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Conjugated Polyelectrolytes

Permanent Link: http://ufdc.ufl.edu/UFE0024832/00001

Material Information

Title: Conjugated Polyelectrolytes Synthesis, Photophysical Studies and Application to Sensors and Biocidal Activity
Physical Description: 1 online resource (167 p.)
Language: english
Creator: Ji, Eunkyung
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: activity, biocidal, conjugated, polyampholytes, polyelectrolytes, sensors
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: CONJUGATED POLYELECTROLYTES: SYNTHESIS, PHOTOPHYSICAL STUDIES AND APPLICATIONS TO SENSORS AND BIOCIDAL ACTIVITY This dissertation is focused on the design, synthesis, photophysical characterization and application of conjugated polyelectrolytes (CPE)s including anionic meta-linked poly(phenylene ethynylene)s (PPEs) such as mPPESO3 and mPPE-SO3-py, and cationic PPEs featuring quaternary ammonium side groups. We also introduce the synthesis and characterization of PPE-based polyampholytes bearing both anionic and cationic side groups. First, we have investigated the application of mPPESO3 for sensing of protease activity based on the amplified fluorescence quenching of the polymer. Since the polymer is folded into a helix in water, we have studied a mechanism for the interaction between the polymer and cationic intercalator quenchers. In this study, Re(dppz)-biotin and biocytin-TMR have been used as biotin-functionalized quenchers. The polymer fluorescence is quenched by both quenchers; however, addition of the target protein avidin does not recover the fluorescence from the quenched polymer. We also found that pre-formed avidin-quencher complexes less efficiently quench the polymer fluorescence compared to the only quenchers. Second, a PPE featuring meta-linked pyridine rings on the polymer backbone (mPPE-SO3-py) was designed and synthesized. The polymer is shown to undergo a conformational change from a random-coil to a helix by solvent polarity, protonation and metal complexation. The polymer also shows high sensitivity and selectivity for the Pd2+ ion. Third, a series of cationic CPEs with quaternary ammonium side groups has been synthesized and examined their biocidal activity. The photophysical studies in solution shows that direct excitation of the polymers produces a triplet state, sensitizing effectively singlet oxygen generation. Therefore, the polymer effectively kills bacteria such as Cobetia marina and Pseudomonas aeruginosa. Finally, a series of new conjugated polyampholytes containing both anionic and cationic side groups has successfully synthesized. These polymers show different behavior depending on the nature of ionic groups, the ratio of anionic to cationic groups, and pH. The polymer with 1:1 ratio of ammonium to sulfonate groups shows very low solubility in water and organic solvents; however, when the ratio is smaller, the polymers behave like CPEs. The polyampholyte bearing carboxyl and ammonium side groups shows pH- dependent photophysical changes.
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 Eunkyung Ji.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Schanze, Kirk S.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024832:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024832/00001

Material Information

Title: Conjugated Polyelectrolytes Synthesis, Photophysical Studies and Application to Sensors and Biocidal Activity
Physical Description: 1 online resource (167 p.)
Language: english
Creator: Ji, Eunkyung
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: activity, biocidal, conjugated, polyampholytes, polyelectrolytes, sensors
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: CONJUGATED POLYELECTROLYTES: SYNTHESIS, PHOTOPHYSICAL STUDIES AND APPLICATIONS TO SENSORS AND BIOCIDAL ACTIVITY This dissertation is focused on the design, synthesis, photophysical characterization and application of conjugated polyelectrolytes (CPE)s including anionic meta-linked poly(phenylene ethynylene)s (PPEs) such as mPPESO3 and mPPE-SO3-py, and cationic PPEs featuring quaternary ammonium side groups. We also introduce the synthesis and characterization of PPE-based polyampholytes bearing both anionic and cationic side groups. First, we have investigated the application of mPPESO3 for sensing of protease activity based on the amplified fluorescence quenching of the polymer. Since the polymer is folded into a helix in water, we have studied a mechanism for the interaction between the polymer and cationic intercalator quenchers. In this study, Re(dppz)-biotin and biocytin-TMR have been used as biotin-functionalized quenchers. The polymer fluorescence is quenched by both quenchers; however, addition of the target protein avidin does not recover the fluorescence from the quenched polymer. We also found that pre-formed avidin-quencher complexes less efficiently quench the polymer fluorescence compared to the only quenchers. Second, a PPE featuring meta-linked pyridine rings on the polymer backbone (mPPE-SO3-py) was designed and synthesized. The polymer is shown to undergo a conformational change from a random-coil to a helix by solvent polarity, protonation and metal complexation. The polymer also shows high sensitivity and selectivity for the Pd2+ ion. Third, a series of cationic CPEs with quaternary ammonium side groups has been synthesized and examined their biocidal activity. The photophysical studies in solution shows that direct excitation of the polymers produces a triplet state, sensitizing effectively singlet oxygen generation. Therefore, the polymer effectively kills bacteria such as Cobetia marina and Pseudomonas aeruginosa. Finally, a series of new conjugated polyampholytes containing both anionic and cationic side groups has successfully synthesized. These polymers show different behavior depending on the nature of ionic groups, the ratio of anionic to cationic groups, and pH. The polymer with 1:1 ratio of ammonium to sulfonate groups shows very low solubility in water and organic solvents; however, when the ratio is smaller, the polymers behave like CPEs. The polyampholyte bearing carboxyl and ammonium side groups shows pH- dependent photophysical changes.
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 Eunkyung Ji.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Schanze, Kirk S.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024832:00001


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1 CONJUGATED POLYELECTROLYTES: SYNTHESIS, PHOTOPHYSICAL STUDIES AND APPLICATIONS TO SENSORS AND BIOCIDAL ACTIVITY By EUNKYUNG JI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Eunkyung Ji

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3 To my family

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4 ACKNOWLEDGMENTS During m y long journey toward this point of my academic career, there are many people who I sincerely want to acknowledg e. First of all, I really woul d like to thank my advisor, Dr. Kirk Schanze for his support, advice and encourag ement. His enthusiasm to science encouraged me to keep on studying fascinating areas of photophysics and conjugated polymers which I barely knew before I joined his group. I also would like to acknowledge all the form er and current members of the Schanze group. When I first started experiments Eric Silverman kindly taught me how to use instruments and Dr. Kye-Young Kim helped me a lot to get used to work in the Schanze group. Dr. Xiaoyong Zhao always shared his skills for polymer synthe sis with me and encouraged me to overcome difficulties in the lab. Dr. Hui Jiang helped me to learn about the photochemistry with pleasure. Dr. Yongjun Li has been a great classmate and la bmate since I joined the chemistry department at UF. Dr. Yan Liu has been a good friend and shared her knowledge for sensor projects. Seoung Ho Lee has always been available for me to discuss about projects. His friendship and encouragement cheer me up to finish study here. I give my thanks to Julia Keller and Abigail Shelton for managing the orders for group member s. Their kindness and smile always make me comfortable during the long tough five years. Jo nathan Sommer and Jarrett Vella have showed their sense of humor, which always make me laugh in the lab. I would like to thank Dr. J on Stewart and Dimitri Dascier. They let me use their instruments to finish my projects. Dimitri Dasc ier also has supported me to overcome difficulties in a different country. I would lik e to express my appreciation to Dr. William Dolbier, one of my committee members, for his kindness in writing a recommendation letter for me. I am also thankful to other committee members, Dr. Lisa McElwee-White, Dr. Valeria D. Kleiman and Dr. Stephen Hagen for their tim e and suggestions.

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5 Finally, I am grateful to my family for thei r support and love. They always have provided me with the best conditions for my education. Without them, I cant continue my education and come to this far. I am sincerely thankful to my two grandmothers for their love. I am also really sorry to them because they passed away when I was here so I couldnt attend their funeral.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF TABLES .........................................................................................................................10LIST OF FIGURES .......................................................................................................................11ABSTRACT ...................................................................................................................... .............16CHAPTER 1 INTRODUCTION .................................................................................................................. 18Conjugated Polyelectrolytes ...................................................................................................18Amplified Fluorescence Quenching of Conjugated Polyelectrolytes .....................................19Fluorescence Quenching ................................................................................................. 19Amplified Quenching ......................................................................................................22Aggregation of Conjugated Polyelectrolytes ...................................................................24Synthetic Helical Conjugated Polyelectrolytes ...................................................................... 26History of Helical Polymers ............................................................................................26Self-Assembly of Meta-Linked Phenylene Ethynylenes: Helical Folding ......................28Application of Conjugate d Polyelectrolytes ...........................................................................34Optical Sensors ............................................................................................................... .34Superquenching mechanism .....................................................................................34Fluorescence resonance energy tr ansfer (FRET) mechanism ..................................35Conformation change ...............................................................................................37Biocidal Activity .............................................................................................................38Scope of the Present Study .................................................................................................... .392 SENSING OF PROTEASE ACTIVI TY USING META-LINKED POL Y (PHENYLENE ETHYNYLENE) SULFONATE ..................................................................41Introduction .................................................................................................................. ...........41Results and Discussion ........................................................................................................ ...43Overview of Protease Turn-on Assay ..............................................................................43Peptidase Activity Sensing ..............................................................................................43Concentration Effect of Polymer a nd Buffer on Fluorescence Quenching ..................... 45Summary and Conclusions .....................................................................................................47Experimental .................................................................................................................. .........48Materials ..................................................................................................................... .....48Instrumentation ............................................................................................................... .48General Methods .............................................................................................................48

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7 3 FLUORESCENCE QUENCHING OF HELICAL CONJUGATED POLYELECTROLYTE BY RHENIUM-BIOTIN COMPLEX AS QUENCHERTETHER-LIGAND (QTL) PROBE .......................................................................................50Introduction .................................................................................................................. ...........50Results .....................................................................................................................................54Synthesis of Re complexes .............................................................................................. 54Photopysical Properties of Re complexes ....................................................................... 54Fluorescence Quenching of mPPESO3 with Re(dppz) and Re(dppz)-biotin ..................55Avidin Interactions with mPPESO3/Re(dppz)-biotin ..................................................... 57Fluorescence Quenching of mPPESO3 with Pre-formed Re(dppz)-biotin/avidin Complex ....................................................................................................................... 58Effect of Specific Protein on Fluores cence Quenching of mPPESO3 by Re(dppz)biotin ........................................................................................................................ ....58Discussion .................................................................................................................... ...........62Summary and Conclusions .....................................................................................................63Experimental .................................................................................................................. .........64Materials ..................................................................................................................... .....64Instrumentation ............................................................................................................... .64General Methods .............................................................................................................64Fluorescence quenching ...........................................................................................64Fluorescence recovery ..............................................................................................65Synthetic Procedures .......................................................................................................654 FLUORESCENCE RESONANCE ENERGY TRANSFER FROM HELI CAL CONJUGATED POLYELECTROLYTE TO CHARGED FLUORESCENT DYELIGAND CONJUGATED (DLC) MOLECULE ................................................................... 68Introduction .................................................................................................................. ...........68Results .....................................................................................................................................70Photophysical Properties. ................................................................................................70FRET from mPPESO3 to Biocytin-TMR ........................................................................71Anisotropy Measurements ............................................................................................... 76Fluorescence Quenching of PPESO3 by Biocytin-TMR ................................................ 77Avidin binding to Biotin on Biocytin-TMR/CPEs complex ...........................................78Fluorescence Quenching of mPPESO3 by Pre-formed Biocytin-TMR/avidin complex ....................................................................................................................... .80Discussion .................................................................................................................... ...........84Summary and Conclusions .....................................................................................................85Experimental .................................................................................................................. .........86Materials ..................................................................................................................... .....86Instrumentation ............................................................................................................... .86General Methods .............................................................................................................87

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8 5 META-LINKED POLY (PHENYLEN E ETHYNYLENE) SULFONATE CONTAINING PYRIDI NE .................................................................................................... 88Introduction .................................................................................................................. ...........88Results and Discussion ........................................................................................................ ...93Synthesis ..................................................................................................................... .....93Photophysical Characterization .......................................................................................93Fluorescence Quenching of mPPE-SO3-py by Metal ions in water ............................... 95Protonation and Metal Complexation with Pd2+ ............................................................. 96Experimental .................................................................................................................. .......100Materials ..................................................................................................................... ...100Instrumentation .............................................................................................................. 100General Methods ...........................................................................................................100Synthetic Procedures .....................................................................................................1016 POLY (PHENYLENE ETHYNYLENE) WI TH TETRA ALKYLAMM ONIUM SIDE GROUPS FOR LIGHT-INDUCED BIOCIDAL ACTIVIY ................................................102Introduction .................................................................................................................. .........102Results and Discussion ........................................................................................................ .107Synthesis ..................................................................................................................... ...107Polymer Characterization .............................................................................................. 1071H NMR and pulse gradient echo (PSGE) NMR ...................................................107Photophysical Characterization ..............................................................................109UV-vis and fluorescence spectroscopy ..................................................................109Transient absorption spectroscopy ......................................................................... 112Singlet Oxygen Production ............................................................................................112Biocidal Activity. ..........................................................................................................116Mechanism for Biocidal Activity .................................................................................. 118Experimental .................................................................................................................. .......121Materials ..................................................................................................................... ...121Instrumentation .............................................................................................................. 121General Methods ...........................................................................................................122Synthetic Procedures .....................................................................................................1237 CONJUGATED POLYAMPHOLYTES BASE D ON POLY(PHENYLENE ETHYNYLENE) .................................................................................................................. 128Introduction .................................................................................................................. .........128Results and Discussion ........................................................................................................ .132Synthesis ..................................................................................................................... ...132Properties of Conjugated Polyampholytes .................................................................... 135Summary and Conclusions ...................................................................................................147Experimental .................................................................................................................. .......147Materials ..................................................................................................................... ...147Instrumentation .............................................................................................................. 148

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9 General Methods ...........................................................................................................148Synthetic Procedures .....................................................................................................1488 CONCLUSIONS .................................................................................................................. 152Intercalation of Intercalator Quenchers to mPPESO3 .......................................................... 152Helical Self-Assembly of mPPESO3-py .............................................................................. 153Biocidal Activity of Cationic CPEs ......................................................................................153Conjugated Polyampholytes ................................................................................................. 153LIST OF REFERENCES .............................................................................................................155BIOGRAPHICAL SKETCH .......................................................................................................167

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10 LIST OF TABLES Table page 2-1 Stern-Volmer Quenching constant s for m PPESO3 and PPESO3 fluorescence quenching by KpNA ......................................................................................................... 473-1 Stern-Volmer quenching constant s for mPPESO3 and PPESO3 fluorescence quenching by Re complexes .............................................................................................. 554-1 Photophysical properties of mPPESO 3, PPESO3 and biocytin-TMR in 1 mM phosphate buffer solution (pH 7.4) ....................................................................................705-1 Photophysical properties of mPPE-SO3-py ....................................................................... 956-1 Photophysical properties of cationic CPEs ...................................................................... 1157-1 Photophysical properties of conjugated polyampholytes ................................................ 1377-2 Stern-Volmer constant ( KSV) for P1 and P2 fluorescence quenching upon addition of MV2+ and NDS in water and DMF. ................................................................................. 140

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11 LIST OF FIGURES Figure page 1-1 Structures of some cla sses of conjugated polym ers ...........................................................181-2 Structures of conjugate d polyelectolytes (CPEs) ............................................................... 201-3 Jablonski diagram ......................................................................................................... .....201-4 Comparison of dynamic and static quenching ................................................................... 221-5 Structures of conjugated pol ymer, monomer and quencher (MV2+) used by Swagers group. ........................................................................................................................ .........221-6 Amplification of fluorescence que nching of MPS-PPV with MV2+ ................................231-7 SternVolmer plots of polyfluorene quenching by 5-nm gold nanoparticles .................... 241-8 Structures of a PPP-type CPE, a PPV-type CPE, and thei r neutral analogous polymers ...................................................................................................................... .......251-9 Structures of PPE-type CPE PPESO3................................................................................271-10 Absorption and fluorescence spectr a of PPE-type CPE PPESO3 in methanol, methanol/water (1:1) and water ......................................................................................... 271-11 Schematic illustration of face-face stacking of PPESO3.............................................271-12 Structures of OPEs studied by Moore s group and a space-filling model showing the conformationalequilibrium fo r OPE of length n = 18 ........................................................ 281-13 Postulated association equilibrium between OPEs and chiral molecules .......................... 291-14 Structures of amphiphilic mPPEs studied by Tews group ............................................... 291-16 UV-vis absorption and emission spectra of mPPESO3 in MeOH/H2O at different compositions .................................................................................................................. ....301-17 Emission spectra of [Ru(bpy)2(dppz)]2+ in absence and pres ence of mPPE-Ala .............. 321-18 Circular dichroism spectra of mPPE-Ala in methanol, water and methanol/water mixtures..............................................................................................................................331-19 Emission quenching of mPPESO3by MV2+ in a 7:3 methanol/water mixture .................. 341-20 Detection of avidin using CPE ba sed on the superquenching mechanism ........................ 351-21 Structures of CPEs studied by Bazan and co-workers ....................................................... 36

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12 1-22 Schematic representation for the use of PF1/PNA-C* to detect a complementary ssDNA ......................................................................................................................... .......361-23 Structures of poly(thiophene) deri vatives used in CPE-based sensor via conformational change mechanism .................................................................................... 371-24 Schematic illustration for the detection of ssDNA sequences using poly(thiophene) ....... 381-25 Structure of cationic CPE used by the Whitten group ....................................................... 391-26 Phase contrast and fluorescence microscope images of CPE-treated E. coli and CPEtreated B. anthracis Stern spores .......................................................................................392-1 Mechanism of the turn-on and t urn-off CPE-based enzyme assays ...........................422-2 Structures of CPEs and quencher substr ates used for CPE-based enzyme assays ............ 422-3 Structures for a polymer and a quencher substrate and mechanism of the turn-on mPPESO3-based sensor .....................................................................................................442-4 Fluorescence spectroscopic cha nges observed during turn-on assay ................................ 442-5 Stern-Volmer plots for fluores cence quenching of mPPESO3 with KpNA and pnitroaniline .................................................................................................................. .......462-6 Stern-Volmer plots of at different concentrations of mPPESO3 with KpNA .................. 473-1 Three binding modes of a metal complex with DNA ........................................................523-2 The light-switch effect of dppz-based metallo-intercalators ............................................. 523-3 Structures and acronyms of polymers and Re complexes .................................................543-4 Fluorescence spectra of mPPESO3 in th e presence of Re(dppz) and Re(dppz)-biotin .....563-5 Stern-Volmer plots for mPPESO3 qu enching by Re(dppz)-biotin, pre-formed Re(dppz)-biotin/avidin complex (avidin: Re(dppz)-biotin = 1:4) and avidin ..................... 593-6 Stern-Volmer plots for PPESO3 quen ching by Re(dppz)-biotin and pre-formed Re(dppz)-biotin-avidin complex (a vidin:Re(dppz)-biotin = 1:4). .....................................593-7 Stern-Volmer plots for mPPESO3 quench ing by different ratios of pre-foremd Re(dppz)-biotin/avidin complex and quenchi ng percent of mPPESO3 by pre-formed Re(dppz)-biotin/avidin comple x containing different concentrations of avidin ................ 603-8 Stern-Volmer plots of mPPESO3 quenching by Re(dppz)-biotin, pre-mixed Re(dppz)-biotin-BSA mixture (BSA: Re (dppz)-biotin = 1:4) and BSA ........................... 613-9 Cross-linking of mPPESO3 and avidin by using Re(dppz)-biotin .....................................63

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13 3-10 Binding of Re(dppz)-biotin and pre-for med Re(dppz)-biotin/avidin complex to mPPESO3 ....................................................................................................................... ...634-1 Structures and acronyms of conjugated polyelectrolytes and dye-ligand conjugate used in this study ............................................................................................................ ....704-2 Energy transfer from mP PESO3 to biocytin-TMR ............................................................714-3 Normalized absorption spectrum of bi ocytin-TMR and normalized fluorescence spectra of biocytin-TMR and CPEs ...................................................................................734-4 Normalized fluorescence spectra of m PPESO3 upon addition of biocytin-TMR .............744-5 Normalized fluorescence spectra of bioc ytin-TMR in the absence and presence of mPPESO3 ....................................................................................................................... ...754-6 Excitation spectrum of mPPE SO3/biocytin-TMR complex .............................................. 764-7 Excitation anisotropy of biocytin-T MR/mPPESO3 complex and biocytin-TMR .............774-8 Normalized fluorescence spectra of PPESO3 upon addition of biocytin-TMR ................ 784-9 Fluorescence spectra of mPPESO3 solu tion upon addition of biocytin-TMR and avidin..................................................................................................................................794-10 Fluorescence spectra of biocytin-TMR upon addition of avidin ..................................... 804-11 Stern-Volmer plots of mPPESO3 and PPESO3 fluorescence quenc hed by biocytinTMR or pre-formed biocytin-TMR/avidin complex .......................................................... 824-12 Sensitized biocytin-TMR fluorescence intens ity at 590 nm after addition of biocytinTMR or pre-formed biocytin-TMR/avidin complex to CPEs ............................................ 824-13 SV plots for fluorescence quenching of mPPESO3 at 450 nm and polymer-sensitized biocytin-TMR fluorescence at 590 nm upon addition of pre-formed biocytinTMR/avidin complex ......................................................................................................... 835-1 Structures of phenylene ethynylene (PE) based macrocyc le and oligomers used for stacked self-assemblies ......................................................................................................895-2 Schematic diagram illustrates the self-a ssembly of disk-shaped phenylene ethynylene macrocycles and oligomers ................................................................................................895-3 Structures of conjugated polymers cont aining bipyridyl and te rpyridyl ligands ...............905-4 Structures of poly(pyridyl/phenyl ethynylene) polymers with varying ratios of para/ meta -linked constitutional units ........................................................................................ 91

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14 5-5 Structures of a poly(p-phenylene ethynylnene) derivative containing meta -substitu ted monopyridyl groups ...........................................................................................................925-6 Structure of a meta -poly(phenylene ethynylene) c ontaining pyridine units ( m -PPY) .......925-7 Synthesis of mPPE-SO3-py ............................................................................................... 935-8 Normalized absorption and emission spect ra of mPPE-SO3-py in water, methanol and mixture of the two solvents ......................................................................................... 945-9 Stern-Volmer plots for mPPE-SO3-py fluor escence quenching by metal ions in water ... 975-10 Fluorescence responses of mPPE -SO3-py to metal ions in water 975-11 Absorption and emission spectra of mPPE -SO3-py in water and in methanol with addition of HCl solution.....................................................................................................985-12 Absorption and emission spectra of mPPE -SO3-py in water and in methanol with addition of Pd2+ ..................................................................................................................996-1 Pathway of type I and Type II reac tion of light absorbing photosensitizer .....................1046-2 Structure of PPE-NMe3-OR8 and phase contrast and fl uorescent microscope images of polymer-treated E. coli and polymer-treated B. anthracis Sterne spores .....................1056-3 Structures and acronyms of cationic conjuga ted polyelectrolytes in vestigated in this study ......................................................................................................................... ........1066-4 General synthetic scheme for monomer 5, 9 and 12 ........................................................ 1086-5 General synthetic scheme for polymer 13a, 14 and 16 .................................................... 1096-6 Absorption and emission spectra of CPEs in methanol and water .................................. 1106-7 Transient absorption spectrocopy of ca tionic CPEs in methanol and water .................... 1136-8 Pathway for PPE-sensitized singlet oxygen generation ...................................................1146-9 Singlet oxygen emission sensitized by CPEs in CD3OD. ................................................ 1146-10 Peroxidation of CH DDE by singlet oxygen (1O2) ........................................................... 1176-11 UV-visible spectra of CHDDE and PPE-NMe3-OR8, PPE-NMe3-Th, PPE-4+ or PPE-C6-NMe3-OR8 in D2O solution containing phosphate buffer (100 M, pH 7) as a function of the irradiation time ..................................................................................... 1176-12 Images of single SGCP-particle with captured bacteria ..................................................1186-13 Confocal fluorescence images of SGCP-13b with C. marina in ambient air ................... 119

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15 6-14 Dead/live ratios of C. marina exposed to 5 m SGCP-13b under various conditions .... 1206-15 Mechanism of biocidal action .......................................................................................... 1207-1 Structures of the four subclasses of polyampholytes ....................................................... 1297-2 Structures of polyampholytes that have been synthesized ...............................................1307-3 Structures of conjugated polyampholytes ........................................................................1317-4 Synthesis of monomers 4, 6, 8, 9 and 13 ......................................................................... 1337-5 Synthesis of conj ugated polyampholytes ......................................................................... 1347-6 1H NMR spectrum of P1 and P2 in DMSO-d6. ................................................................1367-7 Absorption and emission spectra of P1 and P2 in methanol and water ........................... 1387-8 Structures of a cationic and an an ionic quencher used in this study ................................ 1397-9 Emission spectra of P1 and P2 in wate r upon addition of a cationic and an anionic quencher. ..........................................................................................................................1407-10 Absorption and emission spectra of P1 and P2 in DMF .................................................. 1427-11 Emission spectra of P1 and P2 in DMF upon addition of a cationic and an anionic quencher ...........................................................................................................................1437-12 Absorption and emission spectra of PPE -NMe3-COOH as a function of pH in aqueous solution...............................................................................................................1447-13 Absorption and emission spectra of PPE-N Me3-COOH in methanol upon addition of HCl solution .....................................................................................................................1457-14 Emission spectra of PPE-NMe3-COOH upon addition of an anionic quencher, NDS at different pH solutions................................................................................................... 146

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16 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CONJUGATED POLYELECTROLYTES: SYNTHESIS, PHOTOPHYSICAL STUDIES AND APPLICATIONS TO SENSORS AND BIOCIDAL ACTIVITY By Eunkyung Ji August 2009 Chair: Kirk S. Schanze Major: Chemistry This dissertation is focused on the design, s ynthesis, photophysical characterization and application of conjugated polyelect rolytes (CPE)s including anionic meta -linked poly(phenylene ethynylene)s (PPEs) such as mPPESO3 and mPPE-SO3-py, and cationic PPEs featuring quaternary ammonium side groups. We also intr oduce the synthesis and ch aracterization of PPEbased polyampholytes bearing both anionic and cationic side groups. First, we have investigated the application of mPPESO3 for sensing of protease activity based on the amplified fluorescence quenching of the polymer. Since the polymer is folded into a helix in water, we have studied a mechanis m for the interaction between the polymer and cationic intercalator quenchers. In this study, Re(dppz)-b iotin and biocytin-TMR have been used as biotin-functionalized quenchers. The polymer fluorescen ce is quenched by both quenchers; however, addition of the target protein avidin does not recover the fluorescence from the quenched polymer. We also found that pre-formed avidin-quencher complexes less efficiently quench the polymer fluorescence co mpared to the only quenchers. Second, a PPE featuring meta -linked pyridine rings on the polymer backbone (mPPE-SO3py) was designed and synthesized. The polymer is shown to undergo a conformational change

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17 from a random-coil to a helix by solvent polarity, protonation and metal complexation. The polymer also shows high sensitiv ity and selectivity for the Pd2+ ion. Third, a series of cationic CPEs with qua ternary ammonium side groups has been synthesized and examined their biocidal activity. The photophysic al studies in solution shows that direct excitation of the polymers produces a triplet state, sensitizing effectively singlet oxygen generation. Therefore, the polymer effectively kills b acteria such as Cobetia marina and Pseudomonas aeruginosa. Finally, a series of new c onjugated polyampholytes contai ning both anionic and cationic side groups has successfully synthesized. These polymers show different behavior depending on the nature of ionic groups, the ratio of anionic to cationic gro ups, and pH. The polymer with 1:1 ratio of ammonium to sulfonate groups shows very low solubility in water and organic solvents; however, when the ratio is smaller, the polymers behave like CPEs. The polyampholyte bearing carboxyl and ammonium side groups show s pHdependent photophysical changes.

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18 CHAPTER 1 INTRODUCTION Conjugated Polyelectrolytes Conjugated polym ers (CPs) are organic se miconducting polymers containing a base structure of alternating singl e and double/triple bonds. Sin ce Shirakawa, MacDiarmid and Heeger discovered the electrically conducting po lymer, polyacetylene doped with halogen in 1977,1 much effort has been directed to a variety of other CPs with differe nt backbone structures based on aromatic, heteroaromatic, vinylic or acetylenic -systems including poly( para phenylene) (PPP)2, polyfluorene (PF),3 polypyrrole (PPy),4 polythiophene (PT),5 polyaniline (PANI),6 poly(phenylene ethynylene) (PPE),7 poly(phenylene vinylene) (PPV)8 as shown in Figure 1-1. In the past three decades, the CPs have been important materials for applications in light-emitting diodes (LEDs),8 light-emitting electrochemical cells (LECs),9 plastic lasers,10 solar cells,11 field-effect transistors (FETs),12 and sensors.13, 14 Each class of CP is prepared by its own synthetic methodology. Metal-catalyzed reaction ha s been widely used for synthesis of CPs, including PPPs by the Suzuki, Stille and Yamamoto coupling reaction;15 PPVs by the Figure 1-1. Structures of some classes of conjugated polymers.

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19 Wittig-Horner and Heck reactions, or the Gilch and Wessling routes; 16 and PPEs by the Sonogashira coupling and alkyne methathesis reactions.7, 17 The various palladium-catalyzed coupling methods such as Suzuki, Stille, Heck and Sonogashira allow mild reaction conditions, wide functional group tolerance, and flexibility of solvent used for the polymerization. These methods are also used for the synthesi s of CPs featuring ionic side groups which make the CPs soluble in wate r and other polar solvents.18, 19 Shi and Wudl reported the synthesis of water soluble PPV.20 The synthesis of water soluble PPP was reported by Wallow and Novak.21 These early reports were important initial contribution to this area. In the decade, several synthetic methods have been developed, resulting in structurally diverse of conjugated polyelectrolytes (CPEs). Most CPEs contain PPV, PPP or PPE backbone with ionic functional groups such as sulfonate (SO3 -), carboxylate (CO2 -), phosphate (PO4 2-) and ammonium (NR3 +) (Figure 1-2). In addition, CPEs feature strong light absorption and strong fluorescence in solution and in solid state. Amplified Fluorescence Quenching of Conjugated Polyelectrolytes Fluorescence Quenching Recently CP Es have been studied for application in chemoand biosensors13, 22-28 because of their amplified quenching by oppositely charge d small molecules. Before we present the details of amplified quenching of CPEs, it is necessary to provide a brief overview of photophysical processes which occurs between th e absorption and emission of light. These processes can be illustrated w ith a Jablonski Diagram (Figure 1-3). Following light absorption, molecules are excited from singlet ground state of S0 to singlet excited state of S1, or higher levels of S2 or S3. Molecules relax from highe r vibrational level of S2 to the lowest energy level of S0 and this is called internal conversion. This process generally occurs within 10-12 s or less, which is much less than fluorescence lifetime. Th erefore, fluorescence emission generally arises

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20 Figure 1-2. Structures of conj ugated polyelectolytes (CPEs). Figure 1-3. Jablonski diagram. Figure was taken from ref. 29. from the lowest energy vibrational level of S1. Besides fluorescence emission, excited molecule is returned to ground state S0 from the excited state by vari ous competing pathways, including nonradiative decay to release heat and intersystem crossing to a triplet state T1. Emission from T1 is termed phosphorescence. Fluorescence quenchi ng is another important competing process, in which the intensity of fluorescence is decreased by either collision quenching (Eq. 1-1) or static quenching (Eq. 1-2).29 In Eq. 1-1 and 1-2, F* is an excited-state fluorophore, Q is a quencher, kq is the bimolecular quenc hing rate constant and Ka is the association constant for

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21 (1-1) (1-2) formation of the ground-state complex [F, Q]. Collision quenching, which is also called dynamic quenching, occurs when the excite d state fluorophores c ontact with quenchers to deactivate and return to the ground state S0 without fluorescence emission. St atic quenching occurs when a nonfluorescent ground state complex is formed between a fluorophore and a quencher. When this complex absorbs light, it immediately returns to the ground state wit hout fluorescence emission. Both dynamic quenching and sta tic quenching are described by St ern-Volmer (SV) equation: (1-3) where I0 and I are the fluorescence intensities in th e absence and presence of a quencher, respectively, [ Q ] is the quencher concentration and KSV is the Stern-Volmer quenching constant. When the quenching is dominated by a dynamic process, KSV = kq0, where 0 is the fluorescence lifetime of F*; however, when a static quenching is dominant, KSV = Ka. The dependence of dynamic and static quenching on temperature and visc osity, or lifetime meas urement can be used to distinguish between these two quenching mech anisms (Figure 1-4). At higher temperature diffusion is faster, and hence a larger amount of collision quenching occurs whereas weakly bound complex is dissociated, and hence a smaller amount of static quenching occurs. For static quenching 0/ =1. In contrast, for dynamic quenching F0/F = 0/ where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively. Linear SV plots of I0/ I vs [Q] are observed when quenching is dom inated by either purely static or dynamic pathways. However, SV plots are curved upward in many situations due to various processes

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22 including mixed static and dynamic quenching, va riation in the associ ation constant with quencher concentration and chromo phore (or polymer) aggregation. Amplified Quenching Am plified quenching was first investigated by Swager and co-workers.30, 31 They observed amplified fluorescence quenching in a neutral, organic soluble poly(phe nylene ethylene) (PPE) functionalized with cyclophane receptors, whic h form complexes with N,N-dimethyl-4,4bipyridium (MV2+), resulting in fluorescence quenchi ng. This study also showed that the Figure 1-4. Comparison of dynamic and static qu enching. Figure was taken from ref. 29. Figure 1-5. Structures of conjugate d polymer, monomer and quencher (MV2+) used by Swagers group.

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fluoresc e analogo u quenche r polymer Re fluoresc e first rep o fluoresc e PPV) by magnitu d that of f o at low c o 1-7.32-35 T complex migrati o the poly m quenche r Figure 1 e nce of the p u s monomer r and the po l (molecula r cently, amp l e nce of CPE s o rted the am p e nce quench i methyl viol o d e greater th o r stilbene i n o ncentration T he superli n formation b o n within th e m er and the q r .22, 33-37 6. Amplifi c taken fro m p olymer is q u Amplified q l ymer, and r a r wire effect l ified fluore s s is efficien t p lified quen c i ng of poly( 2 o gen (MV2 + an that for d n micelles. I n followed b y n ear quenchi b etween the p e polymer,22, q uenche r 34, c ation of flu o m Chen et a l u enched ab o q uenching i s a pid diffusi o ). s cence quen c t ly quenche d c hing of CP E 2 -methoxy5 + ) (Figure 16 d ilute soluti o n most CPE / y superlinea r ng behavior p olymer an d 33, 34, 37, 38 e f and aggreg a o rescence q u l .22 23 o ut 60 times m s explained b o n and/or de l c hing has b e d by opposit e E s, where K 5 -propyloxy s 6 ).22 The qu e o n of stilben e / quencher s y r behavior a t is attribute d d the quench f ficient long a tion of poly m u enching of m ore effici e b y complex l ocalization e en extende d e ly charged q K SV of 107 M s ulfonate p h e nching co n e and four o r y stems, SV p t high conce n d to several f er,22, 33, 34, 36 range Frs t m er chains i MPS-PPV w e ntly compa r formation b of singlet e x d to CPEs.22 q uencher io n 1 was obser v h enylene vin y n stant is six o r ders of ma g p lots show a n tration as s h f actors incl u efficient si n t er energy tr a i nduced by t w ith MV2+. r ed to an etween the x citon withi n 23 The n s. Chen et. v ed in the y lene) (MP S o rders of g nitude grea t linear beha v h own in Fi g u ding ion-pa i n glet excito n a nsfer betw e t he Figure was n the al. S t er v ior g ure i r n e en

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24 Figure 1-7. SternVolmer pl ots of polyfluorene (1.0 10-6M in monomer repeat units) quenching by 5-nm gold nanoparticles. (Inset) Linear range in the low quencher concentration regime. Figure was taken from Fan et. al.34 Aggregation of Conjugated Polyelectrolytes It is well known that amphiphilic polym ers self-assemble into various supramolecular architectures including micelles, ve sicles and liquid-crystalline phases.39, 40 The amphiphilic characteristics of CPEs also make them self-a ssemble into supramolecular aggregates. When CPEs exist in solution in a non-aggregated state, their photophysical pr operties are similar to those of a neutral analog dissolv ed in a non-polar organic solven t. For example, Reynolds and co-workers reported that sulfona ted PPE-type CPE PPP-OPSO3 (Figur e 1-8) in a dilute aqueous solution displays a fluorescence maximum of 410 nm and quantum yield of 0.60.41 These values are similar to the fluorescence maximum of 406 nm and quantum yield of 0.70 of structurally analogous, neutral PPP with alkyloxy side gr oup, PPP-OR11 (Figure 1-8) in chloroform.42 On the other hand, the aggregation of CPEs in aqueous solution strongly influences the photophysical properties of the polymers. For exam ple, Whitten and co-workers reported that sulfonated PPV (MPS-PPV) (Figure 1-8) exhibits a fluorescence ma ximum that is relatively red-

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25 shifted and has a significantly decreased fluorescence quantum yield compared to the neutral analog MEH-PPV (Figure 1-8) in solution.22, 43 These changes arise due to inter-chain aggregation, producing eximer-like states. Figure 1-8. Structures of a PPP-type CPE, a PPV-type CPE, and their neutral analogous polymers. Previously, our group reporte d that the solvent dependence of the absorption and fluorescence spectra of PPE-type of CPE, PPE-SO3 (Figure 1-9).36 As shown in Figure 1-10, as the amount of water increases, th e absorption red-shifts and narro ws. The fluorescence red-shifts and broadens, and the fluorescence quantum yield decreases with increasing water content. All of these features provide clear evidence that PPE-S O3 aggregates in water, while it exits in a non-aggregated state in methanol Specifically, the absorption changes are consistent with increased structural order and conjugation length in the PPE b ackbone. The red-shifted and broad fluorescence band is attributed to the formation of an excimer-like state, which arises from interchain stacks. In aggregated polymer, the chains align with their long axis parallel and the phenylene rings in each chain must be nearly co-planar to optimize hydrophobic interactions and stacking (Figure 1-11).

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26 Synthetic Helical Conjugated Polyelectrolytes History of Helical Polymers Biopolym ers such as proteins a nd nucleic acids fold into we ll-defined three-dimensional structures in solution. Syntheti c helical polymers have been of interest for understanding the mechanism for helix formation in biopolymers.39, 44 Synthetic helical polymers also have been attractive because of their pot ential applications including molecular recognition, a molecular scaffold function for controlled special ali gnment of functional groups or chromophores, and ordered molecular alignment in the solid phase such as that in liquid crystalline materials.44 The history of helical polymers goes back to the finding of the conformation of same natural polymers. Among the helical polymers, -conjugated materials have been of interest because they are potentially useful in areas such as polarizatio n-sensitive electro-optical materials, asymmetric electro-synthesis, nonlin ear optics, polarized photoand elec troluminescence and enantioslective sensing.45 The materials feature primary structures th at allow folding of backbones into a helical secondary structure. Optically act ive polyacetylenes with chiral si de groups are an early example of this class of materials.46 Since the first report of such polyacetylenes, a variety of monosubstituted47-50 and disubstituted51-54 diacetylenes were synthe sized from the monomers with chiral side groups. Yashima et. al. found th at polyacetylenes bearin g achiral side groups exhibit induced optical activity by in teraction with chiral molecules that selectively binds to one form of the helix.48, 55 In addition to polyacetylenes, vari ous conjugated polymers have been reported to form helical conformation in poor solven ts or in the solid state. The helical polymers includes oligoand polythiophenes,56-60 oligoand polyfluorenes,61-63 poly( p-phenylene)s,64, 65

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Figure 1 Figure 1 Figure 1 9. Structur e 10. Absor p methanol water co n 11. Schem a e s of PPE-t y p tion (left) a n methanol/ w n tent. Figure a tic illustrat i O O PP E y pe CPE PP E n d fluoresce n w ater (1:1) a was taken f i on of facef 27 SO3 -N a SO3 -Na+ E SO3 E SO3. n ce (right) s a nd water. A r f rom Tan et. f ace stac a + s pectra of P P r rows show a l .36 king of PP E P E-type CP E the directio n E SO3. E PPESO3 i n n of change w n w ith

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28 polycarbazoles,66 poly(p-phenylene ethylene)s,67-70 and poly(p-phenylene vinylene)s71-73 with optically active side groups. Self-Assembly of Meta-Linked Phenylene Ethynylenes: Helica l Folding Moore and co-workers demonstrated that meta -linked oligo(phenylen e ethylene)s (OPEs) with longer chains than 8 monome r units fold into a helical conf ormation in a polar solvent such as acetonitrile .74 However, the oligomers exist in an e xpanded form in a non-polar solvent such as chloroform. This helical conformation is thermodynamically driven by solvophobic effects. 74, 75 Figure 1-12 shows the structures of OPEs and a space-filling model of the extended coil form of OPEs in chloroform and a helical conformation in acetonitrile. Their folding process was studied using 1H NMR, UV absorption and fluorescence spectra.74, 75 When the percent of acetonitrile in chloroform (by volume) is increase d, the polar side chains are extended to the environment and the non-polar backbone folds into a helical conformation, which is stabilized by the stacking interactions between the phenyl rings. The folded conformation of OPEs also create a tubular cavity, which acts as a host to recognize different ch iral compounds (Figure 113).76 Reversible 1:1 association of chiral guests to folded OPEs induces circular dichroism (CD) Figure 1-12. Structures of OPEs studied by Moores group and a space-filling model showing the conformationalequilibrium for OPE of length n = 18. Side chains are not shown for clarity. Figure was takenfrom Prince et.al. 75

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29 signal in UV regions where the polymer absorbs li ght. This probes the inte raction of a chiral molecule with a achiral OPE resulti ng in induced chirality on OPEs. Tew and co-workers observed a helical conformation for amphiphilic meta -linked poly(phenylene ethylene)s (mPPEs) c ontaining ionic side groups in 90 % H2O/DMSO solution (Figure 1-14).77 mPPE bearing long alkyl ch ains self-assemble into or dered bilayers. The mPPE is too large for interior cavity formed by the helix; therefore it cannot adopt a helical conformation. In contrast, mPPE bearing no alkyl chains shows similar changes in absorption and fluorescence spectra as spectra obtained by Moore and co-workers, suggesting that this mPPE folds into a helical conformation in 90 % H2O/DMSO. Figure 1-13. Postulated associa tion equilibrium between OPEs and chiral molecules. Figure was taken from Prince et.al .76 Figure 1-14. Structures of amphi philic mPPEs studied by Tews group.77

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O u linked P P Figure 1 polymer s decrease amount o Figure 1 Figure 1 u r group als o P E-type CP E 15. The sol v s fold into a in oscillato r o f water in t h O mPPESO 3 15. Struct u 16. UV-vi s composit i varies wi t o has been i n E s with sulf o v ent effect o helical con fo r strength a n h e solvent m n SO3 -Na+ 3 u res of mPP E s absorption i ons. Inset i l t h solvent c o n terested in t h o nate (SO3 -) o n the absor p fo rmation in n d re d -shift o m ixture (wat e m E studied by and emissio l lustrates ho w o mposition. F 30 h e synthesi s carboxylat e p tion and fl u water (Figu r o f the band ( h e r/methanol ) n CO2 N m PPECO2Schanzes g n spectra o f w the fracti o F igure was t s and photo p e (CO2 -) an d u orescence s u r e 1-16).78-8 0 h ypochrom i ) increases. T N a+ g roup.78-80 f mPPESO3 i o n of the ran d t aken from T p hysical pro p d L-alanine ( A u ggests that 0 In the abs o i c effect) ar e T hese are at t O H N mP P i n MeOH/H 2 d o m -coil co T an et. al.79 p erties of m e A la) as sho w the meta -li n o rption spect e observed a s t ributed to O-Na + O H P E-Ala 2 O at differ e nformation e taw n in n ked ra, a s the n + e nt

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31 stacking interactions in the he lical conformation. The changes in fluorescence spectrum provide additional evidence for the solvent-induced trans ition of mPPEs. With the increase of water amount, the intensity of the structured near-UV emission decreases and it is replaced with a less intense, broad, structureless and red-shifted emi ssion. The broad and struct ureless excimer-like emission band arises due to -stacking interactions in the fold ed, helical confor mation of mPPEs. Given this structural similarity between double-helical DNA and helical conformation of our anionic mPPEs (i.e. both are helical polyani ons which contains aromatic units that are -stacked along the helical axis), small molecule DNA intercal ators were expected to interact in a similar manner with mPPEs. A well-known example of intercalative binding to DNA is the metal complex [Ru(bpy)2(dppz)]2+, where bpy = 2,2 -bipyridine and dppz = dipyrido[3,2-a:2 ,3 c]phenazine. This complex has been of particular in terest due to the light-switch effect. It is non-luminescent in water; however, it shows strong photoluminescence when it is bound with DNA via intercalation of the dppz ligand.81, 82 The luminescence quenching is believed to arise due to H-bond interactions betw een water and nitrogen on the phen azine ring, resulting in rapid non-radiative decay of 3MLCT.79 However, the dppz ligand is shielded from the water when the complex is intercalated, and hence the complex displays strong luminescence. Interestingly, the interaction between the Ru comp lex and mPPEs also shows the light-switch effect. As shown in Figure 1-17, in water [Ru(bpy)2(dppz)]2+ is non-emissive; however, the 3MLCT emission from the complex increases upon addition of mPPEs. The emission from [Ru(bpy)2(dppz)]2+ saturates when a ratio of polymer concentration (i n polymer repeat units, PRU) per [Ru(bpy)2(dppz)]2+ is approximately 6:1. This suggests that the interc alated Ru complex occupies a binding site corresponding to one turn of the mPPE helix (there are 6 PRUs per turn).

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32 Circular dichroism (CD) is the most useful t echnique to study chiral structures. Either an enantiomeric excess of chiral molecules or the exis tence of a chiral environment is required for a CD signal. When achiral oligomers or polymers fold into a helical structure, racemic mixtures of the rightand left-handed forms (P and M forms, respectively) would be expected; therefore, CD signal cannot be observed. To provide a clear probe of the solvent effect on the folding process using CD spectroscopy, chirality is introduced to achir al oligomers or polymers by interacting with chiral molecules. Previously, Moore and co-workers used CD to explain the helical conformation of meta -linked OPEs where chiral guest mol ecules added to the achiral OPEs or chiral groups incorporated into conjugated ol igomer backbones to produce an enantiomeric excess in the helical conformation.76 In our previous studies, a small chiral molecule (-)-pinene Figure 1-17. Emission spectra of [Ru(bpy)2(dppz)]2+ (15 M), in absence (solid black line) and presence of mPPE-Ala (0 -120 M polymer repeat units). The in set illustrates the [Ru(bpy)2(dppz)]2+ emission intensity with increasing the concentr ation of mPPEAla. Figure was taken from Zhao et. al.80

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33 interacts with our two polymers mPPESO 3 and mPPECO2 resulting in CD signals.78 For mPPEAla with chiral and optical active sidechains, a strong bisi gnate CD spectrum was observed because the alanine side chain induces an enan tiomeric excess in the M-form of the helical conformation (Figure 1-18). We also examined the effect of helical conformation on the amplified quenching in mPPESO3. N,N-dimethyl-4,4-bipyridium (MV2+) was added to the random-coil and helical conformation of the polymer. Interestingly, the random-coil conforma tion of mPPESO3 is quenched less strongly than the helical conformation of the polymer at any given [MV2+] (Figure 1-19). This effect might be explained by two factors. The firs t one is an increased exciton length in the helical conformati on due to delocalization between -stacked segments. The second one is the fact that MV2+ quencher can interact with a la rge fraction of the chain when it is folded into the helical conformation.79 Figure 1-18. Circular dichroism spectra of mP PE-Ala in methanol, water and methanol/water mixtures. Figure was taken from Zhao et. al .80

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34 Figure 1-19. Emission quenching of mPPESO3by MV2+ in a 7:3 methanol/water mixture. Arrows show how the intensit y changes with increasing [MV2+]. The inset shows the Stern-Volmer plots of random-coil state a nd helix using emission intensities at 363 nm and 445 nm. Figure was taken from Tan et. al .79 Application of Conjugat ed Polyelec trolytes Optical Sensors One of the most exciting applications of CPEs is highly sensitive fluorescence-based sensors for chemical and biological targets due to amplified quenching by small molecules with opposite charge.14 CPEs have been used as sensors in different formats, including homogeneous aqueous solution,83 glass-slide supported CPEs84 and particle supported CPEs.26 In most of the CPE-based sensors, fluorescence is either enhanc ed (turn-on approach) or quenched (turnoff approach) by interacting with targets. Bo th approaches are based on three mechanisms: superquenching, fluorescence resonance energy transfer (FRET) and conformation change. Superquenching mechanism In the superquenching strategy, a quencher-tether-ligand (QT L ) complex is synthesized by combining a quencher to biologi cally interesting ligands. A fl uorescence response is produced when the ligands bind to their specific targets. Whitten and co-workers re ported the first example

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35 of CPE-based sensor using QTL systems.22 They covalently linked MV2+ to a biotin molecule by a short but flexible tether. Biotin has been well -known as an excellent re ceptor for biotin-binding proteins such as avidin and streptavidin. Addition of biotin-methyl viologen (B-MV) to MPSPPV (Figure 1-8) results in quenching of the polymer fluorescence. The quenched fluorescence is recovered by adding avidin th rough avidin-biotin complex form ation (Figure 1-20). Based on the QTL systems, Whitten and co-workers also developed CPE-coated particles to detect enzymatic activity and DNA hybridization.26 Figure 1-20. Detection of avidin using CPE based on the superquenching mechanism. Figure was taken from Chen et. al .22 Fluorescence resonance energy transfer (FRET) mechanism FRET is the non-radiative transfer of photon en ergy from an excited fluorophore (donor) to another fluorophore (acceptor) via dipole-dipole interaction. The FRET efficiency depends on the distance and orientation of the donor and accep tor, and the overlap between the emission of the donor and the absorption of the acceptor. CPEs have been used as light harvesting molecules that transfer excitations to signaling fluorophores attached to biomolecular probes, therefore providing enhanced signal intensi ties and sensitivities. Specific examples of FRET-based sensors were reported by Bazan and co-workers.24, 83 Cationic CPEs based on polyfluorene (PF) (Figure 1-21) were used for the detection of DNA. Th ese CPEs display relatively high fluorescence quantum yields in aqueous solution compared to other CPEs, therefore increasing sensitivity.

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36 Figure 1-21. Structures of CPEs studied by Bazan and co-workers. Figure 1-22 shows a schematic representation of a sensing system based on FRET. This system contains PF1 and a peptid e nucleic acid (PNA) strand labeled with fluorescein (C*) as a probe. Since PNA-C* is neutral, there is no el ectrostatic interaction between the PF1 and PNAC*, and the distance between them is too la rge for effective FRET. Upon addition of complementary ssDNA into the solution of PNA-C*/PF1, PNA hybridized with ssDNA (shown in red). The hybridizatio n provides a hybrid complex bearing C* with negative charges, resulting in formation of complex between PF1 and the hy brid complex via electro static interaction. Figure 1-22. Schematic representation for the use of PF1/PNA-C* to detect a complementary ssDNA. Figure was taken from Gaylord et. al .24

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37 The distance between PF1 and PNA-C* decr eases allowing for efficient FRET. When noncomplementary ssDNA is added, hybridization does not occur and the distance between PF1 and PNA-C* remains too large for FRET. The enhanced emission of fluorescein (C*) provides a probe for the presence of a target ssDNA. Conformation change This sensing m echanism is based on conforma tional change of the conjugated polymer backbone upon complexation with different analyt es, inducing chromic changes, both in the absorption and fluorescence of CPEs. Water so luble poly(thiophene)s have been the most commonly used (Figure 1-23).85-88 Leclerc and co-workers re ported the detection of ssDNA sequences using cationic poly(thiophene), PT2.85, 86 This sensing system is based on different electrostatic interactions and conformational structures betwee n electroactive and photoactive cationic poly(thiophene) derivatives, and singl e-stranded oligonucleotid es or double-stranded (hybridized) nucleic acids. PT2 exist as a ra ndon coil conformation befo re addition of ssDNA (Figure 1-24). Addition of ssD NA forms an electrostatic complex, called duplex between the PT2 and ssDNA, where PT2 exists as a highly conjugated, planar conformation. When a complementary ssDNA is added to the duplex soluti on, triplex form is observed and it displays Figure 1-23. Structures of poly(thiophene) derivatives used in CPE-based sensor via conformational change mechanism.

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38 less conjugated non-planar conformation. Depend ing on the conformational change, PT2 shows different absorption and fluorescence spectra. By monitoring cha nges in photophysical properties of the polymer, ssDNA sequences can be detected. Figure 1-24. Schematic illustration for the detect ion of ssDNA sequences using poly(thiophene). Figure was taken from Ho et.al .86 Biocidal Activity Whitten and co-workers reported that a cati onic CPE (Figure 1-25) shows light-induced biocidal activity against Gram negative bacteria ( Escherichia coli, E. coli, BL 21) and Gram positive bacterial spores ( Bacillus anthracis Sterne, B. anthracis, Sterne).89 Phase contrast and fluorescence microscopy indicate s that polymer is taken up by both microorganisms, and polymer coated on either bacteria or spores displays strong fluorescence (Figure 1-26). Incubation of bacteria with the polymer under th e light shows reduction of bacterial survival. The biocidal activity arises due to the associa tion of the polymers with the cell surface of the bacteria and light-induced activation of the cell surface coated polymer.

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39 Figure 1-25. Structure of cationic CPE used by the Whitten group. Figure 1-26. Phase contrast and fluores cence microscope images of CPE-treated E. coli (upper panel) and CPE-treated B. anthracis Stern spores (lower panel). Figure was taken from Lu et.al .89 Scope of the Present Study The aim of the present study is the synthe sis of poly(arylene ethynylene) conjugated polyelectrolytes with different chemical stru ctures, the investigation of their photophysical properties in solution, and their application to sens ors and antimicrobials.

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40 Chapter 2 describes a fluorescent assay for the detection of enzymes using mPPESO3. The assay is based on the quenching mechanism. Quencher-tether-ligand (QTL) system is added to the CPE, resulting in fluorescence quenchi ng. The fluorescence is recovered by addition of target enzymes or carbohydrates. Chapter 3 and 4 are focused on fundamental unde rstanding of the mechanism of amplified quenching in helical conformational mPPESO3 w ith cationic intercalator quenchers. Both quenchers are functionalized with biotin ligands, which form st rong complexes with proteins such as avidin and streptavidin. We have also been interested in helical stru ctured CPEs in aqueous solution. In Chapter 5, a meta -linked CPE containg pyridine was synthesized. The self-asse mbly behavior in solution was studied using absorption and emission spectroscopy. The helical structure of the polymer was confirmed by the titration experiment using [Ru(bpy)2(dppz)]2+ and the polymer. Quenching experiments were carried out using various metals. In Chapter 6, a series of cationic CPEs with backbones based on a poly (arylene ethynylene) repeat unit and tetr aalkylammonium side groups were designed and synthesized. Their photophysical properties were studied in solution. We investig ated the biocidal activity of the cationic CPEs and the mechanism for the light induced bacteria l killing by the CPEs. In Chapter 7, we synthesized conjugate d polyampholytes carrying both cationic and anionic groups. Their photophysical properties and quenching behaviors were studied in solution.

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41 CHAPTER 2 SENSING OF PROTEASE ACTIVITY US ING META-LINKED POL Y (PHENYLENE ETHYNYLENE) SULFONATE Introduction Enzym es are important targets for the scre ening of noxious toxins and pathologies associated with their presence, and for the deve lopment of effective and selective therapeutics. Proteases90, 91 are specially relevant because they conduct proteolysis, which is the final step in the expression of the activity of a variety of proteins.92 Colorimetric and fluorescence-based homogeneous method assays have been validated for a variety of proteases.93 These assays are based on the develo pment of color or fluorescence in solution as a result of substrate hydrolysis. Although the methods have been commonly used, their sensitivity is limited to micromolar or submicromolar range, and hence it is difficult to measure the initial rates at ultralow substrat e concentrations or at low enzyme activity. To improve sensitivity in proteolytic enzyme assays, CPE-based enzyme assays have been reported.94, 95 Incorporation of CPEs into assays leads to amplification of sensory response. The strategy using CPEs also contains an enzyme-cle avable peptide substrat e covalently bound to a quencher (an electron and/ or en ergy acceptor) that associates w ith CPEs due to electrostatic interaction, leading to fluorescence quenching of the CPEs. In the presence of enzyme, the substrate is cleaved and the uncharged quenc her is released from CPEs, turning on the fluorescence of CPEs (turn-on mechanism) (Fi gure 2-1). For example, Pinto and co-workers used a cationic peptide labeled with a p-nitroanilide ( p-NA) unit and anionic PPESO3 (Figure 22). The p-NA moiety strongly quenches CPE fluorescen ce via electrostatic interaction between them. Addition of protease induces the hydrolysis of the p-NA group, which is neutral and its quenching ability is eliminated. Pinto and co-wor kers also reported a fluorescence turn-off assay to monitor protease activ ity. In the turn-off assay, the bi s-Arg derivative of Rhodamine 110

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(Rho-Ar g quench P amide li n quenche s Figure 2 +Na-O3S Figure 2 In t mPPES O proteoly t shown i n g -2) was us e P PECO2 flu o n kage in Rh o s the polym e 1. Mechan i O O P P O N H R Rho 2. Structur e t his chapter O 3 in an aqu e t ic enzyme, p n the previo u e d. The Rho o rescence. H o -Arg2, pro d e r fluoresce n i sm of the t SO3 -Na + P ESO3 O O O N H R Arg-2 e s of CPEs a we describ e e ous solutio n p eptidase u s u s study wit h Arg-2 is co l H owever, ad d d ucing a mo n n ce by FRE T t urn-on an d n + N H R R a nd quenche e a CPEb as n This assa y s ing a p -NA h PPESO3. A 42 l orless and n d ition of pr o n o amide d e T (Figure 21 d turn-off C O O C COO-N a PPECO2 O OH O R ho-Argr substrates ed enzyme a y is based o n labeled pep t A mplified q u n on-fluoresc o tease induc e e rivative (R h 1 ). C PEb ased e n C OO-Na+ a + O K B NH2 R=used for CP E a ssay using t n a fluoresc e t ide, L-Lysp u enching of ent and it al s e s the hydro l h o-Arg), wh i e nzyme ass a O 2N K -pNA:R=Lys B z-FVR-pNA: R O NH3 (CH2)3 N E b ased en z t he helical c o e nce turn-on p -nitroanilid the mPPES O s o does not l ysis of one i ch strongly a ys. NH R R =ArgValPhe C N HCH(NH2)2 z yme assays o nformatio n sensor for e ( K p NA) a O 3 fluoresc e of C Bz n of a s e nce

PAGE 43

43 is observed with KpNA. Introduction of peptidase reve rses the fluorescence quenching concomitant with peptide hydrolysis To investigate the effect of helical structure on the polymer fluorescence quenching Stern-Vo lmer quenching constants of helical mPPESO3 by KpNA are compared with those of linear PPESO3 in buffe r solutions of varying concentration. mPPESO3 displays more efficient quenching than PPESO3. Additionally, the less e ffect of concentrated buffer on the fluorescence quenching is observed in mPPESO3 compared to PPESO3. Results and Discussion Overview of Protease Turn-on Assay A fluorescence turn-on sensor was investig ated to monitor enzyme activity with mPPESO3 and a cationic peptide labeled with p -nitroanilide ( p-NA) unit (Figure 2-3). mPPESO3 is an anionic meta-linked poly(phenylene ethylene) (PPE) featuring sulfonate side chains. In aqueous solution, the polymer absorbs at max =320 nm and it exhibits an intense fluorescence at max = 450 nm. The p-NA moiety strongly quenches the polymer fluorescence because the cationic peptide ion-pairs with the anionic polymer. In this investigation, L-Lysp-nitroanilide dihydrobromide (KpNA) is chosen as an enzyme substrate, which contains a cationic amino acid residue, lysine. The amplified quenching of the polymer fluorescence is recovered by the addition of peptidase. Upon introducti on of peptidase to a mixture of the p-NA peptide and mPPESO3 hydrolysis of the p-NA group is induced. Since the cleaved p-NA group is not charged, its quenching ability is eliminated. Ther efore, quenching reversal is observed with the peptide hydrolysis. Peptidase Activity Sensing As shown in Figure 2-4, fluorescence spectral changes are observed after addition of KpNA and peptidas e, respectively. The initial fluorescence from 15 M of mPPESO3 (black

PAGE 44

44 Figure 2-3. (a) Structures for a polymer and a quencher substrate. (b) Mechanism of the turnon mPPESO3-based sensor. Figure 2-4. Fluorescence spectroscopic cha nges observed during tu rn-on assay. Initial fluorescence of mPPESO3 (15 M) ( ) in phosphate buffer solution (1.0 mM, pH 7.1); fluorescence after addition of 2 M of K-pNA ( ). Fluorescence intensity of as a function of time after addition of peptidase (0.13 mg / mL): 5 ( --), 20 ( -), 40 ( ), 60 ( --) min. ex = 320 nm.

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45 solid line) is quenched up to ~ 90 % by 2 M of KpNA (red dotted line). After addition of 0.13 mg/ mL of peptidase to the quenched polymer solution, the hydrolysis of KpNA is detected by an increase in fluorescence intensity as a functio n of incubation time. Approximately 45 % of the initial intensity of mPPESO3 is recovered. Th e fluorescence of mPPESO3 is not completely recovered by addition of peptidase due to nonspeci fic interaction of pep tidase with the polymer. This can be supported by the observation, that addition of peptidas e lowers the initial fluorescence from the polymer by ~ 45 %. Addition of peptidase to a mixture of the polymer and KpNA induces hydrolysis of pNAgroups. The quenching efficiency of p-nitroaniline is determined by Stern-Volmer quenching experiments and then compared w ith the quenching efficiency of KpNA. To obtain the same extent of quenching by KpNA, much higher concentration of p-nitroaniline is required as shown in Figure 2-5. Therefore, the quenching ability of the hydrolyzed pNA group is negligible. Concentration Effect of Polymer a nd Buffer on Fluorescence Quenching Sensitiv ity of mPPESO3 was determined by quenching experiments with KpNA at various concentrations of the polymer solutions. The quenching effi ciency increases with a decrease in the concentration of the polymer as shown in Figure 2-6. 5 M of the polymer solution exhibits the most efficient quenching. To investigate the effect of buffer i ons on fluorescence quenching, the quenching experiments were carried out by using 5 M of PPESO3 or mPPESO3, and KpNA at various concentrations of buffer solutions. Table 21 represents the Stern-Volmer constants ( Ksv) obtained for quenching mPPESO3 or PPE-SO3 by KpNA. Ksv was acquired at low concentration of quencher (linear regime). In the previous study, we reported th at the quenching constants of PPESO3 by KpNA decreased with increasing buffer concentration.94 This arises due to an

PAGE 46

46 electronic screening eff ect of the added ions. However, th e helical conformation of mPPESO3 prevents the effect of increased ions on the mPPESO3 fluorescence quenching by K-pNA. Figure 2-5. Stern-Volmer plots for fluorescence quenc hing of mPPESO3 with KpNA (a) and p nitroaniline. (b). [mPPE-SO3] = 15 M, 1.0 mM Potassium phosphate buffer solution, pH7.1. ( ex = 320 nm em = 450 nm)

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47 Figure 2-6. Stern-Volmer plot s of at different concentr ations of mPPESO3 with KpNA in 1.0 mM of potassium phosphate buffer (pH 7.1) ( ex = 320 nm em = 450 nm). Table 2-1. Stern-Volmer Quenching cons tants for mPPESO3 and PPESO3 fluorescence quenching by KpNA KSV, M-1 [Buffer], mM mPPESO3 PPESO3 1 1.54 107 4.79 106 5 1.60 107 2.18 106 10 1.53 107 8.20 105 100 9.50 105 7.38 104 Summary and Conclusions This chapter dem onstrates the application of mPPESO3 for detec tion of proteolytic enzyme activity. The polymer fluorescence is quenched strongly by KpNA and peptidase reverses the fluorescence from the quenched polymer solution concur rent with peptide hydrolysis. mPPESO3 is more efficiently quenched by KpNA compared to PPESO3. More

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48 interestingly, the polymer still shows amplifie d quenching at even high concentration of buffer solution while PPESO3 shows a reduced quenc hing efficiency with increasing buffer concentration. Typically, CPEs show a significantly reduced quenching efficiency in the concentrated buffer solution because the buffer io ns screen the Coulomb interactions, removing quenchers from the vicinity of CPEs. These tw o results are believed to arise due to the intercalation of quenchers into the helical c onformation of mPPESO3 gi ving less ion-screening effect on the Coulomb interaction as well as an enhanced quenching efficiency. Experimental Materials mPPESO3 was synthesized by the S onogashira r eaction as described in the literature.79 All solutions were prepared by using water that was distilled and then purified by a Millipore purification system. Buffer soluti ons were prepared with reagent grade chemicals before use. Concentrated aqueous polymer solutions were diluted with buffer to a final concentration. Avidin, KpNA, and peptidase were purchased from Si gma-Aldrich Inc. All solutions were prepared in an appropriate buffer soluti on before use in th e fluorescence assay. Instrumentation Absorption spectra were obtained on a Varian-Cary 100 UVvisible absorption spectrom eter. Fluorescence spec tra were recorded on a Jobin Yvon-SPEX Industr ies Fluorolog-3 Model FL3-21 spectrofluorometer and corrected by using correction factors generated in-house with a primary standard lamp. General Methods The solutions of m PPESO3, KpNA, and peptidase in 1.0 mM sodium phosphate buffer (pH 7.1) were prepared before use. Enzyme assay was conducted at 37oC. The 2 mL of polymer solution (15 M) was placed in a poly (methyl methacry late) cuvette with 10 mm path length and

PAGE 49

49 the initial fluorescence intensity was recorde d. The polymer solution was incubated with KpNA solution for 10 minutes and then the fluorescen ce intensity was measured again. A peptidase solution was added into the mixture of the polymer and KpNA. Fluorescence intensity was then recorded at 5 min intervals. Fl uorescence intensity was acquired at 450 nm after it was excited at 320 nm.

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50 CHAPTER 3 FLUORESCENCE QUENCHING OF HELI CAL CONJ UGATED POLYELECTROLYTE BY RHENIUM-BIOTIN COMPLEX AS QUENCHER-TETHER-LIGAND (QTL) PROBE Introduction Since the structure of doubl e helical DNA was elucidated,96 the design of small molecules that recognize and react at specific DNA sites has been of considerable interest.97 Over the past twenty five years, transition metal complexes have been studied as reversible DNA binding agents. Metal complexes bind with DNA in se veral different non-cova lent modes including electrostatic and groove binding, intercalation and insertion (Figure 3-1).98 Transition metal complexes are attractive because of the second specific advantages: (1) they exhibit a welldefined coordination geometry because the meta l center acts as an anchor, holding in place a rigid, three dimensional scaffold of ligands that can, if desire d, bear recognition elements. (2) They also possess unique photophysical and el ectrochemical properties to enhance the functionality of the binding agents.98 The unique properties of tran sition metal complexes have been used in a wide range of areas including fluorescent labe ls, DNA foot-printing agents and electrochemical probes.99 Much of the work in this area origin ated from the earliest study of Pt(II) complexes.100 Stimulated by the early work by Barton, Turro and co-workers, a variety of metal complexes including of those of Ru(II), Rh(III), Cu(I) with polynuclear aromatic chelate ligands such as 1,10-phenanthroline have been investig ated as physical and chemical tools for probing and modifying the structures of nucleic acids.101-112 There has been par ticular interest in transition metal complexes that contain the lig and dipyrido[3,2-a:2,3 -c]phenazine (dppz), which bind strongly to DNA via intercalation of the dppz ligand. One of the interesting properties of these complexes is that intercala tion into DNA, resulting in a change in their photophysical properties.81, 113 Specifically, they exhibit weak luminescence in aqueous solution

PAGE 51

51 due to solvent-induced quenching of the luminescent 3MLCT state; however, when bound to DNA, they are moderately luminescent (Figure 3-2). Previously, we reported that [ fac -(dppz)ReI(CO3)(4-MePy)]+ (where 4-MePy = 4methylpyridine) binds to DNA via in tercalation. By analogy to Ru(bpy)2dppz2+, the Re(I) complex is non-luminescent in water, but becomes emissive from 3,* intraligand excited state upon intercalation.113 In 2004, Lo and co-workers reporte d bifunctional Re(I) complexes that contain both an extended planar diimine ligand and a biotin moie ty and hence they can bind to both nucleic acid and proteins to develop new biorecognition reagents.114 These biofunctional Re(I) complexes display a significant emission e nhancement in the presence of either DNA or avidin. We have been interested in the optical and self-assembly properties of water-soluble conjugated polyelectrolytes (CPEs), which feat ure poly(phenylene ethyn ylene) (PPE) backbones with ionic side chains.23, 36, 79, 80, 115-118 These polymers exhibit str ong light absorption, efficient fluorescence, and good transport properties for char ge carriers and excito ns. One property that has been of particular interest in our studies is solvent-induced self-assembly.36, 79, 80 For example, the anionic conjugated po lyelectrolyte PPESO3, which has a para -linked (linear chain) PPE backbone, self-assembles into -stacked aggregates in H2O (a poor solvent) while it exits in a monomeric state in MeOH (a good solvent).36 This result is based on the study of solvent effects on the spectroscopic properties. The polymer also exhibits a stronger quenching efficiency by oppositely charged quenchers in H2O compared to MeOH. This effect has been termed amplified quenching30 and arises due to rapid interc hain exciton migration in the aggregated PPESO3 chains. The amplified quenc hing allows CPEs to be used as a unique platform for the development of highly sens itive fluorescence-based sensor for biological

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Figure 3 Figure 3 1. Three bi intercalat i and (c) m 2. The lig h Zeglis et. nding mode i on and (c) i m etalloinsert o h t-switch eff e al .98 s of a metal nsertion an d o r. Figure w a e ct of dppzb 52 complex w i d geometrie s a s taken fro m b ased metal l i th DNA: (a ) s of (a) groo v m Zeglis et. l o-intercalat o ) groove bin d v e binder, ( b a l .98 o rs. Figure w d ing, (b) b ) intercalat o w as taken fr o o r o m

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53 targets22, 24, 94, 95, 119-124. In contrast to the para-linked PPESO3, the meta -linked PPE-type polymer mPPESO3 folds into a helical conformation in H2O while it exists in a random coil conformation in MeOH.79 The fluorescence of the helical conformation of the polymer is quenched by oppositely charged quenchers more e fficiently, suggesting eith er an increase of exciton length in the helical conformation, which results from delocalization between -stacked segments, or the fact that the quencher ion can interact with a larger fraction of the chain when it is helical. Given the structural similarity between the helical conformation of mPPESO3 and double helical DNA (i.e., both feature aromatic residues that are -stacked along the helical axis and they present negatively charged side groups to the surrounding solv ent environment), DNA intercalators are expected to interact in a similar manner w ith the polymer. Interestingly, [Ru(bpy)2(dppz)]2+, where bpy = 2,2-bipyridine and dppz = dipyrido[3,2-a:2,3-c]phenazine binds to helical mPPESO3 via inter calation of the dppz ligand into the -stacked phenylene ethynylene units. The intercalated complex is strongly luminescent and it also quenches the polymer fluorescence.79 In this chapter, we investigated the interaction between mPPESO3 and two Re(I) complexes. One complex contains the dppz ligan d and the second contains the dppz ligand and a pyridine ligand covalently linked to bioti n. Both Re(I) complexes bind to mPPESO3 via intercalation of the dppz ligand into the polym er, resulting in amplified quenching of the polymer fluorescence. The Re(I) complex containing biotin ligands specifi cally interact with avidin. Effect of avidin-biotin complexation on the interaction betwee n the polymer and the Re(I)-biotin complex was also investigated by observing changes in the polymer fluorescence. Scheme 4-3 shows the structures of CPEs and Re complexes used in this study.

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54 Figure 3-3. Structures and acronym s of polymers and Re complexes. Results Synthesis of Re complexes Re(dppz) (P F6) was prepared from the reaction of [ fac-(dppz)Re(CO)3](Cl) and 4methylpyridine in DMF containing AgPF6.. Re(dppz)-biotin (PF6) was prepared from the reaction of [ fac-(dppz)Re(CO)3(CH3CN)](CF3SO3) and the ligand py-CH2NH-biotin in THF/MeOH and followed by metathesis with NH4PF6. The PF6 salts of two Re complexes were purified by column chromatography and characterized by 1H NMR and positive-ion ESI-MS, and then metathesized to Clform. Photopysical Properties of Re complexes The Re(dppz) com plex displays moderately in tense absorption band with two maxima at 366 nm and 384 nm in aqueous solution. The absorp tion of Re(dppz)-biotin also appears in the near UV as a moderate intense band with two maxima at 368 nm and 385 nm. Both of Re(dppz)

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55 and Re(dppz)-biotin are non-luminescent in a queous solution because their luminescent 3MLCT and 3,* excited states are strongly quenched in aqueous solution by proton transfer (or H-bond formation) from H2O to one of the phenazine nitrogens in the dppz ligand.81, 82 Fluorescence Quenching of mPPESO3 wi th Re (dppz) and Re(dppz)-biotin Transition metal cations, such as Pd2+, Ru2+, Cu2+, and Pt2+ possess high quenching efficiency to conjugated polymer fluorescen ce via electron transfer or energy transfer mechanism.123, 125, 126 Therefore, cationic transition metal Re( ) was chosen to design Re complex containing a dppz ligand, which can bind to mPPE SO3 through both electrost atic interaction and intercalation of a dppz ligand to the polymer. The intercala ting and quenching properties of Re(dppz) were studied by emission titration experiments. Upon addition of Re(dppz) (0 0.48 M) to mPPESO3 aqueous solution, the polymer emission at 450 nm is quenched (Figure 3-4). Similar changes are also observed for quenchi ng of the polymer emission by Re(dppz)-biotin. The fluorescence of mPPESO3 is strongly quenc hed by both Re(dppz) and Re(dppz)-biotin at low concentration. The fluorescence quenching constants ( Ksv) of mPPESO3 by Re(dppz) and Re(dppz)-biotin are 7.56 x 106 M-1 and 7.38 x 106 M-1, respectively (Table 3-1). The Ksv values were obtained in the linear range of 0 0.32 M for Re(dppz) and 0 0.34 M for Re(dppz)-biotin. Table 3-1. Stern-Volmer quenching constants for mPPESO3 and PPESO3 fluorescence quenching by Re complexes K sv (106 M-1)a Re(dppz)-biotin Re(dppz) Only protein avidin BSA avidin avidin BSA mPPESO3 7.38b 1.37c 9.66c 7.56d 3.41c 0.22e 0.23c PPESO3 1.00f 1.10g a Ksv values are obtained from the linear region of the SV plots. b 0 0.34 M. c 0 0.40 M. d 0 0.32 M. e 0 0.60 M. f 0 1.02 M. g 0 0.50 M

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56 Figure 3-4. Fluorescence spectra of mPPESO3 (15 M) in the pr esence of (a) Re(dppz) (0 0.48 M) and (b) Re(dppz)-biotin (0 0.61 M) in a 1 mM phosphate buffer solution (pH 7.4).

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57 The effect of polymer structure on the fl uorescence of polymer/Re(dppz)-biotin complex was investgated using para -linked PPESO3. PPESO3 self-assembles linearly into -stacked aggregates in contrast to helical structured mPPESO3 in aqueous solution. PPESO3 also contains sulfonate side groups to complex with pos itively charged quenchers via electrostatic interactions.36 The fluorescence quenching experiment was carried out un der previously described conditions for mPPESO3. As shown in Table 3-1, PPESO3 is less strongly quenched by Re(dppz)-biotin compared to mPPESO3. Avidin Interactions with mPPESO3/Re(dppz)-biotin The specific biosensor system QTL (quencher-tether-ligand, QTL)22 has been developed to detect biological targets94, 95 in which organic cations such as MV2+( N ,N -dimethyl-4,4bipyridinium) and MV+( N -methyl-4,4-pyridylpyridium) are frequently used as a quencher. Whittens group reported a violgen-type quencher linked to a biotin molecule, which is an excellent acceptor for proteins such as avidin and streptavidin. Recently, Ais group used metal cation rhenium( ) to design a biotin-rhenium ( ) complex as a QTL probe to detect avidin and streptavidin.125 Both studies demonstrate that the fluo rescence of polymers is quenched by quencher-biotin molecules and then addition of avidin to the quenched solution reverses the quenching. Avidin is known to bind strongly with biotin ( K 1015 M-1).127 In addition, at neutral pH, avidin (pI 10.5)128 is negatively charged and theref ore it can be expected to be electrostatically bound to mPPESO3. Knowing the Re(dppz)-biotin binds to avidin through the biotin moiety, we were interested in the fluorescence change of mPPESO3 upon addition of avidin to the polymer-intercalated Re(dppz)-biotin. For this experiment the polyme r/Re(dppz)-biotin complex was pre-formed to induce quenching polymers fluorescence and then avidin was added to the quenched polymer solution in a 4:1 ratio of Re(dppz)-biotin/avidin. On contrary to expectation, the recovery of

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58 fluorescence from the quenched polymer solution wa s not observed after the addition of avidin (data not shown). We conclude from this result that avidin does not displace biotin-Re(dppz) from its mPPESO3-intercalated form to cause the reversal of fluorescence quenching. Fluorescence Quenching of mPPESO3 with Pre-formed Re(dppz)-biotin/avidin Complex Next, the effect of biotin/avidin complexation on the fluorescence of mPPESO3 was investigated. The pre-formed Re (dppz)-biotin/avidin complex (avidin:biotin = 1:4) was added to the polymer. Interestingly, the polymer is less efficiently quenched by the pre-formed Re(dppz)biotin-avidin complex compared to Re(dppz)-bio tin (Figure 3-5). For example, addition of 0.6 M of Re(dppz)-biotin reduces the initial fluores cence of the polymer more strongly (ca. 5-fold) compared to the pre-formed Re(dppz)-biotin/avi din complex. Pre-formed Re(dppz)-biotin-avidin complex also was added to PPESO3 and then Ksv (1.10 x 106 M-1) was obtained at low concentration of regime by lin ear fit (Figure 3-6). There is little change in the Ksv values compared with the Ksv value of the polymer by onl y Re(dppz)-biotin (1.14 x 106 M-1). Similar quenching experiments of mPPESO3 with different ratios of avidin/Re(dppz)biotin mixtures were conducted. Each Re(dppz)-biotin sample was pre-mixed with different ratios of avidin (avidin:biotin = 1:1, 1:2, 1:4, 1:8) and added to the polymer. Emission at 450 nm was recorded at various concentrations of Re (dppz)-biotin (Figure 3-7). Re(dppz)-biotin mixed with different ratios of avidin (avidin:biot in = 1:1, 1:2, 1:4, and 1:8) does not show the pronounced difference in fluorescence quenching. Effect of Specific Protein on Fluorescence Quenching of mPPESO3 by Re(dppz)-biotin As shown in Table 1, pre-mixing avidin with Re(dppz) has a distin ct effect in the quenching efficiency. This is surprising because th is complex does not contain the biotin moiety. The result shows that there is an non-specific interaction betw een the Re(dppz) and the protein which competes with the Re(dppz) binding to the polymer. Ksv value of pre-mixed Re(dppz)

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59 Figure 3-5. Stern-Volmer plots for mPPESO 3 (15 M) quenching by Re(dppz)-biotin, preformed Re(dppz)-biotin/avidin complex (avidin:Re(dppz)-biotin = 1:4) and avidin in1 mM phosphate buffer solution (pH 7.4). Figure 3-6. Stern-Volmer pl ots for PPESO3 (15 M) quenchi ng by Re(dppz)-biotin and preformed Re(dppz)-biotin-avidin complex (avi din:Re(dppz)-biotin = 1:4) in a 1 mM phosphate buffer solution (pH 7.4).

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60 Figure 3-7. (a) Stern-Volmer pl ots for mPPESO3 (15 M) quenchi ng by different ratios of preforemd Re(dppz)-biotin/avidin complex. (b ) Quenching percent of mPPESO3 (15 M) by pre-formed Re(dppz)-biotin (0.6 M )/avidin complex containing different concentrations of avidin in a 1 mM phosphate buffer solution (pH 7.4).

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61 avidin (3.41 x 106 M-1) is about two times less than that of Re(dppz) (7.56 x 106 M-1). Given that avidn nonspecifically disturbs fluorescence quenching by Re(dppz), we examined influence of protein without biotin binding site on fluores cence quenching by Re(dppz)-biotin (Figure 3-8). BSA (pI 4.7)128 was chosen for this experiment. BSA did not cause significant difference in Ksv values of pre-mixed Re(dppz)-biotin/BSA ( Ksv = 9.66 x 106 M-1) and Re(dppz)-biotin (7.38 x 106 M-1). These results suggest that th e biotin moiety on Re(dppz)-biot in complex specifically binds to avidin, but avidin (pI 10-10.5)128 can non-specifically intera ct with negatively charged mPPESO3 via electrostatic intera ction between charged molecules to disrupt polymer quenching by Re(dppz). Figure 3-8. Stern-Volmer plot s of mPPESO3 (15 M) quenching in a 1 mM phosphate buffer solution (pH 7.4) by Re(dppz)-biotin, pre-mi xed Re(dppz)-biotin-BSA mixture (BSA: Re(dppz)-biotin = 1:4) and BSA.

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62 Discussion Re(dppz) and Re(dppz)-biotin bi nd strongly to mPPESO3 via in tercalation of the extended planar dppz ligand within -stacked phenylene ethynylene residue s in the helical conformation. This interaction between the polymer and Re complexes induces a pronounced quenching for mPPESO3 compared with PPESO3. The same result was seen previously with Ru(bpy)2(dppz ), which quenched strongly the fluorescence of mPPESO3 via the intercalative binding.79 The special quenching effect seen with mPPESO3 prov ides evidence that intercalation of the Re complex into the helical conformation of the polymer results in stronger polymer-quencher complex association and electrostatic interacti on. Therefore, efficient quenching of the polymer fluorescence is observe d from mPPESO3. Addition of avidin to the quenched mPPESO 3 solution does not recover the fluorescence of the polymer even though the bio tin-avidin complexation is strong ( K 1015 M). It can be explained by two possibilities. First, the mPPESO3-intercalated Re(dppz)-biotin complex binds to avidin at the same time (i.e. cross-linking of mPPESO3 and avidin; Figure 3-9).129 When avidin binds to biotin, positively charged avidin sticks to the negatively charged polymer and therefore the quencher remains intercalated to result in no reversal of quenching. Second, Re(dppz)-biotin is hidden due to in tercalation and theref ore the biotin may not be accessible to avidin. Re(dppz)-biotin remains intercalated to the polymer, but it does not bind to avidin. Pre-formed Re(dppz)-biotin/avidin (4/1) co mplex gives weaker quenching of mPPESO3 fluorescence than only Re(dppz)-biotin does. Av idin bound to Re(dppz)-bio tin is positively charged at neutral pH and it is attracted to anionic mPPESO3 el ectrostatically. Therefore, the quencher is close to polymer, but it might not intercalate into the polymer resulting in less efficient quenching (Figure 3-10).

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Figure 3 Figure 3 Re intercala t biotin-p o the earli e First, bi o to acces s for the c o 9. Cross-li n 10. Bindin g mPPESO (dppz) and R t ion and sta t o lymer com p e r work of W o cytin-TMR s to biotin, l e o ncurrent bi n n king of mP g of Re(dpp z 3. R e(dppz)b i o t ic interactio p lex gives li t W hittens gr o is buried wi e ading to re v n ding of the PESO3 and a z )b iotin an d Summar y o tin quench e n. However t tle effect o n o up on QTL thin the hel i v ersal quenc h polymer an 63 a vidin by u s d pre-forme d y and Concl e rs strongly q introducti o n the polym e and MPS-P P i cal structur e h ing. Secon d d avidin. W h s ing Re(dpp z d Re(dppz)b usions q uench the m o n of avidin t e r fluoresce n P V. This su g e s polymer, a d Re(dpp)b h en avidin i s z )-biotin. b iotin/avidi n m PPESO3 f l t o the pre-fo n ce. This res u g gests two p a nd hence a v b iotin serves s previously n complex t o l uorescence rmed Re(dp u lt conflicts p ossibilities. v idin is not a as a cross l i bound to bi o via pz)with a ble i nker otin

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64 Re (dppz)-biotin, the Re(dppz)-biotin-avidin complex gives less effect on the fluorescence quenching because of the electrostatic interaction between the oppositely charged polymer and avidin. Experimental Materials All chem icals used for synthesis were of reagent grade and used without purification. Biotin, N-hydroxysuccinimide, 4-picoline and 4aminomethyl pyridine were purchased from Sigma-Aldrich. 1,10-phenanthro line, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide and ophenyldiamine, and silver hexafluorophosphate were obtained from Acros. Rhenium pentacarbonyl chloride and silver trifluoromethanesulfonate were purchased from Strem. Silica gel (Merck, 230-400 mesh) and neutral alumina (Fisher, Brockman grade ) were used for chromatographic purification of Re(d ppz) and Re(dppz)-biotin, respectively. Instrumentation 1H NMR spectra were recorded on either a Varian VXR 300 or Merc ury-300 spectrometer and chemical shifts are reported in ppm relative to TMS. Absorption spectra were obtained on a Varian-Cary 100 UV-visible absorption spectrome ter. Steady state fluorescence spectra were recorded on a spectrof luorometer from Photon Technology In ternational and corrected by using correction factors generated with a primary standard lamp. General Methods Fluorescence quenching All fluorescence quenching experiments of mPPESO3 and PPESO3 were conducted by titrating the polymer solutions with aliquots of Re(dppz), pre-mixed Re(dppz)-biotin, Re(dppz)biotin, pre-formed Re(dppz)-biotin-avidin comple x and avidin, respectively. The fluorescence intensity was recorded at different co ncentration of the quenchers. For mPPESO3, the

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65 fluorescence intensity was acquired with excita tion and emission wavelengths of 320 nm and 450 nm, respectively; and for PPESO3, excitation and emission wa velengths are 420 nm and 540 nm, respectively. The quenching of polymer fluorescence follows a conventional SternVolmer relationship: ][10QK I Isv (3-1) where I0 and I are the intensities of fluorescence in th e absence and presence of quenchers, and [Q] is the concentration of the quencher. Thus, the quenching constant, Ksv defines quenching efficiency. The quenching consta nts were determined from the low concentration range of the Stern-Volmer plots because deviations from the linearity occurred at high quencher concentration. Fluorescence recovery mPPESO3 was titrated with Re(dppz)-biotin to quench the fluorescence followed by the addition of avidin to the quenched mixture solu tion in a 4:1 ratio of Re(dppz)-biotin/ avidin. Synthetic Procedures [ fac-(dppz )Re(CO)3(4-methylpyridine)][Cl], Re(dppz). This complex was prepared according to a literature procedure.113 The following NMR data were obtained by using the PF6 salt of the complex. 1H NMR (300MHz, CD3CN) 2.16 (s, 3H), 7.06 (d, 2H), 8.12-17 (m, 4H), 8.26 (m, 2H), 8.45 (m, 2H), 9.65 (d, 2H), 9.90 ( d, 2H). Positive-ion ESI-MS calcd for C27H17N5O3Re (M+) 646.089, found 646.085; calcd for C21H10N4O3Re (M+-pyridine) 553.031, found 553.028.

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66 S NH HN H N O O N N N Re (CO)3 N N N N Re CO NCMe CO CO N N CF3SO3 -ClS NH HN HO O O S NH HN O O O N O O S NHHN H N O O N ii N N Re CO CO CO Cl N N 4 1 2 3 i 5 iii iv 6 (Re(dppz)-biotin) i.N-hydroxysuccinimide,1-Ethyl-3-(3-dimethylaminopr opyl)-carbodiimide(EDC),DMF;ii.4-Aminomethylpyridine, triethylamine,DMF;iii.1)Silvertriflate,acetonitrile;2)NH3PF6;iv.1)py-CH2-NH-biotin,THF/methanol(3/1,v/v),heat; 2)anionexchangeresin.Figure 3-11. Synthesis of Re(dppz)-biotin. Biotin succinimide (2). To a solution of biotin (1 g, 4.09 mmol) in anhydrous DMF (25 mL), N-hydroxysuccinimide (0.56 g, 4.91 mm ol) and 1-(3-dimethyaminopropyl)-3ethylcarbodiimide (0.94 g, 4.91 mmol) was added. The solution was stirred for 24 h and was recrystallized in 2-propa nol to collect white powder (0.96 g, 2.82 mmol). 1H NMR (300MHz, DMSOd6) 1.41-1.62 (m, 6H), 2.56-87 (m, 8H), 3.09-3.11 (m, 1H), 4.134.34 (m, 2H), 6.37 (s, 1H), 6.42 (s, 2H). Py-CH2-NH-biotin (3). Compound 2 (115 mg, 0.34 mmol) was dissolved in dry DMF (5mL) and triethylamine (190 L, 1.36 mmol) was added. After addition of 4-aminomethyl

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67 pyridine (35 mg, 0.34 mmol) in DMF (5 mL), th e mixture was stirred under argon at room temperature. After stirring for 24 h, DMF was removed and recrystallized from MeOH/Et2O to get white precipitate (0.17g, 0.24 mmol). 1H NMR (300MHz, DMSOd6) 1.32-1.64 (m, 6H), 2.168 (t, 3H), 2.58 (d, 1H), 2.80 (dd, 1H), 3.09-3.11 (m, 1H), 4.11-4.14 (m, 1H), 4.30-4.32(m, 3H), 6.37 (s, 2H), 6.46 (s, 1H), 7.21 (d, 2H), 8.4-8.48 (m, 3H). [ fac-(dppz)Re(CO)3(py-CH2NH-biotin)][Cl], Re(dppz)-biotin (6). This complex was prepared according to a literature procedure.114 PF6 salt was metathesized to Clform in the same way as Re(dppz).113 The following NMR data wa s obtained by using the PF6 salt of the complex. 1H NMR (300MHz, DMSOd6) 1.23-1.43 (m, 6H), 2.27 (t, 2H), 2.60 (d, 1H), 2.8 (dd, 1H), 3.05 (m, 1H), 4.14 (m, 3H), 4.30 ( m, 1H), 6.40 (m, 2H), 7.18 (d, 2H), 8.17-8.21 (m, 2H), 8.368.51 (m, 7H), 9.79 (d, 2H), 9.88 (d, 2H). Positive-ion ESI-MS calcd for C37H32N8O5ReS (M+) 887.177, found 887.173; calcd for C21H10N4O3Re (M+py CH2NH-biotin) 553.031, found 553.028.

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68 CHAPTER 4 FLUORESCENCE RESONANCE ENERGY TRANSFER FROM HELICAL CONJUGATED POLYELECTROLYTE TO CH AR GED FLUORESCENT DYE-LIGAND CONJUGATED (DLC) MOLECULE Introduction Conjugated polyelectrolytes (CPEs) are -conjugated polym ers with ionic side groups which make them soluble in water.23 These materials exhibit strong light absorption, high fluorescence quantum yield and an enhanced que nching effect compared to low molecular weight fluorescence probes.30, 36 These interesting properties have proven the CPEs to be useful for fabrication of sensors for biological targets. 22, 94Another intrinsic charact eristic of CPEs is their ability to self-assemble in to supramolecular structures in solution. Previously, our group showed that an anionic conjugated po lyelectrolyte mPPESO3, which has a metalinked poly(phenylene ethynylene) (PPE) backbone, self-a ssembles into a helical conformation in H2O (a poor solvent) while it ex its in a random coil conformation in MeOH (a good solvent).79 It was also demonstrated that the cat ionic DNA metallo-in tercalator, [Ru(bpy)2(dppz)]2+ binds to the helical structure of mPPESO3 via in tercalation of the dppz ligand to the -stacked phenylene ethynylene units. This effect arises due to th e structural similarity between the helical conformation of m PPESO3 and double helical DNA (i.e., both feature aromatic residues along the helical axis and they present negatively charged side groups to the surrounding solvent environment). Intercalation induces strong luminescence from the non-luminescent Ru complex as well as fluorescence quenching of the polymer. We have an interest in fundamental unde rstanding of the mech anism of amplified quenching in mPPESO3 fluorescence with cationic intercal ator quenchers. To address this issue, we carried out a detailed photophysical investigati on that probes the interaction of the polymer with a quencher. The structures of two different types of CPEs and the qu encher used for this

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69 study are shown in Figure 4-1. mPPESO3 and PP ESO3 are anionic poly(phenylene ethynylene)s (PPEs) that feature sulfonate side groups. In aqueous solution, mPPESO 3 self-assembles into a helical conformation while PPESO3 self-assemble s into an aggregate where the chains are aligned.36, 79 Biotinylated rhodamine, 5 -(and-6)-tetramethylrhodamine biocytin (biocytin TMR), which is a dye-ligand conjugate was selected for this investigation for several reasons. First, biocytin-TMR is a positively charged dye and th erefore interacts with anionic CPEs, mPPESO3 or PPESO3 by ion pairing, resul ting in fluorescence quenching.32, 36 Second, it is well known that rhodamine can intercalate into th e helical structure of DNA and th erefore it can in tercalate into mPPESO3.130, 131 Third, because biocytin-TMR absorbs strongly in the visible region, it efficiently quenches the fluorescence of the polymer via energy transfer from the polymer to the dye. Finally, biocytin-TMR is a biotin-functionalized fluorophore, which is capable of binding to avidin. Water soluble CPEs and a dye-modified lig and were used as a donor and an acceptor, respectively. In this system, the CPEs act as light-harvest ing units which transfer excitation via FRET to a signaling dye to enhance the fluorescence signals of dye-modified ligand. Biocytin-TMR intercalates into the helical conformational mPPESO3, resulting in a decrease in the polymer fluorescence. Our efforts concerned FRET expe riments to biocytin-TMR upon excitation of mPPESO3 with the fluore scence quenching of the polymer. Th e Stern-Volmer relation, given by I0/I = 1+ Ksv[Q] (where I0 and I are the intensity of fluorescence in the absence and presence of a quencher, respectively, Ksv is the Stern-Volmer constant, an d [Q] is the concentration of a quencher) is used to quantitativ ely measure the quenching effici ency. The efficiency of energy transfer can be determined by the extent of emission quenching and relative intensities of acceptor emission in the donor al one or the donor-acceptor pair.

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70 Figure 4-1. Structures and acronyms of conjugated pol yelectrolytes and dye -ligand conjugate used in this study. Results Photophysical Properties. The photophysical properties of three CPEs in aqueous solution are show n in Table 4-1. In aqueous solution mPPESO3 absorbs with max = 320 nm and shows strong blue fluorescence at max = 450 nm. PPESO3 absorbs in the blue of the visible region ( max = 420 nm) and is Table 4-1. Photophysical properties of mP PESO3, PPESO3 and biocytin-TMR in 1 mM phosphate buffer solution (pH 7.4) max abs/ nm max em/nm fl mPPESO3 320 nm 450 nm 0.17a PPESO3 420 nm 540 nm 0.10b Biocytin-TMR 553 nm 580 nm a Anthracene in EtOH was used as a standard, fl = 0.27, see ref. 132, 133. b From ref. 36

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71 strongly fluorescent at max = 540 nm. For biocytin-TMR, the absorption maximum appears at 553 nm and the emission maximum is at 580 nm. The fluorescence quantum yields for the CPEs vary in the sequence mPPESO3 > PPESO3. FRET from mPPESO3 to Biocytin-TMR O N(CH3)2 C O O C O HN(H2C)5HN C O H C NH3 + HN(HC)4 C O (CH2)4 S N H H N O = FRET 320nm 590nm Figure 4-2. Energy transfer fr om mPPESO3 to biocytin-TMR. Our strategy for CPE-sensitized biocytin-TMR emission is illustrated in Figure 4-2. Rhodamine is known to have a pr opensity to intercalate into DNA130, 131 and therefore biocytinTMR is expected to intercalate into the heli cal structured polymer mPPESO3. This brings biocytin-TMR within close proximity of the polymer. Additionally, biocytin-TMR is a positively charged dye, and hence it forms an electrostatic complex with the polymer. After the excitation of the polymer, biocytin-TMR quenches the fluor escence of mPPESO3 via singlet-singlet energy transfer, where the polymer is the donor and biocytin-TMR is the acceptor. The occurrence of the energy transfer is confirmed by the observa tion of sensitized fluores cence from biocytinTMR concomitant with fluorescence quenching of the polymer. As described by Frster, dipole-dipole inte raction leads to long -range resonance energy transfer from a fluorescent donor to an acceptor. The rate of energy transfer (kt(r)) depends on the donor-acceptor distance ( r ), the orientation factor ( ), and the overlap integral (J ( )) as described in Eq. 4-1:134, 135

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72 (4-1) R= 9000 ln 10 QD2J ( ) 1285NAn4 1/6 (4-2) (4-3) where, D is the lifetime of the donor in the absence of an acceptor, R0 is the Frster distance, QD is the quantum yield of the donor in the absence of an acceptor, NA is the Avogadros number, n is the refractive index of the medium, FD( ) is the donor emission, and A ( ) is the acceptor absorption. The distance between the donor (mPPESO3) and the acceptor (biocytin-TMR) is controlled by intercalation and electrostatic interaction to sa tisfy the distance requirement (< 10 nm) for energy transfer. The overlap inte gral provides the information about how the spectral overlap between the donor emission and the acceptor absorption affects the rate of energy transfer. Figure 4-3 shows the fluorescence of the two CPEs with the absorption and fluorescence of biocytin-TMR in aqueous solution. As a consequence of red-shift of the polymer fluorescence, spectral overlap between the emission of the polymer donor and the absorption of the biocytin-TMR acceptor varies systematically. The PPESO3 fluorescence spectrum is redshifted compared with the mPPESO3 fluorescen ce, and hence the overlap between PPESO3 fluorescence and biocytin-TMR absorption spectrum is larger than that between mPPESO3 and biocytin-TMR as seen in Figure 4-3. Figure 4-4 illustrates the fl uorescence spectra of mPPESO3 (15 M) in aqueous solution during titration with biocytin-TMR (0 to 0.225 M). As biocytin-TMR is added to the polymer solution, the fluorescence of the polymer is quenched ( em = 450 nm) and it is replaced by a red-

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73 Figure 4-3. Normalized absorpti on spectrum of biocytin-TMR () and normalized fluorescence spectra of biocytin-TMR ( --) and CPEs ( ) in 1 mM phosphate buffer (pH 7.4). ex = 553 nm for biocytin-TMR, ex = 320 nm for mPPESO3 and ex = 420 nm for PPESO3.

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74 shifted fluorescence ( em = 590 nm) from biocytin-TMR. Dir ect excitation of the solution containing only biocytin-TMR at 320 nm provi des supplementary information to the energy transfer from the polymer to biocytin-TMR. As seen in Figure 4-5, an 82-fold increase in biocytin-TMR fluorescence intensity is observed in the presence of the polymer, whereas the direct excitation of only biocyt in-TMR solution lead to weak fluorescence. The excitation spectrum of biocytin-TMR-int ercalated mPPESO3 also suppo rts the premise that the fluorescence at 590 nm originates from the pol ymer-biocytin-TMR complex (Figure 4-6). The integrated absorption intensity around 320 nm is 24-fold higher than that around 550 nm. This result is consistent with the amplified fluorescence intensity of biocytin-TMR in the presence of the polymer. Figure 4-4. Normalized fluores cence spectra of mPPESO3 (15 M) upon addition of biocytinTMR (0.225 M) in phosphate buffer (1 mM, pH 7.4). ex = 320 nm.

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75 Figure 4-5. Normalized fluorescen ce spectra of biocytin-TMR (0.225 M) in the absence () and presence ( --) of mPPESO3 (15 M) in phosphate buffer (1 mM, pH 7.4). ex = 320 nm.

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76 Figure 4-6. Excitation spectrum of mPPESO3 (15 M)/ biocytin-TMR (0.225 M) complex in phosphate buffer (1 mM, pH 7.4), em = 590 nm. Anisotropy Measurements Fluorescence anisotropy was used to provide insight into the intera ction between mPPESO3 and bioctyin-TMR (Figure 4-7). Rapid internal rotation and en ergy transfer are two major mechanisms for a loss of anisotropy. The excitation spectra of both mPPESO3/biocytinTMR complex and biocytin-TMR were collected in the region of 400 570 nm at the four different polarization confi gurations: VV, VH, HH, and HV after emission at 580 nm corresponding to the maximum emission wavelengt h of biocytin-TMR. The anisotropy value, r at each wavelength was determined from the standard equation given by r = (IVV GIVH)/(IVV + 2GIVH), where IVV and IVH are the vertical and perpendicular emission intensities when vertically polarized excitation is used, and G is an instrume ntal correction factor, G = IHV/IHH.136 The introduction of the polymer into biocytin-TMR re sults in an increase in the measured anisotropy

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77 in the absorption range (450 nm nm) of bi ocytin-TMR. This is due to the decreased rotational rate of biocytin-TMR when it is intercalated into the polymer.136 The anisotropy is primarily determined by rotational motion of a fluorophore (biocytin-TMR). In the polymer intercalated-biocyitn-TMR, the size of the comp lex results in an increase in the anisotropy between 450 nm and 560 nm. Therefore, the an isotropy observation provides evidences for the intercalation of biocytin -TMR into the polymer. Figure 4-7. Excitation anisotropy of biocytin-TMR/mPPESO3 complex ( ) and biocytinTMR ( ). [mPPESO3] = 15 M, [biocytin-TMR] = 0.225 M in phosphate buffer (1 mM, pH 7.4), em = 580 nm. Fluorescence Quenching of PPESO3 by Biocytin-TMR The effect of polym er structure on the fluorescence of polymer/b iocytin-TMR complex was examined using PPESO3. This experiment was conducted under the previously described conditions for mPPESO3. As mentioned before, para-linked PPESO3 self-assembles linearly into -stacked aggregates in cont rast to helical structured mPPESO3 in aqueous solution. PPESO3 also contains sulfonate side groups to complex with positively charged quenchers via

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78 electrostatic interactions.36 Changes in the fluorescence in tensity of PPESO3 (15 M) upon addition of biocytin-TMR (0 to 2 M) were monitored by excitation of the polymer at 420 nm. As seen in Figure 4-8, the sensitized biocyt in-TMR fluorescence did not appear, whereas a decrease in the polymer fluorescence intensity was observed (Figure 4-8). Figure 4-8. Normalized fluorescence spectra of PPESO3(15 M ) upon addition of biocytinTMR (0 0.2 M) in phosphate buffer (1 mM, pH 7.4). ex = 420 nm. Avidin binding to Biotin on Biocytin-TMR/CPEs complex Quencher-tether-lig and (QTL)-based protein se nsors have been widely used because QTL probes are able to undergo competitive binding between CPEs and the target proteins.22 Biotin and avidin were selected as the ligand and the target protein due to their high affinity binding. Avidin contains four identical binding sites for biotin and the binding affinity is known to be very high (Ka 1015 M-1).127 In previous studies it has been shown that the CPE fluorescence intensity decreases upon addition of the biotin conjugated quencher, as a result of electrostatic interactions between the CPE and the quencher. Addition of avidin disrupts the CPE/QTL

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79 complex due to the strong binding between the avidin and biotin of the QTL. Once the QTL molecule is trapped by avidin, the quenching proce ss is attenuated, resulting in an increase in the CPE fluorescence intensity. The results shown above indicate that the fl uorescence intensity of mPPESO3 is quenched by the biotin-functiona lized QTL probe, biocytin-TMR. Theref ore, the recovery of the quenched fluorescence was expected by speci fic binding between biotin on th e biocytin-TMR and avidin. For the experiments of fluorescence recovery, av idin was added to the quenched biocytinTMR/mPPESO3 complex solution in a 4:1 molar ra tio of biocytin-TMR/avi din (Figure 4-9). In contrast to our expectation, the quenched fluor escence was not recovered. A small decrease in sensitized fluorescence intensity from biocy tin-TMR at 590 nm was observed upon addition of Figure 4-9. Fluorescence spectra of mPPESO3 solution upon additi on of biocytin-TMR () and avidin (---). [mPPESO3] = 15 M,[bioc ytin-TMR] = 0.225 M and [avidin] = 0.065 M in phosphate buffer (1 mM, pH 7.4). ex = 320 nm.

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80 avidin. This reduced fluorescence might arise due to the binding interact ion between biocytinTMR and avidin, resulting in quenching of the bi ocytin-TMR fluorescence as seen in Figure 410. Introduction of avidin (0 to 0.05 M) into on ly biocytin-TMR solution (0.2 M) significantly decreases the fluorescence inte nsity of biocytin-TMR. Figure 4-10. Fluorescence spectra of biocytin-TMR (0.20 M) upon addition of avidin (0 0.05 M) in phosphate buffer (1 mM, pH 7.4). ex = 550 nm. Fluorescence Quenching of mPPESO3 by Pre-formed Biocytin-TMR/a vidin complex Since it was found that addition of avidin di d not induce an increase in the fluorescence from the quenched mPPESO3/biocytin-TMR comple x, we were interested in the interactions between the polymer and a pre-formed biocytin-TMR/avidin complex. For this experiment, the biocytin-TMR/avidin complex (a vidin:biocytin-TMR = 1:4) wa s pre-formed and then it was added to mPPESO3. Changes in mPPESO3 fluorescence intensity at 450 nm were observed after excitation of the polymer at 320 nm. The same experiment was conducted using PPESO3 instead

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81 of mPPESO3. PPESO3 was excited at 420 nm and fluorescence intensities were obtained at 540 nm. Figure 4-11 shows the Stern-Volmer pl ots for quenching of mPPESO3 and PPESO3 fluorescence with KSV values. Addition of biocytin-TMR/a vidin complex strongly quenches the mPPESO3 fluorescence ( KSV = 2.6 106); however the KSV value is 4.2-fold lower than that of the solution containing only biocytin-TMR. For PPESO3, the KSV value of the biocytinTMR/avidin complex is 10-fold lower compared to biocytin-TMR. Figure 4-12 displays the polymer-sensitized biocytin-TMR fluorescence inte nsity as a function of the concentration of biocytin-TMR after addition of biocytin-TMR or biocytin-TMR/avidin complex to the polymers. As described before, PPESO3 did not show the se nsitized biocytin-TMR fluorescence at 590 nm and there is a little decrease in the fluorescence intensity at 5 90 nm by both biocytin-TMR and biocytin-TMR/avidin complex. In contrast, mP PESO3 exhibits a 21-fold increase in the fluorescence intensity at 590 nm after addition of 2.0 M of biocytin-TMR, whereas the same concentration of avidin bound biocytin-TMR induces only a 5-fold enhancement in the fluorescence intensity at 590 nm. To investigate th e influence of an avidin concentration on the polymer fluorescence, different ratios of pre-formed biocytin-TMR/avidin complexes (avidin:biocytin-TMR = 1:1, 1:2, 1:3 and 1:4) were prepared a nd then added to the mPPESO3 solution. Avidin consists of four identical binding sites and each biding site is capable of binding one biotin ligand. Figure 4-13 (a) shows the Stern-Volmer plots for mPPESO3 fluorescence quenched by different ratios of biocytin-TMR to avidin. When four binding sites bind to biocytin-TMR, the least efficient fluorescence qu enching is observed. With an increase in the avidin concentration the extent of the polymer fluorescence quenching increases in the sequence of 1:3 < 1:2 < 1:1. The polymer-sensitized biocytin-TMR fluorescence intensities are also collected at 590 nm. As seen in Figure 4-13 (b ), 1:4 ratio of biocyt in-TMR/avidin complex

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82 Figure 4-11. Stern-Volmer plots of mPPE SO3 (15 M) and PPESO3 (15 M) fluorescence quenched by biocytin-TMR or pre-form ed biocytin-TMR/avidin complex in phosphate buffer (1 mM, pH 7.4). KSV values were calculated in the range of 0 0.2 M except mPPESO3/biocytin-TMR (0-0.1 M). ex = 320 nm and em = 450 nm for mPPESO3. ex = 420 nm and em = 540 nm for PPESO3. Figure 4-12. Sensitized biocytin-TMR fluorescence in tensity at 590 nm after addition of biocytin-TMR or pre-formed biocytin-TMR/avidin complex to CPEs in phosphate buffer (1 mM, pH 7.4). ex = 320 nm for mPPESO3. ex =420 nm for PPESO3.

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83 Figure 4-13. (a) SV plots for fluorescence que nching of mPPESO3 (15 M) at 450 nm and (b) polymer-sensitized biocytin-TMR fluore scence at 590 nm upon addition of preformed biocytin-TMR/avidin complex ([biocytin-TMR] = 0 0.20 M, avidin:biocytin-TMR = 1:1, 1: 2, 1:3 and 1:4) in phospha te buffer (1 mM, pH 7.4). ex = 320 nm.

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84 induces a 5.8-fold increase in the polymer-sensi tized fluorescence intensity. An increase in the concentration of avidin enhances the sensitized fluorescence intens ity at 590 nm in the order of 1:3 < 1:2 < 1:1. This result is c onsistent with the above result where the pre-formed biocytinTMR/avidin less strongly quenches the polymer fluorescence and induces the weaker FRET signal from biocytin-TMR. Discussion Fluorescence quenching and FRET studies shows that CPE-sensiti zed biocytin-TMR fluorescence is different for helical structured m PPESO3 and linearly -stacked PPESO3 under the same experimental conditions. Both polymer s fluorescence intensities are quenched by biocytin-TMR; however, only mPPESO3/biocytin -TMR complex displays the sensitized biocytin-TMR fluorescence at 590 nm. FRET occurs through dipole-dipole interactions and the energy transfer rate is given by Eq. 4-1. The di fference between the two polymers in the FRET result can be explained using this equation. Sin ce the two polymers have different conformations in aqueous solution, different tr ansition moment orientations be tween the polymers and biocytinTMR can be expected. Additionally, the polymers exhibit similar spectral overlaps between the polymer fluorescence and the biocytin-TMR absorption; howe ver, the quantum yield of mPPESO3 is 1.7-fold higher than PPESO3 in aque ous solution. As seen in Eq. 4-2, the quantum yield of the donor (CPEs) QD determines the Frster distance R0, therefore any variations in the donor quantum yield might influence the energy tr ansfer efficiency. The polymer self-quenching upon complexation with biocytin-TMR due to aggr egation of polymer chains gives a negative effect on the polymer-sensitized biocytin-TMR fluorescence.32, 137 More efficient fluorescence quenching and FRET efficiency of mPPESO3 can be explained by the intercalation of biocytin-TMR to the helical polymer. This helix is believed to cause the strong complexation be tween the polymer and biocytin -TMR via interc alation with

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85 electrostatic interaction. Anisotropy measurements support the binding mechanism between mPPESO3 and the positive intercalator quenche r, biocytin-TMR. The polymer-intercalated biocytin-TMR shows an increased anisotropy value due to the change of the complex size via the interaction. Once mPPESO3 fluorescence is quenched by bi ocytin-TMR, the fluorescence cannot be recovered by addition of avidin. Th is suggests that avidin is not able to displace biocytin-TMR from binding to mPPESO3 by intercalation. This result can be explained by two possibilities. First, the mPPESO3-intercalated biocytin-TMR complex binds to avidin at the same time (i.e., cross-linking of m PPESO3 and avidin).129 When avidin binds to biotin, positively charged avidin is attached to the negatively charged polymer and therefore the quencher remains intercalated, resulting in no reversal of quenc hing. Second, biocytin-TMR is hidden due to in tercalation and therefore the biotin may not be accessible to avidin. Biocytin-TMR remains intercalated to the polymer, but it does not bind to avidin. Pre-formed biocytin-TMR/avidin quenches less strongly the two polymer fluorescence intensities. This complex also induces the w eaker sensitized fluorescence from the biocytinTMR compared to the solution c ontaining only biocytin-TMR. Such difference is attributed to the positive charge of avidin at neutral pH. The positively charged avidin interacts with the polymers electrostatically. Therefore, the quenche r might not intercalate to the polymer even though it is close to the polymer. This results in less efficient fluorescence quenching and weaker FRET emission signals. Summary and Conclusions In this study, fluorescence que nching and FRET of the anionic conjugated polyelectrolytes, mPPESO3 a nd PPESO3, were investigated in aq ueous solution to understand the mechanism of amplified quenching of mPPESO3 by a cationic intercalator que ncher. We have shown that

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86 mPPESO3 possibly takes advantage of its helical conformation as a fluorescence resonance gate to transfer the polymer excitati on to biocytin-TMR intercalated within the polymer. Therefore, biocytin-TMR fluorescence can be very efficiently sensitized by mPPESO3 and the energy transfer results in amplified fluorescence quenching of mPPESO3. Once the biocytin-TMR intercalates to mPPESO3, avidin is not able to displace the biocytin-TMR from the polymer. This result suggests that either biocytin-TMR is hidden within th e polymer or serves as a cross linker for the concomitant binding of the polymer and avidin. Pre-formed biocytin-TMR/avidin give a little effect on the fluorescence que nching and FRET because of the electrostatic interaction between the oppositely charged polymer and avidin. Experimental Materials The positively charged 5-(and-6)-tetram ethyl rhodamine biocytin (biocytin TMR) and avidin were purchased from Invitrogen TM a nd Sigma, respectively. All chemicals used for synthesis were of reagent grade and purchase d from Sigma-Aldrich Chemical Company. All sample solutions were prepared by using water th at was distilled and then purified by a Millipore purification system (Millipore Simplicity Ultr apure Water System). Buffer solutions were prepared with reagent-grade materials (Fisher). The polymer stock solution was diluted with buffer to a final concentration of 15 M. All con centrations of polymers ar e provided in polymer repeat unit concentration (PRU). A concentrated stock solution of biocytin-TMR and avidin was prepared in buffer to obtain the desired concentr ations. All assays were conducted in 1.0 mM potassium phosphate buffer (pH 7.4). Instrumentation NMR spectra were recorded on either a Vari an VXR 300 or Mercury-300 spectrom eter and chemical shifts are reported in ppm relative to TMS. Absorption spectra were obtained on a

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87 Varian-Cary 100 UV-visible absorption dual beam spectrophotometer, with a scan rate of 300 nm/min. Steady state fluorescence spectra were recorded on a spectrofluorometer from Photon Technology International and corrected by using correction factors generated with a primary standard lamp. General Methods Fluorescence quenching experim e nts were carried out by micr otitration in a fluorescence cuvette. In titration quenching experiments, 2 mL of polymer solution was placed in 1 cm fluorescence cuvette. Then fluorescence spectra were repeatedly acquired after addition of microliter aliquots of a c oncentrated solution that contained bi ocytin-TMR, avidin or pre-formed biocytin-TMR/avidin complex.

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88 CHAPTER 5 META-LINKED POLY (PHENYLENE ET HYNYLE NE) SULFONATE CONTAINING PYRIDINE Introduction Many natural m olecules self-assemble into diffe rent conformations in solution, such as the helical, double-stranded form of DNA. These ordered confor mations are stabilized by noncovalent interactions incl uding hydrogen bonding, hydrophobic, and van der Waals forces.138 The self-organization of nonbiological macromolecules has b een challenged using the noncovalent interactions. Fold ed structures induced by hydr ogen bonds or metal-ligand coordination interactions have been well understood;139-142 however, there are a few reports on the solvophobic74, 77, 79, 143 or interactions144, 145 used to control ordered conformations. Supramolecular -stacked assemblies have been observed in phenylene ethynylene architectures bearing a flexible polar group substituted to a rigid, arom atic backbone. Figure 5-1 shows representative examples including hexameric macrocycles, para -linked rigid rod segments, and the more conformationally diverse ortho and meta -linked oligomers.146 For example, hexameric macrocycles with appropriate side chains form stacked dimers and higher order aggregates in solution due to -stacking interactions.147 The strength of -stacking interactions has been known to be strongly dependent on solvent polarity.146 For meta -linked oligo(phenylene ethynylene)s with a polar tri(ethylene glycol) ester-linked side chains they selfassemble into a -stacked helical conformation in polar solvents due to solvophobic interactions.75, 146 The conformational transition is mon itored by absorption and fluorescence spectroscopy. Solvent-induced self-assembly of meta -linked PPEs into a helical conformation was also reported in our group.79 On the basis of solvent effects on the spectroscopic properties, we conclude a m eta -linked poly(phenylene ethynylene) ( PPE)-type CPE adopts a random coil

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89 Figure 5-1. Structures of phenyl ene ethynylene (PE) based macroc ycle and oligomers used for -stacked self-assemblies. Fi gure was taken from Prest et.al.146 Figure 5-2. Schematic diagram illustrates the self-assembly of disk-shaped phenylene ethynylene macrocycles (a) and oligomers (b). Figure was taken from Prest et.al.146

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conform a mixture o with a d e emissio n with the due to th Additio n to the de l A v ions, me t the possi oxidatio n containi n and fluo r density c Figure 5 a tion in met h o f the two s o e crease in o s n is also obs e spectra obt a e -stackin g n ally, fluore s l ocalization v ariety of c o t al cations a n bility of tun i n N-alkylat i n g 2,2 b ipy r r escence ba n c hanges wit h 3. Structur e Figure w a h anol and a h o lvent syste m s cillator stre n e rved in the f a ined by Mo o g between p h s cence quen c of singlet e x o njugated p o n d acids. C o i ng their op t i on, and met a r idyl and ter p n ds upon ad d h in the poly m e s of conjug a s taken fro m h elical conf o m increases, n gth. A less f luorescenc e o re and cow h enylene eth y c hing is mor x citon in the o lymers hav e o njugated po t ical propert i a l complex a p yridyl liga n d ition of me t m er backbo n ated polym e m Wang et. a 90 o rmation in a long wav e intense, bro a e spectrum. T w orkers for h y nylene rin g e strongly a m helix. e been studi e lymers with i es by electr o a tion of pyri d n ds (Figure 5 t als.148, 149 T h n e, when the e rs containi n al and Kim u water. As t h e length abs o a d, unstruct u T hese spect r h elical conf o g s in the hel i m plified in t e d as chemo s pyridine u n o n acceptin g d yl nitrogen 5 -3) display h ese change polymers i n n g bipyridyl u ra et. al.148, h e amount o f o rption b and u red and re d r al changes a o rmations. T h i cal confor m t he helical c o s ensory mat e n its in the ba c g ability, Np Conjugate d changes in t s arise due t n teract with m and terpyri d 149 f water in th e is re d -shift e d -shifted a re consiste n h e changes a m ation. o nformatio n e rials for an c kbone pro v p rotonation, d polymers t heir absorp t o the electr o m etal ions. d yl ligands. e e d n t a rise n due v ide Nt ion o n

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91 Incorporation of meta substituents into pol y(phenylene ethynylene) polymers backbone improves the solubility and processability of the polymers.150 This incorporation also can modify the molecular conformation of the polymers from a rigid rod-like conformation into a flexible coil-like conformation. Winter et. al. reported that poly(pyridyl/phenyl ethynylene) polymers showed different rigidity/flexibi lity depending on the ratio of parato meta -pyridine linkages within the polymer backbone (Figure 5-4).151 The polymer containing 100 % para-pyridine linkages shows linear conformati on. With increasing proportion of meta -pyridine linkages, the linearity of the conformation decreased, and henc e blue shift of the ab sorption and the emission maximum was observed. Figure 5-4. Structures of poly( pyridyl/phenyl ethynylene) polymers with varying ratios of para/ meta -linked constitutional units. Fi gure was taken from Winter et.al .151 In 2004, Huang et. al. designed and synt hesized a poly(p-phenylene ethynylnene) derivative containing meta -substituted monopyridyl groups (Polymer 1 in Figure 5-5).152 The polymer binds selectively with Pd2+ ions over other metal ions and forms interpolymer interaction through the palladium -pyridyl coordination, resulti ng in fluorescence quenching.

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92 Three years later, Li et.al. reported a meta -poly(phenylene ethynylene) containing pyridine units ( m -PPY) (Figure 5-6) 153 The polymer undergoes a solventinduced conformational transition from an extended coil structure to a helical stru cture. Interestingly, the polymer also shows the conformational change by protonation or complexation with a metal ion (Ag+). Figure 5-5. Structures of a poly( p-phenylene ethynylnene) derivative containing meta substituted monopyridyl groups. Fi gure was taken from Huang et.al .152 Figure 5-6. Structure of a meta -poly(phenylene ethynylene) c ontaining pyridine units ( m -PPY). Figure was taken from Li et. al.153 In this chapter, we synthesized mPPE-SO3 -py, which is a PPE-type CPE containing meta linked pyridine rings in the pol ymer backbone. The solvent-depe ndent conformational transition of this polymer was studied by absorption and fluorescence spectroscopy. The incorporation of the metasubstituted monopyridyl units can improve the spatial matching for selective binding. The polymer shows a great affinity for the Pd2+ ion. The conformational change was also observed upon protonation and metal complexa tion of the pyridine rings on the polymer backbone.

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93 Results and Discussion Synthesis The synthesis of m PPE-SO3-py containing pyr idyl units in the polymer backbone was carried out with 2,6-pyridyldiacetylene ( 1) and sodium 3-(3,5-diiodophenoxy)propane-1sulfonate ( 2 ) under Sonogashira coupling conditions (Figure 5-7). The structure of the polymer was characterized by using 1H NMR and 13C NMR in DMSO-d6. O SO3 -Na+ N N 1 O I I SO3 -Na+ 2 + Pd(PPh3)4/CuI (i Pr2)NH/DMF/H2O n mPPE-SO3-py Figure 5-7. Synthesi s of mPPE-SO3-py. Photophysical Characterization Previously, the water-m ethanol solvent syst em was used for the investigation of CPE folding behavior.36, 79 The same solvent system was used to investigate solven t-induced helical folding of mPPE-SO3-py. Table 5-1 summarizes the photophysical properties of the polymer. The absorption spectrum of mPPE-SO3-py exhibits two featured absorption bands at 387 nm and 306 nm in methanol (Figure 5-8). With an increasing amount of water in the methanol-water solvent mixture, the oscillator st rength decreases. And a more pronounced decrease is observed in the long wavelength band. Thus, the ratio of oscillator strength for 387 nm and 306 nm is smaller in water than that in methanol. Add itionally, the long wavelength band is red-shifted from 387 nm to 396 nm. These spectral change s support that the polymer undergoes a randomcoil to a helix. The reduced osci llator strength and red-shifted bands (hypochromic effect) is believed to arise due to -stacking interactions in the helix conformation.

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94 Figure 5-8. Normalized absorp tion and emission spectra of mPPE-SO3-py (15 M) in water, methanol and mixture of the two solvents. ex = 390 nm.

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95 Table 5-1. Photophysical pr operties of mPPE-SO3-py max abs/nm max fl/nm b fl MeOH 306, 387 464, 680 0.015 0.001a H2O 310, 396 680 0.003 0.0003a a Coumarine 30 in MeOH as standard, fl = 0.307 (ref.154). The solvent-induced conformational change of the polymer is also supported by the changes in the polymer fluorescence spectru m (Figure 5-8). In me thanol the polymer fluorescence ( fl = 0.015) is characterized by a structured band centered at 464 nm with a weak and very broad excimer like emission band around 680 nm. The fact that the structured emission dominates in methanol solution suggests that the polymer exists mainly in a random coil; however, the broad excimer like emission indicat es that a fraction of the polymer exists in a helical conformation even in pure methanol solution.80 Interestingly, as the amount of water increases in the methanol-water solvent mixtur e, the intensity of structured emission is significantly reduced. In water, the structured emission is completely quenched and the broad emission is dominant ( fl = 0.003). This is consistent w ith the changes in the absorption spectrum. These spectral results suggest that th e polymer predominantly exists in a random coil conformation in methanol whereas the polymer is mostly folded into a helical conformation in water. Fluorescence Quenching of mPPE-SO3-py by Metal ions in w ater The absorption and fluorescence spectra already showed that mPPE-SO3-py folded into a helix by -stacking interactions in water. In this he lix, nitrogen atoms of the pyridine rings are located inside the pore, whereas negatively charged side chains extend outside. Due to the good complexation ability of pyridine nitrogen atoms inside the pore of the polymer helix, the significant response of mPPE-SO3-py to various metal ions was expected. To investigate the

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96 interaction of the polymer with me tal ions, aliquots of solutions of metal ions were added to the polymer solution in water and then changes in th e polymer fluorescence intensity were observed. As seen in Figure 5-9, addition of Pd2+ metal ions to the polymer results in strong fluorescence quenching with an excelle nt selectivity for Pd2+ over other metal ions. This quenching is believed to arise due to a photo-i nduced electron transfer (PET) pr ocess or energy transfer (ET) process.155 Cu2+, Fe3+ and Cr3+ slightly quench the polymer fluorescence compared to Pd2+ whereas Ni+ and Li+ does not show any quenching ability. Additionally, Ag+, Ca2+, Cd2+, Zn2+, K+ and Mg2+ relatively enhanced fluorescence intensity compared with the fluorescence intensity of the polymer in the absence of metal ions Figure 5-10 shows the ratio of fluorescence intensities for the absence (I0) and presence of 20 M of metal ions (I). Protonation and Metal Complexation with Pd2+ As shown in Figure 5-11, protonation does not lead to any effect on the absorption and fluorescence spectrum of the polymer solution in water. The helical structured polymer shows stability to introduction of HCl solution. Howe ver, mPPE-SO3-py in methanol undergoes a significant reduction of oscillat or strength in the long wavele ngth band. Thus, the ratio of oscillator strengths for the 387 nm and 307 nm is smaller with an incr easing concentration of HCl solution. Additionally, with an increasing concentration of HCl solution the polymer emission intensity decreases with appearance of the excimer like emission, which is typical of stacked aromatic residues.156 These spectral observations suggest the conformation transition of the polymer from a random-coil conformation to a helix structure in meth anol by addition of HCl solution. The hypothesis of conformational transition is further supported by the response of mPPESO3-py to Pd2+ ions in methanol. Since a monopyridyl group ha s been known to exhibit a strong affinity for Pd2+ ions and selectively bind by self-assembly,152 titration experiments of Pd2+

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97 Figure 5-9. Stern-Volmer plot s for mPPE-SO3-py fluorescence quenching by metal ions in water. ex = 400 nm and em = 680 nm. Figure 5-10. Fluorescence respons es of mPPE-SO3-py to metal ions (20 M) in water. I0 and I represent the fluorescence intensity in th e absence and presence of a metal ion. ex = 400 nm and em = 680 nm.

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ions to t h induces t shows re nm (hyp o spectra u emissio n dominan t Figure 5 h e polymer w t he spectral c duced oscil l o chromic ef f u pon metal c o n maximum a t These spe c 11. Absor p (c and d) 410 nm i n w ere conduc t c hanges of t h l ator strengt h f ect). mPPE o mplexatio n a t 458 nm is c tral chang e p tion and e m in methanol n methanol. t e d in meth a h e polymer ( h s with reds SO3-py als n indicating progressiv e e s are simila r m ission spect r with additi o 98 a nol. Introd u ( Figure 5-1 2 s hift of the a o exhibits a n the coexist e e ly quenche d r to those o b r a of mPPE o n of HCl s o u ction of P d 2 2 (c) and (d) ) a bsorption m n isosbestic p e nce of two k d and very b r b serve d for t h SO3-py (15 o lution. ex = + ions to th e ) The absor p m aximum fro p oint in the k inds of co n r oad emissi o h e polymer w M) (a an d = 400 nm in w e polymer p tion spectr u m 387 nm t o absorption n formations. o n at 680 n m w hen the a m d b) in water w ater and e u m o 414 The m is m ount and e x =

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of water the com p even in m After ad d spectru m enforces Figure 5 increases in p lexation of m m ethanol b e c d ition of P d 2 m were obser v -stacking i 12. Absor p (c and d) in metha n the methan o m PPE-SO3 c ause the m e 2 + to the pol y v ed (Figure i nteractions a p tion and e m in methanol n ol. o l-water sol v py with Pd 2 e tal comple x y me r solutio n 5-10 (a)) w i a nd stabiliz e m ission spect r with additi o 99 v ent mixtur e 2 + induces th e x favors aro m n in water, s i th those in m e s the helica l r a of mPPE o n of P d 2+. e These res u e helical co n m atic fa c s imilar chan g m ethanol, s u l conformat i SO3-py (15 ex = 400 n m u lts suggest t n formation o c e-to-face in t g es in the a b u ggesting th a i on in water. M) (a an d m in water an t hat suggest o f the polym e t eractions.15 b sorption a t metal co m d b) in water d ex = 408 n that e r 7 m plex and n m

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100 Experimental Materials 2,6-Pyridyldiacetylene (1)158 and sodium 3-(3,5-diiodophe noxy)propane-1-sulfonate ( 2)79 were synthesized according to prev iously described procedures. Pd(PPh3)4 was purchased from Strem Chemical Company and used as received. Th e solutions of metal ions were prepared from their chloride salts, except for AgNO3, ZnBr2 and NiBr2. Except PdCl2, all metal sample solutions were prepared by using water that was distilled and then purified by a Millipore purification system (Millipore Simplicity Ultr apure Water System). A stock solution of PdCl2 was prepared in methanol. The polymer stock solution was diluted with water or methanol to a final concentration of 15 M. All concentrations of polymers are pr ovided in polymer repeat unit (PRU) concentration. Instrumentation NMR spectra were recorded on either a Vari an VXR 300 or Mercury-300 spectrom eter and chemical shifts are reported in ppm relative to TMS. Absorption spectra were obtained on a Varian-Cary 100 UV-visible absorption dual beam spectrophotometer, with a scan rate of 300 nm/min. Steady state fluorescence spectra were recorded on a spectrofluorometer from Photon Technology International and corrected by using correction factors generated with a primary standard lamp. General Methods Titra tion experiments were carried out in a fluorescence cuvette. In a typical titration experiment, 3 mL of a polymer solution was placed in a 1 cm quartz fluorescence cuvette with a small magnetic stir bar. The UV-visible absorp tion and fluorescence spectra were recorded at room temperature. Then fluorescence and/or absorption spectra were repeatedly acquired subsequent to the addition of microliter aliquots of a concen trated solution containing the

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101 quencher. The polymer is soluble in water and insoluble in methanol. For measurements in methanol, stock solution of the polymer is diluted with methanol to a final concentration of 15 M. Synthetic Procedures mPPE-SO3-py. A solution of 2,6-pyridyldiacetylene ( 1) (26 mg, 0.2 mmol) and sodium 3(3,5-diiodophenoxy)prop ane-1-sulfonate ( 2 ) (98 mg, 0.2 mmol) in 7 mL of DMF/H2O/ i Pr2NH (v/v/v = 5/1/1) was placed in side a Schlenk flask. The resul ting solution was degassed with argon for 15 minutes. Then Pd(PPh3)4 (7 mg, 6 mol) and CuI (1 mg, 6 mol) powder were added under the protection of argon. The solution was degassed with argon for 15 minutes and then stirred at 60oC for 24 hours. The reaction mixture was poured into the 200 mL of acetone. The precipitated polymer was dissolved in an aqueous solution containing NaCN and the resulting solution was filtered through a 25 m gla ss filter followed by dialysis against deionized water using 6-8 kD MWCO cellulose membrane for 2 days. The polymer solution was lyophilized to yield a brown solid (36 mg, 50%). 1H NMR (300 MHz, DMSO-d6) 2.09 (broad, 2H), 2.71 (broad, 2H), 4.20 (broad, 2H), 7.82-7.89 (broad, 6H). 13C NMR (75 MHz, DMSO-d6) 25.30, 48.15, 68.23, 85.79, 93.95, 112.99, 117.32, 122.46, 127.28, 137.89, 142. 92, 153. 47.

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102 CHAPTER 6 POLY (PHENYLENE ETHYNYLENE) WITH TETRA ALKYLAMM ONI UM SIDE GROUPS FOR LIGHT-INDUCED BIOCIDAL ACTIVIY Introduction Antim icrobials have gained interest in various areas, such as medical devices, healthcare products, water purification systems, hospital, de ntal office equipment, food packaging, food storage, household sanitation, etc.159, 160 because contamination by microorganisms such as bacteria is a great concern in those areas. Antimicrobials are materials capable of killing pathogenic microorganisms.161 Conventionally, such antimicrobial materials have been usually prepared by incorporation of leach able antiseptic into a polymeric surface matrix. Antimicrobials are gradually released and kill the microorga nism by diffusing into and disrupting the cell.162 Typical leaching microbial materials contain antibiotics, halogens or heavy metals like silver or mercury. However, these conventional antimicrobi al reagents have the limitation of residual toxicity since they are liquids or gases of low molecular weight. Antimicrobial reagents of low molecular weight also suffer from short-term an timicrobial activity as the leachable components eventually become exhausted. Furthermore, th e gradually decreasing le vel of the released compound leads to subinhibitory concentrati on of antimicrobials in the surrounding, which provides conditions for resistance development of bacteria. To overcome these disadvantages of conventional antimicrobials, polymeric materials with antimicrobial activities have been suggested as an alternative. Previous studies have shown that cationic polymers with pendant cationic groups, es pecially quaternary ammonium salts possesses effective antimicrobial activity.163-166 Tilter et al. have found that immobilized cationic groups on the surface of glass or SiO2-coated plastic kill bacteria on contact.167, 168 The biocidal polymers are covalently immobilized onto the surface of substrates, which prevents uncontrolled material release to the environment and enable s them to be reused after cleaning.163, 165, 169, 170

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103 The antibacterial ability of the cationic polym ers has been attributed to penetration of cationic groups into bacteria membrane resul ting in the cell damage and death. The following sequential steps have been proposed for this m echanism: (1) adsorption of cationic groups onto negatively charged cell surfaces; (2) penetration in to the cell wall; (3) binding to the cytoplasmic membrane; (4) disruption of the cyto plasmic membrane; (5) release of K+ ions and constituents of the cytoplasmic membrane; and (6) death of the cell.163, 171, 172 Increasing bacteria resistance to antibiotics re quires the development of new antibacterial strategies. Photodynamic killing of bacteria is one of new promisi ng strategies. It utilizes light with a photosensitizer to indu ce a phototoxic reaction, which is identical to the use of photodynamic therapy for skin.173-175 A large number of compounds with photodynamic activity such as phenothiazines, phthalocyanines and phophyrins have been used as light induced biocides against Gram-positive and Gram-negative bacteria.176-179 Light absorption by a photosensitizer initiates photosensi tization mechanism. Absorbed light excites a photosensitizer to an excited state and the ex cited photosensitizer undergoes an intersystem cross-over to the excited state. From the excited stated photosensitzer either char ge (type I reaction) or energy (type II reaction) is transferre d to substrate or molecular oxygen to generate reactive oxygen species (Figure 6-1). In phot odynamic actions, singlet oxygen (1O2) is considered to play a crucial role via type II reacti on since this reactive form of oxygen initiates further oxidative reaction of its surroundings such as cell wall lipid membranes, enzymes or nucleic acids.180-184 Therefore, photodynamic inactivatio n of bacteria is based on the concept that a photosensitizer can accumulate in significant amount in or at th e cytoplasmic membrane, which is the critical target to induce an irreversible damage in bacteria after irradiation.176 Lu et. al. previously showed that a catio nic conjugated polyelectrolyte (CPE) with

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104 quaternary ammonium groups (PPE-NMe3-OR8) exhibited light-activated biocidal activity against Gram-negative bacteria such as Escherichia coli ( E. coli ) and Gram-positive bacterial spores such as Bacillus anthracis ( B. anthracis ).89 These studies were carried out with an aqueous solution of the polymer and microsphe re-coated suspension of the polymer. Due to limited water solubility of CPEs, they spontaneo usly coat oppositely ch arged surfaces close to monolayer coverage.22, 185, 186 The coating is irreversible, robus t and stable both in the presence and absence of interfacial water. As seen in phase contrast and fluores cent microscope images (Figure 6-2), PPE-NMe3-OR8 is taken by both bacteria and th e polymer coated on the bacteria or spores shows strong fluorescence. The polymer coated on the bacteria effectively kills bacteria upon irradiation with white light. In contrast to under white light, there is little or no biocidal activity under yellow light. Figure 6-1. Pathway of type I a nd Type II reaction of light absorbing photosen sitizer. After light activating of the ground state of photosensi tizer (PS), activated form of PS* can follow two alternative pathways via reactive singlet oxygen (1O2), hydrogen peroxide, hydroxyl radical (type II) or organic substrate (S) (type I). The intermediates react rapidly with their surroundings: cell wall, ce ll membrane, peptides and nucleic acids. Figure was taken from Maisch et al .187

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105 Figure 6-2. Structure of PPE-NMe3-OR8 and phase contrast and fl uorescent microscope images of polymer-treated E. coli (upper panel) and polymer-treated B. anthracis Sterne spores (lower panel). Figure was taken from Lu et al .89 O O O O O 2 O 2 n NMe3 Me3N

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106 In this chapter, we designed and synthesi zed a series of cationic CPEs with backbones based on a poly(phenylene ethynylene) repeat unit and tetraalkylammonium side groups. The biocidal activity of the CPEs and surface graf ted conjugated polymer (SGCP) beads with the same repeat unit as PPE-NMe3-OR11188 was examined. The biocidal effects of the CPEs were tested against two Gram negative bacteria: Cobetia marina a marine bacterium and Pseudomonas aeruginosa strain PA01, a model pathogen. Such biocidal activity is correlated with the photophysical properties of the cat ionic polymers. Therefore, the photophysical properties of the polymer were studied in soluti on. We investigated the role of oxygen in the light induced bacterial killing and proposed a mechanism in the bi ocidal action of the CPEs. The structures of CPEs used for this study are given in Figure 6-3. Figure 6-3. Structures and acronym s of cationic conjugated polyelect rolytes investigated in this study.

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107 Results and Discussion Synthesis Figure 6-4 illustrates synthesi s of the monom ers used to synthesize the polymers. 2,5Diiodohydroquinone 3 was synthesized according to the literature procedure.30 Compounds 4a and 4b were obtained by deprot onation of 2,5-diiodohydroquinone 3 with K2CO3 and subsequent alkylation with 1,3-dibromopropane and 1,6-dibromohexane, respectively. Subsequently, pendant groups were quaternized with trimethyl amine (25 wt. % in water) in a mixture of H2O/ethanol/ acetone (v/v/v/ =2/3/3) to give the desired cationic monomer 5a and 5b in 85% yield. Phenylenediacetylene 9 was made by Sonogashira coupling of trimethylsilylacetylene (TMSA) with compound 8. The silyl protecting groups were next removed with TBAF. The synthesis of 2,5-diethynylthiophene 12 was accomplished by using procedures analogous to those described for the phenyl derivatives.189 The reaction of 2,5-dibromothiophene with TMSA resulted in the formation of compound 11 in 56% yield. Deprotection of the two silyl protecting groups was accomplished by treatment of with dilute KOH in degassed methanol. 2,5Diethynylthiophene 12 was isolated as yellow oil in 73% yield after extraction with pentane. Compound 12 was immediately used for polymerization because it was unstable in air or light. As shown in Figure 6-5, the polymers studied in this chapter were synthesized by polycondensation between bifunctional monome rs under Sonogashira coupling conditions. PPE4+116 and PPE-NEt3-OR11188 were prepared according to literature procedures. Polymer Characterization 1H NMR and pulse gradient echo (PSGE) NMR All polymers were dried by lyophi lization technique. They are so luble in water, methanol and DMSO. The structures of a ll polymers were confirmed by 1H NMR. Due to their limited solubility, only partial 13C NMR was obtained at 54.606 ppm (ammonium met hyl groups). The

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108 OCH3 H3CO OCH3 H3CO I I OH HO I I i ii 41% 85% 1 23 iii 82% O(CH2)mBr Brm(H2C)O I I iv 85% O(CH2)mN(CH3)3Br Br(H3C)3Nm(H2C)O I I 4a:m=3 4b:m=6 HO O O O O OTs O(C2H4O)2CH3 H3C(OC2H4)2O v vi O(C2H4O)2CH3 H3C(OC2H4)2O I I 6 7 8 9a;R=TMS 9b:R=H vii-141% 43% 55% i.KIO3,I2,H2SO4,CH3CO2H,heat;ii.BBr3,CH2Cl2,-78oC0oC;iii.Br(CH2)3BrorBr(CH2)6Br,K2CO3, acetone,heat;iv.25%N(CH3)3,H2O,C2H5OH,CH3COCH3,heat;v.TsCl,NaOH,H2O,THF,RT;vi. 3 ,K2CO3, KI,CH3COC2H5,heat;vii.1)Trimethy lsilylacetylene,Pd(PPh3)4,CuI,Toluene,( i Pr)2NH;2)TBAF,CH3OH,RT; viii.Trimethylsilylacetylene,Pd(PPh3)4,CuI,( i Pr)2NH,0oC75oC;ix.KOH,CH3OH,RT 5a:m=3 5b:m=6 S Br Br S 10 11 TMS TMS S H H 12 viii 56% ix 73% R R vii-2 74% Figure 6-4. General synthetic sc heme for monomer 5, 9 and 12.

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109 O(CH2)nN(CH3)3Br Br(H3C)3Nn(H2C)O I I O(C2H4O)2CH3 H3C(OC2H4)2O 9 n=3;5a n=6;5b 12 OR1 OR2 R2O R1O n + R1=(CH2)mN(CH3)3Br R2=O(C2H4O)2CH3m=3;13a,PPE-NMe3-OR8 m=6;16,PPE-C6-NMe3-OR8 O(CH2)3N(CH3)3Br Br(H3C)3N(H2C)3O I I + i.Pd(PPh3)4,CuI, i Pr2NHorN(C2H5)3,DMF,H2O,heat S H H O(CH2)3N(CH3)3Br Br(H3C)3N(H2C)3O S n 5-1 14,PPE-NMe3-Th i i Figure 6-5. General synthetic sc heme for polymer 13a, 14 and 16. molecular weight of only PPE-NMe3-OR8 (Mn = 53, 600) was determined by pulsed gradient spin echo (PSGE) NMR technique, in proton spectra.79, 190-192 Photophysical Characterization UV-vis and fluorescence spectroscopy Poly(phenylene ethynylene) (PPE)-ty pe CPEs typically absorb strongly in the violet region ( ~ 400 450 nm ) and strongly fluorescen ce in the blue or green regions ( ~ 450 550 nm). The absorption and fluorescence of CPEs are stro ngly solvent-dependent due to aggregation of the polymer chains in different environments (Figure 6-6). PPE-NMe3-OR8. PPE-NMe3-OR8 exhibits an absorption band with max = 414 nm in methanol and max = 417 nm in water. The fluorescence em ission spectrum of the polymer is solvent-dependent. In methanol (a good solven t), the polymer features a narrow emission band with max = 460 nm with a shoulder at 500 nm. In wate r (a poor solvent), the emission band is

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110 Figure 6-6. Absorption and emissi on spectra of CPEs in methanol and water. The spectra are normalized according to extinction coeffici ents (absorption) and relative quantum yield efficiency (emission).

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111 redshifted to max = 500 nm and the relative fluorescence quantum efficiency is lower. The solvent-dependent fluorescence behavi or is attributed to aggrega tion of the polymer chains in water and consequent domination of the fluores cence by interchain exc itons (analogous to excimer).188 PPE-NMe3-Th. As seen in Figure 6-6, a methanol so lution of the polymer exhibits a broad absorption band with max at 422 nm. In water, the absorption maximum red-shifts to 432 nm and also the absorption extinction coefficien t decreases. The emission properties are more dependent on the nature of the solvent. In methanol, the polymer exhibits a narrow emission band at max = 475 nm with a vibronic band at max = 502 nm. In aqueous solution, the emission band becomes broader and the quantum efficiency ( fl = 0.016) is lower than that in methanol ( fl = 0.045). Interestingly, the emission maximum is at the same position as in methanol (475 nm) with a shoulder at 502 nm. Such behavior is different from other PPE-based CPEs, which exhibit a large red-shift of emission band in a poor solvent. Th e lack of a spectral shift for PPENMe3-Th likely is due to the fact that the aggregated state of the polymer has a much lower quantum yield, and therefore its contribution to the total emission spectrum is small. PPE4+. The photophysical properties of PPE4+ we re previously stud ied and reported in the literature.116 PPE-C6-NMe3-OR8. As shown in absorption spectrum of other cationic CPEs, the absorption maximum band of PPE-C6-NMe3-OR8 undergoes a very clear red-shift from 432 nm to 450 nm in water. In methanol the fluorescen ce is strong and it features a structured band at max = 467 nm with a shoulder at 500 nm. In wate r, the quantum efficiency of the polymer decreases and a new red-shifted a nd broad band appears at 505 nm.

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112 Transient absorption spectroscopy Molecular oxygen has the rela tively sm all energy difference (94.2 kJ/ mol) between its ground state (3 g -and excited state (1g), and thus singlet oxygen (1O2 (1g)) can be produced by energy transfer from the lowest triplet state of sensitizers (CPEs) to molecular oxygen. Since the triplet state of CPEs plays an importan t role in singlet-oxygen generation, transient absorption spectroscopy was utilized to investigate the triplet states of the non-phosphorescent cationic CPEs 13-16. Direct excitation of PPE-based conjug ated polymers produces a triplet state (triplet exciton) with a moderate efficiency.193-195 The triplet state is t ypically detected by its characteristic long-lived transient absorption in th e red of the visible region. As shown in Figure 6-7, the triplet states of cationic CPEs 13-16 were confirmed by a broad, intense transient absorption band centered at max ~ 760 nm which is quite sim ilar to other PPE-based CPEs. A lower lifetime of the transient absorption is observ ed in methanol compared to that in water. The spectral appearance and long lifetim e provide strong evidence that th e transient species is due to the triplet states of the cationic CPEs. The lo wer intensity of transient absorption in water suggests that the triplet yield is lower than that in methanol. Such a lower triplet yield in water can be explained by aggregation of the CPEs, resu lting in quenching of the singlet states of CPEs. This result is consistent with reduced fluorescence intensities of CPEs in water compared to in methanol. Singlet Oxygen Production The trans ient absorption results indicate that direct excitation of the cationic CPEs 13-16 affords a triplet state in a moderate yield. Therefor e, we expected that th ese CPEs could sensitize singlet oxygen (1O2) according to the pathway shown in Figure 6-8. Such singlet oxygen sensitization by the cationic CPEs 13-16 was investigated in CD3OD by monitoring the emission

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113 Figure 6-7. Transient absorpti on spectrocopy of cationic CPEs in methanol and water. The Spectra of PPE-NMe3-OR8 and PPE4+ were obtained on the laser system described in ref.196 and those of PPE-NMe3-Th were obtained in the system described in ref.197.

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Figure 6 Figure 6 8. Pathwa y 9. Singlet o emission y for PPE-se n o xygen emi s intensity ve r n sitized sin g s sion sensiti z r sus optical d 114 g let oxygen g z ed by CPE s d ensity of t h g eneration. s in CD3OD. h e polymer s Inset: Inte g s olution. g rated 1O2

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115 at 1260 nm, which results from the deactivati on of singlet oxygen (Figure 6-9). Solvents containing O-H bonds quench the si nglet oxygen emission to great extent since the terminal bonds of the solvents absorb the near-infrared emission according to the respective energies of the highest fundamental vibration of that bond.198 Therefore, solvents containing heavier atoms in the terminal bonds were used for this study. The concentration dependence of the 1O2 emission was investigated by varying the polymer concentration and a linear correlation was observed between the polymer concentration and the 1O2 emission intensity. The quantum yield of sensitized 1O2 emission199 by the CPEs in CD3OD was determined using 2-acetonaphthone as a reference ( = 0.79).200 The quantum yields of singlet oxyg en generation from the CPEs are presented in Table 6-1. This obs ervation clearly indicates that PPE-type CPEs are able to sensitize 1O2 with moderate efficiency. Table 6-1. Photophysical pr operties of cationic CPEs max abs/nm max fl/nm fl c PPE-NMe3-OR8 MeOH 414 460 0.285 0.02a 0.130 0.02d H2O 417 500 0.106 0.01a 0.069 0.004d PPE-NMe3-Th MeOH 422 475, 502 0.045 0.004a 0.112 0.008d H2O 432 475, 502 0.016 0.001a 0.037 0.001d PPE-4+ MeOH 422 441 0.15 0.01 b 0.091 0.005d H2O 394 436 0.047 0.004 b 0.320 0.039d PPE-C6-NMe3-OR8 MeOH 432 467 0.239 0.02a 0.079 0.007d H2O 450 505 0.131 0.01a a Coumarin 30 in MeOH as standard, fl = 0.307 (ref.154). b From ref.116. c Quntaum yield of singlet oxygen. d Oxygen atmosphere. Since the biocidal experiments were carried out in aqueous solution, the same experiments were carried out in D2O to provide evidence that the cat ionic PPE-type CPEs will sensitize 1O2 in

PAGE 116

116 water. However, near-infrared emission spectro scopy did not afford a ny detectable emission from 1O2 in D2O. This is likely due to the fact that the quantum efficiency for 1O2 emission is considerably less in D2O, making it more difficult to detect in this medium.201 Thus, a second approach was used to det ect the sensitization of 1O2 by the polymer. This method is based on the use of a water-soluble chemical trap, 1,3-cycl ohexadiene-1,4-diethanoate (CHDDE), which forms a stable endoperoxide when it reacts with 1O2 (Figure 6-10).201 In these experiments disappearance of CHDDE was monitored by decrease of its absorption at 270 nm as a function of irradiation time (Figure 6-11). The quantum yields of singlet oxygen generation were determined following a literature proce dure using 5,10,15,20-tetrakis(4-sulf onatophenyl)-porphyrin (TPPS) as a reference ( = 0.66).201 This result confirms that the production of 1O2 is sensitized in water, but with reduced efficiency compared to that in methanol. The decreased efficiency is consistent with the photophysical experiments whic h show that the triplet yield of the CPEs is lower in water relative to methanol, presumably due to quenching of the singlet excited state by aggregation. The maximum absorption band for PPE-C6-NMe3-OR8 is reduced with the 1O2 generation and hence the quantum efficiency for 1O2 emission is not obtained in D2O. Biocidal Activity. In collabora tion with Dr. Whitten and his co-wor kers at the University of New Mexico, the biocidal activity of the cationic CPEs was studi ed using silica particles with surface grafted conjugated polyelectrolyte (SGCP) 13b For live-dead assays of b acteria exposed to the CPEcoated particles in the light a nd dark, suspensions of CPE-coated particles, bacteria and two DNA stains, SYTO-60 and SYTOX Green stains were examined by confocal fluorescence microscopy. SYTO 60 is a red fluorescent (~650 nm) nucleic acid stain that is cell permeant and used to stain both live and dead cells. SYTOX Gr een is a green-fluorescent (~ 530 nm) nuclear

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117 Figure 6-10. Peroxidation of CHDDE by singlet oxygen (1O2). Figure 6-11. UV-visible spectra of CHDDE (100 M) and PPE-NMe3-OR8 (2 M), PPE-NMe3Th (10 M), PPE-4+ (2 M) or PPE-C6-NMe3-OR8 (2 M) in D2O solution containing phosphate buffer (100 M, pH 7) as a function of the irradiation time. Inset: decrease of absorbance at 270 nm (%) as a function of irradiation time. and chromosome counterstain that is impermeant to live cells but stains the chromatin of dead cells with comprised membranes, indicat ing cell death. Figure 6-12 shows that C. marina is captured by the particles and several (7) bacteria associate with a single particle. As shown in

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Figure 6 Figure 6 form a c l fluoresc e Li v quantify ambient a dark. Ho w increase d in dark a n activity. Mechan i Fi g particles singlet o x and iii). T with bac t 12. Image s experime n 13(b), irrad i l uster of par t e nce from th v e/dead cou n the dead/liv a ir shows a s w ever, susp e d dead/live r n d when irr a i sm for Bio c g ure 6-15 su m adhere to b a x ygen is pr o T he singlet o t eria resulti n of single S G n ts were do n i ation of the t icles and b a e dye (SYT O n ting assays e ratio unde r s mall incre a e nsions pur g atio. In con t a diated. Thi s c idal Activi t m marizes t h a cteria cell w o duced at th e o xygen or s u n g in bacteri a G CP-particl e n e at the Un i suspension s a cteria. And O X Green) i were perfo r r various co n a se in the nu m g ed with ox y t rast, deoxy g s observatio n ty h e possible m w all and the y e polymer/b a u bsequently a death (iv). 118 e with captu i versity of N s containing most of bac t i s dominant o r med from t h n ditions (Fi g m ber of dea d y gen and irr a g enated susp n clearly in d m echanism o y bind to the a cteria inter f produced re Finally, ba c red bacteria N ew Mexico particles, b a t eria in the c o n the conf o h e confocal f g ure 6-14). L d bacteria c o a diated with ensions exh i d icates the r o o f biocidal a c inner bacte r f ace after irr a active oxyg e c terial remai n Data from r a cteria and d c luster are d e o cal fluores c f luorescenc e L ight-expos e o mpared to s visible ligh t i bit low dea d o le of oxyge n c tion. First, C r ia membra n a diation of t h e n intermed i n s during th e r ef. 202, d yes induce t e ad and gre e c ence image. e images to e d suspensi o s uspension i n t displays la r d /live ratio b n in biocida l C PE-coated n e (i). Seco n h e polymer i ates interac t e physical t o e n o n in n the r ge b oth l n d, (ii t

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Figure. 6 6 -13. Confo c Before ir r green ch a show red were don e c al fluoresc e r adiation, b) a nnel corres p channel cor r e at the Uni v e nce images after irradi a p onding to d r esponding t v ersity of N e 119 of SGCP-1 3 a tion and c) a d ead bacteri a t o live bacte e w Mexico. 3 b with C. m a large field a and polym e ria. Data fr o m arina in a m of view. Le f e r emission; o m ref. 202, m bient air. a) f t panels sh o right panel s experiment s o w s s

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120 Figure 6-14. Dead/live ratios of C. marina exposed to 5 m SGCP-13b under various conditions. Data from ref. 202, experiments were done at the University of New Mexico. Figure 6-15. Mechanism of biocidal action. destruction of the bacteria form large clusters of dead b acteria and particles shown in Figure 6-13 (b) and Figure 6-15 (v). The biocidal activity experiments show that the deaerated suspensions of bacteria and CPE-coated particles induce little bacterial de ath in the dark and when irradiated. On the contrary, the suspensions that were oxygenated and irradiated result in more pronounced

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121 bacterial death. This clearly proves efficient light-induced biocidal activity of CPE-coated particles and the role of oxygen in the bioc idal action. As mentioned before, photophysical studies of CPEs show that dir ect excitation of CPEs produces efficiently a long-lived triplet excited state (exciton) with a m oderate yield, and the triplet st ate can sensitize singlet oxygen. Oxygen is required for the light-i nduced biocidal activity. These results suggest that singlet oxygen generated on the CPE/bacter ia interface is the key role in the light-induced biocidal activity. Experimental Materials Polym er 15,116 and 1,3-cyclohexadiene 1,4-diethanoate (CHDDE)203 were synthesized according to literature procedures. 2 -Acetonaphthone and 5,10,15,20-tetrakis(4sulfonatophenyl)porphyrin (TPPS) were purchased from J. T. Baker and Sigma-Aldrich Chemical Company, respectively. CPE-coated partic les were prepared acco rding to procedures described in the previous literature.188 Instrumentation 1H and 13C NMR spectra were recorded on either a VXR 300 or Va rian Mercury-300 spectrometer and chemical shifts are reported in ppm relative to TMS. UV-vis absorption spectra were obtained with samples contained in a 1 cm quartz cuvette on a Varian Cary 100 spectrometer. Steady state fluorescence emi ssion spectra were recorded on a PTI (Photon Technology International) fluoromet er and corrected by using correc tion factors generated with a primary standard lamp. Transient absorption spectra were collected using laser systems that are described elsewhere.196, 197 The polymer solutions were prepared in water or methanol, and purged with argon for 30 min before each transient absorption measurement. Steady-state nearIR phosphorescence spectra recorded on SPEX-2 fl uorescence spectrophotometer equipped with

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122 an Indium-Gallium-Arsenide (InGaAs) detector A long pass filter (LP850) was placed before the emission monochromator to eliminate UV and visible light. General Methods Singlet Oxy gen Phosphorescence Quantum Yield. Oxygen gas was bubbled into CD3OD solutions of CPEs in the dark for 15 mi n with stirring. The oxygen-saturated polymer solutions were excited at 336 nm and steadystate near-IR phosphorescen ce spectra recorded. The singlet oxygen quantum yield of a sample can be calculated by the Eq. 6-1.199 r s r r s s r sI I I I (6-1) where Is and Ir are the absorbed incident light, I s and I r are the integrated singlet oxygen emission intensities of the sample (CPEs) a nd the reference (2-acetonaphthone), respectively, s and r are the singlet oxygen phosphorescence lifetime in the reference and the sample solvents, and r is the singlet oxygen quantum yield ( = 0.79)200 of the reference compound. Quantum Yields of Singlet Oxygen Generation by Chemical Trap. Oxygen gas was bubbled into aqueous (D2O) solutions containing CHDDE (100 M), phosphate buffer (100 M, pH 7.0), and CPEs in the dark for 15 minutes with stirring. Solutions were irradiated at 365 nm with stirring using a xenon short arc lamp equipped with a monochromator. Duration of irradiation was recorded and UV-visibl e spectra collected on Cary 100 UV-Vis Spectrophotometer. The disappear ance of CHDDE was monitored as a decrease in absorption at 270 nm. The quantum yields of singlet oxygen ge neration were determined following Eq. 6-2 using 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (TPPS) as a reference ( = 0.66).201 s r rst t (6-2)

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123 where tr and ts are irradiation time to induce the oxi dation of the same amount of CHDDE and CPEs, respectively, and r is the singlet oxygen quantum yi eld of the reference compound (TPPS). The irradiation time for 10 % loss of C HDDE absorbance at 270 nm was obtained from the linear plots for both TPPS and for CPEs. And then the quantum yield singlet oxygen generation by CPEs is calculated. Synthetic Procedures 2, 5-Diiodohydroquinone (3). This com pound was synthesized using a literature procedure30 modified for easier work-up. 2,5-Diiodo-1,4-dimethoxybenzene (4.64 g, 11.9 mmol) was dissolved in dry CH2Cl2 (100 mL) in a three-necked fl ask fitted with a condenser and additional flask. The solution was cooled to -78 in a dry ice-acetone bath. Boron tribromide (2.24 mL, 23.8 mmol) solution in CH2Cl2 (20 mL) was added slowly. After addition, the mixture was warmed to room temperature and stirred fo r 24 hours. The resulting solution was hydrolyzed by mixing with 150 mL of water. After addition of water, a white powder was formed and it was recrystallized in benzene. (yield: 3.87 g, 9.87 mmol, 83%). 1H NMR (300 MHz, acetone-d6): 7.28 (s, 2H), 8.98 (s, 2H). 1,4-Bis(3-bromopropoxy)-2,5-diiodobenzene (4a). 1,3-Dibromopropane (4.69 g, 23.22 mmol), K2CO3 (5.35 g, 38.7 mmol), and acetone (150 mL) were added to a three-neck, round bottomed flask equipped with a condenser and an additional flask. 2,5-Diiodohydroquinone (1.4 g, 3.87 mmol) was dissolved in 150 mL of acetone a nd added dropwise to the mixture solution at 70 C. The reaction was stirred overnight and cooled to room temperature. K2CO3 was removed by filtration through Celite and the solvent was re moved. The resulting solid was dissolved in chloroform and washed with 10 % NaOH, water, and saturated NaCl solution. The organic layer was dried with sodium sulfate, filtered and concentrated. The resulting solid was crystallized

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124 from ethylacetate and hexane. The white solid was dissolved in hot etha nol and insoluble solid was removed using hot filtration. The solution was concentrated to yield a white solid (2 g, 82 %). 1H NMR (300 MHz, CDCl3): 2.29 (m, 4H), 3.70 (t, 4H), 4.09 (t, 4H), 7.32 (s, 1H). 1,4-Bis(6-bromohexyloxy)-2,5-diiodobenzene (4b). This compound was synthesized using the same procedure described for compound 4a except replacing 1,3-dibromopropane with 1,6-dibromohexane (Yield: 80%). 1H NMR (300 MHz, CDCl3): 1.52 (m, 8H), 1.87 (m, 8H), 3.41 (t, 3H), 3.92 (t, 4H), 7.17 (s, 1H). 3,3'-[(2,5-Diiodo-1,4-phenylene)bis(oxy)]bis[N ,N,N-trimethylpropan-1-aminium] (5a). Compound 4 (1.56 g, 2.47 mmol) was suspended in 25 % trimethylamine in water (80 mL), ethanol (120 mL), and acetone (120 mL) and heated to 120 C. The reaction was refluxed overnight. The solvent was removed and the white solid recrystallized fr om ethanol to yield 1.52 g (85 %). 1H NMR (300 MHz, CD3OD): 2.26 (m, 4H), 3.15 (s, 18H), 3.57 (m, 4H), 4.04 (t, 4H), 7.30 (s, 1H). 3,3'-[(2,5-Diiodo-1,4-phenylene)bis(oxy)]bis[N ,N,N-trimethylpropan-1-aminium] (5b). This compound was synthesized using th e same procedure described for compond 5a except replacing compound 4a with compound 4b (Yield: 91%). 1H NMR (300 MHz, CD3OD): 1.36 (m, 12H), 1.72 (m, 4H), 3.06 (s, 18H), 3.26 (m, 4H), 3.89 (t, 4H), 7.21 (s, 1H). 1,4-Diiodo-2,5-bis[2-(2-methoxye thoxy)ethoxy]benezene (8). 2,5-Diiodohydroquinone (2.00 g, 5.53 mmol) and diethylene glycol monomethyl ether p-toluenesulfonate (6.07 g, 22.12 mmol) in 120 mL of methylethylketone wa s placed under argon. To this solution K2CO3 (3.06 g, 22.12 mmol) and KI (0.09 g, 0.55 mmol) was added. The reaction mixture was refluxed at 100 C for 48 hours and then cooled to room temper ature. The solvent was removed and the solid was dissolved in CH2Cl2 (200 mL), followed by washing with 100 mL of 10 % KOH solution,

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125 water, and saturated NaCl solution. The organi c layer was dried with sodium sulfate and concentrated to give a gold color oil. Flash chromatography on silica gel (80 % CH2Cl2/ 10 % hexane/ 10% ethyl acetate) yiel ded a white solid (1.35 g, 43 %). 1H NMR (300 MHz, CDCl3): 3.40 (s, 6H), 3.58 (m, 4H), 3.78 (m, 4H), 3.88 (m, 4H), 4.11 (m, 4H), 7.23 (s, 2H). 13C NMR (75 MHz, CDCl3): 59.5, 69.9, 70.7, 71.4, 72.4, 86.7, 123.8, 153.4. 1,4-Bis(trimethylsilyl)ethynyl-2,5-bis[2-(2 -methoxyethoxy)ethoxy]benezene (9a). Schlenk flask equipped with compound 8 (1.29g, 2.27 mmol), CuI (0.013 g, 0.068 mmol), and Pd(PPh3)4 (0.052 g, 0.045 mmol) was placed under argon. And then 20 mL of toluene and 40 mL of diisopropylamine were added and argon bubbled through the solution for 30 minutes. To this solution, (trimethylsilyl)acetylene was a dded and the solution stirred at 70 C for 3 days. The solvent was removed and the residue was dissolved in CH2Cl2 and filtered through one-inch silica gel using ethyl acetate. The filtrate was concentrated and purifie d by flash chromatography on silica gel (8 % CH2Cl2/ 67 % hexane/ 25% ethyl acetate) to yield a gold oil, which solidified slowly at room temperature (0.87 g, 76 %). 1H NMR (300 MHz, CDCl3): 0.25 (s, 18H), 3.93 (s, 6H), 3.56 (m, 4H), 3.77 (m, 4H), 3.80 (t, 4H), 4.17 (t, 4H), 6.92 (s, 2H). 1,4-Diethynyl-2,5-bis[2-(2-methoxy ethoxy)ethoxy]benezene (9b). A two-necked flask with compound 9a (0.8 g, 1.58 mmol, 1 eq.) and metha nol (45 mL) was placed and argon bubbled through the solution for 30 minutes. Tetrabutylammonium fluoride (1M in THF, 3.79 mL) was then added to the flask under the argon a nd the mixture was stirred at room temperature for 9 hours. The solvent was removed and the solid was purified by flash chromatorgraphy on silica gel (5% methanol/95% me thylene chloride) to yield a light yellow solid (0.42 g, 1.16 mmol, 73%). 1H NMR (300 MHz, CDCl3): 3.32 (s, 2H), 3.39 (s, 6H ), 3.55 (m, 4H), 3.74 (m,

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126 4H), 3.86 (t, 4H), 4.15 (t, 4H), 7.00 (s, 2H). 13C NMR (75 MHz, CDCl3): 59.4, 69.9, 69.9, 71.3, 72.4, 79.9, 83.1, 113.9, 118.6, 154.4. 2,5-Bis((trimethylsilyl)ethynyl)thiophene (11). 2,5-Dibromothiophene (4.00 g, 16.53 mmol), CuI (0.38 g, 1.98 mmol), Pd(PPh3)2Cl2 (0.69 g, 0.98 mmol), and 120 mL of diisopropylamine were placed in a Schlenk flask and the solution was degassed with stirring for 30 min under ice-bath by bubbling argon gas. To th is solution, (trimethylsilyl)acetylene (6.49 g, 66.12 mmol) was added. The solution was stirred under an ice-bath for 1 hour. Temperature was raised to room temperature and the mixture was kept stirring for an additional 1 hour. The resulting solution was heated to 75 C and stirred for 20 hours. The solvent was removed and the solid was purified by flash chromatography on si lica gel with hexane to yield a yellow solid 7 (2.54 g, 55.5 %). 1H NMR (300 MHz, CDCl3): 0.24 (s, 18H), 7.04 (s, 2H). 2,5-Diethynylthiophene (12). To a suspension of compound 7 (0.4 g, 1.45 mmol) in deoxygenated methanol (20 mL) was added 0.1 mL of 0.5 M aqueous KOH solution. The mixture was stirred at room temperature under argon for 40 min. The solution was diluted with water (50 mL) and extracted with n-pentane (2 x 50 mL). The combined organic solution was dried over Na2SO4 and the solvent was removed at reduced pressure to yield a viscous oil 8 (0.14 g, 73 %). 1H NMR (300 MHz, CDCl3): 3.32 (s, 2H), 7.09 (s, 1H). 13C NMR (75 MHz, CDCl3): 132.6, 123.6, 82.1, 76.2. PPE-NMe3-OR8 (13a). Compound 5 (0.1 g, 0.14 mmol), compound 10 (0.05 g, 0.14 mmol), DMF (5 mL), and water (5 mL) were pl aced into a Schlenk flask and degassed with argon for 30 min. In a separate flas k, CuI (1 mg, 0.005 mmol) Pd(PPh3)4 (4.8 mg, 0.004 mmol), DMF (2.5 mL), and triethylamine (2.5 mL) were degassed with argon for 30 minutes and added to the degassed solu tion containing compound 5 and compound 10. The reaction mixture was

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127 stirred at 60 C for 22 hours. The DMF solution was added to 200 mL of acetone to form a precipitate. The collected yellow precipitate was dissolved in an aqueous solution containing NaCN, filtered using a 25 m glass filter, and followed by dial ysis against deionized water using 6-8 kD MWCO cellulose membrane for 2 days. The polymer solution was lyophilized to yield a dark yellow solid (72 mg, 62 %). 1H NMR (300 MHz, CD3OD): 2.54 (b, 4H), 3.16 (s, 18H), 3.39 (s, 6H), 3.53 (b, 4H), 3.65 (b, 8H), 3.89 (b, 4H), 4.26 (b, 4H), 7.25 (b, 4H). PPE-NMe3-Th (14). A solution of compound 5a (100 mg, 0.15 mmol), CuI (4 mg, 0.02 mmol), and Pd(PPh3)4 (10 mg, 0.01 mmol) in 8.5mL of DMF/H2O/(iPr)2NH (v/v/v/ = 9/6/2) was deoxygenated with argon for 30 minutes. Then, compound 12 was added to the mixture solution under argon. The resulting so lution was heated at 70 C for 22 hours. The reaction mixture was poured into 200 mL of acetone. Th e precipitate was dissolved in small amount of Millipore water and treated with NaCN, filtered using a 25 m glass filter and followed by dialysis against deionizer water using 6-8 kD MWCO cellulose membrane for 2 days. The polymer solution was lyophilized to yield a yellow -tan solid (46 mg, 51 %). 1H NMR (300 MHz, CD3OD): 2.38 (br, 4H), 3.21 (br, 18H), 3.63 (br, 4H), 4.22 (b r, 4H), 7.23 (br, 2H), 7.33 (br, 2H). PPE-C6-NMe3-OR8 (16). This polymer was synthesized in the same procedure described for PPE-NMe3-OR8 ( 13a ) using compound 5b (78 mg, 0.1 mmol), compound 9b (36 mg, 0.1mmol). Yield: 45 mg, 49%. 1H NMR (300 MHz, DMSO-d6): 1.37 (br, 18H), 3.05 (s, 18H), 3.22 (s, 6H), 3.30 (br, 4H), 3.45 (br, 4H), 3.67 (br, 4H), 3.81 (br, 4H) 4.08 (br, 4H), 4.22 (br, 4H) 7.15 (br, 4H).

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128 CHAPTER 7 CONJUGATED POLYAMPHOLYTES BASED ON POL Y(PHENYLENE ETHYNYLENE) Introduction Polym ers containing ionic groups are divi ded into two groups: polyelectrolytes and polyampholytes.204 The former contain either anionic or cationic groups along the polymer chain while latter contain both anionic and cationic gr oups. Polyelectrolytes show ability to obtain large hydrodynamic volumes in deionized water at low concentrations because of Coulombic repulsion between charged groups along the polymer chain forcing the polymer chain into a rodlike conformation. However, the addition of electrolytes or change s in pH screens the Coulombic repulsions allowing the polymers to more random conformation with a subs equent decrease in the hydrodynamic volume. In contrast to polyelect rolytes, structure-prop erty relationship of polyampholytes is controlled by Coulombic attr action between anionic and cationic groups on different monomer units. When either anionic or cationic groups are in sufficient excess ( 1015 mol%), charge repulsion forces the chains into an extended conformation showing typical behavior of polyelectrolytes. However, when th e molar ratio of anioni c and cationic groups is close to unity, Coulombic interactions induce gl obular-like conf ormation and, in most cases, insolubility in deionized water. The addition of electrolytes and change in pH screens these attractive interactions allowing a random coil conformation, often improv ing solubility. This behavior is called as antipolyel ectrolyte effect. Therefore, th e property of polyampholytes in aqueous solution depends on both the chemical structure and the comp osition of the polymer. Essentially, there are four subc lasses of polyampholytes based on their pH responses (Figure 71).204, 205 First, the polyampholytes contain a carboxyl ate group as the ani onic species and an amine hydrohalide group as the cationic species a nd both species may be neutralized (Figure 71a). Second, the anionic groups may be neutrali zed while the cationic groups remain charged

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129 over the whole range of pH (i.e., quaternary alkyl ammonium groups) (Figure 7-1 b). Third, the cationic groups may be neutra lized while the anionic groups remain unchanged over the whole range of pH (i.e., sulfonate groups) (Figure 7-1c). Finally, both the anionic and cationic groups are insensitive to pH changes. C N O OH3C CH3 H C N O OH3C CH3 CH3 S N O OH3C CH3 H O S N O OH3C CH3 CH3 O a b c d Figure 7-1. Structures of the f our subclasses of polyampholytes. The first synthetic polyampholytes were re ported in 1950s by Alfrey and Katchalsky.206-209 The first synthesized polyampholytes are copoly mers of acrylic (or methacrylic) acid and vinylpyridine. They are typically synthesized via radical copoly merization of acidic and basic monomers because of their different reactivit y. A typical example is copolymerization of 2vinylpyridine (weak base) and meth acrylic acid (weak acid), which results in the formation of statistical copolymers. Since these early reports, many researchers have st udied the synthesis and properties of a wide range of stat istical polyampholytes (Figure 7-2).205 The polyampholytes have been of interest because they are s ynthetic analogous proteins. Therefore they can contribute to understanding the aqueous solution behavior of biological molecules such as proteins and be applied to various areas of biotechnology, medicine and hydrometallurgy.204, 210

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130 Particularly, they have been used to absorb and deliver metals, drugs, amino acids, and nucleic acids.210 Figure 7-2. Structures of polya mpholytes that have been synt hesized. Figure was taken from Lowe et.al.205 The polyampholytes containing both anionic and cationic gro ups along their backbone can form polyanion-polycation complex formation (symplex formation) via two pathways.211 First, homosymplex is formed by interactions be tween the anionic and cationic groups of the polyampholytes without a participation of another macroion. Second, heterosymplex formation occurs by interactions between ei ther the anionic or cationic gr oups of the polyampholytes with an oppositely charged macroion added to the po lyampholytes. These homoand heterosymplex formation are dependent on pH, the kind of ionic groups, the ratio of anionic to cationic groups within the polyampholytes, and the distri bution of both groups along the polyampholyte backbone. The properties of polyampholytes have been investigated mostly by means of

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131 viscometry and turbidimetry.204, 210, 212 Most of synthetic polyamphol ytes posses an isoelectric point (IEP) at given pH. The IEP is determined by a minimum viscosity and at this IEP point there are strong Coulombic inte ractions between oppositely ch arged groups, and hence the polyampholytes are electoneutral and a highly co mpact structure of the polymer is expected. When the pH is changed and either cationic or anionic groups become neutral or when electrolytes are added, Coulombic repulsion induces chain expansion of the polymer. In this chapter, we report that synthesi s of conjugated polya mpholytes carrying both cationic and anionic groups (Figure 7-3). The polyampholytes contai n different molar ratios of O O O O n PPE-SO3-NMe3 O O O O O O O O m n m=0.7,n=0.3:PPE-SO3-OR8-NMe3-1 m=0.3,n=0.7:PPE-SO3-OR3-NMe3-2 NMe3Br SO3 -Na+ BrMe3N +Na-O3S SO3 -Na+ +Na-O3S O O 2 2 NMeBr BrMe3N O O 2 2 O O O O n PPE-NMe3-COOH NMe3Br COOH BrMe3N HOOC Figure 7-3. Structures of conjugated polyampholytes.

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132 anionic to cationic groups and show different be havior in solution. The solution properties of polyampholytes were studied using absorpti on and fluorescence spec troscopy. PPE-NMe3-SO3 with 1:1 ratio of cationic to anionic groups exhibits the low solubility in deioninized water due to the static interaction between oppositely char ged side groups. On the other hand, PPE-SO3NMe-OR8 with low ratio (< 1) of cationic to anioni c groups shows good solubi lity in deionized water with the similar solution properties with those observed for cationic or anionic conjugated polyelectrolytes (CPEs). The highlight of this study is the finding that the competition between homoand heterosymplex formation is depende nt on the pH of the PPE-NMe3-COOH solution containing quaternary ammonium and carboxyl groups. The polymer shows the pH dependent optical properties. Results and Discussion Synthesis Figure 7-4 illustrates the synt hetic route for the monom ers. The synthesis started with commercially available 1,4-dimethoxybenzene 1 which was converted to 2,5diiodohydroquinone 3 according to the procedur es described previously.30 Substitution reaction of 2,5-diiodohydroquinone 3 with bromoacetic acid yielded compound 4 with procedures adapted from the previous literature.189 Reaction of 1,3-dibrom opropane with compound 3 afforded compound 5 and subsequent quaternization of pe ndant groups gave the desired cationic monomer 6 in 85% yield. Substitution of both iodines of 5 by (triiosopropyl)acetylene (TIPSA) was achieved under Sonogashira coupling conditions. Compound 5 was treated with TIPSA in THF with diisopropylamine (iPr)2NH as base and dichlorobis( triphenylphosphine)palladium (Pd(PPh3)2Cl2) and copper iodide (CuI) as catalysts at room temperature and then quaternization of pendant groups was carried out to give the cationic monomer with TIPS protection group 8 in 98 % yield. The anionic monomer with sulfonate 9 was synthesized following the procedures

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133 Figure 7-4. Synthesis of monomers 4, 6, 8, 9 and 13.

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134 Figure 7-5. Synthesis of conjugated polyampholytes. described previously.36 Reaction of compound 11 with 2,5-diiodohydroquinone 3 gave diiodobenzene derivative 12 with di(ethyleneoxide) side chains. Phenylacetylene with di(ethyleneoxide) groups 13 was made by Sonogashira coupli ng of trimethylsilylacetylene (TMSA) with compound 12 and deprotection of the silyl gr oup with TBAF in 55% yield. As shown in Figure 7-5, conjugated polyampholytes were synthesized in the mixture of DMF/H2O/(iPr)2NH or N(C2H5)3 with catalytic amount of Pd(PPh3)4 and CuI. All polymer

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135 solutions were dried by lyophilization process after dialysis against deionized water and characterized by 1H NMR spectroscopy. The solubility of PPE-NMe3-SO3 in various organic solvents as well as water is too low to obtain 1H NMR spectra. For two different PPE-SO3-OR8NMe3, 1H NMR spectra were obtained in DMSO-d6; however, 13C NMR spectra were not able to be measured because of their poor solubi lity. PPE-NMe3-COOH shows limited solubility in both DMSO and D2O (< 1mg/mL). Using a mixture of D2O/DMSO-d6 (v/v = 1:1), 1H NMR spectrum was finally obtained after intensive scanning (1024 scans) at room temperature. Properties of Conjugated Polyampholytes PPE-NMe3-SO3. This polyam pholyte is composed of an equimolar ratio of quaternary ammonium (NR4 +) and sulfonate groups (SO3 -) along the polymer backbone. The sulfonate group is a strong polyelectrolyte group, and hence it remains fully ionized over a wide pH range in aqueous solution. In the previous studies using polyampholytes with a sulfonate group, the polyampholytes showed interesting rheologica l behavior in the pr esence of electrolytes.213-218 Specifically, polyampholytes in which the cationic/ anionic charge ratio is one are insoluble in deionized water. This arises due to the interaction between the an ionic and cationic groups of the polymer to form an ionically crosslinked netw ork (Homosymplex) without participation of a further macroion.211 Addition of a critical concen tration of salt is required to achieve solubility in aqueous solution. 215, 216, 218 Similarly, PPE-NMe3-SO3 is insoluble (shows turbidity) in water and requires addition of salt to make it water-s oluble. This polyampholyte also shows poor solubility in polar organic solvents such as DMSO, DMF and methanol. PPE-NMe3-OR8-SO3. When anionic or cationic gr oups are in sufficient excess ( 10 mol%), polyampholytes exist in an extended conf ormation due to charge repulsion, resulting in rheological behavior typical of polyelectrolytes. Here, conjugat ed polyampholytes with different molar ratios of anionic groups to cationic groups were synthesi zed. PPE-NMe3-OR8-SO3-1 (P1)

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136 and PPE-NMe3-OR8-SO3-2 (P2) contain 3:7 and 7:3 molar ratio of quaternary ammonium to sulfonate groups, respectively. The polymers show good solubility in water, DMSO and DMF whereas they are insoluble in methanol. The ra tio of anionic and cati onic groups on the polymer backbone was confirmed by integration ratio of protons on CH2 right next to anionic and cationic groups in the 1H NMR spectrum. The proton peak next to quaternary ammonium groups appears at 2.2-2.4 ppm while the proton peak next to sulfonate groups shows up around 2.06 ppm (Figure 7-6). Their ratios are about 3:7 and 7:3 for P1 and P2, respectively. Figure 7-6. 1H NMR spectrum of P1 and P2 in DMSO-d6. The absorption and fluorescence spectra of P1 an d P2 in methanol or water are similar to those for conjugated poly electrolytes (CPEs) 23, 35, 36, 79 which contain poly(phenylene

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137 ethynylene) (PPE) backbones. As reported previo usly for PPE-type CPEs, P1 and P2 in water show red-shifted absorption bands from 425 nm to 443 nm for P1 and from 418 nm to 435 nm for P2 with reduced oscillator strength (Figure 7-7). This supports that P1 and P2 exist as aggregates in water like other CPEs. The aggr egation of these two polyampholytes is also evidenced by a broad and red-shifted fluorescence band with a relatively low quantum yield in water (Figure 7-7). By contrast, the polyampholytes are not aggregated in methanol as proved by the fact that the polyampholytes feature a struct ured fluorescence spectrum with a comparatively high quantum yield (Table 7-1). These results suggest that the polyampholytes which contain Table 7-1. Photophysical propertie s of conjugated polyampholytes max abs/nm max fl/nm fl P1 MeOHa 425 470 0.13 0.01 b H2O 443 556 0.08 0.008c P2 MeOHa 418 471, 495 0.088 0.008 b H2O 435 524 0.058 0.005c PPE-NMe3-COOH MeOHa 420 463 0.10 0.01 b H2O 440 561 0.071 0.007 (pH 3.0) b 0.077 0.007 (pH 7.6) b 0.046 0.004 (pH 10.3) b a Microliters of a concentrated polymer so lution in water was diluted with methanol. b Coumarin 30 in MeOH as standard, fl = 0.307 (ref. 154). c Coumarine 6 in EtOH as standard, fl = 0.78 (ref. 219).

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138 Figure 7-7. Absorption and emissi on spectra of P1 (10 M) and P2 (10 M) in methanol and water. Each polymer solution is excited at its own maximum absorption wavelength.

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139 either cationic or anionic groups are in sufficient excess show solvent-dependent photophysical properties similar with those for CPEs. Titration of P1 and P2 in water with aliquots of N,N-dimethyl-4,4 -bipyridinium (MV2+) and sodium 1,4,5,8-naphthalenediimide-N,N-bis (methylsulfonate) (NDS) was conducted to provide the quenching behavior of the two polymers. MV2+ and NDS (Figure 7-8) efficiently quench the fluorescence of CPEs in water. MV2+ and NDS are known to quench the fluorescence of anionic and cationic CPEs by photoinduced electron transfer, respectively. Therefore, the same quenching mechanism was expected for P1 and P2 since they showed the similar solution behavior as CPEs. First, the quenc hing efficiency of P1 and P2 by MV2+ and NDS were investigated in water. As shown in Figure 7-9, P1 in which sulfonate groups are dominant on the polymer backbone is much more efficiently quenched by a cationic quencher, MV2+ (KSV = 2.1 106 M-1) compared to an anionic quencher, NDS (KSV = 4.2 103 M-1). In contrast, P2 in which quaternary ammonium groups are dominant is quenched much more strongly by NDS (KSV =3.56 106 M-1) than MV2+ (KSV = 1.5 103 M-1). The fluorescence quenching data were analyzed using Stern-Volmer (SV) relation, given by I0/I = 1+ Ksv[Q] (where I0 and I are the intensity of fluorescence in the absence and presence of a quencher, respectively, KSV is the Stern-Volmer constant, and [Q] is the concentrat ion of a quencher). The Stern-Volmer constant (Ksv) is used to quantitatively measure the quenching efficiency. Ksv values for P1 and P2 Figure 7-8. Structures of a cationic and an anionic que ncher used in this study.

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140 Figure 7-9. Emission spectra of P1 and P2 in water upon addition of a cationic and an anionic quencher. ex = 440 nm for P1 and ex = 430 nm for P2. The inset shows the SternVolmer plots using emission intensities at 556 nm for P1and 445 nm for P2. Table 7-2. Stern-Vo lmer constant (KSV) for P1 and P2 fluorescence quenching upon addition of MV2+ and NDS in water and DMF. In water In DMF P1-MV2+ 2.1 106 M-1 7.4 105 M-1 P1-NDS 4.2 103 M-1 1.5 103 M-1 P2-MV2+ 1.5 103 M-1 4.0 102 M-1 P2-NDS 3.56 106 M-1 8.3 105 M-1

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141 quenching were obtained in the lin ear region of the SV plots. Table 7-2 summarizes the SternVolmer constants (Ksv). Figure 7-10 shows the absorption and emission spect ra of P1 and P1 in DMF. Compared to the absorption spectra of P1 and P2 in water, relatively blue-shifted absorption maximum appears at 432 nm for P1 and 428 nm for P2. Both polymers feature a well-defined 0-0 band with max = 480 nm for P1 and max = 476 nm with a shoulder band around 510 nm. These spectral properties indicate th e non-aggregated states of both polymers in DMF. The same fluorescence quenching experiments as described above we re carried out in DMF (Figure 7-11 and Table 7-2). The results are consistent with those observed in quenching experiments with P1 and P2 solution in water. Interestingly, at any given [MV2+] or [NDS], the aggregated conformation of P1 and P2 is que nched more strongly compared to the nonaggregated conformation of polymers as previo usly reported for fluorescence quenching of PPEbased CPEs. This is likely due to the ability of the exciton to diffuse rapidly along the polymer chain. These observations provide the evidences for the fact that the polyampholytes with a low anionic-group/cationic-group ratio (< 1.0) on their backbone show similar solution properties with cationic or anionic CPEs. PPE-NMe3-COOH. To investigate the effect of the nature of ionic groups on the ampholyte properties, a sulfonate group was repla ced with a carboxyl group as an anionic side group. The carboxyl group remains charged or neut ralized according to pH changes whereas the quaternary ammonium group is insensitive to pH changes. The spectral changes induced by pH on the absorption and fluorescence spectra of th is polymer are shown in Figure 7-12. For the absorption spectra, oscillator st rength decreases with a decrea se in the pH of the polymer solution. Interestingly, emission intensity and emission quantum efficiency increases in the

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142 Figure 7-10. Absorption and emission spectra of P1 and P2 in DMF. ex = 430 nm for both P1and P2.

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143 Figure 7-11. Emission spectra of P1 and P2 in DMF upon addition of a cationic and an anionic quencher. ex = 430 nm for both P1and P2. sequence of pH 10.3 (fl = 0.046) < pH 3.0 (fl = 0.071) < pH 7.6 (fl = 0.077). In the emission spectra of PPE-NMe3-COOH, as pH decreases from 11 to 8, emission intensity sharply increases. At pH 5-8, the emission intensity remains almost constant. With a decrease in pH from 5 to 3, intensity is progressively reduced. At pH > 8, the COOgroups are involved in the formation of zwitterions on the polymer backbone, and hence the attractive interaction between the negatively charged and positively charged groups forms an ionically crosslinked network that forces the polymer chains into a highly compact structure under this basic condition.204, 212 This might result in the reduced fluorescence intensity. In a pH range of 5-8, two contrary factors influence the polymer solution.220 First, COOCOOand ammonium-ammonium repulsion prevents

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144 Figure 7-12. Absorption and emission spectra of PPE-NMe3-COOH (30.72 M) as a function of pH in aqueous solution. The inset illustrates that emission intensity at 548 nm varies depending on the pH in the polymer solution. ex = 430 nm.

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145 formation of a crosslinked network that induces the compact stru cture of polymer. At the same time, the static inte raction between the car boxyl and ammonium groups restricts extended conformation of the polymer chain. As a result, the fluorescence intensity remains constant. At pH < 4, COOH groups are dominant, reducing the ne gative charge density on the polymer chain. Thus this reduces the attractive intera ctions between the opposit ely charged groups. The polymer shows the similar behavior with that of cationic or anionic CPEs in water. Therefore, the fluorescence intensity of the polymer is reduced due to aggregation via -stacking between adjacent polymer chains at this acidic condition. Figure 7-13. Absorption and emission spectra of PPE-NMe3-COOH (10 M) in methanol upon addition of HCl solution. (a) a nd (b) are spectra after additio n of low concentration of HCl solution(0.0 M). (c) and (d) are spec tra after addition of high concentration of HCl solution (0.2 mM). ex = 420 nm.

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146 The effect of HCl on the optical properties is also investigated using the polymer solution in methanol (Figure 7-13). Since the polymer is insoluble, the polymer aqueous solution (pH 11) is diluted with methanol to get the desired con centration of the polymer solution (10 M). At low concentrations of HCl, the absorption and emi ssion intensity are slightly decreased (Figure 7-13 (a) and (b)). After addition of higher concen tration of HCl solution, the absorption and fluorescence spectra slightly red-shift compared to those in the absence of HCl solution (Figure 7-13 (c) and (d)). This suggests th at introduction of HCl slightly interrupts th e interactions between the oppositely charged side groups, inducing to expand the polymer chains. Figure 7-14. Emission spectra of PPE-NMe3 -COOH (30.72 M) upon addition of an anionic quencher, NDS at different pH solutions. ex = 440 nm.

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147 The quenching efficiency for PPE-NMe3-COOH by an anionic quencher, NDS is observed at different pH (Figure 7-14) Stern-Volmer constants (Ksv) were obtained in a linear region of SV plots. At pH 2.5, most carboxyl groups exist as COOH, and the negative charge density of ammonium groups is dominant in the polymer so lution. Thus, the polymer is the most strongly quenched by NDS (KSV = 4.55 105 M-1). There is little difference between the Ksv values at pH 6.5 and pH 10.0. Summary and Conclusions In this chapter, we illustrated the effects of m olecular architecture of polyampholytes on solution behavior. The nature of charged side groups and composition of the charged groups on the polymer backbone induce different solubili ty and rheological behavior. PPE-NMe3-SO3 bearing an equimolar ratio of ammonium and su lfonate groups shows very poor solubility in water. P1 and P2 containing a low molar ratio of ammonium to sulfonate groups (< 1.0) show similar solution behavior with anionic or ca tionic CPEs. Depending on the dominant charges on the polymer backbone, the polymers are strongl y quenched by oppositely charged molecules. PPE-NMe3-COOH containing carboxyl groups se nsitive to changes in pH behaves as a polyelectrolyte or polyampholyte depending on the pH in aqueous solution. The different solution behavior also leads to changes in th e absorption and emission spectra. The quenching efficiency for PPE-NMe3-COOH varies according to pH. Experimental Materials 2,2' -(2,5-Diiodo-1,4-phenylene)bi s(oxy)diacetic acid ( 4 )189 and 3,3'-(2,5-diiodo-1,4phenylene)bis(oxy)diprop ane-1-sulfonate ( 9 )36 were synthesized according to the literature procedure. 3,3'-[(2,5-Diiodo-1,4phenylene)bis(oxy)]bis[N,N,N -trimethylpropan-1-aminium]

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148 ( 6) and 1,4-Diethynyl-2,5-bis[2-(2-m ethoxyethoxy)ethoxy] benzene ( 13 ) were synthesized according to the procedures described in Chapter 6. Instrumentation 1H and 13C NMR spectra were recorded on either a VXR 300 or Va rian Mercury-300 spectrometer and chemical shifts are reported in ppm relative to TMS. UV-vis absorption spectra were obtained with samples contained in a 1 cm quartz cuvette on a Varian Cary 100 spectrometer. Steady state fluorescence emi ssion spectra were recorded on a PTI (Photon Technology International) fluoromet er and corrected by using correc tion factors generated with a primary standard lamp. General Methods All sam ple solutions were prepared by using water that was distille d and then purified by a Millipore purification system (Millipore Simplicity Ultrapure Water System). The polymer stock solutions were diluted with the deionized water to a final concentration. All concentrations of polymers are provided in polymer repeat unit (PRU) concentration. In titration quenching experiments, 3 mL of polymer solution was placed in a 1cm fluorescence cuvette. Then fluorescence spectra were repeatedly acquired after addition of microliter aliquots of a concentrated solution containing a quencher. A ll experiments using the polymer solutions in methanol, the polymer aqueous solutions were di luted with methanol to obtain the desired concentration. Synthetic Procedures 1,4-Bis(triisopropylsilylethynyl)2,5-Bis(3-bromopr opoxy)benz ene (7). Under an argon atmosphere, THF (20 mL) and diisopropyl amine (2.5 mL) were added to compound 5 (98 mg, 1.56 mmol), Pd(PPh3)2Cl2 (62 mg, 0.09 mmol) and CuI ( 45 mg, 0.234 mmol).The mixture solution was degassed by argon bubbling at room temperature for 20 minutes and this was

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149 followed by the dropwise addition of trisopr opylsilylacetylene (0.63 mL, 2.77 mmol). The solution was stirred at room temperature for 40 hours. The solvent was removed and the solid was purified by flash chromatography on silica gel with hexane to yield a white solid 7 (570 mg, 0.80 mmol 51%). 1H NMR (300 MHz, CDCl3): 1.14 (s, 42H), 2.29 (m, 4H), 3.60 (t, 4H), 4.08 (t, 4H), 6.89 (s, 2H). 3,3'-(2,5-bis((Triisopropy lsilyl)ethynyl)-1,4-phen ylene)bis(oxy)bis(N,N,Ntrimethylpropan-1-aminium) (8). Compound 7 (210 mg, 0.25 mmol) was suspended in 25 % trimethylamine in water (20 mL), ethanol (30 mL), and acetone (30 mL) and heated to 120 C. The reaction was refluxed overnight. The solvent wa s removed and the white solid recrystallized from ethanol to yield 200 mg (0.24 mmol, 98%). 1H NMR (300 MHz, CDCl3): 1.10 (s, 42H), 2.23 (m, 4H), 3.12 (s, 18H), 3.48 (m, 4H), 4.08 (t, 3H), 6.99 (s, 2H). PPE-SO3-NMe3. The solvent mixture (17 mL) of DMF/H2O/(iPr)2NH (v/v/v/ = 9/6/2) was degassed with argon for 15 minutes and followed by the addition of compound 8 (100 mg, 0.12 mmol). After argon bubbling through the solution for 15 minutes, 1.0 M tetrabutylammonium fluoride solution in THF (1.60 mmol) was added to the flask under argon and the mixture was stirred at room temperature for 30 minutes. In a separate flask, a solution of CuI (4 mg, 0.02 mmol) and Pd(PPh3)4 (11 mg, 0.01 mmol) in DMF was degassed with argon for 30 minutes and added to the dega ssed solution co ntaining compound 8. Addition of compound 9 and 15 minute degassing were followed. Finally the solution was stirred under argon atmosphere at 60 C for 24 hours. The reaction mixture was poured into 200 mL of acet one. The precipitate was dissolved in small amount of Millipore wa ter and treated with NaCN, filtered using 25 m glass filter and followed by dial ysis against deionizer water using 6-8 kD MWCO cellulose membrane. The polymer solution was lyophilized to yield a yellow solid (35 mg, 0.036 mmol, 30

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150 %). 1H NMR (300 MHz, CD3OD) and 13C NMR (75 MHz, CD3OD) spectra were not obtained due to the poor solubility of the compound. PPE-SO3-NMe3-OR8-1. A solution of compound 6 (22 mg, 0.03 mmol), compound 9 (46 mg, 0.07 mmol,) and compound 13 (36 mg, 0.1 mmol) in 15 mL of DMF/water/ triethylamine (v/v/v = 3/2/1) were placed to a Schlenk fl ask and degassed with argon for 30 minutes. CuI (2 mg, 0.01 mmol) and Pd(PPh3)4 (7 mg, 0.006 mmol) were added to the mixture solution containing compound 6 and compound 9. The reaction mixture was stirred at 60 C for 26 hours. The resultant solution was added to 200 mL of acetone to form a precipitate. The collected precipitate was dissolved in an aqueous soluti on containing NaCN (8 mg), filtered using a 25 m glass filter and followed by dial ysis against deionized water using 6-8 kD MWCO cellulose membrane for 2 days. The polymer solution wa s lyophilized to yield a yellow solid (50 mg, 57%). 1H NMR (300 MHz, DMSO-d6): 2.06 (br, 2.8H), 2.22 (br, 1.2H), 2.65 (br, 2.8H), 3.01 (br, 5.4 H), 3.25 (br, 6H), 3.43 (br, 4H), 3.66 (br, 5.2 H), 3.79 (b, 4H). 13C NMR (75 MHz, DMSO-d6) spectrum was not obtained due to th e limited solubility of the compound. PPE-SO3-NMe3-OR8-2 This polymer was synthesized by the same procedure described for PPE-SO3-NMe3-OR8-1 using compound 6 (51 mg, 0.07 mmol), compound 9 (20 mg, 0.03 mmol) and compound 13 (36 mg, 0.1 mmol). Yield: 12 mg, 13%. 1H NMR (300 MHz, DMSOd6): 2.06 (br, 1.2 H), 2.24 (br, 2.8H), 2.66 (br, 1.2H), 3.07 (br, 12.6 H), 3.21 (br, 6H), 3.40 (br, 4H), 3.65 (br, 6.8H), 3.79 (br, 4H). 13C NMR (75 MHz, DMSO-d6) spectrum was not obtained due to the limited sol ubility of the compound. PPE-NMe3-COOH. The solvent mixture (17 mL) of DMF/water/diisopropylamine (v/v/v/ = 9/6/2) was degassed with argon for 15 minut es and followed by the addition of compound 8 (79 mg, 0.095 mmol). After argon bubbling through the solution for 15 minutes, 1.0 M

PAGE 151

151 tetrabutylammonium fluoride solution in TH F (0.95 mmol) was then added to the flask under argon and the mixture was stirred at room temp erature for 30 minutes. CuI (2 mg, 0.011 mmol) and Pd(PPh3)4 (7 mg, 0.006 mmol) was added to the mixture solution. After 15 minutes of degassing, compound 4 was added and the reaction mixt ure was stirred under argon at 60 C for 24 hours. The reaction mixture was poured into 200 mL of acetone. The precipitate was dissolved in small amount of Millipore water an d treated with NaCN (8 mg), filtered using 25 m glass filter and followed by dialysis against deionized water using 6-8 kD MWCO cellulose membrane for 2 days. The polymer solution was lyophilized to yield a yellow solid (10 mg, 15 %). 1H NMR (300 MHz, CD3OD/D2O) 2.23 (br, 4H), 3.10 (br, 18 H), 3.59 (br, 4H), 4.23 (br, 4H), 4.62 (br, 4H), 6.82 (br, 4H).13C NMR (75 MHz, CD3OD) spectra were not obtained due to the limited solubility of the compound.

PAGE 152

152 CHAPTER 8 CONCLUSIONS In the previous chapters, the synthesis, photophysical characte rization and applications of poly(arylene ethynylene)-based conjugated polyelectrolytes (CPEs) have been presented. Intercalation of Intercalator Quenchers to mPPESO3 Meta-linked poly(phenylene ethynylene)s bearing ionic groups can fold into a helical conformation in water, which is stabilized by stacking and hydrophobic interactions. The helical polymer was used to sense protease act ivity. The polymer fluorescence is quenched significantly by L-Lys-p-nitroanilide (K-pNA) and peptidase reverses the fluorescence from the quenched polymer solution concurrent with pept ide hydrolysis. More im portantly, the polymer still shows amplified quenching at even high concentration of buffer solution up to 100 mM. Typically, CPEs show a significantly reduced quenching efficiency with increasing buffer concentration because the buffer ions screen th e Coulomb interactions, removing quenchers from the vicinity of CPEs. This resu lt is likely due to intercalation of quenchers into the helical conformation of the polymer, giving less ion-sc reening effect on the Coulomb interaction. By taking the advantage of the he lical structure, the polymer was used as a sensor platform with intercalator quenchers containing ligand. For th is study, biotin and avid in were chosen as a ligand and a target protein, respectively. Biotin -functionalized quenchers strongly quench the polymer fluorescence via intercala tion and static interaction. However, introduction of avidin to the polymer/quencher complexes is not capable of displacing the quenchers from the polymer, which does not increase the polymer fluorescence. Tw o possibilities are suggest ed to explain this result. First, biocytin-TMR is hi dden within the helical polymer, a nd hence avidin is not able to be accessible to the biotin, leading to revers al of quenching. Second, the quenchers serve as crosslinkers for the concurrent binding of the pol ymer and avidin. When avidin is previously

PAGE 153

153 bound to biotin on the quenchers, the quencher/avidin complexe s give less effect on the fluorescence quenching. This might arise due to the electrosta tic interactio n between the oppositely charged polymer and avidin Helical Self-Assembly of mPPESO3-py In m PPESO3, phenylene units were replaced with pyridine rings. PPE-SO3-py shows solvent-induced self-assembly into a helix, which is evidenced by the absorption and emission spectra. The polymer also shows st rong and selective affinity for Pd2+ ions in water. Interestingly, Pd-complexation with the polymer in methanol induces conformation changes into a helix. Changes in the absorp tion and emission spectra are co nsistent with those obtained depending on solvent polarity. Protonation of pyridine rings in methanol also leads to selfassembly of the polymer into a helix. Biocidal Activity of Cationic CPEs PPE-based cationic conjugated polyelectrolyte s with te traalkylamm onium side groups exhibit light-induced biocidal activity. It wa s found that surface grafted conjugated polymer (SGCP) particles entrap and kill bacteria. Singlet oxygen generation at the polymer/bacteria surface is a crucial step in th e biocidal action. This study show s that SGCP particles with bacteria that are oxygenated a nd irradiated exhibit the most efficient bioc idal activity. Photophysical studies of the cationic polymers in solution st rongly support the role of oxygen in the biocidal process. Direct excitation of the polymers generates a long-lived triplet state and the triplet state sensitizes singlet oxygen. For the light-induced bi ocidal activity, oxygen is also required at the polymer/bacteria interface. Conjugated Polyampholytes The effects of m olecular architecture of polyampholytes on solution behavior were investigated by varying the nature of charge d side groups, composition of the charged groups

PAGE 154

154 and changes in pH. As the molar ratio of ani onic to cationic groups is one and both charged groups are insensitive to change s in pH, the polyampholyte shows poor solubility in water. For polyampholytes with a low molar ratio of ani onic to cationic groups, th e polyampholytes show similar solution behavior compared with anio nic or cationic CPEs. De pending on the dominant charges, the polymers are strongly quenched by oppositely charged molecules. The polyampholyte containing charged groups (carboxyl groups) that are sensitive to changes in pH behaves as a polyelectrolyte or polyampholyte depending on the pH in aqueous solution. The different solution behavior also leads to changes in the ab sorption and emission spectra.

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155 LIST OF REFERENCES 1. Shirakawa, H.; Louis, E. J.; Macdiarm id, A. G.; Chiang, C. K.; Heeger, A. J., J. Chem. Soc., Chem. Commun. 1977, (16), 578. 2. Grimsdale, A. C.; Mullen, K., Emissive Materials: Nanomaterials 2006, 199, 1. 3. Scherf, U.; List, E. J. W., Adv Mater 2002, 14, (7), 477. 4. Patil, A. O.; Heeger, A. J.; Wudl, F., Chem Rev 1988, 88, (1), 183. 5. Perepichka, I. F.; Perepichka, D. F.; Meng, H.; Wudl, F., Adv Mater 2005, 17, (19), 2281. 6. MacDiarmid, A. G., Angew Chem Int Edit 2001, 40, (14), 2581. 7. Bunz, U. H. F., Chem. Rev. 2000, 100, (4), 1605. 8. Kraft, A.; Grimsdale, A. C.; Holmes, A. B., Angew. Chem., Int. Ed. 1998, 37, (4), 402. 9. Heeger, A. J., Solid State Commun. 1998, 107, (11), 673. 10. Hide, F.; DiazGarcia, M. A.; Schwartz, B. J.; Heeger, A. J., Accounts Chem Res 1997, 30, (10), 430. 11. Gunes, S.; Neugebauer, H.; Sariciftci, N. S., Chem. Rev. 2007, 107, (4), 1324. 12. Sirringhaus, H., Adv Mater 2005, 17, (20), 2411. 13. McQuade, D. T.; Pullen, A. E.; Swager, T. M., Chem. Rev. 2000, 100, (7), 2537. 14. Thomas, S. W.; Joly, G. D.; Swager, T. M., Chem. Rev. 2007, 107, (4), 1339. 15. Skotheim, T. A.; Elsenbaumer, R. L.; Reynolds, J. R., Handbook of cond ucting polymers. 2nd ed.; M. Dekker: New York, 1998; p 209. 16. Wise, D. L., Photonic polymer systems : fundamentals, methods, and applications. M. Dekker: New York, 1998; p 61. 17. Giesa, R., J. Macromol. Sci., Rev. Macromol. Chem. Phys. 1996, C36, (4), 631. 18. Reddinger, J. L.; Reynolds, J. R., Radical Polymerisation Polyeletrolytes 1999, 145, 57. 19. Schluter, A. D., J. Polym. Sci., Part A: Polym. Chem. 2001, 39, (10), 1533. 20. Shi, S. Q.; Wudl, F., Macromolecules 1990, 23, (8), 2119. 21. Wallow, T. I.; Novak, B. M., J. Am. Chem. Soc. 1991, 113, (19), 7411.

PAGE 156

156 22. Chen, L. H.; McBranch, D. W.; Wang, H. L.; Helgeson, R.; Wudl, F.; Whitten, D. G., Proc. Natl. Acad. Sci. U. S. A. 1999, 96, (22), 12287. 23. Pinto, M. R.; Schanze, K. S., Synthesis-Stuttgart 2002, (9), 1293. 24. Gaylord, B. S.; Heeger, A. J.; Bazan, G. C., Proc. Natl. Acad. Sci. U. S. A. 2002, 99, (17), 10954. 25. Juan, Z.; Swager, T. M., Poly(Arylene Ethynylene)S: From Synthesis to Application 2005, 177, 151. 26. Achyuthan, K. E.; Bergstedt, T. S.; Chen, L.; Jones, R. M.; Kumaraswamy, S.; Kushon, S. A.; Ley, K. D.; Lu, L.; McBranch, D.; M ukundan, H.; Rininsland, F.; Shi, X.; Xia, W.; Whitten, D. G., J. Mater. Chem. 2005, 15, (27-28), 2648. 27. Kim, I. B.; Dunkhorst, A.; Gilbert, J.; Bunz, U. H. F., Macromolecules 2005, 38, (11), 4560. 28. Cabarcos, E. L.; Carter, S. A., Macromolecules 2005, 38, (25), 10537. 29. Lakowicz, J. R., Principles of fluorescence spectroscopy. 3rd ed.; Springer: New York, 2006. 30. Zhou, Q.; Swager, T. M., J. Am. Chem. Soc. 1995, 117, (26), 7017. 31. Zhou, Q.; Swager, T. M., J. Am. Chem. Soc. 1995, 117, (50), 12593. 32. Wang, J.; Wang, D. L.; Miller, E. K.; Moses, D.; Bazan, G. C.; Heeger, A. J., Macromolecules 2000, 33, (14), 5153. 33. Wang, D. L.; Wang, J.; Moses, D.; Bazan, G. C.; Heeger, A. J., Langmuir 2001, 17, (4), 1262. 34. Fan, C. H.; Wang, S.; Hong, J. W.; Bazan, G. C.; Plaxco, K. W.; Heeger, A. J., Proc. Natl. Acad. Sci. U. S. A. 2003, 100, (11), 6297. 35. Tan, C. Y.; Alas, E.; Muller, J. G.; Pint o, M. R.; Kleiman, V. D.; Schanze, K. S., J. Am. Chem. Soc. 2004, 126, (42), 13685. 36. Tan, C. Y.; Pinto, M. R.; Schanze, K. S., Chem Commun 2002 (5), 446. 37. Gaylord, B. S.; Wang, S. J.; Heeger, A. J.; Bazan, G. C., J. Am. Chem. Soc. 2001, 123, (26), 6417. 38. Chen, L. H.; McBranch, D.; Wang, R.; Whitten, D., Chem. Phys. Lett. 2000, 330, (1-2), 27. 39. Cornelissen, J. J. L. M.; Rowan, A. E.; Nolte, R. J. M.; Sommerdijk, N. A. J. M., Chem. Rev. 2001, 101, (12), 4039.

PAGE 157

157 40. Mueller, A.; O'Brien, D. F., Chem. Rev. 2002, 102, (3), 727. 41. Kim, S.; Jackiw, J.; Robinson, E.; Schanze, K. S.; Reynolds, J. R.; Baur, J.; Rubner, M. F.; Boils, D., Macromolecules 1998, 31, (4), 964. 42. Harrison, B. S.; Foley, T. J.; Knefely, A. S. ; Mwaura, J. K.; Cunningham, G. B.; Kang, T. S.; Bouguettaya, M.; Boncella, J. M. ; Reynolds, J. R.; Schanze, K. S., Chem. Mater. 2004, 16, (15), 2938. 43. Samuel, I. D. W.; Crystall, B.; Rumbles, G.; Burn, P. L.; Holmes, A. B.; Friend, R. H., Chem Phys Lett 1993, 213, (5-6), 472. 44. Nakano, T.; Okamoto, Y., Chem. Rev. 2001, 101, (12), 4013. 45. Pu, L., Acta Polym. 1997, 48, (4), 116. 46. Ciardell.F; Lanzil lo, S.; Pieroni, O., Macromolecules 1974, 7, (2), 174. 47. Moore, J. S.; Gorman, C. B.; Grubbs, R. H., J. Am. Chem. Soc. 1991, 113, (5), 1704. 48. Yashima, E.; Matsushima, T.; Okamoto, Y., J. Am. Chem. Soc. 1995, 117, (46), 11596. 49. Nakako, H.; Mayahara, Y.; Nomura R.; Tabata, M.; Masuda, T., Macromolecules 2000, 33, (11), 3978. 50. Shinohara, K.; Yasuda, S.; Kato, G.; Fujita, M.; Shigekawa, H., J. Am. Chem. Soc. 2001, 123, (15), 3619. 51. Aoki, T.; Shinohara, K.; Kaneko, T.; Oikawa, E., Macromolecules 1996, 29, (12), 4192. 52. Aoki, T.; Kobayashi, Y.; Kaneko, T.; Oikawa, E.; Yamamura, Y.; Fujita, Y.; Teraguchi, M.; Nomura, R.; Masuda, T., Macromolecules 1999, 32, (1), 79. 53. Lam, J. W. Y.; Dong, Y. P.; Cheuk, K. K. L.; Tang, B. Z., Macromolecules 2003, 36, (21), 7927. 54. Lam, J. W. Y.; Dong, Y. P.; Cheuk, K. K. L.; Law, C. C. W.; Lai, L. M.; Tang, B. Z., Macromolecules 2004, 37, (18), 6695. 55. Yashima, E.; Matsushima, T.; Okamoto, Y., J. Am. Chem. Soc. 1997, 119, (27), 6345. 56. Sakurai, S.; Goto, H.; Yashima, E., Org. Lett. 2001, 3, (15), 2379. 57. Leclere, P.; Surin, M.; Viville, P.; Lazzaroni, R.; Kilbinger, A. F. M.; Henze, O.; Feast, W. J.; Cavallini, M.; Biscarini, F.; Schenning, A. P. H. J.; Meijer, E. W., Chem. Mater. 2004, 16, (23), 4452. 58. Yashima, E.; Goto, H.; Okamoto, Y., Macromolecules 1999, 32, (23), 7942.

PAGE 158

158 59. Nilsson, K. P. R.; Olsson, J. D. M.; Konradsson, P.; Inganas, O., Macromolecules 2004, 37, (17), 6316. 60. Goto, H.; Yashima, E., J. Am. Chem. Soc. 2002, 124, (27), 7943. 61. Geng, Y. H.; Trajkovska, A.; Katsis, D.; Ou, J. J.; Culligan, S. W.; Chen, S. H., J. Am. Chem. Soc. 2002, 124, (28), 8337. 62. Oda, M.; Nothofer, H. G.; Scherf, U.; Sunjic V.; Richter, D.; Regenstein, W.; Neher, D., Macromolecules 2002, 35, (18), 6792. 63. Craig, M. R.; Jonkheijm, P.; Meskers, S. C. J.; Schenning, A. P. H. J.; Meijer, E. W., Adv Mater 2003, 15, (17), 1435. 64. Chen, H. P.; Katsis, D.; Mastrangelo, J. C.; Marshall, K. L.; Chen, S. H.; Mourey, T. H., Chem. Mater. 2000, 12, (8), 2275. 65. Fiesel, R.; Scherf, U., Acta Polym. 1998, 49, (8), 445. 66. Zhang, Z. B.; Motonaga, M.; Fujiki, M.; McKenna, C. E., Macromolecules 2003, 36, (19), 6956. 67. Fiesel, R.; Scherf, U., Macromol. Rapid Commun. 1998, 19, (8), 427. 68. Wilson, J. N.; Steffen, W.; McKenzie, T. G.; Li eser, G.; Oda, M.; Neher, D.; Bunz, U. H. F., J. Am. Chem. Soc. 2002, 124, (24), 6830. 69. Zahn, S.; Swager, T. M., Angew. Chem., Int. Ed. 2002, 41, (22), 4225. 70. Fiesel, R.; Halkyard, C. E.; Rampey, M. E.; Kloppenburg, L.; Studer-Martinez, S. L.; Scherf, U.; Bunz, U. H. F., Macromol. Rapid Commun. 1999, 20, (3), 107. 71. Peeters, E.; Delmotte, A.; Janssen, R. A. J.; Meijer, E. W., Adv Mater 1997, 9, (6), 493. 72. Peeters, E.; Christiaans, M. P. T.; Janssen, R. A. J.; Schoo, H. F. M.; Dekkers, H. P. J. M.; Meijer, E. W., J. Am. Chem. Soc. 1997, 119, (41), 9909. 73. Satrijo, A.; Swager, T. M., Macromolecules 2005, 38, (10), 4054. 74. Nelson, J. C.; Saven, J. G.; Moore, J. S.; Wolynes, P. G., Science 1997, 277, (5333), 1793. 75. Prince, R. B.; Saven, J. G.; Wolynes, P. G.; Moore, J. S., J. Am. Chem. Soc. 1999, 121, (13), 3114. 76. Prince, R. B.; Barnes, S. A.; Moore, J. S., J. Am. Chem. Soc. 2000, 122, (12), 2758. 77. Arnt, L.; Tew, G. N., Macromolecules 2004, 37, (4), 1283.

PAGE 159

159 78. Tan, C. Ph.D. Dissertation, University of Florida, 2004. 79. Tan, C. Y.; Pinto, M. R.; Kose, M. E.; Ghiviriga, I.; Schanze, K. S., Adv Mater 2004, 16, (14), 1208. 80. Zhao, X. Y.; Schanze, K. S., Langmuir 2006, 22, (10), 4856. 81. Friedman, A. E.; Chambron, J. C.; Sauvage, J. P.; Turro, N. J.; Barton, J. K., J. Am. Chem. Soc. 1990, 112, (12), 4960. 82. Hartshorn, R. M.; Barton, J. K., J. Am. Chem. Soc. 1992, 114, (15), 5919. 83. Liu, B.; Bazan, G. C., Chem. Mater. 2004, 16, (23), 4467. 84. Liu, B.; Bazan, G. C., Proc. Natl. Acad. Sci. U. S. A. 2005, 102, (3), 589. 85. Dore, K.; Dubus, S.; Ho, H. A.; Levesque, I.; Brunette, M.; Corbeil, G.; Boissinot, M.; Boivin, G.; Bergeron, M. G.; Boudreau, D.; Leclerc, M., J. Am. Chem. Soc. 2004, 126, (13), 4240. 86. Ho, H. A.; Boissinot, M.; Bergeron, M. G.; Corbeil, G.; Dore, K.; Boudreau, D.; Leclerc, M., Angew. Chem., Int. Ed. 2002, 41, (9), 1548. 87. Nilsson, K. P. R.; Rydberg, J.; Baltzer, L.; Inganas, O., Proc. Natl. Acad. Sci. U. S. A. 2003, 100, (18), 10170. 88. McCullough, R. D.; Ewbank, P. C.; Loewe, R. S., J. Am. Chem. Soc. 1997, 119, (3), 633. 89. Lu, L. D.; Rininsland, F. H.; Wittenburg, S. K.; Achyuthan, K. E.; McBranch, D. W.; Whitten, D. G., Langmuir 2005, 21, (22), 10154. 90. Hedstrom, L., Chem. Rev. 2002, 102, (12), 4501. 91. Tong, L., Chem. Rev. 2002, 102, (12), 4609. 92. Neurath, H., J. Cell. Biochem. 1986, 32, (1), 35. 93. Eisenthal, R.; Danson, M. J., Enzyme assays : a practical approach. 2nd ed.; Oxford University Press: Oxford, OX ; New York, 2002. 94. Pinto, M. R.; Schanze, K. S., Proc. Natl. Acad. Sci. U. S. A. 2004, 101, (20), 7505. 95. Kumaraswamy, S.; Bergstedt, T.; Shi, X. B. ; Rininsland, F.; Kushon, S.; Xia, W. S.; Ley, K.; Achyuthan, K.; McBranch, D.; Whitten, D., P Natl Acad Sci USA 2004, 101, (20), 7511. 96. Watson, J. D.; Crick, F. H. C., Nature 1953, 171, (4356), 737.

PAGE 160

160 97. Demeunynck, M.; Bailly, C.; Wilson, W. C., Small molecule DNA and RNA binders : from synthesis to nucleic acid complexes. Wiley-VCH: Weinheim, 2003. 98. Zeglis, B. M.; Pierre, V. C.; Barton, J. K., Chem Commun 2007, (44), 4565. 99. Erkkila, K. E.; Odom, D. T.; Barton, J. K., Chem. Rev. 1999, 99, (9), 2777. 100. Jennette, K. W.; Lippard, S. J.; Vassilia.Ga; Bauer, W. R., Proc. Natl. Acad. Sci. U. S. A. 1974, 71, (10), 3839. 101. Pyle, A. M.; Barton, J. K., Prog. Inorg. Chem. 1990, 38, 413. 102. Barton, J. K.; Danishefsky, A. T.; Goldberg, J. M., J. Am. Chem. Soc. 1984, 106, (7), 2172. 103. Barton, J. K.; Dannenberg, J. J.; Raphael, A. L., J. Am. Chem. Soc. 1982, 104, (18), 4967. 104. Barton, J. K.; Goldberg, J. M.; Kumar, C. V.; Turro, N. J., J. Am. Chem. Soc. 1986, 108, (8), 2081. 105. Pyle, A. M.; Long, E. C.; Barton, J. K., J. Am. Chem. Soc. 1989, 111, (12), 4520. 106. Mahnken, R. E.; Billadeau, M. A.; Nikonowicz, E. P.; Morrison, H., J. Am. Chem. Soc. 1992, 114, (24), 9253. 107. Tamilarasan, R.; Mcmillin, D. R., Inorg. Chem. 1990, 29, (15), 2798. 108. Grover, N.; Gupta, N.; Thorp, H. H., J. Am. Chem. Soc. 1992, 114, (9), 3390. 109. Grover, N.; Thorp, H. H., J. Am. Chem. Soc. 1991, 113, (18), 7030. 110. Gupta, N.; Grover, N.; Neyhart, G. A.; Singh, P.; Thorp, H. H., Inorg. Chem. 1993, 32, (3), 310. 111. Kalsbeck, W. A.; Thorp, H. H., J. Am. Chem. Soc. 1993, 115, (16), 7146. 112. Carlson, D. L.; Huchital, D. H.; Mantilla, E. J.; Sheardy, R. D.; Murphy, W. R., J. Am. Chem. Soc. 1993, 115, (14), 6424. 113. Stoeffler, H. D.; Thornton, N. B.; Temkin, S. L.; Schanze, K. S., Journal of the American Chemical Society 1995, 117, (27), 7119. 114. Lo, K. K. W.; Tsang, K. H. K., Organometallics 2004, 23, (12), 3062. 115. Pinto, M. R.; Kristal, B. M.; Schanze, K. S., Langmuir 2003, 19, (16), 6523. 116. Zhao, X. Y.; Pinto, M. R.; Hardison, L. M.; Mwaura, J.; Muller, J.; Jiang, H.; Witker, D.; Kleiman, V. D.; Reynolds, J. R.; Schanze, K. S., Macromolecules 2006, 39, (19), 6355.

PAGE 161

161 117. Jiang, H.; Zhao, X. Y.; Schanze, K. S., Langmuir 2006, 22, (13), 5541. 118. Jiang, H.; Zhao, X. Y.; Schanze, K. S., Langmuir 2007, 23, (18), 9481. 119. Fan, C. H.; Plaxco, K. W.; Heeger, A. J., J Am Chem Soc 2002, 124, (20), 5642. 120. DiCesare, N.; Pinto, M. R.; Schanze, K. S.; Lakowicz, J. R., Langmuir 2002, 18, (21), 7785. 121. Rininsland, F.; Xia, W. S.; Wittenburg, S.; Shi, X. B.; Stankewicz, C.; Achyuthan, K.; McBranch, D.; Whitten, D., P Natl Acad Sci USA 2004, 101, (43), 15295. 122. Liu, Y.; Ogawa, K.; Schanze, K. S., Anal Chem 2008, 80, (1), 150. 123. Zhao, X. Y.; Liu, Y.; Schanze, K. S., Chem Commun 2007, (28), 2914. 124. Gaylord, B. S.; Heeger, A. J.; Bazan, G. C., J Am Chem Soc 2003, 125, (4), 896. 125. Chen, Y. G.; Zhao, D.; He, Z. K.; Ai, X. P., Spectrochim Acta A 2007, 66, (2), 448. 126. Zhao, D.; Du, J.; Chen, Y. G.; Ji, X. H.; He, Z. K.; Chan, W. H., Macromolecules 2008, 41, (14), 5373. 127. Livnah, O.; Bayer, E. A.; Wilchek, M.; Sussman, J. L., Proc. Natl. Acad. Sci. U. S. A. 1993, 90, (11), 5076. 128. Dwight, S. J.; Gaylord, B. S.; Hong, J. W.; Bazan, G. C., J. Am. Chem. Soc. 2004, 126, (51), 16850. 129. Slim, M.; Durisic, N.; Grutter, P.; Sleiman, H. F., ChemBioChem 2007, 8, (7), 804. 130. Hou, X. L.; Xu, M.; Wu, L. X.; Shen, J. C., Colloid Surface B 2005, 41, (2-3), 181. 131. Liu, X. D.; Diao, H. Y.; Nishi, N., Chem. Soc. Rev. 2008, 37, (12), 2745. 132. Dawson, W. R.; Windsor, M. W., J. Phys. Chem. 1968, 72, (9), 3251. 133. Eaton, D. F., Pure Appl. Chem. 1988, 60, (7), 1107. 134. Forster, T., Ann Phys-Berlin 1948, 2, (1-2), 55. 135. Wang, S.; Gaylord, B. S.; Bazan, G. C., J. Am. Chem. Soc. 2004, 126, (17), 5446. 136. Lakowicz, J. R., Principles of fluorescence spectroscopy. 3rd ed.; Springer Verlag: 2006. 137. Liu, B.; Bazan, G. C., J. Am. Chem. Soc. 2006, 128, (4), 1188. 138. Friedman, R. A.; Honig, B., Biophys. J. 1995, 69, (4), 1528.

PAGE 162

162 139. Delnoye, D. A. P.; Sijbesma, R. P.; Ve kemans, J. A. J. M.; Meijer, E. W., J. Am. Chem. Soc. 1996, 118, (36), 8717. 140. Brunsveld, L.; Zhang, H.; Glasbeek, M.; Ve kemans, J. A. J. M.; Meijer, E. W., J. Am. Chem. Soc. 2000, 122, (26), 6175. 141. Bielawski, C.; Chen, Y. S.; Zhang, P.; Prest, P. J.; Moore, J. S., Chem Commun 1998 (12), 1313. 142. Yagi, S.; Sakai, N.; Yamada, R.; Takahashi, H.; Mizutani, T.; Taka gishi, T.; Kitagawa, S.; Ogoshi, H., Chem Commun 1999 (10), 911. 143. Lahiri, S.; Thompson, J. L.; Moore, J. S., J. Am. Chem. Soc. 2000, 122, (46), 11315. 144. Ravve, A., Principles of polymer chemistry. Plenum Press: Ne w York, 1995; p xiv, 496 p. 145. Chan, H. S.; Dill, K. A., Macromolecules 1989, 22, (12), 4559. 146. Prest, P. J.; Prince, R. B.; Moore, J. S., J. Am. Chem. Soc. 1999, 121, (25), 5933. 147. Shetty, A. S.; Zhang, J. S.; Moore, J. S., J. Am. Chem. Soc. 1996, 118, (5), 1019. 148. Wang, B.; Wasielewski, M. R., J. Am. Chem. Soc. 1997, 119, (1), 12. 149. Kimura, M.; Horai, T.; Hanabusa, K.; Shirai, H., Adv. Mater. 1998, 10, (6), 459. 150. Pang, Y.; Li, J.; Hu, B.; Karasz, F. E., Macromolecules 1998, 31, (19), 6730. 151. Winter, D.; Eisenbach, C. D., J. Polym. Sci., Part A: Polym. Chem. 2004, 42, (8), 1919. 152. Huang, H. M.; Wang, K.; Tan, W. H.; An, D. ; Yang, X. H.; Huang, S. S.; Zhai, Q.; Zhou, L.; Jin, Y., Angew Chem Int Edit 2004, 43, (42), 5635. 153. Li, C. H.; Guo, Y. B.; Lv, J.; Xu, J. L.; Li, Y. L.; Wang, S.; Liu, H. B.; Zhu, D. B., J. Polym. Sci., Part A: Polym. Chem. 2007, 45, (8), 1403. 154. Senthilikumar, S.; Nath, S.; Pal, H., Photochem. Photobiol. 2004, 80, (1), 104. 155. Fan, L. J.; Jones, W. E., J. Am. Chem. Soc. 2006, 128, (21), 6784. 156. Birks, J. B., Photophysics of aromatic molecules. Wiley-Interscience: London, New York,, 1970. 157. Blake, A. J.; Baum, G.; Champness, N. R.; Chung, S. S. M.; Cooke, P. A.; Fenske, D.; Khlobystov, A. N.; Lemenovskii, D. A.; Li, W. S.; Schroder, M., J Chem Soc Dalton 2000, (23), 4285. 158. Dana, B. H.; Robinson, B. H.; Simpson, J., J. Organomet. Chem. 2002, 648, (1-2), 251.

PAGE 163

163 159. Patel, M. B.; Patel, S. A.; Ray, A.; Patel, R. M., J. Appl. Polym. Sci. 2003, 89, (4), 895. 160. Park, E. S.; Lee, H. J.; Park, H. Y.; Kim, M. N.; Chung, K. H.; Yoon, J. S., J. Appl. Polym. Sci. 2001, 80, (5), 728. 161. Kenawy, E. R., J. Appl. Polym. Sci. 2001, 82, (6), 1364. 162. Denyer, S. P., Int. Biodeterior. Biodegrad. 1995, 36, (3-4), 227. 163. Cen, L.; Neoh, K. G.; Kang, E. T., Langmuir 2003, 19, (24), 10295. 164. Ikeda, T.; Yamaguchi, H.; Tazuke, S., Antimicrob. Agents Chemother. 1984, 26, (2), 139. 165. Kugler, R.; Bouloussa, O.; Rondelez, F., Microbiology 2005, 151, 1341. 166. Thorsteinsson, T.; Masson, M.; Kristinsson, K. G.; Hjalmarsdottir, M. A.; Hilmarsson, H.; Loftsson, T., J. Med. Chem. 2003, 46, (19), 4173. 167. Tiller, J. C.; Liao, C. J.; Lewis, K.; Klibanov, A. M., Proc. Natl. Acad. Sci. U. S. A. 2001, 98, (11), 5981. 168. Tiller, J. C.; Lee, S. B.; Lewis, K.; Klibanov, A. M., Biotechnol. Bioeng. 2002, 79, (4), 465. 169. Lewis, K.; Klibanov, A. M., Trends Biotechnol 2005, 23, (7), 343. 170. Lin, J.; Tiller, J. C.; Lee, S. B.; Lewis, K.; Klibanov, A. M., Biotechnol. Lett. 2002, 24, (10), 801. 171. Kwon, D. H.; Lu, C. D., Antimicrob Agents Ch 2006, 50, (5), 1623. 172. Johnston, M. D.; Hanlon, G. W.; Denyer, S. P.; Lambert, R. J. W., J. Appl. Microbiol. 2003, 94, (6), 1015. 173. Fink-Puches, R.; Hofer, A.; Smolle, J.; Kerl, H.; Wolf, P., J. Photochem. Photobiol., B 1997, 41, (1-2), 145. 174. Hohenleutner, U.; Baumler, W.; Karrer, S.; Michel, S.; Landthaler, M., Hautarzt 1996, 47, (3), 183. 175. Jeffes, E. W.; McCullough, J. L.; Weinstein, G. D.; Fergin, P. E.; Nelson, J. S.; Shull, T. F.; Simpson, K. R.; Bukaty, L. M.; Hoffman, W. L.; Fong, N. L., Arch Dermatol 1997, 133, (6), 727. 176. Merchat, M.; Bertolini, G.; Giaco mini, P.; Villanueva, A.; Jori, G., J. Photochem. Photobiol., B 1996, 32, (3), 153. 177. Merchat, M.; Spikes, J. D. ; Bertoloni, G.; Jori, G., J. Photochem. Photobiol., B 1996, 35, (3), 149.

PAGE 164

164 178. Minnock, A.; Vernon, D. I.; Schofield, J.; Griffiths, J.; Parish, J. H.; Brown, S. B., J. Photochem. Photobiol., B 1996, 32, (3), 159. 179. Zanin, I. C. J.; Goncalves, R. B.; Brugnera, A.; Hope, C. K.; Pratten, J., J. Antimicrob. Chemother. 2005, 56, (2), 324. 180. Foote, C. S., Photochem. Photobiol. 1991, 54, (5), 659. 181. Jones, L. R.; Grossweiner, L. I., J. Photochem. Photobiol., B 1994, 26, (3), 249. 182. Kilger, R.; Maier, M.; Szeimies, R. M.; Baumler, W., Chem. Phys. Lett. 2001, 343, (5-6), 543. 183. Baumler, W.; Abels, C.; Karrer, S.; Weiss, T.; Messmann, H.; Landthaler, M.; Szeimies, R. M., Br. J. Cancer 1999, 80, (3-4), 360. 184. Halliwell, B.; Gutteridge, J. M. C., Lancet 1984, 1, (8391), 1396. 185. Jones, R. M.; Bergstedt, T. S.; McBranch, D. W.; Whitten, D. G., J. Am. Chem. Soc. 2001, 123, (27), 6726. 186. Lu, L. D.; Jones, R. M.; McBranch, D.; Whitten, D., Langmuir 2002, 18, (20), 7706. 187. Maisch, T.; Szeimies, R. M.; Jori, G.; Abels, C., Photochem. Photobiol. Sci. 2004, 3, (10), 907. 188. Ogawa, K.; Chemburu, S.; Lopez, G. P.; Whitten, D. G.; Schanze, K. S., Langmuir 2007, 23, (8), 4541. 189. Zhao, X. Ph.D. Dissertation, University of Florida, 2007. 190. Johnson, C. S., Prog. Nucl. Magn. Reson. Spectrosc. 1999, 34, (3-4), 203. 191. Antalek, B.; Hewitt, J. M.; Windig, W.; Yacobucci, P. D.; Mourey, T.; Le, K., Magn. Reson. Chem. 2002, 40, S60. 192. Antalek, B., Concepts Magn. Reson. 2002, 14, (4), 225. 193. Walters, K. A.; Ley, K. D.; Schanze, K. S., Chem. Commun. 1998, (10), 1115. 194. Partee, J.; Frankevich, E. L.; Uhlhorn, B.; Shinar, J.; Ding, Y.; Barton, T. J., Phys. Rev. Lett. 1999, 82, (18), 3673. 195. Funston, A. M.; Silverman, E. E.; Schanze, K. S.; Miller, J. R., J. Phys. Chem. B 2006, 110, (36), 17736. 196. Farley, R. T. Ph. D. Dissertation, University of Florida, 2007. 197. Wang, Y. S.; Schanze, K. S., Chem. Phys. 1993, 176, (2-3), 305.

PAGE 165

165 198. Ogilby, P. R.; Foote, C. S., J Am Chem Soc 1983, 105, (11), 3423. 199. Mathai, S.; Smith, T. A.; Ghiggino, K. P., Photochem. Photobiol. Sci. 2007, 6, (9), 995. 200. Wilkinson, F.; Helman, W. P.; Ross, A. B., J. Phys. Chem. Ref. Data 1993, 22, (1), 113. 201. Nardello, V.; Brault, D.; Chavalle, P.; Aubry, J. M., J. Photochem. Photobiol., B 1997, 39, (2), 146. 202. Chemburu, S.; Corbitt, T. S.; Ista, L. K.; Ji, E.; Fulghum, J.; Lopez, G. P.; Ogawa, K.; Schanze, K. S.; Whitten, D. G., Langmuir 2008, 24, (19), 11053. 203. Nardello, V.; Azaroual, N.; Cervoise, I.; Vermeersch, G.; Aubry, J. M., Tetrahedron 1996, 52, (6), 2031. 204. McCormick, C. L.; Kathmann, E. E., Polymeric materials encyclopedia. CRC Press: Boca Raton, 1996; Vol. 7, p 5462. 205. Lowe, A. B.; McCormick, C. L., Chem. Rev. 2002, 102, (11), 4177. 206. Alfrey, T.; Fuoss, R. M.; Morawetz, H.; Pinner, H., J. Am. Chem. Soc. 1952, 74, (2), 438. 207. Alfrey, T.; Morawetz, H., J. Am. Chem. Soc. 1952, 74, (2), 436. 208. Alfrey, T.; Morawetz, H.; Fitzgerald, E. B.; Fuoss, R. M., J. Am. Chem. Soc. 1950, 72, (4), 1864. 209. Katchalsky, A.; Miller, I. R., J. Polym. Sci. 1954, 13, (68), 57. 210. Ibraeva, Z. E.; Hahn, M.; Jaeger, W.; Bimendina, L. A.; Kudaibergenov, S. E., Macromol. Chem. Phys. 2004, 205, (18), 2464. 211. Kotz, J.; Hahn, M.; Philipp, B.; Bekturov, E. A.; Kudaibergenov, S. E., Makromol. Chem. 1993, 194, (2), 397. 212. Lee, W. F.; Tsai, C. C., Polymer 1995, 36, (2), 357. 213. Salamone, J. C.; Watterson, A. C.; Hsu, T. D.; Tsai, C. C.; Mahmud, M. U., J. Polym. Sci.Polym. Lett. Ed. 1977, 15, (8), 487. 214. Salamone, J. C.; Tsai, C. C.; Watterson, A. C., J. Macromol. Sci. Chem. 1979, A13, (5), 665. 215. Salamone, J. C.; Quach, L.; Watterson, A. C.; Krauser, S.; Mahmud, M. U., J. Macromol. Sci. Chem. 1985, A22, (5-7), 653. 216. Corpart, J. M.; Candau, F., Macromolecules 1993, 26, (6), 1333. 217. Mccormick, C. L.; Johnson, C. B., Macromolecules 1988, 21, (3), 694.

PAGE 166

166 218. Mccormick, C. L.; Salazar, L. C., Macromolecules 1992, 25, (7), 1896. 219. Reynolds, G. A.; Drexhage, K. H., Opt. Commun. 1975, 13, (3), 222. 220. Xu, S. M.; Wu, R. L.; Huang, X. J.; Cao, L. Q.; Wang, J. D., J. Appl. Polym. Sci. 2006, 102, (2), 986.

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167 BIOGRAPHICAL SKETCH Eunkyung Ji was born in Onyang in South Korea and grew up there un til she graduated a high school in February 1995. In Marc h of that year she m oved to Seoul, Koreas capital city and started her academic career in chemical engineering. She graduated from Hanynag University with a bachelors degree in ch emical engineering in February 1999 and then she took a job in Seoul. In March 2001, she restarted her educatio n to achieve masters degree in chemical engineering at Hanyang Universit y. Her research was focused on the development of biosensors using polydiacetylene. After obtaining a mast ers degree in February 2003, she moved to Gainesville in August 2004 and continued her educati on in chemistry at the University of Florida toward get Ph. D. During the course of gradua te school, she joined the group of Dr. Kirk Schanze. In the past five years, she did research in the area of water-sol uble conjugated polymers. After her Ph. D., Eunkyung started her postdoctora l research in the group of Dr. David Whitten at the University of New Mexico.