Total synthesis of snake toxin α-bungarotoxin and its analogues by hydrazide-based native chemical ligation

Nicotinic acetylcholine receptors(nAChRs) play important roles in intercellular communications of nerve cells. α-Bungarotoxins(αBtx) is a moderator for the nAChRs. Chemical synthesis provides a promising way to access aBtx and their analogues. Here, we reported a new method for a-bungarotoxin by combining Fmoc-SPPS and peptide hydrazide based ligation strategy. The two-segment ligation method may enable efficient synthesis of aBtx analogues. These synthetic toxin peptides are useful tools for development of imaging or therapeutic reagents.

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

The H3 bivalent modifications of trimethylationat Lys9 and acetylation at Lys18(H3-K9 Me3-K18 Ac) 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-K9 Me3-K18 Ac were indispensable samples.Herein we developed a pseudo dipeptide strategy to efficiently prepare peptide segments,facilitating the chemical synthesis of H3-K9 Me3-K18 Ac at a tens of milligram scale.The synthetic H3-K9 Me3-K18 Ac 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.

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 factorκB(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 describe the total synthesis of TRADD death domain(residues 195-312)with arginine235 NGlcNAcylation(Arg-GIcNAc TRADD(195-312)).Two longish peptidyl fragments of the wild-type primary sequence were obtained by robust,microwave-assisted,highly efficient,solid-phase peptide synthesis(SPPS),the N-GlcNAcylated sector was built by total synthesis and attached specifically to resinbound peptide with an unprotected ornithine residue via silver-promoted on-resin guanidinylation,ArgGlcNAc TRADD(195-312)was constructed by hydrazide-based native chemical ligation(NCL).The facile synthetic strategy is expected to be generally applicable for the rapid synthesis of other proteins with Arg-GIcNAc modification and to pave the way for the related chemically biological study.

Chemical protein synthesis elucidates key modulation mechanism of the tyrosine-O-sulfation in inducing strengthened inhibitory activity of hirudin

Tyrosine sulfation is an important post-translational modification that enhances the inhibitory activity of hirudin.Herein,we developed a facile synthetic strategy to afford the sulfated hirudins with up to three modifications and in multi-milligram scales,after a single HPLC purification step.Through these synthetic proteins,a novel type of modulation mechanism exhibited by tyrosine sulfation was proposed,which would help to delineate the structure-function relationships in other sulfated proteins and more importantly,to serve as a basis for the development of related antithrombotic agents.

Biochemical properties of K_(11,48)-branched ubiquitin chains

As one of the most widely existing post-translational modification models, ubiquitination regulates diverse cellular activities. In eukaryotes, K-branched ubiquitin chains play key roles in cell cycle and protein quality control. However, the structural and biochemical properties of K-branched ubiquitin chains have not been well examined. Here we employed the synthetic linkage-and length-defined K-branched ubiquitin chains to examine their binding and hydrolysis properties in vitro. Quantitatively affinity determination of ubiquitin chains to the proteasome ubiquitin receptor S5 a indicated that the S5 a exhibited preference binding to K-branched chains over K-linked chains, but not K-conjugated chains. In addition, deubiquitination experiments were carried out and the results showed that K-branched chains were preferably hydrolyzed by proteasome-associated deubiquitinase Rpnll than homotypic Kor K-linked chains.

Mechanism of Imidazole-Promoted Ligation of Peptide Phenyl Esters

Imidazole-promoted ligation of peptide phenyl esters was recently found to be a complementary method for protein chemical synthesis. Theoretical calculations have been carried out to understand the detailed mechanism of this particular ligation process. It is found that both the reaction of the phenyl ester with imidazole and the reaction of the acyl imidazole intermediate with cysteine proceed through an addition-elimination mechanism. The cleavage of the C--O bond in the reaction between the phenyl ester and imidazole is the rate-limiting step of the overall liga- tion process. Interestingly, although the imidazole-promoted phenyl ester ligation has a higher free energy barrier than the conventional thiophenol-promoted native chemical ligation for a sterically less hindered C-terminal amino acid （e.g. gylcine）, for a sterically hindered C-terminal amino acid （e.g. proline） the imidazole-promoted phenyl ester ligation is calculated to be more favorable than the conventional thiophenol-promoted native chemical ligation.

On-resin peptide ligation via C-terminus benzvl ester

Here, we report a new approach of on-resin peptide ligation using C-terminal benzyl ester as the stabilized precursor of thioester, which enables both N-terminal elongation and C-terminal peptide ligation on a Rink Amide resin. On-resin native chemical ligation and auxiliary-assisted peptide ligation were successfully achieved. This method is compatible to both protected and unprotected peptide fragments and has potential application in poor water-soluble peptide ligation.

Insertion of unnatural metal ligand in the heme pocket of nitrophorin through protein semi-synthesis:Toward biomimicking binuclear active sites

Herein,we report a semi-synthetic strategy affording a nitrophorin 2(NP2)variant with a N,N'-bis(2-pyridylmethyl)amine(Dpa)ligand as sidechain selectively installed at position 27,which was assembled from a synthetic peptide thioester bearing the Dpa ligand and an expressed protein segment via native chemical ligation.The semi-synthetic NP2 was able to accept the natural heme b cofactor and the Dpa ligand was able to bind Cu(Ⅱ)/Fe(Ⅲ)ions,leading to heteronuclear active site.

Synthetic studies toward human interleukin-5

Interleukin 5(IL-5) is a human cytokine that regulates eosinophil growth and activation, and it plays essential roles in diseases associated with increased level of eosinophils such as asthma. Many studies have been conducted on this important glycoprotein, but all of them utilized recombinantly expressed samples. Here we describe our attempted chemical synthesis of IL-5, using protocols of Fmoc-SPPS and peptidyl hydrazide-based native chemical ligation, where the disulfide-reduced form of IL-5 is assembled from three peptide segments. Reconstitution of the protein under different folding conditions has also been investigated.

Mechanism of N-to-S acyl transfer of N-(2-hydroxybenzyl) cysteine derivatives and origin of phenol acceleration effect

N-（2-Hydroxybenzyl）cysteine derivatives were recently disclosed to be efficient crypto-thioesters for native chemical ligation（NCL）. To elucidate the mechanism of the relevant N-to-S acyl transfer process as well as the origin of the acceleration effect of the phenol substitutes, a density functional theory（DFT）study was performed. It was found that the N-to-S acyl transfer of N-（2-hydroxybenzyl）cysteine derivatives involve four major steps： concerted nucleophilic addition of thiolate/proton transfer,inversion of an amine moiety, water-assisted proton transfer and CààN bond cleavage. The phenol substitutes promote the nucleophilic addition of thiolate by protonating the carbonyl oxygen atom synergistically and the proton transfer from hydroxyl to amide nitrogen atom is the rate-determining step of the N-to-S acyl transfer. By contrast, changing the phenolic hydroxyl to methoxyl was found to significantly slow down the nucleophilic addition of thiolate and thus hinders the N-to-S acyl transfer overall. These computational results are consistent with the observation of previously reported control experiments, by which our proposed mechanism is further validated.

Theoretical investigations on the thiol–thioester exchange steps of different thioesters

As the rate-determining step in native chemical ligation reactions, the thiol–thioester exchange step is important in determining the efficiency of the ligations of peptides. In the present study, systematic theoretical calculations were carried out on the relationships between the structure of different thioesters and the free energy barriers of the thiol–thioester exchange step. According to the calculation results, the thiol–thioester exchange step is disfavored by the steric hindrance around the carbonyl center, while the electronic effect（i.e. conjugation and hyper-conjugation effects） becomes important when the steric hindrance is insignificant.