The central dogma of modern biology underscores the pivotal roles proteins play in diverse biological processes,the study of which necessitates advanced methods to produce proteins with precision and versatility.Chemi...The central dogma of modern biology underscores the pivotal roles proteins play in diverse biological processes,the study of which necessitates advanced methods to produce proteins with precision and versatility.Chemical protein synthesis,a powerful approach utilizing chemical reactions for the de novo construction of structurally accurate proteins,has emerged as a transformative tool for studying proteins and generating protein derivatives/mimics inaccessible by natural biological machinery,including post-translationally modified proteins,proteins comprised of unnatural amino acids,as well as mirror-image proteins.This review summarizes recent strides in synthetic method developments for chemical protein synthesis,including innovative techniques in solid-phase peptide synthesis,the challenges presented by difficult sequences in either synthesis or folding and the exploration of novel ligation reactions using both chemical and enzymatic methods.Furthermore,the review also delves into newly developed protocols for site-selective protein modifications and the generation of stapled or macrocyclized peptides/miniproteins,highlighting the power of chemical methods to make structurally diverse proteins.Recent applications of synthetic proteins in investigating post-translational modifications(phosphorylation,lipidation,glycosylation,ubiquitination,etc.),mirror-image biological processes and drug development are further discussed.Together,these topics provide a comprehensive overview of the current landscape of chemical protein synthesis.展开更多
The 2-hydroxy-4-methoxybenzyl(Hmb)backbone modification can prevent amide bond-mediated sidereactions(e.g.,aspartimide formation,peptide aggregation)by installing the removable Hmb group into a peptide bond,thus impro...The 2-hydroxy-4-methoxybenzyl(Hmb)backbone modification can prevent amide bond-mediated sidereactions(e.g.,aspartimide formation,peptide aggregation)by installing the removable Hmb group into a peptide bond,thus improving the synthesis of long and challenging peptides and proteins.However,its use is largely precluded by the limited Hmb’s installation sites.In this report,an improved installation of Hmb(iHmb)method was developed to achieve the flexible installation and the convenient removal of Hmb.The iHmb method involves two critical steps:(1)oxidative diazotization of the readily installed 2-hydroxy-4-methoxy-5-amino-benzyl(Hmab)to give 2-hydroxy-4-methoxy-5-diazonium-benzyl(Hmdab)by combining soamyl nitrite(IAN)/HBF_(4),and(2)reductive elimination of Hmdab to give the desired Hmb by 1,2-ethanedithiol(EDT).The iHmb method enables the installation of Hmb at any primary amino acid including the highly sterically hindered amino acids(e.g.,valine and isoleucine).The practicality and utility of the iHmb method was demonstrated by one-shot solid-phase synthesis of a challenging aspartimide-prone peptide,the mirror-image version of a hydrophobic peptide and a long-chain peptide up to 76-residue.Furthermore,the iHmb method can be utilized to facilitate chemical protein ligation,as exemplified by the synthesis of the single-spanning membrane protein sarcolipin.The iHmb method expands the toolkit for peptide synthesis and ligation and facilitates the preparation of peptides/proteins.展开更多
Tyrosine sulfation is an important post-translational modification that enhances the inhibitory activity of hirudin.Herein,we developed a facile synthetic strategy to afford the sulfated hirudins with up to three modi...Tyrosine sulfation is an important post-translational modification that enhances the inhibitory activity of hirudin.Herein,we developed a facile synthetic strategy to afford the sulfated hirudins with up to three modifications and in multi-milligram scales,after a single HPLC purification step.Through these synthetic proteins,a novel type of modulation mechanism exhibited by tyrosine sulfation was proposed,which would help to delineate the structure-function relationships in other sulfated proteins and more importantly,to serve as a basis for the development of related antithrombotic agents.展开更多
Chemical synthesis of proteins containing up to 300 amino acids may cover 30%—50%of all the proteins encountered in biomedical studies and may provide an alternate approach to the usually used recombinant expression ...Chemical synthesis of proteins containing up to 300 amino acids may cover 30%—50%of all the proteins encountered in biomedical studies and may provide an alternate approach to the usually used recombinant expression teclmology,vastly expanding the chemical space of the latter.In the present review article,we tried to survey the recent progresses made for more rapid synthesis of increasingly long peptides and more efficient ligation of multiple peptide segments.The developments of seminal methods by many research groups have greatly contributed to the recent breakthroughs in the successful total synthesis of a number of functionally important proteins,such as oligoubiquitins,bacterial GroEL/ES chaperones,and mirror-image DNA polymerases.Through these studies,a potential bottleneck has also been recognized for the chemical synthesis of large proteins,namely,how to ensure that each peptide segment from a large protein avoids unfavorable aggregation when dissolved in aqueous solution.Many new methods,such as removable backbone modification(RBM)strategy have been developed to overcome this bottleneck,while more studies need to be carried out to develop more effective and less costly methods that ultimately,may lead to fully automatable chemical synthesis of customized proteins of 300 amino acids bearing any artificial designs.展开更多
D-peptides are recognized as a new class of synthetic chemical drugs and they possess many interesting advantages such as high enzymatic stability,improved oral bioavailability,as well as high binding affinity and spe...D-peptides are recognized as a new class of synthetic chemical drugs and they possess many interesting advantages such as high enzymatic stability,improved oral bioavailability,as well as high binding affinity and specificity.Recently,D-peptide drugs have been attracting increasing attention in both academic and industrial researches over recent years.One D-peptide etelcalcetide has even entered the market that targets the calcium(Ca2+)-sensing receptor(CaSR) to fight secondary hyperparathyroidism.Effective discovery and optimization of D-peptide ligands that can bind to various disease-related targets with high specificity and potency is of great importance for the development of D-peptide drugs.This review surveys the recent method development in this area especially the chemical protein synthesis-assisted high-throughput screening strategies for D-peptide ligands and their application in drug discovery.展开更多
Small ubiquitin-like modifiers(SUMOs)are protein modifiers that can form polymeric chains.They are important signals in cellular processes,and their study and profiling require the development of molecular tools.Herei...Small ubiquitin-like modifiers(SUMOs)are protein modifiers that can form polymeric chains.They are important signals in cellular processes,and their study and profiling require the development of molecular tools.Herein,the authors have reported an efficient chemical protein synthesis approach for the generation of dimeric SUMO-2-based photoaffinity probes through the ligation of four readily synthesizable peptides.Proteomic studies using this diSUMO-2 probe on HeLa cell nuclear lysate found it to capture a significantly different selection of proteins compared with its monoSUMO counterparts.This resulted in the identification of several previously unknown SUMO chain-specific interacting proteins such as 40S ribosomal protein S3,which showed a significantly higher affinity for polySUMO chains than monomeric SUMO.Collectively,these results emphasize the need to develop SUMO chain-based probes in other species,and to shed light on the important role of polySUMOylation in diseases.展开更多
Guanylate cyclase C(GC-C) is an important receptor protein expressed by intestinal epithelial cells, and its dysregulation leads to severe intestinal diseases. Linaclotide is a 14-amino acid peptide approved by the FD...Guanylate cyclase C(GC-C) is an important receptor protein expressed by intestinal epithelial cells, and its dysregulation leads to severe intestinal diseases. Linaclotide is a 14-amino acid peptide approved by the FDA for the treatment of irritable bowel syndrome with constipation(IBS-C), which activates guanylate cyclase C to accelerate intestinal transit. Drug molecule design based on structural information plays a crucial role and the activity of linaclotide still need to improve, while the structure of linaclotide remains unknown. In this work, linaclotide and its D-enantiomer were obtained through Fmoc solid phase peptide synthesis method and co-crystalized through racemic crystallization. The crystal structure showed that linaclotide has a tight, three-beta turns structure immobilized by three pairs of disulfide bonds.展开更多
Histone H2 A methylation at Gln104(H2AQ104Me) is a new type of histone post-translational modification(PTM) discovered recently. This modification has been found to have significant influence on gene transcription. Ho...Histone H2 A methylation at Gln104(H2AQ104Me) is a new type of histone post-translational modification(PTM) discovered recently. This modification has been found to have significant influence on gene transcription. However, the structural and functional consequence of glutamine methylation on nucleosome remains to be further elucidated. Obtaining of histones with site-specific methylation at glutamine residues might facilitate the studies towards a better understanding of this new PTM. In the present work, total chemical synthesis of H2AQ104 Me was carried out through use of the hydrazide-based native chemical ligation. Synthetic histone H2AQ104 Me could be successfully incorporated into nucleosomes in vitro and showed a negative influence on the nucleosome stability.展开更多
TNFR1-associated death domain protein(TRADD)with arginine N-GlcNAcylation is a novel and structurally unique posttranslational modification(PTM)glycoprotein that blocks the formation of death-inducing signaling comple...TNFR1-associated death domain protein(TRADD)with arginine N-GlcNAcylation is a novel and structurally unique posttranslational modification(PTM)glycoprotein that blocks the formation of death-inducing signaling complex(DISC),orchestrating host nuclear factorκB(NF-κB)signaling in entero-pathogenic Escherichia coli(EPEC)-infected cells.This particular glycosylated modification plays an extremely vital role for the effective colonization and pathogenesis of pathogens in the gut.Herein we describe the total synthesis of TRADD death domain(residues 195-312)with arginine235 NGlcNAcylation(Arg-GIcNAc TRADD(195-312)).Two longish peptidyl fragments of the wild-type primary sequence were obtained by robust,microwave-assisted,highly efficient,solid-phase peptide synthesis(SPPS),the N-GlcNAcylated sector was built by total synthesis and attached specifically to resinbound peptide with an unprotected ornithine residue via silver-promoted on-resin guanidinylation,ArgGlcNAc TRADD(195-312)was constructed by hydrazide-based native chemical ligation(NCL).The facile synthetic strategy is expected to be generally applicable for the rapid synthesis of other proteins with Arg-GIcNAc modification and to pave the way for the related chemically biological study.展开更多
E3 ubiquitin ligases catalyze the final step of ubiquitylation,a crucial post-translational modification involved in almost every process in eukaryotic cells.E3 ubiquitin ligases are key regulators of cellular events,...E3 ubiquitin ligases catalyze the final step of ubiquitylation,a crucial post-translational modification involved in almost every process in eukaryotic cells.E3 ubiquitin ligases are key regulators of cellular events,and the investigation into their functions and functioning mechanisms are research areas with great importance.Synthetic or semi-synthetic tools have greatly facilitated the research about the enzyme activity,distribution in different physiological events,and catalytic mechanism of E3 ubiquitin ligase.In this review,we summarize the development of chemical tools for E3 ubiquitin ligases with an emphasis on the synthetic routes.We show the utility of these chemical tools by briefly discussing their applications in biological research.展开更多
The H3 bivalent modifications of trimethylationat Lys9 and acetylation at Lys18(H3-K9 Me3-K18 Ac) were identified to collectively recruit TRIM33 in the nodal signaling pathway.To understand the underlying mechanism of...The H3 bivalent modifications of trimethylationat Lys9 and acetylation at Lys18(H3-K9 Me3-K18 Ac) were identified to collectively recruit TRIM33 in the nodal signaling pathway.To understand the underlying mechanism of TRIM33 recruitment,the nucleosome core particles(NCPs) containing full-length H3-K9 Me3-K18 Ac were indispensable samples.Herein we developed a pseudo dipeptide strategy to efficiently prepare peptide segments,facilitating the chemical synthesis of H3-K9 Me3-K18 Ac at a tens of milligram scale.The synthetic H3-K9 Me3-K18 Ac was then examined by CD spectroscopy,which demonstrated a prominent shift compared to recombinant H3.Finally,bivalently modified NCPs were assembled and verified by gel mobility shift assay with good homogeneity.展开更多
基金supported by the National Key R&D Program of China(2022YFC3401500)the National Natural Science Foundation of China(22137005,92253302,22227810 to Lei Liu,22177004,92153301,22321005 to Suwei Dong,22277020 to Yiming Li,22022703,22177108,22377118 to Ji-Shen Zheng,92353302,22177059 to Yongxiang Chen,22177035 to Jun Guo,22277029,22077036 to Chunmao He,22077078 to Honggang Hu92353302,92053108 to Yanmei Li,22277015 to Junfeng Zhao)。
文摘The central dogma of modern biology underscores the pivotal roles proteins play in diverse biological processes,the study of which necessitates advanced methods to produce proteins with precision and versatility.Chemical protein synthesis,a powerful approach utilizing chemical reactions for the de novo construction of structurally accurate proteins,has emerged as a transformative tool for studying proteins and generating protein derivatives/mimics inaccessible by natural biological machinery,including post-translationally modified proteins,proteins comprised of unnatural amino acids,as well as mirror-image proteins.This review summarizes recent strides in synthetic method developments for chemical protein synthesis,including innovative techniques in solid-phase peptide synthesis,the challenges presented by difficult sequences in either synthesis or folding and the exploration of novel ligation reactions using both chemical and enzymatic methods.Furthermore,the review also delves into newly developed protocols for site-selective protein modifications and the generation of stapled or macrocyclized peptides/miniproteins,highlighting the power of chemical methods to make structurally diverse proteins.Recent applications of synthetic proteins in investigating post-translational modifications(phosphorylation,lipidation,glycosylation,ubiquitination,etc.),mirror-image biological processes and drug development are further discussed.Together,these topics provide a comprehensive overview of the current landscape of chemical protein synthesis.
基金supported by the National Key Research and Development Program of China(No.2019YFA0706900)the National Natural Science Foundation of China(Nos.22022703 and 22177108)the Collaborative Innovation Program of Hefei Science Center,CAS(No.2022HSC-CIP013).
文摘The 2-hydroxy-4-methoxybenzyl(Hmb)backbone modification can prevent amide bond-mediated sidereactions(e.g.,aspartimide formation,peptide aggregation)by installing the removable Hmb group into a peptide bond,thus improving the synthesis of long and challenging peptides and proteins.However,its use is largely precluded by the limited Hmb’s installation sites.In this report,an improved installation of Hmb(iHmb)method was developed to achieve the flexible installation and the convenient removal of Hmb.The iHmb method involves two critical steps:(1)oxidative diazotization of the readily installed 2-hydroxy-4-methoxy-5-amino-benzyl(Hmab)to give 2-hydroxy-4-methoxy-5-diazonium-benzyl(Hmdab)by combining soamyl nitrite(IAN)/HBF_(4),and(2)reductive elimination of Hmdab to give the desired Hmb by 1,2-ethanedithiol(EDT).The iHmb method enables the installation of Hmb at any primary amino acid including the highly sterically hindered amino acids(e.g.,valine and isoleucine).The practicality and utility of the iHmb method was demonstrated by one-shot solid-phase synthesis of a challenging aspartimide-prone peptide,the mirror-image version of a hydrophobic peptide and a long-chain peptide up to 76-residue.Furthermore,the iHmb method can be utilized to facilitate chemical protein ligation,as exemplified by the synthesis of the single-spanning membrane protein sarcolipin.The iHmb method expands the toolkit for peptide synthesis and ligation and facilitates the preparation of peptides/proteins.
基金The financial support from the National Natural Science Foundation of China(Nos.91853117 and 22077036)the Natural Science Foundation of Guangdong Province(No.2020A1515010766)are greatly acknowledged。
文摘Tyrosine sulfation is an important post-translational modification that enhances the inhibitory activity of hirudin.Herein,we developed a facile synthetic strategy to afford the sulfated hirudins with up to three modifications and in multi-milligram scales,after a single HPLC purification step.Through these synthetic proteins,a novel type of modulation mechanism exhibited by tyrosine sulfation was proposed,which would help to delineate the structure-function relationships in other sulfated proteins and more importantly,to serve as a basis for the development of related antithrombotic agents.
基金Supported by the National Key R&D Program of China(No.2017YFA0505200)the National Natural Science Foundation of China(Nos.21532004,91753205,81621002).
文摘Chemical synthesis of proteins containing up to 300 amino acids may cover 30%—50%of all the proteins encountered in biomedical studies and may provide an alternate approach to the usually used recombinant expression teclmology,vastly expanding the chemical space of the latter.In the present review article,we tried to survey the recent progresses made for more rapid synthesis of increasingly long peptides and more efficient ligation of multiple peptide segments.The developments of seminal methods by many research groups have greatly contributed to the recent breakthroughs in the successful total synthesis of a number of functionally important proteins,such as oligoubiquitins,bacterial GroEL/ES chaperones,and mirror-image DNA polymerases.Through these studies,a potential bottleneck has also been recognized for the chemical synthesis of large proteins,namely,how to ensure that each peptide segment from a large protein avoids unfavorable aggregation when dissolved in aqueous solution.Many new methods,such as removable backbone modification(RBM)strategy have been developed to overcome this bottleneck,while more studies need to be carried out to develop more effective and less costly methods that ultimately,may lead to fully automatable chemical synthesis of customized proteins of 300 amino acids bearing any artificial designs.
基金supported by the National Key R&D Program of China(No.2019YFA0706902)National Natural Science Foundation of China(Nos.U1732161 and 91753120)Science and Technological Fund of Anhui Province for Outstanding Youth(No.1808085J04)。
文摘D-peptides are recognized as a new class of synthetic chemical drugs and they possess many interesting advantages such as high enzymatic stability,improved oral bioavailability,as well as high binding affinity and specificity.Recently,D-peptide drugs have been attracting increasing attention in both academic and industrial researches over recent years.One D-peptide etelcalcetide has even entered the market that targets the calcium(Ca2+)-sensing receptor(CaSR) to fight secondary hyperparathyroidism.Effective discovery and optimization of D-peptide ligands that can bind to various disease-related targets with high specificity and potency is of great importance for the development of D-peptide drugs.This review surveys the recent method development in this area especially the chemical protein synthesis-assisted high-throughput screening strategies for D-peptide ligands and their application in drug discovery.
基金This study was supported by the National Key R&D Program of China(nos.2017YFA0505200 and 2017YFA0505400)the National Natural Science Foundation of China(nos.91753205,21877024,21977089,81621002,and 21621003)the Fundamental Research Funds for the Central Universities(no.JZ2019HGPB0105).
文摘Small ubiquitin-like modifiers(SUMOs)are protein modifiers that can form polymeric chains.They are important signals in cellular processes,and their study and profiling require the development of molecular tools.Herein,the authors have reported an efficient chemical protein synthesis approach for the generation of dimeric SUMO-2-based photoaffinity probes through the ligation of four readily synthesizable peptides.Proteomic studies using this diSUMO-2 probe on HeLa cell nuclear lysate found it to capture a significantly different selection of proteins compared with its monoSUMO counterparts.This resulted in the identification of several previously unknown SUMO chain-specific interacting proteins such as 40S ribosomal protein S3,which showed a significantly higher affinity for polySUMO chains than monomeric SUMO.Collectively,these results emphasize the need to develop SUMO chain-based probes in other species,and to shed light on the important role of polySUMOylation in diseases.
基金supported by the National Natural Science Foundation of China (NSFC No. 21572043)the Fundamental Research Funds for the Central Universities (No. PA2017GDQT0021)
文摘Guanylate cyclase C(GC-C) is an important receptor protein expressed by intestinal epithelial cells, and its dysregulation leads to severe intestinal diseases. Linaclotide is a 14-amino acid peptide approved by the FDA for the treatment of irritable bowel syndrome with constipation(IBS-C), which activates guanylate cyclase C to accelerate intestinal transit. Drug molecule design based on structural information plays a crucial role and the activity of linaclotide still need to improve, while the structure of linaclotide remains unknown. In this work, linaclotide and its D-enantiomer were obtained through Fmoc solid phase peptide synthesis method and co-crystalized through racemic crystallization. The crystal structure showed that linaclotide has a tight, three-beta turns structure immobilized by three pairs of disulfide bonds.
基金supported by the National Basic Research Program of China(2013CB932800)the National Natural Science Foundation of China(21532004,21225207,81621002)
文摘Histone H2 A methylation at Gln104(H2AQ104Me) is a new type of histone post-translational modification(PTM) discovered recently. This modification has been found to have significant influence on gene transcription. However, the structural and functional consequence of glutamine methylation on nucleosome remains to be further elucidated. Obtaining of histones with site-specific methylation at glutamine residues might facilitate the studies towards a better understanding of this new PTM. In the present work, total chemical synthesis of H2AQ104 Me was carried out through use of the hydrazide-based native chemical ligation. Synthetic histone H2AQ104 Me could be successfully incorporated into nucleosomes in vitro and showed a negative influence on the nucleosome stability.
基金the National Natural Science Foundation of China (Nos.91849129,21807112)PLA Youth Medical Science and Technology Youth Development Program (No.16QNP086)Foundation of Second Military Medical University (No.2016JS11)
文摘TNFR1-associated death domain protein(TRADD)with arginine N-GlcNAcylation is a novel and structurally unique posttranslational modification(PTM)glycoprotein that blocks the formation of death-inducing signaling complex(DISC),orchestrating host nuclear factorκB(NF-κB)signaling in entero-pathogenic Escherichia coli(EPEC)-infected cells.This particular glycosylated modification plays an extremely vital role for the effective colonization and pathogenesis of pathogens in the gut.Herein we describe the total synthesis of TRADD death domain(residues 195-312)with arginine235 NGlcNAcylation(Arg-GIcNAc TRADD(195-312)).Two longish peptidyl fragments of the wild-type primary sequence were obtained by robust,microwave-assisted,highly efficient,solid-phase peptide synthesis(SPPS),the N-GlcNAcylated sector was built by total synthesis and attached specifically to resinbound peptide with an unprotected ornithine residue via silver-promoted on-resin guanidinylation,ArgGlcNAc TRADD(195-312)was constructed by hydrazide-based native chemical ligation(NCL).The facile synthetic strategy is expected to be generally applicable for the rapid synthesis of other proteins with Arg-GIcNAc modification and to pave the way for the related chemically biological study.
文摘E3 ubiquitin ligases catalyze the final step of ubiquitylation,a crucial post-translational modification involved in almost every process in eukaryotic cells.E3 ubiquitin ligases are key regulators of cellular events,and the investigation into their functions and functioning mechanisms are research areas with great importance.Synthetic or semi-synthetic tools have greatly facilitated the research about the enzyme activity,distribution in different physiological events,and catalytic mechanism of E3 ubiquitin ligase.In this review,we summarize the development of chemical tools for E3 ubiquitin ligases with an emphasis on the synthetic routes.We show the utility of these chemical tools by briefly discussing their applications in biological research.
基金supported by the National Natural Science Foundation of China(Nos.21708036,31470740,U1732161)Anhui Provincial Natural Science Foundation (No.1808085QC63)。
文摘The H3 bivalent modifications of trimethylationat Lys9 and acetylation at Lys18(H3-K9 Me3-K18 Ac) were identified to collectively recruit TRIM33 in the nodal signaling pathway.To understand the underlying mechanism of TRIM33 recruitment,the nucleosome core particles(NCPs) containing full-length H3-K9 Me3-K18 Ac were indispensable samples.Herein we developed a pseudo dipeptide strategy to efficiently prepare peptide segments,facilitating the chemical synthesis of H3-K9 Me3-K18 Ac at a tens of milligram scale.The synthetic H3-K9 Me3-K18 Ac was then examined by CD spectroscopy,which demonstrated a prominent shift compared to recombinant H3.Finally,bivalently modified NCPs were assembled and verified by gel mobility shift assay with good homogeneity.
基金We thank the National Key R&D Program of China(2017YFA0505200,2016YFA0400903,and 2015CB910103)National Science Foundation of China(91753205,21532004,21761142008,81621002,21621003,91849129,and 21708036)for their financial support.
文摘Mutations in genes encoding PINK1(PTEN-induced kinase 1)and Parkin(E3 ubiquitin ligase)are identified in familial Parkinson’s disease.However,it remains unclear whether the phosphorylated Ub chains activate wild-type Parkin(w-Parkin)or phosphorylated Parkin(p-Parkin),with the consequent expulsion of the damaged mitochondria.