Protein Ligation and Total Synthesis I

Glycoproteins are an important class of naturally occurring biomolecules which play a pivotal role in many biological processes. They are biosynthesized as complex mixtures of glycoforms through post-translational protein glycosylation. This fact, together with the challenges associated with producing them in homogeneous form, has hampered detailed structure-function studies of glycoproteins as well as their full exploitation as potential therapeutic agents. By contrast, chemical synthesis offers the unique opportunity to gain access to homogeneous glycoprotein samples for rigorous biological evaluation. Herein, we review recent methods for the assembly of complex glycopeptides and glycoproteins and present several examples from our laboratory towards the total chemical synthesis of clinically relevant glycosylated proteins that have enabled synthetic access to full-length homogeneous glycoproteins.

Over the past 20 years, native chemical ligation has facilitated the synthesis of numerous complex peptide and protein targets, with and without post-translational modifications, as well as the design and construction of a variety of engineered protein variants. This powerful methodology has also served as a platform for the development of related chemoselective ligation technologies which have greatly expanded the scope and flexibility of ligation chemistry. This chapter details a number of important extensions of the original native chemical ligation manifold, with particular focus on the application of new methods in the total chemical synthesis of proteins. Topics covered include the development of auxiliary-based ligation methods, the post-ligation manipulation of Cys residues, and the synthesis and utility of unnatural amino acid building blocks (bearing reactive thiol or selenol functionalities) in chemoselective ligation chemistry. Contemporary applications of these techniques to the total chemical synthesis of peptides and proteins are described.

In eukaryotic cells, many proteins undergo extensive post-translational modifications (PTMs) such as methylation, acetylation, phosphorylation, glycosylation, and ubiquitination. Among these, ubiquitination is a particularly interesting PTM from both structural and functional viewpoints. In ubiquitination, the C-terminal carboxyl group of the small ubiquitin protein is attached to the ε-amine of a lysine residue of a substrate protein through an isopeptide bond. Ubiquitination has been shown to be involved in the regulation of many cellular processes including protein degradation and gene expression. And dysfunction of these processes is implicated in many human diseases. Despite many years of intensive research, a large number of protein ubquitination events remain poorly characterized. The challenge lies with the tremendous difficulties in isolating homogeneously modified proteins from biological samples for structural and functional studies. Enzymatic ubiquitination in vitro often has limited practical value due to the large number of substrate-specific E3 ligases and the difficulties in identifying or isolating these enzymes. Chemical approaches to the preparation of ubiquitinated proteins provide a powerful solution, and the development of such approaches has been the subject of intense research by many research laboratories. This review summarizes the methodological developments of protein chemical ubiquitination in recent years.

In chemical protein synthesis, peptide building blocks are prepared by solid-phase peptide synthesis (SPPS), and then connected by chemical ligation methods. The peptide thioester is one of key building blocks used in chemical protein synthesis, and improvements in the Fmoc SPPS procedure for preparing such thioesters would be highly desirable. In this review we focus on a method for peptide thioester synthesis based on the use of an intramolecular N to S acyl shift reaction as a key reaction. Amide and thioester forms at the thiol-containing residue are in equilibrium as a result of a reversible intramolecular acyl shift, which is detectable by ¹³C NMR. The amide form is favored under neutral conditions, while the thioester predominates under acidic conditions. Thiol auxiliaries can be employed to facilitate the formation of a thioester from an amide via an intramolecular N–S acyl shift, and the peptide thioester is formed after intermolecular transthioesterification in the presence of excess amounts of thiols. Even under neutral conditions, thiol auxiliary-containing peptides can be ligated with a cysteinyl peptide via an intramolecular N–S acyl shift, followed by native chemical ligation (NCL) in a one-pot reaction. These procedures can be applied to the chemical synthesis of proteins which are post-translationally modified.

Lipidated proteins play a key role in many essential biological processes in eukaryotic cells, including signal transduction, membrane trafficking, immune response and pathology. The investigation of the function of lipidated proteins requires access to a reasonable amount of homogenous lipid-modified proteins with defined structures and functional groups. Chemical approaches have provided useful tools to perform such studies. In this review we summarize synthetic methods of lipidated peptides and developments in the chemoselective ligation for the production of lipidated proteins. We introduce the biology of lipidated proteins and highlight the application of synthetic lipidated proteins to tackle important biological questions.

The utility of native chemical ligation (NCL) in the solution or solid phase synthesis of peptides, cyclic peptides, glycopeptides, and neoglycoconjugates is reviewed. In addition, the mechanistic details of inter- or intra-molecular NCLs are discussed from experimental and computational points of view.