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Total Chemical Synthesis of Proteins
Total Chemical Synthesis of Proteins

Edited by
Ashraf Brik, Philip Dawson, and Lei Liu
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 v

Contents

Preface xvii

1 Characterization of Protein Molecules Prepared by Total

Chemical Synthesis 1
Stephen B. H. Kent
1.1 Introduction 1
1.2 Chemical Protein Synthesis 2
1.3 Comments on Characterization of Synthetic Protein Molecules 8
1.3.1 Homogeneity 8
1.3.2 Amino Acid Sequence 9
1.3.3 Chemical Analogues 10
1.3.4 Limitations of SPPS 10
1.3.5 Folding as a Purification Step 10
1.4 Summary 12
References 12

2 Automated Fast Flow Peptide Synthesis 17

Mark 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. Pentelute
2.1 Introduction 17
2.2 Results 19
2.2.1 Summary 19
2.2.1.1 Mechanical Principles 20
2.2.1.2 Chemical Principles 20
2.2.1.3 User Interface Principles 20
2.2.1.4 Data Analysis Method 20
2.2.1.5 Outcome 21
2.2.2 First-generation Automated Fast Flow Peptide Synthesis 21
2.2.2.1 Key Findings 21
2.2.2.2 Design of First-generation AFPS 21
2.2.2.3 Characterization of First-generation AFPS 23
2.2.3 Second-generation Automated Fast Flow Peptide Synthesis 24
vi Contents

2.2.3.1 Key Findings 24

2.2.3.2 Design of Second-generation AFPS 24
2.2.3.3 Characterization and Use of Second-generation AFPS 26
2.2.4 Third-generation Automated Fast Flow Peptide Synthesis 32
2.2.4.1 Key Findings 32
2.2.4.2 Design of Third-generation AFPS 34
2.2.4.3 Characterization of Third-generation AFPS 39
2.2.4.4 Reagent Stability Study 43
2.2.5 Fourth-generation Automated Fast Flow Peptide Synthesis 45
2.2.5.1 Key Findings 45
2.2.5.2 Effect of Solvent on Fast Flow Synthesis 45
2.2.5.3 Design and Characterization of Fourth-generation AFPS 45
2.3 Conclusions 49
Acknowledgments 53
References 53

3 N,S- and N,Se-Acyl Transfer Devices in Protein Synthesis 59

Vincent Diemer, Jennifer Bouchenna, Florent Kerdraon, Vangelis Agouridas,
and Oleg Melnyk
3.1 Introduction 59
3.2 N,S- and N,Se-Acyl Transfer Devices: General Presentation, Reactivity
and Statistical Overview of Their Utilization 61
3.2.1 General Presentation of N,S- and N,Se-Acyl Transfer Devices 61
3.2.2 Relative Reactivity of N,S- and N,Se-Acyl Transfer Devices 63
3.2.3 A Statistical Overview of the Synthetic Use of N,S- and N,Se-Acyl
Transfer Devices for Protein Total Chemical Synthesis 64
3.3 Preparation of SEA/SeEAoff and SEAlide Peptides 68
3.3.1 Preparation of SEA and SeEA Peptides 68
3.3.2 Preparation of SEAlide Peptides 70
3.4 Redox-controlled Assembly of Biotinylated NK1 Domain of the
Hepatocyte Growth Factor (HGF) Using SEA and SeEA Chemistries 71
3.5 The Total Chemical Synthesis of GM2-AP Using SEAlide-based
Chemistry 75
3.6 Conclusion 79
References 80

4 Chemical Synthesis of Proteins Through Native Chemical

Ligation of Peptide Hydrazides 87
Chao Zuo, Xiaodan Tan, Xianglong Tan, and Lei Liu
4.1 Introduction 87
4.2 Development of Peptide Hydrazide-based Native Chemical Ligation 88
4.2.1 Conversion of Peptide Hydrazide to Peptide Azide 88
4.2.2 Acyl Azide-based Solid-phase Peptide Synthesis 88
4.2.3 Acyl Azide-based Solution-phase Peptide Synthesis 89
4.2.4 The Transesterification of Acyl Azide 90
 Contents vii

4.2.5 Development of Peptide Hydrazide-based Native Chemical Ligation

90
4.3 Optimization of Peptide Hydrazide-based Native Chemical Ligation
91
4.3.1 Preparation of Peptide Hydrazides 91
4.3.1.1 2-Cl-Trt-Cl Resin 91
4.3.1.2 Peptide Hydrazides from Expressed Proteins 92
4.3.1.3 Sortase-mediated Hydrazide Generation 93
4.3.2 Activation Methods of Peptide Hydrazide 94
4.3.2.1 Knorr Pyrazole Synthesis 94
4.3.2.2 Activation in TFA 94
4.3.3 Ligation Sites of Peptide Hydrazide 95
4.3.4 Multiple Fragment Ligation Based on Peptide Hydrazide 96
4.3.4.1 N-to-C Sequential Ligation 96
4.3.4.2 Convergent Ligation 96
4.3.4.3 One-pot Ligation 96
4.4 Application of Peptide Hydrazide-based Native Chemical Ligation 99
4.4.1 Peptide Drugs and Diagnostic Tools 99
4.4.1.1 Peptide Hydrazides for Cyclic Peptide Synthesis 99
4.4.1.2 Screening for D Peptide Inhibitors Targeting PD-L1 99
4.4.1.3 Chemical Synthesis of DCAF for Targeted Antibody Blocking 101
4.4.1.4 Peptide Toxins 101
4.4.2 Synthesis and Application of Two-photon Activatable Chemokine
CCL5 102
4.4.3 Proteins with Posttranslational Modification 103
4.4.3.1 The Synthesis of Glycosylation-modified Full-length IL-6 103
4.4.3.2 The Chemical Synthesis of EPO 105
4.4.3.3 Chemical Synthesis of Homogeneous Phosphorylated p62 105
4.4.3.4 Chemical Synthesis of K19, K48 Bi-acetylated Atg3 Protein 105
4.4.4 Ubiquitin Chains 108
4.4.4.1 Synthesis of K27-linked Ubiquitin Chains 108
4.4.4.2 Synthesis of Atypical Ubiquitin Chains by Using an Isopeptide-linked Ub
Isomer 109
4.4.4.3 Synthesis of Atypical Ubiquitin Chains Using an Isopeptide-linked Ub
Isomer 109
4.4.5 Modified Nucleosomes 110
4.4.5.1 Synthesis of DNA-barcoded Modified Nucleosome Library 110
4.4.5.2 Synthesis of Modified Histone Analogs with a Cysteine
Aminoethylation-assisted Chemical Ubiquitination Strategy 111
4.4.5.3 Synthesis of Ubiquitylated Histones for Examination of the
Deubiquitination Specificity of USP51 111
4.4.6 Membrane Proteins 112
4.4.7 Mirror-image Biological Systems 112
4.5 Summary and Outlook 113
References 114
viii Contents

5 Expanding Native Chemical Ligation Methodology with

Synthetic Amino Acid Derivatives 119
Emma E. Watson, Lara R. Malins, and Richard J. Payne
5.1 Native Chemical Ligation 120
5.2 Desulfurization Chemistries 120
5.3 Aspartic Acid (Asp, D) 122
5.4 Glutamic Acid (Glu, E) 124
5.5 Phenylalanine (Phe, F) 125
5.6 Isoleucine (Ile, I) 127
5.7 Lysine (Lys, K) 130
5.8 Leucine (Leu, L) 133
5.9 Asparagine (Asn, N) 135
5.10 Proline (Pro, P) 138
5.11 Glutamine (Gln, Q) 139
5.12 Arginine (Arg, R) 139
5.13 Threonine (Thr, T) 140
5.14 Valine (Val, V) 142
5.15 Tryptophan (Trp, W) 144
5.16 Application of Selenocysteine (Sec) to Ligation Chemistry 146
5.17 Aspartic Acid (Asp, D) 147
5.18 Glutamic Acid (Glu, E) 148
5.19 Phenylalanine (Phe, F) 149
5.20 Leucine (Leu, L) 151
5.21 Proline (Pro, P) 151
5.22 Serine (Ser, S) 153
References 155

6 Peptide Ligations at Sterically Demanding Sites 161

Yinglu Wang and Suwei Dong
6.1 Introduction 161
6.2 Ligations Using Thioesters 162
6.2.1 Exogenous Additive-promoted Ligations 162
6.2.2 Ligations Using Reactive Thioesters 167
6.2.3 Internal Activation Strategy in Peptide Ligations 169
6.3 Ligations Using Oxo-esters 170
6.4 Peptide Ligations Based on Selenoesters 170
6.5 Microfluidics-promoted NCL 175
6.6 Representative Applications in Protein Synthesis 178
6.7 Summary and Outlook 181
References 181

7 Controlling Segment Solubility in Large Protein

Synthesis 185
Riley J. Giesler, James M. Fulcher, Michael T. Jacobsen, and Michael S. Kay
7.1 Solvent Manipulation 185
 Contents ix

7.2 Isoacyl Strategy 187

7.3 Semipermanent Solubilizing Tags 191
7.3.1 N- or C-Terminal Solubilizing “Tails” 192
7.3.2 Reversible Backbone Modifications as Solubilizing Tags 194
7.3.3 Building Block Solubilizing Tags 195
7.3.4 Extendable Side-chain-based Solubilizing Tags 195
References 198

8 Toward HPLC-free Total Chemical Synthesis of Proteins 211

Phuc Ung and Oliver Seitz
8.1 Introduction 211
8.1.1 Capture and Release Purification 212
8.1.2 Solid-phase Chemical Ligations (SPCL) 212
8.2 Synthesis of Peptide Segments for Native Chemical Ligation 213
8.2.1 HPLC-free Preparation of N-terminal Peptide Segments for NCL 213
8.2.2 HPLC-free Preparation of C-terminal Peptide Segments for NCL 217
8.3 Synthesis of Proteins Using the His6 Tag 220
8.3.1 Reversible His6 -based Capture Tags 220
8.3.2 His6 -based Immobilization for C-to-N Assembly of Crambin 221
8.3.3 His6 -based Immobilization for Assembly of Proteins on Microtiter
Plates 222
8.3.4 His6 and Hydrazide Tags for Sequential N-to-C Capture and
Release 225
8.4 Synthesis of Proteins via Oxime Formation 227
8.4.1 Reversible Oxime-based Capture Tags 227
8.4.2 Oxime-based Immobilization for N-to-C Solid-phase Chemical
Ligations 227
8.4.3 Oxime-based Immobilization for C-to-N Solid-phase Chemical
Ligations 233
8.4.4 Oxime-based C-to-N Solid-phase Chemical Ligations 237
8.5 Synthesis of Proteins via Hydrazone Formation 238
8.5.1 Reversible Hydrazone-based Capture Tags 238
8.5.2 Hydrazone-based Immobilization for Assembly of Proteins on Microtiter
Plates 239
8.6 Synthesis of Proteins Using Click Chemistry 242
8.6.1 Click-based Immobilization for N-to-C Solid-phase Peptide Ligations
Using a Protected Alkyne 242
8.6.2 Click-based Immobilization for N-to-C Solid-phase Peptide Ligations
Using a Sea Group 243
8.7 Synthesis of Proteins Using the KAHA Ligation 244
8.7.1 The KAHA Ligation 244
8.7.2 HPLC-free Synthesis of Proteins Using the KAHA Ligation 245
8.8 Synthesis of Proteins Using Photocleavable Tags 246
8.8.1 Synthesis of Proteins Using a Photocleavable Biotin-based Purification
Tag 246
x Contents

8.8.2 Synthesis of Proteins Using a Photocleavable His6 -based Purification

Tag 247
8.9 Conclusion 249
References 251

9 Solid-phase Chemical Ligation 259

Skander A. Abboud, Agnès F. Delmas, and Vincent Aucagne
9.1 Introduction 259
9.1.1 The Promises of Solid Phase Chemical Ligation (SPCL) 259
9.1.2 Chemical Ligation Reactions Used for SPCL 260
9.1.3 Key Requirements for a SPCL Strategy 261
9.2 SPCL in the C-to-N Direction 262
9.2.1 Temporary Masking Groups to Enable Iterative Ligations 262
9.2.2 Linkers for C-to-N SPCL 264
9.2.2.1 Use of Same Linker and Solid Support for SPPS and SPCL 265
9.2.2.2 Re-immobilization of the C-Terminal Segment 266
9.3 SPCL in the N-to-C Direction 268
9.3.1 Temporary Masking Groups to Enable Iterative Ligations 268
9.3.2 Linkers for N-to-C SPCL 270
9.3.3 Case Study 272
9.3.4 SPCL with Concomitant Purifications 274
9.4 Post-Ligation Solid-Supported Transformations 274
9.4.1 Chemical Transformations 274
9.4.2 Biochemical Transformations 275
9.5 Solid Support 275
9.6 Conclusion and Perspectives 278
Acknowledgment 278
9.A Appendix 278
References 280

10 Ser/Thr Ligation for Protein Chemical Synthesis 285

Carina Hey Pui Cheung and Xuechen Li
10.1 Serine/Threonine Ligation 287
10.2 Epimerization Issue 289
10.3 Other Aryl Aldehyde Esters 289
10.4 Preparation of Peptide Salicylaldehyde Esters 289
10.5 Scope and Limitations 294
10.6 Strategies of Ser/Thr Ligation for Protein Chemical Synthesis 294
10.7 C-to-N Ser/Thr Ligation 294
10.8 N-to-C Ser/Thr Ligation 296
10.9 One-pot Ser/Thr Ligation and NCL 296
10.10 Bioconjugation 296
10.11 Solubility Issues 298
10.12 Extension of Ser/Thr Ligation 298
10.13 Conclusion 302
References 303
 Contents xi

11 Protein Semisynthesis 307

Nam Chu and Philip A. Cole
11.1 Background 307
11.2 Expressed Protein Ligation (EPL) 308
11.2.1 Method Development 308
11.2.2 Applications of EPL for Studying Protein Posttranslational
Modifications 309
11.2.3 Site-specific Protein Labeling with N-Hydroxysuccinimide Esters 311
11.3 Cysteine Modifications 311
11.3.1 Dehydroalanine Generation and Applications in Semisynthesis 312
11.3.2 Cysteine Alkylation-related Methods to Introduce Lys Mimics 313
11.4 Enzyme-catalyzed Protein/Peptide Ligations 314
11.4.1 Sortase 314
11.4.2 Butelase-1 316
11.4.3 Subtiligase 317
11.4.4 Trypsiligase 318
11.5 Enzyme-catalyzed Expressed Protein Ligation 318
11.6 Summary and Outlook 319
Acknowledgments 320
References 320

12 Bio-orthogonal Imine Chemistry in Chemical Protein

Synthesis 327
Stijn M. Agten, Ingrid Dijkgraaf, Stan H. E. van der Beelen, and Tilman M.
Hackeng
12.1 Introduction 327
12.2 Carbonyl Functionalization 328
12.3 Aminooxy, Hydrazine, and Hydrazide Functionalization 335
12.4 Oxime Ligation 337
12.5 Hydrazone Ligation 342
12.6 Pictet–Spengler Reaction 344
12.7 Catalysis of Oxime and Hydrazone Ligations 346
References 348

13 Deciphering Protein Folding Using Chemical Protein

Synthesis 357
Vladimir Torbeev
13.1 Introduction 357
13.2 Modification of Protein Backbone Amides 358
13.3 Insertion of β-turn Mimetics 361
13.4 Inversion of Chiral Centers in Protein Backbone and Side Chains 362
13.5 Modulating cis–trans Proline Isomerization 366
13.6 Steering Oxidative Protein Folding 368
13.7 Covalent Tethering to Facilitate Folding of Designed Proteins 371
13.8 Discovery of Previously Unknown Protein Folds 373
xii Contents

13.9 Site-specific Labeling with Fluorophores 373

13.10 Foldamers and Foldamer–Peptide Hybrids 375
13.11 Conclusions and Outlook 377
Acknowledgement 378
References 378

14 Chemical Synthesis of Ubiquitinated Proteins for Biochemical

Studies 383
Gandhesiri Satish, Ganga B. Vamisetti, and Ashraf Brik
14.1 The Ubiquitin System 383
14.2 Non-enzymatic Ubiquitination: Challenges and Opportunities 386
14.2.1 Chemical Synthesis of Ub Building Blocks 387
14.2.2 Isopeptide Ligation 387
14.2.3 Total Chemical Synthesis of Tetra-Ub Chains 390
14.3 Synthesis and Semisynthesis of Ubiquitinated Proteins 393
14.3.1 Monoubiquitinated Proteins 393
14.3.2 Tetra-ubiquitinated Proteins 395
14.3.3 Modification of Expressed Proteins with Tetra-Ub 400
14.4 Synthesis of Unique Ub Conjugates to Study and Target DUBs 401
14.5 Activity-based Probes 403
14.6 Perspective 405
List of Abbreviations 406
References 407

15 Glycoprotein Synthesis 411

Chaitra Chandrashekar, Kento Iritani, Tatsuya Moriguchi, and Yasuhiro
Kajihara
15.1 Introduction 411
15.2 Total Chemical Synthesis of Glycoproteins 411
15.3 Semisynthesis of Glycoproteins 413
15.4 Chemoenzymatic Synthesis 413
15.5 α-Synuclein 414
15.6 Hirudin P6 415
15.7 Saposin D 416
15.8 Interleukin 2 417
15.9 Interleukin 25 417
15.10 Mucin 1 419
15.11 Crambin 421
15.12 Tau Protein 422
15.13 Chemical Domain of Fractalkine 423
15.14 CCL1 424
15.15 Interleukin 6 424
15.16 Interleukin 8 425
15.17 Erythropoietin 426
15.18 Trastuzumab 430
 Contents xiii

15.19 Antifreeze Glycoprotein 432

15.20 Conclusion 434
References 434

16 Chemical Synthesis of Membrane Proteins 437

Alanca Schmid and Christian F.W. Becker
16.1 Introduction 437
16.2 Solid Phase Synthesis of TM Peptides 438
16.3 Purification and Handling Strategies of TM Peptides 442
16.4 Solubility Tags 443
16.4.1 Terminal Tags 443
16.4.2 Side Chain Tags 445
16.5 Removable Solubilizing Backbone Tags 445
16.6 Chemical Synthesis of Membrane Proteins 449
16.6.1 Proteins With 1 TM Domain 449
16.6.2 Proteins with 2 TM Domains 450
16.6.3 Proteins with 3 and More TM Domains 454
16.7 Outlook 456
References 457

17 Chemical Synthesis of Selenoproteins 463

Rebecca N. Dardashti, Reem Ghadir, Hiba Ghareeb, Orit Weil-Ktorza, and
Norman Metanis
17.1 What are Selenoproteins? 463
17.2 Expression of Selenoproteins 466
17.3 Sec as a Reactive Handle 469
17.4 Synthesis and Semisynthesis of Natural Selenoproteins 473
17.5 Selenium as a Tool for Protein Folding 475
17.6 Conclusions 478
References 478

18 Histone Synthesis 489

Champak Chatterjee
18.1 The Histones and Their Chemical Modifications 489
18.1.1 Histone Proteins 489
18.1.2 Histone Posttranslational Modifications 490
18.2 Chemical Ligation for Histone Synthesis 492
18.2.1 Native Chemical Ligation 492
18.2.2 Expanding the Scope of Native Chemical Ligation With Inteins 494
18.3 Histone Octamer and Nucleosome Core Particle Assembly 494
18.4 Studying the Histone Code With Synthetic Histones 496
18.4.1 Synthesis of Histones Modified by Smaller Functional Groups 497
18.4.1.1 Histone Phosphorylation 497
18.4.1.2 Histone Acetylation 499
18.4.1.3 Histone Methylation 502
xiv Contents

18.4.2 Synthesis of Sumoylated Histones 505

18.5 Conclusions 506
Acknowledgments 506
References 506

19 Application of Chemical Synthesis to Engineer Protein

Backbone Connectivity 515
Chino C. Cabalteja and W. Seth Horne
19.1 Introduction 515
19.2 Backbone Engineering to Facilitate Synthesis 516
19.3 Backbone Engineering to Explore the Consequences of Chirality 517
19.4 Backbone Engineering to Understand and Control Folding 520
19.5 Backbone Engineering to Create Protein Mimetics 522
19.6 Conclusions 525
References 526

20 Beyond Phosphate Esters: Synthesis of Unusually

Phosphorylated Peptides and Proteins for Proteomic
Research 533
Anett Hauser, Christian E. Stieger, and Christian P. R. Hackenberger
20.1 Introduction 533
20.2 General Methods for the Incorporation of Hydroxy-phosphorylated
Amino Acids into Peptides and Proteins 534
20.3 Incorporation of Other Phosphorylated Nucleophilic Amino Acids into
Peptides and Proteins 537
20.3.1 Phosphoarginine (pArg) 537
20.3.2 Phosphohistidine (pHis) 538
20.3.3 Phospholysine (pLys) 539
20.3.4 Phosphocysteine (pCys) 539
20.3.5 Pyrophosphorylation of Serine and Threonine (ppSer, ppThr) 541
20.4 Development of Phospho-analogues as Mimics for Endogenous
Phospho-Amino Acids 541
20.4.1 Analogues of Phosphoserine, Phosphothreonine, and
Phosphotyrosine 541
20.4.2 Stable Analogues of Phosphoaspartate and Phosphoglutamate 543
20.4.3 Stable Analogues of Phosphoarginine 544
20.4.4 Stable Analogues of Phosphohistidine 545
20.4.5 Stable Analogues of Pyrophosphorylated Serine 547
20.5 Conclusion 547
References 547

21 Cyclic Peptides via Ligation Methods 553

Tristan J. Tyler and David J. Craik
21.1 Introduction 553
21.2 Cyclic Peptide Synthesis 554
 Contents xv

21.3 Orbitides 557

21.4 Paws-derived Peptides(PDPs) 559
21.5 Cyclic Conotoxins 561
21.6 θ-Defensins 563
21.7 Cyclotides 563
21.8 Outlook 568
Acknowledgements 568
Funding 568
References 569

Index 579
 xvii

Preface

Chemical protein synthesis enables the generation of proteins of any architecture

and facilitates the site-selective introduction of unnatural amino acids, biophysical
probes, posttranslational modifications, and other functionalities. The preparation
of these protein analogues enables important biochemical and biophysical studies
and contributes to the fundamental understanding of proteins. In this book, we
present a salient collection of articles that cover the modern methods and applica-
tions of the field, along with descriptions of various case studies in which chemical
protein synthesis is leveraged to shed light on fundamental questions in protein
science.
In Chapter 1, Stephen Kent, the pioneer and leading figure in modern chemi-
cal protein synthesis, introduces the essential role of chemical protein synthesis
in developing a fundamental understanding of the principles that give rise to the
structures and biological functions of protein molecules. Strong emphasis was given
to analytical control and documentation of the synthetic process in order to meet
standards, similar to those used to characterize small organic molecules. Character-
ization of a synthetic protein molecule should rigorously characterize the molecular
homogeneity, correct covalent structure, and defined folded structure.
New exciting advances in synthetic methodology have greatly reshaped the
landscape of chemical protein synthesis over the past 10 years. In Chapter 2,
Bradley L. Pentelute et al. describe the new technology of automated flow-based
solid-phase peptide synthesizer (AFPS) that can incorporate each amino acid
residue in as little as 40 seconds. This technology offers a template for accelerating
syntheses of long polypeptide sequences using an automated flow instrument.
This approach may open the chemical biology field to routine manufacture of
custom-made, fully synthetic proteins. In Chapter 3, Vangelis Agouridas, Oleg
Melnyk, and coworkers describe the development of N,S(Se)-acyl shift systems for
linking peptide segments together through the most widely used method of chemi-
cal protein synthesis, namely, native chemical ligation. This strategy is especially
useful for systems that require latent thioesters. Many biologically active proteins
have been successfully synthesized with the SEA (bis(2-sulfanylethyl)amido) and
SEAlide (N-sulfanylethylanilide) chemistries. In Chapter 4, Lei Liu et al. describe
the development and optimization of hydrazide-based native chemical ligation,
as well as the optimization of the preparation, activation, and ligation of peptide
xviii Preface

hydrazides. The hydrazide-based method exhibits several advantages including easy

preparation, high stability, good handling properties, and controllable activation. It
has been successfully used in the synthesis of peptide pharmaceutics, posttransla-
tionally modified proteins, photo-activatable protein tools, membrane proteins, and
mirror-image proteins.
To further expand the scope and utility of native chemical ligation, Richard J.
Payne et al. describe in Chapter 5 the efforts made to develop various thiolated amino
acids, which, with the help of mild, radical-mediated desulfurization conditions,
can enable ligation at 16 of the 20 proteinogenic amino acids. They also describe
their seminal studies on selenocysteine (Sec), which have enabled rapid chemical
protein synthesis through the diselenide–selenoester ligation (DSL) and novel strate-
gies such as “selenol ligation auxiliary.” In Chapter 6, Suwei Dong et al. describe
the recent development on new strategies that aim to facilitate peptide ligations at
sterically demanding sites. These ligation reactions are usually challenging, as the
strong activating conditions may result in competitive hydrolysis. Furthermore, in
Chapter 7, Michael S. Kay et al. describe the recent studies on how to control segment
solubility in large protein synthesis. Various strategies that utilize solvent choice,
isoacyl dipeptides, and semipermanent solubilizing tags are surveyed and analyzed
in depth. This chapter provides very useful guides for researchers to overcome the
often-encountered solubility problems in chemical protein synthesis.
With the advent of fast peptide synthesis and ligation, other limiting factors have
received attentions for chemical protein synthesis. In Chapter 8, Oliver Seitz et al.
describe the current state of the art in the development of methods that allow mini-
mizing the number of high-performance liquid chromatograpohy (HPLC) purifica-
tion steps. These methods typically rely on rapid and HPLC-free assembly of pro-
teins using capture and release purification handles, opening opportunities for the
chemical synthesis of protein arrays under parallel conditions. In Chapter 9, Vincent
Aucagne et al. describe solid-phase peptide chemical ligation (SPCL), which entails
a ligation-based assembly on a solid support to avoid the laborious intermediate
chromatographic purifications. The development of new polymers, new linkers, new
ligations reactions with improved kinetics, and new masking/unmasking strategies
has broadened the scope of SPCL to more ambitious targets. On a different front, to
expand the flexibility of peptide condensation, ligation strategies other than native
chemical ligation need to be developed. In Chapter 10, Xuechen Li et al. describe
the approach of serine/threonine ligation (STL) between peptide C-terminal salicy-
laldehyde esters and N-terminal Ser/Thr residues. This approach takes advantage of
the high abundance of Ser/Thr residues in the protein sequence and has paved the
way for broad applications in proteins synthesis and related constructs.
Modern total chemical synthesis of proteins can now reach approximately
200–400 amino acids. To amplify the ability to make proteins containing unnatural
amino acids and posttranslational modifications, an important strategy is protein
semisynthesis where a biologically produced protein or protein fragment is selec-
tively modified through covalent chemical reactions. In Chapter 11, Philip A. Cole
et al. describe the development of methods for protein semisynthesis including
expressed protein ligation, cysteine modification reactions, and enzyme-catalyzed
 Preface xix

protein/peptide ligations. These methods have been successfully employed to

augment our understanding on protein posttranslational modification. As a unique
example of protein semisynthesis methods, in Chapter 12, Tilman M. Hackeng et al.
describe bio-orthogonal imine chemistry for protein modification. Details were
provided for how to incorporate carbonyl or α-nucleophiles moieties into proteins,
and how to carry out different imine chemistries such as oxime and hydrazone
ligation.
Next are the applications of chemical protein synthesis to solve various bio-
chemical, biophysical, and biomedical problems. In Chapter 13, Vladimir Torbeev
describes how to use chemical protein synthesis to decipher the mechanism of
protein folding. Many interesting methods have been developed including mod-
ification of protein backbone amides, insertion of β-turn mimetics, inversion of
chiral centers in protein backbone and side chains, modulating cis–trans proline
isomerization, covalent tethering to facilitate folding of designed proteins, and
foldamers and foldamer–peptide hybrids. These methods facilitate biophysical
studies on intrinsically disordered proteins (IDPs) that perform important functions
such as gene transcription and chromatin remodeling. They are also helpful to
studies on improper protein folding related to many diseases such as Alzheimer’s
and Parkinson’s disorders.
One of the most exciting applications of chemical protein synthesis is the develop-
ment of chemical tools to study the biological functions and mechanisms of proteins
bearing posttranslational modifications. In Chapter 14, Ashraf Brik et al. described
their contributions in developing innovative methods to construct a variety of ubiq-
uitin conjugates of high purity, in sufficient quantity to facilitate detailed biophysical
and biochemical analysis. As a “game changer” to overcome the inherit limitations
of the enzymatic approaches, chemical protein synthesis has enabled readily con-
struction of versatile ubiquitin conjugates to study and target ubiquitin-processing
enzymes. In Chapter 15 Yasuhiro Kajihara et al. describe the cutting-edge examples
of glycoprotein synthesis employing various synthetic methodologies and ligation
strategies. The combination of both chemical and enzymatic methodologies gives
insight into how glycans function in their biological environment and may eventu-
ally lead to homogeneous glycoprotein pharmaceuticals.
In addition to protein posttranslational modification, chemical protein synthe-
sis also plays important roles in the biochemical studies on some unique protein
families. In Chapter 16, Christian Becker et al. delineate the chemical synthesis of
membrane proteins, a unique protein family comprising about 30% of the proteins
encoded by the human genome. Access to membrane proteins by chemical synthe-
sis has helped to decipher important aspects of membrane protein functions due
to the unique opportunities that chemical synthesis provides in terms of manipula-
tions of the backbone and side chains of proteins. In Chapter 17, Norman Metanis
et al. describe chemical synthesis of selenoproteins, which contain the rare coded
amino acid selenocysteine. Studies on synthetic selenoproteins facilitate elucidation
of the role of selenium in both natural and unnatural processes, providing increasing
insights into this previously mysterious micronutrient’s role in human health and
disease. Furthermore, in Chapter 18, Champak Chatterjee describe the chemical
xx Preface

synthesis of histones bearing various modifications. Due in large part to the addi-
tion of semisynthetic and fully synthetic site-specifically modified histones to the
repertoire of tools available to investigate the histone code hypothesis, our under-
standing of the many mechanistic roles for histones has rapidly expanded in the
past 10 years.
In the last chapters, W. Seth Horne et al. describe in Chapter 19 how to apply
chemical synthesis to engineer protein backbone connectivity. Studies in this area
enable exploration of the structural and functional consequences of protein back-
bone alteration and open the door to new bio-inspired entities with myriad potential
applications. In Chapter 20, Christian Hackenberger et al. describe how to synthe-
size unusually phosphorylated peptides and proteins for proteomic research. Details
are provided on mimics of endogenous phosphate esters as well as rare, naturally
occurring phosphorylations of functional amino acids such as cysteine, lysine, his-
tidine, and arginine . Finally, in Chapter 21, David J. Craik et al. describe how to
synthesize cyclic peptides via ligation methods. Target molecules include orbitides,
paws-derived peptides, cyclic conotoxins, θ-defensin, and cyclotides. These cyclic
peptides have been a topic of considerable interest in recent years due to their unique
structural and pharmacological features that make them excellent starting points in
drug design.
We believe that the 21 chapters exemplifies the multidisciplinary nature of
research in the field of chemical protein synthesis in the twenty-first century. The
readers of the book will be exposed to the state-of-the-art chemistry that the field
has been developing through the last few decades and the remaining challenges
remained to be tackled. The book will also be very useful source for students
and scientists as well to learn about the various synthetic aspects of the peptide
fragments synthesis, peptide ligation based on different strategies.
 1

Characterization of Protein Molecules Prepared by Total

Chemical Synthesis
Stephen B. H. Kent
Department of Chemistry, The University of Chicago, Chicago, IL 60637, USA

“Nevertheless, the chemical enigma of Life will not be solved until organic chem-
istry has mastered another, even more difficult subject, the proteins, in the same
way as it has mastered the carbohydrates.”
Source: Emil Fischer (Nobel Lecture 1902)

1.1 Introduction
Proteins are the “natural prodicts” of the twenty-first century. Protein molecules are
ubiquitous in the biological world, with numerous diverse functions that range from
acting as structural materials such as the keratins, to integral membrane proteins
that serve as ion channels or as active molecular transporters in cells, to proteins
that act as hormonal messengers in higher animal species, and to proteins that reg-
ulate gene expression [1]. The most important function of protein molecules is as
enzymes, the potent and specific catalysts of the chemical reactions of biological
metabolism, without which life would be impossible [2]. Thanks to modern DNA
sequencing methods applied to genome [3] and metagenome [4] sequencing, vast
numbers of proteins are being discovered at the nucleic acid level as open reading
frames that code for a protein’s polypeptide chain.
The central dogma of protein science is that the amino acid sequence of the
polypeptide chain encodes the folded structure of the protein molecule in its natural
environment, and that it is the folded structure of the protein molecule that gives
rise to its biological function(s) [5]. Proteins range in mass from less than 5 kDa to
more than 100 kDa. The median size of globular protein molecules is ∼35–45 kDa,
comprising a polypeptide chain of ∼300–400 amino acid residues. Proteins are
typically made up of two or more domains, autonomous units of folding, each of
∼120–160 amino acid residues [6].
Protein molecules are not simply really big peptides. In its native environment,
each globular protein molecule has a defined folded structure that gives rise to the
Total Chemical Synthesis of Proteins, First Edition. Edited by Ashraf Brik, Philip Dawson, and Lei Liu.
© 2021 WILEY-VCH GmbH. Published 2021 by WILEY-VCH GmbH.
2 1 Characterization of Protein Molecules Prepared by Total Chemical Synthesis

functional properties of that protein, including biochemical and biological activities.

Synthetic proteins are organic molecules of high molecular mass, comprised of lin-
ear polypeptide chains (typically of 50–300 or more amino acid residues) that fold
to form complex, dynamic structures. The large size of proteins together with the
intricacy of their covalent and folded structures creates special challenges in the
characterization of these synthetic molecules. For this reason, it is important for
researchers to rigorously characterize protein molecules prepared by total chemi-
cal synthesis to meet standards similar to those used in synthetic organic chemistry.
Analytical methods and criteria for the rigorous characterization of synthetic pro-
teins have recently been enunciated [7].

1.2 Chemical Protein Synthesis

“The chemical ligation approach . . . . breaks the conceptual shackles imposed
by the peptide bond, frees us from the linear paradigm of the genetic code, and
opens the world of proteins to the entire repertoire of chemistry.”
Stephen Kent (23rd European Peptide Symposium 1994)

Chemistry, enabled by total synthesis, has an essential role to play in developing a

fundamental understanding of the principles that give rise to the structures and bio-
logical functions of protein molecules. Thanks to modern chemical ligation methods
[8–10], the total synthesis of protein molecules in the research laboratory is both
practical and increasingly robust.
Characterization of a protein prepared by total chemical synthesis should begin
with the synthetic process itself. Analytical control of the bond-forming steps, purifi-
cation and analysis of each synthetic intermediate, and folding of the full-length
synthetic polypeptide chain to form a defined tertiary structure are integral to ver-
ifying the structure of the final synthetic protein product. The steps involved in a
typical total chemical synthesis of a protein molecule are listed below. Key aspects
pertinent to characterization of the synthetic protein are in bold:
● Establish a verified amino acid sequence of the polypeptide chain of the
target protein molecule.
⚬ Use a database such as UniProt (www.uniprot.org); include posttranslational
processing where that is known to give the mature protein molecule.
⚬ Resolving data base/literature ambiguities in the reported amino acid sequence
of a protein can be problematic. It is easy to make a mistake and to end up mak-
ing an incorrect target sequence.
● Design a (convergent) synthesis of the protein’s polypeptide chain, starting
from peptide segments containing fewer than 40–50 amino acid residues
(Scheme 1.1).
● Stepwise solid-phase synthesis (SPPS) of peptide segments equipped with suitable
functionalities for chemical ligation.
● Boc chemistry [11] or Fmoc chemistry SPPS [12], with documentation of the
amino acid sequence actually made.
 1.2 Chemical Protein Synthesis 3

H O
O N
O O
H 2N
O R N
H

Polystyrene resin Aminomethyl resin Boc-Xaa-OCH2-Pam-resin

Bio-beads S-X1

O O
Peptide-αthioester S R Peptide-αCOOH O–

–
O HS S HS
O O O O
+H N +
O +H N N H3N
3
Peptide 1 S 3 Peptide 2 S R H Peptide 3 S R Peptide 4 O–

KCL 1. NCL 2. Thz-Cys

SH HS SH
O O O O
+H N +
3
Peptide 1 N Peptide 2 S R H3N Peptide 3 N Peptide 4 O–
H H

NCL

SH SH O SH
O O O
+H N
3
Peptide 1 N Peptide 2 N Peptide 3 N Peptide 4 O–
H H H

Fold

Scheme 1.1 Convergent chemical synthesis of a protein molecule from four synthetic
peptide segments prepared by stepwise SPPS. Key: NCL, native chemical ligation; KCL,
kinetically controlled ligation; Thz-Cys, conversion of N-terminal thiazolidine-CO– to Cys–.
Source: Adapted from Durek et al. [11].

⚬ Purification by preparative high-performance liquid chromatography (HPLC),

which should be performed under displacement mode [13].
⚬ Combined fractions should be checked by a high resolution technique based
on a separation principle distinct from that used in the purification,
such as capillary isoelectric focusing (CIEF) [14]. Because HPLC is used for
the purification of the synthetic peptide segment, it is NOT sufficient to use
analytical HPLC or LCMS run under similar chromatographic conditions to
verify its homogeneity.
⚬ Direct infusion electrospray ionization mass spectrometry (ESI-MS) for check-
ing purified peptide segments for impurities that have different masses.
4 1 Characterization of Protein Molecules Prepared by Total Chemical Synthesis

⚬ Confirmation of the covalent structure of each synthetic peptide seg-

ment.
● Precise mass measurement
– from analytical LCMS, the mass spectrometric data must be collected across
the entire UV absorbing peak corresponding to the purified peptide.
– a correct mass is a necessary but not sufficient analytical criterion.
● Amino acid sequence
– by MALDI-TOF MS ‘ladder sequencing’ [15] of terminated byproducts that are
invariably present at low-levels in crude synthetic peptides made by SPPS.
– by MS-MS of the purified peptide. This process is rendered more straight-
forward because the target sequence of the peptide segment is known.
● Convergent covalent condensation of the peptide segments
⚬ Chemical conversion of cryptic functional groups to the reactive form suitable
for chemical ligation (e.g. Thz–peptide to Cys–peptide; [16] peptide–CONHNH2
to peptide–thioester [17]).
⚬ Native chemical ligation (NCL); [18] pH 7.0; aqueous 6 M Gu.HCL as a
near-universal solvent; ambient temperature; arylthiol catalyst [19].
● Analytical control of synthetic steps
⚬ “Hands-on” real-time analytical LCMS during ligation reactions is an essential
tool for following the course of each bond-forming step.
● Purification and characterization of each intermediate product
⚬ Using the same protocols as those used for the synthetic peptide segments.
● Purification to homogeneity and rigorous characterization of the
full-length synthetic polypeptide chain.
⚬ Purification by preparative HPLC, which should be performed under displace-
ment mode [13].
⚬ Combined fractions should be checked by a high-resolution technique based on
a separation principle distinct from that used in the purification, such as
CIEF [14]. Because HPLC has been used for the purification of the synthetic
polypeptide, it is NOT sufficient to use analytical HPLC or LCMS run under
similar conditions to verify its homogeneity.
⚬ Direct infusion ESI-MS is also a good technique for checking the full-length syn-
thetic polypeptide chain for impurities that have different masses.
● Folding the synthetic polypeptide chain to form the functional protein molecule
⚬ For disulfide-containing proteins, standard thiol redox couple conditions
are used along with moderate amounts of solubility-enhancing agents
(“denaturants”) to keep misfolded polypeptide chains in solution so that they
too are eventually able to fold correctly [20].
⚬ For proteins that do not contain disulfide bonds, it is frequently sufficient to
simply dilute the polypeptide chain into native buffer or to slowly dialyze from
6 M Gu.HCl into native buffer conditions.
● Analytical control of protein folding
⚬ In the case of disulfide-containing proteins, the folded protein molecule will
usually elute earlier than the unfolded polypeptide chain on analytical reverse
phase HPLC, because hydrophobic side chains are less exposed in the folded,
disulfide cross-linked protein molecule [21].
 1.2 Chemical Protein Synthesis 5

⚬ For proteins that do not contain disulfide bonds, other techniques such as
CD-ORD1 or multidimensional nuclear magnetic resonance (NMR) must be
used to monitor the progress of folding.
● Purification of the folded synthetic protein to give a single defined molec-
ular species.
⚬ By one or more of the techniques of reverse-phase HPLC, ion exchange chro-
matography, size exclusion chromatography.
● Homogeneity of the purified synthetic protein must be verified by a
high-resolution technique based on a separation principle distinct from
that used for purification.
⚬ CIEF [14] is the preferred technique for rigorously establishing the homogene-
ity of the purified synthetic protein molecule.
● Precise measurement of the mass of the synthetic protein molecule by
direct infusion ESI-MS (direct infusion ESI-MS).
⚬ Direct infusion ESI-MS provides data representing all the molecular species
present in the final synthetic product. It thus provides both a precise experi-
mental measurement of the mass of the synthetic protein and at the same time
can reveal the presence of any other products that have a mass distinct from
that of the target protein molecule. All measured mass data must include an
experimental uncertainty (Figure 1.1).
⚬ HPLC with on-line ESMS can be used. However, impurity protein species
present in the synthetic product may not be eluted from the reverse phase
support. Mass spectrometric data from LCMS must be acquired over the
entire UV-absorbing peak corresponding to the purified synthetic protein
product.

⚬ **It is NOT acceptable to report a mass determined at a single time point of

the HPLC chromatogram, while ignoring molecular species with different
masses that are present at other time points under the same UV absorbing
peak.**

⚬ Wherever possible, the monoisotopic mass of a synthetic protein should be

reported (together with experimental uncertainty), along with the calculated
monoisiopic mass. An example is shown below (Figure 1.2).
● Verification of the cysteine pairing for disulfide containing proteins.
⚬ Proteolytic digestion followed by LCMS peptide mapping [24].
⚬ Cysteine pairing can also be verified by high resolution X-ray crystallography
(see below).
● Conformational homogeneity of the synthetic protein molecule.
⚬ Multidimensional NMR “fingerprinting” can be used to establish that the
synthetic protein has a single folded structure [11, 25]. Notes: (i) there are
examples of natural protein molecules that form more than one defined folded

1 While a low-resolution technique such as CD-ORD is useful for monitoring the progress of
folding, it must be supplemented with high-resolution characterization of the folded structure of
the synthetic protein by techniques such as NMR and X-ray crystallography.
6 1 Characterization of Protein Molecules Prepared by Total Chemical Synthesis

13H+
1407.0
12H+
1524.1
14H+
1306.6

11H+
15H + 1662.6
1219.5

16H+ 10H+
1143.4 1828.8

1200 1400 1600 1800

m/z

Figure 1.1 Direct infusion ESI-MS data for [Lys24,38,83 ]erythropoietin aglycone prepared by
total chemical synthesis. Impurities of mass less than the target protein molecule are
essentially absent, as shown by the lack of peaks on the low m/z side of each charge state.
The peaks on the high m/z side of each charge state are Na and Ca ion adducts. Source:
Adapted from Liu et al. [22].

0.8

+8

0.6
+7
Absorbance 214 nm

7202.23

+9

0.4 +5
+6

7195 7200 7205 7210 7215 7220 600 800 1000 1200 1400 1600
0.2
m/z m/z

15 20 25 30 35
Time (min)

Figure 1.2 Analytical HPLC and MS characterization of a synthetic protein. With modern
mass spectrometric instrumentation, the monoisotopic mass can be experimentally
determined and compared with the theoretical mass. Source: Adapted from Durek et al. [23].
 1.2 Chemical Protein Synthesis 7

Trp112
Asp53
Glu35

Tyr54
38
N Tyr
C

(a) (b)

Figure 1.3 X-ray diffraction structure of crystalline human lysozyme prepared by total
chemical synthesis. (a) Cartoon representation of the secondary structure of the folded
human lysozyme synthetic protein molecule; (b) 2Fo −F c electron density map , showing the
quality of the data acquired at a resolution of 1.04 Å. Source: Durek et al. [11]. © 2007
National Academy of Sciences.

structure [26]; (ii) intrinsically disordered proteins only fold, if at all, in the
presence of their target molecules [27].
● Determination of the atomic structure of the folded protein molecule.
⚬ X-ray crystallography, including racemic protein crystallography, [28] can be
used to determine the structure of the folded, homogeneous synthetic protein
molecule [29, 30].
⚬ X-ray structural data will also confirm the connectivity of any disulfides that are
present, and at sufficiently high resolution can reveal the presence of aberrant
chemical modifications of the synthetic protein (Figure 1.3).
⚬ Note that because fractional crystallization from protein mixtures is com-
mon, determination of the crystal structure of a synthetic protein by X-ray
diffraction does not in itself establish that all of the synthetic protein molecules
have that structure and are undamaged. Homogeneity of the synthetic
protein molecule must be established before determination of the crystal
structure.
⚬ For small proteins, the folded structure can be determined using natural abun-
dance protein NMR techniques [31].
● Biological/biochemical assays.
⚬ Quantitative assays, especially measurements of the catalytic activity of a syn-
thetic enzyme molecule, can be very informative but are inadequate as proof of
homogeneity or correct structure of the synthetic protein.

The total synthesis of the chemokine CCL2 (MCP-1) by Grygiel et al. [32] is a
near-perfect example of a properly documented total chemical synthesis of a protein
molecule, illustrating essentially all of the synthetic steps described above together
with meticulous and complete characterization of the synthetic protein as a single
molecular species of defined structure (Figure 1.4).
Selected further examples of well-characterized synthetic proteins can be found
in Refs [24, 33–42].
8 1 Characterization of Protein Molecules Prepared by Total Chemical Synthesis

Absorbance (214 nm)

Absorbance (214 nm)

RP-HPLC CE

6 8 10 12 14 8 10 12 14 16 18 20
Time (min) Time (min)
Relative ion absorbance

ESI Qq/TOF MS
(Mcalc. = 8658.39 Da)

8658.21

8500 8600 8700 8800

Mass (Da)

Figure 1.4 Analytical characterization of the protein CCL2 (MCP-1) prepared by total
chemical synthesis. [32] Purity of the synthetic protein was verified by reverse phase HPLC
and by capillary electrophoresis, two high resolution analytical methods based on different
separation principles (hydrophobicity; charge). The monoisotopic mass was measured by
LC-ESI Qq/TOF mass spectrometry. In addition to the data shown here, the disulfide bonds
are verified by enzymatic digestion and LC-(MS-MS) of the resulting peptide fragments, and
the crystal structure of the synthetic protein is determined at a resolution of 1.9 Å. Source:
From Grygiel et al. [32].

1.3 Comments on Characterization of Synthetic Protein

Molecules

“In the field of protein synthesis it is my confident hope that tomorrow’s deeds
will catch up with today’s titles, and that we shall truly be able to obtain enzym-
atically active proteins as synthetic substances: as materials composed of a single
molecular species.”
Josef Rudinger (3rd American Peptide Symposium 1972)

1.3.1 Homogeneity
The single most important aspect of the characterization of a synthetic protein is
rigorous verification of its homogeneity, i.e. that the synthetic product is a single
 1.3 Comments on Characterization of Synthetic Protein Molecules 9

molecular species. In verifying the homogeneity of a synthetic protein it is essential

to use analytical techniques based on separation principles that are distinct from
the purification method(s) used in the synthesis of the protein molecule. Prepara-
tive HPLC is almost invariably used as the final purification step and closely related
by-products from the synthesis may be co-purified with the desired protein product.
For that reason, analytical HPLC is not a sufficient proof of molecular homogeneity
of a synthetic protein product. Direct infusion ESI-MS is a good technique for check-
ing for the presence of protein impurities that have masses that differ from the mass
of the target protein molecule. However, impurities that have the same mass as the
target protein will not be detected.
Characterization of a synthetic protein must also rule out the presence of inad-
vertent modifications of the covalent structure that may have been introduced in
the course of chemical synthesis. For example, chemical manipulations of peptide
chains can lead to the formation of isopeptide bonds (i.e. beta-amide links) at aspar-
tic acid residues, formation of succinimides at Asn residues, deamidations of Asn
and/or Gln side chains, N-to-O acyl shifts at Gly-Ser sequences, and any of a variety
of other possible by-products. These abnormal modifications of the covalent struc-
ture are often not resolved by analytical HPLC and frequently result in small (1 Da)
or zero differences in the observed mass of the synthetic protein molecule, and thus
can be difficult or impossible to detect by mass spectrometry alone.
A state-of-the-art technique for detection of byproducts and for the rigorous
verification of the molecular homogeneity of a synthetic protein is CIEF. CIEF
is a charge-based, ultrahigh-resolution analytical method that can reveal the
presence of impurities that do not directly involve a unit charge change, even subtle
differences as benign as the replacement of an uncharged amino acid residue in
the protein’s polypeptide chain by another uncharged residue. For that reason,
CIEF is to be preferred for documenting the absence of unexpected by-products
and for the verification of the molecular homogeneity of synthetic peptides
and proteins. Recent descriptions of state-of-the-art CIEF can be found here
[43, 44].

The time has come for capillary isoelectric focusing data to be mandatory veri-
fication of homogeneity in the final characterization of a synthetic protein.

1.3.2 Amino Acid Sequence

The amino acid sequence of a protein determines every aspect of its molecular
structure. Ironically, the aspect of the structure of a chemically synthesized protein
molecule that is most often NOT experimentally verified is the amino acid sequence
of a protein‘s polypeptide chain. Because the target amino acid sequence of the
synthetic protein’s polypeptide chain is known, it is straightforward to verify the
amino acid sequence of a synthetic polypeptide chain by enzymatic digestion and
HPLC-(MS-MS) analysis of the peptide fragments [33].
10 1 Characterization of Protein Molecules Prepared by Total Chemical Synthesis

The all-to-frequent failure to verify the amino acid sequence of a synthetic pro-
tein product should be rectified.

1.3.3 Chemical Analogues

An important objective of total protein synthesis is to make uniquely chemical
analogues of the natural protein molecule, including non-coded amino acids
[45], stable analogues of post-translational modifications [46], fixed elements of
secondary structure [47], chemical engineering of the polypeptide chain backbone
[48], site-specific labeling with isotopes [49] or fluorophores [50], glycoproteins
[51, 52] and glycoprotein mimetics [53], novel topologies such as circular [54]
or concatenated polypeptide chains [55], protein diastereomers [56–58], and
mirror-image protein molecules [59]. Thus, in addition to characterizing features of
protein molecular structure found in natural proteins, characterization of synthetic
proteins must include verification of the novel features introduced by chemical
synthesis.

1.3.4 Limitations of SPPS

Stepwise synthesis of polypeptide chains has its limitations. Because of the statistical
accumulation of resin-bound by-products, even the most highly optimized stepwise
SPPS methods should never be used for the preparation of peptide segments longer
than ∼50 amino acid residues. Depending on amino acid composition and the
sequence of the target peptide, the practical limit for preparing homogeneous
synthetic peptides free of closely related contaminant peptide co-products (e.g. dele-
tions, terminations, deamidations, isopeptide bonds, N-to-O acyl shifts, covalent
modifications) can be as short as 40 or even fewer amino acid residues [60]. Effective
purification and careful characterization of each synthetic peptide segment used in
total protein synthesis is essential.

1.3.5 Folding as a Purification Step

The reality is that no matter how meticulously they have been purified, 30–50 amino
acid residue peptide segments prepared by stepwise SPPS will almost always contain
some level of closely related peptide impurities. Even small amounts of impurities
present in the peptide segments used in protein synthesis will carry over in each
bond-forming step and frequently will be found in the full-length synthetic polypep-
tide chain after purification. Fortunately, particularly for disulfide-constrained glob-
ular proteins, the folding step can be an effective further purification in which closely
related polypeptide chains that do not fold correctly are subsequently removed dur-
ing purification of the folded synthetic protein. Examples of LCMS data before and
direct infusion ESI-MS data after folding are shown in Figure 1.5.
Another random document with
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 “He might just be coming out to dinner now,” the boy murmured to
himself, “and I’d be sure to miss him if I left.” But no gardener
appeared till late. The clock had struck six and the streets were full
of workmen returning to their homes when the gate did open and out
stepped the gardener, dinner bucket in hand. He had no sooner
appeared than Jerry met him, outwardly as bold as a lion, but
inwardly anxious.
“Mr. Gardener,” he began.
The man scowled down at him.
“What’s wrong?” he asked. “Out with it.”
“Nothing much,” returned Jerry, “at least, you see—you know me
and my sister were here looking at your garden yesterday.”
“Yes, I remember now. Well?”
“And you know—” Jerry went on to tell his story of the broken
plant, concluding with: “so I thought some time, you know, you might
have an extra plant, just a little bit of a one, that you wouldn’t miss,
and if you’d sell it cheap, I’d work it out, the pay, I mean. I could help
to wheel that sand, you know.”
The man’s face broke into a smile.
“All right, sonny; it’s a bargain. I must go home now, but you come
around Monday, and sister shall get a plant.”
“Shall I come to this gate?” asked Jerry eagerly. “When?”
“No, not here; round at the other side. We don’t often open this
gate, only to take in loads of dirt and such, and when I am late I go
out this way. You go all the way around to the other side and you’ll
see an iron railing; there’s another gate there; go in and knock at the
back door and say you want to see John McClure. Come about
twelve o’clock and bring sissy.” He nodded and passed on, leaving
Jerry in a state of extreme satisfaction, and ready to make for home
with scurrying legs and a large appetite.
IN THE GARDEN
 CHAPTER II
IN THE GARDEN

“Where have you been all day?” Cassy asked as Jerry came
blundering in.
“You can’t guess,” he returned.
“Down by the wharf?”
Jerry shook his head. “Somewhere you like. I stayed outside ’most
all day, but I got in at last; you know where.”
“Not the garden.”
“Yes, sir, the garden, and what’s more we’re going to see it on
Monday. I had a talk with the gardener; his name is John McClure.”
“Really?” Cassy clapped her hands.
“Yes, really.” Jerry winked at his mother. That was not all there was
to tell, but he meant to keep the rest a secret.
“I’m glad it’s Saturday night,” said Cassy after a silence, “for now
I’ll have all the time I want for thinking about it, for I’ll have no
lessons to study and to bother me. Besides, mother won’t have to
work to-morrow and she can tell us all about the house where we
were born. How long has it been since we left it, mother?”
“Six years,” Mrs. Law told her.
“I remember it a little,” said Jerry. “I remember father, too.”
“I wish I did,” said Cassy sorrowfully. “Don’t let’s talk about that
now. Tell us what you did to-day.”
“I went to market and did my errands first, but there were not many
baskets to take home this morning, and then I went and sat out on
the curbstone by the wall and waited. Gee! but that’s a big place; it
takes up ’most a square, and it’s awful pretty up there. I saw a shiny
carriage stop at the door and a lady and a boy got out. I’d like to be
that boy.”
“Was he just your size?” asked Cassy, interested.
“No, lots bigger, but he looked friendly; he kind of smiled when he
saw me there.”
“Come, children, it’s cleaning up time,” said Mrs. Law. “We must
get ready for Sunday; my last buttonhole is finished. I expect Jerry is
as hungry as a bear.”
“I am as hungry as two bears,” Jerry assured her. “What are we
going to have for supper? I don’t care much what it is, so there is
enough of it.”
“Don’t tell him what it is,” said Cassy.
Jerry approached the little stove where something was simmering
and sending out savory odors. He lifted the lid.
“Stew!” he cried.
“Yes, with dumplings in it. You shouldn’t have taken off the lid,
Jerry, it will spoil them.”
“Never mind, it is all ready to dish up,” Mrs. Law told him.
“My, but it smells good,” said Jerry with much satisfaction. “Did you
make plenty of dumplings, mother? They are jolly good with
molasses on them.”
“I hope I made enough,” his mother told him. “Cassy and I did not
take a hearty dinner, for you were not here, and so we decided to
have a hot supper.”
“We don’t have such good things every day,” Jerry remarked,
drawing up his chair. “I wonder if we’ll ever have lots and lots to eat;
meat every day and dessert. My! it must be fine. I’ll bet that boy I
saw to-day has all that.”
“I don’t believe he has dessert every day; I don’t believe anybody
has,” Cassy asserted, eyeing her mother as she dished out a
plentiful supply of stew upon Jerry’s plate.
 “Ho! I’ll bet some people do. Don’t you, mother?”
“Why, yes, of course.”
“Did you use to?” Cassy asked.
“I believe we did.”
“Were we as rich as that?” Cassy looked her surprise.
“We were not rich at all, but we were very comfortable and very
content.” Mrs. Law gave a little sigh.
“Just wait till I grow up, and we will be again,” said Jerry, pausing
with a big piece of dumpling on his fork.
“That’s so long,” sighed Cassy.
But to Jerry with a plentiful meal before him to-morrows were
pleasant anticipations, and he replied: “Pshaw! no it isn’t.”
Cassy glanced up and caught her mother’s tired look.
“Well, no it isn’t,” she agreed; “it won’t be any time, and I’ll be
grown up, too, and mother won’t have a thing to do but——”
“‘Sit on a cushion and sew a fine seam,’” Mrs. Law put in.
“‘And feed upon strawberries, sugar and cream,’” Cassy finished
the line. “I saw strawberries in one of the shops yesterday.”
“I’d rather have dumplings any day,” Jerry decided, having finished
eating his stew, and being now ready to attack the dumplings and
molasses. To tell the truth, the dumplings formed the principal part of
the stew and the meat was very scarce, but the children rather
rejoiced at that, and completed their meal with much satisfaction.
Then there were many little duties to be done, and of all the rooms in
the tenement it is safe to say that Mrs. Law’s was in decidedly the
best order for Sunday.
Cassy could hardly wait till noon time Monday, and though she
was usually a pretty good scholar, she made many mistakes that
morning, and was only aroused to a sense of her inattention when it
suddenly dawned upon her that she might be kept in and that would
be a calamity too dreadful to contemplate.
 At last twelve o’clock came, and she found Jerry waiting outside
the school door.
“Come along,” he cried. “We don’t want to lose any time.” And
catching her by the arm he hurried her along the street till they
reached the long wall.
“Aren’t you going to wait at the gate?” Cassy asked as Jerry,
without pausing, went on.
“No, we are to go around to the other side. ’Way round where the
horses go into the stable.”
They found no difficulty in getting in, and, after walking the length
of the garden path, they came upon their friend, the gardener, sitting
on a wheelbarrow. He looked up as they came near.
“Well, here you are,” he greeted them cordially. “Didn’t forget the
time. Sun’s noon high and a few minutes past. Now then, my little
lass, we’ll go find your plant; I’ve got it safe and sound for you.”
Cassy’s eyes opened wide.
“My little plant?”
“Yes, didn’t brother tell you?”
Cassy shook her head.
“You’re a sly little lad,” he said, pinching Jerry’s ear. “I thought that
was what you came for.”
“I thought it was just to see the flowers,” said Cassy.
“You can do that, too, but we’ll pick out yours first. I slipped a lot of
geraniums a while ago; they’re easy cared for and are good
bloomers; no trouble if you give them a sunny window and a little
water. Now then.” He stopped before a row of potted geraniums
already showing their gay blooms of red and pink. “Take your pick,”
he said.
“Oh!” Cassy crouched down and looked lovingly from one to the
other. How could she decide among so many? However, finally, after
changing her mind frequently, she halted between a crimson and a
lovely pink. Then she sought Jerry’s advice, and he spoke for the red
one, but Cassy thought her mother would like the pink one; it was
such a lovely color, and finally that was selected; Cassy, hugging it to
her, fairly kissed the little flower.
“How good you are,” she said. “Oh, Mr. McClure, what a lovely
father you must be.”
John McClure threw back his head and laughed.
“I’m no father at all,” he said; “I’m a lone man with neither chick nor
child.”
“I think that is a great pity,” said Cassy, gravely. “I have been
thinking of you living in a pretty little house with morning-glories
climbing over the porch.”
“And all the place I’ve got is a room in a workman’s boarding-
house.”
“I wish you did have a cottage.”
“I’ve wished the same more than once, but it doesn’t seem to
come my way. Come now, we’ll go see the rest of the flowers.”
“I’m afraid we shall miss our dinner if we do that,” Jerry put in.
“Oh, I’d rather miss my dinner than not see the flowers,” Cassy
told him.
“You would?” Mr. McClure looked pleased.
Just then they saw a boy coming down the path. He had a cheery
bright face, and Cassy concluded he must be the one of whom Jerry
had told her.
“Well, John,” the boy cried, “I see you have company.”
“Yes, Mr. Rock. This young lass here says she’d rather look at the
flowers than eat her dinner. What do you think of that?”
“That she’s a girl after your own heart. But why can’t she do both?”
The boy smiled down at Cassy as if expecting her to answer.
 “Because we couldn’t get home and back to school in time and
see the flowers too, and I do so want to see the flowers.” She looked
wistfully at Jerry.
“And I suppose your brother would rather eat his dinner,” said
Rock. “I think we can manage it. I’ll run in and get you a sandwich or
something, so you won’t starve.” He was gone like a flash, his long
legs covering the ground with great strides.
“That’s just like Mr. Rock,” said John McClure. “Come along,
children, we’ll be looking at the flowers, and Mr. Rock will see that
you don’t go hungry.”
“But——” Cassy looked confused. “I—mother——Do you think
mother would like it, Jerry?”
“What?” John interrupted. “I’ll venture to say she’ll not object to
your taking a bit of a sandwich from Mr. Rock. Just make yourselves
easy, and if you think there’ll be any trouble I’ll go and explain it to
her myself. By the way, you won’t want to take your geranium to
school, sis; you’d better leave it here and call for it on your way
home. Come now; these are the tulips.” And he began to guide them
around the garden showing them all manner of sweet or showy
flowers.
They were not half way around when Rock appeared bearing a
tray on which were two glasses of milk, a pile of sandwiches and two
generous slices of pie. He set the tray down on a bench under a
spreading tree.
“I say, John, it’s a jolly place to eat, out here, this fine day. I’ve a
mind to bring something for myself. Don’t begin your lunch, children,
till I come back.” And he was off again, returning in a few minutes
with more sandwiches, some crackers and half a pie. “Now,” he said,
“I call this great. Pitch in, youngsters. Come along, John, bring your
dinner-bucket, and we’ll have a lively time.”
Cassy and Jerry were rather shy at first, but Rock soon made
them feel at home, and they thought they had never tasted anything
so good as those chicken sandwiches and that apple pie.
 “There!” exclaimed Rock, as the last crumb disappeared, “I
enjoyed that a great deal more than if I had eaten my lunch indoors. I
went to the country for over Sunday and when I got back this
morning it was too late for school; the train was an hour late. I found
mother wasn’t going to be at home to lunch, so, if you hadn’t been
here to keep me company, I’d have eaten a solitary meal indoors. By
the way, what time do you go back to school?”
Jerry told him, and he pulled out his watch.
“Then you’ll have to scamper,” he cried.
“You’re coming back to get your geranium,” John charged Cassy,
and she smiled up at him with such a sunny expression that John
saw there was little danger of her forgetting.
“Those are nice little things,” said Rock as he watched the two
children depart.
“That they are, Mr. Rock,” returned John.
“I wonder where they live,” said Rock.
“In one of the tenements beyond the square, so they tell me.”
“Pshaw! that’s not a very nice place, and those children seem neat
and well-behaved, and they speak well, too.”
“They’re fatherless,” said John, “and it’s likely their mother has a
hard time to get along, and can afford to live nowhere else, but
they’re different from most of the gang down that way; I saw that the
first day when they stood by the gate and looked in.” And he told
Rock of how he had first met the children.
“I’m going to learn more about them,” Rock declared. “I’ll be here
when they come back after school. That little girl’s face is a perfect
sunbeam when she smiles, and the boy is a manly, honest little
fellow.”
True to his word Rock was there when the children returned.
“Where do you live?” he asked them.
“On Orchard Street,” they told him.
 “Have you always lived there?”
“No,” said Cassy, “we used to live in a lovely little house near the
city, and there were morning-glories growing over the porch.” She
looked at John.
“By the way,” said that worthy, “I told you I’d see about the
morning-glories. I believe I’ve some seed in the tool-house. You’re
welcome to ’em, and if you plant ’em they’ll be likely to grow, and you
can train ’em over your window. Have you a good yard?”
“No,” Cassy said; “we have three rooms on the top floor, one big
room and two little ones. Mother likes it up where we are because it
is nearer the sky, and there is no one above us.”
“Sensible woman,” said John, nodding approvingly.
“And you’ve no yard? Well, you can plant the seeds in a box on
the window-sill, unless you like to have a garden in the common
yard.”
“Oh, we can’t. Billy Miles won’t let us.” And Cassy told the story of
her treasured morning-glory, and of its destruction. Rock and John
listened gravely. “And I was so sorry,” said Cassy, “for I had always
wanted to see a morning-glory, because mother tells how they grew
over our porch where we used to live. We would be there now if
papa had lived.”
“How long since he died?” Rock asked, sympathetically.
“Six years. I wasn’t three years old, and Jerry was about five.
Papa got hurt on the railroad, you know, and he never got well.”
“Yes,” spoke up Jerry. “And mother said some people said she
ought to have lots of money from the railroad, because it was their
fault, but she tried and they put her off, and she couldn’t afford to
have a lawyer, so she just had to give up.”
Rock listened attentively. “I wish she’d come and see papa, he’s a
railroad man, and maybe he could tell her what to do.”
“Mother hasn’t any time,” said Cassy, shaking her head gravely.
“She makes buttonholes all the time; she has to so as to get us
something to eat and to pay the rent, but when we are big we shall
not let her do it.”
“Of course not,” said John. “Well, youngsters, I’ve got to go to
work. You must come around again some day and tell me how the
morning-glories are coming on. There is your geranium, my little
lass.”
“And here’s a bunch of violets for your mother,” said Rock. “Tell me
your mother’s name and just where you live. Some day I might want
to call on you.” He smiled at Cassy as he held out the sweet-smelling
violets, and the children, as happy as lords, went off, Jerry carrying
his own and Cassy’s books and the little girl holding her geranium
carefully with one hand, and in the other bearing the violets which
she sniffed frequently as she went along.
WHERE IS JERRY?
 CHAPTER III
WHERE IS JERRY?

Carrying her plant in triumph, Cassy appeared before her mother.

“See, see,” she cried out, “just see! Isn’t it lovely? And look at
these violets. Oh, mother, we’ve had the loveliest time, and Jerry has
some morning-glory seeds in his pocket. You don’t know all we’ve
been doing. Were you worried that we didn’t come home to dinner?
Did you think we were kept in?”
“No, for I thought it probable that the charms of that garden would
prove too much for you, yet I thought I should have two half-starved
children to come home to supper.”
“But we’re not half-starved. We had—oh, mother, it’s just like a
story. Tell her about it, Jerry, while I put my flower in the window, and
give the violets a drink of water.” She set the flower-pot carefully on
the sill, and then stood off to see the effect. Truly the gay pink
blossom did brighten up the bare room, while the scent of the violets
filled the air. “I feel so rich,” said Cassy. “I never had such a lovely
day.”
“And how about the lessons?” asked Mrs. Law.
Cassy looked a little crestfallen.
“The lessons weren’t quite as good as they are sometimes. You
see,” she came close to her mother and fumbled uneasily with the
hem of her apron. “You see, mother, I couldn’t help thinking about
the garden all the time, and I came near being kept in ’cause I didn’t
pay attention. Wouldn’t that have been dreadful?”
“It would have been pretty bad, for it has never happened to you,
and I would have been very sorry to have had you come home with
such a report.”
“But I remembered just in time, and I did pay attention the rest of
the day. Are you tired, you poor mother, sitting here stitching,
stitching all day long? If I could only have brought you a piece of that
pie.”
“Do you think that would have rested me? I am not so very tired,
for this is only Monday, you know.”
“Oh, Jerry,” Cassy turned to her brother, “we forgot to tell her what
the nice boy said. Is he a boy or a young gentleman?”
“Oh, he’s just a boy,” said Jerry grandly, with the judgment of his
superior years.
“His name is Rock, Rock Hardy, but his mother’s name is Dallas.
That is the old Dallas place, you know, where the garden is, and
Rock—Mr. Rock?” She looked inquiringly at Jerry who answered,
“No, just Rock; he told me to call him that. His real name is Rockwell,
but they call him Rock for short.”
“Well then, Rock said that he wished you would come to see his
father. He is a railroad man and maybe he could get you that
money.”
Mrs. Law shook her head.
“That was very kind, I am sure, but I could not think of troubling a
stranger. No doubt the boy might think his father would be interested,
but that was only his idea, and I couldn’t think of calling on Mr. Dallas
upon such an invitation. I suppose the gentleman is Mr. Dallas, and
Rock Hardy is his stepson.”
“Yes, he is, and I think Mr. Dallas must be very nice, for Rock is so
fond of him.” Cassy looked disappointed that her mother had not
been willing to go right off to see Mr. Dallas. She had dreamed that
great things would come of it, and now her hopes were blasted. But
it did not take from the memory of the day’s pleasure, and she went
about the room, setting the table for supper, and attending to her
little duties, singing softly.
There was not much in the room; a few cheap chairs, one a large
rocker, a table covered with a red cloth, a kitchen safe and a small
cook-stove; the windows were hung with cheap white curtains, but
the floor was bare of carpet, though it had been stained. The house
was an old one, and was let out in rooms to tenants who could afford
only a small rent, consequently the neighborhood was now none of
the best. There was an ill smell of cooking in the halls, and the sound
of a constant banging of doors, and the shuffle of heavy feet on the
bare stairs could always be heard.
The top floor Mrs. Law thought by far the most desirable, although
it was the cheapest, and with her children near her, away from the
confusion and noise below, she felt that it was as much of a home as
she could hope for.
 “Every Now and Then Flora was Carried Over and
Shown the Geranium”

It was hard to keep sturdy Jerry from mixing with the neighborhood
boys, but though he had learned many of their rough ways and much
of their speech, he was not without good principles, and was careful
not to bring the language of the street into his home. His faults were
not such as came from an evil heart, and his love for his mother and
sister would cause any one to forgive him many mistakes.
Cassy was such a mother-child that she shrank from the children
in the house, and when she was at home from school rarely played
with them. She would rather stay with her mother. Her principal
playmate was a battered doll, which she had owned since she was a
baby. It was the last gift from her father, and she prized it above all
her possessions.
The next afternoon she established herself in a corner with her
doll, Flora, and carried on a long whispered conversation with her.
Every now and then Flora was carried over and shown the geranium,
and made to peer into the box which held the morning-glory seeds.
At last the daylight waned and Mrs. Law moved nearer the window.
“It’s ’most bedtime, but I’ll tell you a story before you go to bed,”
she heard Cassy say to her doll. “Listen, and it will give you
something to think about while you are trying to go to sleep. Once
there was a little girl ’bout as big as me, and she had a mother and a
brother and she hadn’t any money at all, but they all wanted some,
so her mother went to see a gentleman who knew where there was
lots of railroad money, and he gave a whole lot of it to her mother
’cause her husband had been hurt in a railroad accident, and so the
little girl had a whole window full of flowers and violets every day,
and chicken sandwiches and apple pie, but she didn’t get a new doll,
only a new silk dress for her old one—a blue silk dress just like the
sky, and oh yes—they had a nice little house with morning-glories
growing all over the porch, and the little girl’s mother didn’t have to
make any more buttonholes or sew any more on the sewing-
machine; she sat on a velvet chair and ate the chicken sandwiches
and apple pie all day.”
At this point Mrs. Law laughed. “Didn’t she get rather tired of that?”
she asked.
“Oh, mother, were you listening?”
 “I couldn’t very well help hearing.”
“That’s a new story,” said Cassy, gravely undressing her doll. “I’ve
never told it to Flora before. It’s not quite a true story, but I wish it
was, don’t you?”
“All but the occupation of the little girl’s mother. I think she would
get dreadfully tired of sitting on a velvet chair, and of eating
sandwiches and pie all day.”
Cassy laughed.
“I don’t believe I’d get tired of them. Come, Flora, you must go to
bed. I’ll give you one more sniff of violets before you go.” And after
being allowed once more to bury her snub nose in the bunch of
violets, Flora was put to bed, her crib being a wooden footstool
turned upside down, and her covers being some old bits of cotton
cloth.
“Go call Jerry and we’ll have supper,” said Mrs. Law.
Cassy placed the violets carefully in the middle of the table, and
leaving her mother to dish up the oat-meal, she went in search of
Jerry. Hearing voices in the back yard she first went there, but there
was no sign of him, and she went next to the front door, which
generally stood wide open. She looked up and down the dingy
street, but saw nothing of her brother. She ran down the steps
looking to right and left. At the corner she saw Billy Miles with a
group of boys.
“Who ye lookin’ fer?” asked Billy.
“I’m looking for Jerry,” Cassy told him. “Have you seen anything of
him?”
“I seen him ’bout an hour ago,” he returned, winking at the other
boys, who broke out into a loud laugh.
Cassy looked at them sharply.
“You know where he is,” she said positively. “I think you might tell
me.”