thesis ppt

transcript

In the name of God

Synthesis and DNA binding studies of a Platinum complex

Containing methyl-substituted 1,10-

phenanthroline

IntroductionMethod & MaterialResults & Discussion Conclusion & suggestion

Introduction

General Medical application of Metals can be traced back almost 5000 years.

The development of modern medicinal inorganic chemistry stimulated by the discovery of cis Platin, has been facilitated by the inorganic chemist’s extensive knowledge of the coordination and redox properties of metal ions.

The pharmaceutical use of Metal Complexes therefore has excellent potential. A broad array of medicinal applications of metal complexes has been investigated, and a lot of different coordination compounds and the mechanism of cytotoxic action have been discussed with regard to the development of new antitumor agents.

Developing metal complexes as drugs, however, is not an easy task. Accumulation of metal ions in the body can lead to deleterious effects. Thus biodistribution and clearance of the metal complexes as well as its pharmacological specificity are to be considered. Favorable physiological responses of candidate drugs need to be demonstrated by in vitro study with targeted biomolecules and tissues as well as in vivo investigation before they enter clinical trials.

Cancer is caused when genetic damage to the cells prevents them from being responsive to normal tissue controls. The cancer spreads when affected cells multiply rapidly, forming tumors of varying degrees.

Cancer

External SourcesTobaccoRadiationChemicalsInfectious Organisms

Internal FactorsInherited MutationsHormonesImmune ConditionsMutations from Metabolism

How is Cancer Treated?

Surgery

Radiotherapy

Chemotherapy

a chemotherapeutic agent is one that kills the

rapidly dividing cells, thus slowing and stopping the cancer from spreading.

In chemotherapy, the key issue is killing the tumor cells, without causing too much harm to healthy cells.

Chemotherapy

Non-metal containing anti cancer drugs Metal based anti cancer drugs

Metal centers being positively charged, are favoured to bind to the negatively charged biomolecules. The constituents of proteins and nucleic acids offer excellent ligands for binding to metal ions…

Ruthenium, Gold, Rhodium, Palladium, Copper, Platinum and etc.

Anti Cancer Drugs

Platinum

Platinum is a chemical element with the atomic symbol Pt and an atomic number of 78. It is in group 10 of the Periodic Table of Elements. A heavy, malleable, ductile, precious, gray-white transition metal. Platinum is resistant to corrosional chemical attack.

Oxidation states are: 0, +1, +2, +3, +4In general, however, Pt(II) complexes are square planar or 5-coordinate and are diamagnetic .The kinetic inertness of the Pt(II) (and also Pt(IV)) complexes has allowed them to play a very important role in the development of coordination chemistry.

Oxidation states of platinum and stereochemistry

Cis Platin Trans Platin

The compounds cis- and trans-dichlorodiammineplatinum(II) were known to chemists even before the geometric characterization work of Alfred Werner in the nineteenth century.

the mid-1960s, Barnett Rosenberg and his co-workers serendipitously discovered that cisplatin exhibited antitumor activity but that the trans isomer, did not.

Rosenberg, a biophysicist, had been studying the growth of bacterial cells in solution in the presence of electrical fields.

He observed elongation of the Escherichia coli bacterial cells, a phenomenon known as filamentous growth, rather than cell division.

Rosenberg also discovered that the supposedly inert platinum electrodes of the electrical circuit were reacting with ammonium and chloride ions in the nutrient broth in the electrical cell forming a number of platinum(II) and platinum(IV) compounds. These platinum compounds, cis-[Pt(NH3)2Cl2] and cis-[Pt(NH3)2Cl4] in particular, were found to cause filamentous growth in cells, absent any electrical current.

Rosenberg made a spectacular ‘‘leap of logic’’ postulating that platinum compounds causing filamentous growth rather than cell division could be antitumor agents.

Despite the success of cisplatin, however, it lacks selectivity for tumor tissue, which leads to severe side effects. These include renal impairment, neurotoxicity and ototoxicity (loss of balance/hearing), which are only partially reversible when the treatment is stopped. To address these problems, modified versions of cisplatin, leading to second and third generation platinum-based drugs have been synthesized over the past 30 years.

cisplatin analogues

Platinum-multimetallic systems: N. Farrell and co-workers have synthesized these compounds. These complexes have been shown to bind DNA, forming primarily interstrand crosslinks in much higher proportions than cisplatin and more significant cytotoxic and tumor activity.

Carboplatin: has fewer toxic side effect than cisplatin and more easily used in combination therapy. Its low reactivity allows a higher close to be administered. Carboplatin is used more for Ovarian cancer treatment.

Oxaliplatin: is known to be most effective in Colon cancer treatment.

Oxaliplatin Carboplatin

BBR 3464

DNA structure

The nucleotides are made up of three parts:

1) One of the pyrimidine or purine “bases”

2) A sugar

3) Phosphoric acid

Nitrogenous bases of DNA

Watson and Crick's base pairs

A DNA molecule has two strands, held together by hydrogen bonding between their bases. As shown in this figure, Adenine can form two hydrogen bonds with Thymine; Cytosine can form three hydrogen bonds with Guanine.

Cellular uptake of cis platin and

Ligand Displacement

cis platin coordinates to DNA mainly through certain nitrogen atoms of the DNA base pairs; these nitrogen atoms (specifically, the N7 atoms of purines) are free to coordinate to cis platin because they do not from bonds with any other DNA bases.

Interaction of cis platin with DNA

Many types of cis platin-DNA coordination complexes, or adducts, can be formed. The most important of these appear to be the ones in which the two chlorine ligands of cis platin are replace by Purine nitrogen atoms on adjacent bases on the same strand of DNA. This formation causes the Purines to become destacked and DNA helix to become kinked.

Methods & Materials

Hydrochloric Acid 37% (HCl)

Nitric Acid 100% (HNO3)

Potassium Chloride (KCl)

Hydrazine dihydrochloride (N2H4.2HCl)

(4,7-dimethyl,1,10-Phenanthroline) (C14H12N2)

Methylene blue (C37H27N3Na2O9S3)

Deoxyribonucleic acid sodium salt from calf thymus (CT DNA)

Tris HCl (C4H11NO3ClH)

Materials:

Heater/Stirrer(MR3002)

pH/EC Meter (3345)

CHN elemental analysis

UV-Visible Spectrophotometer (8453)

Thermostated Bath

CD Spectropolarimeter (J-810)

Fluorescence Spectrophotometer (FP6200)

Viscosimeter (AVS 450)

Apparatus:

synthesis and characterization of complex.

preparation of suitable DNA and complex solutions for analytical experiments.

investigation of metal complex/DNA interactions by a variety of physicochemical techniques including spectrophotometry, DNA melting, circular dichroism, spectrofluorimetery and viscometry.

Index of this investigation

HCl + HNO3 (3:1) + Pt (cooled-condenser with stirring)

stirring (30minutes) and heated to 175ºC

Pt metal solved and reduce to 50 cc ( it take put in ice bath)

KCl was added (KCl/Pt = 2/1 mol)

yellow crysralline salts K2PtCl6

Synthesis & characterization of complex

Synthesis of potassium hexachloroplatinate (IV)

Hydrazine dihydrochloride 1.0 gr (0.01 mol)

suspention of 9.72gr (0.02 mol) of potassium hexachloroplatinate (IV) in 100 ml of water.

The mixture was stirred mechanically while the temperature was raised to 50-65ºC over a period of 5-10 minutes.

the temperature was then raised to 80-90ºC to ensure completion of the reaction.

the mixture was cooled in an ice bath.

filtered to remove unreacted potassium hexachloroplatinate (IV).

Synthesis of potassium tetrachloroplatinate(II)

Boil a solution of K2PtCl4 (1.0 gr) and (4,7-dmphen) ligand (0.5 gr).

in 1.5 l of water containing 0.5ml of HCl.

reflux for 20hr.

the reaction mixture was allowed to cool to room temperature and the light green product that precipitated was filtered out, washed with hot water and dried under vacuum.

Synthesis of [PtCl2(4,7-dmphen)]

Tris HCl (for interaction samples and DNA dissolving) 10mM, pH 7.0 Mw= 157.59 Tris HCl = 0.78795 g H2O…….to 500 mlAt first, dissolve Tris HCl in 200ml distilled water, then adjust the pH using concentrated NaOH to 7.0, at last add distilled water in ballon to determined volume. For confidence adjust the pH, again.

Preparation of Buffers and DNA and Complex solutions for interaction

Preparation of Buffers:

Stock solution of CT DNA was prepared by dissolving a piece of CT DNA fibers in 2 ml Tris HCl (10 mM) and shaked slowly and stored for 24h at 4 ºC. The concentration of DNA solution, was expressed in monomer unit, which was determined by spectrophotometry at 260nm using an extinction coefficient (p) of 6600 M-1cm-1 value and used after no more than 4 days.

Stock solution of [PtCl2(4,7-dmphen)] complex was prepared by dissolving 0.5 mg of the complex in 0.3 ml of DMSO and 1.7 ml of Tris HCl buffer 10 mM (Final concentration =5.27 × 10-4 M).

Preparation of DNA and complex solutions for interaction:

The absorbance measurements were performed in two ways: by keeping the concentration of the CT DNA constant (5.0 × 10-5M) while varying the complex concentrations from 5.0 × 10-6 M to 4.5 × 10-5M.

( ri = [complex]/[DNA] = 0.1,…,0.9)Calculation: e.g. ri = 0.1 = X / (5.0 × 10-5) X = 5.0 × 10-6

For complex: M1V1= M2V2, [Mstock com] × V1= 5.0 × 10-6 × 2ml V1= aFor DNA: M1V1= M2V2, [Mstock DNA] × V1 = 5.0 × 10-5 × 2ml V1 = b(Mstock com , Mstock DNA are known), 2ml – ( a + b ) = V buffer, b is

constant for all samples.

Methods

Electronic absorption measurments

Titration of complex solution by varying amounts of the DNA concentration from 1.5 × 10-6 M to 1.05 × 10-5M

ri = [DNA] / [Complex] = 0.1,…, 0.7

Calculations are similar to previous section.

All samples incubated at 37 ºC for 24h.

Thermal denaturation studies of CT DNA are useful in determining the ability of the present complex to stabilize the double stranded. When DNA molecules are heated and separated in two single strands in occur due to disruption of the intermolecular force a π-π stacking and hydrogen bonding interacting between the DNA base pairs.

DNA concentration is constant and we add various amounts of complex solutions to sample tubes.

ri = [complex]/ [DNA] = 0.2, … , 0.8

Thermal denaturation

Circular Dichroism Spectroscopy

The conformation of a natural nucleic acid depends on salt, solvent, and temperature, and CD has become a favored method for monitoring the changes in secondary structure.

The CD technique is indeed very sensitive to detect minor conformational changes of the DNA conformation produced by ligand binding.

DNA concentration is constant and we add various amounts of complex solutions to sample tubes.

ri = [complex] / [DNA] = 0.2, … , 1.2

Fluorescence spectroscopy

Fluorescence spectroscopy continues its advance to more sophisticated methods and applications.

we have witnessed a rapid migration of the principles of time-resolved fluorescence to cell biology and clinical applications.

Because of the tremendously sensitive emission profiles, spatial resolution, and high specificity of fluorescence investigations, the technique is rapidly becoming an important tool in genetics and cell biology.

MB concentration and DNA concentration are 5 × 10-6 and 5 × 10-5 and we add various amounts of complex solutions to sample tubes.

ri = [complex] / [ DNA] =0.0, 0.3, …, 2.7

Optical physical probes provide necessary but not sufficient dues to support the binding model. Hydrodynamic method such as determination of viscosity, which is exquisitely sensitive to the change of length of DNA, may be the most effective means studying the binding mode of complexes to DNA in absence of X-ray crystallographic or NMR structural data.

The viscosity measurement is based on the flow of a DNA solution through a capillary viscometer.

Viscosimetry

DNA concentration is constant (5.0 × 10-5) and we add various amounts of complex solutions to sample tubes.

ri values are 0.0, 0.2, … , 1.6

Results

Discussion

(HCl + HNO3 (3:1)) + Pt + KCl K2PtCl62 K2 PtC16 + N2H4.2HCl 2 K2PtC14 + N2 + 6 HCl K2PtC14 + (4,7-dmphen) + H+ [Pt(C14H12N2)Cl2

The product of complex synthesis appears as light green product.

characterized by 1H NMR, elemental analysis and UV-Vis spectroscopy.

Synthesis and Characterization

1H NMR

substance Me 2H 3H 5H

PtCl2(NN): 8.3 9.7 8.2 9.07

NN: 9.1 8.3 7.3 8.6

3H5H2H

(9.7) (9.07)(8.2)

Pt(C14H12N2)Cl2 Anal. Calcd.(%) C, 35.5; H, 2.6; N, 5.9 Found (%) C, 36; H, 3.6; N, 6.0

[PtCl2(4,7-dmphen)] shows an intense absorption band at about 278nm

UV of Complex

245 255 265 275 285 295 305

Wavelength (nm)

Elemental analysis and UV-Vis spectroscopy

a)Covalent (DNA alkylation or metallation)

This mechanism of DNA binding involves forming direct bonds to the DNA bases that hold the genetic information and causing distortions to DNA structure. This mode of action is used by the platinum drugs that are among the most widely used anti-cancer agents in the clinic today. cis-platin, carboplatin, oxaliplatin

Modes of DNA binding

b)non-covalent Groove binding

The molecules approaches within van der Waals contact and resides in the DNA groove. Hydrophobic and/or hydrogen-bonding are usually important components of this binding process, and provide stabilisation. Most small molecule drugs fit snugly in the minor groove. The antibiotic netropsin is a model groove-binder.

External binding

This association is electrostatic in nature complexes are 2+ positively charged and the DNA phosphate sugar backbone is negatively charged. This association mode was proposed for complex upon binding to DNA is strongly dependent on the ionic strenght.

Intercalation

This association involves the insertion of a planar fused aromatic ring system between the DNA base pairs, leading to significant p-electron overlap. This mode of binding is stabilised by stacking interactions and is thus less sensitive to ionic strenght relative to the two other binding modes. This mode of binding is usually favoured by the presence of an extended fused aromatic ligands

Investigation of Pt / DNA interaction with analytical methods

UV absorption spectrophotometry

Thermal denaturation of DNA

Fluorescence spectroscopy

Viscosimetry

UV absorption spectrophotometry

Electronic absorption spectra are initially employed to study the binding of complexes to DNA. The binding of intercalative drugs to DNA helix has been characterized classically through absorption spectral titration, by following the changes in absorbance (hypochromism) and shift in wavelength.

Aliquots of the DNA solution at a constant concentration equal to 5×10-5 were incubated with Pt complex at ri values of 0.1-0.6 in 10mm Tris-HCl buffer (pH=7) at 37°C. The UV band of DNA at about 258nm, was monitored in the absence and presence of different amounts of Platinum complex. Hypochromic effect are generally observed. This result indicate a pronounced intraction of Platinum complex with DNA.

UV of DNA

235 245 255 265 275 285

Wavelength (nm)

ri=0.1

ri=0.2

ri=0.3

ri=0.4

ri=0.5

ri=0.6

We have observed significant hypochromicity with minor red shift for the absorption band of the complex. This observation gives a good evidence of the intercalation of the Pt(II) complex through the stacking interaction of aromatic rings of the ligand and the base pairs of DNA and a few of other bindings.

245 255 265 275 285 295 305

Wavelength (nm)

[DNA]/(εa-εf) = [DNA]/(εb-εf)+1/Kb (εb-εf)εa= Aobsd / [Pt]εf = ε for free Platinum complex εb= ε fully bound form, respectively.In plots of [DNA]/(εa-εf) versus [DNA], Kb is given by the ratio of slope to the intercept.

( ε a

- ε f

Kb = [slope] / [Y]

Kb calcd = 6.35 × 104 M-1

Binding Constant (Kb)

The calculated Kb value was found to be (6.35 × 104 M-1). The values of Kb described in the literature for classical intercalators proflavin-DNA, (4.1 × 105 M-1) are at least six order of magnitude higher that that obtained for this Pt(II) complex. In contrast the values of Kb are ten order of magnitude higher than Kb values which found for compound with the mode of groove binding complexes DNA like Cr(III) complexes or Tris (1,10-phen) ruthenium (II) to DNA.

The Kb value obtained for this complex is of the some order of magnitude of that determined in analogues condition for Znl+2 (Kb = 7.35 × 104 M-1) which considered as an intercalating complex.

These results confirm that the Pt(II) complex strongly interact with DNA and suggest that intercalation between base pairs is the main mode of interaction of Pt(II) complex with DNA and a few other interaction also maybe exist.

A curve between absorbance at 258nm and temperature gives a thermal denaturation curve of DNA. The temperature corresponding to the midpoint of the curve is called Melting Temperaure or TM of DNA and denotes the temperature of which 50% of the DNA undergone denaturation.

Thermal denaturation experiments

Our experiments were carried out for CT DNA in the absence and presence of different amounts of Pt(II) complex.

In the presence of intercalators, the Tm rises sharply with low intercalators concentrations. The stabilization which is due to the electrostatic binding increases Tm less steeply.

Experiments showed that the intercalation to DNA increases the stability of the helix and, as a result the melting temperature goes up around 5-12°C.

As shown in figure, this experiment reveal that Tm of the calf thymus DNA was 81.65°C in absence of Pt complex and 89.5°C, 91.5°C, 92°C, 93.5°C in presence of ri=0.2-0.8 of Pt complex, respectively.

The increase in Tm indicates that Pt complex is involved in strong binding to DNA implying that the complex bound DNA is more difficult to melt that the unlabelled DNA. These changes in the Tm indicative that the main mode of interaction is intercalative binding.

Therefore, figure shows the increase in the DNA melting temperature observed for Pt(II) complex = ∆Tm 11.85°C, is consistent with intercalation.

TM of DNA

55 60 65 70 75 80 85 90 95 100 105

Temperature (°C)

Ri=0.2

Ri=0.4

Ri=0.6

Ri=0.8

Linear polarized light can be viewed as a superposition of opposite polarized equal amplitude and phase.

A projection of the combined amplitudes perpendicular to propagation direction thus yields a line. When this light passes through an optically active sample with a different absorbance for two component, the amplitude of the stronger absorbed component will smaller than that of the less absorbed component. So this method measure the differential absorption of right and left circularly polarized light.

This difference is usually expressed in term of the absorption coefficients for L left and R lightεL and εR εL – εR = Δε (circular dichroism)

Δε > 0 Positive peakΔε < 0 Negative peak

236 246 256 266 276 286 296

Wavelength (nm)

With addition of the different concentrations of complex, the ellipticity value of DNA decrease at band 246nm. Suggesting that change of helicity of B DNA to an unknown form occur which unfortunately not be identified so far and ellipticity value of DNA increase at band 276nm indicating that DNA keeps the double helix in the present of Pt complex and complex intercalated with DNA.

The observed CD spectrum of CT DNA consists of a positive band at 273nm (UV:λmax, 260nm) due to base stacking and a negative band at 246nm due to helicity, which is characteristic of DNA in right-handed B form.

236 246 256 266 276 286 296

Wavelength (nm)

ri=0.2

ri=0.4

ri=0.6

ri=0.8

ri=1.0

ri=1.2

Fluorescence is a member of the ubiquitous luminescence family of processes in which susceptible molecules emit light from electronically excited states created by either a physical (for example, absorption of light), mechanical (friction), or chemical mechanism. Generation of luminescence through excitation of a molecule by ultraviolet or visible light photons is a phenomenon termed photoluminescence.

spectrofluorimetery

Some molecules have a few luminescence and it is hard to monitor the interaction of these molecules with DNA by employing direct fluorescence emission methods, but actually possible by using a fluorescence assay of the organic molecule probe examples are Phenothiazinum dyes, such as proflavin, acridine orange, ethidium bromide and methylene blue.

proflavin

acridine orange

ethidium bromide

methylene blue

Extrinsic Fluorescence

In this study, we report the spectroscopic results of interaction between [PtCl2(4,7-dmphen)] complex and DNA by using MB as a fluorescent probe.

Upon binding to DNA the probe fluorescence is efficiently quenched by the DNA bases with no apparent shifts in the emission maximum.

This emission quenching phenomenon also reflects the changes in the excited state electronic structure in consequence of the electronic interactions in the MB-complexes.

Obvious spectroscopic changes of the MB (5mol/l)-DNA (50mol/l) system have been observed after adding [PtCl2(4,7-dmphen)] complex.

Fluorimetry (Competitive binding studies)

Fluorimetry

640 660 680 700 720

Wavelength (nm)

MB+DNA

Fluorimetry

640 660 680 700 720

Wavelength (nm)

MB+DNA

Ri = 0.3

Ri = 0.6

Ri = 0.9

Ri = 1.2

Ri = 2.1

Ri = 2.4

Ri = 2.7

This figure clearly reveals a dramatic increase of the fluorescence intensity of the probe molecule when [PtCl2(4,7-dmphen)] is added. The higher the Pt complex concentrations, the stronger the MB fluorescence intensities.

In contrast, cis platin, which is known to kink DNA through covalent binding, shortening the axial length of the double helix caused a decrease in the relative viscosity of the solution. Partial intercalators also reduce the axial length observed as a reduction in relative viscosity

Viscosimetry

A classical intercalation model demands that the DNA helix must lengthen as base pairs are separated to accommodate the binding ligand, leading to the increase in DNA viscosity.

The data was reported as (η/η°)1/3 vs. [PtCl2(4,7-dmphen)] / [DNA] ratio, where η° is the viscosity of the DNA solution alone, η = ( t-t°) / t° , t = sample flow time, t° = the buffer flow time. Data output of the instrument is flow time (t).

Viscosimetry

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

1/R=[DNA]/[Complex]

(η/η

/3Visc.

In the viscosity curve the results, indicate that absence and the presence of the metal complex have a marked effect on the viscosity of the DNA. The specific viscosity of the DNA sample increases obviously with the addition of the complex.

The viscosity increase of DNA is ascribed to the mainly intercalative binding mode of the complex because this could cause the effective length of the DNA to increase.

To further confirm the interaction mode of the Pt(II) complex with DNA, a comparative viscosity study between Ethidium bromide (EB; classical intercalator) and the title complex shows that the intercalator EB significantly increases the relative specific viscosity of DNA as expected for the lengthening of the DNA double helix resultant from well-characterized intercalation.

Li et al., showed that EB increased the relative viscosity of DNA and the slope of the graph of (η/η°)1/3 vs. 1/R was 0.96 which is very close to value of 1.0 predicted from the theory of Cohn and Eisenberg.

in this study the relative viscosity of DNA increase with a slope of 0.49 and it is reasonably believed that the main interaction between DNA ant titled complex is intercalation and may be other interaction(s) between DNA and the Pt(II) complex occurred; and is responsible for the decrease of the slope.

Conclusion &

suggestion

Various techniques support the conclusion that the Pt(II) complex binds to DNA through intercalation. The findings are as bellow:

• The UV spectra of Pt(II) complex show clearly that addition of DNA results in evident hypochromism and red shift of peaks at 278nm. These results correlate with the strong intercalative interaction.

• The value of Kb ((6.35) × 104 M-1) is of the same order in magnitude of that determined in analogues condition for intercalating complexes.

• The DNA melting temperature, Tm, of about 93.5°C when molar ratio of Pt(II) complex is 0.8, is consistent with intercalation of the complex.

• The CD spectra of CT DNA in the presence of the Pt(II) complex show that the DNA remains right handed and the increase of the intensities of both the bands of CT-DNA confirms the mode of intercalation.

The increase of MB-DNA solution fluorescence bands by addition os the increasing amounts of Pt(II) complex shows that [PtCl2(NN)] complex could release MB completely in this sense. Complete recovery of MB fluorescence is indicative of an intercalative mode of binding.

The viscosity of DNA increase and it is reasonably believed that the Pt(II) complex intercalate intercalates into CT DNA.

My suggestions are:

We can use this ligand with other central metals and study the these new complexes with DNA.

We can change this complex to a cationic form and overcome to solubility problem of this complex in aqua's solution.

Thanks for your attention

thesis ppt

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