Carbohydrate approaches to delivering therapeutic agents across the blood brain barrier
thesisposted on 28.02.2017, 03:04 by Kluvers, Corrine Marie
This thesis describes synthetic approaches for improving the efficiency of transport of small molecules and DNA analogues past the blood brain barrier towards the development of a treatment for motor neurone disease. Chapter one introduces motor neurone disease (MND) and describes potential causes of the disease. A mutation to the antioxidant enzyme, copper zinc superoxide dismutase (SOD1), is known to be associated with familial cases of motor neurone disease, potentially through an oxidative stress and/or a toxic gain of function mechanism. The susceptibility of selective motor neurones to MND could be due to altered AMPA receptor subunit expression. Treatment options include small molecule antioxidants, and antisense agents to block unwanted gene production. Previous work in our laboratory has seen the design, synthesis and evaluation of PNAs aimed to down-regulate an AMPA receptor subunit and the mutated SOD1 enzyme. While promising, the results indicate that the therapeutic potency of PNAs would be enhanced by improved delivery through the blood brain barrier. Chapter two focuses on analogues of vitamin E, a known antioxidant. Natural vitamin E is very lipid soluble and does not cross the BBB, therefore more hydrophilic analogues are required for the treatment of motor neurone disease. The use of silver carbonate as a promoter to form β-orientated glycosyl esters was explored for the production of 6 glycosylated vitamin E derivatives. Various syntheses of glycosyl ester vitamin E derivatives were explored. One method involved the synthesis of and application to the vitamin E carboxylic acid derivative 24. For example, glucosyl ester vitamin E derivative 25 was produced from the silver carbonate glycosylation of 24 in high yield. An alternative, and more appropriate synthetic strategy involved the use of benzyl protecting groups. This required the preparation of the benzyl bromide sugar 34 in situ from the benzyl 4-pentenyl sugar 33. Glucosyl ester vitamin E derivative 35 was produced from the silver carbonate glycosylation of 24. Chapter three examines the synthesis of PNA monomers linked to glucose and galactose as a means to improve the bioavailability of PNA oligomers. Two sites on the PNA structure were explored for modification – the so-called site X and Y. Examples of site X include glycosylation of L-serine with glucose and galactose to produce the glycosylated building blocks 39 and 40. For site Y, three strategies were explored. First, aromatic linking groups were used to produce “rigid-linked” glycosylated monomers, 71 and 72. Incorporation of a flexible linker was achieved using succinic anhydride, producing 75 and 76. However, this pathway was low yielding and complications in removing the C-terminus protecting group were experienced. The third strategy involved the installation of an alkyl-linking group, leading to 85 in good yield. Chapter four consists of three smaller sections, connected by a common theme of peptide nucleic acids (PNA). The first section details the synthesis, purification and characterisation of a library of PNA oligomers that incorporate glycosylated building blocks 39, 40, 71 and 72. The second section describes the use of the quartz crystal microbalance to investigate the interaction of a selection of PNA conjugates with a synthetic lipid bilayer. It was found that all glycosylated PNAs display transmembrane insertion. The third section focuses on the thermodynamic properties of the glycosylated PNA oligomers when binding complementary DNA. Two techniques were used: i) a UV-visible spectroscopic method investigating a thermal melt process between a PNA strand and its complementary DNA sequence; and ii) isothermal titration calorimetry to measure conjugation of the PNAs to complementary DNA. All thermodynamic parameters calculated indicate that binding is not hindered, and in some cases improved, by the attached glycosylated building blocks.