Expanding the Scope of Peptide Ligation Chemistry for Novel Biological Investigations

Abstract

Peptides and proteins are responsible for a plethora of structural and functional roles in the cell and are currently the focus of intensive research. In order to conduct detailed biological studies, access to homogenous samples of peptides and proteins is crucial. This thesis describes important contributions to the field of synthetic peptide and protein chemistry which have enabled the assembly of several previously inaccessible targets. Specifically, this work focuses on overcoming the limitations of Native Chemical Ligation (NCL), the most widely used method for peptide and protein synthesis. Chapter 2 details the synthesis of an unnatural asparagine derivative bearing a β-thiol auxiliary which was utilised in NCL-desulfurisation reactions, including the construction of the polypeptide drug – enfuvirtide. Chapter 3 provides an introduction to the recently developed diselenide-selenoester ligation-deselenisation (DSL) method which is considerably faster than NCL and can be performed chemoselectively in the presence of native Cys residues. The benefits of the DSL manifold are highlighted in Chapter 4 by the development of the first oligonucleotide-templated DSL reaction for rapid, sequence-selective miRNA detection at nanomolar concentrations. To expand the scope of the DSL-deselenisation method, selenolated variants of 3 proteinogenic amino acids (Asp, Glu and Pro) have been developed and employed for the construction of previously intractable protein targets. Finally, Chapter 6 describes the total synthesis of a library of 8 homogeneously sulfated β-synuclein proteins, using DSL and NCL methods in tandem, and investigations into the effect of sulfation on the anti-aggregation properties of the protein. By overcoming the limitations of traditional peptide ligation chemistry, the methods described in this thesis enable the synthesis of complex polypeptides and proteins which will continue to facilitate important biological investigations in the future.