The Experts below are selected from a list of 3159 Experts worldwide ranked by ideXlab platform

Stephen B. H. Kent - One of the best experts on this subject based on the ideXlab platform.

  • Native Chemical Ligation at Asx-Cys, Glx-Cys: Chemical Synthesis and High-Resolution X-ray Structure of ShK Toxin by Racemic Protein Crystallography.
    Journal of the American Chemical Society, 2013
    Co-Authors: Bobo Dang, Kalyaneswar Mandal, Tomoya Kubota, Francisco Bezanilla, Stephen B. H. Kent
    Abstract:

    We have re-examined the utility of Native Chemical Ligation at −Gln/Glu-Cys− [Glx-Cys] and −Asn/Asp-Cys− [Asx-Cys] sites. Using the improved thioaryl catalyst 4-mercaptophenylacetic acid (MPAA), Native Chemical Ligation could be performed at −Gln-Cys− and Asn-Cys− sites without side reactions. After optimization, Ligation at a −Glu-Cys− could also be used as a Ligation site, with minimal levels of byproduct formation. However, −Asp-Cys− is not appropriate for use as a site for Native Chemical Ligation because of formation of significant amounts of β-linked byproduct. The feasibility of Native Chemical Ligation at −Gln-Cys− enabled a convergent total Chemical synthesis of the enantiomeric forms of the ShK toxin protein molecule. The D-ShK protein molecule was ~50,000-fold less active in blocking the Kv1.3 channel than the L-ShK protein molecule. Racemic protein crystallography was used to obtain high resolution X-ray diffraction data for ShK toxin. The structure was solved by direct methods and showed significant differences from the previously reported NMR structures in some regions of the ShK protein molecule.

  • Native Chemical Ligation at Asx-Cys, Glx-Cys: Chemical synthesis and high-resolution X-ray structure of ShK toxin by racemic protein crystallography.
    Journal of the American Chemical Society, 2013
    Co-Authors: Bobo Dang, Kalyaneswar Mandal, Tomoya Kubota, Francisco Bezanilla, Stephen B. H. Kent
    Abstract:

    We have re-examined the utility of Native Chemical Ligation at -Gln/Glu-Cys- [Glx-Cys] and -Asn/Asp-Cys- [Asx-Cys] sites. Using the improved thioaryl catalyst 4-mercaptophenylacetic acid (MPAA), Native Chemical Ligation could be performed at -Gln-Cys- and Asn-Cys- sites without side reactions. After optimization, Ligation at a -Glu-Cys- site could also be used as a Ligation site, with minimal levels of byproduct formation. However, -Asp-Cys- is not appropriate for use as a site for Native Chemical Ligation because of formation of significant amounts of β-linked byproduct. The feasibility of Native Chemical Ligation at -Gln-Cys- enabled a convergent total Chemical synthesis of the enantiomeric forms of the ShK toxin protein molecule. The D-ShK protein molecule was ~50,000-fold less active in blocking the Kv1.3 channel than the L-ShK protein molecule. Racemic protein crystallography was used to obtain high-resolution X-ray diffraction data for ShK toxin. The structure was solved by direct methods and showed significant differences from the previously reported NMR structures in some regions of the ShK protein molecule.

  • A one-pot approach to neoglycopeptides using orthogonal Native Chemical Ligation and click chemistry.
    Organic Letters, 2009
    Co-Authors: Kalyaneswar Mandal, Margaret A. Brimble, Paul W. R. Harris, Stephen B. H. Kent
    Abstract:

    The powerful combination of Native Chemical Ligation and click chemistry has been used to affect a one-pot synthesis of neoglycopeptides from propargyl-containing peptides using GalNAc-N3 as the glycan component. A versatile Chemical toolkit for the fully convergent synthesis of neoglycoproteins using click chemistry, Native Chemical Ligation, and kinetically controlled Ligation is thus demonstrated.

  • A one-pot approach to neoglycopeptides using orthogonal Native Chemical Ligation and click chemistry.
    Organic letters, 2009
    Co-Authors: Dong Jun Lee, Margaret A. Brimble, Kalyaneswar Mandal, Paul W. R. Harris, Stephen B. H. Kent
    Abstract:

    The powerful combination of Native Chemical Ligation and click chemistry has been used to affect a one-pot synthesis of neoglycopeptides from propargyl-containing peptides using GalNAc-N3 as the gl...

  • Direct on-resin synthesis of peptide-αthiophenylesters for use in Native Chemical Ligation
    Organic Letters, 2006
    Co-Authors: Duhee Bang, Zachary P Gates, Bradley L. Pentelute, Stephen B. H. Kent
    Abstract:

    A peptide-αthiophenylester is a key reactant in Native Chemical Ligation. Preformation of the peptide-αthiophenylester could be useful for enhancing the Ligation reaction. We report the direct on-resin preparation of preformed peptide-αthiophenylesters using a simple and efficient method. The peptide-αthiophenylester reacted extremely rapidly with a Cys-peptide when compared to the peptide-αthioalkylester.

Richard J. Payne - One of the best experts on this subject based on the ideXlab platform.

  • Native Chemical Ligation–Photodesulfurization in Flow
    Journal of the American Chemical Society, 2018
    Co-Authors: Timothy S. Chisholm, Jessica Sayers, Daniel Clayton, Luke J. Dowman, Richard J. Payne
    Abstract:

    Native Chemical Ligation (NCL) combined with desulfurization chemistry has revolutionized the way in which large polypeptides and proteins are accessed by Chemical synthesis. Herein, we outline the use of flow chemistry for the Ligation-based assembly of polypeptides. We also describe the development of a novel photodesulfurization transformation that, when coupled with flow NCL, enables efficient access to Native polypeptides on time scales up to 2 orders of magnitude faster than current batch NCL–desulfurization methods. The power of the new Ligation–photodesulfurization flow platform is showcased through the rapid synthesis of the 36 residue clinically approved HIV entry inhibitor enfuvirtide and the peptide diagnostic agent somatorelin.

  • Native Chemical Ligation photodesulfurization in flow
    Journal of the American Chemical Society, 2018
    Co-Authors: Timothy S. Chisholm, Jessica Sayers, Daniel Clayton, Luke J. Dowman, Richard J. Payne
    Abstract:

    Native Chemical Ligation (NCL) combined with desulfurization chemistry has revolutionized the way in which large polypeptides and proteins are accessed by Chemical synthesis. Herein, we outline the use of flow chemistry for the Ligation-based assembly of polypeptides. We also describe the development of a novel photodesulfurization transformation that, when coupled with flow NCL, enables efficient access to Native polypeptides on time scales up to 2 orders of magnitude faster than current batch NCL–desulfurization methods. The power of the new Ligation–photodesulfurization flow platform is showcased through the rapid synthesis of the 36 residue clinically approved HIV entry inhibitor enfuvirtide and the peptide diagnostic agent somatorelin.

  • Rapid and efficient protein synthesis through expansion of the Native Chemical Ligation concept
    Nature Reviews Chemistry, 2018
    Co-Authors: Sameer S. Kulkarni, Jessica Sayers, Bhavesh Premdjee, Richard J. Payne
    Abstract:

    Native Chemical Ligation (NCL) has revolutionized the field of Chemical protein synthesis. This Review discusses milestones such as desulfurization, the development of thiol and selenol analogues of proteinogenic amino acids and novel acyl donors for multi-component iterative Ligations. These have greatly expanded the NCL concept and enabled the synthesis of hitherto inaccessible protein targets.

  • Native Chemical Ligation–Photodesulfurization in Flow
    2018
    Co-Authors: Timothy S. Chisholm, Jessica Sayers, Daniel Clayton, Luke J. Dowman, Richard J. Payne
    Abstract:

    Native Chemical Ligation (NCL) combined with desulfurization chemistry has revolutionized the way in which large polypeptides and proteins are accessed by Chemical synthesis. Herein, we outline the use of flow chemistry for the Ligation-based assembly of polypeptides. We also describe the development of a novel photodesulfurization transformation that, when coupled with flow NCL, enables efficient access to Native polypeptides on time scales up to 2 orders of magnitude faster than current batch NCL–desulfurization methods. The power of the new Ligation–photodesulfurization flow platform is showcased through the rapid synthesis of the 36 residue clinically approved HIV entry inhibitor enfuvirtide and the peptide diagnostic agent somatorelin

  • Modern Extensions of Native Chemical Ligation for Chemical Protein Synthesis
    Topics in Current Chemistry, 2014
    Co-Authors: Lara R. Malins, Richard J. Payne
    Abstract:

    Over the past 20 years, Native Chemical Ligation has facilitated the synthesis of numerous complex peptide and protein targets, with and without post-translational modifications, as well as the design and construction of a variety of engineered protein variants. This powerful methodology has also served as a platform for the development of related chemoselective Ligation technologies which have greatly expanded the scope and flexibility of Ligation chemistry. This chapter details a number of important extensions of the original Native Chemical Ligation manifold, with particular focus on the application of new methods in the total Chemical synthesis of proteins. Topics covered include the development of auxiliary-based Ligation methods, the post-Ligation manipulation of Cys residues, and the synthesis and utility of unnatural amino acid building blocks (bearing reactive thiol or selenol functionalities) in chemoselective Ligation chemistry. Contemporary applications of these techniques to the total Chemical synthesis of peptides and proteins are described.

Ronald Micura - One of the best experts on this subject based on the ideXlab platform.

  • Current Protocols in Nucleic Acid Chemistry - Native Chemical Ligation of Hydrolysis‐Resistant 3′‐NH‐Cysteine‐Modified RNA
    Current protocols in human genetics, 2015
    Co-Authors: Anna‐skrollan Geiermann, Ronald Micura
    Abstract:

    Hydrolysis-resistant RNA-peptide conjugates that contain a 3′-NH linkage between the adenosine ribose and the C-terminal carboxyl group of a peptide moiety instead of the natural ester mimic acylated tRNA termini. Their detailed preparation that combines solid-phase oligonucleotide synthesis and bioconjugation is described here. The key step is Native Chemical Ligation (NCL) of 3′-NH-cysteine-modified RNA to highly soluble peptide thioesters. These hydrolysis-resistant 3′-NH-peptide-modified RNAs, containing the universally conserved 3′-CCA end of tRNA, are biologically active and can bind to the ribosome. They can be used as valuable probes for structural and functional studies of the ribosomal elongation cycle. © 2015 by John Wiley & Sons, Inc. Keywords: oligonucleotide-peptide conjugates; Native Chemical Ligation; nucleoside modification; RNA solid-phase synthesis; peptides

  • Native Chemical Ligation of Hydrolysis-Resistant 3′-Peptidyl–tRNA Mimics
    Journal of the American Chemical Society, 2011
    Co-Authors: Anna‐skrollan Geiermann, Norbert Polacek, Ronald Micura
    Abstract:

    Hydrolysis-resistant 3′-peptidyl–RNA conjugates that mimic tRNA termini represent a remarkable synthetic challenge, particularly if they contain amino acids with complex side-chain functionalities, such as arginines. Here we demonstrate a novel approach that combines solid-phase synthesis and bioconjugation to obtain these derivatives with high efficiency and purity. The key step is Native Chemical Ligation of 3′-cysteinyl-RNA fragments to highly soluble peptide thioesters. The so-prepared 3′-peptidyl–RNA conjugates relate to resistance peptides that can render the ribosome resistant to macrolide antibiotics by a yet unknown ribosomal translation mechanism.

  • Native Chemical Ligation of hydrolysis resistant 3 peptidyl trna mimics
    Journal of the American Chemical Society, 2011
    Co-Authors: Anna‐skrollan Geiermann, Norbert Polacek, Ronald Micura
    Abstract:

    Hydrolysis-resistant 3′-peptidyl–RNA conjugates that mimic tRNA termini represent a remarkable synthetic challenge, particularly if they contain amino acids with complex side-chain functionalities, such as arginines. Here we demonstrate a novel approach that combines solid-phase synthesis and bioconjugation to obtain these derivatives with high efficiency and purity. The key step is Native Chemical Ligation of 3′-cysteinyl-RNA fragments to highly soluble peptide thioesters. The so-prepared 3′-peptidyl–RNA conjugates relate to resistance peptides that can render the ribosome resistant to macrolide antibiotics by a yet unknown ribosomal translation mechanism.

Anna‐skrollan Geiermann - One of the best experts on this subject based on the ideXlab platform.

  • Current Protocols in Nucleic Acid Chemistry - Native Chemical Ligation of Hydrolysis‐Resistant 3′‐NH‐Cysteine‐Modified RNA
    Current protocols in human genetics, 2015
    Co-Authors: Anna‐skrollan Geiermann, Ronald Micura
    Abstract:

    Hydrolysis-resistant RNA-peptide conjugates that contain a 3′-NH linkage between the adenosine ribose and the C-terminal carboxyl group of a peptide moiety instead of the natural ester mimic acylated tRNA termini. Their detailed preparation that combines solid-phase oligonucleotide synthesis and bioconjugation is described here. The key step is Native Chemical Ligation (NCL) of 3′-NH-cysteine-modified RNA to highly soluble peptide thioesters. These hydrolysis-resistant 3′-NH-peptide-modified RNAs, containing the universally conserved 3′-CCA end of tRNA, are biologically active and can bind to the ribosome. They can be used as valuable probes for structural and functional studies of the ribosomal elongation cycle. © 2015 by John Wiley & Sons, Inc. Keywords: oligonucleotide-peptide conjugates; Native Chemical Ligation; nucleoside modification; RNA solid-phase synthesis; peptides

  • Native Chemical Ligation of Hydrolysis-Resistant 3′-Peptidyl–tRNA Mimics
    Journal of the American Chemical Society, 2011
    Co-Authors: Anna‐skrollan Geiermann, Norbert Polacek, Ronald Micura
    Abstract:

    Hydrolysis-resistant 3′-peptidyl–RNA conjugates that mimic tRNA termini represent a remarkable synthetic challenge, particularly if they contain amino acids with complex side-chain functionalities, such as arginines. Here we demonstrate a novel approach that combines solid-phase synthesis and bioconjugation to obtain these derivatives with high efficiency and purity. The key step is Native Chemical Ligation of 3′-cysteinyl-RNA fragments to highly soluble peptide thioesters. The so-prepared 3′-peptidyl–RNA conjugates relate to resistance peptides that can render the ribosome resistant to macrolide antibiotics by a yet unknown ribosomal translation mechanism.

  • Native Chemical Ligation of hydrolysis resistant 3 peptidyl trna mimics
    Journal of the American Chemical Society, 2011
    Co-Authors: Anna‐skrollan Geiermann, Norbert Polacek, Ronald Micura
    Abstract:

    Hydrolysis-resistant 3′-peptidyl–RNA conjugates that mimic tRNA termini represent a remarkable synthetic challenge, particularly if they contain amino acids with complex side-chain functionalities, such as arginines. Here we demonstrate a novel approach that combines solid-phase synthesis and bioconjugation to obtain these derivatives with high efficiency and purity. The key step is Native Chemical Ligation of 3′-cysteinyl-RNA fragments to highly soluble peptide thioesters. The so-prepared 3′-peptidyl–RNA conjugates relate to resistance peptides that can render the ribosome resistant to macrolide antibiotics by a yet unknown ribosomal translation mechanism.

Philip E. Dawson - One of the best experts on this subject based on the ideXlab platform.

  • Native Chemical Ligation of Peptides and Proteins
    Current protocols in chemical biology, 2019
    Co-Authors: Philip A. Cistrone, Michael J. Bird, Dillon T. Flood, Anthony P. Silvestri, Jordi C. J. Hintzen, Darren A. Thompson, Philip E. Dawson
    Abstract:

    For over 20 years, Native Chemical Ligation (NCL) has played a pivotal role in enabling total synthesis and semisynthesis of increasingly complex peptide and protein targets. Classical NCL proceeds by chemoselective reaction of two unprotected polypeptide chains in near-neutral-pH, aqueous solution and is made possible by the presence of a thioester moiety on the C-terminus of the N-terminal peptide fragment and a natural cysteine residue on the N-terminus of the C-terminal peptide fragment. The reaction yields an amide bond adjacent to cysteine at the Ligation site, furnishing a Native protein backbone in a traceless manner. This unit highlights a number of recent and powerful advances in the methodology and outlines their particular uses, facilitating application in the synthesis of challenging protein targets. © 2019 by John Wiley & Sons, Inc.

  • synthesis of peptides and proteins without cysteine residues by Native Chemical Ligation combined with desulfurization
    Journal of the American Chemical Society, 2001
    Co-Authors: Philip E. Dawson
    Abstract:

    The highly chemoselective reaction between unprotected peptides bearing an N-terminal Cys residue and a C-terminal thioester enables the total and semi-synthesis of complex polypeptides. Here we extend the utility of this Native Chemical Ligation approach to non-cysteine containing peptides. Since alanine is a common amino acid in proteins, Ligation at this residue would be of great utility. To achieve this goal, a specific alanine residue in the parent protein is replaced with cysteine to facilitate synthesis by Native Chemical Ligation. Following Ligation, selective desulfurization of the resulting unprotected polypeptide product with H2/metal reagents converts the cysteine residue to alanine. This approach, which provides a general method to prepare alanyl proteins from their cysteinyl forms, can be used to Chemically synthesize a variety of polypeptides, as demonstrated by the total Chemical syntheses of the cyclic antibiotic microcin J25, the 56-amino acid streptococcal protein G B1 domain, and a var...

  • synthesis of peptides and proteins without cysteine residues by Native Chemical Ligation combined with desulfurization
    Journal of the American Chemical Society, 2001
    Co-Authors: Liang Z Yan, Philip E. Dawson
    Abstract:

    The highly chemoselective reaction between unprotected peptides bearing an N-terminal Cys residue and a C-terminal thioester enables the total and semi-synthesis of complex polypeptides. Here we extend the utility of this Native Chemical Ligation approach to non-cysteine containing peptides. Since alanine is a common amino acid in proteins, Ligation at this residue would be of great utility. To achieve this goal, a specific alanine residue in the parent protein is replaced with cysteine to facilitate synthesis by Native Chemical Ligation. Following Ligation, selective desulfurization of the resulting unprotected polypeptide product with H(2)/metal reagents converts the cysteine residue to alanine. This approach, which provides a general method to prepare alanyl proteins from their cysteinyl forms, can be used to Chemically synthesize a variety of polypeptides, as demonstrated by the total Chemical syntheses of the cyclic antibiotic microcin J25, the 56-amino acid streptococcal protein G B1 domain, and a variant of the 110-amino acid ribonuclease, barnase.

  • Protein synthesis by Native Chemical Ligation: Expanded scope by using straightforward methodology
    Proceedings of the National Academy of Sciences of the United States of America, 1999
    Co-Authors: Tilman M Hackeng, John H Griffin, Philip E. Dawson
    Abstract:

    The total Chemical synthesis of proteins has great potential for increasing our understanding of the molecular basis of protein function. The introduction of Native Chemical Ligation techniques to join unprotected peptides next to a cysteine residue has greatly facilitated the synthesis of proteins of moderate size. Here, we describe a straightforward methodology that has enabled us to rapidly analyze the compatibility of the Native Chemical Ligation strategy for X–Cys Ligation sites, where X is any of the 20 naturally occurring amino acids. The simplified methodology avoids the necessity of specific amino acid thioester linkers or alkylation of C-terminal thioacid peptides. Experiments using matrix-assisted laser-desorption ionization MS analysis of combinatorial Ligations of LYRAX-C-terminal thioester peptides to the peptide CRANK show that all 20 amino acids are suitable for Ligation, with Val, Ile, and Pro representing less favorable choices because of slow Ligation rates. To illustrate the method’s utility, two 124-aa proteins were manually synthesized by using a three-step, four-piece Ligation to yield a fully active human secretory phospholipase A2 and a catalytically inactive analog. The combination of flexibility in design with general access because of simplified methodology broadens the applicability and versatility of Chemical protein synthesis.

  • Synthesis of a three zinc finger protein, Zif268, by Native Chemical Ligation
    Biopolymers, 1999
    Co-Authors: Gangamani S. Beligere, Philip E. Dawson
    Abstract:

    Protein synthesis by Native Chemical Ligation has been an effective approach for the synthesis of proteins of moderate size. The utility of this approach for protein synthesis is demonstrated by the synthesis of a transcription factor, Zif 268 that contains three zinc finger domains. This synthesis highlights the modular nature of the Chemical Ligation approach and the ability to synthesize, handle and fold multiple domain proteins.