Preparation of Precise Bioconjugates Using Atom Transfer Radical Polymerization

PREPARATION OF PRECISE BIOCONJUGATES USING ATOM TRANSFER RADICAL POLYMERIZATION

SAADYAH AVERICK Carnegie Mellon University Department of Chemistry

Advisor: Krzysztof Matyjaszewski

July 2014

In partial fulfillment of the requirements for the Ph.D. degree in Chemistry PREFACE

This thesis shows the development of atom transfer radical polymerization (ATRP) for the preparation of biohybrid materials. The general flow of the thesis is an introduction to the field of bioconjugates (Chapter 1), then research that involved growing polymers from biomolecule initiators is covered (Chapter 2-4), followed by projects that conjugated preformed polymers to biomolecules. (Chapters 5 and 6). Chapter 7 covers unique class of biohybrids called polyplexes that are formed by electrostatic interaction between the polymer and biomolecules.

Chapter 1 is a broad overview of the field of polymer bioconjugates (i.e. biohybrids) that are prepared using reversible deactivator radical polymerization methods (RDRP) and the biomolecules covered are proteins, DNA and RNA. Chapter 2 describes the preparation of protein polymer hybrids by “grafting from” a genetically encoded non-canonical amino acid ATRP initiator expressed at the 134 amino acid residue in the green fluorescent protein using normal

ATRP (Chapter 2A) and activators generated by electron transfer (AGET) ATRP (Chapter 2B).

The development of ATRP under biologically relevant conditions (BRC) for the synthesis of well- defined protein polymer hybrids by “by grafting from” a protein initiator using both normal and

AGET ATRP was explored in Chapter 3. DNA is a versatile biomolecules that is synthetically prepared using solid phase synthesis using phosphoramidite coupling chemistry. To incorporate an ATRP initiator into DNA a phosphoramidite ATRP initiator was synthesized and incorporated into DNA and polymers could be “blocked from” DNA both on and of the solid phase bead

(Chapter 4). The preparation of reversible DNA mediated star-polymer assemblies is described in Chapter 5. A self-transfecting nuclease resistant siRNA delivery system was developed by coupling a polymer to both the 5’ and 3’ ends of a passenger RNA strand followed by guide strand

i aneling (Chapter 6). A cationic nanogel was prepared, using AGET ATRP in inverse miniemulsion, and used to form polyplexes with siRNA. The nanogels were capable of binding and delivering siRNA to knockdown a protein of interest (Chapter 7).

[Note]: Chapters In this thesis include published of submitted manuscripts that have been reformatted

Chapter 1: Saadyah Averick and Krzysztof Matyjaszewski Synthesis of Well Defined Biohybrids Using Reversible Deactivation Radical Polymerization Procedures to be submitted to Progress in Polymer Science

Chapter 2: Partially from (Chapter 2A) Jennifer C. Peeler, Bradley F. Woodman, Saadyah Averick, Shigeki J. Miyake-Stoner, Audrey L. Stokes, Kenneth R. Hess, Krzysztof Matyjaszewski, and Ryan A. Mehl “Genetically Encoded Initiator for Polymer Growth from Proteins” Journal of the American Chemical Society, 2010, 132, 13575-13577 DOI: 10.1021/ja104493d and (Chapter 2B) Saadyah Averick, Christopher G. Bazewicz, Bradley F. Woodman, Antonina Simakova, Ryan A. Mehl, and Krzysztof Matyjaszewski “Protein-Polymer Hybrids: Conducting ARGET ATRP from a Genetically Encoded Cleavable ATRP Initiator” European Polymer Journal 2013, 10, 2919–2924 DOI: 10.1016/j.eurpolymj.2013.04.015

Chapter 3: Partially from: Saadyah Averick, Antonina Simakova, Sangwoo Park, Dominik Konkolewicz, Andrew J. D. Magenau, Ryan A. Mehl, and Krzysztof Matyjaszewski "ATRP under Biologically Relevant Conditions: Grafting from a Protein" ACS Macro Letters, 2012, 1, 6-10 DOI: 10.1021/mz200020c

Chapter 4: Partially from: Saadyah Averick, Sourav K. Dey, Debasish Grahacharya, Krzysztof Matyjaszewski and Subha R. Das “Solid Phase Incorporation of an ATRP Initiator for Polymer-DNA Biohybrids” Angewandte Chemie International Edition, 2014, DOI: 10.1002/anie.201308686

Chapter 5: Partially from: Saadyah Averick, Eduardo Paredes, Wenwen Li, Krzysztof Matyjaszewski, and Subha Das “Direct DNA Conjugation of Star Polymers for Controlled Reversible Assemblies” Bioconjugate Chemistry, 2011, 22, 2030–2037 DOI: 10.1021/bc200240q

Chapter 6: Partially from: Saadyah E. Averick, Eduardo Paredes, Sourav K. Dey, Kristin M. Snyder, Nikos Tapinos, Krzysztof Matyjaszewski, and Subha R. Das “Polymer Escorts for siRNA Delivery” Journal of the American Chemical Society 2013, 135, 12508–12511 DOI: 10.1021/ja404520j

Chapter 7: Partially from: Saadyah Averick, Eduardo Pardes, Ainara Irastorza, Abiraman Srinivasan, Daniel J. Siegwart, Andrew J. Magenau, Hong Y. Cho, Arun R. Shrivats, Eric Hsu, Jinku Kim, Shiguang Liu, Jeffrey O. Hollinger, Subha R. Das, and Krzysztof Matyjaszewski “Preparation of Cationic Nanogels for Nucleic Acid Delivery” Biomacromolecules, 2012, 13, 3445–3449 DOI: 10.1021/bm301166s

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Acknowledgments

This thesis is dedicated to my wife Alina Averick for her everlasting patience and being the cornerstone of my world and to my kids Miriam and Eliav for providing constant comic relief to day to day PhD research.

My mentor Krzysztof Matyjaszewski has given ceaseless faith in me allowing me to grow and helping me overcome my faults and reach my potential as a scientist. I never would have made it through graduate school without his guidance and leadership.

Dr Subha Das, my committee chair, has played a major role in helping me develop new and exciting avenues of research. My collaboration with his group has been exciting and fruitful.

Students from the Das laboratory have played a major role in my research and we have collaborated extensively a special thanks goes out to Dr Eduardo Paredes, Sourav Dey and Munira Fonz.

Drs John and Nancy Harrison for their support through the 2012 John & Nancy Harrison Legacy

Graduate Fellowship in Chemistry and Biochemistry. This support enabled me to better focus on my research into preparing biohybrid materials.

Dr Ryan Mehl helped kick start my research into grafting-from proteins. This work has been the starting point for many of my research projects

I am in debt to Dr Roberto Gil and Gaythri Withers from the NMR facility for constantly assiting me and helping me get the best data possible. Dr Alan Russell has helped inspire new avenues of taught and research projects. Antonina Simakova has been a tremendous asset to bounce ideas of and in the design complex challenging experiments that have made up the bulk of my research.

The entire Matyjaszewski has provided me with a tremendous source of inspiration and ideas and my time in this group has been one of the greatest times of personal and professional growth.

I would like to thank my esteemed committee member Dr Marcel Bruchez for his guidance and teaching me what can be done at the interface of polymers and cells.

Dr Katie Whitehead, extended committee member, has shown me how rigorous scientific methods and an engineering approach can be used to create new bioactive materials.

TABLE OF Contents

LIST OF ABBREIVATIONS xii-xiv

LIST OF SCHEMES xv-xviii

LIST OF FIGURES xix-xxxi

LIST OF TABLES xxxii 1. SYNTHESIS OF WELL DEFINED BIOHYBRIDS USING REVERSIBLE DEACTIVATOR MEDIATE RADICAL POLYMERIZATION PROCEDURES 1 1.1. Preface 2 1.2. Biomaterials Introduction 3 1.3.1. Protein Bioconjugates-General Considerations for Synthesis 7 1.3.2. Protein Bioconjugates- “Grafting from” vs. “Grafting to” 9 1.3.3. Protein Bioconjugates- “Grafting to” Lysine 11 1.3.4. Protein Bioconjugates- “Grafting to” Cystine 12 1.3.5. Protein Bioconjugates- “Two Step Grafting to” Lysine 14 1.3.6. Protein Bioconjugates- “Grafting from” 16 1.3.7. Protein Bioconjugates- “Grafting from” Lysine 17 1.3.8. Protein Bioconjugates- “Grafting from” Cysteine 18 1.3.9. Protein Bioconjugates- RDRP Under Biologically Relevant Conditions 19 1.3.10. Protein Bioconjugates- Polymer Based Protein Engineering 21 1.3.11. Protein Bioconjugates- “Grafting from” the N and C termini of a Protein 23 1.3.12. Protein Bioconjugates- “Grafting from” Genetically Encoded Initiators 24 1.4.1. DNA/RNA Bioconjugates- General Considerations 25 1.4.2. DNA Block Copolymers- “Blocking to” DNA in Solution 26 1.4.3. DNA Block Copolymers- “Blocking to” DNA in the Solid Phase 27 1.4.4. DNA Block Copolymers- “Blocking from” DNA in Solution 29 1.4.5. DNA Block Copolymers- “Blocking from” DNA from the Solid Phase 30 1.4.6. RNA Block Copolymers- “Blocking to” in Solution 28 1.5. Conclusions 34

1.6. References 34 2A. A GENETICALLY ENCODED AMIDE BASED ATRP INITIATOR FOR POLYMER GROWTH FROM PROTEINS 47 2A.1. Preface 48 2A.2.A Genetically Encoded Amide Based ATRP Initiator for Polymer Growth from Proteins 50 2A.2.1. Introduction 50 2A.2.2. Experimental 50 2A.2.2.1. Materials and Instruments 50 2A.2.2.2. N-Boc-4-(2’-bromoisobutyramido)-phenylalanine 3 51 2A.2.2.3. 4-(2’-bromoisobutyramido)phenylalanine 1 51 2A.2.2.4. Selection of an aminoacyl-tRNA synthetase specific for 4-(2’- bromoisobutyramido)phenylalanine 1 52 2A.2.2.5. Fluorescence analysis of highest-fluorescing clones 57 2A.2.2.6. Expression and purification of GFP-1 59 2A.2.2.7. MS Analysis of GFP-1 60 2A.2.2.8. ATRP reactions grafting from GFP-wt and GFP-1 61 2A.2.2.9. Characterization of ATRP grafting from GFP-wt and GFP-1 62 2A.2.3 Results and Discussion 63 2A.2.4 Conclusions and Outlook 71 2A.2.5 References 72 2B PROTEIN-POLYMER HYBRIDS: CONDUCTING ARGET ATRP FROM A GENETICALLY ENCODED CLEAVABLE ATRP INITIATOR 75 2B.1. Preface 76 2B.2. Protein-Polymer Hybrids: Conducting ARGET ATRP from a Genetically Encoded Cleavable ATRP Initiator 77 2B.2.1. Introduction 77 2B.2.2. Experimental 79 2B.2.2.1. Materials and Instruments 79 2B.2.2.2. Synthesis of ester amino acid ATRP Initiator 80 2B.2.2.2.1. N-(tert-butoxycarbonyl)-p-iodo-l-phenylalanine t-butyl ester (2) 80

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2B.2.2.2.2. N-(tert-butoxycarbonyl)-p-hydroxymethyl-l-phenylalanine t- butyl ester (3) 80 2B.2.2.2.3. N-(tert-butoxycarbonyl)-p-bromoisobutyryloxymethyl-l- phenylalanine t-butyl ester (4) 81 2B.2.2.2.4. p-Bromoisobutyryloxymethyl-l-phenylalanine hydrochloride salt (5 HCl, biF) 82

2B.2.2.3. Synthesis of GFP-UAAester 82

2B.2.2.4. Grafting from GFP-UAAester 82 2B.2.2.5. Polymer cleavage from protein 83 2B.2.3. Results and Discussion 83 2B.2.4 Conclusions and Outlook 87 2B.2.5 References 87 2.3 Epilogue 95 3 ATOM TRANSFER RADICAL POLYMERIZATION UNDER BIOLOGICALLY RELEVANT CONDITIONS 96 3.1. Preface 97 3.2. ATRP under Biologically Relevant Conditions: Grafting from a Protein 99 3.2.1. Introduction. 99 3.2.2 Experimental 101 3.2.2.1. Materials and Instrumentation 101 3.2.2.2. Preparation of NHS ester initiator 103

3.2.2.3. Synthesis of BSA-O-[iBBr]30 104 3.3.2.4. Polymer cleavage from protein 104

3.2.2.5. Synthesis of POEOMA by ATRP from PEO2000iBBr/BSA-O-[iBBr]30 104

3.2.2.6. AGET ATRP from PEO-iBBr/BSA-O-[iBBr]30 105

3.2.2.7. Synthesis of POEOMA by ATRP in DMSO/water from BSA-O-[iBBr]30 105

3.2.2.8. Synthesis of P(MEO2MA-co-OEOMA475) from BSA-O-[iBBr]30 106 3.2.2.9. Stability of GFP under polymerization conditions 106 3.2.3. Results and Discussion 107 3.2.4 Conclusions and Outlook 123

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3.2.5 References 124 3.3 Epilogue 128 4. SOLID PHASE INCORPORATION OF AN ATRP INITIATOR ONTO NUCLEIC ACIDS AND SMALL MOLECULES FOR DIRECT AND FACILE ACCESS TO POLYMER BIOHYBRIDS 129 4.1. Preface 130 4.2 Solid Phase Incorporation of an ATRP Initiator onto Nucleic Acids and Small Molecules for Direct and Facile Access to Polymer Biohybrids 131

4.2.1. Introduction 131 4.2.2. Experimental 134 4.2.2.1. Materials and Instrumentation 134

4.2.2.2. Synthesis of ATRP initiator phosphoramidite 135

4.2.2.2.1. Synthesis of Hydroxyl functional amide ATRP initiator, 1 135

4.2.2.2.2. Synthesis of ATRP initiator phosphoramidite, 2 135

4.2.2.3. DNA Synthesis 136

4.2.2.4. DNA Sequences 137

4.2.2.5. DNA Block-Copolymer Synthesis - Solution Phase 137

4.2.2.5.1. Preparation of DNA1-b-POEOMA-co-RMA 137

4.2.2.5.2. Preparation of DNA1-b-BnMA-co-RMA 138

4.2.2.6. Preparation of D-TEX particles 138

4.2.2.7. Block-Copolymer Synthesis - Solid Phase 139

4.2.2.7.1. From DNA linked CPG beads 139

4.2.2.7.2. From Biotin linked CPG beads 139

4.2.2.8. Biotin-Block-POEOMA-co-RhMA Avadin Microbead Binding 140

4.2.3. Results and Discussion 140

4.2.4. Conclusions and Outlook 153

4.2.5. References 154

4.3 Epilogue 161

5. DIRECT DNA CONJUGATION TO STAR POLYMERS FOR CONTROLLED REVERSIBLE ASSEMBLIES. 162

5.1. Preface 163

5.2. Direct DNA Conjugation to Star Polymers for Controlled Reversible Assemblies 164

5.2.1. Introduction 164

5.2.2. Experimental 168

5.2.2.1. Materials and Instrumentation 168

5.2.2.2. DNA synthesis 169

5.2.2.3. Star Polymers Synthesis and Characterization 169

5.2.3. Results and Discussion 170

5.2.4. Conclusions and Outlook 178

5.2.5. References 179

5.3. Epilogue 189

6. AUTO-TRANSFECTING SIRNA THROUGH FACILE COVALENT POLYMER ESCORTS 190

6.1. Preface 191

6.2. Auto-transfecting siRNA through Facile Covalent Polymer Escorts 192

6.2.1. Introduction 192

6.2.2. Experimental 193

6.2.2.1. Materials and Instrumentation 193

6.2.2.2. RNA Synthesis 194 6.2.2.3. Polymer Synthesis 195

6.2.2.4. Click Conjugation to Obtain PEp-RNAs 197

6.2.2.5. Annealing Protocol 197

6.2.2.6. Polyacrylamide Gel Electrophoresis of Duplex siRNA and Polymers 198

6.2.2.7. RNase A Stability of PEp-siRNAs 198

6.2.2.8. Dicer Cleavage Study of the PEp-siRNA 199

6.2.2.9. Dual Luciferase Assay for RNAi in Drosophila S2 Cells 199

6.2.2.10. Culture and Transfection of HEK293 Cells 200

6.2.2.11. Western Blot Analysis for Lck 200

6.2.3. Results and Discussion 201

6.2.4. Conclusion and Outlook 209

6.2.5. References 210

6.3 Epilogue 220

7. PREPARATION OF CATIONIC NANOGELS FOR NUCLEIC ACID DELIVERY 221

7.1. Preface 222

7.2. Preparation of Cationic Nanogels for Nucleic Acid Delivery 223

7.2.1. Introduction 211-212

7.2.2. Experimental 224

7.2.2.1. Materials and Instrumentation 224

7.2.2.2. qNG- Synthesis of cationic nanogels by AGET ATRP in inverse miniemulsion 225

7.2.2.3. siRNA transfection using cationic nanogels 226

7.2.2.4. Plasmid transfection using cationic nanogels 226

7.2.2.5. Luciferase assay on TECAN M-1000 227

7.2.3. Results and Discussion 227

7.2.4. Conclusion and Outlook 232

7.2.5. References 234

7.3 Epilogue 239

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LIST OF ABREVIATIONS

3D-Lib Aminoacyl synthetase library 3-HPA 3-hydroxypicolinic acid AA Ascorbic acid ACN Acetonitrile AGET Activators generated by electron transfer ARGET Activators regenerated by electron transfer ATRP Atom Transfer Radical Polymerization b-f Blocking from bFGF Basic fibroblast growth factor BIBAF 4-(2′-bromoisobutyramido)-phenylalanine biF p-bromoisobutyryloxymethyl-L-phenylalanine BPA Bromophenol acetate Bpy 2,2′-bipyridine BSA Bovine serum albumin

BSA-O-[iBBr]30 BSA modifed with 30 ATRP initiators b-t Blocking to CB Carboxybetaine CPG Controlled porosity glass CRP Controlled/living radical polymerizations CTA Chain transfer agent CTA Chymotrypsin CuAAC Copper catalyzed azide-alkyne cycloaddition Da Dalton DCM Dichloromethane DIPEA Diisopropylethylamine DMA Disulfide cross-linker DMAEMA Dimethylaminoethyl methacrylate DMF dDmethylformamide DMSO Dimethyl sulfoxide DMSO Dimethylsulfoxide DNABCp DNA block copolymer DTEX DNA Latex EBiB Ethylbromoisobutryate EGDA ethylene glycol diacrylate ESI Electrospray ionization

Et3N Triethylamine ETOAc Ethyl Acetate

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FC Full complimnet FRET Foster resonance energy transfer g-f Grafting-from GFP Green fluorescent protein GPC Gel permeation chromatography g-RNA guide RNA g-t Grafting-to iBBr 2-bromoisobutyrate IMIA Intein-mediated initiator attachment L Ligand LB Lowry broth LCST Lower critical solution temperature Luc Luceferase MALDI-ToF Matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy MALLS Multi-angle laser light scattering

MEO2MA 2-(2-methoxyethoxy)ethyl methacrylate

Mn Number average molecular weight MPC Methacryloyloxyethylphosphorylcholine MS Mass spectrospy Mw Weight average molecular weight

Mw/Mn Molecular weight distribution MWCO Molecular weight cutoff MWD Molecular weight distribution NA Nucelic acid nCAA Non-canonical amino acid NHS N-hydroxiysuccinimide NIPAM N-isopropylacrylamide NIRF Near infrared flourescent NMP nitroxide mediated polymerization NMR Nuclear magnetic resonance OEOA oligo(ethylene oxide acrylate)

OEOMA300 Oligo(ethylene oxide) Mn = 300

OEOMA475 Oligo(ethylene oxide) Mn = 475 PAC Phenoxyacetyl PBPE Polymer based protein enginering PBS Phosphate buffered saline PBS Phosphate buffered saline PC Partial compliment pDNA plasmid Dioxyribonucelice acid

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PEG Poly(ethylene glycol) PEG Polyethylene glycol PEO Poly(ethylene oxide)

PEO2000iBBr/PEOMI2000 Poly(ethylene oxide)-iBBr Mn = 2000 PEp-siRNA Polymer escorted passanger siRNA PI N-(n-propyl)pyridylmethanimine PMDETA N,N,N’,N”,N”-pentamethyldiethylenetriamine PMMA Poly(methyl methacrylate) PPH Protein polymer hybrids p-RNA passanger RNA PS Poly(styrene) Q-DMAEMA 2-(dimethylamino)ethyl methacrylate quaternized with ethyl bromide qNG Cationic nanogel r.p.m. Rotation per minute RAFT Reversible addition−fragmentation chain-transfer RDRP Reversialbe deactivator radical polmyierzation rh-GH Recombinet human growth hormone RMA RhodamineB methacrylate RNAi RNA interferance ROMP Ring-opening metathesis polymerization S2 Drosophila Schneider 2 SCAm Sulfobetaine methacrylamide SCIA Sortase-catalyzed initiator attachment SDS Sodium dodecyl sulfate SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEC Size exclusion chromotography siRNA Short Interfearing Ribonuclic acid Tet Tetracycline TFA Trifluroacetic acid THF Tetrahydrafuran TLC Thin layer chromotography TPMA Tris(2-pyridylmethyl)amine UTR Untranslated region

LIST OF SCHEMES

Scheme 1.2.1. Biohybrid materials that can be prepared by ATRP. Reprinted with permission from ref. 47. Copyright American Chemical Society

Scheme 1.2.2.(A) Mechanism of ARGET and ICAR ATRP . Reprinted with permission from ref.

50. Copyright American Chemical Society (B) Mechanism for RAFT. Reprinted with permission from ref. 51. Copyright American Chemical Society

Scheme 1.3.2.1. General synthetic strategies to polymer–protein conjugates(CTA= chain transfer agent). Reprinted with permission from ref. 68. Copyright American Chemical Society

Scheme 1.3.3.1. One-Pot polymerization and protein conjugation in ionic liquids. Reprinted with permission from ref. 72. Copyright American Chemical Society

Scheme 1.3.4.1. Synthesis of bFGF-heparin mimic copolymer using RAFT. Reprinted from ref.

75. Copyright Nature Publishing Group

Scheme 1.3.5.1 Synthetic representation of protein−polymer hybrid via bio-orthogonal “Click” reaction with NIRF dye Incorporated living copolymer and BSA. Reprinted with permission from ref. 80. Copyright American Chemical Society

Scheme 1.3.9.1. ATRP under biologically relevant conditions. Reprinted with permission from ref. 68. Copyright American Chemical Society

Scheme 1.3.9.2. General scheme for block copolymerization of polyNIPAAm-block-pDMA from lysosome. Reprinted with permission from ref. 92. Copyright The Royal Society of Chemistry.

Scheme 1.3.10.1. Synthesis of CT-pSBAm-block-pNIPAm Diblock Conjugates Schematic of the hypothesized effect of pSBAm and pNIPAm polymer collapse on substrate affinity (KM). At 25

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°C, both pSBAm and pNIPAm were in their extended conformation and allowed Suc-AAPF-pNA access to CT active site. At temperatures below pSBAm UCST and above pNIPAm LCST, polymer collapse inhibited access to the active site for Suc-AAPF-pNA due to steric blocking. At temperatures below pSBAm UCST, this effect is hypothesized to be more pronounced than at temperatures above pNIPAm LCST, because the pSBAm block was closer to the enzyme core than the pNIPAm block. Reprinted with permission from ref. 94. Copyright The American Chemical

Society.

Scheme 1.3.12.1. Genetic encoding of nCAA-iBBr into GFP at the 134 amino acid residue.

Reprinted with permission from ref. 98.Copyright American Chemical Society.

Scheme 1.4.2.1. Star polymer conjugation to DNA or DNA and other functional molecules using click chemistry. Reprinted with permission from ref. 102. Copyright American Chemical Society.

Scheme 1.4.3.1. Locked nucleic acid polymer amphiphile composition and characterization by electron microscopy. LPAs assemble into spherical micellar nanoparticles as they are released from solid support and dispersed into aqueous solution. The resulting nanoparticles are roughly 20 nm in diameter as evidenced by negative stain TEM. Reprinted with permission from ref. 107.

Scheme 1.4.5.1. Solution-phase synthesis of DNABCp using AGET ATRP. a) After removal of the 5′-ODMT from the DNA (in the CPG bead), the ATRP initiator phosphoramidite was conjugated to 5′-OH. Cleavage from the solid support and removal of the base and cyanoethyl protecting groups yielded the DNA with the ATRP initiator (iBBr-DNA1). b) Direct synthesis of the DNA-polymer hybrid in solution by AGET ATRP using the initiator-modified DNA. Two

xvii different initiator modified DNAs (with either 3′-OH or 3′-Quasar670 dye) were used to synthesize polymers with OEOMA or benzyl methacrylate and rhodamine methacrylate as the monomers.

Scheme 1.4.6.1. Schematic representing polymer escorted passenger siRNA self-transfection and activity in protein knockdown. Reprinted with permission from ref. 114. Copyright American

Chemical Society

Scheme 2A.2.3.1 Synthesis of 4-(2’-bromoisobutyramido)phenylalanine (1)

Scheme 2B.2.3.1. Synthesis of p-bromoisobutyryloxymethyl-L-phenylalanine (biF)

Scheme 2B.2.3.2.ARGET-ATRP from biF-GFP. [biF-GFP]:[OEOMA475]:[CuBr2]:[TPMA]

1/300/0.4/0.05 5% monomer 0.1 mM initiator 1X PBS, 16 ascorbic acid nmol/min. 30C 5 hours

Scheme 3.2.1. Synthesis of PPHs via (AGET) ATRP from [BSA-O-iBBr]30 and Selective

Cleavage of Polymer

Scheme 3.2.3.1. Preparation of the NHS-ester initiator.

Scheme 3.2.3.2. (AGET) ATRP of OEOMA475 from PEO2000iBBr under biologically relevant conditions.

Scheme 4.2.3.1. Synthesis of the ATRP initiator phosphoramidite

AGET ATRP using the initiator modified DNA. Two different initiator modified DNAs (either

xviii with 3'-OH or 3'-Quasar670 dye) were used to synthesize polymers with a mixture of OEOMA or benzyl methacrylate and rhodamine methacrylate as the monomers.

Figure 4.2.3.7. Synthesis of biotin modified polymer on solid support.

Scheme 7.2.3.1. Synthesis of cationic nanogels for nucleotide delivery of siRNA and pDNA using

AGET ATRP

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LIST OF FIGURES

Figure 2A.2.2.4.1 Fluorescence measurements of 92 synthetases with GFP clones. Blue represents colonies induced in media containing 1 mM 1 while red represents colonies induced in the absence of UAA. Expressions of 500 μL were grown for 40 hours before dilution of suspended cells directly from culture 100-fold with phosphate buffer saline (PBS). Fluorescence measurements (arbitrary units) were collected using a HORIBA Jobin Yvon FluoroMax®-4. The emission from 500 to 520 nm (1 nm bandwidth) was summed with excitation at 488 nm (1 nm bandwidth).

Figure 2A.2.2.5.1. Fluorescence measurements of 20 highest-expressing synthetases with GFP clones. Blue represents colonies induced in media containing 1 mM 1 while red represents colonies induced in the absence of UAA. Expressions of 3 mL were grown for 40 hours before dilution of suspended cells directly from culture 100-fold with phosphate buffer saline (PBS). Fluorescence measurements (arbitrary units) were collected using a HORIBA Jobin Yvon FluoroMax®-4. The emission from 500 to 520 nm (1 nm bandwidth) was summed with excitation at 488 nm (1 nm bandwidth).

Figure 2A.2.3.1 Genetic incorporation of ATRP initiator into proteins. (A) Initiator 4-(2’- bromoisobutyramido)phenylalanine 1. (B) The evolved MjRS/tRNACUA pair in pDule-BIBAF allows for site-specific incorporation of 1 in response to an amber codon. Lane 2 shows expression levels of GFP-wt from pBad-GFP-His6. Production of GFP-1 from pBad-GFP-134TAG-His6 is dependent on 1 in the growth media, lane 3 without 1 present, lane 4 with 1 mM 1 present. Protein was purified by Co+2 affinity chromatography, separated by SDS-PAGE and stained with

Coomassie.

Figure 2A.2.3.2 ESI-MS of GFP-wt and GFP-1 proteins demonstrates the efficient high fidelity incorporation of a single 1 in response to an amber stop codon. (A) ESI-MS-Tof analysis of sfGFP shows a single major peak at 27827.0 Da ±1 Da. (B) ESI-MS-Tof analysis of GFP-1 shows a single major peak at 28024.0 Da ±1 Da. This shows the expected molecular weigh difference of 197 Da from native indicating a single efficient incorporation of 1 at the expected site. Each sample did show a small peak at -131 ±1 Da indicating minor amounts of peptidase-based removal of N- terminal methionines and +22 sodium adducts. No other peaks were observed that would correlate with background incorporation of a natural amino acid.

Figure 2A.2.3.3. Isotopic abundance patterns indicate the presence of bromine. (A) Experimental isotopic

2+ 2+ pattern for [M+2H] at 589-592 Da. (B) Predicted isotopic pattern for [C50H77N14O14Br + 2H] as derived from various on-line isotopic pattern generators

4-7). (B & C) SEC of 0.1 mg of desalted reaction time-points on Superdex 200 at flow rate of 0.8 mL/min of PBS buffer monitored at 230 nm. B. GFP wt eluted at the expected volume of 17.3 mL

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(black) and was unaltered by the ATRP reaction (offset green). (C) SEC of GFP-1 ATRP reaction show the protein significantly increasing in size. (black, time=0; green, time=180 min).

Figure 2A.2.3.6. SEC of 0.1 mg of desalted ATRP reaction time-points on Superdex 200 at flow rate of 0.8 mL/min of PBS buffer monitored at 230 nm. SEC of ATRP reaction on GFP-1 show the protein significantly increasing in size with reaction time (Black is time=0 min., Green is time=180 min.). The collected fractions of the SEC separation are indicated by the vertical bars on the chromatogram.

Figure 2A.2.3.7. SDS PAGE analysis of SEC fractionated ATRP reactions. ATRP reactions were separated by SEC, (Figure 4), and the individual fractions were concentrated and separated on a

4-12% gel and stained with Coomassie. The full 3 hr reaction mixture is in lane 6 and fractions 1-

5 are in lanes 1-5 respectively. The GFP-polymer hybrid’s change in size is due to polymer addition as indicated by SEC fractionation and SDS-PAGE separation.

Figure 2B.2.3.1. Characterization of biF-GFP. A) ESI-MS-Tof analysis of GFP-wt shows a single major peak at 27828.0 Da ±1 Da. The sample does show a small peak at -131 ±1 Da indicating minor amounts of peptidase-based removal of N-terminal methionines and +22 sodium adducts.

B) ESI-MS of biF-GFP demonstrates the efficient high fidelity incorporation of biF into site 134 of GFP in response to an amber codon. ESI-MS-Tof analysis of biF-GFP shows a single major peak at 28039.0 Da ±1 Da, which is 211 Da greater than GFP-wt and corresponds to the molecular weight difference between GFP-wt and GFP-5 upon the site-specific incorporation of biF(5) into the 134 site of GFP. The sample does show a small peak at -131 ±1 Da indicating minor amounts of peptidase-based removal of N-terminal methionines and +22 sodium adducts. A small peak

xxii corresponding to hydrolyzed biF at -148 ±1 Da is also observed (~5% hydrolysis) No other peaks were observed that would correlate with background incorporation of a natural amino acid.

Figure 2b.2.3.3. Characterization of bif-GFP-POEOMA. A) GPC traces for cleaved POEOMA from bif-GFP-polymer cleavage conditions 2.5% KOH for 2 hours at 25C. B) Emission spectra of time samples from the polymerization of OEOMA from bif-GFP. Samples were diluted 1:10 in

1X PBS and measured on a TECAN Safire2 plate reader.

Figure 3.2.3.1. Effect of CuCl2:L on GFP (1 mg/ml) stability. [CuCl2]/[L]=1/[bpy],[PI] and

[TPMA] = 2.2 and 1.1 [CuCl2]=19 mM, [OEOMA475]0 = 0.23 M

Figure 3.2.3.2. MALDI-TOF spectra of BSA and BSA-O-[iBBr]30.

Figure 3.2.3.3. Effect of ligand (L = bpy or TPMA) and halide (X = Br or Cl) on ATRP of

OEOMA475 from BSA-O-[iBBr]30 at 30 °C. (A) First order kinetic plot and (B) Mn and Mw/Mn versus conversion plot (C) GPC traces for CuCl/CuCl2/bpy. [OEOMA475]0 = 0.21 M;

[OEOMA475]/[I]/[CuX]/[CuX2]/[L] = 227/1/1/9/11 ([L]: [TPMA] = 2[bpy]).

Figure 3.2.3.4. Effect of copper halide (X=Br or Cl) on ATRP of OEOMA475 under aqueous conditions at 30 °C. (A) First order kinetic plot and (B) Mn and Mw/Mn versus conversion plot; (C)

GPC traces for CuBr/bpy, (D) GPC traces for CuCl/bpy. [PEO2000iBBr]0 = 1 mM, [OEOMA475]0

= 0.45 M and [OEOMA475]/[I]/[L]/[CuX]/[CuX2] = 455/1/11/1/9 ([L]: [TPMA] = 2[bpy]).

Figure 3.2.3.5. GPC traces for CuBr/bpy. [BSA-O-[iBBr]30]0 = 1 mM, [OEOMA475]0 = 0.23 M and [OEOMA475]/[I]/[L]/[CuBr]/[CuBr2] = 227/1/11/1/9 ([L]: [TPMA] = 2[bpy]).

Figure 3.2.3.6. Effect of reducing agent feeding on AGET ATRP of OEOMA475 g-f BSA-O-

[iBBr]30 at 30 °C (reactions 6 – 7, Table 3). (A) First order kinetic plot and (B) Mn and Mw/Mn

xxiii versus conversion plot (C) Corresponding GPC traces. Polymerizations conducted with

[OEOMA475]0 = 0.21 M and [OEOMA475]/[I]/[TPMA]/[CuBr2] = 227/1/11/10. Rate of feeding of

AA 8 nmol/min.

Figure 3.2.3.7. Effect of ligand (L=bpy or TPMA) on AGET ATRP of OEOMA475 under aqueous conditions at 30 °C. (A) First order kinetic plot and (B) Mn and Mw/Mn versus conversion plot.

[PEO2000iBBr]0 = 1 mM, [OEOMA475]0 = 0.45 M and [OEOMA475]/[I]/[CuX2]/[AA] =

455/1/10/0.1 ([L]: [TPMA] = 2[bpy]).

Figure 3.2.3.8. Effect of reducing agent addition time on ATRP of OEOMA475 under aqueous conditions at 30 °C (reactions 3-4, Table 3). (A) First order kinetic plot and (B) Mn and Mw/Mn versus conversion plot. [PEO2000iBBr]0 = 1 mM, [OEOMA475]0 = 0.45 M and

[OEOMA475]/[I]/[TPMA]/[CuBr2]/[AA] = 455/1/11/10/0.03. AA was added step-wise (indicated by black arrows).

Figure 3.2.3.9. ATRP of OEOMA475 in PBS, 30 °C. (A) First order kinetic plot and (B) Mn and

Mw/Mn versus conversion plot; (C) GPC traces. [PEO2000iBBr]0 = 1 mM, [OEOMA475]0 = 0.23 M and [OEOMA475]/[I]/[bpy]/[CuBr]/[CuBr2] = 227/1/22/1/9.

Figure 3.2.3.10. ATRP of OEOMA475 g-f BSA-O-[iBBr]30 at 30 °C in PBS. (A) First order kinetic plot and (B) Mn and Mw/Mn versus conversion plot. [BSA-O-[iBBr]30]0 = 1mM, [OEOMA475]0 =

0.21 M and [OEOMA475]/[I]/[CuBr]/[CuBr2]/[L] = 227/1/1/9/ 21.

Figure 3.2.3.11. AGET ATRP of OEOMA475 in PBS, 30 °C. (A) First order kinetic plot and (B)

Mn and Mw/Mn versus conversion plot; (C) GPC traces. [PEO2000iBBr]0 = 1 mM, [OEOMA475]0 =

0.23 M and [OEOMA475]/[I]/[TPMA]/[CuBr2]/[AA] = 227/1/22/10/0.2. Rate of ascorbic acid addition was 16 nmol/min.

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Figure 3.2.3.12. AGET ATRP of OEOMA475 g-f BSA-O-[iBBr]30 at 30 °C in PBS. (A) First order kinetic plot and (B) Mn and Mw/Mn versus conversion plot (C) Corresponding GPC traces.

[OEOMA475]0 = 0.21 M; [OEOMA475]/[I]/[CuBr2]/[TPMA] = 227/1/10/11. Rate of feeding of AA

8 nmol/min.

Figure 3.2.3.13. AGET ATRP of OEOMA475 in PBS, 30 °C (Table 3, Entry 8). (A) First order kinetic plot, (B) Mn and Mw/Mn versus conversion plot, and (C) GPC traces. [PEO2000iBBr]0 = 1 mM, [OEOMA475]0 = 0.23 M and [OEOMA475]/[I]/[TPMA]/[CuBr2]/[AA] = 227/1/22/10/0.2.

Rate of ascorbic acid addition was 8 nmol/min.

Figure 3.2.3.14. Effect of 10% DMSO (v/v) on ATRP of OEOMA475 g-f BSA-O-[iBBr]30 at 30

°C. (A) First order kinetic plot and (B) Mn and Mw/Mn versus conversion plot (C) GPC traces.

[BSA-O-[iBBr]30]0 = 1 mM, [OEOMA475]0 = 0.21 M and [OEOMA475]/[I]/[CuCl]/[CuCl2]/[bpy]

= 227/1/1/10/21.

Figure 3.2.3.15. Thermo-responsive copolymer g-f BSA-O-[iBBr]30 at 30 °C. (A) Increase of diameter of PPH with temperature (B) GPC of copolymer cleaved from PPH. [OEOMA300]0 = 0.1

M, [MEO2MA]0 = 0.1 M and [MEO2MA]/[OEOMA300]/[I]/[CuCl]/[CuCl2]/[bpy] =

100/100/1/1/10/21.

Figures 4.2.3.1. MALDI mass spectrum of the iBBr-DNA1 after gel purification. Mass calculated:

7320.2; Mass found: 7320.2.

Figure 4.2.3.2. MALDI mass spectrum of the iBBr-DNA1 crude. Mass calculated: 7320.2; Mass found: 7321.4.

Figures 4.2.3.3. Characterization of the DNABCp synthesized using AGET ATRP in solution phase. a. GPC traces of the iBBR-DNA1 (black) and the polymer-DNA conjugates (green, entry

xxv

2 and purple, entry 7 in Table1). The GPC traces show a significant shift of MW after polymerization and also no residual initiator iBBr-DNA1. The GPC characterization of all polymers described in Table1 are in SI Figure S3b. Fluorescence spectra of the different DNA polymer conjugates show that DNA is directly conjugated to the rhodamine containing polymer.

The DNA-polymer conjugate with 3'-OH terminus by itself (purple trace) or when mixed with a free dye (orange trace), shows no energy transfer. However, the DNA polymer conjugate with

Quasar670 dye directly attached to the DNA 3'-terminus shows significant energy transfer (yellow- green trace, arrow) indicating that the Quasar670 dye is in a close proximity to the rhodamine dye from the polymer chain and confirms the integrity of the DNA during polymerization using AGET

ATRP.

Figures 4.2.3.4. Gel permeation chromatography (GPC) traces of all the DNABCps. The entry legends correspond to Table 4.2.3.2

Figure 4.2.3.5. Synthesis and characterization of DNA latex (DTEX) particles to demonstrate its ability of sequence specific recognition. a. After the synthesis polymer from iBBr-DNA1 using

BnMA and RMA as monomers, the DNABCp was dialyzed in water from acetone resulting into aggregation of the hydrophobic polymer chains which formed the core of the particles. The DNA1 remains exposed to water which decorates the the outer surface of the particles. Then the ability of the particles to selectively recognize complementary strands, was demonstrated by binding with a partially complementary sequence DY647-DNA1PC (PC = partial compliment). Furthermore the partically complementary strand was displaced from the DTEX particles using a DNA strand

(DNA1FC) (FC = fully complement). b. The sequence specific binding of the DNA strands with the DTEX particles were demonstrated using flow cytometry. When the DY647-DNA1PC were hybridized with the DTEX particles, a large increse in the fluorescence of the particles indicates

xxvi binding of the DY647 labeled DNA with the particles (Orange line). However when a non- complementary strand (DY647-DNA2) was used for hybridization the Cy5 signal did not increase

(Green line). After displacement of the DY647-DNA1PC from the particles using DNA1FC, the

Cy5 signal is at baseline levels indicating complete removal of the DY647-DNA1PC from the particles. Inset shows the volume distribution of the DTEX particles.

DNA-polymer conjugate was cleaved from the CPG bead and the protecting groups from the DNA were removed using standard conditions to form the functional DNABCp. b. GPC traces of the initiator modified DNA (black) and the DNA-polymer conjugate (red) synthesized on solid support.

Figure 4.2.3.8. GPC trace of the biotin modified polymer. Molecular mass was determined using

DMF GPC with PEO standards.

Figure 4.2.3.9. Synthesis of small molecule modified polymer conjugate on solid support and its characterization. a. Any molecule of interest (MOI) which can be attached to solid support can be functionalized with the ATRP initiator. Following this polymer chain can be grown on solid support using AGET ATRP on solid support. This method will enjoy all the advantages of solid phase syntheis. b. An avidin binding assay was performed to characterize the biotin conjugated polymer using flow cytometry. c. The polymer without the biotin did not bind to the avidin beads giving rise to low fluorescence signal (red trace). When the biotin conjugated polymer was used

xxvii with the avidin beads, a population with significantly higher fluorescence was observed (blue trace) indicating that the rhodamine polymer is directly conjugated to biotin.

Scheme 5.2.3.1. Star polymer conjugation to DNA or DNA and other functional molecules using click chemistry. The inset depicts the structure at the click linkages. The conjugations were performed using azide functionalized star polymers and 5'-alkyne (i) and 3'-alkyne 2 modifiers on the DNA respectively. The 3'-alkyne modifiers allow for 5'-end modification of the DNA with useful probes.

Figure 5.2.3.1. IR spectra of star polymers before (green) and after (red) click reaction with alkyne

DNA

(carrying a Dy547 label) and Cy5 were conjugated to the star polymer, the emission spectrum of the conjugate (black) was a result of FRET in the fluorophore pair confirming that the two were attached to the same star polymer.

Figure 5.3.2.3. Controlled DNA self-assembly of star polymers. (A) Scheme for DNA directed self-assembly of star polymers. (B) DLS scans of star polymers click conjugated to DNA1 (black-

Ai) and DNA 2(blue-Aii). DNA directed hybridization of these two stars yield larger assemblies shown in a 1:1 star/DNA1:star/DNA2 ratio (green-Aiii) and in a 1:10 star/DNA1:star/DNA2 ratio

(purple-Aiv) at 1nM concentrations.

xxviii

Figure 5.2.3.4. DNA Controlled reversal of star polymer-DNA nanoassemblies. (A) Scheme for room temperature disassembly of nanostructures using an invading DNA strand. (B) Mean area plots of DLS scans indicate a large nanoassembly (green bars) formed using a 1:1 ratio of smaller particles of star-DNA3 (black) and star-DNA4 (red) at 10nM concentration can be subsequently gradually disassembled by an invading complementary DNA strand. The smaller assemblies, depicted in yellow and blue bars- are obtained at 1:100 star polymer:invading DNA and 1:300 star polymer:invading DNA ratios, respectively.

Figure 6.2.2.3.1 [N3-PEG3-BPA]0:[OEOMA475]0:[CuBr2]0:[TPMA]0:[Sn(II)] :1:40:0.09:0.27:0.9 in 50% w/vol toluene at 60C for 1 h. Mn= 21, 000 Mw/Mn = 1.13.

Figure 6.2.2.3.2 [N3-PEG3-BPA]0:[OEOMA300]0:[MEO2MA]0:[CuBr2]0:[TPMA]0:[Sn(II)]

:1:70:20:0.09:0.27:0.9 in 50% w/vol toluene at 60C for 1 h. Mn= 20, 500 Mw/Mn = 1.05.

Figure 6.2.2.3.3 [N3-PEG3-BPA]0:[OEOMA475]0: [DMAEMA]0: [CuBr2]0:[TPMA]0:[Sn(II)]

:1:40:10:0.09:0.27:0.9 in 50% w/vol toluene at 60C for 1 h. Mn= 26, 000 Mw/Mn = 1.09.

Figure 6.2.2.6.1. Absence of non-specific binding between duplex siRNA and polymers PM, PT and PN. Non-denaturing gel electrophoresis and EtBr staining shows no shift in duplex siRNA mixed with polymers PM, PT and PN.

Figure 6.2.3.1.. Synthesis of PEp-siRNAs. A. The passenger strand (p-RNA) with bis-alkyne termini was conjugated to azido-functionalized polymers Px (x=M, T, N) and annealed to the guide strand, g-RNA, to form the PxEp-siRNAs (x=M, T, N) B. A non-denaturing polyacrylamide gel

xxix

(Tris; pH 8.5) and EtBr staining confirms that siRNA as well as PxEp-siRNAs include duplex

RNA; polymer PN alone does not stain.

Figure 6.2.3.2. Nuclease stabilty of PxEp-siRNA (x=M,T, N) compared to unmodified siRNA.

Samples were incubated with (+) and without (-) RNase A for 2 hours and run on a non-denaturing polyacrylamide gel and stained with EtBr

Figure 6.2.3.4. Silencing activity of PxEp-siRNAs. The graph indicates Rluc signal relative to control Fluc signal. Following transfection of reporter plasmids, S2 cells were treated with siRNA without (-) or with (+) FuGENE HD for transfection or 50, 125 or 250 nM PxEp-siRNAs.

Luciferase activity was measured after 24 hours; 'cells only' was a control well without siRNA.

Error bars represent the standard deviation from three separate experiments.

Figure 6.2.3.5. Knockdown of an endogenous gene using PNEp-siRNA. A. Western blot analysis of Lck knockdown in Hek293 cells that were plated (cells only) or transfected with 100 nM and

200 nM of Lck-PNEp-siRNA. After 48 hours cell lysates were analyzed for total Lck and actin as a loading control. B. Graph of densitometric quantitation with the Lck signal normalized to actin.

Error bars represent standard deviation from three independent experiments.

Figure 7.2.3.1. Normalized volume distribution of cationic nanogels prepared by AGET ATRP in inverse miniemulsion measured using DLS. Fig. A: volume distribution of purified qNG

xxx prepared from PEOMI2000:OEOMA300:Q-DMAEMA:DMA:Cu(II)Br2:Ascorbic Acid:

1/290/20/4/0.5/0.6/0.3, 55 mg PEGOH2000, in 5% Span80 in cyclohexane for 24 hours at 30C.

Size 350 nm PDI 0.164 Zeta potential 17.9 mV +/-0.902. Fig B: Volume distribution of qNG after incubation with 10 mM glutathione for 4 days.

Figure 7.2.3.2. Agarose gel electrophoresis analysis of polyplex formation and disassociation of qNG and plasmid DNA (LacZ-plasmid). Electrophoresis was conducted for 60 min, 100V and the gels were stained with EtBr and imaged with UV-transillumination. A: Polyplex formation, 500 ng of plasmid was incubated with varying amounts of qNG (R1-R500) 1 hour at 25C and then loaded onto a 0.5% agarose gel electrophoresis. B: Preformed polyplexes of qNG:pDNA (R1-

R200) were incubated with 0.05 g/L Heparin sulphate for 30 min and then examined by gel electrophoresis.

Figure 7.2.3.4. Plasmid DNA delivery using a firefly luciferase reporter assay: Graph of FLuc activity in S2 cells after 24 hour treatment with (N=3): no transfection reagent (negative control, red bar), 20 ng FLuc plasmid with FuGene-HD (positive control, purple bar) or a weight ratio of qNG:pDNA(20 ng) polyplexes at (R1500, R300, R30, R15, R3, R0.3 and R0.03 ) (experimental group, blue bars)

xxxi

9 pmol of duplex siRNA with FuGene-HD (positive control, purple bar) or a weight ratio of qNG:siRNA at R100, R20, R2, R0.2 and R0.02 (experimental group, blue bars).

xxxii

LIST OF TABLES

Table 2A.2.2.4.1 Components for autoinducing and non-inducing mediums, for final volume of

500 mL.

Table 2A.2.2.5.1 Amino acid sequences for synthetase library members

Table 2A.2.2.9.1. Fluorescent measurements of GFP and GFP-polymer hybrids

Table 3.2.3.1. Experimental conditions and results for normal ATRP from PEO2000-iBBr and BSA-

O-[iBBr]30

Table 3.2.3.2. Experimental conditions and results for AGET ATRP from PEO2000-iBBr and BSA-

O-[iBBr]30

Table 4.2.3.1. Reagents and synthesis conditions for solid phase DNA synthesis – 1 μmole scale

Table 4.2.3.2. AGET ATRP conditions for preparing DNABCp (M: OEOMA, RMA: rhodamine methacrylate, TPMA: tris(2-pyridylmethyl)amine), I: iBBr-DNA1 and iBBr-DNA1 with 3'-

Quasar670 dye (entry 8).

Table 5.2.3.1. DNA sequences used in this study

Table 6.2.3.1. Sequence and chemical modifications of the RNAs used in this study. The MALDI mass of the RNAs were used to confirm the successful synthesis of the RNA. (P indicates a 5'- phosphate group).

xxxiii

Chapter 1 Synthesis of Well Defined Biohybrids Using Reversible Deactivation Radical Polymerization Procedures

1 | P a g e

Chapter 1.1. Preface The use of reversible deactivation radical polymerization (RDRP) methods has significantly expanded the field of bioconjugate synthesis. RDRP procedures have allowed the preparation of a broad range of functional materials that could not be realized using prior art ploy(ethylene glycol) functionalization. In this chapter a review of procedures for synthesis of biomaterials is presented with a special focus on the use of RDRP to prepare biohybrids with proteins, DNA and RNA.

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1.2 Biomaterials Introduction

Biomaterials include a full spectrum of natural, synthetic or hybrid materials used for specific, targeted biological applications.[1-3] The development of novel procedures for synthesis of functional hybrid materials within the field of biomaterials has accelerated the treatment of many diseases and provided a huge benefit to patients and basic researchers alike.[4-8] Applications for biomaterials range from sutures (now permanent, bioresorbable or tailored to enhance wound healing),[9] transplanted cell-loaded scaffolds, tissue and cell sheet engineering,[10] drug delivery systems (targeted and untargeted), as well as new diagnostic and therapeutic agents.[11]

The design of highly functional biomaterials requires knowledge of both chemical reactivity and compatibility of reaction conditions with biomolecules that provide optimization of conditions for linkage and retention of biological activity. This requires developing biocompatible reaction conditions and procedure for protein structure function assays. Additional considerations include minimization of experimental errors and overcoming scale up challenges, due to the requirements of stringent purity and minimal batch-to-batch variation from both regulatory agencies and fundamentals of basic research.[12, 13] This synergy between chemistry and biology allows for the creation of new biomaterials with custom designed properties.

A classic biomaterial that exemplifies the combination of chemical and biological reactivity are PEGylated proteins.[14, 15] These biohybrids are prepared by careful selection of reactive groups inherently present on a protein surface with complementary functionality incorporated into the chain-end of PEG to yield a conjugated material with enhanced biological activity. For example, site selective modification of a protein surface,

3 | P a g e critical for maximized bioactivity, can now be accomplished using either non-natural amino acids or careful control of reaction conditions to select for specific amino acid residues to react specifically with selected polymer chain-end functionality.[16-19]

A new generation of polymer modified proteins is being synthesized to generate new or enhanced conjugate properties compared to these first generation PEGylated proteins.

These new polymers include brush like PEG structures (i.e. a molecular architecture that blocks immune recognition and increases biocompatibility) or polymers that can enhance or tailor the bioactivity of the modified biologics.[20, 21] Conjugation with zwitterionic polymers greatly alter the solvation of the protein or polymers and can extend the activity of a protein for longer periods at extreme pHs or at high protease and inhibitor concentrations.[22] These next generation materials have built on traditional biohybrids and advanced the fields of organic and material chemistry.

There are many subdivisions of the field of biomaterials. This chapter will focus on the synthesis of biohybrids comprising DNA, RNA or proteins conjugated with synthetic polymers.[23, 24] The covalent modification of biomolecules requires the presence of a suitable pair of reactive complementary functionalities at specific sites on the biomolecule and the synthetic material. Biomolecules have a palette of functional handles that are found endogenously or introduced using chemical derivation or genetic engineering. Each class of biomolecules has their own endogenous reactive groups. In the case of polysaccharides all have accessible hydroxyl groups that are readily esterified, but individual species have their own unique chemistry. For example, the carboxylic acid groups on hyaluronic acid, which has found application in wound healing, have been used to label this polysaccharide with proteins/polypeptides[25, 26] and methacrylate groups for hydrogel synthesis.[27, 28]

4 | P a g e

Chitosan, whose applications include blood coagulation and antibacterial properties, has a reactive amine group that has been used for modification with solubilization enhancers and polymeric stabilizers for drug delivery applications.[29, 30] Periodate mediated oxidation of dextran produces a poly(aldehyde) that can readily be reacted with amines to form Schiff base moieties.[31, 32] However, the chemical reactivity of polynucleotides such as DNA and RNA is limited due to the labile nature of the phosphate backbone and lack of native synthetic handles. Typically, functional groups are introduced into DNA and RNA during solid phase synthesis at the 5’ and 3’ ends of the nucleic acids using reactive phosphoramidites.[33-35] Once cleaved from the solid support the DNA/RNA can be further derivitized and modified.[36, 37] The most commonly incorporated functional groups for subsequent conjugation with polymers are amines,[38] thiols[39] and alkynes.[40].

Scheme 1.2.1. Biohybrid materials that can be prepared by ATRP. Reprinted with

permission from ref. 47. Copyright American Chemical Society

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A critical requirement for the preparation of bioconjugates is the ability to synthesize well-defined polymers with control over the composition, molecular weight and molecular weight distribution.[41, 42]. The most commonly used methodology to prepare the next generation of functional polymers for biomolecule modification are reversible deactivation radical polymerization (RDRP) procedures.[43] The three most commonly used RDRP methods are nitroxide mediated polymerization (NMP),[44] reversible addition−fragmentation chain transfer polymerization (RAFT)[45, 46] and atom transfer radical polymerization (ATRP).[47-50] ATRP and RAFT (Scheme 1.2.2.) are most commonly used for the preparation of bioconjugates due to their tolerance of functional groups, range of reaction conditions and polymerization media, thereby providing the capability to be conducted under biologically relevant conditions.[51, 52] ATRP, has a distinct advantage over RAFT in that ATRP initiators are more chemically and biologically inert than the chain transfer agents (CTA) used in RAFT and can therefore be used during solid phase synthesis of biomolecules as well as under more extreme pH conditions.[47]

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Scheme 1.2.2.(A) Mechanism of ARGET and ICAR ATRP . Reprinted with permission

from ref. 50. Copyright American Chemical Society (B) Mechanism for RAFT.

Reprinted with permission from ref. 51. Copyright American Chemical Society

1.3.1. Protein Bioconjugates-General Considerations for Synthesis

The most commonly modified biomaterials are proteins, due to their unique ability to act as messengers and enzymes in vivo. Proteins have a diverse range of functional groups that can be modified as they are comprised of amino acids. The dichotomy of an amino acid backbone is that it grants a boon of available ligation chemistries yet procedures for the synthesis of well-defined conjugates are elusive since most amino acid residues are represented multiple times in a single protein. Therefore the following major challenges must be resolved when designing protein based bioconjugates: 1) selection of one specific amino acid residue over another residue on a different region of the same protein; 2) a statistical distribution of products, that is typically observed due to the multiple reactive groups available; and 3) excessive modification of proteins is an additional issue since this can be accompanied by a loss of the modified proteins activity.[53, 54] The most readily modified amino acid is lysine whose amine side chain is used to form amide linked hybrids.

However, due to their hydrophilic nature, lysine residues are commonly found on all surfaces of a protein and therefore site-selective modification is not usually accomplished.[55]

Cysteine is an important structural amino acid that typically forms disulfide bridges that can link protein fragments or lock in a protein’s tertiary structure. Modification of cysteine residues with thiol-terminated polymers, to form disulfides or with maleimides to form thioethers, has the advantage that only one or two sites on a protein are modified by

7 | P a g e conjugation with polymers.[56] However, even these lower levels of modification can sometimes denature a protein, thereby losing bioactivity in the hybrid.[57] Reagents bearing dithiomaleimides have two sites available for reaction with thiols and therefore retention of disulfide bridges can be attained.[58, 59] Recently tyrosine selective oxidation reactions using cerium(IV) salts,[60] 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones [61] and diazonium[62] have been developed. Thus selective modification of tyrosine is a useful bioconjugation strategy, since the total number of these residues on a protein surface is limited. However, the proteins being modified must be oxidatively stable.

Selective modification of a proteins N terminus is a powerful strategy for preparation of a mono-functional bio-hybrid. Exact control of the reaction media’s pH is needed in order to selectively modify the N terminus. One must protonate all available lysine residues while leaving the N terminus in its unprotonated form. An aldehyde or ketone modified polymer is then introduced into the reaction media to form a Schiff base that can then be reduced with sodium cyanoborohydrate to form a stable amide bond.[63, 64] This method has been used to prepare a next generation M.S. drug, Plegridy™, which has superior pharmacokinetics and bioactive properties compared to prior generation materials prepared using non-specific lysine modification.[65]

An emerging strategy to selectively introduce reactive functional groups into proteins is the genetic encoding of non-canonical amino acids (nCAA) into a protein.[66, 67] There are two primary strategies employed for incorporation of nCAA and both utilize amino acid derivatives that bear a new functional group instead of the native side chain. The first method uses site directed mutagenesis of tRNA synthases so that the new side chains can be incorporated into the proteins active site during translation.[66] This method can insert

8 | P a g e a non-natural amino acid at every site that is encoded by the native residue. The second strategy involves the amber (UAG) codon’s tRNA synthetase to recognize a non-natural amino acid. In this strategy, the nCAA is only incorporated when the amber codon is present in the mRNA thereby providing exact control over the nCAA’s residue and stoichiometry.[67] Both of these methods have been used to incorporate azides, alkynes, ketones and aldehydes, among other functionalities, into proteins. The nCAA strategy has the most promise as an applicable procedure for the preparation of discrete bioconjugates with minimal perturbation of protein function. However, since all commercial PEGylated bioconjugates utilize site-specific modification of native amino acid residues the nCAA strategy remains an emerging method that has yet to be proven viable in commercial scale applications.

1.3.2. Protein Bioconjugates- “Grafting from” vs. “Grafting to”

The formation of covalent polymer biomolecule hybrids can be accomplished by grafting a polymer “from” or “to” a protein (Scheme 1.3.2.1.). The “Grafting to” (GT) method uses a pre-formed polymer with a reactive chain-end that is directly conjugated to a suitably reactive protein.[14, 68, 69] A newer, less explored, derivative of the GT method is the “grafting-with” method in which a polymer and biomolecule are both modified with functional groups that selectively form a complex based on non-covalent interactions, such as H-bonding.[70] In the “Grafting-from” (GF) method, a polymerization initiator is attached to a biomolecule and a polymer is grown in situ.[41, 42, 68, 71]

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Scheme 1.3.2.1. General synthetic strategies to form polymer–protein conjugates (CTA=

chain transfer agent). Reprinted with permission from ref. 68. Copyright American

Chemical Society

While the GT allows for the straightforward formation of a functionalized protein and subsequent conjugation of the preformed polymer to proteins, the procedure has several limitations. Typically GT suffers from substantial batch-to-batch variability and production of a broad distribution of products. This variability is due to the presence of a multiplicity of reactive groups on a proteins surface and steric constraints that arise when grafting two large macromolecules together. Consequently, in the purification step there is the need to separate high molecular weight polymer from the biohybrid products and thus purification of these materials may be challenging.

The advantages of the GF approach are high yields, compared to GT, and simple purification procedures, due to the large molecular weight differences between the product and the starting materials. The current limitation is characterization of the grafted polymers

10 | P a g e molecular weight and composition, in the case of copolymers. New advances in both GF and GT procedures are overcoming these challenges and both remain viable methods for the preparation of biohybrids.

1.3.3. Protein Bioconjugates- “Grafting to” Lysine The most direct strategy for GT lysine is the incorporation of an activated ester chain- end, such as an N-hydroxysuccinimide, into the polymer that can be subsequently coupled to the lysine residues of proteins. In one example, ATRP was used to grow polycarboxybetaine (polyCB) with an activated ester chain-end suitable for use in the activated ester strategy.[22] The polyCB was conjugated to trypsin and the activity of the protein was compared to PEGylated trypsin. Two properties of the conjugates were studied and compared: 1) the number of polymers attached to trypsin and 2) the effect of polyCB molecular weight. An increase in the number of conjugated polymers, for both PEG and polyCB, led to an increase in the activity of trypsin compared to native trypsin. It was found that matching the hydrodynamic radius of the polyCB rather than the molecular weight gave dramatically increased activity of the PPH. However, due to the limited solubility of zwitterionic monomers in organic media, only aqueous reaction media could be used for the polymerization, which can lead to hydrolysis of the activated ester chain-ends during polymerization thereby lowering conjugation yields. To overcome this limitation, an activated ester initiator was used to grow poly(methacryloyloxyethylphosphorylcholine)

(polyMPC) in an ionic liquid leading to ~100% retention of the activated ester chain-end

(Scheme 1.3.3.1).[72] The polyMPC was used to prepare a PPH with lysozyme, either after purification of the polymer from the ionic liquid or in a one-pot reaction where a solution of the lysozyme in buffered media was added to the reaction after the polymerization was

11 | P a g e completed. Both methods gave high yields of lysozyme-PPH conjugate. The loading and reaction yields were optimized by changing the activated ester group employed for the bioconjugation. This was demonstrated by using either N-succinimidyl-2-bromopropionate or N-succinimidyl-2-bromoisobutyrate ATRP initiators to prepare a thermoresponsive copolymer of (2-methoxyethoxy)ethyl methacrylate and oligo(ethylene glycol) methyl ether methacrylate for subsequent conjugation to trypsin. Both initiators gave good yields but the N-succinimidyl-2-bromopropionate terminated polymers provided slightly better conjugation efficiency.[73] Lysine modification provides a direct route for the preparation of PPH using readily synthesized reagents (i.e. activated ester initiators).

Scheme 1.3.3.1. One-Pot polymerization and protein conjugation in ionic liquids.

Reprinted with permission from ref. 72. Copyright American Chemical Society

1.3.4. Protein Bioconjugates- “Grafting to” Cysteine Modification of free cysteine residues in a protein presents a method for preparation of better defined bioconjugates. The development of new types of glycoproteins can be used

12 | P a g e to change the biological activity and recognition properties of both the protein and polysaccharide. A protected maleimide ATRP initiator was used to prepare polymers of mannopyranoside and galactopyranoside, either by polymerization of the monosaccharide monomers or through post-synthetic polymer conjugation. After deprotection of the initiator, a PPH with bovine serum albumin (BSA) could be formed by incubation of the maleimide glycopolymer in buffered media.[74] The use of lectins demonstrated that the sugar monomers were still accessible and biologically active after conjugation to the protein. The combination of proteins with biomimetic polymers is one of the more promising strategies for successful development of the next generation of therapeutics. To that end, a heparin mimicking copolymer of oligo(ethylene glycol)methacrylate and 4- styrene sulfonate with a disulfide chain-end was prepared by RAFT.[75] A PPH was formed upon mixing the polymer with basic fibroblast growth factor (bFGF), a protein having a free cysteine residue exposed on the surface. The PPH had the bFGF conjugated with only one poly(oligo(ethylene glycol))methacrylate (polyOEOMA) unit and had significantly improved stability compared to native bFGF.

Scheme 1.3.4.1. Synthesis of bFGF-heparin mimic copolymer using RAFT. Reprinted

from ref. 75. Copyright Nature Publishing Group

The synthesis of environmentally responsive PPHs provides conjugates that have numerous potential applications in the field of sensors and can also generate materials that

13 | P a g e can be used as injectable hydrogels. Poly(N-isopropylacrylamide) (polyNIPAM) is a thermoresponsive polymer that has a lower critical solution temperature (LCST) at ~32°C therefore it has been incorporated into several proteins to enhance the activity of the protein at physiologically relevant temperatures.[76] BSA was selected as a model protein and two strategies were used to conjugate polyNIPAM to a cysteine residue on BSA.[77] In one procedure, a protected disulfide ATRP initiator was used to grow polyNIPAM for subsequent conjugation to BSA through disulfide exchange. The hybrid not only had temperature responsive properties, but the disulfide linkage between the protein and polymer could be cleaved under reducing conditions. In the other approach maleimide functionality was incorporated into the chain-end of polyNIPAM prepared by RAFT by aminolysis of the chain transfer agent (CTA). Reaction with an excess of bis-maleimide gave a thiol reactive chain-end that could be reacted with BSA or ovalbumin (OVA) to yield a thermoresponsive PPH.[78]

The preparation of thermoresponsive star polymers provides a convenient method to prepare responsive multivalent protein clusters. This was exemplified by reaction of lysozyme, with a genetically engineered free cysteine, with a four arm star polymer with maleimide chain-ends.[79] Both of these strategies show the versatility of using surface exposed cysteine residues to control the site-specificity and stoichiometry of PPHs.

1.3.5. Protein Bioconjugates- “Two Step Grafting to” Lysine and Cysteine Although direct modification of amino acid residues with polymers is a straight forward process there are inherent drawbacks encountered during synthesis, including cross- reactivity between the chain-ends with a broad range of monomers including amines, carboxylic acids and thiols. The use of a two-step procedure where the amino acid residues

14 | P a g e on a protein are modified with small molecules that are subsequently reacted with functionalized polymers, has therefore become a popular strategy for forming PPH.

Modification of lysine is readily accomplished by reaction with hetero-bifunctional activated esters. ATRP was used to grow an azido terminated poly(glucose methacrylate) that was subsequently modified by reaction with an excess of dialkyne fluorescein to yield a polymer with one dye unit per poly(glucose) chain and with an alkyne chain-end (Scheme

1.3.5.1).[80] The polymer was conjugated to cowpea mosaic virus, a hollow bionanoparticle, modified with an activated ester-azide using copper(I) catalyzed azide- alkyne cycloaddition (CuAAC) generating a sugarcoated bionanoparticle with potential for drug delivery applications. A similar approach was taken to modify BSA with a sugar-near infrared fluorescent dye (NIRF). The BSA was modified with activated ester-azide and an alkyne terminated copolymer of a near infrared fluorescent dye and glucose amine was conjugated to the BSA using CuAAC.[81] Cysteine is readily modified with functionalized maleimides to yield functional thioethers that can be subsequently reacted with polymers.

A free cysteine residue on BSA was modified with alkyne-maleimide and an azido terminated polyNIPAM, prepared using RAFT was then conjugated to the BSA by

CuAAC.[82] The PPH demonstrated thermoresponsive behavior, forming large aggregates above the PPH’s LCST. The main advantage of modifying amino acids with small molecules for subsequent polymer conjugation stems from the desire to use more efficient ligation protocols (i.e. CuAAC) compared to the direct modification of proteins with reactive polymers. An additional strategy that has not yet been explored for the conjugation of polymers prepared using RDRP to proteins is the genetic encoding of a site specific

15 | P a g e reactive residue (i.e. azide) and conjugation of an alkyne terminated polymer, although this approach has been used for PEGylation.[18]

Scheme 1.3.5.1 Synthetic representation of protein−polymer hybrid via bio-orthogonal

“Click” reaction with NIRF dye Incorporated living copolymer and BSA. Reprinted with

permission from ref. 80. Copyright American Chemical Society

1.3.6. Protein Bioconjugates- “Grafting from” There are several unique parameters that must be considered when preparing PPHs using the GF method. The first is the selection of residues to which the initiator/CTA will be attached. These can be either the lysine or the cysteine residue, the C or N termini, or can be accomplished by genetic encoding of an nCAA ATRP initiator into the protein. An additional challenge of the GF method, due to the formation of a stable conjugate, is the characterization of the grafted polymer using traditional polymer characterization methods, such as gel permeation chromatography (GPC). The GF method is a more recent development that was nearly simultaneously reported by Maynard et al and by

Matyjaszewski in collaboration with the Russell group, in 2005.[83, 84] The Maynard group used a biotinylated ATRP initiator that was mixed with streptavidin forming a

16 | P a g e molecule with 4 initiators per protein and the polymer was GF the site of each initiator.

This work was followed up in a subsequent paper in which a free Cys on BSA was modified using a disulfide exchange reaction to yield a disulfide ATRP initiator that was used to grow polymers from the protein initiator.[83, 85] The work by Matyjaszewski and Russell used an amine reactive ATRP initiator that was immobilized onto chymotrypsin and polymers were GF the protein initiator.[84] This initial research in the field of GF using

ATRP utilized non-optimized polymerization conditions, due to the challenging reaction conditions characteristic of aqueous polymerizations, which led to poor control over the polymerization.

1.3.7. Protein Bioconjugates- “Grafting from” Lysine

GF modified lysine residues has an advantage over other site specific modifications due to the abundance of sites available for modification which leads to a relatively high concentration of initiators per unit of protein, typically 1 mM initiator/CTA per 1 mg of protein. Incorporation of an initiator/CTA at other residues typically results in one or two sites per protein (0.1 mM per mg of protein) which may lead to issues in controlling the target DP and rate of the reaction. The disadvantage of lysine modification is the possible deactivation of protein active sites due to nonspecific modification and dense polymer grafting. An additional challenge of lysine modification is the statistical distribution of initiator/CTA per protein generating a greater heterogeneity in the final PPH. Despite these challenges, lysine modification has been the most widely utilized procedure in the literature to prepare PPH using the GF method.

The modification lysine residues on a Qβ virus capsid with activated ester-azide was used in a two-step reaction to conjugate an ATRP initiator to the virus using CuAAC.[86]

17 | P a g e

PolyOEOMA was copolymerized from the virus particle initiator with an azido-PEG-MA.

The azido residues were functionalized either with Alexaflour488-alkyne, Gd contrast agent-alkyne or an alkyne derivative of the anticancer agent doxorubicin. The drug loaded bionanoparticle-PPH could efficiently deliver doxorubicin in vitro.

1.3.8. Protein Bioconjugates- “Grafting from” Cysteine

As in the GT procedure, the presence of a low number of cysteine residues on the surface of a protein can be utilized for more specific modification of a protein with initiators. BSA was modified with a maleimide functionalized ATRP initiator and OEOMA and rhodamine-MA (rhMA) were copolymerized from the linked initiator to yield a fluorescent PPH.[87] The ability to incorporate a large number of dye molecules into one protein is important for the development of the next generation of sensors that use ultra- bright tags for detecting low analytic concentrations. A P22 bacteriophage capsid was genetically engineered to display a high density of free cysteine residues to which an maleimide functional ATRP initiator was ligated.[88] An amine functional monomer was then grafted from the interior of the capsid and the amines were subsequently modified, either with fluorescein isothiocyanate or a gadolinium magnetic resonance imaging contrast agent, confirming the use of the protein polymer hybrid for encapsulation and delivery of small-molecule cargoes. The selectivity of cysteine residues for initiator ligation provides a useful method for preparing multifunctional PPHs.

1.3.9. Protein Bioconjugates- Conducting RDRP Under Biologically Relevant

Conditions

18 | P a g e

Scheme 1.3.9.1. ATRP under biologically relevant conditions. Reprinted with

permission from ref. 68. Copyright American Chemical Society

The synthesis of well-defined polymers using ATRP in aqueous media is challenging due to poor stability of the deactivator in polar media and very fast rates of polymerization.

Grafting from proteins presents further difficulties due to limited protein stability in media containing high concentrations of the protein and in the presence of organic agents. An additional complication facing the development of ATRP for efficient PPH synthesis is the requirement for buffered reaction conditions which can interfere with catalyst stability leading to a loss of control when grafting polymers from substances in the presence of an excess of counterions.[47]

The development of ATRP under biologically relevant conditions (ATRP BRC)

(Scheme 1.3.9.1.) was critical for overcoming these limitations. ATRP BRC is defined as the ability to conduct an ATRP in buffered solutions at ambient temperatures using a relatively low concentration of protein (~ 5 mg/ml) and less than 20% total organic content,

19 | P a g e i.e. monomer, catalyst and cosolvent. A systematic investigation into selection of suitable ligands and copper species was conducted to develop ATRP BRC.[89]

A protein based initiator (BSA) was prepared with an activated ester ATRP initiator containing a hydrolyzable ester group so that the grafted polymers could be cleaved after formation of the bioconjugate and the polymers directly analyzed using GPC. Two series of reactions using 1 mM of initiator and 10% oligo(ethylene oxide)methacrylate (OEOMA) were conducted in aqueous media and it was determined that a large excess of deactivator to activator (i.e. Cu(II)Cl2:Cu(I)Cl = 9:1 with bipyridine ligand) was required in a traditional ATRP to achieve good control over the aqueous reaction. It was also determined that slow feeding of ascorbic acid reducing agent to the polymerization solution was required when using activators generated by electron transfer (AGET) ATRP to provide good control over the polymerization in addition to using tris(2-pyridylmethyl)amine

(TPMA) as a ligand to form an active catalyst complex. The BRC conditions were then extended to low catalyst ATPR methods; activators regenerated by electron transfer

(ARGET) ATRP[90] and initiators for continuous activator regeneration (ICAR)

ATRP.[91] PolyOEOMA was grafted from recombinant human growth hormone(rh-GH), modified with 4 ATRP initiating sites on lysine residues, to yield a PPH with resistance to denaturation and proteolysis compared to native rh-GH.[21] The rh-GH-polyOEOMA displayed superior in vivo efficacy compared to unmodified rh-GF in a mouse model, demonstrating the therapeutic potential of PPHs prepared using RDRP. In another example an activated ester trithiocarbonate CTA was used to modify lysozyme and RAFT was used to grow a block copolymer of polyNIPAM-b-poly(dimethylacrylamide) from the site of the incorporated CTA (Scheme 1.3.9.2.). A commercially available enzymatic detergent

20 | P a g e

Tergazyme® was used to digest the protein leaving the polymer which could be directly analyzed using GPC.[92]

Scheme 1.3.9.2. General scheme for block copolymerization of polyNIPAAm-block-

pDMA from lysosome. Reprinted with permission from ref. 92. Copyright The Royal

Society of Chemistry.

1.3.10. Protein Bioconjugates- Polymer Based Protein Engineering

A powerful application for PPHs is the use of polymer specific functionality to extend, enhance or tune the activity of a protein. Russell et al have developed a polymer based protein engineering (PBPE) strategy for the design of tailor made PPHs.[93] Chymotrypsin

(CT) was modified with an activated ester ATRP initiator and dimethylaminoethyl methacrylate (DMAEMA) was GF the incorporated initiator. The grafted DMAEMA provided external control over the enzymes activity. At pHs under the pKa of DMAEMA the PPH displayed higher activity compared to native CT, while at pHs above 8 the activity of the PPH drastically decreased due to the change in the protonation state of the

DMAEMA’s units.[93] The concept of PBPE was elegantly extended by grafting a dual thermally responsive block copolymer of poly(sulfobetaine methacrylamide)-block-

21 | P a g e poly(NIPAM) (polySCAm-b-polyNIPAM) from CT modified with ATRP initiators at surface exposed lysine residues (Scheme 1.3.10.1.).[94] The block copolymer had a window of activity between the UCST of polySCAm and the LCST of polyNIPAM. The

PPH retained 60% of its activity after a 3 hour treatment with pepsin, a stomach protease, at pH =1. Under the same conditions native CT was totally denatured and had no residual activity. In many respects, PBPE represents the next generation of PPH technologies, one

where the polymer plays an active role in a proteins in vivo function.

Scheme 1.3.10.1. Synthesis of CT-pSBAm-block-pNIPAm Diblock Conjugates

Schematic of the hypothesized effect of pSBAm and pNIPAm polymer collapse on

substrate affinity (KM). At 25 °C, both pSBAm and pNIPAm were in their extended

22 | P a g e

conformation and allowed Suc-AAPF-pNA access to CT active site. At temperatures

below pSBAm UCST and above pNIPAm LCST, polymer collapse inhibited access to the active site for Suc-AAPF-pNA due to steric blocking. At temperatures below pSBAm

UCST, this effect is hypothesized to be more pronounced than at temperatures above

pNIPAm LCST, because the pSBAm block was closer to the enzyme core than the

pNIPAm block. Reprinted with permission from ref. 94. Copyright The American

Chemical Society.

1.3.11. Protein Bioconjugates- “Grafting from” the N and C termini of a Protein

Modification of the N and C terminals of proteins provides a facile route for the synthesis of discrete conjugates with site specific localization of the polymer chains. In one example of the preparation of an N terminal protein initiator, myoglobin was treated with pyridoxal-5-phosphate, converting the N-terminus to an aldehyde which was then modified with a ATRP initiator containing a hydroxylamine functionality.[95] The aldehyde on the protein was reacted with the hydroxylamine group on the ATRP initiator to attach the initiator via an oxime bond. PolyOEOMA was grafted from the myoglobin-ATRP initiator and the resulting myoglobin-polyOEOMA hybrid had greatly enhanced in vivo circulation times compared to native myoglobin.

Modification of the C terminus of a protein by direct chemical means is challenging.

Two strategies were developed by Chilkoti et al to selectively modify the C terminus of a protein. These methods elegantly combine advances in molecular biology with chemistry to generate C terminal modified initiators. Through the genetic encoding of an intein sequence at the C terminal of a protein post-translational modification can lead to a C- terminal thioether that can subsequently be conjugated to an ATRP initiator using native

23 | P a g e chemical ligation.[96] The intein-mediated initiator attachment (IMIA) protocol was used to conjugate an ATRP initiator to the C terminus of green fluorescent protein (GFP) which was used to grow a polyOEOMA chain without damaging the tertiary structure of the protein, as gauged using fluorescence spectroscopy. The alternative strategy for C terminal modification is sortase-catalyzed initiator attachment (SCIA).[97] The incorporation of sortase’s endogenous peptide substrate, at the C-terminus of a protein, followed by enzymatic digestion of this sequence generates a reactive species at the C-terminus that can be selectively conjugated to an ATRP initiator. SCIA was used to attach an ATRP initiator to the C-terminus of GFP and polyOEOMA was grafted from the functional chain end.

Several factors must be considered when considering N vs C terminal modification methods: does the protein of interest have an active site located at the N or C terminus and are the resources available for genetic modification and expression of proteins with the required C terminal modification tags. When comparing C terminal initiator incorporation methods SCIA typically has higher yields of initiator protein compared to the IMIA strategy.

1.3.12. Protein Bioconjugates- “Grafting from” Genetically Encoded Initiators

Scheme 1.3.12.1. Genetic encoding of nCAA-iBBr into GFP at the 134 amino acid residue. Reprinted with permission from ref. 98.Copyright American Chemical Society.

24 | P a g e

There are two general limitations to the post translational incorporation of initiators/CTA into proteins. The first is that even if only one residue is to be modified on a protein the protein initiator/CTA must be purified to remove any unmodified protein, and the second is that the methods are limited to specific residues and the initiators/CTA cannot be placed at any specific site of interest on a protein. These challenges were resolved by the evolution of a Methanococcus jannaschii tyrosyl-tRNA synthetase/tRNACUA pair to recognize an amide linked ATRP nCAA (nCAA-iBBr) initiator and insert nCAA-iBBr in response to the amber codon (UAG). Using this strategy a nCAA-iBBr was inserted at the

134 amino acid residue of GFP and polyOEOMA was grafted from the initiator using

ATRP (Scheme 1.3.12.1).[98] A limitation inherent to the use of the amide linked ATRP initiator is that the grafted polymer cannot be directly analyzed using GPC. Therefore an ester linked ATRP nCAA initiator was synthesized and inserted into the 134 amino acid residue of GFP in response to the amber codon and polyOEOMA was GF the GFP using

AGET ATRP. The resulting GFP conjugate was shown to be stable throughout the polymerization as measured by fluorescence spectroscopy. The grafted polymer could be cleaved from GFP by treatment with base and analyzed using GPC.[99] The nCAA method represents a convenient and direct method for site-specific incorporation of initiating groups with control over the stoichiometry and homogeneity of the product, i.e. all proteins have a single initiator.

1.4.1. DNA/RNA Bioconjugates- General Considerations

Nucleic acid (i.e. DNA and RNA) block copolymers (NABCp) are a distinct class of biomaterials that have applications in sensing and drug delivery[100] and can be synthesized using “blocking from” (BF) or “blocking to” (BT) methods either in solution

25 | P a g e or directly from controlled porosity glass beads (CPG, solid phase). The exquisite ability to control the sequence and structure of DNA (i.e. branch points) using solid phase synthesis methods combined with high chain-end fidelity allow for efficient loading of polymers, using BT, and incorporation of initiators for BF DNA/RNA. The incorporation of nucleic acid sequences in block copolymers imparts sequence specific recognition properties as well as the ability to form nanoassemblies due to the intrinsic hydrophilicity of nucleic acids.

1.4.2. DNA Block Copolymers- “Blocking to” DNA in Solution

BT DNA has been used to synthesize a wide range of functionalized hybrid materials.

Using a solution phase ligation strategy, a thermoresponsive DNA nanostructured polymer hybrid was prepared by CuAAC between an alkyne DNA tetrahedron and azido- polyNIPAM, prepared using RAFT.[101] The hybrid materials were capable of undergoing reversible assembly above the LCST of polyNIPAM. An ATRP arm-first method was used to prepare an azido functional polyOEOMA star polymer. The star polymer was then conjugated to alkyne functionalized DNA using CuAAC (Scheme 1.4.2.1.).[102] The

DNA star polymers could be annealed to form a multiplexed assembly that could be disassembled using strand invasion of the hybrids. The multivalent nature of the star polymers was used to conjugate Cy3-DNA-alkyne and alkyne-Cy5. This system has potential to find use as a fluorescence resonance energy transfer (FRET) based sensor.

Ring-opening metathesis polymerization (ROMP) was used to prepare an activated ester block PEO which was then reacted with 5’ amine modified DNA. The DNA-ROMP polymer hybrid was able to hybridize into preassembled DNA nanostructures enhancing their nuclease resistance.[103] One limitation of the BT method using solution phase

26 | P a g e techniques is the requirement to remove both the unreacted DNA and polymer from the desired conjugate. However facile purification of hybrid material can readily be accomplished after conducting the BT nucleic acids (NAs) during solid phase synthesis simply by washing the solid phase bead with an excess of solvent prior to deprotection.

Scheme 1.4.2.1. Star polymer conjugation to DNA or DNA and other functional

molecules using click chemistry. Reprinted with permission from ref. 102. Copyright

American Chemical Society.

1.4.3. DNA Block Copolymers- “Blocking to” DNA in the Solid Phase

There are two stringent requirements to prepare NABCp using solid phase methods: 1) the coupling reaction must have very high efficiency, and 2) the polymer formed in the GT reaction has to survive the relatively harsh NA deprotection protocol, i.e. concentrated ammonia for 4 hours. The preferred coupling reaction to achieve high yields of NABCp is the use of phosphoramidite modified polymers, which requires the presence of a free hydroxyl group on the chain-end of the polymer that can be converted to a phosphoramidite thereby precluding the use of polymers with reactive side chains, i.e. hydroxyl, amine,

27 | P a g e carboxylic acid and azid groups among others. The coupling reaction requires a simple incubation of the phosphoramidite polymer with the DNA on a solid phase resin to prepare the NABCp. The procedure was exemplified by the preparation of a hydroxyl functional polystyrene (PS) which was reacted with chlorophosphoramidite and purified by precipitation. The PS-phosphoramidite was coupled to a 20-mer DNA strand on a CPG and a gel shift migration assay found no trace of unreacted DNA after deprotection.[104] The

DNA-b-PS could be assembled into micellar structures with a uniform size. A targeted drug delivery system using a DNABCp was prepared by coupling polypropylene- phosphoramidite with DNA on a CPG bead. Since some cancers overexpress the folic acid receptor, after deprotection and micelle formation, a complimentary strand was labeled with folic acid followed by loading with doxorubicin.[105] The DNABCp drug delivery system showed good uptake into Caco-2 cells and demonstrated good drug delivery capacity.

A significant challenge to the field of polymer chemistry is the efficient synthesis of sequence controlled polymers. Sleiman et al presented an elegant method to prepare sequence defined polymers from DNA using the standard DNA synthetic method.[106]

Two monomers were prepared, a hydrophobic dimethoxytrityl (DMT)-dodecane- phosphoramidite and a DMT-oligo(ethylene glycol)-phosphoramidite (DP = 6). These monomers were sequentially coupled to DNA on a CPG bead. The resultant DNABCp exhibited sequence specific assembly behavior. Gianneschi and coworkers preapred antisense DNABCp against survivin mRNA was prepared by coupling the carboxylic acid functionalized poly((N-Benzyl)-5-norborene-exo-2,3-dicarboximide) and the 5’ amine terminated DNA. The complimentary strand was then annealed to the conjugate. The

28 | P a g e

DNABCp self-assembled into core-shell structures in water and was capable of self- transfecting into cancer cells and knockdown surviving mRNA in a specific manner

(Scheme 1.4.3.1.) .[107]

Scheme 1.4.3.1. Locked nucleic acid polymer amphiphile composition and

characterization by electron microscopy. LPAs assemble into spherical micellar nanoparticles as they are released from solid support and dispersed into aqueous solution.

The resulting nanoparticles are roughly 20 nm in diameter as evidenced by negative stain

TEM. Reprinted with permission from ref. 107. Copyright American Chemical Society.

While, solid phase polymer incorporation provides a more facile synthesis method, there are limitations to the polymers that are amendable to this strategy.

1.4.4. DNA Block Copolymers- “Blocking from” DNA in Solution

BF provides a simple solution to overcome the issue of removing unreacted polymers from DNABCp. The detection of DNA single point mutations is an important diagnostic tool. A sensor for single point mutations was developed by conjugating an activated ester

ATRP initiator to an amine terminated DNA probe.[108] Single point mutations were

29 | P a g e detectable by incubation of the probe compound with DNA on a surface, washing away excess probe, and carrying out a BF the surface by adding hydroxyl ethyl methacrylate and catalyst. Polymer growth was detected visually. DNABCp gold nanoparticle hybrids were prepared by incubating a 5’-ATRP intiator-DNA-thiol-3’, prepared by coupling an activated ester ATRP initiator to the 5’ amine function of a prepared DNA.[109]

PolyOEOMA could be BF the initiator modified gold nanoparticles to form a stable suspended DNA labeled particle with potential application for homogenous sensing. To prepare DNABCp pattered surfaces a 5’-amine-DNA-thiol-3’ was reacted with an activated ester CTA and incubated with a gold surface then OEOMA was BF using

RAFT.[110] The thickness of the DNABCp could be tuned by controlling the surface density of the DNA-CTA. Solution phase initiator/CTA attachment is a convenient modification protocol but the DNA-initiator/CTA must be purified from unreacted DNA and free initiator/CTA.

1.4.5. DNA Block Copolymers- “Blocking from” DNA attached to the Solid Phase

Recently, a direct solid phase method for incorporation of an ATRP initiator onto the

5’-end of DNA on a CPG bead was developed using phosphoramidite chemistry (Scheme

1.4.5.1.).[111] In this procedure an amide linked ATRP initiator with a free hydroxyl group was converted into an ATRP initiator linked to a phosphoramidite and directly incorporated into DNA on a CPG bead. The DNA initiators prepared by this method resulted in a 100% coupling efficiency, as gauged by mass spectroscopy. The conditions for an AGET ATRP were then optimized to grow well-defined polymers from the DNA initiator both on and off the CPG bead. Both hydrophilic, hydrophobic and functional monomers, i.e. monomer units containing amines, acids, alcohols, sugars and dyes, can readily be grown into

30 | P a g e polymers from the DNA using the BF bead method giving access to a wide range of functional DNABCp. Unlike other solid phase polymer coupling strategies, only the initiator unit needs to be modified as a phosphoramidite.

Scheme 1.4.5.1. Solution-phase synthesis of DNABCp using AGET ATRP. a) After

removal of the 5′-ODMT from the DNA (in the CPG bead), the ATRP initiator phosphoramidite was conjugated to 5′-OH. Cleavage from the solid support and removal

of the base and cyanoethyl protecting groups yielded the DNA with the ATRP initiator

(iBBr-DNA1). b) Direct synthesis of the DNA-polymer hybrid in solution by AGET

ATRP using the initiator-modified DNA. Two different initiator modified DNAs (with

either 3′-OH or 3′-Quasar670 dye) were used to synthesize polymers with OEOMA or

benzyl methacrylate and rhodamine methacrylate as the monomers.

1.4.6. RNA Block Copolymers- “Blocking to” in Solution

31 | P a g e

RNABCp have been less widely studied than their DNA counterparts. The primary use for RNABCp is to enhance the efficacy of therapeutic RNA delivery. Short interfering

RNA (siRNA) is being extensively investigated as a therapeutic agent, since the abrogation of endogenous proteins is implicated in many diseases. The direct use of siRNA as a therapeutic agent has been greatly hindered by its poor drug like properties, including rapid enzymatic degradation and clearance as well as its inability to penetrate cells due to its charged anionic phosphodiester backbone. Two polymeric delivery methods have been developed to overcome these challenges: 1) self-assembled anionic siRNA polplexes and

2) covalently modified polymer RNA conjugates. The polyplex method and other delivery vehicles, including viral and liposomal, will not be covered in this review. Thiol modified siRNA, targeting enhanced GFP, was conjugated to pyridyl disulfide-terminated polyOEOA, synthesized using RAFT.[112] The siRNA-polyOEOA displayed greatly enhanced resistance to nuclease mediated degradation. Furthermore, due to the presence of a REDOX sensitive linking group, the disulfide linkage enabled release of the siRNA under reducing conditions. Blending the siRNA-polyOEOA with a short cationic peptide

(KALA) greatly enhanced the transfection efficiency of the conjugate leading to enhanced gene knockdown compared to siRNA-polyOEOA alone and knockdown levels nearly equivalent to lipofectamine-siRNA lipoplexes. The concept of self-transfecting siRNA is attractive, since it precludes the use of additional transfection agents, creating a more molecularly defined therapeutic agent by covalently modifying siRNA with certain polymers, i.e. capable of transfection using active or passive internalization mechanisms.

This property can be imparted to the bioconjugates and two examples are presented.

Hepatocyte targeted polymers, synthesized via cationic polymerization were generated by

32 | P a g e reacting galactose on the side chains, which were then conjugated to thiol functionalized siRNA using disulfide exchange. These so called “Dynamic PolyConjugates™” were capable of the targeted in vivo delivery of siRNA to mice livers.[113]

However, a more generalized strategy is required to prepare siRNA capable of transfecting into any cell type. This was accomplished when an azido terminated poly(OEOMA-co-DMAEMA) was synthesized using ATRP and conjugated to dialkyne passenger RNA using CuAAC. The guide strand was then annealed to form a passenger escorted polymer-siRNA (PEp-siRNA) conjugate(Scheme 1.4.6.1.) .[114] The structure of the PEp-siRNA granted nuclease resistance to the RNA even after extended incubation with RNaseA. PEp-siRNA was also able to knockdown an endogenous protein in HEK cells.

33 | P a g e

Scheme 1.4.6.1. Schematic representing polymer escorted passenger siRNA self- transfection and activity in protein knockdown. Reprinted with permission from ref. 114.

1.5. Conclusions

In conclusion, the refinement of chemical synthesis procedures and an expanded understanding of biological molecule reactivity is part of the new concept of structure-by- design biohybrids. There are many factors that must be globally considered when modifying biomolecules with polymers, including intrinsic reactivity, stability to reaction conditions and reagents as well as complete characterization of the final product. Each

34 | P a g e subdivision of biomolecule hybrids has their own unique opportunities and challenges. The aim of this review is to provide an overview of the nuanced approaches that must be taken when conducting polymer bioconjugation. Rational design of biofunctional materials is now ingrained in the field and these advances can lead to highly optimized formulations for long acting therapeutics.

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Chapter 2A A Genetically Encoded Amide Based ATRP Initiator for Polymer Growth from Proteins

The work presented in this section was reformatted from a previous publication: Jennifer C. Peeler, Bradley F. Woodman, Saadyah Averick, Shigeki J. Miyake-Stoner, Audrey L. Stokes, Kenneth R. Hess, Krzysztof Matyjaszewski, and Ryan A. Mehl “Genetically Encoded Initiator for Polymer Growth from Proteins” Journal of the American Chemical Society, 2010, 132, 13575-13577 DOI: 10.1021/ja104493d

Chapter 2A.1. Preface

The preparation of well-defined discrete polymers protein conjugates is a requirement for the development of new therapeutics. Stoichiometric control over the number of polymers/initiators ligated to a protein is challenging due to the statistical distribution of reaction products associated with typical protein coupling reactions. An additional challenge to preparing well defined conjugates is the precise location of the polymer on the protein. A unique solution to the challenges of discreet protein polymer conjugates is the utilization of non-natural/canonical amino acids that can be incorporated into proteins via amber codon suppression technology.

This method has several major advantages over previously described site selective methods such as incorporation of cysteine residues or modification of the C or N terminus of proteins with polymers or initiators. One advantage is that cysteine incorporation can lead to oxidatively sensitive proteins which may lead to precipitation due to oxidative crosslinking. A second is that modification of the C or N terminus is typically low yielding and challenging reaction conditions, whereas nearly any amino acid residue is available for modification with nCAA using amber codon suppression technology yielding a precise control over the residue and stoichiometry of a bioconjugation reaction.

In this chapter, two different nCAA ATRP initiators were synthesized and a t-RNA synthase was evolved to incorporate these ATRP initiators into super folder GFP in response to an amber codon at its 134 amino acid residue and polymers were grafted from the protein initiator. Historically this project began the Matyjaszewski’s research into the preparation of protein polymer hybrids by grafting-from with a broader focus on controlling ATRP in aqueous

1 | P a g e media. In Chapter 2A the preparation of an amide linked ATRP initiator nCAA, its incorporation into GFP at the 134 amino acid residue, and its subsequent use as an initiator in the polymerization of OEOMA are described. In this chapter, it was found that although polymer could be grafted from the nCAA in GFP the control over the polymerization was poor. This lead to the research described in Chapter 3 to develop ATRP under biologically relevant conditions and extension of those conditions to low PPM ATRP methods. Chapter 2B is the culmination of the ATRP BRC conditions. In this chapter an ester linked ATRP initiator nCAA was prepared and incorporated into GFP. From this ester linked initiator OEOMA was polymerized using

ARGET ATRP and the polymer was cleaved for direct GPC analysis. It was demonstrated that well-defined polymers could be grafted from GFP with a single initiator using ARGET ATRP.

The work in this chapter was done in collaboration with Dr Ryan Mehl, who provided the protein. Antonina Simakova helped with experimental design.

2 | P a g e

Chapter 2A.2.

A Genetically Encoded Amide Based ATRP

Initiator for Polymer Growth from Proteins

2A.2.1. Introduction

The wide functional diversity of proteins—catalysis, regulation, transport, and structure—has made them desirable for integration into materials and medicine. Protein-polymer conjugates have already shown an impressive range of altered or improved properties.1-7 To date, protein polymers have been prepared in two general ways: either by graft-to methods where the functionalized polymer is attached to an amino acid, cofactor or end group, or by the graft-from method where a location(s) on the purified protein is functionalized with an initiator and then the polymer is grown from that site.4, 8-10 Thus far, the graft-from methods employed for residue- specific incorporation of polymerization initiators into proteins are limited to the N-terminal position or specific natural amino-acid directed linkages.2, 3, 11-13 Both methods suffer from challenging purification of intermediates and/or the inability to efficiently control the number or location of polymer connections, which compromises protein structural integrity. While the many graft-to and graft-from experiments using natural amino acids on proteins have illustrated the immense potential impact of well-defined protein-polymer conjugates, their application is limited by technical shortcomings.

2A.2.2. Experimental

2A.2.2.1. Materials and Instruments

3 | P a g e

N-Boc 4-aminophenylalanine, α-bromoisobutyryl bromide THF, dichloromethane, chloroform, ethyl acetate, 4 M HCl in dioxane, pentane and MgSO4 were obtained from Sigma. Fluorescence measurements were collected a HORIBA Jobin Yvon FluoroMax®-4. Mass spectroscopy spectra were obtained on a ESI-Q-Tof Ultima.Size exclusion chromatography was performed on an

Amersham biosciences F.P.L.C. system using a Superdex 200 SEC column at a flow rate of 0.8 mL/min in PBS

2A.2.2.2. N-Boc-4-(2’-bromoisobutyramido)-phenylalanine 3 (Mehl –Labratory)

Commercially obtained N-Boc 4-aminophenylalanine 2 (3.62 g, 0.01447 mol) was dissolved in

50 mL of dry THF. 2-bromoisobutyryl bromide (1.757 mL, 0.01422 mol) was added dropwise over 30-60 seconds with vigorous stirring. The reaction was complete after 10 min (monitored by TLC). After approximately 20 min. the entire reaction mixture (including newly formed ppt) was transferred to a separatory funnel with CHCl3, and approximately 100 mL of H2O. The reaction mixture was extracted with CHCl3 (3× 50 mL). The organic phase was washed with distilled water (2× 50 mL) and brine (50 mL). The organic phase was dried with MgSO4 and evaporated in vacuo to obtain the crude product (4.55 g). The crude solid product was recrystalized in 20-30 mL acetonitrile three times to purify the product. After three recrystalizations, 3 was obtained in 65% yield (3.63 g). 1H NMR (500MHz, DMSO):  9.4 (s, 1

H, COOH),  7.8 (s, 1 H, NH),  7.5 (d, 2 H, Ar H),  7.1 (d, 2 H, Ar H),  6.0 (d, 1 H, NH), 

4.2 (d, 1 H, CH),  3.05 (dd, 2 H, CH2),  2.0 (s, 6 H, CH3),  1.3 (s, 9 H, CH3).

2A.2.2.3. 4-(2’-bromoisobutyramido)phenylalanine 1(Mehl –Labratory)

N-Boc-4-(2’-bromoisobutyramido)-phenylalanine 3 (4.8 g, 0.0112 mol) was dissolved in 50 mL ethyl acetate under argon and dry 4 M HCl in dioxane (50 mL) was subsequently added to the

4 | P a g e solution while stirring at room temperature overnight. The reaction mixture was then evaporated under reduced pressure to a final volume of 5-10 mL. Pentane was then added to the solution, and the precipitate was filtered using an M type filter crucible and dried under reduced pressure.

Compound 1 was present as the HCl salt in 97% yield (3.93 g). 1H NMR (500MHz, DMSO): 

9.8 (s, 1 H, COOH),  8.4 (s, 1 H, NH),  7.6 (d, 2 H, Ar H),  7.2 (d, 2 H, Ar H).  4.1 (bs, 1 H,

13 CH),  3.1 (d, 2 H, CH2),  2.0 (s, 6 H, CH3). C NMR (500MHz, DMSO): 171.0 (1 C), 170.0

(1 C), 138.5 (1 C), 130.9 (1 C), 130.3 (2 C), 121.2 (2 C), 67.0 (dioxane), 61.5(1 C),

-1 53.9(1 C), 35.8(1 C), 31.5.0 (2 C). FT-IR (CH3CN) νmax cm 3374, 2977, 1740, 1664, 1600,

+ 1522, 1416, 1112, 840, 529. LCMS m/z for C13H17BrN2O3 [M+H] : 330.19; found: 329.3 and

331.2. HRMS calculated 329.0501, found 329.0507 (i-Fit 0.7).

2A.2.2.4. Selection of an aminoacyl-tRNA synthetase specific for 4-(2’- bromoisobutyramido)phenylalanine 1 (Mehl –Labratory)

The library of aminoacyl-tRNA synthetases was encoded on a kanamycin (Kn) resistant plasmid

(pBK, 3000 bp) under control of the constitutive Escherichia coli GlnRS promoter and terminator. The aminoacyl synthetase library (3D-Lib) was randomized as follows: Leu65,

His70, Gln155, and Ile159 were randomized to all 20 natural amino acids; Tyr32 was randomized to 15 natural amino acids (less Trp, Phe, Tyr, Cys, and Ile); Asp158 was restricted to

Gly, Ser, or Val; Leu162 was restricted to Lys, Ser, Leu, His, and Glu; and Phe108 and Gln109 were restricted to the pairs Trp-Met, Ala-Asp, Ser-Lys, Arg-Glu, Arg-Pro, Ser-His, or Phe-Gln.

The library plasmid, pBK-3D-Lib, was moved between cells containing a positive selection plasmid (pCG) and cells containing a negative selection plasmid (pNEG).

5 | P a g e

The positive selection plasmid, pCG (10000 bp), encodes a mutant Methanococcus jannaschii (Mj) tyrosyl-tRNACUA, an amber codon-disrupted chloramphenicol acetyltransferase, an amber codon-disrupted T7 RNA polymerase that drives the production of green fluorescent protein, and the tetracycline (Tet) resistance marker. The negative selection plasmid, pNEG

(7000 bp), encodes the mutant tyrosyl-tRNACUA, an amber codon-disrupted barnase gene under control of an arabinose promoter and rrnC terminator, and the ampicillin (Amp) resistance marker. pCG electrocompetent cells and pNEG electrocompetent cells were made from DH10B cells carrying the respective plasmids and stored in 100 μL aliquots at −80 °C for future rounds of selection.

The synthetase library in pBK-3D-Lib was transformed by electroporation into DH10B cells containing the positive selection plasmid, pCG. The resulting pCG/pBK-3D-Lib-containing cells were amplified in 1 L of 2×YT with 50 μg/mL Kn and 25 μg/mL Tet with shaking at 37 °C.

The cells were grown to saturation, then pelleted at 5525 rcf, resuspended in 30 mL of 2×YT and

7.5 mL of 80% glycerol, and stored at −80 °C in 1 mL aliquots for use in the first round of selections.

For the first positive selection, 2 mL of pCG/pBK-3D-Lib cells were thawed on ice before addition to 1.2 L of room temperature 2×YT media containing 50 μg/mL Kn and 25

μg/mL Tet. After incubation (11 h, 250 rpm, 37 °C), a 200 μL aliquot of these cells was plated on eleven 15 cm GMML-agar plates containing 50 μg/mL Kn, 25 μg/mL Tet, and 60 μg/mL chloramphenicol (Cm). The positive selection agar medium also contained 1 mM 1. After spreading, the surface of the plates was allowed to dry completely before incubation (37 °C, 15 h). To harvest the surviving library members from the plates, 10 mL of 2×YT (50 μg/mL Kn, 25

μg/mL Tet) was added to each plate. Colonies were scraped from the plate using a glass

6 | P a g e spreader. The resulting solution was incubated with shaking (60 min, 37 °C) to wash cells free of agar. The cells were then pelleted, and plasmid DNA was extracted. For the first positive selection a Qiagen midiprep kit was used to purify the plasmid DNA. For all other plasmid purification steps a Qiagen miniprep kit was used to purify the plasmid DNA. The smaller pBK-

3D-Lib plasmid was separated from the larger pCG plasmid by agarose gel electrophoresis and extracted from the gel using the Qiagen gel extraction kit.

The purified pBK-3D-Lib was then transformed into pNEG-containing DH10B cells. A

100 μL sample of pNEG electrocompetent cells was transformed with 50 ng of purified pBK-

3D-Lib DNA. Cells were rescued in 1 mL of SOC for 1 h (37 °C, 250 rpm) and the entire 1 mL of rescue solution was plated on three 15 cm LB plates containing 100 μg/mL Amp, 50 μg/mL

Kn, and 0.2% L-arabinose. Cells were collected from plates and pBK-3D-Lib plasmid DNA was isolated in the same manner as described above for positive selections.

For the second round of positive selection, 50 ng of purified library DNA was transformed into 100 μL of pCG competent cells. The transformants were rescued for 1.5 h in 1 mL of SOC (37 °C, 250 rpm). A 50 μL sample of these cells was plated on three plates prepared as described in the first positive selection on LB agar plates.

For the second negative selection, one plate was spread with 250 μL of rescued cells, and two plates were spread with 50 μL of rescued cells and then incubated (12−16 h, 37 °C). For this round, the cells were plated on LB agar containing 100 μg/mL Amp, 50 μg/mL Kn, and 0.04%

L-arabinose.

In order to evaluate the success of the selections based on variation in synthetase efficacy

(as opposed to traditional survival/death results), the synthetases resulting from the selection

7 | P a g e rounds were tested with the pALS plasmid. This plasmid contains the sfGFP reporter with a

TAG codon at residue 150 as well as tyrosyl-tRNACUA. When a pBK plasmid with a functional synthetase is transformed with the pALS plasmid and the cells are grown in the presence of the appropriate amino acid on autoinduction agar, sfGFP is expressed and the colonies are visibly green.

One microliter of each library resulting from the second positive and the second negative rounds of selection was transformed with 60 μL of pALS-containing DH10B cells. The cells were rescued for 1 hr in 1 mL of SOC (37 °C, 250 rpm). A 250 μL and 50 μL of cells from each library were plated on autoinducing minimal media with 25 μg/mL Kn, 25 μg/mL Tet, and 1 mM

1. Plates were grown at 37 °C for 24 hours and then grown on the bench top, at room temperature, for an additional 24 hours.

Table 2A.2.2.4.1 Components for autoinducing and non-inducing mediums, for final volume of 500 mL.

A) Autoinduction B) Non-inducing C) Autoinducing medium medium plates

5% aspartate, pH 7.5 25 mL 25 mL 25 mL

10% glycerol 25 mL - 25 mL

25× 18 amino acid mix 20 mL 20 mL 20 mL

50× M 10 mL 10 mL 10 mL

leucine (4 mg/mL), pH 7.5 5 mL 5 mL 5 mL

20% arabinose 1.25 mL - 1.25 mL

1 M MgSO4 1 mL 1 mL 1 mL

40% glucose 625 μL 6.25 mL 125 μL

Trace metals 100 μL 100 μL 100 μL

8 | P a g e

Autoinducing agar plates were prepared by combining the reagents in Table 2A.2.2.4.1A with an autoclaved solution of 40 g of agarose in 400 mL water. Sterile water was added to a final volume of 500 mL. Antibiotics were added to a final concentration of 25 g/mL Tet and 25

g/mL Kan. Nine plates were poured with 1 mM 1, and nine plates were maintained as controls without UAA.

A total of 92 visually green colonies were selected from the two 1 mM 1 plates and used to inoculate a 96-well plate containing 0.5 mL per well non-inducing minimal Table

2A.2.2.4.1B, with sterile water added to a final volume of 500 mL) with 25 μg/mL Kn, 25 μg/mL

Tet. After 24 hours of growth (37 °C, 250 rpm), 5 μL of these non-inducing samples were used to inoculate 96-well plates with 0.5 mL autoinduction media (Table 2A.2.2.4.1C, with sterile water added to a final volume of 500 mL) containing 25 μg/mL Kn, 25 μg/mL Tet with and without 1 mM 1. Fluorescence measurements of the cultures were collected 40 hours after inoculation using a HORIBA Jobin Yvon FluoroMax®-4. The emission from 500 to 520 nm (1 nm bandwidth) was summed with excitation at 488 nm (1 nm bandwidth). Samples were prepared by diluting suspended cells directly from culture 100-fold with phosphate buffer saline

(PBS). Results of fluorescence measurements are shown in Figure 2A.2.2.4.1.

9 | P a g e

FluorescenceSum

96-Well Block Well Number

Non-inducing cultures (3 mL) with 25 μg/mL Kn and 25 μg/mL Tet were grown to saturation

(37 °C with shaking at 250 rpm) from the 20 highest-fluorescing colonies. Autoinduction cultures (3 mL) with 25 μg/mL Kn and 25 μg/mL Tet were inoculated with 30 μL of non- inducing cultures and grown with and without 1 mM 1 at 37 °C with shaking at 250 rpm. After approximately 40 hours, fluorescence was assessed as described above (Figure 2A.2.2.5.1). The top eight performing clones were sequence revealing five unique members (Table 2A.2.2.5.1).

The top performing clone (G2) was moved from the pBK-G2 plasmid to the pDule plasmid

10 | P a g e

(pDule-BIBAF). pDule plasmid was generated by amplifying the MjYRS gene from the pBK plasmid isolated from the library using primers RSmovef (5’-

CGCGCGCCATGGACGAATTTGAAATG-3’) and RSmover (5’-

GACTCAGTCTAGGTACCCGTTTGAAACTGCAGTTATA-3’). The amplified DNA fragments were cloned in to the respective sites on the pDule plasmids using the incorporated

NcoI and KpnI sites. Fluoresence Sum Fluoresence

96-Well Block Well Number

Table 2A.2.2.5.1 Amino acid sequences for synthetase library members

32 65 108 109 158 159

11 | P a g e

A4 Gly Glu Phe Gln Gly Val

F9 Gly Glu Phe Gln Gly Cys

G2 Gly Glu Trp Met Ser Ile

H2 Gly Glu Phe Gln Gly Leu

H7 Gly Glu Phe Gln Gly Asn

Native Tyr Leu Phe Gln Asp Ile

2A.2.2.6. Expression and purification of GFP-1 (Mehl –Labratory)

DH10B E. coli cells co-transformed with the pBad-sfGFP-134TAG vector and the machinery plasmid pDule-BIBAF were used to inoculate 5 mL of non-inducing medium containing 100

μg/mL Amp and 25 μg/mL Tet. The non-inducing medium culture was grown to saturation with shaking at 37 °C, and 5.0 mL was used to inoculate 0.5 L autoinduction medium with 100 μg/mL

Amp, 25 μg/mL Tet, and 1 mM 1 (0.5 L of media grown in 2 L plastic baffled flasks). After 40 hours of shaking at 37 °C, cells were collected by centrifugation.

The protein was purified using BD-TALON cobalt ion-exchange chromatography. The cell pellet was resuspended in wash buffer (50 mM sodium phosphate, 300 mM sodium chloride, pH 7) containing 1 mg/mL chicken egg white lysozyme, and sonicated 3  1 min while cooled on ice. The lysate was clarified by centrifugation, applied to 6-9 mL bed-volume resin, and bound for 30 min. Bound resin was washed with >50 volumes wash buffer.

Protein was eluted from the bound resin with 2.5 mL aliquots of elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, 150 mM imidazole pH 7) until the resin turned

12 | P a g e pink and the color of the eluent the column was no longer green. The elusions concentrations were check with a Bradford protein assay. The protein were desalted into PBS using PD10 columns and concentrated with 3000 MWCO centrifuge filters

2A.2.2.7. MS Analysis of GFP-1 (Mehl –Labratory)

All protein samples processed for MS analysis were the same proteins used in the subsequent

ATRP reactions. The samples in 50 mM Na H2PO4 pH 6.5 were exchanged into 20 mM ammonium acetate buffer pH 7 using PD10 gel filtration columns. Proteins in 20 mM ammonium acetate buffer were dried overnight on a vacuum-line. Protein samples for full protein mass spectrometry were resuspended in 1:1 water:acetonitrile with 0.2% formic acid. The samples were analyzed at the Mass Spectrometry Facility at University of Illinois Urbana-

Champaign using an ESI-Q-Tof Ultima. Trypsin digestion of GFP and GFP-1 were performed using Trypsin In-Gel Digest Kit from Sigma Aldrich (PP0100). Mass spectral analyses were performed on an Agilent 1100 series LC/MSD SL ion trap mass spectrometer with electrospray ionization and MS/MS capabilities. Ten microliters of the protein digests were injected onto a

Zorbax 300SB-C8 column (narrow-bore 2.1x150mm 5-micron) for separation using a gradient of

5-95% CH3CN (with 0.1% formic acid) in water (with 0.1% formic acid) over 75 min (5-25% 0-

40 minutes, 25-95% 40-60 minutes, 95-5% 60-75 minutes). The flow rate was set to 0.25 mL/min. The SL Trap MS was operated in the SPS mode under the normal scan setting. The dry temperature was 325C, with dry gas flow of 10.0 L/min and a nebulizer pressure of 40 psi. For the MS/MS experiments, the instrument was operated in the auto MS/MS mode selecting two precursor ions with preference given to doubly charged ions while singly charged ions were excluded. The peptide of interest, E(BIBAF)GNILGHK (C50H77N14O14Br, Exact Mass = 1176.49

Da) will generate a [M+2H]2+ peak at 589.2 Da. An extracted ion chromatograph of mass 589

13 | P a g e showed a single peak eluting at 36.3 minutes containing an isotopic cluster with a unique pattern consistent with that expected for the bromine containing peptide. MS/MS analysis of the peptide at 589 Da further confirmed incorporation of the bromine into the peptide.

2A.2.2.8. ATRP reactions grafting from GFP-wt and GFP-1

-3 -4 Initiator stock solution: Bpy (16.70 mg, 1.07*10 mmol) and Cu(II)Br2 (6 mg, 2.68*10 mmol) were dissolved in 10 mL of ddH2O the solution was degassed with nitrogen. Cu(I)Br (3.8 mg,

2.68*10-4 mmol) was added to the mixture.

-2 GFP-wt, 0 and 3 hr ATRP reaction Monomer, OEOMA300 (21 mg, 6.9*10 mmol) was added to

100 μL of GFP-wt (10.2 mg, 3.4*10-2mmol). This solution was degassed with nitrogen for 20 min. and then degassed initiator solution (250 μl) was added to the reaction mixture. The zero time point was removed and the reaction was sealed and mixed for 3 hours then quenched by exposure to air.

-2 GFP-1, 0 and 3 hr ATRP reaction Monomer, OEOMA300 (10 mg, 3.42*10 mmol) was added to

100 μL of GFP-1 (6 mg, 2.14*10-4 mmol). This solution was degassed with nitrogen for 20 min. then degassed initiator solution (100 μL) was added to the reaction mixture. The reaction was sealed and mixed for 3 hours then quenched by exposure to air.

2A.2.2.9. Characterization of ATRP grafting from GFP-wt and GFP-1 (Mehl –Labratory)

All reactions were diluted to 3 mL with PBS and concentrated to 300 μL using a 3000 MWCO centrifuge filter. This process was repeated 3 times for each reaction to insure that all monomer, copper and ligand was removed. All reactions were then diluted to 1.0 mg protein/mL PBS after protein concentration was assessed using BCA protein assay. Crude samples for each reaction (5

14 | P a g e

μg) was analyzed by SDS-PAGE. A 4-12% gel was run at 150 V and was stained with

Coomassie blue.

To verify that the polymers grown from the surface of GFP-1 did not significantly affect the structure of the protein fluorescence/protein concentration ratios were compared. All of the reactions were diluted to 1.0  0.1 mg/mL based on a BCA protein assay. Fluorescence measurements were collected using a HORIBA Jobin Yvon FluoroMax®-4. The emission from

500 to 520 nm (1 nm bandwidth) was summed with excitation at 488 nm (1 nm bandwidth).

Samples were prepared by diluting 100-fold with PBS. Results of fluorescence measurements are shown in Table 2A.2.2.9.1 For SEC analysis 0.1 mg of each reaction was separated on a

Superdex 200 SEC column at a flow rate of 0.8 mL/min in PBS. The samples were monitored at

230 nm and 280 nm Five major fractions were collected from the SEC separation and concentrated for analysis by SDS-PAGE.

Table 2A.2.2.9.1. Fluorescent measurements of GFP and GFP-polymer hybrids

ATRP Reaction GFP Fluorescence GFP-wt, 0 hr 7.6 x 106 GFP-wt, 3 hr 8.6 x 106 GFP-1, 0 hr 7.2 x 106 GFP-1, 3 hr 8.1 x 106

2A.2.3 Results and Discussion

Despite the importance of protein-polymers, there is no general method for producing homogeneous recombinant proteins that contains polymer initiators at defined sites.14 To address this deficiency, we designed the amino acid, 4-(2’-bromoisobutyramido)phenylalanine 1 (Figure

15 | P a g e

2A.2.3.1, Scheme 2A.2.3.1), since it should function as an initiator in atom transfer radical polymerization (ATRP)15-17 and would provide a stable linkage between the protein and growing polymer. We synthesized 1 and evolved a Methanococcus jannaschii (Mj) tyrosyl-tRNA

18 synthetase/tRNACUA pair to genetically encode this initiator in response to an amber codon. To demonstrate the utility of this initiator we produced Green Fluorescent Protein with 1 site- specifically incorporated on its surface (GFP-1). Purified GFP-1 was then used as an initiator under standard ATRP conditions with the monomer, oligo (ethylene oxide) monomethyl ether methacrylate (OEOMA300), efficiently producing a pOEOMA-GFP bioconjugate where the polymer is connected at the selected site on GFP.

Figure 2A.2.3.1 Genetic incorporation of ATRP initiator into proteins. (A) Initiator 4-(2’- bromoisobutyramido)phenylalanine 1. (B) The evolved MjRS/tRNACUA pair in pDule-BIBAF allows for site-specific incorporation of 1 in response to an amber codon. Lane 2 shows expression levels of GFP-wt from pBad-GFP-His6. Production of GFP-1 from pBad-GFP- 134TAG-His6 is dependent on 1 in the growth media, lane 3 without 1 present, lane 4 with 1 mM 1 present. Protein was purified by Co+2 affinity chromatography, separated by SDS-PAGE and stained with Coomassie.

16 | P a g e

Scheme 2A.2.3.1 Synthesis of 4-(2’-bromoisobutyramido)phenylalanine (1) It is important to synthesize 1 in large quantities since relatively large quantities of initiator- containing protein are needed for polymerization experiments. The initiator 1 was synthesized in two steps in 63% yield from commercially available. To evolve the orthogonal

MjTyrRS/tRNACUA pair capable of incorporating 1 in response to an amber codon, we used a library of the synthetase (RS) gene that was randomized for the codons corresponding to six active-site residues (Y32, L65, F108, Q109, D158, I159) within 7 Å of the bound tyrosine.18 We performed two rounds of alternating positive and negative selection on this library. The clones that survived the selection were transformed into cells with a plasmid containing a GFP gene interrupted with an amber codon.19, 20 92 colonies were assessed for UAA-dependent expression of GFP. The top eight performing clones showed greater than 400 mg/L of GFP-1 expression in the presence of 1 and no detectable GFP fluorescence over background in the absence of 1

(Figure 2A.2.2.5.1.). Sequencing these eight clones revealed six different RS sequences (Table

2A.2.2.5.1).

For further characterization of the incorporation of 1 into proteins in response to the amber codon, the most active RS was cloned into a pDule vector that contains one copy of Mj tRNACUA to create pDule-BIBAF.19-21 Expression of GFP gene interrupted by an amber codon at site 134 in

17 | P a g e the presence of pDule-BIBAF was efficient and dependent on the presence of 1 Figure

2A.2.3.1B. Using 1 mM 1, 0.42 g of GFP-1 was purified per liter of media, while GFP-wt yielded 1.27 g/L under similar conditions (no GFP is produced in the absence of 1). To further demonstrate that 1 can be incorporated into recombinant proteins using pDule-BIBAF, we compared the masses of GFP-1 to GFP-wt using ESI-Q-Tof mass analysis to verify that only a single 1 is incorporated in GFP (Figure 2A.2.3.2). The site of 1 incorporation was confirmed by analysis of the tandem mass spectrometry (MS/MS) fragmentation series of the relevant tryptic peptide (Figures 2A.2.3.3 and Figure 2A.2.3.4). Overall, the results of protein expression with affinity purification, SDS-PAGE, and MS analysis demonstrate the high fidelity and efficient incorporation of 1 at a genetically programmed site in GFP using pDule-BIBAF.

18 | P a g e based removal of N-terminal methionines and +22 sodium adducts. No other peaks were observed that would correlate with background incorporation of a natural amino acid.

A. Experimental B. Predicted Relative Relative Intensity

m/e

Figure 2A.2.3.3. Isotopic abundance patterns indicate the presence of bromine. (A) Experimental 2+ isotopic pattern for [M+2H] at 589-592 Da. (B) Predicted isotopic pattern for [C50H77N14O14Br + 2H]2+ as derived from various on-line isotopic pattern generators

19 | P a g e

E(BIBAF)GNILGHK

Figure 2A.2.3.4. MS/MS spectrum of 589 Da. The signal at 580 Da retains the characteristic isotopic pattern associated with the presence of bromine in a 2+ charge state, and is consistent with the doubly charged species resulting from the loss of water, [M-18 + 2H]2+. Loss of water is a recognized low-energy fragmentation pathway for N-terminal glutamic acid peptides The isotopic pattern for the peak at 540 Da indicates a +2 charge absent bromine and is consistent with the loss of HBr from the side chain 4-(2’-bromoisobutyramido)phenylalanine. ATRP’s tolerance of mild temperatures and aqueous conditions has been established for a variety of monomers on many biomolecules. The use of 1 as an initiator has never been reported. Since pOEOMA-protein bioconjugates have shown efficient pharmacokinetics and therapeutic

3-6, 12 potency , the monomer OEOMA300 was selected to demonstrate that the genetically incorporated initiator 1 can be used in ATRP. The GFP-1 initiator and OEOMA300 monomer were degassed with nitrogen and the reaction was initiated by adding a degassed stock solution

20 | P a g e of 2,2’-bipyridine and Cu(I)Br/Cu(II)Br2 (see supporting information for details). The ATRP performed in degassed PBS buffer at 24°C were quenched at different time points to assess polymer growth by SDS-PAGE and SEC (Figure 2A.2.3.5) As expected, polymer growth from

GFP-1 was evident by shifts to higher molecular weight (MW) with increasing ATRP reaction time, whereas no change was evident on GFP-wt under ATRP conditions. While the radical process of ATRP can have early stage termination which would lead to residual unreacted GFP-

1, characterization with SEC showed that 93% of the GFP-1 initiator was incorporated into high

MW polymers at 180 min (Figure 2A.2.3.5 lane 7, Figure 2A.2.3.5 green trace). To verify that the polymers grown from the surface of GFP-1 did not significantly affect the structure of the protein we compared fluorescence/protein concentration ratios, since GFP fluorescence intensity correlates with the structural integrity of the protein (Table 2A.2.2.9.1.)22. To further characterize the pOEOMA-GFP bioconjugate, the 180 min. reaction was fractionated by SEC, yielding purified protein-polymers of different sizes. The fractionated soluble fluorescent pOEOMA-GFP samples exhibited the expected MW increase when characterized by SDS-PAGE

( Figure 2A.2.3.6 and 2A.2.3.7).

21 | P a g e

Figure 2A.2.3.5. Characterization of ATRP grafting from GFP-wt and GFP-1 with OEO300MA monomer in PBS at 24°C. (A) SDS-PAGE of crude time points (5 µg of protein was loaded on each lane of a 4-12% gel). The reaction produced no size change for GFP-wt (Lane 2 and 3), while the majority of GFP-1 showed significant size increases with increasing ATRP reaction time (Lane 4-7). (B & C) SEC of 0.1 mg of desalted reaction time-points on Superdex 200 at flow rate of 0.8 mL/min of PBS buffer monitored at 230 nm. B. GFP wt eluted at the expected volume of 17.3 mL (black) and was unaltered by the ATRP reaction (offset green). (C) SEC of GFP-1 ATRP reaction show the protein significantly increasing in size. (black, time=0; green, time=180 min).

22 | P a g e

SEC of GFP-1 ATRP reactions 390

1 2 3 4 5 290

190

Abs 230 nm (mAU) nm Abs 230

-10 200 300 400 500 600 700 5.2 7.9 10.5 13.2 15.8 18.5

Elution volume (mL)

23 | P a g e

Figure 2A.2.3.7. SDS PAGE analysis of SEC fractionated ATRP reactions. ATRP reactions were separated by SEC, (Figure 4), and the individual fractions were concentrated and separated on a 4-12% gel and stained with Coomassie. The full 3 hr reaction mixture is in lane 6 and fractions 1-5 are in lanes 1-5 respectively. The GFP-polymer hybrid’s change in size is due to polymer addition as indicated by SEC fractionation and SDS-PAGE separation.

2A.2.4 Conclusions and Outlook

A general method for the quantitative, site-specific incorporation of a polymer initiator, 1, into recombinant proteins. While other reports have shown the ability to control and manipulate polymer growth from proteins,3, 12, 17 this method overcomes the technical challenges of attaching an initiator to the protein of interest prior to polymerization and provides facile access to a diversity of sites on proteins. GFP-wt was unable to grow polymers but the single addition of 1 at site 134 allowed for efficient polymer growth. We have demonstrated the utility of this new initiator in a protein by growing polymers of OEOMA300 from GFP-1 and showing that attached polymer does not affect the general structure or solubility of GFP. The resulting amide linkage between the protein and polymer should be stable to drug delivery and material science applications. While we have shown that this initiator on GFP functions well for generating protein-polymers in aqueous conditions by standard ATRP chemistry it should also function with

24 | P a g e other controlled radical polymerization agents as well5, 6, 8. Our future efforts focus on application of this method for preparation and study of unique protein-polymer hybrid materials and pharmaceuticals.

2A.2.5 References

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Concepts to Regulate Functions in Polymer Science. Journal of Polymer Science Part A-

Polymer Chemistry 48, 1-14 (2010).

2. Depp, V., Alikhani, A., Grammer, V. & Lele, B.S. Native protein-initiated ATRP: A

viable and potentially superior alternative to PEGylation for stabilizing biologics. Acta

Biomaterialia 5, 560-569 (2009).

3. Gao, W.P. et al. In situ growth of a stoichiometric PEG-like conjugate at a protein's N-

terminus with significantly improved pharmacokinetics. Proceedings of the National

Academy of Sciences of the United States of America 106, 15231-15236 (2009).

4. Krishna, O.D. & Kiick, K.L. Protein- and Peptide-Modified Synthetic Polymeric

Biomaterials. Biopolymers 94, 32-48 (2010).

5. Lutz, J.-F. & Boerner, H.G. Modern trends in polymer bioconjugates design. Progress in

Polymer Science 33, 1-39 (2008).

6. Nicolas, J., Mantovani, G. & Haddleton, D.M. Living radical polymerization as a tool for

the synthesis of polymer-protein/peptide bioconjugates. Macromolecular Rapid

Communications 28, 1083-1111 (2007)

7. Connor, R.E. & Tirrell, D.A. Non-canonical amino acids in protein polymer design.

Polym Rev 47, 9-28 (2007).

25 | P a g e

8. Liu, J.Q. et al. In situ formation of protein-polymer conjugates through reversible

addition fragmentation chain transfer polymerization. Angewandte Chemie-International

Edition 46, 3099-3103 (2007).

9. Zeng, Q.B. et al. Chemoselective derivatization of a bionanoparticle by click reaction and

ATRP reaction. Chemical Communications, 1453-1455 (2007).

10. Heredia, K.L. et al. In situ preparation of protein - "Smart" polymer conjugates with

retention of bioactivity. Journal of the American Chemical Society 127, 16955-16960

(2005).

11. Le Droumaguet, B. & Velonia, K. In situ ATRP-Mediated hierarchical formation of giant

amphiphile bionanoreactors. Angewandte Chemie-International Edition 47, 6263-6266

(2008).

12. Lele, B.S., Murata, H., Matyjaszewski, K. & Russell, A.J. Synthesis of Uniform

Protein−Polymer Conjugates. Biomacromolecules 6, 3380-3387 (2005).

13. Canalle, L.A., Lowik, D.W.P.M. & van Hest, J.C.M. Polypeptide-polymer bioconjugates.

Chemical Society Reviews 39, 329-353 (2010)

14. Broyer, R.M., Quaker, G.M. & Maynard, H.D. Designed amino acid ATRP initiators for

the synthesis of biohybrid materials. Journal of the American Chemical Society 130,

1041-1047 (2008).

15. Wang, J.-S. & Matyjaszewski, K. Controlled/"living" radical polymerization. atom

transfer radical polymerization in the presence of transition-metal complexes. Journal of

the American Chemical Society 117, 5614-5615 (1995).

16. Matyjaszewski, K. & Xia, J. Atom transfer radical polymerization. Chemical Reviews

101, 2921-2990 (2001).

26 | P a g e

17. Matyjaszewski, K. & Tsarevsky, N.V. Nanostructured functional materials prepared by

atom transfer radical polymerization. Nature Chemistry 1, 276-288 (2009).

18. Xie, J. & Schultz, P.G. An expanding genetic code. Methods 36, 227-238 (2005).

19. Miyake-Stoner, S.J. et al. Generating Permissive Site-Specific Unnatural Aminoacyl-

tRNA Synthetases. Biochemistry 49, 1667-1677 (2010).

20. Stokes, A.L. et al. Enhancing the utility of unnatural amino acid synthetases by

manipulating broad substrate specificity. Molecular Biosystems 5, 1032-1038 (2009).

21. Miyake-Stoner, S.J. et al. Probing Protein Folding Using Site-Specifically Encoded

Unnatural Amino Acids as FRET Donors with Tryptophan. Biochemistry 48, 5953-5962

(2009).

22. Pedelacq, J.D., Cabantous, S., Tran, T., Terwilliger, T.C. & Waldo, G.S. Engineering and

characterization of a superfolder green fluorescent protein. Nature Biotechnology 24, 79-

88 (2006).

27 | P a g e

2.3. Epilogue

The research described in these chapters has garnered much interest in the bioconjugates community and has been featured in several review papers. This work combined protein engineering with polymer chemistry to yield a facile method for preparing protein initiators with exact control over stoichiometry and amino acid residue specificity. Using the methodology developed in Chapter 2 it is expected that new therapeutic protein-polymer hybrids can be developed with enhanced functionality.

95 | P a g e

Chapter 3 Atom Transfer Radical Polymerization Under Biologically Relevant Conditions

The work presented in this section was reformatted from a previous publication: Saadyah Averick, Antonina Simakova, Sangwoo Park, Dominik Konkolewicz, Andrew J. D. Magenau, Ryan A. Mehl, and Krzysztof Matyjaszewski "ATRP under Biologically Relevant Conditions: Grafting from a Protein" ACS Macro Letters, 2012, 1, 6-10 DOI: 10.1021/mz200020c

96 | P a g e

Chapter 3.1. Preface

With the advent of RDRP’s use in bioconjugation reaction there has been a goal to develop novel methodologies to preparing protein polymer hybrids with unique properties compared to simple PEG conjugation. A popular method to preparing PPHs using RDRP is the grafting-from reaction, wherein a protein initiator is prepared and polymer grafted in situ, and although this method has been used to prepare several functional PPHs, it is hampered by a lack of systematic optimization of ATRP in the presence of proteins. To provide a method to control the growth of polymers from proteins using ATRP, a set of conditions for ATRP under biologically relevant conditions was developed. These conditions were defined as: 1) Aqueous buffered media containing no more than 20% organic content (i.e. monomer or cosolvent), 2) Dilute protein-initiator concentration (maximum 5 mg/ml ~0.1-1 mM initiator), 3) Ambient reaction temperatures and 4) Copper ligand species that are stable in the presences of proteins. An additional limitation to preparing well-defined protein-polymer hybrids by grafting-from is the inability to directly characterize the polymer grafted from a protein due to the stable linkage between the protein and polymer. To overcome this challenge an ester based activated ester initiator was prepared and reacted to BSA (BSA-O-[iBBr]30) yielding base cleavable linkages to cleave the polymer from the protein. Using these 4 parameters as a guide Normal and AGET ATRP were optimized in a model system using

PEO2000-iBBr and BSA-O-[iBBr]30. After careful optimization conditions were found, the preparation of well-defined proteins-polymer conjugates by grafting from protein initiators was achieved.

97 | P a g e

A special thanks to my collaborator and labmate Antonina Simakova who conducted and optimized the AGET ATRP conditions used to graft from BSA-O-

[iBBr]30.

98 | P a g e

Chapter 3.2.

ATRP under Biologically Relevant Conditions:

Grafting from a Protein

3.2.1. Introduction

Controlled/living radical polymerizations (CRPs) allow the synthesis of polymers with predefined molecular weights, compositions, architectures, and narrow molecular weight distributions.1 Atom transfer radical polymerization (ATRP) is among the most extensively studied and robust CRP techniques, because it is compatible with a variety of functional monomers and reaction conditions and gives polymers with high chain-end functionality.2, 3 ATRP, along with other CRP techniques, can be used to prepare biohybrids, where synthetic polymers are linked to biomolecules such as peptides, proteins, nucleic acids, and carbohydrates.4

Protein–polymer hybrids (PPHs) are of particular interest in academic and industrial research since they offer improved pharmacokinetics as well as enhanced physical and proteolytic stability.5 Commonly, PPHs are composed of poly(ethylene oxide) (PEO or PEG) polymer segments. Recently, however, a new generation of stimuli responsive polymers are being synthesized and conjugated to proteins to give “smart”

PPHs. For instance, a smart PPH can be made by conjugating thermoresponsive polymers, such as poly(N-isopropylacrylamide) or poly(oligo(ethylene oxide) methacrylate), to a protein.6-11

99 | P a g e

Preparation of well-defined PPHs can be achieved by two methods: “grafting to”

(g-t) and “grafting from” (g-f).9, 12, 13 The g-t approach links a preformed polymer bearing a reactive chain-end to a complimentarily functionalized protein,5, 8, 14-17 whereas the g-f process grows the polymer directly from an initiating site on a protein.18, 19 The g-f method leads to high yields and facile purification of the resulting hybrid,12, 13 although modification of the protein with initiating moieties is required.9, 12 In the g-f method,

ATRP initiating sites (i.e. 2-bromoisobutyrate (iBBr)) can be attached to protein either covalently19 or through strong complexation.20 This technique has been applied to create a variety of PPHs.21-23 Recently, the g-f approach has been extended to activators generated by electron transfer (AGET) ATRP by utilizing ascorbic acid (AA) as reducing agent to synthesize PPHs.18, 24, 25

These advances in the g-f approach have created new opportunities to create innovative therapeutic and diagnostic systems. However, as reported in the literature,12, 26 control over the g-f process is challenging. ATRP in water has previously encountered difficulties from a very high activation rate, dissociation of halide from the X-Cu(II) deactivating species, decreased stability of Cu/ligand complexes, disproportionation of

Cu(I) and hydrolysis of carbon–halogen bonds.27, 28 Typically, polymers grafted from proteins have broad molecular weight distributions (Mw/Mn), substantial tailing to low molecular weights and low initiation efficiencies. Thus, a general set of polymerization conditions for the preparation of PPHs has yet to be established. For the g-f method to become a widely accepted methodology for preparation of PPHs, these challenges must be addressed and optimized conditions must be established. Herein, ATRP methodologies are described for the synthesis of PPHs in aqueous media using the g-f approach under

100 | P a g e biologically relevant conditions (Scheme 3.2.1). The reaction conditions were selected to maintain the protein’s tertiary structure while providing a well-controlled polymerization.

For protein stability, polymerizations were conducted at near ambient temperatures (30

°C), in dilute protein solutions (0.1–3.0 mg/mL), and with at least 80% water content by volume. For analytical purposes, a cleavable ATRP initiator was attached to BSA to facilitate direct gel permeation chromatography (GPC) analysis of the detached polymers

(Scheme 3.2.1). This study investigates the effect of ligand, copper halide and organic cosolvent, and optimizes ATRP under the previously defined biologically relevant conditions for both normal ATRP and AGET ATRP processes. Finally, the conditions developed in this work were used to synthesize a well-defined “smart” PPH which exhibited LCST behavior.

Scheme 3.2.1. Synthesis of PPHs via (AGET) ATRP from [BSA-O-iBBr]30 and Selective Cleavage of Polymer 3.2.2 Experimental

3.2.2.1. Materials and Instrumentation

101 | P a g e

Oligo(ethylene oxide) monomethyl ether methacrylate (average molecular weight ~475,

~300, 188 g/mol, OEOMA475, OEOMA300, MEO2MA respectively), BSA, mono-tert- butyl succinate, N-hydroxysuccinimide (NHS), trifluoroacetic acid, bromoisobutyryl bromide, 2,2’-bipyridine (bpy), , N-(n-propyl)pyridylmethanimine (PI), ascorbic acid

(AA), CuCl, CuCl2, CuBr, and CuBr2 were purchased from Aldrich in the highest available purity. Tris(2-pyridylmethyl)amine (TPMA) was purchased from ATRP

Solutions. Monomers were passed over a column of basic alumina prior to use.

Poly(ethylene oxide) isobutyryl bromide (PEO-iBBr Mn = 2000) was prepared, as previously described.29 GFP was prepared as previously outlined.21 Molecular weight and molecular weight distribution (Mw/Mn) were determined by GPC. The GPC system used a

Waters 515 HPLC Pump and Waters 2414 Refractive Index Detector using PSS columns

(Styrogel 102, 103, 105 Å) in dimethylformamide (DMF) as an eluent at a flow rate of 1 ml/min at 50 °C and in tetrahydrofuran (THF) as an eluent at a flow rate of 1 mL/min at

35 oC. All samples were filtered over anhydrous magnesium sulfate and neutral alumina prior to analysis. The column system was calibrated with 12 linear polystyrene (Mn = 376

1 ~ 2,570,000). Monomer conversion was measured using H NMR spectroscopy in D2O, using a Bruker Avance 300 MHz spectrometer at 27 C. Thermoresponsivity was measured by dynamic light scattering (DLS) on a Zetasizer from Malvern Instruments,

Ltd. The temperature ramp used in this study was from 15 to 64 C at 1 C intervals.

Samples were equilibrated for 2 minutes before measuring particle size. Tangential flow filtration was conducted on a Labscale TFF system from Millipore. Zebra Spin desalting columns were purchased from Fisher and used according to the manufactures

102 | P a g e instructions. The fluorecence spectra for GFP stability testing were obtained on a Tecan

Safire2 using a 384 well plate.

3.2.2.2. Preparation of NHS ester initiator

Mono-tert-butyl succinate-EBiB: Mono-tert-butyl succinate (1.0 g, 5.7×10-3 mol), hydroxyl-EBiB (1.3 g, 6.3×10-3 mol), EDC-HCl (1.4 g, 7.5×10-3 mol) and DMAP (0.1 g,

5.7×10-4 mol) were added to a 100 ml round bottom flask. The reaction mixture was dissolved in 50 ml of dichloromethane and stirred overnight. The reaction mixture was extracted once with 20 ml of water, twice with 1 N HCl, once with 1 N NaOH, once with water and brine. The organic layer was dried over anhydrous sodium sulfate and solvent was removed under reduced pressure. 1H NMR: 1.44 ppm (s, 9H), 1.93 ppm (d, 6H), 2.56 ppm (m, 4H), 4.36 ppm (s, 4H).

COOH- tert-butyl succinate-EBiB: Mono-tert-butyl succinate-EBiB (1.0 g,

2.7×10-3 mol) was dissolved in 50 ml of dichloromethane and trifloroacidic acid (TFA,

2.1 ml, 2.7×10-2 mol) was added dropwise to the reaction mixture. The reaction was stirred for 36 hours, and subsequently extracted 3 times with 30 ml of water and once with brine. The organic phase was dried over anhydrous sodium sulfate, filtered and the solvent was removed under reduced pressure. 1H NMR: 1.94 ppm (d, 6H), 2.68 ppm (m,

4H), 4.38 ppm (s, 4H).

NHS-ester initiator (1): Mono-tert-butyl succinate-EBiB (0.8 g, 2.69×10-3 mol),

EDC-HCl (0.8 g, 4.0×10-3 mol) and NHS (0.5 g, 4.0×10-3 mol) were dissolved in 10 ml of CHCl3 and stirred for 16 hours. 40 ml of ethyl acetate and 30 ml of water were then added to the reaction mixture and stirred for 10 minutes. The organic phase was separated

103 | P a g e and the aqueous phase was washed 3 times with 20 ml of ethyl acetate. The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered and the solvent was removed under reduced pressure. The NHS activated initiator was directly used to modify BSA.

3.2.2.3. Synthesis of BSA-O-[iBBr]30

NHS-ester initiator (1) (1.0 g, 2.5 mmol) was dissolved in 2 ml of DMSO. BSA (1.0 g,

0.5 mmol Lys) was dissolved in 500 ml of 0.1 M PBS (pH 7.4) and 1 was added dropwise. The reaction was stirred overnight and purified using tangential flow filtration with a 30-kDa molecular weight cut off membrane. 15 dia-volumes of water were used to purify BSA-O-[iBBr]30.

3.3.2.4. Polymer cleavage from protein

Polymers were cleaved from proteins by adding 200 l of reaction mixture to 200 l of

5% KOH solution and left at room temperature for 2 hours.

3.2.2.5. Synthesis of POEOMA by ATRP from PEO2000iBBr/BSA-O-[iBBr]30

-3 -3 PEO2000iBBr (10.0 mg 5×10 mmol) or BSA-O-[iBBr]30 (12.5 mg (protein), 5.0×10 mmol (initiator)) was dissolved in 3.5 ml of Millipore water and placed in a 10 ml

Schlenk flask. OEOMA475 (476.2 mg, 1.1 mmol) and 50 l of DMF (internal standard for

NMR) were added dropwise to the initiator solution. The flask was sealed and bubbled for 20 min, while stirring, with nitrogen to deoxygenate the reaction mixture. After the solution was deoxygenated, 1 ml of catalyst stock solution was added via gastight syringe to the reaction mixture to initiate polymerization. The polymerization was carried out at

104 | P a g e

30 C. Samples were taken at allotted times throughout the reaction for GPC and NMR analysis. Stock solutions of CuX/L were prepared in 10 ml of deoxygenated ultra pure

-1 water as follows: X=Br, L=bpy: CuBr (7.2 mg, 0.5×10 mmol), CuBr2 (101.0 mg,

4.5×10-1 mmol), and bpy (164.3 mg, 1.1 mmol). X=Cl, L=bpy: CuCl (5.0 mg, 0.5×10-1 mmol), CuCl2(60.5 mg, 0.3 mmol) and bpy(164.3 mg, 1.1 mmol). X=Br, L=TPMA:

-1 CuBr (7.2 mg, 0.5×10 mmol), CuBr2 (101.0 mg, 0.5 mmol), and TPMA (160.0 mg, 5.5

-1 mmol). X=Cl, L=TPMA: CuCl (5.0 mg, 0.5×10 mmol), CuCl2(60.5 mg, 0.3 mmol) and

TPMA (160.0 mg, 5.5 mmol).

3.2.2.6. AGET ATRP from PEO-iBBr/BSA-O-[iBBr]30

-1 -1 PEO2000iBBr (40.0 mg 0.2×10 mmol) or BSA-O-[iBBr]30 (50.0 mg (protein), 0.2×10 mmol (initiator)), OEOMA475 (2.0 ml, 4.5 mmol), CuBr2 (44.6 mg, 0.2 mmol), and

TPMA (63.8 mg, 0.2 mmol) were dissolved in 18.4 ml of pure water and charged into a

25 ml Schlenk flask. 0.4 ml of DMF was added as internal standard. Next, the reaction mixture was purged with N2 for 20 minutes then placed in an oil bath at 30 C. Then AA was added either at the beginning of the reaction, or slowly fed in via a syringe pump.

3.2.2.7. Synthesis of POEOMA by ATRP in DMSO/water from BSA-O-[iBBr]30

-3 BSA-O-[iBBr]30 (12.5 mg (protein), 5.0×10 mmol (initiator)) was dissolved in 3 ml of

Millipore water and added to a 10 ml Schlenk flask. OEOMA475 (476.2 mg, 1.1mmol) and 50 l of DMF (internal standard for NMR) were dissolved in 500 l of DMSO and added dropwise to the stirring protein solution (to avoid protein precipitation). The flask was sealed and bubbled for 20 min, while stirring, with nitrogen to deoxygenate the reaction mixture. After the solution was deoxygenated, 1 ml of catalyst stock solution

105 | P a g e was added to the reaction mixture to initiate polymerization. The polymerization was carried out at 30 C. Samples were taken several times throughout the reaction for GPC and NMR analysis.

3.2.2.8. Synthesis of P(MEO2MA-co-OEOMA475) from BSA-O-[iBBr]30

-3 BSA-O-[iBBr]30 (12.5 mg (protein), 5.0×10 mmol (initiator)) was dissolved in 3 ml of

Millipore water and added to a 10 ml Schlenk flask. OEOMA475 (144.7 ml, 0.5 mmol),

MEO2MA (92.5 ml, 0.5 mmol) and 50 l of DMF were dissolved in 800 l of DMSO and added dropwise to the well stirred protein solution (to avoid protein precipitation).

The flask was sealed and nitrogen was bubbled for 20 min, with stirring to deoxygenate the reaction mixture. After the solution was deoxygenated, 1 ml of catalyst stock solution

(X=Cl, L=bpy) was added to the reaction mixture to initiate polymerization. Samples were taken at several times throughout the reaction for GPC and NMR analysis. Prior to

DLS analysis samples were passed through a Zebra Spin desalting column to remove solvent, monomer and catalyst species.

3.2.2.9. Stability of GFP under polymerization conditions

-2 The following amounts of ligand were complexed with CuCl2 (5.0 mg, 3.7×10 mmol) respectively: PI(12.2 mg, 8.2×10-2 mmol), bpy(12.8 mg, 8.2×10-2 mmol) and TPMA

-2 (11.9 mg, 4.1×10 mmol). The CuCl2/L were precomplexed in 200 l of water and an additional solution of CuCl2 was prepared in 200 l of water. Once clear solutions of

CuCl2/L and CuCl2 were obtained they were added to 2 ml of GFP solution (1 mg/ml) in

PBS with 10% OEOMA475. The solutions were centrifuged at 4000 r.p.m. to remove any particulates formed (only observed for free CuCl2 and CuCl2/PI). 100 l of each solution

106 | P a g e was removed for analysis using a Tecan Safire2 plate reader with a 384 well plate.

3.2.3. Results and Discussion

Successful g-f ATRP requires the protein to be stable in the presence of Cu complexes.30

The stability of green fluorescent protein (GFP) was evaluated in the presence of various copper-ligand complexes under biologically relevant polymerization conditions (1 mg/mL GFP, 10% monomer in 0.1 M PBS). Initially, three ligands (L) with different activities were selected ranging from strongly activating tris(2-pyridylmethyl)amine

(TPMA), moderately activating 2,2′-bipyridine (bpy), and weakly activating N-(n- propyl)pyridylmethanimine (PI).31

Fluorescence measurements showed that TPMA and bpy CuCl2 complexes had minimal influence on the GFP’s tertiary structure, as indicated by retention of its original emission spectra (Figure 3.2.3.1). In contrast, both CuCl2 or CuCl2/PI species caused the protein to denature, as seen by a 100 fold decrease in GFP’s original fluorescence intensity. Based on these results, bpy and TPMA were selected for the development of

ATRP under biologically relevant conditions.

105 GFP only 40 GFP GFP/TPMA GFP/bpy GFP/TPMA

) 4 GFP/PI

-3 10 30 GFP/bpy GFP/CuCl 2 GFP/PI

x 10 ( GFP/CuCl

20 2

103 Intensity

Intensity 10 102 0 500 525 550 575 600 625 Emmision (=511 nm) Wavelength / nm

107 | P a g e

Figure 3.2.3.1. Effect of CuCl2:L on GFP (1 mg/ml) stability. [CuCl2]/[L]=1/[bpy],[PI] and [TPMA] = 2.2 and 1.1 [CuCl2]=19 mM, [OEOMA475]0 = 0.23 M

To directly analyze the polymer grafted from BSA, the protein was modified with a cleavable ester initiator, designated as BSA-O-[iBBr]30 (Scheme 3.2.3.1, Figure

3.2.3.2). MALDI-ToF analysis of the initiator modified BSA (BSA-O-[iBBr]30) showed an increase in molecular mass by 9.3 kDa compared to the native BSA. This indicates that about 30 initiating sites were added to BSA and no native protein remained. The ester bond linking the initiator to the protein can be selectively cleaved after polymerization using 5% KOH (w/v) solution, without affecting the oligo(ethylene oxide) methyl ether side chains,32 facilitating direct analysis of the g-f polymer by GPC.

Scheme 3.2.3.1. Preparation of the NHS-ester initiator.

108 | P a g e

1.0 BSA BSA-O-iBBr 0.8

0.6

0.4

Intensity (AU)

0.2

0.0 40 50 60 70 80 90 Mass (kDa)

Figure 3.2.3.2. MALDI-TOF spectra of BSA and BSA-O-[iBBr]30.

Initially, normal ATRP was used to synthesize PPHs composed of a BSA protein and POEOMA polymer. Previous work illustrated that in order to achieve a successful

ATRP in protic solvents a high concentration of CuX2 deactivator is required, due to a high activation rate and partial dissociation of X-Cu(II) bond in the deactivator.28 Based on these findings, our system was formulated with 10% of the total copper in the form of

Cu(I). Initial screening experiments were performed with a PEO-macroinitiator (PEO2000- iBBr, degree of polymerization PEO = 45; Scheme 3.2.4.2) and subsequently extended to the protein macroinitiator system (BSA-O-[iBBr]30; Scheme 3.2.1). OEOMA475 was polymerized by ATRP with a monomer volume fraction <20%, using the following molar ratios of [OEOMA475]/[I]/[CuX]/[CuX2] = 455(227)/1/1/9 and CuX to ligand of 1/22 and

1/11 for bpy and TPMA, respectively.

109 | P a g e

Scheme 3.2.3.2. (AGET) ATRP of OEOMA475 from PEO2000iBBr under biologically relevant conditions.

The effect of copper halide on polymerization control was investigated by using either CuBr/CuBr2 or CuCl/CuCl2. Table 3.2.3.1 presents detailed experimental conditions and polymerization results, and Figure 3.2.3.3 shows the first order kinetic plot for g-f BSA-O-[iBBr]30, number average molecular weight (Mn) versus conversion and molecular weight distribution (Mw/Mn) versus conversion. In addition, Figure

3.2.3.3.C shows the GPC traces for normal ATRP using copper chloride salts and bpy.

ATRP initiated by PEO2000-iBBr Figure 3.2.3.4 with both the PEO and BSA initiators essentially displaying the same behavior. In all cases, the CuX/bpy system provided better control and allowed significantly higher conversions than the CuX/TPMA system.

Kinetic analysis revealed that the CuX/TPMA system reached about 5% conversion in the early stages of polymerization, after which point, the polymerization stopped. These results suggest that the TPMA based catalyst is too active, leading to a very high concentration of radicals and significant termination in the early stages of polymerization.

In addition to the ligand, the halide species had a significant impact on polymerization behavior, especially for the CuX/bpy systems. Polymerization kinetics catalyzed by

CuCl/bpy showed a more linear semilogarithmic plot, and narrower polymer distributions compared to the CuBr/bpy system (Figures 3.2.3.3 and 3.2.3.5). This behavior can be partially attributed to the increased activity of the Br-based initiators/chain ends compared to their corresponding Cl analogs, leading to a larger fraction of terminated

110 | P a g e chains.31 Therefore, the optimal condition and results for g-f using normal ATRP are shown in Table 3.2.3.1 (entry 6) and presented in Figure 3.2.3.3 (orange data series and

GPC traces).

Table 3.2.3.1. Experimental conditions and results for normal ATRP from PEO2000-iBBr and BSA-O-[iBBr]30 M ×10- M ×10- M/I/CuX/CuX /L I L X Solvent Time/h Conv./% n,theo n,GPC M /M 2 3 3 w n

1 455/1/1/9/22 PEO bpy Br H2O 4 45 94 108 1.54

2 455/1/1/9/22 PEO bpy Cl H2O 4 27 55 58 1.16

3 455/1/1/9/11 PEO TPMA Br H2O 4 2 40 12 1.27

4 455/1/1/9/11 PEO TPMA Cl H2O 4 2 36 18 1.22

5 227/1/1/9/22 BSA bpy Br H2O 3.5 75 81 73 1.54

6 227/1/1/9/22 BSA bpy Cl H2O 3.5 70 75 70 1.15

7 227/1/1/9/11 BSA TPMA Br H2O 4 5 5 40 1.10

8 227/1/1/9/11 BSA TPMA Cl H2O 4 2 2 35 1.16

9 455/1/1/9/22 BSA bpy Cl DMSO/H2O 3.5 75 81 80 1.22

10 200/1/1/9/22 BSA bpy Cl DMSO/H2O 3.5 82 40 58 1.22 11 227/1/1/9/22 PEO bpy Cl PBS 3 6 6 10 1.19 12 227/1/1/9/22 PEO bpy Br PBS 3 33 36 28 1.19 13 227/1/1/9/22 BSA bpy Br PBS 3 40 43 50 1.26

[I] = 1 mM; 30 °C; entry 1-4,9: 20% [M] (v/v); entry 5-8, 10-13: 10% [M] (v/v); M = OEOMA475, except entry 10: [OEOMA300]/[MEO2MA] = 1/1

(a) (b) M (x10-3) n M /M 80 CuBr/bpy w n 1.5 CuCl/bpy CuBr/TPMA 2.5 CuCl/TPMA ) 60

1.0 /[M]

0 2.0 40

[M] CuBr/bpy ( CuCl/bpy ln 0.5 CuBr/TPMA 1.5 CuCl/TPMA 20

0.0 0 1.0 0 1 2 3 4 0 20 40 60 80 Time / h Conversion / %

(c)

0.5h M :32000, M /M :1.12 n w n 1h M :46000, M /M :1.19 n w n 1.5h M :55000, M /M :1.16 n w n 2h M :62000, M /M :1.14 n w n 3.5h M :70000, M /M :1.15 n w n

104 105 Molecular Weight Figure 3.2.3.3. Effect of ligand (L = bpy or TPMA) and halide (X = Br or Cl) on ATRP of OEOMA475 from BSA-O-[iBBr]30 at 30 °C. (A) First order kinetic plot and (B) Mn and

111 | P a g e

Mw/Mn versus conversion plot (C) GPC traces for CuCl/CuCl2/bpy. [OEOMA475]0 = 0.21 M; [OEOMA475]/[I]/[CuX]/[CuX2]/[L] = 227/1/1/9/11 ([L]: [TPMA] = 2[bpy]).

(A) (B) M (x10-3) M /M n w n 0.8 CuBr/bpy CuBr/TPMA : CuBr/bpy CuCl/bpy CuCl/TPMA 100 : CuCl/bpy : CuBr/TPMA 2.5 0.6 : CuCl/TPMA 80

/[M] 2.0

0 0.4 60

ln[M] 40 0.2 1.5 20 0.0 0 1.0 0 1 2 3 4 0 10 20 30 40 50 Time / h Conversion / %

(C) (D) 0h PEO -Br MI 0h 2000 M : 2000 n 0.5h M : 17300 M /M : 1.28 0.5h n w n M : 77400 M /M : 1.16 n w n 1h M : 24500 M /M : 1.23 1h n w n M : 95200 M /M : 1.32 n w n 1.5h M : 31200 M /M : 1.22 2h n w n M : 103900 M /M : 1.52 n w n 2h M : 37700 M /M : 1.19 3h n w n M : 110300 M /M : 1.70 n w n 3h M : 48800 M /M : 1.16 4h n w n M : 107900 M /M : 1.54 n w n 4h M : 57800 M /M : 1.16 n w n

103 104 105 106 107 103 104 105 106 Molecular Weight Molecular Weight

Figure 3.2.3.4. Effect of copper halide (X=Br or Cl) on ATRP of OEOMA475 under aqueous conditions at 30 °C. (A) First order kinetic plot and (B) Mn and Mw/Mn versus conversion plot; (C) GPC traces for CuBr/bpy, (D) GPC traces for CuCl/bpy. [PEO2000iBBr]0 = 1 mM, [OEOMA475]0 = 0.45 M and [OEOMA475]/[I]/[L]/[CuX]/[CuX2] = 455/1/11/1/9 ([L]: [TPMA] = 2[bpy]).

0.5h M :55000 M /M :1.59 n w n 1h M :68000 M /M :1.57 n w n 1.5h M :72000 M /M :1.45 n w n 2h M :73000 M /M :1.55 n w n 3.5h M :73000 M /M :1.54 n w n

103 104 105 106 Molecular Weight

Figure 3.2.3.5. GPC traces for CuBr/bpy. [BSA-O-[iBBr]30]0 = 1 mM, [OEOMA475]0 = 0.23 M and [OEOMA475]/[I]/[L]/[CuBr]/[CuBr2] = 227/1/11/1/9 ([L]: [TPMA] = 2[bpy]).

112 | P a g e

AGET ATRP was next investigated for the synthesis of PPHs. In this AGET

ATRP approach, the CuX/L activator was generated in situ from oxidatively stable

33 CuX2/L using ascorbic acid (AA). The reaction conditions for AGET ATRP in water from PEO2000-iBBr and BSA-O-[iBBr]30 with AA as a reducing agent are presented in

Table 3.2.3.2 In all experiments, the amount of added AA was from 0.1 to 2.0% of the total amount of CuBr2. A single injection of reducing agent in AGET ATRP showed results similar to that of the TPMA system using normal ATRP. In particular, there was a small monomer conversion in the initial stages, and no further conversion after this initial period (Figures 3.2.3.6 and 3.2.3.7). Despite that monomer conversion was low, the polymer synthesized using TPMA had a lower Mw/Mn value than polymers obtained using bpy (Table 3.2.3.2, entries 1 and 2). Typically, AGET ATRP is performed with the entire charge of reducing agent injected once at the beginning of polymerization.

However, results in this work with PEO2000-iBBr (Figures 3.2.3.8 and 3.2.3.9) and, as in a previous report,34 suggest that continuous and slow feeding of AA in AGET ATRP should lead to lower radical concentrations, diminished termination and promote continued monomer conversion. Figure 3.2.3.6 illustrates that slow addition of 1 mol % of AA (vs CuBr2) leads to 88% conversion in 4 h, compared to 5% conversion when the same amount of AA was added in a single charge. The polymer formed by slowly feeding

AA had high molecular weight and a narrow molecular weight distribution (Mw/Mn =

1.08; Table 3.2.3.2, Figure 3.2.3.6). Because one molecule of ascorbic acid provides two electrons,35 the total amount of AA added after 4 h of polymerization corresponds to no more than 2% of generated Cu(I). Thus, AGET ATRP in water can be used to prepare

113 | P a g e well-defined PPHs using a very small amount of reducing agent added over an extended period of time.

Table 3.2.3.2. Experimental conditions and results for AGET ATRP from PEO2000-iBBr and BSA-O-[iBBr]30 -3 -3 M/I/CuBr2/L /AA I L Solvent Time/h Conv./% Mn,theo×10 Mn,GPC×10 Mw/Mn

1 455/1/10/22/0.1 PEO bpy H2O 6 20 43 25 1.30

2 455/1/10/11/0.1 PEO TPMA H2O 6 15 32 30 1.09

3 455/1/10/11/0.01 PEO TPMA H2O 6 5 11 15 1.09 a 4 455/1/10/11/0.03 PEO TPMA H2O 1 12 26 27 1.10 b 5 227/1/10/11/0.1 PEO TPMA H2O 1.5 78 84 100 1.16

6 227/1/10/11/0.1 BSA TPMA H2O 4 5 5 30 1.10 b 7 227/1/10/11/0.1 BSA TPMA H2O 4 88 95 82 1.08 8 227/1/10/11/0.1b PEO TPMA PBS 4 71 77 93 1.12 9 227/1/10/11/0.1b BSA TPMA PBS 4 75 81 83 1.19 [I] = 1 mM; 30 °C; entry 1-4: 20% [M] (v/v); entry 5-9: 10% [M] (v/v); water, a AA was added stepwise, b AA was fed to the reaction mixture at the rate 8 nmol/min.

(a) (b) 2.5 -3 M (x10 ) M /M Slow feeding n Slow feeding w n One time injection One time injection 2.0 80 2.5

) 1.5 60

2.0

/[M]

1.0 40

[M] (

ln 1.5 0.5 20

0.0 0 1.0 0 1 2 3 4 0 20 40 60 80 100 Time / h Conversion / %

(c) 0.5h M :26400, M /M :1.10 n w n 1h M :40000, M /M :1.15 n w n 1.5h M :54700, M /M :1.13 n w n 2h M :62500, M /M :1.15 n w n 2.5h M :73300, M /M :1.11 n w n 3h M :76200, M /M :1.11 n w n 4h M :83500, M /M :1.08 n w n

104 105 Molecular Weight

Figure 3.2.3.6. Effect of reducing agent feeding on AGET ATRP of OEOMA475 g-f BSA- O-[iBBr]30 at 30 °C (reactions 6 – 7, Table 3). (A) First order kinetic plot and (B) Mn and Mw/Mn versus conversion plot (C) Corresponding GPC traces. Polymerizations conducted with [OEOMA475]0 = 0.21 M and [OEOMA475]/[I]/[TPMA]/[CuBr2] = 227/1/11/10. Rate of feeding of AA 8 nmol/min.

114 | P a g e

(A) (B) 0.4 -3 M (x10 ) M /M bpy n w n 50 bpy 3.0 TPMA TPMA 0.3 40

)

] 2.5

[

0 0.2 30 2.0

[M] ( 20 ln 0.1 1.5 10 0.0 0 1.0 0 1 2 3 4 5 6 7 0 5 10 15 20 25 Time / h Conversion / %

Figure 3.2.3.7. Effect of ligand (L=bpy or TPMA) on AGET ATRP of OEOMA475 under aqueous conditions at 30 °C. (A) First order kinetic plot and (B) Mn and Mw/Mn versus conversion plot. [PEO2000iBBr]0 = 1 mM, [OEOMA475]0 = 0.45 M and [OEOMA475]/[I]/[CuX2]/[AA] = 455/1/10/0.1 ([L]: [TPMA] = 2[bpy]).

(A) (B) 0.20 -3 M /M M (x10 ) w n One initial charge n One initial charge 30 Timed feeding Timed feeding 0.15 2.0

)

]

[

/ 20 0 0.10

[M] ( 1.5

ln 0.05 10

0.00 0 1.0 0.0 0.5 1.0 1.5 0 2 4 6 8 10 12 Time/h Conversion / %

115 | P a g e

(B) (A) -3 M (x10 ) M /M n w n 0.5 50

0.4 40 2.5

0.3 30

/[M] 2.0

0.2 20

ln[M] 1.5 0.1 10

0.0 0 1.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 10 20 30 40 Time / h Conversion / %

(C)

0.3h M :30200, M /M :1.14 n w n 0.5h M :34500, M /M :1.15 n w n 1h M :45800, M /M :1.19 n w n 2h M :49000, M /M :1.31 n w n 3h M :50100, M /M :1.26 n w n

103 104 105 106 Molecular Weight

Figure 3.2.3.9. ATRP of OEOMA475 in PBS, 30 °C. (A) First order kinetic plot and (B) Mn and Mw/Mn versus conversion plot; (C) GPC traces. [PEO2000iBBr]0 = 1 mM, [OEOMA475]0 = 0.23 M and [OEOMA475]/[I]/[bpy]/[CuBr]/[CuBr2] = 227/1/22/1/9.

Polymerization conditions were also investigated for the g-f reaction in phosphate-buffered saline (PBS) solution. PBS (pH = 7.4) is a widely utilized protein buffer36 and served as the reaction medium for the g-f polymerizations. ATRP in PBS is challenging for several reasons. First, copper and phosphate ions can form insoluble

Cu3(PO4)2, and second, chloride anions within the buffer can displace ligands from their copper centers producing a relatively inactive catalyst. To determine optimal conditions for a well-controlled polymerization in PBS, OEOMA475 was polymerized in PBS using both normal ATRP and AGET ATRP. Experimental conditions and polymerization

116 | P a g e results are summarized in Tables 3.2.3.1 (entries 11–13) and Table 3.2.3.2 (entries 8–9) for normal and AGET ATRP, respectively.. The CuCl/CuCl2/bpy catalyzed polymerization in PBS resulted in minimal conversion of monomer after 3 h (Table

3.2.3.1, entry 12), while the CuBr/CuBr2/bpy catalyzed polymerization in PBS (Table

3.2.3.1, entry 11) was approximately 3 times slower than the reaction in water (Table

3.2.3.1, entry 1). Furthermore, the polymerization was well controlled up to 30%, after which time, the semilogarithmic plot became noticeably curved (Figures 3.2.3.10 and

3.2.3.11) Polymers grown from BSA-O-[iBBr]30 in the presence of CuBr/CuBr2/bpy had relatively narrow molecular weight distributions. When AGET ATRP with TPMA ligand was used with slow feeding of AA, there was a linear increase in the first-order kinetic plot up to high monomer conversion for both PEO and BSA macroinitiators (Figures

3.2.3.12 and 3.2.3.13). The feeding rate was the same in both experiments performed in water and PBS. Figure 3.2.3.12B illustrates that AGET ATRP gives a nearly linear increase in molecular weight with conversion while maintaining Mw/Mn below 1.2. The

GPC traces in Figure 3.2.3.12C show monomodal distributions and a gradual shift to higher molecular weights with time. Thus, (AGET) ATRP can be used to create well- controlled PPHs in PBS buffered solutions.

117 | P a g e

(B) (A) -3 M (x10 ) M /M n w n 0.5 50

0.4 40 2.5

0.3 30

/[M] 2.0

0.2 20

ln[M] 1.5 0.1 10

0.0 0 1.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 10 20 30 40 Time / h Conversion / %

(C)

0.3h M :30200, M /M :1.14 n w n 0.5h M :34500, M /M :1.15 n w n 1h M :45800, M /M :1.19 n w n 2h M :49000, M /M :1.31 n w n 3h M :50100, M /M :1.26 n w n

103 104 105 106 Molecular Weight

Figure 3.2.3.10. ATRP of OEOMA475 g-f BSA-O-[iBBr]30 at 30 °C in PBS. (A) First order kinetic plot and (B) Mn and Mw/Mn versus conversion plot. [BSA-O-[iBBr]30]0 = 1mM, [OEOMA475]0 = 0.21 M and [OEOMA475]/[I]/[CuBr]/[CuBr2]/[L] = 227/1/1/9/ 21.

118 | P a g e

(A) (B) M (x10-3) n M /M w n 1.0 70 2.5 0.8 60

] 50

0 0.6

40 2.0

ln[M] 0.4 30 20 1.5 0.2 10 0.0 0 1.0 0 1 2 3 4 0 10 20 30 40 50 60 Time / h Conversion / %

(C) 0.5h M :11800, M /M :1.07 n w n 1h M :15700, M /M :1.09 n w n 2h M :25800, M /M :1.08 n w n 3h M :32500, M /M :1.09 n w n 4h M :36700, M /M :1.09 n w n

103 104 105 Molecular Weight

Figure 3.2.3.11. AGET ATRP of OEOMA475 in PBS, 30 °C. (A) First order kinetic plot and (B) Mn and Mw/Mn versus conversion plot; (C) GPC traces. [PEO2000iBBr]0 = 1 mM, [OEOMA475]0 = 0.23 M and [OEOMA475]/[I]/[TPMA]/[CuBr2]/[AA] = 227/1/22/10/0.2. Rate of ascorbic acid addition was 16 nmol/min.

119 | P a g e

(a) (b)

1.5 -3 M (x10 ) M /M n w n

80 2.5

1.0 ) 60

/[M] 2.0

[M] 40 ( 0.5 ln 1.5 20

0.0 0 1.0 0 1 2 3 4 0 20 40 60

Time / h Conversion / % (c)

0.5h M :31000, M /M :1.09 n w n 1h M :43000, M /M :1.10 n w n 2h M :64900, M /M :1.12 n w n 3h M :74950, M /M :1.14 n w n 4h M :82600, M /M :1.19 n w n

104 105 Molecular Weight

Figure 3.2.3.12. AGET ATRP of OEOMA475 g-f BSA-O-[iBBr]30 at 30 °C in PBS. (A) First order kinetic plot and (B) Mn and Mw/Mn versus conversion plot (C) Corresponding GPC traces. [OEOMA475]0 = 0.21 M; [OEOMA475]/[I]/[CuBr2]/[TPMA] = 227/1/10/11. Rate of feeding of AA 8 nmol/min.

120 | P a g e

(A) (B) -3 M (x10 ) M /M n w n 100 2.5 1.0 ) 80

]

[

/ 0 60 2.0

[M]

(

ln 0.5 40 1.5 20

0.0 0 1.0 0 1 2 3 4 0 20 40 60

Time/h Conversion / %

(C) 0.5h M :34200 M /M :1.05 n w n 1h M :50800 M /M :1.07 n w n 1h M :68500 M /M :1.10 n w n 2h M :82000 M /M :1.11 n w n 3h M :93000 M /M :1.12 n w n

103 104 105 Molecular Weight

Finally, polymerizations were performed with 10% (v/v) dimethyl sulfoxide

(DMSO). The addition of an organic solvent provides a medium suitable for monomers insoluble in purely aqueous systems (e.g., MEO2MA). Furthermore, DMSO is a useful solvent in proteomics,37 because a concentration below 10% (v/v) does not denature proteins.37 Reaction conditions are summarized in the Table 3.2.3.1 (entries 6 and 9), with first-order kinetic plots, molecular weight evolutions with conversion, and molecular weight distribution versus conversion shown in the Figure 3.2.3.14. The rate of polymerization, molecular weight values, and Mw/Mn values were essentially the same as

121 | P a g e those obtained without DMSO. Finally, a well-defined thermoresponsive copolymer of

MEO2MA and OEOMA300 was grafted from BSA-O-[iBBr]30 (Table 3.2.3.1, entry 10) by normal ATRP under these conditions. The polymer grafted from the protein had a Mn(GPC)

= 58000 and Mw/Mn = 1.22. This PPH had an LCST of 52 °C Figure 3.2.3.15 as seen by a reversible change of the PPH’s hydrodynamic diameter from 40 nm to 5 μm, above and below its LCST, respectively.

(A) (B) M (x10-3) n M /M no DMSO No DMSO w n 10% DMSO 100 10% DMSO 2.0 1.0 80

/[M] 60

1.5 0.5 40

ln[M]

0.0 0 1.0 0 1 2 3 0 20 40 60 80 100 Time / h Conversion / %

(C) 0.5h M :61500, M /M :1.17 n w n 1h M :63000, M /M :1.16 n w n 1.5h M :67000, M /M :1.17 n w n 2h M :71000, M /M :1.14 n w n 3h M :84000, M /M :1.22 n w n

104 105 Molecular Weight

Figure 3.2.3.14. Effect of 10% DMSO (v/v) on ATRP of OEOMA475 g-f BSA-O- [iBBr]30 at 30 °C. (A) First order kinetic plot and (B) Mn and Mw/Mn versus conversion plot (C) GPC traces. [BSA-O-[iBBr]30]0 = 1 mM, [OEOMA475]0 = 0.21 M and [OEOMA475]/[I]/[CuCl]/[CuCl2]/[bpy] = 227/1/1/10/21.

122 | P a g e

(A) (B)

104

103

52oC 102

Average Diameter / nm

20 30 40 50 60 70 104 105 106 Temperature / oC Molecular Weight

Figure 3.2.3.15. Thermo-responsive copolymer g-f BSA-O-[iBBr]30 at 30 °C. (A) Increase of diameter of PPH with temperature (B) GPC of copolymer cleaved from PPH. [OEOMA300]0 = 0.1 M, [MEO2MA]0 = 0.1 M and [MEO2MA]/[OEOMA300]/[I]/[CuCl]/[CuCl2]/[bpy] = 100/100/1/1/10/21.

3.2.4 Conclusions and Outlook

This chapter shows how well-defined polymers can be grafted from proteins by (AGET)

ATRP under biologically relevant conditions. These conditions are designed both to maintain protein stability throughout the polymerization and grow polymers with narrow molecular weight distributions. Biologically relevant conditions have been defined as near ambient temperatures (ca. 30 °C), low initiator concentrations (<2 mM), and low monomer and cosolvent concentrations (total organic content should not exceed 20% of the total reaction volume). Furthermore, the catalyst selected must bind to copper sufficiently strongly to prevent protein denaturation. When conducting traditional ATRP, the optimal catalyst was CuX/CuX2/bpy (1/9/22), where X is either Cl or Br. The optimal halide depends upon the reaction media selected: in pure water Cl is preferred, while in

PBS, Br is required to maintain an acceptable polymerization rate. AGET ATRP with slow feeding of ascorbic acid allows for strongly activating TPMA based catalysts to be used at very low ratios of copper(I) to copper(II). Moreover, AGET ATRP with slow

123 | P a g e feeding of AA gives a rapid reaction and well-controlled polymers in both pure water and

PBS. Finally, the use of 10% of an organic solvent (DMSO) expands range of available monomers, giving access to smart biohybrid materials.

3.2.5 References

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New Technique for Preparation of Enhanced Protein−Polymer Conjugates. Bioconjugate

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Protein−Polymer Conjugates. Biomacromolecules 2005, 6, 3380-3387.

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R. A.; Matyjaszewski, K., Covalently incorporated protein–nanogels using AGET ATRP in an inverse miniemulsion. Polymer Chemistry 2011, 2, 1476.

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AGET ATRP using protein as a macroinitiator. ACTA Biomaterialia 2011, 7, 2131-2138.

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3.3 Epilogue

The ATRP BRC conditions developed in Chapter 3 were further expanded to prepare protein polymer hybrids from BSA-O-[iBBr]30 the low ppm catalyzed ATRP methods or

ICAR (Dominik Konkolewicz, Andrew J. D. Magenau, Saadyah Averick, Antonina

Simakova, Hongkun He, and Krzysztof Matyjaszewski “ICAR ATRP with ppm Cu

Catalyst in Water” Macromolecules, 2012, 45, 4461-4468 DOI:10.1021/ma300887r) and

ARGET ATRP (Antonina Simakova, Saadyah Averick, Dominik Konkolewicz,

128 | P a g e

Krzysztof Matyjaszewski "Aqueous ARGET ATRP” Macromolecules, 2012, 45, 6371–

6379 DOI: 10.1021/ma301303b) ATRP. These manuscripts have changed the perspective on grafting-from using ATRP and several reports have been published that cite ATRP BRC as a major breakthrough for preparing protein polymer hybrids. The future for protein-polymer hybrids points toward polymer to engineer specific surface charge/temperature/light/pH responsive behavior into proteins. This can lead to more effective therapeutics and catalysts as is being demonstrated by the Russell and Maynard groups among an ever growing field. Therefore, the need for bio relevant polymerization conditions is critical for synthesizing well-defined biohybrids.

129 | P a g e

Chapter 4 Solid Phase Incorporation of an ATRP Initiator onto Nucleic Acids and Small Molecules for Direct and Facile Access to Polymer Biohybrids

The work presented in this section was reformatted from a previous publication Saadyah Averick, Sourav K. Dey, Debasish Grahacharya, Krzysztof Matyjaszewski and Subha R. Das “Solid Phase Incorporation of an ATRP Initiator for Polymer-DNA Biohybrids” Angewandte Chemie International Edition, 2014, DOI: 10.1002/anie.201308686

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Chapter 4.1. Preface

DNA block copolymers are a unique class of biopolymer-synthetic polymer hybrids that have been used for targeted drug delivery and sensors. In Chapter 4, DNABCp were prepared by directly incorporating a phosphoramidite ATRP initiator into DNA during solid phase synthesis. This method allowed for high yields and purity of the DNA macroinitiators. AGET ATRP was optimized and used to prepare block copolymers of

OEOMA or benzyl methacrylate, both in solution and from the solid phase bead. The

DNA was shown to be stable during polymerization. DNA latex particles were prepared from DNA-block-poly(benzyl methacrylate) and could be used to selectively bind the

DNA’s compliment strand, indicating potential of the DNA latex for DNA pull down experiments. The solid phase initiator incorporation scheme was extended to ligation of small molecule modified onto solid phase beads with initiators and graft polymer in situ.

This provides a tool to prepare chain-end functionalized polymers in high purity. This work was done in collaboration with the Das laboratory.

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Chapter 4.2.

Solid Phase Incorporation of an ATRP

Initiator onto Nucleic Acids and Small

Molecules for Direct and Facile Access to

Polymer Biohybrids

4.2.1. Introduction

The ability to combine polymers with biologically related molecules has spawned a diverse range of tremendously enhanced polymer biohybrid materials.1-6 Among these,

DNA block copolymers (DNABCp) represent a novel class of bioconjugates that have a

DNA segment covalently attached to an organic polymer segment.7-9 By tuning the properties of the DNA and the organic polymer, the properties of the DNABCp can be tailored. Such control has led to DNABCps that form reversible micelles and nanoscale assemblies including some that are responsive to DNA-based recognition events. 10-16

These DNABCp have been used in sensitive biological sensors for single-nucleotide polymorphism detection and scaffolds for organic reactions.17-19 Potent drug-delivery vehicles based on DNABCp have also been prepared for the delivery of antiviral DNA and anticancer therapeutics.20-23 We previously used a DNABCp based linker to prepare non-covalent protein-polymer hybrid.24

131 | P a g e

The primary method to obtain DNABCp involves separate syntheses of the polymer and the DNA segments, followed by a conjugation reaction to covalently attach them.7, 8 This strategy, known as “blocking-to” ('b-t'), offers the advantage that the composition of the polymer segment is well known prior to conjugation to the DNA segment. The DNA segment, available through modern automated solid-phase synthesis methods, can include highly specific sequences as well incorporate various modifications, such as fluorescent dyes or biotin as well as reactive groups for conjugation to the polymer segment. The polymer segment of the DNABCp may be readily prepared by reversible deactivation radical polymerization (RDRP) methods that allow for the preparation of well-defined polymers with precise control of chain-end functionality, molecular weight (MW) and MW distribution (MWD).3, 8, 17, 25-28 One the most common

RDRP method is atom transfer radical polymerization (ATRP) that is tolerant to a broad range of functional groups and chain ends.29-33 Therefore, ATRP has enjoyed wide use in the preparation of a diverse spectrum of biological conjugates such as protein polymer hybrids, polysaccharide polymer conjugates and DNABCp.13, 18, 24, 25, 30, 31, 34-37

An alternate strategy for the synthesis of DNABCps would be “blocking-from” ('b-f'), wherein an initiator is covalently attached to the DNA and the polymer is subsequently synthesized from the initiator using chain extension reactions.38 There are some advantages to this 'b-f' strategy over the 'b-t' approach. Firstly, 'b-f' allows for simpler purification of the low MW monomers and catalyst from the polymer bioconjugates.

Secondly, in the 'b-f' strategy, the reactions are monomer addition reactions, whereas in

'b-t' the conjugation reaction is between two large molecules (the polymer and the DNA) which is sterically more challenging.

132 | P a g e

There have been a few recent reports of the 'b-f' strategy for making DNABCp using either ATRP or reversible addition-fragmentation chain transfer polymerization.18, 39-41 In these reports, the DNA was functionalized with an amine that could be reacted with an activated ester of the initiator. These DNAs were immobilized on different surfaces (e.g. gold surface, gold nanoparticle) followed by polymer growth. As the polymers were grown from surfaces via the attached DNA, this precluded any direct analysis and characterization of the DNABCps. We therefore sought a more facile and direct method to prepare DNA bound initiators with known site(s) of the initiator within the DNA and to characterize the polymer biohybrids so as to assess the degree of control over the 'b-f ' copolymerization. We report here a straightforward method to incorporate an ATRP initiator phosphoramidite to DNA that is compatible with automated DNA synthesis on solid support. Following the coupling of the initiator, polymer synthesis from the DNA initiator can be performed through Activators Generated by Electron Transfer (AGET)

ATRP42-45 in solution or even directly on the solid support prior to deprotection and release of the DNA sequence. We analysed the resultant DNABCps using gel permeation chromatography (GPC) and fluorescence spectroscopy that confirmed that the DNA remained intact under the polymer synthesis conditions and we obtained DNABCp with narrow MWD and control over the desired MW. Our approach also permits ready and rapid functionalization of polymers with the diverse small molecules available for use in solid-phase synthesis of nucleic acids. We also synthesized a biotin modified polymer on solid support using the same ATRP initiator phosphoramidite. Overall, we present a strategy that allows the direct synthesis of DNABCps and other functionalized polymers.

4.2.2. Experimental

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4.2.2.1. Materials and Instrumentation

α-Bromoisobutyryl bromide, 4-amino-1-butanol, benzyl methacrylate, oligo(ethylene oxide) monomethyl ether methacrylate (average molecular mass ~475 g/mol, OEOMA), diphenyl ether, ascorbic acid, acetone, CuBr2, NH4OH and 3-hydroxypicolinic acid (3-

HPA, MALDI matrix) were from Sigma Aldrich in the highest available purity.

RhodamineB methacrylate (RMA) was purchased from Polysciences, Inc. Tris(2- pyridylmethyl)amine (TPMA) was purchased from ATRP Solutions.

Diisopropylethylamine (DIPEA), triethylyamine (Et3N), 1-methyl-imidazole and organic solvents for reaction and chromatography were purchased from VWR. Avidin coated polystyrene particles were obtained from Spherotech. Spectrapore 7 dialysis tubing (25k

MWCO) was purchased from Spectrum Laboratories Inc. Standard DNA phosphoramidites with ultramild protecting groups (dA-PAC, dC-PAC and dG-tBu-PAC) and the 2-cyanoethyl-N,N-diisopropyl-chloro-phosphoramidite were purchased from

Chemgenes (Wilmington, MA, USA). Appropriate reagents for solid phase DNA synthesis (deblock, activator, ultramild CapA, CapB and oxidation reagent) were purchased from Glen Research (Sterling, VA, USA). CPG solid supports for DNA synthesis (both dC and Quasar670) were purchased from Biosearch Technologies

(Novato, CA, USA). Monomers were passed over a column of basic alumina prior to use.

Electrospray ionization (ESI) mass was obtained on a Thermo LCQ ESI/APCI Ion Trap and MALDI mass spectrometry was performed using an Applied Biosystems Voyager

DE-STR MALDI-TOF instrument in positive mode. Fluorescence spectra of the conjugates were collected using a Horiba Jobin Vyon Fluoromax2 steady state fluorimeter. A syringe pump (KDS Scientific, Legato 101) was used for the continuous

134 | P a g e feeding of the reducing agent at the rate of 0.5 μL/min. Molecular weight and dispersities

(Mw/Mn) were determined by GPC. The GPC system used a Waters 515 HPLC Pump and

Waters 2414 Refractive Index Detector using PSS columns (Styrogel 102, 103, 105 Å) in dimethylformamide (DMF) as an eluent at a flow rate of 1 ml/min at 50 °C. The column system was calibrated with 12 linear PEG standards. Flow Cytometry was conducted on a

BD FACSDiva instrument or a BD Accuri C6 flow cytometer.

4.2.2.2. Synthesis of ATRP initiator phosphoramidite

4.2.2.2.1. Synthesis of Hydroxyl functional amide ATRP initiator, 1

4-Amino-1-butanol (5 g, 0.0561 mol) and triethylamine (6.24 g, 0.0624 mol) were dissolved in 20 ml of dichloromethane and α-bromoisobutyryl bromide (12.8 g, 0.0567 mol) was added drop wise. The reaction was stirred for 16 hours. The reaction mixture was filtered and stirred with 20 ml of 5% KOH for 2 hours. The reaction mixture was then added to a separatory funnel and the aqueous layer was separated. The organic layer was then washed with 1N NaOH (25 ml 2X), 1N HCl (25 ml 2X) brine (25 ml 1X) dried

1 over MgSO4 filtered and the solvent was evaporated. H NMR (300 MHz, CDCl3): 7.0 ppm (s 1H) 3.7 ppm (t 2H) 3.3 ppm (t 2H), 2.2 ppm (s 1H), 1.9 ppm (s 6H), 1.6 ppm (m

4H).

4.2.2.2.2. Synthesis of ATRP initiator phosphoramidite, 2 (Das Labartory)

To a solution of alcohol 1 (340 mg, 1.43 mmol) in CH2Cl2 (10 mL), DIPEA (1.24 mL,

7.14 mmol), 2-cyanoethyl-N,N-diisopropyl-chloro-phosphoramidite (478µL, 2.14 mmol) and 1-methyl-imidazole (57µL, 0.713mmol) were added. The mixture was stirred for

30mins at 0 °C and 1.5 hour at r.t. Work up was done with NaHCO3 (saturated)/ EtOAc.

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Column chromatography (EtOAc/Hexane, 1:1) gives the product (515 mg) in 82%

1 isolated yield. H NMR (300 MHz, CDCl3): δ 1.15 (d, J = 2.6 Hz, 6H), 1.17 (d, J = 2.6

Hz, 6H), 1.60-1.69 (m, 4H), 1.93 (s, 6H), 2.63 (app t, J = 6.44 Hz, 2H), 3.26-3.32 (m,

13 2H), 3.52-3.91 (m, 6H), 6.77 (s, 1H); C NMR (75 MHz, CDCl3): δ 20.2, 20.3, 24.4,

24.5, 24.5, 24.6, 25.9, 28.3, 28.4, 32.4, 39.9, 42.8, 43.0, 58.0, 58.3, 62.9, 63.0, 63.1,

31 + 117.5, 171.8; P NMR (127 MHz, CDCl3): 147.65; Mass (ESI): m/z = 439 [M +H], 461

[M++Na].

4.2.2.3. DNA Synthesis (Das Labartory)

Solid phase synthesis of the DNA was performed on a Mermade-4 (Bioautomation,

Plano, TX, USA) automated synthesizer. The reagents and the conditions for solid phase

DNA synthesis is in TableS1. Following the DNA synthesis and incorporation of the initiator, the CPG beads were dried using a stream of argon. Then the CPG beads were transferred to a glass vial and treated with 2mL of concentrated ammonia (~27-30% NH3 basis) for 4 hours at room temperature. Following deprotection, the DNA was desalted using Waters C18 Sep-Pak cartridge (Waters, Milford, MA, USA). The DNA was purified using 15% denaturing polyacrylamide gel electrophoresis (with 8M urea, 1x

TBE). The DNA band in the gel was excised and eluted overnight in TE0.1 buffer (10mM

Tris.HCl, 0.1mM EDTA, pH 7.5). The eluted DNA was desalted again using a

WatersC18 Sep-Pak cartridge (Waters, Milford, MA, USA) and dried down using freeze drying. Finally the DNA was characterized by MALDI mass spectrometry using 3- hydroxypicolinic acid (3-HPA) as matrix.

4.2.2.4. DNA Sequences (Das Labartory)

136 | P a g e iBBr-DNA1: 5'-iBBr-GCA CTG CAG TTG GAT CCC ATA GC-3' iBBr-DNA1-Quasar670: 5'-iBBr-GCA CTG CAG TTG GAT CCC ATA GC-Quasar670-

DY647-DNA1PC: 5'-Dylight647- ATC GA GCT ATG GGA TCC AAC TGC-3'

DNA1FC: 5'-GCT ATG GGA TCC AAC TGC AGT GC-3'

4.2.2.5. DNA Block-Copolymer Synthesis - Solution Phase

Copper stock solution: A catalyst stock solution of CuBr2 (22.3 mg, 0.1 mmol) and

TPMA (232 mg, 0.8 mmol) was prepared in 5 ml of 50:50 ultrapure water:DMF. RMA stock solution was prepared at 5 mg/ml. An iBBr-DNA1 stock solution was prepared at a concentration of 1 mM. A 1M NaCl solution was used to bring reaction mixtures to the desired final NaCl concentration. The ascorbic acid stock solution used to generate the active catalyst species was 5 mM (0.88 mg/ml) in 50 mM NaCl.

4.2.2.5.1. Preparation of DNA1-b-POEOMA-co-RMA

A general reaction protocol is as follows: 25 µL of iBBr-DNA1, 5.5 mg of OEOMA, 5

µL of the RMA stock solution, 5 µL of the catalyst stock solution, 8.8 µL of ultrapure water and 2.2 µL of 1M NaCl were combined in a two necked 2 ml pear shaped flask equipped with a small magnetic stirrer. The reaction was degassed by passing a stream of nitrogen gas over the stirring reaction mixture for 20 min. A degassed ascorbic acid solution was then slow feed into the reaction mixture at a rate of 500 nL/min. The reaction was carried out for 2 hours.

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To screen reaction conditions that lead to well-defined DNABCp, the reaction mixtures were diluted with DMF with 0.5% DPE and analyzed using GPC. The GPC traces of all the DNABCp synthesized are presented in Figures 4.2.3.4.

Samples that were used for analysis (including iBBr-DNA1-Quasar670) were diluted with 1 ml of 1X PBS and purified using membrane dialysis (25k MWCO) into 1X

PBS for 5 solvent exchanges.

4.2.2.5.2. Preparation of DNA1-b-BnMA-co-RMA

50 µL of iBBr-DNA1, 5 µL of benzyl methacrylate, 10 µL of the RMA stock solution, 10

µL of the catalyst stock solution and 25 µL of DMF were added into a two necked 2 ml pear shaped flask equipped with a small magnetic stirrer. The reaction was degassed by passing a stream of nitrogen gas over the stirring reaction mixture for 20 min. A degassed ascorbic acid solution was then slow feed into the reaction mixture at a rate of 500 nL/min. The reaction was carried out for 2 hours.

4.2.2.6. Preparation of D-TEX particles

The DNA-Latex particles (DTEX) were formed by dialysis of the reaction mixture into acetone followed by 2 exchanges into ultra-pure water. Samples were prepared for flow cytometry by mixing 200 µL of the D-Tex particles at 0.1 mg/ml with DNA1 partial compliment- DY647 (DY647-DNA1PC) or a non-specific DNA sequence (DY647-

DNA2) at a final concentration of 100 nM. The samples were annealed at 90°C for 2 min then 65°C for 10 min and the slowly cooled to 25°C. After annealing the samples were analyzed using flow cytometry. The strand displacement experiment was conducted by adding a 10X equivalent of DNA1 full compliment (DNA1FC) to the D-TEX sample

138 | P a g e with DNA1pc-Cy5 and the annealing protocol was repeated and the sample was analyzed using flow cytometry.

4.2.2.7. Block-Copolymer Synthesis - Solid Phase

4.2.2.7.1. From DNA linked CPG beads

2.5 mg of CPG beads with iBBr-DNA1, 2.8 mg of OEOMA, 5 µL of the RMA stock solution, 5 µL of the catalyst stock solution (at 10 mg/ml), 85 µL of ultrapure water and

4.4 µL of 1M NaCl were combined in a two necked 2 ml pear shaped flask equipped with a small magnetic stirrer. The reaction was degassed by passing a stream of nitrogen gas over the stirring reaction mixture for 20 min. A degassed ascorbic acid solution was then slow feed into the reaction mixture at a rate of 0.5 µL/min. The reaction was carried out for 2 hours. The beads were extensively washed and the DNABCp was cleaved and deprotected with ammonium hydroxide for 4 hours at room temperature and lyophilized.

4.2.2.7.2. From Biotin linked CPG beads

5.6 mg of CPG beads with Biotin-iBBr, 14 mg of OEOMA, 20 µL of the RMA stock solution (at 10 mg/ml), 10 µL of the catalyst stock solution (at 10 mg/ml), 55 µL of ultrapure water and 4.4 µL of 1M NaCl were combined in a two necked 2 ml pear shaped flask equipped with a small magnetic stirrer. The reaction was degassed by passing a stream of nitrogen gas over the stirring reaction mixture for 20 min. A degassed ascorbic acid solution was then slow feed into the reaction mixture at a rate of 0.5

µL/min. The reaction was carried out for 1.5 hours. The beads were extensively washed and the Biotin-POEOMA-co-RMA was cleaved with ammonium hydroxide and lyophilized.

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4.2.2.8. Biotin-Block-POEOMA-co-RhMA Avadin Microbead Binding

The Biotin-POEOMA-co-RMA and a control sample (of POEOMA-co-RMA (no biotin)) were dissolved in 1X PBS buffer at a concentration of 1 mg/ml. Biotin-

POEOMA-co-RMA (200 µL) or a control of POEOMA-co-RMA (no biotin) control (200

µL) were mixed with 20 µL of Streptavadin polystyrene beads (5.1 µM), respectively.

The mixture was subjected to 5 min of centrifugation at 2000 rpm and resuspended in 1 ml of ultra-pure water and analyzed using flow cytometry; 10000 beads were counted per run.

4.2.3. Results and Discussion

For DNA synthesis on solid-support, the most common and direct method for attaching functional groups or small molecules is phosphoramidite coupling chemistry.46 However, the incorporated functional group or small molecule needs to be stable under the nucleic acid deprotection conditions. Thereby, we first prepared an ATRP initiator phosphoramidite capable of surviving typical DNA deprotection condition (concentrated ammonia at room temperature for 4 hours). An amide linked initiator, 1, was prepared from 4-aminobutanol and α-bromoisobutyryl bromide (Scheme1). Washing the product with 5% KOH ensured that no ester linked initiator, which might also form through reaction of the 4-aminobutanol hydroxyl group, remained as a contaminant. Following extraction, the intermediate 1 was reacted with 2-cyanoethyl-N,N-diisopropyl-chlorophosphoramidite to yield the ATRP-initiator phosphoramidite 2 (Scheme 4.2.3.1.).

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Scheme 4.2.3.1. Synthesis of the ATRP initiator phosphoramidite

This ATRP initiator phosphoramidite could then be readily coupled to the 5'-hydroxyl of a DNA sequence on solid-support, obtained after acid-deprotection of the terminal dimethoxytrityl (DMT) group (Scheme 4.2.3.2. ). The coupling of 2 was performed in 6 minutes using standard reagent (Table 4.2.3.1.). As a test case, we performed the initiator coupling to 23-mer DNA (DNA1) on a 1 micromole scale controlled pore glass (CPG) support. Following the coupling and incorporation of the initiator, ammonium hydroxide treatment for 4 hours at room temperature cleaved the DNA from the CPG support and removed the cyanoethyl and nucleobase protecting groups, yielding the initiator linked

DNA, iBBr-DNA1 (see section 4.2.2.4. for sequence information). The integrity of the initiator amide linkage on the DNA was confirmed using Matrix-Assisted Laser

Desorption/Ionization time of flight mass spectrometry (MALDI-TOF) (Figures 4.2.3.1. and 4.2.3.2.). Thus with this phosphoramidite coupling approach, an ATRP initiator could be directly incorporated into the DNA sequence during standard solid phase synthesis and afforded concomitant purification of DNA with initiator. Compared to previously reported activated ester couplings that are performed following DNA synthesis, deprotection and purification,18, 39, 40 this method offers both a simplified access and greater yield of the DNA with initiator.

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Scheme 4.2.3.2. Solution phase synthesis of DNABCp using AGET ATRP. A. After removal of the 5'-ODMT from the DNA (in the CPG bead) the ATRP initiator phosphoramidite was conjugated to the 5'-OH. Cleavage from the solid support and removal of the base protecting groups and cyanoethyl groups using standard conditions gave the DNA conjugated to the ATRP initiator (iBBr-DNA1). B. Direct synthesis of the DNA-polymer conjugate in solution phase by AGET ATRP using the initiator modified DNA. Two different initiator modified DNAs (either with 3'-OH or 3'-Quasar670 dye) were used to synthesize polymers with a mixture of OEOMA or benzyl methacrylate and rhodamine methacrylate as the monomers.

Table 4.2.3.1. Reagents and synthesis conditions for solid phase DNA synthesis – 1 μmole scale

Synthesis Reagent Volume Reaction time step

Deblock 3% Trichloroacetic acid 120 μL 2 x 50s

Coupling 0.1(M) DNA/Initiator amidites + 60 μL of 3x70s (for 0.25(M) 5-(Ethylthio)tetrazole activator amidite + 60 DNA amidites) μL of and activator 3x120s (for initiator amidite) Capping CapA: THF/Phenoxyacetic 60 μL of 60s anhydride/Pyridine CapA and 60 CapB: 16% 1-methylimidazole in THF μL of CapB Oxidatio 0.02M I2 in THF/Pyridine/H2O 120 μL 2x50s n

142 | P a g e

(M+H)+

Mass Calculated +2 (M+2H) 7320.2

iBBr-DNA1

Figures 4.2.3.1. MALDI mass spectrum of the iBBr-DNA1 after gel purification. Mass calculated: 7320.2; Mass found: 7320.2.

(M+H)+

Mass Calculated iBBr-DNA1 7320.2

(M+2H)+2

Figure 4.2.3.2. MALDI mass spectrum of the iBBr-DNA1 crude. Mass calculated: 7320.2; Mass found: 7321.4. Recently, procedures have been developed that allow ATRP under biologically relevant conditions using activators generated by electron transfer atom transfer radical polymerization (AGET ATRP).29, 31, 38, 42, 47 There are several advantages of AGET ATRP

143 | P a g e

over traditional ATRP, including addition of the oxidatively stable copper/ligand

complexes to the reaction and control over the reaction rate by controlling the feed rate of

the reducing agent, that lowers the total copper concentration.29, 48 In order to develop

conditions suitable for block copolymer growth from iBBr-DNA1, the initiator

concentration and reducing agent feed rate were kept constant, while varying several key

reaction parameters such as: reaction time, catalyst, monomer and salt concentration

(Table 4.2.3.2.).

Table 4.2.3.2. AGET ATRP conditions for preparing DNABCp (M: OEOMA, RMA: rhodamine methacrylate, TPMA: tris(2-pyridylmethyl)amine), I: iBBr-DNA1 and iBBr- DNA1 with 3'-Quasar670 dye (entry 8). [a] [M]0/[RMA]0/[I]0 NaCl, Cu% Time, m Mn Mw/Mn 3[b] /[TPMA]0/[CuBr2]0 mM *10 1 500/2/1/4/34 50 0.86 120 108 3.5 2 500/2/1/9/69 50 1.7 120 201 1.36 3 200/2/1/4 /34 50 2.2 60 108 1.12 4 200/2/1/4/34 50 2.2 30 110 1.14 5 200/2/1/4 /34 300 2.2 60 No Polymer 6 200/2/1/4/34 100 2.2 60 83 1.48 7 100/2/1/4/34 50 4.3 60 63 1.24 8 625/2/1/4/34 50 1.7 60 58 1.36 The feed rate of ascorbic acid was 2.5 nmol/min. [a] Calculated as mole ratio [b] Mn from DMF GPC with PEO standards.

The monomer chosen for the polymer synthesis was oligo(ethylene oxide)

methacrylate (OEOMA, Mn = 475) due to its biocompatibility and suitability in several

biomedical applications (Scheme 4.2.3.2.).49-51 A small percentage of rhodamine

methacrylate (RMA) was also incorporated into the reaction mixture in order to facilitate

visualization and characterization of the resultant DNABCp (Scheme 4.2.3.2.). With the

iBBr-DNA1 concentration set to 1 mM, a 5 mM solution of ascorbic acid reducing agent

was added to the reaction mixture via syringe pump at a feed rate of 500 nL/min (2.5

nmol/min). One of the variables in the reaction was the NaCl concentration. The addition

144 | P a g e of NaCl to the reaction enhances the concentration of the deactivator leading to a better control over the polymerization.48 Here we varied NaCl concentration from 50 to 300 mM. Another parameter was the concentration of the catalyst, CuBr2:TPMA (1:8), which was varied from ~0.9 % to ~4.5% (by mole to monomer). The targeted degrees of polymerization were from ~100 to ~500 and the total reaction times varied from 0.5 to 2 hours.

Figures 4.2.3.3. Characterization of the DNABCp synthesized using AGET ATRP in solution phase. a. GPC traces of the iBBR-DNA1 (black) and the polymer-DNA conjugates (green, entry 2 and purple, entry 7 in Table1). The GPC traces show a significant shift of MW after polymerization and also no residual initiator iBBr-DNA1. The GPC characterization of all polymers described in Table1 are in SI Figure S3b. Fluorescence spectra of the different DNA polymer conjugates show that DNA is directly conjugated to the rhodamine containing polymer. The DNA-polymer conjugate with 3'- OH terminus by itself (purple trace) or when mixed with a free dye (orange trace), shows

145 | P a g e no energy transfer. However, the DNA polymer conjugate with Quasar670 dye directly attached to the DNA 3'-terminus shows significant energy transfer (yellow-green trace, arrow) indicating that the Quasar670 dye is in a close proximity to the rhodamine dye from the polymer chain and confirms the integrity of the DNA during polymerization using AGET ATRP.

iBBr-DNA1 Entry 1 Entry 2 Entry 3 Entry 4 Entry 6 Entry 7 Entry 8

102 103 104 105 106 107 molecular weight

Figures 4.2.3.4. Gel permeation chromatography (GPC) traces of all the DNABCps. The entry legends correspond to Table 4.2.3.2 To characterize the resulting block copolymers, DNA1-b-POEOMA-co-RMA, the reaction mixtures were diluted in dimethylformamide (DMF) with 0.5% diphenyl ether as internal standard and injected into a DMF GPC which was calibrated using poly(ethylene oxide). Well-defined polymers could be grown from iBBr-DNA1 with good control over targeted MW and narrow MWD (Figures 4.2.3.3. and 4.2.3.4.) through control over the reaction parameters. To determine if the polymerization conditions led to any degradation of the DNA, a second DNA-initiator sequence that included a 3'-terminal fluorescent dye,

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Quasar670 (Q670, a Cy5 equivalent/analogue) was prepared (Scheme 4.2.3.2.) using similar condition (Table 4.2.3.2. Entry 8). If the polymerization conditions led to the cleavage of the DNA, there would be a loss of Quasar670 dye after purification of the reaction mixture. Conversely, if the polymerization conditions did not lead to DNA scission, there would be Fӧrster resonance energy transfer (FRET) from the rhodamine dye in the grafted polymer to the Quasar670 dye at the 3'-end of the DNA. When characterizing the grafted polymer for integrity of the DNA using fluorescence spectroscopy (Figures 4.2.3.3.),the products were purified using dialysis with a 25k molar weight cut off (MWCO) membrane into ultrapure water (18 MΩ cm) to remove any unreacted monomer and catalyst. The fluorescence spectra of the different conjugates show that FRET occurs from the rhodamine to Quasar670 only in the case of the Quasar670 terminated DNABCp (Figures 4.2.3.3.B, yellow-green trace) but not when free dye was mixed with the POEOMA-co-RMA-b-DNA1. This indicated that the

DNA was not degraded during the polymerization.

Functional latex particles comprised of a bioactive surface and a hydrophobic core can be used as biosensors (i.e. able to selectively bind a desired moiety in solution) for flow cytometry analysis of biomolecules. DNA block hydrophobic polymers that could self- assemble into latex particles would be highly useful as biosensors. Due to DNA’s highly charged phosphate backbone it is water soluble therefore the ATRP initiator iBBr-DNA1 can act as a reactive surfactant in an emulsion polymerization to form DNA-latex particles (DTEX). AGET ATRP was used to prepare hydrophobic polymers by growing benzyl methacrylate (BnMA) and RMA copolymers from iBBr-DNA1 resulting in

DNA1-b-BnMA-co-RMA. DTEX particles were formed by dialysis of the DNA1-b-

147 | P a g e

BnMA-co-RMA into acetone then ultra-pure water (Figure 4.2.3.5.) The DTEX particles

(DTEX-DNA1) were characterized by dynamic light scattering and zeta potential analysis which showed well defined particles with a diameter of 1.3±0.09 µM (volume distribution, Figure 4.2.3.5.B Inset) and a zeta potential of -25.8±1 mV.

Figure 4.2.3.5. Synthesis and characterization of DNA latex (DTEX) particles to demonstrate its ability of sequence specific recognition. a. After the synthesis polymer from iBBr-DNA1 using BnMA and RMA as monomers, the DNABCp was dialyzed in water from acetone resulting into aggregation of the hydrophobic polymer chains which formed the core of the particles. The DNA1 remains exposed to water which decorates the the outer surface of the particles. Then the ability of the particles to selectively recognize complementary strands, was demonstrated by binding with a partially complementary sequence DY647-DNA1PC (PC = partial compliment). Furthermore the partically complementary strand was displaced from the DTEX particles using a DNA strand (DNA1FC) (FC = fully complement). b. The sequence specific binding of the

148 | P a g e

DNA strands with the DTEX particles were demonstrated using flow cytometry. When the DY647-DNA1PC were hybridized with the DTEX particles, a large increse in the fluorescence of the particles indicates binding of the DY647 labeled DNA with the particles (Orange line). However when a non-complementary strand (DY647-DNA2) was used for hybridization the Cy5 signal did not increase (Green line). After displacement of the DY647-DNA1PC from the particles using DNA1FC, the Cy5 signal is at baseline levels indicating complete removal of the DY647-DNA1PC from the particles. Inset shows the volume distribution of the DTEX particles.

To test the DNA sequence specific recognition properties of the DTEX particles, imparted by DNA1, the particles were annealed with a DY647 (Cy5 analogue) labeled

DNA (DY647-DNA1PC) which is partially complimentary to DNA1 or a non-specific control DNA sequence (DY647-DNA2) which has no complementarity with DNA1 and analyzed using flow cytometry. The histogram trace for DTEX-DNA1 (red line) shows a large increase in the fluorescence of the particles in the Cy5 channel when annealed with

DY647-DNA1PC (Orange line) and not in the case of Dy647-DNA2 (green line), demonstrating the ability of sequence specific recognition of DNA1. To determine if the

DY647-DNA1PC could be displaced from the particles, a 10X excess of a DNA which is fully complementary to DNA1 (DNA1FC) was annealed to the DTEX particles with

DY647-DNA1PC and analyzed using flow cytometry. The Cy5 signal came back to the baseline levels of DTEX particles without any DY647 indicates complete removal of the

DY647-DNA1PC from the DTEX particles. These experiments demonstrate that the

DNA1 retained its ability of sequence specific strand recognition after the synthesis of the polymer chain.

To further evaluate the power and utility of solid phase initiator incorporation, we attempted to grow polymer from the DNA while still attached to the solid support. We reasoned this method would facilitate not only the rapid preparation, but also the ready

149 | P a g e purification of functional bioconjugates from the unreacted monomers and catalyst.

Therefore, the CPG beads with the protected iBBr-DNA1 sequence were suspended in the polymerization medium and a polymerization was conducted in situ using 5% monomers (OEOMA and RMA) and 1.7% Cu (Figure 4.2.3.6.). After extensive washing of the CPG beads with water to remove any unreacted monomer and catalyst, the beads were bright red, suggesting copolymer growth from the DNA initiator on solid support.

Cleavage from the CPG beads and removal of the protecting groups of the DNA bases using standard DNA deprotection condition yielded the DNABCp. The DNABCp was analyzed using GPC, which indicated a molar mass of Mn = 205 k and Mw/Mn = 1.43

(Figure 4.2.3.6B) with a slight high molecular weight shoulder.

Figure 4.2.3.6. Synthesis and characterization of DNABCp prepared using a solid support. a. After coupling of the initiator phosphoramidite to the 5'-OH of the DNA, the polymer was synthesized using AGET ATRP with the protected DNA still attached to the CPG beads. After ATRP, the DNA-polymer conjugate was cleaved from the CPG bead and the protecting groups from the DNA were removed using standard conditions to form

150 | P a g e the functional DNABCp. b. GPC traces of the initiator modified DNA (black) and the DNA-polymer conjugate (red) synthesized on solid support.

This synthesis exemplifies the direct preparation of well-defined bioconjugates from CPG beads and provides a general approach to the synthesis of biologically related molecule conjugated in polymer hybrids through solid phase incorporation of the initiator. As a large variety and number of small molecule functionalized solid supports are readily available, this method will allow rapid access to small (bio)molecule functionalized polymers. To demonstrate this, we used biotin-CPG and directly coupled with the ATRP initiator 2 and then conducted a 'b-f' copolymerization of OEOMA and

RMA from a solid support (Figure 4.2.3.7.). Although there are alternate methods to incorporate biotin into the chain end of a polymer including utilization of soluble biotin initiators52, 53 and post-synthetic ligation54, 55 our method exemplifies the advantages of solid phase initiator synthesis coupled with polymer growth. After the polymerization, the beads were washed and the biotin functionalized polymer was cleaved from the solid support using ammonium hydroxide. The biotin-polymer was characterized by GPC; the molar mass of the conjugate determined to be Mn =32 k with Mw/Mn = 1.2 (Figure

4.2.3.8.).

Figure 4.2.3.7. Synthesis of biotin modified polymer on solid support.

151 | P a g e

Figure 4.2.3.8. GPC trace of the biotin modified polymer. Molecular mass was determined using DMF GPC with PEO standards. To confirm that the biotin was intact at the chain end of the polymer, the polymer ability to bind to avidin microbeads was assessed using flow cytometry (Figure 4.2.3.9.).

The avidin microbeads themselves or the avidin microbeads with the OEOMA-RMA copolymer (without biotin) showed little fluorescence in the flow cytometry experiment

(Figure 4.2.3.9.B, green and red traces). However, when the biotin terminated polymer was incubated with the avidin microbeads, a significant shift in the peak to higher fluorescence (Figure 4.2.3.9.B, blue trace) indicates binding of the biotin polymer conjugate with the avidin microbeads.

152 | P a g e

4.2.4. Conclusions and Outlook

A robust method for preparing DNABCp compatible with solid phase nucleic-acid synthesis strategies was reported. A phosphoramidite containing an ATRP-initiator was prepared using commonly available commercial reagents in a simple two-step procedure.

This initiator was incorporated into a 23-mer DNA sequence and the stable incorporation was confirmed using mass spectrometry. This DNA-initiator could be used to obtain block copolymers with synthetic polymer segments of OEOMA and RMA using AGET

ATRP. Optimization of the AGET ATRP conditions provided conjugated polymer segments with well-defined MW and narrow MWD. The use of a Qusar670 dye-labelled

DNA macroinitiator confirmed that the AGET ATRP conditions did not damage the

DNA sequence, confirming the development of a polymerization procedure that can be readily applied to other DNA sequences for developing functional DNABCp. We show that this methodology can be readily extended to prepare bioconjugates by directly grafting from CPG beads with attached biomolecules, such as biotin. The approach, we demonstrate here, has two advantages. First, it enables ready access to a diverse and wide range of functional polymers through molecules that are available on solid support.

Second, the ability to synthesize the polymers on solid support allows direct and rapid

153 | P a g e purification of the conjugates. We expect that this ATRP phosphoramidite initiator will find broad applications in the rapid and ready access to a broad range of functional biomaterials.

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4.3 Epilogue

The ability to prepare relatively large amounts of functional DNA macroinitiators in high purity has accelerated the internal development of drug delivery systems using DNA targeting agents. It is expected that the DNA macroinitiator preparation methodology presented, due to its compatibility with solid phase DNA synthetic methods, will find wide spread adoption by research groups interested in developing novel nucleic acid hybrid materials. Potential applications include self-transfecting therapeutic systems, targeted imaging agents and fluorescent sensors. Our current efforts are extending the technology developed for growing DNABCp from CPG beads to preparing RNA block copolymers for self-transfecting siRNA delivery systems.

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Chapter 5 Direct DNA Conjugation to Star Polymers for Controlled Reversible Assemblies

The work presented in this section was reformatted from a previous publication: Saadyah Averick, Eduardo Paredes, Wenwen Li, Krzysztof Matyjaszewski, and Subha Das “Direct DNA Conjugation of Star Polymers for Controlled Reversible Assemblies” Bioconjugate Chemistry, 2011, 22, 2030–2037 DOI: 10.1021/bc200240q

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Chapter 5.1. Preface

The ability to generate functional polymers using ATRP in combination with the recognition properties of DNA can yield bioconjugates with highly tailored properties.

Azido terminated star polymers were prepared using arm first ATRP and alkyne DNA was ligated using Cu(I) catalyzed cycloaddition. These hybrids could form multivalent assemblies that were reversible in the presence of an invading strand. Additionally the multivalent nature of the star was used to form conjugates labeled with DNA and a small molecule dye. This research demonstrates how ATRP can be used to build polymers for functional and responsive bioconjugates. This work was done in collaboration with the

Das laboratory.

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Chapter 5.2.

Direct DNA Conjugation to Star Polymers for

Controlled Reversible Assemblies

5.2.1. Introduction

The marriage of synthetic polymers with naturally occurring macromolecules has led to offspring that display complex bio-macromolecular architectures, revolutionizing the field of bioconjugations.1-6 Bioconjugates can be obtained from copolymers produced through atom transfer radical polymerization (ATRP) as well as other controlled radical polymerization (CRP) procedures, such as reversible addition–fragmentation chain transfer (RAFT) and nitroxide mediated polymerization (NMP).7-12 One of the key features of these CRP procedures is the ability to produce copolymers with well-defined polymer architectures, including linear, brush, and star shaped molecules, with high content of functional chain ends.6, 13-18 The polymer chain ends have recently been used in further conjugations to install biotin for avidin binding,19-21 carbohydrate conjugates for lectin binding,22, 23 or direct protein-polymer hybrids.4, 24-36 A tetra-functional RAFT initiator used in a ‘core-first’ method of star synthesis yielded a tetrafunctional maleimide star polymer that was conjugated to lysozyme, generating a multi-protein star polymer hybrid. In another example, tert-butyl acrylate was polymerized from a tetra-functional

ATRP initiator and its bromo-chain ends were substituted by reaction with sodium azide to produce azide terminated star polymers that were conjugated to a peptide.24 The ease

164 | P a g e of obtaining multivalent functional polymeric architectures by CRP techniques has enriched the field of bioconjugates and has the potential to lead to further novel materials.

Polymer-oligonucleotide hybrids, a relatively new class of biomacromolecules, are bioconjugates of significant promise. Maynard et al37 conjugated siRNA to a thiolated chain of a poly(oligo (ethylene oxide) methacrylate) prepared using RAFT. The disulfide linkage formed in this conjugate was labile under reducing conditions. A more robustly linked polymer-DNA hybrid was prepared from polymers synthesized using ATRP.38

DNA synthesized on beads was directly coupled to a phosphoramidite-terminated polystyrene that has, in previous research, been used as an amphiphilic DNA block copolymer to encapsulate nanoparticles.39 Reaction of maleimide chain ends yielded thioester conjugated DNA aptamers. The aptamers, or single-stranded DNA sequences that bind small-molecules, retained in vitro functionality in binding to their target peptide.

Proposed applications for these polymer-oligonucleotide (DNA or RNA) hybrids have mainly been therapeutic stabilization and delivery of detection agents. However, DNA can provide other functional and structural properties, in vitro, and in vivo,40-42 that can be applied advantageously to polymer hybrids. Polymer-DNA conjugates would thus provide nanoscale macromolecules that could be controlled using sequence specific hybridization and other functional properties of DNA that are highly tunable (Scheme

5.2.1.1.).

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Scheme 5.2.2.1. Strategy for star polymer-DNA hybrids by direct conjugation. These nanostructures would have high tunable control over design parameters. The star polymer-DNA conjugate would combine useful characteristics of each material into one molecule. DNA self-assembly and nanotechnology are well developed fields and DNA can be used to create three-dimensional materials such as nanoscale objects,43-45 macroscopic crystals,46-48 and DNA based gels.49 DNA self-assemblies and origami50 are now moving from static to dynamic systems.51, 52 However, although those designs are based solely or largely on DNA ready access to large (nanoscale) three-dimensional objects and their manipulation remains challenging. Large colloidal metal-based nanoparticles can be improved in their utility with a functional coating of DNA.53-60 Although nanoparticle-

DNA hybrids and nanoscale objects are both easily prepared and are useful, the DNA coating is non-specific and the ability to custom tailor the coat and core is limited. A polymer nanoparticle would provide a highly customizable core material when used with

DNA, including the possibility of concomitant molecules along with the DNA coat through the use of an appropriate conjugation methodology.

One of the most convenient conjugation strategies is the copper catalyzed azide- alkyne cycloaddition (CuAAC) reaction.61, 62 This highly efficient reaction has risen to preeminence among the various 'click' reactions63 because the reacting azide and alkyne

166 | P a g e groups do not cross react with biologically abundant functional groups. The CuAAC reaction thus enables robust bio-orthogonal conjugations in aqueous media.64, 65 This click reaction has been extensively applied to the synthesis of polymers66, 67 and protein- polymer hybrids.1, 4, 9, 68 Besides their use with polymers and biomolecules, particularly proteins, click chemistry has been useful with synthetic and biochemically obtained

DNA. DNA can be obtained with a high density of alkyne groups and alkyne modified

DNA has been used with CuAAC for a wide variety of applications. 54, 69-72 Although

CuAAC has seen widespread use in both polymer chemistry and DNA supramolecular chemistry, the convergence of polymeric material and DNA through click conjugations is limited. 67, 73-75

We show that by using CuAAC, ATRP based multivalent star polymers can be readily conjugated to functionalized DNA. Because click chemistry is used, simultaneous conjugation of these multivalent polymer cores with DNA, as well other molecules, yields multicomponent-armed hybrids. We show that a polymer core armed with DNA provides a novel nanoscale material that can be designed quite simply, such that the DNA arms of the star can be used to engender function. While DNA can provide numerous useful functions, as a simple proof of principle for star-DNA hybrids, we demonstrate the use of hybridization properties of DNA to control the size of the star polymer nanoparticles. The self-assembly of star-DNA particles can be controlled and displacement with complementary DNA can reverse the assemblies. This preliminary study expands the scope of polymer-DNA hybrids by demonstrating how loading of

DNA onto a multivalent architecture can provide control over self-assembly of polymer nanoparticles.

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5.2.2. Experimental

5.2.2.1. Materials and Instrumentation

Commercially available compounds were used without further purification unless otherwise noted. CuBr2 (98%), N,N,N’,N”,N”-pentamethyldiethylenetriamine

(PMDETA, 98%) were purchased from Aldrich. CuBr (98%, Acros) was purified by stirring in acetic acid, filtered, washed with 2-propanol and then dried under vacuum.

Oligo(ethylene oxide) monomethyl ether methacrylate (average molecular weight ~300,

OEOMA) and ethylene glycol diacrylate (EGDA, 90%) were purchased from Aldrich and purified by passing through a column filled with basic alumina to remove the inhibitor and/or antioxidant. Phosphoramidites with labile PAC protecting groups and appropriate reagents for solid phase synthesis of DNA and RNA were purchased from ChemGenes or

Glen Research. RNA and DNA synthesis columns were purchased from Biosearch. The

5'-phosphohexynyl modifier and Dylight 547 phosphoramidites were purchased from

Glen Research. 3'-O-propargyl CPG columns were purchased from ChemGenes. Copper sulfate pentahydrate (CuSO4•5H2O) was purchased from Sigma Aldrich. HPLC grade acetonitrile (ACN) was purchased from Fisher. Sodium ascorbate was purchased from

Alfa Aesar. Other solvents and reagents not otherwise specified were purchased from

Fisher. Molecular weight and polydispersity were measured by GPC (Polymer Standards

Services-PSS) columns (guard, 105, 103, and 102 Å), with THF eluent at 35 °C, flow rate

1.00 mL/min, and differential refractive index (RI) detector (Waters, 2410). Toluene was used as the internal standard to correct for any fluctuation of the THF flow rate. The apparent molecular weights and polydispersity were determined with a calibration based on linear polystyrene standards using WinGPC 6.0 software from PSS. The detectors

168 | P a g e employed to measure the absolute molecular weights (Mw,MALLS) were a triple detector system containing RI detector (Wyatt Technology, Optilab REX), viscometer detector

(Wyatt Technology, ViscoStar) and a multi-angle laser light scattering (MALLS) detector

(Wyatt Technology, DAWN EOS) with the light wavelength at 690 nm. Absolute molecular weights were determined using ASTRA software from Wyatt Technology.

DNAs were synthesized as described below. Infrared spectra (IR) were obtained on a

JASCO FTIR 6300 instrument. UV-vis spectra were obtained on a NanoDrop 1000 spectrophotometer. Emission spectra were obtained on a NanoDrop 3300.

5.2.2.2. DNA synthesis (Das Laboratory)

Solid phase oligonucleotide synthesis was performed on a MerMade 4 instrument

(Bioautomation). Synthesis of the oligonucleotides was conducted on commercially available solid support columns. It was performed with standard commercially available phosphoramidites as directed by the manufacturer. Cleavage off the solid support and base deprotection of the oligonucleotides was performed by using ammonium hydroxide at 65°C for 2 h and standard protocols for PAC protected amidites as recommended by the manufacturer. Desalting and purification was conducted using a C18 desalting column (Waters) using protocols recommended by the manufacturer.

5.2.2.3. Star Polymers Synthesis and Characterization

The azido group containing macroinitiator (N3-POEOMA-Br MI) was obtained through ATRP of

OEOMA from 3-azidopropyl 2-bromophenylacetate according to the procedure previously

76 published Number average molecular weight of linear N3-POEOMA-Br MI Mn=14,700, average degree of polymerization, DP= 49.

169 | P a g e

77 The star polymer was prepared via arm-first method The ratio of reagents [N3-

POEOMA-Br]0/[EGDA]0/[CuBr]0/[CuBr2]0/[PMDETA]0 was 1/10/0.9/0.1/1. A clean and dry

Schlenk flask was charged with N3-POEOMA-Br (0.31 g, 0.026 mmol initiating sites), EGDA

(0.044 mL, 0.26 mmol), CuBr2 (0.0006 g, 0.002 mmol), PMDETA (0.006 mL, 0.026 mmol) and

1.0 mL of DMF. The flask was degassed by five freeze-pump-thaw cycles, during the final cycle the flask was filled with nitrogen, and CuBr (3.6 mg, 0.025 mmol) was quickly added to the frozen mixture. The flask was sealed with a glass stopper and then evacuated and backfilled with nitrogen five times before it was immersed in an oil bath at 60 °C. Samples were withdrawn at timed intervals to measure polymer molecular weight by GPC. The reaction was stopped after 18 h via exposing the catalyst complex to air and dilution with THF. The solution was filtered through a column filled with neutral alumina to remove the copper complex, and then precipitated in diethyl ether to remove the unincorporated MIs. Apparent number average molecular weight of the purified star polymer is 43,000, and the absolute molecular weight of obtained star polymer determined by GPC MALLS is 122,100. The average arm number per star was calculated to be

~7, each arm has one azide group.

5.2.3. Results and Discussion

Multi-arm star polymers with peripheral functional azide terminated arms were prepared by the macroinitiator “arm first” method. The macroinitiator was prepared using

3-azidopropyl 2-bromophenylacetate as an ATRP initiator for polymerization of

OEO300MA (Oligo(ethylene oxide) methacrylate with Mn=300) targeting a degree of polymerization, DP= 49. The macroinitiator was then copolymerized with ethylene glycol diacrylate (EGDA) under dilute conditions. The star polymers were analyzed using multiangle laser light scattering (MALLS) and determined to have Mn=122,100, corresponding to average 7 arms per star. These star polymers with azide terminated arms

170 | P a g e were conjugated with several different DNA strands that bore a reactive alkyne using click chemistry.

Synthetic sequences of DNA with an alkyne, either at the 5'- or 3'-terminus, were obtained using commercially available reagents and protocols recommended by the manufacturer. For these studies several DNA sequences were prepared with either a 5'- phosphohexynyl or 3'-O-propargyl group. In addition, some of the DNA strands included a terminal fluorescent dye that was incorporated during solid phase synthesis using a commercially available phosphoramidite. A summary of the sequences used in this study is presented in Table 5.2.3.1.

Table 5.2.3.1. DNA sequences used in this study

Terminus Name Sequence 5'- 3'-

DNA1 5’-cgc aag aag agc aaa cgc Dy547 O-propargyl

DNA2 5’-gcg ttt gct ctt ctt gcg Dy547 O-propargyl

DNA3 5’-ggc cga cgt gct tcg gct cgt phosphohexynyl OH

DNA4 5’-aat taa cga gcc gaa gca cgt phosphohexynyl OH

5’-acg agc cga agc acg tcg DNA5 OH OH gcc

Using recently optimized conditions to minimize DNA degradation and oxidation78 3'-O-propargyl DNA strands (DNA1or DNA2) were initially clicked to the star polymer. Under these conditions, solutions of DNA and star-polymer in TRIS buffer

(pH 7.5) with sodium ascorbate and acetonitrile (ACN) as a minor co-solvent (2%) were purged with argon gas, followed by addition of a Cu2SO4·5H2O solution. The use of

ACN helps stabilize Cu(I) and prevents DNA degradation due to oxidative damage under

171 | P a g e aqueous conditions, allowing successful conjugation of DNA (Scheme 5.2.3.1.i). This

‘ligandless’ method, using ACN, is in keeping with the philosophy of 'click'-chemistry, which simplifies purification as no additional triazole or other ligand molecules are used in the click-conjugation. Following the click reaction, the star-DNA conjugates were purified by filtration through a 10kDa molecular mass cutoff nanosep filter that removes the solvent and ACN as well as unreacted DNA. The conjugation was verified using IR spectroscopy (Figure 5.2.3.1.). Complete disappearance of the azide peak at 2200 cm-1 suggests that the star-polymers were essentially quantitatively conjugated to the DNA1 and DNA2.

172 | P a g e

Figure 5.2.3.1. IR spectra of star polymers before (green) and after (red) click reaction with alkyne DNA

Because star-polymers include multiple arms, we sought to demonstrate that click chemistry can be used to incorporate multiple groups onto these arms in a one step process. Thus DNA1, that included a 3'-O-propargyl group and 5'-flourescent dye, along with a second fluorescent dye bearing an alkyne were both used for the click reaction with the star polymer (Scheme 5.2.3.1.ii). The two dyes – Dylight547 (Dy547; a

Cyanine3(Cy3) equivalent) on the DNA and Cyanine5 (Cy5) – clicked on the star- polymer have known overlapping emission and absorbance regions respectively, such that when they are close enough, after excitation of the Dy547, the observed emission is from Cy5 due to Fӧrster resonance energy transfer (FRET). Following the click reaction and purification of the stars, to remove unreacted dyes and DNA, we could observe the absorbance peaks of both dyes (Figure 5.2.3.1.A). As the Dy547 dye was at the 5'- terminus of the DNA and the click conjugation was at the 3'-terminus, the presence of the

Dy547 absorbance peak indicated that the DNA strand remained intact. Furthermore, we were able to observe FRET emission of the multiconjugate star-DNA1-Cy5 (Figure

173 | P a g e

5.2.3.1.B). At the sample dilutions at which the fluorescence was measured, even if the statistically highly improbable reaction outcome produced star-polymers conjugates wherein some stars were only DNA-conjugated while other stars were only dye- conjugated, such a solution would not FRET. Solutions with only one dye or even both dyes in solution together do not result in the FRET emission peak. Thus the emission due to FRET in the click-conjugate star-polymer solution strongly indicates that both DNA

(with dye) and the dye molecule were click-conjugated concomitantly onto the same stars. This shows the power of the click chemistry approach to conjugate DNA as well as other molecules directly onto star-polymers together, taking advantage of the multivalency that the arms provide.

Figure 5.2.3.2. Click conjugation permits concomitant loading of molecules along with DNA onto a star polymer. (A) Absorption spectrum of Star/DNA1/Cy3 conjugate. (B) Fluorescence emission spectra showing FRET between fluorophores conjugated to the star polymer. All emission spectra were taken with an excitation wavelength at 470 nm. The emission spectra of Cy5, 62 Cy3 (yellow), and a 1:1 Dy547:Cy5 solution (red) show no FRET between the fluorophores. Once DNA1 (carrying a Dy547 label) and Cy5 were conjugated to the star polymer, the emission spectrum of the conjugate (black) was a result of FRET in the fluorophore pair confirming that the two were attached to the same star polymer. The ability to conjugate DNA by click chemistry onto star-polymers is significant as the functional properties of DNA can be harnessed. While there is a vast array of function that can be derived from DNA, we sought to demonstrate this quite simply by using the

174 | P a g e hybridization properties of DNA. Using star-DNA conjugates, star-DNA1 and star-

DNA2, that include complementary DNA sequences, we obtained supra-molecular star-

DNA architectures. DNA directed assembly was accomplished by heating solutions of mixtures of the two star-DNA conjugates (at 1 nM) at 95C in aqueous buffered salt solution and cooling to room temperature to anneal the DNA strands. (Figure 5.3.2.2.)

We then tested for controlled hybridization behavior by altering the ratios of the star-

DNA conjugates. Hybridization assembly of the nanoparticles was studied by dynamic light scattering (DLS). As expected, a relationship was found between hybridization ratio and size of the final self-assembled supra-molecular architecture. Both star-DNA1 and star-DNA2 averaged approximately 4 nm. The resulting size of the 1:1 star-DNA1:star-

DNA2 hybrid, Figure 5.3.2.2.Aiii, was found to be approximately 9 nm62 while a particle size of approximately 20 nm (purple) was observed in the 1:10 star-DNA1:star-DNA hybrid, Figure 5.3.2.2.Aiv, system. No residual free stars were observed in the DLS traces thereby demonstrating the fidelity of complementary DNA hybridization.

Figure 5.3.2.3. Controlled DNA self-assembly of star polymers. (A) Scheme for DNA directed self-assembly of star polymers. (B) DLS scans of star polymers click conjugated to DNA1 (black-Ai) and DNA 2(blue-Aii). DNA directed hybridization of these two stars yield larger assemblies shown in a 1:1 star/DNA1:star/DNA2 ratio (green-Aiii) and in a 1:10 star/DNA1:star/DNA2 ratio (purple-Aiv) at 1nM concentrations.

175 | P a g e

These results indicate that self-assembled nanoscale structures can be readily created by using simple complementary DNA strands conjugated onto star polymers. By simply controlling the ratios of the star-DNA hybrids one can control the size of the resulting particles. Star polymers provide a powerful tool for obtaining these large assemblies due to the controlled multivalent architecture unavailable to linear DNA systems. Through controlled hybridization these stars can be used as templates for the design of more advanced DNA architectures. Additionally, nanoparticles with DNA of other functions can be designed and obtained.

The ability to controllably assemble star-DNA conjugate particles can be expanded by gaining control over the reverse disassembly process. The ability to control DNA self- assembly using strand invasion techniques has enabled DNA computation.79-82 In this

Scheme, if one of the hybridized strands in a duplex includes a small single-stranded overhang or ‘toehold’, the duplex can be invaded by a complementary DNA strand that includes the toehold region under ambient conditions. This is typically done in linear

DNA systems and this technique was extended to the star-DNA hybrids. DNA3 and

DNA4, that include partially complementary regions, were conjugated to star polymers, respectively. Additionally, the use of the 5'-phosphohexynyl terminated DNA, rather than the 3'-O-propargyl DNA, demonstrated how readily the orientation of the conjugated

DNA strand can be selected, thereby increasing the informational content loading. These

DNA strands were designed such that upon hybridization assembly of the star-DNA hybrids, a short unhybridized ‘toehold’ region would remain available for an invading strand. DNA5, which is fully complementary to DNA3, can bind to the star-DNA3

176 | P a g e conjugate displacing star-DNA4, in effect disassembling the particles. This invasion takes place at room temperature and thus allows for detection using DLS.

Star-DNA3 and Star-DNA4 were hybridized (10 nM) at a 1:1 ratio at 95C in aqueous buffer (Figure 5.2.3.3.; black and red bars). The resulting complex had a mean diameter of 70 nm by DLS (Figure 5.2.3.3.B; green bar). This assembly of hybridized star-DNA3 to star-DNA4 at 10 nM concentrations is large, likely due to the flexibility of the non-binding region that allows for a larger number of Star-DNA to bind to one another. In contrast, the Star-DNA1 Star-DNA2 complexes that were hybridized at 1 nM concentration and where the strands were completely complementary with no flexible single stranded region after hybridization formed smaller assemblies.

177 | P a g e assemblies from the 70 nm mean size to a hybrid of approximately 10 nm (yellow bar).

The excess of the invading DNA5 strand was to ensure adequate invasion of the crowded macromolecular complex at ambient temperature. When additional DNA5 was added, up to a 300-fold excess over star-DNA3 (1:300), the system reduced further in size to a mean of 2 nm (blue bar), close to that of the star-DNA complexes prior to assembly. Thus the resulting particle corresponded to a totally disassembled star-DNA complex. The slightly larger mean size of the final complex can be attributed to the fact that this star-

DNA complex is hybridized to DNA5. The phosphate backbone of the DNA caused the arms of the star to extend further due to charge repulsion, while in the parent non- hybridized stars the single stranded DNA collapsed into the star arms. Overall, these experiments demonstrate that assembled star-DNA hybrids can be selectively invaded.

This can lead to star-DNA based detection systems where not only the presence but also the concentration of an invading strand or other stimulus that DNA is sensitive to can be assayed.

5.2.4. Conclusions and Outlook

This chapter reports successful DNA-polymer conjugation to multifunctional star macromolecules using azide-alkyne copper catalyzed cycloaddition. The application of click chemistry to the growing field of DNA-polymer hybrids represents a breakthrough because of the orthogonal control and high yields of this reaction. It is also useful because of ready availability of well-designed azide terminated polymers, synthesized via

CRP methodology.83 DNA based computing and self-assembly currently relies on either linear DNA with a toehold region82, 84 or hybrid macromolecules with an uncontrolled number of DNA arms formed by addition of DNA to nanoparticle templates.85-87 This

178 | P a g e report demonstrates how DNA based assembly, as well as polymeric systems, can be synergistically expanded in star polymer-DNA hybrids. We have developed a procedure that allows for preparation of star copolymers with a controllable number of azide- terminated arms per star. These peripheral functionalized stars can be clicked to alkyne

DNA producing hybrids with a known number of DNA strands per star, as well as loading arms with DNA and other cargo simultaneously. The control over loading and size distribution is essential for the design and control over self-assembling nanoarchitectures.

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(79)Chen, X.; Ellington, A. D., Shaping up nucleic acid computation. Curr Opin Biotech

2010, 21, 392-400.

(80)Bi, S.; Yan, Y. M.; Hao, S. Y.; Zhang, S. S., Colorimetric Logic Gates Based on

Supramolecular DNAzyme Structures. Angew Chem Int Edit 2010, 49, 4438-4442.

(81)De Silva, A. P., Molecular computation - Molecular logic gets loaded. Nat Mater

2005, 4, 15-16.

(82)Frezza, B. M.; Cockroft, S. L.; Ghadiri, M. R., Modular multi-level circuits from immobilized DNA-Based logic gates. J Am Chem Soc 2007, 129, 14875-14879.

(83)Sumerlin, B. S.; Vogt, A. P., Macromolecular Engineering through Click Chemistry and Other Efficient Transformations. Macromolecules 2010, 43, 1-13.

(84)Konry, T.; Walt, D. R., Intelligent Medical Diagnostics via Molecular Logic. J Am

Chem Soc 2009, 131, 13232-13233.

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(85)Sharma, J.; Chhabra, R.; Yan, H.; Liu, Y., pH-driven conformational switch of "i- motif'' DNA for the reversible assembly of gold nanoparticles. Chem Commun 2007, 477-

479.

(86)Katz, E.; Willner, I., Integrated nanoparticle-biomolecule hybrid systems: Synthesis, properties, and applications. Angew Chem Int Edit 2004, 43, 6042-6108.

(87)Ofir, Y.; Samanta, B.; Rotello, V. M., Polymer and biopolymer mediated self- assembly of gold nanoparticles. Chem Soc Rev 2008, 37, 1814-1823.

5.3. Epilogue

Combining the reactive and versatile nanostructures that can be prepared using ATRP with responsive DNA yields a synergistic approach to building functional nano-systems.

A protein polymer hybrid whose conjugation was mediated by DNA duplex formation was prepared (Saadyah E. Averick, Eduardo Paredes, Debasish Grahacharya, Bradley F.

Woodman, Shigeki J. Miyake-Stoner, Ryan A. Mehl, Krzysztof Matyjaszewski, and

Subha R. Das “A Protein–Polymer Hybrid Mediated By DNA” Langmuir, 2012, 28,

1954–1958 DOI: 10.1021/la204077v). It is envisioned that this DNA linking technology could be extended to multi-valent polymer architectures, that upon strand invasion, that can release their protein cargo. In addition the click conjugation conditions reported here are being employed by several research groups for functional DNA hybrid materials. Our current efforts ate now applying this technology to preparing duplex DNA-brush polymer hybrids for developing ultra-bright sensors-using intercalating dyes. Results so far show that the duplex DNA-brush hybrid with intercalating dyes is not only bright but also retains the intercalating dyes unlike duplex DNA not attached to a brush polymer.

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Chapter 6 Auto-transfecting siRNA through Facile Covalent Polymer Escorts

The work presented in this section was reformatted from a previous publication: Saadyah E. Averick, Eduardo Paredes, Sourav K. Dey, Kristin M. Snyder, Nikos Tapinos, Krzysztof Matyjaszewski, and Subha R. Das “Polymer Escorts for siRNA Delivery” Journal of the American Chemical Society 2013, 135, 12508–12511 DOI: 10.1021/ja404520j

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Chapter 6.1. Preface

The development of novel therapeutic tools based on knockdown of proteins using RNA interference mediated by siRNA is hampered by the poor-drug like properties of this biomolecule. siRNA itself is highly charged, has a short half-life, is readily degraded by nucleases and cannot penetrate cells. To overcome these challenges siRNA was ligated to linear polymers to enable auto-transfection delivery and nuclease resistance of the siRNA. These constructs represent a major advantage over the traditional polyplex delivery approach where cationic polymers are mixed with siRNA to form an assembly.

These polyplex assemblies are unstable and have a distribution of sizes and loadings of siRNA per particle. The polymer siRNA conjugates are single molecular entities whose properties can readily be tuned by changing the polymer composition. Using this approach, a library of polymer siRNA conjugates can be prepared and tested for structure function assays to discover potent conjugates for therapeutic applications. This work was done in collaboration with the Das lab at CMU and Nikos Tapinos from the Geisinger

Clinic.

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Chapter 6.2.

Auto-transfecting siRNA through Facile Covalent

Polymer Escorts

6.2.1. Introduction

RNA interference (RNAi) has altered the landscape of both basic research to examine gene function pathways1-3 and therapeutic paradigms.4-6 RNAi may be initiated by delivery of exogenous short interfering RNA (siRNA) to cells. These are typically delivered in short 21-23 mer duplexes and other forms that are processed by the cellular machinery.3, 7, 8 The duplexes interact with the cellular RNA-induced silencing complex

(RISC) that eventually uses one strand from the duplex, termed the guide or antisense strand, to silence a target mRNA. Barriers to using exogenous short interfering RNA

(siRNA) duplexes are their susceptibility to degradation and their cell impermeability.9, 10

Although chemical modifications can overcome the lability of the native sugar-phosphate backbone towards hydrolysis and nucleases,11 cell permeability still presents a significant challenge.4, 7, 8

Delivery of exogenous siRNA has therefore required complexation with transfection reagents that enhance cell permeability and provide additional protection of the RNA duplex from nuclease degradation.12-14 Non-viral transfection reagents have relied on formation of a non-specific polyplex between cationic lipid nanoparticles12, 15-17 or polymers16, 18-28 and the anionic siRNA. Although widely studied for siRNA delivery,

192 | P a g e these materials have several practical limitations such as relying on ionic interactions to prepare the polyplex which can be destabilized during circulation or in media.13, 29

Alternative methods for siRNA delivery rely on direct covalent modifications of the 5'- and/or 3'-terminus of siRNA with lipid groups,30-32 small molecules such as biotin and folate,33, 34 peptides,35, 36 nanoparticles,14, 37-39 carbon nanotubes,40 nanostructured DNA41 or poly(ethylene glycol) (PEG).42, 43 Modification of siRNA with linear PEG44, 45 or brush

PEG,46, 47 has been accomplished using disulfide formation or a Michael-type addition between a thiol and maleimide group. While the disulfide linkage allows for release of the siRNA duplex following cellular internalization, the generation of redox sensitive thiols and disulfides that can undergo undesired side reactions or premature degradation poses challenges in synthesis and purification of the polymer-siRNA conjugates. These conjugates have enhanced stability, though some require additional transfection agent,46 limiting their overall utility as a stand-alone siRNA delivery system.

Here, we describe straightforward access to siRNA polymer constructs that are stand- alone siRNA delivery vehicles. In the architecture described here, the sense or passenger strand is conjugated to the polymer, with the guide strand simply hybridized to the passenger-polymer conjugate. We reasoned that a suitable polymer directly conjugated to just the passenger strand of the siRNA duplex could confer both desirable properties of nuclease resistance and cell permeability to the ensemble. These stabilized and autotransfecting siRNAs would potentially permit the guide strand to effectuate an RNAi response.

6.2.2. Experimental

6.2.2.1. Materials and Instrumentation

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Oligo(ethylene oxide) monomethyl ether methacrylate (average molecular mass

~475,~300 and 188 g/mol, OEOMA475, OEOMA300, MEO2MA respectively),

Acetonitrile, ascorbic acid, CuBr2 and tin(II) 2-ethylhexanoate were purchased from

Aldrich in the highest available purity. Copper sulfate pentahydrate and sodium ascorbate were purchased from Alfa Aesar. Tris(2-pyridylmethyl)amine (TPMA) was purchased from ATRP Solutions. Standard RNA phosphoramidites with labile phenoxyacetyl (PAC) protecting group, 3'-O-propargyl guanosine CPG column and appropriate reagents for solid phase RNA synthesis were purchased from Chemgenes (Wilmington, MA, USA).

The 5'-hexynyl modifier phosphoramidite was purchased from Glen Research (Sterling,

VA, USA). Monomers were passed over a column of basic alumina prior to use. N3-

48 PEG3-BPA (ATRP initiator) was prepared as previously described. Molecular weight and molecular weight distribution (Mw/Mn) were determined by GPC. The GPC system used a Waters 515 HPLC Pump and Waters 2414 Refractive Index Detector using PSS columns (Styrogel 102, 103, 105 Å) in dimethylformamide (DMF) as an eluent at a flow rate of 1 mL/min at 50 °C and in tetrahydrofuran (THF) as an eluent at a flow rate of 1 mL/min at 35 C. All samples were filtered over anhydrous magnesium sulfate and neutral alumina prior to analysis. The column system was calibrated with 12 linear poly(methyl methacrylate) standards (Mn = 800 ~ 1,820,000).

6.2.2.2. RNA Synthesis (Das Laboratory)

Solid phase synthesis of the RNA was performed on a Mermade-4 (Bioautomation,

Plano, TX, USA) automated synthesizer. Synthesis and deprotection of the RNA was performed with standard protocols following recommendations of the manufacturer.

After deprotection, the RNA was purified using 20% denaturing polyacrylamide gel

194 | P a g e electrophoresis (with 8 M urea). The RNA band in the gel was excised and eluted overnight in TE0.1 buffer (10 mM Tris.HCl, 0.1 mM EDTA, pH 7.5). The eluted RNA was desalted using a C18 Sep-Pak cartridge (Waters, Milford, MA, USA). Finally the

RNA was characterized by MALDI mass spectroscopy using 3-hydroxypicolinic acid as matrix.

6.2.2.3. Polymer synthesis

Monomer, N3-PEG3-BPA, and CuBr2/TPMA (a 10X catalyst solution was prepared in

DMF and aliquot into the reaction mixture) were added to 50% w/v toluene in a 10 mL

Schlenk flask. The flask was sealed and degassed by bubbling with nitrogen for 10 minutes. After degassing the reaction mixture a degased solution of tin(II) 2- ethylhexanoate was injected to generate Cu(I) and the reaction was heated at 60C for 1 h. The reaction was stopped by dilution with tetrahydrofuran and passing the reaction mixture over a short column of basic alumina followed by precipitation into ethyl ether.

The polymer was dried overnight under vacuum and molecular weight was determined using GPC - see Figure 6.2.2.3.1. , 6.2.2.3.2. and 6.2.2.3.3.

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PM : N -POEOMA 3 475 M - 21000 M /M - 1.13 n w n

103 104 105 Molecular Weight

Figure 6.2.2.3.1 [N3-PEG3-BPA]0:[OEOMA475]0:[CuBr2]0:[TPMA]0:[Sn(II)]

:1:40:0.09:0.27:0.9 in 50% w/vol toluene at 60C for 1 h. Mn= 21, 000 Mw/Mn = 1.13.

PT : N -pOEOMA -co-MEO MA 3 300 2 M - 20500 M /M - 1.05 n w n

103 104 105 Molecular Weight

Figure 6.2.2.3.2 [N3-PEG3- BPA]0:[OEOMA300]0:[MEO2MA]0:[CuBr2]0:[TPMA]0:[Sn(II)] :1:70:20:0.09:0.27:0.9 in

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50% w/vol toluene at 60C for 1 h. Mn= 20, 500 Mw/Mn = 1.05.

PN: N -pOEOMA -co-DMAEMA 3 475 M - 26000 M /M - 1.09 n w n

103 104 105 Molecular Weight

Figure 6.2.2.3.3 [N3-PEG3-BPA]0:[OEOMA475]0: [DMAEMA]0:

[CuBr2]0:[TPMA]0:[Sn(II)] :1:40:10:0.09:0.27:0.9 in 50% w/vol toluene at 60C for 1 h.

Mn= 26, 000 Mw/Mn = 1.09.

6.2.2.4. Click Conjugation to Obtain PEp-RNAs

To ensure both the alkynes in the RNA reacted with the azide group on the polymer, a 20 fold excess of the polymer (500 μM) over the p-RNA (25 μM) was used in the click reaction. Click conjugation of the p-RNA to the polymers was performed in Tris buffer

(20 mM, pH 7.5), CuSO4 (250 μM), 0.6% v/v acetonitrile and sodium ascorbate (1mM) in 75 μL final reaction volume. All the reactants except CuSO4 were mixed and degassed by bubbling with argon for five minutes. The reaction was started by the addition of a degassed solution of CuSO4 to the reaction mixture. The reaction was allowed to run for

1.5 hours and the resulting PEp-RNA was separated from unreacted starting materials using an Amicon Ultra-0.5 centrifugal filter device with a 30,000 molecular weight cutoff.48, 49 The amount of RNA was quantitated by absorbance at 260 nm.

6.2.2.5. Annealing Protocol (Das Laboratory)

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Annealing to obtain siRNA and PEp-siRNAs: was performed by heating equimolar g-RNA' or PEp-RNAs and g-RNA at 60C for 5 min and allowing to cool to ambient room temperature (~25 C).

6.2.2.6 Polyacrylamide Gel Electrophoresis of Duplex siRNA and Polymers (Das

Laboratory)

To determine if specific or non-specific aggregation of siRNA to the polymers was occurring, polymers PM, PT and PN (150 pmol) were combined with siRNA duplexes

(150 pmol) in 1X PBS for 10 min and then loaded on a native 10% polyacrylamide gel and stained with EtBr. Alternately, the siRNA (150 pmol) and PEp-siRNAs (150 pmol) were loaded on a 10% polyacrylamide gel (Tris borate buffer; pH 8.5) and stained with

EtBr . The gels show no non-specific aggregation between the polymers and the siRNA

(Figure 6.2.2.6.1. ).

Figure 6.2.2.6.1. Absence of non-specific binding between duplex siRNA and polymers PM, PT and PN. Non-denaturing gel electrophoresis and EtBr

198 | P a g e staining shows no shift in duplex siRNA mixed with polymers PM, PT and PN. 6.2.2.7. RNase A Stability of PEp-siRNAs (Das Laboratory)

PxEp-siRNA (with PM, PT and PN) or siRNA duplex (150 pmol) were incubated with

RNase A (OMEGA-7U) for 2 h at RT. and the reaction mixtures were loaded on a 10 % native polyacrylamide gel and stained with EtBr. The gel shows degradation of the siRNA duplex, but no degradation of either conjugate is observed.

6.2.2.8. Dicer cleavage study of the PEp-siRNA (Das Laboratory)

To study whether these PEp-siRNAs are substrate for Dicer, a Dicer cleavage study was performed with recombinant Dicer enzyme. 300 pmoles of the modified RNA was incubated with 2 units of human recombinant Dicer enzyme (Genlantis, San Diego, CA) in 1x Dicer reaction buffer (110 mM Tris.HCl, 40 mM HEPES (pH=7.6), 200 mM NaCl,

o 2.5 mM MgCl2, 2 mM ATP, 20 µL final volume) for 12hrs at 37 C.The reaction was then stopped with 5 µL of 5 mM EDTA and the 25 µL of native gel loading solution (40% glycerol, 100 mM Tris.HCl, pH=7.5 and 10 mM EDTA) was added to the reaction mixture. Finally the samples were loaded in a 10% non-denaturing polyacrylamide gel and visualized by EtBr staining.

6.2.2.9. Dual Luciferase Assay for RNAi in Drosophila S2 Cells

Drosophila S2 cells (100 l of 200,000 cells per mL) were plated on a 96-well plate in

Schneider's media. On a separate tube, Firefly reporter plasmid (pGL3, Promega—20 ng) and Renilla reporter plasmid (CJ22, Addgene—40 ng) were added to 98 μl of Schneider's media to a final volume of 100 μl. To this solution, 6 l of FuGENE HD reagent was

199 | P a g e added and mixed by pipetting the solution up and down with pipettor. Following a 10 min incubation, 10 μl of this solution was added per well. The 96-well plate was incubated for

3h for the reporter transfection to take place. Following the 3h incubation, The PEp- siRNA against the Renilla reporter (6, 15 or 30 pmol) in 10 µl of 1X PBS added per well, respectively. In the control reactions, the siRNA duplexes (30 pmol) were mixed with either an additional 0.6 µl of FuGENE HD or 0.6 µl of 1X PBS in 10 μl of 1X PBS for

10 minutes and the solution was added to the well. The plates were incubated for 6 h and then 10 μl of 5.5 mM CuSO4 was added per well to induce expression of the reporter genes.50

After 24 h, since initial transfection of the reporter plasmids, 20 μl of 1X Passive Lysis

Buffer (PLB) was added to each well and the plate was shaken on a plate rocker for 15 min. Following lysis, the luciferase activity of each well was read on a TECAN M-1000 with the Dual Luciferase® protocol using 100 μl dispense volumes for each reagent with

2 s delay for a 10 s integration read time.

6.2.2.10. Culture and Transfection of HEK293 Cells (Tapinos Laboratory)

The HEK293 cell line was purchased from ATCC (American Tissue Culture Collection).

HEK293 cells were maintained in 1X MEM (Gibco) supplemented with 10% fetal bovine serum, 1X L-glutamine (Gibco) and 1X MEM non-essential amino acids (Gibco), and were passaged as suggested by the manufacturer. One day prior to transfection, HEK293 cells were moved to 6 well plates. At 50% confluency, approximately 24 h after plating,

100 nM to 200 nM of Lck-PNEp-siRNA was added directly to the media in the 6 well

200 | P a g e plates. The expression level of Lck in the HEK293 cells was determined 48 h after transfection by Western blot analysis.

6.2.2.11. Western Blot Analysis for Lck Tapinos Laboratory

Hek293 cultures were lysed and collected in 1X SDS lysis buffer containing protease and phosphatase inhibitors (Sigma). Lysates were sonicated and debris was collected by centrifugation at 14,000 g for 10 min at room temperature. The supernatant was collected and stored at -80°C. Total protein lysates were quantified by Micro Bicinchoninic Acid

(BCA) (Pierce) colorimetric protein assay. For analysis, 50 μg of each sample was prepared in 1X LDS buffer containing a reducing agent, boiled for 5 minutes at 95°C, separated using a NuPAGE 4-12% Bis-Tris gel (Life Technologies), and transferred onto a nitrocellulose membrane. Membranes were blocked for 1 h at room temperature in 1X

TBS-Tween 20 (TBS-T) with 5% milk and incubated in the primary antibody D88 against Lck (Cell Signaling) overnight in 1X TBS-T with 5% BSA at 4°C with gentle agitation. Membranes were washed with 1X TBS-T and incubated at room temperature for 1 h in HRP conjugated Anti-Rabbit secondary antibody (Cell Signaling) in 1X TBS-T with 5% milk. West Pico ECL (Pierce) was used for signal detection. Membranes were stripped in stripping buffer PLUS (Pierce), washed in 1X TBS-T, and blocked in 1X

TBS-T with 5% milk for 30 minutes at room temperature. Membranes were incubated with a β-Actin primary antibody (Sigma) in 1X TBS-T with 5% milk at room temperature for 45 min, washed, and incubated in HRP conjugated Anti-Mouse secondary for 1 hr at room temperature in 1X TBS-T with 5% milk. Signal was detected with West Pico ECL (Pierce), and results were quantitated using densitometry.

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6.2.3. Results and Discussion

In evaluating rapid and efficient methods for conjugating the RNA,51 we chose the copper catalyzed azide-alkyne cycloaddition (CuAAC) or ‘click’ reaction.52-54 This reaction has seen widespread use for preparing a diverse range of bioconjugates including protein polymer hybrids,55, 56 and DNA and RNA conjugates.48, 51, 57-61 Click-conjugation of small-molecules and lipids to siRNA have been reported62, 63 and the resultant triazole linkage is biocompatible.64-66 We therefore considered efficient click conjugation of polymers to both 5'- and 3'-termini of an RNA (Figure 6.2.3.1.). An extended RNA sequence that included the sense or passenger strand of an siRNA duplex (p-RNA) was synthesized with alkyne groups at both termini using standard commercially available reagents.

Figure 6.2.3.1.. Synthesis of PEp-siRNAs. A. The passenger strand (p-RNA) with bis- alkyne termini was conjugated to azido-functionalized polymers Px (x=M, T, N) and

202 | P a g e annealed to the guide strand, g-RNA, to form the PxEp-siRNAs (x=M, T, N) B. A non- denaturing polyacrylamide gel (Tris; pH 8.5) and EtBr staining confirms that siRNA as well as PxEp-siRNAs include duplex RNA; polymer PN alone does not stain. To generate a series of well-defined azide-terminated polymers for click-conjugation to the bis-alkyne p-RNA, we used AGET ATRP or activators generated by electron transfer atom transfer radical polymerization,48, 50, 67-70 Three biocompatible polymers were considered to probe their ability to confer both nuclease resistance and cell permeability

M to siRNA. The polymers were: a PEG-methacrylate-pOEOMA475 (P ), a temperature

T M responsive copolymer, pOEOMA300-co-MEO2MA (P ) that is more hydrophobic than P

(lower critical solution temperature for PT in water is ca. 39 °C) and a copolymer containing amino groups that can be cationic at neutral pH, pOEOMA475-co-DMAEMA

N (P ). These monoazido-functional polymers all had a molecular weight of Mn~21000 and a narrow molecular weight distribution, Mw/Mn< 1.2 (See Figures 6.2.2.6.1., 6.2.2.6.2 and 6.2.2.6.3). Polymers with similar composition have been successfully used in unconjugated polyplexes and mixtures with siRNA for delivery and have favorable cytocompatible properties.20, 71

These azido-terminated polymers and bis-alkyne terminated p-RNA (Figure 6.2.2.6.1) were conjugated under conditions optimized for oligonucleotide click-reactions.48, 51, 61, 72

A twenty-fold molar excess of azido-polymer to RNA was used to ensure click- conjugation of both termini without RNA degradation. The pseudo-ligandless reaction was in Tris buffer (pH 7.5) with 0.6% acetonitrile as co-solvent. Following a ninety minute reaction, a simple purification step using a 30000 molecular weight cutoff centrifugal filter device, removes catalyst and excess unreacted polymers, as previously shown,61 and provides the PxEp-RNAs (Figure 6.2.3.1.A). This represents the first report of click-conjugation of polymer to RNA. Following the bis-conjugation of the polymers

203 | P a g e to the p-RNA termini, the complementary 21-mer guide strand (g-RNA), was annealed to yield the three PM, PT and PN-escorted duplex siRNA conjugates (PxEp-siRNAs; where x=M, T or N).

To confirm the presence of both strands and integrity of the complex, we visualized the annealed, polymer conjugates by ethidium bromide (EtBr) staining on a non-denaturing polyacrylamide gel (Figure 6.2.3.1.B). The polymer alone was not stained by EtBr, as exemplified by polymer-PN (lane PN), whereas the siRNA duplex alone and in the polymer escort conjugates are stained. The conjugates also displayed a retarded migration through the gel. The gel used Tris borate buffer (pH 8.5) that is well above the pKa of

PDMAEMA (~pH 7.4) to ensure that even the PNEp-RNA entered the gel. No such retarded mobility was observed when the siRNA duplexes were simply mixed but not conjugated to the polymers (Figure 6.2.2.6.1.), indicating that polyplex formation was unlikely. When conjugated in the PEp-siRNAs, the uniform band in the visualized RNA suggested that the flanking polymer escorts were bis-conjugated and homogenous, rather than a mixture of mono- and bis-conjugated RNA. Further, no free siRNA band was observed, indicating that the click-conjugation was efficient and high purity conjugates were prepared.

3'- Mass Name Sequence 5'-terminus Mass terminus Calc 5'-UGG CGG AGG p-RNA UGG GUA UCU GGA Phosphohex O- 11290.5 11268.4 (RLuc) UGU GGU U GG CUC ynyl propargyl (M+Na+) G-3' g-RNA 5'-CUC ACA UUU OH OH 6558.9 6559.9 (RLuc) ACA UAU UCA CAG-

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5'-G UGG GUA UCU g-RNA' GGA UGU GGU U-3' OH OH 6454.8 6457.6 (RLuc) (to make duplex siRNA) 5'-UGU CAU AAG p-RNA CCA UGC CUU CUG Phosphohex O- 10926.4 10927.5 (Lck) CAA UUU GCC UCG ynyl propargyl A-3' 5'-CAA AUU GCA g-RNA GAA GGC AUG GC OH OH 7057.9 7058.5 (Lck) dT dT-3' 5'-AUG ACA UAA GGU GGA AGC CGG Phosphohex O- pRNA1 11860.7 11864.2 GCA UAA CUU AGU ynyl propargyl AAA-3' 5'-GUU AUG CCC gRNA1 GGC UUC CAC C dT OH OH 6554.8 6558.2 dT-3' 5'-GGU GGA AGC gRNA1' CGG GCA UAA C dT OH OH 6784.0 6788.9 dT-3' 5'-P- 5'-P-AUG ACA UAA RNA- GGU GGA AGC CGG O- 11803.7 Phosphate 11780.6 3'- GCA UAA CUU AGU Propargyl (M+Na+) alkyne AAA-3' 5'-AUG CCC GGC cRNA UUC CAC CUU AUG OH OH 8502.1 8503.0 UCA UAG-3'

The covalent polymer modification at both 5'- and 3'-termini of the p-RNA was expected to render it highly resistant to exonuclease. However, whether this resistance is conferred to the g-RNA strand that is simply hybridized within the construct had to be evaluated. We therefore incubated the three PEp-siRNAs with ribonuclease A (RNaseA) that can rapidly degrade both single- and double-stranded RNA. We found that while siRNA (duplex) was almost completely degraded by RNaseA, all the PEp-siRNAs remained intact even after 2 hours (Figure 6.2.3.2.). This result suggests that the flanking escort architecture can be used to sequester and protect not only the directly conjugated

205 | P a g e p-RNA strand, but also the hybridized g-RNA sequence of the siRNA duplex from nuclease mediated degradation.

Figure 6.2.3.2. Nuclease stabilty of PxEp-siRNA (x=M,T, N) compared to unmodified siRNA. Samples were incubated with (+) and without (-) RNase A for 2 hours and run on a non-denaturing polyacrylamide gel and stained with EtBr The protective power of even just one covalent polymer escort also confers the PEp- siRNA with resistance to in vitro processing by the endonuclease dicer (Figure

6.2.3.3.)Dicer processing is required for long RNA duplexes into canonical 21-mer duplexes with overhangs and helps their loading into RISC. However, dicer processing is not required for cleavage of the target mRNA.73 Cleavage of the target mRNA was mediated by argonaute for which the 21-mer g-RNA within the PEp-siRNA would be suitable and sufficient – if the g-RNA was accessible to RISC loading.

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Figure 6.2.3.3. Dicer cleavage of the PNEp-siRNA and related constructs. While construct-C showed Dicer cleavage, all other RNAs did not get cleaved by Dicer. This shows that the PNEp-siRNAs are not substrate for Dicer enzyme to release duplex siRNA which indicates that they will work in RNAi in a Dicer independent pathway plausibly by releasing the guide strand from the conjugate. (For sequence and mass of the RNAs please see Table 6.2.3.1.). Thus, while the PEp-siRNAs were stable to RNaseA and dicer in vitro, the dissociation of the g-RNA from the PEp-siRNA would be necessary in vivo for entry into RISC to induce an RNAi response. We therefore determined the efficiency of the PEp-siRNA conjugates in RNAi mediated knockdown of a target mRNA (Figure 6.2.3.4.).

Drosophila S2 cells transfected with firefly and Renilla luciferase plasmids allow for the evaluation of RNAi-mediated knockdown in a dual luciferase assay. To assess the PEp- siRNAs, the hybridized g-RNA chosen based on a published report on targeting the 3'- untranslated region (3'-UTR) of the Renilla luciferase (RLuc) mRNA.48 Firefly luciferase (FLuc) provides an internal control for transfection efficiency and protein production against to compare the knockdown of the RLuc signal. Following initial transfection of the Fluc and RLuc reporter plasmids with FuGENE HD, a control duplex siRNA (30 pmol; 250 nM) was transfected after three hours, using an additional amount

207 | P a g e of FuGENE HD. This resulted in the knockdown of the RLuc signal (Figure 6.2.3.4.; purple bar) measured after 24 hours. In the absence of the additional FuGENE HD, the effect of the control siRNA was negligible (Figure 6.2.3.4.; red bar), indicating that after the initial transfection of the plasmids, no residual FuGENE HD remained and little non- specific internalization of siRNA occured. In stark contrast, all three PEp-siRNAs required no transfection reagent and resulted in effective knockdowns. The PEp-siRNAs were each tested at 50, 125 and 250 nM concentrations (corresponding to 6, 15 and 30 pmoles of RNA, respectively) and evaluated 24 hours after addition. Each of the PEp- siRNAs resulted in greater knockdown activity than the equivalent or even half the amount of siRNA delivered through the transfection polyplex. Knockdown of the RLuc signal comparable to that with transfected siRNA could be achieved with just one-fifth the amount of RNA (6 pmol; 50 nM) in PNEp-siRNA that incorporates a positively charged DMAEMA in the copolymer segment (see Figure 6.2.2.6.3. for chemical composition).

208 | P a g e a control well without siRNA. Error bars represent the standard deviation from three separate experiments. The success of the covalent polymer escorts for auto-internalization and release of the g-RNA that was effective in RNAi prompted us to test this architecture towards the knockdown of an endogenous gene in human embryonic kidney 293 (HEK293) cells.

Lymphocyte-specific protein tyrosine kinase (Lck) is a member of the Src kinase family that is important in signal transduction events, particularly in T-cells.74 As the PNEp- siRNA was the most effective in the S2 cells, we used the Lck-PNEp-siRNA construct

(see Table 6.2.3.1. for sequences). This was simply added to the media with HEK293 cells. Compared to untreated cells, we observed specific and reproducible knockdown of

Lck protein with the Lck-PNEp-siRNA without any transfection agent (Figure 6.2.3.5.).

In contrast, actin, serving as an internal control for gene expression, was unaffected as assayed by Western blotting (Figure 6.2.3.5.A). Quantitation of the relative protein expression levels indicates that the siRNA polymer-escort architecture is indeed viable in human cells to knockdown expression of an endogenous gene.

Given the viability of these auto-transfecting siRNAs in RNAi across different cell types, we envision a variety of improvements to the architecture to boost efficacy and investigate the mechanisms related to internalization and action. Constructs that include a 5'-phosphate and other chemical modifications for added stability of the g-RNA strand while enhancing release from the duplex as well as well as modifications that enhance

RNA polymer synergy are being designed for further studies.

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Figure 6.2.3.5. Knockdown of an endogenous gene using PNEp-siRNA. A. Western blot analysis of Lck knockdown in Hek293 cells that were plated (cells only) or transfected with 100 nM and 200 nM of Lck-PNEp-siRNA. After 48 hours cell lysates were analyzed for total Lck and actin as a loading control. B. Graph of densitometric quantitation with the Lck signal normalized to actin. Error bars represent standard deviation from three independent experiments.

6.2.4. Conclusion and Outlook

The flanking PEp-siRNA “escort” architecture provides a robust RNAi agent. These

PEp-siRNA hybrids are obtained readily and efficiently by a simple post-synthetic click reaction, filtration and annealing. The PEp-siRNA architecture simultaneously confers nuclease resistance and cell permeability to the RNA. While non-specific polyplexes with

RNA or even disulfide linked polymer or nanostructure siRNA conjugates in the reducing cellular environment release the siRNA duplex, the polymer escorts likely remain covalently conjugated via the triazole linkage to the passenger strand. Thus, rather than releasing the entire duplex siRNA once internalized, the PEp-siRNA retains the ability to deliver only the hybridized guide strand RNA as the payload for effective RNA silencing.

This has significant implications for RNAi, as it simplifies the design and could avoid off-target effects that may arise from the passenger strand. This siRNA architecture that uses polymer escorts is highly amenable to customization and inclusion of other polymer associated moieties for multi-modal delivery of therapeutic agents.

6.2.5. References

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Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes.

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(53)Best, M. D., Click Chemistry and Bioorthogonal Reactions: Unprecedented

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6.3 Epilogue

The PEp-siRNA architecture represents is a very powerful and unique siRNA vector. To ensure that this drug-delivery system achieves its potential extensive optimization studies on both the RNA and polymer architecture and composition are ongoing. Studies on the cellular internalization pathway of these hybrids materials are being conducted to gain deeper insight to the PEp-siRNA mechanism of delivery. Furthermore, new polymer architectures and copolymer compositions are being explored to enhance transfection efficiency.

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Chapter 7 Preparation of Cationic Nanogels for Nucleic Acid Delivery

The work presented in this section was reformatted from a previous publication: Saadyah Averick, Eduardo Pardes, Ainara Irastorza, Abiraman Srinivasan, Daniel J. Siegwart, Andrew J. Magenau, Hong Y. Cho, Arun R. Shrivats, Eric Hsu, Jinku Kim, Shiguang Liu, Jeffrey O. Hollinger, Subha R. Das, and Krzysztof Matyjaszewski “Preparation of Cationic Nanogels for Nucleic Acid Delivery” “Biomacromolecules, 2012, 13, 3445–3449 DOI: 10.1021/bm301166s

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Chapter 7.1.

Preface

This chapter reports the synthesis of reducible cationic nanogels (qNG) by activators generated by electron transfer atom transfer radical polymerization in inverse miniemulsion. These qNG had a well-defined particle size and were degradable using 10 mM glutathione. The ability of the qNG to complex siRNA and plasmid DNA (pDNA) was evaluated using agarose gel electrophoresis and it was found that the qNGs could successfully complex both siRNA and plasmid DNA. Polyplexes of qNG were studied for their capabilities to transfect both pDNA and siRNA into a S2 cell line. S2 cells are an important cell type in studying biological signaling and development pathways. It was found that qNG:pDNA polyplexes were highly effective at transfecting Firefly luciferase.

Using a Dual-glo assay we determined that qNG:siRNA polyplexes could successfully knockdown Renilla luciferase. This work was done in collaboration with the Das and

Hollinger labs.

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Chapter 7.2.

Preparation of Cationic Nanogels for Nucleic Acid

Delivery

7.2.1. Introduction

Short interfering RNA(siRNA) and plasmid DNA (pDNA) have emerged as important agents in both basic research and therapeutic strategies.1-4 They act by affecting the biosynthesis procedures of their targeted proteins either through the introduction of specific units resulting in synthesis of new proteins in the case of pDNA, or targeting of messenger RNAs (mRNAs) in RNA interference (RNAi) in the case of siRNA.5, 6 The selective delivery of siRNA and pDNA has been a challenge due to their degradation in the presence of nuclease and their anionic charge that hinders their cell permeability.7, 8

Delivery of nucleotides into cells is a complex challenge. Solutions to this challenge include cationic carrier systems, such as cationic lipids or polymers, that generate complexes via electrostatic interactions in the form of lipoplexes or polyplexes, respectively, and thus enhance the transfection of nucleic acids into cells.9-23

Nevertheless, the design of efficient polyplex-based siRNA and pDNA delivery systems for transfection is a challenge that limits the full potential of siRNA and pDNA for therapeutic applications.4, 8, 24-27

Previously, the preparation of biodegradable cross-linked nanogels,11 comprising an oligo(ethylene oxide) methacrylate (OEOMA) backbone, was demonstrated using activators generated by electron transfer atom transfer radical polymerization (AGET

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ATRP) in inverse miniemulsion.28-30 The NGs were used to prepare protein-polymer hybrids31, 32 for delivery of small molecules33 and carbohydrates.34 However, in order to use these materials for effective nucleic acid delivery, the site-specific incorporation of cationic monomers for polyplex formation into the predominant OEOMA nanogels is a currently unmet challenge. ATRP35-38 is a versatile polymerization method that can be applied for the synthesis of diverse and complex polymeric architectures, 39-43 including cationic and quaternized nanogels (qNG) via the copolymerization of OEOMA and a cationic monomer, e.g. quaternized dimethylaminoethyl methacrylate.44 A disulfide- based crosslinker9, 29, 45, 46 was utilized to take advantage of the different concentrations of reducing agent contained in the extracellular (~1 μM) vs. intracellular (~10 mM) matrix47,

48 and facilitate the biodegradation of the NG after transfection of the nucleic acid cargo.

In this communication, we report the synthesis and characterization of biodegradable qNG by AGET ATRP in inverse miniemulsion. The qNGs were studied for the complexation and delivery of a pDNA that codes for a firefly luciferase protein and siRNA that targets a renilla luciferase mRNA for a dual luciferase reporter assay. The model cell line selected was the Drosophila Schneider 2 (S2) cell line due to its importance and prevalence in basic biological research and challenges in successfully delivering both siRNA and pDNA to these cells. 49

7.2.2. Experimental

7.2.2.1. Materials and Instrumentation

Oligo(ethylene oxide) monomethyl ether methacrylate (average molecular weight ~300 g/mol, OEO MA 300), tris(2-pyridylmethyl)amine (TPMA), Span-80, ascorbic acid, and

Cu(II)Br2 (98%, Acros) were purchased from Aldrich. Inhibitor was removed from

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OEOMA300 by passing through a basic alumina column (50% weight Basic alumina to monomer). All other reagents and solvents were purchased from Sigma. Q-DMAEMA,

(2-(dimethylamino)ethyl methacrylate quaternized with ethyl bromide) was prepared as described previously.9 Poly(ethylene oxide) monomethyl ether 2-bromoisobutyrate ester

(PEOMI Mn=2000) and poly(ethylene oxide) dimethacrylate (Mn=4000) were prepared as previously described.50 Disulfide cross-linker was prepared as described previously.28 firefly luciferase reporter plasmid, pGL3, and Beta-gal (used in complexation assays) were obtained from Promega and Renilla reporter plasmid, CJ22 from Addgene. Particle size and zeta potential were measured using a Zetasizer Nano from Malvern Instruments. luciferase activity was measured using a Dual-Glo kit from Promega on a Tecan M-1000 plate reader.

7.2.2.2. qNG- Synthesis of cationic nanogels by AGET ATRP in inverse miniemulsion PEOMI2000:OEOMA300:Q-DMAEMA:DMA:Cu(II)Br2:Ascorbic Acid:

1/290/20/4/0.5/0.6/0.3, 55 mg PEGOH2000, in 5% Span80 in Cyclohexane for 24 hours at

30C. Yield~30%, qNGs were prepared through a water-in-oil inverse miniemulsion utilizing AGET ATRP. The inverse miniemulsion was composed of a water phase consisting of Cu(II)Br2 (1.86 mg, 0.013 mmol) / tris(2-pyridylmethyl)amine (TPMA)

(2.91 mg, 0.013 mmol), PEOMI2000 (33.4 mg, 0.025 mmol, Mn=2000), oligo(ethylene oxide)methacrylate (OEOMA300, 1450 mg, 4.3 mmol, Mn=300), Q-DMAEMA (88.9 mg, 0.334 mmol), DMA (84.2 mg, 0.7 mmol) and 55 mg PEO-OH (as a stabilizer) dissolved in 1.4 ml of ultrapure water and emulsified with 20 g of a 0.05% (w/w) of

Span-80 in cyclohexane using ultrasonication to form stable droplets. After degassing, ascorbic acid 200L (4.4 mg/ml degassed) was injected to initiate AGET ATRP

225 | P a g e which was stopped after 15 hours at 30°C. The NGs were purified by precipitation into THF followed by dialysis (50000 MWCO membrane) into water to remove unreacted reagents

7.2.2.3. siRNA transfection using cationic nanogels (Das Laboratory)

S2 cells (100 ul of 200KmL-1) were platted on a 96-well plate. In a separate tube, firefly reporter plasmid (pGL3, Promega—20ng) and Renilla reporter plasmid (CJ22,

Addgene—40ng) were added to 98 μl of Schneider's media to a final volume of 100 μl.

To this solution, 6 ul of FuGENE HD reagent was added and mixed by pipetting the solution up and down with pipettor. Following a 10 min incubation, 10 μl of this solution was added per well. The 96-well plate was incubated for 3h for the reporter transfection to take place. Following the 3h incubation, 3 μl of 3 uM siRNA duplex against the

Renilla reporter in 1X PBS was pre-mixed in a separate tube with 5 μl of 5, 1, 0.1, 0.5,

0.01 or 0.001 mg/mL in 1X PBS for 10 min and added per well. In the control reactions, the siRNA duplexes (9 pmol) were mixed with either an additional 0.6 ul of FuGENE HD or 0.6 ul of 1X PBS in 10 μl of 1X PBS for 10 minutes and the solution was added to the well. The plates were incubated for 6 hours and 10 μl of 5.5 mM CuSO4 was added per well to induce expression of the reporter genes.

7.2.2.4. Plasmid transfection using cationic nanogels (Das Laboratory)

S2 cells (100 ul of 200KmL-1) were platted in a 96-well plate. In a separate tube, firefly reporter plasmid (pGL3, Promega—20ng) was pre-mixed with 5 μl of 5, 1, 0.1, 0.5, 0.01,

0.001, or 0.0001 mg/mL in 1X PBS for 10 min and added per well. In the control reactions, the pGL3 reporter plasmid was transfected with FuGENE HD as before or

226 | P a g e added to the well with no transfection reagent. The plate was incubated for 9 hours and

10 μl of 5.5 mM CuSO4 was added per well to induce expression of the reporter gene.

7.2.2.5. Luciferase assay on TECAN M-1000 (Das Laboratory)

20 μl of 1X Passive Lysis Buffer (PLB) was added to each well and the plate was shaken on a plate rocker for 15 min. Following lysis, The luciferase activity of each well was read on a TECAN M-1000 with the Dual Luciferase® protocol using 100 μl dispense volumes for each reagent with 2 second delay for a 10 second integration read time.

7.2.3. Results and Discussion

Biodegradable qNG were prepared in a water-in-oil inverse miniemulsion utilizing AGET ATRP providing qNGs (Scheme 7.2.3.1.). Cu(II)Br2 / tris(2- pyridylmethyl)amine (TPMA) was used as the ATRP catalytic species, poly(ethylene glycol) methyl ether 2-bromoisobutyrate (PEGMI2000, Mn = 2000) as a macroinitiator, oligo(ethylene oxide)methacrylate (OEOMA300, Mn = 300), and quarternized dimethylaminoethyl methacrylate (Q-DMAEMA, DMAEMA was quaternized with ethyl bromide) as comonomers, and dithiopropionyl poly(ethylene glycol) dimethacrylate

(DMA) (Mn=1260) as a crosslinking agent, and PEG2000OH as stabilizer. They were dissolved in 1.40 ml of ultrapure water and added to a 25 ml solution of 0.05% (w/w) of

Span-80 in cyclohexane. The reaction mixture was emulsified using ultrasonication to form stable water-in-oil droplets. The mixture was degassed with nitrogen and a degassed solution of ascorbic acid was injected into the emulsion to convert Cu(II)Br2 to Cu(I)Br and initiate the AGET ATRP. The polymerization was carried out for 24 hours at 30°C.

The qNGs were purified by precipitation into THF and washed several times with

227 | P a g e ultrapure water followed by dialysis (25 k MWCO membrane) against water to remove all unreacted reagents.

Scheme 7.2.3.1. Synthesis of cationic nanogels for nucleotide delivery of siRNA and pDNA using AGET ATRP The qNGs were characterized by dynamic light scattering (DLS) and zeta potential, revealing a particle size of ca. 350 nm in diameter with a PDI of 0.164 and 17.9 ±0.902 mV, respectively (Figure 7.2.3.1.A). To determine the biodegradability of these qNG under reducing conditions, a 1 mg/ml solutions of qNG in 10 mM glutathione9 was prepared and its particle size was monitored using DLS. The resulting degraded particles had a volume distribution of ca. 4 nm in diameter indicating a successful REDOX mediated degradation of the qNG (Figure 7.2.3.1.B).

Figure 7.2.3.1. Normalized volume distribution of cationic nanogels prepared by AGET ATRP in inverse miniemulsion measured using DLS. Fig. A: volume distribution of purified qNG prepared from PEOMI2000:OEOMA300:Q- DMAEMA:DMA:Cu(II)Br2:Ascorbic Acid: 1/290/20/4/0.5/0.6/0.3, 55 mg PEGOH2000, in 5% Span80 in cyclohexane for 24 hours at 30C. Size 350 nm PDI 0.164 Zeta potential

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17.9 mV +/-0.902. Fig B: Volume distribution of qNG after incubation with 10 mM glutathione for 4 days. First, the ability of the qNG to complex both pDNA and siRNA was investigated using agarose gel shift assay. This assay was used to determine the weight ratio(wt. NG: wt. siRNA/pDNA) (R) at which qNG totally complexed pDNA and siRNA, respectively. 500 ng of pDNA was hybridized with qNG for 1 hour at 25C and then loaded onto a 0.5% agarose gel. Following electrophoresis, the gels were stained with ethidum bromide

(EtBr) and imaged. For pDNA a ratio of R5 (qNG:pDNA) showed a lack of band migration of the qNG:pDNA polyplex, which indicated a near total complexation of the

DNA with the qNG (Figure 7.2.3.2.A). A polyplex disassociation study was conducted, by adding heparin sulphate to the precomplexed qNG:pDNA polyplexes, to determine the reversibility of the complexation between qNG and pDNA.51 At ratios less than R50, the polyplexes could be disassociated using heparin sulphate (0.05 g/l) (Figure 7.2.3.2.B).

This result suggests that although DNA could be complexed it could not be released, under these conditions, when a large excess of qNG was used to complex the pDNA. qNG:siRNA polyplexes were prepared by incubating 300 ng of siRNA with varying ratios of qNG for 1 h at 4C. Band migration of these polyplexes was studied by electrophoresis on a 2% agarose gel followed by EtBr staining and imaged. At a weight ratio of 15:1, qNG:siRNA (i.e. R15) total complexation of siRNA was observed, as indicated by the lack of band migration (Figure 7.2.3.3.). Once the qNGs ability to complex nucleotides was determined, the delivery of the polyplexes of qNG and siRNA and pDNA to S2 cells was studied.

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Figure 7.2.3.2. Agarose gel electrophoresis analysis of polyplex formation and disassociation of qNG and plasmid DNA (LacZ-plasmid). Electrophoresis was conducted for 60 min, 100V and the gels were stained with EtBr and imaged with UV- transillumination. A: Polyplex formation, 500 ng of plasmid was incubated with varying amounts of qNG (R1-R500) 1 hour at 25C and then loaded onto a 0.5% agarose gel electrophoresis. B: Preformed polyplexes of qNG:pDNA (R1-R200) were incubated with 0.05 g/L Heparin sulphate for 30 min and then examined by gel electrophoresis.

Figure 7.2.3.3. Agarose gel electrophoresis complexation assay measured at varying qNG:siRNA weight ratios. Polyplexes were prepared by incubating 300 ng of siRNA with varying amounts of qNG for one hour at 4C, then loaded onto a 2% agarose gel in Tris/Borate/EDTA buffer. After electrophoresis (30 min, 100V) the gel was stained with EtBr and imaged with UV-transillumination. Next, we tested the ability of the qNG to act as gene delivery agents. It is integral to the development of a multivalent delivery platform. pDNA polyplex delivery was investigated using S2 cells.18 A firefly luciferase reporter plasmid (FLuc) polyplex was prepared at different weight ratios of qNG:pDNA and transfected into S2 cells. Different polyplex formation ratios were used to determine the optimal conditions leading to maximum firefly luciferase signal after 24 hours. By screening a range of qNG:pDNA

230 | P a g e polyplex formulations we were able to determine that, of the polyplexes tested, the R30 polyplex resulted in a maximum emission of the FLuc reporter gene signal (Figure

7.2.3.4.).

(Dual-Glo luciferase reporter assay, Promega)52 with S2 cells transfected with both firefly luciferase and Renilla luciferase (RLuc). Three hours after reporter plasmids were transfected, polyplexes of qNG and siRNA against RLuc were formed at R100, R20, R2,

R1, R0.2 and R0.02 to determine the optimal siRNA transfection ratios. After 24 hours the post siRNA transfection RLuc and FLuc signals were measured and the RLuc knockdown was quantified and normalized to the FLuc signal and a control well (N=3).

When no transfection reagent (no Fugene-HD) was used, the siRNA was inactive (Red bar), suggesting the initial transfection agent used to deliver the plasmid had been washed away. Maximum RLuc reporter signal knockdown was observed at a ratio of R0.2, which

231 | P a g e gave more effective knockdown than Fugene-HD (Figure 7.2.3.5.). This result underscores the utility of the qNGs for siRNA delivery.

Figure 7.2.3.5. siRNA delivery using a dual-Luciferase reporter assay: Normalized, to FLuc, RLuc activity in S2 cells after 24 hour treatment with: no transfection reagent (negative control, red bar), 9 pmol of duplex siRNA with FuGene-HD (positive control, purple bar) or a weight ratio of qNG:siRNA at R100, R20, R2, R0.2 and R0.02 (experimental group, blue bars) 7.2.4. Conclusion and Outlook

We have demonstrated that well-defined qNGs of 350 nm (PDI 0.164) can be prepared using AGET ATRP in inverse miniemulsion. The qNGs were degradable. The disulfide crosslinker underwent a REDOX mediated degradation with a model biological relevant reducing agent, glutathione. Moreover, the qNGs complexed pDNA and siRNA at relatively low weight ratios of qNG to DNA (R5) and qNG to siRNA (R15), respectively, as observed in agarose gel electrophoresis. Further, the nanogels provided a robust delivery system for nucleic acids, both plasmid DNA (~5 kb) as well as siRNA. In order to characterize the ability of different ratios of qNG to transfect siRNA, a Dual-luciferase reporter assay was utilized to rapidly and accurately screen knockdown efficiency. A maximum reporter knockdown was obtained at R0.2, suggesting more effective transfection than siRNA-Fugene-HD. For plasmid DNA transfection, the maximum

232 | P a g e firefly luciferase reporter signal was observed at R30. These results confirm that qNGs are a promising platform for pDNA and siRNA delivery and future studies will include clinically relevant mammalian cells treated with the polyplexes.

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7.3. Epilogue

The cationic nanogels siRNA delivery platform has demonstrated good knockdown of proteins in vitro and in vivo (mouse GFP model). Currently there are studies ongoing testing this systems efficacy in knocking down proteins associated with bone development in an in vivo mouse model for the prevention of heterotopic ossification. The nanogels siRNA delivery platform has shown protein knockdown across a range of models both in vitro and in vivo. Further optimization of the nanogel delivery system can possibly achieved by changing the amount of Q-DMAEMA, modifying the cross-linking density of the primary polymer chain and understanding in vivo clearance pathways to design optimal degradable crosslinkers.

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