(2008) the Synthesis of Vinylphosphonate-Linked RNA. Phd Thesis, University of Nottingham

Collis, Alana E.C. (2008) The synthesis of vinylphosphonate-linked RNA. PhD thesis, University of Nottingham. Access from the University of Nottingham repository: http://eprints.nottingham.ac.uk/10541/1/Alana_Collis_Thesis.pdf Copyright and reuse: The Nottingham ePrints service makes this work by researchers of the University of Nottingham available open access under the following conditions. · Copyright and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners. · To the extent reasonable and practicable the material made available in Nottingham ePrints has been checked for eligibility before being made available. · Copies of full items can be used for personal research or study, educational, or not- for-profit purposes without prior permission or charge provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way. · Quotations or similar reproductions must be sufficiently acknowledged. Please see our full end user licence at: http://eprints.nottingham.ac.uk/end_user_agreement.pdf A note on versions: The version presented here may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher’s version. Please see the repository url above for details on accessing the published version and note that access may require a subscription. For more information, please contact [email protected] THE SYNTHESIS OF VINYLPHOSPHONATE-LINKED RNA Alana E. C. Collis, MChem. Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy February 2008 Abstract An introductory chapter discusses the steric block, RNase H and RNA interference antisense mechanisms and the application of antisense nucleic acids as therapeutic agents. Examples of existing chemical modifications of the sugar and backbone regions of nucleic acids are given, followed by the introduction of the vinylphosphonate modification. The vinylphosphonate has previously been examined in DNA and has been synthesised by either Pd(0) catalysed cross- coupling of an H-phosphonate with a vinyl bromide, or by the cross- metathesis of a vinylphosphonate with a terminal olefin. This thesis details the first examples of the vinylphosphonate modification in RNA. The initial aim of this project was the synthesis of a range of nucleosides where the 5’-C-O was replaced by a vinyl bromide carbon-carbon double bond. Starting from -D-glucose, acid catalysed formation of the 1,2:5,6- diisopropylidene -D-glucofuranose was carried out followed by protection of the 3’-OH as an acetate. The 5,6-isopropylidene was then subjected to H5IO6 mediated one-pot hydrolysis-oxidative cleavage to obtain the 5-aldehyde. Wittig olefination using CBr4 and Ph3P led to the dibromo olefin which was then stereoselectively reduced using dimethyl phosphite and diisopropylamine to obtain the pure trans-vinyl bromide. Following hydrolysis of the acetate, the stereochemistry of the 3-OH was then inverted by sequential oxidation and reduction. With the correct stereochemistry, the 3-OH - i - was protected as the 2-methylnaphthyl ether. The 1,2- isopropylidene moiety was then hydrolysed and acetylated to the bis-acetate which was subjected to Vorbrüggen conditions obtaining the uridine (93%), adenosine (77%), cytidine (30) and guanosine (63%) vinyl bromide nucleosides. The 2’-OAc of the nucleosides were hydrolysed to the 2’-OH in yields of 74-92%. The uridine 2’- OH was protected as the 2’-OTBS ether (98%), analogous to the commercially available phosphoramidites used in automated oligonucleotide synthesis. Similarly, the adenosine and uridine nucleosides could also be blocked as the 2’-OMe (59% and 73% respectively). In the case of the uridine vinyl bromide, the 3’-O-(2- methylnaphthyl) protecting group was cleaved using DDQ, this then enabled the vinylphosphonate-linked uridine dinucleotides to be functionalised at the 3’-OH as the cyanoethyl phosphoramidite using N,N-diisopropyl-2-cyanoethyl-chlorophosphoramidite, DIPEA and DMAP in dichloromethane (2’-OTBS 74%, 2’-OMe 41%). These could then be used in automated solid phase oligonucleotide synthesis. The H-phosphonates were prepared in a single step form the commercially available phosphoramidites using a tetrazole. These were then coupled to the vinyl bromide nucleosides using standard conditions of Pd(OAc)2 (0.2 eq.), dppf (0.4 eq.) and propylene oxide (20 eq.) in THF at 70 oC in a sealed vial for 6 hours. A range of vinylphosphonate-linked dinucleotides were accessed in yields of 61- 99%. - ii - A detailed experimental section at the end of this thesis describes the procedures used in the synthesis and the analysis of the structures obtained. - iii - Acknowledgements Throughout my PhD I have received guidance and advice from my supervisor Dr Chris Hayes, for which I am very grateful. The members of the Hayes research group have offered friendship, support and made my PhD both interesting and fun! The technical support staff within the Chemistry Department have always offered assistance. In particular, Dane Toplis has been indispensable in keeping things running. During my PhD I have been faced with additional challenges and I am immensely grateful to everyone who has offered support during this time. This includes everyone in the Organic section, Professor Ivan Powis and University of Nottingham Student Support. I could not have kept going without their support. Unwavering support has also come from my family and friends. They have always been there for me, giving encouragement and support whenever it is needed. Throughout my PhD I have been funded by the EPSRC and towards the end I also received financial support from the University of Nottingham. I am grateful to both of these for the support that has enabled me to complete my PhD. - iv - Table of Contents Abstract i Acknowledgements iv Abbreviations vii 1. Introduction 1 1.1 Therapeutic Agents 2 1.2 Targeting the Nucleic Acids 4 1.3 The Antisense Strategy 5 1.4 Nucleic Acid Modifications 14 1.5 Vinylphosphonate-Linked Nucleic Acids 34 1.6 The Synthesis of Vinylphosphonates 41 1.7 Aims of Research 62 Results and Discussion 65 2.1 Synthesis of the Vinyl Bromide Precursor 66 2.2 Second Generation Synthesis 72 2.3 Vinyl bromide nucleosides 101 2.4 Synthesis of H-Phosphonates 115 2.5 Palladium(0) Cross-Couplings 117 3 Conclusions and Future Work 124 3.1 Conclusions 124 3.2 Future Work 125 4 Experimental 128 - v - 4.1 General Considerations 129 4.2 Synthesis of the Generic Vinyl Bromide 132 4.3 Vinyl Bromide Nucleosides 157 4.4 H-Phosphonates 190 4.5 Vinylphosphonate-linked Dinucleotides 196 5 References 235 6 Appendix 255 - vi - Abbreviations A Adenine AIBN 2,2’-Azoisobutyronitrile AON Antisense oligonucleotide app Apparent aq Aqueous Bhoc Benzyhydroxycarbonyl borsm Based on recovered starting material BSA N,O-bis(trimethylsilyl)acetamide BSTFA N,O-bis(trimethylsilyl)trifluoroacetamide BTEAC Benzyltriethylammonium chloride Bz Benzoyl C Cytosine CAN Cerium ammonium nitrate CE Cyanoethyl CMV Cytomegalovirus DAST Diethylamino sulphurtrifluoride DBU 1,8-Diazobicyclo[5.4.0]undec-7-ene DCA Dichloroacetic acid DCM Dichloromethane dd Double doublet ddd Double double doublet DDQ 2,3-Dichloro-5,6-dicyanobenzoquinone DIPEA Diisopropylethyl amine DMP Dess Martin periodinane DMT Dimethoxytrityl - vii - dppf 1,1’-bis(diphenylphosphino)-ferrocene dsRNA Double stranded RNA ENA Ethylene Nucleic Acid Fmoc Fluorenylmethoxycarbonyl G Guanine HPLC High Performance Liquid Chromatography imid. Imidazole KHMDS Potassium hexamethydisilazide LNA Locked Nucleic Acid MBHA 4-Methyl benzhydrylamine mRNA Messenger RNA Nap Naphthyl nt Nucleotide PDC Pyridinium Dichromate PMB para-methoxy benzyl PNA Peptide nucleic acid pyr Pyridine RISC RNA Induced Silencing Complex RNAi RNA Interference rsm Recovered starting material siRNA Short interfering RNA ssRNA Single stranded RNA T Thymine TBAI Tetra n-butylammonium iodide TBHP tert-Butyl hydroperoxide TIPDS tetraisopropyldisiloxane - viii - Tm Melting temperature TMS Trimethylsilyl TOCSY Total correlation spectroscopy TPAP Tetra n-propylammonium perruthenate U Uracil - ix - - INTRODUCTION - - 1 - Introduction 1. Introduction 1.1 Therapeutic Agents Starting in 1990, the Human Genome Project saw one of the greatest landmarks in scientific history realised in 2003 when the sequence of the 3.7 billion base pairs that make up our human DNA was reported.1 Previous to this the two groups striving to unravel the genetic code; the International Human Genome Project (HGP) and the biotechnology firm Celera both reported outline sequences of the human genome in 2001.2,3 Identifying the 30,000 to 40,000 genes within human DNA, separated by non- coding regions also embraced new technologies.2 Building on this knowledge, it is anticipated that understanding of the proteome and other molecular machinery will increase, enabling scientists to better identify, diagnose and treat disease.4 Traditionally, disease is treated by directing small molecule drugs to target proteins such as enzymes and receptors. Drug discovery is a lengthy, expensive process; in 1997 it was estimated that 100,000 compounds were screened every day and that several million compounds screened, identified a few thousand with the desired characteristic.5 It is estimated that for every 5000-7000 compounds screened, five may progress to clinical trials and only

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