Scheutz_G.pdf

Scheutz_G_pdf.txt

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DYNAMIC INTERACTIONS IN DESIGN AND SYNTHESIS OF POLYMER MATERIALS By GEORG SCHEUTZ 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 2020

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© 2020 Georg Scheutz

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To K.G. and H.B.

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4 ACKNOWLEDGMENTS I would like to thank Prof. Brent Sumerlin for being my mentor, advisor, and boss for the past f ive years. He always provided guidance, encouragement, support, and freedom when needed. Also, I want to thank Brent for constantly motivating me to challenge m yself and to strive for hi g h er goals. I want to express my gratitude to Prof . Ste ph en Miller, P rof . Wolfgang Sigmund, Prof. Adam Veige, and Prof . Ken Wagener for supporting me as my dissertation committee. A special thanks belongs to Prof. Wagener for his advice and encouragement throughout my graduate career and beyond. During my Ph . D . , I was fortu nate to work with great collaborators . I want to thank Dr. Mollie Touve, Dr. Tori Ellison, Dr. Eric Fuller, Jacob Lessard, Dr. Yening Xia , Dr. Fu Sheng Wang, Prof. Thomas Angelini, Prof. Chi How Peng, Prof. Carlos Rinaldi, and Prof. Nathan Giannesch i for t heir contributions and expertise. A big thank you goes to Johnathan Rowell, who was working with me for more than two years as an undergraduate research er. I am grateful for my friends and colleagues in the Sumerlin Research Group, past and present. Their insights , suggestions , and camaraderie have always been a great source of motivation and inspiration. Most of all, I am indebted to the senior graduate students of my first two years: Dr. Adrian Figg, Dr. Megan Hill, Dr. Nick Carmean, Dr. Tomo Kubo, Dr. C harlie Easterling, Dr. Chris Kabb, Dr. Hao Sun, and Dr. Kyle Bentz. I would not have gotten here without them.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 10 LIST OF F IGU RES ................................ ................................ ................................ ........ 11 LIST OF ABBREVIATIONS ................................ ................................ ........................... 17 ABSTRACT ................................ ................................ ................................ ................... 22 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 24 1.1 Dynamic Covalent Chemistry ................................ ................................ ........... 24 1.1.1 Disulfide Exchange ................................ ................................ .................. 25 1.2 S upramolec ular Chemistry ................................ ................................ ................ 29 1.2.1 Solvophobic Interactions ................................ ................................ ......... 29 1. 2.2 Amphiphilic Block Copolymer Assembly ................................ .................. 31 2 RESEARCH OBJECTIVE ................................ ................................ ....................... 33 3 SYNTHESIS OF FUNCTIONAL 1,2 DITHIOLANES FROM 1,3 BIS TERT BUTYL THIOETHERS ................................ ................................ ............................ 35 3.1 O verview ................................ ................................ ................................ ........... 35 3.2 Results and Disc ussion ................................ ................................ ..................... 38 3.3 Summary ................................ ................................ ................................ .......... 47 3.4 Experimental ................................ ................................ ................................ ..... 48 3.4.1 Instrument ation ................................ ................................ ........................ 48 3.4.2 General Procedure for the Synthesis of the 1,2 Dithiolane Derivatives ... 49 3.4.2.1 4 Hydroxy 1,2 dithiolane ( HDL ) ................................ ..................... 50 3.4.2.2 4 n Propyl 4 hydroxy 1,2 dithiolane ( n PrDL ) ................................ . 51 3.4 .2.3 4 Dodecyl 4 hydroxy 1,2 dithiolane ( C12DL ) ................................ . 51 3.4.2.4 4 Isopropyl 4 hydroxy 1,2 dithiolane ( i PrDL ) ................................ . 52 3.4 .2.5 4 Hydroxy 4 phenyl 1,2 dithiolane ( PhDL ) ................................ ..... 52 3.4.2.6 4 Hydroxy 4 (thiophen 3 yl) 1,2 dithiolane ( TphDL ) ...................... 53 3.4.2.7 4 Hydroxy 4 ( 5 bromothiophen 3 yl) 1,2 dithiolane ( BrTphDL ) ...... 53 3.4.2.8 3,3 Dimethyl 4 hydroxy 1 ,2 dithiolane ( DiMeDL ) ........................... 54 3.4.3 Synthesis of 4 Iso butyryl 4 phenyl 1,2 dithiol ane ( 2 ) ............................... 55 3.4.4 Synthesis of 4 Acrylate 4 isopropyl 1,2 dithiolane ( 3 ) ............................. 55 3.4.5 Synthesis of 4 Isopropy l 1,2 dithiolan 4 yl 3 (benzyl thio)propanoate ( 4 ) ................................ ................................ ................................ .................. 56 3.4.6 General Procedure for the Synthesis of tert Butyl Thioethers ................. 57

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6 3.4.6.1 1 ,3 Bis( tert butylthio) 2 phenylp ropan 2 ol ( 1a ) ............................. 57 3.4.6.2 1,3 Bis( tert butylthio) 2 (thiophen 3 yl)propan 2 ol ( 1b ) ................. 58 3.4.6.3 1,3 Bis( tert butylthio) 2 (5 bromothio phen 3 yl)propan 2 ol ( 1c ) .... 58 3.4.6.4 1,3 Bis( tert butylthio) 2 n propylpropan 2 ol ( 1d ) ........................... 59 3.4.6. 5 1,3 Bis( tert butylthio) 2 n dodecy lpropan 2 ol ( 1e ) ........................ 59 3.4.6.6 1,3 Bis( tert butylthio) 2 isopropylpropan 2 ol ( 1f ) .......................... 59 3.4.6.7 1,3 Bis( tert butylthio)propan 2 ol ( 1g ) ................................ ............ 60 3.4.6.8 Alternative route to 1,3 Bis( tert butylthio)propan 2 ol ( 1g ) ............. 60 3.4.6.9 1,3 Bis( tert butylthio) 2 ( tert butyldimethylsiloxy)p ropane ( 1h ) ...... 60 3.4.6.10 1,3 Bis( tert butylthio) 2 acetoxypropane ( 1i ) ................................ 61 3.4.6.11 1,3 Bis( te rt butylthio)acetone ( 1j ) ................................ ................. 61 3.4.6.12 1,3 Bis( tert butylthio) 3 methylbutan 2 one ( 1k ) .......................... 61 3.4.6.13 Synthesis of 1,3 bis( tert butylth io) 3 methylbutan 2 ol ( 1l ) ........... 62 4 HARNESSING STRAINED DISULFIDES FOR PHOTOCURABLE ADAPTABLE HYDROGELS ................................ ................................ ................................ ......... 63 4.1 Overview ................................ ................................ ................................ ........... 63 4.2 Results and Disc ussion ................................ ................................ ..................... 66 4.2.1 1,2 Dithiolane Photochemistry ................................ ................................ . 66 4.2.2 Network Precursor Synthesis ................................ ................................ .. 67 4.2.3 Aggregation and Photolysis of PEG PhDL ................................ .............. 70 4.2.4 UV Photocuring of PEG PhDL ................................ ................................ . 72 4.2.5 Green Li ght Photocuring of PEG PhDL ................................ ................... 76 4.2.6 Dye Release and Cytotoxicity ................................ ................................ .. 79 4.3 Summary ................................ ................................ ................................ .......... 81 4.4 Experimental ................................ ................................ ................................ ..... 81 4.4.1 Instrumentation ................................ ................................ ........................ 82 4.4.2 Procedures ................................ ................................ .............................. 85 4.4.2.1 Synthesis of PEG ditosylate (PEG OTs) ................................ ........ 85 4.4.2.2 Synthesis of PEG diamine (PEG NH 2 ) ................................ ........... 86 4.4.2.3 Synthesis of PEG methyl este r (PEG 0.5) ................................ ..... 86 4.4.2.4 Synthesis of PEG 1.0 ................................ ................................ ..... 86 4.4.2.5 Synthesis of PEG PhDL in DMF at 135 mg/mL ............................. 87 4.4.2.6 Synthesis of 1,3 dichloro 2 phenylpropan 2 ol ............................... 87 4.4.2.7 Synthesis of 4 acrylate 4 phenyl 1,2 di thiolane (PhDLA) ............... 88 4.4.2.8 Gelation of PEG PhDL under UV light ................................ ........... 89 4.4.2.9 Investigation of PEG PhDL photolysis in water .............................. 89 4.4.2.10 Ellm PhDL gels ................................ .............. 89 4.4.2.11 Gelation of PEG PhDL under green light open to air ................... 89 4.4.2.12 Rhodamine 6G release fr om UV cu red gels ................................ 90 5 SOLVENT CONTROLLED PHOTO CROSSLINKING OF COUMARIN BASED SINGLE CHAIN NANOPARTICLES ................................ ................................ ....... 91 5.1 Overview ................................ ................................ ................................ ........... 91 5.2 Results and Discussion ................................ ................................ ..................... 93

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7 5.2.1 Polymer Synthesis ................................ ................................ ................... 93 5.2.2 Solvent Selection ................................ ................................ ..................... 96 5.2.3 Chain Compaction ................................ ................................ ................... 98 5.2.4 Crosslinking Kinetics ................................ ................................ ............. 101 5.3 Summary ................................ ................................ ................................ ........ 104 5.4 Experimental ................................ ................................ ................................ ... 105 5.4.1 Instrumentation ................................ ................................ ...................... 105 5.4.2 Procedures ................................ ................................ ............................ 107 5.4.2.1 Typical RAFT copolymerization of MMA and CMA ...................... 107 5.4.2. 2 General procedure for the methacrylate copolymer RAFT agent removal ................................ ................................ ................................ . 107 5.4.2.3 Synthesis of PMA TTC ................................ ................................ . 108 5.4.2.4 Photoinduced RAFT agent removal of PMA TTC ........................ 108 5.4.2.5 Typical preparation of the acrylate based coumarin copolymers . 109 5.4.2.6 Typical SCNP formati on ................................ ............................... 109 5.4.2.7 Kinetic mo nitoring of the photodimerization of CMOMe ............... 110 5.4.2.8 Estimation of unreacted coumarin units per chain ....................... 111 6 THERANOSTIC NANOCARRIERS COMBINING HIGH DRUG LOADING AND MAGNETIC PARTICLE IMAGING ................................ ................................ ........ 112 6.1 Overview ................................ ................................ ................................ ......... 112 6.2 Results and Discussion ................................ ................................ ................... 115 6.2.1 Synthesis of proDox via Imine Conjugation ................................ ........... 115 6.2.2 Hydrol ysis of proDox ................................ ................................ ............. 116 6.2.3 Iron Ox ide Nanoparticle Synthesis and Characterization ...................... 117 6.2.4 Formulation of proDox Nanocarriers ................................ ...................... 118 6.2.5 Control of proDox Loading ................................ ................................ ..... 119 6.2.6 Characterization of Nanocarrier Size and Morphology .......................... 119 6.2.7 Release Rate of Doxorubicin from Nanocarriers ................................ ... 120 6.2.8 Cellular Internalization and Toxicity of Doxorubicin Nanocarriers .......... 121 6.2.9 MPI of proDox Nanocarriers ................................ ................................ .. 122 6.3 Summary ................................ ................................ ................................ ........ 123 6.4 Experimental ................................ ................................ ................................ ... 123 6.4.1 Instrumentation ................................ ................................ ...................... 123 6.4.2 Procedures ................................ ................................ ............................ 124 6.4.2.1 Synthesi s of proDox ................................ ................................ ..... 124 6.4.2.2 Hydrolysis study ................................ ................................ ........... 125 6.4.2.3 Iron oxide nanoparticle synthesis ................................ ................. 126 6.4.2.4 FNP for pro Dox nanocarriers ................................ ....................... 127 6.4.2.5 Drug loading assay ................................ ................................ ...... 128 6.4.2.6 Doxorubicin release tests ................................ ............................. 128 6.4.2.7 IC 50 toxicity assay and fluorescence Imaging ............................... 129 6.4.2.8 MPI procedure ................................ ................................ ............. 130

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8 7 PROBING THERMORESPONSIVE PO LYMERIZATION INDUCED SELF ASSEMBLY WITH VARIABLE TEMPERATURE LIQUID CELL TRANSMISSION ELECTRON MICROSCOPY ................................ ..................... 131 7.1 Overview ................................ ................................ ................................ ......... 131 7.2 R esults and Discussion ................................ ................................ ................... 135 7.2.1 PISA Polymerization Ki netics and Morphology Evolution ...................... 135 7.2.2 Thermoresponsive Morphology Transiti ons ................................ ........... 138 7.2.3 Observing Crosslinked Worms and Vesicles i n Solution. ...................... 141 7.2.4 Thermally Initiated RAFT Polymerization Inside the Liq uid Cell ............ 143 7.2.5 In Situ Monitoring of Thermally Initiat ed RAFT PISA by LCTEM ........... 146 7.3 Summary ................................ ................................ ................................ ........ 150 7.4 Experimental ................................ ................................ ................................ ... 151 7.4.1 Inst rumentation ................................ ................................ ...................... 152 7.4.2 Procedures ................................ ................................ ............................ 154 7.4.2.1 Synthesis of PDMA macro CTA ................................ ................... 154 7.4.2.2 Polymerization kinetics of DAAm/DMA PISA with a target hydrophobic block DP of 170 ................................ ................................ 154 7.4.2.3 Example procedure for the synthesis of crosslinked polymer assemblies via DAAm/DMA PISA ................................ ......................... 155 7.4.2.4 Synthesis of crosslinked worms and intermediate structu res for LCTEM analysis via photoinitiated RAFT PISA ................................ .... 156 7.4.2.5 Con ventional RAFT polymerization of PDMA for MALDI IMS analysis control ................................ ................................ ..................... 156 7.4.2.6 In situ (i.e., inside the liquid cell) polymerization of PDMA for MALDI IMS analysis ................................ ................................ ............. 156 7.4.2.7 In situ (i.e., inside the liquid cell) thermal RAFT PISA with DAAm/DMA ................................ ................................ .......................... 157 8 INVERTING MONOMER SELECTIVITY WITH P OLYMERIZATION INDUCED SELF ASSEMBLY ................................ ................................ ................................ 158 8.1 Overview ................................ ................................ ................................ ......... 158 8.2 Results and Discussion ................................ ................................ ................... 161 8.2.1 Fundamentals of R adical Polymerization ................................ .............. 161 8.2.2 In Situ Monomer Differentia tion ................................ ............................. 162 8.2.3 End Group Chemistry and New Gradient Block Copolymers ................ 169 8.2.4 Insights into RAFT PISA Kinetics ................................ .......................... 171 8.3 Summary ................................ ................................ ................................ ........ 175 8.4 Experimental ................................ ................................ ................................ ... 176 8.4.1 Instrumentation ................................ ................................ ...................... 177 8.4.2 Procedures ................................ ................................ ............................ 177 8.4.2.1 Synthesis of PEG TTC ................................ ................................ . 177 8.4.2.2 Example RAFT PISA procedure for DAAm65 with a target hydrophobic block DP of 400 ................................ ................................ 177 8.4.2.3 Chain extension of DA Am65 PEG b P(DAAm stat DMA) b P(DAAm grad DMA) with DMA ................................ ............................. 178

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9 8.4.2.4 PEG ester hydrolysis of DAAm65 PEG b P(DAAm stat DMA) b P(DAAm grad DMA) with DMA ................................ ............................. 178 8.4.2.5 Single pot procedure for the generation of a tetrablock gradient copolymer with a target hydrophobic DP of 600 ................................ ... 179 9 CONCLUSION ................................ ................................ ................................ ...... 180 APPENDIX ................................ ................................ ................................ .................. 182 A NMR Spectra Chapter 3 ................................ ................................ ....................... 182 B UV Vis Spectra Chapter 3 ................................ ................................ .................... 195 C NMR Spectra Chapter 4 ................................ ................................ ....................... 196 D SEC Data Chapter 4 ................................ ................................ ............................. 197 E NMR Spectra Chapter 5 ................................ ................................ ....................... 198 F SEC Data Chapter 5 ................................ ................................ ............................. 200 G UV Vis Spectra Chapter 5 ................................ ................................ .................... 201 H NMR Spectra Chapter 6 ................................ ................................ ....................... 203 I NMR Spectra Chapter 7 ................................ ................................ ....................... 204 J SEC Data Chapter 7 ................................ ................................ ............................. 205 K NMR Spectra Chapter 8 ................................ ................................ ....................... 207 L SEC Da ta Chapter 8 ................................ ................................ ............................. 209 LIST OF REFERENCES ................................ ................................ ............................. 211 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 240

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10 LIST OF TABLES Table page 3 1 Optimization of reaction conditions ................................ ................................ ..... 39 3 2 Ring strain calculations ................................ ................................ ....................... 46 5 1 Precursor polymers used for the formation of SCNPs. ................................ ....... 97 5 2 SCNPs formed from various precursor polymers and under different solv ent c onditions ................................ ................................ ................................ ......... 100 8 1 Poly merization kinetics for the DAAm80 system at different particle sizes. ...... 168

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11 LIST OF FIGURES Figure page 1 1 Differentiation between dissociati ve and a ssociative exchange ......................... 25 1 2 Bond exchange equilibria in disulfide crosslinked materials ............................... 27 1 3 Schematic two dimensional depiction of hydrophobic aggregation in water ....... 30 1 4 Cryogenic transmission electron microscopy images (TEM) of assemblies comprised of poly(ethylene oxide) block poly(1,2 bu tadiene) (PEO b PB) in water, sh owing a morphology change ................................ ................................ 32 3 1 Electronic pro perties and synthesis of 1,2 dithiolanes ................................ ........ 36 3 2 The effect of and ring substitution on 1,2 dithiolane reactivity ......................... 37 3 3 Proposed reaction mechanism for the deprotection disulfide formation sequence ................................ ................................ ................................ ............ 40 3 4 1,3 Bis te rt butyl thioether synthesis ................................ ................................ .. 41 3 5 Preparation of hydroxy functional 1,2 d ithiolanes ................................ ............... 42 3 6 Plot of C4C5SS dihedral angle versus C5 SSC3 dihedral angle ......................... 43 3 7 Substituent effect s on the photophysical properties of 1,2 dithiolanes ............... 45 3 8 Investigations of the relat ion ship between max and steric and electronic substituent effects ................................ ................................ ............................... 45 3 9 Downstream functionalization of 1,2 dithiolanes ................................ ................. 46 4 1 Exploiting 1,2 dith iolane photochemistry for disulfide hydrogels ......................... 65 4 2 PEG PhDL network precursor synthesis and characterization ........................... 68 4 3 Spectroscopic evidence for 1,2 dithiolane incorporation ................................ .... 68 4 4 Kinetic profile of the PhDLA conjugation with PEG 1.0 ................................ ...... 69 4 5 Comparison of PEG PhDL SEC traces showing the solvent d ependence on the formation of high molecular weight species ................................ .................. 69 4 6 Schematic depiction of dimeric and tetrameric PEG PhDL acting as more efficient bridging agent s between 1,2 dithiolane aggregates . ............................. 71 4 7 Investigation of PEG PhDL aggregation and photolysis ................................ ..... 71

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12 4 8 Oscillatory shear rheology o f UV cured PEG PhDL hydrogles .......................... 73 4 9 Shape adaptivity of PE G PhDL hydrogels ................................ ......................... 7 5 4 10 Oscillatory shear rheology at 25 °C and 1% strai n of hydrogels formed wit h 0.01 molar equivalents eosin Y ................................ ................................ ........... 77 4 11 Cyclic voltammogram of 2 ................................ ................................ .................. 77 4 12 Thiol triggered dye release from UV cured PEG PhDL gels .............................. 80 4 13 Confocal microscopy images of NIH 3 t3 cells plated with UV cured PEG PhDL hydrogels ................................ ................................ ................................ .. 80 4 14 Lamp emis sion spectra ................................ ................................ ....................... 82 4 15 Synthesis of PEG PhDL . ................................ ................................ .................... 85 5 1 Intrachain cro sslinking of methyl methacrylate and methyl acrylate based copolym ers ................................ ................................ ................................ ......... 92 5 2 Synthesis of 10 MMA and 20 MMA ................................ ................................ .... 94 5 3 Synthesis of 10 MA and 20 MA ................................ ................................ .......... 96 5 4 Static light scattering (SLS) analysis of the 20 MMA model polymer to determine the second virial coefficient B ................................ ............................ 98 5 5 Chain compaction upon intrachain crosslinking . ................................ ................. 99 5 6 Photoinduced [2+2] cycloaddition kinetics for a polymeric and a molecular coumarin system in different solvents ................................ ............................... 102 5 7 The coumarin dimer ization rate of the polymer system ( 20 MMA ) normalized by the rate of 7 methoxy 4 methylcoumarin ( CMOMe) ................................ ..... 103 5 8 Crosslinking efficiency determined by the estimati on of unreacted coumarin units (C units) per chain ................................ ................................ ................... 104 5 9 Determination of the molar extinctio n coefficient ( ) of a coumarin model compound ................................ ................................ ................................ ......... 110 6 1 TEM image and schematic depiction of a nan ocarrier with aggregated proDOX ................................ ................................ ................................ ............ 114 6 2 Synthesis of proDox ................................ ................................ ........................ 115 6 3 Pseudo first order rate analysis of proDox hydrolysis ................................ ..... 116

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13 6 4 FNP mixer setup ................................ ................................ ............................... 118 6 5 Tunab le doxorubicin (Dox) loading in nanocarriers ................................ .......... 119 6 6 TEM images of nanocarriers ................................ ................................ ............. 120 6 7 Release test of the doxorubicin nanoc arriers ................................ ................... 121 6 8 MPI s ignal from doxorubicin nanoca rriers ................................ ........................ 122 6 9 1 H NMR analysis shows the hydrolysis of proDox in DMS O d 6 ....................... 125 7 1 Liquid cell transmissi on electron microscopy (LCTEM) so lution cell ................ 132 7 2 Outline of the polymer ization induced self assembly (PISA) system ................ 135 7 3 Chain Extens ion of PDMA macro CTA with DAAm and DMA via RAFT PISA . 136 7 4 Scenarios upon O alkyl hydroxylamine crosslinker addition at different monomer conversions ................................ ................................ ...................... 137 7 5 Temperature effects on polymer assemblies ................................ .................... 140 7 6 Comparison between LCTEM and dry state TEM images of crosslinked worms and vesicles ................................ ................................ .......................... 142 7 7 Beam induced d amage to vesicles in solutio n during continuous imaging at low electron dose ................................ ................................ .............................. 143 7 8 RAFT polymerization of DMA inside the liquid cell and characterization by matrix assisted laser desorption i onization imaging mass spectrometry .......... 144 7 9 Enlarged MALDI spectrum of PDMA polymerized inside the liquid cell ............ 145 7 10 Control experiments for RAFT polymerizations inside the liquid cell ................ 146 7 11 In situ PISA control experiment without in itiator ................................ ................ 147 7 12 In situ PISA control experiment without macro CTA ................................ ......... 147 7 13 In situ ima ging of thermally initiated RAFT PISA at 11% w/v solids concentration and assembly dissociation upon cooling ................................ .... 149 8 1 Strategic employment of the hydrophobic effect can improve reaction eff iciency and affect product selectivity ................................ ............................ 159 8 2 Polymerization of DAAm/DMA in non selective solvent co nditions ................... 162 8 3 Synthesis of gradient block copolymers via in situ monomer differentiation ...... 163

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14 8 4 Photographs bef ore assembly , at assembly , and after assembly. .................... 164 8 5 Plot of k p,app before and after assembly ................................ ............................. 166 8 6 1 H NMR spectroscop y of DAAm and DMA in the presence of am phiphilic block copolymers ................................ ................................ .............................. 167 8 7 In situ monomer differentiation in PET RAFT PISA ................................ .......... 168 8 8 Chain extension of a DAAm65 gradient block copolymer synthesized via in situ m onomer differentiation ................................ ................................ ............. 169 8 9 Synthesis of a gradient tetrab lock copolymer featuring two distin ct gradient blocks in a single pot ................................ ................................ ........................ 171 8 10 Plot of k p,ap p after assembly normalized by k p,app before assembly at varying DAAm content in the monomer feed ................................ ................................ . 172 8 11 Analysis of the polymerization kinetics during PISA ................................ ......... 174 A 1 Methylene H assignment of PhDL using NOE experim e nts ............................. 182 A 2 H Assignment of DiMeDL using NOE experiment s and J values . .................... 183 A 3 Methylene H assignment of 3 using NOE experiments ................................ .... 183 A 4 1 H NMR spectrum of HDL in CDCl 3 . ................................ ................................ . 184 A 5 13 C NMR spectrum of HDL in CDCl 3. ................................ ................................ 184 A 6 1 H NMR spectrum of n PrDL in CDCl 3 . ................................ ............................. 185 A 7 13 C NMR spec trum of n PrDL in CDCl 3. ................................ ............................ 185 A 8 1 H NMR spectrum of C12DL in CDCl 3 . ................................ ............................. 186 A 9 13 C NMR spectrum of C12DL in CDCl 3. ................................ ............................ 186 A 10 1 H NMR spectrum of i PrDL in CDCl 3 . ................................ ............................... 187 A 11 13 C NMR spectrum of i PrDL in CDCl 3. ................................ .............................. 187 A 12 1 H NMR spectrum of PhDL in CDCl 3 . ................................ ............................... 188 A 1 3 13 C NMR spectrum of PhDL in CDCl 3 . ................................ ............................. 188 A 14 1 H NMR spectrum of TphDL in CDCl 3 . ................................ ............................. 189 A 15 13 C NMR spectrum of TphDL in CDCl 3 . ................................ ........................... 189

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15 A 16 1 H NMR sp ectrum of BrTphDL in CDCl 3 . ................................ ......................... 190 A 17 13 C NMR spectrum of BrTphDL in CDCl 3 . ................................ ....................... 190 A 18 1 H NMR spectrum of DiMeDL in CDC l 3 . ................................ ........................... 191 A 19 13 C NMR spectrum of DiMeDL in CDCl 3 . ................................ ......................... 191 A 20 1 H NMR spectrum of 2 in CDCl 3 . ................................ ................................ ...... 192 A 21 13 C NMR spectrum of 2 in CDCl 3. ................................ ................................ ..... 192 A 22 1 H NMR spectrum of 3 in CDCl 3 . ................................ ................................ ...... 193 A 23 13 C NMR spectrum of 3 in CDCl 3. ................................ ................................ ..... 193 A 24 1 H NMR spectrum of 4 in CDCl 3 . ................................ ................................ ...... 194 A 25 13 C NMR spectrum of 4 in CDCl 3. ................................ ................................ ..... 194 B 1 Normalized UV vis spectra for the 1,2 dithiolane substrates at 10 mM in DMSO ................................ ................................ ................................ ............... 195 C 1 1 H NMR spectrum of PEG PhDL in DMSO d 6 ................................ .................. 196 C 2 1 H NMR spectrum of PEG 1.0 in DMSO d 6 . ................................ ..................... 196 C 3 1 H and 13 C NMR spectrum of PhDLA in CDCl 3 . ................................ ............... 197 D 1 Overlaid size exclusion chromatograms of PEG PhDL ................................ .... 197 E 1 1 H NMR spectra in CDCl 3 of 1 0 MMA DTB and 10 MMA ................................ . 198 E 2 Stability study of 7 hydroxy 4 methylcoumarin via 1 H NMR spectrosc opy ........ 199 E 3 Representative 1 H NMR spectra before and after 6 h irradiation of a solution of CMOMe in DCM/MeOH ................................ ................................ ................ 199 F 1 SEC analysis in DMAc of the RAFT polymer ization of 10 MMA DTB. .............. 200 F 2 SEC analysis in THF of SCNPs formed under varying solvent condit ions ........ 200 G 1 UV vis spectroscopy of linear precursors and SCNPs to determine the final coumarin conversion ................................ ................................ ........................ 201 G 2 UV vis spectrosco py of reaction aliquots during the SCNP formation .............. 202 H 1 1 H NMR spectr oscopy of proDox in DMSO d 6 ................................ ................. 203

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16 H 2 13 C NMR spectrum of proDo x in CDCl 3 ................................ ........................... 204 I 1 1 H NMR spectrum of the PDMA macr o CTA in CDCl 3 ................................ ...... 204 J 1 S EC trace of the PDMA macro CTA in DMAc. ................................ ................. 20 5 J 2 SEC of reaction aliquots of the conventional thermal RAFT PISA using DAAm, DMA and the PDMA macro CTA ................................ .......................... 205 J 3 SEC traces of PDMA b P(DAAm co DMA) generated via DAAm/DMA PISA prior to cro sslinking ................................ ................................ ........................... 206 K 1 1 H NMR spectrum of PEG TTC in DMSO d 6 ................................ .................... 207 K 2 1 H NMR spectrum of DAAm 50 PEG b P(DAAm stat DMA) b P(DAAm grad DMA) in CDCl 3 . ................................ ................................ ................................ . 207 K 3 1 H NMR spectrum of DAAm65 PEG b P(DAAm stat DMA ) b P(DAAm grad DMA) in CDCl 3 . ................................ ................................ ................................ . 208 K 4 1 H NMR spectrum of DAAm80 PEG b P(DAAm sta t DMA) b P(DAAm grad DMA) in CDCl 3 . ................................ ................................ ................................ . 208 K 5 1 H NMR spectrum of DAAm65 PEG b P(DAAm stat DMA) b P(DAAm grad DMA) and P( DAAm stat DMA) b P(DAAm grad DMA) after hydrolysis in DMSO d 6 ................................ ................................ ................................ .......... 209 L 1 Overlaid SEC traces of PEG (MW = 5 kDa) and PEG TTC .............................. 209 L 2 Overlaid SEC traces of PEG ester hydrolysis reaction ................................ ..... 210

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17 LIST OF ABBREVIATIONS Ac A cety l AIBN Azobis(2 methylpropionitrile) ATR Attenuated total reflectance B S econd virial coefficient BDE Bond dissociation energy Bn Benzyl b Broad Br Tph DL 4 Hydroxy 4 (5 bromothiophen 3 yl) 1,2 dithiolane BuSH 1,4 B utanedithiol BuSS 1,2 dithian e C Ch aracteristic ratio C12 DL 4 Dodecyl 4 hydroxy 1,2 dithiolane C3SH 1,3 Dithiol CMOH 7 (2 hydroxyethoxy) 4 methylcoumarin CN Cyano CPADB 4 Cyanopentanoic acid dithiobenzoate CSSC Carbon sulfur sulfur carbon CTA Chain transfer agent Ð Dispersit y d dou blet DAAm Diacetone acrylamide DBU 1,8 Diazabicylcoundec 7 ene DCM Dichloromethane D h Hydrodynamic diameter

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18 DI Deionized DiMeDL 3,3 Dimethyl 4 hydroxy 1,2 dithiolane DIPEA Diisopropylethylamine DL 1,2 Dithiolane DLS Dynamic light scatterin g DMA N ,N Dimethylacrylamide DMAc N,N Dimethylacetamide DMAP 4 Dimethylaminopyridine DMF D imethylformamide DMSO Dimethyl sulfoxide DP Degree of polymerization DTT Dithiothreitol EDC 1 E thyl 3 (3 dimethylaminopropyl)carbodiimide EPHP N ethylpiperid ine hypo phosph i te equiv Equivalent or equivalents EIMS Electrospray ionization mass spectrometry Et 2 O Diethyl ether EtOAc Ethyl acetate EY Eosin Y FNP Flash n ano p recipitation FT Fourier transform G Storage modulus G Loss modulus GC Gas chromato graphy h Hour or hours

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19 HDL 4 Hydroxy 1,2 dithiolane HOMO Highest occupied molecular orbital HRMS High resolution mass s pectrometry Hx Hexanes i Pr DL 4 Isopropyl 4 hydroxy 1,2 dithiolane IR Infrared K O ptical constant k B Boltzmann constant LCST Lo wer critical solution temperature LCTEM Liquid cell transmission electron microscopy LUMO Lowest unoccupied molecular orbital m Multiplet macro CTA Macro chain transfer agent MALDI Matrix assisted las er desorption/i onization MALS Multi angle laser li ght scattering MeOH Methanol MA Methyl acrylate min Minute or minutes MMA Methyl methacrylate M n Number average molecular weight MO Molecular orbital M p A pparent peak molecular weight MPI Magnetic particle imaging MSH 2 Mercaptoethanol M w Weight average molecular weight

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20 MWCO Molecular weight cut off NBS N bromosuccinimide NMR Nuclear magnetic resonance n Pr DL 4 n Propyl 4 hydroxy 1,2 dithiolane PDMA Poly( N,N dimethylacrylamide) PEG Poly(ethy lene glycol) PEG 1.0 A midoamine poly(ethylene glyc ol) PEG NH 2 P oly(ethylene glycol) diamine PET Photoinduced electron/energy transfer PISA Polymerization induced self assembly PhDL 4 H ydroxy 4 phenyl 1,2 dithiolane PLA Poly(lactic acid) PMA poly(met hyl acrylate) PS Polystyrene q Quartet q Quartet q S cattering vector RAFT Reversible addition fragmentation chain transfer R g Radius of gyration R Rayleigh ratio s Singlet S S Sulfur sulfur SAXS S mall angle X ray scattering SEC Size exclusion c hromatography SCE Saturated calomel electrode

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21 SCN P Single chain nanoparticle SLS Static light scattering SPION Superparamagnetic iron oxide nanoparticle t Triplet T Temperature TBD 1,5,7 T riazabicyclo[4.4.0]dec 5 ene t BuSH tert Butylthiol TEA Tri ethylamine TEM Transmission electron microscopy T LC Thin layer chromatography TMS Trimethylsilyl or Tetramethylsilane THF Tetrahydrofuran Tph DL 4 Hydroxy 4 (thiophen 3 yl) 1,2 dithiolane TTC Trithiocarbonate Ts Tosyl UV Ultraviolet vis Visible Molar extinction coefficient max Maximum absorbance/emission wavelength CSSC dihedral angle Angular frequency max Terminal relaxation time

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22 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 INTERACTIONS IN DESIGN AND SYNTHESIS OF POLYMER MATERIALS By Georg Scheutz December 2020 Chair: Brent S. Sumerlin Major: Chemistry W e disclose new strategies for l everaging dynamic covalent bonding and solvophobic interactions to improve material properties, control chemical reactions, and synthesize defined polymers. The utility of our approach was demonstrated with the generation of adaptable polymer networks, d is crete polymer particles, and new copolymer structu res. W e focused on dynamic interactions to control the network strength in hydrogels, modulate photoinduced crosslinking reactions, tune the hydrolysis rate of imine bonds, and regulate monomer sequence i n controlled radical polymerization. Capitalizing on dynamic disulfides, we expanded the synthetic availability of 1,2 dithiolanes, strained disulfides with intricate photoelectronic properties, and i ntegrated this compound class in photocurable one compon en t hydrogels with readily tunable vi scoelastic properties. We leveraged mac romolecular solvophobic interactions to c ontrol the rate of intrachain crosslinking and the construction of discrete polymer nanoparticles. T he versatility of solvophobic interacti on s was explored in complex solvent s ystems that

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23 allowed the generation of organic inorganic hybrid nanocarriers for therapeutic and diagnostic applications. Finally, we used macromolecular assembly, driven by the hydrophobic effect, to develop new in sit u characterization techniques and to synthesize copolymers with unprecedented monomer sequence.

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24 CHAPTER 1 INTRODUCTION Dynamic interactions can control architecture, impart functionality, and provide adaptability in polymer materials. Ranging from stron g covalent bonds to relatively weak supramolecular interactions , the thermodynamic diversity of dyn am ic chemistries can be leveraged to generate a plethora of intricate polymer materials with various properties. This dissertation demonstrates how different d ynamic interactions can be introduced into macromolecular sy stems to generate advanced materials and synthesize function al polymers. 1.1 D ynamic Covalent Chemistry Dynamic covalent chemistry relies on covalent bonds that reversibly exchange under equili br ium control. 1, 2 Such exchange can be where bonds are first broken into their individual reactive functionalities before exchange, or ds in a simultaneous bond forming and bond breaking process (Figure 1 1). 3 The incorporation of dynamic covalent bonds as crosslinks into a polymer network generates structurally dynamic materials that are reprocessable and stimuli responsive, while maintaining mechanical robustness akin to conventional covalent network s . Many ty pes of dynamic cov al ent exchange chemistries have been developed including transesterification, 4, 5 enaminone exchange, 6, 7 or Diels Alder cycloaddition , 8, 9 to name a few. In this dissertation, we used dynamic disulfide linkages for the construction of photocurable adaptable hydro gels (Chapters 3 and 4) .

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25 Figure 1 1. Diffe rentiation between dissociative and associative exchange. The dissociative ex ch ange is exemplified with a maleimide furan Diels Alder reaction and the dissociation of hindered urea linkages. The associative exchange shows enaminone transamination with free amines and transesterification between ester linkages and free alcohols. Rep ri nted with permission from Scheutz , Lessard, et al. , J. Am. Chem. Soc. 2019 , 141 , 16181 16196. Copyright 2019 American Chemical Society. 1.1. 1 Disulfide Exchange The controlled incorporation of d isulfide s in polym er materials started in the early 1840s, w hen Hancock 10 and Goodyear 11 developed t he vulcanization of natural rubber . Their invention constituted the reaction of polyisoprene carbon carbon double bonds in natural rubber with elemental sulfur in the presence o f white lead, a mixture of Pb(CO) 3 and Pb(OH) 2 , to form disulfide and oligosulfide crosslinks. It was also vulcaniz ed rubber that led to the first description and analysis of viscoelasticity in polymer net works: i n 1946, Green and Tobolsky 12 postulated that a continuous exchange of disulfide crosslinks a llow ed for viscous flow and stress relaxation of vulcanized rubber at elevated temperatures. Since the disulfide bond was the weakest linkage i n the

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26 otherwise only hydrocarbon containing system, Green and Tobolsky suggested that the disulfide bond reversib ly dissociated and reform ed , dissipating the applied stress. 13 Tobolsky also no ted that the exchange reaction of propyl disulfide and propanethiol proceeded only slowly even at 140 ° C, which stood in contradiction to the o therwise rather rapid stress relaxation in vulcanized rubbers. However, if sodium butanethiolate or AlCl 3 was add ed to the reaction, the exchange rate increased s ubstantially . Therefore, Tobolsky concluded that ionic impurities in the rubber could cataly ze the disulfide interchange in the crosslinked material. 14 These findings can be con si dered as an early indication for the versatility of disulfide exchange reactions, which can be readily controlled by their chemical environment. Additionally, the equilibrium of such disulfide exchange reactions can be effectively sh ifted by adjusting ex te rnal parameters such as temperature or pH, providing a facile means to tun e the properties of disulfide crosslinked materials . Two major variants of reversible disulfide exchange have been realized in polymer networks : thiol disulfide exchange and disulf id e disulfide exchange (Figure 1 2 ). Thiol disulfide exchange occurs via an S N 2 reaction between a thiolate and the disulfide. The reaction rate is first order each in thiolate and disulfide concentration. The thiolate concentration and therefore the ex ch ange rate is dependent on the p K a of the thiol (the p K a of alkyl thiols i s around 10 and 17 in water and DMSO, respectively) 15 and on the pH of the surrounding medium. At high pH the exchange reacti on is accelerated, whereas at low pH the exchange can be inhibited. 16 For materials, the utility of conventional thiol disulfide exchange is li mi ted due to the inherent sensitivity of free thiols to aerobic oxidation. Go ossens and coworkers created rubbery disulfide -

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27 containing polyether thermosets via the crosslinking of polysulfide glycidyl ether with a tetrathiol crosslinker. 17 The networks exhibited rapid stress relaxation at 60 ° C and good self healing efficiency. However, the dynamic properties of the material deteriorated significantly within two d ays, due to the oxidation of free thiols to disulfides under air. Figure 1 2 . Bond exchange equilibria in disulfide crosslinked materials. Mechanism of (top) thiol disulfide exchange and (bottom) disulfide disulfide exchange with and without reductan t. Disulfide (Figure 1 2 ) of alkyl disulfides proceeds only slowly without external stimuli such as high temperatures, 18 catalysts, 19 or UV light. 20 T his can be beneficial for the generation of polymeric mat erials, which should maintain their mechanical integrity under ambient conditions (i.e., in the absence of an external stimulus ). Leibler and coworkers prepared recyclable epoxidized natural rubber ( ENR) by crosslinking 8 mol% of the ENR epoxide groups

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28 wit h dithiodibutyric acid. 21 At temperatures below 100 ° C, the elastomeric properties of the material were comparab le to those of industrial rubbers. Above 150 ° C, disulfide exchange occu r red, and the rubber network became dynamic. Although some permanent crosslinks were formed due to the reaction of thiyl radi ca ls with double bonds in the polyisoprene backbone, the ma terial could be reprocessed at 180 ° C, without compromising its mechanical properties. E lectron paramagnetic resonance measurements combined with radical trapping experiments suggested that the dis ul fide disulfide exchange proceeds via a radical pathway. 22 H owever, if a suitable reductant is presen t, such as tertiary amines or trialkyl phosphines, the inte rmediate thiyl radical is reduced to the thiolate, which then undergoes thiolate disulfide exchange (Figure 1 2 ) . Capitalizing on the radical nature of disulfide disulfide exchange, the dynamic pr op erties of disulfide materials can be controlled by modulating thiyl radical stability , reflected by the disulfide bond dissociation energy ( BDE ) . For example, materials crosslinked with diphenyl disulfides showed enhanced dynamic propertie s at room tempe ra ture, whereas dialkyl disulfide analo gue s did not undergo exchange reactions at such temperatures. 23 This can be attributed to the diphenyl disulfide BDE of 50 kcal/mol , which is substantially low er t han the BDE o f dialkyl disulfides with 65 kcal/mol. 12, 24 Overall, t he diversity of disulfide crosslinked materials, ranging from soft and ductile elastomers to rigid and durable thermosets, emanates fr om the crosslink excha ng e mechanism, which is dictated by the chemical nature of the disulfide bond and the chemical environment. Here, the intricate properties of strained cyclic disulfides

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29 were investigated (Chapter 3) and applied for the formation of ph otocurable adaptable h yd rogels (Chapter 4). 1.2 Supramolecular Chemistry Supramolecular chemistry, the organization of multiple individual (macro) molecular species guided by noncovalent interactions . 25 N oncovalent interactions, such as hydrogen bonding ( the strength of hydrogen bonds depends on the donor and the acceptor structure; the most common hydrogen bonds are between 0 and 5 kcal/mol ) 25 or interactions ( < 5 kcal/mol), 26 are weaker than covalent bonds and allow for fast bond exchange . 27 For example, damaged hydrogen bonded networks can heal readily through cros slink exchange at room temperature, while comparable dynamic covalent networks would require additional stimuli , such as heat. 28 It is important to note that metal coordinatio n and ionic bond ing are also routinely used to real iz e supramolecular assembly, 29 however, t hose types of bonds can be substantially stronger (> 25 kcal/mol) and often exhibit partial covalent character. 30, 31 P olymer materials crosslinked by supramolecular organization lack the robustness of covalent networks towards deformation, tempera tu re, or solvents, but facile crosslink rearrangements provide a means to readily tunable and adaptable materials . In this dissertation, we leveraged noncovalent , supramolecular solvophobic interactions in combination with reversible covalent bonding to co nt rol chemical reactions and synthesize new macromolecular structures (Chapters 4 8). 1.2.1 Solvophobic Interacti ons The most profound consequences of s olvophobic interactions emerge in aqueous systems. The aggregation of hydrophobic species in water ro phobic

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30 not only dictates fundamental biological processes , such as cell membrane formation 32 or protein fol ding , 33 bu t can also be leveraged to indu ce s electiv ity in molecular transformations . 34, 35 The complexity of the hydrophobic effect has been recognized as early as 1937, when Butler first postulated entropic origins for the low affinity of apolar molecules to water . 36 H owever, even after decades of research, some mechanistic details of the hydrop hobic effect r emain elusive. 37 G solv ) for apolar molecules is dominated by a large S solv ) that outcompetes the also negative solvation H solv ) at room temperature, r es ulting in a positive G solv (Equation 1 1). 38 ( 1 1 ) H solv < 0) can be attributed to the formation of a semi ordered water layer with increased hydrogen bonding around the hydrophobic molecule. 39 The substantially decreased molecular mobility of water in the solvation layer causes a large loss of entropy that ultimately renders hydrophobic hydration endergonic G solv > 0) . 40 Whil e there has been discussion about the exact Figure 1 3. Schematic two dimensional depiction of hydrophobic aggregation in water. In this sim plified model, the reduced surface area after aggregation liberates water molecules from the solvation layer, th us, increasing entropy.

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31 structure of the solvation layer, recent experimental findings 39 from isot opically decoupled infrared spectroscopy corroborated the clathrate like water structures proposed by Evans 41 and Kauzmann 42 in 1945 and 1959, respectively. Considering the thermodynamics of hydrophobe solvation, the aggregation of apolar molecules and polymers in water can be unders tood by the entropic energy gain upon surface area reduction in the aggregated state. The lower surface area of aggregated (macro) molecules resu lts in a decreased solvation layer , liberating water molecules into the bulk solution , and thus, increasing entr opy (Figure 1 3). Solvophobic interactions are most pronounced in water. This is related to the high density of hydrogen bonds per unit volume in water (connected to the small size of the water molecule). 43 However, other cohesive forces, of a dispersive or electrostatic nature, for example, can drive sol vophobic interactions in any solvent system. 44 In this di ssertation, we explored solvophobic interactions of polymers in organic and aqueous solvents to control reaction rates and material struc ture (Chapter 5 and Chapter 6). 1.2.2 Amphiphilic Block Copolymer Assembly Solvophobic interactions drive the assembly of amphiphilic block copolymers in water . By controlling block copolymer composition, concentration, and solvation, discrete and uniform nanoparticles can be obtained, such as spherical micelles, worm like micelles, or vesicles. However, it is the interpla y of chain stretching in the hydrophobic core, interfacial tension on the core surface, and repulsion of the hydrophilic corona chains th at dictate the assembly morphology . 45 This can be illustrated by the assembly of poly(ethylene oxide) block po ly(1,2 butadiene) (PEO b PB) block copolymers at varying block lengths in water (Figure 1 4). 46 Block copolymers with short PEO blocks formed spherical micelles with a high interfacia l curvature to

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32 Figure 1 4. Cryogenic transmission electron microscopy images (TEM) of a ssembl ies comprised of poly( ethylene oxide) block poly(1,2 butadiene) (PEO b PB) in wate r, showing a morphology change from spherical micelles (A) to worm like micelles (B) and vesicles (C). Decreasing the degree of polymerization of the PEO block ( N PEO ) induced the formation of s tru ctures with lower interfacial curvature, such as worm like micelles and vesicles. The block copolymer chain depicted in th e cartoon represents the relative volume fraction of hydrophilic (blue) and hydrophobic block (red), showing that lower curvature stru ctures accommodate block copolymers with higher hydrophobic volume fractions. Cryogenic TEM images were reprinted with per mission from Jain, et al . , Science 2003, 300 , 460 464 . Copyright 2003 AAAS. minimize repulsion between the relatively large hydrophil ic PEO corona chains, outcompeting the entropic penalty from core chain stretching. Upon decreasing the corona chain repul sion by lowering the PEO block length, core stretching becomes more important and the assemblies changed from spherical micelles to st ructures with lower interfacial curvature, such as worm like micelles and vesicles. In this dissertation , w e devised a ne w liquid cell electron microscopy (LCTEM) technique to directly image amphiphilic block copolymer assemblies in a solvated solution st ate (Chapter 7). Finally, we leveraged selective amphiphilic block copolymer organization in polymerization induced self a ssembly (PISA) to control copolymer composition and functionality (Chapter 8 ) .

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33 CHAPTER 2 RESEARCH OBJECTIVE In this research we dev eloped new strategies of leveraging dynamic interactions , specifically dynamic covalent and solvophobic interactions, to control macromolecular reactions, viscoelastic properties, and network topology . In Chapter 3, w e studied synthesis and physicochemica l properties of diversely substituted functional 1,2 dithiolanes , strained five membered cyclic disulfid es. Using X ray crystallography and UV vis spectroscopy, we demonstrate how substituent size and ring substitution pattern can affect the geometry and p hotophysical properties of 1,2 dithiolanes. Capitalizing on the findings from Chapter 3, we employed 1, 2 dithiolane chemistry to generate photocurable dynamic hydrogels in Chapter 4. Specifically, we developed a photomediated disulfide crosslinking strate gy for telechelic network precursors containing strained cyclic disulfides. Gelation was achieved in les s than 10 min with tunable network moduli depending on irradiation time. Investigations into the gelation mechanism suggest the formation of free thiols during light exposure accounting for the dynamic nature of the gels. Chapter 5 and Chapter 6 describe how we used solvophobic interactions in intra and interchain assembly to control chemical reactions. In Chapter 5 , we studied the solvent controlled in trachain photoinduced [2+2] cycloaddition of coumarin units in corporated in methacrylate and acrylate co polymers under varying solvent conditions. W e found that chain compaction and crosslinking rate were highly dependent on copolymer chain solvation. The c omparison between polymeric and molecular coumarin

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34 dimerization kinetics indicated that the reduced cop olymer chain dimensions in poor solvent increased the coumarin dimerization efficiency, akin to a solvophobic effect. In Chapter 6, solvophobic interac tions of poly(ethylene glycol) block poly(lactic acid) (PEG b PLA) and a hydrophobically modified doxoru bicin prodrug (proDox) in water were employed for the generation of magnetic iron oxide nanoparticles with therapeutic and diagnostic properties. We sho wed that the anticancer drug doxorubicin can be released from proDox particles upon imine hydrolysis and that the hydrophobic shielding of the particle core by PEG b PLA can potentially control the hydrolysis rate. Chapter 7 describes our efforts devising a new technique to generate and image amphiphilic block copolymer assemblies via variable temperature liquid cell transmission electron microscopy (VT LCTEM). In combination with traditional ex situ analyses, VT LCTEM enabled not only the characterization of the nanoparticles in situ, but also the observation of a thermal phase transition. Fin ally, in Chapter 8, we used the change of the chemical environment upon polymer aggregation during PISA to induce reactive differentiation in a copolymerization of ot herwise indistinguishable monomers. This study provided not only a new synthetic methodolo gy to new copolymers but also offered unprecedented mechanistic insight into PISA.

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35 CHAPTER 3 S YNTHESIS OF FUNCTIONAL 1,2 DITHIOLANES FROM 1 ,3 BIS TERT BUTYL THIOETH ERS * 3 .1 Overview 1 ,2 Dithiolanes are five membered heterocyclic molecules comprising a d isulfide bond. The intricate reactivity of this class of disulfides, arising from the geometric constraints imposed upon the sulfur sulfur (S S) bond, has been exploit ed for cell uptake applications, 47 52 reversib le protein polymer conjugation, 53 biosensors, 54 dynamic networks, 55 60 and functional polymer synthesis. 61, 62 prote in polymer conjugation, 53 biosens ors, 54 dynamic networks , 55 60 and functional polymer synthesis. 61, 62 Distinct from linear disulfides, which usuall y exhibit CSSC dihedral angles around 90 ° , the five membered cyclic geometry of 1,2 dithiolanes forces t he disulfide scaffold into conformations with CSSC dihedral angles lower than 35 ° (Figure 3 1). 63, 64 At such low dihedral angles, the neighboring fully occupied non bonding sulfur orbitals overlap, 65, 66 causing a destabilizing four electron interact ion , also known as closed shell repulsion (Figure 3 2 ). 67 This stereoelectronic e ffect weakens the S S bond, rendering 1,2 dithiolanes prone to rapid thiol disulfide exchange 16, 68 and ring opening polymerization. 53, 62, 69 Such polydisulfides generated from 1,2 dithiolanes are typically dynamic and can be reversibly depolymerized, 58, 70, 71 a phenomenon that has been exploited for the direct polydisulfide mediated cytosolic delivery of various cargo , 72 such as proteins, 51 quantum dots, 73 or silica particles. 74 * Adapted and reprinted with permission from Org. Biomol. Chem . 20 20 , 18 , 6509 6513. Copyright 2020 Royal Society of Chemistry. Contributions: Johnathan L. Rowell performed parts of the experimental wo rk, Dr. Fu Sheng Wang c ontributed density functional theo ry calculations, and Dr. Khalil A. Abboud acquired the X ray crystal structures.

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36 Figure 3 1. Electronic properties and synthesis of 1,2 dithiolanes. (A) The low CSSC dihedral angle ( ) imparts 1,2 dithiolanes , such as lipoic or asparagusic acid , with un ique re activity. (B) Previous syntheses of 1,2 dithiolanes involved a two step reaction sequence. (C) Our strategy provides hydroxy functional 1,2 dithiolanes in a single step from readily available 1,3 bis tert butyl thioether substrates. In addition to the CSS C dihedral angle, a determining factor for t he reactivity of 1,2 dithiolanes is the ring substitution pattern. 58, 75, 76 For reported profound differences in the polymerization behavior 69 and the cell uptake efficiency 48 of lipoic acid and asparagusic ac id. Whitesides and coworkers showed that higher substituted 1,2 dithiolanes are more resistant towards reduction an d ring opening ( Figure 3 2 ). 16 Considering these results, we believe there is great potential in controll ing 1,2 dithiolane reactivity via tailored substituent selection. However, most application focused reports r evolve around commercially availab le lipoic acid and its amide or ester derivatives . This lack of substrate variety is arguably due to the limited synthetic accessibility of substituted 1,2 dith i olane derivatives with functional handles for

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37 Figure 3 2 . The effect of and ring substitut ion on 1,2 dithiolane reactivity. (A) The simplified molecular orbital diagram for the disulfide bond explains th e increased closed shell repulsion at low , due to the energy penalty from the out of phase (i.e., antibonding; E ) interaction, which outcomp etes the stabilizing in phase (i.e., bonding; E + ) interaction. The increased HOMO energy level at low decreases the energy of the first electronic transition, resulting in a distinct red shift of the disulfide chromophore absorbance. (B) Ring substitutio n can stabilize the ring closed (oxidized) five membered disulfide scaffold. 16 For example, the equilibrium constant ( K ) for the reaction between oxidized dithiothreitol (DTT ox ) and the reduced 1,3 dithiol (DL red ) is 1.3 times higher for 4,4 dimethyl 1,2 dithiolane than for unsubstituted 1,2 dithiolane. (C) Calculated correlation o f MO eigenvalues with S S dihedral angle in HSSH. The shaded and unshaded regions represent MO lobes of different sign. In the ground state, 4b, 5a, and 5b are empty. All other MOs are doubly occupied. Reprinted with permission from Boyd, D. B . J. Am. Chem . Soc. 1972 , 94 , 8799 8804. Copyright 1972 American Chemical Society.

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38 downstream modification. 1,2 Dithiolanes are commonly synthesized in a two step sequence, which includes generation of the 1,3 dithiol via hydrolysis or reduction of suitable precursors, followed by oxidation to the corresponding 1,2 dithiolane ( Figure 3 1 ). However, f ormation of the 1,3 dithiol often requires harsh reaction conditions that limit functional group tolerance and often lead to undesired polydisulfide formation. To overcome t hese synthetic limitations and expand the substrate toolbox for applications of 1,2 dithiolanes, we developed a modular one step synthesis o f diversely substituted 1,2 dithiolanes from readily accessible 1,3 bis tert butyl thioethers. Furthermore, the 1,3 bis tert butyl thioethers were designed to feature a hydroxy group that can be used as a handle for downstream functionalization s of the 1,2 dithiolane product. 3 .2 Results and Discussion tert Butyl protection of thiols is typically known for robustness and stability. 77, 78 However, Mayor 79 and later Feringa 80 leveraged the tert butyl group for the direct transformation of S tert butyl thioethers into thioacetates using acetyl chloride and a catalytic amount of Br 2 or TiCl 4 , respectively. Specifically intriguing to us w observation of disulfide side p roducts during the reaction. 79 Based on these reports, we tested if S tert butyl cleavage in combination w ith intramolecular disulfide formation promoted by electrophilic halogen reagents could provide access to 1,2 dithiolanes from 1,3 bis tert butyl thioethers in a single step. Optimization of the reaction conditions with thioether 1a as a substrate showed t hat Br 2 , in combination with hydrated silica gel, was most effective for the targeted transformation, yielding 4 hydroxy 4 phenyl 1,2 dithiolane PhDL in 77% isolated yield (Table 3 1). The addition of silica gel to the reaction mixture improved the yield ( entries 1 and 2), presumably by scavenging

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39 Table 3 1. Optimization of reaction conditions a Entry Deviation from standard conditions Yield b (%) 1 None 77 2 No silica gel 52 3 c 0 ° C 24 4 d NBS instead of Br 2 28 5 C 2 Cl 4 Br 2 instead of Br 2 n. r. 6 I 2 instead of Br 2 n. r. a All reactions were run to full conversion of 1a unless no reaction (n. r.) was observed. Standard conditions: 1a (1.0 mmol), silica gel ([g silica gel] / [mmol 1a] = 2), DCM (20 mL), room temperature (r. t.). b Isolated yield after column chromatography. c 2.2 equiv Br 2 . d 3 equiv NBS . reactive byproducts, 81 which could be vi sually confirmed by the discoloration of the silica gel over the course of the reaction. While t he slow addition of 1.3 equivalents of Br 2 typically led to full conversion of 1a, lower reaction temperatures required increased amounts of Br 2 for the reactio n to complete (entry 3). Other electrophilic halogen reagents, such as N bromosuccinimide (NBS) or 1,2 dibromotetrachloroethane (C 2 Cl 4 Br 2 ), were less effective or showed no reaction (entries 4 6). Analysis of the crude reaction mixture by 1 H NMR spectros copy and GC MS showed the formation of 1,2 dibromo 2 methylpropane and ter t butyl bromide as the major byproducts. Based on these results, we propose that ring closure proceeds via the initial formation of sulfonium bromide A, 82 followed by elimination of isobutylene (Figure 3 3). The activated sulfenyl bromide B could then undergo intramolecular cyclization to compound C, which yiel ds t he target 1,2 dithiolane after another elimination of isobutylene. Isobutylene (Tb = 6.9 ° C) either evaporates from the reaction mixture or reacts with Br2 and HBr to form 1,2 dibr omo 2 methylpropane and tert butyl bromide, respectively. The higher so lub i lity of isobutylene in the mixture at

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40 Figure 3 3. Proposed reaction mechanism for the deprotection disulfide formation sequence affording 4 hydroxy 4 phenyl 1,2 dithiolane ( PhDL ). After elimination, isobutylene either evaporates or is converted in to b rominated byproducts. lower temperatures would also explain the increased consumption of Br 2 at 0 ° C (Table 3 1, entry 3). Having established this synthetic strategy, we aimed to generate multiple 1,3 bis tert butyl thioether compounds (Figure 3 4 ) f or t he subsequent transformation into 1,2 dithiolanes . Specifically, we synthesized 1,3 bis tert butyl thioethers 1a 1l from various 1,3 dichloropropan 2 ol derivatives, , halogenated ketones, and 2 (chloromethy l)oxiranes with tert butylthiol and K 2 CO 3 as a base in DMF at room temperature. Notably, generation of 1k from 2,4 dibromo 2 methylbutan 3 one involved the conversion of a tertiary bromide into a thioether, which is unlikely to occur via nucleophilic substitution under basic conditions. Since 2,4 dibromo 2 methylbutan 3 one rapidly decomposed upon exposure to K 2 CO 3 and DMF in the absence of t BuSH, we believe that this transformation followed a more complex reacti on sequence, potentially a Favorskii type rearrangement intercepted by ring opening o f the cyclopropanone intermediate with t BuSH . This broad range o f suitable starting materials

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41 Figure 3 4 . 1,3 B is tert butyl thioether synthesis from 1,3 dichloropropan 2 ol derivatives (A) and , halogenated ketones (B). NaBH 4 reduction of 1k provide d hydroxy thioether 1l (C) . The successful synthesis of 1g from 2 (chloromethyl)oxirane suggests that substituted oxiranes can potentially serve as substrates (D). Common protecting groups ( 1h and 1i ) were tolerated under the reaction conditions, showing t hat the hydroxy functionalit y is not essential to the transformation. suggests that a variety of 1,3 bis tert butyl thioether substrates can be readily generated by this protocol. The reaction could be conducted on multigram scales ( 1a and 1f ) and all pro ducts were obtained in good yield and purity, often without the need of chromatographic purification. Following the 1,3 bis tert butyl thioether preparation, we used the o ptimized Br 2 induced ring closure conditions (Table 3 1) to convert 1,3 bis tert buty l thioethers 1a 1f and 1l into 4 hydroxy 1,2 dithiolanes with moderate to good yields (Figure 3 5 ). This approach provided access to seven new 1,2 dithiolane derivatives w ith unprecedented

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42 ring substitution and functionality. Geminal to the hydroxy group, the substituents were var ied from hydrogen ( HDL ) to propyl ( n PrDL ), isopropyl ( i PrDL ), and dodecyl ( C12DL ); phenyl ( PhDL ), thiophenyl ( TphDL ), and bromothiophenyl ( BrTphDL ) groups could be installed as aromatic analogues. Additionally , we created an intrig uing alternative 1,2 dith iolane scaffold with gem dimethyl substitution vicinal to the disulfide bond ( DiMeDL ). Importantly , this reaction proved efficient on multigram sc ales ( i PrDL and PhDL ), which will be beneficial in 1,2 dithiolane applications. F igure 3 5. Preparation of hydroxy functional 1,2 dithiolanes with isolated yields. The lower yield of HDL is likely due to auto polymerization during purification. The X r ay crystal structures of DiMeDL and C12DL show a shortened S S bond length ( d ) and a compressed CSSC dihedral angle ( ). The thermal displacement ellipsoids were drawn at the 50% probability level . For DiMeDL , only the ( S ) enantiomer is shown.

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43 With the co mpounds in hand, we s ought to evaluate th e effect of substituent size and substitution pattern on 1,2 dithiolane ring conformation, since t he geometry and dihedral angle of the disulfide moiety profoundly affects the properties of 1,2 dithiolanes . The crys tal structures of C12DL and DiMeDL ( Figur e 3 5 ) revealed similarly elongated S S bond lengths around 2.06 Å (linear d isulfide bond lengths are typically around 2.03 Å) 63 but differing C SSC dihedral angles of 35.2 and 23.4 ° , respectivel y. Interestingly, Figure 3 6. Plot of C4C5SS dihedral angle versus C5SSC3 dihedral an gle (left) of all available crystal structures of unbridged and monocyclic 1,2 dithiolanes . Expansion for C5SSC3 dihe dral angle above 20 ° (right) revealed an almost li near relationship with substantially eclipsed C4C5SS conformation for 1,2 dithiolanes wit h C5SSC3 dihedral angles around 25 ° . this CSSC dihedral angle reduction coincided with a sharp decrease of the CC SS dihedral angle from 21 to 1 ° . Comparison with all available crystal structures of unbridged and monocyclic 1,2 dithiolanes showed a similar , almost linear relationship between the CSSC and the CC SS dihedral angle s ( Figure 3 6 ). We believe that such eclipse d CC SS conformations in 1,2 dithiolanes with low C SSC dihedral angles could

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44 potentially contribute to the reactivity associated with such c ompounds, warranting future investigations. Next, we turned to UV vis spectroscopy to analyze the maximum absorbance wavelength ( max ) o f the first electronic transition ( S 0 S 1 ) , which provides information about the S S bond geometry in 1,2 dithiolanes. Specifically, t he energy of S 0 S 1 in disulfides is dependent on the overlap of the fully occupied non bonding sulfur orbitals, which in turn is de termined by the CSSC dihedral angle ( Figure 3 2 ). 65, 66 For example, a stronger orbital overlap at lower CSSC dihedral angles raises the HOMO energy while the LUMO remains largely unaffected, 66 thus reducing the photon energy required for the excitation of S 0 S 1 . For the 1,2 dith iolane products tested in this report, we recorded a slight increase of max upon geminal substitution on C4 ( Figure 3 7 ). HDL exhibited a max of 327 nm, whereas derivatives with alkyl and aromatic substituents on C4 showed max values aro und 335 and 339 nm , respectively. The absorbance bands of derivatives with aromatic substituents were generally broader than those of derivatives with alkyl substituents, suggesting differences in the anti bonding character of S 1 . 83 Substitution on C3 in DiMeDL resulted in a large max red shift to 354 nm, which corroborates with the lower CSSC dihedral angle revealed in the crystal structur e. These results suggest that ring substitution affects the geometry and the photophysical properties of 1,2 dithiolanes. Furthermore, based on the linear increase of max with C4 substituent A values (Figure 3 8 ), we propose that the substituent effects i n this position are mostly of steric nature.

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45 Figure 3 7 . Substituent effects on the photophysical properties of 1,2 dithiolanes. (A) Overlay of representative UV vis absorbance spectra taken at 10 mM in DMSO . ( B ) Bar diagram showing the variation of the maximum absorbance wavelength ( max ) with respect to 4 hydroxy 1,2 dithiolane substituent. Figure 3 8. Investigations of the relationship b etween max and steric and electronic substituent effects. (A) max increases linearly with substituent A values , obtained from ref. 86 . (B) No correlation between max and Hammett para parameter 87 was found. A value and para parameter of an ethyl group was used for n Pr in both plots, since no such values are available for n Pr. Typically, l ow CSSC dihedral angles in 1,2 dithiolane compounds are associated with ring strain and S S bond instability. For example, auto polym erization has been commonly observed for 1,2 dithiolanes, 58, 64, 70, 84, 85 an issue we also encountered during purificatio n of the compounds . We note d dramatic stability differences between

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46 HDL and higher substitu ted i PrDL , and C12DL . For example, HDL could be used only in solution due to rapid polymerization upon concentration, whereas i PrDL and C12DL were bench stable over weeks. While the crystalline nature of C12DL could have a stabilizing effect, HDL and i PrD L are liquids and still showed substantial diffe rences in stability. Table 3 2. Ring strain calculations via the isodesmic reaction of 1,4 butanedithiol ( BuSH ) and 1,2 dithiane ( BuSS ) with a 1,2 dithiolane derivative in the ring closed ( DL ) and 1,3 dithio l ( C3SH ) form Compound rxn H (kJ/mol) a max (nm) b H HDL 327 i Pr i PrDL 336 a Calculated via rxn H = f H ( BuSS f H ( C3SH )] f H ( BuSH f H ( DL )] All enthalpies of f H ) were calculated for the gas phase at 298 K using a B3LYP/6 311G** basis set. b Det ermined from UV vis spectroscopy. Higher max values indicate lower CSSC dihedral angles. Figure 3 9. D ownstream functionalization of 1,2 dithiolanes , exploiting the hydroxy functionality. Triethylamine (TEA) was used as a base with 4 dimethylaminopyr idine (DMAP) as a catalyst in the esterification of PhDL . 1,8 Diazabicylcoundec 7 ene (DBU) was employed in the base catalyzed thia Michael addition of 1, 2 dithiolane acrylate 3 with benzyl mercaptan.

PAGE 47

47 To further investigate this observation, we estimated the ring strain for HDL and i PrDL via quantum chemical calculations of the enthalpy of reaction ( rxn H ) for the isodesmic reaction between 1,4 butanedithi ol ( BuSH ) and HDL or i PrDL (Table 3 2 ). The value of rxn H reflects the additional ring strain of the 1,2 dithiolane compound with respect to the relatively unstrained 1,2 dithiane ( BuSS ). The calcu lations revealed a rxn H of 2 7 . 9 kJ/mol for HDL , which is slightly higher than the ring strain for 1,2 dithiolane determined by Sunner 88 via iodine oxidation. U pon geminal substitution on C3 with a n isopropyl group , rxn H was reduced to 2 . 9 kJ/mol , corroborating with the higher stability of i PrDL observed experimentally, emphasizing the stabilizing effect from substitution in cyclic structures. 16, 58, 89, 90 Finally, to test if the hydroxy group incorporated in the 1,2 dithiolane structure was suitable for downstream modification s , we reacted PhDL with isobutyryl chloride , affording 1,2 ditholane ester 2 in good yield ( Figure 3 9 ). Us ing the same strategy, we synthesized 1,2 d ithiolane acrylate 3 from i PrDL and acryloyl chloride. The subsequent b ase catalyzed thia Michael addition between benzyl mercaptan and 3 provided Michael adduct 4 in high yield. We believe that such transformations could be particularly useful in applicat ions that require covalent conjugation of 1,2 dithiolane s to substrates such as proteins or polymers. 3 .3 Summary W e disclosed a scalable and straightforward synth e tic protocol for diversely substituted new 1,2 dithiolane compounds featuring a hydroxy func tionality as a valuable handle for downstream conjugation. X ray crystallography , UV vis spectroscopy , and quantum chemical calculations revealed profound substitu tion effects on the stereoelectronic properties and the stability of t he 1,2 dithiolane deriv atives,

PAGE 48

48 suggesting that 1,2 dithiolane reactivity can be tuned by careful substituent design. We believe this report represents an attractive avenue for the future design of 1,2 dithiolanes in advanced applications, such as cargo delivery and stimuli respo nsive polymer materials . 3 .4 Experimental Reagents and solvents were purchased from commercial sources and used without further purification. Anhydrous THF, Et 2 O, and DCM were obtained by passing the solvent through two sequential activated alumina columns in a MBRAUN solvent purification system . All solvent mixtures are given in volume ratios. Thin layer chromatography (TLC) was performed on SiO 2 60 F254 aluminum p lates with visualization by UV light or staining with KMnO 4 . Flash column chromatography was performed using silica gel (40 The melting points ( T m ) of the solid compounds were determined via differential scann ing calorimetry. 3 .4.1 Instrumentation 500 (125) MHz 1 H ( 13 C) NMR spectra were recorded on an INOVA 500 MHz and referenced to residual protonated solvent purchased from Cambridge Isotope Laboratories, Inc. (CDCl 3 : 1 H 7.26 ppm , 13 C 77.16 ppm). Abbreviations use d are s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), sept (septet), b (broad), and m (multiplet). High Resolution Mass Spectrometr y (HRMS) was conducted on an Agilent 6220 TOF spectrometer with electro spray ionization (ESI).

PAGE 49

49 Gas Chromat ography Electron Ionization Mass Spectrometry (GC EIMS) spectra were recorded on a Thermo Scientific DSQ II after sample introduction via GC (Thermo Scientific Trace GC Ultra). Raman characterization was carried out using a LabRAM ARAMIS (Horiba Jobin Yvo n) with a 633 nm HeNe laser as excitation source. Spectra were recorded with a scans in LabSPEC 5 (Horiba Jobin Yvon). All Raman spectra were baseline subtracted and normalized using Orig inPro 8.5. UV vis characterization was conducted on a Molecular Devices Spectra Max M2 spectrophotometer with Greiner Bio one 96 well clear bottom polypropylene reader plates. Fourier Transform Infrared (FTIR) Spectroscopy was collected on neat samples u sing a PerkinElmer Spectrum One FTIR spectrometer equipped with a PIKE MIRacle single reflection ATR accessory containing a diamond crystal sample p late. Spectra were processed using PerkinElmer Spectrum 10 software. Differential Scanning Calorimetry (DSC ) analysis was performed on a TA Q1000 DSC (TA Instruments, New Castle, DE) equipped with an autosampler and refrigerated cooling system 90, using aluminum hermetic sealed pans . The peak maximum of the endothermic melting peak was used as T m . 3 .4.2 General P rocedure for the S ynthesis of the 1,2 D ithiolane D erivatives Hydrated silica gel was prepared by mixing silica gel with deionized water in a 2:1 (wt/wt, silica/H 2 O) ratio until a free flowing powder was obtained. In a representative procedure, the 1,3 bi s tert butyl t hioether derivative (1 equiv.) and hydrated silica gel ([g] silica gel /[mmol] thiol = 2) were added to DCM. The total volume of DCM was adjusted to

PAGE 50

50 a final 1,3 bis tert butyl thioether concentration of 0.05 M. Under vigorous stirring, Br 2 (1.2 1.5 equiv., 0. 3 M in DCM) was added slowly dropwise until a slightly brownish solution color persisted , which usually coincided with total disappearance of the starting material as determined by TLC analysis . The mixture was filtered through a fritted funn el and methyl acrylate (10 equiv.) was added to scavenge any adventitious thiol impurities. After stirring for 2 h, half of the DCM was evaporated and replaced with hexanes over three evaporation dilution cycles. Importantly, the crude mixture should never be too concen trated because this was found to easily induce polymerization. The crude mixture was then loaded in hexanes onto a silica column and the desired 1,2 dithiolane derivative was obtained after flash column chromatography . 3 .4.2 .1 4 Hydroxy 1,2 d ithiolane ( HDL ) HDL was obtained as a pale yellow liquid after flash column chromatography on silica gel (Et 2 O/hexanes 1/2) in 52 % yield ( 0.663 g, 0.513 mmol). HDL readily polymerizes upon concentration and should only be handled under dilute conditions. R f ~ 0. 10 in EtOAc /hexanes (1/ 7 ) 1 H NMR ( 5 00 MHz, CDCl 3 4.95 (dtt, J = 11.1, 3.7, 1.7 Hz, 1H), 3.20 (dd, J = 11.6, 1.7 Hz, 2H), 3.13 (dd, J = 11.6, 3.8 Hz, 2H), 2.48 (d, J = 11.1 Hz, OH) 13 C NMR (125 MHz, CDCl 3 75.6, 47.1 UV vis ( max , nm; 10 mM in DMSO) 327 Raman (S S max , cm 1 ; neat) 489 FTIR ( max , cm 1 ) 3363, 3310, 2922, 2852, 1406, 1201, 1166, 1017 This compound has been reported before with 1 H and 13 C NMR shifts in DMSO d 6 . 54

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51 3 .4. 2.2 4 n Propyl 4 hydroxy 1,2 dithiolane ( n Pr DL ) n PrDL was obtained as a yellow liquid after flash column chromatography on silica gel (Et 2 O/hexanes 1/10) in 64 % yield ( 0.237 g, 1.44 mmol). n PrDL readily polymerizes upon concentration and should only be handled under dilute conditions. R f ~ 0. 24 in EtOAc /hexanes (1/ 15 ) 1 H NMR ( 5 00 MHz, CDCl 3 3.06 (d, J = 11.2 Hz, 2H), 3.02 (d, J = 11.2 Hz, 2H), 2.95 (b, OH), 1.81 (m, 2H), 1.56 (m, 2H), 0.98 (t, J = 7.3 Hz, 3H) 13 C NMR (125 MHz, CDCl 3 85.3, 49.9, 40.1, 19.0, 14.7 GC EIMS: calculated 164.0, found 164.0 (HRMS in our hands did not yield sufficient ionization.) UV vis ( max , nm; 10 mM DMSO) 333 Raman (S S max , cm 1 ; nea t) 488 FTIR ( max , cm 1 ) 3442, 2958, 2928, 2871, 1465, 1378, 1342, 1207, 1029, 994, 865 3 .4. 2.3 4 Dodecyl 4 hydroxy 1,2 dithiolane ( C12 DL ) C12DL was obtained as a yellow solid after flash column chromatography on silica gel (Et 2 O/hexanes 1/10) in 67 % yield ( 0.099 g, 0.341 mmol). R f ~ 0. 36 in EtOAc /hexanes (1/ 9 ) T m = 43.7 ° C 1 H NMR ( 5 00 MHz, CDCl 3 3.05 (d, J = 11.2 Hz, 2H), 3.02 (d, J = 11.2 Hz, 2H), 2.95 (s, OH), 1.82 (m, 2H), 1.52 (m, 2H), )1.29 (b, 18H), 0.88 (t, J = 6.9 Hz, 3H) 13 C NMR (125 MHz, CDCl 3 85.4, 49.9, 37.9, 32.1, 30.3, 29.80, 29.78, 29.71, 29.65, 29.5, 25.7, 22.8, 14.28 GC EIMS: calculated 290.3 , found 290.2 (HRMS in our hands did not yield sufficient ionization.)

PAGE 52

52 UV vis ( max , nm; 10 mM in DMSO) 336 Raman (S S max , cm 1 ; neat) 500 FTI 1) 3437, 2915, 2848, 1473, 1345, 1202,1067, 1009, 730 3 .4. 2.4 4 Isopropyl 4 hydroxy 1,2 dithiolane ( i Pr DL ) i PrDL was obtained as a yellow liquid after flash column chromatography on silica gel (Et 2 O/hexanes 1/10) in 78 % yield ( 0.193 g, 1.12 mmo l). R f ~ 0. 27 in EtOAc /hexanes (1/ 15 ) 1 H NMR ( 5 00 MHz, CDCl 3 3.08 (d, J = 11.2 Hz, 2H), 3.03 (d, J = 11.1 Hz, 2H), 2.85 (d, J = 0.88 Hz, OH), 2.02 (sept, J = 6.8, 0.84 Hz, 1H), 1.11 (d, J = 6.8 Hz, 6H) 13 C NMR (125 MHz, CDCl 3 88.1, 48.8, 35.6, 19.0 GC EIMS: calculated 164.0 , found 164.0 (HRMS in our hands did not yield sufficient ionization. However, the further functionalized products 3 and 4 were found suitable for HRMS.) UV vis ( max , nm; 10 mM in DMSO) 336 Raman (S S max , cm 1 ; neat) 496 FTIR ( max , cm 1 ) 3477, 2965, 2935, 2878, 1468, 1366, 1217, 1104, 999, 874, 730 3 .4. 2.5 4 Hydroxy 4 phenyl 1,2 dithiolane ( P h DL ) PhDL was obtained as a yellow solid after flash column chromatography on silica gel (Et 2 O/hexanes 1/10) in 77 % yield ( 0.050 g, 0.025 m mol). R f ~ 0. 44 in EtOAc /hexanes (1/ 6 ) T m = 60.1 ° C 1 H NMR ( 5 00 MHz, CDCl 3 7.59 (m, 2H), 7.40 (m, 2H), 7.34 (m, 2H), 3.50 (d, J = 11.5 Hz, 2H), 3.49 (s, OH), 3.27 (d, J = 11.5 Hz, 2H) 13 C NMR (125 MHz, CDCl 3 140.2, 128.7, 120.0, 125.2, 85.3, 52.5

PAGE 53

53 GC EIMS: calculated 198.0 , found 198.0 (HRMS in our hands did not yield suffi cient ionization. However, the further functionalized product 2 was found suitable for HRMS.) UV vis ( max , nm; 10 mM in DMSO) 340 Raman (S S max , cm 1 ; neat) 496 FTIR ( max , cm 1 ) 3446, 3091, 3060, 3025, 2933, 2928, 1600, 1494, 1447, 1350, 1208, 1172, 10 29, 928, 693 3 .4. 2.6 4 Hydroxy 4 (thiophen 3 yl) 1,2 dithiolane ( Tph DL ) TphDL was obtained as a yellow solid after flash column chromatography on silica gel (Et 2 O/hexanes 1/10) in 75 % yield ( 0.048 g, 0.024 mmol). R f ~ 0. 20 in EtOAc /hexanes (1/ 9 ) T m = 82.5 ° C 1 H NMR ( 5 00 MHz, CDCl 3 7.43 (dd, J = 3.1, 1.4 Hz, 1H), 7.36 (dd, J = 5.06, 3.1, Hz, 1H), 7.13 (dd, J = 5.04, 1.4 Hz, 1H) 3.44 (d, J = 11.4 Hz, 2H), 3.27 (d, J = 11.4 Hz, 2H) 13 C NMR (125 MHz, CDCl 3 141.4, 126.8, 125.1, 121.6, 84.2, 51.5 GC EIMS: calculated 204.0 , found 20 4.0 (HRMS in our hands did not yield sufficient ionization.) UV vis ( max , nm; 10 mM in DMSO) 340 Raman (S S max , cm 1 ; neat) 482 FTIR ( max , cm 1 ) 3489, 3087, 2977, 2934, 1405, 1326, 1235, 1157, 1035, 851, 789, 725, 681, 632 3 .4. 2.7 4 Hydroxy 4 (5 bromot hiophen 3 yl) 1,2 dithiolane ( Br Tph DL ) TphDL was obtained as a yellow oil after flash column chromatography on silica gel (Et 2 O/hexanes 1/10) in 57 % yield ( 0.102 g, 0.360 mm ol). R f ~ 0. 30 in EtOAc /hexanes (1/ 9 )

PAGE 54

54 1 H NMR ( 5 00 MHz, CDCl 3 7.32 (d, J = 1.6 H z, 1H), 7.08 (d, J = 1.7, Hz, 1H), 3.41 (s, OH), 3.38 (d, J = 11.46 Hz, 2H), 3.23 (d, J = 11.44 Hz, 2H) 13 C NMR (125 MHz, CDCl 3 141.9, 128.0, 123.0, 113.5, 83.9, 51.4 GC EIMS: calculated 281.9 and 283.9, found 282.0 and 284.0 (HRMS in our hands did not yield sufficient ionization.) UV vis ( max , nm; 10 mM in DMSO) 338 Raman (S S max , cm 1 ; neat) 489 FTIR ( max , cm 1 ) 3435, 3089, 2926, 2856, 1534, 1415, 1322, 1213, 1133, 1027, 994, 963, 868, 830, 724, 628 3. 4. 2.8 3,3 Dimethyl 4 hydroxy 1,2 dithiolane ( Di MeDL ) DiMeDL was obtained as a yellow solid after flash column chromatography on silica gel (Et 2 O/hexanes 1/3) in 85 % yield ( 0.096 g, 0.64 mmol). R f ~ 0. 30 in EtOAc /hexanes (1/ 9 ) T m = 61.2 ° C 1 H NMR ( 5 00 MHz, CDCl 3 4.05 (ddd J = 11.8, 4.0, 1.6 Hz, 1H), 3.33 (dd, J = 11.1, 4.0 Hz, 1H), 3.17 (dd, J = 11.1, 1.6 Hz, 2H), 2.32 (d, J = 11.70 Hz, OH), 1.49 (s, 3H), 1.43 (s, 3H) 13 C NMR (125 MHz, CDCl 3 82.6, 64.9, 43.4, 26.5, 21.4 GC EIMS: calculated 150.0, found 150.1 (HRMS in our hands did not yield suffic ient ionization.) UV vis ( max , nm; 10 mM in DMSO) 354 Raman ( max , cm 1 ; neat) 503, 522, 586 FTIR ( max , cm 1 ) 3273, 2970, 2914, 2854, 1466, 1378, 1311, 1117, 1037, 1002, 863

PAGE 55

55 3 .4. 3 Synthesis of 4 I sobutyryl 4 phenyl 1,2 dithiolane ( 2 ) PhDL (0.10 g, 0.50 m mol), TEA (0.20 g, 2.0 mmol), and DMAP (0.02 5 g, 0 .2 mmol) were dissolved in dry THF (3 mL). Under Ar, isobutyryl chloride (0.22 g, 2.0 mmol) was added dropwise. After 1 h of stirring, the reaction mixture was heated to 50 ° C and stirred for another 12 h. The reaction mixture was diluted with DCM and washed with 1 M HCl, saturated aqueous NaHCO 3 and brine. After drying over MgSO 4 and evaporation of the solvent, 2 was purified via flash column chromatography on silica gel (Et 2 O/hexanes = 1/10) to obtain the pure compound in 9 4 % yield (0.13 g, 0.48 mmol). R f ~ 0.51 in DCM/hexanes (1/1) 1 H NMR (500 MHz, CDCl 3 7.27 (m, 5H), 3.75 (d, J = 12.7 Hz, 2H), 3.66 (d, J = 12.7 Hz, 2H), 2.65 (sept, J = 6.9 Hz, 1H), 1.21 (d, J = 7.0 Hz, 6H). 13 C NMR (125 MHz, CDCl 3 34.5, 19.0. HRMS (ESI TOF): Calculated for [M+Na] + requires 291.0498; found 291.0498. UV vis ( max , nm; 10 mM in DMSO) 329 Raman (S S max , cm 1 ; neat) 508 FTIR ( max , cm 1 ) 2973, 2934, 1736, 1144, 695 3 . 4. 4 Synthesis of 4 A crylate 4 isopropyl 1,2 dithiolane ( 3 ) i PrDL (1.14 g, 6.96 mmol), TEA (2.34 g, 23.1 mmol), DMAP (0.353 g, 2.89 mmol), and a small spatula tip of phenothiazine (as radical inhibitor) were dissolved in dry THF (25 mL). Under Ar, acryloyl chloride (2.09 g, 23 .1 mmol) was added dropwise. After 1 h of stirring, the reaction mixture was heated to 35 ° C and stirred for another 12 h. The reaction mixture was diluted with DCM and washed with 1 M HCl, saturated aqueous NaHCO 3 , and brine. After dry ing over MgSO 4 and e vaporation of the solvent, 2

PAGE 56

56 was purified via flash column chromatography on silica gel (Et 2 O/hexanes = 1/15) to obtain the compound in 3 0 % yield (0.4550 g, 2.084 mmol) with 50% starting material recovery. R f ~ 0.43 in EtOAc/hexanes (1/ 15) 1 H NMR (500 MHz, CDCl 3 J = 17.3, 1.5 Hz, 1H), 6.10 (dd, J = 17. 3 , 10. 4 Hz, 1H), 5.83 (dd, J = 10.4, 1.5 Hz, 1H), 3.53 (d, J = 12.9 Hz, 2H), 3.38 (d, J = 12. 9 Hz, 2H), 3.11 (sept, J = 7.0 Hz, 1H), 1.00 (d, J = 6. 9 Hz, 6H) 13 C NMR (125 MHz, CDCl 3 165.3, 131.1, 12 9.3, 99.3, 47.0, 32.1, 18.3 HRMS (ESI TOF): Calculated for [M+Na] + requires 2 4 1.0 298 ; found 2 41 .0 319 . UV vis ( max , nm; 10 mM in DMSO ) 326 Raman (S S max , cm 1 ; neat) 509 FTIR ( max , cm 1 ) 2968, 2879, 1715, 1634, 1466, 1402, 1282, 1198, 1174, 1044, 974, 8 08 3 .4. 5 Synthesis of 4 I sopropyl 1,2 dithiolan 4 yl 3 ( benzyl thio)propanoate ( 4 ) A solution of 3 (0.050 g, 0.2 3 mmol) and benzyl mercaptan (0.028 g, 0.2 3 mmol ) in DCM (2.1 mL) was purged under Ar for 10 minutes at 0 °C. Then, a solution of DBU (0.0035 g, 0.023 mmol) dissolved in DCM (0.20 mL) was added and the reaction warmed up to room temperature. After 2.5 h, the reaction was complete, as determined by the co mplete disappearance of the starting material by TLC. The reaction was diluted with DCM washed w ith 1 M aqueous HCl, brine, saturated sodium bicarbonate solution, brine, dried over MgSO 4 and passed through a silica plug to give the desired compound in 95% yield (0.0751g, 0.219 mmol). R f ~ 0.21 in EtOAc/hexanes (1/15) 1 H NMR (500 MHz, CDCl 3 ) 7.32 (m , 5H), 3.76 (s, 2H), 3.49 (d, J = 12.8 Hz, 2H), 3.35

PAGE 57

57 (d, J = 12.8 Hz, 2H), 3.06 (sept, J = 6.9 Hz, 1H), 2.69 (t, J = 7.2 Hz, 2H), 2.56 (t, J = 7.2, 2H), 1.01 (d , J = 6.9 Hz, 6H) 13 C NMR (125 MHz, CDCl 3 ) 171.1 138.1, 129.0, 128.7, 127.2, 99.4, 46.8, 36.5, 35.4, 32.1, 26.5, 18.4 HRMS (ESI TOF): Calculated for [M+ H ] + requires 343.0852 ; found 343.0852 UV vis ( max , nm; 10 mM in DMSO) 326 Raman (S S max , cm 1 ; neat) 513 FTIR ( max , cm 1 ) 2986, 2967, 2926, 2876, 1727, 1453, 1409, 1352, 1216, 1135, 984, 978, 69 8 3 .4. 6 General P rocedure for the S ynthesis of tert B utyl T hioethers In a representative procedure, K 2 CO 3 (5 equiv) was dispersed in DMF. The total volume of DMF was adjusted to a final 1,3 dichloropropan 2 ol derivative concentration of 0.3 M. The mixture was sparged with Ar and tert butylthiol (4 equiv) was added, followed by the corresponding 1,3 dichloro 2 propanol derivative (1 equiv). The mixture was stirred vigorously for 24 h, diluted with Et 2 O, poured into aqueous NaOH (5 wt% in water) and extracte d twice with Et 2 O. The organic extract was washed with brine, aqueous 1 M HCl, water (3 times), and brine again. The solution was dried with MgSO 4 and pushed through a silica plug. After solvent evaporation, the desired compound was obtained in most cases with good purity without further column chromatography. 3 .4. 6 .1 1,3 Bis( tert butylthio) 2 phenylpropan 2 ol ( 1a ) 1a was obtained as a white solid in 98 % yield ( 1.25 g, 4.0 mmol). R f ~ 0. 27 in EtOAc /hexanes (1/ 15 ) T m = 50.2 ° C 1 H NMR ( 5 00 MHz, CDCl 3 7.49 (m, 2H), 7.36 (m, 2H), 7.28 (m, 1H), 3.44 (b, OH),

PAGE 58

58 3.18 (d, J = 12.4 Hz, 2 H), 3.06 (d, J = 12.4 Hz, 2 H ), 1.31 (s, 18H) 13 C NMR (125 MHz, CDCl 3 144.9, 128.3, 127.4, 125.5, 74.3, 42.6, 40.8, 31.0 FTIR ( max , cm 1 ) 3473, 3061, 2990, 2961, 2926, 28 63, 1459, 1365, 1340, 1161, 1057, 1030, 735, 697 3 .4. 6.2 1,3 Bis( tert butylthio) 2 (thiophen 3 yl)propan 2 ol ( 1b ) 1b was obtained as a white solid after flash column chromatography on silica gel (EtOAc/hexanes 1/9) in 74 % yield ( 0.76 g, 2.4 mmol). R f ~ 0. 36 in EtOAc /hexanes (1/ 9 ) T m = 54.8 ° C 1 H NMR ( 5 00 MHz, CDCl 3 7.27 (m, 2H), 7.08 (dd, J = 4.2, 2.2 Hz, 1H), 3.48 (s, OH), 3.11 (d, J = 12.4 Hz, 2 H), 3.03 (d, J = 12.4 Hz, 2 H ), 1.30 (s, 18H) 13 C NMR (125 MHz, CDCl 3 146.9, 125.9, 125.8, 121.4, 73.4, 42.7, 40.6, 31.1 FTIR ( max , cm 1 ) 3454, 3102, 2959, 2924, 2862 , 1459, 1364, 1341, 1160, 1058, 859, 800, 738, 650 3 .4. 6.3 1,3 Bis( tert butylthio) 2 (5 bromothiophen 3 yl)propan 2 ol ( 1c ) 1c was obtained as a white solid after flash column chromatography on silica gel (EtOAc/hexanes 1/12) in 73 % yield ( 0.74 g, 1.9 mmol ). R f ~ 0. 28 in EtOAc /hexanes (1/ 12 ) T m = 66.3 ° C 1 H NMR ( 5 00 MHz, CDCl 3 7.15 (d, J = 1.5 Hz, 1H), 7.03 (d, J = 1.6 Hz, 1H), 3.46 (s, OH), 3.04 (d, J = 12.5 Hz, 2 H), 2.97 (d, J = 12.5 Hz, 2 H ), 1.29 (s, 18H) 13 C NMR (125 MHz, CDCl 3 147.2, 128.6, 122.9, 112.5, 73.2, 42.8, 40.3, 31.1 FTIR ( max , cm 1 ) 3486, 3085, 2957, 2920 , 2861, 1458, 1414, 1364, 1330, 1161, 1056, 973, 848, 741, 652

PAGE 59

59 3 .4. 6.4 1,3 Bis( tert butylthio) 2 n propylpropan 2 ol ( 1d ) 1d was obtained as a liquid in 94 % yield ( 0.627 g, 2.25 mmol). R f ~ 0. 35 in EtOAc /hexanes (1/ 15 ) 1 H NMR ( 5 00 MHz, CDCl 3 2.81 (d, J = 12.1 Hz, 2 H), 2.80 (s, OH), 2.62 (d, J = 12.1 Hz, 2H), 1.54 (m, 2H), 1.43 (m, 2H), 1.32 (s, 18H), 0.93 (t, J = 7.2 Hz, 3H) 13 C NMR (125 MHz, CDCl 3 72.7, 42.4, 41.5, 37.9, 31.1, 16.9, 14.6 FTIR ( max , cm 1 ) 3548, 3457, 2923, 2853, 1466, 1437, 1359, 126 1, 1152, 1070, 800, 755, 733 3 .4. 6.5 1,3 Bis( tert butylthio) 2 n dodecylpropan 2 ol ( 1e ) 1e was obtained as a liquid after flash column chromatography on silica gel ( DCM/hexanes = 1/7 gradient to 1/1) in 98 % yield ( 0.867 g, 2.14 mmol). R f ~ 0. 50 in EtOAc /h exanes (1/ 9 ) 1 H NMR ( 5 00 MHz, CDCl 3 2.80 ( d , J = 12.0 Hz, 2 H), 2.79 (s, OH), 2.62 (d, J = 12.1 Hz, 2H), 1.55 (m, 2 H), 1.37 (m, 2H), 1.32 ( s, 18H), 1.27 ( b, 18H), 0.88 (t, J = 6.9 Hz, 3H) 13 C NMR (125 MHz, CDCl 3 72.7, 42.4, 39.2, 37.9, 32.1, 31.1, 30.2, 29.9, 29.79, 29.75, 29.7, 29.5, 23.6, 22.8, 14.26 FTIR ( max , cm 1 ) 3474, 2923, 2853, 1459, 1364, 1162 3 .4. 6. 6 1,3 Bis( tert butylthio) 2 isopropylpropan 2 ol ( 1f ) 1f was obtained as a liquid in 99 % yield ( 0.42 g, 1.5 mmol). R f ~ 0. 23 in DCM /hexanes (1/ 1 ) 1 H NMR ( 5 00 MHz, CDCl 3 2.82 (d , J = 12.3 Hz, 2 H), 2.73 (s, OH), 2.71 (d, J = 12.3 Hz, 2H), 1.92 (sept, J = 6.9 Hz, 1H), 1.32 (s, 18H), 0.98 (d, J = 6.9 Hz, 6H) 13 C NMR (125 MHz, CDCl 3 74.2, 42.4, 36.0, 34.9, 31.1, 17.2 FTIR ( max , cm 1 ) 3480, 2961, 2900, 1459, 1364, 1161, 991

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60 3 .4. 6. 7 1,3 Bis( tert butylthio)propan 2 ol ( 1g ) 1g was obtained as a liquid (solidifies at 4 ° C) in 99 % yield ( 0.917 g, 3.90 mmol). R f ~ 0. 39 in EtOAc /hexanes (1/ 7 ) 1 H NMR ( 5 00 MHz, CDCl 3 3.80 ( tt , J = 7.2, 5.2 Hz, 1 H), 2.79 (dd, J = 12.8, 5.2 Hz, 2H), 2.68 ( dd, J = 12.8, 7.2 Hz, 2H) 1.64 (m, 2 H), 1.33 ( s, 18H) 13 C NMR (125 MHz, CDCl 3 69.9, 42.7, 35.3, 31.2 FTIR ( max , cm 1 ) 3412, 2961, 2900, 2864, 1459, 1364, 1161, 1032 The spectroscopic data agreed with a previous report . 92 3 .4. 6. 8 Alternative route to 1,3 Bis( tert butylthio)propan 2 ol ( 1g ) K 2 CO 3 (2.68 g, 12.4 mmol) was dispersed in DMF (13 mL). The mixture was sparged with Ar and tert butylthiol (1.41 g, 15.5 mmol) was added, followed by epichlorohydrin (0.36 g, 3.9 mmol). The mixture was stirred vigorously for 3 h, diluted with Et 2 O, poured into aqueous NaOH (5 wt% in water) and extracted twice with Et 2 O. The organic extract was washed with brine, aqueou s 1 M HCl, water (3 times), and brine again. The solution was dried with MgSO 4 and pushed through a silica plug. 1g was obtained in 99% yield (0.92 g, 3.9 mmol). 3 .4. 6. 9 1,3 B is( tert butylthio) 2 ( tert butyldimethylsiloxy) propan e ( 1h ) 1 h was obtained as a liquid after flash column chromatography on silica gel (DCM/hexanes 1/15) in 9 6 % yield (0. 138 g, 0.3 9 3 mmol). (Note: T he same reaction conditions with the TMS protected substrate resulted in substantial deprotection) R f ~ 0. 46 in EtOAc /hexanes (1/ 30 ) 1 H NM R ( 5 00 MHz, CDCl 3 3.89 (tt, J = 6.4, 5.4 Hz, 1H), 2.81 (dd, J = 12.6, 6.4 Hz, 2H), 2.61 (dd, J = 12 .6, 5.4 Hz, 2H) (m, 2H), 1.31 (s, 18H), 0.90 (s, 9H), 0.11 (s, 6H) 13 C NMR (125 MHz, CDCl 3 73.0. 42.2, 35.1, 31.2, 26.1, 18.3, 4.2

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61 FTIR ( max , cm 1 ) 2 957, 2928, 2858, 1460, 1364, 1254, 1162, 1083, 1054, 912, 835, 774 3 .4. 6.1 0 1,3 B is( tert butylthio) 2 acetoxy propan e ( 1i ) 1i was obtained as a liquid in 86% yield (0.350 g, 1.26 mmol). R f ~ 0. 47 in EtOAc /hexanes (1/ 7) 1 H NMR ( 5 00 MHz, CDCl 3 5.01 ( p , J = 6.2 Hz, 1 H), 2.86 (dd, J = 13.3, 6.3 Hz, 2 H ), 2.77 (dd, J = 13.3, 6.0 Hz, 2 H ), 2.07 (s, 3H), 1.33 (s, 18H) 13 C NMR (125 MHz, CDCl 3 170.0, 71.9, 42.5, 20.9 FTIR ( max , cm 1 ) 2960, 2900, 2863, 1739, 1460, 1366, 1234, 1162, 1022 3 .4. 6.1 1 1,3 Bis( tert buty lthio)acetone ( 1j ) 1j was obtained as a liquid in 66 % yield (0. 61 g, 2.6 mmol). R f ~ 0. 54 in EtOAc /hexanes (1/ 9) 1 H NMR ( 5 00 MHz, CDCl 3 3.55 (s, 4H), 1.32 (s, 18H) 13 C NMR (125 MHz, CDCl 3 204.9, 43.7, 37.2, 30.9 FTIR ( max , cm 1 ) 2962, 2901, 2865, 17 05, 1459, 1392, 1365, 1248, 1161, 1068 The compound has been reported before, albeit without spectroscopic data. 93 3 .4. 6.1 2 1,3 Bis( tert butylthio) 3 methylbutan 2 one ( 1k ) 1k was obtained as a solid after flash column chromatography on silica g el (DCM/hexanes 1/17 gradient to 1/10) in 63 % yield (0. 68 g, 2.6 mmol). R f ~ 0. 25 in DCM /hexanes (1/ 17) T m = 56.0 ° C 1 H NMR ( 5 00 MHz, CDCl 3 3.90 (s, 2H), 1.52 (s, 6H), 1.35 (s, 9H), 1.31 (s, 9H) 13 C NMR (125 MHz, CDCl 3 207.0, 55.3, 47.0, 42.7, 33.7, 32.4, 30.9, 26.8 FTIR ( max , cm 1 ) 2961, 2922, 2894, 2861, 1696, 1456, 1366, 1155, 1050

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62 3 .4. 6.1 3 Synthesis of 1,3 b is( tert butylthio) 3 methylbutan 2 ol ( 1l ) NaBH 4 (0.044 g, 1.16 mmol) was added to a solution of 1k (0.608 g, 2.32 mmol) in MeOH (10 mL) at 0 ° C. After 2 h, the reaction was diluted with DCM and washed with aqueous 1 M HCl, water and bri ne, followed by drying with MgSO 4 . After flash column chromatography on silica gel ( EtOAc/hexanes = 1/20), 1l was obtained as a colorless oil in 67% yield (0.41 3 g, 1.56 mmol). R f ~ 0. 34 in EtOAc /hexanes (1/ 20) 1 H NMR ( 5 00 MHz, CDCl 3 3.73 (ddd, J = 10.0, 2.3, 2.2 Hz, 1H), 3.09 (dd, J = 2.1, 1.0 Hz, OH), 2.95 (ddd, J = 12.6, 2.4, 1.0 Hz, 1H), 2.59 (dd, J = 12.6, 10.0 Hz, 1H), 1.49 (s, 3H), 1.44 (s, 9H), 1.36 (s, 3H), 1.35 (s, 9H) 13 C NMR (125 MHz, CDCl 3 76.3, 54.6, 46.8, 42.5,33. 5, 3 1.19, 31.16, 26.7, 25.8 FTIR ( max , cm 1 ) 3473, 2962, 2899, 2865, 1459, 1363, 1161, 1116, 1063, 984

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63 CHAPTER 4 HARNESSING STRAINED DISULFIDES FOR PHOTOCURABLE ADAPTABLE HYDROGELS * 4 .1 Overview Hydrogels are widely used in the biomedical field, for exa mple, as material for contact lenses and wound dressings. 94 The recent convergence of dynamic covalent chemistry 2 with hydrogel systems has resulted in a considerable expansion of potential hydrogel applications. 95 Adaptable crosslinks 3 impart dynamic mechanical behavior to hydrogels, which renders these materials as promising platforms for drug delivery or 3D cell encapsulation and culture. 94, 96, 97 The use of light as a stimulus in macromolecular transformations has been an important strategy in the development of adaptable hydrogels. 98 Photomediated reactions can alter gel properties via selective de crosslinking, 99 101 post gelation patterning, 102 104 or network stiffening. 105 107 Ligh t can also be employed for the formation of adaptable hydrogels, for exa mple, via photoinduced thiol ene additions 108, 109 or copper catalyzed alkyne azide reactions 110 between monomers and pre synthesized crosslinkers containing dynamic l inkages or functional groups susceptible to dynamic bonding. 108 However, most of these approaches require photoinitiators, posing potential biocompatibility concerns. Furthermore, only a few gelation approaches exist where the dynamic crosslinks that constitute the netw ork are generated directly upon irradiation in one step. For example, An seth and coworkers recently reported the additive free formation of adaptable hydrazone hydrogels via the condensation of multi * Adapted and reprinted with permission from Macromolecules 20 20 , 53 , 4038 4046. Copyright 2020 American Chemical Soci ety. Contributions: Joh nathan L. Rowell performed parts of the experimental work, Dr. S. Tori Ellison performed cell viability experiments, and John B. Garrison acquired TEM images.

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64 arm hydrazine terminated PEG with photoliberated 2 nitro sobenzaldehyde macromonomers. 111 Herein, we aim to exp and the toolbox of dynamic bond forming photochemistry for the generation of additive free photocurable disulfide crosslinked hydrogels (Figure 4 1). With a bond strength of approximat ely 6 5 kcal/mol, 112 the disulfide bond is among the strongest homonuc lear single bonds; however, disulfides readily exchange with thiols 16 and undergo redox reactions, 113 making th em an attractive dynamic bond for adaptable hydrogels. 1,2 Dit hiolanes, five membered cyclic disulfides, have been recognized for their versatile reactivity arising from a strained disulfide ring system. 76, 114 117 For example, Waymouth and coworkers incorporated the 1,2 dithiolanes methyl asparagusic acid and lipoic acid into amphiphilic triblock copolymers prepared by ring opening polymerization of cyclic carbonates. 57, 58 Under selective solvent conditions, the addition of a small molecule thiol induced crosslinking of the polymer tethered 1,2 dithiolane groups via thiol initiated ring opening, generating disulfide crosslinked hydro gels with viscoelastic properties governe d by a dynamic polymerization depolymerizat i on equilibrium. Apart from their increased reactivity, 1,2 dithiolanes exhibit unusual photoelectronic properties compared to other disulfides, as evidenced in a red shift ed UV vis absorbance. Photolysis of linea r disulfide bonds typically requires UV light at wavelengths below 300 nm. 118 1,2 Dithiolanes, however, can be photolyzed with mil der UV irradiation at wavelengths around 350 nm and above , 119 a characteristic used in small molecule studies 1 19 121 and the crosslinking of as sembled polymer structures 122 and networks. 123

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65 Figure 4 1. Exploiting 1,2 dithiolane photochemistry for disulfide hydrogels. (A) The gelatio n strategy includes the generation of latent telechelic PEG PhDL aggregates in water with hydrophobic 1,2 dithiolane rich cores and poly(ethylene glycol) chains in the corona. Light irra diation generates thiyl radicals that covalently crosslink the network either via ring opening of unreacted 1,2 dithiolane units or recombination with other thiyl radicals. (B) The Fischer projections along the disulfide bond of the unstrained six membered 1,2 dithiane with a CSSC dihedral angle ( ) between 60 and 70 ° and a strained five membered 1,2 dithiolane with lower than 35 ° reveal the sulfur 3p orbital overlap in 1,2 dithiolanes. Both 3p orbitals are fully occupied, thus, the asymmetric splitting into bonding and anti bonding * is overall destabilizing. The HOMO energy is raised, which gives rise to a bathochromic shift of the first electronic transition ( * *), rendering 1,2 dithiolanes susceptible to photolysis with long wave UV irradiation . Herein , we employed our finding s from Chapter 3 and exploited the un ique electronic properties of 1,2 dithiolanes to photocrosslink telechelic polymers into adaptable disulfide hydrogels (Figure 4 1 ). Specifically, we modified PEG with a hydrophobic phenyl substituted 1,2 dithiolane derivative generating th e amphiphilic ne twork precursor, denoted here as P EG phenyl 1,2 dithiolane ( PEG PhDL ) . Immersion of PEG PhDL in water promoted the formation of 1,2 dithiolane aggregates

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66 interlinked by PEG chains. 1,2 Dithiolane photolysis upon UV irradiation ( max = 365 n m) induced radical mediated crosslinking, most likely via ring opening or radical recombination. T o further show the versatility of this hydrogel system, we developed a photocuring strategy with green light under air. The covalently crosslinked , free stand ing hydrogels were shapeable, stimuli responsive, and cell compatible. 4 .2 Results and Discussion 4 .2.1 1,2 Dithiolane Photochemistry The absorbance red shift of five membered disulfide ring systems can be rationalized with simplified molecular orbital (MO ) theory. 124 The Bergson model 124 invoked here involves significant simplifications; however, detailed molecular orbital calculations have verified the results qualitatively. 66 Linear disulfides usually resi de in a gauc he conformation with a CSSC dihedral angle ( ) around 90 ° , minimizing the overlap of the fully occupied sulfur 3p orbitals. 124, 125 When the disulfide bond is incorporated into a cyclic structure, decreases, and the sulfur 3p orbitals begin to overlap. While six membered disulfides ( i.e., 1,2 dithianes) are flexible enough to maintain an almost un s trained conformation with around 60 ° , the 3p orbitals in 1,2 dithiolanes are forced into a nearly eclipsed conformation at values lower than 35 ° , resulting in significant orbital interaction (Figure 4 1). 112 Due to the asymmetric splitting of the fully occupied sulfur 3p orbitals into bonding and anti bonding * MO s, the destabilization of the electrons in * dominates, thus weakening the S S bond. Additionally, the first electronic transition shifts to lower energies (i.e., to longer wavelengths) due to the raised HOM O energy, enabling 1,2 dithiolane photolysis wit h UV irradiation around 365 nm , 124, 126 which we aim to leverage for photoinduced network formation.

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67 4 .2.2 Network Precursor Synthesis PEG PhDL was synthesized via Michael addition between phenyl 1,2 dithiolane acrylate ( PhDLA ) and amidoamine poly(ethylene glycol) ( PEG 1.0 ). PEG 1.0 was readily obtained from poly(ethylene glycol) diamine ( PEG NH 2 ) after a Michael addition aminolysis reaction sequence with methyl acrylate and ethylene diamine (Figure 4 2). Successful 1,2 dithiolane conjugation was confirmed via 1 H NMR, UV vis, and Ram an spectroscopy (Figure 4 3 ). When the reaction was conducted at moderate polymer concentrations (25 mg/mL) in DMF with methanol (MeOH) as a co solvent for better solubility of PEG 1.0 , only the single acrylate amine Michael adduct was obtained eve n after elongated reaction times (30 h) at elevated temperature (50 ° C ; Figure 4 4 ). We attributed this observation to the steric hindrance around the secondary amine after the first addition. Increasing the concentration to 125 mg/mL and switching to DMF as the only solvent resulted in a faster conjugation reaction and higher 1,2 dithiolane incorporation , which should further promote assembly due to the enhanced hydrophobic character of the chain ends and thus facilitate subsequent network formation . Howe ver, the formation of high molecular weight species was observed by size exclusion chromatography (SEC) at longer reaction times (Figure 4 2 ). Peak deconvolution of the SEC trace indicated that dimers and tetramers with the doubled and fourfold molecular w eights r elative to the unimer PEG PhDL were causing the high molecular weight shoulder. Treatment with the disulfide reducing agent tributylphosphine 127 resulted in no significant change of the molecular weight distribution, which le d us to the conclusion that intermolecular disulfide formation via 1,2 dithiolane ring opening is not occurring. Instead, we hypothesize that irreversible amide bond formation via aminolysis of PhDLA amine Michael adducts and unreacted primary amines resul ted in p olymer

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68 Figure 4 2 . PEG PhDL network precursor synthesis and characterization. (A) Schematic reaction sequence for the synthesis of PEG PhDL starting from PEG diamine ( PEG NH 2 ). (B) The evolution of a high molecular weight shoulder during the Mic hael addition of phenyl 1,2 dithiolane acrylate ( PhDLA ) with amidoamine PEG ( PEG 1.0 ) at 125 mg/mL in DMF was attributed to the formation of amides via aminolysis of unhindered single PhDLA Michael adducts with unreacted free amines. (C) Deconvolution of the final molecular weight distribution indicated formation of dimers and tetramers as the coupling products. Apparent peak molecular weights ( M p ) were obtained relative to polystyrene standards. Figure 4 3. Spectroscopic evidence for 1,2 dithiolane in corporation by comparing unfunctionalized PEG 1.0 , model compound 2 , and 1,2 dithiolane functionalized PEG PhDL . (A) The stacked Raman spectra show a distinct signal around 502 cm 1 for PEG PhDL , which is characteristic for the disulfide bond in 1,2 dithio lanes. (B) Stacked UV vis spectra show the appearance of the 1,2 dithiolane absorbance around 335 nm for PEG PhD L.

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69 Figure 4 4 . Kinetic profile of the PhDLA conjugation with PEG 1.0 at 30 ° C. In DMF at high polymer concentration, 68% 1,2 dithiolane incor poration is achieved after 7.5 h. In DMF/MeOH (4:1), the incorporation stalls at 50%, even after elongated reaction times. Figure 4 5 . Comparison of PEG PhDL SEC traces showing the solvent dependence on the formation of high molecular weight species. ( A) Size exclusion chromatogram of PEG PhDL synthesized in DMF at 25 mg/mL PEG 1.0 concentration. The molecular weight and the molecular weight distributions are similar to PEG PhDL formed at 125 mg/mL (Figure 4 2 ). (B) No high molecular weight shoulder was observed if the reaction is carried out in DMF/MeOH (4:1) at the same polymer concentration. These results suggest the formation of the high molecular weight sh oulder is mostly dictated by the solvent composition. chain coupling. Notably, the formation o f high molecular weight species was relatively insensitive to polymer concentration, as indicated by similar SEC patterns observed for reactions carried out at 2 5 and 125 mg/mL in DMF (Figures 4 2 and 4 5 ). On the other hand, monomodal SEC traces were obse rved in DMF/MeOH at 25 mg/mL (Figure 4 5 ),

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70 suggesting that aggregation of PEG 1.0 in DMF, also evident from a hazy reaction mixture, is a determining factor for interchain coupling. We found that multimodal PEG PhDL network precursors obtained from DMF ge nerally yielded hydrogels at much lower polymer loadings than the monomodal PEG PhDL from DMF/MeOH. This is likely due to the dimeric and tetrameric 1,2 dithiola ne conjugates acting as multifunctional bridging units between multiple smaller polymer assembl ies, thus lowering the network percolation threshold (Figure 4 6 ). For consistency, all hydrogels in this report were formed from the same PEG PhDL network precursor containing dimers and tetramers synthesized from DMF with a number average molecular weigh t ( M n ) of 22200 g/mol, a dispersity ( ) of 1.40, and 68% 1,2 dit hiolane incorporation. 4 .2.3 Aggregation and Photolysis of PEG PhDL The accumulation of 1,2 dithiolane moieties in the hydrophobic regions of assembled networks is critical for efficient disu lfide crosslinking. 58 To assess the assembly behavior of PEG PhDL , we performed dynamic light scattering (DLS) on aqueous solutions of PEG PhDL at 1, 2, an d 4 wt% (Figure 4 7 ). At the concentrations tested, DLS showed t he formation of PEG PhDL aggregates with multiple size distributions. Transmission electron microscopy (TEM) of aggregates formed at 1 wt% showed spherical and elongated nanoparticles with dia meters ranging from 20 to 70 nm (Figure 4 7 ), matching with the first major size distribution observed by DLS. These results suggest that the hydrophobicity of the tethered 1,2 dithiolane moieties is sufficient to induce aggregation into nanoparticles with 1,2 dithiolanes units in the core and hydrophilic PEG chains in the corona. Higher concentrations of PEG PhDL

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71 Figure 4 6 . Schematic depiction of dimeric and tetrameric PEG PhDL acting as more efficient bridging agents between 1,2 dithiolane aggregates, resulting in network formation at lower polymer concentrations. Figure 4 7 . Investigation of PEG PhDL aggregation and photolysis. (A) Dynamic light scattering (DLS) of PEG PhDL in aqueous solution showed the formation of polymer aggregates. Larger clu sters of aggregates formed at higher PEG PhDL concentrations. (B) Transmission electron microscopy of a 1 wt% solution of PEG PhDL in water confirmed the presence of nanoparticles. (C) UV vis spectroscopy of PEG PhDL in water at 2 wt% after various irradia tion times. led to larger structures, which can be attributed to the increased clustering of PEG PhDL aggregates via bridging PEG chains. 58 To investigate 1,2 dithiolane photolysis in PEG PhDL aggregates, we monitored the disappearance of the 1,2 dithiolane absorbance band around 330 nm by UV vis spectroscopy after regular time inter vals of UV light exposure under argon atmosphere (Figure 4 7 ). The decreasi ng absorbance with increasing irradiation time confirmed photoinduced cleavage of the strained disulfide bond. 57, 122 The evolution of a new

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72 absorbance band above 300 nm, wh ich most likely arises from the formation of new linear disulfide linkages, prevented absolute quantification of 1,2 dithiolane photolysis. However, we tried to mitigate the impact of this band overlap by determining the relative 1,2 dithiolane absorbance ( A t / A 0 ) after various irradiation times a t a wavelength (i.e., 380 nm) that is separated from the overlap region. Both PEG PhDL concentrations (i.e., 2 and 4 wt%) showed comparable rates of 1,2 dithiolane absorbance decrease, which we believe is due to a similar local concentration of 1,2 dithiol ane units inside the cores of the PEG PhDL aggregates. 4 .2.4 U V Photocuring of PEG PhDL Having established a protocol for 1,2 dithiolane crosslinking at low PEG PhDL concentrations, we increased the polymer concentration to 10 wt%, resulting in macroscopic gelation after 2 min irradiation. The minimum polymer content for gelation was found to be around 7 wt%, and solutions co ntaining less albeit showing an increase in viscosity failed to form macroscopic networks, most likely due to insufficient percola tion of PEG chains between the amphiphilic polymer assemblies. In this report, all hydrogels discussed below were formed a t 10 wt% PEG PhDL in water. No gelation could be observed in non selective solvents such as methanol or dimethyl sulfoxide, which agre es with the findings by Waymouth and coworkers that assembly of 1,2 dithiolanes units is necessary for network formation . 2 9.30 To characterize the crosslinking process, we studied the mechanical properties of PEG PhDL hydrogels as a function of irradiation time using small amplitude oscillatory shear rheology (Figure 4 8 ) . 128 In oscillatory shear measurements, a small sinusoidal deformation is applied to th e sample, resulting in a complex respon se, which can be separated into a storage modulus ( G ) and a loss modulus ( G ). Conceptually, G

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73 Figure 4 8 . Oscillatory shear rheology of UV cured PEG PhDL hydrogles at 25 ° C and 1% strain. (A) Evolution of storage modulus ( G ) and (B) loss modulus ( G ) with increasing irradiation time; the more pronounced increase of G over G is indicative of covalent network formation. is related to the energy stored reversibly in the network (i.e., the elastic response), and G represents the amount of energy bein g dissipated as heat (i.e., the viscous response). After determining the linear viscoelastic region for each sample with strain sweep experiments, we studied the viscoelastic properties of the hydrogels at differ ent irradiation times at 1% strain and 25 ° C via angular frequency ( ) sweeps (Figure 4 8 ). Prior to UV exposure, G was found to be higher than G until the high frequency limit ( ~ 50 rad/s), indicating predominantly viscous behavior. After 1 min of irr adiation, G and G were congruent and exhibited similar frequency s caling (G , G ~ 0.3 ), two characteristics that are typically observed at the gel point of a network. 129 Upon further UV irradiation, G continued to increase more than G , indicating successful covalent network formation. We also tested thiol induced crosslinking via the addition of 10 mol% (with respect to 1,2 dithiolane units) of 2 mercaptoethanol (MSH) , analogous to the report by Waym outh and coworkers. 58 The hydrogels obtained via this method showed almost the same viscoelastic pr operties as the photocured gels, supporting our hypothes is that ring opening and subsequent disulfide crosslinking caused network formation.

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74 Generally, dynamically crosslinked materials exhibit time dependent viscoelastic behavior. 130 At timescales longer t han the terminal relaxation time ( max ), network rearrangements enabled by bond exchange can effectively dissipate energy, resulting in a predominantly viscous response upon deformation (i.e., G < G ). If the deformation is applied on timescales shorter t han max , the lifetime of the dynamic bond is too long to contribute significantly to the relaxation spectrum and the viscoelastic response becomes mostly elastic (i.e., G > G ). Therefore, in an ide al dynamically crosslinked material, we expect the visco elastic spectrum to be separated into a predominantly viscous region at lower frequencies and a mostly elastic region at higher frequencies with a moduli crossover at max . For hydrogels prepared from PEG PhDL after 10 min UV irradiation, G stayed fairly constant, around 1300 Pa across the probed frequency range, and G remained approximately at 200 Pa (Figure 4 8 ). Such a time independent mechanical response is usually associated with elastic materia ls. However, the free standing PEG PhDL hydrogels showed self healing behavior and could be readily shaped into plates (Figure 4 9 ), indicating dynamic bond exchange in the material. Additionally, such a convex shape of G is often observed in frequency sweep curves of dynamic networks at frequencies above the crossover frequency, suggesting that dynamic bond exchange is occurring, b ut not on the investigated timescales. 131 To confirm this hypothesis, we employed step creep recovery experiments, probing the viscoelastic behavior of the PEG PhDL g el after 10 min irradiation at different temperatures at longer experimental timescales (Figure 4 9 ). At 5 ° C, the hydrogel showed little permanent deformation (2% over three steps). Upon increasing the temperature, creep increas ed significantly, and the p ermanent deformation reached

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75 Figure 4 9 . Shape adaptivity of PEG PhDL hydrogels. (A) Photographs of PEG PhDL in water before and after UV irradiation (top); the free standing transparent hydrogels could be shaped into plates ( bottom). (B) Step creep exp eriments at different temperatures indicated temperature dependent bond exchange. ithionitrobenzoic acid ( DTNB) exchanges with thiols forming the colored 2 nitro 5 thiobenzoate. UV vis spectroscopy of Ellman assay on PEG PhDL hydrogels after varying irradiation times showed an increasing absorbance at 412 nm, indicative of thiol formation. 16% at 25 ° C and 63% a t 45 ° C (both over three steps). We believe that this distinct temperature dependence of creep i s most likely due to thermally activated bond exchange. Two mechanisms for bond exchange in disulfide crosslinked materials can be envisioned: disulfide metathe sis and thiol disulfide exchange. Metathesis of alkyl disulfides usually requires higher tempera tures 18, 21, 132 or UV light. 20, 133 Thiol d isulfide exchange, however, is known to occur also at ambient temperatures. 16, 17 Considerin g the substantial creep observed at room temperature, we tested the gels for thiols after assay combined with UV vis spectroscopy (Figure 4 9 ). The increasing absorbance around 412 nm clearly indicated the presence of thiols after irradiation, implying that the dynamic character of PEG PhDL hydrogels can likely be attributed to thiol disulfide exchange. Early work by Barltrop 119 and Brown 120 suggests hydrogen abstraction by thiyl radicals from C H bonds in position to oxygen

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76 atoms, however, the structure of the proposed produ cts was not fully determined. Recently, Bertrand and coworkers unambiguous ly showed that thiyl radicals undergo hydrogen transfer with C H bonds in aliphatic amines, 134, 135 benefiting from favorable pol ar effect s between the electron poor thiyl radical and the electron rich C H bond in position to the nitrogen. 136 Based on these reports we propose that in the PEG PhDL hydrogel system, som e of the thiyl radicals formed via UV induced photolysis of 1,2 dithiolane units are reduced to thiols via C H abstraction from secondary and tertiary amines present in the polymer structure. 4 .2.5 Green Light Photocuring of PEG PhDL Hydrogel crosslinki ng strate gies that employ UV light can substantially limit their utility in biological systems. 100 Moreover, longer wavelength light allows fo r enhanced curing depths. Based on work by Glorius and coworkers, who demonstrated the photocatalytic activation of disulfides under blue light, 137 we hypothesized that the weakened S S bond in 1,2 dithiolanes could undergo cleavage and crossli nking upon photoinduced electron/energy transfer (PET) from a suitable photocatalyst combined with visible light. To test this hypothesis, we employed eosin Y (EY) as a biocompatible 138, 139 and water soluble photo catalyst 140 in 10 wt% P EG PhDL solution with green light irradiation ( max = 51 5 nm). Crosslinked hydrogels formed with 0.01 molar equivalents of EY with respect to 1,2 dithiolane units under an argon atmosphere (Figure 4 10 ). Without the addition of EY, no crosslinking was obse rved even after elongated irradiation times. Si milar to the UV curing approach, we observed a gradual modulus increase with irradiation time, however, the EY gels were significantly weaker than the UV gels. This suggests that the hydrogels generated with g reen light have a lower crosslink density than those prepared during UV irradiation, which is further

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77 Figure 4 10 . Oscillatory shear rheology at 25 ° C and 1% strain of hydrogels formed with 0.01 molar equivalents eosin Y (relative to 1,2 dithiolane unit s ) under green light . (A) Storage ( G ) and loss ( G ) moduli increase with increasing irradiation time, showing covalent crosslinking. (B) Modulus comparison of hydrogels formed under argon, in a degassed solution but open to air (Degassed), and in a non degassed solution open to air (Non Deg assed). Figure 4 1 1 . Cyclic voltamm ogram of 2 (blue), the small molecule model compound for PEG PhDL . The main reduction peak for the 1,2 dithiolane is observed at 1.78 V against Ag/AgCl, which corresponds to 1.83 against SCE. The peak around 0.82 V stems from the reduction of residual oxygen in DMF (grey). reflected in the more pronounced frequency dependence of G in these gels. EY loading had a significant effect on network formation. Specifically, lower EY loadings failed to produce a crosslinked network.

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78 We also observed considerable photobleaching of EY, which does not correlate with a catalytic PET sequence. Since matching redox potentials of the substrate and the excited state photocatalyst are crucial for PET reactions, 141 we conducted cyclic voltammetry experiments on the 1,2 dithiolane small molecule analog 2 (from Chapter 3) to assess the susceptibility of reductive disulfide cleavage in our system (Figure 4 1 1 ). Critically, we found that the peak reduction potential ( E p,red ) of the 1,2 dithiolane model compound ( E p,red = 1.83 V against SCE) is substantially lower than the re ported reduction potential of excited state EY* [ E red (EY ·+ /EY*) = 1.1 V against SCE], 141, 142 which renders a catalytic PET reactio n between those two reaction partners unfavorable. Instead, we believe that EY, in synergy with the amines tethered to the polymer struc ture, acts as a radical photoinitiator for 1,2 dithiolane ring opening. 143 EY amine photoinitiator systems ha ve been widely applied for the polymerization of vinyl monomer based hydrogel films. 144 146 The mechanism of EY amine photoinitiation includes a single electron transfer from the nitroge n lone pair to the excited state EY* followed by proton abstraction from the amino radical cation, with the resulting amino radical initiating polymerization. 147, 148 The ability to crosslink under ambient conditions (i.e., room temperatu re and air atmosphere) is a highly sought after feature in designing novel hydrogel systems. EY is known to bestow oxygen tolerance upon radical polymerizations via the reduction of oxygen to the superoxide anion. 142 Therefore, we tested gelation of PEG PhDL with EY in a degassed solution but in an open vial (Degassed, Figure 4 10 ), and in a non degassed solution open to air (Non Degassed). Bot h conditions led to the formation of hydrogels with similar mechanical properties, albeit with lower moduli than hydrogels

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79 cured with EY under argon atmosphere, which is most likely due to increase d oxygen induced radical termination events. 4 .2.6 Dye Rel ease and Cytotoxicity To further prove the dynamic nature of the PEG PhDL networks, we tested the thiol triggered de crosslinking of the gels by monitoring the release of the dye rhodamine 6G after incorporation into the hydrogel matrix (Figure 4 1 2 ). Dye encapsulated gels were generated by mixing rhodamine 6G into a solution of PEG PhDL (0.02 wt% rhodamine 6G content), followed by curing under UV light. Rhodamine 6G does not absorb light at wavelen gths below 435 nm; thus, no interference with the photocros slinking process was expected, as corroborated by the similar mechanical properties of gels with and without rhodamine 6G. In water, the gel swelled substantially, resulting in mostly diffusive dye release 149, 150 for the first 30 h (~ 65% release). After 30 h, we observed gel disintegration due to slow thiol disulfide exchange in the network originating from residual thiols formed during UV irradiation . In the presence of excess M SH, which readily exchange s with disulfide crosslinks, the gel completely dissolved in 150 min concomitant with a fast release of rhodamine 6G. Notably, the gel seemed to undergo surface erosion rather than bulk swelling, indicating that network de crossli nking through thiol disulfide exchange with excess MSH is faster than water diffusion into the gel. 149, 150 Using the highly potent disulfide reducing agent dithiothreitol (DTT), we observed similarly fas t network dissolution and surface erosion , consistent with rapid disulfide crosslink cleavage by DTT . These results not only provide further evidence for the presence of dynamic disulfide crosslinks in photocured PEG PhDL gels, but also show that network degradation and cargo release can be triggered upon exposure to free thiols.

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80 Figure 4 1 2 . Thiol triggered dye release from UV cured PEG PhDL gels. (A) Photograph of rhodamine 6G encapsulated hydrogels . (B) Chemical structures of the thiol t rigger 2 mercaptoethanol (MSH) and dithiothreitol (DTT). (C) Photographs of the hydrogels immersed in 10 mL H 2 O, aqueous (D) Full dye release profile of the gels. Fig ure 4 1 3 . Confocal microscopy images of NIH 3t3 cells plated with UV cured PEG PhDL hydrogels. Live cells are shown in gr een, and dead cells appear red. (A) Cells were plated with a PEG PhDL gel that has been cured for 10 min with UV light. (B) Image of th e cells depitcted in (A) after 6 h incubation. (C) Cell viability under conditions of (A) and (B) . Finally, we investig ated the cell compatibility of PEG PhDL hydrogels with NIH 3t3 cells (Figure 4 1 3 ) . W hen cells were plated with fully cured PEG PhDL hydrogels, ~80% of the cells were alive after 6 h , suggesting that photocured PEG PhDL hydrogels

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81 could be used in applicati ons that require triggered de crosslinking or the release of a molecular cargo in the presence of living syste ms such as cells. 4 .3 Summary We have demonstrated that the photophysical properties of strained cyclic disulfides in conjunction with telechelic polymer assembly can be leveraged for the design of an adaptable hydrogel system that undergoes a light trigge red disulfide forming crosslinking reaction. Amphiphilic PEG based polymers, modified with 1,2 dithiolane end groups, show covalent network forma tion under UV light without any additives, or under green light in the presence of the photosensitizer EY. The disulfide crosslinked free standing hydrogels exhibit dynamic character at longer timescales due to thiol disulfide exchange. Irradiation time c an be used to tune the mechanical properties of the gels, where longer irradiation times result in stiffer hydr ogels. We believe this spatiotemporal control over crosslinking and network modulus can be a versatile feature in the realm of 3D printing and ph otopatterning. Importantly, this report illustrates how the careful design of macromolecular building blocks e nables the development of advanced soft materials. 151, 152 For the future, we envision that molecular m anipulations of the 1,2 dithiolane groups, 58, 75 together with variations of polymer amphiphilicity, 153, 154 will lead to a diverse design of hydrogel performance, where robustness, biocompatibility, and stimuli responsiveness can be discretely tuned. 4 .4 Experimental Reagents and solvents were purchased from commercial sour ces an d used without further purification, unless noted otherwise. PEG ( M n = 8000 g/mol ) was

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82 purchased from Sigma Aldrich and dried in anhydrous DCM over molecular sieves (4 Ã… ). All solvent mixtures are given in volume ratios. Ethylene diamine (Sigma Aldri ch) wa s distilled under reduced pressure prior to use. UV light ( 4.6 mW/cm 2 ) was supplied from a UV nail gel curing lamp (available online from ad hoc suppliers) with four 9 W bulbs and a peak emission near 365 nm (Figure 4 13) . As the green light source, a phot oreactor with a single LED chip was used giving a peak emission at 515 nm (Figure 4 13). NIH 3t3 cells were 4.5g/L glucose, L glutamine, and sodium pyruvate supplemented with 10% FBS and 1% pen icillin streptomycin and dyed with cell tracker green (CMFDA) (Thermo Fisher, part no. C2925). Figure 4 1 4 . Lamp emission spectra for the (A) UV light and the (B) green light lamp. 4 . 4.1 Instrumentation The same instruments and techniques were used as in Chapter 3 , unless noted otherwise. DLS was performed with a Malvern Zetasizer Nano ZS (Model No. ZEN 3600 , Malvern Instruments Ltd., Worcestershire UK).

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83 Dry State TEM was conducted on a Tecnai G2 Spirit TWIN TEM from Field Electron and Ion Company (FE I Company, Hillsboro, OR, USA) operating at an accelerating voltage of 120 kV. Digital images were acquired wi th a Gatan Ultrascan 1000 2k × 2k CCD camera and DigitalMicrograph 1.93 image acquisition software (Gatan Inc., Pleasanton, CA, USA.) Electron Mic roscopy Sciences Formvar Carbon Film on 400 mesh nickel grids (FCF400 Ni) were used for all trials. A nanopart icle solution (5 µL , 1 mg/mL) was spotted on the grid for 20 s. The excess solvent was wicked off. The grid was gently rinsed with 5 drops of ultr apure water and air dried after wicking off excess solvent . Oscillatory shear rheology was performed on a TA Instruments Discovery Hybrid Rheometer (DHR 2) with a 20 mm parallel plate geometry and a gap size of 1 mm . Strain sweeps of hydrogel samples were conducted at a constant frequency of 1 Hz. Frequency sweeps were performed at 1% strain and 25 °C . Step creep recovery experiments were performed at 5, 25, and 45 ° C at 50 Pa for 2 00 s , followed by 0 Pa for 5 00 s over three steps . All experiments were con ducted using the TRIO S software package from TA . The hydrogel samples were equilibrate d for 15 min and trimmed prior to each ru n. SEC was performed in N , N dimethylacetamide with 50 mM LiCl at 50 °C and a flow rate of 1.0 mL/min (Agilent isocratic pump, de gasser, and autosampler; columns: Viscogel I series 5 µm guard + two ViscoGel I series G3078 mixed bed columns, 3 4 g/mol). Detection consisted of Wyatt Optilab T rEX refractive index detector operating at 658 nm . Relative molecular

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84 weights were obtained through calibration with poly( styrene ) (P S) standards of molecular weights ranging from 1.3 to 327 kDa . CV measurements were performed at 20 ° C using an EG&G Princeton Applied Research Potentiostat/Galvanostat M odel 263A was used to apply the voltage at a scan rate of 0.1 V/s. The analyte concentration was 10 mM in a 0.1 M solution of TBABF 4 in DMF. DMF was degassed for 30 min under vacuum and dried over molecular sieves (3 Å) prior to use. We used a glassy carbo n microelectrode as the working electrode, a platinum counter electrode, and a saturated Ag/AgCl re ference electrode. To qualitatively compare the reduction potentials measured against Ag/AgCl with the EY redox potentials reported against saturated calomel electrode (SCE), the Ag/AgCl potentials were converted to SCE by subtracting 0.045 V. For cell vi ability experiments, we prepared gels were prepared as described under 5.4.2 using glutamine, and sodium pyruvate supplemented with 10% FBS and 1% penicillin streptomycin instead of pure water. The cells were dyed with cell tracker green (CMFDA) and plated with either UV cure d PEG PhDL gels. E thidium homodimer was added to the gel, allowing living cells to fluoresce green and dead cells to fluoresce red and imaged after one hour and after six hours . To asses s percent cell viability, the amount of living cells was determined ba sed on cell fluorescence. All cells in the frame were counted, and then an y cells fluorescing red were counted. The number of cells fluorescing red was subtracted from the total number of cells to determine the number of living cells. The number of living cells was then divided by the total number of cells

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85 and multiplied by 100 to determine the percent of living cells. This was repeated three times per image. 4 .4.2 Procedures Figure 4 1 5 . Synthesis of PEG PhDL . 4 .4.2.1 Syn thesis of PEG ditosylate (PEG OTs) PEG diol ( nominal molecular weight = 8 000 g/mol, M n,SEC,PS = 15000 g/mol, 32.1 g, 4.0 1 mmol) was dissolved in of dry DCM ( 150 mL ) and dried over MS 4Ã… overnight. This solution was then transferred via cannula to a solution of p tosyl chloride (3.06 g, 16. 0 mmol), TEA ( 1.62 g, 16. 0 mmol) and DM AP ( 0.24 g, 1.9 mmol ) in dry DCM ( 20 mL ) . After 24 h, the product was precipitated three tim es into cold Et 2 O. 1 H NMR (500 MHz, DMSO d 6 backbone), 2.41 (s, 6H).

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86 4 .4.2.2 Synthesis of PEG diamine (PEG NH 2 ) PEG OTs (18 . 5 g, 2.3 1 mmol) was dissolved in aqueous NH 4 OH (200 mL , 30 wt%) , and the solution was stirred for 48 h. The aqueous phase was extracted twice with DCM, and the product was precipitated three times into cold Et 2 O. 1 H NMR (500 MHz, DMSO d 6 , 2.65 (t, 4H). M n = 14100 g/mol = 1.10 4 .4.2.3 Synthesis of PEG methyl ester (PEG 0.5 ) PEG NH 2 (12.0 g, 1.50 mmol) was dissolved in 60 mL MeOH, methyl acrylate (27.2 mL, 300 mmol) was added and the solution was stirred at 35 ° C for 48 h. The polymer wa s precipitated twice into cold Et 2 O . 1 H NMR (500 MHz, DMSO d 6 7 ( s , 3H . 70 (t, 2H), 2.56 (t, 1H ) , 2.39 (t, 2H) . M n = 13400 g/mol = 1.10 4 .4.2.4 Synthesis of PEG 1.0 PEG 0.5 (11.6 g, 1.45 mmol) was dissolved in 60 mL MeOH, ethylene diamine (77.5 0 mL, 1160 mmol) was added and the solution was stirred at 35 ° C for 48 h. The p olymer was precipitated twice into cold Et 2 O . 1 H NMR (500 MHz, DMSO d 6 3.22 (br, NH 2 /H 2 O), 3.04 (br, 2H), 2.66 (t, 4 H), 2.55 (m, 1H), 2.19 (br, 2H). M n = 18100 g/mol (Figure S1) = 1.10

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87 4 .4.2.5 Synthesis of P EG PhDL in DMF at 135 mg/mL PEG 1.0 ( 2.00 g, 0. 240 mmol assuming a theoretical molecular weight of 8500 g/mol ) was combined with PhDLA (0. 71 g, 2.8 mmol ) in DMF (1 5 mL) under Ar and stirred at 30 ° C. Aliquots were withdrawn after regular time intervals, precipitated into c old Et 2 O and analyzed by SEC and 1 H NMR. The 1,2 dithiolane incorporation was estimated by comparing the area of the aromatic proton signals at 7. 45 ppm with the PEG backbone. After 7.5 h at 3 0 ° C, 68% of the maximum theoretical 1,2 dithiolane incorporatio n were reached and the polymer was precipitated into cold Et 2 O and cold hexanes . S uccessful conjugation of PhDLA to the polymeric substrate was confirmed by 1 H NMR , UV vis and Raman spectroscopy . M n = 22200 g/mol = 1.40 4 .4.2.6 Synthesis of 1,3 dichloro 2 phenylpropan 2 ol Magnesium turnings (2.11 g, 86. 7 mmol) were placed into a flame dried three neck round botto m flask, equipped with an addition funnel and a reflux condenser under Ar atmosphere. Dry Et 2 O (30 mL) was added, followed by a third of the to tal bromobenzene (13.61 g, 86. 67 mmol) solution in dry Et 2 O (50 mL). The reaction mixture was gently heated until the Grignard reaction started, upon which the remaining bromobenzene solution was added dropwise, maintaining a smooth reflux of the reaction mixture. After complete addition, the mixture was refluxed for 1 h and cooled to 15 ° C in a NaCl/ice bath. 1,3 D ichloroacetone (10.00 g, 78. 76 mmol) in dry Et 2 O (100 mL) was added dropwise. Upon complete addition, t he mixture warmed up to room temperature and was stirred for another hour. The reaction was quenched with aqueous 1M HCl (70 mL) and extracted three time s with Et 2 O. The combined organic extracts

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88 were washed with saturated aqueous NaHCO 3 , brine, and dried over MgSO 4 . Upon evaporation of the solv ent, the product was purified via flash column chromatography on silica gel ( DCM/hexanes = 1/1) . 1,3 D ichloro 2 phenylpropan 2 ol was furnished in 92% yield (14.87 g, 72. 51 mmol). R f ~ 0.35 in DCM/hexanes (1/1) 1 H NMR ( 5 00 MHz, CDCl 3 0 (m, 2 H), 7.41 (m, 2H), 7.36 (m, 1 H), 3.98 ( d, J = 11.6 Hz, 2H), 3.93 (d, J = 11.6 Hz, 2H), 2.89 (s, OH). 13 C NMR (125 MHz, CDCl 3 140.2, 128.7, 128.5, 125.8, 75.6, 50.8. 4 .4.2.7 Synthesis of 4 acrylate 4 phenyl 1,2 dithiolane (PhDLA) PhDL ( synthesized according to Ch apter 4; 0.78 g, 3.9 mmol), TEA (1.98 g, 19. 6 mmol ), DMAP (0.09 g, 0.78 mmol) , and a small spatula tip of phenothiazine (as radical inhibitor) were dissolved in dry THF (16 mL) . Under Ar, acryloyl chloride (1.77 g, 19. 6 mmol) was added dropwise. After 1 h of stirring, the reaction mixture was heated to 50 ° C and stirred for another 12 h. The reaction mixture was diluted with DCM and washed with 1 M HCl, saturated aqueous NaHCO 3 , and brine. After drying over MgSO 4 and evaporation o f the solvent, PhDLA was pu rified via flash column chromatography on silica gel ( DCM /hexanes = 1/2 ) to obtain the pure compound in 46% yield (0.46 g, 1.8 mmol). R f ~ 0.51 in DCM/hexanes (1/1) 1 H NMR (500 MHz, CDCl 3 7.27 (m, 5H), 6.45 (dd, J = 17.33 and 1.3 Hz, 1H), 6.21 (dd , J = 17.3 and 10.4 Hz, 1H), 5.90 (dd, J = 10.4 and 1.3 Hz, 1H), 3.80 (d, J = 12.8 Hz, 2H), 3.71 (d, J = 12.8 Hz, 2H). 13 C NMR (125 MHz, CDCl 3 52.0.

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89 HRMS (ESI TOF): Calculated for [M+NH 4 ] + requires 270.0617; found 270.0612 4 .4.2.8 Gelation of P EG PhDL under UV light In a 20 mL glass vial (25 mm diameter) capped with a rubber septum, PEG PhDL ( 50.0 mg) was dissolved in water (0.45 g, HPLC grade). The viscous solution was purged for 7 min with Ar and i rradiated with UV light ( max = 365 nm , 2.5 cm distance from the light source ) to yield a transparent hydrogel. 4 .4.2.9 Investigation of PEG PhDL photolysis in water In a 10 mL Schlenk flask, PEG PhDL (40 mg) was dissolved in water (1.96 g, HPLC grade) yielding a 2 wt% solution. A fter purging for 10 min, the solution was irradiated with UV light ( max = 365 nm , 2.5 cm distance from the light source ) and aliquots were withdrawn in regular time intervals for UV vis analysis . 4 .4.2.10 PhDL gels reagent solution (10 mM in pH 8 phosphate buffer) were added to UV cured PEG PhDL hdyrogels after 0, 1, 2, 5, and 10 min of UV irradiation. After 5 min incubation time, the s upernatant was pipetted off and analyzed via UV vis . 4 .4.2.11 Gelation of P EG PhD L under green light open to air In a 20 mL glass vial (25 mm diameter) PEG PhDL ( 50.0 mg) was dissolved in water (0.45 mL, HPLC grade). EY (0.18 mg) was added and the mixture was vortexed until a homogeneous solution was obtained. Network formation occurre d upon irradiation with green light ( max = 515 nm , 2.5 cm distance from the light source ) . Note: Assuming 68% 1,2 dithiolane incorporation gives a theoretical PEG PhDL molecu lar weight of 9870 g/mol with 5.4 1,2 dithiolane units per polymer chain. Consequ ently , there are 0.005 mmol PEG PhDL in 50 .0 mg, corresponding to 0.027

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90 mmol of 1,2 dithiolane units. equivalents with respec t to 1,2 dithiolane units. 4 .4.2.12 Rhodamine 6G release from UV cured gels In a 20 mL glass vial (25 mm diameter) capped with a rubber septum, PEG PhDL ( 50.0 mg) was dissolved in water (0.45 g, HPLC grade). Rhodamine 6G (1.0 mg) was added and the mixture was vortexed until a homogeneous solution was obtained. The solution was purged w ith Ar for 7 min and irradiated with UV light ( max = 365 nm , 2.5 cm distance from the light source ) to yield a deep red hydrogel. The gel was cut into three pieces (150 mg each) and one piece was immersed into water (10 mL), one into n (10 ml, 9 tants after regular time intervals for UV the respective aliquot at 527 nm (i.e., max for rhodamine 6G ) by the absorbance value at that wavelength after complete hydro gel disintegration.

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91 CHAPTER 5 SOLVENT CONTROLLED PHOTO CROSSLINKING OF COUMARIN BASED SINGLE CHAIN NANOPARTICLES * 5.1 Overview The properties of polymeric nanomaterials are often defined by the col lective behavior of multiple chains. 155 However, if designed properly, one can leverage intrachain interactions rather than interchain interactions to obtain define d nano structures comprising a single polymer chain. 156 In the past two decades, such single chain nanoparticles (SCNPs) have show n promising performance in catalysis, 157 164 cargo delivery, 165 169 or bulk materials. 170 174 To control SCNP form and function, 175, 176 various covalent intramolecular crosslinking approaches have been develop ed, 177 180 including radical mediated, 181 183 me tal catalyzed, 184 187 or photochemical ligations. 188 191 Additionally, solvent selective intrachain assembly in combination with covalent crosslinking has been introduced t o improve SCNP sphericity 192 or impart more complex structural features. 193 While water based systems are typically used for su ch intrac hain assemblies, Simon and coworkers 194 recently capitalized on the disparate solubility of a norbornene comb copolymer with polyisobutylene side chains to generate more compacted SCNPs in organic solvents. In spired by this work, we sought to further explore the potential of solvent guided intramolecular crosslinking in organic solvents and study the relationship of chain compaction and crosslinking kinetics during SCNP formation. * Contributions: Justine Elgoyhen and Dr. Yening Xia performed parts of th e experimental work; Dr . Kyle C. Bentz c onducted SLS experiments.

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92 Figure 5 1. Intrachain cro sslinking of methyl methacrylate and methyl acrylate based copolymers through the photoinduced dimerization of of coumarin . The addition of poor solvents resulted in faster and more efficient crosslinking due to more compacted polymer chain dimensions. Th e [2+2] coumarin cycloaddition typically yields a mixture of isomers. For clarit y, only the head to tail dimer is show n. Under good solvent conditions, polymer chain s adopt well solvated random coil conformations , wher e monomer solvent interactions are ene rgetically favorable. Therefore, after conventional intrachain crosslinking ( i.e., without secondary effects such as amphiphilic interactions), SCNPs typically show segmented, elongated conformations, far from a compac t spherical particle. 176 Conversely, with decreasing polymer chain solvation upon the addition of poor co solvents and operation under highly dilute condition s to minimize interchain aggregation, a single polymer chain can collapse onto itself, resul ting in a more compacted SCNP upon crosslinking . While the groundwork of this concept has been established experimentally 194, 195 and computationally, 196 198 fundamental st udies of solvent effects on the intrachain crosslinking kinetics have not been investigated. Herein , we demonstrate how polymer chain solvation can affect intrachain crosslinking kinetics and how solvent controlled chain compaction and crosslinking rate ar e interconnected. Specifically, we studied SCNP formation of methacrylate and

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93 acrylate based copolym ers in DCM with MeOH or hexanes (Hx) as poor co solvents (Figure 5 1). We employed the photoinduced [2+2] cycloaddition 199 of coumarin containi ng repeat units for crosslinking , 100, 200 allowing for reaction monitoring via UV vis spectroscopy. Kinetic analysis in combination with static light scattering (SLS) and SEC, revealed that poor co solvents increas ed rate and efficiency of the photoinduced intramolec ular crosslinking reaction due to more compacted SCNP precursor chain conformations. We believe that such straightforward solvent control over chain conformation and reaction kinetics is not only highly desirable and easily implementable in SCNP synthesis but also generally applicable for the design and understanding of macromolecular transformations. 5.2 Results and Discussion 5.2.1 Polymer Synthesis To study the influence of polymer backbone and solven t quality on the photoinduced intrachain crosslinking reaction, we synthesized methyl methacrylate and methyl acrylate based copolymers with 10 mol% and 20 mol% coumarin crosslinker content. The methacrylate series is denoted here as X MMA , and the acryla te series as X MA , with X indicating the molar fracti on of coumarin repeat units. 10 MMA and 20 MMA were synthesized in two steps from reversible addition fragmentation chain transfer ( RAFT ) co polymerizat ion followed by end group removal (Figure 5 2). Sp ecifically, methyl methacrylate ( MMA ) was copolymerized with a 4 methyl coumarin metha crylate derivative ( CMA ) at 70 ° C in 1,4 dioxane using 4 cyanopentanoic acid dithiobenzoate ( CPADB ) as the RAFT agent (Figure 2A). Analysis of the polymerization kinetics revealed similar rates for MMA and CMA vin yl bond consumption, indicating sta tistical incorporation of the two monomers (Figure 5 2), which was further confirmed

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94 Figure 5 2. Synthesis of 10 MMA and 20 MMA . (A) Reversible addition fragmentation chain tr ansfer (RAFT) copolymerization of methyl m ethacrylate (MMA) and a 4 methylcoumarin methacrylate derivative (CMA) with 4 cyanopentanoic acid dithiobenzoate (CPADB), followed by end group removal afforded 10 MMA and 20 MMA with similar degrees of polymerizat ion. (B) Pseudo first order kinetic plot f or MMA and CMA from the polymerization of 10 MMA . (C) Evolution of number average molecular weight ( M n ) and dispersity ( Ð ) with monomer conversion for 10 MMA DTB . (D) Size exclusion chromatogram of the end group re moved 10 MMA. M n and Ð values were obtaine d by SEC in N,N dimethylacetamide coupled with multi angle light scattering detection by 1 H NMR spectroscopy of the final copolymers, showing MMA/CMA repeat unit ratios close the monomer feed ratio. SEC of polymer ization aliquots showed a unimodal shift of the polymer signal to shorter elution times, and the number average molecular weight ( M n ) agreed with the theoretical value (Figure 5 2), suggesting a well controlled polymerization. To avoid cross reactivity of the photoactive dithiobenzoate moie ty 201 during the anticipated photoinduced intrachain crosslinking, we removed the RAFT agent derived end group via aminolys is with hydrazine 202 followed by thia Michael addition with methyl acrylate. 203 1 H NMR spectroscopy verified complete dithiobenz oate removal from the polymers whil e maintaining the coumarin moieties. While the thia -

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95 Michael product is the most probable polymer chain end after this reaction sequence, 203, 204 some cha ins could be terminated by a thiolactone, formed between the intermediate thiolate and the penultimate methyl ester unit. 205 However, both chain ends types should be stable towards UV irradiation at wavelengths over 350 nm and thus not interfere with the photo crosslinking. Furthermore, we conducted control 1 H NMR experiments with 7 hydr oxy 4 methylcoumarin to ensure the stability of the coumarin scaffold towards excess hydrazine and thiols. Finally, after purification via precipitation, we obtained 10 MMA and 20 MMA with narrow Ð and a M n of 19300 and 20700 g /mol, corresponding to an ave rage degree of polymerization (DP) of 162 and 150, respectively. Our attempts to synthesize acrylate based copolymers from methyl acrylate (MA) and a 4 methyl coumarin a crylate derivative by RAFT polymerization resulted in copo lymers with high Ð and uncont rolled molecular weight. We believe this is due to chain transfer and branching via hydrogen abstraction by the more reactive acrylate derived radical from the resonance stabilized exocyclic coumarin me thyl group. However, we were able to generate the acr ylate copolymer series ( 10 MA and 20 MA ) through a combination of RAFT polymeriza tion of MA, photoinduced end group remov al, 206 and a transesterification post polymerizat ion modification of poly(methyl acrylate) (PMA) with 7 (2 hydroxyethoxy) 4 methylcoumarin ( CMOH ) using triazabicyclodecene (TBD) as a catalyst (Figure 5 3) . 207 Upon modification of PMA with CMOH, the SEC peak elu tion time decreased uniformly for 10 MA an d 20 MA , indicating increasing hydrodynamic volume and molecular weight. It is important to note that the DP of 10 MA and 20 MA is defined by their precursor polymer PMA with a DP

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96 Figure 5 3. Synthesis of 10 MA and 20 MA . (A) RAFT polymerization of met hyl acrylate (MA) with 2 ( d odecylthiocarbonothioylthio) 2 methylpropionic acid (Me 2 C 12 TTC) as the RAFT agent, followed by trithiocarbonate (TTC) end group removal using 1 ethylpiperidine hypophosphite (EPHP) and UV light ( max = 365 nm) furnished poly(meth yl acrylate) (PMA) with a degree of polymerization (DP) of 161. PMA was subsequently functionalized with coumarin units using 7 (2 hydroxyethoxy) 4 methylcoumarin ( CMOH ) under triazabicyclodecene (TBD) mediated tra nsesterification cataly sis. (B) SEC analys is of the precursor polymer PMA and the coumarin functionalized polyacrylate polymers 10 MA and 20 MA . (C) 1 H NMR spectrum of 10 MA in CDCl 3 . In the spectrum, (*) denotes residual CHCl 3 at 7.26 ppm and acetone at 2.10 ppm. of 161, which is close to the DP s in the methacrylate series . Analysis of 10 MA and 20 MA by 1 H NMR spectroscopy revealed coumarin incorporations matching the CMOH feed in the transesterification reaction, demonstrati ng the potential of TBD catalyzed transesterification for precisely con trolling the extent of functionalization in post polymerization modification. 208, 209 5.2.2 Solvent S election With the co umarin functional copolymers in hand (Table 5 1 ), we sought to investigate solvent effects o n the chain compaction of X MMA and X MA upon

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97 Table 5 1. Precursor polymers used for the formation of SCNPs. Polymer DP M n,PS (g/mol) c M p,PS (g/mol) c 10 MA 161 a 15000 1.07 16600 20 MA 161 a 16400 1.09 17100 10 MMA 162 b 13300 1.07 14000 20 MMA 150 b 13 500 1.08 14600 a DP set by precursor polymer PMA. b Determined from M n,MALS in DMAc and the copolymer composition as established by 1 H NMR spectroscopy. c Determined by SEC in THF and conventional calibration with PS standards. photoinduced intramolecular coumarin dimerization. Specifically, we envisioned DCM as the good solvent for the copolymer, and 1:1 (v/v) solvent mixtures of DCM with MeOH or Hx for the poor solvent conditions. However, to estimate the solvent quality for the copolymers prior to the cr osslinking experiments, we determined the second virial coefficient ( B ) of a 2 0 MMA model copolymer via SLS and Zimm analysis (Figure 5 4). B relates to the concentration dependence of the scattering and is close to zero for poor solvents but increases for better solvents. 210 Th e 20 MMA model copolymer was synthesized via RAFT copolymerization accordin g to our protocol. We raised the molecular weight of the model copolymer ( M n ,SEC = 63000 g/mol) compared to the SCNP precursor copolymer 20 MMA to increase the inherent scattering i ntensity and improve the sensitivity of the SLS analysis. In DCM, B was 1.1 × 10 6 mol*dm 3 /g 2 and decreased by almost two orders of magnitude to 5.7 × 10 8 mol*dm 3 /g 2 in DCM/MeOH. We attempted SLS in DCM/Hx; however, the poor solubility of the model copolymer in that solvent mixture prohibited analysis at concentrations above 5 mg/mL. These results show that the solvent quality for the model copolymer decreased substantially from DCM to DCM/MeOH and to DCM/Hx. While the higher molecular weight of the model copo lymer might amplify such solubility effects, the overall trend shou ld be applicable to the lower molecular weight SCNP precursors.

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98 Figure 5 4. Static light scattering (SLS) analysis of the 20 MMA model polymer to determine the second virial coefficient B . (A) Molecular weights and Ð obtained from (B) SEC coupled with M ALS detection. Zimm plot of 20 MMA model polymer in (C) DCM and (D) DCM/MeOH, where k is a fudge factor for better readability. The high molecular weight and larger R g in DCM/MeOH indicate aggregation of the polymer chains, likely due to the much higher mo lecular weight used in these SLS studies. 5.2.3 Chain C ompaction Having established a protocol for copolymer synthesis and solvent selection, we tested the intramolecular photo crosslinki ng and chain compaction of 10 MMA , 20 MMA , 10 MA , and 20 MA in DCM, DCM/MeOH, and DCM/Hx ( Figure 5 5 and Table 5 2 ) . The copolymers were irradiated with UV light ( max = 365 nm) at low copolymer concentration (0.1 mg/mL) for 8.5 h and analyzed by UV vis sp ectroscopy to determine coumarin conversion and SEC to verify a hydrodynamic volume reduction ( i.e., chain compaction) upon intramolecular crosslinking. 211, 212 We want to point out that SEC

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99 Figure 5 5. Chain c ompaction upon intrachain crosslinking. (A) Reaction scheme for SCNP formation using UV light ( max = 365 nm). ( B) Representative SEC traces of SCNPs formed in different solvent systems from 20 MMA . Coumarin conversions for the 20 MMA SNCPs shown: 69% (DCM), 86% (DCM/MeOH), 79% (DCM/Hx). (C) Bar graph comparing the relative chain compactions between copolymer series . Peak molecular weight ( M p ) of SCNP and parent polymer (PP) were determined via conventional calibration with polystyrene standards. Coumarin conversions for 10 MMA SCNPS: 54% (DCM), 66% (DCM/MeOH), 66% (DCM/Hx); 10 MA SCNPs: 55% (DCM), 6 6% (DCM/MeOH), 69% (DCM/Hx). bears critical limitations in determining absolute SCNP size, 213 particularly if additional enthalpic interactions between polymer and column material evolve due to changes of the polymer composition upon crosslinking. 195, 214 Here, however, chain compaction occurred without introducing external crosslinker, and thus, the chemical composition should remain relatively constant within the respective copolymer series. All irradiated copolymers exhibited increased elution times compared to their linear parent polymer, indicating intramolecular crosslinking and successful chain compaction (Figure 5 5). Under poor solvent c onditions, the photo crosslinking resulted in mor e pronounced chain compactions, with DCM/Hx causing the most dramatic size reduction.

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100 Table 5 2. SCNPs formed from various precursor polymers and under different solvent conditions. Coumarin conversions we re determined from UV vis spectroscopy after 8.5 h of irradiation, unless specified otherwise . Precursor Solvent M n,PS (g/mol) M p,PS (g/mol) Coumarin conversion M p,PP / M p,SCNP 10 MA DCM 14300 1.10 15900 55% 0.96 DCM/MeOH a 13800 1.09 15000 66% 0.90 DCM/Hx 12900 1.08 14000 69% 0.84 20 MA DCM 15000 1.08 16200 73% 0.95 DCM/MeOH 13200 1.09 14100 85% 0.83 DCM/Hx 12600 1.12 11900 76% 0.70 d 10 MMA DCM 12300 1.08 13500 54% 0.97 DCM/MeOH a 12100 1.08 12800 66% 0.91 DCM/MeOH 12100 1.12 12200 78% 0.87 DCM/Hx 11000 1.09 11700 66% 0.84 20 MMA DCM 11800 1.10 13500 69% 0.93 DCM b 12900 1.08 13800 57% 0.95 DCM/MeOH 11400 1.09 12000 86% 0.82 DCM/Hx 9300 1.14 10800 79% 0.74 DCM/Hx c 11700 1.12 12200 57% 0.84 Molecular weight and Ð determined from SEC in THF and conventional calibration with PS standards. a 5 h of irradiation. b 5.5 h of irradiation. c 3 h of irradiation. d We ex cluded the 20 MA SCNPs from DCM/Hx from the chain compaction analysis due to a substant ial high molecular weight shoulder The final coumarin conversions after 8.5 h varied depending on the solvent condition and the crosslinker content (Table 5 2 ). The l owest conversions were observed in DCM (roughly 55 65%) , followed by DCM/Hx (70 80%), and DCM/MeOH (80 85%) , which is differe nt from the chain compaction trend . The reasons for this result originate from complex solvent effects on the photo crosslinking, w hich will be discussed in detail in the next section. For the chain compaction discussed here, it is important to note that h igher crosslinker conversions could afford denser particles . However, our experiments showed that at constant coumarin conversions, chain compactions with poor co solvent resulted in more collapsed particles . Furthermore, despite lower coumarin conversions, crosslinking in DCM/Hx led to more compacted particles compared to DCM/MeOH. These results show that poor solvent conditions

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101 prom ote the formation of more densely packed SCNPs, corroborating studies by Simon 194 and Lederer. 195 To compare di fferent copolymer series, we defined a measure for the relative size reduction by dividing the SCNP peak molecular weight ( M p ,SCNP ) by the peak molecular weight of the parent cop olymers ( M p ,PP ; Figure 5 5 and Table 5 2 ). Notably, we found similar relative size reductions and coumarin conversions for the methacrylate and the acrylate series with the same coumarin content ( 10 M M A and 10 MA in Figure 5 2 ; 20 M M A and 20 MA in Table 5 2 ). We believe this can be attributed to the similar chain flexibility of X MMA and X MA in solution. T he characteristic ratio ( C ) , which is a PMMA and PMA, with C va lues ranging from 7.3 to 9.0 for PMMA and 6.5 to 8.5 for PMA. 215 217 However, it is possible that such subtle backbone effects require higher polymer molecular weights to become observable in the formation of SCNPs . Alternatively, larger side chains that restrict bond rotation could be introduced to increase C . 5.2.4 Cros slinking K inetics Intrigued by the solvent effects on the coumarin conversion, which appeared to be different from the trends observed for solvent quality and chain compaction, we investigated the detailed crosslinking kinetics of SCNP formati on with 10 MM A and 20 MMA in DCM, DCM/MeOH, and DCM/Hx via UV vis spectroscopy . Furthermore, we subjected 7 methoxy 4 methylcoumarin (CMOMe) to UV irradiation under the same solvent conditions t o distinguish between contributions from coumarin solvent and polymer solve nt interactions. The decrease of coumarin concentration was fitted to a second order rate law, showing the fastest dimerization rate in DCM/MeOH for the

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102 Figure 5 6 . Photoinduced [2+2] cycloaddition kinetics for a polymeric and a molecular coumarin syst em in different solvents. Second order rate analysis for the photo crosslinking of (A) 10 MMA , 20 MMA , and (B) 7 methoxy 4 methylcoumarin ( R 2 > 0.97 for all solvent systems) . Coumarin cycloaddition s typically yield a mixture of isomers. Here, the head to tail dimer is show n. polymeric and the molecular system (Figure 5 6 ) . This could partly be attributed to the polar, protic nature of MeOH, which has been shown to accelerate the photoinduced dimerization of coumarin. 218, 219 However, compared to the dimerizat ion rate in DCM, the relative increase in DCM/MeOH was more pronounced for the copolymers than for the molecular system. Furthermore, in DCM/Hx, 10 MMA and 20 MM A exhibited a 1.5 and 1.7 fold rate increase compared to the dimerization rate in DCM. Converse ly, in the molecular system, no significant difference between DCM and DCM/Hx could be observed. This result indicates that the addition of poor solvent accelerates dimerization due to a solvent induced chain compaction of the linear precursor, arranging t he c oumarin moieties in cl os er proximity and rais ing the reaction rate in a solvophobic effect. 44 Furthermore, we hypothesize that the entropic penalty of intrachain crosslinking is likely reduc ed for a more compacted precursor.

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103 Figure 5 7 . The coumarin dimerization rate of the polymer system ( 20 MMA ) normalized by the rate of 7 methoxy 4 methylcoumarin ( CMOMe) increased linearly with the Hansen solubility parameter difference ( ) between poly(methyl methacrylate) (PMMA) 220 and the co solvent, 221 indicating a more pronounced rate increase at larger solvent solute disparities. We used the dispersion ( d ) and dipolar ( p ) contributions to the Han sen solubility parameters, avoiding the unproportionally large hydrogen bonding term of MeOH. We found that the macromolecular dimerization rate normalized by the rate of the molecular CMOMe system increased li nearly with the Hansen solubility parameter d ifference for poly(methyl methacrylate) and the respective solvent, suggesting that the rate increase can be controlled by inherent solute solvent characteristics (Figure 5 7 ). Finally, to gain further insight i nto the crosslinking efficiency in different solvents, we used the time conversion data of 10 MMA and 20 MMA and estimated the fraction of unreacted coumarin units per polymer chain throughout the photo crosslinking process. We then modelled the decay of u nreacted coumarin units with equation 5 1, de veloped by Duxbury and coworkers 196 to describe the intrachain crosslinking process of SCNPs under consideration of evo lving rigidity effects: (5 1) Here, X t is the fraction of unreacted crosslinkers at time t , C maintains the validity of the equation if t goes to zero, A is related to the crosslinking rate, and X is the fraction of crosslinkers left unreacted at infi nite reaction times due to increased chain

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104 Figure 5 8 . Crosslinking efficiency determined by the estimation of unreacted coumarin units (C units) per chain for (A) 10 MMA and (B) 20 MMA rigidity u pon crosslinking. For both copolymers, X decreased in poor solvents (Figure 5 8 ). After 8.5 h, X t was slightly higher in DCM/Hx than in DCM/MeOH, however, the model revealed the most pronounced decrease of X in DCM/Hx (roughly 50% decrease compared to X in DCM), suggesting that solvent controlled chain dime nsions can be leveraged to overcome chain r igidity during SCNP formation. Overall, these results mirror the solvation order established by SLS and the chain compaction results obtained from SEC. 5.3 Summary We studied solvent effects on the chain compacti on and the intramolecular photo crosslinkin g of coumarin containing acrylate and methacrylate copolymers. Decreasing polymer solvent interactions upon the addition of poor co solvent afforded more compacted SCNPs and substantially accelerated the crosslink ing rate in a solvophobic effect. 44 Comparison to the photo dimerization of a molecular coumarin system indicated that decreased polymer chain solvation resulted in denser chain conformations, l eading to SCNPs with reduced hydrodynamic v olume and more efficient and faster crosslinking.

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105 We believe this study not only showcases the importance of polymer solvation for the formation of SCNPs, but also demonstrates the potential of solvent guided mac romolecular reactions for the construction of advanced nanoscale architectures 5.4 Experimental Reagents and solvents were purchased from commercial sources and used without further purification, unless noted otherwise. DTB, 222 2 ( d odecylthiocarbon othioylthio) 2 methylpropionic acid (Me 2 C 12 TTC), 223 CMOH , 224 CMA , 225 and CMOMe 226 were synthesized according to previous reports. DCM, MeOH, and Hx were obtained from Fisher Scient ific (ACS grade). Anhydrous N,N dimethylformamide (DMF) and tetrahydrofuran (THF) were obtained by passing the solvent through two sequential activated alumina columns in a MBRAUN solvent purification system . All solvent mixtures are give n in volume ratios . UV light ( 4.0 mW/cm 2 ) was supplied from a UV nail gel curing lamp (available online from ad hoc suppliers) with four 9 W bulbs and a peak emission near 365 nm ( Figure 4 13) . 5.4. 1 Instrumentation The same instruments and techniques were used as in Chapte rs 3 and 4, unless noted otherwise. SEC of linear (co)polymers was performed in N , N dimethylacetamide with 50 mM LiCl at 50 °C and a flow rate of 1.0 mL/min (Agilent isocratic pump, degasser, and a utosampler; columns: Viscogel I series 5 µm guard + two Vi scoGel I series G3078 0 k g/mol). Detection consisted of Wyatt Optilab T rEX refractive index detector operating at 658 nm and a Wyatt miniDAWN TREOS light scattering detector operating at 690 nm. Abso lute molecular weights and molecular weight distributions were calculated using the Wyatt

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106 ASTRA software and dn/dc values obtained from 100% mass recovery methods . In the 100% mass recovery methods, the polymer concentration was adjusted to account for sma ll amounts of residual solvent, which was determined by thermogravimetric analysis. SEC of SCNPs was conducted in THF, due to the better solubility. The THF SEC system operated at 35 °C and a flow r ate of 1.0 mL/min (Viscotek GPCmax pump, degasser, and aut D mixed bed columns, molecular weight range 200 400 000 g/mol). Detection consisted of a Wyatt Optilab rEX refractive index detector operating at 658 nm . Rel ative molecular weights were obtained through calibration with poly(styrene) (PS) standards of molecular weights ranging from 1.3 to 327 k g/mol . SLS measurements were performed on an ALV/CGS 3 four angle, compact goniometer system (Langen, Germany), which consisted of a 22 mW HeNe linear polarized laser operating = 42 150° . R adius of gyration ( R g ) , and molecular weight ( M w ) were determined using the Zimm equation, which is a measure of the inverse scattering intensity as a function of the concentration o f solution and scattering angle: (5 2) where K is the optical constant, R is the Rayleigh ratio, B is the second virial coefficient, and c is the concentration of the polymer solution. A double extrap olation to zero concentration and zero angle will produce a y intercept of 1/ M w , while B and R g can be extracted from the slopes of the zero angle and zer o concentration extrapolation lines, respectively. All light scattering samples were performed at 25 ° C, diluted to

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107 filter, and placed into a borosilicate, pre cleaned cuvette for analysis . The dn/dc values used in the analysis were determined with a Thermo Spectronic Refracto meter at 25 ° C from polymer solutions at six different concentrations. 5.4. 2 Procedures 5.4. 2.1 Typical RAFT copolymerization of MMA and CMA CMA (0.48 1 g, 1.67 mmol), MMA (1.50 g, 15.0 mmol) and CPADB ( 0.019 g, 0.0 68 mmol) were dissolved in 4. 2 mL of a mix ture of DMF/dioxane (1/1, v/v) in a 10 mL Schlenk flask. AIBN was added ( 0.001 g, 0.007 mmol ; from a stock solution in DMF ) and the reaction mixture was p urged with Ar for 20 min with stirring. The flask was placed in a preheated oil bath at 70 ° C and reac tion aliquots were withdrawn after regular time intervals and analyzed by 1 H NMR and SEC. At the desired conversion , the reaction was quenched with air, a nd th e polymer w as precipitated into cold Et 2 O three times, followed by drying under vacuum . 10 MMA D TB: Final conversion (520 min): 60% MMA and 66% CMA M n = 18300 g/mol; Ð = 1.01 2 0 MMA DTB: Final conversion (510 min): 54% MMA and 62% CMA M n = 20600 g/mol; Ð = 1.01 5.4. 2.2 General procedure for the methacrylate copolymer RAFT agent removal The polyme r was dissolved in DMF (polymer concentration ~ 150 mg/mL) and thoroughly purged with Ar. Hy drazine monohydrate (5 molar equiv with respect to RAFT end groups) dissolved in degassed DMF (hydrazine concentration ~ 0.2 M) was added

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108 to the reaction mixture. A fter 5 min of stirring, methyl acrylate (100 equiv.) was added, and the reaction was stirred overnight. The polymers were purified by precipitation into cold Et 2 O three times, followed by drying under vacuum. 10 MMA: M n = 18300 g/mol; Ð = 1.01 2 0 MMA: M n = 20700 g/mol; Ð = 1.02 5.4. 2.3 Synthesis of PMA TTC Methyl acrylate (5.00 g, 58. 1 mmol), AIBN ( 0.004 g, 0.0 3 mmol ; from a stock solution in DMF ), and Me 2 C 12 TTC ( 0.0 93 g, 0.2 6 mmol) were dissolved in toluene (14.5 mL) in a 25 mL Schlenk flask. The reactio n mixture was purged with Ar on ice for 20 min and immersed in a preheated oil bath at 60 ° C. After 7 h the polymerization was quenched at 75 % conversion and the resulting polymer was precipitated into cold hexanes thr ee times. PMA TTC : M n = 1 26 00 g/mol, = 1.0 2 5.4. 2.4 Photoinduced RAFT agent removal of PMA TTC According to a published procedure, 206 PMA (2. 0 g) was dissolved in toluene (5.7 mL) with 1 ethylpiperidine hypophosphite (EPHP ; 0.384 g, 2.14 mmol), purged with Ar for 20 min and irradiated with UV light ( max = 365 nm) for 24 h. PMA was precipi tated into cold methanol twice. PMA : M n = 14200 g/mol, = 1.03

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109 5.4. 2.5 Typical preparation of the acrylate based coumarin copolymers In a 10 mL round bottom flask with reflux condenser, PMA (0.25 g, which corresponds to 2.9 mmol methyl acrylate repeat units) was dissolved in 3 mL of a mixt ure of dichlorobenzene/toluene (1/1, v/v). CMOH (0.128 g, 0.58 1 mmol) and TBD ( 0.00 4 g , 0.0 3 mmol; from a stock solution in toluene) were added. After purgi ng with Ar for 15 min, the reaction mixture was heated to 100 ° C for 24 h. The product was purified via dialysis against methanol/acetone 10 MA: M n ,PS = 15000 g/mol; Ð = 1.09 2 0 MA: M n ,PS = 16400 g/mol; Ð = 1.07 5.4. 2.6 Typical SCNP formation The copolyme r ( 1 0 mg) was dissolved in DCM ( 5 0 mL). Methanol ( 5 0 mL) was added slowly under rapid stirring. The reaction mixture was purged with Ar for 15 min before being placed 2 cm from the UV light source ( max = 365 nm) at an intensity of 4.0 mW/cm 2 ( upon irradiation the reaction would heat up to about 35 ° C). After 8.5 h, t he conversion was determined by UV vi s spectroscopy and the SCNPs were obtained after solvent evaporation. For experiments with kinetic monitoring, 2 0 mg of polymer in 200 mL total solvent were used. After regular time intervals, reaction aliquots (3 mL) were withdrawn and analyzed by UV vis spectroscopy. The coumarin concentration was calculated from the absorbance value at 319 nm and the molar extinc tion coefficient in that respective solvent (Figure 5 10).

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110 Figure 5 9 . Determination of the molar extinction coefficient ( ) of a coumarin model compound. We used to obtain coumarin concentration directly from UV vis absorbance in the kinetic experim ents. 5.4. 2.7 Kinetic monitoring of the photodimerization of C M OMe Since the concentration needed for efficient dimerization in molecular coumarin systems was too high for UV vis spectroscopy (beyond the linear regime), we turned to 1 H NMR spectroscopy. CMOMe was weighed into a screwcap vial with 1,3,5 trimethoxybenzene a s an internal standard and purged with Ar. Degassed solven t was added to adjust the CMOMe concentration to 0.15 M. The solution was irradiated with UV light ( max = 365 nm; 4.0 mW/cm 2 ) and after solvent removal the coumarin conversion was determined via 1 H NMR by comparing the coumarin proton resonances at 7.52, 6.88, 6.84, and 6.16 ppm with the internal standard peak at 6.11 ppm. (In a preliminary experiment t he photostability of 1,3,5 trimethoxybenzene was confirmed via 1 H NMR.)

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111 5.4. 2.8 Estimation of unreacted coumarin units per chain The average number of coumarin units per chain ( x 0 ) of 10 MMA (16.7 units) and 20 MMA (31.0 units) was obtained from the DP and the CMA/MMA ratio in the copolymer (determined from 1 H NMR spectroscopy). The n umber of unreacted coumarin units per chain ( x t ) throughout the reaction was then estimated with the conversion at time t : (5 3)

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112 CHAPTER 6 THERANOSTIC NANOCARRIERS COMBINING HIGH DRUG LOADING AND MAGNETIC PARTIC LE IMAGING * 6.1 Overview Theranostic nanoparticles are of interest because of the combined benefit s of therapeutic drug delivery and diagnostic imaging. Imaging with nanoparticles can offer insight into disease state and monitor real time drug delivery to pathology. 227 Nanoparticle drug carriers (or nanocarriers) have potential for improved therapeutic effe ct in the desired region and mitigation of side effects and long term damage to off target areas. 228 This is especially releva nt in the contex t of chemotherapy, which results in long term damage, with comorbidities and a hefty survival burden. 229, 230 Nanocarriers can also shield drugs and biologics from premature degradation 231, 232 and solubilize hydrophobic drugs. 227, 233 Despite these benefits, clinical translation of nanocarriers has been modest. 234 Some challenges in developi ng and translating nanocarriers involve achieving high drug loading, scalable manufacturing , and achieving selective and sufficiently high tumor accumulation. Methods that enable quantitative, unambiguous, and non invasive imaging of nanocarrier biodistr ib ution are valuable in overcoming this challenge. Both liposomal and polymeric nanocarriers typically show drug loadings of 10 20 wt% or less. 235 238 Nanocarriers made from block cop olymers have been commonly loade d through solvent inversion processes. 236, 239 Achieving high drug loading with this technique is challenging due to reliance on phase equilibrium . Mathematical models * Adapted and reprinted with permission from Int. J. Pharm. 20 19 , 5 72 , 118796. Copyright 2019 Elsevier. Contributions: Dr. Eric G. Fuller and I contributed equally. Eric led nanoparticle sy nthesis, characterizati on, and drug rele ase studies, and I was responsible for organic synthesis and polymers. This project was a collaboration with the group of Prof. Carlos Rinaldi at the University of Florida.

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113 predicting these limits align well with experi mental drug loadings of around 20 wt% or less. 240, 241 Low drug loadin gs in nanocarriers make in vivo work difficult because the drug might be prematurely released before reaching the desired region, and large dose s of polymer or lipid must be delivered along with the drug. 242 Flash NanoPrecipitation (FNP) is a novel technique that allows rapid and scalab le formulation of nanocarriers with drug cargo that is kinetically tra pped inside a hydrophobic core, thus overcoming thermodynamic drug loading limits. 233, 243 During the FNP process, block copolymer and hydrophobic components are dissolved in an organic solvent, and then rapidly mi xed with a miscible antisolvent. T o form sta ble nanocarriers with hydrophobic payloads, the logarithmic water octanol partition coefficient (logP) of the molecules to be incorporated generally must be 6 or higher. 233 This limits the number of drug candidates that can be encapsulated via F NP in their active native form. However, a r eversible synthetic modification of the drug to form a more hydrophobic prodrug that hydrolyses into the parent drug can overcome this limitation. While some nanocarrier formulation processes take weeks to compl ete, FNP can be done rapidly and followed wi th dialysis to obtain purified particles in several days. When FNP is used to formulate nano carriers containing magnetic nanoparticles, magnetic filtration can be conducted instead of dialysis and the total time for particle formulation and purification ca n be reduced to hours. 244 Large scale produc tion of some nanocarriers has been challenging, and the rapid formulation and purification of nanocarriers made via FNP with magnetic filtration alleviates these limitations . 245 , 246

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114 Magnetic Particle Imaging (MPI) is a tomographic tracer imaging technology that relies on the response of magnetic nanoparticles to an alternating magnetic field. The concept was first introduced by Gleich and Weiznecker in 2005. 247 In MPI there is no body background signal and the nanoparticle tracer signal is not attenuated by tissue . Further, the strength of the signal is proportional to the concentration of the magnetic nan oparticles. This allows quantitative and unambiguous imaging of the carriers at any location in the body. Combining MPI with drug loaded nanocarriers would enable tracking the biodistribution and pharmacokinetics of the nanocarriers longitudinally in pre c linical models and eventually in patients. With the ability to track the nanocarriers, the regions of the body where the drug is being delivered can be monitored, which offers insight into the therapeutic effects of the nanocarriers. W e report theranostic nanocarriers with high and tunable loading (up to 50 wt%) of doxorubicin via FNP of a hydrophobically modified, hydrolysable doxorubicin prodrug ( proDox ). The na nocarriers consist ed of a hydrophobic core of precipitated proDox together with superparamagne tic iron oxide nanoparticles (SPIONs) and a coating of amphiphilic poly ( ethylene glycol ) block poly ( lactic acid ) (PEG b PLA) block Figure 6 1. TEM image and sc hematic depiction of a nanocarrier with aggregated proDOX around superparamagnetic iron oxide nanoparticles (SPIONs) , encapsulated in a block copolymer shell. In the TEM image the proDOX core appears grey around the dark SPIONs.

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115 copolymer for water solubility and colloidal stability (Figure 6 1) . The SPIONs in the nanocarriers enable d quantitative and unambiguous imaging via MPI. The drug release rate of the nanocarriers was found to be dependent on environmental conditions such as temperature and pH . Furthermore, t he nanocarriers were internalized by cancer cells in vitro and show ed dose dependent toxicity with an IC 50 value of 1.1 µM via metabolic assay. 6.2 Results and Discussio n 6.2.1 Synthesis of p roDox via Imine Conjugation Generally, successfu l incorporation of molecular targets into nanoparticles via FNP requires logP values of 6 and above. 233 The logP value of doxorubicin is 0.57, which is too low for FNP. To make doxorubicin suitable for efficient nanocarrier encapsulation through FNP, 4 (dodecyloxy)benzaldehyde was conjugated via an imine bond to the daunosamine ring of doxorubicin hydrochloride to furnish proDox ( Figure 6 2 ), increasing the logP value to 8.17. The reaction proceeded smoothly in dry DMF over 3 Å molecular sieves to trap water formed during the conde nsation reaction. The structure of the prodrug was Figure 6 2. Synthesis of proDox from doxorubicin hydrochloride and 4 (dodecyloxy)benzaldehyde

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116 confirmed via NMR spectroscopy and HRMS . The use of the aromatic aldehyde was crucial since the same reaction with aliphatic aldehydes resulted in a product mixture of the desired imi ne conjugate and most probably a cyclic hemiaminal ether formed in situ between the Schiff base and the adjacent hydroxyl group. 248 6.2.2 Hydrolysis of p roDox To demo nstrate that imine conjugation is a viable approach to generate a hydrophobically modified doxorubicin prodrug, we investigated the hydrolysis of proDox to its parent drug doxorub i cin at different temperatures and water contents. Keeping the water content at 125 molar equivalents, the dissociation of the imine bond was Figure 6 3. Pseudo first order rate analysis of proDox hydrolysis under various conditions. (A) Variation of temperature from 23, 37, to 47°C with 125 molar equivalents of water. (B) Arrh enius plot of the apparent hydrolysis rate constant at 23, 37 and 47 ° C. (C) Hydrolysis at a fixed temperature of 37 °C wit h three different molar equivalents of water.

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117 monitored via 1 H NMR spectroscopy at 23, 37, and 47 °C in deuterated DMSO (Figure 6 3 ). The rate of hydrolysis followed a pseudo first order rate law with respect to. p roDox, due to the large excess and thus ov erall constant amount of water. As expected, the hydrolysis rate was accelerated at elevated temperatures For example, increasing the temperature from 23 to 47 ° C led to an almost 4 fold higher apparent hydrolysis rate constant. Plotting the apparent rate constants according to Arrhenius law resulted in a linear graph with an activation energy of 44.3 kJ/mol (Figure 6 3 ). Furthermore, we tested the influence of water content on hydrolysis rate at constant temperature and found that hydrolysis increases wit h increasing water content. Although these measurements will not translate quantitatively to hydrolysis rate inside the nanocarriers, due to differences in the chemical environment, they provide evidence that the rate of hydrolysis of the proDox is a funct ion of temperature and water accessibility to the nanocarrier hydrophobic core. 6.2.3 Iron O xide N anoparticle S ynthesis and C haracter ization Iron oxide nanoparticles were synthesized via the thermal decomposition approach, which has become a common route to obtain magnetic nanoparticles with narrow size distributions. An iron oleate precursor was thermally decomposed at 350 °C in a semi batch s ynthesis reactor. To obtain the iron oxide nanoparticles used for MPI, molecular oxygen was added to the thermal decomposition synthesis in slightly super stoichiometric amounts with respect to iron to improve the magnetic properties. 249 The physical size distribution of the particles was evaluated from the TEM images. T he magnetic diameter distribution was obtained by meas uring th e equilibrium magnetization of the sample in a superconducting quantum interference device (SQUID) magnetometer and fitting the curve to the Langevin function weighted using a

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118 lognormal distribution, as has been previously reported. 249 For the particles synthesized without molecular oxygen addition, the mean physical diameter was 25 nm and the mean magnetic diameter was 3.4 nm . For the particles synthesized with the addition of molecular oxygen, the mean physical diameter was approximately 17 nm and the mean magnetic diameter was 17.2 nm . 6.2 . 4 Formulation of proDox Nanocarriers FNP was used as a rapid and scalable method t o pre pare doxorubicin nanocarriers . We found that a four stream setup (Figure 6 4) with two water streams, one DMSO stream, and one THF stream during FNP would provide the best nanoparticle results in terms of reproducibility. Furthermore, the use of aqueo us so dium borate at pH 9 was crucial to ensure high drug loadings, most likely due to the reduced hydrolysis of proDox at alkaline pH. Figure 6 4. FNP m ixer setup for proDox nanocarrier formulation at typical concentrations . DMSO with proDox at 4 mg/mL THF wit h 2 mg/mL SPION, 2 mg/ mL PEG b PLA pH 9 buffer pH 9 buffer Outlet

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119 6.2.5 Control of proDox L oa ding FNP provides for tunable drug loading by varying the concentrations of the constituents in the inlet streams. In our case, the poDox loading was determined by diluting the nanocarriers ten fold in DMSO , followed by centrifugation and quantification o f the supernatant spectrophotometrically. Analysing nanocarriers generated from five different inlet concentrations of proDox ranging from 0.45 to 12 mg/mL , we found highly tunable and reproducible drug loadings. Furthermore, 12 mg/mL proDox in the inlet s tream resulted in a drug loading of about 50 wt% doxorubicin (after accounting for the hydrolysis byproduct) , which is among the highest d rug loading reported for nanoscale doxorubicin carrier s . 235 237, 241 Fig ure 6 5. Tunable doxorubicin (Dox) loading in nanocarriers . By varying the proDox concentration of the inlet stream during FNP, the drug loading of the nanocarriers can be tuned. At the highest concentration of 12 mg/mL proDox in the inlet stream , the resu lting nanocarriers are approximately 50 wt% doxorubicin. Mean ± standard deviation is plotted. 6.2.6 Characterization of Nanocarrier Size and Morphology The nanocarrier h ydrodynamic diameter ( D h ) increased as th e concentration of proDox in the inlet strea m increases. This s uggests the formation of larger hydrophobic cores at increased proDox concentrations in the inlet stream. This was further

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120 corroborated by TEM images of nanocarriers made at 12 mg/mL concentration of proDox in the inlet stream, showing s mal l , high contrast SPIONs surrounded by a large low contrast area of precipitated proDox (Figure 6 6) . The comparison of TEM images of nanocarriers made at different drug loadings shows a gradual decrease of the low contrast region around the SPIONs with decreasing proDox concentr ation in the inlet stream. Figure 6 6 . TEM images of nanocarriers generated at 12 mg/mL proDox inlet stream concentration . TEM images of multiple nanocarriers show evidence of precipitated proDo x in the low contrast areas aroun d the high contrast SPIONs for most nanocarriers. 6.2.7 Release Rate of Doxorubicin from Nanocarriers Drug r elease tests from the nanocarriers were completed with block copolymers having hydrophobic blocks of different glass transition temperatures ( T g ) . Specifically , we used polystyrene (PS , T g ~ 100 ° C ) and polycaprolactone (PCL , T g ~ 50 ° C ), to probe the effect of T g on the release rate. 250 Although the T g of these polymers is likely to be lower when they are incorporated into a nanoparticle as block copolymers, 251 we

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121 still expect a significant T g difference of the two polymers. However, nanocarriers generated with PEG b PS or PEG b PCL did not show a statistically signifi cant difference s in drug release rate. To determine if the release rate from the nanocarriers was dependent on pH, release tests were performed at pH 4, 6, and 7.4 to mimic lysosomal, intratumoral, and vascular environments, respectively. We found that l ower pH promoted faster release (Figure 6 7) . This is likely due to the faster hydrolysis of the proDox at the lower pH values, which is consistent with the pH dependence of imine hydrolysis. 252, 253 For many appli cations a slower release rate from the nanocarriers would be desirable, and we believe this can be achieved by decreasing the hydrolysis rate of the prodrug or substantially inc reasing the hydrophobic polymer shell of the particle . 254 F igure 6 7. Release test of the doxorubicin nanocarriers at 37°C at varying pH . 6.2.8 Cellular Internalization and Toxicity of Doxorubicin Nanocarriers Using fluorescently labeled nanoparticles and a dye selective for the cell me mbrane , we proved that the nanoparticles were internalized by cells . We then determined proDox nanocarrier toxicity against cancer cells via a metabolic assay ,

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122 revealing an IC 50 value of 1 .11 µ M for the proDox nanocarriers , which was slightly lower than th e IC 50 value for free doxorubicin of 0.21 µ M . 6.2.9 MPI of proDox Nanocarriers Owing to the presence of SPIONs in the hydrophobic core, the nanocarriers are potential tracers for MPI. To confirm this , a batch of nanocarriers was prepared under typical FNP conditions with SPIONs having physical and magnetic diameters of 17 nm, as well as a control batch without SPIONs. The MPI images were obtained in i sotropic m ode and the MPI signal was overlaid with the optical image of the sample in its sample holder. A signal confined to the location of the nanocarriers was observed without background signal ( Figure 6 8). The control nanocarriers prepared without SPIONs generated no signal. The limit of detection, found by determining the limit of blank and the deviation of a low concentration sample, was 15 . 4 µg Fe /mL. The volume of the samples imaged was 10 µL, meaning that as little as 154 ng of iron in a 10 µL region could be detected in an MPI scan. This suggests that these particles can be quantitat ively imaged via M PI for further testing of pharmacokinetics and biodistribution in vivo . Figure 6 8. MPI signal from doxorubicin nanocarriers. ( A) Optical image overlaid with MPI signal from Doxorubicin nanocarriers. The signal was only observed at the location of the n anocarriers with no background signal. ( B) Control nanocarriers devoid of SPIONs show ed no MPI signal. A B

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123 6.3 Summary We report the p reparation and characterization of theranostic nanocarriers loaded with doxorubicin that are capable of being quantitatively i maged via MPI. The nanocarriers are loaded with up to 50 wt% doxorubicin, and loading is tunable with the prodrug concentration of the inlet stream. The release rate is dependent on pH and is likely modifiable by tuning the hydrolysis rate of the prodrug. The MPI signal intensity from the nanocarriers varies linearly with nanocarrier concentration. This work shows nanocarriers made w ith high drug loading, suitable for quantitative MPI imaging, and a rapid and scalable formulation process that are promising for future applications. 6.4 Experimental Reagents and solvents were purchased from commercial sources and used without further p urification, unless noted otherwise. Doxorubicin hydrochloride was purchased from Carbosynth. Dialysis membranes (100 500 Da M WCO) were obtained from Spectra/Por. 4 (Dodecyloxy)benzaldehyde was prepared in accordance to a literature procedure. 255 6.4.1 Instrumentation The same instruments and techniques were used as in Chapters 3, 4, and 5, unless noted otherwise. TEM was performed by applying t went y microliters of sample were applied onto a formvar coated 400 mesh Ni grid and evaporated. The grids wer e observed on a Hitachi H7000 microscope operating at 100 kV. The images were recorded with a slow scan CCD camera (Veleta 2k × 2k).

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124 6.4.2 Procedures 6.4. 2.1 Synthesis of proDox Doxorubicin hydrochloride (0.030 g, 0.052 mmol), 4 ( d odecyloxy)benzaldehyde ( 0.075 g, 0.2 6 mmol) , and 3 Ã… molecular sieves were combined in a round bottom flask under argon. Dry DMF (1 ml) was added together with TEA (0.036 mL, 0.2 6 mmol), and the mixture was left on a platform shaker for 72 h. Purification of the product was achie ved via dialysis against 1 L of methanol (two times for 3 h each). The duration of the dialysis should be kept at short due to hydrolysis of the produc t with residual water in the solvent . The desired product was obtained after evaporation of the solvent a s a dark red powder (0.032 g, 74%). 1 H NMR (500 MHz, DMSO d 6 ): (ppm) 14.02 (b, 1H), 13.25 (b, 1H), 8.24 (s, 1H), 7.90 (m, 2H), 7.25 (d, 2H), 7.64 (m, 1H), 6.92 (d, J = 8.3 Hz, 2H), 5.41 (s, 1H), 5.33 (m, 1H), 4.99 (m, 1H), 4.86 (t, J = 6.0 Hz 1H), 4.58 (m, 2H), 4.30 (d, J = 4.7 Hz, 1H), 3.96 (b, 5H), 3.65 (m, 1H), 3 . 50 (m, 1H), 2.97 (s, 2H), 2.18 (m, 3H), 1.68 (m, 2H), 1.31 (b, 22H), 0.84 (t, J = 6.6, 3H) ; 13 C NMR (125 MHz, CDCl 3 ): (ppm) 213.84, 186.96, 186.68, 161.75, 161.11, 156.32, 155.69, 135.82, 135.47, 134.05, 133.61, 132.08, 129.95, 128.38, 120.88, 119.86, 118.56, 114.63, 111.55, 111.41, 101.14, 76.97, 70.19, 69.56, 68.26, 66.93, 65.61, 64.61, 56.75, 35.57, 34.06, 32.02, 30.91, 29.75, 29.73, 29.69, 29.66, 29.47, 29.45, 29.25, 26.09, 22.79, 17.07, 14.23; HRMS (ESI TOF): Calculated for C 46 H 58 NO 12 + [M + H] + r equires 816.3960; found 816.3949.

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125 6.4. 2.2 Hydrolysis s tudy To determine the effect of temperature on hydrolysis rate, proDox (0.012 g, 0.015 mmol) was dissolved in DMSO d 6 (1.5 mL), and water (0.033 g, 1.833 mmol) was added. This solution was split equal ly into three reactions which were placed in oil baths at 23, 37, and 47 °C, respectively. The hydrolysis of proDox was analyzed via 1 H NMR in regular time intervals by mon itoring the disappearance of the imine proton at 8.24 ppm and the appearance of the aldehyde proton at 9.86 ppm with respect to the terminal methyl group at 0.84 ppm (Figure 6 9) . Figure 6 9. 1 H NMR analysis shows the hydrolysis of proDox in DMSO d 6 . ( A) Hydrolysis reaction scheme. (B) Stacked 1 H NMR spectra of pristine proDox (top) a nd after 11.5 h (bottom) with 125 molar equivalents of water at 37 ° C. The reduction of the imine signal at 8.24 ppm and the appearance of the aldehyde proton at 9.86 ppm with respect to the terminal methyl group at 0.84 ppm were used to determine the conv ersion of proDox to doxorubicin

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126 For the effect of water content, a s imilar procedure was applied except all samples were kept at 37 °C at varying water contents (125, 200, and 300 molar equivalents). 6.4. 2.3 Iron oxide nanoparticle synthesis Iron oleate pr ecursor was prepared by reacting 20.15 g (57.13 mmol) of iron acetylacetonate and 80.53 g (284 mmol) of oleic acid in a 500 mL three neck reactor flask. The reaction under 100 sccm of argon was thoroughly mixed using a Caframo compact overhead stirrer at 3 50 rpm. The reaction mixture was heated to about 320 °C at a r amp rate of 8 °C/min using a fabric heating mantle, and temperature was controlled using a Digi sense temperature controller. After 35 min at 320 °C, a dark brown waxy solid was obtained and use d as the precursor for nanoparticle synthesis. For the nanopar ticle synthesis, first, 14.0 g (48.3 mmol) of was initially heated to 350 °C for 50 60 min at a ramp rate of 7 8 °C/min in a 100 mL three neck reaction flask. The rate of addition of argon, the inert gas, was controlled at 100 sccm using mass flow controll ers from Alicat Scientific. Once the reactor reached 350 °C, the controlled addition at 9 mL/hr (using a syringe pump) of 30 mL of iron oleate precursor (0.63 M Fe) mixed with 55 mL of 1 octadec ene was initiated along with oxygen feed of 20% oxygen and 80% argon at a rate of 9.47 sccm, controlled using a mass flow controller (Bronkhorst USA). Uniform mixing at 350 rpm was ensured, and the reaction temperature was controlled at 350 °C for 6 h usin g a Digisense temperature controller. The reaction mixture coo l ed to room temperature, and iron oxide nanoparticles obtained at the end of the reaction were purified by magnetic filtration. For the magnetic filtration, the crude product was diluted 2:1 v/v in hexane, mixed, then flow through a 5 mL syringe placed bet ween permanent magnets and packed to 2 mL at 0.9 g/mL packing

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127 density of steel wool of 0.04 0.05mm diameter. Additional hexane was added to remove excess organics, then the retentate of particle s was recovered with the syringe away from the magnets. The pa rticles were further suspended in THF at required concentrations for preparation of nanocarriers. 6.4. 2.4 F NP for proDox n anocarriers A typical procedure for preparing nanocarriers by FNP was as follows: 2.0 mg of PEG b PLA was weighed in a dry 20 mL glass vial. 0.2 mL of SPIONs at 10 mg/mL in THF were added to the vial as well as 0.8 mL THF. This is the 1 mL THF stream with concentrations of 2 mg/mL for both the PEG PLA and the SPIONs. The DMSO stream with the proDox was made by weighing 4.0 mg of proDox i n dry, powdered form into a dry 20 mL glass vial, then adding 1 mL of DMSO. Both solutions were sonicated in a bath sonicator for 5 to 10 minutes and DLS was done to ensure solutions were well d issolved. FNP was then performed by loading the THF and DMSO s treams each into 5 mL polypropylene syringes (Norm Ject), and 2 opposing syringes with 1 mL pH 9 sodium borate buffer (50 mM) were added as well and pushed through a 4 way mixer at approximately 60 mL/min. The resulting product was caught in 16 mL of pH 9 buffer stirred at 350 rpm with a Caframo mechanical stirrer to give a final organic content of 10% v/v. This product solution was then filtered magnetically by passing it through a Miltenyi Midi Macs LS column or a 5 mL syringe packed with 0.04 0.05 mm in d iameter steel wool which was placed between two permanent magnets. The steel wool was packed to 2 mL with a packing density of 0.91 g/mL, and a polyether ether ketone stopcock was used to contro l the drip rate. The entire 20 mL of product solution was pass ed through the column or syringe at a drip rate of approximately 1 s per drop. To remove excess starting material and organic solvents, 4 mL pH 9 buffer was passed

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128 through the column 5 8 times, and then 4 mL deionized water, followed by removing the column from the magnet and collecting the retentate in 1 mL water . The drug loading assay below was then performed to determine the concentration of doxorubicin in the retentate. 6.4. 2. 5 Drug loading assay Doxorubicin nanocarrier retentate (100 µL) was added to 900 µL of DMSO. At this point, the Dox and PEG PLA were well dissolved in DMSO and the SPIONs were starting to aggregate because of the DMSO. This mixture was centrifuged for 15 min at 20,800 r cf. , and 100uL of supernatant was spotted into a 96 we ll plate in triplicate in addition to a calibration curve of doxorubicin in the same solvent and centrifuged at the same speed and time. All retentate and calibration curve samples were kept covered fro m light to avoid photodegradation of doxorubicin. The absorbance of the retentate samples was quantified against the calibration curve in a SpectraMax M5 spectrometer and reported as mean ± standard deviation. 6.4. 2. 6 Doxorubicin r elease t ests Immediately after purifying nanocarriers and recovering the retent ate, 500 µL samples of the retentate were placed into polypropylene centrifuge vials. A 10 mM sodium acetate buffer was used for pH 4 samples, and 10 mM sodium phosphate buffers were used for pH 6 and p H 7.4 samples. Vials are then placed in a shaking incu bator, covered in aluminum foil to minimize exposure to light at 37 ºC. After each time point measured, vials are taken out of shaking incubator and placed in a centrifuge at 20,800 relative centrifugal force (rcf) for 30 minutes at 4ºC. Supernatant is ali quoted on 96 well plate and read on a spectrometer. Concentration of release samples is quantified via absorbance of the supernatant compared to a calibration curve of

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129 doxorubicin under identical condit ions. To find percent release, the concentration relea sed by the particles in each sample was divided by the maximum amount of drug in the particle, found by the drug loading assay described above. N = 3, mean ± standard deviation is plotted unless otherwi se mentioned. 6.4. 2. 7 IC 50 t oxicity a ssay and f luores cence Imaging MDA MB Modified Eagle Medium (DMEM) in a 96 well plate. 2 days post seeding, doxorubicin or proDox nanocarriers were dis solved in media at the desired concentrations and adde d to the wells, and a negative control of media only and positive control of Triton X 100 solution were included. After 48 hours, all drug and nanocarriers were removed and the MTT assay was performed b y adding 100 µL media with MTT (0.5 mg/mL (3 (4,5 dime thylthiazol 2 yl) 2,5 diphenyl tetrazolium bromide) , incubating for 2 hours at 37°C, removing MTT media, adding 100 µL DMSO, shaking at 300 rpm, and quantifying absorbance in SpectraMax M5 spectrometer. Relatively viability is defined as 100% for the negati ve control of media only and 0% for the positive control of TritonX. For fluorescent imaging, MDA MB 231 cells were seeded at 20,000 cells/well onto an 8 chamber glass slide ( Lab Tek II Chambered Cover glass w/Cover #1.5 Borosilicate 8 well, reference numb er 155409) and grown for 2 days, then doxorubicin nanocarriers were added to the wells at a concentration of 40 µM Dox. The DoxMCNCs were formulated as usual except a PDLLA Cy7 homopolymer (10 15 kDa, P olySciTech) was added in the THF stream at 2 mg/mL and the proDox concentration was 2 mg/mL as well. Images were taken in a Keyence BZ X 710 fluorescent microscope after 5 hours of incubation of the nanocarriers on the cells.

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130 6.4. 2. 8 MPI p rocedure proDox n anocarriers (10 µL) at a concentration of 0.568 mg/mL iron oxide were nanocarriers were made under identical conditions except no SPIONs were added to the THF FNP stream a n d dialysis was done for purification overnight with a 100 kD membrane. Both control and normal nanocarriers were imaged in a 3D printed custom holder cleaned with isopropanol and the image scales for each were set as 1 to 47 arbitrary units in the VivoQu a nt® program. For the quantitative imaging, nanocarriers at the 0.568 mg/mL concentration as well as five other dilutions were imaged in Isotropic Mode and line scans of the intensity values were performed. Peak values for each concentration were found and a linear fit of the data was applied.

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131 CHAPTER 7 PROBING THERMORESPONSIVE POLYMERIZATION INDUCED SELF ASSEMBLY WITH VARIABLE TEMPERATURE LIQUID CELL TRANSMISSION ELECTRON MICROSCOPY * 7.1 Overview First introduced in the 1950s as nonionic surfactants 256, 257 amphiphilic block copolymers have demonstrated widespread utility due to their ability to form an array of int ricate solution assemblies, such as spherical micelles, 45, 258 worms, 259 vesicles, 260, 261 framboidal vesicles, 262 and toroidal structures. 263 The preparation of nanoparticles assembled from amphiphilic block copolymers can be achieved either via post polymerization processing, where block copolymers are treated with selective solvents to in duce self org anization of polymer chains, 45 or via polymerization induced self assembly (PISA), where self assembled structures form during the synthesis of a solvophobic polymer block. 264 For example, during the solvent switch method (a post polymerization approach), amphiphilic diblock copolymers assemble first into spherical micelles and progress to other morphologies as the solvent quality for the solv ophobic block gradually decreases. 265 Most often, the characterization of the assembled structures consists of a combination of bulk solution techniques, s uch as small angle X ray scattering (SAXS) or dynamic light scattering (DLS), with static methods such as transmission electron microscopy (TEM). In situ SAXS techniques, in particular, have shown great potential for the detection of assembly nucleation an d progression . 26 6 268 However, no characterization technique has * This project was a collaboration with the research group of Prof . Nathan Giannesc hi at Northwestern University. Dr. Mollie A. Touve and I contributed equally. Mollie led LC TEM experiments , and I was responsible for polymer synthesis, characterization, and TEM assistance.

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132 be en available for directly imaging the assembly process; expansion of the suite of techniques available for direct real time monitoring of the morphological evol ution of the PISA process is needed. 269 TEM in liquids has been traditionally conducted with an open cell setup; 270, 271 however, recent advances in closed liquid cell technology 272 allow in situ TEM of solution phase samples under high vacuum conditions. 273, 274 Such clos ed liquid cells feature an electron transparent window (commonly silicon nitride) that allows penetration of the sample with the electro n beam while protecting the enclosed sample solution from the internal vacuum of the microscope ( Figure 7 1 ). Liquid cel l transmission electron microscopy (LCTEM) has the potential to provide the spatial and temporal resolution needed for the direct visual ization of block copolymer nanoparticles Figure 7 1. Liquid cell transmission electron microscopy (LCTEM) solution ce ll. (A) Top view of a silicon nitride (SiN x ) chip placed at the tip of an LCTEM holder. The electron in the center of the chip allows imaging of polymer assemblies in the solution between the two chips. (B) Side view of two chips (top and bottom) assembled as a liquid cell containing a sample solution with a liquid thickness of ~200 nm. (C) Cross sectiona l magnified view of the liquid cell window embedded in the chip.

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133 in situ. 275 280 Specifically, real time monitoring by LCTEM has been shown to provide mechanistic information regarding morphological evolution in soft matter. 281, 282 For example, Patterson and coworkers were able to observe th e formation of vesicles from poly(ethylene oxide) block poly( caprolactone) via a solvent switch appro ach inside the liquid cell transmission electron microscope. 283 Time resolved in situ imaging revealed that ves icles are nucleated by liquid liquid phase separ ation, specifically by polymer rich liquid droplets in solution. Similarly, in our own work, phase transitions occurring via a micelle micelle fusion mechanism were captured by direct videography in LCTEM. 275 PISA is a robust and rapid method for accessing assembled polymeric materials with targeted morphologies at high polymer loadings. Reversible addition fragmentation chain transfer (RAFT) polymerization 284 is often employed in PISA to impart control of polymerization rates and chain lengths. 285, 286 PISA can o perate under emulsion and dispersion conditions. 287 For the dispersion ap proach in aqueous solution, a hydrophilic polymer is chain extended with a monomer that is initially water soluble but forms a hydrophobic polymer. As the hydrophobic blo ck length approaches a critical degree of polymerization, the amphiphilic polymer chai ns assemble into micelles, and the homogeneous polymerization turns into a dispersion. During polymerization, a progression through different morphologies occurs, driven by a force equilibrium between core chain stretching, corona chain repulsion, and surf ace tension at the core corona interface. 288, 289 Recently, we reported the in situ formation and observation of spherical micel les using LCTEM. 278 In that work, irradiation with the electron beam induced homolytic cleavage of the carbon sulfur bond of the macro chain transfer agent (macro CTA), leading to initia tion and PISA of the growing amphiphilic block

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134 copolymers . We proposed this mechanism of polymerization to be similar to a photoiniferter polymerization. 290 292 However, using the electron beam to control chemical reactions is challenging due to potential radiolysis side products 293 and concentration gradients resulting from the spatially confined irradi ation of the sample. Moreo ver, it is not directly analogous to reactions occurring during normal benchtop syntheses. Therefore , we employ variable temperature liquid cell transmission electron microscopy (VT LCTEM) 294 to investigate the commonly employed, thermally initiated RAFT PISA process. This was investigated for a robust and reproducible acrylamide based PISA system that allowed thorough characterization of polymerization kine tics and morphology progre ssion. To capture and directly visualize the formation of PISA nanomaterials, we first achieved two key milestones by showing: (1) that higher order assemblies of polymeric amphiphiles (e.g., vesicular phases 283 an d worm like micelles) generated by PISA can be imaged via LCTEM at electron doses that do not damage those structures; and (2) that thermally initiated, controlled radical polymerization via RAFT, can be conducted within the confined TEM liquid cell, yield ing the same materials as can be accessed by standard benchtop syntheses with sufficient exclusion of oxygen. Herein, w e will describe the data obtained with respect to these two milestones and demonstrate thermal initiation and direct imaging of the forma tion of block copolymer assemblies by VT LCTEM. Leveraging this technique further, we were able to probe the effect of temperature on the resulting assemblies, capturing their inherent thermal phase transition behavior in solution by VT LCTEM.

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135 7.2 R esults and Discussion 7.2.1 PISA Polymerization Kinetics and Morphology Evolution Before interrogating PISA by LCTEM, it was c rucial to monitor and image the assembled particles formed during the conventional PISA process ex situ (i.e., outside of the liquid cell ), using standard analytical techniques. We adapted a PISA system recently developed by Figg et al., 295 based on the copolymerization of diacetone acrylamide (DAAm) and N,N dimethylacrylamide (DMA) in a mol ar ratio of 75:25 at 60 ° C using a water soluble poly( N,N dimethylacrylamide) (PDMA) macro CTA with a number average mo lecular weight ( M n ) of 4 kDa (Figure 7 2) . The targeted number average degree of polymerization (DP) at full monomer conversion for the h ydrophobic block was 170. PISA systems are often described with phase diagrams that depict assembly morphologies at ful l monomer conversion at different polymer Figure 7 2. Outline of the polymerization induced self assembly (PISA) system . (A) Synthesis of amphiphilic poly( N,N dimethylacrylamide) 37 block poly(diacetone acrylamide co N,N dimethylacrylamide) 170 [PDMA b P(DAAm co DMA)] using a water soluble poly(N,N dimethylacrylamide) (PDMA) macro chain transfer agent (macro CTA), the azo initiator VA 057, and N,N dimethylacrylamide (DMA) and diacetone acrylamide (DAAm) as monomers. (B) Pr ogression from amphiphilic unimers to vesicles upon chain extension of a hydrophilic PDMA macro CTA with DAAm/DMA as the core forming monomers via RAFT PISA.

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136 Figure 7 3 . Chain Extension of PDMA macro CTA with DAAm and DMA via RAFT PISA . (A) Plot of mono mer conversion (DAAm and DMA averaged) with the corresponding morphology indicated along the top (U = unimers, M = spherical micelles, W = worms, I = intermediate architec tures such as branched worms and jellyfish structures, V = vesicles). The onset of as sembly was determined by the appearance of a slight solution opalescence caused by nanostructures that scatter visible light. (B) Pseudo first order kinetic plot for DAAm and DMA. (C) Dry state TEM analysis of crosslinked morphologies from aliquots taken a t various monomer conversions during polymerization. The respective monomer conversion and predominant morphology observed is indicated at the upper left corner of the TEM image. concentrations and hydrophobic block lengths. However, to gain a more accura te insight into the evolution of morphology within a single polymerization, we monitored morphology with respect to monomer conversion ( Figure 7 3 ). This approach accounts for unreacted monomer that can solvate and plasticize the growing hydrophobic core, thus substantially affecting morphology transitions. Correlating polymerization kinetics

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137 and assembly morphology as determined by dry state TEM analysis, we observed accel erated polymerization kinetics after the onset of assembly, typically observed in PIS A systems. To preserve the morphology of the thermoresponsive polymer assemblies for dry state TEM and DLS analysis, an O alkyl hydroxylamine crosslinker was added at diff erent stages during PISA at 60 ° C to core crosslink the assemblies via oxime formatio n. 295 While critical for post polymerization characterization, this crosslinking procedure proved cumbersome and gave ris e to artifacts. Specifically, unreacted DAAm monomer can interfere with the crosslinking process through undesired consumption of the Figure 7 4. Scenarios upon O a lkyl h ydroxylamine c rosslinker a ddition at d ifferent m onomer c onversions . (A) Crosslinker addition before self assembly at low monomer conversion results in the formation of a macroscopic network. (B) Addition of the crosslinker after assembly at medium monomer conversions provides crosslinked assembled structures, but also ill defined crossli nked Schematic clusters of residual monomers and unimers. (C) At high mo nomer conversion, addition of crosslinker affords predominantly crosslinked assembled structures.

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138 O alkyl hydroxylamine crosslinker via oxime formation and Michael addition, resulting in monomer derived crosslinked clusters ( Figure 7 4) and complicating T EM and DLS analysis. Nonetheless, we were able to observe a morphology progression from spherical micelles (<50% conversion, 20 50 nm diameter) to worms mixed with intermediate structu res such as branched worms and jellyfish (50 85% conversion), and finall y vesicles (300 500 nm diameter) at conversions above 85% (Figure 7 3) . To the best of our knowledge, this is the first report of monitoring morphology progression as a function of mon omer conversion for an acrylamide based thermal dispersion PISA system, thus providing further support for the proposed mechanism of morphology progression during PISA. 22, 296 7.2.2 Thermorespo nsive Morphology Transitions Many amphiphilic block copolymer assemblies generated by PISA show temperature dependent morphology transitions. For example, PISA systems using N isopropy lacrylamide, 297 N,N diethylacrylamide (DEAm), 298 DAAm, 299, 300 2 hydroxypropyl methacrylate, 301 304 2 methoxyethyl met hacrylate, 267 or alkyl hydroxy acrylates 305 as the core forming hydrophobic blocks undergo substantial structural rearrangements upon temperature changes. Although there is great potential in the development of thermoresponsive polymer assemblies, 3 06 308 the temperature induced changes in hydration of the hydrophobic core often impair characterization of the assemblies by techniques that require sample preparation or analysis at room temperature. To preserve assembly morphology for characterizatio n, various crosslinking approaches have been developed. 295, 309 312 However, crosslinking can potentially alter the assembly morphology. Furthermore, crosslinking does not prevent the partial rehydration of the hyd rophobic regions at lower temperatures, which could lead to

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139 swelling and an increase of assembly size, resulting in incorrect assignment of the morphology or particle dimensions during room temperature, post polymerization analysis b y DLS or TEM. Prior to conducting an analysis of the thermal responsiveness of these materials within the liquid cell by TEM, we sought to first investigate temperature induced morphology transitions of non crosslinked PDMA b P(DAAm co DMA) assemblies by DLS ( Figure 7 5 ). Upon cooling from 60 to 20 ° C, the vesicles disassemble into solvated unimers, as indicated by the decrease of the hydrodynamic diameter ( D h ), due to the more favorable hydration of the P(DAAm co DMA) block at lower temperatures. 295, 313 The morphology reversion of non crosslinked vesicles upon cooling coincides with traversing the cloud point of the mixture (Figure 7 5) , evident by the gradual disappearance of solution opacity, 314 whereas a solution of crosslinked vesicles remained opaque at room temperature. Furthermore, we noted that DLS analysi s of vesicles formed at 60 ° C showed assemblies with a hydrodynamic diameter of ~150 nm, which does not match the 300 500 nm diameters observed by dry state TEM for the same assemblies crosslinked at 60 ° C and cooled to room temperature. Since we observed no change of morphology by DLS upon crosslinking at 60 ° C ( Figure 7 5 ), we attribute this size difference to the different extent of hydration of the hydrophobic core at lower temperatures. Upon cooling from 60 ° C to room temperature, the crosslinked hydro phobic block of the polymers swells as it becomes more hydrated, and larger assembly dimensions are recorded by dry state TEM and DLS . Since the hydration of hydrophobic molecules is temperature dependent 37, 315 318 and similar changes in assembly size have been

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140 Figure 7 5. Temperature ef fects on p olymer a ssemblies . (A) Dynamic light scattering (DLS) traces of non crosslinked vesicular assemblies formed at 60 ° C and then gradually cooled to 20 ° C. (B) Cloud poin t determination of PDMA 37 b P(DAAm co DMA) 170 generated through PISA at 11 wt% polymer concentration via UV vis absorbance measurements at a wavelength of 500 nm. (C) DLS trace of vesicles at 60 ° C before (black) and after (blue) the addition of O alkyl hy droxylamine crosslinker . (D) DLS traces of a non crosslinked solution of vesicles at 60 ° C (V, blue) and the same solution of vesicles after crosslinking at 60 ° C, followed by cooling to 25 ° C (X V, black). observed with other PISA systems, 267, 299, 300, 303 305 we believe that these hydration effect s are not unique to our system and generally warrant consideration for careful temperature dependent analysis of crosslinked and non crosslinked PISA assemblies prepared at elevated temperatures.

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141 7.2.3 Observing Crosslinked Worms and Vesicles in Solution. Considering the problems with size determination and tedious sample preparation (e.g., crosslinking steps) during the characterization of thermoresponsive PISA morpholo gies, we recognized the utility of VT LCTEM in imaging polymer assemblies in situ. We ai med first to evaluate if higher order morphologies, such as worms and vesicular assemblies could be imaged at all in the solution state by TEM, and to observe how they compare morphologically to when those same structures are dried and stained for imaging by standard dry state TEM analysis ( Figure 7 6 ). Two solutions, one containing PDMA b P(DAAm co DMA) worms along with intermediate structures and vesicles, and one wit h predominantly vesicles were prepared via RAFT PISA and crosslinked at full monomer conversion. Prepared structures were then loaded into the liquid cell for LCTEM, or solution casted on TEM grids for standard analysis ( Figure 7 6 ). Worms and intermediate structures such as branched worms with Y junctions were identified in the li quid cell. Importantly, worm cross diameter dimensions of about 30 nm were in good agreement with dry state TEM images. Crosslinked vesicular structures showed much lower contrast , perhaps due to their higher water content. However, using NiCl 2 as a LCTEM stain to aid visualization by increased bilayer contrast, 319 we were able to resolve vesicles with diameters between 200 and 600 nm, which matched the overall dimensions of the vesicles observed by dry state TEM. Intriguingly, this experiment reveals LCT EM, akin to cryogenic TEM, 320 can successfully preserve vesicular morphologies ex pected in the solution phase for these materials far better than what is observed by conventional TEM. Drying artifacts of this type are common for vesicles where they often appea r

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142 Figure 7 6. Comparison b etween LCTEM and d ry st ate TEM i mages of c rossl inked w orms and v esicles . (A) Standard dry state TEM image and (B) LCTEM image of a sample containing PDMA 37 b P(DAAm co DMA) 250 worm like micelles, intermediate structures, and v esicles at 0.5% w/v total solid content. Black arrows denote worms, and white arrows denote vesicles. Electron dose used in LCTEM imaging = 0.4 e Ã… 2 s 1 . (C) Standard dry state, uranyl acetate stained TEM image and (D) LCTEM image of a sample containing PD MA 37 b P(DAAm co DMA) 170 vesicles at 0.5% w/v in water. Electron dose used in LCTEM imaging = 0.3 e Ã… 2 s 1 . All inset scale bars = 100 nm . crenated. The fact that we can utilize LCTEM to avoid this entirely should be generalizable to other vesicular stru ctures. With images in hand, we sought to probe the stability of the structures under constant electron beam irradiation to establish imaging conditions that avoid beam damage to the assemblies ( Figure 7 7 ). After 60 s of continuous imaging at low electro n

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143 Figure 7 7. Beam i nduced d amage to v esicles in s olution d uri ng c ontinuous i maging at l ow e lectron d ose . Beam damage is defined here as the onset of nanoparticle change (in terms of contrast and particle features) under electron beam irradiation in th e absence of any other stimuli. (A) Image of crosslinked PDMA b P(DAAm co DMA) assemblies in solution acquired at low instantaneous dose of 0.3 e Å 2 s 1 (and cumulative dose of 6 e Å 2 ). (B) Disintegration of assemblies in the same liquid cell area shown i n (A) after the cumulative electron dose reac hed approximately 18 e Å 2 . Black arrows point to the boundary of the electron beam. dose (0.3 e Å 2 s 1 ), substantial beam induced particle disintegration was apparent. However, shorter imaging times under thes e conditions did not cause detectable damage and allowed reliable visualization of the structures. These short duration, pulsed imaging conditions were then applied for thermally initiated PISA observable by LCTEM ( vide infra ). 7.2.4 Thermally Initiated RA FT Polymerization Inside the Liquid Cell To prove that thermally initiated RAFT polymerization can occur in a controlled manner inside the liquid cell with sufficient exclusion of oxygen, we used matrix assisted laser desorption ionization imaging mass sp ectrometry (MALDI IMS) to analyze polymers fo rmed in situ (Figure 7 8 ). 321, 322 Specifically, DMA was polymerized in water with a water soluble diacid CTA and VA 057 at 70 ° C inside a VT LCTEM liquid cell with a ta rget molecular weight of 6,000 Da. To ensure oxygen free conditions for the radical

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144 Figure 7 8. RAFT p olymerization of DMA i nside the l iquid c ell and c haracterization by m atrix a ssisted l aser d esorption i onizati on i maging m ass s pectrometry (MALDI IMS). (A) Reaction scheme of the polymerization of DMA using a water soluble diacid CTA. (B) Heat map for polymers with 3 kDa obtained by MALDI IMS of the top chip after the polymerization. (C) MALDI mass spectrum of PDM A polymerized in situ using the VT LCTEM holder. The spectrum for the in situ polymerization was acquired in the region boxed in black on the top chip in (B). The inset shows the linear relationship of observed m/z values with DMA repeat units of the polym er. polymerization in the cell, the line s were purged with N 2 and filled with degassed water before the polymerization solution was pipetted onto a bottom LCTEM chip under N 2 flow. After the polymerization, the cell was disassembled, and the cell chips we re air dried prior to MALDI IMS analysis. Mapping of the two chips of the liquid cell by MALDI IMS showed the formation of polymer with a molecular weight around 3,000 Da distributed throughout the heated area of the chip ( Figure 7 8 ). MALDI spectra acqui red in this region revealed a uniform mol ecular weight distribution centered around 3,000 Da, evenly spaced by 99 Da, which corresponds to the molecular weight of one DMA repeat unit . The m/z values observed could be attributed to a vinyl terminated PDMA c hain initiated by a CTA R group but

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145 F igure 7 9. Enlarged MALDI spectrum of PDMA polymerized inside the liquid cell with fragment elucidation showing (a) Na + and (b) K + salt ions of PDMA polymers with CTA R group and vinyl chain ends. If termination via disproportionation was the source for th e unsaturated chains, an equal population of saturated chains should be present in the mass spectrum. However, we find only unsaturation as the predominant type of polymer chain ends. cleaved from the trithiocarbonate group in the middle of the pol ymer chain, which suggests that the molecular weight of the polymer before analysis was close to the target molecular weight of 6,000 Da. While vinyl terminated chain ends could also potentially arise from disproportionation during the radical polymerizati on, our data indicate that the fragmentation occurred in the spectrometer during MALDI IMS analysis via homolytic cleavage of th e polymer CTA carbon sulfur linkage followed by hydrogen abstraction (Figure 7 9) . 323 The great extent of laser induced fragmentation is probably due to the harsh ionizat ion conditions during MALDI IMS, which would also explain the presence of the fragmentation products of lower molecular weight ( < 2500 Da) in the mass spectrum. Critically, the same polymerization conditions without the diacid CTA showed no significant pol ymer formation in the investigated molecular weight range (1.5 10 kDa), indicating the necessity of a CTA for controlled polymer ization (Figure 7 10 ).

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146 Figure 7 10. Control experiments for RAFT polymerizations inside the liquid cell. (A) MALDI IMS analy sis of c ontrol polymerizations for comparison with the in situ (i.e., inside the liquid cell) RAFT polymerization of DMA using the diacid CTA (green). Spectra of the conventional (i.e., ex situ) RAFT polymerization under standard conditions (DMA, diacid CT A, and VA 057) (blue), polymerization without CTA (gray), and solely the DCTB matrix (black). Substantial fragmentation i n the form of a bimodal molecular weight distribution is also present in the MALDI IMS results of the polymer prepared ex situ via conv entional RAFT polymerization (blue), although size exclusion chromatography for the same polymer shows a monomodal molecu lar weight distribution with a M n of 5,900 Da (M n,theo = 6,300 Da) and a low dispersity (shown in B), corroborating with fragmentation occurring during MALDI IMS analysis 7.2.5 In S itu M onitoring of T hermally I nitiated RAFT PISA by LCTEM Following the proc edure for oxygen free conditions described above, we prepared a liquid cell containing an aqueous solution of PDMA macro CTA, DAAm, DM A, and VA 057 in the same ratios as in the conventional RAFT PISA experiments. The liquid cell was sealed, pumped to vacuu m, and inserted into the electron microscope for imaging. For every experiment, initial images were acquired of the liquid cell at low electron dose before heating, to confirm that no structures were present. Then, the temperature was increased by 0.2 ° C/s to 70 ° C. Throughout the experiments,

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147 Figure 7 11. In situ PISA control experiment without initiator. Image of a solution contain ing monomer and macro CTA, but no initiator. (A) Image acquired before and (B) after heating to 70 ° C for 1 h, during whic h no structure formation was observed. Figure 7 12. In situ PISA control experiment without macro CTA. (A) Image acquired of a sol ution containing monomer and initiator, but without macro CTA. Images (B) and (C) acquired of different structures formed in the same liquid cell shown in (A) after heating the solution at 70 ° C for 1 h. Images (D) and (E) acquired of the structures shown in (B) and (C) after cooling the liquid cell back to room temperature. Importantly, the precipitates remain insolub le at room temperature, indicating that irregular aggregates of uncontrolled poly(DAAm co DMA) polymers formed from free radical polymerizati on lack thermoresponsiveness. Instantaneous dose = 0.1 e Å 2 s 1 . Cumulative electron dose = 5 e Å 2 .

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148 imaging was p erformed at regular time intervals with 1 s acquisition times under low electron dose conditions (0.3 e Å 2 s 1 ) with the beam blanked between acquisitions to minimize beam irradiation of the sample. First, we conducted control in situ PISA experiments, wh ere we omitted either the radical initiator or the macro CTA, to show that both components are necessary to achieve RAFT PISA and to verify th at the formation of defined structures inside the cell would only be the result of a controlled PISA process. Inde ed, no polymer assemblies formed after 1 h at 70 ° C if no initiator was present ( Figure 7 1 1 ), and without macro CTA, only irregular poly(DAAm ) co poly(DMA) precipitates lacking the hydrophilic stabilizing block formed from uncontrolled radical polymerizat ion ( Figure 7 1 2 ). With each preliminary milestone met and control reactions completed, RAFT PISA including all the components was conducted inside the liquid cell ( Figure 7 1 3 ). Importantly, no structures were present prior to heating (Figure 7 13 ). Between 30 and 40 min, we observed the local development of an increasingly non uniform background, which we believe can be attributed to the incr eased formation of larger polymer assemblies throughout the volume of the liquid. However, due to the low con trast of PDMA b P(DAAm co DMA) and the stacking of assemblies throughout the cell, we could not infer any conclusive structural information. After 50 min, we observed the formation of spherical nanoparticles with diameters ranging from 120 250 nm. This ass embly onset is earlier than the onset recorded in ex situ experiments, probably due to the higher temperature used in the liquid cell experiments t o counterbalance reduced diffusion. 278, 321 Interestingly, the contrast of those particles continued to increase with reaction time, likely because of the increasing degree of polymerization of the

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149 Figure 7 13. In s itu i maging of t hermally i nitiated RAFT PISA at 11% w/v s olids c oncentration and a ssembly d issociation u pon c ooling . (A) Image acquired of a solution containing macro CTA, acrylamide monomers, and radical initiator in the same ratios as used in the co nventional experiments before heating. (B F) Images acquired upon heating the solution to 70 ° C after periodic time intervals. (G) Image acquired of a new region of the same liquid cell shown in (A F) confirming the presence of nanoparticles throughout the liquid cell. (H) Image acquired after cooling the solution to 30 ° C; the larger particles are likely due to aggregation on the cell window. (I) Image acquired after 20 min without heating at 26 ° C. All images shown at the same magnification. Instantaneous electron dose throughou t imaging = 0.3 e Å 2 s 1 . Cumulative electron dose = 25 e Å 2

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150 hydrophobic block resulting in more densely assembled hydrophobic regions ( Figure 7 13 ). Additionally, the total number of particles also increased over time, while diame ters remained constant. Considering the large diameter of the nanoparticles (> 120 nm), we believe that the structures observed correspond to vesicles or bicontinuous micelles, 275 since even at the unlikely extreme of fully extended polymer chains (i.e., 0.25 nm repeat unit length for vinyl derived polymers), 324 PDMA b P(DAAm co DMA) would only support continuous micelles with a maximum diameter of ~100 nm a t complete monomer conversion (i.e., a total DP of 207). Furthermore, the diameter of the particles correspon ds well to the D h distribution measured by DLS for vesicles at 60 ° C ( Figure 7 5 ). However, continued development of higher contrast imaging method s for soft matter will be required to definitively assign the nanoparticle morphology. 325 After holding the solution at 70 ° C for 90 min, the liquid cell was gradually cooled to room temperature. Within 20 min (i.e., 110 min total), the temperature decreased to 26 ° C, coinciding with a collapse of the particles into dissolved unimers ( Figure 7 1 3 ). Importantly, these findings agree with the DLS results and the cloud point measurement ( Figure 7 5 ), acquired on the materials generated by conventional RAFT PISA. 7.3 Summary Thermoresponsive polymer assemblies bear great potential for the generation of adaptive and functional materials. 326 However, methods to characterize such nano structures in their native solution phase environment ar e limited. In this report, we confirm that higher order polymer assemblies 283 formed via PISA can be imaged in solution by LCTEM and that the observed morphol o gies correlate with, or are of superior structural integrity, to those observed via dry state TEM. Further, we devised a method for conducting thermally initiated RAFT polymerization inside a liquid cell, where

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151 the resulting process can be imaged by LCTEM , illustrating the utility of this approach. Similarly, we probed the thermally responsive behavior of these materials by following their disassembly into unimers. Critically, we investigated the advantages of characterizing polymer assemblies generated a t elevated temperatures in their native solution phase environment by VT LCTEM, as particle dimensions easily change with temperature due to variations in hydration and swelling effects. VT LCTEM will provide insight into the temperature dependent hydropho b ic hydrophilic balance in amphiphilic block copolymers. Such information will influence the design of these materials for noncovalent small molecule encapsulation, target molecule release, or for temperature dependent phase transition behavior. 7.4 Experi m ental Reagents and solvents were purchased from commercial sources and used without further purification, unless noted otherwise 2 (Dodecylthiocarbonothioylthio) 2 methylpropionic acid chain transfer agent (Me 2 C 12 TTC), 223 S , S ' dimethyl acetic acid) trithiocarbonate (diacid CTA), 223 sodium phenyl 2,4,6 trimethylbenzoylphosphinate (SPTP), 327 were synthesized according to previous reports. Diacetone acrylamide (DAAm, TCI Chemicals, > 9 8%) was recrystallized twice from ethyl acetate and once from hexa nes and azobisisobutyronitrile (AIBN, Millipore Sigma, 98%) was recrystallized from ethanol prior to use. N,N Dimethylacrylamide (DMA, Sigma Aldrich, 99%) was filtered through basic alumina prior to use.

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152 7.4.1 Instrumentation The same instruments and techn iques were used as in Chapters 3, 4, 5, and 6 unless noted otherwise. VT LCTEM liquid cells comprised of a top chip (6 x 4.5 mm) and bottom chip (2 x 2 mm), each with a 550 x 50 µm silicon nitride (SiN x ) membrane (Protochips, Morrisville, NC, USA) were fr eshly glow discharged in a PELCO easiGlow glow discharge unit for 30 s. The lines of the VT LCTEM holder (Protochips, Posseidon) were first purged with N 2 and filled with degassed water, the was pipetted onto a bottom LCTEM chip under N 2 flow. Finally, the liquid cell was assembled with the windows aligned in parallel, and the lines of the holder were sealed off without external flow. Imaging was performed on a JEM ARM300F (JEOL, Ltd., Tokyo, Japan) operated at 300 kV. Micrographs were recorded on a 2k × 2k Gatan OneView IS CCD camera (Gatan Inc., Pleasanton, CA, USA) using Gatan Digital Micrograph image acquisition software (Roper Technologies, S arasota, FL). The electron dose values used in LCTEM experiments w ere calculated using the beam current for each aperture selection, as measured by a Faraday Holder through vacuum, and the beam diameter incident upon the sample. Immediately following LCTEM experiments, the SiN x chips were carefully separated and allowed to dry. The cloud point of an 11% w/v solution of PDMA 37 b P(DAAm co DMA) 170 was determined by turbidity measurements with a Molecular Devices Spectra Max M2 spectrophotometer at an irradiat ion wavelength of 500 nm. The polymer solution was transferred fro m the reaction vessel to a cuvette at 70 ° C immediately after synthesis. The temperature of the sample solution in the instrument was then cooled from 40 to 21 ° C in 1 3 ° C increments. After the cooling cycle, the sample was heated from 21 to

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153 40 ° C in 1 3 ° C increments. Turbidity measurements were conducted at each temperature after 7 min equilibration. Each measurement was measured twice, with the values reported being the average. MALDI IM S samples were prepared by adhering the VT LCTEM chips from in sit u polymerizations with their SiN x membranes facing upwards to the conductive face of an ITO (indium tin oxide) coated glass slide with 70 using ~0.5 µL nail polish and allowed to dry. To equalize the height difference from added SiN x chips on the slid e (~0.25 mm), 5 pieces of Scotch tape were applied to both short edges of the slide on the same side. Samples from conventional RAFT polymerizations were deposited as 3 × 2 µL spots onto ITO slide surfaces and allowed to dr y. To each slide was sprayed an e ven thin layer coating of DCTB matrix (20 mg/mL in THF). Briefly, matrix (5 mL) was loaded into the injection loop of an HTX TM Sprayer using a running buffer of THF operated with HTX Imaging software. Matrix sample was int roduced at 0.05 mL/min, through a spray nozzle heated to 55 ° C and sprayed under a constant N 2 gas pressure of 10 Psi at 2 L/min. Patterning was performed at a nozzle velocity of 1200 mm/min, track spacing 3 mm, nozzle height 40 mm, and criss cross pattern over 8 total passes (2 s drying time per pass). MALDI IMS analysis was conducted by mounting ITO slides with samples into an MTP Slide Adapter II and loaded into a Bruker RapifleX MALDI TOF/TOF mass spectrometer for analysis using flexControl software (Bruker Daltonics 8237001). Samples were analyzed by MALDI MS under linear positive mode (2000 10 000 Da) using a 355 nm smartbeam 2 laser with a 50 µm focus diameter, a constant laser power of 50%, and a sum of 125 shots per spectrum. Region of interest mapping and image

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154 analysis was perform ed in flexImaging software (Bruker Daltonics). Raw mass spect ra within a given region of interest were averaged and the baseline subtracted. For each 50 µm diameter pixel, an integrated total signal was generated from integrated mass spectra at each pixel within a defined mass range filter of 3000 (± 500) Da. Visual 2D maps were generated from these pixels and colorized according to 0 100% maximal signal on a logarithmic scale. 7.4.2 Procedures 7.4.2.1 Synthesis of PDMA macro CTA Me 2 C 12 TTC (0.334 g, 0.916 m mol), AIBN (0.0075 g, 0.047 mmol), DMA (4.086 g, 41.22 mmol), and DMF (20.6 mL) were added to a Schlenk flask and purged with Ar for 15 min. Subsequently, the flask was immersed in a preheated oil bath at 60 ° C. After 15 h, at a monomer conversion of 76%, the polymerization was quenched by exposing the mixture to ai r and precipitating the polymer into cold diethyl ether. The polymer was freeze dried after dialyzing against DI water for 24 h using a dialysis membrane with a molecular weight cut off (MWCO) of 1 kDa. M n,theo = 3755 g/mol M n,SEC MALS = 3980 g/mol (calcul ated with a dn/dc of 0.0810 mL/g in DMAc at 50 ° C) Ð = 1.01 7.4.2.2 Polymerization kinetics of DAAm/DMA PISA with a target hydrophobic block DP of 170 PDMA macro CTA (0.025 g, 0.0063 mmol), VA 05 7 (0.0005 g, 0.001 mmol) from an aqueous stock solution, DAAm (0.1355 g, 0.8007 mmol), DMA (0.0265 g, 0.267 mmol), p TSA (0.0007 g, 0.004 mmol; corresponds to a 3:1 molar ratio with respect to VA 057 to ensure an acidic pH of the reaction solution), DMF (2 drops, internal

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155 standard), and DI water (1.51 mL, correspond ing to 11% w/v total polymer content at 100% conversion) were added to a Schlenk flask and purged with Ar for 15 min on ice. Subsequently, the flask was immersed in a preheated oil bath at 60 ° C, stirred at 70 rpm, and aliquots were withdrawn from the reac tion in regular time intervals for SEC and 1 H NMR analysis. Monomer conversions were determined by 1 H NMR spectroscopy by comparing the DMF formamide proton (7.92 ppm) with the vinyl signals of D AAm (5.51 ppm) and DMA (5.65 ppm) in DMSO d 6 . 7.4.2.3 Example procedure for the synthesis of crosslinked polymer assemblies via DAAm/DMA PISA PDMA macro CTA (0.0400 g, 0.0101 mmol), VA 057 (0.0008 g, 0.002 mmol) from an aqueous stock solution, DAAm (0.2170 g, 1.282 mmol), DMA (0.0423 g, 0.427 mmol), p TSA (0.0011 g, 0.0058 mmol; corresponds to a 3:1 molar ratio with respect to VA 057 to ensure an acidic pH of the reaction solution), DMF (3 drops, internal standard), and DI water (2.42 mL, corresponding to 1 1% w/v total polymer content at 100% conversion) were combine d. The solution was split into 6 vials (0.40 mL each), and each vial was purged with Ar for 7 min on ice. The vials were immersed in a preheated oil bath at 60 ° C and stirred at 70 rpm. In regula r time intervals, a vial was opened to air at 60 ° C, and a sm all sample was withdrawn for SEC (Figure S4) and 1 H NMR analysis (Table S1). Still at 60 ° C, O alkyl hydroxylamine crosslinker (10 mol% with respect to DAAm) was added from an aqueous phosphate b uffer stock solution (pH = 7.0), and the reaction mixture was stirred for another 10 min. Subsequently, the crosslinked polymer assemblies were cooled to room temperature and subjected to DLS and TEM analysis.

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156 7.4.2.4 Synthesis of crosslinked worms and int ermediate structures for LCTEM analysis via photoinitiated RAFT PISA PDMA macro CTA (0.0080 g, 0.0020 mmol), SPTP (0.06 mg via stock solution, phosphate buffer (0.76 mL, 50 mM, pH = 7.4, corresponding to 10% w/v total pol ymer content at 100% conversion) were combined. The targeted total hydrophobic block DP was 250 at 75 mol% DAAm. The solution was purged with Ar for 7 min on ice before irradiating with UV light ( max = 365 nm) f or 8 h with constant stirring at 70 rpm. The vial was opened to air, and a small sample was withdrawn for SEC and 1 H NMR analysis (confirming 100% monomer conversion). O Alkyl hydroxylamine crosslinker (10 mol% with respect to DAAm) was added from an aqueo us phosphate buffer stock solution (pH = 7.0 ), and the reaction mixture was stirred for another 10 min. 7.4.2.5 Conventional RAFT polymerization of PDMA for MALDI IMS analysis control Diacid CTA (0.0100 g, 0.0354 mmol), VA 057 (0.0003 g, 0.007 mmol), DMA (0.2106 g, 2.125 mmol), and DI water (0.62 mL) were combined. The solution was purged with Ar for 7 min on ice before immersing in a preheated oil bath at 70 ° C. After 4 h, full conversion was confirmed via 1 H NMR spectroscopy, and the polymer was analyzed by SEC and MALDI IM S . 7.4.2.6 In sit u (i.e., inside the liquid cell) polymerization of PDMA for MALDI IMS analysis The lines of the liquid cell holder were purged with N 2 gas and filled with acid CTA (0.0100 g, 0.0354 mmol), VA 057 (0.0003 g, 0.007 mmol), DMA (0.2106 g, 2.125 mmol), and DI

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157 water (0.62 mL) were pipetted onto the bottom chip of the LCTEM holder under N 2 flow. The liquid cell was assembled and heated at 70 ° C for 12 h. 7.4.2.7 In situ (i.e., inside the liquid cell) thermal RAFT PISA with DAAm/DMA The lines of the liquid cell holder were purged with N 2 gas and filled with degassed DI water. Then, CTA (0.020 g, 0.0050 mmol), VA 057 (0.0004 g, 0.001 mmol), DAAm (0.1084 g, 0.6406 mmol) , DMA (0.0212 g, 0.214 mmol), p TSA (0.0006 g, 0.003 mmol; corresponds to a 3:1 molar ratio with respect to VA 057 to ensure an acidic pH of the reaction solution), and DI water (1.20 mL, corresponding to 11% w/v total solid content at 100% conversion) wer e pipetted onto the bottom chip of the LCTEM holder under N 2 flow. The liquid cell was sealed, pumped to vacuum, and inserted into the transmission electron microscope.

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158 CHAPTER 8 INVERTING MONOMER SELECTIVITY WITH POLYMERIZATION INDUCED SELF ASSEMBLY * 8.1 Overview In organic synthesis, hydrophobic interactions are used to increase reaction rate and improve product selectivity. 34, 40 In a seminal study, Breslow demonstrated that aqueous Diels Alder (DA) cycloadditions proceeded with higher reaction rates 328 and increased preference for the more com p act endo DA adduct when carried out in water compared to bulk conditions or other protic solvents (Figure 8 1). 329 For example, the cycloaddition of cyclop e ntadiene and butanone was 58 fold faster and provided a 2.5 fold increase of the endo/exo product ratio when the reaction was conducted in water as compared to methanol or ethanol. 328, 329 Such waterborne reaction kinetics are often the result of hydroge n bonding and hydrophobic effect combined. However, m any other substrates and transformations with minimal hydrogen bonding susceptibility show advantageous reaction behavior in water, manifesting hydrophobicity as a means to control chemical reactions. 34, 330 In polymer synthesis, the hydrophobic effect is arg uably most relevant for emulsion polymerization techniques. 331, 332 A typical emulsion polymerization in water invol ves the free radical polymerization of hydr ophobic monomer, surfactant, and a water soluble initiator. Driven by the entropically favored reduction of hydrophobic surface area (Chapter 1.2.1), the aggregation of hydrophobic monomers and polymers promotes p article nucleation and monomer imbibition. After nucleation of a reaction * Contributions: Swagata Mondal and Dr. Julia Rh o performed parts of the experimental work; John B. Garrison acquired TEM images.

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159 locus, propagation proceeds compartmentalized in nanoparticles, resulting in polymerization behavior differing from conventional free radical polymerization. Specifically, emulsion p olymerization affords higher molecular weig ht polymers at faster polymerization rates due to the segregation of growing polymer chains in a heterogeneous nanoparticle environment. 331 However, control over molecular weight and monomer sequence is limit ed in such techniques. Figure 8 1. Strat egic employment of the hydrophobic effect can improve reaction efficiency and affect product selectivity. (A) The reaction rate and the endo/exo ratio in the Diels Alder reaction between cyclopentadiene ( 8 1 ) and methyl vinyl ketone ( 8 2 ) increased when conducted in water. (B) Our approach leverages the hydrophobic effect in polymerization induced self assembly (PISA) to synthesize gradient block copolymers with inverted monomer selectivity.

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160 Here, we expand the sc ope of hydrophobicity induc ed reaction selectivity to controlled radical copolymerization using polymerization induced self assembly (PISA; Chapter 7). We show how the hydrophobic effect and the in situ formation of a hydrophobic reaction environment upon assembly promote differenti ation between two monomers with similar reactivities but disparate polarities. Specifically, RAFT PISA 308 with the hydrophilic poly(ethylene glycol) trithiocarbonate (PEG TTC) macro chain transfer agent (macro CTA) and the monomers DAAm and DMA were tested in this study (Figure 8 1). 295, 300 Using PISA , inverted monomer selectivity was achieved, i.e., the faster incorporation of DAAm over DMA. This change if polymerizat ion rates after assembly afforded the autonomous generation of DAAm DMA gradient sequenc es , otherwise inaccessible without outside intervention (e.g., continuous addition of one comonomer during a copolymerization) . While Breslow leveraged the hydrophobic effect in molecular DA reactions , our strategy expands th is approach to increased react ion rates and selectivity in polymer synthesis, specifically controlled radical polymerizations. This chapter will first describe our approach of using PISA to synthesi ze copolymers with unprecedented monomer sequences and accelerated reaction rates. Then, the utility of this polymerization method is tested by generating blocked gradient structures and subjecting the copolymers to down stream reactions and chain extensions. Finally, we use polymerization kinetics to unravel previously poorly understood poly merization phenomena in RAFT PISA.

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161 8.2 Results and Discussion 8.2.1 Fundamentals of Radical Polymerization Our approach capitalizes on manipulating polymerization kinetics through hydrophobicity driven assembly. Therefore, it is necessary first to identif y and analyze the fundamental rate equations that describe radical polymerizations and RAFT PISA. In radic al polymerization, the rate of polymerization ( R p ) is first order in radical chain concentration ([ P M ]) with the prop agation rate constant ( k p ) as the proportionality factor (equation 8 1). In the steady state equilibrium of radical chain termination and initiation, [ P with k p into an apparent propagation rate constant ( k p ,app ). The validity of the steady state assumption is revealed by the linearity of the pseudo first order rate plot, which also allows the determination of k p,app from the slope of the curve (equation 8 2). It is important to note that k p,app contains the initiator efficiency ( f ), the initiator dissociation rate constant ( k d ), the rate of termination ( k t ), and the initiator concentration ([ I ] ), all of which constitute [ P ( equation 8 3). These variables change throughout the polymerization; however, in the steady state, individual fluc tuations are counterbalanced, and k p,app remains constant overall. (8 1) (8 2) (8 3)

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162 8.2.2 In Situ Monomer Differentiation In aqueous radical polymerizations, DAAm and DMA exhibit near identical reactivity . 295 For methanol, Cai and coworkers reported faster consumption of DMA under RAFT conditions. 333 We tested copolymerizations of DAAm/DMA in DMF as a polar aprotic solvent, toluene (PhMe) as a non polar solven t, and a 50:50 DMF/H 2 O mixture (Figure 8 2). Analysis of the polymerization kinetics in the different solvents reve aled the preferential incorporation of DMA in all solvents, indicated by the higher k p,app for DMA. Interestingly, the reactivity di fference decreased when water was present . Intrigued by such solvent effect s, we wanted to test the copolymerization in Figure 8 2. Polymerization of DAAm / DMA in non selective solvent conditions using azobisisobutyronitrile (AIBN) as a radical initiator and 2 ( butylthiocarbonothioylthio)propionic acid (MeC4 TTC). The target DP at full mon omer conversion was 300 at 65 mol% DAAm in the monomer feed. In all solvents, no assembly occurred; the polymer chains remained molecularly dissolved. The pseudo first order rat e plots of monomer consumption in (B) DMF, (C) toluene (PhMe), (D) DMF/H 2 O (1:1 v/v) revealed a faster consumption of DMA compared to DAAm . The ratios of the apparent rate constants ( k p,app ) are given as F DAAm/DMA .

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163 the presence of two reaction media with disparate polarities in the same pot: a hydrophobic local reaction environment in an aqueous continuous phase. A straightforward way to achieve such conditions is aqueous PISA with DAAm / DMA as Figure 8 3. Synthesis of gradient block copolymers via in situ monomer differentiatio n. (A) Copolymer synthesis scheme depicting RAFT PISA of DAAm and DMA with poly(ethylene glycol) with a trithiocarbonate RAFT agent end group (PEG TTC) at three different monomer feed ratios. Pseudo first order rate plots and re p resentative SEC traces of c opolymerizations at a DAAm:DMA molar ratio of (B) 50:50 (DAAm50), (C) 65:35 (DAAm65), and (D) 80:20 (DAAm80). The target hydrophobic block DP at full monomer conversion was 400. The p olymerizations were conducted in triplicate, a nd the data is presented as an average with the error range representing the standard deviation. The ratios of the apparent rate constants ( k p,app ) are given as F DAAm/DMA .

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164 the monomer system (Chapter 7.2.1). 295 DAAm is a water soluble monomer that forms a hydropho bic polymer. When copolymerized with hydrophilic DMA, the hydrophobicity of polymeric DAAm is still sufficient to promote assembly. We copolymerized DAAm and DMA in molar ratios of 50:50 (DAAm50), 65:35 (DAAm65), and 80:20 (DAAm80) in water at 65 ° C using PEG terminated with a trithiocarbonate RAFT agent group ( PEG TTC ) as a hydrophilic macro CTA and VA 057 as an azo initiator (Figure 8 3 ). Across all monomer compositions, at a critical hydrophobic b lock DP (roughly at DP 80, which corresponds to 20% overa ll monomer conversion) PEG b P(DAAm co DMA) assembled in situ with hydrophobic P(DAAm co DMA) cores and a hydrophilic PEG corona. This assembly onset could be determined visually by an abrupt loss o f solution transparency , which continued to decrease with increasing monomer conversion (Figure 8 4). Figure 8 4. Photographs ( left ) before assembly with the reactants molecularly dissolved resulting in a trans parent solution, (middle) at assembly of th e generated amphiphilic block copolymer, and ( right ) after assembly. Throughout all polymerizations, reaction aliquots were withdrawn and analyzed by 1 H NMR spectroscopy and SEC to determine monomer conversion and polymer molecular weight, respectively ( F igure 8 3). As is typical for PISA, 264 we observed a distinct polymerization rate increase after assembly, evident from the increase of k p,app in

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165 th e pseudo first order rate plot. D uring the early stages of the polymeri zation, when the growing polymer chains were molecularly dissolved, DAAm and DMA showed almost identical apparent propagation rate constants ( k p,app ) around 0.003 min 1 , resulting in a s hort statistical block of PDAAm stat PDMA. As the polymer chains assem bled, t he hydrophobic TTC bearing termini of the polymer chains became confined in the cores of the amphiphilic polymer aggregates, causing a dramatic change of the chemical environme n t for the propagating radicals. I n this new polymerization environment , we found that DAAm preferentially incorporated into the polymer chain as indicated by the higher k p,ap p compared to DM A. This rate difference between DAAm and DMA resulted in statisti c al to gradient block copolymers of the general structure P EG b P(DAAm stat DMA) b P(DAAm grad DMA) . Such polymerization behavior is in stark contrast to the copolymerization of DAAm and DMA in non selective solvents (Figure 8 2), which promote faster poly m erization of D MA. Importantly, our results suggest that k p,app , and thus , the gradient sequence are readily tuned by the composition of the polymer aggregates (Figure 8 5 ). Before assembly, k p,app of both monomers remained similar . However, after assembl y , increased DA Am feed and thus presumably the increased hydrophobicity in the assemblies caused a more pronounced differentiation between DAAm and DMA. The k p,app ratio of DAAm and DMA increased from 1.4 to 1.7 upon increasing the DAAm feed to 80 mol% , resulting in a more distinct gradient structure (Figures 8 3 and 8 5).

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166 Figure 8 5. Plot of k p,app before and after assembly. Before assembly DAAm and DMA exhibit similar k p,app values. Polymer assembly promotes differentiation between DAAm and DMA wit h higher k p,app for DAAm. This peculiar polymerization behavior is likely due to the preferential association of DAAm with the hydrophobic cores of the assemblies . 1 H NMR analysis of PISA reaction aliquots with 65 mol% DMA showed broadened peaks and redu c ed integration values for DAAm in the presence of PEG b P(DAAm stat DMA) b P(DAAm grad DMA) assemblies, indicating substantial changes in the chemical environment for DAAm (Figure 8 6). Pinkhassik and coworkers reported similar 1 H NMR results for the poly m erization of methacrylates confined in the bilayer of vesicles. 334 Considering these results, we believe the peak broadening in our experiments can be interpreted as evidence for the increased association of DAAm with the polymer assemblies. We propose that this is due to the h i gher hydrophobicity of DAAm, as suggested by the higher octanol water partition coefficient (cLogP) for DAAm (cLogP = 0.3) compared to DMA (cLopP = 0.2).

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167 Figure 8 6. 1 H NMR spe ctroscopy of DAAm and DMA in the presence of amphiphilic block copolymers i n selective (D 2 O) and non selective (acetone d 6 ) solvent. For better readability PEG b P(DAAm stat DMA) b P(DAAm grad DMA) is abbreviated with PEG b P(DAAm co DMA) in the graphic. The integration values for a set of vinyl protons are indicated above the c o rresponding peaks. The stacked spectra of (black) PEG TTC with DAAM and DMA before polymerization in D 2 O and (blue) after polymerization for 70 min (74% monom er conversion) in acetone d 6 show sharp peaks for both monomers. The same polymerization aliquot a t 74% conversion in D 2 O showed substantially broadened peaks and lower integration values for DAAm. The polymerization kinetics w ere found to be independent of assembly morphology. For example, DAAm65 would progress from micelles to vesicles; however, on l y the assembly onset had a noticeable impact on the polymerization rate . Furthermore, we did not find a dependence of k p,app on particle size (Table 8 1). Und er standard conditions, the typical particle hydrodynamic diameter ( D h ) was around 89 nm. Using l o wer amounts of PEG TTC , while maintaining the same monomer and initiator concentration , resulted in a particle size increase. However, k p,app after assembly d id not change beyond experimental error. The higher k p,app before assembly at lower PEG TTC conce n tration is most likely due to reduced chain transfer.

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168 Table 8 1. Polymerization kinetics for the DAAm80 system at different particle sizes. D h (nm) a k p,app ( before) (min 1 ) k p,app (DAAm) (min 1 ) k p,app (DMA) (min 1 ) 89 ± 2 0.0026 ±0.0002 0.043 ±0.005 0.0 2 5 ±0.003 144 ± 4 0.005 ±0.002 0.049 ±0.008 0.028 ±0.005 a Average hydrodynamic diameter ( D h ) determined by DLS. Polymerizations were conducted in triplicate . T he data is presented as an average with the error range representing the standard deviation. F i nally, we tested DAAm/DMA PISA under alternative polymerization conditions to probe the universality of hydrophobicity induced monomer differenti ation. Specifically, we switched from thermal initiation to room temperature eosin Y catalyzed PET RAFT condit i ons (Figure 8 7). Corroborating the thermal copolymerization results, the pseudo first order rate plot of the PET RAFT copolymerization showed di sparate monomer consumption after assembly, with DAAm being preferentially incorporated into the polymer chain . These results further substantiate our hypothesis that the monomer differentiation between DAAm and DMA in PISA is primarily dictated by polymer assembly and the development of a hydrophobic reaction environment. Figure 8 7. In situ monomer different i ation in PET RAFT PISA. (A) Reaction scheme for the PET RAFT copolymerization of DAAm and DMA using a PDMA macro CTA (PDMA TTC), the photocatalys t eosin Y, and 4 dimethylaminopyridine (DMAP). The molar content of DAAm was 75 mol% at a target DP of 300. Th e repeat unit numbers represent the relative molar ratios in the respective block. (B) The pseudo first order kinetic plot of the polymerization s hows in situ monomer differentiation between DAAm and DMA after assembly.

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169 8.2.3 End Group Chemistry and New G r adient Block Copolymers Having established the fundamental driving force behind hydrophobicity guided copolymer synthesis, we sought to exploit the well defined end group chemistry of the gradient block copolymers generated by this method . For example, s e lective end group modifications could allow the construction of various blocking sequences with disparate functionality. First, we tested the chain extension of a purified DAAm65 gradient triblock copolymer. Green light mediated PET RAFT polymerization of PEG b P(DAAm stat DMA) b P(DAAm grad DMA) with DMA afforded a tetrablock copolymer constituting a blocking order of homo to statistical t o gra dient to homo (Figure 8 8) . The uniform shift to lower elution time by SEC analysis indicated the successful cons t ruction of the terminal PDMA block. Importantly, this experiment proved good retention of the Figure 8 8. Chain extension of a DAAm65 gradient block copolymer syn thesized via in situ monomer differentiation. (A) Synthe sis scheme depicting the PET RAFT p olymerization conditions using DMA, eosin Y, and DMAP in DMSO under green light with a target DMA DP of 500 at full monomer conversion. (B) Overlaid SEC traces (black) before and (green) after chain extension. The dashed line shows the normalized SEC UV d e tector trace at an emission wavelength of 310 nm, which is a characteristic absorption of trithiocarbonate (TTC) groups. The overlap of UV and RI trace suggest ed good TTC group retention during RAFT PISA.

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170 trithiocarbonate RAFT agent moiety at the chai n end th roughout PISA, paving the way for potential chain extensions with various monomers and RAFT polymerization methods. Then, using DAA m65 PEG b P(DAAm stat DMA) b P(DAAm grad DMA) , we developed a mild hydrolysis protocol to remove the hydrophilic PE G chain end to h arvest the pure DAAm/DMA gradient diblock copolymer. The best results were achieved by dispersing the amphiphilic block co polymers in 0.05 M aqueous NaOH solution . Upon hydrolysis of the PEG ester linkage , hydrophobic P(DAAm stat DMA) b P(D A A m grad DMA) copolymer s precipitated and could be collected by centrifugation, while the hydrophilic PEG remained in the supernatant. Finally , considering that block copolymer assembly inverted the monomer preference during copolymerization of DAAm and D M A, we hypothesized that the simple change from selective to non selective solvent during the copolymerization should afford a new polymerization profile and thus, a new block structure. Specifically, PET TTC was polymerized under DAAm65 RAFT PISA conditio n s (Figu re 8 9, regimes A and B ). The addition of DMAc, a good solvent for the PEG and the DAAm/DMA block, at 78% overall monomer conversion resul ted in the immediate dissolution of the polymer assemblies. At this point in the synthesis, the gradient block copolym er structure was PEG b P(DAAm stat DMA) b P(DAAm grad DMA ), analogous to the examples described in Figure 8 3. However, concomitant with t he transition from assembled aqueous to homogenous organic polymerization medium (transition regime B to C in F igure 8 9), the polymerization behavior substantially changed as indicated by the altered k p,app values for DAAm and DMA from the pseudo first or der kinetic plot. In

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171 Figure 8 9. Synthesis of a gradient tetrablock copolymer featuri ng two distinct gradie n t blocks in a single pot. (Left) Synthesis scheme of the tetrablock copolymer with a target hyd rophobic block DP of 600 and (right) pseudo first order rate analysis of the polymerization. The repeat unit numbers represent the relativ e molar ratios in the r espective block. fact, the monomer differentiation reverted from preferential DAAm incorporation to DM A, akin to the polymerization behavior of DAAm/DMA in organic solvents (Figure 8 2). The final block copolymer product featured a homo to statistical to gradient to gradient blocking sequence, with two distinct gradient structures. The first gradient block progressed from DAAm to DMA rich, whereas the second block transitioned from DMA to DAAm rich (Figure 8 9). 8.2.4 Insights into RAFT PISA Kinetics T h e polymerization rate increase after particle nucleation during RAFT PISA has often been ascribed to th e increased monomer diffusion into the hydrophobic assembly cores, resulting in a higher local monomer concentration. 267, 285 Indeed, a higher monomer concentration at the reaction locus directly effects an increased R p (equation

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172 8 1). Furthermore, k p,app although inherently monomer concentration independent (e quation 8 3) increases due to the in situ increa se M 0 at assembly, which is not accounted for in the pseudo first order rate analysis (equation 8 2). Here, we attributed the preferential polymerization of DAAm over DMA post assembly to a hydrophob icity driven increased association of DAAm with th e a ssembl ies (Figure 8 6). However, we were surprised that DMA, as a hydrophilic monomer, also exhibited substantially faster polymerization kinetics after assembly (Figure 8 5). For example, k p,app of DMA increased twofold after assembly in DAAm50, fivefo ld in DAAm65, and tenfold in DAAm80 (Figure 8 10). It seems unlikely that a predominantly hydr ophilic monomer with a negative cLogP would show such a strong driving force to diffuse into increasingly hydrophobic assembly cores . Furthermore, 1 H NMR spectros co p y showed no evidence of DMA association with the polymer assemblies (Figure 8 6). Figur e 8 10. Plot of k p,app after assembly normalized by k p,app before assembly at varying DAAm content in the monomer feed. For clarity, k p,app is depicted as k p in t he graph legend. To investigate the contribution of monomer diffusion to the increase of R p a fter assembly, we tested the polymerization kinetics in a chain extension of PEG TTC with solely DAAm (DAAm100) under RAFT PISA conditions (Figure 8 11). Kinetic an a lysis

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173 revealed a rise of monomer conversion and a 14 fold k p,app increase from 0.0023 to 0. 033 min 1 after assembly. If this k p,app increase is solely due to a higher local monomer concentration, and thus, an instantaneous change of [ M 0 ] in equation 8 2 upon assembly , then k p,app k p,app of the polymerization system. In this case, it should be possible to calculate the increased monomer concentration around the reaction centers after assembly by div id i ng R p obtained from a short k p,app of the system (equation 8 4). k p,app value of 0.0023 min 1 determined from the pseudo first order rate plot before assemb ly , we calculated a monomer concentration of 0.96 M from the R p value of 0.0022 M/min in the pre assembly regime of DAAm100 (Figure 8 11). The starting DAAm concentration of this polymerization was set at 1 M, matching the calculated value. After assembly, w e used the first two concentration data points to estimate an R p of 0.0015 M / min. Using this R p k p,app value, the new local monomer concentration around the reaction centers (i.e., the hydroph obic cores) was estimated to be 6.5 M, which corr e sponds to roughly 1.1 g / mL DAAm. In other words, if k p,app r emained unaffected after assembly, it would take a DAAm concentration of 1.1 g / mL in the assembly cores to sustain the i ncrease of R p . Although no density values have been reported for DAAm, 1.1 g / mL is very likely approaching the molecular density of DAAm . Therefore, such high DAAm concentrations in the polymer particles seem physically impossible. (8 4)

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174 Figure 8 11. Analysis of the polymerization kinetics during PISA. (A) Reaction scheme for RAFT PISA with DAAm and PEG TTC at 65 °C a nd a target DP of 400 at full monomer conversion. (B) Monomer c onversion plot. (C) Pseudo first order rate analysis of the polymerization. (D) Progression of DAAm concentration throughout the polymerization. (E) Pseudo first order rate plot corrected monomer concentration at assembly ([ M 0 ]) of 6.5 M. I t is important to note that R p values obtained from (D) by this method are most accurate if the monomer concentration changes only little over the experime n tal time. In our case, the monomer co ncentration decreased by 22% between the two data points after assembly. Therefore, it is very likely that the actual R p is slightly higher than the estimated value. We want to emphasize that these results do not co nt r adict DAAm accumulation in the polymer particles. 1 H NMR spectroscopy indicated increased interactions

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175 bet ween DAAm and polymer assemblies (Figure 8 6). However, based on our analysis, it is unlikely that increased monomer diffusion alone can account f or the total rate increase after assembly. Furthermore, the construction of a pseudo first order rate plot wit h a corrected [ M 0 ] after assembly (i.e., using a DAAm particle core concentration of 6.5 M at assembly) still revealed a substantially increased k p ,app (Figure 8 11). Recently, Zhang 335 and Pa n 336 have alluded to the role of compartmentalization during PISA in alcoholic media or under photoinitiation conditions, respectively. We believe that our results corrobora te such a compartmentalization hypothesis . Specifically , w e propose that a combination of increased monomer diffusion into the core and a decreased radical termination due to compartmentalization 337, 338 and lower ch a in mobility in the assemblies are the major contributors to the polymerization rate increase in PISA. Compartmentalization effects are characteristic of heterogeneous polymerization systems and have shown precedence in emulsion 338 and dispersion polymerization. 339 Such phenomena would also explain the k p,app increase of DMA with increasing DAAm content in the monomer feed (Figure 8 10). Higher DAAm monomer ratios in the copolymers likely result in more hydroph obic, more dehydrated as s embly cores, leading to diminished chain mobility and lower k t and thus, higher k p,app (equation 8 3). 8.3 Summary We investigated a copolymerization technique that capitalized on the hydrophobic effect to form a selective reaction medium in situ f or th e c ontrolled construction of unprecedented copolymer structures . RAFT PISA with DAAm and DMA resulted in well defined use of hydrophobic interactions for reaction efficiency and product select iv i t y , we

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176 employed hydrophobicity for increased polymerization rates and inverted monomer selectivity in controlled ra dical polymerization. We showed efficient chain extensions and selective Furthermore, judicious solvent selection allowed the construction o f gradient block copolymers with two gradient inversions in a single pot. We believe such consecutive gradient block inversions bear great potential for constructing complex block copolymer interfaces with tailored Flory Huggins interaction parameters, c ri t ical for block copolymer applications. Mechanistic investigations suggested that the differentiation between DAAm and DMA during the copolymerization is based on the preferential associati on of DAAm with the polymer assemblies. Accordingly, our studies in d icate that the polymerization rate increase, characteristic for PISA , is driven by a combination of increased local monomer concentration and compartmentalization effects. Such an improved understanding of the fundamental polymerization behavior in hete ro g eneous systems will guide the future design of material synthesis with increased efficiency and tunable selectivity. 8.4 Experimental Reagents and solvents were purchased from commercial s ources and used without further purification unless noted otherwi se . 2 ( E thyl sulfanylthiocarbonyl sulfanyl) propionic acid (MeC 2 TTC) and 2 (butylthiocarbonothioylthio)propionic acid (MeC 4 TTC) were synthesized according to a previous report. 223 2,2' Azobis[ N (2 carboxyethyl) 2 methylpropionamidine]tetrahydrate (VA 057) was purchased from Wako Chemicals. D AAm ( TCI Chemicals, > 98%) was recrystallized twice from ethyl acetate and once from hexanes . AIBN ( Millipore Sigma, 9 8 %) was recrystallized from ethanol prior to use. DMA ( Sigma Aldrich, 99%) was filtered through basic alumina prior to use. Dry DCM was obtained by passing the

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177 solvent through two sequentia l activated alumina columns in a MBRAUN solvent purification syst em . 8.4.1 Instrumentation The same instruments and techniques were used as described in Chapter 7. 8.4.2 Procedures 8.4.2.1 Synthesis of PEG TTC MeC 2 T TC (0.63 g, 3.0 mmol), PEG (MW = 5 kDa, 3.0 g, 0.60 mmol), N ethyl carbodiimide hydrochloride (0.69 g, 3 . 6 mmol), and DMAP (0.015 g, 0.12 mmol) were combined in a 50 mL round bottom flask under Ar atmosphere. After dry DCM (15 mL) was added on ice, the re action mixture was stirred overnight. The mixture was diluted with DCM (50 mL) and washed with aqueous HC l (1 M), water, and brine. The organic extract was dried with MgSO 4 and precipitated three times into Et 2 O. After drying under vacuum, PEG TTC was obta ined as a yellow solid (2.8 g, 0.56 mmol). 8.4.2.2 Example RAFT PISA procedure for DAAm65 with a target h y drophobic block DP of 400 PEG TTC (0.0 30 g, 0.006 0 mmol), VA 057 (0.000 4 g, 0.001 mmol) from an aqueous stock solution, DAAm (0. 264 g, 1.56 mmol), DMA (0.0 83 g, 0. 84 mmol), p TSA (0.0 60 g ; internal standard) from an aqueous stock solution at pH 3 were d is s olved with DI water to yield a total volume of 2.4 mL. The pH was adjusted to 3 using aqueous HCl (1 M). The solution was transferred to a 10 mL Schle nk flask and sparged with Ar for 15 min on ice. Subsequently, the flask was immersed in a preheated oil b a th at 6 5 ° C, stirred at 28 0 rpm, and aliquots were withdrawn from the reaction in regular time intervals for SEC and 1 H NMR analysis. Monomer conversi ons were determined by 1 H NMR spectroscopy by comparing the p TSA phenyl proton s with the vinyl signals o f

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178 DAAm (5.51 ppm) and DMA (5.65 ppm) in DMSO d 6 or acetone d 6 . For TEM and DLS analysis, a fraction of the reaction solution was crosslinked with O alk yl hydroxylamine crosslinker ( 7 mol% with respect to DAAm) at 65 °C. After the desired monomer conversi on (> 90%) was achieved, the reaction mixture was dialyzed against water for 24 h using a dialysis membrane with a 5 kDa MWCO, and DAAm65 PEG b P(DAAm stat DMA) b P(DAAm grad DMA) as the product was retrieved via lyophilization. 8.4.2.3 Chain extension of D A Am65 PEG b P(DAAm stat DMA) b P(DAAm grad DMA) with DMA DAAm65 PEG b P(DAAm stat DMA) b P(DAAm grad DMA) (0.20 g, 0.0038 mmol assuming M n,th eo of 53300 g/mol), DMA (0.19 g, 1.9 mmol), eosin Y disodium salt DMSO stock solution, and DMF (0.1 mL; internal standard) were dissolved in DMSO to yield a total volume of 1 mL . The reaction mixture was transferred to a 10 mL Schlenk flask, sparged with Ar for 10 min, and irradiated wi th green light for 10 h. The final DMA conversion by 1 H NMR spectroscopy was 95%. The reaction mixture was dialyzed against water for 48 h usin g a dialysis membrane with a 10 kDa MWCO, and PEG b P(DAAm stat DMA) b P(DAAm grad DMA) b PDMA as the product was retrieved via lyophilization. 8.4.2.4 PEG ester hydrolysis of DAAm65 PEG b P(DAAm stat DMA) b P(DAAm grad DMA) with DMA DAAm65 PEG b P(DAAm s tat DMA) b P(DAAm grad DMA) (0.50 g) was dispersed in aqueous NaOH (5 mL, 0.05 M) and left on a plate shaker over night. Upon hydrolysis of the PEG ester linkage pure P(DAAm stat DMA) b P(DAAm grad DMA) precipitated from the solution and was retrieved via c ent rifugation.

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179 8.4.2.5 Single pot procedure for the generation of a tetrablock gradient copolymer with a target h ydrophobic DP of 600 PEG TTC (0.0 30 g, 0.006 0 mmol), VA 057 (0.000 4 g, 0.001 mmol) from an aqueous stock solution, DAAm (0. 396 g, 2.34 mmol), DMA (0. 125 g, 1.26 mmol), p TSA (0.0 60 g ; internal standard) from an aqueous stock solution at pH 3 were dissolved with DI water to yield a total volume of 2.5 mL. The pH was adjust ed to 3 using aqueous HCl (1 M). The solution was transferred to a 10 mL Schlen k flask and sparged with Ar for 15 min on ice. Subsequently, the flask was immersed in a preheated oil bath at 6 5 ° C, stirred at 28 0 rpm, and aliquots were withdrawn from the re action in regular time for 1 H NMR and SEC analysis. At a reaction time of 35 mi n at an overall monomer conversion of approximately 25%, assembly of PEG 113 b P(DAAm stat DMA) 150 occurred. Af ter another 30 min polymerization under reactive monomer differenti ation conditions, at an overall monomer conversion of 77%, DMAc (1 mL) was adde d to dissolve the polymer assemblies and turn the reaction into a homogenous polymerization. At more than 94% overall monomer conversion the final gradient tetrablock copolymer PEG 113 b P(DAAm stat DMA) 150 b P(DAAm grad DMA) 320 b P(DAAm stat DMA) 130 was di alyzed against water for 48 h, and the polymer was retrieved via lyophilization.

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180 CHAPTER 9 CONCLUSION In th is dissertation, the strategic employment of dynamic interactions in polymer chemistry was demonstrated. W e devised a direct one step synthesis t o versatile 1,2 dithiolanes and implemented them into photocurable dynamic covalent hydrogels (Chapter 3 and C hapter 4). The approach described in these chapters capitalized on fundamental physical organic chemistry combined with tailored macromolecular de sign. We envision such synergistic methods to drive the field of dynamic network chemistry in the future. N on covalent solvophobic interactions were employed to control intrachain crosslinking reactions (Chapter 5) and generate multi component hybrid parti cles (Chapter 6) . In both cases, solvent induced aggregation was used to control chemical reactions (i.e., pho toinduced [2+2] cycloaddition and imine hydrolysis). We believe that t he concept of aggregation controlled reactions bears great potential for are as such as drug delivery or stimuli responsive nanomaterials. T he hydrophobic effect was s tudied and used in t he assembly of amphiphilic block co polymer s. We introduced a new technique for the direct imaging of polymer particles in their native solution st ate during polymerization induced self assembly . Leveraging variable temperature liquid ce ll transmission elec tron microscopy, we captured the dynamic hydrophobic to hydrophilic transition of thermoresponsive nanoparticles in real time (Chapter 7). Further more, selective hydrophobic interactions upon assembly enabled the synthesis of unpreceden ted gradient copolym er structures (Chapter 8). The findings in these studies provided a mechanistic understanding of

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181 polymer assembly in solution, resulting in a new avenue to polymer synthesis under improved reaction conditions. We believe that t he design principles and the synthetic methods developed in this dissertation will guide and promote the future use of dynamic interactions for advanced polymer materials.

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182 APPENDIX A. NMR Spectra Chapter 3 Figure A 1. Methylene H assignment of PhDL usin g NOE experiments. The mo re downfield proton is spatially closer to the phenyl ring. The NOE excitation frequency is indicated with the black arrow.

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183 Figure A 2 . H As signment of DiMeDL using NOE experiments and J values. The NOE excitation frequency i s indicated with the blac k arrow. Figure A 3 . Methylene H assignment of 3 using NOE experiments. The more downfield proton is spatially closer to the acrylate group. T he NOE excitation frequency is indicated with the black arrow.

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184 Figure A 4 . 1 H NMR spectrum of HDL in CDCl 3 . Figure A 5. 13 C NMR spectrum of HDL in CDCl 3.

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185 Figure A 6 . 1 H NMR spectrum of n PrDL in CDCl 3 . Figure A 7 . 13 C NMR spectrum of n PrDL in CDCl 3.

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186 Figure A 8 . 1 H NMR spectrum of C12DL in CDCl 3 . Figure A 9 . 13 C NMR spectrum of C12DL in CDCl 3.

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187 Figure A 10 . 1 H NMR spectrum of i PrDL in CDCl 3 . Figure A 11 . 13 C NMR spectrum of i PrDL in CDCl 3.

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188 Figure A 12 . 1 H NMR spectrum of PhDL in CDCl 3 . Figure A 13 . 1 3 C NMR spectrum of PhDL in CDCl 3 .

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189 Figure A 14 . 1 H NMR spectrum of TphDL in CDCl 3 . Figure A 15 . 1 3 C NMR spectrum of TphDL in CDCl 3 .

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190 Figure A 16 . 1 H NMR spectrum of BrTphDL in CDCl 3 . Figure A 17 . 1 3 C NMR spectrum of Br TphDL in CDCl 3 .

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191 Figure A 18 . 1 H NMR spectrum of DiMeDL in CDCl 3 . Figure A 19 . 1 3 C NMR spectrum of DiMeD L in CDCl 3 .

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192 Figure A 20 . 1 H NMR spectrum of 2 in CDCl 3 . Figure A 21 . 13 C NMR spectrum of 2 in CDCl 3.

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193 Figure A 22 . 1 H NMR spectrum of 3 i n CDCl 3 . Figure A 23 . 13 C NMR spectrum of 3 in CDCl 3.

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194 Figure A 24 . 1 H NMR spectrum of 4 in CDCl 3 . F igure A 25 . 13 C NMR spectrum of 4 in CDCl 3.

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195 B. UV Vis Spectra Chapter 3 Figure B 1. Normalized UV vis spectra for the 1,2 dithiolane substrates at 10 mM in DMSO. Notably, esterification of the hydroxyl functionality resulted in a shift of the maximum ab sorbance to lower wavelengths. For example, max of PhDL shifted from 340 to 329 nm upon transformation to the isopropanoate ester 2 . We believe this is due to substantial geometric changes caused by the sterically demanding isopropanoate ester group geminal to the phenyl substituent. However, elect ronic effect s upon conversion of the hydroxy functionality into an ester cannot be ruled out.

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196 C . NMR Spectra Chapter 4 Figure C 1 . 1 H NMR spectrum of PEG PhDL in DMSO d 6 . The PhDLA conjugation was carried out in DMF at 30 ° C and 12 5 mg/mL PEG 1.0 conce ntration. Th e total 1,2 dithiolane incorporation after 7.5 h reaction time is 68 % , as determined by the aromatic signals around 7.4 ppm relative to the PEG backbone signal at 3.55 ppm. Figure C 2 . 1 H NMR spectrum of PEG 1.0 in DMSO d 6 .

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197 Figure C 3. 1 H and 13 C NMR spectr um of PhDLA in CDCl 3 . D. SEC Data Chapter 4 Figure D 1. Overlaid size exclusion chromatograms of PEG PhDL before (black) and after (blue) the addition of the disulfide reducing agent PBu 3 . The minor change of the trace suggests tha t the high molecular weight species are predominantly formed from irreversible polymer chain coupling and not interchain disulfide formation.

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198 E . NMR Spectra Chapter 5 Figure E 1. 1 H NMR spectra in CDCl 3 of (A) 10 MMA DTB and (B) 10 MMA after dithiobenz oate end group removal.

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199 Figure E 2. S tability study of 7 hydroxy 4 methylcoumarin via 1 H NMR spectroscopy. The coumarin proton resonances were retained after 7 h incubation with (A) free thiols and (B) excess hydrazine. Figure E 3. Representative 1 H NMR spectra before (blue) and after (black) 6 h irradiation of a solution of CMOMe in DCM/MeOH. Conversion was determined by com paring the characteristic coumarin proton resonances at 7.52, 6.88, 6.84, and 6.16 ppm with the internal standard (1,3,5 trime thoxybenzene) peak at 6.11 ppm .

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200 F. SEC Data Chapter 5 Figure F 1. SEC analysis in DMAc of the RAFT polymerization of 10 MMA DTB . Average monomer conversions are indicated in the legend. Figure F 2. SEC analysis in THF of SCNPs formed under varyin g solvent conditions. (A) 10 MMA ; coumarin conversions : 54% (DCM), 66% (DCM/MeOH) , 66% (DCM/H x). (B) 10 MA : 55% (DCM), 66% (DCM/MeOH), 69% (DCM/Hx). (C) 20 MA : 73% (DCM), 85% (DCM/MeOH), 76% (DCM/Hx). We excluded the 20 MA SCNPs from DCM/Hx from the chain c ompaction analysis due to the substantial high molecular weight shoulder. (D) 20 MMA : 57 % (D CM), 57 % (DCM/Hx).

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201 G. UV Vis Spectra Chapter 5 Figure G 1. UV vis spectroscopy of linear precursors and SCNPs to determine the final coumarin conversion. (A) 2 0 MMA : 69% (DCM), 86% (DCM/MeOH), 79% (DCM/Hx) . (B) 10 MMA : 54% (DCM), 66% (DCM/MeOH) after 5 h of irradiation*, 78% (DCM/MeOH), 66% (DCM/Hx). (C) 20 MA : 73 % (DCM), 85% (DCM/MeOH), 76% (DCM/Hx) . (D) 10 MA : 55% (DCM), 66% (DCM/MeOH) after 5 h of irradiation * , 69% (DCM/Hx) . See Table S2 for a compilation of UV vis and SEC data.

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202 Figure G 2 . UV vis spectroscopy of reaction aliquots during the SCNP formation with 20 MMA (top) and 10 MMA (bottom).

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203 H. NMR Spectra Chapter 6 Figure H 1. 1 H NMR spec tr oscopy o f pr o Dox in DMSO d 6 indicates successful conjugation of 4 (dodecyloxy)benzaldehyde to doxorubicin via imine bond formation. Particularly, the appearance and integrations of protons b (imine), e and d (phenyl group), and s (terminal methyl group of the dode cyl ch a in) with res pect to the signals of the anthracycline core are indicative for the formation of proDox as the single compound after purification with ~6 mol% residual benzaldehyde starting material. 1 H NMR shifts of the anthracycline core were assigne d acco r ding to Cher if, et al. , J. Med. Chem . 1992 , 35 , 3208 3214.

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204 Figure H 2. 13 C NMR spectrum of proDox in CDCl 3 . I. NMR Spectra Chapter 7 Figure I 1 . 1 H NMR spectrum of the PDMA macro CTA in CDCl 3 .

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205 J. SEC Data Chapter 7 Figure J 1. S EC tra ce of the PDMA macro CTA in DMAc. Figure J 2 . SEC of reaction aliquots of the conventional thermal RAFT PISA using DAAm, DMA and the PDMA macro CTA. (A) Overlaid SEC traces of polymerization aliquots. The traces are monomodal a nd shift uniforml y to lower retention times with increasing monomer conversion. (B) Plot of number average molecular weight ( M n ) and dispersity ( Ð ) with increasing conversion; M n and Ð were obtained by SEC using conventional calibration against PMMA standards.

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206 Figure J 3. S EC tra ces of PDMA b P(DAAm co DMA) generated via DAAm/DMA PISA prior to crosslinking. The traces are all monomodal and shift to lower retention times with increas ing conversion. The respective overall monomer conversion is indicated at the upper right corner of each graph.

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207 K. NMR Spectra Chapter 8 Figure K 1. 1 H NMR spectrum of PEG TTC in DMSO d 6 . Figure K 2. 1 H NMR spectrum of DAAm50 PEG b P(DAAm stat DMA) b P(DAAm grad DMA) in CDCl 3 .

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208 Figure K 3. 1 H NMR spectrum of DAAm65 PEG b P(DAAm sta t DMA) b P( DAAm grad DMA) in CDCl 3 . Figure K 4. 1 H NMR spectrum of DAAm80 PEG b P(DAAm stat DMA) b P(DAAm grad DMA) in CDCl 3 .

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209 Figure K 5. 1 H NMR spectrum of DAAm65 PEG b P(DAAm stat DMA) b P(DAAm grad DMA) (black) and P(DAAm stat DMA) b P(DAAm grad DMA) (blue ) after hydrolysis in DMSO d 6 . The disappearance of the signal at 3.53 ppm is indicative for the removal of PEG. L. SEC Data Chapter 8 Fig ure L 1. Overlaid SEC traces of PEG (MW = 5 kDa) and PEG TTC.

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210 Figure L 2. Overlaid SEC traces of PE G ester hyd rolysis reaction. Figure L 3. SEC analysis of the single pot gradient tetrablock copolymer synthesis.

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240 BIOGRAPHICAL SKETCH Georg Scheutz was born in 1990 in Voggenberg, Austria. After graduating h ig h s chool in Salzburg, h e served for one year as a civil servant in a school for severely disabled children. In the fall of 2009, he moved to Vienna to study c hemistry at the enr olled in the m p rogram for o rganic c hemistry. After an exchange semester at the University of Florida , he quit his studies in Vienna and started his graduate career in the research group of Prof. Brent Sumerlin in the fall of 2015. He received his P h.D. from the Universit y of Florida in 2020.