based Anticancer compound, [(1,10- Phenanthroline)(1S,2S-diaminocyclohexane)platinum(II)]2+(PhenSS)

K2PtCl4(~ 0.4 g) and the ancillary ligand (1S,2S-diaminocycloxane, S,S- dach, 1 mol. equiv.) were stirred together at room temprature in water (30 mL). The stirring was carried out during the practical and the compound was then left in the fridge till the practical session. A yellow precipitate was formed which was collected by filtration, washed with water, ethanol and diethyl ether.

The metal complex [Pt(S,S-datch)Cl2] was then refluxed in 100mL water with the intercalating ligand (1,10-phenanthroline,phen,1.1 mol. equiv. for 24 hours. The clear, yellow solution was then filtered and concentrated by rotary evaporation to 20 mL. A Waters (2g) C18- reverse phase Sep-Pak was activated with methanol (10mL) and washed with water (20 mL) before the metal complex solution is (20 mL) was loaded onto the column and eluted with 30 mL water as a yellow band, leaving an orange/black band on the head of the column. The yellow band was then rotary evaporated evaporated to dryness.

The wavelength of absorption and the molar extinction coefficient of 1,10 phenanthroline and PhenSS was determined in the UV by dissolving a 25 µM sample in 10 mL of water using a volumetric flask. The solution was filled into a cuvette and the spectrum measured. The solution was diluted with distilled water till the maximum absorbance was within the measurable range of spectrometer.

The NMR spectra was also evaluated for the compound.

Determining Partition Coefficient

Preparation of Octanol for Partition Coefficient Experiment:

A solution of sodium carbonate (ca-0.5g) and sodium bicarbonate (ca-0.5g) was prepared in 100 mL of distilled water. 100 mL of n-octanol was mixed with 100 mL of sodium carbonate/ sodium bicarbonate solution and the mixture was allowed to equilibrate by shaking it periodically over a period of 10 mins and then allowing it to stand for 5 min.

Preparation of 56MESS for Partition Coefficient Experiment : -

Two samples of 1,10- phenanthroline were pipetted into separating funnels. In one funnel, 10 mL sample of 2µM solution was placed. In the other 5mL of sample + 5mL of water was placed. 10mL of saturated octanol, prepared earlier was pipetted into each of the separating funnels. They were shaken vigorously for 15 sec at 2 min intervals over a period of 15 min. The mixtures were allowed to stand to ensure complete separation and then a sample of each layer was put into a clean flask, ensuring that there is no contamination of layers within the samples. The process was repeated using phenSS instead of 1,10-phenanthroline.

For each of the extracts, the concentration of the drug in the aqueous extract was determined by running its UV Spectrum and recording the absorbance at the wavelengths corresponding to the peak maxima.

From the concentration of the drug in the aqueous solution the quantity, and hence concentration of the drug in the organic octal phase was determined. The ratio was determined for both the 10 and 5 mL solutions.

Partition Coefficient

P= [Molar Concentration in octanol] / [Molar Concentration in Water]

The Kirby -Bauer Disc Bioassay

The given solution of 25mM solution of phenSS and 56MESS was put into 2 eppindrof tubes containing 750 µL each of 25 and 12.5 solution

Three small absorbent discs were placed into each sample tube and were then placed on the agar plates. A full concentration, a half concentration and a blank were placed on each plate. This was repeated so that there were two replicated sets. The plates were clearly labeled.

A digital camera was used to document the results on the plates. The diameter of the zones of inhibition were measured and recorded.

Binding Studies: Determination Binding Constant By Direct Titration Method

Each solution had 200 µL of DNA and a varying amount of complex. The binding constant was determined using the direct titration method. The solution containing 200 µL and a varying amount of complex was provided. The sample (300µL) was prepared in eppindoff tubes, and then 280 µL of the solution was transferred to a microtitre plate for measurement.

Results and Discussion:

1. Synthesis of Platinum based anti cancer compound. [(1,10- Phenanthroline)(1S,2S-diaminocyclohexane)platinum(II)]2+(PhenSS)

The compound was synthesized successfully and obtained with high degree of purification. The percent yield of the compound was nearly 82 % and this falls within the acceptable range. NMR spectrum and UV spectrums were also analysed and it was clear from these spectrums that the compound synthesized is PhenSS with high degree of purity.

2. Determination of Partition Coefficient.

The hydrophobicity factor was determined quantitatively by using water and octanol as aqueous and organic layers. The partition coefficient is calculated by using the formula:

Partition Coefficient P= [Molar Concentration in octanol] / [Molar Concentration in Water]









Solution A: 10 mL of the drug sample + 10 mL of Octanol

Partition Coefficient P= [Molar Concentration in octanol] / [Molar Concentration in Water]

P = 6.14

Solution B: 5 mL sample drug + 5 mL water + 10 mL Octanol (50 % dilution)

P= 5.31

The Kirby -Bauer Disc Bioassay

The Kirby- Bauer Bioassay was performed to determine the biological activity. Different samples were prepared and all concentrations were tested for displayed inhibition zones. This helps in recording the disc diameter in mm and the concentration in molarity.

Conc (mM)

Experimental Values (mm)

Average (mm)


8, 8, 9, 9, 11, 16, 14



12 ,10, 12 ,14, 8, 8, 8, 8



15, 17, 21, 19, 15, 15, 13, 20,



13, 18, 12, 12,



15, 20, 12,25, 23, 24, 24, 12


Data points in red were not used in the calculation as they are considered as out of range due to experimental errors.

Binding Studies: Determination Binding Constant By Direct Titration Method

The binding constant for Platinum complexes with ct-DNA were calculated by using direct titration methods. The CD of the DNA solution was recorded. The binding constant was determined after finishing the titration and calculating the free complex and bound metal complex. The equilibrium binding constant is the ratio of the concentration of the bound metal complex with the product of the concentration of free metal complex and free DNA concentration.

Kb = [MCb]/[DNAf][MCf]


The relationship between molecular structures and biological activity of Platinum intercalating compounds with ligand 1,10 Phinanthroline is investigated and its influence on the biological activity is studied in this experiment.

The complete experiment is done with a good success rate with some difficulties in handling the DNA and making the stock DNA solution. During the experiment it was ensured that the conditions for determining the partition coefficient should allow the minimum level of ionization. Overall we can say that the experiment is conducted with high success rate and minimal errors.


I am very thankful to the University of Western Sydney for helping me carry out my research in terms of finance and facilities. I am also thankful to _____, _______

(Please fill in the names yourself )