Development and Application of Novel Methods for the Synthesis of Modified Proteins

Abstract

Proteins are essential biomolecules that mediate a range of biological processes in humans. Many proteins contain post-translational modifications (PTMs) that are often vital to their biological activity and these modified proteins have recently attracted significant attention as promising therapeutics for the treatment of a range of diseases. Currently, most protein therapeutics are expressed as mixtures of isoforms containing different PTMs, which have a spectrum of biological activities. Understanding the importance and function of individual PTMs requires access to homogeneous protein isoforms, which can often only be achieved by chemical synthesis. Solid phase peptide synthesis (SPPS) combined with native chemical ligation (NCL)- desulfurization chemistry has led to the synthesis of a large number of protein targets with defined structure and function. In recent years, a new methodology called the diselenide-selenoester ligation (DSL) has proven to be a powerful complementary technology to NCL for the ligation of unprotected fragments to create native amide bonds. However, DSL technology, coupled with deselenization chemistry, is currently limited to ligation at alanine junctions due to a lack of availability of β-selenoamino acids other than selenocysteine (Sec, U). The overarching goal of the research described in this thesis therefore is to expand the utility of DSL through the generation of new β-selenoamino acid building blocks, and to validate their use in DSL through the construction of therapeutic protein targets. By enabling access to two extra ligation junctions (leucine and phenylalanine) for the expedient construction of complex post-translationally modified proteins by DSL-deselenization chemistry, this thesis has made important contributions to the area of protein science.