Fluorescent Dye Labels and Stains. A Database of Photophysical Properties 2022047668, 2022047669, 9781119835134, 9781119835141, 9781119835158, 9781119835165

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Fluorescent Dye Labels and Stains

Fluorescent Dye Labels and Stains A Database of Photophysical Properties

Tarso B. Ledur Kist Federal University of Rio Grande do Sul Brazil

This edition first published 2023 © 2023 John Wiley & Sons Ltd All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions. The right of Tarso B. Ledur Kist to be identified as the author of this work has been asserted in accordance with law. Registered Offices John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK For details of our global editorial offices, customer services, and more information about Wiley products visit us at www.wiley.com. Wiley also publishes its books in a variety of electronic formats and by print-on-demand. Some content that appears in standard print versions of this book may not be available in other formats. Trademarks: Wiley and the Wiley logo are trademarks or registered trademarks of John Wiley & Sons, Inc. and/or its affiliates in the United States and other countries and may not be used without written permission. All other trademarks are the property of their respective owners. John Wiley & Sons, Inc. is not associated with any product or vendor mentioned in this book. Limit of Liability/Disclaimer of Warranty In view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of experimental reagents, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each chemical, piece of equipment, reagent, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. While the publisher and authors have used their best efforts in preparing this work, they make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives, written sales materials or promotional statements for this work. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further information does not mean that the publisher and authors endorse the information or services the organization, website, or product may provide or recommendations it may make. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. Library of Congress Cataloging-in-Publication Data Names: Ledur Kist, Tarso B., 1962- author. | John Wiley & Sons, publisher. Title: Fluorescent dye labels and stains : a database of photophysical properties / Tarso B. Ledur Kist. Description: Hoboken, NJ : John Wiley & Sons, 2023. | Includes bibliographical references and index. Identifiers: LCCN 2022047668 (print) | LCCN 2022047669 (ebook) | ISBN 9781119835134 (hardback) | ISBN 9781119835141 (pdf) | ISBN 9781119835158 (epub) | ISBN 9781119835165 (ebook) Subjects: LCSH: Fluorescent labeling. | Fluorescent probes. | Fluorescence microscopy--Technique. Classification: LCC QH212.F55 L43 2023 (print) | LCC QH212.F55 (ebook) | DDC 570.28/2--dc23/eng/20221117 LC record available at https://lccn.loc.gov/2022047668 LC ebook record available at https://lccn.loc.gov/2022047669 Cover and Author Image: Courtesy of Tarso B. Ledur Kist Cover Design: Wiley Set in 9.5/12.5pt STIXTwoText by Integra Software Services Pvt. Ltd, Pondicherry, India

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Contents Preface   x Acronyms  xii Symbols and Conventions  xiii

1 Introduction  1 2 2.1 2.2 2.3 2.3.1 2.3.2 2.3.3 2.3.4 2.3.5 2.4 2.5 2.5.1 2.5.2 2.5.3 2.5.4 2.5.5 2.5.6 2.6 2.6.1 2.6.2 2.6.3 2.7 2.7.1 2.7.2 2.7.3 2.7.4 2.7.5 2.7.6

Basic Definitions and Fundamentals  5 Introduction  5 Light Sources  5 Filtering and Dispersing Light  6 Absorber Filters  6 Interference Filters  8 Polarizers  8 Prisms  9 Grating  9 Light Detectors  10 Light Beams  12 Radiant Power and Radiance in Space: Divergent and Collimated Beams  12 Radiant Power and Radiance in Time: Continuous, Modulated, and Pulsed  13 Spectral Radiant Power (Emission Spectra) of Lamps, LEDs, and Lasers  14 Light Wavelength, Transmittance, and Absorbance  14 Spontaneous Decay and Stimulated Emission in Lasers and STED Nanoscopy  16 Energy, Momentum, Polarization, Spin, and Angular Momentum  17 Light Collection Set-Ups  17 Microscope Objectives  17 Fluorescence Detection Set-Ups  18 Fluorescence Imaging Set-Ups  18 Fundamentals of Fluorescence  21 Fluorescence: Fields of Application  22 Molar Absorption Coefficient  23 Excitation Spectra  24 Emission Spectra  25 Stokes Shift  25 Fluorescence Quantum Yield  27

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Contents

2.7.7 2.7.8 2.7.9 2.7.10 2.7.10.1 2.7.10.2 2.7.10.3 2.7.10.4 2.7.10.5 2.7.10.6 2.7.10.7 2.8

Brightness  27 Effective Brightness  27 Fluorescence Mean-Lifetime  28 Factors Affecting Fluorescence  29 Effect of Microenvironment  29 Influence of Liquid Viscosity on Fluorescence Quantum Yield and Fluorescence Mean-Lifetime  30 Influence of Electric Permittivity and Hydrogen Bonding  30 Effects of Temperature  31 Quenching  31 Self-Quenching  32 Singlet Oxygen Production by Sensitizer Dyes  32 Photostability  32

3 3.1 3.2 3.2.1 3.2.2 3.3 3.3.1 3.3.1.1 3.3.1.2 3.3.1.3 3.3.2 3.3.3 3.3.4 3.3.5 3.4 3.4.1 3.4.2 3.4.3 3.4.4 3.4.5 3.4.6 3.5

Target-Fluorophore Binding  37 Introduction  37 Choosing the Right Solvent  37 Water and PBS  37 Water Miscible Organic Solvents  39 Fluorogenic Reactions  45 Primary Amines  45 Fluorogenic Reactions of Primary Amines With Homocyclic o-Phthaldihaldehydes  45 Fluorogenic Reactions of Primary Amines With Heterocyclic o-Dicarboxaldehydes  49 Fluorogenic Reactions of Primary Amines With Other Reagents  49 Secondary Amines  52 Thiols  53 Cyanide  53 α-Dicarbonylic Compounds  53 Labeling Reactions  54 Covalent Labeling of Amines  56 Covalent Labeling of Thiols  57 Covalent Labeling of Carboxylic Acids  57 Covalent Labeling of Alcohols  58 Covalent Labeling of Reducing Saccharides  58 Others  60 Immunofluorescence  61

4 4.1 4.2 4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.3 4.3.1 4.3.2

Classes and Molecular Structures  65 Introduction  65 Rhodamines  73 Rhodamines With Absorption Maximum Below 500 nm  73 Rhodamines With Absorption Maximum Between 500 and 550 nm  73 Rhodamines With Absorption Maximum Between 550 and 600 nm  73 Rhodamines With Absorption Maximum Above 600 nm  75 Rhodamines With a High Net Charge  78 HAS-Rhodamines  79 Carbo-Rhodamines  79 Silico-Rhodamines  79

Contents

4.3.3 4.4 4.5 4.6 4.7 4.8 4.8.1 4.8.2 4.8.3 4.8.4 4.9 4.10 4.11 4.12 4.13 4.13.1 4.13.2 4.13.3 4.14 4.14.1 4.14.2 4.14.3 4.14.4 4.15 4.15.1 4.15.2 4.15.3 4.16 4.17 4.18 4.18.1 4.18.2 4.18.3 4.19 4.20 4.21 4.22 4.23 4.23.1 4.23.2 4.24 4.25 4.26 4.26.1 4.26.2 4.26.3 4.27

Other HAS-Rhodamines  79 Pyronines  79 HAS-Pyronines  82 Sulforhodamines  84 HAS-Sulforhodamines  84 Fluoresceins  84 Non-Halogenated Fluoresceins  88 Halogenated Fluoresceines  89 Mercaptofluoresceins  91 Fluorescein-Analogs  91 HAS-Fluoresceins  91 Sulfofluoresceins  91 Fluorones  92 HAS-Fluorones  93 Cyanines  93 Trimethine Cyanines  95 Pentamethine Cyanines  95 Heptamethine Cyanines  97 Borondipyrromethenes  97 Small Water-Soluble Borondipyrromethenes  100 Medium-Sized, Water-Soluble Borondipyrromethenes  104 Large Water-Soluble Borondipyrromethenes  106 Other Classes Derived From Borondipyrromethene  108 Rhodols  109 The First Rhodols Synthesized  109 Rhodols Synthesized More Recently  109 Rhodol Analogs  112 HAS-Rhodols  113 Rosamines  114 HAS-Rosamines  114 Silico-Rosamines  114 Phospha-Rosamines  115 Other HAS-Rosamines  115 Rosols  118 HAS-Rosols  118 Pyrodols and Pyrodones  119 Trianguleniums  120 Acridines  120 Simple Acridines  121 Acridones  122 Merocyanines  122 Phenoxazines  122 Coumarins  125 7-Hydroxy Coumarins  125 Small 7-Amino Coumarins  125 More Elaborated 7-Amino Coumarins  126 Sulforhodols  128

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Contents

4.28 4.29 4.30 4.31 4.32 4.33 4.34 4.35 4.36 4.37 4.38 4.39 4.39.1 4.39.2 4.39.3 4.40 4.40.1 4.40.2 4.40.3 4.41 4.42

Pyrenes  128 Quinolines  129 Benzothiazoles  132 Chromones  133 Naphthalimides  133 Indoles  134 Naphthalenes  135 Squaraines  137 Pteridines  137 Isoquinolines  139 Benzene Derivatives  140 Other Single Structures  140 Small Structures  140 Medium-Sized Structures  142 Large Structures (Na > 80)  142 Hybrid Structures  145 Hybrid Structures: Fusion of Two Existing Dyes  146 Hybrid Structures: Single Bond Connected Dyes  146 Hybrid Structures: Polymethine Bridged Dyes  147 Non-Disclosed Structures  148 Fluorescent Structures Other Than Small-Molecule Organic Dyes  149

5 5.1 5.2 5.2.1 5.2.2 5.2.3 5.2.4 5.2.5 5.2.6 5.2.7 5.2.8 5.3

Scattergrams of the Photophysical Properties  163 Introduction  163 Photophysical Properties Along the Spectrum  164 Molecular Sizes vs. λa,max  164 Molar Absorption Coefficients vs. λa,max  169 Fluorescence Quantum Yield vs. λa,max  169 Brightness vs. λa,max  172 Stokes Shift vs. λa,max  173 Stokes Shift vs. Brightness  174 Fluorescence Mean-Lifetime vs. λa,max  177 Fluorescence Mean-Lifetime vs. Brightness  178 Fluorophore Charges  180

6 6.1 6.2 6.3

Band Shapes and Excitation and Emission Ranges  185 Introduction  185 Typical Absorption and Emission Spectra of Some Classes  187 Coarse Prediction of Excitation and Emission Ranges  191

7 7.1 7.2 7.3

Measuring Photostability and Mitigating Photobleaching  195 Introduction  195 Measuring Photostability  196 Mitigating Photobleaching  202

Contents

Appendix A

A1. Short Name, Name, Class, Molecular Formula, and References  207

Appendix B

B1. Ranked by Excitation Maximum  253

Appendix C

C1. Ranked by Emission Maximum  297

Appendix D

D1. Ranked by Stokes Shifts  335

Appendix E

E1. Ranked by Brightness  371

Appendix F

F1. Ranked by Fluorescence Mean-Lifetime  419

Appendix G

G1. Ranked by Molecular Net Charge  433

Index  479

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Preface This book presents the molecular structures and photophysical properties of over seven hundred fluorescent dyes with medium to high brightness in aqueous solutions. It has been written to be a reliable and useful source of information for users of fluorescent dye labels and stains in general. The specific aqueous solution used to measure the photophysical parameters of each of the seven hundred dyes is clearly provided in this succinct database. The photophysical parameters presented in this database include: wavelengths of absorption and/or excitation maximum, wavelengths of emission maximum, Stokes shifts, excitation ranges, emission ranges, molar absorption coefficients, fluorescence quantum yields, brightnesses, fluorescence mean-lifetimes, sizes (represented by the number of atoms of the structures), and charges in aqueous solutions at neutral pH. Additional interesting properties of some of the fluorescent dyes have also been included, such as photostability and pKa (when present at around neutral pH). Scattergrams are systematically used to give panoramic views of the photophysical data that allow users to find desired photophysical properties within a given spectral range. Previously, this data was only available from varied sources that contained partial lists, were missing data, and, in many cases, omitted descriptions of the liquids in which parameters were measured. It is known that almost all fluorescent dyes that exhibit high molecular fluorescence brightness in aqueous solutions are also bright in organic solvents; however, the opposite is not true. Most dyes that exhibit a high brightness in organic solvents exhibit a lower and, in many cases, a very low brightness in aqueous solutions. Even worse, the great majority of fluorescent dyes available are not soluble enough in aqueous solutions to have their photophysical parameters measured. Hence, the importance of this database, where only dyes whose photophysical parameters were measured in plain water, phosphate-buffered saline, or aqueous buffers (without organic modifiers) are included. Some dyes are no longer commercially available, but are nonetheless kept in the database for completeness and historic record purposes. The author began recording the spectral and photophysical data of fluorescent dyes in his Master’s degree, and has continued throughout his whole academic career. Measurements were collected in practice and from the literature, and now the time has come to share this collection with the whole community in the form of the current printed database. Probably not all bright water-soluble fluorescent dyes are here, as some may have gone unnoticed. Therefore, the author apologizes to the developers of these missing fluorophores for this. The extensive data collection necessary to make this book a reality would not have been possible without the help of so many bright students and colleagues, as well as the professionalism of many companies (suppliers of fluorescent dye labels, stains, and probes). The author would also like to thank Prof A.Z. Khoury, Prof D. Lüdcke, and Prof A. Manz for their many stimulating conversations; Prof J.N. Picada for the suggestion to write this book; and both Dr W. Nietfeld and Prof H. Lehrach from the Max Planck Institute in Berlin, where many photophysical parameters were measured by the author during a post-Doctorate leave.

Preface

Finally, this book is dedicated to all Organic Chemists from all over the world for their talented work that has brought so many interesting fluorescent structures to our daily lives and impressive developments across so many fields. Moreover, these high-tech products are now being developed and synthesized using increasingly sustainable and environmentally-friendly methods. April 2022 

Tarso B. Ledur Kist Porto Alegre

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Acronyms A Acetate buffer ACN Acetonitrile; ethanenitrile ADC Antracene-2,3-dicarboxaldehyde APD Avalanche photodiode B Borate buffer BODIPY 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene C Carbonate buffer CCD Charged coupled device CBQCA 3-(4-Carboxybenxoyl)quinoline-2-carboxaldehyde DMF Dimethylformamide; N,N-dimethylmethanamide DMSO Dimethylsulfoxide; dimethyl(oxido)sulfur FRET Förster resonant energy transfer HAS Heteroatom-substituted HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid IA Iodoacetamido ITC Isothyocianate ME Maleimido NDA Naphthalene-2,3-dicarboxaldehyde NHS N-Hydroxysuccinimidyl ester NIR Near-infrared NMP N-Methyl-2-pyrrolidinone OPA o-Phthaldialdehyde P Phosphate buffer PBS Phosphate-buffered saline (usually: 8.0 g/L NaCl, 0.2 g/L KCl, 1.42 g/L Na2HPO4, and 0.24 g/L KH2PO4) PD Photodiode PC Propylene carbonate; 4-methyl-1,3-dioxolan-2-one PMT Photomultiplier tube RT Room temperature SE N-Hydroxysuccinimidyl ester THF Tetrahydrofuran; 1,4-epoxybutane Tris Tris(hydroxymethyl)aminomethane; 2-amino-2-(hydroxymethyl)propane-1,3-diol UV Ultraviolet UVA UV radiation in the 315 and 400 nm range. Informally also known as “black light” Vis Visible

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Symbols and Conventions Symbol Name and definition [Units] A(λ) Absorbance at wavelength λ. A(λ) = Log10(Po(λ)/P(λ)) = Log10(1/T(λ)). [dimensionless] b Cuvette inner length. [cm] B(λ) Molecular fluorescent brightness at wavelength λ. B(λ) = ε(λ) Φf. [м–1cm–1]. Bmax Molecular fluorescent brightness at wavelength λa,max. Bmax = ε(λx,max) Φf = εmax Φf. [м–1cm–1]. c Amount concentration. Expressed in mol dm–3 or mol L–1, while the non-SI unit м (small cap M) is used as an abbreviation for mol dm–3. [mol dm–3] h Planck constant. h = 6.626069  ×  10–34 J s. [Js] L Radiance. Defined as the radiant power (P) leaving or passing through a small element of surface (dS) divided by the solid angle of this surface (dΩ) and the orthogonally projected area of this element of surface in a plane normal to the beam direction (dS cosθ). L = d2p/(dΩ dS cosθ) [Js–1m–2sr–1 or Wm–2sr–1]. For a parallel beam of radiation the radiance is simple given by L = dp/(dS cosθ). [Js–1m–2 or Wm–2] L(λ) Spectral radiance. Derivative of radiance, L, with respect to wavelength, λ. [Js–1m–2nm–1 or Wm–2nm–1] Lp Photon radiance. Defined as the number of photons leaving or passing through a small element of surface (dS) per second divided by the solid angle of this surface (dΩ) and the orthogonally projected area of this element of surface in a plane normal to the beam direction (dS cosθ). Lp = number of photons per second/(dΩ dS cosθ) [s–1m–2sr–1]. For a parallel beam of radiation the photon radiance is simple given by Lp = number of photons per second/(dS cosθ). [s–1m–2] Lp(λ) Spectral photon radiance. Derivative of photon radiance, Lp, with respect to wavelength, λ. [s–1m–2nm–1] P Radiant power. Power emitted, transferred, or received as electromagnetic radiation. [Js–1 or W] P(λ) Spectral radiant power, at λ. Derivative of radiant power, P, with respect to wavelength, λ. In spectroscopy: P(λ) = {total radiant power between [λ –(Δλ/2)] and [λ +(Δλ/2)] that exits a sample or cuvette filled with a solvent containing the solute}/Δλ. [W nm–1] o P (λ) Spectral radiant power at λ that exits a blank sample or cuvette “filled with solvent only.” Po(λ) = {total radiance, between [λ –(Δλ/2)] and [λ +(Δλ/2)], that exits a blank sample or cuvette filled with solvent only}/Δλ. [W nm–1] Na Number of atoms of the fluorescent structure (counter-ions of salts are not counted) in its prevalent ionic form in an aqueous solution at a specified pH. [dimensionless] Nn Number of negative elementary charges of a fluorescent structure in its prevalent ionic form in an aqueous solution at a specified pH. [dimensionless]

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Symbols and Conventions

Np Number of positive elementary charges of a fluorescent structure in its prevalent ionic form in an aqueous solution at a specified pH. [dimensionless] q Net number of elementary charges of a fluorescent structure in its prevalent ionic form in an aqueous solution at a specific pH (q = Np – Nn). [dimensionless] T Temperature. [oC and sometimes K] T(λ) Transmittance at wavelength λ. T(λ) = P(λ)/Po(λ). [dimensionless] ΔS Stokes shift. ΔS = νx,max – νe,max. [cm–1]. The following are also used:  ΔS = λe,max – λx,max and ΔS = λe,max – λa,max. [nm] ε(λ) Molar decadic absorption coefficient at wavelength λ. ε(λ) = A(λ) b–1c–1. [м–1cm–1]. εmax Molar decadic absorption coefficient at the absorption maximum of the longest-wavelength band. εmax = A(λa,max) b–1c–1. [м–1cm–1]. λ Vacuum wavelength of the electromagnetic radiation. [nm] λa,1 Wavelength of absorption maximum of the longest wavelength absorption band (λa,1 ≡ λa,max). [nm] λa,max Wavelength of absorption maximum of the longest wavelength absorption band (λa,max ≡ λa,1). [nm] λa,–α Wavelength at height α of the left side of the longest wavelength absorption band (0