One-pot multi-segment condensation strategies for chemical protein synthesis

2019 ◽  
Vol 17 (4) ◽  
pp. 727-744 ◽  
Author(s):  
Chong Zuo ◽  
Baochang Zhang ◽  
Bingjia Yan ◽  
Ji-Shen Zheng
Keyword(s):  
Protein Synthesis ◽  
Protecting Group ◽  
One Pot ◽  
Recent Advances ◽  
Kinetically Controlled ◽  
Chemical Protein Synthesis ◽  
Segment Condensation

This paper describes recent advances of one-pot multi-segment condensation strategies based on kinetically controlled strategies and/or protecting group-removal strategies in chemical protein synthesis.

scholarly journals Ru Catalyst Facilitates Total Chemical Protein Synthesis to Investigate the Effect of Epigenetic Modifications Decorated on Linker Histone H1.2 and Heterochromatin Protein 1α (HP1α)

2020 ◽  
Author(s):  
Naoki Kamo ◽  
Tomoya Kujirai ◽  
Hitoshi Kurumizaka ◽  
Hitoshi Murakami ◽  
Gosuke Hayashi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Metal Complexes ◽  
Palladium Complexes ◽  
Protecting Groups ◽  
One Pot ◽  
Heterochromatin Protein ◽  
Site Specific ◽  
Ru Catalyst ◽  
Chemical Protein Synthesis

For epigenetics research, preparing homogeneous proteins bearing site-specific posttranslational modifications (PTMs) is essential to understand the behavior of chromatin. Total chemical protein synthesis is a very powerful method to obtain target proteins with various modifications at site-specific positions. To produce large proteins efficiently, one-pot ligation of multiple peptide fragments was previously reported through repetitive deprotection of protecting groups for N-terminal Cys with palladium complexes. However, this method demanded more than a catalytic amount of metal complexes, and, in general, it had been challenging to achieve catalytic cycles of metal complexes especially for reactions on proteins. Here, we report an efficient and facile method of chemical protein synthesis using Ru catalyst. The use of 10–20 mol% of Ru complexes enabled us to remove the protecting groups on peptides or proteins under peptide ligation conditions, and this complex showed more than 50-fold activity compared to the previous palladium complexes due to the great stability toward thiol moieties. By using this Ru catalyst, we accomplished total chemical synthesis of linker histone H1.2 (212 amino acids) and heterochromatin protein 1a (HP1a) (191 amino acids), which are important components of heterochromatin, through one-pot multiple peptide ligation. This method prompted the preparation of H1.2 and HP1a bearing various patterns of PTMs. Moreover, we found that R53Cit at H1.2 reduced its binding affinity toward nucleosomes and four consecutive phosphorylations at N-terminus HP1a controlled its binding ability against DNA. We envisage that homogeneously modified proteins prepared by our method would facilitate epigenetics research and be applied for the elucidation of various biological phenomena.

scholarly journals Copper-mediated selenazolidine deprotection enables one-pot chemical synthesis of challenging proteins

2019 ◽  
Author(s):  
Zhenguang Zhao ◽  
Norman Metanis
Keyword(s):  
Protein Synthesis ◽  
Catalytic Activity ◽  
Chemical Synthesis ◽  
Total Synthesis ◽  
Active Site ◽  
Synthetic Approach ◽  
One Pot ◽  
Chemical Protein Synthesis ◽  
Key Technologies ◽  
The One

<p>While chemical protein synthesis (CPS) has granted access to challenging proteins, synthesis of longer proteins is often limited by low abundance or non-strategic placement of cysteine (Cys) residues, essential for native chemical ligations (NCL), as well as multiple purification and isolation steps. Selective deselenization and one-pot CPS serve as key technologies to circumvent these issues. Herein, we describe the one-pot total synthesis of human thiosulfate: glutathione sulfurtransferase (TSTD1), a 115-residue protein with a single Cys residue at its active site, and its seleno-analogue. WT-TSTD1 was synthesized in a C-to-N synthetic approach employing multiple NCL reactions, Cu(II)-mediated deprotection of selenazolidine (Sez), and chemoselective deselenization, all in one-pot. In addition, the protein’s seleno analogue (Se-TSTD1), in which the active site Cys is replaced with selenocysteine, was synthesized with a kinetically controlled ligation in a one-pot, N-to-C synthetic approach. TSTD1’s one-pot synthesis was made possible by the newly reported, rapid, and facile copper-mediated selenazolidine deprotection that can be accomplished in one minute. Finally, catalytic activity of the two proteins indicated that Se-TSTD1 possessed only four-fold lower activity than WT-TSTD1 as a thiosulfate: glutathione sulfurtransferase, suggesting that selenoproteins can have physiologically comparable sulfutransferase activity as their cysteine counterparts. </p>

scholarly journals Ru Catalyst Facilitates Total Chemical Protein Synthesis to Investigate the Effect of Epigenetic Modifications Decorated on Linker Histone H1.2 and Heterochromatin Protein 1α (HP1α)

2020 ◽  
Author(s):  
Naoki Kamo ◽  
Tomoya Kujirai ◽  
Hitoshi Kurumizaka ◽  
Hitoshi Murakami ◽  
Gosuke Hayashi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Metal Complexes ◽  
Palladium Complexes ◽  
Protecting Groups ◽  
One Pot ◽  
Heterochromatin Protein ◽  
Site Specific ◽  
Ru Catalyst ◽  
Chemical Protein Synthesis

For epigenetics research, preparing homogeneous proteins bearing site-specific posttranslational modifications (PTMs) is essential to understand the behavior of chromatin. Total chemical protein synthesis is a very powerful method to obtain target proteins with various modifications at site-specific positions. To produce large proteins efficiently, one-pot ligation of multiple peptide fragments was previously reported through repetitive deprotection of protecting groups for N-terminal Cys with palladium complexes. However, this method demanded more than a catalytic amount of metal complexes, and, in general, it had been challenging to achieve catalytic cycles of metal complexes especially for reactions on proteins. Here, we report an efficient and facile method of chemical protein synthesis using Ru catalyst. The use of 10–20 mol% of Ru complexes enabled us to remove the protecting groups on peptides or proteins under peptide ligation conditions, and this complex showed more than 50-fold activity compared to the previous palladium complexes due to the great stability toward thiol moieties. By using this Ru catalyst, we accomplished total chemical synthesis of linker histone H1.2 (212 amino acids) and heterochromatin protein 1a (HP1a) (191 amino acids), which are important components of heterochromatin, through one-pot multiple peptide ligation. This method prompted the preparation of H1.2 and HP1a bearing various patterns of PTMs. Moreover, we found that R53Cit at H1.2 reduced its binding affinity toward nucleosomes and four consecutive phosphorylations at N-terminus HP1a controlled its binding ability against DNA. We envisage that homogeneously modified proteins prepared by our method would facilitate epigenetics research and be applied for the elucidation of various biological phenomena.

Chemical Protein Synthesis by Kinetically Controlled Ligation of Peptide O-Esters

ChemBioChem ◽  
2010 ◽  
Vol 11 (4) ◽  
pp. 511-515 ◽  
Author(s):  
Ji-Shen Zheng ◽  
Hong-Kui Cui ◽  
Ge-Min Fang ◽  
Wei-Xian Xi ◽  
Lei Liu
Keyword(s):  
Protein Synthesis ◽  
Kinetically Controlled ◽  
Chemical Protein Synthesis

scholarly journals Copper-mediated selenazolidine deprotection enables one-pot chemical synthesis of challenging proteins

2019 ◽  
Author(s):  
Zhenguang Zhao ◽  
Norman Metanis
Keyword(s):  
Protein Synthesis ◽  
Catalytic Activity ◽  
Chemical Synthesis ◽  
Total Synthesis ◽  
Active Site ◽  
Synthetic Approach ◽  
One Pot ◽  
Chemical Protein Synthesis ◽  
Key Technologies ◽  
The One

<p>While chemical protein synthesis (CPS) has granted access to challenging proteins, synthesis of longer proteins is often limited by low abundance or non-strategic placement of cysteine (Cys) residues, essential for native chemical ligations (NCL), as well as multiple purification and isolation steps. Selective deselenization and one-pot CPS serve as key technologies to circumvent these issues. Herein, we describe the one-pot total synthesis of human thiosulfate: glutathione sulfurtransferase (TSTD1), a 115-residue protein with a single Cys residue at its active site, and its seleno-analogue. WT-TSTD1 was synthesized in a C-to-N synthetic approach employing multiple NCL reactions, Cu(II)-mediated deprotection of selenazolidine (Sez), and chemoselective deselenization, all in one-pot. In addition, the protein’s seleno analogue (Se-TSTD1), in which the active site Cys is replaced with selenocysteine, was synthesized with a kinetically controlled ligation in a one-pot, N-to-C synthetic approach. TSTD1’s one-pot synthesis was made possible by the newly reported, rapid, and facile copper-mediated selenazolidine deprotection that can be accomplished in one minute. Finally, catalytic activity of the two proteins indicated that Se-TSTD1 possessed only four-fold lower activity than WT-TSTD1 as a thiosulfate: glutathione sulfurtransferase, suggesting that selenoproteins can have physiologically comparable sulfutransferase activity as their cysteine counterparts. </p>

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