Chapter 18 The Chemical Synthesis of Site-Specifically Modified Proteins Via Diselenide-Selenoester Ligation Rhys C. Griffiths and Nicholas J. Mitchell Abstract Peptide ligation techniques enable the controlled chemical synthesis of native and engineered proteins, including examples that display site-specific post-translational modifications (PTMs) and non-proteinogenic functionality. Diselenide-selenoester ligation (DSL) is a recent addition to the synthetic methodology that offers several advantages over existing strategies. The standard DSL reaction involves the additive-free ligation of a peptide carrying an N-terminal selenocysteine (Sec) residue with a fragment bearing a C-terminal selenoester. This operationally simple ligation proceeds rapidly at sterically hindered junctions and is efficient across a broad pH range. The incorporation of deselenization and oxidative deselenization techniques into the DSL protocol enables conversion of the Sec residue at the ligation site to alanine (Ala) and serine (Ser), respectively, thus enhancing the scope and versatility of the method. In this chapter, we describe the application of DSL to the one-pot chemical synthesis of proteins via both two-component and three-component ligation pathways. Key words Peptide, Chemical protein synthesis, Diselenide-selenoester ligation, Selenocysteine, Selenoester, Deselenization, Chemoselective, Native chemical ligation 1 Introduction Peptide ligation techniques enable the chemoselective assembly of multiple peptide fragments to produce proteins bearing native post-translational modifications (PTMs) and non-proteinogenic functionality. The control afforded by employing a fully synthetic approach to protein production ensures the site-specificity of any modifications, providing access to homogenously modified mate- rial for further study and application. The technique of native chemical ligation (NCL) [1] has historically been the most popular ligation method, providing synthetic access to numerous proteins bearing native or non-proteinogenic modifications [2]. Despite the impact of this chemistry, and the development of several advances to enhance the scope of the method [3–8], the NCL technique is Waleed M. Hussein, Rachel J. Stephenson and Istvan Toth (eds.), Peptide Conjugation: Methods and Protocols, Methods in Molecular Biology, vol. 2355, https://doi.org/10.1007/978-1-0716-1617-8_18, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2021 231