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Investigating Boronic Acid-Diol Binding and the Application of Boronic Acids to Stimuli-Responsive Materials

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Title:
Investigating Boronic Acid-Diol Binding and the Application of Boronic Acids to Stimuli-Responsive Materials
Creator:
Brooks, William L
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
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Language:
english
Physical Description:
1 online resource (207 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Chemistry
Committee Chair:
SUMERLIN,BRENT S
Committee Co-Chair:
VEIGE,ADAM S
Committee Members:
MILLER,STEPHEN ALBERT
WAGENER,KENNETH B
ANDREW,JENNIFER
Graduation Date:
12/18/2015

Subjects

Subjects / Keywords:
Borinic acids ( jstor )
Boronic acids ( jstor )
Esters ( jstor )
Gels ( jstor )
Hydrogels ( jstor )
Monomers ( jstor )
pH ( jstor )
Polymerization ( jstor )
Polymers ( jstor )
Titration ( jstor )
Chemistry -- Dissertations, Academic -- UF
acid -- boronic -- diabetes -- glucose -- polymerization -- raft -- rdrp -- responsive -- stimuli -- thermoresponsive
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Chemistry thesis, Ph.D.

Notes

Abstract:
The use of boronic acids has seen significant increase over the last few decades, as researchers have employed the functional group in smart materials ranging from responsive hydrogels, to sensors, to drug delivery devices, as separations media, and targeted cancer treatment. The interest in boronic acids is derived from both the Lewis acidity of the boron center and the ability to form boronate esters with 1,2- and 1,3-diols in solution. As a number of biologically important species contain sugar moieties (which are often diols), the binding of boronic acids with sugars is an important concept for a number of biological applications. In this work, thermoresponsive materials were prepared from a boronic acid monomer bearing an electron withdrawing amide substituent. These materials were shown to have a cloud point transition that could be tuned by controlling the solvent conditions, including solution pH and glucose concentration. With optimization, these polymers have potential application in the controlled release of encapsulated pharmaceuticals under hyperglycemic conditions. As scientists expand the use of boronic acids in the development of diol-dependent medical devices, it is important to understand the relationship between boronic acid structure and diol affinity and how to tune the affinity to a specific diol. The binding of various boronic acids and boronic acid analogues with glucose, fructose, and sorbitol was investigated across a wide pH range. These boronic acids represent the range of boronic acids present in recent boronic acid-containing materials. Interestingly, intramolecularly coordinated Wulff-type boronic acids showed very low affinity for these diols, while heterocyclic benzoxaboroles showed some of the highest glucose binding constants, along with the intramolecularly coordinated 2-formylphenylboronic acid. Finally, novel boronic acid monomers were prepared from 2-aminophenylboronic acid pinacol ester. The amide carbonyl in this monomer was found to coordinate with the boron center. It was also discovered that the removal of the pinacol protecting group resulted in dehydration, yielding a new monomer, 3-vinyl-benzo[c][1,5,2]oxazaborinin-1-ol. Attempts were made to polymerize the new monomer via reversible addition-fragmentation chain transfer polymerization. However, further optimization of the system is necessary to prepared well-defined block copolymers and examine the stimuli-response of the new polymers. ( en )
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.
Thesis:
Thesis (Ph.D.)--University of Florida, 2015.
Local:
Adviser: SUMERLIN,BRENT S.
Local:
Co-adviser: VEIGE,ADAM S.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2016-12-31
Statement of Responsibility:
by William L Brooks.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Embargo Date:
12/31/2016
Classification:
LD1780 2015 ( lcc )

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INVESTIGATING BORONIC ACID DIOL BINDING AND THE APPLICATION OF BORONIC ACIDS TO STIMULI RESPONSIVE MATERIALS BY WILLIAM LLOYD AMBROSE BROOKS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2015

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2015 William Lloyd Ambrose Brooks

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To my wife, Mieu, and my daughter, Linh

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4 ACKNOWLEDGMENTS A number of individuals have been instrumental in molding me into the scientist I am today. First and foremost, I would like to thank Dr. Brent Sumerlin. It has been an honor to work for him over these last few years, and I thank him for the opportunity to join his group. He has been instrumental in guiding my research experience through my graduate career and has been a great mentor. I als o thank him for our many discussions of more abstract project ideas and proposals. Our brainstorming sessions have often left me with a new outlook of project inception and development, along with the chemistry driving the proposal itself. Outside of scien ce, Brent is someone that I will always consider a dear friend and colleague. I am also grateful to my dissertation committee, Prof. Ken Wagener, Prof. Stephen Miller, Prof. Adam Veige, and Prof. Jennifer Andrew, for their advice al ong the way. I particula rly wish to thank Dr. Wagener, who has often served as almost a second mentor in matters of science and life. I want to quickly thank all of the faculty and staff in the Department of Chemistry at Southern Methodist University and the faculty and staff of the Butler Polymer Research Laboratory at the University of Florida. They have been invaluable to my success in graduate school. I owe my gratitude to all of the members of the Sumerlin group, past and present. Drs. Debashish Roy and Abhijeet Bapat have be en great scientific mentors in the lab and friend s outside of the lab. Dr. Jennifer Cambre provided the inspiration and groundwork for much of my research through her investigation of boronic acidderived materials. She also guided me in my pursuit of a wo rk lif e balance. My remaining shreds of sanity are in large part thanks to my many friends within the Sumerlin group. In particular, I want to thank Bryan Tucker for our numerous discussions and debates, both about science and about maintaining a family while in graduate

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5 school (and for taking over the GPC). I am grateful to Chris Deng, who has had the misfortune of having to share a solitary lab with me for the last few years. I a m sure I have annoyed him to his wit ’ s end on multipl e occasions, but in the end you have always been a great friend. I also wish to thank Chris Kabb for his help in the lab and with the preparation of this document. I want to thank my family. Although we did no t have a lot when I was growing up, my parents have always done everyth ing they can to support me in everything I do. I want to thank my extended family, including the members of the Ly family. They have welcomed me into their family with open arms, and I could not ask for better inlaws. Finally, I want to thank my wife, Mie u, and daughter, Linh. It can be tough to balance the rigors of graduate school and the demands of raising a family. They have always been supportive of me and have provided me with the love and devotion that I needed to continue this journey. I love you .

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES .........................................................................................................................10 LIST OF FIGURES .......................................................................................................................11 LIST OF SCHEMES ......................................................................................................................17 LIST OF ABBREVIATIONS ........................................................................................................18 ABSTRACT ...................................................................................................................................21 CHAPTER 1 SYN THESIS AND APPLICATIONS OF BORONIC ACIDCONTAINING POLYMERS ...........................................................................................................................23 1.1 Introduction .......................................................................................................................23 1.2 Boronic Acid Functionalized Hydrogels ..........................................................................28 1.2.1 Dynamic Covalent Boronate Ester Cross Linked Hydrogels ................................28 1.2.2 Thermoresponsive Hydrogels .................................................................................33 1.2.3 HIV Barrier Gels ....................................................................................................37 1.3 Nanomaterials ...................................................................................................................39 1.3.1 Boronic AcidContaining Block Copolymers ........................................................39 1.3.2 Boronate Ester Stabilized Nanoparticles ................................................................45 1.4 Molecular Sensing ............................................................................................................48 1.4.1 Electrochemical Sensors .........................................................................................48 1.4.2 Optical Sensors .......................................................................................................53 1.4.2.1 Surface plasmon resonance ..........................................................................53 1.4.2.2 Reflectance spectroscopy .............................................................................54 1.4.2.3 Holographic devices .....................................................................................55 1.4.2.4 Polymerized crystalline colloidal arrays ......................................................55 1.4.2.5 Absorbance sensors ......................................................................................57 1.4.2.6 Fluorescence sensors ....................................................................................58 1.5 Cell Capture and Culture ..................................................................................................63 1.5.1 Cell Culture Growth ...............................................................................................63 1.5.2 Cell Capture and Rele ase ........................................................................................65 1.5.3 Cell Surface Interactions ........................................................................................67 1.6 Summary and Future Directions .......................................................................................67 2 RESEARCH OBJECTIVE .....................................................................................................70

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7 3 PREPARATION AND CHARACTERIZATION OF GLUCOSE AND THERMORESPONSIVE BLOCK COPOLYMERS EXHIBITING A WIDE CLOUD POINT TRANSITION WINDOW .........................................................................................73 3.1 Introduction .......................................................................................................................73 3.1.1 Stimuli Responsive Materials ................................................................................73 3.1.2 Reversible AdditionFragmentation Chain Transfer (RAFT) polymerization .......77 3.2 Results and Discussion .....................................................................................................79 3.2.1 Synthesis of poly( N,N dimethylacrylamide) block poly( N isopropylacrylamide co 4((2 acrylamidoethyl)carbamoyl)phenyl boronic acid) [PDMA bP(NIPAM co ACPBA)] .............................................................................79 3.2.2 Determination of Cloud Point Transition Temperatures for Copolymers in pH 7.4 Phosphate Buffer Solution .....................................................................................82 3.2.3 Examining the Effect of pH on Cloud Point Transition Temperature ....................85 3.2.4 Examining the Effect of Glucose Concentration on Cloud Point Transition Temperature .................................................................................................................87 3.2.5 Examining the Effect of Polymer Concentration on Cloud Point Transition Temperature O ver Broad Glucose Concentrations ......................................................89 3.3 Conclusions .......................................................................................................................92 3.4 Experimental .....................................................................................................................93 3.4.1 Materials .................................................................................................................93 3.4.2 Characterization ......................................................................................................94 3.4.3 Synthesis of tert butyl (2 aminoethyl)carbamate ...................................................94 3.4.4 Synthesis of tert butyl (2 acrylamidoethyl)carbamate ...........................................95 3.4.5 Synthesis of 2acrylamidoethylammonium trifluoroacetate ..................................95 3.4.6 Synthesis of 4carboxyphenylboronic acid pinacol ester .......................................96 3.4.7 Synthesis of (4(chlorocarbonyl)phenyl)boronic acid pinacol ester ......................96 3.4.8 Synthesis of (4((2 acrylamidoethyl)carbamoyl)phenyl)boronic acid pinacol ester (ACPBAE) ...........................................................................................................96 3.4.9 Synthesis of poly( N,N dimethylacrylamide) [PDMA] ...........................................97 3.4.10 Synthesis of poly( N,N dimethylacrylamide) bpoly( N isopropylacrylamide co (4 ((2 acrylamidoethyl)carbamoyl)phenyl)boronic acid) [PDMA bP(NIPAM co ACPBA] (P1 P4) ...................................................................................97 4 EVALUATION OF DIOL BINDING CONSTANTS WITH VARIOUS BORONIC ACID CLASSES ....................................................................................................................99 4.1 Introduction .......................................................................................................................99 4.2 Results and Discussion ...................................................................................................102 4.2.1 Determination of Boronic Acid pKa Values .........................................................108 4.2.2 A pparent Association Constants for Various Boronic Acid Families ..................111 4.3 Conclusions .....................................................................................................................114 4.4 Experimental ...................................................................................................................115 4.4.1 Materials ...............................................................................................................115 4.4.2 Synthesis of 3Acetamidophenylboronic Acid (3 AcPBA) .................................115 4.4.3 Synthesis of Benzoxaborole (BOB) .....................................................................116 4.4.4 Synthesis of 2((dimethyl amino)methyl)phenylboronic acid (DAPBA) ..............116

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8 4.4.5 pKa Determinations for Free Boronic Acids .........................................................117 4.4.6 Determination of Apparent ARS Boronic Acid Association Constants ( KARS) ...117 4.4.7 Determination of Apparent Diol Boronic Acid Association Constants ( Keq) ......118 5 SYNTHESIS OF MACROMOLECULES CONTAINING INTRAMOLECULAR COORDINATING BORONIC ACIDS AND NOVEL HETEROCYCLIC BORONIC ACID ANALOGUES ...........................................................................................................120 5.1 Introduction .....................................................................................................................120 5.2 Results and Discussion ...................................................................................................121 5.2.1 Polymerization of 2A PBAE with 2 (((Dodecylthio)carbonothioyl)thio) 2methylpropanoic acid monomethoxypolyetheylene glycol ester (PEG DMP) Macro Chain Transfer Agent .....................................................................................121 5.2.2 Polymerization of 2APBAE with Cumyl Dithiobenzoate ..................................125 5.2.3 Copolymerization of 2APBAE and NIPAM and determination of reactivity ratios ...........................................................................................................................127 5.2.4 Deprotection of 2APBAE and the resulting monomer formed ...........................131 5.2.5 RAFT polymerization of 3vinylbenzoxazoborinine ...........................................133 5.3 Conclusions .....................................................................................................................135 5.4 Experimental ...................................................................................................................136 5.4.1 Materials ...............................................................................................................136 5.4.2 Characterization ....................................................................................................137 5.4.3 X Ray Crystallography .........................................................................................137 5.4.4 Synthesis of 2acrylamidophenylboronic acid pinacol ester (2 APBAE) ............138 5.4.5 Synthesis of 3vinyl benzo[c][1,5,2]oxazaborinin1ol (3 VBOB) .....................138 5.4.6 Synthesis of 2(((Dodecylthio)carbonothioyl)thio) 2 methylpropanoic acid monomethoxypolyetheylene glycol ester (PEG DMP) .............................................139 5.4.7 Synthesis of 4Cyano 4 (dodecylsulfanylthiocarbonyl)sulfanyl pentanoic acid (CDTPA) ....................................................................................................................139 5.4.8 Synthesis of Cumyl Dithobenzoate: .....................................................................140 5.4.9 Polymerization of PEG bP(2 APBAE) via RAFT Polymerization with PEG DMP ...........................................................................................................................141 5.4.10 Polymerization of 2APBAE via RAFT Polymerization with CDB ..................141 5.4.11 Copolymerization of PEG bP(2 APBAE co NIPAM) via RAFT Polymerization with PEG DMP ................................................................................142 5.4.12 Determination of Reactivity Ratios between 2acrylamidophenylboronic acid and N isopropylacrylamide ................................................................................142 5.4.13 Polymerization of 3VBOB via RAFT Polymerization with CDTPA ...............143 6 CONCLUSIONS AND FUTURE DIRECTIONS ...............................................................144 APPENDIX...........................................................................................................................148 A.1 Nuclear Magnetic Resonance Spectra ...........................................................................148 A.2 Gel Permeation Chromatography Traces .......................................................................157 A.3 pH Titration Curves .......................................................................................................157 A.4 Solution Conditions for Association Constant Determinations .....................................161

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9 A.5 Association Constant Determination Titrations .............................................................162 A.6 High Resolution Mass Spectrometry Plots ....................................................................193 LIST OF REFERENCES .............................................................................................................194 BIOGRAPHICAL SKETCH .......................................................................................................207

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10 LIST OF TABLES Table page 31 Percent monomer conversion, mol% boronic acid content in copolymer, Mn, Dispersity, and cloud point data for PDMA bP(NIPAM co A CPBA) block copolymers .........................................................................................................................82 41 Measured pKa values for various boronic acids and analogues .......................................109 42 Measured apparent association constants (M1) for various boronic acids with ARS, sorbitol, fructose, and glucose at pH 5.2, 7.4, and 8.7. ....................................................112 51 Experime ntal data for the copolymerization of 2 APBAE and NIPAM. ........................130 52 Calculated values model for the copolymerization of 2 APBAE and NIPAM for the for the inverted FinemannRoss reactivity ratio model ...................................................131 A 1 Concentration (mol/L) of the solution components used to determine apparent association constants for 3 AcPBA, 4 MCPBA, and BOB. ............................................161

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11 LIST OF FIGURES Figure page 11 Various organoboron species that can be prepared from borane and its subsequent decomposition events. ........................................................................................................24 12 The esterification equilibrium between a boronic acid or boronate ani on in aqueous solution ...............................................................................................................................26 13 Ionization equilibrium of various boronic acids and analogues. .......................................27 14 Mechanism of hydrogel formation between poly( N,N dimethylacrylamide co 2acrylamidophenylboronic acid) with either poly(vinyl alcohol) or poly( N,N dimethylacrylamide co N acryloyl dopamine). .................................................................30 15 The structure and gel formation mechanism of 3 aminophenylboronic acid modified hyaluronic acid and maltose modified hyaluronic acid. ....................................................32 16 The structure and shape memory response of calcium cross linked hydrogels prepared from 3aminophenylboronic acid modified sodium alginate and poly(vinyl alcohol). .............................................................................................................................33 17 Swelling (A) and contraction (B) response of NIPAM based nanogels modified with 3aminophenylboronic acid a nd 2aminophenylboronic acid, respectively. .....................36 18 Representation of boronic acidderived HIV barrier gel application and response ...........39 19 Schizophrenic behavior of P(NIPAM b3APBA). ...........................................................42 110 Self assembly of benzoxaborole containing copolymer to encapsulate insulin, followed by disruption of the nanoparticle upon glucose addition to release insulin. .......43 111 Reversible star formation in solution by alternating addition of monoand multi functional diol cross linkers. ..............................................................................................44 112 Encapsulation of insulin in boronic acidcatechol stabilized nanoparticles. .....................46 113 Doxorubicin loading in boronic aciddextran stabilized nanoparticles. ............................47 114 The structure of poly(aniline boronic acid). ......................................................................49 115 Formation of a cross linked network imprinted with dopamine. .......................................50 116 Functionalization of a silica surface with a RAFT chain transfer agent, followed by graftingfrom brush growth to yield boronic acidmodified polymer brushes ..................51 117 Pressure transducer derived glucose sensor. . .....................................................................52

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12 118 Structure of boronic acid modified polylysine and a cyanine dye counter ion. ................57 119 Reversible coordination of a boronic acid and a nitrogen atom in an azobenzenebased m onomer unit. ..........................................................................................................58 120 Disruption of electrostatic interaction between an anionic pyranine and cationic viologen based boronic acid monomer unit. ......................................................................59 121 Fluorescent boronic acid modified colloidosomes ............................................................61 122 Structure of P(Am co 3APBA) and quaternary amine functionalized pyrene. ...............62 123 Layer by layer assembly of alternating b oronic acid modified phospholipidbased polymer and PVA.. ............................................................................................................65 124 Reversible capture of MCF 7 cancer cells through sialic acid containing glycoproteins on the cell surface. .......................................................................................66 31 Representation of the various morphologies possible with the amphiphilic block copolymers described herein. .............................................................................................75 32 1H NMR spectrum of poly( N,N dimethylacrylamide) bpoly( N isopropylacrylamide co (4 ((2 acrylamidoethyl)carbamoyl)phenyl)boronic acid) .............................................80 33 1H NMR spectrum of poly( N,N dimethylacrylamide) bpoly( N isopropylacrylamide co (4 ((2 acrylamidoethyl)carbamoyl)phenyl)boronic acid) .............................................81 34 1H NMR spectrum of poly( N,N dimethylacrylamide) bpoly( N isopropylacrylamide co (4 ((2 acrylamidoethyl)carbamoyl)phenyl)boronic acid) .............................................81 35 Plots for cloud point determination of P1 P4 ....................................................................83 36 Derived count rate measured as a function of solution temperature for sample P3. .........84 37 DLS traces for P1 P4 ... ......................................................................................................85 38 pH dependent cloud point transition temperature for P4 ...................................................87 39 Cloud point determination data ..........................................................................................88 310 Derived count rate measurements for solutions of P4 over wide glucose concentrations. ...................................................................................................................90 311 Glucose dependent cloud point transition temperature for P4 ...........................................92 41 ARS binding and transesterification scheme. ..................................................................101 42 Structures of various boronic acids and boronic acid analogues investigated .................102

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13 51 Kinetics and molecular weight evolution for P(2APBAE) ............................................122 52 Size exclusion chromatography traces of P(2 APBAE) ..................................................122 53 Crystal structure of 2 APBAE, exhibiting coordination between the amide oxygen (O1) and the boron center (B1). .......................................................................................124 54 Kinetics and molecular weight evolution for P(2APBAE) with cumyl dithiobenzoate .126 55 Size exclusion chromatography traces for P(2 APBAE) polymerization wi th cumyl dithiobenzoate ..................................................................................................................126 56 Kinetics and molecular weight evolution for P(2APBAE co NIPAM) .........................128 57 Size exclusion chromatography traces for P(2 APBAE co NIPAM) polymerization with PEG DMP ................................................................................................................129 58 Inverted FinemannRoss plot for the copolymerization of 2 APBAE and NIPAM ........131 59 Kinetics and molecular weight e volution for P(3VBOB) ..............................................135 510 Size exclusion chromatography traces for P(3 VBOB) polymerization with CDTPBA ..........................................................................................................................135 A 1 1H NMR spectrum of tert butyl (2aminoethyl)carbamate in CDCl3..............................148 A 2 1H NMR spectrum of tert butyl (2acrylamidoethyl)carbamate in CDCl3 ......................148 A 3 1H NMR spectrum of 4 carboxyphenylboronic acid pinacol ester in CDCl3 ..................149 A 4 1H NMR spectrum of (4 ((2 acrylamidoethyl)carbamoyl )phenyl)boronic acid pinacol ester (ACPBAE) in CDCl3 ...............................................................................................149 A 5 1H NMR spectrum of poly( N,N dimethylacrylamide) bpoly( N isopropylacrylamide) [PDMA bPNIPAM] (P1) in CD3OD ..............................................................................150 A 6 1H NMR spectrum of 2 ((dimethylamino)methyl)phenylboronic acid in CD3OD .........151 A 7 1H NMR spectrum of 3 acetamidophenylboronic acid in DMSO d6 ..............................152 A 8 1H NMR spectrum of benzoxaborole in DMSO d6 .........................................................153 A 9 1H NMR spectrum of 2 acrylamidophenylboronic acid pinacol ester in CDCl3 .............154 A 10 1H NMR spectrum of 3 vinyl benzo[c][1,5,2]oxazaborinin1ol in CD3OD ..................155 A 11 13C NMR spectrum of 3vinyl benzo[c][1,5,2]oxazaborinin1ol in CD3OD .................156

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14 A 12 Si ze exclusion chromatograms for PDMA macroCTA and block copolymers P1 P4 prepared from a chain extension of PDMA .....................................................................157 A 13 pH titration curve for 3 acetamidophenylboronic acid in 0.1M sodium phosphate buffer; absorbance measured at 219 nm ..........................................................................157 A 14 pH titration curve for 4 methylcarbamoylphenylboronic acid in 0.1M sodium phosphate buffer; absorbance measured at 232 nm .........................................................158 A 15 pH titration curve for benzoxaborole in 0.1M sodium phosphate buffer; absorbance measured at 222 nm .........................................................................................................158 A 16 pH titration curve for 2 formylphenylboronic acid acid in 0.1M sodium phosphate buffer; absorbance measured at 260 nm ..........................................................................159 A 17 pH titration curve for 4 formylphenylboronic acid in 0.1M sodium phosphate buffer; absorbance measured at 245 nm ......................................................................................159 A 18 pH titration curve for 2 ((dimethylamino)methyl)phenylboronic acid in 0.1M sodium phosphate buffer; absorbance measured at 225 nm .........................................................160 A 19 Titration curves to determine KARS of 3 AcPBA at pH 5.2 .............................................163 A 20 Titration curves to determine Keq of 3 AcPBA with sorbitol at pH 5.2 ...........................163 A 21 Titration curves to determine KARS of 3 AcPBA at pH 7.4 .............................................163 A 22 Titration curves to determine Keq of 3 AcPBA with sorbitol at pH 7.4 ...........................164 A 23 Titration curves to determine Keq of 3 AcPBA with fructose at pH 7.4 ..........................164 A 24 Titration curves to determine Keq of 3 AcPBA with glucose at pH 7.4 ...........................165 A 25 Titration curves to determine KARS of 3 AcPBA at pH 8.7 .............................................165 A 26 Titration curves to determine Keq of 3 AcPBA with sorbitol at pH 8.7 ...........................166 A 27 Titration curves to determine Keq of 3 AcPBA with fructose at pH 8.7 ..........................166 A 28 Titration curves to determine Keq of 3 AcPBA with glucose at pH 8.7 ...........................167 A 29 Titration curves to determine KARS of 4 MCPBA at pH 5.2 ............................................167 A 30 Titration curves to determine Keq of 4 MCPBA with sorbitol at pH 5.2 .........................168 A 31 Titration curves to determine KARS of 4 MCPBA at pH 7.4 ............................................168 A 32 Titration curves to determine Keq of 4 MCPBA with sorbitol at pH 7.4 .........................169

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15 A 33 Titration curves to determine Ke q of 4 MCPBA with fructose at pH 7.4 ........................169 A 34 Titration curves to determine Keq of 4 MCPBA wit h glucose at pH 7.4 .........................170 A 35 Titration curves to determine KARS of 4 MCPBA at pH 8.7 ............................................170 A 36 Titration curves to determine Keq of 4 MCPBA with sorbitol at pH 8.7 .........................171 A 37 Titration curves to determine Keq of 4 MCPBA with fructose at pH 8.7 ........................171 A 38 Titration curves to determine Keq of 4 MCPBA with glucose at pH 8.7 .........................172 A 39 Titration curves to determine KARS of BOB at pH 5.2 .....................................................172 A 40 Titration curves to determine Keq of BOB with sorbitol at pH 5.2 ..................................173 A 41 Titration curves to determine KARS of BOB at pH 7.4 .....................................................173 A 42 Titration curves to determine Keq of BOB with sorbitol at pH 7.4 ..................................174 A 43 Titration curves to determine Keq of BOB with fructose at pH 7.4 .................................174 A 44 Titration curves to determine Keq of BOB with glucose at pH 7.4 ..................................175 A 45 Titration curves to de termine KARS of BOB at pH 8.7 .....................................................175 A 46 Titration curves to determine Keq of BOB with sorbitol a t pH 8.7 ..................................176 A 47 Titration curves to determine Keq of BOB with fructose at pH 8.7 .................................176 A 48 Titration curves to determine Keq of BOB with glucose at pH 8.7 ..................................177 A 49 Titration curves to determine KARS of 2 FPBA at pH 5.2 ................................................177 A 50 Titration curves to determine Keq of 2 FPBA with sorbitol at pH 5.2 .............................178 A 51 Titration curves to determine Keq of 2 FPBA with fructose at pH 5.2 ............................178 A 52 Titration curves to determine KARS of 2 FPBA at pH 7.4 ................................................179 A 53 Titration curves to determine Keq of 2 FPBA with sorbitol at pH 7.4 .............................179 A 54 Titration curves to determine Keq of 2 FPBA with fructose at pH 7.4 ............................180 A 55 Titration curves to determine Keq of 2 FPBA with glucose at pH 7.4 .............................180 A 56 Titration curves to determine KARS of 2 FPBA at pH 8.7 ................................................181 A 57 Titration curves to determine Keq of 2 FPBA with sorbitol at pH 8.7 .............................181

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16 A 58 Titration curves to determine Keq of 2 FPBA with fructose at pH 8.7 ............................182 A 59 Titration curves to determine Keq of 2 FPBA with glucose at pH 8.7 .............................182 A 60 Titration curves to determine KARS of 4 FPBA at pH 5 .2 ................................................183 A 61 Titration curves to determine Keq of 4 FPBA with sorbitol at pH 5.2 .............................183 A 62 Titration curves to determine Keq of 4 FPBA with fructose at pH 5.2 ............................184 A 63 Titration curves to determine KARS of 4 FPBA at pH 7.4 ................................................184 A 64 Titration curves to determine Keq of 4 FPBA with sorbitol at pH 7.4 .............................185 A 65 Titration curves to determine Keq of 4 FPBA with fructose at pH 7.4 ............................185 A 66 Titration curves to determine Keq of 4 FPBA with glucose at pH 7.4 .............................186 A 67 Titration curves to determine KARS of 4 FPBA at pH 8.7 ................................................186 A 68 Titration curves to determine Keq of 4 FPBA with sorbitol at pH 8.7 .............................187 A 69 Titration curves to determine Keq of 4 FPBA with fructose at pH 8.7 ............................187 A 70 Titration curves to determine Keq of 4 FPBA with glucose at pH 8.7 .............................188 A 71 Titration curves to determine KARS of DAPBA at pH 5.2 ...............................................188 A 72 Titration curves to determine Keq of DAPBA with sorbitol at pH 5.2 .............................189 A 73 Titration curves to determine Keq of DAPBA with fructose at pH 5.2 ............................189 A 74 Titration curves to determine KARS of DAPBA at pH 7.4 ...............................................190 A 75 Titration curves to determine Keq of DAPBA with sorbitol at pH 7.4 .............................190 A 76 Titration curves to determine Keq of DAPBA with fructose at pH 7.4 ............................191 A 77 Titration curves to determine KARS of DAPBA at pH 8.7 ...............................................191 A 78 Titration curves to determine Keq of DAPBA with sorbitol at pH 8.7 .............................192 A 79 Titration curves to determine Keq of DAPBA with fructose at pH 8.7 ............................192 A 80 Highresolution mass spectrometry trace of 3 VBOB .....................................................193

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17 LIST OF SCHEMES Scheme page 31 Proposed RAFT mechanism ..............................................................................................78 32 Synthesis of PDMA macroCTA and chain extension to prepare PDMA bP(NIPAM co ACPBA) block copolymers ..........................................................................................80 33 Dynamic equilibrium between neutral boronic acid, boronate anion, and boronate ester. ...................................................................................................................................86 41 Equilibrium for the displacement of the boronic acid ARS adduct with a diol ..............105 42 Isomerization of 2 FPBA through ketoenol tautomerization .........................................110 43 pH dependent coordination of an amine to a boron center in Wulff type boronic acids .111 51 RAFT polymerization of 2 APBAE in the presence of a PEG DMP macroCTA ...........122 52 Amide imidic acid tautomerization .................................................................................123 53 Proposed delo calization of radical center in 2 APBAE derived radical fragment ..........124 54 Initialization period for the polymerization of 2 APBAE with a RAFT chain transfer agent. ................................................................................................................................125 55 Copolymerization of 2 APBAE and NIPAM in the presence of PEG DMP ..................127 56 Formation of benzoxaboroles from the dehydration of 2(hydroxymethyl)phenylboronic acid ................................................................................132 57 Proposed route for benzoxazaborininie formation ...........................................................133 58 Proposed delocalization of radical center in 3 VBOB derived radical fragment ............133 59 RAFT polymerization of 3 VBOB with CDTPBA .........................................................134

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18 LIST OF ABBREVIATIONS 2AmPBA 2Aminophenylboronic acid 2APBA 2Acrylamidophenylboronic acid 2APBAE 2Acrylamidophenylboronic acid pinacol ester 3AmPBA 3Aminophenylboronic acid 3APBA 3Acrylamidophenylboronic acid 3VBOB 3Vinyl benzo[c][1,5,2]oxazaborinin1ol ACPBA (4 ((2 A crylamidoethyl)carbamoyl)phenyl)boronic acid ACPBAE (4 ((2 A crylamidoethyl)carbamoyl)phenyl)boronic acid pinacol ester AIBN 2,2' A zobisisobutyronitrile Am Acrylamide ATRP Atom transfer radical polymerization CD3OD Deuterated methanol CDCl3 Deuterated chloroform CIPAAm 2C arboxyisopropylacrylamide CTA Chain transfer agent DCM Dichloromethane Dh Hydrodynamic diameter DLS Dynamic light scattering DMA N,N Dimethylacrylamide DMAc N,N Dimethylacetamide

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19 DMAP 4Dimethylaminopyridine DMAPAA N,N D imethylaminopropyl acrylamide DMF N,N Dimeth yl formamide DMP 2Dodecylsulfanylthiocarbonyl sulfanyl2 methylpropionic acid DMSO Dimethyl sulfoxide FRET Fluorescence resonance energy transfer HIV Human Immunodeficiency Virus HPMA N H ydroxypropylmethacrylamide HRSM Highresolution mass spectrometry LCST Lower critical solution temperature MacroCTA Macro chain transfer agent MBA Methylene bisacrylamide MIP Molecularly imprinted polymer MWCO Molecular weight cut off NIPAM N I sopropylacrylamide NIPMAAm N I sopropylmethacrylamide NMP Nitroxide mediated polymerization NMR Nuclear magnetic resonance OEGMA Oligo(ethylene glycol methacrylate) PABA P oly(aniline boronic acid)

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20 PB Sodium phosphate buffer PDMA Poly( N,N –dimethyl acrylamide) PEG Poly(ethylene glycol) PEGDMA P oly(ethylene glycol) dimethacrylate PET P hotoinduced electron transfer PMMA Poly(methyl methacrylate) PNIPAM P oly( N isopropylacrylamide) PS Polystyrene PVA Poly(vinyl alcohol) RAFT Reversible additionfragmentation chain transfer SEC Size exclusion chromatography SEC MALS Multi angle light scattering size exclusion chromatography SPR Surface plasmon r esonance TEA Triethylamine TFA Trifluoroacetic acid THF Tetrahydrofuran TMS Tetramethylsilane VPBA 4V inylphenylboronic acid VPBAE 4V inylphenylboronic acid pinacol ester VPTT Volume phase transition temperature

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21 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INVESTIGATING BORONIC ACID DIOL BINDING AND THE APPLICATION OF BORONIC ACIDS TO STIMULI RESPONSIVE MATERIALS By William Lloyd Ambrose Brooks December 2015 Chair: Brent S. Sumerlin Major: Chemistry The use of boronic acids has seen significant increase over the last few decades, as researchers have employed the functional group in smart materials ranging from responsive hydrogels, to sensors, to drug deliv ery devices, as separations media, and targeted cancer treatment. The interest in boronic acids is derived from both the Lewis acidity of the boron center and the ability to form bor onate esters with 1,2and 1,3diols in solution. As a number of biologica lly important species contain sugar moieties (which are often diols), the binding of boronic acids with sugars is an important concept for a number of biological applications . In this work, thermoresponsive materials were prepared from a boronic acid monom er bearing an electron withdrawing amide substituent. These materials were shown to have a cloud point transition that could be tuned by controlling the solvent conditions, including solution pH and glucose concentration. With optimization, these polymers have potential application in the controlled release of encapsulated pharmaceuticals under hyperglycemic conditions. As scientists expand the use of boronic acids in the development of diol dependent medical devices, it is important to understand the relationship between boronic acid structure and diol affinity and how to tune the affinity to a specific diol. T he binding of various boronic acids

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22 and boronic acid analogues with glucose, fructose, and sorbitol was investigated across a wide pH range. These bo ronic acids represent the range of boronic acids present in recent boronic acid containing materials. Interestingly, intramolecularly coordinated Wulff type boronic acids showed very low affinity for these diols , while heterocyclic benzoxaboroles showed so me of the highest glucose binding constant s , along with the intramolecularly coordinated 2formylphenylboronic acid. Finally, novel boronic acid monomers were prepared from 2aminophenylboronic acid pinacol ester . T he amide carbonyl in this monomer was found to coordinate with the boron center . It was also discovered that the removal of the pinacol protecting group resulted in dehydration, yielding a new monomer, 3vinyl benzo[c][1,5,2]oxazaborinin1ol . Attempts were made to polymerize the new mono mer via reversible addition fragmentation chain transfer polymerization. However, further optimization of the system is necessary to prepared well defined block copolymers and examine the stimuli response of the new polymers.

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23 CHAPTER 1 SYNTHESIS AND APPLICATIONS OF BORONIC ACIDCONTAINING POLYMERS* 1.1 Introduction Recently, boronic acid containing macromolecules have been utilized in a number of biomedical applications,1 including use in dynamic covalent materials, dual thermo and saccharideresponsive hydrogels, sensors, and nanomaterials, often with the goal of detection and treatment of type 1 diabetes, which requires constant monitoring of blood glucose levels and proactive insulin management. The ability of boronic acids to bind with saccharides and potentially undergo an ionization transition makes the materials ideal for diabetes related applications. Other biomedical applications of boronic acidcont aining macromolecules include use as potential HIV transmission barriers, separations and chromatography, and cell capture and culture. This chapter addresses each of these potential and current areas of application, with particular attention to the fundam ental chemistry involved. Boronic acids can be produced by the successive formal hydrolysis of a borane, a boron center bearing three carbon boron bonds (Figure 11) . The first hydrolysis from a borane results in a borinic acid, which is more stable than a borane but can undergo a second hydrolysis event to form a boronic acid. The boron center of these three species, boranes, borinic acids, and boronic acid, is trivalent.2 While hydrolysis of boranes can be used to prepare boronic acids, there are a number of syntheti c methods to prepare boronic acids including trans metallations, metal halogen exchange, transit ion metalcatalyzed direct bor ylation, an d coupling of dibory l species with aryl halides2. In the neutral form, boronic acids exist with a trigonal planar sp2hybridized bo ron, which shares a bond with either an alkyl or aryl group, along with two * Adapted with permission from Chem. Rev. DOI: 10.1021/acs.chemrev.5b00300. Copyright 2015 American Chemical Society.

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24 hydroxyl groups, giving the boron center six valence electrons. While boronic acids are much more stable than either boranes or borinic acids, the oxidation of a boronic acid yield s boric acid, a low toxicity compound found in many household products. In the solid state, boronic acids often exist as anhydride oligomers, particularly the trimeric anhydride, boroxine. Similarly, borinic acids can form anhydride dimers, known as diboroxanes, with a B O B bridge. Figure 11. Various organoboron species that can be prepared from borane and its subsequent decomposition events. Boroxines are the trimeric anhydride of a boronic acid, and diboroxanes are the dimer anhydride of borinic acid s. Rather than serving as proton donors like most carboxylic acids, boronic acids act primarily as Lewis acids, due to the vacant porbital on the boron center. Boronic acids often form complexes with Lewis bases, such as fluoride or hydroxide anions or e lectron donating centers such as nitrogen or oxygen. Upon complexation, the hybridization of the boron center shifts from sp2 to sp3, with the boronic acid becoming an anionic and tetrahedral hydroxyl coordinate species. In aqueous solution, boronic acids exist in equilibrium between the neutral form and a hydroxyboronate anion after complexation with a hydroxide ion (Figure 12) . The pKa of boronic

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25 acids, where 50% of the boron centers exist as anionic boronate species, can range from 4.0 to 10.5.2 Similar to carboxy lic acids, boronic acids can form esters, most frequently with 1,2 and 1,3diols, allowing boronate ester formation with a number of biologically important species including saccharides, glycoproteins, and dopamine significantly broadening the applicabil ity of boronic acids in biology. As ester formation can be very efficient, being described as a “click” reaction, the use of multifunctional boronic acids and diols have been used to prepared a wide variety of hierarchal structures.3 While diols can form esters with both neutral boronic acids and with boronate anions, the esters formed from the neutral boronic acid are significantly less hydrolyticall y stable than those formed from the boronate species. However, boronic esters often have a p Ka lower than that of free boronic acids. As such, the binding of diols with boronic acids can shift the equilibrium from the neutral species to an anionic boronate ester. This shift in ionization often results in a concomitant shift in some measurable characteristics, such as solubility, fluorescence intensity, or cross link density. The ability to form reversible complexes with both Lewis bases and diols allows for real time molecular recognition of various analytes, with boronic acids being used as sensors for a number of compounds.4 A recent review by James and coworkers highlights much of the research towards recognition of glucose, a key step in the treatment of diabetes mellitus.5

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26 Fig ure 12. The esterification equilibrium between a boronic acid or boronate anion in a queous solution with 1,2 or 1,3 diols, where pKa,BA and pKa,BE are the p Ka values for the boronic acid and boronic ester, respectively. Boronate ester formation is often favored near or above the p Ka of a given boronic acid (Figure 12) . As most boronic acids used in biomedical applications are arylboronic acids, there are a number of modifications that can be made to the aromatic ring and associated substituents to alter the p Ka of the boronic acid and the efficiency of ester formation. For example, the addition of electron withdrawing groups on the aromatic ring can reduce the p Ka through inductive effects, while the addition of electron donating substituents can increase the p Ka. Benzoxaboroles, cyclic analogues of boronic acids (Figure 13C), have similar reactivity as boronic acids towards diols but have lower pKa’s, as the transition from sp2 to sp3 hybridization releases ring strain within the B O heterocycle.6 Wulff and coworkers found that the addition of a nitrogen center adjacent to boron can facilitate the formation of boronate esters.7 As shown in Figure 3D, this observation is thought to be due to formation of a B N dative bond, shifting the boron center towards sp3 hybridization, which allows for the formation of more stable boronate esters. At neutral pH, the deprotonated nitrogen coordinates with the boron center, creating a

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27 tetrahedral boronate anion. At lower pH, the amine is protonated, and the boron shifts to trigonal planar sp2 hybridization. At higher pH, the boronic acid forms a boronate center with a hydroxide anion, releasing the nitrogen. Interestingly, secondary and tertiary amines show similar effects on boronate pKa’s.8 However, recent studies suggest that a solvent insertion mechanism may be favored over B N dative bond formation in protic media, such as methanol or water. This solvent insertion creates a zwitterionic species, with a cationic ammonium center and an anionic boronate center, with the ionic stabilization of the boronate center potential ly being the reason for enhanced binding of diols at neutral pH. 9,10 The real contribution of the B N dative bond is debated, and both in situ and computational examination of the complex point to a number of factors that determine the abu ndance of this dative bond, including solvent, boronic acid structure, steric hindrance on the nitrogen, diol concentration, and solution pH. Similarly, the placement of a carbonyl adjacent to the boron center (Figure 13E) facilitates boronate ester formation across nearly the full pH range as a result of the interaction between boron and oxygen.11 13 Figure 13. Ionization equilibrium of various boronic acids and analogues.

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28 1.2 Boronic Acid Functionalized Hydrogels 1.2.1 Dynamic Covalent Boronate Ester Cross Linked Hydrogels The binding affinity of a diol with a boronic acid is dictated by a number of factors, including diol acidity, boronic acid pKa, solution pH, solution composition, and the dihedral angle of the incoming diol. In an aqueous environment, the free diol is in equilibrium with the bound diol in the boronate ester. As with any equilibrium, there is a kinetically controlled dynamic exchang e between the reactants ( i.e. free diol and boronic acid) and the products ( i.e. boronate ester) when low energy transition state exist s between the two. Due to this exchange, materials prepared from boronate esters constantly underdo dynamic rearrangement . T his relatively facile exchange between the bound and free species allows boronate and boronic esters to be considered “d ynamic covalent ” structures.14,15 In boronate ester hydrogels, this covalent character leads to unique mechanical properties. Hydrogels cross linked via boronate esters are not entirely rigid, but rather can flow under their own weight. This creep is a result of the inevitable rearrangement of boronate esters, in which the esters dissociate by hydrolysis to yield the free boronic acids and diols, with the functional groups potentially diffusing away from each other before reforming boron ate ester cross links with newly adjacent acids and diols. While creep is a potentially unwanted physical property for some applications, the exchange between boronate esters allows for materials to exhibit “self healing” behavior. Damage to the material c an be repaired through the formation of new d ynamic covalent bonds without the need for external stimuli. The following section is meant to serve as an introduction to boronate ester hydrogel formation and response. Applications discussed later will, at times, make use of hydrogels in various architectures to accom plish desired tasks, such as saccharide sensing. A number of approaches have been used in the preparation of boronate ester networks, including mixing of polymeric boronic acids and polymeric diols, mixing of multifunctional

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29 small molecules with a counter functionalized polymer support ( i.e., polymeric boronic acid with multifunctional diols or a polydiol with multifunctional boronic acids) .16 Similarly, boronate ester like gels ca n be prepared from diol functional polymers and borax, a tetrahydroxy boron species.17 Gels can also be prep ared through the cross linking of multifunctional small molecules to form a macroscopic network.14 In an early report of boronate ester hydrogels by Kataoka et al.,18 a copolymer was prepared by a radical polymerization of N vinyl 2pyrrolidone (NVP) and 3acrylamidop henylboronic acid (3APBA), followed by gel formation after mixing with poly(vinyl alcohol) (PVA) of various molecular weights. A number of important factors were found to control the physical properties of the resulting gels. Increasing molecular weight o f the polymers resulted in increased viscosity, an effect attributed to greater chain entanglement, which is enhanced via cross linking. Similarly, increased polymer concentration caused an increase in gel viscosity due to greater chain entanglement. The m olar ratio of boronic acid to diol units was also of importance, since the cross link density is a function of both boronic acid and diol content. Similarly, the modulus of a gel could be tuned by the degree of boronic acid incorporation within a polymer a t a given polymer concentration.19 At higher boronic acid conte nt, the gel cross link density increases, resulting in lower molecular weights between cross links. As boronate esters are dynamic in nature, competitive binding with added diols caused disruption of these hydrogels. The addition of glucose to PVA/P(NVP co 3APBA) hydrogels caused a significant reduction in gel viscosity, suggesting preferential binding with glucose over PVA. This approach provides a potential route for gel disruption under physiological conditions. Our group has recently prepared novel hydrogels that can self heal at both neutral and acidic pH. Copolymers of 2 acrylamidophenylboronic acid (2 APBA) and N,N dimethylacrylamide (DMA) were prepared, and gels were made by mixing the copolymers with

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30 PVA or a dopamine modified polymer (Figure 14) .20 A s the carbonyl oxygen of the amide group was in a position to coordinate with the boron center, the boron existed as the tetrahedral boronate species over a wide pH range. Rheological studies showed that the resulting gels were able to self heal almost ins tantly, even at pH 4.0. Similar self healing hydrogels have been prepared from telechelic boronic acid modified Jeffamine cross linkers.21 The stability of these hydrogels shows promise for applications under more acidic conditions, such as i n the gastrointestinal tract. Figure 14. Mechanism of hydrogel formation between poly( N,N dimethylacrylamide co 2acrylamidophenylboronic acid) with either poly(vinyl alcohol) or poly( N,N dimethylacrylamide co N acryloyl dopamine). Repri nted with permission from ref 20. Copyright 2015 American Chemical Society. Gel strength can be adjusted by coupling multiple complexation mechanisms. The hydrophilic polymer poly(ethylene oxide) (PEO), also known as poly(ethylene glycol) (PEG), has been show to form inclusion complexes with cyclodextrin, allowing for hydrogel formation.22,23 A PEO bPVA block copolym er was mixed with cyclodextrin and homotelechelic boronic acid modified PEO.24 In the resulting gel, the PEO section of the block

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31 copolymer formed an inclusion complex with cy clodextrin, while the boronic acid endgroups formed cross links with PVA units. While boronic acidPVA hydrogels are known to undergo relaxation and creep,25 the addition of the cyclodextrin inclusion complexes inhibited relaxation. With inc reasing cyclodextrin content, the gel strength increased and the material behaved like an elastic solid. The gel was used to encapsulate fluorescein isothiocyanate (FITC)labeled bovine serum albumin, which could be released in the presence of glucose. T his result suggests that gels of this nature can be used as controlled delivery devices for biologically important compounds, including proteins such as insulin. Recently, a boronic acid containing hydrogel was prepared from modified hyaluronic acid, which was chosen as a readily available biopolymer that degrades easily via enzymatic pathways.26 As shown in Figure 15, hyaluronic acid was modified wit h either an aryl boronic acid or a maltose derivative. As hyaluronic acid does not contain cis diol moieties, the polymer side chains were modified with maltose, a disaccharide of glucose. When the boronic acid and maltose modified polymers were mixed, boronate esters formed between the boronic acids and the diols in maltose. The resulting hydrogels exhibited pH dependent self healing behavior and viscoeleastic properties. Given the soft na ture of the materials and the wide distribution of hyaluronic acid in the brain, the hydrogels were suggested for use as soft matrices for neuronal regeneration.

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32 Figure 15. The structure and gel formation mechanism of 3aminophenylboronic acid modified hyaluronic acid and maltosemodified hyaluronic acid. Reprinted with permission from ref 26. Copyright 2014 Wiley VCH Verlag GmbH & Co. KGaA. Boronate ester cross linked hydrogels have been used to prepare shapememory materials. Sodium alginate partially modified with 3aminophenylboronic acid (3AmPBA) was reacted with PVA to form hydrogels (Figure 16) .27 The hydrogels were formed into their permanent shape and cross linked with calcium cations. As the bor onate esters were unstable at low pH, the hydrogel could be temporarily deformed at pH 6.0 before fixing at pH 10.6. The temporary shape could then be reversed either by reducing the pH or by soaking in a saccharide solution.

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33 Figure 16. The structure an d shape memory response of calcium cross linked hydrogels prepared from 3aminophenylboronic acid modified sodium alginate and poly(vinyl alcohol). Reproduced with permission from ref 27. Copyright 2015 Wiley VCH Verlag GmbH & Co. KGaA. 1.2.2 Thermorespon sive Hydrogels Boronic acid modified hydrogels can also be coupled with other stimuli responsive compounds to prepare multi responsive materials. Homopolymers of N isopropylacrylamide (NIPAM) undergo a volume phase transition in aqueous solution when heate d to temperatures above ~32 C.28,29 For polymers that exhibit a lower critical solution temperature (LCST), such as NIPAM, the polymer is soluble in water below the cloud point (the temperature at which polymer transitions from soluble to insoluble upon heating). This behavior arises as a result of hydrogen bonding of the polymer with the surrounding water and limited intra and intermolecular hydrogen bonding between monomer units. Upon heating, the entropic penalty of stabilizing the h ydrophobic portion of the monomer units increases, hydrogen bonds between the polymer and water break, and intra and intermolecular hydrogen bonding dominates. For

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34 polymers exhibiting a LCST like cloud point, the volume phase transition temperature (VPTT) can be adjusted by copolymerization, with hydrophobic comonomers lowering the cloud point and hydrophilic monomers increasing the cloud point.29 As boronic acids can exist as either hydrophobic below the pKa or hydrophilic above the pKa or upon complexation with glucose, the VPTT of boronic acid modified thermoresponsive materials are a function of glucose concentration and pH.30 Kataoka et al. prepared boronic acid modified thermoresponsive hydrogels by copolymerizing NIPAM, methylene bisacrylamide (MBA), and 3APBA, with the resulting gels having 10 mol% boronic acid content.31 The gels were swollen in a pH 9 buffer, and the degree of swelling was measured as a function of temperature and glucose concentration. Because complexation with glucose increases the hydrophilicity of 3 APBA units, increasing glucose concentration caused an increase in the VPTT. The device was employed as an ex situ insulin release device. Fluorescein modified insulin was encapsulated within the gel, and release of the protein was measured as a function of glucose concentration. The swelling of the hydrog els with increasing glucose concentration allowed for the diffusion of insulin from the gel, creating a self regulating insulin delivery device. Cross linked gels were prepared via copolymerizations of either NIPAM or N isopropylmethacrylamide (NIPMAAm) with either 3 APBA or (4 ((2 acrylamidoethyl)carbamoyl)phenyl)boronic acid (ACPBA).32 Gels prepared from NIPAM and 3APBA exhibited enhanced swelli ng upon glucose addition, but only at elevated pH, as the 3APBA copolymer had an apparent pKa of 8.5, much higher than physiological pH. Gels prepared from NIPAM and ACPBA showed enhanced swelling at pH 7.4 because of the lower apparent pKa (8.1) of ACPBA . However, for both gels, complete thermoresponsive deswelling occurred below body temperature, as the VPTT of PNIPAM is already near body temperature before

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35 being depressed by the incorporation of hydrophobic boronic acid comonomers. To shift the response temperature, NIPAM was replaced with NIPMAAm (VPTT of ~ 40 C). Even so, the hydrophobicity of the boronic acids lowered the VPTT of the gel to below body temperature. Copolymerization with a small amount of methacrylic acid was successful in shifting the VPTT to above body temperature upon glucose addition. The resulting gel was collapsed at body temperature, but when glucose was added the gel could swell and release any payload encapsulated within the gel. In a separate study, cross linked hydrogels were prepared from NIPMAAm, ACPBA, and 2 carboxyisopropylacrylamide (CIPAAm).33 Upon reaction with glucose, the hydrogel volume increased by as much as 163%. The volum e increase could potentially serve as a release mechanism for encapsulated insulin, creating an implantable stimuliresponsive insulin delivery device. Thermoresponsive boronic acid modified hydrogels have also been prepared from NIPAM copolymerized with acrylic acid and MBA in water. During the polymerization, the resulting polymers exhibited a volume phase transition, precipitating into microgels that were subsequently cross linked through MBA. After purification, the microgels were modified with 2aminop henylboronic acid (2 AmPBA)34,35 and 3aminophenylboronic acid (3AmPBA)36 through the acrylic acid units. Unlike 3AmPBA conjugates, which have the amide group in the meta position relative to the boronic acid, the amide groups in 2AmPBA conjugates are in the orthoposition. In this geometry, the oxygen of the amide can coordinate with the boron, creating a tetrahedral center. While gels modified with 3 AmPBA expand upon reaction with glucos e, the microgels modified with 2 AmPBA contracted (Figure 17) . The contraction was thought to be the result of glucose forming esters with two boronic units rather than undergoing a single complexation. Previous work showed that if the boronic acids in a hydrogel exist primarily in the

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36 neutral trigonal form, a 1:1 glucose:boronic acid complex is favored, while a 1:2 glucose:boronic acid complex is favored when the boronic acid exists primary in the anionic tetrahedral form.37 As the amide oxygen coordinates with the boron center, the 1:2 complex is favo red and contraction occurs. Figure 17. Swelling (A) and contraction (B) response of NIPAM based nanogels modified with 3aminophenylboronic acid and 2aminophenylboronic acid, respectively. Adapted from ref 34 with permission. Copyright 2014 Royal Society of Chemistry. Zhang and coworkers took a different approach to hydrogel formation. NIPAM and ACPBA were copolymerized in the presence of 3 mercaptopropionic acid to prepare a carboxylic acid functional copolymer.38 NHS coupling linked the polymers covalently to M13 viruses. Upon heating above the VPTT, the M13 virus segments interact and the solution undergoes a sol gel transition. The addition of 25 mM glucose causes a sh ift in the sol gel transition temperature from 18 to 23 C. FTIC labeled insulin was encapsulated within the gel and the release profile was measured with and without glucose. Although insulin was released from the gel without glucose, the release rate increased significantly when glucose was added.

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37 1.2.3 H IV Barrier Gels One of the primary routes of human immunodeficiency virus (HIV) transmission in heterosexual sex occurs through the transfer of virions from seminal fluid to the mucosa of the vaginal wall s. Before intercourse, the vaginal fluid has a pH range of 4 to 5, but the pH increases upon insemination due to the alkaline pH, buffering capacity, and volume of semen. The virions diffuse from the seminal fluid to the vaginal mucosa before penetrating t he mucosal surface. This diffusion occurs within a matter of minutes. After reaching the vaginal mucosa, the virions penetrate the epithelium before infecting CD4+ T cells, macrophages, and dendritic cells in the subepithelium tissue. If the diffusion of HIV virions to the vaginal mucosa can be inhibited, a primary route of HIV infection would be suppressed. This objective could be accomplished through the use of a viscous gel that limits virion diffusion. However, application of a high viscosity gel is problematic, necessitating a lower viscosity during application and higher viscosity during intercourse. Salicylhydroxamic acid has been shown to exhibit weak interactions with boronic acids under acid conditions, while having a much higher binding constant at neutral pH.39 As such, loose gels can be administered at acidic vaginal pH and then become more rigid upon insemination and neutralization (Figure 18) . Jay et al. prepared copolymers of 2 hydroxypropylmethacrylamide (HPMA) with either N [3 (2 methylacryloylamino) propyl] 4amidophenylboronic acid (APMAmPBA) or 4 [(2 methylacryloylamino) methyl] salicylhydroxamic acid (MAAmSHA).40 Mixing the two copolymers at low pH resulted in gels that underwent a sol gel transition at high shear rates, suggesting the boronic ester like cross links were in low concentration. As the pH increased, the boronic acid shifted towards the boronate anion, favoring ester formation. The rheological crossover point shifted to lower shear rate s, and at pH 7.5 the gel behaved purely like a covalently cross linked elastomer.

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38 Diffusion of Gag –Cherrylabeled HIV 1 virions through the gel at various pH was monitored via video particle tracking.41 At pH 4.5, the HIV 1 virions were able to diffuse through the gel due to the low gel viscosity, although virion diffusion may have been retarded due to interactions between the boronic acids in the gel and the protein envelope coating the virions. Binding with surface proteins has been shown to occur in polymers and oligomers functionalized with benzoxaboroles , a boronic acid analogue.42 At higher pH, the virions exhibited limited diffusion, suggesting that higher gel viscosity is the limiting factor to particle diffusion. At pH > 4.8, diffusion of HIV 1 nearly ceased, with the virions diffusing an average of infectious virions.

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39 Figure 18. Representation of boronic acidderived HIV bar rier gel application and response (I) An applicator [a] is used to apply the weak viscoelastic boronic acid salicylhydroxamic acid hydrogel [b]. (II) The gel forms a barrier between the vaginal mucosa and the environment. (III) The gel consists of cross l inks between the two polymers [d] and the diols present on the epithelial surface [c] and in cervical mucus [e] (IV) Upon insemination, vaginal pH is neutralized and the hydrogel undergoes a shift to much higher viscosity, inhibiting diffusion of virions [ g] to the vaginal mucosa. (V) The pH dependent equilibrium between free and boronate cross linked structures with salicylhydroxamic acid. Reprinted with permission from ref 41. Copyright 2011 Elsevier. 1.3 Nanomaterials 1.3.1 Boronic Acid Containing Block Copolymers In aqueous solution, amphiphilic block copolymers can self assemble to form a variety of different nanostructures, including spherical micelles, cylindrical micelles, and vesicles. This self assembly provides a potential route for drug deliver y, as pharmaceuticals can be sequestered within the nanoparticles. By incorporating boronic acids, amphiphilic nanoparticles can be prepared that dissociate in response to changes in solution pH or saccharide

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40 concentration. The inherent ability of block co polymers to self assemble and provide protection for an encapsulated species, while simultaneously allowing for stimuli responsive release of the encapsulated payload, provides a clear advantage over other delivery mechanisms. As such, there is great inter est in employing glucose responsive block copolymers to controllably deliver insulin as a method of treating type 1 diabetes. Boronic acid containing block copolymers have often been prepared by controlled or living polymerization techniques.43 Qin et al. prepared block copolymer of styrene and 4 vinylphenylboronic acid via atom transfer radical polymerizatio n (ATRP).44 Homopolymers of either 4 vinylphenylboronic acid pinacol ester (VPBAE) or a silane functional styrenic monomer were prepared, followed by chain e xtension of the resulting macroinitiator with styrene. The silane functional polymer was converted to the pinacol boronic ester via post polymerization modification. The self assembled morphologies of the pinacol deprotected materials could be tuned through careful control of solution pH and choice of organic cosolvent.45 Water soluble block copolymers have also been prepared by our group.46 VPBAE was polymerized via reversible addition fragmentation chain transfer (RAFT) polymerization, yielding polymers of controlled molecular weight and narrow molecular weight distribution. The PVPBAE homopolymers were chain extended with DMA followed by deprotection of the boronic acid via a transesterification with a heterogeneous boronic acid modified resin. Polymerization of the unprotected styrenic monomer was also possible.43 Due to the hydrophobicity of the boronic acid segment at pH < p Ka, the polymer underwent self assembly in water to form nanostructures with a diameter of ~100 nm. Sugar responsive block copolymers have been prepared from 3 APBA via RAFT polymerization. The free boronic acid, with no protecting group, was polymerized in 95:5 (v/v)

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41 DMF:water.47 The addition of 5% water was necessary to inhibit boronic acid dehydration and boroxine formation.15 After polymerization, the boronic acid homopolymers were chain extended with DMA to prepare the amphiphilic block copolymer. At pH 8.7, below the pKa of the P(3 APBA) block (p Ka ~ 9), the block copolymer formed nanostructures with hydrodynamic diameters of 40 nm. The addition of 45 mM glucose caused the nanoparticles to diss ociate due to the formation of the boronate ester with glucose. Similarly, the nanoparticles were disrupted by increasing the pH to 10.7, ( i.e., pH > pKa of the 3 APBA units ) . The critical concentration of glucose needed to induce nanoparticle dissociation could also be tuned by incorporating DMA as a hydrophilic comonomer within the 3APBA block.48 Our group also combined the pH and sugar responsiveness of 3 APBA with the thermoresponsiveness of NIPAM to prepare schi zophrenic block copolymers.49 3APBA was polymerized via RAFT polymerization , followed by chain ex tension with NIPAM. In aqueous solution, the block copolymer displayed three different morphologies. At high pH and at room temperature, both segments of the block copolymer were hydrophilic, with the boronic acids existing primarily in the anionic tetrahe dral form. Similarly, at a pH near the p Ka of 3 APBA and upon addition of glucose, the polymers transitioned to unimers due to the formation of anionic boronate esters with glucose. If the pH was lowered to below the pKa in the absence of glucose, the majo rity of the boronic acids became neutral and hydrophobic, causing the 3APBA blocks to self assemble in the interior of nanoparticles with NIPAM decorated coronas. At high pH and elevated temperature, the NIPAM segment underwent a transition in solubility, self segregating into the interior of the nanoparticle, while the anionic hydroxyl boronate groups remained in the corona (Figure 19) .

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42 Figure 19. Schizophrenic behavior of P(NIPAM b3APBA). At high pH and low temperature, the polymer exists as unimers. High pH and temperature led to nanoparticle formation with a PNIPAM core, while low temperature and pH leads to nanoparticle formation with a P(3 APBA) core. Reproduced with permissio n from ref 49. Copyright 2009 Royal Society of Chemistry. Most of the previously discussed examples of self assembled boronic acid block copolymers were responsive to the addition of saccharides such as glucose and fructose. However, this response was gen erally only possible at pH higher than physiological pH, as the boronic acids only efficiently bind with diols near or above the pKa of the boronic acid. One route to increasing glucose response at reduced pH is to facilitate boronate anion formation ( i.e., sp3 hybridization) through coordination of Lewis bases with the boron center. The ability to bind with glucose at physiological pH has been obtained through the use of Wulff type boronic acids (Figure 13B), in which a secondary or tertiary amine was use d to weakly coordinate with the boron center.7 Upon coordination, the boronic acid transitions to the boronate anion, facilitating boronate ester formation. Block copolymers of (2 ((dimethylamino)me thyl) 5vinylphenyl)boronic acid with PEG formed nanostructures due to the insolubility of the boronic acid segment.50 The nanostructures dissociated at physiological pH when saccharides were added. Response at physiological pH was also obtained through the use of a para amido substituted boronic acid. 51,52 The electron withdrawing nature of the amide carbonyl reduced the pKa of the arylboronic acid, f acilitating glucose response at physiological pH. Self assembled nanoparticles of PDMA bPACPBA were found to dissociate in the presence of glucose and fructose at pH 7.4. Benzoxaborole s have also been shown to respond at physiological pH.53 An

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43 alternating copolymer of styreneboroxole and oligo(ethylene glycol) maleimide was prepared with a PEG RAFT macro chain transfer agent (Figure 110) .54 The resulting block copolymers were used to encapsulate insulin, with the rate of release being a function of glucose concentration. Figure 110. Self assembly of benzoxaborole containing copolymer to encapsulate insulin, followed by di sruption of the nanoparticle upon glucose addition to release insulin. Reprinted with permission from ref 54. Copyright 2012 American Chemical Society. Block copolymers of boronic acids can also be used in the preparation of dynamic covalent nanostructures. PDMA bP(3 APBA), prepared via RAFT polymerization, was dissolved in methanol and core cross linked stars were prepared through boronic ester formation with multifunctional diols.15 As both segments of the block copolymer were methanol soluble, the polymer existed as unimers in solution with a size of ~5 nm. Upon addition of a multifunctional diol, the unimers began to coalesce, forming polymeric stars (Figure 111) . Various multifunctional cross linkers were examined, with a trifunctional diol prepared from thioglycerol and triacryloylhexahydro 1,3,5 triazine providing the most efficient cross linking, forming stars with a hydrodynamic diameter of ~17 nm as determin ed via dynamic light

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44 scattering. With this tris diol, only 0.3 equivalents of the cross linker was necessary to efficiently form stars with a negligible concentration of free arms remaining. The formation of stars was further verified by 1H NMR spectroscop y, where the attenuation of the aromatic peaks in the stars suggested desolvation of the boronic acid segments. Interestingly, due to the dynamic nature of boronic esters, the stars were easily dissociated upon addition of 2amino 2methyl 1,3propanediol (AMPOH), a diol that competes with the tris diol in boronic ester formation. After dissociation, the stars could be reformed upon addition of more tris diol, even without removal of AMPOH. This star formation dissociation was shown to occur even after 6 cy cles, with the high concentration of both the tris diol and AMPOH having no noticeable deleterious effect on star formation. Figure 111. Reversible star formation in solution by alternating addition of mono and multi functional diol cross linkers. Rep rinted with permission from ref 15. Copyright 2011 American Chemical Society.

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45 1.3.2 Boronate Ester Stabilized Nanoparticles Boronate esters have also been used to stabilize nanoparticle assemblies.55 58 It has previously been shown that boronic acids bind more efficiently with catechol based diols compared to other diol types, including glucose and fructose.59 Lam et al . prepared a series of PEG ylated dendritic cholic acids, referred to as telodendrimers, bearing ei ther phenylboronic acid or catechol functional groups at the junction point between PEG and the dendritic unit.60 Upon mixing, boronate esters formed between the catechol and boronic acid units, with the cross linke d micelles stable upon addition of a physiological concentration of glucose but dissociated when the pH was reduced to 5.0 or upon addition of mannitol, a sugar alcohol. Such micelles show potential for triggered release either in the acidic environment of tumor tissues or upon intravenous injection of mannitol. The in situ stability of the boronate ester cross linked micelles was compared to that of a non cross linked analogue using a fluorescence resonance energy transfer (FRET) reporter system.60 The red orange dye rhodamine B (acceptor) was covalently bound to the dendritic polymer, while green dye DiO (donor) was encapsulated in the micelle. In the nanoparticles, DiO and rhodamine B were in close proximity, allowing energy transfer. However, upon particle dissociation, the FRET ratio was reduced by separation of the donor and acceptor. Solutions of cross linked and noncross linked micelles were injected into the tail vein of mice, and samples of blood were withd rawn periodically for FRET ratio measurement. Non cross linked micelles quickly dissociated, with the FRET ratio falling from 80% pre injection to 46% within 1 minute and to 21% after 24 minutes. The FRET ratio in cross linked micelles decreased much more slowly showing that cross linked micelles had significantly longer blood circulation times compared to their non cross linked analogues. Furthermore, the cross linked micelles were found to accumulate in tumor tissue, potentially allowing for targeted delivery with triggered release as

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46 a result of the lower tissue pH or by injection of mannitol. Similarly, mixtures of catechol and boronic acid functional block copolymers resulted in cross linked nanoparticles that were stable at physiological pH (Figure 112) .57 The nanoparticles were disrupted at low pH, such as that found in an endosome, or upon fructose addition, releasing encapsulated fluoresceinl abeled insulin. Figure 112. Encapsulation of insulin in boronic acidcatechol stabilized nanoparticles. The nanoparticle cross links were disrupted at the lower endosomal pH, resulting in nanoparticle swelling and disassembly, releasing insulin. Adapted with permission from ref 57. Copyright 2013 American Chemical Society. Two sets of doxorubicinloaded nanoparticles were prepared from dextran bpoly(DL lactide) block copolymers.55 One set of nanoparticles was unmodified, while in the other set, the dextran blocks were modified with 3carboxy 5nitrophenylboronic acid which has a relatively low p Ka. Under acidic conditions, both nanoparticle assemblies had similar critical micelle

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47 concentrations. However, at physiological pH, the boronate ester stabilized nanoparticle showed a much lower critical micelle concentration, due to cross links forming between the boronic acid units and the dextran backbone, while simultaneously allowing f or doxorubicin release at the lower pH of cancerous tissue (Figure 113) . Similarly, DOX loaded nanoparticles were prepared from catechol modified PEG bP(L lysine).61 Boronic acid functionalized cholesterol was conjugated to the polymer, facilitating self assembly and thus DOX loading. When the pH was reduced, boronate ester formation became unfavorable and the nanoparticles dissociated. Boronate ester nanoparticles have also been prepared by modification of poly(acrylic acid) with glucosamine, an amine functional monosaccharide, followe d by mixing with PEG bP(AA co 3APBA).58 The resulting nanoparticles dissociated upon a ddition of glucose due to trans esterification with the boronate ester cross links. Figure 113. Doxorubicin loading in boronic acid dextran stabilized nanoparticles. The cross links were disrupted upon endocytosis, releasing doxorubicin in the interior of the cell. Reproduced with permission from ref 55. Copyright 2014 Wiley VCH Verlag GmbH & Co. KGaA.

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48 1.4 Molecular Sensing 1.4.1 Electrochemical Sensors Boronic acids can be used in the analysis of a wide range of substrates, including fluoride,62 saccharides,63 copper,64 67 reactive oxygen species, 68 70 and even water in organic solvents.71,72 Simple glucose electrochemical sensors can be prepared by depositing boronic acid hydrogels onto an electrode surface. For example, a 3APBA based PVA hydrogel was cast on the surface of a platinum electrode and cross linked through the residual hydroxyl groups on PVA.73 Swelling in the presence of glucose yielded a measureable increase in current. The saccharide response could also be measured by Faradaic impedance spectroscopy.74 When an electrode modified with a boronic acid hydrogel was placed in glucose solution, the electrontransfer resistance dropped, suggesting the swollen hydrogels allow for more facile electron transfer between the gold wire and the solution redox labe l. Upon removal of glucose, the resistance increased. A calibration curve based on the reduced electron transfer resistance allowed for the determination of solution glucose concentration and the preparation of glucose sensors. Conductive polymers have be en used to sense a variety of analytes including glucose, fructose, dopamine, and fluoride anions.75 Polyaniline, a highly conductive polymer prepared from the relatively simple monomer aniline through an oxidative polymerization mechan ism, is relatively straightforward to polymerize at the surface of an electrode, allowing for facile probe synthesis. By preparing polyaniline with boronic acid groups, the electrochemical properties of the material change as a function of solution conditi ons,76,77 although binding with diols is highly pH dependent.78 When poly(aniline boronic acid) (PABA) was prepared at the surface of a glassy carbon electrode (Figure 114), the resulting probe exhibited an increased open circuit methyl -Dglucoside, Dglucose, or fructose was added, allowing for

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49 determination of analyte concentrations.79 Inter estingly, the sensitivity of the probes increased significantly when the electrochemical polymerization was carried out in the presence of excess fluoride anion, suggesting side reactions in the absence of fluoride may be detrimental to electrode performan ce.80 Copolymers of aniline and 3 aminophenylboronic acid have also been prepared enzymatically using horseradish peroxidase. At low concentrations, the 3 AmPBA acted as a self dopant, increasing co nductivity. However, at increased concentration, the bulkiness of the boronic acid limited conductivity.81 Films prepared from copolymers of aniline and 3aminophenylboronic acid exhibited fully reversible changes in absorption spectra upon binding with various saccharides.81,82 Increasing the rate of diffusion of analytes into PABA can increase the response of the material.80 In one route, increased diffusion was achieved through th e preparation of PABA nanotubes in the pores of anodized aluminum substrates. The increased porosity and surface area significantly increased response to added saccharides.83 PABA systems have also been used to sense other biologically important compounds, including dopamine84,85 and glycoproteins.86 Figure 114. The structure of poly(aniline boronic acid). The ratio of the repeat units is determined by the oxidation state of the polymer. Adapted with permission from ref 79. Copyright 2001 American Chemical Society. A similar approach was employed to design a molecularly imprinted polymer (MIP) sensor for dopamine.87 Poly(aniline co anthran ilic acid) was modified with 3 AmPBA. Electrochemical sensors were then cast in the presence of dopamine to prepare molecularly

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50 imprinted polymers, increasing sensitivity and specificity towards dopamine over similar compounds. The resulting sensors were used to determine dopamine concentrations in a known sample and in human plasma. Dopamine specific MIP electrochemical sensors have also been prepared from 3APBA with MBA and acrylamide (Figure 115) .88 The resulting sensors responded selectively to dopamine and showed limited interference by ascorbic acid, which has a similar redox potential to dopa mine. Figure 115. Formation of a cross linked network imprinted with dopamine. Removal of the dopamine leaves a cavity with size and shape similar to that of dopamine, increasing sensitivity towards dopamine over other substrates. Adapted with permission from ref 88. Copyright 2013 Wiley VCH Verlag GmbH & Co. KGaA. MIP sensors were also prepared on quartz crystal microbalance (QCM) electrodes using 4methacrylamid ophenylboronic acid and mannose, cross linked with ethylene glycol dimethacrylate.89 Binding with mannose was significantly more favorable than with fructose,

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51 even though fructose generally has a higher association constant.59 The sensor also exhibited binding with immunoglobulin M, a basic antibody that contains mannose groups. QCM based sensors have seen wide use in the analysis of a number of compounds, allowing for rapid, highly sensitive measurements. When modified with boronic acidcontaining polymers, QCM surfaces become reliable sensors for several biologically important compounds, particularly saccharides such as glucose.90 As shown in Figure 116, Klok and coworkers used direct surface RAFT polymerization to grow brushes of 3methacrylamidophenylboronic acid on the surfaces of silica QCM devices, which exhibited reproducible changes in the first harmonic shift in th e presence of glucose. In a different study, copolymers of 3 APBA and various cationic monomers were prepared and placed on QCM electrodes. The QCM hydrogel sensors were then used to measure the capture of nucleotides by the gel.91 Figure 116. Functionalization of a silica surface with a RAFT chain transfer agent, followed by graftingfrom brush growth to yield boronic acidmodified polymer brushes bound to the silica surface. Reproduced with permission from ref 90. Copyr ight 2014 Wiley VCH Verlag GmbH & Co. Pressure transducer based sensors have been prepared from cross linked hydrogels containing 3APBA and N ,N dimethylaminopropyl acrylamide (DMAPAA).92 When

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52 incorporated between a pressure transducer and a semi permeable membrane, the resulting hydro gels shrink in response to increasing glucose concentration, while swelling in response to fructose (Figure 117) . This shrinking was believed to be caused by a combination of boronate ester cross links formed with glucose and the reduced thermodynamic favorability of mixing after glucose binds with the boronic acids.93 The sensors showed little interference from physiological fructose concentrations when measuring glucose response, and the resulting pressure changes were reversible over numerous cycles. Figure 117. Pressure transducer derived glucose sensor. A boronic acid modified hydrogel is pressed between a metal pressure transducer and a semi permeable membrane. If the hydrogel is swollen with a fructose solution, the gel swells upon boronate ester formation, while gels contract in the presence of glucose. Adapted with permission from ref 92. Copyright 2010 Elsevier. There has also been interest in employing boronic acids to identify and quantify endogenous glycated proteins.94 Unlike glycosolation, which is enzymatically driven process and serves a vital role in bodily function, glycation occurs through a nonenzymatic pathway and is considered destructive to the protein. Glycation results in the formation of advanced glycation endproducts (AGEs), many of which are associated with diabetes, including fructosamine and glycated hemoglobin (HbA1c). AGEs are also thought to play a role in other chronic diseases

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53 and the aging process. Boronic acidmodified heterogeneous supports have been used to remove fructosamine, along with glycated lipoprotein and glycated albumin.95 Yoon and coworkers have developed a glucose oxidas e enhanced boronic acid based biosensor for HbA1c.96 The electrodes we re prepared via modification of polyamidoamine dendrimers to the surface of the electrode, followed by covalent attachment of glucose oxidase and 4 formylphenylboronic acid to the dendritic amine groups. As glycation is a relatively slow process, determini ng the blood levels of HbA1c gives a much better understanding of long term glucose management in a diabetic patient. 1.4.2 Optical Sensors The use of optical sensors provides a rapid, relatively simple approach to quantify desired substrates. As such, boronic acids based sensors have been prepared, employing a number of sensing mechanisms, including surface plasmon resonance, reflectance spectroscopy, holographic devices, shifts in absorbance, and changes in fluorescence intensity. While optical sensors can be prepared simply by embedding small molecule boronic acids and esters in polymer based sensors,97,98 covalently linking the boronic acid to a sensor may provide better long term response and stability. 1.4.2.1 Surface plasmon r esonance The use of boronic acids in hydrogels is not limited to cross link formation. By creating permanent covalent hydrogels with boronic acid pendent groups, the resulting materials can be used as sensors for various biological species, including saccharides.99,100 Willner and coworkers examined the glucose bindinginduced swelling of a permanently cross linked hydrogel modified with 3 APBA by determining the shifting reflectance minimum in surface plasmon resonance

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54 (SPR) on a gold modified glass slide.74 As SPR is sensitive to changes in film refractive index, the technique provided an effective means to study hydrogel swelling. Upon submersing the 3APBA hydrogel in a glucose solution, the reflection minimum shifted by an angle of 0.6, corresponding to a film thickness increase of 110 nm. Replacing the glucose solution with a buffer solution reversed the swelling. The increase in film thickness, along wi th increased diffusion of a redox label, suggests glucose responsive hydrogel swelling may be a useful strategy to encapsulate and release target compounds. The use of MIP’s has not been limited to electrochemical sensors, and, interestingly, molecular imp rinting of small molecules can lead to recognition of macromolecular species. SPR was used to investigate the binding of ganglioside, a phospholipid with one or more sialic acid groups.101 MIP crosslinked films of VPBA sialic acid esters were prepared on gold surfaces, after which the sialic acid was removed in an acidic buffer. Although the im print was prepared with sialic acid, ganglioside was found to bind efficiently with the sensor. Binding of sialic acid also occurred, but the molecule was too small to elicit a change in SPR. 1.4.2.2 R eflectance s pectroscopy Optical sensors for glucose have also been prepared through surface modification of porous silicon films. Thiol functional homopolymers of VPBA were grafted to the surface of single layer silicon films and silicon based rugate filters.102 The PVPBA was found to collapse or expand in the pores of the silicon in response to changes in pH and glucose concentration. The sensor response was determined via interferometric reflectance spectroscopy, whereby the effective optical thickness was calculated from the changes in film thickness. The rugate filters had limited applicability, potentially due to degradation of the surfaces, but the boronic acid

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55 modified single layer silicon films exhibited a reversible response to glucose at physiological concentration and pH in both aqueous solution and in glucose spiked wound fluid. 1.4.2.3 H olographic d evices Holographic sensors can provide a readily measureable response to a variety of stimuli. Silver modified holographic films were prepared from 3APBA103,104 and 2 acrylamido 5 fluorophenylboronic acid.104 The sensors exhibited a red shift in the diffraction wavelength with increasing gluc ose concentration. The anionic nature of the 2 acrylamido 5fluorophenylboronic acid units, caused by the coordination of the amide carbonyl with the boron center, led to enhanced selectivity for glucose over lactate, while lactate interacted with the sens ors based solely on 3 APBA. Similar holographic sensors were prepared from 2 APBA, showing rapid response to glucose with limited effect of pH on response.105 Holographic colorimetric sensors of glucose have also been achieved through the preparation of hydrogels containing N,N methylenebisacrylamide (MBA) and (3 acrylamidopropyl)trimethylammon ium chloride with VPBA106 or 3 APBA.107 The resulting hydrogels exhibited holographic fringes that shifted in diffraction wavelength with changing glucose concentration. A calibration curve was prepared by measuring the diffraction wavelength as a function of glucose concentration in plasma solutio ns. To test the applicability of the holograms in realtime continuous glucose monitoring, the diffraction wavelength was measured while fluctuating the concentration of glucose, with all measurements accurate to within 20% error. 1.4.2.4 Polymerized cry stalline colloidal a rrays Colorimetric sensors can be prepared from polymerized crystalline colloidal arrays (PCCA), which consist of colloidal particles packed in a “crystalline” order, similar to that seen

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56 on the atomic scale.108 113 Because the colloidal crystal order is on the size scale of the wavelength of light, crystalline colloidal arrays can diffract light in accordance with Bragg’s law. By varying the distance between colloidal particles, either constructive or destructive interference of electromagnetic waves can cause iridescence. The spacing of particles can be dictated by Coulombic interparticle interactions. Thus, an increase in surface charge on the particles cre ates a Coulombic repulsion, repelling the particles from one another. Particle spacing can also be dictated by matrix swelling. In a PCCA, the particles are unable to diffuse through the matrix, and should the surface charge change, the resulting Donnan po tential within the matrix would shift with the matrix swelling or contracting as a result. This change in matrix volume results in a change of particle spacing, shifting the wavelength of iridescent light. Polymerized crystalline colloidal arrays were prep ared by dispersing boronic acidmodified cross linked particles in cross linked hydrogels.108 113 In an initial study, 3APBA modifi ed polystyrene particles were em bedded in a polyacrylamide matrix. The resulting materials exhibited a red shift in the diffraction wavelength with increasing saccharide concentration as a result of electrostatic repulsion between the boronic acid particles upon boronate ester formation.108 It was proposed that the inability of the boronate anions to diffuse in the gel created a gradient in ionic concentration due to the Donnan effect, causing the gel between the colloidal particles to swell. Subsequent studies employed m onomers that would stabilize the boronate ester109,110 or lower the p Ka of the boronic acid,110,111,113,114 facilitating cross linking via diesterification with glucose. Cross linking caused shrinkage of the gel, and thus a blue shift. As t he color shift covers a wide range of the visible spectrum, it was suggested that these crystalline colloidal arrays may have use in color changing glucose responsive contact lenses. 110,114 Braun and coworkers examined a wide range of functionalized aryl boronic acids to

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57 determine the structure property relationship between glucose response and the chemical propert ies of the boronic acids.113 A number of characteristics were examined, including reaction kinetics, hysteresis, and res ponse mechanism. Boronic acids that contained intramolecularly coordinating carbonyls, such as 2APBA, exhibited a slow, linear response to glucose addition, but also exhibited a large hysteresis, while boronic acids that do not contain coordinating specie s exhibited a nonlinear response to glucose addition with fast kinetics and limited hysteresis. 1.4.2.5 A bsorbance sensors Polymers of Land Dlysine were modified with phenylboronic acids. Ester formation between the boronic acids and saccharides caused a cationic cyanine dye to complex with the anionic boronate center (Figure 118) . Complexation shifted the absorption spectrum of the dye to shorter wavelengths, also causing an increase in the helix content of the poly(lysine) secondary structure a s determined through circular dichroism.115 117 Figure 118. Structure of boronic acid modified polylysine and a cyanine dye counter ion. Upon complexation with glucose, the anionic boronate ester forms an electrostatic complex with the dye, s hifting the max of the dye.115

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58 Shifts in absorbance have also been observed in azobenzene modified boronic acid containing polymers. 118 120 In the absence of diol s, intramolecular coordination occurs between the boronic acid and one of the azobenzene nitrogen centers (Figure 119) . This coordination was disrupted upon binding between the boronic acid and a diol, causing a change in shape o f the absorption spectrum. Polyethyleneimine modified with boronic acid substituted azobenzene groups was manufactured into sugar responsive films via layer by layer assembly with various polyanionic polymers.118 Figure 119. Reversible coordination of a boronic acid and a nitrogen atom in an azobenzene based monomer unit. Glucose binding limits coordination and causes a shift in the absorption spectrum.119 1.4.2.6 F luorescence sensors Glucose sensin g can also be achieved by combining boronic acids with fluorescence quenching. An anionic pyranine derivative and quaternary viologen boronic acid derivatives were cross linked with poly(ethylene glycol) dimethacrylate (PEGDMA). 121 124 The electrostatic interaction between the anionic pyranine and cationic viologenbased boronic acid caused quenching of the pyranine fluorescence. In a model consisting of a m ixture of the hydrogels constituent monomers, the addition of glucose disrupted the electrostatic interaction, leading to

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59 an increase in fluorescence intensity (Figure 120) . A similar response was observed in the resulting hydrogels. The measured fluoresc ence intensity increased with increasing glucose concentration, and the response was reproducible for increasing or decreasing glucose concentrations. The utility of the hydrogel systems was expanded by mounting the materials on the tip of a fiber optic pr obe.122,125 The newly manufactured glucose probe exhibited a nearly ident ical response, although faster, as compared to the mounted film device previously discussed. To determine the extended stability and response of the new sensor, the fiber optic probe was submerged in a circulating solution of glucose. The measured fluoresc ence intensity remained constant over the course of 36 h, proving the probe was stable and that the measured intensity was not affected by extended immersion in glucose solutions. Nonfluorescent fiber optic probes have been prepared from 3APBA hydrogels, based on measuring the changing optical length for several different saccharides over a wide concentration range.126 Figure 120. Disruption of electrostatic interaction between an anionic pyranine and cationic viologen based boronic acid monomer unit. Binding with glucose causes separation of the ionic species and an increase in fluorescence intensity of the pyranine unit. Reprinted with perm ission from ref 122. Copyright 2006 American Chemical Society. Glucose responsive nanogels based on fluorescence resonance energy transfer (FRET) were prepared through a precipitation polymerization with 3APBA, MBA, and NIPAM along with rhodamine (FRET a ccepto r) and fluorescein (FRET donor) based monomers.127 In the absence of glucose, the nanogels were less swollen; the rhodamine and fluorescein were in close

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60 proximity, and fluorescence intensity was reduced. However, upon glucose binding, the gels swelled, se parating the dyes, increasing the fluorescence intensity. As expected, the response was greater at higher pH due to increased boronate ester formation. In another precipitation polymerization of 3 APBA and N ,N dimethylaminoethylacrylate, gold nanoparticles were encapsulated in the nanogel.128 By controllin g the ratio of 3 APBA to DMAEA, the swelling or contraction of the nanogels could be tailored, with gel swelling resulting in a decrease in photoluminescence from the gold nanoparticles and gel shrinkage leading to an increase in photoluminescence. Sacchar ide sensing can also be achieved through photoinduced electron transfer (PET) fluorescence quenching.129 131 Mader et al. used atom transfer radical polymerization (ATRP) to prepare polymers with a Wulff type boronic acidmodified anthracene methacrylate that were used to molecularly imprint fructose. Upon binding with fructose, electron transfer between the nitrogen and anthracene was quenched, causing an increase in anthracene fluorescence intensity.132 A similar approach was taken by Takeuchi and coworkers, whereby fluorescent microgels133 and fiber hydrogels134 we re prepared from PEG modified Wulff type boronic acids bearing anthracene groups. The addition of the PEG spacer allowed for greater mobility of the anthracene units, leading to an enhanced response over a more sterically hindered anthracene group. Both the microgels and fibers showed an increase in fluorescence with the addition of glucose due to the decreased electron transfer from the nitrogen center to the anthracene group. Both materials also exhibited long term in vivo response to glucose concentratio ns in mouse models. However, overtime the microgels were dispersed from the mouse’s ear tissue, necessitating the development of the fiber based sensor, which remained responsive for over four months. Appleton and Gibson saw a similar PET fluorescence resp onse when employing a

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61 naphthalene based Wulff type boronic acid monomer.135 A modular approach has also been used to prepare a series of polymer and small molecule PET based Wulff type boronic acid fluorescent sensors.136 The use of Wulff type PET based glucose sensors is very promising, as shown by the recent successful trial of a implantable fiber optic probe for continuous glucose monitoring.125 Similarly, fluorogenic boronic acids have been covalently attached to the core of agarose colloidosomes through copper(I) catalyzed azide alkyne cycloaddition (Figure 121) .137 The resulting triazole ring coordinated with the boronic acid center, resulting in fluorescence emission from the boronic acid.138 The fluorescence intensity increased upon cis diol binding, allowing for optical determination of saccharide concentrations.138 140 Figure 121. Fluorescent boronic acid modified colloidosomes (A) Fluorogenic boronic acids were covalently attached to the core of agarosefilled colloidosomes. (B) Binding with fructose increased the fluorescence intensity within the core of the colloidosomes. Reproduced from ref 137 with permission. Copyright 2013 Royal Society of Chemistry.

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62 The addition of fluorescent monomers into boronic acid copolymers can also yield saccharideresponsive sensors.141 Copolymers of boronic acid and pyrene based monomers showed sugar responsive fluorescence response. In one example, pyrene aggregation was disrupted upo n saccharide binding, due to electrostatic repulsion between boronate anions. However, when the copolymer was prepared with a quaternary amine monomer, the electrostatic attraction between the ammonium and boronate species increased the pyrene aggregation and thus the fluorescence intensity.130 Fluorescence sensing with boronic acids was also achieved simply through the addition of a quaterna ry amine modified pyrene derivative (Figure 122) .142 In a copolymer of acrylamide (Am) and 3 APBA, the anion formed upon saccharide binding complexed with the cationic pyrene derivative, ca using increased pyrene excimer emission, which allowed ratiometric sensing of glucose concentration. Figure 122. Structure of P(Am co 3APBA) and quaternary amine functionalized pyrene. Electrostatic interactions between the pyrene and the boronic acid upon complexation with a diol resulted in pyrene aggregation and increased pyrene excimer emission.142 Fluorescence sensing has also been achieved in boronic acid modified conjugated polyme rs.143 Zwitterionic boronic acid modified polythiophene was prepared from poly(3bromohexylthiophene). The resulting quaternary pyridine boronic acid exhibited decreasing fluorescence intensity with increasing concentration of Dglucose, lactose, dopamine, and

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63 vitamin C.144 A similar material was prepared from polyfluorene, which showed a fluorescence reduction upon addition of Dglucose, Dfructose, vitamin C or LDOPA.145 Alizarin Red S has frequently been used to quantify saccharide boronic acid affinity through competitive binding.59,146,147 In solution, Alizarin Red S exhibits no fluorescenc e until bound with a boronic acid. Upon addition of a secondary diol, some of the alizarin red S is displaced and the fluorescence intensity is reduced. Tuncel and coworkers have used this reduction in fluorescence intensity to prepare diol sensors.148 Copolymers of NIPAM were prepared with VPBA, followed by conjugation with alizarin red S. Since copolymers of NIPAM are thermoresponsive, the copolymers exhibited limited decrease in fluorescence intensity at 40 C, likely due to the collapsed state of the polymers. However, near room temperature, a reduction in fluorescence int ensity occurred upon addition of various diols, including fructose. Similarly, ARS conjugated boronic acidmodified polystyrene nanoparticles have been used to sense fructose and glucose.149 1.5 Cell Capture a nd Culture 1.5.1 Cell Culture Growth Boronic acids have been used to label cells and viral capsids,150 and have also been shown to bind reversibly with the glycoproteins at cell surfaces.151,152 This interaction can be used in a number of interesting applications including cell culture media, cell capturing, and imaging. Boronic acids have been shown to be effective suppo rts for cell cultures,153 159 even potentially acting as mitogens, or species that promote cell growth.157,158 Cell culture often occurs at hard interfaces, as many cell s are unable to grow in suspension. While cell growth is efficient on cell culture media, detaching cells from surfaces often involves proteases, such as trypsin, which digest the cell attachment proteins and can result in cell damage. The reversible

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64 natur e of boronic esters provides a route for cell attachment for cell growth,154 156,158,159 while simultaneously allowing for cell detachment through a trans esterification with saccharides.154 156,159 Ivanov et al. have undertaken a number of investigations with cell growth occurring directly on boronic acid modified cell culture surfaces,155,156,159 showing that the cell proliferation occurs more efficiently at polymeric brush surfaces rather than chemisorbed organosilane layers or cross linked gels.159 Not only can boronic acid functionalized surfaces facilitate cell growth, they can induce the formation of large tissues ( e.g., in the formation of capillary structures during the growth of endothelial cells on 3APBA surfaces).160,161 Cell proliferation can also be facilitated at protein modified boronic acid surfaces. HeLa Fucci cells were grown on fibronectin, which was covalently bound to a surface coated with a copolymer of 2methacryloyloxyethyl phosphorylcholine, nbutyl acrylate, and 4vinylphenylboronic acid. After incubation, the cells were released from the fibr onectin surface through a trans esterification with fructose or sorbitol. Although the controlled culture of cells is o f great importance, regulation of cell growth is also necessary, particularly in the treatment of cancer. One route to limit tumor growth is through the targeted and controlled release of cytotoxic compounds, an approach that potentially allows for selective treatment of cancerous tissues while limiting deleterious effects in non cancerous tissue. This can be achieved through controlled dissociation of materials that encapsulate pharmaceutical compounds, such as in responsive layer by layer assemblies. Choi and coworkers prepared layer by layer assemblies through alternating deposition of PVA and a water soluble phospholipid polymer bearing VPBA units, with paclitaxel being embedded in the phospholipid layers (Figure 123) .162 The addition of glucose caused dissociation of the layers and release of the encapsulated paclitaxel, leading to cell death. With a slow controlled release, it

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65 was possible to regulate the number of viable cells over an extended period of time. This slow release profile suggests that layer by layer assemblies may serve as effectiv e drugeluting coatings, potentially for arterial stents. Figure 123. Layer by layer assembly of alternating boronic acid modified phospholipidbased polymer and PVA. Paclitaxel (PTX) was loaded in the phospholipid layers and was released upon glucose binding and layer disruption. Reprinted with permission from ref 162. Copy right 2012 Elsevier. 1.5.2 Cell Capture and Release The dynamic covalent nature of boronate esters allows for the selective attachment and detachment of cells from heterogeneous surfaces. Liu et al. grafted 3APBA from the surface of flat and nanowire lik e silica surfaces, allowing MCF 7 cancer cells to be reversibly bound via boronate esters with sialic acid units on the cell surface glycoproteins.163 Because sialic acid has a higher binding constant than glucose, the cells were able to form boronate esters at lower pH, while glucose could not, allowing for efficient capture at pH 6.8. Similarly, without glucose at physiological pH, the cells formed esters with the surface. The addition of an excess of glucose triggered cell release (Figure 124) . This capture and release was shown to be effective over several cycles.

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66 Figure 124. Reversible capture of MCF 7 cancer cells through sialic acid containing glycoproteins on the cell surface. At low pH and in the absence of glucose, the cells were bound to the boronic acid surface . Increasing the pH and the addition of glucose displaced the cells through transesterification. Reprinted with permission from ref 163. Copyright 2013 American Chemical Society. Transport of cells can also be facilitated by cell adhesion to surfaces. Pol y(3 aminophenylboronic acid) was coated onto the surface of Ni/Pt nanotubes through an electrochemical polymerization, with the nanotube acting as a self propelled nanomachine.164 When the nanotube encountered a yeast cell, the cell became bound to the nanotube through esterification with cell membrane glycoproteins and was transported along with the tube. Addition of fructose caused the yeast cells to dissociate from the nanotube surface. Videos of the capture, transport, and release of yeast cells are available in the original manuscript’s supporting information. Lymphocyte growth was increased in solution after complexation with poly(Am co 3APBA) . It is thought that the polymers may mimic lectins ( i.e., sugar binding proteins). Binding of lectin with glycoproteins on the lymphocyte cell membrane causes cross linking of the glycoproteins, starting a cascade of physiological changes resulting in RNA and DNA synthesis, thereby proliferating lymphocyte growth.157

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67 1.5.3 Cell Surface Interactions While it has been shown that attachment of cells to boroni c acid surfaces can be used to regulate cell proliferation, the interaction between boronic acids and cell surfaces can also be used to control the adhesion of cells to other biological materials. Poly(Llysine) was modified with PEG and 4formylphenylboro nic acid, generating a polymer that could bind with the surface of cells through the boronic acid, but also pa ssivate the cell by PEGylation, limiting adhesion of proteins and other cells.165,166 When the polymers were bound to the surface of red blood cells, the modified cells showed limited antibody induced agglutination. This is of great importance to individuals who receive frequent blood transfusions, for example during the treatment for sickle cell anemia. The body’s natural immune response to transfused blood can make finding a suitable donor difficult. PEG ylation of red blood cells through boronate ester coupling may prove to be of great benefit in the attempt to limit immunogenic response. The polymers were also attached to the surface of extracellular matrix and were shown to inhibit attachment of unmodified rabbit lens epithelial cells. 1.6 Summary and Future Directions Boronic acid containing macromolecules have found promise in a wide range of important applications, including as optical and electrochemical sensors for a wide range of biologically relevant materials, separation devices for concentrating and quantifying diol functionalized biomaterials, and in situ treatment or prevention of disorders like diabetes and HIV. Nanomaterials and hydrogels have been modified with boronic acid species for glucose responsive insulin release. Finally, b oronic acids are also effective media for cell manipulation, including capture, culture, and protection of cells.

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68 However, many of the materials investigated in this chapter suffer from a similar shortcoming in the lack of specificity towards desired targets. This has been addressed to a degree with the development of molecularly imprinte d materials, which enhance specificity. Many applications will require greater selectivity towards a desired substrate to see wider clinical use. A variety of strategies can be employed to enhance selectivity of polymers containing boronic acids for specif ic diols. For example, the structural and chemical identity of the boronic acid can be tuned to enhance binding with a diol of a given shape/size or which contains a complimentary structural group to facilitate additional secondary interactions. One way to approach this is by directly modifying the immediate environment of the boronic acid ( e.g., by incorporating additional electron withdrawing/donating or Lewis acid/base functionality on the ring of polymer bound phenyl boronic acid groups). Another method of increasing the diol specificity of boronic acidcontaining polymers is by copolymerization with monomers that contain carefully selected functional groups that enhance diol selectivity by favorably interacting with the boronic acid or diol components. This approach of seeking enhanced selectivity via copolymerization is unique to boronic acid containing polymers and can be exploited to explore the effects of chemical diversity to achieve specificity without tedious multistep syntheses. The examples hig hlighted in this chapter focus on the biological applications of boronic acid containing polymers. While many of the discussed materials have shown limited toxicity in simple cytotoxicity assays, there remains a relative lack of true in vivo evaluation. Mo ving forward, current technologies will need to be refined and the efficacy of the materials tested, first with animal models and finally in human clinical trials. Even so, the use of boronic acids has steadily grown, likely a result of the advantageous sy nergy between boronic acid reactivity and biological composition. The binding of diols, many of which are present in natural systems has

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69 allowed boronic acid containing materials to be of use in many intensely researched fields of biotechnology. With further study and application, boronic acids will likely find much wider use in clinical settings.

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70 CHAPTER 2 RESEARCH OBJECTIVE The purpose of this research was to study the stimuliresponsive nature of boronic acids and analogues and to use knowledge of stimuliresponsive materials to prepare new boronic acid functional materials for glucose responsive applications. Boronic acids have received widespread interest in organic, polymer, and materials chemistry , not only as a functional group that allows for efficient cross coupling reactions, but also as a moiety that instills pH and diol responsive character into materials in which they are incorporated. Boronic acids have found use in a number of interesting applications, ranging from self healing or therm oresponsive hydrogels, to glucose responsive nanostructures for drug delivery applications, or as the foundation of photoor electrochemical sensors. Boronic acids have even seen fringe applications in enzymatic inhibition or in site directed cancer treatment through the use of boron neutron capture therapy. However, there is always room to improve these applications through the use of novel boronic acid functional groups or in the design of materials with enhanced response at lower boronic acid loadi ng. The goal of the research in Chapter 3 was to prepare new thermoresponsive polymers with inherent glucose response through the copolymerization of a thermoresponsive monomer and a boronic acid. To accomplish this , block copolymers were synthesized to contain a permanently hydrophilic poly( N,N dimethylacrylamide) block along with a thermo , pH , and sugar responsive block consisting of monomer units of N isopropylacrylamide (NIPAM) and 2acrylamidoethylcarbamoyl phenyl boronic acid (ACPBA). We found that the volume phase transition temperature, or cloud point, of the copolymers could be tuned by controlling the ratio of NIPAM and ACPBA, with increasing ACPBA content resulting in a lower cloud point transition temperature. The cloud point could then be shif ted to higher temperatures, either

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71 through an increase in solution pH or through the addition of glucose. Interestingly, the polymers exhibited a concentration dependent transition temperature, with decreasing polymer concentration resulting in a n increasi ng cloud point temperature , suggesting that dilution under physiological conditions could shift the cloud point even higher to above physiological temperature. The research in Chapter 4 examines the p Ka and association constants of a number of boronic acid families. Having an understanding of how boronic acid structures affect the binding affinity towards various diols is of vital importance to the design of new materials. In this study, we found that the boronic acids that currently serve as the basis for many applications have high pKa’s and relatively low binding with glucose, a very common diol target for ma n y applications. We also found that Wulff type boronic acids, which are boronic acids with adjacent coordinating nitrogen centers, had the lowest dio l affinity at neutral and basic pH, while performing the best of all the boronic acids investigated at acidic pH. Overall, the boronic acids that provided the greatest glucose binding at physiological pH were benzoxaborole and 2formylphenylboronic acid, w ith the latter molecule containing intramolecular coordination and electron donation to the boron center . Chapter 5 investigates the controlled polymerization of novel boronic acids containing intramolecular coordinating oxygen centers in the form of amide carbonyls. The coordination between the boron center and the oxygen was found to be very stable, likely due to tautomerization of the amide to the imidic acid. Interestingly, the removal of the pinacol protecting group from the boronic acid resulted in a dehydration event involving the amide oxygen, resulting in the formation of a new benzoxazaborinine monomer that has no t previously

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72 been described. Ultimately, controlled polymerization of the monomer proved to be challenging and further optimization is ne cessary to prepare polymers of well defined structure.

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73 CHAPTER 3 PREPARATION AND CHARACTERIZATION OF GLUCOSE AND THERMORESPONSIVE BLOCK COPOLYMERS EXHIBITING A WIDE CLOUD POINT TRANSITION WINDOW 3.1 Introduction 3.1.1 Stimuli Responsive Materials A number of systems have been shown to exhibit reversible changes in conformation or topology in response to environmental change, making these polymers attractive for use in areas like drug delivery, sensors, or tissue engineering.1,167 169. These stimuli responsive or “smart” materials can undergo a number of property changes as a result of various external influences, including nonchemical stimuli, such as light170 or temperature,29,171,172 or through chemical stimuli, such as changes in pH,170,172,173 redox reactions,29,171,172,174 or dynamic covalent175 and supramolecular interactions.167,176 Photoresponsive materials generally demonstrate reversible isomerization up on irradiation and commonly include materials containing azobenzene moieties, which undergo cis trans isomerization,177 or spiropyrans which change from a neutral compound to a zwitterion after UV irradiation.178 Polymers with pH responsive characteristics most often contain we a k acid or base functional groups that lead to ionization or neutralization when exposed to a change in pH. Provided the ionization events are accompanied by a change in solubility or chain dimensions, the polymers can exhibit behaviors such as self assembly or controlled release. Polymers that exhibit redox response have frequently relied on disulfide or thiol groups present in the polymer that reversibly cleave or couple, resulting in gelation, assembly/disassembly, or other morphological changes .179 181 Redoxresponsive ferrocenemodified polymers, coupled with glucose oxidase, have been employed as glucose responsive sensors.182 Dynamic covalent chemistry has also been used to prepare various polymer architectures and to control soluble insoluble transitions in polymeric nanostructures.15,52

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74 Thermal response has been primarily associated with materials that undergo volumephase transitions in solution. Materials exhibi ting a lower critical solution temperature (LCST, maximum temperature for complete miscibility) can undergo a soluble insoluble transition with increasing temperature and materials exhibiting an upper critical solution temperature (UCST, minimum temperatur e for complete miscibility) undergo a soluble insoluble transition with decreasing temperature.29 Because the transition temperatures of thermoresponsive polymers in solution are often concentration dependent, thermal transitions are o ften described as a cloud point (temperature at which a soluble to insoluble phase transition occurs for a given system). In the system described in this report, in the absence of glucose and at physiological pH , the polymers have a n LCST type cloud point, w ith the cloud point increasing with the addition of glucose or increasing pH. This approach allows for the formation of selfassembled nanostructures at room temperature that can dissociate near body temperature in the presence of glucose. Boronic acid d erivatives are of particular interest, as they have been shown to exhibit a number of interesting stimuli responsive behaviors, including the ability to form dynamic covalent boronic (or boronate) esters with 1,2and 1,3diols15 and pH induced solution phase transitions, due to the Lewis acidic nature of the boron center. Many of these applications have focused on the ability of boronic acids to bind various saccharides, primaril y glucose, in aqueous solution. The equilibrium between bound and free glucose is driven by a number of factors, including the p Ka of the boronic acid , the p Ka of the diol, and the diol dihedral angle , with the ratio of bound to free glucose generally incr easing with decreasing pKa of the boronic acid . While phenyl boronic acid has a p Ka value near 9.0, the specific acidity of other phenylboronic acid derivatives is a function of the substituents on the aromatic ring, with electronwithdrawing

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75 groups on the aromatic ring lowering the pKa.2,51,110 The transition temperature of a thermoresponsive polymer is determined by a number of factors, including the m onomer structure and the composition of the block into which the thermoresponsive monomer is incorporated.29 Thus, by incorporating boronic acids within a thermoresponsive polymer, the cloud point of the polymer relies on a synergistic combination of both pH and diol concentration (Figure 3 1) . Figure 31. R epresentation of the various morphologies possible with the amphiphilic block copolymers described herein. The polymers exist as unimers in aqueous solution when the solution tem perature is below the cloud point (CP1). Heating the solution to a temperature greater than CP1 results in self assembly. Increase of solution pH or the addition of glucose shifts the cloud point higher (CP2), causing nanoparticle dissociation. Boronic ac ids and esters have been incorporated into a number of different macromolecular architectures, but controlled release applications have relied on the synthesis of boronic acid containing block copolymers.15,43,46 48,50,51,54,57,183,184 The solution self assembly of such block copolymers allows for the sequestration of materials to the interior of the nanostructures, potentially shielding these materials from degradation or preserving their biological activity while simultaneously increasing circulation lifetime in the blood. By

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76 combining thermoresponsive self assembly and the glucose responsive nature of boronic acids, block copolymers can provide a potential route for the controlled release of bioactive materials , such as insulin under physiological conditions. Multiple synthetic steps must often be taken in the preparation of boronic acid containing monomers and polymers, making these materials expensive and difficult to prepare. However, the incorporation of a secondary stimulus response mechanism to facilitate nanostructure formation can allow for glucoseresponsive nanomaterials with reduced boronic acid content.185 Thus, by incorporating functional groups that allow an orthogonal ( i.e., additional, chemical ly unrelated) stimulus response mechanism, a boronic acidcontaining polymer can be induced to self assemble into a nanostructure when the orth ogonal stimulus is applied. The second response ( i.e., diol binding) can then result in nanostructure dissociation when induced. Through this route, nanoassemblies can be prepared with very broad cloud point windows dependent on the solution pH and glucose concentration. Poly( N isopropylacrylamide), PNIPAM, is one of the most commonly employed thermoresponsive polymers, exhibiting a lower critical solution temperature of approximately 32 C. 28,186 At room temperature, PNIPAM exists as unimers in aqueous solution. Upon heating above the cloud point, dissolution of PNIPAM becomes unfavorable, with the formation of a polymer rich phase, indicated by precipitation for homopolymers or complex nanoassemblies for amphiphilic block copolymers. The copolymers detailed herein were prepared through copolymerization of N isopropylacrylamide (NIPAM ) and 4((2 acrylamidoethyl)carbamoyl)phenyl boronic acid pinacol ester (ACPBAE) to prepare pH , thermo , and glucose responsive block copolymers. The copolymers were then employed to investigate the effect of various pH and glucose concentrations on the t hermoresponsive nature

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77 of the block copolymers, with one copolymer exhibiting a cloud point transition window of nearly 30 C. 3.1.2 Reversible Addition Fragmentation Chain Transfer (RAFT) polymerization Th e copolymers were prepared via reversible deacti vation radical polymerization ( RDRP ), which has been used extensively to prepare many of the architectures described above. Of the various RDRP techniques available, including atom transfer radical polymerization (ATRP)187 and nitroxide mediated radical polymerization (NMP)188, reversible addition fragmentation chain transfer (RAFT) polymerization is particularly su ited to synthesizing well defined block copolymers due to functional group tolerance and high chain end retention. 189,190 The proposed mechanism of a RAFT polymerization is shown in Scheme 31. As with conventional radical polymerization, a RAFT polymerization begins with a traditional initiation step. Following initiation, the monomer derived radical fragment can either continue propagation or add to the c arbon sulfur double bond of a thiocarbonylthio chain transfer agent , producing an intermediate carbon centered radical. This radical then undergoes a degenerative chain transfer process to either reform the original monomer derived radical fragment or to produce a new radical derived from the chain transfer agent R group. This equilibrium is referred to as the pre equilibrium or initialization period. As the concentration of the RAFT agent is much greater than that of the initiator, the majority of polymer chains are in itiated by the R group radical.

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78 Scheme 31. Proposed RAFT mechanism The partition of the intermediate radical is dictated by the relative radical stability and steric factor s of the monomer derived radical and R group radical. To produce polymers of well defined molecular weight, the fragmentation of the intermediate radical should favor fragmentation towards the R group, with the R group undergoing rapid initiation. If the R group is too stable, fragmentation with favor the R group, but reinitiation will be slow, resulting in inhibition of the polymerization. If fragmentation favors regeneration of the monomer derived radical, the polymerization will proceed through a conventional radical polymerization mechanism. Following rei nitiation of the R group radical, the newly formed radical can either continue with propagation or add to a chain transfer agent, entering the main equilibrium. To produce polymers with narrow molecular weight distribution, the main equilibrium should

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79 unde rgo rapid degenerative chain transfer, with each radical produced adding a minimal number of monomer units before adding to another chain transfer agent. It is important to note that all radical polymerizations, including conventional radical polymerizations and RDRP , undergo termination events. The rate of termination is determined by the radical concentration, which is in turn dependent , in part, on the i nitiator concentration at the beginning of the polymerization. However, as the majority of chains are initiated by the CTA derived R group in the RAFT process , there are a limited number of dead polymer chains at the conclusion of the polymerization relative to the total number of polymer chains, with the majority of chains retaining a thiocarbonylthio chain end, allowing for chain extension with a second monomer to produce block copolymers. 3.2 Results and Discussion 3.2.1 Synthesis of poly( N,Ndimethylac rylamide) blockpoly( N isopropylacrylamide co 4 ((2 acrylamidoethyl)carbamoyl)phenyl boronic acid ) [PDMA bP(NIPAM co ACPBA)] The monomer conversion, boronic acid content, Mn, dispersity, and cloud point data for PDMA bP(NIPAM co ACPBA) block copolymers P 1P4 are presented in Table 31. The polymers in this study were prepared by first synthesizing a poly( N,N dimethylacrylamide ) [PDMA] macro chain transfer agent (macroCTA), which was successfully chain extended through a copolymerization of NIPAM and ACPBA E (Scheme 3 2) . The chain extension was evident by the shift in elution volume ( Figure A 1 2) and an increase in molecular weight , as determined by size exclusion chromatography (SEC) . The block copolymer composition was determined via 1H NMR by comparing the methine proton of the NIPAM units to the aromatic protons of the boronic acid ( Figure 32 to Figure 34).

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80 Scheme 3 2. Synthesis of PDMA macroCTA and chain extension to prepare PDMA bP(NIPAM co ACPBA) block copolymers Figure 32. 1H NMR spectr um of poly( N,N dimethylacrylamide) bpoly( N isopropylacrylamide co (4 ((2 acrylamidoethyl)carbamoyl)phenyl)boronic acid) [PDMA bP(NIPAM co ACPBA] (P2) in CD3OD

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81 Figure 33. 1H NMR spectrum of poly( N,N dimethylacrylamide) bpoly( N isopropylacrylamide co (4 ((2 acrylamidoethyl)carbamoyl)phenyl)boronic acid) [PDMA bP(NIPAM co ACPBA] (P3) in CD3OD Figure 34. 1H NMR spectrum of poly( N,N dimethylacrylamide) bpoly( N isopropylacrylamide co (4 ((2 acrylamidoethyl)carbamoyl)phenyl)boronic acid) [PDMA bP(NIPAM co ACPBA] (P4) in CD3OD

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82 Table 31. Percent monomer c onversion, mol% boronic acid content in copolymer, Mn, Dispersity, and c loud point data for PDMA bP(NIPAM co ACPBA) block copolymers PDMA ( equiv.) NIPAM ( equiv.) ACPBAE ( equiv.) AIBN ( equi v .) Conv.a Mn, MALSb b mol% ACPBAc Cloud Point (C) d P1 1 124 0 0.1 >95% 16,100 1.14 0 32 P2 1 110 6 0.1 >95% 19,600 1.27 6 26 P3 1 98 11 0.1 >95% 23,700 1.34 11 19 P4 1 78 20 0.1 >95% 19,700 1.38 21 (7) a ) Conversion determined by 1H NMR spectroscopy by comparing integration of vinyl proton to trioxane internal standard. b ) Absolute molecular weights and dispersity determined via SEC MALS . c ) Boronic acid content determined by comparing integral of NIPAM u nit methi ne proton to aromatic protons. d) Cloud point determined by dynamic light scattering in 0.1 M pH 7.4 PB at 5 mg/mL. The cloud point for P4 was estimated from the linear relationship between cloud point and boronic acid content . 3.2.2 Determination of Cloud Point Transition Temperatures for Copolymers in pH 7.4 Phosphate Buffer Solution As the cloud point of a thermoresponsive polymer can be adjusted by copolymerization with hydrophilic or hydrophobic monomers, the random incorporation of the hydro phobic boronic acid units within the NIPAM block should result in a decrease d cloud point. This was found to be the case in the prepared copolymers, as increasing boronic acid content resulted in a lower cloud point s . The transition temperatures for the co polymers were determined via dynamic light scattering (DLS) . After purification, samples of each polymer were diluted to 5 mg/mL in pH 7.4 sodium phosphate buffer (PB) , cooling if necessary to dissolve the polymer. Each sample was place d in the cavity of t he DLS , and the derived count rate, a numerical representation of solution turbidity, was measured over a wide temperature range. As shown in Figure 35, below the cloud point, the count rate increased slowly with heati ng. However, at the cloud point and upon nanostructure formation, the solution became more turbid due to the higher scattering cross section of the nanoparticles, causing the count rate to increase dramatically with further heating. Therefore, the increase in scattering intensity with tempera ture was used to observe the onset of

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83 self assembly. For the purpose of these measurements, the cloud point was defined as the temperature at which the plot of derived count rate vs. temperature showed an abrupt slope change. Specifically, linear fits of the count rate data were obtained before and after the sharp slope chang e, and the temperature at which these lines intersected was taken as the cloud point. An example is shown in the Figure 36. Figure 35. Plots for cloud point determination of P1 P4 A) Derived count rate plots for P1 P4 in pH 7.4 PBS at 5 mg/mL B) Relationship between cloud point and boronic acid content for PDMA bP(NIPAM co ACPBA) block copolymers . The transition temperature for P4 was below the temperature range of the instrument.

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84 Figure 36. Derived count rate measured as a function of solution temperature for sample P3. The cloud point was taken as the intersection of the linear fits of the blue and red segments before and after the inflection point. Using the linear fit method, the cloud point of P1 was determined to be 32 C, which agrees with literature determination of the cloud point for PNIPAM block copolymers.28,49 As expected, the hydrophobic character of the boronic acid units at neutral pH led to a decrease in the cloud point transition temperature of the NIPAM copolymers. While the cloud point for P4 was below the operation limit of our instrument due to condensation formation on the surface of the cuvette, a linear relationship was found between boronic acid content in the copolymers containing 0, 6, and 11 mol % boronic acid ( Figure 36B) , a characteristic commonly seen in thermoresponsive copolymers .29,191 193 From this linear relationship, the cloud point for P4 can be estimate d to be approximately 7 C, well below room temperature. The self assembled nanostructures exhibited a number average size from 23 26 nm, while the dissociated polymers in glucose solutions had siz es ranging from 7 9 nm (Figure 37) .

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85 Figure 37. DLS trac es for P 1P4 . Each polymer was dissolved in pH 7.4 PBS, forming nanostructures at temperatures greater than the cloud point . For P1, which only contains NIPAM , t he addition of 100 mM glucose disrupted the nanostructures containing boronic acid units, with the size of the assemblies decreas ing from approximately 2326 nm to a size of 7 9 nm. 3.2.3 Examining the Effect of pH on Cloud Point Transition Temperature In solution, boronic acids exist in equilibrium between the neutral trigonal planar geometry and the anionic tetrahedral geometry, with the position of the equilibrium being dictated by the solution pH and diol concentration ( Figure 33) . Anionic boronate esters are much more hydrolytically stable than neutral boro nic esters . Further more , the pKa of a neutral boronic ester is usually lower than the free boronic acid, further facilitating anion formation.

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86 Scheme 3 3. Dynamic equilibrium between neutral boronic acid, boronate anion, and boronate ester. Neutral bor onic ester formation is less favored than boronate ester formation. Because t he hydrophobicity of the boronic acid units is a function of the ioniziation of the boronic acid, the cloud point was measured as a function of solution pH (Figure 38A) . A solution of P4 was prepared in PB at a polymer concentration of 5 mg/mL , after which the pH was adjusted to 7.4 using dilute HCl and NaOH solutions. No cloud point transition above 15 C was observed until pH 8.55, with the cloud point reaching approxim ately 40 C at pH 9.0. As seen in Figure 38B, i ncreasing pH led to a nonlinear increase in cloud point due to the formation of the tetrahedral boronate anion near and above the p Ka of the boronic acid. Since pH is a logarithmic scale, the non linear incr ease in cloud point with increasing solution pH is to be expected. A slight increase in pH can lead to a large shift in boronate anion concentration. This data indicates that the formation of the boronate anion can drastically increase the cloud point tran sition temperature, suggesting increased anionic boronate ester concentration should also lead to an increase in cloud point transition temperature .

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87 Figure 38. pH dependent cloud point transition temperature for P4 A) Count rate as a function of solution temperature and pH for P4 at 5 mg/mL polymer concentration in PBS. B) Non linear relationship between solution pH and cloud point for P4 at 5 mg/mL in PBS . 3.2.4 Examining the Effect of Glucose Concentration on Cloud Point Transition Temperature The effect of glucose concentration on cloud point was also investigated. As the formation of boronic esters with glucose causes a shift in the boronic acid equilibrium towards the anionic boronate ester, the addition of glucose was expected to result in a n increase in anionic charge along the polymer , which in turn should increase the cloud point. Solutions of P2 P4 w ere prepared in pH 7.4 PB at a polymer concentration of 10 mg/mL. An aliquot from each solution was then mixed with an aliquot of 1 M glucose in pH 7.4 PB , and the solutions were diluted with buffer to obtain final polymer concentrations of 5 mg/mL with the various glucose concentration s . After mixing, the cloud points were determined by DLS (Figure 39) .

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88 Figure 39. Cloud point determination data A) Count rate as a function of temperature and glucose concentration for P2 at 5 mg/mL polymer concentration. B) Count rate as a function of temperature and glucose concentration for P3 at 5 mg/mL polymer concentration C) Count rate as a function of temperature and glucose concentration for P4 at 5 mg/mL polymer concentration D) Relationship between cloud point and glucose concentration for P2 P4 in PBS at 5 mg/mL polymer concentration As shown in Figure 39D, increasing glucose concentration led to an increase in the measured cloud points , which is consistent with anionic boronate ester formation. However, for P2 , which had only 6 mol% boronic acid in the thermoresponsive block, the shift in cloud point was minimal, increasing by only approximatel y 4 C over the glucose concentration investigated. With increasing boronic acid content, the temperature range at which the polymers exhibited a cloud point increased. This increased cloud point transition window is due to the higher concentration of boronate ester anion, which increases the hydrophilicity of the NIPAM block. For P4 , which had the highest boronic acid content, the cloud point range was from

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89 approximately 13 to 29 C for the glucose concentrations investigated. All three polymers exhibit a non linear relationship between cloud point and glucose concentration, likely due to near saturation of the boronic acid units at higher glucose concentrations . As the boronic acid units bind with glucose, fewer units are available for further binding, nec essitating a large increase in glucose concentration to measure a small incremental increase in cloud point. 3.2.5 Examining the Effect of Polymer Concentration on Cloud Point Transition Temperature Over Broad Glucose Concentrations M any thermoresponsive polymers exhibit a c loud point transition temperature that varies with polymer concentration. As such, it was necessary to determine if there was a concentration dependence with the system being investigated. Given that P4 exhibited the widest cloud point range over the glucose concentration range investigated , this polymer was chosen to investigate the cloud point transition temperature at various glucose concentrations as a function of polymer concentration ( Figure 310 ) .

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90 Figure 310. Derived count rate measurements for solutions of P4 over wide glucose concentrations. The polymer concentration were A) 10 mg/mL, B) 5.0 mg/mL, and C) 2.5 mg/mL.

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91 As can be seen in Figure 310, the cloud point of P4 exhibits a dist inct concentration depe ndence. Higher polymer concentrations resulted in a more narrow cloud point transition window, with the cloud point ranging from approximately 11 to 20 C at a polymer concentration of 10 mg/mL. Decreasing the polymer concentration led to widening of the c loud point transition window. At a polymer concentration of 2.5 mg/mL , P4 has widest effective cloud point transition windows , with the cloud point ranging from approximately 13 C for the copolymer at 15 mM glucose to 34 C for the copolymer at higher glucose concentration (Figure 311) . For use in controlledrelease applications, a shift in cloud point from below room temperature to above body temperature would provide a facile approach for triggered drug release. These results suggest that under physiolo gical conditions, at which point the polymer would be significantly more dilute than 2.5 mg/mL, P4 may have an even higher cloud point after glucose saturation. Although the largest difference in cloud point was seen at very high glucose concentrations, di lution of the copolymers in the body may result in glucose saturation at lower glucose concentrations. With the cloud point of P4 approaching body temperature, the cloud point range for P4 c ould span from just above the freezing point of water to above bod y temperature, allowing for controlled drug delivery within the body while maintaining nanoparticle integrity at room temperature and below.

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92 Figure 311. Glucose dependent cloud point transition temperature for P4 A) Cloud point measurements for P4 as a function of glucose concentration. The polymer concentration ranged from 2.5 to 10 mg/mL. B) Concentration dependent cloud points for P4 at glucose concentrations ranging from 15 to 500 mM 3.3 Conclusions In this study, thermo , pH , and glucose responsive block copolymers were prepared through copolymerization of NIPAM with ACPBA E with a hydrophilic PDMA macroCTA , followed by deprotection of the boronic acids . The cloud point of the resulting block copolymers can be tuned by controlling t he incorporation of ACPBA within the NIPAM block, with

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93 increasing boronic acid content leading to a lower cloud point at physiological pH . In this system, increasing glucose concentration led to an increase in the solution cloud point due to boronate anion formation upon glucose binding. Polymers with increasing boronic acid content exhibited a wider thermoresponsive temperature window, spanning a cloud point transition temperature window from approximately 7 to 35 C under the conditions examined. The dilution of the boronic acid copolymers led to an increase in the cloud point after glucose binding at higher glucose concentrations , suggesting a concentrationdependent cloud point for the copolymer. Dilution in the body during potential drug delivery applic ations would likely result in higher cloud points after glucose saturation . With further optimization, boronic acid containing thermoresponsive block copolymer systems may provide a route for delivery of biologically important materials, such as insulin, w ith greatly reduced boronic acid content as compared to previously studied systems. 3.4 Experimental 3.4.1 Materials 2(((Dodecylthio)carbonothioyl)thio)2methylpropanoic acid (DMP) was prepared as previously reported.194 N,N dimethylacrylamide ( DMA, Fluka, 98%) was passed through a short column of basic alumina before use to remove inhibitors. N I sopropylacrylamide (NIPAM, TCI, >98%) was recrystallized three times from hexane before use to re move inhibitor. Azobisisobutyronitrile (AIBN, Sigma, 98%) was recrystallized three times from ethanol. N,N D imethylacetamide ( DMAc , Fisher), N,N D imethylformamide (DMF, BDH, 99.8%), 1,3,5trioxane (Alfa Aesar, 98%), acryloyl chloride (Aldrich, >97%), triet hylamine (TEA, Alfa Aesar, 99%), tetrahydrofuran (THF, EMD, 99.5%), toluene (Sigma, 99.5%), sodium hydroxide (Macron), hydrochloric acid (Fisher, ACS grade), anhydrous sodium sulfate (Fisher), basic

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94 alumina (Fisher), ethyl acetate (Fisher, ACS grade), dich loromethane (DCM, Macron), molecular sieves (4, 4 to 8 mesh, Acros Organics), oxalyl chloride (Alfa Aesar, 98%), ethylene diamine (Sigma), di tertbutyl dicarbonate (Fluka, >98%), 4carboxyphenylboronic acid (Combi Blocks, 98%), pinacol (Alfa Aesar, 99%), dialysis membrane (Spectra/Por, 3.5kDa MWCO), deuterated methanol (CD3OD, Cambridge Isotopes), deuterated chloroform (CDCl3, Cambridge Isotopes) and any other chemicals were used as received unless otherwise noted. 3.4.2 Characterization Proton NMR spect roscopy was performed using a Varian Inova 500 spectrometer with either deuterated methanol (CD3OD) or deuterated chloroform (CDCl3) as the solvent. Molecular weights and molecular weight distributions were determined via multi angle laser light scattering size exclusion chromatography (MALS SEC) in N,N dimethylacetamide ( DMAc ) with 50 mM LiCl at 50 C and a flow rate of 1.0 mL/min (Agilent isocratic pump, degasser, and autosampler; series G3078 mixed bed columns, with molecular 3 4 g/mol, respectively). Detection consisted of a Wyatt Optilab T rEX refractive index detector operating at 658 nm and a Wyatt miniDAWN Treos light scattering detector operating at 659 nm. Absolute molecula r weights and polydispersities were calculated using Wyatt ASTRA. Dynamic light scattering (DLS) analysis was conducted with a Zetasizer Nano ZS (Malvern) operating at a wavelength of 633 nm. 3.4.3 Synthesis of tert butyl (2 aminoethyl)carbamate Ethylened iamine (16.0 g, 0.266 mol) was dissolved in THF (250 mL), and a solution of di tert butyldicarbonate (14.3 g, 0.0655g) in THF (50.0 mL) was added dropwise while stirring at room temperature. The reaction was allowed to stir overnight. A white precipitate was

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95 removed via filtration, and the solvent was removed under reduced pressure. The remaining oily solution was dissolved in DI water and the difunctionalized ethylenediamine was removed by filtration. The aqueous solution was saturated with sodium chloride and repeatedly extracted with DCM. The organic layers were collected, dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure to yield the crude product, which was used without further purification. 9.40 g, 90% yield. 3.4.4 Synthesis of tert butyl (2 acrylamidoethyl)carbamate t ert B utyl(2 aminoethyl)carbamate (9.40 g, 0.0587 mol) was dissolved in THF (200 mL), along with triethylamine (5.90 g, 0.0587 mol). The solution was cooled in an ice bath, and acryloyl chloride (5.30 g, 0.0587 g) in THF (50 mL) was added dropwise while maintaining the ice bath. The reaction was allowed to come to room temperature and stir overnight. The resulting TEA HCl salt was removed via filtration , and the reaction solutio n was concentrated under reduced pressure to yield a white powder. The product was used without further purification. 10.0 g, 80% yield. 3.4.5 Synthesis of 2 acrylamidoethylammonium trifluoroacetate Tert butyl (2acrylamidoethyl)carbamate (6.04 g, 0.0282 mol) was dissolved in DCM (30 mL) and trifluoroacetic acid (TFA) (60.0 mL). The reaction was stirred for 3 h until gas evolution had ceased. The excess DCM and TFA were carefully removed via rotary evaporation. Further removal of solvent under high vacuum or excess nitrogen purging was avoided due to the tendency of the material to polymerize. The resulting oil was used without further purification.

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96 3.4.6 Synthesis of 4 carboxyphenylboronic acid pinacol ester Pinacol (10.2 g, 0.0863 mol) was dissolved in dry DCM (250 mL), to which activated molecular sieves (10 g) had also been added. 4C arboxyphenylboronic acid (15.0 g, 0.0904 mol) was added to this solution, solubilizing upon reaction. After stirring at room temperature for 12 hours, the solution was filt ered to remove the molecular sieves and unreacted 4 carboxyphenyl boronic acid. The DCM was removed under reduced pressure, and the resulting white solid was recrystallized from ethyl acetate. 14.6 g, 65% yield. 3.4.7 Synthesis of (4 (chlorocarbonyl)pheny l)boronic acid pinacol ester 4C arboxyphenylboronic acid pinacol ester (6.87 g, 0.0277 mol) was dissolved in DCM (125 mL), to which oxalyl chloride (12.0 mL, 0.140 mol) was added, along with 12 drops of DMF. The reaction was allowed to proceed for several hours, until gas evolution had ceased. Excess DCM and oxalyl chloride were removed under reduced pressure to yield a slightly yellow powder, which was used without further purification. 3.4.8 Synthesis of (4 ((2 acrylamidoethyl)carbamoyl)phenyl)boronic acid pinacol ester (ACPBAE) The 2 a crylamidoethylammonium trifluoroacetate prepared above was dissolved in THF (45.0 mL) and cooled in an ice bath. Triethylamine (45.0 mL) was cooled in an ice bath and then slowly added to the salt while maintaining the ic e bath. (4(C hlorocarbonyl)phenyl)boronic acid pinacol ester, as prepared above, was dissolved in THF (50.0 mL) and added drop wise to the previous solution. The reaction was allowed to come to room temperature and stir overnight. The resulting TEA HCl sal t was removed via filtration, and the reaction solution was concentrated under reduced pressure to yield an off white powder, which was suspended in DI water (200

PAGE 97

97 mL). The pH of the solution was adjusted to 4.0, and the product was recovered by extraction with ethyl acetate (3 x 100 mL). The organic layers were dried over anhydrous sodium sulfate before being filtered and concentrated under vacuum to yield an off white powder. The product was recrystallized from ethyl acetate to yield a white powder. 5.43 g , 57% yield. 3.4.9 Synthesis of poly( N,Ndimethylacrylamide) [PDMA] DMA (10.0 g, 0.101 mol), DMP (0.566g, 1.55 mmol), AIBN (0.0127 g, 0.0773 mmol), and trioxane (0.454 g, 5.04 mmol) were dissolved in toluene (30.0 mL) in a 40 mL septum capped vial. The solution was purged with nitrogen for 30 min before the vial was placed in a preheated block at 70 C. The reaction was quenched after 150 min by exposing to air and chilling in a water bath. The polymerization had reached 76% conversion, as determined by 1H NMR spectroscopy . The product was purified by precipitation into hex ane, first from toluene, before being redissolved in THF and precipitated twice more ( Mn ,MALS . 3.4.10 Synthesis of poly( N,Ndimethylacrylamide) bpoly( N isopropylacrylamide co (4 ((2 acrylamidoethyl)carbamoyl)phenyl)boronic acid) [PDMA b P(NIPAM co ACPBA] (P1 P4) NIPAM (0.264 g, 2.33 mmol) and ACPBAE (0.206 g, 0.598 mmol) were dissolved in DMF (1.00 mL). A stock solution of PDMA (1.43 g, 0.242 mmol), AIBN (0.0090 g, 0.0548 mmol), and 1,3,5trioxane (0.150 g, 1.67 mmol) in DMF (16.4 mL) was prepared, and an aliquot of the stock (2.00 mL) was added the above solution. The mixed solution was purged with nitrogen for 30 min before the vial was placed in a preheated block at 70 C. The reaction was quenched after 24 h by exposing to air and chilling in a water bath. The polymerization had reached greater than 95% conversion. The product was purified to remove residual monomer, solvent, and the pinacol protecting group by dialyzing against pH 10 water for 3 days, followed

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98 by dialysis again st deionized water for 3 days, changing the water twice a day. The product was recovered as a white solid after lyophilization.

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99 CHAPTER 4 EVALUATION OF DIOL BINDING CONSTANTS WITH VARIOUS BORONIC ACID CLASSES 4.1 Introduction B oronic acids and boronic aci d analogues have been employed for a number of biomedical applications , for example, in sensors and drug delivery .195 Sensors provide rapid, quantitative re solution of analytes and are important in the field of diagnostic medicine.5,125 Boronic acid s have been shown to be effective sensors for various saccharides, polysaccharides, glycoproteins, glycated proteins, and dopamine. S ensing occurs primarily through the formation of boronate esters with 1,2or 1,3diols present on the substrate. Careful d esign of macromolecules containing boronic acids can also allow for the controlled release of encapsulated pharmaceuticals.55,196 R elease profiles are generally dictated by formation of boronate esters with the macromol ecules, resulting in a concomitant adjustment in material properties or morphology . For instance, in boronic acidcontaining thermoresponsive hydrogels, the binding of glucose or fructose with boronic acid units along the hydrogel backbone causes a n increa se in the hydrophilicity of the hydrogel , resulting in gel swelling ,32,52 increased pore size, and more rapid diffusion of encapsulated therapeutics through the gel. In boronic acid modified nanoassemblies or in boronate ester stabilized nanoparticles, the binding of saccharides often results in an increase of mater ial solubility, causing a disruption in nanoassemblies and a release of encapsulated therapeutics. Understanding the interactions of boronic acids with various substrates is important for the design of sensors and release devices. D iol binding constants c an easily be determined for boronic acids having absorbance or fluorescence characteristics that depend strongly on ester formation. However, for boronic acids that do not exhibit shifts in spectroscopic properties upon ester formation, determination of binding constants can be less straightforward , often relying on

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100 pH depression methods which require high boronic acid concentrations and assumes that esterificiation always results in anionic boronate ester formation with a negligible concentration of neutra l boronic ester .197,198 The pH depression method also assumes that the boronic esters have a lower p Ka than the free boronic acid, which may not always be the case.199 Wang and Springsteen examined the nature of boronate ester formation and diol binding through the use of Alizarin Red S (ARS), a hydrophilic dye. In solution, ARS exhibits a blue shift in absorbance upon binding with a boronic acid.59,147 Displacement of ARS by a diol results in a red shift back to the characteristic absorbance of unmodified ARS. The binding of ARS with boronic acid s also results in an increase in fluorescence intensity. In solution, excited state proton transfer between the catechol hydroxyl groups and the adjacent ke tone results in quenching of the ARS fluorescence.200 Upon ester formation, this proton transfer is no longer possible , and fluorescence quenching is not observed . To examine the binding of diol substrates wi th various boronic acid s , the transesterification of the ARS boronate ester with the substrate disrupts the ARS ester equilibrium, resulting in reduced fluorescence intensity (Figure 41) . This three component system allows for determination of diol associ ation constants.

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101 Figure 41. ARS binding and trans esterification scheme . A) Upon ester formation with the boronic acid, the ARS adduct becomes fluorescent. B) The fluorescence intensity decreases upon trans esterification with a competing diol. ARS boronate esters have been employed for both the examination of diol association constants61,201 203 and the development of diol sensors.148,149,204 Previous studies have invest igat ed the effects of substituents on boronic acid diol binding, but have not broadly compared various boronic acid classes. Furthermore, apparent diol binding constants as determined by the ARS competitive binding assay have been shown to be dependent on a number of factors , including solution pH, boronic acid structure, and most importantly, buffer composition. As such, it is important to ensure that apparent association constants are determined under identical conditions to allow comparative evaluations of boronic acid spe cies. This study evaluates six boronic acids and boronic acid analogues across a wide pH range and under

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102 identical solution conditions so as to provide a true comparison between boronic acid classes in terms of diol binding. 4.2 Results and Discussion S ix boronic acids and analogues were chosen. (Figure 42) Both 3acetamidophenylboronic acid (3 AcPBA) and 4methylcarbamoylphenylboronic acid (MCPBA) were chosen as analogues of boronic acids used previously in our research48,49,51 and by other groups. 26,27,52,205 Benzoxaboroles (benzoboroxoles) have also seen limited use in self assembled polymeric nanoassemblies.42,53,54,185 Wulff type boronic acids, similar in structure to 2 dimethylaminomethylphenylboronic acid (DAPBA), are of great interest in biomedical applications.21,50,135,206,207 However, there has been very limited investigation of glucose binding with Wulff type boronic acids, which is vital in de velopment of glucose responsive materials.202 Figure 42. Structure s of various boronic acids and boronic acid analogues i nvestigated The determination of boronic acid diol binding constants involves a three component competitive binding assay.59,147,208 The association constant between each boronic acid and ARS ( KARS) must first be determined. To do this, each boronic acid is titrated into a constant

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103 concentration of ARS. Formation of the boron ic acid ARS adduct results in an increase in solution fluorescence intensity. To derive the relationship between the association constant ( KARS) and fluorescence intensity, we first consider the relationship between the association constant and the concent ration of the solution components, given by E quation 41, where [BA ARS] is the concentration of the boronic acid ARS conjugate, [BA] is the free boronic acid concentration, and [ARS] is the free ARS concentration. As the boronic acid is in large excess re lative to ARS, the concentration of the boronic acid is considered constant. = [ BA ARS ] [ BA ] [ ARS ] ( 41) Equation 41 can be rewritten as Equation 42. = [ BA ARS ] [ BA ] ( [ ARS ] [ BA ARS ] ) = [ BA ARS ] [ BA ] rl [ ARS ] m [ BA ARS ] rF 1 rp ( 42) Equation 4.2 can be further simplified to give Equation 4.3 [ ARS ] [ BA ARS ] = [ BA ARS ] [ BA ] + 1 ( 43) The fluorescence intensity for any given concentration of is given by Equation 44, where If is the fluorescence intensity, k is a constant related to instrument parameters such as

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104 monochromator thro ughput, is the quantum yield, Io is the incident light intensity, is the molar absorptivity, and b is the path length. I = k I b [ BA ARS ] ( 44) Rearrangement of Equation 44 allows for determination of concentration, as given by Equation 45. [ BA ARS ] = I k I b ( 45) Substitution of Equation 45 in Equation 43 gives Equation 46. k I b [ ARS ] I = 1 [ BA ] + 1 (4 6) Equation 46 can be rearranged to give Equation 47, the Benesi Hildebrand equation. 1 I = 1 k I b [ ARS ] [ BA ] + 1 k I b [ ARS ] KARS, can be found from the plot of 1/If vs. 1/[BA] for a titration of a boronic acid with a solution of constant ARS concentration. The

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105 association constant is determine by dividing the plot intercept by the slope. Examples of these plots can be found in the appendix (Figure A 19) . Once KARS is known, the apparent boronic aciddiol association constant ( Keq) can be determined through competitive binding assay. Each diol is titrated into a solution with co nstant boronic acid and ARS concentrations. Increasing the diol concentration displaces a fraction of the ARS from the boronic acidARS adduct , resulting in a decr ease in fluorescence intensity. The equilibrium for the displacement of the boronic acidARS adduct with a diol is shown in Scheme 41. Scheme 4 1. Equilibrium for the displacement of the boronic acidARS adduct with a diol The mass balance expressions for the individual components are given by Equations 48 through 410. [ Diol ] = [ Diol ] + [ BA Diol ] ( 48) [ ARS ] = [ ARS ] + [ BA ARS ] ( 4 9 ) [ BA ] = [ BA ] + [ BA Diol ] + [ BA ARS ] ( 410) The association constant for each diol, Keq, is given by Equation 411.

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106 = [ BA Diol ] [ Diol ] [ BA ] ( 411) Combining the mass balance equations with Equations 41 and Equation 411 gives Equation 412. [ BA ] = [ BA ] + [ BA ] [ Diol ] 1 + [ BA ] + [ BA ] [ ARS ] 1 + [ BA ] ( 412) If we define the indicator ratio, Q , by Equation 413, then Equation 412 can be rewritten a s Equation 414. = [ ARS ] [ BA ARS ] ( 413) [ BA ] = 1 + [ Diol ] + + [ ARS ] 1 + = [ BA ] 1 [ ARS ] 1 +

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107 = [ Diol ] + [ Diol ] = + 1 ( 417) Given equation 4 17, the boronic aciddiol association constant, Keq, can be found from a plot of [Diol]o/P vs. Q . The association constant can be determined by dividing KARS by the slope of the plot. The value Q can be obtained from measured fluorescence intensities as given by Equation 418, where I,BAARS is the fluorescence intensity of the boronic acid ARS adduct in the absence of competitive diol, I, ARS is th e fluorescence intensity of ARS , and I is the measured fluorescence intensity in the presence of competitive diol. 202,208 = I , I I I Ka and binding affinity. 6,59,199,202,209 However, these studies have been performed under a number of different conditions, leading to determinations of association constants that can vary between studies .210 In many of these investigations, the binding affinity was found in

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108 aqueous/organic cosolvent s ystems , which was necessary to solubilize the boronic acids in question.202,211 As such, there is a need for a study that examines the association constants of a variety of boronic acid families under identical conditions to better identify the most viable candidates for future applications. 4.2.1 Determination of B oronic A cid p Ka V alues The pKa of a boronic acid is defined as the pH at which 50% of the boronic acid exists as the hydroxyboronate anion species. Many applications of boronic acids rely on a shift in solubility or solute solvent favorability, which is often dictated by the transition from hydrophobic neutral boroni c acid to the anionic hydroxyboronate or boronate ester. As such, employing a boronic acid with a pKa much higher than physiological pH may not yield a material or device with an optimum response profile under physiological conditions. It has been shown th at the binding affinity of a boronic acid is often related to the p Ka of the boronic acid, although the most effective binding is no t always near or above the p Ka of the boronic acid .199 Several studies have proposed that optimal binding should occur at a pH between the p Ka of the boronic acid and of the diol, as given by Equation 419, although this may not account for secondary effects , such as buffer composition , steric hindrance, or solvent system , which can affect the ability to form boronic esters.199 pH = ( p + p ) / 2 (4 19) In this study, the pKa of each boronic acid was determined by monitoring the UV absorption of each molecule as a function of pH . A s the boronic acid transition s from the neutral trigonal planar species to the tetrahedral boronate center , the absorbance at wavelengths ranging

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109 fr om approximately 200 to 260 nm decreases, allowing for facile p Ka determinations . The measured pKa’s are given in Table 41 and e xamples of titration data can be found in the appendix (Figure A1 3Figure A 18) . Table 4 1. Measured pKa values for various boronic acids and analogues Boronic Acid p K a 3AcPBA 8.5 4MCPBA 7.9 4FPBA 7.8 BOB 7.5 2FPBA 7.5 DAPBA 5.3 Unmodified phenylboronic acid has previously been shown to have a pKa of approximately 8.8.9 T o adjust the p Ka, the aromatic ring can be modified with electronwithdrawing or donating substituents. Of the boronic acids investigated, 3acetamidophenylboronic acid (3 AcPBA) was found to have the highest p Ka, while t he electron withdrawing inductive and resonance effects for both 4 methylcarbamoylphenylboronic acid (4 MCPBA) and 4 formylphenylboronic acid (4 FPBA) result in a decrease of p Ka for both boronic

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110 acids . 212 As the aldehyde is slightly more electron withdrawing, 4FPBA has a slightly l ower overall p Ka than 4MCPBA. Benzoxaborole (BOB) is a heterocyclemodified boronic acid, with a p Ka significantly lower than 3AcPBA, 4 MCPBA, or 4 FPBA. The lower p Ka is driven by the release of ring strain in the 5 membered ring as the boron center transitions from a trigonal planar geometry in the neutral form to a tetrahedral geometry in the boronate anion form, with the heterocycle being retained.6 Interestingly, the pKa of 2formylphenyl boronic acid (2FPBA) is similar to that of BOB. This is likely due to intramolecul ar interaction between the carbonyl oxygen and the boron center. It has been shown that in aqueous solution 2FPBA can exist as an isomer of similar structure to BOB, forming a benzoxaborole heterocycle with a hydroxyl substituent on the methylene unit bet ween the aromatic ring and the oxygen derived from the carbonyl. This is due to tautomerization of the carbonyl, followed by a ring closing cyclization.213 The formation of this heterocycle is more favorable as the pH of the solution in creases, resulting in 2FPBA having a p Ka similar to that of BOB. Scheme 4 2. Isomerization of 2FPBA through keto enol tautomerization The boronic acid with the lowest measure p Ka was 2di methylaminomethylphenylboronic a cid (DAPBA). This family of boronic acids, containing an adjacent coordinating amine center , is often referred to as a “Wulff type” boronic acid.7 In these Wulff type boronic acid s , the nitrogen center coordina tes with boron , forming a tetrahedral bo ronate . As such, the pKa of this boronic acid is formally the p Ka of the amine center, as deprotonation of the amine results in the

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111 B N coordinative interaction. I t has been previously shown that both secondary and t ertiary amines have similar effect on boronic acid activity.8 Further increase in solution pH results in formation of the hydroxyboronate anion and release of the adjacent amine. Recently, there has been some eviden ce of a solvent insertion mechanism also taking place with Wulff type boronic acids, whereby a polar protic solve nt such as water or methanol can donate electron density to the boron center . In this mechanism, the solvent insertion results in the formation of an anionic boronate center and a cationic ammoniu m center.9,10 Scheme 4 3. pH dependent coordination of an amine to a boron center in Wulff type boronic acids 4.2.2 Apparent Association Constants for Various Boronic Acid Families The association between a boronic acid and a diol is affected by a number of factors, including boronic acid p Ka, diol pKa, dihedral angle of the diol, steric hindrance, and stabilization of the boronate center. Given the acidity of aromatic hydroxyl groups and the planar nature of the diol, it is common for catechol functional molecules to have high binding constants at relatively low pH. As such, Alizarin Red S, a catechol functional molecule , is often used as a fluorescent reporter, as it becomes fluorescent upon binding with a boronic acid. This provides a direct method for determination of binding constants with ARS. (Table 4 2) The binding of ARS was found to follow a similar pattern for most o f the boronic acids in question , with the highest association constant found near physiological pH. The exceptio n was DAPBA, where the

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112 association constant was significantly higher for DAPBA under acidic conditions, potentially due to an electrostatic attraction between the protonated ammonium center below the amine p Ka and the anion ic sulfonate group on ARS. For the boronic acids with functional groups directly attached or adjacent to boron (BOB, 2 FPBA, and DAPBA), the association constants were much lower than those for the other boronic acids, likely due to steric hindrance at the boron center. For measurements w here the apparent association constant was too low to be reliably determine d, the value is reported as n/d (not determined). Table 4 2. Measured apparent association constants (M1) for various boronic acids with ARS, sorbitol, fructose, and glucose at pH 5.2, 7.4, and 8.7. Under some conditions, the binding constant was too low to accurately measure and was thus not determined ( n/d). Boronic Acid (pKa) 3AcPBA (8.5) 4MCPBA (7.9) 4FPBA (7.8) BOB (7.5) 2FPBA (7.5) DAPBA (5.3) Diol ARS pH 5.2 1220 1720 2580 620 620 17400 pH 7.4 2200 2490 4850 940 760 600 pH 8.7 490 470 720 100 89 43 Sorbitol pH 5.2 6.6 13 22 5.5 9.7 130 pH 7.4 610 980 2100 340 440 380 pH 8.7 3200 4200 3000 1200 1200 230 Fructose pH 5.2 n/d n/d 3.1 n/d 1.9 4 pH 7.4 350 470 1260 230 360 64 pH 8.7 1400 1600 2000 770 790 9.6 Glucose pH 5.2 n/d n/d n/d n/d n/d n/d pH 7.4 8.1 8.8 21 11 22 n/d pH 8.7 20 38 52 42 42 n/d We were interested in investigating the association constant s of the boronic acids and sorbitol, as the sugar alcohol can potentially be have as a multifunctional diol for crosslinking

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113 between boronic acids or as a competitive binding agent . The latter is of great importance in heterogeneous separation devices for release of captured diol functional molecules in affinity chromatography .214 Of the sugar and sugar alcohols investigated, sorbitol had the highest association constants with each boronic acid as compared to fructose and glucose, even though sorbitol, fruc tose, and glucose all have a pKa close to 12.5. This is likely due to the availability of the diols in solution, as both fructose and glucose can exist as various linear and cyclic isomers . Of the possible fructose isomers, D fructofuranose has been show n to have the highest binding constant and an abundance of around 25% in aqueous solution, while the glucose isomer wit h the highest binding constant has been shown to be D glucofuranose which has an abundance of approximately 0.14%.120 For the unhindered boronic acids, the association constant with sorbitol increased with decreasing pKa. This pattern remained true for bo th fruct ose and glucose . Despite the fact that the sterically hindered boronic acids have significantly lower p Ka values than the unhindered boronic acids, it was observed that the association constants for the bulky boronic acids were considerably lower for sorbitol and fructose, with simila r binding constants for glucose . This counterintuitive binding affinity highlights the need an understanding of the e ffect of structure on binding affinity and to not assume that a lower p Ka will consistently result in a higher association constant. The gl ucose association constants for BOB and 2FPBA were amongst the highest values of the various boronic acids , with 2FPBA having a slightly higher association constant than BOB at neutral pH. At higher pH, where intramolecular cyclization of 2 FPBA is more favored, the association constants for 2 FPBA and BOB are very similar . The slightly higher glucose association constants of BOB and 2FPBA, coupled with the lower p Ka of the boronic acids, make these classes of materials fitting for glucoseresponsive sys tems.

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114 DAP BA, the Wulff type boronic acid, which has seen use in self healing hydrogels206 and glucose detection d evices ,125 had the lowest association constants at neutral and basic pH of all the boronic acids investigated. Other groups have observed the lower binding affinity of Wulff type boronic acids at neutral and basic pH .202 Interestingly, the association constants at acid ic pH were notably higher than those for all other boronic acids , likely due to the electrostatic stabilization of the boronate species with the cationic ammonium ion. The ability to stabilize esters at relatively low pH has been utilized to prepared hydrogels at acidic pH.21 4.3 Conc lusions Even with the increased association constants of BOB and 2FPBA, glucose binding is still much lower than the binding of other biologically relevant diols , such as fructose. This may be overcome through the use of specially designed diboronic acid ligands, in which the 3dimensional space between the boronic acids is designed to favor binding of one type of diol over others. A similar strategy is taken in molecularly imprinted network s , where the targeted substrate is incorporated during network for mation, precluding the binding of other diols for purely steric reasons. However, these approaches are either synthetically difficult or may simply not be amenable to a desired application. As such, it is necessary to have knowledge of the characteristics of each boronic acid family, so that the best functional group can be chosen for each application. In the design of boronic acidfunctional materials, it is vital to understand the structure property relationships between the functional groups and the effe ct on boronic acid characteristics , including pKa, binding affinity, and relative selectivity. This study provides a direct comparison of various boronic acid families, allowing for selection of the optimal boronic acid for each application. Many materials intended for physiological application s hav e relied on

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115 boronic acids that are similar in structure to 3 AcPBA or 4MCPBA, while those boronic acid s have been shown to have a pKa much hi gher than physiological pH and glucose association constant s lower than those of other boronic acid families . Wulff type boronic acids have seen relatively widespread use, even though the bulky nature of the amine results in very low diol binding constants at neutral and basic pH . The results of this research indic ate that heterocyclic boronic acids, like benzoxaboroles, or boronic acids with less hindered electron donating species, like 2 formylphenylboronic acid, may provide the best response profile under physiological conditions of the boronic acid families inve stigated. 4.4 Experimental 4.4.1 Materials 4F ormylphenylboronic acid (Combi blocks, 98%), 2formylphenylboronic acid (Combi blocks, 98%), 4(methylcarbamoyl)phenylboronic acid (Combi blocks, 98%), 3aminophenylboronic acid (Combi block, 98%), N,N dimethylammonium chloride (Aldrich, >95%), sodium borohydride (Sigma Aldrich, ACS grade), sodium bicarbonate (Fisher, ACS grade), methanol (Fisher, ACS grade), magnesium sulfate (Alfa Aesar, 99.5%), potassium hydroxide (Fisher, ACS grade), acetic anhydride (Fisher, ACS grade), molecular sieves ( 4, 4 to 8 mesh, Acros Organics ), dichloromethane (DCM, Fisher, ACS grade), sodium sulfate (Sigma Aldrich, ACS grade), diethyl ether (EMD Millipore, ACS), Alizarin Red S (ARS, Acros Organics, Pure Grade) were used a s received unless otherwise noted. 4.4.2 Synthesis of 3 A cetam idophenylboronic Acid (3 AcPBA) In a 100 mL round bottom flask, 3aminophenylboronic acid (2.00 g, 14.6 mmol) was added to an aqueous solution of sodium hydroxide (25 mL, 10 wt %). The solution was cooled in

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116 an ice bath, and acetic anhydride (2.80 mL, 29.7 mmol) was added slowly. After the addition was complete, the solution was allowed to come to room temperature and was stirred for 3 hours. The solution pH was then adjust ed to pH 8 with dilute hydrochloric acid (0.1 M), and a beige precipitate formed. The compound was recovered by vacuum filtration and washed with cold DI water , and was purified via recrystallization from DI water to yield light beige needles. (1.41 g, 54% yield) 4.4.3 Synthes is of Benzoxaborole (BOB ) In a 100 mL round bottom flask, 2formylphenylboronic acid (2.00 g, 13.3 mmol) was added to methanol (30.0 mL). The solution was cooled in an ice bath and sodium borohydride (0.550 g, 14.5 mmol) was added in three portions while maintaining the ice bath. The solution was allowed to come to room temperature and st ir overnight. The solution was concentrated under reduced pressure and hydrochloride acid (3 M, 50 mL) was added. The solution was extracted with diethyl ether (3 x 50 mL) and the organic layers were combined and dried over sodium sulfate. The solvent was removed under reduced pressure and the product was recrystallized from DI water. (1.40 g, 78% yield) 4.4.4 Synthesis of 2((dimethylamino)methyl)phenylboronic acid (DAPBA) In a 200 mL round bottom flask, N,N dimethylammonium chloride (3.26 g, 40.0 mmol) w as added to anhydrous methanol (30.0 mL) , along with potassium hydroxide (2.24 g, 40.0 mmol). After the potassium hydroxide pellets completely dissolved, anhydrous magnesium sulfate (15.0 g) was added as a water scavenger. To this slurry, 2formylphenylbor onic acid (3.00 g, 20.0 mmol) was added and the solution was stirred at room temperature for 2 hours. The solution was cooled in an ice bath, and sodium borohydride (1.13 g, 29.9 mmol) was added in

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117 three portions. The solution was allowed to come to room t emperature and was stirred overnight. The solution was filtered to remove the salts, and the volume was reduced by 2/3 under reduced pressure. To this, DI water (30 mL) and saturated sodium bicarbonate (30 mL) were added and the solution was extracted with (dichloromethante, 3 x 300 mL). The organic phases were combined, dried over sodium sulfate, and concentrated under vacuum to yield a white powder. (3.08 g, 86% yield) 4.4.5 p Ka Determinations for Free Boronic Acids The p Ka of the each boronic acid was determined spectroscopically. E ach compound was dissolved in an acidic sodium phosphate buffer solution (0.1 M) and titrated with dilute NaOH. The absorption spectra were measured over the full titration pH range and an appropriate absorbance wavelength was chosen for each compound. The absorbance at that wavelength was plotted as a function of pH, and the pKa was taken as the midpoint of the titration curve. Absorbance measurements were taken with a Molecular Devices SpectraMax M2 Microplate Reader using 96 well plates. 4.4.6 Determination of Apparent ARS Boronic Acid Association Constants ( KARS) To spectroscopically determine the association constant of ARS with a boronic acid, each boronic acid was titrated into solutions of constant ARS concentration. A stock solution of ARS was prepared in DI water (9.0 x 105 M), along with stock sodium ph osphate buffers (1.0 M, pH 5.2, pH 7.4, or pH 8.7). Stock solutions were also prepared in DI water for each boronic acid (2.0 x 102 M) except for 4 formylphenylboronic acid, which required the measurement of individual aliquots for each measurement due to the limited solubility of 4 FPBA. From these solutions, two separate solutions were prepared in DI water, one containing a boronic acid (2.0 x 103 M),

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118 ARS (9.0 x 106 M), and phosphate buffer (0.1 M) with the second solution containing the same, with the exception of the boronic acid . These solutions were used to perform titrations of constant ARS concentration and decreasin g boronic acid concentration. A nonfluorescent ARS standard was prepared as a solution of ARS and phosphate buffer in the absence of boronic acid and was used as a fluorescence blank . Fluorescence measurements were acquired with a Molecular Devices Spectr aMax M2 Microplate Reader using 96 well plates, with the samples being excited at 468 nm and emission being measured at 572 nm. Deviations in boronic acid concentration were necessary when the KARS for a boronic acid was too low (<100 M1). To determine KA RS for DAPBA at pH 5.2, it was necessary to titrate to a 100x dilution of the boronic acid concentration to determine an accurate association constant. A full presentation of component concentrations for every measurement can be found in the appendix (Tabl e A 1 and A 2) 4.4.7 Determination of Apparent Diol Boronic Acid Association Constants ( Keq) To determine the diol boronic acid association constant of each diol and boronic acid, three solutions were prepared from the previously mentioned stock solutions. The first solution contained the boronic acid (2.0 x 103 M), ARS (9.0 x 106 M), and phosphate buffer (0.1 M), while the second solution also contained the diol in question. The concentration of the diol varied with each boronic acid diol combi nation and at each pH. A third ARS standard solution was prepared containing only ARS (9.0 x 106 M), and phosphate buffer (0.1 M). The first solution was then titrated into the second to prepare solutions of constant ARS and boronic acid concentration but varying diol concentration. The boronic aciddiol free ARS solution was used as a fluorescence blank. Fluorescence measurements were acquired with a Molecular Devices

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119 SpectraMax M2 Microplate Reader using 96 well plates, with the samples being excited at 468 nm and emission being measured at 572 nm.

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120 CHAPTER 5 SYNTHESIS OF MACROMOLECULES CONTAINING INTRAMOLECULAR COORDINATING BORONIC ACIDS AND NOVEL HETEROCYCLIC BORONIC ACID ANALOGUES 5.1 Introduction The development of novel boronic acids is of great impo rtance to the generation of stimuliresponsive sensors or drug delivery applications. One area of particular interest is the development of boronic acids with a response profile that covers a wide pH range. As described in Chapter 4, boronic acids containi ng heterocyclic subunits or intramolecular B coordination show great potential as glucose responsive materials due to their relatively high glucose binding constants. As such, we are interested in the development of novel block copolymer nanoassemblies tha t contain intramolecular coordination or heterocyclic structure. The research detailed below describes our efforts in the design and preparation of (co)polymers derived from 2 acrylamidophenylboronic acid (2 APBA). There has been little discussion of 2APBA modified polymers in the literature.11,20,34,105,113 However , we have recently shown that hydrophilic copolymers of 2APBA can be utilized to prepare d ynamic covalent hydrogels, even at very low pH.20 The hydrogels were found to have very similar performance at neutral and acidic pH, suggesting that self assemble d nanostructures modified with these intramolecularly coordinated boronic acids may prove to be efficient glucose responsive materials. While it is known that boronic acids containing an intramolecularly coordinated carbonyl can undergo isomerization, we were surprised to find that deprotection of 2acrylamidophenylboronic acid pinacol ester (2 APBAE) does not yield the carbonyl coordinated free boronic acid. Instead, the molecule underwent a rapid intramolecular dehydration to form a new heterocyclic species , which has seen very limited mention in the literature.215 217 This

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121 monomer is similar in structure to benzoxaboroles, which w ere found to have effective glucose binding in Chapter 4. 5.2 Results and Discussion 5.2.1 Polymerization of 2APBAE with 2(((D odecylthio)carbonothioyl)thio) 2methylpropanoic acid monomethoxypolyetheylene glycol ester ( PEG DMP ) Macro Chain Transfer Agent Much of our research is dedicated to the s ynthesis and characterization of stimuli responsive amphiphilic block copolymers. Whenever possible, these block copolymers are prepared from PEG derived starting materials, as PEG has been shown to be biocompatible with limited cytotoxicity.218 With that in mind, our first attempt to polymerize 2APBAE was undertaken with the use of a PEG ester macroCTA prepared from 2 (((d odecylthio)carbonothioyl)thio) 2methylpropanoic acid ( DMP ) , a carboxylic acid terminal trithiocarbonate CTA (Scheme 5 1) . The polymerization proceeded with linear pseudo first order kinetics, suggesting a constant radical concentration without a large extent of unwanted chain terminating side reactions (Figure 5 1A) . However, the molecular weight s observed during polymer ization did not increase with conversion as expected (Figure 5 1B) . Rather, the molecular weight was near ly constant through the course of the polymerization (Figure 52) , a characteristic commonly found in conventional radical polymerizations, not in RDRP . The inability to control molecular weight was also observed for polymerizations with the unmodified DMP chain transfer agent. The e ffect of solvent on the polymerization was difficult to investigate , as the monomer was found to have very poor solubility i n nearly every solvent oth er than DMAc .

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122 Scheme 5 1. RAFT polymerization of 2 APBAE in the presence of a PEG DMP macroCTA Figure 51. Kinetics and molecular weight evolution for P(2APBAE) A) Pseudo first order kinetic plot for the RAFT polymerization of 2 APBAE with PEG DMP B) Molecular weight evolution of P(2APBAE) Figure 52. Size exclusion chr omatography traces of P(2 APBAE)

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123 Interestingly, the molecular weight for the monomer , as determined by SEC, was found to be much higher than expect ed , with the monomer exhibiting an apparent molecular weight of approximately 1,300 g/mol when compared to narrow molecular weight polystyrene standa rds. This high apparent molecular weight suggests that in DMAc the monomer is not molecularly dissolved, but rather exists as a multiunit complex. C rystals of 2APBAE grown from wet DMAc via an evaporative crystallization method confirmed that the acrylamide oxygen was in a position to interact with the boron center. This interaction is desired for the final application, but may have been one of the reasons for the lack of polymerization control. Scheme 5 2. Amide imidic acid tautomerization Amide s are known to undergo tautomerization, with two main isomeric forms being po ssible : an amide and an imidic acid (Scheme 5 2) .219 While the amide , containing a carbonoxygen double bond, is often the more stable of the two configurations , the coordination of the carbonyl oxygen with the boron center may help to stabilize the resonance form containing a carbon nitrogen double bond. The crystals grown from wet DMAc were shown to be a monohydrate, with the oxygen of the water m olecule in a position to hydrogen bond with the nitrogen proton (Figure 5 3) . This hydrogen bonding may further stabilize the now slightly more acid ic proton, further stabilizing the resonance form of the monomer containing a carbonnitrogen double bond.

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124 Figure 53. Crystal structure of 2APBAE, exhibiting coordination between the amide oxygen (O1) and the boron center (B1). The delocalization through the carbonnitrogen double bond allows for extended conjugation through the molecule and into the aroma tic ring. As such, when a radical is formed at the carbon adjacent to this carbonnitrogen double bond, the radical can delocalize throughout the molecule (Scheme 5 3) , resulting in a radical center that is much more stable than would be seen in a traditio nal, uncoordinated acrylamide monomer. Scheme 5 3. Proposed delocalization of radical center in 2 APBAE derived radical fragment Due to this increased radical stability, it is believed that the difficulty in controlling molecular weight and molecular w eight distribution may derive from the pre equilibrium or initialization period of the RAFT polymerization (Scheme 5 4) . Following conventional

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125 initiation, the monomer radical fragment adds to the carbon sulfur double bond, resulting in formation of an int ermediate radical. Because of the increased stability of the boronic acid derived radical fragment, the intermediate radical preferentially fragments towards the initial radical species rather than towards the R group. These intermediate radical s are thought to be very short lived under most conditions , resulting in regeneration of the carbonsulfur double bond and the boronic acidderived radical fragment. As the monomer derived radical quickly adds and fragments from the chain transfer agent, the polymeri zation behaves essentially as a conventional radical polymerization. Scheme 5 4. Initialization period for the polymerization of 2 APBAE with a RAFT chain transfer agent. 5.2.2 Polymerization of 2APBAE with Cumyl Dithiobenzoate To test this hypothesis, 2 APBAE was polymerized in the presence of cumyl dithiobenzoate. The cumyl R group produces a much more stable radical species than the gem dimethyl ester of the previous PEG DM P chain transfer agent.189 The inclusion of cumyl dithiobenzoate during the polymerization did lead to more control during the polymerization, with the resulting polymer molecular weights increasing with con version (Figure 5 5B) , although the polymers still exhibited a broad molecular weight distribution and the molecular weight deviated from the theoretical molecular weight at higher monomer conversion .

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126 Figure 54. Kinetics and molecular weight evolution f or P(2 APBAE) with cumyl dithiobenzoate A) Pseudo first order kinetic plot for the polymerization of 2 APBAE with cumyl dithiobenzoate . B) Molecular weight evolution of P(2 APBAE) Figure 55. Size exclusion chromatography traces for P(2 APBAE) polymerization with cumyl dithiobenzoate As mentioned above, the monomer peak of 2APBAE elut es at a lower retention time than what is expected for a molecule of such low molecular weight. This suggests intermolecular interaction of the monomer may be taking place in DMAc that is not otherwise seen with other non intramolecular coo rdinating boronic acids we ha ve studied. Perhaps, if the intramolecular coordination could be disrupted during t he course of the polymerizati on, the monomer would again behave like a conventional acrylamide monomer. Unfortunately, we found no change in

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127 the aggregation behavior of the monomer upon addition of various competitive Lewis bas es, including triethylamine, pyridine, or tetrabutylammonium fluoride (TBAF), where fluoride ion would serve as the competitive coodinating species. Trans esterificati on was also attempted with N methyldiethanolamine , which has been shown to efficient ly displace diol protecting groups as a result of nitrogen coordination with the boronic center.220 Even with a large excess, tran s esterifcation with N methyldiethanolamine was inefficient. The inability of these Lewis bases to coordinate with the boron center suggests that the int ramolecular interaction between the oxygen and boron center is very stable. 5.2.3 Copolymerization of 2APBAE and NIPAM and determination of reactivity ratios As with the investigation in Chapter 2, we were interested in studying the response profil e of NIPAM copolymers with intramolecular coordinating boronic acids. A copolymer of 2APBAE and NIPAM was prepared using the PEG DMP macro chain transfer agent. Although we found that 2APBAE does not controllably homopolymerize with PEG DMP, we hypothesized that the incorporation of NIPAM monomer units would result in favorable R group release and result in a more controlled polymerization (Scheme 5 5) . Scheme 5 5. Copolymerization of 2 APBAE and NIPAM in the presence of PEG DMP Even though the copolymerization of 2 APBAE and NIPAM was carried out with the same PEG DMP macro chain transfer agent that previously yielded an uncontrolled

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128 polymerization with 2 APBAE, the molecular weight of the copolymers increased with conversion (Figure 56B) . This suggests that the incorporation of NIPAM units facilitates fragmentation of the intermediate radical toward s the R group during the initializ ation period . However, the molecular weight at low conversion was higher than the theoretical molecular weight. This is a sign of slow consumption of the chain transfer agent. The slow consumption of the CTA also visible in the SEC traces, as evidenced by the slow reduction in the PEG DMP peak at a retention time of approximately 17 min. (Figure 5 7) The molecular weight distributions were also broad, (Figure 5 6B) which may be a result of poor degenerative chain transfer f or polymers with a terminal 2 APBAE unit. Figure 56. Kinetics and molecular weight evolution for P(2APBAE co NIPAM ) A) Pseudo first order kinetic plots for the copolymerization of 2 APBAE and NIPAM with PEG DMP. B) Molecular weight evolution of P(2AP BAE co NIPAM)

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129 Figure 57. Size exclusion chromatography traces for P(2 APBAE co NIPAM) polymerization with PEG DMP We found that throughout the course of the polymerization, the rate of incorporation of 2 APBAE was significantly higher than the rate of incorporation of NIPAM (Figure 5 6A) . This further suggests that stabilization of the 2 APBAE radical is occurring. The more rapid incorporati on of 2 APBAE yields a gradient copolymer, with the initial segment of the block being primarily 2 APBAE. After much of the 2APBAE has been incorporated, a segment largely consisting of NIPAM monomer units is produced. To quantitatively determine the favorability of incorporation of each monomer unit at the end of a growing chain, the reactivity ratios of 2 APBAE and NIPAM were measured using conventional free radical polymerization. Previous reports have shown that reactivity ratios dete rmine d via conventional radical polymerization do not usually greatly differ from those obtained by RAFT polymerization.221 A number of various models can be chosen to calculate reactivity ratios, including the Kelen Tudos model ,222 the Finemann Ross model,223 and the Inv erted FinemannRoss model.224 Of these three, the inverted Fineman nRoss method was chosen as the primary method for reactivity ratio determination , as it is the least susceptible to errors caused by mo nomer conversion greater than 5 10%. As 2APBAE incorporates

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130 significantly faster than NIPAM, 2APBAE can reach relatively high monomer conversion before NIPAM has been significantly incorporated. To determine the reactivity ratios, smallscale polymerizations were carried out, quenching the polymerization at low conversion. The results of the polymerizations and the reactivity ratio calculations are shown in Table s 5 1 and 52. From the inverted FinemannRoss method , the reactivity ratios were f ound to be r1 = 3.71, as determine from the intercept of the IFR plot, r2 = 0.23, as determined from the slope, and r1r2 = 0.89, where M1 corresponds to 2 APBAE and M2 corresponds to NIPAM (Figure 5 8) . These results are very similar to values calculated u sing the FinemannRoss and Kelen Tudos methods as well . The high r1 value and low r2 value quantitatively proves that the copolymerization of 2 APBAE and NIPAM favors incorporation of 2APBAE over NIPAM , resulting in a random copolymer that heavily incorp orates 2 APBAE early in the polymerization, as opposed to a copolymer that forms blocky regions of one monomer or the other. In a controlled polymerization, this increased incorporation of 2 APBAE results in the formation of a gradient random copolymer. The more favorable addition of 2APBAE over NIPAM further suggests that 2APBAE produces more stable radicals than a traditional acrylamide, like NIPAM, and is further evidence of intramolecular coordination resulting in a radical delocalization in 2 APBAE . Table 5 1. Experimental data for the copolymerization of 2 APBAE and NIPAM. M1, M2, m1, and m2 are values relative to a trioxane internal NMR standard. Sample M1 a (2 APBAE) M2 a (NIPAM) m1 b (2 APBAE) m2 b (NIPAM) Conv.c M1 Conv.c M2 Totalc Conv. 1 0.93 3.04 0.71 2.85 0.24 0.06 0.10 2 1.01 4.44 0.83 4.24 0.18 0.05 0.07 3 0.55 3.33 0.40 3.12 0.27 0.06 0.09 4 0.39 3.78 0.26 3.46 0.34 0.08 0.11 a) initial monomer content relative to non reactive NMR standard where M1 is 2 APBAE and M2 is NIPAM b) monomer content relative to non reactive NMR standard after quenching the polymerization where m1 is 2 APBAE and m2 is NIPAM c) monomer conversion after the polymerization was quenched where M1 is 2 APBAE and M2 is NIPAM d) total monomer conversion

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131 Table 5 2. Calculated values model for the copolymerization of 2 APBAE and NIPAM for the for the inverted FinemannRoss reactivity ratio model M a P b G c H d G/H 1/H 0.31 1.16 0.04 0.08 0.52 12.37 0.23 0.90 0.03 0.06 0.44 17.39 0.17 0.71 0.07 0.04 1.73 26.18 0.10 0.41 0.15 0.03 5.83 39.16 a) M = (M1/M2), b) P = (M1 m1)/(M2 m2), c) G = M (M/P), d) H = (M2)/P Figure 58. Inverted FinemannRoss plot for the copolymerization of 2 APBAE and NIPAM 5.2.4 D eprotection of 2 APBAE and the resulting monomer f ormed As a boronic acid is often less Lewis acidic than its respective boronic esters, we were interested in examining the polymerization behavior of the free boronic acid of 2APBAE. As mentioned above, at tempts to displace pinacol through the use of N methyldiethanolamine were unsuccessful , resulting in incomplete deprotection . Boronic esters have also b een deprotected through a trans esterification with a heterogeneous boronic acid modified resin (which is then deprotected with diethanolamine).46 However, this approach requires refluxing under harsh conditions not amenable to deprotecting a polymerizable molecule. We found the best method to deprotect 2 APBAE was a simple acidic hydrolysis. Initially, the deprotecti on was attempted

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132 through hydrolysi s in a 1:1 mixture of 1 M HCl and THF, followed by oxidative cleavage of the pinacol to acetone using sodium periodate.220 While the deprotection was rapid and quantitative, the resulting boronic acid formed a complex with the excess sodium periodate which made isolation of the product difficult. However, we found that by simply dispersing 2APBAE in THF and adding conc entrated aqueous HCl, 2 APBAE undergoes rapid hydrolysis, becoming temporarily soluble in THF before the product precipitates from solution in very high purity. As others have previously reported the sy nthesis of the este r free 2 acrylamidophenylboronic acid (2 APBA) ,11 we expect ed the hydrolysis of 2 APBAE to yield the free acid. However, this is not what was produced. Rather, the resulting product was found to be a novel heterocyclic monomer not previously reported, 3vinyl benzo[c][1,5,2]oxazaborinin1ol (3 VBOB ) , as verified by highresolution mass spect rometry, where the primary peak correspond to 3 VBOB or anhydrides of 3VBOB , with no major peak found corresponding to 2acrylamidophenylboronic acid . One meth od to prepare benzoxaborole, as described above in section 4.4.3, is through the reduction of 2FPBA. After formation of the primary alcohol from the aldehyde, the molecule undergoes a rapid dehydration to yield the benzoxaborole heterocycle (Scheme 5 6) . This may provide insight in the formation of the benzoxazaborinine . Scheme 5 6. Formation of benzoxaboroles from the dehydration of 2(hydroxymethyl)phenyl boronic acid

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133 As the coordination in 2APBAE of the carbonyl oxygen with the boron center is relatively strong , it is believed that f ollowing hydrolysis of 2 APBAE, the imidic acid isomer undergoes a rapid intramolecular dehydration. This ring formation drives th e amide towards the imidic acid and thus the benzoxazaborinine (Scheme 5 7) . It remains unclear if other reports of 2APBA were truly the free boronic acid , although it is unlikely, as Schofield and coworkers have shown various substituted 2amidophenylboronic acids yielding benzoxazaborinine s upon deprotection .217 Even so, this is the first report of 3 VBOB and preparation of polymers from the monomer. Scheme 5 7. Proposed route for benzoxazaborininie formation 5.2.5 RAFT polymerization of 3 vinylbenzoxazoborinine Given the structure of the resulting monomer, with the internal carbonnitrogen double bond, it is expected that 3VBOB derived radicals would have as much , if n ot more , resonance stabilization than 2APBAE (Scheme 5 8) . Therefore , we chose a RAFT chain transfer agent , CDTPA , with a dialkylcyano R group, which has similar radical stability as a cumyl R group .189 Scheme 58. Proposed delocalization of radical center in 3 VBOB derived radical fragment

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134 As with the polymerization of 2 APBAE, DMAc was chosen as the po lymerization solvent for 3 VBOB (Scheme 5 9) . While the monomer was soluble in methanol, polymerizations carried out in methanol resulted in very slow monomer consumption without the formation of high molecular weight polymer. The nature of the deleterious side reaction that inhibited polymer formatio n is still unknown. Scheme 5 9. RAFT polymerization of 3 VBOB with CDTPBA A s was seen for many of the polymerizations of 2 APBAE, the polymerization of 3 VBOB resulted in polymers with broad molecular weight distribution . Furthermore, the peak molecular weight of polymers formed during the reaction was nearly constant (Figure 5 10) , suggesting that the growth of polymer chains does not proc eed through the RAFT mechanism. (The average molecular weight was artificially inflated for early time points due to cutoff of the overlapped monomer peak.) It seems likely that monomer fragment radical stabilization results in an initialization period that fa vors reformation of the monomer derived radical rather than generation of the R group radical fragment.

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135 Figure 59. Kinetics and molecular weight evolution for P(3VBOB) A) Pseudo first order kinetic plots for the polymerization of 3 VBOB with CDTPBA. B) Molecular weight evolution of P(3 VBOB ) Figure 510. Size exclusion chromatography traces for P(3 VBOB ) polymerization with CDTPBA 5.3 Conclusions In this chapter , we examined the synthesis and controlled polymerization of an intramolecularly coordinated boronic acid, 2APBAE, along with the synthesis and polymerization of the novel heterocyclic 3 vinyl ben zoxazaborinine. We found that for both monomers, 2APBAE and 3 VBOB , the interaction of the amide derived oxygen center and the

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136 boron atom resulted in a monomer structure likely containing an internal carbon nitrogen double bond. The formation of this double bond resulted in conjugation that extended into the aromatic ring, making monomer derived radical fragments very stable. This hinde red our ability to prepare well defined homo and block copolymers with the monomers via RAFT polymerization. We also foun d that 2APBAE does not form a random copolymer when polymerized with NIPAM. Rather, 2APBAE is incorporated at a significantly faster rate than NIPAM, likely resulting in gradient copolymers. As copolymers of 3 VBOB have already been shown to effectively form boronate esters, even under acidic conditions, we feel confident that well defined amphiphilic block copolymers containing 3VBOB could yield glucose responsive nanoassemblies that respond over a wide pH range. Further optimization of the RAFT agent s tructure is necessary, with design of a RAFT agent containing an R group more stable than t he monomer derived radical as a vital factor . 5.4 Experimental 5.4.1 Materials 2A minophenylboronic acid pinacol ester ( Combi blocks, 97%), triethylamine (TEA, Alfa Aesar, 99%), acryloyl chloride (Aldrich, >97%), tetrahydrofuran (THF, EMD, 99.5%), N,N dimethylacetamide ( DMAc , Fisher ), hydrochloric acid ( Fisher, ACS grade ), oxalyl chloride ( Alfa Aesar, 98% ), N,N dimethylform amide (DMF, BDH, 99.8%) , dichloromethane (DCM, Macron) , m onomethoxy polyethylene glycol (PEG, Mn ~ 2 kg/mol, Aldrich) , dodecanethiol (Acros Organics, 98%), sodium hydride (Acros Organics, 60% dispersion in mineral oil ), carbon disu lfide (Alfa Aesar, 99.9% ) , iodine (Alfa Aesar 99.5% ), sodium thiosulfa te (Amresco, Reagent Grade ), magnesium sulfate (Fisher) , 4,4 azobis(4 cyanopentanoic acid) (Aldrich, >98% ), 2,2 -

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137 a zobisisobutyronitrile ( AIBN, Sigma, 98% ) were used as received unl ess otherwise noted. DMP w as prepared as previously described.194 5.4.2 Characterization Proton NMR spectroscopy was performed using a Varian Inova 500 spectrometer wit h deuterated methanol (CD3OD), deuterated chloroform (CDCl3) , or deuterated DMSO (DMSO d6) as the solvent. Molecular weights and molecular weight distributions were determined via conventional calibration relative to narrow molecular weight polystyrene or poly(methyl methacrylate) standards in N,N dimethyla cetamide ( DMAc ) with 50 mM LiCl at 50 C and a column and two ViscoGel I series G3078 mixed bed columns, with molecular weight ranges 3 4 g/mol, respectively). Detection consisted of a Wyatt Optilab T rEX refractive index detector operating at 658 nm . 5.4.3 X Ray Crystallography X Ray Intensity data were collected at 100 K on a Bruker DUO diffractometer using APEXII CCD area detector. Raw data frames were read by program SAINT1 and integrated using 3D profiling algorithms. The resulting data were reduced to produce hkl reflections and their intensities and estimated standard deviations. The data were corrected for Lorentz and polarization effects and numerical absorption corrections were applied based on indexed and measured faces. The structure was solved and refined in SHELXTL2013, using full matrix leastsquares refinement. The nonH atoms were refined with anisotropic thermal parameters and all of the H atoms were calculated in idealized positions and refined riding on their parent atoms. The

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138 asymmetric unit consists of the molecule and a water molecule in general position. The water protons and the amino protons, along with those on C8 and C9 were obtained from a Difference Fourier map and refined freely. In the final cycle of refinement, 3512 reflections (of which 3262 g R1, wR2 and S (goodness of fit) were 6.73%, 17.25% and 1.182, respectively. The refinement was carried out by minimizing the wR2 function using F2 rather than F values. R1 is calculated to provide a reference to the conventional R value but its function is not minimized. 5.4.4 Synthesis of 2 acrylamidophenylboronic acid pinacol ester (2 APBAE) 2Aminophenylboronic pinacol ester (5.00 g, 22.8 mmol) was dissolved in THF (150 mL) along with TEA (3.18 mL, 22.8 mmol), and the resulting solution was chilled to 0 C. A slight excess of acryloyl chloride (2.00 mL, 25.0 mmol) was diluted in THF (50 mL) and added drop wise over the course of 1 h via addition funnel. The solution was allowed to come to room te mperature after the addition and was left to stir for 24 h. The mixture was filtered to remove the TEA salt, and the solvent was removed under reduced pressure to yield a slightly yellow solid. The monomer was recrystallized from toluene (2) to yield a wh ite solid. C rystals of 2APBAE for X r ay crystallography were prepared by solvent evaporation from wet DMAc . 5.4.5 Synthesis of 3vinyl benzo[c][1,5,2]oxazaborinin 1ol (3 VBOB ) 2A crylamidophenylboronic acid pinacol ester ( 0.60 g, 2.2 mmol) was dispersed in THF (25 mL) in a 50 mL round bottom flask. To this, concentrated hydrochloric acid (1.3 mL) was quickly added. After stirring for a few minutes, the starting materials dissolv ed, at which point the product began to precipitate from solution. The mixtur e was stirred for a further 30 minutes

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139 before being stored at 20 C for 6 hours. The product was filtered and washed with cold THF. The powder was dried overnight under vacuum to yield a white solid (0.24 g, 63% yield) 5.4.6 Synthesis of 2(((D odecylthio )carbonothioyl)thio) 2methylpropanoic acid monomethoxypolyetheylene glycol ester ( PEG DMP ) To a 50 mL round bottom flask, 2dodecylsulfanylthiocarbonylsulfanyl2methyl propionic acid (DMP , 0.91 g, 2.5 mmol ) was added, along with DCM (5.0 mL), oxalyl chloride (1.0 mL), and 12 drops of DMF. The reaction was allowed to proceed for 3 hours until gas evolution had ceased. DCM and residual oxalyl chloride were removed under vacuum , and the resulting solid was redissolved in DCM (10 mL). Monomethoxy polyethylen e glycol (PEG, Mn ~ 2 kg/mol, 2.0 g, 1.0 mmol) was dissolved in DCM (12.5 mL), and the resulting solution was added dropwise to the first solution. The reaction was stirred overnight before being precipitated into cold hexane. The precipitate was redissolved in DCM and reprecipitated in hexane three more times. The final product was recovered by centrifugation and dried overnight under vacuum. (2.1 g, 89% yield) 5.4.7 Synthesis of 4Cyano 4(dodecylsulfanylthiocarbonyl)sulfanyl pentanoic acid ( CDTPA) Dodecanethiol (15.4 g, 76 mmol) was added dropwise to a suspension of sodium hydride ( 60% dispersion in mineral oil, 3.2 g, 79 mmol) in diethyl ether (150 mL) at 0 C. A thick, white slurry formed, and carbon disulfide (6.0 g, 79 mmol) was added drop wise while maintaining the temperature at 0 C. The resulting yellow precipitate was isolated by filtration, rinsed with diethyl ether and used without further purification. S dodecyl trithiocarbonate was suspended in ether (150 mL), and iodine (6.3 g, 47 mmol) was added portionwise while stirring. After 1 h, the white sodium iodide precipitate was removed by filtration. The filtrate was washed with aqueous

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140 sodiu m thiosulfate (5 wt%, 150 mL , 1), DI water (150 mL, 2), and brine (150 mL, 2). The orga nic layer was dried over MgSO4, filtered, and concentrated in vacuo to an orange oil. Bis (dodecylsulfanylthiocarbonyl) disulfide (7.4 g, 13 mmol) was dissolved in ethyl acetate (200 mL A zobis(4 cyanopentanoic acid) (5.6 g, 20. mmol) was added and the reaction was heated at reflux for 18 h. The solvent was removed in vacuo and the product was twice recrystallized from hexanes to give a yellow powder (5.1 g, 16% overall yield). 5.4.8 Synthesis of C umyl D ithobenzoate: To a dry three necked roundbottomed flask, s odium methoxide (25% solution in methanol, 51 mL , 0.25 mol ) was added along with a nhydrous methanol (100 mL), followed by rapid a ddition of elemental sulfur (8.0 g, 0.25 mol). Benzyl chloride (18 mL, 0.125 mol) was added dropwise by addition funnel under a nitrogen atmosphere. After addition, t he reaction mixture was heated in an oil bath at 65 C overnight . The reaction mixture was cooled to 0 C, and the precipitated salt was removed by filtration. The solvent was re moved under vacuum, and th e resulting product (sodium dithiobenzoate) was redissolved in DI water and wash three times with DCM in a separatory funnel. These washes were discarded. A nother portion of DCM was slowly added along with concentrated HCl, shakin g frequently to distribute the HCl. The solution color c hange d, turning an opaque pink/purple that partition ed into the DCM layer upon shaking. Concentrated HCl was slowly added until the aqueous layer was nearly transparent and the DCM laye r was a dark co lor. T he DCM layer was removed and the aqueous layer was washed with two more portions of DCM. The organic layers were combined , dried over magnesium sulfate, and the DCM was removed under reduced pressure to recover dithiobenzoic acid, which was used without further purification.

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141 Dithiobenzo ic acid (4.5 g, 0.029 mol) and methylstyrene (3.3 g, 0.027 mol) were diss olved in carbon tetrachloride (50 mL), and the resulting solution was purged with nitrogen and stirred at 70 C overnight . The crude product was obtained as a dark purple oil in 69% yield and was subsequently purified by column chromatography (neutral alumina packing, hexanes as eluent). 5.4.9 Polymerization of PEG b P(2 APBAE) via RAFT P olymerization with PEG DMP To a 20 mL spectrum capped vial, 2 APBAE (0.63 g, 2.3 mmol) was added along with PEG DMP (0.039 g, 0.017 mmol), and trioxane (0.010g, 0.11 mmol). A stock solution of AIBN was prepared (0.030 g, 0.18 mmol, in 10 mL DMAc ), and a portion of the stock solution (0.10 mL) was added to the 20 mL vial. DMAc (5.9 mL) was added, and the vial was purged with nitrogen for 30 min. before being placed in a heating block preheated to 70 C. The reaction was monitored via 1H NMR and SEC , with the reaction being quenched by opening to air. 5.4.10 P olymerization of 2 APBAE via RAFT P olymerization with CDB A 6 mL vial was charged with 2 APBAE (0.050 g, 0.18 mmol) and CDB (0.0015 g, 0.0037 mmol), and 1,3,5trioxane (3.5 mg, 0.039 mmol) , which acted as an internal NMR reference standard. A small m agnetic stir bar was added, and the compone nts were dissolved in DMAc (1.0 mL). A stock solution of AIBN (6.0 mg, 0.036 mmol) was prepared in DMAc (1.0 mL) and a 10 L aliquot was added to the above reaction solution. The via l was sealed with a rubber septum and was degassed by purging with nitrogen gas for 30 minutes. The vial was then placed in a aluminum block, preheated to 60 C. The polymerization was quenched by opening to air.

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142 5.4.11 Copolymerization of PEG b P(2 APBAE co NIPAM) via RAFT P olymer ization with PEG DMP To a 20 mL spectrum capped vial, 2APBAE (0.63 g, 2.3 mmol) was added along with PEG DMP (0.039 g, 0.017 mmol), and 1,3,5trioxane (0.010g, 0.11 mmol). A stock solution of AIBN was prepared (0.030 g, 0.18 mmol, in 10 mL DMAc ), and a portion of the stock solution (0.10 mL) was added to the 20 mL vial. DMAc (5.9 mL) was added, and the vial was purged with nitrogen for 30 min. before being placed in a heating block preheated to 70 C. The reaction was monitored via 1H NMR and SE C , with the reaction being quenched by opening to air. 5.4.12 Determination of R eactivity R atios between 2 acrylamidophenylboronic acid and N isopropylacrylamide Reactivity rations between 2 acrylamidophenylboronic acid and N isopropylacrylamide were determined via conventional free radical copolymerization. Briefly, a stock solut ion of AIBN (4.9 mg, 0.030 mmol in 1.0 mL DMAc ) was prepared, along with a stock solution of trioxane (0.027 g, 0.30 mmol in 11.9 mL DMAc and 0.10 mL AIBN stock solution). A portion of the 1,3,5trioxane solution (2.0 mL) was added to each of four vials containing various ratios of 2 APBAE and NIPAM. Each vial was capped with a rubber septum, and the solutions were purged with nitrogen for 30 minutes . A n aliquot was removed for NMR analysis before the vials were placed in a heating block preheated to 70 C. The reactions were stopped after 30 minutes, and the reactions were quenched by rapidly cooling the vials in an ice bath and opening to air. The monomer conversion for each monomer was determined via 1H NMR by comparing the respective vinyl peaks of each monomer with the trioxane internal standard both at the beginning of the reaction and after the reaction was complete. Reactivity ratios were calculated using the inverted FinemannRoss model .

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143 5.4.13 Polymerization of 3 VBOB via RAFT P olymerization with CDTPA 3VBOB ( 50 m g, 0.29 mmol) was added to a 6 mL vial, along with CDTPBA (1.2 mg, 0.0030 mmol), 1,3,5trioxane (6.5 mg, 0.072 mmol), and DMAc (1.9 mL). A stock solution of AIBN (3.9 mg, 24 mmol) was prepared in DMAc (1.0 mL), and an aliquot of the stock solution (0.10 mL) was transferred to the reaction solution. The vial was sealed with a rubber septum, and the reaction solution was deoxy genated by purged with nitrogen gas for 30 min. After removing an initial aliquot for NMR analysis, the reaction vial was placed in an aluminum block preheated to 70 C. The reaction was monitored via NMR and SEC.

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144 CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTI ONS The research presented i n this dissertation investigated the use of boronic acidcontaining materials and their applicability to novel stimuliresponsive polymer compositions. S ynthesis and character ization of various boronic acid containing materials, inc luding both high molecular weight polymers and small molecule analogues of boronic acids often used in stimuli responsive nanoassemblies and sensors , were described . The purpose of this work is to further develop novel boronic acid polymers and investi gate the response of those polymers to changes in solution conditions, including pH and glucose concentration. Furthermore, a broad study of boronic acid activity was undertaken to investigate the binding affinity of various boronic acids and their potenti al for further investigation in future soft materials. Finally, this research examined novel intramolecularly coordinated boronic acids, whereby an internal carbonyl from an amide group was found to coordinate with the boron center, potentially allowing for ester formation with the boronic acid over a very wide pH range. Many boronic acid functional nanoassemblies require a high molar content of boronic acid to retain the hydrophobic character necessary for preparation of stimuli responsive materials. Previ ous research in our group investigating the copolymerization of boronic acids and hydrophilic comonomers found that less than 50 mol% boronic acid in a copolymerization with DMA led to polymers that were incapable of self assembly. As boronic acids are cos t prohibitive in many situations, the ability to prepare materials that can self assemble while requiring reduced boronic acid content could be of great advantage. The block copolymers described in this dissertation , having a permanently hydrophilic PDMA block and a thermoresponsive block consisting of varying amounts of NIPAM and ACPBA, were shown to exhibit a shifting cloud point, and thus a wide cloud point transition window, that was ultimately dependent on boronic

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145 acid content, solution pH, and glucose concentration. Further optimization of this system, with increased block length for the thermoresponsive block along with the introduction of a boronic acid comonomer with a higher glucose binding constant or lower p Ka could provide a system with an improved response profile. One particularly promising avenue is the development of a carefully designed diboronic acid functional monomer. Various groups have shown that by designing a diboronic acid with just the right spacing between the boronic acids, select ive binding of glucose can be heavily favored over other saccharides , with significantly higher glucose association constants than commonly observed with monofunctional boronic acids . This selective binding has been used almost exclusively in sensing appli cations, but if the monomer were incorporated into a thermoresponsive copolymer, the resulting polymer could potentially have an excellent response profile while maintaining a low overall boronic acid content. With further study of boronic acids as functio nal group s within the area of materials science, the need for a better understanding of the various boronic acids and boronic acid analogues is necessary. Herein , the binding constants of 6 different boronic acids were investigated over a wide pH window an d with three sugars or sugar alcohols that are often of interest in sensing, response, or bulk materials applications. These boronic acids were chosen because they serve as analogues of boronic acids commonly found in stimuli responsive materials, sensors, separations and chromatography, or in the preparation of bulk materials like hydrogels. The improve d understand of the binding profiles of these categories of boronic acids may prove to be valuable for investigators moving forward. As the binding constant s are dependent on solution conditions, including buffer type and concentration, this study compare d materials within a single solvent system rather than the various systems investigated in the literature.

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146 As shown in the binding studies, boronic acids containing heterocycles or intramolecular coordination may be of great interest in new sensing applications or materials development. We have shown through X ray crystallography that 2 acrylamidophenylboronic acid pin acol ester does contain the desired intramolecular coordination. Furthermore, we found that deprotection of 2APBAE resulted in a rapid intramolecular dehydration, producing a previously undescribed monomer, 3 vinyl benzo[c][1,5,2]oxazaborinin1ol (3 VBOB ) . This new heterocyclic monomer may be of great use in future materials. However, controlled polymerization of 3 VBOB through RAFT polymerization proved elusive, likely due to extensive delocalization of the radical center in the monomer derived radical f ragments. Further optimization of RAFT agent design is necessary to prepare well defined macromolecular architectures with 3 VBOB . As we ha ve previously shown that random copolymers containing 3VBOB units can form boronate esters, even under acidic condit ions, nanoassemblies containing 3VBOB derived polymers are of great interest for controlled delivery of insulin for treatment of Type 1 diabetes. Further development of specialty chain transfer agents may be necessary to successfully prepare homo and bl ock copolymers of this novel heterocyclic monomer. As we believe the poor control in RAFT polymerization of these monomers is a result of the pre equilibrium intermediate radical favoring reformation of the monomer derived radical, it may be necessary to prepare a CTA with an R group that mimics the m onomer derived radical. For the best chance of success, the CTA would also benefit from increased steric hindrance at the R group to even further favor radical fragmentation towards the new R group derived radi cal . This could be prepared through amidation of the DMP CTA with 2aminophenylboronic acid. Many groups have applied computational analysis to RAFT polymerizations to better understand the intricacies of the RAFT mechanism and to predict trends in molecul ar weight

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147 growth and molecular weight distribution. Application of that computational analysis to our current attempts to polymerize our boronic acid monomers may provide insight into the cause of our lack of polymerization control. With a better understanding of the reactivity of our monomers and the propensity of monomer derived radicals to partake in the RAFT mechanism, we may be better equipped to design novel RAFT agents that result in better polymerization control. If this methodology continues to prove to be unsuccessful, development of a monomer that separates the reactive moiety of the monomer from the heterocycle may be necessary. To ensure the resulting monomer maintains its hydrophobic nature, it may be necessary to use a more hydrophobic spacer such as 2 carboxyethylacrylate. Following successful preparation of block copolymers containing the new heterocyclic boronic acid, solution studies must be carried out to determine the ability of the resulting polymers to efficiently self assemble and ulti mately dissociate in response to elevated glucose levels. If the results are promising, encapsulation of insulin and in situ animal studies would be the required next step towards development of glucose responsive insulin delivery vehicles.

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148 CHAPTER 7 APPE NDIX A .1 Nuclear Magnetic Resonance Spectra Figure A 1. 1H NMR spectrum of tert butyl (2aminoethyl)carbamate in CDCl3 Figure A 2. 1H NMR spectrum of tert butyl (2acrylamidoethyl)carbamate in CDCl3

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149 Figure A 3. 1H NMR spectrum of 4carboxyphenylboronic acid pinacol ester in CDCl3 Figure A 4. 1H NMR spectrum of (4 ((2 acrylamidoethyl)carbamoyl)phenyl)boronic acid pinacol ester (ACPBAE) in CDCl3

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150 Figure A 5. 1H NMR spectrum of poly( N,N dimethylacrylamide) bpoly( N isopropylacrylamide) [PDMA bPNIPAM] (P1) in CD3OD

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151 Figure A 6. 1H NMR spectrum of 2 ( ( dimethylamino)methyl)phenylboronic acid in CD3OD

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152 Figure A 7. 1H NMR spectrum of 3acetamidophenylboronic acid in DMSO d6

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153 Figure A 8. 1H NMR spectrum of benzoxaborole in DMSO d6

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154 Figure A 9. 1H NMR spectrum of 2 acrylamidophenylboronic acid pinacol ester in CDCl3

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155 Figure A 10. 1H NMR spectrum of 3vinyl benzo[c][1,5,2]oxazaborinin1ol in CD3OD

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156 Figure A 11. 1 3C NMR spectrum of 3vinyl benzo[c][1,5,2]oxazaborinin1ol in CD3OD

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157 A .2 Gel Permeation Chromatography Traces Figure A 12. Size exclusion chromatograms for PDMA macroCTA and block copolymers P1 P4 prepared from a chain extension of PDMA A .3 pH Titration Curves Figure A 13. pH t itration curve for 3 acetamidophenylboronic acid in 0.1M sodium phosphate buffer ; absorbance measured at 219 nm

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158 Figure A 14. pH titration curve for 4methylcarbamoylphenylboronic acid in 0.1M sodium phosphate buffer ; absorbance measured at 232 nm Figur e A 15. pH titration curve for benzoxaborole in 0.1M sodium phosphate buffer ; absorbance measured at 222 nm

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159 Figure A 16. pH titration curve for 2formylphenylboronic acid acid in 0.1M sodium phosphate buffer ; absorbance measured at 260 nm Figure A 17. pH titration curve for 4formylphenylboronic acid in 0.1M sodium phosphate buffer ; absorbance measured at 245 nm

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160 Figure A 18. pH titration curve for 2((dimethylamino)methyl)phenylboronic acid in 0.1M sodium phosphate buffer ; absorbance measured at 2 25 n m

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161 A .4 Solution Conditions for Association Constant Determinations Table A 1. Concentration (mol/L) of the solution components used to determine apparent association constants for 3 AcPBA, 4MCPBA, and BOB. 3 AcPBA 4 MCPBA BOB pH 5.2 Phosphate Buffer 0.10 0.10 0.10 Boronic Acid 2.0 x 10 -3 2.0 x 10 -3 2.0 x 10 -3 ARS 9.0 x 10 -6 9.0 x 10 -6 9.0 x 10 -6 Sorbitol 1.5 2.0 2.0 Fructose 4.0 4.0 4.0 Glucose n/a n/a n/a pH 7.4 Phosphate Buffer 0.10 0.10 0.10 Boronic Acid 2.0 x 10 -3 2.0 x 10 -3 2.0 x 10 -3 ARS 9.0 x 10 -6 9.0 x 10 -6 9.0 x 10 -6 Sorbitol 0.05 0.05 0.05 Fructose 0.1 0.1 0.1 Glucose 3.5 3.5 1.0 pH 8.7 Phosphate Buffer 0.10 0.10 0.10 Boronic Acid 2.0 x 10 -3 2.0 x 10 -3 2.0 x 10 -2 ARS 9.0 x 10 -6 9.0 x 10 -7 9.0 x 10 -6 Sorbitol 0.010 0.010 0.10 Fructose 0.020 0.020 0.10 Glucose 0.40 0.50 2.0

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162 Table A 1. Continued 2 FPBA 4 FPBA DAPBA pH 5.2 Phosphate Buffer 0.10 0.10 0.10 Boronic Acid 2.0 x 10 -3 2.0 x 10 -3 2.0 x 10 -3 ARS 9.0 x 10 -6 9.0 x 10 -6 9.0 x 10 -6 Sorbitol 2.0 2.0 2.0 Fructose 4.0 4.0 4.0 Glucose n/a n/a n/a pH 7.4 Phosphate Buffer 0.10 0.10 0.10 Boronic Acid 2.0 x 10 -3 2.0 x 10 -3 2.0 x 10 -3 ARS 9.0 x 10 -6 9.0 x 10 -6 9.0 x 10 -6 Sorbitol 0.05 0.05 0.1 Fructose 0.1 0.1 0.2 Glucose 1.0 3.5 n/a pH 8.7 Phosphate Buffer 0.10 0.10 0.10 Boronic Acid 2.0 x 10 -2 2.0 x 10 -3 2.0 x 10 -2 ARS 9.0 x 10 -6 9.0 x 10 -6 9.0 x 10 -6 Sorbitol 0.10 0.010 0.10 Fructose 0.10 0.020 1.0 Glucose 2.0 0.5 n/a A .5 Association Constant Determination Titrations

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163 Figure A 19. Titration curves to determine KARS of 3AcPBA at pH 5.2; three replicate titrations Figure A 20. Titration curves to determine Keq of 3 AcPBA with sorbitol at pH 5.2 ; three replicate titrations Figure A 21. Titration curves to determine KARS of 3AcPBA at pH 7.4; three replicate titrations

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164 Figure A 22. Titration curves to determine Keq of 3 AcPBA with sorbitol at pH 7.4 ; three replicate titrations Figure A 23. Titration curves to determine Keq of 3 AcPBA with fructose at pH 7.4; three replicate titrations

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165 Figure A 24. Titration curves to determine Keq of 3 AcPBA with glucose at pH 7.4; three replicate titrations Figure A 25. Titration curves to determine KARS of 3AcPBA at pH 8.7; three r eplicate titrations

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166 Figure A 26. Titration curves to determine Keq of 3 AcPBA with sorbitol at pH 8.7 ; three replicate titrations Figure A 27. Titration curves to determine Keq of 3 AcPBA with fructose at pH 8.7; three replicate titrations

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167 Figure A 28. Titration curves to determine Keq of 3 AcPBA with glucose at pH 8.7; three replicate titrations Figure A 29. Titration curves to determine KARS of 4MCPBA at pH 5.2 ; three replicate titrations

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168 Figure A 30. Titration curves to determine Keq of 4MCPBA with sorbitol at pH 5.2 ; three replicate titrations Figure A 31. Titration curves to determine KARS of 4MCPBA at pH 7.4 ; three replicate titrations

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169 Figure A 32. Titration curves to determine Keq of 4MCPBA with sorbitol at pH 7.4 ; three replicate titrations Figure A 33. Titration curves to determine Keq of 4MCPBA with fructose at pH 7.4; three replicate titrations

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170 Figure A 34. Titration curves to determine Keq of 4MCPBA with glucose at pH 7.4; three replicate titrations Figure A 35. Titration curves to determine KARS of 4MCPBA at pH 8.7 ; three replicate titrations

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171 Figure A 36. Titration curves to determine Keq of 4MCPBA with sorbitol at pH 8.7 ; three replicate titrations Fig ure A 37. Titration curves to determine Keq of 4MCPBA with fructose at pH 8.7; three replicate titrations

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172 Figure A 38. Titration curves to determine Keq of 4MCPBA with glucose at pH 8.7; three replicate titrations Figure A 39. Titration curves to determine KARS of BOB at pH 5.2; three replicate titrations

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173 Figure A 40. Titration curves to determine Keq of BOB with sorbitol at pH 5.2 ; three replicate titrations Figure A 41. Titration curves to determine KARS of BOB at pH 7.4

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174 Figure A 42. Titration curves to determine Keq of BOB with sorbi tol at pH 7.4; three replicate titrations Figure A 43. Titration curves to determine Keq of BOB with fructose at pH 7.4; three replicate titrations

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175 Figure A 44. Titration curves to determine Keq of BOB with glucose at pH 7.4; three replicate titrations Figure A 45. Titration curves to determine KARS of BOB at pH 8.7; three replicate titrations

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176 Figure A 46. Titration curves to determine Keq of BOB with sorbitol at pH 8.7 ; three replicate titrations Figure A 47. Titration curves to determine Keq of BOB with fructose at pH 8.7; three replicate titrations

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177 Figure A 48. Titration curves to determine Keq of BOB with glucose at pH 8.7; three replicate titrations Figure A 49. Titration curves to determine KARS of 2FPBA at pH 5.2; three replicate titrations

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178 Figure A 50. Titration curves to determine Keq of 2 FPBA with sorbitol at pH 5.2 ; three replicate titrations Figure A 51. Titration curves to determine Keq of 2 FPBA with fructose at pH 5.2; three replicate titrations

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179 Figure A 52. Titration curves to determine KARS of 2FPBA at pH 7.4; three replicate titrations Figure A 53. Titration curves to determine Keq of 2 FPBA with sorbitol at pH 7.4 ; three replicate titrations

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180 Figure A 54. Titration curves to determine Keq of 2 FPBA with fructose at pH 7.4; three replicate titrations Figure A 55. Titration curves to determine Keq of 2 FPBA with glucose at pH 7.4; three replicate titrations

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181 Figure A 56. Titration curves to determine KARS of 2FPBA at pH 8.7; three replicate titrations Figure A 57. Titration curves to determine Keq of 2 FPBA with sorbitol at pH 8.7 ; three replicate titrations

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182 Figure A 58. Titration curves to determine Keq of 2 FPBA with fructose at pH 8.7; three replicate titrations Figure A 59. Titration curves to determine Keq of 2 FPBA with glucose at pH 8.7; three replicate titrations

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183 Fi gure A 60. Titration curves to determine KARS of 4FPBA at pH 5.2; three replicate titrations Figure A 61. Titration curves to determine Keq of 4 FPBA with sorbitol at pH 5.2 ; three replicate titrations

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184 Figure A 62. Titration curves to determine Keq of 4 FPBA with fructose at pH 5.2; three replicate titrations Figure A 63. Titration curves to determine KARS of 4FPBA at pH 7.4; three rep licate titrations

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185 Figure A 64. Titration curves to determine Keq of 4 FPBA with sorbitol at pH 7.4 ; three replicate titrations Figure A 65. Titration curves to determine Keq of 4 FPBA with fructose at pH 7.4; three replicate titrations

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186 Figure A 66. Titration curves to determine Keq of 4 FPBA with glucose at pH 7.4; three replicate titrations Figure A 67. Titration curves to determine KARS of 4FPBA at pH 8.7; three replicate titrations

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187 Figure A 68. Titration curves to determine Keq of 4 FPBA with sorbitol at pH 8.7 ; three replicate titrations Figure A 69. Titration curves to determine Keq of 4 FPBA with fructose at pH 8.7; three replicate titrations

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188 Figure A 70. Titration curves to determine Keq of 4 FPBA with glucose at pH 8.7; three re plicate titrations Figure A 71. Titration curves to determine KARS of DAPBA at pH 5.2 ; three replicate titrations

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189 Figure A 72. Titration curves to determine Keq of DAPBA with sorbitol at pH 5.2; three replicate titrations Fig ure A 73. Titration curves to determine Keq of DAPBA with fructose at pH 5.2; three replicate titrations

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190 Figure A 74. Titration curves to determine KARS of DAPBA at pH 7.4 ; three replicate titrations Figure A 75. Titration curves to determine Keq of DAPBA with sorbitol at pH 7.4; three replicate titrations

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191 Figure A 76. Titration curves to determine Keq of DAPBA with fructose at pH 7.4; three replicate titrations Figure A 77. Titration curves to determine KARS of DAPBA at pH 8.7 ; three replicate titrations

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192 Figure A 78. Titration curves to determine Keq of DAPBA with sorbitol at pH 8.7; three replicate titrations Figure A 79. Titration curves to determine Keq of DAPBA with fructose at pH 8.7; three replicate titrations

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193 A .6 High Resolution Mass Spectrometry Plots Figure A 80. Highresolution mass spectrometry trace of 3 VBOB

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207 BIOGRAPHICAL SKETCH William Brooks was born in San Bernardino, California to Edward (Eddie) and Lori Brooks. Throughout his life, he has lived in a number of locations, moving as a child from California to Utah, from Utah to Mississippi, from Mississippi to Wisconsin, and finally from Wisconsin back to Mississippi. (Wisconsin was his favorite.) H e is an alumnus of the Mississippi School for Mathematics and Science (MSMS), having graduated in May of 2006. He continued his education first at Mississippi College, before moving to the University of Southern Mississippi (USM) in the spring of his freshman year. (SMTTT!) There, he received a Bachelor of Science degree (Magna Cum Laude) in P olymer S cience with a M athematics minor. From there, he made the trek to the Lone Star State, beginning graduate school at Southern Methodist University under the dire ction of Dr. Brent S. Sumerlin. He lived in Dallas for two years before moving with the Sumerlin group to the University of Florida in Gainesville, Florida. He completed his Ph.D. in chemistry in the f all of 2015. His research investigated the development of thermo and glucose responsive materials and examined the effect of solution conditions and boronic acid composition on boronic acid reactivity. While a student at USM, William met and fell in love with his future wife, Mieu (Ly) Brooks. They like to jo ke that they “had chemistry in organic chemistry lab.” Together, they have an amazing daughter, Linh. Outside of science, the three of them enjoy outdoor activities, including kayaking, camping, and trips to the springs or to the beach. William also enjoys visiting family and expanding his knowledge of Vietnamese culture. He hopes to one day tra vel the wor l d, including visits to London, England; Paris, France; Mainz, Germany; Shanghai, China; and Ho Chi Minh City , Vietnam. William is a gearhead at heart, having been a Ford Mustang fan since birth.