Description
Book SynopsisFluorescent Analogs of Biomolecular Building Blocks focuses on the design of fluorescent probes for the four major families of macromolecular building blocks. Compiling the expertise of multiple authors, this book moves from introductory chapters to an exploration of the design, synthesis, and implementation of new fluorescent analogues of biomolecular building blocks, including examples of small-molecule fluorophores and sensors that are part of biomolecular assemblies.
Trade Review"This book provides a thorough overview on the design and application of fluorescent analogs of biomolecular building blocks...The way the book is written makes reading enjoyable and relatively easy for readers who already have some knowledge on the subject as well as for beginners...Overall, the book is very well achieved, and I strongly recommend reading." (Angewandte Chemie International Edition May 2017)
Table of ContentsList of Contributors xv
Preface xix
1 Fluorescence Spectroscopy 1
Renatus W. Sinkeldam, L. Marcus Wilhelmsson, and Yitzhak Tor
1.1 Fundamentals of Fluorescence Spectroscopy 1
1.2 Common Fluorescence Spectroscopy Techniques 3
1.2.1 Steady-State Fluorescence Spectroscopy 3
1.2.2 Time-Resolved Fluorescence Spectroscopy 5
1.2.3 Fluorescence Anisotropy 6
1.2.4 Resonance Energy Transfer and Quenching 7
1.2.5 Fluorescence Microscopy and Single Molecule Spectroscopy 8
1.2.6 Fluorescence-Based in vivo Imaging 9
1.3 Summary and Perspective 10
References 10
2 Naturally Occurring and Synthetic Fluorescent Biomolecular Building Blocks 15
Renatus W. Sinkeldam and Yitzhak Tor
2.1 Introduction 15
2.2 Naturally Occurring Emissive Biomolecular Building Blocks 16
2.3 Synthetic Fluorescent Analogs of Biomolecular Building Blocks 18
2.3.1 Synthetic Emissive Analogs of Membranes Constituents 19
2.3.2 Synthetic Emissive Analogs of Amino Acids 22
2.3.3 Synthetic Emissive Analogs of Nucleosides 24
2.4 Summary and Perspective 31
References 32
3 Polarized Spectroscopy with Fluorescent Biomolecular Building Blocks 40
Bo Albinsson and Bengt Nordén
3.1 Transition Moments 40
3.2 Linear Dichroism 41
3.3 Magnetic Circular Dichroism 45
3.4 F̈orster Resonance Energy Transfer (FRET) 46
3.5 Fluorescence Anisotropy 47
3.6 Fluorescent Nucleobases 47
3.7 Fluorescent Peptide Chromophores 48
3.8 Site-Specific Linear Dichroism (SSLD) 50
3.9 Single-Molecule Fluorescence Resonance Energy Transfer (smFRET) 50
3.10 Single-Molecule Fluorescence-Detected Linear Dichroism (smFLD) 51
References 53
4 Fluorescent Proteins: The Show Must go on! 55
Gregor Jung
4.1 Introduction 55
4.2 Historical Survey 55
4.3 Photophysical Properties 57
4.3.1 Absorption Properties and Color Hue Modification 57
4.3.2 Chromophore Formation 61
4.3.3 Fluorescence Color and Dynamics 64
4.3.4 Directional Properties along with Optical Transitions 68
4.3.5 Energy Transfer and Energy Migration 69
4.4 Photochemical Reactions 71
4.4.1 Excited-state Proton Transfer (ESPT) 71
4.4.2 Isomerization Reactions: Reversible Photoswitching 73
4.4.3 Photoconversion: Irreversible Bond Rupture 74
4.4.4 Other Photochemical Reactions 75
4.5 Ion Sensitivity 75
4.5.1 Ground-State Equilibria of Protonation States 75
4.5.2 Quenching by Small Ions 76
4.6 Relation Microscopy–Spectroscopy for Fluorescent Proteins 77
4.6.1 Brightness Alteration from Cuvette to Microscopic Experiments 77
4.6.2 Lessons from Microspectrometry 78
4.6.3 Tools for Advanced Microscopic Techniques 79
4.7 Prospects and Outlook 82
Acknowledgments 82
References 82
5 Design and Application of Autofluorescent Proteins by Biological Incorporation of Intrinsically Fluorescent Noncanonical Amino Acids 91
Patrick M. Durkin and Nediljko Budisa
5.1 Introduction 91
5.2 Design and Synthesis of Fluorescent Building Blocks in Proteins 97
5.2.1 Extrinsic Fluorescent Labels 97
5.2.2 Intrinsic Fluorescent Labels 98
5.2.3 Extrinsic Labels Chemically Ligated using Cycloaddition Chemistry 108
5.2.4 Modification of the Genetic Code to Incorporate ncAAs 109
5.3 Application of Fluorescent Building Blocks in Proteins 111
5.3.1 Azatryptophans 111
5.3.2 FlAsH-EDT2 Extrinsic Labeling System 112
5.3.3 Huisgen Dipolar Cycloaddition System 114
5.4 Conclusions 117
5.5 Prospects and Outlook 118
5.5.1 Heteroatom-Containing Trp Analogs 119
5.5.2 Expanded Genetic Code – Orthogonal Pairs 119
Acknowledgments 120
References 120
6 Fluoromodules: Fluorescent Dye–Protein Complexes for Genetically Encodable Labels 124
Bruce A. Armitage
6.1 Introduction 124
6.2 Fluoromodule Development and Characterization 126
6.2.1 Fluorogenic Dyes 128
6.2.2 Fluorogen-Activating Protein (FAP) Optimization 131
6.2.3 Fluoromodule Recycling 132
6.3 Implementation 132
6.3.1 Fusion Constructs for Protein Tagging 132
6.3.2 Protein Tagging and pH Sensing 133
6.3.3 Super-Resolution Imaging 133
6.3.4 Protease Biosensors 133
6.4 Conclusions 134
6.5 Prospects and Outlook 134
Acknowledgments 134
References 134
7 Design of Environmentally Sensitive Fluorescent Nucleosides and their Applications 137
Subhendu Sekhar Bag and Isao Saito
7.1 Introduction 137
7.1.1 Solvatochromic Fluorophores 138
7.1.2 Origin of Solvatochromism 139
7.2 Solvatochromic Fluorescent Nucleoside Analogs 140
7.2.1 Designing Criteria for Solvatochromic Fluorescent Nucleosides 140
7.3 Fluorescently Labeled Nucleosides and Oligonucleotide Probes: Covalent Attachment of Solvatochromic Fluorophores Onto the Natural Bases 141
7.3.1 Base-Discriminating Fluorescent Nucleosides (BDF) 142
7.4 Nucleosides with Dual Fluorescence for Monitoring DNA Hybridization 153
7.5 Approach for Developing Environmentally Sensitive Fluorescent (ESF) Nucleosides 154
7.5.1 Concept for Designing ESF Nucleosides 154
7.5.2 Examples and Photophysical Properties of ESF Nucleosides 156
7.6 Base-Selective Fluorescent ESF Probe 163
7.6.1 Cytosine Selective ESF Probe 163
7.6.2 Thymine Selective Fluorescent ESF Probe 163
7.6.3 Specific Detection of Adenine by Exciplex Formation with Donor-Substituted ESF Guanosine 165
7.7 Molecular Beacon (MB) and ESF Nucleosides 167
7.7.1 Ends-Free and Self-Quenched MB 167
7.7.2 Single-Stranded Molecular Beacon Using ESF Nucleoside in a Bulge Structure 168
7.8 Summary and Future Outlook 169
Acknowledgments 170
References 170
8 Expanding The Nucleic Acid Chemist’s Toolbox: Fluorescent Cytidine Analogs 174
Kirby Chicas and Robert H.E. Hudson
8.1 Introduction 174
8.2 Design and Characterization of Fluorescent C Analogs 176
8.2.1 1,3-Diaza-2-Oxophenothiazine (tC) 177
8.2.2 1,3-Diaza-2-Oxophenoxazine (tCO) 178
8.2.3 7-Nitro-1,3-Diaza-2-Oxophenothiazine (tCnitro) 179
8.2.4 G-Clamp and 8-oxoG-Clamp 179
8.2.5 Ç and Çf 181
8.2.6 Benzopyridopyrimidine (BPP) 182
8.2.7 Napthopyridopyrimidine (NPP) 183
8.2.8 dChpp 183
8.2.9 dChpd, dCmpp, dCtpp, dCppp 184
8.2.10 dCPPI 184
8.2.11 dxC 185
8.2.12 rxC 186
8.2.13 Methylpyrrolo-dC (MepdC) 186
8.2.14 5-(Fur-2-yl)-2′-Deoxycytidine (CFU) 187
8.2.15 Thiophen-2-yl pC 187
8.2.16 Thiophene Fused pC 188
8.2.17 Thieno[3,4-d]-Cytidine (thC) 189
8.2.18 Triazole Appended 190
8.3 Implementation 190
8.3.1 PNA 192
8.3.2 DNA 196
8.3.3 RNA 200
8.4 Conclusions 202
8.5 Prospects and Outlook 202
Acknowledgments 203
References 203
9 Synthesis and Fluorescence Properties of Nucleosides with Pyrimidopyrimidine-Type Base Moieties 208
Kohji Seio, Takashi Kanamori, Akihiro Ohkubo, and Mitsuo Sekine
9.1 Introduction 209
9.2 Discovery, Design, and Synthesis of Pyrimidopyrimidine Nucleosides 209
9.2.1 Synthesis and Fluorescence Properties of dChpp 209
9.2.2 Design, Synthesis, and Fluorescence Properties of dCPPP, dCPPI, and dCPPI Derivatives 212
9.2.3 Fluorescence Properties of the Oligonucleotides Containing dCPPI 213
9.3 Implementation 215
9.3.1 Application to a DNA Triplex System 215
9.3.2 Double Labeling of an Oligonucleotide with dCPPI and 2-Aminopurine 219
9.4 Conclusions 220
9.5 Prospects and Outlook 221
References 221
10 Förster Resonance Energy Transfer (FRET) Between Nucleobase Analogues – a Tool for Detailed Structure and Dynamics Investigations 224
L. Marcus Wilhelmsson
10.1 Introduction 224
10.2 The Tricyclic Cytosine Family 226
10.2.1 Structural Aspects, Dynamics, and Ability to Serve as Cytosine Analogs 228
10.2.2 Photophysical Properties 231
10.3 Development of the First Nucleic Acid Base Analog FRET Pair 234
10.3.1 The Donor–Acceptor Pair tCO –tCnitro 235
10.3.2 Applications of Tricyclic Cytosines in FRET Measurements 237
10.4 Conclusions 238
10.5 Prospects and Outlook 238
Acknowledgments 239
References 239
11 Fluorescent Purine Analogs that Shed Light on DNA Structure and Function 242
Anaëlle Dumas, Guillaume Mata, and Nathan W. Luedtke
11.1 Introduction 242
11.2 Design, Photophysical Properties, and Applications of Purine Mimics 244
11.2.1 Early Examples of Fluorescent Purine Mimics 245
11.2.2 Chromophore-Conjugated Purine Analogs 246
11.2.3 Pteridines 250
11.2.4 Isomorphic Purine Analogs 251
11.2.5 Fused-Ring Purine Analogs 252
11.2.6 Substituted Purine Derivatives 253
11.3 Implementation 258
11.3.1 Probing G-Quadruplex Structures with 2PyG 259
11.3.2 Energy Transfer Quantification 261
11.3.3 Metal-Ion Localization to N7 264
11.4 Conclusions 265
11.5 Prospects and Outlook 265
Appendix 268
References 268
12 Design and Photophysics of Environmentally Sensitive Isomorphic Fluorescent Nucleosides 276
Renatus W. Sinkeldam and Yitzhak Tor
12.1 Introduction 276
12.2 Designing Environmentally Sensitive Emissive Nucleosides 279
12.2.1 Structural and Electronic Elements that Impart Environmental Sensitivity 279
12.2.2 Sensitivity to Polarity 279
12.2.3 Sensitivity to Viscosity 281
12.2.4 Sensitivity to pH 282
12.3 Two Isomorphic Environmentally Sensitive Designs 282
12.4 Probing Environmental Sensitivity 283
12.4.1 Probing Sensitivity to Polarity 283
12.4.2 Probing Sensitivity to Viscosity 286
12.4.3 Probing Sensitivity to pH 288
12.5 Recent Advancements in Isomorphic Fluorescent Nucleoside Analogs 291
12.6 Summary 293
12.7 Prospects and Outlook 294
Acknowledgments 294
References 294
13 Site-Specific Fluorescent Labeling of Nucleic Acids by Genetic Alphabet Expansion Using Unnatural Base Pair Systems 297
Michiko Kimoto, Rie Yamashige, and Ichiro Hirao
13.1 Introduction 297
13.2 Development of Unnatural Base Pair Systems and Their Applications 299
13.2.1 Site-Specific Fluorescent Labeling of DNA by Unnatural Base Pair Replication Systems 301
13.2.2 Site-Specific Fluorescent Labeling of RNA by Unnatural Base Pair Transcription Systems 307
13.3 Implementation 310
13.3.1 Fluorescence Sensor System Using an RNA Aptamer by Fluorophore-Linked y Labeling 310
13.3.2 Local Structure Analyses of Functional RNA Molecules by s Labeling 313
13.4 Conclusions 315
13.5 Prospects and Outlook 316
Acknowledgments 317
References 317
14 Fluorescent C-Nucleosides and their Oligomeric Assemblies 320
Pete Crisalli and Eric T. Kool
14.1 Introduction 320
14.2 Design, Synthesis, Characterization, and Properties of Fluorescent C-Glycoside Monomers 322
14.2.1 Design of Fluorescent C-Glycoside Monomers 322
14.2.2 Synthesis of Fluorescent C-Glycoside Monomers 323
14.2.3 Characterization and Properties of Fluorescent C-glycoside Monomers 325
14.3 Implementation of Fluorescent C-Glycoside Monomers 327
14.3.1 Environmentally Sensitive Fluorophores 327
14.3.2 Pyrene Nucleoside in DNA Applications 330
14.4 Oligomers of Fluorescent C-Glycosides: Design, Synthesis, and Properties 335
14.4.1 Design of Fluorescent C-Glycoside Oligomers 335
14.4.2 Synthesis of Fluorescent C-Glycoside Oligomers 336
14.4.3 Characterization and Properties of Fluorescent C-Glycoside Oligomers 337
14.5 Implementation of Fluorescent C-Glycoside Oligomers 342
14.5.1 ODFs as Chemosensors in the Solution State 342
14.5.2 ODFs as Sensors in the Solid State 347
14.5.3 Alternative Designs of Oligomeric Fluorescent Glycosides 351
14.5.4 General Conclusions: Oligomers of Fluorescent C-glycosides 352
14.6 Conclusions 353
14.7 Prospects and Outlook 353
Acknowledgments 354
References 354
15 Membrane Fluorescent Probes: Insights and Perspectives 356
Amitabha Chattopadhyay, Sandeep Shrivastava, and Arunima Chaudhuri
Abbreviations 356
15.1 Introduction 357
15.2 NBD-Labeled Lipids: Monitoring Slow Solvent Relaxation in Membranes 358
15.3 n-AS Membrane Probes: Depth-Dependent Solvent Relaxation as Membrane Dipstick 359
15.4 Pyrene: a Multiparameter Membrane Probe 362
15.5 Conclusion and Future Perspectives 362
Acknowledgments 364
References 364
16 Lipophilic Fluorescent Probes: Guides to the Complexity of Lipid Membranes 367
Marek Cebecauer and Radek Šachl
16.1 Introduction 367
16.2 Lipids, Lipid Bilayers, and Biomembranes 368
16.3 Lipid Phases, Phase Separation, and Lipid Ordering 370
16.4 Fluorescent Probes for Membrane Studies 370
16.4.1 Fluorescently Labeled Lipids 371
16.4.2 Environment-Sensitive Membrane Probes 373
16.4.3 Specialized Techniques Using Fluorescent Probes to Investigate Membrane Properties 380
16.5 Conclusions 386
16.6 Prospects and Outlook 386
Acknowledgments 386
References 387
17 Fluorescent Neurotransmitter Analogs 393
James N. Wilson
17.1 Introduction 393
17.1.1 Structure of Neurotransmitters 393
17.1.2 Regulation of Neurotransmitters 394
17.1.3 Native Fluorescence of Neurotransmitters 395
17.1.4 Fluorescent Histochemical Techniques 396
17.2 Design and Optical Properties of Fluorescent Neurotransmitters 397
17.2.1 Early Examples 397
17.2.2 Recent Examples 398
17.3 Applications of Fluorescent Neurotransmitters 400
17.3.1 Probing Binding Pockets with Fluorescent Neurotransmitters 400
17.3.2 Imaging Transport and Release of Fluorescent Neurotransmitters 401
17.3.3 Enzyme Substrates 403
17.4 Conclusions 404
17.5 Prospects and Outlook 405
Acknowledgments 405
References 406
Index 409
