Synthesis and antiviral properties of spirocyclic [1,2,3]-triazolooxazine nucleosides

An efficient synthesis of spirocyclic triazolooxazine nucleosides is described. This was achieved by the conversion of β-D-psicofuranose to the corresponding azido-derivative, followed by alkylation of the primary alcohol with a range of propargyl bromides - obtained via Sonogashira chemistry. The products of these reactions underwent 1,3-dipolar addition smoothly to generate the protected spirocyclic adducts. These were easily deprotected to give the corresponding ribose nucleosides. The library of compounds obtained was investigated for its antiviral activity, using MHV (Mouse Hepatitis Virus) as a model wherein derivative 3f showed the most promising activity and tolerability

Reversible protein affinity-labelling using bromomaleimide-based reagents

Reversible protein biotinylation is readily affected via conjugation with a bromomaleimide-based reagent followed by reductive cleavage. The intermediate biotinylated protein constructs are stable at physiological temperature and pH 8.0. Quantitative reversibility is elegantly delivered under mild conditions of using a stoichiometric amount of a bis-thiol, thus providing an approach that will be of general interest in chemical biology and proteomics

Doctor of Philosophy

dissertationThe nanopore membrane is a single conical-shaped pore in a solid glass or fused quartz membrane at the end of a capillary; it can be used to support a planar lipid bilayer for ion channel recordings with a reconstituted biological nanopore. The work presented here explores the nature of the nanopore membrane and its influence on the suspended bilayer. The nanopore membrane is then used for ion channel recordings with the protein ion channel ?-hemolysin (?-HL) to detect single oxidative damage sites within a DNA sequence. Chemical modifications to the surface of the glass nanopore membrane with hydrophobic silanes (trimethylchlorosilane, n-butyldimethylchlorosilane, and n-octadecyldimethylchlorosilane) are explored to understand their influence on the pore wettability and the bilayer structure (seal resistance, voltage stability, and lifetime). Further, fused quartz was used to fabricate fused quartz nanopore membranes (QNMs) and these were compared with the traditional soda lime glass membranes as bilayer supports. The leakage current across the membrane was compared for fused quartz and soda lime glass capillaries. The structure of the suspended bilayer is investigated as a function of applied pressure across the orifice of a QNM using fluorescence microscopy. Ion channel reconstitution within lipid bilayers suspended across nanopore membranes is a pressure-dependent process; a positive pressure must be applied to the inside of the nanopore relative to the exterior for protein channel insertion to occur. Lastly, the nanopore membrane was used to perform ion channel recordings to detect the presence of a single oxidative damage site within a DNA sequence. The kinetics of the DNA duplex unzipping process within the ?-HL nanopore were monitored to determine the presence of a single DNA lesion, 8-oxo-,8-ihydroguanine (OG). The presence of OG influences the duplex stability which is reflected in the unzipping event duration. Additionally, the detection of a single oxidative damage site is examined using DNA immobilization experiments to determine the presence of the damage site based on the ion channel current. A single OG site within a DNA strand is adducted with a larger molecule and held within the ?-HL protein ion channel. The resultant current blockage level and noise level are shown to be unique to the adducted molecule

Doctor of Philosophy

dissertationThe alpha-hemolysin (a-HL) protein ion channel is a potential next-generation sequencing platform that has been extensively used to study nucleic acids at a singlemolecule level. After applying a potential across a lipid bilayer, the imbedded a-HL allows monitoring of the duration and current levels of DNA translocation and immobilization. Because this method does not require DNA amplification prior to sequencing, all the DNA damage present in the cell at any given time will be present during the sequencing experiment. The goal of this research is to determine if these damage sites give distinguishable current levels beyond those observed for the canonical nucleobases. Because DNA cross-links are one of the most prevalent types of DNA damage occurring in vivo, the blockage current levels were determined for thyminedimers, guanine(C8)-thymine(N3) crosslinks and platinum adducts. All of these crosslinks give a different blockage current level compared to the undamaged strands when immobilized in the ion channel, and they all can easily translocate across the a-HL channel. Additionally, the a-HL nanopore technique presents a unique opportunity to study the effects of DNA cross-links, such as thymine-dimers, on the secondary structure of DNA G-quadruplexes folded from the human telomere sequence. Using this singlemolecule nanopore technique we can detect subtle structural differences that cannot be easily addressed using conventional methods. The human telomere plays crucial roles in maintaining genome stability. In the presence of suitable cations, the repetitive 5'-TTAGGG human telomere sequence can fold into G-quadruplexes that adopt the hybrid fold in vivo. The telomere sequence is hypersensitive to UV-induced thymine-dimer (T=T) formation, and yet the presence of thymine dimers does not cause telomere shortening. The potential structural disruption and thermodynamic stability of the T=T-containing natural telomere sequences were studied to understand how this damage is tolerated in telomeric DNA. The a-HL experiments determined that T=Ts disrupt double-chain reversal loop formation but are well tolerated in edgewise and diagonal loops of the hybrid G-quadruplexes. These studies demonstrated the power of the a-HL ion channel to analyze DNA modifications and secondary structures at a single-molecule level

Plant Defensive β-Glucosidases Resist Digestion and Sustain Activity in the Gut of a Lepidopteran Herbivore

Two-component activated chemical defenses are a major part of many plants’ strategies to disrupt herbivory. The activation step is often the β-glucosidase-catalyzed removal of a glucose moiety from a pro-toxin, leading to an unstable and toxic aglycone. While some β-glucosidases have been well studied, several aspects of their roles in vivo, such as their precise sites of enzymatic activity during and after ingestion, and the importance of particular isoforms in plant defense are still not fully understood. Here, plant defensive β-glucosidases from maize, white mustard and almonds were shown to resist digestion by larvae of the generalist lepidopteran Spodoptera littoralis, and the majority of the ingested activities toward both general and plant pro-toxic substrates was recovered in the frass. Among other proteins potentially involved in defense, we identified specific plant β-glucosidases and a maize β-glucosidase aggregating factor in frass from plant-fed insects using proteomic methods. We therefore found that, while S. littoralis larvae efficiently degraded bulk food protein during digestion, β-glucosidases were among a small number of plant defensive proteins that resist insect digestive proteolysis. These enzymes remain intact in the gut lumen and frass and can therefore further catalyze the activation of plant defenses after ingestion, especially in pH-neutral regions of the digestive system. As most of the ingested enzymatic activity persists in the frass, and only particular β-glucosidases were detected via proteomic analyses, our data support the involvement of specific isoforms (maize ZmGlu1 and S. alba MA1 myrosinase) in defense in vivo

Synthetic studies directed toward azaspirocyclic alkaloids using spironitrone intermediates

HTX 1 is a 6,6-azaspirocyclic alkaloid found to naturally accumulate in the skin of certain frog species and has shown to exhibit activity as a non-competitive inhibitor of neuromuscular and central neuronal nicotinic acetylcholine receptors. A structurally similar, 6,5-azaspirocyclic system, is seen in the marine anti-inflammatory alkaloids pinnaic acid 2 and halichlorine 3. Despite both halichlorine and pinnaic acid having different mechanisms of action, both exhibit anti-inflammatory properties of interest to the academic community. It is due to these properties of interest and the scarcity of this natural product analogue’s availability from their natural sources that have led to synthetic studies being undertaken. This study aimed to develop a common synthetic route to the core structures of all three natural products. The use of this approach towards a formal/total synthesis of all three and libraries of analogues was also investigated. The strategy centred on the use of a 6,5 or 6,6-spitonitrones 105 and 232 respectively. It was planned to access these nitrones by oxidative ring opening of the corresponding isoxazolidines 22 and 23. While the preparation of multigram quantities of nitrone 105 was achieved a similar oxidation of isoxazolidine 23 was unsuccessful. Nevertheless, isoxazolidine 23 was used to access 6,6-azaspirocycles such as 222 and 223. A similar library of 6,5-azaspirocycles was also accessed from the corresponding isoxazolidine 22. Nitrone 105 was used to access the core structures of both pinnaic acid 2 and halichlorine 3 via preparation of the allylated spirocycle 161 accessed via Grignard addition to nitrone 105. Furthermore, addition of a range of Grignard reagents to this nitrone was used to prepare a small library of pinnaic acid analogues. The core structure of pinnaic acid 2 was prepared from allyl-derivative 170 via oxidative cleavage and subsequent Wittig homologation. The quinolizidine core structure 181 of halichlorine was accessed via intramolecular RCM from diene 180 - also prepared from key allylated spirocycle 170. This thesis also investigated the further functionalisation of spirocyclic core 195 towards a formal synthesis of pinnaic acid. While homologation at C11 was achieved the formation of a rigid tricycle 192, to allow stereoselective introduction of the C12 methyl group of pinnaic acid was unsuccessful

The manipulation of inflammation, immunity and infection by novel derivatives of halichlorine

Halichlorine 1 is a marine spirocyclic alkaloid, which has shown to exhibit anti-inflammatory properties.1 Due to the complexity of this structure, and the low abundance in nature, the development of total and partial syntheses of this compound have become of interest to the organic chemist. This project aimed to evaluate the therapeutic potential of this class of compounds by producing a library of simplified halichlorine derivatives by addition of Grignard reagents onto a key spironitrone that maps onto the core structure of halichlorine and thence to monitor potential bioactivity by conducting a series of biological assays to determine what effects these compounds have on human U937 cells. Addition of a wide range of Grignard reagents to spironitrone 128 was successful and generally proceed with high diastereoselectivity. In addition, reductive cleavage of the resulting N-hydroxyspirocycles with Zn/AcOH provided a host of N-acetyl-C7-substiuted spirocyclic derivatives 167-172. Reduction with indium provided free amines 173-181. As additions to spironitrone 128 proceeded with undesired stereoselectivity attempts were made to access O-protected spironitrone 204 by oxidation of spiroamines such as 199. This strategy was unsuccessful. In order to explore alternative spirocyclic derivatives, synthetic studies were also directed in attempts to access un-substituted derivatives by ring closing metathesis (RCM) of diene precursors 222-224. While RCM substrates were accessed cyclisation of these did not proceed. It was discovered that heating 6,5-spiroisoxazolidine 102 under pressure in a microwave reactor provided access to the corresponding 6,6-isomer 164 which maps onto the core structure of the amphibian toxin histrionicotoxin (HTX). Oxidation to 6,6-spironitrone 192, as followed by conversion to cycloadducts 193-195, which represent new analogues of the HTX family of alkaloids. Grignard additions to this nitrone, did not proceed in general. Biological screenings using undifferentiated and LPS activated U937 cells helped to identify a number of biologically active derivatives, when tested in the NO and growth and viability assays. The NO assay using LPS activated cells, identified that the adducts containing larger alkyl or aryl chains, particularly the pentyl, hexyl and benzyl adducts, expressed significant differences in NO inhibition at both 10-4 M and 10-5 M concentrations tested, compared to the untreated cells

Multicomponent Diels-Alder Sequences of 1-Aminodendralenes

This thesis explores the use of in situ generated acyclic 1-aminodendralenes in multicomponent diene-transmissive Diels-Alder (DTDA) reaction sequences. Dendralenes have previously been shown to generate polycyclic frameworks in a step-economic manner. The 1-amino substituent is shown to promote very high levels of site selectivity in these processes. Chapter 1 reviews the Diels-Alder reactions of 1-amino-1,3-butadienes and is divided into three sections. The first two sections cover the Diels-Alder reactions of 1-amino-1,3-butadienes and 1-amino-3-siloxy-1,3-butadienes (Rawal’s dienes) generated with a stoichiometric amount of amine. The third section covers enantioselective Diels-Alder reactions involving 1-amino-1,3-butadienes generated in situ with a catalytic amount of a chiral amine. While there have been many reports of Diels-Alder reactions of 1-amino-1,3-butadienes and 1-amino-3-siloxy-1,3-butadienes, there has been only one involving a semi-cyclic 1-amino[3]dendralene. There have been few examples which combine these Diels-Alder reactions with other transformations in multicomponent reactions to generate polycyclic frameworks. Chapter 2 describes the use of acyclic 1-amino[3]dendralenes in multicomponent reactions to generate a diverse range of heterocyclic structures. The condensation/Diels- Alder reaction sequence was tolerant of a variety of amines as well as carbon and hereoatom-based dienophiles. The Diels-Alder reactions of 1-amino[3]dendralenes were highly site-selective, taking place exclusively at the amine substituted 1,3-butadiene unit. The sequence was extended to a one-pot four-component reaction by including an additional dienophile for a Diels-Alder reaction to take place at the newly generated semi-cyclic diene. These condensation/Diels-Alder/Diels-Alder cycloadducts were generated with high diastereoselectivity, the origins of which were investigated and explained with the use of density functional theory calculations (carried out by Prof Paddon-Row). By reversing the order of events, that is performing a Diels-Alder reaction on the skipped dienal precursor before the condensation/Diels-Alder reaction sequence, constitutional isomers were accessed. The second Diels-Alder reaction could be performed intramolecularly when an amine bearing an alkenyl substituent was used. This condensation/Diels-Alder/intramolecular Diels-Alder reaction sequence furnished a variety of tricyclic and tetracyclic heterocycles. Chapter 3 describes the use of acyclic 1-aminodendralenes bearing chiral amines in organocatalytic, enantioselective Diels-Alder reactions to deliver enantioenriched cycloadducts. The enantioselective Diels-Alder reaction between 1-amino[3]dendralenes, the condensation product of skipped dienals and chiral amines, and various dienophiles followed by elimination of the amine generated trienal cycloadducts in good yield and high enantioselectivity. The reaction tolerates substitution on the skipped dienal as well as dienophiles possessing an aldehyde substituent at the α position. Extension of this methodology by performing Wittig and Diels-Alder reactions on the trienal cycloadducts enabled access to enantioenriched polycyclic products. By using a diene-dialdehyde as the starting precursor, it is anticipated that the Horeau principle would operate in the twofold condensation/Diels-Alder/elimination reaction sequence would furnish the cycloadduct in high enantioselectivity. A preliminary attempt successfully generated the desired cycloadduct as the major product