Facile peptide thioester synthesis via solution-phase tosylamide preparation

Preparation of peptide thioester is essential for native chemical ligation and block condensation. Our novel methodology involves conversion of the carboxylic acid of a peptide into a thioester using p-toluenesulfonyl isocyanate, followed by alkylation, then thiol substitution. Our methodology can also be used for the preparation of glycopeptide thioesters. Furthermore, it is possible to carry out the reaction as a sequential peptide chemical ligation.     

N-Alkyl cysteine-assisted thioesterification of peptides

A new method for the preparation of peptide thioester by the post-solid phase peptide synthesis (SPPS) approach was developed. A series of N-alkyl cysteine derivatives were prepared and used as the C-terminus residue of the peptides prepared by the Fmoc SPPS. The synthetic peptides released from resin by TFA were readily converted to the peptide thioester in aqueous 3-mercaptopropionic acid (MPA) without significant side reactions.     

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.     

An efficient peptide ligation using azido-protected peptides via the thioester method

Azido-protected Fmoc-Lys-OH (Fmoc-Lys(N₃)-OH) was synthesized from Fmoc-Lys-OH by the copper(II)-catalyzed diazo transfer method, and introduced to a peptide by the ordinary Fmoc-based solid-phase peptide synthesis. This azido peptide could be condensed with a peptide thioester by the Ag⁺-free thioester method without any significant side reactions. The azido group was easily reduced to an amino group by Zn powder after peptide condensation.     

The first synthesis of peptide thioester carrying N-linked core pentasaccharide through modified Fmoc thioester preparation: synthesis of an N-glycosylated Ig domain of emmprin

Peptide thioester carrying N-linked core pentasaccharide was prepared by the Fmoc solid-phase method with a combination of the benzyl-protection strategy at the carbohydrate portion. The obtained peptide thioester was successfully used for the synthesis of the first Ig domain of emmprin composed of 61 amino acid residues.     

Application of a novel thioesterification reaction to the synthesis of chemokine CCL27 by the modified thioester method

Aryl thioesters of peptide segments were prepared by the conventional 9-fluorenylmethoxycarbonyl (Fmoc) strategy using a novel N-alkyl cysteine (NAC)-assisted thioesterification reaction. The peptide carrying NAC at its C-terminus was prepared by the Fmoc strategy and converted to the aryl thioester by 4-mercaptophenylacetic acid (MPAA) treatment without significant side reactions. The peptide thioester was used for the efficient preparation of 95-amino acid (AA) chemokine CCL27 by an Ag(+)-free thioester method.     

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).     

Use of thiosulfonate for the protection of thiol groups in peptide ligation by the thioester method

Use of thiosulfonate for protecting thiol (-SH) groups in peptide ligation by the thioester method was examined. Thiosulfonate was introduced and was stable in the presence of silver ion, 4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine, and diisopropylethylamine. Based on these results, a strategy for using the thioester method and the native chemical ligation method in the synthesis of a single polypeptide is described.     

Total chemical synthesis of large CCK isoforms using a thioester segment condensation approach

Silver-ion mediated thioester segment condensation was applied to the chemical synthesis of high molecular weight isoforms of cholecystokinin (CCK). Three building blocks, a C-terminal Tyr(SO₃H)-containing segment and two partially protected thioester segments having a C-terminal Pro residue, were prepared using Fmoc-based chemistry and 2-chlorotrityl chloride (Clt) resin as a solid support. The entire peptide chain was successfully synthesized by two consecutive silver-ion mediated condensation reactions using these building blocks. A brief TFA treatment of the final condensation product gave highly homogeneous CCK-58 in a satisfactory yield. This peptide exhibited glucose-dependent insulinotropic activity at levels comparable to CCK-33. These results demonstrate the usefulness of the silver-ion mediated segment condensation approach in the preparation of large sulfated peptides.     

RNA-catalyzed thioester synthesis

A series of efficient ribozymes with thioester synthetase activities have been isolated from CoA-linked RNA libraries containing four different lengths (30N, 60N, 100N, and 140N) of random nucleotide regions. Competitive evolution of these size-heterogeneous CoA-RNA libraries resulted in an RNA size population in the order of 30N > 60N > 100N > 140N. From isolated clones in the 30N and 60N size groups, two predominant RNA sequences, TES1 (30N) and TES33 (60N), have been shown to catalyze the synthesis of different thioesters using various acyl adenylates as the substrates. Together with our previous findings, the current results demonstrate a CoA thioester synthetic pathway catalyzed by individual metabolic ribozymes, and suggest a likely mechanism for thioester synthesis and utilization in an RNA world.     

Synthesis of thiazolidine thioester peptides and acceleration of native chemical ligation

Thiazolidine thioester peptides were synthesized by reacting bis(2-sulfanylethyl)amido peptides with glyoxylic acid at pH 1. A significant increase in Native Chemical Ligation (NCL) rate was observed with thiazolidine thioesters compared to 3-mercaptopropionic acid-thioester analogues. The method is of particular interest for accelerating valine-cysteine peptide bond formation.     

Sequential peptide chemical ligation by the thioester method and extended chemical ligation

The sequential chemical ligation of peptide thioesters by a combination of the thioester method and extended chemical ligation using a photoremovable auxiliary, 2-mercapto-1-(2-nitrophenyl)ethyl group, is described. The thiazolidine ring was used as a protecting group for the N-terminal 1,2-aminoethanethiol moiety of the auxiliary in the middle peptide thioester. After the first thioester coupling, the thiazolidine ring was opened by treatment with O-methylhydroxylamine. Second coupling by extended chemical ligation followed by UV irradiation gave the target polypeptide.     

An N-protection free ligation of the peptide thioester and the peptide with an N-alkoxy- or N-aryloxyamino group at its N-terminus

Peptides with an N-alkoxy or N-aryloxy amino acid at their N-terminus were synthesized and successfully ligated with a peptide thioester by silver ion activation under a slightly acidic condition without requiring protection of the side chain amino groups. The N-methoxy group was easily cleaved by the SmI₂ reduction in CH₃OH aq. to obtain the desired peptide with a native peptide bond. This method was successfully applied to the synthesis of the human atrial natriuretic peptide showing the efficiency of the novel ligation.     

Efficient sequential segment coupling using N-alkylcysteine-assisted thioesterification for glycopeptide dendrimer synthesis

A highly pure MUC1-derived glycopeptide dendrimer of 22 kDa was prepared by a sequential segment coupling, achieved by an N-alkylcysteine (NAC)-assisted thioesterification. The glycopeptide having C-terminal NAC was prepared by the Fmoc method and converted to the thioester by 3-mercaptopropionic acid treatment. The thioester was condensed with a lysine trimer carrying NAC to afford tetramer, which was then converted to the thioester. Two tetramers were condensed with ethylenediamine to give the octameric glycopeptide dendrimer.     

Protein thioester synthesis enabled by sortase

Proteins containing a C-terminal thioester are important intermediates in semisynthesis. Currently there is one main method for the synthesis of protein thioesters that relies upon the use of engineered inteins. Here we report a simple strategy, utilizing sortase A, for routine preparation of recombinant proteins containing a C-terminal (α)thioester. We used our method to prepare two different anthrax toxin cargo proteins: one containing an (α)thioester and another containing a D-polypeptide segment situated between two protein domains. We show that both variants can translocate through protective antigen pore. This new method to synthesize a protein thioester allows for interfacing of sortase-mediated ligation and native chemical ligation.     

Facile synthesis of C-terminal peptide hydrazide and thioester of NY-ESO-1 (A39-A68) from an Fmoc-hydrazine 2-chlorotrityl chloride resin

The NY-ESO-1 (A39-A68) peptide hydrazide was prepared through 9-fluorenyl-methoxycarbonyl solid-phase peptide synthesis (Fmoc SPPS) from a new 9-fluorenyl-methoxycarbonyl hydrazine 2-chlorotrityl chloride (Fmoc-hydrazine 2CTC) resin. The new resin was ideal for long-term storage and usage in Fmoc SPPS. Besides, the title peptide hydrazide could be transformed nearly quantitatively into the corresponding peptide thioester, which was both isolable and usable directly in native chemical ligation (NCL).     

Peptidyl N,N-bis(2-mercaptoethyl)-amides as thioester precursors for native chemical ligation

With two β-mercaptoethyl groups on the N, a tertiary amide of structure 1 is always poised for intramolecular thioesterification however it flips about the C-N bond. It is shown that a peptide with such a C-terminal N,N-bis(2-mercaptoethyl)-amide (BMEA) can be used directly for native chemical ligation (NCL). These BMEA peptides are easily prepared with standard Fmoc-solid phase peptide synthesis protocols, thus giving a very convenient access to the thioester components for NCL.     

The lyngbyatoxin biosynthetic assembly line: chain release by four-electron reduction of a dipeptidyl thioester to the corresponding alcohol

In comparison with the large number of nonribosomal peptide synthetases (NRPSs) that release their peptide products by hydrolytic cleavage of the peptide carrier protein (PCP) bound thioester, there are relatively few NRPSs that have been shown to use a nicotinamide cofactor to reduce this PCP-peptidyl thioester to an aldehyde or imine moiety. This work describes the first example of a reductase domain within a NRPS scaffold shown to reduce a PCP-peptidyl thioester to the corresponding primary alcohol, via an aldehyde intermediate, using two equivalents of reduced nicotinamide adenine dinucleotide phosphate (NADPH). By employing a ketone mimic of the aldehyde intermediate, as well as a specifically deuterated NADPH, it was further demonstrated that the pro-S hydride of the cofactor is transferred to the re face of the carbonyl group.     

Backbone thioester exchange: a new approach to evaluating higher order structural stability in polypeptides

An amide bond has been replaced by a thioester in bovine pancreatic polypeptide (bPP) to allow rapid and reversible (dynamic) exchange of the alpha-helical segment with other thiols in solution. We have begun to study the higher order structural stability of bPP by measuring the equilibrium constant of the "backbone thioester exchange" (BTE) reaction. The extent to which the equilibrium (KBTE) favors one set of peptides over the other, which can be easily measured, can be directly correlated to the energy gained from favorable noncovalent interactions that occur between peptide segments on either side of the thioester bond (Kfold).     

Precise Synthesis of Poly(thioester)s with Diverse Structures by Copolymerization of Cyclic Thioanhydrides and Episulfides Mediated by Organic Ammonium Salts

The precise synthesis of poly(thioester)s with diverse structures is still a significant challenge in the polymeric materials field. Herein, we report a novel approach to the synthesis of well‐defined poly(thioester)s by the controlled alternating copolymerization of cyclic thioanhydrides and episulfides induced by simple organic ammonium salts. Both the cation and anion have strong effects on the copolymerization. [PPN]OAc ([PPN]=bis(triphenylphosphine)iminium) with a bulky cation was proven to be efficient in initiating this polymerization, yielding poly(thioester)s with a completely alternating structure, controlled molecular weight, and narrow polydispersity. The poly(thioester) obtained from succinic thioanhydride and propylene sulfide is a typical semicrystalline material, possessing a high refractive index of up to 1.78. Because it uses readily available monomers, this method is expected to open up a new route to poly(thioester)s with diverse structures and properties.