Unnatural amino acids(UAAs)have broad applications in pharmaceutical sciences and biological studies.Current synthetic methods for UAAs mainly rely on asymmetric catalysis and often require several steps.There is a la...Unnatural amino acids(UAAs)have broad applications in pharmaceutical sciences and biological studies.Current synthetic methods for UAAs mainly rely on asymmetric catalysis and often require several steps.There is a lack of direct and simple methods.To address this challenge,we designed the LADA(labeling-activation-desulfurization-addition)strategy:selective labeling and activation of cysteine residues,the photocatalytic desulfurization and the subsequent radical addition to alkenes.Although composed of two steps,it is one-pot synthesis and has advantages such as high functional group tolerance,biocompatible reaction condition,and retained stereochemistry.This highly efficient strategy was successfully applied in the direct synthesis of unnatural amino acids and modifications of peptides with more than 50 examples.展开更多
Unnatural amino acids(UAAs)are important building blocks in organic synthesis and drug discovery.They are also frequently integrated into peptides or proteins for biological studies.However,the direct and simplified s...Unnatural amino acids(UAAs)are important building blocks in organic synthesis and drug discovery.They are also frequently integrated into peptides or proteins for biological studies.However,the direct and simplified synthesis of UAAs remains a great challenge.At the same time,vast known peptide modifications are based on carbon-heteroatom bonds.There are no general methods for peptide modifications via the construction of C–C bonds.To address this challenge,herein we propose the LADA strategy,which is composed of two steps:the selective labeling and activation of cysteine residues,the desulfurization to generate carbon-centered radical and the radical addition to alkenes to build C–C bond.This one-pot protocol has obvious advantages such as good functional group tolerance,biocompatible reaction conditions,and retained stereochemistry.This strategy was successfully utilized for the synthesis of unnatural amino acids and direct modifications of peptides.展开更多
Unnatural a-amino acids have been extensively used in the modern drug discovery and protein engineering studies. They have also found applications in the development of chiral molecular catalysts and the total synthes...Unnatural a-amino acids have been extensively used in the modern drug discovery and protein engineering studies. They have also found applications in the development of chiral molecular catalysts and the total synthesis of diverse natural products. Accordingly the development of cost-effective approaches for the preparation of unnatural a-amino acids has received increasing attentions. Among all the available methods for this purpose, direct C–H functionalization of simple amino acids represents one of the most attractive approaches because it exhibits good atom-economy and step-efficiency. In particular, selective functionalization of either the primary or secondary C(sp^3)–H bonds in the amino acids has been explored to make versatile C–C, C–N, C–O, C–B and C–F bonds to modify the side chain of amino acids and even peptides. The present review surveys the recent advances of synthesis of chiral unnatural a-amino acids and peptides through palladium-catalyzed functionalization of un-activated C(sp^3)–H bonds.展开更多
Site-specific incorporation of unnatural amino acids(UNAAs)into proteins using an orthogonal translation system(OTS)has expanded the scope of protein-coding chemistry.The key factor affecting UNAA embedding efficiency...Site-specific incorporation of unnatural amino acids(UNAAs)into proteins using an orthogonal translation system(OTS)has expanded the scope of protein-coding chemistry.The key factor affecting UNAA embedding efficiency is the orthogonality of the OTS.Compared to traditional cell systems,cell-free systems are more convenient to control the reaction process and improve the utilization rate of UNAA.In this study,a linear DNA template-based cell-free unnatural protein synthesis system for rapid high-throughput screening and evolution was proposed.A total of 14 cell extracts were selected for screening out cell extract with high expression level.The result showed that EcAR7ΔAΔSer cell extract was optimal for the cell-free system.In addition,the screening results of four UNAAs,p-propargyloxy-l-phenylalanine(pPaF),p-azyl-phenylalanine(pAzF),p-acetyl-l-phenylalanine(pAcF),and p-benzoyl-l-phenylalanine(pBpF),showed that o-aaRS and o-tRNA of pPaF had good orthogonality.A new pair of corresponding o-aaRS and o-tRNA for pBpF was screened out.These results proved that this method could speed up the screening of optimal OTS components for UNAAs with versatile functions.展开更多
Amber suppression has been widely used to incorporate unnatural amino acids(UNAAs)with unique structures or functional side-chain groups into specific sites of the target protein,which expands the scope of protein-cod...Amber suppression has been widely used to incorporate unnatural amino acids(UNAAs)with unique structures or functional side-chain groups into specific sites of the target protein,which expands the scope of protein-coding chemistry.However,this traditional strategy does not allow multiple-site incorporation of different UNAAs into a single protein,which limits the development of unnatural proteins.To address this challenge,the suppression method using multiple termination codons(TAG,TAA or TGA)was proposed,and cell-free unnatural protein synthesis(CFUPS)system was employed.By the analysis of incorporating 3 different UNAAs(p-propargyloxy-L-phenylalanine,p-azyl-phenylalanine and L-4-Iodophenylalanine)and mass spectrometry,the simultaneous usage of the codons TAG and TAA were suggested for better multiple-site UNAA incorporation.The CFUPS conditions were further optimized for better UNAA incorporation efficiency,including the orthogonal translation system(OTS)components,magnesium ions,and the redox environment.This study established a CFUPS approach based on multiple termination codon suppression to achieve efficient and precise incorporation of different types of UNAAs,thereby synthesizing unnatural proteins with novel physicochemical functions.展开更多
Unnatural amino acids(UAAs)have gained significant attention in protein engineering and drug development owing to their ability to introduce new chemical functionalities to proteins.In eukaryotes,genetic code expansio...Unnatural amino acids(UAAs)have gained significant attention in protein engineering and drug development owing to their ability to introduce new chemical functionalities to proteins.In eukaryotes,genetic code expansion(GCE)enables the incorporation of UAAs and facilitates posttranscriptional modification(PTM),which is not feasible in prokaryotic systems.GCE is also a powerful tool for cell or animal imaging,the monitoring of protein interactions in target cells,drug development,and switch regulation.Therefore,there is keen interest in utilizing GCE in eukaryotic systems.This review provides an overview of the application of GCE in eukaryotic systems and discusses current challenges that need to be addressed.展开更多
Cell-free synthetic biology emerges as a powerful and flexible enabling technology that can engineer biological parts and systems for life science applications without using living cells.It provides simpler and faster...Cell-free synthetic biology emerges as a powerful and flexible enabling technology that can engineer biological parts and systems for life science applications without using living cells.It provides simpler and faster engineering solutions with an unprecedented freedom of design in an open environment than cell system.This review focuses on recent developments of cell-free synthetic biology on biological engineering fields at molecular and cellular levels,including protein engineering,metabolic engineering,and artificial cell engineering.In cell-free protein engineering,the direct control of reaction conditions in cell-free system allows for easy synthesis of complex proteins,toxic proteins,membrane proteins,and novel proteins with unnatural amino acids.Cell-free systems offer the ability to design metabolic pathways towards the production of desired products.Buildup of artificial cells based on cell-free systems will improve our understanding of life and use them for environmental and biomedical applications.展开更多
文摘Unnatural amino acids(UAAs)have broad applications in pharmaceutical sciences and biological studies.Current synthetic methods for UAAs mainly rely on asymmetric catalysis and often require several steps.There is a lack of direct and simple methods.To address this challenge,we designed the LADA(labeling-activation-desulfurization-addition)strategy:selective labeling and activation of cysteine residues,the photocatalytic desulfurization and the subsequent radical addition to alkenes.Although composed of two steps,it is one-pot synthesis and has advantages such as high functional group tolerance,biocompatible reaction condition,and retained stereochemistry.This highly efficient strategy was successfully applied in the direct synthesis of unnatural amino acids and modifications of peptides with more than 50 examples.
基金supported by the Shanghai Jiao Tong University(WF220417003 to Z.S.)。
文摘Unnatural amino acids(UAAs)are important building blocks in organic synthesis and drug discovery.They are also frequently integrated into peptides or proteins for biological studies.However,the direct and simplified synthesis of UAAs remains a great challenge.At the same time,vast known peptide modifications are based on carbon-heteroatom bonds.There are no general methods for peptide modifications via the construction of C–C bonds.To address this challenge,herein we propose the LADA strategy,which is composed of two steps:the selective labeling and activation of cysteine residues,the desulfurization to generate carbon-centered radical and the radical addition to alkenes to build C–C bond.This one-pot protocol has obvious advantages such as good functional group tolerance,biocompatible reaction conditions,and retained stereochemistry.This strategy was successfully utilized for the synthesis of unnatural amino acids and direct modifications of peptides.
文摘Unnatural a-amino acids have been extensively used in the modern drug discovery and protein engineering studies. They have also found applications in the development of chiral molecular catalysts and the total synthesis of diverse natural products. Accordingly the development of cost-effective approaches for the preparation of unnatural a-amino acids has received increasing attentions. Among all the available methods for this purpose, direct C–H functionalization of simple amino acids represents one of the most attractive approaches because it exhibits good atom-economy and step-efficiency. In particular, selective functionalization of either the primary or secondary C(sp^3)–H bonds in the amino acids has been explored to make versatile C–C, C–N, C–O, C–B and C–F bonds to modify the side chain of amino acids and even peptides. The present review surveys the recent advances of synthesis of chiral unnatural a-amino acids and peptides through palladium-catalyzed functionalization of un-activated C(sp^3)–H bonds.
基金supported by the National Key R&D Program of China(2018YFA0901700)National Natural Science Foundation of China(21878173)+2 种基金Beijing Natural Science Foundation(2192023)a grant from the Institute Guo Qiang,Tsinghua University(2019GQG1016)Laboratory Innovation Fund of Tsinghua University.
文摘Site-specific incorporation of unnatural amino acids(UNAAs)into proteins using an orthogonal translation system(OTS)has expanded the scope of protein-coding chemistry.The key factor affecting UNAA embedding efficiency is the orthogonality of the OTS.Compared to traditional cell systems,cell-free systems are more convenient to control the reaction process and improve the utilization rate of UNAA.In this study,a linear DNA template-based cell-free unnatural protein synthesis system for rapid high-throughput screening and evolution was proposed.A total of 14 cell extracts were selected for screening out cell extract with high expression level.The result showed that EcAR7ΔAΔSer cell extract was optimal for the cell-free system.In addition,the screening results of four UNAAs,p-propargyloxy-l-phenylalanine(pPaF),p-azyl-phenylalanine(pAzF),p-acetyl-l-phenylalanine(pAcF),and p-benzoyl-l-phenylalanine(pBpF),showed that o-aaRS and o-tRNA of pPaF had good orthogonality.A new pair of corresponding o-aaRS and o-tRNA for pBpF was screened out.These results proved that this method could speed up the screening of optimal OTS components for UNAAs with versatile functions.
基金the National Key R&D Program of China(Grant No.2018YFA0901700)the National Natural Science Foundation of China(Grant No.21878173)the Beijing Natural Science Foundation(Grant No.2192023).
文摘Amber suppression has been widely used to incorporate unnatural amino acids(UNAAs)with unique structures or functional side-chain groups into specific sites of the target protein,which expands the scope of protein-coding chemistry.However,this traditional strategy does not allow multiple-site incorporation of different UNAAs into a single protein,which limits the development of unnatural proteins.To address this challenge,the suppression method using multiple termination codons(TAG,TAA or TGA)was proposed,and cell-free unnatural protein synthesis(CFUPS)system was employed.By the analysis of incorporating 3 different UNAAs(p-propargyloxy-L-phenylalanine,p-azyl-phenylalanine and L-4-Iodophenylalanine)and mass spectrometry,the simultaneous usage of the codons TAG and TAA were suggested for better multiple-site UNAA incorporation.The CFUPS conditions were further optimized for better UNAA incorporation efficiency,including the orthogonal translation system(OTS)components,magnesium ions,and the redox environment.This study established a CFUPS approach based on multiple termination codon suppression to achieve efficient and precise incorporation of different types of UNAAs,thereby synthesizing unnatural proteins with novel physicochemical functions.
基金This work was supported by the National Key R&D Program of China(Nos.2019YFA0904200 and 2019YFA0906100)the National Natural Science Foundation of China(No.32171464)Shenzhen Science and Technology Innovation Program(JCYJ20180504165501371).
文摘Unnatural amino acids(UAAs)have gained significant attention in protein engineering and drug development owing to their ability to introduce new chemical functionalities to proteins.In eukaryotes,genetic code expansion(GCE)enables the incorporation of UAAs and facilitates posttranscriptional modification(PTM),which is not feasible in prokaryotic systems.GCE is also a powerful tool for cell or animal imaging,the monitoring of protein interactions in target cells,drug development,and switch regulation.Therefore,there is keen interest in utilizing GCE in eukaryotic systems.This review provides an overview of the application of GCE in eukaryotic systems and discusses current challenges that need to be addressed.
文摘Cell-free synthetic biology emerges as a powerful and flexible enabling technology that can engineer biological parts and systems for life science applications without using living cells.It provides simpler and faster engineering solutions with an unprecedented freedom of design in an open environment than cell system.This review focuses on recent developments of cell-free synthetic biology on biological engineering fields at molecular and cellular levels,including protein engineering,metabolic engineering,and artificial cell engineering.In cell-free protein engineering,the direct control of reaction conditions in cell-free system allows for easy synthesis of complex proteins,toxic proteins,membrane proteins,and novel proteins with unnatural amino acids.Cell-free systems offer the ability to design metabolic pathways towards the production of desired products.Buildup of artificial cells based on cell-free systems will improve our understanding of life and use them for environmental and biomedical applications.