Efficient Chemical Protein Synthesis using Fmoc‐Masked N‐Terminal Cysteine in Peptide Thioester Segments

We report an operationally simple method to facilitate chemical protein synthesis by fully convergent and one‐pot native chemical ligations utilizing the fluorenylmethyloxycarbonyl (Fmoc) moiety as an N‐masking group of the N‐terminal cysteine of the middle peptide thioester segment(s). The Fmoc group is stable to the harsh oxidative conditions frequently used to generate peptide thioesters from peptide hydrazide or o‐aminoanilide. The ready availability of Fmoc‐Cys(Trt)‐OH, which is routinely used in Fmoc solid‐phase peptide synthesis, where the Fmoc group is pre‐installed on cysteine residue, minimizes additional steps required for the temporary protection of the N‐terminal cysteinyl peptides. The Fmoc group is readily removed after ligation by short exposure (<7 min) to 20 % piperidine at pH 11 in aqueous conditions at room temperature. Subsequent native chemical ligation reactions can be performed in presence of piperidine in the same solution at pH 7.     

Peptide dithiodiethanol esters for in situ generation of thioesters for use in native ligation

A new approach is described for the general Fmoc-based solid-phase synthesis of C-terminal peptide (thio)esters. One hydroxy group of 2,2-dithiodiethanol (used in large excess) was anchored on trityl resin, and the remaining hydroxy group was loaded with the first amino acid. Standard chain elongation and TFA-based peptide release yielded peptide C-terminal dithiodiethanol esters in good purities. Under standard conditions of native chemical ligation (excess thiol, neutral pH), the dithiodiethanol function is presumably reduced and rearranged (or equilibrated) to the thioester via a 5-membered intermediate. The resulting thioesters are shown to undergo native chemical ligation with N-terminal cysteine peptides. Notably, hydrolysis of the reduced ester is a major competing reaction, especially in the presence of 6 M guanidinium chloride, which is often required for solubilization of large peptide fragments.     

Chemical synthesis of a cyclotide via intramolecular cyclization of peptide O-esters

Cyclotides constitute a fascinating family of circular proteins containing ca.30 amino acid residues.They have a unique cyclic cysteine knot topology and exhibit remarkable thermal,chemical and enzymatic stabilities.These characteristics enable them to have a range of biological activities and promising pharmaceutical and agricultural applications.Here,we present a practical strategy for the chemical synthesis of cyclotides through the intramolecular ligation of fully unprotected peptide O-esters.This strategy involves the mild Fmoc solid-phase peptide synthesis of the peptide O-ester backbone,the head-to-tail cyclization of the cyclotide backbone by native chemical ligation,and the oxidative refolding to yield the natural knot protein.The simplicity and high efficiency of the strategy can be employed in the synthesis of artificial cyclotides for pharmaceutical applications.     

A novel ring opening reaction of peptide N-terminal thiazolidine with 2,2′-dipyridyl disulfide (DPDS) efficient for protein chemical synthesis

In the protein chemical synthesis via native chemical ligation (NCL) method with three peptide segments, the N-terminal cysteine residue of middle segment is generally protected by thiazolidine ring. In this paper, we show the novel method for thiazolidine ring opening using 2,2′-dipyridyl disulfide (DPDS). The N-terminal thiazolidine was converted into S-pyridylsulfenylated cysteine residue with DPDS under acidic conditions, and this N-terminally Cys peptide protected with disulfide was applicable to NCL reaction without purification and deprotection steps. DPDS treatment did not remove other Cys protecting groups generally used for regioselective disulfide bond formation reactions. These results indicate that this thiazolidine ring opening reaction is quite useful for the protein chemical synthesis with three-segment NCL strategy.     

半胱氨酸肽片段连接法合成ω-芋螺毒素MVIIA

ω-芋螺毒素MVIIA是已上市的镇痛药Ziconotide的有效成分.采用标准Fmoc保护策略在聚苯乙烯树脂上合成ω-MVIIA比较困难,是固相合成中的"困难肽".本研究将ω-MVIIA分为N-端15肽硫酯和C-端10肽两个片段采用标准Fmoc保护策略分别合成,再通过半胱氨酸肽片段连接得到全长的ω-芋螺毒素MVIIA肽链.该方法提高了合成ω-芋螺毒素MVIIA产率.该研究为"困难肽"的合成提供了较好的参考方法.     

Efficient Palladium‐Assisted One‐Pot Deprotection of (Acetamidomethyl)Cysteine Following Native Chemical Ligation and/or Desulfurization To Expedite Chemical Protein Synthesis

The acetamidomethyl (Acm) moiety is a widely used cysteine protecting group for the chemical synthesis and semisynthesis of peptide and proteins. However, its removal is not straightforward and requires harsh reaction conditions and additional purification steps before and after the removal step, which extends the synthetic process and reduces the overall yield. To overcome these shortcomings, a method for rapid and efficient Acm removal using Pd^II complexes in aqueous medium is reported. We show, for the first time, the assembly of three peptide fragments in a one‐pot fashion by native chemical ligation where the Acm moiety was used to protect the N‐terminal Cys of the middle fragment. Importantly, an efficient synthesis of the ubiquitin‐like protein UBL‐5, which contains two native Cys residues, was accomplished through the one‐pot operation of three key steps, namely ligation, desulfurization, and Acm deprotection, highlighting the great utility of the new approach in protein synthesis.     

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).     

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     

Bis(2-sulfanylethyl)amino native peptide ligation

The reaction of a peptide featuring a bis(2-sulfanylethyl)amino (SEA) group on its C-terminus with a cysteinyl peptide in water at pH 7 and 37 °C leads to the chemoselective and regioselective formation of a native peptide bond. This method called SEA ligation enriches the native peptide ligation repertoire available to the peptide chemist. Preparation of an innovative solid support which allows the straightforward synthesis of peptide SEA fragments using standard Fmoc/tert-butyl solid phase peptide synthesis procedures is also described.     

Recent advances in the preparation of Fmoc-SPPS-based peptide thioester and its surrogates for NCL-type reactions

Solid phase peptide synthesis (SPPS) based on Fmoc chemistry has become a commonly used technique in peptide chemistry, as it can be easily conducted using automated machine, and not requiring highly toxic HF in comparison to Boc-SPPS. With the fast development in the emerging field of protein chemical synthesis, many efforts have been endeavored aiming to find more efficient methods for preparing peptide fragments required in ligation reactions. This review briefly summarizes recent advances in the engineering and modification of Fmoc-SPPS-derived peptides, which can be used as the N-terminal fragments in a native chemical ligation (NCL) or NCL-type ligation 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.     

A technique for the synthesis of highly-pure, mono-epitopic, multi-valent lipid core peptide vaccines

The synthesis of lipid core peptide (LCP) vaccines using stepwise solid-phase peptide synthesis commonly results in products which are difficult to purify to homogeneity. A new technique for synthesizing highly-pure, mono-epitopic, multi-valent LCP-systems using native chemical ligation is presented. Various conditions were assessed for ligating four copies of a thioester-modified 88/30 serotype group A streptococcal peptide antigen onto an LCP-system featuring four cysteine residues. Overall, the vaccine was synthesized in high purity (>99%), and high yield (90%) when the ligation reaction was performed in the presence of 1% sodium dodecyl sulfate and at elevated temperatures (37 °C).     

On-resin native chemical ligation for cyclic peptide synthesis

A novel cysteine derivative, N(alpha)-trityl-S-(9H-xanthen-9-yl)-l-cysteine [Trt-Cys(Xan)-OH] has been introduced for peptide synthesis, specifically for application to a new strategy for the preparation of cyclic peptides. The following steps were carried out to synthesize the cyclic model peptide cyclo(Cys-Thr-Abu-Gly-Gly-Ala-Arg-Pro-Asp-Phe): (i). side-chain anchoring of Fmoc-Asp-OAl via its free beta-carboxyl as a p-alkoxybenzyl ester to a solid support; (ii). stepwise chain elongation of the peptide by standard Fmoc/tBu solid-phase chemistry; (iii). removal of the N-terminal Fmoc group; (iv). coupling of Trt-Cys(Xan)-OH; (v). selective Pd(0)-promoted cleavage of the C-terminal allyl ester; (vi). coupling of the C-terminal residue, i.e., H-Phe-SBzl, preactivated as a thioester; (vii). selective removal of the N(alpha)-Trt and S-Xan protecting groups under very mild acid conditions; (viii). on-resin cyclization by native chemical ligation in an aqueous milieu; and (ix). final acidolytic cleavage of the cyclic peptide from the resin. The strategy was evaluated for three supports: poly[N,N-dimethacrylamide-co-poly(ethylene glycol)] (PEGA), cross-linked ethoxylate acrylate resin (CLEAR), and poly(ethylene glycol)-polystyrene (PEG-PS) graft resin supports. For PEGA and CLEAR, the desired cyclic product was obtained in 76-86% overall yield with initial purities of approximately 70%, whereas for PEG-PS (which does not swell nearly as well in water), results were inferior. Solid-phase native chemical ligation/cyclization methodology appears to have advantages of convenience and specificity, which make it promising for further generalization.     

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.     

Hydrazine hydrate: A new reagent for Fmoc group removal in solid phase peptide synthesis

In solid-phase peptide synthesis using the Fmoc/tBu strategy (SPPS-Fmoc/tBu), an orthogonal protection scheme of amino acids is used; specifically, the alpha-amine group is protected by the 9-fluorenylmethyloxycarbonyl (Fmoc) group, which is removed by weak bases, while side chains are protected by groups that are acid labile. We demonstrated that hydrazine hydrate is an efficient reagent for eliminating the Fmoc group in SPPS-Fmoc/tBu. First, experimental conditions were established for Fmoc group removal from Fmoc-Val-OH in solution. It was determined that the Fmoc group was completely removed with 16% hydrazine hydrate in DMF after 60?min at rt. Second, SPPS-Fmoc/tBu using hydrazine hydrate for Fmoc group removal was standardized. The Fmoc group removal was completed using 16% hydrazine hydrate in DMF for 10?min at rt (twice). When the reaction of Fmoc group removal was microwave-assisted, the reaction only required 30?s to efficiently remove the Fmoc group in SPPS-Fmoc/tBu. The method reported here can be routinely used, and it is equivalent to conventional SPPS-Fmoc/tBu methodologies where 4-methylpiperidine or piperidine is used.     

Enhancing Auxiliary-Mediated Native Chemical Ligation at Challenging Junctions with Pyridine Scaffolds

To expand the scope of native chemical ligation (NCL) beyond reactions at cysteine, ligation auxiliaries are appended to the peptide N-terminus. After the introduction of a pyridine-containing auxiliary, which provided access to challenging junctions (proline or β-branched amino acids), we herein probe the role of the pyridine-ring nitrogen. We observed side reactions leading to preliminary auxiliary loss. We describe a new easy to attach β-mercapto-β-(4-methoxy-2-pyridinyl)-ethyl (MMPyE) auxiliary, which 1) has increased stability; 2) enables NCL at sterically encumbered junctions (e. g., Leu-Val); and 3) allows removal under mildly basic (pH 8.5) conditions was introduced. The synthesis of a 120 aa long peptide containing eight MUC5AC tandem repeats via ligation of two 60mers demonstrates the usefulness. Making use of hitherto unexplored NCL to tyrosine, the MMPyE auxiliary provided access to a head-to-tail-cyclized 21-mer peptide and a His₆-tagged hexaphosphorylated peptide comprising 6 heptapeptide repeats of the RNA polymerase II C-terminal domain.     

Design of thiol-containing amino acids for native chemical ligation at non-Cys sites

<正>Protein chemical synthesis usually relies on the use of native chemical ligation that couples peptide thioester with a Cys-peptide.A limitation of this method is the difficulty of finding an appropriate Cys ligation site in many synthetic targets.To overcome this problem,the ligation-desulfurization approach has been developed.This approach involves the use of a thiol-containing amino acid as the ligation partner.After the sequence assembly is completed,the thiol group is removed through a desulfurization reaction to generate the standard amino acids.Currently this strategy has been applied to the ligations at a number of amino acids including Ala,Phe,Val,Lys,Thr,Leu,Pro and Gln.The present article reviews the design and synthesis of these thiol-containing amino acids for native chemical ligation at non-Cys sites.     

Synthesis of a selenocysteine-containing peptide by native chemical ligation

[reaction in text] A new method for the synthesis of selenocysteine derivatives and selenocysteine-containing peptides is described. Fmoc-Se-p-methoxybenzylselenocysteine (1) was prepared and used for solid-phase synthesis of peptides with an N-terminal unprotected selenocysteine. Subsequent native chemical ligation with a peptide thioester provided a 17-mer that corresponds to the C-terminus of ribonucleotide reductase with selenocysteine in place of cysteine.     

A New Base-Labile Anchoring Group for Polymer-Supported Peptide Synthesis

A new base-labile anchoring group, derived from 9-(hydroxymethyl)fluorene-4-carboxylic acid (HO₂CFmoH or HOFmCO₂H; 7), for polymer-supported peptide synthesis os described. The synthesis of 7 starting from 2,2′-biphenyldicarboxylic acid ( 1 ) proceeds in an overall yield of 53%. The group HO₂CFmo exhibits properties similar to the well known Fmoc protecting group: It is stable to acidic conditions and cleavable by 15% piperidine in DMF. In combination with acid labile N^α-protecting groups (e.g. Boc, Ddz, Bpoc, Nps etc.), it renders more flexibility to the stepwise synthesis using polymer supports. The versatility of the new anchoring group in solid- and liquid-phase peptide synthesis is demonstrated for the synthesis of a model peptide.     

Synthetic procedure for N-fmoc amino acyl-N-sulfanylethylaniline linker as crypto-Peptide thioester precursor with application to native chemical ligation

N-Sulfanylethylanilide (SEAlide) peptides 1, obtainable using Fmoc-based solid-phase peptide synthesis (Fmoc SPPS), function as crypto-thioesters in native chemical ligation (NCL), yielding a wide variety of peptides/proteins. Their acylating potential with N-terminal cysteinyl peptides 2 can be tuned by the presence or absence of phosphate salts, leading to one-pot/multifragment ligation, operating under kinetically controlled conditions. SEAlide peptides have already been shown to be promising for use in protein synthesis; however, a widely applicable method for the synthesis of N-Fmoc amino acyl-N-sulfanylethylaniline linkers 4, required for the preparation of SEAlide peptides, is unavailable. The present study addresses the development of efficient condensation protocols of 20 naturally occurring amino acid derivatives to the N-sulfanylethylaniline linker 5. N-Fmoc amino acyl aniline linkers 4 of practical use in NCL chemistry, except in the case of the proline- or aspartic acid-containing linker, were successfully synthesized by coupling of POCl(3)- or SOCl(2)-activated Fmoc amino acid derivatives with sodium anilide species 6, without accompanying racemization and loss of side-chain protection. Furthermore, SEAlide peptides 7 possessing various C-terminal amino acids (Gly, His, Phe, Ala, Asn, Ser, Glu, and Val) were shown to be of practical use in NCL chemistry.