Peptide Α-thioester Research Articles

A dual functional peptide-auxiliary conjugate for C-to-N and N-to-C sequential native chemical ligation of glycopeptides

Long, homogeneously glycosylated peptides and proteins can be assembled from multiple segments via sequential chemoselective reactions. The efficiency of the synthesis depends on the effectiveness and number of steps and on their compatibility with glycosylation methods. Here, we present how the combination of auxiliary-mediated native chemical ligation and thioester generation via hydrazinolysis from Wang-type resin enables multiple, sequential N-to-C and C-to-N ligations. The method can be applied to glycosylated peptides and peptide α-thioesters and has the potential to be further extended to sequential glycosylation, thus paving the way to the synthesis of complex homogeneous glycoproteins. We applied this methodology to the synthesis of long MUC1 variants comprising 2, 4 and 6 tandem repeats and three O-glycosylations.

A Facile N-Mercaptoethoxyglycinamide (MEGA) Linker Approach to Peptide Thioesterification and Cyclization.

The C-terminal electrophilic activation of peptides by α-thioesterification requires strongly acidic conditions or complex chemical manipulations, which ultimately limit functional group compatibility and broad utility. Herein, we report a readily accessible N-mercaptoethoxyglycinamide (MEGA) solid-phase linker for the facile synthesis of latent peptide α-thioesters. Incubating peptide-MEGA sequences with 2-mercaptoethanesulfonic acid at mildly acidic pH yielded α-thioesters that were directly used in NCL without purification. The MEGA linker yielded robust access to thioesters ranging in length from 4 to 35 amino acids, and greatly simplified the synthesis of cyclic peptides. Finally, the high utility of MEGA was demonstrated by the one-pot synthesis of a functional analog of the Sunflower Trypsin Inhibitor 1.

Chemical synthesis of a polypeptide backbone derived from the primary sequence of the cancer protein NY-ESO-1 enabled by kinetically controlled ligation and pseudoprolines.

The cancer protein NY-ESO-1 has been shown to be one of the most promising vaccine candidates although little is known about its cellular function. Using a chemical protein strategy, the 180 amino acid polypeptide, tagged with an arginine solubilizing tail, was assembled in a convergent manner from four unprotected peptide α-thioester peptide building blocks and one cysteinyl polypeptide, which were in turn prepared by Boc and Fmoc solid phase peptide synthesis (SPPS) respectively. To facilitate the assembly by ligation chemistries, non-native cysteines were introduced as chemical handles into the polypeptide fragments; pseudoproline dipeptides and microwave assisted Fmoc SPPS were crucial techniques to prepare the challenging hydrophobic C-terminal fragment. Three sequential kinetically controlled ligations, which exploited the reactivity between peptide arylthioesters and peptide alkylthioesters, were then used in order to assemble the more tractable N-terminal region of NY-ESO-1. The ensuing 147 residue polypeptide thioester then underwent successful final native chemical ligation with the very hydrophobic C-terminal polypeptide bearing an N-terminal cysteine affording the 186 residue polypeptide as an advanced intermediate en route to the native NY-ESO-1 protein.

High‐Throughput Synthesis of Peptide α‐Thioesters: A Safety Catch Linker Approach Enabling Parallel Hydrogen Fluoride Cleavage

Peptide α-thioesters are fundamental building blocks in peptide and protein science, providing powerful tools for peptide medicinal chemistry. The application of peptide α-thioesters in native chemical ligation reactions has enabled synthetic access to cysteine-rich peptides and proteins, cyclic peptides as well as labeled and chemically modified biomolecules. An efficient high-throughput synthesis of peptide α-thioester building blocks would be beneficial for many medicinal chemical applications that require peptides and proteins. Herein we present a novel synthetic route to cysteine-rich peptide α-thioesters using a safety catch linker that enables a parallel synthetic strategy for chemical protein synthesis. ACP(68-75), bradykinin and dynorphin(1-13) were synthesized via Boc chemistry in their thioester form on a safety catch amide linker (SCAL), employing polystyrene- or poly(ethylene glycol)-based resins, compartmentalized in tea bags. This compartmentalized resin/linker strategy facilitated a parallel hydrogen fluoride cleavage in which each peptide thioester was subsequently cyclized by native chemical ligation, demonstrating the utility of this approach. A naturally occurring bioactive cyclic peptide, the sunflower trypsin inhibitor SFTI-1, was synthesized to demonstrate the viability of this method to access important peptide biomolecules.

A Parallel Semisynthetic Approach for Structure–Activity Relationship Studies of Peptide YY

The gut hormone peptide YY (PYY) is postprandially secreted from enteroendocrine L cells and is involved in the regulation of energy homeostasis. The N-terminal truncated version PYY(3-36) decreases food intake and has potential as an anti-obesity agent. The anorectic effect of PYY(3-36) is mediated through Y₂ receptors in the hypothalamus, vagus, and brainstem regions, and it is well known that the C-terminal tetrapeptide sequence of PYY(3-36) is crucial for Y2 receptor activation. The aim of this work was to develop a semisynthetic methodology for the generation of a library of C-terminally modified PYY(3-36) analogues. By using an intein-based expression system, PYY(3-29) was generated as a C-terminal peptide α-thioester. Heptapeptides bearing an N-terminal cysteine and modifications at one of the four C-terminal positions were synthesized in a 96-well plate by parallel solid-phase synthesis. In the plate format, an array of [Ala30]PYY(3-36) analogues were generated by ligation, desulfurization, and subsequent solid-phase extraction. The generated analogues, in which either Arg33, Gln34, Arg35, or Tyr36 had been substituted with proteinogenic or non-proteinogenic amino acids, were tested in a functional Y₂ receptor assay. Generally, substitutions of Tyr36 were better tolerated than modifications of Arg33, Gln34, and Arg35. Two analogues showed significantly improved Y₂ receptor selectivity; therefore, these results could be used to design new drug candidates for the treatment of obesity.

Chemical Synthesis of an Erythropoietin Glycoform Containing a Complex‐type Disialyloligosaccharide

New and improved: New reaction conditions for tert-Boc-based solid-phase peptide synthesis make acid-labile sialyloligosaccharyl peptide α-thioesters accessible. To demonstrate this, a sialyloligosaccharyl-erythropoietin glycoform (see picture) with 166 amino acid residues was synthesized.

A Synthetic Approach to a Peptide α‐Thioester from an Unprotected Peptide through Cleavage and Activation of a Specific Peptide Bond by N‐Acetylguanidine

A different route to peptide α-thioesters through a new peptide-bond-cleavage method at a cysteine residue by S-thiocarbonylation and subsequent treatment with N-acetylguanidine is described (see scheme). The resultant peptidyl-N-acetylguanidine can be converted into the corresponding peptide α-thioester and is also usable as an alternative to a peptide α-thioester. This method allows efficient kinetically controlled ligation in the presence of thiols. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Preformed Selenoesters Enable Rapid Native Chemical Ligation at Intractable Sites

Going pro: The first facile Pro-Cys ligation using a preformed prolyl selenoester is reported (see scheme; P=peptide). In a comparative study of peptide selenoesters in native chemical ligation peptide α-selenoesters are shown to be superior acyl donors and result in rate enhancements of at least two orders of magnitude when compared to the well-established peptide α-thioesters. This method permits rapid chemical ligation even at previously intractable sites, such as Pro-Cys.

Theoretical Analysis of the Detailed Mechanism of Native Chemical Ligation Reactions

The method of native chemical ligation between an unprotected peptide α-thioester and an N-terminal cysteine-peptide to give a native peptide in aqueous solution is one of the most effective peptide ligation methods. In this work, a systematic theoretical study was carried out to fully understand the detailed mechanism of ligation. It was found that for the conventional native chemical ligation reaction between a peptide thioalkyl ester and a cysteine in combination with an added aryl thiol as catalyst, both the thiol-thioester exchange step and the transthioesterification step proceed by an anionic concerted S(N)2 displacement mechanism, whereas the intramolecular rearrangement proceeds by an addition-elimination mechanism, and the rate-limiting step is the thiol-thioester exchange step. The theoretical method was then extended to study the detailed mechanism of the auxiliary-mediated peptide ligation between a peptide thiophenyl ester and an N-2-mercaptobenzyl peptide in which both the thiol-thioester exchange step and intramolecular acyl-transfer step proceed by a concerted S(N)2-type displacement mechanism. The energy barrier of the thiol-thioester exchange step depends on the side-chain steric hindrance of the C-terminal amino acid, whereas that of the acyl-transfer step depends on the side-chain steric hindrance of the N-terminal amino acid.

Synthesis of Heavily Glycosylated Peptide αThioester

Erythropoietin (EPO) needs to be heavily glycosylated to exhibit its bioactivity in vivo. In order to synthesize heavily glycosylated EPO analogues, corresponding glycosylated peptide αthioesters are essential to prepare glycosylated whole EPO peptide backbones through native chemical ligation. After construction of the peptide αthioester corresponding to the 1–32 amino acid sequence in EPO, we aimed to incorporate three complex-type biantennary sialyloligosaccharides to this peptide αthioester by the haloacetamide method. The reaction afforded the desired heavily glycosylated peptide αthioester.

High Throughput Synthesis of Peptide α-Thioesters Through the Use of “Volatilizable” Support

High Throughput Synthesis of Peptide α-Thioesters Through the Use of “Volatilizable” Support

Peptide thioester preparation based on an N- S acyl shift reaction mediated by a thiol ligation auxiliary

Formation of peptide thioesters, based on an N to S acyl shift mediated by an auxiliary, N-4,5-dimethoxy-2-mercaptobenzyl (Dmmb) group, under acidic conditions, is described. The protected peptide was assembled on a hydroxymethylphenylacetamidomethyl resin via an N-Dmmb-amino acid residue according to standard Fmoc solid-phase peptide synthesis following treatment with trifluoroacetic acid. The peptide α-thioester was released from the resin by reaction with 2-mercaptoethanesulfonic acid in the presence of N, N-diisopropylethylamine.

Peptide αthioester formation using standard Fmoc-chemistry

A highly efficient and simple Fmoc-based preparation of peptide αthioesters is presented. After Fmoc/ t-butyl solid-phase synthesis on 2-chlorotrityl resin the C-terminal carboxylic group of the protected peptide is directly converted to the corresponding thioester. The method leads to very high yields, shows a low level of epimerization and can be easily applied also for the preparation of long peptide αthioesters as demonstrated for the 41 amino acid N-terminal fragment of pro-neuropeptide Y (proNPY 1–40).

New Directions in Glycoprotein Engineering

Semisynthetic protein engineering techniques can be employed to overcome classic bottlenecks in the total synthesis or recombinant production of proteins bearing natural or unnatural post-translational modifications. Glycoproteins are particularly suited to semisynthesis as homogeneous samples for biological analyses are not readily available using traditional recombinant techniques. Here we report the use of expressed protein ligation for the construction of semi-synthetic glycoproteins, which are assembled when recombinantly derived peptide α-thioesters are covalently joined with synthetic glycopeptides with clustered O-linked glycans by native chemical ligation. We demonstrate how this approach may be employed for the construction of glycoproteins bearing mucin-like domains.

Chemical ligation of unprotected peptides directly from a solid support.

In this article we describe a new, convenient procedure to carry out intramolecular (cyclization) and intermolecular native chemical ligations of unprotected peptides directly from a solid support. Our solid-phase ligation approach eliminates the need to manipulate peptide (alpha)thioacid and peptide (alpha)thioester intermediates in aqueous solution before the ligation step, thereby leading to a reduction in handling losses and significantly increasing the overall efficiency of the chemical ligation strategy. A key step in our ligation scheme is the ability to generate fully unprotected peptides tethered to a solid support through an (alpha)thioester linkage. This can be achieved efficiently using optimized Boc-solid-phase peptide synthesis on a 3-mercaptopropionamide-polyethylene glycol-poly-(N,N-dimethylacrylamide) copolymer support (HS-PEGA). Once the synthesis is complete, the fully protected peptide (alpha)thioester resin is treated with HF to give the corresponding fully unprotected peptide (alpha)thioester resin. Using this procedure several polypeptides ranging from 15 to 47 residues were synthesized successfully. These peptide-resins were then used to perform both intramolecular (head-to-tail cyclizations) and intermolecular solid-phase ligations. The intramolecular solid-phase ligations proceeded much faster than their intermolecular counterparts, but in both cases the reactions were observed to be remarkably clean. The presence of aromatic thiol cofactors significantly accelerated the relatively slow intermolecular ligations. This novel methodology was then extended to provide a general method for performing sequential intermolecular ligations, allowing easy access to much larger polypeptide and protein systems.