We describe an unprecedented reaction between peptide selenoesters and peptide dimers bearing N-terminal selenocystine that proceeds in aqueous buffer to afford native amide bonds without the use of additives. The selenocystine-selenoester ligations are complete in minutes, even at sterically hindered junctions, and can be used in concert with one-pot deselenization chemistry. Various pathways for the transformation are proposed and probed through a combination of experimental and computational studies. Our new reaction manifold is also showcased in the total synthesis of two proteins.

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.

Despite the unique chemical properties of selenocysteine (Sec), ligation at Sec is an under-utilized methodology for protein synthesis. We describe herein an unprecedented protocol for the conversion of Sec to serine (Ser) in a single, high-yielding step. When coupled with ligation at Sec, this transformation provides a new approach to programmed ligations at Ser residues. This new reaction is compatible with a wide range of functionality, including the presence of unprotected amino acid side chains and appended glycans. The utility of the methodology is demonstrated in the rapid synthesis of complex glycopeptide fragments of the epithelial glycoproteins MUC5AC and MUC4 and through the total synthesis of the structured, cysteine (Cys)-free protein eglin C.

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

The convergent assembly of peptide fragments by native chemical ligation has revolutionized the way in which proteins can be accessed by chemical synthesis. A variation of native chemical ligation involves the reaction of peptides bearing an N-terminal selenocysteine residue with peptide thioesters, which proceeds through the same mechanism as the parent reaction. This transformation was first investigated in 2001 for the installation of selenocysteine into peptides and proteins via ligation chemistry. The recent discovery that selenocysteine residues within peptides can be chemoselectively deselenized without the concomitant desulfurization of cysteine residues has led to renewed interest in ligation chemistry at selenocysteine. This review outlines the use of selenocysteine in ligation chemistry as well as recent investigations of chemoselective ligation-deselenization chemistry at other selenol-derived amino acids that have the potential to greatly expand the number of targets that can be accessed by chemical synthesis.

A series of metal-chelating lipid conjugates has been designed and synthesized. Each member of the series bears a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) macrocycle attached to the lipid head group, using short n-ethylene glycol (n-EG) spacers of varying length. Liposomes incorporating these lipids, chelated to Gd3+, 64Cu2+, or 111In3+, and also incorporating fluorescent lipids, have been prepared, and their application in optical, magnetic resonance (MR) and single-photon emission tomography (SPECT) imaging of cellular uptake and distribution investigated in vitro and in vivo. We have shown that these multimodal liposomes can be used as functional MR contrast agents as well as radionuclide tracers for SPECT, and that they can be optimized for each application. When shielded liposomes were formulated incorporating 50% of a lipid with a short n-EG spacer, to give nanoparticles with a shallow but even coverage of n-EG, they showed good cellular internalization in a range of tumour cells, compared to the limited cellular uptake of conventional shielded liposomes formulated with 7% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethyleneglycol)2000] (DSPE-PEG2000). Moreover, by matching the depth of n-EG coverage to the length of the n-EG spacers of the DOTA lipids, we have shown that similar distributions and blood half lives to DSPE-PEG2000-stabilized liposomes can be achieved. The ability to tune the imaging properties and distribution of these liposomes allows for the future development of a flexible tri-modal imaging agent.

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