ISSN:1075-2617    

DOI:10.1002/psc.2607

年代:2014

数据来源: WILEY

摘要:

The convergent assembly of peptide fragments by native chemical ligation has revolutionized the way in which proteins can be accessed by chemical synthesis. A variation of native chemical ligation involves the reaction of peptides bearing an N‐terminal selenocysteine residue with peptide thioesters, which proceeds through the same mechanism as the parent reaction. This transformation was first investigated in 2001 for the installation of selenocysteine into peptides and proteins via ligation chemistry. The recent discovery that selenocysteine residues within peptides can be chemoselectively deselenized without the concomitant desulfurization of cysteine residues has led to renewed interest in ligation chemistry at selenocysteine. This review outlines the use of selenocysteine in ligation chemistry as well as recent investigations of chemoselective ligation–deselenization chemistry at other selenol‐derived amino acids that have the potential to greatly expand the number of targets that can be accessed by chemical synthesis. Copyright © 2013 European Peptide Society and John Wiley&Son

ISSN:1075-2617    

DOI:10.1002/psc.2581

年代:2014

数据来源: WILEY

摘要:

Native chemical ligation (NCL) is a powerful method for the convergent synthesis of proteins and peptides. In its original format, NCL between a peptide containing a C‐terminal thioester and another peptide offering an N‐terminal cysteine has been used to enable protein synthesis of unprotected peptide fragments. However, the applications of NCL extend beyond the scope of protein synthesis. For instance, NCL can be put under the control of template molecules. In such a scenario, NCL enables the design of conditional reaction systems in which, peptide bond formation occurs only when a specific third party molecule is present. In this review, we will show how templates can be used to control the reactivity and chemoselectivity of NCL reactions. We highlight peptide and nucleic‐acid‐templated NCL reactions and discuss potential applications in nucleic acid diagnosis, origin‐of‐life studies and gene‐expression‐specific therapies. Copyright © 2014 European Peptide Society and John

ISSN:1075-2617    

DOI:10.1002/psc.2602

年代:2014

数据来源: WILEY

摘要:

Thioamides can be used as photoswitches, as reporters of local environment, as inhibitors of enzymes, and as fluorescence quenchers. We have recently demonstrated the incorporation of thioamides into polypeptides and proteins using native chemical ligation (NCL). In this protocol, we describe procedures for the synthesis of a thioamide precursor and an NCL‐ready thioamide‐containing peptide using Dawson'sN‐acyl‐benzimidazolinone (Nbz) process. We include a description of the synthesis by NCL of a thioamide‐labeled fragment of the neuronal proteinα‐synuclein. Copyright © 2014 European Peptide Society and John Wi

ISSN:1075-2617    

DOI:10.1002/psc.2589

年代:2014

数据来源: WILEY

摘要:

Protein total chemical synthesis enables the atom‐by‐atom control of the protein structure and therefore has a great potential for studying protein function. Native chemical ligation of C‐terminal peptide thioesters with N‐terminal cysteinyl peptides and related methodologies are central to the field of protein total synthesis. Consequently, methods enabling the facile synthesis of peptide thioesters using Fmoc‐SPPS are of great value. Herein, we provide a detailed protocol for the preparation ofbis(2‐sulfanylethyl)amino polystyrene resin as a starting point for the synthesis of C‐terminalbis(2‐sulfanylethyl)amido peptides and of peptide thioesters derived from 3‐mercaptopropionic acid. Copyright © 2013 European Peptide Society and Jo

ISSN:1075-2617    

DOI:10.1002/psc.2580

年代:2014

数据来源: WILEY

摘要:

Boc‐solid phase peptide synthesis is useful for the preparation of peptide‐α‐thioesters. However, this strategy usually employs hydrogen fluoride for the final deprotection step. These strongly acidic conditions cannot be applied for the synthesis of acid‐labile glycopeptide‐α‐thioesters. The protocol presented here is a modifiedin situ neutralizationBoc‐solid phase peptide synthesis employing 10% sulfuric acid/dioxane conditions for intermediate Boc removal and TfOH for the final side‐chain deprotection step. These conditions were found to be applicable for the synthesis of acid‐labile glycopeptide‐α‐thioesters. In this protocol, a glycopeptide is synthesized asα‐thioester on a thiol linker, and the product glycopeptide‐α‐thioester is released from the resin by thiolysis after side‐chain deprotection step with an acid cocktail containing TfOH instead of hydrogen fluoride. Copyright © 2014 European Pepti

ISSN:1075-2617    

DOI:10.1002/psc.2608

年代:2014

数据来源: WILEY

摘要:

The ligation of peptide hydrazides at a Gly site carrying a removal auxiliary was found to be an efficient process. This technology was successfully used for the synthesis of ubiquitin C‐terminal conjugates. Recombinant Ub(1–75)‐NHNH2was prepared through the hydrozinolysis of the Ub(1–75)‐intein fusion protein. It was ligated with a glycine derivative modified with an acid‐sensitive thiol auxiliary. The final acid treatment produced the desired bioactive ubiquitin conjugates in practical quantities. Thus, the method described here extends the protocols of expressed protein ligation. Copyright © 2013 European Peptide Society and John Wil

ISSN:1075-2617    

DOI:10.1002/psc.2568

年代:2014

数据来源: WILEY

摘要:

Peptide cyclization via chemoselective reactions between side chains has proven a useful strategy to control folded structure. We report here a method for the synthesis of side‐chain to side‐chain cyclic peptides based on the intermolecular reaction between a linear peptide functionalized with two aminooxy or hydrazide side chains and an organic dialdehyde linker. A family of oxime‐based and hydrazone‐based cyclic products is prepared in a modular and convergent fashion by combination of unprotected linear peptide precursors and various small molecule linkers in neutral aqueous buffer. The side‐chain to side‐chain linkages that result can alter peptide folding behavior. The dynamic covalent nature of the Schiff bases in the cyclic products can be utilized to create mixtures where product composition changes in response to experimental conditions. Thus, a linear peptide precursor can select one organic linker from a mixture, and a cyclic product can dynamically exchange the small molecule component of the macrocycle. Copyright © 2014 European Peptide Society and John Wil

ISSN:1075-2617    

DOI:10.1002/psc.2596

年代:2014

数据来源: WILEY

摘要:

Fluorescence resonance energy transfer (FRET) is a valuable tool for studying protein structure, folding and interactions. The steep distance dependence of the FRET efficiency requires the donor and acceptor to be in close proximity (1–7.5 nm) to exhibit sufficient energy transfer. One possibility to overcome this limitation is the usage of a FRET cascade that utilizes more than one FRET pair. Essential for realizing this FRET cascade is the site‐specific introduction of multiple fluorophores to a given protein, which remains a great challenge. In this study, orthogonal labeling techniques, including fluorescent protein tagging, oxime ligation and kinetically controlled cysteine conjugation, are employed to introduce three fluorophores at specific sites of Rab1b GTPase, yielding a triple‐labeled FRET probe. The generated protein probe exhibits efficient energy transfer from the primary donor enhanced green fluorescent protein over the intermediate acceptor rhodamine to the final acceptor Dy630. The labeling strategy opens up a new avenue for multi‐color labeling of proteins, facilitating long‐distance FRET studies. Copyright © 2014 European Peptide Society and John Wil

ISSN:1075-2617    

DOI:10.1002/psc.2590

年代:2014

数据来源: WILEY

摘要:

Ubiquitin and ubiquitin‐like proteins such as SUMO represent important and abundant post‐translational modifications involved in many cellular processes. These modifiers are reversibly attached via an isopeptide bond to lysine side chains of their target proteins by the action of specific E1, E2, and E3 enzymes. A significant challenge in studying ubiquitylation and SUMOylation is the frequently encountered inability to access desired conjugates at a defined position of the target protein and in homogenous form by using enzymatic preparation. In recent years, several chemical conjugation approaches have been developed to overcome this limitation. In this study, we aimed to selectively SUMOylate a 189‐amino acid fragment of human RanGAP1 (amino acids 398–587) at the position of Lys524 by applying two recently reported approaches based on the Cu(I)‐catalyzed alkyne‐azide cycloaddition. Because of low yields observed for the incorporation of an unnatural amino acid with an azide moiety by the tRNA suppression technology, this route was abandoned. However, installing a single cysteine at position 524 and its selective alkylation was successful to introduce the azide group. The triazole‐linked SUMO1**RanGAP1 conjugate could be obtained in good yields, purified, and was shown to specifically interact with RanBP2/Ubc9. Thus, we expand the scope of proteins accessible to chemical conjugation with ubiquitin‐like proteins and underline the importance of having alternative approaches to do so. Copyright © 2013 European Peptide Society and John

ISSN:1075-2617    

DOI:10.1002/psc.2591

年代:2014

数据来源: WILEY