Solid‐Phase Parallel Synthesis of Functionalised Medium‐to‐Large Cyclic Peptidomimetics through Three‐Component Coupling Driven by Aziridine Aldehyde Dimers

The first solid‐phase parallel synthesis of macrocyclic peptides using three‐component coupling driven by aziridine aldehyde dimers is described. The method supports the synthesis of 9‐ to 18‐membered aziridine‐containing macrocycles, which are then functionalized by nucleophilic opening of the aziridine ring. This constitutes a robust approach for the rapid parallel synthesis of macrocyclic peptides.     

P−B Desulfurization: An Enabling Method for Protein Chemical Synthesis and Site‐Specific Deuteration

Cysteine‐mediated native chemical ligation is a powerful method for protein chemical synthesis. Herein, we report an unprecedentedly mild system (TCEP/NaBH₄ or TCEP/LiBEt₃H; TCEP=tris(2‐carboxyethyl)phosphine) for chemoselective peptide desulfurization to achieve effective protein synthesis via the native chemical ligation–desulfurization approach. This method, termed P−B desulfurization, features usage of common reagents, simplicity of operation, robustness, high yields, clean conversion, and versatile functionality compatibility with complex peptides/proteins. In addition, this method can be used for incorporating deuterium into the peptides after cysteine desulfurization by running the reaction in D₂O buffer. Moreover, this method enables the clean desulfurization of peptides carrying post‐translational modifications, such as phosphorylation and crotonylation. The effectiveness of this method has been demonstrated by the synthesis of the cyclic peptides dichotomin C and E and synthetic proteins, including ubiquitin, γ‐synuclein, and histone H2A.     

Cysteine/Penicillamine Ligation Independent of Terminal Steric Demands for Chemical Protein Synthesis

The chemical ligation of two unprotected peptides to generate a natural peptidic linkage specifically at the C‐ and N‐termini is a desirable goal in chemical protein synthesis but is challenging because it demands high reactivity and selectivity (chemo‐, regio‐, and stereoselectivity). We report an operationally simple and highly effective chemical peptide ligation involving the ligation of peptides with C‐terminal salicylaldehyde esters to peptides with N‐terminal cysteine/penicillamine. The notable features of this method include its tolerance of steric hinderance from the side groups on either ligating terminus, thereby allowing flexible disconnection at sites that are otherwise difficult to functionalize. In addition, this method can be expanded to selective desulfurization and one‐pot ligation‐desulfurization reactions. The effectiveness of this method was demonstrated by the synthesis of VISTA (216‐311), PD‐1 (192‐288) and Eglin C.     

Traceless Purification and Desulfurization of Tau Protein Ligation Products

We present a novel strategy for the traceless purification and synthetic modification of peptides and proteins obtained by native chemical ligation. The strategy involves immobilization of a photocleavable semisynthetic biotin–protein conjugate on streptavidin‐coated agarose beads, which eliminates the need for tedious rebuffering steps and allows the rapid removal of excess peptides and additives. On‐bead desulfurization is followed by delivery of the final tag‐free protein product. The strategy is demonstrated in the isolation of a tag‐free Alzheimer's disease related human tau protein from a complex EPL mixture as well as a triphosphorylated peptide derived from the C‐terminus of tau.     

Parallel Chemical Protein Synthesis on a Surface Enables the Rapid Analysis of the Phosphoregulation of SH3 Domains

Analysis of postranslationally modified protein domains is complicated by an availability problem, as recombinant methods rarely allow site‐specificity at will. Although total synthesis enables full control over posttranslational and other modifications, chemical approaches are limited to shorter peptides. To solve this problem, we herein describe a method that combines a) immobilization of N‐terminally thiolated peptide hydrazides by hydrazone ligation, b) on‐surface native chemical ligation with self‐purified peptide thioesters, c) radical‐induced desulfurization, and d) a surface‐based fluorescence binding assay for functional characterization. We used the method to rapidly investigate 20 SH3 domains, with a focus on their phosphoregulation. The analysis suggests that tyrosine phosphorylation of SH3 domains found in Abl kinases act as a switch that can induce both the loss and, unexpectedly, gain of affinity for proline‐rich ligands.     

Aziridine-2-carboxylic acid-containing peptides: application to solution- and solid-phase convergent site-selective peptide modification

The development of a method for site- and stereoselective peptide modification using aziridine-2-carboxylic acid-containing peptides is described. A solid-phase peptide synthesis methodology that allows for the rapid generation of peptides incorporating the aziridine residue has been developed. The unique electrophilic nature of this nonproteinogenic amino acid allows for site-selective conjugation with various thiol nucleophiles, such as anomeric carbohydrate thiols, farnesyl thiol, and biochemical tags, both in solution and on solid support. This strategy, combined with native chemical ligation, provides convergent and rapid access to complex thioglycoconjugates.     

Controlling Peptide Self‐Assembly through a Native Chemical Ligation/Desulfurization Strategy

Self‐assembled peptides were synthesized by using a native chemical ligation (NCL)/desulfurization strategy that maintained the chemical diversity of the self‐assembled peptides. Herein, we employed oxo‐ester‐mediated NCL reactions to incorporate cysteine, a cysteine‐based dipeptide, and a sterically hindered unnatural amino acid (penicillamine) into peptides. Self‐assembly of the peptides resulted in the formation of self‐supporting gels. Microscopy analysis indicated the formation of helical nanofibers, which were responsible for the formation of gel matrices. The self‐assembly of the ligated peptides was governed by covalent and non‐covalent interactions, as confirmed by FTIR, CD, fluorescence spectroscopy, and MS (ESI) analyses. Peptide disassembly was induced by desulfurization reactions with tris(2‐carboxyethyl)phosphine (TCEP) and glutathione at 80 °C. Desulfurization reactions of the ligated peptides converted the Cys and penicillamine functionalities into Ala and Val moieties, respectively. The self‐supporting gels showed significant shear‐thinning and thixotropic properties.     

Microwave Heating Promotes the S-Alkylation of Aziridine Catalyzed by Molecular Sieves: A Post-Synthetic Approach to Lanthionine-Containing Peptides

Aziridine derivatives involved in nucleophilic ring-opening reactions have attracted great interest, since they allow the preparation of biologically active molecules. A chemoselective and mild procedure to convert a peptide cysteine residue into lanthionine via S-alkylation on aziridine substrates is presented in this paper. The procedure relies on a post-synthetic protocol promoted by molecular sieves to prepare lanthionine-containing peptides and is assisted by microwave irradiation. In addition, it represents a valuable alternative to the stepwise approach, in which the lanthionine precursor is incorporated into peptides as a building block.     

Synthesis of l‐ and d‐Ubiquitin by One‐Pot Ligation and Metal‐Free Desulfurization

Native chemical ligation combined with desulfurization has become a powerful strategy for the chemical synthesis of proteins. Here we describe the use of a new thiol additive, methyl thioglycolate, to accomplish one‐pot native chemical ligation and metal‐free desulfurization for chemical protein synthesis. This one‐pot strategy was used to prepare ubiquitin from two or three peptide segments. Circular dichroism spectroscopy and racemic protein X‐ray crystallography confirmed the correct folding of ubiquitin. Our results demonstrate that proteins synthesized chemically by streamlined 9‐fluorenylmethoxycarbonyl (Fmoc) solid‐phase peptide synthesis coupled with a one‐pot ligation–desulfurization strategy can supply useful molecules with sufficient purity for crystallographic studies.     

Site-selective conjugation of thiols with aziridine-2-carboxylic acid-containing peptides

The synthesis and convergent site-selective conjugation of aziridine-2-carboxylic acid-containing peptides with thiols, both in solution and on solid support, are described. The synthesis and use of FmocAzyOH in this capacity demonstrate both the efficient incorporation and tolerance of the Azy moiety in multistep Fmoc solid-phase peptide synthesis (SPPS), as well as the competence of solution and on-bead ligation through a highly regioselective base-promoted aziridine ring-opening process.     

Reversible Deactivation Radical Polymerization of Monomers Containing Activated Aziridine Groups

Polymers bearing activated aziridine groups are attractive precursors to α‐substituted‐β‐amino‐functionalized materials due to the enhanced reactivity of the pendant aziridine functionalities toward ring‐opening by nucleophiles. Two aziridine‐containing styrenic monomers, 2‐(4‐vinylphenyl)aziridine (VPA) and N‐mesyl‐2‐(4‐vinylphenyl)aziridine (NMVPA), were polymerized under a variety of reversible deactivation radical polymerization conditions. Low‐catalyst‐concentration atom transfer radical polymerization (LCC‐ATRP) and reversible addition‐fragmentation chain‐transfer (RAFT) polymerization were ineffective at producing well‐defined polymers from VPA due to side reactions between the aziridine functionalities and the agents controlling the polymerizations (catalysts or chain transfer agents). PolyVPA produced under nitroxide‐mediated polymerization (NMP) conditions had narrow molecular weight distribution at low to moderate conversions of monomer, but branched and eventually cross‐linked polymers were formed at higher conversions due to ring‐opening reactions of the aziridine groups. Most of these undesirable side reactions were eliminated by attaching a methanesulfonyl (mesyl) group to the aziridine nitrogen atom, and well‐defined linear homopolymers with targeted molecular weights were realized from NMVPA under RAFT and NMP conditions; however, side reactions between the aziridine groups and the catalyst in LCC‐ATRP still occured and the polymerization was uncontrolled using this technique.

     

One‐Pot/Sequential Native Chemical Ligation Using N‐Sulfanylethylanilide Peptide

N‐Sulfanylethylanilide (SEAlide) peptides were developed with the aim of achieving facile synthesis of peptide thioesters by 9‐fluorenylmethyloxycarbonyl (Fmoc)‐based solid‐phase peptide synthesis (Fmoc SPPS). Initially, SEAlide peptides were found to be converted to the corresponding peptide thioesters under acidic conditions. However, the SEAlide moiety was proved to function as a thioester in the presence of phosphate salts and to participate in native chemical ligation (NCL) with N‐terminal cysteinyl peptides, and this has served as a powerful protein synthesis methodology. The reactivity of a SEAlide peptide (anilide vs. thioester) can be easily tuned with or without the use of phosphate salts. This interesting property of SEAlide peptides allows sequential three‐fragment or unprecedented four‐fragment ligation for efficient one‐pot peptide/protein synthesis. Furthermore, dual‐kinetically controlled ligation, which enables three peptide fragments simultaneously present in the reaction to be ligated in the correct order, was first achieved using a SEAlide peptide. Beyond our initial expectations, SEAlide peptides have served in protein chemistry fields as very useful crypto‐peptide thioesters. DOI 10.1002/tcr.201200007     

Imidazole‐Aided Native Chemical Ligation: Imidazole as a One‐Pot Desulfurization‐Amenable Non‐Thiol‐Type Alternative to 4‐Mercaptophenylacetic Acid

Various bioactive proteins have been synthesized by native chemical ligation (NCL) and its combination with subsequent desulfurization (e.g., conversion from Cys to Ala). In NCL, excess 4‐mercaptophenylacetic acid (MPAA) is generally added to facilitate the reaction. However, co‐elution of MPAA with the ligation product during preparative high‐performance liquid chromatography sometimes reduces its usefulness. In addition, contamination of MPAA disturbs subsequent desulfurization. Here, we report for the first time that imidazole can be adopted as an alternative to MPAA in NCL using a peptide‐alkylthioester. The efficiency of the imidazole‐aided NCL (Im‐NCL) is similar to that of traditional MPAA‐aided NCL. As model cases, we successfully synthesized adiponectin(19‐107) and [Ser(PO₃H₂)⁶⁵]‐ubiquitin using Im‐NCL with a one‐pot desulfurization.     

Synthesis,Physical Properties,and Cytotoxicity of Nitroxyl–Aziridine Hybrid

Nitroxyl–aziridine hybrid, a candidate for a magnetic resonance imaging (MRI) probe and an anticancer drug, was synthesized by aziridine formation reaction of nitroxyl‐introduced aldehyde and guanidinium ylide in good diastereoselectivity. The relative configuration at aziridine C(2) C(3) bond of the major diastereoisomer was determined to be cis by X‐ray crystallographic analysis. Application of chiral guanidinium ylide resulted in the formation of the corresponding optically active aziridine in 84% ee. Reversible one‐electron redox potential and quantitative spin yield of the hybrid were observed in cyclic voltammogram and electron spin resonance, respectively. However, cytotoxicity of the hybrid against cancer cell lines used was not observed.     

Identification of Core Segment of Amyloidal Peptide Mediated by Chaperone Molecules by using Scanning Tunneling Microscopy

We illustrate in this work that pristine assemblies of amyloidal peptides can be obtained by perturbations of reduced scanning bias, and show a broad distribution in peptide length. In contrast, the chaperone‐mediated peptide co‐assembly presents ordered lamellar structures with a homogeneous distribution in length, which could be attributed to the core segment of the peptide. The efforts are beneficial for gaining insight into the aggregation propensity of peptides and inter‐peptide interactions.     

Constrained Peptides with Target‐Adapted Cross‐Links as Inhibitors of a Pathogenic Protein–Protein Interaction

Bioactive conformations of peptides can be stabilized by macrocyclization, resulting in increased target affinity and activity. Such macrocyclic peptides proved useful as modulators of biological functions, in particular as inhibitors of protein–protein interactions (PPI). However, most peptide‐derived PPI inhibitors involve stabilized α‐helices, leaving a large number of secondary structures unaddressed. Herein, we present a rational approach towards stabilization of an irregular peptide structure, using hydrophobic cross‐links that replace residues crucially involved in target binding. The molecular basis of this interaction was elucidated by X‐ray crystallography and isothermal titration calorimetry. The resulting cross‐linked peptides inhibit the interaction between human adaptor protein 14‐3‐3 and virulence factor exoenzyme S. Taking into consideration that irregular peptide structures participate widely in PPIs, this approach provides access to novel peptide‐derived inhibitors.     

A One‐Pot Chemically Cleavable Bis‐Linker Tether Strategy for the Synthesis of Heterodimeric Peptides

Heterodimeric peptides linked by disulfide bonds are attractive drug targets. However, their chemical assembly can be tedious, time‐consuming, and low yielding. Inspired by the cellular synthesis of pro‐insulin in which the two constituent peptide chains are expressed as a single‐chain precursor separated by a connecting C‐peptide, we have developed a novel chemically cleavable bis‐linker tether which allows the convenient assembly of two peptide chains as a single “pro”‐peptide on the same solid support. Following the peptide cleavage and post‐synthetic modifications, this bis‐linker tether can be removed in one‐step by chemical means. This method was used to synthesize a drug delivery‐cargo conjugate, TAT‐PKCi peptide, and a two‐disulfide bridged heterodimeric peptide, thionin (7‐19)‐(24‐32R), a thionin analogue. To our knowledge, this is the first report of a one‐pot chemically cleavable bis‐linker strategy for the facile synthesis of cross‐bridged two‐chain peptides.     

Effects of peptide acetylation and dimethylation on electrospray ionization efficiency

Peptide acetylation and dimethylation have been widely used to derivatize primary amino groups (peptide N‐termini and the ε‐amino group of lysines) for chemical isotope labeling of quantitative proteomics or for affinity tag labeling for selection and enrichment of labeled peptides. However, peptide acetylation results in signal suppression during electrospray ionization (ESI) due to charge neutralization. In contrast, dimethylated peptides show increased ionization efficiency after derivatization, since dimethylation increases hydrophobicity and maintains a positive charge on the peptide under common LC conditions. In this study, we quantitatively compared the ESI efficiencies of acetylated and dimethylated model peptides and tryptic peptides of BSA. Dimethylated peptides showed higher ionization efficiency than acetylated peptides for both model peptides and tryptic BSA peptides. At the proteome level, peptide dimethylation led to better protein identification than peptide acetylation when tryptic peptides of mouse brain lysate were analyzed with LC‐ESI‐MS/MS. These results demonstrate that dimethylation of tryptic peptides enhanced ESI efficiency and provided up to two‐fold improved protein identification sensitivity in comparison with acetylation. Copyright © 2016 John Wiley & Sons, Ltd.     

A General Strategy for the Synthesis of Enantiomerically Pure Azetidines and Aziridines through Nickel‐Catalyzed Cross‐Coupling

In this communication, we report a straightforward synthesis of enantiomerically pure 2‐alkyl azetidines. The protocol is based on a highly regioselective nickel‐catalyzed cross‐coupling of aliphatic organozinc reagents with an aziridine that features a tethered thiophenyl group. Activation by methylation transforms the sulfide into an excellent leaving group and triggers the formation of the 2‐substituted azetidine core structure by cyclization. In addition, we have expanded this concept to the synthesis of enantiomerically pure, terminal alkyl aziridines. Coupling of a TMS‐protected aziridine alcohol, followed by acidic work‐up to remove the silyl group, provides 1,2‐amino alcohol products that are readily cyclized to aziridines. Both of these sequences display excellent functional group tolerance and deliver the desired azetidine and aziridine products in good to excellent yields.     

Multivalent Design of Apoptosis‐Inducing Bid‐BH3 Peptide–Oligosaccharides Boosts the Intracellular Activity at Identical Overall Peptide Concentrations

Multivalent peptide–oligosaccharide conjugates were prepared and used to investigate the multivalency effect concerning the activity of Bid‐BH3 peptides in live cells. Dextran oligosaccharides were carboxyethylated selectively in the 2‐position of the carbohydrate units and activated for the ligation of N‐terminally cysteinylated peptides. Ligation through maleimide coupling was found to be superior to the native chemical ligation protocol. Monomeric Bid‐BH3 peptides were virtually inactive, whereas pentameric peptide conjugates induced apoptosis up to 20‐fold stronger at identical peptide concentrations. Comparison of lowly multivalent and highly multivalent peptide dextrans proved a multivalency effect in life cells which was specific for the BH3 peptide sequence.