Highly Emissive Chiral Lanthanide(Iii) Complexes for Labelling and Imaging

•••HIGHLY EMISSIVE CHIRAL LANTHANIDE(III) COMPLEXES FOR LABELLING AND IMAGING Andrew Timothy Frawley A thesis submitted for the degree of Doctor of Philosophy 2017 Declaration The work described herein was undertaken at the Department of Chemistry, Durham University between October 2014 and September 2017. All of the work is my own, except where specifically stated otherwise. No part has previously been submitted for a degree at this or any other university. Statement of Copyright The copyright of this thesis rests with the author. No quotation from it should be published without the author’s prior written consent and information derived from it should be acknowledged. iAbstract Sensitised lanthanide complexes based on macrocyclic chelating ligands have been used extensively to study biological function at a cellular level, due to their very bright and long-lived emission, sharp emission bands and high stability to photodegradation. These photophysical properties, in addition to their excellent chiroptical behaviour, also make these complexes promising candidates for security labelling and as anti-counterfeiting tools. Novel highly emissive chiral europium(III) complexes based on macrocyclic ligands have been synthesised and their photophysical properties studied. They possess high molar extinction coefficients and are amenable to excitation using commonly available light sources. These complexes have been resolved by chiral HPLC and the circularly polarised luminescence (CPL) spectra of their enantiomers recorded. They exhibit strong circularly polarised emission in response to excitation using near ultra-violet light, and are stable to thermally-activated racemisation. A simple off-the-shelf camera set up has been developed which is capable of discriminating ‘real’ europium(III) emission from emission from a ‘fake’ marking, based on emission lifetime and wavelength. Additionally, chiroptical discrimination has been achieved using a custom built microscope incorporating a quarter-wave plate and linear polariser. The solvent dependent emission behaviour of a series of C3-symmetric lanthanide(III) complexes has been studied, demonstrating that the form of the total emission and CPL is extremely sensitive to minor changes in the outer solvation sphere of the complex. Finally, macropinocytosis has been identified as the mechanism of cell uptake of this family of complexes in NIH-3T3 cells, and the internalisation and subsequent sub-cellular localisation has been shown to be dependent on complex chirality. ii Acknowledgements I would like to thank the following people for making this work possible: My supervisor, Prof. David Parker, for giving me the opportunity to carry out this project and for his ideas, guidance and support over the last 4 years. Special thanks must go to Dr. Robert Pal for his help with all things both living and optical, specifically cameras, microscopes and cells. Thank you to the following members of the Chemistry Department for their assistance: Dr. Alan Kenwright, Dr. Juan Aguilar, Catherine Heffernan and Dr. Raquel Belda-Vidal for NMR spectroscopy; Dr. Jackie Mosely, Pete Stokes and Dr. Dave Parker for mass spectrometry; Dr. Aileen Congreve and Lenny Lauchlan for HPLC training and for letting me loose in the HPLC lab; Dr. Lars-Olof Pålsson for assistance with low temperature photophysical measurements; Annette Passmoor, Gary Southern and all the stores and technical team for keeping us supplied with equipment and chemicals; the lab attendants for the daily supply of tea and coffee, and specifically to Claire for gently encouraging me to tidy my desk every so often. Thanks also to Dr. Chris Ottley (Dept. of Earth Sciences) for ICP-MS analyses. Thanks to all the Parker Group members, past and present. In particular I’d like to thank Steve, Brian, Kanthi, Cidalia, Nicola, Matthieu, Kevin, Alex, Martina, Emily, Katie, Sergey, Edward, Alice, Laura, Ryan and Hannah for sharing their knowledge and keeping me going over the years. I would also like to acknowledge Durham University for the award of a Durham Doctoral Scholarship which has allowed me to pursue my postgraduate studies. Thanks to all the leaders, parents and young people at 19th Durham Scout Group for providing plenty of fun and adventure (and distraction from chemistry). A special thank you to Siân, Will, Evelyn and Nicholas Greeves for being my ‘Durham family’ for the last 7 years. I really can’t thank you enough for everything you’ve done. Finally, the biggest thanks to Mum, Dad and Helen for their unwavering support in everything I do. iii List of Abbreviations α1-AGP 12-N4 9-N3 A431 AAT alpha-1-acid glycoprotein 1,4,7,10-tetraazacyclododecane 1,4,7-triazacyclononane human epidermoid carcinoma cell line alpha-1-antitrypsin <ul style="display: flex;"><li style="flex:1">AC </li><li style="flex:1">alternating current </li></ul>acac ADP AFM AMP ATP acetylacetonate adenosine diphosphate atomic force microscopy adenosine monophosphate adenosine triphosphate 2,2’-bipyridine-6,6’-dicarboxylate back energy transfer bda BET BINAP BINOL BODIPY BP 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl 1,1'-bi-2-naphthol boron-dipyrromethene band pass <ul style="display: flex;"><li style="flex:1">br </li><li style="flex:1">broad (NMR) </li></ul><ul style="display: flex;"><li style="flex:1">BSA </li><li style="flex:1">bovine serum albumin </li></ul>charge-coupled device Cambridge Crystallographic Data Centre circular dichroism CCD CCDC CD CHO CIE Chinese hamster ovary Commission internationale de l'éclairage (International Commission on Illumination) CPEL CPL circularly polarised electroluminescence circularly polarised luminescence <ul style="display: flex;"><li style="flex:1">chiral stationary phase </li><li style="flex:1">CSP </li></ul>iv <ul style="display: flex;"><li style="flex:1">CW </li><li style="flex:1">continuous wave </li></ul>cyclen d, dd, ddd DC 1,4,7,10-tetraazacyclododecane doublet, doublet of doublets, etc. (NMR) direct current DCM dfppy DFT dichloromethane difluorophenylpyridine density functional theory DMEM DMF DMSO DOTA dpa Dulbecco's modified Eagle's medium N,N-dimethylformamide dimethylsulfoxide 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid dipicolinic acid, pyridine-2,6-dicarboxylic acid 1,1'-bis(diphenylphosphino)ferrocene 1,3-dipyridylbenzene dppf dpyb DSLR DTPA ED digital single lens reflex diethylenetriaminepentaacetic acid electric dipole <ul style="display: flex;"><li style="flex:1">ee </li><li style="flex:1">enantiomeric excess </li></ul>퐸푇푁 Reichardt's normalised solvent polarity <ul style="display: flex;"><li style="flex:1">equivalents </li><li style="flex:1">eq. </li></ul><ul style="display: flex;"><li style="flex:1">ESI </li><li style="flex:1">electrospray ionisation </li></ul><ul style="display: flex;"><li style="flex:1">eT </li><li style="flex:1">electron transfer </li></ul><ul style="display: flex;"><li style="flex:1">ET </li><li style="flex:1">energy transfer </li></ul>facam FOV 3-trifluoroacetylcamphorate field of view FRET FWHM GCMS HEPES hfac Förster/fluorescence resonance energy transfer full width half maximum gas chromatography mass spectrometry 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 1,1,1,5,5,5-hexafluoroacetylacetonate v<ul style="display: flex;"><li style="flex:1">hfbc </li><li style="flex:1">3-heptafluorobutyrylcamphorate </li></ul><ul style="display: flex;"><li style="flex:1">HP-DO3A </li><li style="flex:1">10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7- </li></ul>triacetic acid HPLC HRMS IC high pressure/performance liquid chromatography high resolution mass spectrometry internal conversion IC50 half-maximal inhibitory concentration inductively coupled plasma mass spectrometry internal charge transfer ICP-MS ICT <ul style="display: flex;"><li style="flex:1">IR </li><li style="flex:1">infra-red </li></ul><ul style="display: flex;"><li style="flex:1">ISC </li><li style="flex:1">intersystem crossing </li></ul>iTTL LCMS LED through the lens metering (camera flash unit) liquid chromatography mass spectrometry light emitting diode <ul style="display: flex;"><li style="flex:1">Ln </li><li style="flex:1">lanthanide </li></ul><ul style="display: flex;"><li style="flex:1">LP </li><li style="flex:1">long pass </li></ul>LSCM LTG laser scanning confocal microscopy LysoTracker Green <ul style="display: flex;"><li style="flex:1">m</li><li style="flex:1">multiplet (NMR) </li></ul>m.p. mCPBA MD melting point meta-chloroperbenzoic acid magnetic dipole MOPS MRI Ms 3-(N-morpholino)propanesulfonic acid magnetic resonance imaging methanesulfonyl MTG NIH-3T3 NMR NOTA PBS MitoTracker Green mouse skin fibroblast cell line nuclear magnetic resonance 1,4,7-triazacyclononane-1,4,7-triacetic acid phosphate buffered saline vi pda PEM PET phen PhMoNa PMMA PMT ppm ppy PVC Q 1,10-phenanthroline-2,9-dicarboxylic acid photoelastic modulator positron emission tomography phenanthroline phase modulated nanoscopy poly(methyl methacrylate) photomultiplier tube parts per million phenylpyridine poly(vinyl chloride) quencher <ul style="display: flex;"><li style="flex:1">QR </li><li style="flex:1">quick response </li></ul>QToF Squadrupole time of flight (mass spectrometry) singlet (energy level) <ul style="display: flex;"><li style="flex:1">s</li><li style="flex:1">singlet (NMR) </li></ul>SEM SOM Tscanning electron microscopy single organic molecule triplet (energy level) <ul style="display: flex;"><li style="flex:1">t</li><li style="flex:1">triplet (NMR) </li></ul>TBAF td tetrabutylammonium fluoride triplet of doublets (NMR) <ul style="display: flex;"><li style="flex:1">2,2,2-trifluoroethanol </li><li style="flex:1">TFE </li></ul>THF TLC TQD TTA UPLC UV tetrahydrofuran thin layer chromatography triple quadrupole (mass spectrometry) 2-thenoyltrifluoroacetonate ultra performance liquid chromatography ultra violet <ul style="display: flex;"><li style="flex:1">YAG </li><li style="flex:1">yttrium aluminium garnet, Y3Al5O12 </li></ul>vii Table of Contents <a href="#5_0">Abstrac</a><a href="#5_0">t</a><a href="#5_0">.</a><a href="#5_0">......................................................................................................................</a><a href="#5_0">.</a><a href="#5_0">i</a><a href="#5_0">i </a><a href="#6_0">Acknowledgements..................................................................................................... iii </a><a href="#7_0">List of Abbreviations ................................................................................................... iv </a><a href="#0_0">CHAPTER ONE : INTRODUCTIO</a><a href="#0_0">N</a><a href="#0_0">.</a><a href="#0_0">.................................................................................1 </a> <a href="#0_1">1.1 Introduction</a><a href="#0_1"> </a><a href="#0_1">to lanthanide luminescenc</a><a href="#0_1">e</a><a href="#0_1">.</a><a href="#0_1">....................................................1 </a> <a href="#0_2">1.1.1 Overview</a><a href="#0_2"> </a><a href="#0_2">................................................................................................1 </a><a href="#0_0">1.1.2 Emission</a><a href="#0_0"> </a><a href="#0_0">lifetim</a><a href="#0_0">e</a><a href="#0_0">.</a><a href="#0_0">...................................................................................5 </a><a href="#0_3">1.1.3 Excitation</a><a href="#0_3"> </a><a href="#0_3">of lanthanides........................................................................6 </a><a href="#0_4">1.1.4 Quenching</a><a href="#0_4"> </a><a href="#0_4">processes .............................................................................7 </a><a href="#0_5">1.1.5 Photoluminescence</a><a href="#0_5"> </a><a href="#0_5">quantum yield and brightness ..............................8 </a><a href="#0_6">1.1.6 Ligands</a><a href="#0_6"> </a><a href="#0_6">for Ln(III) complexes..................................................................9 </a> <a href="#0_7">1.2 Introduction</a><a href="#0_7"> </a><a href="#0_7">to circularly polarised luminescence......................................18 </a> <a href="#0_8">1.2.1 Theoretical</a><a href="#0_8"> </a><a href="#0_8">background .......................................................................18 </a><a href="#0_9">1.2.2 Instrumentation</a><a href="#0_9"> </a><a href="#0_9">for the detection of CPL............................................21 </a> <a href="#0_10">1.3 CPL-active</a><a href="#0_10"> </a><a href="#0_10">organic and transition metal systems........................................23 </a> <a href="#0_11">1.3.1 Single</a><a href="#0_11"> </a><a href="#0_11">Organic Molecule (SOM) systems .............................................23 </a><a href="#0_12">1.3.2 Supramolecular</a><a href="#0_12"> </a><a href="#0_12">aggregates .................................................................27 </a><a href="#0_13">1.3.3 Transition</a><a href="#0_13"> </a><a href="#0_13">metal complexe</a><a href="#0_13">s</a><a href="#0_13">.</a><a href="#0_13">................................................................29 </a> <a href="#0_3">1.4 Chiral</a><a href="#0_3"> </a><a href="#0_3">lanthanide complexes .......................................................................32 </a> <a href="#0_14">1.4.1 Design</a><a href="#0_14"> </a><a href="#0_14">and control of chirality .............................................................32 </a><a href="#0_15">1.4.2 Ln(III)</a><a href="#0_15"> </a><a href="#0_15">complexes as CPL probes for biological analytes ......................40 </a> <a href="#0_16">1.5 Cell</a><a href="#0_16"> </a><a href="#0_16">imaging with lanthanide complexes.....................................................51 </a>viii <a href="#0_17">1.5.1 Design</a><a href="#0_17"> </a><a href="#0_17">considerations ..........................................................................51 </a><a href="#0_18">1.5.2 Examples</a><a href="#0_18"> </a><a href="#0_18">of sub-cellular localisation profile</a><a href="#0_18">s</a><a href="#0_18">.</a><a href="#0_18">.....................................52 </a><a href="#0_19">1.5.3 Cell</a><a href="#0_19"> </a><a href="#0_19">uptake mechanisms ......................................................................57 </a> <a href="#0_0">1.6 Lanthanides</a><a href="#0_0"> </a><a href="#0_0">in security labelling .................................................................61 </a><a href="#0_20">1.7 Project</a><a href="#0_20"> </a><a href="#0_20">Aims and Outline.............................................................................69 </a> <a href="#0_0">CHAPTER TWO : CHIRAL Eu(III) COMPLEXES FOR SECURITY LABELLING ...................71 </a> <a href="#0_21">2.1 Requirements</a><a href="#0_21"> </a><a href="#0_21">for a security label ...............................................................71 </a><a href="#0_3">2.2 Synthesis</a><a href="#0_3"> </a><a href="#0_3">of target system</a><a href="#0_3">s</a><a href="#0_3">.</a><a href="#0_3">........................................................................72 </a> <a href="#0_22">2.2.1 Synthesis</a><a href="#0_22"> </a><a href="#0_22">of [EuL</a>1]................................................................................73 <a href="#0_23">2.2.2 Synthesis</a><a href="#0_23"> </a><a href="#0_23">of [EuL</a>2]................................................................................81 <a href="#0_0">2.3 Photophysical</a><a href="#0_0"> </a><a href="#0_0">properties of [EuL</a>1] and [EuL2] ............................................84 <a href="#0_24">2.4 Complex</a><a href="#0_24"> </a><a href="#0_24">chirality and resolution by chiral HPLC.........................................88 </a><a href="#0_25">2.5 CPL</a><a href="#0_25"> </a><a href="#0_25">characterisatio</a><a href="#0_25">n</a><a href="#0_25">.</a><a href="#0_25">...................................................................................94 </a><a href="#0_8">2.6 An</a><a href="#0_8"> </a><a href="#0_8">alternative strategy for the synthesis of enantiopure carboxylate </a><a href="#0_8">complexes ............................................................................................................106 </a><a href="#0_26">2.7 Racemisation</a><a href="#0_26"> </a><a href="#0_26">kinetic</a><a href="#0_26">s</a><a href="#0_26">.</a><a href="#0_26">...............................................................................114 </a><a href="#0_27">2.8 Development</a><a href="#0_27"> </a><a href="#0_27">of time-gated photography in security labellin</a><a href="#0_27">g</a><a href="#0_27">.</a><a href="#0_27">...............121 </a><a href="#0_28">2.9 Time-gated</a><a href="#0_28"> </a><a href="#0_28">chiral contrast microscopy.....................................................133 </a><a href="#0_29">2.10 Conclusions</a><a href="#0_29"> </a><a href="#0_29">and Future Work ...................................................................137 </a> <a href="#0_0">CHAPTER THREE : SOLVENT EFFECTS IN Ln(III) EMISSION .......................................140 </a> <a href="#0_30">3.1 Introduction...............................................................................................140 </a><a href="#0_31">3.2 Solvent</a><a href="#0_31"> </a><a href="#0_31">effects in the emission and CPL of [EuL</a>2]<a href="#0_31">.</a><a href="#0_31">....................................142 </a> <a href="#0_32">3.2.1 Solvent</a><a href="#0_32"> </a><a href="#0_32">effects in the total emission of [EuL</a>2]<a href="#0_32">.</a><a href="#0_32">..................................142 </a><a href="#0_3">3.2.2 Solvent</a><a href="#0_3"> </a><a href="#0_3">effects in the CPL of [EuL</a>2]<a href="#0_3">.</a><a href="#0_3">...................................................148 </a><a href="#0_33">3.2.3 Chiral</a><a href="#0_33"> </a><a href="#0_33">additives...................................................................................152 </a>ix <a href="#0_34">3.3 Solvent</a><a href="#0_34"> </a><a href="#0_34">effects in the total emission and CPL of [EuL</a>6] ............................156 <a href="#0_0">3.3.1 Solvent</a><a href="#0_0"> </a><a href="#0_0">effects in the total emission of [EuL</a>6]<a href="#0_0">.</a><a href="#0_0">..................................157 </a><a href="#0_35">3.3.2 Solvent</a><a href="#0_35"> </a><a href="#0_35">effects in the CPL of [EuL</a>6]<a href="#0_35">.</a><a href="#0_35">...................................................159 </a><a href="#0_36">3.3.3 Specific</a><a href="#0_36"> </a><a href="#0_36">solvent effect of chlorinated solvents ..................................162 </a> <a href="#0_0">3.4 A</a><a href="#0_0"> </a><a href="#0_0">combined emission and NMR study into solvent effects in the [LnL</a>7] <a href="#0_0">series ...................................................................................................................167 </a><a href="#0_37">3.5 Conclusions</a><a href="#0_37"> </a><a href="#0_37">and Future Work ...................................................................182 </a> <a href="#0_0">CHAPTER FOUR : CELL UPTAKE OF CHIRAL Eu(III) COMPLEXES ...............................184 </a> <a href="#0_38">4.1 Introduction...............................................................................................184 </a><a href="#0_0">4.2 Cell</a><a href="#0_0"> </a><a href="#0_0">uptake mechanism of [EuL</a>4]...............................................................186 <a href="#0_9">4.3 Effect</a><a href="#0_9"> </a><a href="#0_9">of chirality on cell uptake and sub-cellular localisation..................191 </a> <a href="#0_0">4.3.1 Cell</a><a href="#0_0"> </a><a href="#0_0">uptake of enantiopure complexes ..............................................193 </a><a href="#0_0">4.3.2 Sub-cellular</a><a href="#0_0"> </a><a href="#0_0">localisation of enantiopure complexe</a><a href="#0_0">s</a><a href="#0_0">.</a><a href="#0_0">.........................198 </a> <a href="#0_18">4.4 Conclusions</a><a href="#0_18"> </a><a href="#0_18">and Future Work ...................................................................205 </a> <a href="#0_0">CHAPTER FIVE : EXPERIMENTAL METHOD</a><a href="#0_0">S</a><a href="#0_0">.</a><a href="#0_0">............................................................208 </a> <a href="#0_38">5.1 General</a><a href="#0_38"> </a><a href="#0_38">Procedures...................................................................................208 </a><a href="#0_3">5.2 HPLC</a><a href="#0_3"> </a><a href="#0_3">analysi</a><a href="#0_3">s</a><a href="#0_3">.</a><a href="#0_3">............................................................................................209 </a><a href="#0_29">5.3 Optical</a><a href="#0_29"> </a><a href="#0_29">measurements ..............................................................................211 </a><a href="#0_29">5.4 Time-gated</a><a href="#0_29"> </a><a href="#0_29">photography...........................................................................212 </a><a href="#0_39">5.5 Circularly</a><a href="#0_39"> </a><a href="#0_39">polarised microscopy ................................................................212 </a><a href="#0_40">5.6 Preparation</a><a href="#0_40"> </a><a href="#0_40">of PMMA films.......................................................................213 </a><a href="#0_41">5.7 Cell</a><a href="#0_41"> </a><a href="#0_41">culture protocols ................................................................................213 </a><a href="#0_42">5.8 Confocal</a><a href="#0_42"> </a><a href="#0_42">microscopy and cell imaging ......................................................215 </a><a href="#0_0">5.9 Synthetic</a><a href="#0_0"> </a><a href="#0_0">procedures.................................................................................217 </a> <a href="#0_0">REFERENCES .............................................................................................................256 </a>

Highly Emissive Chiral Lanthanide(Iii) Complexes for Labelling and Imaging

Details

Download

Copyright

We respect the copyrights and intellectual property rights of all users. All uploaded documents are either original works of the uploader or authorized works of the rightful owners.

Support