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Photoactive Conjugated Polyelectrolytes and Conjugated Polyelectrolyte Dendrimers

Permanent Link: http://ufdc.ufl.edu/UFE0042427/00001

Material Information

Title: Photoactive Conjugated Polyelectrolytes and Conjugated Polyelectrolyte Dendrimers
Physical Description: 1 online resource (195 p.)
Language: english
Creator: Lee, Seoung
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: aggregation, conjugated, dendrimer, fluorescence, mercury, polyelectrolyte, polymer, pyrophosphate, rhodamine, sensor
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: In this dissertation, we primarily focus on the fundamental investigation of the photophysical properties of conjugated polyelectrolytes (CPEs) and conjugated polyelectrolyte dendrimers (CPE-Ds). Also, applications to the selective and sensitive pyrophosphate ions and mercury (II) ion sensors are explored. First, for CPEs, various aromatic moieties including phenyl (Ph), 2,1,3-benzothiadiazole (BTD), and 4,7-bis(2-thienyl)-2,1,3-benzothiadiazole (TBT) units have been incorporated into the polymer backbones. The photophysical properties of CPEs with branched polyionic side chains were investigated in CH3OH and H2O solutions by UV-Vis absorption, steady-state fluorescence, and lifetime spectroscopy. The different arylene units in the backbone led to variation of the HOMO-LUMO energy, resulting in distinctive absorption and fluorescence spectra. Branched polyionic side chains in the conjugated polyelectrolyte give rise to less aggregation even in aqueous solution, leading to higher quantum yields compared to the similar CPEs with linear side chains. Second, we also used the CPE with branched polyionic side chains as a mercury (II) ion sensor. Conjugated polyelectrolyte (CPE)/Rhodamine derivative combination system was designed as a mercury (II) ion sensor with high selectivity and sensitivity. CPE exhibited highly efficient quenching effect by the mercury (II) ion complexed rhodamine derivative via energy and/or charge transfer. The combination system displayed improved sensory response for the mercury (II) ion complex compared to a unitary CPE or rhodamine-based sensor. Third, a fluorescence chemosensor bearing four sodium carboxylates linked to tetra-phenylacetylene substituted pyrene, PyA4, has been designed and developed. PyA4 displays self-assembly behavior with strong intermolecular excimer emission in aqueous buffer solution. Fluorescence changes upon the addition of various metal ions show that PyA4 has high selectivity for the Cu(II) ion over other metal ions tested via fluorescence suppression, i.e. 98.5% fluorescence quenching. We found that more excimer quenching in aqueous solution may be caused by energy migration through the aggregates of PyA4 if the delocalized excited state of the pyrene stack is mobile as in the case of conjugated polymer. That is, the molecular aggregation controls exciton transport and amplified quenching phenomena. This system, the Cu(II) ion complexed to the PyA4, acts as a highly selective and sensitive fluorescent sensor for pyrophosphate, showing fluorescence enhancement which leads to 98% fluorescence recovery. For the bioanalytical applications, the activity of alkaline phosphatase (ALP) was successfully monitored by real-time turn-off assay. Fourth, we have prepared three generation of CPE-Ds (G-1, G-2, and G-3). The phenylacetylene units are connected at the meta-position, and their interior hydrophobic focal point is surrounded by the geometrically increased hydrophilic carboxylate end-groups as the generation increases. GPC analysis of the ester precursors, PG-1, PG-2, and PG-3, clearly demonstrates the monodisperse nature of these macromolecular structures; the three-dimensional structures of CPE-Ds show more spherical shape as the generation increases. Also, AFM images and DLS data suggests that G-2 and G-3 in water solution show intra-dendrimer interactions rather than inter-dendrimer aggregation. The photophysical properties of CPE-Ds revealed that intra-dendrimer interaction becomes stronger in aqueous solution with increasing generation, showing a red-shift in fluorescence spectra. The pH dependent quantum yields provide information for the state of aggregate of CPE-Ds, in which inter-dendrimer aggregate states of G-1 exist while G-2 and G-3 shows intra-dendrimer aggregation at very low pH (pH=3.0). More detail geometric structure of CPE-Ds is investigated by fluorescence lifetime measurement. Fluorescence quenching of G-3 is observed in the presence of cyanine dyes (DOC, DODC, and DOTC). The quenching is independent on the chain length of cyanine dyes. Also, it is attributed to degree of energy transfer, showing different fluorescence enhancement of cyanine dyes. Finally, conjugated polyelectrolyte dendrimers (Th-G-1, Th-G-2, and Th-G-3) containing thienyl (Th) groups in the conjugated backbone have been newly designed and synthesized. The modified convergent approach was used on the dendrimer synthesis. The thienyl group extended conjugated backbone allowed a low energy UV-Vis absorption and fluorescence emission. As the generation increases, intra-dendrimer aggregation is more pronounced, resulting in more red-shifted fluorescence spectra in aqueous solution. Structural peculiarity of the thienyl group induced the lack of inter-dendrimer aggregation in aqueous solution. Dynamic light scattering (DLS) and fluorescence excitation results revealed that even the first generation (Th-G-1) not allows inter-dendrimer aggregation. The fluorescence quenching efficiency of Th-G-n for methyl viologene was more significant with increasing generation in water, and very efficient quenching was observed in Th-G-3. In addition, quenching was more significant in water than methanol. Effective fluorescence enhancement of cyanine was observed by the energy transfer effect from Th-G-3.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Seoung Lee.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Schanze, Kirk S.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042427:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042427/00001

Material Information

Title: Photoactive Conjugated Polyelectrolytes and Conjugated Polyelectrolyte Dendrimers
Physical Description: 1 online resource (195 p.)
Language: english
Creator: Lee, Seoung
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: aggregation, conjugated, dendrimer, fluorescence, mercury, polyelectrolyte, polymer, pyrophosphate, rhodamine, sensor
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: In this dissertation, we primarily focus on the fundamental investigation of the photophysical properties of conjugated polyelectrolytes (CPEs) and conjugated polyelectrolyte dendrimers (CPE-Ds). Also, applications to the selective and sensitive pyrophosphate ions and mercury (II) ion sensors are explored. First, for CPEs, various aromatic moieties including phenyl (Ph), 2,1,3-benzothiadiazole (BTD), and 4,7-bis(2-thienyl)-2,1,3-benzothiadiazole (TBT) units have been incorporated into the polymer backbones. The photophysical properties of CPEs with branched polyionic side chains were investigated in CH3OH and H2O solutions by UV-Vis absorption, steady-state fluorescence, and lifetime spectroscopy. The different arylene units in the backbone led to variation of the HOMO-LUMO energy, resulting in distinctive absorption and fluorescence spectra. Branched polyionic side chains in the conjugated polyelectrolyte give rise to less aggregation even in aqueous solution, leading to higher quantum yields compared to the similar CPEs with linear side chains. Second, we also used the CPE with branched polyionic side chains as a mercury (II) ion sensor. Conjugated polyelectrolyte (CPE)/Rhodamine derivative combination system was designed as a mercury (II) ion sensor with high selectivity and sensitivity. CPE exhibited highly efficient quenching effect by the mercury (II) ion complexed rhodamine derivative via energy and/or charge transfer. The combination system displayed improved sensory response for the mercury (II) ion complex compared to a unitary CPE or rhodamine-based sensor. Third, a fluorescence chemosensor bearing four sodium carboxylates linked to tetra-phenylacetylene substituted pyrene, PyA4, has been designed and developed. PyA4 displays self-assembly behavior with strong intermolecular excimer emission in aqueous buffer solution. Fluorescence changes upon the addition of various metal ions show that PyA4 has high selectivity for the Cu(II) ion over other metal ions tested via fluorescence suppression, i.e. 98.5% fluorescence quenching. We found that more excimer quenching in aqueous solution may be caused by energy migration through the aggregates of PyA4 if the delocalized excited state of the pyrene stack is mobile as in the case of conjugated polymer. That is, the molecular aggregation controls exciton transport and amplified quenching phenomena. This system, the Cu(II) ion complexed to the PyA4, acts as a highly selective and sensitive fluorescent sensor for pyrophosphate, showing fluorescence enhancement which leads to 98% fluorescence recovery. For the bioanalytical applications, the activity of alkaline phosphatase (ALP) was successfully monitored by real-time turn-off assay. Fourth, we have prepared three generation of CPE-Ds (G-1, G-2, and G-3). The phenylacetylene units are connected at the meta-position, and their interior hydrophobic focal point is surrounded by the geometrically increased hydrophilic carboxylate end-groups as the generation increases. GPC analysis of the ester precursors, PG-1, PG-2, and PG-3, clearly demonstrates the monodisperse nature of these macromolecular structures; the three-dimensional structures of CPE-Ds show more spherical shape as the generation increases. Also, AFM images and DLS data suggests that G-2 and G-3 in water solution show intra-dendrimer interactions rather than inter-dendrimer aggregation. The photophysical properties of CPE-Ds revealed that intra-dendrimer interaction becomes stronger in aqueous solution with increasing generation, showing a red-shift in fluorescence spectra. The pH dependent quantum yields provide information for the state of aggregate of CPE-Ds, in which inter-dendrimer aggregate states of G-1 exist while G-2 and G-3 shows intra-dendrimer aggregation at very low pH (pH=3.0). More detail geometric structure of CPE-Ds is investigated by fluorescence lifetime measurement. Fluorescence quenching of G-3 is observed in the presence of cyanine dyes (DOC, DODC, and DOTC). The quenching is independent on the chain length of cyanine dyes. Also, it is attributed to degree of energy transfer, showing different fluorescence enhancement of cyanine dyes. Finally, conjugated polyelectrolyte dendrimers (Th-G-1, Th-G-2, and Th-G-3) containing thienyl (Th) groups in the conjugated backbone have been newly designed and synthesized. The modified convergent approach was used on the dendrimer synthesis. The thienyl group extended conjugated backbone allowed a low energy UV-Vis absorption and fluorescence emission. As the generation increases, intra-dendrimer aggregation is more pronounced, resulting in more red-shifted fluorescence spectra in aqueous solution. Structural peculiarity of the thienyl group induced the lack of inter-dendrimer aggregation in aqueous solution. Dynamic light scattering (DLS) and fluorescence excitation results revealed that even the first generation (Th-G-1) not allows inter-dendrimer aggregation. The fluorescence quenching efficiency of Th-G-n for methyl viologene was more significant with increasing generation in water, and very efficient quenching was observed in Th-G-3. In addition, quenching was more significant in water than methanol. Effective fluorescence enhancement of cyanine was observed by the energy transfer effect from Th-G-3.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Seoung Lee.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Schanze, Kirk S.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042427:00001


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1 PHOTOACTIVE CONJUGATED POLYELECTROLYTES AND CONJUGATED POLYELECTROLYTE DENDRIMERS By SEOUNG HO LEE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Seoung Ho Lee

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3 To my family

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4 ACKNOWLEDGMENTS Foremost, I would like to express my deep and sincere gratitude to my advisor Professor Kirk S. Schanze. My Ph.D. study and research could not have been finished without him who not only served as my supervisor but also encouraged and challenged me throughout my academic program. His guidance helped me in all the time of research and writing of this thesis. I could not have imagined having a better advisor and mentor for my Ph.D study. Besides my advisor, I would like to thank the rest of my thesis committee: Professors Lisa McElweeWhite, Daniel R. Talham, Eric Enholm, and Elliot Douglas, for their encouragement, insightf ul comments, and hard questions. I warmly thank Professors John R. Reynolds and Valeria D. Kleiman for their valuable advice and friendly help, especially for kindness in writing a letter of recommendation. Their extensive discussions around my work and in teresting explorations in operations have been very helpful for this study. My former and current colleagues supported me in my research work. I want to thank them for all their help, support, interest, and valuable hints. Especially I am very much obliged to Dr. Key Young Kim, Dr. Xiaoyong Zhao, Dr. John Peak, Dr. Yan Liu, Dr. Yongjun Li, and Dr. Jerret Vella. I also want to thank Dr. Hui Jiang and Dr. Eunkyung Ji for all discussions about the life as well as projects. I wish to thank Dr. Zhen Fang and Dr. Fude Feng for their guidance and extensive discussions in my researches. Julia Keller and Abigail Shelton were of great helps in difficult times. They looked closely at the thesis for English style and grammar, correcting both and offering suggestions for improvement. I warmly thank Dr. Anand Parthasarathy, Dr. Chen Liao, Emine Demir, Zhuo Chen, Aaron Eshbaugh, Dongping Xie, Randi Price, Amanda

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5 Sylvester, Danlu Wu, and Cheer Yang for their valuable advice and friendship. During my researches I have collaborated with Sevnur Kmrl in Dr. Kleiman group. I would like to show my gratitude to her for sharing her knowledge in photochemistry. This thesis would not have been possible without the love and support of my family. I owe my deepest gratitude to my parent They always encourage and understand me to continue my education abroad. Finally, as always, my biggest thanks are due to my wife Sangmi without whose love and understanding my works could never have been completed. Additionally, I would like to share this pleasure with my son, Junseo.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIS T OF TABLES .......................................................................................................... 10 LIST OF FIGURES ........................................................................................................ 11 ABSTRACT ................................................................................................................... 17 CHAPTER 1 INTRODUCTION .................................................................................................... 21 Conjugated Polyelectrolytes ................................................................................... 21 S ynthetic Methodology ..................................................................................... 23 Functional Group Effects on Aggregation ......................................................... 25 Linear functi onal group effects ................................................................... 26 Branched functional group effects .............................................................. 27 Amplified Quenching of Conjugated Polyelectrolytes ....................................... 30 Stern Volmer fluorescence quenching ....................................................... 30 Molecular wire effect .................................................................................. 31 Applications of Conjugated Polyelectrolytes to Sensors ................................... 33 Conjugated Polyelectrol yte Dendrimers .................................................................. 37 Dendrimers ....................................................................................................... 37 Architecture of Conjugated Dendrimers ........................................................... 38 Synthetic Approaches ...................................................................................... 40 Divergent approach .................................................................................... 41 Convergent approach ................................................................................ 42 Water Soluble Dendrimers ............................................................................... 43 Water Soluble Conjugated Dendrimers ............................................................ 45 Scope of the Present Study .................................................................................... 46 2 WATERSOLUBLE CONJUGATED POLYELECTROLYTES WITH BRANCHED POLYIONIC SIDE CHAINS: SYNTHESIS, CHARACTERIZATION, AND OPTICAL PROPERTIES ........................................................................................ 50 Results and Discussion ........................................................................................... 52 Synthesis and Characterization ........................................................................ 52 Precursor polymers .................................................................................... 54 Hydrolysis of precursor polymers ............................................................... 56 1H NMR spectroscopy ................................................................................ 56 Optical Properties ............................................................................................. 58 pH Dependent Aggregation .............................................................................. 65 Fluorescence Lifetime Sepctroscopy ................................................................ 69 Summary and Conclusions ..................................................................................... 73

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7 Experimental ........................................................................................................... 75 Materials ........................................................................................................... 75 Instrumentation and Methods ........................................................................... 75 Synthetic Procedures ....................................................................................... 76 3 HIGHLY EFFICIENT MERCURY (II) ION SENSOR BASED ON CONJUGATED POLYELECTROLYTERHODAMINE COMBINATION SYSTEM ........................... 84 Results and Discussion ........................................................................................... 85 Synthesis .......................................................................................................... 85 Optical Properties ............................................................................................. 86 Application to Hg2+ ion Sensor ......................................................................... 87 Selectivity ................................................................................................... 87 Sensitivity ................................................................................................... 89 S ensing M echanism ......................................................................................... 90 Compari son of Sensitivity between S Rho/PPE System and S Rho ................ 92 Summary and Conclusions ..................................................................................... 93 Experimental ........................................................................................................... 93 Materials ........................................................................................................... 93 Ins trumentation and Methods ........................................................................... 94 Synthetic Procedures ....................................................................................... 94 4 PHOTOPHYSICAL PROPERTIES OF PYA4 AGGREGATE AND ITS APPLICATION TO PYROPHOSPHATE SENSOR BASED ON TURNON SYSTEM ................................................................................................................. 96 Results and Discussion ........................................................................................... 99 Synthesis .......................................................................................................... 99 Characterization of PyA4 ................................................................................ 100 Quenching with Metal Ions ............................................................................. 104 Application to Pyrophosphat e (PPi) Sensing .................................................. 109 Selective and sensitive detection of PPi .................................................. 109 Real time ALP assay ............................................................................... 112 Plausible mechanism ............................................................................... 113 Summary and Conclusions ................................................................................... 114 Experimental ......................................................................................................... 114 Materials ......................................................................................................... 114 Instrumentation and Methods ......................................................................... 115 Synthetic Procedure ....................................................................................... 115 5 PHOTOPHYSICS AND ENERGY TRANSPORT IN CONJUGATED POLYELECTROLYTE DENDRIMERS ................................................................. 118 Results .................................................................................................................. 120 Synthesis and Characterization ...................................................................... 120 Synthesis of precursor s ........................................................................... 120 Hydrolysis of precursor ............................................................................ 122

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8 Geometric structure of CPE Ds ................................................................ 123 Geometric Properties: CPE Ds Size .............................................................. 124 Dynamic light scattering (DLS) ................................................................. 1 24 Atomic force microscopy (AFM) ............................................................... 126 Optical Properties ........................................................................................... 127 UVVis & fluorescence spectroscopy ....................................................... 127 Fluorescence quantum yield .................................................................... 132 Fluorescence lifetime studies ................................................................... 134 Fluorescence excitation spectroscopy ..................................................... 136 Concentration dependent fluorescence studies ....................................... 136 Fluorescence Quenching of CPE Ds by DOC, DODC, and DOTC ................ 137 Summary and Conclusions ................................................................................... 140 Experimental ......................................................................................................... 141 Materials ......................................................................................................... 141 Ins trumentation and Methods ......................................................................... 142 Synthetic Procedures ..................................................................................... 142 6 DESIGN, SYNTHESIS, AND PHOTOPHYSI CAL STUDIES OF THIENYL GROUP EXTENDED CONJUGATED POLYELECTROLYTE DENDRIMERS ..... 149 Results and Discussion ......................................................................................... 152 Synthesis and Characterization ...................................................................... 152 Optical Properties ........................................................................................... 155 UVVis absorption spectroscopy .............................................................. 155 Fluorescence spectroscopy ..................................................................... 157 Fluorescence quantum yield .................................................................... 158 Fluorescence lifetimes ............................................................................. 158 Chromophore/Dendrimer Aggregation ........................................................... 163 Concentration dependent fluorescence ................................................... 163 Fluorescence excitation spectroscopy ..................................................... 163 Dynamic l ight scattering (DLS) ................................................................. 164 Fluorescence Quenching of ThG n with MV2+ ............................................... 165 FRET from Th G 3 to Cyanine Dyes .............................................................. 167 Summary and Conclusions ................................................................................... 170 Experimental ......................................................................................................... 170 Materials ......................................................................................................... 170 Instrumentation and Methods ......................................................................... 171 Synthetic Procedures ..................................................................................... 172 7 CONCLUSIONS ................................................................................................... 179 Branched Polyionic Effect on Aggregation ............................................................ 179 Mercury (II) ion and Pyrophosphate ion Sensors .................................................. 180 Conjugated Polyelectrolyte Dendrimers ................................................................ 181 APPENDIX

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9 A CONJUGATED POLY ELECTROLYTES WITH BRANCHED POLYCATIONIC SIDE CHAINS ....................................................................................................... 182 B NMR SPECTRA .................................................................................................... 183 LIST OF REFERENCES ............................................................................................. 188 BIOGRAPHICAL SKETCH .......................................................................................... 195

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10 LIST OF TABLES Table page 2 1 Structures of conjugated polyelectrolytes with branched polyionic side chains and GPC analyses for their precursor polymers. ................................................ 53 2 2 UVVis absorption and photoluminescent properties of CPEs containing branched carboxylate or ammonium side chains. ............................................... 58 2 3 Fluorescence lifetimes ( i, ns) and relative amplitudes (RA, %) for PPEAr -bCO2 in MeOH, basic (pH = 9.0), and acdic (pH = 4.5) conditionsa ................... 70 4 1 Ksv a and [Q]90 b for Cu2+ ion quenching of PyA4 in H2O and MeOH ................. 107 5 1 UVVis absorption and photoluminescent properties of CPE Ds (CH3OH and H2O) and their precursors (THF) ................................................................... 127 5 2 Fluorescence lifetimes ( i, ns)a and relative amplitudes (RA, %) for CPE Ds (CH3OH and H2O) and their precursors (THF). ................................................ 134 5 3 Ksv a and [Q]90 b of CPEDs with cyanine dyes in H2O ....................................... 138 6 1 UVVis absorption and photoluminescent properties of ThG n (CH3OH and H2O (pH 8.0) ) and their precursors (THF) ....................................................... 157 6 2 Fluorescence lifetimes ( i, ns) and relative amplitudes (RA, %) for ThPG n and ThG n in THF, CH3OH and H2O (pH = 8 .0) solutions.a ........................... 159 6 3 Ksv a of ThG n with methyl viologen (MV2+) in CH3OH and H2O ...................... 165 6 4 Ksv a of CPEDs with cyanine dyes in H2O ........................................................ 169 A 1 Fluorescence lifetimes ( i, ns) and relative amplitudes (RA, %) for PPEAr -bNH3 + in MeOH, basic (pH = 9.5), and acdic (pH = 4.5) conditionsa ................. 182

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11 LIST OF FIGURES Figure page 1 1 Example of conjugated polyelectrolytes with various ionic side chains. ............. 21 1 2 Structure of watersoluble conducting polymer ( PT 1 and PT 2 ). ....................... 22 1 3 Various aromatic units. ....................................................................................... 22 1 4 Example of two different methods for the synthesis of CPEs (a) direct polymerization of monomers with ionic functional groups; (b) prec ursor route using monomers of organic functional groups. ................................................... 23 1 5 Various functional groups. .................................................................................. 25 1 6 Absorption and fluorescence spectra of PPESO3 -. ............................................ 26 1 7 Structures of P1 P2 and P3 .............................................................................. 28 1 8 UVVis absorption (left) and fluorescence spectra (right) of P1 P3 ................... 29 1 9 Schematic illustration of quenching effect in conjugated polymers with receptor and their fragment via energy migration to a receptor site occupied by PQ2+. .............................................................................................................. 32 1 10 Quenching mechanism of Molecular wire effect in conjugated polymers with receptors. .................................................................................................... 33 1 1 1 Structure of cationic conjugated polyelectrolyte. ................................................. 34 1 1 2 Schematic representation for the use of a cationic water soluble CP with a specific PNAC* optical reporter probe to detect a complementary ssDNA sequence. ........................................................................................................... 35 1 1 3 (a) Structure of PPE, (b) fluorescence intensity changes; A: PPEpapain, B: all 10 metals added to PPEpapain complex, C: Same without Hg2+ ion, and (c) qualitative interpretation of the Hg2+ ion induced agglutination. .................... 36 1 1 4 Structure of D 22 (3rd generation). ...................................................................... 38 1 1 5 Spacefilling models of phenylacetylene tridendrons D 4 D 10, D 22 D 46, and D 94. ............................................................................................................ 39 1 1 6 Synthetic diagram of divergent approach. .......................................................... 41 1 1 7 Synthetic diagram of convergent approach. ....................................................... 42

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12 1 1 8 Schematic representation showing the structureal similarity between the traditional Hartley micelle model and a dendrimer. ............................................. 44 1 1 9 (a) Representative structure of conjugated polyelectrolyte dendrimer (CPE D). (b) CPEDs paired with guest molecules. ..................................................... 45 2 1 Synthesis of 8 .................................................................................................... 52 2 2 Structure of bis ethynylene substituted Ph, BTD, and TBT; synthesis of monomers ( 1 and 2 ). .......................................................................................... 54 2 3 Polymerization through precursor route. ............................................................. 55 2 4 1H NMR spectra of (a) monomer 1 (b) PPEPh -bCO2 tBu and (c) PPE Ph -bCO2 -. ................................................................................................................. 57 2 5 (a) Relative absorption and (b) fluorescence emission spectra of PPE Ph -bCO2 -, PPE BTD-bCO2 -, and PPETBT -bCO2 -. ................................................... 58 2 6 (a) Visual and (b) Fluorescence colors of A: PPEPh -bCO2 -, B: PPEBTD-bCO2 -, and C: PPETBT-bCO2 -. ........................................................................... 59 2 7 Excitation spectra of (a) PPEPh -bCO2 at 430 and 500 nm; (b) PPEBTD-bCO2 at 550 and 700 nm; (c) PPE TBT -bCO2 at 650 and 800 nm. ................... 59 2 8 (a) Relative absorption and (b) fluorescence emission spectra of PPE Ph bNH3 + and PPEBTDbNH3 + in MeOH, MeOH/H2O (1/1, v/v ), and H2O ............. 61 2 9 Fluorescence emission spectra of precursors of branched anionic CPEs in THF; [ P PEAr -bCO2 -] = 5 M; Ar = Ph, BTD, and TBT. ...................................... 65 2 10 Absorption and fluorescence emission spectra of (a, b) PPEPh -bCO2 and (c, d) PPEPh -bNH3 + as pH changes in aqueous solution. ................................. 66 2 11 Absorption and fluorescence emission spectra of (a, b) PPEBTD-bCO2 and (c, d) PPEBTDbNH3 + as pH changes in aqueous solution. .............................. 67 2 12 (a) Absorption and (b) fluorescence emission spectra of PPETBTbCO2 as pH changes in aqueous solution. ........................................................................ 68 3 1 Structure of PPE-b CO2 -, S Rho 1, and S Rho 2. ................................................ 85 3 3 (a) Fluorescence spectra of PPE-bCO2 PPE-bCO2 S Rho 1 and S Rho 1 PPE-bCO2 S Rho 1 Hg2+. ... 87 3 4 Fluorescence changes of S Rho 1/ PPE-bCO2 system in H2O/DMSO (99/1, v/v) upon the addition of various metal ions ...................................................... 88

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13 3 5 Stern Volmer plots of S Rho 1/ PPE-bCO2 ( ) and S Rho 2/ PPE -bCO2 ( ) (PPE: 0.1 M and R hodamine derivatives 1 M) titrated with Hg2+ ion. ............. 89 3 6 Fluorescence changes of S Rho 2/ PPE-bCO2 system upon the addition of various metal ions. .............................................................................................. 90 3 7 Plausible sensing mechanism. ........................................................................... 90 3 8 Fluorescence of FRET donor ( PPE-bCO2 ) and absorption of FRET acceptors ( S Rho 1Hg2+ complex and S Rho 2 Hg2+ complex). ....................... 91 3 9 Fluorescence intensity changes of S Rho 1 upon the addition of various amounts of Hg2+ ion. ........................................................................................... 92 3 10 Titration profile I as a function of [Hg2+] ........................................................... 92 4 1 Structure of PyA4 .............................................................................................. 98 4 2 Synthesis of PyA4 ............................................................................................. 99 4 3 UV/Vis (left) and fluorescence emission (right) spectra of PyA4 in various solutions .......................................................................................................... 101 4 4 Fluorescence excitation and emission spectra of PyA4 (5 M) in 20 mM HEPES buffer at pH 7.5. ................................................................................... 102 4 5 Fluorescence emission spectra of PyA4 in 20 mM HEPES buffer at pH 7.5. ... 102 4 6 1H NMR spectra of PyA4 in (a) D2O/CD3OD (3/1, v/v ) and (b) DMSO d6; denotes solvent peak. ....................................................................................... 103 4 7 (a) Fluorescence emission spect ra of PyA4 solutions with increasing concentration (110 M) in 20 mM HEPES buffer solutions at pH 7.5; (b) ratio of excimer to monomer with increasing concentration (1100 M). .................. 104 4 8 (a) Fl uorescence emission changes of PyA4 (5 M) upon the addition of various metal ions (30 M); (b) Titration of PyA4 (5 M) with Cu2+ ions (020 M); Inset: ratio of excimer to monomer. .......................................................... 105 4 9 S tern Volmer plots of PyA4 (5 M) titrated with Cu2+ ions in 20 mM HEPES buffer at pH 7.5; Excitation at 456 nm, fluorescence intensity was monitored at 497 and 640 nm. ........................................................................................... 105 4 10 Stern Volmer pl ots of PyA4 (1, 5, and 10 M) titrated with Cu2+ ions in MeOH and pure H2O. ................................................................................................... 106 4 1 1 Titration of (a) PyA4 (5 M) and (b) PyE4 (5 M) with Cu2+ ions in MeOH ..... 108

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14 4 1 2 Fluorescence emission changes of PyA4 ( 5 M) Cu2+(20 M) at 640 nm upon the addition of anions (50 M). ................................................................ 109 4 1 3 (a) Titration of PyA4 ( 5 M)Cu2+ (20 M) with PPi (1 30 M) in 20 mM HEPES buffer at pH 7.5; (b) Titration profile with I/I0 ratio represented by the intensity at 640 nm. .......................................................................................... 110 4 1 4 (a) Titration of PyA4 ( 5 M)Cu2+ (30 M) with PPi (10100 M) in 100 mM HEPES buffer at pH 7.5. ................................................................................... 111 4 1 5 Fluorescence intensity changes of (a) PyA4 (b) PyA4 Cu2+ complex, and (c) PyA4 Cu2+ with PPi in 20 mM HEPES buffer at pH 7.5. ................................... 111 4 1 6 Real time ALP assay using PyA4 ( 5 M) Cu2+ (20 M) and PPi (30 M) in 20 mM HEPES buffer at pH 7.5, 37.0 oC. .............................................................. 113 4 1 7 Plausible m echanism of sensing process. ........................................................ 113 5 1 Structure of CPE Ds ( G 1 G 2 and G 3 ) .......................................................... 119 5 2 Synthesis of 5, 7, and 8 ................................................................................... 120 5 3 Synthesis of PG 1, PG 2, and PG 3 ................................................................ 121 5 4 Hydrolysis of branched side chains. ................................................................. 122 5 5 Spacefilling model of G 1 G 2 and G 3 generated by using MM2 molecular mechanics in Chem 3D Pro (version 10.0). ...................................................... 123 5 6 GPC data of precursor of CPE Ds ( PG 1 PG 2 and PG 3 ); polystyrene standards in THF. ............................................................................................. 124 5 7 Hydrodynamic radii obtained from dynamic light scattering (DLS) for G 1 (black), G 2 (red), and G 3 (blue) in H2O ......................................................... 125 5 8 AFM images of (a) G 1 (b) G 2 and (c) G 3 ................................................... 126 5 9 UVVis absorption and fluorescence spectra of CPE Ds ( G 1 G 2 and G 3 ) and their precursors ( PG 1 PG 2 and PG 3 ) in CH3 OH, H2O, and THF, respectively. ...................................................................................................... 129 5 10 UVVis absorption spectra of G 1 in CH3OH and H2O. ..................................... 130 5 11 UVVis abs orption and fluorescence spectra of CPEDs ( G 1 G 2 and G 3 ) and their precursors ( PG 1 PG 2 and PG 3 ). ................................................. 131 5 12 Fluorescence quantum yields changes of G 1, G 2, and G 3 at the pH 3 ~ 9 ... 133

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15 5 1 3 Fluorescence excitation spectra of (a) G 1 at 380, 400, and 450 nm, (b) G 2 at 400, 450, and 500 nm, and (c) G 3 at 450, 500, and 550 nm in H2O. .......... 136 5 1 4 Fluorescence emission spectra of (a) G 1 (b) G 2 and (c) G 3 with increasing concentration in H2O. ...................................................................... 136 5 15 Structure of cyanine dyes (DOC, DODC, and DOTC). ..................................... 137 5 1 6 Fluorescence emission spectra of (a) cyanine dyes only (DOC, DODC, and DOTC: 1.0 M) and G 3 titrated with (b) DOC, (c) DODC, and (d) DOTC in H2O, pH 8.0. ..................................................................................................... 139 5 1 7 Stern Volmer plots of G 3 ; fluorescence quenched by cyanine dyes in H2O ... 140 6 1 Structure of CPE Ds ( ThG 1 ThG 2 and ThG 3 ). ....................................... 151 6 2 Synthesis of (a) focal points and (b) a core unit ................................................ 152 6 3 Synthesis of precursors of CPE Ds ((a) ThPG 1 (b) Th PG 2 and (c) Th PG 3 ). ............................................................................................................... 153 6 4 Hydrolysis of branched side chains. ................................................................. 154 6 5 UVVis absorption and Fluorescence spectra of CPE Ds ( ThG 1 ThG 2 and T h G 3 ) and their precursors ( ThPG 1 ThPG 2 and ThPG 3 ). ............ 156 6 6 Fractional amplitude changes of fluorescence lifetimes of ThG 1 in (a) CH3OH and (d) H2O ThG 2 in (b) CH3OH and (e) H2O and ThG 3 in (c) CH3OH and (f) H2O .......................................................................................... 161 6 7 Fluorescence emission spectra of (a) ThG 1 (b) ThG 2 and (c) ThG 3 with increasing concentration in H2O. ............................................................... 163 6 8 Fluorescence excitation spectra of (a) ThG 1 at 400 and 500 nm, (b) ThG 2 at 400 and 500 nm, and (c) Th G 3 at 450 and 500 nm in H2 O. ....................... 163 6 9 Hydrodynamic radii obtained from dynamic light scattering (DLS) for (a) ThG 1 (b) Th G 2 and (c) ThG 3 in H2O. ........................................................... 164 6 10 Stern Volmer plots of ThG n (a) CH3OH and (b) H2O; fluorescence was quenched by methyl viologen (MV2+). ............................................................... 165 6 1 1 Structure of cyanine dyes (DOC, DODC, and DOTC). ..................................... 167 6 12 Fluorescence of FRET donor ( ThG 3 ) and absorption of FRET acceptors (cyanine dyes). ................................................................................................. 167

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16 6 13 Fluorescence emission spectra of (a) cyanine dyes only (DOC, DODC, and DOTC) and ThG 3 titrated with (b) DOC, (c) DODC, and (d) DOTC in H2O pH 8 ; [ThG 3 ] = 1.0 M; [dye quencher] = 0 ~ 0.3 M .................................... 168 6 14 Stern Volmer plots of ThG 3 in H2O; fluorescence quenched by cyanine dyes : DOC, DODC, and DOTC. ........................................................................ 169 A 1 (a) Visual and (b) Fluorescence colors of A: PPEPh -bNH3 + and B: PPEBTD-bNH3 +; [PPEAr -bNH3 +] = 30 M in H2O. .................................................. 182 B 1 1H NMR (300 MHz, CDCl3) sp ectrum of S Rho 1 (chapter 3). ......................... 183 B 2 1H NMR (300 MHz, CDCl3) spectrum of S Rho 2 (chapter 3). ......................... 183 B 3 1H NMR (300 MHz, CDCl3) spectrum of PyE4 (chapter 4). .............................. 184 B 4 1H NMR (300 MHz, D2O/CD3OD (3/1, v/v ) ) spectrum of PyA4 (chapter 4). ..... 184 B 5 1H NMR (300 M Hz, CDCl3) spectrum of PG 1 (chapter 5). ............................... 185 B 6 1H NMR (300 MHz, CDCl3) spectrum of PG 2 (chapter 5). ............................... 185 B 7 1H NMR (300 MHz, CDCl3) spectrum of PG 3 (chapter 5). ............................... 186 B 8 1H NMR (300 MHz, CDCl3) spectrum of ThPG 1 (chapter 6). ......................... 186 B 9 1H NMR (300 MHz, CDCl3) spectrum of ThPG 2 (chapter 6). ......................... 187 B 10 1H NMR (300 MHz, CDCl3) spectrum of ThPG 3 (chapter 6). ......................... 187

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17 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy PHOTOACTIVE CONJUGATED POLYELECTROLYTES AND CONJUGATED POLYELECTROLYTE DENDRIMERS By Seoung Ho Lee December 2010 C hair: Kirk S. Schanze Major: Chemistry In this dissertation, we primarily focus on the fundamental investigation of the photophysical properties of conjugated polyelectrolytes (CPEs) and conjugated polyelectrolyte dendrimers (CPE Ds) Also, applications t o the selective and sensitive pyrophosphate ions and mer cury (II) ion sensors are explored. First, for CPEs, various aromatic moieties including phenyl (Ph), 2,1,3benzothiadiazole (BTD), and 4,7bis(2 thienyl) 2,1,3benzothiadiazole (TBT) units have been incorporated into the polymer backbones. The photophysical properties of CPEs with branched polyionic side chains were investigated in CH3OH and H2O solutions by UVVis absorption, steady state fluorescence, and lifetime spectroscopy The different aryle ne units in the backbone led to variation of the HOMO LUMO energy, resulting in distinctive absorption and fluorescence spectra. Branched polyionic side chains in the conjugated polyelectrolyte give rise to less aggregation even in aqueous solution, le adin g to higher quantum yields compared to the similar CPEs with linear side chains. Second, we also used the CPE with branched polyionic side chains as a mercury (II) ion sensor. C onjugated polyelectrolyte ( CPE) / Rhodamine derivative combination system was d esigned as a Hg2+ ion sensor with high selectivity and sensitivity CPE

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18 exhibited highly efficient quenching effect by the Hg2+ ion complexed rhodamine derivative via energy and/or charge transfer T he combination system displayed improved sensory response for the Hg2+ ion complex compared to a unitary CPE or rhodaminebased sensor Third, a fluorescence chemosensor bearing four sodium carboxylates linked to tetra phenylacetylene substituted pyrene, PyA4 has been designed and developed. PyA4 displays self assembly behavior with strong intermolecular excimer emission in aqueous buffer solution. Fluorescence changes upon the addition of various metal ions show that PyA4 has high selectivity for the Cu2+ ion over other metal ions tested via fluorescence suppression, i.e. 98.5% fluorescence quenching. We found that more excimer quenching in aqueous solution may be caused by energy migration through the aggregates of PyA4 if the delocalized excited state of the pyrene stack is mobile as in the case of conjugated polymer. That is, the molecular aggregation controls exciton transport and amplified quenching phenomena. This system, the Cu2+ ion complexed to the PyA4 acts as a highly selective and sensitive fluorescent sensor for pyrophosphate, showing fluorescence enhancement which leads to 98% fluorescence recovery. For the bioanalytical applications, the activity of alkaline phosphatase (ALP) was successfully monitored by real time turn off assay. Fourth, we have prepared three generation of CPE Ds ( G 1 G 2 and G 3 ). The phenylacetylene units are connected at the meta position, and their interior hydrophobic focal point is surrounded by the geometrically increased hydrophilic carboxylate endgroups as the generation increases. GPC analysis of the ester precursors, PG 1 PG 2 and PG 3 clearly demonstrates the monodisperse nature of these macromolecular

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19 structures; the threedimensional structures of CPE Ds show more spherical shape as the generation increases ( G1 G2 G3 ). Also, AFM images and DLS data suggests that G 2 and G 3 in H2O show intra dendrimer interactions rather than inter dendrimer aggregation. The photophysical properties of CPE Ds revealed that intra dendrimer interaction becomes stronger in aqueous solution with increasing generation, showing a r edshift in fluorescence spectra. The pH dependent quantum yields provide information for the state of aggregate of CPE Ds, in which inter dendrimer aggregate states of G 1 exist while G 2 and G 3 shows intra dendrimer aggregat ion at very low pH (pH = 3). More detail geometric structure of CPE Ds is investigated by fluorescence lifetime measurement. Fluorescence quenching of G 3 is observed in the presence of cyanine dyes (DOC, DODC, and DOTC). The quenching is independent on the chain length of cyanine dyes. Also, it is attribut ed to degree of energy transfer, showing different fluorescence enhancement of cyanine dyes Finally, conjugated polyelectrolyte dendrimers ( ThG 1 ThG 2 and ThG 3 ) containing thienyl (Th) groups in the conjugated backbone have been newly designed and synthesized. The modified convergent approach was used on the dendrimer synthesis The thie nyl extended conjugated backbone allowed a low energy UVVis absorption and fluorescence emission. As the generation increases ( ThG 1 ThG 2 ThG 3 ) intra dendrimer aggregation is more pronounced, resulting in more redshifted fluorescence spectra in aqueous solution. Structural peculiarity of the thienyl group induced the lack of inter dendrimer aggregation in aqueous solution. Dynamic l ight scattering (DLS) and fluorescence excitation results revealed that even the first generation ( ThG 1 ) not allows inter dendrimer aggregation. The fluorescence

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20 quenching efficiency of ThG n for methyl viologen e (MV2+) was more significant with incre asing generation ( ThG 1 < ThG 2 < Th G 3 ) in H2O, and very efficient quenching was observed in ThG 3 In addition, quenching was more significant in H2O than CH3OH. Effective fluorescence enhancement of cyanine was observed by the energy transfer effect fro m Th G 3

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21 CHAPTER 1 INTRODUCTION Conjugated Polyelectrolytes Conjugated polyelectrolytes (CPEs) are water soluble conjugated polymers (CPs) having ionic functional groups such as sulfonate (SO3 -), carboxylate (CO2 -), phosphate (PO3 2-), and alkyl ammonium (NR3 +) Such ionic functional groups make the CPs soluble in aqueous solution or other polar solvents.12 Some examples of CPEs are shown in Figure 11. These CPEs are of great interest because they provide a number electron polarization seen in organic conjugated polymers, but also environment friendly processing, applications to biological system, and amphiphilic properties.1,37 O O S O3 n O O n N M e3 + N M e3 + n -O2C C O2 S O3 P P E N M e3 +P F C O2 -P P E S O3 Figure 11. Example of conjugated polyelectrolytes with various ionic side chains. Due to such unique properties of CPEs, the researches on conjugated polyelectrolytes have attracted considerable attention, and much effort has been devoted to the development of versatile CPEs. Since Wudl and Heeger et al. reported the results of a pioneering study in 1987, in which water soluble conducting polymers of 3 (2 sulfonatoethyl) substituted ( PT 1 ) and 3(4 sulfonatobutyl) substituted ( PT 2 ) polythiophene were prepared by electropolymerization (Figure 12),8 presently, extensive researches have been performed by many scientists over the world to

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22 develop highly efficient optoelectronic applications using CPEs. In addition, because their photophysical properties can be easily modified by external stimulus, it is considered and developed to be used as sensory materials to detect chemical s (chemosensors) or bioactive species (biosen sors) in aqueous media.9 S S O3 n N a S n S O3 N a P T 1 P T 2 Figure 12. Structure of watersoluble conducting polymer ( PT 1 and PT 2 ). S N S O O N S N N S N S S C6H1 3 C6H1 3 P h P y E D O T T h B T D T B T F Figure 13. Various aromatic units. Systematic modification of both CPEs backbone and functional groups is required f or the effective and advanced applications As a component of CPEs backbone, variable aromatic units such as phenyl (Ph), pyrrole (Py), thiophene (Th), 3,4ethylenedioxythiophene (EDOT), 2,1, 3 benzothiadiazole (BTD), fluorene (F) and their combinations are incorporated into polymer backbones.1011 Various aromatic units are present ed in Figure 13 Also, several cationic and anionic or bulky ionic side chains are

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23 available for efficient doping in the film or as a receptor for chemical or biomelecules as well as overcoming the solubility problems.10 Synthetic Methodology I I O O R R P d ( P P h3)4, C u I H2O / T E A O O R R A r nR = i o n i c f u n c t i o n a l g r o u p s(a ) (b ) I I O O R R H A r H P d ( P P h3)4, C u I T H F / T E AR = o r g a n i c f u n c t i o n a l g r o u p s P o l y m e r i z a t i o n O O R R A r n O O R R A r nR = I o n i c f u n c t i o n a l g r o u p s H y d r o l y s i s1 A ci d i c, b a si c, o r n e u t r a l co n d i t i o n 2 N a2C O3so l n H A r H Po l y m e r i z a ti o n Figure 14. Example of two different methods for the synthesis of CPEs (a) direct polymerization of monomers with ionic f unctional groups; (b) precursor route using monomers of organic functional groups. There are mainly two pathways to synthesize water soluble conjugated polyelectrolytes: one is direct polymerization of conjugated monomer having ionic side chains in aqueous solution or polar solvents (Figure 14a) ; the other is through precursor route in organic solvents (Figure 1 4 b) The former way is simple and fast, but it is limited to estimate polymer size. In addition, electrostatic repulsions between ionic side c hains prevent effective polymerization of monomers in aqueous solution or polar solvents. On the other hand, the latter way is relatively protracted because it requires more synthetic steps. Nevertheless, precursor route is frequently used in CPEs synthesi s because of its easines s to understand conjugated poly me rs characteristics. Indeed, gel permeation chromatography (GPC) measurement of the precursors of CPEs provides useful information such as the number of monomer units and polydispersity

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24 (PDI ) of the conjugated polymer. Furthermore, their NMR spectra and UV Vis & fluorescence spectral data afford comparable information for CPEs a fter the precursor is converted to water soluble one. There are many methodologies for t he extension of conjugated backbone in CPs. Among them, transitionmetal catalyzed cross coupling reactions can easily be considered and widely used nowadays. Stille,12 Heck,13 Suzuki,14 and Sonogashira15 coupling reactions are typical examples. Also, plenty of newly developed coupling re actions were explored during the last decades: most of these reactions are used without amine or become simplified. It is very interesting that all coupling reactions can be perform ed in aqueous solution while many general reactions are affected by extreme ly small amount of oxygen or water. Instead, oxygen is more able to disrupt the coupling reaction. This is attributed that the coupling reactions occur with unsaturated metal complexes that do not have 18 valence electrons. The empty coordination sites of the unsaturated metal ions are usually very reactive with oxygen from the air. In addition, The widely used palladium catalyst, t etrakis(triphenylphosphine)palladium(0) for the coupling reaction is usually prepared in two steps from Pd(II) precursors T he Pd( 0) is easily oxidized to relatively stable Pd(II). In fact, Pd ( 0 ) stabilizing ligands such as triphenylphosphine are frequently used. While CPEs as a result of direct polymerization of monomers with ionic side chains are soluble in water or highly pola r solvents most important issue after polymerization using precursor routes is how organic soluble side chains can be modified to ionic states to dissolve them in aqueous or highly polar solvents. So, many organic soluble functional groups are designed as it can be easily transformed to ionic side groups

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25 through hydrolysis or other hydrolyzing processes. Some example of functional groups that can be readily converted to ionic groups by hydrolysis process is present in Figure 1 5 .These functional groups are hydrolyzed by base, acid, or metal catalyzed process. However, choosing the appropriate method is critical because some may disturb the conjugated backbone during the process. Therefore, attaching functional groups should be systematically designed, consi dering conjugated backbone. R O O R O O R O O O N H O R O N H O R O N H O R Figure 15 Various functional groups. Functional Group Effects on Aggregation The solvent induced aggregation of conjugated polyelectrolytes in solutions has been extensively explored through UV Vis absorption and fluorescence spectral changes in poor solvents or good solvents. Because the hydrophobic backbone and hydrophilic side chain of CPEs induce aggregation in aqueous solution, their photophysical properties can be affected by solvent environment. Also, such aggregation is more significant in the presence of oppositely charged species such as metal ions, anions, and fluorescence dyes. While the aggregation frequently brings the positive effects such as highly efficient response for anal ytes sensing, it also induces low quantum yield, low solubility, and complex sensing behaviors. As a result, to

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26 promote or prevent such aggregation, linear or branched side chains can be incorporated on the conjugated backbone. Linear functional group effects Figure 16 Absorption (left) and fluorescence (right) spectra of PPE SO3 in CH3OH ( ) 1/1 CH3OH/H2O ( ------) and H2O ( ) Figure was taken from Tan et al.16 Conjugated polyelectrolytes with linear side chains tend to aggregate in aqueous solution because of the hydrophobic effect In addition, when conjugated backbones approach closely, electrostatic repulsions between side chains are not significant as much as it prevents aggregation. In our previous work, solvent induced aggregation of PPESO3 was observed as the water ratio increase against CH3OH.16 As seen in inset of Figure 16 its functional group is relatively a linear ( nonbulky) type side chain. As seen in Figure 16 both the absorption and fluor escence properties of PPESO3 are very similar to those exhibited in CH3OH where the polymer chains exist as an unaggregated state.17 Therefore, the aggregation of PPESO3 can be minimized in CH3OH. The absorption spectra in H2O were bathochromically shifted with increasing

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27 water ratio showing narrow absorption spectra. The most significant change is that strong and narrow fluorescence spectra in CH3OH become weak and broad one with bathochromic shift. Consequently, both features in UV Vis and fluorescence spectra prove that PPESO3 in water is strongly aggregated but it exists in monomeric state in CH3OH. Such aggregation of CPEs having linear side chains was also found in several other CPEs having cationic or anionic linear side chains. Another previous work shows polymer aggregation in water solution, in which various aromat ic units such as pyridine (Py), thiophene (Th), 3,4 ethylenedioxythiophene ( EDOT), and 2,1,3 benzothiadiazole (BTD) are incorporated into conjugated backbone, containing anionic sulfonate (R SO3 -) or cationic bis alkylammonium (R N+R N+R) as the side chains.10 It is believed that both anionic ( PPEAr SO3 -) and cationic ( PPEAr ( 4+ )) polymers are well dissolved in CH3OH, but in water. Their amphiphilic character induces polymer aggregation driven by the hydrophobic effect. Branched functional group effects Incorporation of bulky functional group can effectively modulate the degree of aggregation in aqueous solutions. Recently, Huang et. al., reported that fluorinecontaining poly(arylene ethynylene)s with bulky amino functionalized side groups exhibit a gradually decreased degree of aggregation as the number of functional group bec omes larger in aqueous solution, resulting in increased quantum yield.18

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28 I M e E t2N H2C H2C O O C H2C H2N E t2M e I H I M e E t2N C6H1 2 C6H1 2N E t2M e I n I M e E t2N C6H1 2 C6H1 2N E t2M e I n I M e E t2N C6H1 2 C6H1 2N E t2M e I H n P 1 P 2 P 3 Figure 17 Structures of P1 P2 and P3 The structures of water soluble fluor e necontaining poly(arylene ethynylene)s are presented in Figure 17 The structure of P1 is spatially more functionalized compared to the other P2 and P3 and their steric congestion decreases from P1 to P3 UVVis absorption and fluorescence spectra were monitored in CH3OH, H2O, and a mixture of the two solvents. As seen in Figure 18 with an increased volume fraction of H2O in the solvent, P1 shows very subtle wav elength changes in the UVVis absorption and fluorescence spectra except for the dec reased intensity induced by nonradi ative decay in polar solvent. These spectra are almost similar to its organic soluble precursor in THF. This observation suggests that the solution behavior of P1 in H2O may be similar with its precursor in THF, resulting in minimal aggregation. P2 however, exhibited bathochromic shift in UV Vis absorption and fluorescence spectra, and their intensities are reduced. These spectral changes are further pronounced in P3 P2 and P3 show similar behaviors as seen in the result of CPEs with linear side chains. These results support that degree of aggregation gradually increases from P1 to P3 in aqueous solution.

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29 Figure 18 UVVis absorption (left) and fluorescence spectra (right) of P1 P3 in pure CH3OH (gray line), in pure H2O (red line), and in mixtures of the two solvents at different compositions (black lines; 8/2, 7/3, 5/5, and 2/8 CH3OH/H2O from the top to the bottom). Figure was taken f rom Huang et al.18 More evidence regarding degree of aggregation from P1 to P3 was proposed wit h the fluorescence lifetimes in H2O. For P1 in CH3OH and H2O, similar shortlived component ( = 0.35 ns/CH3OH and = 0.32 ns/H2O) were observed with singleexponential fluorescence lifetime. These lifetimes are also very similar to that observed for its organic soluble precursor in THF. These observations suggested that aggregation of P1 is negl igible in H2O as observed in UV Vis and fluorescence spectra. For P2 and P3 in CH3OH, on the other hand, longlived component ( 2 p2 = 1.11 ns and 2 p3 = 0.94 ns) with around 7% amplitude, while fast decay components are predominant with 1 p2 = 0.35 ns (94% ) and 1 p3 = 0.28 ns (93%), respectively. These observations suggested

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30 that the degree of aggregation of P2 and P3 are extremely small in CH3OH. In water, however, the contribution of fast decay components ( 1 p2 = 0.33 ns and 1 p3 = 0.17 ns) decreased and longlived components ( 2 p2 = 0.96 ns and 2 p3 = 1.46 ns) increased, contributing to 23% ( P2 ) and 41% ( P3 ). This increased contribution of longlive decay supports that degree of aggregation increase as the functional groups are less congested. Amplified Quenching of Conjugated Polyelectrolytes One of the major features of CPEs is amplification of the fluorescence quenching when the quenchers are bound to them. This property which is referred to as superquenching or amplified quenching, has attracted much interest because of its usefulness in biological applications. This efficient quenching effect is induced via energy migration through entire conjugated backbone of CPEs, and the polymers provide very effective conduits for energy migration over long dist ances. Stern Volmer fluorescence quenching F* + Q F + Q kqF + Q F + Q Ka [F,Q] [F*,Q] hv (1 1) (1 2) I0/I = 1 + Ksv[Q] (1 3) F luorescence quenching arises by the interaction between fluorophore and quenchers, in which the quenching can be dynamic or static quenching. The dynamic quenching (Eq. 11) is induced by diffusive encounter s while static quenching (Eq. 12) takes place as a result of complex formation. In equation 11, F* is an excitedstate fluorophore, Q is a quencher, and kq is the bimolecular quenching rate constant. Also,

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31 Ka is the association constant for the groundst ate complex formation [F,Q] in equation 1 2. The kinetics of these process follow the SternVolmer19 equation (Eq. 13), where I0 is the intensity without a quencher, I is the intensity with a quencher In the case of purely dynamic quenching, Ksv is equal to kq0, where 0 is the fluorescence lifetime of F*. Since the excited state is quenched by a collision with a quencher (Q), the lifetime is reduced by the addition of quencher. On the other hand, Ksv is equal to association constant Ka, for purely static quenching. The quenching is not affected by the diffus ion rate of the quencher and the fluorescence lifetime is also independent for the quencher. This SV plot is frequently used to determine the difference between materials in the quantitative quenching. I f either static or dynamic quenching is dominant the slope of the S V plot becomes a linear. However, the two processes are competitive in most cases, resulting in nonlinear SV data. Molecular wire effect The amplified quenching was first explored by Swager and co workers.20 The fluorescence chemosensor using the molecular wire approach showed enhanced sensory response.21 To evaluate this concept, the fluorescence quenching of cyclophane containing polymer was compared to that of a low molar mass model (monomer unit of cyclophanecontaining polymer) ( Figure 19 ). Interestingly, greatly enhanced quenching of cyclophanecontaining polymer was monitored while the model compound showed moderate quenching effect in the presence of methyl viologen (MV2+) which is a well known electron transfer quenching agent. As seen in Figure 19 the comparison of the conjugated system to an isolated fluorescence receptor illustr ated how amplified quenching is processed in both systems. In a monoreceptor system, the fluorescence is quenched only for the receptor forming complex with methyl viologen. In

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32 a molecular wire system, however, fluorescence quenching effect occurs throughout entire polymer even though only a part of polymer units is occupied by the quencher. Therefore, the electronic properties of conjugated polymers provide amplified response for analytes through the efficient energy migration to occupied receptor sites. N C H3 n N H3C h 100% reduction in emission N C H3 N H3C h 33% reduction in emission h n h h h + PQ2+PQ2+ + PQ2+PQ2+ h O O O O O O O O O O R R R = C O N ( C8H1 7)2 n n Figure 19 Schematic illustration of quenching effect in conjugated polymers with receptor and their fragment via energy migration to a receptor site occupied by P Q2+. Figure was taken from Swager et al.20 The am plified quenching efficiency in the conjugated polymer is attributed to the molecular wire effect. That is, the extended electronic communication and transport by conjugated polymer chain induce efficient fluorescence quenching. As shown in Figure 1 10, ex cited electron (a bound electronhole) is generated on the polymer backbone upon the absorption of light, and it moves very rapidly along the chain. The conjugated

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33 polymer acts as a conduit for exciton. The fluorescence is quenched when the exciton gets into proximity with the polymer repeat unit which contains a quencher occupied receptor. The extremely efficient exciton migration in the excited state leads to quenching of many repeat units in polymer chains, resulting in amplified quenching for the quench er or analytes. + + eeET Quencher receptor analyte Figure 110 Quenching mechanism of Molecular wire effect in conjugated polymers with receptors. Figure was taken from Swager et al.21 Applications of Conjugated Polyelect rolytes to Sensors Fluorescence is a widely used and rapidly expanding method in chemical and biological sensing. Sensor should selectively recognize the guest molecules, and sensitively translate its recognition to signal. Sensing behaviors display their signal through changes in intensity, wavelength, energy transfer, and fluorescence lifetime. So to improve the sensitivity, such fluorescence signal should be readily perturbed by external stimuli. From this point of view, conjugated polymers (CPs) are one of the most efficient sensors because of their extraordinary sensitivity for the guest molecule

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34 sensing, which is a remarkable advantage of CP based sensors compared to the sensor s using small molecules .2226 Intra or inter molecular energy migration provides very efficient sensing response to even minor perturbation. Recently, water soluble conjugated polymers have attracted intensive attention as a very efficient optical chemical sensor due to their use in analys is and detection of the guest molecules in environmental and biological setting. N H2C H3 H3C H2N n I I Figure 11 1 Structure of cationic conjugated polyelectrolyte. It is reported that cationic water soluble conjugated polymer (CCP) responds to single stranded DNA (ssDNA) in the presence of peptide nucleic acids (PNAs) labeled with a fluorescein at the 5 fluorescein dye.27 Poly(9,9 bis(6 N,N,Ntrimethylammonium) hexyl) fluorene phenylene containing iodide counter anions (Figure 11 1 ), CCP, was used as a donor for fluorescence resonance energy transfer (FRET), and f luorescein dye on the 5 position of PNAs (PNAC*) acts as an energy acceptor. As seen in Figure 11 2 no electrostatic interaction between CCP and PNA C* are observed in the initial solution. Complementary ssDNA hybridizes with the PNA, and it forms a com plex with CCP, resulting in a decrease in the average CCP C* distance (Route A). This complex allows FRET from CCP to fluorecein dye on PNA. However, when a ssDNA does not match

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35 the PNA sequence, hybridization does not take place (Route B). In this case, N o FRET occurs. Figure 11 2 Schematic representation for the use of a cationic water soluble CP with a specific PNAC* optical reporter probe to detect a complementary ssDNA sequence. Figure was taken from Bazan et al.27 To improve the sensory response, frequently, two or more combinations of polymers and/or receptors are used. Bunz et al. also reported that poly (paraphenyleneethynylene) ( PPE) papain system effectively detects Hg2+ ion with high sensitivity compared to PPE.28 Figure 11 3 a shows the structure of PPE containing carboxylate side chains (R CO2 -), which is water soluble. The addition of Hg2+ ion leads to the weak fluorescence quenching of PPE in PIPES (piperazine 1,4bis(2

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36 O O +N a-O O O-N a+ O n (a) (c) (b) Figure 11 3 (a) Structure of PPE, (b) fluorescence intensity changes; A: PPEpapain complex, B: a ll 10 metals added to PPEpapai n complex, C: Same without Hg2+ ion, and ( c) qualitative interpretation of the Hg2+ ion induced agglutination of the PPEpapain complex; top left: PPE alone, top right: electrostatic complex from PPEpapain, bottom: the addition of Hg2+ ion to PPEpapain l eads to its precipitation by cross linking of the papain molecules through Hg2+ ion Figure was taken from Bunz et al.28 ethanesulfonic acid)) buffer at pH 7.2. On the other hand, the detection of Hg2+ ion by PPE papain complex system was more efficient than either PPE or papain alone. PPEpapain complex (A ) shows blue color fluorescence as shown in Figure 11 3 b. Its fluorescence was quenched in the presence of various divalent metal ions including Hg2+ ion (B). However, the fluorescence quenching was not observed upon the addition of other metal ions without Hg2+ ion (C) Figure 11 3 c proposed mechanism of action for this Hg2+ ion sensing. Positively charged papain slightly quenched the fluorescence of PPE. The addition of Hg2+ ion, however, caused precipitation because all of the chain of PPE incorporated into PPEHg2+papain agglutinate, and its solution was fully quenched.

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37 Conjugated Polyelectrolyte Dendrimers Dendrimers Dendrimers are highly branched macromolecules that can be subdivided into three architectural components: a central core, interior branches, and surface functional groups. Since the dendrimer was firstly synthesized by Vgtle in 1978,29 it has been disclosed by Denkewalter at Allied Corporation in 1981, Tomalia at Dow Chemical in 1983 and in 1985,30 Newkom e in 1985,31 and in 1990 Frchet introduced a convergent synthesis.32 In spite of short historical background, dendrimer has been r apidly developed as new research area and attracted great scientific interest because of their unique molecular architecture. At present, the global trend for dendrimer researches is shifted to their properties and potential application from the research r elated to the synthesis. The word dendrimer by Tomalia in 1980 is derived from the Greek words Dendron (tree) and meros (part) because this type of molecule resembles a tree. Dendrimer is symmetric and monodisperse molecules and synthesized through a stepw ise repetitive reaction sequence, which gives rise to different generations of the dendrimers. In addition, their structure is mainly divided into three parts: core, branches, and end groups. Such distinguished frameworks induce relatively rigid conformat ion in terms of size and shape compared to linear polymers.33 Furthermore, geometrically increased number of end groups with increasing generation affect to the surface functionality of the dendrimer, i.e. changing the end groups leads to distinct characteristics of dendrimer. Also, appropriat ely designed high generation dendrimers display a distinctive interior that is sterically encapsulated by external end groups, enabling applications as unimolecular container molecules.34 Such structural features enable the

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38 dendrimers to be used as components in drug or gene delivery, and numerous researches are currently in progress. Architecture of Conjugated Dendrimers D -2 2 Figure 1 1 4 Structure of D 22 (3rd generation). As one of the dendritic systems, phenylacetylene building blocks have been incorporated into dendrimer backbone by Moore and coworkers (Figure 11 4 ).35 Because of their fully conjugated backbone, it has a higher net electron density throughout entire dendrimer, which induces extended electronic state and coherent transfer or enhanced throughbond energy transfer. It is also noted that dendrimer syntheses are frequently limited by their intrinsic poor solubility. Especially in the case of dendrimer having more rigid backbone including phenylacetylene building blocks, designing the dendrimer considering the solubilizing end groups is a principal issue.

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39 Figure 11 5 Spacefilling models of phenylacetylene tridendrons D 4 D 10, D 22, D 46, and D 94. Figure was taken from Moore et al.36 One of the characteristics of dendrimers is the presence of numerous peripheral end groups that all converge to a s ingle core. Such plentiful end groups induce a threedimensional globular architecture of dendrimers, which has limited degrees of conformational flexibility. Figure 11 5 shows the spacefilling models of the five members of the conjugated phenylacetylene dendrimers. This series also show s more spherical shape as the generation increases. The structure of D 96 has a diameter of approximately 5.5 nm.36 Consequently, such structures define the concept of a shapepersistent macromolecules.

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40 Synthetic Approaches Similar to synthesis of conjugated polyelectrolytes, the reactions used in preparation of phenylacetylene dendrimers are activati on process of protected terminal acetylene and its palladium catalyzed cross coupling reaction with aryl halides in the presence of a catalytic amount of copper (I) iodide. As a protecting group of the terminal acetylene, the trimethylsilyl (TMS) group is an excellent masking group which is the most common protecting group on the terminal acetylene group. Necessarily, various protecting groups such as triisopropylsilyl (TIPS), tertbutyldimethylsilyl (TBDMS), and tertbutyldimethylsilyloxymethyl (TOM) are a vailable when the reaction needs selective activation for regioselective substitutions or appropriate reaction conditions. These protecting groups are readily removed by acid or fluoride salts (such as NaF, TBAF, HF Py, or HF NEt3). For the cross coupling reaction, typically two kinds of catalysts can be employed: palladium complexes and a halide salt of copper (I). Several palladium (0) complexes such as tetrakis (triphenylphosphine)palladium (0) and bis (dibenzylideneacetone)palladium (0) as well as palladi um (II) complex, bis (triphenylphosphine)palladium (II) chloride, are available in this reaction. These catalysts activate the aryl halides by oxidative addition into carbonhalogen bond. Also, another catalyst, copper (I) halides, forms aryl alkyneCu complex with terminal alkyne, in which complex formation is promoted by base such as amines (usually diisopropylamine or triethylamine). Finally both activated species generate new carboncarbon bonds. Based on above coupling reaction, various approaches are c onsidered for the combination of phenylacetylene building blocks. Among various synthetic methodologies for dendrimer synthesis, two major approaches known as divergent and

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41 convergent methods have been developed and widely used as foremost means. Although desired dendrimers can be generated by either one approach, the reactions have to be considered by many factors including the solubility, separation from byproducts, and costs. Divergent approach + 3 Coupling Step Activation Step + 6 Coupling Step Repeat Activation and Coupling Step Figure 11 6 Synthetic diagram of divergent approach. Figure was taken from Frchet et al.37 The original divergent method is developed by Newkome and Tomalia, which involves conjunction of repeat units on a core. Figure 11 6 shows stepwise synthetic cartoon for divergent approach. The sequential repetitions of coupling and activation process induce continuous outward of branches with a number of functional groups on the periphery of the dendrimer. Additional coupling and activation steps lead to an geometric increase in the number of branches, providing the abundant reactive site for

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42 the endgroups. Finally, f u nctionalized endgroups are incorporated into each reactive site to have unique properties. Remarkable advantage of the divergent approach is less steric effect in growth of the dendrimer size, which also induce improved yields in growth of dendrimer size. However, this method is limited by the factor that many functional groups should be incorporated at higher generation. This may cause increase of the possibility of molecular imperfection with increasing generation. Poor solubility also makes dendrimer synthesis difficult. Furthermore, it is difficult to selectively substitute the endgroups for exclusive functionality in use. Convergent approach Coupling Step Activation Step Coupling Step Repeat Activation and Coupling Step Figure 11 7 Synthetic diagram of convergent approach. Figure was taken from Frchet et al.37 The convergent approach was first reported by Hawker and Frche t in 19891990. As seen in Figure 11 7 the growth of dendrimer initiates inward from the exterior by coupling end groups to each branch of the monomer. This coupling reaction creates a

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43 single functional group located at the focal point, displaying wedge shaped dendritic fragment. After activation of this dendron, continuous accumulation on an additional monomer units leads to a higher generation dendron. After repetition of this process for desired dendrimer, the dendrons can be incorporated into a core unit, affording a multi functionalized dendrimer. It should be noted that this convergent method is absolutely comparable to its divergent counterpart because only a few reactive sites are involved in this reaction. This approach proffers distinctive features such as involvement of one or two reactive sites for growth, easy organization of end group functionality, and uncomplicated purification and characterization of intermediates. That is, it provides greater control in synthetic and structural manipulati on than the divergent method. Such distinct features enable their functionality to be selectively and strategically modified throughout the molecules. One limitation of this approach is that attaching the dendrons on the core is less straight forward than the divergent method because of steric congestion, causing poor yield. Nevertheless, i t is the most attractive that well defined unsymmetrical dendrimers can be producible with this approach. Also, advanced convergent approaches such as doublestage conver gent method have been developed to overcome such shortcoming, in which flexible multifunctional dendritic core is employed in the final step of the synthesis.36 Water Soluble Dendrimers Water soluble d endrimers are dendrimers having either positively or negatively charged ionic solubilizing groups as an end groups. Their entire structures also are very similar in both size and shape t o that of the Hartley micelle model as seen in Figure 11 8 .38 That is, both models have spherical structure with hydrophobic interior and

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44 hydrophilic exterior. In fact, such similarity between Hartley micelle and unimolecular micelle has been demonstrated by several groups.31,34,3840 The critical difference between these micelles is that water soluble dendrimer is a single molecule incapable of disintegration while the formation of traditional micelle relies on the external surroundings. For instance, the traditional micelles are affected by a variety of factors such as pH changes, ionic strength, solvent polarity, concentration, and temperature. On the other hand, the elemental structures of water soluble dendrimers in solut ion are in dependent on such exterior environments. Namely, water soluble dendrimers retain their structure unless covalent bonds are physically destroyed. O O O O O O O O O O O O O O O O O O O O O O O O H a r t l e y M i c e l l e OO OO OO OO -O O OO -O OO O OO -O OO O O-O OO-O -O OO-O O-O OO O O O O O O O O O OO O O D e n d r i t i c U n i m o l e c u l a r M i c e l l e Figure 11 8 Schematic representation showing the structureal sim ilarity between the traditional Hartley micelle model and a dendrimer. Figure was taken from Moore et al.38 Such water soluble dendrimers have recently attracted intensive attention because of their amphiphilic characteristic.4145 Because of the presence of charged groups, the properties of these dendrimers are strongly affected by the electrostatic interactions on

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45 the dendrimer surface and by the hydrophilic effect in interior branches. Also, although litt le attention is paid to the aggregation behavior of such amphiphilic dendrimers, their higher generation exists as a single molecule capable of molecular inclusion. Such feature is of use to the application such as the drug delivery, molecular encapsulation, charge transpor t, and sensor.46 Water S oluble Conjugated D endrimers H ydrophobic interaction H ydrophilic interaction(a) (b)R = ionic solubilizing groups CPE D R R R R R R R R R R R R Figure 11 9 (a) Representative s tructure of conjugated polyelectrolyte dendrimer ( CPED). (b) CPEDs paired with guest molecules; (left) hydrophobic species is closed to the dendrimer core and (right) hydrophilic species resides in dendrimer periphery. Conjugated polyelectrolyte dendrimers (CPE Ds) are a new class of the water soluble dendrimers carrying ionic solubilizing groups, and their back bones are conjugated with phenyacetylene units (Figure 11 9 a) The most remarkable feature of these CPE Ds is the shapepersistence. In the case of common nonconjugated dendrimers with amphiphil ic feature, their conformations are found to shrink or change size with changing external environment because they are physically flexible systems. Unlike such nonconjugated system s, however, CPEDs retain their certain stiffness because of the regid back bone. Another unique feature of conjugated dendrimers is that their hydrophobic backbone consists of all hydrocarbon without hetero atoms, which

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46 makes the interior more hydrophobic than other systems containing hetero atoms including oxygen and nitrogen. S uch characteristics of the CPE D might provide some unusual properties. It is possible that relatively more rigid and hydrophobic characteristics provide proper geometric structure to the specific molecules and form stable complex with analytes. In additio n, amphiphilic feature allows two different binding modes, so that both hydrophilic species and hydrophobic molecules can affect its photophysical properties or sensing response (Figure 11 9 b). Furthermore, we may expect that CPE Ds having higher net densi ty throughout entire conjugated backbone provide outstanding platform s for energy or electron transport as seen in conjugated polyelectrolytes (CPEs). For this distinctive dendrimer, not many researches have been investigated and developed. Only one exampl e is documented by Moore and co workers, in which synthesis and synthetic characterization are reported. Scope of the Present Study The purpose of the present study is to investigate and elucidate the photophysical properties of conjugated polyelectrolytes (CPEs) and conjugated polyelectrolyte dendrimers (CPE Ds). In this dissertation, CPEs and CPEDs are newly designed and synthesized and their characterizations are well described. Their photophysical properties are studied by the spectroscopic analysis s uch as UV Vis absorption, fluorescence, and fluorescence lifetime spectroscopy Also, AFM images and Dynamic Light Scattering (DLS) measurement are used to define their aggregation property Finally their unique photophysical properties are developed as a metal or bi o molecular sensor. In C hapter 2, branched polyionic side chain effects on the conjugated polymers are examined and compared to linear side chains Since conjugated polyelectrolytes

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47 having linear side chains show aggregation propensity in aqueous solution, their prospective applications are sometimes limited by low quantum yield, low solubility, and unexpected or complicated solution behavior induced by aggregation. To prevent polymer aggregation, relatively bulky polyionic side chains are incorporated to polymer backbone. Comparisons between CPEs having linear or branched polyionic side chains are studied by UV Vis absorption, fluorescence, and lifetime spectroscopy in CH3OH and H2O solutions Aggregation dependent on size of aromatic units in conjugated backbone is also explored. In C hapter 3, CPE based Hg (II) ion sensor is described I n CPE based sensory system, P PE with branched polyionic side chains retaining less aggregation in aqueous solution is used as a signaling moiety and a rhodami ne derivative act s as a Hg (II) ion receptor. This system shows high selectivity and sensitivity for Hg (II) ion upon the addition of various metal ions Such sensing response was identified by fluorescence intensity changes. Also, plausible sensing mechanism is provided, in which a spirotype rhodamine derivative undergoes ringopening by complexation with Hg (II) ion, providing a positive charge on the nitrogen atom. This result leads to the strong electrostatic binding with anionic carboxylate of P PE, re sulting in fluorescence quenching of P PE by the effective energy transfer. In C hapter 4, water soluble conjugated pyrene bearing phenylacetylene units with carboxylate side chains ( PyA4) was synthesized, and it is applied to a pyrophosphate (PPi) sensor. Photophysical properties and solution behaviors of PyA4 were studied in CH3OH and H2O solutions by fluorescence spectroscopy. Selectivity for Cu2+ ion over other metal ions and its quenching mechanism were explained by

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48 comparison of S tern V olmer plots in CH3OH and H2O solutions. PyA4 Cu2+ complex as a PPi sensor was also studied by fluorescence intensity changes upon addition various anions. Furthermore, the activity of alkaline phosphatase (ALP ) was monitored by the real time turn off assay. In C hapter 5, novel water soluble conjugated polyelectrolyte dendrimers (CPE Ds) were synthesized by the convergent method, where the branched carboxylate ionic groups were incorporated as an end group. S tructural configuration of each CPE was characterized by the com putational modeling, AFM and dynamic light scattering (DLS). GPC analysis provides a m onodispersity which one of the notable characteristics of the dendrimers. Because of higher electrostatic repulsion between the branched polyionic side chains and more spherical type with generation, little or no aggregation is expected at higher generation. Such properties of CPE Ds are characterized by UV Vis absorption, fluorescence, and lifetime spectroscopy Quenching and energy transfer effect from CPE Ds to the cy anine dyes (DOC, DODC, and DOTC) were studied with fluorescence measurement and SternVolmer Plot. In C hapter 6, we developed systematically water soluble conjugated polyelectrolyte dendrimers containing thienyl (Th) groups in the center of conjugated bac kbone, which w ere obtained by hydrolysis process using basic condition to dissolve them in aqueous solution. The photophysical properties were carefully studied in CH3OH and H2O solutions using UVVis absorption and fluorescence spectroscopy Intra dendrim er energy transfer from phenyl units to aromatic unit including thienyl (Th) and 2,1,3benzothiadiazole (BTD) was observed. Lifetime spectroscopy provides more

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49 detail photophysical properties of ThCPEDs in CH3OH and H2O solutions More e fficient quenchin g or energy transfer effect was observed at the high er generation.

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50 CHAPTER 2 WATERSOLUBLE CONJUGATED POLYELECTROLYTES WITH BRANCHED POLYIONIC SIDE CHAINS: SYNTHESIS, CHARACTERIZATION, AND OPTICAL PROPERTIES Over the past several decades conj ugated polyelectrolytes (CPEs) have attracted considerable attention and bec o me one of the versatile polymeric materials in optoelectronic devices47 and biochemical26 detection research because they have the exceptional feat ures, such as high fluorescence quantum yield, unique solution behavior ability to interact electrostatically with other oppositely charged species, and extraordinary high sensitivity to fluorescence quenchers due to exciton migration.21,4851 Particularly, high sensitivity to quenchers is amplified in aggregated states because exciton migration occur s through not only intrachain but also inter chain process es when quencher ions are bound to polymer aggregates.16,5253 However, in spite of such potential advantages of CPEs, their solution processing is sometimes limited by a low quantum yield, low solubility, and unexpected sensing behavior induced by a strong propensity to sel f assemble into aggregates in aqueous solutions.11,18,5455 For this reason, much effort has been devoted to retaining nonaggregated state or increasing fluorescence quantum yield in aqueous media by variation of solvent polarity, pH, and ionic strength.5658 In previous studies, however, conditions that are required for nonaggregation of polymers were not sufficient to maintain nonaggregated states or could not be qualifi ed for detecting analytes in biological settings. Furthermore, fluorescence quantum yield showed relatively small enhancement in such conditions.57 Nevertheless, several photochemical sensors have been developed to take advantage of aggregation between water soluble CPEs modulated by addition of analytes, which are based on electron transfer or energy transfer.16,5253,5961 For example, Bazan et al.

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51 observed that significant enhancement of fluorescence emission in polymer aggregates is due to shielding effect of the emitters from water c ontacts.62 This result may encourage development of highly sensitive chemical sensors even in various experimental conditions. According to previous studies, the aggregation occurs by a hydrophobic interaction of the polymer backbones and/or an electrostatic interaction between side chains of polymer in certain conditions.10,58 Furthermore, it is not surprising that such aggregation is also related to the solubility of polymers.11,18,5455 To overcome this drawback in aqueous solutions, recently, Swager reported that introducing relatively bulky fluorophore into the CPEs backbone induce less aggregation because its backbone is a less planar and linear shaped .54 Therefore, we hypothesized that attaching crowded and bul ky ionic functional groups could be an effective way of reducing hydrophobic interaction by increased electrostatic repulsion between polymers as well as it would increase polymers solubility in aqueous solution. Moreover, another distinctive advantage of these CPEs having branched polyionic side chains is threedimensional structure of a receptor, which can provide appropriate geometric cavity to the specific biomolecule and form stable complex with analytes.6364 This might increase their selectivity to the target molecules. In addition, we may expect that highly dense charges in a receptor can enhance binding of the anion, cation or biomolecule. Consequently, these structural advantages with a sensible optical characteristic which can be easily perturbed by external stimuli would show a potent ial possibility as an effective fluorescence sensor in highly sensitive bioassays.

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52 Results and Discussion Synthesis and Characterization In this study, we report the synthesis of water soluble CPEs having branched polyionic side chains (bulky functional gr oups) as shown in Table 1. As a polymer repeat unit, phenyl (Ph), 2,1,3 benzothiadiazole (BTD), or 4,7Bis( 2 thienyl ) 2,1,3benzothiadiazole (TBT) units have been introduced into polymer backbone, and negatively or positively charged water soluble branched polyionic side chains that can structurally hinder the aggregation of the polymer chains were also incorporated to the conjugated polymer backbones, thereby suppressing self quenching of their excited state. O2N C O2H C O2H C O2H O2N C O C l C O C l C O C l O2N N H2 N H2 N H2 O2N N HtB o c N HtB O c N HtB o c H2N N HtB o c N HtB O c N HtB o c 3 H C l 3 4 5 6 8 ( i ) ( i i a ) ( i i i ) ( i v ) ( i i b ) Figure 21 Synthesis of 8 (i) SOCl2, DMF, reflux for 2 hrs.; (ii a) (CH3)3SiN3, dioxane, 80 oC for 2 hrs.; (ii b) acetone, HCl (12 N), 50 oC for 1 hr.; (iii) Boc2O, Et3N, CH3CN, reflux for 7 hrs.; (iv) T 1 Raney nickel, EtOH, H2, 100 psi, 70 oC for 36 hrs.

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53 Table 2 1. Structures of conjugated polyelectrolytes with branched polyionic side chains and GPC analyses for their precursor polymers. Precursor polymers (GPC)a Structure Acronym R Ar Mn (kDa) Mw (kDa) PDI O O N H O R R R H N O R R R nA rR = C O2 -o r N H3 + PPE-Ph -bCO2 CO2 33.2 112.0 3.00 PPE-BTD -bCO2 CO2 N S N 11.7 16.3 1.40 PPE-TBT -bCO2 CO2 N S N S S 16.8 37.8 2.26 PPE-Ph -bNH3 + NH3 + 24.1 105.6 4.40 PPE-BTD -bNH3 + NH3 + N S N 12.3 44.7 3.60 a Estimated by GPC (THF), polystyrene standards.

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54 H2N R R R 7 R C O2 tB u N HtB o c 8 O O R R I I 9 R C O2H C O C l 1 0 ( i ) + O O H N O R R R N H O R R R I I 1 R C O2 tB u N HtB o c 2 ( i i ) N S N N S N S S P h B T D T B T Figure 2 2. Structure of bis ethynylene substituted Ph, BTD, and TBT; synthesis of monomers ( 1 and 2 ) (i) SOCl2, DMF, reflux for 2 hrs; (ii) TEA, DCM, 25 oC for 24 hrs. P recursor polymers For the s ynthesis of monomers 1 and 2 anionic bulky functional group 7 and compound 9 were prepared in a good yield as described in the literature,11,18,5455 and cat ionic bulky functional group 8 was first prepared as depicted in Figure 2 1. Starting from commercially available 4 (2 carboxyethyl) 4 nitroheptanedioic acid ( 3 ), it was converted to 4(3 chloro3 oxopropyl) 4 nitroheptanedioyl dichloride ( 4 ) in an excellent yield by treating with thionyl chloride (SOCl2). The latter was then reacted with trimethylsilyl azide to give 3(2 aminoethyl) 3 nitropentane1,5diamine HCl salt ( 5 ) in 81% yield. After reacti ng with 3 equivalents of di tert butyldicarbonate, compound 6 was obtained in 85% yield. The nitro group in compound 6 was then hydrogenated (60 oC,

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55 100 psi) in the presence of T1 Raney nickel65 to give compound 8 in 93% yield. For the aromatic monomer synthesis, bis ethynylene substituted aromatic units (Ph, BTD, and TBT) were easily synthesized with an excellent yield as described in literatures ( Figure 2 2 left).16,5253 Figure 2 2 shows the synthetic route of monomers 1 and 2 which are protected with butyloxycarbonyl (Boc) groups (after deprotection, side chains of 1 and 2 will be negatively and positively charged, respectively). Compound 9 was converted to 10 by chlorination using SOCl2, which was purified by precipitation with mixture of heptane/toluene (10/1, v/v). Then, reactions of 10 with 7 or 8 afforded monomers 1 and 2 in 76% and 50% yield, respectively. The purity of these monomers was proven by 1H and 13C NMR spectroscopy, elemental analysis, and mass spectrometry O O N H O R R R H N O R R R n O O N H O R R R H N O R R R n A r A r O O N H O R R R H N O R R R I I 1 R C O2 tB u N HtB o c 2 P P E A r -bC O2 tB u P P E A r -bN HtB o c P P E A r -bC O2 -P P E A r -bN H3 +( i ) ( i i a ) A r( i i b ) Figure 2 3 Polymerization through precursor route; Ar = Ph, BTD and TBT. (i) Pd(PPh3)4, CuI, THF/TEA (3/1, v/v ); (iia) Ph and BTD: TFA/DCM for anionic polymer HCl/DMSO for cationic polymer; TBT: ZnBr2, DCM/H2O, (ii b) sat. Na2CO3 aqueous solution for anionic polymer, 4 N HCl, dioxane for cationic polymer. The precursor route for the polymerization w as carried out in organic solutions in order to prevent less polymerization by electrostatic repulsion of ionic charged functional groups during polymerization in aqueous media, and the polymer molecular weight was estimated by GPC analysis. As shown in Figure 2 3 all precursor polymers

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56 were synthesized by S onogashira coupling of a stoichiometric amount of the monomer s ( 1 or 2 ) and a diacetylene aromatic unit using Pd(Ph3)4 and CuI as catalysts in a mixture of THF/TEA (3/1, v/v ) with yield of 5080%. After the reaction was stirred at 60 C for 24 hours, the organic soluble precursors were isolated as solids and purified by repeated precipitation from THF solution into methanol or hexane. Each polymer was characterized by 1H NMR spectroscopy. By GPC analysis using polystyrene standards, the number average molecular weight (Mn), weight average molecular weight ,10 and polydispersity of each polymer were listed in Table 2 1 Hydrolysis of precursor polymers While the hydrolysis of both precursors for anionic and cationic CPEs was easily accomplished in acidic conditions ( TFA/DCM for anionic polymer, HCl/DMSO for cationic polymer), in the c ase of a CPE having TBT as an aromatic unit, which can be decomposed by acid or basic conditions, mild condition using zinc bromide and water was carried out.66 The residues were treated with saturated Na2CO3 solution and then purified by dialysis method using 12 kD molecular weight cutoff (MWCO) dialysis membranes. The water soluble branched poly cationic and anionic side chains attached CPEs were obtained as solids in 90~ 100% yield. 1H NMR spectroscopy Fig ure 24 shows representative 1H NMR spectra of monomer 1 the precursor polymer PPEPh -bCO2 tBu and the water soluble polymer PPEPh -bCO2 ( w here superscript b stands for branched polyionic side chains) Comparison between the spectra of monomer 1 and PPEPh -bCO2 tBu reveals that there is only one new -

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57 phenylene on the polymer backbone. The tertbutyl protons appear as a strong singlet 1 and PPEPh -bCO2 tBu After hydrolysis, the 1H NMR spectroscopy of PPEPh -bCO2 was accomplished in a mixture of DMSOd6/D2O (1/1, v / v). No signals were observed in the 1.4 ~ 1.5 ppm range, indicating that the tert butyl groups were cleaved with an excellent yield (> 95%). The absorp tion band changes corresponding to carbonyl group in IR spectra also support disappearance of tert butyl groups after deprotection process. The similar phenomenon was also found in other deprotected polymers in which more than 95% of the peak of tert butyl groups disappear ed in 1H NMR and IR spectra. 9 8 7 6 5 4 3 2 1 0 ppm (a) (b) (c) CDCl3CDCl3D2O DMSO d6 Ph H CON H Ph H Figure 2 4 1H NMR spectra of (a) monomer 1 (b) PPEPh -bCO2 tBu and (c) PPE Ph -bCO2 -.

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58 Optical P roperties Figure 2 5 (a) Relative absorption and (b) fluorescence emission spectra of PPE Ph -bCO2 (blue), P PEBTD-bCO2 (red), and PPETBT -bCO2 (purple) in MeOH ( 2O (1/1, v/v ) ( ) and H2O ( PPEAr -bCO2 -] = 5 M. Table 2 2. UVVis absorption and photoluminescent properties of CPEs containing branched carboxylate or ammonium side chains. MeOH H2O max abs /nm max em /nm PL max abs / nm max em /nm PL PPE-Ph -bCO2 403 433 0.31a 404 432 0.12a PPE-BTD -bCO2 491 530 (sh) 605 0.04b 490 530 (sh) 623 0.007b PPE-TBT -bCO2 529 698 780 (sh) 0.028b 536 741 780 (sh) 0.004b PPE-Ph b NH3 + 402 432 0.45 a 405 432 0.13 a PPE-BTD -bNH3 + 493 604 0.04b 489 530 (sh) 620 0.004b a Coumarin 102 in EtOH as the standard, FL=0.95. b Ru(bpy)3Cl2 in H2O as standard, FL=0.036 (degassed) The photophysical properties of CPEs having branched poly anionic and cationic side chains were investigated by UV Vis sp ectroscopy and steady state fluorescence measurement in MeOH, MeOH/H2O (1/1, v/v ), and H2O. Figure 2 5 shows systematically red shifted UV Vis absorption and fluorescence spectra of both branched

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59 anionic and cationic side chains attached polymers in the or der Ph < BTD < TBT in all solutions (MeOH, MeOH/H2O (1/1, v/v), and H2O), respectively. Figure 26 illustrates the visual and fluorescence colors of branched anionic side chains attached CPEs as variation of the HOMO LUMO band gap of the polymers; visual and fluorescence color: pale yellow, bright blue for PPEPh -bCO2 -; red, bright red for PPEBTDbCO2 -; purple, dark purple for PPETBT bCO2 (for PPEAr -bNH3 +, see Figure A 1 in the Appendix A). A B C A B C(a) (b) Figure 2 6 (a) Visual and (b) Fluorescence colors of A: PPEPh -bCO2 -, B: PPEBTD-bCO2 -, and C: PPETBT-bCO2 -; [PPEAr -bCO2 -] = 3 0 M in H2O. 260 300 340 380 420 460 500 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Wevelength (nm)Normalized Intensity (a.u.)em = 430 nm em = 500 nm(a) 400 450 500 550 600 650 0.0 0.2 0.4 0.6 0.8 1.0 1.2 (b)Wevelength (nm)Normalized Intensity (a.u.)em = 550 nm em = 700 nm 420 460 500 540 580 620 660 700 0.0 0.2 0.4 0.6 0.8 1.0 1.2 (c)Wevelength (nm)Normalized Intensity (a.u.)em = 650 nm em = 800 nm Figure 2 7 Excitation spectra of (a) PPEPh -bCO2 at 430 and 500 nm; (b) PPEBTD-bCO2 at 550 and 700 nm; (c) PPE TBT -bCO2 at 650 and 800 nm; [ PPEAr -bCO2 -] = 5 M in MeOH. In MeOH, PPEPh -dCO2 exhibited an absorption maximum at 403 nm and a fluorescence emission maximum at 433 nm, and its counterpart CPE, PPEPh -bNH3 +, showed almost similar wavelength maximum ( abs = 402 nm and em = 432 nm) in both spectra (see Table 2 2 ). These wavelength maxima in the UV Vis absorpt ion spectra

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60 were hypsochromically shifted (around 15 to 22 nm shifted) compared to that of PPECO2 or PPESO3 which has the same conjugated backbone but has linear side chains (less bulky than branched side chains).10,6768 Since the degree of polymerization (DP) for both PPEPh -bCO2 and PPEPh -bNH3 + is larger than 10 (approximately 20 arylene ethynylene units), we believe that the coplanarity of the CPEs back bone was disturbed by the increased electronic repulsion between the branched side chains compared to that in PPE CO2 in MeOH, resulting in hypsochromic shift in the UV Vis absorption spectra. In fluorescence emission spectra, however, there is a negligible wavelength difference between PPEPh -bCO2 -, PPEPh -bNH3 + and PPECO2 -. This lack of energy difference in the fluorescence emission indicates that regardless of the state of the CPEs backbone, singlet excitons migrate to the lowest energy level in the excited state by an energy transfer process. Similar to branched polyionic side chains attached PPE Ph series, in MeOH, UV Vis absorption maxima of PPEBTD-bCO2 ( max abs = 491 nm) and PPEBTD -bNH3 + ( max abs = 493 nm) respectively showed around 23 and 21 nm hypsochromic shift s compared to that of PPEBTDSO3 -, while the emission spectra of PPEBTD-bCO2 ( max em = 605 nm) and PPEBTD-bNH3 + ( max em = 604 nm) displayed similar wavelength maxima to PPEBTDSO3 or PPEBTD (4+) .10 These observations support the fact that the polymers optical properties are mainly determined by the struc ture of the conjugated backbone. Unfortunately, for PPETBT -bCO2 we could not compare the optical properties to its counterpart having linear side chains even though this polymer is structurally different from others: there is longer distance between phenyl groups caused by introducing large size of TBT in PPETBT -bCO2 -, which may reduce the electronic repulsion between branched side chains. However, as seen in Figure 2 5

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61 we could observe the wavelength maximum at 698 with the shoulder band around 780 nm in the fluorescence spectrum, thereby indicating that even in MeOH solution there is already a small fraction of aggregated chains which differ from other branched side chains attached CPEs having Ph or BTD backbones. Although we could not observe the shoulder like band in UV spectrum, in MeOH its excitation spectra ( em = 650 nm and em = 800 nm) clearly showed that aggregates exist in the ground state (Figure 2 7 c). On the other hand, no big difference was observed in the excitation spectra of PPEPh -bCO2 ( em = 430 nm and em = 500 nm) and PPEBTD -bCO2 ( em = 550 nm and em = 700 nm) as seen in Figure 2 7 a and 27 b, respectively. This implies little or no aggregation in the ground state. 300 400 500 600 700 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Wavelength (nm)Relative Absorbance(a) 400 500 600 700 800 0.0 0.2 0.4 0.6 0.8 1.0 1.2 (b) Wavelength (nm)Relative Fluorescence Figure 2 8 (a) Relative absorption and (b) fluorescence emis sion spectra of PPE Ph bNH3 + (blue) and PPEBTDbNH3 + (red) in MeOH ( 2O (1/1, v/v ) ( ) and H2O (PPEAr -bNH3 +] = 5 M. The effect of branched side chains has been widely investigated through the UV Vis absorption and fluorescence emission changes in the presence of a relatively poor solvent to a good solvent (MeOH, MeOH/H2O (1/1, v/v ), and H2O). In our previous studies, PPESO3 and PPECO2 -, CPEs with linear anionic side chains, reveal ed strongly solvent dependent optical properties.10,68 In MeOH, the U V Vis absorption and

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62 fluorescence emission spectra display ed structurally analogous spectra with precursor polymer which exists as an unaggregated or less aggregated monomeric state. On the other hand, in aqueous solution it is believed that both polymers are strongly aggregated, showing a relatively redshifted UV Vis absorption spectrum with a shoulder band and an excimer like band in fluorescence emission spectra; this was concluded as the result of coplanarization of the backbones induced by strong aggregation.69 As seen in Figure 2 5 and 2 8 however, PPEPh -bCO2 and PPEPh -bNH3 + showed subtle wavelength changes in both UV/Vis absorption and fluorescence emission spectra as increasing volume fraction of H2O. It is only observed that the fluorescence quantum yield is moderately reduced. In addition, we could not find any shoulder band which PPESO3 and PPECO2 reveal ed in the UVVis absorption spectra. Moreover, the shapes of all the spectra were almost similar to those of their precursor polymer (Figure 29 ) This observation suggests that unlike C PEs which have linear side chains, PPEPh -bCO2 and PPEPh -bNH3 + is possibly less aggregated even in H2O solution because of their torsional strains induced by electrostatic repulsion between branched side chains For branched side chains attached CPEs ha ving BTD unit, PPEBTD -bCO2 and PPEBTD-bNH3 + reveal slightly decreased absorption spectra as increasing H2O ratio, and their fluorescence emission was gradually diminished and a little bit bathochromically shifted (18 and 16 nm, respectively). According to previous report, it is well known that tight stacking or aggregation gives rise to a huge redshift (>30 nm) in both absorption and fluorescence emission spectra.17,70 Our research group reported a similar re sult that PPEBTDSO3 showed more than 40 nm redshifted absorption and

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63 fluorescence emission maxima.10 In addition, another previous studies revealed that relatively modest redshifts (<20 nm) in both UV Vis absorption and fluorescence spectra might be caused by the planarization of the conjugated backbone.18 In present study, however, negligible wavelength changes were observed in UV Vis absorption spectra. These observat ions suggest that PPEBTD-bCO2 and PPEBTD-bNH3 + disfavor strong aggregation in aqueous solution which is attributed to electrostatic repulsion as seen in branched side chains attached CPEPh series. Also, the redshift in fluorescence spectra is possibly due to the intramol ecular charge transfer (ICT) effect between Ph (donor) and BTD (acceptor) in the excited state. It is notified that such solvatochromism is typical in donor acceptor type molecule.71 Accordingly we propose that the branched side chains attached CPEs having BTD backbones may retain their monomeric characteristics in aqueous solution as a result of minimal polymer aggregation. This less aggregation of CPEs having Ph or BTD conjugated backbones is also supported by comparison of the quantum yi eld with CPEs having linear side chains ( Table 2 2 ). The quantum yields of the branched side chains attached CPEs were relatively higher ( 1.3 ~ 21 fold ) than those of CPEs having linear side chains in both MeOH and H2O. Furthermore, the increase of quantum yield of CPEs was stronger in H2O Such increased quantum yield might be due to less aggregation caused by electrostatic repulsion between branched side chains.10,6768 In contrast to branched side chains attached CPEs with Ph or BTD, the UV/Vis absorption and fluorescence spectra of PPETBT -bCO2 were significantly shifted to the red region by 29 nm and 43 nm, respectively with increasing volume fraction of H2O as seen in Figure 25 In addition, its fluorescenc e intensity was gradually decreased and

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64 the shoulder band was relatively enforced at 780 nm which became the fluorescence maximum in aqueous solution. These might be ascribed to the following possible two reasons: first, the chargetransfer character in the excited state led to bathochromical shift in fluorescence spectra as H2O fraction increases. It is well known that the BTD group acts as a strong electron acceptor while phenyl thiophene is electron donor, which gives rise to charge separation in the excited state, and this charge separation is more pronounced in PPETBT -bCO2 than phenyl BTD system (e.g. PPEBTD-bCO2 and PPEBTD -bNH3 +) in high polar solvent.72 Second, the shoulder broad band at 780 nm is presumably exciplex like band induced by hydrophobic inter chain interaction between Ph and TBT groups. The bath ochromic shi ft with increased volume fraction of H2O in absorption spectrum also supports the aggregation characteristic of PPETBT -bCO2 -.16 It is possible that longer distance between phenyl groups caused by introducing TBT may reduce the electronic repulsion between branched side chains. Also, the incorporation of TBT group increases the hydrophobic character of the polymer. The similar result was found in our previous research in which PPECO2 having cofacial factor (additional phenyl group between side functional group substituted phenyl units) displayed alternative type aggregation, showing dominant excimer like emission in the fluorescence spectrum over PPECO2 without additional phenyl group.58,68,73 Therefore, on the basis of spectral changes of the branched side chains attached CPE series having Ph, BTD, and TBT, we propose that less aggregation is dominant in the case of aromatic units (Ph and BTD) in relatively small size while affordable hydrophobic interaction induced by cofacial factors (e.g. TBTPh system) is more considerable in polymer aggregation.

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65 400 500 600 700 800 0.0 0.5 1.0 1.5 Normalized IntensityWavelength (nm) PPE-Ph-bCO2 tBu PPE-BTD-bCO2 tBu PPE-TBT-bCO2 tBu Figure 29 Fluorescence emission spectra of precursors of branched anionic CPEs in THF ; [ PPEAr -bCO2 -] = 5 M ; Ar = Ph (b lack), BTD (red), and TBT (blue) pH Dependent Aggregation In several previous investigations of water soluble anionic CPEs, low pH induced different levels of aggregati on and energy transfer behavior as a result of the decreased electronic repulsion betw een side chains caus ed by the protonation of anionic charged functional group, while the cationic ones react the opposite way.40,58,62 So, we believe that controlling pH of the solution would influence the optical properties of CPEs having branched anionic or cationic side chains. We observed that PPEAr -bCO2 and PPEAr -bNH3 + reveal strongly pH dependent optical properties in the UVVis absorption and fluorescence emission spectra. The pH of aqueous solution was adj usted with HCl and/or NaOH using a pH meter. These conditions encouraged their aggregation or disaggregation, controlling the repulsion of charged side chains.

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66 Relative Fluorescence300 350 400 450 500 550 0.0 0.2 0.4 0.6 pH = 4.5 pH = 5.5 pH = 6.5 pH = 7.5 pH = 8.5 pH = 9.5 pH =10.5(c)400 450 500 550 600 650 700 0 1 2 3 4 (d) pH = 4.5 pH = 5.5 pH = 6.5 pH = 7.5 pH = 8.5 pH = 9.5 pH =10.50.0 0.2 0.4 0.6 (a) pH = 4.5 pH = 5.5 pH = 6.5 pH = 7.5 pH = 8.5 pH = 9.5 pH =10.5 0 1 2 3 4 (b) pH = 4.5 pH = 5.5 pH = 6.5 pH = 7.5 pH = 8.5 pH = 9.5 pH =10.5Wavelength (nm)Absorbance Figure 210 Absorption and fluorescence emission spectra of (a, b) PPEPh -bCO2 and (c, d) P PEPh -bNH3 + as pH changes in aqueous solution; [ PPEPh -bCO2 or bNH3 +] = 5 M. Figure 210 show s the absorption and emission spectra of PPEPh -bCO2 and PPEPh -bNH3 + in aqueous solution as a function of pH At pH 10.5 the absorption spectra of PPEPh -bCO2 showed one band maximized at 404 nm and the emission showed a well defined emission at 435 nm. As the pH decreases, its absorption spectra showed bathochromic shift with strong shoulder band at 435 nm and the fluorescence emission displays excimer like band at 515 nm (pH 4.5) concomitant with fluorescence quenching. Based on many research groups reports about these characteristics, we conclude that these optical behaviors imply the aggregation of CPE in acidic conditions (pH 4.5).58,62 This is ascribed to conformational change of CPE backbone. As pH decreases from 10.5 to 4.5, phenylene ethynylene groups tend to be less twisted in

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67 CPE backbone because electrostatic repulsion between the protonated carboxylate side chains could be minimized. Moreover, the desolvation effect of the neutral and hydrophobic CPE leads to the planarizat ion of the conjugated backbone. It is also very important to note that the series of absorption spectra define an isosbestic point at 410 nm, suggesting that the pH induced change is between two distinct types of chromophores, which are probably the neutral and charged segments. Unlike PPEPh -bCO2 -, positively charged CPE, PPEPh -bNH3 +, showed opposite tendency. That is, we found aggregati on behavior of PPEPh -bNH3 + at high pH (pH 10.5) and it retains monomeric characteristic at low pH (pH 4.5). Especially, in this CPE, we found that a significant decrease occurs in the UVVis absorp tion spectra when the pH reaches around 9.5. This probably indicates that CPE is slightly precipitated at this point. Absorbance 300 400 500 600 700 0.0 0.2 0.4 0.6 (c) pH = 4.5 pH = 5.5 pH = 6.5 pH = 8.5 pH = 9.5Wavelength (nm) 500 600 700 800 0 1 2 pH = 4.5 pH = 6.5 pH = 8.5 pH = 10.5(d)Wavelength (nm) 0 1 2 (b) pH = 4.5 pH = 5.5 pH = 6.5 pH = 7.5 pH = 8.5 pH = 9.5 pH =10.5 0.0 0.2 0.4 0.6 pH = 4.5 pH = 5.5 pH = 6.5 pH = 7.5 pH = 8.5 pH = 9.5 pH =10.5(a) Relative Fluorescence Figure 2 11 Absorption and fluorescence emission spectra of (a, b) PPEBTD-bCO2 and (c, d) PPEBTDbNH3 + as pH changes in aqueous solution; [ PPEAr -bCO2 or bNH3 +] = 5 M.

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68 300 400 500 600 700 800 0.0 0.1 0.2 AbsorbanceWavelength (nm) pH = 4.5 pH = 5.5 pH = 6.5 pH = 8.5 pH =10.5(a) 500 600 700 800 0.0 0.5 1.0 (b) pH = 4.5 pH = 6.5 pH = 8.5 pH = 10.5Wavelength (nm)Relative Fluorescence Figure 2 12 (a) Absorption and (b) fluorescence emission spectra of PPETBT bCO2 as pH changes in aqueous solution; [ PPETBT bCO2 -] = 5 M. The pH dependent absorption and emission changes of PPEBTD-bCO2 and PPEBTD-bNH3 + are shown in Figure 2 11. In the absorption spectra, similar changes to PPEPh -bCO2 and PPEPh -bNH3 + were observed. However, unexpected behavior compared to CPEs having Ph backbone was observed in the emission spectra. The emission band of PPE BTD-bCO2 was narrow and gradually increased in intensit y when the pH decreased. At pH = 4.5, more than 3fold enhancement of fluorescence emission intensity was observed at 640 nm (Figure 211 b) T his phenomenon was also found by Bazan et al., in which aggregated CPEs having BTD and fluorinephenylene units as a conjugated backbone caused fluorescence intensity enhancement. This is due to the result of shielding effect from water within the aggregate structure.62 Interestingly, w e found that the similar appearance was also observed in the fluorescence emission spectra of PPEBTD-bNH3 + under basic conditions (pH = 10.5) (Figure 2 11d ). This observation makes it clear that the fluorescence enhancement of aggregated CPEs is not due to the pho to physical changes of the CPEs backbone affecte d by acidic or basic conditions. As a result, we propose that one possible reason

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69 for this enhancement in fluorescence intensity may be less water contact effect of conjugated backbone induced by aggregation as suggested previously in Bazan and coworkers report.62 Like CPEs having BTD units, when the BTD group was replaced by a TBT unit, PPETBT -bCO2 showed a bathochromic shift in the UV/Vis a bsortption spectra and displayed fluorescence enhancement as the pH decreases from 10.5 to 4.5 as seen in Figure 2 12 In addition, the enhancement of fluorescence intensity was similar to those of CPEs having a BTD unit. However, the UV/Vis absorption spectrum exhibited hypsochromic shift by 16 nm For this observation, we propose that the blue shift in acidic condition was likely a result of the slightly increased torsional conformation of the momomeric units when PPETBT -bCO2 approaches closely, but it is true that the aggregation obviously controls considerable emission efficiency. Fluor escence L ifetime Sepctroscopy To gain more insight into the photophysical properties of the CPEs with branched polyionic side chains pH dependent behaviors of branched side chains attached CPEs were studied by a fluorescence lifetime measurement based on the solvent polarity and pH changes. As seen in Table 2 3, all the CPEs showed relatively complicated multi exponential lifetimes, which is attributed to the existence of nonspecific interaction between CPEs and/or to unexpected results caused by the poly dispersity in the polymer chain length. Therefore, we focus on the highly dominant fluorescence lifetime decays over contribution of amplitudes. For two CPEs having cationic side chains, the fluorescence lifetime decays under acidic or basic conditions showed almost opposite tendencies to their anionic counterpart ( see Table A 1 in the Appendix A ) T herefore, we only present lifetime results for CPEs having anionic carboxylate side chains .

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70 Table 2 3. Fluorescence lifetimes ( i, ns) and relative amplitudes ( RA, %) for PPEAr -bCO2 in MeOH, basic (pH = 9.0), and acdic (pH = 4.5) conditionsa MeOH H2O, pH 9.0 H2O, pH 4.5 Compd. RA (%) RA (%) RA (%) PPE-Ph -bCO2 i (ns) b 430 nm 500 nm i (ns) 430 nm 500 nm i (ns) 430 nm 500 nm 1 = 0.21 32 17 1 = 0.08 55 36 1 = 0.25 94 54 2 = 0.52 64 70 2 = 0.23 42 52 2 = 1.30 5 24 3 = 1.76 3 11 3 = 1.29 2 6 3 = 4.57 <1 22 4 = 4.67 <1 <2 4 = 4.19 <1 <6 2 0.977 1.068 2 1.016 1.112 2 1.173 1.042 PPE-BTD -bCO2 i (ns) 600 nm 650 nm i (ns) 600 nm 650 nm i (ns) 600 nm 650 nm 1 = 0.27 34 28 1 = 0.17 92 93 1 = 0.25 58 59 2 = 0.92 48 47 2 = 1.11 6 4 2 = 0.92 41 39 3 = 2.38 18 25 3 = 5.10 <2 <3 3 = 5.20 1 <2 2 1.105 1.143 2 1.112 1.273 2 1.243 1.153 PPE-TBT -bCO2 i (ns) 650 nm 700 nm i (ns) 700 nm >700 nm i (ns) 700 nm >700 nm 1 = 0.12 17 68 1 = 0.14 87 86 1 = 0.16 75 81 2 = 1.05 51 23 2 = 0.75 1 10 2 = 0.62 6 15 3 = 3.55 32 9 3 = 3.46 12 4 3 = 3.25 19 <4 2 1.234 1.185 2 1.099 1. 080 2 1.223 1.054 aData were collected by global fitting Algorithm bT ypical limits of error on i are less than 3%.

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71 At pH 9.0, PPEPh -bCO2 featured relatively wavelength independent fluorescence decays and two fast components (1 = 80 and 230 ps) were dominant (RA > 88%) when the emission decays were monitored at 430 or 500 nm, which were very similar to that observed in MeOH (1 = 210 and 2 = 520 ps), but the lifetimes at pH 9.0 were faster than those in MeOH. On the other hand, the lifetim es became wavelengthdependent at pH 4.5, in which short lived lifetime (1 = 250 ps) is over 90% at 430 nm whereas at 500nm, the relative amplitude of longlived lifetimes (2 = 1.30 ns and 3 = 4.57 ns) become 46%. These observations support the fact that the aggregation of PPE Ph -bCO2 is minimized at pH 9.0 with repulsion between side chains but is more pronounced in acidic condition (pH 4.5). The similar results of lifetime changes were found in the studies on the aggregation of CPEs having linear side chains, in which the lifetimes were wavelengthdependent, and the amplitude of the longlived component ( > 3 ns) became larger in aqueous solution.10 For PPEBTD -bCO2 -, when emis sion decay was monitored at 600 or 650 nm, relatively short lived lifetimes (1 = 270 and 2 = 920 ps) was over 75% in MeOH Also, lifetime decay of 1 = 170 ps was mostl y dominant (> 90 %) at pH 9.0, which is likely due to rapid decay of the charge transf er state in the polar solvent T hese results propose that less aggregation occurs in both MeOH and basic conditions (pH = 9.0). At pH 4.5, however, when the lifetime components were compared to those at pH 9.0, the amplitudes of 1 = 250 ps decreased from around 90 to 60% while those of 920 ps, which is also relatively short lived lifetime, increased from around 5 to 40% at both 600 and 650 nm. Also, although the amplitude is very small, longlived lifetime (3 = 5.20 ns) exists (around 1%). In addition, we found that these lifetimes reveal wavelength-

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72 independent tendencies. This increased amplitude of 2 = 920 ps is attributable to the distinctive characteristic of PPEBTD-bCO2 via fluorescence enhancement in low pH conditions, and small amplitude of 3 = 5.20 ns is probably due to the presence of very week fluorescence of exciplex like emission in aggregate states.10 The similar results for PPETBT -bCO2 were obtained in aqueous solution (pH 4.5 and 9.0, respectively). Interestingly, the wavelength dependent lifetime decay was observed in MeOH. A t short wavelength (monitored at 650 nm), large amplitude of longlived component (3 = 3.55 ns) was observed (3 > 30%) whereas short lived component (1 = 120 ps) was less than 20 %. In comparison with CPEs having Ph and BTD, PPETBT -bCO2 is in strong donor acceptor structure which may produce longlived charge transfer state in the excited state of the CPE. In this regard, this charge transfer state should return to the ground state very slowly.74 However, when monitored at 700 nm, short lived component (1 = 120 ps) became dominant (around 68%) and contribution of longlived one (3 = 3.55 ns) decreases to less than 10%. This result indicates that even in MeOH aggregation of PPETBT-bCO2 exists at the long wavelength ranges (> 700 nm) which comes from r apid quenching effect of the interchain exciton. W e could find similar phenomenon in lifetime results of precursor polymer ( PPETBT -bCO2 tBu ) where lifetime from non aggregated state of PPE TBT -bCO2 tBu showed a large fraction (around 80%) of the longlived component ( = 2.33 ns) in THF. Furthermore, we could not find large fraction of short component ( < 1 ns) at entire wavelength, indicating that no aggregation exists in THF. In other words, PPETBT -bCO2 is slightly aggregated in MeOH, showing the short lived component at all wavelength.

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73 Summary and Conclusions In this Chapter, we have prepared a new class of CPEs with Ph, BTD, or TBT aromatic units featuring branched anionic (R -dCO2 -) or cationic (R -dNH3 +) side chains. The polymerization was carried out with a prec ursor route in a mixture of THF and Triethylamine using Sonogashira coupling reaction. Hydrolysis of the precursor polymers was completed in either acidic condition for Ph and BTD units or mild condition using ZnBr2/H2O for a TBT unit, which was followed by a solubilization process into water solution using saturated Na2CO3 solution for the anionic polymers and 4 N HCl solution for the cationic polymers. Varying the structure of arylene units in conjugated backbones caused the variation of the HOMO LUMO band gap of the polymers, showing clearly different visual and fluorescence colors. The photophysical properties data suggested that the branched anionic and cationic CPEs with Ph and BTD backbones show less aggregation in aqueous solution, resulting in high er quantum yields than all other CPEs with linear side chains. However, PPETBT -bCO2 showed aggregated aspect in both MeOH and aqueous solution. Probably, this is due to less electrostatic repulsion between side chains induced by the size effect of an ary lene unit of the conjugated backbone, which may cause alternative type aggregation. In this case, we observed the redshift in UV/Vis absorption spectra, and more pronounced ICT effect in aqueous solution particularly led to the significant redshift emiss ion in fluorescence spectra. In addition, exciplex type band strongly suggested inter chain interaction between polymer backbones. The pH dependent spectral changes of all CPEs having branched anionic or cationic side chains clearly showed their photophysical behaviors in both acidic and basic conditions. PPEPh X (X = bCO2 or bNH3 +) showed redshift in UV/Vis absorption spectra at either pH 4.5 for the anionic side chain

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74 or pH 10.5 for the cationic one and displayed strong excimer type band with less efficient fluorescence as the polymers aggregate. On the other hand, monomeric characteristics in both UV/Vis absorption and fluorescence spectra were retained at their opposite pHs (pH = 10.5 for anionic side chain, pH = 4.5 for cationic one). For PPEBT D X we obtained attractive result that aggregation may induce fluorescence enhancement. In addition, the similar result could be also found in the fluorescence spectra of PPETBT-bCO2 -. We proposed that the aggregation of these CPEs reduces water contact of conjugated backbone via decreasing nonradioactive relaxation processes. Particularly, observed blue shift in UV/Vis absorption spectra of PPE TBT -bCO2 as the pH decreases revealed the new insight that conjugated polymer backbone could be twisted form r ather than coplanar one in the aggregated state. The lifetime measurement provides concrete evidence that changing the pH of CPEs having branched side chains in aqueous solution controls the degree of aggregation. The branched anionic CPEs having Ph and B TD backbones exhibited wavelength independent lifetime and showed predominant short lived lifetimes ( < 1 ns) in MeOH and pH 9.0. At pH 4.5, PPEPh -bCO2 revealed longlived lifetimes ( = 1.30 and 4.57 ns) more than 68%. For PPEBTD-bCO2 -, the extremely longlived lifetime ( > 5.00 ns) and dominant lifetime of 920 ps indicated that aggregated polymers exist and their fluorescence intensity increases in acidic condition, respectively. The photophysical behaviors of PPETBT-bCO2 in acidic and basic condi tions showed similar results to those of PPEBTD-bCO2 -. Interestingly, lifetime of PPETBT -bCO2 was wavelength dependent in MeOH. However, more amplitude longlived lifetime ( = 3.55, 32%) of PPETBT -bCO2 is seen at short wavelength (650 nm) while more amplitude

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75 short lived one ( = 0.12, 68%) showed at long wavelength (700 nm). This result is believed to arise because the longlived charge transfer state exists in this system, which causes dominant longlived lifetime decay. Furthermore, unlike PPEBTD -bCO2 -, predominant short lived lifetimes ( = 0.12, 68%) of PPETBT -bCO2 over 700 nm revealed that aggregation exists in MeOH. Experimental Materials All chemicals used in the synthesis were of reagent grade and used without further purification. T 1 Raney nickel, tert butylacrylate, copper iodide, 2 bromoacetic acid, thionyl chloride, 4(2 carboxyethyl) 4 nitroheptanedioic acid, azidotrimethylsilane, di tertbutyl dicarbonate, triethylamine, tetrakis(triphenylphosphine)palladium (0) and diisopropylamine were purchased from SigmaAldrich Chemical Company. Iodine, n itromethane, hydroquinone, and sodium carbonate were bought from Fisher Scientific Company. Zinc bromide was obtained from Acros Chemical Company. THF and DMF were purified by solvent d ispensing system (SDS) Silica gel (Merck, 230400 mesh) was used for chromatographic purification of all of intermediate and target molecules. All other chemicals and solvents were purchased from Sigma Aldrich, Fisher Scientific, or Acros Chemical Company and used as received. Instrumentation and Methods NMR spectra were recorded using a Varian VXR 300 FT NMR, operating at 300 MHz for 1H NMR and at 75 MHz for 13C NMR. Gel permeation chromatography (GPC) analyses were carried out on a system comprised of a Rainin Dy namax SD 200 pump, Polymer Laboratories PL gel mixed D columns, and a Beckman Instruments Spectroflow 757 absorbance detector. P olystyrene standards were used for m olecular

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76 weight calibration. UV/Vis absorption spectra were recorded using a Varian Cary 50 Spectrophotometer. Steady state fluorescence spectra were obtained with a PTI fluor o meter Lifetime measurements were carried out using a PicoQuant FluoTime 100 Compact Fluorescence Lifetime Spectrometer. A 1 cm quartz cuvette was used for all spectral measurements. Stock solutions (1.0 mM ) of all of the CPEs were prepared in H2O and have been stored at 0 oC. The solutions have been kept at the room temperature for one hour before use. The excessive exposure of the stock solution to the room temperature caused slight polymer aggregation in both CH3OH and H2O solutions: the aggregation was detected by excitation measurement at different wavelength, but no definite aggregation was observed in fluorescence emission spectra (no considerable excimer type band). H owever, t his slight aggregation can be overcome by addition of catalytic amount of NaOH. Fluorescence quantum yield are reported relative to know n standards (coumarin 102, = 0.9575 in EtOH; Ru(bpy)3Cl2, = 0.03676 in H2O ). The pH of aqueous solution was adjusted with HCl and/or NaOH using a C orning pH meter 320. Synth etic Procedures Compounds 7 and 9 w ere prepared in a good yield as described in the literature.3 4 (3 Chloro3 oxopropyl) 4 nitroheptanedioyl dichloride (4). A 100 mL round bottom flask was charged with 4.44 g of 4(2 carboxyethyl) 4 nitroheptanedioic acid ( 3 ) (16.0 mmol) and 30 m L of thionyl chloride (SOCl2). Two drops of DMF was added to the suspension and the mixture was then slowly heated up to reflux. After 1 hour, the solution became clear and there was no more gas evolution. The excess SOCl2 was

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77 removed by vacuum distillation. The yellow residue solidified after flushing with nitrogen and used without further purification. 1H NMR (CDCl3, ppm): 2.96 (t, 6H), 2.30 (t, 6H); 13C NMR (300 MHz, CDCl3, ppm): 172.46, 89.91, 41.29, 30.16. 3 (2 Aminoethyl)3 nitropentane1,5diamineHCl salt (5). The acid chloride ( 4 ) obtained from last step was dissolved in dioxane (30 mL) in a threenecked round bottom. Trimethylsilyl azide (6.3 mL, 48.0 mmol) was added to the solution at room temperature under argon gas. The solution was then slowly heated up to 80 oC. When there was no more gas evolution, the reaction mixture was allowed to cool down to 45 oC a nd then added 20 mL of acetone. Concentrated HCl (12 mL) was added to the mixture dropwise. White precipitate formed immediately following the addition. After 1 hour, the reaction mixture was allowed to cool down to room temperature. The white precipitate was collected by vacuum filtration and washed with 200 mL of cold acetone. After drying in the hood overnight, a slightly yellow solid was obtained (yield: 3.86 g, 81%). 1H NMR (300 MHz, DMSO d6, ppm): 8.39 (s, 9H), 2.81 (t, 6H), 2.33 (t, 6H). Compound (6). 3 (2 Aminoethyl) 3 nitropentane1,5diamineHCl salt ( 5 ) (3.0 g, 10.0 mmol) was dissolved in 100 mL of Et3N/CH3CN (1/3, v/v). Then di tertbutyl dicarbonate (10.2 g, 46.8 mmol) was added. The mixture was heated at reflux for 7 hours and then diluted wit h 150 mL of ethyl acetate. The mixture was washed with H2O (250 mL 2). Then the aqueous phase was extracted with ethyl acetate (150 mL 2). The organic phase was combined and dried with anhydrous MgSO4. After the filtration, the solvent was removed in v acuo affording a yellow oil which was solidified under vacuum (yield: 4.2 g, 86%). 1H NMR ( 300 MHz, CDCl3, ppm): 4.81 (s, 3H), 3.13 (m, 6H), 2.16 (t, 6H), 1.40 (s, 27H); 13C NMR (75 MHz, CDCl3, ppm): 155.84, 90.59, 79.63,

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78 35.79, 35.59, 28.34; LR MS: cal cd for C22H42N4O8 [M+H]+ = 491.6, found 491.0; Elemental analysis: calcd for C22H42N4O8: C, 57.86; H, 8.63; N, 11.42. found: C, 53.78; H, 9.03; N, 11.24. Compound (8) A solution of compound 6 (3.1 g, 6.3 mmol) in 200 mL of ethanol with T1 Raney Nickel (3 .0 g) was hydrogenated at 100 psi and 70 oC for 36 hours. The catalyst was removed by filtering the reaction mixture through a bed of celite. The solvent was removed in vacuo, affording a slightly yellow oil, which was solidified as a fluffy white solid un der vacuum (yield: 90%). 1H NMR (300 MHz, CDCl3, ppm): 5.06 (s, 3H), 3.18 (m, 6H), 1.78 (s, 2H), 1.56 (t, 6H), 1.41 (s, 27H); 13C NMR (75 MHz, CDCl3, ppm): 155.99, 79.22, 52.96, 39.37, 36.14, 28.41; LR MS: calcd for C22H44N4O6 [M+H]+ = 461.6, found 461. 0. 2,2' (2,5 Diiodo1,4phenylene)bis(oxy)diacetyl chloride (10). 2,2'(2,5 Diiodo 1,4phenylene)bis(oxy)diacetic acid ( 9 ) (5.8 g, 12.0 mmol) was suspended in 30 mL of SOCl2. After adding 2 drops of DMF, the reaction mixture was heated up and stirred at re flux for 2 hours. Then, the excess SOCl2 was removed by vacuum distillation and the resulting off white solid was crystallized from 200 mL of heptane/toluene (10/1, v/v), affording a slightly yellow crystalline solid (yield: 5.0 g, 80%). 1H NMR (300 MHz, CDCl3, ppm): 7.15 (s, 2H), 4.92 (s, 4H); 13C NMR (75 MHz, CDCl3, ppm): 152.58, 124.41, 113.62, 86.44, 74.05. Compound 1. Compound 7 (1.2 g, 2.9 mmol), 0.4 mL of Et3N and 25 ml of dry CH2Cl2 were placed in a 50 ml round bottom flask and cooled with an ice/water bath. Then 2,2'(2,5 diiodo1,4phenylene)bis(oxy)diacetyl chloride ( 10) (10.67 mg, 1.30 mmol) in 15 mL of CH2Cl2 was added. After 2 hours, the reaction mixture was allowed to

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79 warm to room temperature and further stirred for 24 hours. The solvent was removed in vacuo, the crude product was purified by flash chromatography (silica gel, EtOAc/hexane (1/3, v/v )) to give a white solid (yield: 1.4 g, 78%). 1H NMR (300 MHz, CDCl3, ppm): 7.13 (S, 2H), 6.60 (s, 2H), 4.35 (s, 4H), 2.25 (m, 12H), 2.03 (m, 12H), 1.42 (s, 27H); 13C NMR (75 MHz, CDCl3, ppm): 172.19, 165.73, 151.57, 122.63, 86.29, 80.63, 68.83, 57.80, 30.17, 29.74, 28.09; HR MS: calcd for C54H86I2N2O16 [M+Na]+ = 1295.39, found 1295.39. Compound 2. Compound 8 (1.5 g, 3.3 mmol), 0.45 mL of Et3N (3.2 mmol) and 30 ml of dry CH2Cl2 were placed in a 50 mL round bottom flask, which was cooled in an ice/water bath. To the mixture, a solution of 2,2'(2,5 diiodo1,4phenylene)bis(oxy)diacetyl chloride ( 10) (0.76 g, 1.5 mmol) was added. After 2 hours, the reaction mixture was allowed to warm to room temperature and further stirred for 24 hours. The solvent was removed in vacuo, the crude product was purified by flash chromatography on silica gel with EtOAc/hexane (1/1, v/v ) to give a colorless oil, which solidified as a white solid under vacuum (yield: 1.0 g, 50%). 1H NMR (300 MHz, CDCl3, ppm): 7.15 (s, 2H), 6.71 (s, 2H), 4.80 (s, 6H), 4.36 (s, 4H), 3.19 (m, 12H), 2.03 (m, 12H), 1.42 (s, 27H); 13C NMR (75 MHz, CDCl3, ppm): 166.38, 155.94, 151.61. 122.72, 86.43, 79.33, 68.71, 59.92, 35.76, 35.60, 28.39; HR MS: calcd for C54H92I2N8O16 [M+Na]+ = 1385.46, found 1385.46. General polymerization procedure for P P Es with branched side chains. Monomer 1 or 2 (0.25 mmol) and 0.25 mmol of the other monomers (1,4diethynylbenzene (Ph), 4,7Diethynyl 2,1,3benzothiadiazole (BTD) or 4,7bis[2 (5 ethynyl)thienyl] 2,1,3benzothiadiazole (TBT) ) were dissolved in 16 mL of THF/Et3N

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80 (3/1, v/v ). The r esulting solution was deoxygenated with argon for 15 minutes. Then Pd(PPh3)4 (17.3 mg, 15.0 mol) and CuI (5.7 mg, 30.0 mol) were added to the stirred solution under the protection of argon. The reaction mixture was then heated up to 60 ~ 65 C and stirred for 24 hours. The viscous solution was then poured into 200 mL of methanol. The precipitate was collected by vacuum filtration and washed with methanol (200 mL). After drying under vacuum, the polymer was stored as a solid. Typical reaction yields for the polymerization are 80 ~ 90%. Hydrolysis for P P Es with branched anionic side chains. P P Es having Ph or BTD: the organic precursor polymer (0.25 mmol) was dissolved in 20 mL CH2Cl2 and cooled in an ice/water bath. T rifluoroacetic acid (TFA 20 mL) was added to the polymer solution dropwise. Upon the completion of the addition, the reaction mixture was allowed to warm to room temperature and stirred for another 12 hours. The excess of TFA and the solvent were removed in vacuo; P P E having TBT: to a solution of the organic precursor polymer (0.25 mmol) in 20 mL CH2Cl2/DMSO (3/1, v/v ) ZnBr2 ( 6.0 equiv. ) was added and the solution stirred for 24 hours. At this time, 20 mL of water was added and the mixture was stirred for 1 hour. The layers were separated and the organic solvent was removed in vacuo; the residue was treated with saturated aqueous Na2CO3 solution (10 mL) and stirred at room temperature for 3 hours. The solution was then poured into 200 mL of acetone. The polymer precipitate was then dissolved in water and purified by dialysis using 12 kD MWCO regenerated cellulose membranes (yield: 90 ~ 100%). The water soluble polymers could be either stored as aqueous solutions or as solid powders.

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81 PPEPh -bCO2 tBu 1H NMR (300 MHz, CDCl3, ppm): 7.57 (br, s, 4H), 7.04 (s, 2H), 6.39 (s, 2H), 4.47 (s, 4H), 2.13 (br, m, 12H), 1.96 (br, s, 12H), 1.39 (s, 54 H); GPC (THF, polystyrene standard): Mw = 33, 230, Mn = 101, 210, PDI = 3.00; FTmax, KBr pellet): 3403, 2978, 2935, 2205, 1731, 1692, 1532, 1512, 1484, 1456, 1410, 1393, 1368, 1312, 1282, 1256, 1214, 1154, 1101, 1051, 954, 891, 848, 758. PPEPh -bCO2Na 1H NMR (300 MHz, D2O/DMSOd6 ( v/v 1/1), ppm): 7.58 (br, 4H), 7.16 (s. 2H). 5.25 (s, 4H); FT max, KBr pellet): 3391, 2937, 2202, 1665, 1564, 1404, 1283, 1208, 1099, 1053, 892, 847, 675. PPEBTD-bCO2 tBu 1H NMR (300 MHz, CDCl3, ppm): 7.91 (br, s, 2H), 7.19 (s, 2H), 6.51 (s, 2H), 4.59 (s, 4H), 2.12 (br, m, 12H), 1.94 (br, s, 12H), 1.39 (br, s, 54H); GPC (THF, polystyrene standard): Mw = 16, 250, Mn = 11, 690, PDI = 1.40; FTmax, KBr pellet): 3405, 2978, 2936, 2679, 2494, 2204, 1731, 1693, 1519, 1486, 1457, 1393, 1368, 1312, 1281, 1256, 1213, 1154, 1101, 1056, 954, 847, 758, 721. PPEBTD-bCO2Na 1H NMR (300 MHz, D2O/DMSOd6 ( v/v 1/1), ppm): 7.88 (br, s, 2H), 7.22 (br, s, 2H), 4.83 (s, 4H); FTmax, KBr pellet): 3391, 2951, 2204, 1667, 1566, 1403, 1283, 1207, 1097, 1061, 838, 778, 721, 667. PPETBT-bCO2 tBu 1H NMR (300 MHz, CDCl3, ppm): 8.10 (s, 2H), 7.95 (s, 2H), 7.51 (s, 2H), 7.08 (s, 2H), 6.45 (s, 2H), 4.55 (s, 4H), 2.24 (m, 12H), 2.08 (m, 12H), 1.38 (s, 54H); GPC (THF, polystyrene standard): Mw = 37, 817, Mn = 16, 771, PDI = 2.26. PPETBT-bCO2Na 1H NMR (300 MHz, D2O/DMSO d6 ( v/v, 1/1), ppm): 8.20 (br, 4H), 7.55 (br, 2H), 7.40 (br, 2H), 7.18 (br, 2H), 4.62 (br, 4H), 2.20 (br, 12H), 1.90 (br, 12H).

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82 Hydrolysis for PAEs with branched cationic side chains. The organic polymer (0.25 mmol) was dissolved in 20 mL of dioxane. The polymer solution was then cooled to 0 ~ 5 C using an ice/water bath. Concentrated HCl (7 m L 4 N) was added to the stirred solution dropwise. Upon the completion of the addition, the reaction mixture was allowed to warm to room temperature and stirred for another 12 hours. The polymer was then precipi tated by pouring the solution into a large amount of acetone (200 m L ). The precipitate was collec ted, washed with acetone (100 m L ) and finally dried under vacuum (yield: 90 ~ 100%). The polymer was then dissolved in water and purified by dialysis using 12 kD MWCO regenerated cellulose membranes (yield: 90 ~ 100%). The water soluble polymers could be either stored as aqueous solutions or as solid powders. PPEPh -bNHtBoc 1H NMR (300 MHz, CDCl3, ppm):7.61 (br, s, 4H), 7.05 (s, 2H), 6.60 (br, s, 2H), 4.92 (s, 6H), 4.46 (s, 4H), 3.09 (br, s, 12H), 1.93 (br, 2, 12H), 1.39 (s, 54H); GPC (THF, polystyrene standard): Mw = 105, 640, Mn = 24, 080, PDI = 4. 40); FTmax, KBr pellet): 3393, 2977, 1691, 1517, 1457, 1392, 1367, 1274, 1252, 1170, 1046, 866, 839, 781, 637, 601. PPEPh -bNH3Cl 1H NMR (300 MHz, D2O/DMSOd6 ( v/v 1/1), ppm): 7.61 (br, s, 4H), 7.18 (s, 2H), 4.66 (s, 4H), 2.92 (br, s, 12H), 2.05 (br, 2, 12H); FTmax, KBr pellet): 3392, 3031, 2202, 2002, 1672, 1607, 1516, 1489, 1407, 1281, 1191, 1063, 1017, 966, 906, 842, 786, 721, 548. PPEBTD-bNHtBoc 1H NMR (300 MHz, CDCl3ppm): 7.94 (br, s, 2H), 7.25 (br,s, 2H), 4.97 (s, 6H), 4.58 (s, 4H), 3.09 (s, 12H), 1.94 (s, 12H), 1.40 (s, 54H); G PC (THF, polystyrene standard): Mw = 44, 700, Mn = 12, 320, PDI = 3.60); FTmax, KBr pellet):

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83 3350, 2977, 2939, 2679, 2490, 2203, 1693, 1570, 1458, 1392, 1366, 1279, 1252, 1171, 1041, 966, 892, 866, 780, 634, 564. PPEBTDbNH3Cl 1H NMR (300 MHz, D2O /DMSO d6 ( v/vppm): 8.01 (br, s, 2H), 7.36 (s, 2H), 4.78 (s, 4H), 2.94 (s, 12H), 2.07 (s, 12H); FTmax, KBr pellet): 3394, 3035, 2202, 2011, 1672, 1610, 1542, 1509, 1409, 1342, 1281, 1191, 1067, 1020, 965, 893, 852, 786, 632, 563, 509.

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84 CHAPTER 3 HIGHLY EFFICIENT MERCURY (II) ION SENSOR BASED ON CONJUGATED POLYELECTROLYTERHODAMINE COMBINATIO N SYSTEM Mercury ion is a toxic heavy metal ion which causes serious nervous disorder s such as acrodynia, Hunter Russell syndrome, and Minamat a disease. Wide availability in many areas including medicines, cosmetics, and optics gives rise to the increase of diseases related to such mercury poisoning.77 Accordingly, during the past several decades, numerous fluorescence mercury ion sensors for selective detection have been desi gned and developed.7880 Especially, a great effort has gone into the increase of its sensitivity because even extremely trace amount of mercury ion gives rise to critical issues.8182 Among many fluorescence sensors, conjugated polyelectrolytes (CPEs) have been extensively investigated as potential chemoand biosensor s because of their highly efficient quenching effect known as superquenching or amplified quenching.26,83 Such quenching effect is induced by the various interactions including biotinavidin, anti genantibody, and electrostatic or hydrophobic interactions. Recently, Bunz et al. reported the detection of Hg2+ ion using CPEs in which biotin func t ionalized CPE avidin agglutination showed highly effective quenching effect for Hg2+ ion complexation via not only intrapolymer but also inter polymer exciton migration as the mercury ions are bound to carboxlate side chains of the CPE.82 Also, rhodamine based sensors have been extensively utilized in biolabeling and material science because rhodamine derivatives have high fluorescence quantum efficiency and exhibit long wavelength absorption and fluorescence.78,84 The rhodamine derivatives with spirocyclic form have received increasing attention as a turnon Hg2+ ion sensor with high sensitivity.8081,8590 The s piro type r hodamine derivatives are nonfluorescent and colorless. Ringopening

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85 of spirocyclic form induced by Hg2+ ion complexation, however, leads to significant fluorescence enhancement. We speculate that the positively charged r hodamine derivativeHg2+ ion ( S Rho 1Hg2+ ion) complex would form an electrostatic complex with negatively charged CPE ( P PE-dCO2 -) Also, highly favorable effect of sulfur atom on spirotype rhodamine to complex with Hg2+ ion coupled with the amplified quenching effect of P PE-bCO2 might cause improved sensory response for Hg2+ ion detection. In this system, P PE-bCO2 is used as a signaling unit, and rhodamine acts as a receptor because of the strong thiophilic nature of Hg2+ ion. Results and Discussion Synthesis O O N H H N O O O-N a+ O-N a+ O-N a+ +N a-O +N a-O +N a-O O O O O O O n O S O N H2 H2N O S O N N S R h o 1 S R h o 2 C P E -dC O2 Figure 3 1 Structure of PPE-bCO2 -, S Rho 1, and S Rho 2. Figure 3 1 shows the structure of the CPE with branched poly anionic side chains ( PPE-bCO2 -) and spirocyclic rhodamines ( S Rho 1 and S Rho 2). As discussed in Chapter 2, t he conjugated polyelectrolyte PPE-bCO2 was synthesized through precursor route using a Sonogashira coupling reaction. As a polymer repeat unit,

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86 phenyl group has been in troduced into polymer backbone, and negatively charged water soluble branched side chains that can structurally hinder the aggregation of the polymer chains were also incorporated to the conjugated polymer backbones, thereby suppressing self quenching of t heir excited state. As shown in Figure 3 2 S Rho 1 was synthesized from commercially available r hodamine 110. First, it was reacted with phosphorus oxychloride, which was then treated with excess Na2S in saturated aqueous solution. S Rho 1 was obtained as a colorless powder in 48 % yield. As a reference compound, S Rho 2 was also prepared in a good yield as described in the literatures .88 O S O N H2 H2N S R h o 1 O N H2N C O2H H H R h o 1 1 0 ( i ) ( i i ) C l Figure 3 2 Synthesis of S Rho 1 (i) 1,2 dichloroethane, POCl3, reflux, 4 h rs. ; (ii) excess Na2S saturated aqueous solution, 12 hrs Optical Properties It is well known that CPEs having linear ionic chains tend to be aggregated in aqueous solution because of a hydrophobic interaction of the polymer backbones and an electrostatic interaction between side chains of polymer, showing excimer like fluorescence at l onger wavelength.10 As shown in Figure 3 3 a, however, P PE-bCO2 shows structured fluorescence at max = 433 nm without an excimer like band at the longer wavelength. Unlike CPEs having linear side chains, aggregation effect of P PE-bCO2 which has bulky, branched poly ionic functional groups is minimized because of

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87 the e lectrostatic repulsion between the branched side chains. In addition, a number of ionic side chains are able to increase solubility in aqueous solution. Such features provide higher fluorescence quantum efficiency, so that less aggregated PPE-bCO2 gives r ise to an efficient sensing signal 400 500 600 700 800 0 1 2 3 4 Hg2+ (150 nM) PPE-bCO2(0.1 M) + S-Rho 1 (1 M) Fluorescence Intensity ( a.u. )Wavelength (nm) (b) 400 500 600 700 800 0 1 2 3 4 (a)S-Rho 1 ( 1 M ) /PPE-bCO2( 0.1 M ) PPE-bCO2( 0.1 M ) + Hg2+ ( 300 nM ) Fluorescence Intensity ( a.u. )Wavelength (nm)PPE-bCO2( 0.1 M ) + S-Rho 1 ( 1 M ) PPE-bCO2( 0.1 M ) + Hg2+ ( 300 nM ) S-Rho 1 ( 1 M ) Figure 3 3 (a) Fluorescence spectra of P PE-bCO2 ( 0.1 M) and P PE-bCO2 ( 0.1 M)/ S Rho 1( 1 M) upon the addition of Hg2+ ion (300 nM), and S Rho 1 ( 1 M) in H2O/DMSO (99/1, v/v ); (b) fluorescence intensity changes of P PE -bCO2 ( 0.1 M)/ S Rho 1( 1 M) upon the addition of various amounts of Hg2+ ion; Excitation at 403 nm Application to Hg2+ ion Sensor Selectivity Figure 3 3 a show s the fluorescence intensity of S Rho 1 P PE -bCO2 (0.1 mixture ( S Rho 1/ P PE-bCO2 -) in H2O/DMSO (99/1, v/v ). The addition of S Rho 1 P PE-bCO2 g ive rise to no changes in the fluorescence spectra. Without Hg2+ ion, interaction between S Rho 1 to P PE-bCO2 is negligible. T he addition of Hg2+ ion however, caused the overall intensity of the emission spectra to decrease, resulting in 98. 6 % quenching at 433 nm upon the addition of 300 nM of Hg2+ ion (Figure 3 3 a) On the other hand, PPE-bCO2 displayed no detectable fluorescence quenching by the addition of Hg2+ ion This implies that fluorescence change

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88 by interaction between carboxylate side chains of P PE-bCO2 and Hg2+ io n is negligib e in this concentration level Addition of Hg2+ ion creates a positively charged S Rho 1Hg2+ ion complex by ringopening process80 of S Rho 1, resulting in efficient fluorescence quenching of P PE-bCO2 via energy or charge transfer mechanism. It is evident that fluorescence quenching is static because of the ionpair complex formed between negatively charged P PE -bCO2 and positively charged S Rho 1/Hg2+ ion complex In contrast, the addition of other metal ions (Ag+, Ca2+, Mg2+, Co2+, Ni2+, Mn2+, Fe2+, Zn2+, Cd2+, Pb2+, Cu2+, Ba2+, and Cr3+) showed very subtle fluorescence intensity changes (Figure 3 4 ).91 Thus the selectivity of this system for Hg2+ ion over other metal ions is remarkably high. 0 50 100 150 I0/IMetal Ions ( 1.0 M ) Ag+, Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Ba2+, Pb2+, Cr3+ Hg2+ Figure 3 4 Fluorescen ce changes of S Rho 1/ PPE-bCO2 system in H2O/DMSO (99/1, v/v) upon the addition of various metal ions; Excitation at 403 nm; f luorescence intensity was monitored at 433 nm

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89 0 50 100 150 200 0 5 10 15 S-Rho 1 / PPE-dCO2 S-Rho 2 / PPE-dCO2 -[Hg2+] (nM)I0/I Figure 35 Stern Volmer plots of S Rho 1/ P PE-bCO2 ( ) and S Rho 2 / P PE -bCO2 ( ) ( PPE: 0.1 M and R hodamine derivatives 1 M) titrated with Hg2+ ion i n in H2O/DMSO (99/1, v/v ) ; Excitation at 403 nm ; F luorescence intensity was monitored at 433 nm Sensitivity Figure 3 3 b shows fluorescence intensity change of P PE -bCO2 by the addition of Hg2+ ion in the presence of S Rho 1in H2O/DMSO (99/1, v/v) The Stern Volmer 19 plot wa s obtainemax = 433 nm (Figure 3 5 ). The SV plot for such quenching effect exhibits a linear profile at very low conc entration of Hg2+ ion, where KSV value of 1.4 107 M1 was obtained, which is the highest value among early reported CPE based Hg2+ ion sensors. This quenching profile becomes nonlinear with increasing concentration of Hg2+ ion, indicating amplified quenc hing.83 It is reported the fluorescence of S Rho 2 is efficiently enhanced with increasing Hg2+ ion concentration, which implies that S Rho 2 can act as a good receptor for Hg2+ ion.88 U nlik e S Rho 1/ PPE-bCO2 system, nevertheless, the fluorescence of S Rho 2 / PPE-bCO2 system is not effectively quenched by Hg2+ ion as well as other metal ions

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90 (Figure 3 6 ). Its SV plot is much less sloped than S Rho 1/ PPE-bCO2 system (Figure 3 5 ), in which KS V value is 8.3 105 M1. 0 50 100 150 I0/IMetal Ions ( 1.0 M )Ag+, Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Ba2+, Pb2+, Cr3+ Hg2+ Figure 3 6 Fluorescen ce changes of S Rho 2 / PPE-bCO2 system in H2O/DMSO (99/1, v/v) upon the addition of various metal ions; Excitation at 403 nm; f luorescence intensity was monitored at 433 nm S ensing M echanism The plausible mechanism for sensing Hg2+ ion is displayed in Figure 3 7 A spiro type rhodamine derivative undergoes ringopening by complexation with Hg (II) ion, providing a positive charge on the nitrogen atom. According to the results of comparison + = = +Energy transfer Quenched Fluorescence +Strong Fluorescence Figure 3 7 Plausible sensing mechanism.

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91 400 500 600 700 0.0 0.5 1.0 1.5 FL of PPE-dCO2 Abs of SRho 1-Hg2+ complex Abs of SRho 2-Hg2+ complexNormalized AbsorbanceWavelength (nm) Figure 3 8 Fluorescence of FRET donor ( P PE-bCO2 ) and absorption of FRET acceptors ( S Rho 1Hg2+ complex and S Rho 2 Hg2+ complex). of S Rho 1 and S Rho 2, it is suggested that the proton on the nitrogen atom plays an important role in the complexation with carboxylate ions of P PE-bCO2 -. Figure 3 8 shows a spectral overlap between donor fluorescence ( P PE-bCO2 -) and acceptor absorption ( S Rho 1 Hg2+ ion or S Rho 2 Hg2+ ion complex), in which absorption of S Rho 2 Hg2+ ion complex is less overlapped with fluorescence of P PE-bCO2 than that of S Rho 1 Hg2+. Such less overlapped bands between donor fluorescence ( P PE -bCO2 -) and acceptor absorption ( S Rho 2Hg2+ ion complex) give r ise to less FRET (fluorescence resonance energy transfer) effect, resulting in less efficient quenching effect in S Rho 2/ P PE -bCO2 system. With this reason it is quite understandable that efficient quenching effect is mainly due to FRET effect in S Rho 1/ P PE-bCO2 system. Unfortunately, we could not observe fluorescence increase of the acceptor ( S Rho 1 Hg2+ ion complex) by FRET effect. It is possible relatively small stoichiometric ratio of the donor ( P PE-bCO2 -) is not enough to act as an efficient fluorescence donor in this system.

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92 Comparison of S ensitivity between S Rho/P PE S ystem and S Rho 500 550 600 650 700 0 1x1062x1063x1064x106 S-Rho 1 ( 1 M ) Fluorescence Intensity ( a.u. )Wavelength (nm) 150 0 [ Hg2+] ( nM ) Figure 3 9 Fluorescen ce intensity changes of S Rho 1 in H2O/DMSO (99/1, v/v ) upon the addition of various amounts of Hg2+ ion; Excitatio n at 500 nm 0 50 100 150 0 1 2 3 4 [ Hg2+] (nM) S-Rho 1 PPE/S-Rho 1 Figure 3 10 Titration profile I as a function of [Hg2+]; I : I0I at 433 nm excitation at 403 nm ( S Rho 1/ P PE -bCO2 -) and I I0 at 529 nm excitation at 500 nm ( Rho 1 ); I0: fluorescence emission intensity of S Rho 1 (1 M)/ P PE-bCO2 ( 0. 1 M) or S Rho 1 (1 M) I : fluorescence emission intensity in the presence of Hg2+ ion.

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93 The fluorescence response of S Rho 1/ P PE -bCO2 system for Hg2+ ion was compared to that of S Rho1 alone As expected, the fluorescence intensity of S Rho 1 increased max = 529 nm upon addition of Hg2+ ion (Figure 3 9 ). Figure 3 10 sh ows fluorescence intensity changes of S Rho 1/ P PE-bCO2 system and S Rho1 alone for Hg2+ ion detection. Interestingly, S Rho 1/ P PE-bCO2 system showed larger intensity changes, which implies that S Rho 1 / P PE-bCO2 system is more efficient Hg2+ ion detector than S Rho1 in terms of sensitivity. Summary and Conclusions In this Chapter we have devised a novel combination system for Hg2+ ion detection using PPE-bCO2 and S Rh o 1. The fluorescence intensity of PPE-bCO2 -/ S Rho 1 was selectively and sensitively quenched with Hg2+ ion over other metal ions. We demonstrated that the fluorescence energy transfer is the main factor in the fluorescence quenching. Although this PPE-bCO2 -/ S Rho 1 system is turn off sensor, this sensor showed improved sensory response compared to the unitary rhodamine based sensor that is a widely used one with high sensitivity and strong fluorescence quantum efficiency. Thus, this result proposes that st oichiometric uses of CPEs for combination system may promote sensory response of the other sensors that are presently available. Experimental Materials All chemicals used for the synthesis were of reagent grade and used without further purification. Rhodam ine 110, phosphorus oxychloride, 1,2 dichloroethane, and thiourea were purchased from SigmaAldrich Chemical Company. THF was purified by Solvent Dispensing System (SDS). Silica gel (Merck, 230 400 mesh) was used for

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94 chromatographic purification of all of intermediate and target molecules. All other chemicals and solvents were purchased from SigmaAldrich, Fisher Scientific, or Acros Chemical Company and used as received. Instrumentation and Methods NMR spectra were recorded using a Varian VXR 300 FT NMR, operating at 300 MHz for 1H NMR and at 75 MHz for 13C NMR. UV/Vis absorption spectra were recorded using a Varian Cary 50 Spectrophotometer. Steady state fluorescence spectra were obtained with a PTI fluor o meter A 1 cm quartz cuvette was used for all spec tral measurements. Stock solutions (1.0 mM ) of S Rho 1 and S Rho 2 was prepared in DMSO The chloride salts of Ag+, Ca2+, Mg2+, Co2+, Ni2+, Mn2+, Fe2+, Hg2+, Zn2+, Cd2+, Pb2+, Cu2+ Ba2+, and Cr3+ ions (s tock solutions = 1 0 .0 mM in H2O) were tested to eval uate the metal ion binding properties of P PE-bCO2 ( stock solution = 1.0 mM in H2O). For fluorescence measurements of combination system and S Rho 1, the excitations were made at 403 nm and 500 nm, respectively. Synth etic Procedures Synthesis of rhodamine derivatives. Rhodamine 110 (0.20 g, 0.55 mmol) or rhodamine B base (0.5 g, 1.3 mmol) was dissolved in 20 mL of 1,2dichloroethane. Then phosphorus oxychloride (1 mL) was added dropwise The mixture was heated at reflux for 4 hours and then the solvent was removed in vacuo. The crude acid chloride was dissolved in 5 mL of THF. After the addition of solution of thiourea and triethylamine in THF/ water (20 mL/5 mL) at room temperature, the reaction mixture was stirred for overnight. T he solvent was removed in vacuo and then 30 mL of water was added. The

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95 precipitate was filtered and washed 3 times with water. T he crude product was purified by chromatography (silica gel, CH2Cl2) to give a white solid. S Rho 1 : Yield: 48% ; 1H NMR ( 300 MHz, CDCl3, ppm): 7.84 (d 1H), 7.50 (m, 2H), 7.16 (d, 1H), 6.66 (d, 2H), 6.38 (s, 2H), 6.27 (d, 2H), 4.77 (br, 4H); 13C NMR (75 MHz, CDCl3, ppm): 1605.0, 159.8, 157.0, 153.9, 151.8, 147.5, 134.2, 130.3, 128.4, 127.2, 122.5, 112. 8, 101.5. S Rho 2: Yield: 72% ; 1H NMR ( 300 MHz, CDCl3, ppm): 8.85 (d, 1H), 7.51 (m, 1 H), 7. 43(m, 1 H), 7. 20 (d, 1H), 6. 70 (d, 2H), 6.3 5 (s, 2H), 6.30 (d, 2H), 3.32 ( q 8 H) 1.15 (t, 12 H).

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96 CHAPTER 4 PHOTOPHYSICAL PROPERTIES OF PYA4 AGGREGATE AND ITS APPLICATI ON TO PYROPHOSPHATE SEN SOR BASED O N TURNON SYSTEM In the past decade, many fluorescence sensors for ionic species have been designed and developed due to their use in analysis and detection of metal ions, anions, and biomolecules.19,92 Effective fluorescence chemosensors should selectively recognize guest molecules and easily convert the recognition event into photophysical changes with high sensitivity.93 In recent years many fluorescence sensors having photophysical properties utilizing excimer/exciplex formation, PET (photoindu ced electron transfer), ESPT (excited state proton transfer), MLCT (metal to ligand charge transfer), and FRET (fluorescence resonance energy transfer) mechanisms have been developed.9495 O ne widely used mechanism is the excimer formation of pyrenes because pyrenes display not only a well defined monomer emission but also an efficient excimer emission: after excitation, pyrene displays fluorescence from both the excited state monomer and the excited state dimer (exc imer).96 Because excimer formation is sensitive t o even subtle physical changes of the pyrene moieties induc ed by the environment such as metal ion binding, temperature, and use of viscous solvent, t he Ie/ Im ( the excimer to monomer emission intensity) changes can be an informative parameter in various sensing systems.94 Anions play important roles in biolog y and the environment. As an example, pyrophosphate ion ( PPi) has an important role in many biological processes such as cellular energy metabolism and regulation of enzyme activity.9798 PPi is the product of ATP hydrolysis under cellular conditions, and many of the common enzymes, such as phosphoribosyltransferases and alkaline phosphatase, produce or consume pyrophosphate (PPi), which are also related to the enzyme activity.98 Accordingly,

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97 detecting variation of pyrophos phate concentration in the enzymatic conditions is a general way to monitor the enzymes activity. Fluorescence based pyrophosphate (PPi) sensor s have attracted considerable interest, and m uch effort has been devoted to the development of PPi receptors or sensors,99 since Czarnik et al.100 reported the results of a pioneering study in 1994 in which an anthracene derivative bearing polyamine groups was used as a PPi sensor in 100% aqueous solutions. However, relatively few reports of the effective PPi receptors101 or sensors97,102108 that operate in aqueous solutions were reported because most known anion sensors do not have a sufficiently strong affini ty for anions in aqueous solutions or they have a limit to convert anion recognition into a fluorescence signal.109 Recently, Yoon et al. reported that a chemosensor based on Zn2+ ion complexed DPAs (di 2 p icolylamines) with high selectiv ity for PPi over ATP, ADP and AMP in aqueous solution in which two fluorophores display 2+2 type intermolecular dimer formation upon PPi complexati on, showing excimer type emission at 480 nm.10 4 Fabbrizzi et al. also reported an Off On switchable fluorescence chemosensor for effective PPi sensing with a chemosensing ensemble (CE) paradigm in which an indicator (I: fluorophore) binds to a receptor (R: a dinuclear Cu2+ ion macrocyclic complex) by means of noncovalent interaction, which is able to effectively detect PPi through recovery of the fluorescence emission of the displaced indicator in aqueous solutions.108 In most cases, however, fluorescence emission after PPi recognition was just several fold higher than in the absence of PPi, attributable to comparatively

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98 inefficient quenching effect in aqueous sol utions when quencher is bound. Therefore, the sensitivity for detecting PPi is limited. On the other hand, more recently it was reported that PPE CO2 -which is a conjugated polyelectrolyte (CPE) responds even at very low PPi concentration where the amplifi ed quenching system has been introduced.102 In a molecular wire system, the excit on migra tes throughout the polymer, which makes the quenching of polymer more efficient compared to single molecular system when a quencher is bound.16,21,59 This approach enables sensor to sensitively change the fluorescence emission when the quencher is affected by another analyte. O O O O O-N a+ +N a-O O-N a+ +N a-O O O O O P y A 4 Figure 41 Structure of PyA4 With this background in mind, here we have designed and developed an Off On switchable fluorescence sensing system for PPi that consists of PyA4 (Figure 4 1) and cupric (II) ions, giving sensitive fluorescence changes in 20 mM HEPES buffer

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99 Results and Discussion Synthesis B r B r B r B r T M S T M S T M S T M S 4 5 6 ( i i ) ( i i i ) H H H H 7 ( i v ) H H H H O O O O R R R R O O O O R = O E t R = O-N a+7 P y E4P y A 4 ( v ) ( v i ) O H I + B r O O O I O O 3 1 2 ( i ) O I O O 3+ Figure 4 2 Synthesis of PyA4 (i) K2CO3, CH3CN, reflux for 24 hr s ; ( ii) B r2, nitrobenzene, 120 oC for 24 hr s; (iii) trimethylsilylacetylene, Pd(PPh3)4, CuI, THF/TEA (1/1, v/v), 70 oC for 12 hr s ; TBAF, THF, r.t for 1 hr ; (v) 3 Pd(PPh3)4, CuI, THF/TEA (1/1, v/v ), 70 oC for 12 hr; ( vi ) NaOH, 2 methoxyethanol, reflux for 12 hr s. T he s ynthetic pathway for PyA4 is outlined in Figure 4 2 First, the starting material, 4 (ethyloxycarbonylmethoxy)iodobenzene ( 3 ), was synthesized by the reaction of 4iodophenol ( 1 ) with 1.1 equi valents of ethyl bromoacetate ( 2 ) in the presence of anhydrous K2CO3 as a base in dried CH3CN in moderate yield. Second, c ommercially

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100 available pyrene ( 4 ) was converted to 1,3,6,8tetrabromopyrene ( 5 ) in a 94% yield through bromination using 5.0 equiv alents of bromine in nitrobenzene. Then, 5 was reacted with 4.5 equiv alents of trimethysilylacetylene using a catalytic amount of Pd(PPh3)2Cl2, PPh3 and CuI to gi ve a 1,3,6,8t etrakis(trimethylsilylethynyl) pyrene ( 6 ) in a 21% yield (Sonogashira coupling). After desilylation by treat ment with TBAF ( n tetrabutylammonium fluoride) in THF for 1 h our 1,3,6,8 tetraethynylpyrene ( 7 ) was obtained in a 93% yield. Using the Sonogashira coupling, 7 was then reacted with previously synthesized 3 to give the tetraphenoxymethyl ester bearing tetrae t hynylpyrene ( PyE4 ) with yield of 5 5%. The hydrolysis of PyE4 was easily accomplished by the addition of 5.0 equiv alents of NaOH in 2 methoxyethanol. The further purification was followed by dialysis method using 500 D molecular weight cutoff (MWCO) dialysis membranes. The water soluble PyA 4 was obtained as a dark red crystalline solid in an excellent yield (83% yield) Characterizati on of PyA4 The photophysical properties of the PyA4 were investigated by the UV/Vis spectroscopy and by the fluorescence measurement in MeOH, DMSO, and HEPES buffer (0.02 M, pH 7.5) (HEPES=2 [4 (2 hydroxyethyl) 1 piperazinyl]ethanesulfonic acid) at 25 oC. It is well known that p yrene derivatives show excimer emission in both high ly concentrated solutions and high ly polar solvents, especially in water.96 Figure 4 3 shows UV/Vis spectra (left) and fluorescence em ission spectra (right) of PyA4 in a solution of DMSO, MeOH, and 20 mM HEPES buffer. Unlike in DMSO and MeOH solutions, the absorption spectrum of PyA4 in HEPES buffer at pH 7.5 exhibits a small shoulder band at 518 nm (Figure 4 3 c). Moreover, the f luorescence emission spectrum of PyA4 in HEPES buffer exhibits broad excimer emission at 640 nm (Figure 4 3 c ).

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101 This indicates that PyA4 s in HEPES buffer are stacked not only in the ground state but also in the excited state. This behavior can be ascribed to intermolecular hydrophobic interaction of pyrene centered in aqueous media. In contrast, PyA4 in DMSO an d MeOH exhibited only monomer emission at around 500 nm and 480 nm, respectively, suggesting that no aggregation occurs (Figure 4 3 a and 4 3 b ). 0.0 0.2 0.4 0.0 0.2 0.4 Absobance 300 400 500 600 0.0 0.2 0.4 Wavelength (nm) 0.0 0.5 1.0 0.0 0.5 1.0 Normalized Intensity (a.u.) 500 600 700 800 0.0 0.5 1.0 Wavelength (nm) (a) (a (b (c (b) (c) Figure 4 3 UV/Vis (left) and fluorescence emission (right) spectra of PyA4 in various solutions. (a), (a ) : DMSO; (b), (b ): MeOH; (c), (c ): 20 mM HEPES buffer at pH 7.5.

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102 320 380 440 500 560 620 680 740 800 0.0 0.3 0.6 0.9 1.2 1.5 em = 529 nmWavelength (nm)Normalized Intensity (a.u.) ex = 456 nm em = 640 nm Figure 4 4 Fluorescen ce excitation and emission spectra of PyA4 (5 M) in 20 mM HEPES buffer at pH 7.5; left: excitation spectra ( red dash line, em=529 nm and b lue dot line em=640 nm), right: emission spectrum ( ex=456 nm). 480 520 560 600 640 680 720 760 800 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Wavelength (nm)Normalized Intensity (a.u.) ex = 456 nm ex = 494 nm Figure 4 5 Fluorescen ce emission spectra of PyA4 in 20 mM HEPES buffer at pH 7.5 (blue, ex=456 nm and red, ex=494 nm).

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103 ( b) D M S O d6 ( a ) D2O / C D3O D Figure 4 6 1H NMR spectra of PyA4 in (a) D2O /CD3OD (3/1, v/v ) and (b) DMSO d6; denotes solvent peak. As shown in Figure 4 4 two distinctive excitation spectra were observed from monomer emission ( em=529 nm) and excimer emission ( em=640 nm), respectively. The excitation spectrum from the monomer part ( red dashed line) shows a band which resembles the absorption spectrum of PyA4 in DMSO and MeOH solutions. On the other hand, the excitation spectra monitoring at the excimer emission shows a shoulder band at 494 nm (blue dot line) This is at tributed to direct excitation from aggregated PyA4 in the ground state. In addition, fluorescence emission of PyA4 shows only the excimer band when it was excited at the shoulder band (494 nm) (Figure 4 5 ). As a result, fluorescence excitation spectra ( em=529 and 640 nm) clearly show that two pathways for the excimer band exist: direct excitation from the aggregated ground state and excitation from the nonaggregated ground state. This ground state aggregation is also supported by 1H NMR spectrum, in which broad peaks in D2O/CD3OD (3/1, v/v ) indicate ground state aggregation whereas peaks in DMSO d6 show relatively sharp features (Figure 4 6 ).

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104 500 600 700 800 0 3 6 9 12 15 1 M 5 M 10 MFluorescent Intensity (a.u.) Wavelength (nm) (a) 0 20 40 60 80 100 0 50 100 150 200 (b)[PyA4] ( M)Ie @ 640nm / Im @497 nm Figure 4 7 (a) Fluorescence emission spectra of PyA4 solutions with increasing concentration (110 M) in 20 mM HEPES buffer solutions at pH 7.5; (b) ratio of excimer (640 nm) to monomer (497 nm) with increasing concentration (1 100 M); Excitation at 456 nm. As shown in Figure 4 7 a the fluorescence spectrum ( ex = 456 nm) of PyA4 displays a stronger excimer emiss ion at 640 nm with increasing its concentration in HEPES buffer. F igure 47b shows the intensity ratio of the excimer (640 nm) to monomer (497 nm) for PyA4 with varying concentration. T he ratio increases with increasing concentration of PyA4 indicating that the higher the concentration of PyA4 is, the more the aggregation occurs in HEPES buffer. Quenching with M etal I ons To develop a metal based PyA4 sensor for anion, the quenching efficiency of PyA4 was tested with many divalent metal ions: Ca2+, Mg2+, C o2+, Ni2+, Mn2+, Fe2+, Hg2+, Zn2+, Cd2+, Pb2+, and Cu2+. PyA4 shows a Cu2+ ion selectivity over other metal ions in terms of decreasing fluorescence (Figure 4 8 a ). The fluorescence quenching can be explained as PET95 from PyA4 to the Cu2+ ions bound to carboxylate groups when Cu2+ ion is bound to the carboxylate groups, which efficiently leads to the quenching in this system. As shown in Figure 4 8 b the addition of Cu2+ ions caused the overall

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105 intensity of the emission spectra to decrease, resulting in 98.5% quenching at 640 nm upon the addition of Cu2+ ions ( 20 M ) 480 520 560 600 640 680 720 760 800 0.0 2.0x1054.0x1056.0x1058.0x1051.0x1061.2x106 Cu2+Pb2+Cd2+ PyA4, Ca2+, Mg2+, Co2+, Mn2+, Ni2+, Fe2+, Hg2+ Zn2+Fluorescent Intensity (a.u.)Wavelength (nm) 480 520 560 600 640 680 720 760 800 0.0 2.0x1054.0x1056.0x1058.0x1051.0x1061.2x106 Wavelength (nm)Fluorescent Intensity (a.u.)20 0 PyA 4 Cu 2+ ion (a) (b) Figure 4 8 (a) Fluorescence emission changes of PyA4 (5 M) upon the addition of various metal ions (30 M); (b) Titration of PyA4 (5 M) with Cu2+ ions (020 M); Inset: ratio of excimer (640 nm) to mo nomer (497 nm); Excitation at 456 nm in 20 mM HEPES buffer at pH 7.5. 0 3 6 9 12 15 18 0 2 4 6 8 10 12 Ksv = 1.8 X 104 @ 497 nm Ksv = 9.3 X 104 @ 640 nm[ Cu2+] ( M )I0 / I Figure 49. Stern Volmer plots of PyA4 ( 5 M) titrated with Cu2+ ions in 20 mM HEPES buffer at pH 7.5; Excitation at 456 nm fluorescence intensity was monitored at 497 and 640 nm.

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106 Figure 49 shows the SternVolmer plots of PyA4 (5 M) titrated with Cu2+ ions in 20 mM HEPES buffer at pH 7.5. Both SV plots are linear at very low concentration of Cu2+ ions, but it showed upward curve at the higher concentration of Cu2+ ions It is observed that m ore efficient quenching at excimer emission ( Ksv = 9.3 104 M1) occurs than monomer emission ( Ksv = 1.8 104 M1). This indicates that the excimer (the delocalized excited state) is more efficiently quenched than the monomer emission (single fluorophore component) by Cu2+ ions and its quenching efficiency is amplified in more aggregated states Furthermore, this decrease is more distinguished in higher concentration of PyA4 T his phenomenon was also found by Kool et al., in which the stacked pyrene system in nonconjugated polymer may cause efficient quenching as in conjugated polymer system.110 0 3 6 9 12 15 0 10 20 30 40 MeOH} } 1 5 10 1 5 10 I0/I[ Cu2+] / Mpure H 2 O F igure 4 10 Stern Volmer plots of PyA4 ( 1 5 and 10 M) titrated with Cu2+ io ns in MeOH and pure H2O ; Excitation at 456 nm fluorescence intensity was monitored at 491 nm in MeOH and 640 nm in pure H2O, respectively.

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107 Table 4 1. Ksv a and [Q]90 b for Cu2+ ion quenching of PyA4 in H2O and MeOH [PyA4]/ M Ksv(H2O) /M1 [ Q ]90(H2O) / M Ks v(MeOH)/M-1 [ Q ]90(MeOH) / M 1 9.0 105 2.5 8.1 105 > 3 5 2.8 105 7 1.5 105 > 1 0 10 1.7 105 14 7.7 104 > 35 a Computed from linear fit at low quencher concentration. bQuencher concentration at 90% quenching. Efficient quenching in the aggregate state was further investigated by SternVolmer 19 quenching experiment in MeOH and pure H2O. In both cases, fluorescence intensity was efficiently quenched by the addition of Cu2+ ions The SV plots for Cu2+ ion quenching of PyA4 displayed different features in MeOH and pure H2O, respectively. As expected, i n pure H2O, the SV plot for PyA4 (5 M) is linear at very low concentration of Cu2+ ions, but became nonlinear with incr easing concentration of Cu2+ ions (Figure 4 10), in which KSV value of 2.8 105 M1 was obtained. In MeOH, however, its SV plot ( PyA4 = 5 M ) showed relatively linear at the same range of quencher concentration as in pure H2O (KSV value = 1.5 105 M1 in MeOH). This large KSV value suggests that the quenching results from a ground state complex between PyA4 and Cu2+ ions. In addition, KSV values of PyA4 (1 M and 10 M) decrease with increasing its concentration in both MeOH and pure H2O (Table 4 1) This decrease of the KSV value from 1 M to 10 M of PyA4 is around 5fold in pure H2O which is smaller compared to that in MeOH (10 fold). In other words, the decrease of KSV values by the effect of concentration is less pronounced in pure H2O Furthermore, KSV value of PyA4 in pure H2O is modestly larger than that in MeOH, even though there is more so lvation effect in pure H2O Moreover, the difference of KSV value in between H2O and MeOH is more

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108 distinctive in 10 M than that in 1 M These differences refl ect that PyA4 is more efficiently quenched in aqueous solution by Cu2+ ions. Similar result was observed in the comparison of the relative quenching efficiencies in H2O and MeOH. [Q]90 values for Cu2+ ion in H2O and MeOH were present ed in Table 41 The [ Q]90 values in H2O are lower than MeOH. Also, [Q]90 in MeOH tracks closely with the concentration of PyA4 whereas their difference between 1 M and 10 M in H2O is less significant than MeOH. As a result, we suggest that more excimer quenching in aqueous s olution may be caused by energy migration through the aggregates of PyA4 if the delocalized excited state of the pyrene stack is mobile as in the case of conjugated polymer. However, quenching is still less efficient compared to PPECO2-, reflecting improv ed transport in CPEs.102 480 520 560 600 640 680 720 760 800 0.0 5.0x1051.0x1061.5x1062.0x1062.5x1063.0x106 Cu2+ ( 100 M ) PyE 4 ( 5 M ) / MeOH Wavelength (nm)Fluorescent Intensity (a.u.) 480 520 560 600 640 680 720 760 800 0.0 3.0x1056.0x1059.0x1051.2x1061.5x106 Cu2+ ( 10 M ) Cu2+ ( 5 M ) PyA 4 ( 5 M ) / MeOH Wavelength (nm)Fluorescent Intensity (a.u.) (a) (b) Figure 4 1 1 Titration of (a) PyA4 (5 M) and (b) PyE4 (5 M) with Cu2+ ions in MeOH; Excitation at 456 nm. To know the binding sites of PyA4 in the presence of Cu2+, we tested PyE4 having tetraester groups with Cu2+ ion in MeOH (Figure 4 1 1 ). For PyE4 no remarkable changes were observed, even in spite of the additi on of 200 equivalents Cu2+ ions. PyA4 on the other hand, showed significant quenching in the same media. T hus,

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109 carboxylate groups of PyA4 play an important role as a receptor for binding Cu2+ ions. Unfortunately, we cannot clearly explain the exact binding mode and ratio between PyA4 and Cu2+ ions only by lack of fluorescence emission changes observed for PyE4 upon excess amount of Cu2+ ions in MeOH Application to P yrophosphate (PPi) S ensing Selective and sensitive detection of PPi 0 10 20 30 40 50 60 70 80 ATP ADP AMP CO3 2-SO4 2-NO3 -HCO3 -HSO4 -MeCO2 -PPi H2PO4 -HPO4 2-I-Br-F-I / I 0AnionsClFigure 4 1 2 Fluorescence emission changes of PyA4 ( 5 M ) Cu2+( 20 M) at 640 nm upon the addition of anions (50 M ); Excitation at 456 nm in 20 mM HEPES b uffer at pH 7.5; I0: fluorescence emission intensity of PyA4 Cu2+ complex I : fluorescence emissi on intensity in the presence of anions The anion binding ability of PyA4 Cu2+ ( PyA4 : 5 M and Cu2+: 20 M) with the anions F-, Cl-, Br-, I-, H2PO4 -, HPO4 2-, PPi, AcO-, HSO4 -, NO3 -, HCO3 -, SO4 2-, CO3 2-, AMP, ADP, and ATP (Sodium salts), in 20 mM HEPES buff er at pH 7.5 was investigated using fluorescence emission spectrum ( ex=456 nm). T he overall fluorescence intensity changes upon the addition of various anions (50 M ) are compared in Figure 4 1 2 Interestingly, the PyA4 Cu2+ system is highly selective to

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110 0.0 2.0x10-54.0x10-56.0x10-58.0x10-51.0x10-41.2x10-40 20 40 60 80 100 I / I 0[PPi] (M) 480 520 560 600 640 680 720 760 800 0.0 2.0x1054.0x1056.0x1058.0x1051.0x1061.2x106 PyA 4 + Cu2+ ( 20 M ) Wavelength (nm)Fluorescent Intensity (a.u.)0 M 30 M PyA 4 PPi (a) (b) Figure 4 1 3 (a) Titration of PyA4 ( 5 M ) Cu2+ ( 20 M) with PPi (1 30 M ) in 20 mM HEPES buffer at pH 7.5; (b) Titration profile with I/I0 ratio represented by the intensit y at 640 nm ; I0: fluorescence emission intensity of PyA4 Cu2+ complex I : fluoresc ence emission intensity in the presence of PPi. PPi with a significant enhancement of excimer intensity compared to the other anions Figure 4 1 3 shows fluorescence titration results of PyA4 Cu2+ complex with PPi in 20 mM HEPES buffer at pH 7.5. Upon the addition of 20 M PPi (which is the same concentration as Cu2+ ions complexed), a 54 fold enhancement of fluorescent emission intensity is observed at 640 nm, approximately 77% of the initial fluorescence of PyA4 without Cu2+ ions. The analytical detection limit (ADL) for PPi is 65.6 nM at low range of quencher concentration. W hen 4 0 M of PPi was added to a solution of the PyA4 Cu2+ complex, recovery reached approximately 99 % ( 70fold enhancement) of the fluorescence intensity. We believe that two oxygen anions of PPi are involved in the complexation with the divalent Cu2+ ion by the ratio of 1:2 (Cu2+ : PPi) inducing dissociation of interaction between PyA4 and Cu2+ ions with recovered fluorescence. Further addition of excess PPi to PyA4 Cu2+ complex shows neither any subtle wavelength change nor remarkable emission intensity change (Figure 4 1 3 b ). This observation means that an excess amount of PPi does not affect recovered PyA4

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111 PyA4 was also tested in the high ionic strength buffer solution (100 mM HEPES solutions at pH 7.5). As seen in Figure 4 1 4 increasing ionic strength leads to a reduced affinity of PyA4 Cu2+ complex for PPi. Even so, it showed 91% recovery of the fluorescence intensity upon addition of 60 M PPi in 100 mM HEPES buffer which is a 6 3 fold enhancement Figure 4 1 5 shows the fluorescence intensity changes of PyA4 upon addition of Cu2+ ions, the fluorescence is completely quenched (Figure 4 1 5 b), while the further addition of PPi recovers the fluorescence intensity (Figure 4 1 5 c). 480 520 560 600 640 680 720 760 800 0.0 2.0x1054.0x1056.0x1058.0x1051.0x106 PyA 4 + Cu2+ ( 30 M ) Wavelength (nm)Fluorescent Intensity (a.u.)0 M 100 M PyA 4 PPi Figure 4 1 4 (a) Titration of PyA4 ( 5 M ) Cu2+ ( 3 0 M) with PPi (10 100 M ) in 100 mM HEPES buffer at pH 7.5; Excitation at 456 nm. Figure 4 1 5 Fluorescence intensity changes of (a) PyA4 (b) PyA4 Cu2+ complex, and (c) PyA4 Cu2+ w ith PPi; [ PyA4 ]=10 M ; [Cu2+]=40 M ; [PPi]= 8 0 M in 20 mM HEPES buffer at pH 7.5.

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112 PyA4 also shows no significant changes to 50 M of AMP (adenosine monophosphate) However, for ADP (adenosine diphosphate) and ATP (adenosine triphosphate), PyA4 Cu2+ complex shows smaller recovery compared to PPi (Figure 4 1 2 ). This is attributed to the c omparatively lower anion densities on the phosphorus oxygen of AMP, ADP, and ATP and their steric bulk when approaching to PyA4 Cu2+ complex, resulting in less effective fluorescence recovery (only 5, 15, and 50% for AMP, ADP, and ATP were recovered, respectively). T his is also supported by Hong s and Febbrizzi s works in which di metallic receptor s showed higher affinity for PPi compared to ATP, Pi, and the other anions due to its relatively larger charge density.97,108 Real time ALP assay In order to provide an insight into the bioanalytical applications of PyA4 Cu2+ system for monitoring enzymes activity, the real time assay of pyrophosphate hydrolysis was investigated using alkaline phosphatase (ALP) in HEPES buffer (0.02 M, pH 7.5) at 37 oC. The solution for ALP assay was prepared with 5 M of PyA4 20 M of Cu2+, and 40 M of PPi and incubated for 10 minutes before addition of ALP. As shown in Figure 4 1 6 fluorescence intensity ( ex=456 nm and em=640 nm) has gradually decreased as a function of time in the presence of ALP. As expected, this reaction is catalyzed by ALP, in which PPi is hydrolyzed to Pi. Free PyA4 is re associated with Cu2+ ion because produced Pi shows less affinity for Cu2+ ion, leading to the fluorescence quenching. Its quenching effect was accelerated as the ALP concentration increases. This study clearly shows that PyA4 Cu2+ system can be useful tool for not only ALP assay but also monitoring enzymes activity involving PPi and Pi.

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113 0 200 400 600 800 1000 1200 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 0 30 50 100 150 200 Relative fluorescence intensityTime (s) [ALP]/nM Figure 4 1 6 Real time ALP assay using PyA4 ( 5 M ) Cu2+ ( 20 M) and PPi (30 M ) in 20 mM HEPES buffer at pH 7.5, 37.0 oC; Excitation wavelength = 456 nm, emission monitored = 640 nm. Plausible mechanism Fi gure 41 7 show a plausible mechanism of the entire sensing processes. Quenched fluorescence of the PyA4 aggregates Cu2+ ions complex was revived by the addition of PPi, in which PPi forms complex with Cu2+ ions. Hydrolysis of PPi by ALP produces reassocia tion of PyA4 aggregates and Cu2+ ions, which lea ds to the fluorescenceoff Figure 4 1 7 Plausible mechanism of sensing process

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114 Summary and Conclusions In this Chapter we synthesized a PyA4 that displays self assembly with strong excimer emission in HEPES buffer solution. PyA4 is stacked not only in the ground state but also in the excited state in HEPES buffer PyA4 Fluorescence intensity of PyA4 was selectively quenched with the Cu2+ ion ; this can be explained by the PET effect. Furthermore, the effic ient quenching effect of PyA4 in aqueous HEPES buffer can probably be explained by energy migration. This system, PyA4 Cu2+, sensitively and selectively recognizes PPi over other anions, inducing strong fluorescence recovery by dissociation of the interact ion between the PyA4 and the Cu2+ ion The real time turn off assay was developed to monitor ALP activity. Experimental Materials All chemicals used in the synthesis were of reagent grade and used without further purification. Pyrene, 4 i odophenol, ethyl bromoacetate, bromine, c opper iodide, triethylamine, tetrakis (triphenylphosphine)palladium (0) tetra butylammonium fluoride, and diisopropylamine were purchased from SigmaAldrich Chemical Company. Ethynyltrimethylsilane was bought from GFC Chemical Company Potassium carbonate was obtained from Fisher Scientific Company. THF was purified by Solvent Dispensing System (SDS). Silica gel (Merck, 230 400 mesh) was used for chromatographic purification of all of intermediate and target molecules. All other chemi cals and solvents were purchased from SigmaAldrich, Fisher Scientific, or Acros Chemical Company and used as received.

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115 Instrumentation and Methods NMR spectra were recorded using a Varian VXR-300 FT NMR, operating at 300 MHz for 1H NMR and at 75 MHz for 13C NMR. UV/Vis absorption spectra were recorded using a Varian Cary 50 Spectrophotometer. Steady-state fluorescence spectra were obtained with a PTI fluorometer A 1 cm quartz cuvette wa s used for all spectral measurements. Stock solutions (1.0 mM) of PyA4 was prepared in H2O. The chloride salts of Ca2+, Mg2+, Co2+, Ni2+, Mn2+, Fe2+, Hg2+, Zn2+, Cd2+, Pb2+ and Cu2+ ions (stock solutions = 10.0 mM in H2O) were tested to evaluate the meta l ion binding properties of PyA4 (stock solution = 1.0 mM in H2O). The excitation was 456 nm The sodium salts of F-, Cl-, Br-, I-, H2PO4 -, HPO4 2-, PPi, AcO-, HSO4 -, NO3 -, HCO3 -, SO4 2-, CO3 2-, AMP, ADP, and ATP (stock solutions = 10.0 mM in H2O) were used. For all fluorescence measurements, the excitation was made at 456 nm to give fluorescence intensity at 640 nm at room temperature. Analytical detection limit (ADL) was calculated using the equation ADL = 3 bk/m ( bk= c=0/ ), where c=0 is the standard deviation of the blank and m is the slope of the calibration plot.111 Synthetic Procedure 4-(ethyloxycarbonylmethoxy)iodobenzene (3). To a solution of 4-iodophenol ( 1 ) (10.0 g, 45.5 mmol) in dried CH3CN (50 mL), anhydrous K2CO3 (6.30 g, 45.5 mmol) was added. After stirring for 30 min, ethyl bromoacetate ( 2 ) (8.4 g, 50 mmol) was added to the reaction mixture. The resulting mi xture was vigorously stirred at 80 oC for 24 hours under argon gas. After the reaction mixture was cooled to room temperature, the solvent was removed in vacuo The reaction mixture wa s acidified with 5 % aqueous

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116 HCl solution (100 mL), and then extracted with CH2Cl2 (200 mL). The organic layer was separated and washed with water (100 mL) and dried over anhydrous MgSO4, and the solvent was evaporated to yield a white solid. The pure product was isolated by column chromatography on silica gel using ethyl acetate:hexane (1:3) as the eluent. 61% yield; mp 5860 oC ; 1H NMR (300 MHz CDCl3, ppm): 7.55 (d, 2H, IAr Hortho, J=9.0 Hz), 6.97 (d, 2H, IAr Hmeta, J=9.0 Hz), 4.56 (s, 2H, ArOC H2CO2), 4.25 (q, 2H, CO2C H2CH3, J=7.2 Hz), 1.27 (t, 3H, CO2CH2C H3, J=7.2 Hz) ; 13C NMR (75 MHz, CDCl3ppm): 16 9 4 158 5 139 0 117 8 84.5, 6 6.0 62.0, 14.2. Compounds 5 6 and 7 w ere prepared in a good yield as described in the literatures .1 1,3,6,8Tetrabromopyrene ( 5 ). 94% yield. This solid product was not soluble enough in most of the common organic solvents to take a NMR spectrum. Therefore, it was identi fied after the Sonogashira coupling reaction with trimethylsilylacetylene. 1,3,6,8Tetrakis(trimethylsilylethynyl)pyrene ( 6 ). 21% yield. 1H NMR (300 MHz CDCl3, ppm): 8.59 (s, 4H, p yreneH ), 8.30 (s, 2H, pyrene H ), 0.40 (s, 36H, SiC H3). 1,3,6,8Tetraethynylpyrene ( 7 ). 93% yield. 1H NMR (300 MHz CDCl3, ppm): 8.55 (s, 4H, p yreneH ), 8.31 (s, 2H, pyreneH ), 4.93 (s, 4H, acetyleneH ). 1,3,6,8Tetrakis[{4(ethyloxyc arbonylmethoxy)phenyl}ethynyl]pyrene (PyE4). To a mixture of 1,3,6,8 tetraethynylpyrene ( 7 ) (0.40 g, 1.34 mmol) and 4 (ethyloxycarbonylmethoxy)iodobenzene ( 3 ) (2.05 g, 6.70 mmol) in degassed solution of THF (10 mL) and triethylamine (10 mL), Pd(PPh3)4 (0.0 77 g, 0.067 mmol) and CuI (0.013 g, 0.067 mmol) were added. The resulting mixture was stirred at 80 oC for 12 hours under argon gas. After the reaction mixture was cooled to room temperature, the

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117 solvent was removed in vacuo. The crude mixture was purified by column chromatography on silica gel using CH2Cl2:Hexane (3:7) as the eluent. 55% yield ; mp 1 401 42 oC; 1H NMR (300 MHz, CDCl3ppm): 8.58 ( s, 4H, p yreneH ), 8.30 (s, 2H, pyreneH ), 7.65 (d, 8H, CArHortho, J=8.4 Hz), 6.97 (d, 8H, CArHmeta, J=8.4 Hz) 4.68 (s, 8H, ArOC H2CO2), 4.33 (q, 8H, CO2C H2CH3, J=7.2 Hz), 1.34(t, 12H, CO2CH2C H3, J=7.2 Hz ); 13C NMR (75 MHz, CDCl3ppm): 168. 5 15 8 1 133 5 1 31. 5 12 6 .4, 122 9 119 0 116 8 115.0, 94.9, 87.4, 65. 6 61.7, 14.4; APCI TOF MS ( m/z ): [M+H]+ calcd for C64H50O12, 1011.3375; found, 1011.3367. 1,3,6,8Tetrakis[{4(carboxymethoxy)phenyl}ethynyl]pyrene, tetrasodium salt (PyA4). To a solution of PyE4 (0. 5 0 g, 0.51 mmol) in 2 methoxyethanol (20 mL), NaOH in water (1 mL) was added dropwise. The reaction mixtur e was vigorously stirred at 80 oC for 12 hours. T he reaction mixture was cooled to room temperature, and then poured into a solution of methanol (300 mL) and diethyl ether (100 mL) to give the fine reddish PyA4 precipitate. Further purification of PyA4 precipitate was accomplished by dialysis using nanopure water (Millipore Simplicity water system) and a 500 D MWCO cellulose membrane. After dialysis, the water was removed in vacuo, which gave the crystal line product. 83% yield. 1H NMR (300 MHz; D2O:CD3OD ( 3 :1/ v: v) ppm): 7.65 (br, 6H, p yreneH ), 7.47 (br, 8H, OArHmeta), 6.95 (br, 8H, OArHortho), 4.68 (br, 8H, ArOC H2CO2) ; ESI MS ( m/z ): [M H]calcd for C56H34O12 898.21; found, 898.00.

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118 CHAPTER 5 PHOTOPHYSICS AND ENE RGY TRANSPORT IN CONJUGATED POLYELECTROLYTE DENDR IMERS Since dendrimers have received considerable attention as a potential candidate for optical devices,112 113 light harvesting systems,41 and medical applications,45,114 there is a growing interest in the synthetic methodology and development of new functional materials.37,45 Dendrimers are highly branched threedimensional globular architectures and monodisperse macromolecules, which are structurally distinct from classical linear polymers.34 Also, because of such distinguished framewor ks, their structures in solution are relatively rigid compared to linear polymers that vary in the size and shape depending on the solvent.33 Furthermore, the number of dendrimer endgroups increases exponentially as the generation increases. Such endgroups in dendrimers can be interfaced bet ween the inside of dendrimers and external materials Therefore, their functionality is readily modulated by tuning the end groups. In addition, their interior is encapsulated by external endgroups, which enable one to have two distinctive properties in one molecule.33 Such unique structure m ay prevent inherent problems shown in traditional polymers, such as unexpected behavior induced by aggregation and/or large polydispersity (PDI). These extraordinary features can be utilized in light harvesting system, gene and drug delivery system. Over the past several decades, numerous types of dendritic systems have been developed, and their electronic properties have also been studied.37,45,115116 As one of the dendritic systems, phenylacetylene dendrimers con nected at meta position have been synthesized by Moore and coworkers. Such fully conjugated dendrimers have a higher net density throughout entire dendrimer.117 In addition, electronic communication

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119 between chromophores can lead to extended electronic states and coherent transfer118120 or enhanced throughbond energy transfer.121 O O O R R R O O O O O O R R R R R R O O R R O R O R O R O R O R O R O O R O R O R R G -1 G-2 G -3R = N H O O-L i+ O-L i+ O-L i+ O O O Figure 5 1 Structure of CPE Ds ( G 1 G 2 and G 3 ) In this Chapter, we designed water soluble conjugated polyelectrolyte dendrimers (CPEDs) containing branched carboxy l ate endgroups and meta conjugated phenylacetylene as a core and branched repeat units Figure 5 1 shows the structure of the CPEDs ( G 1 G 2 and G 3 ). As the generation increases, the interior hydrophobic focal point and branches are surrounded by an increasing number of hydrophilic carboxylate endgroups. Interestingly, the G 2 and G 3 systems closely resemble unimolecular micelle s. Unlike traditional micelle all hydrophilic carboxylate end groups are covalently connected to the hydrophobic inner parts. Consequently, this structure displays unimolecular micelle type formation, which retains its structure in various solvents and over the entire range of concentrations.3334

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120 Results Synthesis and Characterization Synthesis of precursor s H2N OtB u OtB u tB u O O O O N H OtB u OtB u OtB u O O O O C l ( i )1 2 I O H I O 3 4 ( i i ) ( i i i ) H N OtB u tB u O tB u O O O O O O 5H N OtB u tB u O tB u O O O O O T M S I I I B r B r T M S 7 8 B r B r B r 6( i i i ) ( i v ) Figure 5 2 Synthesis of 5 7, and 8 (i) chloroacetyl chloride, K2CO3, EtOAc/H2O (1/1 v/v), r.t. for 2 hrs.; (ii) 2 K2CO3, CH3CN, 80 oC for 12 hrs.; (iii) ethynyltrimethylsilane, THF/DIPA (1/1, v/v ), Pd(PPh3)2Cl2, CuI and PPh3, r.t. for 12 hrs ; (iv) CuI, I2, HMPA, reflux The preparation of water soluble conjugated polyelectrolyte dendrimers (CPE Ds) is described in F igure 5 2 ~ 4 Figure 5 2 presents the synthesis of compound 5 Compound 1 was easily synthesized with an excellent yield as described in the

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121 literature .39 It was converted to di tert butyl 4 (3 tertbutoxy 3 oxopropyl) 4 (2 chloroacetamido)heptanedioate ( 2 ) with 95 .0 % yield by the reaction with chloroacetyl chloride in the presence of K2CO3 in EtOAc/H2O (1/1, v/v ) solution. Then, 4iodophenol was ( 3 ) reacted with 2 to give 4 in 72.0% yield. The coupling reaction of 4 with commercially available ethynyltrimethylsilane in the presence of Pd(PPh3)4 and CuI in THF/DIPA (1/3, v/v ) afforded 5 in 62.8% yield. Compound 7 was readily synthesized fro m 1,3,5 tribromobenzene ( 6 ) by Sonogashira coupling reaction. Also, the bromine group of compound 6 was effectively converted to iodine group in the presence of CuI and I2.122123 R O O R R R O R O R O R O R P G 1 P G 2 P G 3 O R R H N O OtBu OtBu O O tB uO O R =T M S 1 0 1 1 H R T M S 1 2 1 3 H R ( i ) ( i i i ) ( i i ) ( i ) ( i i i ) T M S 5 9 H R ( i ) ( i i i ) ( i i ) Figure 5 3 Synthesis of PG 1, PG 2, and PG 3 (i) K2CO3, DCM/CH3OH (1/1 v/v), r.t. for 2 hrs.; (ii) 7 THF/DIPA (1/4 v/v ), Pd(dba)2, PPh3, CuI, 80 oC for 12 hrs.; (iii) 8 THF/DIPA (1/4 v/v ), Pd(dba)2, PPh3, CuI, 80 oC for 12 hrs.

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122 Figure 5 3 shows the synthesis of precursor G 1 G 2 and G 3 ( PG 1 PG 2 and PG 3 ). De protection of trimethylsilyl group of 5 was accomplished by using K2CO3, giving compound 9 in 95% yield. Convergent approach37 was used to extend the size of the dendrimers, and compound 7 was used as a focal point monomer. Coupling of 7 with 2.4 equiv of 9 in the presence of Pd(dba)2 as a catalyst resulted in the dendron 10. The repeat of deprotection o f dendron 10 and accumulation processes of dendron 11 with a focal point monomer 7 gives a more accumulated d endron 12. Compound 13 w as o btained for further reaction by deprotection of the trimethylsilyl group. The coupling reaction of 9 with 1,3,5triiodobenzene ( 8 ) in the presence of CuI and Pd(dba)2 as catalysts in THF/DIPA(1/3, v/v ) gives PG 1 (35 % yield). PG 2 and PG 3 were also obtai ned in 2030% yield by the coupling reaction of dendron 1 1 or 1 3 with 1,3,5triiodobenzene ( 8 ) in the presence of Pd(dba)2, respectively. Further iteration of Dendron 1 3 leads to a highly congested dendron for the next generation ( PG 4 ). Unfortunately, how ever, sterically dens e PG 4 was not synthesized. It is possible that sterically congested carboxylate end groups prevent formation of the next generation ( PG 4 ) as physical limitation on dendrimer size. Hydrolysis of precursor O H N O OtB u OtB u O O tB u O O O H N O O-N a+ O-N a+ O O +N a-O O ( i ) ( i i ) Figure 5 4 Hydrolysis of branched side chains (i) TFA/DCM, r.t. for 2 hrs. ; (ii) sat. Na2CO3 aqueous solution.

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123 Hydrolysis process of organic soluble dendrimers is well described in Figure 5 4 H ydrolysis was easily accomplished in acidic conditions ( TFA/DCM) for 12 hours. The residues were treated with saturated Na2CO3 solution and then purified by dialysis method using 1000 D molecular weight cutoff (MWCO) dialysis membranes. The water soluble anionic conjugated polyelectrolyte dendrimers (CPE Ds: G1 G2 and G3 ) were obtained as solids in 8090% yield. The purity of all compounds was proven by 1H and 13C NMR spectroscopy, and mass spectroscopy. Geometric structure of CPE Ds Figure 5 5 Spacefilling model of G 1 G 2 and G 3 generated by using MM 2 molecular mechanics in Chem 3D Pro (version 10.0). The structure of CPEDs presents fully conjugated phenylacetylene branches at meta position with branched carboxylate end groups. The number of endgroups geometrically increase s with generation, in whic h G 1 G 2 and G 3 posses 9, 18, and 36 carboxylate groups, respectively. It is noted that poor intrinsic solubility of dendrimers especially in higher generation is one of the factors that make dendrimer synthesis difficult. Attaching ample carboxylate endgroups into CPE Ds not only solves such solubility issue, but also affords the sufficient volume to overspread the inside core and branches. It is evident from space filling models that CPEDs take on a more threedimensional spherical shape as the generation increases (Figure 55 ). Another

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124 distinctive character in the dendrimer chemistry is that they are perfect monodisperse macromolecules.37 In a series of CPE Ds, GPC data provide information on relative size of the precursor CPE Ds ( PG 1 PG 2 and PG 3 ) as seen in Figure 56 in which the polydispers ities are close to one. 10 12 14 16 18 20 0.0 0.2 0.4 0.6 0.8 1.0 1.2 precursor G-1 precursor G-2 precursor G-3Elution time (min)Detector output (a.u.) Figure 5 6 GPC data of precursor of CPE Ds ( PG 1 PG 2 and PG 3 ); polystyrene standards in THF. Geometric Properties: CPE Ds Size Dynamic light scattering (DLS) The solution state of the CPEDs was studied by dynamic light scattering (DLS) measurements. This study provides information for the hydrodynamic radius of the molecules in different solvent environments and goes insight to state of aggregation.67 Figure 57 shows the distribution profiles for the populations of scatterers with the hydrodynamic radii in CH3OH and H2O solution. In CH3OH, DLS on G 1 G 2 and G 3 shows 1.61, 1.56 and 1.47 nm as an average size, respectively. Although their average sizes were opposite of the dendrimer generation, the distribution profiles are consider to

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125 be the single dendrimer. As expected, DLS of G 1 in H2O shows a relatively larger size (3.26 nm), and the distribution profile show s a maximum size of approximately 5.5 nm. Thus, based on DLS studies, we believe that G 1 undergoes inter dendrimer aggregation in H2O, but its accumulation is restricted within narrow limits. For G 2 and G 3 DLS data in H2O present 1.04 and 1.03 nm respectively. The single dendrimer structures of G 2 and G 3 retained even in H2O solution. Interestingly, the smaller hydrodynamic radius than those in CH3OH was observed for G 2 and G 3 For this observation, we suggest that intradendrimer s elf aggregation process caused by hydrophobic interaction leads to the contraction of the dendrimer in H2O. 0 1 2 3 4 5 6 0 10 20 30 40 ChannelSize (nm) G-1 G-2 G-3 Figure 5 7 Hydrodynamic radii obtained from dynamic light scattering (DLS) for G 1 (black), G 2 (red), and G 3 (blue) in H2O; [ G n ] = 1.0 M ; average size: 1.61 nm ( G 1 ), 1.56 nm ( G 2 ), and 1.47 nm ( G 3 ).

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126 Atomic force microscopy (AFM) (a) 0 m 1.22 m 2.44 m 0 1.22 2.44 (b) 0 m 1.02 m 0 1.02 2.05 (c) 0 m 0.88 m 1.76 m 0 0.88 1.76 2.05 m 0 nm 11.1 nm 0 nm 4.8 nm 0 nm 6.5 nm Figure 5 8 AFM images of (a) G 1 (b) G 2 and (c ) G 3 To further elucidate the intra or intermolecular aggregation of CPE Ds, the CPEDs were visualized by AFM (Atomic Force Microscope). Figure 58 shows the topographic images of CPE Ds, which were deposited on mica substrates from 1 mM solution in H2O solvent. This confirms the dendrimer shape and size on the Mica surface. As seen in Figure 58 a, the G 1 exhibits the roundtype dendrimers, and their size are analogous (approximately 4.56 nm). Figure 58 b and 5 8 c show a large number of dendrimers ( G 2 and G 3 ) with consistent sizes, respectively. Interestingly, even smaller size of G 2 (approximately 1.32 nm) and G 3 (approximately 0.98 nm) than that of G 1 was observed. The relatively small and constant size of G 2 and G 3 supports that their geometric structures disfavor mutual approaches, resulting in little or no inter dendrimer aggregate formation. On the other hand, the relatively larger size of G 1 reflects inter dendrimer aggregation in the ground state. Also, consistency in aggregate size supports that spatially crowded carboxylate anions in G 1 aggregates prevent additional aggregation. The smaller size of G 3 compared to G 2 is explained by the fact that strong electrostatic interactions between the t erminal endgroups and substrate causes compression, resulting in deformation of surfacebound dendrimers. It is

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127 possible that more endgroup substitution provides better interaction with substrate, resulting in smaller size in AFM images.45 Optical Properties UVVis & fluorescence spectroscopy Table 5 1 UVVis absorption and photoluminescent properties of CPED s (CH3OH and H2O) and their precursors ( THF ) THF CH3OH H2O CDs max abs (nm) max em(nm) PL a CPE -Ds max abs (nm) max em (nm) PL a max abs (nm) max em (nm) PL a PG -1 316 373 0.34 G -1 277 373 0.025 283 390 0.011 PG -2 314 374 0.38 G -2 291 374 0.024 291 416 0.008 PG -3 313 376 0.36 G -3 294 404 0.018 294 435 0.004 a 9,10FL=0.90. The UV Vis absorption and fluorescence spectra of the CPE Ds ( G 1 G 2 and G 3 ) and their precursors ( PG 1 PG 2 and PG 3 ) are shown in Figure 59 and their wavelength maxima are collected in Table 5 1. The UV Vis absorption spectra of PG 1 PG 2 and PG 3 in THF show the two peaks around 303 and 313 nm, respectively. The water soluble CPEDs ( G 1 G 2 and G 3 ) exhibit blue shifted UV Vis absorption spectra compared to their precursor dendrimers ( P G n series) by around 10 ~ 20 nm in both CH3OH and H2O solutions. We believe that these hypsochromic shifts of CPEDs in UVVis absorption spectra are attributed to more twisted states of the branches induced by their hydrophobic effect and electrostatic r epulsion between bulky carboxylate endgroups. It is possible that the contraction of interior branches caused by the hydrophobic effect in CH3OH and H2O solutions induces their twist, and the electrostati c repulsions between negatively charged terminal end groups give rise to the

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128 rotation of single bonds in the branches to minimize the resistance. Such distortion of the CPEDs branches can also be supported by blueshifted absorption spectrum of the monomer unit ( 8 ) compared to those of PG n series, in wh ich the absorption maximum showed at 250 nm in THF. Interestingly, for only G 1 absorption spectra in H2O was bathochromically shifted by 6 nm compared to that in CH3OH, showing narrow absorption spectra (Figure 510). This observation is distinguished fr om that of G 2 or G 3 which shows negligible spectral difference between CH3OH and in H2O solutions. The spectral change of G 1 in CH3OH and in H2O solutions can be ascribed to the ground state aggregation in H2O solution. Also, it should be noted that, for G 2 and G 3 such spectral similarity in CH3OH and H2O solutions implies that solvent polarity does not much affect to photophysical changes in the ground state. As can be seen from Figure 511 the absorbance of CPE Ds and their precursors increases wit h increasing generation. In THF for PG n series, their absorption maxima in the UV Vis absorption spectra are retained for all generations. In other words, the dendrimer size of PG n series in THF does not affect to their wavelength change in the UVVis ab sorption spectra. On the other hand, G n series were gradually redshifted with increasing generation in CH3OH ( = 7 nm from G 1 to G 3 ) and H2O ( = 10 nm from G 1 to G 3 ) solutions as seen in Figure 511. Besides, the wavelength maxima with increasing generation of G n series approach that of PG n series. For this observation, we propose that the red shift is likely a result of the geometrical changes of CPEDs toward decreased torsional conformation of each phenylacetylene unit when branches approach closely: the approaching is due to the increased number of branches and endgroups with increasing generation, and outer hydrophilic solvents

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129 PG-1 (THF) G-1 (MeOH) G-1 (water)( a' ) Normalized Intensity PG-2 (THF) G-2 (MeOH) G-2 (water)( b' ) 300 400 500 600 700 Wavelength (nm) PG-3 (THF) G-3 (MeOH) G-3 (water)( c' ) Absorbance PG-2 (THF) G-2 (MeOH) G-2 (water)(b) 250 300 350 400 Wavelength (nm) PG-3 (THF) G-3 (MeOH) G-3 (water)(c) PG-1 (THF) G-1 (MeOH) G-1 (water)(a) Figure 5 9 UVVis absorption and f luorescence spectra of CPEDs ( G 1 G 2 and G 3 ) and their precursors ( PG 1 PG 2 and PG 3 ) in CH3OH, H2O, and THF, respectively; (a, a ) first generation, ( b b ) second generation, and ( c, c' ) third generation; Excitation wavelength is 300 nm; [ G n ] = [ PG n ] = 1 M. lead to approaching phenylenene units to the adjacent moieti es. It is not expected that such approaching occur s in welldissolved organic soluble PG n series in THF. On the other hand, it is likely that the conformational changes of CPE Ds having two different properties (hydrophobic interior and hydrophilic exteri or) in one molecule are correlated

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130 with solvent effects. This effectiveness is more pronounced as the generation increases, resulting in further bathochromic shift in the UVVis spectra. 200 250 300 350 400 0.0 0.1 0.2 0.3 0.4 AbsorbanceWavelength (nm) Methanol Water Figure 5 10 UVVis absorption spectra of G 1 in CH3OH and H2O; [G 1 ] =5.0 M. In contrast to the UVVis absorption spectra, CPE Ds ( G 1 G 2 and G 3 ) in the fluorescence spectra exhibit bathochromic shift compared to their precursors ( G 1 : = 0 nm/CH3OH, = 17 nm/water; G 2 : = 0 nm/CH3OH, = 42 nm/water; G 3 : = 28 nm/CH3OH, = 59 nm/water), and the spectra become more shifted to red region and broadened as the solvent polarity increases. Furthermore, such spectral shifts were more significant at higher generation. The red shift and broad spectra of G 1 G 2 and G 3 in fluorescence emission might be ascribed to both the solvent effect and excited state dimer formation (excimer) in water solution. It is well known that solvent effect becomes larger as the solvent polarity is increased, resulting in emission at lower energy.124125 Also, the broad long wavelength emission can be the results of ground state and/or excited state complexes of two or more chromophores. For G 2 and G 3

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131 however, no considerable UV Vis absorption changes were observed in water solution, suggesting that ground state strong dendrimer aggregation can be exclusive. ( a' ) PG-1 PG-2 PG-3 ( b' ) G-1 G-2 G-3 Fluorescence Intensity (a.u.) 300 400 500 600 700 (a) G-1 G-2 G-3 Wavelength (nm) (b) G-1 G-2 G-3Absorbance PG-1 PG-2 PG-3(a) 250 300 350 400 (c) G-1 G-2 G-3Wavelength (nm) Figure 5 11 UVVis absorption and fluorescence spectra of CPEDs ( G 1 G 2 and G 3 ) and their precursors ( PG 1 PG 2 and PG 3 ) with increasing generation in (a, a ) THF, (b, b' ) CH3OH, and (c, c ) H2O, respectively; Excitation wavelength is 300 nm; [ G n ] = [ PG n ] = 1 M. Similar to the UVVis absorbance, the fluorescence emission intensities i ncrease with the generation as seen in Figure 5 11. For PG n series ( max = 373~ 376 nm), wavelength changes were not observed with increasing generation in THF, but for the

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132 water soluble G n series the UV Vis absorption is bathochromically shifted in CH3O H ( = 31 nm from G 1 to G 3 ) and H2O ( = 45 nm from G 1 to G 3 ) solutions, respectively. Notably, the photophysical properties of PG n series in the excited state as well as the ground state are unrelated to the dendrimer size in terms of wavelength ch anges. As expected, metalinked phenylacetylene system does not vary with conjugation lengths. Nevertheless, in the fluorescence spectra, more significant redshifts of G n series were observed in more polar solvent s (CH3OH < H2O): some bathochromic shift as seen in the UV Vis absorption spectra were observed in CH3OH while more significant redshift compared to those in UV Vis absorption spectra were observed in H2O. Such remarkable change in highly polar solvent (H2O) with increasing generation compared t o that in UV Vis absorption spectra is ascribed to excited state aggregation of CPE Ds in water solution; the aggregation is more pronounced in higher generation. Consequently, the is more pronounced in the higher generation dendrimer s ( G 1 < G 2 < G 3 ) and in the more polar solvent Fluorescence quantum yield The fluorescence quantum yields for PG n series in THF and G n series in CH3OH and under various pH conditions were obtained as seen in Table 51 and Figure 512. In spite of different size of PG n similar quantum yields for PG 1 PG 2 and PG 3 were observed ( FL= 0.34/ PG 1 0.38/ PG 2 and 0.36/ PG 3 ). Such similarity in quantum yield of PG n series confirms that because of their meta linked system, each chromophore acts independently even in PG 3 as it shows in PG 1. Furthermore, formation of aggregates which can act as an exciton trap is negligible On the other hand, for G n

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133 series the higher generation dendrimers have lower quantum yield in both CH3OH and H2O, which follow the order G 1 > G 2 > G 3 (Table 51 and Figure 512). pH 3 pH 5 pH 7 pH 8 pH 9 0.0 0.5 1.0 1.5 2.0 Quantum Yield (%)Water (pH) G1 G2 G3 Figure 5 12 Fluorescence quantum yields changes of G 1, G 2, and G 3 at the pH 3 ~ 9; 9,10FL=0.90. In addition, G n series showed the consistent quantum yield in various pH ranges (pH 5 ~ 9). These results support that aggregates are more pronounced in the higher generation and H2O, but they ar e not dependent on pH changes (pH 5 ~ 9). At pH 3, the quantum yield of G 1 and G 2 decreased, but it was retained in G 3 ; this decrease displayed higher rate in lower generation ( G 1 > G2 > G 3 ). After all, the degree of aggregation of G 1 was maintained in the range of pH 5 ~ 9. However, highly acidic condition (pH 3) caused more aggregation of G 1 in which hydrogenated carboxylates probably induce hydrogen bonding with adjacent terminal groups. Nevertheless, the quantum yield of G 3 at pH 3 is similar t o those at other pH ranges. Probably more intra dendrimer aggregation is not allowed because of highly dense branched carboxylate side chains. R elatively less branched G 2 might be inter molecularly aggregated in pH 3.

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134 Fluorescence lifetime studies Table 5 2 Fluorescence lifetimes (i, ns)a and relative amplitudes (RA, %) for CPE Ds (CH3OH and H2O) and their precursors ( THF ). THF CH3OH H2O CDs (ns) RA (%) CPE -Ds (ns) RA (%) av (ns) (ns) RA (%) av (ns) PG -1 9.08 100 G -1 1 = 0.62 2= 5.57 32 68 4.00 1 = 1.21 2= 3.23 22 78 2.92 PG -2 8.05 100 G -2 1 = 1.54 2= 6.54 72 28 2.93 1 = 0.94 2= 4.33 43 57 2.88 PG -3 8.20 100 G -3 1 = 1.59 2 = 5.63 66 34 2.96 1 = 0.88 2 = 4.16 47 53 2.39 a Tipical limits o f error on i are less than 3%. Fluorescence lifetimes of G n series and their precursors ( PG n series) were measured in CH3OH, H2O and THF, respectively. The values of the fluorescence lifetime are given in Table 52. The excitation wavelength is 300 nm and emission decays were monitored over 320 nm. Like fluorescence quantum yield, each PG n showed similarity in fluorescence lifetimes in THF. Also, relatively longer lifetimes with single exponential decay were measured ( = 9.08, 8.05 and 8.20 for PG 1 PG 2 and PG 3 respectively) and these are not dependent on dendrimer size. These features indicate that only one component exists in both ground and excited states and both intra and inter dendrimer aggregation of PG n series does not occur in THF. On the other hand, the G n series showed two decay constants in CH3OH and H2O solutions, respectively (Table 52). The emission decay of G 1 in CH3OH features 32% of fast decay (1 = 0.62 ns) and 68% of relatively longlived decay (2 = 5.57 ns). Similar results are obtained in H2O solution, where 22% of fast decay (1 = 1.21) and 78% of longlived one (2 = 3.23 ns) are observed. This suggests that there are two emis sive pathways in this condition. W e suggested that the slow decay component is a

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135 similar state with long lived component seen in PG 1 in THF; faster decay is due to the geometrical change caused by electrostatic repulsion between bulky endgroups It is noted that high polar solvent allows the long lived charge separation state in the excited state. Nevertheless, the average lifetime in more polar solvent (H2O) is about 1.5 times shorter than that in less polar solvent (CH3OH). It is found that energy transfer or quench ing lead to changes on the emission decay times.53,73 The decrease of average lifetime in this case is mainly due to two issues: first, aggregated states exist in H2O, and so it acts as an exciton or energy trap; second, the direct contact of less shield branches of G 1 with water molecules causes enhanced nonradi active deactivation. For G 2 and G 3 in CH3OH, long lived components (2 G 2 = 6.54 ns and 2 G 3 = 5.63 ns) were observed with around 30% amplitude, while fast decay components are predominant with 1 = 1.54 ns (72%) and 1.59 ns (66%) concomitant with the faster average lifetimes (av. G 2 = 2.93 ns and av. G 3 = 2.96 ns) than G 1 (av. = 4.00 ns). The m ore twisted states of the branches allows a larger contribution from the fast decay. In H2O, however, the contribution of fast decay components (1 G 2 = 0.94 and 1 G 3 = 0.88 ns) decreased and longlived components (2 G 2 = 4.33 ns and 2 G 3 = 4.16 ns) are more dominant, contributing to 57% ( G 2 ) and 53% ( G 3 ). The increased contribution of longlived decay supports that torsional conformation of interior branches is reduced where the branches are more like that in PG n series. I nterestingly, the faster decay in the average lifetime of G 3 (av. = 2.39) compared to that of G 2 (av. = 2.88) was observed in H2O. This decrease implies that more interaction between phenylacetylene units occurs in G 3 rather than G 2 Thus, intra dendrimer aggregation is more pronounced at the higher generation.

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136 Fluorescence excitation spectroscopy 250 300 350 400 450 0.0 0.2 0.4 0.6 0.8 1.0 1.2 em= 380 nm em= 400 nm em= 450 nm Normalized Intensity (a.u.)Wavelength (nm)(a) 250 300 350 400 450 0.0 0.2 0.4 0.6 0.8 1.0 1.2 (b) em= 400 nm em= 450 nm em= 500 nmNormalized Intensity (a.u.)Wavelength (nm) 250 300 350 400 450 0.0 0.2 0.4 0.6 0.8 1.0 1.2 (c) em= 450 nm em= 500 nm em= 550 nmNormalized Intensity (a.u.)Wavelength (nm) Figure 5 1 3 Fluorescence excitation spectra of (a) G 1 at 380, 400, and 450 nm, (b) G 2 at 400, 450, and 500 nm, and (c) G 3 at 450, 500, and 550 nm; [ G n ] = 1.0 M in H2O. T he excitation spectra of G 1 ( em = 380, 400, and 450 nm) clearly showed that aggregates exist in the ground state in water solution, in which distinctive excitation spectra were observed (Figure 5 1 3 a ). On the other hand, little diffe rence was observed in the excitation spectra of G 2 ( em = 400, 450, and 500 nm) and G 3 ( em = 450, 500, and 550 nm) in the same media as seen in Figure 5 1 3 b and 1 3 c. This implies that the ground state aggregates of G 1 exist in H2O. In the cases of G 2 and G 3 however, little aggregation occurs in the ground state; excited state complexes are dominant. Concentration dependent fluorescence studies 300 400 500 600 7000 1x1062x1063x1064x1065x106 1 5 10 15 20Fluorescence Intensity (a.u.)Wavelength (nm)30 [G-1] ( M ) /Water (a) 300 400 500 600 700 0.0 4.0x1058.0x1051.2x1061.6x1062.0x1062.4x106 (b)0.5 2 1 3 4 6 8Fluorescence Intensity (a.u.)Wavelength (nm)10 [G-2] ( M ) /Water 300 400 500 600 700 0.0 4.0x1058.0x1051.2x1061.6x1062.0x1062.4x106 (c)0.5 2 1 3Fluorescence Intensity (a.u.)Wavelength (nm) [G-3] ( M ) /Water Figure 5 1 4 Fluorescence emission spectra of (a) G 1 (b) G 2 and (c) G 3 with increasing concentration in H2O; [ G n ] = 1.0 M

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137 As shown in Figure 5 1 4 the fluorescence spectr a ( ex = 300 nm) of all CPEDs display little change in wavelength and bandshape with increasing concentration in water solution, indicating that their state of aggregation is independent of the concentration of CPE Ds. This indicates that the spectral shifts seen in the UVVis absorption and fluorescence spectra are potentially due to intra dendrimer aggregation. It is reported that Intra molecular self association is predominant especially at sterically highly crowded dendrimers.33 As discussed above in UV Vis spectra and fluorescence excitation spectra of G 1 its ground state aggregates exist in H2O. Also, it should be noted that it is difficult to form intradendrimer aggregates in the case of spatially less crowded C PED ( G 1 ). Nevertheless, for G 1 the fluorescence spectra do not vary as the concentration increases (Figure 51 4 a) For this observation, we propose that inter G 1 can be formed, but its size is limited by the proportion of aggregation. The certain degree of aggregation seems to produce another spherical type of dendrimer in which external surface is fully charged, which prevents additional This would be analogous to micelle formation. Fluorescence Quenching of CPEDs by DOC, DODC, and DOTC N O O N C H2C H3 C H2C H3 I n n = 1 : D O C n = 2 : D O D C n = 3 : D O T C Figure 5 1 5 Structure of cyanine dyes (DOC, DODC, and DOTC).

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138 Table 5 3 Ksv a and [Q]90 b of CPEDs with cyanine dyes in H2O Ksv/M-1 CPE -Ds DOC DODC DOTC G -3 1.84 106 1.67 106 7.03 105 a Computed from linear fit at low quencher concentration (0~1.0 M). Fluorescence quenching of G 3 series in H2O was investigated upon the addition of cyanine dyes (DOC, DODC, and DOTC), in which the quenching occurs via energy transfer. Figure 5 1 5 shows the structure of DOC, DODC, and DOTC, in which conjugation chain length are only in difference. As s een in Figure 5 1 6 t he addition of cyanine dyes causes the overall emission intensity of G 3 to decrease. Their SternVolmer 19 plots and KSV values present in Table 53 and Figure 517, respectively Figure 51 6 show s the fluorescence intensity changes of G 3 upon addition of DOC. It is reported in CPEs research areas where the fluorescence quenching is m ore amplified in interchain aggregates or aggregationinduced coplanarized conjugated backbones because singlet excitons migrate more efficiently, leading to upward SV plots.53,6768 S im ilarly the addition of cyani ne dyes causes the amplified fluorescence quenching G 3 resulting in upward curves (Figure 5 1 7 ). A s the chain length of cyanine dyes increases, quenching efficiency of G 3 decreases: SV plots displayed upward curves and KSV values were significant in the sequence DOC > DODC > DOTC (see Table 53 and Figure 51 7 ). These observations conflict with our previous work in which quenching efficiency increased with expanding chain length of cyanine dyes (DOC < DODC < DOTC).53 Such efficient quenching with increasing chain length is due to stabilization of complexes by the solvophobic and interactions in polymer dye electrostatic interaction. Unlike polymers, FRET (Fluorescence Resonance Energy

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139 300 400 500 600 700 800 0 1 2 3 4 DOC DODC DOTCFluorescence Intensity (a.u.)Wavelength (nm) (a) 300 400 500 600 700 800 0 1 2 3 4 (c)Fluorescence Intensity (a.u.)Wavelength (nm)1.0 [DODC] ( M) 0 300 400 500 600 700 800 0 1 2 3 4 (d)Fluorescence Intensity (a.u.)Wavelength (nm)1.0 [DOTC] ( M) 0 300 400 500 600 700 800 0 1 2 3 4 (b)1.0Fluorescence Intensity (a.u.)Wavelength (nm) [DOC] ( M) 0 Figure 5 1 6 Fluorescence emission spectra of (a) cyanine dyes only (DOC, DODC, and DOTC: 1.0 M ) and G 3 titrated with (b) DOC, (c) DODC, a nd (d) DOTC in H2O, pH 8.0; [ G 3 ] = 1 .0 M; [dye quencher] = 0 ~ 1.0 M ; excitation wavelength = 300 nm. Transfer) effect related to chain length is involved in the quenching mechanism. It is well known that the FRET efficiency depends on many parameters s uch as the distance between donor and acceptor and the spectral overlap of the donor emission and the acceptor absorption spectra. In G 3 we observed that degree of spectral overlap increases as the chain length of cyanine dyes decreases. Furthermore, mos t significant fluorescence enhancement was observed upon the addition of DOC (Figure 16 b). Only subtle fluorescence increase was observed for the largest dye, DOTC (Figure 51 6 d). These observations clearly indicate that the degree of FRET determines quenching efficiency of G 3

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140 0.0 0.5 1.0 1.5 2.0 0 2 4 6 8 DOC DOCD DOTCI0/I[ Q ] ( M ) Figure 5 1 7 Stern Volmer plots of G 3 ; fluorescence quenched by cyanine dyes in H2O; [CPEDs] = 1 .0 M; DOC ( ), DODC ( ), and DOTC ( ) Summary and Conclusions In this Chapter we have prepared conjugated polyelectrolyte dendrimers (CPE Ds) G 1 G 2 and G 3 with branched carboxylate side chains, providing globular architecture with increasing generati on. B oth AFM and DLS results suggest ed that the aggregate state of G 2 and G 3 in H2O is likely due to intra dendrimer interactions rather than inter dendrimer aggregation while inter dendrimer aggregation occurs in G 1 The absorption and fluorescence int ensities of CPE Ds increase as the generation increases. In MeOH, absorption and fluorescence spectral bandshape of G n does not change significantly, suggesting that the state of the chromophores does not change much with generation. By contrast, in H2O there is a considerable change in the fluorescence wavelength and band shape with increasing generation. This observation suggests that an aggregate state of CPE Ds develops in the poor solvent environment.

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141 Both comparison of fluorescence spectra in CH3OH a nd H2O and the excitation spectra in H2O reveal ed that G 1 shows inter dendrimer aggregation in the ground state while G 2 and G 3 display intra dendrimer aggregates in the excited state in aqueous solution. Also, the same quantum yields of G 3 at various pH condition (pH 3 ~ 9) support inter dendrimer aggregates do not exist. Fluorescence lifetime also support the aggregation features of CPEDs The quenching from G 3 to cyanine dyes is independent on the chain length of cyanine dyes and is due to primarily energy transfer effects Experimental Materials All chemicals used for the synthesis were of reagent grade and used without further purification. Nitromethane, tertbutylacrylate, T1 Raney nickel, diisopropylamine, chloroacetyl chloride, 4 iodophenol, palladium (0) bis(dibenzylideneacetone) tetrakis(triphenylphosphine)palladium 1,3,5 tribromobenzene, and triphenylphosphine were purchased from SigmaAldrich Chemical Company. S odium carbonate, and copper iodide were obtained from Acros Chemical Company. Ethynyltrimethylsilane was obtained from GFC Chemical Company. trans Dichlorobis(triphenylphosphine)palladium (II) was purchased from St re m Chemical Company. Potassium carbonate and trifluoroacetic acid were obtained from Fisher Scientific Company. THF and DMF were purified by solvent d ispensing system Silica gel (Merck, 230400 mesh) was used for chromatographic purification of all of intermediates and target molecules. All other chemicals and solvents were purchased from either SigmaAldrich or Acros Chemical Company and used as received.

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142 Instrumentation and Methods NMR spectra were recorded using a Varian VXR 300 FT NMR, operating at 300 MHz for 1H NMR and at 75 MHz for 13C NMR. Gel permeation chromatography (GPC) analyses were carried out on a system comprised of a Rainin Dynamax SD 200 pump, Polymer Laboratories PL gel mixed D columns, and a Beckman Instruments Spectroflow 757 absorbance detector. Molecular weight calibration was effected by using polystyrene standards. UV/Vis absorption spectra were recorded using a Varian Cary 50 Spectrophotometer. Steady state fluorescence spectra were obtained with a PTI fluor o meter Lifetime measurements were carried out using a PicoQuant FluoTime 100 Compact Fluorescence Lifetime Spectrometer. A 1 cm quartz cuvet te was used for all spectral measurements. Dynamic light scattering (DLS) experiments were performed with Zeta PALS from Brookhaven Instrument Corporation. Atomic Forece Microscopy (AFM) images were obtained with a Veeco Innova Scanning Probe Microscope. Stock solutions (1.0 mM ) of all of the CPED s were prepared in H2O and were stored at 0 oC. The solutions were maintained at room temperature for one hour before use. Fluorescence quantum yields are reported relative to know n standards ( 9,10diphenyl anthr acene, = 0.9 0 in EtOH). The pH of aqueous solution was adjusted with HCl and/or NaOH using a corning pH meter 320. Synth etic Procedures Compounds 1 7 and 8 were prepared in a good yield as described in the literature.39,122123 Compound 2. To a suspension of 1 (30.0 g, 72.2 mmol) and anhydrous K2CO3 (40.1 g, 296.0 mmol) in a mixture of water (200 mL) and ethyl acetate (200 mL), a solution of chloroacetyl chloride (7.30 mL, 91.7 mmol) in ethyl acetate (50 mL) was

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143 added dropwise. The reaction mixture was then stirred at room temperature for 2 hours, after which the organic phase was separated from the aqueous phase, and dried over anhydrous MgSO4. The solvent was evaporated to give the crude product. Column chromatography using EtOAc:hexane (2:1) as eluent (Rf = 0.80) on silica gel gave 2 as a white solid in a 95.0% yield. mp: 7374 oC; 1H NMR (300 MHz, CDCl3, ppm): 6.45 (s, 1H, CONH ), 3.87 (s, 2H, ArOC H2CO2), 2.15 (m, 6H, NHC(CH2C H2CO2 tBu)3), 1.93 (m, 6H, NHC(C H2CH2CO2 tBu)3), 1.36 (s, 27H, CO2 tBu ); 13C NMR (75 MHz, CDCl3, ppm): 172.5, 165.2, 80.8, 60.5, 58.1, 42.5, 34.5, 29.8, 28.2; LC MS ( m/z ): [M+Na]+ cal cd for C22H42ClNO7, 514.3; found, 514.1. Compound 4. To a solution of 4 iodophenol ( 3 ) (11.6 g, 52.8 mmol) in dried CH3CN (50 mL), anhydrous K2CO3 (8.03 g, 58.1 mmol ) was added. After stirring for 30 min, compound 2 (26.0 g, 52.8 mmol) was added to the reaction mixture. The resulting mixture was vigorously stirred at 80 oC for 12 hours under argon gas. After the reaction mixture was cooled to room temperature, the solvent was removed in vacuo. The reaction mixture was acidified with 5% aqueous HCl solution (200 mL), and then extracted with CH2Cl2 (200 mL). The organic layer was separated and washed with water (200 mL) and dried over anhydrous MgSO4, and the solvent was evaporated to yield a white solid. The crude product was isolated by column chromatography on silica gel using ethyl acetate:hexane (1:2) as the eluent. 72.0% yield; mp 114116 oC; 1H NMR (300 MHz, CDCl3, ppm): 7.62 (d, 2H, IArHortho, J=9.0 Hz), 6.75 (d, 2H, IAr Hmeta, J=9.0 Hz), 6.57 (s, 1H, CON H ), 4.35 (s, 2H, ArOC H2CO2), 2.20 (m, 6H, NHC(CH2C H2CO2 tBu)3), 2.00 (m, 6H, NHC(C H2CH2CO2 tBu)3), 1.44 (s, 27H, CO2 tBu ) ;

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144 13C NMR (75 MHz, CDCl3ppm): 172.7, 167.2, 157.0, 138.7, 117.2, 82.5, 80.9, 67.8, 57.9, 30.1, 29.8, 28.3; LC MS ( m/z ): [M+Na]+ calcd for C30H46INO8, 698.0; found, 698.1. Compound 5. To a solution of compound 4 (25.0 g, 37.0 mmol) in degased 100 mL THF/DIPA (1/3, v/v ), Pd(PPh3)2Cl2 (0.52 g, 0.74 mmol ) and CuI (0.28 g, 1.48 mmol ) were added. Then, ethynyltrimethylsilane (6.32 mL, 44.4 mmol) was added to the reaction mixture. The resulting mixture was vigorously stirred at 70 ~ 75 C for 12 hours under argon gas. After the reaction mixture was cool ed to room temperature, the solvent was removed in vacuo. The crude product was isolated by column chromatography on silica gel using methylene chloride:acetone (10:1) as the eluent. 62.8% yield; mp 148150 oC; 1H NMR (300 MHz, CDCl3, ppm): 7.45 (d, 2H, IAr Hortho, J=9.0 Hz), 6.89 (d, 2H, IAr Hmeta, J=9.0 Hz), 6.51 (s, 1H, CON H ), 4.39 (s, 2H, ArOC H2CO2), 2.20 (m, 6H, NHC(CH2C H2CO2 tBu)3), 2.00 (m, 6H, NHC(C H2CH2CO2 tBu)3), 1.43 (s, 27H, CO2 tBu ), 0.24 (s, 9H, (C H3)3Si) ; 13C NMR (75 MHz, CDCl3ppm): 172.7, 167.5, 157.5, 133.9, 117.0, 114.8, 105.1, 93.8, 80.9, 67.6, 57.9, 30.1, 29.8, 28.3, 0.2; LC MS ( m/z ): [M+Na]+ calcd for C35H55NO8Si, 668.0; found, 668.2. General deprotection process of trimethylsilyl group. A mixture of compound 5 10, or 1 2 (1.0 mmol) and K2CO3 (5.0 equiv.) in a solution mixture of dichlomethane (20 mL) and methanol (20 mL) was stirred at room temperature for 2 hours. The mixutre was washed with water (20 mL 3), and dried over anhydrous magnesium sulfate, then the solvent was removed i n vacuo. The product was used in the coupling reaction without further purification. 80 ~ 90% yield.

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145 9 : mp 126128 oC; 1H NMR (300 MHz, CDCl3, ppm): 7.47 (d, 2H, IArHortho, J=9.0 Hz), 6.92 (d, 2H, IAr Hmeta, J=9.0 Hz), 6.55 (s, 1H, CON H ), 4.39 (s, 2H, A rOC H2CO2), 3.03 (s, 1H, H CCAr), 2.20 (m, 6H, NHC(CH2C H2CO2 tBu)3), 2.00 (m, 6H, NHC(C H2CH2CO2 tBu)3), 1.43 (s, 27H, CO2 tBu ) ; 13C NMR (75 MHz, CDCl3ppm): 172.7, 167.2, 157.5, 134.0, 116.0, 114.9, 83.8, 80.9, 67.5, 57.8, 30.1, 29.8, 28.3; LC MS ( m/z ): [M+ Na]+ calcd for C32H47NO8, 596.0; found, 596.2. 1 1 : mp 7476 oC; 1H NMR (300 MHz, CDCl3, ppm): 7.58 (s, 1H, phenyl H ), 7.54 (s, 2H, phenyl H ), 7.48 (d, 4H, CH2OAr Hmeta, J =9.0 Hz), 6.94 (d, 4H, CH2OAr Hortho, J=9.0 Hz), 6.56 (s, 2H, CON H ), 4.39 (s, 4H, Ar OC H2CO2), 3.09 (s, 1H, H CCAr), 2.20 (m, 12H, NHC(CH2C H2CO2 tBu)3), 2.00 (m, 12H, NHC(C H2CH2CO2 tBu)3), 1.41 (s, 54H, CO2 tBu ) ; 13C NMR (75 MHz, CDCl3, ppm): 172.4, 166.6, 156.7, 132.2, 131.8, 124.2, 116.3, 115.0, 90.3, 87.2, 80.9, 67.5, 57.9, 30.1, 29.8, 28.3 ; LC MS ( m/z ): [M+Na]+ calcd for C72H96N2O16, 1267.0; found, 1267.4. 1 3 : mp 102104 oC; 1H NMR (300 MHz, CDCl3, ppm): 1H NMR (300 MHz, CDCl3, ppm): 7.60 (m, 9H, phenyl H ), 7.55 (d, 8H, CH2OAr Hmeta, J=9.0 Hz), 6.98 (d, 8H, CH2OAr Hortho, J=9.0 Hz), 6. 58 (s, 4H, CON H ), 4.41 (s, 8H, ArOC H2CO2), 3.12 (s, 1H, H CCAr), 2.20 (m, 24H, NHC(CH2C H2CO2 tBu)3), 2.00 (m, 24H, NHC(C H2CH2CO2 tBu)3), 1.42 (s, 108H, CO2 tBu ) ; 13C NMR (75 MHz, CDCl3, ppm): 172.7, 167.1, 157.5, 133.8, 124.5, 116.5, 115.2, 90.3, 87.2, 80. 9, 67.7, 57.8, 30.0, 29.9, 28.3; LC MS ( m/z ): [M+2Na]2+ calcd for C152H194N4O32, 1316.7; found, 1316.8. General coupling reaction procedure. Dendron 9 1 1 or 1 3 (2.2 or 3.3 equiv.) and 0.2 mmol of compound 3,5dibromo1 trimethylsilylethynylbenzene ( 7 ) o r 1,3,5 triiodobenzene ( 8 ) were dissolved in 20 mL of THF/Et3N (1/3, v/v ). The resulting solution

PAGE 146

146 was deoxygenated with argon for 30 minutes. Then Pd(dba)2 (5.8 mg, 10.0 mol), PPh3 (5.2 mg, 20.0 mol) and CuI (1.9 mg, 10.0 mol) were added to the stirred solution under the protection of argon. The reaction mixture was then heated up to 70 ~ 75 C and stirred for 12 hours. The reaction mixture was cooled down to the room temperature and t he solvent was removed in vacuo, affording a pale yellow solid. The cr ude product was isolated by column chromatography on silica gel using methylene chloride:acetone (10:1) as the eluent. 30 ~ 40% yield. 10: mp 8688 oC; 1H NMR (300 MHz, CDCl3, ppm): 7.55 (s, 1H, TMS C2Ar Hpara), 7.52 (s, 2H, TMS C2Ar Hortho), 7.46 (d, 4H, CH2OAr Hmeta, J=9.1 Hz), 6.92 (d, 4H, CH2OAr Hortho, J=9.2 Hz), 6.54 (s, 2H, CON H ), 4.39 (s, 4H, ArOC H2CO2), 2.15 (m, 12H, NHC(CH2C H2CO2 tBu)3), 2.00 (m, 12H, NHC(C H2CH2CO2 tBu)3), 1.41 (s, 54H, CO2 tBu ), 0.23 (s, 9H, (C H3)3Si) ; 13C NMR (75 MHz, CDCl3, ppm): 172.5, 167.1, 157.9, 134.0, 124.0, 116.0, 114.0, 92.0, 86.5, 81.0, 67.5, 57.8, 30.1, 29.9, 28.0, 0.1; LC MS ( m/z ): [M+Na]+ calcd for C75H104N2O16Si, 1339.0; found, 1339.7. 1 2 : mp 108110 oC; 1H NMR (300 MHz, CDCl3, ppm): 7.62 (m, 9H, phenyl H ), 7.53 (d, 8H, CH2OAr Hmeta, J=9.0 Hz), 6.98 (d, 8H, CH2OAr Hortho, J=9.0 Hz), 6.58 (s, 4H, CONH ), 4.42 (s, 8H, ArOC H2CO2), 2.20 (m, 24H, NHC(CH2C H2CO2 tBu)3), 2.00 (m, 24H, NHC(CH2CH2CO2 tBu)3), 1.43 (s, 108H, CO2 tBu ), 0.27 (s, 9H, (C H3)3Si) ; 13C NMR (75 MHz, CDCl3, ppm): 172.7, 167.2, 157.5, 133.8, 124.6, 116.7, 114.9, 90.5, 87.3, 80.9, 67.7, 57.9, 30.2, 29.8, 28.3, 0.2; LC MS ( m/z ): [M+2Na]2+ calcd for C155H202N4O32Si, 1352.7; found, 1352.7. PG 1 : mp 175177 oC; 1H NMR (300 MHz, CDCl3ppm): 7.57 (s, 3H, phenyl H ), 7.50 (d, 6H, CH2OAr Hmeta, J=9.0 Hz), 6.93 (d, 6H, CH2OAr Hortho, J=9.0 Hz), 6.55 (s,

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147 3H, CONH ), 4.40 (s, 6H, ArOC H2CO2), 2.20 (m, 18H, NHC(CH2C H2CO2 tBu)3), 2.00 (m, 18H NHC(CH2CH2CO2 tBu)3), 1.42 (s, 81H, CO2 tBu ) ; 13C NMR (75 MHz, CDCl3, ppm): 172.6, 167.3, 157.5, 133.8, 130.0, 124.2, 116.7, 115.0, 90.2, 87.5, 80.9, 67.5, 57.8, 30.2, 29.9, 28.3; MALDI MS ( m/z ): calcd for C101H141N3O24, 1793.22; found, 1792.30. PG 2 : mp 152154 oC; 1H NMR (300 MHz, CDCl3ppm): 7.68 (s, 3H, phenyl H ), 7.63 (m, 9H, phenyl H ) 7.50 (d, 12H, CH2OAr Hmeta, J =9.0 Hz), 6.96 (d, 12H, CH2OAr Hortho, J=9.0 Hz), 6.58 (s, 6H, CON H ), 4.41 (s, 12H, ArOC H2CO2), 2.20 (m, 36H, NHC(CH2C H2CO2 tBu)3), 2.00 (m, 36H, NHC(CH2CH2CO2 tBu)3), 1.42 (s, 162H, CO2 tBu ) ; 13C NMR (75 MHz, CDCl3, ppm): 172.6, 167.2, 154.4, 133.5, 130.0, 124.4, 116.3, 114.9, 90.3, 87.2, 80.8, 67.5, 57.8, 30.1, 29.7, 28.2; MALDI MS ( m/z ): M+ calcd for C222H288N6O48, 3808.67; found, 3809.15. PG 3 : mp 90 92 oC; 1H NMR (300 MHz, CDCl3, ppm): 7.71 (s, 3H, phenyl H ), 7.68 (s, 9H, phenyl H ), 7.62 (s, 18H, phenyl H ), 7.49 (d, 24H, CH2OAr Hmeta, J=9.0 Hz), 6.94 (d, 24H, CH2OAr Hortho, J=9.0 Hz), 6.55 (s, 12H, CON H ), 4.39 (s, 24H, ArOC H2CO2), 2.20 (m, 72H, NHC(CH2C H2CO2 tBu)3), 2.00 (m, 72H, NHC(C H2CH2CO2 tBu)3), 1.42 (s, 324H, CO2 tBu ) ; 13C NMR (75 MHz, CDCl3, ppm): 172.7, 167.2, 157.5, 133.8, 130.0, 124.2, 116.3, 114.9, 90.6, 87.5, 80. 9, 67.6, 57.9, 30.1, 29.8, 28.2; MALDI MS ( m/z ): M+ calcd for C462H582N12O96, 7 839. 59; found, 7 839. 50. Hydrolysis for CPEDs with branched anionic side chains. Precursors (0.2 g) of G 1 G 2 and G 3 ( PG 1 PG 2 and PG 3 ) were dissolved in 20 mL CH2Cl2 and cooled in an ice/water bath. 20 mL of trifluoroacetic acid (TFA) was added to the dendrimer solution dropwise. Upon the completion of the addition, the reaction mixture was allowed to warm to room temperature and stirred for another 12 hours. The excess

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148 TFA and the solvent were removed in vacuo. The residue was treated with saturated aqueous Na2CO3 solution (10 mL) and stirred at room temperature for 3 hours. The solution was then poured into 50 mL of methanol. The dendrimer precipitate was then dissolved in water and purified by dialysis using 1000 D MWCO regenerat ed cellulose membranes (yield: 90~100%). The water soluble conjugated polyelectrolyte dendrimers could be either stored as aqueous solutions or as solid powders. G 1 1H NMR (300 MHz, D2O/DMSOd6 ( v/v ppm): 8.1 7.2 (br, 18H), 4.65 (br, 6H, ArOC H2CO2), 2.19 (br, 18H, NHC(CH2C H2CO2 tNa )3), 1.98 (br, 18H, NHC(C H2CH2CO2 tNa )3); 13C NMR (75 MHz, CDCl3, ppm): 182.9, 170.0, 162.0, 131.5, 115.0, 67.9, 58.6, 31.8, 30.1; ESI MS ( m/z ): [MH]calcd for C66H69N3O24, 1286.0; found, 1286.2. G 2 1H NMR (300 MHz, D2O/DMSOd6 ( v/v ppm): 8.15 7.05 (br, 42H) 4.65 (br, 12H, ArOC H2CO2), 2.18 (br, 36H, NHC(CH2C H2CO2 tNa )3), 1.98 (m, 36H, NHC(C H2CH2CO2 tNa )3); 13C NMR (75 MHz, CDCl3, ppm): 183.2, 170.0, 162.1, 151.0, 132.0, 112.0, 100.3, 66.5, 59.0, 32.0, 31.0; ESI MS ( m/z ): [M H]calcd for C150H144N6O48, 2796.0; found, 2800.02900.0: With both C8 HPLC analysis and flow injection analyses`, numerous compounds were detected. A number of compounds yielded ions consistent with molecular weights in the 28002900 u range. None yielded ions consistent with the fully protonated or fully sodiated forms of the expected compound. G 3 1H NMR (300 MHz, D2O/DMSOd6 ( v/v ppm): 8.20 7.0 (br, 90H) 4.70 (br, 24H, ArOC H2CO2), 2.10 (br, 144H, NHC(C H2C H2CO2 tNa )3).

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149 CHAPTER 6 DESIGN, SYNTHESIS, AND PHOTOPHYSICAL STU DIES OF THIENYL GROUP EXTENDED CONJUGATED POLYELECTROLYTE DENDRIMERS Conjugated polyelectrol ytes (CPEs) have attracted considerable attention because of their remarkable materials properties. In particular, the CPEs are water soluble and retain the optical and electronic properties characteristic of the conjugated backbone in polar solvents .10 However, the CPEs have a strong propensity to self assemble into aggregates in solution because of their amphiphilic feature. For this reason, much effort has been devoted to retaining nonaggregated state in aqueous media.5658 For instance, in our previous work, CPEs with branched polyio nic side chains shows little or no aggregation because of the electrostatic repulsion between branched polyionic side chains. Nevertheless, the problems including the polydisperse and structure defect of CPEs which can affect to the photophysical studies are still remained Dendrimer chemistry is a fast growing field and its numerous applications ranging from medicine to nano engineering have led to a great interest in the development of novel domain in dendrimer field.37,4142,4445 C onjugated polyelectrolyte dendrimers (CPEDs) are a new class of water soluble dendrimer and monodisperse macromolecules. The CPEDs includ e conjugated backbone at the inner part and ionic solubilizing groups at the periphery preser ving the intrinsic optical and electronic fearues of the the conjugated backbone in aqueous media.38 Also, the large number of ionic peripheries prevents inter dendrimer aggregation in polar solvent s as well as such sufficient side chains is related to the solubi lity of conjugated dendrimers Unlike ordinary nonconjugated dendrimers, the CPE Ds are structurally rigid b ecause of the fully conjugated backbone, and have globular structure with increasing generation. S uch

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150 unique characteristic s of CPEDs enable vari ous photophysical studies and provide possibilities for the applications to the solar cells, electrochromic devices, and sensors.44 The low band gap conjugated molecules exhibit many useful photophysical properties, including broad and long wavelength absorption and fluorescence.126 The absorption of conjugate dendrimer can be tuned via structural modification; one of the synthetic strategies is to introduce electron donor and acceptor units into the conjugated backbone.1,119 Also, One characteristic of dendritic molecules is the presence of numerous peripheral end groups that all converge to a single core. O ne particular design is the socalled extended dendrimer that possesses consecutively increasing conjugation length toward the center of the molecule.119 Such a structure naturally creates energy gradient from the outside branches to the inside branches. Thus, efficient energy transfer can be achieved in the donor acceptor type dendrimer. In the present Chapter, as a D A system, a series of CPE Ds having thienyl (Th) groups in the conjugated backbone was synthesized and characterized. Figure 61 shows the structures of the series of CPEDs having thienyl (Th) groups ( ThG 1 ThG 1 and ThG 3 ). Branched poly ionic side chains were substituted at the peripher ies, which provide more abundant ionic unit s with increasing the generation. Utilizing both divergent and convergent synthetic approaches allows relatively facile preparation of CPEDs. The geometric characteristic o f the series of CPEDs was studied via dynamic light scattering (DLS), and t heir photophysical properties were compared to their organic soluble precursors and also studied in methanol and aqueous solutions To the end, the quenching or energy transfer eff ect s from CPE Ds to methyl viologen or cyanine dyes

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151 were studied using UV Vis absorption, steady state fluorescence, and lifetime spectroscopy S S S O O O O O O O O O O O O R R R R R R R R R R R R S O O S O O S O O R R R R R R S S S O O R R O R R = N H O O-L i+ O-L i+ O-L i+ O O O T h G 1 T h G 2 T h G 3 Figure 61. St ructure of CPE Ds ( ThG 1 ThG 2 and ThG 3 ).

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152 Results and Discussion Synthesis and Characterization B r B r B r B r B r T M S B r T M S T M S + + T M S T M S T M S 1 3 4 5( i ) H H 5 6 H S B r T M S S S S T M S T M S T M S S S S H H H 8 9 7( i i ) ( i ) ( i i )( a ) ( b ) T M S 2 Figure 6 2. Synthesis of (a) focal points and (b) a core unit (i) Pd(PPh3)2Cl2, CuI, THF/DIPA (1/1, v/v ), 80 oC for 12 hrs.; (ii) K2CO3, MeOH/DCM (1/1, v/v) r.t. for 2 hrs Both convergent and divergent methods37 have been used in the synthesis of dendrimers. However, i t should be noted that a conventional divergent or convergent approach is restrictive in the CPE Ds synthesis because of the intrinsic poor solubility of conjugated backbone and highly congested branched polyioni c side chains Thus the combined approach of both methods should be considered in the synthesis of CPE Ds, where a divergent method was used in the core synthesis; dendrons were prepared by two stage convergent methods. As a focal point, mono( 3 ), bis ( 4 ), and tris(trimethyl silylethynyl) group substituted benzene ( 5 ) were prepared by the coupling reaction using commercially available 1,3,5tribromobenzene ( 1 ) and trimethylsilylethylene ( 2 ) in the presence of a catalytic amount of Pd(PPh3)2Cl2 and CuI (Figure 62a). The compounds

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153 I O 1 0 H N O OtB u OtB u O O tB u O O I O H N O O H O H O O H O O I O H N O O O O O O O 1 1 1 2 B r H H 4 B r O O R R 1 3 1 4 T I P S T I P S T M S T I P S T I P S H B r T I P S T I P S T I P S T I P S B r H H H H B r O O O O R R R 3 R 1 6 1 7 1 8 1 9 2 0 ( i ) ( i i ) ( i v ) ( i i i ) ( i i i ) ( i v ) ( i i i ) ( v ) ( i i i ) T h P G 1 ( i i i ) T h P G 2 ( i i i ) T h P G 3 ( i i i )( a ) ( b ) ( c )9 9 9 1 2 1 2 T I P S 1 5 1R = N H O O O O Figure 63. Synthesis of precursors of CPE Ds ((a) ThPG 1 (b) Th PG 2 and (c) Th PG 3 ) (i) TFA, DCM, r.t. for 3 hrs.; (ii) H2SO4, EtOH, 80 oC for 12 hrs.; (iii) Pd(PPh3)2Cl2, CuI, THF/DIPA (1 /1, v/v ), 80 oC for 12 hrs.; (iv) K2CO3, MeOH/DCM (1/1, v/v ) r.t. for 2 hrs.; (v) TBAF, THF, r.t for 1 hr. 3 4 and 5 were isolated using column chromatography as solids in 20~35% yield. Figure 62b illustrates the synthesis of a core having thienyl (Th) units. Trimethylsilyl groups of 5 were readily deprotected by using K2CO3 in CH3OH/H2O (1/1, v/v ) to produce 1,3,5triethynylbenzene ( 6 ). Then deactivated compound 8 was synthesized by the reaction of 6 with monosubstituted ((5 bromothiophen2 yl)ethynyl) trimethylsilane

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154 ( 7 )127 which is previously prepared by the quantitative use of ethynyltrimethylsilane Finally, core ( 9 ) was obtained by deprotection of TMS (trimethylsilyl) protected ethynyl terminal groups. O H N O O-L i+ O-L i+ O O +L i-O O T h T h T h O H N O O O O O O O T h T h T h T h P G n T h G n( i ) Figure 64. Hydrolysis of branched side chains (i) LiOH, THF/water Compound 10 was prepared in good yield as described in our previous work (see Chapter 5). To convert organic sid e chains to ionic side chains, the stability of the conjugated backbone should be considered in the final hydrolysis step. Unfortunately, thienyl units in the conjugated backbone undergo reaction under the acidic conditions used in the hydrolysis process of the tert butyl ester group. Therefore, the tert butyl ester group was modified to the n alkyl ester group which can be hydrolyzed in basic conditions. To change the tert butyl ester group to n alkyl ester group, the tert butyl ester of 10 was first hydrolyzed to carboxylic acid, and then it was converted to the ethyl ester, affording 12 (Figure 63a). As seen in Figure 63b, after deprotection of TMS group of 4 two activated ethyl terminal groups of 13 were reacted with the first dendron ( 12), which produced the second dendron ( 14) in moderate yield. As seen in Figure 63c, for the third dendron, 3 was used as an intermediary compound, which was reacted with ethynyltriisopropylsilane ( 15) to prepare 16, and then its TMS group was deprotected by K2CO3 in t he mixture of MeOH and DCM. The main framework ( 1 8 ) for

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155 the third dendron was successfully synthesized by the coupling reaction of 17 with 1,3,5tribromobenzene ( 1 ), and it s terminal TIPS protected ethynyl groups were readily activated by TBAF in THF solut ion. Then, the coupling reaction of 19 with 12 produced the third d endron ( 20). As precursor dendrimers, ThPG 1 ThPG 2 and ThPG 3 were prepared in 20 3 0% yield by the coupling reaction of dendron 12, 14, or 20 with a core unit ( 9 ) respectively Final ly, we successfully obtained water soluble ThG 1 ThG 2 and ThG 3 by hydrolysis of the ethyl ester groups of each precursor in the presence of LiOH in the solution of THF and water. T he structure of all intermediates, CPE Ds, and their precursors were characterized and confirmed by 1H NMR, 13C NMR, and Mass spectroscopy. Optical Properties UVVis absorption spectroscopy Figure 65 show s the UV Vis absorption and fluorescence spectra of CPE Ds ( ThG 1 Th G 2 and ThG 3 ) and their precursors ( ThPG 1 T h PG 2 and ThPG 3 ), and their wavelength maxima were displayed in Table 61. As seen in Figure 65, both ThPG 2 and ThPG 3 showed strong absorption at 302 nm and shoulder band at 360 nm in THF (Figure 65a). T he absorption at 302 nm increased with incr easing generation while the long wavelength absorption remains approximately constant intensity. We believe that the enhanced absorption intensity at short wavelength is attributed to exponentially increased conjugated branches with increasing generation. Also, the same number of thienyl groups in all CPE Ds induced spectral similarity in the wavelength and absorption intensity. Nevertheless, thienyl moieties of ThPG 1 showed slightly higher intensity and redshifted UV Vis absorption spectrum ( max = 364 nm).

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156 U nlike ThPG 2 and ThPG 3 the alkoxy group at the paraposition of the periphery phenyl groups is believed to the electron density on the thienyl group of the core, ( d ) Th-PG-1 Th-PG-2 Th-PG-3 ( e )Normalized Intensity (a.u.) Th-G-1 Th-G-2 Th-G-3 400 500 600 700 ( f ) Th-G-1 Th-G-2 Th-G-3Wavelength (nm) 0.0 0.2 0.4 (a) Th-PG-1 Th-PG-2 Th-PG-3 0.0 0.2 0.4 Th-G-1 Th-G-2 Th-G-3Absorbance 250 300 350 400 450 500 0.0 0.2 0.4 (c) Th-G-1 Th-G-2 Th-G-3Wavelength (nm) Figure 6 5. UVVis absorption and Fluorescence spectra of CPE Ds ( ThG 1 ThG 2 and ThG 3 ) and their precursors ( ThPG 1 ThPG 2 and ThPG 3 ) in (a, d ) THF, (b, e ) CH3OH, and (c, f ) H2O, respectively; e xcitation wavelength is 360 nm; [Th G n ] = [ ThPG n ] = 1 .0 M. inducing increased intensity and bothochromic shift in the UV Vis ab sorption of ThPG 1 The water soluble ThG n showed negligible spectral changes compared to their organic soluble precursors in the UV Vis absorption spectra (Figure 65b and 5c).

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157 Table 61. UVVis absorption and photoluminescent properties of ThG n (CH3OH and H2O (pH 8.0) ) and their precursors ( THF ) THF CH3OH H2O pH 8.0 CDs max abs (nm) max em (nm) PL a (%) CPE -Ds max abs (nm) max em (nm) PL (%) max abs (nm) max em (nm) PL (%) Th-PG -1 3 64 407 31.2 Th-G -1 360 418 23.4 359 427 2.4 Th-PG -2 302 360 394 37.9 Th-G -2 301 359 390 13.8 302 364 392 2.7 Th-PG -3 302 360 394 27.0 Th-G -3 300 3 62 390 9.6 301 3 70 4 80 1.7 a 9,10FL=0.90. Fluorescence spectroscopy Figure 65 d 5 e and 5f show the fluorescence emission spectra of ThPG n and ThG n in THF, CH3OH and H2O (pH 8.0) solutions res pectively (excitation wavelength is 360 nm). Similar to the UVVis absorption spectra, the fluorescence spectra of ThPG 2 ( max = 394 nm) and ThPG 3 ( max = 394 nm) are similar while that of ThPG 1 ( max = 407 nm) is broader and redshifted compared to the higher generation dendrimer in THF. T he similar fluorescence spectra in ThPG 2 and ThPG 3 imply that the dendrimer size does not affect to photopysical characteristics of thienyl groups However, the effect of the alkoxy substituent to the thienyl gr oups in ThPG 1 is more pronounced in the excited state, resulting in larger bathochromic shift compared to that in the UVVis apsorption spectra. S imilar to the fluorescence of the precursors ThPG n the ThG n series showed negligible fluorescence spect ral changes in CH3OH. O n the other hand, relatively significant spectral changes were observed in water solution. ThG 1 showed a slightly bathochromic shift and broader spectrum as the solvent polarity increases ( max = 418 nm/ CH3OH and 427 nm/ H2O, pH 8.0). It is noted that the more polar solvent induces more positive solvatochromi c effect, resulting in variation in position, intensity and shape of the fluorescence spectra.124125 In MeOH ThG 2

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158 exhibits the well-defined structure in t he fluorescence spectra, and a new emission band at 475 nm appeared in H2O (pH 8.0). For Th-G-3 a similar emission band at 480 nm was observed in MeOH, and it became significant in H2O (pH 8.0). It is proposed that such strong fluorescence at lower energy is ascribed to the intr a-dendrimer aggregation between thienyl groups and phenylethynylene mo ieties in the excited state. This phenomenon was also found in our previous wo rk for compact type CPE-Ds, in which the phenylethynylene units were intra-molecularly aggregated in the excited state, and their aggregation effect was more signific ant at higher generation (see Chapter 5). Fluorescence quantum yield The fluorescence quantum yiel ds of the precursors ( Th-PG-n ) and Th-G-n were obtained in THF, CH3OH, and H2O (pH 8.0) solutions (Table 6-1). The excitation wavelength was 360 nm which corresponds to the thienyl unit. The quantum yields of Th-G-n in H2O are similar and significantly lowered (approximately 5~10 folds) compared to CH3OH. This decrease of the fluoresc ence quantum yield is attributed to the solvent effect rather than the aggregate formation. Fluorescence lifetimes Table 6-2 and Figure 6-6 show the fluor escence lifetimes and their fractional amplitude changes of Th-PG-n (precursors) in THF and Th-G-n in CH3OH, and H2O (pH 8.0), respectively. The excitation wa velength is 370 nm and emission decays of ThPG-n were monitored over 380 nm. Th-G-n s decays were obtained at 400, 450, and 500 nm where the data were collected by global fitting algorithm. Relatively fast decays were observed for Th-PG-1 ( = 0.36 ns), Th-PG-2 ( = 0.30 ns), and Th-PG-3 ( = 0.39 ns), and these are predominant (>94%) and independent from dendrimer size. For this observation, we propose that a single molecular species is predominant in both ground

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159 Table 6 2 Fluorescence lifetimes (i, ns) and relative amplitudes (RA, %) for ThPG n and ThG n in THF, CH3OH, and H2O (pH = 8 .0) solutions.a aData were collected by global fitting Algorithm bT ypical limits of error on i are less than 3%. T HF CH3OH H2O, pH 8.0 RA (%) RA (%) RA (%) Th-PG -1 i (ns) b >380 nm Th-G -1 i (ns) 400 nm 4 5 0 nm 500 nm i (ns) 400 nm 4 5 0 nm 500 nm 1 = 0. 36 100 1 = 0. 39 100 >99 99 1 = 0. 37 >99 >99 93 2 = 1.30 0 <1 1 2 = 1. 57 <1 <1 7 2 1. 06 2 2 1.010 0.953 1.031 2 1.067 1. 009 0.995 Th-PG -2 i (ns) >380 nm Th-G -2 i (ns) 400 nm 4 5 0 nm 500 nm i (ns) 400 nm 4 5 0 nm 500 nm 1 = 0.30 28 1 = 0.32 99 91 6 3 1 = 0.32 13 24 33 2 = 0. 66 6 2 = 0.98 1 8 30 2 = 1.04 86 74 60 3 = 3.06 0 1 7 3 = 3.61 <1 2 7 2 0.968 2 1.160 1. 162 1.084 2 1.035 1.24 1 1.074 Th-PG -3 i (ns) >380 nm Th-G -3 i (ns) 400 nm 4 5 0 nm 500 nm i (ns) 400 nm 4 5 0 nm 500 nm 1 = 0.39 68 1 = 0. 32 99 82 42 1 = 0. 44 66 47 32 2 = 0.78 5 2 = 1.19 <1 15 39 2 = 1.65 27 48 56 3 = 3.64 <1 3 19 3 = 5.35 6 5 12 2 1. 029 2 1.167 0.973 0.921 2 1.123 1.172 0.979

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160 and excited states. S uch simplicity in the emission decays implies absence of inter or intra dendrimer aggregation of ThPG n i n THF. Unlike ThPG n ThG n showed two or three emission decays in CH3OH and H2O (pH 8.0) solutions (Table 62). The emission decay of ThG 1 features two pathways ( 1 = 0.39 ns, 2 = 1. 30 ns in CH3OH and 1 = 0.37 ns, 2 = 1. 57 ns in H2O pH 8.0). One faster component is predominant in both solutions ( 1 at = 400 nm > 99% in CH3OH and 1 at = 400 nm > 98%, in H2O pH8.0). T he amplitude of 2 = 1. 57 ns in H2O (pH 8.0) showed slightly increased aspect at 500 nm ( 2 > 6%) (Figure 66d). We suggest that fast decay component is a similar state with short lived component as seen in its precursor in THF In CH3OH the similar results are obtained for ThG 2 and ThG 3 where the fast decays are predominant ( 1 Th G 2 = 0.32 ns > 99% and 1 Th G 3 = 0.32 ns > 9 9%) at 400 nm. These observations imply that both dendrimer states are very similar to their precursors in THF. These fast components showed decreased contribution at 500 nm, where the amplitudes of ThG 2 and ThG 3 are 63% and 42%, respectively (Figure 66b and 6c) On the other hand, relatively slow decays ( 2 Th G 2 = 0.98 ns and 3 Th G 2 = 3.06 ns ; 2 Th G 3 = 1.19 ns and 3 Th G 3 = 3.64 ns) showed increased amplitudes. Also, it is found that such decrease or increase of the fast or slow decays becomes more significant with increasing generation. Based on above observation in CH3OH we propose that ThG 2 and ThG 3 retain the characteristics of their precursors. H owever, small fractions of intra dendrimer aggregates exist, but these are only detectable at longer wavelength.

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161 400 450 500 0 20 40 60 80 100 120 Th-G-1Fractional Amplitudes (%)Wavelength (nm) = 0.39 = 1.30(a) 400 450 500 0 20 40 60 80 100 120 ( d )Fractional Amplitudes (%)Wavelength (nm) = 0.37 = 1.57Th-G-1 400 450 500 0 20 40 60 80 100 120 Th-G-2 (b) = 0.32 = 0.98 = 3.06Wavelength (nm)Fractional Amplitudes (%) 400 450 500 0 20 40 60 80 100 120 Th-G-2 ( b' ) = 0.32 = 1.04 = 3.61Wavelength (nm)Fractional Amplitudes (%) 400 450 500 0 20 40 60 80 100 120 Th-G-3 (c) = 0.32 = 1.19 = 3.64Wavelength (nm)Fractional Amplitudes (%) 400 450 500 0 20 40 60 80 100 120 Th-G-3 ( c' ) = 0.44 = 1.65 = 5.35Wavelength (nm)Fractional Amplitudes (%) H2O CH3OH Figure 66. Fractional amplitude changes of fluorescence lifetimes of ThG 1 in (a) CH3OH and (d) H2O ThG 2 in (b) CH3OH and (e) H2O and ThG 3 in (c) CH3OH and (f) H2O ; excitation wavelength is 370 nm and life time decays were monitored at 400, 450, and 500 nm; D ata were collected by global fitting a lgorithm More contributions of the decays at 400 nm corresponding to the slow component of ThG 1 were observed in ThG 2 ( 2 = 1.04 ns > 86%) in H2O while the fast ( 1 = 0.32

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162 ns) and longlived decay ( 3 = 3.61 ns) showed relatively lower contributions (Figure 66e) At 500 nm, however, the contribution of 2 = 1.04 ns decrease to around 60% and 32% of the fast decay ( 1 = 0.32 ns) was observed with 7% new longlived decay ( 1 = 3.61 ns). The moderate increase of both a fast decay and a sl ow decay is ascribed to the slight intradendrimer aggregation, which also induced decrease of the component of 2 = 1.04 ns For ThG 3 although the amplitude of 2 = 1.65 ns is relatively small ( 2 > 27%) at 400 nm, that of the fast component ( 1 = 0.44 ns ) decreased to around 66%. As seen in Figue 65 f the emission of ThG 3 at 400 nm in H2O is fully quenched because of intra dendrimer aggregation. It is noted that energy transfer or quenching lead to changes on the emission decay times.53,73 I n this case of ThG 3 in H2O, the relatively large contribution of fast decay ( 1 = 0.44 ns > 66% ) at 400 nm is possibly due to two issues: first, the electron or energy trap caused by intradendrimer aggregation lead to fast decay ; second, the direct contact of thienyl moieties with H2O caused nonradiative decay On the other hand, a t 500 nm, the fast component ( 1 = 0.44 ns) of Th G 3 showed decreased contribution (32 %), and the component of 2 = 1.65 ns and 3 = 5.35 ns increased to around 56% and 13%, respectively. ThG 3 shows strong exciplex type fluorescence for intra dendrimer aggregated state at 500 nm A s a result, the increased contribution of fast decay ( 1 = 0.44 ns) and longlived component ( 3 = 5.35) ns at 500 nm is due to the aggregation effect. Also, it is possible that structure changes induced by the intra dendrimer aggregation give rise to the increase of contribution of the decay ( 2 = 1.65 ns).

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163 Chromophore/Dendrimer Aggregation C oncentration dependent fluorescence 400 450 500 550 600 650 700 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 (a)7 MWavelength (nm)Normalized Intensity 0 M [Th-G-1] 400 450 500 550 600 650 700 0.0 0.5 1.0 1.5 2.0 2.5 3.0 (b)10 MWavelength (nm)Normalized Intensity 0 M [Th-G-2] 400 450 500 550 600 650 700 0.0 0.5 1.0 1.5 2.0 (c)8 MWavelength (nm)Normalized Intensity 0 M [Th-G-3] Figure 67 Fluorescence emission spectra of (a) ThG 1 (b) ThG 2 and (c) ThG 3 with increasing concentration in H2O; [ThG n ] = 1.0 M. A ggregation of ThG n in aqueous solution is further studied by the concentration dependent fluorescence and excitation spectral changes. As the concentration of ThG n increases, the fluorescence spectra ( ex = 360 nm) exhibit no change in the wavelength and bandshape in H2O (pH 8.0) (Figure 67 ). This observation suggests that inter dendrimer aggregation does not occur in ThG n Fluorescence excitation spectroscopy 250 300 350 400 450 500 0.0 0.5 1.0 1.5 2.0 Normalized Intensity Wavelength (nm) em = 400 nm em = 500 nm(a) 250 300 350 400 450 500 0.0 0.5 1.0 1.5 2.0 (b)Normalized Intensity Wavelength (nm) em = 400 nm em = 500 nm 250 300 350 400 450 500 0.0 0.5 1.0 1.5 2.0 (c)Normalized Intensity Wavelength (nm) em = 450 nm em = 500 nm Figure 6 8 Fluorescence excitation spectra of (a) ThG 1 at 400 and 500 nm, (b) ThG 2 at 400 and 5 00 nm, and (c) ThG 3 at 450 and 500 nm; [ ThG n ] = 1.0 M in H2O. As seen in Figure 68 the excitation spectra ( em = 400, 450 or 500 nm) of all ThG n do not display any changes in the excitation s pectra. Although Th G 1 showed

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164 slightly broader fluorescence spectra in water solution, the wavelength independent excitation spectra and constant fluorescence spectra as a function of concentration suggest that it does not aggregate. In addition, excitati on spectral result s of ThG 2 and ThG 3 illustrate that their inter dendrimer aggregation is negligible. Nevertheless, aggregation seen in fluorescence spectra is attributed to the excited state intra dendrimer aggregation. For ThG 1 i t is possible that its inter dendrimer interaction can be fairly minimized by the geometrical peculiarity of the thienyl group. Dynamic l ight s cattering (DLS) 0 2 4 6 8 10 0 10 20 30 40 50 ChannelSize (nm)(a) 0 2 4 6 8 10 0 10 20 30 40 50 (c)ChannelSize (nm) 0 2 4 6 8 10 0 10 20 30 40 50 (b)ChannelSize (nm) Figure 69 Hydrodynamic radii obtained from dynamic light scattering (DLS) for (a) ThG 1 (b) Th G 2 and (c) T h G 3 in H2O; [ G n ] = 1.0 M. Dynamic light scattering (DLS) provide further evidence for the state of aggregation of ThG n in H2O (pH 8 .0 ). Figure 69 shows the distribution profiles of the populations for hydrodynamic radius of ThG n in aqueous soluti on. DLS on Th G 1 ThG 2 and ThG 3 shows 3.42, 3.45, and 2.86 nm as an average size, respectively. Their average sizes are relatively larger than compact type G n which is probably ascribed to the extended size of inner part by incorporating thienyl gr oups into the conjugated backbone. The physical size increases with the generation but smaller size of ThG 3 than the other two dendrimers was observed. It is possible that more hydrophobic conjugated backbones induce stronger contraction. In fact, a stro ng

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165 correlation was established between the solvent polarity and the mean radius of gyration.44,128 For example, the simulations with branched polyelectrolytes showed a drastic contraction as the ionic strength of th e solvent increases.129 Also, unlike compact type G 1 in aqueous solution, DLS result showed narrow and regular size of hydrodynamic radius for ThG 1 This implies the absence of aggregation of ThG 1 in aqueous solution. For ThG 3 although its distribution profile is very broad compared to ThG 1 and ThG 2 the maximum size does not exceed around 5.8 in aqueous solution. Thus, it is conclude d that ground state inter dendrimer aggregation of ThG n does not occur but int ra dendrimer interaction is more pronounced with increasing generation in more polar solvent. Fluorescence Quenching of Th G n with MV2+ 0 5 10 15 20 0 2 4 6 8 10 ( a ) Th-G-1 Th-G-2 Th-G-3I0/I[MV2+] ( M ) 0 5 10 15 20 0 2 4 6 8 10 ( b )I0/I Th-G-1 Th-G-2 Th-G-3[MV2+] ( M ) Figure 610. Stern Volmer plots of ThG n ( a) CH3OH and (b) H2O; fluorescence was quenched by methyl viologen (MV2+) ; ThG 1 ( ),Th G 2 ( ), and ThG 3 ( ) ; [ ThG n ] = 1 .0 M Table 63. Ksv a of ThG n with methyl viologen (MV2+) in CH3OH and H2O CH3OH H2O CPE -Ds Ksv/M-1 Ksv/M-1 Th-G -1 2.4 104 1.4 105 Th-G -2 7.8 105 6.1 105 Th G 3 4.0 10 5 7.3 10 6 a Computed from linear fit at low quencher concentration (0~ 2.5 M).

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166 The fluorescence quenching of ThG n was investigated with methyl viologen (MV2+) in CH3OH and H2O solutions Their SternVolmer (SV) plots and KSV values present in Figure 610 and Table 63 respectively. The SV plots were obtained at the wavelength of maximum fluorescence intensity. As seen in Figure 610, the typical SV plots of ThG n were observed: at very low quencher concentration (< 2.5 M ) SV plot is linear and it becomes upward curved at high concentration (> 5.0 M ) In CH3OH ThG 1 showed much less efficient quenching with a low Ksv value ( Ksv = 2. 4 104 M1) while very similar quenching features w ere observed in ThG 2 ( Ksv = 7.8 105 M1) and ThG 3 ( Ksv = 4.0 105 M1) Based on the similarity in the results of both fluorescence and lifetime decays, we suggest that the quenching effect in ThG 2 and ThG 3 is not related to the dendrimer size. Interestingly, the fluorescence quenching efficiency increased with the generation ( ThG 1 < ThG 2 < Th G 3 ) in H2O (Figure 610b), and the most efficient quenching was observed in ThG 3 ( Ksv = 7.3 106 M1). The Ksv value ( Ksv = 1.4 105 M1) of ThG 1 in H2O showed slightly higher value than that in CH3OH and similar Ksv values ( Ksv = 7.8 105 M1/H2O and Ksv = 6.1 105 M1) of ThG 2 were observed in CH3OH and H2O solutions. T he highest Ksv value of ThG 3 is ascribed to int ra dendrimer aggregati on effect, which is supported by the fact that the quenching is amplified in aggregate states by effective energy or exciton migration between chromophores.1,68 We also propose that static quenching is dominant in a ll processes based on the results of SV quenching.

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167 FRET from ThG 3 to Cyanine Dyes N O O N C H2C H3 C H2C H3 I n n = 1 : D O C n = 2 : D O D C n = 3 : D O T C Figure 6 1 1 Structure of cyanine dyes (DOC, DODC, and DOTC). 300 400 500 600 700 800 0.0 0.2 0.4 0.6 0.8 1.0 1.2 FL of Th-G-3 Abs of DOC Abs of DODC Abs of DOTCIntensity (a.u.)Wavelength (nm) Figure 612. Fluorescence of FRET donor ( ThG 3 ) and absorption of FRET acceptors (cyanine dyes). Fluorescence quenching of ThG 3 with cyanine dyes (DOC, DODC, and DOTC) was studied in H2O (excitation wavelength is 360 nm). The structures of DOC, DODC, and DOTC are present ed in Figure 611. These cyani ne dyes are different in conjugation length, showing the variation of the absorption (Figure 612). Figure 613 shows the overall fluorescence intensity changes upon the addition of DOC, DODC, and DOTC. As seen in Figure 613, all cyanine dyes are nearly nonfluorescent in 0.2~ 0.3

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168 350 400 450 500 550 600 650 700 0 1 2 3 4 Intensity (a.u.)Wavelength (nm) 0.3 M DOC (b) 400 500 600 700 800 0 1 2 3 4 (c)Intensity (a.u.)Wavelength (nm) 0.2 M DODC 400 500 600 700 800 0 1 2 3 4 (d)Intensity (a.u.)Wavelength (nm) 0.2 M DODC 400 500 600 700 800 0 1 2 3 4 (a)DOTC = 0.2 M DOC = 0.3 M DODC = 0.2 M Wavelength (nm)Intensity (a.u.) Figure 6 13 Fluorescence emission spectra of (a) cyanine dyes only (DOC, DODC, and DOTC) and ThG 3 titrated with (b) DOC, (c) DODC, and (d) DOTC in H2O pH 8 ; [ThG 3 ] = 1 .0 M; [dye quencher] = 0 ~ 0.3 M ; excitation wavelength is 360 nm. M concentration range. The fluorescence intensity of ThG 3 was efficiently quenched, which was concomitant with a significant fluorescence enhancement of cyanine dyes via fluorescence energy transfer Figure 61 2 shows the spectral overlap between donor fluorescence ( ThG 3 ) and acceptor absorption (DOC, DODC, or DOTC), in which the degree of overlap is in the order DOC>DODC> DOTC. The more overlap between donor fluorescence and acceptor absorption gives rise to efficient energy transfer fro m ThG 3 to cyanine dyes.

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169 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0 3 6 9 12 DOC DODC DOTCI0/I[Cyanine dyes] ( M ) Figure 614. Stern Volmer plots of ThG 3 in H2O; fluorescence quenched by cyanine dyes ; DOC ( ), DODC ( ), and DOTC ( ) ; [ ThG 3 ] = 1 .0 M Table 64 Ksv a of CPEDs with cyanine dyes in H2O Ksv/M-1 (H2O, pH 8) CPE -D DOC DODC DOTC Th-G -3 1.43 107 1.39 107 1.35 107 a Computed from linear fit at low quencher concentration. Interestingly, the similar the quenching efficiency of ThG 3 for all cyanine dyes was observed. As seen in Figure 61 4 and Table 64 the Stern Volmer (SV) plots of ThG 3 are very similar and their Ksv values are the same ( Ksv 1.35 107). We could not clearly explain the interesting results However, one possible reason is that the charge transfer effect participates in their quenching and its efficiency is compatible with energy transfer effect. Thus, although the energy transfer effect from ThG 3 to DOTC is less

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170 effective than other cyanine dyes, the further fluorescence can be quenched by charge transfer effect S ummary and Conclusions In this Chapter a series of CPEDs having thienyl (Th) groups in the conjugated backbone was systemically prepared via precursor route. In CH3OH, all ThG 1 Th G 2 and ThG 3 showed welldefined UV Vis absorption and fluorescence spectra, implying nonaggregated states in CH3OH On the other hand, the UV Vis absorption and fluorescence spectra were redshifted in H2O, which was more significant with increasing generation. It was proposed that such redshift with generation is due t o intra dendrimer aggregation. Also, fluorescence lifetime decays provide the information for the intra dendrimer aggregation of ThG n in H2O. Methyl viologen and cyanine dyes (DOC, DODC, and DOTC) efficiently quenched the fluorescence of ThG 3 rather than the other generation in H2O, which is due to more efficient energy or charge transfer effect in aggregated state. The similar quenching results for all cyanine dyes illustrated that both energy and charge transfer effects complementarily participate in entire quenching mechanism. Experimental Materials All chemicals used for the synthesis were of reagent grade and used without further purification. Nitromethane, tertbutylacrylate, T1 Raney nickel, diisopropylamine, chloroacetyl chloride, 4 iodophenol, 1,3,5tribromobenzene, 2,5dibromothiophene, and triphenylphosphine were purchased from SigmaAldrich Chemical Company. Sodium carbonate, and copper iodide were obtained from Acros Chemical Company. trans Dichlorobis(triphenylphosphine)palladium (II) was purchased from Sterm Chemical

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171 Company. Ethynyltrimethylsilane and ethynyltriisopropylsilane w ere bought from GFC Chemical Company. Potassium carbonate, ethanol, sulfuric acid, and trifluoroacetic acid were obtained from Fisher Scientific Company. THF was p urified by Solvent Dispensing System (SDS). Silica gel (Merck, 230 400 mesh) was used for chromatographic purification of all of intermediate and target molecules. All other chemicals and solvents were purchased from either SigmaAldrich or Acros Chemical Company and used as received. Instrumentation and Methods NMR spectra were recorded using a Varian VXR 300 FT NMR, operating at 300 MHz for 1H NMR and at 75 MHz for 13C NMR. Gel permeation chromatography (GPC) analyses were carried out on a system comprised of a Rainin Dynamax SD 200 pump, Polymer Laboratories PL gel mixed D columns, and a Beckman Instruments Spectroflow 757 absorbance detector. Molecular weight calibration was effected by using polystyrene standards. UV/Vis absorption spectra were recorded using a Varian Cary 50 Spectrophotometer. Steady state fluorescence spectra were obtained with a PTI fluor o meter Lifetime measurements were carried out using a PicoQuant FluoTime 100 Compact Fluorescence Lifetime Spectrometer. A 1 cm quartz cuvette was used for all spectral measurements. Dynamic light scattering (DLS) experiments were performed with Zeta PALS from Brookhaven Instrument Corporation. Stock solutions (1.0 mM) of all of the CPE Ds were prepared in H2O and have been stored at 0 oC. The soluti ons have been kept at the room temperature for one hour before use. Fluorescence quantum yield are reported relative to known standards (9,10-

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172 diphenyl anthracene, = 0.90 in EtOH). The pH of aqueous solution was adjusted with HCl and/or NaOH using a C orni ng pH meter 320. Synthetic Procedures Compounds 7 and 10 w ere prepared in a good yield as described in literature and Chapter 5, respectively 4 (2 carboxyethyl) 4 (2 (4 iodophenoxy)acetamido)heptanedioic acid (11). To a sloution of 1 0 ( 5 0.0 g, 74.0mmol) in 500 mL of CH2Cl2, 200 mL of trifluoroacetic acid (TFA) was added dropwise. Upon completion of the addition, the reaction mixture was stirred at room temperature for 3 hours. excess TFA and the solvent were removed in vacuo. The crude product was used in the next esterification reaction without further purification. Yield: 98%. Dendron 1 (12). To a solution of compound 11 ( 30.0 g, 59.1 mmol) in EtOH ( 300 mL), 5 mL of H2SO4 was added. The mixture was vigorously stirred at 80 oC for 12 hours under argon gas After the reaction mixture was cooled to room temperature, the solvent was removed in vacuo. The reaction mixture was washed with water (200 mL), and then extracted with CH2Cl2 (200 mL). The organic layer was separated and dried over anhydrous MgSO4, and the solvent was evaporated to yield a white solid. The crude product was isolated by column chromatography on silica gel using ethyl acetate:hexane (1:2) as the eluent. 8 6 % yield; mp 120122 oC; 1H NMR (300 MHz, CDCl3, ppm): 7.42 (d, 2H, IArHortho, J= 8. 9 Hz), 6.8 5 (d, 2H, IAr Hmeta, J= 8.9 Hz), 6.5 0 (s, 1H, CONH ), 4.3 7 (s, 2H, ArOC H2CO2), 4.10 (q, 6H, CO2C H2CH3) 2.25 (m, 6H, NHC(CH2C H2CO2 tEt )3), 2.0 5 (m, 6H, NHC(C H2CH2CO2 tEt )3), 1. 23 ( t 9 H, CO2CH2C H3)

PAGE 173

173 General coupling reaction procedure. T erminal alky nes and aryl halides were dissolved in 20 mL of THF/ DIPA (1/3, v/v ). The resulting solution was deoxygenated with argon for 1 hour Then Pd( PPh3)2Cl2 ( 2 mol% ) and CuI (1.9 mg, 10.0 mol) were added to the stirred solution under the protection of argon. The reaction mixture was then heated up to 70 ~ 75 C and stirred for 12 hours. The reaction mixture was cooled down to the room temperature and t he solvent was removed in vacuo. The crude product was isolated by column chromatography on silica gel using methylene chloride:acetone as the eluent. M ono, di or tri substitution of trimethylsilylethynyl group onto 1,3,5tribromobenzene. 1,3,5tribromobenzene ( 1 ) (30.0 g, 95.3 mmol) and two equivalents of ethynyltrimethylsilane ( 2 ) ( 18.7 g, 190.6 mmol) were used as an aryl halide and terminal alkynes, respectively. The mixture of mono, di or tri trimethylsilylethynyl substituted benzenes w as separated by column chromatography on silica gel using ethyl acetate:hexane as the eluent. ((3,5 dibromophenyl)ethynyl ) trimethylsilane ( 3 ) : Yield: 22%; mp 105106 oC; 1H NMR (300 MHz, CDCl3, ppm): 7.5 6 (s, 1 H, TMS C2Ar Hpara), 7.5 3 (s, 2 H, TMS C2Ar Hortho), 0.2 5 (s, 9 H, (C H3)3Si) (5 bromo1,3phenylene)bis(ethyne2,1diyl)bis(trimethylsilane) (4) : Yield: 35%; mp 125 126 oC; 1H NMR (300 MHz, CDCl3, ppm): 7.5 4 (s, 2 H, Br Ar Hortho), 7.5 3 (s, 1 H, Br Ar Hpara), 0.2 5 (s, 18H, (C H3)3Si) 1,3,5tris((trimethylsilyl)ethynyl)benzene ( 5 ) : Yield: 31%; mp 129 130 oC; 1H NMR (300 MHz, CDCl3, ppm): 7.48 (s, 3 H, benzeneH ), 0.25 (s, 27H, (C H3)3Si) ; 13C NMR (75 MHz, CDCl3, ppm): 135.0, 124.2, 105.5, 92.5, 11.5.

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174 1,3,5tris((5 ((trimethylsilyl)ethynyl)thiophen 2 yl)ethynyl)benzene (8) : ((5 bromothiophen2 yl)ethynyl)trimethylsilane ( 7 ) ( 11.4 g, 43.9 mmol) and 1,3,5triethynylbenzene ( 6 ) ( 2.0 g, 13.3 mmol) were used as an aryl halide and terminal alkynes, respectively. Yield: 54%; mp 132133 oC; 1H NMR (300 MHz, CDCl3, ppm): 7. 61 (s, 3 H, benzeneH ), 7.12 (s, 6 H, thiophene H ), 0.2 6 (s, 27H, (C H3)3Si) ; 13C NMR (75 MHz, CDCl3, ppm): 134.1, 132.2, 132.1, 125.6, 123.9, 123.8, 100.5, 96.8, 92.0, 84.0, 0.1. Dendron 2 (14) : Dendron 1 ( 12) ( 6.4 g, 10,7 mmol) and 1 bromo3,5 diethynylbenzene ( 13) ( 2.0 g, 4.9 mmol) were used as an aryl halide and terminal alkynes, respectively. Yield: 45%; mp 1 45146 oC ;1H NMR (300 MHz, CDCl3, ppm): 7.60 (s, 2 H, Br Ar Hortho), 7.5 8 (s, 1 H, Br Ar Hpara), 7.50 (d, 4H, CH2OAr Hmeta, J=9. 0 Hz), 6.98 (d, 4H, CH2OAr Hortho, J=9. 0 Hz), 6.5 8 (s, 2H, CON H ), 4.42 (s, 4H, ArOC H2CO2), 4.12 (q, 12 H, CO2C H2CH3) 2. 3 (m, 12H NHC(CH2C H2CO2 tEt )3), 2.10 (m, 12H, NHC(C H2CH2CO2 tEt )3), 1. 23 ( t 18 H, CO2CH2C H3) ; 13C NMR (75 MHz, CDCl3, ppm): 173.5 167.3, 157.8, 134.0,133.8, 133.2, 125.9, 122.1, 116.3, 114.9, 90.8, 86.6, 67.5, 61.0, 57.9, 31.0, 30.0, 28.5, 14.2. (5 ((trimethylsilyl)ethynyl)1,3phenylene)bis(ethyne2,1diyl)bis(triisopropylsilane) (16) : ((3,5 dibromophenyl)ethynyl)trimethylsilane ( 3 ) ( 10.0 g, 30.1 mmol) and ethynyltriisopropylsilane ( 15) ( 16.5 g, 90.3 mmol) were used as an aryl halide and terminal alkynes, respectiv ely. The crude product was used in the next deprotection reaction without further purification. Yield: 72% ( 5,5' (5 bromo1,3phenylene)bis(ethyne 2,1diyl)bis(benzene 5,3,1triyl))tetrakis (ethyne2,1diyl)tetrakis(triisopropylsilane) (18) : 1,3,5-

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175 tribromobenzene (1) ( 5.0 g, 15.9 mmol) and (5 ethynyl 1,3phenylene)bis(ethyne2,1diyl)bis(triisopropylsilane) ( 17) ( 16.2 g, 34.9 mmol) were used as an aryl halide and terminal alkynes, respectively. The crude product was used in the next deprotection reaction w ithout further purification. Dendron 3 (20) : Dendron 1 ( 12) ( 5.7 g, 9.7 mmol) and 5,5'(5 bromo 1,3phenylene)bis(ethyne2,1diyl)bis(1,3 diethynylbenzene) ( 19) ( 1 .0 g, 2.2 mmol) were used as an aryl halide and terminal alkynes, respectively. Yield: 32%; mp 1651 66 oC; 1H NMR (300 MHz, CDCl3, ppm): 7.6 4 (m, 9H, phenyl H ), 7.5 2 (d, 8H, CH2OAr Hmeta, J=9. 1 Hz), 6.9 6 (d, 8H, CH2OAr Hortho, J=9.0 Hz), 6.58 (s, 4H, CON H ), 4.42 (s, 8H, ArOC H2CO2), 4.12 (q, 24H, CO2C H2CH3) 2. 3 0 (m, 24H, NHC(CH2C H2CO2 tEt )3), 2. 1 0 (m, 24H, NHC(CH2CH2CO2 tEt )3), 1. 23 ( t 36H, CO2CH2C H3) ; 13C NMR (75 MHz, CDCl3, ppm):173.0, 167.5, 157.8, 133.7, 124.4, 116.2, 114.9, 90.2, 87.3, 67.5, 61.0, 57.9, 30.0, 28.6, 14.3. ThPG 1 : Dendron 1 ( 12) ( 2.1 g, 3.3 mmol) and 1,3,5tris((5 ethynylthi ophen2 yl)ethynyl)benzene ( 9 ) ( 0.5 g, 1.1 mmol) were used as an aryl halide and terminal alkynes, respectively. Yield: 40%; mp 169170 oC; 1H NMR (300 MHz, CDCl3ppm): 7.61 (s, 3H, phenyl H ), 7.5 2 (d, 6H, CH2OAr Hmeta, J=9.0 Hz), 7.17 (dd, 6H, thiopheneH ), 6.95 (d, 6H, CH2OAr Hortho, J=9.0 Hz), 6.5 6 (s, 3H, CON H ), 4.40 (s, 6H, ArOC H2CO2), 4.12 (q, 1 8 H, CO2C H2CH3) 2. 3 0 (m, 18H, NHC(CH2C H2CO2 tEt )3), 2.12 (m, 18H, NHC(C H2CH2CO2 tEt )3), 1. 23 ( t 27 H, CO2CH2C H3) ; 13C NMR (75 MHz, CDCl3, ppm): 173.4, 167.3, 159.3, 157.6, 133.5, 114.4, 114.5, 67.5, 61.2, 58.1, 29.8, 28.7, 14.1; MALDI MS ( m/z ): M+ calcd for C102H111N3O24S3, 1858 68; found, 18 5 7.84.

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176 ThPG 2 : Dendron 2 ( 14) ( 1.6 g, 1.4 mmol) and 1,3,5tris((5 ethynylthiophen2 yl)ethynyl)benzene ( 9 ) ( 0.2 g, 0.4 mmol) were used as an aryl halide and terminal alkynes, respectively. Yield: 27%; mp 18 2 1 8 4 oC; 1H NMR (300 MHz, CDCl3ppm): 7.66 (s, 3H, phenyl H ), 7.6 2 (m, 9H, phenyl H ) 7.5 1 (d, 12H, CH2OAr Hmeta, J=9.0 Hz), 6.9 0 (d, 12H, CH2OAr Hortho, J=9.0 Hz), 6 .5 6 (s, 6H, CON H ), 4.4 0 (s, 12H, ArOC H2CO2), 4.12 (q, 36 H, CO2C H2CH3) 2. 3 0 (m, 36H, NHC(CH2C H2CO2 tEt )3), 2.11 (m, 36H, NHC(C H2CH2CO2 tEt )3), 1. 2 4 ( t 64 H, CO2CH2C H3) ; 13C NMR (75 MHz, CDCl3, ppm): 173.5, 167.8, 159.5, 157.7, 133.9, 114.8, 114.7, 67.5, 61.0, 58.0, 30.0, 28.7, 14.2; MALDI MS ( m/z ): M+ calcd for C204H222N6O48S3, 3621 43; found, 362 2.52. ThPG 3 : Dendron 3 ( 20) ( 3.3 g, 1.4 mmol) and 1,3,5tris((5 ethynylthiophen2 yl)ethynyl)benzene ( 9 ) ( 0.2 g, 0.4 mmol) were used as an aryl halide and termi nal alkynes, respectively. Yield: 24%; mp 189190 oC; 1H NMR (300 MHz, CDCl3, ppm): 7.80 (s, 3H, phenyl H ), 7.72 (s, 9H, phenyl H ), 7.6 5 (s, 18H, phenyl H ), 7.50 (d, 24H, CH2OAr Hmeta, J=9.0 Hz), 6. 96 (d, 24H, CH2OAr Hortho, J=9.0 Hz), 6.50 (s, 12H, CON H ), 4.39 (s, 24H, ArOC H2CO2), 4.15 (q, 72 H, CO2C H2CH3) 2. 31 (m, 72H, NHC(CH2C H2CO2 tEt )3), 2.11 (m, 72H, NHC(CH2CH2CO2 tEt )3), 1. 2 4 ( t 128H, CO2CH2C H3) ; 13C NMR (75 MHz, CDCl 3, ppm): 173.8, 167.9, 160.0, 158.2, 134.5, 115.2, 115.0, 105.4, 100.2, 66.5, 61.3, 58.4, 30.6, 28.9, 14.1; MALDI MS ( m/z ): M+ calcd for C408H444N12O96S3, 7146 95; found, 7145.89. General deprotection process of trimethylsilyl group. A mixture of TMS protected compounds ( 3 4 8 or 16) and K2CO3 (5.0 equiv.) in a solution mixture of C H2Cl2 (20 mL) and CH3OH (20 mL) was stirred at room temperature for 2 hours. The mixutre was washed with water (20 mL 3), and organic layer was separated. I t was

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177 dried over anhydrous magnesium sulfate, and then the solvent was removed in vacuo. The product was used in the coupling reaction without further purification. 80 ~ 90% yield. 1,3,5triethynylbenzene (6) : Yield: 90%; mp 127128 oC; 1H NMR (300 MHz, CDCl3, ppm): 7. 4 5 (s, 3 H, benzeneH ), 3.76 (s, 3H, Ar C2H ). 1,3,5tris((5 ethynylthiophen2 yl)ethynyl)benzene (9) : Yield: 85%; mp 132 1 33 oC; 1H NMR (300 MHz, CDCl3, ppm): 7.6 2 (s, 3 H, benzeneH ), 7.1 5 ( dd, 6 H, thiophene H ), 3.41 (s, 3 H, thipheneC2 H ) ; 13C NMR (75 MHz, CDCl3, ppm): 134.0, 133.5, 132.6, 124.1, 124.0, 123.8, 92.2, 83.8, 83.0, 76.3. 1 bromo3,5diethynylbenzene (13) : Yield: 93%; mp 1 401 41 oC; 1H NMR (300 MHz, CDCl3, ppm): 7.5 5 (s, 2 H, Br Ar Hortho), 7.5 3 (s, 1 H, Br Ar Hpara), 3. 05 (s, 2H, ArC2H ). (5 ethynyl 1,3phenylene)bis(ethyne2,1diyl)bis(triisopropylsilane) (17) : Yield: 8 7 %; mp 1461 47 oC; 1H NMR (300 MHz, CDCl3, ppm): 7.52 (s, 3H, benzeneH ), 3.05 (s, 1H, Ar C2H ), 1.10 ( m, 42H, SiC H (C H3)2). 5,5' (5 bromo 1,3phenylene)bis(ethyne2,1 diyl)bis(1,3 diethynylbenzene) (19). TIPS protected compound ( 18) ( 5.0 g, 4.6 mmol) wa s dissolved in THF, and tetra butylammonium fluoride ( 4.6 mL 4.6 mmol) was added dropwise. Upon the completion of the addition, the reaction mixture stirred for another 30 minutes, and then the solvent was removed in vacuo T he crude product was isolated by flash column chromatography on silica gel using THF as the eluent. Yield: 89%; mp 1861 87 oC; 1H NMR (300 MHz, THF d8, ppm): 7.76 (s, 2H, Br Ar Hortho), 7.70, (s, 1H, BrAr Hpara), 7.64

PAGE 178

178 (s, 4H, BrAr C2Ar Hortho), (s, 2H, BrAr C2Ar Hpara); 13C NMR (7 5 MHz, CDCl3ppm): 136.0, 135.8, 135.7, 134.2, 133.2, 126.0, 124.2, 123.8, 122.5, 82.0, 81.0. Hydrolysis of ethyl ester groups on precursor of CPEDs. To a solution of ester p recursors ThPG 1 Th PG 2 and Th PG 3 in THF (20 mL), Li OH in H2O (1 mL) was added dropwise. The reaction mixture was vigorously stirred at 80 oC for 12 hours. T he reaction mixture was cooled to room temperature, and then poured into a solution of methanol ( 1 00 mL) to give the yellowish precipitate. Further purification of CPEDs was accomplished by dialysis using nanopure water (Millipore Simplicity water system) and a 500 D MWCO cellulose membrane. After dialysis, the water was removed in vacuo, which gave the pale yellow solid powder 85~ 90% yield. ThG 1 : 1H NMR (300 MHz, D2O/ DMSO d6 ( v/vppm): 7 5 0 6 8 0 (br, 22H), 4. 7 5 (br, 6 H, ArOC H2CO2), 2.1 8 (br, 36H, NHC(CH2C H2CO2Na )3) ThG 2 : 1H NMR (300 MHz, D2O/DMSO d6 ( v/vppm): 7.806.90 ( br, 60H), 4. 77 (br, 12H, ArOC H2CO2), 2.1 9 (br, 72H, NHC(CH2C H2CO2Na )3) ThG 3 : 1H NMR (300 MHz, D2O/DMSO d6 ( v/vppm): 8.106.95 (br, 96H), 4. 7 5 (br, 24H, ArOC H2CO2), 2.1 8 (br, 144H, NHC(C H2C H2CO2Na )3)

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179 CHAPTER 7 CONCLUSIONS In conclusions we have seen a tremendous amount of growth in polymer and dendrimer field. Neverthel ess, m any scientists are still looking for the new domains in the polymer and dendrimer field in order to overcome the inveterate drawbacks or to discover potential possibility to advanced application. In this dissertation, newly designed conjugated polyel ectrolytes (CPEs) and conjugated polyelectrolyte dendrimers (CPEDs) were studied and described. In C hapter 2, a series of CPEs having branched polyionic side chains were synthesized and their nonaggregation effects by electrostatic repulsions of side chains were investigated. In C hapter 3, a highly selective and sensitive Hg2+ ion senor was studied using CPE and rhodamine combinative system. In C hapter 4, the aggregation of water soluble pyrene derivatives was characterized and such effect was utilized fo r efficient PPi sensing and to monitor enzyme s activity. In C hapter 5 and 6, two different types (compact and thienyl extended types) of CPE Ds having phenylethylene backbone and branched polyionic side chains were synthesized. The geometric structure a nd freatures of these CPE Ds were investigated, and their phopophysical properties fluorescence quenching, and energy transfer were also explored. Branched Polyionic Effect on Aggregation A new series of water soluble PPE type CPEs (conjugated polyelectrolytes) with branched polyionic side chains featuring less aggregation in aqueous solution have been synthesized and characterized. T he branched polyionic side chains in a conjugated polymer caused a less aggregation even in aqueous solution, which result ed in high quantum yields compared to CPEs with linear side chains. T he pH dependent

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180 results supported their little aggregation effect in netural aqueous solution. M ore aggregations were observed as pH decreases while the photophysical properties exhibited by their organic soluble precursors were retained at higher pH As expected, the CPEs with branched polycationic side chains (R -bNH3 +Cl-) show ed opposite behavior s. A t pH 4.5, aggregated PPEBTD-bCO2 showed a typical feature. On the contrary, both PPEBTD -dCO2 and PPE TBT -dCO2 exhibit unusually enhanced fluorescence in their aggregated form at pH 4.5. We proposed that the aggregation of these CPEs reduces water contact of conjugated backbone, thereby decreasing of a nonradi active process Lifetime measurements also supported the aggregation of the anionic and cationic branched polymers at low pH (~4.5) and high pH (~10.5), respectively. The emission lifetime ( < 1 ns) of CPEs with branched poly anionic side chains are wavelength independent lifetimes in both methanol and aqueous (pH 9.0) solutions. However, it was found that the emission lifetime of PPEAr -dCO2 at pH 4.5 consisted of two wavelength dependent lifetimes : longlived lifetimes ( > 2 ns) and extremely short lived lifetimes ( < 250 ps) S uch observation suggested the existence of aggregated polymers Mercury (II) ion and Pyrophosphate ion Sensors Hg2+ ion was efficiently detected by using the mixture of P PE-dCO2 and S Rho 1 R hodamine derivatives ( S Rho1) have been synthesized and used as a Hg2+ ion receptor. T he strong thiophilic affinity of mercury ion induced complex formation between S Rho 1 and Hg2+ ion, which induced ringopening of spiro structure. I ts complex sensitively quenched P PE -bCO2 through the fluorescence resonance energy tra nsfer, where hydrogen bonding between terminal bCO2 groups and hydrogen on

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181 nigrogen atom of S Rho 1 act as an important role for their complex formation. This combination system showed improved sensory response compared to S Rho 1 alone. W e synthesized a PyA4 that displays self assembly with strong excimer emission in HEPES buffer solution. The f luorescence intensity of PyA4 was most efficiently quenched with the Cu2+ ion and t he efficient quenching effect of PyA4 can be explained by intermolecular energy migration in aggregated PyA4 Taking advantage of the efficient fluorescence quenching of PyA4 by Cu2+, PyA4 Cu2+ complex was used as a fluorescence turnon sensor and it sensitively and selectively recognizes PPi over other anions I t is believed that ef ficient chehelation of the diphophate anion to Cu2+ ion induced higher selectivity Conjugated Polyelectrolyte Dendrimers Three generation of w ater soluble conjugated polyelectrolyte dendrimers (CPE Ds) with branched polyionic side chains have been synthesized and characterized. Compact type CPEDs and CPEDs having thienyl (Th) groups in the conjugated backbone w ere systemically prepared via precursor route. The geometric structure of CPE Ds based on computational modeling, GPC, and AFM studies showed th reedimensional globular architectures and monodisperse macromolecules Photophysical stuies of CPDs revealed that intradendrimer interaction becomes stronger in aqueous solution with generation. Incorporation of thienyl groups induced enhanced optical properties compared to the compact type CPE Ds without thienyl groups. The fluorescence lifetime decays provide information for intra dendrimer aggregation in H2O. Methyl viologen and cyanine dyes (DOC, DODC, and DOTC) efficiently quenched the fluorescence of the third generation CPED rather than the other generation in H2O, which is due to more efficient energy or charge transfer effect in aggregated state.

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182 APPENDIX A CONJUGATED POLYELECT ROLYTES WITH BRANCHED POLYCATIONIC SIDE CHAINS A B A B(a) (b) Figure A 1 (a) Vi sual and (b) Fluorescence colors of A: PPEPh -bNH3 + and B: PPEBTD -bNH3 +; [PPEAr -bNH3 +] = 3 0 M in H2O. Table A 1. Fluorescence lifetimes (i, ns) and relative amplitudes (RA, %) for PPEAr -bNH3 + in MeOH, basic (pH = 9.5), and acdic (pH = 4.5) conditionsa MeOH H2O, pH 9.5 H2O, pH 4.5 Compd. RA (%) RA (%) RA (%) PPE-Ph -bNH3 + i (ns) b 430 nm 500 nm i (ns) 430 nm 500 nm i (ns) 430 nm 500 nm 1 = 0.20 35 14 1 = 0.38 50 18 1 = 0.20 92 58 2 = 0.47 64 66 2 = 1.72 30 41 2 = 0.87 6 17 3 = 1.59 <1 13 3 = 4.52 20 41 3 = 3.71 2 25 4 = 3.97 <1 <7 2 1.183 1.043 2 1.133 1.128 2 1.211 1.108 PPE-BTD bNH3 + i (ns) 600 nm 650 nm i (ns) 600 nm 650 nm i (ns) 600 nm 650 nm 1 = 0.31 35 33 1 = 0.22 48 48 1 = 0.17 92 90 2 = 0.87 44 46 2 = 0.64 39 39 2 = 0.73 7 8 3 = 1.71 21 21 3 = 1.41 12 12 3 = 2.24 <1 <2 4 = 5.34 <1 <1 2 1.128 1.109 2 1.179 1.014 2 1.066 1.262 aData were collected by global fitting Algorithm bTipical limits of error on i are less than 3%.

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183 APPENDIX B NMR SPECTRA Figure B 1. 1H NMR (300 MHz, CDCl3) spectrum of S Rho 1 (chapter 3 ). Figure B 2. 1H NMR (300 MHz, CDCl3) spectrum of S Rho 2 (chapter 3).

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184 Figur e B 3 1H NMR (300 MHz CDCl3) spectrum of PyE4 (chapter 4). Figure B 4. 1H NMR (300 MHz, D2O /CD3OD (3/1, v/v ) ) spectrum of Py A 4 (chapter 4).

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185 Figure B 5. 1H NMR (300 MHz, CDCl3) spectrum of PG 1 (chapter 5). Figure B 6. 1H NMR (300 MHz, CDCl3) spectrum of PG 2 (chapter 5).

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186 Figure B 7. 1H NMR (300 MHz, CDCl3) spectrum of PG 3 (chapter 5). Figure B 8. 1H NMR (300 MHz, CDCl3) spectrum of ThPG 1 (chapter 6).

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187 Figure B 9. 1H NMR (300 MHz, CDCl3) spectrum of ThPG 2 (chapter 6). Figure B 10. 1H NMR (300 MHz, CDCl3) spectrum of Th PG 3 (chapter 6).

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195 BIOGRAPHICAL SKETCH Seoung Ho Lee was born in Seoul, Korea in 1976, and completed his undergraduate studies at Konyang University in Non san He received his bachelor s degree with honors in 2002. Lee continued his graduate studies at the same university under the supervision of Dr. Jong Seung Kim where he studied Host Guest C hemistry He also received the Best Poster Presentati on Award in 2004 Fall Meeting of Korean Chemical Society During his grad uate studies, in 2002 he had a research experience as an exchange student at the University of Louis Pasteur de Strasbourg, France under the supervision of Dr. Jacques Vicens After he received an M .S. degree with honors in 2004, he moved to the Dankook University in Seoul and continued training as a research associate. In 2005, he started his Ph. D. studies at the University of Florida. In the past five years, he carried out the research in the topic of P hotoactive C onjugated P olyelectrolytes and C onjugated P olyelectrolyte D endrimers under the supervision of Dr. Kirk S. Schanze. During the Ph. D. degree, he got married with Sangmi Lee in 2006 and had the first son, Junseo Lee, in the summer of 200 9 In 2011, Seoung Ho will join the group of Dr. Eric T. Kool as a postdoctoral associ ate at Stanford University.