Synthesis of Therapeutic Oligonucleotides (eBook)

Satoshi Obika, Osaka University, JapanMitsuo Sekine, Tokyo Institute of Technology, Japan

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)
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