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Büsch, Florian (2007): Synthese und Untersuchungen eines alpha-konfigurierten, oxidativen DNA-Schadens (alpha-cFaPydG) sowie Entwicklung einer PNA-Templat dirigierten Ligationsstrategie. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Following oxidative stress often two kinds of DNA-lesions can be found: 8-OxodG and the 2,6-Diamino-4-hydroxy-5-formamidopyrimidine (FaPydG). Both derive from the DNA-Nucleobase guanine. The FaPydG-lesion can exist both in the beta-configuration and the alpha configuration. To clarify biological questions concerning the basepairing and the encoding features of both forms it was necessary to synthesise of the alpha-anomer of the carbocyclic FaPydG-DNA-lesion. The synthesis of the beta-cFaPydG-lesion was already finished in 2005 in the Carell group. In a sixteen-step synthesis the alpha-lesion could be prepared in the form of its phosphoramidite component. Oligonucleotides could be prepared on solid support using automated DNA-synthesis. Some of the synthesised DNA-Nucleotides contained the inserted FaPydG-lesion. Others showed an oxidation to alpha-c8-OxodG. This reaction is not known for natural lesion. Just a de-hydration to guanosine under harsh conditions is described. The observations can be ex-plained by formulation of a cyclic intermediate, which is possibly oxidized by the oxidation di-lution during the DNA-synthesis. If the sequence-dependent equilibrium between alpha cFaPydG and alpha c8-Hydro,hydroxydG is at the side of the closed form, an oxidation from this interme-diate directly to 8-OxodG is imaginable. This assumption is proven by the fact that at the same time synthesised and completely identically treated DNA-strands of different sequence didn´t show oxidation products. This could be ascertained by high resolution ESI-FTICR-mesurement. With this analytical method even smallest variations are detectable in a reliable way, whereas the often used MALDI-TOF-mesurements not always show these variations. Thermodynamic studies showed that alpha-cFaPydG doesn´t form a preferred DNA-basepair. All possible basepairs showed a strong destabilisation in comparison to the undamaged, cor-rect basepair. In vitro elongation experiments with S. cerevisiae polymerase Pol eta, G. stearothermophilus DNA-polymerase I and DinB from G. stearothermophilus showed that the alpha-lesion is a definitive block for all mentioned polymerases. Only by applying rigorous re-action conditions an elongation of the primer with BstPol I could be detected. This elongation showed a favored incorporation of dCTP, followed by an incorporation of dATP. In the time to come experiments to avoid the unwanted oxidation of alpha-cFaPydG will be es-sential to promote the cocristallisation of the alpha-cFaPydG-oligomere with the Fpg-protein from Lactococcus lactis and the BstPol I. In the second part of the thesis attempts were taken to prepare tetrafunctional amino acid derivatives, which were planned to be used in a PNA-template directed ligation reaction. Liga-tion takes place between two amino acids, so that for a ligation attempt with this method there is always needed a matching pair of amino acids. This pair requires a connection of the aminoacid to the PNA over the side chain of the amino acid. The N- and C-terminus of the amino acid had to be unprotected for the ultimate ligation reaction. These termini must not react during the solid support PNA-synthesis. After ligation the template should be separated selectively. Thus, the following requirements for the pair of amino acids result: The amino acids have to possess functional side chains, which exist naturally after cleavage of the PNA-template. For the selective cleavage of the template a con-nection orthogonal to acid and base unstable protecting groups is necessary, because these are already needed for the solid support synthesis on the residual three termini of the compound. Imaginable orthogonal cleavable links are allylic compounds, which are cleavable with Pd(0), but also silicon based compounds, which are cleavable with fluoridions, would be appropriate. In the area of the Pd(0)-cleavable link a retrosynthetic cut at this funtionality should be adequate. Here, e.g. a Horner-Wadsworth-Emmons-reaction is imaginable. The aldehydes and phosphorylides needed for this key step could be prepared with good yields, however only one of the two amino acid derivatives was obtained. The synthesis of the other compound didn´t succeed under diverse reaction conditions. Also efforts to prepare analogue Pd(0)-cleavable link molecules through classic Wittig or metathese reactions with Grubbs catalysts of the first and second generation were unsuccessful. The most successful efforts to make a target molecule synthetically accessible for a first liga-tion attempt involved the preparing of a fluorid-cleavable silyl link. Here all requirements stay the same, just instead of allylic compounds silyl ethers are needed. The acid stability of the silyl protecting group depends on the size of the alkyl groups (tert-butyl>isopropyl>ethyl). Firstly, substitution experiments with the compounds Di-tert-butylchlorosilane, Di-tert-butyl-dichlorosilane and Di-tert-butylsilylbis(trifluoromethanesulfonate) were undertaken to estab-lish a connection between PNA and the amino acid through a Di-tert-butyldisilylether. How-ever, the substitutions produced just monosubstituted silanoles, irrespective of the choice of reaction conditions and substitution order. The experiments with the commercially available (3-Cyanopropyl)-diisopropyl-chlorosilane were more successfull. Here the synthesis of one of the target molecules could be achieved.