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Here, we describe the development of a highly efficient one-pot ligationdeselenization technology at aspartate and glutamate that enables the synthesis of polypeptides and proteins on unprecedented timescales. The power of the methodology is showcased through the rapid assembly of three thrombininhibiting tick-derived proteins as well as the synthesis of the 21 kDa homodimeric selenoprotein K. This work lays the foundation for the facile synthesis of a range of bioactive polypeptides and proteins in the future. HIGHLIGHTSRapid one-pot ligationdeselenization at b-selenoaspartate and g-selenoglutamate Methodology enables chemical protein synthesis on unprecedented timescales Synthesis of Selenoprotein K through chemoselective deselenization of b-selenoaspartate Synthesis, purification, and quantification of thrombin inhibitory proteins in 3 hr Mitchell et al., Chem 2, 703-715 May 11, 2017 ª 2017 Elsevier Inc. http://dx. SUMMARYPeptide ligation chemistry has revolutionized protein science by facilitating access to synthetic proteins. Here, we describe the development of additive-free ligation-deselenization chemistry at b-selenoaspartate and g-selenoglutamate that enables the generation of native polypeptide products on unprecedented timescales. The deselenization step is chemoselective in the presence of unprotected selenocysteine, which is highlighted in the synthesis of selenoprotein K. The power of the methodology is also showcased through the synthesis of three tick-derived thrombin-inhibiting proteins, each of which were assembled, purified, and isolated for biological assays within a few hours. The methodology described here should serve as a powerful means of accessing synthetic proteins, including therapeutic leads, in the future.

Polyproline sequences are highly abundant in prokaryotic and eukaryotic proteins, where they serve as key components of secondary structure. To date, construction of the proline−proline motif has not been possible owing to steric congestion at the ligation junction, together with an n → π* electronic interaction that reduces the reactivity of acylated proline residues at the C-terminus of peptides. Here, we harness the enhanced reactivity of prolyl selenoesters and a trans-γ-selenoproline moiety to access the elusive proline−proline junction for the first time through a diselenide−selenoester ligation− deselenization manifold. The efficient nature of this chemistry is highlighted in the high-yielding one-pot assembly of two proline-rich polypeptide targets, submaxillary gland androgen regulated protein 3B and lumbricin-1. This method provides access to the most challenging of ligation junctions, thus enabling the construction of previously intractable peptide and protein targets of increasing structural complexity. 65 donors have been reported to react with N-terminal Cys-66 containing peptides, albeit in the presence of a selenol catalyst 67 and a large molar excess of the acyl donor fragment. More 68 recently, Dong et al. have designed a prolyl thioester whereby 69 the γ-position of the Pro ring is functionalized with a thiol 70 moiety (Scheme 1B). 17 This modified Pro thioester reacts via a 71 bicyclic thiolactone intermediate, which leads to activation of 72 the carbonyl through the generation of a highly strained cyclic 73 thioester. While this is a very elegant strategy, the γ-thiol

A PNA-templated peptide ligation reaction has been developed between selenocystine and selenoesters. The methodology was used for the sequence specific detection of miRNA at low concentrations.