Organocatalyst-mediated cross aldol ligation of proteins

The chemical modification of proteins to produce protein bioconjugates has revolutionised the field of chemical biology, with wide-ranging applications in cell biology and chemical medicine. Of particular note are bioconjugates linked by carbon-carbon (C-C) bonds, which are highly prized due to their hydrolytic stability. Current strategies for their synthesis, however, suffer from a number of practical limitations, such as utilising acidic/basic conditions, showing reduced reactivity in the presence of oxygen, and requiring large concentrations of chemical probe in high molar excess.
This thesis describes the design and development of a novel protein bioconjugation strategy, the Organocatalyst-mediated Protein Aldol Ligation, or “OPAL”. During this project, the OPAL was conceived, designed, and rigorously optimised, and subsequently found to site-selectively modify proteins bearing previously installed α-oxo-aldehyde handles via hydrolytically stable C-C bonds at neutral pH within an hour using minimal molar equivalents of chemical probe through an organocatalyst-mediated process. The OPAL, along with the palladium mediated decaging of thiazolidine-containing unnatural amino acids, was found to be compatible with site-selective affinity tagging and protein pulldown in cell lysate. Additionally, the OPAL products could be site-selectively functionalised further using alternative protein aldehyde ligation strategies through modification of a newly generated β-hydroxy aldehyde handle that results from the initial OPAL modification. The OPAL strategy was applied to the chemical mimicry of lipidated Leishmania surface proteins previously inaccessible through conventional methods in molecular biology. Finally, within the context of protein bioconjugation, further aldol based C-C bond cleaving and forming protocols were designed and investigated.