Synthesis of cholesterol derivatives for exploration of cytochromes P450 from Dioscorea transversa and inhibition of Mycobacterium tuberculosis

Steroids are a class of organic molecule typified by a seventeen carbon skeleton consisting of four fused rings. These molecules have crucial roles in a number of biological processes across both plants and animals such as regulating membrane fluidity, signaling, and hormone biosynthesis. Cholesterol in particular is used by plants as a precursor to biosynthesise steroidal saponins, a group of natural products often found to be the active component of herbal medicines. However, plant extracts often contain a large number of impurities, and these molecules are difficult to chemically synthesise due to their complexity, rendering production of pure steroidal saponins challenging. By identifying the enzymes in this biosynthetic pathway, it may be possible to develop semi-synthetic methods towards these bioactive compounds, vastly cutting both the time and production costs. The Australian yam Dioscorea transversa presents an ideal model plant to study this biosynthetic pathway, as its roots contain only two steroidal saponins. These saponins are thought to be produced via three initial sequential cytochrome P450 mediated hydroxylations at key positions on cholesterol, producing an intermediate that is further cyclised and glycosylated to yield the observed saponins. Previous work has identified several P450 enzymes from D. transversa that may be implicated in this pathway, but in order to determine the substate preferences and products of these enzymes, a suite of 12 hydroxycholesterols were required. Chapters 1-2 detail the synthesis of hydroxycholesterols that may be present in the steroidal saponin biosynthetic pathway in D. transversa. These compounds possess each combination of hydroxylations that could be present in this pathway, and also explore different stereochemistry at two of these positions. By synthesising these compounds from only two starting materials -diosgenin and stigmasterol- the scattered literature synthesis were unified, allowing access to a variety of hydroxysterols from two commercially available materials. Collaborators then used the synthesised steroids as standards to identify enzyme turnover products, as well as substrates for enzyme turnover, allowing elucidation of one of the enzymes in the steroidal saponin biosynthetic pathway of D. transversa. Cholesterol is also used as a carbon and energy source by mycobacteria such as Mycobacterium tuberculosis. This unusual capability is made possible by a large suite of genes devoted to cholesterol catabolism, a process which is initiated by P450 mediated oxidation of the side chain. Previous studies have shown that cholesterol analogues with a degradation resistant side chain can effect micromolar inhibition of Mycobacterium tuberculosis in vitro. The key transformation that caused these compounds to become inhibitors was hydroxylation of the C16 position. Using techniques developed in Chapters 1-2, Chapters 3-4 discuss synthesis of a range of cholesterol derivatives with a variety of functionality at the terminal position of the side chain. In particular, the impact of the C3 and C16 hydroxyl group was explored by comparing compounds with this functionality to those in which it had been removed. A range of side chain moieties were installed, exploring both known P450 inhibitor functionality and groups with demonstrated anti-tubercular activity. The synthesised steroids were then tested by collaborators in the West group for in vitro inhibition against M. tuberculosis. This data confirmed the role of each hydroxyl group and several inhibitors showed significantly improved activity compared to the initial lead.