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.

Native chemical ligation followed by desulfurization is a powerful strategy for the assembly of proteins. Here we describe the development of a high-yielding, one-pot ligation-desulfurization protocol that uses trifluoroethanethiol (TFET) as a novel thiol additive. The synthetic utility of this TFET-enabled methodology is demonstrated by the efficient multi-step one-pot syntheses of two tick-derived proteins, chimadanin and madanin-1, without the need for any intermediary purification.

Madanin-1 and chimadanin are two small cysteine-free thrombin inhibitors that facilitate blood feeding in the tick Haemaphysalis longicornis. Here, we report a post-translational modification-tyrosine sulfation-of these two proteins that is critical for potent anti-thrombotic and anticoagulant activity. Inhibitors produced in baculovirus-infected insect cells displayed heterogeneous sulfation of two tyrosine residues within each of the proteins. One-pot ligation-desulfurization chemistry enabled access to homogeneous samples of all possible sulfated variants of the proteins. Tyrosine sulfation of madanin-1 and chimadanin proved crucial for thrombin inhibitory activity, with the doubly sulfated variants three orders of magnitude more potent than the unmodified inhibitors. The three-dimensional structure of madanin-1 in complex with thrombin revealed a unique mode of inhibition, with the sulfated tyrosine residues binding to the basic exosite II of the protease. The importance of tyrosine sulfation within this family of thrombin inhibitors, together with their unique binding mode, paves the way for the development of anti-thrombotic drug leads based on these privileged scaffolds.

Revolutionary proteomics strategies have enabled rapid profiling of the cellular targets of electrophilic small molecules. However, precise means to directly interrogate how these individual electrophilic modifications at low occupancy functionally reshape signaling networks have until recently been largely limited. Here we highlight new methods that transcend proteomics platforms to forge a quantitative link between protein target-selective engagement and downstream signaling. We focus on recent progress in the study of non-enzyme assisted signaling mechanisms and crosstalk choreographed by native reactive electrophiles. Using this as a model, we offer a long-term vision on how these toolsets along with the fundamental biochemical knowledge of precision electrophile signaling may be harnessed to assist covalent ligand–target matching and ultimately amend a disease-specific signaling dysfunction.

Off-target effects continue to impede disease interventions, particularly when targeting a specific protein within a family of similar proteins, such as kinase isoforms that play tumor-subtype-specific roles in cancers. Exploiting the specific electrophilic-metabolite-sensing capability of Akt3, versus moderate or no sensing, respectively, by Akt2 and Akt1, we describe a first-in-class functionally Akt3-selective covalent inhibitor [MK-H­(F)­NE], wherein the electrophilic core is derived from the native reactive lipid metabolite HNE. Mechanistic profiling and pathway interrogations point to retention of the metabolite’s structureas opposed to implicit electrophilicityas being essential for biasing isoform preference, which we found translates to tumor-subtype specificity against pten-null triple-negative breast cancers (TNBCs). MK-H­(F)­NE further enables novel downstream target identification specific to Akt3-function in disease. In TNBC xenografts, MK-H­(F)­NE fares better than reversible pan-Akt-inhibitors and does not show commonly observed side-effects associated with Akt1-inhibition. Inhibitors derived from native-metabolite sensing are thus an enabling plan-of-action for unmasking kinase-isoform-biased molecular targets and tumor-subtype-specific interventions.

2 Materials and Methods Statistics and data presentation. For experiments involving cultured cells, samples generated from individual wells or plates were considered biological replicates. For zebrafish experiments, each larval fish was considered a biological replicate. In the figure legend for each experiment, the number of independent biological replicates and how the data are presented in the figure (typically mean ± SEM) are clearly indicated. P-values calculated with Student's unpaired t-test are clearly indicated within datafigures. Data were plotted/fit and statistics generated using GraphPad Prism 7 or 8.

The anophelins are small protein thrombin inhibitors that are produced in the salivary glands of the Anopheles mosquito to fulfill a vital role in blood feeding. A bioinformatic analysis of anophelin sequences revealed the presence of conserved tyrosine residues in an acidic environment that were predicted to be post-translationally sulfated in vivo. To test this prediction, insect cell expression of two anophelin proteins, from Anopheles albimanus and Anopheles gambiae, was performed, followed by analysis by mass spectrometry, which showed heterogeneous sulfation at the predicted sites. Homogeneously sulfated variants of the two proteins were subsequently generated by chemical synthesis via a one-pot ligation–desulfurization strategy. Tyrosine sulfation of the anophelins was shown to significantly enhance the thrombin inhibitory activity, with a doubly sulfated variant of the anophelin from A. albimanus exhibiting a 100-fold increase in potency compared with the unmodified homologue. Sulfated anophelins were also shown to exhibit potent in vivo anticoagulant and antithrombotic activity.

Tyrosine (Tyr) sulfation is a common post-translational modification that is implicated in a variety of important biological processes, including the fusion and entry of human immunodeficiency virus type-1 (HIV-1). A number of sulfated Tyr (sTyr) residues on the N-terminus of the CCR5 chemokine receptor are involved in a crucial binding interaction with the gp120 HIV-1 envelope glycoprotein. Despite the established importance of these sTyr residues, the exact structural and functional role of this post-translational modification in HIV-1 infection is not fully understood. Detailed biological studies are hindered in part by the difficulty in accessing homogeneous sulfopeptides and sulfoproteins through biological expression and established synthetic techniques. Herein we describe an efficient approach to the synthesis of sulfopeptides bearing discrete sulfation patterns through the divergent, site-selective incorporation of sTyr residues on solid support. By employing three orthogonally protected Tyr building blocks and a solid-phase sulfation protocol, we demonstrate the synthesis of a library of target N-terminal CCR5(2-22) sulfoforms bearing discrete and differential sulfation at Tyr10, Tyr14, and Tyr15, from a single resin-bound intermediate. We demonstrate the importance of distinct sites of Tyr sulfation in binding gp120 through a competitive binding assay between the synthetic CCR5 sulfopeptides and an anti-gp120 monoclonal antibody. These studies revealed a critical role of sulfation at Tyr14 for binding and a possible additional role for sulfation at Tyr10. N-terminal CCR5 variants bearing a sTyr residue at position 14 were also found to complement viral entry into cells expressing an N-terminally truncated CCR5 receptor.