Synthese und Untersuchungen eines alpha-konfigurierten, oxidativen DNA-Schadens (alpha-cFaPydG) sowie Entwicklung einer PNA-Templat dirigierten Ligationsstrategie

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.