Mycolactone A/B (MycA/B) is the compound which confers virulence to <em>Mycobacterium ulcerans</em> which causes the neglected tropical disease Buruli ulcer. Simmonds discovered that MycA/B inhibits the protein channel Sec61, which is responsible for the movement of proteins into the endoplasmic reticulum (translocation) for incorporation into the cell membrane or secretion from the cell. This inhibition leads to apoptosis which causes skin necrosis. To discover a novel treatment of Buruli ulcer, a small molecule which can prevent MycA/B from binding to Sec61 but still allow translocation is sought. However, its discovery requires the development of an assay to visualise and quantify binding between Sec61 and MycA/B. This can be achieved using a NanoBRET assay which requires a cell line expressing recombinant Sec61-luciferase fusion protein and a probe consisting of a fluorophore conjugated to MycA/B. This project focused on the semi-synthesis of MycA/B for conversion into the probe. Semi-synthesis involves the isolation of the Myc core from MycF from the faster-growing and less hazardous <em>M. marinum</em> and the development of a shorter synthetic route to the southern fatty acid tail of MycA/B.This project began with the development and validation of a Liquid Chromatography-Mass Spectrometry (LC-MS) method to quantify MycF during the extraction process. Two extraction methods, using CHCl₃:MeOH and ethanol were evaluated and a Solid Phase Extraction (SPE) method was developed to reduce the effects of ion suppression. The ethanol method was found to be more effective due to fewer steps and avoiding the use of acetone, which destabilises MycF under light. In this work also explored the synthesis of the penta-ene fragment of MycA/B using Burke’s MIDA boronate methodology. Key steps include the Heck reaction, Suzuki-Miyaura coupling and Negishi coupling. The importance of water in the Suzuki reaction was highlighted and optimised. Efforts to couple the penta-ene with a triol precursor to form the MycA/B tail faced challenges due to steric hindrance and side reactions. Various catalysts, bases and protecting groups were tested. Despite the challenges, this work successfully synthesised the penta-ene and the triol precursor and has made significant progress in the isolation of MycF (and therefore the core) and the addition of the triol precursor to the penta-ene.