Boc Solid-Phase Synthesis of Conjugable Cyclic Depsipeptides for Use as Biological Probes

Research into the mechanism of action of bioactive chemicals, such as drugs, natural products and toxins, can be greatly informed by the application of synthetic chemical probes to biological systems, usually cells. Probes are often conjugates of the active molecule with a 'tracer' in the form of a fluorescent group that allows its location in a cell to be visualised but, more importantly, assays to be developed that show when it is binding to its target (usually a protein) in the cell (e.g. a bioluminescence resonance energy transfer (BRET) assay). In order to convert bioactive molecules into conjugates, they must have a reactive 'handle' incorporated. The aim of this project is to develop methods to rapidly synthesise bioactive cyclic depsipeptides (peptides containing an ester bond) bearing such a handle in the form of the amino group of a lysine (or lysine-like) residue. The synthesis of peptides is most rapidly performed on solid-phase: the first residue is attached, by its carboxylic acid, to an insoluble polymer resin and subsequent iterative deprotection and coupling reactions build up the rest of the peptide without the need to purify after each step - only wash off excess reagents from the resin. In this project, we will take advantage of the older "Boc"-method as it does not involve an alkaline step (Fmoc deprotection by piperidine) which leads to problems in depsipeptide synthesis (hydrolysis of the ester bond). However, the Boc-method is usually avoided due to it requiring dangerous hydrofluoric acid for the final removal of the product from the resin. We will develop alternative linkages to the resin to circumvent the use of this reagent as well as carry out a full investigation of protecting groups to be used as the depsipeptide is built that will allow on-resin cyclisation prior to removal from resin and purification, followed by conversion into chemical probes.