*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
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 nanoparticlesSilane coupling agentsDendrimers and dendronsChemoselective ligationQuantum dotsLanthanide chelatesCyanine dyesDiscrete PEG compoundsBuckyballs,fullerenes, and carbon nanotubesMass tags and isotope tagsBioconjugation in the study of protein interactions
Front Cover 1
Bioconjugate Techniques 4
Copyright Page 5
Detailed Contents 8
Preface to the Second Edition 24
Preface to the First Edition 27
Acknowledgments 29
Health and Safety 30
Intellectual Property 31
PART I: Bioconjugate Chemistry 32
Chapter 1. Functional Targets 34
1. Modification of Amino acids, Peptides, and Proteins 34
1.1. Protein Structure and Reactivity 35
Amino Acids 35
Nucleophilic Reactions and the pI of Amino Acid Side Chains 44
Secondary, Tertiary, and Quaternary Structure 46
Prosthetic Groups, Cofactors, and Post-Translational Modifications 50
Protecting the Native Conformation and Activity of Proteins 52
Oxidation of Amino Acids in Proteins and Peptides 54
Solvent Accessibility of Functional Targets in Proteins 60
1.2. Protein Crosslinking Methods 63
2. Modification of Sugars, Polysaccharides, and Glycoconjugates 66
2.1. Carbohydrate Structure and Functionality 67
Basic Sugar Structure 68
Sugar Functional Groups 70
Polysaccharide and Glycoconjugate Structure 75
2.2. Carbohydrate and Glycan Conjugation Methods 80
3. Modification of Nucleic Acids and Oligonucleotides 81
3.1. Polynucleotide Structure and Functionality 82
Nucleotide Functional Groups 84
RNA and DNA Structure 93
3.2. Polynucleotide Crosslinking Methods 97
4. Creating Specific Functionalities 97
4.1. Introduction of Sulfhydryl Residues (Thiolation) 98
Modification of Amines with 2-Iminothiolane (Traut ’s Reagent) 98
Modification of Amines with SATA 102
Modification of Amines with SATP 105
Modification of Amines with SPDP 107
Modification of Amines with SMPT 108
Modification of Amines with N -Acetyl Homocysteine Thiolactone 111
Modification of Amines with SAMSA 112
Modification of Aldehydes or Ketones with AMBH 114
Modification of Carboxylates or Phosphates with Cystamine 115
Modification of Proteins with Cystamine 118
Modification of Nucleic Acids and Oligonucleotides with Cystamine 118
Use of Disulfide Reductants 118
Complete Reduction of Disulfides in Protein Molecules Using DTT 121
Use of DTT to Cleave Disulfide-Containing Crosslinking Agents 122
Ellman’s Assay for the Determination of Sulfhydryls 131
4.2. Introduction of Carboxylate Groups 132
Modification of Amines with Anhydrides 133
Modification of Sulfhydryls with Iodoacetate 140
Modification of Sulfhydryls with BMPA 142
Modification of Hydroxyls with Chloroacetic Acid 144
4.3. Introduction of Primary Amine Groups 145
Modification of Carboxylates with Diamines 145
Modification of Sulfhydryls with N-(& #914
Modification of Sulfhydryls with Ethylenimine 150
Modification of Sulfhydryls with 2-Bromoethylamine 151
Modification of Sulfhydryls with 2-Aminoethyl-2'-aminoethanethiolsulfonate 152
Modification of Carbohydrates with Diamines 153
Modification of Alkylphosphates with Diamines 155
Modification of Aldehydes with Ammonia or Diamines 155
Introduction of Arylamines on Phenolic Compounds 156
Amine Detection Reagents 158
4.4. Introduction of Aldehyde Residues 160
Periodate Oxidation of Glycols and Carbohydrates 161
Oxidase Modification of Sugar Residues 162
Modification of Amines with NHS-Aldehydes (SFB and SFPA) 163
Modification of Amines with Glutaraldehyde 165
Periodate Oxidation of N-Terminal Serine or Threonine Residues 167
4.5. Introduction of Hydrazine or Hydrazide Functionalities 170
Modification of Aldehydes with Bis-Hydrazide Compounds 171
Modification of Carboxylates with Bis-Hydrazide Compounds 173
Modification of Amines with SANH, SHNH, or SHTH 174
Modification of Alkylphosphates with Bis-Hydrazide Compounds 177
4.6. Introduction of Saccharide or Glycan Groups 178
Modification of Amines with Mono(lactosylamido) mono(succinimidyl)suberate 180
Modification of Amine or Hydrazide Molecules by Carbohydrates and Glycans 181
Labeling Glycans with Fluorescent 2-Aminopyridine, 2-Amino Benzamide, or Anthranilic Acid 184
Synthesis of Glycosylamines for Conjugating Glycans 186
5. Blocking Specific Functional Groups 187
5.1. Blocking Amine Groups 188
Sulfo-NHS Acetate 188
Acetic Anhydride 189
Citraconic Anhydride 190
Maleic Anhydride 190
5.2. Blocking Sulfhydryl Groups 191
N-Ethyl Maleimide 191
Iodoacetate Derivatives 192
Sodium Tetrathionate 192
Methyl Methanethiosulfonate 194
Ellman’s Reagent 195
Dipyridyl Disulfide Reagents 196
5.3. Blocking Aldehyde Groups 197
Reductive Amination with Tris or Ethanolamine 197
5.4. Blocking Carboxylate Groups 198
Tris or Ethanolamine plus EDC 198
Chapter 2. The Chemistry of Reactive Groups 200
1. Amine Reactions 200
1.1. Isothiocyanates 201
1.2. Isocyanates 201
1.3. Acyl Azides 202
1.4. NHS Esters 202
1.5. Sulfonyl Chlorides 204
1.6. Aldehydes and Glyoxals 204
1.7. Epoxides and Oxiranes 205
1.8. Carbonates 206
1.9. Arylating Agents 206
1.10. Imidoesters 207
1.11. Carbodiimides 207
1.12. Anhydrides 209
1.13. Fluorophenyl Esters 210
1.14. Hydroxymethyl Phosphine Derivatives 211
1.15. Guanidination of Amines 212
2. Thiol Reactions 213
2.1. Haloacetyl and Alkyl Halide Derivatives 213
2.2. Maleimides 214
2.3. Aziridines 215
2.4. Acryloyl Derivatives 215
2.5. Arylating Agents 216
2.6. Thiol-Disulfide Exchange Reagents 216
Pyridyl Disulfides 217
TNB-Thiol 218
Disulfide Reductants 218
2.7. Vinylsulfone Derivatives 219
2.8. Metal-Thiol Dative Bonds 219
2.9. Native Chemical Ligation 222
2.10. Cisplatin Modification of Methionine and Cysteine 223
3. Carboxylate Reactions 223
3.1. Diazoalkanes and Diazoacetyl Compounds 224
3.2. Carbonyldiimidazole 225
3.3. Carbodiimides 226
4. Hydroxyl Reactions 226
4.1. Epoxides and Oxiranes 226
4.2. Carbonyldiimidazole 227
4.3. N,N'-Disuccinimidyl Carbonate or N-Hydroxysuccinimidyl Chloroformate 227
4.4. Oxidation with Periodate 228
4.5. Enzymatic Oxidation 229
4.6. Alkyl Halogens 229
4.7. Isocyanates 230
5. Aldehyde and Ketone Reactions 231
5.1. Hydrazine Derivatives 231
5.2. Schiff Base Formation 231
5.3. Reductive Amination 232
5.4. Mannich Condensation 232
6. Active Hydrogen Reactions 233
6.1. Diazonium Derivatives 233
6.2. Mannich Condensation 234
6.3. Iodination Reactions 234
7. Photo-Chemical Reactions 235
7.1. Aryl Azides and Halogenated Aryl Azides 235
7.2. Benzophenones 236
7.3. Anthraquinones 236
7.4. Certain Diazo Compounds 238
7.5. Diazirine Derivatives 239
7.6. Psoralen Compounds 239
8. Cycloaddition Reactions 241
8.1. Diels–Alder Reaction 241
8.2. Complex Formation with Boronic Acid Derivatives 241
8.3. Click Chemistry: Cu[sup(1)]-promoted Azide—Alkyne [3 + 2] Cycloaddition 242
PART II: Bioconjugate Reagents 244
Chapter 3. Zero-Length Crosslinkers 246
1. Carbodiimides 246
1.1. EDC 247
1.2. EDC plus Sulfo-NHS 250
1.3. CMC 254
1.4. DCC 255
1.5. DIC 257
2. Woodward's Reagent K 259
3. N,N'-Carbonyldiimidazole 259
4. Schiff Base Formation and Reductive Amination 262
Chapter 4. Homobifunctional Crosslinkers 265
1. Homobifunctional NHS Esters 266
1.1. DSP and DTSSP 269
1.2. DSS and BS[sup(3)] 272
1.3. DST and Sulfo-DST 274
1.4. BSOCOES and Sulfo-BSOCOES 275
1.5. EGS and Sulfo-EGS 277
1.6. DSG 279
1.7. DSC 280
2. Homobifunctional Imidoesters 281
2.1. DMA 282
2.2. DMP 283
2.3. DMS 284
2.4. DTBP 285
3. Homobifunctional Sulfhydryl-Reactive Crosslinkers 287
3.1. DPDPB 288
3.2. BMH 289
4. Difluorobenzene Derivatives 290
4.1. DFDNB 290
4.2. DFDNPS 291
5. Homobifunctional Photoreactive Crosslinkers 292
5.1. BASED 293
6. Homobifunctional Aldehydes 293
6.1. Formaldehyde 294
6.2. Glutaraldehyde 296
7. Bis-epoxides 299
7.1. 1,4-Butanediol Diglycidyl Ether 300
8. Homobifunctional Hydrazides 300
8.1. Adipic Acid Dihydrazide 301
8.2. Carbohydrazide 302
9. Bis-diazonium Derivatives 302
9.1. o-Tolidine, Diazotized 303
9.2. Bis-diazotized Benzidine 304
10. Bis-alkyl Halides 305
Chapter 5. Heterobifunctional Crosslinkers 307
1. Amine-Reactive and Sulfhydryl-Reactive Crosslinkers 308
1.1. SPDP, LC-SPDP, and Sulfo-LC-SPDP 309
1.2. SMPT and Sulfo-LC-SMPT 312
1.3. SMCC and Sulfo-SMCC 314
1.4. MBS and Sulfo-MBS 317
1.5. SIAB and Sulfo-SIAB 319
1.6. SMPB and Sulfo-SMPB 322
1.7. GMBS and Sulfo-GMBS 323
1.8. SIAX and SIAXX 324
1.9. SIAC and SIACX 326
1.10. NPIA 327
2. Carbonyl-Reactive and Sulfhydryl-Reactive Crosslinkers 328
2.1. MPBH 329
2.2. M[sub(2)]C[sub(2)]H 330
2.3. PDPH 331
3. Amine-Reactive and Photoreactive Crosslinkers 333
3.1. NHS-ASA, Sulfo-NHS-ASA, and Sulfo-NHS-LC-ASA 336
3.2. SASD 337
3.3. HSAB and Sulfo-HSAB 339
3.4. SANPAH and Sulfo-SANPAH 341
3.5. ANB-NOS 343
3.6. SAND 343
3.7. SADP and Sulfo-SADP 345
3.8. Sulfo-SAPB 347
3.9. SAED 347
3.10. Sulfo-SAMCA 350
3.11. p-Nitrophenyl Diazopyruvate 353
3.12. PNP-DTP 354
4. Sulfhydryl-Reactive and Photoreactive Crosslinkers 355
4.1. ASIB 356
4.2. APDP 357
4.3. Benzophenone-4-iodoacetamide 359
4.4. Benzophenone-4-maleimide 361
5. Carbonyl-Reactive and Photoreactive Crosslinkers 361
5.1. ABH 362
6. Carboxylate-Reactive and Photoreactive Crosslinkers 363
6.1. ASBA 364
7. Arginine-Reactive and Photoreactive Crosslinkers 364
7.1. APG 365
Chapter 6. Trifunctional Crosslinkers 367
1. 4-Azido-2-nitrophenylbiocytin-4-nitrophenyl ester 367
2. Sulfo-SBED 368
3. MTS-ATF-Biotin and MTS-ATF-LC-Biotin 372
4. Hydroxymethyl Phosphine Derivatives 373
Chapter 7. Dendrimers and Dendrons 377
1. Dendrimer Construction 377
2. Conjugation to Dendrimers 384
2.1. Coupling to Amine-Dendrimers 387
Modification of Amine-Dendrimers with Sulfo-NHS-LC-SPDP 387
NHS-PEG-Maleimide Coupling to Amine-Dendrimers 390
Coupling Glycoproteins to Amine-Dendrimers by Reductive Amination 392
Blocking of Amines on PAMAM Dendrimers 394
Preparation of Sugar-Dendrimer Derivatives 397
Conjugation of Carboxylate Organic Molecules to Amine-Dendrimers 402
Epoxy Activation of Amine-Dendrimers 404
Biotinylation of Amine-Dendrimers 407
Fluorescent Labeling of Amine Dendrimers 411
3. Dendrimer-Chelate Derivatives for Imaging Applications 414
4. Dendrimer Derivatives as Surface Modification Agents 416
5. Dendrimer Fluorescent Quantum Dots 420
Chapter 8. Cleavable Reagent Systems 422
1. Cleavage of Disulfides by Reduction 423
2. Periodate-Cleavable Glycols 424
3. Dithionite-Cleavable Bonds 425
4. Hydroxylamine Cleavable Esters 425
5. Base Labile Sulfones 426
Chapter 9. Fluorescent Probes 427
1. Fluorescein Derivatives 431
Amine-Reactive Fluorescein Derivatives 432
Sulfhydryl-Reactive Fluorescein Derivatives 437
Aldehyde/Ketone and Cytosine-Reactive Fluorescein Derivatives 443
2. Rhodamine Derivatives 446
Amine-Reactive Rhodamine Derivatives 447
Sulfhydryl-Reactive Rhodamine Derivatives 456
Aldehyde/Ketone and Cytosine-Reactive Rhodamine Derivatives 458
3. Coumarin Derivatives 461
Amine-Reactive Coumarin Derivatives 462
Sulfhydryl-Reactive Coumarin Derivatives 465
Aldehyde- and Ketone-Reactive Coumarin Derivatives 469
4. BODIPY Derivatives 471
Amine-Reactive BODIPY Derivatives 472
Aldehyde/Ketone-Reactive BODIPY Derivatives 475
Sulfhydryl-Reactive BODIPY Derivatives 480
5. Cascade Blue Derivatives 484
Amine-Reactive: Cascade Blue Acetyl Azide 484
Carboxylate-Reactive: Cascade Blue Cadaverine and Cascade Blue Ethylenediamine 486
Aldehyde/Ketone-Reactive: Cascade Blue Hydrazide 487
6. Lucifer Yellow Derivatives 488
Sulfhydryl-Reactive: Lucifer Yellow Iodoacetamide 489
Aldehyde/Ketone-Reactive: Lucifer Yellow CH 490
7. Phycobiliprotein Derivatives 492
8. Cyanine Dye Derivatives 495
Amine-reactive Cyanine Dyes 498
Thiol-reactive Cyanine Dyes 501
Carbonyl-reactive Cyanine Dyes 503
9. Lanthanide Chelates for Time-resolved Fluorescence 505
10. Quantum Dot Nanocrystals 516
Properties of Quantum Dots 516
Conjugation to QDs 524
Conjugation of Proteins to QDs Using EDC 525
Conjugation to QDs Using sulfo-SMCC 527
Chapter 10. Bifunctional Chelating Agents and Radioimmunoconjugates 529
1. DTPA 530
2. DOTA, NOTA, and TETA 531
3. DTTA 532
4. DFA 533
5. Use of Thiolation Reagents for Direct Labeling to Sulfhydryl Groups 534
6. FeBABE 536
Chapter 11. Biotinylation Reagents 537
1. Amine-Reactive Biotinylation Agents 538
1.1. D-Biotin and Biocytin 539
1.2. NHS-Biotin and Sulfo-NHS-Biotin 541
1.3. NHS-LC-Biotin and Sulfo-NHS-LC-Biotin 543
1.4. NHS-Iminobiotin 546
1.5. Sulfo-NHS-SS-Biotin 548
2. Sulfhydryl-Reactive Biotinylation Agents 551
2.1. Biotin-BMCC 551
2.2. Biotin-HPDP 553
2.3. Iodoacetyl-LC-Biotin 555
3. Carbonyl- or Carboxyl-Reactive Biotinylation Agents 556
3.1. Biotin-Hydrazide and Biotin-LC-Hydrazide 557
3.2. Biocytin Hydrazide 559
3.3. 5-(Biotinamido)pentylamine 560
4. Photoreactive Biotinylation Agents 561
4.1. Photobiotin 562
4.2. Psoralen-PEO[sub(3)]-Biotin 564
5. Active Hydrogen-Reactive: p-Aminobenzoyl Biocytin, Diazotized 565
6. Glycan Biotinylation Reagents 568
6.1. Biotinylated Aminopyridine 569
6.2. Biotinyl-L-3-(2-naphthyl)-alanine hydrazide 572
6.3. Biotin-PEG-Phosphine 574
Chapter 12. Iodination Reagents 577
1. Chloramine-T 579
2. Iodobeads 581
3. Iodogen 584
4. Lactoperoxidase-Catalyzed Iodination 586
5. Iodinatable Modification and Crosslinking Agents 587
5.1. Bolton–Hunter Reagent 588
5.2. Iodinatable Bifunctional Crosslinking Agents 591
Chapter 13. Silane Coupling Agents 593
1. Silane Reaction Strategies 596
1.1. Aqueous/Organic Solvent Deposition 597
1.2. Aqueous Deposition 597
1.3. Organic Solvent Deposition 598
1.4. Vapor Phase Deposition 598
2. Functional Silane Compounds 599
2.1. 3-Aminopropyltriethoxysilane and 3-Aminopropyltrimethoxysilane 599
2.2. Carboxyethylsilanetriol 604
2.3. N-(Trimethoxysilylpropyl)ethylenediamine triacetic acid 606
2.4. 3-Glycidoxypropyltrimethoxysilane and 3-Glycidoxypropyltriethoxysilane 608
2.5. Isocyanatopropyltriethoxysilane 610
Chapter 14. Microparticles and Nanoparticles 613
1. Particle Types 613
2. Particle Characteristics and Stability 615
3. Particle Concentration 619
4. Polymeric Microspheres and Nanospheres 619
4.1. Passive Adsorption 621
4.2. Covalent Coupling to Polymeric Particles 625
4.3. Coupling to Carboxylate Particles 626
4.4. Coupling to Amine Particles 630
4.5. Coupling to Amine Particles Using Crosslinking Agents 630
4.6.Glutaraldehyde 632
4.7. SPDP Coupling to Amine Particles 633
4.8. NHS-PEG[sub(n)]-Maleimide Coupling to Amine Particles 635
4.9. Coupling to Hydroxyl Particles 637
4.10. Coupling to Hydrazide Particles 644
4.11. Coupling to Epoxy Particles 646
4.12. Coupling to Aldehyde Particles 648
5. Silica Particles 649
5.1. Fluorescent Silica Particles 651
5.2. Silane Functionalization of Silica Particles 656
Chapter 15. Buckyballs, Fullerenes, and Carbon Nanotubes 658
1. Buckyballs and Fullerenes 658
1.1. Properties of Fullerenes 658
1.2. Modification of Fullerenes 660
2. Carbon Nanotubes 669
2.1. Nanotube Properties 669
2.2. Nanotube Functionalization 671
2.3. Detergent or Lipid Modification of Carbon Nanotubes 671
2.4. Pyrene Modification of Carbon Nanotubes 675
2.5. Modification of Carbon Nanotubes by Cycloaddition 676
Chapter 16. Mass Tags and Isotope Tags 680
1. ICAT Reagents 682
2. ECAT Reagents 688
3. Isobaric Tags 690
Chapter 17. Chemoselective Ligation: Bioorthogonal Reagents 697
1. Diels–Alder Reagent Pairs 698
2. Hydrazine–Aldehyde Reagent Pairs 700
Protocol for Modification of Amine-Oligo with SANH or SFB 705
Protocol for Modification of Protein or Antibody with SANH or SFB 706
Conjugation Using the Aldehyde/Hydrazine Reaction 706
3. Boronic Acid–Salicylhydroxamate Reagent Pairs 707
4. Click Chemistry: Cu(I)-Promoted Azide–Alkyne [3 + 2] Cycloaddition 711
5. Staudinger Ligation 721
6. Native Chemical Ligation 728
6.1. Expressed Protein Ligation and Inteins 732
Chapter 18. Discrete PEG Reagents 738
1. Homobifunctional PEG Crosslinkers 742
1.1. Bis-NHS Ester PEG Compounds 742
1.2. Bis-Maleimide–PEG Compounds 745
BM(PEG)[sub(2)], BM(PEG)[sub(3)], and Bis-MAL–dPEG[sub(3)] 745
2. Heterobifunctional PEG Reagents 749
2.1. Maleimide–PEG[sub(n)]–NHS Ester Compounds 749
2.2. NHS-PEG[sub(n)]-Azide/Alkyne Compounds for Chemoselective Ligation 753
3. Biotinylation Reagents Containing Discrete PEG Linkers 757
3.1. NHS–PEG[sub(n)]–Biotin Compounds 758
3.2. NHS-Chromogenic-PEG[sub(3)]-Biotin 761
3.3. Maleimide-PEG[sub(n)]-Biotin Compounds 763
3.4. Hydrazide-PEG[sub(4)]-Biotin 764
3.5. Biotin-PEG[sub(n)]-Amine Compounds 767
3.6. Biotin–PEG[sub(3)]–Benzophenone 770
4. Discrete PEG Modification Reagents 770
PART III: Bioconjugate Applications 774
Chapter 19. Preparation of Hapten–Carrier Immunogen Conjugates 776
1. The Basis of Immunity 776
2. Types of Immunogen Carriers 778
2.1. Protein Carriers 779
KLH 779
BSA and cBSA 780
Thyroglobulin and OVA 782
Tetanus and Diphtheria Toxoids 784
2.2. Liposome Carriers 784
2.3. Synthetic Carriers 785
3. Carbodiimide-Mediated Hapten–Carrier Conjugation 786
4. NHS Ester-Mediated Hapten–Carrier Conjugation 794
5. NHS Ester-Maleimide Heterobifunctional Crosslinker-Mediated Hapten–Carrier Conjugation 797
6. Active-Hydrogen-Mediated Hapten–Carrier Conjugation 804
6.1. Diazonium Conjugation 804
6.2. Mannich Condensation 807
7. Glutaraldehyde-Mediated Hapten–Carrier Conjugation 810
8. Reductive Amination-Mediated Hapten–Carrier Conjugation 812
Chapter 20. Antibody Modification and Conjugation 814
1. Preparation of Antibody–Enzyme Conjugates 818
1.1. NHS Ester–Maleimide-Mediated Conjugation 819
Activation of Enzymes with NHS Ester–Maleimide Crosslinkers 820
Conjugation with Reduced Antibodies 821
Conjugation with 2-Iminothiolane-Modified Antibodies 824
Conjugation with SATA-Modified Antibodies 826
1.2. Glutaraldehyde-Mediated Conjugation 828
One-Step Glutaraldehyde Protocol 829
Two-Step Glutaraldehyde Protocol 831
1.3. Reductive-Amination-Mediated Conjugation 831
Activation of Enzymes with Sodium Periodate 833
Activation of Antibodies with Sodium Periodate 834
Conjugation of Periodate-Oxidized HRP to Antibodies by Reductive Amination 835
Conjugation of Periodate-Oxidized Antibodies with Amine or Hydrazide Derivatives 836
1.4. Conjugation Using Antibody Fragments 838
Preparation of F(ab')[sub(2)] Fragments Using Pepsin 838
Preparation of Fab Fragments Using Papain 839
1.5. Removal of Unconjugated Enzyme from Antibody–Enzyme Conjugates 843
Immunoaffinity Chromatography 844
Nickel-Chelate Affinity Chromatography 845
2. Preparation of Labeled Antibodies 847
2.1. Fluorescently Labeled Antibodies 848
2.2. Radiolabeled Antibodies 850
2.3. Biotinylated Antibodies 852
Chapter 21. Immunotoxin Conjugation Techniques 855
1. Properties and Use of Immunotoxin Conjugates 858
2. Preparation of Immunotoxin Conjugates 860
2.1. Preparation of Immunotoxin Conjugates via Disulfide Exchange Reactions 864
Pyridyl Disulfide Reagents 865
SPDP 865
SMPT 872
3-(2-Pyridyldithio) Propionate 874
Use of Cystamine, Ellman’s Reagent, or S-Sulfonates 875
2.2. Preparation of Immunotoxin Conjugates via Amine- and Sulfhydryl-Reactive Heterobifunctional Crosslinkers 878
SIAB 878
SMCC 881
MBS 883
SMPB 885
2.3. Preparation of Immunotoxin Conjugates via Reductive Amination 886
Periodate Oxidation of Glycoproteins Followed by Reductive Conjugation 886
Periodate-Oxidized Dextran as Crosslinking Agent 888
Chapter 22. Preparation of Liposome Conjugates and Derivatives 889
1. Properties and Use of Liposomes 889
1.1. Liposome Morphology 889
1.2. Preparation of Liposomes 892
1.3. Chemical Constituents of Liposomes 894
1.4. Functional Groups of Phospholipids 900
2. Derivatization and Activation of Lipid Components 900
2.1. Periodate Oxidation of Liposome Components 901
2.2. Activation of PE Residues with Heterobifunctional Crosslinkers 902
3. Use of Glycolipids and Lectins to Effect Specific Conjugations 908
4. Antigen or Hapten Conjugation to Liposomes 910
5. Preparation of Antibody–Liposome Conjugates 912
6. Preparation of Biotinylated or Avidin-Conjugated Liposomes 914
7. Conjugation of Proteins to Liposomes 916
7.1. Coupling via the NHS Ester of Palmitic Acid 917
7.2. Coupling via Biotinylated PE Lipid Derivatives 919
7.3. Conjugation via Carbodiimide Coupling to PE Lipid Derivatives 919
7.4. Conjugation via Glutaraldehyde Coupling to PE Lipid Derivatives 921
7.5. Conjugation via DMS Crosslinking to PE Lipid Derivatives 923
7.6. Coupling via Periodate Oxidation Followed by Reductive Amination 924
7.7. Conjugation via SPDP-Modified PE Lipid Derivatives 925
7.8. Conjugation via SMPB-Modified PE Lipid Derivatives 926
7.9. Conjugation via SMCC-Modified PE Lipid Derivatives 927
7.10. Conjugation via Iodoacetate-Modified PE Lipid Derivatives 928
Chapter 23. Avidin–Biotin Systems 931
1. The Avidin–Biotin Interaction 931
2. Use of (Strept)avidin–Biotin Interactions in Assay Systems 933
3. Preparation of (Strept)avidin Conjugates 936
3.1. NHS Ester–Maleimide-Mediated Conjugation Protocols 937
3.2. Conjugation Using Periodate Oxidation/Reductive Amination 941
3.3. Glutaraldehyde Conjugation Protocol 944
4. Preparation of Fluorescently Labeled (Strept)avidin 945
4.1. Modification with FITC 946
4.2. Modification with Lissamine Rhodamine B Sulfonyl Chloride 947
4.3. Modification with AMCA–NHS 948
4.4. Conjugation with Phycobiliproteins 949
5. Preparation of Hydrazide-Activated (Strept)avidin 950
6. Biotinylation Techniques 951
7. Determination of the Level of Biotinylation 952
Chapter 24. Preparation of Colloidal Gold-Labeled Proteins 955
1. Properties and Use of Gold Conjugates 955
2. Preparation of Mono-Disperse Gold Suspensions for Protein Labeling 959
2.1. Preparation of 2 nm Gold Particle Sols 959
2.2. Preparation of 5 nm Gold Particle Sols 959
2.3. Preparation of 12 nm Gold Particle Sols 960
2.4. Preparation of 30 nm Gold Particle Sols 960
3. Preparation of Protein A–Gold Complexes 961
4. Preparation of Antibody–Gold Complexes 962
5. Preparation of Lectin–Gold Complexes 963
6. Preparation of (Strept)avidin–Gold Complexes 965
Chapter 25. Modification with Synthetic Polymers 967
1. Protein Modification with Activated Polyethylene Glycols 968
1.1. Trichloro-s-triazine Activation and Coupling 969
1.2. NHS Ester and NHS Carbonate Activation and Coupling 971
1.3. Carbodiimide Coupling of Carboxylate–PEG Derivatives 976
1.4. CDI Activation and Coupling 977
1.5. Miscellaneous Coupling Reactions 979
2. Protein Modification with Activated Dextrans 982
2.1. Polyaldehyde Activation and Coupling 983
2.2. Carboxyl, Amine, and Hydrazide Derivatives 985
2.3. Epoxy Activation and Coupling 987
2.4. Sulfhydryl-Reactive Derivatives 991
Chapter 26. Enzyme Modification and Conjugation 992
1. Properties of Common Enzymes 992
1.1. Horseradish Peroxidase 992
1.2. Alkaline Phosphatase 994
1.3. & #914
1.4. Glucose Oxidase 996
2. Preparation of Activated Enzymes for Conjugation 997
2.1. Glutaraldehyde-Activated Enzymes 997
2.2. Periodate Oxidation Techniques 997
2.3. SMCC-Activated Enzymes 998
2.4. Hydrazide-Activated Enzymes 998
2.5. SPDP-Activated Enzymes 999
3. Preparation of Biotinylated Enzymes 999
Chapter 27. Nucleic Acid and Oligonucleotide Modification and Conjugation 1000
1. Enzymatic Labeling of DNA 1001
2. Chemical Modification of Nucleic Acids and Oligonucleotides 1004
2.1. Diamine or Bis-Hydrazide Modification of DNA 1005
Conjugation via Bisulfite Activation of Cytosine 1005
Conjugation via Bromine Activation of Thymine, Guanine, and Cytosine 1007
Conjugation via Carbodiimide Reaction with the 5' Phosphate of DNA (Phosphoramidate Formation) 1009
2.2. Sulfhydryl Modification of DNA 1011
Cystamine Modification of 5' Phosphate Groups Using EDC 1012
SPDP Modification of Amines on Nucleotides 1013
SATA Modification of Amines on Nucleotides 1015
2.3. Biotin Labeling of DNA 1016
Biotin-LC-dUTP 1016
Photobiotin Modification of DNA 1018
Reaction of NHS-LC-Biotin with Diamine-Modified DNA Probes 1018
Biotin–Diazonium Modification of DNA 1020
Reaction of Biotin–BMCC with Sulfhydryl-Modified DNA 1021
Biotin–Hydrazide Modification of Bisulfite-Activated Cytosine Groups 1021
2.4. Enzyme Conjugation to DNA 1023
Alkaline Phosphatase Conjugation to Cystamine-Modified DNA Using Amine- and Sulfhydryl-Reactive Heterobifunctional Crosslinkers 1024
Alkaline Phosphatase Conjugation to Diamine-Modified DNA Using DSS 1025
Enzyme Conjugation to Diamine-Modified DNA Using PDITC 1027
Conjugation of SFB-Modified Alkaline Phosphatase to Bis-Hydrazide-Modified Oligonucleotides 1029
2.5. Fluorescent Labeling of DNA 1029
Conjugation of Amine-Reactive Fluorescent Probes to Diamine-Modified DNA 1032
Conjugation of Sulfhydryl-Reactive Fluorescent Probes to Sulfhydryl-Modified DNA 1033
Chapter 28. Bioconjugation in the Study of Protein Interactions 1034
1. Homobifunctional Crosslinking Agents 1037
1.1. DSS and BS[sup(3)] 1038
1.2. Heavy Atom, Deuterated Crosslinking Agents 1039
1.3. Formaldehyde 1041
1.4. Protein Interaction Reporters 1042
2. Use of Photoreactive Crosslinkers to Study Protein Interactions 1047
2.1. Sulfo-SAND, SANPAH, and Sulfo-SANPAH 1047
2.2. Sulfo-SFAD 1049
3. Trifunctional Label Transfer Reagents 1051
3.1. Sulfo-SBED 1052
3.2. MTS-ATF-Biotin and MTS-ATF-LC-Biotin 1059
4. Metal Chelates in the Study of Protein Interactions 1063
4.1. FeBABE for Protein Mapping Studies 1063
4.2. Ru(II)bpy[sub(3)][sup(2+)] for Light-Triggered Zero-Length Crosslinking of Protein Interactions 1068
References 1072
Index 1164
A 1164
B 1171
C 1175
D 1181
E 1186
F 1189
G 1191
H 1194
I 1198
J 1201
K 1202
L 1203
M 1205
N 1208
O 1210
P 1211
Q 1218
R 1218
S 1220
T 1228
U 1231
V 1232
W 1232
X 1233
Y 1233
Z 1233
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 (λmax; ε = 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
| Erscheint lt. Verlag | 26.7.2010 |
|---|---|
| Sprache | englisch |
| Themenwelt | Sachbuch/Ratgeber |
| Medizin / Pharmazie | |
| Naturwissenschaften ► Biologie ► Biochemie | |
| Naturwissenschaften ► Chemie ► Organische Chemie | |
| Technik ► Umwelttechnik / Biotechnologie | |
| ISBN-10 | 0-08-056872-6 / 0080568726 |
| ISBN-13 | 978-0-08-056872-0 / 9780080568720 |
| Haben Sie eine Frage zum Produkt? |
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