Fluorescent Analogs of Biomolecular Building Blocks: Design and Applications

Fluorescent 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.

Table of Contents:
List 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

About the Author :
Marcus Wilhelmsson is an Associate Professor of Biophysical Chemistry at Chalmers University of Technology, Göteborg, Sweden.  He earned his Ph.D. in 2003 and thereafter entered the field of DNA-nanotechnology. In 2008 he joined Chalmers University of Technology as an Assistant Professor, where he is now working on the design, characterization and application of novel fluorescent nucleic acid base analogues. Yitzhak Tor is a Professor of Chemistry and Biochemistry and the George and Carol Lattimer Professor at the University of California, San Diego. He earned his Ph.D. in 1990 from the Weizmann Institute of Science and conducted postdoctoral work at the California Institute of Technology. His first faculty appointment was at the University of Chicago, followed by the University of California, San Diego since 1994.

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)