Indoloquinoline-Oligonucleotide Conjugates in the antigene strategy [Elektronische Ressource] : binding and NMR structural studies. / Andrea Eick

Indoloquinoline-Oligonucleotide Conjugates in the Antigene Strategy: Binding and NMR Structural Studies I n a u g u r a l d i s s e r t a t i o n zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) an der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald vorgelegt von Andrea Eick geboren am 02.01.1982 in Königs Wusterhausen Greifswald, September 2011 Dekan: Prof. Dr. Klaus Fesser 1. Gutachter: Prof. Dr. Klaus Weisz 2. Gutachter: Prof. Dr. Gerd Buntkowsky Tag der Promotion: 28. November 2011 ± Table of Contents 1 Introduction .................................................................................................................................................... 1 2 General Concepts ........................................................................................................................................ 11 2.1 Physicochemical Methods in Nucleic Acids Research ....... 11 2.1.1 UV-Vis Spectroscopy ................................................. 11 2.1.2 CD Spectroscopy ....................................................................................... 12 2.1.3 Fluorescence Spectroscopy ....................................... 14 2-Aminopurine - Fluorescent Probe in Nucleic Acids Research .............
Publié le : samedi 1 janvier 2011
Lecture(s) : 26
Source : D-NB.INFO/1018189599/34
Nombre de pages : 134
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Indoloquinoline-Oligonucleotide Conjugates in the Antigene Strategy:
Binding and NMR Structural Studies


I n a u g u r a l d i s s e r t a t i o n


zur


Erlangung des akademischen Grades


doctor rerum naturalium (Dr. rer. nat.)


an der Mathematisch-Naturwissenschaftlichen Fakultät


der


Ernst-Moritz-Arndt-Universität Greifswald



vorgelegt von

Andrea Eick

geboren am 02.01.1982

in Königs Wusterhausen




Greifswald, September 2011


















Dekan: Prof. Dr. Klaus Fesser



1. Gutachter: Prof. Dr. Klaus Weisz


2. Gutachter: Prof. Dr. Gerd Buntkowsky

Tag der Promotion: 28. November 2011




3.5 NMR Structural Studies of PIQ-DNA Complex .................................................................................. 65
3.5.1 Resonance Assignment of Triple-Helical DNA .......... 66
3.5.2 Resonance Assignment of PIQ-DNA Complex .......................................................................... 75
15 13Resonance Assignment of the N-, C-labeled Triplex ............................................................ 75
Resonance Assignment of the PIQ Ligand ................................................................................ 85
3.5.3 Construction of Molecular Models ............................................................. 91

4 Conclusions ................................................................................................................................................. 97

5 Experimental Part ...................................................................... 100
5.1 Materials .......... 100
5.2 Methods ........................................................................... 102
5.2.1 Syntheses ................................................................................................ 102
5.2.2 Mass Spectrometry.................................................................................................................. 105
5.2.3 Flash Chromatography ............................................ 105
5.2.4 High Pressure Liquid Chromatography (HPLC) ....................................... 106
5.2.5 UV Measurements ................................................... 106
5.2.6 CD Measurements 107
5.2.7 Fluorescence Measurements .................................................................................................. 107
5.2.8 Isothermal Titration Calorimetry .............................................................. 110
5.2.9 NMR Spectroscopy.................................................................................................................. 111
5.2.10 Molecular Modeling .............................................. 113

6 Abbreviations............................................. 114

7 Literature .................................................................................................................................................... 115

8 Supplementary Material ............................................................................................................................ 128

Eidesstattliche Erklärung ..................................................................... i



Part of this work has been published or has been submitted for publication:
Eick, A., Riechert-Krause, F., and Weisz, K. (2011) Binding and NMR structural studies on indoloquinoline-
oligonucleotide conjugates targeting duplex DNA, submitted.
Riechert-Krause, F., Eick, A., Grünert, R., Bednarski, P. J., and Weisz, K. (2011) In vitro anticancer activity and
evaluation of DNA duplex binding affinity of phenyl-substituted indoloquinolines. Bioorg. Med. Chem. Lett. 21,
2380-2383.
Eick, A., Riechert-Krause, F., and Weisz, K. (2010) Spectroscopic and calorimetric studies on the triplex formation with
oligonucleotide-ligand conjugates. Bioconjug. Chem. 21, 1105-1114.
Eick, A., Xiao, Z., Langer, P., and Weisz, K. (2008) Spectroscopic studies on the formation and thermal stability of DNA
triplexes with a benzoannulated -carboline-oligonucleotide conjugate. Bioorg. Med. Chem. 16, 9106-9112.

Posters:
Riechert-Krause, F., Autenrieth, K., Eick, A., and Weisz, K. (2011) Studies on the interaction of phenyl-substituted
thindoloquinolines with DNA triplexes by spectroscopic methods. 4 European Conference on Chemistry for Life Sciences,
August 31- September 03, 2011, Budapest/ Hungary.

Weisz, K., Riechert-Krause, F. and Eick, A. (2010) Targeting DNA triplexes with phenyl-substituted indoloquinoline
ligands. International Chemical Congress of Pacific Basin Societies, December 15-20, 2010, Honolulu/ Hawaii.

Riechert-Krause, F., Eick, A. and Weisz, K. (2010) Spectroscopic studies on DNA binding and in vitro anticancer
properties of phenyl-substituted indoloquinolines. IRT 2010 - XIX International Round Table on Nucleosides, Nucleotides
and Nucleic Acids, August 29 - September 03, 2010, Lyon/ France.

Eick, A., Riechert-Krause, F. and Weisz, K. (2009)Targeting double-helical DNA with -carboline derivatives tethered to
rd a triplex forming oligonucleotide. 3 European Conference on Chemistry for Life Sciences, September 02-05, 2009,
Frankfurt am Main/ Germany.

Riechert-Krause, F., Eick, A. and Weisz, K. (2009) Spectroscopic studies on the binding of two benzoannulated -
rdcarbolines to triplex DNA. 3 European Conference on Chemistry for Life Sciences, September 02-05, 2009, Frankfurt
am Main/ Germany.

Eick, A., Riechert, F. and Weisz, K. (2008) Spectroscopic studies of DNA triplex formation with a -carboline-TFO
conjugate. 3. Nukleinsäurechemie-Treffen (Poster and oral presentation), September 05, 2008, Stuttgart/ Germany.

thEick, A. and Weisz, K. (2008) DNA triplex formation with a -carboline-oligonucleotide conjugate. 236 American
Chemical Society National Exposition, August 17-21, 2008, Philadelphia/ USA.

/////1 Introduction
Initially, triple helices were solely considered as being artificial and exotic structural motifs lacking
any physiological relevance. This changed with the finding, however, that an intramolecular triple helical
structure called H-DNA can also be formed in vivo by homopurine-homopyrimidine sequences when
organized in mirror repeats and promoted by negatively supercoiled DNA [10]. Here, one of the single strands
folds back to form a triple helix and the other strand is left unpaired and can be targeted by single strand
binding proteins. This alternative DNA secondary structure has been shown to be involved in the regulation of
DNA replication and in the activation of the cell repair machinery, which may result in DNA translocations,
rearrangements and in the worst case cell lysis [for a review see 11].
In recent years, two strategies for selectively inhibiting gene expression through the use of
oligonucleotides have been developed: the triplex-based antigene strategy and the antisense approach
[12,13]. The antisense strategy utilizes oligonucleotides that specifically bind complementary mRNAs via
conventional Watson-Crick base pairing mostly resulting in arrested translation. The antigene strategy on the
other hand makes use of triplex-forming oligonucleotides (TFOs) and target the gene itself rather than its
mRNA product.
Unfortunately, triplex formation is often hampered by the low thermodynamic stability of the formed
complexes, especially under physiological conditions. Therefore, efforts aimed at the stabilization of triplexes
under physiological conditions are prerequisite for a successful and more general application of an antigene-
based strategy [for a review see 14]. Thus, a modified non-ionic backbone as within peptide nucleic acids
(PNA) decreases the high negative charge density resulting from the association of three anionic strands.
There is also considerable interest in finding base analogues that could replace the positively charged
cytosine in a third homopyrimidine strand to form stable triplexes without the necessity of a low pH. Besides,
2+various sodium salts and divalent cations as Mg are known to stabilize pyrimidine triplexes [15]. Especially
polyamines like the physiologically relevant spermine [16] and the aminogylcoside neomycin [17] are able to
interact electrostatically with the negatively charged nucleic acid backbone and demonstrate increased triplex
stabilities without affecting the overall secondary structure. Another common approach to stabilize triplexes
makes use of triplex-specific binding agents. Whereas numerous natural and synthetic drugs are known to
bind double-helical DNA, only a limited number of triplex-specific agents have been described and

4 1 Introduction
characterized to date [18]. The latter have been found to enhance stabilities of DNA triplexes mostly through
intercalation. Examples are illustrated in Figure 1.3 and 1.4 and include various benzopyridoindoles (BPI) with
a carboline substructure [19-21], benzopyridoquinoxalines (BPQ) [22] as well as benzoquinoquinoxalines
(BQQ) [23]. Also, substituted anthraquinones [24,25], dibenzophenanthrolines [26,27], coralyne [28] and
naphthylquinoline [29] have been studied as triplex-stabilizing drugs. In an attempt to further increase the
triplex stability as well as selectivity, the best triplex-recognizing compounds have also been combined. Thus,
BQQ-neomycin conjugates combine the features of one of the best triplex-specific intercalators with the
negative charge compensating neomycin that selectively binds within the triplex groove [30,31].

Figure 1.3: Chemical structures of some triplex-stabilizing ligands.


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