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Native chemical ligation is a key reaction in the toolbox of chemical methods for the synthesis of native and modified proteins. The catalysis of ligation is commonly performed by using small aryl-thiol molecules added at high concentrations. In this work, we incorporated thiotyrosine, a non-canonical amino acid containing an aryl-thiol moiety, into a designed cyclic protein « sans queue ni tête ». Importantly, the protein environment reduced the pK a of the thiol group to 5.8-5.9, which is significantly lower than the previously reported value for thiotyrosine in a short peptide (pK a 6.4). Furthermore, we demonstrated the catalytic activity of this protein both as hydrolase and in native chemical ligation of peptides. These results will be useful for the development of efficient protein catalysts (enzymes) for protein synthesis and modification., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The reversible nature of disulfide functionality has been exploited to design intelligent materials such as nanocapsules, micelles, vesicles, inorganic nanoparticles, peptide and nucleic acid nanodevices. Herein, we report a new chemical methodology for the construction redox-sensitive protein assemblies using monodisperse facially amphiphilic protein-dendron bioconjugates. The disulfide functionality is strategically placed between the dendron and protein domains. The custom designed bioconjugates self-assembled into nanoscopic objects of a defined size dictated by the nature of dendron domain. The stimuli-responsive behavior of the protein assemblies is demonstrated using a suitable redox trigger., (© 2021 Wiley-VCH GmbH.)

Recently, solubilizing tag methods (Trt-K and Trt-R method) were developed for the challenging synthesis of peptides/proteins by means of native chemical ligation. In this system, the solubilizing tag can be attached to the Cys side chain by simply mixing the tag-introducing reagent under acidic conditions. The tagged peptides/proteins exhibited high water solubility thanks to the introduction of redundant oligo-Lys/Arg. In the final reaction, the tag can be quickly and cleanly detached by a standard deprotection reaction with trifluoroacetic acid. Herein, the development and application of these methods are described., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The universal genetic code of 20 amino acids is the product of evolution. It is believed that earlier versions of the code had fewer residues. Many theories for the order in which amino acids were integrated into the code have been proposed, considering factors ranging from prebiotic chemistry to codon capture. Several meta-analyses combined these theories to yield a feasible consensus chronology of the genetic code's evolution, but there is a dearth of experimental data to test the hypothesised order. We used combinatorial chemistry to synthesise libraries of random polypeptides that were based on different subsets of the 20 standard amino acids, thus representing different stages of a plausible history of the alphabet. Four libraries were comprised of the five, nine, and 16 most ancient amino acids, and all 20 extant residues for a direct side-by-side comparison. We characterised numerous variants from each library for their solubility and propensity to form secondary, tertiary or quaternary structures. Proteins from the two most ancient libraries were more likely to be soluble than those from the extant library. Several individual protein variants exhibited inducible protein folding and other traits typical of intrinsically disordered proteins. From these libraries, we can infer how primordial protein structure and function might have evolved with the genetic code., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bioorthogonal chemistry can be used for the selective modification of biomolecules without interfering with any other functionality that might be present. Recent developments in the field include orthogonal bioorthogonal reactions to modify multiple biomolecules simultaneously. During our research, we observed that the reaction rates for the bioorthogonal inverse-electron-demand Diels-Alder (iEDDA) reactions between nonstrained vinylboronic acids (VBAs) and dipyridyl-s-tetrazines were exceptionally higher than those between VBAs and tetrazines bearing a methyl or phenyl substituent. As VBAs are mild Lewis acids, we hypothesised that coordination of the pyridyl nitrogen atom to the boronic acid promoted tetrazine ligation. Herein, we explore the molecular basis and scope of VBA-tetrazine ligation in more detail and benefit from its unique reactivity in the simultaneous orthogonal tetrazine labelling of two proteins modified with VBA and norbornene, a widely used strained alkene. We further show that the two orthogonal iEDDA reactions can be performed in living cells by labelling the proteasome by using a nonselective probe equipped with a VBA and a subunit-selective VBA bearing a norbornene moiety., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The power of CPS, live! More than 90 attendees from around the world came together in Haifa to present and hear about cutting-edge science in protein chemistry, from advances in synthetic methods to applications in biology and medicine. The meeting was a powerful demonstration that chemical protein synthesis can provide otherwise unattainable insights into protein structure and function., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cyclic peptides have been proposed as privileged scaffolds that might mimic the folding and function of natural proteins. However, simple cyclic peptides typically cannot fold into well-defined structures. Herein, we describe a foldable cyclic peptide scaffold on which functional side chains can be displayed for targeted recognition of biomolecules. The foldable scaffold is based on prolinomycin, a proline-rich analogue of valinomycin. We report synthetic mutants of prolinomycin that retain the metal-assisted folding behavior under physiological conditions. The predictable structure formation of prolinomycin makes it a powerful platform to enable the development of synthetic receptors for biomolecules of interest. We demonstrate the potential of this scaffold by creating prolinomycin mutants that selectively bind anionic vesicles and bacterial cells., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Oxazetidine assisted KAHA ligation was developed by the Bode group as an efficient peptide segment ligation strategy at native serine residues. This milestone achievement should enable the chemical synthesis of difficult-to-prepare proteins for biological studies., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The pyrrolysine translational machinery has been extensively explored for the production of recombinant proteins containing a variety of "site-specific" non-canonical amino acids for both in vitro and in vivo biochemical studies. In this study, we report the first use of this technology for the production of branched cyclic proteins with a tadpole-like topology. As a proof of concept, we fused the well-studied RGD peptide to the C terminus of an mCherry reporter protein. Previous studies have shown that cyclization of the RGD peptide enhances its internalization into cells compared to its linear counterpart. The cellular uptake efficiencies of mCherry-cyclo(RGD), mCherry-linear(RGD), and wild-type mCherry determined by flow cytometry follow the trends expected, thereby confirming the feasibility and potential utility of this cyclization approach., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

With the study of peptides and proteins at the heart of many scientific endeavors, the omics era heralded a multitude of opportunities for chemists and biologists alike. Across the interface with life sciences, peptide chemistry plays an indispensable role, and progress made over the past decades now allows proteins to be treated as molecular patchworks stitched together through synthetic tailoring. The continuous elaboration of sophisticated strategies notwithstanding, Merrifield's solid-phase methodology remains a cornerstone of chemical protein design. Although the non-practitioner might misjudge peptide synthesis as trivial, routine, or dull given its long history, we comment here on its many advances, obstacles, and prospects from a practitioner's point of view. While sharing our perspectives through thematic highlights across the literature, this treatise provides an interpretive overview as a guide to novices, and a recap for specialists., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The antibiotic feglymycin is a linear 13-mer peptide synthesized by the bacterium Streptomyces sp. DSM 11171. It mainly consists of the nonproteinogenic amino acids 4-hydroxyphenylglycine and 3,5-dihydroxyphenylglycine. An alanine scan of feglymycin was performed by solution-phase peptide synthesis in order to assess the significance of individual amino acid side chains for biological activity. Hence, 13 peptides were synthesized from di- and tripeptide building blocks, and subsequently tested for antibacterial activity against Staphylococcus aureus strains. Furthermore we tested the inhibition of peptidoglycan biosynthesis enzymes MurA and MurC, which are inhibited by feglymycin. Whereas the antibacterial activity is significantly based on the three amino acids D-Hpg1, L-Hpg5, and L-Phe12, the inhibitory activity against MurA and MurC depends mainly on L-Asp13. The difference in the position dependence for antibacterial activity and enzyme inhibition suggests multiple molecular targets in the modes of action of feglymycin., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Egged on: Elaborate syntheses of biological macromolecules consisting of more than two different components is developing. Kajihara and co-workers have used a bio-resource to develop a new strategy for the semisynthesis of glycoproteins., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Intramolecular ligation of peptide hydrazides is reported to occur readily, causing the lactamization of fully unprotected peptides in an epimerization-free manner. This method relies on the routine procedures of Fmoc solid-phase peptide synthesis. It can be used to prepare cyclic peptides and cyclic proteins under simpler, mild conditions at lower costs., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Insulin-like peptide 5 (INSL5) was first identified through searches of the expressed sequence tags (EST) databases. Primary sequence analysis showed it to be a prepropeptide that was predicted to be processed in vivo to yield a two-chain sequence (A and B) that contained the insulin-like disulfide cross-links. The high affinity interaction between INSL5 and the receptor RXFP4 (GPCR142) coupled with their apparent coevolution and partially overlapping tissue expression patterns strongly suggest that INSL5 is an endogenous ligand for RXFP4. Given that the primary function of the INSL5-RXFP4 pair remains unknown, an effective means of producing sufficient quantities of this peptide and its analogues is needed to systematically investigate its structural and biological properties. A combination of solid-phase peptide synthesis methods together with regioselective disulfide bond formation were used to obtain INSL5. Both chains were unusually resistant to standard synthesis protocols and required highly optimized conditions for their acquisition. In particular, the use of a strong tertiary amidine, DBU, as N(alpha)-deprotection base was required for the successful assembly of the B chain; this highlights the need to consider incomplete deprotection rather than acylation as a cause of failed synthesis. Following sequential disulfide bond formation and chain combination, the resulting synthetic INSL5, which was obtained in good overall yield, was shown to possess a similar secondary structure to human relaxin-3 (H3 relaxin). The peptide was able to inhibit cAMP activity in SK-N-MC cells that expressed the human RXFP4 receptor with a similar activity to H3 relaxin. In contrast, it had no activity on the human RXFP3 receptor. Synthetic INSL5 demonstrates equivalent activity to the recombinant-derived peptide, and will be an important tool for the determination of its biological function.

Peptides can be designed to form self-assembled monolayers on gold nanoparticles to give nanomaterials with some chemical properties analogous to those of proteins. A variety of molecular-recognition properties are readily integrated within the peptide monolayer. Monofunctionalized nanoparticles are obtained by using separation methods that have been optimized for proteins. Recent applications as artificial enzymes and artificial enzyme substrates are presented. The limitations and long-term potential of peptide-capped nanoparticles as artificial proteins are discussed.