Synthesis of novel aziridine cofactors for labeling DNA and its applications [Elektronische Ressource] / Basar Gider

B.Gider

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2011
• Nombre de lectures: 25
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

SYNTHESIS OF
NOVEL AZIRIDINE COFACTORS
FOR LABELING DNA
AND ITS APPLICATIONS



Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH
Aachen University zur Erlangung des akademischen Grades eines Doktors der
Naturwissenschaften genehmigte Dissertation



vorgelegt von
Basar Gider, M.Sc.,
aus Konya, Türkei

Berichter: Universitätsprofessor Dr. Elmar Weinhold
Univeessor Dr. M. Albrecht

Tag der mündlichen Prüfung: 11.03.2011

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online
verfügbar. Die vorliegende Arbeit wurde im Zeitraum von Oktober 2006 bis März 2010 am Institut für
Organische Chemie der Rheinisch-Westfälischen Technischen Hochschule Aachen unter
Anleitung von Prof. Dr. E. Weinhold angefertigt.

Mein besonderer Dank gilt Prof. Dr. Elmar Weinhold für die interessante Themenstellung, das
entgegengebrachte Vertrauen, das stete Interesse und seine Diskussionsbereitschaft.

Herrn Prof. Dr. M. Albrecht danke ich für die freundlich Übernahme des Koreferates.

Teile dieser Arbeit wurden bereits veröffentlicht:
1. F. H.-G. Schmidt, M. Hueben, B. Gider, F. Renault, M.-P. Teulade-Fichou, E.
Weinhold, “Sequence-specific Methyltransferase-Induced Labelling (SMILing) of
Plasmid DNA for Studying Cell Transfection”, Bioorg. Med. Chem. 2008, 16, 40 – 48.


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Table of Contents

Abbreviations ............................................................................................................................ V

1. Introduction ......................... 1
1.1 Epigenetics and DNA methylation .............................................................................. 1
1.2 DNA methylation and disease susceptibility ............................................................... 3
1.3 DNA methylation detection methods .......... 4
1.4 Enzyme-mediated labeling technologies through cofactor engineering ...................... 7
1.4.1 Protein and carbohydrate Labeling ....................................................................... 7
1.4.2 DNA labeling ..................................... 11
1.4.3 Sequence-specific Methyltransferase-Induced Labeling of DNA
(SMILing DNA) ................................................................. 11
1.5 Road Block approach for DNA methylation detection .............. 15

2. Aim of the projects ........................................................................................................... 16

3. Results and Discussion ..... 18
3.1 Novel aziridine cofactor 6RBAz to detect DNA methylation ................................... 18
3.1.1 Design of the novel cofactor 6RBAz ................................. 18
3.1.2 PPEs: Properties, applications and syntheses ..................... 19
3.1.3 Synthetic strategies for the rigid unit ................................. 23
3.1.3.1 Synthesis of the water soluble diiodo monomer ......... 23
3.1.3.2 Direct Sonogashira coupling ....................................... 24
3.1.3.3 Stoichiometric Sonogashira coupling ......................... 26
3.1.3.4 Controlled extension ................................................................................... 28
3.1.3.5 Extension through polymerization .............................. 32
3.1.4 Synthesis of the recognition unit ........................................................................ 35
3.1.5 Peptide coupling reaction ................... 38
3.1.5.1 Carbodiimides with aromatic additives ....................................................... 39
3.1.5.2 Uronium salts .............................................................. 40
3.1.5.3 Peptide coupling of recognition (26) and rigid (19) unit ............................ 41
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3.1.5.4 Peptide coupling of the recognition unit (26) with elongated
rigid unit (32) .............................................................................................. 45
3.2 Novel fluorescent aziridine cofactor 6TAMRAAz .................... 47
3.2.1 Synthesis of 6TAMRAAz .................................................................................. 47
3.3 SMILing DNA applications ....................... 49
3.3.1 SMILing DNA for photoporation transfection studies ....................................... 49
3.3.2 Amplification of labeled DNA by PCR ............................................................. 51
3.3.3 Quantitative labeling of long plasmid DNA with nanometer precision ............. 55
3.3.4 Synthesis of 6BAz, labeling of pBR322 with 6BAz and M.BseCI .................... 60
3.3.5 Labeling of duplex ODN with 6BAz and M.TaqI or M.HhaI ............................ 62

4. Summary, Conclusions and Outlook ................................................................................ 65
4.1 Novel aziridine cofactor 6RBAz to detect DNA methylation ... 65
4.2 Synthesis of 6TAMRAAz cofactor for DNA labeling .............. 69
4.3 SMILing DNA applications ....................................................................................... 69

5. Materials and Methods ...................................................................................................... 71
5.1 Chemicals .................. 71
5.2 General instrumentation ............................................................................................. 73
5.3 Spectroscopy and spectrometry ................. 73
5.3.1 NMR spectroscopy 73
5.3.2 Mass spectroscopy .............................................................................................. 73
5.3.3 UV-Vis absorption spectroscopy ........................................ 74
5.3.4 Fluorescence spectroscopy ................................................. 74
5.3.5 Infrared spectroscopy ......................................................... 74
5.3.6 Elemental analysis .............................................................. 74
5.4 HPLC ......................................................... 75
5.5 Chromatography ........................................................................................................ 76
5.6 Syntheses ................... 77
5.6.1 1,4-Diiodo-2,5-dimethoxybenzene (10) ............................................................. 77
5.6.2 2,5-Diiodobenzene-1,4-diol (11) ........................................ 79
5.6.3 1,4-Diiodo-2,5-di(propyloxysodiumsulfonate)benzene (12) 80
5.6.4 4-[(Trimethylsilyl)ethynyl]benzoic acid (16) ..................................................... 82
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5.6.5 4-Ethynylbenzoic acid (17) ................................................................................ 84
5.6.6 Disodium 3,3'-[(2,5-bis((4-carboxyphenyl)ethynyl)-1,4 - phenylene)] - bis -
(oxy) dipropane-1-sulfonate (18) ...................................................................... 86
5.6.7 Di(triethylammonium)3,3'-[(2,5-bis((4-carboxyphenyl)ethynyl)-1,4 -
phenylene)] - bis - (oxy) dipropane-1-sulfonate (19) ........ 88
5.6.8 6-Chloro-2´,3´-O-isopropylideneadenosine (21) ................................................ 90
5.6.9 N6-(4''-Aminobutyl)-2',3'-O-isopropylideneadenosine (22) .............................. 92
5.6.10 N6-(4''-(Trifluoracetamido)butyl)-2',3'-O-isopropylideneadenosine (23) .......... 93
5.6.11 N6-(4''-(Trifluoraceyl)-2',3'-O-(isopropylidene)-5'-O-
mesyladenosine (24) .......................................................................................... 94
5.6.12 N6-(4''-(Trifluoracetamido)butyl)-5'-O-mesyladenosine (25) ............................ 95
5.6.13 Aziridine (28) ..................................................................................................... 96
5.6.14 5'-N-Aziridinyl-N6-(4''-aminobutyl)-5'-deoxyadenosine (26) 97
5.6.15 6RBAz (29) ........ 98
5.6.16 6TAMRAAz (31) ............................................................................................. 100
5.6.17 1,4-Diethynylbenzene (14) ............... 101
5.6.18 Di(triethylammonium)-3,3'-(2,5-bis((4-((4-carboxyphenyl)ethynyl) phenyl)
ethynyl)-1,4 phenylene)-bis-(oxy)dipropane-1-sulfonate (24) ......................... 102
5.6.19 PPE-SO (33) (n = 20) ...................................................................................... 104 3
5.6.20 PPE-SO (34) (n = 4) ........................ 106 3
5.6.21 6BAz (35) ......................................................................................................... 108
5.7 DNA labeling experiments ...................... 109
5.7.1 Agarose gel electrophoresis .............. 109
5.7.2 Purification of labeled DNA ............................................................................. 109
5.7.3 Enzymes ........................................... 109
5.7.4 DNAs, DNA ladders, PCR primers .................................................................. 110
5.7.5 Labeling of pAcGFP1-N2 DNA with 6TexAz (47) and M.HhaI ..................... 115
5.7.6 Restriction of pAcGFP1-N2 DNA labeled with 6TexAz (47) and M.HhaI ..... 115
5.7.7 PCR amplification of pAcGFP1-N2 DNA l