Fluorescent Analogs of Biomolecular Building Blocks

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.

• List of Contributors xvPreface xix1 Fluorescence Spectroscopy 1Renatus W. Sinkeldam, L. Marcus Wilhelmsson, and Yitzhak Tor1.1 Fundamentals of Fluorescence Spectroscopy 11.2 Common Fluorescence Spectroscopy Techniques 31.2.1 Steady-State Fluorescence Spectroscopy 31.2.2 Time-Resolved Fluorescence Spectroscopy 51.2.3 Fluorescence Anisotropy 61.2.4 Resonance Energy Transfer and Quenching 71.2.5 Fluorescence Microscopy and Single Molecule Spectroscopy 81.2.6 Fluorescence-Based in vivo Imaging 91.3 Summary and Perspective 10References 102 Naturally Occurring and Synthetic Fluorescent Biomolecular Building Blocks 15Renatus W. Sinkeldam and Yitzhak Tor2.1 Introduction 152.2 Naturally Occurring Emissive Biomolecular Building Blocks 162.3 Synthetic Fluorescent Analogs of Biomolecular Building Blocks 182.3.1 Synthetic Emissive Analogs of Membranes Constituents 192.3.2 Synthetic Emissive Analogs of Amino Acids 222.3.3 Synthetic Emissive Analogs of Nucleosides 242.4 Summary and Perspective 31References 323 Polarized Spectroscopy with Fluorescent Biomolecular Building Blocks 40Bo Albinsson and Bengt Nordén3.1 Transition Moments 403.2 Linear Dichroism 413.3 Magnetic Circular Dichroism 453.4 F̈orster Resonance Energy Transfer (FRET) 463.5 Fluorescence Anisotropy 473.6 Fluorescent Nucleobases 473.7 Fluorescent Peptide Chromophores 483.8 Site-Specific Linear Dichroism (SSLD) 503.9 Single-Molecule Fluorescence Resonance Energy Transfer (smFRET) 503.10 Single-Molecule Fluorescence-Detected Linear Dichroism (smFLD) 51References 534 Fluorescent Proteins: The Show Must go on! 55Gregor Jung4.1 Introduction 554.2 Historical Survey 554.3 Photophysical Properties 574.3.1 Absorption Properties and Color Hue Modification 574.3.2 Chromophore Formation 614.3.3 Fluorescence Color and Dynamics 644.3.4 Directional Properties along with Optical Transitions 684.3.5 Energy Transfer and Energy Migration 694.4 Photochemical Reactions 714.4.1 Excited-state Proton Transfer (ESPT) 714.4.2 Isomerization Reactions: Reversible Photoswitching 734.4.3 Photoconversion: Irreversible Bond Rupture 744.4.4 Other Photochemical Reactions 754.5 Ion Sensitivity 754.5.1 Ground-State Equilibria of Protonation States 754.5.2 Quenching by Small Ions 764.6 Relation Microscopy–Spectroscopy for Fluorescent Proteins 774.6.1 Brightness Alteration from Cuvette to Microscopic Experiments 774.6.2 Lessons from Microspectrometry 784.6.3 Tools for Advanced Microscopic Techniques 794.7 Prospects and Outlook 82Acknowledgments 82References 825 Design and Application of Autofluorescent Proteins by Biological Incorporation of Intrinsically Fluorescent Noncanonical Amino Acids 91Patrick M. Durkin and Nediljko Budisa5.1 Introduction 915.2 Design and Synthesis of Fluorescent Building Blocks in Proteins 975.2.1 Extrinsic Fluorescent Labels 975.2.2 Intrinsic Fluorescent Labels 985.2.3 Extrinsic Labels Chemically Ligated using Cycloaddition Chemistry 1085.2.4 Modification of the Genetic Code to Incorporate ncAAs 1095.3 Application of Fluorescent Building Blocks in Proteins 1115.3.1 Azatryptophans 1115.3.2 FlAsH-EDT2 Extrinsic Labeling System 1125.3.3 Huisgen Dipolar Cycloaddition System 1145.4 Conclusions 1175.5 Prospects and Outlook 1185.5.1 Heteroatom-Containing Trp Analogs 1195.5.2 Expanded Genetic Code – Orthogonal Pairs 119Acknowledgments 120References 1206 Fluoromodules: Fluorescent Dye–Protein Complexes for Genetically Encodable Labels 124Bruce A. Armitage6.1 Introduction 1246.2 Fluoromodule Development and Characterization 1266.2.1 Fluorogenic Dyes 1286.2.2 Fluorogen-Activating Protein (FAP) Optimization 1316.2.3 Fluoromodule Recycling 1326.3 Implementation 1326.3.1 Fusion Constructs for Protein Tagging 1326.3.2 Protein Tagging and pH Sensing 1336.3.3 Super-Resolution Imaging 1336.3.4 Protease Biosensors 1336.4 Conclusions 1346.5 Prospects and Outlook 134Acknowledgments 134References 1347 Design of Environmentally Sensitive Fluorescent Nucleosides and their Applications 137Subhendu Sekhar Bag and Isao Saito7.1 Introduction 1377.1.1 Solvatochromic Fluorophores 1387.1.2 Origin of Solvatochromism 1397.2 Solvatochromic Fluorescent Nucleoside Analogs 1407.2.1 Designing Criteria for Solvatochromic Fluorescent Nucleosides 1407.3 Fluorescently Labeled Nucleosides and Oligonucleotide Probes: Covalent Attachment of Solvatochromic Fluorophores Onto the Natural Bases 1417.3.1 Base-Discriminating Fluorescent Nucleosides (BDF) 1427.4 Nucleosides with Dual Fluorescence for Monitoring DNA Hybridization 1537.5 Approach for Developing Environmentally Sensitive Fluorescent (ESF) Nucleosides 1547.5.1 Concept for Designing ESF Nucleosides 1547.5.2 Examples and Photophysical Properties of ESF Nucleosides 1567.6 Base-Selective Fluorescent ESF Probe 1637.6.1 Cytosine Selective ESF Probe 1637.6.2 Thymine Selective Fluorescent ESF Probe 1637.6.3 Specific Detection of Adenine by Exciplex Formation with Donor-Substituted ESF Guanosine 1657.7 Molecular Beacon (MB) and ESF Nucleosides 1677.7.1 Ends-Free and Self-Quenched MB 1677.7.2 Single-Stranded Molecular Beacon Using ESF Nucleoside in a Bulge Structure 1687.8 Summary and Future Outlook 169Acknowledgments 170References 1708 Expanding The Nucleic Acid Chemist’s Toolbox: Fluorescent Cytidine Analogs 174Kirby Chicas and Robert H.E. Hudson8.1 Introduction 1748.2 Design and Characterization of Fluorescent C Analogs 1768.2.1 1,3-Diaza-2-Oxophenothiazine (tC) 1778.2.2 1,3-Diaza-2-Oxophenoxazine (tCO) 1788.2.3 7-Nitro-1,3-Diaza-2-Oxophenothiazine (tCnitro) 1798.2.4 G-Clamp and 8-oxoG-Clamp 1798.2.5 Ç and Çf  1818.2.6 Benzopyridopyrimidine (BPP) 1828.2.7 Napthopyridopyrimidine (NPP) 1838.2.8 dChpp 1838.2.9 dChpd, dCmpp, dCtpp, dCppp 1848.2.10 dCPPI 1848.2.11 dxC 1858.2.12 rxC 1868.2.13 Methylpyrrolo-dC (MepdC) 1868.2.14 5-(Fur-2-yl)-2′-Deoxycytidine (CFU) 1878.2.15 Thiophen-2-yl pC 1878.2.16 Thiophene Fused pC 1888.2.17 Thieno[3,4-d]-Cytidine (thC) 1898.2.18 Triazole Appended 1908.3 Implementation 1908.3.1 PNA 1928.3.2 DNA 1968.3.3 RNA 2008.4 Conclusions 2028.5 Prospects and Outlook 202Acknowledgments 203References 2039 Synthesis and Fluorescence Properties of Nucleosides with Pyrimidopyrimidine-Type Base Moieties 208Kohji Seio, Takashi Kanamori, Akihiro Ohkubo, and Mitsuo Sekine9.1 Introduction 2099.2 Discovery, Design, and Synthesis of Pyrimidopyrimidine Nucleosides 2099.2.1 Synthesis and Fluorescence Properties of dChpp 2099.2.2 Design, Synthesis, and Fluorescence Properties of dCPPP, dCPPI, and dCPPI Derivatives 2129.2.3 Fluorescence Properties of the Oligonucleotides Containing dCPPI 2139.3 Implementation 2159.3.1 Application to a DNA Triplex System 2159.3.2 Double Labeling of an Oligonucleotide with dCPPI and 2-Aminopurine 2199.4 Conclusions 2209.5 Prospects and Outlook 221References 22110 Förster Resonance Energy Transfer (FRET) Between Nucleobase Analogues – a Tool for Detailed Structure and Dynamics Investigations 224L. Marcus Wilhelmsson10.1 Introduction 22410.2 The Tricyclic Cytosine Family 22610.2.1 Structural Aspects, Dynamics, and Ability to Serve as Cytosine Analogs 22810.2.2 Photophysical Properties 23110.3 Development of the First Nucleic Acid Base Analog FRET Pair 23410.3.1 The Donor–Acceptor Pair tCO –tCnitro 23510.3.2 Applications of Tricyclic Cytosines in FRET Measurements 23710.4 Conclusions 23810.5 Prospects and Outlook 238Acknowledgments 239References 23911 Fluorescent Purine Analogs that Shed Light on DNA Structure and Function 242Anaëlle Dumas, Guillaume Mata, and Nathan W. Luedtke11.1 Introduction 24211.2 Design, Photophysical Properties, and Applications of Purine Mimics 24411.2.1 Early Examples of Fluorescent Purine Mimics 24511.2.2 Chromophore-Conjugated Purine Analogs 24611.2.3 Pteridines 25011.2.4 Isomorphic Purine Analogs 25111.2.5 Fused-Ring Purine Analogs 25211.2.6 Substituted Purine Derivatives 25311.3 Implementation 25811.3.1 Probing G-Quadruplex Structures with 2PyG 25911.3.2 Energy Transfer Quantification 26111.3.3 Metal-Ion Localization to N7 26411.4 Conclusions 26511.5 Prospects and Outlook 265Appendix 268References 26812 Design and Photophysics of Environmentally Sensitive Isomorphic Fluorescent Nucleosides 276Renatus W. Sinkeldam and Yitzhak Tor12.1 Introduction 27612.2 Designing Environmentally Sensitive Emissive Nucleosides 27912.2.1 Structural and Electronic Elements that Impart Environmental Sensitivity 27912.2.2 Sensitivity to Polarity 27912.2.3 Sensitivity to Viscosity 28112.2.4 Sensitivity to pH 28212.3 Two Isomorphic Environmentally Sensitive Designs 28212.4 Probing Environmental Sensitivity 28312.4.1 Probing Sensitivity to Polarity 28312.4.2 Probing Sensitivity to Viscosity 28612.4.3 Probing Sensitivity to pH 28812.5 Recent Advancements in Isomorphic Fluorescent Nucleoside Analogs 29112.6 Summary 29312.7 Prospects and Outlook 294Acknowledgments 294References 29413 Site-Specific Fluorescent Labeling of Nucleic Acids by Genetic Alphabet Expansion Using Unnatural Base Pair Systems 297Michiko Kimoto, Rie Yamashige, and Ichiro Hirao13.1 Introduction 29713.2 Development of Unnatural Base Pair Systems and Their Applications 29913.2.1 Site-Specific Fluorescent Labeling of DNA by Unnatural Base Pair Replication Systems 30113.2.2 Site-Specific Fluorescent Labeling of RNA by Unnatural Base Pair Transcription Systems 30713.3 Implementation 31013.3.1 Fluorescence Sensor System Using an RNA Aptamer by Fluorophore-Linked y Labeling 31013.3.2 Local Structure Analyses of Functional RNA Molecules by s Labeling 31313.4 Conclusions 31513.5 Prospects and Outlook 316Acknowledgments 317References 31714 Fluorescent C-Nucleosides and their Oligomeric Assemblies 320Pete Crisalli and Eric T. Kool14.1 Introduction 32014.2 Design, Synthesis, Characterization, and Properties of Fluorescent C-Glycoside Monomers 32214.2.1 Design of Fluorescent C-Glycoside Monomers 32214.2.2 Synthesis of Fluorescent C-Glycoside Monomers 32314.2.3 Characterization and Properties of Fluorescent C-glycoside Monomers 32514.3 Implementation of Fluorescent C-Glycoside Monomers 32714.3.1 Environmentally Sensitive Fluorophores 32714.3.2 Pyrene Nucleoside in DNA Applications 33014.4 Oligomers of Fluorescent C-Glycosides: Design, Synthesis, and Properties 33514.4.1 Design of Fluorescent C-Glycoside Oligomers 33514.4.2 Synthesis of Fluorescent C-Glycoside Oligomers 33614.4.3 Characterization and Properties of Fluorescent C-Glycoside Oligomers 33714.5 Implementation of Fluorescent C-Glycoside Oligomers 34214.5.1 ODFs as Chemosensors in the Solution State 34214.5.2 ODFs as Sensors in the Solid State 34714.5.3 Alternative Designs of Oligomeric Fluorescent Glycosides 35114.5.4 General Conclusions: Oligomers of Fluorescent C-glycosides 35214.6 Conclusions 35314.7 Prospects and Outlook 353Acknowledgments 354References 35415 Membrane Fluorescent Probes: Insights and Perspectives 356Amitabha Chattopadhyay, Sandeep Shrivastava, and Arunima ChaudhuriAbbreviations 35615.1 Introduction 35715.2 NBD-Labeled Lipids: Monitoring Slow Solvent Relaxation in Membranes 35815.3 n-AS Membrane Probes: Depth-Dependent Solvent Relaxation as Membrane Dipstick 35915.4 Pyrene: a Multiparameter Membrane Probe 36215.5 Conclusion and Future Perspectives 362Acknowledgments 364References 36416 Lipophilic Fluorescent Probes: Guides to the Complexity of Lipid Membranes 367Marek Cebecauer and Radek Šachl16.1 Introduction 36716.2 Lipids, Lipid Bilayers, and Biomembranes 36816.3 Lipid Phases, Phase Separation, and Lipid Ordering 37016.4 Fluorescent Probes for Membrane Studies 37016.4.1 Fluorescently Labeled Lipids 37116.4.2 Environment-Sensitive Membrane Probes 37316.4.3 Specialized Techniques Using Fluorescent Probes to Investigate Membrane Properties 38016.5 Conclusions 38616.6 Prospects and Outlook 386Acknowledgments 386References 38717 Fluorescent Neurotransmitter Analogs 393James N. Wilson17.1 Introduction 39317.1.1 Structure of Neurotransmitters 39317.1.2 Regulation of Neurotransmitters 39417.1.3 Native Fluorescence of Neurotransmitters 39517.1.4 Fluorescent Histochemical Techniques 39617.2 Design and Optical Properties of Fluorescent Neurotransmitters 39717.2.1 Early Examples 39717.2.2 Recent Examples 39817.3 Applications of Fluorescent Neurotransmitters 40017.3.1 Probing Binding Pockets with Fluorescent Neurotransmitters 40017.3.2 Imaging Transport and Release of Fluorescent Neurotransmitters 40117.3.3 Enzyme Substrates 40317.4 Conclusions 40417.5 Prospects and Outlook 405Acknowledgments 405References 406Index 409

"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)