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.
Native chemical ligation (NCL) combined with desulfurization chemistry has revolutionized the way in which large polypeptides and proteins are accessed by chemical synthesis. Herein, we outline the use of flow chemistry for the ligation-based assembly of polypeptides. We also describe the development of a novel photodesulfurization transformation that, when coupled with flow NCL, enables efficient access to native polypeptides on time scales up to 2 orders of magnitude faster than current batch NCL-desulfurization methods. The power of the new ligation-photodesulfurization flow platform is showcased through the rapid synthesis of the 36 residue clinically approved HIV entry inhibitor enfuvirtide and the peptide diagnostic agent somatorelin.
Hematophagous organisms produce a suite of salivary proteins which interact with the host’s coagulation machinery to facilitate the acquisition and digestion of a bloodmeal. Many of these biomolecules inhibit the central blood-clotting serine proteinase thrombin that is also the target of several clinically approved anticoagulants. Here a bioinformatics approach is used to identify seven tick proteins with putative thrombin inhibitory activity that we predict to be posttranslationally sulfated at two conserved tyrosine residues. To corroborate the biological role of these molecules and investigate the effects of amino acid sequence and sulfation modifications on thrombin inhibition and anticoagulant activity, a library of 34 homogeneously sulfated protein variants were rapidly assembled using one-pot diselenide-selenoester ligation (DSL)-deselenization chemistry. Downstream functional characterization validated the thrombin-directed activity of all target molecules and revealed that posttranslational sulfation of specific tyrosine residues crucially modulates potency. Importantly, access to this homogeneously modified protein library not only enabled the determination of key structure–activity relationships and the identification of potent anticoagulant leads, but also revealed subtleties in the mechanism of thrombin inhibition, between and within the families, that would be impossible to predict from the amino acid sequence alone. The synthetic platform described here therefore serves as a highly valuable tool for the generation and thorough characterization of libraries of related peptide and/or protein molecules (with or without modifications) for the identification of lead candidates for medicinal chemistry programs.
The synthesis of a β-thiol asparagine derivative bearing a novel (2,4,6-trimethoxyphenyl)thiazolidine protecting group is described. The efficient incorporation of the amino acid into the N-termini of peptides is demonstrated as well as the utility of the β-thiol asparagine moiety for rapid ligation reactions with peptide thioesters. The streamlined synthesis of native peptide products could be accomplished using a one-pot radical desulfurization of the β-thiol auxiliary following the ligation event. The utility of the amino acid is highlighted in the efficient one-pot assembly of the HIV entry inhibitor enfuvirtide.
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