Synthesis and spectrophotometric studies of interaction between transition metal Schiff base complexes and biomolecules

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CHAPTER ONE: INTRODUCTION ………………………………………

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1.1. Schiff Base…………………………………………………………………

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    1.1.1. Metal Schiff base complexes………………………………………..

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          1.1.1.1. Lanthanide ҆s Schiff base macrocyclic complexes……………..

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1.2. Lanthanide Element………………………………………………………

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    1.2.1. General properties……………………………………………………..

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    1.2.2. The therapeutic and biological applications…………………………

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1.3. The structure of DNA…………………………………………………….

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1.4. DNA-ligand interaction…………………………………………………………….

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   1.4.1. DNA-lanthanide interaction……….…..………………………………..

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1.5. Serum albumin……………………………………………………………

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    1.5.1. The structure of Human and Bovine Serum albumin………………

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      1.5.2. Ligand binding to Serum albumins…………………………………

      1.5.3. Serum albumin-Schiff base interaction…………………………….

  1.6. Fluorescence Quenching…………………………………………………

  1.7. The objection of this project……………………………………………..

  CHAPTER TWO

EXPERIMENTAL………………………………………………………..

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2.1. Materials………….………………………………………………………

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2.2. Analytical Instruments………….……………………………………….

2.3. Synthesis and Characterization of Compounds…………………………

    2.3.1. Template Synthesis of Lanthanide (III) hexaaza macrocyclic Schiff       base complex Ln(L¹)…………………………………………………….

         2.3.1.1. Lanthanum (III) hexaaza Schiff base (La(L¹))………………..

    2.3.2. Template Synthesis of Lanthanide (III) hexaaza macrocyclic Schiff

     base complex Ln(L²)……………………………………………………..

         2.3.2.1. Lanthanum (III) hexaaza Schiff base (La(L²))………………….

2.4. DNA-Binding study………………………………………………………

   2.4.1. Electronic absorption spectroscopy……………………………………

   2.4.2. Fluorescence spectral studies………………………………………….

   2.4.3. Viscosity measurements……………………………………………… 2.5. Protein-Binding study……………………………………………………

   2.5.1. Absorption spectral measurements……………………………………

   2.5.2. Fluorescence quenching spectra………………………………………

   2.5.3. Site marker competitive experiments…………………………………

   2.5.4. Conformational change investigation by synchronous fluorescence

   Spectra measurements……………………………………………………….

2.6. The cell proliferation assay………………………………………………

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CHAPTER THREE

RESULTS AND DISCUSSION……………………………………….……….

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3.1. General Information……………………….……………………………….

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3.2. The Characterization of Compounds………………………………………..

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   3.2.1. ¹H NMR Spectra……………………………………………………….

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   3.2.2. The Elemental analysis (C.H.N)……………………………….………

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   3.2.3. IR Spectra……………………………………………………………..

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  3.2.4. Electronic Spectra………………………………………………………

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3.3. Spectroscopic studies on DNA interaction………………………………..

   3.3.1. Electronic absorption titration…………………………………………

   3.3.2. Fluorescence spectroscopic studies……………………………………

         3.3.2.1. Fluorescence quenching…………………………………..……..

         3.3.2.2. Determination of the Binding constants and the number of

         Binding sites……………………………………………………………..

         3.3.2.3. Binding mode between the complex and DNA…………………

   3.3.3. Analysis of the viscometric results…………………………………….

3.4. Spectroscopic studies on Bovine serum albumin (BSA)-interaction………

  3.4.1. Absorption spectra studies……………………………………………..

  3.4.2. Fluorescence quenching results…………………………………………

  3.4.3. Binding Stoichiometries…………………………………………………

  3.4.4. Analysis of the binding constants and the number of binding sites……..

  3.4.5. Thermodynamic parameters and binding modes…………………………

  3.4.6. Site-Selective binding of La(III) Schiff base complexes on BSA……….

  3.4.7. Energy transfer and binding distance between La(III) Schiff base

  Complexes and BSA……………………………………………………………

  3.4.8. Effect of La(III) Schiff base complexes on the protein conformation……

3.5. Spectroscopic studies on Human serum albumin (HSA)-interaction……….

   3.5.1. Absorption spectra measurements……………………………………….

   3.5.2. Fluorescence studies……………………………………………………..

   3.5.3. Binding Stoichiometries………………………………………………..

   3.5.4. Binding parameters……………………………………………………..

   3.5.5. Determination of the thermodynamic parameters and the nature of the

   Binding site between La(III) Schiff base complexes and HSA………………

   3.5.6. Competition reactions between site markers and La(III) Schiff base

   Complexes for binding to HSA………………………………………………..

   3.5.7. Energy transfer between La(III) Schiff base complexes and HSA……..

   3.5.8. Analysis of HSA conformation after binding…………………………..

3.6. Evaluation of growth inhibitory activity of the Schiff base complexes

Against Jurkat cancer cell line………………………………………………….

3.7. Conclusion………………………………………………………………….

References………………………………………………………………………

Appendix……………………………………………………………………….

3.1. Elemental analytical data of La(L¹) and La(L²) complexes……….……………

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3.2. The binding constants (K_b) values for La(L¹) and La(L²) at 25⁰C…………………

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3.3. The values of Stern- Volmer quenching constants (K_sv) and quenching rate constant (K _q), for the interaction of metal complexes with HS-DNA at different temperatures…

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3.4. Apparent binding constant, binding site and Relative Thermodynamic Parameters of DNA-La(III) Complex systems.……………………………………………………

3.5. The values of Stern-Volmer quenching constants (K_sv) and quenching rate

Constant (K_q), for the interaction of metal complexes with BSA at different    temperatures. …………………………………………………………………………

3.6. The values of the average aggregation number of BSA molecules (<J>) for the interaction of metal complexes with BSA……………………………………………

3.7. Apparent binding constant, binding site and Relative Thermodynamic Parameters for the interaction of La(III) complexes with BSA at different temperatures.……………………………………………………………………………

3.8. Binding constants of competitive experiments of Schiff base complexes–BSA system…………………………………………………………………………………..

3.9. Energy transfer parameters for the interaction of metal complexes with BSA….

3.10. The values of Stern-Volmer quenching constants (K_sv) and quenching rate constant (K_q), for the interaction of metal complexes with HSA at different temperatures……………………………………………………………………………..

3.11. The values of the average aggregation number of HSA molecules (<J>) for the interaction of metal complexes with HSA. (T=300 K)……………………………….

3.12. Apparent binding constant, binding site and thermodynamic parameters for the interaction of La(III) Schiff base complexes with HSA at different temperatures….

3.13. Binding constants of competitive experiments of Schiff base complexes–HSA system…………………………………………………………………………………..

3.14. Energy transfer parameters for the interaction of metal complexes with HSA…

3.15. Cell growth inhibitory activity of compounds in vitro…………………………

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2.1. Synthesis of La(L¹)…………………………………………………………………

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2.2. Synthesis of La(L²)………………………………………………………………..

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1.1. Macrocyclic Schiff base compounds……………………………………………….

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1.2. Schematic diagram of the structure of DNA ……………………………………….

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1.3. Illustrations of binding modes………………………………………………………

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1.4. Domain structure of bovine serum albumin…………………………………………

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1.5. Location of binding sites of serum albumin………………………………………….

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3.1. ¹H NMR spectra of La(L¹) in DMSO………………………………………………..

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3.2. ¹H NMR spectra of La(L²) in DMSO…………………………………………………

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3.3. Infra-red spectra of La(L¹) complex………………………………………………….

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3.4. Infra-red spectra of La(L²) complex………………………………………………….

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3.5. Electronic spectra of La(L¹) complex…………………………………………………

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3.6. Electronic spectra of La(L²) complex…………………………………………………..

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3.7. UV–Vis absorption spectra of titration of La(L¹) (dash line) with various concentrations of HS-DNA at 25 °C……………………………………………………………………

3.8. The plot of [DNA]/(ɛ_a – ɛ_f) versus [DNA] for La(L¹) and La(L²) binding to HS-DNA..

3.9. Optimized molecular structures of La(L²) (a) and La(L¹) (b) complexes…………

3.10. Effect of HS-DNA on fluorescence spectra of La(L¹)(λ_EX=284), C(La(L¹)) = 1×10^-6 mol L^-1/ C(HS-DNA)=1×10^-3 mol L^-1): 0.3, 0.6, 0.9, 1.3, 1.6.(T=310 K)……………………

3.11. The Stern–Volmer plots of La(L¹) and La(L²) binding to HS-DNA………………

3.12. The Stern–Volmer for the quenching of La(L¹) by HS-DNA at three  temperatures. λ_ex = 284  nm; λ_em = 338 nm; C(La(L¹)) = 10^-6mol L^-1………………………………………

3.13. Plot of log(F0-F)/F vs. log[Q]. C(La(L¹)) = 1×10^-6 mol L^-1/ C(HS-DNA)= 1×10^-3mol L^-1): 0.3, 0.6, 0.9, 1.3, 1.6………………………………………………………………..

3.14. The van’t Hoff plots of the interaction of the La(L¹) (a) and La(L²) (b) complexes and HS-DNA……………………………………………………………………………………..

3.15. Effects of increasing amounts of La(L¹) and La(L²) complexes on the viscosity of HS-DNA (1.44×10^-5 M) in tris-HCL , at 25⁰C………………………………………………..

3.16. Effect of increasing amounts of [EB] on the viscosity of DNA………………….

3.17. UV−Vis absorption spectra of BSA in the absence and presence of a) La(L¹) and  b) La(L²). Solid line: the absorption spectrum of protein. Dashed line: the absorption spectrum of protein in the presence of La(L¹) and La(L²)  complexes……………………………

3.18. Effect of La(L²) on fluorescence spectra of BSA(λ_EX=280), C(BSA) = 1×10^-7 mol L^-1/ C(La(L²))=1×10^-4 mol L^-1): 0.3, 0.6, 0.9, 1.3, 1.6, 1.9, 2.2.(T=310 K)………………….

3.19. The Stern–Volmer plots of BSA (1.0×10^-7 molL^-1) binding to La(L¹) and La(L²) complexes λ_Ex=280 nm, λ_Em=345 nm……………………………………………………..

3.20. The Stern–Volmer plots for the quenching of BSA by La(L²) at three temperatures. λ_ex = 280  nm; λ_em = 345 nm; C(BSA) = 10^-7mol L^-1………………………………………

3.21. Determination of the average aggregation number of BSA (<J>) in the presence of La(L¹) and La(L²) complexes. λ_Ex=280 nm, λ_Em=345 nm.(T=310 K)………………………

3.22. Plot of log(F0-F)/F vs. log[Q]. C(BSA) = 1×10^-7 mol L^-1/ C(La(L²))=1×10^-4

mol L^-1): 0.3, 0.6, 0.9, 1.3, 1.6, 1.9, 2.2……………………………………………………

3.23. The van’t Hoff plots of BSA binding to La(L¹) and La(L²) complexes……………

3.24. Quenching effect of BSA (1.0×10^-7 mol dm^-3) binding to La(L²)(1×10^-4 mol dm^-3) in the a) absence of site marker, b) presence of ibuprofen and c) presence of phenyl butazone……………………………………………………………………………………

3.25. Fluorescence change of BSA by La(L²)in 5 mM NaCl , λ_EX=290 (Dashed line), λ_EX=280 (Continuous line)…………………………………………………………………..

3.26. Spectral overlap of La(L²)absorption (a) with BSA fluorescence (b)……………..

3.27. Synchronous fluorescence spectra of BSA (1.00×10^-7 mol L^-1) with Δλ=15nm (a) and Δλ=60nm (b) in the absence (dashed lines) and presence of La(L²)(1×10^-4 mol L^-1) (solid lines)………………………………………………………………………………………..

3.28. UV−Vis absorption spectra of HSA in the absence and presence of M[L]: M= a) La(L¹) b)La(L²). Solid line: the absorption spectrum of protein. Dashed line: the absorption spectrum of protein in the presence of M(III) complexes……………………………….

3.29. Effect of La(L²) on fluorescence spectra of HSA, C(HSA) = 1×10^-7 mol L^-1/ C(La(L²))=1×10^-4 mol L^-1): 0.3, 0.6, 0.9, 1.3, 1.6………………………………………..

3.30. Fluorescence change of HSA by La(L²) in 5 mM NaCl , λ_EX=290 (Dashed line), λ_EX=280 (Continuous line)……………………………………………………………….

3.31. The Stern–Volmer plots of HSA binding to La(L²), λ_Ex = 290 nm, λ_Em = 346 nm…

3.32. Determination of the average aggregation number of HSA (<J>) in the presence of La(III) complexes. λ_Ex = 290 nm, λ_Em = 346 nm………………………………………….

3.33. Plot of log(F0-F)/F vs. log[Q]. C(HSA) = 1×10^-7 mol L^-1/ C(La(L²))=1×10^-4

mol L^-1): 0.3, 0.6, 0.9, 1.3, 1.6………………………………………………………………

3.34. The van’t Hoff plots of HSA binding to La(L¹) and La(L²) complexes…………..

3.35. Quenching effect of HSA (1.0×10^-7 mol dm^-3) binding to La(L²) (1.0×10^-4mol dm^-3) in the a)absence of site marker, b)presence of ibuprofen, c)presence of phenyl butazone.(T=300 K)………………………………………………………………………..

3.36. Spectral overlap of La(L²) absorption (a) with HSA fluorescence (b)…………….

3.37. Synchronous fluorescence spectra of HSA (1.00×10^-7 mol L^-1) with Δλ=15nm (a) and Δλ=60nm (b) in the absence (dashed lines) and presence of La(L²) (1×10^-4 mol L^-1) (solid lines)………………………………………………………………………………………….

3.38. The anti-proliferation activity of the Schiff base complexes (C₁=La(L¹), C₂=La(L²)).

3.39. UV–Vis absorption spectra of titration of La(L²) (dash line) with various concentrations of HS-DNA at 25 °C………………………………………………………

3.40. Effect of HS-DNA on fluorescence spectra of La(L²)(λ_EX=284), C(La(L²)) = 1×10^-6 mol L^-1/ C(HS-DNA)=1×10^-3 mol L^-1): 0.3, 0.6, 0.9, 1.3, 1.6.(T=310 K)………………..

3.41. The Stern–Volmer for the quenching of La(L²) by HS-DNA at three  temperatures. λ_ex = 284  nm; λ_em = 338 nm; C(La(L²)) = 10^-6mol L^-1…………………………………………..

3.42. Plot of log(F0-F)/F vs. log[Q]. C(La(L²)) = 1×10^-6 mol L^-1/ C(HS-DNA)= 1×10^-3mol L^-1): 0.3, 0.6, 0.9, 1.3, 1.6……………………………………………………………………..

3.43. Effect of La(L¹) on fluorescence spectra of BSA(λ_EX=280), C(BSA) = 1×10^-7 mol L^-1/ C(La(L¹))=1×10^-4 mol L^-1): 0.3, 0.6, 0.9, 1.3, 1.6, 1.9, 2.2.(T=310 K)……………………….

3.44. The Stern–Volmer plots for the quenching of BSA by La(L¹) at three temperatures. λ_ex = 280  nm; λ_em = 345 nm; C(BSA) = 10^-7mol L^-1…………………………………………

3.45. Plot of log(F0-F)/F vs. log[Q]. C(BSA) = 1×10^-7 mol L^-1/ C(La(L¹))=1×10^-4

mol L^-1): 0.3, 0.6, 0.9, 1.3, 1.6, 1.9, 2.2………………………………………………….

3.46. Quenching effect of BSA (1.0×10^-7 mol dm^-3) binding to La(L¹)(1×10^-4 mol dm^-3) in the a) absence of site marker, b) presence of ibuprofen and c) presence of phenyl butazone…………………………………………………………………………………….

3.47. Fluorescence change of BSA by La(L¹)in 5 mM NaCl , λ_EX=290 (Dashed line), λ_EX=280 (Continuous line)……………………………………………………………….. 3.48. Spectral overlap of La(L¹) absorption (a) with BSA fluorescence (b)……………

3.49. Synchronous fluorescence spectra of BSA (1.00×10^-7 mol L^-1) with Δλ=15nm (a) and Δλ=60nm (b) in the absence (dashed lines) and presence of La(L¹) (1×10^-4 mol L^-1) (solid lines)…………………………………………………………………………………………

3.50. Effect of La(L¹) on fluorescence spectra of HSA, C(HSA) = 1×10^-7 mol L^-1/ C(La(L¹))=1×10^-4 mol L^-1): 0.3, 0.6, 0.9, 1.3, 1.6……………………………………………

3.51. Fluorescence change of HSA by La(L¹) in 5 mM NaCl , λ_EX=290 (Dashed line), λ_EX=280 (Continuous line)…………………………………………………………………

3.52. The Stern–Volmer plots of HSA binding to La(L¹), λ_Ex = 290 nm, λ_Em = 346 nm……..

3.53. Plot of log(F0-F)/F vs. log[Q]. C(HSA) = 1×10^-7 mol L^-1/ C(La(L¹))=1×10^-4

mol L^-1): 0.3, 0.6, 0.9, 1.3, 1.6…………………………………………………………….

3.54. Quenching effect of HSA (1.0×10^-7 mol L^-1) binding to La(L¹) (1×10^-4 mol L^-1) in the a) absence of site marker, b) presence of ibuprofen and c) presence of phenyl butazone……………………………………………………………………………………

3.55. Spectral overlap of La(L¹) absorption (a) with HSA fluorescence (b)……………

3.56. Synchronous fluorescence spectra of HSA (1.00×10^-7 mol L^-1) with Δλ=15nm (a) and Δλ=60nm (b) in the absence (dotted lines) and presence of Na₂ La(L¹) (1×10^-4 mol L^-1) (solid lines)…………………………………………………………………………………