Hermanson Bioconjugate Techniques

2. Auflage 2010
ISBN: 978-0-08-056872-0
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: Adobe DRM (»Systemvoraussetzungen)

E-Book, Englisch, 1323 Seiten

Bioconjugate Techniques, 2nd Edition, is the essential guide to the modification and cross linking of biomolecules for use in research, diagnostics, and therapeutics. It provides highly detailed information on the chemistry, reagent systems, and practical applications for creating labeled or conjugate molecules. It also describes dozens of reactions with details on hundreds of commercially available reagents and the use of these reagents for modifying or cross linking peptides and proteins, sugars and polysaccharides, nucleic acids and oligonucleotides, lipids, and synthetic polymers.
*A one-stop source for proven methods and protocols for synthesizing bioconjugates in the lab
*Step-by-step presentation makes the book an ideal source for researchers who are less familiar with the synthesis of bioconjugates
*More than 600 figures that visually describe the complex reactions associated with the synthesis of bioconjugates
*Includes entirely new chapters on the latest areas in the field of bioconjugation as follows:
Microparticles and nanoparticles
Silane coupling agents
Dendrimers and dendrons
Chemoselective ligation
Quantum dots
Lanthanide chelates
Cyanine dyes
Discrete PEG compounds
Buckyballs,fullerenes, and carbon nanotubes
Mass tags and isotope tags
Bioconjugation in the study of protein interactions

Autoren/Hrsg.

Hermanson, Greg T.

Inhaltsverzeichnis

1;Front Cover;1
2;Bioconjugate Techniques;4
3;Copyright Page;5
4;Detailed Contents;8
5;Preface to the Second Edition;24
6;Preface to the First Edition;27
7;Acknowledgments;29
8;Health and Safety;30
9;Intellectual Property;31
10;PART I: Bioconjugate Chemistry;32
10.1;Chapter 1. Functional Targets;34
10.1.1;1. Modification of Amino acids, Peptides, and Proteins;34
10.1.1.1;1.1. Protein Structure and Reactivity;35
10.1.1.1.1;Amino Acids;35
10.1.1.1.2;Nucleophilic Reactions and the pI of Amino Acid Side Chains;44
10.1.1.1.3;Secondary, Tertiary, and Quaternary Structure;46
10.1.1.1.4;Prosthetic Groups, Cofactors, and Post-Translational Modifications;50
10.1.1.1.5;Protecting the Native Conformation and Activity of Proteins;52
10.1.1.1.6;Oxidation of Amino Acids in Proteins and Peptides;54
10.1.1.1.7;Solvent Accessibility of Functional Targets in Proteins;60
10.1.1.2;1.2. Protein Crosslinking Methods;63
10.1.2;2. Modification of Sugars, Polysaccharides, and Glycoconjugates;66
10.1.2.1;2.1. Carbohydrate Structure and Functionality;67
10.1.2.1.1;Basic Sugar Structure;68
10.1.2.1.2;Sugar Functional Groups;70
10.1.2.1.3;Polysaccharide and Glycoconjugate Structure;75
10.1.2.2;2.2. Carbohydrate and Glycan Conjugation Methods;80
10.1.3;3. Modification of Nucleic Acids and Oligonucleotides;81
10.1.3.1;3.1. Polynucleotide Structure and Functionality;82
10.1.3.1.1;Nucleotide Functional Groups;84
10.1.3.1.2;RNA and DNA Structure;93
10.1.3.2;3.2. Polynucleotide Crosslinking Methods;97
10.1.4;4. Creating Specific Functionalities;97
10.1.4.1;4.1. Introduction of Sulfhydryl Residues (Thiolation);98
10.1.4.1.1;Modification of Amines with 2-Iminothiolane (Traut ’s Reagent);98
10.1.4.1.2;Modification of Amines with SATA;102
10.1.4.1.3;Modification of Amines with SATP;105
10.1.4.1.4;Modification of Amines with SPDP;107
10.1.4.1.5;Modification of Amines with SMPT;108
10.1.4.1.6;Modification of Amines with N -Acetyl Homocysteine Thiolactone;111
10.1.4.1.7;Modification of Amines with SAMSA;112
10.1.4.1.8;Modification of Aldehydes or Ketones with AMBH;114
10.1.4.1.9;Modification of Carboxylates or Phosphates with Cystamine;115
10.1.4.1.10;Modification of Proteins with Cystamine;118
10.1.4.1.11;Modification of Nucleic Acids and Oligonucleotides with Cystamine;118
10.1.4.1.12;Use of Disulfide Reductants;118
10.1.4.1.13;Complete Reduction of Disulfides in Protein Molecules Using DTT;121
10.1.4.1.14;Use of DTT to Cleave Disulfide-Containing Crosslinking Agents;122
10.1.4.1.15;Ellman’s Assay for the Determination of Sulfhydryls;131
10.1.4.2;4.2. Introduction of Carboxylate Groups;132
10.1.4.2.1;Modification of Amines with Anhydrides;133
10.1.4.2.2;Modification of Sulfhydryls with Iodoacetate;140
10.1.4.2.3;Modification of Sulfhydryls with BMPA;142
10.1.4.2.4;Modification of Hydroxyls with Chloroacetic Acid;144
10.1.4.3;4.3. Introduction of Primary Amine Groups;145
10.1.4.3.1;Modification of Carboxylates with Diamines;145
10.1.4.3.2;Modification of Sulfhydryls with N-(?-Iodoethyl)trifluoroacetamide [Aminoethyl-8];149
10.1.4.3.3;Modification of Sulfhydryls with Ethylenimine;150
10.1.4.3.4;Modification of Sulfhydryls with 2-Bromoethylamine;151
10.1.4.3.5;Modification of Sulfhydryls with 2-Aminoethyl-2'-aminoethanethiolsulfonate;152
10.1.4.3.6;Modification of Carbohydrates with Diamines;153
10.1.4.3.7;Modification of Alkylphosphates with Diamines;155
10.1.4.3.8;Modification of Aldehydes with Ammonia or Diamines;155
10.1.4.3.9;Introduction of Arylamines on Phenolic Compounds;156
10.1.4.3.10;Amine Detection Reagents;158
10.1.4.4;4.4. Introduction of Aldehyde Residues;160
10.1.4.4.1;Periodate Oxidation of Glycols and Carbohydrates;161
10.1.4.4.2;Oxidase Modification of Sugar Residues;162
10.1.4.4.3;Modification of Amines with NHS-Aldehydes (SFB and SFPA);163
10.1.4.4.4;Modification of Amines with Glutaraldehyde;165
10.1.4.4.5;Periodate Oxidation of N-Terminal Serine or Threonine Residues;167
10.1.4.5;4.5. Introduction of Hydrazine or Hydrazide Functionalities;170
10.1.4.5.1;Modification of Aldehydes with Bis-Hydrazide Compounds;171
10.1.4.5.2;Modification of Carboxylates with Bis-Hydrazide Compounds;173
10.1.4.5.3;Modification of Amines with SANH, SHNH, or SHTH;174
10.1.4.5.4;Modification of Alkylphosphates with Bis-Hydrazide Compounds;177
10.1.4.6;4.6. Introduction of Saccharide or Glycan Groups;178
10.1.4.6.1;Modification of Amines with Mono(lactosylamido) mono(succinimidyl)suberate;180
10.1.4.6.2;Modification of Amine or Hydrazide Molecules by Carbohydrates and Glycans;181
10.1.4.6.3;Labeling Glycans with Fluorescent 2-Aminopyridine, 2-Amino Benzamide, or Anthranilic Acid;184
10.1.4.6.4;Synthesis of Glycosylamines for Conjugating Glycans;186
10.1.5;5. Blocking Specific Functional Groups;187
10.1.5.1;5.1. Blocking Amine Groups;188
10.1.5.1.1;Sulfo-NHS Acetate;188
10.1.5.1.2;Acetic Anhydride;189
10.1.5.1.3;Citraconic Anhydride;190
10.1.5.1.4;Maleic Anhydride;190
10.1.5.2;5.2. Blocking Sulfhydryl Groups;191
10.1.5.2.1;N-Ethyl Maleimide;191
10.1.5.2.2;Iodoacetate Derivatives;192
10.1.5.2.3;Sodium Tetrathionate;192
10.1.5.2.4;Methyl Methanethiosulfonate;194
10.1.5.2.5;Ellman’s Reagent;195
10.1.5.2.6;Dipyridyl Disulfide Reagents;196
10.1.5.3;5.3. Blocking Aldehyde Groups;197
10.1.5.3.1;Reductive Amination with Tris or Ethanolamine;197
10.1.5.4;5.4. Blocking Carboxylate Groups;198
10.1.5.4.1;Tris or Ethanolamine plus EDC;198
10.2;Chapter 2. The Chemistry of Reactive Groups;200
10.2.1;1. Amine Reactions;200
10.2.1.1;1.1. Isothiocyanates;201
10.2.1.2;1.2. Isocyanates;201
10.2.1.3;1.3. Acyl Azides;202
10.2.1.4;1.4. NHS Esters;202
10.2.1.5;1.5. Sulfonyl Chlorides;204
10.2.1.6;1.6. Aldehydes and Glyoxals;204
10.2.1.7;1.7. Epoxides and Oxiranes;205
10.2.1.8;1.8. Carbonates;206
10.2.1.9;1.9. Arylating Agents;206
10.2.1.10;1.10. Imidoesters;207
10.2.1.11;1.11. Carbodiimides;207
10.2.1.12;1.12. Anhydrides;209
10.2.1.13;1.13. Fluorophenyl Esters;210
10.2.1.14;1.14. Hydroxymethyl Phosphine Derivatives;211
10.2.1.15;1.15. Guanidination of Amines;212
10.2.2;2. Thiol Reactions;213
10.2.2.1;2.1. Haloacetyl and Alkyl Halide Derivatives;213
10.2.2.2;2.2. Maleimides;214
10.2.2.3;2.3. Aziridines;215
10.2.2.4;2.4. Acryloyl Derivatives;215
10.2.2.5;2.5. Arylating Agents;216
10.2.2.6;2.6. Thiol-Disulfide Exchange Reagents;216
10.2.2.6.1;Pyridyl Disulfides;217
10.2.2.6.2;TNB-Thiol;218
10.2.2.6.3;Disulfide Reductants;218
10.2.2.7;2.7. Vinylsulfone Derivatives;219
10.2.2.8;2.8. Metal-Thiol Dative Bonds;219
10.2.2.9;2.9. Native Chemical Ligation;222
10.2.2.10;2.10. Cisplatin Modification of Methionine and Cysteine;223
10.2.3;3. Carboxylate Reactions;223
10.2.3.1;3.1. Diazoalkanes and Diazoacetyl Compounds;224
10.2.3.2;3.2. Carbonyldiimidazole;225
10.2.3.3;3.3. Carbodiimides;226
10.2.4;4. Hydroxyl Reactions;226
10.2.4.1;4.1. Epoxides and Oxiranes;226
10.2.4.2;4.2. Carbonyldiimidazole;227
10.2.4.3;4.3. N,N'-Disuccinimidyl Carbonate or N-Hydroxysuccinimidyl Chloroformate;227
10.2.4.4;4.4. Oxidation with Periodate;228
10.2.4.5;4.5. Enzymatic Oxidation;229
10.2.4.6;4.6. Alkyl Halogens;229
10.2.4.7;4.7. Isocyanates;230
10.2.5;5. Aldehyde and Ketone Reactions;231
10.2.5.1;5.1. Hydrazine Derivatives;231
10.2.5.2;5.2. Schiff Base Formation;231
10.2.5.3;5.3. Reductive Amination;232
10.2.5.4;5.4. Mannich Condensation;232
10.2.6;6. Active Hydrogen Reactions;233
10.2.6.1;6.1. Diazonium Derivatives;233
10.2.6.2;6.2. Mannich Condensation;234
10.2.6.3;6.3. Iodination Reactions;234
10.2.7;7. Photo-Chemical Reactions;235
10.2.7.1;7.1. Aryl Azides and Halogenated Aryl Azides;235
10.2.7.2;7.2. Benzophenones;236
10.2.7.3;7.3. Anthraquinones;236
10.2.7.4;7.4. Certain Diazo Compounds;238
10.2.7.5;7.5. Diazirine Derivatives;239
10.2.7.6;7.6. Psoralen Compounds;239
10.2.8;8. Cycloaddition Reactions;241
10.2.8.1;8.1. Diels–Alder Reaction;241
10.2.8.2;8.2. Complex Formation with Boronic Acid Derivatives;241
10.2.8.3;8.3. Click Chemistry: Cu[sup(1)]-promoted Azide—Alkyne [3 + 2] Cycloaddition;242
11;PART II: Bioconjugate Reagents;244
11.1;Chapter 3. Zero-Length Crosslinkers;246
11.1.1;1. Carbodiimides;246
11.1.1.1;1.1. EDC;247
11.1.1.2;1.2. EDC plus Sulfo-NHS;250
11.1.1.3;1.3. CMC;254
11.1.1.4;1.4. DCC;255
11.1.1.5;1.5. DIC;257
11.1.2;2. Woodward's Reagent K;259
11.1.3;3. N,N'-Carbonyldiimidazole;259
11.1.4;4. Schiff Base Formation and Reductive Amination;262
11.2;Chapter 4. Homobifunctional Crosslinkers;265
11.2.1;1. Homobifunctional NHS Esters;266
11.2.1.1;1.1. DSP and DTSSP;269
11.2.1.2;1.2. DSS and BS[sup(3)];272
11.2.1.3;1.3. DST and Sulfo-DST;274
11.2.1.4;1.4. BSOCOES and Sulfo-BSOCOES;275
11.2.1.5;1.5. EGS and Sulfo-EGS;277
11.2.1.6;1.6. DSG;279
11.2.1.7;1.7. DSC;280
11.2.2;2. Homobifunctional Imidoesters;281
11.2.2.1;2.1. DMA;282
11.2.2.2;2.2. DMP;283
11.2.2.3;2.3. DMS;284
11.2.2.4;2.4. DTBP;285
11.2.3;3. Homobifunctional Sulfhydryl-Reactive Crosslinkers;287
11.2.3.1;3.1. DPDPB;288
11.2.3.2;3.2. BMH;289
11.2.4;4. Difluorobenzene Derivatives;290
11.2.4.1;4.1. DFDNB;290
11.2.4.2;4.2. DFDNPS;291
11.2.5;5. Homobifunctional Photoreactive Crosslinkers;292
11.2.5.1;5.1. BASED;293
11.2.6;6. Homobifunctional Aldehydes;293
11.2.6.1;6.1. Formaldehyde;294
11.2.6.2;6.2. Glutaraldehyde;296
11.2.7;7. Bis-epoxides;299
11.2.7.1;7.1. 1,4-Butanediol Diglycidyl Ether;300
11.2.8;8. Homobifunctional Hydrazides;300
11.2.8.1;8.1. Adipic Acid Dihydrazide;301
11.2.8.2;8.2. Carbohydrazide;302
11.2.9;9. Bis-diazonium Derivatives;302
11.2.9.1;9.1. o-Tolidine, Diazotized;303
11.2.9.2;9.2. Bis-diazotized Benzidine;304
11.2.10;10. Bis-alkyl Halides;305
11.3;Chapter 5. Heterobifunctional Crosslinkers;307
11.3.1;1. Amine-Reactive and Sulfhydryl-Reactive Crosslinkers;308
11.3.1.1;1.1. SPDP, LC-SPDP, and Sulfo-LC-SPDP;309
11.3.1.2;1.2. SMPT and Sulfo-LC-SMPT;312
11.3.1.3;1.3. SMCC and Sulfo-SMCC;314
11.3.1.4;1.4. MBS and Sulfo-MBS;317
11.3.1.5;1.5. SIAB and Sulfo-SIAB;319
11.3.1.6;1.6. SMPB and Sulfo-SMPB;322
11.3.1.7;1.7. GMBS and Sulfo-GMBS;323
11.3.1.8;1.8. SIAX and SIAXX;324
11.3.1.9;1.9. SIAC and SIACX;326
11.3.1.10;1.10. NPIA;327
11.3.2;2. Carbonyl-Reactive and Sulfhydryl-Reactive Crosslinkers;328
11.3.2.1;2.1. MPBH;329
11.3.2.2;2.2. M[sub(2)]C[sub(2)]H;330
11.3.2.3;2.3. PDPH;331
11.3.3;3. Amine-Reactive and Photoreactive Crosslinkers;333
11.3.3.1;3.1. NHS-ASA, Sulfo-NHS-ASA, and Sulfo-NHS-LC-ASA;336
11.3.3.2;3.2. SASD;337
11.3.3.3;3.3. HSAB and Sulfo-HSAB;339
11.3.3.4;3.4. SANPAH and Sulfo-SANPAH;341
11.3.3.5;3.5. ANB-NOS;343
11.3.3.6;3.6. SAND;343
11.3.3.7;3.7. SADP and Sulfo-SADP;345
11.3.3.8;3.8. Sulfo-SAPB;347
11.3.3.9;3.9. SAED;347
11.3.3.10;3.10. Sulfo-SAMCA;350
11.3.3.11;3.11. p-Nitrophenyl Diazopyruvate;353
11.3.3.12;3.12. PNP-DTP;354
11.3.4;4. Sulfhydryl-Reactive and Photoreactive Crosslinkers;355
11.3.4.1;4.1. ASIB;356
11.3.4.2;4.2. APDP;357
11.3.4.3;4.3. Benzophenone-4-iodoacetamide;359
11.3.4.4;4.4. Benzophenone-4-maleimide;361
11.3.5;5. Carbonyl-Reactive and Photoreactive Crosslinkers;361
11.3.5.1;5.1. ABH;362
11.3.6;6. Carboxylate-Reactive and Photoreactive Crosslinkers;363
11.3.6.1;6.1. ASBA;364
11.3.7;7. Arginine-Reactive and Photoreactive Crosslinkers;364
11.3.7.1;7.1. APG;365
11.4;Chapter 6. Trifunctional Crosslinkers;367
11.4.1;1. 4-Azido-2-nitrophenylbiocytin-4-nitrophenyl ester;367
11.4.2;2. Sulfo-SBED;368
11.4.3;3. MTS-ATF-Biotin and MTS-ATF-LC-Biotin;372
11.4.4;4. Hydroxymethyl Phosphine Derivatives;373
11.5;Chapter 7. Dendrimers and Dendrons;377
11.5.1;1. Dendrimer Construction;377
11.5.2;2. Conjugation to Dendrimers;384
11.5.2.1;2.1. Coupling to Amine-Dendrimers;387
11.5.2.1.1;Modification of Amine-Dendrimers with Sulfo-NHS-LC-SPDP;387
11.5.2.1.2;NHS-PEG-Maleimide Coupling to Amine-Dendrimers;390
11.5.2.1.3;Coupling Glycoproteins to Amine-Dendrimers by Reductive Amination;392
11.5.2.1.4;Blocking of Amines on PAMAM Dendrimers;394
11.5.2.1.5;Preparation of Sugar-Dendrimer Derivatives;397
11.5.2.1.6;Conjugation of Carboxylate Organic Molecules to Amine-Dendrimers;402
11.5.2.1.7;Epoxy Activation of Amine-Dendrimers;404
11.5.2.1.8;Biotinylation of Amine-Dendrimers;407
11.5.2.1.9;Fluorescent Labeling of Amine Dendrimers;411
11.5.3;3. Dendrimer-Chelate Derivatives for Imaging Applications;414
11.5.4;4. Dendrimer Derivatives as Surface Modification Agents;416
11.5.5;5. Dendrimer Fluorescent Quantum Dots;420
11.6;Chapter 8. Cleavable Reagent Systems;422
11.6.1;1. Cleavage of Disulfides by Reduction;423
11.6.2;2. Periodate-Cleavable Glycols;424
11.6.3;3. Dithionite-Cleavable Bonds;425
11.6.4;4. Hydroxylamine Cleavable Esters;425
11.6.5;5. Base Labile Sulfones;426
11.7;Chapter 9. Fluorescent Probes;427
11.7.1;1. Fluorescein Derivatives;431
11.7.1.1;Amine-Reactive Fluorescein Derivatives;432
11.7.1.2;Sulfhydryl-Reactive Fluorescein Derivatives;437
11.7.1.3;Aldehyde/Ketone and Cytosine-Reactive Fluorescein Derivatives;443
11.7.2;2. Rhodamine Derivatives;446
11.7.2.1;Amine-Reactive Rhodamine Derivatives;447
11.7.2.2;Sulfhydryl-Reactive Rhodamine Derivatives;456
11.7.2.3;Aldehyde/Ketone and Cytosine-Reactive Rhodamine Derivatives;458
11.7.3;3. Coumarin Derivatives;461
11.7.3.1;Amine-Reactive Coumarin Derivatives;462
11.7.3.2;Sulfhydryl-Reactive Coumarin Derivatives;465
11.7.3.3;Aldehyde- and Ketone-Reactive Coumarin Derivatives;469
11.7.4;4. BODIPY Derivatives;471
11.7.4.1;Amine-Reactive BODIPY Derivatives;472
11.7.4.2;Aldehyde/Ketone-Reactive BODIPY Derivatives;475
11.7.4.3;Sulfhydryl-Reactive BODIPY Derivatives;480
11.7.5;5. Cascade Blue Derivatives;484
11.7.5.1;Amine-Reactive: Cascade Blue Acetyl Azide;484
11.7.5.2;Carboxylate-Reactive: Cascade Blue Cadaverine and Cascade Blue Ethylenediamine;486
11.7.5.3;Aldehyde/Ketone-Reactive: Cascade Blue Hydrazide;487
11.7.6;6. Lucifer Yellow Derivatives;488
11.7.6.1;Sulfhydryl-Reactive: Lucifer Yellow Iodoacetamide;489
11.7.6.2;Aldehyde/Ketone-Reactive: Lucifer Yellow CH;490
11.7.7;7. Phycobiliprotein Derivatives;492
11.7.8;8. Cyanine Dye Derivatives;495
11.7.8.1;Amine-reactive Cyanine Dyes;498
11.7.8.2;Thiol-reactive Cyanine Dyes;501
11.7.8.3;Carbonyl-reactive Cyanine Dyes;503
11.7.9;9. Lanthanide Chelates for Time-resolved Fluorescence;505
11.7.10;10. Quantum Dot Nanocrystals;516
11.7.10.1;Properties of Quantum Dots;516
11.7.10.2;Conjugation to QDs;524
11.7.10.3;Conjugation of Proteins to QDs Using EDC;525
11.7.10.4;Conjugation to QDs Using sulfo-SMCC;527
11.8;Chapter 10. Bifunctional Chelating Agents and Radioimmunoconjugates;529
11.8.1;1. DTPA;530
11.8.2;2. DOTA, NOTA, and TETA;531
11.8.3;3. DTTA;532
11.8.4;4. DFA;533
11.8.5;5. Use of Thiolation Reagents for Direct Labeling to Sulfhydryl Groups;534
11.8.6;6. FeBABE;536
11.9;Chapter 11. Biotinylation Reagents;537
11.9.1;1. Amine-Reactive Biotinylation Agents;538
11.9.1.1;1.1. D-Biotin and Biocytin;539
11.9.1.2;1.2. NHS-Biotin and Sulfo-NHS-Biotin;541
11.9.1.3;1.3. NHS-LC-Biotin and Sulfo-NHS-LC-Biotin;543
11.9.1.4;1.4. NHS-Iminobiotin;546
11.9.1.5;1.5. Sulfo-NHS-SS-Biotin;548
11.9.2;2. Sulfhydryl-Reactive Biotinylation Agents;551
11.9.2.1;2.1. Biotin-BMCC;551
11.9.2.2;2.2. Biotin-HPDP;553
11.9.2.3;2.3. Iodoacetyl-LC-Biotin;555
11.9.3;3. Carbonyl- or Carboxyl-Reactive Biotinylation Agents;556
11.9.3.1;3.1. Biotin-Hydrazide and Biotin-LC-Hydrazide;557
11.9.3.2;3.2. Biocytin Hydrazide;559
11.9.3.3;3.3. 5-(Biotinamido)pentylamine;560
11.9.4;4. Photoreactive Biotinylation Agents;561
11.9.4.1;4.1. Photobiotin;562
11.9.4.2;4.2. Psoralen-PEO[sub(3)]-Biotin;564
11.9.5;5. Active Hydrogen-Reactive: p-Aminobenzoyl Biocytin, Diazotized;565
11.9.6;6. Glycan Biotinylation Reagents;568
11.9.6.1;6.1. Biotinylated Aminopyridine;569
11.9.6.2;6.2. Biotinyl-L-3-(2-naphthyl)-alanine hydrazide;572
11.9.6.3;6.3. Biotin-PEG-Phosphine;574
11.10;Chapter 12. Iodination Reagents;577
11.10.1;1. Chloramine-T;579
11.10.2;2. Iodobeads;581
11.10.3;3. Iodogen;584
11.10.4;4. Lactoperoxidase-Catalyzed Iodination;586
11.10.5;5. Iodinatable Modification and Crosslinking Agents;587
11.10.5.1;5.1. Bolton–Hunter Reagent;588
11.10.5.2;5.2. Iodinatable Bifunctional Crosslinking Agents;591
11.11;Chapter 13. Silane Coupling Agents;593
11.11.1;1. Silane Reaction Strategies;596
11.11.1.1;1.1. Aqueous/Organic Solvent Deposition;597
11.11.1.2;1.2. Aqueous Deposition;597
11.11.1.3;1.3. Organic Solvent Deposition;598
11.11.1.4;1.4. Vapor Phase Deposition;598
11.11.2;2. Functional Silane Compounds;599
11.11.2.1;2.1. 3-Aminopropyltriethoxysilane and 3-Aminopropyltrimethoxysilane;599
11.11.2.2;2.2. Carboxyethylsilanetriol;604
11.11.2.3;2.3. N-(Trimethoxysilylpropyl)ethylenediamine triacetic acid;606
11.11.2.4;2.4. 3-Glycidoxypropyltrimethoxysilane and 3-Glycidoxypropyltriethoxysilane;608
11.11.2.5;2.5. Isocyanatopropyltriethoxysilane;610
11.12;Chapter 14. Microparticles and Nanoparticles;613
11.12.1;1. Particle Types;613
11.12.2;2. Particle Characteristics and Stability;615
11.12.3;3. Particle Concentration;619
11.12.4;4. Polymeric Microspheres and Nanospheres;619
11.12.4.1;4.1. Passive Adsorption;621
11.12.4.2;4.2. Covalent Coupling to Polymeric Particles;625
11.12.4.3;4.3. Coupling to Carboxylate Particles;626
11.12.4.4;4.4. Coupling to Amine Particles;630
11.12.4.5;4.5. Coupling to Amine Particles Using Crosslinking Agents;630
11.12.4.6;4.6.Glutaraldehyde;632
11.12.4.7;4.7. SPDP Coupling to Amine Particles;633
11.12.4.8;4.8. NHS-PEG[sub(n)]-Maleimide Coupling to Amine Particles;635
11.12.4.9;4.9. Coupling to Hydroxyl Particles;637
11.12.4.10;4.10. Coupling to Hydrazide Particles;644
11.12.4.11;4.11. Coupling to Epoxy Particles;646
11.12.4.12;4.12. Coupling to Aldehyde Particles;648
11.12.5;5. Silica Particles;649
11.12.5.1;5.1. Fluorescent Silica Particles;651
11.12.5.2;5.2. Silane Functionalization of Silica Particles;656
11.13;Chapter 15. Buckyballs, Fullerenes, and Carbon Nanotubes;658
11.13.1;1. Buckyballs and Fullerenes;658
11.13.1.1;1.1. Properties of Fullerenes;658
11.13.1.2;1.2. Modification of Fullerenes;660
11.13.2;2. Carbon Nanotubes;669
11.13.2.1;2.1. Nanotube Properties;669
11.13.2.2;2.2. Nanotube Functionalization;671
11.13.2.3;2.3. Detergent or Lipid Modification of Carbon Nanotubes;671
11.13.2.4;2.4. Pyrene Modification of Carbon Nanotubes;675
11.13.2.5;2.5. Modification of Carbon Nanotubes by Cycloaddition;676
11.14;Chapter 16. Mass Tags and Isotope Tags;680
11.14.1;1. ICAT Reagents;682
11.14.2;2. ECAT Reagents;688
11.14.3;3. Isobaric Tags;690
11.15;Chapter 17. Chemoselective Ligation: Bioorthogonal Reagents;697
11.15.1;1. Diels–Alder Reagent Pairs;698
11.15.2;2. Hydrazine–Aldehyde Reagent Pairs;700
11.15.2.1;Protocol for Modification of Amine-Oligo with SANH or SFB;705
11.15.2.2;Protocol for Modification of Protein or Antibody with SANH or SFB;706
11.15.2.3;Conjugation Using the Aldehyde/Hydrazine Reaction;706
11.15.3;3. Boronic Acid–Salicylhydroxamate Reagent Pairs;707
11.15.4;4. Click Chemistry: Cu(I)-Promoted Azide–Alkyne [3 + 2] Cycloaddition;711
11.15.5;5. Staudinger Ligation;721
11.15.6;6. Native Chemical Ligation;728
11.15.6.1;6.1. Expressed Protein Ligation and Inteins;732
11.16;Chapter 18. Discrete PEG Reagents;738
11.16.1;1. Homobifunctional PEG Crosslinkers;742
11.16.1.1;1.1. Bis-NHS Ester PEG Compounds;742
11.16.1.2;1.2. Bis-Maleimide–PEG Compounds;745
11.16.1.2.1;BM(PEG)[sub(2)], BM(PEG)[sub(3)], and Bis-MAL–dPEG[sub(3)];745
11.16.2;2. Heterobifunctional PEG Reagents;749
11.16.2.1;2.1. Maleimide–PEG[sub(n)]–NHS Ester Compounds;749
11.16.2.2;2.2. NHS-PEG[sub(n)]-Azide/Alkyne Compounds for Chemoselective Ligation;753
11.16.3;3. Biotinylation Reagents Containing Discrete PEG Linkers;757
11.16.3.1;3.1. NHS–PEG[sub(n)]–Biotin Compounds;758
11.16.3.2;3.2. NHS-Chromogenic-PEG[sub(3)]-Biotin;761
11.16.3.3;3.3. Maleimide-PEG[sub(n)]-Biotin Compounds;763
11.16.3.4;3.4. Hydrazide-PEG[sub(4)]-Biotin;764
11.16.3.5;3.5. Biotin-PEG[sub(n)]-Amine Compounds;767
11.16.3.6;3.6. Biotin–PEG[sub(3)]–Benzophenone;770
11.16.4;4. Discrete PEG Modification Reagents;770
12;PART III: Bioconjugate Applications;774
12.1;Chapter 19. Preparation of Hapten–Carrier Immunogen Conjugates;776
12.1.1;1. The Basis of Immunity;776
12.1.2;2. Types of Immunogen Carriers;778
12.1.2.1;2.1. Protein Carriers;779
12.1.2.1.1;KLH;779
12.1.2.1.2;BSA and cBSA;780
12.1.2.1.3;Thyroglobulin and OVA;782
12.1.2.1.4;Tetanus and Diphtheria Toxoids;784
12.1.2.2;2.2. Liposome Carriers;784
12.1.2.3;2.3. Synthetic Carriers;785
12.1.3;3. Carbodiimide-Mediated Hapten–Carrier Conjugation;786
12.1.4;4. NHS Ester-Mediated Hapten–Carrier Conjugation;794
12.1.5;5. NHS Ester-Maleimide Heterobifunctional Crosslinker-Mediated Hapten–Carrier Conjugation;797
12.1.6;6. Active-Hydrogen-Mediated Hapten–Carrier Conjugation;804
12.1.6.1;6.1. Diazonium Conjugation;804
12.1.6.2;6.2. Mannich Condensation;807
12.1.7;7. Glutaraldehyde-Mediated Hapten–Carrier Conjugation;810
12.1.8;8. Reductive Amination-Mediated Hapten–Carrier Conjugation;812
12.2;Chapter 20. Antibody Modification and Conjugation;814
12.2.1;1. Preparation of Antibody–Enzyme Conjugates;818
12.2.1.1;1.1. NHS Ester–Maleimide-Mediated Conjugation;819
12.2.1.1.1;Activation of Enzymes with NHS Ester–Maleimide Crosslinkers;820
12.2.1.1.2;Conjugation with Reduced Antibodies;821
12.2.1.1.3;Conjugation with 2-Iminothiolane-Modified Antibodies;824
12.2.1.1.4;Conjugation with SATA-Modified Antibodies;826
12.2.1.2;1.2. Glutaraldehyde-Mediated Conjugation;828
12.2.1.2.1;One-Step Glutaraldehyde Protocol;829
12.2.1.2.2;Two-Step Glutaraldehyde Protocol;831
12.2.1.3;1.3. Reductive-Amination-Mediated Conjugation;831
12.2.1.3.1;Activation of Enzymes with Sodium Periodate;833
12.2.1.3.2;Activation of Antibodies with Sodium Periodate;834
12.2.1.3.3;Conjugation of Periodate-Oxidized HRP to Antibodies by Reductive Amination;835
12.2.1.3.4;Conjugation of Periodate-Oxidized Antibodies with Amine or Hydrazide Derivatives;836
12.2.1.4;1.4. Conjugation Using Antibody Fragments;838
12.2.1.4.1;Preparation of F(ab')[sub(2)] Fragments Using Pepsin;838
12.2.1.4.2;Preparation of Fab Fragments Using Papain;839
12.2.1.5;1.5. Removal of Unconjugated Enzyme from Antibody–Enzyme Conjugates;843
12.2.1.5.1;Immunoaffinity Chromatography;844
12.2.1.5.2;Nickel-Chelate Affinity Chromatography;845
12.2.2;2. Preparation of Labeled Antibodies;847
12.2.2.1;2.1. Fluorescently Labeled Antibodies;848
12.2.2.2;2.2. Radiolabeled Antibodies;850
12.2.2.3;2.3. Biotinylated Antibodies;852
12.3;Chapter 21. Immunotoxin Conjugation Techniques;855
12.3.1;1. Properties and Use of Immunotoxin Conjugates;858
12.3.2;2. Preparation of Immunotoxin Conjugates;860
12.3.2.1;2.1. Preparation of Immunotoxin Conjugates via Disulfide Exchange Reactions;864
12.3.2.1.1;Pyridyl Disulfide Reagents;865
12.3.2.1.2;SPDP;865
12.3.2.1.3;SMPT;872
12.3.2.1.4;3-(2-Pyridyldithio) Propionate;874
12.3.2.1.5;Use of Cystamine, Ellman’s Reagent, or S-Sulfonates;875
12.3.2.2;2.2. Preparation of Immunotoxin Conjugates via Amine- and Sulfhydryl-Reactive Heterobifunctional Crosslinkers;878
12.3.2.2.1;SIAB;878
12.3.2.2.2;SMCC;881
12.3.2.2.3;MBS;883
12.3.2.2.4;SMPB;885
12.3.2.3;2.3. Preparation of Immunotoxin Conjugates via Reductive Amination;886
12.3.2.3.1;Periodate Oxidation of Glycoproteins Followed by Reductive Conjugation;886
12.3.2.3.2;Periodate-Oxidized Dextran as Crosslinking Agent;888
12.4;Chapter 22. Preparation of Liposome Conjugates and Derivatives;889
12.4.1;1. Properties and Use of Liposomes;889
12.4.1.1;1.1. Liposome Morphology;889
12.4.1.2;1.2. Preparation of Liposomes;892
12.4.1.3;1.3. Chemical Constituents of Liposomes;894
12.4.1.4;1.4. Functional Groups of Phospholipids;900
12.4.2;2. Derivatization and Activation of Lipid Components;900
12.4.2.1;2.1. Periodate Oxidation of Liposome Components;901
12.4.2.2;2.2. Activation of PE Residues with Heterobifunctional Crosslinkers;902
12.4.3;3. Use of Glycolipids and Lectins to Effect Specific Conjugations;908
12.4.4;4. Antigen or Hapten Conjugation to Liposomes;910
12.4.5;5. Preparation of Antibody–Liposome Conjugates;912
12.4.6;6. Preparation of Biotinylated or Avidin-Conjugated Liposomes;914
12.4.7;7. Conjugation of Proteins to Liposomes;916
12.4.7.1;7.1. Coupling via the NHS Ester of Palmitic Acid;917
12.4.7.2;7.2. Coupling via Biotinylated PE Lipid Derivatives;919
12.4.7.3;7.3. Conjugation via Carbodiimide Coupling to PE Lipid Derivatives;919
12.4.7.4;7.4. Conjugation via Glutaraldehyde Coupling to PE Lipid Derivatives;921
12.4.7.5;7.5. Conjugation via DMS Crosslinking to PE Lipid Derivatives;923
12.4.7.6;7.6. Coupling via Periodate Oxidation Followed by Reductive Amination;924
12.4.7.7;7.7. Conjugation via SPDP-Modified PE Lipid Derivatives;925
12.4.7.8;7.8. Conjugation via SMPB-Modified PE Lipid Derivatives;926
12.4.7.9;7.9. Conjugation via SMCC-Modified PE Lipid Derivatives;927
12.4.7.10;7.10. Conjugation via Iodoacetate-Modified PE Lipid Derivatives;928
12.5;Chapter 23. Avidin–Biotin Systems;931
12.5.1;1. The Avidin–Biotin Interaction;931
12.5.2;2. Use of (Strept)avidin–Biotin Interactions in Assay Systems;933
12.5.3;3. Preparation of (Strept)avidin Conjugates;936
12.5.3.1;3.1. NHS Ester–Maleimide-Mediated Conjugation Protocols;937
12.5.3.2;3.2. Conjugation Using Periodate Oxidation/Reductive Amination;941
12.5.3.3;3.3. Glutaraldehyde Conjugation Protocol;944
12.5.4;4. Preparation of Fluorescently Labeled (Strept)avidin;945
12.5.4.1;4.1. Modification with FITC;946
12.5.4.2;4.2. Modification with Lissamine Rhodamine B Sulfonyl Chloride;947
12.5.4.3;4.3. Modification with AMCA–NHS;948
12.5.4.4;4.4. Conjugation with Phycobiliproteins;949
12.5.5;5. Preparation of Hydrazide-Activated (Strept)avidin;950
12.5.6;6. Biotinylation Techniques;951
12.5.7;7. Determination of the Level of Biotinylation;952
12.6;Chapter 24. Preparation of Colloidal Gold-Labeled Proteins;955
12.6.1;1. Properties and Use of Gold Conjugates;955
12.6.2;2. Preparation of Mono-Disperse Gold Suspensions for Protein Labeling;959
12.6.2.1;2.1. Preparation of 2 nm Gold Particle Sols;959
12.6.2.2;2.2. Preparation of 5 nm Gold Particle Sols;959
12.6.2.3;2.3. Preparation of 12 nm Gold Particle Sols;960
12.6.2.4;2.4. Preparation of 30 nm Gold Particle Sols;960
12.6.3;3. Preparation of Protein A–Gold Complexes;961
12.6.4;4. Preparation of Antibody–Gold Complexes;962
12.6.5;5. Preparation of Lectin–Gold Complexes;963
12.6.6;6. Preparation of (Strept)avidin–Gold Complexes;965
12.7;Chapter 25. Modification with Synthetic Polymers;967
12.7.1;1. Protein Modification with Activated Polyethylene Glycols;968
12.7.1.1;1.1. Trichloro-s-triazine Activation and Coupling;969
12.7.1.2;1.2. NHS Ester and NHS Carbonate Activation and Coupling;971
12.7.1.3;1.3. Carbodiimide Coupling of Carboxylate–PEG Derivatives;976
12.7.1.4;1.4. CDI Activation and Coupling;977
12.7.1.5;1.5. Miscellaneous Coupling Reactions;979
12.7.2;2. Protein Modification with Activated Dextrans;982
12.7.2.1;2.1. Polyaldehyde Activation and Coupling;983
12.7.2.2;2.2. Carboxyl, Amine, and Hydrazide Derivatives;985
12.7.2.3;2.3. Epoxy Activation and Coupling;987
12.7.2.4;2.4. Sulfhydryl-Reactive Derivatives;991
12.8;Chapter 26. Enzyme Modification and Conjugation;992
12.8.1;1. Properties of Common Enzymes;992
12.8.1.1;1.1. Horseradish Peroxidase;992
12.8.1.2;1.2. Alkaline Phosphatase;994
12.8.1.3;1.3. ?-Galactosidase;995
12.8.1.4;1.4. Glucose Oxidase;996
12.8.2;2. Preparation of Activated Enzymes for Conjugation;997
12.8.2.1;2.1. Glutaraldehyde-Activated Enzymes;997
12.8.2.2;2.2. Periodate Oxidation Techniques;997
12.8.2.3;2.3. SMCC-Activated Enzymes;998
12.8.2.4;2.4. Hydrazide-Activated Enzymes;998
12.8.2.5;2.5. SPDP-Activated Enzymes;999
12.8.3;3. Preparation of Biotinylated Enzymes;999
12.9;Chapter 27. Nucleic Acid and Oligonucleotide Modification and Conjugation;1000
12.9.1;1. Enzymatic Labeling of DNA;1001
12.9.2;2. Chemical Modification of Nucleic Acids and Oligonucleotides;1004
12.9.2.1;2.1. Diamine or Bis-Hydrazide Modification of DNA;1005
12.9.2.1.1;Conjugation via Bisulfite Activation of Cytosine;1005
12.9.2.1.2;Conjugation via Bromine Activation of Thymine, Guanine, and Cytosine;1007
12.9.2.1.3;Conjugation via Carbodiimide Reaction with the 5' Phosphate of DNA (Phosphoramidate Formation);1009
12.9.2.2;2.2. Sulfhydryl Modification of DNA;1011
12.9.2.2.1;Cystamine Modification of 5' Phosphate Groups Using EDC;1012
12.9.2.2.2;SPDP Modification of Amines on Nucleotides;1013
12.9.2.2.3;SATA Modification of Amines on Nucleotides;1015
12.9.2.3;2.3. Biotin Labeling of DNA;1016
12.9.2.3.1;Biotin-LC-dUTP;1016
12.9.2.3.2;Photobiotin Modification of DNA;1018
12.9.2.3.3;Reaction of NHS-LC-Biotin with Diamine-Modified DNA Probes;1018
12.9.2.3.4;Biotin–Diazonium Modification of DNA;1020
12.9.2.3.5;Reaction of Biotin–BMCC with Sulfhydryl-Modified DNA;1021
12.9.2.3.6;Biotin–Hydrazide Modification of Bisulfite-Activated Cytosine Groups;1021
12.9.2.4;2.4. Enzyme Conjugation to DNA;1023
12.9.2.4.1;Alkaline Phosphatase Conjugation to Cystamine-Modified DNA Using Amine- and Sulfhydryl-Reactive Heterobifunctional Crosslinkers;1024
12.9.2.4.2;Alkaline Phosphatase Conjugation to Diamine-Modified DNA Using DSS;1025
12.9.2.4.3;Enzyme Conjugation to Diamine-Modified DNA Using PDITC;1027
12.9.2.4.4;Conjugation of SFB-Modified Alkaline Phosphatase to Bis-Hydrazide-Modified Oligonucleotides;1029
12.9.2.5;2.5. Fluorescent Labeling of DNA;1029
12.9.2.5.1;Conjugation of Amine-Reactive Fluorescent Probes to Diamine-Modified DNA;1032
12.9.2.5.2;Conjugation of Sulfhydryl-Reactive Fluorescent Probes to Sulfhydryl-Modified DNA;1033
12.10;Chapter 28. Bioconjugation in the Study of Protein Interactions;1034
12.10.1;1. Homobifunctional Crosslinking Agents;1037
12.10.1.1;1.1. DSS and BS[sup(3)];1038
12.10.1.2;1.2. Heavy Atom, Deuterated Crosslinking Agents;1039
12.10.1.3;1.3. Formaldehyde;1041
12.10.1.4;1.4. Protein Interaction Reporters;1042
12.10.2;2. Use of Photoreactive Crosslinkers to Study Protein Interactions;1047
12.10.2.1;2.1. Sulfo-SAND, SANPAH, and Sulfo-SANPAH;1047
12.10.2.2;2.2. Sulfo-SFAD;1049
12.10.3;3. Trifunctional Label Transfer Reagents;1051
12.10.3.1;3.1. Sulfo-SBED;1052
12.10.3.2;3.2. MTS-ATF-Biotin and MTS-ATF-LC-Biotin;1059
12.10.4;4. Metal Chelates in the Study of Protein Interactions;1063
12.10.4.1;4.1. FeBABE for Protein Mapping Studies;1063
12.10.4.2;4.2. Ru(II)bpy[sub(3)][sup(2+)] for Light-Triggered Zero-Length Crosslinking of Protein Interactions;1068
13;References;1072
14;Index;1164
14.1;A;1164
14.2;B;1171
14.3;C;1175
14.4;D;1181
14.5;E;1186
14.6;F;1189
14.7;G;1191
14.8;H;1194
14.9;I;1198
14.10;J;1201
14.11;K;1202
14.12;L;1203
14.13;M;1205
14.14;N;1208
14.15;O;1210
14.16;P;1211
14.17;Q;1218
14.18;R;1218
14.19;S;1220
14.20;T;1228
14.21;U;1231
14.22;V;1232
14.23;W;1232
14.24;X;1233
14.25;Y;1233
14.26;Z;1233

Leseproben

4. Creating Specific Functionalities
It is often desirable to alter the native structure of a macromolecule to provide functional targets for modification or conjugation. The use of most reagent systems requires the presence of particular chemical groups to effect coupling. For instance, heterobifunctional crosslinkers contain two different reactive species that are directed against different functionalities. One target molecule has to contain chemical groups able to react with one end of the crosslinker, while the other target molecule must contain groups able to react with the other end. Occasionally, the required chemical groups are not present on one of the target molecules and must be created. This usually can be done by reacting an existing chemical group with a modification reagent that contains or produces the desired functionality upon coupling. Thus, an amine can be “changed” into a sulfhydryl or a carboxylate can be altered to yield an amine simply by using the appropriate reagent. This same type of modification strategy also can be used to create highly reactive groups from functionalities of rather low reactivity. For instance, carbohydrate chains on glycoproteins can be modified with sodium periodate to transform their rather unreactive hydroxyl groups into highly reactive aldehydes. Similarly, cystine or disulfide residues in proteins can be selectively reduced to form active sulfhydryls, or 5'-phosphate groups of DNA can be transformed to yield modifiable amines. Alternatively, spacer arms can be introduced into a macromolecule to extend a reactive group away from its surface. The extra length of a spacer can provide less steric hindrance to conjugation and often yields more active complexes. The use of modification reagents to create specific functionalities is an important technique to master. In one sense, the process is like using building blocks to construct on a target molecule any desired functional groups necessary for reactivity. The success of many conjugation schemes depends on the presence of the correct chemical groups. Care should be taken in choosing a modification strategy, however, since some chemical changes will radically affect the native structure and activity of a macromolecule. A protein may lose its capacity to bind a specific ligand. An enzyme may lose the ability to act upon its substrate. A DNA probe may no longer be able to hybridize to its complementary target. In many cases, the potential for inactivation relates to changing conformational structures, blocking active sites, or modifying critical functional groups. Trial and error and careful literature searches are often necessary to optimize any modification tactic. 4.1. Introduction of Sulfhydryl Residues (Thiolation)
The sulfhydryl group is a popular target in many modification strategies. Crosslinking agents that have more than one reactive group often employ a sulfhydryl-reactive functionality at one end to direct the conjugation reaction to a particular part of a target macromolecule. The frequency of sulfhydryl occurrence in proteins or other molecules is usually low (or nonexistent) compared to other groups like amines or carboxylates. The use of sulfhydryl-reactive chemistries thus can restrict modification to only a limited number of sites within a target molecule. Limiting modification greatly increases the chances of retaining activity after conjugation, especially in sensitive proteins like some enzymes. Unfortunately, sulfhydryl groups often need to be generated (from reduction of indigenous disulfides) or created (from use of the appropriate thiolation reagent systems). The following sections describe the most popular techniques of creating these functionalities. Some of these reagent systems are specifically designed to form —SH groups, while others are crosslinkers that also can serve the dual purpose of sulfhydryl-generating agents. Sulfhydryl groups are susceptible to oxidation and formation of disulfide crosslinks. To prevent disulfide bond formation, remove oxygen from all buffers by degassing under vacuum and bubbling an inert gas (i.e., nitrogen) through the solution. In addition, EDTA (0.01–0.1 M) may be added to buffers to chelate metal ions, preventing metal-catalyzed oxidation of sulfhydryls. Some proteins of serum origin (particularly bovine serum albumin (BSA)) contain so much contaminating metal ions (presumably iron from hemolyzed blood) that 0.1 M EDTA is required to prevent this type of oxidation. Modification of Amines with 2-Iminothiolane (Traut’s Reagent)
Perham and Thomas (1971) originally prepared an imidoester compound containing a thiol group, methyl 3-mercaptopropionimidate hydrochloride. The imidoester group can react with amines to form a stable, charged linkage (Chapter 2, Section 1.10), while leaving a sulfhydryl group available for further coupling (Figure 1.59). Traut et al. (1973) subsequently synthesized an analogous reagent containing one additional carbon, methyl 4-mercaptobutyrimidate. Later, this compound was found to cyclize as a result of the sulfhydryl group reacting with the intrachain imidoester, forming 2-iminothiolane (Jue et al., 1978). The cyclic imidothioester still can react with primary amines in a ring-opening reaction that regenerates the free sulfhydryl (Figure 1.60). Figure 1.59 Thiolation of an amine-containing compound with methyl 3-mercaptopropionimidate. The modification preserves the positive charge on the primary amine. Traut’s reagent is fully water-soluble and reacts with primary amines in the range of pH 7–10. The cyclic imidothioester is stable to hydrolysis at acid pH values, but its half-life in solution decreases as the pH increases beyond neutrality. However, even at pH 8.0 in 25 mM triethanolamine the rate of sulfhydryl formation without added primary amine was found to be negligible. Upon addition of dipeptide amine, the reagent reacted quickly as evidenced by the production of Ellman’s reagent color. The rate of reaction also can be followed by 2-iminothiolane’s absorbance at 248 nm (?max; e = 8,840 M-1cm-1). As the cyclic imidate reacts with amines, its absorbance at this wavelength decreases. With addition of the dipeptide glycylglycine, the starting absorbance of a solution of Traut’s reagent decreased over 80 percent within 20 minutes (Jue et al., 1978). Thus, protein modification with 2-iminothiolane is very efficient and proceeds rapidly at slightly basic pH. At high pH (10.0), Traut’s reagent also is reactive with aliphatic and aromatic hydroxyl groups, although the rate of reaction with these groups is only about 0.01 that of primary amines. In the absence of amines, however, carbohydrates such as agarose or cellulose membranes can be modified to contain sulfhydryl residues (Alagon and King, 1980). Polysaccharides modified in this manner are effective in covalently crosslinking antibodies for use in immunoassay procedures. Figure 1.60 Methyl 4-mercaptobutyrimidate forms 2-iminothiolane, which can react with a primary amine to create a sulfhydryl group. The modification preserves the positive charge of the original amine. Proteins modified with 2-iminothiolane are subject to disulfide formation upon sulfhydryl oxidation. This can cause unwanted conjugation, potentially precipitating the protein. The addition of a metal-chelating agent such as EDTA (0.01–0.1 M) will prevent metal-catalyzed oxidation and maintain sulfhydryl stability. In the presence of some serum proteins (i.e., BSA) a 0.1 M concentration of EDTA may be necessary to prevent metal-catalyzed oxidation, presumably due to the high contamination of iron from hemolyzed blood. Traut’s reagent has been used successfully in the investigation of ribosomal proteins (Sun et al., 1974; Jue et al., 1978; Kenny et al., 1979; Lambert et al., 1983; Blattler et al., 1985b), RNA polymerase (Hillel and Wu, 1977), progesterone receptor subunits (Birnbaumer et al., 1979), and in the synthesis of enzyme-labeled DNA hybridization probes (Ghosh et al., 1990). It is an excellent thiolation reagent for use in the preparation of immunotoxins (Section 3.3). It also has been used to modify and introduce sulfhydryls into oligosaccharides from asparagine-linked glycans (Tarentino et al., 1993). Side reactions other than oxidation to disulfides also can occur using Traut’s reagent. Once an amine on a protein is modified with 2-iminothiolane, the terminal thiol can recyclize by attacking the amidine carbon (Figure 1.61). This then can rearrange into an iminothiolane derivative, which effectively ties up the thiol (Singh et al., 1996; Mokotoff et al., 2001). Proteins and other molecules thiolated using Traut’s reagent can loose substantial amounts of available thiol to recyclization in just hours. For this reason, the thiolated product of a Traut’s reaction should be used immediately in a conjugation reaction to avoid significant loss of activity. Figure 1.61 Traut’s reagent can undergo side reactions after the modification of an amine-containing molecule. The terminal thiol group can recyclize to create another iminothiolane derivative that effectively ties up the thiol. Protocol 1. Prepare the protein or macromolecule to be thiolated in a non-amine containing buffer at pH 8.0. For the modification of ribosomal...