University of Florida Thesis Or Dissertation Formatting

STIMULI-RESPONSIVE AND BIODEGRADABLE POLYMERS

MEGAN RAE HILL

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2018

To solving science

ACKNOWLEDGMENTS

First, I would like to thank my mother for inspiring me to become a strong, hardworking, and independent person. Her support and encouragement have allowed me to chase my dreams in both education and travel. I am grateful for the love and support of my sister, whose kindness and compassion continues to inspire me every day. I thank the friends that I have met throughout my life that make each day enjoyable and who continue to inspire me to become a better person – Alexander Pemba, Morgan

Brett, Katie Fisher, Matthew Jaoudi, Carolyn Averback, and Lindsey DeRatt. I am especially grateful for Noah Burrell, for his limitless support, for his patience and compromise, and for being the best partner in life’s adventures.

I am thankful for my entire graduate experience, which would not have been possible without my advisor, Prof. Brent Sumerlin. His continued belief in my abilities over the last seven years not only inspired me to attend graduate school, but also has made graduate school a successful and rewarding experience. Additionally, I want to thank Brent for the occasional music recommendations over the years, which were usually pretty good. I am grateful to George and Josephine Butler for their establishment of the Butler Polymer Research Laboratory, which continually promotes a collaborative and supportive environment, and to the Department of Chemistry at the

University of Florida for the opportunity to earn my Ph.D. and for financial support. I thank Prof. Ken Wagener for his commitment to us as students, and never failing to make us feel important and included in the Butler Polymer Research Laboratory. I thank the rest of my committee members as well, Prof. Stephen Miller, Prof. Daniel Talham, and Prof. Jennifer Andrew, for their instruction and time.

I would like to thank the members of the Sumerlin research group, for their

constant support, encouragement, and collaboration. I am grateful for the open atmosphere that allowed me to discuss many of the obstacles I reached in both research and life. I would like to thank Dr. Bryan Tucker, Dr. William Brooks, Nick

Carmean, Tomo Kubo, Charles Easterling, and Georg Schulz in particular, for their helpful discussions concerning research, and the development of new research projects. I would like to thank Dr. Bryan Tucker for his friendship from our very first day of graduate school. Our daily conversations truly made me aspire to become a better scientist and human being. I would like to thank all of those who were involved in my research projects, and in particular, the graduate and undergraduate students who worked with me during my doctoral research, Sofia Goodrich, Courtney Ligon, Elliot

Mackrell, Carl Forsthoefel, and Andrew Turner.

Lastly, I would like to thank Prof. Philip Costanzo for his mentorship during my undergraduate career at California Polytechnic State University. I would have never understood the splendor of polymers if he had not taken me into his research group and patiently mentored me for four years. Some of my best memories originate from the time I spent in the lab with my ‘polymer family’ that I first established in his group.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF FIGURES ...... 9

LIST OF SCHEMES ...... 11

LIST OF ABBREVIATIONS ...... 12

ABSTRACT ...... 14

CHAPTER

1 STIMULI-RESPONSIVE AND BIODEGRADABLE POLYMERS ...... 16

1.1 Overview ...... 16 1.2 Stimuli-Responsive Materials ...... 16 1.3 Biodegradable Polymers ...... 18 1.4 Responsive and Biodegradable Natural Polymers ...... 19 1.5 Responsive and Biodegradable Synthetic Polymers ...... 23 1.5.1 Step-Growth Polymers ...... 25 1.5.2 Chain-Growth Polymers ...... 28 1.6 Properties and Applications of Responsive and Degradable Polymers ...... 32 1.7 Conclusion and Future Outlook ...... 34

2 RESEARCH OBJECTIVE ...... 36

3 BIODEGRADABLE AND pH-RESPONSIVE NANOPARTICLES DESIGNED FOR SITE-SPECIFIC DELIVERY IN AGRICULTURE ...... 39

3.1 Overview ...... 39 3.2 Results and Discussion ...... 43 3.2.1 Synthesis of Amphiphilic PSI Copolymers and Nanoparticle Formation .. 43 3.2.2 Loading of Model Hydrophobic Molecule Nile Red ...... 47 3.2.3 Response of the Nanoparticle to Alkaline pH and Release of Nile Red... 49 3.2.4 Plant Toxicity ...... 51 3.3 Conclusion ...... 53 3.4 Materials and Methods ...... 53

4 ALTERNATING RADICAL RING-OPENING COPOLYMERIZATION AFFORDS HOMOGENEOUS INCORPORATION OF DEGRADABLE UNITS ...... 59

4.1 Overview ...... 59 4.2 Results and Discussion ...... 62 4.3 Conclusion ...... 73

4.3 Materials and Methods ...... 74 4.3.1 Materials ...... 74 4.3.2 Analytical Techniques ...... 74 4.3.3 Experimental Procedures ...... 75

5 TUNABLE AND FUNCTIONAL POLYESTER COPOLYMERS SYNTHESIZED BY ALTERNATING RADICAL RING-OPENING COPOLYMERIZATION OF 5,6- BENZO-2-METHYLENE-1,3-DIOXEPANE AND N-SUBSTITUTED MALEIMIDES ...... 80

5.1 Overview ...... 80 5.2 Results and Discussion ...... 82 5.3 Conclusion ...... 91 5.4 Materials and Methods ...... 91 5.4.1. Materials ...... 91 5.4.2 Analytical Techniques ...... 92 5.4.3 Experimental Procedures ...... 94

6 CONCLUSION ...... 100

APPENDIX

LOADING OF MODEL HYDROPHOBIC MOLECULE NILE RED ...... 102

MALDI-ToF OF DEGRADED P(MPDL-ALT-NETMI) ...... 103

REACTION OF BMDO WITH MALEIC ANHYDRIDE ...... 104

ANALYSIS OF BMDO AND N-SUBSTITUTED MALEIMIDE COPOLYMERS ...... 105

LIST OF REFERENCES ...... 108

BIOGRAPHICAL SKETCH ...... 121

LIST OF TABLES Table page

3-1 Nanoparticle diameter and loading capacity of Nile red ...... 49

4-1 Polymerizations of MPDL and NEtMI ...... 67

5-1 Copolymerization of BMDO with N-substituted maleimides ...... 88

5-2 Functionalization of poly(BMDO-alt-TCTMI) with model thiols and amines ...... 89

LIST OF FIGURES

Figure page

1-1 Stimuli-responsive polymers ...... 17

1-2 Chemical structures of some natural biodegradable polymers ...... 20

1-3 Stiffness-changing polymer cellulose nanocrystal (CNC) nanocomposites...... 21

1-4 Mechanism of the assembly and disassembly of ELP-CLP vesicles...... 23

1-5 Structures of some synthetic biodegradable polymers ...... 24

1-6 Step-growth polymerization of functional AB and AA + BB monomers ...... 26

1-7 Thiol-ene step-growth polymerization...... 27

1-8 Ring-opening polymerization of functional cyclic monomers ...... 29

1-9 Synthesis of thermoresponsive oligo(ethylene glycol) (OEG) polylactides ...... 31

3-1 Preparation of polysuccinimide (PSI)-based pH-responsive nanoparticles for controlled release of loaded compounds at alkaline pH...... 40

3-2 1H NMR spectra of PSI-HA copolymers with functionalization 7-92%...... 45

3-3 PSI-nanoparticles formed by nanoprecipitation of amphiphilic PSI-copolymer solutions into DI water ...... 47

3-4 Release of Nile Red from PSI-based copolymer nanoparticles ...... 50

3-5 Plant tissue toxicity study of various polymer concentrations after 21 days of incubation ...... 52

4-1 Synthesis of alternating degradable polymers from conventional and RAFT- mediated radical ring-opening copolymerization of MPDL and NEtMI...... 61

4-2 Copolymerization of MPDL and maleic anhydride ...... 63

4-3 Copolymerization of MPDL and N-ethylmaleimide ...... 63

4-4 Bond-forming initiation theory of donor-acceptor (D-A) monomer pairs ...... 64

4-5 Reaction of MPDL with catalytic maleic anhydride ...... 66

4-7 RAFT polymerization of MPDL and NEtMI in toluene (1 M) at 70 °C with [MPDL]:[NEtMI]:[CTA] = 50:50:1, in the absence and presence of AIBN...... 69

4-8 Incorporation of MPDL in copolymerizations ...... 70

13 4-9 C NMR spectrum of P(MPDL-alt-NEtMI) copolymer in CDCl3...... 72

4-10 Chain-extension of P(MPDL-alt-NEtMI) and subsequent degradation of P(MPDL-alt-NEtMI)-b-PS...... 73

5-1 Alternating radical ring-opening copolymerization of CKAs with N-substituted maleimides...... 81

5-2 Alternating radical ring-opening copolymerization of CKA 5,6-benzo-2- methylene-1,3-dioxepane (BMDO) with N-substituted maleimides ...... 82

5-3 Copolymerization of BMDO and N-ethylmaleimide ...... 83

5-4 Copolymerization of BMDO and N-benzylmaleimide ...... 83

5-5 MALDI-ToF MS spectrum of RAFT-derived P(BMDO-alt-NBnMI) ...... 85

5-6 MALDI-ToF MS spectrum of RAFT-derived poly(BMDO-alt-NEtMI)...... 85

5-7 RAFT polymerization of BMDO and NEtMI ...... 86

5-8 Functionalization of poly(BMDO-alt-TCTMI) with furfuryl thiol and benzyl amine...... 90

1 5-9 H NMR spectra of BMDO in CDCl3 ...... 95

1 5-10 H NMR spectra of TCT-MI in CDCl3 ...... 96

A-1 Nile red calibration and nanoparticle loading...... 102

B-1 MALDI-ToF spectrum of degraded P(MPDL-alt-NEtMI)...... 103

B-2 MALDI-ToF spectrum...... 103

1 C-1 H NMR spectra of BMDO and maleic anhydride in CDCl3...... 104

D-1 Poly(BMDO-co-NEtMI) ...... 105

D-2 Poly(BMDO-co-NEG3MI)...... 105

D-3 Poly(BMDO-co-NPhMI)...... 106

D-4 Poly(BMDO-co-NBnMI) ...... 106

D-5 RAFT-derived poly(BMDO-co-NBmMI)...... 107

D-6 Poly(BMDO-co-NEtMI-co-NBnMI-co-NEG3) ...... 107

10 LIST OF SCHEMES Scheme page

3-1 Preparation of PSI from acid-catalyzed condensation of L-aspartic acid and hydrolysis to PASP ...... 41

3-2 Proposed PSI-based nanodelivery system ...... 43

3-3 Preparation of amphiphilic PSI copolymers by reaction with 2(2- aminoethoxy)ethanol (2AEE), NaOH, or hexylamine (HA)...... 44

LIST OF ABBREVIATIONS

AIBN 2,2’-Azobisisobutryonitirle

BMDO 5,6-benzo-2-methylene-1,3-dioxepane

CKA Cyclic ketene acetal

CTA Chain transfer agent

D-A Donor-acceptor

Dh Hydrodynamic diameter

DLS Dynamic light scattering

DMAc N,N-Dimethylacetamide

DMF N,N-Dimethylformamide

DMSO Dimethylsulfoxide

DSC Differential scanning calorimetry

GPC Gel permeation chromatography

MacroCTA Macro chain transfer agent

MALS Multi-angle light scattering

MAnh Maleic Anhydride

MDO 2-Methylene-1,3-dioxepane

MI Maleimide

Mn Number average molecular weight

MPDL 2-Methylene-4-phenyl-1,3-dioxolane

MW Molecular weight

Mw Weight average molecular weight

MWCO Molecular weigh cutoff

MWD Molecular weight distribution

NIPAM N-Isopropylacrylamide

NMR Nuclear magnetic resonance spectroscopy

OEG Oligoethylene glycol

PEG Polyethylene glycol

PS Polystyrene

RAFT Reversible addition-fragmentation chain transfer

RDRP Reversible-deactivation radical polymerization

ROP Ring-opening polymerization rROP Radical ring-opening polymerization

SEC Size exculsion chromatography

Td Degradation temperature

TEA Triethylamine

TEM Transmission electron microscopy

Tg Glass transition temperature

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

STIMULI-RESPONSIVE AND BIODEGRADABLE POLYMERS

Megan Rae Hill

May 2018

Chair: Brent S. Sumerlin Major: Chemistry

The use of stimuli-responsive, “smart,” polymers in biology, medicine, and materials have contributed to some of the most groundbreaking advancements over the last decade. Imparting degradability onto responsive systems is important to reduce the concern of the eventual environmental and biological fate of these materials, and enable their application to new fields or functions. We first utilize the concept of polymers that are both responsive and degradable to develop a polymeric drug-delivery system for applications in agriculture. Unlike drug-delivery to humans, plants do not have an excretory system to rid its system of any remaining materials after the delivery event; therefore any materials used must be innocuous and biodegradable. The system was designed for specific delivery to plant phloem, the vascular tissue in plants that transports nutrients and photosynthates, due to its ability to access many areas within the plant, as well as its susceptibility to plant pathogens and diseases. While the majority of plant tissue exhibits a slightly acidic pH (~5-6), the phloem exhibits a slightly alkaline pH (~7.5-8.5). Therefore, pH-responsive nanocarriers were developed to release their loaded components under an alkaline environment similar to plant phloem.

Next, a novel route to develop degradable polymers with the ability to easily tune polymer properties and add functionality was realized using radical-ring opening polymerization (rROP) of cyclic ketene acetals (CKAs). CKAs have been widely used to synthesize polyesters or to incorporate degradable ester moieties onto vinyl polymers under radical conditions. However, copolymerization of CKAs with most vinyl monomers results in poor incorporation of the ester moiety due to low reactivity of CKAs and the propensity to copolymerize through the vinyl bond, without ring opening to the ester moiety. Following the concepts of donor-acceptor radical copolymerization (e.g., styrene-maleic anhydride), we found that the copolymerization of CKAs with maleimides proceeds in an alternating fashion, with 70% and 100% ring-opening of the CKA, depending on the CKA used, producing alternating polymers with a homogenous incorporation of degradable ester. The polymer properties could additionally be easily tuned by modification of the N-substituent of the maleimide, providing a straightforward route to degradable polyester copolymers for many different applications.

CHAPTER 1 STIMULI-RESPONSIVE AND BIODEGRADABLE POLYMERS

1.1 Overview

This dissertation focuses on the synthesis of responsive and degradable

polymeric materials from i) the step-growth polymerization of L-aspartic acid, with

applications in controlled drug-delivery in agriculture, and ii) the chain-growth radical

ring-opening copolymerization of cyclic-ketene acetals and N-substituted maleimides. A

brief overview of the synthesis of stimuli-responsive and degradable polymers is

presented in this chapter.

1.2 Stimuli-Responsive Materials

Stimuli-responsive, or “smart,” polymers are designed to change a specific property in response to a change in their surrounding environment. These materials

have been designed to respond to a variety of stimuli including temperature, pH, light,

redox potential, and small molecules, with a resulting chemical or physical property

change (e.g., chain conformation, solubility, topology, charge density, etc.) (Figure 1-

1).1–3 Such “smart” polymers have become instrumental for a range of applications

including controlled drug-delivery, (bio)sensing, adaptive shape memory materials, and

tissue engineering.4–6

One of the most studied applications of responsive polymers is in targeted and

controlled drug-delivery. Drug nanocarriers that can be programmed to target diseased

areas in the body are desirable to decrease nonspecific interactions with healthy tissue

and to increase the biodistribution time, which prevents excretion from the body before

reaching its targeted area.5 Polymer-based nanocarriers typically utilize responsive

polymers that transition from hydrophobic to hydrophilic in the presence of a specific stimuli, inducing the disassembly of the nanocarrier to release the cargo. These drug- delivery systems can be programmed to respond to exogenous stimuli such as light, ultrasound, or magnetic fields; or to endogenous stimuli, such as the variations of local pH in specific tissue, or an increase in glutathione concentration.

Figure 1-1. Stimuli-responsive polymers

The most well-known and studied responsive polymers are derived from radical

polymerization and typically contain non-degradable carbon-carbon backbones (e.g., pH-responsive poly[(meth)acrylic acid] and poly[N,N-dimethylaminoethyl (meth)acrylate]

and temperature responsive poly[N-isopropylacrylamide] and poly[oligo(ethylene glycol)

(meth)acrylate], etc.). However, imparting degradability onto these responsive, “smart,” polymeric systems has significant potential for reducing the concern of the environmental and biological fate of these materials,7 as well as enabling their

application in new fields or functions.

1.3 Biodegradable Polymers

Biodegradable polymers can be divided into two groups: natural and synthetic.

Natural polymers are derived from plants, animals, and microbes and are typically polysaccharide- or protein-based in composition.8 Many natural polymers are appealing

as they are available from renewable biosources and typically demonstrate good

biocompatibility. Synthetic polymers, on the other hand, are more versatile in their

applications, with the ability to tailor specific properties and alter the degradation rate

with the chemistry that is used.9

The inherent ability of biodegradable polymers to breakdown over time has led to

their prolific use in applications such as biomaterials and pharmaceuticals. Additionally,

as the accumulation of plastics becomes more obvious and the concern for the

environmental fate of materials increases, the development of degradable polymeric

packaging, housewares, and personal care products has become more important.7 The basic criteria for biodegradable materials include (1) the ability to match the mechanical properties of the intended application, (2) a degradation profile in concomitant with its function, (3) the break down of the material into non-toxic degradation products, (4) suitable shelf-life and stability, (5) processability, and (6) cost.10 Given the wide-ranging use of biodegradable polymers, many different polymers have been modified and developed to achieve the properties needed for the intended applications. Herein, we will highlight select examples of stimuli-responsive systems built from a platform of biodegradability.

1.4 Responsive and Biodegradable Natural Polymers

While many polymers exist in nature, the most used naturally derived polymers are easily isolated, abundant, and biocompatible. Some of the most useful natural polymers have proven to be polysaccharide-based polymers, such as cellulose, starch, alginate, chitosan, hyaluronic acid, and chondroitin sulphate; protein-based polymers, including collagen, gelatin, elastin-like peptides (ELPs), albumin, fibrin, and naturally occurring poly(amino acids) (i.e., poly(γ-glutamic acid)); and microbial-synthesized poly(hydroxyalkanoates) (Figure 1-2).8,11 Unlike synthetic polymers, the chemical composition and structure of naturally occurring polymers has already been set by nature. Response to stimuli is therefore either inherent to the polymer structure (e.g., containing pH-responsive acidic or basic groups or thermoresponsive peptide sequences) or arises from chemically modifying the polymer. Additionally, it is common for an inherently responsive material to be further modified to enhance its response or to be programmed to respond to multiple stimuli.

Figure 1-2. Chemical structures of some natural biodegradable polymers

Cellulose, for example, contains hydroxyl groups, which participate in hydrogen bonding. Inherent stimuli-response can therefore be induced by increasing or decreasing the amount of hydrogen bonding (e.g., by introducing competitive hydrogen bonding molecules), which can affect the strength, or self-healing properties of cellulose-based material. Alternatively, the hydroxyl groups can be used as a chemical handle for the incorporation of responsive moieties.12

Initially inspired by the sea cucumber and its ability to reversibly alter the stiffness

of its connective tissue, the groups of Weder and Rowan have studied mechanically-

responsive materials derived from cellulose nanocrystals (CNCs).13 They demonstrated

that incorporation of CNCs into rubbery polymer matrices (1:1 ethylene oxide-

epichlorohydrin copolymer or poly(vinyl acetate)) results in nanocomposites that can change their modulus in response to a competitive hydrogen bonding solvent (Figure 1-

2 A). This response arises due to the hydrogen bonding between the hydroxyl moieties on the surface of the CNCs. An extensive degree of hydrogen bonding between CNCs results in a nanocomposites with high moduli. However, introduction of a competitive hydrogen-bonding solvent disrupts the intermolecular hydrogen bonding, and reduces the overall modulus of the nanocomposite. The nanocomposite can therefore reversibly change from a stiff material with high modulus to a soft material with lower modulus in

response to hydrogen bonding solvent. Additionally, modifying the CNCs with carboxyl

and amine groups can induce the same type of reversible changes in stiffness in

response to changes in pH (Figure 1-2 B).14

Figure 1-3. Stiffness-changing polymer cellulose nanocrystal (CNC) nanocomposites. A) Pictures of a sea cucumber in its relaxed state and in its stiff state, the structures of cellulose and the rubbery polymers used, and a schematic representation of the mechanism whereby the CNCs hydrogen bonding interactions can be switched on and off with competing solvent. Reproduced with permission from ref. 13. Copyright 2008 American Association for the Advancement of Science. B) Modification of CNC nanocomposites to produce pH-responsive stiffness changing nanocomposites. Reprinted with permission from ref. 14. Copyright 2012 American Chemical Society.

Elastin-like polypeptides (ELPs), are another class of natural polymers that have gained increasing attention recently. ELPs are derived from tropoelastin and demonstrate thermoresponsive behavior.15 The general structure of ELPs consist of

pentatpeptide repeat Val-Pro-Gly-Xaa-Gly (VPGXG) sequences, where Xaa represents

a variable amino acid that can be used to manipulate the specific transition

temperature.16 ELPs demonstrate a lower critical solution temperature (LCST) (also

referred to as an inverse transition temperature) in aqueous media in which below their

LCST, they exist as linear monomeric polymers, and above their LCST, the ELPs

collapse into an aggregated, coacervate phase. Furthermore, ELPs have been modified

to respond to other stimuli such as pH,17 redox triggers,18 and light.19 Within the last

decade, responsive-ELPs have been extensively studied for applications in drug

delivery, tissue engineering, and mechanical actuation.

Recently, Luo and Kiick developed thermoresponsive conjugates of ELPs and

collagen-like peptides (CLPs), demonstrating the ability to further manipulate the

thermoresponsive behavior of ELPs.20 CLPs interact reversibly to form triple helices, similar to native collagen below a melting temperature (Tm), which is dependent on the

specific CLP sequence. The CLP-ELP conjugates demonstrated a significantly lower

LCST temperature compared to the unconjugated ELP (a difference of about 80 °C) and

assembled into uniform vesicles at temperatures as low at 4 °C. Additionally, the CLP

Tm of the conjugate was much higher than the CLP alone. It is expected that the ELPs

and CLPs work cooperatively within the conjugate, where at lower temperatures, three

CLPs come together to form a helix, which induces coacervation of the ELPs at a

temperature much lower than their unconjugated form. Similarly, at higher

temperatures, the aggregated ELPs stabilize the CLP helix to a much higher Tm

compared to the unconjugated form.

While many useful materials have been synthesized from natural polymers, they continue to suffer from difficult isolation procedures, batch-to-batch variation, and

antigenicity.8 Synthetic polymers are therefore desirable to tailor the polymer properties

for specific applications in a reproducible manner.

1.5 Responsive and Biodegradable Synthetic Polymers

The majority of biodegradable polymers rely on degradation by hydrolysis. The rate of degradation and the degradation mechanism (e.g., surface versus bulk erosion)

plays a significant role in the possible applications of the material. A drug-delivery nanocarrier, for example, should degrade within a few days into byproducts that can be easily excreted by the body. Degradable plastics used for water bottles, however, should take many years or specific stimuli to degrade to preserve its shelf life and

intended use. Control over degradation rate relies heavily on the type of hydrolyzable

bonds incorporated in the backbone (i.e., esters, carbonates, anhydrides, phosphates, acetals, urethanes, etc.), where increased hydrolysis occurs with esters, phosphates, and acetals moieties, and very stable, long-lasting linkages are formed by urethanes and amides. Aside from the hydrolysis rate of the backbone linkages, degradation is further modulated by the ability of water to diffuse into the material, polymer crystallinity, the size and shape of the device, and the solubility and diffusion of the degraded byproducts. Additionally, while many polymers have been synthesized with specific mechanical properties and degradation profiles, polymers with FDA or clinical approval are the polymers of choice for most applications. Biomaterials are therefore dominated by FDA-approved polylactide and polyglycolide (co)polymers.21 Nevertheless, the last

decade has brought about new synthetic techniques with the ability to produce a wide

array of novel stimuli-responsive degradable materials, as will be outlined in the

following sections.

Figure 1-5. Structures of some synthetic biodegradable polymers

1.5.1 Step-Growth Polymers

The majority of commercially available degradable polymers are prepared by step- growth or condensation polymerization, such as poly(ethylene terephthalate) (PET) and polycarbonate (PC) (derived from bisphenol A). Step-growth polymerization is an appealing method to prepare responsive polymers due to the versatility of attainable backbone chemistry. Many step-growth polymerization techniques traditionally required high temperatures and low pressures for removal of small molecules byproducts (e.g.,

water, alcohols, acids) to reach the high monomer conversion required to produce high

molecular weight polymers,22 thereby limiting the incorporation of many functional

groups. Recently, the use of click and multi-component chemistry to generate step- growth polymers has greatly extended the breadth of functional and responsive polymers attainable through step-growth polymerization (Figure 1-6). These reactions are typically highly efficient with high atom economy, exceptionally modular, proceed under mild conditions, and often do not require removal of a byproduct to drive the reaction forward. Many click reactions have recently been employed for the synthesis of step-growth polymers including copper-catalyzed azide-alkyne cycloaddition

(CuAAc),23–25 thiol-ene and thiol-yne coupling,26,27 the Diels-Alder reaction,28 and oxime

formation.29 Both reactive AB and AA + BB difunctional monomers can be used to synthesize step-growth polymers (Figure 1-6A); however most examples utilize AA and

BB comonomers to prevent oligomer formation before polymerization or the use of protection/deprotection chemistry, and to expand the wealth of achievable backbone chemistries by employing two different comonomers.

Figure 1-6. Step-growth polymerization of functional AB and AA + BB monomers: A) general mechanism for step-growth polymerization; b) examples of “click” chemistry to synthesize step-growth polymers; c) examples of multicomponent reactions to synthesize step-growth polymers.

The mild reaction conditions employed in click reactions allow for the synthesis of functional and responsive step-growth polymers under ambient reaction conditions, often in the presence of water and oxygen, and with access to a vast assortment of polymerizable monomers. For example, thiol-ene chemistry has been used to generate high molecular weight polymers with carbonate, zwitterionic, and alcohol backbone functionality under ambient conditions by synthesizing dithiol or diene monomers containing the desired functionality (Figure 1-7 A).27 The formation of backbone thioethers further demonstrated redox sensitivity, with controlled oxidation to sulfoixdes or sulfones, drastically changing the polarity of polymer backbone. The mild and high- yielding conditions afforded by thiol-ene step-growth polymerization has furthermore been used to incorporate temperature-responsive azo-functionality into polymer backbones, producing poly(β-thioester)s with temperature-dependent degradation

(Figure 1-7 B).30 Given the sensitivity to high heat, an azo-functional polymer could not

have been achieved by traditional condensation polymerization.

Figure 1-7. Thiol-ene step-growth polymerization: A) Step-growth from divinyl and dithiol AA + BB monomers to yield thiol-ether polymers with the ability to undergo selective oxidation. Reprinted with permission from ref. 27. Copyright 2016 Wiley-VCH. B) Thermally-responsive azo-containing polymer synthesized via thiol-ene step-growth polymerization. Reprinted with permission from ref. 30. Copyright 2016 American Chemical Society.

Multicomponent reactions (MCRs) have more recently become utilized in the

synthesis of step-growth polymers, with successful polymerization being achieved by the Passerini,31 Ugi, Biginelli,32 Kabachnik-Fields,33 Cu-catalyzed reactions34,35 as well

as other notable MCRs (Figure 1-6 B).36 Similar to “click” reactions, MCRs exhibit high

efficiency, high atom economy, and modularity, which allows for mild reaction

conditions, high yield, and incorporation of a diverse array of functionality. Theato et al.,

for example, demonstrated a step-growth MCR polymerization using the Kabachnik-

Fields MCR with dialdehydes, diamines, and phosphites to synthesize highly functional

polymers, including zwitterionic and photodegradable polymers, by utilizing α-amino phosphonates and photolabile dialdehyde monomers, respectively.33 Although this reaction produces water as a byproduct, the reaction proceeded to high conversion without the need to remove water. Additionally, while click reactions allow the synthesis

of a library of monomers derived from one to two comonomers, MCRs are based on three or more starting monomers, further expanding the possible polymer backbone chemistries accessible through step-growth.

1.5.2 Chain-Growth Polymers

Despite the advances in step-growth polymerization methods, which have allowed nearly an infinite number of possible polymer backbone chemistries to be achieved, the step-growth mechanism inherently leads to different molecular weights and large polydispersity values. The use of chain-growth mechanisms is therefore

desirable to achieve polymers with predetermined molecular weights, narrow molecular

weight distributions, and access to (multi)block copolymers. The most straightforward

way to achieve degradable chain-growth polymers is through ring-opening

polymerization (ROP) of cyclic monomers containing heteroatoms. Many types of cyclic monomers have been developed to ROP, with some of the most common including:

lactones, lactams, carbonates, disulfides, anhydrides, silicones, and phosphates, and

can proceed under various mechanisms (cationic, anionic, methathesis, metal and

organo-catalyzed, radical, etc.) (Fig. 1-8).

Figure 1-8. Ring-opening polymerization of functional cyclic monomers: A) General ring- opening polymerization (ROP) mechanism with examples of cyclic monomers for the synthesis of b) polyesters, c) polycarbonates, d) polyamides, e) and others. Initiating and propagating species (*) may be anionic, cationic, radical, or metal-coordinated centers.

Generating polymers with response to stimuli by ROP generally requires functionalization of the cyclic monomer with the desired responsive group. Many

examples of temperature, redox, pH-responsive, etc. degradable polymers have been synthesized accordingly. However, synthesis of functional monomers can be very challenging, often requiring multiple-step syntheses with the possibility of altering the ring-strain of the monomer, which may reduce the propensity to ring-open and polymerize. Additionally, many desired responsive groups might be incompatible with the polymerization mechanism, rendering direct polymerization of many responsive groups impossible.

Alternatively, incorporation of a functional group that can be modified post- polymerization is attractive to introduce multiple different functionalities, including functionality that may be incompatible with the polymerization mechanism or characterization techniques, for example, GPC. A wide range of “click” functionalities have therefore been incorporated into ROP monomers, including allyl, alkyne, azide, norbornene, activated ester, and maleimide groups.9 This chemistry allows flexibility in

polymer composition by allowing covalent attachment of various functional molecules

post-polymerization, and without backbone degradation, due to the mild reaction

conditions, orthogonality, and simple work-up procedures.

For example, Baker et al. synthesized thermoresponsive polylactides with

pendent oligo(ethylene glycol) (OEG) substituents from direct polymerization of glycolides functionalized with different OEG chain-lengths.37 Four different glycolides

were synthesized, each requiring a five-step synthesis with moderate yield (Figure 1-9

A). However, only two of the four OEG-polylactides demonstrated any thermoresponse,

and due to the lack of functionality, the polymer properties could not be altered after

polymerization. A few years later, the same group reported a dipropargyl-functionalized

glycolide that could be functionalized with alkyl and OEG azides post-polymerization via copper-catalyzed azide-alkyne cycloaddition (CuAAc).38 The dipropargyl glycolide was

synthesized in four steps with a good yield and easy scalability, to enable gram-scale

synthesis of propargyl functional polyester. The resulting polymer was functionalized

using copper-catalyzed “click” chemistry with a variety of OEG and alkyl azides to obtain

the desired thermoresponse. This post-polymerization mechanism allowed for facile tuning of the thermoresponsive behavior, demonstrated by the ability to access polymers with a range of lower critical solution temperatures (LCSTs) from 22-69 °C, as well as enabled and self-assembly of the polymers into stable nanoparticles.

Figure 1-9. Synthesis of thermoresponsive oligo(ethylene glycol) (OEG) polylactides from A) direct polymerization of OEG-functionalized glycolides and B) post- polymerization “click” functionalization of propargyl-functional glycolides. Adapted from ref. 37 and 38.

Many ROP methods have been combined with click chemistry techniques to obtain diverse, functional, and responsive degradable materials, much of which can be found in detail in the literature, including overviews on functional polyesters,39

polycarbonates,40,41 N-carboxyanhydride-derived polypeptides,42 and

poly(phosphoester)s.43 As such, responsive polymers with degradable backbones can

be synthesized by direct incorporation of the responsive group onto the polymer

backbone, or by post-polymerization modification using efficient chemistry to modulate

the resulting response for specific applications.

1.6 Properties and Applications of Responsive and Degradable Polymers

Incorporation of functionality and responsiveness into degradable polymers has

allowed tuning of properties for a vast array of potential applications. The most widely

studied application of responsive and biodegradable polymers is in biomaterials.

Previous work in this area focused on the development of biocompatible and nontoxic

materials for a variety of functions from tissue engineering to drug delivery. Of the

promising materials for biomedical purposes, aliphatic polyesters, specifically poly(lactic

acid) and poly(lactide), poly(lactide-co-glycolide), and poly(ε-caprolactone) have been

among the most heavily studied.44 These materials are derived from readily available

and low cost starting materials and display low toxicity of the polymers and their

degradation products. Poly(lactic acid), for example, is hydrolyzed to lactic acid which

can be removed in the Krebs cycle.45

Current work in the field has focused on controlling the degradation rate,

manipulating mechanical properties for use in various applications (for both hard and

soft tissue), and incorporating biologically active molecules for targeting or tissue

regeneration. Many of these goals can be achieved by tuning the polymer backbone

chemistry, controlling copolymer sequence, and by incorporating molecules onto the

polymer side-chains. Polymer degradation, for example, can be tuned by the

incorporation of specific backbone chemistry (e.g., esters degrade faster than carbonates, and polypeptides are stable within our lifetimes), the polymer molecular weight and degree of crystallization, manipulation of the comonomer sequence (e.g., in

poly(lactide-co-glycolide)), tuning of the hydrophilic/hydrophobic balance, and by chain-

end functionalization.10,46,47

The ability to control chemical functionality has been particularly useful in the

design of polymeric drug delivery systems. For example, polymer gene delivery

systems, in which polymers can complex to and deliver RNA and DNA, have significant

potential in treating genetic disorders, cancer, and viral infections.48 However, these

systems require a careful balance of cationic and hydrophobic functionality to aid with

complexation of the DNA or RNA, internalization into the cell, and finally, escape from

the endosome after uptake. The use of post-polymerization modification has enabled the synthesis of a library of degradable cationic polymers for screening of polymeric structures with optimized delivery. For example, Cheng et al. developed poly(γ-4- vinylbenzyl)-L-glutamate) (PVBLG) with alkene functionality placed 11 σ-bonds from the polypeptide backbone, which allowed incorporation of a high density of cationic charge without disrupting the secondary α-helix.49 Many known cell-penetrating peptides adopt

secondary α-helices, which allows interactions with the lipid bilayers of the cell

membrane. Using PVBLG as a reactive scaffold, the Cheng group synthesized a library

of cationic, helical polypeptides by ozonolysis and subsequent aminolysis with 31

different primary and secondary amines.50 Polypeptides with high transfection efficiency

of DNA and low cytotoxicity were identified and, interestingly, transfection efficiency

showed a strong correlation with the ability of the polycation to form helices. Similarily,

Siegwart et al. synthesized more than 130 lipocationic polyesters from direct functionalization of an ene-functional lactone (5,6-dihydro-2H-pyran-2-one) with amino and alkyl thiols, demonstrating the necessity of optimized amino and hydrophobic monomers for high delivery activity of short interfering RNA.51

1.7 Conclusion and Future Outlook

Natural and synthetic biodegradable polymers have been extensively utilized and

studied for applications in biomaterials and pharmaceuticals. More recently, the need to

replace non-degradable plastic packaging, housewares, and personal care products

has become apparent as the accumulation of non-degradable plastics becomes

problematic. The last decade has built upon efficient chemistry to develop routes to

highly functional, responsive, and degradable materials. “Smart” properties can

therefore be easily screened and manipulated by tuning the ratio or composition of

different functionalities. These smart materials have allowed for the modulation of

precise responses to specific environments, and importantly, the ability to synthesize

polymer precursors that can be modified and applied to a variety of applications.

There is still much to be explored with respect to the synthesis and application of

responsive degradable materials; including moving towards sustainably sourced

materials for monomer and catalyst development to reduce dependency on petroleum

and coal-based resources, expanding the scope of backbone functionality achievable

through ring-opening polymerization, and access to functional degradable polymers with

properties that can be easily be manipulated for a multitude of applications, from

replacement of current non-degradable polymers in packaging and manufacturing, to synthetic tissue scaffolds and drug-delivery systems.

CHAPTER 2 RESEARCH OBJECTIVE

The purpose of this research was to develop novel responsive and degradable

materials. Responsive polymers have played an integral role in advanced “smart”

materials; however, the majority of responsive polymers are synthesized with non-

degradable carbon-carbon backbones. My research goals were to develop a responsive

and degradable drug-delivery system that could be applied to agriculture for site-specific drug-delivery and to develop new methods to access degradable and responsive

materials. These endeavors will be discussed in the following Chapters.

The development of a pH-responsive and biodegradable polymeric drug

nanocarrier for site-specific delivery in plants is described in Chapter 3.

Polysuccinimide, synthesized from condensation polymerization of L-aspartic acid, was

used as the pH-responsive polymer for the delivery system. The nanocarriers

demonstrated controlled release of encapsulated molecules at high pH, which is

desirable for specific delivery within the plant phloem – the vasculature tissue that

transports nutrients and photosynthates in plants. Importantly, the L-aspartic acid

derived polymers showed minimal toxicity to plant tissue.

In Chapters 4 and 5, we demonstrate the use of radical polymerization to

synthesize degradable ester-containing copolymers. Radical polymerization is a

desirable technique to use in polymer synthesis due to its robust and mild reaction

conditions and the availability of a wide variety of vinyl monomers. While radical

polymerization typically results in non-degradable carbon-carbon backbones, radical

ring-opening polymerization involves cyclic monomers with exo-methylene bonds that

ring-open upon radical addition, enabling the synthesis of polymer backbones with

degradable moieties. We employed radical ring-opening polymerization with cyclic ketene acetal (CKA) monomers, which yield polymers with esters in the backbone. The use of radical ring-opening polymerization in copolymerizations has been sought after in attempts to impart degradability onto typical vinyl polymers. Copolymerization of CKAs, however, typically results in low incorporation of the CKA comonomer due to its low reactivity and propensity to polymerize through the vinyl bond, without ring-opening.

We became interested in the copolymerization of CKAs with electron deficient comonomers, such as maleic anhydride and maleimides. Given the electron-rich nature of CKAs, we hypothesized that the copolymerization would proceed in an alternating fashion, incorporating a high degree of CKA comonomer. In Chapter 4, we utilize CKA

2-methylene-4-phenyl-1,3-dioxolane (MPDL) and demonstrate the alternating tendency of copolymerization with N-ethylmaleimide, with 70% ring-opening of the CKA comonomer, providing a high and regular distribution of ester bonds along the polymer backbone. Using reversible addition-fragmentation chain transfer (RAFT) polymerization, copolymers and block copolymers were synthesized with predetermined molecular weights and narrow molecular weight distributions. Given the high content of ester, copolymers were rapidly degraded under basic conditions. We additionally found that copolymerization with maleic anhydride led to spontaneous oligomerization and degradation of MPDL, due to the significant electron disparity between the two monomers.

Finally, in Chapter 5, we optimized radical ring-opening copolymerization by employing the CKA 5,6-benzo-2-methylene-1,3-dioxepane (BMDO), which led to quantitative ring-opening and access to a fully polyester-based copolymer. We

demonstrate the facile tuning of polymer properties by altering the N-substituent on the maleimide comonomer, as well as the incorporation of functionality, by copolymerizing a cyanuric chloride (TCT)-functional maleimide for subsequent post-polymerization modification.

CHAPTER 3 BIODEGRADABLE AND pH-RESPONSIVE NANOPARTICLES DESIGNED FOR SITE- SPECIFIC DELIVERY IN AGRICULTURE

3.1 Overview

Although pH-responsive materials have been extensively studied in the realm of

medicine, less attention has been given to the application of these adaptive materials in

agriculture.52–54 However, the efficiency of fertilizer remains low even in modern

agriculture, and pesticides often fail to kill pathogens, as only a small portion of the

effective compounds ever reach their targeted sites.55,56 Despite the relative lack of

attention in agricultural sciences, responsive polymeric nanoparticles have significant

potential to enhance the delivery efficacy of pesticides, nutrients, and drugs, which can

in turn provide valuable benefits to help cure deadly plant diseases.57–59 Specific

delivery to the phloem, the vascular tissue in plants that aids in the transport of nutrients

and photosynthates, is desirable not only because of its critical role in carrying nutrients

but also because many plant pathogens reside in the phloem (i.e., phloem-limited

pathogens) and are responsible for numerous deadly crop diseases, such as citrus

huanglongbing (HLB).60 While most plant tissue exists in a slightly acidic environment,

the phloem exhibits a higher, slightly alkaline pH.61 Thus, much like pH-responsive

nanoparticles designed to exploit the low pH of cancer cells, a nanodelivery system

designed to respond to the higher pH of the phloem may be useful for site-specific

delivery in plants, thereby potentially enhancing the efficiency of delivered components

(Figure 3-1).1

Figure 3-1. Preparation of polysuccinimide (PSI)-based pH-responsive nanoparticles for controlled release of loaded compounds at alkaline pH.

Many fundamental questions arise when applying stimulus- responsive62 polymers for delivery in plants. For instance, additional consideration must be given to how the nanoparticles enter plant cells and are subsequently transported to targeted sites. As opposed to a cell membrane, which takes in materials of various sizes by endocytosis, plants possess a cell wall, which is more ordered and exhibits specific

63 pore diameters of ∼30 nm. Therefore, it is important to carefully control the size of polymer nanoparticles so they can readily pass through the cell wall and reach the plasma membrane. Once in the plasma membrane, the loaded nanoparticles can be further transported to the targeted sites along apoplastic and symplastic pathways by diffusion or electrochemical gradients.

Given the lack of an excretory system in plants, the fate of materials used for such applications is another important concern. While the most well-known and studied pH-responsive polymers {e.g., poly[(meth)acrylic acid] and poly[N,N- dimethylaminoethyl(meth)acrylate]} have proven to be effective in a number of physiological applications, they typically contain nondegradable all-carbon backbones, which limits their use in plants. Because biodegradability is of utmost importance for the delivery to plants to reduce concerns about environmental fate and sustainability, new types of stimulus-responsive biodegradable materials are needed.6,52,64 The

construction of nanoparticles suitable for delivery to plant phloem thus becomes more complicated. The nanoparticles must be (i) responsive to the basic pH found in the phloem, (ii) small enough to enter the plant cell through cell wall junctions, and (iii) biodegradable to reduce the extent of accumulation over time. Additionally, these nanoparticles would ideally be equipped with moieties to facilitate transport along electrochemical gradients and, most importantly, be capable of encapsulating guest compounds, including hydrophobic and hydrophilic small molecules or drugs.

Scheme 3-1. Preparation of PSI from acid-catalyzed condensation of L-aspartic acid and hydrolysis to PASP

Polysuccinimide (PSI) has attracted attention for many years because of the biodegradable and hydrophilic nature of its derivatives, namely, poly(aspartic acid)

(PASP) and poly(hydroxyethylaspartimide) (PHEA).65–70 PSI is derived from the ring- closing condensation polymerization of L-aspartic acid. Subsequent hydrolysis of the polymeric repeat units under mildly basic conditions results in the hydrophilic derivative,

PASP, with both α and β ring-opened units (Scheme 3-1).71 The biodegradability of

PASP derived from the hydrolysis of PSI has been previously documented, although longer degradation periods compared to those of other poly(amino acids) were necessary, which is likely due to the presence of a mix of L- and D-aspartic acid units as well as the β-hydroxyl structures in the backbone.67,72–74 Nevertheless, the degradation is still expected to progress as opposed to that of polymers prepared radically with

polymethylene backbones. Because of the reactivity of PSI toward primary amines, previous reports have involved various moieties being readily incorporated onto the PSI backbone to give fully functionalized PASP derivatives. Alternatively, PSI has been partially functionalized, with the remaining succinimidyl units being hydrolyzed to give

PASP copolymers.75,76 Additionally, many groups have incorporated stimulus- responsive moieties onto the PSI backbone (i.e., hydrazone bonds,77–79 amines,65,68,80 thiols and disulfides,81–83 carboxyls,75,84imidazole,85 etc.) to impart responsiveness onto the biodegradable PASP backbone.

We, however, are interested in exploring the inherent pH-responsive nature of

PSI. Because PSI is hydrolyzed at elevated pH to form water-soluble and biodegradable

PASP, we envisioned employing PSI as a potential platform for the development of a site-specific delivery system for agricultural applications. Thus, utilizing PSI as our pH- responsive and hydrophobic scaffold, we aimed to prepare a nanosized delivery system to capitalize on the higher pH of the phloem (Scheme 3-2). Our approach includes partially functionalizing the backbone of PSI to form self-assembled nanoparticles and relying on the hydrolytic lability of the remaining succinimidyl groups for stimulus responsiveness. While PSI is not water-soluble at neutral pH, its succinimidyl groups are hydrolyzed at elevated pH to yield derivatives of water-soluble PASP.86 This pH- driven solubility transition may provide a convenient mechanism for inducing supramolecular dissociation of PSI-based polymeric assemblies. We reasoned that this transition in solubility could be exploited to allow PSI-based polymers to serve as a platform for site-specific, pH-responsive guest molecule release. Moreover, because the resulting PASP-based copolymers are known to be biodegradable, the change in water

solubility may simultaneously increase the rate of degradation of the polymeric

byproduct. Although previous reports have utilized PSI as a precursor for the

development of stimulus-responsive (co)polymer materials, to the best of our

knowledge, the inherent pH-responsiveness of PSI itself has yet to be investigated.

Scheme 3-2. Proposed PSI-based nanodelivery system: (a) PSI is synthesized through the step-growth condensation reaction of L-aspartic acid and (b) functionalized with hydrophilic primary amines to prepare amphiphilic and pH- responsive PSI copolymers. (c) Amphiphilic PSI copolymers are assembled into nanoparticles and, (d) loaded with hydrophobic molecules, (e) which may disassemble and release loaded components at elevated pH, leaving behind the water-soluble and biodegradable poly(aspartic acid) derivative polymer.

3.2 Results and Discussion

3.2.1 Synthesis of Amphiphilic PSI Copolymers and Nanoparticle Formation

PSI was synthesized by acid-catalyzed condensation polymerization of L-aspartic

71,86 acid (Mn = 13 500; Mw/Mn = 1.52). The resulting polymer was partially functionalized

by reaction with N-alkyl primary amines or via partial hydrolysis with NaOH to yield

amphiphilic PSI derivatives (Scheme 3-3). The resulting polymers were dissolved in

DMF and precipitated into water to form stable polymeric nanoparticles. A carefully controlled balance of hydrophilic and hydrophobic character was required to obtain PSI- based polymers capable of assembling in aqueous medium to form PSI-based nanoparticles, as previously demonstrated.87,88

Scheme 3-3. Preparation of amphiphilic PSI copolymers by reaction with 2(2- aminoethoxy)ethanol (2AEE), NaOH, or hexylamine (HA).

We first explored the functionalization of PSI with 2-(2-aminoethoxy)ethanol

(2AEE). The degree of functionalization for all of the copolymers was determined by 1H

NMR spectroscopy by comparing the backbone succinimidyl proton (-CHCON-) (5.2 ppm) to the shift of the backbone methine proton on the opened PASP unit (4.5 ppm)

(Figure 3-2). We found that incorporation of >5 mol % 2AEE rendered the entire polymer chain hydrophilic to the extent that it was completely water-soluble and unable to form nanoparticles. Similarly, PSI modified by partial hydrolysis of the succinimidyl

units by titrating the dissolved polymer with a low mole percent of NaOH also showed that hydrolysis of >5 mol % led to a completely water-soluble polymer that did not assemble into nanoparticles. However, amphiphilic copolymers were readily accessible by limiting the functionalization of 2AEE (PSI-2AEE) or NaOH hydrolysis (PSI-PASP) to

<5 mol %. We hypothesized that the incorporation of hydrophilic moieties in the functionalizing agent and the concomitant increase in hydrophilicity of the backbone unit as hydrophobic succinimide units were converted to hydrophilic aspartamide/aspartic acid units could be responsible for the increased solubility observed at this low degree of functionalization. Therefore, we reasoned that it might be possible to tune the amphiphilicity of PSI by partial functionalization with hydrophobic amines, relying only on ring opening of succinimidyl units to impart hydrophilicity.

Figure 3-2. 1H NMR spectra of PSI-HA copolymers with functionalization 7-92%.

To explore higher degrees of functionalization, we employed hexylamine (HA) to

modify PSI. The more hydrophobic nature of HA compared to 2AEE allowed the

preparation of amphiphilic copolymers from 1 to 100% functionalization.87 Both the

degree of functionalization and the solution concentration of the copolymers prior to

precipitation were explored as variables for controlling nanoparticle size. Contrary to

previous reports, we found little to no relationship between the degree of

functionalization and nanoparticle size. However, nanoparticle size was dependent on

the concentration of the copolymer solution, with smaller nanoparticles being formed as

the solution became more dilute, as determined by DLS and TEM analysis (Figure 3-3).

While previous methods have been used to prepare PASP nanoparticles with diameters

89,90 of >100 nm, we found that using precipitation concentrations of ∼20 mg/mL produced nanoparticles with diameters of ≤30 nm, which may potentially allow

nanoparticles of this type to diffuse through the cell wall of plant cells.

Additionally, copolymers both functionalized with HA (15 mol %) and partially

hydrolyzed with NaOH (1 mol %) were capable of forming stable nanoparticles. The

presence of the carboxylate groups on the nanoparticle surface was supported by zeta

potential measurements. While the control 15% PSI-HA exhibited a zeta potential of 0.0

± 0.54 mV, the partially hydrolyzed 15% PSI-HA nanoparticle revealed a zeta potential

of −33.0 ± 1.9 mV (pH 4.5). Incorporating carboxylate groups into the nanoparticle shell

could be valuable for chelating water-soluble nutrients or facilitating the transport of the

nanoparticle along electrochemical gradients present within plant tissues.

Figure 3-3. PSI-nanoparticles formed by nanoprecipitation of amphiphilic PSI-copolymer solutions into DI water. The size of the nanoparticle could be controlled by the copolymer solution concentration before precipitation, as depicted by: a) Z- average hydrodynamic diameter as a function of solution concentration for several copolymers, which shows an increasing Dh with increasing solution concentration; b) DLS overlay of PSI-HA (55% HA functionalization) nanoparticles prepared from different solution concentrations (100 mg/mL, 50 mg/mL, 20 mg/mL, 10 mg/mL, and 5 mg/mL); and c) TEM image of nanoparticles formed at 50 mg/mL.

3.2.2 Loading of Model Hydrophobic Molecule Nile Red

With the method of generating stable nanoparticles of controlled size determined, we next sought to evaluate the capacity of the nanoparticles to store and release hydrophobic molecules. Nile red, a hydrophobic dye that served as a model active

compound,91 was dissolved in acetone and added to an aqueous solution of PSI

nanoparticles. After the mixture had been stirred in an open vessel to allow the

evaporation of acetone, unencapsulated dye was removed by filtration, leaving a

transparent purple solution of dye-loaded nanoparticles. TEM and DLS showed that the

dye loading did not significantly alter the nanoparticle size or shape (Figure A-1). The

difference in absorption maximum was observed from 540 to 575 nm for the

nanoparticles, with the variations depending on the hydrophobic environment of each

nanoparticle, as has been noted in the literature.92 For example, PSI-HA nanoparticles

with 5, 50, and 100% functionalization gave absorption maxima of 575, 560, and 555

nm, respectively. To determine loading capacity, the solutions of Nile red-loaded

nanoparticles were freeze-dried and dissolved in DMF to release all encapsulated dye.

A calibration of the fluorescence of Nile red in DMF at varying concentrations allowed

the concentration of dye to be calculated for each nanoparticle solution (Figure A-1).

The loading capacity of nanoparticles with various sizes and functionalization ranged

from 1 to 33 wt % with an initial dye:polymer ratio of 1:1 (w/w) (Table 3-1).

Table 3-1. Nanoparticle diameter and loading capacity of Nile red

a Loading Dh b c Copolymer PDI capacity [nm] [%]

1% PSI-HA (PSI137-co-(PSI-HA)2) 37 0.25 7.3

1% PSI-PASP (PSI137-co-PASP2) 65 0.20 24

1% PSI-2AEE (PSI137-co-(PSI-2AEE)2) 27 0.30 12

5% PSI-HA (PSI132-co-(PSI-HA)7) 27 0.27 9.8

10% PSI-HA (PSI125-co-(PSI-HA)14) 29 0.24 12

30% PSI-HA (PSI97-co-(PSI-HA)42) 78 0.12 6.1

50% PSI-HA (PSI70-co-(PSI-HA)69) 83 0.11 20

100% PSI-HA (PSI-HA)139 75 0.16 4.8

10% PSI-HA (PSI125-co-(PSI-HA)14) 83 0.39 16

10% PSI-HA (PSI125-co-(PSI-HA)14) 8 0.31 33 aHydrodynamic diameter as measured by DLS (Z-Avg). bPolydispersity. cLoading capacity ((grams Nile red/ grams polymer) × 100)

3.2.3 Response of the Nanoparticle to Alkaline pH and Release of Nile Red

To study the pH-responsiveness and the drug release of the PSI-based nanoparticles, the loaded nanoparticles were exposed to buffered solutions, and the change in fluorescence intensity was monitored over 72 h. Because Nile red is hydrophobic in water and fluoresces only within the hydrophobic interior of the nanoparticle, fluorescence is expected to decrease as the succinimidyl units are hydrolyzed and the nanoparticles disassemble to release the dye. While the hydrolysis of PSI under basic conditions is well known, we expected some hydrolysis would still occur under neutral conditions, albeit at a reduced rate. We thus first explored the hydrolysis kinetics at various pH values with 1% PSI-2AEE. As expected, the release rate was rapid at pH 8.5 and 8 because of the hydrolysis of the succinimidyl backbone

of the PSI units, with approximately 80 and 60%, respectively, of the dye being released at 30 h. On the other hand, PSI is more stable under neutral and acidic conditions; therefore, at pH 7 and 6, less than 40 and 20%, respectively, of the dye was released over 72 h (Figure 3-4a).

Figure 3-4. Release of Nile Red from PSI-based copolymer nanoparticles (a) with 1% PSI-2AEE in buffers at various pHs (6, 7, 8, and 8.5); (b) with varying degrees of functionalization of PSI-HA (5, 10, and 50%); (c) with varying functionalizing moieties (1% PSI-HA, PSI-2AEE, and PSI-PAsp); and (d) with varying nanoparticle sizes at pH 8.5.

Release studies also showed that the functionalizing moiety (2AEE, HA, and

NaOH) did not significantly affect the release rate (Figure 3-4c), but incorporating high

degrees of functionality, in the case of PSI-HA, slowed the release or prevented

nanoparticle disassembly, with minimal release being observed in alkaline environments

(Figure 3-4b). We hypothesize that the hydrophobicity of the hexyl chains kept the

copolymer sufficiently amphiphilic to maintain nanoparticle stability, even after complete

hydrolysis. Greater than 15% functionalization with HA appeared to render the material

sufficiently hydrophobic to prevent disassembly and any dye release. Lastly, a copolymer of 10% PSI-HA was precipitated at different concentrations to produce nanoparticles of varying sizes (13, 28, and 83 nm). The release at pH 8.5 suggested hydrolysis was slightly faster with smaller nanoparticles (Figure 3-4d), which is potentially due to the increased surface area. Although a small amount of hydrolysis occurs at neutral pH, almost no hydrolysis occurs in acidic environments. Because plant tissue is slightly acidic (pH ∼5–6) except in the phloem (pH ∼8), the nanoparticles offer considerable promise for site-specific delivery in agricultural applications.

3.2.4 Plant Toxicity

To evaluate any possible toxicity of the polymers toward plant tissue, a method was developed using living plant tissue. Citrus seeds were planted on germination medium and cultured in the dark at 25 °C for 5 weeks, until the seedling reached the length of the culture tube. Each seedling was then cut into fragments and seeded on plates filled with MSBC medium with varying concentrations of the polymers (PASP,

PSI, and PSI-HA) (Figure 3-5 a-c). The plates were then put into a growth chamber with alternating 12 h lighting cycles and analyzed after 8 and 21 days to determine the percent of living tissue (Figure 3-5 d). As shown in Figure 3-5 d, PASP, PSI, and PSI-

HA showed limited toxicity up to concentrations of 196 μg/mL. While extremely high concentrations of PSI and PSI-HA showed toxicity (0% tissue viability at 385 μg/mL PSI-

HA), PASP at 385 μg/mL showed no toxicity. It should be noted that although PASP dissolved easily in the MSBC medium, the more hydrophobic PSI homopolymer and

PSI-HA copolymer required DMSO to fully dissolve into the medium, which may have influenced the results of the toxicity assays for these (co)polymers at high

concentrations. Nevertheless, the relatively low toxicity of the PSI-based polymers to plant tissue provides further evidence of their promising potential for future applications in agriculture.

Figure 3-5. Plant tissue toxicity study of various polymer concentrations after 21 days of incubation. Images of plant tissue on germination medium with varying concentrations of (a) PASP (b) 10% PSI-HA, and (c) PSI; and (d) percent tissue viability versus polymer concentration.

3.3 Conclusion

Responsive nanoparticles were developed to capitalize on the higher pH of plant phloem for the design of a site-specific delivery system to plants. Amphiphilic copolymers based on PSI were synthesized by functionalization with various amines that provided a convenient means of tuning the hydrophilic–hydrophobic balance needed for nanoparticle formation. Controlling the degree of functionalization and nanoprecipitation conditions proved to be viable methods of programming nanoparticle size, and could prove useful when developing new systems for delivery applications.

The nanoparticles were loaded with a model hydrophobic compound and showed controlled release at alkaline pH, with increased rates at higher solution pH and lower degrees of functionalization. Lastly, the toxicity of the polymers was tested on plant tissue, with only minimal toxicity being observed at reasonable concentrations of the polymers.

3.4 Materials and Methods

3.4.1 Materials

L-Aspartic acid (98%), o-phosphoric acid (85%), hexylamine (99%), 2-(2- aminoethoxy)ethanol (98%), and Nile red were purchased from Sigma-Aldrich.

Potassium phosphate monobasic (Fisher) was used to prepare 0.1 M phosphate buffers with adjusted pH values for release studies. Murashige and Skoog basal salt mixture

(MS salts) purchased from Phytotechnology Laboratories. Benzyl adenine (BA), myoinositol, Claforan (cefotaxime), and a plant cell viability assay kit were obtained from

Sigma-Aldrich. All organic solvents were used as received.

3.4.2 Characterization

1H NMR spectroscopy was performed using a Varian Mercury 300 or Varian

Inova 500 spectrometer with deuterated dimethyl sulfoxide (DMSO-d6) as the solvent.

The molecular weight and polydispersity were determined by size exclusion

chromatography (SEC) in N,N-dimethylacetamide (DMAc) with 50 mM LiCl at 50 °C and

a flow rate of 1.0 mL min–1 (Agilent isocratic pump, degasser, and autosampler; PLgel 5

μm guard column and two ViscoGel I-series G3078 mixed bed columns, with molecular

weight ranges 0–20 × 103 and 0–100 × 104 g/mol, respectively). Detection consisted of

a Wyatt Optilab T-rEX refractive index detector operating at 658 nm and a Wyatt

miniDAWN Treos light scattering detector operating at 659 nm. Absolute molecular

weights and polydispersities were calculated using Wyatt ASTRA. Transmission

electron microscopy (TEM) was conducted with a Hitachi H7000 microscope operating

at 100 kV. A freshly glow discharged (Pelco easiGlow, Ted Pella, Inc.) Formvar-coated

200-mesh Cu grid was placed on a 0.1 mg/mL drop of solution for 30 s. The grid was

dried and stained with a 0.5% aqueous uranyl acetate solution. Dynamic light scattering

(DLS) analysis was conducted at room temperature on a Zetasizer Nano-ZS (Malvern)

operating at a wavelength of 633 nm. UV–vis and fluorescence spectroscopy

measurements were taken on a Molecular Devices SpectraMax M2Multimode

Microplate reader with a λexcitation of 530 nm and a λemission of 650 nm.

3.4.3 Synthesis of Polysuccinimide (PSI)

Polysuccinimide was prepared as previously reported.66,77 Briefly, L-aspartic acid

(20 g, 0.15 mol) and phosphoric acid (10 g, 0.10 mol) were added to a 500 mL round-

bottom flask. The reaction vessel was placed under nitrogen and heated to 180 °C while its contents were being stirred for 2 h. The product was dissolved in N,N- dimethylformamide (DMF, 100 mL) and precipitated into cold methanol. The product was then collected via vacuum filtration and washed with additional methanol and water to remove any remaining DMF.

3.4.4 Preparation of Functionalized Nanoparticles

The nucleophile (HA, 2-AEE, or NaOH) was added to a solution of PSI dissolved in DMF and stirred at room temperature overnight. The functionalized copolymer was then added dropwise to a beaker of stirring deionized water. For example, for a copolymer functionalized with 20% HA (20% PSI-HA), PSI (498 mg, 5.15 mmol) was dissolved in DMF (5 mL), and HA (0.13 mL, 1.0 mmol) was added dropwise to the solution. The solution was stirred at room temperature overnight and precipitated into deionized water (200 mL). The aqueous nanoparticle solution was transferred to dialysis tubing (Spectra/Por, molecular weight cutoff of 3500) and placed in deionized water, which was replenished daily for 1 week.

3.4.5 Loading and Release of Nile Red

A solution of Nile red dissolved in acetone was added to an aliquot of a nanoparticle solution and stirred for several hours (1:1 dye:polymer weight ratio). The acetone was allowed to evaporate freely from the solution, and any unencapsulated Nile red was removed by filtration. The loading efficiency was calculated via fluorescence spectroscopy using a calibration curve of Nile red in DMF. Loaded samples were

freeze-dried and dissolved in DMF before being measured. Similarly, loading capacity

was defined as where Mencapsulated is the mass of dye

encapsulated by the nanoparticles and Msystem represents the mass of the nanoparticles.

To study the release of Nile red, the nanoparticle solutions were placed in 0.1 M

phosphate buffers at varying pH values. The fluorescence of the solutions was

monitored with a λexcitation of 530 nm and a λemission of 650 nm over 72 h.

3.4.6 Preparation of Germination Medium for the Plant Toxicity Assay

MS salts (2.15 g), myoinsoitol (50 mg), FM stock (1.865 g of Na2EDTA and

FeSO4·7H2O (1.390 g) into 500 mL; 5 mL), and sucrose (15 g) were added to an

autoclaved beaker. The solution pH was adjusted to 5.7, and the volume was brought to

1 L. Agar (7 g) was added to the medium and heated for 30 min to obtain the final

germination medium.

3.4.7 Preparation of Citrus Seeds

Germination medium (12 mL) was dispensed into autoclaved culture tubes.

Healthy and viable citrus seeds from grapefruit (Citrus paradisi) and sweet orange

(Citrus sinensis) were selected, and the outer seed coats were removed. Seed kernels

were kept moist at all times. Each seed was placed in a sterile autoclaved beaker with a

stir bar and stirred in 300 mL of the following solutions for the indicated time intervals:

70% alcohol (2 min), 10% sodium hypochlorite (10 min), and three sterile DI water

rinses (2 min).

3.4.8 Culture of Citrus Seeds

One seed was inserted into each germination medium-filled culture tube. The test tube racks were wrapped with plastic wrap and doubly wrapped with aluminum foil to minimize light exposure. Finally, the test tube racks with seeds were placed on the bottom of a growth chamber for 5 weeks, when the etiolated seedlings were used for toxicity screening.

3.4.9 Preparation of MSBC medium

MS salts (4.3 g), myoinositol (100 mg), GM stock [10 mL of a solution of glycine

(20 mg), nicotinic acid (50 mg), pyridoxine HCl (100 mg), and thiamine HCl (100 mg) in

DI water (500 mL)], sucrose (30 g), and BA (2 mg) were dissolved in DI water. The pH of the solution was adjusted to 5.7, and additional DI water was added to bring the volume to 1 L. Agar (8 g) was added and the solution autoclaved for 25 min. After the solution had cooled, 1 mL of filtered and sterilized (500 mg/mL) Claforan stock was added to obtain 500 μg/mL MSBC medium. The medium was dispensed into sterilized culture dishes, and different concentrations of polymers were added before solidification when the MSBC medium was in liquid phase near room temperature.

3.4.10 Toxicity assessment by tissue culture

Citrus plants were cut into 1–2 cm segments and placed on an appropriate

MSBC medium-filled culture dish. The dishes were then put into a growth chamber with

alternating 12 h light and dark periods for 21 days. Alive and dead segments were counted after 8 and 21 days.

CHAPTER 4 ALTERNATING RADICAL RING-OPENING COPOLYMERIZATION AFFORDS HOMOGENEOUS INCORPORATION OF DEGRADABLE UNITS

4.1 Overview

2Controlling monomer sequence in chain-growth polymerization has been a

continuous challenge in the attempt to synthetically mimic the precise nature of many

biological macromolecules.93,94 Although alternating polymerization of two monomers

provides a rather simple primary sequence (-A-B-A-B-), the synthesis of nearly perfectly alternating polymers is readily achievable through radical polymerization of donor- acceptor (D-A) monomer pairs.95 Furthermore, employing reversible-deactivation radical

polymerization (RDRP) techniques96–100 imparts additional control over polymer synthesis through predetermined chain lengths, simultaneous growth of all polymer chains, and narrow molecular weight distributions.

Maleic anhydride (MAnh) and N-substituted maleimides (MI) are widely used as acceptor monomers in alternating polymerizations.101 The vinyl bonds of MAnh and MI

are electron poor and sterically hindered, thereby discouraging homopolymerization or

lowering the homopropagation rate. This combined electronic deficiency and steric

hindrance increases the alternating tendency in copolymerizations with electron rich

monomers (e.g., styrene, vinyl acetate, etc.). Lutz et al. have extensively demonstrated

the ability to synthesize precision polymers using the copolymerization of styrene and

MIs with nitroxide-mediated polymerization or atom transfer radical polymerization.102–

110 In particular, Lutz has utilized the increased cross-propagation rate of D-A monomer

pairs to place MIs in specific regions of growing polystyrene (PS) chains, creating polymers with precisely placed functionality. MIs have additionally been employed to synthesize 1:2 (ABB) sequence-controlled chain-growth polymers by copolymerizing with limonene.111–113 The extensive range of available or easily accessible MIs,114 along

with the tendency to form alternating copolymers with most donor monomers, gives rise

to a library of easily accessible precision and sequence-controlled polymers.

D-A radical copolymerization affords a versatile and robust route to functional,

responsive, and precision polymers; however, the use of radical polymerization

traditionally leads to polymers with non-degradable carbon-carbon backbones. The

biological and environmental fate of polymeric materials is a significant concern, and the

ability for polymers to break down after use has become increasingly important. We

therefore hoped to extend the versatility of D-A copolymerizations to afford copolymers

that contained both alternating repeat units and a degradable backbone.

The use of radical ring-opening polymerization (rROP) offers an effective route to

incorporate degradable linkages into the backbone of vinyl polymers.115,116 The rROP mechanism relies on cyclic monomers with exomethylene groups that are susceptible to radical addition and subsequent ring-opening to impart degradable moieties onto the polymer backbone. Cyclic ketene acetals (CKAs), in particular, have been used to synthesize polyesters or to incorporate degradable ester moieties in vinyl polymers under radical conditions.117 However, with the exception of less-activated monomers

such as vinyl acetate,118,119 copolymerization of CKAs with most vinyl monomers (e.g.,

styrenics, acrylates, methacrylates, acrylamides, etc.) typically results in a low

incorporation of CKA into the copolymer.120 High equivalents of CKA relative to the

comonomer must therefore be used to achieve a reasonable degree of degradable units into the polymer, and even then the CKA likely incorporates in a gradient fashion (during

RDRP) rather than randomly along the backbone. However, to achieve homogeneous degradation, the incorporation of randomly and/or regularly placed degradable moieties is necessary. Synthesis of degradable polymers by copolymerization of CKAs is further complicated by the fact that ring-opening of the CKA is required to yield the desired ester moiety needed for degradability. However, in many cases 1,2-addition across the

vinyl group of the CKA (i.e., without ring opening) is highly competitive. Therefore, an

ideal scenario for preparing degradable polymers from CKAs would be one where there

is a high and uniform distribution of CKA units along the backbone and where the

fraction of CKA units that undergo ring opening is high.

Figure 4-1. Synthesis of alternating degradable polymers from conventional and RAFT- mediated radical ring-opening copolymerization of MPDL and NEtMI.

Given the electron-rich nature of CKAs, we postulated that copolymerization with

electron deficient MAnh and MIs could result in alternating copolymers with a regular

and high incorporation of degradable moieties. While 2-methylene-1,3-dioxepane

(MDO, also termed MDP) is often employed in rROP copolymerization to mimic poly(ε-

caprolactone), we believed 2-methylene-4-phenyl-1,3-dioxolane (MPDL)121 would be a

better candidate given its higher propensity for ring opening. Additionally, the resulting

ring-opened benzylic radical (analogous to propagating styrenics),122–124 may increase

the alternating tendency of copolymerization with acceptor monomers (Fig. 1). Herein,

we demonstrate that the large electron disparity between MPDL and cyclic anhydrides

elicits spontaneous reactions and enhances the alternating tendency of

copolymerization, thereby enabling the synthesis of alternating copolymers with a high

incorporation of backbone degradable moieties via D-A radical polymerization.

4.2 Results and Discussion

In the first attempt to synthesize alternating MPDL-containing copolymers, we

employed MAnh as the electron deficient comonomer due to its well-known tendency to

alternate with electron-rich monomers and its inability to homopolymerize. However,

mixing equimolar MPDL and MAnh in dioxane led to an exothermic reaction and rapid

color change (from clear, to red, then brown) at room temperature. Attempts at free

radical copolymerization of MPDL and MAnh led to low yielding (<20% mass) oligomers,

and 1H NMR spectroscopy showed degradation of MPDL at room temperature (Fig. 4-

2). Interestingly, replacing MAnh with N-ethyl maleimide (NEtMI) did not result in any

spontaneous reaction or degradation at room temperature, and high molecular weight

polymer was produced under conventional radical polymerization with 2,2’-

azobis(isobutyronitrile) (AIBN) as the initiating source (Fig. 4-3).

1 Figure 4-2. Copolymerization of MPDL and maleic anhydride (MAnh): H NMR in CDCl3 spectra of a) MPDL, b) MPDL and MAnh polymerization solution before polymerization and c) MPDL and MAnh post polymerization and purification; d) SEC trace of polymer precipitate.

Figure 4-3. Copolymerization of MPDL and N-ethylmaleimide (NEtMI): 1H NMR spectra of MPDL and NEtMI polymerization solution a) in CDCl3 before polymerization, and b) in DMSO-d6 post polymerization and purification; c) SEC trace of polymer precipitate.

We hypothesized that the vastly different reaction of MPDL with MAnh and NEtMI may be due to the increased electrophilicity of MAnh as compared to MIs, especially given the electron rich nature of CKA methylene bonds.125 Indeed, several reports have

demonstrated that spontaneous polymerizations and cyclization reactions can occur

between CKAs and electrophilic compounds including cyanoallene,126 β-

propiolactone,127 tulipalin A,128 cyanoacrylate,126 and methyl methacrylate.129 The

generally accepted mechanism of these reactions is believed to follow the ‘bond-forming

initiation theory’ established by Hall in the 1980s, in which charge-transfer complexes

initiate ionic or radical reactions via a tetramethylene intermediate (Fig. 4-4).130 The type

of reaction that occurs (ionic/radical, polymerization/cycloaddition) largely depends on

the level of electron disparity between the donor and acceptor molecules, as well as the

bond geometry, solvent polarity, and the temperature of the reaction.

Figure 4-4. A) Bond-forming initiation theory of donor-acceptor (D-A) monomer pairs in which charge-transfer complexes form zwitterionic or diradical tetramethylene intermediates that can initiate spontaneous polymerizations and cycloadditions.B) Potential tetramethylene formed between MPDL and cyclic anhydrides and resulting polymerization from zwitterionic or diradical intermediate (* * = diradical or zwitterionic pair intermediate).

To determine whether the electron disparity or charge-transfer complex between

MPDL and MAnh/MI could be leading to spontaneous reactions as outlined by the bond- forming initiation theory, the monomers were mixed together with and without solvent and monitored by 1H NMR. Given that MPDL can polymerize cationically or radically to

give two different polymers (a homopolymer of ring-retaining poly(cyclic acetal) or a

(co)polymer of ring-opened polyester, respectively, Fig. 4-4B),131 the resulting polymers

can provide insight into the nature of the initiating species. First, a catalytic amount of

MAnh was added to MPDL. After 2 h at room temperature, a homopolymer of the ring-

retaining poly(cyclic acetal) MPDL was isolated (Fig. 4-5). This indicates that the

addition of MAnh to a solution of MPDL leads to an ionic species, which can initiate the

cationic polymerization of MPDL. When higher amounts of MAnh were used, complete

degradation of MPDL was observed in all solvents and at low temperatures (0 °C) and

low concentrations (to 0.1 M). We believe the degradation arises from the

oligomerization of MPDL via a cationic species, rather than cycloadditions with MAnh,

as little consumption of MAnh is observed.

Figure 4-5. Reaction of MPDL with catalytic maleic anhydride (MAnh) at room 1 temperature: a) H NMR spectrum in DMSO-d6; b) SEC trace of purified 1,2- 13 vinyl ring-retaining PMPDL; and c) C NMR in spectrum in CDCl3.

Conversely, spontaneous reactions with NEtMI did not proceed at room

temperature for catalytic or stoichiometric amounts of NEtMI with MPDL. However,

heating an equimolar solution of MPDL and NEtMI in toluene above 60 °C produced a

copolymer of NEtMI and ring-opened MPDL (Fig. 4-6 and Table 4-1 entry 2). This indicated the spontaneous formation of a radical species upon heating MPDL and

NEtMI. In fact, a similar spontaneous radical polymerization between MDO and N- phenyl maleimide was previously demonstrated by Shi and Agarwal.132 Spontaneous

alternating copolymerization occurred between MDO and N-phenyl maleimide at high

temperatures. However most of the incorporated MDO was polymerized by traditional

vinyl polymerization, without ring-opening. The above findings are consistent with the

bond-forming initiation theory, demonstrating increased electron disparity leads to spontaneous ionic reactions (MPDL and MAnh), while moderate electron disparity leads to radical reactions upon heating (MPDL and MIs).

Figure 4-6. Reaction of MPDL and N-ethylmaleimide (NEtMI) at 70 °C in toluene: a) 1H NMR spectrum in DMSO-d6 and b) SEC trace of purified P(MPDL-co-NEtMI)

Table 4-1. Polymerizations of MPDL and NEtMI a b c Entry MPDL:NEtMI :CTA:AIBN Mn,theo Mn Mw/Mn molar equiv. (g mol-1) (g mol-1) 1 50:50:0:1 - 26,100 2.18 2 50:50:0:0 - 33,020 2.74 3 50:50:1:0.1 10,990 11,400 1.24 4 50:50:1:0 7,900 7,880 1.23 5 60:40:1:0.1 11,300 12,260 1.26 6 80:20:1:0.1 5,840 7,870 1.24 7 20:80:1:0.1 4,430 5,410 1.18 aAll polymerizations proceeded at a total monomer concentration of 1 M in dry toluene at 70 °C with 2 wt% pyridine. bTheoretical molecular weight was calculated using conversion of NEtMI by 1H NMR, assuming alternating polymerization and therefore that conversion of MPDL was equal to conversion of NEtMI cMolecular weights and molecular weight distributions were obtained using a polystyrene calibration curve.

To achieve copolymers of MPDL and NEtMI with predetermined molecular weights and narrow molecular weight distributions, reversible addition-fragmentation chain transfer (RAFT) polymerization was performed with and without an exogenous initiating source (AIBN) utilizing 4-cyano-4-

[(dodecylsylfanylthiocarbonyl)sulfanyl]pentanol as a chain transfer agent (CTA).

Toluene was used as the polymerization solvent, and total monomer concentrations were kept at 1 M. Pyridine (1-2 wt%, relative to MPDL) was also added to the polymerization to discourage cationic homopolymerization of MPDL.133 Under these

conditions at 70 °C, good control over polymerization was observed as shown by the

linear pseudo-first-order kinetic plot, increasing Mn with conversion, and the low Mw/Mn

of the resulting polymers (Fig. 4-7, Table 4-1 entry 3 and 4) for copolymerizations with

and without AIBN. RAFT polymerization in the absence of AIBN exhibited a much

longer inhibition time, likely due to the slow generation of radicals from the charge-

transfer complex, but otherwise produced well-defined copolymers. Changing the initial

monomer feed to non-stoichiometric amounts additionally gave good control and good

correlation to theoretical molecular weights by monitoring conversion of NEtMI and

assuming alternating polymerization (conversion of NEtMI = conversion of MPDL)

(Table 4-1, entries 5-7).

Figure 4-7. RAFT polymerization of MPDL and NEtMI in toluene (1 M) at 70 °C with [MPDL]:[NEtMI]:[CTA] = 50:50:1, in the absence and presence of AIBN (0.1 equiv.). A) Polymerization scheme; B) size exclusion chromatography (SEC) traces for conventional and RAFT polymerization with and without addition of AIBN; C) pseudo-first-order kinetic plots for RAFT polymerizations with and without AIBN; D) conversion versus molecular weight for RAFT polymerizations with and without AIBN.

Analysis of the 1H NMR spectrum of the purified copolymers revealed a 1:1 incorporation of MPDL and NEtMI by integrating the methyl protons of the maleimide (-

NCH2CH3) at 0.9 ppm compared to the aromatic protons of MPDL at 7.2 ppm (Fig. 4-

8A). Monomer reactivity ratios provide fundamental insight into specific chain microstructure and monomer addition to the propagating chain end. Therefore, to determine reactivity ratios and the alternating tendency of the copolymerization, varying molar ratios of monomers (0.2:0.8 to 0.8:0.2 MPDL:NEtMI) were subjected to RAFT polymerization with AIBN. Copolymerizations were quenched at low monomer conversion (≤20%) to maintain a relatively constant monomer feed ratio, and a high monomer to CTA ratio (200:1) was used to facilitate the isolation of polymer via precipitation after low monomer conversion. Final monomer composition was determined by 1H NMR spectroscopy and plotted against the initial monomer feed. As

shown in Figure 4-8B, the composition of the copolymers was consistently 1:1, with the exception of higher NEtMI feed, which resulted in a small excess of incorporated NEtMI.

Using the non-linear least squares method,134,135 reactivity ratios were determined to be

rNEtMI = 0.199 and rMPDL = 0.022, indicating that while NEtMI can homopolymerize, both

monomers prefer cross-propagation over homopropagation.

Figure 4-8. Incorporation of MPDL in copolymerizations: A) 1H NMR spectrum of P(MPDL-alt-NEtMI) in DMSO-d6; B) copolymer composition curve for the copolymerization of NEtMI and MPDL in toluene at 70 °C with [MPDL]:[NEtMI]:[CTA]:[AIBN] = 100:100:1:0.1. Polymerizations were terminated at conversion ≤20%. The grey line corresponds to the non-linear least squares fitting curve where rNEtMI = 0.199 and rMPDL = 0.022. C) Degradation of P(MPDL-alt-NEtMI) in 1 wt% KOH MeOH/THF (1/10 v/v) after t = 0 min, t = 5 min, and t = 10 min.

Given the high incorporation of the ring-opened MPDL, we expected rapid

degradation of the copolymer backbone under basic conditions. To demonstrate this,

100 mg of copolymer was dissolved in 1 mL of tetrahydrofuran, and 0.1 mL of 10 wt%

KOH in methanol was added. The solution rapidly turned dark red, indicative of the

136 formation of a maleimide enolate anion. SEC revealed a decreased Mn from 12,200

-1 to 2,100 g mol after 5 min, and full degradation was observed in 10 min, with a final Mn

of ~500 g mol-1 being observed (Fig. 4-7C). While it is expected that hydrolysis of

polymers with precisely alternating ester functionality would degrade into a single

molecule, we observed by SEC that full degradation was not achieved, and limiting

molecular weights of ~500 to 1,300 g mol-1 were often reached after extended exposure

to basic conditions. We suspected that this could be due to incomplete degradation

resulting from 1,2-vinyl incorporation of MPDL into the polymer. In fact, while original

reports by Bailey and Cho indicate 100% ring-opening at all temperatures and with all

comonomers tested,131,133 later reports demonstrated that about 20-30% of the 1,2- vinyl-polymerized, ring-retaining MPDL monomer was often incorporated into the final polymer backbone.137,138 Indeed, 13C NMR spectroscopy revealed that approximately

30% of ring-retaining MPDL was incorporated into the alternating polymer (Fig. 4-9).

However, given the low Mn oligomers after degradation, incorporation of degradable,

ring-opened MPDL units are likely to be randomly dispersed along the backbone

polymer. Degraded polymers were further subjected to matrix-assisted laser desorption-

ionization time-of-flight (MALDI-ToF) mass spectrometry, which showed mass relating

to alternating units of 3-6 ring-closed units (Fig. A-2 and A-3). This further corroborated

the alternating structure as well as a moderate amount of 1-2 vinyl, ring-retaining MPDL

moieties.

13 Figure 4-9. C NMR spectrum of P(MPDL-alt-NEtMI) copolymer in CDCl3.

Lastly, to demonstrate CTA chain-end retention after the copolymerization,

P(MPDL-alt-NEtMI) was chain-extended with styrene via RAFT at 70 °C for 24 h to

produce block copolymer P(MPDL-alt-NEtMI)-block-polystyrene (P(MPDL-alt-NEtMI)-b-

PS) (Fig. 4-10). 1H NMR spectroscopy of the purified copolymer showed the appearance of PS peaks after chain-extension (Fig. 4-10 B), and SEC showed a clear

-1 -1 shift in molecular weight from Mn = 10,500 g mol to 15,100 g mol with an Mw/Mn =

1.17 (Fig. 4-11 C), confirming the retention of the RAFT CTA. After exposing the block

copolymer to basic conditions, P(MPDL-alt-NEtMI)-b-PS was hydrolyzed to produce a

homopolymer of PS, thereby demonstrating near-complete degradation of the P(MPDL-

alt-NEtMI) block. 1H NMR spectroscopy showed the disappearance of P(MPDL-alt-

NEtMI) peaks, and SEC revealed a low molecular weight polymer with a Mn = 7,100 g

-1 mol and Mw/Mn = 1.08.

Figure 4-10. Chain-extension of P(MPDL-alt-NEtMI) and subsequent degradation of P(MPDL-alt-NEtMI)-b-PS; A) polymerization and degradation scheme; B) 1H NMR spectra and C) SEC trace of P(MPDL-alt-NEtMI) chain-extension with styrene and degradation of P(MPDL-alt-NEtMI)-block-polystyrene.

4.3 Conclusion

In conclusion, we have demonstrated the polymerization of a donor, CKA monomer, MPDL, and an acceptor maleimide monomer, NEtMI, to produce an alternating and degradable polymer using RAFT polymerization. Due to the electron rich nature of the MPDL, attempts at copolymerization with MAnh led to homopolymerization of MPDL or complete degradation/oligomerization of MPDL when equivalent amounts of

MAnh were introduced. Due to the lower electron disparity between NEtMI and MPDL, spontaneous reactions at room temperature did not proceed, however an increase in alternating tendency of the copolymerization and the ability to produce charge-transfer complex-derived radicals were realized. RAFT polymerization led to controlled copolymerization of MPDL and NEtMI, which could then be chain-extended to produce

degradable-block-nondegradable copolymers. The high incorporation of randomly positioned degradable ester bonds resulted in rapid degradation of the alternating homopolymer and block copolymers to polystyrene.

4.3 Materials and Methods

4.3.1 Materials

Cyclic ketene acetal (CKA), 2-methylene-4-phenyl-1,3-dioxolane (MPDL) was synthesized according to previous literature methods.121 Maleic anhydride

(recrystallized from methanol and chloroform), anhydrous toluene (99.8%), anhydrous

1,4-dioxane (99.8%), anhydrous N,N-dimethylformamide (DMF, 99.8%), 4-cyano-4-

[(dodecylsylfanylthiocarbonyl)-sulfanyl]pentanol and azobisisobutyronitrile (AIBN,

recrystallized from methanol) were purchased from Sigma-Aldrich. N-ethylmaleimide was purchased from Sigma-Aldrich (98+%) or VWR (Alfa Aesar, 98+%).

4.3.2 Analytical Techniques

1H and 13C NMR spectroscopy were recorded on a Bruker Avance 300

spectrometer or on an Inova 500 MHz spectrometer at 25 °C in CDCl3 or DMSO-d6.

SEC analysis was performed on two different systems: (1) In chloroform (HPLC grade) at 35 °C at a flow rate of 1.0 mL min-1 using a Tosoh ECO SEC HLC-8320 GPC with two columns from Polymer Laboratories (PL-gel MIXED-D 300 x 7.5 mm, beads diameter 5 micron; linear part 400 to 4 x105 g mol-1). Toluene was used as a flow-rate marker. The samples were filtered with a 0.2 micron PTFE filter before analysis. The calibration curve was based on polystyrene (PS) standards from Polymer Laboratories.

The EcoSEC analysis software enabled the determination of the number-average molar mass Mn, the weight-average molar mass Mw and the dispersity (Mw/Mn). (2) In N,N-

dimethylacetamide with 50 mM LiCl 50 °C and a flow rate of 1.0 mL min−1 using an

agilent isocratic pump, degasser, and autosampler with Viscogel I-series 10 μm guard +

two ViscoGel I-series G3078 mixed bed columns: molecular weight range 0−20 × 103 and 0−100 × 104 g mol−1 columns. Detection consisted of a Wyatt Optilab T-rEX refractive index detector operating at 658 nm and a Wyatt miniDAWN Treos light scattering detector operating at 659 nm. The system was calibrated using 10 polystyrene (PS) standards from 9.88 x 105 to 602 g.mol-1. Matrix assisted laser

desorption/ionization time-of-flight (MALDI-TOF) was performed on a AB Sciex 5800

MALDI TOF/TOF (Framingham, MA) spectrometer operated in linear, positive ion mode

with a 1 KHz N2 OptiBeam™ on-axis laser. Laser power was used at the threshold level

required to generate signal. Spectra were analyzed using Polymerix Version 3 software

(Sierra Analytics). Samples were prepared by mixing solutions of trans-2-[3-(4-t- butylphenyl)-2-methyl-2-propenylidine]malononitrile (DCTB, Santa Cruz Biotechnology,

≥99%) matrix (10 mg/mL in THF) and degraded product (1 mg/mL in THF) at a v:v ratio of 5:1 matrix:product and 2.00 µL were spotted and air dried on a polished stainless steel Bruker plate (residual potassium from polymer degradation was used as the ionizing salt).

4.3.3 Experimental Procedures

4.3.3.1 Conventional polymerization of MPDL and MAnh

MAnh (305 mg, 3.11 mol) and AIBN (10.0 mg, 0.061 mmol), were dissolved in

2.0 mL dioxane in a 20 mL vial. MPDL (507 mg, 3.12 mmol) in 2.0 mL dioxane was added and immediate color change (orange to red to brown) was observed. An aliquot of the solution was removed for 1H NMR spectroscopy analysis. The solution was placed in an oil bath at 80 °C for 2 h and precipitated into diethyl ether. Precipitate was collected by filtration and analyzed by 1H NMR spectroscopy and SEC.

4.3.3.2 Conventional polymerization with MPDL and NEtMI

NEtMI (388 mg, 3.10 mol) and AIBN (10.1 mg, 0.06 mmol), were dissolved in 2.0

mL dioxane in a 20 mL vial. MPDL (503 mg, 3.11 mmol) in 2.0 mL dioxane was added

to the vial, the solution remained colorless. An aliquot of the solution was removed for

1H NMR analysis. The solution was placed in an oil bath at 80 °C for 2 h and precipitated

into diethyl ether. Precipitate was collected by filtration and analyzed by 1H NMR

spectroscopy and SEC.

4.3.3.3 Polymerization of MPDL with catalytic MAnh

MAnh (1.9 mg, 1.9 μmol) was added to MPDL (170 mg, 1.04 mmol) and stirred at room temperature. After 2 h, crude 1H NMR spectroscopy and SEC showed presence of

polymer. The polymer was precipitated into cold methanol and further analyzed by 1H

NMR spectroscopy and SEC.

4.3.3.4 Reaction of MPDL and NEtMI

NEtMI (43 mg, 3.4 mmol) and MPDL (56 mg, 3.5 mmol) were dissolved in

toluene (6.0 mL) and stirred at room temperature or at 70 °C for 2 h. The reaction

solution was analyzed by 1H NMR spectroscopy and SEC and showed polymer

formation for reaction at 70 °C, but only monomer for reaction at room temperature. The

polymer formed at 70 °C was precipitated in cold ether and analyzed by 1H NMR

spectroscopy and SEC.

4.3.3.5 Typical RAFT polymerization of MPDL and NEtMI

In a typical RAFT polymerization, NEtMI (283 mg, 2.26 mmol), AIBN (0.74 mg,

0.0045 mmol), CTA (18.3 mg, 0.045 mmol), and 2.2 mL toluene were added to a 20 mL vial and purged with argon for 15-25 min. In a separate 20 mL vial, MPDL (367 mg, 2.26 mmol) in 2.2 mL toluene was purged with argon for 15-25 min. The MPDL solution was then added via a purged syringe into to the NEtMI, AIBN, and CTA solution and placed in an oil bath at 70 °C. Monomer conversion was monitored using 1H NMR spectroscopy

of sample aliquots, by monitoring the disappearance of the vinyl peaks of MPDL and

NEtMI. Polymerizations were conducted for 2-24 hours. NEtMI conversion was used for

kinetic data due to the propensity of MPDL to undergo degradation reactions and hydrolysis during polymerization and/or during sample characterization (e.g., small amounts of water in NMR solvents), leading to inconsistent integrations of MPDL during the polymerization. Polymerizations were terminated by removing from heat and exposing to air and purified by precipitating into diethyl ether 2-3 times.

4.3.3.6 Chain-extension with styrene

Macro-CTA (P(MPDL-co-NEtMI)) (49 mg, 0.0051 mmol) and AIBN (0.08 mg, 0.5

μmol) were dissolved in 0.5 mL toluene and purged with argon for 15-25 min. Styrene

(57 mg, 0.55 mmol) was added to the solution and placed in an oil bath at 80 °C for 24

hours. Polymerizations were terminated by removing from heat and exposing to air and

purified by precipitating into methanol 2-3 times.

4.3.3.7 Degradation of copolymers and block copolymers

Copolymers and block copolymers were degraded by dissolving P(MPDL-co-

NEtMI) or p(MPDL-alt-NEtMI)-block-polystyrene (50 mg) in THF (2 mL) and adding

potassium hydroxide solution (KOH, 1%) in methanol (1 mL). The solution was stirred at

room temperature. Kinetic samples were dried under vacuum, diluted with chloroform,

and filtered to remove salts. SEC was used to analyze degradation products and

complete degradation was achieved in 10 minutes. The presence of acid chain ends

after degradation can be responsible for aggregation of polymer chains due to their low

solubility in chloroform, which would lead to a larger apparent molecular weight. This

problem was overcome by adding 0.1 % (v/v) of TFA in chloroform to dissolve the

polymer and as eluent.

CHAPTER 5 TUNABLE AND FUNCTIONAL POLYESTER COPOLYMERS SYNTHESIZED BY ALTERNATING RADICAL RING-OPENING COPOLYMERIZATION OF 5,6-BENZO-2- METHYLENE-1,3-DIOXEPANE AND N-SUBSTITUTED MALEIMIDES

5.1 Overview

The prolific production and accumulation of non-degradable polymers over the

last century has emphasized the importance of developing useful polymers with the

ability to breakdown and degrade within our lifetime.7 Ideally, instilling degradability

should proceed without sacrificing specific polymer properties, performance, or

established synthetic methods.

Radical ring-opening polymerization (rROP), in which a cyclic vinyl monomer

undergoes ring-opening under radical conditions, is an attractive route to synthesize

degradable polymers using radical polymerization techniques.115,117 In particular, the

ability to copolymerize with typical vinyl monomers to impart degradability onto standard

vinyl polymers is an appealing aspect of rROP. The most studied monomers employed

in rROP are cyclic ketene acetals (CKAs), which ring-open to give ester moieties in the

polymer backbone. Copolymerization of CKAs with most vinyl monomers, however,

typically results in low incorporation of the degradable moiety. This is due to both the

low reactivity of most CKAs in copolymerizations, and to the propensity of CKAs to

polymerize through the vinyl bond (resulting in the non-degradable ring-retaining

structure) rather than ring-opening to the ester.

Recently, we demonstrated that the CKA 2-methylene-4-phenyl-1,3-dioxolane

(MPDL) undergoes alternating copolymerization with N-ethylmaleimide (NEtMI), following the concepts of donor-acceptor radical copolymerization (e.g., styrene-maleic

anhydride).139 While this alternating monomer sequence provides high and regular

incorporation of degradable moieties, about 30% of the incorporated CKA polymerized

through the vinyl bond rather than ring-opening to the degradable ester. Agarwal et al. has additionally reported the alternating copolymerization of the CKA 2-methylene-1,3- dioxepane (MDO) with N-phenyl maleimide (NPhMI), however, the CKA polymerized mostly through the vinyl-bond without ring-opening, unless high temperatures (120 °C) and high feed ratios of CKA (9:1 CKA:maleimide) were used (Figure 5-1 A).132

Figure 5-1. Alternating radical ring-opening copolymerization of CKAs with N-substituted maleimides. A) Previous work on alternating radical ring-opening copolymerization of cyclic ketene acetals with N-substituted maleimides to afford alternating CKA-MI copolymers, with ring-retaining acetal structures and B) alternating radical ring-opening copolymerization of CKA 5,6-benzo-2- methylene-1,3-dioxepane (BMDO), with quantitative ring-opening to polyester.

While the aforementioned copolymerizations provide a method to produce copolymers with a high degree of CKA incorporation, full ring-opening is desirable to

achieve a truly alternating polyester copolymer with ester groups precisely placed along

the polymer backbone. Control of monomer sequence has been a coveted goal in polymer chemistry, not only in the attempt to synthetically mimic information-carrying biomacromolecules like RNA, DNA, and protiens;93 but, additionally, due to the

considerable effects monomer sequence imparts on macroscopic properties such as

crystallinity,107 optoelectronic properties,140 and degradability.46,141 We therefore hoped

to develop a system that would lead to quantitative ring-opening of the CKA comonomer

to impart a higher degree of monomer sequence control and, subsequently, a higher

degree of degradability (figure 5-2).

Figure 5-2. Alternating radical ring-opening copolymerization of CKA 5,6-benzo-2- methylene-1,3-dioxepane (BMDO) with N-substituted maleimides

5.2 Results and Discussion

We hypothesized that alternating copolymerizations of CKAs and maleimides

could proceed with quantitative ring-opening of the CKA with a few optimizations. First,

CKA 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) replaced MPDL as the CKA comonomer. BMDO has a higher propensity to ring-open during copolymerizations due to the release of strain of the 7-membered ring and the stable resulting benzylic radical after ring opening.142,143 Second, higher temperatures (80-90 °C) and more dilute

solution conditions (0.5 M total monomer in toluene) were used to further favor ring- opening. Under these conditions, we found that copolymerization of BMDO with equimolar NEtMI or N-benzylmaleimide (NBnMI) resulted in high MW copolymer with a

1:1 incorporation of both BMDO and MI by 1H NMR spectroscopy (Fig. 5-3 and Fig. 5-

4). Analysis of the 13C NMR spectrum revealed the absence of any ketal carbons from

100 – 110 ppm, confirming the quantitative ring-opening of BMDO. Furthermore, size exclusion chromatography (SEC) showed complete degradation of the copolymers in 1

-1 wt % KOH MeOH/THF (1/10 v/v) to Mn = ~400 g mol by SEC (expected degraded

-1 alternating unit Mn = 305 g mol ) (Figure 5-3 A).

Figure 5-3. Copolymerization of BMDO and N-ethylmaleimide. A) SEC trace of polymer precipitate and degraded polymer solution; B) 1H NMR spectrum of 13 poly(BMDO-alt-NEtMI) precipitate in DMSO-d6; and C) C NMR spectrum of poly(BMDO-alt-NEtMI) precipitate in CDCl3

Figure 5-4. Copolymerization of BMDO and N-benzylmaleimide. A) SEC trace of polymer precipitate and degraded polymer solution; B) 1H NMR spectrum of 13 poly(BMDO-alt-NBnMI) precipitate in DMSO-d6; and C) C NMR spectrum of poly(BMDO-alt-NBnMI) precipitate in CDCl3

To gain insight into the copolymer monomer sequence, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-ToF MS) was employed to analyze P(BMDO-alt-NBnMI) and P(BMDO-alt-NEtMI) synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization to access low molecular weight copolymers. As shown in Figure 5-5, the MALDI-ToF mass spectrum of poly(BMDO-alt-NBnMI) revealed 5 polymer distributions with a repeat unit of 349.5 Da, the expected repeat unit molecular weight for the alternating copolymer. Distribution 1 is attributed to equal amounts of BMDO and NBnMI incorporation. Distributions 2 and 3 are attributed to polymer chains with one extra BMDO or NBnMI, respectively. These extra MI additions could arise from defects in the alternating sequence, or, more likely, due to an extra monomer at either the alpha or omega chain end. The minor distributions 4 and 5, however, arise from two and three extra NBnMI additions, which are due to defects in the alternating sequence. Polymer chains with higher MI to BMDO content are expected, however, given the previously reported reactivity ratios of CKAs

132,139 and MIs (rCKA ~ 0, rMI ~ 0.2). MALDI-ToF MS of P(BMDO-alt-NEtMI) demonstrated

a similar trend, but revealed an additional polymer distribution with 4 extra MI

monomers, which is consistent with previous work demonstrating the propensity of MI

homopolymerization based on the N-substituent of the maleimide (Fig. 5-6).144

Figure 5-5. MALDI-ToF MS spectrum of RAFT-derived P(BMDO-alt-NBnMI), showing alternating polymerization product with 5 distributions: (1) equal incorporation of BMDO and NBnMI; (2) one excess BMDO incorporated; (3) one excess NBnMI; (4) two excess NBnMI; (5) three excess NBnMI.

Figure 5-6. MALDI-ToF MS spectrum of RAFT-derived poly(BMDO-alt-NEtMI), showing 6 distributions with the expected repeat unit mass (287.3 Da): (1) equal incorporation of BMDO and NEtMI; (2) one excess BMDO incorporated; (3) one excess NEtMI; (4) two excess NEtMI; (5) three excess NEtMI.

Access to high MW RAFT-derived polymers were additionally obtained as shown in figure 5-7. Copolymerization of BMDO proceeded with good control over

polymerization as demonstrated by the linear pseudo-first-order kinetic plot and

increasing Mn with conversion. However, it should be noted that copolymerizations were

consistently ~20% lower than theoretical molecular weight. We suspect this was the

result of cycloadditions between BMDO and the maleimide comonomers, which often

accompany alternating copolymerizations due to the disparate electron density between

the monomers and the formation of charge-transfer-complexes.130 Additionally, after

attempts at copolymerizing BMDO with maleic anhydride, we found that incubation of

the monomers in CDCl3 overnight led to ~40% cycloadduct (Fig. A-4). We believe that

the reduced electron disparity between BMDO and maleimides results in less

cycloaddition, but that cycloaddition occurs concurrent to alternating polymerization.

Nevertheless, well-defined polymers were prepared using RAFT polymerization, albeit

with lower than expected molecular weights.

Figure 5-7. RAFT polymerization of BMDO and NEtMI. A) Pseudo-first-order kinetic plots; B) size exclusion chromatograph (SEC) kinetic traces during polymerization; C) conversion versus molecular weight.

To demonstrate the copolymerization versatility, BMDO was copolymerized with

a variety of N-substituted maleimides (Fig. A-5—A-10). As shown in Table 5-1, all

copolymerizations resulted in high molecular weight (MW) copolymer with ~1:1 incorporation of CKA and MI. Additionally, the copolymers demonstrated a diverse range of Tg’s, from 127 °C when N-phenyl maleimide (NPhMI) was employed as the comonomer (table 5-1, entry 3) to 3 °C with N-(methoxy triethylene glycol) maleimide

(NEG3MI) as the comonomer (table 5-1, entry 2). Furthermore, copolymerization with

two or more maleimides significantly altered the Tg compared to the original copolymers

(table 5-1, entry 6), demonstrating the ability to easily tune the copolymer thermal

properties by incorporating various maleimides. One significant challenge in ring-

opening polymerization (ROP) of cyclic esters is producing polyesters with high thermal

and mechanical properties. This is due to the difficulty in synthesizing cyclic monomers that incorporate rigid backbone functionality, particularly backbone aromatic groups. rROP of BMDO with maleimides provides a platform for incorporating backbone aromatic groups from BMDO and facile tuning of the resulting polymer properties by manipulation of the N-substituent on the maleimide comonomers, allowing access to a range of thermal and mechanical properties, including high thermal properties when rigid aromatic groups are incorporated.

Table 5-1: Copolymerization of BMDO with N-substituted maleimides Mn,MALS %BMDO Entry R Mw/Mn Tg (°C) Td (°C) g mol-1 [c] incorp.[d] 1 Et 12,220 1.6 49.6% 121 277

2 EG3 13,190 1.63 47.5% -17 282 3 Ph 7,980 1.59 49.5% 147 258 4 Bn 13,510 1.27 48.4% 113 287 5 Bn[a] 7,240 1.15 48.2% 97 265 [b] 6 Et, EG3, Bn 10,050 1.32 44.6%* 67 279 Unless otherwise noted, reactions were run in dry toluene with a total monomer concentration of 0.5 M at 80 °C with 2 mol% AIBN and BMDO:maleimide of 1:1 [a] Polymerized under RAFT conditions with CTA dodecylsulfanylthiocarbonylsulfanyl-2-methyl-propionic acid in a ratio BMDO:NBzMI:CTA:AIBN = 60:40:1:0.1 at 85 °C. [b] [BMDO]:[MIs] = 1:1; [BMDO]:[NEtMI]:[NEG3MI]:[NBnMI] = 3:1:1:1 [c] Determined by 100 % mass recovery [d] Determined by 1H NMR.

While the array of available or easily synthesized N-substituted maleimides

provides abundant opportunities for tuning the resulting alternating BMDO copolymer

properties, post-polymerizaiton of polymers enables the incorporation of functionality

that may be incompatible with the polymerization conditions. We therefore synthesized

a maleimide containing two pendent electrophilic sites using cyanuric chloride, or 2,4,6-

trichloro-1,3,5-triazine (TCT). We have previously reported on the facile and efficient modification of TCT end- and side-chain-functional polymers with amines and thiols.145,146 To demonstrate its applicability in alternating BMDO polyesters, the TCT-

maleimide (TCTMI) conjugate was copolymerized with BMDO, producing high MW

functionalizable polyester copolymers (Table 5-2, polymer 1). Subsequent nucleophilic aromatic substitution reactions on the pendent triazine using model nucleophiles afforded the incorporation of two distinct groups within polymer side chains.

Table 5-2: Functionalization of poly(BMDO-alt-TCTMI) with model thiols and amines

-1 Polymer Nucleophile [a]M0 (g mol ) Mn,theoretical (g/mol) [a]Mn,MALS (g/mol) Mw/Mn

1 -- 451.26 -- 14,180 1.66 2 511.92 16,110 16,300 1.63

3 528.96 16,650 16,190 1.43

4 582.62 18,330 15,700 1.34

5 599.66 18,870 21,340 1.35

Functionalization reactions were run in THF at 0 to 80 °C with [TCT]:[nucleophile]:;DIPEA = 1:1.2:1.5 [a] Theoretical molecular weight of TCT-MI repeat unit [b] Determined by 100 % mass recovery.

TCT-functional copolymers were first conjugated with either furfuryl amine or

furfuryl thiol from 0 °C to room temperature to achieve selective single substituition to

the TCT group. Incorporation of one equivalent of the furfuryl group was confirmed by

the appearance of furfuryl proton peaks in the 1H NMR spectrum, as shown in Figure 5-

7 A, and the increase in molecular weights by SEC (Table 5-2, polymers 2, 3). Benzyl

amine was subsequently used for the second conjugation at 80 °C. Appearance of

benzyl proton peaks in the 1H NMR spectrum confirms the incorporation onto the TCT

ring, however, conjugation of benzyl amine to the furfuryl amine-TCT conjugate polymer

resulted in a decrease in molecular weight by SEC (Table 5-2, polymer 4). Previously,

we demonstrated that functionalization of TCT with amines led to reduced

electrophilicity of the triazine due to good π-orbital overlap of the ring with the lone pair

of the nitrogen. Conversely, with thiols, the larger size of sulfur decreases electron density donation of the lone pairs into the triazine, thereby causing the triazine ring to be more eletrophilic. We therefore believe that the deactivation of the triazine when nitrogen nucleophiles are added first enables aminolysis of the ester backbone, rather than the preferential reaction with the triazine. As shown in Figure 5-7, functionalization

of polymer 3 (furfuryl thiol-conjugated TCT-copolymer), proceeded with quantitative

functionalization and no appreciable aminolysis of the polymer backbone. The

molecular weight of polymer 5 is higher than expected, likely due to a loss of low MW

sample during purification by dialysis. Given the sensitivity of CKAs to many reactive

moieties, post-polymerization of TCT-functional copolymers enables the synthesis of responsive or functional moieties that would otherwise be incompatible with the initial polymerization.

Figure 5-8. Functionalization of poly(BMDO-alt-TCTMI) with furfuryl thiol and benzyl amine. A) Reaction scheme of heterodifunctionalization of TCT-functional polyester and B) 1H NMR spectrum overlay of polymer functionalizations, 1 – poly(BMDO-alt-TCTMI), 3 – functionalization with furfuryl thiol, and 5 – functionalization with benzyl amine.

5.3 Conclusion

In conclusion, we demonstrated a synthetic route to alternating, degradable, and

tunable polyester copolymers using radical ring-opening copolymerization. Quantitative

ring-opening of the CKA to the ester was realized with optimized conditions using

BMDO as the CKA comonomer and was confirmed by 13C NMR spectroscopy. The

alternating tendency of the copolymerization was demonstrated by MALDI-ToF MS analysis of RAFT-derived BMDO copolymers with NEtMI and NBnMI. The ability to tune polymer thermal properties was demonstrated by altering the N-substituent of the maleimide, providing access to polyesters with Tg’s ranging from 3 – 127 °C. Finally, we developed a functional polyester copolymer using a TCT-conjugated maleimide comonomer, allowing the incorporation of two distinct nucleophiles post-polymerization.

The TCT-functional polyester provides a route for the synthesis of a library of functional or responsive degradable copolymers, or the incorporation of functionality that may be incompatible with the polymerization or the CKA comonomer.

5.4 Materials and Methods

5.4.1. Materials

147 N-(methoxy triethylene glycol) maleimide (NEG3MI), N-(2-hydroxyethyl)-

maleimide,148 and dodecylsulfanylthiocarbonylsulfanyl-2-methyl-propionic acid chain transfer agent149 (CTA) were prepared as previously reported. Diethyl ether (Fisher,

ACS grade), anhydrous tetrahydrofuran (THF, Fisher, HPLC grade), anhydrous toluene

(Fisher, HPLC grade), bromoacetaldehyde dimethyl acetal (Acros Organic, 97%),

lithium aluminum hydride (LiAlH4, Sigma-Aldrich, 95%), dimethyl phthalate (Alfa Aesar,

99%) Dowex50WX8 (H+) resin (Alfa Aesar), Aliquat ® 336 (Alfa Aesar), potassium tert- butoxide (Chem-Impex, 99.28%), furan (Alfa Aesar, 99%), maleimide (Acros Organic,

98%), triethylene glycol monomethyl ether (TCI, 98.0%), 4-toluenesulfonyl chloride

(Acros Organic, >99%), 4-dimethylaminopyridine (DMAP, Acros, 99%), N-ethyl maleimide (NEtMI, Alfa Aesar, 98%), N-benzyl maleimide (NBzMI, Aldrich, 99%), N- phenyl maleimide (NPhMI, Aldrich, 97%), 2,4,6-trichloro-1,3,5-triazine (TCT, Sigma-

Aldrich, 99%), chloroform-d (CDCl3, 99.5%, Cambridge Isotope Laboratories,

dimethylsulfoxide-d6 (DMSO-d6, 99.9%, Cambridge Isotope Laboratories) were used as

received. 2,2’-Azobisisobutyronitrile (AIBN, 98%, Sigma-Aldrich) was recrystallized from

ethanol and maleic anhydride was recrystallized from methanol and chloroform.

5.4.2 Analytical Techniques

1H and 13C NMR spectroscopy were recorded on an Inova 500 MHz

spectrometer at 25 °C in CDCl3 or DMSO-d6. Size exclusion chromatography (SEC)

analysis was performed in N,N-dimethylacetamide with 50 mM LiCl 50 °C and a flow

rate of 1.0 mL min−1 using an agilent isocratic pump, degasser, and autosampler with

Viscogel I-series 10 μm guard + two ViscoGel I-series G3078 mixed bed columns:

molecular weight range 0−20 × 103 and 0−100 × 104 g mol−1 columns. Detection

consisted of a Wyatt Optilab T-rEX refractive index detector operating at 658 nm and a

Wyatt miniDAWN Treos light scattering detector operating at 659 nm. The system was

calibrated using 10 polystyrene (PS) standards from 9.88 x 105 to 602 g.mol-1. Matrix

assisted laser desorption/ionization time-of-flight (MALDI-TOF) was performed on an AB

Sciex 5800 MALDI TOF/TOF (Framingham, MA) spectrometer operated in linear,

positive ion mode with a 1 KHz N2 OptiBeam™ on-axis laser. Laser power was used at

the threshold level required to generate signal. Spectra were analyzed using Polymerix

Version 3 software (Sierra Analytics). Samples were prepared by mixing solutions of

trans-2-[3-(4-t-butylphenyl)-2-methyl-2-propenylidine]malononitrile (DCTB, Santa Cruz

Biotechnology, ≥99%) matrix (10 mg/mL in THF) and degraded product (1 mg/mL in

THF) at a v:v ratio of 5:1 matrix:product and 2.00 µL were spotted and air dried on a

polished stainless steel Bruker plate (residual potassium from polymer degradation was

used as the ionizing salt). Thermogravimetric analyses (TGA) were measured under

nitrogen with a TGA Q5000 from TA Instruments (10 °C/min). Decomposition

temperature (Td) is determined as the temperature at which 5% mass loss is observed.

Differential scanning calorimetry (DSC) theromograms were obtained with a DSC

Q1000 from TA instruments. Reported data are from the second heating cycle (10

°C/min). Dynamic light scattering (DLS) analysis was conducted at room temperature on

a Zetasizer Nano-ZS (Malvern) operating at a wavelength of 633 nm. UV−vis and

fluorescence spectroscopy measurements were taken on a Molecular Devices

SpectraMax M2Multimode Microplate reader with a λexcitation of 530 nm and a

λemission of 650 nm.

5.4.3 Experimental Procedures

5.4.3.1 Synthesis of BMDO

Synthesis of 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) was modified from

previous procedures.142,143 1,2-Benzenedimethanol was first synthesized by placing

LiAlH4 (5.0 g, 0.13 mol) into a dry three-neck round bottom flask with an addition funnel

and condenser. THF was added (100 mL) and the system was purged with argon.

Dimethyl phthalate (18 mL, 0.11 mol) in 100 mL THF was added and let reflux overnight

(16 h). The mixture was worked up by adding 50 mL acidic water (25% H2SO4 v/v), then

extracting with diethyl ether x3), washing with water, and drying over magnesium

sulfate. Diethyl ether was removed under vacuum and the product was recrystallized

from hexane and chloroform (1:1) in a 65.2 % yield. Next, 1,2-benzenedimethanol (10.3

mL, 68.8 mmol) was added a round bottom flask and heated with bromoacetaldehyde

dimethyl acetal (9.5 g, 68.8 mmol) at 120 °C with Dowex 50 (H+) resin (100 mg) for 15

h, using a distillation apparatus to collect methanol. The Dowex resin was filtered off

and all remaining methanol was removed by rotary evaporation. Fractional distillation

was used to purify the product, 5,6-benzo-2(bromomethyl)-1,3-dioxepane in a 68.3 %

yield. BMDO was then synthesized by mixing 5,6-benzo-2(bromomethyl)-1,3-dioxepane

(8.35 g, 34.4 mmol) and Aliquat ® 336 (289 mg, 0.688 mmol) in 50 mL anhydrous THF.

The flask was cooled to 0 °C and potassium tert-butoxide (4.63 g, 41.2 mmol) was

slowly added through a powder funnel and stirred for 2 h at room temperature. Diethyl ether was added and filtered using vacuum filtration. The collect liquid was concentrated by rotary evaporation and purified by fractional distillation to give the product, BMDO, as

a white solid in an 87.9 % yield (Fig. 5-9).

1 Figure 5-9. H NMR spectra of BMDO in CDCl3

5.4.3.2 Synthesis of 2,4,6-trichloro-1,3,5-triazine (TCT)-maleimide conjugate

TCT (1.96 g, 10.6 mmol) was added to a stirred solution of N-(2-hydroxyethyl)- maleimide (1.00 g, 7.09 mmol) and DIPEA (1.85 mL, 10.6 mmol) in THF (35 mL) at 0

°C. The solution was stirred for 16 h at room temperature. The reaction solution was filtered and concentrated, and the crude material was purified by flash chromatography to yield the product as an off-white solid.

1 Figure 5-10. H NMR spectra of TCT-MI in CDCl3

5.4.3.3 Conventional copolymerization of BMDO with N-ethyl maleimide

NEtMI (125.2 mg, 1.54 mol) and AIBN (5.1 mg, 0.03 mmol), were dissolved in 2.0 mL toluene in a 20 mL vial. BMDO (253 mg, 1.55 mmol) in 2.0 mL toluene was added to the vial. An aliquot of the solution was removed for 1H NMR analysis. The solution was

placed in an oil bath at 85 °C for 3 h and precipitated into diethyl ether. Precipitate was

collected by filtration and analyzed by 1H NMR spectroscopy and SEC.

5.4.3.4 Conventional copolymerization of BMDO with N-substituted maleimides

Copolymerizations of BMDO and maleimides were synthesized using the ratio

BMDO:maleimide:AIBN = 1:1.5:0.02 in toluene at 85 °C with a total monomer concentration of 0.5 M. The reactions were monitored by 1H NMR spectroscopy and

stopped by removing from heat and exposing to air once monomer conversion reached

> 90% (typically 2-4 hours). The solution was precipitated into diethyl ether (x2) and

analyzed by 1H NMR spectroscopy and SEC.

5.4.3.5 Degradation of copolymers

Copolymers and block copolymers were degraded by dissolving P(BMDO-co-MI)

(50 mg) in THF (2 mL) and adding potassium hydroxide solution (KOH, 10%) in

methanol (1 mL). The solution immediately turned dark red upon addition of KOH. SEC was used to analyze degradation products and complete degradation was achieved in

10 minutes.

5.4.3.6 Functionalization of Poly(BMDO-alt-TCT-MI)

Synthesis of 2

Furfurylamine (0.046 mL, 0.52 mmol) was added to a stirred solution of polymer

1 (250 mg, 0.523 mmol) and DIPEA (0.096 mL, 0.55 mmol) in THF (2.5 mL) at 0 °C.

The reaction mixture was stirred at 0 °C for 3 h, warmed to room temperature over ca. 1 h, and stirred for an additional 12 h. The solution was dialyzed against THF and concentrated. The resulting material was dissolved in THF, precipitated into cold diethyl ether, filtered, and dried to yield polymer 2 as an off-white solid.

Synthesis of 3

Furfuryl mercaptan (0.053 mL, 0.52 mmol) was added to a stirred solution of polymer 1 (250 mg, 0.523 mmol) and DIPEA (0.096 mL, 0.55 mmol) in THF (2.5 mL) at

0 °C. The reaction mixture was stirred at 0 °C for 3 h, warmed to room temperature over ca. 1 h, and stirred for an additional 12 h. The solution was dialyzed against THF and concentrated. The resulting material was dissolved in THF, precipitated into cold diethyl ether, filtered, and dried to yield polymer 3 as an off-white solid.

Synthesis of 4 Benzylamine (0.040 mL, 0.37 mmol) was added to a stirred solution of polymer 2

(180 mg, 0.334 mmol) and DIPEA (0.063 mL, 0.37 mmol) in dioxane (1.8 mL). The

reaction mixture was stirred at 80 °C for 16 h. The solution was cooled to room

temperature, dialyzed against THF, and concentrated. The resulting material was

dissolved in THF, precipitated into cold diethyl ether, filtered, and dried to yield polymer

4 as an off-white solid.

Synthesis of 5 Benzylamine (0.039 mL, 0.36 mmol) was added to a stirred solution of polymer 3

(180 mg, 0.324 mmol) and DIPEA (0.062 mL, 0.36 mmol) in dioxane (1.8 mL). The

reaction mixture was stirred at 80 °C for 16 h. The solution was cooled to room

temperature, dialyzed against THF, and concentrated. The resulting material was

dissolved in THF, precipitated into cold diethyl ether, filtered, and dried to yield polymer

5 as an off-white solid.

5.4.3.7 RAFT copolymerization of BMDO and N-ethylmaleimide (NEtMI)

In a typical RAFT polymerization, NEtMI (260 mg, 2.07 mmol), AIBN (0.74 mg,

0.0045 mmol), CTA (17.0 mg, 0.046 mmol), and 4.5 mL toluene were added to a 20 mL vial and purged with argon for 15-25 min. In a 25 mL schlenk flask, BMDO (369 mg,

2.22 mmol) in 4.5 mL toluene was purged with argon for 15-25 min. The NEtMI, AIBN,

and CTA solution was then added to the schlenk flask via a purged syringe and placed

in an oil bath at 85 °C. Monomer conversion was monitored using 1H NMR spectroscopy

of sample aliquots, by monitoring the disappearance of the vinyl peaks of BMDO and

NEtMI. NEtMI conversion was used for kinetic data as BMDO degradaded during the

reaction upon adding NMR solvent. Polymerizations were terminated by removing from

heat and exposing to air and purified by precipitating into diethyl ether 2-3 times.

CHAPTER 6 CONCLUSION

The increased utilization of responsive polymers in medicine and materials has highlighted the necessity of the eventual breakdown and degradability of these materials within our lifetimes. The goal of this dissertation was to develop useful responsive and degradable polymer-based systems.

Many polymeric drug-delivery vehicles have been synthesized for the site- specific release of drugs to a targeted area within the human body. Here, polymeric drug-delivery vehicles were synthesized for the site-specific release of drugs into the phloem of plants. The increased pH of phloem tissue compared to the rest of plant tissue was used as the endogenous stimulus for which our delivery system could respond. The delivery system, derived from L-aspartic acid, demonstrated controlled release at pH greater or equal to 7, and exhibited minimal toxicity to plant tissue. The responsive drug-delivery system described has significant potential in applications in agriculture given the straight-forward synthesis using the inexpensive and sustainable amino acid, L-aspartic acid, the response to alkaline pH, and the resulting polymer after delivery, water-soluble and degradable polyaspartic acid.

In order to encourage the increased production of responsive materials with degradable polymer backbones, it is necessary to have access to various synthetic techniques to achieve the desired polymer properties and functionality. Here, the synthesis of degradable and tunable polyester-based copolymers was demonstrated using radical ring-opening polymerization. Radical polymerization is desirable due to its relatively mild and robust reaction conditions, and the ability to employ many of the

RDRP methods that have emerged over the last 20 years. Cyclic ketene acetals (CKAs)

100

were copolymerized with maleimides, following the concepts of donor-acceptor radical copolymerization (e.g. styrene-maleic anhydride), and were demonstrated to proceed in an alternating fashion. The high incorporation of CKAs and the high degree of ring- opening to esters led to rapid degradation of the copolymers and block-copolymers.

Finally, the ability to easily tune the polymer properties or to incorporate functionality was demonstrated by altering the N-substituent of the maleimide comonomer. The ability to use one copolymerization system to access degradable polymers with a range of thermal properties and functionality is highly desirable to streamline the synthesis of degradable polymers that can be applied to many different applications.

101

APPENDIX A LOADING OF MODEL HYDROPHOBIC MOLECULE NILE RED

Figure A-1. (a) Fluorescence calibration of Nile Red in DMF with λexcitation = 530 nm and λemission = 650 nm, (b) Fluorescence intensity of loaded 1% PSI-HA nanoparticles compared to Nile red in water and, (c) TEM images of 50% PSI- HA nanoparticles before Nile red loading (left, clear), and after loading (right, pink).

102

APPENDIX B MALDI-ToF OF DEGRADED P(MPDL-ALT-NETMI)

Figure B-1. MALDI-ToF spectrum of degraded P(MPDL-alt-NEtMI) (conventionally polymerized with excess MPDL, [MPDL]:[NEtMI]:[AIBN] = 70:30:2).

Figure B-2. MALDI-ToF spectrum, showing different degrees of hydrolyzed maleimide.

103

APPENDIX C REACTION OF BMDO WITH MALEIC ANHYDRIDE

Equimolar amounts of MAnh (48.9 mg, 0.5 mmol) was and BMDO (81.15 mg, 0.50 1 mmol) were mixed in CDCl3 and let sit overnight at room temperature. H NMR spectroscopy showed consumption of BMDO into degradation products or cycloreactions with maleic anhydride.

1 Figure C-1. H NMR spectra of BMDO and maleic anhydride in CDCl3 A) immediately after mixing together and B) after 16 hours at room temperature.

104

APPENDIX D ANALYSIS OF BMDO AND N-SUBSTITUTED MALEIMIDE COPOLYMERS

1 Figure D-1. Poly(BMDO-co-NEtMI): A) H NMR spectrum in DMSO-d6; B) SEC trace; C) TGA thermogram; and D) DSC curve.

1 Figure D-2. Poly(BMDO-co-NEG3MI): A) H NMR spectrum in DMSO-d6; B) SEC trace; C) TGA thermogram; and D) DSC curve.

105

1 Figure D-3. Poly(BMDO-co-NPhMI): A) H NMR spectrum in DMSO-d6; B) SEC trace; C) TGA thermogram; and D) DSC curve.

1 Figure D-4. Poly(BMDO-co-NBnMI): A) H NMR spectrum in DMSO-d6; B) SEC trace; C) TGA thermogram; and D) DSC curve.

106

1 Figure D-5. RAFT-derived poly(BMDO-co-NBmMI): A) H NMR spectrum in DMSO-d6; B) SEC trace; C) TGA thermogram; and D) DSC curve.

1 Figure D-6. Poly(BMDO-co-NEtMI-co-NBnMI-co-NEG3): A) H NMR spectrum in DMSO-d6; B) SEC trace; C) TGA thermogram; and D) DSC curve.

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BIOGRAPHICAL SKETCH

At age 11, Megan moved to Davis, California with her mother and her sister.

While she was born and lived in Salt Lake City, Utah prior to then, she refers to herself as a Californian – as many people who have ever spent any time living in California do.

Davis lies 20 minutes west of the state’s Capitol, Sacramento, and 45 minutes east of the state’s grapes, Napa. The town is centered around University of California, Davis, which puts an interesting academic and technological presence in the middle of an otherwise free-loving, tree-hugging, environmentally-conscious city.

After graduating Davis High School in 2007, she attended California Polytechnic

State University (Cal Poly) in San Luis Obispo, California, majoring in Chemistry. Rather than quickly switching majors after her first semester of classes, as her mother expected, she became even more enthralled and involved in chemistry. She became a resident advisor in the science and math dorms, joined the chemistry fraternity Alpha

Chi Sigma, and began research in the laboratory of Prof. Philip Costanzo on the synthesis of functional polymers. During her undergraduate career, Megan was also able to spend a year in Uppsala, Sweden as an exchange student, where she primarily engaged in fika, but was able to occasionally dabble with the ring-opening polymerization of lactides under the guidance of Prof. Jöns Hilborn.

Upon her return to California, Megan continued her work in the Costanzo lab.

During her last summer at Cal Poly, she conducted a Research Experience for

Undergraduates program at Southern Methodist University, in Dallas, Texas, in the laboratory of Prof. Brent Sumerlin. During her stay, Prof. Sumerlin swindled Megan into going to graduate school and working under him at the University of Florida. Megan was granted the National Science Foundation for Graduate Research Fellows during her

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second year at the University of Florida, and spent her graduate career researching the development of responsive and degradable polymeric materials. She completed her

Ph.D. in chemistry in the spring of 2018.

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