Hermanson G. Bioconjugate Techniques, Second Edition

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Bioconjugate

Techniques

2nd Edition

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Bioconjugate

Techniques

2nd Edition

Greg T. Hermanson

Pierce Biotechnology,

Thermo Fisher Scientiﬁ c,

Rockford, Illinois, USA

AMSTERDAM • BOSTON • HEIDELBERG • LONDON • NEW YORK • OXFORD

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Academic Press is an imprint of Elsevier

The molecular model on the cover is a nitrite reductase enzyme obtained from the RCSB Protein

Data Bank (2afn), as determined by Murphy, M.E., Turley, S., Kukimoto, M., Nishiyama, M.,

Horinouchi, S., Sasaki, H., Tanokura, M., and Adman, E.T. (1995) Structure of Alcaligenes

faecalis nitrite reductase and a copper site mutant, M150E, that contains zinc. Biochemistry 34,

12107–12117. The space-ﬁ lling model was created from the coordinate ﬁ le using PovChem and

the ﬁ nal image ray-traced using POV-Ray

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First edition 1996

Second edition 2008

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Contents Overview

PART I

Bioconjugate Chemistry

1. Functional Targets 3

2. The Chemistry of Reactive Groups 169

PART II

Bioconjugate Reagents

3. Zero-Length Crosslinkers 215

4. Homobifunctional Crosslinkers 234

5. Heterobifunctional Crosslinkers 276

6. Trifunctional Crosslinkers 336

7. Dendrimers and Dendrons 346

8. Cleavable Reagent Systems 391

9. Fluorescent Probes 396

10. Bifunctional Chelating Agents and Radioimmunoconjugates 498

11. Biotinylation Reagents 506

12. Iodination Reagents 546

13. Silane Coupling Agents 562

14. Microparticles and Nanoparticles 582

15. Buckyballs, Fullerenes, and Carbon Nanotubes 627

16. Mass Tags and Isotope Tags 649

17. Chemoselective Ligation: Bioorthogonal Reagents 666

18. Discrete PEG Reagents 707

PART III

Bioconjugate Applications

19. Preparation of Hapten–Carrier Immunogen Conjugates 745

20. Antibody Modiﬁ cation and Conjugation 783

21. Immunotoxin Conjugation Techniques 824

22. Preparation of Liposome Conjugates and Derivatives 858

23. Avidin–Biotin Systems 900

24. Preparation of Colloidal Gold-Labeled Proteins 924

25. Modiﬁ cation with Synthetic Polymers 936

26. Enzyme Modiﬁ cation and Conjugation 961

27. Nucleic Acid and Oligonucleotide Modiﬁ cation and Conjugation 969

28. Bioconjugation in the Study of Protein Interactions 1003

vii

Detailed Contents

Preface to the Second Edition xxiii

Preface to the First Edition xxvi

Acknowledgments xxviii

Health and Safety xxix

Intellectual Property xxx

PART I

Bioconjugate Chemistry

1. Functional Targets

1. Modiﬁ cation of Amino acids, Peptides, and Proteins 3

1.1. Protein Structure and Reactivity 4

Amino Acids 4

Nucleophilic Reactions and the pI of Amino Acid Side Chains 13

Secondary, Tertiary, and Quaternary Structure 15

Prosthetic Groups, Cofactors, and Post-Translational Modiﬁ cations 19

Protecting the Native Conformation and Activity of Proteins 21

Oxidation of Amino Acids in Proteins and Peptides 23

Solvent Accessibility of Functional Targets in Proteins 29

1.2. Protein Crosslinking Methods 32

2. Modiﬁ cation of Sugars, Polysaccharides, and Glycoconjugates 35

2.1. Carbohydrate Structure and Functionality 36

Basic Sugar Structure 37

Sugar Functional Groups 39

Polysaccharide and Glycoconjugate Structure 44

2.2. Carbohydrate and Glycan Conjugation Methods 49

3. Modiﬁ cation of Nucleic Acids and Oligonucleotides 50

3.1. Polynucleotide Structure and Functionality 51

Nucleotide Functional Groups 53

RNA and DNA Structure 62

3.2. Polynucleotide Crosslinking Methods 66

4. Creating Speciﬁ c Functionalities 66

4.1. Introduction of Sulfhydryl Residues (Thiolation) 67

Modiﬁ cation of Amines with 2-Iminothiolane (Traut ’s Reagent) 67

Modiﬁ cation of Amines with SATA 71

Modiﬁ cation of Amines with SATP 74

viii Detailed Contents

Modiﬁ cation of Amines with SPDP 76

Modiﬁ cation of Amines with SMPT 77

Modiﬁ cation of Amines with N -Acetyl Homocysteine Thiolactone 80

Modiﬁ cation of Amines with SAMSA 81

Modiﬁ cation of Aldehydes or Ketones with AMBH 83

Modiﬁ cation of Carboxylates or Phosphates with Cystamine 84

Modiﬁ cation of Proteins with Cystamine 87

Modiﬁ cation of Nucleic Acids and Oligonucleotides with Cystamine 87

Use of Disulﬁ de Reductants 87

Complete Reduction of Disulﬁ des in Protein Molecules Using DTT 90

Use of DTT to Cleave Disulﬁ de-Containing Crosslinking Agents 91

Ellman’s Assay for the Determination of Sulfhydryls 100

4.2. Introduction of Carboxylate Groups 101

Modiﬁ cation of Amines with Anhydrides 102

Modiﬁ cation of Sulfhydryls with Iodoacetate 109

Modiﬁ cation of Sulfhydryls with BMPA 111

Modiﬁ cation of Hydroxyls with Chloroacetic Acid 113

4.3. Introduction of Primary Amine Groups 114

Modiﬁ cation of Carboxylates with Diamines 114

Modiﬁ cation of Sulfhydryls with N -(  -Iodoethyl)triﬂ uoroacetamide [Aminoethyl-8] 118

Modiﬁ cation of Sulfhydryls with Ethylenimine 119

Modiﬁ cation of Sulfhydryls with 2-Bromoethylamine 120

Modiﬁ cation of Sulfhydryls with 2-Aminoethyl-2  -aminoethanethiolsulfonate 121

Modiﬁ cation of Carbohydrates with Diamines 122

Modiﬁ cation of Alkylphosphates with Diamines 124

Modiﬁ cation of Aldehydes with Ammonia or Diamines 124

Introduction of Arylamines on Phenolic Compounds 125

Amine Detection Reagents 127

4.4. Introduction of Aldehyde Residues 129

Periodate Oxidation of Glycols and Carbohydrates 130

Oxidase Modiﬁ cation of Sugar Residues 131

Modiﬁ cation of Amines with NHS-Aldehydes (SFB and SFPA) 132

Modiﬁ cation of Amines with Glutaraldehyde 134

Periodate Oxidation of N-Terminal Serine or Threonine Residues 136

4.5. Introduction of Hydrazine or Hydrazide Functionalities 139

Modiﬁ cation of Aldehydes with Bis-Hydrazide Compounds 140

Modiﬁ cation of Carboxylates with Bis-Hydrazide Compounds 142

Modiﬁ cation of Amines with SANH, SHNH, or SHTH 143

Modiﬁ cation of Alkylphosphates with Bis-Hydrazide Compounds 146

4.6. Introduction of Saccharide or Glycan Groups 147

Modiﬁ cation of Amines with Mono(lactosylamido) mono(succinimidyl)suberate 149

Modiﬁ cation of Amine or Hydrazide Molecules by Carbohydrates and Glycans 150

Labeling Glycans with Fluorescent 2-Aminopyridine, 2-Amino Benzamide, or

Anthranilic Acid 153

Synthesis of Glycosylamines for Conjugating Glycans 155

5. Blocking Speciﬁ c Functional Groups 156

5.1. Blocking Amine Groups 157

Detailed Contents ix

Sulfo-NHS Acetate 157

Acetic Anhydride 158

Citraconic Anhydride 159

Maleic Anhydride 159

5.2. Blocking Sulfhydryl Groups 160

N -Ethyl Maleimide 160

Iodoacetate Derivatives 161

Sodium Tetrathionate 161

Methyl Methanethiosulfonate 163

Ellman’s Reagent 164

Dipyridyl Disulﬁ de Reagents 165

5.3. Blocking Aldehyde Groups 166

Reductive Amination with Tris or Ethanolamine 166

5.4. Blocking Carboxylate Groups 167

Tris or Ethanolamine plus EDC 167

2. The Chemistry of Reactive Groups

1. Amine Reactions 169

1.1. Isothiocyanates 170

1.2. Isocyanates 170

1.3. Acyl Azides 171

1.4. NHS Esters 171

1.5. Sulfonyl Chlorides 173

1.6. Aldehydes and Glyoxals 173

1.7. Epoxides and Oxiranes 174

1.8. Carbonates 175

1.9. Arylating Agents 175

1.10. Imidoesters 176

1.11. Carbodiimides 176

1.12. Anhydrides 178

1.13. Fluorophenyl Esters 179

1.14. Hydroxymethyl Phosphine Derivatives 180

1.15. Guanidination of Amines 181

2. Thiol Reactions 182

2.1. Haloacetyl and Alkyl Halide Derivatives 182

2.2. Maleimides 183

2.3. Aziridines 184

2.4. Acryloyl Derivatives 184

2.5. Arylating Agents 185

2.6. Thiol-Disulﬁ de Exchange Reagents 185

Pyridyl Disulﬁ des 186

TNB-Thiol 187