A Native Chemical Ligation Handle that Enables the Synthesis of Advanced Activity‐Based Probes: Diubiquitin as a Case Study

We present the development of a native chemical ligation handle that also functions as a masked electrophile that can be liberated during synthesis when required. This handle can thus be used for the synthesis of complex activity‐based probes. We describe the use of this handle in the generation of linkage‐specific activity‐based deubiquitylating enzyme probes that contain substrate context and closely mimic the native ubiquitin isopeptide linkage. We have generated activity‐based probes based on all seven isopeptide‐linked diubiquitin topoisomers and demonstrated their structural integrity and ability to label DUBs in a linkage‐specific manner.     

Efficient Synthesis of Peptide and Protein Functionalized Pyrrole-Imidazole Polyamides Using Native Chemical Ligation

The advancement of DNA-based bionanotechnology requires efficient strategies to functionalize DNA nanostructures in a specific manner with other biomolecules, most importantly peptides and proteins. Common DNA-functionalization methods rely on laborious and covalent conjugation between DNA and proteins or peptides. Pyrrole-imidazole (Py–Im) polyamides, based on natural minor groove DNA-binding small molecules, can bind to DNA in a sequence specific fashion. In this study, we explore the use of Py–Im polyamides for addressing proteins and peptides to DNA in a sequence specific and non-covalent manner. A generic synthetic approach based on native chemical ligation was established that allows efficient conjugation of both peptides and recombinant proteins to Py–Im polyamides. The effect of Py–Im polyamide conjugation on DNA binding was investigated by Surface Plasmon Resonance (SPR). Although the synthesis of different protein-Py–Im-polyamide conjugates was successful, attenuation of DNA affinity was observed, in particular for the protein-Py–Im-polyamide conjugates. The practical use of protein-Py–Im-polyamide conjugates for addressing DNA structures in an orthogonal but non-covalent manner, therefore, remains to be established.     

Combination of Thiol‐Additive‐Free Native Chemical Ligation/Desulfurization and Intentional Replacement of Alanine with Cysteine

We report a novel strategy for native chemical ligation (NCL). Alanines not located at a ligation site are temporarily replaced with cysteines, and this enables efficient thiol‐additive‐free NCL, with subsequent desulfurization to regenerate the target peptide. We synthesized stresscopin‐related peptide and neuroendocrine regulatory peptide‐2 (NERP‐2) by this method. We confirmed that both conventional alkyl thioester and thioester‐equivalent N‐acyl‐N′‐methyl‐benzimidazolinone (MeNbz) can be adopted as thioester components for thiol‐additive‐free NCL of multi‐Cys‐containing peptides.     

Cyclization of the Antimicrobial Peptide Gomesin with Native Chemical Ligation: Influences on Stability and Bioactivity

Gomesin is an 18‐residue peptide originally isolated from the hemocytes of the Brazilian spider Acanthoscurria gomesiana. A broad spectrum of bioactivities have been attributed to gomesin, including in vivo and in vitro cytotoxicity against tumour cells, antimicrobial, antifungal, anti‐Leishmania and antimalarial effects. Given the potential therapeutic applications of gomesin, it was of interest to determine if an engineered version with a cyclic backbone has improved stability and bioactivity. Cyclization has been shown to confer enhanced stability and activity to a range of bioactive peptides and, in the case of a cone snail venom peptide, confer oral activity in a pain model. The current study demonstrates that cyclization improves the in vitro stability of gomesin over a 24 hour time period and enhances cytotoxicity against a cancer cell line without being toxic to a noncancerous cell line. In addition, antimalarial activity is enhanced upon cyclization. These findings provide additional insight into the influences of backbone cyclization on the therapeutic potential of peptides.     

Exploring the Functional Consequences of Protein Backbone Alteration in Ubiquitin through Native Chemical Ligation

Ubiquitin (Ub) plays critical roles in myriad protein degradation and signaling networks in the cell. We report herein Ub mimetics based on backbones that blend natural and artificial amino acid units. The variants were prepared by a modular route based on native chemical ligation. Biological assays show that some are enzymatically polymerized onto protein substrates, and that the resulting Ub tags are recognized for downstream pathways. These results advance the size and complexity of folded proteins mimicked by artificial backbones and expand the functional scope of such agents.     

Chemical Synthesis of A Pore‐Forming Antimicrobial Protein,Caenopore‐5, by Using Native Chemical Ligation at a Glu‐Cys Site

The 2014 report from the World Health Organization (WHO) on antimicrobial resistance revealed an alarming rise in antibiotic resistance all around the world. Unlike classical antibiotics, with the exception of a few species, no acquired resistance towards antimicrobial peptides (AMPs) has been reported. Therefore, AMPs represent leads for the development of novel antibiotics. Caenopore‐5 is constitutively expressed in the intestine of the nematode Caenorhabditis elegans and is a pore‐forming AMP. The protein (82 amino acids) was successfully synthesised by using Boc solid‐phase peptide synthesis and native chemical ligation. No γ‐linked by‐product was observed despite the use of a C‐terminal Glu‐thioester. The folding of the synthetic protein was confirmed by ¹H NMR spectroscopy and circular dichroism and compared with data recorded for recombinant caenopore‐5. The permeabilisation activities of the protein and of shortened analogues were evaluated.     

Native Chemical Ligation Directed by Photocleavable Peptide Nucleic Acid (PNA) Templates

A novel peptide–peptide ligation strategy is introduced that has the potential to provide peptide libraries of linearly or branched coupled fragments and will be suited to introduce simultaneous protein modifications at different ligation sites. Ligation is assisted by templating peptide nucleic acid (PNA) strands, and therefore, ligation specificity is solely encoded by the PNA sequence. PNA templating, in general, allows for various kinds of covalent ligation reactions. As a proof of principle, a native chemical ligation strategy was elaborated. This PNA‐templated ligation includes easy on‐resin procedures to couple linkers and PNA to the respective peptides, and a traceless photocleavage of the linker/PNA oligomer after the ligation step. A 4,5‐dimethoxy‐2‐nitrobenzaldehyde‐based linker that allowed the photocleavable linkage of two bio‐oligomers was developed.     

A Modular Ligation Strategy for Asymmetric Bivalent Nucleosomes Trimethylated at K36 and K27

In nature, individual histones in the same nucleosome can carry identical (symmetric) or different (asymmetric) post-translational modification (PTM) patterns, increasing the combinatorial complexity. Embryonic stem cells exhibit “bivalent” nucleosomes, some of which are marked by an asymmetric arrangement of H3K36me3 (an activating PTM) and H3K27me3 (a repressive PTM). Here we describe a modular synthetic method to access such asymmetrically modified nucleosomes and show that H3K36me3 inhibits the activity of the methyltransferase PRC2 locally while still prolonging its chromatin binding time.     

Capture‐Tag‐Release: A Strategy for Small Molecule Labeling of Native Enzymes

A strategy for labeling native enzymes in a manner that preserves their activity is reported: capture–tag–release (CTR). Key to this approach is the small molecule CTR probe that contains an enzyme inhibitor, benzophenone crosslinker, and aryl phosphine ester. After UV‐derived capture of the enzyme, addition of an azide‐containing tag triggers a Staudinger ligation that labels the enzyme. A further consequence of the Staudinger ligation is fragmentation of the CTR probe, thus releasing the inhibitor and restoring enzymatic activity. As a proof‐of‐principle, the CTR strategy was applied to the hydrolase β‐galactosidase. The enzyme was efficiently labeled with biotin, and the kinetic data for the biotinylated enzyme were comparable to those for unlabeled β‐galactosidase. The CTR probe exhibits excellent targeting specificity, as it selectively labeled β‐galactosidase in a complex protein mixture.     

Chemical Synthesis of Integral Membrane Proteins: Methods and Applications

The development of efficient chemical methods for total synthesis or semisynthesis of integral membrane proteins is an important challenge at the interface between chemistry and biology. This review outlines the recent advances in the synthesis of integral membrane proteins, with particular focus on the methods for difficult peptide synthesis, purification, and enhancement of peptide solubility under the ligation conditions. The applications of these methods to the synthesis of integral membrane proteins with one or multiple transmembrane domains are also described.     

Facile One‐Step Assembly of Bona Fide SUMO Conjugates by Chemoenzymatic Ligation

The post‐translational conjugation of the small ubiquitin‐like modifiers (SUMOs) to target proteins occurs through a complex machinery that involves sequential action of at least three enzymes. SUMOylation performs crucial regulatory functions in several cellular processes. The availability of well‐defined SUMO conjugates is necessary for untangling the mechanism of SUMOylation. However, assembly of homogeneous SUMO conjugates represents a challenge because of the multi‐step synthesis involved and the unwieldiness of the reconstituted biosynthetic systems. Here we describe a simple one‐step chemoenzymatic strategy for conjugating engineered SUMO (eSUMO) proteins to a prefabricated isopeptide‐linked SUMO target peptide. Notably, the eSUMOs were efficiently recognized by the enzymes of the SUMOylation machinery and the SUMO conjugates served as bona fide substrates for DeSUMOylating enzymes.     

Sortase‐Mediated Ligation: A Gift from Gram‐Positive Bacteria to Protein Engineering

A new enzymatic protein ligation tool , sortase, has recently emerged from Gram‐positive bacteria. This article outlines the technique, sortase‐mediated ligation, and its applications in protein engineering, which include the introduction of unnatural molecules into proteins, protein immobilization, protein–protein conjugation, protein cyclization, as a self‐cleavable tag for protein expression, protein–PNA hybrids, neoglycoconjugates, and cell‐surface protein labeling, etc.

     

Selective Desulfurization Significantly Expands Sequence Variety of 3'-Peptidyl-tRNA Mimics Obtained by Native Chemical Ligation

Accessible: Functionally complex 3'-peptidyl-RNA conjugates can be efficiently desulfurized under free radical reaction conditions as demonstrated here. One-pot procedures for native chemical ligation (NCL) and desulfurization enable sequences for this class of bioconjugates that are inaccessible by other methods.     

Tailored Synthesis of 162‐Residue S‐Monoglycosylated GM2‐Activator Protein (GM2AP) Analogues that Allows Facile Access to a Protein Library

A synthetic protocol for the preparation of 162‐residue S‐monoglycosylated GM2‐activator protein (GM2AP) analogues bearing various amino acid substitutions for Thr69 has been developed. The facile incorporation of the replacements into the protein was achieved by means of a one‐pot/N‐to‐C‐directed sequential ligation strategy using readily accessible middle N‐sulfanylethylanilide (SEAlide) peptides each consisting of seven amino acid residues. A kinetically controlled ligation protocol was successfully applied to the assembly of three peptide segments covering the GM2AP. The native chemical ligation (NCL) reactivities of the SEAlide peptides can be tuned by the presence or absence of phosphate salts. Furthermore, NCL of the alkyl thioester fragment [GM2AP (1–31)] with the N‐terminal cysteinyl prolyl thioester [GM2AP (32–67)] proceeded smoothly to yield the 67‐residue prolyl thioester, with the prolyl thioester moiety remaining intact. This newly developed strategy enabled the facile synthesis of GM2AP analogues. Thus, we refer to this synthetic protocol as “tailored synthesis” for the construction of a GM2AP library.