To My Family List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Upadhayaya, R.*; Deshpande, S. G.*; Li, Q.*; Kardile, R. A.; Sayyed, A. Y.; Kshirsagar, E. K.; Salunke, R. V.; Dixit, S. S.; Zhou, C.; Földesi, A.; Chattopadhyaya, J. Carba-LNA-5MeC/A/G/T Modified Oligos Show Nucleobase- Specific Modulation of 3′-Exonuclease Activity, Thermody- namic Stability, RNA Selectivity, and RNase H Elicitation: Synthesis and Biochemistry *Co-first authorship with Upadhayaya, R and Deshpande, S. G J. Org. Chem. 2011, 76, 4408-4431. II Li, Q.; Yuan, F.; Zhou, C.; Plashkevych, O.; Chattopadhyaya, J. Free-Radical Ring Closure to Conformationally Locked α-L- Carba-LNAs and Synthesis of Their Oligos: Nuclease Stability, Target RNA Specificity, and Elicitation of RNase H J. Org. Chem. 2010, 75, 6122-6140. III Li, Q.; Plashkevych, O.; Upadhayaya, R.; Deshpande, S. G.; Földesi, A.; Chattopadhyaya, J. The Physicochemical Properties of DNA-RNA Duplexes Con- taining Pure 7′R-Me- or 7′S-Me-Carba-LNA Derivatives of A, G, 5-MeC or T in the DNA Strand: Diastereomer Specific Com- parison of The 3′-Exonuclease Stability and RNase H Elicita- tion Submitted 2012. Reprints were made with permission from the respective publishers. Contribution Report The author wishes to clarify his contributions to the research papers pre- sented in the thesis: Paper I: Designed research with RamShankar Upadhayaya, Sachin Gangad- har Deshpande and Prof. Jyoti Chattopadhyaya (J. C.). Synthesized modified oligonucelotides, performed studies of thermal denaturation, SVPDE, blood serum, and RNase H digestions. Interpreted the data obtained from enzy- matic studies and wrote enzymatic part of the manuscript. Paper II: Designed research with Prof. Jyoti Chattopadhyaya (J. C.). Synthe- sized, purified and characterized all the intermediates for α-L-carba-LNA derivatives. Incorporated them into oligos and performed all the physico- chemical and biochemical studies (including thermal denaturation, circular dichroism, SVPDE, blood serum and RNase H digestions) towards modified oligonucleotides. Interpreted the data and wrote the first draft of the manu- script. [Note: Dr. Oleksandr Plashkevych (O. P.) contributed to the solvation free energy calculations and Dr. Chuanzheng Zhou (C. Z.) helped to revise and correct the manuscript] Paper III: Designed research with Prof. Jyoti Chattopadhyaya (J. C.). Syn- thesized, purified and characterized all the intermediates for 7′-Me-carba- LNA-A, -G, -MeC and -T analogues. Incorporated them into oligos and per- formed all the physicochemical (Tm measurement and thermodynamic study) and enzymatic studie (SVPDE, blood serum, E. coli RNase H1). Interpreted the data and wrote the first draft of the manuscript. Computational calcula- tions were performed by Dr. Oleksandr Plashkevych (O. P.) in collaboration with Qing Li (Q. L.) and Prof. Jyoti Chattopadhyaya (J. C.). Contents 1. Introduction...............................................................................................11 1.1 General introduction to nucleic acids.................................................11 1.2 Components of nucleic acids..............................................................12 1.3 Structural properties of nucleotides and nucleic acids .........................13 1.3.1 Nucleotide conformation ............................................................13 1.3.2 Structural features of nucleic acids.............................................14 1.4 Nucleic acids for therapeutic application ...........................................16 1.4.1 Nucleic acid-based therapeutics .................................................16 1.4.1.1 Antisense oligonucleotide...................................................16 1.4.1.2 Triple-helix forming oligonucleotide..................................16 1.4.1.3 Ribozyme and DNAzyme ...................................................17 1.4.1.4 RNA interference (siRNA and miRNA).............................17 1.4.1.5 Nucleic acid aptamers .........................................................18 1.4.2 Chemical synthesis of oligonucleotides......................................18 1.4.3 Chemical modifications in oligonucleotides ..............................19 1.4.3.1 Backbone modifications......................................................20 1.4.3.2 Sugar Moiety modifications................................................20 1.4.3.3 Base modifications..............................................................21 1.5 Overview of the thesis........................................................................22 2. Conformationally constrained nucleosides: introduction, synthesis and structural characterization.............................................................................23 2.1 Brief introduction of conformationally constrained nucleos(t)ides....23 2.1.1 Recent advances of intramolecular free-radical cyclization reactions on pentose sugars .................................................................25 2.2 Synthesis and structural elucidation of 7'Me-cLNA-A, -G, 5MeC, and T as well as (6'OH,7'Me)-α-L-carba-LNA-T nucleosides (Paper I- III) ............................................................................................................26 2.2.1 Synthesis of diastereomerically pure (7'S- or R-Me)-cLNA-A, -G, -5MeC, and -T nucleosides and their phosphoramidites..................26 2.2.2 Structural evidence of free-radical ring closure products in the synthesis of cLNA-A, -G, -MeC and -T nucleosides ............................29 2.2.2.1 Confirmation of bicyclic systems in 5-exo cyclization products...........................................................................................29 2.2.2.2 Stereochemistry of cLNA nucleosides................................30 2.2.3 Synthesis of diastereomerically pure (6'-OH,7'-Me)-α-L- carba-LNA-T nucleosides....................................................................31 2.2.4 NMR characterization of key intermediates involved in the synthesis of α-L-carba-LNA analogues...............................................33 2.2.4.1 Confirmation of bicyclic and tetracyclic systems by HMBC and COSY experiments...................................................................33 2.2.4.2 Stereochemistry of key intermediates in the synthesis of α- L-carba-LNA analogues..................................................................34 2.2.5 Mechanism of the free-radical cyclization and radical rearrangement involved in the synthesis of α-L-carba-LNA analogues .............................................................................................36 3. Antisense properties of modified AONs containing α-L-carba-LNAs and 7'-Me-carba-LNAs (Paper I-III).............................................................38 3.1 Thermo-stabilities of chemically modified AONs toward the RNA and DNA targets.......................................................................................38 3.1.1 Binding affinity of AONs containing α-L-carba-LNA and α-L- LNA thymines toward complementary RNA and DNA......................38 3.1.2 Binding affinity and thermodynamic properties of 7'-Me- cLNA-A, -G, -MeC and -T modified AONs toward complement- ary RNA and DNA strands........................................................................40 3.2 Nuclease stabilities of modified AONs..............................................46 3.2.1 Nucleolytic stabilities of AONs modified with α-L-carba-LNA derivatives and α-L-LNA ....................................................................46 3.2.2 Nucleolytic stabilities of AONs containing 7'R- and S-Me- cLNA-A, -G, -MeC and -T nucleosides ................................................47 3.3 RNase H-mediated RNA degradation in modified AON/RNA hybrids......................................................................................................50 3.3.1 RNase H elicitation induced by AONs modified with α-L- carba-LNA and α-L-LNA analogues...................................................50 3.3.2 RNase H elicitation induced by AONs modified with 7'-Me- cLNA-A, -G, -MeC, -T and LNA-A, -G, -C, -T analogues...................52 Sammanfattning............................................................................................54 Acknowledgements.......................................................................................56 References.....................................................................................................58 Abbreviations 1D One dimensional 2D Two dimensional A Adenosine Ade Adenine AIBN Azobisisobutyronitrile AMD Age-related macular degeneration AONs Antisense oligonucleotides aza-ENA 2′-N,4′-C-Ethylene bridged nucleic acid BNA Bridged nucleic acid Bu3SnH Tributyltinhydride C Cytidine CEM 2-Cyanoethoxymethyl cENA Carbocyclic 2′-O,4′-C-ethylene bridged nucleic acid cLNA Carbocyclic locked nucleic acid COSY Correlation spectroscopy Cyt Cytosine DEPT Distortionless enhancement by polarization transfer DMTr 4′,4′-Dimethoxytrityl DNA Deoxyribonucleic acid dsRNA Double-stranded RNA ENA 2′-O,4′-C-Ethylene bridged nucleic acid FDA Food and drug administration G Guanosine Gua Guanine HMBC Heteronuclear multiple bond correlation HMQC Heteronuclear multiple quantum coherence HNA Hexitol nucleic acid LNA Locked nucleic acid miRNA Micro-RNA MOE 2′-O-Methoxyethyl mRNA Messenger RNA ncRNA Non-coding RNA NMO N-Methylmorpholine-N-oxide NMR Nuclear magnetic resonance NOE Nuclear Overhauser effect PAGE Polyacrylamide