Towards sequence-selective DNA recognition withdesigned major groove binders

M.Sc Hu, Wenbin

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This thesis is mainly focused on the design, synthesis and DNA binding studies of aminobenzyl and guanidinium calix[4]arene dimers. The work consists of two parts. The first part of my Ph.D. work was focused on the synthesis and DNA binding studies of Dimer A and Dimer B. In order to broaden the scope of the investigation, the corresponding monomeric calixarenes (Monomer A and Monomer B) were also synthesized. Compared with anilino-calix[4]arenas (R. Zadmard, T. Schrader, Angew. Chem. Int. Ed. 2006, 45, 2703 –2706), these dimers and monomers are water-soluble, and bind to DNA with much higher affinity (binding constants: 1000000–100000000 M-1 in 2 mM Hepes buffer with 150 mM NaCl).
When investigating the properties of new DNA binding molecules, one initial goal is to establish their mode of binding to DNA. Because direct structure information from a crystal or NMR structure was not yet available in my work, several other biophysical experiments have been designed and performed between calixarenes and different nucleic acids with varying base composition and conformation. The following results from established binding assays strongly support a major groove binding mode:
(1). The DAPI displacement assay indicates that the calixarene dimers and DAPI can simultaneously bind to poly (dAdT) – poly (dAdT). Because DAPI occupies the minor groove, the dimeric calixarenes should reside in the major groove.
(2). Ethidium bromide displacement assays, fluorescence titrations, and circular dichroism measurements indicate that calixarene dimers strongly prefer nucleic acids with a wide, shallow or even major groove; while low affinities are observed for nucleic acids with a narrow, deep or rugged major groove, which must be widened before complexation occurs. This characteristic also implies that the accessible area for our ligands is on the major groove side, contrary to the well-known slim oligoamide binders, which target the minor groove.
In line with these observations, it was noticed that changes in shape and width of the minor groove had no influence on the ligands’ affinities.

Dimer A

Dimer B

Monomer A

Monomer B

Unfortunately, Dimer A and Dimer B showed similar affinities for different DNA duplexes. Therefore, we planed to replace the simple alkyl bridge between both calixarenes by a fragment which should be able to bind specific sequences of DNA in the promoter region of each gene. Heterocyclic oligoamides were found out. They consist of N-methylpyrrole, 3-hydroxypyrrole and N-methyl imidazole amino acid building blocks (P. B. Dervan, Bioorganic & Medicinal Chemistry 2001, 9, 2215–2235.).

In the second part of my Ph.D. work, a calixarene dimer with a triimidazole bridge (Dimer C) was synthesized.

Dimer C
Because in minor grooves, N-methylimidazoles of polyamides could recognize the positive electrostatic potential of amino groups of guanine bases via hydrogen bonds, it was assumed that Dimer C, which contained a triimidazole bridge, could prefer a major groove with large positive potentials. Moreover, because the triimidazole bridge is slim, it was also assumed that Dimer C should prefer a narrow major groove, which could offer the appropriate shape for the bridge’s recognition. In view of the above-mentioned assumptions, we found that the major groove of poly dG – poly dC fulfilled both requirements. Therefore, it was predicted that Dimer C should show the high affinity for poly dG – poly dC.
Ethidium bromide displacement assays, fluorescence titrations, and circular dichroism measurements imply that Dimer C is a major groove binder. Furthermore, as we expected, these experiments indicate that Dimer C showed stronger preference for this specific DNA duplex: poly dG – poly dC.
It was also observed that Dimer C frequently showed reduced affinity to other DNA duplexes. Probably, the decrease in affinity is related to their wider major grooves, in which the slim triimidazole bridge will not produce extensive close van der Waals contacts, and the whole ligand itself shows orientational disorder. Another explanation is the unfavorable electrostatic potentials, because these DNA duplexes have more neutral areas in major grooves than does poly dG – poly dC.
Herein, we present a proposed mechanism of DNA binding by Dimer C: the cationic moieties on the upper rim of the calixarenes form hydrogen bonds with nucleobases as well as phosphate groups of the nucleic acid, the butoxy tail is toward the free solution, and the triimidazole bridge binds along the major groove and recognizes the amino groups of nucleobases.
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Wissenschaftliche Abschlussarbeiten » Dissertation
Fakultät / Institut:
Fakultät für Chemie » Organische Chemie
Dewey Dezimal-Klassifikation:
500 Naturwissenschaften und Mathematik » 540 Chemie » 547 Organische Chemie
major groove binding, dimeric calixarene, polyamide, sequence selectivity, binding affinity, minor groove binding, DAPI, ethidium bromide, circular dichroism spectroscopy, fluorescence titration
Prof. Dr. Schrader, Thomas [Gutachter(in), Rezensent(in)]
Prof. Dr. Schmuck, Carsten [Gutachter(in), Rezensent(in)]
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