Download Click, Double Click and Chelating Click Reactions Performed on Base

Click, Double Click and Chelating Click Reactions Performed on BaseModified Nucleosides and Oligonucleotides: Fluorescence Properties of
Pyrene Conjugates
Click, Doppel-CIick und Chelat-Click Reaktionen an basenmodifizierten
Nukleosiden und Oligonukleotiden: Fluoreszenz-Eigenschaften von
Pyren-Konjugaten
dem Fachbereich Biologie/Chemie
der Universitat Osnabruck
zur Erlangung des Grades eines
Doktors der Naturwissenschaflen
-Dr. rer. nat.-
vorgelegte Dissertation
-Thesis-
von
Sachin Asaram Ingale
aus Parbhani, Maharashtra, Indien
Osnabruck 2013
http://d-nb.info/1051644380
Click, Double Click and Chelating Click Reactions Performed on Base-Modified
Nucleosides and Oligonucleotides: Fluorescence Properties of
Pyrene Conjugates
Table of Contents
1 '
Introduction
1
1.1
7-Deazapurine (Pyrrolo[2,3-</]pyrimidine) Nucleosides
1
1.2
Copper(I)-catalyzed Azide-alkyne Cycloaddition (CuAAC) Reaction
2
1.3
Pyrene
5
2
Subject of the Work
8
2.1
Click and Double Click Reactions Performed on 7-Deazaguanine
Nucleosides and Oligonucleotides with 1-Azidomethyl Pyrene
2.2
Nucleobase Controlled Fluorescence Quenching of 7-Deazapurine and
8-Aza-7-deazapurine Nucleoside and Oligonucleotide Pyrene Click Conjugates
2.3
10
Stepwise Functionalization of DNA by the Click Reaction with Chelating
Bi factional Azides
11
2.5
A Ratiometric Fluorescent Chemosensor (On-OfI) for
2.6
Ethynyl Side Chain Hydration During Synthesis and Work-up of "Clickable"
Zn2+ Ions
Oligonucleotides
3
9
Nucleoside and Oligonucleotide Pyrene Conjugates with 1,2,3-Triazolyl or
Ethynyl Linkers
2.4
8
12
12
"Double Click" Reaction on 7-Deazaguanine DNA: Synthesis and Excimer
Fluorescence of Nucleosides and Oligonucleotides with Branched Side Chains
Decorated with Proximal Pyrenes
14
3.1
Background
14
3.2
Synthesis of the Phosphoramidite Building Block of 7-Deaza-7-tripropargylamine-2'-deoxyguanosine
15
3.3
Synthesis of Nonbranched, Branched and Abasic Pyrene Click Conjugates
15
3.4
Synthesis and Duplex Stability of Alkynylated Oligonucleotides and
Oligonucleotide Pyrene Click Conjugates
16
3.4.1
Synthesis and Characterization of Alkynyl Modified Oligonucleotides
16
3.4.2
Synthesis and Characterization of Pyrene Modified Oligonucleotides
17
3.4.3
Duplex Stability of Alkynyl Modified Oligonucleotides and Their Pyrene Click
Conjugates
3.5
18
Photophysical Properties-of Nucleoside and Oligonucleotide Pyrene Click
Conjugates
19
3.5.1
Nucleoside Pyrene Click Conjugates
19
3.5.2
Oligonucleotide Pyrene Click Conjugates
19
4
7-Deazapurine and 8-Aza-7-deazapurine Nucleoside and Oligonucleotide
Pyrene "Click" Conjugates: Synthesis, Nucleobase Controlled Fluorescence
Quenching, and Duplex Stability
24
4.1
Background
24
4.2
Synthesis of Nucleoside Pyrene Click Conjugates with Octadiynyl and
Tripropargylamine Side Chains
25
4.3
Synthesis and Duplex Stability of Oligonucleotide Pyrene Click Conjugates
26
4.4
Fluorescence Properties of Nucleoside Pyrene Click Conjugates
29
4.5
Fluorescence Study of 7-Deazapurine and 8-Aza-7-deazapurine Oligonucleotide
Pyrene Click Conjugates
5
31
Nucleoside and Oligonucleotide Pyrene Conjugates with 1,2,3-Triazolyl or
Ethynyl Linkers: Synthesis, Duplex Stability and Fluorescence Changes
Generated by the DNA-Dye Connector
34
5.1
Background
34
5.2
Synthesis of Nucleoside Pyrene Conjugates and Phosphoramidites
35
5.2.1
Synthesis of Pyrene Conjugates Containing a 1,2,3-Triazolyl Moiety
35
5.2.2
Synthesis of Pyrene Conjugate with an Ethynyl Linker
37
5.3
Synthesis and Duplex Stability of Oligonucleotide Pyrene Conjugates
39
5.4
Influence of Linkers on the Fluorescence Properties of Nucleoside and
Oligonucleotide Pyrene Conjugates
5.4.1
Monomeric 7-Deazaguanine Pyrene Conjugates
5.4.2
Oligonucleotide Pyrene Conjugates
40
:41
42
6
Stepwise Click Functionalization of DNA Through a Bifunctional Azide
with a Chelating and a Nonchelating Azido Group
47
6.1
Background
47
6.2
Stepwise Chemoselective Click Reaction on Alkynyl Modified Nucleosides
48
6.3
Stepwise Chemoselective Click Reaction on Alkynyl Modified Oligonucleotides....49
6.4
Duplex Stability of Monofunctionalized and Bifunctionalized Oligonucleotides
7
A Ratiometric Fluorescent On-OfT Zn2+ Chemosensor Based on a
SI
Tripropargylamine Pyrene Azide Click Adduct
53
7.1
Background
53
7.2
Synthesis of Tripropargylamine and Dipropargylamine Pyrene Click Adducts
53
7.3
Cation-Binding Properties of the Tripropargylamine Pyrene Click Adducts
54
8
Ethynyl Side Chain Hydration During Synthesis and Work-up of "Clickable"
Oligonucleotides: Bypassing Acetyl Group Formation by Triisopropylsilyl
Protection
59
8.1
Background
59
8.2
Synthesis of the 7-Deazaguanine Phosphoramidite with an Ethynyl Side Chain
60
8.3
Synthesis and Characterization of Ethynyl Modified Oligonucleotides
61
8.4
Acid-Catalyzed Hydration of Ethynyl Modified Nucleosides
63
8.5
Stability of Ethynyl Modified Nucleosides in Concentrated Aqueous Ammonia
64
8.6
Comparison of Side Products Obtained from the Enzymatic Hydrolysis of
Oligonucleotides with Those Obtained by Synthesis
66
8.7
Synthesis of Triisopropylsilyl Protected Phophoramidites
67
8.8
Synthesis of TIPS Modified Oligonucleotides
68
8.9
Synthesis of Ethynyl Oligonucleotides by Deprotecting Triisopropylsilyl Group
from TIPS Modified Oligonucletotides
69
9
Results
71
10
Nomenclature and Abbreviations
84
11
Experimental Part (Unpublished)
87
12
References
90
13
List of Publications
99
Acknowledgements... .•
101
Curriculum Vitae
102
Statement
103
Attached Manuscripts 13.1-13.11