scholarly journals Selenolysine: A New Tool for Traceless Isopeptide Bond Formation

2019 ◽  
Author(s):  
Rebecca Notis Dardashti ◽  
Shailesh Kumar ◽  
Shawn M. Sternisha ◽  
Post Sai Reddy ◽  
Brian G. Miller ◽  
...  
Keyword(s):  
Synthetic Peptides ◽  
Bond Formation ◽  
Native Chemical Ligation ◽  
Cysteine Residues ◽  
Expression Systems ◽  
Chemical Ligation ◽  
Design And Synthesis ◽  
Isopeptide Bond ◽  
Chemical Protein Synthesis ◽  
Conventional Expression

<p>Despite their biological importance, post-translationally modified proteins are notoriously difficult to produce in a homogeneous fashion using conventional expression systems. Chemical protein synthesis or semi-synthesis offers a solution to this problem; however, traditional strategies often rely on sulfur-based chemistry that is incompatible with the presence of multiple cysteine residues in the target protein. To overcome these limitations, we present the design and synthesis of γ-selenolysine, a selenol-containing form of the commonly modified proteinogenic amino acid, lysine. The utility of γ-selenolysine is demonstrated with the traceless ligation of the small ubiquitin-like modifier protein, SUMO-1, to a peptide segment of human glucokinase. The resulting polypeptide is poised for native chemical ligation and selective deselenization in the presence of unprotected cysteine residues. Selenolysine’s straightforward synthesis and incorporation into synthetic peptides marks it as a universal handle for conjugating any ubiquitin-like modifying protein to its target.</p>

scholarly journals Selenolysine: A New Tool for Traceless Isopeptide Bond Formation

2019 ◽  
Author(s):  
Rebecca Notis Dardashti ◽  
Shailesh Kumar ◽  
Shawn M. Sternisha ◽  
Post Sai Reddy ◽  
Brian G. Miller ◽  
...  
Keyword(s):  
Synthetic Peptides ◽  
Bond Formation ◽  
Native Chemical Ligation ◽  
Cysteine Residues ◽  
Expression Systems ◽  
Chemical Ligation ◽  
Design And Synthesis ◽  
Isopeptide Bond ◽  
Chemical Protein Synthesis ◽  
Conventional Expression

<p>Despite their biological importance, post-translationally modified proteins are notoriously difficult to produce in a homogeneous fashion using conventional expression systems. Chemical protein synthesis or semi-synthesis offers a solution to this problem; however, traditional strategies often rely on sulfur-based chemistry that is incompatible with the presence of multiple cysteine residues in the target protein. To overcome these limitations, we present the design and synthesis of γ-selenolysine, a selenol-containing form of the commonly modified proteinogenic amino acid, lysine. The utility of γ-selenolysine is demonstrated with the traceless ligation of the small ubiquitin-like modifier protein, SUMO-1, to a peptide segment of human glucokinase. The resulting polypeptide is poised for native chemical ligation and selective deselenization in the presence of unprotected cysteine residues. Selenolysine’s straightforward synthesis and incorporation into synthetic peptides marks it as a universal handle for conjugating any ubiquitin-like modifying protein to its target.</p>

scholarly journals Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I)

2020 ◽  
Author(s):  
Patrick Erickson ◽  
James Fulcher ◽  
Michael Kay
Keyword(s):  
Synthetic Peptides ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
Ribosomal Subunit ◽  
Side Reaction ◽  
Native Chemical Ligation ◽  
One Pot ◽  
Direct Production ◽  
Chemical Ligation ◽  
Peptide Cleavage

<div><div><div><p>Chemoselective ligation reactions, such as native chemical ligation (NCL), enable the assembly of synthetic peptides into proteins. However, the scope of proteins accessible to total chemical synthesis is limited by ligation efficiency. Sterically hindered thioesters and poorly soluble peptides can undergo incomplete ligations, leading to challenging purifications with low yields. This work describes a new method, ClickAssisted NCL (CAN), which overcomes these barriers. In CAN, peptides are modified with traceless “helping hand” lysine linkers that enable addition of dibenzocyclooctyne (DBCO) and azide handles for strain-promoted alkyne-azide cycloaddition (SPAAC) reactions. This cycloaddition templates the peptides to increase their effective concentration and greatly accelerate ligation kinetics. After ligation, mild hydroxylamine treatment tracelessly removes the linkers to afford the native ligated peptide. Although DBCO is incompatible with standard Fmoc solid-phase peptide synthesis (SPPS) due to an acid-mediated rearrangement that occurs during peptide cleavage, we demonstrate that copper(I) protects DBCO from this side reaction, enabling direct production of DBCO-containing synthetic peptides. Excitingly, low concentrations of triazole-linked model peptides reacted ~1,200-fold faster than predicted for non-templated control ligations, which also accumulated many side products due to the long reaction time. Using the E. coli ribosomal subunit L32 as a model protein, we further demonstrate that the SPAAC, ligation, desulfurization, and linker cleavage steps can be performed in a one-pot fashion. CAN will be useful for overcoming ligation challenges to expand the reach of chemical protein synthesis.</p></div></div></div>

Chemical Protein Synthesis by Native Chemical Ligation and Variations Thereof

2019 ◽  
Vol 37 (11) ◽  
pp. 1181-1193 ◽  
Author(s):  
Siyao Wang ◽  
Yogesh Abaso Thopate ◽  
Qingqing Zhou ◽  
Ping Wang
Keyword(s):  
Protein Synthesis ◽  
Native Chemical Ligation ◽  
Chemical Ligation ◽  
Chemical Protein Synthesis

Design and Synthesis of Lipopeptide - Carbohydrate Assembled Multivalent Vaccine Candidates Using Native Chemical Ligation

2009 ◽  
Vol 62 (9) ◽  
pp. 993 ◽  
Author(s):  
Wei Zhong ◽  
Mariusz Skwarczynski ◽  
Yoshio Fujita ◽  
Pavla Simerska ◽  
Michael F. Good ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Native Chemical Ligation ◽  
High Yield ◽  
Antigenic Peptides ◽  
Chemical Ligation ◽  
Vaccine Candidates ◽  
Multivalent Vaccine ◽  
Peptide Antigens ◽  
Design And Synthesis ◽  
Α Helix

Development of a synthetic vaccine against group A streptococcal infection is increasingly paramount due to the induction of autoimmunity by the main virulent factor – M protein. Peptide vaccines, however, are generally poorly immunogenic, necessitating administration with carriers and adjuvants. One of the promising approaches to deliver antigenic peptides is to assemble peptides on a suitable template which directs the attached peptides to form a well defined tertiary structure. For self-adjuvanting human vaccines, the conjugation of immunostimulatory lipids has been demonstrated as a potentially safe method. This study describes the design and optimized synthesis of two lipopeptide conjugated carbohydrate templates and the assembling of peptide antigens. These lipopeptide–carbohydrate assembled multivalent vaccine candidates were obtained in high yield and purity when native chemical ligation was applied. Circular dichroism studies indicated that the template-assembled peptides form four α-helix bundles. The developed technique extends the use of carbohydrate templates and lipopeptide conjugates for producing self-adjuvanting and topology-controlled vaccine candidates.

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