The synthesis of vinylphosphonate-linked RNA

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 alpha-D-glucose, acid catalysed formation of the 1,2:5,6-diisopropylidene alpha-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 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 Vorbruggen 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%. A detailed experimental section at the end of this thesis describes the procedures used in the synthesis and the analysis of the structures obtained

The synthesis of vinylphosphonate-linked RNA

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 alpha-D-glucose, acid catalysed formation of the 1,2:5,6-diisopropylidene alpha-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 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 Vorbruggen 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%. A detailed experimental section at the end of this thesis describes the procedures used in the synthesis and the analysis of the structures obtained.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Extracting valuable compounds from straw degraded by Pleurotus ostreatus

This work aims to the recovery of lignocellulosic waste in an environmentally friendly process, as an alternative to the energy- intensive technologies: steam explosion, subcritical and/or supercritical water treatment, gasification through pyrolisis, etc. A study was made to optimize the extraction conditions of potentially valuable compounds in straw degraded by the fungus Pleurotus ostreatus. The effects of solvent nature, temperature and extraction time were quantified by material balances with a special view to the extracts obtained. Confirmation of the effectiveness of the operations was done by spectrophotometric, HPLC and LC-MS analyses. Following this study, a technology localized to the farm was conceived, requiring few craftsmanship and no special utilities, to obtain a semi-product for further processing. A centralized technology could be also taken into account to process the straw by direct extraction with hot solvents, in order to obtain products yields three times higher than in the case of the aqueous extraction followed by solvent extraction at 20oC

2-Aryl propionamides via 1,4-aryl radical migration from N-arylsulfonyl-2-bromopropionamides

Reaction of N-alkyl-N-arylsulfonyl-2-halo-propionamides with pentamethyldiethylenetriamine and either CuBr or CuCl leads to 2-aryl propionamides via initial radical generation, 1,4-aryl migration with loss of SO2 and reduction of the intermediate amidyl radical in 40-99% yields. (C) 2009 Elsevier Ltd. All rights reserved

1,4-Aryl migration under copper(I) atom transfer conditions

Reaction of N-alkyl-N-(trichloroacetyl)arylsulfonamides with CuCl/amines leads to N-alkyl-N-(dichloroacetyl)-arylsulfonamides via reduction or N-alkyl-aryldichloroacetamides via 1,4-aryl migration with loss of SO2. The ratio of reduction to aryl migration is dependent upon the temperature and the ligand utilised. Along with amide bond hydrolysis these reactions may compete when carrying out slow atom transfer radical cyclisation reactions using sulfonamides. (C) 2009 Elsevier Ltd. All rights reserved

Integrating medicinal plants extraction into a high-value biorefinery : an example of Artemisia annua L.

A principle of biorefining is extended to medicinal plants with the view of developing a more sustainable business model for biomass producers and extractors. This is demonstrated for Artemisia annua L. currently cultivated or harvested in the wild for extraction of a single compound, artemisinin, comprising on average 1 wt% dry weight of the plant biomass. We scaled extraction of artemisinin by a non-toxic to bacterial fermentation solvent tetrafluoroethane to a 5 L pilot scale. We identified a number of co-metabolites that could be extracted from the plant along with artemisinin and describe the multi-step extraction-fractionation sequence that potentially could be transferred to a large-scale multi-step extraction process. We also show possible routes to higher-value compounds on the basis of A. annua secondary metabolites, exemplified by the conversion of flavonoids to monomers

Wealth out of waste

Emerging legislation and increasing social and political demand for more ethical and sustainable manufacturing routes has led researchers to investigate alterations to methods used to produce everyday items. Addressing this problem requires a multi-disciplinary approach to enable a variety of novel technologies. Conventional biorefineries use traditional fermentation to convert biomass to feedstocks such as bioethanol, however there is an untapped resource for sustainable materials being wasted. Straw is made up of three main constituents: cellulose, hemi-cellulose and lignin. Whilst the first two can be readily converted to bioethanol, lignin remains under-exploited. The chemicals structure of lignin has been shown to yield many interesting breakdown products potentially useful on an industrial scale. The use of straw is advantageous due to the surplus currently produced in the U.K. The overriding objective of this program is to develop novel materials from plant waste by utilizing natural mechanisms in a truly innovative solid-state biorefinery. Bacterial and fungal species have been shown to target ligno-cellulosic breakdown followed by extractions to produce metabolic profiles of available chemicals. The chemicals are then able to be converted to or utilized as useful materials such as nutraceuticals, polymers and other high value products. This program is developing underpinning science and technology that can be applied at a local level, but on a global scale, allowing the utilization of local resource to produce sustainable high value products

Atom-Transfer Cyclization with CuSO4/KBH4: A Formal "Activators Generated by Electron Transfer" Process Also Applicable to Atom-Transfer Polymerization

The 4-exo and 5-exo-trig atom-transfer cyclizations of 1, 8a–e, 9, 12, and 13 can be mediated with as little as 0.05 mol % of Cu(TPMA)SO4·5H2O in the presence of 2.5 mol % of borohydride salts in 10 min at room temperature in air. This formal “activators generated by electron transfer” (AGET) procedure utilizes a cheap and oxidatively stable copper source (CuSO4·5H2O) and can be carried out in environmentally benign solvents (EtOH). It is possible to alter the product distribution in the 5-endo radical–polar crossover reactions of 10a,b and 11 by tailoring the amount of borohydride. Cyclization onto alkynes 14 and 15 is also possible in only 20 min. Controlled radical polymerization of styrene, with increased rates over conventional atom-transfer radical polymerization (ATRP), can be carried out in a controlled fashion (Mn, PDI) using either CuBr or CuSO4·5H2O and Bu4NBH4