Chemical Ligation Tools for Biomolecule Synthesis and Modification

Luca D. D'Andrea, PhD, is Research Scientist at the Institute of Biostructures and Bioimaging, CNR Naples, Italy. His scientific interests are in the field of peptide and protein chemistry. His research activity focuses on design, synthesis, and structural characterization of peptide/proteins as therapeutic/diagnostic agents.

Alessandra Romanelli, PhD, is assistant professor of General Chemistry at Department of Pharmacy, University of Naples "Federico II", Italy. She actively works in the field of peptides and peptide-based molecules (such as peptide nucleic acids) as tools for chemical biology.

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

1.2 Chemical Ligation Chemistries 6

1.3 Imine Ligations 7

1.4 Serine/Threonine Ligation (STL) 21

1.5 Thioether Ligation 24

1.6 Thioester Ligation 25

1.7 α�–Ketoacid�–Hydroxylamine (KAHA) Ligation 49

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

2.4 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

3.2 Serine/Threonine Ligation (STL) 130

3.3 Application of STL in Protein Synthesis 140

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

4.3 Beyond Native Chemical Ligation at Cysteine – The Development of Thiolated Amino Acids and Their Application in Protein Synthesis 174

4.4 Ligation–Deselenization in the Chemical Synthesis of Proteins 211

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 MUC2 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

7.3 Ligation and Refolding 273

7.4 Illustrative Examples 276

References 280

8 Chemoselective Modification of Proteins 285
Xi Chen, Stephanie Voss, and Yao-Wen Wu

8.1 Chemical Protein Synthesis 285

8.2 Chemoselective and Bioorthogonal Reactions 287

8.3 Site-Selective Protein Modification Approaches 307

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

10.6 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

11.2 Macrocyclic Organo-Peptide Hybrids as Natural-Product-Inspired Macrocycles 396

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

13.3 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

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