Total Chemical Synthesis of Proteins

Ashraf Brik, PhD, is a Professor of Chemistry at the Schulich Faculty of Chemistry in the Technion-Israel Institute of Technology. Philip E. Dawson, PhD, is a Professor of Chemistry at Scripps Research, CA, where he is also the Dean of Graduate and Postdoctoral Studies. Lei Liu, PhD, is a Professor of Chemistry at Tsinghua University.

• Preface xvii1 Characterization of Protein Molecules Prepared by Total Chemical Synthesis 1Stephen B. H. Kent1.1 Introduction 11.2 Chemical Protein Synthesis 21.3 Comments on Characterization of Synthetic Protein Molecules 81.3.1 Homogeneity 81.3.2 Amino Acid Sequence 91.3.3 Chemical Analogues 101.3.4 Limitations of SPPS 101.3.5 Folding as a Purification Step 101.4 Summary 12References 122 Automated Fast Flow Peptide Synthesis 17Mark D. Simon, Alexander J. Mijalis, Kyle A. Totaro, Daniel Dunkelmann, Alexander A. Vinogradov, Chi Zhang, Yuta Maki, Justin M. Wolfe, Jessica Wilson, Andrei Loas, and Bradley L. Pentelute2.1 Introduction 172.2 Results 192.2.1 Summary 192.2.1.1 Mechanical Principles 202.2.1.2 Chemical Principles 202.2.1.3 User Interface Principles 202.2.1.4 Data Analysis Method 202.2.1.5 Outcome 212.2.2 First-generation Automated Fast Flow Peptide Synthesis 212.2.2.1 Key Findings 212.2.2.2 Design of First-generation AFPS 212.2.2.3 Characterization of First-generation AFPS 232.2.3 Second-generation Automated Fast Flow Peptide Synthesis 242.2.3.1 Key Findings 242.2.3.2 Design of Second-generation AFPS 242.2.3.3 Characterization and Use of Second-generation AFPS 262.2.4 Third-generation Automated Fast Flow Peptide Synthesis 322.2.4.1 Key Findings 322.2.4.2 Design of Third-generation AFPS 342.2.4.3 Characterization of Third-generation AFPS 392.2.4.4 Reagent Stability Study 432.2.5 Fourth-generation Automated Fast Flow Peptide Synthesis 452.2.5.1 Key Findings 452.2.5.2 Effect of Solvent on Fast Flow Synthesis 452.2.5.3 Design and Characterization of Fourth-generation AFPS 452.3 Conclusions 49Acknowledgments 53References 533 N,S- and N,Se-Acyl Transfer Devices in Protein Synthesis 59Vincent Diemer, Jennifer Bouchenna, Florent Kerdraon, Vangelis Agouridas, and Oleg Melnyk3.1 Introduction 593.2 N,S- and N,Se-Acyl Transfer Devices: General Presentation, Reactivity and Statistical Overview of Their Utilization 613.2.1 General Presentation of N,S- and N,Se-Acyl Transfer Devices 613.2.2 Relative Reactivity of N,S- and N,Se-Acyl Transfer Devices 633.2.3 A Statistical Overview of the Synthetic Use of N,S- and N,Se-Acyl Transfer Devices for Protein Total Chemical Synthesis 643.3 Preparation of SEA/SeEAoff and SEAlide Peptides 683.3.1 Preparation of SEA and SeEA Peptides 683.3.2 Preparation of SEAlide Peptides 703.4 Redox-controlled Assembly of Biotinylated NK1 Domain of the Hepatocyte Growth Factor (HGF) Using SEA and SeEA Chemistries 713.5 The Total Chemical Synthesis of GM2-AP Using SEAlide-based Chemistry 753.6 Conclusion 79References 804 Chemical Synthesis of Proteins Through Native Chemical Ligation of Peptide Hydrazides 87Chao Zuo, Xiaodan Tan, Xianglong Tan, and Lei Liu4.1 Introduction 874.2 Development of Peptide Hydrazide-based Native Chemical Ligation 884.2.1 Conversion of Peptide Hydrazide to Peptide Azide 884.2.2 Acyl Azide-based Solid-phase Peptide Synthesis 884.2.3 Acyl Azide-based Solution-phase Peptide Synthesis 894.2.4 The Transesterification of Acyl Azide 904.2.5 Development of Peptide Hydrazide-based Native Chemical Ligation 904.3 Optimization of Peptide Hydrazide-based Native Chemical Ligation 914.3.1 Preparation of Peptide Hydrazides 914.3.1.1 2-Cl-Trt-Cl Resin 914.3.1.2 Peptide Hydrazides from Expressed Proteins 924.3.1.3 Sortase-mediated Hydrazide Generation 934.3.2 Activation Methods of Peptide Hydrazide 944.3.2.1 Knorr Pyrazole Synthesis 944.3.2.2 Activation in TFA 944.3.3 Ligation Sites of Peptide Hydrazide 954.3.4 Multiple Fragment Ligation Based on Peptide Hydrazide 964.3.4.1 N-to-C Sequential Ligation 964.3.4.2 Convergent Ligation 964.3.4.3 One-pot Ligation 964.4 Application of Peptide Hydrazide-based Native Chemical Ligation 994.4.1 Peptide Drugs and Diagnostic Tools 994.4.1.1 Peptide Hydrazides for Cyclic Peptide Synthesis 994.4.1.2 Screening for D Peptide Inhibitors Targeting PD-L1 994.4.1.3 Chemical Synthesis of DCAF for Targeted Antibody Blocking 1014.4.1.4 Peptide Toxins 1014.4.2 Synthesis and Application of Two-photon Activatable Chemokine CCL5 1024.4.3 Proteins with Posttranslational Modification 1034.4.3.1 The Synthesis of Glycosylation-modified Full-length IL-6 1034.4.3.2 The Chemical Synthesis of EPO 1054.4.3.3 Chemical Synthesis of Homogeneous Phosphorylated p62 1054.4.3.4 Chemical Synthesis of K19, K48 Bi-acetylated Atg3 Protein 1054.4.4 Ubiquitin Chains 1084.4.4.1 Synthesis of K27-linked Ubiquitin Chains 1084.4.4.2 Synthesis of Atypical Ubiquitin Chains by Using an Isopeptide-linked Ub Isomer 1094.4.4.3 Synthesis of Atypical Ubiquitin Chains Using an Isopeptide-linked Ub Isomer 1094.4.5 Modified Nucleosomes 1104.4.5.1 Synthesis of DNA-barcoded Modified Nucleosome Library 1104.4.5.2 Synthesis of Modified Histone Analogs with a Cysteine Aminoethylation-assisted Chemical Ubiquitination Strategy 1114.4.5.3 Synthesis of Ubiquitylated Histones for Examination of the Deubiquitination Specificity of USP51 1114.4.6 Membrane Proteins 1124.4.7 Mirror-image Biological Systems 1124.5 Summary and Outlook 113References 1145 Expanding Native Chemical Ligation Methodology with Synthetic Amino Acid Derivatives 119Emma E. Watson, Lara R. Malins, and Richard J. Payne5.1 Native Chemical Ligation 1205.2 Desulfurization Chemistries 1205.3 Aspartic Acid (Asp, D) 1225.4 Glutamic Acid (Glu, E) 1245.5 Phenylalanine (Phe, F) 1255.6 Isoleucine (Ile, I) 1275.7 Lysine (Lys, K) 1305.8 Leucine (Leu, L) 1335.9 Asparagine (Asn, N) 1355.10 Proline (Pro, P) 1385.11 Glutamine (Gln, Q) 1395.12 Arginine (Arg, R) 1395.13 Threonine (Thr, T) 1405.14 Valine (Val, V) 1425.15 Tryptophan (Trp,W) 1445.16 Application of Selenocysteine (Sec) to Ligation Chemistry 1465.17 Aspartic Acid (Asp, D) 1475.18 Glutamic Acid (Glu, E) 1485.19 Phenylalanine (Phe, F) 1495.20 Leucine (Leu, L) 1515.21 Proline (Pro, P) 1515.22 Serine (Ser, S) 153References 1556 Peptide Ligations at Sterically Demanding Sites 161Yinglu Wang and Suwei Dong6.1 Introduction 1616.2 Ligations Using Thioesters 1626.2.1 Exogenous Additive-promoted Ligations 1626.2.2 Ligations Using Reactive Thioesters 1676.2.3 Internal Activation Strategy in Peptide Ligations 1696.3 Ligations Using Oxo-esters 1706.4 Peptide Ligations Based on Selenoesters 1706.5 Microfluidics-promoted NCL 1756.6 Representative Applications in Protein Synthesis 1786.7 Summary and Outlook 181References 1817 Controlling Segment Solubility in Large Protein Synthesis 185Riley J. Giesler, James M. Fulcher, Michael T. Jacobsen, and Michael S. Kay7.1 Solvent Manipulation 1857.2 Isoacyl Strategy 1877.3 Semipermanent Solubilizing Tags 1917.3.1 N- or C-Terminal Solubilizing “Tails” 1927.3.2 Reversible Backbone Modifications as Solubilizing Tags 1947.3.3 Building Block Solubilizing Tags 1957.3.4 Extendable Side-chain-based Solubilizing Tags 195References 1988 Toward HPLC-free Total Chemical Synthesis of Proteins 211Phuc Ung and Oliver Seitz8.1 Introduction 2118.1.1 Capture and Release Purification 2128.1.2 Solid-phase Chemical Ligations (SPCL) 2128.2 Synthesis of Peptide Segments for Native Chemical Ligation 2138.2.1 HPLC-free Preparation of N-terminal Peptide Segments for NCL 2138.2.2 HPLC-free Preparation of C-terminal Peptide Segments for NCL 2178.3 Synthesis of Proteins Using the His6 Tag 2208.3.1 Reversible His6-based Capture Tags 2208.3.2 His6-based Immobilization for C-to-N Assembly of Crambin 2218.3.3 His6-based Immobilization for Assembly of Proteins on Microtiter Plates 2228.3.4 His6 and Hydrazide Tags for Sequential N-to-C Capture and Release 2258.4 Synthesis of Proteins via Oxime Formation 2278.4.1 Reversible Oxime-based Capture Tags 2278.4.2 Oxime-based Immobilization for N-to-C Solid-phase Chemical Ligations 2278.4.3 Oxime-based Immobilization for C-to-N Solid-phase Chemical Ligations 2338.4.4 Oxime-based C-to-N Solid-phase Chemical Ligations 2378.5 Synthesis of Proteins via Hydrazone Formation 2388.5.1 Reversible Hydrazone-based Capture Tags 2388.5.2 Hydrazone-based Immobilization for Assembly of Proteins on Microtiter Plates 2398.6 Synthesis of Proteins Using Click Chemistry 2428.6.1 Click-based Immobilization for N-to-C Solid-phase Peptide Ligations Using a Protected Alkyne 2428.6.2 Click-based Immobilization for N-to-C Solid-phase Peptide Ligations Using a Sea Group 2438.7 Synthesis of Proteins Using the KAHA Ligation 2448.7.1 The KAHA Ligation 2448.7.2 HPLC-free Synthesis of Proteins Using the KAHA Ligation 2458.8 Synthesis of Proteins Using Photocleavable Tags 2468.8.1 Synthesis of Proteins Using a Photocleavable Biotin-based Purification Tag 2468.8.2 Synthesis of Proteins Using a Photocleavable His6-based Purification Tag 2478.9 Conclusion 249References 2519 Solid-phase Chemical Ligation 259Skander A. Abboud, Agnès F. Delmas, and Vincent Aucagne9.1 Introduction 2599.1.1 The Promises of Solid Phase Chemical Ligation (SPCL) 2599.1.2 Chemical Ligation Reactions Used for SPCL 2609.1.3 Key Requirements for a SPCL Strategy 2619.2 SPCL in the C-to-N Direction 2629.2.1 Temporary Masking Groups to Enable Iterative Ligations 2629.2.2 Linkers for C-to-N SPCL 2649.2.2.1 Use of Same Linker and Solid Support for SPPS and SPCL 2659.2.2.2 Re-immobilization of the C-Terminal Segment 2669.3 SPCL in the N-to-C Direction 2689.3.1 Temporary Masking Groups to Enable Iterative Ligations 2689.3.2 Linkers for N-to-C SPCL 2709.3.3 Case Study 2729.3.4 SPCL with Concomitant Purifications 2749.4 Post-Ligation Solid-Supported Transformations 2749.4.1 Chemical Transformations 2749.4.2 Biochemical Transformations 2759.5 Solid Support 2759.6 Conclusion and Perspectives 278Acknowledgment 2789.A Appendix 278References 28010 Ser/Thr Ligation for Protein Chemical Synthesis 285Carina Hey Pui Cheung and Xuechen Li10.1 Serine/Threonine Ligation 28710.2 Epimerization Issue 28910.3 Other Aryl Aldehyde Esters 28910.4 Preparation of Peptide Salicylaldehyde Esters 28910.5 Scope and Limitations 29410.6 Strategies of Ser/Thr Ligation for Protein Chemical Synthesis 29410.7 C-to-N Ser/Thr Ligation 29410.8 N-to-C Ser/Thr Ligation 29610.9 One-pot Ser/Thr Ligation and NCL 29610.10 Bioconjugation 29610.11 Solubility Issues 29810.12 Extension of Ser/Thr Ligation 29810.13 Conclusion 302References 30311 Protein Semisynthesis 307Nam Chu and Philip A. Cole11.1 Background 30711.2 Expressed Protein Ligation (EPL) 30811.2.1 Method Development 30811.2.2 Applications of EPL for Studying Protein Posttranslational Modifications 30911.2.3 Site-specific Protein Labeling with N-Hydroxysuccinimide Esters 31111.3 Cysteine Modifications 31111.3.1 Dehydroalanine Generation and Applications in Semisynthesis 31211.3.2 Cysteine Alkylation-related Methods to Introduce Lys Mimics 31311.4 Enzyme-catalyzed Protein/Peptide Ligations 31411.4.1 Sortase 31411.4.2 Butelase-1 31611.4.3 Subtiligase 31711.4.4 Trypsiligase 31811.5 Enzyme-catalyzed Expressed Protein Ligation 31811.6 Summary and Outlook 319Acknowledgments 320References 32012 Bio-orthogonal Imine Chemistry in Chemical Protein Synthesis 327Stijn M. Agten, Ingrid Dijkgraaf, Stan H. E. van der Beelen, and Tilman M. Hackeng12.1 Introduction 32712.2 Carbonyl Functionalization 32812.3 Aminooxy, Hydrazine, and Hydrazide Functionalization 33512.4 Oxime Ligation 33712.5 Hydrazone Ligation 34212.6 Pictet–Spengler Reaction 34412.7 Catalysis of Oxime and Hydrazone Ligations 346References 34813 Deciphering Protein Folding Using Chemical Protein Synthesis 357Vladimir Torbeev13.1 Introduction 35713.2 Modification of Protein Backbone Amides 35813.3 Insertion of β-turn Mimetics 36113.4 Inversion of Chiral Centers in Protein Backbone and Side Chains 36213.5 Modulating cis–trans Proline Isomerization 36613.6 Steering Oxidative Protein Folding 36813.7 Covalent Tethering to Facilitate Folding of Designed Proteins 37113.8 Discovery of Previously Unknown Protein Folds 37313.9 Site-specific Labeling with Fluorophores 37313.10 Foldamers and Foldamer–Peptide Hybrids 37513.11 Conclusions and Outlook 377Acknowledgement 378References 37814 Chemical Synthesis of Ubiquitinated Proteins for Biochemical Studies 383Gandhesiri Satish, Ganga B. Vamisetti, and Ashraf Brik14.1 The Ubiquitin System 38314.2 Non-enzymatic Ubiquitination: Challenges and Opportunities 38614.2.1 Chemical Synthesis of Ub Building Blocks 38714.2.2 Isopeptide Ligation 38714.2.3 Total Chemical Synthesis of Tetra-Ub Chains 39014.3 Synthesis and Semisynthesis of Ubiquitinated Proteins 39314.3.1 Monoubiquitinated Proteins 39314.3.2 Tetra-ubiquitinated Proteins 39514.3.3 Modification of Expressed Proteins with Tetra-Ub 40014.4 Synthesis of Unique Ub Conjugates to Study and Target DUBs 40114.5 Activity-based Probes 40314.6 Perspective 405List of Abbreviations 406References 40715 Glycoprotein Synthesis 411Chaitra Chandrashekar, Kento Iritani, Tatsuya Moriguchi, and Yasuhiro Kajihara15.1 Introduction 41115.2 Total Chemical Synthesis of Glycoproteins 41115.3 Semisynthesis of Glycoproteins 41315.4 Chemoenzymatic Synthesis 41315.5 α-Synuclein 41415.6 Hirudin P6 41515.7 Saposin D 41615.8 Interleukin 2 41715.9 Interleukin 25 41715.10 Mucin 1 41915.11 Crambin 42115.12 Tau Protein 42215.13 Chemical Domain of Fractalkine 42315.14 CCL1 42415.15 Interleukin 6 42415.16 Interleukin 8 42515.17 Erythropoietin 42615.18 Trastuzumab 43015.19 Antifreeze Glycoprotein 43215.20 Conclusion 434References 43416 Chemical Synthesis of Membrane Proteins 437Alanca Schmid and Christian F.W. Becker16.1 Introduction 43716.2 Solid Phase Synthesis of TM Peptides 43816.3 Purification and Handling Strategies of TM Peptides 44216.4 Solubility Tags 44316.4.1 Terminal Tags 44316.4.2 Side Chain Tags 44516.5 Removable Solubilizing Backbone Tags 44516.6 Chemical Synthesis of Membrane Proteins 44916.6.1 Proteins With 1 TM Domain 44916.6.2 Proteins with 2 TM Domains 45016.6.3 Proteins with 3 and More TM Domains 45416.7 Outlook 456References 45717 Chemical Synthesis of Selenoproteins 463Rebecca N. Dardashti, Reem Ghadir, Hiba Ghareeb, Orit Weil-Ktorza, and Norman Metanis17.1 What are Selenoproteins? 46317.2 Expression of Selenoproteins 46617.3 Sec as a Reactive Handle 46917.4 Synthesis and Semisynthesis of Natural Selenoproteins 47317.5 Selenium as a Tool for Protein Folding 47517.6 Conclusions 478References 47818 Histone Synthesis 489Champak Chatterjee18.1 The Histones and Their Chemical Modifications 48918.1.1 Histone Proteins 48918.1.2 Histone Posttranslational Modifications 49018.2 Chemical Ligation for Histone Synthesis 49218.2.1 Native Chemical Ligation 49218.2.2 Expanding the Scope of Native Chemical LigationWith Inteins 49418.3 Histone Octamer and Nucleosome Core Particle Assembly 49418.4 Studying the Histone CodeWith Synthetic Histones 49618.4.1 Synthesis of Histones Modified by Smaller Functional Groups 49718.4.1.1 Histone Phosphorylation 49718.4.1.2 Histone Acetylation 49918.4.1.3 Histone Methylation 50218.4.2 Synthesis of Sumoylated Histones 50518.5 Conclusions 506Acknowledgments 506References 50619 Application of Chemical Synthesis to Engineer Protein Backbone Connectivity 515Chino C. Cabalteja and W. Seth Horne19.1 Introduction 51519.2 Backbone Engineering to Facilitate Synthesis 51619.3 Backbone Engineering to Explore the Consequences of Chirality 51719.4 Backbone Engineering to Understand and Control Folding 52019.5 Backbone Engineering to Create Protein Mimetics 52219.6 Conclusions 525References 52620 Beyond Phosphate Esters: Synthesis of Unusually Phosphorylated Peptides and Proteins for Proteomic Research 533Anett Hauser, Christian E. Stieger, and Christian P. R. Hackenberger20.1 Introduction 53320.2 General Methods for the Incorporation of Hydroxy-phosphorylated Amino Acids into Peptides and Proteins 53420.3 Incorporation of Other Phosphorylated Nucleophilic Amino Acids into Peptides and Proteins 53720.3.1 Phosphoarginine (pArg) 53720.3.2 Phosphohistidine (pHis) 53820.3.3 Phospholysine (pLys) 53920.3.4 Phosphocysteine (pCys) 53920.3.5 Pyrophosphorylation of Serine and Threonine (ppSer, ppThr) 54120.4 Development of Phospho-analogues as Mimics for Endogenous Phospho-Amino Acids 54120.4.1 Analogues of Phosphoserine, Phosphothreonine, and Phosphotyrosine 54120.4.2 Stable Analogues of Phosphoaspartate and Phosphoglutamate 54320.4.3 Stable Analogues of Phosphoarginine 54420.4.4 Stable Analogues of Phosphohistidine 54520.4.5 Stable Analogues of Pyrophosphorylated Serine 54720.5 Conclusion 547References 54721 Cyclic Peptides via Ligation Methods 553Tristan J. Tyler and David J. Craik21.1 Introduction 55321.2 Cyclic Peptide Synthesis 55421.3 Orbitides 55721.4 Paws-derived Peptides(PDPs) 55921.5 Cyclic Conotoxins 56121.6 θ-Defensins 56321.7 Cyclotides 56321.8 Outlook 568Acknowledgements 568Funding 568References 569Index 579

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