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Dynamic-Covalent Macromolecular Architectures Composed of Reversible Oxime Bonds

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Title:
Dynamic-Covalent Macromolecular Architectures Composed of Reversible Oxime Bonds
Creator:
Mukherjee, Soma
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[Gainesville, Fla.]
Florida
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University of Florida
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Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Chemistry
Committee Chair:
SUMERLIN,BRENT S
Committee Co-Chair:
WAGENER,KENNETH B
Committee Members:
MILLER,STEPHEN ALBERT
SAVIN,DANIEL ANDREW
ANDREW,JENNIFER
Graduation Date:
8/8/2015

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Subjects / Keywords:
Block copolymers ( jstor )
Copolymers ( jstor )
Disulfides ( jstor )
Gels ( jstor )
Hydrogels ( jstor )
Monomers ( jstor )
Oximes ( jstor )
Polymerization ( jstor )
Polymers ( jstor )
Self healing materials ( jstor )
Chemistry -- Dissertations, Academic -- UF
dynamic -- oxime -- reversible -- thermoreversible
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bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Chemistry thesis, Ph.D.

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Abstract:
Interest in reversible macromolecular architectures is burgeoning with a rapidly growing number of applications envisioned in degradable, self-healing, and responsive materials. Oxime formation has been utilized as an efficient tool for post-polymerization modification, conjugating biomolecules to polymers and hydrogels, and engineering cell surfaces for tissue engineering. We utilized the reversible assembly of amphiphilic keto-functional block copolymers prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization to construct star polymers via oxime formation. The reversible nature of the oxime linkage was demonstrated by competitive exchange with an excess of a monofunctional alkoxyamine or monofunctional aldehyde under acidic conditions to result in the dissociation of the stars. We have also utilized the inherent reversibility of oxime bonds to synthesize self-healing hydrogels. Keto-functional hydrophilic copolymers were crosslinked with difunctional alkoxyamines to obtain hydrogels via oxime formation. A gel to sol transition was induced in the presence of monofunctional alkoxyamines and an acid catalyst. The mechanical properties of this hydrogels were characterized by rheology. Physical gelation of the copolymers was also demonstrated above their cloud points. These rehealable and thermo-responsive hydrogels may have potential applications in coatings for medical devices, sensors, scaffolds for biomolecules, and network scaffolds for tissue engineering. Orthogonal multi-functionalization strategy has emerged in recent years as a powerful technique to obtain novel polymers with diverse composition and topology. Presence of multiple functional groups on a single polymer backbone enables the polymer to respond to external stimuli in different ways. We combined orthogonal oxime and Diels-Alder chemistry to yield a linear step-growth polymer which could disassemble under two unique sets of conditions. An AB monomer containing furfuryl, maleimido, and latent oxime bonds was synthesized and polymerized by Diels-Alder reaction to obtain the polymer that was capable of reversible disassembly by retro-Diels-Alder and oxime exchange. These remendable polymers offer great potential for designing sensors and constitutionally dynamic polymers. While the research described here demonstrates the benefit of having reversible links in the polymers to construct reversible branched architecture, similar strategy can be used to design more complex adaptive polymeric architectures. ( en )
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In the series University of Florida Digital Collections.
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Includes vita.
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Includes bibliographical references.
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Description based on online resource; title from PDF title page.
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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: WAGENER,KENNETH B.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2016-02-29
Statement of Responsibility:
by Soma Mukherjee.

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2/29/2016
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1 DYNAMIC COVALENT MACROMOLECULAR ARCHITECTURES COMPOSED OF REVERSIBLE OXIME BONDS By SOMA MUKHERJEE 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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2 © 2015 Soma Mukherjee

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3 To my family, friends, and my teachers who guided me throughout this journey to graduate school

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4 ACKNOWLEDGMENTS This dissertation would not have been complete without the sincere contributions and assistance of the many individuals who have been a part of my journey through graduate school. I would like to thank my advisor , Prof. Brent Sumerlin , for the opportunity to work with him, and the endless support and encouragement he provided throughout the last five years. I am also very grateful to my advisor for giving me the freedom and motivation to be innovative and to pursue my own research ideas , which helped me to become the scientist I am today. I am grateful to my dissertation committee, Prof. Ken Wagener, Prof. Stephen Miller, Prof. Daniel Savin, and Prof. Jennifer Andrew, for their valuable time and advices. My sincere gratitude goes to a former graduate student in the Sumerlin group and my mentor, Dr. Abh ijeet P. Bapat for providing me invaluable guidance, assistance, and motivation throughout graduate school. I am also thankful to the former post doctoral fellow , Dr. Debashish Roy for his advice and encouragement. I would like to thank the current graduat e students in the Sumerlin group, especially Megan Hill for helping with the TEM and SEM analysis , Yuqiong Dai (Daily) and William Brooks for helping me with research and providing valuable suggestions, and Hao Sun and Jessica Cash for their advice regarding research, and also all for sharing good times with me. I could n o t be thankful enough to my parents for their sincere effort to provide me a higher education and my entire family and close relatives for alw ays being encouraging. I am very fortunate to have friends in India and in the USA who have provided constant support during the last five years. It would not be possible for me to pursue my dream of becomin g a polymer scientist and earn a doctoral degree without the emotional support and motivation provided by my family and friends.

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5 My sincere thank goes to the entire faculty and staff of the Department of Chemistry at the University of Florida for providing me help whenever I needed. I am also very thank ful to the faculty and staff in the Department of C hemistry at Southern Methodist University (SMU), especially Prof. Nicolay V. Tsarevsky , Prof. David Y. Son , Prof. Patty J. Wisian Neilson , and Prof. Brian Zoltowski for serving on my PhD committee at SMU a nd for offering their valuable suggestions for my research, and Laurieann Ram Kern for helping m e with non research related issues at school, and to everyone for providing a friendly atmosphere in my first two years of graduate school in SMU. This materia l is based upon work supported by the National Science Foundation (DMR 1410223 and CAREER DMR 0846792 ) and the Alfred P. Sloan Foundation. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author and do not necessarily reflect the views of the National Science Foundation or the Alfred P. Sloan Foundation.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ........................... 8 LIST OF FIGURE S ................................ ................................ ................................ ......................... 9 LIST OF SCHEMES ................................ ................................ ................................ ...................... 12 LIST OF ABBREVIATIONS ................................ ................................ ................................ ........ 13 ABSTRACT ................................ ................................ ................................ ................................ ... 16 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ ................... 18 1. 1 Alkoxyamine bonds ................................ ................................ ................................ .......... 20 1.2 Diels Alder linkages ................................ ................................ ................................ ......... 23 1.3 Disulfide linkages ................................ ................................ ................................ ............. 29 1.4 Imine linkages ................................ ................................ ................................ ................... 33 1.5 Acyl hydrazone bonds ................................ ................................ ................................ ...... 37 1.6 Oxime bonds ................................ ................................ ................................ ..................... 42 2 RESEARCH OBJECTIVE ................................ ................................ ................................ ..... 46 3 DYNAMIC MACROMOLECULAR CORE CROSSLINKED STARS WITH REVERSIBLE OXIME LINKS ................................ ................................ ............................. 49 3.1 Overview ................................ ................................ ................................ ........................... 49 3.2 Results and Discussion ................................ ................................ ................................ ..... 51 3.2.1 Synthesis of block copolymers containing reactive keto functionality via RAFT ................................ ................................ ................................ ........................... 52 3.2.2 Functionalization of PDMA 110 b PAB 37 (P3) with model alkoxyamines ............. 55 3.2.3 Core crosslinked star formation ................................ ................................ ............. 60 3.2.4 Disassembly of core crosslinked oxime stars via oxime exchange ........................ 69 3.3 Conclusions ................................ ................................ ................................ ....................... 71 3.4 Experimental Section ................................ ................................ ................................ ........ 72 3.4.1 Materials ................................ ................................ ................................ ................. 72 3.4.2 Instrumentation and Analysis ................................ ................................ ................. 73 3.4.3 Synthesis and Experimental Procedures ................................ ................................ . 74 4 SELF HEALING HYDROGELS WITH TUNABLE GELATION AND DEGRADATION ................................ ................................ ................................ ................... 82

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7 4.1 Overview ................................ ................................ ................................ ........................... 82 4.2 Results and Discussion ................................ ................................ ................................ ..... 85 4. 2.1 Synthesis of keto functional copolymers and formation of supramolecular hydrogel ................................ ................................ ................................ ....................... 86 4.2.2 Hydrogels from P(DMA m stat DAA n ) and difunctional alkoxyamines ............... 90 4.2.3 Gel to sol transition ................................ ................................ ................................ 97 4.2.4 Self healing behavior ................................ ................................ .............................. 98 4.3 Conclusion ................................ ................................ ................................ ...................... 102 4.4 Experimental Section ................................ ................................ ................................ ...... 103 4.4.1 Materials ................................ ................................ ................................ ........ 103 4.4.2 Instrument and analysis ................................ ................................ ................. 103 4.4.3 Synthesis and Experimental Procedures ................................ ....................... 104 5 DYNAMIC DUO: DOUBLY DYNAMIC COVALENT POLYMERS COMPOSED OF OXIME AND OXY NORBORNENE LINKS ................................ ................................ .... 110 5.1 Overview ................................ ................................ ................................ ......................... 110 5.2 Results and Discussion ................................ ................................ ................................ ... 115 5.2.1 Synthesis of AB monomer ................................ ................................ .................... 116 5.2.2 Step growth polymerization of the AB monomer ................................ ................ 117 5.2.3 One pot post polymerization modification by thiol Michael addition reaction ... 119 5.2.3 Polymer degradation by oxime exchange ................................ ............................. 122 5.2.4 Polymer degradation by retro Diels Alder ................................ ........................... 123 5.3 Conclusion ................................ ................................ ................................ ...................... 124 5.4 Experimental Section ................................ ................................ ................................ ...... 125 5.4.1 Materials ................................ ................................ ................................ ............... 125 5.4.2 Instru mentation and analysis ................................ ................................ ................ 125 5.4.3 Synthesis and Experimental Procedures ................................ ............................... 126 6 CONCLUSIONS AND FUTURE DIRECTIONS ................................ ................................ 131 REFERENCE LISTS ................................ ................................ ................................ ................... 133 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ....... 144

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8 LIST OF TABLES Table page 3 1 Results for synthesis of block copolymers with N,N dimethyl acrylamide (DMA), 4 acryloyloxy 2 butanone (AB), and diacetone acrylamide (DAA) ............................... 55 3 2 Results for f unctionalization of PDMA 110 b PAB 37 with m onofunctional a lkoxyamines. ................................ ................................ ................................ .................... 58 3 3 Molecular weight and size results of purified core crosslinked oxime stars obtained from block copolymers of varying composition and [ketone]:[alkoxyamin e] ratios ........ 68 4 1 Copolymers with N,N d imethylacrylamide (DMA) and d iacetone a crylamide (DAA) ... 88 5 1 Step growth polymers with an AB monomer ( 5 ) ................................ ............................ 119

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9 LIST OF FIGURES Figure page 1 1 Formation of reversible crosslinked polymers by nitroxide exchange between two different polymers. ................................ ................................ ................................ ............. 20 1 2 Synthesis and thermal reorganization of alkoxyamine bearing polyesters.. ...................... 21 1 3 Brush copolymer containing thermoreversible alkoxyamine units, prepared by surface initiated Atom Transfer Radical Polymerization (ATRP). ................................ .... 23 1 4 Self healing of Diels Alder network polymer.. ................................ ................................ . 25 1 5 Synthesis of hydrogels by Diels Alder cycloaddition of furan and maleimide derivatives.. ................................ ................................ ................................ ........................ 26 1 6 Formation and disassembly of core crosslinked macromolecular star polymers with reversible Diels Alder linkages. ................................ ................................ ......................... 28 1 7 Formation of reversible disulfide crosslinked star polymer gel and self healing study of the thin film containing disulfide links.. ................................ ................................ ........ 31 1 8 Formation of photoresponsive reorganizable linear polymer and its reorganization upon irradiation with UV light at 365 nm. ................................ ................................ ......... 33 1 9 Formation and dissociation of multi stimuli responsive nanoparticles. ............................ 35 1 10 Formation and dissociation of multi stimuli responsive shell crosslinked (SCL) micelles.. ................................ ................................ ................................ ............................ 36 1 11 Formation of the single chain nanoparticles (SCNPs) via acyl hydrazone formation, and the reversible transformation of the SCNPs to hydrogel. ................................ ........... 39 1 12 Assembly of the PS CH=N NH COO PEG copolymers into a cylindrical morphology and porosity generation.. ................................ ................................ ............... 41 1 13 Liposome liposome fusion and liposome cell fusion to adorn cell surface with reactive functionality. ................................ ................................ ................................ ........ 45 3 1 1 H NMR spectra of acryloyl choride, 4 hydroxy 2 butanone, and 4 acryloyloxy 2 butanone in CDCl3. ................................ ................................ ................................ ........... 53 3 2 Size exclusion chromatography traces of chain extension of PDMA macroCTA with AB and DAA.. ................................ ................................ ................................ .................... 54 3 3 Results from the model reactions of PDMA 110 b PAB 37 ( P3 ) with O allyl hydroxylamine. ................................ ................................ ................................ .................. 56

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10 3 4 Functionalization of PDMA 110 b PAB 37 (P3) with O (tetrahydro 2 H pyran 2 yl)hydroxylamine . ................................ ................................ ................................ .............. 57 3 5 Reaction between PDMA 127 b PDAA 39 and O (tetrahydro 2 H pyran 2 yl)hydroxylamine. ................................ ................................ ................................ .............. 58 3 6 Reaction between RAFT CTA and O (tetrahydropyran 2 H pyran 2 yl)hydroxylamine.. ................................ ................................ ................................ ............. 59 3 7 Core crosslink star formation via an arm first approach. ................................ .................. 6 2 3 8 1 H NMR spectra of core crosslinked oxime stars ( Star 1 , red) and PDMA 127 b PDAA 14 ( P10 , blue) in CDCl 3 . ................................ ................................ ........................... 63 3 9 Core crosslinked star formation in organic solvent (methanol). ................................ ....... 64 3 10 Effect of [ketone]:[alkoxyamine] on star formation kinetics and star size. ....................... 66 3 11 Effect of polymer concentration on star formation kinetics and star size. . ........................ 67 3 12 Star dissociation via oxime exchange with furfuraldehyde.. ................................ ............. 70 3 13 Star dissociation in presence of excess monofunctional alkoxyamine.. ............................ 71 4 1 1 H NMR spectrum of poly( N,N dimethylacrylamide stat diacetone acrylamide) (P(DMA 0.72 stat DAA 0.28 )). ................................ ................................ ............................... 87 4 2 Determination of cloud point (CP). ................................ ................................ ................... 88 4 3 Reversible sol to gel transitions of P(DMA 0.52 stat DAA 0.48 ) ................................ .......... 89 4 4 Reversible sol to gel transitions of P(DMA 0.60 stat DAA 0.40 ). ................................ ......... 90 4 5 Hydrogel formation with P(DMA 0.68 stat DAA 0.32 ) at polymer concentrations 0.2 g/mL and [ketone]:[alkoxyamine] = 1:1 at 25 °C. ................................ ............................. 91 4 6 Dynamic time sweep experiments with P(DMA 0.68 stat DAA 0.32 ) at different concentrations ( i.e ., 0.05 g/mL, 0.1 g/mL, and 0.2 g/mL) and different [ketone]: [alkoxyamine] ratios ( i.e ., 1.5:1 and 1:1) at 1% strain, 10 rad/s, and 25 °C.. ..... 93 4 7 Dynamic time sweep experiments for hydrogel s formed from P(DMA 0.68 stat DAA 0.32 ) at different concentrations ( i.e., 0.05, 0.1, and 0.2 g/mL) and different [ketone]:[alkoxyamine] ratios ( e.g., 1.5:1 and 1:1) (data presented for longer reaction time). ................................ ................................ ................................ ................................ .. 94 4 8 Dynamic time sweep experiments for hydrogels formed from P(DMA 0.6 stat DAA 0.4 ) at different concentrations ( i.e., 0.05, 0.1, and 0.2 g/mL) and different [ketone]:[alkoxyamine] ratios ( e.g., 1.5:1 and 1:1). ................................ ........................... 95

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11 4 9 Rheological experiments of P(DMA m stat DAA n ) copolymers with varying DAA content. ................................ ................................ ................................ ............................... 96 4 10 SEM images of hydrogels prepared with 32, 40, and 48% DAA containing P(DMA m stat DAA n ) copolymers.. ................................ ................................ .................. 96 4 11 Mass swelling ratio of hydrogels with different DAA content of P(DMA m stat DAA n ). ................................ ................................ ................................ ............................... 97 4 12 Gel to sol transition of oxime containing hydrogel prepared at [P(DMA 0.68 stat DAA 0.32 )] = 0.2 g/mL and [ketone]:[alkoxyamine] = 1:1. ................................ ................. 98 4 13 Room temperature healing test of hydrogels prepared with P(DMA 0.68 stat DAA 0.32 ) and [ketone]:alkoxyamine] = 1:1.. ................................ ................................ ................... 100 4 14 Self healing of hydrogels after fracture. ................................ ................................ .......... 101 4 15 Self healing of hydrogels after fracture.. ................................ ................................ ......... 102 5 1 Synthesis of doubly dynamic covalent polymer containing reversible oxime and oxynorbornene links. ................................ ................................ ................................ ........ 115 5 2 Synthesis and characterization of AB monomer. A) Synthesis of AB monomer and B) 1 H NMR spectrum of the purified monomer in DMSO d 6 . ................................ ........ 117 5 3 Polymerization of AB monomer by step growth Diels Alder cycloaddition. ................. 119 5 4 Functionalization of polymer ( P3 ) with 7 mercapto 4 methylcoumarin. ........................ 121 5 5 Polymer degradation by oxime exchange.. ................................ ................................ ...... 122 5 6 Degradation of the polymer by retro Diels Alder reaction.. ................................ ............ 124

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12 LIST OF SCHEMES Schemes page 1 1 Examples of A) reversible non covalent, and B) reversible covalent linkages to prepare dynamic covalent polymeric materials. ................................ ................................ 19 1 2 Example of nitroxide exchange between t wo alkoxyamine molecules. ............................ 20 1 3 Thiol disulfide and disulfide disulfide exchange. ................................ ............................. 30 1 4 The formation and exchange reactions of imines. ................................ ............................. 34 1 5 Acyl hydrazone formation with a carbonyl compound (aldehyde or ketone) and an acyl hydrazine molecule ................................ ................................ ................................ .... 38 1 5 Oxime formation between a carbonyl compound and an alkoxyamine. . ........................... 43 3 1 Synthesis of 4 acryloyloxy 2 butanone (AB) ................................ ................................ ... 52 3 2 Synthesis of poly( N, N dimethylacrylamide) (PDMA) and its subsequent chain extension with diacetone acrylamide (DAA) and 4 acryloxy 2 butanone (AB) to pr epare PDMA b PDAA and PDMA b PAB respectively ................................ ............. 54 3 3 Model reactions of PDMA 110 b PAB 37 (P3) with O allyl hydroxylamine and O (tetrahydro 2 H pyran 2 yl)hydroxylamine ................................ ................................ ..... 56 3 4 Formation of core crosslinked micelles via self assembly and oxime formation ............. 61 3 5 Disassembly of core crosslinked oxime stars by competitive exchange with monofunctional aldehydes/ketones or monofunctional alkoxyamines .............................. 69 4 1 Synthesis of P(DMA m stat DAA n ) ................................ ................................ .................... 87 5 1 Functionalization of P3 with 7 mercapto 4 methylcoumarin. ................................ ......... 120

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13 LIST OF ABBREVIATIONS AB 4 acryloyloxy 2 butanone (AB) AFM Atomic force microscopy AIBN 2,2' azobisisobutyronitrile AO Alkoxyamine ATRP Atom transfer radical polymerization CP Cloud point CTA Chain transfer agent DA Diels Alder DAA Diacetone acrylamide DART Direct analysis in real time DCM Dichloromethane D h Hydrodynamic diameter DLS Dynamic light scattering DMA N,N Dimethylacrylamide DMAc Dimethylacetamide DMF Dimethyl formamide DMP 2 Dodecylsulfanylthiocarbonyl sulfanyl 2 methylpropionic acid DMAP Dimethyl aminopyridine DMSO Dimethyl sulfoxide

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14 DOTAP 1,2 dioleoyl 3 trimethylammonium propane DSDMA bis(2 methacryloyloxyethyl disulfide) DSPES Disulfide containing poly ester EDC chloride 1 (3 dimethylaminopropyl) 3 ethylcarbodiimide, HCl HRMS High resolution mass spectrometry GPC Gel permeation chromatography HQ Hydroquinone LCST Lower critical solution temperature M a n Maleic anhydride MAEBA p (2 metharyloxyethoxy)benzaldehyde M acroCTA Macro chain transfer agent MWCO Molecular weight cut off NMP Nitroxide mediated polymerization NMR Nuclear magnetic resonance OEGMA Oligo ( ethylene glycol methacrylate ) PA prednisolone 21 acetate PBS Phosphate buffer saline PDMA Poly( dimethyl acrylamide) PEG Poly(ethylene glycol) PMMA Poly(methyl methacrylate)

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15 POPC 1 palmitoyl 2 oleoylphosphatidylcholine PPFS poly(2,3,4,5,6 pentafluorostyrene) PS Polystyrene Q Quinone RAFT Reversible addition fragmentation chain transfer SCL Shell crosslinked micelles SCNP Single chain nanoparticle SEC Size exclusion chromatography TAD Terephthaldicarboxaldehyde TCEP tris(2 carboxyethyl)phosphine TEMPO (2,2,6,6 Tetramethylpiperidin 1 yl)oxyl radical TEA Triethylamine TFA Trifluoroaceticacid THF Tetrahydrofuran TMS Tetramethylsilane

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16 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 DYNAMIC COVALENT MACROMOLECULAR ARCHITECTURES COMPOSED OF REVERSIBLE OXIME BONDS By Soma Mukherjee August 2015 Chair: Brent S. Sumerlin Major: Chemistry Interest in reversible macromolecular architectures is burgeoning with a rapidly growing number of applications envisioned in degradable, self healing, and responsive materials. Oxime formation has been utilized as an efficient tool for post polymerization modification, conjugating biomolecules to polymers and hydrogels, and engineering cell surfaces for tissue engineering. We utilized the reversible assembly of amphiphilic keto functional block copolymers prepared by reversible addition fragmentation chain transfer (RAFT) polymerization to construct star polymers via oxime formation. The reversible nature of the oxime linkage was demonstrated by competitive exchange with an excess of a monofunctional alkoxyamine or monofunctional aldehyde under acidic condi tions to result in the dissociation of the stars. We have also utilized the inherent reversibility of oxime bonds to synthesize self healing hydrogels. Keto functional hydrophilic copolymers were crosslinked with difunctional alkoxyamines to obtain hydrog els via oxime formation. A gel to sol transition was induced in the presence of monofunctional alkoxyamines and an acid catalyst. The mechanical properties of this hydrogels were characterized by rheology. Physical gelation of the copolymers was also demon strated above their cloud points. These rehealable and thermo responsive hydrogels may

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17 have potential applications in coatings for medical devices, sensors, scaffolds for biomolecules, and network scaffolds for tissue engineering. Orthogonal multi function alization strategy has emerged in recent years as a powerful technique to obtain novel polymers with diverse composition and topology. Presence of multiple functional groups on a single polymer backbone enables the polymer to respond to external stimuli in different ways . We combined orthogonal oxime and Diels Alder chemistry to yield a linear step growth polymer which could disassemble under two unique sets of conditions. An AB monomer containing furfuryl, maleimido, and latent oxime bonds was synthesized and polymerized by Diels Alder reaction to obtain the polymer that was capable of reversible disassembly by retro Diels Alder and oxime exchange. These remendable polymers offer great potential for designing sensors and consti tutionally dynamic polymers. While the research described here demonstrates the benefit of having reversible links in the polymers to construct reversible branched architecture, similar strategy can be used to design more complex adaptive polymeric architectures.

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18 CHAPTER 1 INTRODUCTION Polymers have traditionally served as building blocks to prepare materials with enhanced mechanical, thermal, and electronic properties for a specific application. Incorporation of covalent crosslinking in the polymer matrix was used primarily to prevent degradation of the been a growing interest in preparing selectively tailored polymeric assemblies and interfaces having dynamic or reversible bonds, which can be broken and reformed by exposure to an appropriate stimulus and can produce a macroscopic response. 1 , 2 Dynamic bonds encompass two broad ca tegories: supramolecular or non covalent interactions, and reversible covalent or dynamic covalent interactions. Hydrogen bonding, 3 4 electrostatic interaction (ion ion and ion dipole), 5 and metal ligand coordination 6 , 7 are some o f the non covalent interactions that have been utilized to construct reversible macromolecular architectures. 7 , 8 Non covalent int eractions are the basis of supramolecular chemistry, which emerged spectacularly in the area of molecular recognition and recognition based self organization in the past few decades. 1 Dr. Donald J. Cram, Dr. Jean Marie Lehn, and Dr. Charles J. Pederson shared the Nobel Prize in chemistry in 1987 for their pioneering work in the area of molecular recognition, specifically for the discovery and investigation of cryptands, a family of syn thetic bi and polycyclic multidentate ligands for a variety of cations. 1 , 5 Their seminal works in the field of of reversible covalent bonds carried ou t under thermodynamic equilibrium conditions. 7 Some commonly explored dynamic covalent bonds are thermally cleavable alkoxyamine, 9 , 10 furan A portion of this ch apter wa s submitted to Wiley. Copyright 2015 Wiley.

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19 maleimide Diels Alder linkage, 11 , 12 es ter, 13 disulfide, 14 , 15 , 16 , 17 boronic ester, 18 imine, 19 oxime 20 23 and hydrazone ( Scheme 1 1 ). 7 Incorporation of dy namic bonds in macromolecules allows reversible disassembly and reassembly of the molecule by changes in certain environmental conditions, such as in temperature, pH, light intensity, magnetic field, and redox potential. The adaptable nature of the reversi ble linkages combined with their stimuli responsive behavior offer numerous applications in the areas of self healing, shape memory polymers, molecular receptors, catalysis, responsive biointerfaces, sensors, and actuators. 2 Scheme 1 1 . Examples of A) reversible non covalent, and B) reversible covalent linkages to prepare dynamic covalent polymeric materials. This chapter describes examples of polymeric materials that contain some commonly employed covalent bonds that are reversible under specific stimuli and their application.

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20 1.1 Alkoxyamine bonds The fundamental radical exchange reaction of 2,2,6,6 tetramethylpiperidine 1 oxy (TEMPO), as a radical scavenger during a free radical polymerization, led to the discovery of nitroxide mediated polymerization (NMP), in which TEMPO acts as the reacti ve initiating radical and the stable mediating radical. 24 , 25 Scheme 1 2. Example of nitroxide exchange between two alkoxyamine molecules. Figure 1 1 . Formation of reversible crosslinked polymers by nitroxide exchange between two different polymers. A) The mixture of polymers a and b in anisole was heated to 100 °C, resulting in a crosslinked gel. B) Pictures of the mixture of polymer a and b before (left) and after heating (right). Adapted from Y. Higaki, H. Otsuka and A. Takahara, Macromolecules , 2 006, 39 , 2121 2125. Copyright 2006 American Chemical Society.

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21 The nitroxide radicals are stable at room temperatures but ~90 °C and above they undergo exchange with another nitroxides or H abstraction. 25 The exchange reaction be tween different nitroxides (Scheme 1 2) in two alkoxyamine bearing polymers was first reported in 1996 by Hawker et al. 9 Figure 1 2 . Synthesis and thermal reorganization of alkoxyamine bearing polyesters. A) Polycondensation of a diol and adipoyl chloride to yield alkoxyamine bearing polyester, and degradation of the polymer by nitroxide exchange upon heating to 100°C. B) Nitroxide exchange between alkoxyamine polyesters (polymers a and b), and SEC traces (PS standards, THF) of the mixture of polymers a and b before heating (left) and the resultant polymer after heating (right) in anisole at 100 °C. Adapted from, H. Otsuka, K. Aotani, Y. Higaki, Y. Amamoto and A. Takahara , Macromolecules , 2007, 40 , 1429 1434. Copyright 2006 American Chemical Society. Later Turro et al. , 26 reported that the terminal nitroxide of the polystyrene prepared by NMP was capable of undergoing exchange with an excess amount of other nitroxides. The authors functionalized the terminus of a polymeric alkoxyamine with a chromophore via

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22 nitroxide exchan ge with TEMPO terminated polystyrene and a chromophore bearing nitroxide. In an early attempt to synthesize a dynamic covalent polymers by the nitroxide exchange process, Takahara et al., mixed two different alkoxyamine containing poly(methyl methacrylate)s and heated the reaction mixture to 100 °C to afford crosslinked polymeric gel (Fi g. 1 1 ). 27 The de crosslinking of the ge l was carried out by heating the gel in the presence of small alkoxyamine molecules like TEMPO or styryl TEMPO. The same group has taken this concept further to prepare linear polymers having the alkoxyamine functionality in the polymer backbone by step gr owth polymerization of a nitroxide containing diol and adipoyl chlor ide in dichloromethane (Fig. 1 2 A). 28 The authors u sed nitroxide exchange to tune the molecular weights of the polymers by simply mixing (in anisole) equal quantities of two polymers having different molecular weights, and upon heating the mixture to 100 °C for 12 h. A single peak for the resultant mixture was obtained in the SEC trace, confirming that the nitroxide exchange led to reorgani zation of the polymers (Fig. 1 2 B). There are several other examples of incorporation of thermoreversible alkoxyamines in more complex macromolecular systems, such as bru sh copolymers to reversibly alternate silica surface properties, 29 , 30 core crosslink ed star polymers, 31 , 32 , 33 and cyclic polymers. 34 In a recent report, Otsuka and Takahara et al. , synthesized poly(methyl methacrylate) b ased brush copolymers containing exchangeable alkoxyamine units by surface initiated atom transfer radical polymerization (ATRP). 30 The authors were able to reduce the surface free energy of the brush copolymer by grafting poly(2,3,4,5,6 pentafluorostyrene) (PPFS) (prepared by nitroxide mediated polymerization) onto the brush copolymer by nitroxide exchange (Fig. 1 3 ). The de grafting of the PPFS from the brush surface was carried out with excess small alkoxyamine molecule via nitroxide exchange. Successful de grafting was confirmed by contact angle

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23 measurements and X ray photoelectron spectroscopy. This is an excellent example of how exchangeability of dynamic bonds could lead to smart surfaces and reversible surface property control. Continuing progress in this area of dynamic covalent chemistry is expected to create a new class of dynamic soft materials. Figure 1 3 . Brush copolymer containing thermoreversible alkoxyamine units, prepared by surface initiated Atom Transfer Radical Polymerization (ATRP). Poly(2,3,4,5,6 pentafluorostyrene) (PPFS) was grafted to the brush copolymer by nitroxide exchange at 100 °C and de grafting was achieved by nitroxide exchange with excess small alkoxyamine mol ecules. Adapted from T. Sato, Y. Amamoto, H. Yamaguchi, T. Ohishi, A. Takahara and H. Otsuka, Polym. Chem. , 2012, 3 , 3077 3083, with permission from The Royal Society of Chemistry. 1.2 Diels Alder linkages Diels Alder (DA) cycloaddition is another powerful synthetic technique to prepare mendable polymeric materials. The reaction does not require a catalyst, and the equilibrium can

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24 be tuned to favor the forward or reverse direction by changing the reaction temperature. The Diels Alder reaction involves [2+4] cycloaddition of an electron rich diene and an electron poor dienophile. Lehn and coworkers reported one of the first examples of using Diels Alder reaction between fulvene derivatives and cyanolefins to generate a dynamic library of Diels Alder adducts t hat can exchange with each other reversibly between 25 and 50 °C. 35 Due to the inherent reversibility of some Diels Alder adducts, for example furan maleimide adducts, this reaction has been widely used to prepare smart healable materials. The first Diels Alder self healing systems was devised by Wudl et al., who employed a tetra furan molecule and a tri m aleimide compound as precursors to construct a crosslinked macromolecular network (Fig. 1 4 A). 36 The authors prepared a thin film by casting a solution of the furan and maleimide derivatives, and heating the film to undergo Diels Alder reaction. The specimen was used for repeatable self healing experiments by a compact tension test. Healing was obser ved by SEM, as a result of Diels Alder reaction between de bonded furan and maleimide moieties at 120 150 °C, with an average men ding efficiency of 57% (Fig. 1 4 B). These results were remarkable, given that the healing was achieved without any additional c omponent, and the mechanical properties (compression strength, compression modulus, and flexural strength) of the network film were comparable to those of a similar epoxy system ( e.g ., tensile strength as high as 68 MPa). In 2003, the authors addressed the limitation of healing efficiency by using a lower melting maleimide precursor to afford network formation in the bulk. 37 In their specimens for healing test, a drilled hole was introduced before cutting the sample to limit the crack propagation during the test and also to ensure that the sample remained in one piece after fracture. Indeed, their new stra tegies led to higher healing efficiencies of 80% on average, and the healing by Diels Alder and retro -

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25 Diels Alder was repeatable multiple times, as evidenced by 78% healing efficiency in the second cycle . Figure 1 4 . Self healing of Diels Alder network polymer. A) Synthesis of Diels Alder network with tetra furan and tri maleimide derivatives. B) Mending efficiency obtained by fracture toughness test of a compact tension test specimen of the network. C) Image of the damaged specimen before thermal treatm ent. D) Image of the damaged specimen after thermal treatment. E) SEM image of the healed sample: left side was as healed surface and right side was the scraped surface. F). Enlarged image of the boxed area in E. Adapted from, X. Chen, M. A. Dam, K. Ono, A . Mal, H. Shen, S. R. Nutt, K. Sheran and F. Wudl, Science , 2002, 295 , 1698 1702, with permission from The American Association for the Advancement of Science .

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26 Figure 1 5 . Synthesis of hydrogels by Diels Alder cycloaddition of furan and maleimide derivatives. A) Thiol Michael addition reaction between a multifunctional maleimide (in excess) and thiol PEG macromere. B) Release of the furan containing peptide sequence (furan RGDS) by retro Diels Alder reaction. C) Schematic representation of the thio l Michael addition and Diels Alder reaction employed to create the hydrogel. D) Representation of the overall network obtained by thiol Michael addition and Diels Alder reaction. E) Release study of furan RGDS by retro Diels Alder reaction initially, and F ) over a longer period of time, at 37 °C (red circle), 60 °C (blue square), and 80 °C (orange triangle). Adapted from, K. C. Koehler, K. S. Anseth and C. N. Bowman, Biomacromolecules , 2013, 14 , 538 547. Copyright 2013 American Chemical Society. Knowledge of the inherently reversible nature of Diels Alder reaction led to many interesting works such as thermo responsive organic inorganic hybrid optical polymers, 38 modular polymeric color switches employing hetero Diels Alder reaction, 39 rewritable surface

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27 coating, 40 and polymeric thin film for scanned probed data storage and lithography applications. 41 Recently both Bowman et al ., 42 and Goepferich et al. , 43 demonstrated that poly(ethylene glycol) (PEG) based hydrogels having furan maleimide lin kages could degrade at human body temperatures by the retro Diels Alder reaction. Bowman et al. , utilized a carboxyfluorescein labelled 3 furan RGDS peptide sequence (furan RGDS) to create hydrogels by Diels Alder reaction with the maleimide units in a PEG b ased thiol ene network (Fig. 1 5 A D). The thiol ene Michael addition was performed with excess maleimide compound, so that unreacted maleimide sites remained for subsequent Diels Alder reaction with furan RGDS. Creating the network with both permanent (t hiol ene) and reversible or labile (furan maleimide link) bonds allowed control of the release rate of furan RGDS by simply varying the available maleimide units for Diels Alder reaction. The authors introduced cysteine, a monothiol, for capping the availa ble maleimide functionality by thiol ene reaction, to control the number of available maleimide groups, while maintaining a cons tant crosslink density (Fig. 1 5 E, F). Exposing the hydrogel to different temperatures ( 37 °C, 60 °C, and 80 °C ) resulted in a t emperature dependent release via Diels Alder mechanism (Fig. 1 5 E, F). This observation, along with their diffusion control experiments, suggested that the release of furan RGDS occurred via retro Diels Alder reaction. Recently our group, employed the Diels Alder reaction to prepare core crosslinked star polymers by an arm first approach, 44 and segmented hyperbranched polymers by self condensation vinyl polymerization. 45 For preparation of the core crosslinked star polymers, well defined block copolymers of styrene (s) and maleic anhydride (MAn), [P(S alt MAn ) b PS] were prepared via a one pot cascade approach by reversible addition fragmentation chain transfer (RAFT) polymerization.

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28 Figure 1 6 . Formation and disassembly of core crosslinked macromolecular star polymers with reversible Diels Alder linkages. A) Core crosslinked star polymers were prepared by Diels Alder reaction of the pendent furan groups of a block copolymer [PS b P(S alt Man)] ( M n = 12.8 kg/mol) and a bis maleimide. The star polymers were disassembled by retro Diels Alder reaction of the f uran maleimide adducts in the core. B) Kinetics of star formation with furan functionalized block copolymers at different polymer concentrations (30, 50, and 100 mg/mL). The % arm calculated from the area under the SEC trace of reaction solution using: % arm = Area arm /(Area arm +Area star ) C) TEM images of the star polymers at 15 k (scale bar = 100 nm) and 25 k (scale bar = 50 nm). D) SEC traces of the unimers (solid lines) and the star polymers (dashed lines) obtained during the formation and dissociation of the star polymers. E) Solution size measured by DLS during the formation and dissociation of the star polymers over multiple cycles. Adapted from, A. P. Bapat, J. G. Ray, D. A. Savin, E. A. Hoff, D. L. Patton and B. S. Sumerlin, Polym. Chem. , 2012, 3 , 311 2 3120, with the permission from The Royal Society of Chemistry. The maleic anhydride rings were opened with furfuryl amine to yield the functionalized polymer with the pendent furan groups, which were subsequently crosslinked with a bis maleimide compound by Diels Alder reaction to yield the core crosslinked star polymers (Fig. 1 -

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29 6 A, C). Due to the reversible nature of the furan maleimide links, the core crosslinked polymers could be degraded and reformed over multiple cycles as demonstrated by the SEC and dynamic light scattering (D LS) size distributions (Fig. 1 6 D, E). Interestingly the block copolymers could be self assembled in a selective solvent (toluene) into micelles, and a dramatic increase in the rate of crosslinking was observed when the pendent furan molecules in the core of the micelles were allowed to react with bis maleimide molecules. The strategy of preorientation of block copolymers in a selective solvent before crosslinking was also adopted by others to enhance the rate of star formation, as well as the yield, and size dispersity of the resultant star polymers. 20 , 46 1.3 Disulfide linkages Disulfides are commonly found in natural syst ems, and are known for their reversibility and exchange behavior. Disulfide bonds play an important role in biological systems; for example, in the develoment of the secondary structure of proteins. 47 The disulfide links between the cysteine residues in proteins, frequently undergo disulfide exchange to attain the lo west energy folded structure. 48 In most cases, the thiol disulfide exchange process proceeds through the base catalyzed pathway by generating thiolate anion (Fig. 1 9). 49 The disulfide exchange, on the other hand, could occur via a radical pathway through generating a sulfenyl radical by photoirradiation. 50 Considerable work has been devoted to prepare reversible polymeric material using both route. 1 , 51 Matyjaszewski et al ., prepared self healing polymeric materials by employing a redox responsive thiol disu l fide exchange reaction. 52 , 53 In one of their reports the authors chose a multiarm star polymer precursor containing thiol groups, considering the advantage of achieving the low viscosity (for the flow properties necessary to induce healing) of the branched star polymers compared to their linear counterparts. 52

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30 Scheme 1 3 . Thiol disulfide and disulfide disulfide exchange. The star polymers were synthesized by ATRP via a core first approach, and subsequently the star polymers were chain extended with a divinyl compound, bis(2 methacryloyloxyethyl disulfide) (DSDMA) to induce macroscopic crosslinking which resulted in gel formation. 16 The disulf ide links in the crosslinked gel were cleaved by reduction with tri butyl phosphine, and the thiol functionalized polymer was deposited on a silicon wafer to form a thin film. The thiol groups in the thin film were oxidized with either FeCl 3 or I 2 to obtain a disulfide crosslinked film, which was insoluble in chloroform and THF, and the disulfide formation was confirmed by Raman spectroscopy. For control experiments, a thin film containing permanent crosslink was prepared by reacting the free thiol gro ups with 1,2,7,8 diepoxyoctane u nder basic conditions (Fig. 1 7 A). The self healing study was performed by damaging the thin film with an AFM tip or a penetrating cut by a microscratching method. The healing process was monitored by AFM a nd optical microsc opy (Fig. 1 7 B, C). The damaged film self healed at room temperature with no external stimulus. The AFM study showed that damage at the center position was not fully healed after 70 min, but damage at the edge was completely healed after 70 min, suggesting that the healing was initiated from the bottom of the cut and the edge of the cut due to the accessibility of cut surfaces to each other.

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31 Figure 1 7 . Formation of reversible disulfide crosslinked star polymer gel and self healing study of the thin fil m containing disulfide links. A) Reduction of a disulfide containing multiarm star polymer to obtain thiol functionalized star polymer for thin film preparation. The thiols in the thin film were oxidized to disulfide with I 2 or FeCl 3 . Another thin film was prepared with permanent crosslink for control experiment. B) Healing response of the thin film accelerated by AFM tip stroke with contact mode scanning: 1) original cut, 2) after the five series of strokes at the position shown by the red arrow, 3) after the second series of strokes marked by the blue arrow, and 4) HarmoniX mode modulus map in logarithmic scale. The brightest regions in the the bottom of the deep cut. C) Op tical microscopy images of self healing response of the thin film after different time intervals for a penetrating cut. Adapted from, J. A. Yoon, J. Kamada, K. Koynov, J. Mohin, R. Nicola, Y. Zhang, A. C. Balazs, T. Kowalewski and K. Matyjaszewski, Macrom olecules , 2012, 45 , 142 149. Copyright 2012 American Chemical Society. To bring about the contact of the damaged areas, the AFM tip was used to stroke the damaged surface in the direction perpendicular to the damaged area (Fig. 1 7 B). Immediate

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32 smoothenin tapping mode) of the healed area was found to be comparable to the modulus of the undamaged area. The healing capacity of the thin film was found to be dependent on the initial film t hickness and the width of the damage. A thicker penetrating cut resulted in rapid healing as observed by optical microscopy (Fig. 1 7 C). No self healing was observed in the control film sample due to the absence of any exchangeable links to cause healing. This study showed that reversible branched polymeric materials hold tremendous promise for self healing applications, due to their low viscosity and accessibility to higher functional groups. Takahara and coworkers reported the first example of a photoresp onsive reorganizable linear polymer utilizing the radical exchange reaction between disulfides (disulfide metathesis). 49 The authors synthesized a disulfide containing polyester (DSPES) by polycondensation of adipoyl chloride and 2 hydroxyethyldisulfide in dichloromethane in the presence of pyridine. Due to the presence of the disulfide links in the polymer backbone, the polymer was susceptible to disulfide exchange by UV irradiation. To test the hypothesis, two polyesters having different molecular weights (h DSPES: M n = 60 400, M w / M n = 1.06, l DSPES: M n = 8700, M w / M n = 1.54) were synthesized and mixed in equal quantities in solution to cast a film. The polymer film was irradiated with UV light (365 nm), and after 60 min the SEC traces of the two polymers (blue traces) were f used in one (red trace), confirming that the two polymers were reorganized via disulfide exchange (Fig. 1 8 ).

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33 Figure 1 8 . Formation of photoresponsive reorganizable linear polymer and its reorganization upon irradiation with UV light at 365 nm. The SEC traces of the two different polymers (blue) were fused into one peak (red) due to the exchange between the disulfide links in the polymers. Adapted from, H. Otsuka, S. Nagano, Y. Kobashi, T. Maeda and A. Takahara, Chem. Commun. , 2010, 46 , 1150 1152, with permission from The Royal Society of Chemistry. 1.4 Imine linkages The condensation reaction of an aldehyde or ketone with a primary amine results in an imine compound, often referred to as Schiff base ( Scheme 1 4 ). 54 The reaction equilibrium of imine formation is sensitive to environmental conditions such as pH, temperature, and reactant or product concentration. By careful selection of the electronic structures of the starting materials, it is possible to control the optimum pH at which the reaction occurs. 55 Due to the facile reversibility of the imines, they are capable of exchanging partn ers in a multi component mixture

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34 of imines via transimination, and this exchange reaction has been utilized extensively in creating libraries of constitutionally dynamic compounds ( Scheme 1 4 ). 56 , 57 Scheme 1 4. The formation and exchange reactions of imines. Lehn and coworkers studied the equilibrium constant of imine formation of different aldehyde and amine compounds in aqueous media and found that the equilibrium constant was higher for basic amines. 54 The same group also combined metal ligand coordination with imine formation to construct a morphological switch involving metal ion assisted interconversion between a polymer and macrocycles. 58 This example demonstrated that the dynamic exchange of reversible bonds could be a useful tool to induce morphological changes in macromolecular architecture. Dynamic exchange of imines has also been widely used in preparing stimuli responsive dynamic co valent polymeric materials. Recently, Fulton and coworker constructed core crosslinked star polymers and nanogel assemblies by the reaction of an aldehyde functional block copolymer with an amine containing block copolymer. 59 The star polymers were able to dissociate into unimers reversibly via transamination with a small amine molecule in the presence of an acid catalyst. In ano ther interesting work, Fulton et al. , employed two orthogonal dynamic covalent bonds, imine and disulfide, to construct double stimuli re sponsive nanoparticles (Fig. 1 9 ). 60 The crosslink density in the core can be modulated by either maintaining pH 5.5 to cause hydrolysis of imine bonds, or by reducing the disulfide bonds with tris(2 carboxyethyl)phosphine (TCEP). With the dissocia tion of the imine and disulfide

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35 crosslinks, the nanoparticles disassembled, as indicated by the shift of the SEC trace to higher elution time for N6 (Fig. 1 9 C). Figure 1 9 . Formation and dissociation of multi stimuli responsive nanoparticles. A) An al dehyde functional block copolymer ( P1 ) was reacted with an amine containing block copolymer ( P2b ) to prepare the nanoparticle in aqueous media. Both the polymers contained disulfide bonds. Nile red was encapsulated in the nanoparticles. The release of Nile red was afforded by simultaneous application of two different stimuli, low pH (5.5) and a reducing environment (TCEP). B) Normalized SEC refractive index traces of the polymers ( P1 and P2 ) and the resulting nanoparticle ( N1 ). C) Normalized SEC refractive index traces of nanoparticles N2 and N3 . D) Normalized SEC refractive index traces of the nanoparticle N5 and degraded unimers N6 . E) Release kinetics of Nile red over time by fluorescence emission spectroscopy. Adapted from, A. W. Jackson and D. A. Fulton, Macromolecules , 2012, 45 , 2699 2708. Copyright 2012 American Chemical Society. Nile Red was encapsulated in the nanoparticle ( N5 ), and the release of the Nile red was afforded by simultaneous application of acidic pH (5.5) a nd reducing conditions (Fig. 1 9 A). The release kinetics of Nile Red was monitored with fluorescence spectroscopy (Fig. 1 9 C). This is an excellent example of a multifunctional system in which only specific functional groups

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36 respond to an external stimulus while others remain unaffected. This specificity of response depending on the environmental conditions could be useful for designing sensors and receptors. Figure 1 1 0 . Formation and dissociation of multi stimuli responsive shell crosslinked (SCL) mice lles. A) Micelle formation with triblock copolymers in aqueous media at pH 9 below the LCST of PNIPAM, and the shell crosslinking of the micelles by reacting them with a di aldehyde compound. Degradation of the shell crosslinked micelle at acidic pH (5.5) and recrosslinking of the dissociated unimers at pH 9 and elevated temperature (50 °C). B) Dynamic light scattering size distribution of the tri block copolymer, shell crosslinked micelles, dissociated unimers, and re crosslinked micelles under various con ditions. C) Release kinetics of PA over time by UV Vis spectroscopy. Adapted from, X. Xu, J. D. Flores and C. L. McCormick, Macromolecules , 2011, 44 , 1327 1334. Copyright 2011 American Chemical Society. A common approach to prepare nanoparticle for drug de livery applications is use of a crosslinked micelle as the drug carrier. Release of the drug is achieved by dissociation of the micelles only under a specific stimulus that is present in the targeted cellular environment.

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37 McCormick and coworkers utilized t his concept to prepare shell crosslinked micelles using a triblock copolymer containing amine groups in the middle block. 61 The authors synthesized poly(ethyleneglycol) b poly(3 aminopropyl methacrylamide) b poly( N isopropylacrylamide) (PEG b PAPMA b PNIPAM) via RAFT polymerization (Fig. 1 1 0 A). The triblock copolymer was shown to self assemble at higher temperature (50 °C, pH 9) as the PNIPAM blocks collapsed due to volume phase transition above the lower critical solution temperature (Fig. 1 10 C). The shell, containing the PAPMA and PEG blocks, was crosslinked with terephthaldicarboxaldehyde (TAD) via imine formation. A model hydrophobic anti inflammatory drug, prednisolone 21 acetate (PA), was encapsulated in the core of the shell crosslinked micelles (Fig. 1 10 B). A di aldehyde was used to crosslin k the shell of the micelles via imine formation. The release of the hydrophobic drug was afforded by lowering the temperature to 25 °C, so that the PNIPAM core became hydrophilic, and lowering the pH (5.5) to hydrolyze the imine bonds in the micelle core. The release of the drug was monitored by UV Vis spectroscopy. The authors observed an enhanced rate of release of the hydrophobic drug at lower pH (5) irrespective of the temperature, confirming the dissociation of the shell crosslinked micelles was govern ed in large part by imine hydrolysis (Fig. 1 1 0 D). The encouraging results of this study led to further development in the area of stimuli responsive polymeric nanoparticle for various applications. 55 1.5 Acyl hydrazone bonds Acyl hydrazone ( C=N NH (C=O) ) is another dynamic covalent bond which has been widely used to construct dynamic combinatorial libraries and reversible poly meric materials. 5 , 56 Acyl hydrazones are formed by the reaction of a carbonyl compound and an acyl hydrazine molecule (R 3 CONHNH 2 , Scheme1 5 ). Due to the reduced nucleophilicity of the NH 2 group in the acyl hydrazine compound, the reaction often needs an acid catalyst, and the optimum rates of

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38 hydrolysis and exchange ocuurs at pH 4.4. Lehn and coworkers demonstrated the utility of hydrazone exchange using trifluoroacetic acid (TFA) as a cata lyst to prepare dynamic covalent noncovalent interactions ( e.g. , hydrogen bonding) able to fold into well defined conformations, sheets. 62 , 63 Scheme 1 5. Acyl hydrazone formation with a carbonyl compound (aldehyde or ketone) and an acyl hydrazine molecule In a seminal work, Fulton and coworkers prepared a polymer scaffolded dynamic co mbinatorial library of acyl hydrazone functional polymers, and the authors showed that hydrazone exchange took place at acidic pH. 64 This polymer bas ed dynamic combinatorial library has generated considerable interest in the design of molecules that can mimic natural biomolecules, and polymer based receptors for small molecules or proteins. Later the same group harnessed the reversibility of acyl hydra zone bonds along with the thermoresponsive behavior of a polymer to prepare single chain nanoparticles (SCNPs) that can undergo reversible macroscopic transformation into a hydrogel network. 65 Oligoethylene glycol methacrylate (OEGMA 300 ) and p (2 metharyloxyethoxy)benzaldehyde (MAEBA) having similar molecular weights ( M w = 37 61 kDa) were copolymerized via RAFT polymerization (Fig. 1 11 ). The linear polymers displayed thermoresponsive behavior due to their lower critical solution temperature (LCST) ranging from 32 52 °C, depending on the OEGMA content in the polymers. The pendent aldehyde groups of the copolymers (polymer concentration = 0.1 1 wt % in AcOH/AcONH 4

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39 buffer) were intramolecularly crosslinked at pH 4.5 with a di acyl hydrazine (or hydrazide) crosslinker by hydrazone formation. As the crosslinking progressed the intramolecularly crosslinked polymer chai ns collapsed leading to lower hydrodynamic volume and the SEC trace of the copolymers were shifted t o higher elution time (Fig. 1 11 B). Figure 1 1 1 . Formation of the single chain nanoparticles (SCNPs) via acyl hydrazone formation, and the reversible tra nsformation of the SCNPs to hydrogel. A) Aldehyde functional polymers ( P1 P4 ) were intramolecularly crosslinked with a dihydrazide to yield single chain nanoparticles (SCNPs) ( NP1 NP4 ) at pH 4.5. Upon heating the SCNP solution above the LCST of the corresp onding polymer, hydrogel formation took place. The reverse transformation of the hydrogel to the SCNPs (NP1' NP4') was carried out by cooling to 25 °C. B) Dynamic light scattering size distribution of the tri block copolymer, shell crosslinked micelles, d issociated unimers, and re crosslinked micelles under various conditions. C) Normalized SEC refractive index traces of aldehyde functional polymer ( P2 , 47.1 kg/mol), SCNP prepared from P2 ( NP2 ), and SCNPs after the hydrogel (prepared from NP2 ) transformed to sol and equilibrated ( NP2' ). Adopted from, D. E. Whitaker, C. S. Mahon and D. A. Fulton, Angew. Chem. Int. Ed. , 2013, 52 , 956 959, Copyright 2013 Wiley. Upon heating the crosslinked polymer solution above the LCST, the crosslinked single chain nanoparti cles aggregated and the highly localized nanoparticle concentration resulted in

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40 intermolecular reorganization of the acyl hydrazone crosslinks at the molecular level. As a result, the intramolecular crosslinks were replaced by the interchain crosslinks, an d the supramolecular interaction between the reorganized polymer chains induced hydrogel formation. The reverse transformation of the hydrogel to the SCNPs was triggered by cooling the hydrogel to 25 °C and equilibrated to the sol state af ter 0.5 20 days ( Fig. 1 11 ). Their hypothesis of the reversible transformation was supported by the fact that with decrease in amount of crosslinking in the nanoparticles, the time required to equilibrate to the corresponding SCNPs also decreased. Khan et al. combined the facile reversibility of acyl hydrazone linkages and the self assembly of block copolymers to prepare a nanoporus membrane. 66 Nanoporus materials are useful in the areas of separation, water purification, catalysis, and fuel cell design. 67 The authors polymerized styrene (s) via atom transfer radical polymerization (ATRP) using a PEG macroinitiator containing a hydrazone linkage to yield a block copolymer PEG b PS. The block copolymer so lution in benzene was spin coated onto a silicon surface to yield a 100 nm film. Solvent annealing and exposure to humidity (90%) resulted in a film in which the self organized and hexagonal packed arrays of cylindrical PEG domains (25 nm) were dispersed i n a continuous PS phase with a lon g range lateral order (Fig. 1 12 ). The film was immersed into a water methanol mixture (2:1 vol/vol) or alternatively into an acidic water bath (pH 4) to generate porosity by hydrolyzing the acyl hydrazone bonds connecting the styrene and ethylene glycol blocks, and simultaneously dissolving the PEG hydrazide block. To confirm the removal of the PEG hydrazide, the recovered porous thin film was exposed to the vapors of methylamine in methanol, and imine formation with the a ldehyde groups in the PS matrix was confirmed by 1 H NMR spectroscopy. The mild conditions employed to prepare porous thin films offers great potential to design highly ordered functional nanoporous materials.

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41 Acyl hydrazone bonds were also utilized by other groups to form self healing polymeric materials by others. Chen et al. did a detailed investigation of the reversible sol to gel transition and self healing properties of organo /hydrogels having acyl hydraz one bonds, and the authors reported the gelation kinetics and mechanical properties of acyl hydrazone containing organogels. 68 70 Figure 1 12 . Assembly of the PS CH=N NH COO PEG copolymers into a cylindrical morphology and porosity generation. A) Copolymer of ethylene glycol and styrene. B) The minor PEG block forms a cylinder in the continuous matrix of PS upon solvent annealing and humidity exposure. Subsequent removal of the PEG CO NHNH 2 by immersing the film into acidic water or water methanol mixture resulted in a PS based porous matrix. The free aldehyde groups on the PS matrix were further functionalized wi th methylamine. C) AFM height, and D) phase images (1 mm x 1 mm) of the polymer thin film on a silicon wafer after solvent annealing. Inset shows the corresponding Fourier transform. E) TEM (scale bar = 200 nm), and F) SEM (scale bar = 100 nm) images of th e porous thin film. Adopted from, J. Rao and A. Khan, Polym. Chem. , 2013, 4 , 2691 2695. Copyright 2013 Wiley.

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42 The authors later combined the reversibility of acyl hydrazone bonds and disulfide linkages to prepare hydrogels capable of sol to gel transition upon exposure of two different stimuli. 68 The requirement of mild acidic condition for hydrazone exchange offers tremendous possibilities for design of self mendable polymeric m aterials. 1.6 Oxime bonds Oxime ligation has proven promising in the preparation of functionalized polymer scaffolds, surface modification of proteins, and cell surface engineering, due to the high reaction efficiency, benign side product ( i.e. , water), and ambient reaction conditions. 22 , 71 77 The condensation reaction between an aldehyde or ketone and an alkoxyamine yields either an aldoxime or a ketoximes. Oxime formation was utilized by Maynard et al. , to p repare micropatterned alkoxyamine containing polymer films to conjugate an ketoamide protein by oxime formation using a photolithographic technique. 78 The authors also demonstrated immobilization of site selectively modified protein onto a gold surface by bio orthogonal click react ions, including oxime formation. 79 Francis et al. , employed oxime ligation to construct thermoresponsive protein polymer conjugates, 80 and protein functional hydrogels, 71 and to functionalize a polymer coating with an an ti freeze protein for devices that operate at low temperature without ice build up. 72

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43 Scheme 1 5 . Oxime formation between a carbonyl compound and an alkoxyamine. Dynamic oxime exchange (acid catalyzed) between oxime and alkoxyamine o r carbonyl compounds. Reversibility of oxime formation has received less attention, especially in the realm of polymer chemistry, possibly due to the greater hydrolytic stability of oximes compared to imines or hydrazones. 23 In fact, the enhanced hydrolytic stability of oxime renders the possibility of utilizing oxime formation to create functional materials needing superior aqueous stability. In contrast to hydrolytic stability, oxime bonds are suseptable to dynamic exchange with either an alkoxyamine molecule or a carbonyl compound in presence of catalytic acid ( Scheme 1 5) . Eliseev and coworkers have studied the reaction kinetics and mechanism of imine/oxime exchange between different aliphatic or aryl alkoxyamine molecules , and the authors suggested the use of oxime exchange to create a library of oxime compounds capable of constitutional rearrangement under specific condition.. 22 Raines and coworkers reported a detailed study of the hydrolysis mechanism and the effects of the electronic structures of the carbonyl compounds and the alkoxyamines on the rates of hydrolysis and exchange 23 Recently the redox responsive nature of oxime bonds was harnessed by Yousuf et al. , who employed a liposome based methodology to functionalize cell surfaces with dynamic oxime links for application in stem cell

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44 differentiation and tissue engineering. 21 , 81 The authors synthesized a hydroquinone (HQ) and aminoxy alkane or alkoxyamine (AO), and incorporated the m into different liposomes. 21 The hydroquinone was transformed into an active quinone (Q) state by applying mild oxidation potential (either chemically or electrochemically) and was mixed with the AO containing liposomes to react with the alko xyamine to generate oxime via liposome liposome fusion, as observed by the TEM images (Fig. 1 1 2 A, B). This liposome liposome fusion methodology was employed to tailor cell surfaces via oxime formation. An HQ alkane was mixed with 1 palmitoyl 2 oleoylphosphatidylcholine (POPC) and DOTAP (a cationic lipid, 1,2 dioleoyl 3 trimethylammonium propane) in a 10:88:2 ratio, and the AO was mixed with POPC and DOTAP in a 5:93:2 ratio. After their addition to the cells (Swiss 3T3T fibroblast (Fbs)) in culture, the liposome fused and delivered the chemical functionality on the cell surfaces. The oxime bonds were selectively cleaved by applying mild redox potential ( 100 Mv, 10 S, PBS, pH = 7.4) with the working electrod, resulting in the release of ligand from the cell surface. This dynamic cycle was found non toxic and, most importantly, could be performed in situ and under physiological conditions.

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45 Figure 1 1 3 . Liposome liposome fusion and liposo me cell fusion to adorn cell surface with reactive functionality. A) A hydroquinone containing liposome was fused with an alkoxyamine containing liposome via oxime ligation. B) TEM images of multiadherent (left), partially fused (middle), and completely fu sed (right) liposomal structures. C) The HQ functional liposome was mixed with AO containing liposome, and then the HQ and AO containing liposomes were mixed with the cells in culture. The HQ groups were activated to Q by applying mild oxidation potential. The ketone groups in Q were conjugated with the alkoxyamine (AO) through oxime formation. The oxime bonds were cleaved by applying mild reduction potential to detach the ligand from the cell. Adapted from, A. Pulsipher, D. Dutta, W. Luo and M. N. Yousaf, Angew. Chem. Int. Ed. , 2014, 53 , 9487 9492. Copyright 2014 Wiley. These reports clearly demonstrate that the inherently reversible exchangability of the oxime link can be a useful tool in the design of complex platforms for drug delivery, cell surface modi fication, protein modification, and tissue engineering purposes.

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46 CHAPTER 2 RESEARCH OBJECTIVE The purpose of the present research was to synthesize and investigate linear and branched polymeric materials that contain reversible covalent linkages such as oximes and oxy norbornene groups, by virtue of which the polymeric architectures can break and reconstruct themselves under the influence of suitable stimuli. We have seen tremendous growth in the development of well defined functional polymers that exhibi t dramatic property changes under the exposure of various stimuli. The ability of reversible polymeric systems to reorganize themselves at a molecular level via the cleavage and exchange of their component when expos ed to specific stimuli, offers immense p otential to create the next generation of smart materials. Research in this area is still in progress. A variety of reversible bonds has been used to access reversible polymeric materials, but the reversibility of oxime bonds has received less attention. W e investigated the ability of oxime bonds to undergo dynamic exchange in the presence of excess alkoxyamine or carbonyl compounds, to prepare reversible linear and branched polymeric materials. Three different polymerization techniques ( i.e ., conventional radical, reversible addition fragmentation chain transfer (RAFT), and step growth) were employed to access functional polymers during the course of this work. The ketone containing copolymers were prepared by conventional radical and RAFT polymerizations, and crosslinked via oxime formation to yield different branched architectures ( e.g. , star polymers, hydrogels), and their reversibility was examined by competitive oxime exchange. The Diels Alder reaction was also employed to polymerize an AB monomer that contained oxime bonds and oxy norbornene links, which allowed the polymer to undergo reversible disassembly under exposure to two different stimuli.

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47 Specific objectives of each research project and a brief summary of our results are given below. The goal of the research described in Chapter 3 was to synthesize and investigate oxime functional macromolecular star polymers that had the ability to dissociate and reconstruct upon application of a stimulus. RAFT polymerization was utilized to prepare w ell defined amphiphilic block copolymers of N,N dimethylacrylamide (DMA) and di a cetone acrylamide (DAA). The hydrophobic keto functional block in the resulting block copolymer allowed self assembly into micelles in water. Adding a difunctional alkoxyamine small molecule to these solutions resulted in crosslinking of the micelles to yield high molecular weight macromolecular stars. The reversible nature of the O alkyl oxime linkages was demonstrated via competitive exchange in the presence of competitive car bonyl compounds or monofunctional alkoxyamines under acidic conditions and at elevated temperatures to cause dissociation of the stars to unimolecular oxime functional polymer chains. This was one of the first in depth investigations into the reversibility of oxime linkages in macromolecular systems (Chapter 3). The objective of the research described in Chapter 4 was to exploit the reversibility of oximes for the preparation of self healing hydrogels with tunable gelation and degradation behavior. Keto fu nctional copolymers were prepared by conventional radical polymerization of DMA and DAA. The resulting water soluble copolymers (P(DMA stat DAA)) were chemically crosslinked via oxime formation by reacting with difunctional alkoxyamines to obtain hydrogels . Gel to sol transitions were induced by the addition of excess monofunctional alkoxyamines to promote competitive oxime exchange under acidic conditions at 25 °C. The hydrogel could autonomously heal after it was damaged due to the dynamic nature of its o xime crosslinks. In addition to their chemo responsive behavior, the P(DMA stat DAA) copolymers exhibited

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48 cloud points which vary with the DAA content in the copolymers. This thermo responsive behavior of the P(DMA stat DAA) was exploited to prepare physic al hydrogels at elevated temperatures. Therefore, these materials proved to be capable of forming either dynamic covalent or physically crosslinked gels, both of which demonstrate reversible gelation behavior. These results represent the first time oxime f ormation has been employed to prepare self healing materials, and the potential application of these systems range from drug delivery and tissue engineering to cosm etics and self healing coatings (Chapter 4). The goal of the research described in Chapter 5 was to synthesize a polymer capable of reversible disassembly under two orthogonal sets of conditions ( i.e. , oxime exchange and retro Diels Alder). Two sets of reversible covalent linkages distributed in series along a polymer backbone were used to prepar e a new class of doubly dynamic covalent polymers capable of reversibly dissociating via two distinct pathways. These self repairable linear polymers were prepared via step growth Diels Alder polymerization of an AB monomer that contained furfuryl and mal eimido groups linked by an oxime bond. Both the oxime and oxy norbornene links lent reversible character to the polymer backbone. The sequentially distributed oxime bonds in the polymer were capable of dynamic oxime exchange in the presence of a competitiv e monofunctional alkoxyamine under acidic conditions, while the oxy norbornene linkages were susceptible to cleavage via retro Diels Alder reactions at higher temperatures (160 °C) and recombination upon lowering the temperature to 130 °C (Chapter 5). Thes e results are significant in that they represent the first time two reversible covalent linkages have been combined within a singl e of conditions.

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49 CHAPTER 3 DYNA MIC MACROMOLECULAR CORE CROSSLINKED STARS WITH REVERSIBLE OXIME LINKS 3.1 Overview Macromolecules containing readily reversible covalent bonds offer potential as new degradable, responsive, and adaptable materials. A variety of reversible linkages have bee n used mines and acyl hydrazones having proven particularly promising. 56 , 59 , 62 , 68 , 82 , 83 In contrast, the reve rsibility of oxime bonds has received less attention, possibly due to their greater stability as compared to imines or hydrazides. 23 However, it is exactly this enhanced stability, especially with respect to hydrolysis, which confers considerable promise on oxime formation f or the construction of dynamic covalent materials that can be triggered to exchange their components only und er a specific set of conditions . 22 Post p olymerization modification of aldehyde or ketone containing polymers with alkoxyamines has also received significant attention due to high reaction yields, catalyst free mild reaction conditions (ambient temperature, aqueous environment), and benign side product (water). 23 , 84 Given its high efficiency and selectivity, oxime formation should be a valuable method to facilitate the synthesis of polymers with complex macromolecular architecture. 84 We have recently reported reversible addition fragmentation chain transfer (RAFT) polymerization of an alkoxyamine containing monomer and subsequent functionalization of the polymers with small molecule aldehydes and ketones. 75 Theato et al. reported cont rolled polymerization of acetone oxime acrylate and then used N isopropylamine to partially convert the acetone oxime groups in the polymer to yield thermoresponsive poly( N isopropylamine co acrylamide). 85 An alternative approach would be to include the requisite aldehyde or ketone functionality in the Reprinted from Polym. Chem. , 2014 , 5 , 6923 6 9 3 1 , with permission from the Royal Society of Chemistry.

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50 polymer and to functionalize with small molecule alkoxyamines. Aldehyde containing polymers have been demonstrated to be useful reactive scaffolds, and keto functional polymers have recently begun receiving similar interests. 55 , 84 , 86 88 , 89 It can be argued that the greater stability of ketones toward oxidation as compared to aldehydes in aqueous media renders them more sui ted for biological applications . 86 For this reason, we have chosen to focus here on well defined ketone containing polymers as reactive scaffolds for functionalization with low molecular weight O alkoxyamines. Control over molecular weight and dispersity of the reactive functional polymer scaf folds is desired to permit use in areas such as therapeutics, crystal engineering, coating, membrane technology, electronics, organo /hydrogels, detergent formulations, personal care products, and as lubricant additives. 84 , 90 Controlled radical polymerization techniques have been used to prepare well defined keto functional polymer scaffolds for modification via oxime and hydrazone formation reactions. 84 , 87 , 91 The condensation product of a ketone and an alkoxyamine ( i.e. , a ketoxime) generally possesses superior hydrolytic stability to the condensation product of an aldehyde and alkoxyamine ( i.e. , an aldoxime). As a result, ketoxime formation has been employed for protein modification, 92 to prepare oxime functional polyketoesters, 93 and to obtain biospecific and chemoselective surface gradients. 94 We were intrigued by the possibility of creating branched architectures containing ketoxime linkages and then capitalizing on their inherent reversibility to induce disassembly and reassembly. We reasoned that reversibility of oxime linked macromolecules could be achieved by competitive exchange of the otherwise hydrolytically stable ketoxime in the presence of small molecule alkoxyamine or carbonyl compounds. Incorporating such stable yet exchangeable linkages in macromolecular branched architectures could lead to robust dynamic materials with potential use in encapsulating cargo ( e.g., drug, dyes, and fragrances), as lubricant additives, and

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51 in coating formulations. 7 , 56 , 89 , 95 As one example of a regularly branched macromolecule, wel l defined star polymers are generally prepared via 96 There is considerable interest in the preparation of star architectures that possess reversible linkages, such as alkoxyamines, 31 , 33 , 97 imines, 59 , 82 , 98 acylhydrazones, 14 , 99 boroxines, 100 , 101 boronic esters, 18 disulfides, 14 , 15 , 16 , 17 and Diels Alder linkages. 44 , 102 , 103 Herein, we demonstrate the synthesis of well defined keto functional block copolymers by RAFT polymerization and their subsequent functionalization with small molecule alkoxyamines. Addition of difunctional alkoxyamines to an aqueous solu tion of keto functional block copolymers led to core crosslinked oxime stars by an arm first method. Due to the reversible nature of the oxime units, 22 star dissociation was induced by competitive exchange with monofunctional alkoxyamines or monofunctional aldehydes and ketones in presence of an acid catalyst. 3.2 Results and Discussion We reasoned that combining the high efficiency of oxime formation w ith the reversibility that arises in the presence of a competitive aminooxy compound could lead to new adaptive nanoparticles and fundamental insight into the dynamic nature of oxime containing materials. Two different ketone containing monomers, namely 4 acryloyloxy 2 butanone (AB) and diacetone acrylamide (DAA), were polymerized by RAFT and chain extended with N , N dimethylacrylamide (DMA). The resulting block copolymers were amphiphilic and contained one hydrophobic (ketone containing) block susceptible to reaction with alkoxyamines and a second unreactive, hydrophilic block. After self assembly into micelles in w ater, addition of difunctional alkoxyamines led to confined crosslinked networks that were stabilized by the passivating non reactive blocks. These core crosslinked stars, which can also be referred to as eaved and dissociated to unimers by the

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52 addition of a monofunctional alkoxyamine that was capable of displacing the crosslinker by oxime exchange. 22 An alternative approach to dissociate the core involved the addition of a monofunctional aldehyde or ketone to effectively scavenge the crosslinker under acidic aqueous conditions. 3.2.1 Synthesis of block copolymers containing reactive keto functionality v ia RAFT Keto functional monomer AB was prepared by the reaction with acryloyl chloride and 4 hydroxy 2 butanone and characterized by 1 H NMR spectroscopy (Scheme 3 1, Fig. 3 1). Both AB and DAA were used to prepare ketone containing block copolymers by chai n extension of a poly( N,N dimethylacrylamide) macro chain transfer agent (PDMA macroCTA). A PDMA 1 23 macroCTA was chain extended with 4 acryloyloxy 2 butanone to yield the PDMA 123 b PAB 18 ( P1 ) block copolymer that contained the keto groups need ed for oxime formation (Scheme 3 2 ). The number average molecular weight ( M n,NMR ) of the resulting block copolymers was estimated by 1 H NMR spectroscopy by comparing the area of methylene protons ( OC H 2 , = 4.2 ppm) of the pendent AB units to the area of the termina l methyl protons of the dodecyl group ( (CH 2 ) 11 C H 3 , = 0.85 ppm) (Table 1 ). Scheme 3 1. Synthesis of 4 acryloyloxy 2 butanone (AB) A PDMA 115 macroCTA was also chain extended with DAA to prepare the PDMA 115 b PDAA 7 ( P2 ) block copolymer. The M n of the block copolymers was determined by comparing the area of the methyl protons ( (C=O) C H 3 , = 2.15 ppm) of the pendent DAA units to the terminal methyl protons of the dodecyl group ( (CH 2 ) 11 C H 3 , = 0.85 ppm). The discrepancy

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53 between theoretical mo lecular weight based on the monomer conversion ( M n,theo ) and M n,NMR for PDMA 127 b PDAA 14 ( P10 ), PDMA 127 b PDAA 39 ( P11 ), and PDMA 127 b PDAA 71 ( P12 ) could be a result of the error involved in calculating the conversion, as the broad 1 H NMR signal of ((C=O)N H ) in each DAA unit ( = 6.09 ppm) increasingly overlapped with the 1 H NMR signals of the vinyl peaks of DAA as the polymerization proceeded. Efficient chain extension of PDMA to yield PDMA 123 b PAB 18 ( P1 ) and PDMA 115 b PDAA 7 ( P2 ) was confirmed from the c lean shift of the unimodal SEC trace of the macroCTA to a lower elution time for the block copolymer (Fig. 3 2 ). Figure 3 1. 1 H NMR spectra of acryloyl choride, 4 hydroxy 2 butanone, and 4 acryloyloxy 2 butanone in CDCl3.

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54 Scheme 3 2. Synthesis of poly( N, N dimethylacrylamide) (PDMA) and its subsequent chain extension with diacetone acrylamide (DAA) and 4 acryloxy 2 butanone (AB) to prepare PDMA b PDAA and PDMA b PAB respectively Figure 3 2. Size exclusion chromatography traces of chain extension of PDMA macroCTA with AB and DAA. Normalized refractive index traces from size exclusion chromatography of (A) poly( N,N dimethylacrylamide) 110 (PDMA 110 ) macroCTA and PDMA 110 b PAB 37 and (B) PDMA 115 ma croCTA and PDMA 115 b PDAA 7 .

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55 Table 3 1. Results for synthesis of block copolymers with N,N d imethyl acrylamide (DMA), 4 acryloyloxy 2 butanone (A B), and diacetone acrylamide (DAA) Entry Polymer M n,theo a (kg/mol) M n,NMR b (kg/mol) M n, SEC c (kg/mol) M w, SEC c (kg/mol) M w / M n c P1 PDMA 123 b PAB 18 17.5 15.1 17.4 20.6 1.20 P2 PDMA 115 b PDAA 7 14.1 13.5 13.0 13.7 1.10 P3 PDMA 110 b PAB 37 17.6 16.6 15.6 19.1 1.22 P4 PDMA 115 b PDAA 4 12.7 12.5 11.1 11.4 1.03 P5 PDMA 110 11.0 11.3 11.3 12.4 1.10 P6 PDMA 115 11.0 11.8 10.7 11.0 1.10 P7 PDMA 123 13.0 12.5 12.5 12.9 1.10 P8 PDMA 127 9.1 13.0 9.8 10.0 1.10 P9 PDMA 127 b PDAA 7 14.4 14.2 11.0 11.0 1.10 P10 PDMA 127 b PDAA 14 20.6 15.7 15.2 15.4 1.10 P11 PDMA 127 b PDAA 39 28.4 19.1 19.2 19.6 1.10 P12 PDMA 127 b PDAA 71 34.5 17.1 24.3 25.0 1.10 a Calculated from monomer conversion determined by 1 H NMR spectroscopy. b Calculated using 1 H NMR spectroscopy by end group analysis. c Determined by SEC with multiangle light scattering detection. 3.2.2 Functionalization of PDMA 110 b PAB 37 (P3) with model alkoxyamines While the focus of our research was to prepare adaptive nanoparticles crosslinked via oxime linkages, we expected keto functional polymers could also be utilized as versatile scaffolds for post polymerization modification. Hence, we first investigated reactions of low molecular weight alkoxyamines with our keto functional block copolymers.

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56 Scheme 3 3. Model reactions of PDMA 110 b PAB 37 (P3) with O allyl hydroxylamine and O (tetrahydro 2 H pyran 2 yl)hydroxylamine Figure 3 3. Results from the model reactions of PDMA 110 b PAB 37 ( P3 ) with O allyl hydroxylamine. (A) 1 H NMR spectra of PDMA 110 b PDAA 37 ( P3 ) before (blue) and after (green) functionalization in DMSO d 6 , (B) SEC overlay (normalized refractive index signal) of PDMA 110 b PDAA 37 ( P3 ) and functionalized PDMA 110 b PDAA 37 ( P3a ).

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57 Figure 3 4. Functionalization of PDMA 110 b PAB 37 (P3) with O (tetrahydro 2 H pyran 2 yl)hydroxylamine (A) 1 H NMR spectra of PDMA 110 b PDAA 37 before (blue) and after functionalization (green) in DMSO d 6 . (B) Overlay SEC refractive index traces of PDMA 110 b PDAA 37 (P3) and functionalized PDMA 110 b PDAA 37 (P3b). O Allyl hydroxylamine and O (tetrahydro 2 H pyran 2 yl) hydroxylamine were chosen as model reactants for oxime formation reaction because of the ease with which their final products could be characterized by 1 H NMR spectroscopy. Efficient functionalization with O allyl hydroxylamine (Scheme 3 3 ) was suggested by the upfield shift of the peak corresponding to the keto methyl protons in P3a from 2.21 ppm to 1.85 ppm ( a in Fig. 3 3 A). The degree of functionalizati on (>95%) was calculated from the peak integral ratio for the O C H 2 CH=CH 2 allylic protons and the O C H 2 CH 2 C(CH 3 )=N O methylene protons (Fig. 3 3 A) in the functionalized polymer. SEC analysis (Fig. 3 3 B) indicated a slight increase in the molecular we ight of the block copolymer after the reaction (Table 3 2 ). Efficient functionalization (>95%)

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58 with O (tetrahydro 2 H pyran 2 yl) hydroxylamine (Scheme 3 3 ) was also confirmed by 1 H NMR spectro scopy and SEC analysis (Table 3 2 and Fig. 3 4 ). Table 3 2 . Results for f unctionalization of PDMA 110 b PAB 37 with m onofunctional a lkoxyamines . Polymer Monofunctional alkoxyamine used [ketone] : [alkoxyamine] % Functionalization M n, NMR (g/mol) of functionalized polymers PDMA 110 b PAB 37 (P3) O allyl hydroxyl amine hydrochloride 1 : 2 99 17,800 ( P3a ) PDMA 110 b PAB 37 (P3) O (tetrahydro 2 H pyran 2 yl) hydroxylamine 1 : 2 99 18,800 ( P3b ) Figure 3 5. Reaction between PDMA 127 b PDAA 39 and O (tetrahydro 2 H pyran 2 yl)hydroxylamine . (A) UV Vis spectra of PDMA 127 b PDAA 39 ( P11 ) in MeOH before (control) and after its reaction with O (tetrahydro 2 H pyran 2 yl)hydroxylamine . (B) SEC overlay (normalized refractive index signal) of PDMA 127 b PDAA 39 ( P11 ) before and after its reaction with O (tetrahyd ro 2 H pyran 2 yl)hydroxylamine at [keto ne]:[alkoxyamine] = 1:1 and 1:2. Primary amines are well known to attack thiocarbonyl groups of RAFT generated polymers to yield thiol end groups. 104 Therefore, we were curious whether the thiocarbonylthio groups at the terminus of the RAFT polymers would also be susceptible to cleavage via

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59 nucleophilic attack by the alkoxyamines. Such a side reaction would be expected to convert the RAFT generated end groups to their corresponding thiols. Figure 3 6. R eaction between RAFT CTA and O (tetrahydropyran 2 H pyran 2 yl)hydroxylamine. Photographs of (A) solution of 2 dodecylsulfanylthiocarbonylsulfanyl 2 methyl propionic acid (DMP) in methanol, (B) reaction solution of DMP with 1 equiv of O (tetrahydropyran 2 H pyran 2 yl)hydroxylamine in methanol, and (C) reaction solution of DMP with 2 equiv of O (tetrahydropyran 2 H pyran 2 yl)hydroxylamine in methanol after 24 h at 25 °C. (D) UV Vis spectra of DMP in MeOH before and after the reaction with O (tetrahydro 2 H pyran 2 yl)hydroxylamine. To investigate this possibility, model reactions were conducted between PDMA 127 b PDAA 39 ( P11 ) and O (tetrahydro 2 H py ran 2 yl)hydroxylamine at 25 °C at [ketone]:[alkoxyamine] ratios of 1:1 and 1:2. Interestingly, no significant reduction of the characteristic absorbance of the trithiocarbonate end group ( max 24 h, even in the case of the excess alkoxyamine (Fig. 3 5A ). Additionally, the SEC traces of the products from both the reactions remained mono modal after 24 h, suggesting the absence of

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60 polymer polymer coupling via disulfide formation, which might be expected if significant cleavage of the trithiocarbonate moiety had occurred (Fig. 3 5 B). The conservation of the thiocarbonylthio end group in the presence of alkoxyamines was further confirmed by a model reaction between the original low molecular weight RAFT CTA (2 dodecylsulfanylthiocarbonylsulfanyl 2 methyl propionic acid ) and O (tetrahydro 2 H pyran 2 yl)hydroxylamine. Similarly, no noticeable change in the UV absorbance ( max occurred in this case (Fig. 3 6 ). These results suggest that end group cleavage via aminolysis by O alkyl alkoxyamines is not a complicating factor. 3.2.3 Core crosslinked star formation Given the demonstrated high efficiency of oxime formation with these newly prepared keto functional block copolymers, we turned our attention to the possibility of creating reversible core crosslinked stars by reaction with difunctional alkoxyamines. Selective crosslinking of th e reactive block of a diblock copolymer with a multifunctional crosslinker is a convenient route for the synthesis of stars. 17 , 18 , 31 , 59 , 100 , 103 We recently observed that pre orientation of the block copolymers into micellar aggregates in a sele ctive solvent prior to the crosslinking reaction drastically improves the yield and size dispersity of the resulting core crosslinked stars. 44 , 46 PDAA demonstrates poor water solubility, making it an ideal monome r for the synthesis of amphiphilic block copolymers in which the reactive ketone is contained in the aggregated hydrophobic block. 86 , 105 Moreover, as compared to the acryloyl units in the AB containing block copolymer, the acrylamido DAA units, with their adjacent gem dimethyls, are expected to have enhanced hydrolytic stability. Therefore, we decided to l imit our star formation studies in aqueous media to the PDAA containing block copolymers.

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61 Scheme 3 4. Formation of core crosslinked micelles via self assembly and oxime formation Self assembly of the PDMA b PDAA block copolymers was achieved by controlled dilution of a solution of the block copolymer in THF in to PBS buffer (pH 7.4) (Scheme 3 4 ). SEC, light scattering, and TEM were used to characterize the resulting micelles. Depending on the length of the hydrophobic PDAA and the hydrophilic PDMA blocks, DLS measurements indicated a hydrodynamic diameter ( D h ) of 17 to 36 nm for the micelles (Table 3 3 ). For example, DLS analysis of the micelles obtained from PDMA 127 b PDAA 14 ( P10 , 20 mg/mL in PBS) yielded a D h of 26 nm, while the precursor block copolymer existed as completely dissolved unimers ( D h = 6 nm) in methanol, a non selective solvent (Fig. 3 7D ). Addition of a difunctional alkoxyamine to the micelle solution led to crosslinking of the keto functional hydrophobic core via oxime formation a t 25 ° C to yield core crosslinked stars ( Star 1 ). The stars were observed to have similar sizes as their original micelle precursors, as observed by DLS and TEM (Fig. 3 7D, E). The covalently crosslinked nature of the stars was confirmed by their stability in methanol, a non selective solvent that would be expected to dissociate non crosslinked micelles to their corresponding unimers (Fig. 3 7D)

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62 Figure 3 7. Core crosslink star formation via an arm first approach. (A) Normalized refractive index (RI) SEC t races showing the progress of star formation between PDMA 127 b PDAA 14 ( P10, 20 mg/mL) and 1,3 propanediylbishydroxylamine in PBS at [ketone]:[alkoxyamines] = 1:1 equiv. (B) Example of a deconvoluted SEC RI trace to obtain star and unimer areas. (C) Ov erlay of normalized SEC RI traces of PDMA 127 b PDAA 14 (red) and purified oxime stars ( Star 1 , blue). (D) DLS size distribution for PDMA 127 b PDAA 14 in MeOH (red), as micelles formed in PBS (blue) and as purified core crosslinked stars in MeOH (green). (E) TEM image of purified core crosslinked star (ne gative stain, scale bar: 50 nm). SEC analysis of the core crosslinked nanostructures revealed a new peak at a lower elution volume as compared to the original unimers, indicating a significant increase in mole cular weight upon assembly and crosslinking. By observing the relative deconvoluted peaks areas of the stars and unimers as a function of time, it was possible to monitor the kinetics of the crosslinking reaction (Fig. 3 7 A, B). In all cases, star formation was observed to be highly efficient, with typically less than 10% residual unimers being present after crosslinking. These unreacted unimers were readily removed by dialyzing the reaction solution against deionized

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63 water and isolating the purified stars by lyophilization. SEC MALS was used to calculate the molecular weight of the resulting purified stars. The corresponding numbers of arms per star was estimated by the ratio of molecular weight of stars ( M w,star ) to the mo lecular weight of the unimers obtained prior to crosslinking ( M w,arm ) (Table 3 3 ). 1 H NMR spectra of the purified stars ( Star 1 ) showed attenuation of the keto methyl peak [ (C=O)C H 3 ] (2.11 ppm) and backbone protons (~1.35 ppm and 1.88 ppm) of the PDAA uni ts, suggesting a highly crosslinked and partially desolvated core in CDCl 3 (Fig. 3 8 ). Fig ure 3 8. 1 H NMR spectra of core crosslinked oxime stars ( Star 1 , red) and PDMA 127 b PDAA 14 ( P10 , blue) in CDCl 3 . We also studied star formation by selective crosslinking of the ketone containing block of PDMA b PDAA in a non selective solvent. 1,3 Propanediylbishydroxylamine was added to a solution of molecularly dissolved PDMA 127 b PDAA 14 (60 mg/mL) in methanol with [ketone]:[alkoxyamine] = 1:2 equiv.

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64 Fig ure 3 9. Core crosslinked star formation in organic solvent (methanol). (A) Normalized SEC refractive index traces showing progress of star formation reaction between PDMA 127 b PDAA 14 ( P10 , 60 mg/mL) and 1,3 propanediylbishydroxylamine at [ketone] :[alkoxyamine] = 1:2 equiv in MeOH. (B) DLS solution size distribution of PDMA 127 b PDAA 14 in MeOH (red) and as core crossli nked oxime stars in MeOH (blue). As expected, the kinetics of star formation in MeOH was found to be slower than the star formation carried out in aqueous media (Fig. 3 9 ). These results highlight the benefit afforded by pre orientation of the reactive block copolymers into micelles prior to intermolecular crosslinking. To study the star formation process in more detail, two sets of experiments were carried out to elucidate the effect of ( i ) ketone: alkoxyamine ratio, and ( ii ) the overall block copolymer concentration on the reaction kinetics and size of the resulting star nanostructures. PDMA 127 b PDAA 14 was chosen for this study con sidering that the star formation reaction with this block copolymer was found to achieve completion within approximately 24 h, even at the lowest crosslinker equivalence used during our preliminary studies.

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65 First, the rate of crosslinking was studied at 25 ° C in PBS with a constant polymer concentration ([PDMA 127 b PDAA 14 ] = 20 mg/mL) using [ketone]:[alkoxyamine] equiv = 1:1, 1:2, 1:4, and 1:6). About ~53% of the micelle arms crosslinked after ~3 h with 1 equiv of difunctional alkoxyamines, whereas ~73% of micelle arms were incorporated into stars after 3 h in the presence of 2 equiv of difunctional alkoxyamines (Fig. 3 10 ). When the amount of difunctional alkoxyamine was further increased to 4 equiv, the conversion increased to ~81% after just 2 h and ~91% within 6 h. No significant change in the crosslinking rate was observed when the amount of difunctional alkoxyamine was further increased to 6 equiv. Almost complete conversion of micelles to stars was observed after ~25 h regardless of the amount of cross linker used. The solution sizes (average hydrodynamic diameter, D h ) of the stars obtained after purification for all the four [ketone]:[alkoxyamine] ratios was found to be similar by both DLS ( D h = 26 nm, Table 3 3 ) and SEC (Fig. 3 10 B). The M w of the star s ranged between 660 700 kg/mol corresponding to 43 46 arms per star. These findings indicate that while increasing the amount of crosslinker increases the rate of core crosslinking, completely crosslinked cores are obtained even at [ketone]:[alkoxyamine] = 1:1 equiv. The efficiency and kinetics of star formation was also investigated as a function of block copolymer composition and concentration. Micellization and star formation was studied for several block copolymers having the same length of the passive PDMA 127 block and different lengths of the reactive PDAA block at a [ketone]: [alkoxya mine] ratio of 1:2 equiv (Fig. 3 11 ).

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66 Figure 3 10 . Effect of [ketone]:[alkoxyamine] on star formation kinetics and star size. (A) Kinetics of star formation ( Star 1 4 , Table 3 3 ) determined by deconvolution of the normalized SEC refractive index traces obtained during the reaction between PDMA 127 b PDAA 14 ( P10 ) and 1,3 propanediylbishydroxylamine dihydrochloride at varying [ketone]:[alkoxyamine] ratios. (B) Normal ized SEC refractive index traces of PDMA 127 b PDAA 14 and the resulting purified stars ( Star 1 4 , Table 3 3 ). However, a larger increase in initial reaction rate and final extent of star formation was observed for the block copolymer with the shortest PDAA segment (PDMA 127 b PDAA 7 ) on increasing the polymer concentration from 10 to 20 mg/mL. For example, at 10 mg/mL only 22% of the block copolymer arms were incorporated into the stars in ~ 27 h, whereas at 20 mg/mL the conversion of arms to stars was 65% in the same time. While the final conversion to stars was almost quantitative in the case of the block copolymers with degrees of polymerization for the PDAA block between 14 to 71, only 86% conversion was observed for PDMA 127 b PDAA 7 , even after 96 h ([PDMA 1 27 b PDAA 7 ] = 20 mg/mL).

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67 Figure 3 11. Effect of polymer concentration on star formation kinetics and star size. Kinetics of star formation for PDMA b PDAA block copolymers determined by deconvolution of SEC refractive index traces at a block copolymer concentration of (A) 10 mg/mL and (B) 20 mg/mL . SEC (normalized refractive index) traces of purified stars obtained from PDMA b PDAA block copolymers at concentrations of (C) 10 mg/mL and (D) 20 mg/mL. The solution size and molecular w eight of the stars were also found to increase with increasing length of the rea ctive PDAA block length (Table 3 3 ). Interestingly, no change in D h and M w was observed with increasing polymer concentration. It should be noted that in all cases the solution sizes of the stars were almost the same as those of the uncrosslinked micelles from which they were prepared. These observations indicate that the average number of arms for stars obtained by crosslinking of pre organized micelles is dictated primarily by the equilibrium size of the

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68 micelles and likely not by the concentration of polymer and crosslinker. Thus, the morphology of the self assembled aggregates of the reactive block copolymers is likely preserved after crosslinking, and various reversibly crosslinked nano /micro aggregates like micelles, vesicles, rods etc ., can possibly be obtained by using this protocol of self assembly followed by a covalent crosslinking pro cess. Table 3 3 . Molecular weight and size results of purified core crosslinked oxime stars obtained from block copolymers of varying composition and [ketone]:[alkoxyamine] ratios Entry Block copolymer (star precursor) [ketone]: [alkoxyamine] equiv Block copolymer concentration (mg/mL) D h,micelle (nm) c M w,star (kg/ mol) a Arms per star ( M w,star / M w,arm ) b D h,star (nm) c Star 1 PDMA 127 b PDAA 14 1:1 20 26 660 43 26 Star 2 PDMA 127 b PDAA 14 1:2 20 26 714 46 26 Star 3 PDMA 127 b PDAA 14 1:4 20 26 700 45 26 Star 4 PDMA 127 b PDAA 14 1:6 20 26 660 43 26 Star 5 PDMA 127 b PDAA 7 1:2 10 18 192 14 19 Star 6 PDMA 127 b PDAA 7 1:2 20 18 205 14 19 Star 7 PDMA 127 b PDAA 14 1:2 10 25 700 45 25 Star 8 PDMA 127 b PDAA 39 1:2 10 30 1600 81 32 Star 9 PDMA 127 b PDAA 39 1:2 20 34 1620 83 31 Star 10 PDMA 127 b PDAA 71 1:2 10 34 2510 101 36 Star 11 PDMA 127 b PDAA 71 1:2 20 35 2550 102 37 Star 12 PDMA 115 b PDAA 7 1:2 20 19 340 26 24 a Weight average molecular weight of the stars determined by SEC using multiangle light scattering (MALS). b Weight average number of arms per star determined by dividing M w,star by M w,arm . c Hydrodynamic diameter determined by dynamic light scattering (DLS) in methanol. The results also indicate that a fine balance between the number of reactive functional groups in the block copolymer and polymer concentration leads to higher efficiency of the

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69 crosslinking reaction. Certainly, a higher number of reactive groups per polym er chain leads to more efficient crosslinking, as it ensures the presence of a sufficient number of functional groups for intermolecular crosslinking despite unavoidable intramolecular reactions. The concentration of block copolymers and the stoichiometry employed in this study were sufficient to achieve near quantitative conversion of the micelles to stars in most cases. 3.2.4 Disassembly of core crosslinked oxime stars via oxime exchange While the enhanced stability and controlled exchange of oxime linkag es has been utilized in analytical chemistry for isolation of small molecules, 106 synthesis of polymer scaffolds, and main chain oxime linked amphiphilic copolymers, 107 reversible assembly of oxime containing polymers has remained largely unexplored. Sc heme 3 5. Disassembly of core crosslinked oxime stars by competitive exchange with monofunctional aldehydes/ketones or monofunctional alkoxyamines

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70 Figure 3 12. Star dissociation via oxime exchange with furfuraldehyde. (A) Overlay of SEC refractive index traces. (B) Kinetics of star dissociation determined by deconvolution of the SEC refractive index traces during the reaction between a core crosslinked oxime star ( Star 12 ) and furfuraldehyde in presence of TFA at 60 °C. (C) DLS size distribution of oxime stars (red), unimers (blue), and unimers after star dissociation (green). Oxime bonds provide the opportunity for reversible disassembly via competitive oxime exchange under acidic conditions. 22 , 106 We reasoned that star dissociatio n could be triggered in the presence of added monofunctional alkoxyamines or monofunctional aldehydes/ketones that compete for binding with the polymer or difunctional alkoxyamine crosslinker (Scheme 3 5 ). To investigate this possibility, a solution of pur ified stars was treated with furfuraldehyde in the presence of trifluroacetic acid (TFA) at 60 ºC. The pH of the reaction solution was found to be

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71 ~4 after addition of TFA. Under these conditions, approximately 82 and 100% disassociation of the stars was o bserved after 10.5 h and 24 h, respectively (Fig. 3 12A, B). DLS confirmed complete dissociation of the stars ( D h = 24 nm) to individual PDMA 115 b PDAA 7 chains ( D h = 5 nm) (Fig. 3 12C ). Stars could also be dissociated via competitive exchange in the presence of monofunctional alkoxyamines under similar experimental conditions ( Fig. 3 13 ). Figure 3 13. Star dissociation in presence of excess monofunctional alkoxyamine. (A) Normalized refractive index SEC traces of purif ied oxime star (Star 2, green), dissociated unimers (red) after 48 h of reaction between oxime stars and O allyl hydroxylamine in presence of TFA at 60 °C, and PDMA 127 b PDAA 14 ( P10 , blue). (B) DLS solution size distribution of PDMA 127 b PAB 14 in MeOH (blue), dissociated unimers in MeOH (red), and purified core crosslinked oxime stars in MeOH (green). 3.3 Conclusions Oxime formation between ketone containing block copolymers and difunctional alkoxyamines is clearly a viable means to prepare co re crosslinked stars. A variety of factors control the efficiency of star formation, including solvent quality ( i.e ., selective vs . non -

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72 selective), stoichiometry, and block copolymer composition/concentration. An increase in the rate of core crosslinking w as observed with increasing block copolymer concentration. The star formation kinetics at a constant block copolymer concentration and stoichiometry was also found to be accelerated when the reactive ketone containing block length was varied from 7 to 71. These results clearly demonstrate the efficiency of oxime formation is convenient for the facile preparation of core crosslinked star polymers. The susceptibility of the stars to disassembly suggests oximes are useful linkages to employ in the construction of dynamic covalent materials. The reversible nature of oxime linkages in the crosslinked core made it possible to disassemble the stars under conditions that promote oxime exchange in the presence of small alkoxyamine or carbonyl compounds. To our knowle dge, the reversible nature of oxime containing polymeric architectures has not been previously explored. The tunable reversibility of the oxime equilibrium can potentially be utilized for transporting and releasing cargo of fragrances, dyes, and drugs. Mor eover, even though the work described here relies on assemblies in solution, application of oxime chemistry could be extended to rehealable gels and bulk materials by careful selection of reagents and controlling branching properties. 3.4 Experimental Sect ion 3.4.1 Materials 2 Dodecylsulfanylthiocarbonylsulfanyl 2 methyl propionic acid (DMP) was prepared as previously reported. 107 N,N Dimethylacrylamide (DMA, Fluka, 98%) was passed through a small column of basic alumina to remove inhibitor prior to polymerization. Diacetone acrylamide (DAA, 99%, Sigma Aldrich) was recrystallized from hexane. Azobisisobutyronitrile (AIBN, Sigma Aldrich, 98%) was recrystallized from ethanol. O (Tetrahydro 2 H pyran 2 yl)hydroxylamine (Sigma Aldrich, 96%), O allyl hydroxylamine

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73 1,3 propanediylbishydroxylamine dihydrochlorid e (Sigma Aldrich, >99%), acryloyl chloride (Alfa Aesar, 96%), 4 hydroxy 2 butanone (Alfa Aesar, 95%), trifluroacetic acid (TFA, 99.5%, EMD Millipore), triethyl amine (TEA, Fisher Chemical, 99%), 1,3,5 trioxane (Acros Organics, 99.5%), sodium hydrogen carbo nate (Acros Organics, 99.5%), N,N dimethylformamide (DMF, EMD, 99.9%), dimethyl acetamide (DMAc, Sigma Aldrich, 99.9%), tetrahydrofuran (THF, EMD, 99.5%), dichloromethane (DCM, 99.5%, BDH), 1,4 dioxane (Fisher Chemicals, 99%), phosphate buffered saline (P BS, pH 7.4, Sigma Aldrich), diethyl ether (Fisher Chemicals), methanol (Mallinckrodt), ethyl acetate (99.9%, Fisher chemicals), hexane (98.5%, BDH), dimethylsulfoxide d 6 (DMSO d 6 , Cambridge Isotope, 99.9% D), CDCl 3 (Cambridge Isotope, 99% D), and molecular sieves 4Å (Acros Organics) were used as received . 3.4.2 Instrumentation and Analysis Molecular weight and molecular weight dispersity were determined by size exclusion chromatography (SEC). (i) SEC in DMF (with 0.05 M LiBr) was conducted at 55 °C with a flow rate of 1.0 mL/min (Viscotek SEC Pump; Columns [(ViscoGel I Series G3000 and G4000 mixed bed columns: molecular weight range 0 60 x 10 3 and 0 400 x 10 3 g/mol, respectively), or (Polymer Laboratories; PolarGel M mixed bed columns: molecular weight rang e 0 60 x 10 3 and 0 2000 x 10 3 = 660 nm, a Viscotek UV platform, consisting of a laser light scattering detection angles of 7 and 90 °) and a four capillary viscometer. Molecular weights were determined by the triple detection method assuming 100% mass recovery. (ii) SEC in DMAc with 0.05 M LiCl at 55 °C and a fl ow rate of 1.0 mL/min (Viscotek SEC pump, columns: Guard + two ViscoGel I series G3078 mixed bed columns: molecular weight range 0 20 × 10 3 and 0

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74 100 × 10 4 g/mol; or Agilent isocratic pump, degasser, and autosampler, columns: PLgel guard + two ViscoG el I series G3078 mixed bed columns: molecular weight range 0 20 × 10 3 and 0 100 × 10 4 g/mol). Detection either consisted of a Viscotek VE 3580 refractive index detector operating at 660 nm, or a Wyatt Optilab T rEX refractive index detector operating at 6 58 nm and a Wyatt miniDAWN TREOS light scattering detector operating at 659 nm. Molecular weights were determined using narrowly dispersed polystyrene standards for column calibration. Absolute molecular weights and molecular weight dispersities were calcu lated using the Wyatt ASTRA software. Dynamic light scattering (DLS) was conducted at 173° with a Malvern Zetasizer Nano ZS equipped with a 4 mW, 633 nm He Ne laser and an Avalanche photodiode detector. UV Vis spectroscopic measurements were conducted with Varian Cary 500 Scan UV Vis NIR spectrophotometer. 1 H NMR spectra were recorded using JEOL Delta 500 or Inova spectrometers operating at 500 MHz. Chemical shifts are reported in parts per million (ppm) downfield relative to tetramethylsilane (TMS, 0.0 ppm ). Multiplicities are reported using the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Transmission electron microscopy (TEM) images were taken on an H 7000 TEM microscope (Hitachi, Japan) at a working vo ltage of 100 kV. Samples (0.1 mg/mL or 1 mg/mL in methanol) were prepared using copper grids and 0.5% uranyl acetate stain. Fisher Scientific single syringe pump (95 120V/60Hz) was employed for controlled addition of PBS (pH 7.4) to prepare micellar soluti ons. 3.4.3 Synthesis and Experimental Procedures Synthesis of 4 acryloyloxy 2 butanone (AB) . 4 hydroxy 2 butanone (24 mL, 0.28 mol), triethylamine (78 mL, 0.56 mol, dried over molecular sieves), dichloromethane (DCM, 100 mL, dried over molecular sieves), and anhydrous sodium sulfate (20 g) were added to a 1000 mL round bottom flask equipped with a magnetic stir bar. The contents were allowed to cool below 5

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75 °C while stirring in an acetone ice bath. Acryloyl chloride (27.8 mL, 0.556 mol) was dissolved in dichloromethane (DCM, 100 mL) and added drop wise through an addition funnel over 2 h. The tempe rature of the reaction mixture was maintained below 5 °C during addition. Stirring continued overnight at 25 °C, and the mixture was filtered under vacuum and washed with DCM several times. The filtrate was transferred to a separating funnel and washed wit h deionized water (×3), a saturated solution of sodium bicarbonate (×3), and brine (×3). The crude monomer was purified by flash chromatography using a gradient solvent composition of ethyl acetate/hexanes (15/85, 40/60, and 50/50 v/v). Yield: 10 15% in di fferent batches. 1 H NMR (500 MHz, CDCl 3 ): = 6.25, 5.72 (dd each, 2H, vinyl C H 2 ), 5.97 (t, H, vinyl C H ), 4.29 (t, 2H, O C H 2 CH 2 ), 2.70 (t, 2H), 2.08 (s, 3H). 13 C NMR (500 MHz, CDCl 3 ): = 130.9 ( C H 2 =CH ), 127.9 (CH 2 = C H ), 165.9 ( CH C OO ), 59.2 ( COO C H 2 ), 41.9 ( C H 2 COCH 3 ), 205.7( CH 2 C OCH 3 ), 30.3( CO C H 3 ); HRMS + C 7 H 10 O 3, Theoretical: 142.1530. Actual: 142.0582. Synthesis of PDMA macro chain transfer agent (macroCTA) by RAFT polymerization of N,N dimethylacrylamide . A typical procedure for the synthesis of PDMA macroCTA is as follows. DMA (14.43 g, 0.1456 mol), DMP (0.288 g, 0.786 m mol), AIBN (0.0065 g, 0.039 mmol), s trioxane (0.355 g, 3.94 mmol), and 1,4 dioxane (16.5 mL) were sealed in a 50 mL round bottom flask with a rubber septum and purged with nit rogen for 40 min. The reaction flask was then placed on a preheated silicon oil bath at 60 °C. Samples were removed periodically by degassed syringe to determine monomer conversion by 1 H NMR spectroscopy. The polymerization was quenched after 2.6 h at 58% monomer conversion by removing the round bottom flask from the oil bath and opening it to expose its contents to atmospheric oxygen. The reaction solution was dialyzed in DI water through a regenerated cellulose dialysis tubing having molecular weight cut off (MWCO) = 3.5 kg/mol, and the

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76 product was isolated by lyophilization resulting in poly( N,N dimethylacrylamide) (PDMA 115 macroCTA, P6 ) ( M n, NMR = 11,800 g/mol, and degree of polymerization ( DP n ) = 115; M n,SEC = 10,700 g/mol; M w , SEC MALS = 11,000 g/mol an d M w / M n = 1.10). A similar procedure was followed to prepare PDMA 110 ( P5 ) , PDMA 123 ( P7 ) , and PDMA 127 ( P8 ) . RAFT polymerization of 4 acryloyloxy 2 butanone (AB) using PDMA 123 macroCTA (PDMA 123 b PAB 18 , P1) . 4 Acryloyloxy 2 butanone (1.42 mL, 9.99 mmol), PDMA 123 macroCTA (2.5 g, 0.20 mmol), AIBN (0.003 g, 0.02 mmol), s trioxane (0.09 g, 1 mmol), and 1,4 dioxane (7.5 mL) were sealed in a 20 mL vial with screw septa and purged with nitrogen for 30 min. The reaction vial was then placed on a preheated heating block at 70 °C. The reaction was quenched by removing the reaction vial from the heating block after 4.3 h at ~70% monomer conversion and opening to expose its contents to atmospheric oxygen. The reaction solution was precipitated in diethyl ether (×3) an d dried under vacuum ( M n ,NMR = 15,100 g/mol and DP n = 18; M n, SEC = 17,400 g/mol, M w /M n = 1.20). Similar procedure was followed to prepare polymer P3. RAFT polymerization of diacetone acrylamide using PDMA 115 macroCTA (PDMA 115 b PDAA 7 , P2) . Diacetone acryl amide (1.31 g, 7.74 mmol), PDMA 115 macroCTA (3.02 g, 0.256 mmol), AIBN (0.0028 g, 0.017 mmol), s trioxane (0.121 g, 1.34 mmol), and 1,4 dioxane (12 mL) were sealed in a 20 mL vial with screw septa and purged with nitrogen for 30 min. The reaction vial was then placed on a preheated heating block at 60 °C. The reaction was quenched by removing the vial from the heating block after 6 h at 4 5% monomer conversion and opening it to expose its contents to atmospheric oxygen. The reaction solution was dialyzed against deionized water. The reaction solution was dialyzed through a regenerated cellulose dialysis tubing (MWCO = 3.5 kg/mol) and the pr oduct was isolated by lyophilization resulting

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77 PDMA 115 b PDAA 7 , P2 ( M n,NMR = 13,500 g/mol; M n, SEC = 13,000 g/mol, M w,MALS = 13700 g/mol; M w / M n = 1.05). Similar procedures were followed to prepare polymer P4 and P9 P12 . Functionalization of PDMA 110 b PAB 3 7 (P3) with O allyl hydroxylamine PDMA 110 b PAB 37 (0.100 g, 0.223 mmol) was dissolved in THF (1 mL) in an 8 mL glass vial containing molecular sieves and a magnetic stir bar. O Allyl hydroxylamine hydrochloride (0.049 g, 0.45 mmol) was dissolved in anhydro us THF (1 mL) along with TEA (186 µL, 1.34 mmol) and DI water (0.2 mL) and the resulting solution was added to the polymer solution under stirring. The reaction vial was kept under stirring at 25 °C for 6 h. The solution was then precipitated in cold hexan e (×3), and dried under vacuum at 40 °C. Functionalization of PDMA 110 b PAB 37 (P3) with O (tetrahydro 2 H pyran 2 yl)hydroxylamine . PDMA 110 b PAB 37 (0.100 g, 0.223 mmol) was dissolved in THF (1 mL) in an 8 mL glass vial containing molecular sieves and magnetic stir bar. O (Tetrahydro 2 H pyran 2 yl)hydroxylamine (0.053 g, 0.45 mmol) was dissolved in anhydrous THF (1 mL) and the resulting solution was added to the polymer solution under stirring. The reaction solution was continued to stir for 8 h at 25 °C. The solution was then precipitated in cold hexane (×3), and the product was dried under vacuum at 40 ° C. UV Vis spectroscopy studies of the reaction of PDMA 127 b PDAA 39 (P11) and O (tetrahydro 2 H pyran 2 yl)hydroxylamine. Three solutions of PDMA 127 b PDAA 39 ( P11 ) (0.1 g, 0.2 mmol each) were prepared in methanol (3 mL each) in three different 4 mL glass vials containing magnetic stir bars (solutions A, B, and C). A stock solution of O (tetrahydro 2 H pyran 2 yl)hydroxylamine (0.10 g, 0.85 mmol) in methanol (1 mL) was added in two different portions (0.23 mL and 0.47 mL) to polymer

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78 solutions B and C. The mixed contents were stirred at 25 °C for 24 h. Sol ution A, without any added alkoxyamine, was used as the blank solution. The reactions were monitored periodically using UV Vis spectroscopy by diluting the reaction solution aliquot (55 µL) with methanol (5 mL) before analysis. UV Vis spectroscopy studies of the reaction of DMP and O (tetrahydro 2 H pyran 2 yl)hydroxylamine. Three solutions of DMP (0.020 g, 0.055 mmol each) were prepared in methanol (0.50 mL, 0.44 mL, 0.37 mL) in 4 mL glass vials containing magnetic stir bars (solutions A, B, and C respectiv ely). A stock solution of O (tetrahydro 2 H pyran 2 yl)hydroxylamine (0.05 g, 0.43 mmol) in methanol (0.5 mL) was added in two different portions (0.064 mL and 0.13 mL) to solutions B and C, respectively. The mixed contents were stirred at 25 °C for 24 h . S olution A, without any added alkoxyamine, was used as the blank solution. The reaction was monitored using UV Vis spectroscopy by diluting the reaction solution aliquot (5 µL) with methanol (5 mL) before analysis. Star formation and dissociation experiments with PDMA b PDAA Investigating the effect of the block copolymer concentration on star formation. To study the nature of the star formation in a selective solvent such that the block copolymers form micelles before cross linking, PDMA 127 b PDAA 7 ( P9 ) (0.400 g, 0.197 mmol) was dissolved in THF (5 mL) in a 40 mL vial. After complete dissolution, PBS (pH 7.4, 20 mL) was added at a rate of 9 mL/h using a syringe pump, and the resulting micellar solution was allowed to stir at 25 °C in an open vessel for 12 h . The solution was sonicated for 30 min, and the size of the micelles was determined using DLS. 1,3 P ropanediylbishydroxylamine dihydrochloride (0.050 g, 0.28 mmol) was added to PBS (pH 7.4, 1 mL), and an aliquot of the resulting solution (0 .710 mL) was transferred

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79 to the micellar solution under stirring to obtain a final PDMA 127 b PDAA 7 concentration of 20 mg/mL . The reaction vial was kept at 25 °C under stirring and was monitored periodically using SEC, with the extent of star formation bei ng calculated based on deconvolution via Gaussian multi peak fitting. The reaction solution was dialyzed through a regenerated cellulose dialysis tubing (MWCO = 50 kg/mol), and the product was isolated by lyophilization. Similarly the star formation was st udied at [PDMA m b PDAA n ] = 10 mg/mL and 20 mg/mL for other block copolymers ( m = 127 and n = 7, 14, 39, 71). Effect of [ketone]:[alkoxyamine] on star formation . A micellar solution of PDMA 127 b PDAA 14 ( P10 ) (0.24 g, 0.22 mmol) in PBS (pH 7.4, 12 mL) was prepared in a manner similar to that described above, and the solution (3 mL each) was transferred into four different 4 mL vials equipped with magnetic stir bars. O,O 1,3 Propanediylbishydroxylamine dihydrochloride (0.050 g, 0.28 mmol) was added to PBS (pH 7.4, 1 mL) and aliquots (0.097 mL, 0.20 mL, 0.39 mL, and 0.59 mL) of the resulting solution were added to the four different vials containing the micellar solution under stirring to obtain final PDMA 127 b PDAA 14 concentration of 20 mg/mL. The reaction vials were kept at 25 °C under stirring and were monitored periodically using SEC, with the extent of star formation being calculated based on the Gaussian multi peak fitting. The reaction solutions were dialyzed with regenerated cellulose dialysis tubing (MWCO = 50 kg/mol), and the products were isolated by lyophilization. Kinetics of star formation with PDMA m b PDAA n . To study the kinetics of the star formation in an aqueous system, micellar solutions of PDMA m b PDAA n (10 mg/mL and 20 mg/mL) were prepared in PBS (pH 7.4) in a manner similar to that described before. The sizes of the micelles were determined by DLS. The kinetics of star formation were studied at polymer

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80 concentrations of 10 mg/mL and 20 mg/mL, and stoichiometries of [ketone]:[alkoxyamine] = 1:1 and 1:2 for various block copolymers (PDMA m b PDAA n ; m = 127 and n = 7, 14, 39, 71). O,O 1,3 Propanediylbishydroxylamine dihydrochloride (0.050 g, 0.28 mmol) was added to PBS (pH 7.4, 1 mL) and aliquots (0.0530 mL, 0.106 mL) of were added to two diff erent vials containing micellar solutions of PDMA 127 b PDAA 7 (3 mL, 10 mg/mL; 3 mL, 20 mg/mL, respectively) under stirring. The mixed contents were stirred at 25 °C, and the reaction progress was monitored periodically using SEC. The kinetics of star forma tion were studied in a similar method for other block copolymers PDMA m b PDAA n (m = 127 and n = 7, 14, 39, 71). Kinetics of star formation with PDMA m b PDAA n in a non selective solvent . To study the kinetics of the star formation in a non selective solve nt, PDMA 127 b PDAA 14 (0.09 g, 0.08 mmol) was dissolved in MeOH (1.2 mL) in a 4 mL vial equipped with a stir bar. O,O 1,3 Propanediylbishydroxylamine dihydrochloride (0.050 g, 0.28 mmol) was added to a mixture of MeOH (0.9 mL) and TEA (0.078 mL, 0.56 mmol) under stirring. An aliquot (0.30 mL) of the resulting solution was added to the polymer solution under stirring to obtain the final PDMA 127 b PDAA 14 concentration of 60 mg/mL. The reaction vial was kept at 25 °C under stirring and the reaction progre ss was monitored periodically using SEC, with the extent of star formation being calculated based on deconvolution via Gaussian multi peak fitting. The reaction solution was dialyzed with a regenerated cellulose dialysis tubing (MWCO = 50 kg/mol) and the p roduct was isolated by lyophilization. Kinetics of Oxime Star Dissociation. Star dissociation experiments were carried out using furfuraldehyde, acetone, and O allyl hydroxylamine hydrochloride. Star dissociation with monofunctional alkoxyamine: Purified oxime stars ( Star 2) (0.010 g, 0.015 mmol) was dissolved in PBS (pH 7.4, 1.0 mL) in a 4 mL vial containing a magnetic stir

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81 bar. A solution of O allyl hydroxylamine hydrochloride (0.084 g, 0.77 mmol) in PBS (0.84 mL) was added to the star solution along wit h trifluroacetic acid (TFA, 60 µL). The reaction vial was kept on preheated hot plate at 60 °C under stirring. The progress of the reaction was monitored periodically by SEC. An aliquot of the reaction solution (60 µL) was diluted with methanol (1 mL) prio r to analysis by DLS. Star degradation with furfuraldehyde: Purified oxime stars ( Star 12) (0.0153 g, 0.0920 mmol) was dissolved in PBS (pH 7.4, 1.5 mL), and furfuraldehyde (0.38 mL, 0.0046 mol) was added to this solution along with TFA (10 µL). Th e reaction vial was kept on a preheated hot plate at 60 °C under stirring. The progress of the reaction was monitored periodically by SEC. An aliquot of the reaction solution (60 µL) was diluted with methanol (1 mL) prior to analysis by DLS. Star degradati on study with acetone was performed in a similar manner.

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82 CHAPTER 4 SELF HEALING HYDROGELS WITH TUNABLE GELATION AND DEGRADATION 4.1 Overview Polymers have become one of the most essential components in microelectronics, surface coatings, lightweight aerospace materials, drug delivery systems, regenerative medicine, dental fillings, prosthetic limbs, and artificial heart valves. 108 However, polymeric engineering materials u sed for such purposes often suffer mechanical failure leading to limited life span because of the continuous exposure to chemical and mechanical stresses. 108 , 109 To extend the l ife span of polymeric materials, research in the area of self mendable or self healing polymers is growing rapidly. Self healing is one of the most fascinating features of biological systems, such as skin, bones, and muscle tissues, and this phenomena has inspired the scientific community for decades to design synthetic auto repairable materials. 110 The ability of self healing polymers to reorganize themselves at a molecular level after mechanical damage renders them suitable for sensing applications, designing shape memory materials, and increasing the life time of surface coatings. 111 , 112 The pro cess of healing cracks in natural systems often involves an energy dissipation mechanism, due to the presence of sacrificial bonds that can break and reform dynamica lly before or while the failure occurs. 113 Several covalent ( e.g ., Diels Alder linkages, hydrazone bonds, alkoxyamines, and disulfide linkages) 12 , 68 , 114 and non covalent interactions ( e.g., ligand coordination) 115 , 116 have been employed to prepare self healing polymers. However, the use of reversible covalent or dynamic covalent interactions to prepare such materials remains a particularly interesting Reprinted from Soft Matt . 2015 , 11 , 6152 6161 , with permission from the Royal Society of Chemistry .

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83 possibility for creating robust materials that are covalently crosslinked, while maintaining the dynamic nature that allows healing by bond reorganization . 95 , 117 Moreover, incorporation of labile covalent bonds into densely crosslinked m acromolecules allows not only tailoring of their mechanical properties but also tuning of their macroscopic response when exposed to a stimulus. Among the various branched/crosslinked polymeric architectures, hydrogels have been studied extensively for diverse biomedical applications. 68 , 69 , 116 , 118 , 119 , 120 Hydrogels are three dimensional crosslinked polymeric networks swollen in large amounts of water that have many of the characteristics of cellular environments, such as their high water content, the diffusivity of small molecules in the hydrogel matrix, and comparable elasticity and mechanical prope rties that are often similar to those of soft tissue . 121 , 122 These materials are often considered as prime candidates for biosensors, delivery vehicles for drugs and biomolecules, and carriers or matrices for cells in tissue engineering. Traditionally, hydrogels have been synthesized by non covalen t and covalent crosslinking in attempts to mimic biological environments. However, the majority of these hydrogels lack the dynamic properties necessary for many complex tissue processes. Recent attempts to introduce dynamic complexity into hydrogels, more specifically, responsiveness to biological stimuli or sol to gel transition behavior, have beg u n to address very fundamental questions about how synthetic hydrogels function in cellular environments. 121 For example, introduction of hydrolytically degradable components in PEG and hyaluronic based hydrogels containing encapsulated mesenchymal stem cells for cartilage tissue engineering can lead to more uniform tissue dist ribution and cellular organization due to the improved distribution of chondroitin sulfate , an extra cellular matrix molecule in the cartilage . 123 Recently Jiang et al. demonstrated a liquid lens system consisting of a pH and temperature responsive hydrogel which could actuate adjustments to the shape and , accordingly, the focal length and

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84 autonomous focus of a liquid droplet contained in the hydrogel by sensing the change in pH/temperature of the medium. 124 Hydrogels have also been designed to sense other environmental changes, such as light intensity, 125 electric signal s , 126 redox po tential s , 120 , 127 and variations in glucose concentration. 128 Incorporation of reversible bonds in hydrogels allows post synthetic tailoring of network properties, such as crosslink density, swelling, and mechanical strength. 129 A variety of reversible linkages have been utilized in the past to prepare stimuli responsive hydrogels having self healing capabilities. For example, Chen et al. investigated the reversible sol to gel transition and self healing properties o f organo /hydrogels having acyl hydrazone bonds, and reported the gelation kinetics and mechanical properties of acyl hydrazone containing organogels. 68 70 In another report Anseth et al have shown that the temperature and pH of the reaction medium influenced the reaction rate constant of acyl hydrazone formation, and the reverse reaction rate constant a ffected the s tress relaxation characteristics of the acyl hydrazone containing hydrogel. 130 Fulton and coworkers, demonstrated an interesting reversible transformation from acyl hydrazone con taining single chain nanoparticles (SCNP) to chemically crosslinked hydrogels, facilitated by intermolecular reorganization of the collapsed polymer chains when the SCNPs were heated above their lower critical solution temperature. 65 Diels Alder chemistry is another widely used method to design degradable hydrogels, and recently both Bowman et al . an d Goepferich et al. demonstrated that poly(ethylene glycol) based hydrogels containing furan maleimide Diels Alder linkages could degrade at human body temperatures by the retro Diels Alder mechanism. 42 , 4 3 Several other reversible bonds, such as imines, 119 disulfides, 131 boronate esters , 132 , 133 a nd thermoreversible alkoxyamines 134 have been used to construct dynamic covalent hydrogels, however, the reversibility of oxime containing hydrogels has yet to be explored.

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85 Oxime ligation has proven particular ly promising for bioconjugation, due to the high reaction efficiency, ambient reaction conditions, benign side product ( i.e. , water), and most importantly, the superior hydrolytic stability of oxime bonds. 22 , 71 , 72 , 74 , 75 Maynard et al. incorporated oxime bonds into hydrogels that can encapsulate stem cells and support cell adhesion. 135 Oxime ligation was also utilized by Becker et al. to prepare poly(ethylene glycol) based hydrogels for three dimensional patterning of peptides onto a hydrogel matrix by photoinitiated thiol ene chemistry . 76 However, there are limited examples of exploiting the controlled exchange behavior of oxime linkages, even in small molecule chemistry, 21 , 22 , 81 , 136 and it is only recently that our group has demonstrated the synthesis and characterization of oxime containing core crosslinked star polymers that can degrad e by competitive oxime exchange within the core. 20 Inspired by our recent success with core crosslinked star polymers, 17 , 18 , 20 , 44 we reasoned that the controlled exchange behavior of oxime bonds could be tuned to obtain self repairable hydrogels having reversible sol to gel transition capabilities under suitable conditions. The covalent nature of the oxime bond is expected to contri bute to the mechanical integrity of the hydrogel matrix, while the reversibility of oxime bonds will allow the components to reshuffle at the molecular level when u nder stress to effect healing. Herein , we describe the synthesis of novel hydrogels that contain oxime crosslinks and are capable of autonomous healing due to their dynamic nature. The reversible sol to gel transition was induced in the presence of excess monofunctional alkoxyamine and an acid catalyst. 4.2 Results and Discussion We were interested in creating crosslinked polymeric materials by incorporating reversible linkages that were hydrolytically stable under most conditions in aqueous media but were susceptible to dynamic exchange when triggered by a change in pH or the addition of

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86 specific small molecules. This dynamic behavior is the result of linkages that can be reversibly disassembled and reformed under thermodynamic equilibrium. 137 Incorporation of the dynamic or reversible oxime bonds in the hydrogels was expected to not only induce reversible sol to gel transition by competitive exchange with excess aminooxy compo unds, but also to enable the system to heal autonomously after damage. Diacetone acrylamide (DAA) and N,N dimethylacrylamide (DMA) were copolymerized by conventional radical polymerization to obtain keto functional hydrophilic copolymers with varying DAA c ontent (Scheme 4 1). The resulting ketone containing copolymers were crosslinked with a difunctional alkoxyamine in aqueous media to result in hydrogels containing the condensation product of the pendent ketone and alkoxyamine ( i.e ., ketoximes). The revers ible nature of the hydrogels was first demonstrated by the gel to sol transition in the presence of excess monofunctional alkoxyamine. As detailed below, the hydrogels were able to self repair by oxime exchange after damage occurred. The mechanical propert ies of the hydrogels were studied by rheometry. 4.2.1 Synthesis of keto functional copolymers and formation of supramolecular hydrogel DAA was chosen as a comonomer due to its keto functional group, which could be modified with an alkoxyamine compound by the highly efficient oxime formation reaction. DAA was copolymerized with DMA by conventional radical polymerization to obtain P(DMA m stat DAA n ) with the mole fraction of DAA ( m ) content varying from 0.28 to 0.48 (Table 4 1). The number average molecular w eight ( M n ) of the polymers was determined by SEC. The amount of DAA incorporated in the copolymer was obtained by 1 H NMR spectroscopy by comparing the area of the methyl protons in the DMA unit ( N (C H 3 ) 2 , = 2.90 ppm) and the area of the methyl protons ( (C=O) C H 3 , = 2.04 ppm) in the DAA unit in the P(DMA stat DAA) copolymers (Fig. 4 1 ).

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87 Scheme 4 1. Synthesis of P(DMA m stat DAA n ) Recently Cai et al . reported DAA containing copolymers having tunable aq ueous solubility in response to temperature . 86 We were interested in investigating whether the DAA within our P(DMA stat DAA) copolymers could impart composition dependent thermoresponsive behavior to our hydrogels. 138 Interestingly, the presence of the DAA unit imparted thermoresponsive behavior to the copolymers. For example, the copolymers P(DMA 0.52 stat DAA 0.48 ) and P(DMA 0.6 stat DAA 0.4 ) exhibited cloud points of 34 and 61 °C, respectively, for solutions of 0.5 mg/mL (Fig. 4 2 ). Figure 4 1. 1 H NMR spectrum of poly( N,N dimethylacrylamide stat diacetone acrylamide) (P(DMA 0.72 stat DAA 0.28 )).

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88 However, copolymers with lower contents of DAA did not demonstrate thermoresponsive behavior, with solutions (0.5 mg/mL) of P(DMA 0.68 stat DAA 0.32 ) and P(DMA 0.72 stat DAA 0.28 ) remaining homogeneous up to 70 °C. Figure 4 2. Determination of cloud point (CP) . CP was determined by turbidi ty by monitoring the change in transmission (%T) with increasing temperature of (A) P(DMA 0.6 stat DAA 0.4 ) (B) P(DMA 0.52 stat DAA 0.48 ) (0.5 mg/mL in deionized water). Table 4 1 . Copolymers with N,N d imethyl acrylamide (DMA) and d iacetone a crylamide (DAA) Entry Polymer M n,SEC a (kg/mol) M w,SEC a (kg/mol) M w / M n a DAA mole% b P1 P(DMA 0.72 stat DAA 0.28 ) 250 370 1.48 28 P2 P(DMA 0.68 stat DAA 0.32 ) 309 412 1.33 32 P3 P(DMA 0.60 stat DAA 0.40 ) 271 371 1.37 40 P4 P(DMA 0.52 stat DAA 0.48 ) 277 395 1.42 48 a Determined by SEC with multiangle light scattering detection. b Calculated from 1 H NMR spectroscopy. When the aqueous solutions of P(DMA 0.6 stat DAA 0.4 ) and P(DMA 0.52 stat DAA 0.48 ) (0.2 g/mL in PBS, pH 7) were heated, the copolymers demonstrated temperature responsive solubility. Interestingly, at this relatively high concentration, the solutions became opaque as the

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89 polymer chains collapsed and the solutions gelled, as con firmed b y vial inversion (Fig. 4 3 and Fig. 4 4 ). To understand the supramolecu lar gelation process further, rheological temperature step experiment s were carried out. Figure 4 3. Reversible sol to gel transitions of P(DMA 0.52 stat DAA 0.48 ) (A ) by vial inversion and ( B ) by temperature step rheology experiments at 10 rad/s and 1% strain. Upon heating, the thermoresponsive nature of these copolymers led to reversible physical gelation. The polymer solution was heated and cooled in cycles, and the change in modulus was phenomenon was demonstrated by carrying out the te mperature step experi ment over several cycles (Fig. 4 3B and Fig. 4 4B behaviour is possibly d ue to the presence of residual physical aggregation and chain entanglement that persists after cooling below the cloud point of the polymer. The kinetics of

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90 demixing and remixing and the elastic properties of physical hydrogels can be complicated by a vari ety of factors, including heating rate and the onset of phase separation and sedimentation above the cloud point. 139 Even though a thorough and more detailed investigation is required to interpret the na ture of the supramolecular gelation process, the current result nicely shows that the DAA polymers could be well utilized to form physically crosslinked hydrogels. Figure 4 4. Reversible sol to gel transitions of P(DMA 0.60 stat DAA 0.4 0 ) ( A ) by vial inve rsion and ( B ) by temperature step rheology experiments at 10 rad/s and 1% strain. Upon heating, the thermoresponsive nature of these copolymers led to reversible physical gelation. 4.2.2 Hydrogels from P(DMA m stat DAA n ) and difunctional alkoxyamines Given the high efficiency of oxime formation in aqueous media, as demonstrated previously by our group and others, 20 , 72 , 84 , 140 we believed the reaction of polymer bound ketones and multifunctional alkoxyamines could be a viable means to prepare densely crosslinked polymeric networks, such as hydrogels or organogels. T he minimum concentration of P(DMA m

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91 stat DAA n ) and the [ketone]:[alkoxyamine] ratio required for gel formation were determined to be 0.05 g/mL and 1.5:1, respectively, in aqueous and organic (methanol) media. Considering the utility of hydrogels for many biological applications, we decided to focus o n gel formation in aqueous media. Hydrogel formation was triggered by mixing the polymer solution in PBS (pH 7) with a solution of O,O 1,3 propanediylbishydroxylamine dihydrochlorid e in PBS (pH 7) at 25 °C (Fig. 4 5 ). Figure 4 5 . Hydrogel formation wi th P(DMA 0.68 stat DAA 0.32 ) at polymer concentrations 0.2 g/mL and [keton e]:[alkoxyamine] = 1:1 at 25 °C. To obtain more insight into the effect of polymer concentration and stoichiometry on the hydrogel formation kinetics, we carried out rheology experimen ts employing three different polymer concentrations ( i.e., 0.05, 0.1, and 0.2 g/mL) and two different stoichiometries ( i.e., [ketone]:[alkoxyamine] = 1:1 and 1.5:1) (Fig. 4 6 ). Gel formation was also confirmed more qualitatively by the vial inversion metho d ( Fig. 4 5 ). For rheological experiments, a dynamic strain sweep was carried out to determine the linear viscoelastic region of the gel, and a dynamic

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92 time sweep was conducted to observe the elastic modulus ( G ) and viscous modulus ( G ) for hydrogels form ed at different polymer concentrations and stoichiometries. For example, solutions of P(DMA 0.68 stat DAA 0.32 ) in PBS were mixed with solutions of the difunctional alkoxyamine crosslinker ([ketone]:[alkoxyamine] = 1:1 or 1.5:1), and the rheological measurem ents were began after a brief period of mixing. In all cases, G increased with time and eventually exceeded G , indicating the approximate onset of gelation. The value of crossover time for the hydrogel formation reaction was found to decrease with increasing polymer concentration due to the enhanced rate of the crosslinking reaction (Fig. 4 6 ). For example, the value of crossover time was 102 s for the hydrogel prepared with P(DMA 0.68 stat DAA 0.32 ) at 0.1 g/mL and [ketone]:[alkoxyamine] = 1.5:1, whil e the hydrogel formation carried out at a concentration of 0.2 g/mL copolymer with a similar stoichiometry led to a crossover time of 38 s. However, no significant change in was noticed when the crosslinker equivalence alo ne was increased (Fig. 4 6 ), su ggesting the concentration of crosslinker had less impact on gelation kinetics than the concentration of copolymer . of the hydrogels reached a near constant value (Fig. 4 7 ).

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93 Figure 4 6 . Dynamic time swe ep experiments with P(DMA 0.68 stat DAA 0.32 ) at different concentrations ( i.e ., 0.05 g/mL, 0.1 g/mL, and 0.2 g/mL) and different [ketone]:[alkoxyamine] ratios ( i.e ., 1.5:1 and 1:1) at 1% strain, 10 rad/s, and 25 °C. These results indicate gelation was faste r with increasing polymer concentration, while the ratio of [ketone]:[alkoxyamin e] groups had less of an effect. The ketone content in the copolymer available for crosslinking with the dialkoxyamine had a significant impact on the gelation kinetics and hyd rogel properties. Gelation was significantly faster with increasing DAA content in the parent copolymer. In fact, the copolymer with the highest DAA content (P(DMA 0.52 stat DAA 0.48 )) gelled immediately upon addition of the difunctional alkoxyamine, even be fore the time sweep experiment could be started. Ketone content also played a key role in determining hydrogel strength. For example, under identical conditions (0.2 g/mL copolymer, [ketone]:[alkoxyamine] = 1:1, 25 min), the equilibrium storage

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94 modulus of the hydrogels observed during the dyna mic time sweep experiment were 4.1 , 13, and 75 kPa for the hydrogels prepared from copolymers containing 32, 40, and 48% DAA, respectively. Figure 4 7 . Dynamic time sweep experiments for hydrogels formed from P(DMA 0.6 8 stat DAA 0. 32 ) at different concentrations ( i.e., 0.05, 0.1, and 0.2 g/mL) and different [ketone]:[alkoxyamine] ratios ( e.g., 1.5:1 and 1:1) (d ata presented for longer reaction time ) .

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95 Figure 4 8 . Dynamic time sweep experiments for hydrogels formed fr om P(DMA 0.6 stat DAA 0.4 ) at different concentrations ( i.e., 0.05, 0.1, and 0.2 g/mL) and different [ketone]:[alkoxyamine] ratios ( e.g., 1.5:1 and 1:1). This result is consistent with increased gel strength with increasing crosslink density. In all cases, th ly with frequency, suggesting t he frequency scale probe (>0.1 rad/s) was faster than the rate needed for the oxime links to rearrange themselves (Fig. 4 9 ). 68

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96 Figure 4 9 . Rheological experiments of P(DMA m stat DAA n ) copolym ers with varying DAA content. (A exper iment ([copolymer] = 0.2 g/mL, [ketone]:[alkoxyamine] = 1:1, 10 rad/s, 1% strain, and 25 °C). (B frequency sweep experiment ([copolymer] = 0.2 g/mL, [ketone]:[alkoxyamine] = 1:1, 10 % strain and 2 5 °C). The hydrogels were also imaged by SEM to observe the effect of ketone content on pore structure. As expected, higher DAA content in the copolymers ( i.e. , increased crosslink density in the gels) resulted in smaller pore sizes (Fig. 4 10 ) and reduc ed swelling ratios (Fig. 4 11 ). Figure 4 10 . SEM images of hydrogels prepared with 32, 40, and 48% DAA containing P(DMA m stat DAA n ) copolymers. Increasing DAA content led to increased crosslink density and decreased pore size.

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97 Figure 4 11 . Mass swelling ratio of hydrogels with different DAA content of P(DMA m stat DAA n ). Mass swelling ratio = (Mass of swollen gel Mass of dry gel)/Mass of dry gel. 4.2.3 Gel to sol transition While oxime functional hydrogels have been utilized for patter n formation and peptide encapsulation, 76 stem cell encapsulation, 135 and as an injectable delivery systems, 77 their reversible gel to sol transition and self mending ability has remained largely unexplored. Oxime containing polymers can be rendered dynamic, as we recentl y reported for the reversible disassembly of core crosslinked star polymers containing oxime functional cores. 20 We envisioned that incorporation of such reversible bonds in a hydrogel matrix would allow the gels to undergo a gel to sol transition upon cleavage of the crosslinks during addition of excess monofunctional alkoxyamin e that would compete for binding with the polymer bound ketones. Indeed, addition of 20 equiv O (tetrahydro 2 H pyran 2 yl)hydroxylamine along with catalytic TFA led to dissociation of hydrogels prepared with P(DMA 0.68 stat DAA 0.32 ) at 0.2 g/mL polymer conc entration and [ketone]:[alkoxyamine] = 1:1 within 2 h. When only 5 equiv of the monofunctional alkoxyamine was employed, 24 h was required for the gel to sol transition at 25 °C (Fig. 4 12 ). Similarly, the gel to sol transition became slower as the DAA con tent in the

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98 parent copolymer increased. The hydrogel prepared with P(DMA 0.6 stat DAA 0.4 ) required 24 h to become a solution with 20 equiv of monofunctional alkoxyamine, and the hydrogel prepared with P(DMA 0.52 stat DAA 0.48 ) never turned into a solution aft er several weeks at 25 °C, even with 50 equiv of monofunctional alkoxyamine. This observation is likely due to the relative hydrophobicity or the significantly reduced pore sizes within the highly crosslinked hydrogels prepared with P(DMA 0.52 stat DAA 0.48 ) , which limited diffusion of the monofunctional alkoxyamine through the collapsed hydrogel matrix to retard the gel to sol transition. Figure 4 12 . Gel to sol transition of oxime containing hydrogel prepared at [P(DMA 0.68 stat DAA 0.32 )] = 0.2 g/mL and [ketone]:[alkoxyamine] = 1:1. Addition of the monofunctional alkoxyamine led to crosslin k cleavage via transoximination. 4.2.4 Self healing behavior While considerably more stable than imines, many oximes remain susceptible to cleavage under specific reac tion conditions ( e.g., in presence of excess aldehyde/ketone and/or acid catalyst). 23 Moreover, hydrolytic cleav age of the oxime bond is reversible, which suggests that under carefully tuned aqueous conditions, the direction of the equilibrium between oximes and their alkoxy amine and ketone/aldehyde components can be tuned to permit facile oxime

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99 exchange. 22 We reasoned that reversible covalent bonds of this type could be u sed to induce self healing behavior . 141 To qualitatively assess the potential for self healing, two round hydrogel samples were prepared with P(DMA 0.68 stat DAA 0.32 ) and one equivalent of the difunctional alkoxyamine crosslinker. E ach hydrogel sample was cut into two equal pieces. One half of each respective hydrogel was brought in contact with the other and allowed to remain in contact in a Petri dish containing sufficient mo isture to avoid dehydration. We anticipated the intimate contact of the two hydrogel pieces wo uld promote oxime exchange across the damage interface to effect covalent healing. 133 , 141 Healing occurred within 2 h, as evidenced by the gel qualitatively regaining its original mechanical strength, being capable of withstanding stretching with a tensile force applied perpendicular to the cut line. The gels were also capable of supporting their own weight when suspended under gravity for extend ed periods of time (Fig. 4 13 ). A similar process was repeated three additional times with the same piece of previously healed hydrogel . S uccessful healing was observed after every cycle. This behavior suggested healing occurs by oxime bond formation acros s the cut interface as the chemical functionalities gradually diffuse across the interface of the cut pieces. 23 The self healing experiment was also performed with a hydrogel sample prepared with a different polymer concentration [P(DMA 0.68 stat DAA 0.32 )] = 0.05 g/mL) and stoichiometry ([ketone]:[alkoxyamine] = 1.5:1). Healing was observed after the cut pieces were brought together and kept in intimate contact for 2 h, suggesting that self healing can be achieved with a variety of hydrogel compositions. Interestingly , hydro gels prepared with P(DMA 0.6 stat DAA 0.4 ) and P(DMA 0.52 stat DAA 0.48 ) did not heal under similar conditions even after being kept in contact for several days. The densely crosslinked network in those

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100 hydrogels may have hindered the diffusion of chains requi red to bridge the damage interface and bring about healing. Figure 4 13 . Room temperature healing test of hydrogels prepared with P(DMA 0.68 stat DAA 0.32 ) an d [ketone]:alkoxyamine] = 1:1. A ) Two previously cut pieces of distinct hydrogel samples (a dye w as added to one gel for clarity). B ) and B were placed in contact for 2 h. C ) and C stretched with tweezers with tensile force being applied perpendicularly to the original cut. D ) and D aled gels suspended under gravity. e) After maturing the healed hydrogel for 24 h, the hydrogel was cut again along the previous cut line. To obtain quantitative insight into the dynamic or reversible nature of oxime exchange, a hydrogel sample was prepared on the rheometer plate and matured for 2 h before being intentionally damaged during a strain sweep experiment (0.1 1000% strain), such that the elastic modulus became less than the viscous modulus u pon strain induced failure (Fig. 4 14 ). After reducing the strain to 50%, the crosslinks rapidly recoupled in less than 17 s to restore conditions

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101 Figure 4 14 . Self he aling of hydrogels after fracture. The top plot shows the change in modulus (red and blue) during the strain ramp (green) described by the bottom plot. The hydrogel fractured during the strain ramp up to 387% and rapidly healed after the strain was reduced . The strain ramp was conducted at constant angular frequency of 10 rad/s and a constant temperature 25 °C. [P(DMA 0.68 stat DAA 0.32 )] = 0.2 g/mL, and [ketone]:[alkoxyamine] = 1:1. However, the moduli did not completely recover to their original values, which may indicate that the strain induced failure also led to some irreversible carbon carbon bond scission and/or partial loss of the sample during the strain sweep . Similar behavior was observed for the hydrogel prepared with 0.05 g/mL polymer concentra tion and [ketone]:[alkoxyamine] = 1.5:1 (Fig. 4 15).

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102 Fig ure 4 15 . Self healing of hydrogels after fracture. The top plot shows the change in modulus (red and blue) during the strain ramp (green) described by the bottom plot. The hydrogel fractured during the strain ramp up to 630% and rapidly healed after the strain was reduced. The strain ramp was conducted at constant angular frequency of 10 rad/s and a constant temperature 25 °C. [P(DMA 0.68 stat DAA0.32)] = 0.05 g/mL, and [ketone]:[alkoxyamine] = 1.5:1 using a 50 mm CP geometry . 4.3 C onclusion We demonstrated that ketoxime formation is an efficient route to prepare hydrogels that were both self healing and stimuli responsive. Several factors such as polymer composition, polymer concentration, stoichiometry, and temperature can be tuned to contro l the hydrogel properties. A significant rate of crosslinking was observed as the polymer concentration and DAA content in the polymer was increased, whereas the stoichiometry of the crosslinker seemed to have minimal effect on the gelation kinetics. The r eversible nature of oxime linkages led to reversible gel to sol transitions of the hydrogels in the presence of excess monofunctional

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103 alkoxyamine at ambient temperature and promoted autonomous healing in damaged hydrogel samples. Additionally, w e observed that higher incorporation of crosslinks resulted in a stronger hydrogels but restricted the polymer chain mobility necessary to cause healing in a damaged hydrogel. We believe that t he tunable degradation and healing behavior of these oxime hyd rogels may o ffer a viable mean to design biosensors, shape memory materials, and delivery vehicles for dyes and drugs. 4.4 E xperimental Section 4.4.1 Materials N,N Dimethylacrylamide (DMA, Fluka, 98%) was passed through a small column of basic alumina to remove inhibitor prior to polymerization. Diacetone acrylamide (DAA, Sigma Azobisisobutyronitrile (AIBN, Sigma Aldrich, 98%) was recrystallized from ethanol. O (Tetrahydro 2 H pyran 2 yl)hydroxylamine (Sigma Aldri ch, 96%), O 1,3 propanediylbishydroxylamine dihydrochloride (Sigma Aldrich, >99%), cresol red sodium salt (Alfa Aesar), trifluoroacetic acid (TFA, EMD Millipore, 99.5%), triethyl amine (TEA, Fisher C hemical, 99%), 1,3,5 trioxane (Acros Organics, 99.5%), N,N dimethylformamide (DMF, EMD, 99.9%), N,N dimethyl acetamide (DMAc, Sigma Aldrich, 99.9%), tetrahydrofuran (THF, EMD, 99.5%), dichloromethane (DCM, BDH, 99.5%), 1,4 dioxane (Fisher Chemicals, 99%), phosphate buffered saline (PBS, pH 7, Sigma Aldrich), methanol (Mallinckrodt, 99.8%), ethyl acetate (Fisher chemicals, 99.9%), hexane (BDH, 98.5%,), dimethylsulfoxide d 6 (DMSO d 6 , Cambridge Isotope, 99.9% D), and CDCl 3 (Cambridge Isotope, 99% D) were used as received. 4.4.2 Instrument and analysis Molecular weights and molecular weight dispersities were determined by size exclusion chromatography (SEC). SEC was performed with 0.05 M LiCl in DMAc at 55 °C and a flow rate

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104 of 1.0 mL/min (Agilent isocratic pum p, degasser, and autosampler, columns: PLgel + two ViscoGel I series G3078 mixed bed columns: molecular weight range 0 20 × 10 3 and 0 100 × 10 4 g/mol). Detection consisted of a Wyatt Optilab T rEX refractive index detector operating at 658 nm a nd a Wyatt miniDAWN TREOS light scattering detector operating at 659 nm. Absolute molecular weights and molecular weight dispersities were calculated using the Wyatt ASTRA software. Dynamic light scattering (DLS) was conducted at 173° with a Malvern Zetas izer Nano ZS equipped with a 4 mW, 633 nm He Ne laser and an Avalanche photodiode detector. UV Vis spectroscopic measurements were conducted with a Varian Cary 500 Scanning UV Vis NIR spectrophotometer. Proton NMR spectra were recorded using Inova spectrom eters operating at 500 MHz. Chemical shifts are reported in parts per million (ppm) downfield relative to tetramethylsilane (TMS, 0.0 ppm). The lyophilized hydrogel samples were examined and digital micrographs acquired by field emission scanning electron microscope (S 4000, Hitachi High Technologies America, Inc. Clarksburg, MD USA). The hydrogel samples were mounted on carbon adhesive tabs on aluminum specimen mounts. Samples for SEM were rendered conductive with an Au/Pd sputter coater (DeskV, Denton Vac uum, Moorestown, NJ USA). The mechanical properties of the hydrogels were determined by rheological experiments on a TA Instruments ARES LS1 rheometer using 25 and 50 mm cone and plate geometries for gelation kinetics and parallel plate geometries for pre formed gels. A solvent trap was used to minimize solvent loss during long experiments. 4.4.3 Synthesis and Experimental Procedures Conventional radical polymerization of DMA and DAA (P(DMA stat DAA), P 2 ) . A typical procedure for the synthesis of P(DMA stat DAA) was as follows. DMA (14.0 g, 0.194 mol), DAA (3.64 g, 0.0215 mol), AIBN (0.0214 g, 0.129 mmol), s trioxane (0.193 g, 2.16 mmol), and 1,4 dioxane (25 mL) were sealed in a 100 mL round bottom

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105 flask with a rubber septum and purged with nitrogen for 40 min. The reaction flask was then placed on a preheated silicon oil bath at 60 °C. Samples were removed periodically using a degassed syringe to determine monomer conversion by 1 H NMR spectroscopy. The polymerization was quenched after 52 min at 48% convers ion of DMA by removing the round bottom flask from the oil bath and opening it to expose its contents to atmospheric oxygen. The reaction solution was dialyzed in DI water through a regenerated cellulose dialysis tubing having a molecular weight cut off (M WCO) = 3.5 kg/mol, and the product was isolated by lyophilization resulting in poly( N,N dimethylacrylamide stat diacetone acrylamide) (P(DMA 0.68 stat DAA 0.32 ), (i.e. , a copolymer with mole fractions of DMA and DAA equal to 0.68 and 0.32, respectively) P2 ) ( M n, SEC MALS = 309 kg/mol and M w / M n = 1.33). A similar procedure was followed to prepare P(DMA 0.72 stat DAA 0.28 ) ( P1 ) , P(DMA 0.60 stat DAA 0.40 ) ( P3 ), and P(DMA 0.52 stat DAA 0.48 ) ( P4 ). Preparation of organogel with P(DMA stat DAA) . To study gel formation in an organic medium such as methanol, P(DMA 0.52 stat DAA 0.48 ) ( P4, 0.050 g, 0.18 mmol) was dissolved in methanol (0.25 mL) in a 4 mL vial. O,O 1,3 Propanediylbishydroxylamine dihydrochloride (0.055 g, 0.31 mmol) was added to a mixt ure of methanol (1 mL) and TEA (0.086 mL, 0.62 mmol) under stirring. An aliquot (0.214 mL) of the resulting solution was added to the polymer solution, and the reaction vial was kept on a mechanical shaker at 25 °C until the solution gelled and stopped flo wing. The gel formation was confirmed by vial inversion. Organogels with other polymers at different polymer concentrations (0.1 g/mL and 0.05 g/mL) and different stoichiometries ( i.e ., [ketone]:[alkoxyamine] = 1:1) were prepared in a manner similar to th at described above.

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106 Preparation of hydrogel with P(DMA stat DAA) . To study the hydrogel formation, P(DMA 0.52 stat DAA 0.48 ), P4 (0.050 g, 0.18 mmol) was dissolved in PBS (pH 7, 0.25 mL) in a 4 mL vial. O,O 1,3 Propanediylbishydroxylamine dihydrochloride ( 0.050 g, 0.28 mmol) was dissolved in PBS (1 mL), and an aliquot (0.21 mL) of the resulting solution was added to the polymer solution. The reaction vial was kept on a mechanical shaker at 25 °C until the solution turned into a gel and stopped flowing. Gel formation was confirmed by vial inversion and characterized by rheometry. Hydrogels with other polymers at different polymer concentrations ( e.g., 0.1 g/mL and 0.05 g/mL) and different stoichiometries ( i.e ., [ketone]:[alkoxyamine] = 1:1) were prepared and characterized in a manner similar to that described above. Determination of mass swelling ratio of hydrogels . The hydrogels used for determining swelling ratio were prepared in a manner similar to that described above and were dialyzed against deionized w ater. The increase in hydrogel weight was monitored until there was no further change in weight. The hydrogels were lyophilized to obtain the dry gel. The experiment was repeated with 3 4 hydrogel samples. Mass swelling ratio was determined by Mass swelli ng ratio = (Mass of swollen gel Mass of dry gel)/Mass of dry gel. Determination of the cloud point of P(DMA stat DAA) by turbidity . P(DMA 0.6 stat DAA 0 .4 ), P3 (0.5 mg) was dissolved in deionized water (1 mL), and the resulting solution was transferred in to a glass cuvette for turbidity studies using a UV Vis spectrophotometer. The change in transmittance values with increasing temperature was recorded to obtain the cloud point, defined here as the temperature corresponding to 50% reduction of transmission at 500 nm of the polymer.

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107 Preparation of thermoresponsive hydrogel with P(DMA stat DAA) . P(DMA 0.6 stat DAA 0.4 ), P3 (0.10 g, 0.31 mmol) was dissolved in PBS (pH 7, 0.5 mL), and the solution was heated above the cloud point of the polymer ( i.e ., 61 °C) to induce gelation. Gel formation was confirmed by the vial inversion and oscillatory rheological measurements. Thermoresponsive hydrogel with P(DMA 0.52 stat DAA 0.48 ) was prepared in a similar manner. Hydrogel to sol transition . P(DMA 0.68 stat DAA 0. 32 ), P2 (0.10 g, 0.26 mmol) was dissolved in PBS (pH 7, 1 mL), and the resulting solution (0.3 mL) was transferred to a 4 mL vial. O,O 1,3 Propanediylbishydroxylamine dihydrochloride (0.050 g, 0.28 mmol) was dissolved in PBS (1 mL), and the resulting solu tion (0.098 mL) was added to the polymer solution in the vial. The reaction solution was kept on a mechanical shaker for four days to mature the resulting hydrogel. O (Tetrahydro 2 H pyran 2 yl)hydroxylamine (0.20 g, 1.7 mmol) was dissolved in PBS (pH 7), t he resulting solution (0.16 mL) and TFA (10 µL) were added to the hydrogel, and the reaction vial was placed on a mechanical shaker. The change in viscosity of the reaction mixture was observed visually at different time intervals to monitor the gel to sol transition. The other hydrogels were investigated in a similar manner. Rheological investigation of gelation kinetics and mechanical properties . Two different geometries (either 25 or 50 mm cone and plate) were used to study the hydrogel formation kinetic s using a TA ARES LS1 rheometer. The viscoelastic region for each hydrogel was determined by a dynamic strain sweep experiment with a preformed hydrogel at a frequency of 10 rad/s and 25 °C. For a typical dynamic time sweep experiment P(DMA 0.68 stat DAA 0.3 2 ) (0.020 g, 0.053 mmol) was dissolved in PBS (pH 7, 0.1 mL), and the solution was transferred onto the 25 mm plate. O,O 1,3 Propanediylbishydroxylamine dihydrochloride (0.050 g, 0.28 mmol) was dissolved in PBS (1 mL) and an aliquot (0.095 mL) of the resu lting solution was

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108 added to the polymer solution on the plate before mixing homogeneously for 43 45 s. A solvent trap was used during the test to prevent solvent loss. The time sweep experiment was carried out at a frequency of 10 rad/s, 1% strain at 25 °C to obtain the crossover point of the storage and loss modulus values ( and , respectively). A frequency sweep test was conducted with a preformed gel at 25 °C and a constant strain (1 0 % s train). All the experiments were repeated 2 3 times. A similar procedure was followed to characterize the other hydrogels. Rheological studies of thermoresponsive hydrogels . P(DMA 0.6 stat DAA 0.4 ) ( P3 , 0.10 g, 0.31 mmol) was dissolved in PBS (pH 7, 0.5 mL), and the resulting solution (0.2 mL) was transferred to the 25 mm plate. A dynamic temperature step test was carried out at a frequency of 10 rad/s, 1% strain, from 25 to 65 °C in 3 consecutive cycles. A dynamic temperature step experiment with P(DMA 0.52 stat DAA 0.48 ) was carried out in a similar manner. Self healing study of hydrogels . Self healing was carried out with preformed hydrogels prepared at either [P(DMA 0.68 stat DAA 0.32 )] = 0.05 g/mL and [ketone]:[alkoxyamine] = 1.5:1 or [P(DMA 0.68 stat DAA 0.32 )] = 0.2 g/mL and [ketone]:[alkoxyamine] = 1:1. P(DMA 0.68 stat D AA 0.32 ) (0.20 g, 0.53 mmol) was dissolved in PBS (pH 7, 1 mL), and two portions of the resulting solution (0.18 mL each) were transferred into two silicone molds on a glass plate. A solution of cresol red (0.015 mL, 0.050 g/mL) was added to one of the mold s containing polymer solution and mixed well. O,O 1,3 Propanediylbishydroxylamine dihydrochloride (0.050 g, 0.28 mmol) was dissolved in PBS (1 mL), and two portions (0.17 mL each) of the resulting solution were added to both polymer solutions in the mold and mixed well. The molds were kept in a closed Petri dish saturated with moisture and allowed to mature for four days before study. The hydrogels were cut in half, and the cut surface of one half of the clear hydrogel was kept in

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109 contact with one half of the colored hydrogel. Healing was confirmed by stretching the healed hydrogel with tweezers from both sides of the cut after 2 h and also by the ability of the healed hydrogel to hold its structure when suspended under gravity for months. The experiment wa s repeated three more times by cutting the healed sample along the same line of the previous cut and also in a perpendicular direction. Each time the healed hydrogel was matured for 24 h before being cut again. Self recovery study using rheometer . A prefor med hydrogel prepared with P(DMA 0.68 stat DAA 0.32 ) ( P2 , 0.2 g/mL) and 1 equivalence of the dialkoxyamine crosslinker (0.05 g/mL) in a manner similar to that described above was used for the strain sweep test (0.1 1000% strain). The hydrogel was matured for 2 h before intentionally damaging it during a strain sweep test. The hydrogel was broken with high strain, and the recovery of the hydrogel was observed with a time sweep at 1% strain, 10 rad/s frequency at 25 °C using a 25 mm CP geometry. A similar proce dure was adopted for the hydrogel prepared with P(DMA 0.68 stat DAA 0.32 ) ( P2 , 0.05 g/mL) at [ketone]:[alkoxyamine] = 1.5:1 using a 50 mm CP geometry. The hydrogel was matured for 3 h before beginning the strain sweep experiment.

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110 CHAPTER 5 DYNAMIC D UO : D OUBLY D YNAMIC COVALENT P OLYMERS COMPOSED OF OXIME AND OXY NORBORNENE LINKS 5.1 Overview Combining the dynamic na ture of supramolecular chemistry with the robustness of covalent bonds has led to the development of covalent chemistry , which refers to reactions carried out reversibly under equilibrium conditions . 101 , 137 A tremendous amount of progress has been made in designing reversible polymeric materials for the purpose of molecular recognition, drug delivery, and self reorganization by utilizing dynamic covalent bonds. 1 The a bility of dynamic covalent linkages to u ndergo reversible formation and dissociation al lows the creat ion of dynamic polymer s that can adapt their structures and composition in response to external stimul i . 95 , 112 The scope of dynamic covalent chemistry has been expanded further in the /stereospecific chemica l reactions ( e.g. , site specific protein modification, selective modification of living cells) reversibly in the presence of multiple reactive functionalities, by employing orthogonal chemistries. 142 The concept of orthogonal chemistry was first coined by Merrifield and Barany in 1977 in the context of protecting group removal strategy for peptide synthesis. 143 Since that time, the concept of orthogonality has been exploited for synthesizing complex biomolecules such as natural products, oligosaccharides, and glycoproteins, and to decorate polymers with multiple functionalities , as well as for catalysis and surface modification. 144 The orthogonal functionalization strat egy not only allows the synthesis of complex macromolecules by multifunctionalization of polymers in one pot but also often eliminates the need for a protecting 1 A portion of this material was submitted to Polym. Chem. 2015 and reprinted with permission from the Royal Society of Chemistry.

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111 group. One of the seminal examples of an orthogonal coupling was by Ogawa et al. for the synthesis of long chain oligosaccharides by employing two sets of glycosyl donors and two different anomeric groups ( i.e. , phenylthio and fluoro) that could be activated by different conditions without interfering with each other. 145 This concept was extended to solid phase oligosaccharide synthesis 146 and to the creation of a combinatorial library of oligosaccharides. 147 A similar strategy was applied by Frechet et al. to synthesize dendrimers by the convergent approach using two different AB 4 type monomers by highly efficient orthogonal coupling reactions ( i.e ., Mitsunobu esterification and Sonogashira coupling ). Each orthogonal reaction added two layers to the dendrimers, and this method remains the most rapid synthetic pathw ay to dendrimers, requiring only three synthetic steps from monomer to sixth generation dendrimers of molecular weight ~ 21 kg/mol. 148 Maynard et al. demonstrated iterative electron beam lithography patterning combined with two orthogonal click reactions oxime ligation and Cu catalyzed alkyne azide cycloaddition to immobilize two different proteins ( i.e ., myoglobin and ubiquitin) on silicon surface s. 149 Several other orthogonal coupling reactions such as Diels Alder, 42 , 44 , 103 , 150 , 151 thiol ene, 152 hydrazone formation, 153 disulfide reduction, 17 , 68 boronic ester formation, 18 , 133 , 154 and nitroxide radical coupling 155 have also been employed to prepare complex polymeric materials. 156 Addition of the d imension of reversibility, particularly under a specific stimulus, to the orthogonal multi functionalization approach is fairly new. The ability to tune the response of a material in presence of multiple stimuli can lead to higher specificity with respect to the site of the response and its mechanism, a feature that would be highly beneficial in the field of drug delivery and sensing applications. The sequential use of multiple non covalent reversible interactions to prepare well defined self assemblies was first realized in 1997 by Reinhoudt et

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112 al ., who utilized hydrogen bonding and metal ligand coordination to prepare self assembled nanosized metallodendrimers as large as 28 kg/mol. 157 More recently Schubert et al ., used small molecules consisting of multiple recognition units for hydrogen bonding (u reidopyrimidinone moeity) and terpyridine metal ligand coordination to prepare linear polymers. 158 Lehn and coworkers combined a reversible covalent (imine exchange) and a reversible non covalent interaction (metal ligand coordination) to create double level orthogonal dynamic combinatorial libraries. 159 The same group later utilized reversible imine linkages and biodegradable polyester units to prepare a green dynamer which exhibited good mechanical properties, moldability, mendability and biodegradability. 160 In another interesting example Yang et al. , combined two orthogonal reversi ble chemistries ( i.e. , anthracene dimerization and host guest interaction ) to obtain smart responsive linear polymers. 161 Anthracene functional pillar[5]arene was dimerized upon exposure to UV irradiation (350 nm). The pillar[ 5]arene functional dimers then yielded supramolecular polymers when mixed with bisimidazole alkane via a host guest interaction. The supramolecular polymer could be dissociated through either an increase in temperature or exposure to UV irradiation (< 300 nm), followed by reformation upon re irradiating with UV light (> 350 nm) or cooling to 15 ° C. This dynamic polymerization was carried over several cycles. Another example of combination of stimuli responsive covalent and non covalent linkages was reported by Thayumanavan et al. , who incorporated multiple stimuli responsive orthogonal units in block copolymers comprised of an acid labile hydrophobic block, temperature sensitive hydrophilic block and a redox responsive disulfide interface. 162 The block copolymers self assembled into micelles capable o f encapsulating Nile red in aqueous media. Responsive release was induced by either lowering the pH of the medium or creating a reducing environment to cleave the disulfide linkages between two blocks. More recently, Fulton et al .,

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113 reported an excellent ex ample of the use of two reversible covalent bonds ( i.e ., disulfide and imine linkages) to prepare doubly stimuli responsive nanoparticles which could be disassembled by simultaneous application of acidic pH and reducing agents. 60 Sanders and Otto on the other hand, demonstrated the preparation of a dynamic combinatorial library by combining two communicating exchange process es, in cluding a monotopic acetyl group acting as a labile capping group for thiol s through thioester exchange and a ditopic dithiol monomer capable of disulfide and thioester exchange. 163 The thioester and d isulfide exchange process could be carried out sequentially by tuning the pH of the medi a from mildly acidic to neutral, and by allowing exposure to atmospheric oxygen at neutral pH. With our recent success of demonstrating of the reversibility of oxime bonds in core crosslinked star polymers 20 and hydrogels, 164 we were interested in exploring the possibility of combining oxime ligation with an orthogonal reversible chemistry to prepare functional polymers that could disassemble under two orthogonal conditio ns. Oxime bond formation is highly efficient, orthogonal, hydrolytically tolerant, and requires ambient reaction conditions. 22 , 71 , 72 , 74 , 75 Oxime ligation has recently received considerable attention for bioconjugation, surface modification with proteins, and cell surface engineering. 21 , 73 , 76 , 135 , 165 However, only relatively recently has the reversibility of oxime formation been expl oited for synthetic and materials applications. 20 , 164 , 166 In this report we combine the orthogonal furfuryl maleimide Diels Alder reaction with oxime ligation to prepare linear polymers capable of disassembly under two different sets of conditions. Diels Alder cycloaddition is a powerful synthetic method to pre pare functional materials and can be done without any need of catalyst. 150 , 167 Perhaps most importantly the reaction equilibrium can be tuned to favor either the forward or reverse reaction simply by changing the reaction te mperature. Diels Alder

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114 cycloaddition has been utilized to prepare self healing hydrogels, 42 , 43 thermoresponsive polyurethane two pack surface coating system from renewable resource such as soybean oil, 12 polymeric color switches, 39 and rewritable surface coating. 40 The oxime exchange process, on the other hand, can be carried out in the presence of competitive monofunctional alkoxyamines, aldehydes, or ketones, which compete for binding with existing oxime bonds under acidic conditions. 20 , 22 , 23 An AB type monomer was synth esized and used to prepare a novel linear polymer capable of disassembly and reassembly over multiple cycles. The monomer contains a central oxime unit with furfuryl and latent maleimido groups at the two ends. The polymerization proceeds by a step growth Diels Alder mechanism. The free maleimide end groups in the polymer were functionalized by thiol ene modification reaction in one pot immediately after the polymerization was quenched. The disassembly of the polymer was carried out by either oxime exchange with excess monofunctional alkoxyamines, or by a retro Diels Alder reaction. To our knowledge, this is the first example of a polymer that contains a backbone composed of oxime and oxy norbornene, two distinct types of readily reversible covalent bonds.

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115 Figure 5 1. Synthesis of doubly dynamic covalent polymer containing reversible oxime and oxynorbornene links . 5.2 Results and Discussion We anticipated that incorporation of two highly orthogonal reversible linkages in a polymer could lead to new adaptive smart materials with potential utility in an environment where chemical specificity is desired. An AB monomer was synthesized that has a reactive furfuryl and a latent maleimide unit connected by an oxime bond. The furfuryl and maleimide moieties undergo step growth polymerization to result in polymer containing both the reversible oxy norbornene and oxime bonds. The polymer was modified with thiol ene chemistry in one pot directly after the polymerization was quenched. The polymer was disassembled by oxim e exchange in presence of an acid catalyst at 60 °C. The successive disassembly and reassembly of the polymer was carried out by retro Diels Alder and Diels Alder reactions, respectively.

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116 5.2.1 Synthesis of AB monomer Our goal was to prepare a monomer tha t contains multiple orthogonal functionalities and would polymerize by step growth to yield a polymer possessing oxy norbornene and oxime bonds distributed sequentially throughout the backbone. A carboxyl containing alkoxyamine molecule ( 1 ) was functionali zed with furfural ( 2 ) by oxime formation. The remaining carboxyl group in 1 was modified by EDC coupling with a hydroxyl functional furan maleimide adduct ( 4 ) in the same pot to obtain the AB monomer ( 5 ). The maleimido group in 4 was protected with furan t o avoid undesired Michael addition to the maleimide double bond during EDC coupling. The oxime formation and EDC coupling reactions occurred at room temperature to yield the monomer ( 5 ), which was characterized by proton and carbon NMR spectroscopy (Fig. 2), mass spectrometry, and elemental analysis. The monomer was readily soluble in chloroform, DMAc, DMSO, and DMF, and had limited solubility in toluene.

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117 Figure 5 2 . Synthesis and characterization of AB monomer. A) Synthesis of AB monomer and B) 1 H NMR spectrum of the purified monomer in DMSO d 6 . 5.2.2 Step growth polymerization of the AB monomer Initiatlly, we considered the polymerization of the deprotected version of the furfuryl maleimide monomer. A solution of 5 in DMSO was heated to 100 ° C under constant nitrogen purging to induce the retro Diels Alder reaction needed to deprotect the maleimide group. Under these conditions, we expected the furan protecting group would be removed over the course of the reaction due to its volatility. Deprotection was indeed successful, as judged by the disappearance of the peak k in the 1 H NMR spectrum of the monomer (Fig. 5 2 B). However, we also observed simultaneous polymer formation by GPC, suggesting that deprotection was accompanied by step growth polymerizati on of the newly liberated maleimide groups with the furfuryl groups on the monomer. Therefore, we reasoned that it might be possible to combine the

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118 deprotection and polymerization steps into a one pot cascade like process. Evidence of the simultaneous depr otection polymerization process was confirmed by the size exclusion chromatography (SEC) traces (Fig. 5 3 B) of the reaction solution and the broadening of the 1 H NMR signals (Fig. 5 3 C) with increasing time. The area under the monomer peak (~19.9 min) in t he SEC chromatogram decreased gradually as the polymerization proceeded, and a new peak appeared at ~17 min shifted to lower elution time as the reaction progressed (Fig. 5 3 B), suggesting an increase in molecular weight. The monomer concentration, reactio n temperature, and reaction time influenced the molecular weight of the polymer . For example, higher molecular weight polymers were obtained when the monomer concentration was 0.50 g/mL ( P1) as compared to 0.38 g/mL ( P2 ) . At first it may be surprising that polymerization occurs efficiently under conditions at which deprotection of the maleimide occurs, considering the latter relies on a retro Diels Alder reaction that might be expected to limit the molecular weight of the polymer being formed due to concurr ent retro Diels Alder degradation of the newly formed oxy norbornene groups in the polymer backbone. However, as compared to typical furan maleimide reactions that would be relatively inefficient at elevated temperatures, at 100 ° C the Diels Alder reaction needed for polymerization of 5 seems to favor the forward reaction because of the oxime bond on furfural in the monomer serving to reduce the electron density of the diene. In fact, the molecular weight of the polymer ( P4 ) was found to be even higher when the reaction was carried out at 130 ° C for longer time. Despite the relatively slow polymerization kinetics under all the conditions considered, the polymerizations seemed to achieve relatively high conversions (Fig 5 3 A, Table 5 1).

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119 Figure 5 3 . Polymerization of AB monomer by step growth Diels Alder cycloaddition. A) Step growth polymerization of an AB monomer ( 5 ), B) Overlay of normalized refractive index traces from size exclusion chromatography and C) 1 H NMR spectr a of the purified polymer of P2 . Table 5 1 . Step growth polymers with an AB monomer ( 5 ) Polymer Monomer concentration (g/mL) Reaction temperature (°C) Reaction time (days) M n,SEC a (g/mol) DP c P1 0.38 100 9 3500 19 P2 0.50 100 9 6800 38 P3 0.50 130 10 8500 47 P4 0.50 130 17 13000 72 a,b Determined by SEC using PMMA calibration method in DMAc with 0.05M LiCl. c Number average degree of polymerization ( DP n ) = M n /(monomer molecular weight/2). 5.2.3 One pot post polymerization modification by thiol Michael addition reaction The presence of the maleimido end group in the polymer provided the opportunity for post polymerization modification via thiol Michael addition. We chose UV active 7 mercapto 4 -

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120 methylcoumarin for functionalizing the maleimi do end group of the polymer, as a UV active moiety at the chain end could be useful for labelling and imaging purposes. The model thiol we employed, 7 Mercapto 4 methylcoumarin (Cm SH), has been used as a standard substrate for monitoring S glucuronidation 168 and as a reporter of thiol binding to CdSe ZnS quantum dots. 169 Scheme 5 1. Functionalization of P3 with 7 mercapto 4 methylcoumarin. The thiol Michael addition of Cm SH to the polymer was carried out in the same pot after the polymerization was quenched. The polymerization solution of P3 was diluted with DMSO, and Cm SH and TEA were added (Scheme 5 1 ). The reaction was readily monitored by UV Vis spectroscopy, as the max of Cm SH in DMSO was 326 nm and P3 did not absorb in the same region. After the reaction, the functionalized polymer demonstrated the characteristic coumarin absorption (Fig. 5 4 D), indicating that the Cm SH was success fully attached to the polymer. The appearance of the characteristic peaks (a ' , b ', c', d', and f') of Cm SH in the 1 H NMR spectrum also confirmed the successful functionalization of the polymer (Fig 5 4 A). The SEC trace of the functionalized polymer shifte d to slightly lower elution time than the polymer

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121 ( P3 ), suggesting a slight increase in molecular weight occurred upon conjugation of Cm SH (Fig. 5 4C) . The facile reaction set up and mild reaction conditions of this model thiol Michael addition suggest th at this functionalization strategy could be an excellent avenue to conjugate other thiols or amines to the polymer for labelling and imaging purposes. Fig ure 5 4 . Functionalization of polymer ( P3 ) with 7 mercapto 4 methylcoumarin . (A) 1 HNMR spectra of the functionalized P3 in DMSO d 6 , (B) 1 HNMR spectra of the functionalized P3 in DMSO d 6 , (C) Normalized SEC refractive index traces of P3 (red) and purified functionalized P3 , and (D) UV Vis spectrum of DMSO (grey), and Cm SH (green), P3 (r ed) , and functionalized P3 (blue) in DMSO.

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122 5.2.3 Polymer d egradation by oxime exchange The reversibility and controlled exchange behavior of oxime bonds have been utilized in a number of areas, including determination of the position of carbonyl groups in steroids, 136 to engineer cell surfaces by attaching cells to an aminoxy patterned substrate by oxime formation and subsequently releasing the cells by oxime cleavage, 21 and also to prepare degradable core crosslinked star polymers containing oxime core, 20 and to prepare degradable core crosslinked star polymers 20 and self healing hydrogels. 164 Figure 5 5 . Polymer degradation by oxime exchange . A) Degradation of polymer P2 by oxime exchange, B) Overlay of the SEC refractive index traces of the degradation of polymer ( P2 ) by oxime exchange. The oligomer peaks at 19.7 min of the SEC traces of 4 and 6 days were normalized to the polymer peak (red dash line). Oxime linkages provide scope for reversible disassembly via competitive oxime exchange with excess monofunctional aldehydes, ketones, or alkoxyamines in the presence of an acid catalyst. 22 , 170 To investig ate the possibility of cleaving of the sequentially distributed oxime

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123 bonds within the polymer of monomer 5 , the polymer ( P2 ) was treated with an excess of monofunctional alkoxyamine in the presence of TFA at 60 ° C. As expected, the SEC trace shifted to hi gher elution time during the reaction as polymer gradually degraded to oligomers and monomer (Fig 5 5 B). 5.2.4 Polymer de gradation by retro Diels Alder The Diels Alder reaction is one of the most widely used chemistries to prepare thermally sensitive coval ent materials due to its facile reversibility, versatility, and easy reaction set up. 12 , 36 , 45 , 171 We envisioned that incorporation of a Diels Alder reactive unit in the polymer back bone would allow for thermal reversibility over several cycles and also would be an ideal ortho gonal functionality to oxime linkages, as oxy norbornenes remain inert under the reaction conditions of oxime exchange. To test our hypothesis, polymer (P1) was dissolved in DMSO d 6 , and the solution was heated to 160 °C. The progress of the retro Diels Al der reaction was monitored by SEC (Fig. 5 6 B). As the polymer was thermally degraded, the SEC traces shifted to higher elution times, confirming the generation of low molecular weight oligomers and monomers. Upon quenching the reaction solution after 9 day s and raising the temperature to 130 ° C, the oligomeric units and deprotected monomers combined to yield the healed polymer. The repolymerization reaction was observed for 7 days.

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124 Figure 5 6 . Degradation of the polymer by retro Diels Alder reaction. A ) Degradation of the polymer of 5 by retro Diels Alder and reformation of the polymer by Diels Alder; B) Overlay of the normalized SEC refractive index traces of the degradation and reformation cycles by retro Diels Alder and Diels Alder, respectively. 5. 3 Conclusion We employed oxime formation and the Diels Alder reactions to prepare a linear step growth polymer that can be induced to dissociate under two distinct sets of conditions. Due to the presence of reversible oxime links along the backbone, the polymer was cap able of undergoing oxime exchange with excess monofunctional alkoxyamine. The linear polymer was also self repairable over multiple cycles via Diels Alder and retro Diels Alder reactions. The oxime formation/exchange and Diels Alder reactions are orthogona l and can therefore be used to induce dissociation of specific bonds within a single polymer. This kind of polymeric material could be a platform to design even more complex polymeric architectures and to allow response to multiple stimuli. To our knowledg e, this is the first example of a polymer that contains a

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125 backbone composed of oxime and oxy norbornene, two distinct types of readily reversible covalent bonds. 5.4 Experimental Section 5.4.1 Materials Furan (Alfa Aesar, 99%), maleic anhydride (Alfa Aesar, 99%), ethanolamine (Sigma Aldrich, 99%), furfural (Sigma Aldrich, 98%), O (carboxymethyl)hydroxylamine hemihydrochloride (TCI, 98%), O (tetrahydro 2 H pyran 2 yl)hydroxylamine (Sigma Aldrich, 96%), 7 me rcapto 4 methylcoumarin (Cm SH, Sigma Aldrich, 97%), trifluoroacetic acid (TFA, EMD Millipore, 99.5%), triethylamine (TEA, Fisher Chemical, 99%), 1 (3 dimethylaminopropyl) 3 ethylcarbodiimide, HCl (EDC chloride, Combi Blocks, 95%), 4 (dimethylamino)pyridin e (DMAP, Alfa Aesar, 99%), benzyl mercaptan (Alfa Aesar, 99%), N,N dimethylformamide (DMF, EMD, 99.9%), N,N dimethylacetamide (DMAc, Sigma Aldrich, 99.9%), 1,4 dioxane (Fisher Chemicals, 99%), methanol (Mallinckrodt, 99.8%), ethanol (Fisher chemicals, 99.9 %), hexane (BDH, 98.5%), acetone (Fisher Chemicals, 99%), dichloromethane (DCM, BDH, 99.5%), dimethyl sulfoxide (Fisher Chemicals, 99.9%), dimethyl sulfoxide d 6 (DMSO d 6 , Cambridge Isotope, 99.9% D), and CDCl3 (Cambridge Isotope, 99% D) were used as receiv ed. 5.4.2 Instrumentation and analysis Molecular weights and molecular weight dispersities were determined by size exclusion chromatography (SEC). SEC was performed in 0.05 M LiCl in DMAc at 55 °C and a flow rate of 1.0 mL/min (Agilent isocratic pump, dega sser, and autosampler, columns: PLgel + two ViscoGel I series G3078 mixed bed columns: molecular weight range 0 20 × 10 3 and 0 100 × 10 4 g/mol). Detection consisted of a Wyatt Optilab T rEX refractive index detector operating at 658 nm and a Wy att miniDAWN TREOS light scattering detector operating at 659

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126 nm. Relative molecular weights were obtained through calibration with poly(methyl methacrylate) (PMMA) standards. UV Vis spectroscopic measurements were conducted with a Molecular Devices Spectr aMax M2Multimode Microplate reader using 0.45 mg/mL of sample solution in DMSO. The sample proton and carbon NMR spectra were recorded using Inova spectrometers operating at 500 MHz and 300 MHz. Chemical shifts are reported in parts per million (ppm) downf ield relative to tetramethylsilane (TMS, 0.0 ppm). Multiplicities are reported using the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. High Resolution Mass Spectrometry (HRMS) was conducted with an Agilen t 6220 TOF MS mass spectrometer in the Direct Analysis in Real Time (DART) mode with the IonSense DART source. 5.4.3 Synthesis and Experimental Procedures Synthesis of 3,6 epoxy 1,2,3,6 tetrahydrophthalic anhydride. Maleic anhydride (73 g, 0.74 mol) was d ispersed in diethyl ether (180 mL) in a 1 L round bottom flask equipped with a magnetic stirrer, and furan (300 mL, 4.13 mol) was added to the flask. Within a few minutes, the maleic anhydride had dissolved and the reaction was allowed to stir overnight at 25 °C. The desired product was crystallized from the ether. The solid was filtered and rinsed with an excess of ether before drying under vacuum overnight. (Yield: 95%). 1 H NMR (300 MHz, CDCl 3 ): (ppm) = 6.58 (m, 2H, C H =C H ), 5.46 (m, 2H, C H O C H ), 3.18 (m, (C=O) C H C H (C=O) ). 13 C NMR (500 MHz, CDCl3): (ppm) = 169.80 ( 2C, ( C =O) CH CH ( C =O) ), 136.90 (2C, C H = C H ), 82.10 (2C, C H O C H ), 48.60 (2C, (C=O) C H C H (C=O) ). 3,6 epoxy 1,2,3,6 tetrahydrophthalic anhydride

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127 Synthesis of N hydroxyethyl 3,6 epoxy 1,2,3,6 tetrahydrophthalimide . 3,6 Epoxy 1,2,3,6 tetrahydrophthalic anhydride (50 g, 0.30 mol) was dispersed in methanol (200 mL) in a 500 mL round bottom flask equipped with a magnetic stirrer, and ethanolamine (18.4 mL, 0.305 mol) was added to the flask. The solution was heated to 60 °C in an oil bath for 5 h. After heating, the solution was cooled to 20 °C overnight, causing the product to crystallize from solution. The solid was filtered and washed with a minimal amount of cold methanol. The filtrate was concentrated and cooled to 20 °C overnight to recover more product, that was subsequently filtered and washed with a minimal amount of cold methanol before drying under vacuum overnight. (Yield: 65%). 1 H NMR (500 MHz, CDCl 3 ): (ppm) = 6.51 (m, 2H, C H =C H ), 5.27 (m, 2H, C H O C H ), 2.88 (m, 2H, (C=O) C H C H (C=O) ), 3.75 (t, 2H, N CH 2 C H 2 OH), 3.69 (t, 2H, N C H 2 CH 2 OH), 2.31 ( 1H, CH 2 CH 2 O H ). 13 C NMR (500 MHz, CDCl3): (ppm) = 176.90 (2C, ( C =O) CH CH ( C =O) ), 136.90 (2C, C H = C H ), 81.2 0 (2C, C H O C H ), 60.20 ( C H 2 OH), 47.80 ( C H 2 CH 2 OH), 41.80 (2C, (C=O) C H C H (C=O) ). N hydroxyethyl 3,6 epoxy 1,2,3,6 tetrahydrophthalimide Synthesis of AB monomer (Furan 2 ylmethyleneaminooxy) acetic acid 2 (3,5 dioxo 10 oxa 4 aza tricyclo[5.2.1.02,6]dec 8 en 4 yl) ethyl ester). O Carboxymethyl)hydroxylamine hemihydrochloride (9.44 g, 86.4 mmol) was mixed with DCM (50 mL) and TEA (15.0 mL, 86.4 mmol) in a 1 L round bottom flask, and the resulting solution was con tinued to stir for 1 h at 25 °C. The reaction flask was cooled in an ice water bath to 10 °C before the addition of f urfural (12.0 mL, 0.145 mol). The reaction solution was stirred for 2 h. EDC chloride (50.1 g, 0.261

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128 mol), DMAP (3.18 g, 0.259 mol), and D CM (500 mL) were added to the reaction mixture. The flask was cooled to 5 °C in an acetone ice bath while stirring. N hydroxyethyl 3,6 epoxy 1,2,3,6 tetrahydrophthalimide (15 g, 0.072 mol) was added to the reaction mixture, and the reaction solution was c ontinued to stir for 3 more days at 25 °C. The reaction solution was concentrated by rotary evaporation, and the product was isolated by recrystallizing from ethanol at 58 ° C. Yield = 27%. 1 H NMR (500 MHz, DMSO): (ppm) = 2.91, 2.92 (d each, 2H, (C=O) C H C H (C=O) ), 3.64 (t, 2H, COO CH 2 C H 2 ), 4.20 (t, 2H, COO C H 2 CH 2 ), 4.72 (m, 2H, N O C H 2 COO ), 5.13 (m, 2H, C H O C H ), 6.54 (m, C H =C H CH O ), 8.23 (m, 1H, C H =N O ), 7.67, 7.80, 7.86 (m, 1H, C O C H =CH ), 6.84, 7.24 (m, 1H, C H =C( O )CH=N , 6.61, 6 .68 (m, CH C H =CH O ). 13 C NMR (500 MHz, CDCl 3 ): (ppm) = 176.78 ( ( C =O) N ( C =O) ), 169.70 ( CH 2 C OO ), 145.80 ( C C H=N ), 136.90 ( C= C H O C ), 115.40 ( O C H= C H CH ), 112.5 ( O C H 2 COO ), 80.80 ( C H O C H ), 70.60 ( COO C H 2 C H 2 ), 60.96 ( COO C H 2 C H 2 ), 47.70 ( N (C=O) C H C H ) , 37.30 ( (C=O) C H C H(C=O) ). (Furan 2 ylmethyleneaminooxy) acetic acid 2 (3,5 dioxo 10 oxa 4 aza tricyclo[5.2.1.02,6]dec 8 en 4 yl) ethyl ester Deprotection of the furan protected maleimido group in the monomer and concurrent polymerization (P2) . The monomer ( 1.0 g, 4.2 mmol ) was di ssolved in DMSO (2 mL) in a 20 mL septa vial equipped with a magnetic stirrer. The reaction vial was placed in an oil bath preheated at 100 ° C and continued to stir under nitrogen for 9 days. Aliquot s were

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129 removed periodically to monitor the reaction by SEC. The reaction was quenched by freezing the reaction solution in liquid nitrogen and precipitating in cold methanol (X 3). The product was dried under vacuum. ( P2 , M n (PMMA calibration) = 6800 g/mol , number average degree of polymerization ( DP n ) = 38). A similar procedure was followed to prepare P1 , P3 , and P4 , unless otherwise mentioned. One pot post polymerization modification by base catalysed thiol ene reaction. The polymer was modified by thiol ene Micheal addition directly after the polymerization was quenched in the same reaction vial. A typical procedure was as follows. The polymerization reaction of P3 was quenched by freezing the reaction solution in liquid nitrogen and then thawing to room temperature. The thawed reaction solution of P3 (0.60 mL, 0.055 mmol) was diluted with 1.6 mL DMAc in a 4 mL vial and the solution was purged with nitrogen for 30 min. 7 Mercapto 4 methylcoumarin (0.14 g, 0.75 mmol) and T EA (0.01 mL, 0.07 mmol) were added to the reaction mixture and the content was stirred at 25 °C for 24 h under nitrogen. The reaction solution was dialyzed against a mixture of 1,4 dioxane and DCM (90:10) through a regenerated cellulose dialysis tube havin g molecular weight cut off (MWCO) = 1 kg/mol. The solution in dialysis tubing was filtered to remove any insoluble Cm SH or its dimer. The filtrate was concentrated and precipitated in cold methanol (x2) to remove any trace amount of small molecule in the product. The product was vacuum dried. Degradation of linear polymer by oxime exchange. The polymer ( P2 , 0.015 g, 0.083 mmol) was dissolved in DMSO (1.5 mL) in a 4 mL vial equipped with a magnetic stirrer, and O (tetrahydro 2H pyran 2 yl)hydroxylamine (0.4 88 g, 4.16 mmol) and TFA (250 µL) were added to the solution. The reaction vial was placed in an oil bath preheated to 60 °C and the reaction

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130 solution stirred for 7 days. Samples were removed periodically to mon itor reaction progress by SEC. Reversibility of polymer over multiple cycles by repeated Diels Alder and retro Diels Alder reactions. Polymer ( P1 , 0.0760 g, 0.413 mmol) was dissolved in DMSO d 6 in a 4 mL vial equipped with a magnetic stirrer. The reaction vial was placed in a silicon oil bath prehea ted to 160 °C and the reaction solution stirred for 9 days to allow the retro Diels Alder reaction. Aliquots were removed periodically from the reaction vial to monitor the progress of the reaction by SEC. The reaction was quenched by cooling the reaction mixture with liquid nitrogen. For repolymerization, the frozen reaction solution was thawed and allowed to equilibrate to room temperature. The reaction vial was then placed in a silicone oil bath preheated to 130 °C, and Samples were removed periodically to mon itor reaction progress by SEC.

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131 CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS The research presented in this dissertation is meant to highlight the benefits of endowing macromolecules with reversible linkages. As compared to most polymers t hat are constructed exclusively by covalent bonds that are relatively stable or for which chain cleavage is meant to proceed irreversibly ( e.g ., degradable polymers), dynamic covalent polymers offer the potential for dramatic, though reversible, transforma tions in properties. The work contained herein demonstrates the synthesis of linear and branched polymers composed of reversible covalent bonds such as oxime and oxy norbornene Diels Alder adducts. Conventional radical, RAFT and step growth polymerizations were employed to prepare the polymers. Well defined block copolymers were prepared by RAFT polymerization and the selective crosslinking of the keto functional hydrophobic blocks with a difunctional alkoxyamine in aqueous media yielded core crosslinked st ar polymers by highly efficient oxime formation. The preassembly of the block copolymers into micelles in aqueous media resulted in high conversion of unimers to star polymers. The nanosized star polymers were disassembled by dynamic oxime exchange within the core in the presence of excess alkoxyamine or carbonyl compounds (aldehyde or ketone) and an acid catalyst at 60 °C. Similar synthetic protocol can be adopted to synthesize star polymers in which the core and shell impart different properties ( e.g. , cr ystallinity or response to different stimuli) by careful selection of the monomer and other starting materials. Moreover, the thermoresponsive nature of the diacetone acrylamide can be exploited to obtained various reversibly crosslinked nano architectures using self assembly and controlled polymerization. The keto functional statistical copolymers prepared by conventional radical polymerization, were crosslinked with a difunctional alkoxyamine to form hydrogels. The hydrogels were capable of autonomous he aling ( i.e. , without external intervention) and could

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132 undergo reversible sol to gel transition in the presence of excess monofunctional alkoxyamine and an acid catalyst at 25 °C. The tunable gelation and degradation behavior combined with the self mendable nature of the hydrogel may offer a viable means to design biosensors, shape memory materials, and delivery vehicle for dyes and drugs. Diels Alder step growth polymerization was employed to prepare a reversible polymer that could disassemble under two un ique sets of conditions due to the presence of oxime bonds and oxy norbornene links on the polymer backbone. Given the orthogonal nature of oxime formation and Diels Alder ligations, both reactions could be carried out in one pot. Such multifunctional reve rsible polymeric systems offer tremendous potential for designing sensors, molecular logic gates, and reorganizable polymers. While the research described here signifies the utility of dynamic covalent chemistry to synthesize simple linear and branched po lymers, similar synthetic strategies can be employed to construct more complex branched architectures ( e.g ., hyperbranched, star branched, and brush copolymers). Branched polymers with tunable crosslink density can be obtained by incorporating reversible c ovalent links which are responsive to different stimuli.

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144 BIOGRAPHICAL SKETCH Soma Mukherjee was born in Kankinara, West Bengal in eastern India. She received her Bachelor of Science degree in c hemistry (Honours) in June 2002 from Rishi Bankim Chandra College (Calcutta University ), Naihati, West Bengal. Soma obtained her Bachelor of Technology (B.Tech) in polymer science and technology in June 2005 from University College of Science and Technology (Calcutta University) , and was supervised by Prof. Bibek Das on the research project p reparation and characterization of poly(lactic acid) amylose conjugate as a drug delivery vehicle in Maharashtra (India) s Research Association as a junior research fellow in July 2005 . She also worked at PPG Asian Paints Pvt. Ltd. , an automotive paint company, as a technical officer. After spending about 5 years in industry Soma decided to go back to school to earn her PhD degree in Chemistry. Soma joined Pr of. Brent Sumerlin in Southern Methodist University in Dallas, TX in the fall of 2010 and transferred to University of Florida in the fall of 2012 with Prof. Brent Sumerlin. During the course of her graduate research s he worked on various linear and branch ed polymeric architectures which were capable of reversible structural transformations under specific stimuli and may potentially be used in designing sensors, carriers for dyes and drugs, and imaging agents. She participated in various conferences and del ivered oral and poster presentations of her research. She also participated in various outreach activities for popularizing science among local pre school to high school students. She received her PhD in chemistry in the summer of 2015. Apart from explorin g sci ence in academia and industry, S oma also took interest in Hindustani Classical Music and other forms of Indian music since her childhood. She obtained

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145 her Sangeet Visharad Pratham Khanda in classical vocal from Pracheen Kala Kendra, West Bengal in 199 9, and Madhyama Purna in classical vocal from A khil B haratiya Gandharva M ahavidyalaya , Mumbai in 2009. She continues to take opportunities to intersperse music throughout her daily routine by performing at local cultural programs.