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Synthesis of Therapeutic Oligonucleotides
von: Satoshi Obika, Mitsuo Sekine
Springer-Verlag, 2018
ISBN: 9789811319129 , 280 Seiten
Format: PDF, Online Lesen
Kopierschutz: Wasserzeichen
Preis: 171,19 EUR
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Preface
5
Contents
6
Part I: Synthesis of Natural Oligonucleotides
8
Non-protected Synthesis of Oligonucleotides
9
1 Introduction
9
2 Development of Proton-Block Strategy for the Synthesis of Oligonucleotides Without Base Protection
10
3 Development of the Activated Phosphite Method Using N-Unprotected Phosphoramidites
13
4 Mechanism of the Activated Phosphite Method
17
5 Synthesis of RNA Oligomers Using the Activated Phosphite Method
18
6 Synthesis of Phosphoramidite Monomer Building Blocks
20
7 Conclusion
20
References
20
Various Coupling Agents in the Phosphoramidite Method for Oligonucleotide Synthesis
23
1 Introduction
23
2 Coupling Agents in the Phosphoramidite Method
26
2.1 1H-Tetrazole and Its Derivatives
26
2.1.1 1H-Tetrazole
26
2.1.2 5-Ethylthio-1H-tetrazole
28
2.1.3 5-Benzylthio-1H-tetrazole
29
2.1.4 5-[3,5-Bis(trifluoromethyl)phenyl]-1H-tetrazole (Activator 42)
32
2.2 4,5-Dicyanoimidazole
33
2.3 Carboxylic Acids
33
2.4 Acid/Azole Complexes
35
3 Conclusion
40
References
41
Recent Development of Chemical Synthesis of RNA
46
1 Introduction
46
2 Basic Principle of Solid-Phase Synthesis of DNA/RNA in Phosphoramidite Approach
47
3 Current RNA Synthesis Using TBDMS as 2?-Hydroxyl Protecting Group
48
4 RNA Synthesis Using Acid-Labile 2?-Hydroxyl Protecting Groups
49
5 RNA Synthesis Using 2?-Protecting Groups Having an Acetal Skeleton
51
5.1 (2-Nitrobenzyl)oxymethyl (NBOM) Group
51
5.2 2-(Trimethylsilyl)ethoxymethyl (SEM) Group
52
6 RNA Synthesis Using the Triisopropylsilyloxymethyl (Tom) Group
53
7 Cyanoethoxy-1-Methylethyl (CEE) and Cyanoethoxymethyl (CEM) Groups
54
8 RNA Synthesis Using 4-Methylphenylsulfonylethoxymethyl (TEM) Group
58
9 RNA Synthesis Using tert-Butyldithiomethyl (DTM) Group
58
10 RNA Synthesis Using [[2-(Methylthio)phenyl]thio]methyl (MPTM) Group
59
11 RNA Synthesis Using (N-Dichloroacetyl-N-methyl)aminobenzyloxylmethyl (DCMABOM) Group
60
12 RNA Synthesis Using the Acetal Levulinyl Ester (ALE) Group
62
13 RNA Synthesis Using the Cyanoethyl (CE) Group
63
14 RNA Synthesis Without Using Base Protection
65
15 Recent Studies on RNA Chemical Synthesis
65
16 Summary and Perspectives
66
References
67
RNA Synthesis Using the CEM Group
71
1 Introduction
71
2 Synthesis of CEM Amidites
72
3 Synthesis of RNA
73
4 Experimental Section
73
4.1 Preparation of CEM-SMe
73
4.2 Synthesis of U-CEM Phosphoramidite (5a.)
73
4.2.1 3?,5?-O-(Tetraisopropyldisiloxane-1,3-diyl)-2?-O-(2-cyanoethoxymethyl)uridine (2a.)
73
4.2.2 2?-O-(2-Cyanoethoxymethyl)uridine (3a.)
74
4.2.3 5?-O-(4,4?-Dimethoxytrityl)-2?-O-(2-xyanoethoxymethyl)uridine (4a.)
75
4.2.4 5?-O-(4,4?-Dimethoxytrityl)-2?-O-(2-cyanoethoxymethyl)uridine 3?-O-(2-Cyanoethyl N, N-diisopropylphosphoramidite) (5a.)
75
4.3 Synthesis of C-CEM Phosphoramidite (5b.)
76
4.3.1 4-N-Acetyl-3?, 5?-O-(tetraisopropyldisiloxane-1,3-diyl)-2?-O-(2-cyanoethoxymethyl)cytidine (2b.)
76
4.3.2 4-N-Acetyl-2?-O-(2-cyanoethoxymethyl)cytidine (3b.)
76
4.3.3 4-N-Acetyl-5?-O- (4,4?-dimethoxytrityl) -2? -O- (2-cyanoethoxymethyl)cytidine (4b.)
77
4.3.4 4-N-Acetyl-5?-O-(4,4?-dimethoxytrityl)-2?-O-(2-cyanoethoxymethyl)cytidine 3?-O-(2-cyanoethyl N, N-diisopropylphosphoramidite) (5b.)
77
4.4 Synthesis of A-CEM Phosphoramidite (5c.)
78
4.4.1 6-N-Acetyl-3?,5?-O-(tetraisopropyldisiloxane-1,3-diyl)-2?-O-(2-cyanoethoxymethyl)adenosine (2c.)
78
4.4.2 6-N-Acetyl-2?-O-(2-cyanoethoxymethyl)adenosine (3c.)
78
4.4.3 6-N-Acetyl-5?-O-(4,4?-dimethoxytrityl)-2?-O- (2-cyanoethoxymethyl)adenosine (4c.)
79
4.4.4 6-N-Acetyl-5?-O-(4,4?-dimethoxytrityl)-2?-O-(2-cyanoethoxymethyl)adenosine 3?-O-(2-Cyanoethyl N, N-diisopropylphosphoramidite) (5c.)
79
4.5 Synthesis of CEM-G Phosphoramidite (5d.)
80
4.5.1 2N-Phenoxyacetyl-3?,5?-O-(tetraisopropyldisiloxane-1,3-diyl)-2?-O-(2-cyanoethoxymethyl)guanosine (2d.)
80
4.5.2 2N-Phenoxyacetyl-2?-O-(2-cyanoethoxymethyl)guanosine (3d.)
80
4.5.3 2N-Phenoxyacetyl -5?-O- (4,4?-dimethoxytrityl) -2? -O- (2-cyanoethoxymethyl)guanosine (4d.)
81
4.5.4 2N-Phenoxyacetyl-5?-O-(4,4?-dimethoxytrityl)-2?-O-(2-cyanoethoxymethyl)guanosine 3?-O-(2-Cyanoethyl N, N-diisopropylphosphoramidite) (5d.)
81
4.6 Synthesis of CEM-I Phosphoramidite (5e.)
82
4.6.1 3?,5?-O-(Tetraisopropyldisiloxane-1,3-Diyl)-2?-O-(2-cyanoethoxymethyl)inosine (2e.)
82
4.6.2 2?-O-(2-Cyanoethoxymethyl)inosine (3e.)
82
4.6.3 5?-O- (4,4?-Dimethoxytrityl) -2? -O-(2-cyanoethoxymethyl)inosine (4e.)
82
4.6.4 5?-O-(4,4?-Dimethoxytrityl)-2?-O-(2-cyanoethoxymethyl)inosine 3?-O-(2-Cyanoethyl N, N-diisopropylphosphoramidite) (5e.)
83
4.7 Synthesis of Oligoribonucleotide
83
4.8 Cleavage and Deprotection
84
References
85
Liquid-Phase Synthesis of Oligonucleotides
86
1 Introduction
86
2 PEG-Based Liquid-Phase Synthesis
87
3 Non-polymeric Anchor-Assisted Synthesis
89
3.1 Ionic Liquid Tag-Assisted Synthesis
89
3.2 Fluorous Tag-Assisted Synthesis
90
3.3 Tetravalent Cluster Approach
90
3.4 Adamntylmethylester Synthesis
91
3.5 Alkyl Chain-Assisted Synthesis
91
4 Other Approaches
91
4.1 Product Anchored Sequential Synthesis (PASS) Method
91
4.2 Solution-Phase Synthesis Using Polymer-Supported Reagents
92
5 AJIPHASE® for Oligonucleotide Synthesis
93
6 Conclusion
96
References
96
Large-Scale Oligonucleotide Manufacturing
99
1 Manufacturing Process for Therapeutic Oligonucleotides
99
1.1 Oligonucleotide Synthesis
100
1.1.1 Solid Support
100
1.1.2 Synthesizer
102
1.2 Oligonucleotide Cleavage and Deprotection
103
1.3 Oligonucleotide Chromatography
104
1.4 Oligonucleotide Ultrafiltration and Diafiltration
106
1.5 Oligonucleotide Lyophilization
107
2 Small-Scale Modeling for Oligonucleotide Manufacturing
108
2.1 Case Study 1 (Synthesis)
109
2.2 Case Study 2 (C&D)
110
2.3 Case Study 3 (Purification)
112
References
113
Part II: Synthesis and Properties of Artificial Oligonucleotides
115
Nucleosides and Oligonucleotides Incorporating 2-Thiothymine or 2-Thiouracil Derivatives as Modified Nucleobases
116
1 Purposes of the Thio Modification of Uracil and Thymine
117
2 Physicochemical Similarities and Differences Between Oxygen and Sulfur
118
2.1 Atom Sizes
118
2.2 Electronic Properties
119
3 Physicochemical Properties of 2-Thiouracil and 2-Thiothymine
119
3.1 Intrinsic Hydrogen Bonding Ability of s2Ura and s2Thy
119
3.2 Stacking Interactions of s2Ura
121
3.3 ‘Rigid’ Sugar Conformation of 2-Thiouridine (s2U) and 2-Thiothymidine (s2T) Derivatives
122
3.4 Conformational Properties of Single Stranded Oligonucleotides Incorporating 2-Thiouridine
122
3.5 Hybridization Ability of Oligonucleotides Incorporating 2-Thiouridine Derivatives
123
3.6 Base Discrimination of 2-Thiouridine Derivatives in a Duplex
124
3.7 Application of 2-Thiouridine Derivatives as Nucleic Acid Drugs
125
4 Chemical Synthesis of 2-Thiouridine Derivatives and Their Incorporation into Oligonucleotides
125
4.1 Synthesis of 2-Thiouridine and 2-Thiothymidine by Glycosylation
125
4.2 Synthesis of 2-Thiouridine Derivatives Form Uridine Derivatives
126
4.3 Synthesis of the Phosphoramidites of 2-Thiothymidine and 2-Thiouridine Derivatives and Their Use in Oligonucleotide Synthesis
128
References
130
Site-Specific Modification of Nucleobases in Oligonucleotides
132
1 Introduction
132
2 Modifications at the 2- or 4-Positions of Pyrimidine Nucleobases
134
3 Modifications at the 2- or 6-Positions of Purine Nucleobases
136
4 Other Modifications in Nucleobase Units
137
5 Experimental Example: N,N-Disubstituted Cytosine Nucleobases
139
6 Summary
142
References
142
Four-Hydrogen-Bonding Base Pairs in Oligonucleotides: Design, Synthesis, and Properties
147
1 Introduction
148
2 Designing Four-H-Bonding DNA Base Pairs
150
2.1 Size-Expanded Im:Im Base Pairs
150
2.2 Design of Im:Na Base Pairs with Comparable Shape Complementarity to That of WC Base Pairs
153
3 Creation of a Thermally Stabilized Decoy Molecule with Im:Na Pairs
155
4 Polymerase Reactions Involving the Im:Na Pair
157
4.1 Enzymatic Replication of Im:Na Pairs by DNA Polymerases
157
4.2 Transcription System with an Alternative Genetic Set Im:Na Pair
163
5 Conclusion and Perspective
165
References
166
Photo-Cross-Linkable Artificial Nucleic Acid: Synthesis and Properties of 3-Cyanovinylcarbazole-Modified Nucleic Acids and Its Photo-Induced Gene-Silencing Activity in Cells
170
1 Introduction
170
2 Synthesis of 3-Cyanovinylcarbazole-Based Photo-Cross-Linker
172
2.1 Synthetic Procedures of CNVK and Its Phosphoramidite Monomer
174
2.1.1 3-Iodocarbazole (2)
174
2.1.2 3-Cyanovinylcarbazole (3)
174
2.1.3 3-Cyanovinylcarbazole-9-yl-1?-?-deoxyriboside-3?,5?-di-(P-toluoyl)ester (4)
174
2.1.4 3-Cyanovinylcarbazole-9-yl-1?-?-deoxyriboside (5)
175
2.1.5 5?-O-(4,4?-Dimethoxytrityl)-3-cyanovinylcarbazole-9-yl-1?-? -deoxyriboside (6)
175
2.1.6 5?-O-(4,4?-Dimethoxytrityl)-3-cyanovinylcarbazole-9-yl-1?-? -deoxyriboside-3?-O-(cyanoethoxy-N,N-diisopropylamino)phosphoramidite (7)
175
2.2 Synthetic Procedures of CNVD and Its Phosphoramidite Monomer
175
2.2.1 Ethyl 3-cyanovinylcarbazol-9-yl-acetate (8)
175
2.2.2 3-Cyanovinylcarbazol-9-yl-acetic Acid (9)
176
2.2.3 N-(3-Cyanovinylcarbazol-9-yl-acetyl)-D-threoninol (10, CNVD)
176
2.2.4 N-(3-Cyanovinylcarbazol-9-yl-acetyl)-1?-O-(4,4?-dimethoxytrityl)-D-threoninol (11)
176
2.2.5 N-(3-Cyanovinylcarbazol-9-yl-acetyl)-1?-O-(4,4?-dimethoxytrityl)-D-threoninol 3?-O-(Cyanoethoxy-N,N-diisopropylamino)phosphoramidite (12)
176
2.3 Synthesis of the Oligonucleotide Having CNVK or CNVD
177
2.4 Further Modification of CNVK- or CNVD-Modified ODNs
177
3 Inter-Strand Photo-Cross-Linking Using ODNs Having CNVK or CNVD
177
3.1 Properties of the Inter-Strand Photo-Cross-Linking Reaction of CNVK and CNVD in Nucleic Acid Double Strands
178
3.2 Light Source
179
3.3 Structural Insight of the DNA Duplex Including CNVK
179
4 Gene-Silencing Using CNVK Modified Antisense ODNs
180
4.1 Design of the Photoreactive Antisense ODNs Having CNVK
181
4.2 Evaluation of the Photo-Cross-Linking Reaction with mRNA
181
4.3 Photo-Induced Gene Silencing in Cells
182
5 Summary
183
References
183
Effects of 2?-O-Modifications on RNA Duplex Stability
186
1 Introduction
186
2 Effects of Modifications on RNA Duplex Stability
187
2.1 Preorganization of RNA
187
2.2 Hydration Effect
188
2.3 Electrostatic Effect
189
2.4 Effect of Substituent Size
189
3 Computational Approach to Design Novel Modifications
190
4 Discussion
194
5 Conclusion
195
References
195
2?,4?-Bridged Nucleic Acids Containing Plural Heteroatoms in the Bridge Moiety
199
1 Introduction
199
2 Five-Membered Bridged Nucleic Acids
200
3 Six-Membered Bridged Nucleic Acids
207
4 Seven-Membered Bridged Nucleic Acids
212
5 Summary
215
References
215
Synthesis and Therapeutic Applications of Oligonucleotides Containing 2?-O,4?-C-Ethylene- and 3?-O,4?-C-Propylene-Bridged Nucleotides
220
1 Introduction
221
1.1 Structural Properties of 2'-O,4'-C-Ethylene-Bridged Nucleic Acids (ENA) and 2'-O,4'-C-Propylene-Bridged Nucleic Acids (PrNA) Residues
221
1.2 Properties of Oligonucleotides Containing ENA and PrNA Residues
222
1.3 Therapeutic Applications of Oligonucleotides Containing ENA Residues
224
1.4 The Development of Novel 2-5A Analogs Containing 3'-O,4'-C-Alkylene-Bridged Nucleosides as a Therapeutic Reagent
224
1.5 Synthesis of 3'-O,4'-C-Propylene Adenosine as the Potent Modified Unit for 2-5A Analog
225
1.6 Synthesis of 2-5A Analog 1 Using DNA/RNA Autosynthesizer
226
1.7 In Vitro Activity of 2-5A Analog 1 in Cancer Cells
227
References
228
RNA Bioisosteres: Chemistry and Properties of 4?-thioRNA and 4?-selenoRNA
230
1 Introduction
231
2 Chemistry and Properties of 4?-thioRNA
232
2.1 Stereoselective Synthesis of 4'-thioribonucleosides
232
2.2 Synthesis and Properties of 4?-thioRNA
233
3 Biological Applications of 4?-thioRNA
236
3.1 Application of 4'-thioRNA for Chemically Modified siRNA
237
3.2 Application to Isolation of 4'-thioRNA Aptamers
240
4 Chemistry for the Synthesis of 4?-selenoRNA
242
4.1 Practical Synthesis of 4'-selenoribonucleosides
242
4.2 Synthetic Study of 4'-selenoRNA
244
5 Conclusion and Perspective
246
References
247
Development of Triplex Forming Oligonucleotide Including Artificial Nucleoside Analogues for the Antigene Strategy
250
1 Introduction
250
2 Design of W-shaped Nucleoside Analogues (WNAs) for TA Inversion Site
252
2.1 Synthesis of Oligonucleotides Including WNAs (WNA-?T)
253
2.2 Evaluation of Triplex Formation
254
2.3 Antiploriferative Effect and Inhibition of Gene Expression Product for A549 Cells
255
3 Design of Pseudo-dC Derivatives (MeAP-?dC) for CG Inversion Site
257
3.1 Synthesis of Oligonucleotides Including ?dC Derivatives (MeAP-?dC)
258
3.2 Evaluation of Triplex Formation
259
3.3 Speculation of the Recognition Model of MeAP-?dC/CG Triplet
260
3.4 Inhibition of Transcription of the hTERT Gene in HeLa Cells
261
4 Conclusion and Perspectives
264
References
264
Chemical Synthesis of Boranophosphate Deoxy-ribonucleotides
267
1 Introduction
267
2 Synthesis of Boranophosphate DNA by the Phosphoramidite Approach
268
3 Synthesis of Boranophosphate DNA by the H-phosphonate Approach
271
4 Synthesis of Boranophosphate DNA by the Boranophospho-Triester Approach
273
5 Synthesis of Boranophosphate DNA by the H-boranophospho-nate Approach
274
6 Stereocontrolled Synthesis of Boranophosphate DNA by the Oxazaphospholidine Approach
275
7 Summary and Perspectives
278
References
278