Rapid and efficient protein synthesis through expansion of the native chemical ligation concept

The growing interest in proteins, both in fundamental research and in drug discovery, has fuelled demand for efficient synthetic methods to access these biomolecules. Although solid-phase synthesis serves as the workhorse for accessing peptides up to 50 amino acids in length, ligation technologies have underpinned protein synthesis. Native chemical ligation (NCL) represents the most widely used method and relies on the reaction of a peptide bearing an N-terminal cysteine residue with a peptide thioester. While the seminal methodology was limited to reaction at N-terminal cysteine residues, the NCL concept has recently been extended with a view to improving reaction efficiency and scope. Specifically, the discovery that cysteine residues can be desulfurized to alanine has led to the development of a range of thiol-derived variants of the proteinogenic amino acids that can be employed in protein synthesis under a ligation-desulfurization manifold. Furthermore, a number of important technologies have been developed to access larger targets via multi-fragment assembly, including methods for latent thioester activation and orthogonal protecting group strategies. Very recently, the amino acid selenocysteine, together with selenylated proteinogenic amino acid variants, has been shown to facilitate rapid ligation with peptide selenoesters. The large rate accelerations of these ligations have enabled access to proteins on unprecedented timescales, while chemoselective deselenization chemistry renders hitherto unobtainable targets accessible. This Review highlights innovative developments that have greatly expanded the NCL concept, allowing it to serve as a rapid and efficient means of conquering more challenging synthetic protein targets in the near future.