Presenting a wide array of information on chemical ligation one of the more powerful tools for protein and peptide synthesis this book helps readers understand key methodologies and applications that include protein therapeutic synthesis, drug discovery, and molecular imaging.

List of Figures xiii List of Plates xxiii List of Contributors xxix Preface xxxiii 1 Introduction to Chemical Ligation Reactions 1 Lucia De Rosa, Alessandra Romanelli, and Luca Domenico D’Andrea 1.1 Introduction 1 1.1.1 Chemical Synthesis of Proteins: From the Stepwise Synthesis to the Chemical Ligation Approach 2 1.1.2 Chemical Modification of Proteins: From Conventional Methods to Chemoselective Labeling by Chemical Ligation 5 1.2 Chemical Ligation Chemistries 6 1.3 Imine Ligations 7 1.3.1 Oxime Ligation 7 1.3.2 Hydrazone Ligation 13 1.3.3 Pictet–Spengler Ligation 15 1.3.4 Thiazolidine Ligation 19 1.4 Serine/Threonine Ligation (STL) 21 1.5 Thioether Ligation 24 1.6 Thioester Ligation 25 1.6.1 Native Chemical Ligation (NCL) 26 1.6.2 Expressed Protein Ligation (EPL) 40 1.6.2.1 Protein trans‐Splicing (PTS) 43 1.6.3 Thioacid‐Mediated Ligation Strategies 44 1.7 α‐Ketoacid‐Hydroxylamine (KAHA) Ligation 49 1.7.1 Acyltrifluoroborates and Hydroxylamines Ligation 51 1.8 Staudinger Ligation 52 1.9 Azide–Alkyne Cycloaddition 57 1.10 Diels–Alder Ligation 61 References 64 2 Protein Chemical Synthesis by SEA Ligation 89 Oleg Melnyk, Claire Simonneau, and Jérôme Vicogne 2.1 Introduction 89 2.2 Essential Chemical Properties of SEA Group 93 2.3 Protein Total Synthesis Using SEA Chemistry – SEAon/off Concept 97 2.3.1 Synthesis of SEA off Peptide Segments 97 2.3.2 SEAon/off Concept and the Design of a One-Pot Three Peptide Segment Assembly Process 99 2.3.3 SEAon/off Concept and the Solid-Phase Synthesis of Proteins in the N-to-C Direction 103 2.4 Chemical Synthesis of HGF/SF Subdomains for Deciphering the Functioning of HGF/SF-MET System 106 2.5 Conclusion 114 References 114 3 Development of Serine/Threonine Ligation and Its Applications 125 Tianlu li and Xuechen li 3.1 Introduction 125 3.1.1 Protein Synthesis by SPPS 125 3.1.2 Native Chemical Ligation (and Extended Desulfurization) 125 3.1.3 KAHA Ligation 128 3.2 Serine/Threonine Ligation (STL) 130 3.2.1 SAL Ester Preparation 130 3.2.2 N‐Terminal‐Protecting Group for Successive C‐to‐N Ser/Thr Ligations 136 3.2.3 Scope and Limitations 137 3.2.3.1 Effect of Side‐Chain‐Unprotected Lys Residue 137 3.2.3.2 Effect of the C‐Terminal Amino Acid at Ligation Site 138 3.3 Application of STL in Protein Synthesis 140 3.3.1 Consecutive STL of Peptides/Proteins 140 3.3.1.1 Teriparatide (Forteo) 140 3.3.1.2 hGH‐RH 141 3.3.1.3 Human Erythrocyte Acylphosphatase (ACYP1) 142 3.3.1.4 MUC1 Glycopeptides 143 3.3.2 STL‐Mediated Peptide Cyclization 143 3.3.2.1 STL in Head‐to‐Tail Tetrapeptide Cyclization 143 3.3.2.2 STL in Head‐to‐Tail Cyclization of Peptides of Various Sizes 145 3.3.2.3 Total Synthesis of Daptomycin via Serine‐Ligation‐Mediated Peptide Cyclization 145 3.3.3 Thiol SAL Ester‐Mediated Aminolysis in Peptide Cyclization 147 3.3.4 A Fluorogenic Probe for Recognizing 5‐OH‐Lys Inspired by STL 149 3.3.5 Expressed Protein Semisynthesis via Ser/Thr Ligation 151 3.4 Conclusion and Outlook 154 References 154 4 Synthesis of Proteins by Native Chemical Ligation–Desulfurization Strategies 161 Bhavesh Premdjee and Richard J. Payne 4.1 Introduction 161 4.2 Ligation–Desulfurization and Early Applications 162 4.2.1 Metal‐Free Desulfurization 164 4.2.2 Ligation–Desulfurization toward the Synthesis of Proteins 166 4.3 Beyond Native Chemical Ligation at Cysteine – The Development of Thiolated Amino Acids and Their Application in Protein Synthesis 174 4.3.1 Phenylalanine 174 4.3.2 Valine 178 4.3.3 Lysine 179 4.3.4 Threonine 188 4.3.5 Leucine 188 4.3.6 Proline 193 4.3.7 Glutamine 195 4.3.8 Arginine 198 4.3.9 Aspartic Acid 198 4.3.10 Glutamic Acid 202 4.3.11 Tryptophan 206 4.3.12 GlcNAc‐Asparagine 206 4.3.13 Asparagine 206 4.4 Ligation–Deselenization in the Chemical Synthesis of Proteins 211 4.4.1 Selenol Amino Acids 214 4.5 Conclusions and Future Directions 216 References 218 5 Synthesis of Chemokines by Chemical Ligation 223 Nydia Panitz and Annette G. Beck‐Sickinger 5.1 Introduction – The Chemokine–Chemokine Receptor Multifunctional System 223 5.2 Synthesis of Chemokines by Native Chemical Ligation 224 5.3 Synthesis of Chemokines by Alternative Chemical Ligation 231 5.4 Semisynthesis of Chemokines by Expressed Protein Ligation 233 5.5 Prospects 241 References 243 6 Chemical Synthesis of Glycoproteins by the Thioester Method 251 Hironobu Hojo 6.1 Introduction 251 6.2 Ligation Methods and Strategy of Glycoprotein Synthesis 252 6.3 The Synthesis of the Extracellular Ig Domain of Emmprin 254 6.4 Synthesis of Basal Structure of MUC 2 256 6.5 N‐Alkylcysteine‐Assisted Thioesterification Method and Dendrimer Synthesis 257 6.6 Synthesis of TIM‐3 260 6.7 Resynthesis of Emmprin Ig Domain 262 6.8 Conclusion 264 References 264 7 Membrane Proteins: Chemical Synthesis and Ligation 269 Marc Dittman and Martin Engelhard 7.1 Introduction 269 7.2 Methods for the Synthesis and Purification of Membrane Proteins 270 7.2.1 Synthesis of Hydrophobic Peptides 270 7.2.2 Purification of Hydrophobic Peptides 272 7.3 Ligation and Refolding 273 7.3.1 Ligation Strategies 273 7.3.2 Refolding of Chemically Synthesized Hydrophobic Peptides and Membrane Proteins 275 7.4 Illustrative Examples 276 7.4.1 Diacylglycerol Kinase (DAGK) 276 7.4.2 Semisynthesis of the Sensory Rhodopsin/Transducer Complex 278 7.4.3 Semisynthesis of the Functional K + Channel KcsA 279 References 280 8 Chemoselective Modification of Proteins 285 xi Chen, Stephanie Voss, and Yao-wen Wu 8.1 Chemical Protein Synthesis 285 8.1.1 Native Chemical Ligation (NCL) and Expressed Protein Ligation (epl) 285 8.1.2 Traceless Staudinger Ligation 286 8.2 Chemoselective and Bioorthogonal Reactions 287 8.2.1 Oxime/Hydrazone Ligation 287 8.2.2 Staudinger Ligations 294 8.2.3 Copper-Catalyzed Azide–Alkyne Cycloaddition (CuAAC) 294 8.2.4 Strain-Promoted Azide–Alkyne Cycloaddition (SPAAC) 297 8.2.5 Inverse Electron-Demand Diels–Alder Cycloaddition (DA INV) 300 8.2.6 Light-Induced Click Reactions 303 8.2.7 1,2-Aminothiol Condensation 304 8.2.8 Transition-Metal-Catalyzed Couplings 305 8.2.9 Miscellaneous Protein-Labeling Reactions 306 8.3 Site-Selective Protein Modification Approaches 307 8.3.1 Site-Selective Modification of Native Proteins 307 8.3.1.1 Cysteine (Cys), dehydroalanine (Dha), and disulfides 307 8.3.1.2 N-Terminal Protein Labeling 309 8.3.1.3 Kinetically-Controlled Protein Labeling (KPL) 309 8.3.1.4 Affinity Labeling for Site-Specific Labeling of Native Proteins 310 8.3.2 Chemical Tags for Labeling Proteins in Live Cells 311 8.3.2.1 Self-Labeling Peptide Tags 312 8.3.2.2 Ligand-Binding Domains 317 8.3.2.3 Self-Labeling Enzymatic Domains 317 8.3.2.4 Enzymatic Modifications 318 8.3.2.5 Metal Chelation 318 8.3.3 Unnatural Amino Acid Mutagenesis 319 8.3.3.1 Residue-Specific UAA Mutagenesis Via SPI 319 8.3.3.2 Site-Specific UAA Incorporation 322 References 322 9 Stable, Versatile Conjugation Chemistries for Modifying Aldehyde-Containing Biomolecules 339 Aaron E. Albers, Penelope M. Drake and David Rabuka 9.1 Introduction 339 9.2 Aldehyde as a Bioorthogonal Chemical Handle for Conjugation 339 9.3 Aldehyde Conjugation Chemistries 340 9.4 The Pictet–Spengler Ligation 341 9.5 The Hydrazinyl-Iso-Pictet–Spengler (HIPS) Ligation 341 9.6 The Trapped-Knoevenagel (thioPz) Ligation 343 9.7 Applications – Antibody–Drug Conjugates 346 9.8 Next-Generation HIPS Chemistry – AzaHIPS 348 9.9 Applications – Protein Engineering 349 9.10 Applications – Protein Labeling 349 9.11 Conclusions 351 References 351 10 Thioamide Labeling of Proteins through a Combination of Semisynthetic Methods 355 Christopher R. Walters, John J. Ferrie, and E. James Petersson 10.1 Introduction 355 10.2 Thioamide Synthesis 356 10.3 Thioamide Incorporation into Peptides 357 10.4 Synthesis of Full‐Sized Proteins Containing Thioamides 360 10.5 Applications 368 10.5.1 Structural Studies 368 10.5.2 Use as Photoswitches 371 10.5.3 Site‐Specific Circular Dichroism Labels 373 10.5.4 Fluorescence Quenching 374 10.5.5 Protein Folding in Model Systems 375 10.5.6 Monitoring Proteolysis 377 10.5.7 α‐Synuclein Misfolding Studies 379 10.6 Conclusions 381 Acknowledgments 381 References 382 11 Macrocyclic Organo-Peptide Hybrids by Intein-Mediated Ligation: Synthesis and Applications 391 John R. Frost and Rudi Fasan 11.1 Introduction 391 11.1.1 Naturally Occurring Macrocyclic Peptides 392 11.1.2 Natural Product Analogs via Reengineering of NRPS and PRPS Biosynthetic Pathways 395 11.2 Macrocyclic Organo-Peptide Hybrids as Natural-Product-Inspired Macrocycles 396 11.2.1 MOrPHs via CuAAC/Hydrazide-Mediated Ligation 398 11.2.2 Catalyst-Free MOrPH Synthesis via Oxime/AMA-Mediated Ligation 401 11.2.3 Structure–Reactivity Relationships in MOrPH Synthesis 401 11.2.4 Synthesis of MOrPH Libraries 404 11.2.5 Macrocyclization Mechanism 405 11.2.6 Bicyclic Organo-Peptide Hybrids 406 11.3 Application of MOrPHs for Targeting α-Helix-Mediated Protein– Protein Interactions 406 11.4 Conclusions 410 References 410 12 Protein Ligation by HINT Domains 421 Hideo Iwaï and A. Sesilja Aranko 12.1 Introduction 421 12.2 Protein Ligation by Protein Splicing 423 12.3 Naturally Occurring and Artificially Split Inteins for Protein Ligation 424 12.4 Conditional Protein Splicing 427 12.5 Inter- and Intramolecular Protein Splicing 429 12.6 Protein Ligation by Other HINT Domains 430 12.7 Bottleneck of Protein Ligation by PTS 432 12.8 Comparison with Other Enzymatic Ligation Methods 432 12.9 Perspective of Protein Ligation by HINT Domains 437 12.10 Conclusions and Future Perspectives 438 Acknowledgment 438 References 438 13 Chemical Ligation for Molecular Imaging 447 Aurélien Godinat, Hacer Karatas, Ghyslain Budin, and Elena A. Dubikovskaya 13.1 Introduction 447 13.2 Chemical Ligation 448 13.2.1 Classical Chemical Ligation 448 13.2.2 Bioorthogonal Chemistry 450 13.2.2.1 Bioorthogonal Chemistry for Optical Imaging 454 13.2.2.2 Bioorthogonal Chemistry for Nuclear Imaging (PET, SPECT) 462 13.2.2.3 Bioorthogonal Chemistry for Magnetic Resonance Imaging (mri) 469 13.3 Conclusion 470 References 473 14 Native Chemical Ligation in Structural Biology 485 Lucia De Rosa, Alessandra Romanelli, and Luca Domenico D’Andrea 14.1 Introduction 485 14.2 Protein (Semi)synthesis for Molecular Structure Determination 486 14.3 Protein (Semi)Synthesis for Understanding Protein Folding, Stability, and Interactions 494 14.4 Protein (Semi)Synthesis in Enzyme Chemistry 501 References 506 Index 517

Chemical biology deals with the use and development of chemical tools to solve biological problems, and chemical ligation fits within this paradigm as a set of techniques used for creating long peptide or protein chains. The practices involved represent a powerful enhancement of traditional solid-phase peptide synthesis – allowing the chemical preparation of proteins, biomolecule synthesis, protein labeling or immobilization, and preparation of proteins with unnatural amino acids. These molecular tools are useful for understanding biological systems and preparing novel bio- and nanomaterials or synthetizing bioactive molecules. Until recently restricted to use by specialized scientists, chemical ligation methods are now in widespread use across interdisciplinary research groups and to different scientific areas. There is a clear need for a single-source resource and reference about these techniques and that is where Chemical Ligation: Tools for Biomolecule Synthesis and Modification comes in. Presenting a wide array of information on chemical ligation, this book guides readers between different chemical ligation methodologies and applications – from the basics to more recent and sophisticated applications. The chapters move from the fundamental to applied aspects, so that novice readers can follow the entire book and apply these reactions in the laboratory. The authors reserve attention for synthetic aspects, so the book also serves as a valuable reference for experimental work. Additionally, a selection of outstanding applications – including protein therapeutic synthesis, drug discovery, and molecular imaging – provides an overview of chemical ligation's potential and get other scientists involved bringing new ideas and applications.