Convergent synthesis of peptide nucleic acids by native chemical ligation

[reaction: see text] A convergent strategy for synthesizing long contiguous PNA by a native chemical ligation-like technique of PNA segment couplings is presented. This approach required the synthesis of a new PNA-monomer featuring a 1-amino-2-thiol group. It is shown that the additional mercaptomethyl group leaves the hybridization properties of PNA ligation products unaffected. Furthermore, rapid and efficient fluorescence labeling of the ligation products is demonstrated.     

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.     

Rapid synthesis of acyl transfer auxiliaries for cysteine-free native glycopeptide ligation

[reaction: see text] Rapid, facile routes to the TFA-cleavable 4,5,6-trimethoxy-2-mercaptobenzyl and 1-(2,4-dimethoxyphenyl)-2-mercaptoethyl classes of auxiliaries for cysteine-free native chemical ligation are described. Rapid synthesis, coupled with mild cleavage conditions will undoubtedly broaden the utility of such auxiliaries, particularly where chemically fragile peptide modifications such as glycosylation are present.     

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.     

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.     

A designed well-folded monomeric four-helix bundle protein prepared by Fmoc solid-phase peptide synthesis and native chemical ligation

The design and total chemical synthesis of a monomeric native-like four-helix bundle protein is presented. The designed protein, GTD-Lig, consists of 90 amino acids and is based on the dimeric structure of the de novo designed helix-loop-helix GTD-43. GTD-Lig was prepared by the native chemical ligation strategy and the fragments (45 residues long) were synthesized by applying standard fluorenylmethoxycarbonyl (Fmoc) chemistry. The required peptide-thioester fragment was prepared by anchoring the free gamma-carboxy group of Fmoc-Glu-allyl to the solid phase. After chain elongation the allyl moiety was orthogonally removed and the resulting carboxy group was functionalized with a glycine-thioester followed by standard trifluoroacetic acid (TFA) cleavage to produce the unprotected peptide-thioester. The structure of the synthetic protein was examined by far- and near-UV circular dichroism (CD), sedimentation equilibrium ultracentrifugation, and NMR and fluorescence spectroscopy. The spectroscopic methods show a highly helical and native-like monomeric protein consistent with the design. Heat-induced unfolding was studied by tryptophan absorbance and far-UV CD. The thermal unfolding of GTD-Lig occurs in two steps; a cooperative transition from the native state to an intermediate state and thereafter by noncooperative melting to the unfolded state. The intermediate exhibits the properties of a molten globule such as a retained native secondary structure and a compact hydrophobic core. The thermodynamics of GuHCl-induced unfolding were evaluated by far-UV CD monitoring and the unfolding exhibited a cooperative transition that is well-fitted by a two-state mechanism from the native to the unfolded state. GTD-Lig clearly shows the characteristics of a native protein with a well-defined structure and typical unfolding transitions. The design and synthesis presented herein is of general applicability for the construction of large monomeric proteins.     

Fmoc-based synthesis of peptide thioesters for native chemical ligation employing a tert-butyl thiol linker

tert-Butyl thioesters display an astonishing stability toward secondary amines in basic milieu, in contrast to other alkyl and aryl thioesters. Exploiting this enhanced stability, peptide thioesters were synthesized in a direct manner, applying a tert-butyl thiol linker for Fmoc-based solid-phase peptide synthesis.     

Total chemical synthesis of bivalently modified H3 by improved three-segment native chemical ligation

The H3 bivalent modifications of trimethylation at Lys9 and acetylation at Lys18 (H3-K9Me3-K18Ac) were identified to collectively recruit TRIM33 in the nodal signaling pathway. To understand the underlying mechanism of TRIM33 recruitment, the nucleosome core particles (NCPs) containing full-length H3-K9Me3-K18Ac were indispensable samples. Herein we developed a pseudo dipeptide strategy to efficiently prepare peptide segments, facilitating the chemical synthesis of H3-K9Me3-K18Ac at a tens of milligram scale. The synthetic H3-K9Me3-K18Ac was then examined by CD spectroscopy, which demonstrated a prominent shift compared to recombinant H3. Finally, bivalently modified NCPs were assembled and verified by gel mobility shift assay with good homogeneity.     

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.     

Total chemical synthesis by native chemical ligation of the all-D immunoglobulin-like domain 2 of Axl

Axl is a tyrosine kinases receptor playing crucial role in several cellular responses. The deregulation of Axl signaling has been associated to many high impact diseases ranging from cancer to multiple sclerosis. We report the successful procedure for the chemical synthesis of the Ig2 domain of Axl, one of the key extracellular regions of the receptor involved in ligand binding. The protein was synthesized in its d-enantiomeric form (D-Axl-2), opening the way to the selection of D-peptides selectively targeting Axl receptor through the mirror-image phage display peptide library screening approach.     

Strategy for the synthesis of multivalent peptide-based nonsymmetric dendrimers by native chemical ligation

A strategy for the synthesis of multivalent peptide-based nonsymmetric dendrimers by native chemical ligation using poly(lysine) dendritic wedges as scaffolds is presented.     

Direct on-resin synthesis of peptide-alphathiophenylesters for use in native chemical ligation

[reaction: see text] A peptide-(alpha)thiophenylester is a key reactant in native chemical ligation. Preformation of the peptide-(alpha)thiophenylester could be useful for enhancing the ligation reaction. We report the direct on-resin preparation of preformed peptide-(alpha)thiophenylesters using a simple and efficient method. The peptide-(alpha)thiophenylester reacted extremely rapidly with a Cys-peptide when compared to the peptide-(alpha)thioalkylester.     

Total synthesis of TRADD death domain with arginine N-GlcNAcylation by hydrazide-based native chemical ligation

TNFR1-associated death domain protein(TRADD) with arginine N-GlcNAcylation is a novel and structurally unique posttranslational modification(PTM) glycoprotein that blocks the formation of death-inducing signaling complex(DISC),orchestrating host nuclear factor κB(NF-κB) signaling in entero-pathogenic Escherichia coli(EPEC)-infected cells.This particular glycosylated modification plays an extremely vital role for the effective colonization and pathogenesis of pathogens in the gut.Herein we descri...