Synthesis of Therapeutic Oligonucleotides

Preface

Contents

Part I: Synthesis of Natural Oligonucleotides

Non-protected Synthesis of Oligonucleotides

1 Introduction

2 Development of Proton-Block Strategy for the Synthesis of Oligonucleotides Without Base Protection

3 Development of the Activated Phosphite Method Using N-Unprotected Phosphoramidites

4 Mechanism of the Activated Phosphite Method

5 Synthesis of RNA Oligomers Using the Activated Phosphite Method

6 Synthesis of Phosphoramidite Monomer Building Blocks

7 Conclusion

References

Various Coupling Agents in the Phosphoramidite Method for Oligonucleotide Synthesis

1 Introduction

2 Coupling Agents in the Phosphoramidite Method

2.1 1H-Tetrazole and Its Derivatives

2.1.1 1H-Tetrazole

2.1.2 5-Ethylthio-1H-tetrazole

2.1.3 5-Benzylthio-1H-tetrazole

2.1.4 5-[3,5-Bis(trifluoromethyl)phenyl]-1H-tetrazole (Activator 42)

2.2 4,5-Dicyanoimidazole

2.3 Carboxylic Acids

2.4 Acid/Azole Complexes

3 Conclusion

References

Recent Development of Chemical Synthesis of RNA

1 Introduction

2 Basic Principle of Solid-Phase Synthesis of DNA/RNA in Phosphoramidite Approach

3 Current RNA Synthesis Using TBDMS as 2?-Hydroxyl Protecting Group

4 RNA Synthesis Using Acid-Labile 2?-Hydroxyl Protecting Groups

5 RNA Synthesis Using 2?-Protecting Groups Having an Acetal Skeleton

5.1 (2-Nitrobenzyl)oxymethyl (NBOM) Group

5.2 2-(Trimethylsilyl)ethoxymethyl (SEM) Group

6 RNA Synthesis Using the Triisopropylsilyloxymethyl (Tom) Group

7 Cyanoethoxy-1-Methylethyl (CEE) and Cyanoethoxymethyl (CEM) Groups

8 RNA Synthesis Using 4-Methylphenylsulfonylethoxymethyl (TEM) Group

9 RNA Synthesis Using tert-Butyldithiomethyl (DTM) Group

10 RNA Synthesis Using [[2-(Methylthio)phenyl]thio]methyl (MPTM) Group

11 RNA Synthesis Using (N-Dichloroacetyl-N-methyl)aminobenzyloxylmethyl (DCMABOM) Group

12 RNA Synthesis Using the Acetal Levulinyl Ester (ALE) Group

13 RNA Synthesis Using the Cyanoethyl (CE) Group

14 RNA Synthesis Without Using Base Protection

15 Recent Studies on RNA Chemical Synthesis

16 Summary and Perspectives

References

RNA Synthesis Using the CEM Group

1 Introduction

2 Synthesis of CEM Amidites

3 Synthesis of RNA

4 Experimental Section

4.1 Preparation of CEM-SMe

4.2 Synthesis of U-CEM Phosphoramidite (5a.)

4.2.1 3?,5?-O-(Tetraisopropyldisiloxane-1,3-diyl)-2?-O-(2-cyanoethoxymethyl)uridine (2a.)

4.2.2 2?-O-(2-Cyanoethoxymethyl)uridine (3a.)

4.2.3 5?-O-(4,4?-Dimethoxytrityl)-2?-O-(2-xyanoethoxymethyl)uridine (4a.)

4.2.4 5?-O-(4,4?-Dimethoxytrityl)-2?-O-(2-cyanoethoxymethyl)uridine 3?-O-(2-Cyanoethyl N, N-diisopropylphosphoramidite) (5a.)

4.3 Synthesis of C-CEM Phosphoramidite (5b.)

4.3.1 4-N-Acetyl-3?, 5?-O-(tetraisopropyldisiloxane-1,3-diyl)-2?-O-(2-cyanoethoxymethyl)cytidine (2b.)

4.3.2 4-N-Acetyl-2?-O-(2-cyanoethoxymethyl)cytidine (3b.)

4.3.3 4-N-Acetyl-5?-O- (4,4?-dimethoxytrityl) -2? -O- (2-cyanoethoxymethyl)cytidine (4b.)

4.3.4 4-N-Acetyl-5?-O-(4,4?-dimethoxytrityl)-2?-O-(2-cyanoethoxymethyl)cytidine 3?-O-(2-cyanoethyl N, N-diisopropylphosphoramidite) (5b.)

4.4 Synthesis of A-CEM Phosphoramidite (5c.)

4.4.1 6-N-Acetyl-3?,5?-O-(tetraisopropyldisiloxane-1,3-diyl)-2?-O-(2-cyanoethoxymethyl)adenosine (2c.)

4.4.2 6-N-Acetyl-2?-O-(2-cyanoethoxymethyl)adenosine (3c.)

4.4.3 6-N-Acetyl-5?-O-(4,4?-dimethoxytrityl)-2?-O- (2-cyanoethoxymethyl)adenosine (4c.)

4.4.4 6-N-Acetyl-5?-O-(4,4?-dimethoxytrityl)-2?-O-(2-cyanoethoxymethyl)adenosine 3?-O-(2-Cyanoethyl N, N-diisopropylphosphoramidite) (5c.)

4.5 Synthesis of CEM-G Phosphoramidite (5d.)

4.5.1 2N-Phenoxyacetyl-3?,5?-O-(tetraisopropyldisiloxane-1,3-diyl)-2?-O-(2-cyanoethoxymethyl)guanosine (2d.)

4.5.2 2N-Phenoxyacetyl-2?-O-(2-cyanoethoxymethyl)guanosine (3d.)

4.5.3 2N-Phenoxyacetyl -5?-O- (4,4?-dimethoxytrityl) -2? -O- (2-cyanoethoxymethyl)guanosine (4d.)

4.5.4 2N-Phenoxyacetyl-5?-O-(4,4?-dimethoxytrityl)-2?-O-(2-cyanoethoxymethyl)guanosine 3?-O-(2-Cyanoethyl N, N-diisopropylphosphoramidite) (5d.)

4.6 Synthesis of CEM-I Phosphoramidite (5e.)

4.6.1 3?,5?-O-(Tetraisopropyldisiloxane-1,3-Diyl)-2?-O-(2-cyanoethoxymethyl)inosine (2e.)

4.6.2 2?-O-(2-Cyanoethoxymethyl)inosine (3e.)

4.6.3 5?-O- (4,4?-Dimethoxytrityl) -2? -O-(2-cyanoethoxymethyl)inosine (4e.)

4.6.4 5?-O-(4,4?-Dimethoxytrityl)-2?-O-(2-cyanoethoxymethyl)inosine 3?-O-(2-Cyanoethyl N, N-diisopropylphosphoramidite) (5e.)

4.7 Synthesis of Oligoribonucleotide

4.8 Cleavage and Deprotection

References

Liquid-Phase Synthesis of Oligonucleotides

1 Introduction

2 PEG-Based Liquid-Phase Synthesis

3 Non-polymeric Anchor-Assisted Synthesis

3.1 Ionic Liquid Tag-Assisted Synthesis

3.2 Fluorous Tag-Assisted Synthesis

3.3 Tetravalent Cluster Approach

3.4 Adamntylmethylester Synthesis

3.5 Alkyl Chain-Assisted Synthesis

4 Other Approaches

4.1 Product Anchored Sequential Synthesis (PASS) Method

4.2 Solution-Phase Synthesis Using Polymer-Supported Reagents

5 AJIPHASE® for Oligonucleotide Synthesis

6 Conclusion

References

Large-Scale Oligonucleotide Manufacturing

1 Manufacturing Process for Therapeutic Oligonucleotides

1.1 Oligonucleotide Synthesis

1.1.1 Solid Support

1.1.2 Synthesizer

1.2 Oligonucleotide Cleavage and Deprotection

1.3 Oligonucleotide Chromatography

1.4 Oligonucleotide Ultrafiltration and Diafiltration

1.5 Oligonucleotide Lyophilization

2 Small-Scale Modeling for Oligonucleotide Manufacturing

2.1 Case Study 1 (Synthesis)

2.2 Case Study 2 (C&D)

2.3 Case Study 3 (Purification)

References

Part II: Synthesis and Properties of Artificial Oligonucleotides

Nucleosides and Oligonucleotides Incorporating 2-Thiothymine or 2-Thiouracil Derivatives as Modified Nucleobases

1 Purposes of the Thio Modification of Uracil and Thymine

2 Physicochemical Similarities and Differences Between Oxygen and Sulfur

2.1 Atom Sizes

2.2 Electronic Properties

3 Physicochemical Properties of 2-Thiouracil and 2-Thiothymine

3.1 Intrinsic Hydrogen Bonding Ability of s2Ura and s2Thy

3.2 Stacking Interactions of s2Ura

3.3 ‘Rigid’ Sugar Conformation of 2-Thiouridine (s2U) and 2-Thiothymidine (s2T) Derivatives

3.4 Conformational Properties of Single Stranded Oligonucleotides Incorporating 2-Thiouridine

3.5 Hybridization Ability of Oligonucleotides Incorporating 2-Thiouridine Derivatives

3.6 Base Discrimination of 2-Thiouridine Derivatives in a Duplex

3.7 Application of 2-Thiouridine Derivatives as Nucleic Acid Drugs

4 Chemical Synthesis of 2-Thiouridine Derivatives and Their Incorporation into Oligonucleotides

4.1 Synthesis of 2-Thiouridine and 2-Thiothymidine by Glycosylation

4.2 Synthesis of 2-Thiouridine Derivatives Form Uridine Derivatives

4.3 Synthesis of the Phosphoramidites of 2-Thiothymidine and 2-Thiouridine Derivatives and Their Use in Oligonucleotide Synthesis

References

Site-Specific Modification of Nucleobases in Oligonucleotides

1 Introduction

2 Modifications at the 2- or 4-Positions of Pyrimidine Nucleobases

3 Modifications at the 2- or 6-Positions of Purine Nucleobases

4 Other Modifications in Nucleobase Units

5 Experimental Example: N,N-Disubstituted Cytosine Nucleobases

6 Summary

References

Four-Hydrogen-Bonding Base Pairs in Oligonucleotides: Design, Synthesis, and Properties

1 Introduction

2 Designing Four-H-Bonding DNA Base Pairs

2.1 Size-Expanded Im:Im Base Pairs

2.2 Design of Im:Na Base Pairs with Comparable Shape Complementarity to That of WC Base Pairs

3 Creation of a Thermally Stabilized Decoy Molecule with Im:Na Pairs

4 Polymerase Reactions Involving the Im:Na Pair

4.1 Enzymatic Replication of Im:Na Pairs by DNA Polymerases

4.2 Transcription System with an Alternative Genetic Set Im:Na Pair

5 Conclusion and Perspective

References

Photo-Cross-Linkable Artificial Nucleic Acid: Synthesis and Properties of 3-Cyanovinylcarbazole-Modified Nucleic Acids and Its Photo-Induced Gene-Silencing Activity in Cells

1 Introduction

2 Synthesis of 3-Cyanovinylcarbazole-Based Photo-Cross-Linker

2.1 Synthetic Procedures of CNVK and Its Phosphoramidite Monomer

2.1.1 3-Iodocarbazole (2)

2.1.2 3-Cyanovinylcarbazole (3)

2.1.3 3-Cyanovinylcarbazole-9-yl-1?-?-deoxyriboside-3?,5?-di-(P-toluoyl)ester (4)

2.1.4 3-Cyanovinylcarbazole-9-yl-1?-?-deoxyriboside (5)

2.1.5 5?-O-(4,4?-Dimethoxytrityl)-3-cyanovinylcarbazole-9-yl-1?-? -deoxyriboside (6)

2.1.6 5?-O-(4,4?-Dimethoxytrityl)-3-cyanovinylcarbazole-9-yl-1?-? -deoxyriboside-3?-O-(cyanoethoxy-N,N-diisopropylamino)phosphoramidite (7)

2.2 Synthetic Procedures of CNVD and Its Phosphoramidite Monomer

2.2.1 Ethyl 3-cyanovinylcarbazol-9-yl-acetate (8)

2.2.2 3-Cyanovinylcarbazol-9-yl-acetic Acid (9)

2.2.3 N-(3-Cyanovinylcarbazol-9-yl-acetyl)-D-threoninol (10, CNVD)

2.2.4 N-(3-Cyanovinylcarbazol-9-yl-acetyl)-1?-O-(4,4?-dimethoxytrityl)-D-threoninol (11)

2.2.5 N-(3-Cyanovinylcarbazol-9-yl-acetyl)-1?-O-(4,4?-dimethoxytrityl)-D-threoninol 3?-O-(Cyanoethoxy-N,N-diisopropylamino)phosphoramidite (12)

2.3 Synthesis of the Oligonucleotide Having CNVK or CNVD

2.4 Further Modification of CNVK- or CNVD-Modified ODNs

3 Inter-Strand Photo-Cross-Linking Using ODNs Having CNVK or CNVD

3.1 Properties of the Inter-Strand Photo-Cross-Linking Reaction of CNVK and CNVD in Nucleic Acid Double Strands

3.2 Light Source

3.3 Structural Insight of the DNA Duplex Including CNVK

4 Gene-Silencing Using CNVK Modified Antisense ODNs

4.1 Design of the Photoreactive Antisense ODNs Having CNVK

4.2 Evaluation of the Photo-Cross-Linking Reaction with mRNA

4.3 Photo-Induced Gene Silencing in Cells

5 Summary

References

Effects of 2?-O-Modifications on RNA Duplex Stability

1 Introduction

2 Effects of Modifications on RNA Duplex Stability

2.1 Preorganization of RNA

2.2 Hydration Effect

2.3 Electrostatic Effect

2.4 Effect of Substituent Size

3 Computational Approach to Design Novel Modifications

4 Discussion

5 Conclusion

References

2?,4?-Bridged Nucleic Acids Containing Plural Heteroatoms in the Bridge Moiety

1 Introduction

2 Five-Membered Bridged Nucleic Acids

3 Six-Membered Bridged Nucleic Acids

4 Seven-Membered Bridged Nucleic Acids

5 Summary

References

Synthesis and Therapeutic Applications of Oligonucleotides Containing 2?-O,4?-C-Ethylene- and 3?-O,4?-C-Propylene-Bridged Nucleotides

1 Introduction

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

1.2 Properties of Oligonucleotides Containing ENA and PrNA Residues

1.3 Therapeutic Applications of Oligonucleotides Containing ENA Residues

1.4 The Development of Novel 2-5A Analogs Containing 3'-O,4'-C-Alkylene-Bridged Nucleosides as a Therapeutic Reagent

1.5 Synthesis of 3'-O,4'-C-Propylene Adenosine as the Potent Modified Unit for 2-5A Analog

1.6 Synthesis of 2-5A Analog 1 Using DNA/RNA Autosynthesizer

1.7 In Vitro Activity of 2-5A Analog 1 in Cancer Cells

References

RNA Bioisosteres: Chemistry and Properties of 4?-thioRNA and 4?-selenoRNA

1 Introduction

2 Chemistry and Properties of 4?-thioRNA

2.1 Stereoselective Synthesis of 4'-thioribonucleosides

2.2 Synthesis and Properties of 4?-thioRNA

3 Biological Applications of 4?-thioRNA

3.1 Application of 4'-thioRNA for Chemically Modified siRNA

3.2 Application to Isolation of 4'-thioRNA Aptamers

4 Chemistry for the Synthesis of 4?-selenoRNA

4.1 Practical Synthesis of 4'-selenoribonucleosides

4.2 Synthetic Study of 4'-selenoRNA

5 Conclusion and Perspective

References

Development of Triplex Forming Oligonucleotide Including Artificial Nucleoside Analogues for the Antigene Strategy

1 Introduction

2 Design of W-shaped Nucleoside Analogues (WNAs) for TA Inversion Site

2.1 Synthesis of Oligonucleotides Including WNAs (WNA-?T)

2.2 Evaluation of Triplex Formation

2.3 Antiploriferative Effect and Inhibition of Gene Expression Product for A549 Cells

3 Design of Pseudo-dC Derivatives (MeAP-?dC) for CG Inversion Site

3.1 Synthesis of Oligonucleotides Including ?dC Derivatives (MeAP-?dC)

3.2 Evaluation of Triplex Formation

3.3 Speculation of the Recognition Model of MeAP-?dC/CG Triplet

3.4 Inhibition of Transcription of the hTERT Gene in HeLa Cells

4 Conclusion and Perspectives

References

Chemical Synthesis of Boranophosphate Deoxy-ribonucleotides

1 Introduction

2 Synthesis of Boranophosphate DNA by the Phosphoramidite Approach

3 Synthesis of Boranophosphate DNA by the H-phosphonate Approach

4 Synthesis of Boranophosphate DNA by the Boranophospho-Triester Approach

5 Synthesis of Boranophosphate DNA by the H-boranophospho-nate Approach

6 Stereocontrolled Synthesis of Boranophosphate DNA by the Oxazaphospholidine Approach

7 Summary and Perspectives

References