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Carlos F. Barbas - One of the best experts on this subject based on the ideXlab platform.
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facile and stabile linkages through tyrosine bioconjugation strategies with the tyrosine click reaction
Bioconjugate Chemistry, 2013Co-Authors: Masanobu Nagano, Julia Gavrilyuk, Wataru Hakamata, Tsubasa Inokuma, Carlos F. BarbasAbstract:The scope, chemoselectivity, and utility of the click-like tyrosine labeling reaction with 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones (PTADs) is reported. To study the utility and chemoselectivity of PTAD derivatives in peptide and Protein Chemistry, we synthesized PTAD derivatives possessing azide, alkyne, and ketone groups and studied their reactions with amino acid derivatives and peptides of increasing complexity. With Proteins we studied the compatibility of the tyrosine click reaction with cysteine and lysine-targeted labeling approaches and demonstrate that chemoselective trifunctionalization of Proteins is readily achieved. In particular cases, we noted that PTAD decomposition resulted in formation of a putative isocyanate byproduct that was promiscuous in labeling. This side reaction product, however, was readily scavenged by the addition of a small amount of 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) to the reaction medium. To study the potential of the tyrosine click reaction to introduce...
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tyrosine bioconjugation through aqueous ene type reactions a click like reaction for tyrosine
Journal of the American Chemical Society, 2010Co-Authors: Julia Gavrilyuk, Carlos F. BarbasAbstract:A new and versatile class of cyclic diazodicarboxamides that reacts efficiently and selectively with phenols and the phenolic side chain of tyrosine through an ene-like reaction is reported. This mild aqueous tyrosine ligation reaction works over a broad pH range and expands the repertoire of aqueous chemistries available for small molecule, peptide, and Protein modification. The tyrosine ligation reactions are shown to be compatible with the labeling of native enzymes and antibodies in a buffered aqueous solution. This reaction provides a novel synthetic approach to bispecific antibodies. We believe this reaction will find broad utility in peptide and Protein Chemistry and in the Chemistry of phenol-containing compounds.
Akira Otaka - One of the best experts on this subject based on the ideXlab platform.
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preparation of peptide thioesters from naturally occurring sequences using reaction sequence consisting of regioselective s cyanylation and hydrazinolysis
Biopolymers, 2016Co-Authors: Rin Miyajima, Tsubasa Inokuma, Akira Shigenaga, Yusuke Tsuda, Miki Imanishi, Shiroh Futaki, Akira OtakaAbstract:The vital roles of peptide/Protein thioesters in Protein Chemistry, including chemical or semi-synthesis of Proteins, have encouraged studies on the development of methods for the preparation of such chemical units. Biochemical protocols using intein or sortase have proved to be useful in Protein Chemistry as methods suitable for naturally occurring sequences, including recombinant Proteins. Although chemical protocols are potential options for thioester preparation, only a few are applicable to naturally occurring sequences, because standard chemical protocols require an artificial chemical device for producing thioesters. In this context, the chemical preparation of thioesters based on a reaction sequence consisting of regioselective S-cyanylation and hydrazinolysis was investigated. Regioselective S-cyanylation, which is required for cysteine-containing thioesters, was achieved with the aid of a zinc-complex formation of a CCHH-type zinc-finger sequence. Free cysteine residues that are not involved in complex formation were selectively protected with a 6-nitroveratryl group followed by S-cyanylation of the zinc-binding cysteine. Hydrazinolysis of the resulting S-cyanopeptide and subsequent photo-removal of the 6-nitroveratryl group yielded the desired peptide hydrazide, which was then converted to the corresponding thioester. The generated thioester was successfully used in N-to-C-directed one-pot/sequential native chemical ligation using an N-sulfanylethylanilide peptide to give a 64-residue peptide toxin. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 531-546, 2016.
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one pot sequential native chemical ligation using n sulfanylethylanilide peptide
Chemical Record, 2012Co-Authors: Akira Otaka, Kohei Sato, Hao Ding, Akira ShigenagaAbstract: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
Henning D Mootz - One of the best experts on this subject based on the ideXlab platform.
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synthetic histone code
Current Opinion in Chemical Biology, 2015Co-Authors: Wolfgang Fischle, Henning D Mootz, Dirk SchwarzerAbstract:Chromatin is the universal template of genetic information in all eukaryotic cells. This complex of DNA and histone Proteins not only packages and organizes genomes but also regulates gene expression. A multitude of posttranslational histone modifications and their combinations are thought to constitute a code for directing distinct structural and functional states of chromatin. Methods of Protein Chemistry, including Protein semisynthesis, amber suppression technology, and cysteine bioconjugation, have enabled the generation of so-called designer chromatin containing histones in defined and homogeneous modification states. Several of these approaches have matured from proof-of-concept studies into efficient tools and technologies for studying the bioChemistry of chromatin regulation and for interrogating the histone code. We summarize pioneering experiments and recent developments in this exciting field of chemical biology.
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Recent progress in intein research: from mechanism to directed evolution and applications
Cellular and Molecular Life Sciences, 2013Co-Authors: Gerrit Volkmann, Henning D MootzAbstract:Inteins catalyze a post-translational modification known as Protein splicing, where the intein removes itself from a precursor Protein and concomitantly ligates the flanking Protein sequences with a peptide bond. Over the past two decades, inteins have risen from a peculiarity to a rich source of applications in biotechnology, biomedicine, and Protein Chemistry. In this review, we focus on developments of intein-related research spanning the last 5 years, including the three different splicing mechanisms and their molecular underpinnings, the directed evolution of inteins towards improved splicing in exogenous Protein contexts, as well as novel applications of inteins for cell biology and Protein engineering, which were made possible by a clearer understanding of the Protein splicing mechanism.
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engineering artificially split inteins for applications in Protein Chemistry biochemical characterization of the split ssp dnab intein and comparison to the split sce vma intein
Biochemistry, 2006Co-Authors: Steffen Brenzel, Thomas Kurpiers, Henning D MootzAbstract:In Protein trans-splicing, an intein domain split into two polypeptide chains mediates linkage of the flanking amino acid sequences, the N- and C-terminal exteins, with a native peptide bond. This process can be exploited to assemble Proteins from two separately prepared fragments, e.g., for the segmental labeling with isotopes for NMR studies or the incorporation of chemical and biophysical probes. Split inteins can be artificially generated by genetic means; however, the purified inteinN and inteinC fragments usually require a denaturation and renaturation treatment to fold into the active intein, thus preventing their application to Proteins that cannot be refolded. Here, we report that the purified fragments of the artificially split DnaB helicase of Synechocystis spp. PCC6803 (Ssp DnaB) intein are active under native conditions. The first-order rate constant of the Protein trans-splicing reaction was 7.1 x 10(-4) s(-1). The previously described split vacuolar ATPase of Saccharomyces cerevisiae (Sce VMA) intein is the only other artificially split intein that is active under native conditions; however, it requires induced complex formation of the intein fragments by auxiliary dimerization domains for efficient Protein trans-splicing. In contrast, fusion of the dimerization domains to the split Ssp DnaB intein fragments had no effect on activity. This difference was also reflected by a higher thermostability of the split Ssp DnaB intein. Further investigations of the split Sce VMA intein under optimized conditions revealed a first-order rate constant of 9.4 x 10(-4) s(-1) for Protein trans-splicing and 1.7 x 10(-3) s(-1) for C-terminal cleavage involving a Cys1Ala mutant. Finally, we show that the two split inteins are orthogonal, suggesting further applications for the assembly of Proteins from more than two parts.
Akira Shigenaga - One of the best experts on this subject based on the ideXlab platform.
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preparation of peptide thioesters from naturally occurring sequences using reaction sequence consisting of regioselective s cyanylation and hydrazinolysis
Biopolymers, 2016Co-Authors: Rin Miyajima, Tsubasa Inokuma, Akira Shigenaga, Yusuke Tsuda, Miki Imanishi, Shiroh Futaki, Akira OtakaAbstract:The vital roles of peptide/Protein thioesters in Protein Chemistry, including chemical or semi-synthesis of Proteins, have encouraged studies on the development of methods for the preparation of such chemical units. Biochemical protocols using intein or sortase have proved to be useful in Protein Chemistry as methods suitable for naturally occurring sequences, including recombinant Proteins. Although chemical protocols are potential options for thioester preparation, only a few are applicable to naturally occurring sequences, because standard chemical protocols require an artificial chemical device for producing thioesters. In this context, the chemical preparation of thioesters based on a reaction sequence consisting of regioselective S-cyanylation and hydrazinolysis was investigated. Regioselective S-cyanylation, which is required for cysteine-containing thioesters, was achieved with the aid of a zinc-complex formation of a CCHH-type zinc-finger sequence. Free cysteine residues that are not involved in complex formation were selectively protected with a 6-nitroveratryl group followed by S-cyanylation of the zinc-binding cysteine. Hydrazinolysis of the resulting S-cyanopeptide and subsequent photo-removal of the 6-nitroveratryl group yielded the desired peptide hydrazide, which was then converted to the corresponding thioester. The generated thioester was successfully used in N-to-C-directed one-pot/sequential native chemical ligation using an N-sulfanylethylanilide peptide to give a 64-residue peptide toxin. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 531-546, 2016.
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one pot sequential native chemical ligation using n sulfanylethylanilide peptide
Chemical Record, 2012Co-Authors: Akira Otaka, Kohei Sato, Hao Ding, Akira ShigenagaAbstract: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
E Bursaux - One of the best experts on this subject based on the ideXlab platform.
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human erythrocyte band 3 polymorphism band 3 memphis characterization of the structural modification lys 56 glu by Protein Chemistry methods
Blood, 1991Co-Authors: Drakoulis Yannoukakos, Corinne Vasseur, Catherine Driancourt, Henri Wajcman, Y Blouquit, Jacques Delaunay, E BursauxAbstract:Band 3 variants occur rather frequently in different populations. Based on sodium dodecyl sulfate (SDS)-polyacrylamide electrophoretic properties, a widespread polymorphism (band 3 Memphis) has been previously described. It corresponds to a Protein that has been hypothesized to be elongated in its N-terminal cytoplasmic domain. Band 3 from a heterozygote subject for this polymorphism and that displays a normal reactivity towards stilbene disulfonates has been isolated and its primary structure determined by Protein Chemistry. Reverse-phase high performance liquid chromatography tryptic peptide mapping showed, as the only difference with controls, that the enzymatic cleavage between the two N-terminal peptides did not occur, yielding a 69 residue-long fragment. Further cleavages of this peptide (cyanogen bromide, V8 protease), amino acid composition, and sequence analyses demonstrated that the lysine at position 56 was replaced by a glutamic acid. Thus, surprisingly, a single amino acid change is responsible for the large difference in the electrophoretic behavior. This result suggests that single amino acid substitutions may similarly be involved in the structural modification of several other Protein variants, described as elongated or shortened based only on SDS-polyacrylamide electrophoresis studies. When deletions/insertions were confirmed by sequence analysis, their extent was often different from that expected from electrophoresis.
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three regions of erythrocyte band 3 Protein are phosphorylated on tyrosines characterization of the phosphorylation sites by solid phase sequencing combined with capillary electrophoresis
Biochimica et Biophysica Acta, 1991Co-Authors: Drakoulis Yannoukakos, Corinne Vasseur, Catherine Driancourt, Henri Wajcman, Helmut E. Meyer, E BursauxAbstract:Abstract The major part of band 3 phosphorylation was recently shown to concern the first tryptic peptide of the Protein (Yannoukakos et al. (1991) Biochim. Biophys. Acta 1061, 253–266). Tyrosine 8 is the prevalent site of phosphorylation, but other phosphorylated regions were found which could not be analyzed with certainty. Direct characterization of the phosphorylated residues in all these phosphorylated fragments was made possible due to recent advances in Protein Chemistry techniques, such as solid phase sequence analysis and capillary electrophoresis. The present report establishes that band 3 phosphorylation occurs predominantly on tyrosines: besides tyrosine 8 already known in the N-terminal region, two other tyrosines are demonstrated to be targets for the tyrosine kinase, tyrosine 359 and tyrosine 904. These residues lie in regions of band 3 exposed to the cytoplasm, the junction of the cytoplasmic and the membrane-spanning domains, and the C-terminal end of the Protein which is also cytosolic, respectively.
