Fmoc-based synthesis of peptide alpha-thioesters using an aryl hydrazine support

C-Terminal peptide thioesters are key intermediates in the synthesis/semisynthesis of proteins and of cyclic peptides by native chemical ligation. They are prepared by solid-phase peptide synthesis (SPPS) or biosynthetically by protein splicing techniques. Until recently, the chemical synthesis of C-terminal alpha-thioester peptides by SPPS was largely restricted to the use of Boc/Benzyl chemistry due to the poor stability of the thioester bond to the basic conditions required for the deprotection of the N(alpha)-Fmoc group. In the present work, we describe a new method for the SPPS of C-terminal thioesters using Fmoc/t-Bu chemistry. This method is based on the use of an aryl hydrazine linker, which is totally stable to conditions required for Fmoc-SPPS. When the peptide synthesis has been completed, activation of the linker is achieved by mild oxidation. This step converts the acyl hydrazine group into a highly reactive acyl diazene intermediate which reacts with an alpha-amino acid alkyl thioester (H-AA-SR) to yield the corresponding peptide alpha-thioester in good yield. This method has been successfully used to prepare a variety of peptide thioesters, cyclic peptides, and a fully functional Src homology 3 (SH3) protein domain.     

Transfer allyl esters to thioesters in solid phase condition: synthesis of peptide thioesters by Fmoc chemistry

A method to transfer allyl esters to thioesters under a solid phase condition has been developed to synthesize peptide thioesters. A Fmoc chemistry has been applied to synthesize the peptide allyl esters, which are selectively transferred to the expected peptide thioesters under solid phase synthesis conditions successfully.     

9-Fluorenylmethoxycarbonyl-based solid-phase synthesis of peptide α-thioesters

Peptide thioesters play a key role in convergent protein synthesis strategies such as native chemical ligation, traceless Staudinger ligation, and Ag(+) -mediated thioester ligation. The Boc-based solid-phase synthesis provides a very reliable access to peptide thioesters. However, the acid lability of many peptide modifications and the requirements of most parallel peptide synthesizers call for the milder Fmoc-based solid-phase synthesis. The Fmoc-based synthesis of peptide thioesters is more cumbersome and typically proceeds with lower yields than the synthesis of peptide acids and peptide amides. The success of native chemical ligation and related technologies has sparked intensive research effort devoted to the development of new methods. The recent progress in this rapidly expanding field is reviewed.     

Development of a Chemical Methodology for the Preparation of Peptide Thioesters Applicable to Naturally Occurring Peptides Using a Sequential Quadruple Acyl Transfer System

Peptide thioesters are very useful in protein chemistry, and chemistry- and biochemistry-based protocols are used for the preparation of thioesters. Among such protocols, only a few biochemistry-based approaches have been use for naturally occurring peptide sequences. The development of chemistry-based protocols applicable to natural sequences remains a challenge, and the development of such methods would be a major contribution to protein science. Here, we describe the preparation of peptide thioesters using innovative methodology that features nickel(II)-mediated alcoholysis of a naturally occurring peptide sequence, followed by O−N and N−S acyl transfers. This protocol involves sequential quadruple acyl transfer, termed SQAT. Notably, the SQAT system consists of sequential chemical reactions that allow naturally occurring peptide sequences to be converted to thioesters without requiring an artificial chemical unit.     

The reduction of oxidized methionine residues in peptide thioesters with NH4I-Me2S

Oxidized methionine residues in peptide thioesters can be reduced rapidly with NH4I to the corresponding sulfide by using Me2S as coreductant. Comparative reduction studies employing a 28-amino acid peptide thioester with an N-terminal methionine oxide as model system revealed the importance of the Me2S addition to avoid hydrolysis of the reactive thioester functionality. In addition, an NH4I-Me2S containing cleavage cocktail has been used for the global deprotection of various thioesters which revealed no hydrolysis or oxidative side products. These results demonstrate the general applicability of sulfoxides as protecting groups in advanced peptide synthesis techniques by facilitating the preparation and handling of methionine containing peptide thioesters for native chemical ligation (NCL).     

Fmoc solid-phase synthesis of peptide thioesters using an intramolecularn,S-acyl shift

[reaction: see text] We describe the Fmoc solid-phase synthesis of peptide thioesters based on the alkylation of the safety-catch sulfonamide linker with a protected 2-mercaptoethanol derivative. The thioester is generated on the solid phase after the peptide chain assembly as a consequence of an intramolecular N,S-acyl shift. Depending on the stability of the spacer separating the sulfonamide linker from the resin toward TFA, treatment of the peptidyl resin with TFA led to a soluble or supported deprotected thioester.     

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     

Solid-phase synthesis of peptide and glycopeptide thioesters through side-chain-anchoring strategies

An efficient new strategy for the synthesis of peptide and glycopeptide thioesters is described. The method relies on the side-chain immobilization of a variety of Fmoc-amino acids, protected at their C-termini, on solid supports. Once anchored, peptides were constructed using solid-phase peptide synthesis according to the Fmoc protocol. After unmasking the C-terminal carboxylate, either thiols or amino acid thioesters were coupled to afford, after cleavage, peptide and glycopeptide thioesters in high yields. Using this method a significant proportion of the proteinogenic amino acids could be incorporated as C-terminal amino acid residues, therefore providing access to a large number of potential targets that can serve as acyl donors in subsequent ligation reactions. The utility of this methodology was exemplified in the synthesis of a 28 amino acid glycopeptide thioester, which was further elaborated to an N-terminal fragment of the glycoprotein erythropoietin (EPO) by native chemical ligation.     

Facile, fmoc-compatible solid-phase synthesis of peptide C-terminal thioesters

A short route to peptide C-terminal thioesters was developed that does not require the use of special linkers or resins and is compatible with standard Fmoc chemistry. Following conventional solid-phase peptide synthesis, an excess of Me(2)AlCl and EtSH in dichloromethane cleaves peptides from Wang or Pam resins to give the corresponding thioesters directly in good yield and purity.     

Fmoc solid-phase synthesis of peptide thioesters by masking as trithioortho esters

[reaction: see text] Total chemical synthesis of proteins by chemoselective ligation relies on C-terminal peptide thioesters as building blocks. Their preparation by standard Fmoc solid-phase peptide synthesis is made difficult by the lability of thioesters to aminolysis by the secondary amines used for removal of the Fmoc group. Here we present a novel backbone amide linker (BAL) strategy for their synthesis in which the thioester functionality is masked as a trithioortho ester throughout the synthesis.     

Fmoc-SPPS chemistry compatible approach for the generation of (glyco)peptide aryl thioesters

A new approach is described for the general Fmoc-based solid-phase synthesis of (glyco)peptide aryl thioesters. A peptide alkyl oxoester obtained by standard Fmoc-based chain elongation undergoes an O-to-S acyl shift, and is followed by alkyl thioester exchanges with a large excess of aryl thiol, affording the corresponding peptide aryl thioester. The newly developed methodology is useful for the chemical synthesis of post-translationally modified proteins because of its compatibility with standard Fmoc-SPPS conditions. In addition, the peptide aryl thioesters are essential intermediates for chemical synthesis of proteins by kinetically controlled convergent strategy.     

HF‐Free Boc Synthesis of Peptide Thioesters for Ligation and Cyclization

We have developed a convenient method for the direct synthesis of peptide thioesters, versatile intermediates for peptide ligation and cyclic peptide synthesis. The technology uses a modified Boc SPPS strategy that avoids the use of anhydrous HF. Boc in situ neutralization protocols are used in combination with Merrifield hydroxymethyl resin and TFA/TMSBr cleavage. Avoiding HF extends the scope of Boc SPPS to post‐translational modifications that are compatible with the milder cleavage conditions, demonstrated here with the synthesis of the phosphorylated protein CHK2. Peptide thioesters give easy, direct, access to cyclic peptides, illustrated by the synthesis of cyclorasin, a KRAS inhibitor.     

Letter: collision-induced dissociation of peptide thioesters: influence of the peptide length on the fragmentation

Five peptide thioesters of increasing length were fragmented under two processes, in-source and in- collision cell fragmentation, using an electrospray source coupled to a triple quadrupole. Comparison of their fragmentations was made in regard to the length. The two fragmentation conditions show that the peptide length has no influence on structural information and that the fragmentation efficiency is higher for the smallest peptides than for the longest. The particularity of these peptide thioesters consists on the neutral loss of ethanethiol. The absence of the a3 fragment ion and the presence of the (a3-17) ion on the CID mass spectra are noted.     

Peptide thioester 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).     

Peptide o‐Aminoanilides as Crypto‐Thioesters for Protein Chemical Synthesis

Fully unprotected peptide o‐aminoanilides can be efficiently activated by NaNO₂ in aqueous solution to furnish peptide thioesters for use in native chemical ligation. This finding enables the convergent synthesis of proteins from readily synthesizable peptide o‐aminoanilides as a new type of crypto‐thioesters. The practicality of this approach is shown by the synthesis of histone H2B from five peptide segments. Purification or solubilization tags, which are sometimes needed to improve the efficiency of protein chemical synthesis, can be incorporated into the o‐aminoanilide moiety, as demonstrated in the preparation of the cyclic protein lactocyclicin Q.     

Leveraging the Knorr Pyrazole Synthesis for the Facile Generation of Thioester Surrogates for use in Native Chemical Ligation

Facile synthesis of C‐terminal thioesters is integral to native chemical ligation (NCL) strategies for chemical protein synthesis. We introduce a new method of mild peptide activation, which leverages solid‐phase peptide synthesis (SPPS) on an established resin linker and classical heterocyclic chemistry to convert C‐terminal peptide hydrazides into their corresponding thioesters via an acyl pyrazole intermediate. Peptide hydrazides, synthesized on established trityl chloride resins, can be activated in solution with stoichiometric acetyl acetone (acac), readily proceed to the peptide acyl pyrazoles. Acyl pyrazoles are mild acylating agents and are efficiently exchanged with an aryl thiol, which can then be directly utilized in NCL. The mild, chemoselective, and stoichiometric activating conditions allow this method to be utilized through multiple sequential ligations without intermediate purification steps.     

Stereoselective Metal‐Free Synthesis of β‐Amino Thioesters with Tertiary and Quaternary Stereogenic Centers

β‐Amino thioesters are important natural building blocks for the synthesis of numerous bioactive molecules. An organocatalyzed Mannich reaction was developed which provides direct and highly stereoselective access to acyclic β²‐ and β^2,3,3‐amino thioesters with adjacent tertiary and quaternary stereocenters. Mechanistic studies showed that the stereochemical course of the reaction can be controlled by the choice of the substrates. The β‐amino thioesters were further functionalized by, for example, stereoselective decarboxylation to access β^2,3‐frameworks. In addition, the value of the β‐amino thioesters was shown in coupling‐reagent‐free peptide synthesis.     

Solid-phase synthesis of peptide thioacids through hydrothiolysis of resin-bound peptide thioesters

We report that solid-phase hydrothiolysis is an efficient method to convert resin-bound peptide thioesters to thioacids in aqueous buffer by using a total PEG-based resin. Also demonstrated is the use of the so-prepared peptide thioacids in chemoselective amide bond formation reactions.     

Selenocysteine‐Mediated Native Chemical Ligation

C‐Terminal peptide thioesters are shown to react efficiently with peptide fragments containing an N‐terminal selenocysteine to give selenoproteins. In analogy to the native chemical ligation of thioesters and peptides containing N‐terminal cysteines, the selenol presumably attacks the thioester nucleophilically to give a selenoester intermediate that subsequently rearranges to give a native chemical bond. The utility of this procedure was demonstrated by the synthesis of a selenium‐containing derivative of bovine pancreatic trypsin inhibitor (BPTI) in which Cys³⁸ is replaced by selenocysteine. The artificial selenoprotein folds into a conformation similar to that of wild‐type BPTI and inhibits trypsin and chymotrypsin with unaltered affinity.     

Cysteine-derived s-protected oxazolidinones: potential chemical devices for the preparation of peptide thioesters

[reaction: see text]. An N-S acyl-transfer-mediated preparation of peptide thioesters using the S-protected oxazolidinone derived from cysteine has been developed and applied to the synthesis of a 32-mer biologically active peptide by native chemical ligation protocols.