**5.2 DNA binding studies through cyclic voltammetry**

To demonstrate the approach of interaction and the DNA binding constraints, different techniques are employed. Cyclic voltammetry is proved to be one of the most sophisticated and sensitive techniques to carryout DNA binding studies. Investigation of mode of interaction between DNA and derivative compounds is determined through shift in peak potential. Indication of intercalation of derivative compounds into double helix structured DNA comes from slightly positive shift of the peak potential. The ratio of binding of oxidized and reduced molecules is calculated using the following equation Eq. (1) [78, 79]:

$$\stackrel{\circ}{Eb}^{\circ} - \stackrel{\circ}{Ef}^{\circ} = 0.05916 \log \left(\frac{K\_{md}}{K\_{ad}}\right) \tag{1}$$

where *Eb*° and *Ef*° are proper potentials of bound and free drug candidates, correspondingly. Positive shift is indicative for intercalation of derivative compounds with DNA. The formation of a supramolecular complex due to drug diffusion into DNA results in dropping of current in electrochemical analysis. Drop off in current is observed depending upon the number of transferred electrons, which is decreased upon formation of a supramolecular complex. Binding constant is calculated using the following equation Eq. (2) [80]:

$$\frac{1}{[DNA]} = \frac{K[(1 - A) \quad \text{ } -K \quad \text{ }]}{\mathbf{1} - i/i\_o} - K \tag{2}$$

where is the binding constant, and *io* represent peak currents in the presence and absence of DNA, and is the proportionality constant. The plot of 1/[DNA] versus 1 <sup>−</sup> *<sup>i</sup>*/*io* produces binding constants.

Following are the examples of cyclic voltammetric results in **Figures 12** and **13** for the characteristic ferrocenyl thioureas discussed in Section 3.2 and their molecular structures in **Figures 9** and **10**.

#### **Figure 12.**

*Cyclic voltammograms of 1 mM B16 at a 0.05 V s<sup>−</sup><sup>1</sup> potential sweep rate on a glassy-carbon electrode at 298 K with and without incorporation of 1 mL of CT-DNA by its increasing concentrations (i.e. 10, 20, and 60 μM) in a 20% aq. DMSO buffer at pH 6.0; supporting electrolyte 0.1 M TBAP [60].*

#### **Figure 13.**

*Cyclic voltammograms of 1 mM B3 at a 0.05 V s<sup>−</sup><sup>1</sup> potential sweep rate on a glassy-carbon electrode at 298 K with and without incorporation 1 mL of CT-DNA by its increasing concentrations (i.e. 10, 20, and 60 μM) in a 20% aq. DMSO buffer at pH 6.0; supporting electrolyte 0.1 M TBAP [60].*

Cyclic voltammetric analysis of 1 mM of B16 and B3 was carried out incorporating and without incorporating calf-thymus DNA (CT-DNA). After the addition of CT-DNA, an obvious positive shift in formal potential was observed for B16, which indicated the intercalative mode of interaction, whereas for B3, a formal potential shift toward negative side was observed, which is credited to electrostatic interactions among compound and DNA [60].

#### **5.3 DNA binding studies through viscometry**

Viscosity measurement is another beneficial technique to demonstrate intercalation of derivative compounds with DNA. It is sensitive to change in DNA length as base pair active pockets are broadened to provide lodging to binding molecule that ultimately results in lengthening of DNA helix. This technique is considered

**61**

in a thermostatic bath [81].

interactions [82].

**Figure 14.**

**Figure 15.**

**5.4 Molecular docking**

*Supramolecular Chemistry and DNA Interaction Studies of Ferrocenyl Ureas and Thioureas*

*Relative viscosity versus [Drug]/[DNA] representative plot for mode of interaction determination.*

as a least abstruse and utmost precarious test for binding mode determination in solution phase under suitable conditions, i.e., constant temperature at 25.0 ± 0.1°C

*Effect of increasing concentration of compound 1,1′-(4,4′-di-ferrocenyl)di-phenylthiourea on relative viscosity* 

Following is the representative plot (**Figure 14**) [82] which exhibits the relative viscosity (η/ηo) against [compound/[DNA] concentrations to examine the mode of interaction. Derivative compounds have shown that upon increasing binding ratio, relative viscosity decreases which is a representative for electrostatic

**Figure 15** [81] is another example of a viscosity measurement result of a ferrocenyl urea derivative compound that shows the consequence of increment in

Molecular docking approach is one of the most frequently employed approaches in structure-based drug designing, owing to its capability to predict conformation

concentration of derivative compound on viscosity [81].

*of DNA at 25ᵒC. [DNA] = 30 μM and [compound] = 5–25 μM.*

*DOI: http://dx.doi.org/10.5772/intechopen.84412*

*Supramolecular Chemistry and DNA Interaction Studies of Ferrocenyl Ureas and Thioureas DOI: http://dx.doi.org/10.5772/intechopen.84412*

#### **Figure 14.**

*Photophysics, Photochemical and Substitution Reactions - Recent Advances*

Cyclic voltammetric analysis of 1 mM of B16 and B3 was carried out incorporating and without incorporating calf-thymus DNA (CT-DNA). After the addition of CT-DNA, an obvious positive shift in formal potential was observed for B16, which indicated the intercalative mode of interaction, whereas for B3, a formal potential shift toward negative side was observed, which is credited to electrostatic interac-

*with and without incorporation 1 mL of CT-DNA by its increasing concentrations (i.e. 10, 20, and 60 μM) in a* 

*with and without incorporation of 1 mL of CT-DNA by its increasing concentrations (i.e. 10, 20, and 60 μM)* 

 *potential sweep rate on a glassy-carbon electrode at 298 K* 

 *potential sweep rate on a glassy-carbon electrode at 298 K* 

Viscosity measurement is another beneficial technique to demonstrate intercalation of derivative compounds with DNA. It is sensitive to change in DNA length as base pair active pockets are broadened to provide lodging to binding molecule that ultimately results in lengthening of DNA helix. This technique is considered

**60**

**Figure 13.**

**Figure 12.**

tions among compound and DNA [60].

*Cyclic voltammograms of 1 mM B3 at a 0.05 V s<sup>−</sup><sup>1</sup>*

*Cyclic voltammograms of 1 mM B16 at a 0.05 V s<sup>−</sup><sup>1</sup>*

*in a 20% aq. DMSO buffer at pH 6.0; supporting electrolyte 0.1 M TBAP [60].*

**5.3 DNA binding studies through viscometry**

*20% aq. DMSO buffer at pH 6.0; supporting electrolyte 0.1 M TBAP [60].*

*Relative viscosity versus [Drug]/[DNA] representative plot for mode of interaction determination.*

#### **Figure 15.**

*Effect of increasing concentration of compound 1,1′-(4,4′-di-ferrocenyl)di-phenylthiourea on relative viscosity of DNA at 25ᵒC. [DNA] = 30 μM and [compound] = 5–25 μM.*

as a least abstruse and utmost precarious test for binding mode determination in solution phase under suitable conditions, i.e., constant temperature at 25.0 ± 0.1°C in a thermostatic bath [81].

Following is the representative plot (**Figure 14**) [82] which exhibits the relative viscosity (η/ηo) against [compound/[DNA] concentrations to examine the mode of interaction. Derivative compounds have shown that upon increasing binding ratio, relative viscosity decreases which is a representative for electrostatic interactions [82].

**Figure 15** [81] is another example of a viscosity measurement result of a ferrocenyl urea derivative compound that shows the consequence of increment in concentration of derivative compound on viscosity [81].

#### **5.4 Molecular docking**

Molecular docking approach is one of the most frequently employed approaches in structure-based drug designing, owing to its capability to predict conformation

#### **Figure 16.**

*(a) Docked conformation of representative P3Cl compound with 1-BNA; P3Cl is shown in green color while 1-BNA exhibited a ribbon structure, (b) surface outlook of docked-P3Cl with 1-BNA (color code: grey-carbon, red-oxygen, and blue-nitrogen) and (c) 3D-model representing interactions of P3Cl and DNA [82].*

responsible for binding of small molecular entities, i.e., ligands to a suitable target active binding site [83].

The example illustrated in **Figure 16(a)** exhibits the representative docked conformation of compound 1-(3-chlorobenzoyl)-3-(4-ferrocenylphenyl)urea (**P3Cl**) with DNA attributed to have the lowest binding energy, recommended by AutoDock [84], whereas **Figure 16b** depicts the surface view of docked conformation of the represented compound and it is clearly indicated from **Figure 16b** that ferrocenyl group of docked P3Cl is in close interaction with O-atom which is linked to sugar phosphate DNA backbone. This, in another way, suggested that electrostatic interaction force exist among Fe and O-atoms of sugar and phosphate backbone [82]. **Figure 16c** is the close view of DNA atoms that clearly interact with the active surface of the P3Cl compound, and O-atom of the sugar-phosphate backbone can be seen, which prevails among deoxyadenosine (DA)-18 and DA-17, showing electrostatic interactions with ferrocenyl group [85]. The presence of hydrogen bond is also observed in the structure among one of the O atoms of P3Cl and H-atom attached to N-atom of DA5 [86].

### **5.5 Dynamic light scattering (DLS)**

Dynamic light scattering (DLS) approach is employed to govern size distribution of small particles in suspension or polymers in solution. This method is used in medicine to detect molecular changes in the cornea in biology to measure the rate of diffusion on proteins, and in material science to study the orientational fluctuation in the liquid crystals [87].

DLS is, in principle, capable of distinguishing whether a protein is a monomer or dimer; it is much less accurate for distinguishing small oligomers than is classical light scattering or sedimentation velocity. The advantage of using dynamic scattering is the possibility to analyze samples containing broad distributions of species of widely differing molecular masses (e.g. a native protein and various sizes of aggregates), and to detect very small amounts of the higher mass species (<0.01% in many cases) [88].

**63**

**Figure 18.**

**Figure 17.**

*Supramolecular Chemistry and DNA Interaction Studies of Ferrocenyl Ureas and Thioureas*

For example, expansion of DNA helix occurs upon intercalation of a small molecule into DNA helix, as cavities are created to lodge small molecules between the bases which minimize the hydrodynamic radius (Rh) as shown in **Figure 17** [61].

UV-vis spectroscopy is used frequently for studying ferrocene and ferrocenyl derivative compounds owing to their fairly high stability under visible irradiation, and hence, they are widely used in luminescent systems. They are classical quenchers of excited states. Both energy and electron transfer may be involved, depending on the nature of the excited species [89]. The color of the ferrocene greatly changes upon oxidation, hence permitting spectroscopic measurements in the visible range. UV-vis spectroscopy is proved to be an effective approach for calculation of binding strength of DNA with derivative compounds. **Figure 18** depicts a representative UV-vis plot of ferrocenyl urea depicting a noticeable hypochromic and a slender blue peak shift representing adducts of drug-DNA comparative to free drug entities,

which approves the electrostatic nature of interactions (**Figure 18a**) [38].

static type of interactions and remarkable free-radical scavenging capability [90].

*Decrease in hydrodynamic radius (Rh) upon intercalation of complex in DNA helix.*

*(a) Representative plots of absorbance versus wavelength of 25 μM 1-(3-bromobenzoyl)-3-(4-ferrocenylphenyl)*

*/A Ao*

 *versus* 

*urea in ethanol with DNA's increasing concentration from (4.5–17 μM) and (b) plot of Ao*

*1/[DNA] for determining binding constant of DNA attached to the compound [38].*

Substantial binding of derivative compounds with DNA can be observed via electro-

*DOI: http://dx.doi.org/10.5772/intechopen.84412*

**5.7 Free radical scavenging activity**

**5.6 UV-vis spectroscopy**

*Supramolecular Chemistry and DNA Interaction Studies of Ferrocenyl Ureas and Thioureas DOI: http://dx.doi.org/10.5772/intechopen.84412*

For example, expansion of DNA helix occurs upon intercalation of a small molecule into DNA helix, as cavities are created to lodge small molecules between the bases which minimize the hydrodynamic radius (Rh) as shown in **Figure 17** [61].

## **5.6 UV-vis spectroscopy**

*Photophysics, Photochemical and Substitution Reactions - Recent Advances*

responsible for binding of small molecular entities, i.e., ligands to a suitable target

*(a) Docked conformation of representative P3Cl compound with 1-BNA; P3Cl is shown in green color while 1-BNA exhibited a ribbon structure, (b) surface outlook of docked-P3Cl with 1-BNA (color code: grey-carbon,* 

*red-oxygen, and blue-nitrogen) and (c) 3D-model representing interactions of P3Cl and DNA [82].*

The example illustrated in **Figure 16(a)** exhibits the representative docked conformation of compound 1-(3-chlorobenzoyl)-3-(4-ferrocenylphenyl)urea (**P3Cl**) with DNA attributed to have the lowest binding energy, recommended by AutoDock [84], whereas **Figure 16b** depicts the surface view of docked conformation of the represented compound and it is clearly indicated from **Figure 16b** that ferrocenyl group of docked P3Cl is in close interaction with O-atom which is linked to sugar phosphate DNA backbone. This, in another way, suggested that electrostatic interaction force exist among Fe and O-atoms of sugar and phosphate backbone [82]. **Figure 16c** is the close view of DNA atoms that clearly interact with the active surface of the P3Cl compound, and O-atom of the sugar-phosphate backbone can be seen, which prevails among deoxyadenosine (DA)-18 and DA-17, showing electrostatic interactions with ferrocenyl group [85]. The presence of hydrogen bond is also observed in the structure among one of the O atoms of P3Cl

Dynamic light scattering (DLS) approach is employed to govern size distribution of small particles in suspension or polymers in solution. This method is used in medicine to detect molecular changes in the cornea in biology to measure the rate of diffusion on proteins, and in material science to study the orientational fluctuation

DLS is, in principle, capable of distinguishing whether a protein is a monomer or dimer; it is much less accurate for distinguishing small oligomers than is classical light scattering or sedimentation velocity. The advantage of using dynamic scattering is the possibility to analyze samples containing broad distributions of species of widely differing molecular masses (e.g. a native protein and various sizes of aggregates), and to detect very small amounts of the higher mass species (<0.01%

**62**

active binding site [83].

**Figure 16.**

and H-atom attached to N-atom of DA5 [86].

**5.5 Dynamic light scattering (DLS)**

in the liquid crystals [87].

in many cases) [88].

UV-vis spectroscopy is used frequently for studying ferrocene and ferrocenyl derivative compounds owing to their fairly high stability under visible irradiation, and hence, they are widely used in luminescent systems. They are classical quenchers of excited states. Both energy and electron transfer may be involved, depending on the nature of the excited species [89]. The color of the ferrocene greatly changes upon oxidation, hence permitting spectroscopic measurements in the visible range. UV-vis spectroscopy is proved to be an effective approach for calculation of binding strength of DNA with derivative compounds. **Figure 18** depicts a representative UV-vis plot of ferrocenyl urea depicting a noticeable hypochromic and a slender blue peak shift representing adducts of drug-DNA comparative to free drug entities, which approves the electrostatic nature of interactions (**Figure 18a**) [38].

### **5.7 Free radical scavenging activity**

Substantial binding of derivative compounds with DNA can be observed via electrostatic type of interactions and remarkable free-radical scavenging capability [90].

**Figure 17.** *Decrease in hydrodynamic radius (Rh) upon intercalation of complex in DNA helix.*

#### **Figure 18.**

*(a) Representative plots of absorbance versus wavelength of 25 μM 1-(3-bromobenzoyl)-3-(4-ferrocenylphenyl) urea in ethanol with DNA's increasing concentration from (4.5–17 μM) and (b) plot of Ao /A Ao versus 1/[DNA] for determining binding constant of DNA attached to the compound [38].*

**Figure 19.**

*Electronic absorption spectra of [1-(2-florobenzoyl)-3-(2-chloro, 4-ferrocenylphenyl)thiourea (***2F***)] (3.125–100 μg mL<sup>−</sup><sup>1</sup> ) representing free-radical scavenging outline.*

The free-radical scavenging activity of compound [1-(2-florobenzoyl)-3-(2-chloro, 4 ferrocenylphenyl)thiourea **(2F)]** molecular structure is given in Section 3.2 in **Figure 6**, which exhibits a sheer increment in % inhibition by increment in its concentration. It had been found that with increasing concentration of ferrocenyl thioureas, the % inhibitory activity is increased as depicted in the representative plot in **Figure 19** [40].
