Comparative Binding Analysis of Pyrimidine Derivative to BSA : Equilibrium , FTIR and Acoustical Study

This paper presented the comparative binding interaction of ethyl-4-(4-hydroxyphenyl)-6-methyl-2-oxo-1,2,3,4tetrahydropyrimidine-5-carboxylate (4-HP2OTP) and ethyl-4-(2-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4tetrahydropyrimidine-5-carboxylate (2-HP2STP) to bovine serum albumin (BSA) in 1,4-dioxane, DMSO and DMF by equilibrium dialysis, FT-IR and acoustical study at physiological pH. The binding data obtained was interpreted by scatchard plot, which gives the association constants. An increase in association constants is observed with increase in temperature and concentration. FT-IR study explains the binding through shifting in peak positions of amide I and II. It explained the changes in secondary structure of BSA on binding with the drugs. The free energy (∆G), enthalpy (∆H) and entropy (∆S) values were calculated by using van’t Hoff equation. The negative ∆G showed the spontaneous process and positive values of ∆H and ∆S showed endothermic interaction between ligands and BSA. ∆G becomes more negative with increased in temperature, indicated feasibility of binding interaction at high temperature. The positive values of ∆H and ∆S also showed specific electrostatic and hydrophobic interaction between ligand and BSA.


Introduction
Serum albumins are the most abundant proteins in the circulatory system of wide variety of organisms, being the major macromolecules contributing to the osmotic blood pressure [1].Their functional and physiological properties have been studied over several decades [2].These proteins have long been used as model proteins in both industrial and academic research areas [3].The protein is single polypeptide chain of 585 amino acids with a large helical triple domain structure with the concentration of 0.63 mM in the blood.Protein binds relatively a number of insoluble endogenous drugs such as unesterified fatty acids, bilirubin and bile ducts and thus facilitates their transport.A variation in temperature is found to be a key factor in binding affinities of proteins [4] as evident from the drugs ligustrazine [5], ciprofloxacin [6], methotrexate [7] and cisplatin [8].4-HP2OTP and 2-HP2STP are the poly-functionalized dihydropyrimidine compounds exhibiting a broad range of therapeutic and pharmacological [9], anticarcinogenic [10], antihypertensive, antiviral, antitumor, antibacterial, anti-inflammatory, calcium channel modulators [11], antimycobacterial and anticonvulsant [12], anticancer [13] properties.Human serum albumin (HSA) and BSA exhibit similar chemical properties due to high percentage of sequence identities.BSA in lieu of HSA was used in this study because of low cost and easy availability.In BSA varying binding sites are available for ligands [14][15].Ranges of techniques are available to monitor the binding interactions of ligands to protein viz.NMR [16], isothermal titration calorimetry (ITC) [17], UV-visible absorbance [18], fluorescence [19], FT-IR and CD spectroscopy [20].In the view of above considerations, present study demonstrates the effect of drug concentration, temperature and polar/non-polar solvent on binding interaction of ligands to BSA at physiological pH and determination of thermodynamic parameters like free energy, enthalpy and entropy.

Equilibrium dialysis
The binding parameters of 4-HP2OTP-BSA and 2-HP2STP-BSA complexes have been determined using scatchard analysis.Scatchard curves obtained by plotting the absorbance and specific binding against the concentrations of ligands.Different observations have noticed for both the drugs in all the solvents.The association constants for 4-HP2OTP are found to be 0.7845 (±0.0005), 0.7185 (±0.0005), 0.7305 (±0.0005) in 1,4-dioxane, DMSO and DMF respectively.While the association constants for 2-HP2STP are in 1,4-dioxane, DMSO and DMF are 0.7405 (±0.0005), 0.7265 (±0.0005), 0.7265 (±0.0005) respectively.It has been seen that, the binding affinity of 4-HP2OTP is slightly more than 2-HP2STP in 1,4-dioxane than DMSO and DMF.The scatchard analysis of binding of 4-HP2OTP & 2-HP2STP with BSA at pH 7.4 in all the solvents were provided a non-linear curve.This suggests the presence of at least two binding sites for the binding of the ligands to BSA.Figures 3 and 4 shows the scatchard plots of BSA-ligands complexes in 1,4-dioxane, DMSO and DMF.

Effect of foreign particles
The binding analysis of 4-HP2OTP and 2-HP2STP to BSA is also studied in presence of foreign particles.Results obtained are interpreted by the Scatchard plot (Figure 5 and 6).The association constants for 4-HP2OTP-BSA and 2-HP2STP-BSA complexes in presence of foreign particles are 0.7006 (±0.0005) & 0.5757 (±0.0005) for Hg and 0.6782 (±0.0005) & 0.6855 (±0.0005) respectively.The association constant for 4-HP2OTP and 2-HP2STP in absence of impurities of foreign particles were 0.7305 (±0.0005) and 0.7261 (±0.0005).It is noticed that the binding of these ligands is decreased in presence of foreign particles may be due to packing of binding sites in the BSA.

FT-IR analysis
The protein drug binding by FT-IR spectroscopy is analyzed by shifting of amide bands in BSA.The amide I band at 1635 cm -1 is due to C=O stretching and amide II band at 1543 cm -1 is due to C-N stretch coupled with N-H bending.A change in frequency of these bands is observed on binding of ligands to BSA.Similar to ultrasonic and equilibrium dialysis study, more binding is observed in case 1,4-dioxane than DMSO & DMF.The peak position of amide I is shifted from 1635 to 1650 cm -1 in 4-HP2OTP and to 1644 cm -1 in 2-HP2STP as compared to BSA.However, a very small change is observed in the shifting of amide II band in the complex.It is noticed that, as the concentration of ligands increases the frequency of the binding of amide I bans is also increases (Figure 7 and 8).As amide I band is more sensitive to the changes of secondary structure of BSA than amide II therefore increase in binding is characterized by shift in amide I band.Similarly, BSA-ligands binding are not as much as significant in case of DMSO and DMF.

Ultrasonic study
In present study, ultrasonic velocities of 0.15 µM BSA are measured at temperature 298, 303 and 308K and are found to be 1390.301,1393.720 and 1398.105m/s respectively.Ultrasonic velocities of 4-HP2OTP-BSA and 2-HP2STP-BSA complexes are also measured at various concentrations and temperatures (Table 1  and 2).It is observed that different values are obtained at different concentrations and temperatures for the ligands.
The Scatchard graph has plotted against ultrasonic velocity and specific binding versus percent ligand fraction.Binding parameters of 4-HP2OTP and 2-HP2STP to BSA have been determined using Scatchard plot.The Scatchard analysis gave different association constants at different temperatures and solvents.It is found that the association constants in 1, 4-dioxane at 308K is higher than 298K and 303K than DMSO and DMF.It means that the association constants for binding are more significant in 1,4-dioxane at high temperature, concluding that the binding increases with the increased in temperature.Binding is more significant in 1,4-dioxane than DMSO and DMF which is due to aprotic and nonpolar nature of 1,4-dioxane.Comparatively binding is more significant in 4-HP2OTP than 2-HP2STP.From the structural analysis it is evident that, more binding of 4-HP2OTP is may be due to greater hydrogen bonding of oxygen in 4-HP2OTP than sulphur in 2-HP2STP.This increase of association constants in 1, 4-dioxane at high temperature clearly indicates the endothermic nature of reaction.This supports the interaction of ligands to BSA by means of Vander Waal's interactions and hydrogen bonds in the hydrophobic packet of binding sites.It is also observed that binding affinity increased as the concentration of the ligands increases; this probably enhances the pharmacological activity of the drug.

Thermodynamic study
In order to elucidate the interaction of ligands to the BSA, the thermodynamic parameters (∆G, ∆H and ∆S) have been calculated by using van't Hoff equation at the temperatures 298, 303 and    Positive values of ∆H and ∆S indicate that ligands interaction to BSA are enthalpy and entropic driven.Positive values of entropy indicate that there is unfolding of BSA.For unfolding, process must be endothermic which is indicated by positive values of enthalpy and entropy (table 3 and 4).The specific electrostatic interaction is also characterized by the positive values of enthalpy and entropy.The negative value of ∆G indicates that the ligands-BSA complexation is a spontaneous process.As the temperature increased the negative value of ∆G is also increases, which concluded the ligands -BSA interaction is more feasible at high temperature.So, the hydrogen bonding, electrostatic and hydrophobic interactions are supposed to be possible factors contributing binding of the ligands to BSA.Slightly greater free energy values for 4-HP2OTP than 2-HP2STP is due to greater intermolecular hydrogen bonding of oxygen than Sulphur.

Material and Methods
Dialysis membrane (molecular weight cut off 3500) used in the experiment purchased from Sigma Chemical Co.
(USA).UV-VIS spectrophotometer (UV-1800, Shimadzu, Japan) and metabolic shaking incubator (REMI RS-24AC) used in the experiment.FT-IR measurements were taken at room temperature on a Bruker FT-IR spectrometer (Alpha model, Germany) equipped with Zn-Se attenuated total reflection (ATR) accessory.Multi-frequency ultrasonic interferometer (VI microsystem, Chennai, India), BSA (essential fatty acid free) purchased from Chemsworth Chemicals Ltd (India) and used without further purification.Basic buffer selected to maintain the physiological pH.For the synthesis, all the chemicals used are of A.R. grade of Merck India Limited and purchased from commercial suppliers.The purity of the synthesized compound was ascertain by thin layer chromatography on silica gel G in petroleum ether and ethyl acetate (7:3) mixture, melting point recorded using digital melting point apparatus Equiptronics (EQ 730). 1 H NMR spectra of the compound recorded in CDCl3 on NMR instrument (500MHz) using TMS as an internal standard from SAIF, CDRI Lucknow.

Optimization study
4-HP2OTP and 2-HP2STP are not completely soluble in buffer.Hence mixture of buffer with nonaqueous solvent such as 1,4-dioxane, DMF & DMSO used to dissolve 4-HP2OTP and 2-HP2STP.Different ratio of buffer: non-aqueous solvent tried, but the complete solubility of these drugs was obtained at optimum ratio 30:70:nonaqueous solvent:buffer.

Measurements of binding affinity
For the Scatchard analysis, binding affinity of BSA and 4-HP2OTP and 2-HP2STP is expressed as an association constant or binding constant which is derived from the law of mass action.BSA (B) interacts with the ligands (L) i.e. 4-HP2OTP and 2-HP2STP to form the complex as given below.
B + L ⇌ BL Hence, association constant Binding strength of the ligand to BSA is a measure of association constants.

Equilibrium dialysis
Different concentrations (1×10 -3 M to 3.5×10 -3 M) of 4-HP2OTP and 2-HP2STP in 1,4-dioxane, DMSO, DMF (30:70:solvent:buffer) were prepared and mixed separately with 0.15 µM BSA solution.These solutions were allowed to stand at room temperature for the maximum binding of ligands to BSA.From each mixture 3.5 ml solution was poured into previously prepared semipermeable membrane and both the ends were sealed properly.The membrane tubes having 4-HP2OTP-BSA and 2-HP2STP-BSA complex solutions were immersed in a 100 ml conical flask containing 40 ml buffer solution each.These conical flasks placed in a metabolic shaker for dialysis for 12 hrs at room temperature.After dialysis, absorbance's of bound fraction of 4-HP2OTP and 2-HP2STP to BSA were measured on a UV spectrophotometer (λmax 520 nm).

Effect of foreign particles
The binding study of 4-HP2OTP-BSA and 2-HP2STP-BSA is carried out in presence of foreign particles in 1,4-dioxane.0.1M solution of arsenic and mercury salts were prepared and mixed with same solutions of ligands and BSA.These mixed complex solutions were kept some time to check the effect of foreign particles on binding.

FT-IR spectroscopic study
Different concentrations of 4-HP2OTP and 2-HP2STP and the BSA as mentioned above mixed and allowed to stand at room temperature for maximum binding.FT-IR measurements were carried out at room temperature on FT-IR spectrometer equipped with Zn-Se attenuated total reflection (ATR) method.All spectra taken via the ATR method with a resolution of 4 cm -1 and 60 scans in the region 1800-1300 cm -1 .Absorbances of complexes were measured at room temperature.

Ultrasonic study
Ultrasonic is a versatile non-destructive and highly investigatory technique.Ultrasonic absorption a medium provides important tools for the evaluation of the structural, chemical and physical properties of the medium [22].Initially ultrasonic interferometer set at 1MHz.Different concentrations of 4-HP2OTP and 2-HP2STP mixed with BSA and allowed to stand for some time to get maximum binding, ultrasonic velocities of these complex solutions were recorded at 298K.Similar steps were performed at 303K and 308K to determine ultrasonic velocities of the complexes.Specific binding and association constants are determined using Scatchard plot.

Conclusions
In the present study, the binding interaction of 4-HP2OTP and 2-HP2STP to BSA has been studied by equilibrium dialysis, FT-IR spectroscopy and acoustical study at physiological pH in various solvents.The scatchard analysis provided a non-linear curve on binding of ligands to the BSA, suggested the presence of at least two binding sites in BSA.The experimental result by equilibrium dialysis and acoustical study clearly indicate that 4-HP2OTP and 2-HP2STP interact to BSA by means of Vander Waal's interactions and hydrogen bonds in the hydrophobic packet of binding sites.It is also observed that binding affinity increases with increased in the concentrations and temperatures; this probably enhances the pharmacological activity of the drugs.FT-IR spectroscopy showed the binding mainly through amide I site by hydrophobic interaction, which changes the secondary structure of BSA.The greater binding is observed in 4-HP2OTP than 2-HP2STP due to greater intermolecular hydrogen bonding of oxygen than sulphur.Aprotic and nonpolar nature of 1,4-dioxane supports the binding of 4-HP2OTP and 2-HP2STP to BSA than DMSO and DMF.The thermodynamic parameters also indicated that the hydrogen bonding, electrostatic and hydrophobic interactions induce alterations in secondary structure of the BSA.
Figures 9 to 11 shows the Scatchard plots of BSA-4-HP2OTP binding in 1,4-dioxane, DMSO and DMF at 298, 303 and 308 K respectively.Similarly figures 12 to14 shows the Scatchard plots of BSA-2-HP2STP binding in 1,4dioxane, DMSO and DMF at 298, 303 and 308 K, respectively.The effect of temperature on BSAligands binding is summarized in van't Hoff equation.
308 K.The enthalpy change is calculated from the slope of the van't Hoff relationship.Graph plotted between lnk vs 1/T shows straight line with positive slope (Figure 15 and 16).