Science, Faculty of
Chemistry, Department of
DSpace
UBCV
Arsenault, Nicole Evelyn
Doctor of Philosophy - PhD
University of British Columbia
Herein an investigation of the photophysical properties of stimuli-responsive molecules incorporating a triarylborane Lewis acid and phosphine oxide Lewis base pair on a bithiophene backbone, called flexible Lewis pairs (FlexLPs), is presented. The FlexLPs change conformation between the Lewis acid-base bound adduct form and unbound open form with surrounding solvent, where strongly hydrogen bond donating solvent stabilizes the open form through formation of a hydrogen bond with the Lewis base. The ability to synthesize diverse stimuli-responsive molecular motifs incorporating FlexLPs is achieved by installing a reactive terminal alkyne functional group on a FlexLP molecule. The application of this FlexLP molecule as a versatile stimuli-responsive ligand that can be incorporated into more complex molecular architectures, such as dimers, transition-metal complexes, and functionalized nanoparticle materials, is thus demonstrated by utilizing different alkyne coupling chemistry. Synthesis of a dimeric molecule with two FlexLP pairs connected through a conjugated bithiophene-alkyne-alkyne-bithiophene backbone is described and compared to a monomeric FlexLP molecule. The monomer and dimer FlexLP demonstrate changing emission colours and lifetimes with varying solvent, proposed to be from a change in FlexLP conformation. In the closed form a π–π* energy transition occurs resulting in blue-green emission colour, while in the unbound form a charge transfer event occurs resulting in yellow-orange emission colour. Monometallic and bimetallic Pt(terpyridine)-containing complexes functionalized with the FlexLP ligand are explored as solvent-responsive optical switches. A low energy CT band between the acid-base pair ligand and terpyridine ligand appears for both complexes, Absorption spectroscopy reveals this CT band is solvent- and temperature-responsive, and is dependent on the FlexLP ligand conformation. Investigation of the photophysical properties of two emissive Pt-functionalized FlexLP complexes reveal both complexes fluoresce despite being able to populate the excited triplet states of the complexes. Transient absorption spectroscopy reveals that the FlexLP ligand conformation has a significant effect on the triplet-state lifetime. Functionalization of FlexLPs onto nanoparticles is explored for applications as water-sensing emissive fluorophore tags for biomedical imaging applications. The emission colour and lifetime change in response to water concentration in surrounding solvent, evidenced by fluorescence and emission lifetime spectroscopy. Preliminary fluorescence microscopy studies are also performed.
SYMMETRIC AND NON-SYMMETRIC STIMULI-RESPONSIVE FLEXIBLE LEWIS PAIR SYSTEMS by Nicole Evelyn Arsenault B.Sc., University of Prince Edward Island, 2015 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) July 2020 © Nicole Evelyn Arsenault, 2020 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Symmetric and Non-Symmetric Stimuli-Responsive Flexible Lewis Pair Systems submitted by Nicole Evelyn Arsenault in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry Examining Committee: Michael Wolf, Chemistry Supervisor Zachary Hudson, Chemistry Supervisory Committee Member W. Russell Algar, Chemistry University Examiner Derek Gates, Chemistry University Examiner Additional Supervisory Committee Members: Chris Orvig, Chemistry Supervisory Committee Member Martin Tanner, Chemistry Supervisory Committee Member iii Abstract Herein an investigation of the photophysical properties of stimuli-responsive molecules incorporating a triarylborane Lewis acid and phosphine oxide Lewis base pair on a bithiophene backbone, called flexible Lewis pairs (FlexLPs), is presented. The FlexLPs change conformation between the Lewis acid-base bound adduct form and unbound open form with surrounding solvent, where strongly hydrogen bond donating solvent stabilizes the open form through formation of a hydrogen bond with the Lewis base. The ability to synthesize diverse stimuli-responsive molecular motifs incorporating FlexLPs is achieved by installing a reactive terminal alkyne functional group on a FlexLP molecule. The application of this FlexLP molecule as a versatile stimuli-responsive ligand that can be incorporated into more complex molecular architectures, such as dimers, transition-metal complexes, and functionalized nanoparticle materials, is thus demonstrated by utilizing different alkyne coupling chemistry. Synthesis of a dimeric molecule with two FlexLP pairs connected through a conjugated bithiophene-alkyne-alkyne-bithiophene backbone is described and compared to a monomeric FlexLP molecule. The monomer and dimer FlexLP demonstrate changing emission colours and lifetimes with varying solvent, proposed to be from a change in FlexLP conformation. In the closed form a π–π* energy transition occurs resulting in blue-green emission colour, while in the unbound form a charge transfer event occurs resulting in yellow-orange emission colour. Monometallic and bimetallic Pt(terpyridine)-containing complexes functionalized with the FlexLP ligand are explored as solvent-responsive optical switches. A low energy CT band between the acid-base pair ligand and terpyridine ligand appears for both complexes, Absorption iv spectroscopy reveals this CT band is solvent- and temperature-responsive, and is dependent on the FlexLP ligand conformation. Investigation of the photophysical properties of two emissive Pt-functionalized FlexLP complexes reveal both complexes fluoresce despite being able to populate the excited triplet states of the complexes. Transient absorption spectroscopy reveals that the FlexLP ligand conformation has a significant effect on the triplet-state lifetime. Functionalization of FlexLPs onto nanoparticles is explored for applications as water-sensing emissive fluorophore tags for biomedical imaging applications. The emission colour and lifetime change in response to water concentration in surrounding solvent, evidenced by fluorescence and emission lifetime spectroscopy. Preliminary fluorescence microscopy studies are also performed. v Lay Summary The ability to control the movement of objects at the molecular scale to produce a response visible by eye is an ongoing challenge for scientists developing dynamic materials and sensors. This work presents a new molecule that changes shape and emits different coloured light dependent on its surroundings, and can be modified to have one or two reactive ends. By having a reactive end, the molecule can be attached to many other molecules in one step in order to produce a variety of complex shape-dependent emissive materials. This allows more complex materials to have similar controllable properties. This thesis demonstrates the versatility of this shape-dependent molecule to react with a broad scope of chemical structures, including organic, metal, and nanomaterials, and discusses a variety of potential applications that could benefit from these dynamic materials, such as sensing or biomedical imaging. vi Preface In all chapters I am the principle author of the work, carried out under the supervision of Prof. Michael O. Wolf. I performed all the experiments except those contributions noted below. Sections of Chapter 2 regarding the synthesis and characterization of compound 24 and a version of Chapter 3 have been previously published1 as a communication written by myself with contributions from M. O. Wolf. DFT calculations were performed by Dr. Zhen (Allen) Xu. Variable Temperature NMR experiments were performed by myself with assistance from Dr. Maria B. Ezhova. The contents of Chapter 4 are included in a manuscript in preparation, written by myself with contributions from M. O. Wolf. Transient absorption measurements were performed by Dr. Saeid Kamal, and analyzed by myself. A version of Chapter 5 has been previously published2 as a communication written by myself with contributions from M. O. Wolf. TEM images were taken by Dr. Yiling Dai, and pXRD data collected by Anita Lam. Variable pH emission studies were performed by Kathleen Downey. A list of publications arising from work presented in this thesis: 1. Arsenault, N. E.; Xu, Z.; Wolf, M. O. Inorg. Chem., 2019, 58, 65–68. 2. Arsenault, N. E.; Downey, K. T.; Wolf, M. O. Chem Commun., 2020, 56, 5981-5984. vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ............................................................................................................................... xii List of Figures ............................................................................................................................. xiv List of Symbols and Abbreviations ........................................................................................ xxvi Acknowledgements ................................................................................................................ xxxiv Dedication .................................................................................................................................xxxv Chapter 1: Introduction ................................................................................................................1 1.1 Overview ............................................................................................................. 1 1.2 Introduction to Photophysical Processes ............................................................ 1 1.2.1 Charge Transfer Processes and Solvatochromism .......................................... 4 1.3 Photophysics of Conformational Switches ......................................................... 7 1.3.1 Organic-Based π-Conjugated Molecular Switches ......................................... 7 1.3.2 Transition Metal-Containing Molecular Switches .......................................... 9 1.4 Boron-Functionalized Emissive Materials ........................................................ 10 1.4.1 Emissive Properties of Three-Coordinate Triarylborane Materials .............. 10 1.4.2 Stimuli-Responsive Borane-Functionalized Materials ................................. 11 1.5 Lewis Pair Chemistry ........................................................................................ 13 1.5.1 Lewis Adducts .............................................................................................. 13 viii 1.5.2 Frustrated Lewis Pairs................................................................................... 14 1.5.3 Flexible Lewis pairs (FlexLPs) ..................................................................... 15 1.5.3.1 Phosphine Oxide Boron Mesityl FlexLP System ................................... 18 1.6 Goals and Outline of Thesis .............................................................................. 21 Chapter 2: Photophysical Properties of a Flexible Lewis Pair Dimer Synthesized Using Alkyne-Alkyne Coupling Chemistry ..........................................................................................24 2.1 Introduction ....................................................................................................... 24 2.1.1 Dimeric Stimuli-Responsive Molecules ....................................................... 24 2.1.2 Goals ............................................................................................................. 28 2.2 Synthesis and Characterization ......................................................................... 30 2.3 Photophysical Data ........................................................................................... 34 2.3.1 Absorption and Emission Properties of FlexLP-TBS and Dimer ................. 34 2.3.2 Absorption and Emission Properties of the Monomer and Dimer Molecule in THF-MeOH Solvent Gradients ................................................................................. 39 2.3.3 Emission Lifetime Data of FlexLP-TBS and Dimer Molecule .................... 42 2.4 Synthetic Attempts to Increase Solubility of FlexLP Dimer ............................ 47 2.5 Conclusion ........................................................................................................ 51 2.6 Experimental Details ......................................................................................... 52 2.6.1 General Procedures ....................................................................................... 52 2.6.2 Synthesis of New Compounds ...................................................................... 53 Chapter 3: Solvent- and Temperature-Responsive Platinum(II)-Functionalized Flexible Lewis Pairs ....................................................................................................................................61 3.1 Introduction ....................................................................................................... 61 ix 3.1.1 Platinum(II)-Containing Molecular Switches ............................................... 61 3.1.2 Metal-Functionalized FlexLP Systems ......................................................... 63 3.2 Synthesis and Characterization of Pt(II)-FlexLP Complexes ........................... 65 3.3 Room Temperature Photophysical Characterization of Pt(II)-FlexLP Complexes ..................................................................................................................... 68 3.4 Computational Data .......................................................................................... 72 3.5 Variable Temperature Photophysical Characterization .................................... 75 3.6 Conclusion ........................................................................................................ 78 3.7 Experimental Details ......................................................................................... 78 3.7.1 General .......................................................................................................... 78 3.7.2 Synthetic Procedures of New Molecules ...................................................... 80 Chapter 4: Singlet and Triplet Energy Dynamics of Emissive Platinum-FlexLP Complexes .....................................................................................................................................89 4.1 Introduction ....................................................................................................... 89 4.1.1 Triplet State Properties of Platinum(II) Complexes ..................................... 89 4.1.1.1 Differences between Singlet and Triplet Excited State Localization ..... 90 4.1.1.2 Emissive and Non-Emissive Triplet Decay Rates of Metal Complexes 91 4.1.2 Photophysics of Pyrene ................................................................................. 95 4.1.3 Purpose .......................................................................................................... 97 4.1.4 Transient Absorption Spectroscopy .............................................................. 99 4.2 Synthesis and Characterization of Emissive Platinum(II) FlexLP Complexes ………….. ................................................................................................................... 101 4.3 Photophysical Data ......................................................................................... 102 x 4.3.1 Absorption Spectroscopy Measurements .................................................... 102 4.3.2 Emission Spectroscopy Measurements ....................................................... 104 4.4 Transient Absorption Spectra ......................................................................... 108 4.4.1 Picosecond Transient Absorption Spectroscopy Measurements ................ 108 4.5 Conclusion ...................................................................................................... 115 4.6 Experimental Details ....................................................................................... 117 4.6.1 General Synthetic Procedures and Characterization ................................... 117 4.6.2 Synthesis of New Complexes ..................................................................... 118 4.6.3 Additional Spectra ...................................................................................... 120 Chapter 5: Environment-Responsive Flexible Lewis Pair Fluorophore-Modified Nanoparticles for Fluorescence Imaging .................................................................................123 5.1 Introduction ..................................................................................................... 123 5.1.1 Fluorescence Microscopy ........................................................................... 124 5.1.1.1 Epifluorescence Microscopy ................................................................ 125 5.1.1.2 Fluorescence Lifetime Imaging Microscopy........................................ 126 5.1.2 FlexLP Systems as Fluorescence Microscopy Dyes ................................... 128 5.2 Synthesis and Characterization of FlexLP Fluorophore and Functionalized Nanoparticles .............................................................................................................. 130 5.3 Photophysical Data ......................................................................................... 137 5.4 Concentration and pH Studies ........................................................................ 142 5.5 Fluorescence Microscopy Data ....................................................................... 146 5.6 Conclusions ..................................................................................................... 148 5.7 Experimental Details ....................................................................................... 149 xi 5.7.1 Synthetic Procedures ................................................................................... 149 5.7.1.1 Synthesis of new compounds ............................................................... 149 5.7.1.2 Synthesis of nanoparticles .................................................................... 152 5.7.2 Structural Characterization Techniques ...................................................... 153 5.7.3 Photophysical Measurements and Microscopy Techniques ....................... 154 5.7.4 Additional Emission Lifetime Data ............................................................ 155 Chapter 6: Conclusions and Future Work ..............................................................................157 Bibliography ...............................................................................................................................160 Appendix: Proton NMR Spectra of Select New FlexLP-Containing Molecules ..................172 xii List of Tables Table 2-1. FlexLP-TBS and dimer lifetime and percent contribution (in parenthesis) data with varying ratio of THF:MeOH solvent system. ............................................................................... 46 Table 3-1. Comparison of absorption data of complex Pt-FlexLP and Pt-FlexLP-Pt with (OOC-tpy)Pt-T and (OOC-tpy)Pt-T2. ...................................................................................................... 71 Table 3-2. Calculated dihedral angles (°) and orbital energies (eV) of complex Pt-FlexLP and Pt-FlexLP-Pt. ..................................................................................................................................... 73 Table 3-3. Excited states of complex Pt-FlexLP and Pt-FlexLP-Pt in the open and closed forms calculated at the TD-DFT B3LYP/(6-31G**). Oscillator strengths for calculated excited states are reported in square brackets. .................................................................................................... 75 Table 4-1. Emission lifetime data for Pt(FlexLP)2, Pt(FlexLP)(Py), and FlexLP-TBS in acetone and MeOH. .................................................................................................................................. 107 Table 4-2. Overall transient absorption lifetimes of both complexes in acetone and MeOH, in air and inert atmosphere, from 440 –730 nm. Parentheses indicate the percent contribution to the overall lifetime. ........................................................................................................................... 110 Table 4-3. Transient absorption decay data for specific wavelengths, including the lifetimes (τ1 and τ2, in μs) and percent contribution (in parenthesis, see section 4.7.1 for more details). ...... 112 Table 5-1. Weight % loading of NHS-FlexLP onto the nanoparticles was determined by comparing the weight % loss from TGA data. ........................................................................... 133 Table 5-2. Small angle pXRD data of mesoporous nanoparticles. ............................................. 134 Table 5-3. Emission lifetimes (ns) and relative percentage values (% in parenthesis) for NHS-FlexLP and NP-FlexLP-10 in THF and MeOH .......................................................................... 138 xiii Table 5-4. Emission lifetimes (ns) and relative percentage (% in parenthesis) values of NP-FlexLP-10 in varying pH environments. .................................................................................... 146 Table 5-5. Emission lifetime values (ns) and relative percentage values (% in parenthesise) of NP-FlexLP samples in dioxane-water mixed solvent environments. ......................................... 155 Table 5-6. Emission lifetime values (ns) and relative percentage values (% in parenthesis) of NP-FlexLP samples at three different concentrations in 50:50 dioxane-water mixed solvent environment. ............................................................................................................................... 156 xiv List of Figures Figure 1-1. Simplified Jablonski energy diagram with different energy transitions. ..................... 2 Figure 1-2. Solvatochromism effect on the absorption wavelength of CT transitions. .................. 5 Figure 1-3. Effect of surrounding solvent (small circles) polarity on the excited states of an emissive molecule (grey circle) with a stronger dipole moment (black arrow) in the excited state. Figure adapted from Ref.15 ............................................................................................................. 6 Figure 1-4. Conformational switching in dithienylethenes and spiropyrans using light or heat. ... 7 Figure 1-5. Effect of increasing conjugation length on the HOMO-LUMO energy gap in organic molecular systems. .......................................................................................................................... 9 Figure 1-6. Overlapping of π orbitals from pendent aryl groups with the empty p orbital on the boron center leads to CT transitions. Electron donating or accepting groups can significantly change the CT energy and photophysical properties. ................................................................... 10 Figure 1-7. Dimesitylborane attached to a bithiophene backbone with varying functional groups reported by the Yamaguchi group.28. The emission red-shifts with increasing conjugation and electron donating functional groups. ............................................................................................. 11 Figure 1-8. Switching between three- and four-coordinate boron center changes the availability of a CT event to the boron center to occur. ................................................................................... 12 Figure 1-9. Addition of fluoride anions induces increased blue emission from increased CT to the naphthyl linker in a boron-functionalized molecule.30 ................................................................. 13 Figure 1-10. Common Lewis adduct and FLP molecular structure (a) showing the sterically bulky groups around the Lewis pairs which prevent Lewis adduct formation, and early examples of H2 activation by FLPs (b).37,38 .................................................................................................. 14 xv Figure 1-11. Small molecule FlexLP systems containing boron Lewis acid functional groups... 17 Figure 1-12. Polymeric systems containing FlexLP groups. ........................................................ 18 Figure 1-13. Previously studied bithiophene-based FlexLP incorporating diphenylphosphine oxide Lewis base and dimesitylboron Lewis acid, with accompanying HBD strength values of commonly used solvents.48,50 ........................................................................................................ 19 Figure 1-14. One-pot synthesis of FlexLP groups from a bithiophene backbone with R groups of different conjugation length and electron donating strength.52 ..................................................... 20 Figure 1-15. General scheme of symmetrical and non-symmetrical alkyne-functionalized FlexLP, and the possible FlexLP motifs achieved through terminal alkyne coupling reactions with different functional groups (R). ............................................................................................ 22 Figure 2-1. Binding cooperativity in enzyme-susbtrate pairs, where the enzyme has two binding sites. .............................................................................................................................................. 25 Figure 2-2. Robin-Day classification describing the mixed valency of the homonuclear dimetallic complexes, and the corresponding degree of electronic communication between metal centers. 26 Figure 2-3. Pt complex containing two DTE ligands which can switch between fully open and fully closed conformations through a closed-open intermediate that can be spectroscopically observed.25 .................................................................................................................................... 27 Figure 2-4. Boron-thiadiazole FlexLP exhibiting negative cooperativity characteristics, where binding of one boron-nitrogen pair inhibits the binding affinity of the second boron-nitrogen pair.42 ............................................................................................................................................. 28 Figure 2-5. Eglinton (a), Glaser (b), and Cadiot-Chodkiewicz (c) alkyne-alkyne coupling reactions. ....................................................................................................................................... 28 xvi Figure 2-6. Schematic of FlexLP-TBS 24 and FlexLP dimer 25 in equilibrium between the three states. ............................................................................................................................................. 29 Figure 2-7. 1H NMR spectra of compound FlexLP-TBS 24 (red) and doubly deprotected FlexLP 27 (black) in CDCl3 showing the terminal acetylene protons....................................................... 30 Figure 2-8. HSQC (top) and HMBC (bottom, purple) spectra with HSQC (red) overlap of FlexLP-TBS in CDCl3. Starred peak is residual solvent. ............................................................. 31 Figure 2-9. Schematic depicting the synthesis of dimer 25, using Glaser coupling reaction conditions. ..................................................................................................................................... 33 Figure 2-10. 31P{1H} NMR spectra of FlexLP-TBS and dimer in varying deuterated solvents. . 34 Figure 2-11. Absorption (a) and emission (b) spectra of the dimer and FlexLP-TBS in various solvents. ........................................................................................................................................ 35 Figure 2-12. Schematic of possible open and closed forms of the dimer and the areas where the HOMO and LUMO MOs reside (indicated in colour). Blue highlight indicates potential HOMO-LUMO π-π* transition, where red highlight indicates potential CT transition. ........................... 36 Figure 2-13. Schematic depicting strong vibronic transitions of bithiophene dimer control molecule, 28. ................................................................................................................................. 37 Figure 2-14. Absorption and emission profiles comparing the FlexLP dimer 25 with the control dimer 28 without Lewis acid-base pairs (1 x 10-6 M solutions, emission/excitation spectra taken in THF solvent using λex = 415 nm (28) and λex = 450 nm (dimer 25) ). ..................................... 38 Figure 2-15. Absorbance spectra of FlexLP-TBS (a) and dimer (b) in mixed THF:MeOH solvent ratios, with increasing % MeOH (5 × 10-6 M (FlexLP-TBS) and 1 × 10-6 M (dimer) solutions used). ............................................................................................................................................. 39 xvii Figure 2-16. Absorbance at 475 nm, assumed to be absorption of solely closed-closed form of the dimer with increasing MeOH (1 × 10-6 M). ............................................................................ 40 Figure 2-17. Emission spectra of FlexLP-TBS (a, λex = 335 nm, 5 × 10-6 M) and dimer (b, λex = 345 nm, 1 × 10-6 M) in mixed THF:MeOH solvent (% MeOH by volume indicated). Above are shown the respective samples excited with a 365 nm UV handlamp, showing the changing emission colour from left to right with increasing MeOH. ........................................................... 41 Figure 2-18. Emission lifetime graphs of FlexLP-TBS in THF (a) and MeOH (b) solvents. ...... 43 Figure 2-19. Emission lifetime graphs of 29, in THF and MeOH solvents. No significant change in emission lifetime is seen. .......................................................................................................... 44 Figure 2-20. Emission lifetime decay curves of dimer, comparing the fitting of 3 exponentials (a) to 2 exponentials (b)...................................................................................................................... 45 Figure 2-21. Relative percent contributions of emission lifetimes with varying THF:MeOH solvent ratio for FlexLP-TBS (a) and dimer (b). No clear trend could be seen between percent contribution of all lifetimes and % MeOH for the dimer.............................................................. 47 Figure 2-22. Scheme of synthetic attempts to add solubilizing OEG groups to FlexLP molecules........................................................................................................................................................ 48 Figure 2-23. Scheme of synthetic attempts to add solubilizing ethylene glycol oligomer groups to either side of a FlexLP molecule. ................................................................................................. 49 Figure 2-24. Attempted synthesis of an OEG-functionalized FlexLP dimer (9), and successful synthesis of FlexLP2 dimer with terminal alkynes (10). ............................................................... 50 Figure 2-25. Full synthetic scheme of FlexLP-TBS and FlexLP dimer. ...................................... 53 Figure 2-26. Synthetic scheme for the synthesis of control dimer 28 without functionalization of Lewis pairs .................................................................................................................................... 58 xviii Figure 3-1. Dithienylethene-functionalized Pt-tpy complex which switches from open to closed form by excitation of the Pt-tpy chromophore.87 .......................................................................... 62 Figure 3-2. Bimetallic Pt-tpy complex with a terthiophene linker switches conformation with surrounding solvent.88 ................................................................................................................... 62 Figure 3-3. Previous metal-containing FlexLP systems.101,102 The open and closed forms of both complexes were observed by variable temperature NMR, where it was shown that the complexes respond with temperature. ............................................................................................................. 64 Figure 3-4. Structures of monometallic Pt-FlexLP and bimetallic Pt-FlexLP-Pt investigated in this Chapter. .................................................................................................................................. 64 Figure 3-5. Synthesis of monometallic Pt-FlexLP and bimetallic Pt-FlexLP-Pt complexes. ....... 65 Figure 3-6. 31P{1H} NMR spectra of (a) complex Pt-FlexLP and (b) complex Pt-FlexLP-Pt in solvents of varying HBD strength at 25 °C (400 MHz). .............................................................. 66 Figure 3-7. NOESY spectrum of Pt-FlexLP-Pt in THF-d8. Correlation between H10 and H12/H13 is observed (black rectangle). The starred peak is residual THF solvent. .................... 67 Figure 3-8. ROESY spectrum of Pt-FlexLP-Pt in CD3OD. Correlation between H10 and H12/H13 is not observed. ............................................................................................................. 68 Figure 3-9. Control complexes Pt-T2 and Pt-T2-Pt. ...................................................................... 69 Figure 3-10. Absorption spectra of complexes Pt-FlexLP (a), Pt-FlexLP-Pt (b), Pt-T2 (c), and Pt-T2-Pt (d) in solvents with varying HBD strength (1.0 × 10-5 M). The LLCT/LMCT band is slightly blue-shifted in ACN due to the increased polarity compared to acetone and MeOH. Complexes Pt-T2 and Pt-T2-Pt did not dissolve in THF. .............................................................. 70 Figure 3-11. Complexes (OOC-tpy)Pt-T and (OOC-tpy)Pt-T2 synthesized and characterized by the Yam group.106.......................................................................................................................... 70 xix Figure 3-12. Optimized geometry of complex Pt-FlexLP and Pt-FlexLP-Pt in open and closed forms. The B-O bond length of both Pt-FlexLP and Pt-FlexLP-Pt in the closed form is calculated to be 1.63 Å. .................................................................................................................................. 72 Figure 3-13. Complex Pt-FlexLP orbital projections of the HOMO (left), LUMO (middle), and LUMO+1 (right) energy levels in the open (top) and closed (bottom) forms. ............................. 74 Figure 3-14. Complex Pt-FlexLP-Pt orbital projections of the HOMO (left), LUMO (middle), and LUMO+1 (right) energy levels in the open (top) and closed (bottom) forms. ...................... 74 Figure 3-15. 31P NMR{1H} of complex Pt-FlexLP-Pt in CD3OD with decreasing temperature. The peak at 30 ppm shifts downfield and broadens with decreasing temperature, until -35 °C. At -45 °C the peak narrows and becomes increasingly sharp due to decreased vibration and rotation in the molecule. ............................................................................................................................. 76 Figure 3-16. Variable temperature absorption spectra of complex Pt-FlexLP-Pt (a-b, 5 × 10-6 M) and Pt-T2-Pt (c-d, 1 × 10-5 M) in MeOH/EtOH (1:4 v/v) (a,c), and in acetone (5 × 10-6 M) (b,c)........................................................................................................................................................ 77 Figure 3-17. Full synthetic scheme of Pt-FlexLP and Pt-FlexLP-Pt complexes. ......................... 80 Figure 3-18. Full synthetic scheme of Pt-T2 and Pt-T2-Pt complexes. ......................................... 81 Figure 4-1. Energy diagram describing singlet oxygen production. Ground state dissolved oxygen, which exists in the triplet form, collides with photosensitized molecules in their triplet state, quenching the photosensitized molecule and producing singlet oxygen. ............................ 90 Figure 4-2. Examples of Pt-aryl polymers previously studied (a) and the singlet and triplet (b) excited state localizations along the polymer backbone.120–122 ..................................................... 91 Figure 4-3. Energy diagram of zero-field splitting of triplet energy substates. ............................ 92 xx Figure 4-4. Energy diagrams depicting temperature-independent (a) and temperature-dependent (b) non-radiative decay pathways. ................................................................................................ 94 Figure 4-5. Schematic of oxygen-induced enhanced intersystem crossing in pyrene. ................. 95 Figure 4-6. Phosphorescent pyrene-functionalized Pt(II) complexes, with varying emission decay rates. An intermediate 3MLCT state is thought to increase the rate of ISC to the pyrene-localized T1.132 .............................................................................................................................. 97 Figure 4-7. Structure of emissive Pt(FlexLP) complexes investigated in this Chapter. ............... 98 Figure 4-8. Simplified diagram comparing the MC d orbital energies in Pt-tpy and Pt-phosphine complexes. .................................................................................................................................... 99 Figure 4-9. Schematic of transient absorption spectroscopy depicting the theory (a), and its use for assigning excited state transitions (b-d), including the experimental set-up (b) in relation to the excitation of a population of molecules (c), and the resulting transient absorption graph (d)...................................................................................................................................................... 100 Figure 4-10. Synthesis of Pt(FlexLP)2 and Pt(FlexLP)(Py) complexes. ..................................... 102 Figure 4-11. Absorption spectra of Pt(FlexLP)2 and Pt(FlexLP)(Py) complexes, and FlexLP-TBS in acetone (a), and MeOH (b). .................................................................................................... 103 Figure 4-12. Emission spectra of Pt(FlexLP)2 (2 × 10-6 M, λex(acetone) = 405 nm, λex(MeOH) = 335 nm) and Pt(FlexLP)(Py) (2 × 10-6 M, λex(acetone) = 395 nm, λex(MeOH) = 385 nm) complexes, and FlexLP-TBS (5 × 10-6 M, λex(acetone) = 390 nm, λex(MeOH) = 335 nm), in acetone (blues) and MeOH (pinks). Emission spectra of FlexLP-TBS and Pt(FlexLP)2 in MeOH were stopped at 650 nm to prevent detection of excitation wavelength due to Bragg’s Law. ............................................... 104 Figure 4-13. Emission spectra of Pt(FlexLP)(Py) under air and inert atmosphere (2 × 10-6 M, λex = 335 nm). ................................................................................................................................... 105 xxi Figure 4-14. Transient absorption decay curves of complexes in air (a) and inert (b-d) atmosphere in acetone (b) and MeOH (c,d). In air, all complexes fully decay monoexponentially after 2 μs, while in inert atmosphere the complexes decay biexponentially over longer timescales...................................................................................................................................................... 110 Figure 4-15. Transient absorption decay curves for Pt(FlexLP)2 in acetone (a) and MeOH (c) and Pt(FlexLP)(Py) in acetone (b) and MeOH (d) analyzed at specific wavelengths. Higher energy wavelength (450 nm, blue) has a slightly longer decay time than lower energy (red) wavelengths...................................................................................................................................................... 111 Figure 4-16. Simplified diagram of the triplet state energy transitions between open and closed conformation. .............................................................................................................................. 113 Figure 4-17. Schematic of possible resonance structures for the open form of Pt(FlexLP)2. The second resonance structure (middle) undergoes the same arrow pushing mechanism to afford the same negatively charged FlexLP ligand, but was not shown for simplicity. Similar resonance structures for Pt(FlexLP)(Py) are also possible when the FlexLP ligand is in the open form. ... 114 Figure 4-18. Energy diagram depicting the IC from pyrene to FlexLP triplet states in Pt(FlexLP)(Py). ........................................................................................................................... 115 Figure 4-19. Emission decay spectra for Pt(FlexLP)2 under air (a) and argon (b) in acetone and MeOH solvents. .......................................................................................................................... 120 Figure 4-20. Emission decay spectra of Pt(FlexLP)(Py) in acetone and MeOH, under air and argon atmosphere. ....................................................................................................................... 121 Figure 4-21. Transient absorption spectra of Pt(FlexLP)2 (a, c) and Pt(FlexLP)(Py) (b, d) in deaerated acetone (a,b) and deaerated MeOH (c, d) at different delay times. ............................ 122 xxii Figure 5-1. Schematic of an inverted epifluorescence microscope, equipped with a blue cut-off excitation filter and green band-pass emission filter. Though the specimen emits across the red and green visible light range, the green band-pass emission filter prevents red light from hitting the detector, and therefore the fluorescence image depicts a green sample. .............................. 125 Figure 5-2. (a) Structures of FlexLP dyes investigated and (b) schematic and (c) fluorescence microscopy image of adipocyte tissue showing yellow emission from FlexLP-(NMe3)22+ dye on the periphery of lipid droplets, and blue emission from FlexLP-(Bu)2 dye inside the lipid droplets (scale bar = 100 μm) (© Y. Cao, 2016, used with permission from Ref).157 .............................. 129 Figure 5-3. (a) Schematic of brain neuron and (b) fluorescence microscopy image of fixed brain slices showing the cross-section of axons stained with FlexLP-(NMe2)2 (scale bar = 20 μm) (© Y. Cao, 2016, used with permission).157 ..................................................................................... 130 Figure 5-4. Synthetic route to FlexLP-functionalized mesoporous silica nanoparticles, NP-FlexLP. ........................................................................................................................................ 131 Figure 5-5. TGA spectra comparing weight % loss of various samples with increasing temperature. ................................................................................................................................ 132 Figure 5-6. Small angle pXRD spectra of nanoparticle samples. ............................................... 133 Figure 5-7. TEM image of unfunctionalized mesoporous silica nanoparticle, NP (a), and NP-NH2 (b) samples. ................................................................................................................................. 134 Figure 5-8. TEM image of NP-FlexLP-3 (a), NP-FlexLP-5 (b), and NP-FlexLP-10 (c) samples...................................................................................................................................................... 134 Figure 5-9. FT-IR spectra of NP, NP-NH2, NHS-FlexLP, and NP-FlexLP-10 samples. Inset shows a zoomed-in region highlighting the amide carbonyl C=O peak at 1650 cm-1 for Np-xxiii FlexLP-10 sample. NHS-FlexLP trace intensity in the insert was reduced by a factor of 5 to easily compare peak wavenumbers. ............................................................................................ 135 Figure 5-10. UV-Vis absorption spectra (a) of NP-FlexLP-10 (0.6 mg / 10 mL THF) and NP+FlexLP control sample 70 (0.6 mg / 10 mL THF). UV-Vis absorption spectra (b) of FlexLP “leached” out of NP-FlexLP-10 after 1 day in THF. Inset shows a vial of NP-FlexLP-10 sample after 5 days in THF. Emission of the nanoparticles shows they have settled to the bottom of the vial, and no observable free FlexLP has leached into the surrounding solvent. Refracted emission can be seen at the meniscus. ....................................................................................................... 136 Figure 5-11. UV-Vis absorption spectra of NHS-FlexLP (4 x 10-6 M) (a) and of NP-FlexLP samples (b, 5 x 10-5 M FlexLP added) in THF (normalized at maximum peak wavelength) and MeOH (normalized at 325 nm) solvent. ..................................................................................... 137 Figure 5-12. Normalized emission spectra (a) of NHS-FlexLP and NP-FlexLP samples in THF (λex = 400 nm) and MeOH (λex = 325 nm) solvent. Emission lifetime (b) graphs of NHS-FlexLP and NP-FlexLP-10 samples in THF and MeOH, with overlapping fitted traces in corresponding colours. ........................................................................................................................................ 138 Figure 5-13. Emission spectra of NP-FlexLP-3 in dioxane with increasing % by volume of water (λex = 410 nm, left) (λex = 325 nm, right). Spectra were normalized at 515 nm, which is the wavelength where the emission peaks from the open and closed structure of free NHS-FlexLP intersect. ...................................................................................................................................... 139 Figure 5-14. Emission spectra of NP-FlexLP-5 in dioxane with increasing % by volume of water (λex = 410 nm) (λex = 325 nm, right). Spectra were normalized at 515 nm. ............................... 140 Figure 5-15. Emission spectra of NP-FlexLP-10 in dioxane with increasing % by volume of water (λex = 400 nm) (λex = 325 nm, right). Spectra were normalized at 515 nm. ..................... 140 xxiv Figure 5-16. Emission lifetime graphs of NP-FlexLP-3 (left), NP-FlexLP-5 (middle), and NP-FlexLP-10 (right) in dioxane with increasing water content (% by volume). ............................ 141 Figure 5-17. Relative percentages of long emission lifetime component of NP-FlexLP samples in dioxane-water mixed solvent ratios. ....................................................................................... 142 Figure 5-18. Emission spectra of NP-FlexLP-3 (a), -5 (b), and -10 (c) samples in 50:50 dioxane:water (v/v), with varying concentrations of sample (mg/10 mL) excited at λex = 325 nm. Spectra were normalized at 515 nm. Variations in the ratio of open to closed form is predominantly affected by the weight percent loading of NHS-FlexLP, not concentration. Emission lifetime spectra of NP-FlexLP-3 (d), -5 (e), and -10 (f) samples in 50:50 dioxane:water (v/v), with varying concentrations of sample (mg/10 mL). ........................................................ 143 Figure 5-19. Relative percentages of the long emission lifetime component versus short emission lifetime component for NP-FlexLP samples of different concentrations in 50:50 (v/v) dioxane-water mixed solvent environments. .............................................................................. 144 Figure 5-20. Normalized emission spectra (λex = 325 nm) (a) and emission lifetime graphs (b) of NP-FlexLP-10 in different buffer solutions of varying pH. Spectra were normalized 515 nm, the wavelength where the emission spectra of open and closed forms intersect. ............................. 145 Figure 5-21. Relative percentage (%) of short emission lifetime and long emission lifetime of NP-FlexLP-10 plotted against changing pH. .............................................................................. 145 Figure 5-22. Fluorescence microscopy images of NP-FlexLP-10 in dioxane (a) and water (b)...................................................................................................................................................... 147 Figure 5-23. Fluorescence microscopy image of NP-FlexLP-10 in dioxane (a), and separated red (b), green (c), and blue (d) channels showing the relative emission intensity of the three channels. Scale bar = 129 μm. .................................................................................................................... 147 xxv Figure 5-24. Fluorescence microscopy image of NP-FlexLP-10 in water (a), along with separated red (b), green (c), and blue (d) channels showing the relative emission intensity of the three channels. Scale bar = 129 μm. .................................................................................................... 148 Figure A-1. 1H NMR spectrum of complex FlexLP-TBS 24 (CDCl3, 400 MHz, 25 °C). ......... 172 Figure A-2. 1H NMR spectrum of complex FlexLP dimer 25 (CDCl3, 400 MHz, 25 °C). Starred peaks are solvent residue. ........................................................................................................... 172 Figure A-3. 1H NMR spectrum of complex Pt-FlexLP 45 (CDCl3, 400 MHz, 25 °C). Insert is of aromatic region. .......................................................................................................................... 173 Figure A-4. 1H NMR spectrum of complex Pt-FlexLP-Pt 46 (CDCl3, 400 MHz, 25 °C). Insert is of aromatic region. ...................................................................................................................... 173 Figure A-5. 1H NMR spectrum of Pt(FlexLP)2 61 (CD2Cl2, 400 MHz, 25 °C). ........................ 174 Figure A-6. 1H NMR spectrum of Pt(FlexLP)(Py) 62 (CD2Cl2, 400 MHz, 25 °C). ................... 174 Figure A-7. 1H NMR spectrum of NHS-FlexLP 66 (CDCl3, 400 MHz, 25 °C). ........................ 175 xxvi List of Symbols and Abbreviations * excited state 1 singlet state 2D two-dimensional 3 triplet state 1H proton 13C carbon-13 31P phosphorus-31 31P{1H} phosphorus-31 proton decoupled Å Angström A amplitude ACN acetonitrile ATR attenuated total reflection BBB blood-brain barrier Bu butyl δ chemical shift (ppm) ºC degrees Celsius cat catalyst or catalytic amount CCD charge-coupled device CDCl3 deuterated chloroform CD2Cl2 deuterated dichloromethane CD3CN deuterated acetonitrile xxvii CD3OD deuterated methanol cm–1 wavenumber CT charge transfer CuAAC copper azide-alkyne click reaction Δ difference or heat ΔOD optical density difference d doublet splitting DCM dichloromethane DFT density functional theory DMF dimethylformamide DNA deoxyribonucleic acid DTE dithienylethene E entgegen isomer E energy EA elemental analysis EDC N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide EDG electron-donating group EPR enhanced permeability and retention ESI electrospray ionization Et2O diethyl ether EtOH ethanol e- electron eqiv. equivalent xxviii eV electron volts EWG electron-withdrawing group FlexLP flexible Lewis pair FLIM fluorescence lifetime imaging microscopy FLP frustrated Lewis pair FRET Förster resonance energy transfer fs femtosecond (10–15 seconds) FT-IR Fourier transform infrared spectroscopy g grams GFP green fluorescent protein GS ground state h hours HBD hydrogen bond-donating HMBC heteronuclear multiple-bond correlation spectroscopy HOMO highest occupied molecular orbital HRMS high resolution mass spectrometry HSQC heteronuclear single-quantum correlation spectroscopy Hz hertz I emission intensity IC internal conversion i-Pr iso-propyl IR infrared ISC intersystem crossing xxix J coupling constant K Kelvin kS21 rate constant of energy transfer from S2 to S1 kS10 decay rate constant from S1 to S0 kT10 decay rate constant from T1 to S0 kT12 rate constant of energy transfer from T1 to T2 kISC rate constant of intersystem crossing knr non-radiative decay rate constant kr radiative decay rate constant kV kilovolts λ wavelength λem wavelength of emission λex wavelength of excitation LA Lewis acid LB Lewis base LC ligand-centered LLCT ligand-to-ligand charge transfer LMCT ligand-to-metal charge transfer LUMO lowest unoccupied molecular orbital µs microsecond m multiplet m/z mass-to-charge ratio M molar xxx MALDI matrix-assisted laser desorption/ionization MC metal-centered Me methyl MECP minimum energy crossing point MeOH methanol Mes mesityl MHz megahertz (106 Hz) min minutes mL milliliter MLCT metal-to-ligand charge transfer MO molecular orbital mol mole MS mass spectrometry n population of molecules NanoLED nano-light emitting diode n-BuLi n-butyllithium NHS succinimidyl ester nm nanometer NMR nuclear magnetic resonance nOe nuclear Overhauser effect NOESY nuclear Overhauser effect spectroscopy ns nanosecond (10–9 seconds) OAc acetate xxxi OEG ethylene glycol oligomer OLED organic light-emitting diode OTf triflate PCM polarized continuum model PDMS polydimethylsiloxane Ph phenyl ppm parts per million ps picosecond (10–12 seconds) PS polystyrene PTI Photon Technology International pXRD powder X-ray diffraction Py pyrene Φ quantum yield q quintet Ref reference rpm rotations per minute RT room temperature ROESY rotating frame nuclear Overhauser effect spectroscopy S0 ground-state S1 first excited singlet state S1′ Frank-Condon excited state S2 second excited singlet state s singlet peak xxxii SCRF self-consistent reaction field SOC spin-orbit coupling SPS solvent purification system ϴ angle of diffraction t triplet or time τ lifetime T temperature T1 first excited triplet state TADF thermally activated delayed fluorescence TBAF tetrabutylammonium fluoride TBS tert-butyldimethylsilyl tBu tert-butyl TCI Tokyo Chemical Industry Co., Ltd. TCSPC time-correlated single photon counting TD-DFT time-dependent density functional theory TEM transmission electron microscopy TES triethylsilyl Tg glass transition temperature TGA thermogravimetric analysis THF tetrahydrofuran TMEDA tetraethylenediamine TMS trimethylsilyl TOF time of flight xxxiii tpy 2,2′:6′,2′′-terpyridine UBC University of British Columbia UV ultraviolet v/v volume/volume Vis visible W watts w/w weight/weight XRD X-ray difraction ZFS zero-field splitting Z zusamenn isomer xxxiv Acknowledgements First, I would like to thank my supervisor Mike Wolf for his guidance and support throughout the years. Thank you for encouraging my ideas and helping transform them into meaningful pursuits, and keeping me motivated to stay on track to achieve my goals. Good stuff! Thank you to the faculty on my supervisory committee for your guidance and interest in my research, especially Zac Hudson for being my reader and offering suggestions. Thank you to the staff I have had the pleasure of working with at UBC, including Marshall, Ken, and Raz, for always being so friendly and available to fix any problems. Thank you to Maria and Paul in the NMR facility, Anita in the Crystallography facility, and Elena and Jessie in the Biological Services facility, for their training and help with my research. I would also like to thank Saeid Kamal in the LASIR lab for his immense support and always having time to try another experiment these last few months. To all my colleagues in the Wolf group over the years, thank you for making this experience so enjoyable. Special thanks to Yang for your mentorship, training, and continued guidance throughout the years, including laying the groundwork for a lot of the chemistry in this thesis. Thank you to Katy for your amazing motivation and spirit as a summer undergraduate student. To all of my senior Wolf members, thank you for always being there to help whenever I stumbled with my chemistry or whenever a pitcher of T-bird needed finishing. To all the current Wolf members, thank you for being the encouraging, supportive bunch that you are, and for making the last couple of years as enriching as the first. Lastly, I would like to thank my mom and dad, for their unconditional love, and Keegan, for his patience and support no matter the time or distance. xxxv Dedication To Mom and Dad 1 Chapter 1: Introduction 1.1 Overview The ability to control the movement of atoms at the molecular scale to elicit a macroscale response is an active challenge that may be solved by using stimuli-responsive materials. Stimuli-responsive materials are those which undergo a response to an external stimulus that is applied. Common types of stimuli include physical stimuli such as temperature, pressure, light, or voltage, or chemical stimuli such as pH or other small molecules. The responses are usually easily measured, such as a change in colour, emission, rigidity, or elasticity. Due to the large variety of controllable stimuli that can be applied, and range of responses that can be observed, stimuli-responsive materials have been utilized in many fields of chemistry, such as in the application of biological imaging and drug delivery,1–5 the design of smart coatings and self-healing polymers,6–9 and the development of anti-counterfeit technologies.10–12 This thesis aims to investigate one such stimuli-responsive molecule incorporating a Lewis acid and base pair, where manipulation of the molecular conformation by external stimuli induces a change in photophysical properties, often observable by eye. This molecule has been incorporated into metal complexes, organic dimeric systems, and nanomaterials, and the photophysical properties studied. 1.2 Introduction to Photophysical Processes When a molecule becomes exposed to electromagnetic radiation (photons) of certain wavelengths, many physical processes can occur. These processes are shown below with a classical Jablonski diagram, first illustrated by Aleksander Jablonski in 1933 (Figure 1-1).13 For 2 an isolated molecule in its lowest energy state (ground state), radiative excitation (absorption) of an electron to a higher electronic state is often the first photophysical process to occur, if the photons are of appropriate energy. Absorption events occur readily between energy states of the same multiplicity (10-15 seconds) because this is a spin-allowed transition. Therefore, for molecules which have a singlet ground state multiplicity (S0), absorption occurs to a higher singlet excited state (S1, S2, etc.). Figure 1-1. Simplified Jablonski energy diagram with different energy transitions. Once in the singlet excited state, a number of processes can occur, ultimately in effort to return the excited electron back to the singlet ground state. One process is vibrational relaxation between vibronic energy states within the same electronic state, which occurs readily (10-14 to10-11 seconds). Another process is internal conversion (IC) between singlet excited states, where the transition occurs between isoelectronic vibration levels between a higher excited state (S2) to a 3 lower excited state (S1), also on a fast timescale (10-14 to 10-9 seconds). Both of these transition pathways are non-radiative transitions, because a photon is not released during the process. Vibrational relaxation and IC occur on similar timescales where the excited state electron decays to the lowest vibrational level of the lowest excited state (S1) before proceeding with other photophysical processes. From the S1 state, radiative (fluorescence) or non-radiative decay can occur. Non-radiative decay is more efficient for molecules with many vibrational modes, however for rigid structures, such as conjugated materials, fluorescence where decay occurs with the emission of a photon, is possible. Because fluorescence from the S1 state to the S0 ground state is a spin-allowed transition, the timescale between initial absorption and fluorescence is relatively fast (10-10 to 10-7 seconds). In some cases, transition from the S1 state to an isoenergetic excited triplet state (T1, T2, etc.) can occur through intersystem crossing (ISC). Decay to the ground state through non-radiative decay or phosphorescent emission can occur. ISC to the triplet state is generally a spin-forbidden transition because it requires a change in spin multiplicity of the excited-state electron. Therefore, phosphorescence occurs less readily and on a longer timescale (10-6 to 102 seconds). The probability of ISC and phosphorescence occurring can be increased by minimizing vibrational relaxation by decreasing the temperature, or by incorporating a heavy atom, such as a transition metal, into the molecular system. The latter is known as the “heavy atom effect”, where the increased size of the transition metal nucleus increases the spin-orbit coupling effect between its nuclear magnetic field and neighboring electrons.14 This increased spin-orbit coupling effect can induce a spin flip of the excited state electron, thus inducing ISC. Once in the triplet excited state, the electron can radiatively decay to the ground state through phosphorescence. 4 1.2.1 Charge Transfer Processes and Solvatochromism The physical and chemical properties of a molecule can be greatly influenced by the location within the molecule that electronic transitions occur. For many fluorescent -conjugated organic molecules, the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) are located in the same π-conjugated portion of the molecule, and thus fluorescence is said to occur from the π*-π molecular orbital (MO) transition. The location of the HOMO and LUMO can be predicted through density functional theory (DFT) for simple organic molecules. Because the transition occurs in the same region of the molecule, often the vibrational wave functions involved have similar nuclear geometries, and therefore the transitions occur rapidly, with very little energy dissipation occurring between absorption and emission energy. This energy dissipation, or energy difference between absorption (excitation) and emission energy is known as the Stokes shift and can be determined by a combination of absorption and fluorescence spectroscopies. If the HOMO and LUMO reside on different areas on the molecule, the transition between the two is called a charge transfer (CT) transition. A CT transition between HOMO and LUMO occurs if there is some orbital spatial overlap between the ground state HOMO and LUMO, therefore the probability of a CT event occurring in a purely organic molecule is lower than compared to a π-π* event because of the reduced spatial overlap. However, in some organic molecules that contain highly electron accepting or electron donating functional groups, a CT event may occur. CT transitions often occur in coordination complexes, because of the strong Lewis acidic character of metals. CT transitions in coordination complexes may be classified further as either ligand-to-ligand CT (LLCT), metal-to-ligand CT (MLCT), ligand-to-metal CT (LMCT), or more 5 complex combinations. Often in coordination complexes, the CT band is lower in energy than the π-π* absorption energy transitions of the surrounding ligands and are usually weaker in intensity due to decreased probability of MO overlap between the HOMO and LUMO. Figure 1-2. Solvatochromism effect on the absorption wavelength of CT transitions. Many molecules that exhibit a CT event are solvatochromic, meaning their MO energies are affected by the surrounding solvent polarity, and thus their optical properties change based on the polarity of the solvent used. In solution, the dipole moment of the surrounding solvent changes direction to stabilize the energy of either the ground state or excited state of the molecule. With increasingly polar solvent, the difference in energy between the ground state and excited state changes more drastically (Figure 1-2). In a positive solvatochromic system, the excited state of the molecule has a greater dipole moment compared to the ground state, and therefore polar solvents will have a greater stabilizing effect on the excited state compared to the ground state. In this scenario the energy difference between the two states decreases with increasingly polar solvents, and the absorption red-shifts. In a negative solvatochromic system, the ground state is more polar than the excited state, and therefore the polarity of the solvent has a greater stabilizing 6 effect on the ground state compared to the excited state. This results in a blue-shift in absorption with increasingly polar solvents. Figure 1-3. Effect of surrounding solvent (small circles) polarity on the excited states of an emissive molecule (grey circle) with a stronger dipole moment (black arrow) in the excited state. Figure adapted from Ref.15 The time delay between excitation (10-15 seconds) and solvent reorganization through vibrational and rotational movements (10-12 to 10-10 seconds) is significant, and therefore the solvent and the excited state nuclei immediately after absorption do not significantly change in position. Consequently, the dipole moment of the solvent sphere initially around the excited state is similar to the dipole moment around the ground state, and the molecule is in an initial higher energy excited state called the Franck Condon excited state (S1’, Figure 1-3). If the lifetime of the excited state is sufficiently long, solvent reorganization can occur to stabilize the energy of the excited state, followed by emission. Thus the Stokes shift between excitation and emission 7 wavelength can be significantly larger for molecules with highly polarized excited states, such as those which exhibit CT transitions, compared to molecules with π–π* transitions which have generally less polarized states. This large Stokes shift can be useful when designing emissive materials, because it reduces spectral overlap between absorption and emission wavelengths, reducing emission quenching from intermolecular energy transfer processes or reabsorption, and increasing overall emission efficiency. 1.3 Photophysics of Conformational Switches 1.3.1 Organic-Based π-Conjugated Molecular Switches Many stimuli-responsive materials undergo a conformational change when a stimulus is applied, which causes a change in property, such as absorption or emission colour. Conformational changes can include ring formation or breaking, flipping between E- and Z- isomers, or switching between atom connectivity. For organic-based conformational switches involving extended π-conjugated frameworks, a change in length or position of the π-conjugated framework can occur upon conformational switching. Examples include dithienylethenes or spiropyrans, which undergo a ring opening or closing response upon activation (Figure 1-4).16 Figure 1-4. Conformational switching in dithienylethenes and spiropyrans using light or heat. 8 For dithienylethenes in the ring open conformation, the π-conjugated framework is mainly situated on the thiophene rings, and the absorption spectra are often similar to those of functionalized thiophene compounds.17,18 In the ring closed conformation, the π-conjugated framework extends across both thiophenes, resulting in a red-shifted absorption spectrum. For spiropyrans, the ring closed conformation is significantly twisted between the indoline and chromene moieties, so that the π-conjugated framework is localized on either moiety, and the absorption bands are at higher energies.19 Upon activation to the ring open conformation, the π-conjugated framework is delocalized across both moieties and the absorption spectrum is considerably red-shifted. Often the changes in photophysical properties due to extended delocalization of the π-conjugated framework can be related to the particle-in-a-box model, where with increasing conjugation length the absorption and emission energies decrease, and thus the absorption and emission colours shift towards the red region of the visible spectrum (Figure 1-5). This effect is due to the increased contribution of π MOs in the system and π electrons with increasing conjugation length. As more π MOs are added into the system, the energy gap between the HOMO and LUMO levels decreases. Consequently, the energy needed for an electron to transition from the HOMO to LUMO level decreases with larger conjugated systems, and thus the absorption spectrum of the ground state of the system red-shifts towards lower energy wavelengths. The same can be said about the emission spectrum, where increasing conjugation length often red-shifts the emission spectrum to lower energies. The resulting changes in absorption and emission properties have made π-conjugated molecular switches particularly attractive to researchers investigating new materials for optoelectronic devices,20 fluorescent sensors,21 or emissive dyes.22 9 Figure 1-5. Effect of increasing conjugation length on the HOMO-LUMO energy gap in organic molecular systems. 1.3.2 Transition Metal-Containing Molecular Switches Photophysical properties can often be accessed by combining organic ligands onto transition metal centers, which cannot be accessed by pure organic molecules. One example is metal-ligand charge transfer (MLCT) transition events between metal and organic ligand, which are often intensely coloured and lower energy than ligand π- π* transitions. This can be beneficial when designing conductive optoelectronic materials, or photosensitizers, which generally require high absorptivity for increased efficiency. Increased probability of phosphorescence can also occur with coordination complexes because of the metal center’s heavy atom effect which induces ISC. This is important for designing efficient OLEDs due to the increased spin statistics that access to a triplet excited state affords.23 Hence, many OLED materials incorporate transition metals to increase light efficiency and stability. Molecular switches incorporating transition metals have been designed, where the conformational change occurs either on the organic ligand, or by changing the atom connectivity 10 between ligand and metal center. The optical properties can often be tuned, where a change in intensity or energy of the absorption or emission properties occur.24,25 An example of an iridium complex with a triarylborane ligand demonstrated turn on/off phosphorescence, where addition of fluoride ions binding to the boron center caused quenching of the phosphorescence.24 Interestingly the boron-fluoride bond could be broken when exposed to an electric field, allowing phosphorescence to occur in the system, demonstrating reversible turn on/off phosphorescence. Other examples are discussed further in Chapter 3. 1.4 Boron-Functionalized Emissive Materials 1.4.1 Emissive Properties of Three-Coordinate Triarylborane Materials Boron has been incorporated into a wide variety of π-conjugated systems as an effective strategy in designing stimuli-responsive emissive materials.26,27 The electron deficiency of boron, specifically its unoccupied p orbital in the three-coordinate neutral state, makes boron highly Lewis acidic. When functionalized with aryl groups, a CT transition between the π orbitals of the aryl system to the empty p orbital on the boron can occur, often exhibiting emission (Figure 1-6). Figure 1-6. Overlapping of π orbitals from pendent aryl groups with the empty p orbital on the boron center leads to CT transitions. Electron donating or accepting groups can significantly change the CT energy and photophysical properties. 11 The energy of this CT transition can be altered by modifying the Lewis basicity of the aryl groups (ie. increasing the electron donating ability). Yamaguchi and co-workers incorporated a dimesitylborane group onto bithiophene in the 3-position (1), which gave blue emission attributed to a bithiophene-borane CT event (Figure 1-7).28 When the terminal ends of the bithiophene were functionalized with increasingly conjugated and electron donating groups, the emission colour red-shifted from blue to green to red in both the solution and solid state. It was determined that the red-shift in emission is a result from an increase in HOMO energy, with little to no change in LUMO energy. Figure 1-7. Dimesitylborane attached to a bithiophene backbone with varying functional groups reported by the Yamaguchi group.28. The emission red-shifts with increasing conjugation and electron donating functional groups. 1.4.2 Stimuli-Responsive Borane-Functionalized Materials Boron’s empty p orbital allows for acceptance of electron density from nearby electron donating groups, often resulting in a four-coordinate boron species. Small ions or molecules, such as fluoride or pyridine, can bind to boron inter- or intramolecularly, allowing for stimuli-responsive switching between bound and unbound states; finding potential applications in optical 12 sensor for small molecule detection.29 A change in emission properties can occur upon forming the four-coordinate species, often from the charge transfer (CT) pathway being quenched (Figure 1-8). More information and examples of stimuli-responsive intramolecular binding to three-coordinate borane centers in molecular systems are described in section 1.5.3. Figure 1-8. Switching between three- and four-coordinate boron center changes the availability of a CT event to the boron center to occur. An example of a dimesitylboryl-diphenylphosphine-Lewis pair bridged by a phenyl-naphthyl-phenyl U-shaped spacer was reported by the Wang group,30 which displayed ratiometric fluorescence dependent on the exposure to fluoride (Figure 1-9). In the absence of fluoride, the system displayed dual emission from a through-space CT state, involving a CT event from the phosphine to boron center and a CT event between the mesityl π to boron-phenyl π* LUMO, exhibiting relatively low quantum yields in DCM (Φ = 0.13). In the presence of fluoride, emission from the low-energy through-space phosphine-boron CT state diminished, while the emission from a higher-energy CT state appeared with increased efficiency (Φ = 0.29). This high energy CT state was proposed to be from either a CT transition involving the fluorinated dimesitylborane-phenyl group or the diphenylphosphine-phenyl group to the naphthyl π* LUMO. 13 Figure 1-9. Addition of fluoride anions induces increased blue emission from increased CT to the naphthyl linker in a boron-functionalized molecule.30 1.5 Lewis Pair Chemistry 1.5.1 Lewis Adducts In 1923, Gilbert N. Lewis first described acids and bases as electron acceptors and electron donors, where a Lewis acid is able to accept an electron pair and a Lewis base is able to donate an electron pair.31 This definition generalized the classification of acids and bases from the Arrhenius and Brønsted-Lowry definitions, which allowed for new insights into chemical reactions that did not occur in aqueous media or involve a proton transfer. By classifying chemical compounds as Lewis acids or bases many chemical reactions can be mechanistically explained as a Lewis acid and base binding together to form a stable, unreactive Lewis adduct, demonstrating their importance in chemistry.32–34 14 1.5.2 Frustrated Lewis Pairs Lewis adduct formation is often the major product in a reaction, however, if the Lewis acid and base are surrounded by significantly bulky groups, the surrounding steric bulk prevents the Lewis pairs from reacting with one another to make a classical Lewis adduct.35 Instead, they are said to be ‘frustrated’ Lewis pairs (FLPs), because the Lewis acid and base centers remain highly reactive (Figure 1-10a).36 Common Lewis acids incorporated into FLPs include bulky triarylboranes due their air and moisture stability, and tunable Lewis acidity, by variation of the aryl group. Commonly used Lewis bases include bulky phosphines, carbenes, and nitrogen-containing bases. Figure 1-10. Common Lewis adduct and FLP molecular structure (a) showing the sterically bulky groups around the Lewis pairs which prevent Lewis adduct formation, and early examples of H2 activation by FLPs (b).37,38 In 2006, the Stephan group reported the first FLP which could activate H2 gas in a metal-free, room temperature reaction at atmospheric pressure, and subsequently expel H2 gas at higher temperatures (Figure 1-10b).37 Soon following, the Erker group contributed to FLP small 15 molecule activation by reporting a simple FLP with high H2 activation yield (Figure 1-10c), which could then reduce benzaldehyde.38 These early reports contributed to the rapid expansion of research on FLP molecules for metal-free small molecule activation, where predominantly arylborane-containing FLPs have been used to activate CO2, CO, NO, SO2, and H2, and for the catalytic hydrogenation of unsaturated olefins, alkynes, imines, and carbonyl compounds.35,39,40 It has also been proposed that FLPs can be used in sensors due the ability of the Lewis pairs to interact with a variety of small molecules.41 1.5.3 Flexible Lewis pairs (FlexLPs) The ability to switch between a bound Lewis adduct and an unbound Lewis pair using an external stimulus offers the ability to dynamically control the properties of the Lewis pair system, such as reactivity, energy, or emission lifetime. Our group has studied such systems which can easily interchange between the two conformations and have termed them “flexible” Lewis pairs or FlexLPs. Much of the research on FlexLP systems has focused on incorporating different electron donating nitrogen-containing heterocycles as the Lewis base and using electron accepting triarylborane Lewis acids, because of their well-known ability to coordinate through a B-N bond. Additionally, the Lewis acid and base pairs are often incorporated on the same molecule through a conjugated spacer, which allows for easy switching between closed and open form. One example from the Murata group (Figure 1-11a) incorporated a dimesitylboryl group attached to a benzothiadiazole through a thiophene linker, which could reversibly switch between bound and unbound form through a temperature change in the solid state.42 The two forms emit different 16 colours, with the open form being bright yellow, and the closed form being blue. In solution, the compound exists in equilibrium between the two forms. The Wang group has also reported a temperature-responsive B-N FlexLP, incorporating a dimesitylboryl group on a phenyl backbone with a secondary amine in the ortho position, and a tertiary amine in the para position relative to the boryl group (Figure 1-11b).43 When cooled to 263 K the system is in the closed form and exhibits green emission, however with increasing temperature (to 313 K) the system switches to the open form and exhibits blue emission and higher overall emission intensity. This system also demonstrated CO2 binding in the open form, with a fluorescence colour change upon binding, demonstrating potential for application in CO2 sensors. Another FlexLP system from the Wang group uses a similar dimesitylboryl-phenyl-dimethylamine scaffold, with a carbonyl Lewis base group in the ortho position relative to the boryl Lewis acid group, where the carbonyl oxygen was able to reversibly bind to the boron by varying the solvent, temperature, or pressure (Figure 1-11c).44 The system was shown to have conformation-dependent emission colour and quantum yield, and could be used as an erasable emissive ink to create solvent-dependent erasable emissive images. Other FlexLP systems have been developed to respond to light, including an azobenzene-containing FlexLP system from the Kawashima group, where UV or visible light was able to switch the azobenzene group between the Z and E isomers, respectively, where the E isomer binds to the boryl group through a B-N bond (Figure 1-11d).45 The two isomers have different reactivity towards pyridine, where the Z isomer could more readily coordinate to pyridine through the three-coordinate boron center. 17 Figure 1-11. Small molecule FlexLP systems containing boron Lewis acid functional groups. Polymeric analogues of FlexLPs where a Lewis acid and base are incorporated onto the polymer backbone have been designed to make dynamic macromolecular systems. One example from the Jäkle group had pyridine groups added to the terminal ends of long polydimethylsiloxane (PDMS) polymer chains, and triarylboryl groups onto polystyrene (PS) polymers, and combined them in solution to make a PDMS-PS dynamic polymer blend with thermo-responsive self-healing properties (Figure 1-12a).46 Another example from Wang and coworkers attached the carbonyl-dimesitylboryl FlexLP system as pendent groups along a non-conjugated polymer backbone, which changed emission colour dependent on the ratio of open to closed forms along the polymer chain (Figure 1-12b).47 18 Figure 1-12. Polymeric systems containing FlexLP groups. 1.5.3.1 Phosphine Oxide Boron Mesityl FlexLP System Previously our lab has been able to synthesize a solvent- and temperature-responsive FlexLP, incorporating a diphenylphosphine oxide Lewis base and bulky boron mesityl Lewis acid onto the 3 and 3 position of a bithiophene backbone (Figure 1-13).48 The FlexLP is in equilibrium between closed Lewis adduct and unbound open Lewis pair state, dependent on the hydrogen bond donating (HBD) strength of the surrounding solvent, as classified by the Kamlet-Taft parameter.49 Kamlet and coworkers correlated overall solvent polarity of bulk solvents by measuring separately the polarizability, hydrogen bond accepting, and HBD parameters. The HBD parameter was measured using multiple linear regression analysis to construct the HBD scale, by determining the average HBD value from a number of indicators such as changes in 13C and 19F NMR shifts and changes in electronic transition properties of different hydrogen bond acceptors.49,50 It should be noted that because these parameters are calculated based on an average of values, the exact HBD values may change with improved analysis methods, however the relative HBD strengths of various solvents are known with relative certainty. 19 When exposed to non-HBD environments the Lewis pairs are bound together through a B-O bond, making the closed form. When in HBD environments, such as water or methanol, the phosphine oxide Lewis base is able to hydrogen bond with the surrounding solvent, which shifts the equilibrium to the open unbound conformation, facilitated by the low torsional energy barrier between the thiophene units on bithiophene.51 Figure 1-13. Previously studied bithiophene-based FlexLP incorporating diphenylphosphine oxide Lewis base and dimesitylboron Lewis acid, with accompanying HBD strength values of commonly used solvents.48,50 The two conformations have drastically different photophysical properties due to the different dihedral angles along the bithiophene backbone and the change in boron coordination number. In the closed form, the HOMO and LUMO resides predominantly on the bithiophene backbone, with a π-π* transition to the singlet excited state and blue emission with a small Stokes shift (5050 cm-1). In the open form, the HOMO is predominantly on the bithiophene backbone, while the LUMO is centered on the three-coordinate boron, allowing a charge transfer absorption transition to occur, resulting in a polar singlet excited state. This polar excited state induces a large Stokes shift (13400 cm-1) between the excitation and emission wavelength, resulting in yellow 20 emission from the compound. This FlexLP system can be switched from the open form to the closed form with decreasing temperature, making this FlexLP system temperature-sensitive. It was proposed that this may be due to a decrease in entropy in the system with increased rigidity of the closed form. Figure 1-14. One-pot synthesis of FlexLP groups from a bithiophene backbone with R groups of different conjugation length and electron donating strength.52 A wide range of FlexLP compounds with varying bithiophene backbones were prepared and investigated, including the effect of increased conjugation along the bithiophene backbone (R), and of increased electron donating ability of the R groups by first synthesizing the bithiophene backbone through predominantly Suziki or Sonogashira cross-coupling reactions (Figure 1-14).52 Both increasing conjugation length and electron donating strength of the functional groups on the 21 terminal ends of the bithiophene backbone resulted in a red shift in the absorption and emission of both the open and closed forms. These findings showed that the Lewis pairs could be incorporated onto a large variety of bithiophene backbones, and exhibit emission colour tunability spanning the visible spectrum from blue to red. 1.6 Goals and Outline of Thesis Previous FlexLP molecules were synthesized by first building the bithiophene backbone of the FlexLP through predominantly lithium-halogen exchange reactions, followed by addition of the Lewis pair as the final step. This approach allows for a variety of small, stable FlexLP molecules to be synthesized, however in order to expand the molecular design possibilities of FlexLP-containing molecules the synthesis of a FlexLP molecule containing reactive terminal groups, specifically alkyne groups, that could be further functionalized was sought. This new approach would allow synthetic access to symmetrical FlexLP molecules, where terminal groups are the same, and non-symmetrical FlexLP molecules, where terminal groups are different (Figure 1-15). This thesis aims to broaden the scope of stimuli-responsive FlexLP chemistry by exploring the synthetic possibilities of a reactive alkyne-functionalized FlexLP compound which undergoes post-modification with metallic or organic groups by utilizing different terminal alkyne coupling reactions (Figure 1-15). Examples of Cu(I) catalytic coupling reactions will be demonstrated and discussed, showcasing the versatility of the terminal alkyne handle to afford different FlexLP motifs. 22 Figure 1-15. General scheme of symmetrical and non-symmetrical alkyne-functionalized FlexLP, and the possible FlexLP motifs achieved through terminal alkyne coupling reactions with different functional groups (R). Chapter 2 describes the synthesis of a reactive terminal alkyne-functionalized FlexLP molecule, and a FlexLP dimer synthesized using Glaser coupling conditions from the alkyne-functionalized FlexLP molecule. The reactivity and photophysical properties of these FlexLP molecules are investigated. Chapter 3 explores the synthesis of monometallic and bimetallic FlexLP-functionalized Pt(II) complexes, and their optical properties in response to solvent and temperature. These complexes were synthesized using Cu(I) coupling reactions. Chapter 4 investigates emissive Pt(II)-FlexLP complexes, including their fluorescence response and excited state dynamics with solvents of varying HBD strength. A bis-FlexLP-Pt(II) complex and a pyrene-containing Pt(II)-FlexLP complex were analyzed by fluorescence and transient absorption microscopy, to elucidate singlet and triplet excited state dynamics. 23 Finally, Chapter 5 explores the use of a N-hydroxysuccinimidyl ester-functionalized FlexLP system as a stimuli-responsive fluorophore for potential biological imaging. The N-hydroxysuccinimidyl ester-functionalized FlexLP system was synthesized using azide-alkyne Cu(I) click chemistry, and then further functionalized onto amine-functionalized mesoporous silica nanoparticles as a biologically-relevant substrate for fluorescence imaging. 24 Chapter 2: Photophysical Properties of a Flexible Lewis Pair Dimer Synthesized Using Alkyne-Alkyne Coupling Chemistry 2.1 Introduction 2.1.1 Dimeric Stimuli-Responsive Molecules Stimuli-responsive dimeric molecules (or “dimers”), which consist of two covalently attached molecules, are interesting molecular motifs because of their potential ability to have two individual stimuli-responsive active sites that may exhibit dynamic reactivity, where the activation of one stimuli-responsive site influences the reactivity of the other active site. Additionally, dimers are the first synthetic progression towards longer oligomers and polymers, and therefore can be investigated and compared to their monomeric counterparts to predict the properties that might be observed in oligomeric molecules. The concept of a dimer having dynamic reactivity upon initial activation is analogous to the cooperative binding effects studied in enzyme-substrate binding kinetics research, where the enzyme may be thought of as a large dimer (or trimer, tetramer, etc.) unit that is responsive to surrounding substrate.54 For instance, many examples of enzyme-substrate and other receptor-ligand systems have been shown to have changing binding affinities upon initial binding, which can be modelled to assign positive, negative, or non-cooperativity.55–59 For a receptor exhibiting non-cooperativity there is no change in binding affinity towards ligand upon initial binding, and the fraction of filled binding sites at equilibrium increases in a hyperbolic fashion with increasing concentration of ligand according to Michaelis-Menten kinetics. Positive cooperativity exhibits increased binding affinity towards ligand upon initial binding, while a system with negative 25 cooperativity will have a decreased binding affinity towards ligand upon initial binding (Figure 2-1). A number of different models have been developed that are able to assign the different binding characteristics of receptor-ligand pairs with varying binding sites, shapes, and thermodynamic-sensitive properties.56,57,60,61 Figure 2-1. Binding cooperativity in enzyme-susbtrate pairs, where the enzyme has two binding sites. Similar cooperativity effects for small molecules are greatly influenced by the degree of electronic delocalization or communication between the two stimuli-responsive sites, particularly for highly conjugated molecules. For example, the metal oxidation potentials of dinuclear metal complexes can vary due to the degree of electronic communication between the two metals.62 The degree of electronic communication between the two metals, referenced by the Robin-Day 26 classification system (Figure 2-2),63 is a particularly important property when designing Wolf Type III metallopolymers for application as molecular wires, which require a high degree of electronic conjugation between metal centers for efficient electron conductivity to occur.64,65 Figure 2-2. Robin-Day classification describing the mixed valency of the homonuclear dimetallic complexes, and the corresponding degree of electronic communication between metal centers. Previously our group has synthesized a dithienylethene (DTE)-functionalized Pt complex (Figure 2-3), where the DTE ligands can be switched between open and closed form with light, where the formation of the three possible isomers were observed and analyzed by 31P NMR, absorption spectroscopy, and electrochemistry.25 In previously reported organic DTE-oligomers, the closed-open conformation experiences an energy transfer from the singlet S1 state situated on the open DTE chromophore to the closed DTE chromophore, which inhibits formation of the fully closed-closed conformation.66,67 For the DTE-functionalized Pt complex, the Pt center is thought 27 to effectively reduce the electron delocalization between DTE ligands, slowing the rate of energy transfer from the singlet excited state of the open DTE chromophore to the closed DTE chromophore in the closed-open form, allowing access to the closed-closed conformation with exposure to UV light. Figure 2-3. Pt complex containing two DTE ligands which can switch between fully open and fully closed conformations through a closed-open intermediate that can be spectroscopically observed.25 An example of a stimuli-responsive molecule was reported by Shimogawa and coworkers, where a B-N FlexLP containing a thiadiazole with two pendent boron-functionalized thiophene groups was synthesized (Figure 2-4).42 The molecule switched from open-open to open-closed forms with temperature, with no observed formation of the closed-closed conformation. DFT calculations confirmed that the Gibbs free energy for the closed-closed conformation is significantly higher compared to the closed-open form, due to the reduced nucleophilicity of the uncoordinated nitrogen and the steric bulk around the boron mesityl groups. 28 Figure 2-4. Boron-thiadiazole FlexLP exhibiting negative cooperativity characteristics, where binding of one boron-nitrogen pair inhibits the binding affinity of the second boron-nitrogen pair.42 2.1.2 Goals As discussed in Chapter 1, a FlexLP molecule with terminal alkyne reactive functional group was sought in order to allow for further functionalization. Alkyne-alkyne coupling chemistry is one example of a coupling reaction that incorporates terminal alkynes, which allows for the design of increasingly conjugated molecules without adding steric bulk.68–70 Three common alkyne-alkyne coupling reactions are the Eglinton,71,72 Glaser,69,73 and Cadiot-Chodkiewicz70,74, all of which use Cu metal under mild reaction conditions to afford symmetric or non-symmetrical alkyne-alkyne coupled molecules (Figure 2-5). Figure 2-5. Eglinton (a), Glaser (b), and Cadiot-Chodkiewicz (c) alkyne-alkyne coupling reactions. 29 Figure 2-6. Schematic of FlexLP-TBS 24 and FlexLP dimer 25 in equilibrium between the three states. An interesting molecular motif to explore would be an organic FlexLP dimer, whereby alkyne-alkyne coupling between two FlexLP molecules would afford a molecule with two FlexLP moieties connected along a fully conjugated organic backbone. By comparing the photophysical properties of the FlexLP monomeric unit (FlexLP-TBS, 24) with the new proposed dimer 25, the aim of investigating the effect of having two stimuli-responsive FlexLP groups conjugated to one another could be achieved, to determine if the conformation of one FlexLP group affects the interaction of the second FlexLP group with its surroundings (Figure 2-6). By having two distinct stimuli-responsive FlexLP groups, three different conformational states (closed-closed, closed-open, and open-open) may be accessed, with distinct photophysical characteristics possible for each of the three states. By first investigating the properties of the dimer, the photophysical 30 properties and stimuli-responsive nature of oligomeric or polymeric FlexLP products may be predicted. 2.2 Synthesis and Characterization A key element towards the synthesis of the FlexLP dimer 25 is selective deprotection of the silyl group closest to the phosphine oxide in the silyl-acetylene functionalized FlexLP (TBS-FlexLP-TBS, 26) using one equivalent of TBAF in good yield (77%) (See Experimental Section for full synthetic scheme). The 1H NMR spectra of FlexLP-TBS shows a single acetylene proton peak, indicating efficient mono-deprotection of the compound (Figure 2-7). Figure 2-7. 1H NMR spectra of compound FlexLP-TBS 24 (red) and doubly deprotected FlexLP 27 (black) in CDCl3 showing the terminal acetylene protons. 31 Figure 2-8. HSQC (top) and HMBC (bottom, purple) spectra with HSQC (red) overlap of FlexLP-TBS in CDCl3. Starred peak is residual solvent. 32 Figure 2-8 depicts the 2D HSQC and HMBC NMR spectra of FlexLP-TBS in CDCl3. The bithiophene proton H4 peak is split by coupling with the 31P nucleus, and coupled to the carbons at 75 ppm (C2) and 121 ppm (C3, doublet split by 31P). Proton H5 is directly bonded to the carbon at 83.47 ppm (C1), with strong 2J coupling to the C2 carbon, and coupled to the C3 carbon, indicating the H5 acetylene proton is situated on the phosphine oxide Lewis basic side of FlexLP-TBS. The 1H NMR spectra in Figure 2-7 show that the downfield shifted alkyne proton for double deprotected FlexLP is on the phosphine oxide side of the molecule, indicating that this proton is more deshielded and therefore more acidic than the alkyne proton nearest the boron Lewis acid. The more acidic character of the proton nearest the phosphine oxide side may explain the highly selective nature of the decoupling reaction to afford FlexLP-TBS, where the F- anion in TBAF reacts first on the phosphine oxide side to produce FlexLP-TBS, with doubly-deprotected FlexLP 27 as the minor product. This synthetic route is advantageous for selective reaction of the FlexLP-TBS with itself to form a symmetric dimer with phosphine oxide Lewis base groups nearest the center of the molecule and dimesitylboron Lewis acid groups near the ends of the molecule. Of note is the stability to air and light of FlexLP-TBS which simplifies purification and characterization. The FlexLP dimer was made through alkyne-alkyne coupling chemistry (Figure 2-9). After some experimental attempts, it was determined that the Glaser Cu(I) coupling method gave the best yield, however this was still relatively poor (10%). The poor yield was thought to be due to difficulty in solubility and purification by column chromatography. 33 Figure 2-9. Schematic depicting the synthesis of dimer 25, using Glaser coupling reaction conditions. The isolated product had poor solubility in many solvents, however was soluble in THF and chloroform, therefore proton and phosphorus NMR spectra could be obtained. 1H NMR did not show terminal alkyne proton peak, and MS gave the expected theoretical dimer mass, suggesting that the product was the FlexLP dimer. The 31P{1H} NMR spectrum of FlexLP-TBS shows a downfield shift in solvents with decreasing HBD strength, similar to the observation for other FlexLP molecules (Figure 2-10).48,52 The downfield shift is attributed to the decreased electron density around the oxygen atom upon binding to the boron center to make the closed FlexLP form, which deshields the phosphorus nucleus. Because the equilibrium between the two conformations of the FlexLP is faster than the NMR timescale, an overall average magnetic field is felt for the molecule in solution, and therefore a single phosphorus peak is observed. Comparing the 31P{1H} NMR spectra of FlexLP-TBS and the dimer in different solvents, the dimer’s 31P peak shifts upfield in chloroform compared to THF, similar to the monomer (Figure 2-10). This large difference in chemical shift with solvent suggests that the dimer’s FlexLP conformation is also solvent-responsive, where in non-HBD solvent the FlexLP is in the closed conformation, and in solvents with weak HBD strength the equilibrium shifts towards the open form. However, other solvents with varying HBD strength could not be analyzed because of 34 poor solubility issues, therefore 31P{1H} NMR spectroscopy could not be used to confirm a change in conformation. Figure 2-10. 31P{1H} NMR spectra of FlexLP-TBS and dimer in varying deuterated solvents. 2.3 Photophysical Data 2.3.1 Absorption and Emission Properties of FlexLP-TBS and Dimer Absorption and emission spectroscopy studies were performed in order to determine whether a change in photophysical properties occurred with changing HBD strength of the surrounding medium. THF and chloroform were chosen as the non-HBD and weakly HBD solvents, respectively, according to their Kamlet-Taft parameters.49 Due to poor solubility in protic solvents, the dimer could not be analyzed using a pure solvent with high HBD strength, therefore 35 mixed MeOH/chloroform (1:1 v/v) solvent system was used to gain insight on the photophysical properties of the dimer in relatively high HBD strength surroundings. The absorption spectra of the dimer were red-shifted in energy in all solvents compared to FlexLP-TBS monomer unit, and the molar absorptivity of the dimer was significantly higher in value compared to FlexLP-TBS, due to the increased conjugation length along the backbone (Figure 2-11). Comparison of the absorption spectra of the dimer in various solvents shows a similar trend to FlexLP-TBS, where with increasing HBD strength of the solvent, a low energy absorption band decreases, and a higher energy absorption band (325-350 nm) increases in intensity. This similarity between dimer and FlexLP-TBS suggests that the dimer also undergoes a change in structural conformation with varying HBD strength solvent. Figure 2-11. Absorption (a) and emission (b) spectra of the dimer and FlexLP-TBS in various solvents. The emission spectra of the two molecules also show similarities with one another (Figure 2-11). The emission spectra for FlexLP-TBS in the two different solvents shows different emission peaks that are well separated in energy and have similar intensities; in the closed form 36 (in THF) the emission peak is blue-shifted (λem = 475 nm) compared to the open form (in MeOH, λem = 550 nm). The emission peak for the dimer is also blue-shifted in THF compared to in MeOH-containing solvents, indicating a solvent-induced emission response. In THF, the dimer’s emission is red-shifted compared to FlexLP-TBS, as expected from the longer conjugation length, suggesting that there is a significant portion of HOMO and LUMO density along the bithiophene-bithiophene backbone of the dimer which contributes to the significantly red-shifted emission. For MeOH-containing solvents the maximum emission peak is not significantly shifted in energy from FlexLP-TBS. This may be an indicator that the HOMO does not span the full bithiophene-bithiophene backbone, perhaps due to thiophene-thiophene twisting from a change in conformation, which decreases the backbone conjugation (Figure 2-12). Figure 2-12. Schematic of possible open and closed forms of the dimer and the areas where the HOMO and LUMO MOs reside (indicated in colour). Blue highlight indicates potential HOMO-LUMO π-π* transition, where red highlight indicates potential CT transition. The emission profiles of the dimer in the two different solvents significantly overlap with one another, and the intensity of the emission in MeOH/chloroform solvent greatly diminishes. 37 These photophysical characteristics, along with the known poor solubility of the dimer in protic solvents, pose challenges on assigning conformation of the FlexLP groups within the dimer molecule. Nevertheless, the changes in both absorption and emission profiles of the dimer with different solvents indicates that the dimer has solvent-responsive photophysical properties. An interesting feature to note in the absorption spectrum of the dimer in THF solvent is that it shows distinct absorption shoulder peaks at 475 nm and 400 nm, which are not apparent for FlexLP-TBS in THF solvent, or for the dimer in other solvents. The dimer also exhibits two distinct emission peaks in THF that are not apparent for any other samples. A dimer control molecule 28 (Figure 2-13, see experimental section 2.6.2 for full synthetic details) with two bithiophene groups conjugated together by an alkyne-alkyne spacer was synthesized in order to determine whether these multiple absorption and emission peaks for the dimer in THF were due either to two species in equilibrium in THF, the closed-closed and closed-open forms, or from strong vibrational transitions. The control dimer 28 was expected to have strong alkyne-alkyne vibronic stretching modes that may affect the absorption and emission profiles, similar to previous bithiophene-alkyne molecules reported previously.75 Figure 2-13. Schematic depicting strong vibronic transitions of bithiophene dimer control molecule, 28. 38 The control molecule also exhibited similar shoulder peaks in the absorption spectra, in both THF and MeOH (99:1 MeOH/THF) solvent (Figure 2-14). The control also exhibited two distinct emission peaks, with a similar profile as the dimer emission in THF (Figure 2-14). Excitation spectra for the control 28 and dimer 25 molecule showed that emission of the two peaks were a result of the two low energy absorption peaks (450 nm and 410 nm for control molecule, and 475 nm and 450 nm for dimer molecule). Therefore, it is highly likely that the two emission peaks for the dimer in THF are from strong vibronic transitions, likely from the strong alkyne-alkyne stretching vibrations. As well, it is likely that the dimer is in the closed-closed FlexLP conformation, with HOMO and LUMO MOs spanning along the full length of the conjugated backbone, considering the similarities in absorption and emission profiles of the dimer to control 28, and the non-HBD character of THF solvent. Figure 2-14. Absorption and emission profiles comparing the FlexLP dimer 25 with the control dimer 28 without Lewis acid-base pairs (1 x 10-6 M solutions, emission/excitation spectra taken in THF solvent using λex = 415 nm (28) and λex = 450 nm (dimer 25) ). 39 2.3.2 Absorption and Emission Properties of the Monomer and Dimer Molecule in THF-MeOH Solvent Gradients Further investigation of the dimer in mixed THF-MeOH solvent gradients was performed to compare the solvent equilibrium effects between dimer and FlexLP-TBS. The absorption spectra of both molecules in THF with increasing MeOH % show isosbestic points at ~350 nm and ~385 nm for FlexLP-TBS and dimer respectively (Figure 2-15). Because the dimer exhibits only one isosbestic point, distinct absorption profiles for each potential conformation, closed-closed, closed-open, and open-open, cannot be distinguished. Therefore, though it is highly likely that in THF the dimer is in the closed-closed position, it is unclear whether the dimer is predominantly in the closed-open or open-open form in MeOH (99%) solvent. Figure 2-15. Absorbance spectra of FlexLP-TBS (a) and dimer (b) in mixed THF:MeOH solvent ratios, with increasing % MeOH (5 × 10-6 M (FlexLP-TBS) and 1 × 10-6 M (dimer) solutions used). From Figure 2-15, it is clear that the shoulder absorption peak at 475 nm in pure THF (0% MeOH) does not significantly overlap with the absorption profile in 99% MeOH, therefore a graph of absorbance at 475 nm versus MeOH % in solution can be prepared to determine the overall 40 concentration of closed-closed dimer with increasing MeOH solvent, with the assumptions that the dimer is 100% in the closed form in 0% MeOH and the closed-open form does not significantly absorb at 475 nm. The concentration of closed-closed dimer decreases linearly with increasing MeOH (Figure 2-16), which may be an indication of the dimer not exhibiting positive or negative cooperative effects. Additionally, the linear decrease in closed-closed dimer concentration suggests that the equilibrium lies heavily towards the closed-closed form, with incomplete switching to 100 % open-open form. However it should be noted that this analysis relies heavily on the assumption that absorption at 475 nm only occurs for the closed-closed form of the dimer; in reality the closed-open form of the dimer may also absorb at this wavelength. Therefore, it remains uncertain whether formation of the open-open dimer form occurs. Figure 2-16. Absorbance at 475 nm, assumed to be absorption of solely closed-closed form of the dimer with increasing MeOH (1 × 10-6 M). Emission properties of the two molecules were analyzed in varying THF:MeOH solvent ratios. When excited at 335 nm, the emission peak at 475 nm from the closed form of FlexLP-TBS decreases with increasing MeOH concentration, and the emission peak at 550 nm increases, 41 as expected (Figure 2-17). Similarly, for the dimer, the emission peaks at 500 and 540 nm decrease and the broad emission peak at 560 nm increases with increasing MeOH percentage. A gradient from blue to yellow, and green to yellow emission colours for FlexLP-TBS and the dimer respectively with increasing MeOH can be seen when using a UV handlamp with excitation wavelength 365 nm, showing that the change in emission colour can be monitored easily using a single excitation wavelength due to sufficient overlap between the absorption peaks at 365 nm. Figure 2-17. Emission spectra of FlexLP-TBS (a, λex = 335 nm, 5 × 10-6 M) and dimer (b, λex = 345 nm, 1 × 10-6 M) in mixed THF:MeOH solvent (% MeOH by volume indicated). Above are shown the respective samples excited with a 365 nm UV handlamp, showing the changing emission colour from left to right with increasing MeOH. Although no solids were observed in solution at low concentrations for the dimer, the formation of nanoscopic solid aggregates in high MeOH % solvent may be possible because of the known 42 poor solubility of the dimer in MeOH. Most FlexLP molecules in the solid form exhibit yellow emission when excited using a UV handlamp, however further solid-state photophysical studies have not been performed thus far. Therefore, the change in emission colour with increasing MeOH due to solid aggregates forming in solution cannot be dismissed. Future studies on the solid-state emission properties of these FlexLP molecules may help corroborate whether the emission peak at 560 nm is from the dimer exhibiting solid-state emission, or whether the dimer undergoes a significant change in conformation with increasing MeOH. 2.3.3 Emission Lifetime Data of FlexLP-TBS and Dimer Molecule A solution containing two or more molecules with overlapping emission energies can be analyzed by emission lifetime to discern between the two molecules if the molecules have significantly different emission lifetimes. In attempt to further elucidate the structural conformation of the dimer in MeOH-containing solvents, where perhaps three distinct emission lifetimes would be seen for the dimer corresponding with the three possible conformations, emission lifetimes were analyzed and compared with FlexLP-TBS. It was observed that FlexLP-TBS had different emission lifetimes depending on the structural conformation, where the closed form had a significantly shorter lifetime (~1 ns) compared to the open form (5-15 ns) (Figure 2-18, Table 2-1). This is thought to be due to the different HOMO-LUMO energy transitions that occur between the two forms, where the open form undergoes a CT transition, which may help stabilize the lifetime of the singlet excited state energy. 43 Figure 2-18. Emission lifetime graphs of FlexLP-TBS in THF (a) and MeOH (b) solvents. In order to further confirm that this change in emission lifetime was due to the change in conformation, and not solvent, a previously reported bithiophene molecule with Lewis basic dimesitylphosphine oxide and Lewis acidic dimesitylboron groups (29)48 was analyzed (Figure 2-19). The bulky mesityl groups around the phosphine prevent the molecule from switching to the closed form. As shown in Figure 2-19, the difference in lifetime between the two solvents for this non-stimuli-responsive molecule is significantly less drastic, with a slight change in emission lifetime thought to be due to slight differences in solvent polarity. Therefore, it is concluded that the very short emission lifetime for the stimuli-responsive FlexLP-TBS monomer in THF is due to the change in conformation, and not due to the change in solvent polarity. 44 Figure 2-19. Emission lifetime graphs of 29, in THF and MeOH solvents. No significant change in emission lifetime is seen. The emission lifetime spectra of the dimer showed three different emission lifetimes for all solvent systems, τ1, τ2, and τ3 (Figure 2-20, Table 2-1). Three exponentials were chosen as they gave the best fit, particularly in MeOH solvent, compared to fitting with two exponentials, although it should be noted that the percent contribution from the longer lifetime τ3 is low. 45 Figure 2-20. Emission lifetime decay curves of dimer, comparing the fitting of 3 exponentials (a) to 2 exponentials (b). Lifetimes τ1 and τ2 did not significantly change in value with varying MeOH, with the exception of τ2 at 100% THF, however τ3 did increase significantly with increasing MeOH. This change in τ3 emission lifetime may be attributed to a more accurate computational fit with the increased signal of τ3. As shown in Figure 2-21 and Table 2-1, the relative percent contribution of τ3 is small (< 5 %) in solvent mixtures containing more THF therefore the fit of the curve will have greater uncertainty due to the small signal to noise ratio. The longer emission lifetime of τ3 suggests that it may be associated with emission from the open conformation of the FlexLP based on the lifetime trends of previous FlexLP molecules. If τ3 is associated with the open-form FlexLP, which emits at 560 nm at lower intensity compared to the closed-closed form, then the relatively low percent contribution of τ3 may be attributed to the lower emission intensity of the open form, rather than the open form being in significantly low concentration in MeOH solution compared to the closed form. 46 Table 2-1. FlexLP-TBS and dimer lifetime and percent contribution (in parenthesis) data with varying ratio of THF:MeOH solvent system. FlexLP-TBS dimer % MeOH τ1 (ns) τ2 (ns) τ1 (ns) τ2 (ns) τ3 (ns) 0 1.02 (98 %) 11.1 (2 %) 0.460 (91 %) 1.01 (7.5 %) 3.83 (1.5 %) 20 0.98 (92 %) 9.36 (8 %) 0.395 (63 %) 0.686 (34 %) 4.24 (2 %) 40 0.94 (82 %) 9.77 (18 %) 0.456 (86 %) 0.963 (12 %) 6.03 (2 %) 60 0.91 (67 %) 9.97 (33 %) 0.409 (63 %) 0.722 (34 %) 6.64 (3 %) 80 0.91 (47 %) 10.1 (53 %) 0.444 (70 %) 0.778 (26 %) 7.46 (4 %) 95 0.96 (27 %) 9.86 (73 %) 0.459 (72 %) 0.808 (23 %) 7.84 (5 %) 100 0.91 (26%) 10.5 (74 %) -- -- -- When plotting the percent contribution of each of the lifetimes (relative amplitude) with increasing % MeOH a trend is observed for FlexLP-TBS, where increasing % MeOH solvent increases the percent contribution of the longer lifetime, τ2 (Figure 2-21). For the dimer, a slight increase in the percent contribution of the longest lifetime, τ3, was seen with increasing % MeOH, however the increase was less than 5%, and no clear opposing trend was seen with either τ1 or τ2. Therefore, based on this data it is inconclusive whether the three different lifetimes are associated with the three possible dimer conformations, or if each conformation has multiexponential emission decay characteristics. As well, the data shown does not eliminate the possibility that solid-state aggregates form in high MeOH percentage solvent mixtures. Therefore, attempts to increase the solubility of the dimer before performing further experiments were made. 47 Figure 2-21. Relative percent contributions of emission lifetimes with varying THF:MeOH solvent ratio for FlexLP-TBS (a) and dimer (b). No clear trend could be seen between percent contribution of all lifetimes and % MeOH for the dimer. 2.4 Synthetic Attempts to Increase Solubility of FlexLP Dimer Attempts were made to increase the solubility of the dimer in protic solvents by adding ethylene glycol chains (OEGs), to mitigate the possibility that any photophysical characteristics were due to solid-state aggregates forming from poor solubility in solution. OEG groups were chosen because of their high polarity and previous success as hydrophilic solubilizing groups for many hydrophobic materials and drugs in biochemistry.8,76 Reaction of an OEG group functionalized with a terminal alkyne (OEG) with FlexLP 27 under Eglinton (1) and Glaser (2) alkyne-alkyne coupling conditions was attempted, however these reactions were not successful (Figure 2-22). Reaction attempts using brominated OEG and FlexLP-TBS under Pd-assisted Cadiot-Chodkiewicz reaction conditions (similar to Sonogashira reaction conditions) were carried out in an attempt to better induce unsymmetrical bisacetylene formation using Pd(PPh3)2Cl2 and CuI (3), however the desired product did not form. After 48 switching the Pd source to PdCl2 to reduce steric bulk around the Pd center and promote transmetallation and isomerization, the desired product was formed (4) (Figure 2-22). Figure 2-22. Scheme of synthetic attempts to add solubilizing OEG groups to FlexLP molecules. After deprotection of the remaining TBS group, reaction of the OEG-functionalized FlexLP with another brominated OEG group under the same PdCl2/CuI reaction conditions was attempted, however the reaction was unsuccessful (5) (Figure 2-23). Addition of Lewis acid and base pairs onto OEG-functionalized dibromobithiophene using n-BuLi also proved unsuccessful (6). It was thought that perhaps the long ethylene glycol chains interact with the n-BuLi, therefore tetraethylenediamine, TMEDA, was added to the reaction to increase the reactivity of the n-BuLi reagent, however this also proved unsuccessful (7). When attempting to add the Lewis pairs in two separate steps using the n-BuLi-TMEDA reaction conditions, the Lewis base could be added in low yield (33, 16%), however further attempts to add the Lewis acidic dimesitylboron were unsuccessful (8). 49 Figure 2-23. Scheme of synthetic attempts to add solubilizing ethylene glycol oligomer groups to either side of a FlexLP molecule. Reaction of OEG-FlexLP monomer with itself to afford OEG-terminated FlexLP dimer was unsuccessful under Glaser reaction conditions (9) (Figure 2-24). The doubly-deprotected dimer (FlexLP2) could be synthesized using 2 equivalents of TBAF and isolated in good yield (10). It should be noted that FlexLP2 also had poor solubility in protic solvents, and therefore was not characterized further by absorption or emission spectroscopy. Reaction of FlexLP2 dimer with OEG was not attempted due to the availability of only a limited amount of FlexLP2 and the 50 unsuccessful development of equivalent reaction conditions using FlexLP to add OEG to the terminal alkyne nearest the dimesitylborane group. Figure 2-24. Attempted synthesis of an OEG-functionalized FlexLP dimer (9), and successful synthesis of FlexLP2 dimer with terminal alkynes (10). It is postulated that the Lewis acidity of the boron center decreases when in the closed form in non-HBD solvents, resulting in an increase in the electronegativity of the terminal alkyne carbon after reaction with the amine base. This increased electronegativity of the carbon may increase the coordination strength to the Cu catalyst, decreasing the efficiency of transmetallation in reactions (3) and (5), and reductive elimination in reaction (9). It should be noted that reaction of doubly-deprotected FlexLP with Cu(I) and i-Pr2NH in THF to make a dimetallic Pt-functionalized FlexLP complex is possible (described in Chapter 3), and therefore it is not likely that the dimesitylboron group hinders oxidative addition of the FlexLP to the Cu catalyst. For future attempts to add solubilizing groups, it is suggested that other terminal alkyne-containing reactions that couple strongly electrophilic groups in effort to increase reaction with the 51 electronegative alkyne carbon be attempted, perhaps by copper azide-alkyne click reaction77,78 or aldehyde-alkyne-amide A3 coupling.79,80 Both of these reactions encompass electrophilic nitrogen-containing reagents, which may help promote further reactivity of the alkyne after oxidative addition to the Cu catalyst. 2.5 Conclusion A FlexLP dimer was synthesized using an alkyne-alkyne coupling reaction, which saw solvent-responsive tunable emission colour. Although differences in absorption and emission spectra were observed, at this time it cannot be confirmed whether these differences are due to a change between the possible three FlexLP conformations, or solid-state aggregation occurring. Future studies on the solid-state properties of these FlexLP systems may help elucidate the dimer solution-state conformation. Comparison of the emission characteristics of the dimer and FlexLP-TBS monomer in THF and MeOH suggests that with increasing FlexLP units, a red-shift in emission in THF would occur, with little to no change in emission wavelength when in MeOH. Therefore, future studies of FlexLP oligomers and polymers may see reduced emission colour differences with solvents of varying HBD strength. The reactivity of the terminal alkyne nearest the Lewis acidic boron was reduced when attempting to add solubilizing groups and functionalization through alkyne-alkyne coupling mechanisms was unsuccessful. It is suggested that future functionalization methods should be explored which include strong electrophiles, such as azide-alkyne click or A3 coupling reaction, which may allow for increased solubility of future FlexLP dimer molecules. 52 2.6 Experimental Details 2.6.1 General Procedures 1H, 13C, 31P{1H}, HSQC, and HMBC NMR experiments were carried out on a Bruker Avance 400 spectrometer. 31P{1H} NMR spectra are referenced to 85% H3PO4 as an external standard. 1H and 13C NMR spectra are referenced to residual 1H and 13C solvent peaks respectively. 1H assignments are made using HSQC and HMBC NMR experiments. Absorption spectra were obtained using a Varian Cary 5000 UV-vis-NIR spectrophotometer, and emission spectra were obtained using a Photon Technology International (PTI) QuantaMaster 50 fluorimeter fitted with double excitation monochromator and 75 W Xe arc lamp as the source, respectively. Emission lifetime data were obtained using a Horiba Yvon Fluorocube TCSPC apparatus, using a 370 nm NanoLED excitation source with a pulse width of 1.2 ns, pulsing at a repetition rate of 1 MHz, and using a 399 nm long-pass filter between sample and detector. Lifetime data were fitted against the NanoLED source background using the DAS6 Data Analysis software package, to afford sub-nanosecond deconvoluted lifetime data. Electrospray ionization (ESI) mass spectrometry data were obtained on a Waters/Micromass LCT TOF-MS spectrometer. Elemental analysis (EA) data were obtained using a Thermo Flash 2000 Elemental Analyzer. Synthetic procedures involving air or moisture-sensitive reagents were carried out using standard Schlenk techniques under inert N2 atmosphere. Dry THF and Et2O solvents were obtained from an Inert PureSolv MD5 Solvent Purification System (SPS). H2O2 solution (30% wt. in H2O), n-BuLi (1.6 M in hexane), diisopropylamine, chlorodiphenylphosphine, copper(I) iodide, copper(I)chloride, copper(II) acetate monohydrate, bipyridine, tetrabutylammonium fluoride (TBAF, 1.0 M in THF), and 3,3',5,5'-tetrabromo-2,2'-bithiophene were purchased from Sigma 53 Aldrich and used as received. Trans-bis(triphenylphosphine) palladium(II) dichloride was purchased from Strem Chemicals Inc. and used as received. Dimesitylfluoroborane, (triethylsilyl)acetylene, and (tert-butyldimethylsilyl)acetylene were purchased from Tokyo Chemical Industry Co., Ltd. (TCI) and used as received. 2.6.2 Synthesis of New Compounds Figure 2-25. Full synthetic scheme of FlexLP-TBS and FlexLP dimer. 54 TBS-T2Br2-TBS, 36. Copper(I) iodide (0.100 g, 0.500 mmol), trans-bis(triphenylphosphine) palladium(II) dichloride (0.350 g, 0.500 mmol), and 3,3',5,5'-tetrabromo-2,2'-bithiophene 35 (3.00 g, 6.23 mmol) were dissolved in dry diisopropylamine/THF solution (60 mL, 1:3 v/v) in a 100 mL round bottom flask equipped with a Schlenk arm under an N2 atmosphere. (tert-Butyldimethylsilyl)acetylene (2.45 mL, 13.1 mmol) was added and the reaction stirred at room temperature for 16 h. Hydrochloric acid (0.3 mL, 3 M) was added to quench the reaction, and THF removed under reduced pressure. The organic layer was extracted with Et2O, washed with H2O and brine, and dried over MgSO4. The solvent was removed under reduced pressure and the crude precipitate purified by column chromatography using hexanes. The product was collected as a light yellow powder. Yield: 2.90 g, 77%. 1H NMR (CDCl3, 400 MHz, 25 °C): δ 7.17 (s, 2H, H1), 1.01 (s, 18H, H3), 0.21 (s, 12H, H2). HRMS (ESI) m/z: calc. C24H33Si2S2Br2 [M+H]+: 598.9929, found 598.9933. TBS-FlexLP-TBS, 26. Compound TBS-T2Br2-TBS 36 (1.33 g, 2.22 mmol) was added to a 100 mL round bottom flask with Schlenk arm under N2. Dry Et2O (40 mL) solution was added and the reaction cooled to –78 °C. n-BuLi (1.5 mL, 1.6 M in hexane) was added and the reaction stirred at –78 °C for 1 h. 55 Chlorodiphenylphosphine (0.45 mL, 2.4 mmol) was added dropwise, and the reaction stirred for 2 h at room temperature. n-BuLi (1.67 mL, 1.6 M in hexane) was added and the reaction stirred for 1 h at –78 °C. Dimesitylfluoroborane (0.780 g, 2.91 mmol) was rapidly added to the reaction under positive nitrogen pressure and the reaction stirred for 16 h at room temperature under N2. The reaction turned from dark yellow to red in colour. H2O2 (0.3 mL, 30% w/w in H2O) was added to the reaction and the reaction stirred for 2 h at room temperature. The product was extracted using Et2O and washed with H2O and brine, and dried using MgSO4. The solvent was removed at reduced pressure and the orange solid was purified by column chromatography using CH2Cl2/hexanes (1:2 v/v) as the eluting solvent. The product was collected and the solvents removed under reduced pressure to afford an orange precipitate. Yield: 0.770 g, 40%. 1H NMR (CDCl3, 400 MHz, 25 °C): δ 7.52-7.57 (m, 2H, H3), 7.34-7.41 (m, 8H, H1+H2), 6.87 (s, 1H, H10), 6.57 (d, 3JPH = 4.3 Hz, 1H, H4), 6.49 (s, 4H, H8), 2.18 (s, 6H, H9), 1.86 (s, 12H, H7), 0.95 (s, 18H, H6+H12), 0.13 (s, 12H, H5+H11). 31P{1H}NMR (CDCl3, 162 MHz, 25 °C): δ 32.14. HRMS (ESI) m/z: calc. C54H65OPSi2S210B [M+H]+: 890.3882, found 890.3907. FlexLP-TBS, 24. 56 Compound TBS-FlexLP-TBS 26 (0.722 g, 0.811 mmol) was added to a 20 mL glass vial under N2 atmosphere. N2-sparged THF was added (10.0 mL) and the solution stirred. H2O (0.3 mL) was added dropwise to the reaction, where no visible colour change occurred. TBAF (0.9 mL, 1.0 M in THF, 0.9 mmol) solution was added dropwise and the reaction stirred at room temperature for 16 h. During this time the reaction slowly turned from orange to red, then to yellow. The solvent was removed at reduced pressure, and the product extracted using Et2O and washed with H2O and brine. The organic layer was dried using MgSO4, and then filtered using vacuum filtration. The Et2O was removed at reduced pressure, and the crude product purified by column chromatography using CH2Cl2/hexanes (3:2 v/v). The product was collected and the solvents evaporated under reduced pressure to afford a yellow solid. Attempts to grow single crystals were unsuccessful. Yield: 0.480 g, 77%. 1H NMR (CDCl3, 400 MHz, 25 °C): δ 7.53-7.61 (m, 2H, H3), 7.34-7.41 (m, 8H, H1 + H2), 6.90 (s, 1H, H9), 6.62 (d, 3JPH = 4.3 Hz, 1H, H4), 6.52 (s, 4H, H7), 3.35 (s, 1H, H5), 2.19 (s, 6H, H8), 1.90 (s, 12H, H6), 0.95 (s, 9H, H11), 0.13 (s, 6H, H10). 13C{1H} NMR (CDCl3, 100 MHz, 25 °C): δ 159.4 (C11), 151.5 (C10), 144.9 (C13), 142.0 (C19), 141.4 (C14), 137.1 (C12), 136.9 (C4, d split by 31P), 135.9 (C17), 134.0 (C6), 132.5 (C9, d split by 31P), 132.3 (C8, d split by 31P), 128.6 (C16), 128.4 (C7, d split by 31P), 124.6 (C20), 123.0 (C5, d split by 31P), 121.3 (C3, d split by 31P), 99.0 (C22), 98.1 (C21), 83.5 (C1), 75.0 (C2), 26.08 (C25), 24.1 (C15), 20.8 (C18), 16.6 (C24), -4.8 (C23). 31P{1H}NMR (CDCl3, 162 MHz, 25 °C): δ 31.37. HRMS (ESI) m/z: calc. C48H51SiOPS210B [M+H]+: 776.3018, found 776.3022. 57 FlexLP dimer, 25. FlexLP-TBS 24 (113 mg, 0.160 mmol), CuCl (2 mg, 0.02 mmol), and bipyridine (33 mg, 0.020 mmol) were added to a 5 mL round bottom flask with stir bar, along with 2 mL of DMF. The reaction stirred at room temperature under air atmosphere for 5 hours, before removing organic solvent by reduced pressure. The crude product was extracted using DCM, and washed with deionized H2O, before drying the organic layer with MgSO4. The organic layer was filtered to remove MgSO4, and solvent removed by reduced pressure. The crude product was purified by silica gel column chromatography using a gradient solvent system starting with 3:2 v/v hexane:DCM to 100 % DCM. The final emissive yellow spot was collected as an orange solid. Attempts to grow single crystals were unsuccessful. Characterization by 13C NMR was attempted but unsuccessful due to solubility issues. Yield: 22 mg, 10 %. 1H NMR (CDCl3, 400 MHz 7.54-7.57 (m, 4H), 7.34-7.39 (m, 16H), 6.89 (s, 2H), 6.66 (d, J = 4.1 Hz, 2H), 6.52 (s, 8H), 2.19 (s, 12H), 1.89 (s, 24H), 0.94 (s, 18H), 0.13 (s, 12H). 31P{1H} NMR (CDCl3, 162 MHz): δ 28.4. Low Resolution MS (EI-MS) m/z: [M + Na]+ calcd for C96H98B2O2P2S4Si2Na: 1574.6; found 1574.7. 58 Figure 2-26. Synthetic scheme for the synthesis of control dimer 28 without functionalization of Lewis pairs Br-T2-tBu, 38. A similar synthetic procedure to that used for TBS-T2Br2-TBS 36 was used to synthesize Br-T2-tBu 38, using copper(I) iodide (0.015 g, 0.080 mmol), trans-bis(triphenylphosphine)palladium(II) dichloride (0.044 g, 0.060 mmol), 5,5'-dibromo-2,2'-bithiophene (0.496 g, 1.53 mmol), and tert-butylacetylene (0.19 mL, 1.54 mmol) as the alkyne reagent. The reaction was stirred at room temperature under an N2 atmosphere for 1.5 h. The product was collected as a light yellow powder. Yield: 0.224 g, 45%. 1H NMR (CDCl3, 400 MHz, 25 °C): δ 6.97 (d, 3J = 3.8 Hz, 1H, H1/H4), 6.95 (d, 3J = 3.9 Hz, 1H, H2/H3), 6.92 (d, 3J = 3.8 Hz, 1H, H2/H3), 6.87 (d, 3J = 3.9 Hz, 1H, H1/H4), 1.31 (s, 9H, H5). HRMS (ESI) m/z: calc. C14H14S279Br [M+H]+: 324.9720, found 324.9729. 59 TBS-T2-tBu, 39. A similar synthetic procedure to that used for TBS-T2Br2-TBS 36 was used to synthesize TBS-T2-tBu 39, using copper(I) iodide (0.009 g, 0.05 mmol), trans-bis(triphenylphosphine)palladium(II) dichloride (0.034 g, 0.050 mmol), Br-T2-tBu 38 (0.197 g, 0.606 mmol), and (tert-butyldimethylsilyl)acetylene (0.12 mL, 0.64 mmol) as the alkyne reagent. The product was collected as a light yellow powder. Yield: 0.232 g, 99%. 1H NMR (CDCl3, 400 MHz, 25 °C): δ 7.10 (d, 3J = 3.8 Hz, 1H, H2/H4), 6.98 (s, 2H, H1+H3), 6.96 (d, 3J = 3.8 Hz, 1H, H2/H4), 1.31 (s, 9H, H5), 0.98 (s, 9H, H7), 0.18 (s, 6H, H6). HRMS (ESI) m/z: calc. C22H29SiS2 [M+H]+: 385.1480, found 385.1478. T2-tBu, 40. A similar synthetic procedure to that used for FlexLP-TBS 24 was used to synthesize T2-tBu 40, using TBS-T2-tBu 39 (0.233 g, 0.606 mmol), H2O (0.3 mL), and TBAF (0.65 mL, 1.0 M in THF, 0.65 mmol). The product was collected as a yellow solid. Product quickly decomposes in air and light, therefore was used the same day without characterization by HRMS. Yield: 0.152 g, 93%. 1H NMR (CDCl3, 400 MHz, 25 °C): δ 7.16 (d, 3J = 3.8 Hz, 1H, H1), 6.99 (m, 3H, H2+H3+H4), 3.40 (s, 1H, H6), 1.31 (s, 9H, H5). 60 Control dimer, 28. T2-tBu 40 (150 mg, 0.550 mmol) and Cu(OAc)2•H2O (5 mg, 0.03 mmol) were added to a 20 mL vial with stir bar and dissolved using 6.5 mL of DCM. Piperidine (0.07 mL, 0.7 mmol) was added dropwise, and the orange solution stirred at room temperature under air atmosphere for 18 hours. Over this time the orange solution turned dark red. The red solution was washed with deionized H2O, and the organic layer dried using MgSO4, filtered, and DCM removed using reduced pressure. The crude product was dry loaded onto a silica column and purified by column chromatography using a 5:1 v/v hexane/DCM. The second spot was collected as a yellow solid. Yield: 80 mg, 27 %. 1H NMR (CDCl3, 400 MHz 7.23 (d, J = 3.9 Hz, 2H), 7.03 (d, J = 3.8 Hz, 2H), 7.01 (d, J = 3.9 Hz, 2H), 6.99 (d, J = 3.8 Hz, 2H), 1.31 (s, 18H). 61 Chapter 3: Solvent- and Temperature-Responsive Platinum(II)-Functionalized Flexible Lewis Pairs 3.1 Introduction 3.1.1 Platinum(II)-Containing Molecular Switches A wide variety of platinum(II)-containing coordination complexes have been investigated because of the square-planar d8 electronic configuration of Pt(II) complexes,81 which offers interesting photophysical properties when paired with conjugated organic ligands. The square-planar d8 electronic configuration hinders spin-forbidden d-d transitions from occurring, which can often quench emission pathways,82 while their heavy atom effect and metal character can promote new emission characteristics, such as MLCT or LMCT events or phosphorescence.83 Terpyridine (tpy) is a common ligand for Pt(II) complexes because of the well-known capability of Pt(II)-tpy complexes to exhibit strong MLCT absorption characteristics in the visible region, and ability to tune the absorption intensity or energy of the CT transition by changing the ancillary ligand.82 Application of stimuli-responsive ancillary ligands on Pt(II) centers has been investigated in order to obtain tunable CT states, which may be useful for developing tunable photosensitizers.84–86 An example from our group utilizes dithienylethene as an ancillary ligand on a Pt(II)-tpy complex to investigate the role of ligand conformation on the MLCT intensity (Figure 3-1).87 When the dithienylethene was in the closed form, a low energy absorption peak appeared, attributed to the π-π* transition of the ring-closed dithienylethene. The ring-closing process could be triggered by exciting the dithienylethene chromophore directly using UV light (302 nm), or 62 indirectly by exciting the lower energy MLCT/LLCT absorption band (465 nm) attributed to the Pt-tpy chromophore. In this example, the added Pt-tpy chromophore significantly lowers the energy required to switch the dithienylethene to the closed form, demonstrating the synergistic effect of conjugating the two chromophores to one another. Another example is shown in Figure 3-2 where a terthiophene ligand bridges two Pt(II)-tpy centers, where the solvent induces a conformational change on the bimetallic complex which can be monitored by the energy of the CT band in the absorption spectrum.88 By changing the proximity of the Pt centers to one another, a shift in MLCT energy was observed. Figure 3-1. Dithienylethene-functionalized Pt-tpy complex which switches from open to closed form by excitation of the Pt-tpy chromophore.87 Figure 3-2. Bimetallic Pt-tpy complex with a terthiophene linker switches conformation with surrounding solvent.88 63 3.1.2 Metal-Functionalized FlexLP Systems Stimuli-responsive chromic and fluorescent materials have gained attention because of their potential uses in sensing, displays, and imaging technologies.3,89–92 The reversible response to controlled external stimuli, including light,22,93 temperature,94,95 solvent,21,96 or redox potential,97,98 and the ease by which the optical response may be monitored, make these “smart” materials attractive. A class of stimuli-responsive compounds are flexible Lewis pairs (FlexLPs), where Lewis basic and Lewis acidic moieties exist in an equilibrium between an open Lewis pair conformation and closed Lewis adduct conformation, which may be controlled through external stimuli. Thus far, the majority of FlexLP systems have been constructed using conjugated organic backbones substituted with varying functional groups.42,48,53,99,100 Incorporation of transition metal complexes into FlexLPs may allow the electronic, optical and redox properties of the transition metal centers to be coupled to the FlexLP. An example of a ferrocene-framework FlexLP with boron and pyridine Lewis pair (43) was reported by Jäkle and coworkers,101 demonstrating a dynamic equilibrium between closed and open forms in solution with heating at elevated temperatures (Figure 3-3a). Another system reported by Erker and coworkers also contained a ferrocene complex functionalized with a boron and phosphine Lewis pair (44) which could also switch between open and closed form with temperature, observed through variable temperature NMR experiments.102 The reactivity of the open form of the ferrocene-boron-phosphine complex towards small molecules was further investigated (Figure 3-3b). 64 Figure 3-3. Previous metal-containing FlexLP systems.101,102 The open and closed forms of both complexes were observed by variable temperature NMR, where it was shown that the complexes respond with temperature. To date, there have been limited studies in which FlexLP systems are coupled to metal centers and show changes in optical properties.101–103 In this Chapter, an optically active metal-functionalized FlexLP system, and investigation of the influence of incorporation of a transition metal on the switching behavior and optical properties at different temperature and in varying solvents, is reported. The strategy involves appending terpyridyl-functionalized platinum(II) metal centers to the FlexLP backbone previously established in our group to give monometallic and bimetallic platinum(II)terpyridine (Pt-tpy) FlexLP complexes (Figure 3-4). Pt-tpy metal complexes are known to exhibit intense and tunable charge transfer absorption bands,88,104,105 which are anticipated to allow for monitoring of electronic differences between open and closed structures. Figure 3-4. Structures of monometallic Pt-FlexLP and bimetallic Pt-FlexLP-Pt investigated in this Chapter. 65 3.2 Synthesis and Characterization of Pt(II)-FlexLP Complexes Figure 3-5. Synthesis of monometallic Pt-FlexLP and bimetallic Pt-FlexLP-Pt complexes. Copper(I) catalyzed coupling between t-Bu3tpy-Pt(II) chloride and terminal alkyne-functionalized FlexLP gave monometallic (Pt-FlexLP) and bimetallic (Pt-FlexLP-Pt) Pt-containing FlexLPs (Figure 3-5). Complete synthetic procedures can be found at the end of this Chapter. Of note is the stability to air and light of the terminal alkyne-functionalized FlexLP precursors (FlexLP-TBS and FlexLP), which simplifies purification and characterization, previously described in Chapter 2. Characterization of Pt-FlexLP and Pt-FlexLP-Pt by 31P{1H} NMR spectroscopy in deuterated solvents of varying Kamlet-Taft HBD strengths49 was carried out to assess the effect of the platinum centers on the switching of the Lewis pairs between open and closed forms (Figure 3-6). A downfield shift in the 31P peak spanning 10 ppm was observed with decreasing HBD strength for both complexes, with similar downfield shifts observed in non-metallated FlexLP systems with decreasing HBD solvents.48 The downfield shift is a result of the phosphorus nucleus 66 becoming increasingly deshielded due to the neighboring oxygen donating electron density to the boron center as increasing amounts of the FlexLP in the closed form are present, previously described in Chapter 2. 31P{1H} NMR spectra of Pt-FlexLP and Pt-FlexLP-Pt suggest both complexes experience similar solvent effects, where the HBD strength of the solvent results in a shift in the equilibrium between the two conformations either to the open or closed form. Broadening of the 31P NMR peak is observed for Pt-FlexLP-Pt in CD3OD, which was not observed for any other of the compounds, nor was there broadening observed in the 1H NMR spectrum of Pt-FlexLP-Pt in CD3OD. It is speculated that the broadening of the 31P NMR peak of Pt-FlexLP-Pt in CD3OD originates from a difference in spin-spin relaxation rate rather than a difference in forward and reverse reaction rates between open and closed forms. Figure 3-6. 31P{1H} NMR spectra of (a) complex Pt-FlexLP and (b) complex Pt-FlexLP-Pt in solvents of varying HBD strength at 25 °C (400 MHz). 67 To further confirm the solvent dependent behavior of these FlexLP complexes, NOESY/ROESY NMR experiments of Pt-FlexLP-Pt in THF-d8 and CD3OD were performed, in order to examine differences in the correlation between the phenyl protons attached to the phosphine group and the ortho-CH3 mesityl protons attached to the boron center. In non-HBD THF-d8 a correlation peak between these protons is observed, indicating the phenyl and ortho-CH3 mesityl protons are close enough to each other to experience a through-space nOe (Figure 3-7). This suggests Pt-FlexLP-Pt exists primarily in the closed form in the non-HBD solvent THF-d8. In HBD CD3OD, however, a correlation is not observed, indicating the phenyl and mesityl protons are further away from one another and the FlexLP exists largely in the open form (Figure 3-8). Figure 3-7. NOESY spectrum of Pt-FlexLP-Pt in THF-d8. Correlation between H10 and H12/H13 is observed (black rectangle). The starred peak is residual THF solvent. 68 Figure 3-8. ROESY spectrum of Pt-FlexLP-Pt in CD3OD. Correlation between H10 and H12/H13 is not observed. 3.3 Room Temperature Photophysical Characterization of Pt(II)-FlexLP Complexes Absorption and emission spectroscopy experiments were performed on the two complexes in varying solvents, however it should be noted that no emission was observed for either complex at room temperature or lower temperatures. Differences in UV-Vis absorption with solvents of varying HBD strength were observed for Pt-FlexLP and Pt-FlexLP-Pt (Figure 3-10). The low energy LLCT/LMCT band (450-550 nm) is red-shifted (15-34 nm) for Pt-FlexLP-Pt compared to Pt-FlexLP in all solvents tested. A blue shift in the LLCT/LMCT bands of both complexes occurred with increasing HBD solvents. The absorption band attributed to the bithiophene π-π* transition (360–440 nm) for complex Pt-FlexLP-Pt decreased in extinction coefficient and shifted to slightly higher energy with increasing HBD solvents. 69 Figure 3-9. Control complexes Pt-T2 and Pt-T2-Pt. Pt-FlexLP and Pt-FlexLP-Pt were compared with mono- and bimetallic Pt(tpy)-bithiophene control complexes Pt-T2 and Pt-T2-Pt, which did not contain Lewis pairs (Figure 3-9). The LLCT/LMCT band of complex Pt-T2-Pt is red-shifted compared to the band of Pt-T2 in all solvents examined, similar to the behavior observed for Pt-FlexLP and Pt-FlexLP-Pt (15-20 nm, Figure 3-10). In contrast to the behavior of Pt-FlexLP and Pt-FlexLP-Pt, the LLCT/LMCT bands of Pt-T2 and Pt-T2-Pt do not undergo a significant shift in energy with solvents of varying HBD strength. These observations indicate the shift observed for the FlexLP systems is due to the difference in open and closed conformations of the FlexLPs, with a blue-shift in the LLCT/LMCT band observed with increasing amounts of the open form. Consequently, the ability to tune the charge transfer energies of complexes Pt-FlexLP and Pt-FlexLP-Pt is possible by controlling the equilibrium between open and closed states of the FlexLP ligands. 70 Figure 3-10. Absorption spectra of complexes Pt-FlexLP (a), Pt-FlexLP-Pt (b), Pt-T2 (c), and Pt-T2-Pt (d) in solvents with varying HBD strength (1.0 × 10-5 M). The LLCT/LMCT band is slightly blue-shifted in ACN due to the increased polarity compared to acetone and MeOH. Complexes Pt-T2 and Pt-T2-Pt did not dissolve in THF. Figure 3-11. Complexes (OOC-tpy)Pt-T and (OOC-tpy)Pt-T2 synthesized and characterized by the Yam group.106 71 Table 3-1. Comparison of absorption data of complex Pt-FlexLP and Pt-FlexLP-Pt with (OOC-tpy)Pt-T and (OOC-tpy)Pt-T2. Sample Solvent Absorption λmax [nm] (εmax × 10-4 [M-1cm-1]) Pt-FlexLP MeOH 340 (3.40), 398 (2.05), 460 (0.85) THF 345 (2.26), 402 (3.03), 477 (0.90) Pt-FlexLP-Pt MeOH 340 (4.96), 405 (2.27), 475 (1.50) THF 344 (3.09), 419 (3.53), 511 (1.35) (OOC-tpy)Pt-T 106 MeOH 336 (1.83), 356 (2.07), 400 (0.55), 460 (0.57) (OOC-tpy)Pt-T2 106 MeOH 356 (3.65), 406 (0.63), 492 (0.62) Terpyridyl-functionalized Pt(II)acetylene monometallic complexes containing thiophene ((OOC-tpy)Pt-T) and bithiophene ((OOC-tpy)Pt-T2) were previously synthesized by Yam and coworkers (Figure 3-11, Table 3-1),106 where the absorption energy of the LLCT/MLCT band shifted to lower energy in conjunction with increasing π-conjugation length on the acetylene group. Comparing the behavior of complexes Pt-FlexLP and Pt-FlexLP-Pt to (OOC-tpy)Pt-T and (OOC-tpy)Pt-T2, Pt-FlexLP and Pt-FlexLP-Pt experience a blue-shift in the LLCT/LMCT band when in the open structure, resulting from the increased torsional rotation between the thiophene rings. It is proposed that the increased rotation along the backbone of Pt-FlexLP and Pt-FlexLP-Pt in the open form alters the π-interaction between the FlexLP backbone and Pt metal center, perhaps by altering the Pt-alkyne backbonding strength, thus increasing the LUMO energies of Pt-FlexLP and Pt-FlexLP-Pt in the open form. 72 3.4 Computational Data Complex Pt-FlexLP open form Complex Pt-FlexLP closed form Complex Pt-FlexLP-Pt open form Complex Pt-FlexLP-Pt closed form Figure 3-12. Optimized geometry of complex Pt-FlexLP and Pt-FlexLP-Pt in open and closed forms. The B-O bond length of both Pt-FlexLP and Pt-FlexLP-Pt in the closed form is calculated to be 1.63 Å. DFT and TD-DFT calculations were performed on the optimized geometries of Pt-FlexLP and Pt-FlexLP-Pt in their open and closed forms (Figure 3-12, Table 3-2). The S–C–C–S dihedral angles of complexes Pt-FlexLP and Pt-FlexLP-Pt in the closed form are calculated to be 26.15° and 25.11°, respectively, with a B–O bond length of 1.63 Å for both complexes. The dihedral angle in the open forms are significantly higher, 122.96° and 121.37° for complexes Pt-FlexLP and Pt-FlexLP-Pt respectively. This indicates decreased π-orbital overlap between the thiophene units along the backbone in the open form. 73 Table 3-2. Calculated dihedral angles (°) and orbital energies (eV) of complex Pt-FlexLP and Pt-FlexLP-Pt. Complex Pt-FlexLP Complex Pt-FlexLP-Pt Open Form Closed Form Open Form Closed Form S-C-C-S dihedral angle 122.96 26.15 121.37 25.11 LUMO+2 –2.15 –2.15 –2.59 –2.96 LUMO+1 –2.57 –2.88 –2.95 –3.25 LUMO –2.96 –3.24 –2.97 –3.32 HOMO –5.58 –5.45 –5.44 –5.38 HOMO-1 –6.15 –5.74 –6.00 –5.86 HOMO-2 –6.25 –5.89 –6.18 –5.99 For complexes Pt-FlexLP and Pt-FlexLP-Pt, the HOMO is located on the bithiophene-acetylene unit for both conformations, with a small proportion of orbital density on the Pt centers (Figure 3-13, Figure 3-14). The LUMO and LUMO+1 of complex Pt-FlexLP are delocalized over the Pt-tpy center (Figure 3-13), while the LUMO for Pt-FlexLP-Pt is delocalized over the Pt-tpy center on the phosphorus side, and the LUMO+1 is delocalized over the Pt-tpy center on the boron side (Figure 3-14). 74 HOMO LUMO LUMO+1 Figure 3-13. Complex Pt-FlexLP orbital projections of the HOMO (left), LUMO (middle), and LUMO+1 (right) energy levels in the open (top) and closed (bottom) forms. HOMO LUMO LUMO+1 Figure 3-14. Complex Pt-FlexLP-Pt orbital projections of the HOMO (left), LUMO (middle), and LUMO+1 (right) energy levels in the open (top) and closed (bottom) forms. 75 The percent contribution of the first (HOMOLUMO) and second (HOMOLUMO+1) excited states are similar for both the open and closed forms of each complex (Table 3-3). The HOMOs for Pt-FlexLP and Pt-FlexLP-Pt are similar in energy regardless of whether the complex is in the open or closed form, however the LUMO and LUMO+1 energies differ substantially between the two conformations. The LUMO and LUMO+1 are significantly higher in energy for the open form compared to the closed form, which agrees with observed trends in the absorption spectra for the LLCT/LMCT band. Table 3-3. Excited states of complex Pt-FlexLP and Pt-FlexLP-Pt in the open and closed forms calculated at the TD-DFT B3LYP/(6-31G**). Oscillator strengths for calculated excited states are reported in square brackets. Sample Energy transition Excited state Energy Pt-FlexLP open S1 H  L (96.9 %) [0.0366] 2.20 eV S2 H  L+1 (96.7 %) [0.0013] 2.60 eV Pt-FlexLP closed S1 H  L (97.5 %) [0.2414] 1.83 eV S2 H  L+1 (98.3 %) [0.0106] 2.24 eV Pt-FlexLP-Pt open S1 H  L (81.5 %) [0.1126] 2.07 eV S2 H  L+1 (82.5 %) [0.1341] 2.10 eV Pt-FlexLP-Pt closed S1 H  L (96.6 %) [0.0520] 1.70 eV S2 H  L+1 (97.3 %) [0.3028] 1.77 eV 3.5 Variable Temperature Photophysical Characterization Studies were conducted on complex Pt-FlexLP-Pt to determine whether a metallated FlexLP could retain the temperature-responsive switching observed in non-metallated FlexLPs.13 76 It is proposed that at lower temperature the equilibrium shifts from open to closed forms in HBD solvents due to the decrease in entropy within the system, favoring the more-rigid closed form.48 Variable temperature 31P{1H} NMR spectroscopy in CD3OD of Pt-FlexLP-Pt (Figure 3-15) shows a downfield shift with decreasing temperature, indicating in HBD solvent the equilibrium shifts from open to closed, and that the Pt-tpy centers do not inhibit the temperature-dependent equilibrium of the FlexLP. Figure 3-15. 31P NMR{1H} of complex Pt-FlexLP-Pt in CD3OD with decreasing temperature. The peak at 30 ppm shifts downfield and broadens with decreasing temperature, until -35 °C. At -45 °C the peak narrows and becomes increasingly sharp due to decreased vibration and rotation in the molecule. Variable temperature absorption experiments of Pt-FlexLP-Pt and control complex Pt-T2-Pt in MeOH/EtOH (1:4 v/v) and acetone were carried out to further explore the effect of temperature on the FlexLP system compared with the control complex Pt-T2-Pt. The LLCT/LMCT band of complex Pt-FlexLP-Pt in strong HBD solvent red-shifted with decreasing temperature from ~480 nm to ~500 nm, while the peak at ~410 nm experienced a similar red-shift with decreasing temperature, becoming increasingly similar to the absorption spectrum of Pt-FlexLP-Pt in the closed form at room temperature (Figure 3-16). In low-HBD acetone, the 77 LLCT/LMCT band did not undergo a significant shift with decreasing temperature, nor did the peak at around 415 nm significantly change, suggesting that the FlexLP only switches from the open to the closed form with decreasing temperature in strong HBD solvent (Figure 3-16). Complex Pt-T2-Pt did not show a significant shift in the energy of the LLCT/LMCT band when subjected to decreasing temperatures in either MeOH/EtOH or acetone solvents (<5 nm shift, Figure 3-16), further supporting that the red-shift in LLCT/LMCT band observed for Pt-FlexLP-Pt is due to the shift in equilibrium from the open form to the closed form with decreasing temperature. Figure 3-16. Variable temperature absorption spectra of complex Pt-FlexLP-Pt (a-b, 5 × 10-6 M) and Pt-T2-Pt (c-d, 1 × 10-5 M) in MeOH/EtOH (1:4 v/v) (a,c), and in acetone (5 × 10-6 M) (b,c). 78 3.6 Conclusion In summary, transition metal-functionalized FlexLPs were synthesized using a one-step copper(I) catalytic reaction under mild conditions from terminal alkyne-functionalized FlexLP precursors and halogenated Pt metal complexes. Both the monometallic and bimetallic Pt-tpy-functionalized FlexLP complexes exhibited solvent-responsive conformational changes. In non-HBD solvent, such as THF, both FlexLP complexes exist in the closed form, while in HBD solvent the complexes switched to the open form. With decreasing temperature, bimetallic FlexLP complex Pt-FlexLP-Pt switched from open to closed geometries in a HBD solvent. Our findings demonstrate that both monometallic and bimetallic transition metal-functionalized FlexLPs retain the stimuli-responsive equilibrium effects observed in non-metallated FlexLP systems of the same type,48 demonstrating the versatility of these FlexLP systems. The observed shifts in the LLCT/LMCT bands demonstrate that the FlexLP ligands can effect reversible changes in the excited state electronic properties of the metal centers. This may allow solvent and temperature control of photoredox catalysis and excited state electron transfer involving the metal centers. Exploration of other transition metal-functionalized FlexLPs, such as iron or ruthenium-functionalized complexes, may encourage the discovery of redox-active FlexLPs, expanding the potential applications for FlexLPs as stimuli-responsive conductive materials. 3.7 Experimental Details 3.7.1 General 1H, 13C, 31P{1H}, HSQC, HMBC, NOESY, and ROESY NMR experiments were carried out on a Bruker Avance 400 spectrometer. 31P{1H} NMR spectra are referenced to 85% H3PO4 as an external standard. 1H and 13C NMR spectra are referenced to residual 1H and 13C solvent peaks 79 respectively. 1H assignments are made using HSQC and HMBC NMR experiments. Absorption spectra were obtained using a Varian Cary 5000 UV-vis-NIR spectrophotometer, with variable temperature measurements obtained using an Oxford Instruments OptistatDN liquid N2 cryostat. Electrospray ionization (ESI) mass spectrometry data were obtained on a Waters/Micromass LCT TOF-MS spectrometer. Elemental analysis (EA) data were obtained using a Thermo Flash 2000 Elemental Analyzer. Density functional theory (DFT) calculations were carried out using the Gaussian 16 Rev.a03 suite of programs.107 The B3LYP functional with 6-31G** basis set (for C, H, N, S, P, B, Si and O atoms)108–110 and the LANL2DZ effective-core pseudopotential (for Pt)111 was employed to optimize the geometry of the ground-states (S0) with no symmetry restrictions. All the calculations were performed in the presence of the solvent (methanol and tetrahydrofuran). Solvent effects were considered within the self-consistent reaction field (SCRF) theory using the polarized continuum model (PCM) approach.112,113 Time-dependent DFT (TD-DFT) calculations were also performed to understand the nature of singlet excited states.114–116 Synthetic procedures involving air or moisture-sensitive reagents were carried out using standard Schlenk techniques under inert N2 atmosphere. Dry THF and Et2O solvents were obtained from an Inert PureSolv MD5 Solvent Purification System (SPS). H2O2 solution (30% wt. in H2O), n-BuLi (1.6 M in hexane), diisopropylamine, chlorodiphenylphosphine, copper(I) iodide, tetrabutylammonium fluoride (TBAF, 1.0 M in THF), and 3,3',5,5'-tetrabromo-2,2'-bithiophene were purchased from Sigma Aldrich and used as received. Trans-bis(triphenylphosphine) palladium(II) dichloride was purchased from Strem Chemicals Inc. and used as received. Dimesitylfluoroborane, (triethylsilyl)acetylene, and (tert-butyldimethylsilyl)acetylene were purchased from Tokyo Chemical Industry Co., Ltd. (TCI) and used as received. Chloro(4,4',4''-tri-80 tert-butyl-2,2':6',2''-terpyridine)platinum(II) triflate, [Pt(tBu3tpy)Cl]OTf, was synthesized using previous literature methods,117 and identity and purity verified by 1H NMR spectroscopy and ESI-MS. Purifications via column chromatography were carried out over silica gel (SiliaFlash F60). The procedures for the synthesis of FlexLP-TBS 24, and T2-tBu 40 and precursors were described in Chapter 2. 3.7.2 Synthetic Procedures of New Molecules Figure 3-17. Full synthetic scheme of Pt-FlexLP and Pt-FlexLP-Pt complexes. 81 Figure 3-18. Full synthetic scheme of Pt-T2 and Pt-T2-Pt complexes. TES-T2Br2-TES, 51 A similar synthetic procedure to that used for TBS-T2Br2-TBS (36) in Chapter 2 was used to synthesize TES-T2Br2-TES (51), where copper(I) iodide (0.039 g, 0.20 mmol), trans-bis(triphenylphosphine)palladium(II) dichloride (0.112 g, 0.170 mmol), 3,3',5,5'-tetrabromo-2,2'-bithiophene (1.00 g, 2.10 mmol), were dissolved in dry diisopropylamine/THF solution (60 mL, 1:3 v/v) in a 100 mL round bottom flask equipped with a 82 Schlenk arm under an N2 atmosphere. (Triethylsilyl)acetylene (0.77 mL, 4.3 mmol) was added and the reaction stirred at room temperature for 16 h. Hydrochloric acid (0.3 mL, 3 M) was added to quench the reaction, and THF removed under reduced pressure. The organic layer was extracted with Et2O, washed with H2O and brine, and dried over MgSO4. The solvent was removed under reduced pressure and the crude precipitate purified by column chromatography using hexanes. The product was collected as a light yellow powder. Yield: 1.10 g, 87%. 1H NMR (CDCl3, 400 MHz, 25 °C): δ 7.17 (s, 2H, H1), 1.05 (t, 3J = 7.8 Hz, 18H, H3), 0.69 (q, 3J = 7.8 Hz, 12H, H2). HRMS (ESI) m/z: calc. C24H33Si2S2Br2 [M+H]+: 598.9929, found 598.9916. TES-T2-TES, 53 A similar synthetic procedure to that used for TES-T2Br2-TES (51) was used to synthesize TES-T2-TES (53), using copper(I) iodide (0.027 g, 0.14 mmol), trans-bis(triphenylphosphine)palladium(II) dichloride (0.088 g, 0.17 mmol), 5,5'-dibromo-2,2'-bithiophene (0.498 g, 1.54 mmol), and (triethylsilyl)acetylene (0.60 mL, 3.34 mmol) as the alkyne reagent. The product was collected as a light yellow powder. Yield: 0.585 g, 86%. 1H NMR (CDCl3, 400 MHz, 25 °C): δ 7.11 (d, 3J = 3.8 Hz, 2H, H1/H2), 6.99 (d, 3J = 3.8 Hz, 2H, H1/H2), 1.04 (t, 3J = 7.9 Hz, 18H, H4), 0.68 (q, 3J = 7.9 Hz, 12H, H3). HRMS (ESI) m/z: calc. C24H35Si2S2 [M+H]+: 443.1719, found 443.1717. 83 TES-FlexLP-TES, 52 A similar synthetic procedure to that used for TBS-FlexLP-TBS (26) in Chapter 2 was used to synthesize TES-FlexLP-TES (52), where TES-T2Br2-TES (51) (1.05 g, 1.80 mmol) was added to a 100 mL round bottom flask with Schlenk arm under N2. Dry Et2O (40 mL) solution was added and the reaction cooled to –78 °C. n-BuLi (1.2 mL, 1.6 M in hexanes, 1.9 mmol) was added and the reaction stirred at –78 °C for 1 h. Chlorodiphenylphosphine (0.37 mL, 2.0 mmol) was added dropwise, and the reaction stirred for 2 h at room temperature. n-BuLi (1.3 mL, 1.6 M hexanes, 0.81 mmol) was added and the reaction stirred for 1 h at –78 °C. Dimesitylfluoroborane (0.64 g, 2.4 mmol) was rapidly added to the reaction under positive nitrogen pressure and the reaction stirred for 16 h at room temperature under N2. The reaction turned from dark yellow to red in colour. H2O2 (0.3 mL, 30% w/w in H2O) was added to the reaction and the reaction stirred for 2 h at room temperature. The product was extracted using Et2O and washed with H2O and brine, and dried using MgSO4. The solvent was removed at reduced pressure and the orange solid was purified by column chromatography using CH2Cl2/hexanes (1:2 v/v) as the eluting solvent. The product was collected and the solvents removed under reduced pressure to afford a yellow powder. Yield: 0.68 g, 44%. 1H NMR (CDCl3, 400 MHz, 25 °C): δ 7.52-7.57 (m, 2H, H3), 7.34-7.39 (m, 8H, H1+H2), 6.87 (s, 1H, H10), 6.57 (d, 3JPH = 4.3 Hz, 1H, H4), 6.49 (s br, 4H, H8) 2.18 (s, 6H, H9), 1.87 (s, 12H, H7), 1.00 (t, 3J = 7.9 Hz, 9H, H6/H12), 0.99 (t, 3J = 7.9 Hz, 9H, H6/H12), 0.64 (q, 3J = 7.9 Hz, 6H, H5/H11), 0.63 (q, 3J = 7.9 Hz, 6H, H5/H11). 31P{1H}NMR (CDCl3, 162 MHz, 25 °C): δ 32.13. HRMS (ESI) m/z: calc. C54H65Si2OPS211B [M+H]+: 891.3846, found 891.3839. 84 FlexLP, 27. A similar synthetic procedure to that used for FlexLP-TBS (24) in Chapter 2 was used to synthesize FlexLP (27), where TES-FlexLP-TES (52) (0.674 g, 0.756 mmol) was added to a 20 mL glass vial under N2 atmosphere. N2-sparged THF was added (10.0 mL) and the solution stirred. H2O (0.3 mL) was added dropwise to the reaction, where no visible colour change occurred. TBAF (1.80 mL, 1.0 M in THF, 1.8 mmol) solution was added dropwise and the reaction stirred at room temperature for 16 h. During this time the reaction slowly turned from orange to red, then to yellow. The solvent was removed at reduced pressure, and the product extracted using Et2O and washed with H2O and brine. The organic layer was dried using MgSO4, and then filtered using vacuum filtration. The Et2O was removed at reduced pressure, and the crude product purified by column chromatography using CH2Cl2/hexanes (3:2 v/v). The product was collected and the solvents evaporated under reduced pressure to afford a yellow solid. Attempts to grow single crystals were unsuccessful. Yield: 0.470 g, 94%. 1H NMR (CDCl3, 400 MHz): δ 7.51-7.58 (m, 2H, H3), 7.35-7.42 (m, 8H, H1 + H2), 6.92 (s, 1H, H9), 6.63 (d, 3JPH = 4.4 Hz, 1H, H4), 6.52 (s broad, 4H, H7), 3.36 (s, 1H, H5), 3.31 (s, 1H, H10), 2.18 (s, 6H, H8), 1.90 (s, 12H, H6). 13C{1H} NMR (CDCl3, 100 MHz, 25 °C): δ 160.0 (C11), 151.5 (C10), 145.2 (C13), 143.1 (C19), 141.5 (C14), 137.0 (C12), 136.9 (C4, d split by 31P), 136.0 (C17), 132.7 (C9, d split by 31P), 132.5 (C8, d split 85 by 31P), 130.4 (C6), 128.9 (C16), 128.6 (C7, d split by 31P), 123.7 (C5), 123.1 (C20), 121.7 (C3, d split by 31P), 83.9 (C1), 82.9 (C22), 77.2 (C21), 75.1 (C2), 24.4 (C15), 21.3 (C18). 31P{1H}NMR (CDCl3, 162 MHz, 25 °C): δ 31.21. HRMS (ESI) m/z: calc. C42H36ONaPS211B [M+Na]+: 685.1936, found 685.1928. T2, 54. A similar synthetic procedure to that used for FlexLP (27) was used to synthesize T2 (54), using TES-T2-TES (53) (0.487 g, 1.10 mmol), H2O (0.3 mL), and TBAF (2.2 mL, 1.0 M in THF, 2.2 mmol). The product was collected as a yellow solid. The product quickly decomposes in air and light, therefore was used the same day without characterization by HRMS. Yield: 0.219 g, 94%. 1H NMR (CDCl3, 400 MHz): δ 7.17 (d, 3J = 3.8 Hz, 2H, H1/ H2), 7.03 (d, 3J = 3.8 Hz, 2H, H1/H2), 3.41 (s, 2H, H3). Pt-FlexLP, 45. Compound FlexLP-TBS (24) (0.124 g, 0.160 mmol) and [Pt(tBu3tpy)Cl]OTf (136 mg, 0.170 mmol) were added to a 100 mL round bottom flask under N2 atmosphere. A N2-sparged solution of diisopropylamine/CH2Cl2 (40 mL, 1:4 v/v) was added and the reaction stirred. Copper(I) iodide (0.003 g, 0.02 mmol) was quickly added under positive N2 pressure and the reaction turned dark red. The reaction stirred under nitrogen atmosphere for 16 h. The solvent was removed at reduced pressure, and the product purified by 86 column chromatography using CH2Cl2/acetone (5:1 v/v). The product was collected and the solvent removed at reduced pressure to afford a dark red solid. Attempts to grow single crystals were unsuccessful. Yield: 0.162 g, 66%. 1H NMR (CDCl3, 400 MHz): δ 8.98 (d, 3J = 6.0 Hz, 2H, H6), 8.47 (s, 2H, H3), 8.40 (d, 4J = 2.0 Hz, 2H, H4), 7.61 (dd, 3J = 6.0, 4J =2.0 Hz, 2H, H5), 7.54-7.58 (m, 2H, H14), 7.37-7.40 (m, 8H, H12+H13), 6.86 (s, 1H, H8), 6.51 (d, 3JPH = 4.3 Hz, 1H, H7), 6.49 (s, 4H, H9), 2.19 (s, 6H, H11), 1.86 (s, 12H, H10), 1.58 (s, 9H, H2), 1.49 (s, 18H, H1), 0.95 (s, 9H, H16), 0.14 (s, 6H, H15). 31P{1H}NMR (CDCl3, 162 MHz): δ 34.43. HRMS (ESI) m/z: calc. C75H84N3OS2Si194Pt10BP [M-OTf]+: 1369.5319, found 1369.5330. EA calc. 59.99 % C, 5.56 % H, 2.76 % N, 6.32 % S, found 59.49 % C, 5.74 % H, 2.72 % N, 6.19 % S. Pt-FlexLP-Pt, 46. A similar synthetic procedure to that used for Pt-FlexLP (45) was used to synthesize Pt-FlexLP-Pt (46), using FlexLP (27) (0.113 g, 0.171 mmol), [Pt(tBu3tpy)Cl]OTf (0.253 g, 0.323 mmol), and copper(I) iodide (0.002 g, 0.007 mmol). A dark red product was collected. Attempts to grow single crystals were unsuccessful. Yield: 0.242 g, 35%. 1H NMR (CDCl3, 400 MHz): δ 9.08 (d, 3J = 6.0 Hz, 2H, H15), 9.00 (d, 3J = 6.0 Hz, 2H, H6), 8.45 (s, 2H, H3), 8.43 (s, 2H, H18), 8.39 (d, 4J = 2.0 Hz, 2H, H4), 8.36 (d, 4J = 2.0 Hz, 2H, H17), 7.63 (t, 3J = 6.0 Hz, 4J = 2.0 Hz, 2H, H16), 7.62 (dd, 3J = 6.0 Hz, 4J = 2.0 Hz, 2H, H5), 7.55-7.59 (m, 2H, H14), 7.35-7.45 (m, 8H, H12+H13), 6.81 (s, 1H, H8) 6.52 (s, 1H, H7), 6.51 (s, 87 4H, H9), 2.20 (s, 6H, H11), 1.94 (s broad, 12H, H10), 1.57 (s, 18H, H2+H19), 1.49 (s, 36H, H1+H20). 31P{1H}NMR (CDCl3, 162 MHz): δ 33.97. Positive MALDI-TOF m/z: calc C97H104BF3N6O4PPt2S3 [M-OTf]+: 2001.6, found 2002.6. EA calc. 54.69 % C, 4.87 % H, 3.91 % N, 5.96 % S, found 54.16 % C, 5.09% H, 3.59 % N, 5.79 % S. Pt-T2, 47. A similar synthetic procedure to that used for Pt-FlexLP (45) was used to synthesize Pt-T2 (47), using T2-tBu (40) (0.026 g, 0.096 mmol), [Pt(tBu3tpy)Cl]OTf (0.156 g, 0.199 mmol), and copper(I) iodide (0.002 g, 0.01 mmol). A dark red product was collected. Yield: 0.071 g, 73%. 1H NMR ((CD3)2CO, 400 MHz): δ 9.20 (d, 3J = 6.0 Hz, 2H, H6), 8.84 (s, 2H, H3), 8.79 (d, 4J = 2.0 Hz, 2H, H4), 8.03 (dd, 3J = 6.0 Hz, 4J = 2.0 Hz, 2H, H5), 7.22 (d, 3J = 3.8 Hz, 1H, H7/H8/H9/H10), 7.16 (d, 3J = 3.8 Hz, 1H, H7/H8/H9/H10), 7.10 (d, 3J = 3.8 Hz, 1H, H7/H8/H9/H10), 7.09 (d, 3J = 3.8 Hz, 1H, H7/H8/H9/H10), 1.56 (s, 9H, H2), 1.51 (s, 18H, H1), 1.34 (s, 9H, H11). HRMS (ESI) m/z: calc. C43H48N3S2194Pt [M-OTf]+: 864.2916, found 864.2919. 88 Pt-T2-Pt, 48. A similar method as for Pt-FlexLP (45) was followed, using T2 (54) (0.063 g, 0.29 mmol), [Pt(tBu3tpy)Cl]OTf (0.476 g, 0.620 mmol), and copper (I) iodide (0.006 mg, 0.03 mmol). The product was collected as a dark red solid. Yield: 0.155 g, 31%. 1H NMR ((CD3)2CO, 400 MHz): δ 9.21 (d, 3J = 6.0 Hz, 4H, H6), 8.83 (s, 4H, H3), 8.79 (d, 4J = 2.0 Hz, 4H, H4), 8.03 (dd, 4J = 2.0 Hz, 3J = 6.0 Hz, 4H, H5), 7.19 (d, 3J = 3.8 Hz, 2H, H7/H8), 7.11 (d, 3J = 3.8 Hz, 2H, H7/H8), 1.54 (s, 18H, H2), 1.50 (s, 36H, H1). HRMS (ESI) m/z: calc. C67H74N6O3F3S3194Pt2 [M-OTf]+: 1551.4190, found 1551.4205. 89 Chapter 4: Singlet and Triplet Energy Dynamics of Emissive Platinum-FlexLP Complexes 4.1 Introduction 4.1.1 Triplet State Properties of Platinum(II) Complexes As discussed in Chapter 1, heavy atoms can induce ISC from a singlet to a triplet state. By accessing the triplet state, different physical and reactive properties can occur that are not possible in the singlet state, such as phosphorescence or singlet oxygen (1O2) production (Figure 4-1). Singlet oxygen can be produced when ground state triplet oxygen reacts with excited molecules in their triplet state, called photosensitizers (Figure 4-1).118,119 Since singlet oxygen is highly reactive it reacts readily with a range of cellular components, such as proteins, lipids, and DNA, making singlet oxygen an effective tool in cancer-fighting research. Additionally, singlet oxygen can be produced in highly specific areas of the body, because it relies on using light to excite triplet state photosensitizers, therefore singlet oxygen production can be spatially controlled by only applying light in select diseased areas where tumours are located. Research on the relationship between structure and triplet state energy and dynamics is often challenging because of the added ISC requirement for triplet state population compared to population of the singlet state. Additionally, the triplet state is often non-emissive, and requires advanced laser spectroscopy techniques to observe the population and dynamics of these “dark” triplet states. Despite these challenges, understanding of the triplet state energy, lifetime, and ISC dynamics in metal complexes is important for designing complexes to be used as phosphorescent materials for OLEDs, photosensitizers for solar cells, or singlet oxygen producers. 90 Figure 4-1. Energy diagram describing singlet oxygen production. Ground state dissolved oxygen, which exists in the triplet form, collides with photosensitized molecules in their triplet state, quenching the photosensitized molecule and producing singlet oxygen. 4.1.1.1 Differences between Singlet and Triplet Excited State Localization Understanding the structural effect of π-conjugated ligands on the localization of singlet and triplet energy states of materials is important when tailoring phosphorescent emitters with specific energies. Research by the Schanze group has elucidated the relationship between chemical structure and excited state energies, particularly in Pt-containing metallic polymers. They determined that for Pt-alkyne-phenyl oligomers and polymers the MO of the singlet S1 state is significantly delocalized across multiple Pt-alkyne-phenyl repeat units, causing the fluorescence emission to decrease in energy with increasing oligomer length (Figure 4-2).120 However the triplet state phosphorescence did not show this same decrease in energy with increasing oligomer length, and therefore the triplet state was determined to be more localized on a single π-conjugated ligand unit. 91 Research on thiophene-containing Pt complexes and polymers also saw a similar trend where the singlet state was delocalized over multiple repeat units, however the T1 triplet state is significantly localized on a single thiophene ring.121,122 When increasing the length of the thiophene linker to bithiophene and terthiophene, the energy of the T1 state decreased, and remained localized on a single organic linker, with increasingly organic-like properties. Figure 4-2. Examples of Pt-aryl polymers previously studied (a) and the singlet and triplet (b) excited state localizations along the polymer backbone.120–122 4.1.1.2 Emissive and Non-Emissive Triplet Decay Rates of Metal Complexes The overall rate of decay of an excited molecule relies on the rates of emissive and non-emissive transitions, therefore an understanding of the factors which effect these rates in molecules is essential, particularly when designing metal complexes for applications which require specific triplet state lifetime values for increased efficiency. 92 The Yersin group has related phosphorescence lifetimes of different emissive organometallic complexes, including Pt(II) complexes, with their corresponding zero-field splitting values.23 Briefly, zero-field splitting (ZFS) is when degenerate energy states split into distinct substates with different energies when in the presence of more than one unpaired electron (Figure 4-3). The most common example is the Zeeman effect for spin ½ nuclei in NMR spectroscopy, which split into their two substates when in a magnetic field. This same effect occurs in molecules, where the excited triplet state can split into three substates when in a molecule with relatively high ZFS value.123,124 The ZFS value of a molecule is related to the geometry, and the spin-spin and SOC strength within the molecule.125 Figure 4-3. Energy diagram of zero-field splitting of triplet energy substates. Organic molecules often have low or negligible SOC, and therefore only spin-spin coupling between electrons in the π and π* states occurs, giving relatively small splitting energies. Metallic complexes often have high SOC strength due to the heavy atom effect, and therefore can have relatively high ZFS values. It has been shown by the Yersin group that the geometries and energy transition type of metallic complexes also contribute to the overall ZFS value.23,125 Octahedral 93 complexes often exhibit 3MLCT transitions, which allows for efficient SOC because of the large metal contribution. This high SOC efficiency generally increases the ZFS value.126 Additionally, many octahedral complexes are distorted from perfect 90° geometry due to bulky organic ligands, decreasing the symmetry within the complex either in the axial or equatorial plane, which can also contribute to the high ZFS value.127 For square planar complexes, the triplet state is often LC in nature, which decreases SOC efficiency because of the reduced contribution of the metal center to the transition, and thus ZFS values tend to be lower. A correlation between geometry, energy transition type, ZFS value, and overall triplet emission decay rate has been investigated by the Yersin group.23 They reported that the triplet emission decay time exponentially decreases with higher ZFS value, attributed to increased 3MLCT energy transitions compared to complexes with low ZFS value, attributed to 3LC energy transitions. For the non-emissive triplet decay process of a molecule there are two components: the temperature independent decay rate, and the temperature-dependent decay rate.128–130 The temperature-independent decay rate is primarily influenced by the Franck-Condon overlap between triplet and ground state; with larger structural distortions the energy gap between triplet and ground state is smaller, causing faster decay rates (Figure 4-4a). Large structural distortions could arise from CT type transitions, or large differences in vibrational modes, which would alter the geometry of the molecule between its excited and ground state energies. The temperature-dependent decay rate is associated with the thermal population of metal-centered (MC) excited states, which is one of the main causes for emission quenching of a complex at room temperature. Decay to the ground state through the minimum energy crossing point (MECP) between the MC and S0 potential energy surfaces occurs through this pathway (Figure 94 4-4b). The temperature-dependent decay rate can be orders of magnitude larger than the temperature-independent non-emissive decay rate if the MC excited state is of appropriate energy to allow for IC between the T1 state and MC state. The temperature-dependent decay rate can be minimized by using strong-field ligands that increase the MC energy state, thus increasing the required thermal energy to induce IC from the T1 state.130 These findings are significant for researchers designing complexes for OLED applications, which require rapid phosphorescence decay times, or for photosensitizers for electronic devices, which require long triplet lifetimes to increase the probability of charge separation occurring. These results may also aid in elucidating the type of energy transition occurring within complex metallic systems that contain multiple transition pathways. Figure 4-4. Energy diagrams depicting temperature-independent (a) and temperature-dependent (b) non-radiative decay pathways. 95 4.1.2 Photophysics of Pyrene Pyrene is a common π-conjugated ligand that has been used to study room temperature phosphorescence and triplet state dynamics. This is because molecular pyrene has many interesting photophysical properties, such as its ability to exhibit excimer emission in concentrated solutions, and its ability to effectively undergo ISC to its triplet state when conjugated to a transition metal. The increased efficiency of ISC is caused by the increased spin-orbit coupling from the heavy atom effect of the transition metal. However, ISC can also occur in pyrene by undergoing oxygen-induced enhanced intersystem crossing (Figure 4-5).131 This is due to pyrene’s long-lived singlet excited state (~100 ns), which can interact with ground state triplet oxygen to afford a triplet pyrene-triplet oxygen complex intermediate, resulting in excited state triplet pyrene. This can be observed by the change in fluorescence intensity between samples in air versus inert atmosphere. Figure 4-5. Schematic of oxygen-induced enhanced intersystem crossing in pyrene. 96 In addition, pyrene-functionalized metal complexes are known to exhibit room-temperature phosphorescence when ISC occurs to the pyrene ligand centered triplet state. This has allowed researchers to conjugate pyrene onto metal centers with new ancillary ligands in order to investigating the structure and electronic effects of ancillary ligands on triplet state dynamics, by strategically using pyrene with its known triplet state phosphorescence energy. For example, the Castellano group has previously been able to investigate the ultrafast excited state dynamics of pyrene-functionlized Pt complexes with and without bipyridine ancillary ligand to observe the effect of MLCT energy states on the rate of excited state transitions and phosphorescence (Figure 4-6).132 By using ultrafast transient absorption, it was shown that both complexes exhibited room temperature phosphorescence from the pyrene triplet energy state, however the bipyridine-containing complex exhibited a significantly increased rate of ISC to the pyrene triplet state by over an order of magnitude. This was thought to be due to fast ISC between the singlet MLCT and the triplet MLCT located on the bipyridine, followed by fast IC between the triplet MLCT and triplet ligand-centered pyrene excited state. 97 Figure 4-6. Phosphorescent pyrene-functionalized Pt(II) complexes, with varying emission decay rates. An intermediate 3MLCT state is thought to increase the rate of ISC to the pyrene-localized T1.132 4.1.3 Purpose Based on the photophysics of pyrene-functionalized Pt(II) complexes described above, as well as their ability to exhibit room temperature phosphorescence, it was proposed that by functionalizing a Pt-pyrene square planar complex with a FlexLP ligand, the effect of the FlexLP conformation on the triplet state energies and dynamics could be explored, and possibly produce a phosphorescent Pt-FlexLP complex. Thus, this chapter focuses on the design, synthesis, and photophysics of two new emissive Pt-FlexLP complexes in order to analyze the FlexLP triplet state energies and dynamics for both the open and closed FlexLP ligand conformation (Figure 98 4-7). A common way to probe triplet state energy dynamics is by using transient absorption spectroscopy, therefore a short description of the technique is described in section 4.1.4. Figure 4-7. Structure of emissive Pt(FlexLP) complexes investigated in this Chapter. In Chapter 3, Pt(II)-tpy complexes functionalized with FlexLP ligands were synthesized, however were non-emissive. This is not surpising, as Pt(II)-tpy complexes are often non-emissive due to slight distortions of the N-Pt-N bond angle from 90°, inducing low-lying metal-centered (MC) d-d transitions which rapidly decay non-radiatively, therefore quenching emission.133 To achieve emissive Pt(II) complexes, phosphine functional groups (PR3) are incorporated as ancillary ligands due to the strong field ligand character of phosphines, which increases the energy difference between the d orbital energy states (Figure 4-8). 99 Figure 4-8. Simplified diagram comparing the MC d orbital energies in Pt-tpy and Pt-phosphine complexes. 4.1.4 Transient Absorption Spectroscopy Transient absorption is a time-resolved pump-probe technique where the absorption properties of different excited states of a molecule can be observed. This is in contrast to steady-state absorption spectroscopy, where only the absorption properties of the molecular ground state are observed. During transient absorption, a laser at a specific wavelength and pulse time is used to excite (pump) a small percentage of the population of molecules (M) in solution to an excited state at time zero, t0 (Figure 4-9a). Both the excited molecules (M*) and molecules in the GS are probed using white light at a specific time, tn, after the pump light, creating an absorption spectrum of GS and excited state molecules. By taking the difference between the GS absorption spectrum and the absorption spectrum created by the probe light, GS absorption peaks and excited state absorption peaks can be seen. The GS absorption peaks (called GS bleach) will appear in the 100 negative ΔA region because the overall population of GS molecules will be less after initiation of the pump light. The excited state absorption peaks will appear as positive peaks if they do not overlap with the GS absorption energies. Figure 4-9. Schematic of transient absorption spectroscopy depicting the theory (a), and its use for assigning excited state transitions (b-d), including the experimental set-up (b) in relation to the excitation of a population of molecules (c), and the resulting transient absorption graph (d). 101 By changing the time between pump light and probe light, different excited states can be observed based on the different energy transitions that occur within the timeframe (Figure 4-9b). For example, if the time between the pump and probe pulse is relatively short, excited state absorption transitions will be observed for fast energy transitions, such as singlet excited state IC transitions (Figure 4-9c). If the time between pump and probe pulses is longer, ISC between singlet and triplet state may occur within the timeframe between pump and probe, and the absorption spectrum for the triplet excited state may be observed. By plotting the relationship between absorption intensity, absorption wavelength, and time, the lifetime dynamics of different excited states can be analyzed (Figure 4-9d). 4.2 Synthesis and Characterization of Emissive Platinum(II) FlexLP Complexes To obtain an emissive FlexLP-functionalized Pt(II) complex, the dichlorobis(tributylphosphine)platinum(II) complex Pt(PBu3)2Cl2 was used as the platinum reagent because alkylphosphine ligands generally do not directly contribute to the HOMO or LUMO MOs, and therefore alkylphosphine-functionalized Pt(aryl) complexes usually retain the emissive properties associated with the aryl ligand. To make complex Pt(FlexLP)2, Pt(PBu3)2Cl2, CuI, and 2 equivalents of FlexLP-TBS were added together in solution under inert atmosphere in a Cu(I)-coupling reaction (see section 4.6.2 for synthesis details, Figure 4-10). Additionally, in an attempt to obtain a phosphorescent FlexLP system, pyrene was chosen as a second ligand that is known to phosphoresce at room temperature. First, one equivalent FlexLP ligand was added to the Pt-center to afford Pt(FlexLP)Cl with could be further functionalized with alkyne-functionalized pyrene to yield Pt(FlexLP)(Py) (Figure 4-10). The 102 two complexes, Pt(FlexLP)2 and Pt(FlexLP)(Py), were further characterized by 1H NMR, 31P{1H} NMR, HRMS, and EA, to confirm purity. Figure 4-10. Synthesis of Pt(FlexLP)2 and Pt(FlexLP)(Py) complexes. 4.3 Photophysical Data 4.3.1 Absorption Spectroscopy Measurements The absorption spectra of complexes Pt(FlexLP)2 and Pt(FlexLP)(Py) were determined in both non-HBD acetone and HBD MeOH solvent, where it was shown that the spectra for the complexes were red-shifted compared to the free FlexLP-TBS ligand for both solvents (Figure 4-11). Additionally, neither complex exhibits a low energy, broad peak, typical of Pt-containing complexes with CT bands, such as the broad LLCT band seen for the complexes in Chapter 3. For complex Pt(FlexLP)2, the absorption spectra have similar features to the absorption spectra of FlexLP-TBS, in acetone and MeOH solvents, however the extinction coefficient is more than double the magnitude of the value for the free ligand. This is not unexpected, and is common for complexes containing two of the same chromophores. The similarities between the free 103 FlexLP-TBS and Pt(FlexLP)2 complex suggests that the conformation of the FlexLP ligand switches between closed and open form with solvent. The absorption spectra for Pt(FlexLP)(Py) have additional finger-like peaks in both acetone and MeOH between 350-400 nm, which are not shifted between the two solvents. These vibrational splitting peaks are attributed to the pyrene absorption bands, as they also appear for other pyrene-functionalized Pt complexes. In acetone, the spectra shows a shoulder peak at 430 nm, attributed to the red-shifted closed FlexLP absorption band. In MeOH, this shoulder peak is significantly diminished, but still apparent. The intense higher energy peak at 335 nm attributed to the bithiophene-boron transition on the free FlexLP is not shown for Pt(FlexLP)(Py). This may suggest that the thiophene-thiophene torsion angle is significantly more twisted in the open form of the Pt(FlexLP)(Py) complex compared to the open forms of the free FlexLP and Pt(FlexLP)2 complex, breaking the orbital overlap between the thiophene units. Attempts to obtain the crystal structure of the complex to determine the exact structure were unsuccessful. Figure 4-11. Absorption spectra of Pt(FlexLP)2 and Pt(FlexLP)(Py) complexes, and FlexLP-TBS in acetone (a), and MeOH (b). 104 4.3.2 Emission Spectroscopy Measurements The emission spectra of the complexes were collected in acetone and MeOH, in air atmosphere. In acetone, both complexes exhibited slightly red-shifted fluorescence (490 nm) compared to FlexLP-TBS compound (470 nm) (Figure 4-12). This peak was attributed to emission from the closed FlexLP ligand. In MeOH, emission from the complexes was also slightly red-shifted compared to FlexLP-TBS. For Pt(FlexLP)2, when excited at 335 nm there was a single emission peak at 580 nm, attributed to emission from the open form FlexLP. The single emission peak suggests that both FlexLP ligands are predominantly in the open form when in MeOH, and that having two FlexLP ligands within the same complex does not inhibit either FlexLP ligand from switching between open and closed forms with change in solvent. Figure 4-12. Emission spectra of Pt(FlexLP)2 (2 × 10-6 M, λex(acetone) = 405 nm, λex(MeOH) = 335 nm) and Pt(FlexLP)(Py) (2 × 10-6 M, λex(acetone) = 395 nm, λex(MeOH) = 385 nm) complexes, and FlexLP-TBS (5 × 10-6 M, λex(acetone) = 390 nm, λex(MeOH) = 335 nm), in acetone (blues) and MeOH (pinks). Emission spectra of FlexLP-TBS and Pt(FlexLP)2 in MeOH were stopped at 650 nm to prevent detection of excitation wavelength due to Bragg’s Law. 105 For Pt(FlexLP)(Py) complex in MeOH, when excited at 385 nm, there are two distinct emission peaks at 575 nm and 490 nm, attributed to emission from the open and closed FlexLP conformation, respectively. The ratio between open and closed emission intensity (I575:I490) was ~ 4:3 when excited at 385 nm (Figure 4-12). When the complex was excited using higher energy 335 nm light, the two emission peaks remained, with a slight change in ratio of open:closed emission intensity, I575:I490 = 2:1 (Figure 4-13). Additionally, when the complex was excited using higher energy light, vibrational emission peaks at 380-425 nm appeared, attributed to pyrene emission, as the emission peaks had similar energies and appearance as other pyrene-functionalized Pt complexes.134 Figure 4-13. Emission spectra of Pt(FlexLP)(Py) under air and inert atmosphere (2 × 10-6 M, λex = 335 nm). The intensity of the pyrene vibrational emission peaks for the Pt(FlexLP)(Py) complex significantly increased under inert atmosphere, while the emission intensity attributed to the 106 FlexLP emission did not change (Figure 4-13). This increase in the intensity of pyrene fluorescence emission under inert atmosphere is most likely due to the lack of oxygen in the system. Oxygen is known to induce ISC of pyrene-containing systems, increasing triplet state population and decreasing fluorescence quantum yield, therefore, for Pt(FlexLP)(Py), it is highly likely that population of the triplet state occurs, with subsequent non-radiative decay to the ground state. It is postulated that IC to a lower triplet excited state associated with the FlexLP ligand is occurring, followed by non-radiative decay. When the complexes were analyzed under inert atmosphere, no additional peaks attributed to room temperature phosphorescence appeared. To confirm phosphorescence peaks were not hidden underneath the fluorescence peaks, room temperature time-resolved emission spectroscopy experiments under inert atmosphere were performed, and no emission signal was observed. Therefore, it is concluded that neither Pt(FlexLP)2 nor Pt(FlexLP)(Py) phosphoresces at room temperature. This was unexpected for Pt(FlexLP)(Py), as pyrene-functionalized Pt(II) complexes are known to phosphoresce at room temperature. Emission lifetime measurements were obtained for both complexes under air and inert atmosphere, and compared with FlexLP-TBS (Table 4-1, see section 4.6.3 for complete emission decay spectra). For FlexLP-TBS, in acetone where the conformation is in the closed form, the decay profile showed a biexponential decay, with the major contribution from a short component (0.98 ns) associated with emission from the closed form. In MeOH, the decay profile is also biexponential, with the major contribution from a much longer lifetime component (10.45 ns) associated with the open form. A discussion of emission lifetimes of FlexLP-containing organic fluorophores may be found in Chapter 2, therefore discussion will focus on the Pt-containing complexes in this Chapter. 107 Table 4-1. Emission lifetime data for Pt(FlexLP)2, Pt(FlexLP)(Py), and FlexLP-TBS in acetone and MeOH. Solvent Conditions Lifetimes (ns) and Percent Contributions FlexLP-TBS Pt(FlexLP)2 Pt(FlexLP)(Py) Acetone - Air 0.98 (98%) 5.81 (2%) 0.03 (100%)a 0.07 (97%) a 4.1 (3%) Acetone - Inert -- 0.09 (98%)a 2.6 (2%) 0.07 (97%) a 7.1 (3%) MeOH – Air 0.91 (26%) 10.45 (74%) 0.23 (31%) 1.1 (65%) 4.3 (4%) 0.07 (96%) a 4.5 (4%) MeOH - Inert -- 0.26 (32%) 1.1 (64%) 4.8 (4%) 0.06 (96%)a 4.1 (3%) 32 (1%) a Lifetimes are on a similar timescale to the minimum timescale the instrument can calculate, and therefore have a greater uncertainty. Samples were excited with a 370 nm laser, and emission detected from >400 nm. Both complexes exhibited significantly shorter emission lifetimes compared to FlexLP-TBS, suggesting that ISC occurs for the complexes at a similar timescale as emission decay. It should be noted that many of the emission lifetimes are on a similar timescale to the minimum timescale that the instrument can accurately determine by deconvoluting between sample and background response, and therefore may be faster than what is reported. Pt(FlexLP)2 has a faster emission decay in acetone compared to in MeOH, similar to FlexLP-TBS, associated with the difference in conformation in the two solvents. In inert 108 atmosphere the emission decay is slightly longer in acetone, but remained the same when in MeOH. For Pt(FlexLP)(Py), in both acetone and MeOH, there are both short and long lifetime components, which do not significantly change in lifetime or percent contribution in air. A slight increase in the long lifetime component is observed in acetone under inert atmosphere, changing from 4.1 ns to 7.1 ns. In MeOH, a third emission lifetime (32 ns) is observed under inert atmosphere, with small percentage contribution. Considering that the 370 nm excitation laser used is high enough energy to excite the complex into the pyrene LC singlet state, it is possible that a small portion of the emission associated with the pyrene emission above 400 nm is observable. It is suggested that the long lifetime component is associated with the pyrene emission because of the small percent contribution, which increases in lifetime under inert atmosphere due to the lack of quenching from oxygen-induced enhanced ISC. Importantly, under inert atmosphere, the emission lifetime does not increase (to > 100’s ns) for either complex, indicating thermally activated delayed fluorescence from reverse ISC from the triplet state to the singlet state does not occur. 4.4 Transient Absorption Spectra 4.4.1 Picosecond Transient Absorption Spectroscopy Measurements In an attempt to confirm whether triplet state population occurs to a low lying 3FlexLP excited state, transient absorption spectroscopy was carried out. Additionally, transient absorption lifetimes of both Pt(FlexLP)2 and Pt(FlexLP)(Py) complexes in acetone and MeOH were analyzed to determine whether FlexLP conformation affects overall triplet state lifetime. Using a streak camera system equipped with a 355 nm pump laser (picosecond pulse width), the overall 109 decay rate of a dark excited state was determined for Pt(FlexLP)2 and Pt(FlexLP)(Py) complexes over the range 440 nm – 730 nm associated with the positive transient absorption peak (see section 4.6.3 for transient absorption spectra), in acetone and MeOH, under both inert and air atmosphere (Figure 4-14, Table 4-2). For all complexes, the overall transient absorption lifetimes were relatively long in inert atmosphere (10-7 to 10-5 seconds) indicating that both complexes undergo ISC to populate a dark triplet state, in both solvents. Additionally, when the complexes were in air atmosphere, the lifetimes of the complexes were shorter (10-7 seconds) suggesting oxygen is able to interact and quench the excited triplet state of the complex, and possibly produce singlet oxygen. It is possible that the effects of the complexes’ conformations on singlet oxygen production may show that these complexes behave as fluorescent stimuli-responsive singlet-oxygen generators, however further work will be needed to explore this possibility. The behavior of a complex to both generate singlet-oxygen and fluorescently emit would be beneficial for development of cancer-fighting drugs, as the singlet-oxygen producing molecule could be tracked by fluorescence microscopy. 110 Figure 4-14. Transient absorption decay curves of complexes in air (a) and inert (b-d) atmosphere in acetone (b) and MeOH (c,d). In air, all complexes fully decay monoexponentially after 2 μs, while in inert atmosphere the complexes decay biexponentially over longer timescales. Table 4-2. Overall transient absorption lifetimes of both complexes in acetone and MeOH, in air and inert atmosphere, from 440 –730 nm. Parentheses indicate the percent contribution to the overall lifetime. Complex Air Atmosphere (μs) Inert Atmosphere (μs) Acetone MeOH Acetone MeOH Pt(FlexLP)2 0.31 0.19 7.0 (97%), 34 (3%) 0.48 (99%), 4 (1%) Pt(FlexLP)(Py) 0.24 0.14 6.5 (97%), 30 (3%) 0.26 (99%), 2 (1%) 111 Figure 4-15. Transient absorption decay curves for Pt(FlexLP)2 in acetone (a) and MeOH (c) and Pt(FlexLP)(Py) in acetone (b) and MeOH (d) analyzed at specific wavelengths. Higher energy wavelength (450 nm, blue) has a slightly longer decay time than lower energy (red) wavelengths. Both complexes exhibit a long lifetime component that contributes considerably less to the overall triplet lifetime. By analyzing the lifetime at specific wavelengths attributed to the excited state absorption maximum and broad shoulder features (Figure 4-21 in section 4.6.3), it was determined for both complexes the longer component is from a transient absorption feature at higher energy (Figure 4-15). The decay curve for wavelength 450 nm is shown to have a slightly 112 longer lifetime and a higher percentage contribution of the long component compared to lower energy wavelengths when fitted to a biexponential curve (Table 4-3). It is not known what energy transition gives this longer lifetime feature, however considering the longer lifetime feature is from a high energy absorption, it could be a transition from the T1 state to a higher T3 state. Table 4-3. Transient absorption decay data for specific wavelengths, including the lifetimes (τ1 and τ2, in μs) and percent contribution (in parenthesis, see section 4.7.1 for more details). Wavelength Pt(FlexLP)2 Pt(FlexLP)(Py) Acetone (μs) MeOH (μs) Acetone (μs) MeOHa (μs) 450 nm τ1: 7.7 (92 %) τ2: 38 (8 %) τ1: 0.49 (98 %) τ2: 3.3 (2 %) τ1: 8.2 (100 %) τ2: -- τ1: 0.27 (100 %) τ2: 2.2 (< 1 %) 535 nm τ1: 7.2 (98 %) τ2: 41 (2 %) τ1: 0.5 (98 %) τ2: 1.6 (2 %) τ1: 5.8 (94 %) τ2: 19 (6 %) τ1: 0.26 (100 %) τ2: 2.0 (< 1 %) 640 nm τ1: 7.0 (100 %) τ2: -- τ1: 0.49 (100 %) τ2: -- τ1: 6.9 (100 %) τ2: -- τ1: 0.24 (100 %) τ2: 1.8 (< 1 %) a. Biexponential fit was used in order to achieve good fit, however percent contribution was less than 1 %. Most apparent is the stark difference in triplet state lifetimes of the complexes in different solvents. In acetone, where the FlexLP ligand is predominantly in the closed form, both complexes have lifetimes on the order of tens of μs, which is over an order of magnitude longer than the lifetime of the complexes in MeOH (100s of ns). This relatively long non-emissive triplet state lifetime suggests that the T1 state for both complexes in acetone does not undergo significant structural distortions between the excited state and ground state, and therefore likely involves a 3LC transition (Figure 4-16). 113 Figure 4-16. Simplified diagram of the triplet state energy transitions between open and closed conformation. In MeOH, when the FlexLP ligand is predominantly in the open form, the triplet state lifetime is much shorter and on the order of 100s of ns for both complexes. Recently reported triarylborane-functionalized iridium complexes had similar emissive triplet state lifetimes on the order of 100s of ns, associated with a 3MLCT transition from the metal center to the triarylborane ligand.135 Other triarylborane-functionalized Pt(II) complexes also demonstrate strong MLCT transitions between the Pt and boron center, due to boron’s Lewis acidity.136 Additionally, the open conformation of the FlexLP has resonance structures involving the Pt and boron center (Figure 4-17). Considering the strong Lewis acidic character of the triarylborane, it is likely that this MLCT resonance state is relatively more stable compared to the possible resonance structure involving the Pt and FlexLP in the closed form. Based on these findings, and the known CT character associated with the S1 state of the FlexLP ligand in the open form, it is suggested that the T1 state of both complexes in MeOH involves a CT transition to the boron center on the FlexLP ligand, with significant contribution from the Pt metal center (Figure 4-16). This CT transition may force a large change between the 114 triplet and ground state geometries, leading to a fast temperature-independent non-radiative decay rate. In all, the FlexLP ligand conformation has a significant effect on the overall triplet state lifetime of both complexes. Figure 4-17. Schematic of possible resonance structures for the open form of Pt(FlexLP)2. The second resonance structure (middle) undergoes the same arrow pushing mechanism to afford the same negatively charged FlexLP ligand, but was not shown for simplicity. Similar resonance structures for Pt(FlexLP)(Py) are also possible when the FlexLP ligand is in the open form. When comparing the triplet state lifetimes of Pt(FlexLP)(Py) to Pt(FlexLP)2, it is found that Pt(FlexLP)(Py) has a slightly faster decay rates in both solvents, under both inert and air atmosphere. With 355 nm pump laser light the 1pyrene excited state would be significantly populated based on the absorption data of the complex in both solvents, followed most likely by population of a 3pyrene state Tn state based on the oxygen enhanced ISC fluorescence emission data. Previous 3pyrene T1 lifetimes of pyrene-functionalized Pt complexes have been reported to be ~5 μs,132 significantly different from the triplet lifetime of the complex in the open form. Therefore, if excited state decay to the ground state from the 3pyrene Tn state and the FlexLP T1 state were occurring, the overall transient absorption decay rate would likely be biexponential under inert atmosphere, with a significant contribution from a longer lived triplet state associated with the 3pyrene Tn state (Figure 4-18). Considering the overall decay rate in MeOH is faster for 115 the pyrene-functionalized complex compared to Pt(FlexLP)2, it is suggested that the pyrene ligand increases the rate of triplet state population to the FlexLP T1 state, likely through IC from the higher energy 3pyrene state to the FlexLP T1 state (Figure 4-18). The lack of phosphorescence associated with decay from the 3pyrene state also suggests IC from 3pyrene to the lower energy FlexLP T1 state is occurring. This is interesting considering the fluorescence emission spectra did not indicate IC from the higher energy 1pyrene state to the lower energy 1FlexLP state occurs, despite organometallic singlet states generally being more delocalized compared to organometallic triplet states,120 and thus increased probability of orbital overlap and IC between singlet states. Figure 4-18. Energy diagram depicting the IC from pyrene to FlexLP triplet states in Pt(FlexLP)(Py). 4.5 Conclusion In summary, two new Pt complexes containing FlexLP functional groups as ligands have been prepared, which exhibited solvent-responsive fluorescence emission, attributed to the change in conformation of the FlexLP ligand. The Pt(FlexLP)(Py) complex also exhibited higher energy fluorescence from the pyrene ligand when excited with higher energy wavelengths, which exhibited oxygen-dependent pyrene emission intensity, whereas the FlexLP emission intensity was 116 not dependent on oxygen. This observation suggests that IC from the singlet pyrene to the singlet FlexLP excited state does not occur significantly. Neither complex exhibited phosphorescence, although transient absorption experiments showed that population of a dark triplet state occurred for both complexes. It is suggested that a lower energy triplet excited state situated on the FlexLP ligand is populated, and therefore IC from the triplet pyrene excited state to the triplet FlexLP excited state occurs, followed by non-radiative decay. Transient absorption experiments concluded that the triplet lifetimes of the two conformations of the Pt(FlexLP)(Py) complex are slightly shorter than the Pt(FlexLP)2 complex, indicating the pyrene ligand increases the rate of non-radiative triplet decay to the ground state. Finally, a large difference in the overall non-radiative decay rate of the triplet state between the open and closed forms of both complexes was observed, which is suggested to be caused by the difference in energy transfer between the two conformations. For the closed form, the triplet state lifetime is on the order of μs, however in the open form the triplet state lifetime is significantly shorter, on the order of ns. This relatively short lifetime for the open form was suggested to be caused by the T1 state having CT character between the Pt and boron centers, increasing the SOC effect from the increased heavy Pt metal contribution to the T1 state, and increasing the geometry distortion between T1 state and ground state. Both of these effects could conceivably contribute to the increased decay rate. For the closed form, the T1 state is thought to be localized on the FlexLP ligand, with significantly less geometry distortion between T1 state and ground state, causing the non-radiative triplet decay rate to be significantly longer. DFT studies to elucidate the T1 MOs are needed to confirm whether the difference in T1 lifetime between open and closed FlexLP form is from a difference in energy transition. 117 4.6 Experimental Details 4.6.1 General Synthetic Procedures and Characterization Synthesis procedures involving air or moisture-sensitive reagents were carried out using standard Schlenk techniques under inert N2 atmosphere. Diisopropylamine, DCM, THF, CuI, and TBAF (1.0 M solution in THF) were purchased from Sigma Aldrich and used as received except where noted. Pt(PBu3)2Cl2,137 and Pyrene-TMS138 were synthesized according to previous literature methods. Synthesis of FlexLP-TBS has been reported in Chapter 2.139 Solution-state 1H, and 31P{1H} NMR experiments were carried out on a Bruker Avance 400MHz instrument at room temperature, and peak shifts referenced with residual solvent peaks. High resolution mass spectrometery were obtained using a Waters/Micromass LCT spectrometer, and elemental analysis performed on a Thermo Flash 2000 Elemental Analyzer. Absorption spectroscopy and emission spectroscopy measurements were obtained using a Varian Cary 5000 UV-Vis-NIR spectrophotometer and a Photon Technology International (PTI) QuantaMaster 50 fluorimeter fitted with double excitation monochromator and 75 W Xe arc lamp as the source, respectively. Emission lifetime data were obtained using a Horiba Yvon Fluorocube TCSPC apparatus, using a 370 nm NanoLED excitation source with a pulse width of 1.2 ns, pulsing at a repetition rate of 1 MHz, and using a 399 nm long-pass filter between sample and detector. Lifetime data were fitted against the NanoLED source background using the DAS6 Data Analysis software package, to afford sub-nanosecond deconvoluted lifetime data. Transient absorption spectra were obtained using a streak camera set up, equipped with 355 nm pump laser with a pulse width of 35 ps, and analyzed using HPD-TA and OriginPro graphing software. Decay curves were fitted using either monoexponential (air samples) or biexponential (inert atmosphere samples) decay functions (Equations 4-1 and 4-2) to obtain 118 lifetimes (τn) and amplitudes (A). All fitted functions had coefficient of determination (r2) values greater than 0.998. Percent contributions of each lifetime were determined by calculating the ratio of the amplitude A to the sum of the A values, according to Equation 4-3. 𝑓(𝑥) = 𝐴1𝑒(𝑥𝜏1) (Equation 4-1) 𝑓(𝑥) = 𝐴1𝑒(𝑥𝜏1)+ 𝐴2𝑒(𝑥𝜏2)+ 𝑦0 (Equation 4-2) 𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑐𝑜𝑛𝑡𝑟𝑖𝑏𝑢𝑡𝑖𝑜𝑛 𝑜𝑓 𝜏1 = 𝐴1𝐴1+𝐴2× 100% (Equation 4-3) 4.6.2 Synthesis of New Complexes Pt(FlexLP)Cl, 63 Pt(PBu3)2Cl2 (59 mg, 0.087 mmol), FlexLP-TBS (63 mg, 0.081 mmol), and CuI (2 mg, 0.01 mmol) were added to a 100 mL round bottom flask equipped with Schlenk arm and stir bar, and purged with N2. Degassed DCM/diisopropylamine (2:1 v/v, 50 mL) was added by syringe, and the reaction stirred under inert atmosphere at room temperature for 16 hours. Solvent was removed under reduced pressure and the orange product was purified by silica gel column chromatography using DCM solvent. The product was collected as a yellow powder. Yield: 42 mg, 37%. 1H NMR (CD2Cl2, 400 MHz):  7.53-7.57 (m, 2H), 7.28-7.38 (m, 8H), 6.64 (s, 1H), 6.45 (s, 4H), 6.20 (d, J = 4.1 Hz, 1H), 2.16 (s, 6H), 1.89-1.96 (m, 12H), 1.81 (s, 12H), 1.44-1.56 (m, 12H), 1.32-1.44 (td, J = 7.1 Hz, 12H), 0.95 (s, 9H), 0.88 (t, J = 7.2 Hz, 18H), 0.13 (s, 6H). 31P{1H} NMR (CD2Cl2, 162 MHz): δ 7.5, 35.9. 119 Pt(FlexLP)2, 61 A similar procedure as for Pt(FlexLP)Cl was used, using Pt(PBu3)2Cl2 (62 mg, 0.090 mmol), FlexLP-TBS (143 mg, 0.184 mmol), and CuI (5 mg, 0.03 mmol). The product was collected as a yellow powder. Attempts to grow single crystals were unsuccessful. Yield: 94 mg, 47%. 1H NMR (CD2Cl2, 400 MHz): 7.49-7.53 (m, 4H), 7.31 (m, 16H), 6.64 (s, 2H), 6.44 (s, 8H), 6.36 (d, J = 4.2 Hz, 2H), 2.16 (s, 12H), 1.87-1.94 (m, 12H), 1.81 (s, 24H), 1.35-1.47 (m, 24H), 0.94 (s, 18H), 0.90 (t, J = 7.0 Hz, 18H), 0.13 (s, 12H). 31P{1H} NMR (CD2Cl2, 162 MHz): δ -2.8, 36.3. Positive MALDI-TOF m/z: calc C120H152B2NaO2P4PtS4Si2 [M+Na]+: 2173.9, found 2173.7. Elemental analysis calcd for C120H152B2O2P4PtS4Si2: C 66.99, H 7.12, found: C 67.00, H 7.21. Pt(FlexLP)(Py), 62 Pt(FlexLP)Cl (40 mg, 0.028 mmol) and CuI (1 mg, 0.005 mmol) were added to a 100 mL round bottom flask equipped with Schlenk arm and stir bar, and purged with N2. Degassed THF/diisopropylamine solution (1:1 v/v, 10 mL) was added to the flask by syringe. To a separate 25 mL round bottom flask, Pyrene-TMS (9.5 mg, 0.032 mmol) was added, along with THF (10 mL). The solution was sparged with N2 for 30 min, and then TBAF (0.03 mL, 1.0 M in THF) was added by syringe. The solution stirred for 30 min under inert atmosphere. The pyrene solution was then transferred to the Pt(FlexLP)Cl reaction flask by syringe, and the reaction stirred at room 120 temperature under inert atmosphere for 16 hours. The solvent was removed under reduced pressure, and the precipitate purified by silica gel column chromatography using DCM solvent. The product was collected as a yellow solid. Attempts to grow single crystals were unsuccessful. Yield: 37 mg, 88%. 1H NMR (CD2Cl2, 400 MHz 8.67 (d, J = 8.1 Hz, 1H), 8.12-8.15 (m, 2H), 7.92-8.04 (m, 6H), 7.54-7.58 (m, 2H), 7.31-7.38 (m, 8H), 6.70 (s, 1H), 6.46 (s, 4H), 6.26 (d, J = 4.2 Hz, 1H), 3.37 (s, 1H), 2.17 (s, 6H), 2.11-2.15 (m, 12H), 1.82 (s, 12H), 1.59-1.67 (m, 12H), 1.37-1.46 (td, J = 7.2 Hz, 12H), 0.89 (t, J = 7.3 Hz, 18H). 31P{1H} NMR (CD2Cl2, 162 MHz): δ 4.6, 37.1. HRMS (EI-MS) m/z: calcd for C84H99OS2P3BPt [M + H]+ : 1486.6091; found 1486.6123. Elemental analysis calcd for C84H98OS2P3BPt: C 67.87, H 6.64, found: C 67.86, H 6.81. 4.6.3 Additional Spectra Figure 4-19. Emission decay spectra for Pt(FlexLP)2 under air (a) and argon (b) in acetone and MeOH solvents. 121 Figure 4-20. Emission decay spectra of Pt(FlexLP)(Py) in acetone and MeOH, under air and argon atmosphere. 122 Figure 4-21. Transient absorption spectra of Pt(FlexLP)2 (a, c) and Pt(FlexLP)(Py) (b, d) in deaerated acetone (a,b) and deaerated MeOH (c, d) at different delay times. 123 Chapter 5: Environment-Responsive Flexible Lewis Pair Fluorophore-Modified Nanoparticles for Fluorescence Imaging 5.1 Introduction The efficient delivery of cancer-fighting drugs to their intended targets is an ongoing challenge because of the poor selectivity of therapeutics towards cancerous tissue over healthy tissue. One approach to combat this is through nanomedicine, where nanoparticles are loaded with a cargo of cancer-fighting drugs, in order to allow the active species to efficiently accumulate in tumours. This can be done through passive targeting, such as through the enhanced permeability and retention (EPR) effect, or through active targeting where tumour-targeting functional groups are added to the nanoparticles, increasing the efficiency of endocytic pathways to occur.140–144 However, recent reports suggest that the efficiency of nanoparticle delivery towards tumour cells is still significantly poor,145,146 emphasizing the continuing need to understand the physical and chemical factors limiting the ability of nanoparticles to act as efficient drug transporters.147–149 In order to adequately design efficient drug carriers, it is important to understand the chemical properties of target tissues, such as pH and hydrophobicity. In particular, hydrophobicity plays an important role in drug delivery because drugs must be sufficiently stable to be transported along hydrophilic blood pathways, while being able to cross hydrophobic membranes to reach their targets. This is especially relevant for the design of drugs and drug carriers which are required to cross the blood-brain barrier (BBB),150 an ongoing challenge due to the efficacy of the BBB in preventing compounds from diffusing between brain and blood.151 124 The ability to easily assess the hydrophobicity of the regions where drugs and drug carriers are travelling to and accumulating in tissues would provide valuable information for pharmaceutical engineering, such as in the early stages of research and development of new drug carriers. In particular, “smart” fluorescent molecules, which show a readily detectable fluorescence response to external stimuli, represent an attractive route towards specific sensing for biomedical applications due to the high sensitivity, rapid response, and ease of detection of microscopy techniques.1 Smart fluorophores for application as cell-environment sensors include those which change in emission intensity or colour based on surrounding polarity as a way to quantify the hydrophobicity of the probes’ environment.152–154 However, complications arise when attempting to quantify polarity and hydrophobicity in a heterogeneous polarity microenvironment within cells due to the presence of charged species and strong local electric fields on similar size scales to the fluorescent sensor.155 Therefore, there is incentive to develop smart fluorophores which are stimulated by other known physical properties of water in order to deduce the water content in microenvironments. 5.1.1 Fluorescence Microscopy Fluorescence microscopy is a useful tool allowing the detection of microscopic emissive objects, which can be imaged and analyzed by their emissive properties, such as colour or lifetime. The high sensitivity, rapid response, and ease of detection of fluorescence microscopy techniques have made fluorescence microscopy one of the most widely used imaging techniques in biochemical and biomedical research.2,156 In 2008, the Nobel Prize in Chemistry was awarded to Osamu Shimomura, Martin Chalfie, and Roger Y. Tsien for their discovery and development of green fluorescent protein (GFP), which can be used as an imaging dye with fluorescence 125 microscopy to investigate different biological processes through tagging GFP to non-emissive proteins. As well, in 2014, the Nobel Prize in Chemistry was awarded to Erik Betzig, Stefan W. Hell, and William E. Moerner for their development of super-resolution fluorescence microscopy, which can image systems in the nanometer scale, solving the subwavelength resolution challenge known as the Abbe diffraction limit. With these fluorescence microscopy advancements, researchers have been able to investigate many different biochemical processes. 5.1.1.1 Epifluorescence Microscopy Figure 5-1. Schematic of an inverted epifluorescence microscope, equipped with a blue cut-off excitation filter and green band-pass emission filter. Though the specimen emits across the red and green visible light range, the green band-pass emission filter prevents red light from hitting the detector, and therefore the fluorescence image depicts a green sample. 126 A common fluorescence microscopy technique is epifluorescence microscopy, where a sample is exposed to an excitation light, and a camera detector detects the emission colour and/or intensity (Figure 5-1). By using specific excitation and emission filters within a filter cube, epifluorescence microscopy can separate multi-colour emission from a sample into different distinct colour blocks. This is useful for visualizing a sample that has two or more different coloured dyes, called multiplexing, because the dyes can be imaged independently to gain insight on the position of each dye within the sample. Most epifluorescence microscopes can house multiple filter cubes, which allows for fast and easy detection of different coloured dyes by switching between filter cubes. The different dyes can then be layered atop one another to compare the position of certain dyes and elucidate different structures within the same specimen. 5.1.1.2 Fluorescence Lifetime Imaging Microscopy One of the challenges of using different coloured dyes within a sample is that many dyes can spectrally overlap with one another, which cannot be separated unambiguously with filter cubes. As well, there are only a finite number of filter cubes that can be housed within a microscope. Therefore, in order to increase the number of components that can be imaged within the same sample, investigation into other properties of emissive dyes needs to be developed. One such microscopy technique is fluorescence lifetime imaging microscopy, FLIM,157,158 which detects the emission lifetime of dyes on a 2D plane by applying pulsed excitation light on an xy grid and scanning across the plane. The emission lifetime of a molecule is defined as the time that a molecule remains in the excited state, and is influenced by both the radiative and non-radiative decay rates. Small changes in the microenvironment of a molecule, such as presence of oxygen or viscosity, can affect either the radiative or non-radiative decay rate, and therefore can 127 influence the overall emission lifetime of a molecule. Since a population of molecules decay exponentially, emission lifetime (τ) is calculated as the time (t) it takes for 1/e of the original population (n) of excited molecules to decay to the ground state (Equation 5-1). This can be expressed in terms of emission intensity, I, where I will also decrease exponentially with time (Equation 5-2). 𝑛𝑡𝑛𝑜= 𝑒−𝑡/𝜏 Equation 5-1 𝐼𝑡 = 𝛼𝑒−𝑡/𝜏 Equation 5-2 A common method to detect the emission lifetime is to use time-correlated single photon counting (TCSPC), where excitation light is pulsed at time zero onto a sample, and the emitted photons are detected with respect to time. This is repeated thousands to hundreds of thousands of times and plotted as a histogram of Counts versus Time to get a statistical average of the emission decay time. For a dye with a single lifetime, the emission lifetime can be determined from fitting the histogram to Equation 5-2. FLIM uses the TCSPC method at individual pixel sites to create a 2D image of lifetime vs position, where lifetime is depicted as a colour gradient. An advantage of probing emission lifetime is that it allows for the collection of quantitative information, such as lifetime values of individual emissive dyes, which can be beneficial for multiplex bioimaging. Additionally, lifetime values of an emissive dye are not influenced by sample concentration, therefore FLIM can be used to image dyes that may have relatively low quantum yields, or poor solubility in various cellular areas. It should be noted that using low quantum yield dyes is not ideal because it requires longer 128 experimental times to gain the required photon counts to have a representative emission decay curve with high signal-to-noise ratio, however it is possible. This microscopy technique has been used to track changes in microenvironments of dyes, such as changes in lysosomal viscosities, and cellular pH.159,160 In addition to emission lifetime values, FLIM can also image samples based on the relative contribution ratio of two individual lifetimes, a1/a2. This can give more information on the relative concentration of different fluorophores, such as the concentration of interacting versus non-interacting FRET-sensitive fluorophores, to gain insight on the interactions between cellular components.158,161 5.1.2 FlexLP Systems as Fluorescence Microscopy Dyes Our group has previously developed FlexLP molecules which can be used as fluorescent dyes to image fixed biological tissues, where emission colour contrast between fatty hydrophobic tissues and aqueous hydrophilic areas is observed, due to the difference in HBD strength between water and lipid environments.53 Three FlexLP molecules were synthesized (Figure 5-2a), which had differing solubility preferences to lipid and aqueous medium. The FlexLP molecules had primarily blue-green/yellow-orange colour contrast, which could be separated using long pass (yellow) or band-pass (blue) filters, in order to determine the hydrophobic (blue-green) and hydrophilic (yellow-orange) areas. It was determined that using two complimentary dyes – a lipophilic dye and hydrophilic dye – together could stain both the inside of lipid droplets and the aqueous periphery of adipocytes (Figure 5-2). 129 Figure 5-2. (a) Structures of FlexLP dyes investigated and (b) schematic and (c) fluorescence microscopy image of adipocyte tissue showing yellow emission from FlexLP-(NMe3)22+ dye on the periphery of lipid droplets, and blue emission from FlexLP-(Bu)2 dye inside the lipid droplets (scale bar = 100 μm) (© Y. Cao, 2016, used with permission from Ref).157 As well, a single FlexLP dye could be used to stain brain tissue, where colour contrast could be seen between fatty myelin sheath and aqueous axon center in preliminary studies (Figure 5-3). However, there is an ongoing challenge for these FlexLP dyes to provide good colour contrast with high emission quantum yields in both lipophilic and hydrophilic areas. Currently, these FlexLP dyes either preferentially accumulate in either hydrophilic or lipophilic areas due to their solubility preferences. 130 Figure 5-3. (a) Schematic of brain neuron and (b) fluorescence microscopy image of fixed brain slices showing the cross-section of axons stained with FlexLP-(NMe2)2 (scale bar = 20 μm) (© Y. Cao, 2016, used with permission).157 Because of this challenge in solubility, it was proposed that FlexLP molecules would be better suited for applications as fluorophore tags, which could bind to a specific biologically-relevant target, such as an antibody, protein, or nanoparticle, in small quantities as to not interfere with the target’s solubility. Herein a target-specific stimuli-responsive FlexLP tag that binds to mesoporous silica nanoparticles is demonstrated, providing a way to directly image and assess the hydrophobicity of the nanoparticles microenvironment through changes in fluorescence. Mesoporous silica nanoparticles were chosen as a substrate for the FlexLP tag because of their promising use as drug-carrying substrates due to their low toxicity, size and surface tunability, high surface area and pore volumes, and ability to encapsulate a wide variety of therapeutic agents.163,164 5.2 Synthesis and Characterization of FlexLP Fluorophore and Functionalized Nanoparticles The FlexLP tag comprises a reactive succinimidyl ester (NHS) functional group known to react with primary amines in good yields under mild conditions. Synthesis of the FlexLP tag, NHS-131 FlexLP, was accomplished through an azide-alkyne copper click (CuAAC) reaction between previously reported alkyne-terminated FlexLP (FlexLP-TBS)139 and NHS-functionalized azide molecule (NHS-N3) with good yield (70 %), affording the succinimidyl-ester functionalized FlexLP tag (Figure 5-4). NHS-FlexLP was then attached to amine-functionalized mesoporous silica nanoparticles, NP-NH2, by stirring the two compounds in ethanol in air overnight (see Experimental Section for more details on synthesis and purification of NP-NH2 from mesoporous silica nanoparticles, NP (69)). Three different Np-FlexLP samples were made with varying NHS-FlexLP percent weight loading, NP-FlexLP-3 for 3 % weight loading, NP-FlexLP-5 for 5 %, and NP-FlexLP-10 for 10 %. Functionalized fluorescent nanoparticles were gathered by centrifugation, and structurally characterized by Fourier-transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), small angle powder X-ray diffraction (pXRD), and transmission electron microscopy (TEM). Figure 5-4. Synthetic route to FlexLP-functionalized mesoporous silica nanoparticles, NP-FlexLP. 132 TGA was employed to assess the experimental weight % loading of NHS-FlexLP on the nanoparticles by comparing the weight loss upon heating with NP-NH2. TGA data confirms that NHS-FlexLPs are attached onto the nanoparticles with good agreement with theoretical values (Figure 5-5, Table 5-1). Small angle pXRD spectra show a broad peak near 1.0° for NP-NH2 and NP-FlexLP samples, characteristic of disordered mesoporosity (Figure 5-6). The d spacing increases with more functionalization of the FlexLP as the distance between the center of neighbouring pores becomes wider, while the intensity of the peak decreases, likely due to increasingly clogged pores (Table 5-2). TEM images also show disordered mesoporous nanoparticles, with no significant change in overall shape for all samples (Figure 5-7, Figure 5-8). Figure 5-5. TGA spectra comparing weight % loss of various samples with increasing temperature. 133 Table 5-1. Weight % loading of NHS-FlexLP onto the nanoparticles was determined by comparing the weight % loss from TGA data. Sample % Weight Loss 25-100 °C % Weight Loss 100-700 °C Weight % Amine group Weight Loss % FlexLP NP 1.64 0.02 -- -- NP-NH2 1.34 11.95 11.93 -- NHS-FlexLP 0.53 88.99 -- -- NP-FlexLP-3 2.96 15.69 11.70 3.96 NP-FlexLP-5 2.61 17.68 11.60 6.07 NP-FlexLP-10 5.08 21.73 11.35 10.36 Figure 5-6. Small angle pXRD spectra of nanoparticle samples. 134 Table 5-2. Small angle pXRD data of mesoporous nanoparticles. Sample 2ϴ (°) d(Å) NP 1.0 87.4 NP-NH2 0.98 90.1 NP-FlexLP-3 0.87 101.7 NP-FlexLP-5 0.82 107.1 NP-FlexLP-10 0.80 110.4 Figure 5-7. TEM image of unfunctionalized mesoporous silica nanoparticle, NP (a), and NP-NH2 (b) samples. Figure 5-8. TEM image of NP-FlexLP-3 (a), NP-FlexLP-5 (b), and NP-FlexLP-10 (c) samples. 135 Figure 5-9. FT-IR spectra of NP, NP-NH2, NHS-FlexLP, and NP-FlexLP-10 samples. Inset shows a zoomed-in region highlighting the amide carbonyl C=O peak at 1650 cm-1 for Np-FlexLP-10 sample. NHS-FlexLP trace intensity in the insert was reduced by a factor of 5 to easily compare peak wavenumbers. FT-IR spectroscopy of NP-FlexLP-10 shows a peak at 1650 cm-1 which may be attributed to the new amide C=O group; this is not present in the spectra of NP-NH2, NHS-FlexLP, or unsubstituted nanoparticles (Figure 5-9). The strong succinimidyl C=O peaks between 1740-1820 cm-1 for NHS-FlexLP are not apparent for NP-FlexLP, however given the low molar ratio of FlexLP to nanoparticle, other vibrational peaks are difficult to assign. A control experiment was conducted to assess the possibility that the NHS-FlexLP was adsorbed into the pores of the nanoparticles rather than covalently bonded to the surface. NHS-136 FlexLP was reacted in a 10% by weight loading concentration with mesoporous silica nanoparticles (NP) that had not been functionalized with amines (70, see Experimental Section for more details). This NP+FlexLP control sample 70 gave white nanoparticles which did not emit under UV irradiation, and the absorption spectrum does not show the characteristic absorption peak of the flexLP fluorophore at 410 nm (Figure 5-10a). To confirm no free NHS-FlexLP was trapped in the pores of the fluorescent NP-FlexLP-10 sample which could leach out, NP-FlexLP-10 was dispersed in THF. The suspension was left for 1 day, during which time the nanoparticles settled to the bottom of the vial, and the THF was then analyzed by absorption spectroscopy. No absorption signal was observed, indicating NHS-FlexLP had not leached out of the nanoparticle during this time and that the sample is stable in THF (Figure 5-10b). These results together support the conclusion that NHS-FlexLP reacts with NP-NH2 through amide bond formation. Figure 5-10. UV-Vis absorption spectra (a) of NP-FlexLP-10 (0.6 mg / 10 mL THF) and NP+FlexLP control sample 70 (0.6 mg / 10 mL THF). UV-Vis absorption spectra (b) of FlexLP “leached” out of NP-FlexLP-10 after 1 day in THF. Inset shows a vial of NP-FlexLP-10 sample after 5 days in THF. Emission of the nanoparticles shows they have settled to the bottom of the vial, and no observable free FlexLP has leached into the surrounding solvent. Refracted emission can be seen at the meniscus. 137 5.3 Photophysical Data Comparison of the absorption and emission spectra of the NHS-FlexLP tag and functionalized NP-FlexLP samples show good preservation of photophysical characteristics between free NHS-FlexLP and tagged NP-FlexLP, with no significant shifts in absorption (Figure 5-11) or emission (Figure 5-12a) wavelengths between the samples. Both NHS-FlexLP and NP-FlexLP samples exhibit blue emission in non-HBD solvent (THF) attributed to the closed form, and yellow emission in HBD solvent (MeOH) attributed to the open form of the Lewis pair.48 Comparison of the normalized emission intensities in THF and MeOH shows a trend, where increasing functionalization of the nanoparticles shifts the equilibrium towards the open form. This results in broadening of the tail of the emission peak in THF, and lowered intensity of the emission shoulder at 480 nm in MeOH, with increasing functionalization (Figure 5-12a). Figure 5-11. UV-Vis absorption spectra of NHS-FlexLP (4 x 10-6 M) (a) and of NP-FlexLP samples (b, 5 x 10-5 M FlexLP added) in THF (normalized at maximum peak wavelength) and MeOH (normalized at 325 nm) solvent. 138 Figure 5-12. Normalized emission spectra (a) of NHS-FlexLP and NP-FlexLP samples in THF (λex = 400 nm) and MeOH (λex = 325 nm) solvent. Emission lifetime (b) graphs of NHS-FlexLP and NP-FlexLP-10 samples in THF and MeOH, with overlapping fitted traces in corresponding colours. Table 5-3. Emission lifetimes (ns) and relative percentage values (% in parenthesis) for NHS-FlexLP and NP-FlexLP-10 in THF and MeOH Sample THF MeOH <1 nsa Short Long <1 nsa Short Long NHS-FlexLP -- 2.25 (100) -- -- 1.90 (58.6) 7.41 (41.4) NP-FlexLP 0.51 (9.3) 1.85 (80.1) 7.11 (10.6) 0.09 (27.9) 2.23 (13.2) 7.64 (59.0) a. Very short lifetime is thought to be due to scattering from aggregated nanoparticles. The emission lifetimes of NP-FlexLP-10 are similar to the emission lifetime values of NHS-FlexLP in THF and MeOH (Figure 5-12b). In THF, a short emission lifetime (~2 ns) feature predominates, attributed to the π-π* transition on the bithiophene backbone of the closed form for both NHS-FlexLP and NP-FlexLP-10.48 In MeOH, the emission lifetime is longer (~7.5 ns) for the open structure, due to the bithiophene-boron charge transfer nature of the emission. 139 The effect of mixed non-HBD/water solvent systems on the emission spectra and lifetimes of the NP-FlexLP samples was explored, to gain insight into the sensitivity of the NP-FlexLP’s photophysical characteristics to the water content of the surrounding environment. NP-FlexLP samples were dispersed in dioxane-water mixed solvent systems. All samples showed decreasing emission at 480 nm and increasing emission at 560 nm with increasing amounts of water, indicative of the equilibrium shifting from predominantly closed form in dioxane to open form in water (Figure 5-13–15). Additionally, the emission intensity ratio (I560 nm/I480 nm) varies over the full range from 0% water to 100% water. This suggests that the nanoparticles may be used to sense water concentrations in fluorescence imaging over the full range of water concentrations, without the change in intensity ratio reaching a maximum at a specific water concentration beyond which no further changes are observed. Figure 5-13. Emission spectra of NP-FlexLP-3 in dioxane with increasing % by volume of water (λex = 410 nm, left) (λex = 325 nm, right). Spectra were normalized at 515 nm, which is the wavelength where the emission peaks from the open and closed structure of free NHS-FlexLP intersect. 140 Figure 5-14. Emission spectra of NP-FlexLP-5 in dioxane with increasing % by volume of water (λex = 410 nm) (λex = 325 nm, right). Spectra were normalized at 515 nm. Figure 5-15. Emission spectra of NP-FlexLP-10 in dioxane with increasing % by volume of water (λex = 400 nm) (λex = 325 nm, right). Spectra were normalized at 515 nm. Emission lifetime data in dioxane-water mixtures show the lifetime values for the open form (~6-8 ns) and closed form (~2 ns) remain relatively constant with increasing percentage of water (Table 5-5, Table 5-6). This consistency in lifetime values may be useful for fluorescence lifetime imaging microscopy (FLIM), because it eliminates ambiguity in whether the fluorophore 141 is in the open or closed state, since the two species always significantly differ from one another in lifetime regardless of changes in polarity and dielectric constant in mixed solvent systems.165 Emission lifetime plots of the NP-FlexLPs show the long lifetime component increases in amplitude with increasing water content, and the short lifetime component decreases (Figure 5-16), as the percentage of open FlexLP form increases relative to the closed form. This is evident when plotting the relative percentages of short and long components versus water content (Figure 5-17). Figure 5-16. Emission lifetime graphs of NP-FlexLP-3 (left), NP-FlexLP-5 (middle), and NP-FlexLP-10 (right) in dioxane with increasing water content (% by volume). The effect of varying FlexLP functionalization on the relative percentage of the short and long lifetime components is also shown in Figure 5-17. Comparison of the data for NP-FlexLP-3 to NP-FlexLP-10 indicates that for all water percentages, more of the shorter lifetime component (closed form) is observed for the sample with lower coverage. This is the same trend observed in the emission spectral data shown in Figure 5-12; increasing coverage leads to a shift towards the open form. The difference in Figure 5-17 between NP-FlexLP-3 and NP-FlexLP-5 is less pronounced. 142 Figure 5-17. Relative percentages of long emission lifetime component of NP-FlexLP samples in dioxane-water mixed solvent ratios. 5.4 Concentration and pH Studies In imaging of biological tissues, where nanoparticles and other fluorescent imaging dyes may preferentially collect in certain areas, it is important to understand any concentration dependent emission effects. To probe this, samples of NP-FlexLP-3, -5, and -10 were prepared in a 50:50 water-dioxane mixed solvent in three different concentrations (0.6 mg, 1.2 mg and 2.0 mg of NP-FlexLP sample in 10 mL solvent), and the emission intensities and lifetimes analyzed. The emission profiles of the samples showed that the NP-FlexLP-3 samples showed significantly higher emission intensity at 480 nm compared to the NP-FlexLP-10 samples when normalized at 515 nm, however concentration did not have a great effect on the emission profiles with individual samples (Figure 5-18). The emission intensity at 480 nm for the NP-FlexLP-5 samples was between the intensities for NP-FlexLP-3 and -10. Likewise, the relative amplitude of the long emission lifetime component did not change significantly with concentration for all three NP-FlexLP samples (Figure 5-20), where for all concentrations NP-FlexLP-10 had the highest 143 relative percentages for the long component, and NP-FlexLP-3 had the lowest relative percentages. Therefore, significant variations in the emission colour or lifetimes due to concentration differences is not expected if the dye-modified particles are used to image media such as biological tissues. Figure 5-18. Emission spectra of NP-FlexLP-3 (a), -5 (b), and -10 (c) samples in 50:50 dioxane:water (v/v), with varying concentrations of sample (mg/10 mL) excited at λex = 325 nm. Spectra were normalized at 515 nm. Variations in the ratio of open to closed form is predominantly affected by the weight percent loading of NHS-FlexLP, not concentration. Emission lifetime spectra of NP-FlexLP-3 (d), -5 (e), and -10 (f) samples in 50:50 dioxane:water (v/v), with varying concentrations of sample (mg/10 mL). 144 Figure 5-19. Relative percentages of the long emission lifetime component versus short emission lifetime component for NP-FlexLP samples of different concentrations in 50:50 (v/v) dioxane-water mixed solvent environments. The effect of pH on the relative emission intensities and emission lifetimes of NP-FlexLP-10 was also investigated to gain insight into the stability of the fluorescent nanoparticles under these conditions. Experiments were carried out with the help of Kathleen Downey, where a sample of NP-FlexLP-10 was dispersed in different buffer solutions of varying pH from 3.0 – 10.8. No significant shifts in emission wavelength, or changes in the ratio of the 480 nm and 560 nm emission intensities were observed over the full range of pHs (Figure 5-20). In addition, no major changes in emission lifetimes, or the relative percentages of short and long lifetime components, were found (Figure 5-20–21). It is concluded that pH does not have an effect on the equilibrium between the open and closed forms of the FlexLPs. This is particularly encouraging for application in fluorescence microscopy and FLIM of biological samples, in which environments with varying pH may be found, because it allows for a pH-independent assessment of the hydrophobicity of the biological environment being imaged. 145 Figure 5-20. Normalized emission spectra (λex = 325 nm) (a) and emission lifetime graphs (b) of NP-FlexLP-10 in different buffer solutions of varying pH. Spectra were normalized 515 nm, the wavelength where the emission spectra of open and closed forms intersect. Figure 5-21. Relative percentage (%) of short emission lifetime and long emission lifetime of NP-FlexLP-10 plotted against changing pH. 146 Table 5-4. Emission lifetimes (ns) and relative percentage (% in parenthesis) values of NP-FlexLP-10 in varying pH environments. pH <1 nsa lifetime Short lifetime Long lifetime 3.0 0.70 (17.3) 4.07 (24.0) 8.56 (58.7) 4.0 0.31 (8.6) 3.53 (20.4) 8.56 (71.0) 5.0 0.29 (8.7) 3.44 (19.8) 8.52 (71.4) 6.2 0.23 (11.1) 3.08 (23.9) 7.83 (65.0) 7.0 0.24 (10.5) 3.15 (22.4) 8.08 (67.1) 8.0 0.26 (10.3) 3.33 (22.1) 8.24 (67.5) 9.2 0.29 (12.1) 3.11 (22.1) 8.10 (65.8) 10.1 0.29 (12.4) 3.12 (23.4) 8.06 (64.1) 10.8 0.28 (12.9) 2.99 (23.5) 8.00 (63.7) a. Very short lifetime is thought to be due to scattering from aggregated nanoparticles. 5.5 Fluorescence Microscopy Data To demonstrate that the nanoparticles can be imaged by fluorescence microscopy, NP-FlexLP-10 was added in dioxane and water and the samples were imaged on glass slides using an inverted epifluorescence microscope. Figure 5-22 shows the different emission colours of the sample in the two solvents, demonstrating good colour contrast between the two images. Fluorescence microscopy images of the nanoparticles comparing the separated red, green, and blue channels also shows the difference in relative emission intensity between channels when in different solvent. Emission from the sample in dioxane (Figure 5-23) is apparent everywhere in green and blue channels but less so in the red channel, whereas in water (Figure 5-24) the sample 147 is seen under red and green channels everywhere, but emission from the blue channel is only seen for the larger sized samples. Figure 5-22. Fluorescence microscopy images of NP-FlexLP-10 in dioxane (a) and water (b). Figure 5-23. Fluorescence microscopy image of NP-FlexLP-10 in dioxane (a), and separated red (b), green (c), and blue (d) channels showing the relative emission intensity of the three channels. Scale bar = 129 μm. 148 Figure 5-24. Fluorescence microscopy image of NP-FlexLP-10 in water (a), along with separated red (b), green (c), and blue (d) channels showing the relative emission intensity of the three channels. Scale bar = 129 μm. 5.6 Conclusions New environment-responsive fluorescent mesoporous silica nanoparticles are investigated which undergo a change in emission colour and lifetime in response to hydrogen bonding environments. The photophysical properties of the NP-FlexLPs are concentration and pH stable, demonstrating that the change in emission colour and lifetime is specific to the HBD strength of the surrounding environment, highlighting the potential application of this NHS-FlexLP fluorophore tag to assess water content surrounding biologically-relevant materials. Fluorescence microscope imaging of the nanoparticles reveals distinctly different emission colours in different environments. Using similar chemistry, other primary amine-containing species, such as proteins or antibodies, or other potential drug-carrying nanoparticles may be tagged. Further application of 149 the FlexLP tag system for imaging drug-carrying nanoparticles inside cells may aid in determining the mechanism of movement of nanoparticles within cellular components. For example, visualization of the nanoparticles traveling i) between hydrophilic endosome lumen and hydrophobic organelles within the cell, or ii) passively through the EPR effect, may be possible to discern.166,167 5.7 Experimental Details 5.7.1 Synthetic Procedures 5.7.1.1 Synthesis of new compounds Synthesis procedures involving air or moisture-sensitive reagents were carried out using standard Schlenk techniques under inert N2 atmosphere. Dry dichloromethane (DCM) was obtained from an Inert PureSolv MD5 Solvent Purification System (SPS). N-hydroxysuccinimide, N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC•HCl), CuI, and ascorbic acid were purchased from Sigma Aldrich and used as received. Azide-functionalized carboxylic acid168 (Please see safety note below!) and FlexLP-TBS139 were synthesized according to previous literature methods. Safety Note: It should be noted that the synthesis of azide-functionalized carboxylic acid requires the use of sodium azide, a very reactive/explosive and acutely toxic (oral and dermal) inorganic compound! Extreme caution should be used when using sodium azide! This includes reading the appropriate safety literature, using small amounts, ensuring that the compound does not come into contact with halogenated solvents, acids, metals (including drain pipes and metal spatulas), dimethyl sulfate, or water, limiting use with ground glass joints and elevated 150 temperatures, avoiding concentrating azide-containing compounds by rotary evaporation, and knowing appropriate quenching and storing methods. NHS-N3, 65 Azide-functionalized carboxylic acid (330 mg, 1.37 mmol), N-hydroxysuccinimide (190 mg, 1.65 mmol), and EDC•HCl (430 mg, 2.25 mmol) were added to a 20 mL vial equipped with stir bar, and purged with N2. Dry DCM (8 mL) was added to the reaction by syringe to dissolve the compounds, and the reaction stirred overnight under inert atmosphere at room temperature. The reaction was extracted with EtOAc and washed with H2O and brine, and the organic layer dried over Na2SO4. The organic layer was filtered to remove Na2SO4, and the product collected by removal of solvent under reduced pressure. The product was collected as a white solid. Yield: 440 mg, 99%. 1H NMR (CDCl3, 400 MHz): δ 1.24-1.42 (m, 12H, H4-H9), 1.59 (quintuplet, 2H, H3/H10), 1.74 (quintuplet, 2H, H3/H10), 2.60 (t, 2H, H2/H11), 2.83 (s, 4H, H1), 3.25 (t, 2H, H2/H11).13C{1H} (CDCl3,100 MHz): δ 24.7 (CH2), 25.7 (CH2), 26.8 (CH2), 28.9 (CH2), 29.0 (CH2), 29.14 (CH2), 29.2 (CH2), 29.3 (CH2), 29.5 (CH2), 31.1 (CH2), 51.6 (CH2), 168.8 (C4), 169.4 (C4). HRMS (EI-MS) m/z: [M + Na]+ calcd for C15H24N4O4Na: 347.1695; found 347.1705. 151 NHS-FlexLP, 66 NHS-N3 (103 mg, 0.32 mmol), FlexLP-TBS (135 mg, 0.170 mmol), CuI (15 mg, 0.080 mmol), and ascorbic acid (3 mg, 0.02 mmol) were added to a 50 mL round bottom flask equipped with Schlenk arm and stir bar, and purged with N2. THF (20 mL, degassed with N2 for 1 hr) was added by syringe, and the reaction stirred for 2 days under inert atmosphere at room temperature. The THF was removed under reduced pressure, the residue extracted using EtOAc and washed with H2O and brine, and the organic layer dried using MgSO4. The organic layer was filtered and EtOAc solvent removed under reduced pressure. The crude product was purified by silica gel column chromatography using EtOAc/hexane (1:1 v/v), and the second major spot collected to afford a yellow solid. Yield: 122 mg, 70%. 1H NMR (CDCl3, 400 MHz): δ 0.14 (s, 6H, H19), 0.95 (s, 9H, H20), 1.30-1.39 (m, 13H, H3-H9), 1.73 (m, 3H, H3/H10), 1.88 (s, 12H, H15), 2.16 (s, 6H, H17), 2.59 (t, J = Hz, 2H, H2), 2.83 (s, 4H, H1), 4.33 (t, J = Hz, 2H, H11), 6.48 (s, 4H, H16), 6.88-6.89 (m, 2H, H13 + H18), 7.38 (m, 8H, H14), 7.54 (m, 3H, H14 + H12). 13C{1H} (CDCl3,100 MHz): δ -4.5 (CH3), 16.9 (C4), 21.0 (CH3), 24.5 (CH3), 24.7 (CH2), 25.7 (CH2), 26.4 (CH3), 26.6 (CH2), 28.8 (CH2), 29.0 (CH2), 29.1 (CH2), 29.3 (CH2), 29.3 (CH2), 30.4 (CH2), 31.1 (CH2), 50.7 (CH2), 98.7 (C4), 98.9 (C4), 119.6 (CH), 122.4 (C4), 123.9 (C4), 127.6 (C4), 127.8 (C4), 128.6 (d, J = 13 Hz, CH), 128.9 (CH), 129.6 (d, J = 113 Hz, C4), 132.3 (d, J = 17 Hz, CH), 132.6 (d, J = 11 152 Hz, CH), 132.7 (d, J = 3 Hz, CH), 135.5 (C4), 137.2 (C4), 141.8 (CH), 141.9 (d, J = 166 Hz, C4), 150.3 (C4), 151.8 (C4), 160.0 (C4), 168.8 (C4), 169.3 (C4). 31P{1H} NMR (CDCl3, 162 MHz): δ 33.0. HRMS (EI-MS) m/z: [M + H]+ calcd for C63H75O5S2N4PSi11B: 1101.4779; found 1101.4774. Elemental analysis calcd for C63H74O5S2N4PSi11B: C 68.71, H 6.77, N 5.09, found: C 68.58, H 6.98, N 4.98. 5.7.1.2 Synthesis of nanoparticles Synthesis of NP-FlexLP (68) with varying weight percentage of NHS-FlexLP to NP-NH2 were synthesized in the following manner. Amine-functionalized silica nanoparticles (synthesized according to previous literature methods)169 (NP-NH2 (67), 40 mg) and the appropriate weight percentage of NHS-FlexLP (1.2 mg, 2.0 mg, and 4.0 mg for 3% (NP-FlexLP-3), 5% (NP-FlexLP-5), and 10% (NP-FlexLP-10) samples respectively) were added to a 100 mL round bottom flask with a stir bar, and 40 mL of EtOH was added. The reaction stirred in the dark for 16 hours at room temperature in air. The reaction was centrifuged at 2790 rpm for ten minutes, and the EtOH supernatant pipetted out and collected. Fresh EtOH was used to re-suspend the nanoparticles and centrifugation/EtOH supernatant collection was repeated an additional 3 times to thoroughly wash away unconjugated NHS-FlexLP. The fluorescently labelled nanoparticles were dried under reduced pressure to afford a free-flowing light-yellow powder. NP+FlexLP control, 70 A similar procedure as for the NP-FlexLP samples was followed using unfunctionalized mesoporous silica nanoparticles (synthesized according to previous literature methods)170 (NP 153 (69), 40 mg) and NHS-FlexLP (4.0 mg). The nanoparticle powder was collected as a free-flowing white powder after washing with EtOH 3 times and collecting by centrifugation. 5.7.2 Structural Characterization Techniques Solution-state 1H, 31P{1H}, and 13C NMR experiments were carried out on a Bruker Avance 400MHz instrument at room temperature, and peak shifts referenced with residual solvent peaks. Infrared spectroscopy was performed on an attenuated total reflection (ATR) crystal using a PerkinElmer Frontier FT-IR spectrometer on dry sample. Thermogravimetric analysis (TGA) was performed on a Pyris 6 TGA instrument, using a platinum crucible loaded with 4-5 mg of sample. Measurements were performed under a synthetic air atmosphere (20 mL/min flow) with increasing temperature at a heating rate of 5 °C/min from 25 to 700 °C. Weight % loading of fluorophore was calculated by assuming 100% loss of propyl-amine from NP-NH2 sample when heated to 700 °C, and converting weight % loss of propyl-amine functional group to moles of total propyl-amine on 1 mg of NP sample. This value could be used to calculate the free propyl-amine functional groups (Table 5-1, column 4) and the propyl-amine groups reacted with NHS-FlexLP for each NP-FlexLP sample, to obtain the weight % loss of propyl-amide-FlexLP fluorophore (Table 5-1, column 5). Small angle powder X-ray diffraction (pXRD) patterns of nanoparticle samples were obtained using a Bruker D8-Advance X-ray diffractometer with Cu Kα radiation at 40 mA and 40 kV, scanned from 0.5 – 10 2ϴ. Transmission electron microscopy (TEM) images were collected with a Hitachi H7600 electron microscope with an acceleration voltage of 80 kV. Samples were prepared by sonicating approximately 2 mg of nanoparticles in 0.5 mL of ethanol for 10 min. 154 5.7.3 Photophysical Measurements and Microscopy Techniques Absorption spectroscopy and emission spectroscopy measurements were obtained using a Varian Cary 5000 UV-Vis-NIR spectrophotometer and a Photon Technology International (PTI) QuantaMaster 50 fluorimeter fitted with double excitation monochromator and 75 W Xe arc lamp as the source, respectively. All NP-FlexLP samples were sonicated for 2 minutes before spectroscopy measurements were obtained to prevent settling of the nanoparticles. Emission lifetime experiments were carried out on a Horiba Yvon Fluorocube TCSPC apparatus, using a 370 nm NanoLED excitation source with a pulse width of 1.2 ns, pulsing at a repetition rate of 1 MHz, and using a 399 nm long-pass filter between sample and detector. Lifetime data were fitted against the NanoLED source background using the DAS6 Data Analysis software package, to afford sub-nanosecond deconvoluted lifetime data. All nanoparticle samples were fitted to a triexponential function, to afford chi-square values under 1.0. Multiple trials of emission lifetime experiments at 0%, 50%, and 100% water percentage confirmed the relative amplitude (%) of lifetimes to be within ± 5 %. pH stability studies were conducted on NP-FlexLP-10. Three different buffer systems were used to afford a pH range from 3.0 – 10.8. Citric acid-sodium phosphate buffer was used for pH ranges 3.0 – 5.0, potassium phosphate buffer for pH ranges 6.2 – 8.0, and carbonate-bicarbonate buffer for pH ranges 9.2 – 10.8. The solutions were prepared by dissolving the indicated salts in distilled water to achieve stock solutions with ionic strengths of 0.15 M. NP-FlexLP-10 (0.6 mg) was dispersed in 10 mL of buffer solution of known pH. Fluorescence microscopy images were collected on an Olympus IX70 inverted fluorescence microscope fitted with an Olympus DP80 dual CCD 12.5 megapixel colour camera, using a 400 nm longpass filter. Images were taken with a 37 ms exposure time.155 5.7.4 Additional Emission Lifetime Data Table 5-5. Emission lifetime values (ns) and relative percentage values (% in parenthesise) of NP-FlexLP samples in dioxane-water mixed solvent environments. % Water NP-FlexLP-3 NP-FlexLP-5 NP-FlexLP-10 <1 nsa Short Long <1 nsa Short Long <1 nsa Short Long 0% 0.44 (23.5) 1.72 (64.3) 6.07 (12.2) 0.18 (54.3) 1.72 (37.3) 4.90 (8.4) 0.17 (41.2) 1.57 (44.7) 6.11 (14.1) 20% 0.45 (22.3) 1.83 (64.6) 5.84 (13.2) 0.33 (15.0) 1.88 (69.3) 6.17 (15.8) 0.32 (26.3) 1.73 (50.4) 6.25 (23.3) 40% 0.41 (28.5) 1.74 (51.8) 5.70 (19.7) 0.31 (20.0) 1.85 (60.9) 6.33 (19.1) 0.19 (38.8) 1.55 (37.2) 5.68 (24.1) 50% 0.42 (25.6) 1.90 (51.8) 6.73 (22.6) 0.32 (25.1) 1.89 (46.6) 7.07 (28.4) 0.28 (28.3) 1.73 (36.4) 7.02 (35.3) 60% 0.33 (31.5) 1.87 (40.6) 6.89 (27.9) 0.27 (28.5) 1.79 (35.7) 7.25 (35.9) 0.24 (31.7) 1.59 (25.1) 7.14 (43.2) 80% 0.29 (27.9) 1.99 (35.3) 7.13 (36.8) 0.17 (35.0) 1.72 (24.8) 7.37 (40.3) 0.20 (35.7) 1.78 (20.2) 7.24 (44.2) 100% 0.30 (18.2) 2.60 (22.4) 8.41 (59.4) 0.11 (35.2) 2.13 (16.9) 7.94 (47.9) 0.15 (24.5) 2.99 (17.6) 8.29 (57.9) a. Very short lifetime is thought to be due to scattering from aggregated nanoparticles. 156 Table 5-6. Emission lifetime values (ns) and relative percentage values (% in parenthesis) of NP-FlexLP samples at three different concentrations in 50:50 dioxane-water mixed solvent environment. Concentration (mg / 10 mL) NP-FlexLP-3 NP-FlexLP-5 NP-FlexLP-10 <1 nsa Short Long <1 nsa Short Long <1 nsa Short Long 0.6 0.45 (28.3) 1.82 (54.5) 5.99 (17.2) 0.37 (26.3) 1.71 (51.5) 6.11 (22.3) 0.28 (28.3) 1.73 (36.4) 7.02 (35.3) 1.2 0.40 (26.6) 1.78 (55.3) 6.00 (18.1) 0.32 (25.1) 1.89 (46.6) 7.07 (28.4) 0.39 (30.5) 1.86 (29.7) 6.86 (39.8) 2.0 0.42 (25.6) 1.90 (51.8) 6.73 (19.7) 0.44 (27.2) 1.82 (47.1) 6.62 (25.8) 0.40 (31.6) 1.94 (29.4) 6.89 (39.0) a. Very short lifetime is thought to be due to scattering from aggregated nanoparticles. 157 Chapter 6: Conclusions and Future Work The development of stimuli-responsive molecules with tunable photophysical properties is an ongoing pursuit for many sought-after applications that involve advanced sensing and imaging. To this end, development and understanding of stimuli-responsive molecules that can be easily modified and applied to a variety of chemistry fields is an attractive endeavor, as it allows for the rapid advancement of new materials that retain the properties of the stimuli-responsive core. This thesis introduced a FlexLP chromophore TBS-FlexLP-TBS 26 incorporating alkyne groups on either end of the FlexLP scaffold, where reaction of this chromophore with stoichiometric amounts of TBAF resulted in selective deprotection to make FlexLP-TBS 24 and FlexLP 25 in good yields. Functionalization of these FlexLP molecules using three different Cu(I) catalytic reaction conditions showcased their versatility as stimuli-responsive ligands that could be effective in a range of chemistry fields and potential applications, including the synthesis and investigation of organic dimers, organometallic complexes, and functionalized nanomaterials. In Chapter 2, FlexLP-TBS 24 with a reactive terminal alkyne group was synthesized and photophysical properties characterized. Further functionalization of this molecule through Glaser chemistry conditions gave a dimer that had similar solvent-dependent absorption and emission properties to that of the monomeric 24, with no definitive indication of cooperativity effects between the two FlexLP chromophores occuring. The dimer exhibited red shifted emission in MeOH compared to in THF solvent, similar to the monomer. Differences were found between dimer and monomer when comparing the change in emission energy between the two solvents; the emission of the dimer was significantly red shifted in energy compared to the monomer in THF, however in MeOH the dimer and monomer had similar emission energies. This is an important 158 finding for potential future investigations on polymeric FlexLPs, because it suggests that if this FlexLP system is to be incorporated into polymeric materials and maintain the stimuli-responsive emission properties a reasonable path would be to incorporate the FlexLP as pendent groups, or separate the FlexLP groups using multiple monomers to afford a non-conjugated copolymer. Investigation of the reactivity of the FlexLP monomer and dimer suggest that if a polymer where the FlexLP is within the backbone were to be made, this might be achieved using other alkyne-coupling reactions, such as azide-alkyne click chemistry or A3 coupling, as alkyne-alkyne coupling on the boron Lewis acidic side was unsuccessful. Platinum complexes functionalized with one or two FlexLP ligands were explored as solvent- and temperature-responsive coordination complexes. In Chapter 3, it was shown that the Pt(terpyridine) metal center does not hinder the stimuli-responsive nature of the FlexLP ligand, and that both monometallic and bimetallic Pt-FlexLP complexes had new CT bands not seen in the organic FlexLP proligand, that could be tuned in energy based on the conformation of the FlexLP ligand with applied solvent or temperature. This offers an interesting potential route towards stimuli-responsive redox catalysis, where using a redox active metal, such as Fe or Ru, may exhibit a tunable MLCT band and change in redox behavior dependent on FlexLP ligand conformation. Chapter 4 described the properties of two emissive Pt-FlexLP complexes, Pt(FlexLP)2 61 and Pt(FlexLP)(Py) 63, including the excited triplet state properties using transient absorption spectroscopy. Both complexes exhibited solvent-responsive fluorescence emission attributed to the FlexLP conformation. Additionally, complex Pt(FlexLP)(Py) also exhibited pyrene-localized fluorescence emission at a higher energy, indicating the singlet MOs of the two chromophores are electronically separated and that sufficient IC between the MO excited singlet states to prevent 159 fluorescence from the pyrene chromophore does not occur. The use of the FlexLP ligand may prove an interesting concept for designing dual-emissive complexes that have solvent-responsive emissive properties. Transient absorption spectroscopy indicated that population of the complexes’ triplet states occurs, and the FlexLP ligand conformation greatly influences the overall triplet state lifetimes of the complexes. The FlexLP ligand may prove useful in the design of stimuli-responsive triplet photosensitizers with tunable lifetimes, as the triplet state lifetime of a complex is an important property when designing photosensitizers for catalysis or photodynamic therapy. Lastly, FlexLP-TBS 24 was functionalized with an NHS group by CuAAC reaction conditions in Chapter 5 to afford NHS-FlexLP 66, which could be tagged onto a biologically relevant mesoporous silica nanoparticle substrate. The NP-FlexLP samples had tunable emission colour and lifetime with solvent of varying water content, and the different emission colour could be imaged using fluorescence microscopy, demonstrating their water-sensing abilities. The emission properties of the nanoparticle samples were also stable to pH and varying concentration, which is important when designing fluorescent labels for biomedical imaging applications. These results suggests that further fluorescence microscopy and FLIM studies of the NP-FlexLP samples as stimuli-responsive emissive drug-delivery materials may be of interest. As well, investigation of NHS-FlexLP 66 as a water-sensing fluorescent tag for a broad scope of biologically relevant substrates, such as proteins or antibodies, may also be of interest. 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C 2016, 63, 393–410. 172 Appendix: Proton NMR Spectra of Select New FlexLP-Containing Molecules Figure A-1. 1H NMR spectrum of complex FlexLP-TBS 24 (CDCl3, 400 MHz, 25 °C). Figure A-2. 1H NMR spectrum of complex FlexLP dimer 25 (CDCl3, 400 MHz, 25 °C). Starred peaks are solvent residue. 173 Figure A-3. 1H NMR spectrum of complex Pt-FlexLP 45 (CDCl3, 400 MHz, 25 °C). Insert is of aromatic region. Figure A-4. 1H NMR spectrum of complex Pt-FlexLP-Pt 46 (CDCl3, 400 MHz, 25 °C). Insert is of aromatic region. 174 Figure A-5. 1H NMR spectrum of Pt(FlexLP)2 61 (CD2Cl2, 400 MHz, 25 °C). Figure A-6. 1H NMR spectrum of Pt(FlexLP)(Py) 62 (CD2Cl2, 400 MHz, 25 °C). 175 Figure A-7. 1H NMR spectrum of NHS-FlexLP 66 (CDCl3, 400 MHz, 25 °C).
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Symmetric and non-symmetric stimuli-responsive flexible Lewis pair systems
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