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    Isoxazole Linked siRNA Cholesterol Conjugates by Catalyst Free Nitrile Oxide/Alkyne Cycloadditions

    Algay, Virginie (2012) Isoxazole Linked siRNA Cholesterol Conjugates by Catalyst Free Nitrile Oxide/Alkyne Cycloadditions. PhD thesis, National University of Ireland Maynooth.

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    The ultimate goal of the novel work discussed in this Ph.D. thesis was the synthesIs of a range of chemically modified siRNAs by catalyst free Click Cycloaddition Chemistry. The first chapter overviews the structure and biological function of RNA, before turning to a discussion on the synthetic value of dipolar cycloaddition chemistry of nitrones and nitrile oxides with alkene and alkyne dipolarophiles Chapter 2 presents the somewhat disappointing findings on the cycloaddition reactions of maleimides. Reactions with “simple” maleimides suffered from poor yield and/or poor diastereoselectivity, and synthetic access to the desired sugar maleimides was plagued by poor chemoselectivity. Following a protection/ deprotection strategy a glucofuranoside with a pendant maleimide was formed, and its cycloaddition potential explored with nitrones and nitrile oxides. Unfortunately the pyrrolo-isoxazole skeleton resulting from cycloaddition to the maleimides was unstable to the standard conditions of oligonucleotide cleavage and thus Chapter 2 concludes that the plan to prepare chemically modified oligonucleotides by cycloaddition to maleimides should be abandoned. The third chapter reports on the successes of solution phase cycloaddition between thymidine-alkynes, as model oligonucleotide substrates, and nitrile oxide dipoles using a chloramine-T protocol. The fourth chapter presents the synthesis of isoxazole linked cholesterol conjugates by nitrile oxide/alkyne cycloadditions. Novel alkyne and oxime modified cholesterol derivatives were prepared and demonstrated to be efficient dipolarophiles and dipole precursors. Chapter 5 embraces the synthetic success discussed in chapters 3 and 4 and applies them to oligonucleotide conjugation on the solid phase. Isoxazole linked aryl and cholesterol conjugates were formed by a convenient and fast protocol. Two distinct formations of siRNAs duplexes have been synthetized, siRNAs with 3’-overhang where strands were modified by addition of an extra U(2’-Chol) or U(3’-Chol) at the 3’- or 5’-terminus, and bluntmers with no overhang. The goal was to evaluate the biological consequences of the chemical modifications introduced. Unfortunately, the Luciferase assay did not show any evidence of activity. However, a real gene knockdown resulted from the siRNA transfection into CHO-K1 cells for eGFP gene. The sixth chapter details the experimental procedures together with full characterization of all the new compounds described in this thesis., It is followed by an appendix which carries a detailed description of structural determination of the protected maleimido-sugar, as representative example, a listing of the presentations delivered by the author and a copy of the first peer reviewed paper arising from this authors work. Finally, a complete bibliography of articles referred to in this thesis is included.

    Item Type: Thesis (PhD)
    Keywords: Isoxazole; siRNA; Cholesterol Conjugates; Catalyst Free Nitrile Oxide/Alkyne Cycloadditions;
    Academic Unit: Faculty of Science and Engineering > Chemistry
    Item ID: 4739
    Depositing User: IR eTheses
    Date Deposited: 29 Jan 2014 14:04
      Use Licence: This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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