Syntheses Of The Metal Complexes Involving Benzimidazole And Their Interaction With DNA

Benzimidazole functions as active group in anticancer, antitumor, antibacterial drugs and in a variety of biologically important molecules. Its particular π-conjugate system and high sequence-selective DNA binding make its derivatives as a probe for DNA structure detection. A more profound study on the mechanism of its interaction of the benzimidazole derivatives with DNA, and on the relationships between the molecular structure and biological activity will not only help us to understand the process of life on a molecular level and pathogenic mechanism of some illness, but also provide rationales for new drug design, as well as a means to develop novel powerful agents for probing and targeting nucleic acids.In this thesis, we have synthesized a series of benzimidazole derived metal complexes, characterized their structures with the methods of elemental analyses and X-ray crystallography. We also investigated the interaction of these complexes with DNA by using UV, fluorescent spectra, viscosity measurements, equilibrium dialysis and gel electrophoresis. We found that the other complexes cannot cleave DNA, except BBAMn(II) can cleave DNA via a hydrolytic mechanism;the complexes containing two benzimidazole groups form a near planar geometry and are mainly electrostatically bound to DNA;the complexes involving four benzimidazole groups form a distorted octahedral geometry and interact with DNA by partial insertion and electrostatic binding;the complexes can selectively bind with DNA. The results are:Two ligands containing respectively two benzimidazole rings, Bis(2-benz imidazolylmethyl)amine(BBA) and N-(2-hydroxyethyl)bis(2-benzimidazolyl methyl)amine(BBAOH), have been synthesized, and their metal complexes,BBACu(II), (BBA)2Mn(II) and BBAOHCu(II) have been determined by X-ray crystallography. The BBA acts in a tridentate manner, all taking N as the donors, namely, two imidazole-N atoms from two benzimidazole groups and one N from a second amine which acting as a bridge to link the two benzimidazole rings. While in Mn(II) complex (BBA)2Mn(II), bis[bis(benz imidazol-2-ylmethyl)amine]-A:3A^A^')A^";/c2Ar)Ar'-chloromanganese(II), a imida-zole N atom was replaced by Cl". The second ligand, BBAOH, acts as a quadridentate, more specifically, a hydroxyethyl O atom, two benzimidazole N atoms and a tertiary amine N atom. The interaction of BBACu with CT(calf thymus) DNA has been investigated with UV, fluorescent spectra, viscosity, Scatchard plots. The experimental results show that the acting mode of the complex to the negatively charged oxygen of phosphodiester of DNA is mainly electrostatic. Studies on the inhibition of the restriction enzymes Dral and Sma\ revealed that the interaction of the complex BBACu(II) preferring to AT-rich sequences than to CG-rich sequences. Applying polyelectrolyte theory we analyze the fluorescence and equilibrium dialysis values, and obtained the binding constant of BBACo(II) with CT DNA as 1.96xl04L/mol. Combined with viscosity and gel electrophoresis results, we conclude that complex BBACo(II) is essentially electrostatically bound to DNA. We also found that complex BBAMn(II) can act as an effective artificial nuclease, promoting the hydrolysis of plasmid DNA under the physiological pH and temperature. Studies on the mechanism of DNA cleavage, resorting to several methods, including the addition of radical scavengers, strict anaerobic condition, malondialdehyde-like products analysis, and religation assays implicate that DNA cleavage mediated by BBAMn(II) occurs via a hydrolytic path. The catalytic rate constant (Kca[) for the decrease of form I, valued as 0.72 ± 0.02 h"'(pH=8, 37 °C), was obtained from a best-fit line of the apparent rate constant versus complex concentration, based on the transformation of the Michaelis-Menten equation. In BBAOHCu(II) crystal, the Cu(II) ion is five-coordinated, adopting a distorted square-pyramidal geometry, and taking its coordination set by threeN and one O from the ligand BBAOH and one another O from water molecule;The BBAOHCu(II) molecules are interleaved to form a n-n stack. The T4 DNA ligase circularization assay indicates that the complex can partially intercalate into the double helix of DNA. Studies on the inhibition of the restriction enzymes Dral and Smal have shown that the complex prefers the AT-rich sequences.Two ligands, 7V,Af,Ar,A^-tetrakis(2-benzimidazolylmethyl)-l,2-ethanediam ine)(EBA) and N,N,N ,N-tetrakis[(2-benzimidazolyl)methyl]-l ,3-diaminopro pane(PBA) have been synthesized, and the crystals of EBAZn(II), PBAZn(II), PBACu(II) and PBANi(II) have been obtained. Both ligands contain two bis(2-benzimidazolylmethyl)amine groups. The difference between them is that the number of the C atoms in the bridging chain is 2 in EBA, and 3 in PBA. Both ligands are sexadentate, taking four benzimidazole N atoms and two amine N atoms as their coordination sets, and bind metal ions to form a distorted octahedral geometry. The differences of the metal ions and the coordinated structure of the complexes lead to the different experimental results concerning the interaction of the complexes with DNA. Our investigation of the interaction of EBAZn, PBAZn, PBACu and PBANi with DNA showed that the DNA hyperchromicity in PBAZn is larger than that of EBAZn, the latter two lead to a hypochromism from DNA;The four complexes make the m.p. of DNA increase by 1.9 °C, 2.2 °C, 1.7 °C and 4 °C, respectively, suggesting the order of binding intensity: PBANi > PBAZn > EBAZn > PBACu;Scatchard plots show that the binding mode of complex EBAZn to DNA is electrostatic in main, and the latter three is a mixed-mode (partial insertion and electrostatic binding);T4 DNA ligase circularization assay gave an order for unwinding of the double helix as: PBANi > PBAZn ~ PBACu > EBAZn, this can be explained with the differences of their structure, the dihedral angels formed by the optimum benzimidazole ring for intercalating into the double helix of DNA and the adjacent two benzimidazole rings in these complexes (67.4° and 90 ° for EBAZn, 96.9° and 69.2° for PBAZn, 97.3° and 72.8° for PBACu, 118.8° and94.4 ° for PBANi, respectively). Compared to the above dihedral angels, we conclude that PBANi molecule can more deeply intercalate into the double helix of DNA, which leads to the maximal degree of unwinding of the double helix. Studies on the inhibition of the restriction enzymes Dra\ and Sma\ have shown that EBAZn can nonselectively bind with DNA;compared to the first cleavage site of Dral, the binding of PBAZn with DNA inclines to the second cleavage site;PBACu and PBANi prefers the CG-rich sequences. In addition, DNA make the fluorescence intensity of EBAZn, PBAZn decrease, the binding constants calculated from the fluorescence are 4.7 xlO4 L/mol (EBAZn) and 7.2 xlO4 L/mol(PBAZn). The binding constant for PBACu with DNA obtained from equilibrium dialysis is 1.21 * 104 L/mol.l,3-bis(benzimidazol-2-yl)-2-oxapropane(OBA) has been synthesized, and the crystals of (OBA)2Zn(II) and OBACu(H) have been obtained. Compared to the BBA, the bridged N atom was replaced by O atom in the OBA. It acts in a tridentate manner, namely two imidazole N atoms and one O atom. The Zn(II) environmental in (OBA)2Zn(II) is distorted octahedral. The binding constants of the complex with DNA obtained from UV and fluorescent spectra are consistent in general, both are closed to 1 xlO4 L/mol. The interaction between the complex and DNA make the m.p. of DNA increase 2.4 °C, resonance light scattering enhance, and the DNA's relative viscosity decrease. Scatchard plots show a mixed mode of the (OBA)2Zn(II) binding to DNA, viz. partial insertion and electrostatic binding. The Cu(II) ion in OBACu(II) crystal is five-coordinated by two benzimidazole N atoms, one O atom of the ligand OBA and two Cl" ions.The theoretical studies on the complex OBACu(II) are carried out with DFT method at B3LYP/ LanL2DZ level. Calculations show that the ligand makes major contributions to the HOMO and the LUMO, and indicate the coordination ability between the OBA and Cu(II) in OBACu2+ get stronger than that in OBACuCI2. The T4 DNA ligase circularization assay indicates that the interaction mode between the complex and DNA is the electrostatic binding, which is similar to that of BBACu(II). Studies on the inhibition ofthe restriction enzymes Dra\ and Smal have shown that the complex tends to bind with the AT-rich base sequence.