Protein Semisynthesis Provides Access to Tau Disease-Associated Post-translational Modifications (PTMs) and Paves the Way to Deciphering the Tau PTM Code in Health and Diseased States - Haj-Yahya, Mahmood; Lashuel, Hilal A. (Journal of the American Chemical Society, 2018)

Protein Semisynthesis Provides Access to Tau Disease-Associated Post-translational Modifications (PTMs) and Paves the Way to Deciphering the Tau PTM Code in Health and Diseased States - Haj-Yahya, Mahmood; Lashuel, Hilal A. (Journal of the American Chemical Society, 2018)

Single Posttranslational Modifications in the Central Repeat Domains of Tau4 Impact its Aggregation and Tubulin Binding - Ellmer, Doris; Brehs, Manuel; Haj‐Yahya, Mahmood; Lashuel, Hilal A.; Becker, Christian F. W. (Angewandte Chemie International Edition, 2019)

Single Posttranslational Modifications in the Central Repeat Domains of Tau4 Impact its Aggregation and Tubulin Binding - Ellmer, Doris; Brehs, Manuel; Haj‐Yahya, Mahmood; Lashuel, Hilal A.; Becker, Christian F. W. (Angewandte Chemie International Edition, 2019)

Engineering the Glutamate Transporter Homologue Glt Ph Using Protein Semisynthesis - Focke, Paul J.; Annen, Alvin W.; Valiyaveetil, Francis I. (Biochemistry, 2015)

Engineering the Glutamate Transporter Homologue Glt Ph Using Protein Semisynthesis - Focke, Paul J.; Annen, Alvin W.; Valiyaveetil, Francis I. (Biochemistry, 2015)

Total chemical synthesis of a thermostable enzyme capable of polymerase chain reaction - Xu, Weiliang; Jiang, Wenjun; Wang, Jiaxing; Yu, Linping; Chen, Ji; Liu, Xianyu; Liu, Lei; Zhu, Ting F (Cell Discovery, 2017)

Total chemical synthesis of a thermostable enzyme capable of polymerase chain reaction - Xu, Weiliang; Jiang, Wenjun; Wang, Jiaxing; Yu, Linping; Chen, Ji; Liu, Xianyu; Liu, Lei; Zhu, Ting F (Cell Discovery, 2017)

A thermostable d-polymerase for mirror-image PCR - Pech, Andreas; Achenbach, John; Jahnz, Michael; Schülzchen, Simone; Jarosch, Florian; Bordusa, Frank; Klussmann, Sven (Nucleic Acids Research, 2017)

A thermostable d-polymerase for mirror-image PCR - Pech, Andreas; Achenbach, John; Jahnz, Michael; Schülzchen, Simone; Jarosch, Florian; Bordusa, Frank; Klussmann, Sven (Nucleic Acids Research, 2017)

Efficient Semisynthesis of a Tetraphosphorylated Analogue of the Type I TGFβ Receptor - Flavell, Robert R.; Huse, Morgan; Goger, Mike; Trester-Zedlitz, Michelle; Kuriyan, John; Muir, Tom W. (Organic Letters, 2002)

Synthesis and folding of a mirror-image enzyme reveals ambidextrous chaperone activity - Weinstock, M. T.; Jacobsen, M. T.; Kay, M. S. (Proceedings of the National Academy of Sciences, 2014)

Efficient Semisynthesis of a Tetraphosphorylated Analogue of the Type I TGFβ Receptor - Flavell, Robert R.; Huse, Morgan; Goger, Mike; Trester-Zedlitz, Michelle; Kuriyan, John; Muir, Tom W. (Organic Letters, 2002)

Synthesis and folding of a mirror-image enzyme reveals ambidextrous chaperone activity - Weinstock, M. T.; Jacobsen, M. T.; Kay, M. S. (Proceedings of the National Academy of Sciences, 2014)

A semisynthetic Atg3 reveals that acetylation promotes Atg3 membrane binding and Atg8 lipidation - Li, Yi-Tong; Yi, Cong; Chen, Chen-Chen; Lan, Huan; Pan, Man; Zhang, Shao-Jin; Huang, Yi-Chao; Guan, Chao-Jian; Li, Yi-Ming; Yu, Li; Liu, Lei (Nature Communications, 2017)

A semisynthetic Atg3 reveals that acetylation promotes Atg3 membrane binding and Atg8 lipidation - Li, Yi-Tong; Yi, Cong; Chen, Chen-Chen; Lan, Huan; Pan, Man; Zhang, Shao-Jin; Huang, Yi-Chao; Guan, Chao-Jian; Li, Yi-Ming; Yu, Li; Liu, Lei (Nature Communications, 2017)

Chemical Synthesis of Ubiquitinated Peptides with Varying Lengths and Types of Ubiquitin Chains to Explore the Activity of Deubiquitinases - Bavikar, Sudhir N.; Spasser, Liat; Haj-Yahya, Mahmood; Karthikeyan, Subramanian Vedhanarayanan; Moyal, Tal; Ajish Kumar, K. S.; Brik, Ashraf (Angewandte Chemie, 2012)

Chemical Synthesis of Ubiquitinated Peptides with Varying Lengths and Types of Ubiquitin Chains to Explore the Activity of Deubiquitinases - Bavikar, Sudhir N.; Spasser, Liat; Haj-Yahya, Mahmood; Karthikeyan, Subramanian Vedhanarayanan; Moyal, Tal; Ajish Kumar, K. S.; Brik, Ashraf (Angewandte Chemie, 2012)

An amalgamation of solid phase peptide synthesis and ribosomal peptide synthesis - Ottesen, Jennifer J.; Bar-Dagan, Maya; Giovani, Baldissera; Muir, Tom W. (Biopolymers, 2008)

An amalgamation of solid phase peptide synthesis and ribosomal peptide synthesis - Ottesen, Jennifer J.; Bar-Dagan, Maya; Giovani, Baldissera; Muir, Tom W. (Biopolymers, 2008)

Nonenzymatic Polyubiquitination of Expressed Proteins - Hemantha, Hosahalli P.; Bavikar, Sudhir N.; Herman-Bachinsky, Yifat; Haj-Yahya, Najat; Bondalapati, Somasekhar; Ciechanover, Aaron; Brik, Ashraf (Journal of the American Chemical Society, 2014)

Nonenzymatic Polyubiquitination of Expressed Proteins - Hemantha, Hosahalli P.; Bavikar, Sudhir N.; Herman-Bachinsky, Yifat; Haj-Yahya, Najat; Bondalapati, Somasekhar; Ciechanover, Aaron; Brik, Ashraf (Journal of the American Chemical Society, 2014)

Reconstitution of the Catalytic Core of F-ATPase (αβ) 3 γ from Escherichia coli Using Chemically Synthesized Subunit γ - Wintermann, Frank; Engelbrecht, Siegfried (Angewandte Chemie International Edition, 2013)

Cysteine-Aminoethylation-Assisted Chemical Ubiquitination of Recombinant Histones - Chu, Guo-Chao; Pan, Man; Li, Jiabin; Liu, Sanling; Zuo, Chong; Tong, Ze-Bin; Bai, Jing-Si; Gong, Qingyue; Ai, Huasong; Fan, Jian; Meng, Xianbin; Huang, Yi-Chao; et al. (Journal of the American Chemical Society, 2019)

Ligation of Soluble but Unreactive Peptide Segments in the Chemical Synthesis of Haemophilus Influenzae DNA Ligase - Zhang, Baochang; Deng, Qiang; Zuo, Chong; Yan, Bingjia; Zuo, Chao; Cao, Xiu‐Xiu; Zhu, Ting F.; Zheng, Ji‐Shen; Liu, Lei (Angewandte Chemie International Edition, 2019)

Copying Life: Synthesis of an Enzymatically Active Mirror-Image DNA-Ligase Made of D-Amino Acids - Weidmann, Joachim; Schnölzer, Martina; Dawson, Philip E.; Hoheisel, Jörg D. (Cell Chemical Biology, 2019)

Semisynthesis of unnatural amino acid mutants of paxillin: Protein probes for cell migration studies - Vogel, Elizabeth M.; Imperiali, Barbara (Protein Science, 2007)

Semisynthesis of unnatural amino acid mutants of paxillin: Protein probes for cell migration studies - Vogel, Elizabeth M.; Imperiali, Barbara (Protein Science, 2007)

HF-Free Boc Synthesis of Peptide Thioesters for Ligation and Cyclization - Raz, Richard; Burlina, Fabienne; Ismail, Mohamed; Downward, Julian; Li, Jiejin; Smerdon, Stephen J.; Quibell, Martin; White, Peter D.; Offer, John (Angewandte Chemie International Edition, 2016)

HF-Free Boc Synthesis of Peptide Thioesters for Ligation and Cyclization - Raz, Richard; Burlina, Fabienne; Ismail, Mohamed; Downward, Julian; Li, Jiejin; Smerdon, Stephen J.; Quibell, Martin; White, Peter D.; Offer, John (Angewandte Chemie International Edition, 2016)

Diverse fate of ubiquitin chain moieties: The proximal is degraded with the target, and the distal protects the proximal from removal and recycles - Sun, Hao; Mali, Sachitanand M.; Singh, Sumeet K.; Meledin, Roman; Brik, Ashraf; Kwon, Yong Tae; Kravtsova-Ivantsiv, Yelena; Bercovich, Beatrice; Ciechanover, Aaron (Proceedings of the National Academy of Sciences, 2019)
One of the enigmas in the ubiquitin (Ub) field is the requirement for a poly-Ub chain as a proteasomal targeting signal. The canonical chain appears to be longer than the distance between the two Ub-binding proteasomal receptors. Furthermore, genetic manipulation has shown that one receptor subunit is sufficient, which suggests that a single Ub can serve as a degradation signal. To shed light on this mystery, we chemically synthesized tetra-Ub, di-Ub (K 48 -based), and mono-Ub adducts of HA-α-globin, where the distal or proximal Ub moieties were tagged differentially with either Myc or Flag. When incubated in a crude cell extract, the distal Ub moiety in the tetra-Ub adduct was mostly removed by deubiquitinating enzymes (DUBs) and reconjugated to other substrates in the extract. In contrast, the proximal moiety was most likely degraded with the substrate. The efficacy of degradation was proportionate to the chain length; while tetra-Ub globin was an efficient substrate, with mono-Ub globin, we observed rapid removal of the Ub moiety with almost no degradation of the free globin. Taken together, these findings suggest that the proximal moieties are necessary for securing the association of the substrate with the proteasome along the proteolytic process, whereas the distal moieties are important in protecting the proximal moieties from premature deubiquitination. Interestingly, when the same experiment was carried out using purified 26S proteasome, mono- and tetra-Ub globin were similarly degraded, highlighting the roles of the entire repertoire of cellular DUBs in regulating the degradation of proteasomal substrates.

Diverse fate of ubiquitin chain moieties: The proximal is degraded with the target, and the distal protects the proximal from removal and recycles - Sun, Hao; Mali, Sachitanand M.; Singh, Sumeet K.; Meledin, Roman; Brik, Ashraf; Kwon, Yong Tae; Kravtsova-Ivantsiv, Yelena; Bercovich, Beatrice; Ciechanover, Aaron (Proceedings of the National Academy of Sciences, 2019)
One of the enigmas in the ubiquitin (Ub) field is the requirement for a poly-Ub chain as a proteasomal targeting signal. The canonical chain appears to be longer than the distance between the two Ub-binding proteasomal receptors. Furthermore, genetic manipulation has shown that one receptor subunit is sufficient, which suggests that a single Ub can serve as a degradation signal. To shed light on this mystery, we chemically synthesized tetra-Ub, di-Ub (K 48 -based), and mono-Ub adducts of HA-α-globin, where the distal or proximal Ub moieties were tagged differentially with either Myc or Flag. When incubated in a crude cell extract, the distal Ub moiety in the tetra-Ub adduct was mostly removed by deubiquitinating enzymes (DUBs) and reconjugated to other substrates in the extract. In contrast, the proximal moiety was most likely degraded with the substrate. The efficacy of degradation was proportionate to the chain length; while tetra-Ub globin was an efficient substrate, with mono-Ub globin, we observed rapid removal of the Ub moiety with almost no degradation of the free globin. Taken together, these findings suggest that the proximal moieties are necessary for securing the association of the substrate with the proteasome along the proteolytic process, whereas the distal moieties are important in protecting the proximal moieties from premature deubiquitination. Interestingly, when the same experiment was carried out using purified 26S proteasome, mono- and tetra-Ub globin were similarly degraded, highlighting the roles of the entire repertoire of cellular DUBs in regulating the degradation of proteasomal substrates.

Selenolysine: A New Tool for Traceless Isopeptide Bond Formation - Dardashti, Rebecca Notis; Kumar, Shailesh; Sternisha, Shawn M.; Reddy, Post Sai; Miller, Brian G.; Metanis, Norman (Chemistry – A European Journal, 2020)

Selenolysine: A New Tool for Traceless Isopeptide Bond Formation - Dardashti, Rebecca Notis; Kumar, Shailesh; Sternisha, Shawn M.; Reddy, Post Sai; Miller, Brian G.; Metanis, Norman (Chemistry – A European Journal, 2020)

Chemical Synthesis of Diubiquitin-Based Photoaffinity Probes for Selectively Profiling Ubiquitin-Binding Proteins - Liang, Jun; Zhang, Lin; Tan, Xiang-Long; Qi, Yun-Kun; Feng, Shan; Deng, Haiteng; Yan, Yijing; Zheng, Ji-Shen; Liu, Lei; Tian, Chang-Lin (Angewandte Chemie International Edition, 2017)

Chemical Synthesis of Diubiquitin-Based Photoaffinity Probes for Selectively Profiling Ubiquitin-Binding Proteins - Liang, Jun; Zhang, Lin; Tan, Xiang-Long; Qi, Yun-Kun; Feng, Shan; Deng, Haiteng; Yan, Yijing; Zheng, Ji-Shen; Liu, Lei; Tian, Chang-Lin (Angewandte Chemie International Edition, 2017)

Protein Semisynthesis Provides Access to Tau Disease-Associated Post-translational Modifications (PTMs) and Paves the Way to Deciphering the Tau PTM Code in Health and Diseased States - Haj-Yahya, Mahmood; Lashuel, Hilal A. (Journal of the American Chemical Society, 2018)

Protein Semisynthesis Provides Access to Tau Disease-Associated Post-translational Modifications (PTMs) and Paves the Way to Deciphering the Tau PTM Code in Health and Diseased States - Haj-Yahya, Mahmood; Lashuel, Hilal A. (Journal of the American Chemical Society, 2018)

Single Posttranslational Modifications in the Central Repeat Domains of Tau4 Impact its Aggregation and Tubulin Binding - Ellmer, Doris; Brehs, Manuel; Haj‐Yahya, Mahmood; Lashuel, Hilal A.; Becker, Christian F. W. (Angewandte Chemie International Edition, 2019)

Single Posttranslational Modifications in the Central Repeat Domains of Tau4 Impact its Aggregation and Tubulin Binding - Ellmer, Doris; Brehs, Manuel; Haj‐Yahya, Mahmood; Lashuel, Hilal A.; Becker, Christian F. W. (Angewandte Chemie International Edition, 2019)

Engineering the Glutamate Transporter Homologue Glt Ph Using Protein Semisynthesis - Focke, Paul J.; Annen, Alvin W.; Valiyaveetil, Francis I. (Biochemistry, 2015)

Engineering the Glutamate Transporter Homologue Glt Ph Using Protein Semisynthesis - Focke, Paul J.; Annen, Alvin W.; Valiyaveetil, Francis I. (Biochemistry, 2015)

Total chemical synthesis of a thermostable enzyme capable of polymerase chain reaction - Xu, Weiliang; Jiang, Wenjun; Wang, Jiaxing; Yu, Linping; Chen, Ji; Liu, Xianyu; Liu, Lei; Zhu, Ting F (Cell Discovery, 2017)

Total chemical synthesis of a thermostable enzyme capable of polymerase chain reaction - Xu, Weiliang; Jiang, Wenjun; Wang, Jiaxing; Yu, Linping; Chen, Ji; Liu, Xianyu; Liu, Lei; Zhu, Ting F (Cell Discovery, 2017)

A thermostable d-polymerase for mirror-image PCR - Pech, Andreas; Achenbach, John; Jahnz, Michael; Schülzchen, Simone; Jarosch, Florian; Bordusa, Frank; Klussmann, Sven (Nucleic Acids Research, 2017)

A thermostable d-polymerase for mirror-image PCR - Pech, Andreas; Achenbach, John; Jahnz, Michael; Schülzchen, Simone; Jarosch, Florian; Bordusa, Frank; Klussmann, Sven (Nucleic Acids Research, 2017)

Efficient Semisynthesis of a Tetraphosphorylated Analogue of the Type I TGFβ Receptor - Flavell, Robert R.; Huse, Morgan; Goger, Mike; Trester-Zedlitz, Michelle; Kuriyan, John; Muir, Tom W. (Organic Letters, 2002)

Efficient Semisynthesis of a Tetraphosphorylated Analogue of the Type I TGFβ Receptor - Flavell, Robert R.; Huse, Morgan; Goger, Mike; Trester-Zedlitz, Michelle; Kuriyan, John; Muir, Tom W. (Organic Letters, 2002)

Synthesis and folding of a mirror-image enzyme reveals ambidextrous chaperone activity - Weinstock, M. T.; Jacobsen, M. T.; Kay, M. S. (Proceedings of the National Academy of Sciences, 2014)

Synthesis and folding of a mirror-image enzyme reveals ambidextrous chaperone activity - Weinstock, M. T.; Jacobsen, M. T.; Kay, M. S. (Proceedings of the National Academy of Sciences, 2014)

A semisynthetic Atg3 reveals that acetylation promotes Atg3 membrane binding and Atg8 lipidation - Li, Yi-Tong; Yi, Cong; Chen, Chen-Chen; Lan, Huan; Pan, Man; Zhang, Shao-Jin; Huang, Yi-Chao; Guan, Chao-Jian; Li, Yi-Ming; Yu, Li; Liu, Lei (Nature Communications, 2017)

A semisynthetic Atg3 reveals that acetylation promotes Atg3 membrane binding and Atg8 lipidation - Li, Yi-Tong; Yi, Cong; Chen, Chen-Chen; Lan, Huan; Pan, Man; Zhang, Shao-Jin; Huang, Yi-Chao; Guan, Chao-Jian; Li, Yi-Ming; Yu, Li; Liu, Lei (Nature Communications, 2017)

Chemical Synthesis of Ubiquitinated Peptides with Varying Lengths and Types of Ubiquitin Chains to Explore the Activity of Deubiquitinases - Bavikar, Sudhir N.; Spasser, Liat; Haj-Yahya, Mahmood; Karthikeyan, Subramanian Vedhanarayanan; Moyal, Tal; Ajish Kumar, K. S.; Brik, Ashraf (Angewandte Chemie, 2012)

Chemical Synthesis of Ubiquitinated Peptides with Varying Lengths and Types of Ubiquitin Chains to Explore the Activity of Deubiquitinases - Bavikar, Sudhir N.; Spasser, Liat; Haj-Yahya, Mahmood; Karthikeyan, Subramanian Vedhanarayanan; Moyal, Tal; Ajish Kumar, K. S.; Brik, Ashraf (Angewandte Chemie, 2012)

An amalgamation of solid phase peptide synthesis and ribosomal peptide synthesis - Ottesen, Jennifer J.; Bar-Dagan, Maya; Giovani, Baldissera; Muir, Tom W. (Biopolymers, 2008)

An amalgamation of solid phase peptide synthesis and ribosomal peptide synthesis - Ottesen, Jennifer J.; Bar-Dagan, Maya; Giovani, Baldissera; Muir, Tom W. (Biopolymers, 2008)

Nonenzymatic Polyubiquitination of Expressed Proteins - Hemantha, Hosahalli P.; Bavikar, Sudhir N.; Herman-Bachinsky, Yifat; Haj-Yahya, Najat; Bondalapati, Somasekhar; Ciechanover, Aaron; Brik, Ashraf (Journal of the American Chemical Society, 2014)

Nonenzymatic Polyubiquitination of Expressed Proteins - Hemantha, Hosahalli P.; Bavikar, Sudhir N.; Herman-Bachinsky, Yifat; Haj-Yahya, Najat; Bondalapati, Somasekhar; Ciechanover, Aaron; Brik, Ashraf (Journal of the American Chemical Society, 2014)

Reconstitution of the Catalytic Core of F-ATPase (αβ) 3 γ from Escherichia coli Using Chemically Synthesized Subunit γ - Wintermann, Frank; Engelbrecht, Siegfried (Angewandte Chemie International Edition, 2013)

Cysteine-Aminoethylation-Assisted Chemical Ubiquitination of Recombinant Histones - Chu, Guo-Chao; Pan, Man; Li, Jiabin; Liu, Sanling; Zuo, Chong; Tong, Ze-Bin; Bai, Jing-Si; Gong, Qingyue; Ai, Huasong; Fan, Jian; Meng, Xianbin; Huang, Yi-Chao; et al. (Journal of the American Chemical Society, 2019)

Ligation of Soluble but Unreactive Peptide Segments in the Chemical Synthesis of Haemophilus Influenzae DNA Ligase - Zhang, Baochang; Deng, Qiang; Zuo, Chong; Yan, Bingjia; Zuo, Chao; Cao, Xiu‐Xiu; Zhu, Ting F.; Zheng, Ji‐Shen; Liu, Lei (Angewandte Chemie International Edition, 2019)

Copying Life: Synthesis of an Enzymatically Active Mirror-Image DNA-Ligase Made of D-Amino Acids - Weidmann, Joachim; Schnölzer, Martina; Dawson, Philip E.; Hoheisel, Jörg D. (Cell Chemical Biology, 2019)

Synthesis and Crystal Structure of a Phosphorylated Estrogen Receptor Ligand Binding Domain - Möcklinghoff, Sabine; Rose, Rolf; Carraz, Maëlle; Visser, Arie; Ottmann, Christian; Brunsveld, Luc (ChemBioChem, 2010)

A Semisynthesis Platform for Investigating Structure−Function Relationships in the N-Terminal Domain of the Anthrax Lethal Factor - Pentelute, Brad L.; Barker, Adam P.; Janowiak, Blythe E.; Kent, Stephen B. H.; Collier, R. John (ACS Chemical Biology, 2010)

Reconstitution of the Catalytic Core of F-ATPase (αβ) 3 γ from Escherichia coli Using Chemically Synthesized Subunit γ - Wintermann, Frank; Engelbrecht, Siegfried (Angewandte Chemie International Edition, 2013)

Total chemical synthesis of SUMO-2-Lys63-linked diubiquitin hybrid chains assisted by removable solubilizing tags - Bondalapati, Somasekhar; Eid, Emad; Mali, Sachitanand M.; Wolberger, Cynthia; Brik, Ashraf (Chemical Science, 2017)
We report the first total chemical synthesis of four different SUMO-2-Lys63-linked di-ubiquitin hybrid chains, in which the di-ubiquitin is linked to different lysines in SUMO. , Small ubiquitin like modifier (SUMO) proteins are known to regulate many important cellular processes such as transcription and apoptosis. Recently, hybrid SUMO-ubiquitin chains containing SUMO-2 linked to Lys63-di-ubiquitin were found to play a major role in DNA repair. Despite some progress in understanding the role of these hybrid chains in DNA repair, there are various fundamental questions remaining to be answered. To further investigate the importance of hybrid SUMO-ubiquitin chains in DNA repair, the homogenous material of these chains, and their unique analogues, are needed in workable quantities. By applying advanced chemical strategies for protein synthesis, we report the first total chemical synthesis of four different SUMO-2-Lys63-linked di-ubiquitin hybrid chains, in which the di-ubiquitin is linked to different lysines in SUMO. In these syntheses, the usefulness of removable solubilizing tags is demonstrated, and two different approaches were examined in terms of reliability and efficiency. In the first approach, a poly-Arg tag was attached to the C-terminus of SUMO via a 3,4-diaminobenzoic acid cleavable linker, whereas in the second we attached the tag via a phenylacetamidomethyl linker, which can be cleaved by PdCl 2 . The comparison between these different strategies offers guidelines for future scale-up preparation of these analogues and other proteins, which currently use synthetic peptide intermediates that are difficult to handle and purify. The availability of the SUMO-ubiquitin hybrid chains opens up new opportunities for studying the role of these chains in DNA repair and other cellular processes.

Cysteine-Aminoethylation-Assisted Chemical Ubiquitination of Recombinant Histones - Chu, Guo-Chao; Pan, Man; Li, Jiabin; Liu, Sanling; Zuo, Chong; Tong, Ze-Bin; Bai, Jing-Si; Gong, Qingyue; Ai, Huasong; Fan, Jian; Meng, Xianbin; Huang, Yi-Chao; et al. (Journal of the American Chemical Society, 2019)

Ligation of Soluble but Unreactive Peptide Segments in the Chemical Synthesis of Haemophilus Influenzae DNA Ligase - Zhang, Baochang; Deng, Qiang; Zuo, Chong; Yan, Bingjia; Zuo, Chao; Cao, Xiu‐Xiu; Zhu, Ting F.; Zheng, Ji‐Shen; Liu, Lei (Angewandte Chemie International Edition, 2019)

Copying Life: Synthesis of an Enzymatically Active Mirror-Image DNA-Ligase Made of D-Amino Acids - Weidmann, Joachim; Schnölzer, Martina; Dawson, Philip E.; Hoheisel, Jörg D. (Cell Chemical Biology, 2019)

Photo-cleavable purification/protection handle assisted synthesis of giant modified proteins with tandem repeats - Liu, Xueyi; Liu, Jiazhi; Wu, Zhichao; Chen, Liangbiao; Wang, Siyao; Wang, Ping (Chemical Science, 2019)
A new N-terminal protection/purification handle (PPH), which contained a His 6 tag for purification and a photo-cleavable linker, facilitates the efficient synthesis of large proteins with tandem repeats. , Proteins with tandem repeats have essential physical or biological roles in cells and have been widely investigated as biomaterials or vaccines. Chemically derived proteins with tandem repeats would be beneficial for research, owing to their accurate structures, possibly with precise modifications, produced by chemical synthesis. Traditional protein synthesis often leads to severe handling loss due to multiple ligations and HPLC purifications. To improve the protein synthesis efficiency, we developed a purification/protection handle consisting of a His 6 tag and a photo-labile linker. This handle has great potential to facilitate purification with immobilized metal affinity chromatography techniques and also provides orthogonal protection for N-terminal Cys. The synthesis of the model proteins Muc1 and antifreeze glycoprotein mimics shows that the handle decreases the requirement for HPLC and enables both convergent and sequential assembly of peptide segments.

Total Chemical Synthesis of ISGylated-Ubiquitin Hybrid Chain Assisted by Acetamidomethyl Derivatives with Dual Functions - Eid, Emad; Boross, Gábor N.; Sun, Hao; Msallam, Muna; Singh, Sumeet K.; Brik, Ashraf (Bioconjugate Chemistry, 2020)

Synthesis and Crystal Structure of a Phosphorylated Estrogen Receptor Ligand Binding Domain - Möcklinghoff, Sabine; Rose, Rolf; Carraz, Maëlle; Visser, Arie; Ottmann, Christian; Brunsveld, Luc (ChemBioChem, 2010)

A Semisynthesis Platform for Investigating Structure−Function Relationships in the N-Terminal Domain of the Anthrax Lethal Factor - Pentelute, Brad L.; Barker, Adam P.; Janowiak, Blythe E.; Kent, Stephen B. H.; Collier, R. John (ACS Chemical Biology, 2010)

Synthesis of Poly-Ubiquitin Chains Using a Bifunctional Ubiquitin Monomer - van der Heden van Noort, Gerbrand J.; Kooij, Raymond; Elliott, Paul R.; Komander, David; Ovaa, Huib (Organic Letters, 2017)

Total chemical synthesis of SUMO-2-Lys63-linked diubiquitin hybrid chains assisted by removable solubilizing tags - Bondalapati, Somasekhar; Eid, Emad; Mali, Sachitanand M.; Wolberger, Cynthia; Brik, Ashraf (Chemical Science, 2017)
We report the first total chemical synthesis of four different SUMO-2-Lys63-linked di-ubiquitin hybrid chains, in which the di-ubiquitin is linked to different lysines in SUMO. , Small ubiquitin like modifier (SUMO) proteins are known to regulate many important cellular processes such as transcription and apoptosis. Recently, hybrid SUMO-ubiquitin chains containing SUMO-2 linked to Lys63-di-ubiquitin were found to play a major role in DNA repair. Despite some progress in understanding the role of these hybrid chains in DNA repair, there are various fundamental questions remaining to be answered. To further investigate the importance of hybrid SUMO-ubiquitin chains in DNA repair, the homogenous material of these chains, and their unique analogues, are needed in workable quantities. By applying advanced chemical strategies for protein synthesis, we report the first total chemical synthesis of four different SUMO-2-Lys63-linked di-ubiquitin hybrid chains, in which the di-ubiquitin is linked to different lysines in SUMO. In these syntheses, the usefulness of removable solubilizing tags is demonstrated, and two different approaches were examined in terms of reliability and efficiency. In the first approach, a poly-Arg tag was attached to the C-terminus of SUMO via a 3,4-diaminobenzoic acid cleavable linker, whereas in the second we attached the tag via a phenylacetamidomethyl linker, which can be cleaved by PdCl 2 . The comparison between these different strategies offers guidelines for future scale-up preparation of these analogues and other proteins, which currently use synthetic peptide intermediates that are difficult to handle and purify. The availability of the SUMO-ubiquitin hybrid chains opens up new opportunities for studying the role of these chains in DNA repair and other cellular processes.

Photo-cleavable purification/protection handle assisted synthesis of giant modified proteins with tandem repeats - Liu, Xueyi; Liu, Jiazhi; Wu, Zhichao; Chen, Liangbiao; Wang, Siyao; Wang, Ping (Chemical Science, 2019)
A new N-terminal protection/purification handle (PPH), which contained a His 6 tag for purification and a photo-cleavable linker, facilitates the efficient synthesis of large proteins with tandem repeats. , Proteins with tandem repeats have essential physical or biological roles in cells and have been widely investigated as biomaterials or vaccines. Chemically derived proteins with tandem repeats would be beneficial for research, owing to their accurate structures, possibly with precise modifications, produced by chemical synthesis. Traditional protein synthesis often leads to severe handling loss due to multiple ligations and HPLC purifications. To improve the protein synthesis efficiency, we developed a purification/protection handle consisting of a His 6 tag and a photo-labile linker. This handle has great potential to facilitate purification with immobilized metal affinity chromatography techniques and also provides orthogonal protection for N-terminal Cys. The synthesis of the model proteins Muc1 and antifreeze glycoprotein mimics shows that the handle decreases the requirement for HPLC and enables both convergent and sequential assembly of peptide segments.

Total Chemical Synthesis of ISGylated-Ubiquitin Hybrid Chain Assisted by Acetamidomethyl Derivatives with Dual Functions - Eid, Emad; Boross, Gábor N.; Sun, Hao; Msallam, Muna; Singh, Sumeet K.; Brik, Ashraf (Bioconjugate Chemistry, 2020)

Synthesis of Poly-Ubiquitin Chains Using a Bifunctional Ubiquitin Monomer - van der Heden van Noort, Gerbrand J.; Kooij, Raymond; Elliott, Paul R.; Komander, David; Ovaa, Huib (Organic Letters, 2017)

Chemical Synthesis of Natural Polyubiquitin Chains through Auxiliary-Mediated Ligation of an Expressed Ubiquitin Isomer - Xu, Ling; Huang, Jian-Feng; Chen, Chen-Chen; Qu, Qian; Shi, Jing; Pan, Man; Li, Yi-Ming (Organic Letters, 2018)

Advanced Hierarchical Vesicular Carbon Co-Doped with S, P, N for High-Rate Sodium Storage - Zou, Guoqiang; Hou, Hongshuai; Foster, Christopher W.; Banks, Craig E.; Guo, Tianxiao; Jiang, Yunling; Zhang, Yun; Ji, Xiaobo (Advanced Science, 2018)

Chemical Synthesis of Natural Polyubiquitin Chains through Auxiliary-Mediated Ligation of an Expressed Ubiquitin Isomer - Xu, Ling; Huang, Jian-Feng; Chen, Chen-Chen; Qu, Qian; Shi, Jing; Pan, Man; Li, Yi-Ming (Organic Letters, 2018)

Advanced Hierarchical Vesicular Carbon Co-Doped with S, P, N for High-Rate Sodium Storage - Zou, Guoqiang; Hou, Hongshuai; Foster, Christopher W.; Banks, Craig E.; Guo, Tianxiao; Jiang, Yunling; Zhang, Yun; Ji, Xiaobo (Advanced Science, 2018)

Quasi-Racemic X-ray Structures of K27-Linked Ubiquitin Chains Prepared by Total Chemical Synthesis - Pan, Man; Gao, Shuai; Zheng, Yong; Tan, Xiaodan; Lan, Huan; Tan, Xianglong; Sun, Demeng; Lu, Lining; Wang, Tian; Zheng, Qingyun; Huang, Yichao; Wang, Jiawei; et al. (Journal of the American Chemical Society, 2016)

Efficient Semi‐Synthesis of Atypical Ubiquitin Chains and Ubiquitin‐Based Probes Forged by Thioether Isopeptide Bonds - Chu, Guo‐Chao; Hua, Xiao; Zuo, Chong; Chen, Chen‐Chen; Meng, Xian‐Bin; Zhang, Zhongping; Fu, Yao; Shi, Jing; Li, Yi‐Ming (Chemistry – A European Journal, 2019)

Total chemical synthesis of ester-linked ubiquitinated proteins unravels their behavior with deubiquitinases - Sun, Hao; Meledin, Roman; Mali, Sachitanand M.; Brik, Ashraf (Chemical Science, 2018)
The novel synthetic strategy for preparation of ester linked ubiquitinated proteins was developed. We found that the ester linkage could be cleaved by deubiquitinases with different efficiency relative to the isopeptide-linked substrate. , Ester-linked ubiquitinated proteins have been reported by several groups to be involved in ubiquitin signalling. However, due to the lack of the suitable tools to homogeneously produce such conjugates, their exact physiological roles and biochemical behavior remain enigmatic. Here, we report for the first time on the development of a novel synthetic strategy based on total chemical synthesis of proteins to construct ubiquitinated proteins, where ubiquitin is linked to the substrate via an ester bond. In this study, we prepared ester- and isopeptide-linked ubiquitinated α-globin and examined their relative behaviors with various deubiquitinases. We found that deubiquitinases are able to cleave the ester linkage with different efficiency relative to the isopeptide-linked substrate. These results may indicate that ester-linked ubiquitinated proteins are natural substrates for deubiquitinases.

Quasi-Racemic X-ray Structures of K27-Linked Ubiquitin Chains Prepared by Total Chemical Synthesis - Pan, Man; Gao, Shuai; Zheng, Yong; Tan, Xiaodan; Lan, Huan; Tan, Xianglong; Sun, Demeng; Lu, Lining; Wang, Tian; Zheng, Qingyun; Huang, Yichao; Wang, Jiawei; et al. (Journal of the American Chemical Society, 2016)

Efficient Semi‐Synthesis of Atypical Ubiquitin Chains and Ubiquitin‐Based Probes Forged by Thioether Isopeptide Bonds - Chu, Guo‐Chao; Hua, Xiao; Zuo, Chong; Chen, Chen‐Chen; Meng, Xian‐Bin; Zhang, Zhongping; Fu, Yao; Shi, Jing; Li, Yi‐Ming (Chemistry – A European Journal, 2019)

A bi-terminal protein ligation strategy to probe chromatin structure during DNA damage - Kilic, Sinan; Boichenko, Iuliia; Lechner, Carolin C.; Fierz, Beat (Chemical Science, 2018)
A convenient method to bi-terminally modify proteins using recombinant masking groups reveals that H2A.X ubiquitylation opens chromatin during DNA repair. , The cellular response to DNA damage results in a signaling cascade that primes chromatin for repair. Combinatorial post-translational modifications (PTMs) play an important role in this process by altering the physical properties of chromatin and recruiting downstream factors. One key signal integrator is the histone variant H2A.X, which is phosphorylated at a C-terminal serine (S139ph), and ubiquitylated within its N-terminal tail at lysines 13 and 15 (K13/15ub). How these PTMs directly impact chromatin structure and thereby facilitate DNA repair is not well understood. Detailed studies require synthetic access to such N- and C-terminally modified proteins. This is complicated by the requirement for protecting groups allowing multi-fragment assembly. Here, we report a semi-synthetic route to generate simultaneously N- and C-terminally modified proteins using genetically encoded orthogonal masking groups. Applied to H2A.X, expression of a central protein fragment, containing a protected N-terminal cysteine and a C-terminal thioester masked as a split intein, enables sequential C- and N-terminal protein modification and results in the convergent production of H2A.X carrying K15ub and S139ph. Using single-molecule FRET between defined nucleosomes in synthetic chromatin fibers, we then show that K15 ubiquitylation (but not S139ph) impairs nucleosome stacking in tetranucleosome units, opening chromatin during DNA repair.

Total chemical synthesis of ester-linked ubiquitinated proteins unravels their behavior with deubiquitinases - Sun, Hao; Meledin, Roman; Mali, Sachitanand M.; Brik, Ashraf (Chemical Science, 2018)
The novel synthetic strategy for preparation of ester linked ubiquitinated proteins was developed. We found that the ester linkage could be cleaved by deubiquitinases with different efficiency relative to the isopeptide-linked substrate. , Ester-linked ubiquitinated proteins have been reported by several groups to be involved in ubiquitin signalling. However, due to the lack of the suitable tools to homogeneously produce such conjugates, their exact physiological roles and biochemical behavior remain enigmatic. Here, we report for the first time on the development of a novel synthetic strategy based on total chemical synthesis of proteins to construct ubiquitinated proteins, where ubiquitin is linked to the substrate via an ester bond. In this study, we prepared ester- and isopeptide-linked ubiquitinated α-globin and examined their relative behaviors with various deubiquitinases. We found that deubiquitinases are able to cleave the ester linkage with different efficiency relative to the isopeptide-linked substrate. These results may indicate that ester-linked ubiquitinated proteins are natural substrates for deubiquitinases.

Aromatic thiol-mediated cleavage of N–O bonds enables chemical ubiquitylation of folded proteins - Weller, Caroline E.; Dhall, Abhinav; Ding, Feizhi; Linares, Edlaine; Whedon, Samuel D.; Senger, Nicholas A.; Tyson, Elizabeth L.; Bagert, John D.; Li, Xiaosong; Augusto, Ohara; Chatterjee, Champak (Nature Communications, 2016)

A bi-terminal protein ligation strategy to probe chromatin structure during DNA damage - Kilic, Sinan; Boichenko, Iuliia; Lechner, Carolin C.; Fierz, Beat (Chemical Science, 2018)
A convenient method to bi-terminally modify proteins using recombinant masking groups reveals that H2A.X ubiquitylation opens chromatin during DNA repair. , The cellular response to DNA damage results in a signaling cascade that primes chromatin for repair. Combinatorial post-translational modifications (PTMs) play an important role in this process by altering the physical properties of chromatin and recruiting downstream factors. One key signal integrator is the histone variant H2A.X, which is phosphorylated at a C-terminal serine (S139ph), and ubiquitylated within its N-terminal tail at lysines 13 and 15 (K13/15ub). How these PTMs directly impact chromatin structure and thereby facilitate DNA repair is not well understood. Detailed studies require synthetic access to such N- and C-terminally modified proteins. This is complicated by the requirement for protecting groups allowing multi-fragment assembly. Here, we report a semi-synthetic route to generate simultaneously N- and C-terminally modified proteins using genetically encoded orthogonal masking groups. Applied to H2A.X, expression of a central protein fragment, containing a protected N-terminal cysteine and a C-terminal thioester masked as a split intein, enables sequential C- and N-terminal protein modification and results in the convergent production of H2A.X carrying K15ub and S139ph. Using single-molecule FRET between defined nucleosomes in synthetic chromatin fibers, we then show that K15 ubiquitylation (but not S139ph) impairs nucleosome stacking in tetranucleosome units, opening chromatin during DNA repair.

Semisynthesis of human thymidine monophosphate kinase - Gogolin, Lars; Seidel, Ralf; Engelhard, Martin; Goody, Roger S.; Becker, Christian F. W. (Biopolymers, 2010)

Aromatic thiol-mediated cleavage of N–O bonds enables chemical ubiquitylation of folded proteins - Weller, Caroline E.; Dhall, Abhinav; Ding, Feizhi; Linares, Edlaine; Whedon, Samuel D.; Senger, Nicholas A.; Tyson, Elizabeth L.; Bagert, John D.; Li, Xiaosong; Augusto, Ohara; Chatterjee, Champak (Nature Communications, 2016)

The convergent chemical synthesis of histone H3 protein for site-specific acetylation at Lys56 and ubiquitination at Lys122 - Qi, Yun-Kun; He, Qiao-Qiao; Ai, Hua-Song; Guo, Jing; Li, Jia-Bin (Chemical Communications, 2017)
The first total chemical synthesis of modified H3 bearing Lys56 acetylation and Lys122 ubiquitination. , Deposition of (H3–H4) 2 tetramers is believed to be the critical step in nucleosome assembly. Site-specific acetylation and ubiquitination of histone H3 have been speculated to synergistically facilitate the formation and deposition of (H3–H4) 2 tetramers. Here we report our endeavors toward the first chemical synthesis of homogenous histone H3, which bears Lys56 acetylation and Lys122 ubiquitination, for in vitro biochemical and biophysical studies.

Convergent Hybrid Phase Ligation Strategy for Efficient Total Synthesis of Large Proteins Demonstrated for 212-residue Linker Histone H1.2 - Hong, Ziyong Z.; Yu, Ruixuan R.; Zhang, Xiaoyu; Webb, Allison M.; Burge, Nathaniel L.; Poirier, Michael G.; Ottesen, Jennifer J. (Biochemistry, 2019)
Abstract Simple and efficient total chemical synthesis of large proteins remains a significant challenge. Here, we report development of a convergent hybrid phase native chemical ligation (CHP-NCL) strategy that should be generally applicable for facile preparation of large proteins. Key to the strategy is the use of sequential ligation on the solid phase for the directed assembly of ~100-residue segments from short, synthetically accessible peptide components. These segments can then be assembled via convergent solution phase ligation, exploiting o-aminoaniline as a chemically flexible cryptic thioester with multiple activation modalitiies on resin and in situ . We demonstrate the feasibility of our approach through the total synthesis of 212-residue linker histone H1.2 in unmodified, phosphorylated, and citrullinated forms, each from eight component peptide segments. We further demonstrate that fully synthetic H1.2 replicates the binding interactions of linker histones to intact mononucleosomes, as a proxy for the essential function of linker histones in the formation and regulation of higher order chromatin structure.

Semisynthesis of human thymidine monophosphate kinase - Gogolin, Lars; Seidel, Ralf; Engelhard, Martin; Goody, Roger S.; Becker, Christian F. W. (Biopolymers, 2010)

Synthesis of ubiquitylated histone H3 using a thiirane linker for chemical ligation: Synthesis of ubiquitylated histone H3 using a thiirane linker - Kawakami, Toru; Mishima, Yuichi; Hojo, Hironobu; Suetake, Isao (Journal of Peptide Science, 2017)

The convergent chemical synthesis of histone H3 protein for site-specific acetylation at Lys56 and ubiquitination at Lys122 - Qi, Yun-Kun; He, Qiao-Qiao; Ai, Hua-Song; Guo, Jing; Li, Jia-Bin (Chemical Communications, 2017)
The first total chemical synthesis of modified H3 bearing Lys56 acetylation and Lys122 ubiquitination. , Deposition of (H3–H4) 2 tetramers is believed to be the critical step in nucleosome assembly. Site-specific acetylation and ubiquitination of histone H3 have been speculated to synergistically facilitate the formation and deposition of (H3–H4) 2 tetramers. Here we report our endeavors toward the first chemical synthesis of homogenous histone H3, which bears Lys56 acetylation and Lys122 ubiquitination, for in vitro biochemical and biophysical studies.

Chemical synthesis of the ubiquitinated form of histone H3 and its effect on DNA methyltransferase 1 - Kawakami, Toru; Mishima, Yuichi; Takazawa, Masaya; Hojo, Hironobu; Suetake, Isao (Journal of Peptide Science, 2019)

Convergent Hybrid Phase Ligation Strategy for Efficient Total Synthesis of Large Proteins Demonstrated for 212-residue Linker Histone H1.2 - Hong, Ziyong Z.; Yu, Ruixuan R.; Zhang, Xiaoyu; Webb, Allison M.; Burge, Nathaniel L.; Poirier, Michael G.; Ottesen, Jennifer J. (Biochemistry, 2019)
Abstract Simple and efficient total chemical synthesis of large proteins remains a significant challenge. Here, we report development of a convergent hybrid phase native chemical ligation (CHP-NCL) strategy that should be generally applicable for facile preparation of large proteins. Key to the strategy is the use of sequential ligation on the solid phase for the directed assembly of ~100-residue segments from short, synthetically accessible peptide components. These segments can then be assembled via convergent solution phase ligation, exploiting o-aminoaniline as a chemically flexible cryptic thioester with multiple activation modalitiies on resin and in situ . We demonstrate the feasibility of our approach through the total synthesis of 212-residue linker histone H1.2 in unmodified, phosphorylated, and citrullinated forms, each from eight component peptide segments. We further demonstrate that fully synthetic H1.2 replicates the binding interactions of linker histones to intact mononucleosomes, as a proxy for the essential function of linker histones in the formation and regulation of higher order chromatin structure.

Semisynthetic prion protein (PrP) variants carrying glycan mimics at position 181 and 197 do not form fibrils - Araman, Can; Thompson, Robert E.; Wang, Siyao; Hackl, Stefanie; Payne, Richard J.; Becker, Christian F. W. (Chemical Science, 2017)
Semisynthesis and characterization of homogeneously mono- and di-PEGylated full length PrP variants to study the impact of PEGylation (as N -glycan mimics) on protein folding and aggregation. , The prion protein (PrP) is an N -glycosylated protein attached to the outer leaflet of eukaryotic cell membranes via a glycosylphosphatidylinositol (GPI) anchor. Different prion strains have distinct glycosylation patterns and the extent of glycosylation of potentially pathogenic misfolded prion protein (PrP Sc ) has a major impact on several prion-related diseases (transmissible spongiform encephalopathies, TSEs). Based on these findings it is hypothesized that posttranslational modifications (PTMs) of PrP influence conversion of cellular prion protein (PrP C ) into PrP Sc and, as such, modified PrP variants are critical tools needed to investigate the impact of PTMs on the pathogenesis of TSEs. Here we report a semisynthetic approach to generate PrP variants modified with monodisperse polyethyleneglycol (PEG) units as mimics of N-glycans. Incorporating PEG at glycosylation sites 181 and 197 in PrP induced only small changes to the secondary structure when compared to unmodified, wildtype PrP. More importantly, in vitro aggregation was abrogated for all PEGylated PrP variants under conditions at which wildtype PrP aggregated. Furthermore, the addition of PEGylated PrP as low as 10 mol% to wildtype PrP completely blocked aggregation. A similar effect was observed for synthetic PEGylated PrP segments comprising amino acids 179–231 alone if these were added to wildtype PrP in aggregation assays. This behavior raises the question if large N-glycans interfere with aggregation in vivo and if PEGylated PrP peptides could serve as potential therapeutics.

Synthesis of ubiquitylated histone H3 using a thiirane linker for chemical ligation: Synthesis of ubiquitylated histone H3 using a thiirane linker - Kawakami, Toru; Mishima, Yuichi; Hojo, Hironobu; Suetake, Isao (Journal of Peptide Science, 2017)

Chemical synthesis of the ubiquitinated form of histone H3 and its effect on DNA methyltransferase 1 - Kawakami, Toru; Mishima, Yuichi; Takazawa, Masaya; Hojo, Hironobu; Suetake, Isao (Journal of Peptide Science, 2019)

Utility of the Phenacyl Protecting Group in Traceless Protein Semisynthesis through Ligation-Desulfurization Chemistry - Matveenko, Maria; Hackl, Stefanie; Becker, Christian F. W. (ChemistryOpen, 2018)

Semisynthetic prion protein (PrP) variants carrying glycan mimics at position 181 and 197 do not form fibrils - Araman, Can; Thompson, Robert E.; Wang, Siyao; Hackl, Stefanie; Payne, Richard J.; Becker, Christian F. W. (Chemical Science, 2017)
Semisynthesis and characterization of homogeneously mono- and di-PEGylated full length PrP variants to study the impact of PEGylation (as N -glycan mimics) on protein folding and aggregation. , The prion protein (PrP) is an N -glycosylated protein attached to the outer leaflet of eukaryotic cell membranes via a glycosylphosphatidylinositol (GPI) anchor. Different prion strains have distinct glycosylation patterns and the extent of glycosylation of potentially pathogenic misfolded prion protein (PrP Sc ) has a major impact on several prion-related diseases (transmissible spongiform encephalopathies, TSEs). Based on these findings it is hypothesized that posttranslational modifications (PTMs) of PrP influence conversion of cellular prion protein (PrP C ) into PrP Sc and, as such, modified PrP variants are critical tools needed to investigate the impact of PTMs on the pathogenesis of TSEs. Here we report a semisynthetic approach to generate PrP variants modified with monodisperse polyethyleneglycol (PEG) units as mimics of N-glycans. Incorporating PEG at glycosylation sites 181 and 197 in PrP induced only small changes to the secondary structure when compared to unmodified, wildtype PrP. More importantly, in vitro aggregation was abrogated for all PEGylated PrP variants under conditions at which wildtype PrP aggregated. Furthermore, the addition of PEGylated PrP as low as 10 mol% to wildtype PrP completely blocked aggregation. A similar effect was observed for synthetic PEGylated PrP segments comprising amino acids 179–231 alone if these were added to wildtype PrP in aggregation assays. This behavior raises the question if large N-glycans interfere with aggregation in vivo and if PEGylated PrP peptides could serve as potential therapeutics.

Examination of the Deubiquitylation Site Selectivity of USP51 by Using Chemically Synthesized Ubiquitylated Histones - Ai, Huasong; Guo, Yu; Sun, Demeng; Liu, Sanling; Qi, Yunkun; Guo, Jing; Qu, Qian; Gong, Qingyue; Zhao, Suwen; Li, Jiabin; Liu, Lei (ChemBioChem, 2019)

Utility of the Phenacyl Protecting Group in Traceless Protein Semisynthesis through Ligation-Desulfurization Chemistry - Matveenko, Maria; Hackl, Stefanie; Becker, Christian F. W. (ChemistryOpen, 2018)

Convergent Chemical Synthesis and Crystal Structure of a 203 Amino Acid “Covalent Dimer” HIV-1 Protease Enzyme Molecule - Torbeev, Vladimir Yu.; Kent, Stephen B. H. (Angewandte Chemie International Edition, 2007)

Convergent Versus Sequential Protein Synthesis: The Case of Ubiquitinated and Glycosylated H2B - Seenaiah, Mallikanti; Jbara, Muhammad; Mali, Sachitanand M.; Brik, Ashraf (Angewandte Chemie International Edition, 2015)

Convergent Chemical Synthesis of Histone H2B Protein for the Site-Specific Ubiquitination at Lys34 - Siman, Peter; Karthikeyan, Subramanian Vedhanarayanan; Nikolov, Miroslav; Fischle, Wolfgang; Brik, Ashraf (Angewandte Chemie International Edition, 2013)

Examination of the Deubiquitylation Site Selectivity of USP51 by Using Chemically Synthesized Ubiquitylated Histones - Ai, Huasong; Guo, Yu; Sun, Demeng; Liu, Sanling; Qi, Yunkun; Guo, Jing; Qu, Qian; Gong, Qingyue; Zhao, Suwen; Li, Jiabin; Liu, Lei (ChemBioChem, 2019)

Convergent Chemical Synthesis and Crystal Structure of a 203 Amino Acid “Covalent Dimer” HIV-1 Protease Enzyme Molecule - Torbeev, Vladimir Yu.; Kent, Stephen B. H. (Angewandte Chemie International Edition, 2007)

Chemical Synthesis of K34-Ubiquitylated H2B for Nucleosome Reconstitution and Single-Particle Cryo-Electron Microscopy Structural Analysis - Li, Jiabin; He, Qiaoqiao; Liu, Yuntao; Liu, Sanling; Tang, Shan; Li, Chengmin; Sun, Demeng; Li, Xiaorun; Zhou, Min; Zhu, Ping; Bi, Guoqiang; Zhou, Zhenghong; et al. (ChemBioChem, 2017)

Convergent Versus Sequential Protein Synthesis: The Case of Ubiquitinated and Glycosylated H2B - Seenaiah, Mallikanti; Jbara, Muhammad; Mali, Sachitanand M.; Brik, Ashraf (Angewandte Chemie International Edition, 2015)

Palladium Mediated Rapid Deprotection of N-Terminal Cysteine under Native Chemical Ligation Conditions for the Efficient Preparation of Synthetically Challenging Proteins - Jbara, Muhammad; Maity, Suman Kumar; Seenaiah, Mallikanti; Brik, Ashraf (Journal of the American Chemical Society, 2016)

Convergent Chemical Synthesis of Histone H2B Protein for the Site-Specific Ubiquitination at Lys34 - Siman, Peter; Karthikeyan, Subramanian Vedhanarayanan; Nikolov, Miroslav; Fischle, Wolfgang; Brik, Ashraf (Angewandte Chemie International Edition, 2013)

Total chemical synthesis of a biologically active and homogeneous analog of Human Growth Hormone [Nle 14,125,170 ,Glu 29,91 ,Gln 74 ,Asn 107 ,Asp 109 ]hGH-NH 2 by sequential native chemical ligation - Sueiras-Diaz, Javier; Zhang, Ying; Velentza, Anastasia; Santoso, Buyung; Yang, Suijin (Tetrahedron Letters, 2017)

Chemical Synthesis of K34-Ubiquitylated H2B for Nucleosome Reconstitution and Single-Particle Cryo-Electron Microscopy Structural Analysis - Li, Jiabin; He, Qiaoqiao; Liu, Yuntao; Liu, Sanling; Tang, Shan; Li, Chengmin; Sun, Demeng; Li, Xiaorun; Zhou, Min; Zhu, Ping; Bi, Guoqiang; Zhou, Zhenghong; et al. (ChemBioChem, 2017)

The discovery of a freezing-induced peptide ligation during the total chemical synthesis of human interferon-ε - Yang, Yin-He; Di, Bin; Yang, Da-Song (Organic & Biomolecular Chemistry, 2018)
The first example of a counterintuitive freezing-induced peptide ligation was discovered during the total synthesis of human interferon-ε (187 amino acids) which blocks HIV infection through unique mechanisms. , A counterintuitive freezing-induced peptide ligation was discovered during the total synthesis of human interferon-ε (hIFN-ε) which blocks HIV infection through unique mechanisms. The successful synthesis of hIFN-ε (187 amino acids) in this research laid the foundation for related anti-AIDS drug development. Moreover, alanine mutation based on sequence alignment to solve the maldistribution of the ligation site and freezing-induced dominant conformation that facilitates peptide ligation are expected to be helpful for the synthesis of macrobiomolecules.

Palladium Mediated Rapid Deprotection of N-Terminal Cysteine under Native Chemical Ligation Conditions for the Efficient Preparation of Synthetically Challenging Proteins - Jbara, Muhammad; Maity, Suman Kumar; Seenaiah, Mallikanti; Brik, Ashraf (Journal of the American Chemical Society, 2016)

Chemical synthesis of a polypeptide backbone derived from the primary sequence of the cancer protein NY-ESO-1 enabled by kinetically controlled ligation and pseudoprolines: Chemical Synthesis of a Polypeptide Backbone - Harris, Paul W. R.; Brimble, Margaret A. (Biopolymers, 2015)

Total chemical synthesis of a biologically active and homogeneous analog of Human Growth Hormone [Nle 14,125,170 ,Glu 29,91 ,Gln 74 ,Asn 107 ,Asp 109 ]hGH-NH 2 by sequential native chemical ligation - Sueiras-Diaz, Javier; Zhang, Ying; Velentza, Anastasia; Santoso, Buyung; Yang, Suijin (Tetrahedron Letters, 2017)

Total Chemical Synthesis of SUMO and SUMO-Based Probes for Profiling the Activity of SUMO-Specific Proteases - Mulder, Monique P. C.; Merkx, Remco; Witting, Katharina F.; Hameed, Dharjath S.; El Atmioui, Dris; Lelieveld, Lindsey; Liebelt, Frauke; Neefjes, Jacques; Berlin, Ilana; Vertegaal, Alfred C. O.; Ovaa, Huib (Angewandte Chemie International Edition, 2018)

Chemical Synthesis of the 20 kDa Heme Protein Nitrophorin 4 by α-Ketoacid-Hydroxylamine (KAHA) Ligation - He, Chunmao; Kulkarni, Sameer S.; Thuaud, Frédéric; Bode, Jeffrey W. (Angewandte Chemie International Edition, 2015)

The discovery of a freezing-induced peptide ligation during the total chemical synthesis of human interferon-ε - Yang, Yin-He; Di, Bin; Yang, Da-Song (Organic & Biomolecular Chemistry, 2018)
The first example of a counterintuitive freezing-induced peptide ligation was discovered during the total synthesis of human interferon-ε (187 amino acids) which blocks HIV infection through unique mechanisms. , A counterintuitive freezing-induced peptide ligation was discovered during the total synthesis of human interferon-ε (hIFN-ε) which blocks HIV infection through unique mechanisms. The successful synthesis of hIFN-ε (187 amino acids) in this research laid the foundation for related anti-AIDS drug development. Moreover, alanine mutation based on sequence alignment to solve the maldistribution of the ligation site and freezing-induced dominant conformation that facilitates peptide ligation are expected to be helpful for the synthesis of macrobiomolecules.

Chemical synthesis of a polypeptide backbone derived from the primary sequence of the cancer protein NY-ESO-1 enabled by kinetically controlled ligation and pseudoprolines: Chemical Synthesis of a Polypeptide Backbone - Harris, Paul W. R.; Brimble, Margaret A. (Biopolymers, 2015)

Total Chemical Synthesis of All SUMO-2/3 Dimer Combinations - Bouchenna, Jennifer; Sénéchal, Magalie; Drobecq, Hervé; Vicogne, Jérôme; Melnyk, Oleg (Bioconjugate Chemistry, 2019)

Total Chemical Synthesis of SUMO and SUMO-Based Probes for Profiling the Activity of SUMO-Specific Proteases - Mulder, Monique P. C.; Merkx, Remco; Witting, Katharina F.; Hameed, Dharjath S.; El Atmioui, Dris; Lelieveld, Lindsey; Liebelt, Frauke; Neefjes, Jacques; Berlin, Ilana; Vertegaal, Alfred C. O.; Ovaa, Huib (Angewandte Chemie International Edition, 2018)

Chemical Synthesis of the 20 kDa Heme Protein Nitrophorin 4 by α-Ketoacid-Hydroxylamine (KAHA) Ligation - He, Chunmao; Kulkarni, Sameer S.; Thuaud, Frédéric; Bode, Jeffrey W. (Angewandte Chemie International Edition, 2015)

Semisynthesis of Biologically Active Glycoforms of the Human Cytokine Interleukin 6 - Reif, Andreas; Siebenhaar, Sebastian; Tröster, Andrea; Schmälzlein, Marina; Lechner, Carolin; Velisetty, Phanindra; Gottwald, Karen; Pöhner, Claudia; Boos, Irene; Schubert, Volker; Rose-John, Stefan; Unverzagt, Carlo (Angewandte Chemie International Edition, 2014)

Total Chemical Synthesis of All SUMO-2/3 Dimer Combinations - Bouchenna, Jennifer; Sénéchal, Magalie; Drobecq, Hervé; Vicogne, Jérôme; Melnyk, Oleg (Bioconjugate Chemistry, 2019)

Mutagenesis Studies of Substrate Recognition and Catalysis in the Sortase A Transpeptidase from Staphylococcus aureus - Bentley, Matthew L.; Lamb, Erin C.; McCafferty, Dewey G. (Journal of Biological Chemistry, 2008)

Semisynthesis of Biologically Active Glycoforms of the Human Cytokine Interleukin 6 - Reif, Andreas; Siebenhaar, Sebastian; Tröster, Andrea; Schmälzlein, Marina; Lechner, Carolin; Velisetty, Phanindra; Gottwald, Karen; Pöhner, Claudia; Boos, Irene; Schubert, Volker; Rose-John, Stefan; Unverzagt, Carlo (Angewandte Chemie International Edition, 2014)

Mutagenesis Studies of Substrate Recognition and Catalysis in the Sortase A Transpeptidase from Staphylococcus aureus - Bentley, Matthew L.; Lamb, Erin C.; McCafferty, Dewey G. (Journal of Biological Chemistry, 2008)

A simple and traceless solid phase method simplifies the assembly of large peptides and the access to challenging proteins - Ollivier, N.; Desmet, R.; Drobecq, H.; Blanpain, A.; Boll, E.; Leclercq, B.; Mougel, A.; Vicogne, J.; Melnyk, O. (Chemical Science, 2017)
We show that the combination of solid phase and solution ligation techniques facilitates the production of a challenging and biologically active protein made of 180 amino acids. , Chemical protein synthesis gives access to well-defined native or modified proteins that are useful for studying protein structure and function. The majority of proteins synthesized up to now have been produced using native chemical ligation (NCL) in solution. Although there are significant advantages to assembling large peptides or proteins by solid phase ligation, reports of such approaches are rare. We report a novel solid phase method for protein synthesis which relies on the chemistry of the acetoacetyl group and ketoxime ligation for the attachment of the peptide to the solid support, and on a tandem transoximation/rearrangement process for the detachment of the target protein. Importantly, we show that the combination of solid phase and solution ligation techniques facilitates the production of a challenging and biologically active protein made of 180 amino acids. We show also that the solid phase method enables the purification of complex peptide segments through a chemoselective solid phase capture/release approach.

Accelerating chemoselective peptide bond formation using bis(2-selenylethyl)amido peptide selenoester surrogates - Raibaut, Laurent; Cargoët, Marine; Ollivier, Nathalie; Chang, Yun Min; Drobecq, Hervé; Boll, Emmanuelle; Desmet, Rémi; Monbaliu, Jean-Christophe M.; Melnyk, Oleg (Chemical Science, 2016)
SeEA latent selenoester: go fast by switching to selenium. , Given the potential of peptide selenoesters for protein total synthesis and the paucity of methods for the synthesis of these sensitive peptide derivatives, we sought to explore the usefulness of the bis(2-selenylethyl)amido (SeEA) group, i.e. the selenium analog of the bis(2-sulfanylethyl)amido (SEA) group, for accelerating peptide bond formation. A chemoselective exchange process operating in water was devised for converting SEA peptides into the SeEA ones. Kinetic studies show that SeEA ligation, which relies on an initial N , Se -acyl shift process, proceeds significantly faster than SEA ligation. This property enabled the design of a kinetically controlled three peptide segment assembly process based on the sequential use of SeEA and SEA ligation reactions. The method was validated by the total synthesis of hepatocyte growth factor K1 (85 AA) and biotinylated NK1 (180 AA) domains.

Mirror‐Image 5S Ribonucleoprotein Complexes - Ling, Jun‐Jie; Fan, Chuyao; Qin, Hong; Wang, Min; Chen, Ji; Wittung‐Stafshede, Pernilla; Zhu, Ting F. (Angewandte Chemie International Edition, 2020)

A simple and traceless solid phase method simplifies the assembly of large peptides and the access to challenging proteins - Ollivier, N.; Desmet, R.; Drobecq, H.; Blanpain, A.; Boll, E.; Leclercq, B.; Mougel, A.; Vicogne, J.; Melnyk, O. (Chemical Science, 2017)
We show that the combination of solid phase and solution ligation techniques facilitates the production of a challenging and biologically active protein made of 180 amino acids. , Chemical protein synthesis gives access to well-defined native or modified proteins that are useful for studying protein structure and function. The majority of proteins synthesized up to now have been produced using native chemical ligation (NCL) in solution. Although there are significant advantages to assembling large peptides or proteins by solid phase ligation, reports of such approaches are rare. We report a novel solid phase method for protein synthesis which relies on the chemistry of the acetoacetyl group and ketoxime ligation for the attachment of the peptide to the solid support, and on a tandem transoximation/rearrangement process for the detachment of the target protein. Importantly, we show that the combination of solid phase and solution ligation techniques facilitates the production of a challenging and biologically active protein made of 180 amino acids. We show also that the solid phase method enables the purification of complex peptide segments through a chemoselective solid phase capture/release approach.

Protein chemical synthesis by α-ketoacid–hydroxylamine ligation - Harmand, Thibault J; Murar, Claudia E; Bode, Jeffrey W (Nature Protocols, 2016)

Accelerating chemoselective peptide bond formation using bis(2-selenylethyl)amido peptide selenoester surrogates - Raibaut, Laurent; Cargoët, Marine; Ollivier, Nathalie; Chang, Yun Min; Drobecq, Hervé; Boll, Emmanuelle; Desmet, Rémi; Monbaliu, Jean-Christophe M.; Melnyk, Oleg (Chemical Science, 2016)
SeEA latent selenoester: go fast by switching to selenium. , Given the potential of peptide selenoesters for protein total synthesis and the paucity of methods for the synthesis of these sensitive peptide derivatives, we sought to explore the usefulness of the bis(2-selenylethyl)amido (SeEA) group, i.e. the selenium analog of the bis(2-sulfanylethyl)amido (SEA) group, for accelerating peptide bond formation. A chemoselective exchange process operating in water was devised for converting SEA peptides into the SeEA ones. Kinetic studies show that SeEA ligation, which relies on an initial N , Se -acyl shift process, proceeds significantly faster than SEA ligation. This property enabled the design of a kinetically controlled three peptide segment assembly process based on the sequential use of SeEA and SEA ligation reactions. The method was validated by the total synthesis of hepatocyte growth factor K1 (85 AA) and biotinylated NK1 (180 AA) domains.

Fully Synthetic Granulocyte Colony-Stimulating Factor Enabled by Isonitrile-Mediated Coupling of Large, Side-Chain-Unprotected Peptides - Roberts, Andrew G.; Johnston, Eric V.; Shieh, Jae-Hung; Sondey, Joseph P.; Hendrickson, Ronald C.; Moore, Malcolm A. S.; Danishefsky, Samuel J. (Journal of the American Chemical Society, 2015)

Mirror‐Image 5S Ribonucleoprotein Complexes - Ling, Jun‐Jie; Fan, Chuyao; Qin, Hong; Wang, Min; Chen, Ji; Wittung‐Stafshede, Pernilla; Zhu, Ting F. (Angewandte Chemie International Edition, 2020)

A synthetic molecular system capable of mirror-image genetic replication and transcription - Wang, Zimou; Xu, Weiliang; Liu, Lei; Zhu, Ting F. (Nature Chemistry, 2016)

Semisynthesis of Erythropoietin Analog Having Three Oligosaccharides - Hirano, Kiriko; Izumi, Masayuki; Macmillan, Derek; Tezuka, Katsunari; Tsuji, Takashi; Kajihara, Yasuhiro (Journal of Carbohydrate Chemistry, 2011)

Total chemical synthesis of a functional interacting protein pair: The protooncogene H-Ras and the Ras-binding domain of its effector c-Raf1 - Becker, C. F. W.; Hunter, C. L.; Seidel, R.; Kent, S. B. H.; Goody, R. S.; Engelhard, M. (Proceedings of the National Academy of Sciences, 2003)

Protein chemical synthesis by α-ketoacid–hydroxylamine ligation - Harmand, Thibault J; Murar, Claudia E; Bode, Jeffrey W (Nature Protocols, 2016)

Fully Synthetic Granulocyte Colony-Stimulating Factor Enabled by Isonitrile-Mediated Coupling of Large, Side-Chain-Unprotected Peptides - Roberts, Andrew G.; Johnston, Eric V.; Shieh, Jae-Hung; Sondey, Joseph P.; Hendrickson, Ronald C.; Moore, Malcolm A. S.; Danishefsky, Samuel J. (Journal of the American Chemical Society, 2015)

Chemical Synthesis of Homogeneous Human Glycosyl-interferon-β That Exhibits Potent Antitumor Activity in Vivo - Sakamoto, Izumi; Tezuka, Katsunari; Fukae, Kazuhiro; Ishii, Kazuyuki; Taduru, Keisuke; Maeda, Masatoshi; Ouchi, Masaki; Yoshida, Kenta; Nambu, Yuri; Igarashi, Jun; Hayashi, Naohiro; Tsuji, Takashi; et al. (Journal of the American Chemical Society, 2012)

A synthetic molecular system capable of mirror-image genetic replication and transcription - Wang, Zimou; Xu, Weiliang; Liu, Lei; Zhu, Ting F. (Nature Chemistry, 2016)

Chemical Synthesis of an Erythropoietin Glycoform Containing a Complex-type Disialyloligosaccharide - Murakami, Masumi; Okamoto, Ryo; Izumi, Masayuki; Kajihara, Yasuhiro (Angewandte Chemie, 2012)

Semisynthesis of Erythropoietin Analog Having Three Oligosaccharides - Hirano, Kiriko; Izumi, Masayuki; Macmillan, Derek; Tezuka, Katsunari; Tsuji, Takashi; Kajihara, Yasuhiro (Journal of Carbohydrate Chemistry, 2011)

At Last: Erythropoietin as a Single Glycoform - Wang, Ping; Dong, Suwei; Brailsford, John A.; Iyer, Karthik; Townsend, Steven D.; Zhang, Qiang; Hendrickson, Ronald C.; Shieh, JaeHung; Moore, Malcolm A. S.; Danishefsky, Samuel J. (Angewandte Chemie International Edition, 2012)

Total chemical synthesis of a functional interacting protein pair: The protooncogene H-Ras and the Ras-binding domain of its effector c-Raf1 - Becker, C. F. W.; Hunter, C. L.; Seidel, R.; Kent, S. B. H.; Goody, R. S.; Engelhard, M. (Proceedings of the National Academy of Sciences, 2003)

Semisynthesis of H-Ras with a glutamic acid methylester at position 61 - Milić, Jelena; Seidel, Ralf; Becker, Christian F. W.; Goody, Roger. S.; Engelhard, Martin (Biopolymers, 2008)

Chemical Synthesis of Homogeneous Human Glycosyl-interferon-β That Exhibits Potent Antitumor Activity in Vivo - Sakamoto, Izumi; Tezuka, Katsunari; Fukae, Kazuhiro; Ishii, Kazuyuki; Taduru, Keisuke; Maeda, Masatoshi; Ouchi, Masaki; Yoshida, Kenta; Nambu, Yuri; Igarashi, Jun; Hayashi, Naohiro; Tsuji, Takashi; et al. (Journal of the American Chemical Society, 2012)

Erythropoietin Derived by Chemical Synthesis - Wang, P.; Dong, S.; Shieh, J.-H.; Peguero, E.; Hendrickson, R.; Moore, M. A. S.; Danishefsky, S. J. (Science, 2013)

Chemical Synthesis of an Erythropoietin Glycoform Containing a Complex-type Disialyloligosaccharide - Murakami, Masumi; Okamoto, Ryo; Izumi, Masayuki; Kajihara, Yasuhiro (Angewandte Chemie, 2012)

At Last: Erythropoietin as a Single Glycoform - Wang, Ping; Dong, Suwei; Brailsford, John A.; Iyer, Karthik; Townsend, Steven D.; Zhang, Qiang; Hendrickson, Ronald C.; Shieh, JaeHung; Moore, Malcolm A. S.; Danishefsky, Samuel J. (Angewandte Chemie International Edition, 2012)

Design and Synthesis of a Homogeneous Erythropoietin Analogue with Two Human Complex-Type Sialyloligosaccharides: Combined Use of Chemical and Bacterial Protein Expression Methods - Hirano, Kiriko; Macmillan, Derek; Tezuka, Katsunari; Tsuji, Takashi; Kajihara, Yasuhiro (Angewandte Chemie International Edition, 2009)

Reconciling past changes in Earth’s rotation with 20th century global sea-level rise: Resolving Munk’s enigma - Mitrovica, Jerry X.; Hay, Carling C.; Morrow, Eric; Kopp, Robert E.; Dumberry, Mathieu; Stanley, Sabine (Science Advances, 2015)
In 2002, Munk defined an important enigma of 20th century global mean sea-level (GMSL) rise that has yet to be resolved. First, he listed three canonical observations related to Earth’s rotation [(i) the slowing of Earth’s rotation rate over the last three millennia inferred from ancient eclipse observations, and changes in the (ii) amplitude and (iii) orientation of Earth’s rotation vector over the last century estimated from geodetic and astronomic measurements] and argued that they could all be fit by a model of ongoing glacial isostatic adjustment (GIA) associated with the last ice age. Second, he demonstrated that prevailing estimates of the 20th century GMSL rise (~1.5 to 2.0 mm/year), after correction for the maximum signal from ocean thermal expansion, implied mass flux from ice sheets and glaciers at a level that would grossly misfit the residual GIA-corrected observations of Earth’s rotation. We demonstrate that the combination of lower estimates of the 20th century GMSL rise (up to 1990) improved modeling of the GIA process and that the correction of the eclipse record for a signal due to angular momentum exchange between the fluid outer core and the mantle reconciles all three Earth rotation observations. This resolution adds confidence to recent estimates of individual contributions to 20th century sea-level change and to projections of GMSL rise to the end of the 21st century based on them.

Semisynthesis of H-Ras with a glutamic acid methylester at position 61 - Milić, Jelena; Seidel, Ralf; Becker, Christian F. W.; Goody, Roger. S.; Engelhard, Martin (Biopolymers, 2008)

Erythropoietin Derived by Chemical Synthesis - Wang, P.; Dong, S.; Shieh, J.-H.; Peguero, E.; Hendrickson, R.; Moore, M. A. S.; Danishefsky, S. J. (Science, 2013)

Chemical synthesis of erythropoietin glycoforms for insights into the relationship between glycosylation pattern and bioactivity - Murakami, Masumi; Kiuchi, Tatsuto; Nishihara, Mika; Tezuka, Katsunari; Okamoto, Ryo; Izumi, Masayuki; Kajihara, Yasuhiro (Science Advances, 2016)
The role of sialyloligosaccharides on the surface of secreted glycoproteins is still unclear because of the difficulty in the preparation of sialylglycoproteins in a homogeneous form. We selected erythropoietin (EPO) as a target molecule and designed an efficient synthetic strategy for the chemical synthesis of a homogeneous form of five EPO glycoforms varying in glycosylation position and the number of human-type biantennary sialyloligosaccharides. A segment coupling strategy performed by native chemical ligation using six peptide segments including glycopeptides yielded homogeneous EPO glycopeptides, and folding experiments of these glycopeptides afforded the correctly folded EPO glycoforms. In an in vivo erythropoiesis assay in mice, all of the EPO glycoforms displayed biological activity, in particular the EPO bearing three sialyloligosaccharides, which exhibited the highest activity. Furthermore, we observed that the hydrophilicity and biological activity of the EPO glycoforms varied depending on the glycosylation pattern. This knowledge will pave the way for the development of homogeneous biologics by chemical synthesis.

Design and Synthesis of a Homogeneous Erythropoietin Analogue with Two Human Complex-Type Sialyloligosaccharides: Combined Use of Chemical and Bacterial Protein Expression Methods - Hirano, Kiriko; Macmillan, Derek; Tezuka, Katsunari; Tsuji, Takashi; Kajihara, Yasuhiro (Angewandte Chemie International Edition, 2009)

Reconciling past changes in Earth’s rotation with 20th century global sea-level rise: Resolving Munk’s enigma - Mitrovica, Jerry X.; Hay, Carling C.; Morrow, Eric; Kopp, Robert E.; Dumberry, Mathieu; Stanley, Sabine (Science Advances, 2015)
In 2002, Munk defined an important enigma of 20th century global mean sea-level (GMSL) rise that has yet to be resolved. First, he listed three canonical observations related to Earth’s rotation [(i) the slowing of Earth’s rotation rate over the last three millennia inferred from ancient eclipse observations, and changes in the (ii) amplitude and (iii) orientation of Earth’s rotation vector over the last century estimated from geodetic and astronomic measurements] and argued that they could all be fit by a model of ongoing glacial isostatic adjustment (GIA) associated with the last ice age. Second, he demonstrated that prevailing estimates of the 20th century GMSL rise (~1.5 to 2.0 mm/year), after correction for the maximum signal from ocean thermal expansion, implied mass flux from ice sheets and glaciers at a level that would grossly misfit the residual GIA-corrected observations of Earth’s rotation. We demonstrate that the combination of lower estimates of the 20th century GMSL rise (up to 1990) improved modeling of the GIA process and that the correction of the eclipse record for a signal due to angular momentum exchange between the fluid outer core and the mantle reconciles all three Earth rotation observations. This resolution adds confidence to recent estimates of individual contributions to 20th century sea-level change and to projections of GMSL rise to the end of the 21st century based on them.

Chemical synthesis of erythropoietin glycoforms for insights into the relationship between glycosylation pattern and bioactivity - Murakami, Masumi; Kiuchi, Tatsuto; Nishihara, Mika; Tezuka, Katsunari; Okamoto, Ryo; Izumi, Masayuki; Kajihara, Yasuhiro (Science Advances, 2016)
The role of sialyloligosaccharides on the surface of secreted glycoproteins is still unclear because of the difficulty in the preparation of sialylglycoproteins in a homogeneous form. We selected erythropoietin (EPO) as a target molecule and designed an efficient synthetic strategy for the chemical synthesis of a homogeneous form of five EPO glycoforms varying in glycosylation position and the number of human-type biantennary sialyloligosaccharides. A segment coupling strategy performed by native chemical ligation using six peptide segments including glycopeptides yielded homogeneous EPO glycopeptides, and folding experiments of these glycopeptides afforded the correctly folded EPO glycoforms. In an in vivo erythropoiesis assay in mice, all of the EPO glycoforms displayed biological activity, in particular the EPO bearing three sialyloligosaccharides, which exhibited the highest activity. Furthermore, we observed that the hydrophilicity and biological activity of the EPO glycoforms varied depending on the glycosylation pattern. This knowledge will pave the way for the development of homogeneous biologics by chemical synthesis.

Nutritional input from dinoflagellate symbionts in reef-building corals is minimal during planula larval life stage - Kopp, Christophe; Domart-Coulon, Isabelle; Barthelemy, Dominique; Meibom, Anders (Science Advances, 2016)
Dispersion of larval offspring is of fundamental ecological importance to sessile marine organisms. Photosymbiotic planulae emitted by many reef-forming corals may travel over large distances before settling to form a new colony. It is not clear whether the metabolic requirements of these planula larvae are met exclusively with lipid and protein reservoirs inherited from the mother colony or when metabolic inputs from their endosymbiotic dinoflagellates become important. Pulse-chase experiments using [ 13 C]bicarbonate and [ 15 N]nitrate, combined with subcellular structural and isotopic imaging of freshly emitted symbiotic larvae from the coral Pocillopora damicornis , show that metabolic input from the dinoflagellates is minimal in the planulae compared with adult colonies. The larvae are essentially lecithotrophic upon emission, indicating that a marked shift in metabolic interaction between the symbiotic partners takes place later during ontogeny. Understanding the cellular processes that trigger and control this metabolic shift, and how climate change might influence it, is a key challenge in coral biology.

Nutritional input from dinoflagellate symbionts in reef-building corals is minimal during planula larval life stage - Kopp, Christophe; Domart-Coulon, Isabelle; Barthelemy, Dominique; Meibom, Anders (Science Advances, 2016)
Dispersion of larval offspring is of fundamental ecological importance to sessile marine organisms. Photosymbiotic planulae emitted by many reef-forming corals may travel over large distances before settling to form a new colony. It is not clear whether the metabolic requirements of these planula larvae are met exclusively with lipid and protein reservoirs inherited from the mother colony or when metabolic inputs from their endosymbiotic dinoflagellates become important. Pulse-chase experiments using [ 13 C]bicarbonate and [ 15 N]nitrate, combined with subcellular structural and isotopic imaging of freshly emitted symbiotic larvae from the coral Pocillopora damicornis , show that metabolic input from the dinoflagellates is minimal in the planulae compared with adult colonies. The larvae are essentially lecithotrophic upon emission, indicating that a marked shift in metabolic interaction between the symbiotic partners takes place later during ontogeny. Understanding the cellular processes that trigger and control this metabolic shift, and how climate change might influence it, is a key challenge in coral biology.

Past and future rainfall in the Horn of Africa - Tierney, Jessica E.; Ummenhofer, Caroline C.; deMenocal, Peter B. (Science Advances, 2015)
The recent decline in Horn of Africa rainfall during the March–May “long rains” season has fomented drought and famine, threatening food security in an already vulnerable region. Some attribute this decline to anthropogenic forcing, whereas others maintain that it is a feature of internal climate variability. We show that the rate of drying in the Horn of Africa during the 20th century is unusual in the context of the last 2000 years, is synchronous with recent global and regional warming, and therefore may have an anthropogenic component. In contrast to 20th century drying, climate models predict that the Horn of Africa will become wetter as global temperatures rise. The projected increase in rainfall mainly occurs during the September–November “short rains” season, in response to large-scale weakening of the Walker circulation. Most of the models overestimate short rains precipitation while underestimating long rains precipitation, causing the Walker circulation response to unrealistically dominate the annual mean. Our results highlight the need for accurate simulation of the seasonal cycle and an improved understanding of the dynamics of the long rains season to predict future rainfall in the Horn of Africa.

Past and future rainfall in the Horn of Africa - Tierney, Jessica E.; Ummenhofer, Caroline C.; deMenocal, Peter B. (Science Advances, 2015)
The recent decline in Horn of Africa rainfall during the March–May “long rains” season has fomented drought and famine, threatening food security in an already vulnerable region. Some attribute this decline to anthropogenic forcing, whereas others maintain that it is a feature of internal climate variability. We show that the rate of drying in the Horn of Africa during the 20th century is unusual in the context of the last 2000 years, is synchronous with recent global and regional warming, and therefore may have an anthropogenic component. In contrast to 20th century drying, climate models predict that the Horn of Africa will become wetter as global temperatures rise. The projected increase in rainfall mainly occurs during the September–November “short rains” season, in response to large-scale weakening of the Walker circulation. Most of the models overestimate short rains precipitation while underestimating long rains precipitation, causing the Walker circulation response to unrealistically dominate the annual mean. Our results highlight the need for accurate simulation of the seasonal cycle and an improved understanding of the dynamics of the long rains season to predict future rainfall in the Horn of Africa.

Total Chemical Synthesis and Folding of All- l and All- d Variants of Oncogenic KRas(G12V) - Levinson, Adam M.; McGee, John H.; Roberts, Andrew G.; Creech, Gardner S.; Wang, Ting; Peterson, Michael T.; Hendrickson, Ronald C.; Verdine, Gregory L.; Danishefsky, Samuel J. (Journal of the American Chemical Society, 2017)

Total Chemical Synthesis and Folding of All- l and All- d Variants of Oncogenic KRas(G12V) - Levinson, Adam M.; McGee, John H.; Roberts, Andrew G.; Creech, Gardner S.; Wang, Ting; Peterson, Michael T.; Hendrickson, Ronald C.; Verdine, Gregory L.; Danishefsky, Samuel J. (Journal of the American Chemical Society, 2017)

Studies on the Precise Chemical Synthesis of Human Glycoproteins - Kajihara, Yasuhiro (Bulletin of the Chemical Society of Japan, 2016)

Studies on the Precise Chemical Synthesis of Human Glycoproteins - Kajihara, Yasuhiro (Bulletin of the Chemical Society of Japan, 2016)

A synthetic approach to ‘click’ neoglycoprotein analogues of EPO employing one-pot native chemical ligation and CuAAC chemistry - Lee, D. J.; Cameron, A. J.; Wright, T. H.; Harris, P. W. R.; Brimble, M. A. (Chemical Science, 2019)
The batch-wise variability of commercial erythropoietin (EPO) preparations warrants development of more advanced synthetic methodologies. We have developed a diverse chemical toolkit to prepare ‘click’ neoglycoprotein variants of EPO. , The clinical significance of batch-wise variability on the pharmacokinetics and potency of commercial erythropoietin (EPO), prepared recombinantly as a heterogeneous mixture of glycoforms, necessitates the development of synthetic strategies to afford homogenous EPO formulations. Herein we present a previously unexplored and divergent route towards ‘click’ neoglycoprotein analogues of EPO, employing one-pot native chemical ligation (NCL) of alkynylated peptides and copper-catalysed azide–alkyne cycloaddition (CuAAC) with azido monosaccharides. By design, our synthetic platform permits glycosylation at virtually any stage, providing flexibility for the synthesis of various glycoforms for biological analysis. Insights obtained from attempted folding of our ‘click’ neoglycoprotein EPO analogue, bearing four different neutral sugar moieties, highlight the important role played by the charged oligosaccharides present in native EPO glycoproteins.

A synthetic approach to ‘click’ neoglycoprotein analogues of EPO employing one-pot native chemical ligation and CuAAC chemistry - Lee, D. J.; Cameron, A. J.; Wright, T. H.; Harris, P. W. R.; Brimble, M. A. (Chemical Science, 2019)
The batch-wise variability of commercial erythropoietin (EPO) preparations warrants development of more advanced synthetic methodologies. We have developed a diverse chemical toolkit to prepare ‘click’ neoglycoprotein variants of EPO. , The clinical significance of batch-wise variability on the pharmacokinetics and potency of commercial erythropoietin (EPO), prepared recombinantly as a heterogeneous mixture of glycoforms, necessitates the development of synthetic strategies to afford homogenous EPO formulations. Herein we present a previously unexplored and divergent route towards ‘click’ neoglycoprotein analogues of EPO, employing one-pot native chemical ligation (NCL) of alkynylated peptides and copper-catalysed azide–alkyne cycloaddition (CuAAC) with azido monosaccharides. By design, our synthetic platform permits glycosylation at virtually any stage, providing flexibility for the synthesis of various glycoforms for biological analysis. Insights obtained from attempted folding of our ‘click’ neoglycoprotein EPO analogue, bearing four different neutral sugar moieties, highlight the important role played by the charged oligosaccharides present in native EPO glycoproteins.

Convergent Chemical Synthesis of [Lysine24, 38, 83] Human Erythropoietin - Liu, Suhuai; Pentelute, Brad L.; Kent, Stephen B. H. (Angewandte Chemie International Edition, 2012)

Convergent Chemical Synthesis of [Lysine24, 38, 83] Human Erythropoietin - Liu, Suhuai; Pentelute, Brad L.; Kent, Stephen B. H. (Angewandte Chemie International Edition, 2012)

Total chemical synthesis of human interferon alpha-2b via native chemical ligation: SYNTHESIS OF IFNΑ2B VIA NATIVE CHEMICAL LIGATION - Li, Jing; Lehmann, Clara; Chen, Xishan; Romerio, Fabio; Lu, Wuyuan (Journal of Peptide Science, 2015)

Total chemical synthesis of human interferon alpha-2b via native chemical ligation: SYNTHESIS OF IFNΑ2B VIA NATIVE CHEMICAL LIGATION - Li, Jing; Lehmann, Clara; Chen, Xishan; Romerio, Fabio; Lu, Wuyuan (Journal of Peptide Science, 2015)

The total chemical synthesis of the monoglycosylated GM2 ganglioside activator using a novel cysteine surrogate - Sato, Kohei; Kitakaze, Keisuke; Nakamura, Takahiro; Naruse, Naoto; Aihara, Keisuke; Shigenaga, Akira; Inokuma, Tsubasa; Tsuji, Daisuke; Itoh, Kohji; Otaka, Akira (Chemical Communications, 2015)
We describe a novel peptide ligation/desulfurization strategy using a β-mercapto- N -glycosylated asparagine derivative. The strategy is successfully applied to the total chemical synthesis of GM2 ganglioside activator protein. , We describe a novel peptide ligation/desulfurization strategy using a β-mercapto- N -glycosylated asparagine derivative. The newly developed procedure was successfully applied to the total chemical synthesis of the GM2 ganglioside activator protein bearing a monosaccharide on the native glycosylation site.

The total chemical synthesis of the monoglycosylated GM2 ganglioside activator using a novel cysteine surrogate - Sato, Kohei; Kitakaze, Keisuke; Nakamura, Takahiro; Naruse, Naoto; Aihara, Keisuke; Shigenaga, Akira; Inokuma, Tsubasa; Tsuji, Daisuke; Itoh, Kohji; Otaka, Akira (Chemical Communications, 2015)
We describe a novel peptide ligation/desulfurization strategy using a β-mercapto- N -glycosylated asparagine derivative. The strategy is successfully applied to the total chemical synthesis of GM2 ganglioside activator protein. , We describe a novel peptide ligation/desulfurization strategy using a β-mercapto- N -glycosylated asparagine derivative. The newly developed procedure was successfully applied to the total chemical synthesis of the GM2 ganglioside activator protein bearing a monosaccharide on the native glycosylation site.

Chemical Synthesis of Biologically Active Monoglycosylated GM2-Activator Protein Analogue Using N -Sulfanylethylanilide Peptide - Sato, Kohei; Shigenaga, Akira; Kitakaze, Keisuke; Sakamoto, Ken; Tsuji, Daisuke; Itoh, Kohji; Otaka, Akira (Angewandte Chemie International Edition, 2013)

Chemical Synthesis of Biologically Active Monoglycosylated GM2-Activator Protein Analogue Using N -Sulfanylethylanilide Peptide - Sato, Kohei; Shigenaga, Akira; Kitakaze, Keisuke; Sakamoto, Ken; Tsuji, Daisuke; Itoh, Kohji; Otaka, Akira (Angewandte Chemie International Edition, 2013)

A novel post-ligation thioesterification device enables peptide ligation in the N to C direction: synthetic study of human glycodelin: CHEMICAL SYNTHESIS OF HUMAN GLYCODELIN - Takenouchi, Takaomi; Katayama, Hidekazu; Nakahara, Yuko; Nakahara, Yoshiaki; Hojo, Hironobu (Journal of Peptide Science, 2014)

A novel post-ligation thioesterification device enables peptide ligation in the N to C direction: synthetic study of human glycodelin: CHEMICAL SYNTHESIS OF HUMAN GLYCODELIN - Takenouchi, Takaomi; Katayama, Hidekazu; Nakahara, Yuko; Nakahara, Yoshiaki; Hojo, Hironobu (Journal of Peptide Science, 2014)

Tailored Synthesis of 162-Residue S -Monoglycosylated GM2-Activator Protein (GM2AP) Analogues that Allows Facile Access to a Protein Library - Nakamura, Takahiro; Sato, Kohei; Naruse, Naoto; Kitakaze, Keisuke; Inokuma, Tsubasa; Hirokawa, Takatsugu; Shigenaga, Akira; Itoh, Kohji; Otaka, Akira (ChemBioChem, 2016)

Tailored Synthesis of 162-Residue S -Monoglycosylated GM2-Activator Protein (GM2AP) Analogues that Allows Facile Access to a Protein Library - Nakamura, Takahiro; Sato, Kohei; Naruse, Naoto; Kitakaze, Keisuke; Inokuma, Tsubasa; Hirokawa, Takatsugu; Shigenaga, Akira; Itoh, Kohji; Otaka, Akira (ChemBioChem, 2016)

Auxiliary-assisted chemical ubiquitylation of NEMO and linear extension by HOIP - Burlina, Fabienne; Abdel-Aal, Abu-Baker M.; Raz, Richard; Pinzuti, Irene; Papageorgiou, George; Li, Jiejin; Antrobus, Robin; Martin, Stephen R.; Kunzelmann, Simone; Stieglitz, Benjamin; Offer, John (Communications Chemistry, 2019)

Auxiliary-assisted chemical ubiquitylation of NEMO and linear extension by HOIP - Burlina, Fabienne; Abdel-Aal, Abu-Baker M.; Raz, Richard; Pinzuti, Irene; Papageorgiou, George; Li, Jiejin; Antrobus, Robin; Martin, Stephen R.; Kunzelmann, Simone; Stieglitz, Benjamin; Offer, John (Communications Chemistry, 2019)

Chemical Synthesis of Atomically Tailored SUMO E2 Conjugating Enzymes for the Formation of Covalently Linked SUMO–E2–E3 Ligase Ternary Complexes - Zhang, Yinfeng; Hirota, Tsuyoshi; Kuwata, Keiko; Oishi, Shunsuke; Gramani, Subramanian G.; Bode, Jeffrey W. (Journal of the American Chemical Society, 2019)

Chemical Synthesis of Atomically Tailored SUMO E2 Conjugating Enzymes for the Formation of Covalently Linked SUMO–E2–E3 Ligase Ternary Complexes - Zhang, Yinfeng; Hirota, Tsuyoshi; Kuwata, Keiko; Oishi, Shunsuke; Gramani, Subramanian G.; Bode, Jeffrey W. (Journal of the American Chemical Society, 2019)

Exploring neoglycoprotein assembly through native chemical ligation using neoglycopeptide thioesters prepared via N→S acyl transfer - Richardson, Jonathan P.; Chan, Chung-Hei; Blanc, Javier; Saadi, Mona; Macmillan, Derek (Organic & Biomolecular Chemistry, 2010)

Exploring neoglycoprotein assembly through native chemical ligation using neoglycopeptide thioesters prepared via N→S acyl transfer - Richardson, Jonathan P.; Chan, Chung-Hei; Blanc, Javier; Saadi, Mona; Macmillan, Derek (Organic & Biomolecular Chemistry, 2010)

Access to phosphoproteins and glycoproteins through semi-synthesis, Native Chemical Ligation and N→S acyl transfer - Masania, Jinit; Li, Jiejin; Smerdon, Stephen J.; Macmillan, Derek (Organic & Biomolecular Chemistry, 2010)

Access to phosphoproteins and glycoproteins through semi-synthesis, Native Chemical Ligation and N→S acyl transfer - Masania, Jinit; Li, Jiejin; Smerdon, Stephen J.; Macmillan, Derek (Organic & Biomolecular Chemistry, 2010)

Synthesis of tumor necrosis factor α for use as a mirror-image phage display target - Petersen, Mark E.; Jacobsen, Michael T.; Kay, Michael S. (Organic & Biomolecular Chemistry, 2016)
Chemical synthesis of TNFα, a central regulator of inflammation, for use as a mirror-image phage display target. , Tumor Necrosis Factor alpha (TNFα) is an inflammatory cytokine that plays a central role in the pathogenesis of chronic inflammatory disease. Here we describe the chemical synthesis of l -TNFα along with the mirror-image d -protein for use as a phage display target. The synthetic strategy utilized native chemical ligation and desulfurization to unite three peptide segments, followed by oxidative folding to assemble the 52 kDa homotrimeric protein. This synthesis represents the foundational step for discovering an inhibitory d -peptide with the potential to improve current anti-TNFα therapeutic strategies.

Synthesis of tumor necrosis factor α for use as a mirror-image phage display target - Petersen, Mark E.; Jacobsen, Michael T.; Kay, Michael S. (Organic & Biomolecular Chemistry, 2016)
Chemical synthesis of TNFα, a central regulator of inflammation, for use as a mirror-image phage display target. , Tumor Necrosis Factor alpha (TNFα) is an inflammatory cytokine that plays a central role in the pathogenesis of chronic inflammatory disease. Here we describe the chemical synthesis of l -TNFα along with the mirror-image d -protein for use as a phage display target. The synthetic strategy utilized native chemical ligation and desulfurization to unite three peptide segments, followed by oxidative folding to assemble the 52 kDa homotrimeric protein. This synthesis represents the foundational step for discovering an inhibitory d -peptide with the potential to improve current anti-TNFα therapeutic strategies.

Total chemical synthesis of murine ISG15 and an activity-based probe with physiological binding properties - Xin, Bo-Tao; Gan, Jin; Fernandez, Daniel J.; Knobeloch, Klaus-Peter; Geurink, Paul P.; Ovaa, Huib (Organic & Biomolecular Chemistry, 2019)
The linear synthesis of the N-terminal domain of mISG15 has been developed which enables the synthesis of full-length mISG15 and the activity-based probe Rho-mISG15-PA via native chemical ligation. , The linear synthesis of the N-terminal domain of mISG15 has been developed which enables the synthesis of full-length mISG15 and the activity-based probe Rho-mISG15-PA via native chemical ligation. Pilot experiments showed that the synthetic proteins were properly folded and recognized by endogenous enzymes. Our synthesis strategy allows the generation of other mISG15-based probes and reagents that can accelerate the research in this field.

Total chemical synthesis of murine ISG15 and an activity-based probe with physiological binding properties - Xin, Bo-Tao; Gan, Jin; Fernandez, Daniel J.; Knobeloch, Klaus-Peter; Geurink, Paul P.; Ovaa, Huib (Organic & Biomolecular Chemistry, 2019)
The linear synthesis of the N-terminal domain of mISG15 has been developed which enables the synthesis of full-length mISG15 and the activity-based probe Rho-mISG15-PA via native chemical ligation. , The linear synthesis of the N-terminal domain of mISG15 has been developed which enables the synthesis of full-length mISG15 and the activity-based probe Rho-mISG15-PA via native chemical ligation. Pilot experiments showed that the synthetic proteins were properly folded and recognized by endogenous enzymes. Our synthesis strategy allows the generation of other mISG15-based probes and reagents that can accelerate the research in this field.