Thermodynamic and NMR structural studies on the DNA binding of bifunctional drugs with alkylating activity [Elektronische Ressource] / vorgelegt von Michael Rettig

Thermodynamic and NMR Structural Studies on the DNA Binding
of Bifunctional Drugs with Alkylating Activity


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 Michael Rettig
geboren am 06.09.1980
in Berlin


Greifswald, Januar 2010















Dekan: ....................................................................................................................


1. Gutachter : ....................................................................................................................

2. Gutachter: ....................................................................................................................


Tag der Promotion:






Hiermit erkläre ich, dass diese Arbeit bisher von mir weder an der Mathematisch-
Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald noch einer anderen
wissenschaftlichen Einrichtung zum Zwecke der Promotion eingereicht wurde.
Ferner erkläre ich, dass ich diese Arbeit selbständig verfasst und keine anderen als die darin
angegebenen Hilfsmittel benutzt habe.




Unterschrift des Promovenden





Parts of this thesis were published:


Rettig, M., Kamal, A., Ramu, R., Mikolajczak, J., and Weisz, K. (2009) Spectroscopic and
calorimetric studies on the DNA recognition of pyrrolo[2,1-c][1,4]benzodiazepine
hybrids, Bioorganic & Medicinal Chemistry 17, 919-928.

Rettig, M., Weingarth, M., Langel, W., Kamal, A., Kumar, P. P., and Weisz, K. (2009)
Solution Structure of a Covalently Bound Pyrrolo[2,1-c][1,4]benzodiazepine-
Benzimidazole Hybrid to a 10mer DNA Duplex, Biochemistry 48, 12223-12232.

Rettig, M., Langel, W., Kamal, A. and Weisz, K. (2010) NMR Structural Studies on the
Covalent DNA Binding of a Pyrrolobenzodiazepine-Naphthalimide Conjugate, Organic &
Biomolecular Chemistry (submitted).


Table of Contents
Table of Contents
ACKNOWLEDGEMENTS ........................................................................................................................... 1
1 INTRODUCTION ................................................................................................................................ 2
2 THEORY ............................................................................................................................................. 11
2.1 DNA MELTING CURVES .................................................................................................................... 11
2.2 CD SPECTROSCOPY OF NUCLEIC ACIDS ............................................................................................. 12
2.3 FLUORESCENCE TITRATIONS ............................................................................................................. 13
2.4 ISOTHERMAL TITRATION CALORIMETRY ........................................................................................... 14
2.5 DNA RESONANCE ASSIGNMENT STRATEGIES .................................................................................... 16
312.6 P NMR OF DNA ............................................................................................................................ 17
2.7 MOLECULAR MODELING .................................................................................................................. 18
2.7.1 Molecular Modeling using AMBER........................................................................................ 18
2.7.2 Explicit and implicit solvent ................................................................................................... 20
2.7.3 Determination of partial charges ........................................................................................... 21
2.8 NMR RESTRAINTS IN MOLECULAR MODELING .................................................................................. 22
2.9 CLUSTERING ..................................................................................................................................... 23
3 RESULTS ............................................................................................................................................ 25
3.1 SPECTROSCOPIC AND CALORIMETRIC STUDIES ON THE DNA-DRUG INTERACTION ............................ 25
3.1.1 UV melting experiments ......................................................................................................... 25
3.1.2 CD spectroscopy ..................................................................................................................... 29
3.1.3 UV titration ............................................................................................................................. 33
3.1.4 Fluorescence titration ............................................................................................................ 35
3.1.5 Isothermal titration calorimetry ............................................................................................. 36
3.2 NMR STRUCTURAL STUDIES ON THE DNA-DRUG COMPLEX ............................................................. 38
3.2.1 NMR titration ......................................................................................................................... 38
3.2.2 Resonance assignment of the DNA-drug complex .................................................................. 41
3.2.3 Evaluation of drug-DNA interactions ..................................................................................... 53
3.2.4 Stereochemistry at C11 ........................................................................................................... 58
3.2.5 NOE restraints ........................................................................................................................ 61
3.3 MOLECULAR MODELING .................................................................................................................. 62
3.3.1 Restraint MD simulations ....................................................................................................... 62
4 DISCUSSION ...................................................................................................................................... 69
4.1 MODE OF BINDING ............................................................................................................................ 69
4.2 KINETICS OF BINDING ....................................................................................................................... 69
4.3 STRUCTURAL ANALYSIS OF THE DNA-DRUG COMPLEXES ................................................................ 70
4.3.1 Stereochemistry at C11 and orientation of the drug ............................................................... 70
4.3.2 PBD-naphthalimide DNA adduct ........................................................................................... 72
I Table of Contents
4.3.2.1 General characterization of the refined structure .......................................................................... 72
4.3.2.2 Drug-DNA interactions ................................................................................................................ 75
4.3.2.3 Ring flipping of the naphthalimide chromophore ......................................................................... 78
4.3.3 PBD-benzimidazole DNA adduct ............................................................................................ 78
4.3.3.1 General characterization of the refined structure78
4.3.3.2 Drug-DNA interact....... 81
4.3.3.3 Conformation of the N-methylpiperazine ring .............................................................................. 83
4.4 SEQUENCE SELECTIVITY .................................................................................................................... 84
4.5 THERMODYNAMICS OF BINDING ........................................................................................................ 88
5 CONCLUSIONS .................................................................................................................................. 95
6 MATERIALS AND METHODS ........................................................................................................ 97
6.1 MATERIALS ....................................................................................................................................... 97
6.2 UV MELTING EXPERIMENTS ............................................................................................................... 97
6.3 CIRCULAR DICHROISM ....................................................................................................................... 98
6.4 FLUORESCENCE MEASUREMENTS ...................................................................................................... 98
6.5 ISOTHERMAL TITRATION CALORIMETRY ............................................................................................ 99
6.6 NMR ....